Dabigatran etexilate activation is affected by the CES1 genetic polymorphism G143E (rs71647871) and gender

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.

Keywords: Activation; Carboxylesterase 1; Carboxylesterase 2; Dabigatran etexilate; Gender difference; Pharmacogenetics.

Copyright © 2016 Elsevier Inc. All rights reserved.

Figures

Figure 1

Activation of DABE, M1, and M2 by recombinant human CES1 and CES2. The concentrations of active metabolite DAB were determined after incubation of DABE, M1 or M2 with CES1 and CES2 at 37 °C for 5 min. The final protein concentration of the enzymes was 25 ng/μl, and the concentration of all three substrates was 200 nM. Data are expressed as the rates of DAB formation (Mean ± S.D., n=3).

Figure 2

Correlations between DABE, M1, M2 activations and CES1 and CES2 activities in 104 individual human liver samples. CES1 and CES2 activities were measured using the selective CES1 substrate oseltamivir and the selective CES2 substrate fluorescein diacetate, respectively. Data are the means from two independent experiments. (A) DABE vs oseltamivir; (B) M1 vs oseltamivir; (C) M2 vs oseltamivir; (D) DABE vs fluorescein diacetate; (E) M1 vs fluorescein diacetate; (F) M2 vs fluorescein diacetate.

Figure 3

No associations between the CES1 SNPs rs71647871, rs2244613, and rs9182935 and CES1 protein expression in human livers. Absolute CES1 protein expression was determine in 104 human livers by a targeted MS-based targeted proteomics assay. Data are the means from duplicate measurements.

Figure 4

The CES1 SNPs rs2244613 and rs8192935 were not associated with metabolism of DABE, M1, and M2 in human livers. The formation of the active metabolite DAB was determined after incubation of DABE (A), M1 (B), and M2 (C) with 104 HLS9 fractions. Data are the means from two independent experiments.

Figure 5

The effect of the CES1 variant G143E on DABE, M1, and M2 activation in human livers. The formation rates of DAB following incubation of DABE (A), M1 (B), and M2 (C) with HLS9 samples were compared between five 143G/E heterozygotes and 99 non-carriers. The comparison between the two genotypes was also conducted after the metabolic rates of DABE (D), M1 (E), and M2 (F) were normalized to CES1 protein expression. The study was duplicated, and the mean values are presented.

Figure 6

Determination of the function of the CES1 variant G143E on DABE, M1, and M2 activation using transfected cell lines. No appreciable activation of DABE (A), M1 (B), and M2 (C) was observed after incubation of the substrates with the S9 fractions from the G143E transfected cells, whereas S9 fractions from the WT CES1 transfected cells readily activated all three substrates. Data are means ± S.D. (n=3).

Figure 7

Hepatic absolute CES1 protein expression was significantly higher in females than males. Sample size: female: 56; male: 46.

Figure 8

Gender differences in DABE, M1, and M2 activation in human livers. The activation rates of DABE (A), M1 (B), and M2 (C) in human livers were compared between males and females. The data were also reanalyzed after the activation rates were normalized to the CES1 protein expression levels (D, E, and F). Data are presented as the means from two independent experiments.

Figure 9

Activation pathway of DABE in humans. DABE is extensively metabolized to the intermediate metabolite M2 by CES2 in the intestine during absorption. M2 and remaining DABE are then metabolized to the active metabolite DAB by CES1 in the liver. A small portion of DABE enters into peripheral circulation system after escaping from fist-pass metabolism, and is eventually activated to DAB by CES1 in the liver.