Human Liver Microsomal Samples Research Articles

Cytochrome P450 Family 4F2 and 4F11 Haplotype Mapping and Association with Hepatic Gene Expression and Vitamin K Hydroxylation Activity.

This study evaluated the underlying mechanistic links between genetic variability in vitamin K metabolic pathway genes (CYP4F2 and CYP4F11) and phylloquinone hydroxylation activity using genotype- and haplotype-based approaches. Specifically, we characterized genetic variability in the CYP4F2/CYP4F11 locus and compared common single allele genotypes and common haplotypes as predictors of hepatic gene expression, enzyme abundance, and phylloquinone (VK1) ω-hydroxylation kinetics. We measured CYP4F2 and CYP4F11 mRNA levels, CYP4F2 and CYP4F11 protein abundances, and the VK1 concentration-dependent ω-hydroxylation rate in matched human liver nucleic acid and microsome samples, utilizing a novel in vitro population modeling approach. Results indicate that accounting for the CYP4F2*3 allele alone is sufficient to capture most of the genetic-derived variability in the observed phenotypes. Additionally, our findings highlight the important contribution that CYP4F11 makes toward vitamin K metabolism in the human liver.

Metabolism of dipentylone in zebrafish and human liver microsomes determined by liquid chromatography–high resolution mass spectrometry

The consumption of novel psychoactive substances (NPS) is exceedingly prevalent in society, as these substances are sold and distributed as “legal highs.” One novel synthetic cathinone emerging in the market is 1-(1,3-benzodioxol-5-yl)–2-(dimethylamino) pentan-1-one (dipentylone). The goal of this work was to study the in vivo and in vitro metabolism of dipentylone in zebrafish and human liver microsomes (HLMs) by liquid chromatography-high resolution mass spectrometry (LC-HRMS). The zebrafish and HLM samples contained 14 dipentylone metabolites, specifically 12 phase Ⅰ metabolites and 2 phase Ⅱ metabolites. The main metabolic pathways included monohydroxylation (M1 and M2), N-dealkylation (M3), hydroxylation of the aromatic ring and dealkoxylation of M3 (M4), O-dealkylation (M5), N-dealkylation of M5 (M6), reduction of carboxide (M7), monohydroxylation of M5 (M8), dehydrogenation (M9), dealkoxylation (M10), N-dealkylation of M10 (M11), dealkoxylation of M9 (M12), glucuronidation of M5 (M13), and sulfation (M14). The monohydroxylated metabolite (M2) can be recommended as metabolic markers for dipentylone. This study is the first to identify a target compound for monitoring the abuse of dipentylone and to determine the essential chemical structure of the metabolites for further toxicological research.

Identification of HSD17B12 as an enzyme catalyzing drug reduction reactions through investigation of nabumetone metabolism

Nabumetone, a nonsteroidal anti-inflammatory prodrug, is converted to a pharmacologically active metabolite, 6-methoxy-2-naphthylacetic acid (6-MNA); however, it is 11-fold more efficiently converted to 4-(6-methoxy-2-naphthyl)butan-2-ol (MNBO) via a reduction reaction in human hepatocytes. The goal of this study was to identify the enzyme(s) responsible for MNBO formation from nabumetone in the human liver. MNBO formation by human liver microsomes (HLM) was 5.7-fold higher than in the liver cytosol. In a panel of 24 individual HLM samples with quantitative proteomics data, the 17β-hydroxysteroid dehydrogenase 12 (HSD17B12) protein level had the high correlation coefficient (r = 0.80, P < 0.001) among 4457 proteins quantified in microsomal fractions during MNBO formation. Recombinant HSD17B12 expressed in HEK293T cells exhibited prominent nabumetone reductase activity, and the contribution of HSD17B12 to the activity in the HLM was calculated as almost 100%. MNBO formation in HepG2 and Huh7 cells was significantly decreased by the knockdown of HSD17B12. We also examined the role of HSD17B12 in drug metabolism and found that recombinant HSD17B12 catalyzed the reduction reactions of pentoxifylline and S-warfarin, suggesting that HSD17B12 prefers compounds containing a methyl ketone group on the alkyl chain. In conclusion, our study demonstrated that HSD17B12 is responsible for the formation of MNBO from nabumetone. Together with the evidence for pentoxifylline and S-warfarin reduction, this is the first study to report that HSD17B12, which is known to metabolize endogenous compounds, such as estrone and 3-ketoacyl-CoA, plays a role as a drug-metabolizing enzyme.

