British Journal of Pharmacology最新文献

Background and purpose: KCNT1 encodes a sodium-activated potassium channel (Slack channel), and its mutation can cause several forms of epilepsy. Traditional antiepileptic medications have limited efficacy in treating patients with KCNT1 mutations. Here, we describe one heterozygous KCNT1 mutation, M267T, in a patient with EIMFS. The pathological channel properties of this mutation and its effect on neuronal excitability were investigated. Additionally, this study aimed to develop a medication for effective prevention of KCNT1 mutation-induced seizures.

Experimental approach: Wild-type or mutant KCNT1 plasmids were expressed heterologously in Xenopus laevis oocytes, and channel property assessment and drug screening were performed based on two-electrode voltage-clamp recordings. The single-channel properties were investigated using the excised inside-out patches from HEK293T cells. Through in utero electroporation, WT and M267T Slack channels were expressed in the hippocampal CA1 pyramidal neurons in male mice, followed by the examination of the electrical properties using the whole-cell current-clamp technique. The kainic acid-induced epilepsy model in male mice was used to evalute the antiseizure effects of carvedilol.

Key results: The KCNT1 M267T mutation enhanced Slack channel function by increasing single-channel open probability. Through screening 16 FDA-approved ion channel blockers, we found that carvedilol effectively reversed the mutation-induced gain-of-function channel properties. Notably, the KCNT1 M267T mutation in the mouse hippocampal CA1 pyramidal neurons affected afterhyperpolarization properties and induced neuronal hyperexcitability, which was inhibited by carvedilol. Additionally, carvedilol exhibited antiseizure effects in the kainic acid-induced epilepsy model.

Conclusion and implication: Our findings suggest carvedilol as a new potential candidate for treatment of epilepsies.

Background and purpose: FGF, VEGFR-2 and CSF1R signalling pathways play a key role in the pathogenesis of multiple sclerosis (MS). Selective inhibition of FGFR by infigratinib in MOG_35-55-induced experimental autoimmune encephalomyelitis (EAE) prevented severe first clinical episodes by 40%; inflammation and neurodegeneration were reduced, and remyelination was enhanced. Multi-kinase inhibition of FGFR1-3, CSFR and VEGFR-2 by fexagratinib (formerly known as AZD4547) may be more efficient in reducing inflammation, neurodegeneration and regeneration in the disease model.

Experimental approach: Female C57BL/6J mice were treated with fexagratinib (6.25 or 12.5 mg·kg^-1) orally or placebo over 10 days either from time of EAE induction (prevention experiment) or onset of symptoms (suppression experiment). Effects on inflammation, neurodegeneration and remyelination were assessed at the peak of the disease (Day 18/20 post immunization) and the chronic phase of EAE (Day 41/42).

Key results: In the prevention experiment, treatment with 6.25 or 12.5 mg·kg^-1 fexagratinib prevented severe first clinical episodes by 66.7% or 84.6% respectively. Mice treated with 12.5 mg·kg^-1 fexagratinib hardly showed any symptoms in the chronic phase of EAE. In the suppression experiment, fexagratinib resulted in a long-lasting reduction of severe symptoms by 91 or 100%. Inflammation and demyelination were reduced, and axonal density, numbers of oligodendrocytes and their precursor cells, and remyelinated axons were increased by both experimental approaches.

Conclusion and implications: Multi-kinase inhibition by fexagratinib in a well-tolerated dose of 1 mg·kg^-1 in humans may be a promising approach to reduce inflammation and neurodegeneration, to slow down disease progression and support remyelination in patients.

Background and purpose: DT-678 is a novel antiplatelet prodrug, capable of releasing the antiplatelet active metabolite of clopidogrel (AM) upon exposure to glutathione. In this study, we investigated factors responsible for clopidogrel high on-treatment platelet reactivity (HTPR) in acute coronary syndrome (ACS) patients and evaluated the capacity of DT-678 to overcome HTPR.

Experimental approach: A total of 300 consecutive ACS patients naive to P2Y₁₂ receptor inhibitors were recruited and genotyped for CYP2C19 alleles. Blood samples were drawn before and after administration of 600-mg clopidogrel. Platelet reactivity index (PRI) and plasma AM concentrations were determined and grouped according to their CYP2C19 genotypes. DT-678 was applied ex vivo to whole blood samples to examine its inhibitory effects. To further examine the antiplatelet effectiveness of DT-678 in vivo, 20 healthy human subjects were recruited in a Phase I clinical trial, and each received a single dose of either 3-mg DT-678 or 75-mg clopidogrel. The pharmacokinetics and pharmacodynamics in different CYP2C19 genotype groups were compared.

Key results: Statistical analyses revealed that CYP2C19 genotype, body mass index, hyperuricaemia, and baseline PRI were significantly associated with a higher risk of clopidogrel HTPR in ACS patients. The addition of DT-678 ex vivo decreased baseline PRI regardless of CYP2C19 genotypes, overcoming clopidogrel HTPR. This observation was further confirmed in healthy volunteers receiving 3 mg of DT-678.

