Metabolites In Human Liver Microsomes Research Articles

In vitro and in vivo pharmacokinetics, disposition, and drug-drug interaction potential of tinengotinib (TT-00420), a promising investigational drug for treatment of cholangiocarcinoma and other solid tumors

Early-stage clinical evaluation of tinengotinib (TT-00420) demonstrated encouraging preliminary efficacies in multiple types of refractory cancers, including fibroblast growth factor receptors (FGFR) inhibitors relapsed cholangiocarcinoma (CCA), castrate-resistant prostate cancer (CRPC), and HR+/HER2- breast cancer and triple negative breast cancer (TNBC). To further evaluate drug-like properties of the drug candidate, it is imperative to understand its metabolism and pharmacokinetic properties. This manuscript presented the investigation results of in vitro permeability, plasma protein binding, metabolic stability, metabolite identification, and drug-drug interaction of tinengotinib. Preclinical ADME (absorption, distribution, excretion, and metabolism) studies in rats and dogs was also conducted using a radioactive labeled tinengotinib, [14C]tinengotinib. Tinengotinib was found to have high permeability and high plasma protein binding and equally distributed between blood and plasma. There were no unique metabolites in human liver microsomes and tinengotinib showed moderate hepatic clearance. Tinengotinib is neither a potential inhibitor nor an inducer of P450 enzymes at clinically relevant concentrations, and unlikely to cause drug-drug interactions when used in combination with other drugs mediated by a key transporter, either as victim or perpetrator. Taken together, tinengotinib demonstrated a minimal risk of clinically relevant drug-drug interactions. Tinengotinib showed good oral bioavailability and dose-dependent exposures in both rat and dog after oral administration. The total radioactivity was largely distributed in the gastrointestinal system and liver, and tinengotinib could not easily pass through the blood-brain barrier. The major drug-related component in rat and dog plasma was unchanged drug (>89 %) with primary route of elimination via feces (>93 % of the dose) and minor via renal excretion (<4 % of the dose). Tinengotinib metabolism is mediated largely by CYP3A4, with minor contributions from CYP2D6 and CYP2C8. Major metabolic pathways include oxidation, oxidative cleavage of the morpholine ring, glucuronide and glutathione conjugations. The overall preclinical pharmacokinetics profile supported the selection and development of tinengotinib as a clinical candidate.

Identification of Cytochrome P450 Enzymes Responsible for Oxidative Metabolism of Synthetic Cannabinoid (1-Hexyl-1H-Indol-3-yl)-1-naphthalenyl-methanone (JWH-019)

(1-Hexyl-1H-indol-3-yl)-1-naphthalenyl-methanone (JWH-019) is one of the second-generation synthetic cannabinoids which as a group have been associated with severe adverse reactions in humans. Although metabolic activation can be involved in the mechanism of action, the metabolic pathway of JWH-019 has not been fully investigated. In the present study, we aimed to identify the enzymes involved in the metabolism of JWH-019. JWH-019 was incubated with human liver microsomes (HLMs) and recombinant cytochrome P450s (P450s or CYPs). An animal study was also conducted to determine the contribution of the metabolic reaction to the onset of action. Using an ultra-performance liquid chromatography system connected to a single-quadrupole mass detector, we identified 6-OH JWH-019 as the main oxidative metabolite in HLMs supplemented with NADPH. JWH-019 was extensively metabolized to 6-OH JWH-019 in HLMs with the KM and Vmax values of 31.5 µM and 432.0 pmol/min/mg. The relative activity factor method estimated that CYP1A2 is the primary contributor to the metabolic reaction in the human liver. The animal study revealed that JWH-019 had a slower onset of action compared to natural and other synthetic cannabinoids. CYP1A2 mediates the metabolic activation of JWH-019, contributing to the slower onset of its pharmacological action.

Identification of the metabolites of neocnidilide in rat, monkey and human liver microsomes by liquid chromatography coupled to benchtop Orbitrap mass spectrometry.

