The glutathione transferase Mu null genotype leads to lower 6-MMPR levels in patients treated with azathioprine but not with mercaptopurine.

The conversion of azathioprine (AZA) to mercaptopurine (MP) is mediated by glutathione transferase Mu1 (GSTM1), alpha1 (GSTA1) and alpha2 (GSTA2). We designed a case-control study with data from the TOPIC trial to explore the effects of genetic variation on steady state 6-methylmercaptopurine ribonucleotide (6-MMPR) and 6-thioguanine nucleotide (6-TGN) metabolite levels. We included 199 patients with inflammatory bowel disease (126 on AZA and 73 on MP). GSTM1-null genotype carriers on AZA had two-fold lower 6-MMPR levels than AZA users carrying one or two copies of GSTM1 (2239 (1006-4587) versus 4371 (1897-7369) pmol/8 × 108 RBCs; P<0.01). In patients on MP (control group) 6-MMPR levels were comparable (6195 (1551-10712) versus 6544 (1717-11600) pmol/8 × 108 RBCs; P=0.84). The 6-TGN levels were not affected by the GSTM1 genotype. The presence of genetic variants in GSTA1 and GSTA2 was not related to the 6-MMPR and 6-TGN levels.

Azathioprine Therapy in a Pediatric TPMT-Deficient Patient-Still an Option.

We describe the case of a pediatric patient on azathioprine therapy with previously undiagnosed homozygote thiopurine S-methyltransferase (TPMT) deficiency, resulting in myelotoxic thiopurine metabolite levels. The patient was successfully treated with a very low azathioprine dose of 50 mg once a week (4% of standard dose), guided by frequent thiopurine metabolite measurement and a close clinical surveillance. We demonstrate that azathioprine therapy still might be an effective and safe therapeutic option in pediatric thiopurine S-methyltransferase-deficient IBD patients.

Extended thiopurine metabolite assessment during 6-thioguanine therapy for immunomodulation in Crohn's disease.

The proposed metabolic advantage of 6-thioguanine (6-TG) is the direct conversion into the pharmacologically active 6-thioguaninenucleotides (6-TGN). The authors assessed metabolic characteristics of 6-TG treatment in patients with Crohn's disease (N = 7) on therapy with 20 mg 6-TG. 6-thioguanine-monophosphate (6-TGMP), 6-thioguanine-diphosphate (6-TGDP), and 6-thioguanine-triphosphate (6-TGTP) were measured by high-performance liquid chromatography analysis in erythrocytes. Thiopurine S-methyltransferase activity and total 6-TGN levels were determined by standard methods. High interindividual variance in metabolite measurements was observed. Main metabolites were 6-TGTP (median = 531 pmol/8 x 10(8) red blood cells) and 6-TGDP (median = 199 pmol/8 x 10(8) red blood cells). Traces of 6-TGMP (median = 39 pmol/8 x 10(8) red blood cells) and 6-TG (2 patients) could be detected. 6-TGN levels correlated with 6-TGTP levels (r = 0.929, P = .003) and with the sum of separate nucleotides (r = 0.929, P = .003). No correlations were established between TPMT activity (median = 13 pmol/h/10(7)) and 6-TG metabolites. The 1-step metabolism of 6-TG still leads to high interindividual variance in metabolite concentrations. Total 6-TGN level monitoring may suffice for clinical practice.

Pancytopenia due to high 6-methylmercaptopurine levels in a 6-mercaptopurine treated patient with Crohn's disease.

