ORIGINAL ARTICLE: GASTROENTEROLOGY
Thiopurine Metabolite Ratios for Monitoring Therapy in Pediatric Crohn Disease
U. Kopylov, yD. Amre, yY. Theoret, yC. Deslandres, and E.G. Seidman
ABSTRACT Objectives: Thiopurines (azathioprine, 6-mercaptopurine) are a mainstay of treatment in Crohn disease (CD). Monitoring intracellular metabolite (6-thioguanine nucleotides [6-TGN] and 6-methylmercaptopurine [6-MMP]) levels can help optimize therapeutic efficacy and minimize potential toxicity. Determination of 6-MMP/6-TGN ratios may provide additional useful information, such as the identification of individuals with excessive thiopurine methyltransferase activity and disadvantageous 6-MMP overproduction. These patients are at increased risk of therapeutic failure and hepatotoxicity. The aim of the study was to evaluate the correlation of 6-MMP/6-TGN ratios with therapeutic efficacy and risk of hepatotoxicity in CD. Methods: The present study was a single-center cross-sectional study including pediatric patients with CD studied prospectively with clinical and laboratory assessments along with serial measurements of 6-MMP and 6-TGN. Clinical response was determined using established clinical indices. Results: The study included 238 pediatric patients with CD with a total of 1648 evaluation points. The patients were in steroid-free remission at 59.1% of the evaluation points. 6-MMP/6-TGN ratios of 4 to 24 were protective against relapse (odds ratio [OR] 0.52, 95% confidence interval [CI] 0.39 to 0.69, P ¼ 0.001). Hepatotoxicity was associated with high 6-MMP levels (>3919 pmol/8 108 red blood cell count: OR 7.65, 95% CI 3.7–15.9, P ¼ 0.001) and high 6-MMP/6-TGN ratios (>24: OR 5.35, 95% CI 3.43 to 8.43, P ¼ 0.001). Conclusions: We observed significant associations between 6-MMP/6-TGN ratios and clinical response, and risk of hepatotoxicity. Our results suggest that determination of thiopurine metabolite ratios is a valuable tool for identification of patients at increased risk of therapeutic failure and hepatotoxicity. Key Words: azathioprine, Crohn disease, drug monitoring, 6-methylmecaptopurine, 6-thioguanine, thiopurines
(JPGN 2014;59: 511–515)
Received April 1, 2014; accepted June 5, 2014. From the Division of Gastroenterology, McGill University Health Center, and the yDepartment of Pediatrics, Research Center, Sainte Justine Hospital, University of Montreal, Quebec, Canada. Address correspondence and reprint requests to Dr Uri Kopylov, Division of Gastroenterology, McGill University Health Center, 1650 Cedar Ave, Montreal, QC H3G 1A4, Canada (e-mail: [email protected]). E.G.S. is the recipient of a Canada Research Chair in Immune-Mediated Bowel Disorders and the Bruce Kaufman Chair in IBD at McGill. U.K. received the McGill IBD fellowship, supported by Janssen Inc. D.A. is the recipient of the senior researcher award from the Fonds de la Recherche´ en Sante´ du Que´bec (FRSQ). The authors report no conflicts of interest. Copyright # 2014 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition DOI: 10.1097/MPG.0000000000000455
JPGN
Volume 59, Number 4, October 2014
T
hiopurine medications (6-mercaptopurine [6-MP] and azathioprine [AZA]) have long been a mainstay of therapy in Crohn disease (CD). In the Cochrane database meta-analyses, AZA had a pooled efficacy of 54% and 71% for induction and maintenance of CD remission, respectively (1,2). Approximately 50% of patients with inflammatory bowel disease (IBD) are treated with thiopurines (3,4). Thiopurine treatment is associated with adverse effects in 15% to 39% of patients, leading to its discontinuation (4,5). In addition, treatment is associated with loss of response over time. The therapeutic benefit and toxicity of thiopurines have been shown to be mediated by the levels of their principal intracellular metabolites (Fig. 1), 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP). The key enzyme is thiopurine methyltransferase (TPMT), which methylates 6-MP to form 6-MMP. Intracellular metabolite levels have been associated with both therapeutic efficacy and risk of hepatotoxicity. Dubinsky et al first reported a therapeutic benefit of a 6-TGN level between 235 and 450 pmol/8 108 erythrocytes and the association of 6-MMP levels >5700 pmol/8 108 with an increased risk (18%) of hepatotoxicity in pediatric patients with IBD (6,7). A pediatric