Selected Abstracts from Pharmacology 2024

16

The effect of fluoxetine on diffused intrinsic pontine glioma (DIPG), ICR-B169 cells and HSJD-DIPG-007 cells

F. Javid

Department of Pharmacy, University of Huddersfield

Introduction

Diffuse intrinsic pontine glioma (DIPG) is an aggressive glial tumour with a median survival of 9–12 (Malbari, 2021; Van Genechten et al., 2024). Our previous studies have shown that fluoxetine (Prozac), the well-known antidepressant which is a selective serotonin uptake inhibitor (SSRI), induced cytotoxicity in human colon carcinoma cells (Marcinkute et al., 2019). The aim of the present study was to explore if fluoxetine could also induce cytotoxicity in patient-derived ICR-B169 2D and HSJD-DIPG-007 cells. These cells represent a type of diffuse intrinsic pontine glioma (DIPG). The cells were kindly donated by ICR.

Method

The ICR-B169 and HSJD-DIPG-007 cells were maintained and subcultured as per recommended guidelines. When cells reached 70% confluence, they were seeded in 96-well plates and treated with fluoxetine and temozolomide at different concentrations (1 nM–100 μM) or vehicle control. After 96 h contact time, cell viability was assessed using Cell-Titre Glo-2D assay. Experiments were repeated independently four times. Cell viability at each concentration was recorded and half maximal inhibitory concentration (IC50) was calculated. Results were expressed as the mean ± s.e. mean of N = 5.

Results

Pre-treatment with fluoxetine (1 nM–100 μM) induced cytotoxicity in a concentration dependent manner in both cell lines. The cell viability was reduced when compared to control. The cytotoxicity was induced at micromolar range. The IC50s were 16.1 ± 1.9 and 11.3 ± 3.3 μM, compared with IC50s induced by temozolomide, 37.8 ± 2.4 and 55.61 ± 11.4, in ICR-B169 and HSJD-DIPG-007 cells, respectively.

Conclusions

Fluoxetine induced significant cytotoxicity at micromolar concentrations in brain cancer cells. Further studies should be carried out to investigate the mechanisms of action that underpin the observed in vitro cytotoxic effect.

References

1. Malbari F. (2021). Pediatric Neuro-Oncology. 39(3):829-845.

2. Marcinkute M., et al. (2019). Fluoxetine selectively induces p53-independent apoptosis in human colorectal cancer cells. Eur J. Pharm., 857: 172441-172450.

3. Van Genechten T. et al., (2024). Adjuvant Wilms' tumour1-specific dendritic cell immunotherapy complementing conventional therapy for paediatric patients with high -grade glioma and diffused intrinsic pontin glioma: protocol of a monocentric phase I/II clinical trial Belgium. BMJ Open. 14(3):e077613.

64

Modifying tumour blood vessels to improve cancer immunotherapy

M. Hillgaertner¹, A. Gallimore¹, R. Andrews¹, A. Godkin¹, S. Milutinovic¹, S. Lauder¹, K. Smart¹, C. Von Ruhland² and M. Somerville¹

¹Systems Immunity Research Institute, School of Medicine, Cardiff University; ²Central Biotechnology Services, School of Medicine, Cardiff University

Introduction/Aims

High endothelial venules (HEV) are specialised blood vessels found in secondary lymphoid organs and involved in lymphocyte trafficking. HEV can develop in human solid tumours (TA-HEV), where they are associated with high numbers of infiltrating T-cells and improved immunotherapy effectiveness [1]. Our lab uses a mouse model (C57BL/6) of carcinogen-induced tumours where depletion of immunosuppressive regulatory T-cells results in two groups: ‘Regressors’, characterised by TA-HEV development and tumour growth control, and ‘Non-regressors’ lacking both TA-HEV and tumour control. The aim of this project is to determine the role of TA-HEV and how they can be harnessed to improve immunotherapy success.

Methods

TA-HEV, basal tumour blood vessels (TA-BV) and lymph node HEV (LN-HEV) were compared using a variety of methods. Transmission electron microscopy (TEM) was conducted to examine vessel morphology. Two tumours and three LN were imaged. Bioinformatic analyses of RNA sequencing data (RStudio) were performed to identify differentially expressed genes and select candidate genes of interest. Immunofluorescence staining was done to quantify expression of candidate proteins. Staining was carried out on five tumours and three LN. Qupath was used to generate pixel classifiers to quantify protein expression.

Results

TEM revealed that TA-HEV differed from LN-HEV in that they were smaller and contained fewer cells than LN-HEV (Figure 1A + B) and had fewer lymphocytes crossing the HEV endothelium (Figure 1C + D). Bioinformatic analysis revealed differential expression of genes related to lymphocyte adhesion and trafficking (Emcn, Lgals3, Selp, Ednrb, Cxcl9 and Cxcl12) between TA-HEV and TA-BV. Immunostaining of endomucin, encoded by Emcn, revealed that endomucin expression was significantly lower on TA-HEV compared to TA-BV but higher on TA-HEV compared to LN-HEV (Figure 2).

Conclusions

The results obtained in this study indicate that whilst TA-HEV are associated with higher frequencies of tumour infiltrating lymphocytes, they may not support lymphocyte migration as effectively as LN-HEV. Preliminary data demonstrate that TA-HEV but not LN-HEV express endomucin, an adhesion molecule previously shown to negatively affect T-cell trafficking. Endomucin may therefore represent a target whereby lymphocyte trafficking via TA-HEV can be improved.

Reference

1. Milutinovic S & Gallimore A (2023) The link between T cell activation and development of functionally useful tumour-associated high endothelial venules. Discov Immunol. 2, (1), kyad006. https://doi.org/10.1093/discim/kyad006.

118

Determining which β-blocker may have the best molecular pharmacology properties as an anti-cancer agent

J. Baker

University of Nottingham

Introduction/ Background and Aims

Many cancers express β2-ARs (adrenoceptors) and some express β1-ARs. β-agonists increase cancer growth and metastasis and β-antagonists (β-blockers) reduce these. β-blockers (particularly propranolol) are being investigated as potential adjuvant cancer therapy [1]. To minimise both tumour growth and metastatic potential, theoretically, the ideal anti-cancer β-blocker may be one with high affinity, no partial agonism, and long duration of action at β2 and ideally β1-AR. This study assessed clinically available β-blockers for these properties.

Method/Summary of Work

CHO cell lines stably expressing the human β1 or β2-AR and a CRE-SPAP reporter gene were used. Ligands affinity was assessed by 3H-CGP12177 competition binding in whole cells where competing ligand and 3H-CGP12177 were are incubated together for 2 h [2]. Duration of binding was assessed by washout: competing ligand was incubated for 2 h, washed out, then radioligand incubated alone for 2 h [2]. The degree of rightward shift relates to the duration of binding (shorter duration ligands washed out giving larger shift). Function was determined using CRE-SPAP reporter assay.

Results

Antagonist KD values are given in Table 1. Some ligands were readily washed out (e.g., nadolol, Figure 1, Table 1). Other ligands inhibited 3H-CGP12177 even after washout demonstrating their residual presence (long duration, e.g., carvedilol, Figure 1). For a few ligands, the washout curve was biphasic (e.g., timolol, Figure 1). The reason for this is unknown. The more promising ligands were assessed for agonist responses (Table 2).

Conclusions

Propranolol, bupranolol and nadolol had high affinity and little agonist action, but a short duration of action. Bucindolol and cyanopindolol had high affinity and long duration however their substantial partial agonist activity may risk stimulating cancer growth. Carvedilol, whose low level of partial agonism appears not clinically relevant in cardiovascular systems [3] and is in widespread clinical use, may have better overall balance of these properties as an anti-cancer agent than propranolol.

References

1. Carnet Le Provost K, Kepp O, Kroemer G, Bezu L (2023) Trial watch: Beta-blockers in cancer therapy. Oncoimmunology. 12(1):2284486.

2. Baker JG, Proudman RGW, Hill SJ (2015) Salmeterol's extreme β2-selectivity is due to residues in both extracellular loops and transmembrane domains. Mol Pharmacol. 87: 103-120.

3. Baker JG, Kemp P, March J, Fretwell L, Hill SJ, Gardiner SM (2011) Predicting in vivo cardiovascular properties of β-blockers from cellular assays: A quantitative comparison of cellular and cardiovascular pharmacological responses. FASEB J 25: 4486-4497.

128

Evaluation of an adenovirus-10 precision virus directed enzyme prodrug therapy (VDEPT) system for use in breast cancer

T. Hepburn^1,2, E. Bates¹ and A. Parker¹

¹Cardiff University; ²St James University Hospital

Introduction

Cancer is a cause of death worldwide. Current cancer adenoviral therapies use Human adenoviruses (HAdV) as biological vectors. These DNA viruses have a capsid with fibre proteins which bind, and aid internalisation of the virus to a target cell. An amino acid sequence (A20) can be genetically inserted into the fibre protein so it will target avβ6-expressing cells, such as breast cancer cells. Upon replication, it will transcribe genes to produce proteins. The transgene FCU1, a cytodeaminase fusion protein from yeast, can be genetically inserted to transcribe a specific pro-drug activating enzyme. When these transduced cells are treated with the pro-drug, 5-Flurouracil (5-FC), FCU1 mediates the conversion into its toxic metabolites which kills the cell.

Currently, therapy is limited due to a naturally high pre-existing immunity to the common HAdV5 vector. We aimed to evaluate the effect of a novel precision virus, HAdV10.A20.FCU1, in combination with 5-FC, on breast cancer cell viability and evaluate the effect of human serum on the virus, compared to HAdV5.

Methods

The vector was created by replacing the E1 region with the transgene FCU1 under control of the CMV promoter, and the A20 peptide was inserted into the DG loop of the fibre knob. This virus was added to breast (BT20) and lung (A549) cells and incubated with 5-FC. The CellTitreGlo cell viability assay was then used to measure cell killing. Controls used were HAdV10.GFP (contained the GFP fluorescence transgene) and HAdV10.FCU1 (contained FCU1 but not specific to avβ6 expressing cells), and lung cancer cells (did not express avβ6 for HAdv10.A20 to enter).

Results

IC50 values identified the concentration (mM) of 5-FC required to reduce cell viability to 50%. A lower IC50 value meant the virus worked better to kill cells. In breast cancer cells, HAdV10.A20.FCU1 had the lowest IC50 (0.06 mM) compared to HAdV10.FCU1 (0.54 mM), HAdV10.GFP (cell viability > 50%) and HAdV5.FCU1 (4.5 mM). HAdV10.A20.FCU1 and HAdV5.FCU1 were neutralised by human serum (with cell viability > 50%). HAdV10.A20.FCU1 also killed lung cancer cells (IC50: 0.04 mM), showing it was not exclusively selective to breast cancer cells.

Conclusions

This study has shown promising data that HAdV10.A20.FCU1 can reduce breast cancer cell viability. The neutralising serum experiments should be repeated with serum from multiple donors to better represent HAdV10 pre-existing immunity in the population. To improve the vectors, they could be made more specific to breast cancer cells by ablating CAR binding.

156

Modelling the relationship between erlotinib and transforming growth factor beta: An important cytokine for interstitial lung disease

L. Wanika, N. Evans and M. Chappell

University of Warwick

Introduction

Approximately 1% of patients who are diagnosed with non-small cell lung cancer (NSCLC) and are treated with tyrosine kinase inhibitors (TKIs), such as erlotinib, may develop interstitial lung disease (ILD). Unfortunately, the mechanism for TKI induced ILD is not fully understood [1]. A potential pathway for erlotinib induced ILD can be simulated through increased expression of transforming growth factor beta (TGFβ). TGFβ has been shown to promote pulmonary fibrosis [2]. The aim of study is to model erlotinib induced increased TGFβ expression using in vitro data and mixed effects modelling within Monolix.

Method

Published phosphorylation assays and gene expression analyses from multiple NSCLC cell lines were used to build the erlotinib dataset. An in vitro PK/PD model was developed to simulate the following postulated pathway:

Erlotinib inhibits the phosphorylation of epidermal growth factor receptor (EGFR) and subsequently protein kinase B (AKT). A decrease in phosphorylated AKT decreases the expression of the mouse double minute 2 homolog gene (MDM2) which increases the expression of tumour protein P53. The increased expression of P53 leads to an increased expression of thrombospodin-1 (TSP1) and TGFβ.

Results

The model predicted higher production rates of phosphorylated EGFR and AKT compared to their respective degradation rates.

Table 1. Summary of the estimated population parameter values.

Conclusion

The mixed effects model was able to estimate population parameter values with RSE values less than 50% for the erlotinib induced increased TGFβ expression pathway. Further work will explore the incorporation of other growth factor receptors as well as other cytokines which may play a key role in ILD.

References

1. Higenbottam T, Kuwano K, Nemery B, Fujita Y. Understanding the mechanisms of drug-associated interstitial lung disease. British Journal of Cancer. 2004;91(2):S31-S37

2. Penn JW, Grobbelaar AO, Rolfe KJ. The role of the TGF-β family in wound healing, burns and scarring: A review. Int J Burns Trauma. 2012;2(1):18-28.

191

Antiproliferative effect of Alpinia galanga against human non-small-cell lung cancer cell lines (A549)

P. Katanyutanon

School of Medicine, University of St Andrews

Introduction and aim

Cancer is the type of disease that results from cells mutating—most cancer is diagnosed when the disease gets out of control. Pulmonary cancer is the type of cancer that causes the most deaths worldwide. There are currently many chemotherapeutic drugs used for cancer treatment; however, cancer cells can become resistant to these drugs. Therefore, an effort to explore novel drugs to fight against cancer is still required. With this in mind, along with the rapid progress in the phytochemical studies of plants, many functional foods are becoming popular due to their anticancer effects. This study aims to determine anticancer activity of Alpinia galangal (A. galangal) extract against non-small-cell lung cancer cell lines (NCI-H460).

Methods

In this study, A. galanga extract was extracted using 90% ethanol. This extract was then studied to see the cytotoxic activity by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The A. galanga extract was incubated at increasing concentration (0–320 μg·ml⁻¹) for 24, 48 and 72 h, respectively, against pulmonary cancer cells. 5-Fluorouracil was used as a positive control in this study.

Results

The result showed that the half-maximal inhibitory concentration (IC50) of A. galangal extract at 24, 48 and 72 h were 39.56 ± 0.361, 25.87 ± 0.204 and 11.25 ± 0.104 μg·ml⁻¹, respectively.

Conclusions

The findings suggested that A. galanga possessed potent anticancer activity against pulmonary cancer. Further studies regarding the mechanism of action would be required to ensure the safety of using this extract as a novel drug, especially with people who may have other underlying diseases or are on a steady prescription.

Keywords

Alpinia galanga; anticancer activity; antiproliferative activity; MTT assay; pulmonary cancer

References

1. Siegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. CA: a cancer journal for clinicians. 2024 1;74(1).

2. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68(6):394-424. https://doi.org/10.3322/caac.21492. PMID: 30207593.

3. Herbst RS, Morgensztern D, Boshoff C. The biology and management of non-small cell lung cancer. Nature. 2018;553(7689):446-454. https://doi.org/10.1038/nature25183. PMID: 29364287.

4. Planchard D, Besse B. Lung cancer in 2015: Lung cancer in 2015: Progress, but still a lot more to do! Nat Rev Clin Oncol. 2016;13(2):71-2. https://doi.org/10.1038/nrclinonc.2015.229. PMID: 26750815.

5. Holohan C, Van Schaeybroeck S, Longley DB, Johnston PG. Cancer drug resistance: An evolving paradigm. Nat Rev Cancer. 2013;13(10):714-26. https://doi.org/10.1038/nrc3599. PMID: 24060863.

6. Gottesman MM. Mechanisms of cancer drug resistance. Annu Rev Med. 2002;53:615-27. https://doi.org/10.1146/annurev.med.53.082901.103929. PMID: 11818492.

211

Farnesoid X receptor: A potential modulator of Treg polarization and immune metabolic rewiring in hepatocellular carcinoma

Y. Attia¹, A. Ali², R. Tawfiq¹, O. Hammam³ and M. Elmazar¹

¹Pharmacology Department, Faculty of Pharmacy, The British University in Egypt; ²Health Research Center of Excellence, Drug Research and Development Group, Faculty of Pharmacy, The British University in Egypt; ³Pathology Department, Theodor Bilharz Research Institute

Introduction

The liver, once considered ‘immune-privileged’, harbours a dynamic immune landscape. In hepatocellular carcinoma (HCC), the tumour microenvironment is often immunologically ‘cold’, due to T regulatory cells (Tregs) that subdue antitumor immune responses by promoting a tolerogenic milieu via immunosuppressive cytokines, particularly, IL-10 and TGF-β. While the farnesoid X receptor (FXR) has gained attention for its ‘metabolic’ and ‘immunomodulatory’ roles in various settings, how it might impact Tregs in HCC remains unresolved. This study, therefore, aims to decipher whether FXR modulation, by obeticholic acid (OCA), can influence Treg polarization in experimental HCC.

Method

HCC was induced in 3-week-old Swiss albino male mice using diethylnitrosamine (DEN; single i.p. dose of 1 mg·kg⁻¹) and carbon tetrachloride (CCl4; twice/week i.p. injections of 0.2 ml·kg⁻¹). After 20 weeks, mice received OCA (10 mg·kg⁻¹·day⁻¹, p.o., for 84 days). Histopathological examination besides alpha-fetoprotein (AFP) immunohistochemical analysis were performed. To assess FXR activation, hepatic CYP7A1, an FXR target gene, was measured using ELISA. The impact of FXR activation on Treg polarization was explored by measuring hepatic gene expression of Treg markers, Foxp3 and IL-2RA. Hepatic levels of TGF-β1 and its signalling activity, p-SMAD2/3, along with IL-10, were measured using ELISA. Data are presented as mean ± SD (n = 5–6). Statistical significance (P < 0.05) was determined using one-way ANOVA followed by Tukey's post hoc test.

Results

Histopathological examination demonstrated OCA's potential to alleviate HCC in DEN + CCl4-treated mice. Moreover, AFP immunoreactivity was curbed in the OCA-treated group (14 ± 4.18 versus 43 ± 5.7% in DEN + CCl4 control group). OCA also curbed hepatic CYP7A1 levels (2 ± 0.27 versus 6.74 ± 1.43 ng·mg⁻¹ protein in control), confirming FXR activation. Consistent with an immunosuppressive tumour microenvironment, the DEN + CCl4 group depicted increased hepatic expression of the Treg markers, Foxp3 (4.82 ± 0.57-fold change from normal) and IL-2RA (6.1 ± 0.56-fold change from normal), implying Treg enrichment. Furthermore, OCA down-regulated the gene expression of Foxp3 (2.39 ± 0.41-fold change) and IL-2RA (2.58 ± 0.8-fold change), suggesting a mitigation of Treg polarization (Figure 1). Additionally, OCA treatment reduced the hepatic levels of TGF-β1 (145.5 ± 5.52 versus 438.2 ± 9.7 pg·mg⁻¹ protein in control), p-SMAD2/3 (3.89 ± 0.44 versus 10.36 ± 0.13 pg·mg⁻¹ protein in control) and IL-10 (23.44 ± 3.45 versus 244.5 ± 6.54 pg·mg⁻¹ protein in control), alluding to a shift away from an immunosuppressive milieu.

Conclusion

223

Identification of potential clinical drug candidates for repurposing against prostate cancer: In vitro and in vivo efficacy in zebrafish xenografts

G. Brokalakis, S. Stavrou, C. Assiotis, G. Zinonos, T. Balabanidou, G. Koumourou, M. Phiniotou and N. Dietis

University of Cyprus Medical School

Introduction

Despite the growing interest in drug repurposing as a strategy to expedite therapeutic development, its application in prostate cancer remains underexplored. In this proof-of-concept study, we leveraged drug informatics databases and generative AI tools to identify clinically approved compounds with structural and pharmacodynamic similarities to standard prostate cancer treatments, evaluating their in vitro potential as anticancer agents.

Method

We combined data from the NIH Inxight Drugs database and ChatGPT-4 analysis module with appropriate prompting to identify clinical drugs with structural similarities with known anticancer agents. Thirteen clinical drugs were identified in silico as potential anticancer agents. We assessed their cytotoxic, antimigratory and antiproliferative efficacy in vitro against PC3 cells using the AlamarBlueHS, the scratch and the clonogenic assays, respectively, at different concentrations. The top-3 ranking drugs (lowest IC50 values or the lowest area under the curve from dose-response inhibitory graphs) were selected for in vivo drug screening against 2-day old zebrafish larvae xenografts injected with fluorescent-labelled PC3 cells (100 cells/animal). Animal viability at 5 days and metastatic index were assessed after treatment compared to vehicle control. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test, against appropriate controls (P < 0.05 significance).

Results

Paroxetine, piperidolate and prednisone demonstrated a dose-response inhibitory effect against PC3 cells and presented the highest cytotoxic efficacy compared to control (IC50 36.5, 48.7 and 152 μM, respectively; P < 0.01). The same drugs exhibited the highest inhibitory effect in cell migration compare to control (gap filled reduced by 72%, 65% and 55%, respectively; P < 0.01). Healthy 2-day-old zebrafish larvae treated by immersion with 300 μM of each drug showed no apparent behavioural and morphological toxicity, while none of them affected their overall viability (15-day cut-off). When drugs were administered at 30, 100 and 200 μM to 2-day-old zebrafish xenografts injected with PC3 cells, they showed a variable moderate inhibition against metastasis (20%–40% reduction in metastatic index; P < 0.05) but a significant increase in animal viability at cut-off (45%–60% increase in overall survival; P < 0.01). Paroxetine and piperidolate showed the highest anticancer effects both in vitro and in vivo in every assessed metric.

Conclusion

While paroxetine's in vitro anticancer effects have been previously demonstrated, this is the first pilot study to report its in vivo efficacy in zebrafish cancer xenografts. Additionally, we present novel evidence of piperidolate's antitumor potential both in vitro and in vivo against prostate cancer.

225

NRAS mutational status impacts FLT3-ITD mutant AML

F. Healy, V. Marensi, A. Turner, J. Woolley and D. MacEwan

University of Liverpool

Introduction

Acute myeloid leukaemia (AML) presents a significant clinical problem, with considerable heterogeneity and a lack of targeted therapeutic strategies contributing to poor prognosis and high mortality. Mutations in the myeloid-specific receptor tyrosine kinase FLT3 are most common, occurring in ~28% patients. Therefore, FLT3 presents a good therapeutic target, and small molecule inhibitors of FLT3 are now FDA-approved. However, resistance is emerging and has been attributed to the presence of NRAS mutations (McMahon et al., 2019). Here, we discuss the differences in phenotypic impacts conveyed by key clinical NRAS mutations in FLT3-ITD+ AML and assess potential risk to patients.

Methods

NRAS wild-type (WT), G12C, G12D and Q61K over-expressing MOLM-13 cells were generated using lentiviral transduction and selected using puromycin. Cytotoxicity assays were performed using 0.01–10 μM quizartinib (FLT3 inhibitor), cytarabine (AML chemotherapeutic) or sotorasib (KRAS G12C inhibitor), assessed by Annexin V-FITC/propidium iodide staining after 48 h. Western blotting was used to assess signalling alterations. RNASeq was performed to assess transcriptional alterations, with Fragments per Kilobase per Million counts used to represent gene expression level and calculate differences between cell lines. Colony forming assays were performed in 2.1% methylcellulose/RPMI-1640 + 10% FBS and counted 10 days post-plating.

Results

NRAS G12C, G12D and Q61K over-expression conferred increased proliferation 3-fold and increased colony forming capacity ~2.5-fold, compared to MOLM-13 cells or NRAS-WT over-expressing MOLM-13 cells. Transcriptomic profiling revealed distinct differences between mutational hotspot sites, with mutations occurring at G12 causing alterations to cellular redox activity, as opposed to mutations at Q61 affecting cytokine and receptor expression, stimulation and secretion. This included down-regulation of FLT3 in MOLM-13-NRAS-Q61K cells, verified by RNASeq and Western blotting, which was accompanied by resistance to quizartinib (MOLM-13 IC50 < 0.01 μM, MOLM-13-NRAS-Q61K IC50 > 10 μM). MOLM-13-NRAS-G12C cells exclusively exhibited sensitivity to sotorasib, which is known to act on multiple Ras isoforms, despite its initial designation as KRAS-only. Sensitivity to cytarabine was not significantly affected by NRAS over-expression. MAPK signalling (measured by phosphorylated ERK) increased in MOLM-13-NRAS-WT, MOLM-13-NRAS-G12C and MOLM-13-NRAS-Q61K, and PI3K-AKT (AKT) signalling increased in NRAS-WT over-expressing cells only, all compared to control.

Conclusions

Overall, NRAS-mutated AML confers a significantly increased proliferative capacity compared to NRAS-WT AML. This can be considered due to increased MAPK pathway signalling. Presence of NRAS-Q61K reduces FLT3 expression in FLT3-ITD+ AML, thereby decreasing sensitivity to FLT3 inhibitors and potentially rendering FLT3-ITD/NRAS-Q61K patients unsuitable for FLT3 inhibitor therapy.

Reference

1. McMahon. et al., Cancer Discov. 2019;9(8):1050-1063. https://doi.org/10.1158/2159-8290.CD-18-1453.

228

Bad to the bone: An RNA-sequencing study identifying a role for IKK alpha in osteosarcoma cell division and progression

K. Tinto, M. Bonfanti, M. Cunningham and R. Plevin

University of Strathclyde

Introduction

Osteosarcoma (OS) is the most prevalent primary malignant bone tumour generally made up of osteoblasts and is diagnosed in one to three individuals per million people every year [1]. Despite research efforts, there has been little improvement to OS treatment and prognosis in the last 30 years [2]. Given these marginal improvements, research at a molecular level is imperative to enable discovery of therapeutic drug targets for OS.

Methods

Our present study utilises short-read RNA-sequencing techniques (Illumina) to a human OS cell line, U2OS, with and without IKKα deletion by CRISPR-Cas9 knockdown, and in the absence or presence of IL-1β (10 ng·ml⁻¹) stimulation for 8 or 24 h (n = 3). Following RNA-sequencing analysis, we further validated gene expression of genes of interest by RT-qPCR and at protein level by utilising immunofluorescence techniques and SDS-PAGE and western blotting.

Results

Our findings highlight that IKKα knockdown significantly decreases expression of cell division genes associated with OS progression, including AURKA, AURKB, TPX2, BIRC5, GTSE1, E2F2, FOXM1 and SPC24 (****P < 0.0001). Interestingly, IKKα knockdown increased osteoclast-associated receptor (OSCAR) gene expression (****P < 0.0001), which is a central receptor in bone degradation processes. Osteoclast activity is associated with decreased OS-derived metastasis, and hence this implies a role for IKKα in promoting metastasis. Additionally, with IL-1β (10 ng·ml⁻¹) stimulation for 8 h, we identified that there are several IL-1β-dependent, IKKα-dependent genes associated with OS tumour progression including CXCL5 and GAS7 [3]. These results were validated in the laboratory by RT-qPCR. This reveals an important role for IKKα in osteogenesis mediated by the CXCR2 axis and GAS7 (Figure 1).

Conclusions

References

1. Huang X, Wang L, Guo H, Zhang W. Single-cell RNA sequencing reveals SERPINE1-expressing CAFs remodelling tumour microenvironment in recurrent osteosarcoma. Clin Transl Med. 2024;14(1):e1527.

2. Gianferante DM, Mirabello L, Savage SA. Germline and somatic genetics of osteosarcoma—Connecting aetiology, biology and therapy. Nature Reviews Endocrinology. 2017;13(8):480-91.

3. Chen H, Wang J, Xie J, Li J, Wang F, Liu S. GAS7 expression and its significance in human osteosarcoma. The Chinese-German Journal of Clinical Oncology. 2008;7(2):118-20.

234

Patient-derived zebrafish xenografts: A translational splash in personalized oncology

N. Dietis

University of Cyprus Medical School

Introduction

The zebrafish (Danio rerio) model has emerged as a pivotal tool in translational cancer research, offering several advantages over traditional in vivo models due to its rapid development, optical transparency and genetic similarity to humans. Zebrafish patient-derived xenografts (zPDX) have gained particular attention as a co-clinical trial platform, where patient-derived cancer cells are xenografted into zebrafish larvae, providing a fast-track avenue for personalized medicine. The aim of this talk is to showcase how zebrafish zPDX models can serve as valuable translational asset for clinicians and scientists in making informed, patient-specific treatment decisions.

Discussion

Zebrafish models present a unique combination of advantages for cancer research, particularly in the use of patient-derived xenografts. With 70% of human genes having a zebrafish orthologue and 84% of human disease-related genes mirrored in zebrafish, these models offer significant genetic and physiological parallels critical for translational research. zPDX systems allow for rapid tumour growth assessment, metastasis tracking and vascularization analysis using real-time imaging techniques that are non-invasive and cost-effective. Key endpoints such as tumour size, drug response and metastasis can be evaluated within days. The high-throughput capability of zebrafish models further enhances their efficiency, allowing simultaneous testing of multiple therapeutic regimens.

Co-clinical trials, where zebrafish models are run in parallel with human clinical trials, provide a cutting-edge approach for optimizing cancer treatments. These trials involve transplanting patient-derived cancer cells into zebrafish embryos, which are then subjected to the current or experimental therapeutic regimens. The zPDX models are evaluated for tumour response, drug sensitivity and toxicity, providing an early indication of how the patient might respond to treatment. These models work on the principle of rapid, real-time feedback, allowing for continuous adjustments in the patient's treatment plan as new data from zebrafish trials emerge. The integration of zPDX models into clinical trial designs offers a practical, scalable method for precision oncology, especially in cancers where the window for treatment decisions is narrow, thus increasing the likelihood of better patient outcomes.

Conclusion

Zebrafish patient-derived xenografts are rapidly becoming a cornerstone in the field of precision oncology, bridging the gap between preclinical drug testing and patient-specific treatment. The ability to generate quick, reliable data on drug efficacy and toxicity in a cost-effective manner positions zebrafish models as a valuable asset in co-clinical trials.

241

Repurposing of atovaqoune for treatment of FLT3-ITD quizartinib sensitive and resistant acute myeloid leukaemia

F. Rabia, F. Healy, A. Chadwick, M. Bosakhar, V. Marensi and D. MacEwan

University of Liverpool

Introduction

FLT3-ITD is the most common somatic mutation in acute myeloid leukaemia, affecting 28% of patients. Clinically. It is highly correlated with high recurrence rate and poor survival. Despite availability of targeted FLT3 inhibitors, including the recently FDA-approved quizartinib, patient outcomes are poor due to therapy resistance. Therefore, new therapeutic approaches are urgently required to improve the therapeutic outcome in FLT3-ITD. Atovaquone is an FDA-approved, anti-malarial drug, shown to also have anti-cancer properties [1]. It acts mainly by inhibiting oxidative phosphorylation and cell metabolism. Here, we explore the effect of Atovaquone on FLT3-ITD quizartinib sensitive and resistant cells.

Methods

MOLM-13 quizartinib sensitive and resistant cell lines were treated with 1, 3, 10 and 30 μM atovaqoune for up to 96 h. Apoptosis was measured using annexin v and 7-AAD staining, quantified by flowcytometry. Microscope images were taken. 24 h post-treatment, caspase 3 activity was measured by Caspase-3 assay kit (Abcam). ROS quantified by CelloRox green kit (Thermofischer). Mitochondrial respiration was measured by Seahorse XF96 Cell Mito Stress Test. Total RNA was extracted using Monarch total RNA miniprep kit (Biolabs). Samples was sequenced by Novogene (Cambridge Cancer Res Centre, UK). Data were analysed by GraphPad Prism software, using one-way ANOVA followed by Tukey test. N > 3. Values presented as mean ± SEM. Data showed a normal distribution (P > 0.05).

Results

96 h post-treatment, 30 μM atovaquone significantly induced apoptosis by >80% in both cell lines. Differences were observed in cellular appearance and morphology. Caspase 3 activity increased by >100% in both cell lines. However, this was not statistically significant. Atovaquone induced a dose-dependent reduction in the oxygen consumption rate (OCR) accompanied by reduction in basal respiration, maximal mitochondrial respiration and ATP production (Table 1). ROS elevated by 50-fold on MOLM-13 sensitive (P < 0.0001) and 29-fold in MOLM-13 resistant (P < 0.001). Genetic analysis shows cell cycle and oxidative phosphorylation as the main down-regulated pathways in both cell lines, respectively.

Conclusions

Results demonstrated that at a clinically relevant concentration, atovaquone exerts its antileukemic effect in FLT3-ITD quizartinib sensitive and resistant cells by inhibiting cell cycle and mitochondrial oxidative phosphorylation, respectively, thus increasing oxidative stress leading to cell death.

Reference

1. James Coates, et al. Potent inhibition of tumour cell proliferation and immunoregulatory function by mitochondria-targeted atovaquone. Cell Death Discovery. (2020) 6:110.

261

Differential effects of CA3-mediated hippo pathway modulation on AIF expression in luminal A versus triple-negative breast cancer cells

M. Bal Albayrak¹, T. Korak¹, S. Yanar² and N. Kayır^3,4

¹Faculty of Medicine, Department of Medical Biology, Kocaeli University; ²Faculty of Medicine, Department of Histology and Embryology, Sakarya University; ³Faculty of Medicine, Department of Medical Pharmacology, Medipol University; ⁴Department of Medical Oncology, Derince Education and Training Hospital

Background

Apoptosis-inducing factor (AIF) is a key regulator of caspase-independent apoptosis, impacting cancer cell survival and proliferation. The Hippo signalling pathway, which controls cell proliferation and apoptosis, is involved in the development of cancers like breast cancer. This study examines the effect of Hippo pathway modulation on AIF expression in breast cancer cells using the Hippo modulator CA3.

Methods

MCF7 cells (luminal A subtype, ER+) and MDA-MB-231 cells (triple-negative breast cancer-TNBC) were treated with 2 and 4 μM of CA3 for 24 h, respectively, based on IC50 values from previous studies. AIF expression levels were quantified by Western blot, with β-actin as the reference protein. Statistical analyses were performed using one-way ANOVA, with a P-value of <0.05 considered significant.

Results

CA3 treatment significantly decreased AIF expression in both cell lines. In MCF7 cells, a 2.25-fold decrease was observed (P < 0.05), while in MDA-MB-231 cells, AIF expression was almost entirely suppressed (91.5-fold decrease, P < 0.001). This indicates a differential response to Hippo pathway modulation by CA3 between these breast cancer subtypes.

Discussion

The differential impact of CA3 on AIF expression suggests that oestrogen receptor status may modulate the response to Hippo pathway modulation. While CA3 effectively reduces AIF in Luminal A subtype cells, its near-complete suppression in TNBC cells positions CA3 as a promising candidate for targeted therapy in aggressive cancers. Future studies should explore combination therapies to enhance apoptotic responses and overcome resistance in difficult-to-treat breast cancer subtypes.

Keywords

AIF, apoptosis, breast cancer, CA3, Hippo pathway, Luminal A, TNBC

272

Investigating acquired resistance in non-small cell lung cancer

J. Bugeja Wettinger

University of Malta

Introduction

Lung cancer is one of the leading causes of death in Malta, while 80% of all lung cancer cases are NSCLC [1]. The EGFR is a crucial target in NSCLC as it signals several pathways of cell growth and proliferation. Third generation EGFR-TKI osimertinib inhibits the EGFR and in turn, inhibiting the growth of cancerous cells.

Aim

To sensitize two adenocarcinoma cell lines, namely, A549 (EGFR wild-type) and HCC827 (EGFR-mutant) by using osimertinib, a third generation EGFR-TKI.

Methods

PrestoBlue^TM cell viability assays were carried out to evaluate the optimum concentrations of osimertinib on each cell line. Then, wound healing assays were carried out to assess the metastatic potential of each cell line treated with different concentrations of the third generation EGFR-TKI osimertinib.

Results

A concentration-dependent decrease in percentage cell viability was observed in the A549 cell line. The one-way ANOVA test was done and the 25 and 50 μM concentrations were significant at all time points (P < 0.05). However, this was not the case with the EGFR-mutated HCC827 cell line; hence, this cell line is not sensitive to osimertinib. The one-way ANOVA test was done and the 10 and 25 μM concentrations were significant at 48 h (P < 0.05). The wound healing assays showed that osimertinib decreased the metastatic potential of the A549 cells. An independent samples T-test was done, and all the concentrations were significant at each time point (P < 0.05) with the exception of the 1 μM concentration at 48 h (P > 0.05).

Conclusions

Osimertinib reduces proliferation and migration potential in the A549 adenocarcinoma cells but not in the HCC827 cell line, which reflects adenocarcinoma with an EGFR mutation. This research is a preliminary step to studying the effect of novel combinatory treatment with two ASOs for TCTP and HSP27. A combination which may possibly overcome the non-sensitivity seen by HCC827 to osimertinib, which may suggest resistance. Additionally, it can increase the effectiveness of osimertinib seen in A549 cells.

References

1. Zappa C, Mousa SA. Non-small cell lung cancer: Current treatment and future advances. Translational lung cancer research. 2016;5(3):288–300. https://www.ncbi.nlm.nih.gov/pubmed/27413711. https://doi.org/10.21037/tlcr.2016.06.07.

282

Investigating the effectiveness and possible toxicity of novel lung cancer treatment

E. Farrugia¹, R. Kelly-laubscher², N. Vella¹, V. Petroni Magri¹ and A. Fenech¹

¹Department of Clinical Pharmacology and Therapeutics, Faculty of Medicine & Surgery, University of Malta; ²Department of Pharmacology and Therapeutics, University College Cork

Introduction

Globally, lung cancer persists as the leading cause of cancer-related deaths with non-small cell lung cancer (NSCLC) exhibiting a 5-year survival rate of only 15%. The dysregulation and aberrant signalling of the PI3k/Akt/mTOR pathway, together with VEGF/VEGFR signalling interplay, commonly leads to NSCLC initiation and progression, potentiating an attractive therapeutic approach through the inhibition of this cascade.

This research aims to analyse the anticancer efficacy of INK128, a dual mTOR inhibitor, and ramucirumab, an anti-VEGFR2 monoclonal antibody, independently and in combination, in A549 NSCLC adenocarcinoma cells. The modes of action of INK128 and ramucirumab target the dysregulated, malignant PI3k/Akt/mTOR pathway at different levels; hence, their combination poses a possible therapeutic advantage over limitations of monotherapies. The rationale behind this combination is therefore aiming towards augmenting anticancer efficacy of treatment through the simultaneous inhibition of VEGFR-2 and mTOR. Additionally, we aim to investigate possible cardiotoxic effects of both mono- and combination therapies in vitro, moving towards holistic translational medicine research, in light of cardiovascular disease persisting as the leading cause of long-term morbidity and non-cancer related mortality in cancer survivors.

Methods

Changes in cell viability were assessed via rezasurin-based assays on A549 cells and H9C2 cardiomyocytes following INK128 (0.01, 0.1, 0.3, 0.5, 1, 2, 3 and 5 μM solubilized in DMSO) and ramucirumab (1, 5, 10, 15 and 20 nM solubilized in PBS) treatment, both separately and in combination at 24, 48 and 72 h post-treatment. Moreover, wound healing assays were used to study effects of the combinatory treatment approach (using increasing concentration of ramucirumab with 0.01 μM INK128 pre-treatment) on cellular proliferation over a period of 72 h.

Results

Both INK128 and ramucirumab monotherapies caused a concentration-dependent decrease in the viability of A549 cells, exhibiting the highest efficacy at 72 h (45% viability) and at 24 h (47% viability), respectively. Results of the combinatory treatment exhibit similar trends to INK128 monotherapy, with A549 dose-response curves exhibiting an overall lower viability at all time-points, when compared to individual treatments. Treatments did not cause a significant decrease in cardiomyocyte viability, pointing towards the treatments' safety in terms of cardiotoxicity. Of interest, variability in both H9C2 cell viability and proliferation was observed with INK128 treatment at the 48-h time-point (P < 0.05), suggesting the need for further investigation.

Conclusions

This study therefore serves foundational to translational medicine research, evaluating novel combinational oncopharmacology and possible toxicities, holistically sustaining hope in better clinical outcomes and disease-free survival thereafter.

284

Role of FXR activation in modulating stemness in oestrogen-dependent breast cancer: Impact on the PI3K/AKT/mTOR signalling axis

A. Ali¹, O. Hammam², M. Galal³ and Y. Attia⁴

¹Health Research Center of Excellence; Drug Research and Development Group, Faculty of Pharmacy, The British University in Egypt; ²Pathology Department, Theodor Bilharz Research Institute; ³Pharmacology & Toxicology Department, Faculty of Pharmacy, Cairo University; ⁴Pharmacology Department, Faculty of Pharmacy, The British University in Egypt

Introduction

Breast cancer stem cells (BCSCs) are pivotal to breast cancer initiation and progression owing to their self-renewal, differentiation and metastasis, hence tumour aggressiveness. Farnesoid X receptor (FXR) is a nuclear receptor that represents a significant target for metabolic reprogramming yet its role in breast cancer has always been an area of intense debate. This study, therefore, is spurred to investigate the possible therapeutic mechanisms behind FXR activation by obeticholic acid (OCA), in breast cancer mechanistic insights into its effects on BCSCs and the underlying molecular pathways.

Methods

MCF7 cells were treated with OCA and the FXR antagonist Guggulsterone (GUGG). OCA-treated MCF-7 cells were preincubated with GUGG for IC₅₀ calculation. Gene expression of BAX, BCL2, PI3K, AKT and mTOR was assessed by qRT-PCR. A scratch wound assay was conducted, with wound areas measured at 24 and 48 h. Mammosphere formation assays were performed and respective volumes were recorded. An in vivo model of dimethylbenz[a]-anthracene/medroxyprogesterone acetate-induced breast cancer in rats was employed, with daily administration of 5 mg·kg⁻¹ OCA starting 6 months post-induction for 8 weeks.

Results

Our findings showed that FXR agonism rather than antagonism can suppress MCF-7 cell viability and proliferation, where the IC₅₀ of OCA and GUGG were 26.18 ± 5.27 and 1178 ± 14.85 μM, respectively. Preincubation with GUGG significantly mitigated OCA's cytotoxicity, raising the IC₅₀ by 86% (P = 0.009, n = 3). This highlights the critical role of FXR activation in the observed anticancer effects. OCA increased the BAX/BCL2 ratio by 7.3-fold compared to control MCF-7, and disrupted the key players in the PI3K/AKT/mTOR pathway (Figure 1). This was mirrored by a reduced wound closure percentage of up to 48 h, (72.35% ± 3.4, n = 3), compared to (100% ± 0.05, n = 3) of control MCF-7 (Figure 2). OCA also induced 64% reduction in mammospheres volume (42 ± 38. n = 3) compared to untreated MCF-7 (115.5 ± 53.2, n = 3) (Figure 3). In vivo, OCA-treated rats showed less pronounced alterations in mammary glandular tissue architecture compared to untreated counterparts (Figure 4).

Conclusions

These results suggest that FXR activators could serve as a promising therapeutic strategy in oestrogen-dependent cancer by inhibiting both tumour growth and metastasis via interfering with the PI3K/AKT/mTOR pathway.

292

Simvastatin disrupts cholesterol-driven mitochondrial biogenesis and cancer stemness in vitro and impedes tumour growth in breast cancer model in vivo

Y. Amin^1,2, O. Hammam³, M. Khattab⁴ and Y. Attia^1,2

¹Pharmacology Department, Faculty of Pharmacy, The British University in Egypt; ²Health Research Center of Excellence, Drug Research and Development Group, Faculty of Pharmacy, The British University in Egypt; ³Pathology department, Theodor Bilharz Research Institute; ⁴Pharmacology Department, Faculty of Pharmacy, Cairo University in Egypt

Introduction

Emerging evidence suggests a significant role of cholesterol in breast cancer pathogenesis. The peroxisome proliferator-activated receptor-γ co-activator-1 (PGC-1) and the orphan nuclear oestrogen-related receptor alpha (ERR-α) axis has been implicated in breast cancer development and progression, primarily through regulating mitochondrial biogenesis, a crucial orchestrator of cellular energy and carcinogenesis. Cancer stem cells (CSCs) enhance cancer aggressiveness by maintaining metabolic homeostasis through mitochondrial biogenesis. Cholesterol, identified as a potential ligand for ERR-α, suggests statins as a therapeutic target for this pathway. This study investigates the potential of simvastatin (SIM) to target breast CSCs via mitochondrial biogenesis and the PGC-1/ERR-α axis in vitro along with an in vivo breast cancer model.

Methods

The effects of SIM on cell viability were assessed by calculating IC50 values in MCF-7 and MDA-MB-231 breast cancer cell lines, with and without cholesterol enrichment, following MTT assays. Mitochondrial biogenesis was assessed by measuring PGC1-α and NRF-1 protein levels along with TFAM gene expression. The impact of SIM on CSCs was determined using mammosphere formation assays and by measuring ALDH-1 and SOX-2 protein levels as markers of stemness. An in vivo rat model of breast cancer induced by dimethylbenz(a)anthracene/medroxyprogesterone acetate was implemented, with a daily oral dose of 40 mg·kg⁻¹ SIM starting 6 months post-induction for 8 weeks.

Results

Cholesterol-treated MCF-7 and MDA-MB-231 cells displayed enhanced viability than their untreated counterparts with P-values of 0.0216 (n = 6) and <0.0001 (n = 6), respectively. SIM significantly suppressed cell proliferation in both cell lines regardless of cholesterol status. Cholesterol increased ERR-α expression (2.19 ± 0.19, n = 3) and (3.001 ± 0.09, n = 3) as compared to control cells (1.002 ± 0.07, n = 3) and (1.001 ± 0.03, n = 3), whereas SIM significantly reduced it in MCF-7 and MDA-MB-231 cells, respectively. Similarly, cholesterol up-regulated PGC1-α (2473 ± 19.59) and (2852 ± 29.72), NRF-1 (636.1 ± 24.38) and (492.1 ± 12.17) and TFAM (1.62 ± 0.3) and (1.99 ± 0.1), respectively, relative to their untreated counterparts. Whereas, SIM decreased these markers in both cell lines. Cholesterol also enhanced ALDH-1 and SOX-2 levels, whereas SIM significantly reduced them alongside mammosphere diameters (Figure 1). Regarding the in vivo model, histopathological evaluation exhibited tumour regression in SIM-treated rats compared to their untreated counterparts (Figure 2). Additionally, the incidence was 1.7 folds higher in the positive control group compared to the SIM-treated group.

Conclusions

SIM might impede cholesterol-driven mitochondrial biogenesis, curb stemness and proliferative capacity in breast cancer.

32

MARCKS-PIP2: A potential novel pathway for understanding pulmonary artery hypertension

Ali Alattar¹, Mahya Shenasa¹, Dalal Zainal¹, Hamad Alobaidli¹, Aidan Conway³, Luca McDonald⁴, Anthony Albert² and Yousif Shamsaldeen¹

¹University of Brighton; ²St. George's University of London; ³Exeter University; ⁴University of Manchester

Introduction

Pulmonary arterial hypertension (PAH) is a progressive, fatal disorder marked by increased pulmonary arterial pressure and vascular resistance which leads to right ventricular failure. Current treatments fall short due to their diverse side effects and lack of optimal symptoms control. This has caused a move towards costly combination therapies with increased side effects, thus prompting a need for novel treatment strategies with improved efficacy and tolerability 1. L-type voltage-gated calcium channels (CaV1.2) play a crucial role in the regulation of pulmonary artery vascular tone 2. Recent studies suggest that the release of phosphatidylinositol 4,5-bisphosphate (PIP2) from myristoylated alanine-rich C kinase substrate (MARCKS) influences CaV1.2 activity 3. This study aims to explore the expression and function of MARCKS, PIP2 and CaV1.2 in the context of PAH.

Method

Guinea pigs (females and males) weighing between 300 and 400 g at weaning age 3–4 weeks were euthanised schedule 1 (AWERB approval: 2023-12489). Freshly dissected guinea pig pulmonary artery for myography. Moreover, pulmonary artery segments were lysed using liquid nitrogen and RIPA buffer for Western blotting. Additionally, vascular smooth muscle cells were enzymatically dispersed for immunocytochemistry and proximity ligation assay (PLA).

Results

Western blot has shown the expression of three cellular components: MARCKS, PIP2 and CaV1.2. Furthermore, myography experiments showed MARCKS inhibitor (MANS peptide)-induced vasoconstriction was significantly inhibited (P < 0.0001) by 70% when pulmonary artery rings were pre-incubated with CaV1.2 blocker (nifedipine 100 μM). PLA showed significant (P < 0.01) reduction by approximately 50% in colocalization of MARCKS and PIP2 when cells were stimulated with MANS peptide (100 μM). Moreover, MANS peptide (100 μM) showed significant (P < 0.05) increase in PIP2 and CaV1.2 colocalization by approximately 70%. Additionally, immunocytochemistry showed MANS peptide treatment (100 μM) significantly reduces MARCKS intensity (P < 0.01) by approximately 55%.

Conclusions

Our findings support the proposal that MARCKS, as a PIP2 buffer may play major roles in VSMCs contraction and Cav1.2 channels activity in PAH, which sets a foundation for further research for further investigation to understand the molecular pathophysiology of PAH through MARCKS-PIP2 pathway.

References

1. Lan NS, Massam BD, Kulkarni SS, Lang CC. Pulmonary arterial hypertension: Pathophysiology and treatment. Diseases. 2018;6(2):38.

2. Gamper N, Shapiro MS. Target-specific PIP2 signalling: How might it work? The Journal of physiology. 2007;582(3):967-975.

3. Jahan KS, Shi J, Greenberg HZ, et al. MARCKS mediates vascular contractility through regulating interactions between voltage-gated Ca2+ channels and PIP2. Vascular Pharmacology. 2020;132:106776.

65

Effect of 3-mercaptopyruvate sulfotransferase (3-MST) inhibitors on contraction of porcine coronary arteries

Richard Roberts and Sahar Alharthi

University Of Nottingham

Introduction

Hydrogen sulphide (H₂S) is synthesised from L-cysteine through cystathionine γ lyase, cystationine β synthase, and 3-mercaptosulfurtransferase (3-MST). Although studies have indicated that H₂S is a vasorelaxant, the majority of studies have investigated the effects of exogenously applied H2S on vascular tone [1]. The aim of this study was to determine the effect of 3-MST inhibitors on contractile responses in porcine coronary artery.

Method

Hearts from pigs of both sexes were obtained from a local abattoir and coronary arteries (PCAs) set up for isometric tension recording in Krebs-Henseleit buffer gassed with carbogen. PCAs were exposed to 3-MST inhibitors I3MT-3 (50 μM; [2]) or 1-(3,4-dihydroxyphenyl)-2-[(4-hydroxy-6-methyl-2-pyrimidinyl) sulfanyl] ethanone (DPHE) (100 μM) or 0.1% v/v DMSO for 1 h. Cumulative concentration–response curves to the thromboxane receptor agonist U46619 (1 to 300 nM) or the acetylcholine receptor agonist carbachol (1 nM to 3 μM) were then carried out. In some experiments the endothelium was removed by rubbing the lumen with forceps. Contractile responses were expressed as a percentage of the response to 60 mM KCl.

Results

I3MT-3 and DPHE significantly reduced U46619-induced contractions (contraction to 100 nM U46619 117.0 ± 16.2% in control compared to 31.9 ± 8.1% with DPHE and 50.0 ± 17.9% with I3MT-3 [mean ± SEM, n = 6–8, P < 0.05 vs. control, ANOVA followed by Tukey's multiple comparisons test]). A similar effect was seen on carbachol-induced contractions. In the absence of the endothelium, both DPHE and I3MT-3 still inhibited the contraction to U46619. In the absence of extracellular calcium, contractions to U46619 were reduced (8.3 ± 1.1% in control, n = 6). I3MT-3 inhibited the contraction to U46619 in the absence of extracellular calcium (1.0 ± 0.1%, P < 0.05 Tukey's multiple comparison's test).

Conclusion

3-MST inhibitors inhibit contractile responses in the porcine coronary artery through an endothelium-independent pathway. DPHE appears to inhibit a calcium-dependent pathway, whereas I3MT-3 may inhibit both a calcium-dependent and calcium-independent pathway. The data suggest that 3-MST may be involved in vascular contraction.

References

1. Dunn W. R., Alexander S. P. H., Ralevic V., et al. Effects of hydrogen sulphide in smooth muscle. Pharmacology and Therapeutics. 2016; 158, 101-113.

2. Hanoaka K., Sasakura K., Suwanai Y., et al. Discovery and mechanistic characterization of selective inhibitors of H2S-producing enzyme: 3-Mercaptopyruvate sulfurtransferase (3MST) targeting active-site cysteine persulfide. Scientific Reports. 2017; 7: 40227.

78

Dapagliflozin mitigates doxorubicin-induced cardiac and vascular toxicity in zebrafish

Amira Mohamed, Hevna Dhulkifle, Abdulla Alyafei, Safer Al-Hajri, Faisal Al-Hammadi, Abdulaziz Al-Zaraa and Zaid H. Almaayah

Department of Pharmaceutical Sciences, College of Pharmacy, QU Health, Qatar University, Doha, Qatar

Introduction

Doxorubicin (DOX) is a potent chemotherapeutic agent used for various cancers. However, its clinical use is limited due to its potential toxic impact on the cardiovascular system. Therefore, it is vital to combat DOX's detrimental cardiovascular impacts to improve the long-term health of cancer patients. Recent evidence shows that SGLT2 inhibitors like dapagliflozin (DAPA) have demonstrated protective effects in several models of cardiovascular disease. While some studies have shown that DAPA protects against DOX-induced cardiotoxicity [1], the effect of DAPA on DOX-induced vascular toxicity has yet to be investigated. Thus, given the beneficial impact of DAPA on the cardiovascular system [1], we aimed to examine the effect of DAPA on DOX-induced cardiac and vascular toxicity in zebrafish.

Method

We used a zebrafish model of DOX-induced cardiotoxicity, as described previously [2]. Briefly, at 24 h post-fertilization (24 hpf), the fish embryos were randomly segregated into four groups that were incubated with either vehicle (n = 10), 100 μM of DOX (n = 10), 50 μM of DAPA (n = 10) or a combination of 100 μM of DOX and 50 μM of DAPA (n = 10) for 72 h. The cardiovascular parameters, such as blood flow velocity and vessel diameter, were measured in the dorsal aorta and the posterior cardinal vein using the Viewpoints MicroZebralab version 3.6 software. We used quantitative real-time PCR to measure the mRNA expression level of cardiotoxicity, inflammatory and oxidative stress markers. GraphPad Prism (version 7.04) was used to conduct a one-way analysis of variance (ANOVA) followed by Tukey Kramer's post hoc test.

Results

Our results showed that DAPA significantly improved cardiac oedema and reduced the expression of the cardiotoxicity marker myh7 by about 50% and 80%, respectively, compared to DOX alone. Furthermore, DAPA significantly improved cardiac output, blood flow velocity, and blood vessel diameter by approximately 70%, 40% and 30%, respectively, compared to DOX alone. Mechanistically, DAPA significantly normalized DOX-induced mRNA expression of the inflammatory marker, interleukin 1b, and oxidative stress marker, glutathione peroxidase, in our zebrafish model.

Conclusions

Our data indicate that DAPA reduces inflammatory and oxidative stress markers and improves DOX-induced cardiac and vascular toxicity in zebrafish.

References

1. Dabour, M.S., et al., The cardioprotective and anticancer effects of SGLT2 inhibitors: JACC: CardioOncology state-of-the-art review. JACC CardioOncol, 2024. 6(2): p. 159-182.

2. Liu, Y., et al., Visnagin protects against doxorubicin-induced cardiomyopathy through modulation of mitochondrial malate dehydrogenase. Sci Transl Med, 2014. 6(266): 266ra170.

116

β-Phenylethylamine-induced-vasodilator responses are mediated by an intracellular receptor

Harrison Broadley¹, William Ford² and Alexander Voisey³

¹Cardiff School of Pharmacy and Pharmaceutical Sciences; ²RCSI Bahrain; ³Cardiff School of Medicine

Introduction

β-Phenylethylamine (β-PEA), a trace amine, classically induces vasoconstriction. However, in pre-constricted-isolated blood vessels causes vasodilatation [1]. Studies have suggested this is caused by inhibition of α₁-adrenoceptors [2]. However, β-PEA is also an agonist of trace amine-associated receptor 1 (TAAR1), found intracellularly [3]. Outside the vasculature, organic cation transporters (OCT1–3) have been demonstrated to facilitate uptake of trace amines [3]. In this study, we aim to demonstrate that dilation is mediated by an intracellular receptor and not via inhibition of α1-adrenoceptors.

Method

Aortic rings (0.5 cm) from adult male Sprague–Dawley rats were mounted onto fixed and mobile hangers and were immersed in Kreb's bicarbonate solution gassed with CO₂/O₂ (5%/95%) at 37°C, under 1.5 g of resting tension. Isometric tension was measured using Power Lab (ADInstruments, Oxfordshire, UK). After the equilibration period, 60 mM KCl in Krebs was added to the organ bath causing maximum contraction to confirm tissue viability. At the peak of contraction, the presence of endothelium was confirmed by relaxation to acetylcholine (1 mM).

Cumulative-concentration response curves (CRC's) for vasodilator responses to β-PEA were obtained in aortic rings pre-constricted with phenylephrine (0.3–1 μM) or 60 mM KCl. CRC's were also obtained in the presence of the OCT1–3 transporter inhibitor decynium-22 (1 μM).

Results

Decynium-22 (1 μM) abolished β-PEA-induced-vasodilation of pre-constricted tissues, with 1 mM β-PEA instead inducing a significant vasoconstriction (124 ± 29%, n = 4). In the absence of decynium-22, β-PEA (1 mM) induced a statistically significant dilation (34 ± 12%, n = 4).

In tissues pre-constricted with 60 mM KCl, β-PEA caused dose-dependent vasodilation of rat aortic rings. The maximum relaxation reached was 61 ± 4% (n = 6) of the KCl-induced vasoconstriction.

Conclusions

β-PEA moves into intracellular compartments via a decynium-22-sensitive transporter to cause vasodilation. As β-PEA induces vasodilation in tissues pre-constricted with KCl, vasodilatation is clearly independent of α₁-adrenoceptor antagonism, possibly via TAAR1.

References

1. Anwar et al. 2012. Vasoconstrictor and vasodilator responses to tryptamine of rat-isolated perfused mesentery: Comparison with tyramine and β-phenylethylamine. Br. J. Pharmacol. 165(7), pp. 2191–2202. Available at: https://doi.org/10.1111/j.1476-5381.2011.01706.x.

