Stereochemistry of Mitoglitazone Carbonyl Reduction in Rat, Monkey and Man

The stereochemistry of the carbonyl reduction of mitoglitazone, a thiazolidinedione insulin sensitizer drug candidate, was investigated in rat, monkey and man following repeated-dose oral administration of mitoglitazone. A chiral HPLC-UV analytical method that resolved all four diastereomers of hydroxymitoglitazone in plasma was used in this investigation. Sodium heparin plasma samples from subchronic toxicity studies in male and female CD® [Crl:CD®(SD)] rats and cynomolgus monkeys, and from a repeated-dose tolerance study in human volunteers were obtained after the first and last doses of mitoglitazone. Calibration standards and the analytical internal standard (structural analog) were prepared in diluent solution (methanol:deionized water, 1:1, v:v). Aliquots of plasma (500 µL) were fortified with 50 µL of the appropriate calibration standard or diluent (quality control and incurred samples) and 60 µL of internal standard. The samples were buffered with 0.5 mL 0.1M potassium phosphate, vortex mixed, and transferred to SPE cartridges that had been activated by successive addition of 1 mL acetonitrile and 0.1 M potassium phosphate. After gravity elution, the cartridges were successively washed with 2 mL methanol:potassium phosphate (30:70, v:v) and 1 mL 0.1 M potassium hydrogen phosphate before drying. Analytes were eluted by successive addition of 500 µL of acetonitrile:deionized water (40:60, v:v) and 500 µL acetonitrile:deionized water (50:50, v:v). The eluate was evaporated under nitrogen at 40°C to dryness and reconstituted in 200 µL ethanol:methanol (50:50, v:v) with sonication and vortex mixing. Aliquots (125 µL) of reconstituted samples were chromatographed on a CHIRALPAK® IA column (Chiral Technologies, Inc., West Chester, PA) at a flow rate of 1 mL/min and gradient elution chromatography using mobile phases containing methanol:ethanol:diethylamine (50:50:0.2) and methanol:ethanol:methane sulfonic acid (50:50:0.2). Analytes were detected by UV at 265 nm and quantitation was accomplished using peak height ratios (analyte/internal standard). The analytical range of the method was 50 - 1000 ng/mL. Comparison of hydroxymitoglitazone concentrations determined by a non-chiral LC-MS/MS method and the chiral HPLV-UV method demonstrated good agreement in total hydroxymitoglitazone concentrations for the two analytical methods; mean total hydroxymitoglitazone concentrations determined by the chiral method in rat, monkey and man were 100%, 106% and 108%, respectively, of the LC-MS/MS method. The R-diastereomers were the major hydroxymitoglitazone species observed in all three species, with mean percentages of R-hydroxymitoglitazone in rat, monkey and man being 97%, 61% and 90%, respectively. Except for the rat, the levels of the R,R- and R,S-diastereomers were equivalent and the levels of the S,S- and S,R-diastereomers were equivalent. In the rat, the ratio of the R,R- and R,S-diastereomers differed by approximately 2-fold.