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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Background Measurement of thiopurine metabolite levels may be useful as a clinical tool to optimize thiopurine treatment of paediatric inflammatory bowel disease (IBD).

Aim The authors evaluated correlations between 6-thioguanine nucleotide (6-TGN) and therapeutic response, metabolite levels and drug toxicity.

Methods Fifty-six paediatric IBD patients treated with thiopurines had 326 metabolite level measurements and were retrospectively reviewed. Clinical status and laboratory parameters were compared with metabolite levels.

Results There was significant correlation between 6-TGN levels and therapeutic response, with higher median 6-TGN levels among patients with therapeutic response than those with non-therapeutic response (194 vs. 146 pmol/8 × 108 RBC; P = 0.0004). Patients with 6-TGN levels >235 pmol/8 × 108 RBC were more likely to achieve therapeutic response than those below the cut-off (odds ratio, 2.5; 95% CI, 1.5–4.1). Patients who developed leukopenia tended to have higher median 6-TGN levels than those without leukopenia (261 vs. 160 pmol/8 × 108 RBC) but the difference was not statistically significant. There was no correlation between 6-methylmercaptopurine levels and hepatotoxicity. Two patients developed acute pancreatitis. Metabolite level measurements were helpful in identifying non-compliance in nine patients.

Conclusions Monitoring of thiopurine metabolite levels is useful to guide and optimize dosing, as an adjunct to clinical judgement, blood count and liver biochemistry measurements to minimize the risk of drug toxicity and to confirm non-compliance.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The therapeutic benefits of thioguanine precursor derivatives, azathioprine (AZA) and mercaptopurine (MP), are well documented in the treatment of inflammatory bowel disease (IBD).1, 2 AZA and MP are important therapies as steroid-sparing agents, in inducing and maintaining remission in steroid-dependent and in chronic active IBD1–4 and limiting development of tolerance to biological agents.5 However, there remain concerns of drug-induced toxicity while attempting to optimize AZA and MP dosing. Adverse effects of AZA/MP include bone marrow depression, hepatotoxicity and idiosyncratic reactions such as pancreatitis, fever, rash, malaise and gastrointestinal intolerance.6

The metabolism of AZA/MP is a well-studied complex pathway.6, 7 AZA/MP is converted to 6-thiouric acid (6-TUA), 6-methylmercaptopurine (6-MMP), and 6-thioguanine nucleotide (6-TGN). Among these metabolites, 6-MMP and 6-TGN have gained the most interest because 6-TUA is believed to be inactive. Although 6-MMP is also inactive, it is thought to be hepatotoxic.8 AZA and MP mediate their cytotoxic and immunosuppressive properties via 6-TGN, which incorporates into DNA leading to DNA breakage and inhibition of lymphocyte proliferation. The side effects of bone marrow depression and secondary infections are attributed to this effect. It is well recognized that some patients are more susceptible to bone marrow depression while on AZA/MP therapy. This susceptibility is generally due to the inter-individual variation in thiopurine methyl transferase (TPMT) enzyme activity consequent upon genetic polymorphism of high- and low-metabolising alleles. Patients with negligible (0.3% of patients) or low (11%) enzyme activity levels have an increased risk of bone marrow depression, as MP metabolism is shunted towards excessive 6-TGN production.8, 9 These patients should be considered for alternative forms of therapy or have lower doses of thiopurine.

Azathioprine and MP doses of 2.0–3.0 mg/kg/day and 1.5 mg/kg/day, respectively, are effective for treatment of Crohn’s disease (CD).1 MP, similarly, is effective in ulcerative colitis (UC).2 Many clinicians commence at approximately half the target dose and gradually titrate the dose to a desired level of 6-TGN and 6-MMP, while monitoring the full blood count (FBC) and liver biochemistry. It has been suggested that clinicians aim for a 6-TGN level of >235 pmol/8 × 108 red blood cells (RBC) and 6-MMP level of <5700 pmol/8 × 108 RBC.8, 10 6-TGN levels >235 pmol/8 × 108 RBC are associated with increased therapeutic efficacy but these higher concentrations are also linked to bone marrow suppression.6, 8 A threefold increase in risk of hepatotoxicity is noted with 6-MMP levels >5700 pmol/8 × 108 RBC.8 Other clinicians prefer a pharmacogenetic-based dosing approach by assessing the TPMT in vitro enzyme activity or genotype before starting treatment and using the results to determine dosing.11, 12 Prior to the availability of metabolite levels and TPMT activity or genotype measurements, a falling white cell count (WCC) was used as a marker of adequate AZA/MP dosing.

