Characterization and partial purification of the rat and human enzyme systems active in the reduction of N-hydroxymelagatran and benzamidoxime.

The enzymic basis for intracellular reduction of N-hydroxylated amidines to their corresponding amidines, and hydroxylamines to their corresponding amines, is unknown. The hydroxylated amidines can be used as prodrug moieties, and an understanding of the enzyme system active in the reduction can contribute to more efficient drug development. In this study, we examined the properties of this enzyme system using benzamidoxime and N-hydroxymelagatran as substrates. In rats and humans, the hepatic enzyme system was localized in mitochondria as well as in microsomes, using preferably NADH as cofactor. Potassium cyanide, N-methylhydroxylamine, p-hydroxymercuribenzoate, and desferrioxamine were efficient inhibitors, whereas typical cytochrome P450 (P450) inhibitors were ineffective. In rats, the highest specific activity was found in liver, adipose tissue, and kidneys, whereas in humans, the specific activity in the preparations of adipose tissue examined was lower. A sex difference was observed in rat liver, where 4-fold higher activity was seen in microsomes from female rats. No gender differences were present in any other tissue investigated. Partial purification of the hepatic system was achieved using polyethylene glycol fractionation followed by Octyl Sepharose chromatography at low detergent concentrations, whereas the enzyme was denatured after complete solubilization. The unique appearance of the enzyme activity in adipose tissue, together with the cyanide sensitivity and the failure of typical P450 inhibitors to impede the reaction, indicates that the enzyme system active in reduction of benzamidoxime and N-hydroxymelagatran formation is not of cytochrome P450 origin, but likely consists of an NADH-dependent electron transfer chain with a cyanide-sensitive protein as the terminal component.

Time-dependent pharmacokinetics and drug metabolism of atovaquone plus proguanil (Malarone) when taken as chemoprophylaxis.

OBJECTIVE: To determine the pharmacokinetic profiles of atovaquone (ATO), proguanil (PROG) and its active metabolite cycloguanil (CYCLO) with respect to possible accumulation and kinetic interaction upon repeated dosing with Malarone. METHODS: Thirteen healthy volunteers first received a single dose and then after 1 week, repetitive daily doses of Malarone (one tablet) for 13 days. For analysis of plasma drug concentrations, blood samples were collected at regular intervals over 8 days after a single dose and over 12 days after the last day of multiple dosing. Single-dose and steady-state pharmacokinetic parameters were determined for each individual. Genotyping of the gene coding for CYP2C19, a major enzyme catalyzing PROG metabolism, was performed using polymerase chain reaction, and in vitro enzyme kinetic experiments were carried out to study the possible effect of ATO on the catalytic activities of CYP2C19 and 3A4 using fluorometric assays. RESULTS: For ATO, the ratio of the area under the concentration-time curve (AUC) during the last dose interval to the AUC after the single dose (AUC(0- tau)/AUC(0- infinity)) was found to be 0.90 [95% confidence interval (CI) 0.56, 1.24] indicating absence of undue accumulation. AUC(0- tau), and peak plasma concentration at steady state (C(max,ss)) values were, however, threefold lower than those reported in human immunodeficiency virus-infected subjects after 12 multiple daily doses of 250 mg ATO alone. Four volunteers, with mean CYCLO/PROG AUC(0-tau) of 0.03 (-0.23, 0.09) were classified as poor metaboliser (PM) phenotypes. There was a significant increase in the AUC of PROG at steady state with a PROG AUC(0-tau)/AUC(0-infinity) ratio of 1.38 (1.07, 1.69) in extensive metaboliser (EM) phenotypes. CYCLO/PROG AUC ratios were significantly lower 0.67 (0.54, 0.81) at steady state than that after the first single dose in EM phenotypes. The in vitro kinetic experiments on recombinant enzymes (CYP2C19 and CYP3A4) suggested a possible inhibition of catalytic activity of CYP3A4 by ATO. CONCLUSIONS: There was no unexpected accumulation of ATO following repeated administrations of the combination. In EM phenotypes, PROG elimination was reduced at steady state. Also, at steady state, either the elimination of CYCLO was increased or its formation clearance decreased the latter possibly by inhibition of CYP3A4 by ATO.