Experimental design of phenotyping probe drugs with emphasis on CYP1A2 : their use in studies on genetic and environmental regulation of drug metabolism

Sammanfattning: Assessment of the cytochrome (CYP) P450 enzyme activity or phenotyping is a method to characterise the real activity of an enzyme by the use of a marker probe drug. The information of the phenotype is of clinical relevance because it reflects the combined effects of genetic, environmental e.g. smoking and endogenous factors such as disease on CYP activity. The overall aim of this thesis was to evaluate probe drugs to be used in phenotyping studies of the main drug metabolising CYP enzymes. A special emphasis was to characterise the interindividual variation in CYP1A2 activity with a robust phenotyping method. Caffeine has been used to assess CYP1A2 activity. A single oral dose of 100 mg caffeine followed by a 4-hour blood and saliva sample as well as a urinary collection was validated. Caffeine oral clearance (CL) in plasma correlated with that assessed in saliva (rs=0.97). The total oral caffeine CL correlated with the paraxanthine / caffeine concentration ratio in plasma (rs=0. 84), saliva (rs=0. 82) and the urinary ratio of N-3-demethylated metabolites / caffeine collected for 8 or 24 hours (rs=0.63-0.70). The use of a 36-hour caffeine abstinence period was appropriate to avoid the influence of caffeine ingested before the study. Melatonin metabolism is catalysed by CYP1A2. It has not been validated as a probe for CYP1A2. After a single oral dose, the apparent melatonin CL correlated with the caffeine CL (rs=0.748). A plasma sample obtained 1.5 hours after melatonin intake correlated also with caffeine CL (rs=-0.62). These results indicate that melatonin may serve as an alternative to caffeine in phenotyping the CYP1A2 activity. Fluvoxamine is a strong inhibitor of CYP1A2, but it also blocks the metabolism of other CYPs in a dose-dependent manner. The administration of very low doses (10 and 25 mg) of fluvoxamine could therefore possibly separate this inhibitory effect on different CYPs. Caffeine and omeprazole were used as probe drugs for CYP1A2 and CYP2C19 activity. Their phenotype indices were inhibited to a similar extent (80 %) on the high dose. With the lower dose the results were similar, but with a less pronounced inhibition (40 %). These results show that fluvoxamine is not useful as a selective CYP inhibitor. With these low doses, CYP2D6 was not important for the disposition of fluvoxamine, as shown by no difference between the dose-adjusted fluvoxamine concentrations in poor and extensive metabolisers of CYP2D6. Simultaneous administration of specific probe drugs for the assessment of CYP1A2 (caffeine), CYP2C9 (losartan), CYP2C19 (omeprazole), CYP2D6 (debrisoquine) and CYP3A4 (quinine) activity was validated in a phenotyping method ("cocktail") with as few sampling occasions as possible. The administration of the cocktail showed no analytical interactions, but the metabolism of debrisoquine was inhibited by some of the other drugs. Quinine was however, ingested after the elimination of debrisoquine. Therefore, debrisoquine may be given before the other drugs to avoid interaction and ensure its elimination in future studies. The whole procedure is simple and requires only 2 blood samples and 2 urine collections. The method may be applied in drug development to study the effects (induction or inhibition) of a new chemical entity on individual CYPs. Environmental factors such as smoking and use of oral contraceptives were shown to heavily induce and inhibit the caffeine metabolism (CYP1A2 activity), respectively. No relationship between phenotype and genotype was established based on 3 mutations in the promoter region of the CYP1A2 gene. The mutations were all linked together suggesting that haplotypes may be more important than a single nucleotide polymorphism in exploring the genetic influence on CYP1A2 activity and importance in drug interactions.