Results

A total of 951 patients with IBD had TPMT activity measured between 1992 and June 1998. These patients' medical records were reviewed during the fall of 1998 for eligibility criteria. We identified 376 patients who had either started or continued AZA or 6-MP at the time of their last Mayo Clinic evaluation. These 376 patients were contacted by mail regarding the study, of whom 217 initially agreed to participate and 170 actually returned the questionnaires and submitted blood specimens; 575 patients were ineligible. Detailed records of the reasons for ineligibility were not kept but reasons included: patient not started on AZA or 6-MP; patient no longer taking AZA or 6-MP because of failure to respond; AZA or 6-MP discontinued after operation; patient discontinued AZA or 6-MP because of toxicity; and other miscellaneous reasons.

ACTIVITY OF INFLAMMATORY BOWEL DISEASE

Among all study participants, mean (SD) IBDQ score was 176 (32) (range 75–223) (table 1). There was no significant correlation between disease activity (IBDQ point score) and whole blood 6-TGN concentration (r _s=−0.09, p=0.24), as shown in fig 1. Comparing 56 patients with more active disease (IBDQ <170) versus 114 patients in clinical remission (IBDQ ⩾170), median whole blood 6-TGN concentrations were similar (139 v131 pmol/8×10⁸ RBCs, respectively; p=0.26) (table4). Similarly, there was no correlation between disease activity and current (study) TPMT activity level (r _s=−0.05, p=0.55). Mean (SD) current TPMT activity was 17.3 (3.6) U/ml among the 56 patients with active disease compared with 17.3 (3.9) U/ml among the 114 patients in clinical remission. Furthermore, 33% of patients with TPMT activity in the homozygous normal range had active disease compared with 39% of patients with TPMT activity in the heterozygous intermediate range. There was also no significant correlation between IBDQ score and RBC 6-MMP concentration, AZA or 6-MP dose (mg/kg/day), or WBC or neutrophil count at the time blood was obtained for the study.

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Figure 1

Relationship between inflammatory bowel disease questionnaire (IBDQ) score and red blood cell (RBC) 6-thioguanine nucleotide (6-TGN) concentration in patients with active disease and in those in remission.

Discussion

In patients with ALL receiving 6-MP chemotherapy, the active metabolite 6-TGN is considered to exert its therapeutic effect through incorporation into DNA, with subsequent disruption of cell replication. Routine measurement of RBC 6-TGN concentration has been advocated as a means of assessing 6-MP drug effect, and paediatric patients with ALL receiving 6-MP at a dose of 75 mg/m² are more likely to relapse if RBC 6-TGN concentration falls below the median value of 275 pmol/8×10⁸ RBCs.12 Likewise, this patient group is more likely to experience leucopenia if RBC 6-TGN concentrations are elevated.7

Our study suggests that some caution should be used in extrapolating these findings to patients with IBD receiving immune modifier therapy with AZA or 6-MP. One recent study of paediatric patients with IBD treated with 6-MP reported that the median RBC 6-TGN concentration in patients with clinical response was 312 pmol/8×10⁸ RBCs whereas the median RBC 6-TGN concentration in non-responders was 199 pmol/8×10⁸ RBCs.9 These authors suggested a target RBC 6-TGN concentration exceeding 230 pmol/8×10⁸RBCs to optimise induction and maintenance of remission. In contrast, in our study we found no correlation between whole blood 6-TGN concentrations and remission and no difference in median whole blood 6-TGN concentrations in patients in clinical remission compared with those with active disease (131 v 139 pmol/8×10⁸ RBCs). While the median whole blood 6-TGN concentration of 136 pmol/8×10⁸ RBCs in our study was lower than the RBC 6-TGN concentrations reported in previous studies which used a different assay (see below),8 ,9 the majority of the patients in our study (67%) were in clinical remission, suggesting that the AZA and 6-MP doses used, and whole blood 6-TGN concentrations achieved, were sufficient to achieve the desired clinical effect in most patients. In addition, there was no correlation between whole blood 6-TGN concentrations and total WBCs. Our data are similar to those of another recent study that assessed the efficacy of an intravenous loading dose of AZA compared with standard oral dosing.11 In this latter study, in which mean steady state RBC 6-TGN concentrations for the intravenous loading dose and oral AZA groups were 164 and 124 pmol/8×10⁸ RBCs, respectively, there was no correlation between RBC 6-TGN concentrations and either clinical response or occurrence of leucopenia.

