Author Report for: Zhu HJNo contact information in database for Zhu HJ
Wang X, Shi J, Zhu HJ Ref: Methods Mol Biol, 2603:269, 2023 : PubMed ESTHER: Wang_2023_Methods.Mol.Biol_2603_269 PubMedSearch: Wang 2023 Methods.Mol.Biol 2603 269 PubMedID: 36370287 Abstract Mass spectrometry (MS)-based proteomics has been increasingly used for targeted absolute protein quantifications in both basic and clinical research. There is a great need to overcome some pitfalls of current MS-based targeted absolute protein quantification methods, such as high inter-assay variability and high cost associated with the use of synthesized isotopic peptides/proteins. Here we describe a targeted absolute protein quantification method utilizing SILAC internal standards and unlabeled full-length protein calibrators (TAQSI). The method has proven accurate, precise, reproducible, and cost-effective. Notably, the method is resistant to the variabilities caused by protein extraction and digestion. Moreover, it avoids measurement errors due to nonsynonymous mutations. This versatile method can be used for determining the absolute expressions of numerous proteins in various biological samples. As a proof-of-concept, this method was successfully applied to absolutely quantitate the protein expressions of carboxylesterase 1 (CES1) in human liver tissues. Title: Identification of regulatory variants of carboxylesterase 1 (CES1): A proof-of-concept study for the application of the Allele-Specific Protein Expression (ASPE) assay in identifying cis-acting regulatory genetic polymorphisms Her L, Shi J, Wang X, He B, Smith L, Jiang H, Zhu HJ Ref: Proteomics, :e2200176, 2022 : PubMed ESTHER: Her_2022_Proteomics__e2200176 PubMedSearch: Her 2022 Proteomics e2200176 PubMedID: 36413357 Gene_locus related to this paper: human-CES1 Abstract It is challenging to study regulatory genetic variants as gene expression is affected by both genetic polymorphisms and non-genetic regulators. The mRNA allele-specific expression (ASE) assay has been increasingly used for the study of cis-acting regulatory variants because cis-acting variants affect gene expression in an allele-specific manner. However, poor correlations between mRNA and protein expressions were observed for many genes, highlighting the importance of studying gene expression regulation at the protein level. In the present study, we conducted a proof-of-concept study to utilize a recently developed allele-specific protein expression (ASPE) assay to identify the cis-acting regulatory variants of CES1 using a large set of human liver samples. The CES1 gene encodes for carboxylesterase 1 (CES1), the most abundant hepatic hydrolase in humans. Two cis-acting regulatory variants were found to be significantly associated with CES1 ASPE, CES1 protein expression, and its catalytic activity on enalapril hydrolysis in human livers. Compared to conventional gene expression-based approaches, ASPE demonstrated an improved statistical power to detect regulatory variants with small effect sizes since allelic protein expression ratios are less prone to the influence of non-genetic regulators (e.g., diseases and inducers). This study suggests that the ASPE approach is a powerful tool for identifying cis-regulatory variants. This article is protected by copyright. All rights reserved. Title: Physiologically-based pharmacokinetic modeling to predict methylphenidate exposure affected by interplay among carboxylesterase 1 pharmacogenetics, drug-drug interactions, and sex Xiao J, Shi J, Thompson BR, Smith DE, Zhang T, Zhu HJ Ref: J Pharm Sci, :, 2022 : PubMed ESTHER: Xiao_2022_J.Pharm.Sci__ PubMedSearch: Xiao 2022 J.Pharm.Sci PubMedID: 35526575 Abstract BACKGROUND AND OBJECTIVE: The pharmacokinetics (PK) of methylphenidate (MPH) differ significantly among individuals. Carboxylesterase 1 (CES1) is the primary enzyme metabolizing MPH, and its function is affected by genetic variants, drug-drug interaction (DDI), and sex. The object of this study is to evaluate CES1 pharmacogenetics as related to MPH metabolism using human liver samples and develop a physiologically-based pharmacokinetic (PBPK) modeling approach to investigate the influence of CES1 genotypes and other factors on MPH PK. METHODS: The effect of the CES1 variant G143E (rs71647871) on MPH metabolism was studied utilizing 102 individual human liver S9 (HLS9) fraction samples. PBPK models were developed using the population-based PBPK software PK-Sim(a) by incorporating the HLS9 incubation data. The established models were applied to simulate MPH PK profiles under various clinical scenarios, including different genotypes, drug-alcohol interactions, and the difference between males and females. RESULTSL: The HLS9 incubation study showed that subjects heterozygous for the CES1 variant G143E metabolized MPH at a rate of approximately 50% of that in non-carriers. The developed PBPK models successfully predicted the exposure alteration of MPH from the G143E genetic variant, ethanol-MPH DDI, and sex. Importantly, the study suggests that male G143E carriers who are alcohol consumers are at a higher risk of MPH overexposure. CONCLUSION: PBPK modeling provides a means for better understanding the mechanisms underlying interindividual variability in MPH PK and PD and could be utilized to develop a safer and more effective MPH pharmacotherapy regimen. Title: Transcriptional regulation of carboxylesterase 1 (CES1) in human liver: role of the nuclear receptor NR1H3 (LXRalpha) and its splice isoforms Collins JM, Lu R, Wang X, Zhu HJ, Wang D Ref: Drug Metabolism & Disposition: The Biological Fate of Chemicals, :, 2021 : PubMed ESTHER: Collins_2021_Drug.Metab.Dispos__ PubMedSearch: Collins 2021 Drug.Metab.Dispos PubMedID: 34697082 Gene_locus related to this paper: human-CES1 Abstract Carboxylesterase 1 (CES1) is the predominant carboxylesterase in the human liver, involved in metabolism of both xenobiotics and endogenous substrates. Genetic or epigenetic factors that alter CES1 activity or expression are associated with changes in drug response, lipid, and glucose homeostasis. However, the transcriptional regulation of CES1 in the human liver remains uncertain. By applying both the random forest and Sobol's Sensitivity Indices (SSI) to analyze existing liver RNA expression microarray data (GSE9588), we identified NR1H3 (LXRalpha) as a key factor regulating constitutive CES1 expression. This model prediction was validated using siRNA knockdown and CRISPR-mediated transcriptional activation of NR1H3 in Huh7 and HepG2 cells. We found that NR1H3's activation of CES1 is splice isoform-specific, namely that increased expression of the NR1H3-211 isoform increased CES1 expression while NR1H3-201 did not. Also, in human liver samples, expression of NR1H3-211 and CES1 are correlated, while NR1H3-201 and CES1 are not. This trend also occurs during differentiation of induced pluripotent stem cells (iPSCs) to hepatocytes, where only expression of the NR1H3-211 isoform parallels expression of CES1 Moreover, we found that treatment with the NR1H3 agonist T0901317 in HepG2 cells had no effect on CES1 expression. Overall, our results demonstrate a key role of NR1H3 in maintaining the constitutive expression of CES1 in the human liver. Furthermore, our results support that the effect of NR1H3 is splice isoform-specific and appears to be ligand independent. Significance Statement Despite the central role of CES1 in metabolism of numerous medications, little is known about its transcriptional regulation. Here we identify NR1H3 as a key regulator of constitutive CES1 expression, and therefore is a potential target for future studies to understand inter-person variabilities in CES1 activity and drug metabolism. Title: Effect of CES1 genetic variation on enalapril steady-state pharmacokinetics and pharmacodynamics in healthy subjects Her LH, Wang X, Shi J, Choi HJ, Jung SM, Smith LS, Wu AH, Bleske BE, Zhu HJ Ref: British Journal of Clinical Pharmacology, :, 2021 : PubMed ESTHER: Her_2021_Br.J.Clin.Pharmacol__ PubMedSearch: Her 2021 Br.J.Clin.Pharmacol PubMedID: 33963573 Abstract BACKGROUND AND OBJECTIVE: Enalapril is a prodrug and needs to be activated by carboxylesterase 1 (CES1). A previous in vitro study demonstrated the CES1 genetic variant, G143E (rs71647871), significantly impaired enalapril activation. Two previous clinical studies examined the impact of G143E on single-dose enalapril PK (10 mg); however, the results were inconclusive. A prospective, multi-dose, pharmacokinetics, and pharmacodynamics (PK/PD) study was conducted to determine the impact of the CES1 G143E variant on enalapril steady-state PK and PD in healthy volunteers. METHODS: Study participants were stratified to G143E non-carriers (n=15) and G143E carriers (n=6). All the carriers were G143E heterozygotes. Study subjects received enalapril 10 mg daily for seven consecutive days prior to a 72h PK/PD study. Plasma concentrations of enalapril and its active metabolite enalaprilat were quantified by an established LC-MS/MS method. RESULTS: The CES1 G143E carriers had 30.9% lower enalaprilat C(max) (P = 0.03) compared to the non-carriers (38.01 