Dr Y. Horsmans, Department of Gastroenterology, St Luc Hospital, Avenue Hippocrate, 10, B-1200 Brussels, Belgium. E-mail: Horsmans@gaen.ucl.ac.be
The inhibition of thiopurine methyltransferase activity, one of the enzymes responsible for azathioprine metabolism, by aminosalicylates has been described in an in vitro study. This could result in a higher risk of bone marrow depression when using the two drugs together.
To investigate the in vivo interaction between azathioprine and aminosalicylates in quiescent Crohn’s disease by measuring 6-thioguanine nucleotide levels, thiopurine methyltransferase activity and the plasma levels of the acetylated metabolite of 5-aminosalicylic acid.
Sixteen patients taking a stable dose of azathioprine, plus sulfasalazine or mesalazine, were enrolled and completed the study. They were not taking any drugs interfering with azathioprine metabolism. Four visits every 4 weeks were held over a 3-month period. Aminosalicylate administration was withdrawn after the second visit. At each visit, the blood cell count, inflammatory parameters, levels of 6-thioguanine nucleotide and the acetylated metabolite of 5-aminosalicylic acid and thiopurine methyltransferase activity were determined.
After aminosalicylate withdrawal, mean 6-thioguanine nucleotide levels decreased significantly from 148 pmol (57–357 pmol) to 132 pmol (56–247 pmol) per 8 × 108 red blood cells (P=0.027), without significant changes in thiopurine methyltransferase activity or biological parameters.
This in vivo study favours the existence of an interaction between azathioprine and aminosalicylates through a mechanism which remains unclear. This drug–drug interaction should be taken into account when using azathioprine and aminosalicylates simultaneously.
Azathioprine and its metabolite, 6-mercaptopurine, are effective in achieving and maintaining remission in patients with Crohn’s disease.1 Their indications are wide: refractory disease, fistulating Crohn’s disease, steroid dependency, maintenance of long-term remission, etc.
Azathioprine, a thiopurine analogue, is rapidly converted non-enzymatically into 6-mercaptopurine which, in turn, is converted into the active moiety, 6-thioguanine nucleotide, by the hypoxanthine phosphoribosyltransferase pathway. There are two other enzymatic pathways, leading to inactive metabolites: one using xanthine oxidase and leading to 6-thiouric acid, and the second using thiopurine methyltransferase and leading to 6-methylmercaptopurine.2,3
Various side-effects of azathioprine have been reported:4 fever, rash, nausea, diarrhoea, opportunist infections, pancreatitis, hepatic and bone marrow toxicity. Myelosuppression seems to be directly related to increased red blood cell levels of 6-thioguanine nucleotide.5,6
High 6-thioguanine nucleotide levels can be observed in patients with low thiopurine methyltransferase activity, such as, for example, patients with a genetic deficiency of this enzyme.5,7,8 The expression of the enzyme is inherited in an autosomal codominant fashion, and consequently varies within the population. In Caucasians, 11% of the population are heterozygous and 0.3% are homozygous with respect to thiopurine methyltransferase deficiency, leading to an intermediate or low thiopurine methyltransferase activity, respectively. In these patients, azathioprine metabolism is shunted towards an increased production of 6-thioguanine nucleotide.9
Certain drugs might also interfere with azathioprine metabolism. Allopurinol inhibits xanthine oxidase, blocking one of the metabolic pathways of azathioprine.10 Other drugs may also interfere with thiopurine methyltransferase activity, such as non-steroidal anti-inflammatory drugs,11 diuretics11 and derivatives of benzoic acid, e.g. sulfasalazine or mesalazine.12
The last two drugs are commonly used as first-line treatment in inflammatory bowel disease, and therapy is sometimes maintained in refractory patients after the initiation of azathioprine therapy.
