Biological & pharmaceutical bulletin最新文献

Mirabegron, a β₃-adrenoceptor agonist used to treat overactive bladder, is associated with increased heart rate; however, the mechanism underlying its cardiac effect remains unclear. In this study, we investigated the β-adrenoceptor subtypes that are involved in the chronotropic effects of mirabegron in mouse and guinea pig atria. Mirabegron (0.03-10 μM) produced concentration-dependent positive chronotropic effects in both species, with maximum effects of 75.9% in mice and 27.7% in guinea pigs. Isoproterenol (0.1-100 nM), a nonselective β-adrenoceptor agonist, dobutamine (0.001-100 μM), a β₁-adrenoceptor agonist, and salbutamol (0.001-100 μM), a β₂-adrenoceptor agonist, induced concentration-dependent positive chronotropic effects, with the potency order of isoproterenol > dobutamine > mirabegron ≥ salbutamol in mice, and isoproterenol > dobutamine ≥ salbutamol > mirabegron in guinea pigs. Mirabegron-induced positive chronotropic effects were unaffected by 0.1% dimethyl sulfoxide in the mouse atria, but non-competitively antagonized by the selective β₃-adrenoceptor antagonist L748337 (100-1000 nM), with the slope of the Schild plot being 0.65. The selective β₂-adrenoceptor antagonist ICI118551 (30 nM) attenuated the positive chronotropic effects of mirabegron, producing a 3-fold rightward shift in the concentration-response curve. Conversely, the selective β₁-adrenoceptor antagonist CGP20172A (0.03-3 nM) strongly and non-competitively antagonized mirabegron-induced positive chronotropic effects in mouse and guinea pig atria. These results suggest that the positive chronotropic effects of mirabegron are primarily mediated through β₁-adrenoceptors, with minimal or no involvement of β₂- and β₃-adrenoceptors.

Matching transformation system (MA-T), an on-demand aqueous chlorine dioxide production solution is utilized in various applications, including antibacterial and cancer therapies. Herein, we focused on the ability of MA-T in oxidizing human transient receptor potential cation channel subfamily V member 4 (hTRPV4), which functions as a redox sensor, using Ca²⁺ imaging and patch-clamp experiments. We found that 10 ppm MA-T activated hTRPV4 in HEK293T cells. Moreover, MA-T induced increase in cytosolic calcium concentration through hTRPV4 activation in human non-pigmented ciliary epithelial cells. Our study findings suggest MA-T as a potential agent for TRPV4-related diseases.

Nuclear factor erythroid-derived 2 like 1 (NFE2L1) is reported to be embedded in the endoplasmic reticulum (ER) membrane and subsequently undergo N-glycosylation at several asparagine residues as well as other ER-resident factors including cAMP response element binding protein 3 (CREB3)/ATF6 family members. In this study, we investigated the regulation of NFE2L1 protein expression by treating wild-type HEK293 cells and HEK293 cells deficient in selected ER-associated degradation (ERAD) factors with various reagents. NFE2L1 protein expression in wild-type HEK293 cells was negligible, but MG132/bortezomib treatment induced Endo H-resistant two bands. Suppressor/enhancer of lin-12-like (SEL1L)/hydroxymethylglutaryl-CoA (HMG-CoA) reductase degradation 1 (Hrd1) loss increased NFE2L1 protein expression without any stimuli. In these deficient cells, the band shift of NFE2L1 by MG132 was mostly suppressed. Treatment with the valosin containing protein (VCP) inhibitor CB-5083 increased NFE2L1 expression, but deficiencies in other ERAD-associated factors (ER degradation-enhancing α-mannosidase-like protein 2 (EDEM2), thioredoxin domain-containing protein 11 (TXNDC11), gp78, ring finger protein 5 (RNF5), ring finger protein 185 (RNF185), and USP19) did not affect its expression. Comparing the stability of the two intrinsic NFE2L1, which increases with proteasome inhibition, the higher molecular weight form corresponding to full-length form, was more unstable. Therefore, we constructed NFE2L1 genes with mutations in the site where NFE2L1 is cleaved by DDI2 and in the four asparagine residues where N-glycosylation occurs, and found that the high molecular weight form, especially a hypoglycosylated mutant, tended to be more unstable. Taken together, this study using several ERAD disordered models shows that the regulation of NFE2L1 is different in some ways from the regulation of CREB3/ATF6 family, and these findings implicate the diversity of N-glycosylated protein regulation in the ER.