vs. 55.01 ng/mL). The carrier group had 27.5% lower AUC(0-) (P = 0.02) of plasma enalaprilat compared to the non-carriers (374.29 vs. 515.91 ng*hr/mL). The carriers also had a 32.3% lower enalaprilat-to-enalapril AUC(0-) ratio (P = 0.003) relative to the non-carriers. The average maximum reduction of systolic blood pressure in the non-carrier group was approximately 12.4% at the end of the study compared to the baseline (P = 0.001). No statistically significant blood pressure reduction was observed in the G143E carriers. CONCLUSIONS: The CES1 loss-of-function G143E variant significantly impaired enalapril activation and its systolic blood pressure-lowering effect in healthy volunteers. Title: Activation of Tenofovir Alafenamide and Sofosbuvir in the Human Lung and Its Implications in the Development of Nucleoside/Nucleotide Prodrugs for Treating SARS-CoV-2 Pulmonary Infection Li J, Liu S, Shi J, Zhu HJ Ref: Pharmaceutics, 13:, 2021 : PubMed ESTHER: Li_2021_Pharmaceutics_13_ PubMedSearch: Li 2021 Pharmaceutics 13 PubMedID: 34683949 Gene_locus related to this paper: human-CTSA Inhibitor(s) related to this paper: Sofosbuvir Abstract ProTide technology is a powerful tool for the design of nucleoside/nucleotide analog prodrugs. ProTide prodrug design improves cell permeability and enhances intracellular activation. The hydrolysis of the ester bond of a ProTide is a determinant of the intracellular activation efficiency and final antiviral efficacy of the prodrug. The hydrolysis is dictated by the catalytic activity and abundance of activating enzymes. The antiviral agents tenofovir alafenamide (TAF) and sofosbuvir (SBV) are typical ProTides. Both TAF and SBV have also been proposed to treat patients with COVID-19. However, the mechanisms underlying the activation of the two prodrugs in the lung remain inconclusive. In the present study, we profiled the catalytic activity of serine hydrolases in human lung S9 fractions using an activity-based protein profiling assay. We evaluated the hydrolysis of TAF and SBV using human lung and liver S9 fractions and purified enzymes. The results showed that CatA and CES1 were involved in the hydrolysis of the two prodrugs in the human lung. More specifically, CatA exhibited a nearly 4-fold higher hydrolytic activity towards TAF than SBV, whereas the CES1 activity on hydrolyzing TAF was slightly lower than that for SBV. Overall, TAF had a nearly 4-fold higher hydrolysis rate in human lung S9 than SBV. We further analyzed protein expression levels of CatA and CES1 in the human lung, liver, and primary cells of the two tissues using proteomics data extracted from the literature. The relative protein abundance of CatA to CES1 was considerably higher in the human lung and primary human airway epithelial cells than in the human liver and primary human hepatocytes. The findings demonstrated that the high susceptivity of TAF to CatA-mediated hydrolysis resulted in efficient TAF hydrolysis in the human lung, suggesting that CatA could be utilized as a target activating enzyme when designing antiviral ester prodrugs for the treatment of respiratory virus infection. Title: Contributions of Cathepsin A and Carboxylesterase 1 to the hydrolysis of Tenofovir Alafenamide in the Human Liver, and the Effect of CES1 Genetic Variation on Tenofovir Alafenamide Hydrolysis Li J, Shi J, Xiao J, Tran L, Wang X, Zhu HJ Ref: Drug Metabolism & Disposition: The Biological Fate of Chemicals, :, 2021 : PubMed ESTHER: Li_2021_Drug.Metab.Dispos__ PubMedSearch: Li 2021 Drug.Metab.Dispos PubMedID: 34933885 Gene_locus related to this paper: human-CES1, human-CTSA Abstract The prodrug tenofovir alafenamide (TAF) is a first-line antiviral agent for the treatment of chronic hepatitis B infection. TAF activation involves multiple steps, and the first step is an ester hydrolysis reaction catalyzed by hydrolases. This study was to determine the contributions of carboxylesterase 1 (CES1) and cathepsin A (CatA) to TAF hydrolysis in the human liver. Our in vitro incubation studies showed that both CatA and CES1 catalyzed TAF hydrolysis in a pH-dependent manner. At their physiological pH environment, the activity of CatA (pH 5.2) was approximately 1,000-fold higher than that of CES1 (pH 7.2). Given that the hepatic protein expression of CatA was approximately 200-fold lower than that of CES1, the contribution of CatA to TAF hydrolysis in the human liver was estimated to be much greater than that of CES1, which is contrary to the previous perception that CES1 is the primary hepatic enzyme hydrolyzing TAF. The findings were further supported by a TAF incubation study with the CatA inhibitor telaprevir and the CES1 inhibitor bis-(p-nitrophenyl) phosphate. Moreover, an in vitro study revealed that the CES1 variant G143E (rs71647871) is a loss-of-function variant for CES1-mediated TAF hydrolysis. In summary, our results suggest that CatA may play a more important role in the hepatic activation of TAF than CES1. Additionally, TAF activation in the liver could be affected by CES1 genetic variation, but the magnitude of impact appears to be limited due to the major contribution of CatA to hepatic TAF activation. Significance Statement Contrary to the general perception that carboxylesterase 1 (CES1) is the major enzyme responsible for tenofovir alafenamide (TAF) hydrolysis in the human liver, the present study demonstrated that cathepsin A (CatA) may play a more significant role in TAF hepatic hydrolysis. Furthermore, the CES1 variant G143E (rs71647871) was found to be a loss-of-function variant for CES1-mediated TAF hydrolysis. Title: Plasma carboxylesterase 1 predicts methylphenidate exposure: a proof-of-concept study using plasma protein biomarker for hepatic drug metabolism Shi J, Xiao J, Wang X, Jung SM, Bleske BE, Markowitz JS, Patrick KS, Zhu HJ Ref: Clinical Pharmacology & Therapeutics, :, 2021 : PubMed ESTHER: Shi_2021_Clin.Pharmacol.Ther__ PubMedSearch: Shi 2021 Clin.Pharmacol.Ther PubMedID: 34743324 Abstract Hepatic drug-metabolizing enzymes (DMEs) play critical roles in determining the pharmacokinetics and pharmacodynamics of numerous therapeutic agents. As such, noninvasive biomarkers capable of predicting DME expression in the liver have the potential to be used to personalize pharmacotherapy and improve drug treatment outcomes. In the present study, we quantified carboxylesterase 1 (CES1) protein concentrations in plasma samples collected during a methylphenidate (MPH) PK study. CES1 is a prominent hepatic enzyme responsible for the metabolism of many medications containing small ester moieties, including MPH. The results revealed a significant inverse correlation between plasma CES1 protein concentrations and the area under the concentration-time curves (AUCs) of plasma d-MPH (p = 0.014, r = -0.617). In addition, when plasma CES1 protein levels were normalized to the plasma concentrations of 24 liver-enriched proteins to account for potential interindividual differences in hepatic protein release rate, the correlation was further improved (p = 0.003, r = -0.703), suggesting that plasma CES1 protein could explain approximately 50% of the variability in d-MPH AUCs in the study participants. A physiologically based pharmacokinetic (PBPK) modeling simulation revealed that the CES1-based individualized dosing strategy might significantly reduce d-MPH exposure variability in pediatric patients relative to conventional fixed dosing trial and error regimens. This proof-of-concept study indicates that the plasma protein of a hepatic DME may serve as a biomarker for predicting its metabolic function and the pharmacokinetics of its substrate drugs. Title: Impact of carboxylesterase 1 genetic polymorphism on trandolapril activation in human liver and the pharmacokinetics and pharmacodynamics in healthy volunteers Wang X, Her L, Xiao J, Shi J, Wu AH, Bleske BE, Zhu HJ Ref: Clin Transl Sci, :, 2021 : PubMed ESTHER: Wang_2021_Clin.Transl.Sci__ PubMedSearch: Wang 2021 Clin.Transl.Sci PubMedID: 33660934 Abstract Trandolapril, an angiotensin-converting enzyme inhibitor prodrug, needs to be activated by carboxylesterase 1 (CES1) in the liver to exert its intended therapeutic effect. A previous in vitro study demonstrated that the CES1 genetic variant G143E (rs71647871) abolished CES1-mediated trandolapril activation in cells transfected with the variant. This study aimed to determine the effect of the G143E variant on trandolapril activation in human livers and the pharmacokinetics (PKs) and pharmacodynamics (PDs) in human subjects. We performed an in vitro incubation study to assess trandolapril activation in human livers (5 G143E heterozygotes and 97 noncarriers) and conducted a single-dose (1 mg) PK and PD study of trandolapril in healthy volunteers (8 G143E heterozygotes and 11 noncarriers). The incubation study revealed that the mean trandolapril activation rate in G143E heterozygous livers was 42% of those not carrying the variant (p = 0.0015). The clinical study showed that, relative to noncarriers, G143E carriers exhibited 20% and 15% decreases, respectively, in the peak concentration (C(max) ) and area under the curve from 0 to 72 h (AUC(0-72 h) ) of the active metabolite trandolaprilat, although the differences were not statistically significant. Additionally, the average maximum reductions of systolic blood pressure and diastolic blood pressure in carriers were ~ 22% and 23% less than in noncarriers, respectively, but the differences did not reach a statistically significant level. In summary, the CES1 G143E variant markedly impaired trandolapril activation in the human liver under the in vitro incubation conditions; however, this variant had only a modest impact on the PK and PD of trandolapril in healthy human subjects. Title: Carboxylesterase 1 and Precision Pharmacotherapy: Pharmacogenetics and Nongenetic Regulators Her L, Zhu HJ Ref: Drug Metabolism & Disposition: The Biological Fate of Chemicals, 48:230, 2020 : PubMed ESTHER: Her_2020_Drug.Metab.Dispos_48_230 PubMedSearch: Her 2020 Drug.Metab.Dispos 48 230 PubMedID: 31871135 Gene_locus related to this paper: human-CES1 Abstract Carboxylesterase (CES) 1 is the most abundant drug-metabolizing enzyme in human livers, comprising approximately 1% of the entire liver proteome. CES1 is responsible for 80%-95% of total hydrolytic activity in the liver and plays a crucial role in the metabolism of a wide range of drugs (especially ester-prodrugs), pesticides, environmental pollutants, and endogenous compounds. Expression and activity of CES1 vary markedly among individuals, which is a major contributing factor to interindividual variability in the pharmacokinetics (PK) and pharmacodynamics (PD) of drugs metabolized by CES1. Both genetic and nongenetic factors contribute to CES1 variability. Here, we discuss genetic polymorphisms, including single-nucleotide polymorphisms (SNPs), and copy number variants and nongenetic contributors, such as developmental status, genders, and drug-drug interactions, that could influence CES1 functionality and the PK and PD of CES1 substrates. Currently, the loss-of-function SNP G143E (rs71647871) is the only clinically significant CES1 variant identified to date, and alcohol is the only potent CES1 inhibitor that could alter the therapeutic outcomes of CES1 substrate medications. However, G143E and alcohol can only explain a small portion of the interindividual variability in the CES1 function. A better understanding of the regulation of CES1 expression and activity and identification of biomarkers for CES1 function in vivo could lead to the development of a precision pharmacotherapy strategy to improve the efficacy and safety of many CES1 substrate drugs. SIGNIFICANCE STATEMENT: The clinical relevance of CES1 has been well demonstrated in various clinical trials. Genetic and nongenetic regulators can affect CES1 expression and activity, resulting in the alteration of the metabolism and clinical outcome of CES1 substrate drugs, such as methylphenidate and clopidogrel. Predicting the hepatic CES1 function can provide clinical guidance to optimize pharmacotherapy of numerous medications metabolized by CES1. Title: Chemoproteomic Identification of Serine Hydrolase RBBP9 as a Valacyclovir-Activating Enzyme Shenoy VM, Thompson BR, Shi J, Zhu HJ, Smith DE, Amidon GL Ref: Mol Pharm, 17:1706, 2020 : PubMed ESTHER: Shenoy_2020_Mol.Pharm_17_1706 PubMedSearch: Shenoy 2020 Mol.Pharm 17 1706 PubMedID: 32196348 Gene_locus related to this paper: human-RBBP9 Abstract Prodrug discovery and development in the pharmaceutical industry have been hampered by a lack of knowledge of prodrug activation pathways. Such knowledge would minimize the risks of prodrug failure by enabling proper selection of preclinical animal models, prediction of pharmacogenomic variability, and identification of drug-drug interactions. Technologies for annotation of activating enzymes have not kept pace with the growing need. Activity-based protein profiling (ABPP) has matured considerably in recent decades, leading to widespread use in the pharmaceutical industry. Here, we report the extension of competitive ABPP (cABPP) to prodrug-activating enzyme identification in stable isotope-labeled cell lysates using a modified fluorophosphonate probe. Focusing on the antiviral ester prodrug valacyclovir (VACV), we identified serine hydrolase RBBP9 as an activating enzyme in Caco-2 cells via shotgun proteomics, validating the activity via the selective inhibitor emetine (EME). Kinetic characterization of RBBP9 revealed a catalytic efficiency (k(cat).K(M)(-1) = 104 mM(-1).s(-1)) comparable to that of BPHL, the only known VACV-activating enzyme prior to this work. EME incubation in wild-type and Bphl-knockout jejunum and liver lysates demonstrated the near-exclusivity of VACV activation by RBBP9 in the intestine. Additionally, these studies showed that RBBP9 and BPHL are the two major and coequal VACV-activating enzymes in the liver. Single-pass intestinal perfusions of VACV +/- EME in mice showed EME coperfusion significantly inhibited the intestinal activation of VACV, implying the in vivo relevance of RBBP9-mediated VACV activation. We envision that others might use the cABPP approach in the future for global, rapid, and efficient discovery of prodrug-activating enzymes. Title: FRACPRED-2D-PRM: A fraction prediction algorithm-assisted two-dimensional liquid chromatography-based parallel reaction monitoring-mass spectrometry approach for measuring low-abundance proteins in human plasma Shi J, Xiao J, Li J, Wang X, Her L, Sorensen MJ, Zhu HJ Ref: Proteomics, :e2000175, 2020 : PubMed ESTHER: Shi_2020_Proteomics__e2000175 PubMedSearch: Shi 2020 Proteomics e2000175 PubMedID: 33085175 Abstract Multidimensional fractionation-based enrichment methods improve the sensitivity of proteomic analysis for low-abundance proteins. However, a major limitation of conventional multidimensional proteomics is the extensive labor and instrument time required for analyzing many fractions obtained from the first dimension separation. Here, we present a fraction prediction algorithm-assisted two-dimensional LC-based parallel reaction monitoring-mass spectrometry (FRACPRED-2D-PRM) approach for measuring low-abundance proteins in human plasma. Plasma digests were separated by the first dimension high-pH RP-LC with data-dependent acquisition (DDA). We then used the FRACPRED algorithm to predict the retention time of undetectable target peptides according to those of other abundant plasma peptides during the first dimension separation. Fractions predicted to contain target peptides were analyzed by the second dimension low-pH nano RP-LC PRM. We demonstrated the accuracy and robustness of fraction prediction with the FRACPRED algorithm by measuring two low-abundance proteins, aldolase B and carboxylesterase 1, in human plasma. The FRACPRED-2D-PRM proteomics approach demonstrated markedly improved efficiency and sensitivity over conventional 2D-LC proteomics assays. We expect that this approach will be widely used in the study of low-abundance proteins in plasma and other complex biological samples. This article is protected by copyright. All rights reserved. Title: Label-free absolute protein quantification with data-independent acquisition He B, Shi J, Wang X, Jiang H, Zhu HJ Ref: J Proteomics, 200:51, 2019 : PubMed ESTHER: He_2019_J.Proteomics_200_51 PubMedSearch: He 2019 J.Proteomics 200 51 PubMedID: 30880166 Abstract 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. SIGNIFICANCE: Data 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. Title: Effect of biphenyl hydrolase-like (BPHL) gene disruption on the intestinal stability, permeability and absorption of valacyclovir in wildtype and Bphl knockout mice Hu Y, Epling D, Shi J, Song F, Tsume Y, Zhu HJ, Amidon GL, Smith DE Ref: Biochemical Pharmacology, 156:147, 2018 : PubMed ESTHER: Hu_2018_Biochem.Pharmacol_156_147 PubMedSearch: Hu 2018 Biochem.Pharmacol 156 147 PubMedID: 30121252 Gene_locus related to this paper: human-BPHL Abstract Biphenyl hydrolase-like protein (BPHL) is a novel human serine hydrolase that was originally cloned from a breast carcinoma cDNA library and shown to convert valacyclovir to acyclovir and valganciclovir to ganciclovir. However, the exclusivity of this process has not been determined and, indeed, it is possible that a number of esterases/proteases may mediate the hydrolysis of valacyclovir and similar prodrugs. The objectives of the present study were to evaluate the in situ intestinal permeability and stability of valacyclovir in wildtype (WT) and Bphl knockout (KO) mice, as well as the in vivo oral absorption and intravenous disposition of valacyclovir and acyclovir in the two mouse genotypes. We found that Bphl knockout mice had no obvious phenotype and that Bphl ablation did not alter the jejunal permeability of valacyclovir during in situ perfusions (i.e., 0.54x10(-4) in WT vs. 0.53x10(-4)cm/s in KO). Whereas no meaningful changes occurred between genotypes in the gene expression of proton-coupled oligopeptide transporters (i.e., PepT1, PepT2, PhT1, PhT2), enzymatic upregulation of Cyp3a11, Cyp3a16, Abhd14a and Abhd14b was observed in some tissues of Bphl knockout mice. Most importantly, we found that valacyclovir was rapidly and efficiently hydrolyzed