systematic review dedicated to thiopurine metabolite monitoring in pediatric IBD reported heterogeneous results in regard to the correlation of 6-TGN levels with clinical remission, with stronger evidence for the correlation of excessive 6-MMP levels with hepatotoxicity (8). The level of TPMT activity is controlled by common genetic polymorphisms, leading to a trimodal distribution of TPMT activity in the general population, with absent to low activity in 0.3% of individuals with a homozygous mutation, intermediate enzyme activity among heterozygotes in 11%, and normal or high activity in approximately 89% of the patients. Patients with low or intermediate TPMT activity phenotypes who are treated with standard thiopurine doses are at risk of severe myelosuppression caused by excessive accumulation of 6-TGN (9). In our experience, in up to 20% of patients failing therapy, thiopurine metabolism is skewed toward excessive production of 6-MMP (‘‘excessive TPMT’’) (10). In these patients it is difficult to achieve therapeutic 6-TGN levels without accumulating toxic 6-MMP levels. Further elevation of a thiopurine dosage usually results in a steep increase in 6-MMP production without a significant rise in 6-TGN levels (11), leading to discontinuation of these medications because of lack of efficacy or hepatotoxicity (12). Prompt identification of this unique subgroup is crucially important for both safety and efficacy considerations, because these patients may benefit from thiopurines if combined with medications that affect TPMT activity such as allopurinol or 5-aminosalicylates (13,14). We hypothesized that 6-MMP/6-TGN ratios are significantly associated with the risk of relapse and hepatotoxicity, and may provide valuable clinical data particularly important for patients with excessive TPMT activity. In the present study, we aimed to evaluate the clinical utility of 6-MMP/6-TGN ratios for
511
Copyright 2014 by ESPGHAN and NASPGHAN. Unauthorized reproduction of this article is prohibited.
Kopylov et al
JPGN Thiopurine metabolic pathway 6-TG nucleotides
6-TU
XO 6-MP
6-TImP
TPMT
TPMT 6-MMP ribonucleotides
6-MMP
Circulation
RNA
IMPDH
HPRT AZA
DNA
Intracellular
FIGURE 1. Principal thiopurine metabolic pathways. Azathioprine (AZA) is rapidly converted to 6-mercaptopurine (6-MP) by a nonenzymatic process. 6-MP is subsequently metabolized to immunologically inactive 6-methylmercaptopurine (6-MMP) metabolite ribonucleotides by thiopurine methyltransferase (TPMT). The alternative competing pathway is conversion to 6-thioinosine 5-monophosphate (6-TImP) by intracellular hypoxanthine-guanine phosphoribosyltransferase (HPRT) and then further enzymatic transformation by 2 separate metabolic pathways to produce 6-thianoguanine metabolites (6-TGN) through an enzymatic cascade including inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate synthase (GMPS) or, alternatively, by TPMT to 6-MMP.
prediction of clinical remission and hepatotoxicity in patients with IBD treated with thiopurines.
METHODS We carried out a prospective study that included pediatric patients with CD recruited from the gastroenterology clinics of Sainte Justine Hospital in Montreal (1996–2005). Diagnosis of CD was determined using standard clinical, radiological, endoscopic, and histological criteria. Patients treated with a thiopurine for at least 3 months were included. TPMT activity was determined before administration of a thiopurine, as reported earlier (12). Patients treated with concomitant biologics or methotrexate were excluded. Clinical information collected during an 18-month follow-up included the following: patient-related data including sex, age at diagnosis, disease location, and phenotype; disease activity calculated at each visit using the Harvey-Bradshaw index (15); medication-related data including age at initiation, indication, and dose; and toxicity-related data including complete and differential blood counts, serum alanine transaminase, aspartate transaminase, g-glutamyl transpeptidase, and bilirubin.
Measurement of 6-MP Metabolite Levels 6-MP metabolites were measured prospectively at 3- to 4-monthly intervals, or at the time of a clinical relapse or adverse events. Erythrocyte 6-TGN and 6-MMP concentrations (picomoles per 8 108 RBC) were measured by high-performance liquid chromatography, as described earlier (7).
