Alflutinib (AST2818), primarily metabolized by CYP3A4, is a potent CYP3A4 inducer.

Alflutinib (AST2818) is a third-generation epidermal growth factor receptor (EGFR) inhibitor that inhibits both EGFR-sensitive mutations and T790M mutations. Previous study has shown that after multiple dosages, alflutinib exhibits nonlinear pharmacokinetics and displays a time- and dose-dependent increase in the apparent clearance, probably due to its self-induction of cytochrome P450 (CYP) enzyme. In this study, we investigated the CYP isozymes involved in the metabolism of alflutinib and evaluated the enzyme inhibition and induction potential of alflutinib and its metabolites. The data showed that alflutinib in human liver microsomes (HLMs) was metabolized mainly by CYP3A4, which could catalyze the formation of AST5902. Alflutinib did not inhibit CYP isozymes in HLMs but could induce CYP3A4 in human hepatocytes. Rifampin is a known strong CYP3A4 inducer and is recommended by the FDA as a positive control in the CYP3A4 induction assay. We found that the induction potential of alflutinib was comparable to that of rifampin. The Emax of CYP3A4 induction by alflutinib in three lots of human hepatocytes were 9.24-, 11.2-, and 10.4-fold, while the fold-induction of rifampin (10 µM) were 7.22-, 19.4- and 9.46-fold, respectively. The EC50 of alflutinib-induced CYP3A4 mRNA expression was 0.25 µM, which was similar to that of rifampin. In addition, AST5902 exhibited much weak CYP3A4 induction potential compared to alflutinib. Given the plasma exposure of alflutinib and AST5902, both are likely to affect the pharmacokinetics of CYP3A4 substrates. Considering that alflutinib is a CYP3A4 substrate and a potent CYP3A4 inducer, drug-drug interactions are expected during alflutinib treatment.

Differences in the In Vivo and In Vitro Metabolism of Imrecoxib in Humans: Formation of the Rate-Limiting Aldehyde Intermediate.

Imrecoxib is a typical cyclooxygenase-2 inhibitor and the benzylic carbon motif is its major site of oxidative metabolism, producing a hydroxymethyl metabolite (M1) and a carboxylic acid metabolite (M2). The plasma exposure of M2 is four times higher than those of both M0 and M1 in humans. However, this metabolite is rarely formed in in vitro experiments. Therefore, this study aims to investigate the formation mechanism of M2 and to further elucidate the reason for the discrepancy between in vitro and in vivo metabolic data. By employing human hepatocytes, human liver microsomes (HLMs), human liver cytosols (HLCs), recombinant enzymes, and selective enzyme inhibitors, the metabolic map of imrecoxib was elaborated as follows: the parent drug was initially hydroxylated to form M1 in HLMs, mainly mediated by CYP3A4 and CYP2D6, and to subsequently form aldehyde imrecoxib (M-CHO) in HLMs and HLCs. The latter process is the rate-limiting step in generating the end-product M2. In further M-CHO metabolism, two opposite reactions (namely, rapid oxidation catalyzed by CYP3A4, CYP2D6, and cytosolic aldehyde oxidase to form M2 versus reduction to regenerate M1 mediated by NADPH-dependent reductases in HLMs and HLCs, such as cytochrome P450 reductase) led to marked underestimation of the M2 amount in static in vitro incubations. The findings provided a possible explanation for the difference between in vitro and in vivo metabolism of imrecoxib, suggesting that the effect of competitive reduction on the static oxidation metabolism in in vitro metabolic experiments should be considered.

Metabolism of MRX-I, a novel antibacterial oxazolidinone, in humans: the oxidative ring opening of 2,3-Dihydropyridin-4-one catalyzed by non-P450 enzymes.