Metabolism of the Synthetic Cathinone Alpha-Pyrrolidinoisohexanophenone in Humans Using UHPLC--MS-QToF.

Alpha-pyrrolidinoisohexanophenone (α-PHiP/α-PiHP) is a synthetic drug structurally related to cathine, a natural psychoactive alkaloid, isolated from Khat plant. The α-PiHP is a structural isomer of α-PHP, and both α-PHP and α-PiHP could be considered an analog of α-PVP, a Schedule I drug under the Convention on Psychotropic Substances by the United Nations. This α-pyrrolidinophenone was first reported to European Monitoring of Drug and Drug Addiction by Slovenia in December 2016. In Hungary, it was initially reported in August 2016, and until 2021, it had been detected in seizures only twice and never been identified in biological samples. However, in 2021, its consumption became prevalent in Hungary. This study aims to investigate the α-PiHP metabolites by performing in vitro and in vivo metabolite identification studies of human liver microsome (pHLM), S9 fraction (pS9) and urine samples (from control and users), using liquid chromatography in conjunction with high-resolution mass spectrometry. Ten in vivo urinary metabolites of α-PiHP were tentatively identified and confirmed by in vitro metabolites detected in pHLM and pS9 samples. Among the eight Phase I and the two Phase II metabolites, five were more abundant in urine than the parent compound. The two major metabolites via reduction of the keto moiety (M01) and via oxidation of the pyrrolidine ring combined with aliphatic hydroxylation and keto reduction (M06) were identified. The metabolites via the combination of keto reduction and aliphatic hydroxylation (M04), via ring-opening followed by carboxylation (M09) and via glucuronidation of the keto reduced metabolite (M07) were also dominant. The minor metabolites were one Phase II metabolite (M08), two metabolites via aliphatic hydroxylation (M02 and M03), one metabolite via the combination of keto reduction and oxidation of the pyrrolidine ring (M05) and one metabolite via oxidation of the pyrrolidine ring (M10).

Label-Free but Still Constrained: Assessment of Global Proteomic Strategies for the Quantification of Hepatic Enzymes and Transporters.

Building and refining pharmacology models require "system" data derived from tissues and in vitro systems analyzed by quantitative proteomics. Label-free global proteomics offers a wide scope of analysis, allowing simultaneous quantification of thousands of proteins per sample. The data generated from such analysis offer comprehensive protein expression profiles that can address existing gaps in models. In this study, we assessed the performance of three widely used label-free proteomic methods, "high N" ion intensity approach (HiN), intensity-based absolute quantification (iBAQ) and total protein approach (TPA), in relation to the quantification of enzymes and transporters in 27 human liver microsomal samples. Global correlations between the three methods were highly significant (R2 > 0.70, P < 0.001, n = 2232 proteins). Absolute abundances of 57 pharmacokinetic targets measured by standard-based label-free methods (HiN and iBAQ) showed good agreement, whereas the TPA overestimated abundances by two- to threefold. Relative abundance distribution of enzymes was similar for the three methods, while differences were observed with TPA in the case of transporters. Variability (CV) was similar across methods, with consistent between-sample relative quantification. The back-calculated amount of protein in the samples based on each method was compared with the nominal protein amount analyzed in the proteomic workflow, revealing overall agreement with data from the HiN method with bovine serum albumin as standard. The findings herein present a critique of label-free proteomic data relevant to pharmacokinetics and evaluate the possibility of retrospective analysis of historic datasets. SIGNIFICANCE STATEMENT: This study provides useful insights for using label-free methods to generate abundance data applicable for populating pharmacokinetic models. The data demonstrated overall correlation between intensity-based label-free proteomic methods (HiN, iBAQ and TPA), whereas iBAQ and TPA overestimated the total amount of protein in the samples. The extent of overestimation can provide a means of normalization to support absolute quantification. Importantly, between-sample relative quantification was consistent (similar variability) across methods.

Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Crosslinking Mass Spectrometry.