In a 23-year-old female with colonic Crohn's disease 6-mercaptopurine 100 mg daily (1.7 mg/kg) was added to mesalamine and prednisolone therapy because of ongoing disease activity. One month later she had fever and a pancytopenia. 6-methylmercaptopurine ribonucleotides levels were extremely elevated (57,000 pmol/8x10(8) red blood cells) and 6-thioguanine nucleotides levels were subtherapeutically (126 pmol/8x10(8) red blood cells). Genotyping showed a wildtype thiopurine S-methyltransferase TPMT(H/H) (*1/*1) genotype and a wildtype inosine triphosphate pyrophosphatase gene. TPMT and inosine triphosphate pyrophosphatase activity were normal. The pancytopenia recovered spontaneously within a few weeks, parallel with decreasing 6-methylmercaptopurine ribonucleotides levels after interrupting 6-mercaptopurine treatment. Epstein-Barrvirus, Cytomegalovirus and Herpesvirus infections were excluded by serology. This is the first report of pancytopenia due to extremely high 6-methylmercaptopurine ribonucleotides levels. No relation was found with the genotype of TPMT and inosine triphosphate pyrophosphatase enzymes, which play key roles in the thiopurine metabolic pathway. Apparently, 6-methylmercaptopurine ribonucleotides metabolites can cause pancytopenia, as was already known for 6-thioguanine nucleotides.

Toxicity of 6-thioguanine: no hepatotoxicity in a series of IBD patients treated with long-term, low dose 6-thioguanine. Some evidence for dose or metabolite level dependent effects?

BACKGROUND: 6-Thioguanine is used in inflammatory bowel disease since 2001, with promising short-term results. In 2003, liver histology of some 6-thioguanine treated patients showed nodular regenerative hyperplasia. Recently, magnetic resonance imaging revealed nodular regenerative hyperplasia in patients with normal histology. AIMS: Investigating the presence of nodular regenerative hyperplasia in long-term 6-thioguanine treated patients. PATIENTS AND METHODS: Inflammatory bowel disease patients, using 6-thioguanine minimally 24 months, were asked to undergo liver biopsy and magnetic resonance imaging. RESULTS: Fourteen patients used 6-thioguanine minimally 24 months, 13 participated. Mean 6-thioguanine therapy duration, daily dose and 6-thioguanine nucleotide levels were: 36 months, 18.8 mg (0.28 mg/kg) and 705 pmol/8x10(8) erythrocytes, respectively. Liver histology and magnetic resonance imaging showed no nodular regenerative hyperplasia. DISCUSSION: Liver biopsy and magnetic resonance imaging showed no nodular regenerative hyperplasia in these long-term 6-thioguanine treated inflammatory bowel disease patients. 6-thioguanine dose and metabolite levels were lower compared with previous nodular regenerative hyperplasia reports, suggesting dose or metabolite level-dependent effects. Otherwise, nodular regenerative hyperplasia is related with inflammatory bowel disease itself and immunosuppressives, including azathioprine and 6-mercaptopurine. CONCLUSION: 6-Thioguanine is debated due to nodular regenerative hyperplasia. We found no nodular regenerative hyperplasia in inflammatory bowel disease patients with long-term, low dosed 6-thioguanine, suggesting metabolite level-dependent effects. Therefore, 6-thioguanine still seems useful, but in selected patients, intolerant for other immunosuppressives, low dosed and under close surveillance of metabolite levels and hepatotoxity.

Risk factors for thiopurine-induced myelosuppression and infections in inflammatory bowel disease patients with a normal TPMT genotype.