2. Narang et al. Modulation of resistance artery tone by the trace amine β-phenylethylamine: Dual indirect sympathomimetic and α₁-adrenoceptor blocking actions. J. Pharmacol. Exp. Ther. 2014, 351 (1) 164-171. https://doi.org/10.1124/jpet.114.216523

3. Berry et al. 2016. Pharmacological characterization of a high-affinity p-tyramine transporter in rat brain synaptosomes. Scientific Reports. 6(1), 38006. Available at: https://doi.org/10.1038/srep38006.

142

Determining the kinetics of GPVI and CLEC-2 ligand-induced clustering

Joanne Clark

University Of Birmingham

Introduction

The platelet receptors, glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2) are promising targets in treating thrombosis. Using fluorescence correlation spectroscopy (FCS), we have shown that GPVI and CLEC-2 are present on the membrane as a mixture of monomers and dimers in HEK cells when expressed at a similar level to that in platelets. Receptor activation is mediated by clustering but its regulation and how it influences activation and signalling is not fully understood. The aims were to measure the kinetics of GPVI and CLEC-2 clustering, including cluster lifetime in the membrane using single particle tracking.

Methods

We used two-colour single particle tracking (SPT) [1] using total internal reflection fluorescence (TIRF) microscopy to image Snaptag and Halotag versions of GPVI and CLEC-2 in resting and stimulated transfected live CHO cells. Advanced computational analyses were performed to generate receptor trajectories and determine kinetic outputs such as diffusion coefficients, mobile/immobile fractions, cluster lifetime, association and dissociation rates.

Results

SPT experiments were performed on cells expressing low levels (0.06–1.08 receptors μm⁻²) of GPVI or CLEC-2 to visualise individual receptors (n = 6). Under basal conditions, productive interactions for GPVI and CLEC-2 were not observed suggesting the receptors are monomers at this level of expression. A proportion of GPVI (42.2%) and CLEC-2 (46.9%) receptors showed Brownian (free) diffusion at basal level with similar rates of movement of 0.12 + 0.09 and 0.14 + 0.09 μm² s⁻ª, respectively. The remaining GPVI and CLEC-2 receptors had immobile motion (26.9%; 18.9%), confined motion (24.8%; 27.3%) or directional motion (6.1%; 7.0%), respectively. Upon addition of activating trivalent nanobodies to the two receptors (100 nM), productive interactions (clustering) were detected for GPVI and CLEC-2 (n = 6). The receptors became immobile with an increase in the immobile fraction of GPVI and CLEC-2 from 26.9% to 41.9% and 18.9% to 40.7%, respectively. The cluster lifetime for GPVI and CLEC-2 ligand-induced clustering was determined to be 0.6 (dissociation rate koff = 1.57 s⁻¹; confidence interval: 1.47–1.66) and 6.8 (koff = 0.15 s⁻¹; confidence interval: 0.07–0.22) seconds, respectively.

Conclusions

We have monitored single receptor movement in the membrane to measure interactions and their associated kinetic information. The results show that GPVI and CLEC-2 are monomeric at low receptor levels. Ligand addition causes receptors to cluster and become immobile. GPVI ligand-induced clusters are short-lived compared to CLEC-2 clusters, and therefore, targeting clustering may be more effective for CLEC-2 than GPVI.

Reference

1. Sungkaworn T, Jobin ML, Burnecki K, Weron A, Lohse MJ, Calebiro D. Single-molecule imaging reveals receptor-G protein interactions at cell surface hot spots. Nature. 2017;550(7677):543-7. https://doi.org/10.1038/nature24264

165

Endothelial cell proliferation, migration and nitric oxide (NO) production induced by AP39, a mitochondria-targeted hydrogen sulfide (H2S) donor

Leonardo da Costa Marques¹, Sophia Machado da Veiga¹, Luisa Silva², Niels Camara², Soraia Costa¹ and Marcelo Muscara¹

¹Department of Pharmacology-Institute of Biomedical Sciences, University of Sao Paulo; ²Department of Immunology-Institute of Biomedical Sciences, University of Sao Paulo

Introduction

H2S signalling affects angiogenesis, and the main enzyme source of the endogenous production of H2S in the cardiovascular system is cystathionine beta-synthase (CSE). Different types of H2S-donors are currently under development in order to circumvent the low bioavailability of H2S and NO observed in microvascular dysfunction conditions (such as ageing, abnormal vasa vasorum, etc.), as the crosstalk between NO and H2S signalling is involved in vascular control mechanisms. Considering that impaired mitochondrial function of endothelial cells contributes to microvascular dysfunctions, targeting this organelle may represent a new therapeutic strategy. Compound AP39 has been described as a mitochondria-targeted H2S-donor with beneficial antioxidant and vascular effects, both in vivo and in vitro. Since, to the best of our knowledge, no evidences of its effects on NO-production or angiogenesis are available to date, we aimed to study the effects of AP39 on human umbilical vein endothelial cells (HUVECs) in culture.

Methods

HUVECs were treated with either AP39 (10, 30 and 100 nM) or the control moieties (ADT-OH and AP219, at equimolar concentrations) for 48 h; for all the procedures, the number of replicates were 4–6 for each experimental group. Cell viability was evaluated by MTT reduction, and cellular proliferation and migration were assessed by wound closure (WC) in the scratch wound assay. After 1 h exposure of the cells to the agents, NO production was assessed using the fluorescent probe DAF-DA, and O₂ consumption rate was measured using the Agilent® Seahorse XFp96 system. Differences among the groups were analysed by one-way ANOVA followed by the Bonferroni's test. Significant differences were expressed as *P < 0.05, **P < 0.01 or ***P < 0.001.

Results

After 48 h incubation, 100 nM AP39, but not ADT-OH or AP219, significantly increased MTT reduction (129 ± 7.4%** relative to vehicle-treated cells). VEGF-induced proliferation (at 1 μg·mL⁻¹: WC = 77.5 ± 6.9%**) was significantly attenuated in the presence of 100 μM L-NAME (WC = 40 ± 3.35%***) or 1 mM of the selective CSE inhibitor PGly (WC = 46.1 ± 2.85%***). Furthermore, AP39 (at both 10 and 30 nM) induced HUVEC proliferation (WC = 25.35 ± 3.5%* and 33.8 ± 8.6%**, respectively) whereas no significant effect was elicited by ADT-OH or AP219. However, in the presence of the cell proliferation inhibitor mitomycin (M; at 5 ng·mL⁻¹), both AP39 and ADT-OH (at 100 nM) induced significant HUVEC migration after 48 h (AP39 + M = 41.8 ± 3.9%***, ADT + M = 38 ± 4.5%***). After 1 h incubation with AP39, but not with ADT-OH or AP219, significant NO production was measured in a concentration-dependent manner (i.e., 100 nM AP39 = 124.5 ± 4.5%*** of the basal response). After 1 h incubation, AP39 (at 30 or 100 nM) significantly increased the maximal respiration rate, spare capacity and non-mitochondrial O₂ consumption rate.

Conclusions

These results highlight the interaction between H2S signalling and NO production in HUVECs. However, the link between the mitochondrial and angiogenic effects of AP39-derived H2S is still unclear, thus deserving further investigation, considering the potential use of this type of compounds in the treatment of vascular dysfunctions.

180

Effect of metformin on platelet interaction with healthy and diabetic blood outgrowth endothelial cells under hyperglycaemic conditions in vitro

Kareem Imad Fanous¹, Yazan Emad Kaddorah¹, Aimen Javed¹, Hong Ding¹, Chris R. Triggle¹ and Isra Marei^1,2

¹Weill Cornell Medicine Qatar; ²Imperial College London

Introduction

Type 2 diabetes mellitus (T2D) is a major risk factor of thrombosis and cardiovascular complications. This risk is correlated with hyperglycaemia-induced endothelial dysfunction, which impairs endothelium's ability to regulate platelets functions. Endothelial progenitor cells (EPCs) are a crucial component of the mechanism of vascular repair [1]. Recent studies showed positive benefits for metformin in reducing stroke risk in diabetic patients [2]. This project aimed to investigate (i) the interaction of platelets with the EPCs subtype and blood outgrowth endothelial cells (BOECs) in diabetic milieu in an in vitro co-culture model and (ii) the effect of metformin on this interaction and its implications on the control of thrombotic events.

Methods

BOECs were isolated form healthy and T2D patients using selective plating [1]. Platelets' concentrates were purchased from Human Cells Biosciences. Co-culture of BOECs and platelets were conducted using Transwells (0.4 μm pores size) with normal (5.5 mM) or high glucose (25 mM) endothelial growth media-2 in the presence or absence of metformin (50 μm, solvent: H₂O), with or without activation using collagen type I (10 μg·mL⁻¹). Media supernatants and cell lysates were collected and used for ELISA to detect the release of platelet granules content (platelet factor 4, PF4) and expression of adhesion molecules (VCAM-1 and p-selectin). Data were analysed using ANOVA followed by Bonferroni corrections, n = 3.

Results

ELISA revealed a reduction of induced PF4 release in healthy BOECs when treated with metformin (50 μm) for 24 h, under both normal and hyperglycaemic conditions. Treatment of T2D cells with high glucose media alone induced PF4 release, which was reduced when treating with PF4. Similar effects were observed in high glucose treated cells in presence of collagen activation. Expression of VCAM-1 and p-selectin was significantly induced in T2D cells in all treatments when compared to healthy cells. Treatment of co-cultures with metformin significantly reduced the expression of VCAM-1 in T2D BOECs under high glucose conditions and in presence of collagen activation. Treatments with metformin significantly reduced p-selectin expression under high glucose conditions both in presence and absence of platelets (Figure 1).

Conclusion

Our findings indicate that BOECs adhesion molecules expression is altered in T2D in BOECs/platelets co-culture systems. Furthermore, our findings indicate some positive effects of metformin on platelets activation and interaction with BOECs.

References

1. Ahmetaj-Shala B, Kawai R, Marei I, et al. A bioassay system of autologous human endothelial, smooth muscle cells, and leukocytes for use in drug discovery, phenotyping, and tissue engineering. FASEB J. 2020;34(1):1745-1754.

2. Triggle CR, et al. Metformin: Is it a drug for all reasons and diseases? Metabolism. 2022;133:155223.

189

The cardiac glycoside ouabain differentially modulates relaxations to arachidonic acid in rat aortae with intact and suppressed endothelial function

Chunrong He and Susan W. S. Leung

Department of Pharmacology and Pharmacy, University of Hong Kong

Introduction

In hypertensive patients, an increase in plasma level of ouabain (or ouabain-like substances) is detected [1]. Ouabain belongs to the family of cardiac glycosides, which are inhibitors of sodium/potassium-ATPase (Na/K-ATPase) [2]. In view of the multiple downstream signalling of Na/K-ATPase and its presence in vascular cells, the present study aims to determine how ouabain modulates vascular responses, particularly during endothelial dysfunction, which is a characteristic of cardiovascular diseases such as hypertension.

Methods

All the animal care and experimental procedures were approved by the committee on the Use of Live Animals in Teaching and Research of the University of Hong Kong. Male Sprague Dawley rats, at 10 to 12 weeks old, were anaesthetized with an overdose of pentobarbital sodium, and their aortae were isolated for the measurement of isometric tension in the classical organ chamber setups.

Results

In rat aortae, with endothelium and contracted with phenylephrine, acetylcholine (muscarinic receptor agonist), UK14304 (α₂-adrenergic receptor agonist) and arachidonic acid (a vasoactive fatty acid) induced concentration-dependent relaxations which were abolished by the nitric oxide synthase inhibitor L-NAME or the soluble guanylyl cyclase inhibitor ODQ. While intermediate-conductance calcium-activated potassium channel blocker, TRAM-34, had minimal effect on relaxations to acetylcholine or UK14304, it abolished relaxations to arachidonic acid. Ouabain (200 μM) slightly inhibited relaxations to acetylcholine and UK14304 (Table 1) and abolished arachidonic acid-induced relaxation. Aortic relaxations to arachidonic acid were inhibited by the cyclooxygenase inhibitor indomethacin but were not affected by the cytochrome P450 epoxygenases and ω-hydrolases inhibitor 17-ODYA or the 12-/15-lipoxygenase inhibitor CDC. In the presence of L-NAME and TRAM-34 plus the small-conductance calcium-activated potassium channel blocker UCL1684 [to inhibit endothelium-dependent hyperpolarization], ouabain potentiated arachidonic acid-induced relaxations; the potentiation was inhibited by CDC, but not by indomethacin or 17-ODYA.

Conclusion

References

1. Hauck C, Frishman WH. Systemic hypertension: the roles of salt, vascular Na+/K+ ATPase and the endogenous glycosides, ouabain and marinobufagenin. Cardiol Rev 2012;20:130-8.

2. Liu J, Xie ZJ. The sodium pump and cardiotonic steroids-induced signal transduction protein kinases and calcium-signaling microdomain in regulation of transporter trafficking. Biochim Biophys Acta 2010;1802:1237-45.

192

The role of HMGB1 in experimental myocardial infarction

Martina Cebova^1,2, Andrej Barta¹ and Olga Pechanova^1,2

¹Centre of Experimental Medicine Slovak Academy of Sciences; ²Institute of Pathophysiology, Faculty of Medicine, Comenius University

Introduction

Myocardial infarction (MI) followed by reperfusion triggers a complex sequence of pathophysiological responses. High mobility group box 1 (HMGB1) is a multifunctional DNA-binding protein which plays a crucial role in various cellular processes. It is also released into the extracellular space during heart ischaemia, where it exerts potent pro-inflammatory effects. The aim of the study was to evaluate the effects of anti-HMGB1 protein on biochemical and morphological parameters after myocardial infarction in 12-week-old male WKY rats.

Methods

MI was induced by ligation of the left descending coronary artery. Prior to reperfusion, anti-HMGB1 protein was administrated i.v. Ligation was released twenty minutes after MI was induced. 7 days after MI, nitric oxide synthase (NOS) activity was determined by conversion of 3[H] arginine to 3[H] citrulline in the aorta and in the ischaemic, border and non-ischaemic region of the heart. HMGB1, nuclear factor kappa B (NFκB), inducible NOS (iNOS) and endothelial NOS (eNOS) expression were determined by Western blot. TTC-staining procedure was used for morphological analyses. Cytokine levels were investigated using the Bio-Plex Pro Cytokine kit in the plasma.

Results

The expression of HMGB1 after MI was significantly up-regulated in both tissues. Anti-HMGB1 protein increased NOS activity in both ischaemic and border heart zone, as well as in the aorta. The same pattern was found in eNOS expression level. Anti-HMGB 1 protein administration decreased iNOS and NFκB expression in the ischaemic zone as well as TNF-alpha and IL-6 level in plasma. Simultaneously, anti-HMGB1 protein decreased area of ischaemic part as well as border region of the heart. The concentration of conjugated dienes as a marker of oxidative damage was significantly decreased after the administration of anti-HMGB1 protein in heart, kidney, and liver tissue compared to both the control group and the group with experimental MI.

Conclusion

HMGB1 was up-regulated after myocardial infarction. Administration of anti-HMGB1 antibodies attenuated the inflammatory response in rats with MI by suppressing the NFκB pathway. This resulted in a reduction of infarct size. These findings suggest that targeting HMGB1 could be a promising therapeutic strategy for mitigating the adverse effects of myocardial infarction.

Supported by: APVV-22-0271; VEGA 2/0131/24

194

Cardiomyocyte-specific overexpression of GTP cyclohydrolase 1 rescues ageing-associated adverse cardiac remodelling

Chang Liu¹, Wen Sheng Qi¹ and Yin Cai¹

¹The Hong Kong Polytechnic University; ²Department of Anesthesiology, The First Hospital of Jilin University

Introduction

Cardiac ageing involves adverse remodelling, leading to increased incidence of heart failure and cardiovascular disease in the elderly. A significant factor is the increase in oxidative stress, causing cellular damage and impaired cardiac function. GTP cyclohydrolase 1 (GCH1), the rate-limiting enzyme in tetrahydrobiopterin biosynthesis, mitigates oxidative stress by elevating tetrahydrobiopterin levels. Despite its protective role in different cardiac pathological models, the impact of GCH1 on cardiac ageing remains underexplored. This study investigates whether cardiomyocyte-specific overexpression of GCH1 (GCH1-tg) can alleviate cardiac ageing and explores the involvement of oxidative stress in the underlying mechanisms.

Methods

Male wildtype (WT, 8 weeks old) and age-matched GCH1-tg mice were administered D-galactose (150 mg·kg⁻¹·day⁻¹, intraperitoneally) for 8 weeks to induce ageing. Cardiac function was assessed using echocardiography. Blood pressure was measured using tail-cuff technique. Protein expression levels of myocardial p21 and p53 (cellular ageing biomarkers), MMP9 and collagen III (markers of cardiac fibrosis) and catalase were detected by Western blotting. Reactive oxygen species (ROS) levels were measured using dihydroethidium (DHE) staining. Additionally, to evaluate the impact of GCH1 on age-related physiological decline, we compared cardiac function via echocardiography between male WT and age-matched GCH1-tg mice at 6, 12, and 18 months of age.

Results

We successfully established a cardiac ageing model using D-galactose, characterized by cardiac senescence, fibrosis and impaired cardiac function. GCH1-tg significantly mitigated D-galactose-induced ageing-associated adverse cardiac remodelling. This was evidenced by reduced protein expression of myocardial p21, p53, MMP9 and collagen III, reduced blood pressure, as well as improved cardiac function. Specifically, GCH1-tg led to increased ejection fraction (EF) and fraction shortening (FS), a reduced E/E′ ratio and decreased global longitudinal strain (GLS) compared to WT mice (P < 0.05, n = 5–7). These improvements were accompanied by reduced cardiac ROS generation and catalase expression, a key component of the cellular antioxidant defence system. To further substantiate the cardioprotective role of GCH1 on cardiac ageing, we employed a physiological ageing murine model. While no differences were observed between GCH1 and WT mice at 6 and 12 months of age, GCH1-tg mice exhibited significantly superior systolic and diastolic function compared to WT mice at 18 months of age (P < 0.05, n = 7).

Conclusion

GCH1-tg alleviates ageing-associated adverse cardiac remodelling in both physiological and D-galactose-induced ageing models. The protective effect may be attributed to the anti-oxidative properties of GCH1. Taken together, these findings suggest that GCH1 may be a potential target for prevention and treatment of cardiac ageing-related disease.

197

Control of the TMEM16A channel by GPCR pathways and its implication for the control of artery tone

Catherine Choi, Paolo Tammaro and Rumaitha Al Hosni

Department of Pharmacology, University of Oxford, Oxford, UK

Introduction/Background and Aims

Vascular TMEM16A Ca2+-activated Cl- channels (CaCCs) open in response to 3-phosphate (IP3)-mediated Ca2+ release during Gq protein-coupled receptor (GqPCR) activation [1]. TMEM16A opening promotes smooth muscle cell (SMC) depolarisation and contraction. TMEM16A is a proposed drug target for hypertension and stroke [2]. Whether G-protein βγ (Gβγ) subunits control TMEM16A activity is unestablished. Here, we explore the possible contribution of Gβγ subunits to the modulation of TMEM16A channel function.

Method/Summary of Work

Heterologous TMEM16A currents in human embryonic kidney 293T (HEK-293T) cells were recorded during whole-cell patch-clamp. HEK-293T cells were co-transfected with TMEM16A and either the α1 (α1R) or the β2 (β2R) adrenoreceptors to study the effects of GPCR activation on TMEM16A channel activity in the absence or presence of gallein, a Gβγ inhibitor. Isometric tension (wire myography) of rat aortae was used to investigate the vessel response to phenylephrine (PE), a contractile agonist acting on α1R, in the absence or presence of gallein and specific TMEM16A inhibitors. Statistical significance was determined with paired or unpaired t tests or One-Way ANOVA with appropriate post-test.

Results/Discussion

Co-expression of TMEM16A with the α1 adrenoreceptor (α1R) gave rise to currents of 140 ± 31 pA/pF (n = 30) at +100 mV, in response to PE. Acute application of gallein (50 μM) did not alter the TMEM16A current in both the absence or presence of unstimulated α1R. When α1R was stimulated with PE, gallein dampened TMEM16A currents by 1.7 ± 1.6 fold (n = 27) at +100 mV. Inclusion of Gβγ subunits in the pipette solution potentiated TMEM16A currents by 3.6 ± 0.2 fold (n = 22). In contrast, TMEM16A currents measured in cells co-transfected with the β2R were not affected by gallein implying that the channel is specifically activated by Gβγ subunits released by the GqPCR, but not GsPCR. Treatment of isolated rat aortic rings with gallein did not alter the aortic response to PE.

Conclusions

Gβγ subunits modulate the TMEM16A channel in response to α1R activation. Inhibition of Gβγ subunits did not alter the response of isolated aortae to PE, possibly because multiple membrane currents are modulated by Gβγ subunits counteracting their contribution to vessel tone.

References

1. Arreola J, et al. Insights into the function and regulation of the calcium-activated chloride channel TMEM16A. Cell Calcium. 2024 121:102891.

2. Al-Hosni R, Kaye R, Choi CS, Tammaro P. The TMEM16A channel as a potential therapeutic target in vascular disease. Curr Opin Nephrol Hypertens. 2023 33(2):161-169.

200

Sodium glucose cotransporter 2 inhibitors as a novel therapeutic for pulmonary arterial hypertension

Kate Sloan, Kathryn Wilson and David Welsh

Glasgow Caledonian University

Introduction/ Background and Aims

Pulmonary arterial hypertension (PAH) is a disease with an unmet need for effective novel therapies. Current approved treatments target the narrowing pulmonary vasculature, but patients continue to die of right heart failure caused by maladaptive right ventricular remodelling. Sodium glucose cotransporter 2 inhibitors (SGLT2i) are a class of oral antidiabetic drug which regulate glycaemic parameters. SGLT2i have shown promising off target cardioprotective effects, confirmed by numerous cardiovascular outcome trials in left heart failure [1]. I hypothesise that SGLT2i could also be effective at attenuating right ventricular dysfunction in PAH. An in vitro hypoxic cell culture model of PAH was used to test this hypothesis, determining the effects of SGLT2i on the proliferative and migratory responses which contribute to detrimental vascular and cardiac remodelling in PAH.

Methods

Proliferation assays were used to determine the effects of SGLT2 inhibition (Canagliflozin, 0.1, 1 and 100 μM) in a hypoxic cell culture model (5% O₂, 5% CO₂) of PAH using primary rat pulmonary artery fibroblasts (RPAF) isolated from healthy Sprague–Dawley rats. Cell proliferation was quantified using a Countess 3 Automated Cell Counter. Scratch assays were used to determine the migratory responses of RPAF to SGLT2 inhibition (Canagliflozin, 100 μM).

Results/Discussion

RPAF exposed to hypoxic conditions resulted in increased proliferation (P < 0.01) (Figure 1) and increased migration (P < 0.0001) (Figure 2) compared to those maintained in normoxia. Canagliflozin was able to inhibit proliferation of RPAF in hypoxia at both 1 and 100 μM (P < 0.01) (Figure 1). Migration of RPAF in hypoxia was inhibited by canagliflozin at 100 μM (P < 0.0001) (Figure 2).

Conclusions

Reference

1. Wiviott SD, Raz I, Bonaca MP, et al; DECLARE–TIMI 58 investigators. Dapagliflozin and cardiovascular outcomes in type 2 diabetes. N Engl J Med. 2019;380(4):347-357. https://doi.org/10.1056/NEJMoa1812389

207

H2S is a key mediator in the vascular protective effects of osthole against oxidative stress

Elif Alan Albayrak, Ozan Mert and Gulnur Sevin

Faculty of Pharmacy, Department of Pharmacology, Ege University

Introduction

Oxidative stress impairs endothelium/NO-mediated relaxations and causes vascular dysfunction. H₂S produced from L-cysteine plays a compensatory role in NO deficiency and exhibits antioxidant effects [1]. Osthole, a natural coumarin, has potent relaxant and antioxidant effects [2]. It has been observed that the pharmacological effects of osthole occur through common mechanisms with H₂S. However, the osthole-H₂S relationship has not been previously studied. Our study investigated the role of H₂S in the protective effects of osthole on oxidative stress-induced vascular dysfunction.

Method

Swiss albino mouse (male and female, 8–9 weeks old) aorta segments (MAs) were mounted on a ring myograph. Osthole/vehicle (1–300 μM)-induced relaxations were obtained in the presence/absence of aminooxyacetic acid (AOAA, H₂S synthesis inhibitor, 10 mM, 30 min) and Nω-nitro-L-arginine methyl ester (L-NAME, NO synthesis inhibitor, 300 μM, 30 min). L-Cysteine (300 μM-100 mM)-induced relaxations were investigated in the presence/absence of pyrogallol (induces oxidative stress, 100 μM, 5 min), osthole/vehicle (10 μM, 15 min) and AOAA. Basal and L-cysteine-induced H₂S productions were investigated by measuring real-time H₂S synthesis using an H₂S microsensor in the presence/absence of pyrogallol, osthole/vehicle and AOAA in MA homogenates. We measured reactive oxygen species (ROS) using chemiluminescence assays (luminol/lucigenin) in the presence/absence of pyrogallol, osthole/vehicle and AOAA in MA. Statistical significance was determined using ANOVA followed by a Bonferroni post hoc test. Ethical approval was obtained from the Ege University Local Ethics Committee of Animal Experiments (Approval number: 2021-052).

Results

AOAA and L-NAME inhibited osthole-induced relaxations in healthy MA (P < 0.001) (Table 1). Osthole augmented the relaxation response to L-cysteine in healthy MA (P < 0.05) (Table 1). Osthole protected from pyrogallol-induced decrease in L-cysteine-induced relaxations and AOAA inhibited this effect of osthole (P < 0.001) (Table 1). Osthole augmented L-cysteine-induced H₂S production in healthy MA (P < 0.01) and inhibited the pyrogallol-induced reduction in H₂S production (P < 0.05) (Table 2). Osthole decreased the pyrogallol-induced increase in ROS levels (P < 0.05) (Figure 1). These effects of osthole were inhibited by AOAA (P < 0.05).

Conclusion

In conclusion, our study demonstrated that osthole protects vascular function from oxidative stress by inducing endogenous H₂S production. Further studies are required to determine whether osthole could be a potential drug for oxidative stress-induced endothelial dysfunction-related diseases.

Acknowledgements

References

1. Citi V, Martelli A, Gorica E, Brogi S, Testai L, Calderone V. Role of hydrogen sulfide in endothelial dysfunction: Pathophysiology and therapeutic approaches. J Adv Res.2021;27:99-113.

2. Zhang ZR, Leung WN, Cheung HY, Chan CW. Osthole: A review on its bioactivities, pharmacological properties, and potential as alternative medicine. Evidence-based Complement Altern Med. 2015;2015:919616.

209

Neurokinin 1 receptor inhibition is protective against pulmonary arterial hypertension and pulmonary fibrosis

Kara Hetherington^1,2, Himanshu Chuglani¹, Jordyn Nelson¹, Giannie Barsha^1,2, Jordyn Thomas^1,2, Barbara Kemp-Harper¹, Stephen Nicholls² and Kristen Bubb^1,2

¹Biomedicine Discovery Institute, Monash University; ²Victorian Heart Institute, Monash University

Introduction

Pulmonary hypertension (PH) can develop spontaneously, or in response to chronic lung fibrosis, heart or thromboembolic disease, connective tissue disorders, or after exposure to certain substances/drugs. Ultimately, PH leads to increased right ventricular (RV) pressure, hypertrophy and eventually RV failure. Substance P promotes vasoconstriction and inflammation in the lungs and has been associated with PH. Some evidence shows that inhibition of neurokinin 1 (NK1) receptor-mediated effects of substance P can lower pulmonary pressure and organ (cardiac/liver) fibrosis. However, little is known about whether NK1 receptor blockade can alter pulmonary vascular structure, fibrosis or RV function in PH. We hypothesized that by blocking NK1 receptors, pulmonary vascular remodelling and interstitial fibrosis would be attenuated in mouse models of PH and pulmonary fibrosis. We aimed to examine PH parameters and the effects of NK1 receptor deficiency using either a selective antagonist or by assessing mice with global knockout of the NK1 receptor (Tacr1^−/−).

Methods

C57BL6/J or Tacr1^−/− mice (n = 7–9) were treated with bleomycin sulphate by oropharyngeal inhalation to trigger pulmonary fibrosis over 2 weeks. PH was also induced in C57BL6/J or Tacr1^−/− mice with injections of vascular endothelial growth factor inhibitor, SU5416 (20 mg·kg⁻¹·week⁻¹ 3×, s.c.) and exposure to chronic hypoxia (10% oxygen) for 5 weeks. C57BL6/J mice were treated with NK1 receptor antagonist, aprepitant (1.2 mg·kg⁻¹·day⁻¹, oral) or vehicle (1.5% DMSO in saline) for 2 weeks (prevention) or for the last 3 weeks of hypoxia (intervention). RV systolic pressure (RVSP, indwelling catheter) and RV function (echocardiography-tricuspid annular plane systolic excursion [TAPSE]) were measured in anaesthetized mice (isoflurane 1–3%). Lung sections were prepared post-mortem and stained with Masson's trichrome (pulmonary vascular remodelling) and picrosirius red (lung interstitial fibrosis). Data are presented as mean ± SEM and were analysed by one-way ANOVA.

Results

RVSP was elevated in Tacr1^+/+ hypoxic mice (46.8 ± 1.7 mmHg) vs. normoxic controls (24.7 ± 0.9 mmHg) and this was attenuated in Tacr1^−/− mice (39.9 ± 1.0 mmHg; n = 7; P < 0.001). Treatment of hypoxic mice with aprepitant led to a lowering of RVSP (mmHg: vehicle 44.3 ± 1.8 vs. aprepitant 37.8 ± 1.9, n = 8–9, P < 0.05). Pulmonary vascular remodelling measured by media:lumen ratio was attenuated after aprepitant (%: normoxia vehicle 1.94 ± 0.06, hypoxia vehicle 4.13 ± 0.31, hypoxia aprepitant 2.46 ± 0.13; n = 7–8; P < 0.001, Figure 1). In turn, RV function, was improved with aprepitant treatment (TAPSE (mm): normoxia vehicle 0.99 ± 0.02, hypoxia vehicle 0.68 ± 0.02, hypoxia aprepitant 0.92 ± 0.02; n = 5; P < 0.001).

Conclusion

NK1 receptor inhibition offers a potential new treatment target to improve pulmonary hypertension and fibrosis.

210

Exploring the role of osthole in preventing vascular dysfunction in high-glucose environments

Tugba Hilal Kilic, Elif Alan Albayrak, Erenay Altunsayar, Gulcan Demir and Gulnur Sevin

Department of Pharmacology, Faculty of Pharmacy, Ege University

Introduction

One of the most significant chronic diseases that compromise vascular function is diabetes. High glucose (HG) levels lead to vascular dysfunction by disrupting NO production/bioavailability through oxidative stress [1]. Developing agents that protect against HG-induced vascular endothelial dysfunction and oxidative stress is crucial for preventing diabetic microvascular/macrovascular complications. Although osthole has been reported as antioxidant and antidiabetic, its role in diabetes-induced vascular dysfunction has not yet been investigated [2]. We investigated the protective effects of osthole against HG-induced vascular dysfunction in this study.

Method

In the literature, vascular tissues have been incubated with HG under ex vivo conditions to mimic diabetes. We cultured aorta segments isolated from Sprague-Dawley rats (male and female, 10–12 weeks old) for 24 h in the presence/absence of osthole/vehicle (30 μM) and high glucose (HG, 30 mM). The incubation period of osthole was started 2 h before HG. At the end of the incubation period, the segments were mounted on PanLab organ baths; KCl (20 mM-120 mM)- and Phenylephrine (Phe, 0.01–100 μM)-induced contractions and Acetylcholine (ACh, 0.03–100 μM) and sodium nitroprusside (SNP, 0.001–100 μM)-induced relaxations were recorded. Additionally, by measuring luminol and lucigenin chemiluminescence reactive oxygen species (ROS) in the aorta segments were determined. Data were analysed using Bonferroni post-hoc test, two-way ANOVA and Student's t-test (unpaired) as applicable. N is the number of animals. Ethical approval was obtained from the Ege University Local Ethics Committee of Animal Experiments (approval number: 2023-085).

Results

Aorta segments incubated with HG showed increased contraction responses to KCl (P < 0.05, N = 5) and Phe (P < 0.05, N = 4) and decreased relaxation responses to ACh (P < 0.05, N = 5) and SNP (P < 0.05, N = 5) compared to the control/vehicle. Osthole protected vascular tissue from HG-induced increase in KCl- (P < 0.05, N = 4) and Phe-induced (P < 0.05, N = 4) contractions. Additionally, osthole inhibited HG-induced decrease in relaxations to ACh (P < 0.01, N = 5) and SNP (P < 0.001, N = 5) (Table 1). Moreover, osthole reduced the increased ROS production in the presence of HG (Figure 1).

Conclusion

Osthole exhibited a protective effect against HG-induced vascular endothelial and smooth muscle dysfunction and oxidative stress. The vascular protective effect of osthole, a promising multi-target drug, should be further evaluated in vivo diabetes models.

Acknowledgements

This study is supported by the Scientific and Technological Research Council of Türkiye (TUBITAK-2209, 1919B012302025).

References

1. Yang DR, Wang MY, Zhang CL, Wang Y. Endothelial dysfunction in vascular complications of diabetes: A comprehensive review of mechanisms and implications. Front Endocrinol (Lausanne) 2024;15:1359255.

2. Zafar S, Sarfraz I, Rasul A, et al. Osthole: A multifunctional natural compound with potential anticancer, antioxidant and anti-inflammatory activities. Mini Rev Med Chem.2021;21(18):2747-2763.

212

Therapeutic potential of abatacept in rat model of cardiac hypertrophy via CTLA-4: A exploration of signalling pathways

Vaishali Prajapati, Vipin Kumar Verma, Jagriti Bhatia and D. Sarya

All India Institute of Medical Sciences

Introduction

Abatacept (a synthetic CTLA4 analogue) inhibits the co-stimulation of T-cells during their activation and minimize inflammation through infiltration of immune cell at the site of injury. The drug is mainly used in psoriatic diseases and Rheumatoid arthritis [1]. Because of its mechanism of action, we have investigated its role in the isoproterenol-induced cardiac hypertrophy model of rat which is caused by increased cardiac workload [2]. This study hypothesized that Abatacept may reduce cardiac T-cells and further macrophage entry into the myocardium, which can prevent cardiac hypertrophy in male albino Wistar rat model of isoproterenol (ISO)-induced cardiac hypertrophy was assessed after abatacept administration through haemodynamic, structural, biochemical and molecular investigations.

Methods

In this study male Wistar albino rats were divided into six groups (n = 6): Group 1 Control (PBS administration); Group 2 disease control (ISO at 3 mg/kg s.c. was induced to induce hypertrophy); Groups 3, 4 and 5 are experimental groups. Abatacept at doses of 2.5, 5 and 10 mg·kg⁻¹ were administered daily along with the dose of ISO at 3 mg·kg⁻¹ s.c. to induce hypertrophy. In Group 6 only abatacept was injected s.c. at 10 mg/kg dose each day. The experimental drug administration duration was 42 days to investigate the effect of abatacept in pathological Cardiac Hypertrophy. On day 43rd, all the rats were weighed and anaesthetized with sodium pentobarbital (60 mg·kg⁻¹, i.p.). After haemodynamic assessments, the blood was collected and the heart was excised to investigate physiological parameters, cardiac injury markers, oxidant-antioxidant assessments, inflammation, immune cell infiltration and apoptosis.

Results

Abatacept effectively reduced cardiac hypertrophy by stabilizing heart function. It showed positive effects on antioxidants, minimizing cardiac injury markers (CK-MB, LDH), histopathological evidence (H&E and MT staining), along with decreased inflammatory cytokines and inflammation formation. Immunofluorescence staining of T-cell sub-populations was also assessed using co-staining and showed a dose-dependent reduction in activation of T cells. The analysis of anti-apoptotic, apoptotic, necrosis, autophagy and inflammatory (MAPK/Nrf-2-HO1/Smad-Tgf-beta) supports hypothesis of abatacept cardio-protective action.

Conclusions

Abatacept diminishes ISO-induced cardiac hypertrophy by inhibiting oxidative stress, inflammation and infiltration, however, enhancing cardiac function and myocardium architecture. It could be a potential preventive drug for cardiac hypertrophy or for individuals at risk of developing it.

References

1. Blair HA, Deeks ED. Abatacept: A review in rheumatoid arthritis. Drugs. 2017;77(11):1221-1233. https://doi.org/10.1007/s40265-017-0775-4

2. Weisman MH, Durez P, Hallegua D, et al. Reduction of inflammatory biomarker response by abatacept in treatment of rheumatoid arthritis. J Rheumatol. 2006;33(11):2162-2166.

233

Novel NLRP3 inflammasome inhibitor in a mouse model of isoproterenol-induced heart failure

Magalì Giordano¹, Claudia Penna¹, Pasquale Pagliaro¹, Massimo Bertinaria² and Stefano Toldo³

¹Department of Clinical and Biological Sciences, University of Turin; ²Department of Drug Science and Technology, University of Turin; ³Robert M. Berne Cardiovascular Research Center, Department of Medicine, Division of Cardiovascular Medicine, University of Virginia

Introduction

Heart failure (HF) is a clinical syndrome characterized by low cardiac output or increased left ventricular (LV) filling pressures. Increased sympathetic nerve activation and β-adrenergic stimulation promote the development of HF [1]. Chronic administration of isoproterenol (ISO), a non-selective β-adrenergic receptor agonist, can induce HF in mice. This model is useful for studying the role of β-adrenergic signalling and testing cardioprotective drugs. An inflammatory response, particularly through the NLRP3 inflammasome, is activated under chronic β-adrenergic stimulation, contributing to LV remodelling and HF [2]. We hypothesize that INF195, a novel NLRP3 inhibitor that stabilizes the self-inhibited form of its NACHT domain and effective in an ischaemia/reperfusion injury model [3], protects mice from ISO-induced HF.

Methods

Eight-week-old male C57BL/6 mice (N = 6/group) were intraperitoneally injected with ISO (5 mg·kg⁻¹·day⁻¹) for 21 days. INF195 (30 mg·kg⁻¹), or an equal volume of vehicle (100 μL, 25% ethanol-25% chemophor, 50% injectable saline) were injected once daily. We used trans-thoracic echocardiography to measure LV ejection fraction (LVEF) and size, pulse-wave Doppler echocardiography to assess diastolic function, and LV catheterization to evaluate LV end-diastolic pressure (LVEDP). Cardiomyocyte cross-sectional area was measured in tissue sections stained with wheat germ agglutinin (WGA). p-AKT/AKT and p-ERK/ERK were evaluated using western blot. Statistical analysis was performed using one-way ANOVA followed by Tukey's and Dunn's tests.

Results

Isoproterenol decreased the LVEF and increased the LV mass and heart weight. In addition, Isoproterenol increased E/E′ and isovolumetric relaxation time (IRT) and LVEDP, signs of diastolic dysfunction and HF. INF195 preserved the LVEF, the LV mass, E/E′, IRT, and LVEDP (Table 1). INF195 also reduced Isoproterenol-induced cardiomyocyte hypertrophy (Table 2). IFN195 also reduced the changes in p-AKT/AKT and p-ERK/ERK induced by isoproterenol (Table 3).

Conclusions

INF195 protects mice from isoproterenol-induced HF. Additional studies are needed to define the pathophysiological mechanisms mediated by NLRP3 and how INF195 protects the heart from LV remodelling and dysfunction.

References

1. McDonagh TA, Metra M, Adamo M, et al. 2021 ESC guidelines for the diagnosis and treatment of acute and chronic heart failure. Eur Heart J. 2021;42(36):3599-3726. https://doi.org/10.1093/eurheartj/ehab368.

2. Mezzaroma E, Toldo S, Farkas D, et al. The inflammasome promotes adverse cardiac remodeling following acute myocardial infarction in the mouse. Proc Natl Acad Sci U S A. 2011;108(49):19725-30. https://doi.org/10.1073/pnas.1108586108.

3. Gastaldi S, Giordano M, Blua F, et al. Novel NLRP3 inhibitor INF195: Low doses provide effective protection against myocardial ischemia/reperfusion injury. Vascul Pharmacol. 2024;156:107397. https://doi.org/10.1016/j.vph.2024.107397.

236

Ethanolamine as a cardioprotective agent for doxorubicin-induced cardiotoxicity

Réalta Victory¹, Ellena O'Keeffe^1,2, Emily Farrugia³, Eli O'Driscoll¹, Orla Barry¹ and Róisín Kelly-Laubscher¹

¹Department of Pharmacology and Therapeutics, School of Medicine, College of Medicine and Health, University College Cork, Department of Oncology; ²Department of Clinical Pharmacology and Therapeutics, University of Oxford; ³University of Malta

Introduction

Doxorubicin, an anthracycline chemotherapeutic drug, is associated with significant cardiotoxicity. Ethanolamine has been shown to protect the isolated heart against ischaemia-reperfusion injury; however, its use as a cardioprotective agent against doxorubicin-induced cardiotoxicity has not been assessed [1]. Therefore, this study investigates the effects of ethanolamine on doxorubicin-induced cardiotoxicity.

Methods

The dose-dependent effects of ethanolamine (3 μM-300 mM) on doxorubicin (5 or 10 μM)-induced cytotoxicity were determined in H9c2 cardiomyoblasts, NIH3T3 fibroblasts, isolated murine cardiomyocytes and H9c2/fibroblast coculture. Cell viability was determined using MTT and LDH assays for H9c2, fibroblasts and coculture experiments, and trypan blue exclusion and LDH assays in murine cardiomyocytes. Coculture experiments tested whether ethanolamine protected cardiac cells by causing release of factors from fibroblasts, using H9c2 cocultured with H9c2s as a negative control. Statistical significance was determined using one-way ANOVA followed by Dunnett's post hoc test for individual cell culture experiments and two-way ANOVA followed by Tukey's post hoc test for coculture experiments.

Results

A significant increase in metabolic activity was observed for NIH3T3 cells pretreated for 24 h with 30 μM (94.80 ± 18.68%), 300 μM (89.45 ± 29.56%), and 3 mM (100.40 ± 16.52%) ethanolamine compared to 5 μM doxorubicin alone (14.96 ± 14.37% P < 0.05; Figure 1a); no significant changes in LDH release were seen. No significant effects of ethanolamine on cell viability were seen in H9c2 or primary cardiomyocytes when treated 24 h before, during, or 24 h after doxorubicin treatment. Co-culturing of NIH3T3 and H9c2 cells pretreated for 24 h with 3 μM ethanolamine increased metabolic activity of cells (546.10 ± 299.83%) compared to doxorubicin (5 μM) alone (69.18 ± 7.53%, P ≤ 0.001; Figure 1b); no significant changes in LDH release were observed. Unfortunately, this protection was also seen in H9c2 cells cocultured with H9c2 cells (Dox; 69.72 ± 10.99% vs. ethanolamine; 782.20 ± 432.08% P < 0.0001).

Conclusion

The results from this study are mixed, with ethanolamine protecting fibroblasts but not cardiomyocytes. However, coculture with H9c2 or 3 T3 cells confers protection of H9c2 at low concentrations, suggesting that additional cells may absorb and release ethanolamine slowly into the culture The data from these experiments will inform the development of treatment strategies for subsequent in vivo studies.

Funding

Reference

1. Kelly RF, Lamont KT, Somers S, et al. Ethanolamine is a novel STAT-3 dependent cardioprotective agent. Basic Res Cardiol. 2010;105(6):763-770.https://doi.org/10.1007/s00395-010-0125-0

267

A role for endothelial PKGIα in pulmonary artery

Hala Alhabashneh¹, Alison Gurney² and Adam Greenstien²

¹Amman Arab University; ²University of Manchester

Introduction

Protein kinase G (PKG) is the end effector kinase in vasodilation mediated by the NO/cGMP pathway. PKG is also activated by oxidation of cysteine residues in the PKGIα subunit, which in smooth muscle induces relaxation [1]. PKG is also present in endothelium where its function is poorly understood. This study investigated the role of oxidant-activated, endothelial PKG in regulating pulmonary arterial tone.

Method

Endothelium-dependent relaxation evoked by carbachol or theTRPV4 agonist, GSK1016709A, was compared in intra-pulmonary arteries from wild type (WT) mice and PKG[C42S]KI mice lacking the cysteine-based oxidant sensor [1], using wire myography. Endothelial Ca²⁺ signalling was compared in fluo4-loaded en-face preparations using spinning-disc confocal microscopy. All procedures met the requirements of the Animals (Scientific Procedures) Act 1986/Amendment Regulations 2012. Data are given as mean ± SEM and compared using two-tailed unpaired t-tests

Results

U46619 (30 nM)-contracted arteries from WT and PKG[C42S]KI mice relaxed equally to carbachol, with pEC50 values of 5.9 ± 0.1 (n = 9) and 5.8 ± 0.1 (n = 7), respectively, and maxima of 38 ± 9% and 40 ± 9%. Arteries from PKG[C42S]KI mice were less sensitive to GSK1016790A (pEC50 = 7.48 ± 0.08, n = 10) than WT arteries (pEC50 = 8.0 ± 0.1, n = 8; P = 0.0005), but reached a similar maximum relaxation (WT = 91 ± 2%; PKG[C42S]KI = 86 ± 3%). Endothelium removal suppressed relaxation to carbachol in both tissues, but inhibited responses to GSK1016790A only in WT arteries. Carbachol (10 μM) increased the frequency of Ca²⁺ pulsars from 0.6 ± 0.2 Hz to 1.5 ± 0.3 Hz (n = 11, P = 0.016) in WT endothelium, indicating enhanced Ca²⁺ release from the endoplasmic reticulum (ER). Pulsar frequency in PKG[C42S]KI endothelium reached a similar level (1.3 ± 0.4 Hz, n = 7) after adding carbachol, from a baseline of 0.8 ± 0.2 Hz. Endothelial Ca²⁺ influx via TRPV4 channels (sparklets) was recorded in the presence of cyclopiazonic acid (1 μM) to block ER Ca²⁺ storage. The number of sites and frequency of sparklets recorded in the presence of 10 nM GSK1016790A was lower in the endothelium of PKG[C42S]KI arteries (15 ± 2 sites/5 μm², 0.24 ± 0.02 Hz, n = 5) compared with WT arteries (92 ± 27 sites/5 μm², 1.5 ± 0,4 Hz, n = 5, P = 0.02).

Conclusion

The loss of GSK1016790A-induced Ca²⁺ sparklets and endothelium-dependent relaxation in PKG[C42S]KI arteries indicates that oxidative activation of PKGIα facilitates TRPV4-mediated vasodilation. As carbachol retained its ability to stimulate Ca²⁺ pulsars and relaxation in PKG[C42S]KI arteries, muscarinic vasodilation did not employ TRPV4 channels.

Reference

1. Burgoyne, J. R., Madhani, M., Cuello, F., Charles, R. L., Brennan, J. P., Schröder, E, Browning, D. D., & Eaton, P. (2007). Cysteine redox sensor in PKGIa enables oxidant-induced activation. Science, 317(5843), 1393–1397. https://doi.org/10.1126/science.1144318

281

The effect of α1-adrenoceptor antagonists on the positive chronotropic effect induced by catecholamines in the rat isolated atrium

Bruna Lourenconi Alves¹, Jose Britto-Junior^1,2, Denis Oliveira Lima¹, Antonio Tiago Lima¹, Edson Antunes¹ and Gilberto De Nucci^1,3

¹University of Campinas; ²King's College; ³University of São Paulo

Introduction

Alpha-adrenoceptor antagonists are known to exhibit the ‘first-dose phenomenon’, by which following oral administration there is a sudden and severe fall in blood pressure, especially during postural change [1]. Endothelium-derived 6-nitrodopamine has a potent positive chronotropic effect on the rat-isolated right atrium, and it presents remarkable synergism with the classical catecholamines dopamine, noradrenaline, and adrenaline [2]. Here, we evaluated the effect of selective α1-adrenoceptor antagonist tamsulosin, doxasozin, and alfuzosin on the positive chronotropic effect induced by 6-ND.

Method

Adult male Wistar rats (280–320 g) were euthanized by isoflurane overdose (>5%) until one minute after breathing ceased. The heart was removed and the right atrium was isolated and mounted between two metal hooks in 10 mL glass chambers filled with Krebs-Henseleit solution heated (37°C) and gassed (95%O₂:5%CO₂). The α1-adrenergic receptor antagonists alfuzosin, doxazosin, and tamsulosin (100 nM) were added to the organ bath after a 30 min equilibration period, and changes in atrial rate were monitored for 30 min. The increase in atrial rate induced by 6-ND (1 pM) was evaluated with and without the antagonists (100 nM). Cumulative concentration-response curves for noradrenaline (0.1 nM-100 μM), adrenaline (0.1 nM–100 μM), and dopamine (1 nM–1 mM) were performed in the absence and presence (30 min) of each antagonist. Curves were analysed using sigmoidal concentration-response model to determine EC50 and maximum response (Emax). Statistical significance was determined using student t test.

Results

Alfuzosin at 100 nM (30 min; Figure 1A) caused significant fall in the atrial frequency. It also reduced the atrial rate of D-NAME (100 μM) pretreated atria (Figure 1B), but it had no effect on L-NAME (100 μM) pretreated atria (Figure 1C), or in atria harvested from animals chronically treated with L-NAME (Figure 1D). Alfuzosin significantly reduced the increase in atrial rate induced by 6-ND (1pM; Figure 1E). Pre-incubation with Alfusozin followed by concentration-response curves to noradrenaline, adrenaline, and dopamine did not affect the increases in atrial rate induced by these catecholamines in a concentration-dependent manner (Figure 1F–H). Similar results were obtained with doxazosin (100 nM; Figure 2) and tamsulosin (100 nM; Figure 3).

Conclusion

Together the results indicate that the fall in atrial rate induced by α1-adrenoceptor antagonists may be due to blockade of 6-ND positive chronotropic action.

References

1. Elliott HL, McLean K, Sumner DJ, Meredith PA, Reid JL. Immediate cardiovascular responses to oral prazosin-Effects of concurrent β-blockers. Clin Pharmacol Ther. 1981;29(3):303-309. https://doi.org/10.1038/clpt.1981.40

2. Britto-Júnior J, Lima AT, Fuguhara V, Monica FZ, Antunes E, De Nucci G. Investigation on the positive chronotropic action of 6-nitrodopamine in the rat isolated atria. Naunyn Schmiedebergs Arch Pharmacol. 2023;396(6):1279-1290. https://doi.org/10.1007/s00210-023-02394-9

299

Chronic L-NAME hypertension model abolishes 6-nitrodopamine and dopamine positive chronotropic effect in anaesthetized rats

Vivian Fuguhara¹, Mariana Gonçalves de Oliveira², Carlos Alberto Aguiar da Silva³, Pedro Renato Guazzelli¹ and Gilberto De Nucci¹

¹State University of Campinas; ²São Francisco University; ³University of São Paulo

Introduction

In vitro studies showed that 6-nitrodopamine (6-ND) has a potent positive chronotropic effect, surpassing that of classical catecholamines, adrenaline (ADR), noradrenaline (NA) and dopamine (DA) [1]. This study compares the potency of 6-ND and classical catecholamines in the heart rate (HR) of control and hypertensive rats.

Methods

Adult male Wistar rats were divided in two groups: Control (n = 109) and L-NAME (n = 56). The L-NAME group was treated with Nω-nitro-L-arginine methyl ester (L-NAME; 20 mg per rat·day⁻¹), a nitric oxide synthase inhibitor, dissolved in the drinking water for 4 weeks [2]. Subsequently, the animals were initially sedated with isoflurane (5% for 1 min) and anaesthetized with sodium thiopental (40 mg·kg⁻¹, i.p.) and ketamine (70 mg·kg⁻¹, i.p.). The right femoral vessels were cannulated with a polyethylene PE10 catheter and heparin (600 UI/kg, s.c.) was administered. The artery catheter was coupled to a pressure transducer (MLT0699 Disposable BP Transducer) connected to a data acquisition device, PowerLab, with the LabChart software (ADInstruments). 6-ND, ADR, NA, DA, and the vehicle (saline, Sodium Chloride 0.9%) were injected through intravenous bolus (15 to 25 μL) and monitored for 30 min, the doses used for each drug are presented in the graphics (Figures 1 and 2).

Results

In control rats, all drugs induced positive chronotropic effect. 6-ND increased HR at doses starting from 0.3 pmol·kg⁻¹, ADR at 30 pmol·kg⁻¹, NA at 3 nmol·kg⁻¹ and DA at 300 nmol·kg⁻¹ (Figure 1). However, in L-NAME chronically treated rats, 6-ND and DA did not show any significant effect in HR; in contrast, ADR and NA maintained their effect (Figure 1). Saline did not induce any alterations in both groups Control (n = 9) and L-NAME (n = 6).

Conclusions

References

1. Britto-Júnior J, deOliveira MG, dosReis Gati C, et al. 6-Nitrodopamine is an endogenous modulator of rat heart chronotropism. Life Sci. 2022;307. https://doi.org/10.1016/j.lfs.2022.120879

2. Ribeiro MO, Antunes E, deNucci G, Lovisolo SM, Zatz R. Chronic inhibition of nitric oxide synthesis. A new model of arterial hypertension. Hypertension. 1992;20(3):298-303. https://doi.org/10.1161/01.HYP.20.3.298

307

A comparative analysis of Kv1.5 Markov models: Ellinwood vs. Almquist for atrial fibrillation drug targets

Katie Abraham, Hilary Hunt, Michael Clerx and Gary Mirams

University of Nottingham

Introduction

As drug development becomes increasingly resource-intensive, researchers are looking towards computational modelling to predict drug-protein interactions prior to commencing in vitro experiments, saving resources. Mathematical models of ionic currents can streamline testing and save time. We compare two such models of Kv1.5, an atrial-specific potassium channel responsible for the ultrarapid repolarization of the atria. The absence of Kv1.5 expression in the ventricles makes it a favourable target for treating atrial fibrillation due to the reduced risk of ventricular proarrhythmic side effects such as Torsade de points. We compared the Ellinwood (1) and Almquist (2) models (Figure 1), both are Markov chain-derived models. The critical difference is the number of different states and drug-binding mechanisms; the Almquist model is a 6-state model with the drug binding to the blocked state, and the Ellinwood model allows more flexibility for drugs binding to open, closed, inactive states or to a combination.

Methods

Data from Lagrutta et al. (3) was used, collected via voltage-clamp protocols on CHO cells stably transfected with human Kv1.5 channels (Figure 2). Simulations were performed in Myokit (4) using the calculated Kon and Koff values from Lagrutta to compare the normalized voltage-current relationship of each model. Optimization protocols were run on SciPy fmin optimizer (1.14.0) and data was plotted with Python (3.12.4).

Results

We compared the Kon and Koff values produced after fitting each model to the data of Kv1.5 current with 0.03 and 0.1 μM concentrations of DPO-1 applied, respectively. After comparing the outputs from both to work out the optimized Kon and Koff values (Table 1), our results demonstrate that the model allowing drug binding to both the open and inactive states provides the most accurate fit to the experimental data. Due to the conclusions by Lagrutta et al. that the closed state block plays little to no role in channel kinetics, we didn't include this in our simulations.

Conclusions

References

1. Ellinwood N, Dobrev D, Morotti S, Grandi E. Revealing kinetics and state-dependent binding properties of IKur-targeting drugs that maximize atrial fibrillation selectivity. Chaos: An Interdisciplinary Journal of Nonlinear Science. 2017;27(9).

2. Almquist J, Wallman M, Jacobson I, Jirstrand M. Modeling the effect of Kv1.5 block on the canine action potential. Biophysical Journal. 2010;99(9):2726-36.

3. Lagrutta A, Wang J, Fermini B, Salata J. Novel, potent inhibitors of human Kv1.5 K+ channels and ultrarapidly activating delayed rectifier potassium current. The Journal of pharmacology and experimental therapeutics. 2006;317:1054-63.

4. Clerx M, Collins P, deLange E, Volders PGA. Myokit: A simple interface to cardiac cellular electrophysiology. Progress in Biophysics and Molecular Biology. 2016;120(1-3):100-14.

22

Molecular docking analysis of kaempferol and quercetin from Moringa oleifera with diabetic wound healing-associated vascular endothelial growth factor (VEGF) protein: An in silico approach

Abubakar Muhammad Amali, Amina Yusuf Jega and Sharida Fakurazi

Usmanu Danfodiyo University, Sokoto

Background

Diabetic foot disease has become a global concern. People with diabetes have a significant percentage lifetime risk of developing foot ulcer. High prevalence rates of diabetes in many countries of the world make foot ulcers a major and increasing public health problem. Foot ulcers are known to cause substantial morbidity, impair quality of life, endangering high treatment costs. The unavailability of cost-effective therapeutic agents for diabetic wound healing is of great concern. The aim of this study is to perform in silico molecular docking and ADMET analysis of some bioactive compounds identified from Moringa oleifera against one of the important targets of wound healing protein the vascular endothelial growth factor (VEGF) towards enhancement of wound healing in diabetes.

Methods

The two bioactive compounds from M. oleifera (kaempferol and quercetin) earlier identified and reported in our previous study were screened against VEGF proteins using AutoDock Vina, while the ADMET analysis was performed using swissADME and admetSAR.

Results

The in silico computational studies revealed that the compounds (kaempferol and quercetin) from M. oleifera can effectively bind with high affinity and lower energy values to the VEGF, which may be a target for enhancing wound healing in diabetes. ADMET analysis revealed that the compounds possess wound healing activity.

Conclusion

The findings of this study have shown that the plant M. oleifera contains effective ligands for VEGF and may therefore be considered effective in enhancing wound healing in diabetes.

29

ModuMelt™: Allosteric modulator hit confirmation and characterization

Owen Underwood, David A. Sykes and Dmitry B. Veprintsev

Z7 Biotech Ltd

Sensitive protein stability assays are crucial to structural and biophysical studies. Conventionally high quantities of purified protein are required.

Here, we describe novel high-throughput 384-well BRET-based thermostability assay allowing for the ultrasensitive determination of GPCR stability without any requirement for protein purification or a receptor specific tracer, for example, radioligand.

HEK293 membranes expressing cannabinoid receptors (CB1R, CB2R) with N-terminal Sluc fusions were solubilized in different detergent conditions and centrifuged to remove insoluble membranes. Sulfo-Cy3 maleimide dye was added to solubilized receptors alongside test ligands and subjected to a temperature gradient for 30 min. Samples were treated with furimazine and read on the BMG Labtech PHERAstar FSX. Melt curves were fit to a Boltzmann sigmoidal equation to obtain Tm values.

Melt curves were generated using the CB1-Sluc receptor with the agonists CP55940 and 2-AG, as well as the inverse agonist rimonabant. All compounds exhibited Tm shifts versus a DMSO control (4.79, 2.56, and 5.06°C, respectively). Similarly, when added in the presence of a known PAM, Org27569, these shifts were further increased, despite Org27569 exhibiting no stabilizing effect alone.