Aiming to elucidate the system-wide effects of the alcohol-induced increase in the content of cytochrome P450 2E1 (CYP2E1) on drug metabolism, we explored the array of its protein-protein interactions (interactome) in human liver microsomes (HLM) with chemical crosslinking mass spectrometry (CXMS). Our strategy employs membrane incorporation of purified CYP2E1 modified with photoreactive crosslinkers benzophenone-4-maleimide and 4-(N-succinimidylcarboxy)benzophenone. Exposure of bait-incorporated HLM samples to light was followed by isolating the His-tagged bait protein and its crosslinked aggregates on Ni-NTA agarose. Analyzing the individual bands of SDS-PAGE slabs of thereby isolated protein with the toolset of untargeted proteomics, we detected the crosslinked dimeric and trimeric complexes of CYP2E1 with other drug-metabolizing enzymes. Among the most extensively crosslinked partners of CYP2E1 are the cytochromes P450 2A6, 2C8, 3A4, 4A11, and 4F2, UDP-glucuronosyltransferases (UGTs) 1A and 2B, fatty aldehyde dehydrogenase (ALDH3A2), epoxide hydrolase 1 (EPHX1), disulfide oxidase 1α (ERO1L), and ribophorin II (RPN2). These results demonstrate the exploratory power of the proposed CXMS strategy and corroborate the concept of tight functional integration in the human drug-metabolizing ensemble through protein-protein interactions of the constituting enzymes.

Probing functional interactions between cytochromes P450 with principal component analysis of substrate saturation profiles and targeted proteomics

We investigated the correspondence between drug metabolism routes and the composition of the P450 ensemble in human liver microsomes (HLM). As a probe, we used Coumarin 152 (C152), a fluorogenic substrate metabolized by multiple P450 species. Studying the substrate-saturation profiles (SSP) in seven pooled HLM preparations, we sought to correlate them with the P450 pool's composition characterized by targeted proteomics. This analysis, complemented with the assays with specific inhibitors of CYP3A4 and CYP2C19, the primary C152 metabolizers, demonstrated a significant contrast between different HLM samples. To unveil the source of these differences, we implemented Principal Component Analysis (PCA) of the SSP series obtained with HLM samples with a known composition of the P450 pool. Our analysis revealed that the parameters of C152 metabolism are primarily determined by the content of CYP2A6, CYP2B6, CYP2C8, CYP2E1, and CYP3A5 of those only CYP2B6 and CYP3A5 can metabolize C152. To validate this finding, we studied the effect of enriching HLM with CYP2A6, CYP2E1, and CYP3A5. The incorporation of CYP3A5 into HLM decreases the rate of C152 metabolism while increasing the role of CYP2B6 in its turnover. In contrast, incorporation of CYP2A6 and CYP2E1 reroutes the C152 demethylation towards some P450 enzyme with a moderate affinity to the substrate, most likely CYP3A4. Our results reveal a sharp non-additivity of the individual P450 properties and suggest a pivotal role of P450–P450 interactions in determining drug metabolism routes. This study demonstrates the high potential of our new PCA-based approach in unveiling functional interrelationships between different P450 species.

Effects of alcohol-induced increase in CYP2E1 content in human liver microsomes on the activity and cooperativity of CYP3A4

We investigate the effect of the alcohol-induced increase in the content of CYP2E1 in human liver microsomes (HLM) on the function of CYP3A4. Membrane incorporation of the purified CYP2E1 into HLM considerably increases the rate of metabolism of 7-benzyloxyquinoline (BQ) and attenuates the homotropic cooperativity observed with this CYP3A4-specific substrate. It also eliminates the activating effect of α-naphthoflavone (ANF) seen in some HLM samples. To probe the physiological relevance of these effects, we compared three pooled preparations of HLM from normal donors (HLM-N) with a pooled preparation from ten heavy alcohol consumers (HLM-A). The composition of the P450 pool in all samples was characterized by the mass-spectrometric determination of 11 cytochrome P450 species. The fractional content of CYP2E1 in HLM-A was from 2.0 to 3.4 times higher than in HLM-N. In contrast, the content of CYP3A4 in HLM-A was the lowest among all samples. Despite that, HLM-A exhibited a much higher metabolism rate and a lower homotropic cooperativity with BQ, similar to CYP2E1-enriched HLM-N. To substantiate the involvement of interactions between CYP2E1 and CYP3A4 in these effects, we probed hetero-association of these proteins in CYP3A4-containing Supersomes™ with a technique employing CYP2E1 labeled with BODIPY-618 maleimide. These experiments evinced the interactions between the two enzymes and revealed an inhibitory effect of ANF on their association. Our results demonstrate that the functional properties of CYP3A4 are fundamentally dependent on the composition of the cytochrome P450 ensemble and suggest a possible impact of chronic alcohol exposure on the pharmacokinetics of drugs metabolized by CYP3A4.

