UGT1A7 and UGT1A9 Polymorphisms Predict Response and Toxicity in Colorectal Cancer Patients Treated with Capecitabine/Irinotecan

1226 Vol. 11, 1226–1236, February 1, 2005 Clinical Cancer Research

Leslie E. Carlini,1 Neal J. Meropol,1 John Bever,2 irinotecan. Specifically, patients with genotypes conferring Michael L. Andria,2 Todd Hill,2 Philip Gold,3 low UGT1A7 activity and/or the UGT1A9 (dT)9/9 genotype may be particularly likely to exhibit greater antitumor Andre Rogatko,1 Hao Wang,1 and response with little toxicity. Rebecca L. Blanchard1 1 2 Fox Chase Cancer Center, Philadelphia, Pennsylvania; Roche INTRODUCTION Laboratories, Inc., Nutley, New Jersey; and 3Swedish Cancer Institute, Seattle, Washington Colorectal cancer (CRC) is the third most common cancer in both men and women and the third most prevalent cause of cancer-related death (1). A common approach to the systemic ABSTRACT management of metastatic colorectal cancer is a combination Purpose: Capecitabine and irinotecan are commonly of fluoropyrimidine (e.g., 5-fluorouracil or capecitabine) used in the treatment of metastatic colorectal cancer (CRC). and irinotecan (reviewed in ref. 2). Fluoropyrimidines act We hypothesized that germline polymorphisms within genes primarily through the inhibition of thymidylate synthase (TS), related to drug target (thymidylate synthase) or metabolizing resulting in impaired DNA synthesis and cell death. Irinotecan is enzymes (UDP-glucuronosyltransferase, UGT) would impact a camptothecin compound that inhibits DNA topoisomerase I, response and toxicity to the combination of capecitabine plus resulting in an accumulation of DNA damage and cell death. irinotecan (CPT-11). Irinotecan is a prodrug that undergoes conversion by liver Experimental Design: Sixty-seven patients with measur- carboxylesterases to form the active compound SN-38, 7-ethyl- able CRC were treated with irinotecan i.v. (100 or 125 mg/m2) 10-hydroxy-camptothecin (2, 3). SN-38 is largely metabolized on days 1 and 8 and capecitabine orally (900 or 1,000 mg/m2, to the inactive glucuronide, SN-38G, via the action of several twice daily) on days 2 through 15 of each 3-week cycle. UDP-glucuronosyltransferase (UGT) including hepatic Genomic DNA was extracted from peripheral blood and ge- UGT1A1, UGT1A6, and UGT1A9 and extrahepatic UGT1A7 notyped using Pyrosequencing, GeneScan, and direct se- (4–7). quencing (Big Dye terminator) technologies. The existence of significant patient variability in response Results: The overall objective response rate was 45% to fluoropyrimidines and irinotecan, coupled with knowledge with 21 patients (31%) exhibiting grade 3 or 4 diarrhea and of common genetic polymorphisms within genes important to 3 patients (4.5%) demonstrating grade 3 or 4 neutropenia in the pharmacology of these drugs, suggests that pharmacogenetic the first two cycles. Low enzyme activity UGT1A7 genotypes, studies could identify individuals likely to benefit from treatment UGT1A7*2/*2 (six patients) and UGT1A7*3/*3 (seven or develop severe toxicity. Genetic variation within the promoter patients), were significantly associated with antitumor and the 3V-untranslated region (UTR) of the human TS gene has response ( p = 0.013) and lack of severe gastrointestinal been previously linked to the efficacy of 5-fluorouracil-related toxicity ( p = 0.003). In addition, the UGT1A9 118 (dT)9/9 drugs (reviewed in ref. 8). The polymorphic UGT1A family genotype was significantly associated with reduced toxicity members may govern variability in patient response to irinotecan ( p = 0.002) and increased response ( p = 0.047). There were (reviewed in ref. 9). A common TA repeat within the promoter of no statistically significant associations between UGT1A1, the human UGT1A1 gene has been associated with CPT-11- UGT1A6, or thymidylate synthase genotypes and toxicity or related toxicity, most predominantly neutropenia (10–12). tumor response. Functionally significant genetic variation has also been described Conclusions: These data strongly suggest that UGT1A7 for human UGT1A6, UGT1A7, and UGT1A9 (6, 13–17). In this and/or UGT1A9 genotypes may be predictors of response study, we correlate the efficacy and toxicity of combined and toxicity in CRC patients treated with capecitabine plus capecitabine/CPT-11 therapy with genetic variation in genes important in the metabolism of CPT-11 (UGT1A1, UGT1A6, UGT1A7, and UGT1A9). Given the narrow therapeutic index, Received 9/2/04; revised 11/3/04; accepted 11/9/04. frequent resistance, and expanding options for treatment of CRC, Grant support: Roche Laboratories, Inc. the need for predictive markers of response and toxicity has The costs of publication of this article were defrayed in part by the assumed increased importance. payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: Presented in part at the 40th Annual Meeting of the American MATERIALS AND METHODS Society of Clinical Oncology, June 5-8, 2004, New Orleans, LA. Patient Eligibility. This pharmacogenetic analysis was a Request for reprints: Rebecca L. Blanchard, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111-2497. Phone: 215-728- secondary objective of a multicenter phase II trial of capecitabine/ 3141; Fax: 215-728-4333; E-mail: [email protected]. CPT-11 combination therapy in patients with metastatic CRC. #2005 American Association for Cancer Research. The primary clinical objective was determination of objective