MRX-I is an analog of linezolid containing a 2,3-dihydropyridin-4-one (DHPO) ring rather than a morpholine ring. Our objectives were to characterize the major metabolic pathways of MRX-I in humans and clarify the mechanism underlying the oxidative ring opening of DHPO. After an oral dose of MRX-I (600 mg), nine metabolites were identified in humans. The principal metabolic pathway proposed involved the DHPO ring opening, generating the main metabolites in the plasma and urine: the hydroxyethyl amino propionic acid metabolite MRX445-1 and the carboxymethyl amino propionic acid metabolite MRX459. An in vitro phenotyping study demonstrated that multiple non-cytochrome P450 enzymes are involved in the formation of MRX445-1 and MRX459, including flavin-containing monooxygenase 5, short-chain dehydrogenase/reductase, aldehyde ketone reductase, and aldehyde dehydrogenase (ALDH). H2 (18)O experiments revealed that two (18)O atoms are incorporated into MRX445-1, one in the carboxyethyl group and the other in the hydroxyl group, and three (18)O atoms are incorporated into MRX459, two in the carboxymethyl group and one in the hydroxyl group. Based on these results, the mechanism proposed for the DHPO ring opening involves the metabolism of MRX-I via FMO5-mediated Baeyer-Villiger oxidation to an enol lactone, hydrolysis to an enol, and enol-aldehyde tautomerism to an aldehyde. The aldehyde is reduced by short-chain dehydrogenase/reductase, aldehyde ketone reductase, ALDH to MRX445-1, or oxidized by ALDH to MRX459. Our study suggests that few clinical adverse drug-drug interactions should be anticipated between MRX-I and cytochrome P450 inhibitors or inducers.

Simultaneous determination of unecritinib (TQ-B3101) and its active metabolite crizotinib in rat plasma by LC-MS/MS：An application to pharmacokinetic studies.

Unecritinib (TQ-B3101) is a selective tyrosine kinase receptor inhibitor. In the study, in vitro metabolic experiments revealed that the hydrolysis of TQ-B3101 was mainly catalyzed by carboxylesterase 2 (CES2), followed by CES1. Next, a sensitive and reliable LC-MS/MS method was established for the simultaneous determination of TQ-B3101 and its metabolite crizotinib in rat plasma. To prevent in vitro hydrolysis of TQ-B3101, sodium fluoride, the CESs inhibitor at a concentration of 2 M, was immediately added after whole blood collection. Plasma samples were extracted by acetonitrile-induced protein precipitation method, and chromatographically separated on a Gemini C18 column (50 mm × 2.0 mm i.d., 5 µm) using gradient elution with a mobile phase of 0.1% formic acid and 5 mmol/L ammonium acetate with 0.1% formic acid. The retention times for TQ-B3101 and crizotinib were 2.61 and 2.38 min, respectively. The analytes were detected with tandem mass spectrometer by positive electrospray ionization, using the ion transitions at m/z 492.3 → 302.3 for TQ-B3101, m/z 450.3 → 260.3 for crizotinib, and m/z 494.0 → 394.3 for imatinib (internal standard). Method validation was conducted in the linear range of 1.00-800 ng/mL for the two analytes. The precision, accuracy and stabilities all met the acceptance criteria. The pharmacokinetic study indicated that TQ-B3101 was rapidly hydrolyzed to crizotinib with the elimination half-life of 1.11 h after a single gavage administration of 27 mg/kg to Sprague-Dawley rats, and the plasma exposure of TQ-B3101 was only 2.98% of that of crizotinib.

In vitro studies on the oxidative metabolism of 20(s)-ginsenoside Rh2 in human, monkey, dog, rat, and mouse liver microsomes, and human liver s9.