A novel mass spectrometry method for the absolute quantification of several cytochrome P450 and uridine 5'-diphospho-glucuronosyltransferase enzymes in the human liver.

Cytochrome P450 (CYP450) and 5'-diphosphate glucuronosyltransferases (UGT) are the two major families of drug-metabolizing enzymes in the human liver microsome (HLM). As a result of their frequent abundance fluctuation among populations, the accurate quantification of these enzymes in different individuals is important for designing patient-specific dosage regimens in the framework of precision medicine. The preparation and quantification of internal standards is an essential step for the quantitative analysis of enzymes. However, the commonly employed stable isotope labeling-based strategy (QconCAT) suffers from requiring very expensive isotopic reagents, tedious experimental procedures, and long labeling times. Furthermore, arginine-to-proline conversion during metabolic isotopic labeling compromises the quantification accuracy. Therefore, we present a new strategy that replaces stable isotope-labeled amino acids with lanthanide labeling for the preparation and quantification of QconCAT internal standard peptides, which leads to a threefold reduction in the reagent costs and a fivefold reduction in the time consumed. The absolute amount of trypsin-digested QconCAT peptides can be obtained by lanthanide labeling and inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis with a high quantification accuracy (%RE < 20%). By taking advantage of the highly selective and facile ICP-OES procedure and multiplexed large-scale absolute target protein quantification using biological mass spectrometry, this strategy was successfully used for the absolute quantification of drug-metabolizing enzymes. We obtained good linearity (correlation coefficient > 0.95) over concentrations spanning 2.5 orders of magnitude with improved sensitivity (limit of quantification = 2fmol) in nine HLM samples, indicating the potential of this method for large-scale absolute target protein quantification in clinical samples. Graphical abstract.

Potential Regulation of UGT2B10 and UGT2B7 by miR-485-5p in Human Liver

The UDP-glucuronosyltransferase (UGT) family of enzymes is important in the metabolic elimination of a variety of endogenous compounds such as bile acids, steroids, and fat-soluble vitamins, as well as exogenous compounds including many pharmaceuticals. The UGT2B subfamily is a major family of UGT enzymes expressed in human liver. The identification of novel mechanisms including post-transcriptional regulation by microRNA (miRNA) contributes to interindividual variability in UGT2B expression and is a crucial component in predicting patient drug response. In the present study, a high-resolution liquid chromatography–tandem mass spectrometry method was employed to measure UGT2B protein levels in a panel of human liver microsomal samples (n = 62). Concurrent in silico analysis identified eight candidate miRNAs as potential regulators of UGT2B enzymes. Comparison of UGT2B protein expression and candidate miRNA levels from human liver samples demonstrated a significant inverse correlation between UGT2B10 and UGT2B15 and one of these candidate miRNAs, miR-485-5p. A near-significant correlation was also observed between UGT2B7 and miR-485-5p expression. In vitro analysis using luciferase-containing vectors suggested an interaction of miR-485-5p within the UGT2B10 3′-untranslated region (UTR), and significant reduction in luciferase activity was also observed for a luciferase vector containing the UGT2B7 3′-UTR; however, none was observed for the UBT2B15 3′-UTR. UGT2B10 and UGT2B7 activities were probed using nicotine and 3′-azido-3′-deoxythymidine, respectively, and significant decreases in glucuronidation activity were observed for both substrates in HuH-7 and Hep3B cells upon overexpression of miR-485-5p mimic. This is the first study demonstrating a regulatory role of miR-485-5p for multiple UGT2B enzymes.SIGNIFICANCE STATEMENTThe purpose of this study was to identify novel epigenetic miRNA regulators of the UGT2B drug-metabolizing enzymes in healthy human liver samples. Our results indicate that miRNA 485-5p is a novel regulator of UGT2B7 and UGT2B10, which play an important role in the metabolism of many commonly prescribed medications, carcinogens, and endogenous compounds. This study identified potential miRNA-UGT2B mRNA interactions using a novel proteomic approach, with in vitro experiments undertaken to validate these interactions.

