Development of an UPLC-MS/MS method for quantitative analysis of abexinostat levels in rat plasma and application of pharmacokinetics.

Broad-spectrum histone deacetylase inhibitors (HDACi) have excellent anti-tumor effects, such as abexinostat, which was a novel oral HDACi that was widely used in clinical treatment. The purpose of this study was to establish a rapid and reliable method for the detection of abexinostat concentrations in rat plasma using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The mobile phase we used was acetonitrile and 0.1% formic acid, and the internal standard (IS) was givinostat. Selective reaction monitoring (SRM) was used for detection with ion transitions at m/z 397.93 → 200.19 for abexinostat and m/z 422.01 → 186.11 for givinostat, respectively. The intra-day and inter-day precision of abexinostat were less than 11.5% and the intra-day and inter-day accuracy ranged from - 10.7% to 9.7% using this method. During the analysis process, the stability of the test sample was reliable. In addition, the recovery and matrix effects of this method were within acceptable limits. Finally, the method presented in this paper enabled accurate and quick determination of abexinostat levels in rat plasma from the pharmacokinetic study following gavage at a dose of 8.0 mg/kg abexinostat.

Inhibitory effect of napabucasin on arbidol metabolism and its mechanism research.

As a broad-spectrum antiviral, and especially as a popular drug for treating coronavirus disease 2019 (COVID-19) today, arbidol often involves drug-drug interactions (DDI) when treating critical patients. This study established a rapid and effective ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to detect arbidol and its metabolite arbidol sulfoxide (M6-1) levels in vivo and in vitro. In this study, a 200 µL incubation system was used to study the inhibitory effect of the antitumor drug napabucasin on arbidol in vitro, with IC50 values of 2.25, 3.91, and 67.79 µM in rat liver microsomes (RLMs), human liver microsomes (HLMs), and CYP3A4.1, respectively. In addition, we found that the mechanism of inhibition was non-competitive inhibition in RLM and mixed inhibition in HLM. In pharmacokinetic experiments, it was observed that after gavage administration of 48 mg/kg napabucasin and 20 mg/kg arbidol, napabucasin inhibited the metabolism of arbidol in vivo and significantly changed the pharmacokinetic parameters of arbidol, such as AUC(0-t) and AUC(0-∞), in rats. We also found that napabucasin increased the AUC(0-t) and AUC(0-∞) of M6-1, the main metabolite of arbidol. This study provides a reference for the combined use of napabucasin and arbidol in clinical practice.

Exposure to 4-bromodiphenyl ether during pregnancy blocks testis development in male rat fetuses.

4-Bromodiphenyl ether (BDE3) is a photodegradation product of higher polybrominated diphenyl ether flame retardants and is known as an endocrine disruptor. However, it is unclear whether and how BDE3 affects the development of fetal testes. This study aimed to investigate the effect of in utero exposure to BDE3 on fetal testicular development in rats. From gestational day (GD) 12-21, BDE3 (0, 50, 100, and 200 mg/kg) was daily gavaged to female pregnant Sprague Dawley rats. BDE3 significantly reduced serum testosterone levels of male pups starting at 50 mg/kg. BDE3 reduced fetal Leydig cell number at a dose of 200 mg/kg without affecting fetal Leydig cell cluster frequency and Sertoli cell number. In addition, BDE3 down-regulated the expression of fetal Leydig cell genes (Cyp11a1, Hsd3b1, Cyp17a1, and Hsd17b3) and their proteins at 100 and/or 200 mg/kg. RNA-seq analysis showed that genes responsive to cAMP (Ass1, Gpd1, Rpl13a) were down-regulated and hypoxia-related genes (Egln3 and P4ha1) were up-regulated at 200 mg/kg. In utero exposure to BDE3 can promote autophagy and apoptosis of fetal Leydig cells via increasing the levels of Beclin1, LC3-II, BAX, and by decreasing the levels of p62 and BCL2. In conclusion, in utero exposure to BDE3 blocks the development of fetal rat testes.

Neurotrophin-3 stimulates stem Leydig cell proliferation during regeneration in rats.

Neurotrophin-3 (NT-3) acts as an important growth factor to stimulate and control tissue development. The NT-3 receptor, TRKC, is expressed in rat testis. Its function in regulation of stem Leydig cell development and its underlying mechanism remain unknown. Here, we reported the role of NT-3 to regulate stem Leydig cell development in vivo and in vitro. Ethane dimethane sulphonate was used to kill all Leydig cells in adult testis, and NT-3 (10 and 100 ng/testis) was injected intratesticularly from the 14th day after ethane dimethane sulphonate injection for 14 days. NT-3 significantly reduced serum testosterone levels at doses of 10 and 100 ng/testis without affecting serum luteinizing hormone and follicle-stimulating hormone levels. NT-3 increased CYP11A1-positive Leydig cell number at 100 ng/testis and lowered Leydig cell size and cytoplasmic size at doses of 10 and 100 ng/testis. After adjustment by the Leydig cell number, NT-3 significantly down-regulated the expression of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Hsd11b1, Insl3, Trkc and Nr5a1) and the proteins. NT-3 increased the phosphorylation of AKT1 and mTOR, decreased the phosphorylation of 4EBP, thereby increasing ATP5O. In vitro study showed that NT-3 dose-dependently stimulated EdU incorporation into stem Leydig cells and inhibited stem Leydig cell differentiation into Leydig cells, thus leading to lower medium testosterone levels and lower expression of Lhcgr, Scarb1, Trkc and Nr5a1 and their protein levels. NT-3 antagonist Celitinib can antagonize NT-3 action in vitro. In conclusion, the present study demonstrates that NT-3 stimulates stem Leydig cell proliferation but blocks the differentiation via TRKC receptor.