Deepak Kumar Bhatt

Age-Dependent Absolute Abundance of Hepatic Carboxylesterases (CES1 and CES2) by LC-MS/MS Proteomics: Application to PBPK Modeling of Oseltamivir In Vivo Pharmacokinetics in Infants

The age-dependent absolute protein abundance of carboxylesterase (CES) 1 and CES2 in human liver was investigated and applied to predict infant pharmacokinetics (PK) of oseltamivir. The CES absolute protein abundance was determined by liquid chromatography-tandem mass spectrometry proteomics in human liver microsomal and cytosolic fractions prepared from tissue samples obtained from 136 pediatric donors and 35 adult donors. Two surrogate peptides per protein were selected for the quantification of CES1 and CES2 protein abundance. Purified CES1 and CES2 protein standards were used as calibrators, and the heavy labeled peptides were used as the internal standards. In hepatic microsomes, CES1 and CES2 abundance (in picomoles per milligram total protein) increased approximately 5-fold (315.2 vs. 1664.4) and approximately 3-fold (59.8 vs. 174.1) from neonates to adults, respectively. CES1 protein abundance in liver cytosol also showed age-dependent maturation. Oseltamivir carboxylase activity was correlated with protein abundance in pediatric and adult liver microsomes. The protein abundance data were then used to model in vivo PK of oseltamivir in infants using pediatric physiologically based PK modeling and incorporating the protein abundance–based ontogeny function into the existing pediatric Simcyp model. The predicted pediatric area under the curve, maximal plasma concentration, and time for maximal plasma concentration values were below 2.1-fold of the clinically observed values, respectively.

Critical Issues and Optimized Practices in Quantification of Protein Abundance Level to Determine Interindividual Variability in DMET Proteins by LC‐MS/MS Proteomics

Protein quantification data on drug metabolizing enzymes and transporters (collectively referred as DMET proteins) in human tissues are useful in predicting interindividual variability in drug disposition. While targeted proteomics is an emerging technique for quantification of DMET proteins, the methodology involves significant technical challenges especially when multiple samples are analyzed in a single study over a long period of time. Therefore, it is important to thoroughly address the critical variables that could affect DMET protein quantification.

Novel Interactions between Gut Microbiome and Host Drug-Processing Genes Modify the Hepatic Metabolism of the Environmental Chemicals Polybrominated Diphenyl Ethers

The gut microbiome is a novel frontier in xenobiotic metabolism. Polybrominated diphenyl ethers (PBDEs), especially BDE-47 (2, 2′, 4, 4′-tetrabromodiphenyl ether) and BDE-99 (2, 2′, 4, 4′,5-pentabromodiphenyl ether), are among the most abundant and persistent environmental contaminants that produce a variety of toxicities. Little is known about how the gut microbiome affects the hepatic metabolism of PBDEs and the PBDE-mediated regulation of drug-processing genes (DPGs) in vivo. The goal of this study was to determine the role of gut microbiome in modulating the hepatic biotransformation of PBDEs. Nine-week-old male C57BL/6J conventional (CV) or germ-free (GF) mice were treated with vehicle, BDE-47 or BDE-99 (100 μmol/kg) for 4 days. Following BDE-47 treatment, GF mice had higher levels of 5-OH-BDE-47 but lower levels of four other metabolites in liver than CV mice; whereas following BDE-99 treatment GF mice had lower levels of four minor metabolites in liver than CV mice. RNA sequencing demonstrated that the hepatic expression of DPGs was regulated by both PBDEs and enterotypes. Under basal conditions, the lack of gut microbiome upregulated the Cyp2c subfamily but downregulated the Cyp3a subfamily. Following PBDE exposure, certain DPGs were differentially regulated by PBDEs in a gut microbiome–dependent manner. Interestingly, the lack of gut microbiome augmented PBDE-mediated upregulation of many DPGs, such as Cyp1a2 and Cyp3a11 in mouse liver, which was further confirmed by targeted metabolomics. The lack of gut microbiome also augmented the Cyp3a enzyme activity in liver. In conclusion, our study has unveiled a novel interaction between gut microbiome and the hepatic biotransformation of PBDEs.