Neocnidilide, a bioactive component isolated from Angelica sinensis (Oliv.) Diels, displayed anti-inflammatory activity. The present work was performed to investigate in vitro metabolism of neocnidilide using liver microsomes. Neocnidilide (10μM) was incubated with NADPH-supplemented rat monkey and human liver microsomes. To identify the reactive metabolites, glutathione (GSH) was included in the incubations. Liquid chromatography coupled to an Orbitrap mass spectrometer was used to detect and identify the metabolites. The structures of the metabolites were characterized by accurate masses and fragmentation patterns. A total of six hydroxylation metabolites and nine GSH conjugates were tentatively identified characterized. The metabolic pathways included hydroxylation, dehydrogenation and GSH conjugation. M6 was the major metabolite in human liver microsomes. CYP1A2 (25%), 2B6 (31.6%), 2C9 (10.5%) and 3A4 (18%) were the predominant isoenzymes governing its formation. This study provides valuable information on the in vitro metabolism of neocnidilide, which is indispensable for the further safety assessment of this compound.

In Vitro Identification of Potential Metabolites of Plinabulin (NPI 2358) in Hepatic Preparations Using Liquid Chromatography-Ion Trap Mass Spectrometry.

Plinabulin (1, NPI2358), a vascular disrupting agent (VDA) molecule, is a synthetic analogue of the natural product phenylahistin (2, NPI 2350), which is isolated from Aspergillus ustus. Evaluation of the in vitro metabolic profile of VDA plinabulin using human liver microsomes (HLMs) and HepaRG Cells Cryopreserved is described. HLMs and HepaRG Cells Cryopreserved were prepared in-house and incubated with plinabulin according to published methodologies. The incubated mixtures were analyzed by liquid chromatography–ion trap mass spectrometry to identify possible metabolic products. The incubated plinabulin (1) revealed the presence of several peaks representing 19 tentative metabolites in HLMs and HepaRG Cells Cryopreserved in the presence of NADPH (nicotinamide adenine dinucleotide phosphate) and in the absence of NADPH-generating system, respectively. However, in NADPH absence, no metabolites and microsomes were generated for 1 in incubated HLMs, indicating a likely involvement of CYP450 enzymes in the metabolism. The metabolite structures, obtained from HLMs and HepaRG Cells Cryopreserved incubations, were elucidated by LC–MS/MS fragmentation study. Seventeen phase-I metabolites were proposed to be the results of isomerization, hydroxylation, hydration, and oxygenation of 1 in HLMs and two isomeric phase-II sulfate conjugate metabolites of 1 in HepaRG Cells Cryopreserved incubation.

Transesterification of Indazole-3-carboxamide Synthetic Cannabinoids: Identification of Metabolite Biomarkers for Diagnosing Co-abuse of 5F-MDMB-PINACA and Alcohol.

Concurrent use of alcohol with synthetic cannabinoids (SCs) has been widely recorded among drug abusers. The susceptibilities of three indazole-3-carboxamide type SCs with methyl ester moiety, 5F-MDMB-PINACA, 5F-MMB-PINACA, and MMB-FUBINACA, to transesterification in the presence of ethanol warranted further investigation in view of probable augmented toxicity. In vitro metabolite identification experiments were first performed using human liver microsomes (HLMs) to characterize the novel metabolites of the three parent SCs in the presence of ethanol. Formation of transesterified metabolite, hydrolyzed metabolite, and several oxidative metabolites in HLM in the presence of alcohol was further determined for each parent SC and the respective ethyl ester analog, 5F-EDMB-PINACA, 5F-EMB-PINACA, and EMB-FUBINACA, to quantitatively elucidate transesterification and hydrolysis activities. Our results suggested that all three SCs undergo carboxylesterase-mediated transesterification to their respective ethyl ester analog in the presence of ethanol, which was incubation time- and ethanol concentration-dependent. Each ethyl ester metabolite was sequentially and readily metabolized to novel oxidative metabolites with the intact ethyl ester moiety and the same hydrolyzed metabolite as derived from its parent SC. A smaller extent of transesterification was non-enzymatically driven. Notably, we proposed 5F-EDMB-PINACA oxidative defluorination metabolite as the biomarker for diagnosing the potential co-abuse of 5F-MDMB-PINACA and alcohol. Due to the comparable pharmacological activities between each SC and its ethyl ester metabolite, augmented toxicity associated with co-abuse of SCs and alcohol is probable and deserves further investigation.

In Vitro Characterization of Ertugliflozin Metabolism by UDP-Glucuronosyltransferase and Cytochrome P450 Enzymes.