Proton pump inhibitors (PPIs) are implicated in the progression of chronic kidney disease (CKD), but the mechanism behind this is unclear. PPIs are known to inhibit organic anion transporters (OATs) and affect intestinal microbiota. PPI use may influence serum concentrations of indoxyl sulfate (IS), a uremic toxin that contributes to CKD. This study compares serum IS concentrations between Japanese patients receiving long-term PPI prescriptions and those receiving non-PPIs, evaluating the effect of long-term PPI therapies on serum IS concentrations. This single-center, cross-sectional study included patients with an estimated glomerular filtration rate (eGFR) of 15 to 44 mL/min/1.73 m,² who attended an outpatient visit at a nephrology and/or diabetes department between October 2022 and December 2023. Serum IS concentrations were measured by HPLC. The analysis included 29 patients in the PPI group and 28 in the non-PPI group; serum IS concentrations were significantly higher in the PPI group [median (interquartile range) 15.37 (9.69-19.80) µM] and non-PPI group [median (interquartile range) 10.66 (6.97-14.19) µM], p = 0.03. Multiple regression analysis was performed, linking prescription of PPIs and lower eGFR to higher serum IS levels. This study highlights an apparent association of long-term prescriptions of PPIs with high serum IS concentrations. However, more detailed studies are required to evaluate the contribution of intestinal microbiota and diet to this phenomenon.

Immune checkpoint inhibitors (ICIs), essential in cancer therapy, can cause severe immune-related adverse events (irAEs), including myocarditis with a high fatality rate. Currently, the pathogenesis, biomarkers, and risk factors of ICI-induced myocarditis (ICIM) are not fully understood. This exploratory study aimed to develop machine learning-based models to predict the onset of ICIM within 3 months of starting ICI therapy, using a large health insurance database. The models were constructed using the Light Gradient Boosting Machine (LightGBM) and Random Forest algorithms, incorporating clinical variables such as comorbidities and prior medication classifications. In this study, a strategy combining undersampling and bagging was used to minimize the impact of highly imbalanced datasets. The Random Forest model demonstrated superior performance compared with the LightGBM model, and the SHapley Additive exPlanations (SHAP) analysis for the Random Forest model revealed that the concurrent use of ICIs was the most important variable for predictions. Although predictive performance remains limited (AUROC ≈ 0.63), this exploratory framework demonstrates the feasibility of developing data-driven risk prediction models for ICIM. Future studies with expanded datasets and integration of laboratory parameters are warranted to improve predictive accuracy and potential clinical applicability.

Oral administration is the preferred route for drug administration owing to a high level of patient compliance and suitability for long-term treatment. However, drugs with a molecular weight of >1000 Da such as peptide-based therapeutics generally exhibit poor absorption from the gastrointestinal tract. In this study, we evaluated the potential of ionic liquids to enhance the oral absorption of mid-sized molecules such as dextran, which has a size of 10 kDa. In this study, the ionic liquids we tested enhanced the oral absorption of 10 kDa mid-sized dextran. Among the ionic liquids we tested, d/l-Lactic acid-trometamol ([Lac][Tris])-based ionic liquids consistently yielded higher values for maximum concentration (C_max; 153.9 ± 11.0 nmol/L) of dextran as well as for the area under the curve representing 0-2 h (AUC_{0-2 h}; 228.9 ± 54.1 nmol × h/L) compared with that of the other ionic liquids tested. The [Lac][Tris] = 5 : 1 ionic liquid achieved relatively higher values for both C_max and AUC_{0-2 h}. Interestingly, the observed oral absorption enhancement effect by the [Lac][Tris] = 5 : 1 ionic liquid for mid-sized molecules depended on the concentration of dextran (10 kDa). These results show that ionic liquid formulations can overcome the intestinal barrier and lead to systemic absorption of mid-sized molecules with molecular weights. Although further optimizations aimed at translating ionic liquid technology to the oral delivery of peptide drugs must be required, our results could expand the utility and possibility of ionic liquids as an oral absorption enhancer for mid-sized molecules such as peptides.