A further selection of reported cannabinoid PAMs were assessed in a similar manner, each showing varied shifts in Tm when compared to CP55940 alone (see below).

Compounds exhibiting PAM activity in the CB1 assay described were used to treat CB2 receptors in an identical manner. No compounds exhibited differences in Tm versus CP55940 alone; however, fenofibrate did exhibit a Tm shift of approximately 2°C alone, indicating orthosteric binding.

ModuMelt™ can identify allostery in compounds and determine selectivity and probe dependency using previously described ThermoBRET1 techniques. This method is ideal for hit conformation and the identification of orphan or allosteric ligands, two areas where receptor specific probes are lacking.

Reference

1. Hoare BL, Tippett DN, Kaur A, et al. ThermoBRET: a ligand-engagement nanoscale thermostability assay applied to GPCRs. ChemBioChem 2023;25(2). doi: https://doi.org/10.1002/cbic.202300459

52

Development of C5aR1 negative allosteric modulators with potential benefits for neuroinflammation

Ian Winfield¹, Alison Holiday¹, Kamini Magon¹, Jonathan Powell¹, Iwona Ziomkiewicz², David Dexter² and Janusz Kulagowski²

¹Domainex; ²Parkinson's UK

Introduction

Complement 5a receptor 1 (C5aR1) is a GPCR activated via binding C5a and is expressed on microglia [1]. C5a is an inflammatory peptide produced upon complement activation, and elevated levels initiate a feedforward loop of inflammation via recruitment of microglia to sites of injury, leading to neuronal damage and death [2]. Preventing C5aR1 activation may reduce neuroinflammation resulting in disease-modifying effects. Here, we present a drug discovery programme (Figure 1), utilizing medicinal and computational chemistry, in vitro pharmacology and ADME/PK, which successfully identified and characterized lead-like negative allosteric modulators (NAMs) of the C5aR1.

Method

HTRF and β-galactosidase complementation assays measured cAMP and β-arrestin activity in CHO-K1–C5aR1 cells, stimulated with EC₈₀ C5a and 10-point CRCs of compounds. Allostery was confirmed using curve shift assays, 10-point CRCs of C5a in the presence of increasing concentrations of compounds, fitted with the operational model of allostery [3] to calculate α and β values. Live cell imaging measured chemotaxis of iPSC microglia towards C5a (EC₈₀) in the presence of 8-point CRCs of compounds, over 24 h. Functional effects of NAMs were validated using whole blood from humanized C5aR1 C57/Bl6 mice. Using flow cytometry, CD11b activation, in CD45+ cells, was measured in response to EC₈₀ C5a and 7-point compound CRCs. Physicochemical and ADME properties were assessed in a variety of assays (Figure 1). Select compounds were also profiled in mouse PK experiments to determine Kpuu.

Results

Using our screening cascade (Figure 1), we identified compounds with inhibitory activity in cAMP and β-arrestin recruitment assays, in iPSC microglia chemotaxis and in CD11b activation assays. The allosteric nature of these compounds was confirmed using cAMP and β-arrestin assays. Select compounds were advanced to in vivo PK experiments in which initial Kpuu values of >0.3 were observed. The profile of our lead candidate is identified in Table 1.

Conclusions

We have presented a fully integrated drug discovery programme that identified brain-penetrant novel NAMs of the C5aR1. Further work will focus on the refinement of compound drug-like properties along with subsequent testing in in vivo models of neuroinflammation.

References

1. Schartz ND, Liang HY, Carvalho K, Chu SH, Mendoza-Arvilla A, Petrisko TJ, Gomez-Arboledas A, Mortazavi A, Tenner AJ. C5aR1 antagonism suppresses inflammatory glial responses and alters cellular signaling in an Alzheimer's disease mouse model. Nat Commun 2024;15:7028.

2. Carvalho K, Schartz ND, Balderrama-Gutierrez G, Liang HY, Chu SH, Selvan P, Gomez-Arboledas A, Petrisko TJ, Fonseca MI, Mortazavi A, Tenner AJ. Modulation of C5a-C5aR1 signaling alters the dynamics of AD progression. J Neuroinflammation 2022;19(1):178.

3. Jakubík J, Randáková A, Chetverikov N, El-Fakahany EE, Doležal V. The operation model of allosteric modulation of pharmacological agonism. Sci Rep 2020;10:14421.

57

The state of the art in secondary pharmacology and its impact on the safety of new medicines

Andrew Brown

Ikherma Consulting Ltd

Introduction

Secondary pharmacology screening of investigational small-molecule drugs for potentially adverse off-target activities has become standard practice in pharmaceutical research and development, and regulatory agencies are increasingly requesting data on activity against targets with recognized adverse effect relationships. However, the screening strategies and target panels used by pharmaceutical companies may vary substantially.

Method

To help identify commonalities and differences, as well as to highlight opportunities for further optimization of secondary pharmacology assessment, we conducted a broad-ranging survey across 18 companies under the auspices of the DruSafe Leadership Group of the International Consortium for Innovation and Quality in Pharmaceutical Development.

Results

Based on our analysis of this survey and discussions and additional research within the group, we present here an overview of the current state of the art in secondary pharmacology screening. We discuss best practices, including additional safety-associated targets not covered by most current screening panels, and present approaches for interpreting and reporting off-target activities. We also provide an assessment of the safety impact of secondary pharmacology screening and a perspective on opportunities and challenges in this rapidly developing field [1].

Conclusion

In vitro secondary pharmacology profiling can affect the clinical safety of drugs, as indicated by a marked decline in drug off-target promiscuity over the past decade, which correlated with a reduction in serious AEs for drugs on the market. Most companies apply secondary pharmacology screening against targets associated with clinically proven side effects, with a high coverage of aminergic GPCRs and highly translatable cardiac ion channels. About one-third of the companies surveyed apply their core testing during drug lead optimization, and over 90% of the tests are conducted before drug candidate selection. The addition of further targets beyond a core panel may now be justified, and the data from the survey support the inclusion of a safety-oriented kinase panel.

Reference

1. Brennan RJ, Jenkinson S, Brown AJ, Delaunois A, Dumotier B, Pannirselvam M, Rao M, Rosenbrier Ribeiro L, Schmidt F, Sibony A, Timsit Y, Toledo Sales V, Armstrong D, Lagrutta A, Mittlestadt SW, Naven R, Peri R, Roberts S, Vergis JM, Valentin J-P. Nat Rev Drug Discov 2024; 23: 525-545.

62

Antiplasmodial efficacy of ethanol leaf extract of Lecaniodiscus cupanioides in Plasmodium berghei-infected mice

Adeyinka Aderinola¹, Jane Ejiofor², Luqman Ogunjimi¹ and Akanji Murtala¹

¹Olabisi Onabanjo University; ²Ahmadu Bello University

Introduction

Malaria, a tropical disease resulting from the bite of an infected female anopheline mosquito, has been a predominant cause of hospitalization and mortality in numerous tropical and subtropical African regions due to the emergence of drug-resistant strains of Plasmodium falciparum (1). According to the WHO report in 2023, more than 249 million cases of malaria with about 608,000 deaths were recorded globally, with Nigeria accounting for 27% of cases and 182,400 deaths (2). For many years, vector control and anti-malarial drug treatment have been the primary approaches to malaria control and prevention. Unfortunately, the effectiveness of these strategies has been compromised by mosquitoes' resistance to insecticides and Plasmodium's resistance to most available anti-malarial medications. The development of two anti-malarial drugs (quinine and artemisinin) from natural products has prompted the need to search for more drugs from plant sources. Thus, this study aimed to explore the anti-plasmodial efficacy of ethanol leaf extract of Lecaniodiscus cupanioides in murine models.

Method

Acute toxicity study (oral median lethal dose [LD50]) of the ethanol leaf extract of L. cupanioides was determined in mice using Lorke's method. The in vivo anti-malarial activity of the leaf extract against the Plasmodium berghei (NK65) strain was assessed through the 4-day suppressive test, prophylactic test and curative test at doses of 200, 500 and 800 mg/kg (3).

Following a 7-day treatment period, five mice were sacrificed from the curative group under light ether, and their serum was utilized to evaluate liver enzymes, haematological parameters and inflammatory biomarkers associated with the severity of malaria. All animal experimentation was conducted in accordance with the EU Directive 2010/EU/23.

Results

No deaths were observed up to a 5000 mg/kg dose of the extract; this suggests that the extract is relatively safe. A significant (P < 0.05) dose-dependent suppression of parasitaemia levels, with a more pronounced effect at the highest dose, was observed in all three mouse models compared to the untreated P. berghei-infected control group. The group treated with chloroquine exhibited superior chemosuppression compared to the group treated with the extract. At all administered doses, the extract successfully normalized aberrations in haematological parameters, reduced elevated liver enzymes and mitigated inflammatory biomarkers induced by P. berghei infection in the mice.

Conclusion

References

1. Saba N, Balwan WK, Mushtaq F. Burden of malaria-a journey revisited. Sch J App Med Sci 2022;6:934-939.

2. WHO. World malaria report 2023. Geneva, World Health Organization; 2022. [cited 2024 Feb 7]. https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2023.

3. Ryley JF, Peters W. The antimalarial activity of some quinolone esters. Ann Trop Med Parasitol 1970;64(2):209-222.

68

Investigating the in vivo effects of the synthetic cannabinoid, O-1918, using Lumbriculus variegatus

Megan Flanagan, Grace Hawkes, James McRobbie-Aston, Benjamin Williams, Georgeena Jomy, Nia Davies, Lisa Wallace and Aidan Seeley

Swansea Worm Integrative Research Laboratory (SWIRL), Swansea University

Introduction

Compounds from Cannabis sativa and their derivatives have seen increasing interest due to their therapeutic potential. Lumbriculus variegatus is a species of aquatic, asexual, regenerative worm found in shallow ponds, lakes and marshes and has been utilized in previous pharmacological studies [1]. As invertebrates, L. variegatus are not subject to regulation by the Animal (Scientific Procedures) Act 1986. Here, we examine the effects of the synthetic cannabinoid, O-1918, in L. variegatus.

Methods

O-1918 was dissolved in 100% DMSO before dilution in artificial pond water [1] for a final DMSO concentration of 0.5%. O-1918 toxicity was determined by exposure of L. variegatus to 0–50 μM O-1918 for 24 h with tissue pallor and/or tissue decomposition used as identifier of toxicity. The effect of 24-h exposure to 0–5 μM O-1918 on locomotor activity and the effect of tactile stimulation to elicit stereotypical behaviours was conducted as previously described [1]. The effect of 0–5 μM O-1918 on the regenerative capacity of L. variegatus determined by bisection of L. variegatus and quantification of tissue growth, using a Nikon SMZ1270i stereomicroscope, up to 72-h post-amputation (HPA).

Results

O-1918 displayed toxicity in 50% of the test population at 15.84 μM (95% CI: 12.88–19.22 μM, n = 6), with a lowest observed adverse effect level observed at 5 μM. Exposure to 0–5 μM O-1918 for 24 h had no effect on locomotor activity of L. variegatus (P > 0.05, n = 8), but ≥2.5–μM O-1918 resulted in a significant decrease in L. variegatus response to tactile stimulating stereotypical behaviours of body reversal and helical swimming (P < 0.05, n = 8). Response to tactile stimulation remained decreased following removal of 5 μM O-1918 and incubation in artificial pond water only (P < 0.05, n = 8) but tactile response recovered in L. variegatus exposed to 2.5 μM O-1918 after 24 h in artificial pond water only (P > 0.05, n = 8). Regeneration of L. variegatus was not affected by O-1918 exposure (P > 0.05, n = 15), but L. variegatus displayed significant regeneration 72 HPA (P < 0.0001, n = 15).

Conclusion

We demonstrate that the synthetic cannabinoid, O-1918, is toxic to L. variegatus at >5 μM and that exposure to O-1918 does not affect locomotory activity, or regenerative capacity, of this organism. However, O-1918 does reduce the response following tactile stimulation suggesting the potential role of endocannabinoid-like system in sensing stimuli within this model organism.

Reference

1. Seeley A, Bellamy C, Davies NA, Wallace MJ. Lumbriculus variegatus: a novel organism for in vivo pharmacology education. Pharmacol Res Perspect 2021;9:e00853. https://doi.org/10.1002/prp2.853

80

Optimizing binding kinetics to develop insurmountable MC2 receptor antagonists for the treatment of congenital adrenal hyperplasia

Mark Soave¹, Kathy Sengmany¹, Rose Wilcox¹, Karolina Gherbi¹, Ali Jazayeri¹, Laia Malet-Sanz¹ and Steven J. Charlton^1,2

¹OMass Therapeutics; ²School of Life Sciences, University of Nottingham

Introduction

Congenital adrenal hyperplasia (CAH) is a collection of genetic disorders characterized by an inability to synthesize cortisol. The resulting loss of cortisol-driven negative feedback causes excess diurnal adrenocorticotropic hormone (ACTH) secretion by the pituitary gland (especially in the morning), causing adrenal hyperplasia and androgen accumulation, leading to virilization and early-onset puberty. ACTH is selective for the melanocortin type 2 receptor (MC2R) [1]; therefore, an MC2R antagonist represents a promising CAH therapeutic agent. The morning surge of ACTH in CAH could outcompete a rapidly equilibrating MC2R antagonist, such as atumelnant [2]. We have optimized binding kinetics to develop an insurmountable MC2R antagonist with a long residence time (Compound1) to treat the pathophysiological actions of excess ACTH in CAH.

Methods

HEK293 membranes expressing human MC2R tagged at the N-terminus with SNAP-tag (SNAP-MC2R) were used in kinetic binding experiments as previously described [3]. For functional antagonism CHO-K1 cells stably expressing SNAP-MC2R (CHO-MC2R) were used with the PerkinElmer cAMP HiRange kit following manufacturer's instructions. For in vivo pharmacodynamic studies, Sprague–Dawley rats (9–11 weeks, n = 4 animals/group) were treated with Compound1 or atumelnant (0.2–100 mg/kg) 6 h p.o. prior to 10μg/kg ACTH 1–24 i.v. Whole blood was collected for 2 h after ACTH 1–24 infusion for plasma corticosterone measurements. In vitro data are mean ± SEM from n separate experiments performed in duplicate.

Results

Compound1 and atumelnant showed specific binding to human SNAP-MC2R, and using competitive kinetic analysis, the residence times (TR) of these ligands were determined (Table 1). The longer TR of Compound1 resulted in insurmountability following acute challenge of MC2R with ACTH 1–24 in vitro, with significant reductions in E_max with Compound1 (Table 1), whereas atumelnant was a surmountable MC2R antagonist. In an acute rodent pharmacodynamic model, MC2R antagonism caused a significant reduction in corticosterone produced by 10 μg/kg ACTH 1–24 infusion with rank efficacy in line with compound TR in vitro (Table 1). Efficacy differences between compounds were most pronounced at the early timepoints (Table 1). Combining multiple studies concentration–response relationships were constructed to obtain IC₅₀ values for corticosterone inhibition (Table 1).

Conclusions

These data show a clear association between the residence time of compounds tested and their efficacy inhibiting acute agonist challenge in vitro and in vivo. These data strongly support the development of long residence time antagonists to inhibit MC2R during the ACTH surge seen in CAH.

References

1. Novoselova TV, King PJ, Guasti L, Metherell LA, Clark AJL, Chan LF. Endocr Connect 2019; 8: R122-R130.

2. Kim et al. ACS Med Chem Lett 2024; 15(4): 478-85.

3. Sykes et al. Mol Pharmacol 2019; 96: 378-92.

97

Anti-breast cancer potentials of Monodora myristica and Xylopia aethiopica aqueous extracts in 7,12-dimethylbenz[a]anthracene-induced female Wistar rats

Moses Aziakpono¹, Udom Godswill², Joseph Oyepata¹ and Theophilus Adegbuyi¹

¹Federal University of Oye-Ekiti; ²Kampala international University

Background

Cancer is a collection of illnesses that can affect any part of the body. Metastasis is the primary cause of death from cancer. Various parts of the body that can be affected by cancer include breast, colon, prostate gland, ovary, stomach, skin, pancreas and lung, among others. Some limitations of orthodox anticancer drugs are not being readily available, costly and having lots of serious adverse effects.

Objective/Aim

This study therefore evaluated the anti-breast cancer potentials of Monodora myristica (MM) and Xylopia aethiopica (XA) aqueous extracts administered separately and in combination in 7,12-dimethylbenz[a]anthracene (DMBA)-induced female Wistar rats.

Method

After extraction, acute toxicity and phytochemical analysis of MM and XA aqueous extracts were performed separately. Evaluation of the anti-breast cancer potency in DMBA-induced female rats. Cancer potency testing involved inducing cancer in female albino Wistar rats and testing for breast cancer marker, notably CA-15.

Results

Group 8, which was treated with MM and XA aqueous in the ratio of 6:4 (318:212 mg/kg body weight), exhibited the most antioxidant, anti-inflammatory and anti-breast cancer effects.

Conclusion

The herbs MM and XA aqueous exhibited the best anti-breast cancer activities when given in a proportion of MM:XA = 6:4; this implied 318:212 mg/kg body weight of the extracts. The combination MM:XA = 6:4 had the best antioxidative and anti-inflammatory effects. The herbs MM and XA aqueous extracts exhibited a remarkable anti-breast cancer activity. This potential was paramount when the two herbs were given in combination in a proportion of MM:XA = 6:4; this implied 318:212 mg/kg body weight of the extracts. This combination had anti-breast cancer effect that was similar to that of the standard drug doxorubicin.

129

Development of a cellular PPI assay enables profiling of molecular glues as a novel approach to targeting pathways implicated in cancer.

Afshan Ahmed, Taiaina Maia De Oliveira and Lori Chan

AstraZeneca

A number of established kinase signalling pathways are mutated in cancer, and conserved members of core components are considered important therapeutic targets. The clinical progress of such targets however is limited due to lack of specificity and by the redundant pathways that drive cell proliferation in aggressive settings. 14-3-3 proteins are universal chaperone proteins, and the majority of 14-3-3 isoforms have been reported to be elevated in cancers. Here, we describe a novel therapeutic strategy to target an important protein-14-3-3 interaction that is tightly regulated by phosphorylation. Binding of 14-3-3 to our protein target of interest at the N-terminal inhibitory phosphorylation site inhibits protein dimerization and consequent activity. By developing a cellular protein–protein interaction assay using NanoBRET™ technology, we have successfully profiled cell active molecular glues that stabilize the protein-14-3-3 inhibitory monomeric complex, thus inhibiting the oncogenic activity of this key signalling pathway.

149

Punicalagin: A novel PAR2 antagonist with therapeutic potential for atopic dermatitis

Hyejin Jeon and Wan Namkung

College of Pharmacy and Yonsei Institute of Pharmaceutical Sciences, Yonsei University

Introduction

Atopic dermatitis is a common chronic inflammatory skin disease worldwide. Currently available therapies have limited efficacy and significant side effects, necessitating novel therapeutic approaches [1]. Protease-activated receptor 2 (PAR2) plays an important role in atopic dermatitis pathophysiology [2]. This study evaluated the mechanism of action of punicalagin, a selective PAR2 antagonist [3], and its potential as a treatment for atopic dermatitis.

Methods

Punicalagin (≥98% purity, pomegranate extract, Sigma-Aldrich) was evaluated for PAR2 inhibitory effect by intracellular calcium measurement in human dermal fibroblasts (HDF) and HaCaT cells (n = 5). PAR2 selectivity was confirmed by receptor internalization observation in HT29 cells expressing EGFP-tagged PAR2 or PAR1 (n = 3). ERK and NF-κB signalling pathways were analysed by western blot (n = 3). In vivo efficacy was verified through PAR2-AP-induced itch model (ICR mice, 1, 3 and 10 mg/kg, n = 3) and DNFB-induced atopic dermatitis model (C57BL/6 mice, 3 mg/kg IP, daily for 10 days). Punicalagin's therapeutic effect was analysed by evaluating skin lesions, pruritus, skin barrier thickness, serum TSLP levels and calcium responses in DRG neurons. Statistical analysis used GraphPad Prism with one-way ANOVA followed by Tukey's post hoc test (P < 0.05 considered significant).

Results

Punicalagin demonstrated remarkable selectivity and potent inhibitory activity against PAR2. Notably, this study uncovered a novel mechanism whereby punicalagin induces selective internalization of PAR2, a unique feature distinguishing it from existing PAR2 antagonists (Figure 1).

In vivo studies corroborated these findings, showing punicalagin's effectiveness in attenuating PAR2-mediated pruritus and eliciting broad therapeutic effects in the atopic dermatitis model, including enhanced skin barrier function, mitigated inflammation and alleviated pruritus.

Conclusion

We identified a novel mechanism by which punicalagin acts as a potent and selective PAR2 antagonist, prominently featuring its ability to induce selective PAR2 internalization. This unique action, combined with punicalagin's superior therapeutic effect in atopic dermatitis, distinguishes it from existing therapies. These results suggest punicalagin's potential as a novel, potent and highly selective treatment for atopic dermatitis targeting PAR2, potentially providing an innovative approach to PAR2-related diseases.

References

1. Lee SE, Jeong SK, Lee SH. Protease and protease-activated receptor-2 signaling in the pathogenesis of atopic dermatitis. Yonsei Med J 2010;51(6):808.

2. Bieber T. Atopic dermatitis: an expanding therapeutic pipeline for a complex disease. Nat Rev Drug Discov 2022;21(1):21-40.

3. Seo Y, Mun CH, Park SH, Jeon D, Kim SJ, Yoon T, Ko E, Jo S, Park YB, Namkung W, Lee SW. Punicalagin ameliorates lupus nephritis via inhibition of PAR2. Int J Mol Sci 2020;21(14):4975.

186

Prediction of Bruton's tyrosine kinase structures: Frequent mutations and key post-translational modifications

Brandyn Lotter

University of Chester

Introduction

We have identified frequent Bruton's tyrosine kinase (BTK) mutations and key post-translational modifications (PTMs), as well as experimental BTK structures. Using a deep learning structural prediction programme, this study will compare experimental structures to a predicted BTK structure and investigate the effects of PTMs and mutations on the predicted BTK structure.

Methods

Pathogenic BTK mutations were identified using ClinVar, a genetic variation database [1], and the most frequent was determined through appearance in literature. Relevant PTMs of BTK were identified using UniProt [2], a protein annotation database, and those with an associated publication were selected. The sequences with the highest resolution for each BTK region were identified using PDB, a database of experimental protein structures [3], and AlphaFold Server (BETA) was used to access AlphaFold 3 for the generation of the predicted BTK structure (Q06187-1) [4], as well as the predicted structures of frequent BTK mutations and key PTMs. PDB Pairwise Structure Alignment was used to compare the experimental structures to the predicted BTK structure (Q06187-1) [3], as well as the predicted BTK structure (Q06187-1) to the predicted mutation and PTM structures.

Results

The predicted template modelling (pTM) score of each aligned structure (pTM > 0.50) indicates the predicted structures are likely similar to the ‘true’ structures (Table 1), while the TM score of each residue (TM > 0.5) indicates the predicted structures are similar to the predicted Q06187-1 structure. The root mean square deviation (RMSD) varies across the predicted structures (Table 1)—with PTM structures (RMSD = 2.1) deviate less on average than the mutation structures (RMSD = 4.0) and therefore more accurate to the predicted Q06187-1 structure. pSer21, pSer180 and pTyr617 + pSer623 are the most similar to the Q06187-1 predicted structure (RMSD < 2 Å), while the rest of the structures show differences in protein folding (RMSD > 3 Å). The predicted mutation structures have higher average aligned residues (~496, 75.3%) than the predicted PTM structures (~464, 70.5%) (Table 1).

Conclusions

References

1. Landrum MJ, Chitipiralla S, Brown GR, et al. ClinVar: improvements to accessing data. Nucleic Acids Res 2019;48(D1):D835-D844. https://doi.org/10.1093/nar/gkz972

2. Bateman A, Martin MJ, Orchard S, et al. UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res 2020;49(D1):D480-D489. https://doi.org/10.1093/nar/gkaa1100

3. Bittrich S, Segura J, Duarte JM, Burley SK, Rose Y. RCSB protein data bank: exploring protein 3D similarities via comprehensive structural alignments. Bioinformatics 2024;40(6). doi:https://doi.org/10.1093/bioinformatics/btae370

4. Abramson J, Adler J, Dunger J, et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 2024;630:1-3. https://doi.org/10.1038/s41586-024-07487-w

193

Machine learning-based drug repositioning of novel human aromatase inhibitors utilizing ADMET screening, molecular docking and molecular dynamic simulation

Samuel Bodun Damilola¹, Ayinde Adeniyi², Ibukunoluwa Temitope Adegbenro³, Olayinka Chidinma Emidun⁴, Samuel Aduramurewa Osunnaya⁵, Oluwatominsin Alonge Mary¹, Olaide Diyaolu Zainab⁶ and Muhammad Temitope Abdulazeez⁷

¹Department of Biochemistry, Adekunle Ajasin University; ²Bayero University, Kano; ³Faculty of Pharmacy, College of Medicine, University of Lagos; ⁴Department of Chemistry, Adekunle Ajasin University; ⁵Nigerian Institute of Medical Research; ⁶Department of Zoology, Lagos State University; ⁷Department of Physics and Electronics, Adekunle Ajasin University

Introduction

Breast cancer has become a major public health challenge. In 2022, this disease caused 670,000 global mortality in 157 countries out of 185 [1]. Current medication approaches including chemotherapy, hormonal therapies and targeted biological therapies still present some significant challenges such as cancer recurrence and side effects. Repurposed compounds from ChEMBL database offer a novel and promising approach for the inhibition of human aromatase involved in breast cancer, yet underexplored. This study aims to develop an in silico technique for the repurposing of small molecules to find an alternative and effective therapy against the aromatase enzyme.

Method

ML-based model training with reference human aromatase inhibitors was performed using scikit-learn library (Figure 1). This was followed by screening over 1.5 million compounds contained in a large small-molecule ChEMBL library to identify candidate human aromatase inhibitors. Top-ranked compounds totalling 148 from the target library with a predicted pIC₅₀ value above 8.5 nM were exported to a .CSV file and later converted into .SDF format using the DataWarrior software before importing them into the Maestro Schrödinger Software for molecular docking (Figure 2). The top 5 analogues from molecular docking as well as the co-crystallized ligand (androstenedione) were analysed for their absorption, distribution, metabolism, excretion and toxicity (ADMET) properties using the admetSAR webserver (Figure 3). Lastly, molecular dynamics (MD) simulation was performed to analyse the root mean square fluctuations (RMSF), root mean square deviation (RMSD) and protein–ligand contacts using the Desmond-Schrödinger suite.

Results

Out of the 1.5 million compounds screened, CHEMBL502014, CHEMBL1672975, CHEMBL1392432, CHEMBL1429417 and CHEMBL1410750 showed high potential for human aromatase enzyme inhibition with good XP binding energies ranging from −5.925 to −9.51 kcal/mol (Table 1). The ADMET parameters and drug-likeness properties of the compounds were also found to be favourable. The RMSF (Figure 4), RMSD (Figure 5) and protein–ligand contact (Figure 6) results obtained following a 100 ns MD simulation confirmed better stability of CHEMBL502014 compared to the co-crystallized ligand in the defined active site of aromatase.

Conclusions

We employed a machine learning model, followed by detailed structure-based screening, for the repurposing of CHEMBL compounds, among which CHEMBL502014, a current inhibitor of the SHP1/SHP2 genes overexpressed in breast cancer, showed the most promising prediction. We propose further in vitro and in vivo experimental tests to validate the potential of this compound and the other top 4 compounds reported in this study.

Reference

1. World Health Organizations. Breast cancer. https://www.who.int/news-room/fact-sheets/detail/breast-cancer. Accessed September 9, 2024.

243

Beneficial effects of elafibranor, a PPAR agonist exerting effects on PPAR-α and PPAR-δ, in primary biliary cholangitis: Mechanistic insights from literature evidence and preclinical data analysis

Jacquie Maignel¹, Aurélie Martin¹, Lesley Millatt² and Bart Staels³

¹Ipsen, 5 Avenue du Canada; ²Genfit SA, Parc Eurasanté, 885 Avenue Eugène Avinée, 59120; ³Univ. Lille, Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, F-59000

Introduction

Elafibranor is a PPAR (peroxisome proliferator-activated receptor) agonist exerting effects on PPAR-α and PPAR-δ, approved by the FDA in June 2024 for the treatment of primary biliary cholangitis (PBC). This work aims to illustrate how, by activating PPAR-α and PPAR-δ elafibranor impacts bile acid homeostasis, inflammation, and fibrosis, the hallmarks of PBC.

Method

Evidence was collected from the literature showing that the activation of multiple cellular pathways by PPAR-α and PPAR-δ agonists is beneficial to pathophysiological events observed in PBC. Then, histological and transcriptomic data were extracted from 25 preclinical studies evaluating elafibranor, where PPAR-α and PPAR-δ activation was evidenced at the functional and gene expression level.

Results

A large amount of literature evidence was identified, supporting the fact that complementary activation of PPAR-α and PPAR-δ improves bile acid homeostasis, inflammation and fibrosis. The preclinical data show that chronic (6 months) treatment with elafibranor induces a dose-dependent and reversible increased expression of several PPAR-α target genes in rats, such as ACOX (acyl-CoA oxidase), EHHADH (enoyl-CoA hydratase/3-hydroxyacyl CoA dehydrogenase), ACAA2 (acetyl-CoA acyltransferase 2) and CPT1 (carnitine palmitoyltransferase 1). In another set of experiments, the anti-inflammatory and anti-fibrotic properties of chronic elafibranor treatment in PPAR-α-KO mice were not as marked as in wild-type C57Bl/6J mice, underlining the involvement of PPAR-α as well as PPAR-δ activation in elafibranor's therapeutic effects. Although the in vitro pharmacology studies detected PPAR-γ activation by elafibranor, toxicology studies in rats and monkeys showed none of the adverse effects that are usually associated with PPAR-γ activation.

Pathway analysis of mouse liver gene expression profiles after elafibranor treatment showed the up-regulation of 15 genes and the down-regulation of 28 genes. Among these 43 genes, 20 are known to be target genes for PPARs, mainly PPAR-α and PPAR-δ. Additionally, interspecies consistency was found in the regulation of pathophysiological pathways when comparing human, rat and mouse data, highlighting the translational value of the preclinical data (with the exception of the expected PPAR-α-associated effects on peroxisome proliferation genes occurring only in rodents).

Conclusion

The literature provides broad evidence for the strong therapeutic value of dual PPAR-α/δ activation in PBC, while experimental data support elafibranor's agonism for PPAR-α and PPAR-δ. In conclusion, the properties of elafibranor present the advantages of targeting more than one PPAR isoform, providing a cumulative effect in treating PBC by decreasing cholestasis, inflammation and fibrosis.

253

A novel gene treatment approach for non-small cell lung cancer

Nathan Vella¹, Anthony G. Fenech¹, Palma Rocchi² and Vanessa Petroni Magri¹

¹Department of Clinical Pharmacology and Therapeutics, Faculty of Medicine and Surgery, University of Malta; ²Cancer Research Centre of Marseille (CRCM)

The PI3K/Akt/mTOR pathway is highly deregulated in several cancers including lung cancer. AZD2014 (vistusertib), a dual mTOR inhibitor, targets both mTORC1 and mTORC2 complexes within this pathway. Translationally controlled tumour protein (TCTP) is an anti-apoptotic protein involved in various oncogenic processes and known to be overexpressed in various cancers including lung cancer. Heat shock protein 27 (Hsp27) also plays a crucial role in cancer progression, promoting cell survival and enhancing drug resistance. Both TCTP and Hsp27 interplay with the PI3K/Akt/mTOR pathway, leading to eIF4E hyperactivity, causing enhanced cell growth and proliferation. Our rationale is conceptualized in the fact that by targeting several proteins on a common pathway, treatment dosages can be reduced while potentially enhancing efficacy and minimizing adverse effects in prospective in vivo studies. We aimed to study and explore the therapeutic potential of the small-molecule AZD2014 in conjunction with antisense oligonucleotides (ASO) designed to target the mRNA of TCTP and HSP27 (apatorsen; OGX-427) cancer-related proteins in non-small cell lung cancer (NSCLC) cells. Cell viability analysis was carried out using a range of AZD2014 concentrations (0–30 μM) on A549, H460 and H520 NSCLC cell lines at 24-, 48- and 72-h post-treatment. Magnetofection was used as the mode of ASO transfection. Cellular uptake of different concentrations of these ASOs into NSCLC cells was investigated using flow cytometry and confirmed by fluorescence imaging. Real-time PCR (qPCR) was performed to analyse TCTP and HSP27 gene expression, achieving a knockdown of at least 50% with each respective ASO. Western blots were carried out at selected ASO concentrations and pre-determined timepoints to confirm inhibition of protein expression. These data were used to identify the minimum concentration of both TCTP and Hsp27 ASOs required to achieve a therapeutic response. Additionally, combining AZD2014 (10 μM) with TCTP ASO (50 nM), resulted in synergistic effects on cell viability following 24-, 48-, 72- and 96-h treatments. Currently, the addition of Hsp27 ASO to the combination is being explored to study both the anti-cancer effects and determine the safety profiles of this novel NSCLC treatment, with the aim of translating this research to 3D lung cancer spheroids. Therefore, these promising results are indicative of the effectiveness of this novel combinatory treatment, which is known to act on separate targets all converging on a common -point (eIF4E). Upon translating this research to an in vivo scenario, the results may potentially include enhanced therapeutic outcomes with improved safety profiles.

257

Exploiting minable connectivity from the IUPHAR/BPS Guide to PHARMACOLOGY to an expanding range of ligands, targets, publications and patents

Christopher Southan, Simon Harding, Elena Faccenda, Jamie Davies, Antony Davenport, Jane Armstrong, Stephen Alexander and Michael Spedding

University of Edinburgh

Introduction

The IUPHAR/BPS Guide to PHARMACOLOGY (GtoPdb; www.guidetopharmacology.org) (1) is an open-access, expert-curated, FAIR-compliant (Findable, Accessible, Interoperable, Reusable) online database of pharmacological targets and the substances that act on them. Its reputation for quality is recognized by a Hidden REF award and selection as both an ELIXIR-UK and Global Core Biodata Resource. We continue to expand connectivity of our curated entities to external resources in other quality databases. These consequently offer an expanding range of powerful navigation and data mining options across chemical structures, sequences and documents that users may not be aware of but will be outlined here.

Methods

GtoPdb regularly submits ligand substances (SIDs) to PubChem, the majority of which have defined chemical structures (CIDs) and a range of useful mappings to many PubChem resources. We have introduced SID tagging for uniquely specific ligand sets that can be used for further analyses. For targets, we maintain a UniProt cross-reference set. For journal publications, we have enabled the NCBI to provide LinkOuts to all our curated references and also submitted a subset of these that document quantitative interaction data to European PubMed Central (EPMC).

Results

Of the 12,744 GtoPdb substances 10565 form CIDs that are connected by identity, extensive annotations and similarity neighbours within the vast PubChem system. Useful intersects include 86% BioAssay results, 83% to patent documents, 23% to PDB entries and 82% vendor offerings. Our expanding SID tags retrieve 1995 approved drugs, 393 clinical antibodies, 136 entries to the Guide to Malaria Pharmacology (GtoMPdb), 1477 from Guide to Immunopharmacology (GtoImmuPdb), 561 antibacterials and 355 natural products. For literature connectivity, NCBI LinkOut includes 33,093 GtoPdb-indexed PubMed IDs. Of these 8418 are indexed in EPMC. The GtoPdb chemistry-to-target cross-references in UniProt are up to 2272 sequences including human (1647), rat (323) and mouse (278).

Conclusions

GtoPdb provides users with expanding connectivity out to the pharmacology informatics ecosystem. While querying and navigating between ligands, documents and targets is complex, the ability to ‘slice-dice-and-compare’ across these key entities is powerful and provides unique insights. We are now documenting how to exploit this connectivity in GtoPdb.

Reference

1. Harding SD, Armstrong JF, Faccenda E, Southan C, Alexander SPH, Davenport AP, Spedding M, Davies JA. The IUPHAR/BPS guide to PHARMACOLOGY in 2024. Nucleic Acids Res 2024;52(D1):D1438-D1449.

266

Evaluation of preclinical antipsychotic models used to support first-in-human clinical trials

John Kelly, Ha Thi-Viet Nguyen and Declan McKernan

University of Galway

Introduction

Schizophrenia is a heterogeneous psychiatric disorder that is inadequately treated with current antipsychotic drugs due to insufficient effectiveness and/or side effects, representing a need for novel antipsychotics.¹ Preclinical testing plays a pivotal role in evaluating novel antipsychotics, and a range of models have been developed to mimic certain features of schizophrenia.² Thus, the aim of this study was to appraise animal models used to assess antipsychotic efficacy in new drug applications (NDAs) submitted to the US Food and Drug Administration (FDA) for approval, as well as those used for novel investigational agents in support first-in-human clinical trials.

Method

We identified preclinical (i.e. rodent) tests used to evaluate the efficacy of all marketed antipsychotics from the past 30 years by consulting the NDAs that were reviewed by the Centre for Drug Evaluation and Research (CDER) of the FDA. Likewise, we investigated novel drugs that have undergone premarketing clinical development by consultation of the Clinical Trials repository, from which comparable preclinical data were obtained from the published literature.

Results

We identified 11 antipsychotic drugs that have been marketed over the last 30 years. These drugs primarily target dopaminergic and/or serotonergic receptors, and the preclinical rodent models used reflect this by employing dopaminergic and serotonergic agonist challenges (for 11 and 6 drugs, respectively; see Table 1). Additionally, we identified 20 other drugs that have at least reached phase 2/3 clinical trials for which preclinical data were available. These drugs have various mechanisms of action, including dopamine and/or serotonin, glutamate, acetylcholine and histamine receptors and phosphodiesterase inhibition. Despite these varied targets, tests have focused on dopaminergic (13 drugs) and serotonergic (two drugs) agonist challenges, respectively, with additional use of glutamatergic receptor challenge (17 drugs). However, many of these novel investigational agents have failed in subsequent clinical phases of development due to lack of efficacy. The conditioned avoidance response (CAR) model was the only behavioural animal model that does not involve any pharmacological challenge (used in eight marketed drugs and six investigational drugs).

Conclusions

Preclinical evaluation of antipsychotic activity has to date focused on a limited number of tests that challenge dopaminergic, serotonergic and glutamatergic receptors. Greater diversity in the preclinical models may improve the detection rate of novel antipsychotics that might be more likely to be effective clinically.

References

1. Jauhar S, Johnstone M, McKenna PJ. Schizophrenia. Lancet 2022;399(10323):473-486. https://doi.org/10.1016/S0140-6736(21)01730-X

2. Spark DL, Fornito A, Langmead CJ, Stewart GD. Transl Psychiatry 2022;12(1):147. https://doi.org/10.1038/s41398-022-01904-2

271

Luteolin modulates the TGFB1/PI3K/PTEN axis in hormone-induced uterine leiomyomas: Insights from a rat model

Lenah Binmahfouz, Amina Bagher and Abdullah Al Otaibi

Department of Pharmacology and Toxicology, Faculty of Pharmacy, King Abdulaziz University

Introduction

Uterine leiomyomas (UL), or fibroids, are non-cancerous smooth muscle tumours of the uterus, affecting approximately 70% of women of childbearing age. They are the most prevalent solid tumours in the gynaecological tract and a major indication for hysterectomy [1]. The pathogenesis of UL involves uterine inflammation, uncontrolled cell division and inhibited apoptosis. This study investigated the protective effects of luteolin, a flavonoid known for its anti-inflammatory and antioxidant properties, against hormone-induced UL in female rats.

Methods

Twenty-four female Wistar rats were divided into four groups (n = 6): (1) control, (2) luteolin (10 mg/kg), (3) UL induced with ß-oestradiol (1.35 mg/kg) and progesterone (1 mg/kg, thrice weekly) [2] and (4) UL + luteolin (10 mg/kg). After 28 days of treatment, assessments included body and uterus weight measurements, visual inspection, histological and immunohistochemical examinations and RT-PCR analysis. Data are presented as the mean ± standard deviation (SD). Statistical analysis was conducted using a one-way ANOVA parametric test followed by Tukey's post hoc test for multiple comparisons. A P-value of less than 0.05 was considered statistically significant.

Results

The UL group experienced a significant uterine weight increase, with 525% over controls and 414% over the luteolin group. Luteolin administration reduced this increase by approximately 45% (P < 0.05). Histologically, the control and luteolin groups maintained normal uterine structure, whereas the UL group showed notable neoplastic cell growth and fibrosis, which luteolin significantly reduced. Luteolin decreased α-SMA expression by 2.5% compared to the UL group, signifying its effectiveness in inhibiting fibrotic pathways (P < 0.05). It also lowered MDA levels by 37% and increased SOD and CAT activities by 44% and 46%, respectively, in the UL group (P < 0.05). Additionally, inflammatory markers in the UL group were elevated but decreased by 15% for IL-6, 11% for TNF-α and 17% for NF-κB with luteolin treatment (P < 0.05). Apoptosis analysis showed a 108% increase in Bax and a 28% decrease in Bcl-2 in the luteolin + UL group, enhancing the Bax/Bcl-2 ratio by 150% compared to the UL group (P < 0.05).

Conclusion

Luteolin effectively mitigates the pathological changes in hormone-induced UL in rats, demonstrating its potential as a therapeutic agent through modulating fibrotic pathways, reducing oxidative stress and regulating inflammatory and apoptotic processes.

References

1. Lewis TD, Malik M, Britten J, San Pablo AM, Catherino WH. A comprehensive review of the pharmacologic management of uterine leiomyoma. Biomed Res Int 2018;2018:e2414609.

2. Zhao H, Li Y, Xu Q, et al. Establishment of a rat model for uterine leiomyomas based on western and traditional Chinese medicine theories. Braz J Med Biol Res 2018;51:e7627.

306

Multiple dosing long-term toxicity study of a new compound for Alzheimer's disease treatment

Henrique Atalaia-barbacena, Mafalda Ferreira-Manso, Sara Inteiro-Oliveira, Leonor Rodrigues, Tiago Coelho, Rui Pinto and Maria José Diógenes

Faculdade De Medicina Da Universidade De Lisboa

Introduction

In Alzheimer's disease (AD), the accumulation of amyloid-beta (Ab) plaques disrupts brain-derived neurotrophic factor (BDNF) signalling, involved in neuronal survival, differentiation and synaptic plasticity [1–3], by promoting a calpain-mediated TrkB receptor cleavage, which is a pathological basis for disease progression [4–6]. We designed a new compound (TAT-TrkB; Figure 1) able to compete for calpain cleavage, re-establishing BDNF signalling [7]. In vivo studies in AD models show promising results in reversing cognitive deficits [7]. Toxicity studies are mandatory to further evaluate compound's viability. Therefore, to evaluate the potential of prolonged exposed toxicity of this compound, a multiple dosing toxicity assay was performed.

Methods

TAT-TrkB (25 mg/kg/day, 5 days per week) was intraperitoneally (IP) administered to the AD mice model 5xFAD from week 16 to week 25 of age. Animals were then euthanized by anaesthetic overdose, a comprehensive necropsy was made, and blood, liver and kidneys were collected. Organs were immersed in formaldehyde 10% for 24 h and processed for paraffin embedding, sectioned at 3 μm, stained with haematoxylin and eosin and evaluated by a pathologist for toxicity findings. Blood was processed, and liver transaminases, creatinine kinase, serum creatinine, blood urea, red blood cells, haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and leukocyte and platelet count were measured.

Results

No histological differences were encountered in livers and kidneys between the different groups evaluated, and all organs were normal (Figure 2A–G). These results were in accordance with measurements of liver transaminases (Figure 2H,I) and serum creatinine and urea (Figure 2J,K), with no differences between groups. As for haematological toxicity, a statistically relevant decrease in red blood cells and haematocrit were encountered in the groups exposed to the drug (Figure 2L,M). Although no differences in haemoglobin levels were seen (Figure 2N), MCV and MCH were also significantly higher in the groups exposed to the drug (Figure 2O,P). No differences in leukocyte and platelet counts were seen in the different groups (Figure 2Q,R).

Conclusions

TAT-TrkB does not have liver or kidney toxicities. However, it caused a significant decrease in red blood cells, together with an increase in MCV and MCH, therefore suggesting a compromised effect in haematopoiesis, although without differences encountered in haemoglobin levels. Further studies are necessary to discern the specific effect the drug has in red blood cells maturation and survival.

Financial Disclosures

References

1. Huang EJ, Reichardt LF. Neurotrophins: roles in neuronal development and function. Annu Rev Neurosci 2001;24:677-736.

2. Minichiello L. TrkB signalling pathways in LTP and learning. Nat Rev Neurosci 2009;10:850-860.

3. Minichiello L, Korte M, Wolfer D, Kühn R, Unsicker K, Cestari V, Rossi-Arnaud C, Lipp HP, Bonhoeffer T, Klein R. Essential role for TrkB receptors in hippocampus-mediated learning. Neuron 1999;24:401-414.

4. Fonseca-Gomes J, Jerónimo-Santos A, Lesnikova A, Casarotto P, Castrén E, Sebastião AM, Diógenes MJ. TrkB-ICD fragment, originating from BDNF receptor cleavage, is translocated to cell nucleus and phosphorylates nuclear and axonal proteins. Front Mol Neurosci 2019;7:12-22.

5. Connor B, Young D, Yan Q, Faull RL, Synek B, Dragunow M. Brain-derived neurotrophic factor is reduced in Alzheimer's disease. Brain Res Mol Brain Res 1997;49:71-81.

6. Ferrer I, Marín C, Rey MJ, Ribalta T, Goutan E, Blanco R, Tolosa E, Martí E. BDNF and full-length and truncated TrkB expression in Alzheimer disease. Implications in therapeutic strategies. J Neuropathol Exp Neurol 1999;58:729-39.

7. Fonseca-Gomes J, Costa-Coelho T, Ferreira-Manso M, et al. A small TAT-TrkB peptide prevents BDNF receptor cleavage and restores synaptic physiology in Alzheimer's disease, Mol Ther 2024.

314

Measuring drug-P2X7 ion channel interactions on live cells with saturation transfer difference NMR spectroscopy

Leanne Stokes¹, Serena Monaco¹, Jacob Browne¹, Jesus Angulo², Matthew Wallace¹ and Elizabeth Allum¹

¹University of East Anglia; ²CSIC-University of Seville

Introduction

Pharmacological measurements involve assessment of ligand affinity/efficacy through indirect binding or functional assays. When designing novel pharmacological tools, structure–activity relationships are used to modify the ligand to impact potency and/or efficacy. Computational docking and receptor mutagenesis are often used to deduce binding sites. X-ray crystallography or cryo-EM delivers the most information on drug–receptor complexes, but can be challenging with ion channels. Saturation-transfer difference (STD) NMR spectroscopy is a powerful method for gaining structural information on protein–ligand interactions. STD NMR on living cells (on-cell) allows structural information to be gathered when in the receptor's native environment. This study is the first demonstration of on-cell STD NMR to define a ligand binding epitope on an ion channel.

Methods

HEK-293 cells overexpressing human P2X7 and mutant P2X7 were used in this study plus non-transfected HEK-293 cells. AZ10606120 and JNJ-47966567 and one positive allosteric modulator of P2X7, ginsenoside F2, were used at a concentration of 300 μM for STD NMR experiments (800 MHz). Antagonist concentration–response curves were generated using a fura-2 calcium influx assay and IC₅₀ values calculated using GraphPad Prism v6.0.

Results

On-cell STD NMR allowed a reproducible binding epitope to be identified for two antagonists, AZ10606120 and JNJ-47965567, at hP2X7 by subtracting non-specific signals (non-transfected cells). The central moiety of AZ10606120 and key aromatic groups in JNJ-47065567 are important for P2X7 antagonism. We confirmed that mutations around this binding pocket affected the potency of both antagonists [1]. IC₅₀ values for AZ10606120 were significantly altered in mutant hP2X7 (WT hP2X7 −7.9 nM, F88 > A 392 nM, M105 > A 266 nM and F103 > A 6170 nM) as were IC₅₀ values for JNJ47965567 (WT hP2X7 11.7 nM, F88 > A 125 nM, M105 > A 45 nM and F103 > A 161 nM; n = 3). We show that ligand binding epitopes for AZ10606120 and JNJ-47965567 are altered across species (human compared to rodent—ratP2X7 and mouseP2X7). We explored a binding pocket for positive allosteric modulators at hP2X7 [2] and determined a binding epitope for ginsenoside F2. STD signals were enhanced by adding the P2X7 agonist ATP, suggesting this PAM site is more accessible in the open channel state. Mutations of P2X7 in the central vestibule altered the F2 ligand binding epitope.

Conclusions

On-cell STD NMR provides new insights into chemical groups important for pharmacological activity in combination with computational docking and is an important new rapid and cost-effective tool for knowledge-based drug design.

References

1. Karasawa A & Kawate T. elife 2016 5:e22153.

2. Bidula SM et al. Sci Rep 2019 9:3231.

318

Grb2-FAK interaction as a drug target for cancer and proliferative heart disease

Vasundhara Singh¹, Pallavi Patanik², Yatender Kumar¹ and Sonika Bhatnagar¹

¹Netaji Subhas University of Technology; ²Netaji Subhas Institute of Technology

Growth factor receptor-bound protein 2 (Grb2) is an adaptor protein that plays a crucial role in cellular processes such as proliferation, differentiation, angiogenesis and survival. Due to its role in proliferation, Grb2-SH2 domain is a target for proliferative diseases like various malignancies and stress-induced cardiac hypertrophy. It specifically binds to the phosphopeptide motif pYXNX found in the intermediate conformation of the FAT domain of focal adhesion kinase (FAK) that facilitates phosphorylation by Src kinases and interaction with the Grb2-SH2 domain. A comprehensive binding site analysis of Grb2-SH2 was conducted. The intermediate conformation of the FAT peptide, identified using targeted molecular dynamics (MD), was used as a pharmacophore to discover small-molecule inhibitors of FAK by virtual screening of a library of 140,000 synthesizable lead-like analogues. Following this, the top five compounds, which featured novel scaffolds and favourable AutoDock binding affinities, were selected based on their absorption, distribution, metabolism, excretion and toxicity (ADMET) properties. MD simulations (100 ns), molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) analysis, dynamic cross-correlation matrix (DCCM) and principal component analysis (PCA) further confirmed the stability of these compounds' interactions with the Grb2-SH2 domain. In vitro testing of the selected compounds using surface plasmon resonance (SPR) and enzyme-linked immunosorbent assay (ELISA) showed promising results. SPR analysis revealed that all compounds had dissociation constants (Kd) in the nanomolar range, lower than those of the tyrosine phosphopeptide substrate. All compounds exhibited concentration-dependent binding to the Grb2-SH2 domain. The results from multiple analytical techniques validate the potential of these novel scaffold compounds to selectively target the Grb2-SH2 domain, positioning them as promising anti-proliferative therapeutic candidates.

70

Embedding equity, diversity and inclusion in curriculum design: A case study from Kaduna State University

Kenneth Bitrus David^1,2, Saheed Ekundayo Sanyaolu², Zichat Blessing Kuyet^1,2, David Adelekan Dada^1,2, Zigwai Gloria Kuyet², Cynthia Agmada Yusuf², Caleb Kehinde Okegbemi², Ahmed Danbala Ahmed¹, Naomi Bitrus-David² and Basira Kankia Lawal¹

¹Kaduna State University; ²Pharmafluence Education Advancement Network (PEAN)

Background and Aims

Equity, diversity and inclusion (EDI) are critical factors that influence knowledge acquisition and learning outcomes, particularly in higher education. Despite the global emphasis on EDI, there is a scarcity of scientific evidence regarding its implementation in Nigerian universities [1]. This study aims to assess the current state of EDI compliance within Kaduna State University (KASU) by exploring the perceptions and experiences of undergraduate students. The goal is to inform curriculum design and enhance teaching practices in the field of pharmacology by embedding EDI principles.

Summary of Work and Outcomes

This study employed a validated questionnaire comprising 18 questions, distributed to eligible undergraduate students (aged 18 and above, enrolled in science-based courses for the 2023/2024 academic session) via online platforms, primarily WhatsApp. The data collected from 551 respondents were analysed using IBM® SPSS V24. Descriptive statistics were utilized for quantitative data, while thematic analysis was conducted for qualitative data. The findings revealed that 39.4% of respondents had experienced some form of discrimination on campus, including religious discrimination, tribalism, nepotism, victimization, institutional disparities and gender bias. Also, the study identified significant barriers to EDI implementation, such as the lack of diversity among decision-makers, limited financial resources, inadequate external support and the perceived complexity of EDI initiatives.

Discussion

The study highlights the challenges and obstacles to achieving EDI at KASU, which are reflective of broader issues within the Nigerian higher education system. The findings suggest that discrimination and bias negatively impact student experiences and learning outcomes, particularly in science-based disciplines like pharmacology. Addressing these issues requires a comprehensive approach to curriculum design that integrates EDI principles, fostering a more inclusive and supportive learning environment.

Conclusion

The moderate level of EDI compliance at KASU underscores the need for coordinated strategies and policy implementation to combat discrimination and bias. By embedding EDI in curriculum design, especially in pharmacology, educators can improve student engagement, enhance learning outcomes and contribute to the development of a more equitable educational landscape. Further research is needed to develop and evaluate specific EDI interventions within the curriculum.

Reference

1. Wolbring G, Nguyen A. Equity/equality, diversity and inclusion, and other EDI phrases and EDI policy frameworks: a scoping review. Trends High Educ 2023;2:168-237. https://doi.org/10.3390/higheredu2010011.

3.2 General perceptions of respondents to the university's commitment to EDI

3.3 Personal experience of discrimination or bias at the university

Based on the 59 responses received to the open-ended question regarding the nature of discrimination or bias experiences, six themes were deduced. Sample responses are provided to highlight the identified themes. Some have been slightly revised for grammar and clarity (Figure 1)

3.4 Barriers to establishing EDI initiatives in the institution

3.5 Challenges faced by underrepresented/minority groups at the university

74

Student and staff perceptions of pharmacology teaching in the first 2 years of an MBBS course

Eleanor Renee Smith², Maximilian Paley³ and John Broad¹

¹King's College London; ²Maidstone Hospital, Maidstone and Tunbridge Wells NHS Trust; ³Tunbridge Wells Hospital, Maidstone and Tunbridge Wells NHS Trust

Introduction

‘Tomorrow's Doctors’ [1] stated that ‘factual information must be kept to the essential minimum that students need at this stage of their medical education’. However, we previously demonstrated [2] that students are introduced to over 1000 individual drugs or drug classes in the first 2 years of the Bachelor of Medicine, Bachelor of Surgery (MBBS) course at Queen Mary University of London (QMUL). 13.6% of these drugs were not found in the British National Formulary. In this study, we wanted to explore the QMUL MBBS student and staff perspectives of pharmacology teaching.

Methods

Students were invited to join focus groups. Senior tutors on the MBBS course were invited to attend semi-structured interviews. The audio recordings of these sessions were transcribed and underwent thematic analysis [2] to allow a deep understanding of the perceptions of students and staff to the teaching of pharmacology in the first 2 years of the MBBS programme.

Results

From three focus groups each containing four students, after coding and thematic analysis, three main themes were identified from seven subthemes: ‘the integrated curriculum’, ‘depth or breadth of teaching’ and ‘what do students value?’. Four senior tutor interviews were completed, and four main themes were developed from 11 sub-themes: ‘current pharmacology teaching’, ‘improving pharmacology teaching’, ‘identifying struggling students’ and ‘the senior tutor programme’. The number of drugs and drug names was a common issue with pharmacology identified by both students and staff, alongside a lack of focus on important drugs and/or pharmacological concepts. Another common issue raised by both groups was the lack of identity of pharmacology in the integrated curriculum, although one tutor saw this as a positive aspect.

Conclusions

When considering early year pharmacology education for medical students, a greater focus of important, clinically relevant drugs and concepts would be appreciated by students and staff. Most students and staff would prefer pharmacology to regain an identity within the curriculum. Limitations of this study include the relatively low sampling depth of students and that this study has only been performed in one institution.

Ethics Statement

This study was approved by the Institute of Health Sciences Education Research Ethics Committee at Queen Mary University of London (IPRCDec2021; QMERC22.117 and IPREC221111.SMI).

References

1. General Medical Council. Tomorrow's Doctors. London: General Medical Council;1993.

2. Smith ER et al. Curriculum analysis - the what, where, how and why of pharmacology teaching in phase 1 of the MBBS at QMUL. Br J Pharmacol 2023;180:554.

3. Braun V, Clarke V. Using thematic analysis in psychology. Qual Res Psychol 2006;3(2):77-101.

76

Structured support for undergraduate laboratory report writing skills.

Christine Edmead, Gwen Scott and Paul Mitchell

Department of Life Sciences, University of Bath

Background and Aims

Concise, accurate reporting of laboratory data is an essential skill for all undergraduate STEM students [1]. Prior to curriculum transformation (CT), our first-year pharmacology practical skills unit focused on robust experimental design/data analysis, since it was assumed (incorrectly) that students had developed basic reporting skills during pre-university studies. Consequently, only one 2-h workshop (semester 1, year 1) was dedicated to laboratory report writing before submission of a full laboratory report. However, the quality of reports was severely lacking in structure and content despite timely feedback before and during semester 2. Consequently, the revised undergraduate programme took a more structured, streamlined approach to supporting the development of laboratory reports.

Summary of Work and Outcomes

Following CT, students were exposed to identical practical sessions as in previous years (see Table 1). However, the first semester experimental sessions were followed by two workshops focused solely on data handling and summary figures. Students were given 1 week to prepare figures based on their data before a follow-up workshop where their figures were self- and peer-assessed with tutor guidance. The second semester practical sessions were followed by six workshops (summarized in Table 1) focused on different sections of a full laboratory report, thereby giving students the opportunity to practise writing each section and receiving guided feedback from the tutor and their peers.

After the final workshop, students submitted a full laboratory report summarizing all their practical data. The impact of this pilot study was assessed through comparative statistical analysis of report marks and also by anonymous qualitative feedback from the students.

Discussion and Conclusion

In 2022/2023, there was a small, significant improvement in marks over the year. However, the low marks were demotivating with poor report writing skills evident. Additionally, the assessment load for staff and students was considerable. Data gained post CT demonstrated that providing iterative learning opportunities and practice, incorporating the benefits of peer-assessment [2], resulted in significant increases in summative report marks while also considerably decreasing staff and student workload. These data are only from single-year groups, but this structured approach appears beneficial in supporting skill development and will be continued and further developed in future years.

References

1. Scientific enhancement programme STEM learning skill 5: scientific writing. 2000. Accessed 8/23/24. https://www.stem.org.uk/resources/collection/3713/skill-5-scientific-writing.