response. Eligible patients were at least 18 years old with PCR reaction mixture included 20 mmol/L Tris-HCl (pH 8.3), histologically confirmed metastatic colorectal adenocarcinoma. 50 mmol/L KCl, 1.5 mmol/L MgCl2,0.2mmol/Leach Previous cytotoxic chemotherapy was not permitted except for deoxynucleotide triphosphate (dNTP), 0.2 Amol/L each primer, neoadjuvant or adjuvant treatment completed 12 months before 1.25 units Platinum Taq polymerase (Invitrogen, Carlsbad, CA), study enrollment. Patients were required to be ambulatory with a and 20 ng genomic DNA in a 50 AL reaction volume. The PCR Karnofsky performance status of z70%. Required laboratory profile included 1-minute denaturation at 94jC, 34 cycles of 30 9 values for inclusion included neutrophil count z1.5 10 , seconds at 94jC, 30 seconds at 52jC, and 30 seconds at 72jC, 9 platelet count z100 10 /L, serum creatinine V1.5 upper limit with a final 5-minute extension at 72jC in a Perkin-Elmer 9700 z normal, estimated creatinine clearance 50 ml/min, serum Thermal Cycler (Perkin-Elmer, Boston, MA). Fluorescently bilirubin V1.25 upper limit normal, ALAT and ASAT V2.5 labeled products were electrophoretically separated on a 4% upper limit normal (<5 with liver metastasis or <10 with bone polyacrylamide gel on an ABI 377 DNA Sequencer. Fluorescent V metastasis), or alkaline phosphatase 2.5 upper normal limit bands were analyzed using GENESCAN 2.1 software to (<5 with liver metastasis or <10 with bone metastasis). Exclusion determine fragment length (Applied Biosystems). Genotypes criteria included known Gilbert’s disease, pregnancy, central were assigned as UGT1A1*1 (reference), UGT1A1*36, nervous system metastasis, active cardiac disease, or myocardial UGT1A7*28, and UGT1A7*37 for TA repeat numbers of 6, 5, infarction within the previous 12 months, active infections, 7, and 8, respectively. physical disorders of the gastrointestinal tract, or problems with UGT1A6. UGT1A6 genotype was determined with a malabsorption. Written informed consent was required, and the PCR-based assay that uses Pyrosequencing technology (Pyrose- study was approved by the institutional review boards at all of the quencing, Westborough, MA). UGT1A6 alleles are defined by participating sites in accord with an assurance filed with and permutations of three single-nucleotide polymorphisms (SNPs) approved by the U.S. Department of Health and Human Services. that alter the encoded amino acid sequence at nucleotide Drug Administration. Capecitabine (Xeloda, Roche positions 19T > G, 541A > G, and 552A > C (for amino acids Laboratories, Inc., Nutley, NJ) was given orally at a dosage of S7A, T181A, and R184S, respectively; ref. 15). For detection of 1,000 mg/m2 (cohort 1, 15 patients) or 900 mg/m2 (cohort 2, 52 the 19T > G SNP, a 238-bp fragment was amplified using primer patients) twice daily on days 2 through 15 of 3-week cycles. F-53 (5V-GATTTGGAGAGTGAAAACTCTTT-3V) and R184 Irinotecan (Camptosar, Pfizer, Inc., New York, NY) was given at (5V-biotin-CAGGCACCACCACTA-CAATCTC-3V). For the a dosage of 125 mg/m2 (cohort 1, 15 patients) or 100 mg/m2 remaining two SNPs, a 215-bp fragment was amplified using (cohort 2, 52 patients) as a 90-minute i.v. infusion on days 1 and primer F414 (5V-biotin-CTTTAAGGAGAGCAAGTTTGATG- 8 of each cycle. The doses of capecitabine and irinotecan were 3V) and R628 (5V-CCACTCGTTG-GGAAAAAGTC-3V). Ap- decreased as described for cohort 2 after the first 15 patients proximately 25 to 50 ng of genomic DNA was amplified in a (cohort 1) because of unacceptable toxicity. The maximum reaction mixture containing 20 mmol/L Tris-HCl (pH 8.4), 50 number of cycles given was 12. mmol/L KCl, 1.5 mmol/L MgCl , 0.2 mmol/L dNTPs, 0.2 Amol/ Evaluation of Response and Toxicities. Baseline tumor 2 L primers, and 2.5 units of Platinum Taq polymerase in a 50 AL measurements were obtained within 21 days before initial volume. Reactions were performed in a Perkin-Elmer 9700 treatment. Response assessments were obtained every two cycles Thermal Cycler with 45-second denaturation at 94jC, followed (6 weeks) with confirmation of response after 4 weeks according by 35 cycles of 94jC for 30 seconds, 56jC for 30 seconds, and to RECIST criteria (18). Toxicity was assessed weekly during the 72jC for 50 seconds. A final 3-minute extension at 72jC first two cycles of treatment according to the National Cancer completed the amplification. Institute Common Toxicity Criteria, version 2.0 (National Cancer Amplicons were prepared for automatic Pyrosequencing Institute Common Toxicity Criteria, http://ctep.cancer.gov). All SNP analysis on the PSQ 96 system using reagents from the toxicities evaluated in this study occurred during the first two PSQ96 SNP Reagent Kit (Pyrosequencing). A 25-AL volume of cycles of treatment. double-stranded biotinylated amplicon was incubated with 100 Ag Genomic DNA Preparation and Genotyping Assays. of streptavidin-coated M280 DynaBeads (Dynal, Brown Deer, Blood samples were collected for isolation of genomic DNA WI) in binding buffer [5 mmol/L Tris (pH 7.6), 1 mol/L NaCl, 0.5 during venipuncture for other diagnostic labs at least 1 week mmol/L EDTA, 0.05% Tween 20] and incubated at 65jC for 15 before starting treatment. Genomic DNA was prepared from minute followed by denaturation in 0.5 mol/L NaOH. Single- peripheral WBCs and suspended in 10 mmol/L Tris with 50 stranded biotinylated DNA was transferred to annealing buffer mmol/L EDTA (TE; Covance, Princeton, NJ). DNA was isolated [20 mmol/L Tris Acetate (pH 7.6) and 5 mmol/L Mg(OAc)2] for from 66 of the 67 subjects and was used to identify UGT1A1, 1 minute then transferred to sequencing primer solution UGT1A6, UGT1A7, UGT1A9, and thymidylate synthase poly- containing annealing buffer and 10 pmol of the appropriate morphisms. For each batch of assays, appropriate positive and sequencing primer. The 19T > G primer was 5V-GAT- negative controls of established genotype were assayed. GGCCTGCCTCCTT-3V, whereas the 541A > G and 552A > C UGT1A1. The UGT1A1 variable length (TA)n repeat primer was 5V-AGGACACAGGGTCTG-3V. The nucleotide dis- polymorphism (n = 5-8) was evaluated using Genescan pensation sequence for the 19T > G SNP was TCGCTGACA technology (Applied Biosystems, Foster City, CA; refs. 19, 20). with the underlined nucleotide representing the negative control A 254-bp region of the UGT1A1 gene was amplified using a and those in bold representing the polymorphic site. The hexachloro-6-carboxyfluorescein fluorescently tagged antisense nucleotide dispensation sequence for the remaining SNPs was primer (5V-ATCAACAGTATCTTCCCAG-3V) and a nonfluores- CGCTGACTGCTGATGTCAT. Genotypes were assigned cent sense primer (5V-TATCTCTGAAAGTGAACTC-3V). The as UGT1A6*1 (S7, T181, and R184 reference), UGT1A6*2