Characterization of the Ontogeny of Hepatic UDP-Glucuronosyltransferase Enzymes Based on Glucuronidation Activity Measured in Human Liver Microsomes.

An understanding of the postnatal development of hepatic UDP-glucuronosyltransferase (UGT) enzymes is required for accurate prediction of the age-dependent changes in pharmacokinetics of many drugs used in children. However, the maturation rate of hepatic UGT isoforms remains a major knowledge gap. This study aimed to establish the age-associated changes in glucuronidation activity of 10 major hepatic UGT isoforms in humans, namely, UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B4, UGT2B7, UGT2B10, UGT2B15, and UGT2B17. Human liver microsomes from pediatric and adult donors were incubated under optimized incubation conditions to assess the activity rates of hepatic UGT isoforms using a panel of 19 in vitro UGT probe substrates and clinically used drugs. Statistically strong correlations of glucuronidation activities allowed the ontogeny of UGT1A1, UGT1A4, UGT2B7, UGT2B10, and UGT2B15 to be established using multiple selective UGT substrates and matched human liver microsome samples. The postnatal development of hepatic UGTs is isoform-dependent using either individual or cross-correlated selective isoform substrates. Maximal adult activity was reached at different times ranging from within a month (UGT1A1, UGT2B4, UGT2B7, UGT2B10, and UGT2B15), during infancy (UGT1A3, UGT1A4, and UGT1A9), to adolescence (UGT1A6 and UGT2B17). This study provides an extensive characterization of the postnatal ontogeny profiles of hepatic UGT enzymes that are instrumental for predicting drug disposition via in vitro-in vivo extrapolation algorithms and verifying pharmacokinetic predictions against in vivo observations via pediatric physiologically based pharmacokinetic modeling in pediatric patients.

Impact of cytochrome P450 variation on meperidine N-demethylation to the neurotoxic metabolite normeperidine

1. Meperidine is an opioid analgesic that undergoes N-demethylation to form the neurotoxic metabolite normeperidine. Previous studies indicate that meperidine N-demethylation is catalyzed by cytochrome P450 2B6 (CYP2B6), CYP3A4, and CYP2C19.2. The purpose of this study was to examine the relative P450 contributions to meperidine N-demethylation and to evaluate the effect of CYP2C19 polymorphism on normeperidine generation. Experiments were performed using recombinant P450 enzymes, selective chemical inhibitors, enzyme kinetic assays, and correlation analysis with individual CYP2C19-genotyped human liver microsomes.3. The catalytic efficiency (kcat/Km) for meperidine N-demethylation was similar between recombinant CYP2B6 and CYP2C19, but markedly lower by CYP3A4.4. In CYP2C19-genotyped human liver microsomes, normeperidine formation was significantly correlated with CYP2C19 activity (S-mephenytoin 4´-hydroxylation).5. CYP2C19 inhibitor (+)-N-3-benzylnirvanol and CYP3A inhibitor ketoconazole significantly reduced microsomal normeperidine generation by an individual donor with high CYP2C19 activity, whereas donors with lower CYP2C19 activity were sensitive to inhibition by ketoconazole but not benzylnirvanol.6. These findings demonstrate that the relative CYP3A4, CYP2B6, and CYP2C19 involvement in meperidine N-demethylation depends on the enzyme activities in individual human liver microsomal samples. CYP2C19 is likely an important contributor to normeperidine generation in individuals with high CYP2C19 activity, but additional factors influence inter-individual metabolite accumulation.