20(S)-Ginsenoside Rh2 (Rh2)-containing products are widely used in Asia, Europe, and North America. However, extremely limited metabolism information greatly impedes the complete understanding of its clinical safety and effectiveness. The present study aims to systematically investigate the oxidative metabolism of Rh2 using a complementary set of in vitro models. Twenty-five oxidative metabolites were found using liquid chromatography-electrospray ionization ion-trap mass spectrometry. Six metabolites and a metabolic intermediate were synthesized. The metabolites were structurally identified as 26-hydroxy Rh2 (M1-1), (20S,24S)-epoxydammarane-12,25-diol-3-ß-d-glucopyranoside (M1-3), (20S,24R)-epoxydammarane-12,25-diol-3-ß-d-glucopyranoside (M1-5), 26,27-dihydroxy Rh2 (M3-6), (20S,24S)-epoxydammarane-12,25,26-triol-3-ß-d-glucopyranoside (M3-10), (20S,24R)-epoxydammarane-12,25,26-triol-3-ß-d-glucopyranoside (M3-11), and 26-aldehyde Rh2 on the basis of detailed mass spectrometry and nuclear magnetic resonance data analysis. Double-bond epoxidation followed by rearrangement and vinyl-methyl group hydroxylation represent the initial metabolic pathways generating monooxygenated metabolites M1-1 to M1-5. Further sequential metabolites (M2-M5) from the dehydrogenation and/or oxygenation of M1 were also detected. CYP3A4 was the predominant enzyme involved in the oxidative metabolism of Rh2, whereas alcohol dehydrogenase and aldehyde dehydrogenase mainly catalyzed the metabolic conversion of alcohol to the corresponding carboxylic acid. No significant differences were observed in the phase I metabolite profiles of Rh2 among the five species tested. Reactive epoxide metabolite formation in both humans and animals was evident. However, GSH conjugate M6 was detected only in cynomolgus monkey liver microsomal incubations. In conclusion, Rh2 is a good substrate for CYP3A4 and could undergo extensive oxidative metabolism under the catalysis of CYP3A4.

Quantification of the major circulating metabolite of BS1801, an ebselen analog, in human plasma.

BS1801 contains two selenium atoms in its structure, which is a specific inhibitor of thioredoxin reductase intended to treat fibrotic interstitial pneumonia (control pulmonary fibrosis) and liver fibrosis. It is currently in phase I clinical trial. However, there was no report about the metabolic transformation and pharmacokinetics of BS1801. In this study, BS1801 metabolites were characterized in the hepatocytes of different species (monkey, dog, mouse, rat, and human) and plasma specimens using the ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC/Q-TOF MS) method. After incubation, BS1801 could not be detected in the hepatocytes of different species and human plasma. Five metabolites were identified based on the characteristic peak clusters of selenium atoms in the mass spectrum, combined with the product ions obtained by MS-MS through collision-induced-dissociation (CID), including M1 (reduction metabolite), M2 (reduction and Se-methylation metabolite), M4 (M2 further oxidized metabolite) and M5 (Se-methylation and Se-glucuronidation conjugation metabolite), of which the amount of M2 was the highest. By comparing the LC-MS information with the synthesized reference substance, the structure of M2 was confirmed. The principal BS1801 metabolic pathways were identified as reduction and Se-methylation in humans. Subsequently, an accurate and fast LC-MS/MS method was established to verify the major metabolite M2 in human plasma. Acetonitrile-induced protein precipitation was employed to extract M2 from human plasma. The metabolite was separated through XDB-C18 (4.6 × 50 mm, 1.8 µm) under isocratic elution with ammonium acetate (5 mM) containing 0.1% formic acid solution (A) and acetonitrile (B) as the mobile phases. A deuterated internal standard for M2 was prepared to overcome the influence of matrix effects during the detection. The bioanalytical method was shown to be precise, specific, accurate, and good linearity over the range of 3.00-3000 ng/mL, and was implemented to assess the pharmacokinetic profiles of M2 in healthy volunteers following a single oral administration of 450 mg BS1801. This is the first-ever study to identify and quantify the major circulating metabolite of ebselen analogs in human plasma.

Investigating the Metabolic Mechanisms of Butaselen, An Ebselen Analog.

