Development, validation, and interlaboratory comparison of an HMG-CoA reductase inhibition assay for quantitation of atorvastatin in plasma matrices.

An HMG-CoA reductase inhibition assay was developed and validated for quantitation of atorvastatin in human, dog, rat, and mouse plasma. Atorvastatin was isolated from plasma by protein precipitation. Rat-liver microsomes were used to provide the reductase enzyme. The method was validated by assaying calibration standards and quality controls in triplicate on each of the 3 days. A customized computer program was used for data calculation. Quantitation of the assay ranged from 0.36 to 16 ng/ml of atorvastatin in different plasma matrices. Assay precision and accuracy, based on the coefficient of variation and percent relative error, respectively, of quality controls were 10.4% to 14.5% and within +/- 6.25% in human; 4.89% to 10.6% (+/- 8.13%) in dog; 2.68% to 8.62% (+/- 5.00%) in rat; and 3.68% to 8.96% (+/- 5.38%) in mouse plasma. The method has been applied to pharmacokinetic studies of atorvastatin in human and toxicokinetic studies in dog, rat, and mouse after atorvastatin administration. Atorvastatin equivalent concentrations in a set of plasma samples from subjects receiving single and multiple doses of atorvastatin were determined by validated HMG-CoA reductase inhibition assays at four different laboratories. Results were compared using linear regression and concordance correlation statistical procedures. Good agreements among these data indicated that results from different laboratories with the same validated method can be used interchangeably.

Determination of 2,2-dimethyl-N-(2,4,6-trimethoxyphenyl) dodecanamide, CI-976, in rat plasma by reversed-phase high-performance liquid chromatography.

A quantitative reversed-phase high-performance liquid chromatographic procedure was developed to facilitate the preclinical development of a new Acyl-CoA:cholesterol acyltransferase inhibitor, CI-976 (I). This procedure has a lower quantitation limit of 0.06 micrograms/ml and a quantitation range of 0.06 to 8.0 micrograms/ml of I in rat plasma. The method was applied to pharmacokinetic and toxicokinetic studies of I in rat. With minor modifications, it has also been employed for analysis of I in human, monkey, and rabbit plasma.

Biodisposition studies with the acyl-coenzyme A: cholesterol acyltransferase inhibitor 2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide, CI-976.

2,2-Dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide, CI-976, is a newly developed fatty acid anilide being evaluated as a plasma lipid regulator and antiatherosclerotic agent. Disposition studies with CI-976 were conducted in rats and monkeys. In rats, CI-976 (suspension, 50 mg/kg) was approximately 30% bioavailable, with 46% of the administered radioactivity recovered in urine suggesting presystemic metabolism. The intravenous elimination half-life of CI-976 in rats was approximately 8 hr. Radioactivity derived from [14C]CI-976 was almost completely recovered in rats after either oral or intravenous administration. In bile duct-cannulated rats receiving either an oral or intravenous dose, biliary excretion was the major route of drug-derived radioactivity elimination. There was no evidence for unchanged drug in urine or bile. In monkeys, 13% urinary recovery and 5% bioavailability was achieved after a single 50 mg/kg suspension dose. Similar values were obtained following a 250 mg/kg dose. Monkeys displayed a CI-976 elimination half-life of 0.6 hr after a 2 mg/kg intravenous dose. No unchanged drug was excreted in urine. In summary, CI-976 exhibits moderate absorption and bioavailability in the rat, but lower absorption and bioavailability in monkey. A special difference in CI-976 elimination half-life was observed consistent with in vitro metabolism results.

Biodehalogenation and metabolism of 125[I]-4-iodobiphenyl.

[125I]-4-Iodobiphenyl has been used as a model compound to investigate the in vivo metabolism of iodinated aromatic compounds in the rat. Material balance studies showed that 57.6% of the injected dose was excreted via feces and 41.2% via urine. Distribution studies indicated uptake of inorganic iodide in the thyroid. In the feces, 2.2% of the dose was unmetabolized iodobiphenyl, 2.6% was 2-hydroxy-4'-iodobiphenyl, 21.9% was 4-hydroxy-4'-iodobipheny, and 20.9% was a polar fraction of which only 10.3% could by silylated. In urine, 1.6% of the dose was 2-hydroxy-4'-iodobiphenyl, 4.9% was 4-hydroxy-4'-iodobiphenyl, 1.9% was 2-hydroxy-4'-iodobiphenyl, 17.7% was 4-hydroxy-4'-iodobiphenyl glucuronide and sulfate conjugates, and 8.2% was inorganic iodide. About 3.0% of the dose in the urine polar fraction could be methylated.

Determination of iodide in metabolic studies of 125I-labeled compounds.

Methods capable of measuring inorganic iodide in the presence of other highly polar metabolites were developed in support of studies concerning the metabolism of 125I-labeled compounds. The methods included separation of iodine on a weakly basic resin paper followed by gamma-counting, methyl iodide exchange, and reverse isotopic dilution.