(A7, A181, and S184), UGT1A6*3 (A7) and UGT1A6*4 subjects: 118 (dT)n repeat, 87G > A, and the intron 1 SNPs (A7 and S184). I143C > T, I152G > A, I219A > T, and I313C > A. UGT1A7. UGT1A7 genotype was determined by direct Thymidylate Synthase Promoter. TS genotypes were sequencing of a PCR product that spans all of the polymorphic determined using a combination of gel electrophoresis–based sites. The UGT1A7 alleles are defined by permutations of six and direct DNA sequencing techniques. Common functional SNPs (342G > A, 387T > G, 391C > A and 392G > A, 417G > C, polymorphisms in the TS gene include a 28-bp variable and 622T > C) that alter the encoded amino acid sequences tandem repeat (n = 2, 3, 4, 5, or 9) within an enhancer of the (G115S, N129K, R131K, E139D, and W208R, respectively; ref. promoter and a SNP within the 5V-UTR of the three repeat 14). A 415-bp fragment from UGT1A7 was amplified using primer F277 (5V-TTTGCCGATGCTCGCTGGACG-3V)and (3R) allele (58G > C; refs.23, 24). The variable tandem R692 (5V-GCTAT-TTCTAAGACATTTTTGAAAAAATAGGG- repeat was detected by PCR amplification with F-220 (5V- 3V). The PCR reaction mixture included JumpStart REDTaq GTGGCTCCTGCGTTTCCCCC-3V) and R + 1 primers (5V- ReadyMix PCR reaction mix (Sigma, St. Louis, MO) containing TCCGAGCCGGCCACAGGCAT-3V)thatflanktherepeat 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 2 mmol/L region. The PCR reaction mixture included 10 mmol/L Tris- MgCl2, 0.2 mmol/L each dNTP, and 0.03 units/Taq polymerase HCl (pH 8.3), 50 mmol/L KCl, 2 mmol/L MgCl2, 5% DMSO, with the addition of 0.2 Amol/L each primer and 40 ng genomic 0.2 mmol/L each dNTP, 0.2 Amol/L each primer, 1.5 units DNA in a 50-AL reaction volume. The PCR conditions included Platinum Taq polymerase (Invitrogen), and 40 ng genomic j j 1-minute denaturation at 94 C, 35 cycles of 30 seconds at 94 C, A j j DNA in a 50- L reaction volume (25). The PCR conditions 30 seconds at 56 C, and 30 seconds at 72 C, with a final 5- j j j included 1-minute denaturation at 94 C, 34 cycles of 94 C minute extension at 72 C in a Perkin-Elmer 9700 Thermal j j Cycler. Amplified products were sequenced using an ABI Prism for 40 seconds, 70 C for 40 seconds and 72 C for 40 j 377 DNA Sequencer (Applied Biosystems) and primers F292 seconds, with a final 5-minute extension at 72 C in a Perkin- (5V-TGGACGGCACCATTG-3V) and R675 (5V- Elmer 9700 Thermal Cycler. After separating the gene TTTGAAAAAATAGGGGCAA-3V). Genotypes were assigned fragments via gel electrophoresis, fragment lengths were as UGT1A7*1 (G115, N129, R131, and W208 reference), determined relative to known molecular markers. Direct UGT1A7*2 (K129 and K131), UGT1A7*3 (K129, K131, and sequencing of the amplified products allowed identification R208), UGT1A7*4 (R208). of the 5V-UTR SNP. Genotypes were assigned according to UGT1A9. Five UGT1A9 polymorphisms were evaluated repeat lengths and the existence of the SNP: one repeat (1R), by direct DNA sequencing of a PCR amplicon spanning all of two repeats (2R reference), three repeats (3RC or 3RG), four the polymorphic sites. Polymorphisms of interest included a repeats (4R), five repeats (5R), or nine repeats (9R). 118 (dT)n repeat (n = 9 or 10), C3Y (8G > A), M33T (98T > Thymidylate Synthase 3V-UTR. The presence or absence C), Y242X (726T > G), and D256N (766G > A; refs.14, 21, 22). of the 6-bp sequence TTAAAG at position 1494 of the TS We also detected the following promoter: and intronic SNPs: mRNA was detected by PCR amplification with F3VutrTS (5V- 87G > A, and intron 1 I143C > T, I152G > A, I201A > C, CAAATCTGAGGGAGCTGAGT-3V) and R3VutrTS (5V-CAGA- I219T > A, and I313A > C. The 118 (dT)9/10 polymorphism TAAGTGGCAGTACAGA-3V) to produce either 148- or 142-bp has been previously localized to nucleotides 98 through 109 fragments (26). The PCR reaction mixture included 10 mmol/L (16, 17). However, we believe it is most accurate to assign the Tris-HCl (pH 8.3), 50 mmol/L KCl, 2.5 mmol/L MgCl , localization of this poly d(T) tract as spanning nucleotides 118 2 0.2 mmol/L each dNTP, 0.2 Amol/L each primer, 1.5 units through 129 (based on assigning the ‘‘A’’ in the ‘‘ATG’’ Platinum Taq polymerase (Invitrogen), and 40 ng genomic DNA translation start codon as +1) and we refer to the polymorphisms in a 50 AL reaction volume (26, 27). The PCR profile included as 118 (dT) or 118 (dT) . 9 10 1-minute denaturation at 94jC, 35 cycles at 94jC for 30 A 1.4-kb fragment from UGT1A9 was amplified using j j primer F-273 (5V-AAACTTAACATTGCAGCACAGGGC-3V) seconds, 58 C for 45 seconds, and 72 C for 45 seconds, and a j and RI337 (intron 1, 5V-CTAAGACCATTT-CCTCTGGGGC- final 5-minute extension at 72 C in a Perkin-Elmer 9700 3V). The PCR reaction mixture included 10 mmol/L Tris-HCl Thermal Cycler. Amplified products were separated via gel electrophoresis on 3.5% Metaphor agarose and fragment lengths (pH 8.3), 50 mmol/L KCl, 2 mmol/L MgCl2, 0.2 mmol/L each dNTP, 0.2 Amol/L each primer, 5% DMSO, 1.5 units Platinum were determined relative to known molecular markers. Geno- Taq polymerase (Invitrogen), and 40 ng genomic DNA in a 50- types were assigned according to the presence or absence of the AL reaction volume. The PCR conditions included 1-minute 6-bp sequence as determined by fragment lengths: 6 bp (148 bp, denaturation at 94jC, 35 cycles of 94jC for 30 seconds, 55jC reference) or 0 bp (142 bp, deletion). for 30 seconds, and 72jC for 1.5 minutes, with a final 5-minute Statistical Methods. The sample size for this trial extension at 72jC in a Perkin-Elmer 9700 Thermal Cycler. (ultimately cohort 2) was governed by the primary objective of Polymorphisms were detected by direct sequencing (ABI 377 response rate (complete or partial) and powered to reject the null DNA Sequencer, Applied Biosystems) of amplified sequences hypothesis of 30% with a one-sided significance level of 5% (a using primers F-273, R361 (5V-AAAAATAAGT- = 0.05). This provided at least 80% power to reject the null CAAAAATGTC-ATTGT-3V), Fsp (5V-GGAGGAACATTTAT- hypothesis at a response rate of 50% with a sample of 45 TATGCCACCG-3V), and RI337 primers. patients. The overall significance level was 0.028 and the overall Observed haplotypes were designated UGT1A9HI power was 0.814. A total of 50 patients allowed for an (10GCGAC), UGT1A9HII (9GCGTA), UGT1A9HIII approximate dropout or nonevaluable rate of 10%. (9GCATA), UGT1A9HIII (9ACGTA), and UGT1A9HIV Two-sided tests of statistical significance were used to (9GTGTA) for the following polymorphisms detected in 66 determine statistically significant P values (P < 0.05) using SAS