2. Topping K. Peer assessment between students in colleges and universities Rev Educ Res, 1998;68 (3);249-276.

92

Design and delivery of a pharmacology capstone course: A New Zealand case study

Rachel Cameron, Leslie Schwarcz and Malcolm Tingle

University of Auckland

Background and Aims

Undergraduate students may struggle to connect concepts learned across their degree and consequently cannot articulate their knowledge and skills to potential employers. To address these challenges, the University of Auckland introduced a compulsory student-led capstone course, requiring the integration and application of discipline-specific knowledge, skills and attitudes to real-world problems. In 2020, we used the British Pharmacological Society's undergraduate curriculum to design and deliver the University of Auckland's first pharmacology capstone course. Four years later, we outline our design and implementation process.

Summary of Work and Outcomes

The course was first run as a pilot, with two students, and three offerings since (including during the COVID-19 pandemic) with 27–40 students per year. Course learning outcomes were defined by combining the British Pharmacological Society's undergraduate curriculum and the University of Auckland's graduate profile. Mastery was determined using two authentic assessments: completion of an ethics application for a student-designed clinical trial and an employability task requiring completion of a CV plus a 1-min video showcasing their skills to an employer. To prepare for these assessments, we used a social-constructivist approach [1] to design team-based learning activities and demonstrate real-world applications of their learning, for example, connections with clinical trial employers, opportunities to act as University of Auckland Open Day ambassadors and pathways to post-graduate research.

Discussion

Assessment performance demonstrated that capstone students can integrate and apply their pharmacology knowledge and skills, and students consistently surpassed grade predictions based on more traditional assessment methods. Additionally, while we have understood progression into post-graduate study at our university, the capstone course clarified the pathways taken to study at other universities as well as the places our graduates gain employment. This has allowed us to refine our other pharmacology courses, provide better career advice to prospective students and enable connections with relevant employers.

Conclusion

A thoughtfully designed capstone course can connect current students to their learning and improve the delivery and content of pre-capstone pharmacology courses to produce confident, competent graduates.

Reference

1. Vygotsky LS. Mind in Society. Cambridge, MA: Harvard University Press; 1978.

133

Active learning to consolidate skills in experimental design and statistical analysis

Paul Mitchell¹ and John Kelly²

¹University of Bath; ²Department of Pharmacology and Therapeutics, University of Galway

Background and Aims

Knowledge of experimental design and statistical analysis is essential in pharmacology in order to ensure that experimental results and conclusions are confidently reported. Unfortunately, both undergraduate and post-graduate students often fail to see the relevance of such knowledge leading to a limited ability to apply data analysis in an appropriate fashion. We have developed an educational programme in experimental design and statistical analysis that uses the principles of active learning to promote retention and enhance engagement [1].

Summary of Work and Outcomes

This work describes a further extension of our programme to 14 international PhD students who received a wholly on-line version, consisting of pre-recorded lectures followed by post-lecture workshops and exercises (see Table 1), and undertook a 32-item MCQ baseline assessment. The lectures linked factual knowledge of experimental design and statistics with data analysis [2], while statistical strategies were taught by directed study hands-on data analysis workshops. Subsequent exercises encouraged the application of analysis skills to further consolidate their working knowledge. Post-programme student performance was assessed by a final 72-item MCQ paper.

Comparison of the MCQ scores showed that prior to the programme student performance was poor (mean ± SEM correct scores = 52.78‰ ± 4.51), but performance significantly improved in the final assessment (mean ± SEM correct scores = 82.11‰ ± 2.32; paired t(12) = 6.138, P < 0.0001), demonstrating the programme's effectiveness.

Discussion and Conclusion

The data presented here demonstrate how a well-constructed programme of study may successfully deliver a range of skills related to an understanding of robust experimental design and rigorous statistical analysis, resulting in consolidated working knowledge essential for today's research environment. Perhaps most importantly the study showcases how on-line asynchronous learning may be turned into active learning with successful engagement by coupling lecture material with application-based workshops and exercises.

References

1. Khan A, Egbue O, Palkie B, Madden J. Active learning: engaging students to maximize learning in an online course. Electron J e-Learn 2017;15:107-115.

2. Mitchell PJ. Experimental Design and Statistical Analysis for Pharmacology and the Biomedical Sciences. John Wiley and Sons Ltd; 2022.

190

The ‘Drug Index Visual Map’: Creating an open, digital, customizable visual mapping tool for pharmacology teaching and Learning

Nikolas Dietis, Michael Temvriotis and Andriana Hadjiyianni

University of Cyprus Medical School

Background and Aims

Students express a positive attitude towards the integration of digital technologies in their learning activities [1]. ‘Visual mapping’ is a visualization technique that displays complex information by graphical organization [2]. Students believe visual maps can be an effective pedagogical tool in pharmacology teaching and learning [3]. However, an open resource that utilizes visual mapping in pharmacology is missing. To address this, we created the first ever visual drug map in pharmacology as an open, digital, customizable educational tool.

Summary of Work and Outcomes

We used a platform that incorporates intelligent non-linear note management and powerful animated visual network, called TheBrain™. We created a network of drug categories using the Anatomical Therapeutic Chemical (ATC) index (‘therapeutic category’) and their stated mechanism of action in the FDA Pharmacological Classification (FPC) index (‘activity category’). For the nomenclature of targets and ligands, we applied the ‘IUPHAR/BPS Guide to Pharmacology’ terminology. Categories and drugs that share commonalities, either in terms of mechanism of action or therapeutics, were visually linked together in the map. For each drug class and individual drug, we created a corresponding landing ‘page’ with drug informatics from different resources (i.e. PubChem, DrugBank, DrugCentral and TDD) and a variety of online educational material (i.e. tables, website links and videos).

Discussion

Visual maps are graphical tools that present organized information in a way that can enhance retention and learning. We created a unique visual map of drug classification using a platform that allows the construction of animated visual maps that change conformation with every user interaction, thus creating a sense of a centred navigation within a concept pharmacology map. The incorporation of drug informatic resources and educational material within this navigation-based knowledge map formed a unique educational tool that can be used in pharmacology learning and teaching.

Conclusion

We created the first digital visual map in pharmacology as an open resource for learning and teaching.

References

1. Plch L. Perception of technology-enhanced learning by medical students: an integrative review. Med Sci Educ 2020;30(4):1707-1720.

2. Davies M. Concept mapping, mind mapping and argument mapping: what are the differences and do they matter? High Educ 2011;62(3):279–301.

3. Qadir F, Zehra T, Khan I. Use of concept mapping as a facilitative tool to promote learning in pharmacology. J Coll Physicians Surg Pak 2011, 21(8):476–81.

208

Creating changemakers: The impact and value of portfolios of capstone projects on learners and educators

David Lewis

University of Leeds

Capstone projects combine the UK undergraduate research project with the US capstone experience—their purpose to equip learners with the workplace experiences and competencies they need to succeed in their careers. The School of Biomedical Sciences has progressively developed a portfolio of 19 different research, workplace or social justice opportunities. Recognizing their potential, educators globally are increasingly implementing capstone projects into programmes. The aim of this study was to investigate the impact of capstones on learners and the challenges faced by educators in their implementation.

Data were obtained through thematic analysis of learner reflective blogs (n = 81, 31% cohort) and quantitative analysis of historical assessment data (n = 699, 93% cohort) and from surveys of alumni (n = 113) and non-Leeds educators (n = 67).

Capstone projects far exceeded learner expectations, in particular, their impact on personal and professional development (62% vs. 22%, post vs. pre). They promote inclusive academic learning gain (6.4× vs. Lv 5), with no difference between genders, ethnicity or socio-economic background. Learners also developed a broad portfolio of workplace competencies including planning and organization (n = 60%), time/deadline management (n = 58%) and confidence (n = 46%), all of which are required by alumni in their current roles. Only 15% of alumni had entered careers in research, the majority undertaking non-research-related science (43%) or non-science (27%) graduate roles. During the pandemic, bioscience educators (n = 67) reported they switched to offering alternative opportunities, with only 8% able to offer laboratory-based capstones. Post-pandemic, the majority (87%) have retained their expanded portfolios of capstone projects. This broadening of portfolios of capstone opportunities has brought challenges for educators including culture, learner experience, scaffolding and support, quality assurance, resourcing and organizational processes.

This study has evidenced the inclusive, transformative and translational potential of capstones. To better support learners in transitioning into the diversity of careers/roles they go onto, educators need to provide more workplace and social justice opportunities. The comparative analysis of competencies with alumni highlighted the need to create opportunities which develop leadership and teamworking competencies and to further enhance communication competencies. The solutions to many challenges faced by educators include changing mindsets or cultures, new ways of working, providing appropriate scaffolding and support and ensuring a high-quality academically robust learner experience.

Capstone projects have huge benefits, impact and value for all stakeholder irrespective of discipline, the potential to create changemakers. However, we are very much at the start of a global journey, with HE yet to fully realize their transformative and translational potential.

214

Design of skills and a virtual practical session for higher education students to learn of inflammatory oedema formation: Reducing mouse use via an unconventional methodology

Dibesh Thapa¹, Ria Fisher², Julie Keeble³ and Susan Brain¹

¹Department of Pharmacology & Therapeutics, King's College London; ²Faculty of Life Sciences and Medicine, King's College London; ³Biological Services, King's College London

Introduction

Oedema formation is a key component of inflammation and an important topic taught in immuno-pharmacology, as utilized in research [1]. With the health and safety concern of animal allergies and challenges caused by COVID, there is a need to rethink on how we deliver laboratory teaching in universities. To meet this challenge and practice the principles of 3Rs, we designed a new educational experience for level 6/7 immuno-pharmacology students that would meet the UK Professional Standard Frameworks. The aim was to design a new practical that provides students with learnings on oedema formation but without the use of live mice as in the traditional practical.

Summary of Work and Outcomes

The practical consisted of a skills session and a virtual practical session. In the skills sessions, students pinned the dorsal skin over a dummy mouse (Figure 1A) and performed intradermal injections(0.05 ml/site) with various concentrations of dye(Evans Blue) and recorded qualitative and quantitative ‘responses’. This was followed by a virtual practical session where students discussed the results and watched a video of the traditional practical involving the anaesthetized mouse and strategic differences discussed. They were then given results from a real experiment that formed part of an experimental practical write-up. The students were surveyed to determine skills and learnings from the practical. The results showed students achieved similar learning to the traditional practical where they learnt key skills and increased their learning on oedema formation (Figure 1B).

Discussion

The new practical significantly reduced the number of mice, reduced the allergy concerns associated with the traditional practical and enabled students to practise the 3Rs while achieving the primary educational objective. The practical also promotes inclusive education as students are not restricted by the home office (HO) licence, meaning more students can participate. Additionally, our simple design of dummy mouse model can be transferred to other teaching/research activities such as preclinical practices like suturing, mini-surgeries, which could have a huge financial benefit to educational/research institutes.

Conclusion

The newly designed practical is a simple but effective pedagogical endeavour. It provides an effective alternative educational experience without live animals, and its benefit is not restricted to pharmacology education as the model can be applied to all fields of bioscience, both in education and research.

Reference

1. Zarban AA, Chaudhry H, Sousa Valente J, Argunhan F, Ghanim H, Brain SD. Elucidating the ability of CGRP to modulate microvascular events in mouse skin. Int J Mol Sci2022;23(20):12246.https://doi.org/10.3390/ijms232012246

60

Gαq coupled AT1R is indispensable for MAPK activation in arterial smooth muscle cell

Saad Alqahtani^1,2 and Jonathon Willets¹

¹Department of Molecular and Cell Biology, Henry Wellcome Building, University of Leicester, Lancaster Road, Leicester, LE1 7RH, UK; ²Department of biochemistry, College of Science, King Saud University

Mitogen-activated protein kinases (MAPK) including ERK1/2 have been linked to cardiovascular diseases including hypertension when challenged with mitogens such as angiotensin II [1]. We have previously elucidated the mechanisms underpinning angiotensin II type 1 receptor-mediated cell proliferation in rat arterial smooth muscle cells [2]. Moreover, the majority of early ERK phosphorylation (≤2 min) has been shown to be induced via the G protein-dependent pathway, while sustained activity (>5 min) is regulated by arrestin-dependent pathway in overexpressed model cell systems [3]. However, the spatiotemporal control of MAPKs including ERK1/2 phosphorylation remains undeciphered at endogenously expressed AT1 receptor in the vasculature. Therefore, we sought to identify the cut-off time point between G protein-dependent and arrestin-dependent pathways in rat aortic smooth muscle cells (RASM) via the inhibition of Gαq using YM-254890 (selective Gαq inhibitor). Agonist-driven ERK phosphorylation was determined via standard western blotting techniques using a specific anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) antibody. To ensure that all samples contained the same levels of protein, membranes were washed and re-probed for total ERK immunoreactivity using an anti-ERK antibody. Protein expression was quantified by densitometry with the use of ImageJ (version 1.51, National Institutes of Health, Bethesda, MD). Stimulation of RASM with 100 nM Ang II induced rapid increases in ERK phosphorylation peaking at 5 min, followed by a sustained phase of signalling over the 30 min experimental time course (basal 1022 ± 477, 5 min 10404 ± 730, P < 0.001, 30 min 6398 ± 1224, P < 0.01, one-way ANOVA, Dunnett's post hoc test, mean ± SEM, n = 5). Pre-incubation with YM-254890 (1 μM, 15 min) virtually abolished peak 5 min (from 10404 ± 730 to 937 ± 384, P < 0.001, two-way ANOVA, Sidak's post hoc test; mean ± SEM, n = 4–5) and the sustained phase of AngII-stimulated ERK phosphorylation (from 7206 ± 1300 to 475 ± 201, P < 0.001, two-way ANOVA, Sidak's post hoc test; mean ± SEM, n = 5). Similarly, stimulation of RASM cells with TRV055 caused time-dependent increases in pERK immunoreactivity, which peaked between 2 and 5 min, and gradually declined between 15 and 30 min (basal 2208 ± 499, 5 min 9660 ± 1908, P < 0.05, 30 min 3159 ± 1072, one-way ANOVA, Dunnett's post hoc test; mean ± SEM, n = 4). Peak (5 min) TRV055-stimulated ERK phosphorylation (from 9660 ± 1908 to 891 ± 518, two-way ANOVA, Sidak's post hoc test; mean ± SEM, n = 4), and sustained phase >5 min was completely suppressed following pre-incubation with YM-254890. Application of TRV027 triggered slow peak increases in ERK phosphorylation at 5 min and returned to the basal after 15 min (basal 2133 ± 843, 5 min 7410 ± 656, P < 0.001, 30 min 1964 ± 604, one-way ANOVA, Dunnett's post hoc test; mean ± SEM, n = 4). YM-254890 pre-treatment attenuated the peak (5 min) TRV027 stimulated pERK activation (from 7410 ± 656 to 3701 ± 404, P < 0.001, two-way ANOVA, Sidak's post hoc test; mean ± SEM, n = 4). Together, these results suggest that TRV027 is a partial agonist in comparison to AngII and TRV055. Furthermore, it seems that arrestin recruitment likely requires Gq activation in RASM as ERK phosphorylation was completely abolished when RASM were pre-incubated with YM-254890 and stimulated by either full or biased G protein agonists. Hence, our next step is to deplete arrestin2/3 to determine the divergent point between G protein and arrestin signalling as such signalling pathway outcomes are crucial in health and disease.

References

1. Molnar P, Perrault R, Louis S, Zahradka P. The cyclic AMP response element-binding protein (CREB) mediates smooth muscle cell proliferation in response to angiotensin II. J Cell Commun Signal 2014;8(1):29-37.

2. Alonazi A, Nash CA, Wang C-H, Christofidou E, Challiss RAJ, Willets JM. GRK2 expression and catalytic activity are essential for vasoconstrictor/ERK-stimulated arterial smooth muscle proliferation. Biochem Pharmacol 2023;216:115795.

3. Kim J, Ahn S, Rajagopal K, Lefkowitz RJ. Independent β-arrestin2 and Gq-protein kinase Cζ pathways for ERK stimulated by angiotensin type 1A receptors in vascular smooth muscle cells converge on transactivation of the epidermal growth factor receptor. J Biol Chem 2009;284(18):11953-62.

75

A 3D adipocyte spheroid model to investigate the role of metabolite-sensing GPCRs in metabolic disorders

Elaine Duncan¹, Catherine Berry², Matthew Dalby² and Brian Hudson¹

¹Centre for Translational Pharmacology, University of Glasgow; ²Centre for the Cellular Microenvironment, University of Glasgow

Introduction

Metabolic disorders such as obesity and type 2 diabetes are a global healthcare and economic challenge affecting millions of individuals worldwide. However, much remains unknown about their underlying biology. Chronic low-level inflammation of adipose tissue is an important hallmark of these disorders, and there is growing evidence that a group of metabolite-sensing G protein-coupled receptors (GPCRs) play a role in metabolic-immune interactions [1]. However, it has been challenging to dissect these complex signalling pathways using traditional 2D cell culture or in vivo experimental models. Here, we describe a novel 3D adipocyte spheroid model, which can be used to investigate the role of metabolite-sensing GPCRs in a physiologically relevant in vitro system

Methods

Adipocyte spheroids are generated by seeding human-derived SGBS pre-adipocytes [2] into ultra-low adhesion plates and differentiating them into adipocytes. Whole or sectioned spheroids were imaged using various techniques. Gene and protein expression of adipogenic markers and metabolite-sensing GPCRs were determined by RT-qPCR and immunocytochemistry. Lipolysis was measured as glycerol release using Glycerol-Glo™ reagent, and glucose uptake was measured using a Glucose Uptake-Glo™ kit (Promega). Statistical comparisons were made using unpaired t-tests or one-way ANOVA with Tukey's multiple comparisons.

Results

Imaging of differentiated SGBS spheroids shows accumulation of characteristic lipid droplets, confirmed using a fluorescent lipid dye. Compared to undifferentiated spheroids, expression of key adipogenic markers PPARG, GLUT4, adiponectin and FABP1 were significantly upregulated in differentiated spheroids (Table 1; n = 3–5, P < 0.05). Furthermore, expression of several metabolite-sensing GPCRs including SUCNR1, FFA4 and HCA2 were also significantly increased compared to day 0 (Table 2; n = 3–4, P < 0.001).

Critically, the differentiated spheroids show characteristic adipocyte functions. Lipolysis was increased in response to treatment with isoprenaline, a β-adrenoceptor agonist, with 13.8-fold increase over baseline at 1 μM concentration and pEC50 of 9.14 ± 0.17 (n = 5–6). A significant increase in glucose uptake of 73.5% was observed following stimulation with 1 μM insulin (n = 4, P < 0.01).

Conclusions

This SGBS adipocyte spheroid model therefore provides a 3D in vitro platform that can be used to investigate how metabolite-sensing GPCRs control adipogenesis and adipocyte function in a more physiologically relevant microenvironment than traditional 2D cell culture. This platform will help to further dissect the complex GPCR signalling networks within adipocytes and better understand how these receptors can be targeted to treat metabolic disease.

References

1. Duncan EM, Vita L, Dibnah B, Hudson BD. Front Endocrinol 2023;14:1197102.

2. Wabitsch M, Brenner RE, Melzner I, Braun M, Möller P, Heinze E, Debatin KM, Hauner H. Int J Obes 2001;25(1):8-15.

94

Antibody tethering of ligands induces selective and logic-gated GPCR signalling

Shivani Sachdev¹, Swarnali Roy¹, Brendan Creemer¹, Thomas J. Gardella² and Ross Cheloha¹

¹National Institutes of Diabetes, Digestive, and Kidney Diseases; National Institutes of Health; ²Massachusetts General Hospital and Harvard Medical School

Introduction

G protein-coupled receptors (GPCRs) are the targets of 35% of approved drugs and control many important physiological processes. Studying GPCR function relies on the use of ligands that selectively activate or block receptor activity. However, ligands derived from nature often show promiscuity, activating multiple GPCRs and GPCR-coupled pathways, complicating their application in mechanistic studies and for therapeutic development. These challenges are further accentuated by the expression of individual GPCRs in different tissues. Antibodies (Abs) are excellent tools for targeting cell surface proteins; however, developing GPCR-targeted Abs that induce activation remains challenging. Camelid single-domain antibodies (or nanobodies [Nbs]) offer some advantages over conventional antibodies, although identifying Nb agonists of GPCRs remains extremely challenging.

Experimental Approach

We have developed methodology to link GPCR (PTHR1)-binding Nbs with synthetic agonist ligands to provide semi-synthetic conjugates that directly modulate GPCR function1. Conjugates were prepared through a combination of enzymatic protein labelling, solid-phase peptide (ligand) synthesis and chemoselective conjugation chemistry.¹ The activity of Nb–ligand conjugates were assessed in a series of pharmacological assay, including second messenger (cAMP) production, G-protein dissociation and β-arrestin translocation assays.

Results

Linking a weakly active PTHR1 ligand fragment (PTH1-11) to a Nb that binds to the same receptor resulted in a substantial enhancement in potency for signalling through the Gαs pathway (EC₅₀ = 3 nM). In contrast, these conjugates displayed a drastically impaired capacity to induce recruitment of β-arrestin to activated receptor (EC₅₀ = nd). This is in stark contrast to conventional PTHR1 ligands, which non-selectively activate all PTHR1-engaged pathways (EC₅₀ = 62 nM). Mechanistic studies revealed that the Nb-PTH1-11 conjugates induce signalling through a mode that involves two receptor protomers (‘activation in trans’). We further elaborated on this platform to assess whether Nb–ligand conjugates could target receptor heteromeric complexes. By linking PTH1-11 to a Nb that targets a GPCR distinct from that bound by ligand (such as A2AR), we produce conjugates that exhibit activity only when both targets are co-expressed in a single-cell population. Such conjugates do not show activity when either the ligand or Nb targets are expressed individually, thus demonstrating logic-gated behaviour.

Conclusions

The platform described here has the potential to advance our understanding of diseases associated with GPCR by enabling selective modulation of individual signalling pathways. This approach also offers a path towards tissue-specific pharmacology, with implications for therapies with reduced side effects.

Reference

1. Sachdev S, Creemer BA, Gardella TJ, et al. Highly biased agonism for GPCR ligands via nanobody tethering. Nat Commun 2024;15:4687.

153

A new reverse-engineered kinetic operational model of agonism

Lloyd Bridge

University of the West of England

Introduction

The operational model of agonism (OMA) [1] has become a ubiquitous tool throughout quantitative pharmacology in analysing steady functional response E as a function of ligand concentration A. Equilibrium assumptions underlying the OMA limit its applicability. Given the acknowledged importance of kinetics in modern therapeutic development, an updated kinetic operational model of agonism (KOMA) is sought.

Recently presented ordinary differential equation (ODE)-based KOMAs have yielded powerful pharma-analysis tools [2,3], but none of these agree exactly with the original OMA at equilibrium.

We present a new KOMA that is an exact kinetic counterpart of the original OMA.

Methods

We derive an ODE model for ligand binding and signalling dynamics by ‘reverse-engineering’; the signalling rate is chosen as a quantity, which is zero according to the OMA. We show that the resulting system is a plausible bio-model for signal transduction and interpret new model parameters.

Solutions are developed using:

•asymptotic analysis to find analytical solutions E(t) in terms of elementary functions, under limiting conditions on transduction and binding parameters;

•computational ODE solvers;

•partial symbolic computation to solve the full model to give a uniformly valid E(t) expression.

Simulations are implemented in MATLAB.

Results

Our KOMA models ligand binding and signal transduction kinetics, via a single ODE for E(t). This ODE admits a closed-form expression for E(t), which may be implemented in MATLAB (Figure 1a) and GraphPad Prism (Figure 1c). Sensitivity analysis revealed response features' dependence on transduction ratio and kinetic parameters (Figure 1a). Agreement is shown between numerical solutions, closed-form solutions and approximate solutions. We observe good fits to experimental data, and kinetic parameters are successfully estimated using pseudo-experimental data (Figure 1b).

Conclusions

Our KOMA yields an exact solution that agrees with numerical simulations, which may be implemented in both MATLAB and GraphPad Prism. Since the KOMA is an exact kinetic counterpart of the original equilibrium OMA, we propose its use as an extension of the modern analytical pharmacology toolkit.

References

1. Black JW, Leff P. Operational models of pharmacological agonism. Proc R Soc Lond B Biol Sci 1983;220(1219):141-62.

2. Hoare SR, Pierre N, Moya AG, Larson B. Kinetic operational models of agonism for G-protein-coupled receptors. J Theor Biol 2018;446:168-204.

3. Hoare SR, Tewson PH, Quinn AM, Hughes TE, Bridge LJ. Analyzing kinetic signaling data for G-protein-coupled receptors. Sci Rep 2020;10(1):12263.

231

Investigating mouse FFA4 expression and localization in ex vivo lung tissue

Dominic Crossley¹, Natasja Barki¹, Bethany Strellis¹, Hannah K. Bayes², Andrew B. Tobin¹ and Graeme Milligan¹

¹University of Glasgow; ²Glasgow Royal Infirmary

Introduction

Free fatty acid receptor 4 (FFA4) has been shown to have a uniquely high expression in the lungs in both mice and humans [1]. FFA4 has been seen to have an anti-inflammatory role [2], and further investigation could allow targeting the receptor for therapies of inflammatory lung diseases. It has not been fully explored as to whether FFA4 expression is different between the lung lobes, and the precise subcellular location of the receptor in lung tissue has yet to be elucidated. To investigate this, we aimed to analyse receptor expression and localization in ex vivo lung tissue.

Methods

Here, we use FFA4-HA transgenic animals, expressing a C-terminal HA epitope tag to facilitate detection, and an FFA4-KO animal where receptor has been genetically replaced with β-galactosidase [3].

To probe for possible receptor expression difference between lobes, RNA was extracted from individual lobes and trachea of the mice. qPCR for the receptor was performed using Fast Sybr with triplicate conditions and a total of four independent runs performed.

Exploring receptor localization in the lungs begun with tissue being fixed prior and embedded in optimal cutting temperature compound (OCT) prior to sectioning using a cryostat. Sections were incubated in antibodies against HA epitope (receptor marker), CC10 (epithelial marker) and α-actin (smooth muscle marker). Sections were mounted on slides and imaged using LSM 880 upright microscope.

Results

qPCR analysis revealed that the expression of mFFA4 mRNA was not significantly different throughout the lobes of the lung and the trachea (P > 0.05, two-way ANOVA). A significant difference was seen between the FFA4-KO and the FFA4-HA animals in their receptor expression (P < 0.0001, two-way ANOVA). Further analysis by immunohistochemistry using the FFA4-HA animals and probing for the HA epitope tag indicates that the receptor is expressed around the airway epithelium, as evidenced by colocalization with the CC10 epithelial cell marker.

Conclusions

Mouse FFA4 seems to be uniformly expressed throughout the different lobes of the lung and trachea and seems to be exclusively expressed in the airways of the lungs rather than the surrounding parenchyma or airway smooth muscle. Localizing this receptor to the airways in the lungs could allow for therapeutic targeting of the receptor for inflammatory lung diseases.

References

1. Hirasawa A, Tsumaya K, Awaji T, Katsuma S, Adachi T, Yamada M, Sugimoto Y, Miyazaki S, Tsujimoto G. (2005) Nat Med 11(1): 90-94.

2. Son SE, Park SJ, Koh JM, Im DS. (2020) Acta Pharmacol Sin 41(10): 1337-1347.

3. Bjursell M, Xu X, Admyre T, Böttcher G, Lundin S, Nilsson R, Stone VM, Morgan NG, Lam YY, Storlien LH, Lindén D, Smith DM, Bohlooly-Y M, Oscarsson J. (2014) PLoS ONE 9(12): e114942.

296

Development of a conformational biosensor to measure FFA4 receptor activation in 3D cultured adipocytes

Luca Vita¹, Beth Dibnah¹, Emily Russell² and Brian Hudson¹

¹University of Glasgow; ²BioAscent Drug Discovery

Introduction

Since deorphanization in 2005, the free fatty acid receptor 4 (FFA4) has generated interest as a target for various metabolic diseases.¹ Some of its beneficial effects are believed to be related to the function of FFA4 in adipocytes; however, better research tools are needed to fully appreciate how FFA4 functions in adipose tissue. Here, we develop a novel bioluminescence resonance energy transfer (BRET) biosensor to assess FFA4 activation in adipocytes. The biosensor is then combined with 3D cultured adipocytes to interrogate FFA4 function in an adipose tissue-like microenvironment. Finally, we will use this combined FFA4 biosensor-3D model to investigate the pharmacology of a range of fatty acids.

Methods

An intermolecular FFA4 BRET biosensor was developed by tagging the hFFA4 receptors' third intracellular loop with NanoLuc-luciferase and an enhanced yellow fluorescent protein on the c-terminus. To study this sensor's function in adipocytes, 3T3-L1 cells stably overexpressing the sensor were differentiated over 14-day period, and BRET was used to assess FFA4 activation. 3D, 3T3-L1 spheroids were cultured using 96-well, U-bottom, ultra-low-adhesive plates. Differentiation of 3T3-L1s was confirmed via lipid staining, expression of adipogenic markers and, when cultured in 3D, an increase in spheroid size.

Results

The biosensor was validated in a Flp-In™ T-REx™ cell line, where TUG-891, a selective FFA4 agonist, potently activated the receptor (pEC50 = 6.64 ± 0.13, n = 4) producing a 15.48% BRET reduction, while the fatty acid ligand, α-linolenic-acid (aLA), produced a 30.67% BRET reduction (pEC50 4.34 ± 0.1, n = 4).

In the stably expressing 3T3-L1, 2D cell model, the potency of both TUG-891 and aLA decrease as cells transition from an undifferentiated to a differentiated state. For TUG-891, the potency shifts from pEC50 = 6.37 ± 0.1 (n = 3) to pEC50 = 5.93 ± 0.2 (n = 3), while for aLA, it shifts from pEC50 = 4.95 ± 0.1 (n = 3) to pEC50 = 4.27 ± 0.1 (n = 3).

A similar trend is observed in the 3D cell model; however, the reduction in potency from undifferentiated to the differentiated cells is exaggerated. This has led to currently undetermined pEC50 values in the differentiated cells as the E_max has not been reached at the tested concentrations; however, there is an obvious shift in potency for both ligands.

Conclusion

This novel biosensor, combined with 3D cell culture, is a valuable tool to investigate FFA4 activation in an adipose tissue-like microenvironment, highlighting differences in cell models and aiding to understand FFA4 function in metabolic disease.

Reference

1. Duncan EM, Vita L, Dibnah B, Hudson BD. Metabolite-sensing GPCRs controlling interactions between adipose tissue and inflammation. Front Endocrinol (Lausanne) 2023;14:1197102. https://doi.org/10.3389/FENDO.2023.1197102/BIBTEX

15

Gas chromatography–mass spectrometry analysis, toxicological and anti-inflammatory potential of Citrus medica L.

Oyepata Simeon Joseph

Federal University Oye Ekiti

Introduction

Inflammation is a common, complex and distressing biological condition that has a profound impact on individuals [1]. However, long-term administration of NSAID may induce severe effects. Conventional anti-inflammatory drugs possesses mild to severe side-effects. Citrus medica L is a plant use for many folkloric purposes. The present study was undertaken to carryout gas chromatography–mass spectrometry analysis, acute toxicity and to assess traditional claim of inflammatory effect of Citrus medica L .juice on rats using carrageenan-induced and egg albumin-induced inflammation on albino rats.

Method

Mass spectra of the individual Gas chromatography peaks were identified by a computer search of the commercial libraries. The median lethal dose (LD50) for Citrus medical fruit juice was determined using modified Lorke’s method (1983). Sixteen (16) ,mice were used for the determination of the median lethal dose. Four groups of three rats each were used for the first phase and received 10, 100, 200 and 400 mL·kg⁻¹, respectively. Subsequently, four groups of one rat each were used for the second phase and given 500, 1000, 1500 and 2000 mL·kg⁻¹. The animals were monitored for changes in behaviour and mortality within 24 hrs. For each antiinflammatory study, 30 male Wistar rats were divided into six (6) groups. Group 1 and 2 received normal saline and 0.1 mL of 1% w/v suspension of carrageenan/egg albumin. Group 3 received diclofenac 50 mg·kg⁻¹ while group 4, 5 and 6 received 250, 500 and 1000 mL/kg Citrus medica L. juice, respectively.

Results

Significant metabolites were identified in Gas chromatography–mass spectrometry profile. No deaths were recorded after 24 h of administration of various doses of the extract. Citrus medica L. fruit exerted a significant (P < 0.05, P < 0.01, P < 0.001) anti-inflammatory effect in a dose dependent manner when compared to the control and carrageenan group. Citrus medica L. fruit also caused significant anti-inflammatory (P < 0.05, P < 0.01, P < 0.001) effect in dose dependent manner when compared to the group that received egg albumin alone. The result obtained in both models were comparable to the standard drug, diclofenac, at 50 mg·kg⁻¹ dose.

Conclusion

The study shows that is safe Citrus medica L. fruit juice is safe. Citrus medica L. fruit juice bears significant anti-inflammatory activities in both models, which agrees with it traditional claim for management of inflammation.

Reference

1. Benyamin, R., Trescot, A.M., Datta, S., Buenaventura, R., Adlaka, R., Sehgal, N., Glaser, S.E., Vallejo, R., 2008 Opioid complications and side effects. Pain Physician Journal. 22: S105-S120.

90

Effect of carbachol (CCh) on pro-inflammatory gene expression changes in BEAS-2B bronchial epithelial cells: Interactions with the long-acting, β2-adrenoceptor agonist (LABA), indacaterol (Ind)

Varuna Jayasinghe¹, Radhika Joshi¹, Taruna Joshi¹, Tamkeen Paracha¹, Cora Kooi¹, Mahmoud Mostafa¹, Carla Bauer², Steven Charlton³, Oleg Iartchouk², Ashley Maillet², Melody Morris², Vera Ruda², David Sandham², Yanqun Wang², Robert Newton¹ and Mark Giembycz¹

¹Department of Physiology & Pharmacology, University Of Calgary; ²Novartis Biomedical Research; ³University of Nottingham

Introduction

Muscarinic receptor antagonists protect against COPD exacerbations, suggesting that endogenous ACh up-regulates pro-inflammatory genes in the airways [1]. Similarly, Gram-negative bacterial infections may increase exacerbation risk by stimulating pro-inflammatory catecholamines release from infiltrating phagocytes [2]. The appearance of catecholamines in the lungs is informative because they may initiate detrimental genomic responses to respiratory health. ACh and catecholamines activate the transcription factor cAMP response element (CRE)-binding protein, which may represent a node of signal integration that fine-tunes genomic responses. Herein, we explored the pro-inflammatory potential of CCh in BEAS-2B human bronchial epithelial cells and its interaction with the LABA, Ind. This is important because LABAs are recommended for COPD treatment, but could mimic the adverse, β2-adrenoceptor-mediated effects of catecholamines derived from pulmonary phagocytes.

Methods

An 8×6-point concentration-response checkerboard assay was performed with CCh and Ind in BEAS-2B cells expressing a CRE-luciferase reporter, generating a 3D-landscape plot and isobologram. Cells were cultured (1–18 h) with Ind (100 nM) and CCh (10 μM) alone and in combination and processed for mRNA-seq. Immunoblotting was used to determine if gene expression changes were replicated at the protein level.

Results

CCh and Ind produced a concentration-dependent increase in BEAS-2B CRE reporter activity ([A]50 = 263 nM; Emax: 2.0-fold and [A]50 = 0.41 nM; Emax: 8.17-fold, respectively). When combined, these stimuli interacted supra-additively (Figure 1). mRNA-seq determined that CCh was a weak stimulus, affecting only 20 genes. Conversely, Ind regulated (q ≤ 0.05) 880 mRNAs (624 induced [≥1.5 fold]; 256 repressed [≤0.67-fold]), which increased to 1043 (691 induced; 352 repressed) in the presence of CCh. Thus, CCh enhanced the transcriptional signature of Ind (Figure 2). Of the 624 Ind-induced genes, 39 behaved similarly to the CRE reporter, which was reproduced at the protein level (Figure 3). On remaining genes, CCh and Ind behaved additively or infra-additively, implicating multiple mechanisms of gene regulation. Functional annotation highlighted transcription and signalling as dominant themes, which were populated with gene ontology terms associated with inflammatory and immune processes (Figure 4).

Conclusion

If a genomic interaction between a LABA and endogenous ACh occurs in vivo then, paradoxically, this could maintain facets of airway pathology in COPD.

References

1. Kistemaker LE, Bos I S, Hylkema MN, et al. Muscarinic receptor subtype-specific effects on cigarette smoke-induced inflammation in mice. Eur Respir J. 2013; 42:1677-1688.

2. Dickson RP, Erb-Downward JR, Prescott HC, et al. Intraalveolar catecholamines and the human lung microbiome. Am J Respir Crit Care Med. 2015; 192:257-259.

100

Development of a novel macrophage cell line to investigate the importance of formyl peptide receptor 2 regulation for immune cell function

Emily Cope, James Hislop and Dawn Thompson

University of Aberdeen

Introduction

Formyl peptide receptor 2 (FPR2), a GPCR expressed on immune cells, is critical for regulating the switch between pro-inflammatory and pro-resolution signalling, and thus is an attractive therapeutic target [1]. Here, we sought to advance our understanding of FPR2 signalling and trafficking by generating novel macrophage cell lines expressing wildtype FPR2 or GPCR kinase-phosphorylation deficient mutant (ΔABC), to probe the effect of impaired desensitization on immune cell function.

Method

FLAG-tagged FPR2 or ΔABC were expressed in either RAW 264.7 or HEK cells. Cells were stimulated with 100 nM WKYMVm and p-ERK measured by Western blot. Receptor internalization and colocalization with transferrin was observed using confocal microscopy, and expression of inflammatory and cholesterol transport regulators analysed by RT-qPCR. Data were analysed using GraphPad Prism and presented as mean ± SEM (n = 3–4 independent experiments). Statistical significance was analysed by one-way ANOVA or unpaired Student’s two-tailed t-test.

Results

FPR2 expressing RAW cells exhibited robust p-ERK signalling at 5 min WKYMVm treatment, returning to baseline 30 min post-treatment (P < 0.05, 5 min vs. 0 min). This correlated with receptor internalization after 30 min WKYMVm treatment. RAW-ΔABC cells exhibited prolonged and enhanced p-ERK levels even after 30 min stimulation (P < 0.05 vs. 0 min), and, indeed, reduced internalization compared to wildtype receptor. Following internalization, FPR2 strongly colocalized with transferrin. RAW-FPR2 cells had elevated Il6 levels, whilst RAW-ΔABC had higher Il10 expression; both had elevated Cd163, a marker of pro-resolution M2 macrophages, and anti-inflammatory Ppara and Pparg (P < 0.05 vs. RAW). RAW-FPR2 cells had lower levels of ATP-binding cassette transporter Abca1, a regulator of cholesterol efflux, than RAW and RAW-ΔABC cells, while both cell lines exhibited altered expression of mediators of cholesterol uptake, with reduced macrophage scavenger receptor 1 and elevated oxidized low density lipoprotein receptor 1 (P < 0.05 vs. RAW).

Conclusions

Here, we describe impaired FPR2 desensitization in RAW cells results in altered signalling, trafficking and immune response. In addition, overexpression of wildtype or mutant receptors results in alteration to inflammatory markers and cholesterol trafficking regulators, and thus may have implications for efferocytosis. An improved understanding of FPR2 regulation and its role in efferocytosis may yield more targeted therapeutics for atherosclerosis.

Reference

1. Cooray SN, Gobbetti T, Montero-Melendez T, et al. Ligand-specific conformational change of the G-protein–coupled receptor ALX/FPR2 determines proresolving functional responses. Proc Natl Acad Sci U S A. 2013;110(45):18232-18237. https://doi.org/10.1073/pnas.1308253110

126

Immunogenic response to tetanus from intradermal delivery of standard and reduced pentavalent vaccine doses using novel microneedles in mice

Ishumeet Kaur Bajwa, Joseph L. Mathew, Yashwant Kumar, Naresh Sachdeva, Smita Pattanaik and Monica Anand

Post Graduation Institute Of Medical Education And Research (pgimer) Chandigarh

Introduction

Microneedles facilitate vaccine delivery into the dermis layer of skin with minimal pain. However, their safety and efficacy need to be confirmed. Objective. We evaluated the safety and effectiveness of indigenously developed microneedles by administering standard and reduced doses of the vaccine through intramuscular (IM), intradermal (ID), and microneedle (MN) method and tetanus immune cell response, antibody kinetics and anamnestic responses were compared.

Method

We utilized 750 μm long, 200 μm bore microneedles to assess the immunogenic response to tetanus in BALB/c mice. Eighty-four female BALB/c mice (aged 6–8 weeks, 20–25 g) were randomly assigned to three groups (n = 28 per group) and received a 0.2 mL dose of standard pentavalent vaccine containing 60 IU of tetanus toxoid. The vaccine was administered intramuscularly (IM) via a 23G needle, intradermally (ID) via a 26G needle, or through microneedles (MN) [1]. Blood samples were collected at days 0, 7, 14, and 28 to analyse antibody kinetics. A booster dose was administered on day 84, followed by additional blood sampling on day 112 to assess the anamnestic response. For immune cell analysis, 28 mice (n = 7 per group for IM, ID, and MN) were sacrificed on day 7. Spleens were harvested, and single-cell suspensions were prepared using RBC lysis. T and B cell populations were analysed using flow cytometry, with markers including CD45, CD3, CD4, CD8, CD69, CD19, and CD38 [2]. In a separate cohort of 56 mice, a reduced vaccine dose (0.1 mL) was administered either intradermally (ID) or via microneedles (MN) (n = 28 per group). Blood samples were collected at the same time points as the original cohort for antibody kinetic analysis. Fourteen mice (n = 7 per group) were sacrificed on day 7 for immune cell analysis, and spleens were processed similarly for T and B cell enumeration using flow cytometry. All antibody titers were quantified using the ELISA method.

Results

Microneedle (MN) injections were well-tolerated, with no adverse effects such as bleeding, erythema, swelling, or pain, demonstrating a favourable safety profile. Across days 0, 7, and 14, no statistically significant differences in total IgG titers against tetanus were observed among the MN, intradermal (ID), and intramuscular (IM) groups. However, by day 28, anti-tetanus IgG titers in the MN group (56.30 ± 9.36 ng mL) remained statistically comparable to IM (68.21 ± 10.24 ng mL) (P = 0.5046), while ID titers were significantly lower (32.63 ± 17.96 ng mL) (P = 0.0034 for IM vs. ID; P = 0.0740 for MN vs. ID). Following the booster dose, no significant differences in IgG titers were noted at day 84 across the groups. By day 112, however, both MN (226.99 ± 29.44 ng mL) and IM (251.80 ± 44.58 ng mL) showed significantly higher IgG titers compared to ID (176.62 ± 59.85 ng mL) (P < 0.0001 for IM vs. ID; P = 0.0558 for IM vs. MN; P < 0.0001 for MN vs. ID). In terms of immune cell response, while the MN group exhibited a higher percentage of T and B cell populations than both the IM and ID groups, no statistically significant differences were detected between IM, ID, and MN. In the reduced dose cohort, total IgG titers showed no significant differences between MN and ID groups at days 0, 7, and 14. By day 28, titers were comparable between MN (29.99 ± 18.18 ng mL) and ID (16.15 ± 10.73 ng mL) (P = 0.6687). For the anamnestic response at day 112, MN (129 ± 42 ng mL) demonstrated significantly higher IgG titers than ID (85.27 ± 42 ng mL) (P = 0.0002). For immune cell response in this cohort, the MN group also showed a higher percentage of T and B cells compared to the ID group, although no statistically significant difference was observed (ID vs. MN).

Conclusion

These data confirm that intradermal injection through microneedles is safe and efficacious, paving the way for clinical studies in human subjects.

References

1. Waghchaure M, Govardhane S, Shende P. Enhancement of immunopotentiation using tetanus toxoid-based nanoparticulate dissolvable microneedles. Biomedical Microdevices. 2021;23:1-9.

2. Hiraishi Y, Nakagawa T, Quan YS, Kamiyama F, Hirobe S, Okada N, Nakagawa S. Performance and characteristics evaluation of a sodium hyaluronate-based microneedle patch for a transcutaneous drug delivery system. International journal of pharmaceutics. 2013;441(1-2):570-9.

143

Raloxifene ameliorates SARS-CoV-1 spike protein-induced inflammation in BEAS-2B human lung epithelial cells

Misturah Adana^1,2 and Olumayokun Olajide¹

¹University of Huddersfield; ²University of Ilorin

Introduction

Studies have suggested that the protective roles of oestrogens in severe COVID-19 [1] may be linked to their anti-inflammatory activity in COVID-19 cytokine storm [2]. In this study, we investigated the effects of raloxifene on inflammation induced by SARS-CoV-2 spike protein (S1) in human lung (BEAS-2B) epithelial cells.

Methods

Cultured BEAS-2B cells were treated with raloxifene (1, 10 and 100 nM) prior to stimulation with S1 (1 μg·mL⁻¹) for 24 h. Cell supernatants were analysed for levels of tumour necrosis factor-alpha (TNF-α), interleukins-6, -1β and -8 (IL-6, IL-1β and IL-8), using ELISA. Cells were also incubated with S1 for 15 min and lysates analysed for protein levels of phospho-p65 and phospho-IkBα. Reporter gene assays were carried out following transfection of BEAS-2B cells with NF-kB-bearing luciferase plasmid, and then treated with raloxifene (1, 10 and 100 nM) followed by stimulation with S1 for 4 h. Data were analysed with one-way ANOVA. Mean ± SEM values with P < .05, in comparison with S1 stimulation were statistically significant.

Results

Analysis of supernatants revealed that stimulation of BEAS-2B cells with S1 (1 μg·mL⁻¹) resulted in significant (P < .001) elevation in the production of TNF-α, IL-6, IL-1β and IL-8. Pretreatment with raloxifene (1 nM) did not cause significant (P < .05) reduction in the release of mediators. However, on increasing the concentration of raloxifene to 10 nM, there were ~1.6-, ~1.5-, ~1.5-, and ~1.7-fold reduction in S1-induced increased production of TNF-α, IL-6, IL-1β and IL-8, respectively. In the presence of raloxifene (100 nM), there were ~1.9-, ~2.4-, ~2.5-, and ~2.6-fold decrease, respectively. Similarly, significant and concentration-dependent reductions in phospho-p65 and phospho-IkBα protein levels were observed with 10 and 100 nM of raloxifene. Results of reporter gene assays showed a significant (P < 0.05) reduction in NF-kB luciferase activities in the presence of 10 and 100 nM of raloxifene but not with 1 nM.

Conclusion

Raloxifene produced anti-inflammatory activity in S1-induced elevated secretion of pro-inflammatory mediators in BEAS-2B cells through NF-kB-dependent mechanisms. We propose that this drug could be repurposed for treating cytokine storms in COVID-19 and related respiratory virus infections.

References

1. Costa AJ, Lemes RMR, Bartolomeo CS, et al. Overexpression of estrogen receptor GPER1 and G1 treatment reduces SARS-CoV-2 infection in BEAS-2B bronchial cells. Mol Cell Endocrinol. 2022; 558:111775.

2. Al-Kuraishy HM, Al-Gareeb AI, Faidah H, Al-Maiahy TJ, Cruz-Martins N, Batiha GE. The looming effects of estrogen in Covid-19: A rocky rollout. Front Nutr. 2021; 8:649128.

148

Targeting mucus to improve antiviral activity of therapeutic proteins in the nasal cavity

Joelton Rocha Gomes, Nathalie Lejal, Audrey Saint-Albin-Deliot, Amélie Donchet, Sophie Le-Poder, Bernard Klonjkowski, Bernard Delmas and Nicolas Meunier

Inrae

Viral respiratory infections are major contributors to mortality worldwide in young and elderly people. The primary entry and reservoir for the dissemination of respiratory virus such as SARS-CoV-2 is the nasal cavity where respiratory viruses infect epithelial cells through specific receptor interaction. SARS-CoV-2 spike protein interacts with host cells receptors through its receptor binding domain (RBD). In order to develop drugs to block infection in the upper airways, our team has validated proteins against the RBD for intranasal administration, called αReps [1]. They showed excellent inhibitory effect in the nM range in vitro. However, in a hamster model, it was rapidly absorbed by the nasal epithelium, causing a low intranasal residence time and partial protection against infection. Therefore, the aim of this study was to improve the efficacy of αReps to block infection in the nasal cavity. For that, we fused the αRep C2 to a mucin-binding domain to form a mucoadhesive protein. This could help to retain αReps in the mucus at the surface of the epithelium, avoiding absorption and retaining the proteins in the lumen of the nasal cavity. The fused mucoadhesive protein conserved the neutralization activity on pseudo viruses expressing SARS-CoV-2 spikes of different variants and on SARS-CoV-2 wild-type. In mice (n = 4), the intranasal residence time of the was increased significantly 1 h and 6 h after nasal instillation thanks to the addition of the mucin binding domain. The mucoadhesive antiviral protein almost fully protected healthy hamsters (n = 6) co-housed for 12 h with infected SARS-CoV-2 hamsters with twice-a-day treatment (0.05 μg of antiviral protein). These results demonstrate the efficiency of targeting mucus to improve antiviral treatments of respiratory viruses in the nasal cavity.

Reference

1. Thébault S, Lejal N, Dogliani A, Donchet A, Urvoas A, Valerio-Lepiniec M, Lavie M, Baronti C, Touret F, Da Costa B, Bourgon C. Biosynthetic proteins targeting the SARS-CoV-2 spike as anti-virals. PLoS Pathogens. 2022;18(9):e1010799. https://doi.org/10.1371/journal.ppat.1010799.

160

Assessment of the biological activity and phenolic composition of ethanol extracts of Ardisia polycephala leaves

Pawanpat Katanyutanon

Cheltenham Ladies’ College

Introduction

The increasing interest in the pharmacological potential of medicinal plants has prompted further investigation into their bioactive properties. This study aimed to explore the antioxidant and anti-inflammatory activities of Ardisia polycephala, alongside its total phenolic and flavonoid content.

Method

The plant's dried powder was extracted using 90% ethanol, and its antioxidant activity was assessed through DPPH and ABTS assays, while anti-inflammatory effects were evaluated using the nitric oxide (NO) inhibition assay via the Griess reaction. Additionally, cytotoxic activity was measured through the MTT assay.

Results

Results demonstrated that A. polycephala exhibited significant anti-inflammatory effects, with an IC50 value of 31.05 ± 0.314 μg·mL⁻¹, and potent antioxidant activity, with IC50 values of 25.549 ± 0.364 mg·mL⁻¹ in the DPPH assay and 19.412 ± 0.258 mg·mL⁻¹ in the ABTS assay. Furthermore, A. polycephala showed the highest inhibition of NO production at 40 μg·mL⁻¹.

Conclusion

In conclusion, A. polycephala displays both strong antioxidant and anti-inflammatory activities, offering valuable insights into its potential therapeutic applications. These findings contribute to the expanding body of research within pharmacology, and serve as a foundation for further studies to elucidate its mechanisms of action.

References

1. Kobayashi H, deMejía EG. The genus Ardisia: A novel source of health-promoting compounds and phytopharmaceuticals. J Ethnopharmacol. 2005;96(3):347-54. https://doi.org/10.1016/j.jep.2004.09.037.

2. deMejía EG, Ramírez-Mares MV. Ardisia: Health-promoting properties and toxicity of phytochemicals and extracts. Toxicol Mech Methods. 2011;21(9):667-74. https://doi.org/10.3109/15376516.2011.601355.

3. Chatatikun M, Chiabchalard A. Comparative evaluation of antioxidant and anti-inflammatory activity of active compounds identified in Ardisia elliptica extracts. BMC Complement Altern Med. 2017;17(1):487. https://doi.org/10.1186/s12906-017-1994-7.

4. Sen S, De B, Devanna N, Chakraborty R. Total phenolic, total flavonoid content, and antioxidant capacity of the leaves of Meyna spinosa Roxb., an Indian medicinal plant Chin J Nat Med. 2013;11(2):149-57. https://doi.org/10.1016/S1875-5364(13)60042-4.

5. Buraphaka H, Puttha W, Putalun W. Comparative evaluation of antioxidant and anti-inflammatory activity of active compounds identified in Ardisia elliptica extracts from different plant parts. Chemistry & Biodiversity. 2022;19(2):e202100796.

6. De Mejía EG, Ramírez-Mares MV. Ardisia: Health-promoting properties and toxicity of phytochemicals and extracts. Toxicology Mechanisms and Methods. 2011;21(9):667-74.

164

Roles of the interleukin-1 receptor-associated kinases (IRAKs), IRAK1 and IRAK4, in the context of interleukin (IL)-1β signalling to nuclear factor (NF)-κB

Keerthana Kalyanaraman, Mahmoud Mostafa and Robert Newton

Lung Health Research Group, Snyder Institute for Chronic Diseases and Department of Physiology & Pharmacology

Background and Aims

IRAK4 and IRAK1 are thought to play coordinated roles in IL1/ toll-like-receptor (TLR) signalling. Upon IL-1β binding to IL1-receptor-1 and IL1-receptor-accessory-protein, IRAK4 and IRAK1 oligomerize with MYD88 at the cytokine-receptor complex. This assembly activates transforming growth factor-β-activated kinase 1 (TAK1) and the IκB kinases (IKKs) to promote NF-κB dependent inflammatory gene expression [1]. However, roles for IRAK4 and IRAK1 in IL-1β-mediated NF-κB activation remain unclear. Since IL-1β expression is upregulated in inflammatory diseases, including asthma, this study aims to address this key knowledge gap.

Methods

A549 cells were used to model signalling and gene expression in epithelial cells. RNA-sequencing and immunoblotting were employed to investigate IRAK mRNA and protein expression, respectively, at various times post IL-1β (1 ng mL) treatment. NF-κB-dependent transcription was assessed in cells harbouring an NF-κB luciferase reporter. Cells were transfected with IRAK4 and IRAK1-targeting siRNAs or pretreated with IRAK1 and IRAK4-selective inhibitors (solubilized in DMSO). Cells were harvested 6 h following stimulation with IL-1β (1 ng mL) or tumour necrosis factor α (TNFα) (10 ng mL) for luciferase assay.

Results and Discussion

IL-1β had minimal impact on mRNA expression for both IRAKs, but IRAK1 protein was lost 0.5–24 h post-IL-1β treatment (n ≥ 6). Respective targeting siRNAs achieved >90% knockdown of IRAK4 and IRAK1, while control siRNAs had no effect (n ≥ 6). A subtle (~20–30%) reduction in IL-1β-induced NF-κB reporter activity was observed with IRAK4-targeting siRNAs (1–10 nM) (n = 6). IRAK1-targeting siRNAs (1–10 nM) also showed significant (~25–50%) losses in reporter activity, but this did not correlate with protein knockdown (n = 8). IRAK1/4-inhibitor-1 (selectivity: IRAK1, IRAK4), AS2444697, and PF6650833 (selectivity: IRAK4), revealed partial repression (~30%) of IL-1β-induced reporter activity (pEC50 = 5.6, 5.8 and 7.1, respectively), while JHX-119-01 (selectivity: IRAK1) pretreatment had no effect. However, as IRAKs are not involved in TNFα signalling, changes observed in TNFα-induced reporter activity suggest off-target effects for many of these compounds (n ≥ 8).

Conclusion

These results suggest a partial role for IRAK4 in activating NF-κB by IL-1β. With possible off-target effects of siRNAs and a lack of validation of JHX-119-01 in these cells, roles for IRAK1 remain unclear. However, IRAK1 may not be a major contributor to IL-1β-induced NF-κB activation in these cells. Confirmation of these findings would suggest key roles for kinases other than IRAK1 and IRAK4 in the activation NF-κB by IL-1β.

Reference

1. Vollmer S, Strickson S, Zhang T, et al. The mechanism of activation of IRAK1 and IRAK4 by interleukin-1 and Toll-like receptor agonists. Biochemical Journal. 2017;474(12):2027-2038. https://doi.org/10.1042/bcj20170097

226

The Nrf2 activator, oltipraz inhibits the release of pro-inflammatory mediators in LPS-activated BV-2 microglia

Marvellous Adewale and Olumayokun Olajide

University of Huddersfield

Introduction

The nuclear factor erythroid 2- related factor (Nrf2) regulates neuroinflammation in response to microglia activation through transcriptional control of cytoprotective antioxidant genes [1]. This potential crosstalk between neuroinflammation and Nrf2 activation has prompted investigations of compounds which can activate Nrf2 to inhibit excessive release of pro-inflammatory mediators. Oltipraz is a synthetic dithiolethione which has been reported to activate Nrf2 [2]. This study evaluated the effects of oltipraz on the release of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated BV-2 microglia.

Method

BV-2 microglia cells were treated with oltipraz (5, 10, and 20 μM) prior to stimulation with LPS (100 ng·mL⁻¹) for 24 h. Culture supernatants were analysed for levels Tumour Necrosis Factor- alpha (TNF-α) and interleukin-6 (IL-6) using ELISA, while the Griess assay was used to detect nitrite production. Prostaglandin E2 (PGE2) levels in were determined using enzyme immunoassay. Cell lysates were analysed for protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) using mouse ELISA.

Results

Stimulation of BV-2 cells with LPS (100 ng·mL⁻¹) produced significant (P < 0.0001) increase in the levels of TNF-α and IL-6 when compared with unstimulated cells. However, pretreatment with oltipraz (5, 10, and 20 μM) prior to LPS stimulation significantly (P < 0.01) reduced TNF-α and IL-6 levels. Similarly, treatment with oltipraz (5, 10, and 20 μM) significantly (P < 0.01) reduced levels of nitrite in LPS-stimulated BV-2 microglia. Levels of PGE2 in supernatants were reduced to 59.1%, 48% and 33.9% when cells were pretreated with 5, 10 and 20 μM, respectively in comparison with LPS stimulation alone. Analyses of cell lysates revealed that pretreatment with oltipraz (5, 10, and 20 μM) resulted in a significant (P < 0.001) reduction in LPS-induced increase in iNOS protein levels. Similarly, oltipraz (5, 10, and 20 μM) reduced LPS-stimulated increased COX-2 protein expression by 32.2%, 46.8% and 59.7%, respectively.

Conclusion

This study suggests that oltipraz inhibits neuroinflammation in LPS-activated BV-2 microglia, possibly due to Nrf2 activation in the microglia.

References

1. Park YJ, Yang HJ, Li W, Oh YC, Go Y. Menthae herba attenuates neuroinflammation by regulating CREB/NRF2/HO-1 pathway in BV2 microglial cells. Antioxidants. 2022;11(4):649.

