6-Mercaptopurine (6-MP) and azathioprine (AZA) are

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1 GASTROENTEROLOGY 2006;130: Association of 6-Thioguanine Nucleotide Levels and Inflammatory Bowel Disease Activity: A Meta-Analysis MARK T. OSTERMAN,*, RABI KUNDU, GARY R. LICHTENSTEIN,* and JAMES D. LEWIS*, *Division of Gastroenterology, and the Center for Clinical Epidemiology and Biostatistics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania; and the Division of Gastroenterology, Graduate Hospital, Philadelphia, Pennsylvania See editorial on page 1352; CME Quiz on page Background & Aims: 6-Thioguanine nucleotide (6-TGN) levels have been proposed to correlate with inflammatory bowel disease (IBD) activity among patients treated with azathioprine or 6-mercaptopurine (6-MP). Previous studies, most with small sample sizes, yielded conflicting conclusions. Our aim was to pool the available data to provide a more precise estimate of the association between 6-TGN levels and IBD activity. Methods: We searched Medline and PubMed (from 1966 to November 2004) and reviewed the reference lists of selected articles. Fixed and random-effects models were used to test whether mean/median 6-TGN levels differed among patients with active disease vs remission and whether 6-TGN levels above a threshold of pmol/ red blood cells were associated with clinical remission. When studies reported multiple 6-TGN threshold values, we used the data for the lower value. Results: We identified 55 articles, 12 of which contained data sufficient for inclusion. The mean/median 6-TGN levels were higher among patients in remission than in those with active IBD (pooled difference, 66 pmol/ red blood cells; 95% confidence interval, ; P.006), but with significant heterogeneity. Excluding the 1 outlier study eliminated this heterogeneity. Patients with 6-TGN levels above the threshold value were more likely to be in remission (62%) than those below the threshold value (36%) (pooled odds ratio, 3.3; 95% confidence interval, ; P <.001), but with significant heterogeneity. Again, excluding the 1 outlier study eliminated this heterogeneity. Conclusions: Although prior studies yielded inconsistent conclusions, this analysis strongly supports that higher 6-TGN levels are associated with clinical remission. 6-Mercaptopurine (6-MP) and azathioprine (AZA) are potent immunomodulators used to treat patients with inflammatory bowel disease (IBD) to induce and maintain remission, as steroid-sparing agents, and to limit loss of tolerance to biological agents. The mechanisms of action of 6-MP and AZA likely are multifactorial. At the molecular level, 6-thioguanine nucleotides (6-TGNs) have the ability to interfere with both DNA and RNA synthesis. 1 AZA and 6-MP also are believed to work at the cellular level by inhibiting the proliferation of both T and B lymphocytes, decreasing suppressor T-lymphocyte function and cellular immunity, inducing apoptosis of T lymphocytes, and interfering with the cytotoxic ability of natural killer cells. 1 3 About 30% of patients with Crohn s disease fail to respond to the standard doses used in most published series. 4 Concerns regarding both short- and long-term toxicity are the most common explanation for their restricted use. 5 6-MP and its nitroimidazole derivative, AZA, are members of the thiopurine class of medications. They are both inactive prodrugs that require extensive chemical alteration by a number of different enzymes to achieve their active forms (Figure 1). On oral administration, AZA is absorbed into the plasma and cleaved rapidly to 6-MP and glutathionyl imidazole by a nonenzymatic reaction occurring within erythrocytes. Three major pathways then ensue to convert 6-MP into its various metabolites. 1 The 3 critical enzymes corresponding to these pathways are xanthine oxidase, thiopurine methyltransferase (TPMT), and hypoxanthine phosphoribosyltransferase. Xanthine oxidase, which is present in high concentrations within enterocytes and hepatocytes, rapidly and extensively metabolizes 6-MP into the inactive compound 6-thiouric acid. As a result, the absolute oral bioavailability of 6-MP is only 5% 37%. 6 The anabolic conversion of 6-MP into its active metabolites occurs along 2 competing pathways via the enzymes TPMT and hypoxanthine phosphoribosyltransferase. TPMT catalyzes 2 reactions, resulting in the formation of 6-methylmercaptopurine and 6-methylmercaptopurine ribonucleotides (6-MMPR). 6-methylmercaptopurine and 6-methylmercaptopurine ribonucleotides are thought to be responsible for some of the toxic effects (particularly Abbreviations used in this paper: AZA, azathioprine; CI, confidence interval; 6-MP, 6-mercaptopurine; OR, odds ratio; 6-TGN, 6-thioguanine nucleotide; TPMT, thiopurine methyltransferase by the American Gastroenterological Association Institute /06/$32.00 doi: /j.gastro