software. A permutation procedure based on an exact test was Table 1 Demographics, overall response and toxicity of study subjects applied to check Hardy-Weinberg equilibrium of each marker, Characteristics Eligible patients (N = 67) and P < 0.05 indicated a lack of agreement with Hardy-Weinberg Subjects equilibrium (28). Haplotype frequencies and their corresponding Assessable for genotyping* 66 variance were estimated by expectation-maximization algorithm Assessable for responsey 56 and jackknife method, respectively. Toxicity was dichotomized Assessable for toxicityz 66 with toxicity grades 3 or 4 as one category and grade V2as Median age (range), y 61 (40-81) another. Statistical associations between genotypes and drug Race/ethnicity Caucasian 55 response or toxicity were examined using a two-sided Fisher’s African American 9 exact test for each individual marker. The exact test of Cochran- Samoan 1 Armitage trend test was done to detect the direction of the Hispanic 1 relationship between toxicity or response rate and genotype Sex Male 36 activity bins. Associations between genotype groups and clinical Female 30 measurements were examined using the nonparametric Kruskal- Median treatment cycles (range) 9 (1-12) Wallis test and the Jonckheere-Terpstra test. Karnofsky performance status (%) For the purposes of genotype/response analyses, we binned 100 36 genotypes into functional categories based on published 90 23 80 6 descriptions of the alleles or genotypes of interest. UGT1A1 70 1 genotypes were grouped into high (UGT1A1 5/6 and UGT1A1 Liver metastasis 46 6/6), moderate (UGT1A1 6/7), and low (UGT1A1 7/7 and Overall response rate (n = 67) 45% (30) Cohort 1 (n = 15) 47% (7) UGT1A1 7/8) activities (19, 20). UGT1A6 was defined by high Cohort 2 (n = 52) 44% (23) (UGT1A6*2/*2), moderate (UGT1A6*1/*1), low (UGT1A6*1/ Grade 3 or 4 diarrhea alone 29% (19) *2), and undefined/unknown activities (UGT1A6*1/*3 and Cohort 1 53% (8) UGT1A6*1/*4; 15). UGT1A7 genotypes were categorized into Cohort 2 25% (13) Grade 3 or 4 neutropeniax alone 1% (1) high (UGT1A7*1/*1), moderate (UGT1A7*1/*2 and Grade 3 or 4 diarrhea and Grade 3 or 4 3% (2) UGT1A7*1/*3), low (UGT1A7*2/*2 and UGT1A7*3/*3), and neutropenia undefined/unknown (UGT1A7*2/*3) activity (14, 29). For Grade 3 or 4 diarrhea and/or neutropenia 33% (22) Cohort 1 53% (8) UGT1A9, genotypes and polymorphisms were analyzed indi- Cohort 2 27% (14) vidually for associations with toxicity and response due to the *Subjects (n = 66) were genotyped (DNA was not obtained from lack of functional data regarding these variations. However, a one subject). recent publication reported 2.6-fold higher transcriptional yResponse status was evaluated for subjects receiving at least two activity associated with the 118 (dT)10 allele compared with cycles of treatment and with follow-up data (10 subjects failed to the 118 (dT) allele based on in vitro transcriptional reporter complete at least two cycles of treatment). 9 zReported toxicities were evaluated for the first two cycles of assays (16). The TS promoter activities were defined as high treatment. (3G/3G), moderate (2/3G and 3G/3C), and low activity (2/2, xGrade 3 or 4 neutropenia was observed only in cohort 2. 2/3C, and 3C/3C). A separate analysis of high (3G/3G, 2/3G, and 3G/3C) and low activity (2/2, 2/3C, and 3C/3C) was also completed due to discrepancies in the literature (23, 24). Finally, Allele and Genotype Frequencies and Hardy-Weinberg V the 3 -UTR TS binning included high (6/6), moderate (0/6), and Equilibrium. We evaluated the predictive value of genotypes low (0/0) activities (26, 30). within the UDP-glucuronosyltransferases (UGT1A1, UGT1A6, UGT1A7, and UGT1A9) as well thymidylate synthase. Table 2 lists the observed allele frequencies, whereas Table 3 lists RESULTS genotype frequencies. There were no significant differences in A total of 67 patients with metastatic colorectal cancer allele frequencies between cohorts 1 and 2. Therefore, these were enrolled in this prospective phase II trial, and germline cohorts were combined for genotype/phenotype analyses. With DNA was available for 66 of these patients. Of those 66 the exception of UGT1A9 and TS promoter polymorphisms, all subjects, response and toxicity data were collected from 56 and genotypes were observed in Hardy-Weinberg equilibrium 66 subjects, respectively. Fifty-five subjects (83%) were Cau- (Table 2). The fairly rare UGT1A1 TA and TA alleles were casian, nine (14%) were African American, one was Samoan, 5 8 and one was Hispanic (Table 1). The overall objective response observed only in African American patients at frequencies rate (partial or complete response) was 45% (30 of 67 patients). consistent with published values (19, 31). The genotype The incidence of severe or life-threatening diarrhea or frequencies observed for UGT1A1 6/6, UGT1A1 6/7, and neutropenia (grade 3 or 4, National Cancer Institute Common UGT1A1 7/7 (Table 3) agreed with that reported in a random Toxicity Criteria version 2.0) was 33% (22 of 66 subjects). population (13, 20) despite the exclusion of patients with Nineteen of 66 subjects (29%) developed grade 3 or 4 diarrhea elevated bilirubin, a phenotype associated with the UGT1A1 TA7 alone, one subject (1%) exhibited grade 3 or 4 neutropenia allele (10, 32). Hence, the distribution of UGT1A1 alleles alone, and two subjects (3%) experienced both diarrhea and followed Hardy-Weinberg equilibrium (Table 2). Allele and neutropenia within the first two cycles of treatment. genotype frequencies for UGT1A6 (Tables 2 and 3) agreed with