The primary objective of the current study was to evaluate the use of AZA/MP metabolite level measurements in optimising the management of IBD in children, specifically by defining the correlation between 6-TGN levels and therapeutic response and evaluating the correlation between metabolite levels and drug toxicity due to accumulation of toxic metabolites. Secondary objectives were to assess correlation between drug dose and 6-TGN levels and to evaluate the role of metabolite levels in detecting or confirming non-compliance.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Patient selection

Patients were retrospectively identified by cross-referencing a database of all paediatric gastroenterology patients who had AZA/6MP metabolites measured by South Eastern Area Laboratory Services between January 2002 and October 2004. Patients less than 19 years of age with IBD on AZA/MP who had metabolite measurements taken at any time point between October 2002 and October 2004 were eligible for inclusion. It was standard practice during that period for all IBD patients on AZA/MP to have regular metabolite level measurements. Diagnoses of CD, UC or indeterminate colitis (IDC) were established by standard clinical, endoscopic and histological criteria.13,14 These patients have been followed up at the multidisciplinary IBD clinic at Sydney Children’s Hospital. Disease activity was assessed at each visit using the Pediatric Crohn’s Disease Activity Index (PCDAI)15 for patients with CD. This score was also used in patients with UC and IDC as no other validated paediatric disease activity score was available for these groups. FBC and liver biochemistry were routinely evaluated concurrently with AZA/MP metabolite levels. Amylase levels were not routinely measured unless indicated clinically.

Clinical information collected at each point of metabolite measurement were: (i) patient-related data including gender and age at diagnosis and time of metabolite level taken; (ii) disease-related data of disease activity at time of metabolite level; (iii) drug-related data including type of thioguanine drug, indication, dose and concurrent use of other medications for IBD; (iv) toxicity-related data including WCC, neutrophil count (in the presence of leukopenia), platelet count, aspartate transaminase (AST), alanine transaminase (ALT) and amylase were indicated and presence of clinical side-effects; and (v) non-compliance if suspected clinically and supported by metabolite levels. The data on type and site of IBD were collected at a single time point.

AZA/MP metabolite assay

Heparinized blood was obtained during scheduled IBD clinic visits or hospitalisation. 6-TGN and 6-MMP in washed red cells were hydrolysed by acid, extracted with phenylmercuric acetate/ethyl acetate and measured by high performance liquid chromatography using a Waters NovaPak C18 3.9 × 150 mm column, a Shimadzu SPD10Avp UV-VIS Detector and Shimadzu LC-10ATvp Pump (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan), which uses a mobile phase consisting of methanol/triethylamine/dithiothreitol. 6-TGN is detected at a wavelength of 342 nm and 6-MMP at 290 nm. The assay was calibrated using 6-TGN and 6-MMP standards obtained from Sigma-Aldrich (Sigma-Aldrich, Castle Hill, New South Wales, Australia) and sulfamethoxazole was used as the internal standard.16, 17

Outcome definitions

Therapeutic response to AZA/MP therapy was defined by PCDAI score of 15 or less for all types of IBD and non-therapeutic response if PCDAI remained greater than 15. Patients were excluded if on parenteral corticosteroids, oral prednisone (or equivalent) of 10 mg/day or more, infliximab, tacrolimus, methotrexate or ciclosporin. Metabolite levels were also excluded if they were taken less than 1 month of commencement of AZA/MP or if the interval between metabolite levels were less than 1 month apart. Therapeutic 6-TGN level was defined as levels >235 pmol/8 × 108 RBC, while 6-MMP level <5700 pmol/8 × 108 RBC was considered safe. Leukopenia was defined as WCC < 4 × 109/L, neutropenia was defined as neutrophil count <2 × 109/L and thrombocytopenia was defined as platelet count <150 × 109/L. Hepatotoxicity and pancreatitis were defined as a rise in AST/ALT and amylase concentrations greater than two times upper limit of respective reference ranges.

Statistical methods

Statistical analysis was performed using Graph Pad Prism computer software (version 4.00 for Windows, Graph Pad Software, San Diego, California, USA). A Pearson’s correlation coefficient test was used to consider the relationship between variables. Group differences were evaluated based on two-sample Student’s t-test. All calculated P values were two-sided and P values <0.05 were considered statistically significant.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Patient characteristics

The medical records of 56 patients with 326 metabolite measurements were reviewed. 58.9% of the patients were male (Table 1). Median age at diagnosis was 8.9 years (range 0.7–15.8), while median age at time of study was 12.4 years (range 1.5–17). The median number of metabolite measurements in this patient population was four (range 1–17). The majority of patients 75% had CD, 17.9% had IDC and 7.1% had UC.