2 1048 OSTERMAN ET AL GASTROENTEROLOGY Vol. 130, No. 4 Figure 1. Metabolism of 6-MP and AZA. XO, xanthine oxidase; 6-TU, 6-thiouric acid; 6-MMP, 6-methylmercaptopurine; 6-MMPR, 6-methylmercaptopurine ribonucleotides; HPRT, hypoxanthine phosphoribosyltransferase; 6-TIMP, 6-thioinosine 5=-monophosphate; IMPDH, inosine monophosphate dehydrogenase; 6-TXMP, 6-thioxanthosine 5=-monophosphate; GMPS, guanosine monophosphate synthetase. hepatotoxicity) of 6-MP and AZA. Hypoxanthine phosphoribosyltransferase begins the process of converting 6-MP into its therapeutically active metabolites, the 6-TGNs, which helps quell the inflammatory response in IBD but may lead to the untoward effect of myelosuppression, which can affect all 3 bone marrow cell lines. The TPMT gene, located on chromosome 6, is inherited as an autosomal-codominant trait. An apparent genetic polymorphism has been observed in TPMT activity, resulting in a trimodal distribution. Approximately 0.3% of the general population shows low to absent activity as a result of inheritance of 2 mutant copies of the TPMT gene (TPMT L /TPMT L ). Roughly 11% of the population is heterozygous for the mutation (TMPT H / TPMT L ), and therefore has intermediate enzyme activity. These 2 groups of individuals are particularly susceptible to myelosuppression after administration of 6-MP or AZA. Fortunately, however, the vast majority of the population (89%) is homozygous for the wild-type of TPMT (TPMT H /TPMT H ), with normal to high activity. 7 Several prior studies have suggested that higher levels of 6-TGN are associated with both clinical response and toxicity (myelosuppression, primarily leukopenia) in IBD (Figure 2). However, the majority of these studies were underpowered to detect small differences, and the studies frequently came to different conclusions. The aim of our meta-analysis was to examine the relationship between 6-TGN levels and clinical response in IBD by pooling the results of studies reporting data on 6-TGN levels and disease activity. Materials and Methods Inclusion/Exclusion Criteria Inclusion and exclusion criteria were delineated before the commencement of the literature search. Studies were included in this meta-analysis if they presented data on 6-TGN levels in IBD patients taking 6-MP or AZA. Studies also were required to measure disease activity and to specify how remission and active disease states were ascertained. Both pediatric and adult data were included. Only studies published in English were included. Studies not meeting the aforementioned criteria were excluded. In addition, studies published only in abstract form were excluded from the primary analysis but were examined in a secondary analysis. Literature Search Medline and PubMed Plus from 1966 through November 2004 were used to identify potential studies for inclusion. Multiple search strategies were used; keywords were used in preference to MeSH terms to increase the sensitivity of our search. The following key words were used alone and in multiple combinations: 6-thioguanine, 6-thioguanine level(s), 6-thioguanine nucleoside(s), 6-thioguanine nucleoside level(s), 6-TG, 6-TG level(s), 6-TG nucleoside(s), 6-TG nucleoside level(s), 6-TGN, 6-TGN level(s), azathioprine, azathioprine metabolite(s), 6-mercaptopurine, 6-mercaptopurine metabolite(s), 6-MP, 6-MP metabolite(s), inflammatory bowel disease, ulcerative colitis, and Crohn s disease. Reference lists of all studies identified were reviewed comprehensively to ensure completeness of the search. Statistical Analysis Data were abstracted independently from all included studies by 2 investigators (M.T.O. and R.K.). Discrepancies in data interpretation were resolved by discussion and re-review of the studies and consultation with the senior author (J.D.L.) when necessary. We conducted 3 primary analyses to address our aim. First, we explored differences in mean or median 6-TGN levels between patients in clinical remission and those with active disease. For the sake of pooling means and medians of 6-TGN levels and calculating 95% confidence intervals (CIs) for the analysis, medians with ranges were considered to approximate means 1.96 SDs. Fixed and random-effects models were used to test whether pooled mean/median 6-TGN levels differed between patients with active and inactive disease, exe- Figure 2. Metabolite levels of 6-MP and AZA in relationship to clinical efficacy and toxicities. All metabolite units are in pmol/ RBCs.

