Association of CYP2C8, CYP3A4, CYP3A5, and ABCB1 Polymorphisms with the Pharmacokinetics of Paclitaxel

Cancer Therapy: Clinical

Association of CYP2C8, CYP3A4, CYP3A5,andABCB1 Polymorphisms with the Pharmacokinetics of Paclitaxel Anja Henningsson,1Sharon Marsh,2 Walter J. Loos,3 Mats O. Karlsson,1Adam Garsa,2 Klaus Mross,4 Stephan Mielke,5 LuciaVigano`,6 Alberta Locatelli,6 Jaap Verweij,3 Alex Sparreboom,3,7 and Howard L. McLeod2

Abstract Purpose: To retrospectively evaluate the effects of six known allelic variants in the CYP2C8, CYP3A4, CYP3A5,andABCB1 genes on the pharmacokinetics of the anticancer agent paclitaxel (Taxol). Experimental Design: A cohort of 97 Caucasian patients with cancer (median age, 57 years) received paclitaxel as an i.v. infusion (dose range, 80-225 mg/m2). Genomic DNA was analyzed using PCR RFLP or using Pyrosequencing. Pharmacokinetic variables for unbound paclitaxel were estimated using nonlinear mixed effect modeling. The effects of genotypes on typical value of clearance were evaluated with the likelihood ratio test within NONMEM. In addition, relations between genotype and individual pharmacokinetic variable estimates were evaluated with one- way ANOVA. Results: The allele frequencies for the CYP2C8*2, CYP2C8*3, CYP2C8*4, CYP3A4*3, CYP3A5*3C,andABCB1 3435C>T variants were 0.7%, 9.2%, 2.1%, 0.5%, 93.2%, and 47.1%, respectively, and all were in Hardy-Weinberg equilibrium.The population typical value of clearance of unbound paclitaxel was 301L/h (individual clearance range, 83.7-1055 L/h).The CYP2C8 or CYP3A4/5 genotypes were not statistically significantly associated with unbound clearance of paclitaxel. Likewise, no statistically significant association was observed between the ABCB1 3435C>Tvariant and any of the studied pharmacokinetic variables. Conclusions: This study indicates that the presently evaluated variant alleles in the CYP2C8, CYP3A4, CYP3A5,andABCB1 genes do not explain the substantial interindividual variability in paclitaxel pharmacokinetics.

Interindividual variability in drug efficacy and toxicity, result- now well recognized that in addition to environmental and ing in unpredictable patient responses, is commonly observed physiologic factors, such interpatient variation is commonly in all therapeutic areas (1, 2). However, these differences are associated with polymorphisms in genes encoding drug- particularly important in the field of cancer therapy because metabolizing enzymes, drug transporters, and/or drug targets. many anticancer agents have a narrow therapeutic index. It is To date, most studies have focused on the analysis of single- phased single nucleotide polymorphisms (SNP) in specific genes. However, there is currently much effort directed towards an understanding of genetic variation within the entire Authors’ Affiliations: 1Department of Pharmaceutical Biosciences, Faculty of biological and pharmacologic pathways (3). 2 Pharmacy, Uppsala University, Uppsala, Sweden; Department of Medicine, Among anticancer drugs, paclitaxel is of particular interest Washington University School of Medicine, St. Louis, Missouri; 3Department of Medical Oncology, Erasmus MC - Daniel den Hoed Cancer Center, Rotterdam, the because of its broad spectrum of activity against malignant solid Netherlands; 4Tumor Biology Center at the Albert-Ludwigs University, 5Department tumors, including breast, lung, and ovarian cancer (4). In the of Hematology and Oncology, University of Freiburg Medical Center, Freiburg im conventional thrice-weekly treatment regimen, dose-limiting 6 Breisgau, Germany; Division of Medical Oncology, Istituto Nazionale Tumori, side effects include hematologic toxicity (mainly neutropenia), Milan, Italy ; and 7Clinical Pharmacology Research Core, National Cancer Institute, Bethesda, Maryland whereas peripheral neuropathy becomes dose limiting in the Received 5/26/05; revised 7/22/05; accepted 8/18/05. more dose-dense, weekly regimens (5). Although paclitaxel- Grant support: Cornelis Vrolijk Development Fund, IJmuiden, the Netherlands induced toxicity is dose dependent, the individual susceptibility (J.Verweij); Swedish Cancer Society (M.O. Karlsson); and NIH Pharmacogenetics to side effects varies considerably. As for most other anticancer Research Network grant U01GM63340 (H.L. McLeod). agents, the administered dose of paclitaxel is normalized by a The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance patient’s body surface area. However, the routine use of body with 18 U.S.C. Section 1734 solely to indicate this fact. surface area as the only independent variable considered in drug Requests for reprints: Alex Sparreboom, Clinical Pharmacology Research Core, dosing is questionable, and overall, pharmacokinetic/pharma- Medical Oncology Branch, National Cancer Institute, 9000 Rockville Pike, Building codynamic variability for paclitaxel remains large (6). Previous 10, Room 5A01, Bethesda, MD 20892. Phone: 301-402-9498; Fax: 301-402- 8606; E-mail: [email protected]. studies have revealed relations between paclitaxel pharmacoki- F 2005 American Association for Cancer Research. netic variables and the likelihood of toxicity and tumor response doi:10.1158/1078-0432.CCR-05-1152 in non–small cell lung cancer with standard doses of paclitaxel

