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Background Thiopurines are used in the treatment of inflammatory bowel disease. They are metabolised via methylation by thiopurine-S-methyltransferase (TPMT), which displays a genetically determined polymorphic activity. Subjects with reduced TPMT activity have a higher concentration of active thiopurine metabolites and may be at increased risk of bone-marrow suppression. Aims To evaluate the relevance of TPMT genotyping in the management of thiopurines therapy in inflammatory bowel disease patients. Patients and methods Adverse effects and clinical response were determined retrospectively and correlated with TPMT genotype in 70 paediatric inflammatory bowel disease patients. Results Nineteen patients (27.1%) developed adverse effects; of the 51 who did not, 34 (66.7%) responded to treatment. Five patients (7.1%) were heterozygous for a variant TPMT allele; two of these (40%) were intolerant to thiopurines, compared to 17 of the 65 patients (26.2%) with a wild type gene (O.R. 1.88, 95% CI 0.29–12.2, p = 0.61); among the 34 responders, the median dosage of the drug required to obtain remission was lower for mutated than for wild type patients (1.6 mg kg−1 day−1 versus 2.0 mg kg−1 day−1, p = 0.043). Conclusions There was no significant association between adverse effects of thiopurines and TPMT heterozygous genotype, but TPMT genotyping could be useful in establishing the most appropriate dose of thiopurines to start treatment.

In this study we verified whether high performance liquid chromatography with UV detection (HPLC-UV) could be optimised and hence become a useful method for measuring ticlopidine concentrations in patient blood. The extraction step was improved by adding isoamylic alcohol in the extraction mixture, resulting in a better recovery. Moreover, the extraction efficiency was consistently ameliorated by evaporation to dryness of the organic extract; we could therefore utilise a lower (213 nm) wavelength, corresponding to the maximum of absorption, achieving a better sensitivity. With these technical improvements, the limit of quantitation is 0.02 microgram/ml, and the limit of detection is 0.01 microgram/ml, hence comparable to levels obtained with gas-liquid chromatography with nitrogen detection (GC-NPD). In addition, HPLC method, if compared with GC-NPD, is simpler and can be easily used for routine determinations of a lot of serum samples.

The contribution of candidate genetic variants involved in azathioprine biotransformation on azathioprine efficacy and pharmacokinetics in 111 young patients with inflammatory bowel disease was evaluated. Azathioprine doses, metabolites thioguanine-nucleotides (TGN) and methylmercaptopurine-nucleotides (MMPN) and clinical effects were assessed after at least 3 months of therapy. Clinical efficacy was defined as disease activity score below 10. Candidate genetic variants (TPMT rs1142345, rs1800460, rs1800462, GSTA1 rs3957357, GSTM1, and GSTT1 deletion) were determined by polymerase chain reaction (PCR) assays and pyrosequencing. Statistical analysis was performed using linear mixed effects models for the association between the candidate variants and the pharmacological variables (azathioprine doses and metabolites). Azathioprine metabolites were measured in 257 samples (median 2 per patient, inter-quartile range IQR 1-3). Clinical efficacy at the first evaluation available resulted better in ulcerative colitis than in Crohn&rsquo, s disease patients (88.0% versus 52.5% responders, p = 0.0003, linear mixed effect model, LME). TGN concentration and the ratio TGN/dose at the first evaluation were significantly higher in responder. TPMT rs1142345 variant (4.8% of patients) was associated with increased TGN (LME p = 0.0042), TGN/dose ratio (LME p &lt, 0.0001), decreased azathioprine dose (LME p = 0.0087), and MMPN (LME p = 0.0011). GSTM1 deletion (58.1% of patients) was associated with a 18.5% decrease in TGN/dose ratio and 30% decrease in clinical efficacy. GSTA1 variant (12.8% of patients) showed a trend (p = 0.049, LME) for an association with decreased clinical efficacy, however, no significant effect on azathioprine pharmacokinetics could be detected. In conclusion, GSTs variants are associated with azathioprine efficacy and pharmacokinetics.

Azathioprine is a thiopurine immunosuppressive antimetabolite used to chronically treat inflammatory bowel disease and autoimmune hepatitis. Azathioprine treatment is a long-term therapy and therefore it is at risk for non-adherence, which is considered an important determinant of treatment inefficacy. Measurement of 6-thioguanine and 6-methylmercaptopurine nucleotides has been recently suggested as a screener for non-adherence detection. We describe four young patients in which non-adherence to azathioprine therapy was detected only through the measurement of drug metabolite concentrations, and the criterion for non-adherence was undetectable metabolite levels. After the identification of non-adherence, patients and their families were approached and the importance of a correct drug administration was thoroughly enlightened and discussed; this allowed obtaining a full remission in all subjects. Our observations support the use of undetectable metabolite levels as indicators of non-adherence to therapy in azathioprine treated patients. The additional level of medical supervision given by this assay allows getting a better adherence to medical treatment, which results in an improvement in the response to therapy; these benefits may justify the costs associated with the assay.

We report a case of a patient with ulcerative colitis receiving maintenance therapy consisting of a combination of azathioprine and mesalamine, whose disease relapsed when the treatment with the salicylate was interrupted. An analysis of azathioprine metabolites present in the blood of the patient revealed that the concentration of the active metabolites of thiopurine had dropped below the therapeutic range following interruption of the mesalamine therapy and that this could have caused the relapse. Therapeutic monitoring of azathioprine metabolites would therefore seem to be advisable in patients starting on or interrupting a salicylate treatment regimen.