BACKGROUND: Leucopenia is a common side effect in patients treated with thiopurines. Variants in the thiopurine S-methyltransferase (TPMT) gene are the best-known risk factor, but only explain up to 25% of leucopenia cases. AIM: To identify the clinical risk factors for thiopurine-induced leucopenia in patients without a common TPMT variant, and explore if these patients are at increased risk for infections. METHODS: Post hoc analysis of the Thiopurine response Optimisation by Pharmacogenetic testing in Inflammatory bowel disease Clinics (TOPIC) trial. For this analysis, patients without a variant in TPMT (*2, *3A or*3C) were included. Uni- and multivariate Cox-proportional hazard models were used to identify risk factors for leucopenia and infections. Leucopenia was defined as a white blood cell (WBC) count <3.0 × 109 /L and infections were classified according to the Common Terminology Criteria for Adverse Events. RESULTS: Sixty hundred and ninety-five patients (90.6%) included in the TOPIC-trial had no variant in TPMT, of which 45 (6.5%) developed leucopenia. Median time to leucopenia was 56 (29-112) days. Multivariate analysis showed that use of mercaptopurine compared to azathioprine was associated with leucopenia (hazard ratio [HR] 2.61 [95% CIs, 1.39-4.88; P < .01]) and a higher baseline WBC count was protective (HR 0.80 [95% CIs, 0.71-0.89; P < .01]). Risk factors for infections were older age (per 10 year; HR 2.07 [95% CIs, 1.18-3.63; P = .01]) and concomitant use of biologic drugs (HR 2.15 [95% CIs, 1.14-4.07; P = .02]). CONCLUSIONS: Low baseline WBC count and mercaptopurine, due to a relatively higher dose, were risk factors for thiopurine-induced leucopenia in patients without a TPMT variant.

Early prediction of thiopurine-induced hepatotoxicity in inflammatory bowel disease.

BACKGROUND: Hepatotoxicity, gastrointestinal complaints and general malaise are common limiting adverse reactions of azathioprine and mercaptopurine in IBD patients, often related to high steady-state 6-methylmercaptopurine ribonucleotide (6-MMPR) metabolite concentrations. AIM: To determine the predictive value of 6-MMPR concentrations 1 week after treatment initiation (T1) for the development of these adverse reactions, especially hepatotoxicity, during the first 20 weeks of treatment. METHODS: The cohort study consisted of the first 270 IBD patients starting thiopurine treatment as part of the Dutch randomised-controlled trial evaluating pre-treatment thiopurine S-methyltransferase genotype testing (ClinicalTrials.gov NCT00521950). Blood samples for metabolite assessment were collected at T1. Hepatotoxicity was defined by alanine aminotransaminase elevations >2 times the upper normal limit or a ratio of alanine aminotransaminase/alkaline phosphatase ≥5. RESULTS: Forty-seven patients (17%) presented hepatotoxicity during the first 20 weeks of thiopurine treatment. A T1 6-MMPR threshold of 3615 pmol/8 × 108 erythrocytes was defined. Analysis of patients on stable thiopurine dose (n = 174) showed that those exceeding the 6-MMPR threshold were at increased risk of hepatotoxicity: OR = 3.8 (95% CI: 1.8-8.0). Age, male gender and BMI were significant determinants. A predictive algorithm was developed based on these determinants and the 6-MMPR threshold to assess hepatotoxicity risk [AUC = 0.83 (95% CI: 0.75-0.91)]. 6-MMPR concentrations above the threshold also correlated with gastrointestinal complaints: OR = 2.4 (95% CI: 1.4-4.3), and general malaise: OR = 2.0 (95% CI: 1.1-3.7). CONCLUSIONS: In more than 80% of patients, thiopurine-induced hepatotoxicity could be explained by elevated T1 6-MMPR concentrations and the independent risk factors age, gender and BMI, allowing personalised thiopurine treatment in IBD to prevent early failure.

Review article: thiopurines in inflammatory bowel disease.

BACKGROUND: In the past 10-20 years, knowledge of both thiopurine pharmacology and -pharmacogenetics has been extended dramatically and used to develop new strategies to improve efficacy and reduce toxicity. AIM: To review thiopurine efficacy, toxicity, pharmacology, pharmacogenetics, interactions in patients with inflammatory bowel disease. Special attention was paid to new strategies for optimization of pharmacotherapy. METHODS: To collect relevant scientific articles, a Pubmed search was performed from 1966 through January 2006 with the following key words (MeSH terms preferentially) in multiple combinations: 'azathioprine', '6-mercaptopurine', '6-MP', '6-thioguanine', '6-TG', 'thiopurine(s)', 'metabolites', 'level(s)', 'TDM', 'TMPT', 'ITPA', 'genotype(s)', 'phenotype(s)', 'inflammatory bowel disease', 'Crohn('s) disease', 'ulcerative colitis'. RESULTS: Strategies for optimization of pharmacotherapy include therapeutic drug monitoring of thiopurine metabolites, geno- or phenotyping crucial enzymes in thiopurine metabolism like thiopurine S-methyltransferase and inosine triphosphate pyrophosphatase, and the use of thioguanine as such. CONCLUSIONS: Thiopurine S-methyltransferase genotyping and therapeutic drug monitoring are useful instruments for individualizing thiopurine pharmacotherapy of inflammatory bowel disease. Inosine triphosphate pyrophosphatase genotyping may be helpful in case of unexplainable myelotoxicity. In case of azathioprine- or mercaptopurine-intolerance, thioguanine seems a promising alternative. However, more knowledge needs to be gathered about its potential hepatotoxicity.