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(7, 8). Hence, identification of factors that are associated with schedule); or at 1, 22, 23, 23.92, 24.08, 24.15, 25, 26, 27, 30, 36, and the clearance of paclitaxel could aid in predicting or adapting 45 hours after infusion (24-hour schedule). Determination of total appropriate, individualized doses of paclitaxel. paclitaxel concentrations in plasma was done by high-performance The primary routes of paclitaxel elimination consist of liquid chromatography with UV detection at 227 nm according to a validated published procedure (17). The lower limit of quantification successive hydroxylation reactions mediated by the cytochrome using 1,000-AL plasma samples was 10 ng/mL, corresponding to 0.0117 P450 CYP2C8, CYP3A4, and CYP3A5 isoforms (9, 10). Another Amol/L (accuracy, 99.7%). For interassay precision, quality control pathway of elimination is through hepatobiliary and intestinal samples containing 40, 200, or 400 ng/mL (corresponding to 0.05, secretion of the parent drug by the ABCB1 gene product 0.23, or 0.47 Amol/L) of paclitaxel were used, resulting in coefficients of P-glycoprotein (11). It has been suggested that variability in variation of 7.3%, 6.3%, and 2.2% and accuracies of 99.1%, 98.5%, and expression and/or function of these proteins accounts for the 100.5%, respectively. Determination of the fraction of unbound substantial interindividual differences in drug clearance and paclitaxel was done using equilibrium dialysis with a tritiated paclitaxel thereby contributes to the variability in severity of drug-induced tracer (Moravek Biochemicals, Brea, CA; ref. 18). neutropenia as well as efficacy and survival (12). However, In view of the profound nonlinear disposition of total paclitaxel in nothing is currently known in the Caucasian population about humans (19), the pharmacologic consequences of the multiple variant genotypes cannot be predicted based on total plasma levels alone when the role of genetic variation in the pharmacokinetics of different dose groups are taken into consideration. Because the area paclitaxel. In the present study, we retrospectively evaluated under the plasma concentration-time curve of unbound paclitaxel is a whether known variant CYP2C8, CYP3A4, CYP3A5,and linear function of the dose administered (see below; ref. 14), we ABCB1 alleles are associated with the pharmacokinetic profile focused here on unbound paclitaxel between the genotype groups. of paclitaxel in adult Caucasian cancer patients. Pharmacokinetic variables for unbound paclitaxel were estimated in a nonlinear mixed effects analysis using a linear three-compartment model (14). Empirical Bayesian estimates were obtained and are Materials and Methods hereafter referred to as individual estimates. Relations between clearance and the demographic factors described in Table 1, including Patient selection. Eligibility criteria have been defined by the age, sex, body surface area, liver enzymes, bilirubin, and hematocrit, individual study protocols, as reported previously (6, 7, 13–16). were also tested for statistical significance within NONMEM. In Briefly, eligible patients had at least a pathologically confirmed addition, dose, rate of infusion, and infusion duration were tested for diagnosis of cancer for which paclitaxel was a therapeutic option. statistical significance. All analyses were done with the first-order Additional common eligibility criteria included (a) a life expectancy of conditional estimation method with interaction in the NONMEM z12 weeks; (b) a WHO performance status of V2; (c) no chemotherapy, hormonal therapy, radiotherapy, within 4 weeks before treatment; (d) age above 18 years; (e) adequate contraception for women of child- Ta b l e 1. Baseline patient characteristics bearing potential; and (f) adequate bone marrow function (absolute 9 9 neutrophil count, >1.5 10 /L; platelet count, >75 10 /L), renal Characteristic Value function (serum creatinine, V1.5 the upper limit of normal), and hepatic function (serum bilirubin, V1.5 the upper limit of normal; Baseline screening alanine aminotransferase and aspartate aminotransferase, <2.5 the Total studied 97 upper limit of normal; and alkaline phosphatase, V5 the upper limit Age (y) 57 (24-81) of normal in the presence of only bone metastases and in the absence of Sex (male/female) 40 (41)/57 (59) any liver disorders). Simultaneous use of any medication, dietary Body surface area (m2) 1.78 (1.30-2.37) supplements, or other compounds known to inhibit affect the Primary tumor site pharmacokinetics of paclitaxel was not allowed. The study protocols Bladder 5 (5) were approved by the local ethical review boards, and all patients Breast 39 (40) provided written informed consent before study entry. Drug administration. Paclitaxel (Bristol Myers Squibb, Wallingford, Esophagus 17 (17) CT) was supplied as a concentrated sterile solution in a mixture of Lung 10 (10) Cremophor EL and ethanol (1:1, v/v) at 6 mg/mL (Taxol). In 82 Ovary 18 (18) patients, the drug was administered as a single agent, whereas another Miscellaneous 8* 15 patients received paclitaxel immediately followed by a 1-hour Pretherapy chemistry and hematology infusion of carboplatin. Prior work has shown that carboplatin does not Alanine aminotransferase (units/L) 20 (2.0-264) affect the pharmacokinetic profile of unbound paclitaxel (16). Paclitaxel Alkaline phosphatase (units/L) 121 (38-1030) was given i.v. over 1 hour (n = 42), 3 hours (n = 49), or 24 hours (n =6) Aspartate aminotransferase (units/L) 21 (6.0-187) at doses of 50 mg/m2 (n = 1), 60 mg/m2 (n = 2), 70 mg/m2 (n = 1), 80 g-Glutamyltransferase (units/L) 35 (9.0-1567) mg/m2 (n = 12), 100 mg/m2 (n = 42), 130 mg/m2 (n = 1), 135 mg/m2 2 2 2 Serum creatinine (Amol/L) 72 (20-145) (n = 5), 150 mg/m (n = 3), 175 mg/m (n = 27), or 225 mg/m (n = 3). Provided toxic effects were not prohibitive, patients were eligible to Total bilirubin (Amol/L) 7.0 (3.0-24) continue treatment until there was evidence of progressive disease. Hematocrit (L/L) 0.37 (0.20-0.45) Pharmacokinetic analysis. During the first course of treatment, WBC ( 10 9 per L) 7.2 (2.9-32) blood samples were collected in glass or polypropylene tubes Absolute neutrophil count (10 9/L) 4.7 (0.90-14) containing lithium heparin as anticoagulant and immediately centri- fuged (at least 2,000 g at 4jC for 10 minutes) to separate plasma, NOTE: Continuous data are given as median with range in bracket, and which was stored at 20jC or below until analysis. These blood categorical data as number of patients with percentage of the total population samples were obtained at the following time points: immediately before in parenthesis. infusion and 0.5, 1, 1.5, 2, 2.5, 3, 5, 7, 13, 25, 33, and 49 hours after the *Primary sites include adenocarcinoma of unknown primary site (3), head and start of paclitaxel infusion (1-hour schedule); 1, 2, 3, 3.08, 3.25, 3.5, 4, neck (1), kidney (1), tongue (2), penis (1). 4.5, 5, 7, 9, 15, 21, 27, 35, and 51 hours after infusion (3-hour