to acyclovir in the absence of BPHL, and that hydrolysis was more extensive after the oral vs. intravenous route of administration (for both genotypes). Taken as a whole, BPHL is not obligatory for the conversion of valacyclovir to acyclovir either presystemically or systemically. Title: Functional study of Carboxylesterase 1 protein isoforms Wang X, Shi J, Zhu HJ Ref: Proteomics, :e1800288, 2018 : PubMed ESTHER: Wang_2018_Proteomics__e1800288 PubMedSearch: Wang 2018 Proteomics e1800288 PubMedID: 30520264 Abstract Carboxylesterase1 (CES1) is a primary human hepatic hydrolase involved in hydrolytic biotransformation of numerous medications. Considerable interindividual variability in CES1 expression and activity has been consistently reported. Four isoforms of the CES1 protein are produced by alternative splicing (AS). In the current study, we examined the activity and expression of each CES1 isoform using transfected cell lines and determined CES1 isoform composition and its impact on CES1 activity in human livers. In transfected cells, isoforms 3 and 4 showed mRNA and protein expressions comparable to isoforms 1 and 2, but had significantly impaired activity when hydrolyzing enalapril and clopidogrel. In individual human liver samples, isoforms 1 and 2 were the major forms, contributing 73%-90% of total CES1 protein expression. In addition, the protein expression ratios of isoforms 1 and 2 to isoforms 3 and 4 were positively associated with CES1 activity in the livers, suggesting that CES1 isoform composition is a factor contributing to the variability in hepatic CES1 function. Further investigations of the regulation of CES1 AS would improve our understanding of CES1 variability and help develop a strategy to optimize the pharmacotherapy of many CES1 substrate medications. This article is protected by copyright. All rights reserved. Title: A Comprehensive Functional Assessment of Carboxylesterase 1 Nonsynonymous Polymorphisms Wang X, Rida N, Shi J, Wu AH, Bleske BE, Zhu HJ Ref: Drug Metabolism & Disposition: The Biological Fate of Chemicals, 45:1149, 2017 : PubMed ESTHER: Wang_2017_Drug.Metab.Dispos_45_1149 PubMedSearch: Wang 2017 Drug.Metab.Dispos 45 1149 PubMedID: 28838926 Gene_locus related to this paper: human-CES1 Abstract Carboxylesterase 1 (CES1) is the predominant human hepatic hydrolase responsible for the metabolism of many clinically important medications. CES1 expression and activity vary markedly among individuals; and genetic variation is a major contributing factor to CES1 interindividual variability. In this study, we comprehensively examined the functions of CES1 nonsynonymous single nucleotide polymorphisms (nsSNPs) and haplotypes using transfected cell lines and individual human liver tissues. The 20 candidate variants include CES1 nsSNPs with a minor allele frequency >0.5% in a given population or located in close proximity to the CES1 active site. Five nsSNPs, including L40Ter (rs151291296), G142E (rs121912777), G147C (rs146456965), Y170D (rs148947808), and R171C (rs201065375), were loss-of-function variants for metabolizing the CES1 substrates clopidogrel, enalapril, and sacubitril. In addition, A158V (rs202121317), R199H (rs2307243), E220G (rs200707504), and T290M (rs202001817) decreased CES1 activity to a lesser extent in a substrate-dependent manner. Several nsSNPs, includingL40Ter (rs151291296), G147C (rs146456965), Y170D (rs148947808), and R171C (rs201065375), significantly reduced CES1 protein and/or mRNA expression levels in the transfected cells. Functions of the common nonsynonymous haplotypes D203E-A269S and S75N-D203E-A269S were evaluated using cells stably expressing the haplotypes and a large set of the human liver. Neither CES1 expression nor activity was affected by the two haplotypes. In summary, this study revealed several functional nsSNPs with impaired activity on the metabolism of CES1 substrate drugs. Clinical investigations are warranted to determine whether these nsSNPs can serve as biomarkers for the prediction of therapeutic outcomes of drugs metabolized by CES1. Title: Ethanol Interactions With Dexmethylphenidate and dl-Methylphenidate Spheroidal Oral Drug Absorption Systems in Healthy Volunteers Zhu HJ, Patrick KS, Straughn AB, Reeves OT, 3rd, Bernstein H, Shi J, Johnson HJ, Knight JM, Smith AT and Markowitz JS <1 more author(s)> Zhu HJ, Patrick KS, Straughn AB, Reeves OT, 3rd, Bernstein H, Shi J, Johnson HJ, Knight JM, Smith AT, Malcolm RJ, Markowitz JS (- 1) Ref: J Clin Psychopharmacol, 37:419, 2017 : PubMed ESTHER: Zhu_2017_J.Clin.Psychopharmacol_37_419 PubMedSearch: Zhu 2017 J.Clin.Psychopharmacol 37 419 PubMedID: 28590363 Abstract BACKGROUND/PURPOSE: Ethanol coadministered with immediate-release dl-methylphenidate (dl-MPH) or dexmethylphenidate (d-MPH) significantly increases the geomean maximum plasma concentration (Cmax) of d-MPH 22% and 15%, respectively, and elevates overall drug exposure and psychostimulant effects. We asked the question: Are these ethanol-MPH interactions based more fundamentally on (1) inhibition of postabsorption d-MPH metabolism or (2) acceleration of MPH formulation gastric dissolution by ethanol in the stomach? This was investigated using the pulsatile, distinctly biphasic, spheroidal oral drug absorption systems of dl-MPH and d-MPH. Title: Regulatory effects of genomic translocations at the human carboxylesterase-1 (CES1) gene locus Sanford JC, Wang X, Shi J, Barrie ES, Wang D, Zhu HJ, Sadee W Ref: Pharmacogenet Genomics, 26:197, 2016 : PubMed ESTHER: Sanford_2016_Pharmacogenet.Genomics_26_197 PubMedSearch: Sanford 2016 Pharmacogenet.Genomics 26 197 PubMedID: 26871237 Gene_locus related to this paper: human-CES1 Abstract OBJECTIVE: CES1 encodes carboxylesterase-1, an important drug-metabolizing enzyme with high expression in the liver. Previous studies have reported a genomic translocation of the 5' region from the poorly expressed pseudogene CES1P1, to CES1, yielding the structural variant CES1VAR. The aim of this study was to characterize this translocation and its effect on CES1 expression in the human liver. MATERIALS AND Title: Sacubitril Is Selectively Activated by Carboxylesterase 1 (CES1) in the Liver and the Activation Is Affected by CES1 Genetic Variation Shi J, Wang X, Nguyen J, Wu AH, Bleske BE, Zhu HJ Ref: Drug Metabolism & Disposition: The Biological Fate of Chemicals, 44:554, 2016 : PubMed ESTHER: Shi_2016_Drug.Metab.Dispos_44_554 PubMedSearch: Shi 2016 Drug.Metab.Dispos 44 554 PubMedID: 26817948 Gene_locus related to this paper: human-CES1 Abstract Sacubitril was recently approved by the Food and Drug Administration for use in combination with valsartan for the treatment of patients with heart failure with reduced ejection fraction. As a prodrug, sacubitril must be metabolized (hydrolyzed) to its active metabolite sacubitrilat (LBQ657) to exert its intended therapeutic effects. Thus, understanding the determinants of sacubitril activation will lead to the improvement of sacubitril pharmacotherapy. The objective of this study was to identify the enzyme(s) responsible for the activation of sacubitril, and determine the impact of genetic variation on sacubitril activation. First, an incubation study of sacubitril with human plasma and the S9 fractions of human liver, intestine, and kidney was conducted. Sacubitril was found to be activated by human liver S9 fractions only. Moreover, sacubitril activation was significantly inhibited by the carboxylesterase 1 (CES1) inhibitor bis-(p-nitrophenyl) phosphate in human liver S9. Further incubation studies with recombinant human CES1 and carboxylesterase 2 confirmed that sacubitril is a selective CES1 substrate. The in vitro study of cell lines transfected with wild-type CES1 and the CES1 variant G143E (rs71647871) demonstrated that G143E is a loss-of-function variant for sacubitril activation. Importantly, sacubitril activation was significantly impaired in human livers carrying the G143E variant. In conclusion, sacubitril is selectively activated by CES1 in human liver. The CES1 genetic variant G143E can significantly impair sacubitril activation. Therefore, CES1 genetic variants appear to be an important contributing factor to interindividual variability in sacubitril activation, and have the potential to serve as biomarkers to optimize sacubitril pharmacotherapy. Title: Dabigatran etexilate activation is affected by the CES1 genetic polymorphism G143E (rs71647871) and gender Shi J, Wang X, Nguyen JH, Bleske BE, Liang Y, Liu L, Zhu HJ Ref: Biochemical Pharmacology, 119:76, 2016 : PubMed ESTHER: Shi_2016_Biochem.Pharmacol_119_76 PubMedSearch: Shi 2016 Biochem.Pharmacol 119 76 PubMedID: 27614009 Gene_locus related to this paper: human-CES1 Abstract The oral anticoagulant prodrug dabigatran etexilate (DABE) is sequentially metabolized by intestinal carboxylesterase 2 (CES2) and hepatic carboxylesterase 1 (CES1) to form its active metabolite dabigatran (DAB). A recent genome-wide association study reported that the CES1 single nucleotide polymorphisms (SNPs) rs2244613 and rs8192935 were associated with lower DAB plasma concentrations in the Randomized Evaluation of Long-term