The pharmacokinetic effect of discontinuation of mesalazine on mercaptopurine metabolite levels in inflammatory bowel disease patients.

BACKGROUND: In vitro studies suggest interactions between mesalazine (mesalamine) and thiopurines by thiopurine S-methyltransferase (TPMT) inhibition, influencing the balance of hepatotoxic 6-methylmercaptopurine ribonucleotide and immunosuppressive tioguanine (thioguanine) metabolites. AIM: To examine the in vivo pharmacokinetic interaction between mesalazine and mercaptopurine. METHODS: A prospective study was performed in quiescent inflammatory bowel disease patients using the combination of mercaptopurine and mesalazine. Laboratory parameters, 6-methylmercaptopurine ribonucleotide and tioguanine levels and thiopurine S-methyltransferase activity in erythrocytes were measured at stable medication, after mesalazine discontinuation and mesalazine reintroduction, further mercaptopurine was continued. RESULTS: Seventeen patients were participated. Mean mercaptopurine dose was 0.78 mg/kg/day and median of mesalazine dose was 3000 mg/day. After mesalazine discontinuation, mean tioguanine levels changed significantly from 262 to 209 pmol/8 x 10(8) red blood cell, increasing to 270 after reintroduction. Mean 6-methylmercaptopurine ribonucleotide levels were 1422, 2149 and 1503 pmol/8 x 10(8) red blood cell respectively. Mean 6-methylmercaptopurine ribonucleotide/tioguanine ratio increased significantly from 6.3 at baseline to 11.2. Mean baseline thiopurine S-methyltransferase activity was 0.58 pmol/10(6) red blood cell/h and stable. All patients had wild-type thiopurine S-methyltransferase genotypes however, leucocyte counts were stable. DISCUSSION: A significantly higher tioguanine levels and improving 6-methylmercaptopurine ribonucleotide/tioguanine ratio were found during mesalazine/mercaptopurine combination. Theoretically, mesalazine inhibits thiopurine S-methyltransferase activity. In vivo thiopurine S-methyltransferase activity did not change, however. CONCLUSION: Mesalazine has synergistic effects on mercaptopurine therapy, but the mechanism is unclear. Combining these drugs may be further indication for mesalazine in inflammatory bowel disease treatment.

Effect of heparin on digitoxin protein binding.

Reduction of digitoxin binding to plasma proteins after heparin has been reported. Our aim was to determine whether this reduction is an in vivo effect or occurs only after blood collection as a result of heparin-induced lipolysis that increases levels of nonesterified fatty acids in vitro. The effect of heparin on digitoxin protein binding was studied in 10 patients undergoing hemodialysis receiving digitoxin maintenance therapy. Digitoxin free fraction increased after heparin, from 2.5% +/- 0.7% to 4.4% +/- 1.1%, but after inhibition of in vitro lipolysis with diethyl p-nitrophenyl phosphate (2mM), a potent lipase inhibitor, there was no increase in the free fraction (2.3% +/- 0.4% before heparin and 2.4% +/- 0.5% after heparin). Digitoxin salivary levels were also unchanged (0.41 +/- 0.08 ng/ml before heparin and 0.41 +/- 0.08 ng/ml after heparin [n = 8]). These data indicate that the binding of digitoxin to plasma proteins in vivo is not altered by heparin. The reduced binding reported elsewhere was a result of heparin-induced in vitro lipolysis.