ClinCancerRes2005;11(22)November15,2005 8098 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2005 American Association for Cancer Research. Paclitaxel Pharmacogenetics program, version V/VI beta (20), and graphical diagnostics was genotypes, a one-way ANOVA was done using logarithmically trans- obtained using the Xpose program (21). formed individual pharmacokinetic variable estimates. Accordingly, the Pharmacogenetic analysis. Genomic DNA was extracted from 1 mL difference in the mean values between wild type and presence of a of whole blood (n = 5) or plasma (n = 92) using the Gentra PureGene variant allele corresponds to the ratio between the groups on the Blood Kit (Gentra, Minneapolis, MN) and the QIAamp DNA Blood original scale. The 95% CI for the difference was calculated from the SE midi kit (Qiagen, Inc., Valencia, CA), respectively, following the of the differences (common variance), which represents the 95% CI of manufacturer’s instructions, and was reconstituted in a buffer contain- the ratio on the original scale. Statistical evaluations of individual ing 10 mmol/L Tris (pH 7.6) and 1 mmol/L EDTA. SNPs in CYP2C8 variable estimates (individuals with missing genotype values were not were identified from the literature (22, 23). Variations in the genes of included) were done using S-PLUS 6.1 for Windows Professional interest [CYP2C8*2 (805A>T, I269F), CYP2C8*3 (416G>A, R139K), Edition (Insightful Corp., Seattle, WA). CYP2C8*4 (792C>G, I264M), CYP3A4*3 (1334T>C, M445T), CYP3A5*3C (6986A>G, splice variant), and ABCB1 3435C>T Results (I1145I)] were analyzed using Pyrosequencing. Analysis of the CYP3A4, CYP3A5, and ABCB1 genes was done as previously described (24). PCR Patients and treatment. Complete pharmacokinetic data primers were designed using Primer Express version 1.5 (ABI, Foster were available for a total of 97 adult Caucasian patients with City, CA) for CYP2C8*2 (forward, 5V-biotin-CCATGATGTTTAGTG- cancer (40 males and 57 females) with a median age of 57 years CAGGCC-3V; reverse, 5V-CTCTACTCATTGATTGTTTCCCAGG-3V), CYP2C8*3 (forward, 5V-biotin-ACGCAGAGTAGAGTCACCCACC-3V; re- (range, 24-81 years; Table 1). The most frequent primary tumor verse, 5V-ATTCTCCCAGTTTCTGCCCC-3V), and CYP2C8*4 (forward, 5V- types were breast cancer (n = 39), ovarian cancer (n = 18), and TGTCAAGCATTACTGGCCTG-3V; reverse, 5V-biotin-CTCTACTCATT- esophageal cancer (n = 17). GATTGTTTCCCAGG-3V). The Pyrosequencing primers were designed Paclitaxel pharmacokinetics. Plasma concentration-time using the Pyrosequencing SNP Primer Design Version 1.01 software profiles of unbound paclitaxel were adequately described by a (http://www.pyrosequencing.com). CYP2C8 Pyrosequencing primer linear three-compartment model based on a previously sequences were as follows: CYP2C8*2 reverse, 5V-TTATC- published population pharmacokinetic model (14), as indicat- GATTGCTTCCTG-3V; CYP2C8*3 reverse, 5V-TGGGATGGGGAAGA-3V; ed by goodness-of-fit plots (Fig. 1). These plots indicate that and CYP2C8*4 forward, 5V-TTGATCAGGAAGCAATC-3V.PCRwas there is no bias in the model predictions at any level of the carried out using Amplitaq Gold PCR master mix (ABI), 5 pmol of predicted concentration. The left-hand graph further indicates each primer, and 5 to 10 ng of DNA isolated from whole blood or that no multimodality or pronounced skewness in interindi- DNA from 1 AL of undiluted plasma. Pyrosequencing was carried out using the Pyrosequencing hsAPSQ96 instrument and software (Biotage, vidual variability is present. Uppsala, Sweden). The genotype was called variant if it differed In this study, we reestimated the population variables and from the Refseq consensus sequence for the SNP position (http:// modified the stochastic model. The population variables are www.ncbi.nlm.nih.gov/RefSeq/). Genotype-frequency analysis of presented in Table 2. The typical value of clearance (301 L/h) Hardy-Weinberg equilibrium was carried out using Clump version was consistent with earlier data (14, 28). Paclitaxel disposition 1.9 (25). was highly variable in the studied cohort, with an estimated Statistical evaluation. The association of the various genotypes with coefficient of variation of 44% and individual clearance values clearance was evaluated within NONMEM. For CYP2C8, CYP3A4, and ranging from 83.7 to 1,055 L/h. Body surface area and bilirubin ABCB1, the associations between clearance and genotypes heterozygous were found to be significant covariates for unbound paclitaxel or homozygous were expressed as clearance = TVCL (1 + B ) and Wt het pharmacokinetics (P < 0.01), with positive and negative clearance = TVCLWt (1 + Bvar), respectively, where TVCLWt is the typical value of clearance in the wild-type group and B is the estimated relations to clearance, respectively. However, when infusion covariate effect. For CYP3A5*3C, the homozygous variants are most duration was included (categorized as short or long infusion common and was therefore chosen as typical. The covariate effect was duration), only bilirubin was statistically significant. Clearance considered an effect of presence of functional allele and the association decreased with increasing bilirubin (2% per Amol/L bilirubin), was estimated as clearance = TVCLVar (1 + Bhet) and clearance = and typical values ranged from 205 to 348 L/h. The typical TVCLVar (1 + Bwt), respectively. value of clearance for the 24-hour infusions was 46.1% lower For individuals with missing genotype information, a mixture model than for the other schedules. However, inclusion of these for the covariate effects was used. In a mixture model, the likelihood covariates did not reduce unexplained interindividual variabil- contribution from an individual is the sum of the likelihoods under ity in paclitaxel clearance and was therefore not included in the each model times the probability (size) of the subpopulation. The final model. In a separate analysis (data not shown), it was observed genotype frequencies were used as estimates for the probabilities, and the magnitude of the covariate effects were assumed found that these covariate relations did not affect the results or to be the same as for the subjects where genotype information existed. conclusions of the study when included together with the The likelihood ratio test was used to evaluate statistical significance genotypes. The administered dose and rate of infusion were not of the covariate effects. The objective function value (OFV) obtained in statistically significant covariates for clearance (P = 0.77 and P = NONMEM is proportional to 2 log-likelihood, and the difference 0.14, respectively) when tested within NONMEM. in OFV (DOFV) between two nested models is approximately m2 Genotyping. Six SNPs were analyzed in four genes of distributed. Accordingly, a DOFV of >3.84, >6.63, and >10.83, between putative relevance for paclitaxel disposition (Table 3). For two two nested models differing in one variable, corresponds to P < 0.05, SNPs in the CYP2C8 gene (i.e., CYP2C8*2 and CYP2C8*4), no P < 0.01, and P < 0.001, respectively. The 95% confidence intervals homozygous variants were observed, as expected based on (95% CI) for the covariate effects were assessed by log-likelihood previously published data obtained from Caucasian individuals profiling, which is one alternative method to estimate confidence limits that does not assume symmetry around the estimate, as opposed to (23). In the other main CYP2C8 variant studied, CYP2C8*3 calculating confidence limits from the SE obtained in NONMEM (26, 27). (represented by the R139K polymorphism), the frequency of In addition, an assessment of the association of the multiple variant the rarest allele (q) was 9.2% (Table 3). All genotype genotypes with individual pharmacokinetic variable estimates was frequencies were found to be in Hardy-Weinberg equilibrium. done. To relate the pharmacokinetic variable means with the different The allele frequencies for CYP3A4*3, CYP3A5*3C, and ABCB1