Outcome Definitions A clinical evaluation point was designated as a clinical visit corresponding to each 6-MP metabolite measurement. Clinical
512
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remission was defined as Harvey-Bradshaw index 2 times the upper limit of normal for age. Adherence to treatment was defined as 6-TGN level 234 pmol/ 8 108 with risk of relapse, and for 6-MMP >5700 pmol/8 108 with hepatotoxicity). In the multivariate analysis, a single model for each metabolite and their ratio was fit after accounting for 6-MP dose, age at diagnosis, and sex. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were estimated. Two-sided P values 0.05 were considered to represent statistical significance. The quality of the models was evaluated using the Akaike information criterion (AIC) and the Bayesian information criterion (BIC) when correlation was similar, with lower values suggesting a better quality of the model.
Ethical Considerations The study received approval from the Sainte Justine Hospital institutional review board, and informed consent was acquired from the patients or their guardians.
RESULTS A total of 237 patients were evaluated for the metabolite levels and clinical outcomes at 1648 time points during follow-up. The clinical and demographic characteristics of the patients are shown in Table 1. The mean thiopurine dose administered was 1.21 mg/kg (0.34) of 6-MP (AZA dose was converted to a corresponding 6-MP dose for analysis). No TPMT homozygotes were included in the cohort; 7.7% of the patients were TPMT heterozygotes. The patients were found to be in steroid-free remission at 59.1% of the clinical evaluation points. At least 1 episode of hepatotoxicity was documented in 27.1% of patients (8.6% of the evaluation points). The mean ( standard deviation) 6-MMP/6TGN ratio was 18.9 (25.7). In TPMT heterozygotes, the mean ratio was 1.73 1.34, whereas in wild-type homozygotes 18.6 22.3 (P ¼ 0.001).
Correlation Between 6-MP Metabolite Levels and Ratios and Risk of Relapse The risk of relapse was associated with a trend toward lower erythrocyte 6-TGN levels (24: OR 5.35, 95% CI 3.43–8.43, P < 0.001; AIC ¼ 625.5, BIC ¼ 650.4) (Table 3); 6-MMP/6-TGN ratio >24 was observed in 25% of the patients. A combination of elevated 6-MMP levels with metabolite ratio >24 was significantly associated with the risk of hepatotoxicity (OR 4.75, 95% CI 3–7.53, P < 0.001; AIC ¼ 638.4, BIC ¼ 658.8). The quality of the models was similar, with marginally improved accuracy for 6-MMP/ 6-TGN levels for prediction of hepatotoxicity, as manifested by lower AIC/BIC. Thiopurine dose was not significantly associated with the risk of hepatotoxicity (OR 0.74, 95% CI 0.3–1.43, P ¼ 0.36).
DISCUSSION
42
Elevated 6-MMP levels (>5700 pmol/108 RBC) were associated with a higher risk for hepatotoxicity (OR 5.03, 95% CI 3.16–7.98, P < 0.001; AIC ¼ 633.8, BIC ¼ 658.8) (Table 3).
In the present large pediatric cohort, we observed a significant association between 6-MMP/6-TGN ratios and risk of relapse and hepatotoxicity. For steroid-free remission, the optimal 6-MMP/ 6-TGN ratio was in the range of 4 to 24. The risk of hepatotoxicity was increased significantly when the ratio exceeded 24. For prediction of relapse, metabolite ratios performed significantly better then 6-TGN levels; for hepatotoxicity, the strength of correlation was similar for 6-MMP levels >5700 pmol/8 108 and 6-MMP/ 6-TGN ratio >24. In the present study, higher 6-TGN levels were not statistically associated with steroid-free remission. This result may be because clinicians at our IBD clinic routinely use 6-MP metabolites in clinical practice, possibly leading to a potential bias in that the 6-MP dose would often be adjusted to try to achieve 6-TGN levels >235 pmol/108 erythrocytes. More important, such correlation was not confirmed by all pediatric studies (8,17,18). Although the majority of patients with CD achieve long-term remission on thiopurine therapy, at least 9% fail to achieve any initial response. Up to 39% of the patients discontinue the treatment during 8 years because of adverse effects, and additional 16% because of gradual loss of response (19). Frequent etiologies for insufficient response to thiopurines include underdosing and adverse effects (primarily myelotoxicity and hepatotoxicity) (4). Adherence is also a critical factor in achieving therapeutic effect; a recent study suggested that 6-TGN levels were not correlated with the prescribed dose; however, the correlation improved significantly when corrected for adherence (20). Monitoring thiopurine metabolites provides an important clue for identification of the etiology of nonresponse to thiopurines. The common reasons for nonresponsiveness include underdosing, lack of adherence, predominant TPMT profile, and ‘‘true’’ drug nonresponsiveness. Each one of these etiologies is characterized by a pathognomonic metabolic profile (Table 4). Nonadherent patients are easily identified based on spuriously low levels of both metabolites, with little improvement in response to dose escalation.