There are a variety of potential explanations for these discrepant results. Previous studies were smaller, ranging in size from 25 to 96 patients.8 ,9 ,11 Two of the previous studies evaluated only paediatric patients8 ,9 whereas another study11 and our current study included only adults. In the current study, disease activity was measured by the IBDQ score whereas in previous studies disease activity was measured by the Harvey-Bradshaw index8 ,9 and the CD activity index.11 In the two studies that measured disease activity with the Harvey-Bradshaw index, different definitions of remission were used (score <4 and score <5).8 ,9 The correlations between IBDQ and both the Harvey Bradshaw index and CD activity index are only −0.55 and −0.67, respectively,13 although all three have been well validated as measures of disease activity. In one of the previous studies, patients were studied for the first four months of AZA therapy.11 In two other studies patients were treated with 6-MP for a minimum of four months (thus patients with early dose dependent toxicity from AZA or 6-MP were not included). In the current study, patients currently receiving AZA or 6-MP for any period of time were offered enrolment but no patients treated for less than 3.5 months participated. The majority of patients in the current study and two of the previous studies8 ,9 were in symptomatic remission on maintenance therapy with AZA or 6-MP, in comparison with patients in another previous study who were starting AZA for active disease.11 Patients with low or intermediate TPMT activity were excluded from one previous study.11 Physicians caring for patients in the current study may have elected to use lower initial doses of AZA or 6-MP in patients with intermediate or low normal TPMT activity (this is nearly impossible to determine after the fact because the decision may not be recorded in the medical record). Multiple sampling of patients in one of the studies may have led to an overestimate of the beneficial effect of RBC 6-TGN monitoring.9 These differences in study design, with differing results, make it difficult to determine precisely the role of 6-TGN monitoring at the present time in patients with IBD treated with AZA or 6-MP. The negative outcome of our study suggests that additional research is needed before this strategy for optimising therapy for patients with IBD is adopted into routine clinical practice.

Studies of 6-TGN therapeutic drug monitoring in patients with IBD which reported an association between elevated 6-TGN concentrations and both efficacy and leucopenia used an assay for RBC 6-TGN developed by Lennard and Maddocks.19 We used a different RBC 6-TGN assay developed by Erdmann and colleagues15 and adapted it to whole blood, and thus our results are not directly comparable. However, we demonstrated a high degree of correlation in a blinded comparison of the two assays, with a conversion factor of approximately 1.6 to convert Erdmann assay results into Lennard assay results; this difference is apparently due to differences in hydrolysis times (unpublished data). Mean whole blood 6-TGN concentration of 163 pmol/8×10⁸ RBCs observed in our study would thus be comparable with a mean RBC 6-TGN concentration of 261 pmol/8×10⁸ RBCs using the Lennard assay.

In our study, patients with intermediate TPMT activity had a significantly greater mean RBC 6-TGN concentration compared with patients with normal TPMT activity (253 v151 pmol/8×10⁸ RBCs; p=0.002). However, although an inverse relationship between TPMT activity and RBC 6-TGN concentration was found, patients with intermediate TPMT activity did not exhibit an increased frequency of leucopenia. This lack of leucopenia is not surprising as no patient with AZA or 6-MP treatment duration less than 3.5 months participated (thus no patients with early leucopenia due to decreased TPMT activity participated). As discussed above, clinicians may have elected to use a lower dose of AZA or 6-MP in patients with known intermediate TPMT activity.

Benzoic acid derivatives such as 5-ASA,N-acetyl-5-ASA, sulphasalazine, olsalazine, and balsalazide cause in vitro inhibition of TPMT activity; IC₅₀ values for 5-ASA, sulphasalazine, olsalazine, and balsalazide are 1380, 104, 31, and 197 μM, respectively.20 ,21 Coadministration of olsalazine and 6-MP in a patient with intermediate TPMT activity led to bone marrow suppression.22 We found that patients receiving AZA or 6-MP and mesalamine, sulphasalazine, or olsalazine had a higher mean whole blood 6-TGN concentration and lower mean WBC count than patients receiving AZA or 6-MP alone. Mean plasma concentrations of 5-ASA,N-acetyl 5-ASA, sulphasalazine, and olsalazine observed in our study were somewhat below the IC₅₀ values for these compounds. Nevertheless, given the trend towards an increase in whole blood 6-TGN (pharmacokinetic effect) that occurred in our study, and the significant decrease in mean WBC (pharmacodynamic effect), it appears that the concentrations of 5-ASA and related compounds found in our study may be sufficient to inhibit TPMT activity in vivo. We have demonstrated elsewhere that addition of mesalamine, sulphasalazine, or balsalazide to stable therapy with AZA or 6-MP in patients with CD results in an increase in whole blood 6-TGN concentrations and a high frequency of leucopenia.10

In the largest study to date, we were unable to confirm the usefulness of therapeutic drug monitoring with 6-TGN in patients with IBD treated with AZA or 6-MP. There are methodological differences between our study and previous ones that demonstrated that measuring 6-TGN was useful in this setting. We confirmed the findings of other reports demonstrating an inverse relationship between TPMT activity and whole blood 6-TGN, and the occurrence of a drug interaction between mesalamine, sulphasalazine, or olsalazine and AZA or 6-MP, leading to increased whole blood 6-TGN and a lower mean WBC. With the exception of a single drug interaction study,10 all studies conducted to date are observational and do not prospectively test the hypothesis that dose adjustment of AZA or 6-MP to a target whole blood or RBC 6-TGN concentration will improve treatment outcomes in patients with IBD. Our study should serve as a cautionary note that adoption of 6-TGN monitoring into routine clinical practice may be premature at the present time. A randomised, double blind, controlled trial comparing AZA or 6-MP dose optimisation based on 6-TGN concentrations with standard therapy (standard initial dose adjusted only for toxicity) should be conducted. In addition, studies investigating the precise cellular and molecular mechanisms of action of AZA and 6-MP should be undertaken.