3 April TGN AND IBD 1049 Table 1. Descriptive Statistics Study N Adult/pediatric % Remission % Crohn s disease a index used Disease activity Achkar et al, Adult Global assessment Goldenberg et al, Adult HBI, Powell Tuck Hindorf et al, Adult CDAI, Seo Wright et al, Adult CDAI, WBI Mardini et al, Adult Global assessment Dubinsky et al, Adult HBI, UCDAI Belaiche et al, Adult CDAI Cuffari et al, Adult HBI, Lichtiger Gupta et al, Pediatric PCDAI, TW Lowry et al, Adult IBDQ Dubinsky et al, Pediatric HBI, TW Cuffari et al, Pediatric HBI HBI, Harvey Bradshaw Index; CDAI, Crohn s Disease Activity Index; WBI, Walmsley Simple Index; UCDAI, Ulcerative Colitis Disease Activity Index; PCDAI, Pediatric Crohn s Disease Activity Index; TW, Truelove and Witts; IBDQ, Inflammatory Bowel Disease Questionnaire. a Some patients in the studies had ulcerative colitis, some of the patients with Crohn s disease had fistulizing disease, and others had luminal disease. cuted by the metan command in STATA (Stata Corp, College Station, TX). We decided a priori that patients labeled as partial responders were considered to have active disease for this meta-analysis. The studies by Achkar et al 8 and Belaiche et al 9 included patients who were partial responders. However, in the Achkar et al 8 study, insufficient data were provided to calculate a grouped SD, and in the Belaiche et al 9 study, insufficient data were provided to calculate a grouped median 6-TGN level. For these reasons, only complete responders and complete nonresponders were used to compare mean/median 6-TGN levels in these studies, which led to the exclusion of 7 patients from the Achkar et al 8 study and 6 patients from the Belaiche et al 9 study. The study by Wright et al 10 provided data on mean/median 6-TGN levels on only the 131 patients who had achieved steady-state concentration, thus excluding 28 patients. Therefore, we were limited to including the data on only these 131 patients in our analysis. The study by Dubinsky et al 11 from 2000 did not provide independent data on 6-TGN levels for all patients and instead used 1 sample for 46 patients and roughly 3 samples from each of the other 46 patients, thus yielding 173 samples that were not independent. We included these results in our analysis because the investigators stated in their publication that nearly identical results were observed in analyses using the nonindependent and independent samples. A second set of analyses were performed to determine whether certain threshold values of 6-TGN levels were associated with clinical remission. Odds ratios (ORs) with 95% CIs for remission were calculated using Mantel Haenszel methods for all studies in which sufficient data were provided. When studies reported more than one 6-TGN threshold value 8,9, we calculated ORs based on each value. Separate pooled ORs were generated based on the lowest and highest threshold values for each study using pooled Mantel Haenszel methods with the metan command in STATA. However, because the pooled results based on the lowest and highest threshold values were nearly identical, only results based on the lowest threshold values are reported here. Both fixed and random-effects models were used. For each study that reported threshold values of the 6-TGN level, we calculated the proportion of patients above and below the threshold values who were in remission. We pooled these results separately for the lowest and highest threshold values using fixed and random-effects models. Again, because the results were nearly identical, only results based on the lowest threshold values are reported. In every pooled analysis, the weight assigned to each study in the pooling process was inversely proportional to the variance of the particular study. In all cases, fixed and randomeffects models yielded nearly identical results, with the random-effects models showing more conservative estimates. For this reason, only random-effects results are reported. We examined the studies for evidence of heterogeneity using a 2 test for heterogeneity. A P value of less than.05 was considered indicative of statistically significant heterogeneity. We performed sensitivity analyses in which we excluded each study individually to determine the effect on the test of heterogeneity and the overall pooled estimates. In additional sensitivity analyses, we included studies published only as abstracts. The likelihood of publication bias was assessed via the Begg and Egger tests. All statistical analyses were performed using STATA version 8.0. Results We identified 55 studies, 12 of which contained data sufficient for inclusion (Table 1) The 12 studies varied widely in sample size, in the proportion of patients who were in remission, their duration of disease, their duration of 6-MP or AZA use, and by the indices used to assess disease activity. The studies appeared less heterogeneous in other aspects because the majority of patients in each study were adults with Crohn s disease