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Fig. 1. Observed unbound concentrations of paclitaxel versus predictions based on population model variables (left)and predictions based on empirical Bayesian estimates (right). Solid lines represent lines of identity.

3435C>T of 0.5%, 93.2%, and 47.1%, respectively, were very CYP3A4, CYP3A5, and ABCB1 genes. The CYP2C subfamily is similar to those reported in previous studies involving responsible for the metabolism of f20% of clinically used Caucasians individuals (29–31). drugs, including the 6a-hydroxylation of paclitaxel by the Genotype-phenotype relations. Because the mechanistic basis CYP2C8 isoform (10, 32). This subfamily consists of four for associations of the studied genetic variants would (primarily) members, including CYP2C8, CYP2C9, CYP2C18, and be with clearance, we focused on presenting these results. No CYP2C19, and the corresponding genes are clustered on statistically significant relations were found in the likelihood chromosome 10q24. Whereas polymorphisms in the CYP2C9 ratio test within NONMEM (DOFV was between 0.028 and and CYP2C19 genes have been shown to result in altered 1.508). The estimated covariate effects with 95% CIs are presented in Table 4. The covariate effects were generally low, and the 95%CI excluded relative differences of a factor two or more for all but two genotypes. The two variants with a possibly Ta b l e 2 . Population variable estimates for unbound larger difference were based on only single subjects. For example, paclitaxel the group heterozygous for CYP2C8*3 had lower typical value of clearance compared with wild type (mean percent difference Variable Estimate RSE (%) from wild type, 3.2%; 95% CI, 21.5 to 19.5%), and the CL (L/h) 301 4.3 homozygous for the variant allele had higher typical value of V1 (L) 225 6.0 clearance (mean, 3.19%; 95% CI, 28.6% to 49.9%). V2 (L) 3450 7.8 Individual clearance values as a function of the variant V3 (L) 303 6.6 genotypes are shown in Fig. 2. Beside the genotypes included in Q2 (L/h) 132 6.6 Fig. 2, the single individual heterozygous for CYP3A4*3 had a Q3 (L/h) 151 6.5 clearance of 301 L/h compared with a mean value in patients Residual error (%) 21.1 4.9 with the wild-type sequence of 333 L/h (n = 93), and the single IIVCL (CV %) 44 18* individual heterozygous for CYP2C8*2 had a clearance of 323 IIV V1 (CV %) 50 27* L/h compared with a mean value in patients with the wild-type IIV V2 (CV %) 38 37* sequence of 347 L/h (n = 69). All the 95% CIs of the ratio for IIVQ2 (CV %) 65 19* each genotype included 1, indicating no statistically significant IIVQ3 (CV %) 37 33* difference between the groups (Fig. 3). In addition, for the CorrIIV CL-V1 0.41 33* genotypes where data from patients with a homozygous variant CorrIIV CL-Q2 0.71 20* genotype were available, the three groups were tested using CorrIIV CL-Q3 0.55 33* one-way ANOVA, but no significant relations were found (P > CorrIIV V1-Q2 0.44 37* 0.69). Differences in other pharmacokinetic variables were also CorrIIV V1-Q3 0.38 50* not statistically significantly different between the different CorrIIV Q2-Q3 0.89 22* genotype groups (data not shown).