Ertugliflozin is primarily cleared through UDP-glucurosyltransferase (UGT)-mediated metabolism (86%) with minor oxidative clearance (12%). In vitro phenotyping involved enzyme kinetic characterization of UGTs or cytochrome P450 enzymes catalyzing formation of the major 3-O-β-glucuronide (M5c) and minor 2-O-β-glucuronide (M5a), monohydroxylated ertugliflozin (M1 and M3), and des-ethyl ertugliflozin (M2) metabolites in human liver microsomes (HLMs). Fractional clearance (fCL) from HLM intrinsic clearance (CLint) indicated a major role for glucuronidation (fCL 0.96; CLint 37 µl/min per milligram) versus oxidative metabolism (fCL 0.04; CLint 1.64 µl/min per milligram). Substrate concentration at half-maximal velocity (Km), maximal rate of metabolism (Vmax), and CLint for M5c and M5a formation were 10.8 µM, 375 pmol/min per milligram, and 34.7 µl/min per milligram and 41.7 µM, 94.9 pmol/min per milligram, and 2.28 µl/min per milligram, respectively. Inhibition of HLM CLint with 10 µM digoxin or tranilast (UGT1A9) and 3 µM 16β-phenyllongifolol (UGT2B7/UGT2B4) resulted in fraction metabolism (fm) estimates of 0.81 and 0.19 for UGT1A9 and UGT2B7/UGT2B4, respectively. Relative activity factor scaling of recombinant enzyme kinetics provided comparable fm for UGT1A9 (0.86) and UGT2B7 (0.14). Km and Vmax for M1, M2, and M3 formation ranged 73.0-93.0 µM and 24.3-116 pmol/min per milligram, respectively, and was inhibited by ketoconazole (M1, M2, and M3) and montelukast (M2). In summary, ertugliflozin metabolism in HLMs was primarily mediated by UGT1A9 (78%) with minor contributions from UGT2B7/UGT2B4 (18%), CYP3A4 (3.4%), CYP3A5 (0.4%), and CYP2C8 (0.16%). Considering higher ertugliflozin oxidative metabolism (fCL 0.12) obtained from human mass balance, human systemic clearance is expected to be mediated by UGT1A9 (70%), UGT2B7/UGT2B4 (16%), CYP3A4 (10%), CYP3A5 (1.2%), CYP2C8 (0.5%), and renal elimination (2%). SIGNIFICANCE STATEMENT: This manuscript describes the use of orthogonal approaches (i.e., enzyme kinetics, chemical inhibitors, and recombinant enzymes) to characterize the fraction of ertugliflozin metabolism through various UDP-glucuronosyltransferase (UGT) and cytochrome P450 (CYP) enzyme-mediated pathways. Phenotyping approaches routinely used to characterize CYP hepatic fractional metabolism (fm) to estimate specific enzymes contributing to overall systemic clearance were similarly applied for UGT-mediated metabolism. Defining the in vitro metabolic disposition and fm for ertugliflozin allows risk assessment when considering potential victim-based drug-drug interactions perpetrated by coadministered drugs.

Identification of suberosin metabolites in human liver microsomes by high-performance liquid chromatography combined with high-resolution quadrupole-orbitrap mass spectrometer.

Suberosin is a natural prenylated coumarin derivative isolated from Citropsis articulata. It has various pharmacological properties, especially as an anticoagulant, for which it has been used since antiquity. However, its metabolic pathway and metabolites have not yet been studied. Therefore, this study characterizes its metabolic pathway and metabolites in human liver microsomes (HLMs) using high-resolution quadrupole-orbitrap mass spectrometry (HRMS/MS). Eight metabolites (M1-M8) were found, including three monohydroxylated (M1-M3), one hydrated (M4), three dihydroxylated (M5-M7), and one glucuronide conjugate (M8). Furthermore, forms of cytochrome P450 (CYPs) responsible for suberosin metabolism in HLMs were characterized. CYP1A2 was identified as a major enzyme for the production of M1 and M5 metabolites. The M2, M3, and M7 metabolites were predominantly generated by CYP2B6. M8 was the only phase II metabolite, identified as a glucuronide conjugate from either M1 or M2. This glucuronide conjugate may be the only promising metabolite from phase II metabolism. Phase I metabolism, especially hydroxylation, was found to provide a predominant metabolic pathway of suberosin in HLMs. Further studies should be conducted to explore the metabolites, examining their efficacy and their toxicity in an in vivo system.