TABLE 2. Thiopurine metabolites levels and ratios and the odds of relapse
TABLE 3. Thiopurine metabolites levels and ratios and the risk of hepatotoxicity
52.1 31.4 27 11.4 7.7
5-ASA ¼ 5-aminosalicylic acid; EIM ¼ extraintestinal manifestations; TPMT ¼ thiopurine methyltransferase. Montreal classification for Crohn disease: location—L1 ¼ ileal, L2 ¼ colonic, and L3 ¼ ileocolonic; disease behavior—B1 ¼ nonstricturing, nonpenetrating, B2 ¼ stricturing, B3 ¼ penetrating, and p ¼ perianal disease. Location of disease for ulcerative colitis: E1 ¼ proctitis, E2 ¼ left-sided (distal) disease, and E3 ¼ pancolitis.
difference did not achieve statistical significance (Table 2). The cutoff level of 234 pmol/108 RBC was not significantly associated with a steroid-free remission (OR 0.89, 95% CI 0.69–1.14, P ¼ 0.36). 6-MMP/6-TGN ratios between 4 and 24 were significantly protective against relapse compared with other values (OR 0.52, 95% CI 0.31–0.81, P ¼ 0.001; AIC ¼ 1426, BIC ¼ 1451). The combination of optimal ratio (4–24) with therapeutic 6-TGN levels was protective of relapse (OR 0.50, 95% CI 0.31–0.81, P ¼ 0.005; AIC ¼ 1439, BIC ¼ 1463), with similar OR and model quality to that of the ratios alone. Thiopurine dose was not significantly correlated with steroid-free remission (OR 1.0, 95% CI 0.71– 1.46, P ¼ 0.91).
Correlation Between 6-MP Metabolite Levels and Ratios and Steroid-Free Remission
OR þ 95% CI
6-TGN 6-TGN >234 pmol/108 RBC 6-MMP/6-TGN ratio 4–24 6-TGN >234 pmol/108 RBC and 6-MMP/6-TGN 4–24
0.75 0.89 0.52 0.50
(0.55–1.03) (0.89–1.14) (0.39–0.69) (0.31–0.81)
0.07 0.36 0.001 0.05
CI ¼ confidence interval; 6-MMP ¼ 6-methylmercaptopurine; OR ¼ odds ratio; RBC ¼ red blood cell count; 6-TGN ¼ 6-thioguanine nucleotides. Tertile 1 versus 2.
www.jpgn.org
OR þ 95% CI
P
6-MMP 6-MMP >5700 pmol/108 RBC 6-MMP/6-TGN ratio >24 6-TGN >5700 pmol/108 RBC and 6-MMP/6-TGN >24
7.65 5.03 5.35 4.75
(3.7 15.9) (3.16–7.98) (3.43–8.43) (3–7.53)
P 0.008 0.001 0.001 0.001
CI ¼ confidence interval; 6-MMP ¼ 6-methylmercaptopurine; OR ¼ odds ratio; RBC ¼ red blood cell count; 6-TGN ¼ 6-thioguanine nucleotides. Quartile 1 versus 3.
513
Copyright 2014 by ESPGHAN and NASPGHAN. Unauthorized reproduction of this article is prohibited.