Table 1.   Characteristics of paediatric IBD patients treated with AZA/MP
CharacteristicsResults
  1. AZA, azathioprine; MP, mercaptopurine; IBD, inflammatory bowel disease; CD, Crohn’s disease; UC, ulcerative colitis; IDC, indeterminate colitis.

Sex, n (%)
 Male33 (58.9%)
 Female23 (41.1%)
Age at diagnosis (years)
 Mean (s.d.)8.6 (4.4)
 Range0.7–15.8
Age at time of study (years)
 Mean (s.d.)11.2 (4.4)
 Range1.5–17.0
Type of IBD and location, n (%)
 CD42 (75%)
 Small bowel37
 Small bowel alone5
 Ileocolon21
 Colon33
 Perianal disease22
 UC4 (7.1%)
 Left sided2
 Pan-colon2
 IDC10 (17.9%)

AZA/MP dose and metabolite response

Fifty-four patients (96.4%) were treated with AZA and two patients (3.6%) were treated with MP. 80.3% of patients had been commenced on AZA/MP because of steroid dependence and 3.6% in patients with steroid-resistant disease. AZA/MP was used in 16.1% of patients already in remission, in the absence of corticosteroid use, to maintain remission and avoid need for further corticosteroids. Mean (s.d.) AZA and MP doses were 2.0 (0.6) and 1.9 (0.4) mg/kg/day, respectively. There was poor correlation between AZA dose and 6-TGN levels (r = 0.12, r2 = 0.015). No patient had AZA/MP ceased or changed to the other type of thioguanine drug.

6-TGN levels and therapeutic response

6-thioguanine nucleotide levels of >235 pmol/8 × 108 RBC were found in 90 (27.6%) measurements. Fifty-eight (64.4%) of these corresponded to therapeutic response. The other 32 (35.6%) therapeutic 6-TGN levels were associated with non-therapeutic response. There were 236 sub-therapeutic 6-TGN level measurements. 100 (42.4%) of these were associated with therapeutic response. The odds ratio (OR) of a therapeutic response for therapeutic 6-TGN levels was 2.5 (95% CI, 1.5–4.1; P = 0.005).

6-thioguanine nucleotide levels were significantly higher in patients who achieved therapeutic response compared with those who did not (Table 2). The median (range) 6-TGN level for all patients in the study was 160 (10–732) pmol/8 × 108 RBC. The median (range) of 6-TGN levels for responders and non-responders were 194 (22–668) and 146 (10–732) pmol/8 × 108 RBC, respectively. Mean (s.d.) of 6-TGN levels for these two groups of patients were 220 (131) and 171 (110), respectively. The 6-TGN levels differed in responders from levels in non-responders (P = 0.0004). When patients with different types of IBD were analysed, significantly higher 6-TGN levels were also noted in patients with CD and UC with therapeutic response (Table 2). This difference was not found in patients with IDC.

Table 2.   6-TGN levels and therapeutic response in IBD, CD, UC and IDC (n = metabolite measurements)
Response6-TGN Level (pmol/8 × 108 RBC)
IBDCDUCIDC
  1. 6-TGN, 6-thioguanine nucleotide; IBD, inflammatory bowel disease; CD, Crohn’s disease; UC, ulcerative colitis; IDC, indeterminate colitis.

Therapeutic response, n1581042232
 Median194209225156
 Range22–66822–52738–66859–545
 Mean (s.d.)220 (131)220 (129)250 (171)196 (110)
Failed response, n1681391613
 Median146158109142
 Range10–73210–73248–26442–326
 Mean (s.d.)171 (110)178 (115)124 (64)159 (81)
P-value0.00040.00980.00870.28

Drug toxicity and metabolite levels

Among the 56 patients reviewed, 12 patients (21.4%) experienced adverse effects attributable to AZA/MP therapy. No patients had AZA/MP therapy discontinued.

Leukopenia Ten patients developed leukopenia; in nine (16.1%) leukopenia was attributed to AZA/MP therapy. One individual was transiently leukopenic in association with an intercurrent viral illness. There were 12 episodes of drug-induced leukopenia from the nine patients. The median (range) WCC in leukopenic events was 3.7 × 109/L (1.8–3.9 × 109/L). The neutrophil counts in these 12 episodes of leukopenia were also low to borderline normal, with a median (range) count of 1.7 × 109/L (0.7–2.1 × 109/L).

