Genotype–phenotype associations for common CYP3A4 and CYP3A5 variants in the basal and induced metabolism of midazolam in European- and African-American men and women Michael D. Floyd  , Guillermo Gervasini  , Andrew L. Masica  , Gail Mayo, Alfred L. George Jr, Kolari Bhat, Richard B. Kim and Grant R. Wilkinson CYP3A activity in adults varies between individuals and it has been suggested that this has a genetic basis, possibly related to variant alleles in CYP3A4 and CYP3A5 genes. Accordingly, genotype–phenotype associations were investigated under constitutive and induced conditions. Midazolam’s systemic and oral clearances, and the erythromycin breath test (ERBT) were determined in 57 healthy subjects: 23 (11 men, 12 women) European- and 34 (14 men, 20 women) African-Americans. Studies were undertaken in the basal state and after 14 – 15 days pretreatment with rifampin. DNA was characterized for the common polymorphisms CYP3A4  1B, CYP3A5  3, CYP3A5  6 and CYP3A5  7 by direct sequencing, and for exon 21 and exon 26 variants of MDR1 by allele-specific, real-time polymerase chain reaction. In 95% of subjects, the basal systemic clearance of midazolam was unimodally distributed and variability was less than four- fold whereas, in 98% of the study population, oral clearance varied five-fold. No population or sex-related differences were apparent. Similar findings were observed with the ERBT. Rifampin pretreatment markedly increased the systemic (two-fold) and oral clearance (16-fold) of midazolam, and the ERBT (two-fold) but the variabilities were unchanged. No associations were noted between these phenotypic measures and any of the studied genotypes, except for oral clearance and its fold-increase after rifampin. These were related to the presence of CYP3A4  1B and the inversely linked CYP3A5  3 polymorphism, with the extent of induction being approximately 50% greater in CYP3A5  3 homozygotes compared to wild-type subjects. In most healthy subjects, variability in intestinal and hepatic CYP3A activity, using midazolam as an in-vivo probe, is modest and common polymorphisms in CYP3A4 and CYP3A5 do not appear to have important functional significance. Pharmacogenetics 13:595–606 & 2003 Lippincott Williams & Wilkins Pharmacogenetics 2003, 13:595–606 Keywords: CYP3A, midazolam, polymorphisms, CYP3A4  1B, CYP3A5  3, CYP3A5  6, CYP3A5  7 Divisions of Clinical Pharmacology and Genetic Medicine, Departments of Pharmacology and Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, USA. Sponsorship: This work was supported in part by USPHS grants GM-31304 and RR-00095. Correspondence and requests for reprints to Grant R. Wilkinson, Division of Clinical Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232-6602, USA. Tel: +1 615 322 3033; fax: +1 615 343 6334; e-mail: grant.wilkinson@vanderbilt.edu Received 11 April 2003 Accepted 24 June 2003 Introduction Enzymes of the CYP3A subfamily are involved in the metabolism of numerous drugs and a number of endo- genous compounds, and four members have been described in humans (http://www.imm.ki.se/CYPalleles/). CYP3A4 is the major form present in the liver and the intestinal epithelium and, along with CYP3A5, which is polymorphically found in these organs as well as some other tissues, essentially accounts for the collective catalytic activity of this subfamily in adults. This is because CYP3A43 does not appear to be present to any significant extent [1,2], and CYP3A7 is primarily a fetal enzyme [3] that has markedly less catalytic activity than either CYP3A4 or CYP3A5 [4]. CYP3A-mediated metabolism is generally characterized as having considerable inter-individual variability which, in part, reflects its ready modulation by inhibi- tion and induction, including that associated with drug–drug interactions and other environmental agents [5]. A genetic factor(s) is also thought to be constitu- tively involved [6] although, by contrast to the activity of several other CYPs [7], that of CYP3A is distributed in a unimodal fashion [8]. This has led to the search for, and identification of, single nucleotide polymorph- isms (SNPs) in the coding and non-coding regions of both CYP3A4 and CYP3A5 (http://www.imm.ki.se/ CYPalleles/). Most of these appear to be uncommon (frequency , 1–2%) and/or selectively distributed to specific populations. However, with CYP3A4, a 392 A.G transition in the so-called nifedipine specific Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.  These individuals contributed equally to the research. Original article 595 0960-314X & 2003 Lippincott Williams & Wilkins DOI: 10.1097/01.fpc.0000054118.14659.48