Anticoagulation Therapy (RE-LY) study participants. In addition, gender differences in exposure to DAB were observed in clinical studies. The aim of this study was to examine the effect of CES1 genetic polymorphisms and gender on DABE activation using several in vitro approaches. The genotypes of the CES1 SNPs rs2244613, rs8192935, and the known loss-of-function CES1 variant rs71647871 (G143E), and the activation of DABE and its intermediate metabolites M1 and M2 were determined in 104 normal human liver samples. DABE, M1, and M2 activations were found to be impaired in human livers carrying the G143E variant. However, neither rs2244613 nor rs8192935 was associated with the activation in human livers. The incubation study of DABE with supernatant fractions (S9) prepared from the G143E-transfected cells showed that the G143E is a loss-of-function variant for DABE metabolism. Moreover, hepatic CES1 activity on M2 activation was significantly higher in female liver samples than male. Our data suggest that CES1 genetic variants and gender are important contributing factors to variability in DABE activation in humans. A personalized DABE treatment approach based on patient-specific CES1 genotypes and sex may have the potential to improve the efficacy and safety of DABE pharmacotherapy. Title: Association of Oseltamivir Activation with Gender and Carboxylesterase 1 Genetic Polymorphisms Shi J, Wang X, Eyler RF, Liang Y, Liu L, Mueller BA, Zhu HJ Ref: Basic Clin Pharmacol Toxicol, 119:555, 2016 : PubMed ESTHER: Shi_2016_Basic.Clin.Pharmacol.Toxicol_119_555 PubMedSearch: Shi 2016 Basic.Clin.Pharmacol.Toxicol 119 555 PubMedID: 27228223 Gene_locus related to this paper: human-CES1 Abstract Oseltamivir, an inactive anti-influenza virus prodrug, is activated (hydrolysed) in vivo by carboxylesterase 1 (CES1) to its active metabolite oseltamivir carboxylate. CES1 functions are significantly associated with certain CES1 genetic variants and some non-genetic factors. The purpose of this study was to investigate the effect of gender and several CES1 genetic polymorphisms on oseltamivir activation using a large set of individual human liver samples. CES1-mediated oseltamivir hydrolysis and CES1 genotypes, including the G143E (rs71647871), rs2244613, rs8192935, the -816A>C (rs3785161) and the CES1P1/CES1P1VAR, were determined in 104 individual human livers. The results showed that hepatic CES1 protein expression in females was 17.3% higher than that in males (p = 0.039), while oseltamivir activation rate in the livers from female donors was 27.8% higher than that from males (p = 0.076). As for CES1 genetic polymorphisms, neither CES1 protein expression nor CES1 activity on oseltamivir activation was significantly associated with the rs2244613, rs8192935, -816A>C or CES1P1/CES1P1VAR genotypes. However, oseltamivir hydrolysis in the livers with the genotype 143G/E was approximately 40% of that with the 143G/G genotype (0.7 +/- 0.2 versus 1.8 +/- 1.1 nmole/mg protein/min, p = 0.005). In summary, the results suggest that hepatic oseltamivir activation appears to be more efficient in females than that in males, and the activation can be impaired by functional CES1 variants, such as the G143E. However, clinical implication of CES1 gender differences and pharmacogenetics in oseltamivir pharmacotherapy warrants further investigations. Title: Targeted absolute quantitative proteomics with SILAC internal standards and unlabeled full-length protein calibrators (TAQSI) Wang X, Liang Y, Liu L, Shi J, Zhu HJ Ref: Rapid Commun Mass Spectrom, 30:553, 2016 : PubMed ESTHER: Wang_2016_Rapid.Commun.Mass.Spectrom_30_553 PubMedSearch: Wang 2016 Rapid.Commun.Mass.Spectrom 30 553 PubMedID: 26842578 Abstract RATIONALE: Liquid Chromatography/Mass Spectrometry (LC/MS)-based proteomics for absolute protein quantification has been increasingly utilized in both basic and clinical research. There is a great need to overcome some major hurdles of current absolute protein quantification methods, such as significant inter-assay variability and the high cost associated with the preparation of purified stable-isotope-labeled peptide/protein standards. Title: CES1 genetic variation affects the activation of angiotensin-converting enzyme inhibitors Wang X, Wang G, Shi J, Aa JY, Comas R, Liang Y, Zhu HJ Ref: Pharmacogenomics J, 16:220, 2016 : PubMed ESTHER: Wang_2016_Pharmacogenomics.J_16_220 PubMedSearch: Wang 2016 Pharmacogenomics.J 16 220 PubMedID: 26076923 Gene_locus related to this paper: human-CES1 Abstract The aim of the study was to determine the effect of carboxylesterase 1 (CES1) genetic variation on the activation of angiotensin-converting enzyme inhibitor (ACEI) prodrugs. In vitro incubation study of human liver, intestine and kidney s9 fractions demonstrated that the ACEI prodrugs enalapril, ramipril, perindopril, moexipril and fosinopril are selectively activated by CES1 in the liver. The impact of CES1/CES1VAR and CES1P1/CES1P1VAR genotypes and diplotypes on CES1 expression and activity on enalapril activation was investigated in 102 normal human liver samples. Neither the genotypes nor the diplotypes affected hepatic CES1 expression and activity. Moreover, among several CES1 nonsynonymous variants studied in transfected cell lines, the G143E (rs71647871) was a loss-of-function variant for the activation of all ACEIs tested. The CES1 activity on enalapril activation in human livers with the 143G/E genotype was approximately one-third of that carrying the 143G/G. Thus, some functional CES1 genetic variants (for example, G143E) may impair ACEI activation, and consequently affect therapeutic outcomes of ACEI prodrugs. Title: CES1P1 variant -816A>C is not associated with hepatic carboxylesterase 1 expression and activity or antihypertensive effect of trandolapril Zhu HJ, Langaee TY, Gong Y, Wang X, Pepine CJ, Cooper-DeHoff RM, Johnson JA, Markowitz JS Ref: European Journal of Clinical Pharmacology, 72:681, 2016 : PubMed ESTHER: Zhu_2016_Eur.J.Clin.Pharmacol_72_681 PubMedSearch: Zhu 2016 Eur.J.Clin.Pharmacol 72 681 PubMedID: 26915813 Gene_locus related to this paper: human-CES1 Abstract PURPOSE: The majority of angiotensin-converting enzyme inhibitors (ACEIs) are synthesized as ester prodrugs that must be converted to their active forms in vivo in order to exert therapeutic effects. Hepatic carboxylesterase 1 (CES1) is the primary enzyme responsible for the bioactivation of ACEI prodrugs in humans. The genetic variant -816A>C (rs3785161) is a common variant located in the promoter region of the CES1P1 gene. Previous studies report conflicting results with regard to the association of this variant and therapeutic outcomes of CES1 substrate drugs. The purpose of this study was to determine the effect of the variant -816A>C on the activation of the ACEI prodrug trandolapril in human livers and the blood pressure (BP)-lowering effect of trandolapril in hypertensive patients. Title: Carboxylesterase 1-Mediated Drug-Drug Interactions between Clopidogrel and Simvastatin Wang X, Zhu HJ, Markowitz JS Ref: Biol Pharm Bull, 38:292, 2015 : PubMed ESTHER: Wang_2015_Biol.Pharm.Bull_38_292 PubMedSearch: Wang 2015 Biol.Pharm.Bull 38 292 PubMedID: 25747989 Abstract Patients with coronary artery disease often receive concurrent treatment with clopidogrel and a hydroxymethylglutaryl (HMG)-CoA reductase inhibitor medication. Accordingly, potential drug-drug interactions associated with the concomitant use of these agents present an area of concern. Both CYP enzymes and carboxylesterase 1 (CES1) are involved in the metabolism of clopidogrel, while CES1 is believed to be the enzyme responsible for the activation of simvastatin. Some in vitro studies have suggested that simvastatin could attenuate clopidogrel activation via inhibiting CYP3A activity. However, these findings have not found support in several recently published clinical investigations. The present study addresses these inconsistencies by exploring the potential role of CES1 in the metabolism of clopidogrel and simvastatin. Our in vitro human liver s9 fraction incubation study demonstrated that simvastatin significantly enhanced the formation of the intermediate metabolite 2-oxo-clopidogrel, and inhibited the CES1-mediated hydrolysis of clopidogrel, 2-oxo-clopidogrel, and the active metabolite. However, the production of the active metabolite remained unchanged. Conversely, clopidogrel was not found to influence the CES1 mediated hydrolysis (activation) of simvastatin. Moreover, we provided evidence that CES1 is not an efficient enzyme for catalyzing simvastatin activation. In summary, the inhibitory effect of simvastatin on the hydrolysis of clopidogrel and its principal metabolites may have offset the influence of simvastatin-mediated inhibition of CYP3A, and permitted the unaltered formation of the clopidogrel active metabolite. These data help explain the conflicting accounts in previous reports regarding clopidogrel and simvastatin interactions by taking into consideration CES1; they suggest that the interactions are unlikely to significantly influence clinical outcomes. Title: Clopidogrel Bioactivation and Risk of