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Effect of Common CYP3A4 and CYP3A5 Variants on the Pharmacokinetics of the Cytochrome P450 3A Phenotyping Probe Midazolam in Cancer Patients Erin R

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Effect of Common CYP3A4 and CYP3A5 Variants on the Pharmacokinetics of the Cytochrome P450 3A Phenotyping Probe Midazolam in Cancer Patients Erin R Cancer Therapy: Clinical Effect of Common CYP3A4 and CYP3A5 Variants on the Pharmacokinetics of the Cytochrome P450 3A Phenotyping Probe Midazolam in Cancer Patients Erin R. Lepper,1 Sharyn D. Baker,5 Matt Permenter,2 Nicole Ries,5 Ron H.N. van Schaik,7 Paul W. Schenk,7 Douglas K. Price,2 Danielle Ahn,2 Nicola F. Smith,2 George Cusatis,5 Roxann G. Ingersoll,6 Susan E. Bates,3 Ron H.J. Mathijssen,8 Jaap Verweij,8 William D. Figg,2,4 and Alex Sparreboom4 Abstract Purpose:To evaluate the effect of naturally occurring variants in genes encoding the cytochrome P450 (CYP) isoforms CYP3A4 and CYP3A5 in patients with cancer receiving midazolam as a phenotyping probe. Experimental Design: Five variants in CYP3A4 and CYP3A5 were evaluated in 58 patients (21women and 37 men) receiving a short i.v. bolus of midazolam (dose, 0.0145 or 0.025 mg/kg). Midazolam concentrations in plasma were determined using liquid chromatography-mass spec- trometry, and pharmacokinetic variables were calculated using noncompartmental analysis. Genomic DNA was characterized for the variants by PCR-RFLP, and all genotypes were con- firmed by direct nucleotide sequencing. Results:The mean clearance of midazolam was 24.4 F 9.12 L/h, and phenotypic CYP3A activity varied about 4-fold in this population (range, 10.8-44.3 L/h). There were six carriers of the CYP3A4*1B allele (allele frequency, 0.061). No variant alleles for CYP3A4*17, CYP3A4*18A , or CYP3A5*6 were identified. Forty-eight of the 58 patients were homozygous variant for CYP3A5*3C, eight were heterozygous, and two were homozygous wild type (allele frequency, 0.897). No associations were noted between any of the studied genotypes and the phenotypic measures (P z 0.16).Likewise, a common variant in exon 26 in the gene encoding P-glycoprotein [i.e., ABCB1 (MDR1) 3435C>T] that was previously reported to be linked to CYP3A4 mRNA levels was unrelated to any of the studied phenotypic measures (P z 0.49). Conclusions.The studied genetic variants in CYP3A4 and CYP3A5 are unlikely to have an impor- tant functional significance to phenotypic CYP3A activity in patients with cancer. Isoforms of the cytochrome P450 (CYP) 3A subfamily are the (1). In adults, CYP3A4 and CYP3A5 are predominant among most abundantly expressed CYP enzymes in the human liver the four known isoforms (CYP3A4, CYP3A5, CYP3A7, and CYP3A43) in the liver and intestines (1). However, metabol- ically active CYP3A5 is expressed only by an estimated 10% to Authors’ Affiliations: 1Science Applications International Corporation-Frederick, 30% of Caucasians and 50% to 60% of African Americans 2Molecular Pharmacology Section, 3Cancer Therapeutics Branch, 4Clinical (2, 3). The genetic basis for polymorphically expressed CYP3A5 5 Pharmacology Research Core, National Cancer Institute, Bethesda, Maryland; The was recently reported to be associated with a single nucleotide Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, 6Institute of Genetic Medicine,The Johns Hopkins University School of Medicine, Baltimore, polymorphism (SNP) in intron 3 of the CYP3A5 gene Maryland; Departments of 7Clinical Chemistry, Erasmus MC and 8Medical (CYP3A5*3C; refs. 4, 5). This SNP causes alternative splicing Oncology,Erasmus MC-Danielden Hoed Cancer Center, Rotterdam, the Netherlands and truncation of CYP3A5 protein and provides the basis for Received 3/9/05; revised 5/10/05; accepted 5/18/05. the absence of CYP3A5 protein in most individuals of Grant support: National Cancer Institute contract N01-CO-12400 (E.R. Lepper) and the CornelisVrolijk Development Fund, IJmuiden, the Netherlands (J.Verweij). Caucasian descent. In people with at least one CYP3A5*1A The costs of publication of this article were defrayed in part by the payment of page wild-type allele, CYP3A5 accounts for at least 50% of the total charges. This article must therefore be hereby marked advertisement in accordance CYP3A content, and results in f2- to 3-fold higher total CYP3A with 18 U.S.C. Section 1734 solely to indicate this fact. activity in vitro (4, 6). The most prevalent polymorphism in Disclaimer: The content of this publication does not necessarily reflect the views or CYP3A4 (CYP3A4*1B) occurs in the 5V flanking region of the policies of the Department of Health and Human Services nor does mention of trade names, commercial products, or organization imply endorsement by the U.S. gene. The frequency of this allele ranges from 2% to 9.6% in Government. Caucasians to 35% to 67% in African Americans (1). Requests for reprints: Alex Sparreboom, Clinical Pharmacology Research Core, A wide variation in CYP3A expression and activity exists in National Cancer Institute, Room 5A01, Building 10, 9000 Rockville Pike, Bethesda, humans, which is reflected by extensive