Conclusion and implications: These results suggest that DT-678 effectively overcomes clopidogrel HTPR resulting from genetic and/or clinical factors in Chinese ACS patients, demonstrating its potential to improve antiplatelet therapy.

Background and purpose: Fentanyl analogues have been implicated in many cases of intoxication and death with overdose worldwide. The aim of this study is to investigate the pharmaco-toxicology of two fentanyl analogues: butyrylfentanyl (BUF) and 4-fluorobutyrylfentanyl (4F-BUF).

Experimental approach: In vitro, we measured agonist opioid receptor efficacy, potency, and selectivity and ability to promote interaction of the μ receptor with G protein and β-arrestin 2. In vivo, we evaluated thermal antinociception, stimulated motor activity and cardiorespiratory changes in female and male CD-1 mice injected with BUF or 4F-BUF (0.1-6 mg·kg^-1). Opioid receptor specificity was investigated using naloxone (6 mg·kg^-1). We investigated the possible role of stress in increasing cardiorespiratory toxicity using the corticotropin-releasing factor 1 (CRF₁) antagonist antalarmin (10 mg·kg^-1).

Key results: Agonists displayed the following rank of potency at μ receptors: fentanyl > 4F-BUF > BUF. Fentanyl and BUF behaved as partial agonists for the β-arrestin 2 pathway, whereas 4F-BUF did not promote β-arrestin 2 recruitment. In vivo, we revealed sex differences in motor and cardiorespiratory impairments but not antinociception induced by BUF and 4F-BUF. Antalarmin alone was effective in blocking respiratory impairment induced by BUF in both sexes but not 4F-BUF. The combination of naloxone and antalarmin significantly enhanced naloxone reversal of the cardiorespiratory impairments induced by BUF and 4F-BUF in mice.

Conclusion and implications: In this study, we have uncovered a novel mechanism by which synthetic opioids induce respiratory depression, shedding new light on the role of CRF₁ receptors in cardiorespiratory impairments by μ agonists.

Background and purpose: Transient receptor potential vanilloid 2 (TRPV2) is a Ca²⁺-permeable non-selective cation channel. Despite the significant roles of TRPV2 in immunological response, cancer progression and cardiac development, pharmacological probes of TRPV2 remain to be identified. We aimed to discover TRPV2 inhibitors and to elucidate their molecular mechanism of action.

Experimental approach: Fluorescence-based Ca²⁺ assay in HEK-293 cells expressing murine TRPV2 was used to identify plumbagin as a novel TRPV2 inhibitor. Patch-clamp, in silico docking and site-directed mutagenesis were applied to investigate the molecular mechanisms critical for plumbagin interaction. ELISA and qPCR were used to assess nitric oxide release and mRNA levels of inflammatory mediators, respectively. si-RNA interference was used to knock down TRPV2 expression, which was validated by western blotting. Neurological and histological analyses were used to examine brain injury of mice following middle cerebral artery occlusion/reperfusion (MCAO/R).

Key results: Plumbagin is a potent TRPV2 negative allosteric modulator with an IC₅₀ value of 0.85 μM, exhibiting >14-fold selectivity over TRPV1, TRPV3 and TRPV4. Plumbagin suppresses TRPV2 activity by decreasing the channel open probability without affecting the unitary conductance. Moreover, plumbagin binds to an extracellular pocket formed by the pore helix and flexible loop between transmembrane helices S5 and S6 of TRPV2. Plumbagin effectively suppresses LPS-induced inflammation of BV-2 microglia and ameliorates brain injury of MCAO/R mice.

Conclusion and implications: Plumbagin is a novel pharmacological probe to study TRPV2 pathophysiology. TRPV2 is a novel molecular target for the treatment of neuroinflammation and ischemic stroke.

Background and purpose: GABAergic neurons in mouse ventral tegmental area (VTA) exhibit elevated activity during withdrawal following chronic ethanol exposure. While increased glutamatergic input and decreased GABA_A receptor sensitivity have been implicated, the impact of inhibitory signaling in VTA GABA neurons has not been fully addressed.

Experimental approach: We used electrophysiological and ultrastructural approaches to assess the impact of chronic intermittent ethanol vapour exposure in mice on GABAergic transmission in VTA GABA neurons during withdrawal. We used CRISPR/Cas9 ablation to mimic a somatodendritic adaptation involving the GABA_B receptor (GABA_BR) in ethanol-naïve mice to investigate its impact on anxiety-related behaviour.

Key results: The frequency of spontaneous inhibitory postsynaptic currents was reduced in VTA GABA neurons following chronic ethanol treatment and this was reversed by GABA_BR inhibition, suggesting chronic ethanol strengthens the GABA_BR-dependent suppression of GABAergic input to VTA GABA neurons. Similarly, paired-pulse depression of GABA_A receptor-dependent responses evoked by optogenetic stimulation of nucleus accumbens inputs from ethanol-treated mice was reversed by GABA_BR inhibition. Somatodendritic currents evoked in VTA GABA neurons by GABA_BR activation were reduced following ethanol exposure, attributable to the suppression of GIRK (K_ir3) channel activity. Mimicking this adaptation enhanced anxiety-related behaviour in ethanol-naïve mice.

