Jacqueline Ramírez

Genetic Variants in the UDP-glucuronosyltransferase 1A1 Gene Predict the Risk of Severe Neutropenia of Irinotecan

PURPOSE Severe toxicity is commonly observed in cancer patients receiving irinotecan. UDP-glucuronosyltransferase 1A1 (UGT1A1) catalyzes the glucuronidation of the active metabolite SN-38. This study prospectively evaluated the association between the prevalence of severe toxicity and UGT1A1 genetic variation. PATIENTS AND METHODS Sixty-six cancer patients with advanced disease refractory to other treatments received irinotecan 350 mg/m(2) every 3 weeks. Toxicity and pharmacokinetic data were measured during cycle 1. UGT1A1 variants (-3279G>T, -3156G>A, promoter TA indel, 211G>A, 686C>A) were genotyped. RESULTS The prevalence of grade 4 neutropenia was 9.5%. Grade 4 neutropenia was much more common in patients with the TA indel 7/7 genotype (3 of 6 patients; 50%) compared with 6/7 (3 of 24 patients; 12.5%) and 6/6 (0 of 29 patients; 0%) (P =.001). The TA indel genotype was significantly associated with the absolute neutrophil count nadir (7/7 < 6/7 < 6/6, P =.02). The relative risk of grade 4 neutropenia was 9.3 (95% CI, 2.4 to 36.4) for the 7/7 patients versus the rest of the patients. Pretreatment total bilirubin levels (mean +/- standard deviation) were significantly higher in patients with grade 4 neutropenia (0.83 +/- 0.08 mg/dL) compared to those without grade 4 neutropenia (0.47 +/- 0.03 mg/dL; P <.001). The -3156G>A variant seemed to distinguish different phenotypes of total bilirubin within the TA indel genotypes. The -3156 genotype and the SN-38 area under the concentration versus time curve were significant predictors of ln(absolute neutrophil count nadir; r(2) = 0.51). CONCLUSION UGT1A1 genotype and total bilirubin levels are strongly associated with severe neutropenia, and could be used to identify cancer patients predisposed to the severe toxicity of irinotecan. The hypothesis that the -3156G>A variant is a better predictor of UGT1A1 status than the previously reported TA indel requires further testing.

UGT1A1*28 polymorphism as a determinant of irinotecan disposition and toxicity

The metabolism of irinotecan (CPT-11) involves sequential activation to SN-38 and detoxification to the pharmacologically inactive SN-38 glucuronide (SN-38G). We have previously demonstrated the role of UGT1A1 enzyme in the glucuronidation of SN-38 and a significant correlation between in vitro glucuronidation of SN-38 and UGT1A1 gene promoter polymorphism. This polymorphism (UGT1A1*28) is characterized by the presence of an additional TA repeat in the TATA sequence of the UGT1A1 promoter, ((TA)7TAA, instead of (TA)6TAA). Here we report the results from a prospective clinical pharmacogenetic study to determine the significance of UGT1A1*28 polymorphism on irinotecan disposition and toxicity in patients with cancer. Twenty patients with solid tumors were treated with a 90 min i.v. infusion of irinotecan (300 mg m−2) once every 3 weeks. The frequency of UGT1A1 genotypes was as follows: 6/6—45%, 6/7—35% and 7/7—20%, with allele frequencies of 0.375 and 0.625 for (TA)7TAA and (TA)6TAA, respectively. Patients with the (TA)7TAA polymorphism had significantly lower SN-38 glucuronidation rates than those with the normal allele (6/6>6/7>7/7, P = 0.001). More severe grades of diarrhea and neutropenia were observed only in patients heterozygous (grade 4 diarrhea, n = 1) or homozygous (grade 3 diarrhea/grade 4 neutropenia, n = 1 and grade 3 neutropenia, n = 1) for the (TA)7TAA sequence. The results suggest that screening for UGT1A1*28 polymorphism may identify patients with lower SN-38 glucuronidation rates and greater susceptibility to irinotecan induced gastrointestinal and bone marrow toxicity.

Phenotype‐genotype correlation of in vitro SN‐38 (active metabolite of irinotecan) and bilirubin glucuronidation in human liver tissue with UGT1A1 promoter polymorphism

Hepatic uridine diphosphate glucuronosyltransferase (UGT) isoform 1A1 (UGT1A1) is primarily responsible for the glucuronidation of SN‐38 (7‐ethyl‐10‐hydroxycamptothecin), the active metabolite of the anticancer agent irinotecan. UGT1A1, also catalyzing the glucuronidation of bilirubin, has been shown to have reduced activity in Gilberts syndrome. The presence of an additional TA repeat [(TA)7 TAA] in the TATA sequence of UGT1A1 has been associated with Gilberts syndrome.