Bleeding in Patients Cotreated With Angiotensin-Converting Enzyme Inhibitors After Myocardial Infarction: A Proof-of-Concept Study Kristensen KE, Zhu HJ, Wang X, Gislason GH, Torp-Pedersen C, Rasmussen HB, Markowitz JS, Hansen PR Ref: Clinical Pharmacology & Therapeutics, 96:713, 2014 : PubMed ESTHER: Kristensen_2014_Clin.Pharmacol.Ther_96_713 PubMedSearch: Kristensen 2014 Clin.Pharmacol.Ther 96 713 PubMedID: 25222620 Abstract Clopidogrel is an oral antiplatelet prodrug, the majority of which is hydrolyzed to an inactive metabolite by hepatic carboxylesterase 1 (CES1). Most angiotensin-converting enzyme inhibitors (ACEIs) are also metabolized by this enzyme. We examined the effects of ACEIs on clopidogrel bioactivation in vitro and linked the results with a pharmacoepidemiological study. In vitro, ACEIs inhibited CES1-mediated hydrolysis of a model substrate, and trandolapril and enalapril increased formation of clopidogrel active metabolite. In 70,934 patients with myocardial infarction, hazard ratios for clinically significant bleeding in ACEI-treated patients cotreated with or without clopidogrel were 1.10 (95% confidence interval (CI): 0.97-1.25, P = 0.124) and 0.90 (95% CI: 0.81-0.99, P = 0.025), respectively, as compared with patients who did not receive ACEIs. This difference was statistically significant (P = 0.002). We conclude that cotreatment with selected ACEIs and clopidogrel may increase the risk of bleeding. Combination of in vitro and pharmacoepidemiological studies may be a useful paradigm for assessment of drug-drug interactions. Title: Isopropylphenidate: an ester homolog of methylphenidate with sustained and selective dopaminergic activity and reduced drug interaction liability Markowitz JS, Zhu HJ, Patrick KS Ref: J Child Adolesc Psychopharmacol, 23:648, 2013 : PubMed ESTHER: Markowitz_2013_J.Child.Adolesc.Psychopharmacol_23_648 PubMedSearch: Markowitz 2013 J.Child.Adolesc.Psychopharmacol 23 648 PubMedID: 24261661 Abstract Abstract Objective: The most widely utilized pharmacological treatment of attention-deficit/hyperactivity disorder (ADHD) is the psychostimulant methylphenidate (MPH). Most MPH formulations consist of the racemic mixture of d-threo-(R, R)-MPH and l-threo-(S, S)-MPH isomers. MPH is characterized by its low bioavailability and short half-life (2-3 hours). Additionally, significant inter-individual variability in MPH pharmacokinetics has been consistently documented. Accordingly, efforts have been directed at developing alternatives to MPH as therapeutic agents. A wide range of MPH analogues (dl-alpha-[2-piperidyl]-phenylacetic acid esters) have been synthesized with the dopamine transporter (DAT) and norepinephrine transporter (NET) as principle neuropharmacological targets. The present study investigated the metabolic profiles and pharmacological activity of the isopropyl ester derivative of MPH, dl-isopropylphenidate (IPH), both in vitro and in vivo. Methods: The synthesis, monoaminergic transporter binding, cellular uptake profiles, and assessment of metabolic hydrolysis and transesterification in the presence of ethanol are described using MPH as a comparator. Additionally, an in vivo assessment of IPH stimulant effects (vs. saline) in rats was performed with locomotor activity as a pharmacodynamic outcome. Results: IPH displayed unique pharmacological characteristics including greater DAT than NET binding and cellular uptake activity, and greater resistance to hydrolysis and transesterification via carboxylesterase 1 relative to MPH. Further, sustained psychostimulant properties offer the prospect of an enhanced duration of action. Conclusions: Our findings are consistent with IPH exhibiting attributes distinguishing it from MPH and warranting further study and development of IPH as a novel psychotherapeutic agent. Title: Carboxylesterase 1 (CES1) genetic polymorphisms and oseltamivir activation Zhu HJ, Markowitz JS Ref: European Journal of Clinical Pharmacology, 69:733, 2013 : PubMed ESTHER: Zhu_2013_Eur.J.Clin.Pharmacol_69_733 PubMedSearch: Zhu 2013 Eur.J.Clin.Pharmacol 69 733 PubMedID: 22847618 Title: Carboxylesterase 1 as a determinant of clopidogrel metabolism and activation Zhu HJ, Wang X, Gawronski BE, Brinda BJ, Angiolillo DJ, Markowitz JS Ref: Journal of Pharmacology & Experimental Therapeutics, 344:665, 2013 : PubMed ESTHER: Zhu_2013_J.Pharmacol.Exp.Ther_344_665 PubMedSearch: Zhu 2013 J.Pharmacol.Exp.Ther 344 665 PubMedID: 23275066 Gene_locus related to this paper: human-CES1 Abstract Clopidogrel pharmacotherapy is associated with substantial interindividual variability in clinical response, which can translate into an increased risk of adverse outcomes. Clopidogrel, a recognized substrate of hepatic carboxylesterase 1 (CES1), undergoes extensive hydrolytic metabolism in the liver. Significant interindividual variability in the expression and activity of CES1 exists, which is attributed to both genetic and environmental factors. We determined whether CES1 inhibition and CES1 genetic polymorphisms would significantly influence the biotransformation of clopidogrel and alter the formation of the active metabolite. Coincubation of clopidogrel with the CES1 inhibitor bis(4-nitrophenyl) phosphate in human liver s9 fractions significantly increased the concentrations of clopidogrel, 2-oxo-clopidogrel, and clopidogrel active metabolite, while the concentrations of all formed carboxylate metabolites were significantly decreased. As anticipated, clopidogrel and 2-oxo-clopidogrel were efficiently hydrolyzed by the cell s9 fractions prepared from wild-type CES1 transfected cells. The enzymatic activity of the CES1 variants G143E and D260fs were completely impaired in terms of catalyzing the hydrolysis of clopidogrel and 2-oxo-clopidogrel. However, the natural variants G18V, S82L, and A269S failed to produce any significant effect on CES1-mediated hydrolysis of clopidogrel or 2-oxo-clopidogrel. In summary, deficient CES1 catalytic activity resulting from CES1 inhibition or CES1 genetic variation may be associated with higher plasma concentrations of clopidogrel-active metabolite, and hence may enhance antiplatelet activity. Additionally, CES1 genetic variants have the potential to serve as a biomarker to predict clopidogrel response and individualize clopidogrel dosing regimens in clinical practice. Title: Prediction and in vitro evaluation of selected protease inhibitor antiviral drugs as inhibitors of carboxylesterase 1: a potential source of drug-drug interactions Rhoades JA, Peterson YK, Zhu HJ, Appel DI, Peloquin CA, Markowitz JS Ref: Pharm Res, 29:972, 2012 : PubMed ESTHER: Rhoades_2012_Pharm.Res_29_972 PubMedSearch: Rhoades 2012 Pharm.Res 29 972 PubMedID: 22161308 Abstract PURPOSE: To predict and determine whether the protease inhibitors (PIs) nelfinavir, amprenavir, atazanavir, ritonavir, and saquinavir could serve as metabolic inhibitors of the human CES1 (hCES1) using both molecular modeling techniques and in vitro inhibition assays. METHODS: Initially, a molecular modeling approach was utilized to predict whether the selected PIs could serve as hCES1 inhibitors. The inhibitory effects of these PIs on hCES1 activity were then further evaluated utilizing previously established in vitro assay. RESULTS: Pharmacophore and 2D-QSAR modeling predicted that nelfinavir would serve as a potent hCES1 inhibitor. This hypothesis was validated by in vitro hCES1 inhibition studies. Other PIs (amprenavir, atazanavir, ritonavir, saquinavir) were evaluated and also shown to be hCES1 inhibitors in vitro, although substantially less potent relative to nelfinavir. CONCLUSION: Computational molecular modeling is a valid approach to identify potential hCES1 inhibitors as candidates for further assessment using validated in vitro techniques. DDIs could occur when nelfinavir is co-administered with drugs metabolized by hCES1. Title: A discriminative analytical method for detection of CES1A1 and CES1A2/CES1A3 genetic variants Zhu HJ, Brinda B, Froehlich TE, Markowitz JS Ref: Pharmacogenet Genomics, 22:215, 2012 : PubMed ESTHER: Zhu_2012_Pharmacogenet.Genomics_22_215 PubMedSearch: Zhu 2012 Pharmacogenet.Genomics 22 215 PubMedID: 22237548 Gene_locus related to this paper: human-CES1 Abstract Human carboxylesterase 1 (hCES1), encoded by the CES1 gene, is the predominant hepatic hydrolase responsible for the metabolism of many therapeutic agents, toxins, and endogenous substances. Genetic variants of CES1 can affect hCES1 function and expression and ultimately influence clinical response to drugs serving as hCES1 substrates. The CES1 gene consists of three isoforms including the functional CES1A1 and CES1A2 genes and the nonfunctional pseudogene CES1A3. Natural variants of these isoforms exert differing impacts on hCES1 function. However, the existing CES1 genotyping methods are incapable of determining whether these variants belong to CES1A1, CES1A2, or CES1A3 because of the high similarity among these three genes, as a consequence they are unable to discriminate between heterozygotes and homozygotes. We report the development of a novel long-range PCR-based, discriminative genotyping assay