ABSTRACT
Title
Role of genetic polymorphisms of enzymes involved in azathioprine activation and correlation with blood metabolite concentrations in pediatric patientswith IBD
Authors
E. Cuzzoni1, S. De Iudicibus2, G. Stocco1, R. Franca2, F. Bartoli2, S. Martellossi2, A. Ventura2, G. Decorti1
1Department of Life Sciences, University of Trieste, Trieste, Italy; 2Department of Reproductive and Developmental Sciences and IRCCS, Istituto per l'Infanzia Burlo Garofolo, Clinica Pediatrica, Trieste, Italy.
Abstract
The thiopurine azathioprine (AZA) is widely used as an immunosuppressive agent in various chronic inflammatory diseases, including inflammatory bowel disease (IBD), particularly to maintain remission in patients with refractory diseases and as steroid-sparing agent (Barabino A, 2002).
AZA is a pro-drug and requires in vivo conversion to its active form: the first step on its bio-transformation involves conjugation with sulphide groups and the formation of 6MP, which has no intrinsic activity, but is activated by the enzymes of the purine salvage pathway to thioguanine nucleotides (TGN). 6MP is inactivated mainly by oxidation to thiouric acid, catalyzed by xanthine oxidase or through methylation of the thiol to 6-methylmercaptopurine, catalyzed by thiopurine-S-methyl transferase (Lennard L, 1992).
Polymorphisms of enzymes involved in the metabolism of AZA influence the efficacy and toxicity of the treatment. In particular patients with inactivating mutations of thiopurine-S-metyl-transferase (TPMT) have an increased production of the active metabolites TGN and polymorphisms in genes encoding the inosine triphosphate pyrophosphatase (ITPA) enzyme have been associated with AZA toxicity. Glutathione-S-transferases (GST) may also have a role in AZA metabolism: indeed, AZA is converted to 6MP mainly through a reaction with glutathione and, although this reaction is considered to be a non-enzymatic conversion, some studies have shown that GST might be involved (Pierik M, 2006).
The aim of this study was to investigate, in pediatric patients with IBD treated with AZA, the correlation between candidate genetic polymorphisms of proteins involved in AZA metabolism, the concentration of the main metabolites of AZA, TGNs and the methylated nucleotides (MMPN) and the dose of the medication.
77 patients with IBD treated with AZA at least from 3 months (median age: 15 years, 46 with Crohn’s disease, 30 with ulcerative colitis, 1 with indetermined colitis) were enrolled. Concentrations of TGN and MMPN metabolites were measured by HPLC from 156 blood samples. Genotyping of GST-M1, GST-T1, GST-P1, ITPA, TPMT genes was performed using PCR-RFLP or PCR-ASO assays; while genotyping of GST-A2 and AOX was performed using Taqman assay.
Analysis was done considering the median value of the phenotype for each patient and the effect of the genotypes was evaluated using Wilcoxon’s test. TPMT polymorphism had a significant effect on MMPN (p=0.018), AZA dose (p=0.02) and the ratio between TGN metabolites and AZA dose (p=0.005), confirming data already described in the literature. GST-M deletion had significant effects on TGN nucleotides (p=0.033), dose of AZA (p=0.033) and the ratio TGN/dose (p=0.001). An association between the C94A polymorphism of the ITPA gene and the concentration of MMPN (p=0.037) was also observed. Analyzing the clinical response to AZA and the concentration of nucleotides with a logistic regression model, a correlation between clinical response, TGN concentration (p=0.065) and TGN/dose (p=0.0048) was observed.
In conclusion, this study has shown the effect of several genetic polymorphisms on the pharmacokinetics and clinical response to AZA of patients with IBD and contributes to the development of pharmacogenetic strategies of therapy personalization in these patients, in order to improve efficacy and reduce adverse events of AZA treatment.
Barabino A, Torrente F, Ventura A, Cucchiara S, Castro M, Barbera C. “Azathioprine in paediatric inflammatory bowel disease: an Italian multicentre survey.” Aliment Pharmacol Ther 2002;16:1125-30.