Characterization of hydrocoptisonine metabolites in human liver microsomes using a high-resolution quadrupole-orbitrap mass spectrometer

Hydrocoptisonine is a new compound that has been isolated from the rhizomes of Coptis chinensis, which belongs to the Ranunculaceae family of Chinese medicines. Although studies on C. chinensis have been reported, the metabolic pathway of hydrocoptisonine in human liver microsomes (HLMs) remains unelucidated. We identified 13 metabolites in HLMs, including six Phase I metabolites and seven glucuronide conjugates, using a high-resolution quadrupole-orbitrap mass spectrometer. The major metabolic pathway was the O-demethylation and mono-hydroxylation of hydrocoptisonine in HLMs. Notably, M3 metabolite was O-demethylated in dioxolane structures (cyclohexa-3,5-diene-1,2-dione), which was mediated by cytochrome P450 1A2. The locations of hydroxylation and hydroxyl-glucuronidation were identified by analyzing the signature fragments generated as a result of tandem mass spectrometry, indicating hydroxylation at an aliphatic chain or aromatic ring. We determined whether the hydroxylation and glucuronidation occurred in an aromatic moiety (M5 and M12) or an aliphatic moiety (M6 and M13), respectively, based on signature fragments of the metabolites.

Assessment of synthetic cannabinoid FUB-AMB and its ester hydrolysis metabolite in human liver microsomes and human blood samples using UHPLC-MS/MS.

FUB-AMB, an indazole carboxamide synthetic cannabinoid recreational drug, was one of the compounds most frequently reported to governmental agencies worldwide between 2016 and 2019. It has been implicated in intoxications and fatalities, posing a risk to public health. In the current study, FUB-AMB was incubated with human liver microsomes (HLM) to assess its metabolic fate and stability and to determine if its major ester hydrolysis metabolite (M1) was present in 12 authentic forensic human blood samples from driving under the influence of drug cases and postmortem investigations using UHPLC-MS/MS. FUB-AMB was rapidly metabolized in HLM, generating M1 that was stable through a 120-min incubation period, a finding that indicates a potential long detection window in human biological samples. M1 was identified in all blood samples, and no parent drug was detected. The authors propose that M1 is a reliable marker for inclusion in laboratory blood screens for FUB-AMB; this metabolite may be pharmacologically active like its precursor FUB-AMB. M1 frequently appears in samples in which the parent drug is undetectable and can point to the causative agent. The results suggest that it is imperative that synthetic cannabinoid laboratory assay panels include metabolites, especially known or potential pharmacologically active metabolites, particularly for compounds with short half-lives.

In vitro evaluation of reactive nature of E- and Z-guggulsterones and their metabolites in human liver microsomes using UHPLC-Orbitrap mass spectrometer

Guggulipid is known to be useful for hypercholesterolemia, arthritis, acne, and obesity. These activities are attributed to its two principal isomeric active constituents, viz., E- and Z-guggulsterones. There are several side effects reported for guggulipid, which include widespread erythematous papules in a morbilliform pattern and macules localized to the arms; swelling and erythema of the face with burning sensation; pruritis; and bullous lesions on the lower legs with associated headaches, myalgia and itching. We hypothesized that one probable reason for these toxic reactions could be the formation of electrophilic reactive metabolites (RMs) of guggulsterones and their subsequent reaction with cellular proteins. Unfortunately, no report exists in the literature highlighting detection of RMs of guggulsterone isomers. Accordingly, the present study was undertaken to investigate the potential of E- and Z-guggulsterones to form RMs in human liver microsomes (HLM) using glutathione (GSH) and N-acetylcysteine (NAC) as trapping agents. The generated samples were analysed using ultra-high performance liquid chromatography (UHPLC) coupled to an Orbitrap mass spectrometer. The analysis of incubations with trapping agents highlighted that hydroxylated metabolites of guggulsterone isomers showed adduction with GSH and NAC. Even direct adducts of guggulsterone isomers were observed with both the trapping agents. The in silico toxicity potential of E- and Z-guggulsterones and their RMs was predicted using ADMET Predictor™ software and comparison was made against reported toxicities of guggulipid.