The majority of therapeutic responses occurred without leukopenia (95.6%). Patients with drug-induced leukopenia had higher 6-TGN levels (median, 261 pmol/8 × 108 RBC; range, 107–440 pmol/8 × 108 RBC) than patients without drug-induced leukopenia (median, 160 pmol/8 × 108 RBC; range, 15–732 pmol/8 × 108 RBC). The difference was not statistically significant (P = 0.1). Seven of the 12 episodes of leukopenia (58.3%) were associated with 6-TGN levels of >235 pmol/8 × 108 RBC. AZA doses were also not significantly higher in patients with leukopenia compared with those without leukopenia. The mean (s.d.) AZA doses in patients with and without leukopenia were 2.4 (0.5) and 2.0 (0.7) mg/kg/day, respectively (P = 0.059).

Thrombocytopenia Mild thrombocytopenia was detected in one patient (platelet count of 144 × 109/L) during AZA/MP treatment. This change was associated with a 6-TGN level of only 149 pmol/8 × 108 RBC.

Hepatotoxicity Four patients had elevated transaminases during this study period. However, all four patients had proven IBD-associated liver disease and hepatic enzyme abnormality was not thought to be attributable to AZA/MP therapy. There were eight episodes of altered transaminases in these four patients, with ALT concentrations more than two times the upper limit of the reference range (median 149 U/L; range 105–301 U/L). None of the corresponding 6-MMP levels were at levels considered to be unsafe (median, 803 pmol/8 × 108 RBC; range 395–2865 pmol/8 × 108 RBC; mean, 1154 pmol/8 × 108 RBC; s.d., 830 pmol/8 × 108 RBC). There were 10 episodes (3.1%) of elevated 6-MMP levels (median, 6845 pmol/8 × 108 RBC; range, 6125–11 195 pmol/8 × 108 RBC) but none were associated with abnormal transaminases or other adverse events.

Pancreatitis Two patients (3.6%) experienced acute pancreatitis with abdominal pain, vomiting and elevated serum amylase each during the fourth week of AZA therapy.

Allergic reactions No patient from the study population experienced allergic drug reactions to AZA/MP.

Non-compliance Clinical suspicion of non-compliance was supported by measurement of metabolite levels in nine patients. All except one patient was above 12 years of age: the median age of presumed non-compliant patients was 14.8 (range 6.4–16.5). These patients had very low concentrations of both 6-TGN and 6-MMP. Further decreases in both 6-TGN and 6-MMP levels were noted in three patients despite repeated encouragement to comply.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

There have been conflicting recommendations in the literature regarding the use of AZA/MP metabolite levels in clinical practice. The findings of this study support the monitoring of AZA/MP metabolite levels by demonstrating a significant correlation between 6-TGN levels and therapeutic response in paediatric IBD patients. Median 6-TGN levels in this study were higher in patients with therapeutic response (194 pmol/8 × 108 RBC) than in those with non-therapeutic response (146 pmol/8 × 108 RBC). This relationship was not only noted in patients with CD but also in those with UC.

There are at least three previous studies of 6-TGN levels in paediatric IBD patients receiving AZA/MP therapy.8, 10, 18 Dubinsky et al.8 and Cuffari et al.10 showed that 6-TGN levels positively correlate with therapeutic response. They showed that patients with higher median levels of 6-TGN are more likely to respond to AZA/MP treatment. Both studies have suggested the utilisation of a target 6-TGN level of >235 and 250 pmol/8 × 108 RBC, respectively, to optimize induction and maintenance of remission. Although the study by Gupta and colleagues18 did not find a correlation between 6-TGN levels and clinical response, they found that with serial monitoring of metabolite levels, higher 6-TGN levels were more likely to be associated with disease remission. This suggests that metabolite level measurements can be used to increase the dose of AZA/MP in order to optimize the rate of therapeutic response in patients with active disease and sub-therapeutic levels of metabolites. Further support that 6-TGN levels are correlated with therapeutic response in IBD patients on AZA/MP therapy was demonstrated in a recent systematic analysis. Osterman and co-authors19 performed a meta-analysis of 12 adult and paediatric studies to correlate 6-TGN levels and IBD activity, including retrospective and cross-sectional studies. They found IBD patients in remission had significantly higher 6-TGN levels than those with active disease, with a pooled difference of 66 pmol/8 × 108 RBC (95% CI, 18-113; P = 0.006).