Stability of amphotericin B in CAPD fluid.

Amphotericin B is the drug of choice in continuous ambulatory peritoneal dialysis (CAPD) associated fungal peritonitis and is usually administered intraperitoneally. The drug is stated to be incompatible with anions. All CAPD fluids contain chloride and lactate anions. Therefore, the physical and chemical compatibility of amphotericin B with dextrose 5%, Dianeal 1.36% CAPD fluid, and Dianeal 1.36% peritoneal effluent was studied at amphotericin B concentrations of 1, 2, and 5 mg/L. Amphotericin B was most stable in Dianeal CAPD fluid. The rate of degradation was concentration dependent in dextrose 5% and peritoneal effluent. The higher the concentration, the lower the rate of degradation. After an incubation of 6 h at 37 degrees C, no significant decomposition was found at all concentrations studied in Dianeal CAPD fluid whereas 12-18% decomposition was found in effluent. No physical incompatibility with any solution was observed.

Intranasal estradiol administration to oophorectomized women.

An open, dose-finding study into the effects of a new intranasal 17 beta-estradiol formulation using dimethyl-beta-cyclodextrin as a solubilizer was conducted in nine hysterectomized and oophorectomized patients with symptoms of estrogen deficiency. After nasal delivery of 0.34 mg estradiol in three patients, concentration-time curves of estradiol in serum and its metabolite estrone in plasma were established. High initial estradiol serum levels were found in the order of 5 nmol/l. Estrone/estradiol ratios were below unity, whereas, in contrast, estrone/estradiol ratios after oral administration of estradiol are known to exceed unity. Biological activity, as reflected by suppression of FSH and LH, was manifest. In nine patients symptoms of estrogen deficiency were assessed by a questionnaire. After 3 and 6 months of treatment, a clinically and statistically significant decrease (P less than 0.01) of the total score according to this questionnaire was found compared to the beginning of the study. Thus nasal delivery of estradiol using this cyclodextrin formulation has potential for further clinical evaluation.

Intranasal administration of estradiol in combination with progesterone to oophorectomized women: a pilot study.

A pilot study was done to investigate the pharmacokinetics and acceptability of an intranasal 17 beta-estradiol/progesterone formulation. This formulation contained dimethyl-beta-cyclodextrin as a solubilizer and absorption enhancer of the steroid hormones. The study was performed in four oophorectomized and hysterectomized patients. After nasal delivery of 0.34 mg estradiol and 0.85 mg progesterone, concentration-time curves of progesterone, estradiol and its metabolite estrone were established. Rapid absorption of progesterone and estradiol was demonstrated. Progesterone peak-levels (3.9-6.7 nmol/l) exceeded the levels which are found in the follicular phase of the menstrual cycle, but did not reach the levels of the luteal phase. Comparison with an earlier reported study in which only estradiol was administered shows that the addition of progesterone does not alter the absorption and pharmacokinetics of estradiol. The nasal formulation was well accepted by the four patients and no adverse effects were noted.

Therapeutic drug monitoring in patients with inflammatory bowel disease and established azathioprine therapy.

