Fig. 31.1
Azathioprine (AZA) metabolism (XO xanthine oxidase; 6-TU 6-thiouric acid; 6-TIMP 6-thioinosine monophosphate)
An apparent genetic polymorphism has been observed in TPMT activity in both the Caucasian and African-American population. Negligible activity was noted in 0.3% and low levels (<5 U/mL of blood) in 11% of individuals [40]. TPMT enzyme deficiency is inherited as an autosomal recessive trait, and to date, ten mutant alleles and several silent and intronic mutations have been described [41]. In patients with heterozygous TPMT genotype, 6-MP metabolism is shunted preferentially into the production of 6-TG nucleotides. Although 6-TG nucleotides are thought to be lymphocytotoxic, and beneficial in the treatment of patients with leukemia and lymphoma, patients with low (<5) TPMT activity are at risk for bone marrow suppression by achieving potentially toxic erythrocyte 6-TGn levels on standard doses of 6-MP [36]. Despite low TPMT enzyme activity levels, therapeutic erythrocyte 6-TGn metabolite levels can still be achieved without untoward cytotoxicity by lowering the dose of 6-MP 10- to 15-fold [37].
Recent studies have also shown that one of these 6TGn ribonucleotides, 6-TGTP, induces the apoptosis of both peripheral blood and intestinal lamina propria T cell lymphocytes through the inhibition of Rac-1, a GTPase that inhibits apoptosis. The specific blockade of CD-28 dependent Rac 1 activation by 6-TGTP is the proposed molecular target of 6-MP and its pro-drug AZA (Fig. 31.1) [3].
The intracellular build-up of this specific 6-TGn metabolite may also be dependent on other, as yet undefined inherent genetic polymorphisms. Our recent studies have also proposed that there may also exist pharmacogenetic differences in the intracellular transport of 6-MP in peripheral blood lymphocytes that could potentially affect responsiveness to antimetabolite therapy. Our studies have shown an inherent variability in the transport of 6-MP in immortalized lymphocytes derived from patients with IBD. In these studies, seven inward and eight outward transporters were tested. One patient demonstrated the least amount of intracellular transport of 6-MP that correlated with the lowest susceptibility to 6-MP cytotoxicity. In this particular patient, multiple inward transporters, including concentrative nucleoside transporter (CNT)-1, CNT-3, equilibrative nucleosider transporter (ENT)-3, and ENT-4, were notably low in expression. In comparison, a second patient exhibited robust 6-MP transport and an increased susceptibility to 6-MP cytotoxicity, and a decreased expression of CNT-1 and ENT-3, but an increased level of expression of another inward transporter, such as ENT-4. Although no single transporter was either under- or over-expressed to explain these patterns of 6-MP transport, a correlation was shown between intracellular drug levels and the in vitro susceptibility to 6-MP-induced cytotoxicity. Interestingly, these differences were independent of 6-MP dose or erythrocytes 6-MP metabolite levels that were monitored clinically. Ongoing studies will also attempt to correlate these differences in drug transport with clinical responsiveness to antimetabolite therapy and drug metabolite levels. Identification of such transporters prior to initiating therapy may allow physicians to tailor therapy more effectively in patients with steroid-dependent IBD [42].
6-MP Metabolite Monitoring in IBD
The measurement of the erythrocyte 6-MP metabolites 6-TGn and 6-MMP has been proposed as a useful clinical tool for measuring clinical efficacy, documenting patient compliance to therapy, and explaining some drug-induced toxicity in patients with IBD. In our preliminary study in 25 adolescent patients with CD on long-term 6-MP therapy, high-performance liquid chromatography measurement of erythrocyte 6-TG metabolite levels showed an inverse correlation with disease activity. Although a wide range of metabolite levels was associated with a favorable clinical response, patients with high 6-TGn levels (>250 pmoles/8 × 108RBCs) were uniformly asymptomatic [43]. Similar results have been reported in 93 pediatric and 45 adult patients with IBD in whom disease remission correlated well with erythrocyte 6-TGn levels between 230 and 260 pmoles/8 × 108 RBCs, respectively [10,11,44]. Although these studies and others would support the notion of a therapeutic index of clinical responsiveness based on the measurement of erythrocyte 6-TGn levels, several studies have shown no clinical correlation with metabolite monitoring (Table 31.1) [12,45–47]. This lack of consensus may in part be due to the heterogeneous nature of CD and UC, and may also be dependent on disease severity. In a study of adult patients with CD, high (>290) erythrocyte 6-TGn levels showed a positive predictive value of 86% of obtaining a favorable clinical response to induction AZA therapy in patients with steroid refractory CD (Table 31.2) [41].