Hepatic Abundance and Activity of Androgen- and Drug-Metabolizing Enzyme UGT2B17 Are Associated with Genotype, Age, and Sex

The major objective of this study was to investigate the association of genetic and nongenetic factors with variability in protein abundance and in vitro activity of the androgen-metabolizing enzyme UGT2B17 in human liver microsomes (n = 455). UGT2B17 abundance was quantified by liquid chromatography-tandem mass spectrometry proteomics, and enzyme activity was determined by using testosterone and dihydrotestosterone as in vitro probe substrates. Genotyping or gene resequencing and mRNA expression were also evaluated. Multivariate analysis was used to test the association of UGT2B17 copy number variation, single nucleotide polymorphisms (SNPs), age, and sex with its mRNA expression, abundance, and activity. UGT2B17 gene copy number and SNPs (rs7436962, rs9996186, rs28374627, and rs4860305) were associated with gene expression, protein levels, and androgen glucuronidation rates in a gene dose-dependent manner. UGT2B17 protein (mean ± S.D. picomoles per milligram of microsomal protein) is sparsely expressed in children younger than 9 years (0.12 ± 0.24 years) but profoundly increases from age 9 years to adults (∼10-fold) with ∼2.6-fold greater abundance in males than in females (1.2 vs. 0.47). Association of androgen glucuronidation with UGT2B15 abundance was observed only in the low UGT2B17 expressers. These data can be used to predict variability in the metabolism of UGT2B17 substrates. Drug companies should include UGT2B17 in early phenotyping assays during drug discovery to avoid late clinical failures.

Genetic and Nongenetic Factors Associated with Protein Abundance of Flavin-Containing Monooxygenase 3 in Human Liver.

Hepatic flavin-containing mono-oxygenase 3 (FMO3) metabolizes a broad array of nucleophilic heteroatom (e.g., N or S)-containing xenobiotics (e.g., amphetamine, sulindac, benzydamine, ranitidine, tamoxifen, nicotine, and ethionamide), as well as endogenous compounds (e.g., catecholamine and trimethylamine). To predict the effect of genetic and nongenetic factors on the hepatic metabolism of FMO3 substrates, we quantified FMO3 protein abundance in human liver microsomes (HLMs; n = 445) by liquid chromatography-tandem mass chromatography proteomics. Genotyping/gene resequencing, mRNA expression, and functional activity (with benzydamine as probe substrate) of FMO3 were also evaluated. FMO3 abundance increased 2.2-fold (13.0 ± 11.4 pmol/mg protein vs. 28.0 ± 11.8 pmol/mg protein) from neonates to adults. After 6 years of age, no significant difference in FMO3 abundance was found between children and adults. Female donors exhibited modestly higher mRNA fragments per kilobase per million reads values (139.9 ± 76.9 vs. 105.1 ± 73.1; P < 0.001) and protein FMO3 abundance (26.7 ± 12.0 pmol/mg protein vs. 24.1 ± 12.1 pmol/mg protein; P < 0.05) compared with males. Six single nucleotide polymorphisms (SNPs), including rs2064074, rs28363536, rs2266782 (E158K), rs909530 (N285N), rs2266780 (E308G), and rs909531, were associated with significantly decreased protein abundance. FMO3 abundance in individuals homozygous and heterozygous for haplotype 3 (H3), representing variant alleles for all these SNPs (except rs2066534), were 50.8% (P < 0.001) and 79.5% (P < 0.01), respectively, of those with the reference homozygous haplotype (H1, representing wild-type). In summary, FMO3 protein abundance is significantly associated with age, gender, and genotype. These data are important in predicting FMO3-mediated heteroatom-oxidation of xenobiotics and endogenous biomolecules in the human liver.

