Pharmacology Journal

The thiazolidinediones rosiglitazone and pioglitazone are peroxisome proliferator-activated receptor-γ agonists, with insulin-sensitizing properties, used in the treatment of Type 2 diabetes. The oral bioavailability of rosiglitazone is nearly 100% and that of pioglitazone >80%. Both drugs are extensively metabolized in the liver. Rosiglitazone is mainly biotransformed by N-demethylation and pyridine ring hydroxylation and pioglitazone by hydroxylation and oxidation. In vitro studies suggest that these reactions are catalysed mainly by CYP2C8, with minor contributions from CYP2C9 for rosiglitazone and CYP3A4 for pioglitazone. All circulating metabolites of rosiglitazone are less potent than the parent drug and are not thought to have substantial effects on blood glucose concentrations, whereas the main metabolites of pioglitazone (M3 and M4) are pharmacologically active, and their plasma concentrations are equal to or greater than those of the parent pioglitazone. The elimination half-life of rosiglitazone is about 3–6 h and that of pioglitazone is about 4–9 h.

SLCO1B1 encodes the organic anion transporting polypeptide 1B1 (OATP1B1) transporter, which is present at the basolateral membrane of hepatocytes and mediates uptake of its substrates from sinusoidal blood. Its substrates include endogenous compounds, such as bilirubin and bile acids, as well as various drugs, such as statins. A common single nucleotide polymorphism (SNP) in SLCO1B1, c.521T→C (p.Val174Ala), has been associated with reduced activity of OATP1B1 in vitro. Studies in humans have revealed that the pharmacokinetics of, for example, the antidiabetic repaglinide, as well as fexofenadine, simvastatin acid and pravastatin, have been significantly associated with SLCO1B1 polymorphism. In particular, the AUC of these compounds has been markedly higher in subjects with the c.521CC genotype than in those with the c.521TT genotype. In Whites, the c.521T→C SNP exists in four major haplotypes, differentiated by the g-11187G→A, g-10499A→C and c.388A→G SNPs: *16 (g-11187G/g-10499C/c.388G/c.521C), *17 (AAGC), *5 (GAAC) and *15 (GAGC).

Rosiglitazone and pioglitazone are potent competitive inhibitors of OATP1B1 in vitro and could thus be its substrates. Moreover, in an in silico pharmacophore modelling study, rosiglitazone and pioglitazone have been identified as possible substrates of OATP1B1. In vivo in humans, gemfibrozil, an inhibitor of CYP2C8 and OATP1B1, has considerably increased the plasma concentrations of rosiglitazone and pioglitazone. Although this evidence suggests that rosiglitazone and pioglitazone could be substrates of OATP1B1, it is not known whether SLCO1B1 genotype affects the pharmacokinetics of rosiglitazone or pioglitazone. The aim of this study was to investigate the effects of SLCO1B1 polymorphism on the pharmacokinetics of rosiglitazone and pioglitazone in a prospective genotype panel study. Because rosiglitazone and pioglitazone are metabolized via CYP2C8 and CYP2C9, the study was controlled for CYP2C8*3, CYP2C9*2 and CYP2C9*3, the major functionally significant alleles of these CYP enzymes.