Conclusions and implications: Chronic ethanol weakens the GABAergic regulation of VTA GABA neurons in mice via pre- and postsynaptic mechanisms, likely contributing to their elevated activity during withdrawal and expression of anxiety-related behaviour. As anxiety can promote relapse during abstinence, interventions targeting VTA GABA neuron excitability could represent new therapeutic strategies for treatment of alcohol use disorder.

This brief review highlights some of the structure-activity relationships of classic serotonergic psychedelics. In particular, we discuss structural features of three chemotypes: phenethylamines, ergolines and certain tryptamines, which possess psychedelic activity in humans. Where they are known, we point out the underlying molecular mechanisms utilized by each of the three chemotypes of psychedelic molecules. With a focus on the 5-HT_2A receptor subtype, a G-protein coupled receptor known to be the primary target of psychedelics, we refer to several X-ray and cryoEM structures, with a variety of ligands bound, to illustrate the underlying atomistic basis for some of the known pharmacological observations of psychedelic drug actions.

Background and purpose: Diabetic nephropathy (DN) is a leading cause of chronic kidney disease (CKD), which is characterized by mesangial matrix expansion that involves dysfunctional mesangial cells (MCs). However, the underlying mechanisms remain unclear. This study aims to delineate the spatiotemporal contribution of adrenergic signalling in diabetic kidney fibrosis to reveal potential therapeutic targets.

Experimental approach: A model of diabetic nephropathy was induced by in db/db mice. Gene expression in kidneys was profiled by RNA-seq analyses, western blot and immunostaining. Subcellular-localized fluorescence resonance energy transfer (FRET) biosensors determined adrenergic signalling microdomains in MCs. Effects of oral rolipram, a phosphodiesterase 4 (PDE4) inhibitor, on the model were measured.

Key results: Our model exhibited impaired kidney function with elevated expression of adrenergic and fibrotic genes, including Adrb1, PDEs, Acta2 and Tgfβ. RNA-seq analysis revealed that MCs with dysregulated YAP pathway were crucial to the extracellular matrix secretion in kidneys from diabetic nephropathy patients. In cultured MCs, TGF-β promoted profibrotic gene transcription, which was regulated by nuclear-localized β-adrenoceptor signalling. Mechanistically, TGF-β treatment diminished nuclear-specific cAMP signalling in MCs and reduced PKA-dependent phosphorylation of YAP, leading to its activation. In parallel, db/db mouse kidneys showed increased expressions of PDE4B and PDE4D. Treatment with oral rolipram alleviated kidney fibrosis in db/db mice.

Conclusion and implications: Diabetic nephropathy impaired nuclear-localized β₁-adrenoceptor-cAMP signalling microdomain through upregulating PDE4 expression, promoting fibrosis in MCs via PKA dephosphorylation-dependent YAP activation. Our results suggest PDE4 inhibition as a promising strategy for alleviating kidney fibrosis in diabetic nephropathy.

Background and purpose: The pharmacology of flavonoids on β-cell function is largely undefined especially in the context of defective secretion of insulin. We sought to identify flavonoids that increased the insulin-secreting function of β-cells and to explore the underlying mechanisms.

Experimental approach: INS-1 β-cells in culture and islets of Langerhans isolated from control and diabetic male rats were used for insulin secretion experiments. Pharmacological and electrophysiological approaches were used for mechanistic studies.

Key results: Among a set of flavonoids, exposure of INS-1 β-cells to resokaempferol (ResoK) enhanced glucose-stimulated insulin secretion and therefore we further characterised its activity and its pharmacological mechanism. ResoK glucose-dependently enhanced insulin secretion in INS-1 β-cells and pancreatic islets isolated from rats. Mechanistically, whole cell patch clamp recordings in INS-1 cells showed that ResoK rapidly and dose-dependently enhanced the L-type Ca²⁺ current whereas it was inactive towards T-type Ca²⁺ current. Accordingly, pharmacological inhibition of L-type Ca²⁺ current but not T-type Ca²⁺ current blocked the effects of ResoK on glucose-stimulated insulin secretion. ResoK was still active on dysfunctional β-cells as it ameliorated glucose-stimulated insulin secretion in glucotoxicity-induced dysfunctional INS-1 cells and in pancreatic islets isolated from diabetic rats.

Conclusion and implications: ResoK is a glucose-dependent activator of insulin secretion. Our results indicated that the effects of ResoK on insulin secretion involved its capacity to stimulate L-type Ca²⁺ currents in cultured β-cells. As ResoK was also effective on dysfunctional β-cells, our work provides a new approach to stimulating insulin secretion, using compounds based on the structure of ResoK.