Cytochrome P450 3A5 polymorphism affects the metabolism of sorafenib and its toxicity for hepatocellular carcinoma cells in vitro

Objective Cytochrome P450 3A5 ( CYP3A5) is a highly polymorphic gene and the encoded protein variants differ in catalytic activity, leading to inter-individual variation in metabolic ability. The aim of the current study was to investigate the effects of seven allelic variants on the ability of CYP3A5 to metabolize sorafenib in vitro and further explore the impacts of CYP3A5 polymorphism on the proliferation and apoptosis of hepatocellular carcinoma cell line (HepG2) induced by sorafenib. Methods Wild-type and variant CYP3A5 enzymes were expressed in Spodoptera frugiperda insect cells using a baculovirus dual-expression system, and protein expression was checked by western blot. The enzymes were incubated with sorafenib at 37°C for 30 min, and formation of the major metabolite sorafenib N-oxide was assayed using ultra-performance liquid chromatography and tandem mass spectrometry. Intrinsic clearance values (V max/ K m) were calculated for each enzyme. Additionally, recombinant HepG2 cells transfecting with CYP3A5 variants were used to investigate the effects of sorafenib on the proliferation of HepG2 cells. Results Intrinsic clearance of the six variants CYP3A5*2, CYP3A5*3A, CYP3A5*3C, CYP3A5*4, CYP3A5*5, and CYP3A5*7 was 26.41–71.04% of the wild-type ( CYP3A5*1) value. In contrast, the clearance value of the variant CYP3A5*6 was significantly higher (174.74%). Additionally, the decreased ATP levels and cell viability and the increased cell apoptosis in HepG2 cells transfected with CYP3A5*2, CYP3A5*3A, CYP3A5*3C, CYP3A5*4, CYP3A5*5, and CYP3A5*7 were observed, whereas, the increased ATP levels and cell viability and the reduced cell apoptosis in HepG2 cells transfected with CYP3A5*6 were also investigated when compared to CYP3A5*1. Conclusion Our results suggest that CYP3A5 polymorphism influences sorafenib metabolism and pharmacotherapeutic effect in hepatic carcinomas . These data may help explain differential response to drug therapy for hepatocellular carcinoma, and they support the need for individualized treatment.

Effects of 31 recombinant CYP2C19 variants on clomipramine metabolism in vitro

Background: CYP2C19 is an important member of the cytochrome P450 enzyme superfamily. We recently identified 31CYP2C19 alleles in the Han Chinese population; studying the effects of CYP2C19 on drug metabolism can help reduce adverse drug reactions and therapeutic failure. Aim: The aim of this study was to assess the catalytic activities of 31 allelic isoforms and their effects on the metabolism of clomipramine in vitro. Methods: The wild-type and 30 CYP2C19 variants were expressed in insect cells, and each variant was characterized using clomipramine as the substrate. Reactions were performed at 37°C with 5–150 μmol/L substrate for 30 min. By using ultra-high-performance liquid chromatography-mass spectrometry to detect the products, the kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of N-desmethyl clomipramine were determined. Results: Among the CYP2C19 variants tested, CYP2C19*29, L16F, and T130M showed extremely increased intrinsic clearance of clomipramine, CYP2C19*3C, and N277K showed similar intrinsic clearance (Vmax/Km) values with CYP2C19*1, while the intrinsic clearance values of other variants were significantly decreased (from 0.65% to 63.28%). In addition, CYP2C19*3 and 35FS could not be detected because they have no detectable enzyme activity. Conclusions: As the first report of 31 CYP2C19 alleles for clomipramine metabolism, our study could provide corresponding reference for clomipramine for further studies in vivo and offer valuable information relevant to the personalized medicine for CYP2C19-metabolized drug.

Persistent RNA virus infection is short-lived at the single-cell level but leaves transcriptomic footprints

Several RNA viruses can establish life-long persistent infection in mammalian hosts, but the fate of individual virus-infected cells remains undefined. Here we used Cre recombinase–encoding lymphocytic choriomeningitis virus to establish persistent infection in fluorescent cell fate reporter mice. Virus-infected hepatocytes underwent spontaneous noncytolytic viral clearance independently of type I or type II interferon signaling or adaptive immunity. Viral clearance was accompanied by persistent transcriptomic footprints related to proliferation and extracellular matrix remodeling, immune responses, and metabolism. Substantial overlap with persistent epigenetic alterations in HCV-cured patients suggested a universal RNA virus-induced transcriptomic footprint. Cell-intrinsic clearance occurred in cell culture, too, with sequential infection, reinfection cycles separated by a period of relative refractoriness to infection. Our study reveals that systemic persistence of a prototypic noncytolytic RNA virus depends on continuous spread and reinfection. Yet undefined cell-intrinsic mechanisms prevent viral persistence at the single-cell level but give way to profound transcriptomic alterations in virus-cleared cells.