The Promises of Quantitative Proteomics in Precision Medicine

Precision medicine approach has a potential to ensure optimum efficacy and safety of drugs at individual patient level. Physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) models could play a significant role in precision medicine by predicting interindividual variability in drug disposition and response. In order to develop robust PBPK/PD models, it is imperative that the critical physiological parameters affecting drug disposition and response and their variability are precisely characterized. Currently used PBPK/PD modeling software, for example, Simcyp and Gastroplus, encompass information such as organ volumes, blood flows to organs, body fat composition, glomerular filtration rate, etc. However, the information on the interindividual variability of the majority of the proteins associated with PK and PD, for example, drug metabolizing enzymes, transporters, and receptors, are not fully incorporated into these PBPK modeling platforms. Such information is significant because the population factors such as age, genotype, disease, and gender can affect abundance or activity of these proteins. To fill this critical knowledge gap, mass spectrometry-based quantitative proteomics has emerged as an important technique to characterize interindividual variability in the protein abundance of drug metabolizing enzymes, transporters, and receptors. Integration of these quantitative proteomics data into in silico PBPK/PD modeling tools will be crucial toward precision medicine.

Age-dependent protein abundance of cytosolic alcohol and aldehyde dehydrogenases in human liver

Hepatic cytosolic alcohol and aldehyde dehydrogenases (ADHs and ALDHs) catalyze the biotransformation of xenobiotics (e.g., cyclophosphamide and ethanol) and vitamin A. Because age-dependent hepatic abundance of these proteins is unknown, we quantified protein expression of ADHs and ALDH1A1 in a large cohort of pediatric and adult human livers by liquid chromatography coupled with tandem mass spectrometry proteomics. Purified proteins were used as calibrators. Two to three surrogate peptides per protein were quantified in trypsin digests of liver cytosolic samples and calibrator proteins under optimal conditions of reproducibility. Neonatal levels of ADH1A, ADH1B, ADH1C, and ALDH1A1 were 3-, 8-, 146-, and 3-fold lower than the adult levels, respectively. For all proteins, the abundance steeply increased during the first year of life, which mostly reached adult levels during early childhood (age between 1 and 6 years). Only for ADH1A protein abundance in adults (age > 18 year) was ∼40% lower relative to the early childhood group. Abundances of ADHs and ALDH1A1 were not associated with sex in samples with age > 1 year compared with males. Known single nucleotide polymorphisms had no effect on the protein levels of these proteins. Quantification of ADHs and ALDH1A1 protein levels could be useful in predicting disposition and response of substrates of these enzymes in younger children.

Abundance of Phase 1 and 2 Drug-Metabolizing Enzymes in Alcoholic and Hepatitis C Cirrhotic Livers: A Quantitative Targeted Proteomics Study

To predict the impact of liver cirrhosis on hepatic drug clearance using physiologically based pharmacokinetic (PBPK) modeling, we compared the protein abundance of various phase 1 and phase 2 drug-metabolizing enzymes (DMEs) in S9 fractions of alcoholic (n = 27) or hepatitis C (HCV, n = 30) cirrhotic versus noncirrhotic (control) livers (n = 25). The S9 total protein content was significantly lower in alcoholic or HCV cirrhotic versus control livers (i.e., 38.3 ± 8.3, 32.3 ± 12.8, vs. 51.1 ± 20.7 mg/g liver, respectively). In general, alcoholic cirrhosis was associated with a larger decrease in the DME abundance than HCV cirrhosis; however, only the abundance of UGT1A4, alcohol dehydrogenase (ADH)1A, and ADH1B was significantly lower in alcoholic versus HCV cirrhotic livers. When normalized to per gram of tissue, the abundance of nine DMEs (UGT1A6, UGT1A4, CYP3A4, UGT2B7, CYP1A2, ADH1A, ADH1B, aldehyde oxidase (AOX)1, and carboxylesterase (CES)1) in alcoholic cirrhosis and five DMEs (UGT1A6, UGT1A4, CYP3A4, UGT2B7, and CYP1A2) in HCV cirrhosis was <25% of that in control livers. The abundance of most DMEs in cirrhotic livers was 25% to 50% of control livers. CES2 abundance was not affected by cirrhosis. Integration of UGT2B7 abundance in cirrhotic livers into the liver cirrhosis (Child Pugh C) model of Simcyp improved the prediction of zidovudine and morphine PK in subjects with Child Pugh C liver cirrhosis. These data demonstrate that protein abundance data, combined with PBPK modeling and simulation, can be a powerful tool to predict drug disposition in special populations.