Table 2 Observed allele frequencies population (n = 53) of mixed ethnicity, mainly comprised of 4 Allele Caucasians (59.4% for n = 9 and 40.6% for n = 10 repeats). Nomenclature Polymorphism frequencies (%) HWE P We identified two novel alleles within the TS promoter, defined by one repeat (1R) from a Caucasian subject and a 2R UGT1A1*1 (TA)6 repeat 65.9 0.51 UGT1A1*28 (TA)7 repeat 30.3 allele with a novel SNP (30C > T) in the second repeat from UGT1A1*36 (TA)5 repeat 1.5 a patient of undefined ethnicity (Tables 2 and 3). The rare 4R UGT1A1*37 (TA)8 repeat 2.3 and 9R alleles were detected in two subjects of non-Caucasian UGT1A6*1 S7, T181, R184 64.4 0.61 UGT1A6*2 A7, A181, S184 25.0 ethnicity (31). Allele (Table 2) and genotype (Table 3) UGT1A6*3 A7 6.1 frequencies for the TS 3V-UTR polymorphism were within UGT1A6*4 A7, S184 4.5 the ranges reported by previous studies (26, 27). However, the UGT1A7*1 N129, R131, W208 43.2 0.39 linkage disequilibrium reported between the 3R TS promoter UGT1A7*2 K129, K131 25.8 V UGT1A7*3 K129, K131, R208 31.1 and 0 bp 3 -UTR polymorphisms (26, 30) was not observed in UGT1A7*4 R208 0 this study. UGT1A9 HI 10GCGAC 43.2 0.02 Genotype Correlations with Tumor Response. We UGT1A9 HII 9GCGTA 34.1 observed an association between the low activity UGT1A7*2/*2 UGT1A9 HIII 9GCATA 17.4 and UGT1A7*3/*3 genotypes and the UGT1A9 (dT) UGT1A9 HIV 9ACGTA 3.0 9 UGT1A9 HV 9GTGTA 2.3 polymorphism and increased tumor response (complete or UGT1A9 n = 9 repeat 56.8 partial, Tables 5 and 6). Among individuals with low activity 118 (dT)9y UGT1A7*2/*2 and UGT1A7*3/*3 genotypes, the response rate UGT1A9 n = 10 repeat 43.2 was 85% (P = 0.013, 11 of 13 patients responded compared 118 (dT)10 TS 3VUTR (bp) 6-bp deletion 0.56 with 44% or 19 of 43 patients with other genotypes, Table 5). 6 TTAAAG present 63.6 Furthermore, a statistically significant trend was observed for 0 TTAAAG absent 36.4 increased response across UGT1A7 genotypes (p for trend = TS promoter 28 bp repeats and 0.0001 0.017; Table 5). A statistically significant trend was also 58G > C observed between the UGT1A9 118 (dT) repeat and tumor 1R 1 repeat 0.8 n 2R 2 repeats 42.4 response (p for trend = 0.033, Table 6). The 118 (dT)9/9 3R 3 repeats 53.0 genotype was significantly associated with efficacious tumor 3RG 3 repeats and 34.3 response when compared with all other genotypes (p = 0.047; 58G SNP 74% or 14 of 19 patients with 118 (dT) versus 43% or 16 3RC 3 Repeats and 17.9 9/9 58C SNP of 37 patients with other genotypes). 3RC/3RG 3 repeats, ratio 54.5 We found no significant association between UGT1A1 of SNPs genotype and tumor response. Although not statistically 4R 4 repeats 0.8 significant, subjects with low UGT1A1 enzyme activity 9R 9 repeats 0.8 responded better to treatment (83% for UGT1A1 7/7 or NOTE. UGT1A9 haplotypes were defined by the following UGT1A1 7/8 genotype relative to 46% for high activity sequence polymorphisms: 118 (dT)n repeat (n = 9 or 10), 87G >Ay, and intronic SNPs I143C > Ty, I152G > Ay, I219A > Ty, and UGT1A1 5/6 or UGT1A1 6/6, Table 7). No significant associ- I313C > Ay (y, not previously reported in the literature) as also ations or trends were observed between UGT1A6 genotype described in Table 4. A total of 66 subjects, including 55 Caucasians, (Table 8), TS polymorphisms, or overall UGT1A9 haplotypes were genotyped. P < 0.05 indicate a lack of Hardy-Weinberg and tumor response. equilibrium (HWE, values were calculated for Caucasians). Genotype Correlation with Toxicity. Twenty-two sub- yThe 118 (dT)n polymorphism has been designated UGT1A9*22 by the UGT Nomenclature Committee (http://som.flinders.edu.au/FUSA/ jects developed grade 3 or 4 diarrhea or neutropenia (33%, ClinPharm/UGT/). Table 1). The predominant toxicity observed was diarrhea (21 subjects) as opposed to neutropenia (three subjects). We observed a striking correlation between the low activity UGT1A7 genotypes (UGT1A7*2/*2 and UGT1A7*3/*3) and our previous study (15). UGT1A7 allele and genotype lack of drug toxicity ( p = 0.003, Table 5). The UGT1A9 frequencies were as expected from the literature (13, 14), 118 (dT) genotype was also associated with a lower although we did not observe UGT1A7*4 (Table 3). 9/9 incidence of toxicity ( p = 0.002, Table 6). We found no We did not detect the C3Y, M33T, Y242X, or D256N statistically significant association between UGT1A1 genotype polymorphisms described for UGT1A9 (14, 21, 22). However, and toxic events. However, none of the six subjects with low we did identify the 118 (dT) polymorphism as well as four n activity UGT1A1 genotypes (UGT1A1 7/7 or UGT1A1 7/8) SNPs within intron sequences of the UGT1A9 gene (Tables 2 experienced toxicity (Table 7). There was no significant and 3) that defined a total of five haplotypes (Table 4). The 118 association between UGT1A6 genotype and incidence of (dT)n repeat and intron 1 SNPs I219 and I313 were completely linked in this study population. UGT1A9 genotypes were in toxicity, but none of the five patients with high activity agreement with Hardy-Weinberg equilibrium (Table 4), although the haplotype frequencies were not (Table 2). Although this study was completed in a diseased population, haplotype frequencies closely resembled those observed within a healthy 4 Unpublished data.

