¹Discovery Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Groton, CT, USA

Correspondence: Dr MB Fisher, Discovery Pharmacokinetics, Dynamics and Metabolism, Pfizer, Inc., PGRD, Eastern Point Road, Groton, CT 06340, USA. Tel: +1 860 715 2421; Fax: +1 860 715 4695; E-mail: michael_fisher@groton.pfizer.com

We have followed with great interest the growing number of publications reporting the varying degrees of importance that cytochrome P450 3A5 (CYP3A5) plays in human drug metabolism. In vitro studies have identified a growing number of substrates for which CYP3A5 contributes significantly to their metabolism. Williams et al¹ report that the contribution of CYP3A5 to midazolam hydroxylation can be readily measured due to the high turnover of midazolam by the enzyme. They also report significant CYP3A5 in vitro clearance values for alprazolam and triazolam, both benzodiazepines, diltiazem, a benzothiazepine, and tamoxifen, an antiestrogen commonly used in therapy for breast cancer. When these in vitro data are extrapolated to an in vivo outcome, the models confirm a significant pharmacokinetic difference between patients homozygous for the CYP3A5^*3 allele (expressing very little to no CYP3A5) and patients homozygous for the CYP3A5^*1 allele (expressing significant levels of CYP3A5).²

To date, there appears to be a growing contingent of researchers who are questioning the metabolic role of CYP3A5 relative to cytochrome P450 3A4 (CYP3A4) in vivo based on clinical studies. For example, Wong et al³ report that an observed interpatient variability in midazolam and vinorelbine clearances in chemotherapy patients cannot be related to the CYP3A5 genotype. Fukada et al⁴ claim to show evidence that large intragroup variations in nifedipine clearance in healthy male Japanese subjects have no relation to the CYP3A5 genotype, and thus conclude that the overall relevance of the CYP3A5 polymorphism in clinical trials needs to be re-evaluated. Furthermore, in a very thorough review of the literature, Thummel⁵ seems to imply that the previously assumed importance of the CYP3A5 polymorphism in the clinic may have been overstated.

It appears to us, however, that the majority of these manuscripts questioning the relevance of CYP3A5 in vivo, along with a number of other publications on the topic, are missing a major aspect of the CYP3A5 polymorphism. In the vast majority of these studies, the variation in clearance of a reported CYP3A substrate is compared among individuals who are heterozygous CYP3A5^*1/^*3 carriers and those who are homozygous for the CYP3A5^*3 allele.^{3, 4, 6} This selection of populations to compare in clinical trials of CYP3A substrates more often than not leads to the conclusion that the CYP3A5 polymorphism does not play a significant role in interpatient clearance variations in the clinic. Further, it has been shown that the higher incidence of the CYP3A5^*1 allele in African Americans (q=0.45) vs Caucasians (q=0.15) correlates with the higher expression levels of the CYP3A5 protein in African Americans over Caucasians (>50 vs 30%).⁷ It is our opinion that, by not including a subset of patients who are homozygous for the CYP3A5^*1 allele, these conclusions may be overlooking a significant portion of the general population that may be most likely to experience clinical variations in the clearance of CYP3A substrates, namely African Americans, for reasons detailed below.

While prevalent around the world, studies in the past 10 years have shown that certain diseases such as end-stage renal disease disproportionately affect some subsets of the population over others. More specifically, Klag et al⁸ in the Journal of the American Medical Association report that African American men have a four-fold higher incidence of end-stage renal disease than white men of comparable age. Furthermore, it has been recently reported that, in certain areas of the United States, African Americans may account for over 70% of individuals awaiting a kidney transplant.⁹ Johnson also reports that actions are being taken to make kidney transplants more readily available to minority populations. One can thus speculate that such populations may someday make up the majority of patients receiving immunosuppressive therapies.