4 1050 OSTERMAN ET AL GASTROENTEROLOGY Vol. 130, No. 4 Table 2. Differences in Mean/Median 6-TGN Levels Between Patients With Active and Inactive Disease Study N 6-TGN level remission (pmol/ RBC) 6-TGN Level Active (pmol/ RBC) % Weight Achkar et al, a Mean, 320 Mean, SD, 22 SD, 20 Goldenberg et al, Mean, 325 Mean, SD, 284 SD, 159 Hindorf et al, Median, 288 Median, SD, 196 SD, 116 Wright et al, b Median, 236 Median, Range, Range, Dubinsky et al, Median, 306 Median, Range, Range, Belaiche et al, c Median, 166 Median, Range, Range, Lowry et al, Mean, 250 Mean, SD, 186 SD, 142 Dubinsky et al, d Median, 312 Median, Range, Range, Pooled difference 66 (18,113) (95% CI) P.006 NOTE: Random-effects results shown only. a Based on patients with only active disease or remission (7 partial responders excluded because SD could not be calculated). b Based on 131 patients with steady-state values. c Based on patients with only active disease or remission (6 partial responders excluded because median value could not be calculated). d Based on 173 samples from patients with active disease and remission (NB: samples are not independent). who had been using 6-MP or AZA at the target dose for at least 10 weeks. In addition, assays for 6-TGN levels were performed in a uniform fashion in 10 of the 12 studies 8 12,14 17,19 via a modification of the high-performance liquid chromatography assay developed initially by Lennard and Singleton 20 ; in 5 of these studies the assays were performed at Prometheus Laboratories in San Diego, CA. 8,12,14,15,17 The study by Lowry et al 18 used the Erdmann method, which required a conversion factor of 1.6 to convert Erdmann assay results into Lennard assay results. The study by Hindorf et al 13 used an assay developed by Bruunshuus and Schmeigelow, which required a conversion factor of 2.0 to convert into Lennard assay results. Eight studies reported differences in mean (with SDs) or median (with ranges) 6-TGN levels between patients with active and inactive disease, thus allowing the calculation of a pooled difference with 95% CIs (Table 2). 8 13,15,18 Mean/median 6-TGN levels were higher among patients in remission than in those with active disease with a pooled difference of 66 pmol/ red blood cells (RBCs) (95% CI, ; P.006). We observed significant heterogeneity among studies when pooling means/medians (P.001). We performed sensitivity analyses excluding each study individually to determine whether there was any effect on the heterogeneity or the pooled estimate of the difference in mean/ median 6-TGN levels. In all cases except 1, the exclusion of each study individually neither eliminated the heterogeneity nor changed the pooled estimate appreciably (pooled difference range, pmol/ RBCs). The exclusion of the study by Lowry et al, 18 however, eliminated the significant heterogeneity (P.09) and increased the pooled estimate of the difference in the mean/median 6-TGN levels between patients with active and inactive disease (pooled difference, 84 pmol/ RBCs; 95% CI, ). It is interesting to note that this study was 1 of the 2 studies using a different assay for 6-TGN levels. With respect to the association between dose of immunomodulator and 6-TGN levels, 7 of these 8 studies formally tested for correlation in this capacity via a Spearman correlation coefficient. 8 13,15 All 7 studies found no correlation between the drug dose and 6-TGN level. The same 7 studies also reported data on the mean or median dose of immunomodulator in patients with active disease and those in remission. Four studies observed no significant difference, 10,11,13,15 2 studies did not perform a test of significance, 9,12 and 1 study found that patients in remission were taking a significantly higher dose of 6-MP or AZA. 8 Six studies reported sufficient data on threshold values of 6-TGN level to allow a calculation of both the proportion of patients above and below the threshold values who were in remission and ORs of remission based on each threshold value. 8,9,11,12,16,17 Threshold values ranged