Abbreviations: RSE, relative standard error (%); CL, clearance; V,volumeof Discussion distribution; Q, intercompartmental clearance; IIV, interindividual variability; CV, coefficient of variation; Corr, correlation between individual estimates. In this study, we tested the hypothesis that the disposition of *RSE is related to the corresponding variance or covariance term. paclitaxel is dependent on genetic variability in the CYP2C8,

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Ta b l e 3 . Genotype and allele frequencies for the studied variant genes

c b Polymorphism* Nomenclature Effect Genotype frequencies Allele frequenciesx Wt Het Var p q CYP2C8 805A>T CYP2C8*2 I269F 69 (98.6) 1 (1.43) 0 (0) 0.993 0.007 CYP2C8 416G>A CYP2C8*3 R139K 78 (84.8) 11(11.9) 3 (3.26) 0.908 0.092 CYP2C8 792C>G CYP2C8*4 I264M 90 (95.7) 4 (4.26) 0 (0) 0.979 0.021 CYP3A4 13 3 4 T>C CYP3A4*3 M445T 93 (98.9) 1 (1.06) 0 (0) 0.995 0.005 CYP3A5 6986A>G CYP3A5*3C Splice variant 1 (1.05) 11(11.6) 83 (87.4) 0.068 0.932 ABCB1 3435C>T NA I1145I 22 (25.3) 48 (55.2) 17(19.5) 0.529 0.471