Subjects

A total of 32 healthy White volunteers (19 men and 13 women) participated in this study after giving written informed consent. The participants were recruited from a pool of subjects genotyped for SLCO1B1, CYP2C8 and CYP2C9 SNPs. All genotyping was performed by TaqMan allelic discrimination with an Applied Biosystems 7300 Real-Time Polymerase Chain Reaction System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions with a reaction volume of 10 µl. Genotyping for SLCO1B1 SNPs was carried out as described previously. Genotyping for the CYP2C9*2 (c.430C→T) and CYP2C9*3 (c.1075A→C) alleles was performed using TaqMan^® Pre-Developed Assay Reagents for Allelic Discrimination (Applied Biosystems). Genotyping for the CYP2C8*3 allele (c.416G→A, c.1196A→G) was carried out using Custom TaqMan^® SNP genotyping assays (Applied Biosystems). CYP2C8 genotyping was validated against a previously described method. Only noncarriers of the CYP2C8*3, CYP2C9*2 and CYP2C9*3 alleles were recruited. The participants were selected on the basis of the SLCO1B1 c.521T→C SNP as well as the g-11187G→A, g-10499A→C and c.388A→G SNPs and were allocated to one of three groups according to the genotype. Haplotypes were assigned as described previously. The control group comprised 16 participants (five women, 11 men) with the homozygous reference genotype at each position (c.521TT group). Their mean ± SD age was 23 ± 2 years, height 177 ± 9 cm and weight 73 ± 10 kg. The second group included 12 participants (six women, six men) heterozygous for the c.521T→C SNP (c.521TC group). Their mean ± SD age was 22 ± 2 years, height 175 ± 11 cm and weight 68 ± 11 kg. Four c.521TC participants had the *15 haplotype (three *1A/*15, one *1B/*15 diplotype), four had the *16 haplotype (one *1A/*16, three *1B/*16) and four had the *17 haplotype (two *1A/*17, two *1B/*17). The third group consisted of four participants (one woman, three men) with the homozygous c.521CC genotype (c.521CC group). Their mean ± SD age was 23 ± 3 years, height 179 ± 9 cm and weight 78 ± 14 kg. The SLCO1B1 diplotypes of the participants with the c.521CC genotype were *5/*15, *15/*16, *16/*16 and *16/*17. The subjects were ascertained to be healthy by medical history, physical examination and routine laboratory tests before enrolment. None of the subjects was a tobacco smoker or taking any continuous medication, including oral contraceptives. Use of other drugs was prohibited for 1 week, use of grapefruit products for 3 days and use of alcohol for 1 day before the day of administration of rosiglitazone or pioglitazone.

Study design

The study protocol was approved by the Coordinating Ethics Committee of the Helsinki and Uusimaa Hospital District and the National Agency for Medicines (Helsinki, Finland). Following an overnight fast, the subjects ingested a single 4-mg dose of rosiglitazone (one Avandia 4-mg tablet; GlaxoSmithKline, Brentford, UK) and, after a wash-out period of at least 1 week, a single 15-mg dose of pioglitazone (one Actos 15-mg tablet; Takeda, London, UK). The study drugs were administered with 150 ml water at 08.00 h. Subjects received a standardized warm meal 4 h after the administration of study medication and a standardized snack after 7 and 10 h. Timed blood samples (5–10 ml each) were drawn from a cannulated forearm vein into tubes containing ethylenediamine tetraaceticacid before administration of rosiglitazone or pioglitazone and at 15, 30, 60, 90 min and at 2, 3, 4, 5, 7, 9 and 12 h, after which the subjects were discharged from the clinical research unit. In addition, blood samples were drawn by venepuncture 24 and 48 h after rosiglitazone, and 24, 48 and 72 h after pioglitazone administration. Plasma was stored at −70°C until analysis.