Szumlanski and Weinshilboum12 reported an in vitro inhibition of thiopurine methyltransferase with sulfasalazine and 5-aminosalicylic acid derivatives. The potential clinical consequence is a higher risk of bone marrow depression when using combined therapy. This has been described in inflammatory bowel disease case reports.13,14 In one of these, a patient developed two episodes of severe bone marrow depression while being treated with olsalazine (a 5-aminosalicylic acid derivative) and 6-mercaptopurine. A similar phenomenon has also been observed in an adult with Still’s disease,15 in which a decrease in leucocyte counts to subnormal levels occurred after the addition of sulfasalazine to long-term azathioprine therapy. In this patient, the drug combination resulted in agranulocytosis.15
In this context, we performed an in vivo study to evaluate the possible drug–drug interaction between azathioprine and sulfasalazine or mesalazine in patients with Crohn’s disease.
PATIENTS AND METHODS
The study protocol was approved by the ethical committee of the Catholic University of Louvain, and all patients gave written informed consent before being enrolled in the study.
Sixteen patients (nine women and seven men) with inactive Crohn’s disease (Crohn’s disease activity index, < 150) on azathioprine (Imuran, GlaxoSmithKline, Dartford, UK) and sulfasalazine (Salazopyrine, Pharmacia-Upjohn, Sweden) (eight patients) or azathioprine and mesalazine (Claversal, Tramedico, Wülfing Pharma, Gronau/Leine, Germany) (eight patients) were enrolled in the study. Eligible patients were at least 18 years of age and had been on a stable regimen of medication for at least 3 months. The mean age was 43 years (23–66 years), and mean duration of azathioprine therapy was 6.4 years (2–11 years). The azathioprine mean daily dosage was 1.46 mg/kg (0.85–2.36 mg/kg). Drugs with the potential to interfere with azathioprine metabolism (warfarin,16 allopurinol, non-steroidal anti-inflammatory drugs, etc.) were not allowed nor was any change in azathioprine regimen. All other drugs taken were recorded. Low doses of steroids (less than 15 mg prednisolone/day) without dose changes were allowed during the study period. Clinical data on the patients and their therapeutic regimen are shown in Table 1.
Table 1. Baseline characteristics of patients
The study period was 3 months. Each patient was seen four times at 4-week intervals. At each visit, clinical examination, Crohn’s disease activity index and blood sampling were obtained. In blood samples, the cell count, inflammatory parameters, red blood cell 6-thioguanine nucleotide levels, plasma levels of 5-aminosalicylic acid and its acetylated metabolite and thiopurine methyltransferase activity were measured. After visit 2 (1 month after study inclusion), the aminosalicylate (sulfasalazine or mesalazine) was stopped, dividing our study into two phases with each patient being his or her own control: phase 1, azathioprine + aminosalicylate; phase 2, azathioprine alone.
The total concentration of 6-thioguanine nucleotide in red blood cells, based on the conversion of 6-thioguanine nucleotide to the free 6-thioguanine base, was assayed by high performance liquid chromatography according to the method of Lennard and Singleton17 using single wavelength detection at 342 nm. Two quality control samples of 6-thioguanine nucleotide at 119 and 299 pmol/8 × 108 red blood cells were analysed during each run. The interday coefficients of variation were 6.8% (116 ± 7.9 pmol/8 × 108 red blood cells) and 4.6% (305 ± 13.9 pmol/8 × 108 red blood cells), respectively (n=25). The limit of detection was 5 pmol/8 × 108 red blood cells.
The activity of thiopurine methyltransferase was determined according to the method described by Weinshilboum et al.18 One unit corresponds to the formation of methylmercaptopurine at a rate of 1 pmol/h per millilitre of packed red blood cells.
The high performance liquid chromatographic method described by Bystrowska et al.,19 measuring the concentration of 5-aminosalicylic acid and its acetylated metabolite in plasma, was used with a few modifications: ethylanthranilate was used as an internal standard and methanol–acetonitrile–water (32:20:48, v/v) as a solvent. The within-run relative standard deviations were below 5.3% (range 30–1500 ng/mL) and the between-run relative standard deviations were below 10% for the same range. The original sample of 5-acetylated metabolites of 5-aminosalicylic acid was a generous gift from Dr Bystrowska (Poland).