Label-free absolute protein quantification with data-independent acquisition

Despite data-independent acquisition (DIA) has been increasingly used for relative protein quantification, DIA-based label-free absolute quantification method has not been fully established. Here we present a novel DIA method using the TPA algorithm (DIA-TPA) for the absolute quantification of protein expressions in human liver microsomal and S9 samples. To validate this method, both data-dependent acquisition (DDA) and DIA experiments were conducted on 36 individual human liver microsome and S9 samples. The MS2-based DIA-TPA was able to quantify approximately twice as many proteins as the MS1-based DDA-TPA method, whereas protein concentrations determined by the two approaches were comparable. To evaluate the accuracy of the DIA-TPA method, we absolutely quantified carboxylesterase 1 concentrations in human liver S9 fractions using an established SILAC internal standard-based proteomic assay; the SILAC results were consistent with those obtained from DIA-TPA analysis. Finally, we employed a unique algorithm in DIA-TPA to distribute the MS signals from shared peptides to individual proteins or isoforms and successfully applied the method to the absolute quantification of several drug-metabolizing enzymes in human liver microsomes. In sum, the DIA-TPA method not only can absolutely quantify entire proteomes and specific proteins, but also has the capability quantifying proteins with shared peptides. SignificanceData independent acquisition (DIA) has emerged as a powerful approach for relative protein quantification at the whole proteome level. However, DIA-based label-free absolute protein quantification (APQ) method has not been fully established. In the present study, we present a novel DIA-based label-free APQ approach, named DIA-TPA, with the capability absolutely quantifying proteins with shared peptides. The method was validated by comparing the quantification results of DIA-TPA with that obtained from stable isotope-labeled internal standard-based proteomic assays.

Pharmacokinetics, tissue distribution and excretion of ACT001 in Sprague-Dawley rats and metabolism of ACT001

This study investigated pharmacokinetics, tissue distribution and excretion of ACT001 in Sprague-Dawley rats. Stability study and metabolism study of ACT001 are conducted. The absolute bioavailability of ACT001 is 50.82%. ACT001 has no accumulation effect and displayed wide tissue distribution. ACT001 can be rapidly distributed to tissues after oral administration and can diffuse through the blood-brain barrier. The total cumulative excretion of ACT001 in feces, urine and bile were found to be 0.05, 3.42 and 0.012%, respectively. UPLC/ESI–QTOF–MS coupled with MetaboLynx XS software was utilized to detect the metabolites of ACT001 in vitro. Five metabolites (M1, M2, M3, M4 and M5) were detected. M2 wasn't discovered in human liver microsome samples and bile samples. M1 and M2 weren't discovered in rat plasma and human plasma. M3, M4 and M5 weren't discovered in bile samples. M5 is an active metabolite named micheliolide (MCL). There is no significant difference in half-life, type of identified metabolites and the amount of each metabolites between using rat plasma and human plasma. Owing to the species differences of hepatomicrosome enzymes, significant differences were shown in half-life, type of identified metabolites and the amount of each metabolites between using rat liver microsome and human liver microsome.

Development and validation of an HPLC\u2013MS/MS method for the determination of arginine-vasopressin receptor blocker conivaptan in human plasma and rat liver microsomes: application to a metabolic stability study

PurposeTo develop and validate a bio-analytical HPLC–MS/MS method for the determination of conivaptan (CVA) an arginine-vasopressin receptor blocker in human plasma and in rat liver microsomes (RLMs).MethodsAnalytes were separated on a reversed phase C18 column (50 mm × 2.1 mm, 1.8 μm). The mobile phase was a mixture of acetonitrile and 10 mM ammonium formate (40:60 v/v, pH 4.0) and was pumped isocratically for 4 min at a flow rate of 0.2 ml/min. Multiple reaction monitoring in positive ionization mode was used for the assay.ResultsThe method yielded a linear calibration plot (r2= 0.9977 and 0.9998) over 5–500 ng/ml with a limit of detection at 1.52 and 0.88 ng/ml for human plasma and RLMs, respectively. The reproducibility of detection of CVA in human plasma and RLMs was found to be in an acceptable range.ConclusionThe method developed in this study is applicable for accurately quantifying CVA in human plasma and rat liver microsomal samples. The optimized procedure was applied to study of metabolic stability of CVA. Conivaptan concentration rapidly decreased in the first 2 min of RLMs incubation and the conversion reached a plateau for the remainder of the incubation period. The in vitro half-life (t1/2) was estimated at 11.51 min and the intrinsic clearance (CLin) was 13.8 ± 0.48 ml/min/kg.

Confounding effect of UGT1A1 interindividual variability on inhibition of its in vitro activity by kinase inhibitors.

