J.-G. Shin

Genetic variants of the organic cation transporter 2 influence the disposition of metformin.

Genetic variants of the organic cation transporter 2 (protein, OCT2; gene, SLC22A2) were evaluated for their contribution to the variations in the pharmacokinetics of metformin, especially to its renal elimination. Genetic variants of SLC22A2 (c.596C>T, c.602C>T, and c.808G>T) showed significant differences in metformin pharmacokinetics when compared with the reference genotype, with higher peak plasma concentration (Cmax) and area under the curve (AUC) and lower renal clearance (Clrenal), thereby suggesting that a decrease in transport function associated with the SLC22A2 variants results in reduced Clrenal of metformin and consequently leads to increased plasma concentrations.

Integration of genetic, clinical, and INR data to refine warfarin dosing

Well‐characterized genes that affect warfarin metabolism (cytochrome P450 (CYP) 2C9) and sensitivity (vitamin K epoxide reductase complex 1 (VKORC1)) explain one‐third of the variability in therapeutic dose before the international normalized ratio (INR) is measured. To determine genotypic relevance after INR becomes available, we derived clinical and pharmacogenetic refinement algorithms on the basis of INR values (on day 4 or 5 of therapy), clinical factors, and genotype. After adjusting for INR, CYP2C9 and VKORC1 genotypes remained significant predictors (P < 0.001) of warfarin dose. The clinical algorithm had an R2 of 48% (median absolute error (MAE): 7.0 mg/week) and the pharmacogenetic algorithm had an R2 of 63% (MAE: 5.5 mg/week) in the derivation set (N = 969). In independent validation sets, the R2 was 26–43% with the clinical algorithm and 42–58% when genotype was added (P = 0.002). After several days of therapy, a pharmacogenetic algorithm estimates the therapeutic warfarin dose more accurately than one using clinical factors and INR response alone.

CYP2D6 Genotype and Adjuvant Tamoxifen: Meta‐Analysis of Heterogeneous Study Populations

The International Tamoxifen Pharmacogenomics Consortium was established to address the controversy regarding cytochrome P450 2D6 (CYP2D6) status and clinical outcomes in tamoxifen therapy. We performed a meta‐analysis on data from 4,973 tamoxifen‐treated patients (12 globally distributed sites). Using strict eligibility requirements (postmenopausal women with estrogen receptor–positive breast cancer, receiving 20 mg/day tamoxifen for 5 years, criterion 1); CYP2D6 poor metabolizer status was associated with poorer invasive disease–free survival (IDFS: hazard ratio = 1.25; 95% confidence interval = 1.06, 1.47; P = 0.009). However, CYP2D6 status was not statistically significant when tamoxifen duration, menopausal status, and annual follow‐up were not specified (criterion 2, n = 2,443; P = 0.25) or when no exclusions were applied (criterion 3, n = 4,935; P = 0.38). Although CYP2D6 is a strong predictor of IDFS using strict inclusion criteria, because the results are not robust to inclusion criteria (these were not defined a priori), prospective studies are necessary to fully establish the value of CYP2D6 genotyping in tamoxifen therapy.

Development of the inje cocktail for high-throughput evaluation of five human cytochrome P450 isoforms in vivo

To develop and validate an in vivo cocktail method for high‐throughput phenotyping of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A, 12 healthy subjects received five probe drugs alone or simultaneously. The in vivo phenotyping index of CYP2C9, the ratio of 8 h urine concentration of losartan to its metabolite after a single administration of losartan, was not significantly different from that obtained using the five‐drug cocktail. Similarly, the ratios of [omeprazole]/[5‐hydroxyomeprazole] (CYP2C19) and [paraxanthine]/[caffeine] (CYP1A2) in 4 h plasma samples and the log ratio of [dextromethorphan]/[dextrorphan] (CYP2D6) in 8 h urine samples and the 4 h plasma concentrations of midazolam (CYP3A) after single administration or well‐established three‐drug cocktail of caffeine, omeprazole, and dextromethorphan were not significantly different from those after the new five‐drug cocktail. In conclusion, the new five‐drug cocktail regimen, named the “Inje cocktail,” can be used as a tool to phenotype in vivo enzyme activities of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A with only 4 h blood sampling and 8 h urine collection following simultaneous administration of the five probe drugs.

