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CYP2C19 is a polymorphic enzyme that plays a pivotal role in the metabolism of 10% of clinically used drugs worldwide. The CYP2C19∗3 allele is characterized by a premature stop codon that leads to a truncated nonfunctional protein and consequently a poor metabolizer phenotype. Aminoglycoside antibiotics have been shown to induce readthrough of premature stop codons and partial restoration of protein function. We investigated the ability of the aminoglycosides gentamicin and G418 to induce readthrough of CYP2C19∗3 premature stop codon in human cells.A CYP2C19∗3 expression model in HeLa cells was used in all experiments. CYP2C19-EGFP expression was assessed by flow cytometry, immunoblotting, and fluorescence microscopy, whereas CYP2C19 enzymatic activity was quantified by hydroxylation of 3-cyano-7-ethoxycoumarin.G418 and gentamicin promoted readthrough of the CYP2C19∗3 premature stop codon in a concentration-dependent manner. Flow cytometry revealed a maximum 23% protein restoration with the highest aminoglycoside concentrations tested, namely 300 mg/ml G418 and 1000 mg/ml gentamicin. At these concentrations, G418 was more effective than gentamicin in restoring CYP2C19 expression in immunoblotting and fluorescence microscopy assays, as well as in restoring CYP2C19 enzymatic activity.This is the first demonstration of readthrough of a stop codon in a pharmacogenetic target of clinical relevance, namely CYP2C19∗3. The experimental models may be adapted to explore readthrough of stop codons in other genes of pharmacogenetic interest. [ABSTRACT FROM AUTHOR]

Genetic variability in hepatic uptake was recently shown to influence the disposition and cholesterol-lowering effects of statins. Ezetimibe, an inhibitor of the intestinal cholesterol uptake protein Niemann-Pick C 1 like 1, is another drug for which genetic polymorphisms of hepatic organic anion transporting polypeptides (OATPs) are expected to be of clinical relevance because ezetimibe undergoes intensive enterohepatic circulation for which hepatic uptake transporters may be rate-limiting determinants.Using OATP1B3-, OATP2B1-, and OATP1B1-transfected HEK cells, including the OATP1B1 variants OATP1B1∗1b and OATP1B1∗5, we measured the uptake of ezetimibe and its glucuronide and we analyzed the competition with the common OATP-substrate bromosulfophthalein. Disposition and sterol-lowering effects of 20-mg ezetimibe were measured in 35 healthy participants genotyped for OATP1B1, ABCB1, ABCC2, and UGT1A1.Ezetimibe glucuronide inhibited bromosulfophthalein uptake in all OATP-transfected cells (50% inhibitory concentration (IC50): 0.14-0.26 μmol/l) whereas ezetimibe was 30-100 times less potent. Only the glucuronide was accumulated significantly in cells expressing OATP1B1 and OATP2B1. Its uptake in cells expressing OATP1B1∗1b and ∗5 was reduced. In-vivo studies showed there was a gene-dose-dependent decrease in the area under the curve of ezetimibe in participants with the OATP1B1∗1b protein (∗1a/∗1a, N=12, 112±66 ng×h/ml vs. ∗1a/∗1b, N=8, 88±39 ng×h/ml vs. ∗1b/∗1b, N=5, 55±18 ng×h/ml; Jonkheere-Terpstra, P=0.041) and a tendency for increased glucuronide exposure (704±296 vs. 878±369 vs. 1059±363 ng×h/ml; P=0.092). Fecal ezetimibe excretion was significantly decreased whereas renal glucuronide excretion was increased in carriers of ∗1b/∗1b. Fecal excretion was also diminished in carriers of OATP1B1∗5 and ∗15. The sterol-lowering effect of ezetimibe was not influenced by OATP1B1 polymorphisms.Pharmacokinetics of ezetimibe is influenced by OATP1B1 polymorphisms in healthy participants after single dose administration. [ABSTRACT FROM AUTHOR]