CES1 genetic variation affects the activation of angiotensin-converting enzyme inhibitors

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.

Figure 1.

Hydrolysis (activation) of enalapril (A), ramipril (B), perindopril (C), moexipril (D) and fosinopril (E) by human liver, intestine, and kidney s9 fractions. The ACEI prodrugs (100 μM) were incubated with 3 different concentrations of HLS9, HIS9 and HKS9. Catalytic activity was determined based on the formation of respective hydrolytic products. Data are expressed as mean ±S.D. from 3 independent experiments.

Figure 2.

Kinetics of the hydrolysis of enalapril (A), ramipril (B), perindopril (C), moexipril (D) and fosinopril (E) by human liver s9 fractions. Various concentrations of the ACEI prodrugs were incubated with HLS9 samples in order to determine kinetic parameters of hydrolysis of the ACEI prodrugs. The incubation time was 10 min for enalapril, moexipril and fosinopril, and 5 min for ramipril and perindopril. Data are mean ±S.D. (n=3).

Figure 3.

Effect of CES1/CES1VAR (A) and CES1P1/CES1P1VAR (B) genotypes on enalapril activation rates (left y axis) and CES1 expressions (right y axis) in 100 human liver samples. Total protein staining was used as the loading control for semi-quantification of CES1 protein expression. Horizontal bars represent means values in each group.

Figure 4.

Influence of CES1 diplotypes on CES1 activity on enalapril activation (left) and CES1 expression (right) in human liver samples (n=100). Divided by the middle dotted line, the activity data were plotted with the left y axis, while the expression levels were plotted with the y axis on the right. CES1 protein expression was semi-quantified using total protein staining as the loading control. Horizontal bars represent mean values in each group.

Figure 5.

Enzymatic activity of wild type CES1 and the variant G143E on the hydrolysis of enalapril (A), ramipril (B), perindopril (C), moexipril (D) and fosinopril (E). The ACEI prodrugs (100 μM) were incubated with 3 different concentrations of the s9 fractions prepared from the WT CES1 and G143E cells. The data are mean ±S.D. from three independent experiments.

Figure 6.

Kinetics of the hydrolysis of enalapril (A), ramipril (B), perindopril (C), moexipril (D) and fosinopril (E) catalyzed by WT CES1 and the variant G143E. Hydrolytic (active) metabolites of the ACEI prodrugs were determined after incubation of various concentrations of the prodrugs with the cell s9 fractions. The incubation time was 10 min for enalapril and fosinopril, and 5 min for the rest three ACEI prodrugs. Data are means ±S.D. (n=3).

Figure 7.

Activation of enalapril by CES1 and its variants G19V, S83L, A270s and G143E. Enalapril (500 μM) was incubated with the s9 fractions prepared from the WT CES1 and the variants (G143E, G19V, S83L and A270s) transfected cells. Activation rate was determined by measuring the formation of the hydrolytic metabolite enalaprilat. Data are expressed as mean± S.D. (n=3). **** P<0.0001 (G143E vs wild type CES1).

Figure 8.

Comparison of hepatic CES1 activity and expression between the G143E heterozygotes and non-carriers. CES1 activity on enalapril activation was impaired in the subjects with 143G/E genotype while the expressions were comparable between the 2 genotypes in 102 human livers. Total protein staining was used as the loading control for the CES1 protein expression assay. ** P<0.01, G143E heterozygotes (G/E) vs non-carriers (G/G).