Contribution of cytochrome P450 3A4 and 3A5 to the metabolism of atorvastatin

Atorvastatin is a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor that is mainly metabolized by cytochrome P450 (CYP) 3A4. A recent study showed that the lipid-lowering effect of statins is affected by the CYP3A5 polymorphism. Therefore, it was investigated whether CYP3A5 contributes to the metabolism of atorvastatin. Two metabolites of atorvastatin, para- and ortho-hydroxyatorvastatin, were produced by human liver microsomes and human recombinant CYP3A enzymes, and the enzyme kinetic pattern exhibited substrate inhibition. The intrinsic clearance (CLint) rates of para- and ortho-hydroxyatorvastatin by CYP3A4 were 2.4- and 5.0-fold of the respective CLint rates of CYP3A5, indicating that CYP3A4 is the major P450 isoform responsible for atorvastatin metabolism. These results suggest that atorvastatin is preferentially metabolized by CYP3A4 rather than by CYP3A5, and thus the genetic CYP3A5 polymorphism might not be an important factor in the inter-individual variation of atorvastatin disposition and pharmacodynamics in human.

Genetic variants of organic cation transporter 2 (OCT2) significantly reduce metformin uptake in oocytes.

1. The authors sought to evaluate the contribution of organic cation transporters (OCTs) to the renal tubular transport of metformin using LLC-PK1 cells as an in vitro model for the renal proximal tubule, and to investigate the effects of three non-synonymous genetic variants of OCT2 on the transport activity of metformin in vitro using an oocyte over-expression system. 2. The basolateral-to-apical transport of metformin was significantly greater than the apical-to-basolateral transport and showed concentration dependency with the kinetic parameters: maximum transport rate (Vmax), 922 pmol min−1 per 5 × 105 cells; Michaelis–Menten constant (Km), 393 µM; intrinsic clearance (CLint), 2.35 µl min−1 per 5 × 105 cells; and diffusion constant (Kd), 0.33 µl min−1 per 5 × 105 cells. The basolateral-to-apical transport of metformin was inhibited by phenoxybenzamine, an inhibitor of OCTs, but not by cyclosporine A, MK571, or fumitremorgin C, which are inhibitors of P-glycoprotein, multidrug resistance proteins (MRPs), and breast cancer resistance protein (BCRP), respectively, suggesting that OCTs play a role in renal tubular secretion of metformin. 3. Metformin uptake was much greater in oocytes expressing OCT2-wild type (OCT2-WT) than OCT1-WT compared with uptake in water-injected oocytes. Uptake was significantly decreased in oocytes expressing OCT2-T199I, -T201M, and -A270S compared with that in OCT2-WT, suggesting that metformin is a better substrate for OCT2 than for OCT1 and that the amino acid-substituted variants of OCT2 cause a functional decrease in metformin uptake. 4. In conclusion, the genetic variants of OCT2 (OCT2-T199I, -T201M, and -A270S) decreased the transport activity of metformin and thus may contribute to the inter-individual variation in metformin disposition as OCT2 plays a pivotal role in renal excretion, the major disposition route of metformin.

Stereoselective inhibition of cytochrome P450 forms by lansoprazole and omeprazole in vitro

The stereoselectivity of the inhibitory interaction potential of lansoprazole and omeprazole isomers on six human cytochrome P450 forms was evaluated using human liver microsomes. Lansoprazole enantiomers showed stereoselective inhibition of CYP2C9-catalysed tolbutamide 4-methylhydroxylation, CYP2C19-catalysed S-mephenytoin 4′-hydroxylation, CYP2D6-catalysed dextromethorphan O-demethylation, CYP2E1-catalysed chlorzoxazone 6-hydroxylation and CYP3A4-catalysed midazolam 1-hydroxylation, whereas omeprazole only inhibited CYP2C19 stereoselectively. Of the P450 forms tested, CYP2C19-catalysed S-mephenytoin 4′-hydroxylation was extensively inhibited by both the lansoprazole and omeprazole enantiomers in a competitive and stereoselective manner; the S-enantiomers of both drugs inhibited the hydroxylation more than the R-enantiomers. The estimated Ki values determined for CYP2C19-catalysed S-mephenytoin 4′-hydroxylation were 0.6, 6.1, 3.4 and 5.7 µM for S-lansoprazole, R-lansoprazole, S-omeprazole and R-omeprazole, respectively. The results indicate that although both lansoprazole and omeprazole are strong inhibitors of CYP2C19, the inhibition of CYP2C19 by lansoprazole is highly stereoselective, whereas the inhibition by omeprazole is less stereoselective. In addition, S-lansoprazole, the most potent CYP2C19 inhibitor, is not a good CYP2C19-selective inhibitor owing to its inhibition of other P450 forms.