5 April TGN AND IBD 1051 Table 3. Pooled Proportions Based on Threshold Values of 6-TGN Levels Study N 6-TGN threshold (pmol/ RBC) Proportion above threshold in remission Proportion below threshold in remission Achkar et al, Goldenberg et al, Belaiche et al, Cuffari et al, Gupta et al, Dubinsky et al, a Pooled proportions (95% CI) (.43.80) (.25.48) NOTE. Random-effects results based on lowest threshold values shown only. a Based on 173 samples from 92 patients (NB: samples are not independent). from 230 to 260 pmol/ RBCs. A pooled analysis showed that 62% of patients above the threshold value were in remission, compared with 36% of patients below the threshold value who were in remission (Table 3). Patients in remission were more likely to have 6-TGN levels above the threshold value in all studies based on calculated ORs; statistical significance was reached in 3 of the 6 studies when considered individually (Figure 1). A pooled analysis showed that patients in remission were more likely to have 6-TGN levels above the threshold value, with a pooled OR of 3.27 (95% CI, ; P.001). We again observed significant heterogeneity in analyses pooling ORs (P.04). In all cases except 1, the exclusion of each study individually neither eliminated the heterogeneity nor changed the pooled estimate appreciably (pooled OR range, ). Exclusion of the study by Cuffari et al, 16 however, did eliminate the significant heterogeneity (P.17) and also mildly decreased the pooled OR (pooled OR, 2.65; 95% CI, ). This study yielded an OR that was noticeably disparate from the others and thus may represent an outlier. Figure 3. Pooled ORs based on threshold values of 6-TGN levels. Random-effects results based on lowest threshold values shown only. In addition, we identified 2 additional studies that would have been included in our primary analysis had they not been published only in abstract form. 21,22 Inclusion of these studies yielded nearly identical results (pooled difference, 66 pmol/ RBCs, 95% CI, ; pooled OR, 3.57; 95% CI, ). Finally, the possibility of publication bias was assessed using both the Begg and Egger tests, neither of which suggested that publication bias was present (P.88 and.87, respectively). Discussion Our pooled analyses showed that IBD patients in remission had significantly higher 6-TGN levels than those with active disease with a pooled difference of 66 pmol/ RBCs. We also found that patients with 6-TGN levels above threshold values of pmol/ RBCs were significantly more likely to be in clinical remission with an OR of In addition, 62% of patients above these threshold values were in remission, compared with only 36% of patients who were in remission at 6-TGN levels below the threshold values. We observed significant heterogeneity when pooling both mean/median 6-TGN levels and ORs. In both cases, we performed multiple sensitivity analyses in which each study was excluded individually to see whether the heterogeneity and pooled estimates would be affected. In the case of mean/median 6-TGN levels, only excluding the study by Lowry et al 18 eliminated the heterogeneity. This study was the only one to report lower 6-TGN levels in patients in remission and thus may be considered a potential outlier study. In addition, this study was only 1 of 2 studies that did not use the modified Lennard and Singleton 20 assay to measure 6-TGN levels. In the case of pooling ORs, only excluding the study by Cuffari et al eliminated the heterogeneity. This study reported a substantially higher OR of remission above the threshold value than the other study and thus may be considered a