Determination of drug concentrations

Plasma rosiglitazone and N-desmethylrosiglitazone were measured by use of an API 2000 Q TRAP liquid chromatography-tandem mass spectrometry system (Sciex Division of MDS Inc., Toronto, Ontario, Canada). The reversed-phase chromatographic separation was achieved on a XBridge C18 column (internal diameter 100 × 2.1 mm and particle size 3.5 µm) (Waters Corp., Milford, MA, USA) using a mobile phase consisting of 10 mmol l^−1 ammonium acetate (pH 9.5, adjusted with 25% ammonium hydroxide solution) and acetonitrile at a ratio of 70 : 30 (v/v). A 10-µl aliquot was injected and the mobile phase was delivered at a flow rate of 200 µl min^−1, yielding a total chromatographic run time of 7 min. Pioglitazone served as an internal standard for both analytes. The mass spectrometer was operated in positive TurboIonSpray^® mode, and the samples were analysed via selected reaction monitoring by use of the transition of the [M+H]+ precursor ion to product ionfor each analyte and internal standard. The selected ion transitions monitored were as follows: m/z 358 to m/z 135 for rosiglitazone, m/z 344 to m/z 121 for N-desmethylrosiglitazone, and m/z 357 to m/z 134 for pioglitazone. The limit of quantification of plasma rosiglitazone was 0.25 ng ml^−1 and the day-to-day coefficient of variation (CV) was 6.3% at 0.3 ng ml^−1, 5.1% at 3.0 ng ml^−1, 5.6% at 30 ng ml^−1 and 3.9% at 300 ng ml^−1 (n = 6). The plasma concentrations of pioglitazone and its metabolites were determined as described previously. The limit of quantification of plasma pioglitazone and its M3 metabolite was 0.3 ng ml^−1 and that of M4 was 1 ng ml^−1. The day-to-day CV was 4.3% at 20 ng ml^−1, 4.5% at 200 ng ml^−1 and 4.9% at 2000 ng ml^−1 of pioglitazone, 9.5% at 10 ng ml^−1, 8.8% at 100 ng ml^−1 and 5.5% at 1000 ng ml^−1 of M3 and 14.1% at 10 ng ml^−1 and 5.7% at 100 ng ml^−1 of M4 (n = 16). Because authentic reference compounds were not available, the concentrations of N-desmethylrosiglitazone and the M5 metabolite of pioglitazone are given in arbitrary units relative to the ratio of the peak area of the metabolite to that of the internal standard. The detector response for these metabolites was confirmed to be linear over the relevant concentration range by means of sample dilution (1–125-fold dilutions, r > 0.995), and a signal to noise ratio (S/N) of 10 : 1 was used as the determination limit.

Pharmacokinetic analysis

The pharmacokinetics of rosiglitazone and pioglitazone were characterized by the peak concentration in plasma (C_max), time to C_max (t_max), elimination half-life (t_1/2) and area under the plasma concentration–time curve (AUC) from 0 to 48 (rosiglitazone) or 72 h (pioglitazone) and AUC from time 0 to infinity (AUC_{0–∞}). The terminal log-linear part of each concentration–time curve was identified visually, and the elimination rate constant (k_e ) was determined from natural log-transformed data with linear regression analysis. The t_1/2 was calculated by the equation t_1/2 = ln2/k_e . AUC was calculated by a combination of the linear (for increasing concentrations) and log-linear (for decreasing concentrations) trapezoidal rules, with extrapolation to infinity, when appropriate, by division of the last measured concentration by k_e . Weight-adjusted AUC_{0–∞} was calculated as follows: AUC_{0–∞} · weight (kg)/70 kg. The apparent formation rate constant (k_f ) of N-desmethylrosiglitazone and the metabolites of pioglitazone was determined by the method of residuals from the ascending part of the metabolite concentration–time curve.

Statistical analysis

Results are expressed as mean ± SD in the text and tables. Statistical comparisons of the pharmacokinetic variables of rosiglitazone and pioglitazone and their metabolites between subjects with the SLCO1B1 c.521TT, c.521TC and c.521CC genotypes were performed using analysis of variance (anova) and post hoc testing with the Tukey test (equal variances) or the Games–Howell test (unequal variances). Equality of group variances was tested with the Levene statistics and compatibility of the residuals with normal distribution with the Shapiro–Wilk test. AUC and C_max data were logarithmically transformed before analysis. The 95% confidence intervals (CI) were calculated on the geometric mean ratio of the C_max and AUC values for the c.521TC/c.521TT and c.521CC/c.521TT pairs, and on the mean difference of the t_1/2 and k_f values. The t_max data were analysed by the Kruskal–Wallis test. The number of subjects in each genotype group was estimated to be sufficient to detect a 50% greater mean AUC_{0–∞} of rosiglitazone and pioglitazone in subjects with the c.521TC genotype than in those with the c.521TT genotype, as well as a 100% greater mean AUC_{0–∞} of rosiglitazone and pioglitazone in subjects with the c.521CC genotype than in those with the c.521TT genotype, with a power of at least 80% (α level of 0.05). All data were analysed with the statistical program SPSS 11.0 for Windows (SPSS Inc., Chicago, IL, USA). Differences were considered statistically significant at P < 0.05.

Filed under: Pharmacogenetics