Comprehensive Pharmacogenetic Analysis of Irinotecan Neutropenia and Pharmacokinetics

PURPOSE We aim to identify genetic variation, in addition to the UGT1A1*28 polymorphism, that can explain the variability in irinotecan (CPT-11) pharmacokinetics and neutropenia in cancer patients. PATIENTS AND METHODS Pharmacokinetic, genetic, and clinical data were obtained from 85 advanced cancer patients treated with single-agent CPT-11 every 3 weeks at doses of 300 mg/m(2) (n = 20) and 350 mg/m(2) (n = 65). Forty-two common variants were genotyped in 12 candidate genes of the CPT-11 pathway using several methodologies. Univariate and multivariate models of absolute neutrophil count (ANC) nadir and pharmacokinetic parameters were evaluated. RESULTS Almost 50% of the variation in ANC nadir is explained by UGT1A1*93, ABCC1 IVS11 -48C>T, SLCO1B1*1b, ANC baseline levels, sex, and race (P < .0001). More than 40% of the variation in CPT-11 area under the curve (AUC) is explained by ABCC2 -24C>T, SLCO1B1*5, HNF1A 79A>C, age, and CPT-11 dose (P < .0001). Almost 30% of the variability in SN-38 (the active metabolite of CPT-11) AUC is explained by ABCC1 1684T>C, ABCB1 IVS9 -44A>G, and UGT1A1*93 (P = .004). Other models explained 17%, 23%, and 27% of the variation in APC (a metabolite of CPT-11), SN-38 glucuronide (SN-38G), and SN-38G/SN-38 AUCs, respectively. When tested in univariate models, pretreatment total bilirubin was able to modify the existing associations between genotypes and phenotypes. CONCLUSION On the basis of this exploratory analysis, common polymorphisms in genes encoding for ABC and SLC transporters may have a significant impact on the pharmacokinetics and pharmacodynamics of CPT-11. Confirmatory studies are required.

Pharmacokinetic and pharmacodynamic evaluation of the topoisomerase inhibitor irinotecan in cancer patients

PURPOSE We conducted a pharmacokinetic and pharmacodynamic evaluation of irinotecan (CPT-11) and determined the effect of race and sex on disposition and toxicity of CPT-11. We tested the efficacy of acetaminophen (AAP) to phenotype SN-38 glucuronidation. PATIENTS AND METHODS Forty patients received a dose of 145 mg/m2 of CPT-11 as a 90-minute infusion. Total CPT-11, SN-38, and SN-38G were quantitated in plasma and urine samples. Following administration of 1 g AAP, urinary concentrations of AAP and AAP-glucuronide (AAP-G) were assessed. RESULTS CPT-11 exhibited a mean elimination half-life (t1/2) of 8.8 hours, an average clearance (CL) of 14.6 L/h/m2, and a mean volume of distribution at steady-state (Vdss) of 136 L/m2. SN-38 and SN-38G had low plasma availabilities (3% and 10% relative to CPT-11), with mean t1/2 values of 11.6 and 10.5 hours, respectively. Urinary recovery accounted for 15% of the dose. Race and sex had no effect on the plasma availability of CPT-11, SN-38, and SN-38G. The applicability of biliary index (BI) in predicting dose-limiting intestinal toxicity was validated. Patients who developed grade 3 or 4 toxicity had significantly higher index values compared with patients with grade 0 to 2 toxicity (P = .001). There was no difference in the incidence and severity of toxicity based on race and sex. AAP was a poor predictor of SN-38 glucuronidation. CONCLUSION The high degree of interpatient variability in parameter estimates suggests pharmacogenetic variation or differential induction or inhibition of the sequential metabolic pathway of CPT-11, as well as variability in transport systems. The low urinary recovery indicates substantial biliary excretion and supports the significant correlation between intestinal toxicity and BI. Black patients are not at increased risk of toxicity. An assessment of individual differences in SN-38 conjugation remains to be established.