BACKGROUND AND OBJECTIVE: Azathioprine is widely used in the treatment of corticosteroid-dependent and refractory inflammatory bowel disease (IBD). The efficacy of this treatment is based on the production of 6-thioguanine nucleotides, but extremely elevated levels may cause bone marrow suppression. Other azathioprine metabolites, 6-methylmercaptopurine ribonucleotides, are associated with hepatotoxicity. Therapeutic drug monitoring (TDM) may be of help in optimising azathioprine treatment, but data on TDM in established azathioprine therapy are lacking. We therefore measured metabolite levels in a small cohort of patients established on azathioprine therapy. PATIENTS AND METHODS: 6-Thioguanine (6-TGN) and 6-methylmercaptopurine (6-MMP) levels in erythrocytes were measured in 15 IBD outpatients established on azathioprine therapy for at least 3 months at baseline and 1, 4 and 8 weeks after inclusion (mean duration of treatment 28 months; range 7-67 months). Disease activity was evaluated by the Crohn's Disease Activity Index (Crohn's disease) or Truelove-Witts (ulcerative colitis) scores. Metabolite levels were measured by modified high-performance liquid chromatography assay (HPLC). Primary outcome measures were 6-TGN and 6-MMP metabolite levels and 95% confidence intervals (CIs). SECONDARY OUTCOMES were correlations between metabolite levels, drug dose, disease activity and laboratory parameters and compliance. RESULTS: One patient had active disease during the study period. Eleven of 15 patients (73%) completed the 8-week study period. Dropout reasons were noncompliance in three patients (20%) and intolerance in one patient (7%). PRIMARY OUTCOMES: At baseline mean 6-TGN levels were 158 (95% CI 113, 203) pmol/8.10(8) RBC (red blood cells), steadily increasing over the 8-week study period, but not significantly. Two patients had zero levels. Another two had significantly increasing levels also suggesting noncompliance. Mean 6-MMP levels showed almost a similar pattern. At baseline, levels were 2213 (95% CI 722, 3704) pmol/8.10(8) RBC. SECONDARY OUTCOMES: A correlation was found between all RBC 6-MMP levels and azathioprine dose (mg/kg bodyweight) [r = 0.43, p = 0.001] and also between the 6-MMP/6-TGN ratio and azathioprine dose (mg/kg) [r = 0.36, p = 0.010). There was no correlation between RBC 6-TGN or 6-MMP levels and haematological parameters or disease activity scores. No hepatic, pancreatic or myelotoxicity occurred.Thirteen of 15 patients (87%) had baseline steady-state 6-TGN levels below the therapeutic threshold of 235 pmol/8.10(8) RBC. Forty percent (6/15) of our patients were noncompliant; TDM revealed this noncompliance in four of the six patients (27% of all patients). CONCLUSION: Our small study demonstrates that TDM may provide insight into individual pharmacokinetics. However, TDM does not seem to be useful in patients with IBD established on azathioprine therapy and without disease activity, although it may be helpful in cases of worsening IBD activity to elucidate noncompliance or inefficient treatment.

Safety of thiopurines in the treatment of inflammatory bowel disease.

BACKGROUND: Thiopurines have proven efficacy in inflammatory bowel disease. However, concerns regarding toxicity have limited the use of these agents as first line of medical therapy. METHODS: Review of the literature regarding metabolism, efficacy and side effects. RESULTS: In clinical trials, up to 15% of patients discontinued 6-mercaptopurine or its pro-drug azathioprine prematurely due to adverse events. These events may be divided into dose-independent idiosyncratic reactions and dose-related, pharmacologically explainable toxicity. Dose-independent reactions include skin rash, fever, diarrhoea and pancreatitis. Most frequently observed dose-dependent adverse events are nausea, malaise and myelotoxicity. Furthermore, dose-dependent and dose-independent hepatotoxicity may occur. Recent insights obtained by therapeutic drug monitoring in patients on azathioprine or 6-mercaptopurine have led to strategies to reduce toxicity. One strategy is to detect poor metabolisers of thiopurines by establishing the activity of the key enzyme thiopurine methyltransferase. However, the clinical relevance of this strategy is still a point of debate. Another strategy is to administer 6-thioguanine, which is an agent close to the effective 6-thioguanine nucleotides. CONCLUSION: Therapeutic drug monitoring of thiopurines resulted in strategies to reduce toxicity. The value of these strategies has yet to be proven in prospective randomized trials.

The pharmacokinetic effect of adalimumab on thiopurine metabolism in Crohn's disease patients.

