Characteristics of cytochrome P450-catalysed drug metabolism with focus on a black Tanzanian population

The cytochrome P450 (CYP) enzymes display interindividual and interethnic variability that may alter the disposition of substances metabolised by these enzymes. Both genetic and environmental factors such as concomitant drug use and concurrent diseases influence the activities of these enzymes. The overall aim of this thesis was to study variability in drug metabolism catalysed by cytochrome P450 enzymes in a Tanzanian population and to assess the consequences of such variability.

In a phenotyping study in Tanzanians, we detected lower metabolic activity of CYP2D6 in Tanzanians compared to Caucasians. The low capacity could not be explained by the partially or fully detrimental CYP2D6 gene mutations analysed for. Moreover, we found that none of the poor metabolisers of debrisoquine was homozygous for defective CYP2D6 genes.

A novel CYP2D6 allele (CYP2D6*29) was found at a high allele frequency in the Tanzanian population. The presence of this allele significantly caused a diminished rate of debrisoquine metabolism in vivo. Using the CYP2D6 substrate bufuralol, it was shown that CYP2D6.29 had only 26% of the catalytic activity of the wild-type enzyme (CYP2D6.1) in a mammalian cell expression system and also lower activity (63% of CYP2D6.1) when debrisoquine was used as substrate.

We studied the effect of the African-specific CYP2D6*17 and *29 alleles in healthy Tanzanian subjects on the metabolism of four CYP2D6 probe drugs (codeine, debrisoquine, dextromethorphan, metoprolol). Carriers of CYP2D6*17 had significantly decreased rate of metabolism for two (debrisoquine and dextromethorphan) of the four probe drugs and CYP2D6*29 caused a reduced metabolism compared with CYP2D6*1 and *2 for dextromethorphan and metoprolol but not for codeine and debrisoquine.

The effect of the antimalarial drug amodiaquine on the activities of four P450 enzymes and the recovery of any such effect was studied in healthy Swedish subjects. We found that amodiaquine, its desethylated metabolite, or both inhibited the CYP2D6 (debrisoquine 4-hydroxylation) and CYP2C9 (losartan oxidation) enzymes. This effect was selective affecting neither the CYP2C19 (omeprazole 5-hydroxylation) nor CYP1A2 (caffeine N3-demethylation) enzymes. The inhibitory effect did not persist one week after drug intake.

We validated a novel liquid chromatography-mass spectrometry method, which enabled us to quantify alprazolam, 4-hydroxyalprazolam and alpha-hydroxyalprazolam in all healthy subjects up to at least 24 hours after drug intake. The plasma concentration ratios (alprazolam/respective metabolite) of samples collected between 1 and 48 hours correlated significantly with the ratios of area under curve values of alprazolam/4-hydroxyalprazolam and alprazolam/alpha-hydroxyalprazolam. Hence, we suggest that it is possible to estimate alprazolam 4- and alpha-hydroxylation activities in subjects with a single plasma sample.

In summary, two African-specific CYP2D6 alleles encode enzymes that exhibit altered substrate specificity compared to the functional enzyme both in vivo and in vitro. This may preclude extrapolation of results from one specific CYP2D6-drug to another. The antimalarial drug amodiaquine inhibited the metabolism of the CYP2D6 and CYP2C9 enzymes in vivo. The 4- and á-hydroxylations of alprazolam can be assessed using a single plasma sample, which makes the method well suited for studies of CYP3A activity in larger populations.

List of scientific papers

I. Wennerholm A, Johansson I, Massele AY, Lande M, Alm C, Aden-Abdi Y, Dahl ML, Ingelman-Sundberg M, Bertilsson L, Gustafsson LL (1999). Decreased capacity for debrisoquine metabolism among black Tanzanians: analyses of the CYP2D6 genotype and phenotype. Pharmacogenetics. 9(6): 707-14.
https://pubmed.ncbi.nlm.nih.gov/10634133

II. Wennerholm A, Johansson I, Hidestrand M, Bertilsson L, Gustafsson LL, Ingelman-Sundberg M (2001). Characterization of the CYP2D6*29 allele commonly present in a black Tanzanian population causing reduced catalytic activity. Pharmacogenetics. 11(5): 417-27.
https://pubmed.ncbi.nlm.nih.gov/11470994

III. Wennerholm A, Dandara C, Sayi J, Svensson JO, Abdi YA, Ingelman-Sundberg M, Bertilsson L, Hasler J, Gustafsson LL (2002). The African-specific CYP2D617 allele encodes an enzyme with changed substrate specificity. Clin Pharmacol Ther. 71(1): 77-88.
https://pubmed.ncbi.nlm.nih.gov/11823760

IV. Wennerholm A, Pihlsgard M, Rais M, Bertilsson L, Gustafsson LL (2003). Amodiaquine, its desethylated metabolite, or both inhibit the metabolism of debrisoquine (CYP2D6) and losartan (CYP2C9) in vivo. [Manuscript]

V. Wennerholm A, Allqvist A, Svensson JO, Gustafsson LL, Bertilsson L, Mirghani RA (2003). Alprazolam as a probe for CYP3A in man using a single blood sample: Development of an LC/MS method and kinetics of parent drug, alpha- and 4-hydroxy metabolites in healthy subjects. [Submitted]