6 1052 OSTERMAN ET AL GASTROENTEROLOGY Vol. 130, No. 4 possible outlier. Thus, in both cases, heterogeneity resulted from studies that were apparent outliers. However, pooled mean/median 6-TGN levels and pooled ORs were similar whether or not these outlier studies were included. Our study, however, is not without limitations. A meta-analysis by definition is inherently limited by the studies included. In our case, many of the studies were descriptive, either cross-sectional or retrospective, and had relatively small sample sizes. In addition, of the 12 studies suitable for inclusion in the meta-analysis, only 8 provided sufficient data to calculate pooled mean/median 6-TGN levels and only 6 provided sufficient data to allow computation of pooled ORs based on threshold 6-TGN values. Thus, some information potentially was lost. Finally, there was significant heterogeneity among the studies when pooling data. However, despite this heterogeneity, similar results were reported by most studies. The fact that studies with different designs, sample sizes, and patient populations generally reached the same conclusions could be viewed as confirmation of the association between 6-TGN level and disease activity in IBD patients taking 6-MP and AZA. Recently, Roblin et al 23 published a study examining 6-TGN levels and steroid-free clinical remission in 106 IBD patients. By using a threshold 6-TGN value of 250 pmol/ RBCs, they reported a multivariable adjusted OR of clinical remission of 11.2 (95% CI, ; P.001) in patients above this threshold 6-TGN level. In fact, no patient with a 6-TGN level less than 250 pmol/ RBCs attained steroid-free remission. Because this study was published after our predefined time period, it was not included in our analysis. However, inclusion of this study in our analysis only would have strengthened the association we found. In addition, the study by Cuffari et al, who reported a similar OR of remission of 11.3 using a threshold 6-TGN level of 250 pmol/ RBCs, would have seemed less of an outlier. The overlying issue that remains concerns the role of monitoring 6-TGN levels in clinical practice. Although this meta-analysis was not designed specifically to address this issue, our results still convey some valuable information. 6-TGN levels do seem to be associated with clinical remission, with higher levels observed in patients with inactive disease. Threshold 6-TGN values of pmol/ RBCs may serve as useful therapeutic targets, especially in patients with active disease. For instance, a patient with active disease who is above the threshold 6-TGN value is likely a nonresponder to 6-MP or AZA. On the other hand, a patient below the threshold value may require an increase in dose, which may lead to induction of remission. Dubinsky et al 15 also suggested that monitoring the 6-TGN level may be useful to identify noncompliers, in which case 6-TGN levels would be negligible. Another potential value of 6-TGN testing concerns the timing of the testing. If testing is performed relatively early in the treatment course and a patient is identified as a nonresponder, exposure to immunomodulator therapy could be minimized, thus potentially decreasing the risk for adverse events such as infection, myelosuppression, hepatotoxicity, and lymphoma. Likewise, early testing (after 2 4 weeks of therapy) could identify patients likely to benefit from a dose increase. Although some argue that these medications should be dosed according to body weight, prior research has documented a poor correlation between dose on a per kilogram basis and 6-TGN levels. 8,10 13,17 Thus, even in patients prescribed the recommended dose of these medications, the measurement of 6-TGN levels may provide additional useful information. Two prospective trials addressing this issue currently are under way. In conclusion, this meta-analysis of retrospective and cross-sectional studies suggests a strong association between 6-TGN levels and induction of remission among patients with IBD. Despite the differences in the populations studied, the pooled results were relatively consistent. Several outlier studies were identified that contributed to the heterogeneity of the published results. Whether these outliers resulted from chance or from differences in study design could not be ascertained with certainty. The results of 2 prospective trials are awaited eagerly to confirm the results of this meta-analysis. Until those studies are completed, this meta-analysis provides strong evidence that 6-TGN levels are associated with clinical response in IBD patients taking 6-MP or AZA and thus may be useful in the management of these patients, particularly those with disease refractory to their current dose of AZA or 6-MP. 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