Cocktail-substrate assay system for mechanism-based inhibition of CYP2C9, CYP2D6, and CYP3A using human liver microsomes at an early stage of drug development.

We established a mechanism-based inhibition cocktail-substrate assay system using human liver microsomes and drug-probe substrates that enabled simultaneous estimation of the inactivation of main cytochrome P450 (CYP) enzymes, CYP2C9, CYP2D6, and CYP3A, in drug metabolism. The inactivation kinetic parameters of typical mechanism-based inhibitors, tienilic acid, paroxetine, and erythromycin, for each enzyme in the cocktail-substrate assay were almost in agreement with the values obtained in the single-substrate assay. Using this system, we confirmed that multiple CYP inactivation caused by mechanism-based inhibitors such as isoniazid and amiodarone could be detected simultaneously. Mechanism-based inhibition potency can be estimated by the determination of the observed inactivation rate constants (k(obs)) at a single concentration of test compounds because the k(obs) of eleven CYP3A inactivators at 10 microM in the assay system nearly corresponded to k(inact)/K(I) values, an indicator of a compound's propensity to alter the activity of a CYP in vivo (R(2) = 0.97). Therefore, this cocktail-substrate assay is considered to be a powerful tool for evaluating mechanism-based inhibition at an early stage of drug development.

Pharmacokinetics and metabolism of TR-14035, a novel antagonist of a4ss1/a4ss7 integrin mediated cell adhesion, in rat and dog.

The pharmacokinetics and disposition of N-(2,6-dichlorobenzoyl)-4-(2,6-dimethoxyphenyl)-L-phenylalanine (TR-14035), a novel a4ss1/a4ss7 antagonist, were investigated in the rat and dog. Results indicate extensive clearance of TR-14035 and low oral bioavailability, 17% and 13% in the rat and dog, respectively, at an oral dose of 10 mg/kg. At least 63% of the oral dose was absorbed from the gastrointestinal tract in the rat, and about one-third of the intravenous dose was excreted into bile as unchanged drug in the rat and dog. These data indicate that the oral bioavailability of TR-14035 was limited due to significant first-pass metabolism and biliary excretion in the liver. A species-dependent difference in metabolism was observed. The principal metabolite, O-desmethyl TR-14035, observed in rat, dog and probably human, was further conjugated with sulfate in the rat, but never in dog and human, based on in vitro metabolism and in vivo metabolite profile studies. Urinary excretion was a minor elimination route, but an interesting species difference was recognized. TR-14035 was reabsorbed from the rat renal proximal tubules, and by contrast, secreted into the tubules in the dog, probably via active transport systems.