interindividual differ- MD 20892. Phone: 301-402-9498; Fax: 301-402-8606; E-mail: SparrebA@ mail.nih.gov. ences in the clearance of CYP3A substrate drugs and phenotyp- F 2005 American Association for Cancer Research. ing probes like midazolam (7). For many anticancer drugs used doi:10.1158/1078-0432.CCR-05-0520 therapeutically, pharmacokinetic variability is a significant ClinCancerRes2005;11(20)October15,2005 7398 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2005 American Association for Cancer Research. CYP3A Phenotype/Genotype Relations contributor to the variability of both the severity of side effects reaction volume of 20 AL. The temperature profile for the PCR reaction and therapeutic response (8). Furthermore, the metabolism of was one cycle at 94jC for 5 minutes followed by 20 cycles of 94jC for j j 37% of all currently approved cytostatic and/or cytotoxic 30 seconds, 62 C for 30 seconds, and 72 C for 30 seconds, with a final j anticancer agents is known to be mediated, at least in part, by 7-minute cycle at 72 C. After initial amplification, 3 AL of amplified product were removed and an additional PCR amplification was done the CYP3A isoforms. Hence, identification of factors affecting using 40 pmol of the primers 5V-GCTCTGTCTGTCTGGGTTTGG-3V the clearance of CYP3A substrates could aid in predicting or (CYP3A4*1B F4) and 5V-CACACCACTCACTGACCTCCT-3V adapting appropriate, individualized doses of anticancer drugs. (CYP3A4*1B R4) in a 50-AL reaction. PCR conditions were 94jC for Here, we evaluated the effect of common naturally occurring 5 minutes followed by 40 cycles of 94jC for 30 seconds, 64jC for variants in genes encoding CYP3A4 and CYP3A5 in cancer 30 seconds, and 72jC for 30 seconds, with a final 7-minute cycle at 72jC. patients receiving midazolam as a phenotyping probe. Sequencing was done using the following primers: (CYP3A4*1B F5) 5V-AGGTGTGGCTTGTTGGGATG-3V for the forward strand and (CYP3A4*1B R5) 5V-TCAGAAACTCAAGTGGAGCC-3V for the reverse Materials and Methods strand. For the CYP3A5*3C allele, primers were designed and chosen as Patient selection. This study was conducted under two Institutional follows: 5V-TCCTCAGAATCCACAGCGCTG-3V(CYP3A5*3C F1) and 5V- Review Board–approved protocols and all patients signed an informed TTTATGTGCTGGAGAAGGACG-3V (CYP3A5*3C R1). These primers consent form before treatment. Patients with a histologically or were used in a 20-AL reaction that contained 1Â PCR buffer (Perkin- cytologically confirmed diagnosis of malignant solid tumor were eligible Elmer), 1.5 mmol/L MgCl2, 2 mmol/L deoxynucleotide triphosphates, for two clinical trials in which patients underwent CYP3A phenotyping and 2 units of Platinum Taq polymerase (Invitrogen). The cycle with i.v. given midazolam (9, 10). Eligibility criteria included age >18 conditions were: one cycle at 94jC for 5 minutes followed by 20 cycles years; performance status V1; and adequate hematopoietic (leukocytes, of 94jC for 30 seconds, 64jC for 30 seconds, and 72jC for 30 seconds, z4.0 Â 109/L; neutrophils, z1.5 Â 109/L; and platelets, z100 Â 109/L), ending with a final 7-minute cycle at 72jC. After initial amplification, hepatic (bilirubin within normal limits; transaminases, V2times 3 AL of amplified product were removed and an additional PCR the upper limit of normal), and renal function (creatinine clearance, amplification (as described above in a total of 50 AL final volume) was z50 mL/min). All patients were required to have an estimated life done using 40 pmol of primers 5V-AGCACTTGATGATTTACCTGCC-3V expectancy of >12 weeks and no previous chemotherapy was allowed for (CYP3A5*3C F2) and 5V-CCAGGAAGCCAGACTTTGATC-3V at least 4 weeks before enrollment. The patients were asked to abstain (CYP3A5*3C R2). PCR conditions for this reaction were 94jC for from substances known to affect the function and/or expression of 5 minutes followed by 40 cycles of 94jC for 30 seconds, 62jC for CYP3A and/or ABCB1 for a period of 2 weeks before, during, and up to 30 seconds, and 72jC for 30 seconds, with a final 7-minute cycle at 72jC. 3 weeks after the administration of midazolam. Sequencing primers were as follows: (CYP3A5*3C F3) 5V-AGTGGCA- Blood sampling and processing. Blood samples were collected in glass TAGGAGATACCCAC-3V and (CYP3A5*3C R3) 5V-AGGTTCTAGTTCAT- tubes containing heparin. Samples were drawn at 5 and 30 minutes and TAGGGTG-3V. 1, 2, 3, 4, and 5 hours following bolus i.v. administration of midazolam For the CYP3A5*6 allele, an initial PCR was done using the sample at a dose of either 0.0145 or 0.025 mg/kg. In about half of the patients, DNA and 20 pmol of each primer, 5V-AGGGAGTAGATGGAAGAT- additional samples were obtained at 15 minutes and 6 hours after GATTC-3V(CYP3A5*6 F1) and 5V-AGTTGATTATTGGATGCTTAGGGC-3V midazolam administration. Immediately after collection, each sample (CYP3A5*6 R1). These primers were used in the initial reaction along was centrifuged for 10 minutes at 2,000 Â g (4jC), and plasma with 1Â PCR buffer (Perkin-Elmer), 1.5 mmol/L MgCl2, 2 mmol/L supernatants were stored at À80jC until the day of analysis.
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