Pregnancy Increases Norbuprenorphine Clearance in Mice by Induction of Hepatic Glucuronidation

Norbuprenorphine (NBUP) is the major active metabolite of buprenorphine (BUP) that is commonly used to treat opiate addiction during pregnancy; it possesses 25% of BUP’s analgesic activity and 10 times BUP’s respiratory depression effect. To optimize BUP’s dosing regimen during pregnancy with better efficacy and safety, it is important to understand how pregnancy affects NBUP disposition. In this study, we examined the pharmacokinetics of NBUP in pregnant and nonpregnant mice by administering the same amount of NBUP through retro-orbital injection. We demonstrated that the systemic clearance (CL) of NBUP in pregnant mice increased ∼2.5-fold compared with nonpregnant mice. Intrinsic CL of NBUP by glucuronidation in mouse liver microsomes from pregnant mice was ∼2 times greater than that from nonpregnant mice. Targeted liquid chromatography tandem-mass spectrometry proteomics quantification revealed that hepatic Ugt1a1 and Ugt2b1 protein levels in the same amount of total liver membrane proteins were significantly increased by ∼50% in pregnant mice versus nonpregnant mice. After scaling to the whole liver with consideration of the increase in liver protein content and liver weight, we found that the amounts of Ugt1a1, Ugt1a10, Ugt2b1, and Ugt2b35 protein in the whole liver of pregnant mice were significantly increased ∼2-fold compared with nonpregnant mice. These data suggest that the increased systemic CL of NBUP in pregnant mice is likely caused by an induction of hepatic Ugt expression and activity. The data provide a basis for further mechanistic analysis of pregnancy-induced changes in the disposition of NBUP and drugs that are predominately and extensively metabolized by Ugts.

PBDEs Altered Gut Microbiome and Bile Acid Homeostasis in Male C57BL/6 Mice

Polybrominated diphenyl ethers (PBDEs) are persistent environmental contaminants with well characterized toxicities in host organs. Gut microbiome is increasingly recognized as an important regulator of xenobiotic biotransformation; however, little is known about its interactions with PBDEs. Primary bile acids (BAs) are metabolized by the gut microbiome into more lipophilic secondary BAs that may be absorbed and interact with certain host receptors. The goal of this study was to test our hypothesis that PBDEs cause dysbiosis and aberrant regulation of BA homeostasis. Nine-week-old male C57BL/6 conventional (CV) and germ-free (GF) mice were orally gavaged with corn oil (10 mg/kg), BDE-47 (100 μmol/kg), or BDE-99 (100 μmol/kg) once daily for 4 days (n = 3–5/group). Gut microbiome was characterized using 16S rRNA sequencing of the large intestinal content in CV mice. Both BDE-47 and BDE-99 profoundly decreased the alpha diversity of gut microbiome and differentially regulated 45 bacterial species. Both PBDE congeners increased Akkermansia muciniphila and Erysipelotrichaceae Allobaculum spp., which have been reported to have anti-inflammatory and antiobesity functions. Targeted metabolomics of 56 BAs was conducted in serum, liver, and small and large intestinal content of CV and GF mice. BDE-99 increased many unconjugated BAs in multiple biocompartments in a gut microbiota-dependent manner. This correlated with an increase in microbial 7α-dehydroxylation enzymes for secondary BA synthesis and increased expression of host intestinal transporters for BA absorption. Targeted proteomics showed that PBDEs downregulated host BA-synthesizing enzymes and transporters in livers of CV but not GF mice. In conclusion, there is a novel interaction between PBDEs and the endogenous BA-signaling through modification of the “gut-liver axis”.