Utility of in vitro drug metabolism data in predicting in vivo metabolic clearance.

Fluvoxamine is a potent inhibitor of cytochrome P4501A2

Objectives
The effect of aging on drug metabolism in humans has not yet been completely described.

Methods
Two hundred twenty-six patients with equal histopathologic conditions were investigated. The cytochrome P450 contents in the liver biopsy samples, the plasma antipyrine clearance rates after oral administration and, as an independent control of vitality, serum testosterone levels were determined.

Results
Cytochrome P450 content in subjects from 20 to 29 years of age was 7.2 ± 2.6 nmol · gm−1, increased during the fourth decade (+ 7.2%, p = NS), declined after 40 years (−16%, p < 0.01) to a level that remained unaltered up to 69 years, and declined further after 70 years (−32%, p < 0.001). The antipyrine (phenazone) clearance rate in young subjects was 46.4 ± 18.5 ml · min−1, remained unaltered during the fourth decade, and declined after 40 years by a rate of 0.34 ml · min−1 per year toward old age (−29%, p < 0.001). The half-life in young subjects was 9.5 ± 2.0 hours and increased after 30 years toward old age (+26%, p < 0.001). The volume of antipyrine distribution, 0.46 ± 0.12 L · kg−1 in young subjects, decreased after 30 years (−11%). In line with the testosterone content, the decrease in drug metabolism was equal in both sexes.

Conclusion
This study shows a reduction of in vitro and in vivo drug metabolism with age in humans. The data suggest that at least three age groups—young, middle-aged, and elderly—should be included in the evaluation of the pharmacokinetics of a new drug. The reduction of drug metabolism (−30%) after 70 years of age indicates that care is needed in the prescription of drugs for elderly subjects.

Clinical Pharmacology & Therapeutics (1997) 61, 331–339; doi:

Age and cytochrome P450-linked drug metabolism in humans: An analysis of 226 subjects with equal histopathologic conditions

Purification and characterization of the human liver cytochromes P-450 involved in debrisoquine 4-hydroxylation and phenacetin O-deethylation, two prototypes for genetic polymorphism in oxidative drug metabolism.

Pharmaceutical industry investigators routinely evaluate the potential for a new drug to modify cytochrome p450 (p450) activities by determining the effect of the drug on in vitro probe reactions that represent activity of specific p450 enzymes. The in vitro findings obtained with one probe substrate are usually extrapolated to the compound's potential to affect all substrates of the same enzyme. Due to this practice, it is important to use the right probe substrate and to conduct the experiment under optimal conditions. Surveys conducted by reviewers in CDER indicated that the most common in vitro probe reactions used by industry investigators include the following: phenacetin O-deethylation for CYP1A2, coumarin 7-hydroxylation for CYP2A6, 7-ethoxy-4-trifluoromethyl coumarin O-dealkylation for CYP2B6, tolbutamide 4'-hydroxylation for CYP2C9, S-mephenytoin 4-hydroxylation for CYP2C19, bufuralol 1'-hydroxylation for CYP2D6, chlorzoxazone 6-hydroxylation for CYP2E1, and testosterone 6 beta-hydroxylation for CYP3A4. We reviewed the validation information in the literature on these reactions and other frequently used reactions, including caffeine N3-demethylation for CYP1A2, S-mephenytoin N-demethylation for CYP2B6, S-warfarin 7'-hydroxylation for CYP2C9, dextromethorphan O-demethylation for CYP2D6, and midazolam 1'-hydroxylation for CYP3A4. The available information indicates that we need to continue the search for better probe substrates for some enzymes. For CYP3A4-based drug interactions it may be necessary to evaluate two or more probe substrates. In many cases, the probe reaction represents a particular enzyme activity only under specific experimental conditions. Investigators must consider appropriateness of probe substrates and experimental conditions when conducting in vitro drug interaction studies and when extrapolating the results to in vivo situations.

Evaluation of cytochrome P450 probe substrates commonly used by the pharmaceutical industry to study in vitro drug interactions.

Biphasic O-deethylation of phenacetin and 7-ethoxycoumarin by human and rat liver microsomal fractions.

Antipyrine is oxidised to three main metabolites in man. There is evidence that the different metabolites are products of different forms of cytochrome P-450. The effect of rifampicin administration for two weeks on the rates of formation of these metabolites was investigated in healthy volunteers. Rifampicin increased antipyrine clearance and shortened its half-life. Two weeks after stopping rifampicin the induction had largely been reversed. Clearance to all three metabolites was increased by rifampicin. Clearance to 3-hydroxymethylantipyrine was increased from 7.8±0.9 ml/min to 13.3±1.3 ml/min, to norphenazone from 5.8±0.6 ml/min to 19.3±2.1 ml/min and to 4-hydroxyantipyrine from 14.3±2.2 ml/min to 21.9±3.9 ml/min. Thus clearance to norphenazone was increased to a much greater extent than to either of the other two metabolites. It is concluded that this provides evidence for the involvement of at least two different forms of cytochrome P-450 in antipyrine metabolism in man.

Differential induction of antipyrine metabolism by rifampicin.

The effects of liver disease and environmental factors on hepatic microsomal cytochrome P-450 content, NADPH-cytochrome c reductase (reductase) activity and aryl hydrocarbon hydroxylase (AHH) activity have been simultaneously investigated in 70 patients undergoing diagnostic liver biopsy. The activity of reductase was not significantly affected by the presence of liver disease or any of the environmental factors studied. Cytochrome P-450 content decreased with increasing severity of liver disease whereas AHH activity was only significantly reduced in biopsies showing hepatocellular destruction. None of the parameters of monooxygenase activity varied significantly with the age or sex of the patients. Alcohol excess was associated with decreased cytochrome P-450 content and AHH activity and this effect was independent of the histological status of the biopsy. Both high caffeine intake and cigarette smoking increased AHH activity in the absence of any change in cytochrome P-450 content. There was a positive correlation between the number of meat meals eaten per week and cytochrome P-450 content. Chronic treatment with enzyme-inducing anticonvulsants appeared to increase both cytochrome P-450 content and AHH activity. Despite differential effects of liver disease and environmental influences on cytochrome P-450 content and AHH activity there was a highly significant correlation between the two parameters. The results of the present study correlate well with the known effects of disease and environment on drug metabolism in vivo.

Influence of liver disease and environmental factors on hepatic monooxygenase activity in vitro.

The selective activation of cytochrome P-450 dependent microsomal hydroxylases in human and rat liver microsomes.

A radiometric high-pressure liquid chromatography assay for the simultaneous determination of the three main oxidative metabolites of antipyrine in studies in vitro.

M. J. Brodie

Papers

Biphasic O-deethylation of phenacetin and 7-ethoxycoumarin by human and rat liver microsomal fractions.

Differential induction of antipyrine metabolism by rifampicin.

Influence of liver disease and environmental factors on hepatic monooxygenase activity in vitro.

The selective activation of cytochrome P-450 dependent microsomal hydroxylases in human and rat liver microsomes.

A radiometric high-pressure liquid chromatography assay for the simultaneous determination of the three main oxidative metabolites of antipyrine in studies in vitro.