Importance of OATP1B1 and 1B3 in the Liver Uptake of Luteolin and Its Consequent Glucuronidation Metabolites

Luteolin is a typical flavonoid and broadly distributed in the plants. Oral bioavailability of luteolin is low owing to extensive metabolism. Regioselective glucuronidation by UDP-glucuronosyltransferases (UGTs) and liver uptake by organic anion transporting polypeptides (OATPs) of luteolin and consequent glucuronidation metabolites were studied. Luteolin-3'-O-glucuronide (L-3'-G) and luteolin-7-O-glucuronide (L-7-G) were the major metabolites in human liver microsomes. Further study demonstrated that UGT1A9 played a predominant role in the glucuronidation of luteolin. Transporter study showed that OATP1B1- and 1B3-transfected cells selectively uptake L-3'-G into cells but not luteolin or L-7-G. After intravenous administration of luteolin to mice, the area under the curve of L-3'-G in the plasma was the highest among luteolin, L-3'-G, and L-7-G. In the liver, the concentration of L-3'-G was significantly greater than L-7-G. In conclusion, OATP1B1 and OATP1B3 play an important role in the liver disposition of luteolin and its glucuronidation metabolites.

Synthesis of [13 C2,15 N]-1,3-2H-1-benzyl-(Z)-3-(benzylidene)indolin-2-one.

Parkinson disease (PD) is a neurodegenerative disorder characterized by the accumulation of α-synuclein into Lewy bodies. 3-Benzylidine-indolin-2-one represents a class of compounds, which are known to inhibit the accumulation of α-synuclein. In this paper, we report the synthesis of [13 C] and [15 N] labelled 1-benzyl-(Z)-3-(benzylidene)indolin-2-one from commercially available [13 C2 ]-chloroacetic acid and [15 N]-aniline in five steps. The product will be used to study its metabolites in human liver microsomes by liquid chromatography-tandem mass spectrometry.

Truncated Itraconazole Analogues Exhibiting Potent Anti-Hedgehog Activity and Improved Drug-like Properties.

We conducted a structure-activity relationship study to explore simplified analogues of the itraconazole (ITZ) scaffold for their ability to inhibit the hedgehog (Hh) signaling pathway. These analogues were based on exploring the effects of chemical modifications to the linker and triazolone/side chain region of ITZ. Analogue 11 was identified as the most potent compound in our first generation, with an IC50 value of 81 nM in a murine Hh-dependent basal cell carcinoma. Metabolic identification studies led us to identify truncated piperazine (26) as the major metabolite in human liver microsomes (HLMs) and an improved Hh pathway inhibitor (IC50 = 22 nM). This work verifies that continued truncation of the ITZ scaffold is a practical method to maintain potent anti-Hh activity while also reducing the molecular weight for the ITZ scaffold and achieving improved pharmacokinetic properties.

In vitro characterization of glycyrol metabolites in human liver microsomes using HR-resolution MS spectrometer coupled with tandem mass spectrometry

1. Glycyrol is a coumestan derivative that is isolated from roots of Glycyrrhiza uralensis. Glycyrol exhibits several biological effects, including anti-oxidative and anti-inflammatory effects.2. Herein, we characterized glycyrol metabolism by cytochrome P450 enzymes (CYPs) and UDP-glucuronosyltransferases (UGTs) using human liver microsomes (HLM), human liver cytosol, human intestinal microsomes, or human recombinant cDNA-expressed CYPs and UGTs. The analysis was conducted using high resolution mass spectroscopy (HR-MS) on a Q ExactiveTM HF Hybride Quadrupole-Orbitrap mass spectrometer.3. NADPH-supplemented HLM generated six glycyrol metabolites (M1–M6) via hydroxylation, oxidation, and hydration; both NADPH- and UDPGA-supplemented liver microsomes generated three glucuronides (M7–M9). Reaction phenotyping revealed that CYP1A2 is the primary enzyme responsible for phase I metabolism, with minor involvement of the CYP3A4/5, CYP2D6, and CYP2E1 enzymes. Glucuronidation of glycyrol was primarily mediated by UGT1A1, UGT1A3, UGT1A9, and UGT2B7.4. In conclusion, glycyrol undergoes the efficient metabolic hydroxylation and glucuronidation reactions in human liver microsomes, which are predominantly catalyzed by CYP1A2, UGT1A1/3/9, and UGT2B7.