Table 3 Observed genotype frequencies Genotype % Frequency (n) Genotype % Frequency (n) UGT1A1 5/6 [*36/*1] 1.5 (1) UGT1A6*1/*1 42.4 (28) UGT1A1 5/8 [*36/*37] 1.5 (1) UGT1A6*1/*2 31.8 (21) UGT1A1 6/6 [*1/*1] 42.4 (28) UGT1A6*1/*3 7.6 (5) UGT1A1 6/7 [*1/*28] 43.9 (29) UGT1A6*1/*4 4.5 (3) UGT1A1 6/8 [*1/*37] 1.5 (1) UGT1A6*2/*2 7.6 (5) UGT1A1 7/7 [*28/*28] 7.6 (5) UGT1A6*2/*3 3.0 (2) UGT1A1 7/8 [*28/*37] 1.5 (1) UGT1A6*3/*4 1.5 (1) UGT1A6*4/*4 1.5 (1) UGT1A7*1/*1 15.2 (10) UGT1A9 HI/HI 15.2 (10) UGT1A7*1/*2 24.2 (16) UGT1A9 HI/HII 37.9 (25) UGT1A7*1/*3 31.8 (21) UGT1A9 HI/HIII 18.2 (12) UGT1A7*2/*2 9.1 (6) UGT1A9 HII/HII 9.1 (6) UGT1A7*2/*3 9.1 (6) UGT1A9 HII/HIII 7.6 (5) UGT1A7*3/*3 10.6 (7) UGT1A9 HII/HIV 3.0 (2) UGT1A9 HIII/HIII 4.5 (3) TS 3VUTR 6/6 37.9 (25) UGT1A9 HIII/HV 1.5 (1) TS 3VUTR 0/6 51.5 (34) UGT1A9 HIV/HIV 1.5 (1) TS 3VUTR 0/0 10.6 (7) UGT1A9 HV/HV 1.5 (1) TS promoter 1Ry/2R 1.5 (1) 2R/2R total 19.7 (13) 2R/2RC 18.2 (12) 2R/2RTy 1.5 (1) 2R/3R total 45.5 (30) 2R/3RC 19.7 (13) 2R/3RG 25.8 (17) 2R/4R 1.5 (1) 2R/9R 1.5 (1) 3R/3R total 30.3 (20) 3RC/3RG 1.5 (1) 3RC/3RC 7.6 (5) 3RG/3RG 21.1 (14) NOTE. UGT1A1 allele designations are presented as TA repeat numbers and cross-referenced with the nomenclature suggested by the UGT Nomenclature Committee in square brackets (http://som.flinders.edu.au/FUSA/ClinPharm/UGT/). yNovel alleles not previously reported in the literature.

UGT1A6*2/*2 genotype experienced toxicity (Table 8). There for the UGT1A9 118 (dT)n repeat. Two of the subjects with was no association between TS polymorphisms and incidence neutropenia also exhibited grade 3 diarrhea; both patients of toxicity. possessed the UGT1A7*1/*2 genotype. Because the UGT1A1 promoter polymorphism has been Linkage Disequilibrium/Haplotyping Analysis. We ob- associated with neutropenia, it is of interest to report the served UGT1A haplotype frequencies similar to those obtained by genotypes specifically of the individuals that experienced Kohle et al. (13) for UGT1A1, UGT1A6, and UGT1A7 (Table 9). neutropenia. Three subjects (4.5%) developed grade 3 Additionally, we expanded our haplotype analysis to include the neutropenia. Two subjects possessed the UGT1A1 6/7 UGT1A9 118 (dT)n repeat. We considered only this polymor- genotype coupled with either the UGT1A7*1/*2 or phism for UGT1A9 because the observed UGT1A9 haplotypes UGT1A7*1/*3 genotype, whereas the other subject possessed were not in Hardy-Weinberg equilibrium (Tables 2 and 4). We UGT1A1 6/8 and UGT1A7*1/*2. All three were heterozygous discovered that the low activity UGT1A7*2 and UGT1A7*3

Table 4 UGT1A9 haplotype analysis Nucleotide position

Haplotype 118 (dT)n 87 I143 I152 I219 I313 Frequency (%) [95% confidence limits] I 10 G C G A C 42.5 [34.7, 50.4] II 9 G C G T A 34.3 [26.2, 42.5] III 9 G C A T A 17.9 [11.0, 24.8] IV 9 A C G T A 3.0 [0, 6.5] V 9 G T G T A 2.2 [0, 6.8] HWE p 0.59 0.058 1.0 0.35 0.59 0.59 NOTE. Haplotype assignment was completed using the expectation-maximization algorithm from the SAS statistical package; all observed haplotypes are shown. p < 0.05 suggests deviance from Hardy-Weinberg equilibrium (HWE was calculated for Caucasians only, n = 55). Haplotypes were assigned according to relative observed frequencies (Table 2) that were nearly identical to the estimated frequencies shown above.

Table 5 UGT1A7 genotypes with reduced enzyme activity (UGT1A7*2/*2 and UGT1A7*3/*3) are associated with improved efficacy and reduced toxicity UGTIA7 Genotype bins 12 34 *1/*1 *1/*2 and *1/*3 *2/*2 and *3/*3 *2/*3 Total Outcome Nonresponder 6 (67%) 15 (54%) 2 (15%) 3 (50%) 26 (46%) Responder* 3 (33%) 13 (46%) 11 (85%) 3 (50%) 30 (54%) Total 9 [16%] 28 [50%] 13 [23%] 6 [11%] 56 [100%] No toxicity 7 (70%) 19 (51%) 13 (100%) 5 (83%) 44 (67%) Toxicityy 3 (30%) 18 (49%) 0 (0%) 1 (17%) 22 (33%) Total 10 [15%] 37 [56%] 13 [20%] 6 [9%] 66 [100%]