The cytosolic translocation of the membrane-associated CYP3A2 protein was examined in male and female rats to evaluate the ubiquitin-dependent protein degradation. In the experiments, the cytosol and membrane fractions were prepared from pooled rat liver tissues, and the amount of CYP3A2 protein in those fractions was determined by Western immunoblotting. The CYP3A2 protein detected in the cytosol fraction was shown to be ubiquitinated, and the apparent translocation ratio of the CYP3A2 protein from the membrane to the cytosol was calculated to be 0.58 in male control rats. The translocation ratio was then evaluated in male rats undergoing CYP3A2 induction. When male rats were treated with 80 mg/kg of dexamethasone (DEX), the translocation ratio decreased to 0.05, but the ratio was barely affected in male rats treated with 1.6 mg/kg of DEX, indicating that the cytosolic translocation of the CYP3A2 protein had a saturable nature. After that, the CYP3A2 translocation was examined in female rats with induced CYP3A2 protein, as the protein was known to be expressed in a male-specific manner. In female rats treated with 80 mg/kg of DEX, the translocation ratio was 0.04, but in those treated with 1.6 mg/kg of DEX, the ratio increased to 1.24, approximately 2 times larger than that in male control rats. It appeared that the DEX-induced CYP3A2 protein in female rats underwent expeditious degradation, presumably reflecting a difference in the mechanisms regulating the hepatic expression of the CYP3A2 protein in male and female rats.

Leydig cells are testosterone synthesis cells in testes, a process tightly regulated by luteinizing hormone (LH) through the activation of steroidogenic enzymes such as steroidogenic acute regulatory protein (StAR) and 3-beta-hydroxy-Delta5-steroid dehydrogenase (3β-HSD). While calcium/calmodulin-dependent protein kinase 2 (CAMK2) is known to modulate diverse cellular processes, including hormone signaling, its role in testosterone production remains unclear. In this study, we investigated the expression and functions of CAMK2 in mouse testes, focusing on its potential involvement in testosterone synthesis. Our findings demonstrate that CAMK2 expression progressively increases from postnatal day 1 (PND 1) to adulthood. Pharmacological inhibition of CAMK2 with KN-62 markedly reduced serum testosterone levels and downregulated the expression of key steroidogenic enzymes, including StAR and 3β-HSD, at both mRNA and protein levels. In vitro experiments using primary Leydig cells further confirmed that CAMK2 inhibition suppressed testosterone production and steroidogenic enzyme expression, particularly after prolonged (12-24 h) KN-62 treatment. Additionally, CAMK2 expression was upregulated in response to LH stimulation, suggesting its involvement in LH-mediated signaling pathways, potentially through modulation of the epidermal growth factor receptor (EGFR)/extracellular signal-regulated kinase 1/2 (ERK1/2) cascade. These findings demonstrate that CAMK2 positively regulates testosterone synthesis in Leydig cells, likely via the EGFR/ERK1/2 cascade. The results of this study enhance our understanding of the regulation of testosterone synthesis and identifies CAMK2 as a potential therapeutic target for male reproductive endocrine disorders.

Using a large health insurance database in Japan, we examined the real-world usage of budesonide enteric-coated capsules (BUD) in treating Crohn's disease. We analyzed data from the Japan Medical Data Center claims database for Crohn's disease patients prescribed BUD from April 2016 to March 2021, focusing on prescription status, adverse events (AEs), monitoring tests, and concomitant medications over 2 years following BUD initiation. Patients were categorized into two groups based on BUD usage duration: ≤1 year and >1 year. Of the 7364 registered patients, 1049 (14.2%) were prescribed BUD. Among the 562 followed for 2 years, 505 (89.9%) used BUD for ≤1 year and 57 (10.1%) for >1 year. Over 70% of the patients used at least one biologic, and more than 20% used at least two. The proportions of new thiopurine initiation were 22 and 9% in the ≤1-year and >1-year groups, respectively (p = 0.0181). We did not identify any obvious increase in AEs from long-term BUD use within the confines of our study design. However, regardless of prescription duration, over half of the patients lacked hepatitis B virus screening, glycated hemoglobin measurement, adrenal function quantification, or bone densitometry. Usage of strong CYP3A4 inhibitors was more frequent among patients in the BUD >1-year group. This study revealed that numerous Japanese patients received long-term BUD prescriptions. Although no apparent increase in AEs from long-term BUD was detected, we identified inadequate monitoring of AEs and drug interactions, as well as insufficient use of steroid-sparing agents.