Background and ObjectiveInhibition of UGT1A1 by kinase inhibitors (KIs) has been shown to cause mild to serve hyperbilirubinemia in cancer patients1. UGT1A1 is highly polymorphic and displays variable expression. The specific aims of this study were i) to stratify human liver microsomes (HLMs) based on UGT1A1 abundance, activity and genotype, ii) to screen 10 clinically used KIs for their UGT1A1 inhibition potential, and iii) to determine inhibition potential (IC50) of the top three potent KIs in high and low UGT1A1 expressing HLM samples.MethodsTargeted genotyping of liver tissue donors (N=445) was performed; and UGT1A1 protein abundance and activity in HLM samples were quantified using a validated proteomics method and using bilirubin or estradiol as probe substrates, respectively. The mono and di‐bilirubin glucuronides and estradiol 3‐β‐D‐glucuronide (E3βG) were monitored by LC‐MS/MS. Association analysis of UGT1A1 genotype with protein abundance or activity was performed. Screening of UGT1A1 inhibition by KIs (at 1 and 20 uM) was performed using a high UGT1A1 expressing (46.7 ± 4.6 pmol/mg) HLM sample. IC50 values of KIs for UGT1A1 were determined using above mentioned high and a low (10.1 ± 1.6 pmol/mg) expressing HLM samples.ResultsUGT1A1 abundance and activity exhibited age‐dependent maturation with 8‐fold increase from neonates to adults. UGT1A1 SNPs rs10929302, rs887829, rs111741722 and rs4124874, are associated with decreased UGT1A1 abundance and activity, whereas rs8175347 was only associated with decreased abundance. Glucuronidation of both substrates was inhibited by the KIs in a substrate‐independent manner in the following order (% inhibition of E3βG activity at 1 and 20 μM in parenthesis): dasatanib (46 and 91)&gt; sorafenib (11 and 74)&gt; temsirolimus (50 and 71)&gt; sirolimus (0 and 63) &gt; sunitinib (21 and 38) &gt; nilotinib (22 and 34) &gt; gefitinib (0 and 29) &gt; pazopanib = imatinib = lapatinib (0). The top three KIs showed differential IC50 values for E3βG inhibition in high and low UGT1A1 expressers, i.e., dasatanib (2.0, and 4.0), sirolimus (IC50= 7.6, and 32.6), and sorafenib (0.95, and 18.0), respectively.DiscussionUGT1A1 abundance is highly variable with significant effect of age and genotype. Inter‐individual variability in UGT1A1 plays a confounding role in predicting drug‐endobiotic or drug‐drug interactions (DDIs) mediated by this enzyme. Identification of genetic or non‐genetic factors associated with UGT1A1 activity will allow better prediction of UGT1A1 mediated drug metabolism and toxicity.Support or Funding InformationThis work was primarily supported by the National Institutes of Health [Grant no. R01 HD081299]. NA and AB contributed equally to this work. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CYP-Mediated Sulfoximine Deimination of AZD6738.

In hepatic S9 and human liver microsomes (HLMs) the sulfoximine moiety of the ATR inhibitor AZD6738 is metabolized to its corresponding sulfoxide (AZ8982) and sulfone (AZ0002). The initial deimination to AZ8982 is nominally a reductive reaction, but in HLMs it required both NADPH and oxygen and also was inhibited by 1-aminobenzotriazole at a concentration of 1 mM. Studies conducted in a panel of 11 members of the cytochrome P450 (P450) family (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 CYP2J2, CYP3A4, and CYP3A5) confirmed that deimination was an oxidative process that was mediated largely by CYP2C8 with some CYP2J2 involvement, whereas the subsequent oxidation to sulfone was carried out largely by CYP2J2, CYP3A4, and CYP3A5. There was no measureable metabolism in flavin-containing monooxygenase (FMO) enzymes FMO3, FMO5 or NADPH cytochrome C reductase. Studies using Silensomes, a commercially available HLM in which specific members of the P450 family have been inhibited by selective mechanism-based inhibitors, showed that when CYP2C8 was inhibited, the rate of deimination was reduced by 95%, suggesting that CYP2J2 is only playing a minor role in HLMs. When CYP3A4 was inhibited, the rate increased by 58% due to the inhibition of the subsequent sulfone formation. Correlation studies conducted in HLM samples from different individuals confirmed the role of CYP2C8 in the deimination over CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A. Hence, although nominally a reduction, the deimination of AZD6738 to its sulfoxide metabolite AZ8982 is an oxidation mediated by CYP2C8, and this metabolite is subsequently oxidized to the sulfone (AZ0002) largely by CYP3A.