Statistical analysis Response Toxicity p for all genotype binsz 0.063 0.005 p of bin 3 versus all other genotypesz 0.013 0.003 p of trend test across genotype bins 1, 2, and 3x 0.017 0.085 NOTE. No. subjects per genotype bin is shown with % indicated outcome shown in parentheses. The square brackets denote % total population with the indicated genotype. p < 0.05 is considered statistically significant. *Responder = complete or partial objective response. yToxicity = grade 3 or 4 diarrhea or neutropenia. zFisher’s exact test. xExact test of Cochran-Armitage trend test.

alleles were completely associated with the UGT1A9 118 (dT)9 genes predicted for tumor response as well as development of allele, whereas the high activity UGT1A7*1 allele was linked with diarrhea during combination irinotecan/capecitabine therapy and

the UGT1A9 118 (dT)10 allele (Table 9, likelihood ratio test of second, low activity UGT1A1 alleles did not predict for the association p < 0.0001). Such clearly defined associations across development of diarrhea. We observed associations between haplotypes were not observed for UGT1A1 or UGT1A6. Finally, UGT1A7 and UGT1A9 genotypes with both tumor response and a statistically significant association was identified between toxic events (Tables 5 and 6). We also found that the haplotype I and patient response and toxicity ( p = 0.04 and UGT1A9 118 (dT)9/9 genotype was predictive for efficacious p = 0.035, respectively, Table 9). tumor response with lower incidence of toxicity (Table 6), whereas the UGT1A9 118 (dT)10/10 genotype predicted for poor tumor response. The functional consequences of the DISCUSSION UGT1A9 promoter polymorphism are not well established. This study was significant for two major observations. However, the 118 (dT)10 allele has recently been associated First, genetic variation in the human UGT1A7 and UGT1A9 with 2.6-fold greater transcriptional activity than the 118 (dT)9

Table 6 UGT1A9 118 (dT)9/9 genotype is associated with improved efficacy and reduced toxicity

Genotype bins for UGT1A9 118 (dT)n 123 9/9 9/10 10/10 Total Outcome Nonresponder 5 (26%) 15 (54%) 6 (67%) 26 (46%) Responder* 14 (74%) 13 (46%) 3 (33%) 30 (54%) Total 19 [34%] 28 [50%] 9 [16%] 56 [100%] No toxicity 18 (95%) 19 (51%) 7 (70%) 44 (67%) Toxicityy 1 (5%) 18 (49%) 3 (30%) 22 (33%) Total 19 [29%] 37 [56%] 10 [15%] 66 [100%]

Statistical analysis Response Toxicity p for all genotype binsz 0.081 0.002 p of bin 1 versus all other genotypesz 0.047 0.002 p of trend test across genotype bins 1, 2, and 3x 0.033 0.07 NOTE. No. subjects per genotype bin is shown with % indicated outcome shown in parentheses. The square brackets denote the % total population with the indicated genotype. Ps < 0.05 are considered statistically significant. *Responder = complete or partial objective response. yToxicity = grade 3 or 4 diarrhea or neutropenia. zFisher’s exact test. xExact test of Cochran-Armitage trend test.

Table 7 UGT1A1 genotypes did not significantly associate with efficacy and toxicity UGTIA1 Genotype bins 123 5/6 and 6/6 6/7 7/7 and 7/8 Total Outcome Nonresponder 13 (54%) 10 (42%) 1 (17%) 24 (44%) Responder* 11 (46%) 14 (58%) 5 (83%) 30 (56%) Total 24 [44%] 24 [44%] 6 [12%] 54 [100%] No toxicity 19 (66%) 18 (62%) 6 (100%) 43 (67%) Toxicityy 10 (34%) 11 (38%) 0 (0%) 21 (33%) Total 29 [45%] 29 [45%] 6 [10%] 64 [100%]

Statistical analysis Response Toxicity p for all genotype binsz 0.25 0.28 p of bin 3 versus all other genotypesz 0.21 0.17 p of trend test across genotype bins 1, 2, and 3x 0.15 0.41 NOTE. No. subjects per genotype bin is shown with % indicated outcome shown in parentheses. The square brackets denote % total population with the indicated genotype. Ps < 0.05 are considered statistically significant. *Responder = complete or partial objective response. yToxicity = grade 3 or 4 diarrhea or neutropenia. zFisher’s exact test. xExact test of Cochran-Armitage trend test. allele (16). If these in vitro studies are predictive of in vivo polymorphisms and the incidence of irinotecan toxicity or tumor function (which remains to be proven), then we would predict response. that alleles containing the UGT1A9 (dT)9 polymorphism might The present study allowed us to evaluate UGT1A confer low activity relative to alleles defined by the UGT1A9 haplotypes (Table 9). This is an important component of this (dT)10 polymorphism. study because the human UGT1A genes comprise a unique Only one previous study has examined the association of nested gene structure on human chromosome 2 allowing for UGT1A7 pharmacogenetics in patients receiving irinotecan and linkage disequilibrium among these genes (13). This raises the those investigators reported no significant association between possibility that association between UGT1A alleles and specific UGT1A7 genotype and the occurrence of toxicity (33). However, phenotypes may be due to linkage disequilibrium. We observed it is important to note that the frequencies of the UGT1A7*2 and complete linkage between the UGT1A7 low activity alleles UGT1A7*3 alleles are significantly lower in the Japanese (UGT1A7*2 and UGT1A7*3) and the UGT1A9 (dT)9 polymor- population, so that the power to detect associations in that study phism (Table 9). Both enzymes have been reported to catalyze may have been compromised. To our knowledge, no previous the glucuronidation of SN-38 with significant efficiency (6, studies have addressed the association between UGT1A9 7, 34). UGT1A9 is expressed in the colon and liver and might be

Table 8 UGT1A6 genotypes did not significantly associate with efficacy and toxicity UGTIA6 Genotype bins 123 4 *2/*2 *1/*1 *1/*2 *1/*3 and *1/*4 Total Outcome Nonresponder 2 (40%) 12 (50%) 8 (47%) 3 (50%) 25 (48%) Responder* 3 (60%) 12 (50%) 9 (53%) 3 (50%) 27 (52%) Total 5 [10%] 24 [46%] 17 [33%] 6 [11%] 52 [100%] No toxicity 5 (100%) 19 (68%) 12 (57%) 4 (50%) 40 (65%) Toxicityy 0 (0%) 9 (32%) 9 (43%) 4 (50%) 22 (35%) Total 5 [8%] 28 [45%] 21 [34%] 8 (13%) 62 [100%]

Statistical analysis Response Toxicity p for all genotypesz 1.0 0.26 p of bin 1 versus other genotypesz 1.0 0.15 p of trend test across genotype bins 1, 2, and 3x 1.0 0.11 NOTE. No. subjects per genotype bin is shown with % indicated outcome shown in parentheses. The square brackets denote % total population with the indicated genotype. Ps < 0.05 are considered statistically significant. *Responder = complete or partial objective response. yToxicity = grade 3 or 4 diarrhea or neutropenia. zFisher’s exact test. xExact test of Cochran-Armitage trend test.

