CES1 Genetic Variation Affects the Activation of Angiotensin-Converting Enzyme Inhibitors

The Pharmacogenomics Journal (2016) 16, 220–230 © 2016 Macmillan Publishers Limited All rights reserved 1470-269X/16 www.nature.com/tpj ORIGINAL ARTICLE CES1 genetic variation affects the activation of angiotensin-converting enzyme inhibitors X Wang1,2, G Wang2, J Shi1,JAa2, R Comas1, Y Liang1,2 and H-J Zhu1 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. The Pharmacogenomics Journal (2016) 16, 220–230; doi:10.1038/tpj.2015.42; published online 16 June 2015 INTRODUCTION such as methylphenidate and clopidogrel.12,13 Additionally, CES1 Angiotensin-converting enzyme inhibitors (ACEIs) are among the is critical for the activation of a number of prodrugs such as most prescribed medications in the world, and are the corner- oseltamivir and several ACEIs including trandolapril, benazepril, 14–16 stone for the treatment of patients with hypertension, heart failure quinapril, temocapril, cilazapril, delapril and imidapril. CES2 is and chronic kidney diseases. The responses to ACEIs therapy vary the enzyme responsible for the metabolism of several ester fi medications, such as aspirin and irinotecan, but is not involved in signi cantly within individual patients. A meta-analysis concluded 15,17,18 that relative to other antihypertensives, the use of ACEIs was the activation of several ACEI prodrugs studied previously. associated with wider interindividual variations in systolic blood CES1 is encoded by the CES1 gene. CES1 consists of 14 exons pressure.1 In fact, target blood pressure was not achieved in located on chromosome 16q13-q22.1. The CES1P1 gene is a approximately 50% of the intent-to-treat patients receiving nonfunctional pseudogene located in proximity with the CES1 2–7 gene. The CES1P1VAR is a functional variant of CES1P1, which is ACEIs. Additionally, unacceptable side effects are commonly identical to the CES1 gene except the differences of five reported in patients treated with ACEIs.8 The current clinical nucleotides in the exon 1. The CES1P1VAR encodes for the same management of ACEI pharmacotherapy is largely based on an protein as the CES1 gene. Interestingly, the exon 1 of CES1 gene empirical 'trial and error' approach owing to the lack of reliable can be converted to that of CES1P1VAR resulting in the CES1 predictors of drug response. Therefore, there is a pressing need to variant CES1VAR.19 The CES1 and CES1P1 gene structures and identify the factors contributing to interindividual variability in nomenclature were illustrated in the Supplementary Figure S1. It responses to ACEI therapy. has been speculated that the CES1/CES1VAR and CES1P1/ With the exception of lisinopril and captopril, all ACEIs are CES1P1VAR genotypes could affect expression levels of CES1, formed as ester prodrugs to improve otherwise poor bioavail- and consequently affect the metabolism of CES1 substrate drugs. ability. The activation of ACEI prodrugs is fundamental for However, the findings from the published studies were incon- successful ACEI pharmacotherapy because the active metabolites sistent or even contradictive.20,21 – are 10 1000 times more potent on ACE inhibition compared with Besides the CES1/CES1VAR and CES1P1/CES1P1VAR genotypes, their respective parent compounds. Carboxylesterase 1 (CES1) is over 1000 single nucleotide polymorphisms (SNPs) have the major hydrolase in humans, contributing to 80–95% of total been identified in the coding and non-coding regions of CES1 9 hepatic hydrolytic activity, while carboxylesterase 2 (CES2), gene. Some of these variants, such as the G143E (rs71647871) another primary hydrolase involved in drug metabolism in originally discovered in our laboratory, markedly affected CES1 10 humans, attributes to the residual 5–20% activity in the liver. activity,13,15,22,23 and significantly altered pharmacokinetics and/or CES1 and CES2 exhibit distinct substrate specificity, that is, CES1 is pharmacodynamics of several drugs metabolized by CES1, highly efficient for hydrolyzing the substrates with small alcohol including methylphenidate, clopidogrel and oseltamivir.23–26 group and large carboxyl group, whereas CES2 prefers to Therefore, these functional CES1 SNPs may have the potential to hydrolyze the esters with bulky alcohol group.11 CES1-mediated impair the activation of ACEI prodrugs, and lead to therapeutic hydrolysis is involved in the deactivation of many medications failure. 