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The pharmacokinetics and hepatic metabolism of [3H] ivermectin (IVM) and [3H]cyclosporin A (CSA) were investigated in a subpopulation of the CF-1 mouse stock naturally deficient in mdr1a p-glycoprotein (PGP). A survey of key drug-metabolizing activities in liver fractions from PGP-deficient (-/-) or wild-type (+/+) animals indicated the two subpopulations are not different in hepatic metabolic activity and capacity. Intravenous pharmacokinetics of CSA were identical between the two groups, and results from microsomal incubations indicated similar biotransformation of IVM and CSA in liver. Intestinal excretion of [3H]IVM and [3H]CSA was enhanced in PGP (+/+) animals. Absence of PGP resulted in higher blood concentrations of IVM after oral dosing, suggesting enhanced absorption of IVM in (-/-) mice. Concentrations of [3H]IVM and [3H]CSA were always greater in the brains of (-/-) mice compared with (+/+) mice after either i.v. or oral administration. In contrast, liver concentrations of either compound were not different between (+/+) and (-/-) animals after an i.v. dose. These results show the PGP (-/-) and (+/+) subpopulations of CF-1 mice are useful for studying the role of mdr1a PGP in systemic exposure and tissue disposition of PGP substrates in the absence of metabolism differences.

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Pyridyloxypropanolamines L-749,372 (8, beta 3 EC50 = 3.6 nM) and L-750,355 (29, beta 3 EC50 = 13 nM) are selective partial agonists of the human receptor, with 33% and 49% activation, respectively. Both stimulate lipolysis in rhesus monkeys (ED50 = 2 and 0.8 mg/kg, respectively), with minimal effects on heart rate. Oral bioavailability in dogs, 41% for L-749,372 and 47% for L-750,355, is improved relative to phenol analogs.

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L-368,899 is a potent, orally-active oxytocin antagonist that completed phase I clinical trials for the prevention of preterm labor. The pharmacokinetics and disposition of L-368,899 were studied in rats (female and male) and dogs (female), the two species used in the toxicology studies. L-368,899 exhibited similar pharmacokinetics in rats and dogs. After iv dosing at 1, 2.5, and 10 mg/kg, the compound had a t1/2 of approximately 2 hr and plasma clearance between 23 and 36 ml/min/kg at all doses and in both species. The exception was female rats at the 10 mg/kg dose where plasma clearance decreased to 18 ml/min/kg. The Vdss was between 2.0 and 2.6 liters/kg for rats and 3.4 to 4.9 liters/kg for dogs. After oral doing, L-368,899 was rapidly absorbed. Mean Cmax values were achieved at <1 hr at the low doses (25 mg/kg in rats and 5 mg/kg in dogs) and between 1 and 4 hr at the higher doses (100 mg/kg in rats and 33 mg/kg in dogs). In bile duct-cannulated female rats, approximately 70% of a radioactive 28 mg/kg dose was recovered in bile and urine within 72 hr post dose. Plasma drug concentrations were higher in female than in male rats especially at the 25 mg/kg dose, where mean AUC values were 4.5-fold higher in the females. In both rats and dogs, plasma drug levels increased more than proportionally with increasing oral dose. In female rats, the mean AUC increased by approximately 8-fold between 25 and 100 mg/kg, while in female dogs, the mean AUC at the 33 mg/kg dose was 12-fold higher than that at 5 mg/kg. Oral bioavailability was estimated at 14% and 18% for the 5 mg/kg dose in female and male rats, respectively, 41% for the 25 mg/kg dose in male rats and 17% and 41%, respectively, for the 5 and 33 mg/kg doses in dogs. Owing to nonlinear kinetics, bioavailability could not be calculated for the other oral doses. L-368,899 was metabolized extensively in both species after iv and oral dosing, with <10% of the dose excreted unchanged. The main route of elimination was via the feces, which contained >70% of the radioactive dose by 48 hr, primarily as metabolites. The gender and dose dependence of the pharmacokinetics of L-389,899 in rats were attributed to gender differences in metabolizing capacity and saturation of hepatic metabolism, respectively. This conclusion was based primarily on results from experiments comparing the rate of in vitro metabolism of L-368,899 in liver microsomes, which showed that the Vmax and KM values for L-368,899 were 4-fold lower in female than in male rat liver microsomes.

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Drug discovery is a process involving multiple disciplines and interests. During the research phase of drug discovery, usually a large number of compounds are evaluated for biological activity and toxicological potential in animal species. Various types of problems with respect to pharmacodynamics, pharmacokinetics, and toxicity are commonly encountered at this stage. Drug metabolism, as a discipline participating in a drug discovery team, can play an important role in identifying factors underlying the problems, facilitate the optimal selection of compounds for further development, provide information on metabolites for possible improvement in drug design, and contribute to the identification of the appropriate animal species for subsequent toxicity testing. During the process of evaluating oxytocin receptor antagonists for further development for treatment of preterm labor, in vivo and in vitro drug metabolism studies conducted in rats, dogs, and monkeys contributed to the selection of L-368,899 as the development candidate on the basis of pharmacokinetic and metabolism observations. The presence of active N-demethylated metabolites of two other equipotent compounds in rats and dogs was found to be the major factor responsible for the discrepancy between oral bioavailability and efficacies observed for these 2 compounds. For L-368,899, a compound that demonstrated 20-40% oral bioavailability in rats, dogs, and chimpanzees, extensive first-pass metabolism rather than absorption was determined as the major factor responsible for the poor bioavailability (< 1%) in rhesus monkeys. In vitro metabolism studies with hepatic microsomes from rats, dogs, monkeys, and humans substantiated the conclusion that the rate of hepatic metabolism of L-368,899 in monkeys is faster than in the other species.