Abbreviations: Wt, homozygous wild type patient; Het, heterozygous patient; Var, homozygous variant patient; NA, not available. *Number represents position in nucleotide sequence. cNumber represents amino acid codon. bNumber represents number of patients with percentage in parenthesis; the difference in the total number of patients is due to the fact that not all samples yielded Pyrosequencing data or showed PCR amplification. xHardy-Weinberg notation for allele frequencies (p, frequency for wild-type allele; q, frequency for variant allele). toxicity of certain anticancer drugs in carriers of a variant allele CYP2C8*3 variant (containing 416G>A; R139K) has an allele (33), much less is known about the functional role of variants frequency of up to 14% in Caucasians (34, 35) but is very rare in CYP2C8. The CYP2C8*2 and CYP2C8*4 alleles have been in African Americans and Asians (36). In vitro studies have found predominantly in African Americans but not in the shown substantially decreased activity of the recombinant Japanese and Caucasians (23), which is consistent with the CYP2C8.2 and CYP2C8.3 enzymes in the metabolism of currently observed frequencies of 0.7% and 2.1% for paclitaxel (22, 23, 37, 38). Recent clinical investigations also CYP2C8*2 and CYP2C8*4, respectively. In contrast, the suggest that the CYP2C8*3 variant allele is associated with altered pharmacokinetics of the CYP2C8 substrate drugs repaglinide (39) and ibuprofen (40). In the present analysis, however, CYP2C8*3 was not significantly associated with the Ta b l e 4 . Estimated covariate effects (Bi)with95%CI pharmacokinetics of paclitaxel. This discrepancy with earlier findings for repaglinide (39) and ibuprofen (40) is possibly Genotype Bi (95% CI) related to the fact that, in contrast to paclitaxel, repaglinide is CYP2C8*2 not a substrate for CYP3A4 (39); that ibuprofen is also a Heterozygous 0.313* (0.287, 1. 485) substrate for CYP2C9 (40); and that the CYP2C9*2 allele is in Homozygous NA linkage disequilibrium with CYP2C8*3 (35). Because the CYP2C8*3 expression and activity of CYP2C8 in the human intestine Heterozygous 0.0320 (0.215, 0.195) and liver is relatively low (41), it is possible that individuals Homozygous 0.0319 (0.286, 0.499) carrying the CYP2C8*3 allele still can efficiently eliminate CYP2C8*4 paclitaxel through other compensatory mechanisms, including Heterozygous 0.146 (0.185, 0.615) elimination via CYP3A4 or through hepatobiliary excretion, Homozygous NA and that the associations of different genotypes with clearance CYP3A4*3 might not become evident until substrates are coadministered Heterozygous 0.054* (0.484, 1.14) with inhibitors of these pathways. Although direct evidence for Homozygous NA this hypothesis is lacking, this possibility is supported by a CYP3A5*3C previous observation that 6a-hydroxypaclitaxel, formed via Heterozygous 0.0764 (0.129, 0.326) CYP2C8, is not the predominant paclitaxel metabolite in all Wild type 0.289* (0.651,0.391) individuals (42). Besides the fact that paclitaxel undergoes ABCB1 3435C>T several routes of elimination that possibly could compensate Heterozygous 0.0912(0.085, 0.297) for any deficiencies, the reason for not seeing associations of Homozygous 0.103 (0.108, 0.362) these different genotypes with clearance could also be due to the fact that the frequency of the homozygous variant CYP2C8 genotypes in the currently studied population is low (i.e., NOTE: Clearance = TVCLWt (1 + Bi). T VCLWT, the typical value of clearance <3.26%). As the heterozygous genotypes have one fully in wild-type group, was 300, 302, 299, 301, and 282 L/h for genotypes CYP2C8*2, CYP2C8*3, CYP2C8*4, CYP3A4*3,andABCB1, respectively. functional allele, it is possible that the regulation of that allele For CYP3A5*3C, the majority of individuals in the studied population was is altered to compensate for the deficient allele. homozygous for the variant allele. Hence, the covariate effect was considered In adults, CYP3A4 and CYP3A5 are the most important as an effect of having a functional allele (i.e., related to the typical value of among the four CYP3A subfamily members for CYP3A- clearance in the homozygous variant group): clearance =300 (1 + Bi). Abbreviation: NA, not available. mediated drug metabolism (i.e., CYP3A4, CYP3A5, CYP3A7, *Based on one individual. and CYP3A43; ref. 43). Because of the genetic diversity in the genes encoding these proteins (44), it has been proposed that

