Improvement in the gastrointestinal absorption of troglitazone
Troglitazone, 4-thiazolidinedione, an insulin action enhancing agent, is one of a new class of agents, the thiazolidinediones, currently in clinical development for the treatment of non insulin dependent diabetes (NIDDM). Animal models of NIDDM provide evidence that troglitazone suppresses hepatic gluconeogenesis and improves insulin sensitivity and responsiveness by potentiating insulin-induced peripheral glucose uptake. In vitro studies have shown that troglitazone acts directly on muscle and liver cells to increase glucose utilisation and reduce glucose production. Furthermore, troglitazone may have beneficial effects on abnormal lipid levels such as triglycerides, high density lipoprotein (HDL) cholesterol and non-esterified fatty acids which are implicated as risk factors for cardiovascular disease in NIDDM. In clinical studies, troglitazone is well tolerated and achieves good glycaemic control in NIDDM patients, accompanied by favourable changes in abnormal lipid profiles.
Troglitazone is a white to pale yellow crystalline powder (pKa 6.1, 12.0) which is practically insoluble in water (solubility approximately 0.02 mg ml−1 ). It has an oil/water partition coefficient of 2.4 at pH 7 and is absorbed largely via the small intestine. Following oral administration, troglitazone is primarily metabolised to a sulphate conjugate and an oxidative quinone metabolite. A glucuronide conjugate is also formed. Troglitazone has a half-life of approximately 10–15 h and that of its major metabolite is approximately 20 h [unpublished data]. A high degree of variability in area under the plasma concentration-time curve (AUC) occurs (approximate CV of 30%), [unpublished data].
It is important therefore to assess the effect of food on the gastrointestinal absorption of troglitazone. This study was designed to investigate the effect of concomitant food intake and food intake 30 min prior to dosing on the pharmacokinetic profile of troglitazone.
hirteen healthy males (mean age 31 years, range 20–41 years) were enrolled in this open, randomized, three-way, cross-over study. They weighed between 64.4 kg and 95.2 kg (mean 79.1 kg), and ranged in height from 1.70–1.86 m (mean 1.76 m). Each subject received one tablet of troglitazone (400 mg) following an overnight fast 1) alone, 2) concomitantly at the start of a standardized diabetic breakfast and 3) 30 min after a standardized diabetic breakfast. All treatments were given with water (200 ml) and were separated by a minimum of 7 days. Apart from the standardized breakfast (approximately 64 g carbohydrate, 25 g fat, 20 g protein and 600 calories) of cornflakes (35 g), skimmed milk (150 ml), a slice of wholemeal toast (25 g) and butter (25 g) and an apple, food was not allowed until 4 h after dosing; a light lunch was then provided.
All subjects were free from any significant medical condition, received no regular medication for 4 weeks prior to the study and no drug treatment for 48 h prior to the study. Subjects were not allowed to consume food or drink (apart from water) from 22.00 h on the night prior to the study. Water was permitted up to midnight prior to dosing. Smoking, alcohol and strenuous exercise were not permitted from the evening prior to drug administration to after the last blood sample on each occasion. Subjects returned between 7 and 10 days following the end of the study for laboratory safety tests.
The study protocol was reviewed and approved by the Ethics Review Committee of Glaxo Wellcome Research and Development Ltd. All subjects gave written consent to participate and the study was conducted at the department of Clinical Pharmacology, Glaxo Wellcome Research and Development Ltd, Ware, Hertfordshire, UK in accordance with the provisions of the revised Declaration of Helsinki (1964).
Assessments
Blood samples (5 ml) were taken pre-dose and at the following nominal times post-dose: 15, 30, 45, 60 and 90 min and 2, 3, 4, 8, 12, 24 and 48 h. Blood samples were centrifuged at 1500 g for 10 min and the resultant plasma frozen at −20° C until required for assay. Plasma samples (300 μl) were assayed by h.p.l.c. with ultraviolet detection (230nm) following liquid/liquid extraction with ethyl acetate:hexane (3ml, 90:10 v/v). The organic phase was dried down under nitrogen and the samples were re-constituted in ethanol (100 μl) prior to injection of an aliquot (30 μl) onto the h.p.l.c. column (YMC-Pack ODS A-314G, 300×6 mm) which was maintained at 35° C and eluted at a flow rate of 1.2ml min−1 with a mobile phase consisting of acetonitrile/water/phosphoric acid (60:40:0.08 v/v). Chromatographic peak height ratios of each analyte relative to the internal standard (9-acetylanthracene) were quantified by reference to linear calibration lines prepared freshly on each occasion of assay. A calibration range of 0.1 to 6.4 μg ml−1 and a limit of quantification of 0.1 μg ml−1 for both troglitazone and the sulphate metabolite were used. Assay performance was monitored by quantifying three quality control samples in duplicate which were required to be within 15% of their nominal concentrations.
The assay was validated and the intra assay precision ranged from 1.7 to 28% for troglitazone and from 1.1 to 13% for the sulphate with a bias of −12 to +3% for troglitazone and +0.8 to +22% for the sulphate. The inter assay variation was determined for three analyte concentrations on seven occasions and precision varied from 13 to 14.8% for troglitazone (bias +0.4 to +7.8%) and from 4.2 to 16.2% for the sulphate (bias −0.4 to +9.5%). Troglitazone and the sulphate metabolite were stable in plasma for up to 12 months and three freeze-thaw cycles had little effect on their concentrations.
Data analysis
The following parameters were derived for each subject from the plasma troglitazone and sulphate metabolite concentration data. The maximum observed plasma concentration (Cmax ) and the time at which Cmax was reached (tmax ) were noted directly. For troglitazone the AUC to the last measurable time point (AUClast ) was calculated by the log-linear trapezoidal rule and the lag-time before appreciable absorption of troglitazone occurred (tlag ) was recorded as the last timepoint with a value below the limit of quantification prior to absorption. For the sulphate metabolite, the terminal elimination rate constant (λz ) was calculated by linear least-square regression using logarithmically transformed points in the terminal phase. The terminal phase plasma half-life (t1/2 ) was calculated by ln(2)/λz. AUC was extrapolated to infinity (AUC∞ ) by adding the ratio of the last measurable concentration divided by the elimination rate constant. The plasma concentrations of troglitazone were not adequately defined in the terminal phase and therefore λz and the corresponding t1/2 and AUC parameters could not be measured.
The pharmacokinetic parameter values, Cmax and AUC for troglitazone and the sulphate metabolite were analysed using analysis of variance allowing for subjects, periods and treatments. Separate tests for treatment by period interaction and for carry-over effects were performed for each of the variables analysed parametrically. Where there was evidence of a carry-over effect, this was included in the statistical model. The pharmacokinetic parameter t1/2 for the sulphate metabolite was therefore analysed using analysis of variance allowing for effects due to subject, period, carryover and treatment. A log transformation was performed for each parameter in order to satisfy the constant variance assumption for the analysis of variance. Geometric mean values for each of the treatments were evaluated together with ranges. Estimates of pairwise treatment differences were calculated together with 95% confidence intervals.
Values of tmax and tlag were summarized by the treatment medians and compared between pairs of treatments using the Wilcoxon signed rank test, with corresponding 95% confidence intervals for the estimates of treatment difference derived.
Safety
Adverse events occurring during the study and laboratory parameters for clinical chemistry, haematology and urinalysis were recorded at screening, pre-dose, 48 h post-dose and between 7 and 10 days following the end of the study.
Filed under: Clinical Pharmacology