In vitro metabolism of a novel PPARγ agonist, KR-62980, and its stereoisomer, KR-63198, in human liver microsomes and by recombinant cytochrome P450s

1. KR-62980 and its stereoisomer KR-63198 are novel and selective peroxisome proliferator-activated receptor gamma (PPARγ) modulators with activity profiles different from that of rosiglitazone. This study was performed to identify the major metabolic pathways for KR-62980 and KR-63198 in human liver microsomes. 2. Human liver microsomal incubation of KR-62980 and KR-63198 in the presence of a β-nicotinamide adenine dinucleotide phosphate (NADPH)-generating system resulted in hydroxy metabolite formation. In addition, the specific cytochrome P450s (CYPs) responsible for KR-62980 and KR-63198 hydroxylation were identified by using a combination of chemical inhibition in human liver microsomes and metabolism by recombinant P450s. It is shown that CYP1A2, CYP2D6, CYP3A4, and CYP3A5 are the predominant enzymes in the hydroxylation of KR-62980 and KR-63198. 3. The intrinsic clearance through hydroxylation was consistently and significantly higher for KR-62980 than for KR-63198, indicating metabolic stereoselectivity (CLint of 0.012 ± 0.001 versus 0.004 ± 0.001 μl min−1 pmol−1 P450, respectively). 4. In a drug–drug interaction study, KR-62980 and KR-63198 had no effect on the activities of the P450s tested (IC50 > 50 μM), suggesting that in clinical interactions between KR-62980 and KR-63198 the P450s tested would not be expected.

Effect of renal impairment and haemodialysis on the pharmacokinetics of gemigliptin (LC15-0444).

This study evaluated the effects of renal impairment (RI) and haemodialysis (HD) on the pharmacokinetics of gemigliptin, a novel dipeptidyl peptidase‐4 (DPP‐4) inhibitor. After a 100 mg administration to subjects with normal renal function (n = 23) or RI (n = 24), plasma, urine or dialysate samples were analysed. Control subjects were matched to patients based on age, gender and body mass index. Patients with mild, moderate, severe RI and end‐stage renal disease (ESRD) showed 1.20, 2.04, 1.50 and 1.66‐fold (1.10, 1.49, 1.22 and 1.21‐fold) increase of mean area under the time‐plasma concentration curve from 0 to infinity (AUCinf) [maximum plasma concentration (Cmax)] of gemigliptin, respectively. Pharmacokinetics of gemigliptin was comparable between HD and non‐HD periods in ESRD patients. Less than 4% of the dose was removed by 4 h HD. RI appeared to have modest effect on the gemigliptin disposition. No dose adjustment in patients with RI is proposed on the basis of exposure–response relationship. Impact of HD on the removal of gemigliptin was negligible.

Dietary salt does not influence the disposition of verapamil enantiomers in relation to efflux transporter ABCB1 genetic polymorphism in healthy Korean subjects.

To evaluate the effects of dietary salt on the stereoselective disposition of verapamil enantiomers in relation to the transporter ABCB1 2677GG/3435CC and 2677TT/3435TT haplotypes, ten healthy subjects were asked to take diets of three different salt levels for 7 days in a randomized, three-way crossover manner. The plasma concentrations of verapamil and norverapamil enantiomers were determined after a single oral dose of 240 mg verapamil on the last day of each phase. Pharmacokinetic parameters were calculated by non-compartmental analysis techniques and compared among the three different dietary salt phases. Compared with the medium salt diet, the high and low salt diets had no significant effect on the disposition of verapamil enantiomers. Moreover, the ABCB1 haplotypes did not alter the impact of dietary salt, although ABCB1 2677TT/3435TT subjects had slightly, but not significantly, higher Cmax and area under the curve (AUC) and lower Tmax for the verapamil enantiomers than did 2677GG/3435CC subjects in each salt phase.