Simultaneous Quantification of Eleven Thiopurine Nucleotides by Liquid Chromatography-Tandem Mass Spectrometry

The prodrugs azathioprine and 6-mercaptopurine, which are well-established anticancer and immunosuppressive agents, are extensively metabolized by activating and inactivating enzymes. Whereas the 6-thioguanine nucleotides (TGN) are currently being considered as major active metabolites, methylthioinosine nucleotides seem to contribute to the cytotoxic effect as well. Thiopurine-related adverse drug reactions and thiopurine failure are frequent. Thus, therapeutic monitoring of TGN and methylthioinosine derivatives has been suggested to improve thiopurine therapy, however with limited success. To elucidate systematically underlying molecular mechanisms as potential explanation for interindividual variability of thiopurine response, we developed a novel highly specific and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantitation of eleven mono-, di-, and triphosphates of thioguanosine, methylthioinosine, methylthioguanosine, and thioinosine. Using stable isotope-labeled analogues as internal standards obtained by chemical synthesis, an intra- and interassay variability below 8% and an accuracy of 92% to 107% were achieved in spiked quality control samples with known standards. All eleven metabolites could be determined in red blood cells from patients with inflammatory bowel diseases and long-term azathioprine therapy. Thus, our novel method opens a new avenue for the understanding of the thiopurine metabolism by quantitation of all important thiopurine nucleotide metabolites in one run

Simultaneous Quantification of Eleven Thiopurine Nucleotides by Liquid Chromatography-Tandem Mass Spectrometry

The prodrugs azathioprine and 6-mercaptopurine, which are well-established anticancer and immunosuppressive agents, are extensively metabolized by activating and inactivating enzymes. Whereas the 6-thioguanine nucleotides (TGN) are currently being considered as major active metabolites, methylthioinosine nucleotides seem to contribute to the cytotoxic effect as well. Thiopurine-related adverse drug reactions and thiopurine failure are frequent. Thus, therapeutic monitoring of TGN and methylthioinosine derivatives has been suggested to improve thiopurine therapy, however with limited success. To elucidate systematically underlying molecular mechanisms as potential explanation for interindividual variability of thiopurine response, we developed a novel highly specific and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantitation of eleven mono-, di-, and triphosphates of thioguanosine, methylthioinosine, methylthioguanosine, and thioinosine. Using stable isotope-labeled analogues as internal standards obtained by chemical synthesis, an intra- and interassay variability below 8% and an accuracy of 92% to 107% were achieved in spiked quality control samples with known standards. All eleven metabolites could be determined in red blood cells from patients with inflammatory bowel diseases and long-term azathioprine therapy. Thus, our novel method opens a new avenue for the understanding of the thiopurine metabolism by quantitation of all important thiopurine nucleotide metabolites in one run

Infliximab or cyclosporine as rescue therapy in hospitalized patients with steroid-refractory ulcerative colitis: A retrospective observational study

Background: Cyclosporine (CsA) or infliximab (IFX) are used as rescue therapies in steroid-refractory, severe attacks of ulcerative colitis (UC). There are no data comparing the efficacy of these two alternatives. Methods: Outcome of rescue therapy was retrospectively studied in two cohorts of patients hospitalized due to steroid-refractory moderate to severe UC: 1) a Swedish-Danish cohort (n 49) treated with a single infusion of IFX; 2) an Austrian cohort (n 43) treated with intravenous CsA. After successful rescue therapy, maintenance immunomodulator treatment was given to 27/33 (82%) of IFX patients and to 31/40 (78%) of CsA patients. Endpoints were colectomy-free survival at 3 and 12 months. Kaplan-Meier and Cox regression models were used to evaluate the association between treatment groups and colectomy. Results: At 15 days, colectomy-free survival in the IFX cohort was 36/49 (73%) versus 41/43 (95%) in the CsA cohort (P = 0.005), at 3 months 33/49 (67%) versus 40/43 (93%) (P = 0.002), and at 12 months 28/49 (57%) versus 33/43 (77%) (P = 0.034). After adjusting for potential confounding factors, Cox regression analysis yielded adjusted hazard ratios for risk of colectomy in IFX-treated patients of 11.2 (95% confidence interval [CI] 2.4-53.1, P = 0.002) at 3 months and of 3.0 (95% CI 1.1-8.2, P = 0.030) at 12 months in comparison with CsA-treated patients. There were no opportunistic infections or mortality. Conclusions: Colectomy frequencies were significantly lower after rescue therapy with CsA than with a single infusion of IFX both at 3 and 12 months' follow-up. The superiority of CsA was seen principally during the first 15 days