Adedayo Adedoyin

OBJECTIVES: To determine whether the probe drugs caffeine, chlorzoxazone, dapsone, debrisoquin (INN, debrisoquine), and mephenytoin can be simultaneously administered as a metabolic cocktail to estimate in vivo cytochrome P450 (CYP) and N-acetyltransferase enzyme activities. METHODS: Fourteen healthy nonsmoking male volunteers (mean age +/- SD, 21.6 +/- 2.2 years) received 100 mg caffeine, 250 mg chlorzoxazone, 100 mg dapsone, 10 mg debrisoquin, and 100 mg mephenytoin individually and in four and five-drug combinations in a randomized manner using a 7 x 7 Latin square. Each drug or drug combination was given orally after an overnight fast, with a minimum 1-week washout between administrations. In each session, urine was collected from 0 to 8 hours and plasma was obtained at 4 and 8 hours after drug administration. Plasma and metabolite concentrations were used to estimate phenotypic trait measures for the efficiency of each drug's metabolism. RESULTS: The phenotypic indexes determined for caffeine, chlorzoxazone, dapsone, debrisoquin, and mephenytoin were not significantly different when given alone than when given in combination. The median percentage change of the trait measures observed during administration of all five compounds compared with individual administration ranged from -10.7% for the 6-hydroxychlorzoxazone to chlorzoxazone plasma ratio to +2.2% for the debrisoquin recovery ratio. CONCLUSIONS: The results of this study show that caffeine, chlorzoxazone, dapsone, debrisoquin, and mephenytoin in low doses can be simultaneously administered without metabolic interaction. This cocktail approach can thus simultaneously provide independent in vivo phenotypic measures for multiple CYP enzymes and N-acetyltransferase.

The recent guidance on "Safety Testing of Drug Metabolites" issued by the U.S. Food and Drug Administration, Center for Drug Evaluation and Research (CDER) has highlighted the importance of identifying and characterizing drug metabolites as early as possible in drug discovery and development. Furthermore, upon identifying significant circulating metabolites in human plasma, it has become important to demonstrate that these metabolites are present at an equal or greater exposure level (area under the curve, AUC) in any one of the preclinical species used in safety testing. Frequently, synthetic standards of metabolites are not available, and hence, obtaining their AUC values can be a challenge. In this report, we demonstrate how combinations of nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography/ultraviolet/mass spectrometry (LC/UV/MS), and plasma pooling methods were used to obtain reliable AUC values of metabolites present in the plasma of preclinical species from short-term safety studies. Plasma pooling methods were compared to the traditional approaches of obtaining quantitative information on the levels of circulating metabolites in preclinical species. The exposure values obtained via sample pooling were comparable to those obtained by traditional methods of analyzing samples individually. In the absence of synthetic chemical standards, calculations of AUC values of metabolites, using either sample pooling or traditional approaches, were achieved through the use of UV detectors. In cases where the UV properties of metabolites were significantly different from their parent compounds, NMR was used as a quantitative tool to obtain exposure values. NMR was found to be useful in quantitating biologically produced metabolites, which could subsequently be used as reference compounds for further quantitative studies. The limitations of UV detectors to obtain exposure estimates are discussed. A practical solution is presented that will enable us to obtain a quantitative assessment of metabolite exposure in humans and coverage in toxicology species, hence, circumventing the use of radiolabeled compounds or authentic chemically synthesized standards of metabolites.

BACKGROUND AND OBJECTIVES: Drug metabolism is influenced by liver disease because of the central role that the liver plays in metabolic activities in the body. However, it is still unclear how activities of specific drug-metabolizing enzymes are influenced by the presence and severity of liver disease. As a consequence, alteration in metabolism of specific drugs cannot be easily predicted or appropriate dosage adjustment recommendations made. METHODS: The activities of cytochromes P450 (CYP) 2C19 and 2D6 were investigated in a group of patients with mild or moderate liver disease (n = 18) and a group of healthy control subjects (n = 10). The disposition of racemic mephenytoin for CYP2C19 and debrisoquin for CYP2D6 were characterized in plasma and urine samples collected over 192 hours. RESULTS: The elimination of S-mephenytoin was severely reduced among patients with liver disease, resulting in a 79% decrease in plasma clearance for all patients combined. This reduction was related to the severity of disease, patients with moderate disease being affected more severely than patients with mild disease. Similar differences were observed in the urinary excretion of 4'-hydroxymephenytoin metabolite. By contrast, there was no effect on the disposition of R-mephenytoin or debrisoquin. CONCLUSION: These results show selectivity in the effect of liver disease on activities of specific metabolizing enzymes, CYP2C19 being more sensitive than CYP2D6. They suggest that recommendations for modification in drug dosage in the presence of liver disease should be based on knowledge of the particular enzyme involved in metabolism of the drug. The results emphasize the need for further studies of each specific drug-metabolizing enzyme in the presence of liver disease.