Kopylov et al
JPGN
Volume 59, Number 4, October 2014
TABLE 4. Algorithm for 6-MP metabolite measurement and ratios to help explain thiopurine failures in IBD Etiology of thiopurine failure
6-TGN level
6-MMP level
6-MMP/6-TGN ratio
Proposed treatment strategy Increase dose TPMT modulation by the addition of allopurinol, or 5-ASA, dose splitting Verify adherence Alternative therapy
Inadequate dose Excessive TPMT
Low (
Thiopurine Metabolite Ratios for Monitoring Therapy in Pediatric Crohn Disease
U. Kopylov, yD. Amre, yY. Theoret, yC. Deslandres, and E.G. Seidman
ABSTRACT Objectives: Thiopurines (azathioprine, 6-mercaptopurine) are a mainstay of treatment in Crohn disease (CD). Monitoring intracellular metabolite (6-thioguanine nucleotides [6-TGN] and 6-methylmercaptopurine [6-MMP]) levels can help optimize therapeutic efficacy and minimize potential toxicity. Determination of 6-MMP/6-TGN ratios may provide additional useful information, such as the identification of individuals with excessive thiopurine methyltransferase activity and disadvantageous 6-MMP overproduction. These patients are at increased risk of therapeutic failure and hepatotoxicity. The aim of the study was to evaluate the correlation of 6-MMP/6-TGN ratios with therapeutic efficacy and risk of hepatotoxicity in CD. Methods: The present study was a single-center cross-sectional study including pediatric patients with CD studied prospectively with clinical and laboratory assessments along with serial measurements of 6-MMP and 6-TGN. Clinical response was determined using established clinical indices. Results: The study included 238 pediatric patients with CD with a total of 1648 evaluation points. The patients were in steroid-free remission at 59.1% of the evaluation points. 6-MMP/6-TGN ratios of 4 to 24 were protective against relapse (odds ratio [OR] 0.52, 95% confidence interval [CI] 0.39 to 0.69, P ¼ 0.001). Hepatotoxicity was associated with high 6-MMP levels (>3919 pmol/8 108 red blood cell count: OR 7.65, 95% CI 3.7–15.9, P ¼ 0.001) and high 6-MMP/6-TGN ratios (>24: OR 5.35, 95% CI 3.43 to 8.43, P ¼ 0.001). Conclusions: We observed significant associations between 6-MMP/6-TGN ratios and clinical response, and risk of hepatotoxicity. Our results suggest that determination of thiopurine metabolite ratios is a valuable tool for identification of patients at increased risk of therapeutic failure and hepatotoxicity. Key Words: azathioprine, Crohn disease, drug monitoring, 6-methylmecaptopurine, 6-thioguanine, thiopurines
(JPGN 2014;59: 511–515)
Received April 1, 2014; accepted June 5, 2014. From the Division of Gastroenterology, McGill University Health Center, and the yDepartment of Pediatrics, Research Center, Sainte Justine Hospital, University of Montreal, Quebec, Canada. Address correspondence and reprint requests to Dr Uri Kopylov, Division of Gastroenterology, McGill University Health Center, 1650 Cedar Ave, Montreal, QC H3G 1A4, Canada (e-mail: [email protected]). E.G.S. is the recipient of a Canada Research Chair in Immune-Mediated Bowel Disorders and the Bruce Kaufman Chair in IBD at McGill. U.K. received the McGill IBD fellowship, supported by Janssen Inc. D.A. is the recipient of the senior researcher award from the Fonds de la Recherche´ en Sante´ du Que´bec (FRSQ). The authors report no conflicts of interest. Copyright # 2014 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition DOI: 10.1097/MPG.0000000000000455
JPGN
Volume 59, Number 4, October 2014
T
hiopurine medications (6-mercaptopurine [6-MP] and azathioprine [AZA]) have long been a mainstay of therapy in Crohn disease (CD). In the Cochrane database meta-analyses, AZA had a pooled efficacy of 54% and 71% for induction and maintenance of CD remission, respectively (1,2). Approximately 50% of patients with inflammatory bowel disease (IBD) are treated with thiopurines (3,4). Thiopurine treatment is associated with adverse effects in 15% to 39% of patients, leading to its discontinuation (4,5). In addition, treatment is associated with loss of response over time. The therapeutic benefit and toxicity of thiopurines have been shown to be mediated by the levels of their principal intracellular metabolites (Fig. 1), 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP). The key enzyme is thiopurine methyltransferase (TPMT), which methylates 6-MP to form 6-MMP. Intracellular metabolite levels have been associated with both therapeutic efficacy and risk of hepatotoxicity. Dubinsky et al first reported a therapeutic benefit of a 6-TGN level between 