2. Sun Q, Shen X, Ma J, Lou H, Zhang Q. Activation of Nrf2 signaling by oltipraz inhibits death of human macrophages with mycobacterium tuberculosis infection. Biochemical and Biophysical Research Communications. 2020;531(3):312-319.

237

Use of the unified information devices (UID) matrix system for non-invasive monitoring of thermoregulation and activity in plasmodium-infected C57BL/6 mice to refine model endpoints

Oluwaseun Abigail Adenigbagbe², Jyothsna Ramesh Kumar¹, Fabrizio Scorrano¹ and Colin Osborne¹

¹Novartis; ²University of York

Introduction

Malaria, caused by Plasmodium parasites, is an infectious disease causing 240 million cases globally each year and animal models are an important tool for understanding the pathogenesis of malaria and the exploration of possible treatments (1,2, 3). This study investigates the effects of Plasmodium chabaudi and Plasmodium berghei infection on the thermoregulation and activity of C57BL/6 mice with the aim of optimizing animal study endpoints for profiling of antimalarial compounds.

Method

All animal procedures were carried out under protocols approved by the Institutional Animal Care and Use Committee, BioMedical Research. Animals were implanted with a subcutaneous transponder and maintained in the Unified Information Devices (UID) matrix system, an RFID-enabled set-up that allows real-time recording and monitoring of location, movement and temperature for animals in their home-cage environment. Mice (C57BL/6, male and female, n = 6/group) were then infected with 200 μL of 5 × 106 infected erythrocytes or Dulbecco’s phosphate buffer saline (control) via intraperitoneal injection. Body weights, clinical observations and peripheral blood parasitemia (% infected erythrocytes) were measured periodically in addition to the UID monitoring. Results were plotted as mean +/- SEM and data analysed using a one-way ANOVA and a mixed two-way ANOVA with post-hoc comparisons via Tukey's test to determine any significance between the values of the infected and control groups.

Results

As parasitemia increased, there was a strong correlation with reduction in body temperature (P = 0.003) and activity (P = 0.0008) and to a lesser degree with body weight (P = 0.02) but not with clinical observations, e.g., a drop of approximately 6 °C from day 4 to day 7 corresponded to an increase in parasitemia from 10 to 20% in P. berghei-infected male mice (Figure 1).

Conclusions

References

1. Abdalal, S.A., Yukich, J., Andrinopoulos, K. et al. Livelihood activities, human mobility, and risk of malaria infection in elimination settings: A case–control study. Malar J 22, 53 (2023).

2. Savi MK. An overview of malaria transmission mechanisms, control, and modeling. Med Sci (Basel). 2022;11(1):3

3 Simwela NV, Waters AP. Current status of experimental models for the study of malaria. Parasitology. 2022;149(6):1-22.

249

Sex-dependent behavioural and inflammatory effects of neonatal lypopolysaccharide exposure in rats

Nicola Opallo¹, Adriano Lama¹, Claudio Pirozzi¹, Federica Comella¹, Filomena Del Piano², Stefania Melini¹, Nicole Pia Navatti¹, Rosaria Meli¹ and Giuseppina Mattace Raso¹

¹Department Of Pharmacy, School Of Medicine, University Of Naples Federico II; ²Department of Veterinary Medicine and Animals Production, University of Naples Federico II

Introduction

Early-life immune activation resulting from perinatal exposure to various endotoxins has been implicated in the development of numerous neuropsychiatric disorders, collectively known as Pediatric Acute-onset Neuropsychiatric Syndrome (PANS) [1]. While PANS has traditionally been linked to streptococcal infections, a range of pathogens are now associated with neuropsychiatric symptoms [2]. These disorders exhibit a broad spectrum of complex and heterogeneous symptoms, characteristic of obsessive-compulsive disorder, autism spectrum disorder, ADHD, and schizophrenia. Patients diagnosed with PANS between the ages of 6 and 14 years display mild cognitive impairments along with mood disturbances, hyperactivity, and stereotyped behaviours.

Aims

This study aims to confirm that postnatal lipopolysaccharide (LPS) administration induces stereotyped behaviours and hyperactivity in young adult rats while investigating sex-related mechanisms.

Method

Male and female Wistar rats were injected with LPS (1 mg·kg⁻¹) on postnatal day (PND) 3 and underwent behavioural assessments (marble burying, self-grooming, and open field tests) on PND42-44. Rats were sacrificed on PND45, and serum and brain tissues were collected for analysis.

Results

Female rats exposed to LPS demonstrated a pronounced increase in both the number of buried marbles and the time spent in self-grooming. Conversely, the OFT revealed that LPS-challenged rats of both sexes exhibited hyperactivity, characterized by increased total distance traveled, absence of freezing and reduced time spent in the centre of the arena. Serum cytokine analysis performed with Bio-Plex assay revealed elevated levels of GM-CSF, Gro-KC, IL-1b, IL-7, MIP-1a, and MCP-1 in female rats, while male rats showed higher Gro-KC and M-CSF levels. Furthermore, LPS administration led to significant alterations in mRNA expression of markers related to inflammation within the prefrontal cortex (PFC) and striatum. IIn the PFC, male-LPS Wistar rats exhibited an upregulation of Tnf compared to control males. Additionally, Il1b transcription was significantly enhanced only in the LPS-treated females rats compared to female controls. In the striatum, the LPS challenge induced the same inflammatory profile for Il1b and Tnf already observed in PFC. In summary, these results indicate that neonatal exposure to LPS triggers sex-specific behavioural and molecular changes aligned with PANS, along with related systemic and neuroinflammatory reactions.

References

1. Endres D, Pollak TA, Bechter K, et al. Immunological causes of obsessive-compulsive disorder: Is it time for the concept of an “autoimmune OCD” subtype?. Transl Psychiatry. 2022;12(1):5. https://doi.org/10.1038/s41398-021-01700-4

2. Efe A. SARS-CoV-2/COVID-19 associated pediatric acute-onset neuropsychiatric syndrome a case report of female twin adolescents. Psychiatry Res Case Rep. 2022;1(2):100074. https://doi.org/10.1016/j.psycr.2022.100074

289

Valorization of agricultural biomass for pharmaceutical applications

Ashitha Preman Karayil

University Of Huddersfield

Helianthus tuberosus L. commonly known as Jerusalem Artichoke (JA), which is also called sunchoke belongs to Helianthus genus from Asteraceae family. It is a perennial plant which has strong medicinal properties. However, the effects of Jerusalem Artichoke on human HaCaT keratinocytes and against UVB induced skin damage is not reported yet. UV radiation increases the long-term damages to skin such as photoaging, photo-immunosuppression, carcinogenesis, inflammatory dermatoses (including atopic dermatitis).5- 10% of children and 2- 10% of adults worldwide suffer from atopic dermatitis (AD), which is an inflammatory and itchy skin disorder that are caused by the exposure of solar radiation. The aim of the study is to evaluate the effects of extracts of JA leaves in human HaCaT keratinocytes and the effects of extracts on UVB induced damage HaCaT keratinocytes. Anti-inflammatory activity will be evaluated by measuring the levels of pro-inflammatory mediators TNF-alpha, IL-1α, IL-1β, IL-6 using Enzyme Linked Immuno-Sorbent Assay (ELISA), Polymerase Chain Reaction (PCR) and Western Blotting. Effects of extracts on UVB damage will be assessed by evaluating effects on the generation of reactive oxygen species (ROS) and cell viability.

293

The role of spinal oxytocin receptor in the lipopolysaccharide-induced mechanical hypersensitivity in male and female rats Abimael Gonzalez-Hernandez, Antonio Espinosa de los Monteros-Zúñiga, Guadalupe Martínez-Lorenzana and Miguel Condes-Lara

INb - UNAM

Introduction

The administration of lipopolysaccharide (LPS) at the spinal level promotes neuroinflammation and subsequent sensitization to tactile stimuli, owing to the release of proinflammatory mediators by glia. Activating oxytocin receptors (OTR) at the spinal cord level promotes antinociception, probably in a biased signalling fashion. Hence, the spinal effects of oxytocin and biased OTR agonists (carbetocin or atosiban) on LPS-induced sensitization were analysed in male and female rats.

Methods

In Wistar rats (250–300 g) behavioural (flinches induced by formalin and mechanical hyperalgesia using von Frey filaments), in vivo electrophysiological recordings (extracellular unitary recordings of spinal dorsal horn wide dynamic range [WDR] cells) and immunofluorescence (against OTR and microglia) assays were performed. Behavioural dose-response curves were constructed for LPS and oxytocin in males and females. Furthermore, the effect of LPS on spinal WDR activity was performed. Carbetocin and atosiban were used to reveal the potential intracellular mechanisms induced by OTR activation. The different treatments were administered 15 min after LPS intrathecal injection. Nocifensive behaviour induced by LPS administration was analysed using von Frey filaments to quantify the paw withdrawal threshold within 6 h after LPS administration.

Results

The administration of LPS increased sensitivity to tactile stimuli in a dose-dependent manner, with females (0.01 ng; ED75) being more sensitive than males (≈10 ng; ED75). The ED75 dose of males or females inhibited the neuronal discharge of spinal WDR. While oxytocin administration decreased LPS-induced inflammation in a dose-dependent manner (1–10 nmol), females were less sensitive to oxytocin (10 nmol) than males (1 nmol); in both cases, the effect was reversed by L-368,899 (10 nmol). Interestingly, although atosiban (1 nmol; biased OTR-Gi ligand) inhibited LPS-induced nociception in both sexes, carbetocin (1 nmol; biased OTR-Gq ligand) had no effect. Atosiban-induced antinociception was prevented by intrathecal pretreatment with pertussis toxin (an inhibitor of the Gi pathway).

Conclusions

These data suggest that OTR activation decreases LPS-induced hypersensitivity via the OTR-Gi pathway.

315

Decoding the anti-inflammatory potential of Aspergillus unguis SP51-EGY: A novel TLR4 inhibitor with therapeutic promise

Anwar Abdelnaser

Institute of Global Health and Human Ecology, School of Sciences and Engineering, The American University in Cairo (AUC), Cairo, 11835, Egypt

Introduction

Chronic inflammation is linked to various diseases such as cancer, autoimmune disorders, and sepsis. Targeting inflammatory pathways like TLR4 holds therapeutic potential. This study examines the anti-inflammatory properties of Aspergillus unguis isolate SP51-EGY, hypothesizing that it modulates key inflammatory pathways through TLR4 inhibition.

Method

RAW 264.7 macrophages were cultured and stimulated with LPS (10 ng·mL⁻¹) and treated with fungal extracts (10 μg·mL⁻¹). Nitric oxide (NO) production was measured using the Griess method, and mRNA levels of iNOS, COX-2, TNF-α, and IL-6 were quantified via qPCR. Q-TOF LC-HRMS was used for chemical profiling. Statistical analysis was performed using one-way ANOVA (n = 3, P < 0.05).

Results

Through real-time qPCR, we assessed the expression levels of pivotal inflammatory genes, including iNOS, COX-2, TNF-α, and IL-6. Remarkably, our fungal extracts significantly diminished NO production and showed noteworthy reductions in the mRNA expression levels of the genes mentioned above. Furthermore, while Nrf2 is typically associated with modulating inflammatory responses, our findings indicate that the anti-inflammatory effects of our extracts are not Nrf2-dependent. Moreover, the chemical diversity of the potent extract (B Sh F) was elucidated using Q-TOF LC-HRMS, identifying 54 compounds, some of which played vital roles in suppressing inflammation. Most notably, compounds like granisetron, fenofibrate, and umbelliprenin were found to downregulate TNF-α, IL-1β, and IL-6 through the NF-κB signalling pathway.

Conclusion

Aspergillus unguis isolate SP51-EGY”, isolated from the Red Sea, Egypt, has been unveiled as a promising TLR4 inhibitor with significant anti-inflammatory potentials, presenting novel insights for their potential therapeutic use in inflammation.

References

1. Chen L, Deng H, Zhu X, et al. Inflammatory responses and diseases. Oncotarget. 2017;9(6):7204-7218.

2. Saleh HA, Yousef MH, Abdelnaser A. The anti-inflammatory properties of phytochemicals. Front Immunol. 2021;12:606069.

3. Newton K, Dixit VM. Signaling in innate immunity. Cold Spring Harb Perspect Biol. 2012;4(3).

316

The specialized pro-resolvin mediators protectin D1 and maresin 1 dampen airway hyperreactivity induced by IL-13 in isolated human airways

Willem Abma^1,2, Jesper Säfholm¹, Craig Wheelock¹, Henric Olsson², Mikael Adner¹ and Sven-Erik Dahlén¹

¹Karolinksa Institutet; ²AstraZeneca

Introduction

Inflammation and airway hyperreactivity are key, interrelated processes in allergic asthma, with IL-13 serving as a pivotal mediator. No treatment to date can completely reverse airway hyperreactivity. Specialized pro-resolving mediators (SPMs) are lipid mediators with anti-hyperreactive potential. Our previous research demonstrated that the docosahexaenoic acid (DHA) derived SPM MCTR3 can attenuate IL-13 induced hyperreactivity (1), however it remains unclear if this is a general effect for DHA-derived SPMs. We therefore tested maresin 1 (Mar1) and protectin D1 (PD1) for their potential anti-hyperreactive effect in our IL-13 driven translational model of airway hyperreactivity in isolated human bronchi (2).

Method

Human bronchi were isolated from lung tissue obtained from patients undergoing lobectomy and divided into segments. These segments were incubated in DMEM/F12 for 48 h in presence or absence of IL-13 (100 ng·mL⁻¹) and SPMs (100 nM). Following incubation, concentration-response curves for histamine (1 nM–0.1 μM) and LTD4 (10 pM–30 nM) were obtained using myography. Responses were normalized to 60 mM KCl.

Results

Compared to controls, incubation for 48 h with IL-13 caused an increased maximal contraction (Emax) of LTD4 (108.2 ± 4.9% vs. 89.0 ± 4.2%, P < 0.05). Treatment with PD1 reduced the Emax to 88.2 ± 4.0%, P < 0.05 vs. IL-13 (figure 1A). The potency (pEC50) of LTD4 was also altered in PD1 treated segments compared to IL-13 treated segments (8.7 ± 0.1 vs. 9.2 ± 0.1, P < 0.05). IL-13 induced a left shift of the pEC50 of histamine compared to controls (7.5 ± 0.2 vs. 6.6 ± 0.2, P < 0.05) and while PD1 partially reversed this shift (6.9 ± 0.2), the change did not reach statistical significance (P = 0.13, figure 1B). In experiments with MaR1, MaR1 reverted IL-13-induced hyperreactivity of LTD4 (114.6 ± 4.7% vs. 82.1 ± 6.1%, P < 0.05, figure 1C). For histamine, the Emax of MaR1 treated segments was lower compared to IL-13 approaching statistical significance (100.9 ± 10.4% vs. 123.6 ± 5.9%, P = 0.07, figure 1D).

Conclusions

References

1. Safholm J, Abma W, Bankova LG, Boyce JA, Al-Ameri M, Orre AC, et al. Cysteinyl-maresin 3 inhibits IL-13 induced airway hyperresponsiveness through alternative activation of the CysLT(1) receptor. Eur J Pharmacol. 2022;934:175257.

2. Manson ML, Safholm J, James A, Johnsson AK, Bergman P, Al-Ameri M, et al. IL-13 and IL-4, but not IL-5 nor IL-17A, induce hyperresponsiveness in isolated human small airways. J Allergy Clin Immunol. 2020;145(3):808-17 e2.

24

Tirzepatide, GIP(1–42) and GIP(1–30) display unique signalling profiles at two common GIP receptor variants, E354 and Q354

Tayla Rees^1,2,3, Benjamin Buttle², Zoe Tasma^2,3, Paul Harris^2,3 and Christopher Walker^2,3

¹King's College London; ²The University of Auckland; ³Maurice Wilkins Centre for Molecular Biodiscovery

Introduction

Type 2 diabetes (T2D) and obesity are widespread metabolic disorders affecting millions globally. Tirzepatide, a dual agonist of the gastric inhibitory polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) receptors, shows greater clinical efficacy than GLP-1 receptor agonists. The potent activity of tirzepatide at the GIP receptor appears to account for this increased efficacy. However, the underlying mechanisms are not fully understood. The GIP receptor is activated by two endogenous forms of GI: GIP(1–42) and GIP(1–30). Furthermore, signalling at the E354 and Q354 GIP receptor variants is poorly characterized. This is particularly important as the Q354 variant is linked to higher T2D risk and lower body mass index. This study aimed to characterize and compare the pharmacological profiles of GIP(1–42), GIP(1–30) and tirzepatide at both GIP receptor variants to better understand GIP receptor signalling.

Methods

The signalling profiles of the human E354 or Q354 GIP receptor variants were examined in transiently transfected Cos7 cells. Intracellular signalling responses to GIP(1–42), GIP(1–30) or tirzepatide were measured for cAMP and IP1 accumulation (n = 5) and AKT, ERK1/2 and CREB phosphorylation (n = 3). Concentration–response curves were fitted using the four-parameter logistic equation. Data are expressed as a percentage of the curve fitted maximum (Emax) and minimum (Emin) responses produced by GIP(1–42). Emax values were derived from raw, non-normalized values (cAMP and IP1) or were normalized and expressed as a percentage of the control GIP(1–42) E_max (pAKT, pERK1/2 and pCREB). Biased signalling was then quantified using the operational model of agonism and compared between the two variants. All data were plotted and analysed using Prism GraphPad 10.0. Data are mean ± standard error of mean combined from three or five independent experiments. Comparisons of the pEC₅₀, E_max, Δlog(τ/KA) or ΔΔlog(τ/KA) of peptides or pathways at a receptor variant were analysed by ratio paired one-way ANOVA with post hoc Dunnett's test. Comparisons of the pEC₅₀, E_max, Δlog(τ/KA) or ΔΔlog(τ/KA) of the peptide or pathway between the receptor variants were analysed by unpaired Student's t-test, with the exception of the raw E_max values compared by ratio paired t-test.

Results

GIP(1–42) and GIP(1–30) displayed equipotent induction for the majority of signalling pathways at both receptor variants, excluding CREB phosphorylation where GIP(1–30) was more potent than GIP(1–42) at the Q354 variant (pEC₅₀; 10.6 ± 0.30 GIP(1–42) vs. 11.5 ± 0.37 GIP(1–30)). Tirzepatide had a lower pEC₅₀ than GIP(1–42) for all pathways at both receptor variants and a reduced E_max for IP1 accumulation (P < 0.05 by one-way ANOVA with Dunnett's post hoc test). At the E354 variant, cAMP signalling was favoured for all three agonists. At the Q354 variant, cAMP signalling was favoured for GIP(1–42) and tirzepatide, whereas CREB was sevenfold more efficacious than cAMP and was favoured for GIP(1–30). When the signalling of the two GIP receptor variants was compared, significantly greater pEC₅₀ were observed for GIP(1–42) (E354 8.62 ± 0.09 vs. Q354 9.53 ± 0.17) and GIP(1–30) (E354 8.79 ± 0.10 vs. Q354 9.28 ± 0.10) for IP1 accumulation and GIP(1–30) for CREB phosphorylation (E354 10.2 ± 0.12 vs. Q354 11.5 ± 0.37) at the Q354 variant, subsequently translating to a small but significant increase in efficacy (log(τ/KA)). No differences in receptor pharmacology or efficacy were observed for tirzepatide between the two GIP receptor variants.

Conclusions

This study demonstrates that tirzepatide is a biased agonist towards Gαs signalling and equally activates the E354 and Q354 GIP receptor variants. Differences in GIP receptor pharmacology with endogenous peptides may explain phenotypic variations. This research enhances understanding of GIP receptor signalling and supports developing therapies for T2D and obesity.

44

The controversial role of roxadustat in gentamicin-induced acute kidney Injury: Worsening renal function via hypoxia-inducible factor stabilization

Rania Salama¹, Omar Ashraf¹, Sara Ghobish² and Mona Abd-Elgalil³

¹Faculty of Pharmacy, Misr International University; ²College of Science and Engineering, James Cook University; ³Faculty of Medicine (Girls), Al Azhar University

Introduction

Gentamicin (GEN) is known for its nephrotoxic properties, contributing significantly to acute kidney injury (AKI), a condition with serious clinical implications. The involvement of hypoxia-inducible factor-1α (HIF-1α) in AKI is debated, with conflicting evidence on whether it exacerbates renal damage or offers protection [1]. Roxadustat (RXD) works by stabilizing HIF and preventing its degradation. Given the uncertainties surrounding HIF-1α's role in AKI, the study aimed to evaluate the effects of RXD in GEN-induced AKI.

Methods

Twenty-four Wistar rats were divided into four groups (n = 6): a control group (receiving RXD's vehicle; 1 ml/kg of 0.5% DMSO, i.p., every other day), an AKI group (receiving GEN at 100 mg/kg/day, i.p., for 7 days) and two treatment groups receiving RXD (10 and 20 mg/kg, i.p., every other day for 14 days) [2], with concurrent daily GEN administration from day 8 to day 14. After the last dose, rats were placed in metabolic cages to collect 24-h urine samples and estimate urine volume and flow rate. Lastly, after anaesthesia with ketamine/xylazine cocktail (87/13 mg/kg; at the dose of 1 ml/kg, i.p.), rats were sacrificed by decapitation, and then the kidneys were dissected and weighed to calculate the kidney/body weight ratio (renal somatic index). Histopathological analysis was also performed using haematoxylin and eosin (H&E) staining. Molecular docking studies using Discovery Studio V4.5 software were conducted on two enzymes, PHD (PDB:6BZN) and E3 ubiquitin ligase (PDB:8QNH), and protocols were validated using self-docking of crystallized inhibitors and RMSD calculations.

Results

RXD treatment at both doses failed to improve urine output and flow rate (Table 1). Moreover, histopathological examination revealed significant glomerular and tubular damage (Figure 1). The docking studies of RXD on two regulatory enzymes, PHD and E3 ubiquitin ligase, showed high affinity and good binding to the active sites according to cDocker energy scores and multiple hydrogen and hydrophobic interactions (Figures 2 and 3).

Conclusions

Our study demonstrated that RXD exacerbated GEN-induced renal injury in rats rather than offering protection. Ongoing biochemical investigations aim to elucidate the role of HIF-1α stabilization and the mechanisms underlying RXD's adverse effects.

References

1. Zhang H, Xu R, Wang Z. Contribution of oxidative stress to HIF-1-mediated profibrotic changes during the kidney damage. Oxidative Med Cell Longev 2021;2021:6114132. https://doi.org/10.1155/2021/6114132

2. Lan Q, Wang K, Meng Z, et al. Roxadustat promotes hypoxia-inducible factor-1alpha/vascular endothelial growth factor signalling to enhance random skin flap survival in rats. Int Wound J 2023;20(9):3586-3598. https://doi.org/10.1111/iwj.14235

53

Investigating the agonistic and antagonistic effects of guaifenesin, flurbiprofen and dextromethorphan on TRP channels in cough receptors

Kenneth Bitrus David¹, Laura Sadofsky² and Oluwajoba Adegoke³

¹Kaduna State University; ²University of Hull; ³Reckitt

Introduction

Transient receptor potential (TRP) ion channels, including hTRPA1, hTRPM8, hTRPV1 and hTRPV4, are known to be involved in the mechanisms of cough reflexes [1]. These channels are activated by various stimuli, such as cold temperatures, chemicals and mechanical forces, making them potential targets for cough treatments. Despite their widespread use in over-the-counter cough medications, the precise mechanisms by which active pharmaceutical ingredients (APIs) like dextromethorphan, flurbiprofen and guaifenesin modulate these TRP channels remain unclear [2]. This study aims to elucidate the effects of these APIs on TRP channels to better understand their roles in cough suppression.

Methods

The study employed calcium signalling assays using FlexStation molecular devices to evaluate the effects of the APIs on hTRPA1, hTRPM8, hTRPV1 and hTRPV4 channels expressed in HEK293 cells. Changes in receptor-mediated calcium influx were measured following exposure to standard agonists (GSK1016790A, capsaicin, WS5, cinnamaldehyde) and varying concentrations of the APIs. Dose–response curves were plotted to determine the extent of modulation, with a focus on both agonistic and antagonistic effects.

Results

Dextromethorphan exhibited significant antagonistic effects across all TRP channels studied, inhibiting receptor activity by over 30%, with further inhibition observed at higher concentrations. For TRPM8, dextromethorphan reduced agonist-induced responses by up to 20% at 100 μM, with receptor desensitization occurring at concentrations between 300 μM and 1 mM. Flurbiprofen and guaifenesin also demonstrated antagonistic effects on most TRP channels but showed a slight augmentation (approximately 10%) of agonist effects on TRPM8.

Conclusion

The findings indicate that dextromethorphan, flurbiprofen and guaifenesin modulate TRP channels involved in cough mechanisms, with dextromethorphan showing the strongest antagonistic action. These results provide new insights into how these APIs contribute to the effectiveness of over-the-counter cough medicines, potentially influencing their therapeutic efficacy. Further research is necessary to explore the clinical implications of these interactions and their role in cough suppression.

References

1. Millqvist E. TRPV1 and TRPM8 in treatment of chronic cough. Pharmaceuticals (Basel) 2016;9(3):45. https://doi.org/10.3390/ph9030045.

2. Smith SM, Schroeder K, Fahey T. Over-the-counter (OTC) medications for acute cough in children and adults in community settings. Cochrane Database Syst Rev 2014;2014(11):CD001831. https://doi.org/10.1002/14651858.CD001831.pub5.

55

Adriamycin resistance observed in monocytic-like cells derived from AML-M5-iPSC upon integration of reprogramming transgenes

Amy Saik, Kit Li Kim, Pooi Pooi Leong and Soon Keng Cheong

Universiti Tunku Abdul Rahman (UTAR)

Introduction

We had previously generated AML-M5-specific-induced pluripotent stem cells (AML-M5-iPSCs) using THP-1 cells obtained from a patient [1]. These AML-M5-iPSCs were induced with specific growth supplements to enter haematopoietic differentiation, generating monocytic-like cells. We noticed that reprogramming transgenes Oct3/4, Sox2 and c-Myc were unintentionally integrated into the genome of AML-M5-iPSC during reprogramming. Out of scientific interest, the effects of reprogramming transgenes integration on drug responses to adriamycin in differentiated monocytic-like cells were investigated.

Method

AML-M5-iPSCs were differentiated as described in [1]. Phagocytotic activities of cells were quantified across 5-, 10-, 15-, 20- and 25-day post-differentiation. Carboxylate-modified red fluorescent latex beads were added at the ratio of 1:400 for 2 h followed by fluorescent quantification at 575 nm. Cytotoxicity of adriamycin on both cells were investigated with CCK-SK viability assay. Cells were seeded at 5000 cells/well and treated with 0.5, 1, 2 or 4 μM Adriamycin for 24 h. Semi-log growth–response curve was plotted to determine the IC₅₀ value. For cell apoptosis assay, after treatment with adriamycin for 24 h, cells were washed with ice-cold PBS before being incubated with Annexin-FITC and propidium iodide. The DNA contents were measured by flow cytometry. All assays were conducted in triplicate and n = 3. Statistical significance was determined using an ANOVA followed by a Tukey's post hoc test.

Results

The phagocytotic activity was similar between THP-1 and monocytic-like cells from day 5 to 25 post-differentiation, with no significant difference (P > 0.05) observed, suggesting that both cells were comparable functionally (Figure 1). However, the IC50 value determined for THP-1 cells after adriamycin treatment for 24 h was 0.59 μM, but the IC₅₀ value for monocytic-like cells could not be determined (Figure 2), suggesting that the monocytic-like cells were significantly (P < 0.05) more resistant to Adriamycin than the THP-1 cells. Upon similar treatment conditions, 92.5±3.9% of THP-1 cells entered late apoptosis stage. However, for monocytic-like cells, only 0.3% ± 0.2% entered late apoptosis stage, 37.2% ± 1.5% entered necrosis stage, and 62.5% ± 1.6% remained in viable stage (Figure 3).

Conclusion

Reference

1. Chiew MY, Boo NY, Voon K, Cheong SK and Leong PP. Generation of a MLL-AF9-specific stem cell model of acute monocytic leukemia. Leuk Lymphoma 2017;58(1):162-170.

71

Differences in uptake kinetics of clozapine, clozapine-N-oxide and N-desmethylclozapine into H9C2 rat cardiomyocytes

Ellen Kingston¹, Kathryn Burns¹, Nuala Helsby² and Malcolm Tingle¹

¹Department of Pharmacology and Clinical Pharmacology, The University Of Auckland; ²Department of Molecular Medicine and Pathology, The University of Auckland

Introduction

Clozapine is an atypical antipsychotic medication under-prescribed due to a high risk of serious adverse effects, including myocarditis, which is considered to be rare. However, a retrospective analysis in the United Kingdom identified an 11.9% incidence of confirmed antipsychotic-related myocarditis, with the majority on clozapine [1]. Our work identified that the ratio of the clozapine-N-oxide/N-desmethylclozapine metabolites is elevated in patients who develop clozapine-induced myocarditis and that cardio-selective CYP isoforms catalyse cycling of clozapine and clozapine-N-oxide [2]. This implicates clozapine metabolites in cardiotoxicity. Clozapine likely accumulates in cardiac tissue, although the transport of clozapine or its metabolites into cardiomyocytes is unknown. We aimed to elucidate the kinetics of clozapine, N-desmethylclozapine and clozapine-N-oxide transport into H9C2 rat cardiomyocytes.

Method

The time course (0–60 min) and kinetics of transport of clozapine, clozapine-N-oxide and N-desmethylclozapine into H9C2 cardiomyocytes were assessed at substrate concentrations (0–30 μM) at 37°C. Efflux was assessed following a 60-min uptake period followed by replacement with drug-free media and then further incubation up to 60 min. Passive diffusion into and out of cells at 4°C was also assessed. Intracellular concentrations were quantified with LCMS. Michaelis–Menten non-linear regression was used to estimate V_max and K_m. Assays were conducted in triplicate, and experiments were repeated (n ≥ 6).

Results

N-desmethylclozapine accumulated in H9C2 cardiomyocytes with threefold greater uptake than clozapine, with classical features of active transport kinetics at 37°C. In contrast, clozapine-N-oxide uptake was minimal and was linear (Figure 1A).

However, at 4°C, the intracellular concentration, particularly for N-desmethylclozapine, was increased compared with uptake at 37°C (Figure 1B). V_max increased to 23.4 (95% CI: 15.4–52.27) compared with 4.11 (95% CI: 3.35–5.63) pmol/10⁶ viable cells/min at 37°C (Table 1).

Upon drug withdrawal, the half-time to maximum efflux was N-desmethylclozapine (3 min) > clozapine (12 min) > clozapine-N-oxide (>60 min and linear). During the incubations, metabolic reduction of clozapine-N-oxide into clozapine was observed.

Conclusions

These combined data suggest that N-desmethylclozapine, in particular, is subject to a mixture of active uptake and efflux from H9C2 cardiomyocytes. In contrast, clozapine-N-oxide appears to undertake passive diffusion.

References

1. Segev A, Iqbal E, McDonagh TA, et al. Clozapine-induced myocarditis: electronic health register analysis of incidence, timing, clinical markers and diagnostic accuracy. Br J Psychiatry 2021;219(6):644-651. https://doi.org/10.1192/bjp.2021.58

2. Kingston E, Tingle M, Bellissima BL, Helsby N, Burns K. CYP-catalysed cycling of clozapine and clozapine-N-oxide promotes the generation of reactive oxygen species in vitro. Xenobiotica 2023;1-12. https://doi.org/10.1080/00498254.2023.2294473

73

Native and polyubiquitinated forms of dihydroceramide desaturase are differentially linked to human embryonic kidney cell survival

Mariam Alsanafi^1,2, Samuel Kelly^3,4, Karawan Jubair², Melissa McNaughton², Rothwelle Tate², Alfred Merrill^3,4, Susan Pyne² and Nijel Pyne²

¹Department of Pharmacy Practice, College of Pharmacy, Kuwait University; ²Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde; ³School of Biological Sciences, Georgia Institute of Technology; ⁴Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology

There is controversy concerning the role of dihydroceramide desaturase (Degs1) in regulating cell survival, with studies showing that it can both promote and protect against apoptosis. We have therefore investigated the molecular basis for these opposing roles of Degs1. Treatment of HEK293T cells with the sphingosine kinase inhibitor SKi [2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole] or fenretinide, but not the Degs1 inhibitor GT11 {N-[(1R,2S)-2-hydroxy-1-hydroxymethyl-2-(2-tridecyl-1-cyclopropenyl)ethyl]octanamide}, induced the polyubiquitination of Degs1 (Mr 40–140 kDa) via a mechanism involving oxidative stress, p38 mitogen-activated protein kinase (MAPK) and Mdm2 (E3 ligase). The polyubiquitinated forms of Degs1 exhibit ‘gain of function’ and activate prosurvival pathways, p38 MAPK, c-Jun N-terminal kinase (JNK) and X-box protein 1s (XBP-1s). In contrast, another sphingosine kinase inhibitor, ABC294640 [3-(4 chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide], at concentrations of 25–50 μM failed to induce formation of the polyubiquitinated forms of Degs1. In contrast to SKi, ABC294640 (25 μM) promotes apoptosis of HEK293T cells via a Degs1-dependent mechanism that is associated with increased de novo synthesis of ceramide. These findings are the first to demonstrate that the polyubiquitination of Degs1 appears to change its function from proapoptotic to prosurvival. Thus, polyubiquitination of Degs1 might provide an explanation for the reported opposing functions of this enzyme in cell survival/apoptosis.

82

The novel catecholamine 6-nitrodopamine potently induces release of intracellular calcium (Ca2+i) from human aortic smooth muscle cells: Comparison with dopamine, noradrenaline and adrenaline

José Britto-Júnior¹, Shuaihua Qiao¹, Ron Jacob¹, Gilberto De Nucci² and Albert Ferro¹

¹King's College London; ²University of São Paulo

Introduction

Mammalian and reptilian vascular tissues exhibit basal release of 6-nitrodopamine, which is reduced when the tissues are pre-incubated with the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester or when the endothelium is mechanically removed (1). The recently described endogenous catecholamine 6-nitrodopamine induces vasorelaxation in pre-contracted vascular rings by antagonizing the dopaminergic D2-like receptor. In the rat isolated heart, 6-nitrodopamine is significantly more potent as both a positive chronotropic and inotropic agent than noradrenaline, adrenaline and dopamine (2). Here, we compared the effect of 6-nitrodopamine on intracellular calcium (Ca2+i) levels in human aortic smooth muscle cells with that induced by the classical catecholamines.

Methods

Human aortic smooth muscle cells (HASMCs) at passage 3 or 5 were cultured in smooth muscle cell medium, supplemented with smooth muscle cell growth supplement, 5% fetal bovine serum and 20 units/ml penicillin/streptomycin, and were seeded at a density of >5 × 10⁵ cells/ml, followed by incubation for 48 h at 37°C in a 95% air/5% CO₂ atmosphere before live cell Ca2+i assay. For these experiments, HASMCs were loaded with 1 μM fura-2 AM for 45 min at 37°C, followed by washing, addition of 100 μl Hank's Balanced Salt Solution and excitation at 340 and 380 nm in a plate reader, all at 37°C (3).

Results

Incubation with 6-nitrodopamine (0.1 nM, Panel A), dopamine (100 nM, Panel D), noradrenaline (1 nM, Panel G) and adrenaline (100 nM, Panel J) did not alter intracellular calcium levels (Ca²⁺i) in HASMCs. However, incubation with concentrations of 6-nitrodopamine (0.3–1 nM, Panels B and C), dopamine (0.3–1 μM, Panels E and F), noradrenaline (3–10 nM, Panels H and I), and adrenaline (0.3–1 μM, Panels K and L) resulted in significant increases in Ca²⁺i in HASMCs.

Conclusion

References

1. Zatz R, De Nucci G. Endothelium-derived dopamine and 6-nitrodopamine in the cardiovascular system. Physiology 2024;39:44-59.

2. Britto-Júnior J, deOliveira MG, dosReis Gati C, et al. 6-Nitrodopamine is an endogenous modulator of rat heart chronotropism. Life Sci2022;307:120879.

3. Morgan AJ, Jacob R. Differential modulation of the phases of a Ca2+ spike by the store Ca2+-ATPase in human umbilical vein endothelial cells. J Physiol 1998;15:83-101.

89

Regulation of β2-adrenoceptor-mediated gene expression changes and wound healing in human bronchial epithelial Cells by cAMP-dependent protein kinase

Tamkeen Paracha¹, Omar Hamed², Varuna Jayasinghe¹ and Mark Giembycz¹

¹Department of Physiology & Pharmacology, University of Calgary; ²Ted Rogers Centre for Heart Research, Translational Biology and Engineering Program, University of Toronto

Introduction

β₂-Adrenoceptor-mediated cAMP signalling is extensively studied. However, current information regarding the functional roles of the various catalytic (C) subunits of cAMP-dependent protein kinase (PKA) is largely restricted to the α-isoform, Cα [1]. Indeed, another major variant, Cβ, is often neglected even though it is highly expressed in many tissues including airway epithelial cells [2]. Herein, we tested the hypothesis that Cα and Cβ play distinct functional roles in β₂-adrenoceptor-mediated gene expression changes and wound healing in human airway epithelial cells.

Methods

The human BEAS-2B airway epithelial cell line was used in all experiments. siRNA-mediated gene knockdown (KD) and CRISPR/Cas9-mediated gene knockout (KO) technologies were employed to explore the roles PKA-Cα and PKA-Cβ in formoterol-induced genomic responses in native cells and those harbouring a cAMP-response element (CRE) luciferase reporter gene. The impact of formoterol (1 nM) in a tissue repair model of mechanical injury was assessed in wild-type and KO mutants with the IncuCyte live cell imaging system using epidermal growth factor (EGF; 10 ng/ml) as a positive control.

Results

In CRE BEAS-2B cells, KD of PKA-Cα reduced reporter activation induced by high (>100 pM) but not low (<30 pM) concentrations of formoterol, whereas KD of PKA-Cβ had little effect. Simultaneous KD of both C subunits reduced reporter drive to a greater degree than either subunit alone (Figure 1A). Similar data were obtained in cells deficient in PKA-Cα and PKA-Cβ (Figure 1B,C). Formoterol-induced genomic responses were also inhibited in cells deficient in PKA-Cα. Indeed, a continuum of activity was observed across induced and repressed mRNAs alike, with changes in the expression of some transcripts being markedly inhibited by the deletion and others considerably less affected (Figure 2). In contrast, cells lacking PKA-Cβ had no impact on formoterol-induced gene expression changes. In the mechanical injury model, formoterol retarded constitutive wound closure, which was lost in clones lacking PKA-Cα but not PKA-Cβ (Figure 3).

Conclusion

References

1. Taylor SS, Søberg K, Kobori E, et al. The tails of protein kinase A. Mol Pharmacol 2022;101:219-225.

2. Hamed O, Joshi R, Mostafa MM, Giembycz MA. α and β catalytic subunits of cAMP-dependent protein kinase regulate formoterol-induced inflammatory gene expression changes in human bronchial epithelial cells. Br J Pharmacol 2022;179:4593-4614.

99

Constitutive internalization of the formyl peptide receptor 3 (FPR3) is mediated by the third transmembrane domain and GRK phosphorylation of and the C-terminal region

Christina Thomson, Dawn Thompson and James Hislop

University of Aberdeen

Introduction

The formyl peptide receptor family (FPR1-3) remain at the forefront of ‘resolution pharmacology’. FPR1 and FPR2 have been extensively studied and have well-defined roles in the inflammatory response and multiple diseases; however, little is known about FPR3. Thus, uncovering the molecular mechanisms that govern FPR3 behaviour may provide key insights into its function and therapeutic potential.

Methods

Experiments were performed in either HEK293 cells or those CRISPR/Cas9 edited to remove GPCR-regulated kinases (GRKs) or β-arrestins and transiently or stably expressing FPR2/FPR3 or mutant constructs. Internalization and ligand binding samples were assessed using confocal microscopy and flow cytometry. Interaction with either β-arrestin 1/2 or G-protein was determined by BRET [1] using luciferase-tagged FPR3 and signalling by bystander BRET [2]. Downstream signalling was measured by Western Blotting. Data are presented as the mean ± SEM (n = 3–4 independent experiments) and analysed by either one-way or two-way ANOVA followed by multiple comparisons t-tests or unpaired two-tailed t-tests using GraphPad 8.0 software.

Results

FPR3 was constitutively internalized in the absence of agonist unlike either FPR1 or FPR2 that reside on the surface. This constitutive internalization was accompanied with β-arrestin 2 recruitment, but not Gi coupling and dependent on GPCR-regulated kinases (GRKs). Importantly, although β-arrestins were associated with FPR3 in the absence of agonist, internalization was not inhibited in CRISPR/Cas9 edited cells. Mutation of putative C-tail phosphorylation sites (ΔABCD) attenuated constitutive internalization (** where P ≤ 0.01 vs. WT FPR3) and blunted β-arrestin recruitment (P ≤ 0.0001 vs WT FPR3) but did not enhance basal Gi coupling. However, after exposure to the endogenous peptide F2L, ΔABCD internalized coupled to Gi. Finally, mutation of residues in the third transmembrane domain of FPR3 (DAC), consistent with the previously reported ‘ionic lock’ region of GPCRs, prevented β-arrestin recruitment (P ≤ 0.05 vs. WT FPR3) but did not facilitate enhanced Gi coupling.

Conclusion

Taken together, these data have uncovered several unknown molecular mechanisms governing the molecular pharmacology of FPR3 that reveal critical information regarding regulation that could be harnessed for future therapeutics.

References

1. Wan Q, Okashah N, Inoue A, et al. Mini G protein probes for active G protein–coupled receptors (GPCRs) in live cells. J Biol Chem 2018;293(19):7466-7473. https://doi.org/10.1074/jbc.RA118.001975

2. Avet C, Mancini A, Breton B, et al. Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs. elife. 2022;11:e74101. https://doi.org/10.7554/eLife.74101

122

Affinity and selectivity of caffeine at the four human adenosine receptors: Potential impact for bronchopulmonary dysplasia

Emily Cash and Jillian Baker

University Of Nottingham

Introduction/Background and Aims

Bronchopulmonary dysplasia (BPD), the most common respiratory condition of prematurity, affects 45% of infants born at 29 weeks or earlier. High-dose caffeine (an adenosine receptor antagonist and phosphodiesterase inhibitor) is administered intravenously to neonates to reduce this risk [1]; however, its mechanism of action is unknown. Here, the affinity of caffeine was determined and compared with known adenosine receptor antagonists at the four human adenosine receptors (non-selective XAC, DPCPX for A1, SCH58261 for A2A, PSB603 for A2B, MRS1220 for A3) and its closely related compound theophylline.

Method/Summary of Work

CHO cell lines, each stably transfected with a CRE-SPAP reporter gene and one subtype of human adenosine receptor, were used [2]. NECA was the agonist throughout and K_d values calculated from parallel shifts of the NECA response as in [2].

Results

NECA stimulated an agonist response in all four adenosine receptor cell lines with stimulatory Gs-coupled responses at A2A (log EC₅₀ −7.30 ± 0.14, n = 43 and A2B −5.58 ± 0.09, n = 32) and Gi-coupled inhibition of forskolin-stimulated responses at adenosine A1 (log IC50 −8.65 ± 0.13, n = 23 and A3 −8.43 ± 0.10, n = 31). These NECA responses were inhibited by subtype-selective antagonists, shown in the table. XAC has a non-selective relatively high affinity at all receptor subtypes. Caffeine and theophylline were found to have non-selective, very low affinity for all four receptors (see following table). No response was seen to NECA in the parent CHO cell line stably expressing the CRE-SPAP reporter gene but no transfected receptor.

Conclusion

Caffeine is a non-selective low-affinity antagonist of all four human adenosine receptors. The therapeutic serum concentration of caffeine used in neonates is 5–20 mg/L (= 25–100 μM) [3]. Thus, adenosine receptor antagonism could be the potential mechanism of action of caffeine action in BPD although there is potential to develop higher affinity and/or selective antagonists that may have less side effects or developmental risks than current very high-dose caffeine.

References

1. Schmidt B, Roberts RS, Davis P, Doyle LW, Barrington KJ, Ohlsson A, Solimano A, Tin W. Caffeine therapy for apnea of prematurity. N Engl J Med 2006;354(20):2112-2121.

2. Baker JG, Hill SJ. A comparison of the antagonist affinities for the Gi- and Gs-coupled states of the human adenosine A1-receptor. J Pharmacol Exp Ther 2007;320(1): 218-228.

3. Natarajan G, Botica ML, Thomas R, Aranda JV. Therapeutic drug monitoring for caffeine in preterm neonates: an unnecessary exercise? Pediatrics 2007;119(5): 936-940.

145

FXR activation modulates Treg polarization and immune metabolism in hepatocellular carcinoma

Yasmeen Attia¹, Rasha Darwish¹, Aya Ali¹, Olfat Hammam² and Mohamed Elmazar¹

¹Department of Pharmacology, Faculty of Pharmacy, The British University in Egypt; ²Theodor Bilharz Research Institute

Introduction

The liver, once considered ‘immune-privileged’, harbours a dynamic immune landscape. In hepatocellular carcinoma (HCC), the tumour microenvironment is often immunologically ‘cold’, due to T regulatory cells (Tregs) that subdue antitumour immune responses. While the farnesoid X receptor (FXR) has gained attention for its ‘metabolic’ and ‘immunomodulatory’ roles in various settings, how it might impact Tregs in HCC remains unresolved. This study, therefore, aims to decipher whether FXR modulation, by obeticholic acid (OCA), can influence Treg polarization in a diethylnitrosamine (DEN)-induced HCC mouse model.

Method

Induction of HCC was performed in mice using DEN and carbon tetrachloride (CCl4). After 20 weeks, mice received 10 mg/kg/day of OCA for 84 days. Liver sections underwent histopathological examination besides alpha-fetoprotein (AFP) immunohistochemical analysis. To assess FXR activation, hepatic protein levels of CYP7A1, an FXR target gene, were measured. The impact of FXR activation on Treg polarization was explored by measuring hepatic gene expression of Treg markers, Foxp3 and IL-2RA. Additionally, hepatic levels of TGF-β1 and its signalling activity, p-SMAD2/3, along with IL-10 were measured by ELISA.

Results

AFP immunoreactivity and histopathological examination demonstrated OCA's potential to alleviate HCC in DEN-treated mice. OCA also curbed hepatic CYP7A1 levels confirming FXR activation. Consistent with an immunosuppressive tumour microenvironment, the DEN + CCl4 group demonstrated increased hepatic expression of the Treg markers, Foxp3 and IL-2RA, indicating Treg enrichment. Notably, OCA significantly reduced Foxp3 and IL-2RA expression, suggesting a decrease in the Treg population. Additionally, OCA treatment significantly reduced the levels of TGF-β1, p-SMAD2/3 and IL-10 in the liver, indicating a shift away from an immunosuppressive milieu.

Conclusion

Overall, FXR can likely orchestrate the metabolic fitness and function of Tregs within the tumour microenvironment. Targeting FXR represents a promising approach for interfering with the immunosuppressive environment in HCC and enhancing antitumour immunity.

147

Development of a chloride-sensitive BRET biosensor to measure the activity of KCC2 modulators in living cells in real time

Charles Lay and Steven Charlton

Omass Therapeutics

Introduction

In Rett syndrome, lower expression of the chloride exporter KCC2 leads to higher neuronal chloride concentrations and disruption of signalling. The development of KCC2 agonists has therefore been suggested as a treatment for this disease. While there have been advances in chloride imaging using FRET biosensors [1], there is no methodology amenable to high-throughput screening for drug discovery. In this study, a novel BRET-based biosensor was developed by fusing nanoluciferase to a chloride sensitive GFP mutant. The resulting biosensor termed glorider (glowing chloride biosensor) was then used to measure the activity of chloride modulators in living cells in real time.

Method

HEK jump in cells inducibly expressing KCC2 and stably expressing the glorider biosensor were plated in a white 384-well microplate with 0.08 ng/μl doxycycline and incubated for 24 h at 37°C and 5% CO₂. Media was removed from the plates and replaced with 20 μl HBSS with 1 in 250 diluted furimazine and 30 μM extracellular NanoLuc inhibitor. After a 20-min equilibration, an additional 20 μl of HBSS was added to the plate containing titrations of compounds of interest. The BRET signal was then measured at 440+/−30 and 535+/−30 nm for 80 min. The BRET ratio was then converted to intracellular chloride concentration using a standard curve generated in lysed cells.

Results

A range of chloride modulators were tested in the glorider system and were found to modulate cellular chloride levels (Figure 1 and Table 1). When KCC2 antagonist VU0463271 was applied to cells, the chloride levels were observed to increase as export of chloride was blocked; when a reported KCC2 agonist compound 12 [2] was applied to cells, the chloride level was reduced.

Conclusions

The glorider assay is capable of measuring changes in cellular chloride levels in real time in response to pharmacological modulation of chloride transporters. This assay is therefore amenable to screening for novel KCC2 agonists and other chloride-modulating compounds.

References

1. Sulis Sato S, Artoni P, Landi S, et al. Simultaneous two-photon imaging of intracellular chloride concentration and pH in mouse pyramidal neurons in vivo. PNAS 2017;114(41):E8770-E8779.

2. Jarvis R, Bürli RW. Fused pyrimidines as KCC2. International Patent WO2021180952. 2021.

158

Multi-coloured sequential resonance energy transfer for simultaneous ligand binding at G protein-coupled receptors

Brian Hudson¹, Alice Valentini², Bethany Dibnah¹, Marjia Ciba², Trond Ulven² and Elisabeth Rexen Ulven²

¹University of Glasgow; ²University of Copenhagen

Background

G protein-coupled receptors (GPCR) are the largest family of signalling proteins and most successful drug targets. Most GPCR drugs were produce effects through the orthosteric binding site; however, advances in structural biology have identified additional allosteric sites. Allosteric GPCR ligands modulate binding to the orthosteric site. There is a need for methods that measure how ligands binding to these different sites interact. We developed a novel approach to measure multiple ligand binding to the same receptor. We use this approach to gain insight into allosteric ligand interactions at the clinically relevant FFA1 free fatty acid receptor.

Methods

Flp-In T-REx 293 cells expressing the FFA1 receptor were used in all experiments. Calcium assays were employed to identify two novel FFA1 fluorescent tracer agonists binding to two distinct sites: (1) a red SulfoCy5 tracer binding to one site and (2) a green NBD tracer binding to a second site. Independent binding of these tracers to FFA1 was confirmed using NanoBRET, measuring energy transfer from a nanoluciferase (Nluc) fused to the receptor. To measure simultaneous binding of both tracers, sequential energy transfer from Nluc to the green tracer then on to the red ligand was measured. For all binding experiments, luminescent emissions for the Nluc, green and red tracers were measured using a CLARIOStar reader (BMG Labtech).

Results

In calcium assays, the red (pEC₅₀ = 6.47 ± 0.16; n = 3) and green (pEC₅₀ = 7.27 ± 0.15; n = 3) tracers were potent FFA1 agonists. Although the green tracer was more potent, when measuring affinity using NanoBRET, the red tracer (K_d = 200 nM; n = 3) had higher affinity than the green tracer (K_d = 390 nM; n = 3). When both ligands were added together, energy transfer from the Nluc to the green and ultimately the red tracer was observed. Red tracer emission was confirmed through luminescent emission consistent with the emission of SulfoCy5 (~690 nm). Data were fit to a saturation binding model to establish red ligand affinity for receptors with green ligand co-bound, indicating a K_d of 100 nM (n = 4). Kinetic analysis of binding indicated on and off rates for the red tracer decreased when the green tracer was bound (K_on = 92000 M⁻¹ min⁻¹, K_off = 0.11 min⁻¹; n = 3), compared to when the red tracer bound to receptors without the green tracer bound (K_on = 1,900,000 M⁻¹ min⁻¹, K_off = 0.24 min⁻¹; n = 3).

Conclusion

We have developed a novel approach using sequential energy transfer to measure simultaneous binding of ligands to two distinct sites on a GPCR. This has provided new insights into FFA1 ligand binding and will help to understand GPCR allosteric ligands.

163

Regulation and roles for cAMP-response element-binding (CREB)-regulated transcription coactivators (CRTCs) in β2-adrenoreceptor-mediated signalling and gene expression in airway epithelial cells

Priyanka Chandramohan, Mark A. Giembycz and Robert Newton

Department of Physiology and Pharmacology, and Lung Health Research Group, Snyder Institute of Chronic Diseases, Cumming School of Medicine, University of Calgary

Introduction

β₂-Adrenoceptor agonists (β₂A) bind β₂-adrenoceptors and lead to activation of cAMP-response element-binding (CREB)-regulated transcription coactivators (CRTCs) 1–3. CRTCs are suggested to interact with CREB and other basic leucine zipper (bZIP) transcription factors that bind to cAMP response elements (CRE) in gene promoters to drive β₂A-responsive gene transcription [1]. Despite their sequence homology, CRTC1–3 are believed to have distinct effects on gene expression through mechanisms that remain unclear. This study explores the roles and mechanisms by which CRTCs regulate β₂A signalling and transcription in airway epithelial cells.

Methods

Human BEAS-2B airway epithelial cells were treated for 1, 2, 6, 18 and 24 h with maximally effective concentrations of the long-acting β₂As, formoterol (10 mM) and indacaterol (100 nM), each solubilized in DMSO. Cells were harvested for western blotting and RNA-sequencing to characterize CRTC expression. siRNA-mediated silencing was used to assess the impact of the CRTCs on CRE-dependent transcription using CRE luciferase reporter cells. Nuclear translocation was assessed by sub-cellular fractionation and western blotting. All data are from ≥4 independent experiments, and significance was assessed with ANOVA. P ≤ 0.05 was taken as significant.

Results

mRNA (n = 4) and total protein expression (n ≥ 7) data showed that indacaterol and formoterol had minimal impact on CRTC1-3 expression in BEAS-2B cells for up to 18 and 24 h, respectively. Despite little change in total protein, formoterol caused a mobility shift to a lower molecular mass for the upper band of CRTC1 and CRTC2, as well as the single band of CRTC3 (n ≥ 4). Formoterol also induced nuclear translocation of CRTC1-3 within 10 min, with significant nuclear presence at 30 min for CRTC1 and CRTC3, and by 1 h for CRTC2 (n ≥ 4). Silencing of CRTC2 and CRTC3 each reduced formoterol-induced CRE-reporter activity by >70% (n ≥ 5), with combined knockdown having a more pronounced effect. CRTC1 silencing had minimal impact on CRE-reporter activity in preliminary experiments.

Conclusion

These findings indicate that CRTC2 and CRTC3, but likely not CRTC1, play significant, non-redundant roles in β₂A-induced CRE-dependent transcriptional activity. This raises the possibility that each CRTC could interact with distinct transcription factors and/or may contribute to gene-specific transcriptional responses. Since β₂A are key bronchodilators used in asthma treatments, an improved understand of their genomics effects may provide insights to improve therapeutic efficacy.

Reference

1. Altarejos JY, Montminy M. CREB and the CRTC co-activators: sensors for hormonal and metabolic signals. Nat Rev Mol Cell Biol 2011;12(3):141–151. https://doi.org/10.1038/nrm3072

173

Comparison of the affinity, duration of action and intrinsic efficacy of short-acting, long-acting and ultra-long-acting β2-agonists in clinical use

Richard Proudman and Jillian Baker

University of Nottingham

Introduction/Background and Aims

β-Agonists are widely used treatments for asthma and COPD. After the 1960s' development of short-acting β-agonists (SABAs) salbutamol and terbutaline, long-acting β-agonists (LABAs) were developed in the 1980s to allow better overnight symptom control and reduce frequency to twice-daily administration [1]. Recently, ultra-long-acting β-agonists (uLABAs) were developed that require once daily administration [1]. This study directly compared the molecular pharmacological properties of these compounds.

Method/Summary of Work

CHO cell lines stably expressing the human β2, β1 or β2-adrenoceptor with two point mutations (β2-H296K-K305D, the known salmeterol exocite [2]) and a CRE-SPAP reporter gene were used. Ligand affinity was assessed by 3H-CGP12177 whole-cell binding and duration via washout whereby short-acting ligand are readily washed out, giving a larger shift of the binding curve [2]. Carvedilol (with very little washout/shift) was used as a long-acting control. Function was determined using CRE-SPAP reporter assay.

Results

The affinity of 3H-CGP12177 was β2 0.16 ± 0.2 nM (157 ± 25 fmol/mg protein, n = 12), β1 0.42 nM (1146 fmol/mg protein [3]) and β2-H296K-K305D 0.13 ± 0.2 nM (332 ± 62 fmol/mg protein). As expected SABAs had low affinity and a short duration of binding (large log shift during washout; Table 1). LABAs and uLABAs had higher affinity and a longer duration, but not as long carvedilol. Agonist responses (Table 2) and efficacy ratios (K_D/EC₅₀; [3]) demonstrated a range of intrinsic efficacy. At the β2-H296K-K305D receptor (Table 3), the β2-affinity of vilanterol was reduced by 676-fold (salmeterol by 616-fold) with little change for any other compound.

Conclusion

There is considerable variation in the binding affinity, selectivity and intrinsic efficacy of current clinical β2-agonists. Indacaterol is highly efficacious but with only 35-fold β2-selectivity, while vilanterol and salmeterol have lower efficacy but are highly β2-selective (>1000-fold). No β2-agonist has as long a duration as carvedilol in this assay. Salmeterol and structurally related vilanterol use the same exocite for very high β2-selectivity, while the ligand–receptor interactions important for the β2-selectivity of other ligands (e.g. formoterol and olodaterol) remain unknown.

References

1. Baker JG, Shaw DE. Asthma and COPD: a focus on β-agonists – past, present and future, Handb Exp Pharmacol 2024;285:369-451.

2. Baker JG, Proudman RGW, Hill SJ. Salmeterol's extreme β2-selectivity is due to residues in both extracellular loops and transmembrane domains. Mol Pharmacol 2015;87:103-120.

3. Baker JG. The selectivity of β-adrenoceptor agonists at the human β1, β2 and β3 adrenoceptors. Br J Pharmacol 2010;160:148-161.

224

Unpicking the idiopathic pulmonary fibrosis (IPF) puzzle: Steroids and senescence

Rebecca Stinson, Laura Sadofsky and Simon Hart

Hull York Medical School, University of Hull

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease with an unknown aetiology, complex clinical needs and poor prognosis, typically affecting individuals aged over 65. With limited treatment options, median survival time after diagnosis is 2–5 years. Cellular senescence is linked to the natural ageing process; however, in IPF, senescence may be accelerated [1]. Interestingly, the PANTHER-IPF trial showed conventionally used corticosteroid treatments negatively impacted patient outcomes. Suggesting, corticosteroid treatments in IPF may accelerate fibrosis development through increased cellular senescence [2]. Thus, there exists a clinical need to identify the mechanisms involved in IPF and potential drug targets. Here, we focus on the extent of cellular senescence in response to corticosteroid treatments.