In Vitro Metabolism of E2, G2: Novel Bile Acid-Coupling Camptothecin Analogues, in Rat Liver Microsomes

Background: E2 (Camptothecin - 20 (S) - O- glycine - deoxycholic acid), and G2 (Camptothecin - 20 (S) - O - acetate - deoxycholic acid) are two novel bile acid-derived camptothecin analogues by introducing deoxycholic acid in 20-position of CPT(camptothecin) with greater anticancer activity and lower systematic toxicity in vivo. Objective: We aimed to investigate the metabolism of E2 and G2 by Rat Liver Microsomes (RLM). Methods: Phase Ⅰ and Phase Ⅱ metabolism of E2 and G2 in rat liver microsomes were performed respectively, and the mixed incubation of phase I and phase Ⅱ metabolism of E2 and G2 was also processed. Metabolites were identified by liquid chromatographic/mass spectrometry. Results: The results showed that phase I metabolism was the major biotransformation route for both E2 and G2. The isoenzyme involved in their metabolism had some difference. The intrinsic clearance of G2 was 174.7mL/min. mg protein, more than three times of that of E2 (51.3 mL/min . mg protein), indicating a greater metabolism stability of E2. 10 metabolites of E2 and 14 metabolites of G2 were detected, including phase I metabolites (mainly via hydroxylations and hydrolysis) and their further glucuronidation products. Conclusion: These findings suggested that E2 and G2 have similar biotransformation pathways except some difference in the hydrolysis ability of the ester bond and amino bond from the parent compounds, which may result in the diversity of their metabolism stability and responsible CYPs(Cytochrome P450 proteins).

Quasi-Irreversible Inhibition of CYP2D6 by Berberine

In our previous study, Hwang-Ryun-Hae-Dok-Tang, which contains berberine (BBR) as a main active ingredient, inhibited cytochrome P450 (CYP) 2D6 in a quasi-irreversible manner. However, no information is available on the detailed mechanism of BBR-induced CYP2D6 inhibition. Thus, the present study aimed to characterize the inhibition mode and kinetics of BBR and its analogues against CYP2D6 using pooled human liver microsomes (HLM). BBR exhibited selective quasi-irreversible inhibition of CYP2D6 with inactivation rate constant (kinact) of 0.025 min−1, inhibition constant (KI) of 4.29 µM, and kinact/KI of 5.83 mL/min/µmol. In pooled HLM, BBR was metabolized to thalifendine (TFD), demethyleneberberine (DMB), M1 (proposed as demethylene-TFD), and to a lesser extent berberrubine (BRB), showing moderate metabolic stability with a half-life of 35.4 min and a microsomal intrinsic clearance of 7.82 µL/min/mg protein. However, unlike BBR, those metabolites (i.e., TFD, DMB, and BRB) were neither selective nor potent inhibitors of CYP2D6, based on comparison of half-maximal inhibitory concentration (IC50). Notably, TFD, but not DMB, exhibited metabolism-dependent CYP2D6 inhibition as in the case of BBR, which suggests that methylenedioxybenzene moiety of BBR may play a critical role in the quasi-irreversible inhibition. Moreover, the metabolic clearance of nebivolol (β-blocker; CYP2D6 substrate) was reduced in the presence of BBR. The present results warrant further evaluation of BBR–drug interactions in clinical situations.

Rifampicin Transport by OATP1B1 Variants

ABSTRACT Single nucleotide polymorphisms in the OATP1B1 transporter have been suggested to partially explain the large interindividual variation in rifampicin exposure. HEK293 cells overexpressing wild-type (WT) or OATP1B1 variants *1b, *4, *5, and *15 were used to determine the in vitro rifampicin intrinsic clearance. For OATP1B1*5 and *15, a 36% and 42% reduction in intrinsic clearance, respectively, compared to WT was found. We consider that these differences in intrinsic clearance most likely have minor clinical implications.