Identification of the Metabolic Profile of the α-Tubulin-Binding Natural Product (-)-Pironetin.

Pironetin, the only crystallographically confirmed natural product to target α-tubulin, displays potent cytotoxic activity against sensitive and resistant A2780 ovarian cancer cell lines but is only marginally active in vivo. We now report that pironetin has a short half-life (<7 min) in human liver microsomes, suggesting that its limited in vivo efficacy is due to rapid metabolism. Further, we describe the discovery of epoxypironetin as pironetin's major metabolite in human liver microsomes.

P.3.10 The role of CYP2D6 gene in treatment resistant schizophrenia and the effectiveness of treatment resistant schizophrenia

l-Corydalmine (l-CDL) was under development as an oral analgesic agent, exhibiting potent analgesic activity in preclinical models. The objective of this study was to compare metabolic profiles of l-CDL in liver microsomes from mouse, rat, monkey, dog and human. Six metabolites (M1–M6) were identified using LC-Q/TOF in liver microsomes from the five species. The metabolism of l-CDL included O-demethylation (M1–3) and hydroxylation (M4–6). The desmethyl metabolites were the major ones among the five species, which accounted for more than 84%. Data from chemical inhibition in human liver microsomes (HLM) and human recombinant CYP450s demonstrated that CYP2D6 exhibited strong catalytic activity towards M1 and M2 formations, while CYP2C9 and CYP2C19 also catalyzed M2 formation. Formations of M3 and hydroxyl metabolites (M4 and M5) were mainly catalyzed by CYP3A4. Further studies showed that M1 and M2 were main metabolites in HLM. The kinetics of M1 and M2 formations in HLM and recombinant CYP450s were also investigated. The results showed that M1 and M2 formations in HLM and recombinant CYP2D6 characterized biphasic kinetics, whereas sigmoid Vmax model was better used to fit M2 formation by recombinant CYP2C9 and CYP2C19. The contributions of CYP2D6 to M1 and M2 formations in HLM were estimated to be 75.3% and 50.7%, respectively. However, the contributions of CYP2C9 and CYP2C19 to M2 formation were only 5.0% and 4.1%, respectively. All these data indicated that M1 and M2 were main metabolites in HLM, and CYP2D6 was the primary enzyme responsible for their formations.

The Metabolism of Separase Inhibitor Sepin-1 in Human, Mouse, and Rat Liver Microsomes.

Separase, a known oncogene, is widely overexpressed in numerous human tumors of breast, bone, brain, blood, and prostate. Separase is an emerging target for cancer therapy, and separase enzymatic inhibitors such as sepin-1 are currently being developed to treat separase-overexpressed tumors. Drug metabolism plays a critical role in the efficacy and safety of drug development, as well as possible drug–drug interactions. In this study, we investigated the in vitro metabolism of sepin-1 in human, mouse, and rat liver microsomes (RLM) using metabolomic approaches. In human liver microsomes (HLM), we identified seven metabolites including one cysteine–sepin-1 adduct and one glutathione–sepin-1 adduct. All the sepin-1 metabolites in HLM were also found in both mouse and RLM. Using recombinant CYP450 isoenzymes, we demonstrated that multiple enzymes contributed to the metabolism of sepin-1, including CYP2D6 and CYP3A4 as the major metabolizing enzymes. Inhibitory effects of sepin-1 on seven major CYP450s were also evaluated using the corresponding substrates recommended by the US Food and Drug Administration. Our studies indicated that sepin-1 moderately inhibits CYP1A2, CYP2C19, and CYP3A4 with IC50 < 10 μM but weakly inhibits CYP2B6, CYP2C8/9, and CYP2D6 with IC50 > 10 μM. This information can be used to optimize the structures of sepin-1 for more suitable pharmacological properties and to predict the possible sepin-1 interactions with other chemotherapeutic drugs.

Charactering the metabolism of cryptotanshinone by human P450 enzymes and uridine diphosphate glucuronosyltransferases in vitro.