To investigate corticosteroid treatment effects on lung epithelial cells, A549 cells were treated with various concentrations of corticosteroids for 48 h. Cell area measurements determined dose dependent effect and optimum working concentrations. Subsequently, cells were treated for 48 h with either dexamethasone (DEX) (10 μM), aldosterone (100 μM), methylprednisolone (100 μM), etoposide, vehicle or DMEM. Additionally, to determine if corticosteroid treatment effects were reversible, widely used corticosteroid inhibitors were tested. Cells were treated with DEX ± RU486 or spironolactone (all 10 μM), inhibitors alone or vehicle for 48 h. Effects were measured by staining for senescence associated β-galactosidase (β-gal) expression.

Treatment of A549 cells with all corticosteroids resulted in a dose-dependent increase in cell size compared to control (n = 3). Similarly, all corticosteroid treatments caused an increased in β-gal expression compared to control, significantly methylprednisolone with a 4.27-fold increase (P = 0.0043) (n = 6). Introduction of corticosteroids inhibitors resulted in a reduction of β-gal expression compared to DEX alone, the mineralocorticoid inhibitor spironolactone resulted in a 25% reduction (P = 0.0086), while the glucocorticoid inhibitor RU486 resulted in a 59% reduction (P < 0.0001) (n = 6).

Cells treated with corticosteroids showed characteristic morphological changes and increased β-gal expression, which could be partially reversed through inhibitor treatment, particularly the glucocorticoid inhibitor. Our findings suggest that susceptible epithelial cells can be induced into a senescent state when treated with corticosteroids; however, this can be limited through inhibitor addition. Future work will aim to further develop this characterization of cellular senescence, considering potential profibrotic and pro-inflammatory effects of corticosteroids in lung fibrosis.

References

1. Han S, Lu Q, Liu X. Advances in cellular senescence in idiopathic pulmonary fibrosis (review). Exp Ther Med 2023:25(4):145. https://doi.org/10.3892/etm.2023.11844.

2. Idiopathic Pulmonary Fibrosis Clinical Research Network. Raghu G, Anstrom KJ, King TE Jr, Lasky JA, Martinez FJ. Prednisone, azathioprine, and N-acetylcysteine for pulmonary fibrosis. N Engl J Med 2012;366(21):1968-77. https://doi.org/10.1056/NEJMoa1113354.

232

Inhibition of neuroinflammation by artemisinin in BV-2 microglia activated with polyinosinic:polycytidylic acid.

Victoria Iwuanyanwu and Olumayokun Olajide

University of Huddersfield

Introduction

During neuroinflammation, microglia tend to produce excess amounts of pro-inflammatory mediators. This may result in neuronal apoptosis. Studies have established a link between neuroinflammation and the pathogenesis of some viral infections, thus providing reliable targets for treating this CNS condition [1]. Research suggests that the antimalarial drug artemisinin has anti-inflammatory properties [2]. This study aimed to evaluate the modulatory effects of artemisinin in polyinosinic:polycytidylic acid [poly (I:C)]-activated BV-2 microglia.

Method

Cultured BV-2 microglia cells were pre-treated with artemisinin (1.25, 2.5, 5 and 10 μM) followed by a 24-h stimulation with 20 μg/ml of poly (I:C). Levels of nitrite in culture supernatants were measured using Griess assay. Levels of pro-inflammatory cytokines (IL-6 and TNF-α) were analysed using mouse ELISA (Invitrogen). Protein expressions of phosphorylated IκB and NF-κB-p65 were also determined using ELISA (Cell Signaling). Protein levels of iNOS and phospho-p38 MAPK were determined using western blotting. NF-κB-mediated gene expression was determined using transient transfection and luciferase reporter gene assay (Promega). Data were analysed using ANOVA with Tukey's post hoc test for multiple comparisons.

Results

Results showed that IL-6 and TNF-α production was significantly increased (P < 0.001) when cells were stimulated with 20 μg/ml of poly (I:C) compared to negative control cells. However, artemisinin (1.25, 2.5, 5 and 10 μM) significantly (P < 0.01) inhibited TNF-α and IL-6 secretion by ~ 0.18-fold, ~0.3-fold, ~ 0.6-fold and ~ 0.8-fold reduction and ~10%, ~15%, ~20% and ~35%, respectively. Poly (I:C)-induced an increase in iNOS protein expression (P < 0.001). Pre-treatment with artemisinin (1.25, 2.5, 5 and 10 μM) decreased iNOS by ~70%, ~65%, ~60% and ~55%.

Furthermore, poly (I:C) significantly (P < 0.001) up-regulated phospho-IκB expression. However, artemisinin (1.25, 2.5, 5 and 10 μM) markedly (P < 0.01) produced 55 %, 60 %, 50 % and 40 % reduction. Artemisinin produced 1.25 μM ~0.6-fold, 2.5 μM ~0.4-fold, 5 μM ~1.0-fold and 10 μM ~1.1-fold reduction of p65 phosphorylation. Also, DNA binding results showed a ~1.0-fold, ~1.8-fold, ~2.3-fold and ~3.5-fold reduction. Transactivation of NF-κB showed a 1.25 μM ~1.5-fold, 2.5 μM, ~1.6-fold, 5 μM ~3.0-fold and 10 μM ~3.5-fold reduction. Pre-treatment with artemisinin produced a marked reduction (P < 0.05) in p-p38 protein level by 1.25 μM ~0.4-fold decrease, 2.5 μM ~0.8-fold decrease, 5 μM ~1.0-fold decrease and 10 μM ~1.2-fold decrease, respectively.

Conclusion

Results of this study suggests that reduction in polyinosinic:polycytidylic acid-induced inflammatory responses by artemisinin may have the potential to be re-purposed as adjuncts in the treatment of viral infections.

References

1. Cheng Y, Sun F, Wang L, et al. Virus-induced p38 MAPK activation facilitates viral infection. Theranostics 2020;10(26):12223-12240.

2. Xie K, Li Z, Zhang Y, Wu H, Zhang T, Wang W. Artemisinin and its derivatives as promising therapies for autoimmune diseases. Heliyon. 2024;10(7):e27972.

246

Novel insights into the effect of SGLT-2 inhibitor empagliflozin on hepatic damage in diabetic obese Zucker rats

Stefania Melini¹, Claudio Pirozzi¹, Federica Comella¹, Filomena Del Piano², Nicola Opallo¹, Alana Aragon Herrera³, Nicole Pia Navatti¹, Giuseppina Mattace Raso¹, Oreste Gualillo⁴, Francisca Lago Paz³ and Rosaria Meli¹

¹Department of Pharmacy, University of Naples Federico II; ²Department of Veterinary Medicine and Animal Productions, University of Naples Federico II; ³Cellular and Molecular Cardiology Unit and Department of Cardiology, Institute of Biomedical Research of Santiago de Compostela (IDIS-SERGAS); ⁴SERGAS (Servizo Galego de Saude) and NEIRID Lab (Neuroendocrine Interactions in Rheumatology and Inflammatory Diseases), Research Laboratory 9, IDIS (Instituto de Investigación Sanitaria de Santiago)

Introduction

The sodium-glucose cotransporter 2 inhibitor empagliflozin (EMPA) is a hypoglycaemic drug, considered a useful pharmacological tool for patients with type 2 diabetes mellitus (T2D), cardiovascular disease and other metabolic disorders [1]. Several evidence have demonstrated the efficacy of EMPA in improving liver disease in patients with T2D [2,3]. However, its effects in counteracting metabolic-associated fatty liver disease are poorly understood.

Method

Our study aimed to evaluate the impact of a 6-week EMPA treatment on hepatic dysfunction observed in diabetic obese Zucker diabetic fatty (ZDF) rats. Glucose, lipid metabolism and inflammatory and pro-fibrotic markers were evaluated in the liver of ZDF mice by real-time PCR and western blot analysis. Histological and serological evaluations were also performed. Statistical t-test analysis was performed for all obtained results (at least n = 6 each group).

Results

We found that EMPA induced the activation of hepatic insulin signalling and contextually counteracted gluconeogenesis process (Figure 1). Consistently, EMPA-treated animals showed a lower degree of steatosis accompanied by a trend of increased glycogen content, confirming gluconeogenesis reduction and a greater hepatic storage of glycogen (Figure 2). Therefore, EMPA improved hepatic lipid metabolism altered in ZDF rats, increasing the phosphorylation of AMPK and differently modulating key mediators of fatty acid metabolism and catabolism (Figure 1). EMPA also increased the expression of the uncoupling protein (UCP)2 and the mitochondrial transporter ATP-binding cassette (ABCG)1, suggesting an improvement of hepatic mitochondrial functions compromised in diabetic rats (Figure 3). Then, we demonstrated EMPA effect against hepatic inflammation and fibrosis associated with insulin resistance (Figure 4). In this context, a fascinating scenario opens up on the potential effect of EMPA as a pro-resolving agent, since the increased immune cell recruitment combined with the induction of resolvins including annexin A1 (Figure 4), an important factor involved in inflammatory resolution in different pathologies including diabetes and obesity. Finally, in the liver of EMPA-treated mice, we found a different transcriptional and protein expression of SGLT-2, suggesting the involvement of other converging mechanisms beyond the pharmacologically established one (Figure 5).

Conclusions

References

1. Frampton JE. Empagliflozin: a review in type 2 diabetes. Drugs 2018;78(10):1037-1048. https://doi.org/10.1007/s40265-018-0937-z

2. Abdelgani S, Khattab A, Adams J, et al. Empagliflozin reduces liver fat in individuals with and without diabetes. Diabetes Care 2024;47(4):668-675. https://doi.org/10.2337/dc23-1646

3. Aragón-Herrera A, Feijóo-Bandín S, Otero Santiago M, et al. Empagliflozin reduces the levels of CD36 and cardiotoxic lipids while improving autophagy in the hearts of Zucker diabetic fatty rats. Biochem Pharmacol 2019;170:113677. https://doi.org/10.1016/j.bcp.2019.113677

248

Oleoylethanolamide attenuates cardio-renal damage secondary to obesity in mice: Translational perspectives for cardiovascular–kidney–metabolic (CKM) syndrome

Federica Comella¹, Alana Aragon Herrera², Nicola Opallo¹, Stefania Melini¹, Adriano Lama¹, Filomena Del Piano³, Nicole Pia Navatti¹, Evaristo Di Napoli³, Rosaria Meli¹, Francisca Lago Paz², Giuseppina Mattace Raso¹ and Claudio Pirozzi¹

¹Department of Pharmacy, University of Naples Federico II; ²Cellular and Molecular Cardiology Unit and Department of Cardiology, Institute of Biomedical Research of Santiago de Compostela (IDIS-SERGAS); ³Department of Veterinary Medicine and Animal Productions, University of Naples Federico II

Introduction and Method

The pathophysiological interrelationship between obesity and diabetes with chronic kidney disease and cardiovascular disorders has been conceptualized as cardiovascular–kidney–metabolic (CKM) syndrome [1]. Metabolic alterations, particularly obesity, cause derangements of the heart and kidney, including insulin resistance (IR), lipotoxicity, inflammation and fibrosis [2]. Oleoylethanolamide (OEA), a noncanonical endocannabinoid and peroxisome proliferator-activating receptor (PPAR)-α agonist, has been extensively studied for its metabolic properties [3]. This study aimed to investigate the beneficial effects of OEA (2.5 mg/kg i.p. daily for 8 weeks) on high-fat diet (HFD)-induced cardio-renal damage secondary to obesity and metabolic syndrome in C57Bl6/J male mice. One- or two-way ANOVA analyses of variance were performed for all obtained results (at least n = 5–6 animals each group).

Results

In obese animals, OEA treatment improved the metabolic pattern, limiting weight gain compared to untreated mice and reducing IR, as shown by in vivo oral glucose tolerance test (Figure 1). OEA treatment restored serum creatinine and BUN, markers of tubular function, which were altered by HFD feeding. OEA also reduced heart weight and serum creatine kinase-myocardial band, a marker of cardiac damage (Figure 2). As known, obesity-driven release of cytokines, chemokines and pro-fibrotic mediators contributes to tissue damage progression in both heart and kidney. OEA exerted a marked anti-inflammatory and antifibrotic effect as showed by haematoxylin and eosin and Masson's trichrome histological staining of cardiac and renal tissues, further confirmed by the reduced transcription of pro-inflammatory and pro-fibrotic markers in both tissues (Figures 3–5). Furthermore, OEA modified cardiac lipid profile of obese animals as demonstrated by metabolomic analysis of different lipids including triglycerides, glycerophospholipids and sphingomyelins; consistently, OEA normalized cardiac metabolic factors reducing the expression of fatty acid translocase CD36 and regulating glucose homeostasis by activating AMPK/AKT/AS160 pathway (Figure 6), which converges in GLUT4 increased transcription. Likewise, OEA improves renal lipid metabolism, reducing the diacylglycerol O-acyltransferase (DGAT)1, which regulates triglycerides' trafficking. Finally, in heart and kidney, OEA significantly increased PPAR-α transcription altered by HFD, suggesting its possible direct involvement in OEA cardio-renal protective effects.

Conclusions

These results indicate that OEA may be a promising molecule for restraining CKM alterations associated with obesity and following metabolic disorders.

References

1. Ndumele CE, Neeland IJ, Tuttle KR, et al. A synopsis of the evidence for the science and clinical management of cardiovascular-kidney-metabolic (CKM) syndrome: a scientific statement from the American Heart Association. Circulation 2023;148(20):1636-1664. https://doi.org/10.1161/CIR.0000000000001186.

2. Al-Chalabi S, Syed AA, Kalra PA, Sinha S. Mechanistic links between central obesity and cardiorenal metabolic diseases. Cardiorenal Med 2024;14(1):12-22. https://doi.org/10.1159/000535772.

3. Bowen KJ, Kris-Etherton PM, Shearer GC, West SG, Reddivari L, Jones PJH. Oleic acid-derived oleoylethanolamide: a nutritional science perspective. Prog Lipid Res 2017;67:1-15. https://doi.org/10.1016/j.plipres.2017.04.001.

270

Characterization of differential Shh-mediated activation of SMO receptor variants implicated in basal cell carcinoma and their inhibition by SMO receptor antagonists

Elvira Diamantopoulou, Giles Brown, Steven Charlton, Ali Jazayeri and Karolina Gherbi

OMass Therapeutics

Introduction

The Smoothened (SMO) receptor plays a central role in the highly conserved Hedgehog (HH) signalling pathway. Abnormal activation of the HH pathway can lead to a number of pathological conditions, and SMO antagonists have been approved for the treatment of basal cell carcinoma (BCC) (vismodegib, sonidegib) and acute myeloid leukaemia (glasdegib) [1]. However, observed resistance to these first-generation SMO inhibitors has been linked to various SMO receptor mutations in BCC, with mutations either affecting the ligand binding pocket or causing constitutive receptor activation, and has instigated the development of second-generation SMO inhibitors [2]. SMO receptor activation is repressed by Patched 1 (PTCH1) and its endogenous ligand Sonic Hedgehog (Shh) with Shh binding causing PTCH1 repression of SMO to be lifted to activate SMO. Here, we have determined the Shh-driven SMO receptor drive for eight SMO variants and have further profiled the inhibitory effects of first- and second-generation SMO receptor antagonists at those receptor variants to differentiate these antagonists in their potential ability to combat SMO resistance.

Methods

SMO knock-out mouse embryonic fibroblasts (MEF-SMO−/−) were transduced with BacMam virus containing the desired hSMO variant sequence. Transduced cells were treated with antagonists (1-h pre-incubation) and mShh, before being incubated for 24 h at 37°C in a humidified 95% air/5% CO₂ atmosphere. After 24 h, the Cells-to-CT™ 1-Step TaqMan® kit was used as per manufacturer's instructions in conjunction with TaqMan probes to detect Gli1 and Gusb genes. Four experimental replicates (two cell replicates, two qPCR replicates) were performed for each condition tested, and data were analysed using the CFX Maestro software (Bio-Rad).

Results

Differential baseline Gli1 mRNA levels and mShh potencies and efficacies have been determined for human SMO-WT and a panel of receptor variants (Table 1). Using equi-effective mShh for each receptor variant, IC₅₀ values were determined for SMO antagonists against eight receptor variants. The activating receptor variant SMO-W535L showed no further increase in Gli1 mRNA levels in the presence of mShh; however, interestingly, all SMO antagonists tested against SMO-W535L in the presence and absence of mShh showed weaker inhibition in the presence of mShh.

Conclusions

These data show differential Shh-mediated receptor drive for BCC relevant SMO variants and further highlight the importance of considering Shh concentrations when determining the pharmacological profile of improved SMO inhibitors for BCC.

References

1. Jing J, Wu Z, Wang J, et al. Signal Transduct Target Ther 2023;8(1):315.

2. Sharpe HJ, Pau G, Dijkgraaf GJ, et al. Cancer Cell 2015;27(3):327-341.

285

Effects of omega-3 on human coronary vascular tone induced by Neurotransmitters

Gaelle Merheb^1,2, Hichem Badji¹, Zhipeng Li¹, Dan Longrois^1,3, Marianne Abifadel^1,2 and Xavier Norel¹

¹Université Paris Cité and Université Sorbonne Paris Nord, INSERM, LVTS, F-75018, Paris, France; ²LBTM, Faculty of Pharmacy, Pôle Technologie-Santé, Saint Joseph University, Beirut, Lebanon; ³AP-HP, Hôpital Bichat-Claude Bernard, Department of Anesthesia and Intensive Care, Université Paris Cité, Paris, France

Background

Coronary artery diseases (CAD) are characterized by chronic inflammation and increased production of neurotransmitters such as serotonin (5-HT) and acetylcholine. Inflammatory processes also raise the levels of pro-inflammatory lipid mediators, such as prostaglandin E₂ (PGE₂) and thromboxane A₂ (TxA₂), contributing to vascular dysfunction through enhanced vasoconstriction. Using data from the GOED (Global Organization for EPA and DHA) clinical study database, we examined the role of omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and their derivatives, specialized pro-resolving mediators (SPMs), in resolving inflammation in cardiovascular diseases. Recent findings from our group show that DHA and its metabolites (resolvin D1, resolvin D5 and maresin 1) reduce PGE2-induced contractions in human coronary arteries (HCA).¹ On the other hand, RvD5 and Mar1 production by human vagus nerve has been measured²; their impact on the cardiac neuronal system remains unexplored.

Aims

This study aims to investigate the impact of omega-3s on the release and effects of neurotransmitters, such as acetylcholine and 5-HT, in HCA using insights from the GOED database.

Methods

HCA were isolated from human hearts (n = 6, obtained with ethic committee agreement and informed consent of the patient) post-transplantation at Bichat Hospital and placed in an organ bath system. Electrical field stimulation (EFS) at varying voltages was used to induce neurotransmitter release, before and after 1 h of incubation with DHA or EPA (0.1 mM). To assess the effect after 1 or 18 h of incubation with DHA, EPA or docosapentaenoic acid (DPA) (0.01 mM) on exogenous neurotransmitters, dose–response curves with 5-HT and acetylcholine were generated. Vascular tone was analysed using the Iox software.

Results

EFS induced voltage-dependent contractions in HCA. These contractions were partly blocked by tetrodotoxin (10 μM), indicating a neuronal component. DHA (0.1 mM) reduced the contractions induced by stimulations at 10 and 30 V by 56% and 31%, respectively. Exogenous 5-HT and acetylcholine also induced contractions. After 18 h of DHA incubation, acetylcholine-induced vasoconstrictions were reduced, while 5-HT-induced contractions remained unaffected by DHA, EPA or DPA.

Conclusion

Our results suggest that omega-3s, particularly DHA, may influence neuronal activity in HCA, opening the potential of new therapeutic strategies for cardiovascular diseases.

References

1. Bouhadoun A, Manikpurage HD, Deschildre C, et al. DHA, RvD1, RvD5, and MaR1 reduce human coronary arteries contractions induced by PGE2. Prostaglandins Other Lipid Mediat 2023;165:106700. https://doi.org/10.1016/j.prostaglandins.2022.106700

2. Serhan CN, de laRosa X, Jouvene CC. Cutting edge: human vagus produces specialized proresolving mediators of inflammation with electrical stimulation reducing proinflammatory eicosanoids. J Immunol 2018;201(11):3161-3165. https://doi.org/10.4049/jimmunol.1800806

14

Ethylacetate extract of Chlorophytum alismifolium improves serum magnesium levels and retinal histomorphology in diabetic rats

Abdulhakim Abubakar¹, Abdullahi Balarabe Nazifi², Abdulazeez Jimoh¹, Fatima Ismail Hassan¹, Matthew Ijarafu Michael¹, Ikram Ezzeldin Abdulrahman¹, Halima Muhammad Shehu¹, Rabiu Nuhu Danraka¹, Abdulmumin Zaid Abubakar¹ and Idris Maje Mohammed¹

¹Department of Pharmacology and Therapeutics; ²Bayero University Kano

Introduction

Chlorophytum alismifolium is widely used in the management of diabetes mellitus and its complications [1]. Diabetic retinopathy (DR) encompasses a complex pathology and a leading cause of blindness [2]. The pathogenesis of DR is complex, and mechanisms are also implicated in its development [3]. This study focused on the role of ethylacetate extract of Chlorophytum alismifolium (EACA) in improving serum magnesium levels and histomorphology of the retina in diabetic rats.

Methods

Diabetic retinopathy was evaluated after induction of hyperglycaemia in Wistar rats using streptozotocin. The serum magnesium levels were evaluated after the administration of EACA at the doses of 150, 300 and 600 mg·kg⁻¹ using diagnostic kits followed by the histology of the retina [4,5]. Statistical significance was established at P < 0.05 using ANOVA followed by Bonferroni's post hoc test.

Results

Induction of diabetes significantly (P < 0.05) reduced the serum magnesium level in the diabetic rats compared to the normal control. The EACA at 150 and 600 mg·kg⁻¹ significantly (P < 0.05) elevated the levels of serum magnesium in comparison to the diabetic control (Table 1). Examination of retinal sections of the diabetic rats showed severe distortion of the outer and inner cells of the retinal layers. Distortions were also observed in the epithelial and the ganglionic layers. Treatment with EACA at the dose of 600 mg·kg⁻¹ showed slight distortion of the outer and inner cells layers with preservation of the epithelial and ganglionic layers (Plate I).

Conclusions

The findings showed that of ethylacetate extract of Chlorophytum alismifolium ameliorates diabetic retinopathy by improving serum magnesium levels and retinal histomorphology.

References

1. Abubakar, A., Nazifi, A.B., Maje, I.M., Tanko, Y., Anuka, J.A. and Abdurahman, E.M. (2021a). Antihyperglycaemic activity of ethylacetate extract of Chlorophytum alismifolium in type 2 diabetes. The involvement of peroxisome proliferator activated receptor-γ and dipeptidyl peptidase-4 Journal of Integrative Medicine, 19 (1), 78-84.

2. Whitehead, M., Wickremasinghe, S., Osborne, A., Van Wijngaarden, P., and Martin, K. R. (2018). Diabetic retinopathy: A complex pathophysiology requiring novel therapeutic strategies. Expert Opinion on Biological Therapy, 18(12), 1257-1270.

3. Kowluru, R. A., Kowluru, A., Mishra, M. and Kumar, B. (2015). Oxidative stress and epigenetic modifications in the pathogenesis of diabetic retinopathy. Progress in Retinal and Eye Research, 48, 40-61.

4. Halim E. M. and Ali H. (2002). Reversal of diabetic retinopathy in streptozotocin induced diabetic rats using Indian anti-diabetic plant Azadirachta indica (L). Indian Journal of Clinical Biochemistry, 17(2) 115-123.

35

Bombax ceiba flower petals (BC): A natural modulator of MAPK/NF-κBp65/TNF-α and Nrf-2/HO-1 signalling in myocardial ischaemia-reperfusion injury

Anil Kumar Sahu, Vipin Kumar Verma, D. Sarya and Jagriti Bhatia

AIIMS, New Delhi

Introduction

Myocardial infarction (MI) is a consequence of sudden diminished blood supply to the heart leading to ischaemia and necrosis of the affected cardiac tissue. Paradoxically therapeutic reperfusion also causes damage to the heart tissue due to production of reactive oxygen species (ROS). This study was conducted to evaluate the potential effects and underlying mechanisms of BC (an important medicinal plant with potent antioxidant and cardioprotective properties) on myocardial ischaemia-reperfusion (IR) injury in rats.

Method

Healthy adult male albino Wistar rats (n = 30) were randomly assigned into 4 groups: sham (n = 6), IR-control (n = 8), BC-400 + IR (n = 8) and BC-400 per se (n = 8), respectively. The study followed ethical approval (221/IAEC-1/2019) and INSA-CPCSEA guidelines (Registration No. 10/GO/ReBi/S/99/CPCSEA) for animal care. BC was administered orally daily for 28 days. On the 29th day, rats were anaesthetized using pentobarbitone sodium (60 mg·kg⁻¹ i.p.), and ischaemia was developed on the 29th day by the occlusion of the LAD coronary artery for 60 min, followed by reperfusion for 60 min, and simultaneously, haemodynamic parameters were recorded. The rats were then sacrificed, their hearts excised, and further biochemical, inflammatory, morphological and molecular studies were done [1]. Statistics were determined using one-way ANOVA with Tukey-Kramer post hoc tests, analysed with GraphPad Prism 10.

Results

Pretreatment with BC 400 mg·kg⁻¹ dose significantly improved the ventricular function such as +LV dp/dt (1990.14 ± 19.35 vs. 1707.75 ± 26.18 mm of hg/sec) (P ≤ 0.001), −LV dp/dt (1663.50 ± 23.31 vs. 1353.22 ± 17.16 mm of hg/sec) (P ≤ 0.001), LVEDP, serum CK-MB and LDH levels as compared to IR-control group (Figures 1 and 2 and Table 1). Significant anti-oxidant properties were observed via an increase in levels of GSH and SOD and decrease in MDA (Table 1). Histopathological analysis showed preserved morphology (Figure 3). The TUNEL assay revealed decreased DNA fragmentation. Further, expressions of Bax were reduced while Bcl2 expressions increased, and inhibition of Nrf-2/HO-1 pathways was observed. Additionally, a reduction in the level of inflammatory markers (TNF-α and IL-6) was also observed. Also, inhibition of the MAP kinase and NF-κB pathway was observed.

Conclusion

In summary, pretreatment of rats reduced myocardial injury by preserving redox balance and modulating inflammatory responses and key pathways (MAPK/NF-κBp65/TNF-α and Nrf-2/HO-1), indicating BC's promise for myocardial infarction and ischaemia-reperfusion injury.

Results

Reference

1. Verma VK, Malik S, Mutneja E, Sahu AK, Bhatia J, Arya DS. Attenuation of ROS-mediated myocardial ischemia–reperfusion injury by morin via regulation of RISK/SAPK pathways. Pharmacol Rep. 2020; 72:877-89.

63

Betulin, a compound isolated from Crinum asiaticum bulbs exerted anti-silicosis and pulmonoprotective effects through the inhibition of NF-κB activation in a rat model

Michael Ofori¹, Cynthia Amaning Danquah², Joshua Asante² and Williams Adu Asamoah³

¹Dr Hilla Limann Technical University; ²Kwame Nkrumah University of Science and Technology; ³Sunyani Technical University

Introduction

Silicosis is a lung disease with no effective treatment, causing shortness of breath, cough, fever and blue skin. Betulin (BET), isolated from Crinum asiaticum bulbs (CAE), has potent pharmacological effects. This study examines the anti-silicosis and pulmonoprotective effects of betulin and CAE in a rat model, examining the mechanism for reducing silicosis in crystalline silica-induced silicosis. Rats were examined closely for morphological alterations before being sacrificed. The lungs were taken for biochemical and histological examination.

Method

In this study, anti-silicosis and pulmonoprotective effects of BET and CAE were investigated after rat models were subjected to lung injury through an intratracheal administration of crystalline silica. Rats were critically observed for morphological changes and were sacrificed. Lungs were harvested for biochemical and histological analysis.

Results

The results showed that CAE and BET reduced significantly (****P < 0.0001) the levels of nuclear factor kappa-B (NF-κB), tumour necrosis factor alpha (TNF-α), interleukin-1B (IL-1β), interleukin-6 (IL-6), hydroxyproline and collagen types I and III when compared with the negative control group. On bronchoalveoli lavage fluid (BALF) biomarkers such as macrophages, lymphocytes, monocytes and neutrophils, CAE and BET were able to reduce their levels significantly (****P < 0.0001). The CAE and BET were investigated for their anti-oxidant activity and were shown to increase the levels of catalase (CAT) and superoxide dismutase (SOD) while lowering the level of malondialdehyde (MDA). There was also an improvement in lung function when lung tissues were examined histologically.

Conclusion

67

Investigating the effects of cannabidiol (CBD) and 7-hydroxy-cannabidiol (7-OH-CBD) on the regeneration of Lumbriculus variegatus

Georgeena Jomy, Benjamin Williams, Megan Flanagan, Grace Hawkes, James McRobbie-Aston, Nia Davies, Lisa Wallace and Aidan Seeley

Swansea Worm Integrative Research Laboratory (SWIRL), Swansea University

Introduction

Cannabidiol (CBD) is a non-psychoactive cannabinoid from Cannabis sativa which is metabolised to 7-hydroxy-cannabidiol (7-OH-CBD) in humans [1]. Here, we examine the behavioural and regenerative effects of CBD and 7-OH-CBD in the regenerative Annelid worm, Lumbriculus variegatus, which are exempt from the Animal (Scientific Procedures) Act 1986.

Methods

CBD and 7-OH-CBD were dissolved in 100% DMSO or methanol, respectively, before dilution in artificial pond water [2] for a solvent concentration of 0.5%. Toxicity was determined by exposure of L. variegatus to 0–25 and 0–15 μM, respectively, for 24 h with tissue pallor and/or tissue decomposition used as identifiers of toxicity. Effects of 24-h exposure to 0–5 μM CBD or 7-OH-CBD on locomotor activity and tactile stimulation to elicit stereotypical behaviours was conducted as previously described [2]. Effects of 0–5 μM CBD and 7-OH-CBD on the regenerative capacity of L. variegatus determined by bisection of L. variegatus and quantification of tissue growth, using a Nikon Nikon SMZ1270i stereomicroscope, up to 72-h post-amputation (HPA).

Results

CBD and 7-OH-CBD displayed toxicity in 50% of the test population at 14.12 μM (95% CI: 12.28–15.90 μM, n = 6) and 11.29 μM (95% CI: 10.53–12.09 μM, n = 6), respectively. 24-h exposure to CBD decreased tactile stimulation response to elicit body reversal at ≥2.5 μM (P < 0.05, n = 8) and helical swimming at ≥0.5 μM (P < 0.05, n = 8), while 7-OH-CBD only inhibited these responses at 5 μM (P < 0.05, n = 8). 7-OH-CBD has no observed effect on locomotor activity of L. variegatus (P > 0.05, n = 8) while 24-h exposure to 5 μM CBD resulted in 54.88 ± 11.23% decrease in locomotor activity (P = 0.002, n = 8). Moreover, we observed that exposure to 7-OH-CBD had no effect on regenerative capacity of L. variegatus (P > 0.05, n = 18) while CBD was shown to have no effect on regeneration of L. variegatus anterior tissue (P > 0.05, n = 18) but ≥2.5 μM decreased tail regeneration (P < 0.05, n = 18).

Conclusion

We demonstrate that CBD and 7-OH-CBD is toxic to L. variegatus at >5 μM and that exposure to both compounds can reduce the response to tactile stimulation, with only CBD affecting locomotor activity. Moreover, ≥2.5 μM CBD was also shown to significantly impair tail regenerative capacity in L. variegatus suggesting a potential role of an endocannabinoid-like system in this model organism.

References

1. Zhang, Q. et al. (2024) Pharmacokinetic variability of oral cannabidiol and its major metabolites after short-term high-dose exposure in healthy subjects. Med Cannabis Cannabioids; 7(1). Doi: https://doi.org/10.1159/000535726.

2. Seeley, A. et al. (2021) Lumbriculus variegatus: A novel organism for in vivo pharmacology education. Pharmacol. res. Perspect; 9:e00853. https://doi.org/10.1002/prp2.853.

79

The propagating effects of griseofulvin on erectile dysfunction: A comprehensive computational and molecular docking study on human phosphodiesterase 5 proteins (1UDT and 1UDU)

John Shinggu, Emmanuel Etim, Samuel Humphrey and Bulus Bako

Federal University Wukari

Erectile dysfunction (ED) is a prevalent condition affecting a significant portion of the male population. This research delves into the potential link between Griseofulvin, a known antifungal medication, and its impact on erectile function. A comprehensive computational approach was employed. Optimization of griseofulvin was carried out using the highly reputable density functional theory (DFT) with the B3LYP functional and 6-31*G(d,p) using water and ethanol as the solvents of interest. We explored the interactions of Griseofulvin with Human Phosphodiesterase 5 proteins (PDE5), specifically targeting the crystal structures 1UDT and 1UDU. Molecular docking studies provided valuable insights into the binding mechanisms of Griseofulvin with PDE5, shedding light on potential allosteric modulation and conformational changes. Further molecular docking studies were carried out on other popular antifungal drugs like amphotericin, terbinafine and ketoconazole in order to compare their interactions with 1UDT and 1UDU with that of griseofulvin. Through an array of computational analyses, including molecular dynamics simulations and binding free energy calculations, we aimed to elucidate the propagating effects of Griseofulvin on the catalytic activity and structural stability of PDE5. The findings from this research could contribute to a deeper understanding of the molecular mechanisms underlying Griseofulvin's impact on erectile function, potentially opening avenues for the development of novel therapeutic interventions for ED.

114

Wound healing potential of quercetin by modulating glycaemic levels, lipid profile and improving oxidative status in type 2 diabetes

Habiba Oussedik-Oumehdi and Lilia Mouhouche

Laboratory of Cellular and Molecular Biology-Tamayouz, University of Science and Technology Houari Boumediene (USTHB)

Introduction

Type 2 diabetes (T2D) accounts for 90%–95% of diabetes cases, with a higher prevalence in developed countries. This chronic disorder, marked by elevated glucose levels, generates excessive reactive oxygen species, impairs cellular function and exacerbates insulin resistance [1]. These factors contribute to complications like diabetic foot ulcers, which results from impaired wound healing, increased infection risk, leading to higher incidence of lower limb amputations [2]. Natural compounds with anti-inflammatory and antioxidative properties could help to manage T2D and its complications, including diabetic wounds. This study investigates the effects of quercetin, a bioactive flavonoid, on glycaemic control, lipid metabolism, oxidative stress and wound healing in a murine model of T2D.

Method

Male Balb/c mice (n = 25) were injected intraperitoneally with streptozotocin (60 mg·kg⁻¹) and nicotinamide (110 mg·kg⁻¹) to induce T2D. They were divided into five groups (n = 5 per group): control, untreated diabetic and three diabetic groups receiving, by oral route, quercetin at 10, 20 and 50 mg·kg⁻¹·day⁻¹ for 16 days, following a full thickness excision skin wound (6 × 6 mm) on the shaved backs under anaesthesia (ketamine 80 mg·kg⁻¹; xylazine 10 mg·kg⁻¹, intraperitoneally). Blood glucose levels and glucose tolerance were assessed at the end of the treatment. Serum lipid profiles (cholesterol, triglycerides, LDL and HDL) were measured. Wound healing was assessed over the 16-day period, and oxidative stress markers (H₂O₂, NO, GSH) were measured in skin tissue. Data were analysed using ANOVA and post hoc tests.

Results

Quercetin, mainly at 50 mg·kg⁻¹·day⁻¹, significantly reduced fasting blood glucose levels (202.8 ± 54.88 mg·dL⁻¹ vs. 459.5 ± 71.97 mg·dL⁻¹ in the untreated diabetic group, P < 0.01) and enhanced glucose tolerance, as indicated by lower glucose levels throughout the glucose tolerance test, particularly at the 120-min mark (257.4 ± 73.9 mg·dL⁻¹ vs. 494.6 ± 8.64 mg·dL⁻¹, P < 0.01). Triglycerides and LDL levels were significantly lowered (2.17 ± 0.34 mg·dL⁻¹ vs. 3.43 ± 0.79 mg·dL⁻¹, P < 0.001 and 0.83 ± 0.24 mg·dL⁻¹ vs. 1.15 ± 0.13 mg·dL⁻¹, P < 0.01, respectively), while HDL levels increased (0.68 ± 0.19 mg·dL⁻¹ vs. 0.41 ± 0.05, P < 0.05). In addition, redox status was improved. Results indicated decreased H₂O₂ and NO levels and increased GSH levels (P < 0.05). Quercetin, at 50 mg·kg⁻¹, also accelerated wound healing, yielding the most significant effect (P < 0.01) (Table 1).

Conclusion

Quercetin showed a promising therapeutic potential in diabetes management, by improving glycaemic control, lipid metabolism, oxidative stress and wound healing. These findings suggest its potential use as a treatment for diabetic foot and other complications associated with impaired wound healing in T2D.

References

1. Dhanya R. Quercetin for managing type 2 diabetes and its complications, an insight into multitarget therapy. Biomed Pharmacother; 2022, 146: 112560. https://doi.org/10.1016/j.biopha.2021.112560

2. Jeffcoate WJ, Harding KG. Diabetic foot ulcers. Lancet Lond Engl; 2003, 361(9368):1545-1551. https://doi.org/10.1016/S0140-6736(03)13169-8

154

Metabolic and calcium modulatory effect of ethanol fraction of Parquetina nigrescens in pancreatic β-cells.

Fatimoh Ojuade¹, Joanne Roberts², Steven Patterson¹ and Sharron Dolan¹

¹Department of Biological and Biomedical Sciences, Glasgow Caledonian University; ²Department of Applied Science, Glasgow Caledonian University

Introduction/Background and Aims

Global access to diabetes medicines is inequitable. As such, natural products are a mainstay of treatment in many countries. Parquetina nigrescens (PN) is indigenous to West Africa and traditionally used for managing type 2 diabetes (T2D). Crude extract of PN has previously been shown to ameliorate hyperglycaemia in an animal model of T2D [1]; however, the underlying mechanisms are unresolved. This study assessed the secondary metabolites present in the ethanolic fraction of Parquetina nigrescens (EFOPN), their effects on digestive enzymes and pancreatic β-cell insulin release and calcium dynamics.

Methods

PN leaves were collected in Nigeria and authenticated at the University of Ilorin (voucher number: UILH/01/019/876). EFOPN was prepared by fractionating crude extract in 80% ethanol and concentrated in rotary evaporator. Characterization of phytochemical constituents was carried out using standard procedures and liquid chromatography-mass spectrometry (LC-MS). Inhibitory effects of EFOPN (0.02, 0.2 mg·mL⁻¹) on alpha-amylase, alpha-glucosidase and pancreatic lipase were evaluated. Insulin secretory effects of EFOPN were measured at basal (1.1 mM) and stimulatory (16.7 mM) glucose concentrations in a rat INS-1 832/13 β-cells and quantified by insulin ELISA. Cell loaded with FURA2-AM was used to determine intracellular calcium responses to EFOPN alone and in the presence of specific calcium channel blockers.

Results

Phytochemical analysis of EFOPN identified alkaloids, flavonoids, steroids, phenolics and tannins. LC-MS characterization confirmed the presence of rutin (flavonol), apigenin and luteolin (flavone) (0.024, 0.13 and 0.092 mg·g⁻¹ of standard, respectively). EFOPN significantly (P ˂ 0.0001) inhibited α-amylase, α-glucosidase and pancreatic lipase (Figure 1). EFOPN (0.2 mg·mL⁻¹) significantly (P ˂ 0.0001) increased insulin secretion by 5.02- and 4.68-fold at basal and stimulatory glucose levels, respectively, compared to control (Figure 2). Under stimulatory glucose, EFOPN (0.02 and 0.2 mg·mL⁻¹) significantly (P ˂ 0.0001) increased intracellular calcium by 58.0% and 86.4%, respectively, compared to KCl (30 mM) which was considered 100% response. This effect was inhibited by pre-incubation with verapamil and SN-6 (P ˂ 0.001) but not thapsigargin, SKF-96365 and mibefradil (Figure 3).

Conclusion

Ethanol fraction of PN may reduce glycaemia via inhibition of digestive enzymes. PN may also have additional glycaemic lowering effects via enhancing pancreatic β-cell insulin release, modulated by opening of L-type calcium channels and an increase in reverse mode action of calcium-sodium exchanger to elevate intracellular calcium.

Reference

1. Ojuade FI, Olorundare OE, Akanbi OB, Afolabi SO, Njan AA. Antidiabetic and antihyperlipidemic effects of aqueous extract of Parquetina nigrescens in streptozotocin–nicotinamide induced type 2 diabetic rats. Heliyon. 2021;7(6). https://doi.org/10.1016/j.heliyon.2021.e07363

166

Investigations on hepatoprotective activity and HPTLC analysis of fractions of Erythroxylum monogynum methanolic leaf extract on paracetamol induced hepatic damage

Ajay Namdeo¹ and Sabeena Syed²

¹Department of Pharmaceutical Sciences, Hemwati Nandan Bahuguna Garhwal Central University, Srinagar; ²School of Pharmacy, Vishwakarma University

Introduction

Erythroxylum monogynum Roxb. (Erythroylaceae) (E. monogynum) is a well-known plant in traditional medicine found in southern parts of India. We have scientifically reported the hepatoprotective action of methanolic extract of leaves of E. monogynum (MEEM) [1]. The present study was aimed to isolate the active fraction(s) of (MEEM) by fractionation and screening of the different fractions thereof for hepatoprotective action against paracetamol induced hepatotoxicity in rats. The active fraction obtained was further analysed by HPTLC technique.

Methods

Wistar albino rats weighing 200–250 g of either sex were maintained under standard conditions of temperature (24 ± 2°C) and relative humidity (55 ± 5%) under 12 h light/dark cycles. They were fed with standard pellet diet and water ad libitum. The animal studies were approved Institutional Animal Ethics Committee (CPCSEA/38/2014).

Methanolic extracts of leaves of E. monogynum were given in doses of 100, 200 and 400 mg·kg⁻¹ for 7 days and toxicity was induced by paracetamol (2 mg·kg⁻¹) on Day 8. Silymarin (50 mg·kg⁻¹) was used as reference standard. After 24 h of toxicity induction, blood samples were collected from retro-orbital plexus and analysed for serum parameters like serum glutamic pyruvic transaminase, serum glutamic oxaloacetate transminase, alkaline phosphatase and total bilirubin. Liver isolated were studied for histopathological changes.

Fractions derived from (MEEM) (Pet. ether, chloroform and hydroalcoholic) were screened for hepatoprotective activity. Doses of 100 and 200 mg·kg⁻¹ of different fractions administered for seven days, and on 8th day, toxicity by paracetamol was induced. Levels of biochemical markers along with histopathological changes were monitored to evaluate the extent of hepatoprotection after 24 h of toxicity induction.

Results

Phytochemical analysis of (MEEM) showed the presence of carbohydrates, flavonoids, phenols and saponins. Prior administration of this extract restored the elevated levels of serum markers as compared to toxic group which is also confirmed by histopathological changes observed.

A significant decrease in the biochemical parameters was evident by the hydroalcoholic fraction as compared to the toxic group which is also confirmed by histopathological changes observed. HPTLC analysis of hydroalcoholic fraction confirmed the presence of rutin, a flavonoidal glycosides besides other phytochemicals.

Conclusions

References

1. Sabina S. Syed and Ajay G. Namdeo, Hepatoprotective effect of leaves of Erythroxylum monogynum Roxb. on paracetamol induced toxicity. Asian Pacific Journal of Tropical Biomedicine 2013; 3(11):877-881.

2. Domitrovic R, Jakovac H, Marchesi VV, Knezevic SV, Cvijanovic O, Tadic Z, et al. Differential hepatoprotective mechanism of rutin and quercetin in CCl4 intoxicated BALB/Cn mice. Acta Pharmacol Sin 2012;33:1260-70.

169

Phytoestrogens remodel the gene expression landscape in blood of healthy rats

Barbara Stefanska, Cayla Boycott, Yuexi Ma, Huiying Amelie Zhang and Tony Yang

UBC

Introduction

The haematopoietic function declines with age, which leads to the deterioration of immune and metabolic functioning. Hence, blood may reflect systemic effects of ageing as demonstrated in transcriptomics, proteomics and metabolomics studies [1]. Transcriptomic profiling of human longevity and biological age in blood samples has identified differentially expressed genes that are involved in regulation of the immune system, inflammatory processes and metabolic pathways [1]. Importantly, polyphenols from the phytoestrogen group, including pterostilbene (PTS), have been shown to exert effects on gene expression profiles in blood of patients with obesity/diabetes and heart disease, affecting anti-inflammatory pathways. However, it remains unknown whether phytoestrogens impact the landscape of gene expression in blood in a healthy state and what processes may be affected.

Method

In the present study, we conducted RNA sequencing to determine transcriptome-wide changes in gene expression in whole blood of healthy rats consuming diets supplemented with phytoestrogens as compared with rats on control chow diet (n = 5/group). Ortholog cell deconvolution was applied to analyse the omics data.

Results

We discovered that PTS leads to differential changes in the gene expression landscape of blood. PTS-target genes (FDR threshold less or equal 0.05; absolute fold change greater than 2) are associated with functions counteracting impaired autophagy, mitochondrial dysfunction and loss of proteostasis. These are major processes that emerge in the immune system and drive age-related decline of the immune functions, leading to low-grade chronic inflammation, so-called inflammaging, in the blood and peripheral tissues. In turn, inflammaging results in disturbances in metabolism, including immune cell metabolism, which further exacerbates inflammatory responses. Most changes in gene expression induced by PTS are linked to a decreased inflammatory response, particularly of the innate immune system and interferon pathway. Indeed, many interferon-related genes were found to be down-regulated by PTS in this study (e.g., Oas1a, Oas2, Irf7, Ifi27, Lgals3bp). Importantly, PTS-mediated changes in gene expression functionally show anti-inflammatory effects through multiple pathways, including immunometabolism where changes in cellular metabolism (e.g., ribosome biogenesis) impact the immune system.

Conclusions

Our findings provide a rationale for pre-clinical and clinical longevity studies and encourage investigations on PTS in maintaining cellular homeostasis, decelerating the process of ageing, and improving conditions with chronic inflammation.

Reference

1. Mitchell CA, Verovskaya EV, Calero-Nieto FJ, Olson OC, Swann JW, Wang X, et al. Stromal niche inflammation mediated by IL-1 signalling is a targetable driver of haematopoietic ageing. Nat Cell Biol. 2023;25:30-41.

219

Effect of Cleistopholis patens stem bark extract on paracetamol-induced hepatotoxicity in Sprague Dawley rats

George Owusu, Baaba Musah, Gilbert Nyamedi, Caren Achiaa Fosu and Kwabena Kyereh Amofa

University of Energy and Natural Resources

Introduction/Background/Aim

Despite advancements in medicine, treating acute liver injuries still needs to improve. Due to unwanted side effects associated with orthodox medications, herbal products are being explored as supplementary or alternative to the conventional treatment of liver diseases [1]. This study explores the ameliorative effect of hydroethanolic leaf extract of Cleistopholis patens stem bark extract (CPE) on paracetamol-induced liver injury in Sprague Dawley rats.

Method

Rats were put into I–VII groups (n = 5): I (10 mL·kg⁻¹ saline), II (10 mL·kg⁻¹ saline), III (100 mg·kg⁻¹ silymarin), IV (silymarin + 50 mg·kg⁻¹ CPE) and V–VII (100, 200 and 400 mg·kg⁻¹ CPE). Silymarin and CPE were administered (PO/OD) from day 0 to day 6. From day 4 to day 6, hepatotoxicity was induced in groups II–VII by daily oral administration of paracetamol (3 g·kg⁻¹). On day 7, rats were sacrificed, and their blood and livers were collected. Serum levels of AST, ALT, albumin, globulin, total protein, total bilirubin and direct bilirubin were assessed. A portion of the liver was homogenized to assess oxidative stress biomarkers while the other was sectioned for histopathology [2]. Qualitative phytochemical tests and FTIR were performed to identify the secondary metabolites and their functional groups in the extract [3].

Result/Discussion

Compared to the disease control, CPE significantly reversed the paracetamol-induced changes in levels of liver enzymes and proteins (P < 0.05) (Table 1). Also, normal levels of GSH, GPX, SOD, CAT and MDA in the hepatocytes of CPE-treated rats were restored (Table 2). These biochemical findings were corroborated by the histopathology which shows reduced damage to the hepatocytes of CPE-treated rats compared to the control (Figure 1). Alkaloids, saponins, flavonoids and tanning were tested positive in the extract. The main functional groups identified from the FTIR were as follows: –OH (alcohol), C–O stretching, C = C stretching (aromatic ring), C = C (alkene) and C–H stretching. The extract may contain benzylic alcohol such as phenols (Figure 2).

Conclusion

It is evidenced that Cleistopholis patens extract ameliorates paracetamol-induced hepatoprotection in rats. However, further studies with different models of liver damage are needed to understand the precise molecular and biochemical mechanisms involved and to confirm its therapeutic benefit in humans.

References

1. Lancaster EM, Hiatt JR, Zarrinpar A (2015) Acetaminophen hepatotoxicity: An updated review. Arch Toxicol. 89: 193-199.

2. Abirami A, Nagarani G, Siddhuraju P (2015) Hepatoprotective effect of leaf extracts from Citrus hystrix and C. maxima against paracetamol-induced liver injury in rats. Food Science and Human Wellness 4: 35-41

3. Balamurugan V, Fatima SMA, Velurajan S (2019) A guide to phytochemical analysis. IJARIIE. 5 (1): 2395-4396

239

Novel synthetic isoindolinones target protein kinases and promotes cellular growth

Finley Cockshott², Luiz Pollo³, Fiona Healy¹, Maique Biavatti³ and Vanessa Marensi¹

¹University of Liverpool; ²University of Chester; ³Universidade Federal de Santa Catarina

Molecules derived from natural products provided a breakthrough in many areas of pharmacology and still play a pivotal therapeutic role in many pathologies. Isoindole derivatives proved to be of much therapeutic use, including anti-inflammatory and anti-cancer activity. 1-Isoindoline scaffolds are bioactive molecules naturally present in plants. It can be fully synthesized, generating synthetic molecules of potential therapeutic use [1]. This study identifies the intracellular targets of four novel isoindolinones derived from isoindole scaffold and proposes therapeutic application.

Three isoindolinones with side chains and one isoindolinone fused to a tetrahydropirane ring by a spiro carbon as synthesized from the 1-isoindoline scaffold using Castro-Stephens and Sonogashira methodologies. UV spectra were recorded with a PDA/UPLC H-class system. NMR chemical shifts were acquired, and LC-HRESIMS spectra were measured with a Waters Xevo G2-S QToF mass spectrometer coupled to a UPLC H-class system, using C18 column. Phase separation was performed by column chromatography (CC) and solid-phase extraction (SPE). Kinase binding prediction was performed using SwissTargetPrediction (http://www.swisstargetprediction.ch/). The first 100 hits were considered and analysed. A table was created, and results were plotted in GraphPad. Kinase activity was validated using 33P ATP assay (Kinase Profiling Unit, University of Dundee), and 20 μM of each 1-isoinloline was used. Results were analysed and plotted in GraphPad. Cell proliferation as performed using CFSE and analysed by flow cytometry (attune) and colony forming assay was measured using light microscope.

Novel isoindolinones derivates were synthesized and the most likely protein target was predicted. Protein kinases were unanimously the most likely substrate, suggesting these isoindoles act by targeting signalling cascades. JAK and stress kinases were among the highest hits. Cyclization of R1 and R2 predicts loss of affinity for kinase. Most targets fall in the CMGC (including cyclin-dependent kinases [CDKs], mitogen-activated protein kinases [MAP kinases], glycogen synthase kinases [GSK] and CDK-like kinases and tyrosine kinases families).

In vitro analysis of the kinase activity identified and confirmed JAK3, JNK1/3, p38B and HIPK3 were down-regulated by the selected 1-isoindiline, whereas spleen associated tyrosine kinase (SYK), insulin receptor-related receptor (IIR), tousled like kinase 1 (TLK1) and ephrin type-A receptor 4 (EPH-A4) were among the enzymes with highly up-regulated activity. Viability was increased in cell treated with all four molecules derived from 1-isoindoline scaffolds, suggesting that this molecule have potential to provide cell regeneration and improve survival, morphology and growth.

This study identifies novel synthetic isoindolinones that provides growth advantage and potential use in regenerative medicine.

Reference

1. Upadhyay, S. P., Thapa, P., Sharma, R. & Sharma, M. 2020. 1-Isoindolinone scaffold-based natural products with a promising diverse bioactivity. Fitoterapia, 146, 104722.

273

Phosphodiesterase inhibitory effect of diosmetin—A flavonoid in thyme extract for possible antispasmodic mechanism

Sara Naqvi^1,2, Syed Hani Abidi³, Najeeb Ur Rehman Rehman⁴, Dr Iqbal Azhar¹ and Amber Palla⁵

¹Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Sciences, University of Karachi; ²Faculty of Pharmacy, Iqra University North Campus; ³Department of Biomedical Sciences, Nazarbayev University School of Medicine, Astana, Kazakhstan; ⁴Department of Pharmacology and Toxicology, College of Pharmacy, Prince Sattam Bin Abdulaziz University, Al Kharj, Saudia Arabia; ⁵Department of Biological and Biomedical Sciences, The Aga Khan University, Karachi, Pakistan

Introduction

Functional gastrointestinal disorders (FGID) are cluster of gastrointestinal (GI) symptoms [1] manifested by diarrhoea and/or constipation and usually triggered by anxiety. Management of gut motility disorders is primarily symptomatic, often requiring life-long management and therefore adding to the non-compliance. Natural products are known to have ‘effect enhancing and side-effect neutralizing’ potential. However, often their effectiveness is not scientifically appreciated due to lack of pharmacological validation. Amongst the natural products, Thymus vulgaris belongs to the family Lamiaceae commonly known as thyme has been used in management of gut motility [2] and anxiety [3]. However, limited pharmacological evidence is available regarding its spasmolytic mechanisms. The current study was aimed to understand the mechanism of spasmolytic effect of thyme and one of its active constituents diosmetin which is a methoxyflavone compound.

Methods

The hydro alcoholic extract of thyme (Tv.Cr) was analysed through reverse-phase high pressure liquid chromatography (RP-HPLC). Spasmolytic mechanisms were studied ex vivo on isolated rabbit jejunum. In vivo studies on Tv.Cr and diosmetin were done to confirm the antidiarrheal effect and mechanisms. Phosphodiesterase receptor's interactions were studied with diosmetin using the swissprot online software and visualized with the BIOVIA Discovery Studio Visualizer.

Results

RP-HPLC analysis confirmed the presence of diosmetin in Tv.Cr. Ex vivo studies showed that Tv.Cr and diosmetin relaxed the spontaneously contracting rabbit jejunal tissues completely at 5 mg·mL⁻¹ and 1*10⁻⁴ μM, respectively. Tv.Cr also relaxed Carbachol (CCh; 1 μM), low K+ (25 mM) and high K+(80 mM)-mediated contraction. The CCh-mediated contraction was further explored for the involvement of phosphodiesterase (PDE) receptors' involvement. Tv.Cr shifted the isoprenaline curves to the left dose-dependently, similar to the standard PDE inhibitor rolipram. The possible role of diosmetin in PDEI effect was explored using ligand–protein interaction using autodocking tools that showed the inhibitory effect of PDE-4B isozyme. Inhibition of PDE4 isozyme. This indicates the possible role of diosmetin in relaxing the gut muscles through PDE-4b enzyme inhibition, warranting its use as an antidiarrheal. Tv.Cr and diosmetin also protected the BALB/c mice against castor oil-induced diarrhoea. This mechanism was attributed to reduction in GI motility in a charcoal meal assay in BALB/c mice. This PDEI is mediated by diosmetin with binding affinity of −6.141 Kcal·mol⁻¹, which was similar to the standard PDEI rolipram with −6.141 Kcal·mol⁻¹.

Conclusions

Tv.Cr effectiveness in reducing gut motility is evident by its antidiarrheal activity and reduced intestinal transit in BALB/c mice. One of the possible antispasmodic mechanisms of Tv.Cr is by PDE inhibition.

References

1. Sperber, A. D., Bangdiwala, S. I., Drossman, D. A., Ghoshal, U. C., Simren, M., Tack, J., … Fukudo, S. (2021). Worldwide prevalence and burden of functional gastrointestinal disorders, results of Rome Foundation Global Study. Gastroenterology, 160(1), 99-114. e113.

2. Che, C.-T. (2018). Review of Duke's handbook of medicinal plants of the bible. ACS Publications.

3. Komaki, A., Hoseini, F., Shahidi, S., & Baharlouei, N. (2016). Study of the effect of extract of Thymus vulgaris on anxiety in male rats. Journal of traditional and complementary medicine, 6(3), 257-261.

274

Neuroprotective effects of Pistacia terebinthus L. leaves

Gokay Albayrak¹, Halil Koyu¹, Fadime Aydin Kose², Elif Alan Albayrak³ and Sinem Ezgi Turunc Ozoglu²

¹Department of Pharmaceutical Botany, Faculty of Pharmacy, Izmir Katip Celebi University; ²Department of Biochemistry, Faculty of Pharmacy, Izmir Katip Celebi University; ³Department of Pharmacology, Faculty of Pharmacy, Ege University

Introduction

Pistacia terebinthus L. (Pt) leaves have been traditionally used to enhance memory function [1]. Additionally, various extracts from the Pistacia species have demonstrated antioxidant and anti-cholinesterase properties [2]. However, the neuroprotective potential of the leaves of Pt has not been evaluated comprehensively.

Methods

Air-dried leaves (50 g) were extracted using a 50:50 H₂O:EtOH mixture in an ultrasonic bath for four cycles, each lasting 3 h. The resulting extracts were suspended in water and partitioned sequentially with n-hexane (Pth), chloroform (Ptc) and n-butanol (Ptb) in a 1:2 ratio. The components of Pth, Ptc, Ptb, the water fraction and the total extract (Ptt) were analysed via LC-MS/MS. The inhibitory activities against tyrosinase and acetylcholinesterase-butyrylcholinesterase were determined spectrophotometrically, while antioxidant activities were measured through in vitro (DCFDA, SH-SY5Y cells) and ex vivo (chemiluminescence in mice brain tissue) assays. Sirtuin expression levels were analysed by real-time PCR, and statistical significance was determined using ANOVA (n = 5).