235 and 450 pmol/8 108 erythrocytes and the association of 6-MMP levels >5700 pmol/8 108 with an increased risk (18%) of hepatotoxicity in pediatric patients with IBD (6,7). A pediatric systematic review dedicated to thiopurine metabolite monitoring in pediatric IBD reported heterogeneous results in regard to the correlation of 6-TGN levels with clinical remission, with stronger evidence for the correlation of excessive 6-MMP levels with hepatotoxicity (8). The level of TPMT activity is controlled by common genetic polymorphisms, leading to a trimodal distribution of TPMT activity in the general population, with absent to low activity in 0.3% of individuals with a homozygous mutation, intermediate enzyme activity among heterozygotes in 11%, and normal or high activity in approximately 89% of the patients. Patients with low or intermediate TPMT activity phenotypes who are treated with standard thiopurine doses are at risk of severe myelosuppression caused by excessive accumulation of 6-TGN (9). In our experience, in up to 20% of patients failing therapy, thiopurine metabolism is skewed toward excessive production of 6-MMP (‘‘excessive TPMT’’) (10). In these patients it is difficult to achieve therapeutic 6-TGN levels without accumulating toxic 6-MMP levels. Further elevation of a thiopurine dosage usually results in a steep increase in 6-MMP production without a significant rise in 6-TGN levels (11), leading to discontinuation of these medications because of lack of efficacy or hepatotoxicity (12). Prompt identification of this unique subgroup is crucially important for both safety and efficacy considerations, because these patients may benefit from thiopurines if combined with medications that affect TPMT activity such as allopurinol or 5-aminosalicylates (13,14). We hypothesized that 6-MMP/6-TGN ratios are significantly associated with the risk of relapse and hepatotoxicity, and may provide valuable clinical data particularly important for patients with excessive TPMT activity. In the present study, we aimed to evaluate the clinical utility of 6-MMP/6-TGN ratios for
511
Copyright 2014 by ESPGHAN and NASPGHAN. Unauthorized reproduction of this article is prohibited.
Kopylov et al
JPGN Thiopurine metabolic pathway 6-TG nucleotides
6-TU
XO 6-MP
6-TImP
TPMT
TPMT 6-MMP ribonucleotides
6-MMP
Circulation
RNA
IMPDH
HPRT AZA
DNA
Intracellular
FIGURE 1. Principal thiopurine metabolic pathways. Azathioprine (AZA) is rapidly converted to 6-mercaptopurine (6-MP) by a nonenzymatic process. 6-MP is subsequently metabolized to immunologically inactive 6-methylmercaptopurine (6-MMP) metabolite ribonucleotides by thiopurine methyltransferase (TPMT). The alternative competing pathway is conversion to 6-thioinosine 5-monophosphate (6-TImP) by intracellular hypoxanthine-guanine phosphoribosyltransferase (HPRT) and then further enzymatic transformation by 2 separate metabolic pathways to produce 6-thianoguanine metabolites (6-TGN) through an enzymatic cascade including inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate synthase (GMPS) or, alternatively, by TPMT to 6-MMP.
prediction of clinical remission and hepatotoxicity in patients with IBD treated with thiopurines.
METHODS We carried out a prospective study that included pediatric patients with CD recruited from the gastroenterology clinics of Sainte Justine Hospital in Montreal (1996–2005). Diagnosis of CD was determined using standard clinical, radiological, endoscopic, and histological criteria. Patients treated with a thiopurine for at least 3 months were included. TPMT activity was determined before administration of a thiopurine, as reported earlier (12). Patients treated with concomitant biologics or methotrexate were excluded. Clinical information collected during an 18-month follow-up included the following: patient-related data including sex, age at diagnosis, disease location, and phenotype; disease activity calculated at each visit using the Harvey-Bradshaw index (15); medication-related data including age at initiation, indication, and dose; and toxicity-related data including complete and differential blood counts, serum alanine transaminase, aspartate transaminase, g-glutamyl transpeptidase, and bilirubin.
Measurement of 6-MP Metabolite Levels 6-MP metabolites were measured prospectively at 3- to 4-monthly intervals, or at the time of a clinical relapse or adverse events. Erythrocyte 6-TGN and 6-MMP concentrations (picomoles per 8 108 RBC) were measured by high-performance liquid chromatography, as described earlier (7).
