THE ROLE OF CYP 3 A 4 / 5 IN ALPRAZOLAM METABOLISM

Cytochrome P450 3A (CYP3A) enzyme family is involved in the metabolism of about 50 % of all drugs in clinical use. Among CYP3A, CYP3A4 and CYP3A5 are the major enzymes in adults; CYP3A5 is polymorphic and primarily expressed in black populations. CYP3A5 may therefore contribute significantly to the metabolism of CYP3A substrates in African populations. The impact of CYP3A5 expression on drug metabolism by CYP3A is fairly unknown. Therefore, a tool for assessment of both CYP3A4 and CYP3A5 using a single probe drug is of interest. Overlapping substrate specificities between CYP3A4 and CYP3A5 have made it difficult to differentiate the two enzymes using probe drugs. However, experimental in vitro data have shown a preference for formation of 4hydroxyalprazolam by CYP3A4 and that of α-hydroxyalprazolam by CYP3A5. The aim of the present thesis was to investigate the role of CYP3A4 and CYP3A5 in alprazolam metabolism as well as to evaluate if alprazolam may serve as a probe drug for these two enzymes. A liquid chromatography-mass spectrometry method for determination of alprazolam and the two metabolites 4and α-hydroxyalprazolam was developed. When using this method we were able to analyze samples for pharmacokinetic and metabolism studies. The parent drug/metabolite AUC ratio has been proven to reflect the enzymatic activity; metabolic ratio calculated from a single plasma sample can be used in phenotyping studies. Alprazolam metabolism was shown to be catalyzed by both CYP3A4 and CYP3A5 in vitro. Formation rates of 4-hydroxyalprazolam in human liver microsomes and recombinant CYP3A4 and CYP3A5 was higher than that of α-hydroxyalprazolam, confirming that 4-hydroxylation is the most important metabolic pathway of alprazolam. The relative formation of α-hydroxyalprazolam was 3-fold higher for recombinant CYP3A5 compared to CYP3A4. We thus have confirmed that CYP3A5 catalyses alprazolam metabolism, in vitro. We then evaluated the role of CYP3A4 and CYP3A5 in alprazolam metabolism in vivo in Tanzanian and Swedish healthy subjects who express CYP3A5 and those who do not. As there were no significant differences in MR between subjects with different CYP3A5 genotypes it shows that CYP3A5 plays no significant role in alprazolam metabolism. Our results show that CYP3A4 is the major enzyme in the metabolism of alprazolam in vivo. LIST OF PUBLICATIONS I. Annika Allqvist, Agneta Wennerholm, Jan-Olof Svensson, Rajaa A. Mirghani Simultaneous quantification of alprazolam, 4and αhydroxyalprazolam in plasma sample using liquid chromatography mass spectrometry Journal of Chromatography B, (2005) 814: 127-131 II. Agneta Wennerholm, Annika Allqvist, Jan-Olof Svensson, Lars L. Gustafsson, Rajaa A. Mirghani, Leif Bertilsson Alprazolam as a probe for CYP3A using a single blood sample: pharmacokinetics of parent drug, and of αand 4-hydroxy metabolites in healthy subjects Eur J Clin Pharmacol, (2005) 61: 113-118 III. Annika Allqvist, Jun Miura, Leif Bertilsson, Rajaa A. Mirghani Inhibition of CYP3A4 and CYP3A5 catalyzed metabolism of alprazolam and quinine by ketoconazole as racemate and four different enantiomers Eur J Clin Pharmacol, (2007) 63: 173-179 IV. Annika Allqvist, Jun Miura, Eleni Aklilu, Mary Jande, Filip Josephson, Margarita Mahindi, Rajaa A Mirghani, Agneta Wennerholm, Jane Sayi, Jolanta Widén, Ulf Diczfalusy, Lars L. Gustafsson and Leif Bertilsson The role of CYP3A4/5 in alprazolam metabolism in Swedes and Tanzanians