capable of specifically detecting the variants among CES1A1, CES1A2, and CES1A3 genes. The comparison of the genotyping results between this novel assay and those previously reported methods highlighted the necessity of applying the discriminative genotyping assay in pharmacogenetic studies involving CES1 gene. Title: Enantiospecific determination of dl-methylphenidate and dl-ethylphenidate in plasma by liquid chromatography-tandem mass spectrometry: Application to human ethanol interactions Zhu HJ, Patrick KS, Markowitz JS Ref: Journal of Chromatography B Analyt Technol Biomed Life Sciences, 879:783, 2011 : PubMed ESTHER: Zhu_2011_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_879_783 PubMedSearch: Zhu 2011 J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci 879 783 PubMedID: 21402502 Abstract In humans, concomitant DL-methylphenidate (DL-MPH) and ethanol results in the carboxylesterase 1 (hCES1) mediated biotransformation of MPH to the transesterification metabolite DL-ethylphenidate (DL-EPH). The separate enantiomers of MPH and EPH are found at low ng/ml to pg/ml plasma concentrations. Substantial pharmacological differences exist between D- and L-isomers of MPH and EPH, both in terms of pharmacological potencies and receptor selectivity, as well as in pharmacokinetic properties. Accordingly, a sensitive, accurate and precise enantiospecific analytical method is required in order to fully explore pharmacokinetic-pharmacodynamic correlations regarding the MPH-ethanol interaction. The present study describes a novel liquid chromatographic-tandem mass spectrometric method for simultaneous analysis of D- and L-MPH as well as D- and L-EPH concentrations from human plasma. This assay provides baseline resolution of the individual MPH and EPH isomers utilizing a vancomycin-based chiral column. The lower limit of quantification was 0.025 ng/ml for each isomer when extracting 0.5 ml plasma aliquots. Calibration curves were linear over the range from 0.025 ng/ml to 25 ng/ml for all analytes (r(2)>0.995). Assay accuracy and precision were excellent and stability studies and assessment of potential matrix effects contributed to the validation of the method. Application of the method to human plasma samples collected after the administration of dl-MPH with or without ethanol is included, and the implications of this pharmacokinetic drug interaction discussed. Title: Identification of selected therapeutic agents as inhibitors of carboxylesterase 1: potential sources of metabolic drug interactions Zhu HJ, Appel DI, Peterson YK, Wang Z, Markowitz JS Ref: Toxicology, 270:59, 2010 : PubMed ESTHER: Zhu_2010_Toxicology_270_59 PubMedSearch: Zhu 2010 Toxicology 270 59 PubMedID: 20097249 Abstract A series of studies were designed and carried out in order to explore the potential for the major human hepatic hydrolase, carboxylesterase 1 (hCES1), to serve as a target of metabolic inhibition by a variety of medications. The risk of adverse drug-drug interaction(s) is present when metabolic inhibitors are combined with known or suspected substrates of a given enzyme. In the present report the abundantly expressed hepatic enzyme, hCES1, was examined as a potential target of metabolic inhibition by a number of routinely prescribed medications. hCES1 has been seldom assessed in this regard despite its role in the metabolism and detoxification of many compounds. The psychostimulant methylphenidate (MPH) was chosen as an hCES1 selective substrate. In vitro studies were performed using previously developed cell lines which overexpress hCES1 with both p-nitrophenyl acetate and d-MPH serving as known substrates. Aripiprazole, perphenazine, thioridazine, and fluoxetine were determined to be the potent hCES1 inhibitors. A complementary animal study followed in vitro screening studies to further evaluate the inhibitory effect of aripiprazole on CES1 activity in FVB mice. The results suggest that the concurrent administration of racemic (i.e. dl-) MPH with aripiprazole significantly increased the plasma concentrations of both total MPH as well as the less active l-isomer. The ratio of d-MPH and l-MPH plasma concentrations was significantly decreased in the mice treated with aripiprazole compared to the control animals, indicating an overall decrease of CES1 catalytic activity in aripiprazole treated animals. Additionally, a quantitative structure-activity relationship based analysis identified a number of structural similarities of CES1 inhibitors. In conclusion, drug-drug interactions with MPH are likely mediated via CES1 inhibition as a result of concomitant drug therapies. CES1 inhibition represents an overlooked and little studied source of variability in MPH disposition, tolerability, and response. Title: Role of carboxylesterase 1 and impact of natural genetic variants on the hydrolysis of trandolapril Zhu HJ, Appel DI, Johnson JA, Chavin KD, Markowitz JS Ref: Biochemical Pharmacology, 77:1266, 2009 : PubMed ESTHER: Zhu_2009_Biochem.Pharmacol_77_1266 PubMedSearch: Zhu 2009 Biochem.Pharmacol 77 1266 PubMedID: 19185566 Gene_locus related to this paper: human-CES1 Abstract Carboxylesterase 1 (CES1) and carboxylesterase 2 (CES2) are the major hydrolytic enzymes responsible for the metabolism of numerous therapeutic agents as well as endogenous substrates. CES1 and CES2 differ distinctly in their substrate specificity and tissue distribution. In this study, we investigated the role of CES1 and CES2 in converting the antihypertensive prodrug trandolapril to its more active form trandolaprilat, and determined the influence of two newly identified CES1 mutations p.Gly143Glu and p.Asp260fs on trandolapril metabolism. Western blot analysis demonstrated that CES1 is expressed in human liver microsomes (HLM) but not in human intestinal microsomes (HIM). In vitro incubation studies were conducted to contrast the enzymatic activity of HLM as well as HIM upon trandolapril hydrolysis. Trandolapril was rapidly hydrolyzed to its principal active metabolite trandolaprilat after incubation with HLM. In contrast, in HIM, where CES2 is predominantly expressed, incubations did not produce any detectable trandolapril hydrolysis. Furthermore, hydrolysis of trandolapril catalyzed by wild type (WT) and mutant CES1 were assessed utilizing transfected Flp-In-293 cells stably expressing WT CES1 and two variants. WT CES1 efficiently hydrolyzed trandolapril to trandolaprilat with V(max) and K(m) values of 103.6+/-2.2 nmole/min/mg protein and 639.9+/-32.9muM, respectively. However, no appreciable trandolapril hydrolysis could be found after incubation with both p.Gly143Glu and p.Asp260fs variants. Thus, trandolapril appears to be a CES1 selective substrate while CES2 exerts little to no catalytic activity towards this compound. CES1 mutations p.Gly143Glu and p.Asp260fs are essentially dysfunctional enzymes with regard to the conversion of trandolapril to its more active metabolite trandolaprilat. Title: Activation of the antiviral prodrug oseltamivir is impaired by two newly identified carboxylesterase 1 variants Zhu HJ, Markowitz JS Ref: Drug Metabolism & Disposition: The Biological Fate of Chemicals, 37:264, 2009 : PubMed ESTHER: Zhu_2009_Drug.Metab.Dispos_37_264 PubMedSearch: Zhu 2009 Drug.Metab.Dispos 37 264 PubMedID: 19022936 Gene_locus related to this paper: human-CES1 Abstract Oseltamivir phosphate is an ethyl ester prodrug widely used in the treatment and prevention of both Influenzavirus A and B infections. The conversion of oseltamivir to its active metabolite oseltamivir carboxylate is dependent on ester hydrolysis mediated by carboxylesterase 1 (CES1). We recently identified two functional CES1 variants p.Gly143Glu and p.Asp260fs in a research subject who displayed significant impairment in his ability to metabolize the selective CES1 substrate, methylphenidate. In vitro functional studies demonstrated that the presence of either of the two mutations can result in severe reductions in the catalytic efficiency of CES1 toward methylphenidate, which is required for hydrolysis and pharmacological deactivation. The aim of the present study was to investigate the function of these mutations on activating (hydrolyzing) oseltamivir to oseltamivir carboxylate using the cell lines expressing wild type (WT) and each mutant CES1. In vitro incubation studies demonstrated that the S9 fractions prepared from the cells transfected with WT CES1 and human liver tissues rapidly convert oseltamivir to oseltamivir carboxylate. However, the catalytic activity of the mutant hydrolases was dramatically hindered. The V(max) value of p.Gly143Glu was approximately 25% of that of WT enzyme, whereas the catalytic activity of p.Asp260fs was negligible. These results suggest that the therapeutic efficacy of oseltamivir could be compromised in treated patients expressing either functional CES1 mutation. Furthermore, the potential for increased adverse effects or toxicity as a result of exposure to high concentrations of the nonhydrolyzed prodrug should be considered. Title: Age- and sex-related expression and activity of carboxylesterase 1 and 2 in mouse and human liver Zhu HJ, Appel