Outcome Definitions A clinical evaluation point was designated as a clinical visit corresponding to each 6-MP metabolite measurement. Clinical
512
Volume 59, Number 4, October 2014
remission was defined as Harvey-Bradshaw index 2 times the upper limit of normal for age. Adherence to treatment was defined as 6-TGN level 234 pmol/ 8 108 with risk of relapse, and for 6-MMP >5700 pmol/8 108 with hepatotoxicity). In the multivariate analysis, a single model for each metabolite and their ratio was fit after accounting for 6-MP dose, age at diagnosis, and sex. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were estimated. Two-sided P values 0.05 were considered to represent statistical significance. The quality of the models was evaluated using the Akaike information criterion (AIC) and the Bayesian information criterion (BIC) when correlation was similar, with lower values suggesting a better quality of the model.
Ethical Considerations The study received approval from the Sainte Justine Hospital institutional review board, and informed consent was acquired from the patients or their guardians.
RESULTS A total of 237 patients were evaluated for the metabolite levels and clinical outcomes at 1648 time points during follow-up. The clinical and demographic characteristics of the patients are shown in Table 1. The mean thiopurine dose administered was 1.21 mg/kg (0.34) of 6-MP (AZA dose was converted to a corresponding 6-MP dose for analysis). No TPMT homozygotes were included in the cohort; 7.7% of the patients were TPMT heterozygotes. The patients were found to be in steroid-free remission at 59.1% of the clinical evaluation points. At least 1 episode of hepatotoxicity was documented in 27.1% of patients (8.6% of the evaluation points). The mean ( standard deviation) 6-MMP/6TGN ratio was 18.9 (25.7). In TPMT heterozygotes, the mean ratio was 1.73 1.34, whereas in wild-type homozygotes 18.6 22.3 (P ¼ 0.001).
Correlation Between 6-MP Metabolite Levels and Ratios and Risk of Relapse The risk of relapse was associated with a trend toward lower erythrocyte 6-TGN levels (24: OR 5.35, 95% CI 3.43–8.43, P < 0.001; AIC ¼ 625.5, BIC ¼ 650.4) (Table 3); 6-MMP/6-TGN ratio >24 was observed in 25% of the patients. A combination of elevated 6-MMP levels with metabolite ratio >24 was significantly associated with the risk of hepatotoxicity (OR 4.75, 95% CI 3–7.53, P < 0.001; AIC ¼ 638.4, BIC ¼ 658.8). The quality of the models was similar, with marginally improved accuracy for 6-MMP/ 6-TGN levels for prediction of hepatotoxicity, as manifested by lower AIC/BIC. Thiopurine dose was not significantly associated with the risk of hepatotoxicity (OR 0.74, 95% CI 0.3–1.43, P ¼ 0.36).
DISCUSSION
42
Elevated 6-MMP levels (>5700 pmol/108 RBC) were associated with a higher risk for hepatotoxicity (OR 5.03, 95% CI 3.16–7.98, P < 0.001; AIC ¼ 633.8, BIC ¼ 658.8) (Table 3).
In the present large pediatric cohort, we observed a significant association between 6-MMP/6-TGN ratios and risk of relapse and hepatotoxicity. For steroid-free remission, the optimal 6-MMP/ 6-TGN ratio was in the range of 4 to 24. The risk of hepatotoxicity was increased significantly when the ratio exceeded 24. For prediction of relapse, metabolite ratios performed significantly better then 6-TGN levels; for hepatotoxicity, the strength of correlation was similar for 6-MMP levels >5700 pmol/8 108 and 6-MMP/ 6-TGN ratio >24. In the present study, higher 6-TGN levels were not statistically associated with steroid-free remission. This result may be because clinicians at our IBD clinic routinely use 6-MP metabolites in clinical practice, possibly leading to a potential bias in that the 6-MP dose would often be adjusted to try to achieve 6-TGN levels >235 pmol/108 erythrocytes. More important, such correlation was not confirmed by all pediatric studies (8,17,18). Although the majority of patients with CD achieve long-term remission on thiopurine therapy, at least 9% fail to achieve any initial response. Up to 39% of the patients discontinue the treatment during 8 years because of adverse effects, and additional 16% because of gradual loss of response (19). Frequent etiologies for insufficient response to thiopurines include underdosing and adverse effects (primarily myelotoxicity and hepatotoxicity) (4). Adherence is also a critical factor in achieving therapeutic effect; a recent study suggested that 6-TGN levels were not correlated with the prescribed dose; however, the correlation improved significantly when corrected for adherence (20). Monitoring thiopurine metabolites provides an important clue for identification of the etiology of nonresponse to thiopurines. The common reasons for nonresponsiveness include underdosing, lack of adherence, predominant TPMT profile, and ‘‘true’’ drug nonresponsiveness. Each one of these etiologies is characterized by a pathognomonic metabolic profile (Table 4). Nonadherent patients are easily identified based on spuriously low levels of both metabolites, with little improvement in response to dose escalation.