Cryptotanshinone (CT) is the main active component in the root of Salvia miltiorrhiza Bunge (SMB) that displays antibacterial, anti-inflammatory and anticancer activities. In this study, we characterized phase I and phase II metabolism of CT in human liver microsomes in vitro and identified the metabolic enzymes (CYPs and UGTs) involved. The metabolites of CT generated by CYPs were detected using LC-MS/MS and the CYP subtypes involved in the metabolic reactions were identified using chemical inhibitors of CYP enzymes and recombinant human CYP enzymes (CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). Glucuronidation of CT was also examined, and the UGT subtypes involved in the metabolic reactions were identified using recombinant human UGT enzymes (1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17). After adding NADPH to the human liver microsomes incubation system, CT was transformed into 6 main dehydrogenation and hydroxylation metabolites. CYP2A6, CYP3A4 and CYP2C19 were the major contributors to the transformation of its hydroxylation metabolites. CYP2C19, CYP1A2 and CYP3A4 were the major contributors to the transformation of its hydrogenation metabolites in human liver microsomes. This study showed that the metabolites at m/z of 473 were mediated by UGT1A9 and that the metabolites at m/z of 489 were mediated by UGT2B7 and UGT2B4. CT was extensively metabolized by UGTs following metabolism by CYPs in the liver.

Inhibition of CYP3A by Antimalarial Piperaquine and Its Metabolites in Human Liver Microsomes With IVIV Extrapolation

The potential of the antimalarial piperaquine and its metabolites to inhibit CYP3A was investigated in pooled human liver microsomes. CYP3A activity was measured by liquid chromatography-tandem mass spectrometry as the rate of 1′-hydroxymidazolam formation. Piperaquine was found to be a reversible, potent inhibitor of CYP3A with the following parameter estimates (%CV): IC50 = 0.76 μM (29), Ki = 0.68 μM (29). In addition, piperaquine acted as a time-dependent inhibitor with IC50 declining to 0.32 μM (28) during 30-min pre-incubation. Time-dependent inhibitor estimates were kinact = 0.024 min−1 (30) and KI = 1.63 μM (17). Metabolite M2 was a highly potent reversible inhibitor with estimated IC50 and Ki values of 0.057 μM (17) and 0.043 μM (3), respectively. M1 and M5 metabolites did not show any inhibitory properties within the limits of assay used. Average (95th percentile) simulated in vivo areas under the curve of midazolam increased 2.2-fold (3.7-fold) on the third which is the last day of piperaquine dosing, whereas for its metabolite M2, areas under the curve of midazolam increased 7.7-fold (13-fold).

The Cytochrome P450 Enzyme Responsible for the Production of (Z)-Norendoxifen in vitro.

Norendoxifen, an active metabolite of tamoxifen, is a potent aromatase inhibitor. Little information is available regarding production of norendoxifen in vitro. Here, we conducted a series of kinetic and inhibition studies in human liver microsomes (HLMs) and expressed P450s to study the metabolic disposition of norendoxifen. To validate that norendoxifen was the metabolite of endoxifen, metabolites in HLMs incubates of endoxifen were measured using a HPLC/MS/MS method. To further probe the specific isoforms involved in the metabolic route, endoxifen was incubated with recombinant P450s (CYP 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, 3A5 and CYP4A11). Formation rates of norendoxifen were evaluated in the absence and presence of P450 isoform specific inhibitors using HLMs. The peak of norendoxifen was found in the incubations consisting of endoxifen, HLMs, and cofactors. The retention times of norendoxifen, endoxifen, and the internal standard (diphenhydramine) were 7.81, 7.97, and 5.86 min, respectively. The Km (app) and Vmax (app) values of norendoxifen formation from endoxifen in HLM was 47.8 μm and 35.39 pmol min-1 mg-1 . The apparent hepatic intrinsic clearances of norendoxifen formation were 0.74 μl mg-1 min. CYP3A5 and CYP2D6 were the major enzymes capable of norendoxifen formation from endoxifen with the rates of 0.26 and 0.86 pmol pmol-1 P450 × min. CYP1A2, 3A2, 2C9, and 2C19 also contributed to norendoxifen formation, but the contributions were at least 6-fold lower. One micromolar ketoconazole (CYP3A inhibitor) showed an inhibitory effect on the rates of norendoxifen formation by 45%, but 1 μm quinidine (CYP2D6 inhibitor) does not show any inhibitory effect. Norendoxifen, metabolism from endoxifen by multiple P450s that including CYP3A5.