Results

Major phenolic compounds, including gallic acid, tannic acid, quinic acid and protocatechuic acid, were identified. Pth, Ptc and Ptb were selected for chemiluminescence experiments due to their superior antioxidant, anti-tyrosinase and anti-cholinesterase activities (Tables 1 and 2). All three fractions significantly mitigated pyrogallol-induced oxidative stress in chemiluminescence assays (P < 0.01) (Figure 1). Furthermore, relative mRNA expression levels of sirtuin1 and sirtuin3 were notably increased in Pth (5 μg·mL⁻¹) compared to the control (1% DMSO).

Conclusion

In conclusion, Pth, Ptc and Ptb fractions exhibited significant neuroprotective effects, which may be attributed to their high phenolic content. These findings highlight the potential of Pistacia terebinthus as a source of bioactive compounds for neuroprotection.

Acknowledgements: This study is supported by the Scientific Research Projects Coordinatorship of Izmir Katip Celebi University (Grant number: 2022-GAP-ECZF-0015).

References

1. Hayta S, Polat R, Selvi S. Traditional uses of medicinal plants in Elazıǧ (Turkey). J Ethnopharmacol. 2014;154(3):613–23.

2. Moeini R, Memariani Z, Asadi F, Bozorgi M, Gorji N. Pistacia genus as a potential source of neuroprotective natural products. Planta Med. 2019;85(17):1326–50.

300

Alpha glucosidase inhibitory activities of extracts of Carpobrotus edulis (L.) bolus and Sclerocarya birrea (A. Rich) Hochst. subsp. caffra (Sond.) Kokwaro

Thandokuhle Gama and Motlalepula Matsabisa

University of The Free State

Introduction

Diabetes mellitus is a chronic disease in which the body is unable to regulate blood glucose levels, leading to abnormally high glucose levels in the blood. The plants Carpobrotus edulis and Sclerocarya birrea are traditionally known to be used by different cultural groups to combat diabetes. The plant extracts were investigated for their alpha glucosidase inhibitory activity.

Methods

C. edulis plant material (leaves and stems, juice and roots) from Bloemfontein, South Africa, was extracted in 70% ethanol/water (yields: 22.57%, 5.79% and 4.91%, respectively). S. birrea bark was extracted in 95% ethanol/water and ethanol separately (yields: 8.23% and 9.09%, respectively). Phytochemical analysis tests were performed according to a method described by Pandey and Tripathi (2014) [1]. Qualitative analysis of the plant extracts was performed using thin-layer chromatography and high-performance liquid chromatography. Cytotoxicity tests of the extracts were performed on the HEK-293 human kidney normal cell line, at concentrations 25, 50, 100 and 200 μg·mL⁻¹. Doxorubicin was used as the positive control. Alpha glucosidase inhibition assays were performed at concentrations 0.25, 0.5, 1, 2, 5 and 10 μg·mL⁻¹, using acarbose as the positive control. The results were corrected for using the vehicle control.

Results

C. edulis plant material was found to contain the phytochemicals: flavonoids, tannins, phenols, steroids, terpenoids and alkaloids, while S. birrea contained saponins, tannins, phenols, steroids, terpenoids and alkaloids. Through HPLC analysis, polyphenols quercetin and vanillic acid were identified in the C. edulis extracts, whilst in S. birrea extracts, quercetin, vanillic acid and gallic acid were identified. The extracts were found to be non-toxic to human kidney cells at concentrations 50 and 25 μg·mL⁻¹. C. edulis extracts were also non-toxic at 100 μg·mL⁻¹, as shown in Figure 1. S. birrea extracts showed the maximum alpha glucosidase inhibitory activity, as shown in Figure 2. C. edulis leaves and stems and juice extracts also exhibited alpha glucosidase inhibitory activity, while C. edulis roots showed the lowest inhibitory activity.

Conclusions

Reference

1. Pandey A, Tripathi S. Concept of standardization, extraction and pre phytochemical screening strategies for herbal drug. J Pharmacogn Phytochem 2014:115-119.

18

Metformin and dapagliflozin prevent methylglyoxal-induced cytotoxicity in human brain neuronal cells (SH-SY5Y)

Zainab Quraishi, Samantha Victor-Sami, Ali Kamali-Roosta and Yousif Shamsaldeen

University of Brighton

Introduction

Diabetes mellitus is a metabolic disease characterized by chronic blood glucose elevation (hyperglycaemia).¹ A wide range of complications associated with uncontrolled diabetes includes increasing the risk of dementia.² Chronic hyperglycaemia induced the formation of the reactive aldehyde, methylglyoxal (MGO).³ This study examines the neuroprotective effects of metformin and dapagliflozin on SH-SY5Y cells exposed to MGO, which highlights the therapeutic potential against diabetes-induced neurotoxicity.

Methods

SH-SY5Y cells were cultured in DMEM/F12 culture media and subjected to one of the following treatment groups: control (untreated); MGO (1 μM); MGO (100 μM), metformin (100 μM) + MGO (100 μM) and dapagliflozin (10 μM) + MGO (100 μM). Several assays were conducted to explore the effect of the treatment groups on the SH-SY5Y cells. These included MTT assay LDH assay, peroxynitrite fluorescence assay and laser scanning confocal microscopy (LSCM).

Results

MTT assay showed significant reduction (P ˂ 0.0001) in cell viability by approximately 75% when SH-SY5Y cells were treated with MGO (100 μM), but no such a reduction was observed in cells treated with MGO (1 μM). Co-incubation of MGO (100 μM) with metformin (100 μM) or dapagliflozin (10 μM) showed significant increase in cell viability by approximately 75% (P ˂ 0.001) and 50% (P ˂ 0.05), respectively. Moreover, incubating cells with metformin (100 μM) or dapagliflozin (10 μM) reduced MGO (100 μM)-induced LDH activity by approximately 75% (P ˂ 0.0001), which was in parallel to significant protection (P ˂ 0.05) against MGO (100 μM)-induced cells loss. These protective effects were accompanied by significant reduction in peroxynitrite generation as metformin (100 μM) completely abolished MGO (100 μM)-induced peroxynitrite generation, while there was approximately 50% reduction in (10 μM)-dapagliflozin-treated cells.

Conclusions

These findings suggest that elevated MGO may induce neurotoxicity and hence brain neurons loss. Therefore, targeting elevated MGO may prevent diabetic complications such as dementia underlining the potential beneficial effects of metformin and dapagliflozin in reducing the risk of dementia in diabetes.

References

1. Banday MZ, Sameer AS, Nissar S. Pathophysiology of diabetes: an overview. Avicenna J Med 2020;10(4):174-188.

2. Shamsaldeen AYS, Mackenzie LA, Lione LD, Benham C. Methylglyoxal, a metabolite increased in diabetes is associated with insulin resistance, vascular dysfunction and neuropathies. Curr Drug Metab 2016;17(4):359-367.

3. Mukhtar Y, Galalain A, Yunusa U. A modern overview on diabetes mellitus: a chronic endocrine disorder. Eur J Biol 2020;5(2):1-14.

19

Neuroinflammation of Porphyromonas gingivalis

Alaa Al-hindawi

University of Central Lancashire

Introduction

Periodontal disease (PD) is a chronic inflammatory disease caused by bacteria such as Porphyromonas gingivalis and characterized by alveolar bone resorption (Gangula et al., 2015). During routine oral activities such as brushing and flossing teeth, P. gingivalis gains access to vascular circulation and may participate in inflammation at locations remote from the oral cavity (Forner et al., 2006). Increasing evidence indicates the correlation between chronic periodontitis and dementia (Chen et al., 2017). Therefore, this study evaluated the neuroinflammatory activity of P. gingivalis in BV-2 microglia.

Methods

BV2 microglia cells were treated with different concentrations of P. gingivalis LPS. Cell viability was assessed by XTT assay, while levels of nitrite production were detected with the Griess assay. Secretion of pro-inflammatory cytokines were measured by ELISA. Furthermore, the western blot technique was used to detect the expression of iNOS protein. Data were expressed as mean ± SEM and analysed by one-way ANOVA, followed by Dunnett's multiple comparison test.

Results

BV2 cells that were treated with 0.1, 0.5, 1 and 10 μg/ml of P. gingivalis LPS showed non-significant changes in viability compared with negative control. Interestingly, results from the Griess assay showed that nitrite production was significantly elevated in BV2 cells treated with 0.5, 1 and 10 μg/ml of P. gingivalis LPS. In addition, a significant increase in TNF-α and IL-6 levels have been noticed in P. gingivalis LPS-treated BV2 cells (0.5, 1 and 10 μg/ml). P. gingivalis LPS caused a significant increase in iNOS protein expression at 1 and 10 μg/ml in BV2 microglia by utilizing the western blot technique.

Conclusion

In this study, P. gingivalis LPS enhanced neuroinflammation in BV2 microglia cells, which represent one of the major players in the pathogenicity of most neurodegenerative diseases. These results suggest that P. gingivalis might cause periodontal disease-related neural damage such as Alzheimer's disease and dementia.

References

1. Chen CK, Wu YT, Chang YC. Association between chronic periodontitis and the risk of Alzheimer's disease: a retrospective, population-based, matched-cohort study. Alzheimers Res Ther 2017;9:1–7.

2. Forner L, Larse, T, Kilian M, Holmstrup P. Incidence of bacteremia after chewing, tooth brushing and scaling in individuals with periodontal inflammation. J Clin Periodontol, 2006;33(6):401–407.

3. Gangula P, Ravella K, Chukkapalli S, Rivera M, Srinivasan S, Hale A, Channon K, Southerland J, Kesavalu L. Polybacterial periodontal pathogens alter vascular and gut BH4/nNOS/NRF2-phase II enzyme expression. PLoS ONE 2015;10(6):e0129885.

21

Effect of adenosine receptor modulation on caffeine-induced motor activity in rats

Romany Gerges

Faculty of Medicine, Aqaba Medical Sciences University

Introduction

Coffee confers many diverse health effects; some are beneficial and others are deleterious. It contains not only caffeine but other bioactive polyphenolic compounds. Controversies regarding benefits and risks of coffee consumption still exist, but the limitless health-promoting benefits of coffee outclass its few reported toxic effects. This study is devoted to the investigation of a potential inhibitory effect of adenosine and its analogues on the enhanced motor activity induced by caffeine. This was carried out by studying the effect of pretreatment of rats with adenosine and its analogues on caffeine enhanced spontaneous coordinate locomotor activity and forced motor performance in rats.

Materials and Methods

Materials

Chemicals:

The following chemicals were used and obtained from the sources indicated:

1. Adenosine (ADO) (ICN Biomedicals, Inc). ADO is fairly soluble in cold water, soluble in room temperature water, freely soluble in hot water and soluble in I N hydrochloric acid (50 mg/ml; clear and colourless) and can be suspended in 8% Tween 20 and insoluble in alcohol.

2. N6-cylcopentyl adenosine (CPA) (A1 agonist) (ICN Biomedicals, Inc). CPA is soluble in ethanol and moderately soluble in water.

3. 5'-(N-Cyclopropyl)carboxamidoadenosine (CPCA) (A2 agonist) (ICN Biomedicals, Inc). CPCA is moderately soluble in ethanol, slightly soluble in warm water and very soluble in dilute aqueous acid and can be suspended in 8% Tween 20.

4. Caffeine (ICN Biomedicals, Inc). Caffeine is moderately soluble in water at room temperature (2 g/100 ml) and also moderately soluble in ethanol (1.5 g/100 ml).

5. Pentylenetetrazole (PTZ) (Sigma, USA). PTZ is soluble in normal saline and water.

All drugs were used as freshly prepared solutions in distilled water except CPA, which was dissolved in 8% ethanol, and CPCA was suspended in 8% Tween 20.

Animals:

Adult male rats weighing 150–200 g were used. The animals were group housed in plastic cages and maintained under standard laboratory conditions with a natural light–dark cycle. Rats were left to acclimatize to the environment for at least a week before the experiments. Food and water were allowed ad libitum.

Effect of adenosine and its analogues on motor activity of rats:

Five groups of rats, each consisting of five animals.

Treatment schedules:

Group A: was given i.p. 0.5 ml of 8% Tween 20.

Group B: was given i.p. 0.5 ml of 8% ethanol.

Group C: was given adenosine i.p. in a dose of 100 mg/kg.

Group D: was given CPA i.p. in a dose of 10 mg/kg.

Group E: was given CPCA i.p. in a dose of 10 mg/kg.

The motor activity was determined by:

1.Activity cages (for screening of locomotor activity):

Rats were placed inside an acrylic transparent cage that rests on a sensor platform. It detects ambulatory movements as well as stereotypic activity like grooming, scratching, digging, etc. Vibrations caused by the animal activity produce proportional electrical signals. These are electrically processed to generate trigger pulses and drive a digital counter. Every count registered is accompanied by a flash. Activity recording was continued for 180 min. Activity records were taken for 1 min each at 1, 5, 30, 60, 120 and 180 min after giving the drugs mentioned above (Paul & Kazi, 1992).

2.Rotarod test (for screening of forced motor performance):

Rats were allowed to remain on a rotating rod until falling off. The length of time the rat remained on the rod was recorded. The falling latency was recorded for each group at 1, 5, 15, 30, 60, 120 and 180 min after giving the drug (Dunham & Miya, 1957).

Effect of adenosine receptor modulation on motor activity of rats induced by caffeine:

Seven groups of rats, each consisting of five animals.

Treatment schedules:

Group A: was given caffeine in therapeutic doses (100 mg/kg).

Group B: was given 0.5 ml of 8% Tween 20.

Group C: was given ADO 100 mg/kg 5 min before caffeine 50 mg/kg.

Group D: was given 0.5 ml of 8% ethanol.

Group E: was given CPA 10 mg/kg 60 min before caffeine 50 mg/kg.

Group F: was given CPCA 10 mg/kg 60 min before caffeine 50 mg/kg. The motor activity of each group was determined as discussed above.

Results

Intraperitoneal injection of CPCA in a dose of 10 mg/kg showed no significant changes in the spontaneous activity of rats.

Intraperitoneal injection of CPCA in a dose of 10 mg/kg 60 min before caffeine in a dose of 50 mg/kg does not affect the action of caffeine. That means that caffeine produced the same significant increase of the spontaneous activity, starting 1 min after its injection and continuing for 1 h, as when injected alone.

Intraperitoneal injection of CPCA in a dose of 10 mg/kg produced no significant changes in the forced motor performance in rats.

Intraperitoneal injection of CPCA in a dose of 10 mg/kg 60 min before caffeine 50 mg/kg does not affect significantly the forced motor performance induced by caffeine. This means that caffeine showed the same increase in the motor performance, which reached its maximal 1 min after caffeine injection. But it was observed that the increase in motor performance lasted for 1 h only, instead of 3 h as when it was administered alone.

In conclusion, adenosine elicited a rapid inhibitory effect on spontaneous motor activity and forced performance of rats. CPA, an A1 agonist, exerted a long-lasting inhibitory effect on motor activity, while CPCA, an A2 agonist, did not cause any change in motor activity. On the contrary, caffeine, a CNS stimulant, produced profound CNS excitability, restlessness and marked increase of motor activity of rats. This stimulant effect was opposed by pretreating rats with adenosine and CPA but not with CPCA. The above results indicate that the CNS depressant activity of adenosine implies that A1 receptors are involved in the control of motor activity while excluding any role of A2 receptors.

References

1. McLellan TM, Caldwell JA, Lieberman HR. A review of caffeine's effects on cognitive, physical and occupational performance. Neurosci Biobehav Rev 2016;71:294-312. https://doi.org/10.1016/J.NEUBIOREV.2016.09.001

2. Rivera-Oliver M, Díaz-Ríos M. Using caffeine and other adenosine receptor antagonists and agonists as therapeutic tools against neurodegenerative diseases: a review. Life Sci 2014;101(1-2):1-9. https://doi.org/10.1016/J.LFS.2014.01.083

3. Ballesteros-Yáñez I, Castillo CA, Merighi S, Gessi S. The role of adenosine receptors in psychostimulant addiction. Front Pharmacol 2018;8:985. https://doi.org/10.3389/FPHAR.2017.00985/

4. Almosawi S, Baksh H, Qareeballa A, et al. Acute administration of caffeine: the effect on motor coordination, higher brain cognitive functions, and the social behavior of BLC57 mice. Behav Sci 2018;8:65. https://doi.org/10.3390/BS8080065

5. Muñiz JA, Prieto JP, González B, et al. Cocaine and caffeine effects on the conditioned place preference test: Concomitant changes on early genes within the mouse prefrontal cortex and nucleus accumbens. Front Behav Neurosci 2017;11:200. https://doi.org/10.3389/FNBEH.2017.00200/

6. Rendón-Ochoa EA, Padilla-Orozco M, Calderon VM, et al. Dopamine D2 and adenosine A2A receptors interaction on Ca2+ current modulation in a rodent model of parkinsonism. ASN Neuro 2022;14. https://doi.org/10.1177/17590914221102075

7. Saadawi SS, Alennabi KA, Baayo S, Fares A, Alosta N, Aburawi SM. Effect of caffeine at different concentrations on behavior and motor activity in mice. J Adv Med Pharm Sci, 2020:1-11. https://doi.org/10.9734/JAMPS/2020/V22I330159

8. Fredholm BB, Svenningsson P. Why target brain adenosine receptors? A historical perspective Parkinsonism Relat Disord 2020;80:S3-S6. https://doi.org/10.1016/j.parkreldis.2020.09.027

9. Olopade FE, Femi-Akinlosotu OM, Adekanmbi AJ, Ighogboja OO, Shokunbi MT. Chronic caffeine ingestion improves motor function and increases dendritic length and arborization in the motor cortex, Striatum, and cerebellum. J Caffeine Adenosine Res 2021;11(1):3-13. https://doi.org/10.1089/CAFF.2020.0017

10. Tallis J, Duncan MJ, James RS. What can isolated skeletal muscle experiments tell us about the effects of caffeine on exercise performance? Br J Pharmacol 2015;172(15):3703. https://doi.org/10.1111/BPH.13187

31

N-Methyl-D-aspartate receptor (NMDAR) blockers improved depressive behaviour initiated by levetiracetam administration in mice

Azadeh Mesripour and Tanin Ahmadi

Isfahan University of Medical Sciences

Background and Aim

Antiepileptic drugs, for instance, levetiracetam can exacerbate depression in epileptic patients, apart from epilepsy itself. Epilepsy augments indoleamine 2,3-dioxygenase (IDO) enzyme activity, resulting in the formation of end-product quinolinic acid, that is, a N-methyl-D-aspartate receptor (NMDAR) agonist responsible in neurotoxic effects related to depression. Thus, the aim was evaluating the effect of NMDAR blockers on levetiracetam induced depression [1].

Methods

Male NMRI mice (25 ± 3 g, 6–8 weeks old) were used, seven in each group. Animals were daily injected with levetiracetam (20 mg/kg) for 14 consecutive days; pretreatments with dextromethorphan (30 mg/kg), MK801 (dizocilpine) (0.075 mg/kg) or imipramine (10 mg/kg) were performed 30 min before levetiracetam administration starting from day 8. The control group received normal saline (1 ml/100 g) all the drugs were injected intraperitoneally. The locomotor test, forced swimming test (FST) and the novelty suppressed feeding test (NSFT) were performed to assess depressive-like behaviour [2]. Statistical significance was determined using an ANOVA followed by a Tukey's post hoc test.

Results

Following dextromethorphan pretreatment immobility time during FST was significantly lower (44.29 ± 5.6 s) than levetiracetam alone (161.4 ± 11.8 s, P < 0.001) and the control group (109.4 ± 6.06, P < 0.001). MK801 significantly reduced immobility time (53.0 ± 7.04 s, P < 0.001 compared to levetiracetam). There were no significant changes in the locomotor activity among diverse treatment groups. While levetiracetam increased latency and decreased food intake in NSFT, pretreatment with dextromethorphan and MK801 reversed these depressant effects (Figure 1a,b). These behavioural changes were similar to levetiracetam-imipramine group.

Figure 1. Effect of drugs on latency (a) and food intake (b) during NSFT. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control group, #P < 0.05, ###P < 0.001 compared with Lev alone group. Dxt, dextromethorphan; Imi, imipramine; Lev, levetiracetam; MK, MK801.

Conclusion

References

1. Mesripour A, Ahmadi T. Depression-like effects of levetiracetam was halted by pretreatment with N-methyl-D-aspartate receptor (NMDAR) blockers in mice. Bull Pharm Sc (Assiut University) 2023; 46(1):517-527.

2. Mesripour A, Golbidi M, Hajhashemi V. Dextromethorphan improved cyclosporine-induced depression in mice model of despair. Res Pharm Sci 2020;15(5):447–453.

33

Studies on the protective effects of mangiferin and its interaction with nitric oxide (NO) modulators in animal model of Alzheimer's disease

Rishi Pal¹, Pryanshu Pradhan¹, Manju J. Chaudhary² and Rajendra Nath¹

¹Department of Pharmacology & Therapeutics, King George's Medical University; ²Department of Physiology, Dr. BRRA Government Medical College

Objective

The study was designed to evaluate role of mangiferin and its interaction with L-arginine (NO donor) and 7-nitroindazole (nNOS inhibitor) in AlCl₃-induced Alzheimer's disease.

Methods

A total of 60 Wistar rats were used in the study and divided into 10 groups (n = 6/group). All animal groups were received AlCl₃ (100 mg/kg, i.p.) for a duration of 28 days daily except control group. mangiferin at doses of (20–60 mg/kg, p.o.) alone and in combination with NO modulators, L-arginine (100 mg/kg) and 7-NI (10 mg/kg) were administered, while memantine (2 mg/kg) used as standard drug and control group received normal saline from day 21 to 28 days daily at morning hours. On 28th day, behavioural tests were performed using Morris's water maze and Y-maze used to assess memory and other cognitive and locomotor parameters were recorded using EPM and open field tests. After the behavioural and memory assessment their brain were collected under anaesthesia (pentobarbitone, 60 mg/kg, i.p.) for biochemical, immunological and histochemical markers in the brain homogenate. Behavioural data obtained was statistically analysed by using Mann–Whitney U-test. One-way ANOVA followed by Newman–Kewl post hoc statistical test were used for biochemical analysis. A P-value of <0.05 was considered as significant.

Results

The mangiferin group (60 mg/kg) showed a significant reduction in the transfer latency in the Elevated Plus maze (TL: 20.67 ± 1.97 vs. AlCl₃-only group: 29.50 ± 1.76, P < 0.001), time taken to reach the platform in Morris water maze (t: 58.33 ± 2.66 vs. AlCl₃-only group: 70.83 ± 4.35, P < 0.05) and increase in the percentage alternations in the Y-maze (% altrntns: 50.33 ± 4.89 vs. AlCl₃-only group: 45.50 ± 6.09, P < 0.05). Moreover, there was significant reduction in brain MDA levels (6.30 ± 1.92 vs. AlCl₃ only group: 9.29 ± 0.81, P < 0.001) and increase in SOD levels (16.88 ± 1.90 vs. AlCl₃-only group: 10.89 ± 1.44, P < 0.00). The inflammatory markers also showed significant reduction in TNF-α: 170.01 ± 17.06 vs. AlCl₃-only group: 225.23 ± 8.00, P < 0.001; IL-6: 475.34 ± 97.02 vs. AlCl₃-only group: 579.71 ± 73.18, P < 0.05, and NF-κB: 7.10 ± 1.12 vs. AlCl₃-only group: 9.38 ± 0.39, P < 0.05. However, there was no significant difference in the mangiferin (20 mg/kg) and (40 mg/kg) in the above parameters as compared to the AlCl₃-only group. The reduction of Aβ-proteins by mangiferin (60 mg/kg) was not significant as compared to the AlCl₃ group.

The combination of n-NOS inhibitor (7-NI 10 mg/kg) with mangiferin (40 mg/kg) enhanced the behavioural (transfer latency in elevated plus maze: 17.16 ± 2.71 vs. AlCl₃-only group: 29.50 ± 1.76, P < 0.001; time to reach platform in Morris water maze: 48.5 ± 3.31 vs. AlCl₃-only group: 70.83 ± 4.35, P<0.001; percentage alternations in Y-maze: 60.33 ± 3.39 vs. AlCl₃-only group: 45.50 ± 6.09, P < 0.001; oxidative stress markers MDA levels: 5.15 ± 1.07 vs. AlCl₃-only group: 9.29 ± 0.81, P < 0.001; SOD levels: 22.22 ± 1.69 vs. AlCl₃-only group: 10.89 ± 1.44, P < 0.05) and anti-inflammatory TNF-α: 124.98 ± 7.79 vs. AlCl₃-only group: 225.23 ± 8.00, P < 0.001; IL-6: 289.17 ± 29.98 vs. AlCl₃ group: 579.71 ± 73.18, P < 0.001; NF-κB: 5.73 ± 1.89 vs. AlCl₃-only group: 9.38 ± 0.39, P < 0.001 and improve in the Aβ-42: 400.19 ± 48.59 vs. 759.00 ± 88.23, P < 0.001, whereas the L-arg (100 mg/kg) with mangiferin (40 mg/kg) combination group does not show significant changes in the above parameters as compared to the AlCl₃ group.

Conclusions

The current study shown that mangiferin at high dose ameliorates aluminium chloride induced changes in AD model on memory, cognitive behaviour, oxidative stress and inflammatory markers in brain. NO modulators, specifically nNOS inhibitor 7-NI, enhanced protective effects of mangiferin in AD. Results of this study suggest that neuronal NO plays an important role in Alzheimer's disease, which may be prevented by mangiferin administration with n NOS inhibitor.

37

Neuroprotective effects of fluoxetine on molecular markers of circadian rhythm, cognitive deficits, oxidative damage and biomarkers of Alzheimer's disease under chronic constant light regime

Ashish Sharma, Ashu Mohammad, Adesh Saini and Rohit Goyal

Shoolini University

Introduction

There is mounting evidence of circadian rhythm disruption in Alzheimer's disease (AD) [1]; however, the cause-and-effect relationship between them is not understood. Chronic constant light exposure effectively disrupts circadian rhythm in rats.

Methods

We hypothesized that chronic constant light exposure might contribute significantly to development of AD-like-phenotype in rats and that fluoxetine (Flx) treatment might protect the brain against it [2]. Adult male rats were exposed to normal light-dark cycles, constant light (LL), constant dark and LL + Flx (5 mg/kg/day, ZT5) for 4 months.

Results

The expression of molecular markers of circadian rhythm, Per2 transcripts, and protein expression of peroxiredoxin-1 (PRX1) and hyperoxidized peroxiredoxins (PRX-SO2/3) were significantly dysregulated in the suprachiasmatic nuclei (SCN) of LL rats, which was prevented with concomitant fluoxetine administration. The levels of glutamate and γ-aminobutyric acid were dysregulated, and oxidative damage was observed in the SCN and hippocampi of LL rats. Fluoxetine treatment conferred protection against oxidative damage in LL rats. Constant light exposure also impaired rats' performance on Y-maze, Morris maze and novel object recognition test, which was prevented with fluoxetine administration. A significant elevation in soluble Aβ1–42 levels, which strongly correlated with up-regulation of Bace1 and Mgat3 transcripts, was observed in the hippocampus of LL rats. Further, the expression of anti-ageing gene Sirt1 was down-regulated, and neuronal damage indicator Prokr2 was up-regulated in hippocampus. Fluoxetine rescued Aβ1–42 up-regulation and AD-related genes' dysregulation.

Conclusion

Our findings show that circadian disruption by exposure to chronic constant light may contribute to progression of AD, which can be prevented with fluoxetine treatment [3].

DOI: 10.1021/acschemneuro.1c00238 (ACS Chemical Neuroscience)

References

1. Musiek ES, Lim MM, Yang G, Bauer AQ, Qi L, Lee Y, Roh JH, Ortiz-Gonzalez X, Dearborn JT, Culver JP, et al. Circadian clock proteins regulate neuronal redox homeostasis and neurodegeneration. J Clin Invest 2013;123(12):5389−400.

2. Sharma A, Sethi G, Tambuwala MM, Aljabali AAA, Chellappan DK, Dua K, Goyal R. Circadian rhythm disruption and Alzheimer's disease: The dynamics of a vicious cycle. Curr Neuropharmacol 2020;19:248.

3. Zhou CN, Chao FL, Zhang Y, Jiang L, Zhang L, Fan JH, Wu YX, Dou XY, Tang Y. Fluoxetine delays the cognitive function decline and synaptic changes in a transgenic mouse model of early Alzheimer's disease. J Comp Neurol 2019;527(8):1378−1387.

56

Comparative analysis of the anti-inflammatory potential of pterostilbene with other SIRT1 activators and its role in ameliorating cafeteria diet obesity-induced depression: In silico, in vitro and in vivo perspective

Rashmi Patil¹, Urmila Aswar¹ and Nishant Vyas²

¹Poona College of Pharmacy, Bharati Vidyapeeth (deemed To Be)university; ²Logical Lifesciences

Introduction

Adolescent obesity due to westernized food consumption is increasing at an alarming rate and leading to depression [1]. Cytokines are the key players in the development of IR, hyperlipidaemia, oxidative stress, obesity and associated depression [2]. Pterostilbene (PTE), a SIRT1 activator, plays an important role as an antiobesity and neuroprotective agent and displays favourable pharmacokinetic profile [3]. In present study, the role of PTE in obesity-induced depression (OID) was evaluated.

Method

Comparative in silico (IL-6, TNF-α, NF-κB, SIRT1) and in vitro (ThP1 cell line-IL-6, TNF-α) analysis of PTE was carried out with other SIRT1 activators: resveratrol (RES) and curcumin (CUR). It was further checked for its role in cafeteria diet (CD)-induced obesity in adolescent Swiss albino mice.10 weeks of administration of CD led to the development of OID in mice. For 4 weeks, OID mice were administered the test drug PTE (10, 20 and 40 mg/kg), cetilistat (CET 10 mg/kg) and fluoxetine (FLX 10 mg/kg). Inflammatory mediators (IL-6, TNF-α, NF-κB), cortisol, insulin resistance (IR), lipid profile, behavioural parameters, histopathological examination (brain and adipose tissue) and gene expression analysis of SIRT1, leptin and ghrelin receptors in the brain were carried out.

Results

PTE exhibited potent anti-inflammatory potential by favourable in silico docking profile and in vitro THP-1 cell line compared to RES and CUR. Obesity-associated adipose tissue macrophages (ATM) are a hallmark of inflammation and produce NF-κB, TNF α and IL-6. In an in vivo study in OID mice, PTE decreased the elevated cytokine levels, thus inhibiting the crosstalk and halting the advancement of obesity, insulin resistance, HPA axis dysregulation, oxidative stress and hyperlipidaemia. It was evident by reduced body weight, IR, cortisol, lipid profile and improved behavioural parameters, histopathological features and gene expression. Since SIRT1, leptin and ghrelin signalling play important roles in mood along with maintaining metabolic homeostasis, up-regulation by PTE might have played a role in the amelioration of OID.

Conclusion

The beneficial effect of PTE in the attenuation of OID can be attributed to its anti-inflammatory, antioxidant, amelioration of HPA axis dysregulation, up-regulation of SIRT1 and leptin and ghrelin gene expression.

References

1. Lalanza J.F.and Snoeren E. M.(2021). The cafeteria diet: A standardized protocol and its effects on behavior. Neurosci Biobehav Rev 122: 92-119.

2. Milano W., Ambrosio P., et.al (2020). Depression and obesity: analysis of common biomarkers. Diseases, 8(2), 23.

3. Pan, M. H., Wu, J. C., et al (2018) Antiobesity molecular mechanisms of action: Resveratrol and pterostilbene. Biofactors, 44(1), 50-60.

72

Multiplexed iPSC assays as a tool for studying the effects of neurotensin on neuroinflammation

Alison Holiday and Ian Winfield

Domainex

Introduction

Chronic neuroinflammation is implicated in many neurological diseases including ALS, neurodegeneration and multiple sclerosis whereby the immune response becomes excessive and perpetuates cell death rather than protecting against it. Microglia are the innate immune cells of the immunologically privileged CNS. Like macrophages, they scavenge for damaged neurons and synapses, plaques and infectious agents via chemotaxis, cytokine release and phagocytosis. Neurotensin is a neuropeptide distributed throughout the CNS that increases microglial motility, releases proinflammatory molecules and is associated with neuroinflammation in autism spectrum disorder [1]. Here, we employ neurotensin as a tool to demonstrate how iPSC microglia can be employed to study all aspects of neuroinflammation.

Methods

Live cell imaging over 24 h using a Transwell format was employed to study chemotactic response of NucLight Rapid Red-stained microglia to an 8-point neurotensin CRC. Cells were also stimulated with EC₈₀ of neurotensin and 8-point CRC of pan-neurotensin receptor inhibitor, SR142948. In a multiplexed assay IL-1β, TNF-α, ROS and cell viability were measured after 16-h treatment using alphaLISAs for cytokine release, live cell dye DCFDA for ROS production and luminescent CellTiter-Glo for viability in response to 12-point neurotensin and LPS (positive control) CRCs. Finally, the impact of neurotensin on microglial phagocytosis was measured by incubating cells with pHrodo Green Staphylococcus aureus bioparticles after 8-point neurotensin CRC pre-incubation (16 h).

Results

Neurotensin induced an inflammatory response in iPSC microglia. Neurotensin induced chemotaxis, plateauing at 13 h, which was inhibited by SR142948 (IC₅₀ 21 nM), caused release of pro-inflammatory cytokines IL-1β and TNF-α comparable to LPS and increase ROS production without impacting cell viability after 16 h. Phagocytosis was increased in response to neurotensin after 16-h pre-incubation before addition of bioparticles, suggesting this action is downstream of chemotaxis and cytokine release.

Conclusions

We employed iPSC microglia and live cell imaging to establish a series of functional assays to measure the effects of neuroinflammation. Multiplexing imaging and non-imaging experiments can give a wealth of knowledge from fewer cultures, and by taking these methods together, we are able to measure the main immune functions of microglia in an efficient and cost-effective manner. This work demonstrates neurotensin as a key regulator in neuroinflammation and provides a platform for the development of novel therapeutics.

Reference

1. Tsilioni I, Patel AB, Pantazopoulos H, et al. IL-37 is increased in brains of children with autism spectrum disorder and inhibits human microglia stimulated by neurotensin. Proc Natl Acad Sci USA 2019;116(43):21659-21665.

106

TrkC has distinct spatiotemporal dynamics compared to TrkA and TrkB

Ryan Duffy, Stephen Hill and Chloe Peach

School of Life Sciences, Centre of Membrane Proteins and Receptors (COMPARE), University of Nottingham, Queen's Medical Centre

Introduction

Neurotrophins are a family of proteins crucial in the development and maintenance of the nervous system (1). Neurotrophins include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3) and neurotrophin-4 (NT4), which interact with a class of receptor tyrosine kinases (RTKs), known as tropomyosin receptor kinases (Trk), specifically TrkA, TrkB and TrkC. Despite the profound role neurotrophins have on the nervous system, little work has been done to characterize their molecular pharmacology with spatial and temporal resolution independently of disease models. Here, we have utilized bioluminescence resonance energy transfer (BRET) as a proximity-based measure (2) to characterize Trk receptor dimerization and trafficking in response to NGF, BDNF, NT3 and NT4.

Methods

Real-time dimerization was measured as BRET between an N-terminal luciferase-tagged Trk receptor (NanoLuc) and a fluorophore-tagged Trk receptors (SnapTag) (3). HEK293 cells in 96-well plates were transiently transfected using polyethylenimine with NanoLuc-TrkA/TrkB/TrkC (20 ng/well) and SnapTag-TrkA/TrkB/TrkC (40 ng/well), respectively. Following 48 h, cells were incubated with membrane-impermeant SnapTag-AlexaFluor488 (250 nM, 1 h) and then washed in assay buffer (Hanks buffered saline solution/0.1% bovine serum albumin, pH 7.4). The NanoLuc substrate furimazine (10 μM) was added (5 min, 37°C). Real-time trafficking was measured as BRET assay between a fluorescent marker of the plasma membrane (CAAX) and a C-terminal luciferase-tagged Trk receptor (RLuc8). HEK293 cells were transfected in a similar manner with TrkA/TrkB/TrkC-Rluc8 (20 ng/well) and RGFP-CAAX (20 ng/well). The Rluc8 substrate coelenterazine purple (10μM) was added (5 min, 37°C). In both dimerization and trafficking studies, BRET ratios were recorded using a BMG Pherastar. Following five baseline reads, increasing concentrations (1 pM–30 nM) of NGF, BDNF, NT3 and NT4 were added to triplicate wells. BRET ratios were calculated every 45 s for 20 min for dimerization and every 74 s for trafficking.

Results

Using these novel BRET approaches across the Trk family, both real-time dimerization and trafficking of Trk receptors could be measured. Temporally, TrkC produced the quickest dimerization response, as well as the highest BRET ratio. TrkA and TrkB dimerization, on the other hand, was lower. TrkA, TrkB and TrkC dimerization in response to aforementioned ligands was concentration dependent with a potency ranging from 0.46 to 32 nM (Table 1; n = 5).

Ligand-induced TrkA, TrkB and TrkC trafficking was then measured as a reduction in BRET as Rluc8-tagged receptors moved away from the plasma membrane marker (RGFP-CAAX). TrkC was distinct in its trafficking profile with a minimal change in BRET and slower kinetics compared than TrkA and TrkB. These trafficking responses were concentration dependent, with a potency ranging from 1.0 to 28 nM (Table 2; n = 5).

Conclusions

Studying real-time Trk receptor dimerization and trafficking revealed that these receptors can respond to their ‘non-canonical’ growth factor ligand where, in some cases, the response is comparable to the canonical growth factor. The dynamics of the ligand–receptor relationship is unique across the Trk receptors, with TrkC exhibiting the most distinctive properties.

References

1. Conroy JN, Coulson EJ. High-affinity TrkA and p75 neurotrophin receptor complexes: a twisted affair. J Biol Chem 2022;298(3):101568.

2. Stoddart LA, Kilpatrick LE, Hill SJ. NanoBRET approaches to study ligand binding to GPCRs and RTKs. Trends Pharmacol Sci 2018;39(2):136–47.

3. Peach CJ, Tonello R, Gomez K, Calderon-Rivera A, Bruni R, Bansia H, et al. Neuropilin-1 is a co-receptor for NGF and TrkA-evoked pain. bioRxiv : the preprint server for biology. United States; 2024.

115

Control of irinotecan-induced emesis by a 5-HT3 and NK1 receptor antagonist regimen in combination with olanzapine

John A. Rudd¹, Zengbing Lu¹, Julia Y. H. Liu¹, Dexuan Cui¹, Man-Pui Ngan¹, Xiaofei Huang¹ and Yasuhiro Nakagami²

¹Chinese University of Hong Kong; ²Daiichi Sankyo Co., Ltd

Introduction/Background and Aims

Irinotecan (7-ethyl-10-[4-(l-piperidono)-1-piperidino]carbonlyoxycamtothecin) is a camptothecin derivative used in the treatment of cancer. Unfortunately, it may be associated with the side effects of acute and delayed nausea and emesis [1]. We recently showed that Irinotecan induces emesis over a 3-day period in Suncus murinus, with the most intense response occurring during the first 4 h via the involvement of the abdominal vagi and 5-HT3 but not NK1 receptors [2]. The present studies were designed to investigate if irinotecan-induced emesis is better controlled by a combination of the 5-HT3 receptor antagonist, palonosetron, and the NK1 receptor antagonist, aprepitant. We also investigated the anti-emetic potential of olanzapine, which is useful to improve the control of nausea and emesis in patients receiving 5-HT3 and NK1 receptor antagonist regimens.

Methods/Summary of Work

Adult male S. murinus (60–80 g) were used. Animals were administered palonosetron (0.01 mg kg⁻¹, p.o.), aprepitant (1 mg kg⁻¹, p.o.), olanzapine (1 mg kg⁻1, s.c.) or their respective vehicles, alone or in combination 1 h before irinotecan (75 mg kg⁻¹, p.o.). All behavioural recordings were conducted in a whole-body plethysmography chambers, with assessment of body weight, and food and water intake made at 24-h intervals for up to 72 h.

Results/Discussion

Irinotecan induced 34.1 ± 6.8 retches + vomits (RV) in the first 24 h and 17.3 ± 8.0 RV during the 24- to 72-h period; 60.3 % of the response occurred in the first 4 h following a latency of 1.2 h (interquatile:0.74–2.67 h). Palonosetron and olanzapine when administered alone reduced RV by 55.7% (P < 0.05) and 71.8 % (P < 0.05) during first 24 h, but aprepitant was ineffective. However, the combination of palonosetron and aprepitant reduced RV by 76.6 % (P < 0.05), and when olanzapine was added to the palonosetron and aprepitant regimen, RV was reduced further to 95.6 % (P < 0.05), a similar pattern to reduce RV was observed over the entire 0- to 72-h period (P < 0.05). None of the treatment alone or in combination could reduce the RV during the 24- to 72-h period, or the body weight, and food and water intake of Irinotecan-treated animals. Irinotecan and/or the drug/vehicle combinations did not affect any of the respiratory parameters that we recorded during the 72-h observation period.

Conclusions

The combined regimen of palonosetron and aprepitant with olanzapine is useful to control the emesis induced by irinotecan in S. murinus, with the benefits mainly being observed during the acute phase.

References

1. Hesketh PJ, Bosnjak SM, Nikolic V, Rapoport B. Incidence of delayed nausea and vomiting in patients with colorectal cancer receiving irinotecan-based chemotherapy. Support Care Cancer 2011;19(12):2063-2066.

2. Rudd JA, Lingqing Y, Zengbing L, Liu JYH, Ngan MP, Cui D, Nakagami Y. Investigation into the mechanism of action of irinotecan to induce emesis in Suncus murinus. Cancer Supportive Care 2004;32:5278.

120

Effects of ginsenoside Rb1 and Rg1 on alcohol-induced reinforcement and rewarding effects

Wun A. Kook, Youyoung Lee, Eugene Sin, Seok-Yong Lee and Choon-Gon Jang

Sungkyunkwan University

Introduction/Background and Aims

Alcohol use disorder (AUD) is a chronic disease characterized by compulsive alcohol-seeking behaviours such as craving [1]. This study examined the potential of ginsenosides, major components of Korean red ginseng, as therapeutic agents to attenuate alcohol-induced addictive-like behaviours in male mice. Additionally, the study investigated the neuronal mechanisms by which ginsenosides inhibit these alcohol-induced behaviours.

Method/Summary of Work

Male C57BL/6J mice were used for all experiment. To evaluate whether ginsenosides inhibit alcohol-induced addictive-like behaviours, alcohol oral self-administration (SA) and the conditioned place preference (CPP) test were performed. Ethanol 10% (for SA, dissolved in distilled water) and 20% (for CPP, in saline) were used to establish the alcohol dependence model in mice. Mice were administered ginsenoside Rb1 and Rg1 (25, 50 and 100 mg/kg, i.p., in saline) 1 h before each experimental session. After the behavioural experiments were completed, all mice were sacrificed, and their brain tissues were used for western blot analysis and neurotransmitter enzyme-linked immunosorbent assay (ELISA). Statistical significance was determined using ANOVA followed by a Fisher's LSD or Tukey post hoc test.

Results/Discussion

In the SA studies, treatment with ginsenosides Rb1 and Rg1 significantly attenuated alcohol-induced self-administration under a fixed ratio 4 and progressive ratio schedule of reinforcement (Table 1). In the CPP studies, both ginsenosides Rb1 and Rg1 (50 mg/kg) significantly inhibited the alcohol-induced rewarding effect (n = 10/group; P < 0.01). Furthermore, our results showed that ginsenosides modulate alcohol-driven change in the glutamatergic and DAergic systems in the brains of mice after the CPP schedule injection (n = 7/group; P < 0.001).

Conclusions

Reference

1. Mason BJ. Emerging pharmacotherapies for alcohol use disorder. Neuropharmacology 2017;122:244-253. https://doi.org/10.1016/j.neuropharm.2017.04.032

125

Addictive potential and dopaminergic mechanisms of 4-methylmethyphenidate (4-MMP): Evidence from behavioural and neurochemical studies in mice

Youyoung Lee¹, Wun-A Kook¹, Eugene Sin¹, Kyeong-Man Kim² and Choon-Gon Jang¹

¹Sungkyunkwan University; ²Chonnam National University

Introduction

4-Methylmethyphenidate (4-MMP) is an emerging central nervous system (CNS) stimulant that serves as a replacement for methylphenidate [1] and has seen increasing misuse globally [2]. Despite its prevalence, there is a lack of in vivo scientific evidence regarding its addictive potential, and its pharmacological profile remains largely unclear.

Method

In this study, we investigated the addictive properties of 4-MMP through conditioned place preference (CPP) and behavioural sensitization (BS) tests in mice. To elucidate the role of dopaminergic pathways, we employed dopamine receptor antagonists (SCH23390 and haloperidol) during CPP tests and used chemogenetic techniques to inhibit specifically medium spiny neurons expression dopamine D1 receptors (D1R-MSNs) in the nucleus accumbens (NAc). We also assessed the dopamine transporter (DAT) inhibition potency of 4-MMP in vitro. Additionally, we evaluated dopamine (DA) concentrations, DA-related protein expressions in the NAc and neuronal activation in the ventral tegmental area (VTA) by analysing c-fos immunoreactivity in mice injected with 4-MMP.

Results

Our results demonstrated that 4-MMP significantly enhanced CPP (P < 0.05 vs. vehicle) and induced BS, also showing bidirectional cross-sensitization with methamphetamine (P < 0.05 vs. vehicle and vs. day 3, when first drug injection day). Notably, CPP induced by 4-MMP was completely blocked by SCH23990 but not by haloperidol (Table 1), while clozapine (CNO)-mediated inhibition of D1-MSNs effectively prevented CPP acquisition (P < 0.05 vs. 4-MMP). In vitro studies revealed that 4-MMP exhibited over a 10-fold greater DAT inhibition (IC₅₀ = 70.89 nM) compared to methamphetamine (IC₅₀ = 878.5 nM) in HEK-293 cells (n = 3 independent assays per drug). Furthermore, 4-MMP administration led to increased DA levels, up-regulation of D1DR and p-CREB/CREB in the NAc and elevated c-fos expression in VTA DA neurons (P < 0.05 vs. vehicle).

Conclusion

These results indicate that 4-MMP has a high potential for abuse in mice, likely due to significant alterations in the dopaminergic system, particularly involving D1-MSNs, and associated neural plasticity.

Supported by grant from Ministry of Food and Drug Safety 23212MFDS218.

References

1. Beharry S, Gibbons S. An overview of emerging and new psychoactive substances in the United Kingdom. Forensic Sci Int 2016;267:25-34.

2. Carlier J, Giorgetti R, Varì MR, et al. Use of cognitive enhancers: methylphenidate and analogs. Eur Rev Med Pharmacol Sci 2019;23(1):3-15.

127

Fluclotizolam and flualprazolam, novel synthetic benzodiazepines, elicit psychological and physical dependence in rodents

Eugene Sin, Youyoung Lee, Wun-A Kook and Choon-Gon Jang

Sungkyunkwan University

Introduction/Background and Aims

Fluclotizolam (FCZ) and flualprazolam (FAZ) are benzodiazepine (BDZ) type novel psychoactive substances. For BDZ's sedative and hypnotic effects, diverse BDZ-type designer drugs are illegally synthesized and introduced to street drug market [1]. Especially, FCZ and FAZ are synthetic derivates of fluorinated triazolodiazepine that are more addictive and toxic than alprazolam. However, FCZ and FAZ are regulated in only a few countries. In this study, to propose scientific evidences for the regulation of FCZ and FAZ, somatic withdrawal symptoms and reinforcing effect are evaluated.

Method/Summary of Work

FCZ was diluted with vehicle solution (DMSO 5% and Tween 80 5% in saline 90%) for intravenous self-administration (IVSA) (0.01, 0.03 and 0.1 mg/kg/inf) and withdrawal (WD) test (3 and 6 mg/kg). FAZ was diluted with the vehicle for IVSA (0.01, 0.03 and 0.1 mg/kg/inf) and WD test (0.3 and 0.6 mg/kg). For positive control, diazepam (DZP) (3 mg/kg for WD test and 0.06 mg/kg/inf for IVSA) diluted with the vehicle was used. For the IVSA test, Sprague−Dawley rats (4 weeks old) were used to investigate the reinforcing effect of the drugs. C57BL/6J male mice (7–8 weeks old) were used for WD test to evaluate the physical dependence of FCZ and FAZ. Each of the drugs was injected intraperitoneally twice a day for 7 days. All data were analysed using ANOVA with Fisher's LSD post hoc test.

Results/Discussion

For the FCZ IVSA, all concentration groups exhibited significantly higher values of the number of infusions and active lever pressing for average of 7 days than the vehicle group. In the WD test, chronic use of FCZ 6 mg/kg induced somatic WD symptoms, such as forepaw tremor. For the FAZ IVSA, 0.01 and 0.03 mg/kg groups represented significantly increased values of the number of infusions and active lever pressing for average of 7 days than the vehicle group. Also, in the WD test, 0.6 mg/kg FAZ induced WD symptoms in forepaw tremor, writhing, body tremor and piloerection.

Conclusions

Reference

1. Manchester KR, Lomas EC, Waters L, Dempsey FC, Maskell PD. The emergence of new psychoactive substance (NPS) benzodiazepines: a review [published correction appears in Drug Test Anal. 2018 Feb;10(2):392-393. doi: 10.1002/dta.2349]. Drug Test Anal. 2018;10(1):37-53. https://doi.org/10.1002/dta.2211

146

The transient receptor potential ankyrin 1 (TRPA1) expressed by the peptidergic Edinger–Westphal nucleus (EWcp) in the mouse brain plays a role in neurodegeneration

Erika Pintér, Viktória Kormos, Petra Prókay, János Konkoly, Maja Payrits, Eva Borbely, Balázs Gaszner and Dóra Zelena

University of Pécs

Introduction/Background and Aims

The transient receptor potential ankyrin 1 (TRPA1) is involved in pain and inflammation. However, little is known about its expression patterns and functions in the brain.

Earlier, we found that WT mice injected with amyloid-beta1-42 exhibited cholinergic cell and fibre loss, which was attenuated in TRPA1−/− animals. Memory loss was observed in amyloid beta1–42 injected TRPA1+/+ mice, but not in the TRPA1−/− group. Elderly KO mice showed significantly milder memory loss [1].

Recently, we have shown by RNAscope in situ hybridization technique that the EWcp area is the site of abundant Trpa1 mRNA expression in the mouse CNS, localizing to peptidergic, UCN1-containing neurons [2]. Given that the EW is affected by AD and TRPA1 is highly expressed here, we hypothesized that TRPA1 plays a role in AD-associated neurodegeneration of EW peptidergic neurons. We also presented evidence that TRPA1 may be involved in stress adaptation, mood regulation and loss of olfaction, which are early signs of several neurodegenerative disorders.

Method/Summary of Work

In the present study, we used triple transgenic (3xTg) animals overexpressing three genes (amyloid precursor protein (APP), presenilin-1 (PSEN1) and tau protein) each of them predisposing to AD. The Trpa1 mRNA expression and the UCN1 peptide content were assessed in five age groups of 3xTg and C57BL6 mice (2, 6, 9, 12 and 18 months) using RNAscope in situ hybridization (ISH) technique combined with immunofluorescence labelling in the EWcp. Experiments were carried out on intact male 3xTg and control C57BL/6J mice of the same age (n = 4–6 per group). For Trpa1 mRNA signal quantification, the copy number per cell was manually determined in three slices per animal, with 5–10 neurons per slice. UCN1 immunohistochemistry was used to identify UCN1-positive cells.

Results/Discussion

Higher Trpa1 expression was observed in the 2- and 6-month-old control groups compared to the 3xTg counterparts; however, no genotype-dependent differences were detectable in the elder groups due to the progressive age-related reduction of Trpa1 copy number in the C57BL6 strain. Trpa1 expression of transgenic mice persisted at basally lower levels that was not affected by ageing (Figure 1).

Conclusions

References

1. Payrits M, Borbely E, Godo S, et al. Mech Ageing Dev. 2020;189:111268. https://doi.org/10.1016/j.mad.2020.111268.

2. Kormos V, Kecskés A, Farkas J, et al. J Psychiatry Neurosci. 2022;47(3):E162-E175. https://doi.org/10.1503/jpn.210187.

150

Comparison of (±)MDMA, fluoxetine and dexfenfluramine to inhibit uptake and/or stimulate release of [3H]5-HT in rat frontal cortical synaptosomes

Isobel Jones, Ian Davies, Sharon Cheetham, Wioletta Pijacka, Steve Vickers, Naheed Mirza and Elizabeth Jagger

Sygnature Discovery

Background and Aims

The empathogen/psychedelic MDMA (3,4-methylenedioxymethamphetamine) has been tested in phase 3 clinical trials in PTSD patients in combination with psychotherapy. MDMA exerts its effects primarily through potentiation of 5-HT release. In this study, we have evaluated the ability of (±)MDMA, fluoxetine and dexfenfluramine to stimulate release and inhibit uptake of [3H]5-HT from/into rat brain synaptosomes. Synaptosomes are re-sealed pre-synaptic terminals that serve as a functional model of intact neurons as they retain the morphology and molecular machinery required for neurotransmission.

Methods

Frontal cortex from male Sprague–Dawley rats (126-150 g) was dissected and placed in sucrose (0.32 M). Tissue was homogenized (0.5 mm clearance, 12 strokes, 800 rpm) and centrifuged (1500 × g, 10 min, 4°C). The pellet (P1) was discarded, and the supernatant was re-centrifuged (18,000 × g, 10 min, 4°C). The synaptosomal pellet (P2) was resuspended in Krebs–Henseleit (126.5 mM NaCl, 27.5 mM NaHCO₃, 2.4 mM KCl, 0.83 mM MgCl₂, 0.5 mM KH2PO4, 0.5 mM Na₂SO₄, 1.1 mM CaCl₂ and 5.6 mM glucose, pH 7.4) or Krebs-phosphate (126.5 mM NaCl, 13.7 mM Na₂HPO₄, 2.4 mM KCl, 0.83 mM MgCl₂, 0.5 mM KH₂PO₄, 0.5 mM Na2SO4, 1.1mM CaCl2, 11.1mM Glucose, 1 mg/ml ascorbic acid and 50μM pargyline, pH 7.4) buffer equivalent to 8.3mg tissue/mL. Compounds were assessed at ten concentrations (10⁻⁴ to 10⁻¹¹ M). [3H]5-HT uptake: synaptosomes were pre-incubated in a shaking water bath (15 min, 37°C, 80 oscillations/min). Aliquots (150 μL) were added to Krebs–Henseleit buffer (275 μL), buffer (total uptake, 50 μL), compound (50 μL) or fluoxetine (non-specific uptake, 10⁻⁵ M, 50 μL). Uptake was initiated by the addition of [3H]5-HT (2 nM, 25 μL) and continued for 5 min at 37°C. [3H]5-HT release: synaptosomes were pre-incubated with [3H]5-HT (2 nM) in a shaking water bath (20 min, 37°C, 80 oscillations/min). Aliquots were incubated for 5 min with Krebs-phosphate buffer (275 μL), buffer (50 μL, total uptake), compound (50 μL) or dexfenfluramine (non-specific uptake, 10⁻⁵ M; 50 μlL). Assays were terminated by filtration through Whatman GF/C filters, pre-soaked in 0.5% polyethylenimine, using a Skatron cell harvester. Radioactivity was quantified by liquid scintillation counting.

Results

(±)MDMA and dexfenfluramine were 5-HT releasing agents. In contrast, fluoxetine was a potent 5-HT reuptake inhibitor, as summarized in Table 1.

Conclusions

Our data are consistent with literature findings. Using our experimental paradigm, we are able to discriminate between reuptake inhibitors and releasing agents [1].

Reference

1. Partilla JS, Baumann MH, Decker AM, Blough BE, Rothman RB. Chapter 3: Interrogating the activity of ligands at monoamine transporters in rat brain synaptosomes. In: Neurotransmitter Transporters: Investigative Methods, Neuromethods. Vol. 118. Humana Press;2016:41-52.

151

Binding affinities of a range of psychedelics for 5-HT1A, 5-HT2A and 5-HT2C receptors in rodent brain

Albert Carter, Isobel Jones, Keely Porter, Max Hopewell, Michael Burnett, Ian Davies, Wioletta Pijacka, Steve Vickers, Naheed Mirza, Sharon Cheetham and Elizabeth Jagger

Sygnature Discovery

Background and Aims

The pharmacological mode of action of psychedelics such as psilocin, lysergic acid diethylamide (LSS) and 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) has focused on agonism at 5-HT receptors, predominantly 5-HT2A receptors. The aim of this study was to determine the affinity of a range of psychedelics for 5-HT1A, 5-HT2A and 5-HT2c receptors using [3H]8-OH-DPAT, [3H]Cimbi-36 and [3H]mesulergine, respectively, in rodent brain and compare their receptor binding profiles to non-psychedelic compounds.

Methods

Compounds were tested at 10 concentrations (10⁻⁴ to 10−¹¹ M) in the following assays: (i) mouse hippocampal membranes (400 μL, 1.25 mg wet weight tissue/tube) were incubated with [3H]8-OH-DPAT (0.5 nM, 50 μL) and either assay buffer (50 mM Tris, pH 7.7, 4 mM CaCl₂, 0.1% ascorbic acid, 10 μM pargyline, 50 μL), compound (50 μL) or WAY-100635 (1 μM; 50 μL) for 30 min at 25°C; (ii) mouse frontal cortex membranes (400 μL, 1 mg wet weight tissue/tube) were incubated with [3H]Cimbi-36 (0.06 nM, 50 μL) and either assay buffer (50 mM Tris, pH 7.4, 4 mM CaCl₂, 50 μL), compound (50 μL) or 25CN-NBOH (1 μM, 50 μL) for 90 min at 25°C; (iii) rat choroid plexus membranes (400 μL, 0.625 mg wet weight tissue/tube) were incubated with [3H]mesulergine (0.76 nM, 50 μL) and either assay buffer (50 mM Tris, pH 7.4, 4 mM CaCl₂, 0.1% ascorbic acid, 10 μM pargyline, 50 μL) compound (50 μL) or RS-102221 (10 μM, 50 μL) for 30 min at 37°C. Membrane-bound radioactivity was recovered by filtration under vacuum through Whatman GF/A filters, pre-soaked in 0.5% polyethylenimine using a Skatron cell harvester. Filters were rapidly washed with ice-cold wash buffer and radioactivity quantified by liquid scintillation counting. Inhibition constants (K_i values) were calculated by non-linear regression analysis. All assays were conducted in triplicate (n = 3).

Results

Data are summarized in Table 1.

