Crohn's disease is one of the most common forms of autoimmune chronic inflammatory bowel diseases. An important therapeutic approach to maintain the disease in remission is azathioprine therapy. Due to the narrow therapeutic window of this immunosuppressive drug, severe side effects, such as myelosuppression and hepatotoxicity, may develop during therapy. To avoid this, it is important to plan an effective and safe therapy by determining the appropriate individual dose of azathioprine for the patient based on the following diagnostic approaches: genotyping and measurement of the activity of the enzyme thiopurine S-methyltransferase (TPMT) prior to treatment, and determination of the levels of the main metabolites of azathioprine - 6-thioguanosine nucleotides (6-TG) and 6-methylmercaptopurine (6-MMP) – 4–6 weeks after the initiation of therapy.
The master's thesis included analyses on blood samples from Crohn's disease patients referred from the UKC Ljubljana, Department of Gastroenterology to the Laboratory of Molecular Diagnostics at the Faculty of Pharmacy in order to determine the genotype and activity of TPMT and to analyse the major metabolites of the drug. After DNA isolation from the patients' blood, genotyping of the most frequent TPMT alleles (TPMT *2, *3A, *3C) was performed based on PCR using hydrolysable probes. In a subset of samples, TPMT enzyme activity was further determined in red blood cell lysates based on the conversion of 6-mercaptopurine to 6-MMP using an established HPLC method. The presence of the most common allelic variants TPMT*3A and TPMT*3C was identified in 6.25% and 1.56% of the subjects, respectively; the most common allelic variants TPMT*3A and TPMT*3C were identified in 6.25 % and 1.56 % of the subjects.
We also investigated the influence of the TPMT genotype on the major metabolites and their ratio on previously determined azathioprine metabolites (6-TG and 6-MMP). The presence of a variant TPMT allele was typically associated with higher 6-TG concentrations and a low 6-MMP : 6-TG ratio. We further tested the predictive value for the occurrence of adverse effects based on 6-TG, 6-MMP concentrations and their ratio. According to the literature, a 6-TG concentration > 450 pmol x (8 x 10^8 erythrocytes)^-1 correlates with an increased risk of myelosuppression, and a 6-MMP concentration > 5700 pmol x (8 x 10^8 erythrocytes)^-1 and a 6-MMP : 6-TG ratio > 11 with hepatotoxicity. In our population, one in three subjects had at least one risk factor for azathioprine adverse effects present at the first sample collection. Myelosuppression could be manifested in 20.31 % of subjects and hepatotoxicity in 23.44 % of subjects. By measuring metabolites during the course of therapy, physicians were able to lower these values in most patients when appropriate measures were introduced into the therapy.
The importance of correct dosing and treatment modification based on TPMT genotyping or metabolite measurement was illustrated in two clinical cases. In the case of myelotoxicity, the dosage of azathioprine was switched to a biologic, and in the case of hepatotoxicity, the dose of azathioprine was reduced according to guidelines when allopurinol was introduced.
The results of this Master thesis underline the importance of genotyping and activity determination of TPMT and close monitoring of thiopurine metabolites during treatment. These diagnostic approaches can prevent serious side effects or take appropriate measures leading to disease remission without serious complications.
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