In the current study, patients with 6-TGN level of >235 pmol/8 × 108 RBC were also found to be more likely to achieve therapeutic response with OR of 2.5. Osterman et al.19 reported an OR of 3.8 (95% CI, 1.7–6.3, P < 0.001) in their study of 193 children and 244 adults. In addition, the poor correlation between AZA dose and 6-TGN levels in the current study further supports the monitoring of 6-TGN levels.

Due to the nature of the study design, the positive correlation between 6-TGN levels and therapeutic response in paediatric IBD patients may be potentially explained by various factors. Patients who achieve therapeutic response and have higher 6-TGN levels may have milder forms of disease with better absorption of medication or may be compliant to treatment. The timing and frequency of blood sampling for metabolite levels were also heterogeneous and this may have influenced the findings of this study.

AZA/MP therapy was safe and well tolerated in this study population. AZA/MP drug toxicity occurred in 21.4% of patients. However, only the two patients (3.6%) who developed pancreatitis were symptomatic of toxicity. AZA doses were higher in patients with leukopenia compared with those without. All leukopenic and thrombocytopenic episodes were asymptomatic and only one leukopenic episode was considered severe (WCC < 2.0 × 109/L). Apart from the two episodes of pancreatitis, idiosyncratic reactions were not recorded.

The literature reports that adverse effects of AZA/MP occur in 15–39% of patients6, 8 and this was consistent with our finding of 21.4%. Adverse effects such as bone marrow depression are reported to occur in 2–14%, while hepatic enzyme derangement occurs in 0.3–17%.6, 8, 18 Infections secondary to leukopenia is reported to occur in 2.5–7.5%.6 Idiosyncratic reactions such as pancreatitis occur in 3%, fever, rash and malaise in 2% and gastrointestinal intolerance in 10%.6 In the present study, patients with drug-induced leukopenia had higher median 6-TGN levels (261 pmol/8 × 108 RBC) than patients without drug-induced leukopenia (160 pmol/8 × 108 RBC). However, the difference was not statistically significant (P = 0.1). This is in contrast to a previous published finding of an association between leukopenia and higher 6-TGN levels.8

The current study also showed that leukopenia can develop at a variable range of 6-TGN level (107–440 pmol/8 × 108 RBC). This finding supports the routine use of serial FBC as there are limitations with follow-up based solely on metabolite level measurements.

None of the 10 episodes of elevated 6-MMP levels (median, 6845 pmol/8 × 108 RBC) in the current study was associated with elevated transaminases. However, the high incidence of hepatotoxicity reported in another study8 suggests that monitoring of liver biochemistry while on AZA/MP should continue. The lack of association between metabolite levels and the adverse effects of bone marrow suppression and hepatotoxicity may relate to the small number of episodes where high 6-TGN and 6-MMP levels occurred in this cohort.

The role of TPMT enzyme activity or TPMT genotype-based dosing remains unclear.7 None of patients from the reported study population had TPMT activity or genotype measurement. Metabolite levels, FBC and liver biochemistry would still need to be monitored once AZA/MP is commenced regardless of whether low TPMT activity is detected. Until more studies are undertaken, pharmacogenetic-based dosing is not warranted.

Due to the retrospective nature of this study, it was difficult to determine how clinical practice related to dose management of AZA/MP (i.e. decisions to increase, decrease or maintain current dosage). However, our retrospective review of the clinical notes gave a strong impression that metabolite levels were particularly useful to the treating clinicians in helping adjust the doses upwards or downwards. This may be more suitably assessed in a prospective study.

In conclusion, monitoring of AZA/MP metabolite levels was useful to guide and optimize the dose of AZA/6-MMP therapy in a cohort of paediatric IBD patients. The results of this study suggest that higher 6-TGN levels are associated with therapeutic response. The monitoring of metabolite levels complements the clinical approach to AZA/MP dose titration, together with regular monitoring of FBC and liver biochemistry to minimize the risk of drug toxicity. Further research such as a prospective, randomized, controlled trial evaluating the clinical outcomes of patients on AZA/MP with and without metabolite level monitoring should be undertaken to help refine the role of this tool in appropriate patient populations.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Declaration of personal interests: None.

Declaration of funding interests: Dr Ooi was funded by a Sydney Children's Hospital Foundation Fellowship.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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