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Fig. 2. Individual clearance values of unbound paclitaxel as a function of variant genotypes: Wt, wild-type patient; Het, heterozygous variant ^ type patient; Var, homozygous variant ^ type patient.

genotyping for CYP3A4 and CYP3A5 variants may be useful for ABCB1) have been described that may affect transporter prediction of total CYP3A activity. Over 30 SNPs in CYP3A4 expression or function (58). The most extensively studied have been published; however, most are unlikely to affect ABCB1 variant to date is a common synonymous mutation at CYP3A4 activity in vivo (44–46). Due to the very low allele the transition C to T at nucleotide position 3435 at a wobble frequency of the CYP3A4*3 variant evaluated in the present position in exon 26 (59). Although this transition does not study (0.5%), this polymorphism most likely has no relevance change its encoded amino acid, recent findings suggest that this to CYP3A4 activity and function in the Caucasian population. variant is associated with altered protein expression in different In contrast to CYP3A4, the CYP3A5 protein isoform is known human tissues (60), and this may result in decreased hepato- to be expressed in only a small percentage of Caucasian biliary and/or intestinal secretion of substrate drugs like individuals, and this has been linked to a common transition in intron 3 of the CYP3A5 gene (CYP3A5*3C), which introduces a frameshift during translation and results in a truncated, nonfunctional protein (47, 48). Approximately 85% to 95% of Caucasian subjects are homozygous variant for CYP3A5*3C and thus are deficient in functionally active CYP3A5 (49), which is consistent with the currently observed genotype frequency of 87.4%. In the present study, no association was noted between the CYP3A5*3C variant and paclitaxel pharmacokinetics. This is similar to findings obtained in healthy subjects and cancer patients using the CYP3A phenotyping probes midazolam (50–53), erythromycin (51, 54), and nifedipine (55), although one recent study involving a predominantly Caucasian population of cancer patients observed a 1.4-fold higher midazolam clearance in four patients with the CYP3A5*1/*3C genotype compared with 39 patients with the CYP3A5*3C/*3C genotype (56). The extent to which the CYP2C8 and CYP3A isoforms metabolize paclitaxel is likely to be determined, at least in part, by intracellular drug concentrations in the liver and, to a lesser extent, the intestine. This process, in turn, is partially dependent on the efflux transporter P-glycoprotein, which is localized in the apical membrane of hepatocytes and enterocytes. It has been suggested that drug accumulation in these cells inversely reflects Fig. 3. Observed ratios of mean (dots) clearance values of unbound paclitaxel in the different genotype groups (presence of variant allele/wild type) and the the activity of P-glycoprotein (57). In recent years, various 95% CI (bars). For CYP3A5*3C, the ratio represents presence of wild-type allele/ genetic variants in the gene encoding P-glycoprotein (i.e., homozygous variants.

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paclitaxel. However, in this study, the ABCB1 3435C>T genotype The lack of statistically significant relations in this study was not associated with paclitaxel pharmacokinetics. It should does not necessarily mean that there are none, especially in be noted, however, that the lack of relationships with this SNP is light of the few individuals studied with a homozygous consistent with preclinical observations that Cremophor EL variant genotype. However, the 95% CIs of the genotype inhibits ABCB1 function (61), and also the notion that meta- effects are sufficiently narrow and/or genotype heterogeneity bolism rather than transport is the prominent elimination sufficiently small to conclude that the studied variants in pathway for paclitaxel (62). Moreover, even the complete the CYP2C8, CYP3A4, CYP3A5, and ABCB1 genes do not absence of P-glycoprotein in mice did not have any substantial cause a substantial interindividual difference in paclitaxel influence on the plasma area under the plasma concentration- clearance. Moreover, additional genetic variants or haplotypes time curve of total paclitaxel after the administration of Taxol, on of importance to paclitaxel pharmacokinetics may yet be the cumulative urinary excretion, or on cumulative biliary discovered. Further investigation is needed to determine the secretion (11). This suggests that the involvement of P- relative role of genetic variation and environmental variables glycoprotein in the disposition of paclitaxel may be relatively (e.g., concomitant drugs or herbs) on the pharmacokinetics of unimportant regardless of ABCB1 genotype status. paclitaxel.

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Anja Henningsson, Sharon Marsh, Walter J. Loos, et al.

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