1Department of Clinical, Social, and Administrative sciences, University of Michigan, Ann Arbor, MI, USA and 2The Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing, China. Correspondence: Dr G Wang, State Key Laboratory of Natural Medicines, Key Lab of Drug Metabolism & Pharmacokinetics, China Pharmaceutical University, Tongjiaxiang 24, Nanjing 210009, China or Dr H-J Zhu, Department of Clinical, Social and Administrative Sciences, University of Michigan College of Pharmacy, 428 Church Street, Room 3567A CCL, Ann Arbor, MI 48109-1065, USA. E-mail: [email protected] or [email protected] Received 14 September 2014; revised 7 April 2015; accepted 28 April 2015; published online 16 June 2015 CES1 variation affects ACE inhibitor activation X Wang et al 221 In the present study, we demonstrated that hepatic CES1 is the of variants located in the exon 1 of the genes.21 To determine CES1/ enzyme responsible for the activation of the ACEI prodrugs CES1VAR genotypes, PCR was carried out to amplify the exon 1 of the CES1 enalapril, ramipril, perindopril, moexipril and fosinopril. We then and CES1VAR genes using the PCR conditions and primers outlined in the assessed the effect of the CES1/CES1VAR and CES1P1/CES1P1VAR Supplementary Table S1 and Supplementary Table S2. The PCR products fi fi genotypes, diplotypes and several selected CES1 nonsynonymous were puri ed with Pure Link Quick PCR Puri cation Kit (Life technologies), then subjected to Sanger sequencing. The first batch of 10 samples were SNPs on the activation of ACEI prodrugs utilizing transfected cell bidirectionally sequenced using the same primers for the PCR reactions lines and a large set of individual human liver samples. (Supplementary Table S2). The data form the two sequencing directions were in full agreement with each other. Thereafter, the samples were sequenced using the forward primers. MATERIALS AND METHODS It is challenging to determine the CES1P1/CES1P1VAR genotypes owing Materials to high similarity of DNA sequences between the CES1 and CES1P1 genes. Enalapril maleate, moexipril hydrochloride, perindopril erbumine and 5- A restriction fragment length polymorphism (RFLP) method was developed and utilized for the determination of CES1P1/CES1P1VAR genotypes hydroxy omeprazole were purchased from Sigma-Aldrich (St. Louis, MO, 21,28 USA). Fosinopril sodium salt and ramipril were products from Toronto in two previously published studies. This assay was based upon the Research Chemicals (Toronto, Canada). The enalapril hydrolytic metabolite differences of several nucleotides in the exon 5 between the CES1P1 and enalaprilat dehydrate was purchased from Sellechchem (Houston, TX). The CES1P1VAR (Supplementary Figure S2). However, the CES1P1 genotype is fi hydrolytic metabolites of other tested ACEI prodrugs including ramipril, de ned by the presence of a stop codon in exon 3 of the gene, and the perindopril, moexipril and fosinopril were obtained via incubation of the stop codon cannot be directly detected by this RFLP assay. Though the μ μ μ μ authors claimed that the nucleotides for differentiating CES1P1 from parent compounds (100 M,30 M,40 M and 40 M, respectively) with the 21 cell s9 fractions (10 mg protein ml − 1) prepared from the transfected cells CES1P1VAR are in complete linkage with the CES1P1 stop codon, we feel stably expressing wild-type (WT) CES1.23 The hydrolytic reactions for that the assay needs to be validated independently. Accordingly, we ramipril, perindopril, moexipril and fosinopril were completed at 37 °C developed a novel RFLP CES1P1/CES1P1VAR genotyping method by taking in 20 min, 1 h, 24 h and 24 h, respectively. The completion of the advantage of the differences of exon 3 between the CES1P1 and CES1-mediated biotransformation of those ACEI prodrugs was confirmed CES1P1VAR. In brief, a set of primers were designed based on the shared sequences flanking the exon 3 of the CES1, CES1VAR, CES1P1 and by monitoring the reduction of the parent compounds via an fi high performance liquid chromatography–tandem mass spectrometry CES1P1VAR

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