TABLE 2. Thiopurine metabolites levels and ratios and the odds of relapse
TABLE 3. Thiopurine metabolites levels and ratios and the risk of hepatotoxicity
52.1 31.4 27 11.4 7.7
5-ASA ¼ 5-aminosalicylic acid; EIM ¼ extraintestinal manifestations; TPMT ¼ thiopurine methyltransferase. Montreal classification for Crohn disease: location—L1 ¼ ileal, L2 ¼ colonic, and L3 ¼ ileocolonic; disease behavior—B1 ¼ nonstricturing, nonpenetrating, B2 ¼ stricturing, B3 ¼ penetrating, and p ¼ perianal disease. Location of disease for ulcerative colitis: E1 ¼ proctitis, E2 ¼ left-sided (distal) disease, and E3 ¼ pancolitis.
difference did not achieve statistical significance (Table 2). The cutoff level of 234 pmol/108 RBC was not significantly associated with a steroid-free remission (OR 0.89, 95% CI 0.69–1.14, P ¼ 0.36). 6-MMP/6-TGN ratios between 4 and 24 were significantly protective against relapse compared with other values (OR 0.52, 95% CI 0.31–0.81, P ¼ 0.001; AIC ¼ 1426, BIC ¼ 1451). The combination of optimal ratio (4–24) with therapeutic 6-TGN levels was protective of relapse (OR 0.50, 95% CI 0.31–0.81, P ¼ 0.005; AIC ¼ 1439, BIC ¼ 1463), with similar OR and model quality to that of the ratios alone. Thiopurine dose was not significantly correlated with steroid-free remission (OR 1.0, 95% CI 0.71– 1.46, P ¼ 0.91).
Correlation Between 6-MP Metabolite Levels and Ratios and Steroid-Free Remission
OR þ 95% CI
6-TGN 6-TGN >234 pmol/108 RBC 6-MMP/6-TGN ratio 4–24 6-TGN >234 pmol/108 RBC and 6-MMP/6-TGN 4–24
0.75 0.89 0.52 0.50
(0.55–1.03) (0.89–1.14) (0.39–0.69) (0.31–0.81)
0.07 0.36 0.001 0.05
CI ¼ confidence interval; 6-MMP ¼ 6-methylmercaptopurine; OR ¼ odds ratio; RBC ¼ red blood cell count; 6-TGN ¼ 6-thioguanine nucleotides. Tertile 1 versus 2.
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OR þ 95% CI
P
6-MMP 6-MMP >5700 pmol/108 RBC 6-MMP/6-TGN ratio >24 6-TGN >5700 pmol/108 RBC and 6-MMP/6-TGN >24
7.65 5.03 5.35 4.75
(3.7 15.9) (3.16–7.98) (3.43–8.43) (3–7.53)
P 0.008 0.001 0.001 0.001
CI ¼ confidence interval; 6-MMP ¼ 6-methylmercaptopurine; OR ¼ odds ratio; RBC ¼ red blood cell count; 6-TGN ¼ 6-thioguanine nucleotides. Quartile 1 versus 3.
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Kopylov et al
JPGN
Volume 59, Number 4, October 2014
TABLE 4. Algorithm for 6-MP metabolite measurement and ratios to help explain thiopurine failures in IBD Etiology of thiopurine failure
6-TGN level
6-MMP level
6-MMP/6-TGN ratio
Proposed treatment strategy Increase dose TPMT modulation by the addition of allopurinol, or 5-ASA, dose splitting Verify adherence Alternative therapy
Inadequate dose Excessive TPMT
Low (