Psilocin, lisuride and LSD exhibit equipotent affinity for all three receptors. 5-MeO-DMT, 5-HT and WAY-100635 have high affinity for 5-HT1A receptors and low-to-moderate affinity for 5-HT2A and 5-HT2C receptors. 25CN-NBOH, DOI and ketanserin demonstrate high affinity for 5-HT2A receptors, moderate-to-low affinity for 5-HT2C receptors and little or no affinity for 5-HT1A receptors. RS-102221 exhibits 19-fold higher affinity for 5-HT2C receptors compared to the 5-HT2A receptor and no significant affinity for 5-HT1A receptors.

Conclusions

198

Netupitant prevents apomorphine-induced emesis, but not all the associated physiological changes indicative of nausea, in ferrets

John A. Rudd¹, Zengbing Lu¹, Longlong Tu¹, Man-Pui Ngan¹ and Wendy Winchester²

¹Chinese University of Hong Kong; ²Nxera Pharma UK Ltd

Introduction/Background and Aims

Netupitant is a tachykinin NK1 receptor antagonist that has well-known anti-emetic properties [1]. However, it was shown that while it can prevent apomorphine-induced emesis in humans, the associated nausea was not blocked [2]. Assaying nausea in animals is problematic, because they are unable to communicate their emotions. Physiological changes associated with nausea include changes behaviour (e.g. altered locomotor activity), gastric myoelectric activity (GMA), heart rate variability (HRV) and temperature, and these can be recorded in preclinical studies by observation and using radiotelemetry and other technologies [3]. The aim of the present studies is investigate if netupitant can prevent both emesis and physiological changes indicative of nausea (PCIN) induced by apomorphine in ferrets.

Methods/Summary of Work

Under general anaesthesia, eight castrated male ferrets (1.16–1.75 kg) were surgically implanted with DSI radiotelemetry transmitters (HD-S11, DSI) to record blood pressure (GMA) and core body temperature (CBT). They were allowed 7 days to recover and then randomized to a crossover design (1-week interval) to receive netupitant (3 mg kg⁻¹, p.o.) or saline (1 mL kg⁻¹, i.p.) 2 h prior to apomorphine (0.25 mg kg⁻¹, s.c.); recordings continued for 30 min post-apomorphine administration.

Results/Discussion

During the 30 min before the administration of apomorphine, the vehicle-treated animals had a diastolic and systolic blood pressures of 88.15 ± 1.93 and 145.69 ± 3.19 mmHg, respectively. Heart rate, HRV and the dominant frequency (DF) of GMA were 222.19 ± 7.50 bpm, 0.075 ± 0.0060 and 9.68±0.29 cpm, respectively; CBT was 38.05 ± 0.098°C. Backward walking and lip licking did not occur. The subsequent administration of apomorphine induced 36.3 ± 7.5 retches and 2.9 ± 0.7 vomits following a latency of 3.8 min; it also induced 24.5 ± 5.3 lip licking episodes (P < 0.01) and 3.1 ± 1.1 backward walking episodes (P < 0.05) and reduced HRV to 0.024 ± 0.0026 (P < 0.001), without affecting significantly blood pressure, GMA or CBT. Netupitant did not have any impact on any of the variables recorded during the pretreatment period, but reduced apomorphine-induced retching and vomiting by 97.6% (P < 0.05) and 96.6 % (P < 0.05), respectively; there was also a 51.8% reduction of lip licking episodes (P < 0.05). The effect of apomorphine to reduce HRV was not antagonized by netupitant. However, the netupitant treated animals had a significant ~1°C lower CBT relative to the vehicle-treated animals that received apomorphine. This may be because they were protected from the physical act of retching and vomiting.

Conclusions

Apomorphine reliably induced retching and vomiting and increased lip licking and backwards walking, as well causing a reduction of HRV. Netupitant predictably reduced apomorphine-induced and retching and vomiting and increases in lip licking activity but did not affect the other PCIN measures. However, as both dopamine D2 and NK1 receptors exist in salivary glands [4, 5], the reduction of apomorphine-induced lip licking by netupitant may not necessarily relate to ‘nausea’. The failure of netupitant to reduce the apomorphine-induced backward walking, or the associated decreases of HRV, may tentatively suggest that the animals are experiencing ‘nausea’.

References

1. Rudd JA, Ngan MP, Lu Z, Higgins GA, Giuliano C, Lovati E, Pietra C. Profile of antiemetic activity of netupitant alone or in combination with palonosetron and dexamethasone in ferrets and Suncus murinus (house musk shrew). Front Pharmacol 2016;7:263.

2. Beitez J. Approval package: application number 205718Orig1S000. 2004. Akynzeo. https://www.accessdata.fda.gov/drugsatfda_docs/nda/2014/205718Orig205711s205000Approv.pdf.

3. Tu L, Lu Z, Dieser K, Schmitt C, Chan SW, Ngan MP, Andrews PLR, Nalivaiko E, Rudd JA. Brain activation by H1 antihistamines challenges conventional view of their mechanism of action in motion sickness: a behavioral, c-Fos and physiological study in Suncus murinus (house musk shrew). Frontiers in physiology 2017;8:412.

4. Tomassoni D, Traini E, Mancini M, Bramanti V, Mahdi SS, Amenta F. Dopamine, vesicular transporters, and dopamine receptor expression in rat major salivary glands. Am J Physiol Regul Integr Comp Physiol 2015;309:R585-593.

5. Beaujouan JC, Saffroy M, Torrens Y, Sagan S, Glowinski J. Pharmacological characterization of tachykinin septide-sensitive binding sites in the rat submaxillary gland. Peptides 1999;20:1347-1352.

242

Salicylaldehyde benzoyl hydrazone alleviates microglial activation in vitro and neuroinflammatory dysfunction in zebrafish in vivo

Niamh Clarke^1,3, Bernadette S. Creavan⁴, Lasse Jensen^5,6, Alison L. Reynolds^2,3 and Derek Costello^1,3

¹School of Biomolecular & Biomedical Science; ²School of Veterinary Medicine; ³Conway Institute; ⁴School of Chemical and BioPharmaceutical Science; ⁵Department of Medical and Health Sciences; ⁶BioReperia AB

Introduction

Neurodegenerative diseases are associated with complex pathological changes in the brain. These include chronic inflammation due to uncontrolled microglial activation and oxidative stress-mediated neuronal cell death. In recent years, the accumulation of iron has been shown to accelerate these changes, predisposing the brain to neurodegeneration. Salicylaldehyde benzoyl hydrazone (SBH) is a Schiff base compound that functions as a tridentate chelating agent, with specific affinity for iron. It has shown efficacy as an anti-bacterial agent and readily complexes with transition metals. More recently, promising anti-inflammatory and anti-oxidant properties have also been revealed. This study sought to explore whether SBH may exert neuroprotective properties in vitro and in vivo.

Methodology

BV2 microglia were challenged with the TLR2 agonist lipoteichoic acid (LTA; 5 μg/mL) in the presence and absence of SBH (10 μM). Inflammatory changes were measured by the release of nitrite (Griess assay) and pro-inflammatory cytokines TNFα and interleukin (IL)-6 (ELISA). SH-SY5Y neuronal cells were exposed to H2O2 and co-incubated with SBH. Cytotoxicity was determined using lactate dehydrogenase (LDH) assay. To examine the impact of SBH in vivo, zebrafish (Danio rerio) larvae 4 days post-fertilization (dpf) were exposed to lipopolysaccharide (LPS; 20 μg/mL; 24 h) in the presence and absence of SBH (10 μM). Larvae were assessed for survival and evidence of toxicity, indicated by gross morphological malformations and loss of the touch startle response. Statistical differences were determined by one-way or two-way ANOVA, followed by Tukey's post-tests.

Results

LTA exposure significantly enhanced the microglial expression of NO, TNFα and IL-6. Co-incubation with SBH significantly attenuated nitrite and TNFα, compared with LTA alone (Table 1; n = 12 replicates, n = 4 independent experiments). H₂O₂ promoted cytotoxicity of SH-SY5Y cells (P < 0.0001), which was significantly alleviated by co-application with SBH (P < 0.01; n = 15). Exposure to LPS reduced survival of zebrafish larvae (P < 0.0001) and increased the incidence of morphological malformations (77.8%) relative to controls (n = 45). This was accompanied by an impairment in the touch startle response (LPS: 45.7 ± 2.4%; n = 35, Control: 100%; n = 45). Co-application of SBH significantly improved survival (P < 0.0001; n = 45) and reduced the incidence of morphological malformations compared with LPS alone (33.3% vs. 77.8%; n = 45). Touch startle was also improved in larvae treated with SBH + LPS (74.4 ± 2.1%; n = 43), compared with LPS (45.7 ± 2.4%; P < 0.0001; n = 35).

Conclusions

SBH alleviated microglial activation and oxidative stress-induced neurotoxicity. Exposure to SBH restored the negative impact of LPS-induced inflammation in zebrafish larvae in vivo. These findings support the further exploration of SBH as a potential multifunctional therapeutic for neurodegenerative disease.

244

Activation of autophagy by tat-beclin 1 reveals a protective mechanism in chemotherapy-induced neuropathic pain

Sofia Fontana-Giusti, Gary Stephens and Maria Maiaru

University of Reading

Introduction

Cancer is the second leading cause of death worldwide, with most treatment approaches relying on chemotherapy drugs. Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating and painful adverse effect of commonly used anticancer drugs such as paclitaxel. Neurotoxic chemotherapeutic drugs are thought to induce molecular changes within nociceptive pathways leading to CIPN, some of which have been linked to autophagy—an essential physiological mechanism contributing to protein and organelle degradation, cellular remodelling and survival. Moreover, there is evidence that autophagy has a role in chronic pain states [1]. We have found that autophagy is disrupted in the spinal dorsal horn of mice with CIPN at the peak of measured pain-like behaviours. The aim of this study was to explore whether inducing autophagy in vivo could improve the CIPN pain phenotype, using tat-beclin 1 [2], a cell permeant analogue of beclin 1, a key component of the class III phosphatidylinositol 3-kinase complex that activates autophagy.

Methods

Eight- to twelve-week-old adult female C57BL6/J mice (Charles River UK) received intraperitoneal injections of paclitaxel (4 mg/kg) on alternating successive days for 8 days [3] according to Animals (Scientific Procedures) Act 1986. Paclitaxel derived from Taxus brevifolia was dissolved 1:1 with ethanol and kolliphor and diluted in physiological saline. Tat-beclin 1 and the lysosomal blocker chloroquine were diluted in saline. Behavioural testing was followed throughout using von Frey filaments for mechanical allodynia and thermal plate preference testing for cold hypersensitivity.

Results

Inducing autophagy with a single 20 mg/kg dose of tat-beclin 1 at the peak of the pain state (40 days post-paclitaxel) provided long-lasting relief from mechanical hypersensitivity lasting nearly 30 days (two-way ANOVA, P < 0.001, n = 5.5). During this period, maximum mechanical hypersensitivity was maintained in the control group, indicating that this was likely not due to any recovery from CIPN pain, which we have characterized as persisting for up to 120 days post-paclitaxel. Tat-beclin 1 had no significant effect on cold hypersensitivity (two-way ANOVA, P = 0.057, n = 5.5). The antinociceptive effects of tat-beclin 1 were abolished in mice receiving 50 mg/kg of chloroquine over three days, further confirming the involvement of autophagy pathways.

Conclusion

Stimulation of autophagy in chronic CIPN via beclin 1 induction causes long-lasting amelioration of mechanical hypersensitivity, which is reversed by autophagy block. These data suggest that pharmacological agents that target such autophagic pathways may have therapeutic utility in chronic neurodegenerative pain states.

References

1. Berliocchi et al. Molecular Pain 2015;11 (1):3.

2. Shoji-Kawata et al. Nature, 2013;494(7436):201-6.

3. Maiarù et al. Pain 2018;159, 1224-1234.

252

Glutamatergic pharmacology and receptor modifications by knocking down of ESCRT proteins

Mohamed Shalaby, Samantha McLean and Harsha Kentameneni

School of Pharmacy and Medical Sciences, University of Bradford, UK

Introduction

As evidence for the N-methyl-D-aspartate (NMDA) receptor hypofunction theory of schizophrenia grows, more attention is being paid to restoring glutamatergic signalling as a therapeutic option to develop novel antipsychotics. The endosomal sorting complexes required for transport (ESCRT) proteins groups are involved in sorting ubiquitinated membrane receptors to lysosomes, which is a key method for reducing cell surface receptor signalling. In this study, we aimed to investigate the effects of knocking down of Tsg101 and Vps4a genes on the expression and functions of ionotropic glutamate receptors.

Methods

NR1a/NR2a and GluK2 transfected in HEK293t cells, treated with shRNA Tsg101 and negative dominant of Vps4a. Mutant receptors were expressed in HEK293t cells, confirmed by western blot, and the surface expression was detected by the live cell biotinylation and immunocytochemistry staining methods. The functions of the mutant receptors were tested by NMDA and kainate agonists binding using calcium fluorescence and whole cell patch clamp recording.

Results

Increased epidermal growth factor receptor (EGFR) accumulation is observed in cells treated with the negative dominant of Vps4a and shTsg101 plasmids. The rising of the binding site mutations, due to the knockdown of Tsg101, is mostly reflected in more binding to the agonists NMDA and kainate, eliminating functional responses to glutamate. Immunocytochemistry staining and cell biotinylation studies showed that the mutant receptors were accumulated intracellularly and trafficked to the plasma membrane. Knocking down of Tsg101 showed a high level of calcium signalling of NMDA receptors, which was sufficient to alleviate the action of phencyclidine (PCP, NMDAR antagonist), and the IC₅₀ of PCP was increased for cells treated with shRNA Tsg101 (19.6 ± 1.6 μm vs. 73 ± 6.1 μm, n = 3, P < 0.01). The overexpression of Gluk2 receptors showed its protective role in decreasing calcium signalling. According to the net results, overexpression of ionotropic glutamate receptors acts to stabilize the neuronal circuits and plasticity.

Conclusions

Our findings suggest that Tsg101 acts as a novel regulator of neuronal membrane receptors trafficking, which may provide a new therapeutic strategy for treating neurodegenerative and psychiatric diseases.

References

1. Collingridge GL, Isaac JT, Wang YT. Receptor trafficking and synaptic plasticity. Nat Rev Neurosci. 2004;5(12):952-962. https://doi.org/10.1038/nrn1556

2. Katzmann DJ, Odorizzi G, Emr SD. Receptor downregulation and multivesicular-body sorting. Nat Rev Mol Cell Biol. 2002;3(12):893-905. https://doi.org/10.1038/nrm973

3. Tanaka N, Kyuuma M, Sugamura K. Endosomal sorting complex required for transport proteins in cancer pathogenesis, vesicular transport, and non-endosomal functions. Cancer Sci. 2008;99(7):1293-1303. https://doi.org/10.1111/j.1349-7006.2008.00825.x

258

Innovative cellular therapy for stroke: Primed MSCs mitigating neuroinflammation and promoting neuroprotection

Maryam Adenike Salaudeen, Stuart Allan and Emmanuel Pinteaux

Division of Neuroscience, Faculty of Biology, Medicine, and Health, University of Manchester

Introduction

Neuroinflammation and disruption of the blood–brain barrier are hallmark features of both ischaemic and haemorrhagic stroke. Mesenchymal stem cell (MSC) therapies present a promising strategy to manage these critical symptoms. This study investigates the therapeutic potential of conditioned media (CM) derived from primed bone marrow-derived MSCs (hBMSCs) in mitigating inflammation and ischaemic damage in microglial cells.

Methods

hBMSCs at P5 were primed with interleukin-1α (10 ng/mL) and cobalt chloride (CoCl₂) (100 μM), either independently or in combination, for 24 h at 37°C and 5% CO₂. After priming, the media (MesenPro supplemented with 1% penicillin/streptomycin and 2% growth serum) was replaced with a fresh one, and CM was harvested 24 h later. BV2 microglia cells (ATCC) were subjected to inflammatory stimulation using endotoxin-free lipopolysaccharide (LPS) (1 μg/mL) and treated with various CM formulations. After 24 h, the supernatants were analysed for inflammatory markers, and cell viability was assessed using lactate dehydrogenase (LDH) assays (Promega, UK).

To mimic ischaemic conditions, BV2 cells were exposed to oxygen and glucose deprivation (OGD) using glucose-free RPMI media (supplemented with 10% heat-inactivated FBS, 1% L-glutamine, 1% penicillin/streptomycin and 100 μM CoCl₂), concurrently treated with the CM from primed hBMSCs. This condition was maintained for 2 h at 37°C and 5% CO₂. Reperfusion injury was simulated by restoring oxygen and glucose for 24 h. Inflammatory cytokines, including interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α) and interleukin-10 (IL-10), were measured, while cell death was assessed using LDH release. Immunocytochemistry was performed to evaluate matrix metalloproteinase-9 (MMP-9) expression following reperfusion.

All experiments were performed in four replicates.

Results

In both LPS-induced inflammation and OGD/reperfusion models, CM from primed hBMSCs significantly (P < 0.05) reduced the levels of pro-inflammatory cytokines IL-6, TNF-α and IL-1β while enhancing anti-inflammatory IL-10 production (Figures 1 and 2). Moreover, MMP-9 expression was markedly decreased in BV2 cells treated with CM from both naïve and primed hBMSCs (Figure 3). Although CM from all hBMSCs reduced cell death, only dual-primed CM demonstrated a significant protective effect in reducing cell death (Figure 2D).

Conclusions

Priming MSCs with inflammatory and hypoxic cues enhances their therapeutic potential, making them a promising approach for treating neuroinflammation and reperfusion injury in stroke. These findings support the use of MSC-based therapies in neurological conditions characterized by such pathological features.

54

Technical and reporting quality in rodent studies on diabetic nephropathy reported in 2018–2022

Aqsa Ashfaq¹, Oyku Bese^1,2 and Martin Michel^1,3

¹Johannes Gutenberg University; ²Ankara University; ³Partnership for the Assessment and Accreditation of Scientific Practice

Introduction

Many publications lack crucial information required for replication and limited measures and reporting to reduce biases (Ramirez et al., 2017). This may contribute to an overall limited robustness of published work. As the last report on this is from 2017, we wished to explore the reporting quality in more recent articles.

Methods

A recent systematic review of renal effects of SGLT2 inhibitors in rodent models of diabetes had identified 102 studies published in 2018–2022 (Ashfaq et al., 2023). We analysed the reporting in those reports in four major categories including general transparency of reporting, technical reproducibility, general factors related to robustness and statistical analysis reporting.

Results

Data for all statements are shown in Table 1. The general reporting quality was good, that is, about 90% included the necessary information on animal committee approval and disclosure of funding and conflicts of interest; however, only a quarter of studies included statements on raw data availability. Providing of information to technically enable reproducibility was heterogeneous: Information required in the ARRIVE guidelines ranged from 2.9% (housing conditions) to 96.0% (sex), and the specific animal strain was identifiable in 53.9%. In studies using antibodies, the specific antibody was identifiable in 12.7%; RRIDs were provided in 2.9%. Related to data robustness, 76.4% reported specific sample sizes per group. While 9.8% reported randomization, 5.8% disclosed the randomization method. For statistical analysis, sample size justification 4.9% and handling of outliers were reported in 5.8% studies. The specific statistical test was identifiable for each parameter in 65.6%. The denominator used for normalization was disclosed in 72.5%, but quantification of the denominator in each group was not reported in any study. Importantly, none of the 102 studies had a prespecified primary endpoint or a statistical analysis plan.

Conclusions

Despite being discussed as relevant for more than 20 years, reporting quality remains limited. This specifically includes adherence to the ARRIVE guidelines. While all studies included some type of statistical analysis and had assessed multiple parameters, none of them identified a primary endpoint or had a prespecified statistical analysis plan. We conclude that the reporting quality in animal studies must improve further.

References

1. Ashfaq et al. Pharmacol Ther 2023;239: e108503.

2. Ramirez et al. Circ Res 2017;120:1916-1920.

69

Disulfiram inhibits locomotor activity and induces body fragmentation of Lumbriculus variegatus

Grace Labdon, Yasmin Sofi, Matthew McKinlay, Nia Davies, Lisa Wallace and Aidan Seeley

Swansea Worm Integrative Research Laboratory (SWIRL), Swansea University

Introduction

Disulfiram is used in the treatment of alcohol use disorder (AUD) and exerts its pharmacological effect through inhibition of aldehyde dehydrogenase (ALDH). We demonstrate that disulfiram has an effect on Lumbriculus variegatus, an aquatic, asexually reproducing and regenerative annelid worm. We describe the behavioural effects of disulfiram exposure and the occurrence of body fragmentation when exposed to disulfiram.

Method

Disulfiram was dissolved in 100% DMSO before dilution in artificial pond water [2] for a final DMSO concentration of 0.5%. Toxicity was determined by exposure of L. variegatus to 0–50 μM disulfiram for 24 h with tissue pallor and/or tissue decomposition used as identifier of toxicity. L. variegatus which displayed fragmentation, whereby L. variegatus split into two or more fragments was also recorded. The effect of 24-h exposure to 0–1 μM disulfiram on locomotor activity and the effect on tactile stimulation to elicit stereotypical behaviours was conducted as previously described [2].

Results

Disulfiram displayed toxicity in 50% of the test population at 14.05 μM (95% CI: 9.92–19.92 μM, n = 7), with a no observable adverse effect level at 1 μM. Moreover, there was a significant induction of L. variegatus fragmentation at 10 μM (P < 0.05, n = 7). Twenty-four-hour exposure to disulfiram 0–1 μM showed that 1 μM decreased locomotor activity of L. variegatus (P = 0.049, n = 8) with effects persisting 24 h after removal from disulfiram (P = 0.035, n = 8). Exposure to disulfiram for 24 h had no effect on the ability of tactile stimulation to elicit body reversal or helical swimming movements (P > 0.05, n = 8). After removal from disulfiram and incubation in artificial pond water only, ≥0.5 μM disulfiram was shown to decrease responses to tactile stimulation (P < 0.05, n = 8).

Conclusion

Here, we demonstrate that disulfiram is capable of reducing L. variegatus locomotor activity and response to tactile stimulation. Additionally, we observe that disulfiram induces L. variegatus fragmentation, which is primarily a mechanism for survival and asexual reproduction, through an as-yet unidentified mechanism.

References

1. Sapi E, Biniaz-Harris N, Kuvaldina M, et al. Disulfiram: mechanisms, applications, and challenges. Antibiotics (Basel) 2023;12(3). doi: https://doi.org/10.3390/antibiotics12030524

2. Seeley A, Bellamy C, Davies NA, Wallace MJ. Lumbriculus variegatus: a novel organism for in vivo pharmacology education. Pharmacol Res Perspect 2021;9:e00853. https://doi.org/10.1002/prp2.853

96

Fostering independent academic research and forging new academic/pharma partnerships: Case studies and outcomes from Boehringer Ingelheim's opnMe Platform

Markus Koester, Menorca Chaturvedi, Sven Thamm, Florian Montel, Deepa Ghosh, Claudia Heine, Michaela Walter, Martin Graf, George Augustine, Judith Schweimer, Thomas Wollmann, Heike Schauerte and Georg Rast

Boehringer Ingelheim

Introduction

Aiming to facilitate the diffusion of academic and industry research, Boehringer Ingelheim's open innovation platform, opnMe.com, has shared >2400 well-characterized chemical probes with scientists in 53 countries for free. This led to >180 publications and enabled >120 research collaborations to date. We focus on opnMe's impact in fostering novel insights by highlighting research examples, covering independent use of chemical probes as well as collaborative research.

Results

The first study introduces an enzyme-responsive hydrogel for on-demand release of BI-4394, a potent MMP-13 blocker, to treat early-stage osteoarthritis (OA) [1]. The hydrogel, made with triglycerol monostearate, showed reduced inflammation and bone erosion in a rat OA model compared to weekly BI-4394 injections. The hydrogel increased collagen-2 and aggrecan levels while reducing MMP-13, indicating effective cartilage degradation prevention.

The second study used optogenetics technologies to study complex neuronal interconnectivity within the claustrum. In the claustrum, two different output neuron populations respond in opposite ways to acetylcholine and GABA co-released by the cholinergic system [2]. This differentially alters neuronal gain and dynamic range in the two neuron types and revealed a microcircuit basis for attention- and learning-related cholinergic computations within the claustrum that increases the flexibility to the cholinergic system.

In the final study, ‘DeepRod’, a human-in-the-loop system for automated rodent behaviour analysis, was designed to aid in preclinical drug discovery [3]. The system uses active learning and machine learning to identify and classify behaviours from video data, significantly improving efficiency and accuracy. The system has proven effective in discovering and annotating rare behaviour types, improving model accuracy and broadening the spectrum of detectable behaviours.

Conclusions

Initiatives such as opnMe foster an environment that accelerates innovation and discovery. A wide spectrum of research fields benefitted from this approach and may translate into novel therapeutic options.

References

1. Roy HS, Murugesan P, Kulkarni C, Arora M, Kumar Nagar G, Guha R, Chattopadhyay N, Ghosh D. On-demand release of a selective MMP-13 blocker from an enzyme-responsive injectable hydrogel protects cartilage from degenerative progression in osteoarthritis. J Mater Chem B 2024;12:5325. https://doi.org/10.1039/d3tb02871b

2. Nair A, Teo YY, George J, Augustine GJ, Graf M. A functional logic for neurotransmitter corelease in the cholinergic forebrain pathway. PNAS 2023;120(28):e2218830120. https://doi.org/10.1073/pnas.2218830120

3. Loy A, Garafolj M, Schauerte H, Behnke H, Charnier H, Schwarz P, Rast G, Wollmann T. DeepRod: a human-in-the-loop system for automatic rodent behavior analysis. bioRxiv preprint article. 2024. https://doi.org/10.1101/2024.01.04.572506.

134

Glycogen content in detrusor muscle of the bladder in five diabetic rodent models

Participant Öykü Deniz Bese^1,2, Ebru Arioglu-Inan², Ralf Elvert³, Martin C. Michel¹ and Myriam Meineck¹

¹Johannes Gutenberg University; ²Ankara University; ³Sanofi Research and Development

Introduction

Glycogen content has been suggested as a potential marker for assessing the extent of deterioration in urinary bladder function (de Jong et al., 2008) and is typically assessed by periodic acid Schiff (PAS) staining. Although bladder dysfunction is one of the most common complications in diabetes, most of the findings on glycogen content are derived from animal models of bladder outlet obstruction (Mitsogiannis et al., 2022), whereas none has focused on the diabetic bladder. Therefore, we have explored PAS staining of bladder sections generated from four studies representing five distinct rodent models of diabetes.

Methods

Bladder tissue was obtained from existing tissue of models of both type 1 (T1DM) diabetes and type 2 (T2DM) diabetes; thus, no additional animal was treated or killed for the purpose of this study. The rodent groups were (I) female Sprague-Dawley rats treated with intraperitoneal streptozotocin (STZ) injection (T1DM) and treatment with empagliflozin in some rats; (II) female Wistar rats induction on a high fat diet (HFD), followed by low-dose intraperitoneal STZ injection (T2DM) and treatment with valsartan in some rats; (III) male Zucker fatty and spontaneously hypertensive (ZSF) rats (T2DM); and (IV) male and female ob/ob and db/db mice (T2DM). Ten images were taken randomly at 40× magnification from each of the bladder sections and scored by an investigator blinded to group allocation. Ten images were analysed based on how many of them showed glycogen accumulation. This was converted into a score where 0, 1, 2 and 3 mean 0–1, 2–4, 5–7 or 8–10 out of 10 positive fields. Data were analysed by non-parametric tests (Mann–Whitney for ZSF study; Kruskal–Wallis followed by Dunn's multiple comparison tests for all others); based on the exploratory nature of the study, the resulting P-values should not be interpreted as hypothesis-testing.

Results

The PAS score was increased in all five diabetes models relative to the respective controls with a low P-value < 0.05 (I, II, IV) in although it was 0.0671 for ZSF rats (Figure 1). While treatment with empagliflozin abolished the increase in the PAS score in STZ rats, treatment with valsartan had no effect in HFD/STZ rats.

Conclusions

An increased glycogen content of the bladder as assessed by PAS staining was a consistent feature across five rodent models of diabetes. These results encourage further examination of glycogen accumulations in bladder tissue to confirm it as characteristic for type 1 and 2 diabetes, as well as potentially correlating their emergence to disease severeness. Future studies to understand the mechanisms behind increased glycogen content and its possible contribution to diabetes-associated bladder dysfunction are to be considered.

References

1. deJong BW, Wolffenbuttel KP, Scheepe JR, Kok DJ. The detrusor glycogen content of a de-obstructed bladder reflects the functional history of that bladder during PBOO. Neurouol Urodyn 2008;27(5):454-460.

2. Mitsogiannis I, Komninos C, Karakosta A, Papatsoris A, Skolarikos A, Tzelves L. Glycogen deposition in the detrusor muscle of patients with bladder outlet obstruction (BOO) due to benign prostate hyperplasia (BPH); correlation with the urodynamic parameters. World J Urol 2022;40(12):3029-3034.

136

Optimization of specimen handling and mitochondrial analysis in patient skeletal muscle biopsies

Maheen Wahid¹, Graeme MacKenzie¹, Liam Rooney¹, Emilie Combet², Stuart Gray³, James Murray⁴, Gwyn Gould¹ and Margaret Cunningham¹

¹University of Strathclyde; ²School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow; ³School of Cardiovascular and Metabolic Health, University of Glasgow; ⁴Swansea University Medical School, Swansea University

Introduction

Skeletal muscle biopsies are valuable in pharmacological research for identifying drug targets in muscle-related conditions. Traditional freezing methods cause artefacts that can lead to misinterpretation of research findings [1]. Proper handling of muscle biopsies is critical for accurate histopathological and mitochondrial analysis. Preserving the entire tissue is essential, especially for small needle biopsies. While most research focuses on mitochondrial analysis in cells, there are few studies on tissue samples [2]. This study aimed to improve cryopreservation techniques for muscle biopsies and create a reliable method for mitochondrial analysis in muscle tissues.

Methods

Human and rat muscle samples were preserved with different concentrations of formaldehyde after freezing with liquid nitrogen to study effects of freeze–thaw cycles. We compared the edge and belly of muscle samples embedded in optimal cutting temperature compound (OCT) to see how OCT affects ice crystal formation. Rat muscle biopsies were frozen using direct liquid nitrogen immersion, liquid nitrogen with OCT dip, liquid nitrogen in a histocassette, pre-cooled isopentane immersion, pre-cooled isopentane with OCT dip and pre-cooled isopentane in a histocassette. The effectiveness of these six methods was evaluated using histological and immunohistochemical staining. Mitochondrial analysis in type I and II myofibres was attempted by employing the Trainable Weka Segmentation plugin using Fiji.

Results

Picrosirius red-stained human tissue sections showed that freeze–thaw led to freezing artefacts, disrupted endomysium and widely spaced cells. Quantitative differences in ice crystals between the edge and belly of rat whole muscle samples demonstrated effects of OCT in crystal formation. Picrosirius red and haematoxylin and eosin-stained tissue sections from rat muscle biopsies frozen in six different cryopreservation techniques revealed that only isopentane/histocassette combination preserved tissue integrity in both core and periphery of tissue sections. Moreover, an optimized Fiji workflow enabled quantification and mapping of mitochondrial networks.

Conclusions

The isopentane/histocassette combination was the most effective cryopreservation method, ensuring artefact-free preservation of both the core and periphery of the tissue sections. Our workflow utilizing Trainable Weka Segmentation plugin provided a reliable method for mitochondrial analysis in skeletal muscle tissues, facilitating future studies in muscle research.

References

1. Meng H, Janssen PML, Grange RW, Yang L, Beggs AH, Swanson LC, Cossette SA, Frase A, Childers MK, Granzier H, Gussoni E, Lawlor MW. Tissue triage and freezing for models of skeletal muscle disease. J Vis Exp 2014;(89). https://doi.org/10.3791/51586.

2. Hemel IMGM, Engelen BPH, Luber N, Gerards M. A hitchhiker's guide to mitochondrial quantification. Mitochondrion 2021;59:216-24. https://doi.org/10.1016/j.mito.2021.06.005.

137

6-Nitrodopamine: A novel and potent endogenous modulator of mouse urinary bladder relaxation

Mariana G. de Oliveira², José Britto-Júnior¹, Douglas R. M. Dias², Luise G. S. Pereira², Silvana Chiavegatto³, Idam Hermawan⁴, Hiroaki Shimokawa, Hiroaki Shimokawa⁵, Masato Tsutsui⁴ and Gilberto De Nucci^1,3

¹University of Campinas; ²Sao Francisco University; ³University of Sao Paulo; ⁴University of the Ryukyus; ⁵International University of Health and Welfare

Introduction

6-Nitrodopamine (6-ND) modulates vas deferens [1] and seminal vesicles [2] contractility; however, its role in lower urinary tract organs has not been evaluated.

Methods

Male and female C57BL/6 (10–15 weeks old) wild type (WT) mice, along with male mice knockouts to endothelial (eNOS−/−), neuronal (nNOS−/−), inducible (iNOS−/−) or triple (n/i/eNOS−/−) nitric oxide synthase (NOS), were used. Basal release of catecholamines was determined by liquid chromatography coupled to tandem mass spectrometry, with or without sodium channel blocker tetrodotoxin (10 μM) [1,2]. Concentration–response curves to 6-ND were generated in carbachol-pre-contracted bladders, with or without the NOS inhibitor L-NAME or the soluble guanylyl cyclase inhibitor ODQ. Concentration–response curves to acetylcholine were generated following 6-ND pretreatment (0.1–10 μM). Electrical field stimulation (EFS; 1–16 Hz) was used to assess bladder contractility.

Results

Male and female WT mice showed 6-ND release comparable to dopamine and adrenaline, while noradrenaline levels were below the LOQ in all tested samples (Figure 1A–D). 6-ND release was similar in eNOS−/− (Figure 1E) or iNOS−/− mice (Figure 1F) compared to WT but significantly reduced in nNOS−/− (Figure 1G) and abolished in n/i/eNOS−/− (Figure 1H) knockout mice. Tetrodotoxin reduced 6-ND release in WT bladders (P < 0.05, n = 5), but not in nNOS−/− (n = 5). 6-ND induced concentration-dependent bladder relaxations (19.5% ± 3.7%, n = 5; Figure 2A), independently of eNOS (23.3% ± 5.0%, n = 5; Figure 2A) or sGC (17.5% ± 1.53%, n = 5; Figure 2A) inhibition, demonstrating high potency (pEC50: 8.04 ± 0.86). Additionally, 6-ND significantly reduced the maximal response to acetylcholine in a concentration-dependent manner (n = 5; Figure 2B). Bladders from nNOS−/− and n/i/eNOS−/− mice exhibited higher contractile responses to EFS than eNOS−/−, iNOS-/- or WT bladders (P < 0.05), which was reversed by co-incubation (30 min) with the bladder mucosal layer from a donor WT mouse (P < 0.05, n = 5).

Conclusions

6-ND is released in the urinary bladders of both male and female mice, reduced in nNOS−/− mice, and abolished in triple NOS−/− knockout mice. As a novel endogenous relaxant, 6-ND is 10 times more potent than noradrenaline in inducing relaxation [3]. Inhibition of its release is linked to bladder hypercontractility.

References

1. Britto-Júnior J, Nacário Silva SG, Lima AT, et al. The pivotal role of neuronal nitric oxide synthase in the release of 6-nitrodopamine from mouse isolated vas deferens. Nitric Oxide 2023;1-8. https://doi.org/10.1016/j.niox.2023.12.002.

2. Britto-Júnior J, Uramoto EHS, Lima AT, et al. Epithelium-derived 6-nitrodopamine modulates noradrenaline-induced contractions in human seminal vesicles. Life Sci 2024;122695. https://doi.org/10.1016/j.lfs.2024.122695.

3. Canda A, Chapple C, Chess-Williams R. Pharmacologic responses of the mouse urinary bladder. Open Medicine 2009;4(2):192-197. https://doi.org/10.2478/s11536-008-0082-2.

229

The IUPHAR/BPS Guide to PHARMACOLOGY: Open, accessible and expert-curated pharmacology

Simon Harding¹, Jane Armstrong¹, Elena Faccenda¹, Chris Southan¹, Stephen Alexander², Anthony Davenport³, Michael Spedding⁴ and Jamie Davies¹

¹University of Edinburgh; ²School of Life Sciences, University of Nottingham; ³Experimental Medicine and Immunotherapeutics, University of Cambridge; ⁴Spedding Research Solutions SAS

Introduction

The IUPHAR/BPS Guide to PHARMACOLOGY (GtoPdb; www.guidetopharmacology.org) (1) is an open-access, online database of pharmacological targets and the substances that act on them. Our expert-driven curatorial processes, selection stringency and regular update schedule differentiate the GtoPdb from resources that cover similar subject areas.

GtoPdb contains succinct overviews and key references for ~2000 human protein targets and data on ≥12,700 ligand molecules including approved drugs, small molecules, peptides and antibodies.

Here, we present updates on recent curation, including a focus on collaborations that improve coverage of antibacterial and natural products.

Method

The development of GtoPdb is overseen by the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), data being selected by its subcommittees and expert curators to include established drug targets as well as those of emerging interest for drug discovery. Curation and database development is conducted by the University of Edinburgh GtoPdb Curation Team, with regular database updates. GtoPdb is recognized as both an ELIXIR-UK and Global Core Biodata resources.

Results

GtoPdb continues its collaboration with AntibioticDB (www.antibioticdb.com) to identify and improve the antibacterial compound coverage in GtoPdb and to build reciprocal links between the two resources. Currently, over 560 antibacterial compounds have been curated in GtoPdb. This work is supported by the Global Antibiotic Research and Development Partnership (GARDP), whose mission is to combat the worldwide crisis of antibiotic resistance.

Natural products (NPs) offer an invaluable source of biologically active compounds and are well recognized for their potential in drug discovery and development. Safe and effective drug development using NPs requires validation of the pharmacological profile of any potentially useful NP. To achieve these objectives IUPHAR (https://iuphar.org/) and the Italian Society of Pharmacology (SIF; https://www.sifweb.org/) are collaborating to provide an expert-driven project to curate NPs as a resource within the Guide to PHARMACOLOGY.

GtoPdb's coverage of drugs in development has been extended by including resources such as ‘first disclosure’ sessions at scientific conferences and the most recent lists of proposed INNs. In the last year, we have added over 800 quantitative interactions to GtoPdb, and full details of new additions and updates for each version of the database are collated in our freely accessible blog posts (https://blog.guidetopharmacology.org/category/database-updates).

Reference

1. Harding SD, Armstrong JF, Faccenda E, et al. The IUPHAR/BPS guide to PHARMACOLOGY in 2024. Nucleic Acids Res 2024;52(D1):D1438-D1449. https://doi.org/10.1093/nar/gkad944

240

opnMe.com: The open innovation portal of Boehringer Ingelheim

Sven Thamm, Menorca Chaturvedi, Oliver Kraemer, Florian Montel and Markus Koester

Boehringer Ingelheim

Introduction

Open science is a set of principles and practices that aim to make scientific research from all fields accessible to everyone for the benefits of scientists and society as a whole. Open science is about making sure not only that scientific knowledge is accessible but also that the production of that knowledge itself is inclusive, equitable and sustainable. Through its online platform opnMe.com, Boehringer Ingelheim aims to foster innovation, attract talent and enhance our scientific reputation. Its effectiveness has been demonstrated through various initiatives designed to increase its appeal externally. By its seventh anniversary in November 2024, opnMe will have established over 120 research partnerships through platforms like Molecules for Collaboration (M4C), opn2EXPERTS and techMATCH.

Our Molecules to Order (M2O) programme has shipped over 7000 molecule batches to scientists in 50+ countries, leading to more than 180 publications. Molecules to order comply with criteria for high-quality chemical probes as laid out by Arrowsmith et al. [1] and are also made available through the Structural Genomic Consortium's (SGC) Donated Chemical Probes (DCP) initiative.

opnMe's greatest value lies in its impact on therapeutic area pipelines. The Molecules for Collaboration (M4C) programme has led to 30 collaborations, validating six out of 13 new therapeutic concept (NTC) hypotheses and initiating nine exploratory projects. This has indirectly supported 14 in-house projects, resulted in two publications so far and fostered collaborations with external experts.

While it is early for a comprehensive review of the opn2EXPERTS programme launched in 2020, it is already impacting our pipeline, as shown by a call on Crohn's disease and the development of a platform using computer vision approaches for rodent behaviour analysis. We have also launched techMATCH in 2024 to access innovative technologies, proving successful with a call on sustainable drug delivery devices.

Conclusions

Initiatives such as opnMe foster an environment that accelerates innovation and discovery. A wide spectrum of research fields benefitted from this approach and may translate into novel therapeutic options.

Reference

1. Arrowsmith C, Audia J, Austin C, et al. The promise and peril of chemical probes. Nat Chem Biol 2015;11:536–541. https://doi.org/10.1038/nchembio.1867

287

Investigation of the relaxing effect of cryptotanshinone on isolated guinea pig tracheal smooth muscle: Role of epithelial NO

Naima Rodwan¹, Aysegul Koc², Saliha Ayşenur C. A. M. Ozunlu², Fatma Uysal², Muhammet Zahit Celik², Halil Kara² and Seyfullah Oktay Arslan²

¹Institute of Health Sciences, Ankara Yıldırım Beyazıt University; ²Department of Medical Pharmacology, Faculty of Medicine, Ankara Yıldırım Beyazıt University

Introduction

Asthma is a respiratory disease characterized by airway inflammation, airway obstruction and increased airway hyperresponsiveness expressed by airway smooth muscle hyperconstriction [1]. Cryptotanshinone is a bioactive terpenoid molecule with a variety of biological activities including vasodilator, anti-inflammatory, antioxidant, anti-angiogenic and antiproliferative properties [2–6]. This study aims to examine the relaxing effects of cryptotanshinone on isolated guinea pig tracheal muscles and identify possible underlying mechanisms.

Methods

The trachea was obtained from guinea pigs following euthanasia with ketamine/xylazine (40 mg/kg/5 mg/kg, i.p.). The trachea was carefully dissected, and six to eight strips were obtained from each animal. Strips were mounted in a 10-mL organ bath containing Krebs–Henseleit solution for isometric tension studies. Baths were aerated with 95% O₂ and 5% CO₂ at 37°C. Isolated trachea tissues were pre-contracted by carbachol (CCh, 1 μM) and KCl (80 mM). Effective concentrations of cryptotanshinone were based on our preliminary studies (n = 3). Cumulative doses of cryptotanshinone (10⁻⁷ to 3 × 10⁻⁴ M) on CCh-mediated contractile responses in NO-dependent and NO-independent (L-NAME, 10⁻⁴ M) were evaluated. The relaxation effect was expressed as the percentage decrease of tonic contraction induced by CCh (1 μM). Concentration–relaxation curves were generated cumulatively.

Results

Cryptotanshinone showed a significant relaxant effect in tracheal strips contracted by CCh (1 μM). In NO-dependent and NO-independent groups, cryptotanshinone (10⁻⁷ to 3 × 10⁻⁴ M) significantly reduced CCh-induced contractile response. In the NO-dependent group, Cryptotanshinone at concentrations of 10⁻⁴ and 3 × 10⁻⁴ M reduced contraction responses by 30.4% ± 2.73 and 41.9% ± 2.58, respectively (P < 0.05; n = 8). In the NO-independent group, cryptotanshinone at concentrations of 10⁻⁴ and 3 × 10⁻⁴ M decreased contractile responses by 24.3% ± 2.91 and 33.5% ± 1.37, respectively, in a concentration-dependent manner (P < 0.05; n = 8). Cryptotanshinone-induced relaxations at higher concentrations (10⁻⁴ and 3 × 10⁻⁴ M) were significantly less in the NO-independent group compared to the NO-dependent group (P < 0.01). Moreover, the relaxing effect of Cryptotanshinone at the cumulative concentration on the carbachol-induced contraction of the tracheal ring was more potent than that induced by high K+ (80 mM). Statistical comparison between the groups was performed by the non-parametric Mann–Whitney U test.

Conclusions

This study has shown that cryptotanshinone has a relaxing effect on tracheal smooth muscle. This effect may be mediated by nitric oxide in airway smooth muscle. These results may establish the basis for employing cryptotanshinone in the therapeutic management of respiratory diseases, like asthma, that are characterized by the hypercontractility of the smooth muscle in the airways.

References

1. Martinez FD, Vercelli D. Asthma. Lancet (London, England) 2013;382(9901):1360-1372. https://doi.org/10.1016/S0140-6736(13)61536-6

2. Wang Y, Lu HL, Liu YD, et al. Cryptotanshinone sensitizes antitumor effect of paclitaxel on tongue squamous cell carcinoma growth by inhibiting the JAK/STAT3 signaling pathway. Biomed Pharmacother 2017;95:1388-1396. https://doi.org/10.1016/J.BIOPHA.2017.09.062

3. Qi P, Li Y, Liu X, et al. Cryptotanshinone suppresses non-small cell lung cancer via microRNA-146a-5p/EGFR axis. Int J Biol Sci 2019;15(5):1072. https://doi.org/10.7150/IJBS.31277

4. Chen L, Yang Q, Zhang H, et al. Cryptotanshinone prevents muscle wasting in CT26-induced cancer cachexia through inhibiting STAT3 signaling pathway. J Ethnopharmacol 2020;260. https://doi.org/10.1016/J.JEP.2020.113066

5. Luo Y, Song L, Wang X, et al. Uncovering the mechanisms of cryptotanshinone as a therapeutic agent against hepatocellular carcinoma. Front Pharmacol 2020;11. https://doi.org/10.3389/FPHAR.2020.01264/FULL

6. Lam FFY, Yeung JHK, Chan KM, Or PMY. Mechanisms of the dilator action of cryptotanshinone on rat coronary artery. Eur J Pharmacol 2008;578(2-3):253-260. https://doi.org/10.1016/J.EJPHAR.2007.09.040

66

The effects of environmentally relevant concentrations of CBD and CBD-related compounds using the invertebrate model Lumbriculus variegatus

Benjamin Williams, Georgeena Jomy, Megan Flanagan, Grace Hawkes, James McRobbie-Aston, Nia Davies, Lisa Wallace and Aidan Seeley

Swansea Worm Integrative Research Laboratory (SWIRL), Swansea University

Introduction

Cannabidiol (CBD) is a major non-psychoactive phytocannabinoid that has been detected in wastewater [1]. However, CBD's ecotoxicological effects remain unknown. We examine the in vivo effects of environmentally relevant concentrations of CBD [1] and its metabolite 7-hydroxycannabidiol (7-OH-CBD) [2] using the ecological indicator species, Lumbriculus variegatus.

Methods

CBD and 7-OH-CBD were dissolved in 100% DMSO or methanol, respectively, before dilution in artificial pond water [3] for a solvent concentration of 0.5% to give a final concentration of 0–5 μM. The ability of tactile stimulation to elicit stereotypical behaviours and the effect on unstimulated locomotor activity were conducted as previously described [3]. Oxygen consumption following exposure to 0–5 μM CBD or 7-OH-CBD was measured using Jenway 352012, and effects on blood vessel pulse rate was determined using the Nikon SMZ1270i. Energy reserves in L. variegatus homogenate was determined by the phenol-sulphuric acid method for carbohydrates, the vanillin-sulphuric acid assay for lipids and the Bradford assay for proteins.

Results

24-h exposure to CBD or 7-OH-CBD decreased tactile stimulation response to elicit stereotypical behaviours ≥2.5 μM or 5 μM, respectively. 5 μM CBD resulted in a significant decrease in locomotor activity (P = 0.002, n = 8), while no effect on locomotor activity was observed following 24-h exposure to 7-OH-CBD (P > 0.05, n = 8). 0–5 μM CBD had no effect on L. variegatus oxygen consumption (P > 0.05, n = 3), but ≥2.5 μM significantly reduced dorsal blood vessel pulsation rate (P < 0.05, n = 3). Conversely, L. variegatus oxygen consumption increased after 24-h exposure to 5 μM 7-OH-CBD (P = 0.034, n= 3) with no significant effect on pulse rate (P > 0.05, n = 3). Exposure to ≤2.5 μM 7-OH-CBD for 72 h did not affect energy reserves in L. variegatus homogenate, while 2.5 μM CBD resulted in a significant decrease in carbohydrates (P = 0.025, n = 6), increased lipids (P = 0.040, n = 6) and no effect on proteins (P >0 .05, n = 6).

Conclusion

We demonstrate that environmentally relevant concentrations of CBD can reduce L. variegatus behaviours, decrease pulsation rates and alter energy reserves in vivo. Therefore, these compounds, once released into the environment, merit further study to minimize ecological effects.

References

1. Mastroianni N, Postigo C, deAlda ML, Barcelo D. Illicit and abused drugs in sewage sludge: method optimization and occurrence. J Chromatogr A 2013;1322. https://doi.org/10.1016/j.chroma.2013.10.078.

2. Zhang Q, Melchert PW, Markowitz JS. Pharmacokinetic variability of oral cannabidiol and its major metabolites after short-term high-dose exposure in healthy subjects. Med Cannabis Cannabioids 2024;7(1). doi: https://doi.org/10.1159/000535726.

3. Seeley A, Bellamy C, Davies NA, Wallace MJ. Lumbriculus variegatus: a novel organism for in vivo pharmacology education. Pharmacol Res Perspect 2021;9:e00853. https://doi.org/10.1002/prp2.853.

157

Protective potentials of rituximab, prednisolone and lisinopril on acrylamide-induced nephropathy: a comparative study in male Wistar rats

Olufunke Olorundare¹, Adeoye Idris² and Adejuwon Adeneye³

¹Department of Pharmacology and Therapeutics, Faculty of Basic Clinical Sciences, College of Health Sciences, University of Ilorin; ²Department of Pharmacology and Therapeutics, College of Health Sciences, Ladoke Akintola University of Technology; ³Department of Pharmacology, Therapeutics and Toxicology, Faculty of Basic Clinical Sciences, Lagos State University College of Medicine

Introduction

Acrylamide (ACR), a carcinogen formed in carbohydrate- and asparagine-rich foods exposed to high temperatures, presents serious health risks due to its neurotoxic, mutagenic, carcinogenic and nephrotoxic effects [1,2]. This study investigated the ameliorative potential of rituximab, prednisolone and lisinopril on ACR-induced nephropathic Wistar rats.

Methods

Forty rats were divided into five groups: a control group, an ACR-induced group and three treatment groups receiving either rituximab, prednisolone or lisinopril after ACR exposure. Throughout the 28-day experiment, weekly body weight and daily urine volume were measured. Renal function tests included urine protein, plasma urea, creatinine, cystatin C and KIM-1 levels using ELISA kits. Immunology markers (C3 and C4) and inflammatory markers (CRP, IL-1β and TNF-α) were assessed using standard methods [3]. The immunohistochemistry of kidney tissues was analysed using caspase-9, caspase-3, BCL-2 and BAX expression, while the histopathological evaluation examined tubular and glomerular injury [4]. Statistical analysis was done using one-way ANOVA with Tukey's post hoc test, considering differences significant at P ≤ 0.05.

Results

ACR significantly caused kidney damage, weight loss, reduced urine output, increased proteinuria, urea and creatinine levels, along with elevated inflammatory and apoptotic markers. Rituximab significantly reversed ACR-induced nephropathy by reducing proteinuria, lowering serum urea, creatinine and inflammatory markers (CRP, TNF-α and IL-1β) and mitigating kidney injury molecule (KIM-1) levels. It also restored C3, C4 and suppressed caspase-3, caspase-9 and BAX, while histopathology showed improved kidney structure. Prednisolone and lisinopril exhibited less efficacy in reversing ACR-induced renal damage, with lisinopril showing minimal impact on apoptotic markers.

Conclusions

The study concluded that rituximab's protective effect against ACR-induced nephropathy involves anti-inflammatory, anti-apoptotic and immune-modulating mechanisms, repositioning it as a potential therapeutic agent for ACR-induced renal damage.

References

1. Kandemir FM, Yıldırım S, Kucukler S, Caglayan C, Darendelioğlu E, Dortbudak MB. Protective effects of morin against acrylamide-induced hepatotoxicity and nephrotoxicity: a multi-biomarker approach. Food Chem Toxicol 2020;138:111190.

2. Başaran B, Çuvalcı B, Kaban G. Dietary acrylamide exposure and cancer risk: A systematic approach to human epidemiological studies. Foods 2023;12(2):346.

3. Idris AO, Olorundare O. Chloroquine attenuates acrylamide-induced nephropathy in male Wistar rats. J Clin Nephrol Res 2024;11(2):1121.

4. Olorundare OE, Akinsola AO, Ajayi AM, Atolani O, Soyemi SS, Mgbehoma AI, Albrecht RM. Anti-apoptotic and antioxidant mechanisms may underlie the abrogative potential of Ocimum gratissimum Linn. leaf extract and fractions against trastuzumab-induced cardiotoxicity in Wistar rats. Toxicol Rep 2024;12:200-214.

215

Amodiaquine induces visual impairment in zebrafish larvae through disruption of outer retinal histology and phototransduction and circadian rhythm pathways

Qi Lu¹ and Alison Reynolds^1,2

¹School of Veterinary Medicine, University College Dublin; ²UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin

Introduction

Amodiaquine is a 4-aminoquinoline used as an anti-malarial medicine. Previous studies have indicated that medications in this family can cause ocular adverse effects in humans, for example, Bull's eye maculopathy and cone-rod dystrophy similar to the juvenile Stargardt disease are typical retinopathies caused by 4-aminoquinoline toxicity [1].

This study continued our previous work to study whether amodiaquine exerts retinotoxic effects in zebrafish, similar to those retinopathies seen in humans. We examined visual behaviour, ultrastructural changes in the retina and transcriptomic analysis to identify amodiaquine-related changes in ocular gene expression.

Method

Wild-type (Tübingen) zebrafish were treated at 1-day post-fertilization (dpf) by immersion for 96 h with amodiaquine (10, 25, 50, 75 and 100 μM). Survival was recorded and 50% lethal dose (LD₅₀) calculated. At 5 dpf, larvae were characterized for gross morphology, retinal histology and visual behaviour. RNA was extracted from pooled eye samples at 5 dpf (n = 30 zebrafish/group in triplicate), and RNAseq was performed to find ocular gene expression patterns and related pathways involved in amodiaquine-induced visual impairment.

Results

The LD₅₀ of amodiaquine was determined at 19.25 μM. Several adverse effects were seen in amodiaquine-treated (10 μM) larvae, which exhibited smaller eyes, shorter body length, curved tails, failure to inflate the swim bladder and necrosis in the yolk sac. In the visual behaviour assays, amodiaquine-treated larvae displayed attenuated visual responses. A dose-dependent reduction was observed in optokinetic response test (OKR) with a 25.8% reduction in the number of saccades (P < 0.05 vs. vehicle) compared to vehicle control. Visual motor response (VMR) results showed that amodiaquine induced decreased locomotor activities towards light-on and light-off changes. Amodiaquine-treated larvae displayed thinner outer retinal layers and ultrastructural defects in the photoreceptors and RPE cells. RNAseq analysis identified down-regulation of phototransduction pathways and genes controlling circadian rhythms and up-regulation of pathways associated with lysosome, proteasome and phagosome in the amodiaquine-treated group.

Conclusions

Similar to the retinotoxic effects seen in humans, amodiaquine-treatment can cause visual impairment in zebrafish larval (Table 1) as demonstrated by an attenuation of visual behaviour and ultrastructural defects in the outer retina. RNAseq analysis identified the down-regulation of pathways involved in phototransduction and circadian rhythm, whereas pathways involved in protein degradation were up-regulated.

Reference

1. Nõupuu K, Lee W, Zernant J, Greenstein VC, Tsang S, Allikmets R. Recessive Stargardt disease phenocopying hydroxychloroquine retinopathy. Graefes Arch Clin Exp Ophthalmol 2016;254(5):865-872. https://doi.org/10.1007/s00417-015-3142-8

250

Polychlorinated biphenyls promote metabolic dysfunctions in mature 3T3-L1 adipocytes: role of aquaglyceroporins

Filomena Del Piano¹, Adriano Lama², Claudio Pirozzi², Stefania Melini², Nicola Opallo², Federica Comella², Nicole Pia Navatti², Giuseppina Mattace Raso², Rosaria Meli² and Maria Carmela Ferrante¹

¹Department of Veterinary Medicine and Animal Productions, University of Naples Federico II; ²Department of Pharmacy, University of Naples Federico II

Introduction

Obesity is a global health issue, and its pathogenesis is related to many factors among which environmental ones. Persistent organic pollutants, including polychlorinated biphenyls (PCBs), can affect adipose tissue development and functioning, acting as chemical obesogens [1]. Despite that their production has been banned, PCBs are still detected in human and animal tissues worldwide. Due to their hydrophobicity and resistance to enzymatic degradation, PCBs accumulate in fat deposits contributing to the onset of metabolic diseases [2]. Aquaglyceroporins (AQPs) are transmembrane channels facilitating the transport of water and small solutes across biological membranes. AQP3, AQP7 and AQP9 mediate the release and uptake of glycerol in adipose tissue and are involved in several diseases, including obesity [3]. We aimed to investigate the impact of PCBs on AQP levels in adipocytes and the following effects on metabolism.

Methods

3T3-L1 cells were differentiated into mature adipocytes and then exposed to 1 μM PCB 101, 153 or 180 for 48 h.

Results

PCBs reduced the protein expression of AQPs involved in glycerol release, that is, AQP3 and AQP7, and increased levels of AQP9 involved in glycerol uptake. This modulation suggested a greater accumulation of glycerol in treated adipocytes, confirmed by the reduction of free glycerol in the culture media. PCB 153 increased levels of GyK gene, which mediates the conversion of glycerol into glycerol-3-phosphate, as well as of key factors involved in lipid uptake and storage (i.e. Fabp4 and Pparg), without modifying those involved in de novo lipogenesis (i.e. Fasn and ACC). Moreover, cells treated with PCB 153 showed increased levels of key enzymes involved in triglyceride synthesis from glycerol-3-phosphate and free fatty acids, that is, Dgat1 and Agpat9. All these alterations suggested an increased fat storage in treated adipocytes, confirmed by Oil Red O staining. The role of AQPs in the above effect was confirmed by pre-treating cells with phloretin, a well-known AQP9 inhibitor.

Conclusions

The obtained results showed the involvement of AQPs and glycerol in PCB-induced metabolic dysfunctions in adipocytes, contributing to better defining the mechanisms underlying their obesogenic effect.

References

1. Aaseth J, Javorac D, Djordjevic AB, et al. The role of persistent organic pollutants in obesity: a review of laboratory and epidemiological studies. Toxics 2022;10(2):65.

2. Gao X, Yan D, Li G, et al. Polychlorinated biphenyls and risk of metabolic syndrome and comparison with the risk of diabetes: a systematic review and meta-analysis. Sci Total Environ 2023;900:165773.

3. Calamita G, Perret J, Delporte C. Aquaglyceroporins: drug targets for metabolic diseases? Front Physiol 2018;9:851.