AN EVALUATION OF CYTOCHROME-P450 ISOFORM ACTIVITIES IN THE FEMALE DARK AGOUTI (DA) RAT - RELEVANCE TO ITS USE AS A MODEL OF THE CYP2D6 POOR METABOLIZER PHENOTYPE

The female dark agouti (DA) rat lacks CYP2D1, the equivalent enzyme in the rat to human CYP2D6 (debrisoquine hydroxylase), and shows impaired metabolism of a number of CYP2D6 substrates. However, from the data available in the literature it is not entirely clear whether the enzyme deficiency in the DA rat is restricted to CYP2D1, and whether factors such as age and substrate concentration are important determinants of interstrain differences in the activity of this enzyme. Given that the female DA rat is used as a model of the human CYP2D6 poor metaboliser phenotype, there is a need for a systematic evaluation of the P450 activities in the DA rat, and of its suitability as a model of the PM phenotype. In the present study metoprolol was used as a probe substrate to investigate CYP2D1 activity since both the alpha-hydroxylation and O-demethylation of this drug are catalysed by CYP2D6 in man. Formation of alpha-hydroxymetoprolol (AHM) and O-demethylmetoprolol (ODM) was 10- and 2.5-fold lower in liver microsomes from female DA rats compared with microsomes from age-matched female Wistar rats, the latter representing the extensive metaboliser strain. Kinetic analysis suggested that in both strains of rat both the alpha-hydroxylation and O-demethylation of metoprolol were catalysed by more than one enzyme. By using quinine as a specific inhibitor of the enzyme, CYP2D1 was identified as an intermediate affinity site in the Wistar strain and was shown to have impaired activity in the DA strain. The activities of lower and higher affinity sites were similar in the two strains. Thus, the only difference between the two strains with respect to both routes of metoprolol metabolism appeared to be in the activity of CYP2D1. Interstrain differences were found to be highly dependent on the choice of substrate concentration, being more marked at lower concentrations. We have also investigated the metabolism of a number of probe compounds for some of the other P450 isoforms commonly involved in drug metabolism to determine the selectivity of the deficiency in the DA strain. p-Nitrophenol hydroxylation and erythromycin N-demethylation were catalysed at higher rates by DA than by Wistar liver microsomes, indicating higher levels of activity of CYP2E1 and CYP3A in the former strain. Felodipine oxidation, tolbutamide hydroxylation and both the hydroxylation and N-demethylation of S-mephenytoin were catalysed at similar rates by microsomes from the two strains, indicating similar activities of enzymes in the CYP2C and CYP3A families. However, both the hydroxylation and N-demethylation of R-mephenytoin were impaired in the DA strain. This indicates that at least one other isoform of P450, thought also to be a member of the CYP2C or CYP3A families, in addition to CYP2D1 is deficient in the DA strain. Our findings indicate that whilst the female DA rat could be used as a preliminary screen to identify CYP2D6 substrates, because of interspecies differences in metabolism it could not be used to provide quantitative information regarding the contribution of CYP2D6 to an oxidation in man. In addition, a small number of false positives would be identified owing to other enzyme deficiencies; no false negatives would be expected. Comparisons between strains should be performed using female, age-matched animals and low substrate concentrations.