BACKGROUND: Butaselen is an ebselen analog that is under clinical trials for treating hepatic and pulmonary fibrosis. Our previous studies showed that butaselen is mainly present in human plasma in the form of M2, a free Se-methylated metabolite. OBJECTIVE: This study aimed to investigate the metabolic mechanisms of butaselen. METHODS AND RESULTS: Butaselen was incubated with human plasma. Butaselen immediately disappeared, and the butaselen-HSA (human serum albumin) adduct was detected by HPLC-HRMS, showing that butaselen covalently binds to HSA. The butaselen-HSA adduct was precipitated using acetonitrile and then incubated with PBS, Cys, and GSH for 1 hour. The product was M1, a reduced form of butaselen. The results indicated that HSA, Cys, and GSH can reduce the butaselen-HSA covalent bond. The binding site for butaselen could be the cysteine-34 residue of HSA through pronase and trypsin hydrolysis. Incubating butaselen with cysteine, butaselen-Cys, butaselen-2Cys, and M1 were generated, indicating the covalent binding and reduction of butaselen by cysteine. We incubated liver microsomes and cytosol with butaselen, 6.22 and 246 nM M2 were generated, respectively. The results demonstrated that cytosolic enzymes are mainly involved in M2 production. The amount of M2 in the liver cytosol decreased from 246 nM to 2.21 nM when 10 mM m-anisic acid (a specific TPMT enzyme inhibitor) was added, showing that TPMT is responsible for M2 formation. CONCLUSION: Butaselen was covalently bound to HSA, and the binding site was the cysteine-34 residue of HSA. The butaselen-HSA adduct was reduced by free thiol compounds to generate M1. M1 was further metabolized to M2 by cytosolic TPMT. This study provides a basis for studying the pharmacokinetics of selenium-containing drugs.

Metabolism and bioactivation of famitinib, a novel inhibitor of receptor tyrosine kinase, in cancer patients.

BACKGROUND AND PURPOSE: Famitinib is a novel multi-targeted receptor tyrosine kinase inhibitor under development for cancer treatment. This study aims to characterize the metabolic and bioactivation pathways of famitinib. EXPERIMENTAL APPROACH: The metabolites in human plasma, urine and feces were identified via ultra-high performance liquid chromatography-quadrupole-time of flight-mass spectrometry and confirmed using synthetic standards. Biotransformation and bioactivation mechanisms were investigated using microsomes, recombinant metabolic enzymes and hepatocytes. KEY RESULTS: Famitinib was extensively metabolized after repeated oral administrations. Unchanged famitinib was the major circulating material, followed by N-desethylfaminitib (M3), whose steady-state exposure represented 7.2 to 7.5% that of the parent drug. Metabolites in the excreta were mainly from oxidative deamination (M1), N-desethylation (M3), oxidative defluorination (M7), indolylidene hydroxylation (M9-1 and M9-5) and secondary phase-II conjugations. CYP3A4/5 was the major contributor to M3 formation, CYP3A4/5 and aldehyde dehydrogenase to M1 formation and CYP1A1/2 to M7, M9-1 and M9-5 formations. Minor cysteine conjugates were observed in the plasma, urine and feces, implying the formation of reactive intermediate(s). In vitro microsomal studies proved that famitinib was bioactivated through epoxidation at indolylidene by CYP1A1/2 and spontaneously defluorinated rearrangement to afford a quinone-imine species. A correlation between famitinib hepatotoxicity and its bioactivation was observed in the primary human hepatocytes. CONCLUSION AND IMPLICATIONS: Famitinib is well absorbed and extensively metabolized in cancer patients. Multiple enzymes, mainly CYP3A4/5 and CYP1A1/2, are involved in famitinib metabolic clearance. The quinone-imine intermediate formed through bioactivation may be associated with famitinib hepatotoxicity. Co-administered CYP1A1/2 inducers or inhibitors may potentiate or suppress its hepatotoxicity.