DI, Jiang Y, Markowitz JS Ref: Drug Metabolism & Disposition: The Biological Fate of Chemicals, 37:1819, 2009 : PubMed ESTHER: Zhu_2009_Drug.Metab.Dispos_37_1819 PubMedSearch: Zhu 2009 Drug.Metab.Dispos 37 1819 PubMedID: 19487248 Abstract Carboxylesterase (CES) 1 and CES2 are two major hepatic hydrolases responsible for the metabolism of numerous endogenous and exogenous compounds. In this study, age- and sex-dependent expression and activity of CES1 and CES2 were investigated using both animal models and individual human liver s9 samples. The expression and activity of mouse CES1 (mCES1) and mCES2 in the liver were markedly lower in newborns relative to adults and increased gradually with age, approximating levels of adult animals by age 2 to 4 weeks. Likewise, the average human CES1 (hCES1) expression in the subjects <1 year of age was significantly lower than that of pooled samples. In particular, hCES1 expression in the 13-day and 1-month-old subjects was just 20.3 and 11.1%, respectively, of the pooled sample values. In addition, the subjects <1 year of age exhibited a trend suggestive of low hCES2 expression, but this difference failed to reach statistical significance because of large interindividual variability. The expression and activity of mCES1 and mCES2 were not significantly altered after the animals were treated with human growth hormone, indicating growth hormone may not be associated with the low level of CES expression during early developmental stages. No significant differences of the expression and activity of mCES1 and mCES2 were observed between sexually mature male and female mice. In conclusion, the expression and activity of CES1 and CES2 are age-related but independent of growth hormone level. Sex seems to be an unlikely factor contributing to the regulation of CES1 and CES2. Title: Derivatives of vibralactone from cultures of the basidiomycete Boreostereum vibrans Jiang MY, Wang F, Yang XL, Fang LZ, Dong ZJ, Zhu HJ, Liu JK Ref: Chem Pharm Bull (Tokyo), 56:1286, 2008 : PubMed ESTHER: Jiang_2008_Chem.Pharm.Bull.(Tokyo)_56_1286 PubMedSearch: Jiang 2008 Chem.Pharm.Bull.(Tokyo) 56 1286 PubMedID: 18758102 Inhibitor(s) related to this paper: Vibralactone Abstract Four new natural products possessing vibralactone skeleton, 1,5-secovibralactone (1), vibralactone B (2), vibralactone C (3) and acetylated vibralactone (4), together with known compound vibralactone (5), had been isolated from cultures of the basidiomycete Boreostereum vibrans. The structures of 1-4 were elucidated on the basis of spectroscopic methods. The absolute configuration of 1 was suggested to be S by computational methods. Title: Enantiospecific gas chromatographic-mass spectrometric analysis of urinary methylphenidate: implications for phenotyping LeVasseur NL, Zhu HJ, Markowitz JS, DeVane CL, Patrick KS Ref: Journal of Chromatography B Analyt Technol Biomed Life Sciences, 862:140, 2008 : PubMed ESTHER: Levasseur_2008_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_862_140 PubMedSearch: Levasseur 2008 J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci 862 140 PubMedID: 18155648 Abstract A chiral derivatization gas chromatographic-mass spectrometric (GC-MS) method for urine methylphenidate (MPH) analysis was developed and validated to investigate preliminary findings regarding a novel MPH poor metabolizer (PM). Detection was by electron impact (EI) ionization-selected ion monitoring of the N-trifluoroacetylprolylpiperidinium fragments from MPH and the piperidine-deuterated MPH internal standard. The PM eliminated approximately 70 times more l-MPH in urine (9% of the dose over 0-10h), and approximately 5 times more of the d-isomer (10% of the dose), than the mean values determined from 10 normal metabolizers of MPH. Only minor amounts of the metabolite p-hydroxy-MPH were found in the urine of both the PM and normal metabolizers, while the concentration of MPH lactam was not high enough to be detectable. The described method indirectly gauges the functional carboxylesterase-1 status of patients receiving MPH based on the evaluation of relative urine concentrations of d-MPH:l-MPH. Clinical implications concerning rational drug selection for an identified or suspected MPH PM are discussed. Title: Protective effect of an endothelial lipase gene variant on coronary artery disease in a Chinese population Tang NP, Wang LS, Yang L, Zhou B, Gu HJ, Sun QM, Cong RH, Zhu HJ, Wang B Ref: J Lipid Res, 49:369, 2008 : PubMed ESTHER: Tang_2008_J.Lipid.Res_49_369 PubMedSearch: Tang 2008 J.Lipid.Res 49 369 PubMedID: 17986713 Gene_locus related to this paper: human-LIPG Abstract The aim of the present study was to assess the influence of the endothelial lipase (EL) gene 584C/T variant, which results in a change at codon 111 of the EL gene from threonine to isoleucine, on the risk of coronary artery disease (CAD) in a Chinese population. The study population consisted of 265 CAD patients and 265 age- and sex-matched control subjects. The T allele frequency was significantly lower among CAD patients than among control subjects (18.3% vs. 29.8%; P < 0.001). In both the CAD and control groups, the T allele carriers had higher high density lipoprotein cholesterol (HDL-C) levels than homozygote C allele carriers. In a multiple logistic regression model adjusted for age, sex, body mass index, smoking, hypertension, diabetes, hyperlipidemia, and low density lipoprotein cholesterol, a significantly decreased risk of developing CAD was found in subjects carrying a variant CT or TT genotype (odds ratio = 0.496, 95% confidence interval = 0.341-0.723; P < 0.001), and the significance persisted after further adjustment for HDL-C. In conclusion, our observation that the EL 584T allele was associated with protection from CAD in this Chinese population replicates the findings in a Japanese study, which found a similar association of this allele with acute myocardial infarction, independent of HDL-C levels. Title: Two CES1 gene mutations lead to dysfunctional carboxylesterase 1 activity in man: clinical significance and molecular basis Zhu HJ, Patrick KS, Yuan HJ, Wang JS, Donovan JL, DeVane CL, Malcolm R, Johnson JA, Youngblood GL and Markowitz JS <2 more author(s)> Zhu HJ, Patrick KS, Yuan HJ, Wang JS, Donovan JL, DeVane CL, Malcolm R, Johnson JA, Youngblood GL, Sweet DH, Langaee TY, Markowitz JS (- 2) Ref: American Journal of Human Genetics, 82:1241, 2008 : PubMed ESTHER: Zhu_2008_Am.J.Hum.Genet_82_1241 PubMedSearch: Zhu 2008 Am.J.Hum.Genet 82 1241 PubMedID: 18485328 Gene_locus related to this paper: human-CES1 Abstract The human carboxylesterase 1 (CES1) gene encodes for the enzyme carboxylesterase 1, a serine esterase governing both metabolic deactivation and activation of numerous therapeutic agents. During the course of a study of the pharmacokinetics of the methyl ester racemic psychostimulant methylphenidate, profoundly elevated methylphenidate plasma concentrations, unprecedented distortions in isomer disposition, and increases in hemodynamic measures were observed in a subject of European descent. These observations led to a focused study of the subject's CES1 gene. DNA sequencing detected two coding region single-nucleotide mutations located in exons 4 and 6. The mutation in exon 4 is located in codon 143 and leads to a nonconservative substitution, p.Gly143Glu. A deletion in exon 6 at codon 260 results in a frameshift mutation, p.Asp260fs, altering residues 260-299 before truncating at a premature stop codon. The minor allele frequency of p.Gly143Glu was determined to be 3.7%, 4.3%, 2.0%, and 0% in white, black, Hispanic, and Asian populations, respectively. Of 925 individual DNA samples examined, none carried the p.Asp260fs, indicating it is an extremely rare mutation. In vitro functional studies demonstrated the catalytic functions of both p.Gly143Glu and p.Asp260fs are substantially impaired, resulting in a complete loss of hydrolytic activity toward methylphenidate. When a more sensitive esterase substrate, p-nitrophenyl acetate was utilized, only 21.4% and 0.6% catalytic efficiency (V(max)/K(m)) were determined in p.Gly143Glu and p.Asp260fs, respectively, compared to the wild-type enzyme. These findings indicate that specific CES1 gene variants can lead to clinically significant alterations in pharmacokinetics and drug response of carboxylesterase 1 substrates. Title: Vibralactone: a lipase inhibitor with an unusual fused beta-lactone produced by cultures of the basidiomycete Boreostereum vibrans Liu DZ, Wang F, Liao TG, Tang JG, Steglich W, Zhu HJ, Liu JK Ref: Org Lett, 8:5749, 2006 : PubMed ESTHER: Liu_2006_Org.Lett_8_5749 PubMedSearch: Liu 2006 Org.Lett 8 5749 PubMedID: 17134263 Inhibitor(s) related to this paper: Vibralactone Abstract The structure and absolute configuration of vibralactone (1) from the cultures of the Basidiomycete Boreostereum vibrans were established by spectroscopic methods and computational methods. Vibralactone, an unusual fused beta-lactone-type metabolite, was found to inhibit pancreatic lipase with an IC50 of 0.4 microg/mL. [structure: see text] | |||||||
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