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Racemic sulfonylated 2,5-diaryltetrahydrofuran [L-668,750, (+-)-trans-2-[3-methoxy-5-(2-hydroxy)ethylsulfonyl-4-n-propoxy]-p henyl-5-(3,4,5-trimethoxyphenyl)-tetrahydrofuran, I] is a potent, specific and orally active platelet-activating factor (PAF) receptor antagonist. Its (-)-(2S,5S) enantiomer [L-680,573, (S)-I] exhibited higher PAF antagonistic potency than the (+)-(2R,5R) enantiomer [L-680,574, (R)-I] in vitro and in animal models. For assay of drug concentrations in plasma of rats dosed intravenously or orally with tritium-labeled I, we have developed a high-performance liquid chromatographic (HPLC) method which directly resolved the two enantiomers. The column contained alpha 1-acid glycoprotein as the chiral stationary phase and was eluted with phosphate buffer, methanol and ethanol at neutral pH. The concentration of each enantiomer in the plasma was then determined by reverse isotope dilution assay. Results showed that the plasma clearance rate of the more potent (S)-I enantiomer was more than ten-fold faster than that of the (R)-I enantiomer; the enantioselective clearance resulted in nearly ten-fold higher concentrations of the latter in plasma at all time points regardless of the dosing route. This paper describes the HPLC chiral resolution method and its application in plasma analysis.

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Ronidazole protein-bound adducts were generated by the in vitro anaerobic incubation of [2-methylene-14C]ronidazole with microsomes from the livers of male rats. Acid hydrolysis of the protein adducts yielded an imidazole ring fragment bearing the radiolabel and an amino acid residue derived from the proteins. This fragment has been identified as carboxymethylcysteine by co-chromatography of the amino acid and its dansyl derivative with known standards under a variety of conditions. The carboxymethylcysteine was estimated to represent at least 15% of the radioactivity derived from the protein-bound adducts and provides unequivocal evidence that nucleophilic attack by protein cysteine thiols occurred at the 2-methylene position of ronidazole.

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In vivo experiments were conducted with ronidazole radiolabelled in the 2-14CH2-, 4,5-14C-, N-14CH3- and 4-3H-positions. The hepatic protein-bound residues, assessed by the radioactivity of exhaustively washed protein samples, were independent of the radiolabel position and occurred with 4-3H loss (greater than 80%) in excellent agreement to previous results obtained in vitro with anaerobic incubations of liver microsomes (Miwa et al., Chem. Biol. Interact., 41 (1982) 297). HPLC analysis of acid hydrolyzed in vivo protein-bound residues, obtained from [2-14CH2] ronidazole, produced a radiochromatographic profile which was virtually identical to that obtained from a similarly treated in vitro sample. Moreover, almost quantitative (76-96%) liberation of radiolabelled methylamine was obtained from hydrolysates of in vivo and in vitro residue samples formed from [N-14CH3] ronidazole. With 4,5-ring labeled ronidazole the distribution of total radioactivity of the protein hydrolysate on cation exchange resin and the fraction of the residue recovered as oxalic acid were nearly identical for the in vivo and in vitro products. We interpret these data to indicate that ronidazole alkylates proteins with retention of most of the carbon framework of the molecule, in vivo. It is also concluded that the in vitro model, previously used to examine the mechanism of protein alkylation, accurately reflects the salient process initially occurring in the intact animal during the formation of protein-bound residues of this drug.

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When ronidazole (1-methyl-5-nitroimidazole-2-methanol carbamate) is reduced by either dithionite or rat liver microsomal enzymes in the presence of cysteine, ronidazole-cysteine adducts can be isolated. Upon reduction with dithionite ronidazole can react with either one or two molecules of cysteine to yield either a monosubstituted ronidazole-cysteine adduct substituted at the 4-position or a disubstituted ronidazole-cysteine adduct substituted at both the 4-position and the 2-methylene position. In both products the carbamoyl group of ronidazole has been lost. The use of rat liver microsomes to reduce ronidazole led to the formation of the disubstituted ronidazole-cysteine adduct. These data indicate that upon the reduction of ronidazole one or more reactive species can be formed which can bind covalently to cysteine. The proposed reactive intermediates formed under these conditions may account for the observed binding of ronidazole to microsomal protein and the presence of intractable drug residues in the tissues of animals treated with this compound. They may also account for the mutagenicity of this compound in bacteria.

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When radioactive 1-methyl-5-nitroimidazole-2-methanol carbamate, ronidazole, labeled at the 4,5-ring positions was administered orally to germ-free and conventional rats, a much larger fraction of the radioactivity was excreted in the feces of the conventional animals. Determination of the total radioactive residues present in the carcass, blood, plasma, liver, fat and kidney 5 days after dosing indicated that the carcass of the germ-free animals contained a greater quantity of residue than that of conventional rats. On the other hand, the blood of the conventional animals contained a much higher level of radioactivity than that of the germ-free animals. These results show that while the microflora influence the distribution of the drug their presence is not obligating for the formation of persistent tissue residues in rats dosed with ronidazole.

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The user of cycloheximide to distinguish between covalently-bound drug residues in animals and residues due to the incorporation of drug fragments into endogenous molecules was explored. The results indicated that cycloheximide prevented the absorption of both glycine and ronidazole from the gastro-intestinal tract, an effect that complicates its use in the characterization of drug residues in animals.

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