The levels of 6-thioguanine nucleotide, 5-aminosalicylic acid and its acetylated metabolite and the thiopurine methyltransferase activity of the four samples of each patient were analysed in the same run after study completion; the laboratory technicians performing the assay were blind to the patient’s treatment status.
For each patient, the mean values of the first two visits (phase 1) were compared with the mean values of the last two visits (phase 2). A paired Wilcoxon rank test was used to compare the mean data obtained during phase 1 and phase 2. P < 0.05 was considered to be statistically significant.
Sixteen patients were screened and enrolled: eight in the group taking sulfasalazine and eight in the group taking mesalazine.
Considering all patients, no changes were observed between the two phases with respect to clinical examination, Crohn’s disease activity index, inflammatory parameters and blood cell counts.
The 6-thioguanine nucleotide levels and thiopurine methyltransferase activity are shown in Table 2 and Figures 1 and 2.
Table 2. 6-Thioguanine nucleotide (6-TGN) level and thiopurine methyltransferase (TPMT) activity at phase 1 (azathioprine + aminosalicylate) and phase 2 (azathioprine alone). Values are the mean of each phase. The difference between the two phases was significant for 6-TGN level (P=0.027) but not for TPMT activity (P=0.245, N.S.)
During phase 1, in the whole group, the median concentration of the acetylated metabolite of 5-aminosalicylic acid was 379 ng/mL (range, 105–942 ng/mL); it was 279 ng/mL (105–849 ng/mL) for the sulfasalazine subgroup and 480 ng/mL (214–942 ng/mL) for the mesalazine subgroup.
Monitoring of the acetylated metabolite of 5-aminosalicylic acid during phase 2 showed that all patients had effectively stopped aminosalicylate therapy.
The 5-aminosalicylic acid levels obtained in some samples varied from 49 to 780 ng/mL.
In the whole group, the mean level of 6-thioguanine nucleotide before aminosalicylate withdrawal was 148 pmol/8 × 108 red blood cells (57–357 pmol). This level was 148 pmol/8 × 108 red blood cells for the sulfasalazine subgroup and 149 pmol/8 × 108 red blood cells for the mesalazine subgroup.
After aminosalicylate withdrawal and considering the whole group, the mean 6-thioguanine nucleotide level decreased significantly (P=0.027) to 132 pmol/8 × 108 red blood cells (56–247 pmol). However, this statistically significant effect disappeared when the data from one of the patients (patient 1, 5, 6, 7, 8, 10, 11, 12, 14 or 15), with a decrease in 6-thioguanine nucleotide level, were suppressed. The 6-thioguanine nucleotide level dropped to 130 and 136 pmol/8 × 108 red blood cells in the sulfasalazine and mesalazine subgroups, respectively. No statistically significant differences between these levels were observed comparing the mesalazine and sulfasalazine subgroups. Considering the individual results, the 6-thioguanine nucleotide level decreased in 12 patients, remained stable in two patients and increased in two patients (one in each subgroup).
In the whole group, the thiopurine methyltransferase activity before withdrawal of aminosalicylate showed normal values (mean, 12.29 units; range, 8.25–16.85 units). The mean thiopurine methyltransferase activity was 12.14 units in the sulfasalazine subgroup and 12.43 units in the mesalazine subgroup.
After aminosalicylate withdrawal, the mean thiopurine methyltransferase activity did not change significantly. For the whole group, it decreased to 11.41 units (7.3–14.5 units) (P=0.245, N.S.), with a mean activity of 11.43 units in the sulfasalazine subgroup and 11.39 units in the mesalazine subgroup.
This study, performed in quiescent Crohn’s disease patients, showed an in vivo interaction between azathioprine and aminosalicylates in the absence of inhibition of thiopurine methyltransferase activity.