Table 9 UGT1A haplotype analysis UGT haplotype P

1A1 (TA)n 1A6 1A7 1A9 (dT)n Frequency (%), [95% confidence interval] Response Toxicity I 6 *1 *1 10 40.9 [32.4, 49.5] 0.04 0.035 II 7 *2 *3 9 23.4 [16.0, 30.9] III 6 *1 *2 9 16.6 [9.7, 23.5] IV 6 *3 *3 9 6.1 [2.1, 10.0] V 7 *4 *2 9 3.8 [0, 7.6] VI 7 *1 *2 9 3.1 [.13, 6.1] VII 8 *1 *1 10 2.2 [0, 6.4] VIII 6 *2 *3 9 1.6 [0, 3.7] IX 5 *1 *2 9 1.5 [0, 3.8] X 6 *4 *2 9 0.8 [0, 2.3] XI 8 *1 *2 9 0.03 [0, 2.1]

UGT haplotype

UGT1A7 UGT1A9 (dT)n Frequency (%), [95% confidence interval] I *1 10 43.2 [35.3, 51.1] II *3 9 31.1 [22.9, 39.2] III *2 9 25.8 [17.8, 33.7] NOTE. The haplotype analysis was completed using the expectation-maximization algorithm from the SAS statistical package. All alleles and polymorphisms are in Hardy-Weinberg equilibrium; all derived haplotype frequencies and their 95% confidence limits are shown. Only statistically significant Ps are shown for haplotype associations with response and toxicity.

predicted to affect plasma levels of SN-38 (35, 36). UGT1A7 is We suggest that different dosing regimens of irinotecan not expressed in the liver, but is expressed in the proximal predispose patients to different toxicities. Our study differed gastrointestinal tract and may influence the disposition of SN-38 from the previous studies in that subjects received combination within the gut (35, 36). Based on our current knowledge of tissue irinotecan (100 or 125 mg/m2 on days 1 and 8) with oral expression patterns, one might speculate that UGT1A9 is more capecitabine therapy. With this regimen, we observed diarrhea likely to affect irinotecan disposition than is UGT1A7; however, to be the predominant toxicity (22 subjects with grade 3 or 4 the UGT1A7 alleles are clearly functionally significant and it toxicity: 19 with diarrhea alone, two with diarrhea and is possible that UGT1A7 is expressed (either constitutively or neutropenia, and one with neutropenia alone). This provided inducibly) in as yet undetermined tissues that might influence us with a unique opportunity to study the association between SN-38 disposition. Therefore, it is premature at this time to UGT alleles and specifically irinotecan-induced diarrhea. We predict which of the two genes is more likely the biological observed that low activity alleles protected subjects from connection to the observed responses to irinotecan. Overall, we developing diarrhea, rather than predisposing for toxicity as determined that low activity UGT genotypes were associated has been observed for irinotecan-associated neutropenia. with better tumor response (Tables 5 and 6). This is consistent This apparent dichotomous set of observations is plausible with the notion that low UGT activity might predict for higher when one considers the complex pharmacology of irinotecan. concentrations of plasma SN-38 and increased tumor concen- Glucuronidation represents an elimination pathway from the tration of SN-38. plasma. Hence, low capacity to glucuronidate results in Previous studies have described an association between the increased plasma levels of SN-38 (11) and increased suscep- UGT1A1 TA7 promoter polymorphism and the occurrence of tibility to systemic toxicities such as neutropenia (10–12, 33). irinotecan-induced toxicity (10–12). We were surprised that we However, glucuronidation also represents a route of delivery of did not observe the expected association between UGT1A1 low SN-38 to the gastrointestinal tract. In humans, renal excretion activity genotypes (UGT1A1 7/7 or UGT1A1 7/8) and increased of irinotecan and its metabolites accounts for <20% of the dose incidence of toxicity (Table 7). Indeed, although not statistically with most of the remaining elimination, particularly for SN- significant, we observed the opposite trend. Of the six subjects 38G, occurring via the biliary route (37–39). It is well with low activity genotypes, none experienced toxicity. The documented that bacterial glucuronidases within the gut distribution of UGT1A1 genotypes in this study was reflective of catalyze the deglucuronidation of many drugs, including SN- a random population although patients were excluded with high 38G (reviewed in ref. 40). Therefore, glucuronidation of SN-38 serum bilirubin levels. A careful study of the primary literature directs its delivery to the gut where the toxic SN-38 molecule regarding UGT1A1 pharmacogenetics suggests a clear link can be regenerated to promote lesions within the mucosa of the between low activity alleles and plasma ratio of SN-38/SN-38G gastrointestinal tract leading to diarrhea. Thus, glucuronidation as well as the development of neutropenia (10–12, 33), but the detoxifies SN-38 in the liver, contributing to lower systemic association between these alleles and the development of circulation of SN-38 and protects against systemic toxicity diarrhea has been much weaker. That is true, in part, because including neutropenia; but the glucuronidation pathway, in neutropenia was the predominant dose-limiting toxicity observed directing SN-38G to the gut, may predispose for irinotecan- in these studies. induced diarrhea.

Clinical and animal studies seem to support this hypothesis. metabolism of irinotecan (CPT-11). Role of uridine diphosphate At least two clinical pharmacokinetic studies suggested that glucuronosyltransferase isoform 1A1 in the glucuronidation of its active neutropenia, but not diarrhea, was significantly associated with metabolite (SN-38) in human liver microsomes. J Clin Invest 1998;101:847–54. the AUC for CPT-11 and/or SN-38 (11, 41). In rats, the activity 5. Ciotti M, Basu N, Brangi M, Owens IS. Glucuronidation of 7-ethyl- of bacterial h-glucuronidase, the enzyme that generates SN-38 10-hydroxycamptothecin (SN-38) by the human UDP-glucuronosyl- from SN-38G in the gut, correlated with irinotecan-induced cecal transferases encoded at the UGT1 locus. Biochem Biophys Res Commun damage and was attenuated by administration of antibiotics (42) 1999;260:199–202. or of a specific h-glucuronidase inhibitor (43). Furthermore, 6. 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