Lennard L. “The clinical pharmacology of 6-mercaptopurine.” Eur J Clin Pharmacol 1992;43:329-39
Pierik M, Rutgeerts P, Vlietinck R, Vermeire S. “Pharmacogenetics in inflammatory bowel disease.” World J Gastroenterol 2006;12:3657-67
AZA is a pro-drug and requires in vivo conversion to its active form: the first step on its bio-transformation involves conjugation with sulphide groups and the formation of 6MP, which has no intrinsic activity, but is activated by the enzymes of the purine salvage pathway to thioguanine nucleotides (TGN). 6MP is inactivated mainly by oxidation to thiouric acid, catalyzed by xanthine oxidase or through methylation of the thiol to 6-methylmercaptopurine, catalyzed by thiopurine-S-methyl transferase (Lennard L, 1992).
Polymorphisms of enzymes involved in the metabolism of AZA influence the efficacy and toxicity of the treatment. In particular patients with inactivating mutations of thiopurine-S-metyl-transferase (TPMT) have an increased production of the active metabolites TGN and polymorphisms in genes encoding the inosine triphosphate pyrophosphatase (ITPA) enzyme have been associated with AZA toxicity. Glutathione-S-transferases (GST) may also have a role in AZA metabolism: indeed, AZA is converted to 6MP mainly through a reaction with glutathione and, although this reaction is considered to be a non-enzymatic conversion, some studies have shown that GST might be involved (Pierik M, 2006).
The aim of this study was to investigate, in pediatric patients with IBD treated with AZA, the correlation between candidate genetic polymorphisms of proteins involved in AZA metabolism, the concentration of the main metabolites of AZA, TGNs and the methylated nucleotides (MMPN) and the dose of the medication.
77 patients with IBD treated with AZA at least from 3 months (median age: 15 years, 46 with Crohn’s disease, 30 with ulcerative colitis, 1 with indetermined colitis) were enrolled. Concentrations of TGN and MMPN metabolites were measured by HPLC from 156 blood samples. Genotyping of GST-M1, GST-T1, GST-P1, ITPA, TPMT genes was performed using PCR-RFLP or PCR-ASO assays; while genotyping of GST-A2 and AOX was performed using Taqman assay.
Analysis was done considering the median value of the phenotype for each patient and the effect of the genotypes was evaluated using Wilcoxon’s test. TPMT polymorphism had a significant effect on MMPN (p=0.018), AZA dose (p=0.02) and the ratio between TGN metabolites and AZA dose (p=0.005), confirming data already described in the literature. GST-M deletion had significant effects on TGN nucleotides (p=0.033), dose of AZA (p=0.033) and the ratio TGN/dose (p=0.001). An association between the C94A polymorphism of the ITPA gene and the concentration of MMPN (p=0.037) was also observed. Analyzing the clinical response to AZA and the concentration of nucleotides with a logistic regression model, a correlation between clinical response, TGN concentration (p=0.065) and TGN/dose (p=0.0048) was observed.
In conclusion, this study has shown the effect of several genetic polymorphisms on the pharmacokinetics and clinical response to AZA of patients with IBD and contributes to the development of pharmacogenetic strategies of therapy personalization in these patients, in order to improve efficacy and reduce adverse events of AZA treatment.
Barabino A, Torrente F, Ventura A, Cucchiara S, Castro M, Barbera C. “Azathioprine in paediatric inflammatory bowel disease: an Italian multicentre survey.” Aliment Pharmacol Ther 2002;16:1125-30.
Lennard L. “The clinical pharmacology of 6-mercaptopurine.” Eur J Clin Pharmacol 1992;43:329-39
Pierik M, Rutgeerts P, Vlietinck R, Vermeire S. “Pharmacogenetics in inflammatory bowel disease.” World J Gastroenterol 2006;12:3657-67