Mean 6-thioguanine nucleotide levels decreased significantly when aminosalicylate was stopped. However, this decrease was rather small (around 10%) and not clinically relevant in our group of 16 compliant patients. This slight decrease was observed in the majority of patients, but not in all. Moreover, the statistically significant effect disappeared when the data from one of the patients, with a decrease in 6-thioguanine nucleotide level, were omitted. This might be explained by the low azathioprine dose used. The difference seemed to be more important for higher initial 6-thioguanine nucleotide levels (patients 8 and 10). In contrast, the two patients in whom the 6-thioguanine nucleotide levels remained stable during the two phases had very low levels (patients 2 and 4), suggesting that the interaction might be more prominent with a higher dose of azathioprine. Another explanation for the small decrease might be the long course of combined therapy in our patients, with a possible exhausting or accommodating effect on drug–drug interaction.
In our patients, low steady state levels of 5-aminosalicylic acid were found in plasma, whereas the 5-acetamido metabolite was found to be 2–5 times more abundant, as previously described.20 The high sensitivity and specificity of the methods allowed us to check the compliance with aminosalicylate therapy. In our patients, the mean plasma 5-acetamido metabolite concentration was relatively low, but similar to that found in patients taking doses of aminosalicylate between 750 and 1500 mg/day.21 Plasma 5-aminosalicylic acid concentrations were 225–3900 times lower than the IC50 determined in the in vitro study (190 000 ng/mL) showing an interaction between 5-aminosalicylic acid and thiopurine methyltransferase activity.12 Such a low level of 5-aminosalicylic acid could explain the absence of thiopurine methyltransferase inhibition observed in our patients.
In our study, the interaction between azathioprine and aminosalicylate is not caused by an interaction between 5-aminosalicylic acid and thiopurine methyltransferase activity. This suggests that another mechanism might play a role in inducing a decrease in 6-thioguanine nucleotide levels observed after aminosalicylate withdrawal. Aminosalicylate could interact with other azathioprine metabolic pathways, causing hypoxanthine phosphoribosyltransferase or xanthine oxidase modulation. Another potential site of interaction could be the non-enzymatic azathioprine conversion into 6-mercaptopurine, a transformation which might be blocked or delayed by aminosalicylate.
As our patients had a normal thiopurine methyltransferase activity phenotype, we cannot exclude an inhibiting effect of thiopurine methyltransferase by aminosalicylate in patients with the lowest activity of this enzyme.
After absorption, most of the aminosalicylate is acetylated. The aminosalicylate acetylator phenotype of the patients was not determined, and this might be an influencing factor, with regard to the 5-aminosalicylic acid half-life, its concentration and the duration of a possible interaction. It could be hypothesized that the inhibiting effect of the acetylated metabolite on different enzymes (including thiopurine methyltransferase) could differ from that of the aminosalicylate itself. Individuals who are slow acetylators experience a higher incidence of aminosalicylate dose-dependent adverse effects.22 In these subjects, it has been suggested that a high concentration of total sulfapyridine may induce haematological adverse effects.23
The design of the study, which involved the withdrawal of aminosalicylates in patients already on azathioprine, was inspired by routine clinical practice in which it is common to discontinue aminosalicylates in patients already on azathioprine therapy.
It should be noted that aminosalicylate withdrawal had no effect on the clinical and biological evolution of these Crohn’s disease patients on azathioprine. Relapse of the disease was not observed, and there were no significant changes in the Crohn’s disease activity index, inflammatory parameters or white cell counts during the 3-month study period. Despite the relatively short follow-up period (2 months), it is debatable whether both families of drugs should be maintained in patients with quiescent Crohn’s disease.
In conclusion, in a group of 16 quiescent Crohn’s disease patients, an interaction between azathioprine and aminosalicylate derivatives was observed which seemed to be independent of an inhibitory effect on thiopurine methyltransferase activity. This might be of clinical relevance in inflammatory bowel disease, especially in refractory disease where a high dose (> 2 mg/kg) of azathioprine is given. The clinician should be aware of this drug–drug interaction when treating patients with combined therapy.