BACKGROUND AND AIMS: A drug interaction between infliximab and azathioprine has previously been reported in Crohn's disease patients: the concentration of the main active thiopurine metabolites, the 6-thioguanine nucleotides (6-TGN), increased 1-3 weeks after the first infliximab infusion by 50% compared to baseline. The aim of this prospective study was to determine the effect of adalimumab on thiopurine metabolism in Crohn's disease patients, evaluated by 6-TGN and 6-methylmercaptopurine ribonucleotides (6-MMPR) concentration measurement. METHODS: Crohn's disease patients on azathioprine or mercaptopurine maintenance therapy starting with concomitant adalimumab treatment were included. 6-TGN and 6-MMPR concentrations were determined before initiation of adalimumab and after 2, 4, 6 and 12 weeks of combination therapy. The activity of three essential enzymes involving thiopurine metabolism, thiopurine S-methyltransferase (TPMT), hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and inosine-triphosphate pyrophosphatase (ITPase), was evaluated at baseline and week 4. Clinical outcome was evaluated by the Crohn's disease activity index and C-reactive protein concentrations at baseline, week 4 and week 12. RESULTS: Twelve Crohn's disease patients were analyzed. During the follow-up period of 12 weeks the median 6-TGN and 6-MMPR concentrations did not significantly change compared to baseline. TPMT, ITPase and HGPRT enzyme activity did not change either after 4 weeks. In two patients (17%) myelotoxicity was observed within 2-4 weeks, in whom both low therapeutic 6-TGN and 6-MMPR concentrations were found. CONCLUSIONS: In this study in Crohn's disease patients no pharmacokinetic interaction was shown between adalimumab and the conventional thiopurines, azathioprine and mercaptopurine.

Influence of 5-aminosalicylic acid on 6-thioguanosine phosphate metabolite levels: a prospective study in patients under steady thiopurine therapy.

BACKGROUND AND PURPOSE: 5-aminosalicylate (5-ASA) raises levels of 6-thioguanine nucleotides (6-TGN), the active metabolites of thiopurines such as azathioprine (AZA). Changes in levels of each individual TGN - 6-thioguanosine mono-, di- and triphosphate (6-TGMP, 6-TGDP, 6-TGTP) - and of 6-methylmercaptopurine ribonucleotides (6-MMPR) after 5-ASA are not known. EXPERIMENTAL APPROACH: Effects of increasing 5-ASA doses on AZA metabolites were investigated prospectively in 22 patients with inflammatory bowel disease in 4-week study periods. Patients started with 2 g 5-ASA daily, and then were increased to 4 g daily and followed by a washout period. Thiopurine doses remained unchanged throughout the entire study. Levels of 6-TGMP, 6-TGDP, 6-TGTP and 6-MMPR as well as of 5-ASA and N-acetyl-5-aminosalicylic acid (N-Ac-5-ASA) were determined each study period. KEY RESULTS: Median baseline levels in 17 patients of 6-TGDP, 6-TGTP and 6-MMPR were 52, 319 and 1676 pmol per 8 x 10(8) red blood cells respectively. After co-administration of 2 g 5-ASA daily, median 6-TGDP and 6-TGTP levels increased but median 6-MMPR levels were unchanged. Increasing 5-ASA to 4 g daily did not affect median 6-TGDP and 6-TGTP levels, but median 6-MMPR levels decreased. After discontinuation of 5-ASA, both 6-TGDP and 6-TGTP levels decreased and median 6-MMPR levels increased. The 6-TGTP/(6-TGDP+6-TGTP)-ratio did not change during the study, but 6-MMPR/6-TGN ratios decreased. CONCLUSIONS AND IMPLICATIONS: Individual 6-TGN metabolites increased after addition of 5-ASA, but 6-MMPR-levels and the 6-MMPR/6-TGN ratios decreased. Further studies are needed to decide whether this pharmacokinetic interaction would result in improvement of efficacy and/or increased risk of toxicity of AZA.