Pharmacogenomic and Pharmacokinetic Determinants of Erlotinib Toxicity

PURPOSE To assess the pharmacogenomic and pharmacokinetic determinants of skin rash and diarrhea, the two primary dose-limiting toxicities of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib. PATIENTS AND METHODS A prospective clinical study of 80 patients with non-small-cell lung cancer, head and neck cancer, and ovarian cancer was performed. Detailed pharmacokinetics and toxicity of erlotinib were assessed. Polymorphic loci in EGFR, ABCG2, CYP3A4, and CYP3A5 were genotyped, and their effects on pharmacokinetics and toxicities were evaluated. RESULTS A novel diplotype of two polymorphic loci in the ABCG2 promoter involving -15622C/T and 1143C/T was identified, with alleles conferring lower ABCG2 levels associated with higher erlotinib pharmacokinetic parameters, including area under the curve (P = .019) and maximum concentration (P = .006). Variability in skin rash was best explained by a multivariate logistic regression model incorporating the trough erlotinib plasma concentration (P = .034) and the EGFR intron 1 polymorphism (P = .044). Variability in diarrhea was associated with the two linked polymorphisms in the EGFR promoter (P < .01), but not with erlotinib concentration. CONCLUSION Although exploratory in nature, this combined pharmacogenomic and pharmacokinetic model helps to define and differentiate the primary determinants of skin and gastrointestinal toxicity of erlotinib. The findings may be of use both in designing trials targeting a particular severity of rash and in considering dose and schedule modifications in patients experiencing dose-limiting toxicities of erlotinib or similarly targeted agents. Further studies of the relationship between germline polymorphisms in EGFR and the toxicity and efficacy of EGFR inhibitors are warranted.

A pharmacogenetic study of uridine diphosphate-glucuronosyltransferase 2B7 in patients receiving morphine

We investigated the variation in the uridine diphosphate–glucuronosyltransferase 2B7 (UGT2B7) gene in patients receiving patient‐controlled analgesia with morphine. UGT2B7 was sequenced in phenotypic extremes (n = 12) of the distribution of morphine‐6‐glucuronide/morphine plasma ratios. A new −161C/T promoter variant was in complete linkage disequilibrium with the 802C/T variant and was more frequent in low glucuronidators (P = .039). Both variants were genotyped in all patients (n = 86), and complete linkage disequilibrium was confirmed. Trend analysis showed reduced morphine‐6‐glucuronide/morphine ratios in patients with T/T, C/T, and C/C genotypes (T/T > C/T > C/C) (P = .031). Morphine levels were lower in T/T patients (median, 18 ng/mL [range, 18–1490 ng/mL]) as compared with C/T and C/C patients combined (median, 66 ng/m; range, 18–3995 ng/mL) (P = .04). Morphine‐6‐glucuronide and morphine‐3‐glucuronide concentrations were significantly lower in C/C patients (median, 18 ng/mL; range, 0–66 ng/mL; and median, 152 ng/mL; range, 30–434 ng/mL; respectively) compared with C/T and T/T patients combined (median, 43 ng/mL; range, 0–193 ng/mL; and median, 242 ng/mL; range, 33–1381 ng/mL; respectively) (P = .045 and P = .004, respectively). Interindividual differences in morphine glucuronidation may be the result of genetic variation in UGT2B7, and further studies are indicated.

Haplotype structure of the UDP-glucuronosyltransferase 1A1 promoter in different ethnic groups.

Genetic variation in UDP-glucuronosyltransferase 1A1 (UGT1A1)expression has several important clinical implications. UGT1A1 basal transcription is affected by a polymorphic (TA)n repeat, and another important regulatory element is the phenobarbital-responsive enhancer module (PBREM) which might contain variants affecting inducible gene expression. We assessed the extent of linkage disequilibrium between the (TA)n polymorphism and variants in the PBREM and UGT1A1 promoter. We also investigated the relationship between PBREM-(TA)n haplotypes and the glucuronidation rate of the UGT1A1 substrate SN-38. DNAs from 83 human livers were genotyped for the (TA)n polymorphism and microsomes from the same livers were phenotyped for SN-38 glucuronidation. The (TA)n polymorphism was genotyped in 24 additional African-Americans included in the Human Variation Panel (Coriell Institute). A 606-bp region spanning the PBREM was sequenced in 81 liver and a subset of 22 Human Variation Panel DNAs and six variants were found. The -3279G T and -3156G A variants are common (0.39 and 0.30, respectively). -3279G T is more common in Caucasians than African-Americans (P = 0.001). In Caucasians, linkage disequilibrium was highly significant between sites -3279, -3156, and the (TA)n polymorphism (P < 0.0001). In contrast, in African-Americans, only marginal levels of significance were observed between (TA)n and -3279 (P = 0.02) and between -3279 and -3156 (P = 0.04). Ten promoter haplotypes were identified. Haplotype I is the most common (0.39), from which haplotype II (0.15) differs at position -3279. SN-38G formation rates were correlated with (TA)n genotypes. This study showed that (i) common promoter variants are in linkage disequilibrium and (ii) the haplotype structure of promoter is probably different between Caucasians and African-Americans.