Interestingly, studies that have genotyped this subset of the population have concluded that African Americans are more likely to be homozygous carriers of the CYP3A5^*1 allele than other ethnic groups. Approximately 25% of the African American livers genotyped by Kuehl et al⁷ were found to be homozygous for the CYP3A5^*1 allele vs 0% of the Caucasian livers genotyped. Furthermore, Kuehl et al report that this polymorphic distribution of the CYP3A5^*1 allele results in high levels of metabolically active CYP3A5 protein being expressed in over 50% of the African American population. They conclude that these findings indicate the possibility of CYP3A substrates, including many immunosuppressive agents, being cleared more rapidly in a non-Caucasian population. While population stratification may prevent us from ever conclusively linking CYP3A5^*1 homozygosity and end-stage renal disease, one cannot ignore the fact that the occurrence of patients homozygous for the CYP3A5^*1 allele may be inflated in a transplant population. This becomes a significant clinical situation when one realizes that most components of the immunosuppressive polypharmacy at a physician's disposal are primarily CYP3A substrates (cyclosporin, tacrolimus, sirolimus, everolimus, corticosteroids). Such a situation may also hold true for other immunosuppressive agents, such as azathioprine, which are metabolized by polymorphic enzymes other than CYP3A5.

Fortunately, there have been studies which have compared the clearance of CYP3A substrates in patients homozygous for the CYP3A5^*1 allele vs patients genotyped as CYP3A5^*1/^*3 or CYP3A5^*3/^*3. Thervet et al¹⁰ report that there was a significant difference in the mean dose levels of tacrolimus, an immunosuppressive agent, required to achieve the desired drug levels in patients homozygous for the CYP3A5^*1 allele and those homozygous for the CYP3A5^*3 allele. Patients with the CYP3A5^*1 allele required on average higher daily doses than those patients homozygous for the CYP3A5^*3 allele. In a study of immunosuppression following tacrolimus or cyclosporine administration in African Americans and Caucasians, Neylan¹¹ observed that the African American patients required a 37% mean higher dose of tacrolimus than their Caucasian counterparts. While the study did not explore the CYP3A5 genotypes of the patients, one can hypothesize that the difference in required dose levels may be due to an increased frequency of the CYP3A5^*1 allele in the African American patients. Finally, Hesselink et al,¹² in a study on the importance of CYP3A4, CYP3A5 and MDR-1 gene polymorphisms to the pharmacokinetics of cyclosporine and tacrolimus, conclude that overall lower doses of tacrolimus are required to reach target trough concentrations in patients homozygous for the CYP3A5^*3 allele when compared with patients carrying the CYP3A5^*1 allele. Additionally, if one looks at the comparison of tacrolimus dose levels in CYP3A5^*1/^*3 vs CYP3A5^*3/^*3 patients, the results tend to agree with the conclusions put forth in the manuscripts by Fukuda, Wong and Shih, in that there is no significant difference in the doses required to achieve targeted drug concentrations between these two groups. Thus, even in this study that found that homozygous CYP3A5^*1 individuals display altered pharmacokinetics and require a dose adjustment when compared with homozygous CYP3A5^*3 individuals, there was no significant difference between ^*1/^*3 and ^*3/^*3 patients.

It is our opinion, then, that the subsets of patients selected to participate in a study aimed at determining the relative clinical importance of the CYP3A5 polymorphism should include a group genotyped to be homozygous for the CYP3A5^*1 allele and of sufficient numbers to allow for statistical significance, before any firm conclusions can be made. Studies performed mainly on Caucasian or Japanese populations, as many to date have been, would provide a disservice to other populations such as African Americans, since the overall expression of CYP3A5 protein differs significantly in these populations.⁹ It may also be worthwhile to segregate the data from different populations in order to avoid a potential 'population dilution,' which may be a confounding factor in determining the importance of an enzymatic polymorphism in a given population. For example, a recent study examining the metabolism of midazolam in 23 European-American and 34 African American subjects concluded that the CYP3A5 polymorphism did not appear to have important significance in the clearance of the compound, though data from each population were not looked at on an individual basis.¹³

With the near-term possibility of African Americans making up the majority of individuals waiting for a kidney transplant, the overall number of individuals awaiting a transplant who are homozygous for the CYP3A5^*1 allele may be dramatically increased. While these suggested modifications in the design of clinical trials aimed at assessing the disposition of immunosuppressive agents will not completely solve the problem of variable pharmacokinetics (CYP3A4 and MDR-1 polymorphisms considerably add to its complexity^{12, 14}), they will help in determining the importance of such polymorphisms in populations gaining importance in the field of immunosuppressive therapies. With the knowledge that many immunosuppressive agents are cleared by CYP3A, we feel that it is thus only prudent to enrich such a population in clinical trials assessing the in vivo importance of CYP3A5.