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A sensitive high-performance liquid chromatographic (HPLC) method was developed and validated to separate and quantitate the levels of L-696,229 (I), a novel human immunodeficiency virus type I non-nucleoside reverse transcriptase inhibitor, and its hydroxy metabolites (II and III) in plasma samples. The procedure involves the addition of a constant known quantity of internal standard to the biological specimen followed by extraction of the compounds of interest into methyl tert.-butyl ether. The organic phase is evaporated to dryness under a gentle stream of nitrogen. The residue is then dissolved in methanol and water and injected onto a reversed-phase HPLC column. A gradient HPLC method is used to elute the compounds which are monitored using UV detection at 319 nm. Absolute calibration factors (from the standard curve) were calculated by analyzing standards, and these factors were used to determine the concentration of drug (I) and its hydroxy metabolites (II and III) in the samples using the internal standard method. The method was linear using a standard concentration range of 50 to 20,000 ng/ml. The limit of quantitation was 50 ng/ml using 200 microliters plasma. The procedure was utilized to monitor plasma levels of I, II and III in acute and chronic toxicity studies in several animal species.

A method for the determination of a 2-pyridinone-based specific HIV-1 reverse transcriptase inhibitor in human plasma is described. Plasma samples are extracted using phenyl solid phase extraction columns. The extract is analyzed via HPLC using a column-switching system to remove interferences from late-eluting endogenous components. Detection is based on UV absorbance at 314 nm. The assay was linear in the concentration range of 10-500 ng/ml, when 1-ml aliquots of plasma were extracted. The mean precision of the assay, expressed as the coefficient of variation, was 3.8%. The assay has been validated and utilized to support human pharmacokinetic studies.

A rapid method for measuring pyridinone HIV-1 reverse transcriptase inhibitor plasma levels was necessary for identifying potential clinical candidates and for choosing a clinical formulation. Due to its sensitivity to the pyridinones, ability to tolerate extraneous material, and its capability for rapid, high through-put screening, the HIV-1 RT assay was developed into a bioassay for determining plasma levels of the pyridinones in Rhesus monkey plasma. With this assay, dose proportionality of L-697, 639 was established. Formulation studies using L-697, 639 indicated that the plasma levels achieved in Rhesus monkeys with the clinical formulation (peak levels = 3.9 microM at 30 min) fall between the levels achieved with polyethylene glycol 300 and hydroxypropyl methyl cellulose formulations (peak levels = 8.9 microM and 0.4 microM respectively, at 60 min). Two other pyridinones, L-696, 229 and L-697, 661, administered as the clinical formulation, had peak plasma levels of 1.6 microM (30 min) and 0.3 microM (60 min), respectively. In Rhesus monkeys, the bioavailabilities of these compounds (administered as the clinical formulation) ranged from 11 to 24% and their half-life values ranged from 24 to 120 min. The results of oral studies in Rhesus monkeys with these compounds were very similar to initial results of studies in humans.

A method for the determination of a non-nucleoside HIV-1 reverse transcriptase inhibitor in human plasma is described. Plasma samples are extracted using phenyl solid-phase extraction columns. The extract is analyzed by high-performance liquid chromatography with a polybutadiene-coated alumina column and a mobile phase of methanol-0.025 M pH 8 dibasic sodium phosphate buffer (1:1, v/v). Detection is based on ultraviolet absorbance at 326 nm. The assay was validated in the concentration range 10-500 ng/ml when 1-ml aliquots of plasma are extracted. The assay has been utilized to support human pharmacokinetic studies.