Modulation of glucuronidation of SN-38, the active metabolite of irinotecan, by valproic acid and phenobarbital

Abstract Purpose: Irinotecan (CPT-11) is hydrolyzed to its active metabolite SN-38 which is subsequently conjugated by uridine diphosphate glucuronosyl transferase (UDP-GT) to the glucuronide (SN-38G). Both preclinical and clinical data indicate that conjugation is a primary clearance mechanism for SN-38 with the plasma glucuronide levels being substantially higher than those of SN-38. This investigation was designed to determine the possibility of modulation of glucuronidation of SN-38 and its effect on the disposition of the parent drug and metabolites. Methods: Female Wistar rats were pretreated with 200 mg/kg valproic acid (VPA), an inhibitor of glucuronidation, 5 min prior to the administration of 20 mg/kg irinotecan. The control rats were given 20 mg/kg irinotecan only. To study the effect of inducers of UDP-GT activity, rats were pre- treated with phenobarbital (PB) before irinotecan administration. Results: Pretreatment with VPA caused a 99% inhibition in the formation of SN-38G leading to a 270% increase in the area under plasma concentration-time curve (AUC) of SN-38 compared with the control rats. The irinotecan estimations were unchanged in the two groups. PB pretreatment caused a 1.7-fold increase in the AUC of SN-38G and a concomitant 31% and 59% reduction in the AUCs of SN-38 and irinotecan, respectively. Conclusions: The most plausible explanation for the alterations in SN-38G disposition is inhibition of SN-38 conjugation by VPA and induction of the conjugation by PB.

Single nucleotide polymorphism discovery and functional assessment of variation in the UDP-glucuronosyltransferase 2B7 gene

Objective UDP-glucuronosyltransferase 2B7 (UGT2B7) plays a central role in the liver-mediated biotransformation of endogenous and exogenous compounds. The genetic basis of interindividual variability in UGT2B7 function is unknown. This study aimed to discover novel gene variants of functional significance. Methods Caucasian human livers (n=54) were used. UGT2B7 was resequenced in 12 samples [(six highest and six lowest for the formation of morphine-3-glucuronide (M3G)]. Haplotype-tagging single nucleotide polymorphisms were genotyped in the entire sample set. Samples were phenotyped for mRNA expression. Results 10 haplotype-tagging single nucleotide polymorphisms were identified and their haplotypes were inferred. Haplotype 4 (-45597G; -6682_-6683A; 372A; IVS1+9_IVS1+10A; IVS1+829T; IVS1+985G; IVS1+999C; IVS1+1250G; 801T; IVS4+185C) (frequency of 0.12) was associated with an increase in enzyme activity and gene expression. The 1/4 and 4/6 diplotypes had higher M3G formation compared with 1/1 (P<0.05) and 2/3 (P<0.01) diplotypes. Diplotypes containing haplotype 4 resulted in a significant 45% average increase in the formation of M3G compared with diplotypes without haplotype 4 (P=0.002). There was also an association between haplotype 4 and increased mRNA expression. IVS1+985A>G, 735A>G, and 1062C>T are the putative functional variants of haplotype 4. We also identified two mRNA splicing variants (UGT2B7_v2 and UGT2B7_v3) splicing out exon 1, 4, 5, and 6 but sharing exons 2 and 3 with the involvement of additional 5′ exons. UGT2B7_v2 was detected in all livers tested, but UGT2B7_v3 was present at much lower levels compared with UGT2B7_v2. The UGT2B7 reference sequence mRNA is now named UGT2B7_v1. Conclusion UGT2B7 haplotype 4 is functional and its effects on the biotransformation of UGT2B7 substrates should be tested in controlled clinical trials. Biochemical studies should investigate the functional role of the newly discovered mRNA splicing variants.