UHPLC-Q-TOF-MS/MS-based screening and characterization of metabolites of cnidilin in human liver microsomes.

Cnidilin is an active natural furocoumarin ingredient originating from well-known traditional Chinese medicine Radix Angelicae Dahuricae. In the present study, an efficient approach was developed for the screening and identification of cnidilin metabolites using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. In this approach, an on-line data acquisition method multiple mass defect filter combined with dynamic background subtraction was developed to trace all probable metabolites. Based on this analytical strategy, a total of 24 metabolites of cnidilin were detected in human liver microsomal incubation samples and the metabolic pathways were proposed. The results indicated that oxidation was the main biotransformation route for cnidilin in human liver microsomes. In addition, the specific cytochrome P450 (CYP) enzymes involved in the metabolism of cnidilin were identified using chemical inhibition and CYP recombinant enzymes. The results showed that CYP1A2 and CYP3A4 might be the major enzymes involved in the metabolism of cnidilin in human liver microsomes. The relationship between cnidilin and the CYP450 enzymes could provide us a theoretical basis of the pharmacological mechanism.

Metabolism studies on hydroxygenkwanin and genkwanin in human liver microsomes by UHPLC-Q-TOF-MS

Hydroxygenkwanin (HYGN) and genkwanin (GN) are major constituents of Genkwa Flos for the treatment of edema, ascites, cough, asthma and cancer. This is a report about the investigation of the metabolic fate of HYGN and GN in human liver microsomes and the recombinant UDP-glucuronosyltransferase (UGT) enzymes by using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). An on-line data acquisition method multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS) was developed to trace all probable metabolites. Based on this analytical strategy, three phase I metabolites and seven glucuronide conjugation metabolites of HYGN, seven phase I metabolites and 12 glucuronide conjugation metabolites of GN were identified in the incubation samples of human liver microsomes. The results indicated that demethylation, hydroxylation and o-glucuronidation were main metabolic pathways of HYGN and GN. The specific UGT enzymes responsible for HYGN and GN glucuronidation metabolites were identified using recombinant UGT enzymes. The results indicated that UGT1A1, UGT1A3, UGT1A9, UGT1A10 and UGT2B7 might play major roles in the glucuronidation reactions. Overall, this study may be useful for the investigation of metabolic mechanism of HYGN and GN, and it can provide reference and evidence for further experiments.

Characterization of Atomoxetine Biotransformation and Implications for Development of PBPK Models for Dose Individualization in Children.

Atomoxetine (ATX) is a second-line nonstimulant medication used to control symptoms of attention deficit hyperactivity disorder (ADHD). Inconsistent therapeutic efficacy has been reported with ATX, which may be related to variable CYP2D6-mediated drug clearance. We characterized ATX metabolism in a panel of human liver samples as a basis for a bottom-up PBPK model to aid in ATX exposure prediction and control. Km, Vmax, and Clint values in pooled human liver microsomes (HLMs) were 2.4 µM, 479 pmol/min/mg protein, and 202 µl/min/mg protein, respectively. Mean population values of kinetic parameters are not adequate to describe variability in a population, given that Km, Vmax, and Clint values from single-donor HLMs ranged from 0.93 to 79.2 µM, 20.0 to 1600 pmol/min/mg protein, and 0.3 to 936 µl/min/mg protein. All kinetic parameters were calculated from 4-hydroxyatomoxetine (4-OH-ATX) formation. CYP2E1 and CYP3A contributed to 4-OH-ATX formation in livers with CYP2D6 intermediate and poor metabolizer status. In HLMs with lower CYP2D6 activity levels, 2-hydroxymethylatomoxetine (2-CH2OH-ATX) formation became a more predominant pathway of metabolism, which appeared to be catalyzed by CYP2B6. ATX biotransformation at clinically relevant plasma concentrations was characterized in a panel of pediatric HLM (n = 116) samples by evaluating primary metabolites. Competing pathways of ATX metabolism [N-desmethylatomoxetine (NDM-ATX) and 2-CH2OH-ATX formation] had increasing importance in livers with lesser CYP2D6 activity, but, overall ATX clearance was still compromised. Modeling ATX exposure to individualize therapy would require comprehensive knowledge of factors that affect CYP2D6 activity as well as an understanding of competing pathways, particularly for individuals with lower CYP2D6 activity.