Table 31.1
Clinical responsiveness to 6-MP and AZA therapy based on threshold (235–250*) erythrocyte 6-TGn metabolite levels
Study | Patients | 6-TGn response threshold | Odds ratio (response) | |
|---|---|---|---|---|
Above | Below | |||
Dubinsky [44] | 92(30) | 0.78 | 0.40 | 5.0 |
Gupta [45] | 101(47) | 0.56 | 0.43 | 1.7 |
Belaiche [46] | 28(19) | 0.75 | 0.65 | 1.6 |
Cuffari [10] | 82(47) | 0.86 | 0.35 | 11.6 |
Achkar [11] | 60(24) | 0.51 | 0.22 | 3.8 |
Lowry [12] | 170(114) | 0.64 | 0.68 | 0.9 |
Goldenberg [47] | 74(14) | 0.24 | 0.18 | 1.5 |
Table 31.2
A proposed guide to antimetabolite therapy based on 6-MP metabolite monitoring
Metabolite profiles | Clinical impression | Therapeutic decision | |
|---|---|---|---|
Group A | Absent/very low (<50) 6-TGn absent 6-MMP | Non-adherence | Patient education |
Group B | Low (<250) 6-TGn; low (<2,500) 6-MMP | Subtherapeutic dose | Dose titration |
Group C | Low (<250) 6-TGn; high (>5,700) 6-MMP | Rapid-metabolizer | Switch therapy vs. allopurinol |
Group D | High (>400) 6-TGn; High (>5,700) 6-MMP | Thiopurine resistant | Switch therapy |
TPMT Activity
Genetic polymorphism in TPMT enzyme activity can be quantitatively measured by TPMT phenotype testing. One in 300 patients have absent TPMT enzyme activity, and are at risk for severe bone marrow suppression [36]. There have been a number cases of irreversible bone marrow suppression both in patients with IBD and in patients with leukemia on standard doses of 6-MP or AZA therapy (personal communications). 6-MP metabolism is clearly influenced by inherent differences in TPMT activity present within the population. In a prospective open-labeled study in patients with IBD, the response rate to induction AZA therapy was highest in patients with less than average (<12 U/mL blood) TPMT activity. Clinical response also correlated well with achieving high erythrocyte (>250) 6-TGn metabolite levels. Indeed, the knowing of the low (<5) TPMT activity before initiating AZA therapy in two patients, led to a low dosing strategy (1 mg/Kg/day) with a favorable clinical response without untoward SEs (Table 31.2) [48]. Moreover, a recent study by Kaskas and coworkers would also suggest that an effective low (0.25 mg/Kg/day) treatment approach can be safely and effectively adopted in patients with the homozygous recessive genotype [49]. Both of these studies would support the contention that a low-dose treatment strategy may be used to treat those patients with low TPMT activity.
In comparison, 10% of the population is considered to be rapid metabolizers of 6-MP, and in theory would require larger than standard doses of drug in order to achieve any therapeutic drug benefit. In these patients, 6-MP metabolism is shunted away from 6-TGn production and into the formation of 6-MMP. A study in children with IBD showed that a subgroup of these patients remains refractory to therapy despite a dose-optimizing treatment strategy [50]. This may in part be due to high hepatic TPMT activity that may draw most of the 6-MP from the plasma, thereby limiting the amount of substrate available for the bone marrow and peripheral leukocytes. A similar study in adult patients with IBD was also able to identify these rapid metabolizers based on the measure of erythrocyte TPMT activity levels. In that study, patients with above-average (>12) TPMT activity levels were less likely to respond to AZA therapy, and more likely to require higher dosages (2 mg/Kg/day) of AZA from the outset in order to optimize erythrocyte 6-TGn metabolite levels. Moreover, patients with above-average (>12) TPMT activity had a mean erythrocyte 6-TGn levels that leveled off below a presumed therapeutic (<250) treatment level after 8 weeks of continuous AZA therapy. Sixty-nine percent of patients with TPMT activity levels ≤12 U/mL blood achieved a clinical response compared to just 30% of patients with above-average (>12) TPMT activity after 4 months of continuous therapy. This study was the first to suggest that the pretreatment knowledge of TPMT activity may allow physicians to predict clinical response, and effectively dose AZA in order to maximize efficacy while minimizing the risk of toxicity. Indeed, in that study, patients with a TPMT enzyme activity less than 15 U/mL of blood were six times (OR:6.2) more likely to show a favorable response to AZA therapy [48]. A recent study exploited the use of allopurinol, a potent inhibitor of xanthine oxidase, in patients with high (>16) TPMT activity who shunt 6-MP metabolism away from the production of 6-TGn metabolites, and remain refractory to presumed therapeutic antimetabolite drug dosing. In that study, the concomitant use of allopurinol with either AZA or 6-MP in patients with presumed high TPMT activity levels achieved a significant increase in erythrocyte 6-TGn metabolite levels and the subsequent induction of disease remission. Although this treatment strategy led to a decrease in total leukocyte count, no patient developed clinical signs of toxicity (Table 31.2) [51].
6-MP Toxicity
Many pediatricians have been reluctant to prescribe 6-MP on account of potential drug-related toxicity including pancreatitis 3%, bone marrow depression 2%, superinfection 7%, and hepatitis 0.3% [32]. Severe SEs are either idiosyncratic or related to generic polymorphism as described above. Although Black and coworkers suggested the notion that pharmacogenetic differences in 6-MP metabolism influence a patient’s risk of drug toxicity [44], TPMT polymorphism accounts for only 25% of all 6-MP-induced SE [52]. However, genetic polymorphisms may play a role in determining the long-term risk for malignancy. In several pediatric oncology studies, the risk for secondary malignancies, including acute myeloblastic leukemia and myelodysplasia was higher in those children with low TPMT activity levels with acute lymphocytic leukemia on maintenance 6-MP therapy [53]. Larger longitudinal studies are necessary in order to draw conclusions about the long-term risk of antimetabolite-induced malignancy, especially in the context of concurrent biological therapies.
Conclusions
6-MP and AZA have proven efficacy in the maintenance of disease remission in children with IBD. The application of Pharmacogenomics and metabolite testing in clinical practice has helped to improve the overall clinical response to antimetabolite therapy in children with IBD and reduce the risk of antimetabolite-induced SEs. Several guidelines have been provided for consideration (Table 31.2). The careful monitoring of complete blood counts, and erythrocyte 6-TG metabolite levels are indicated in patients with either low (<5) or above average (>12) TPMT levels; and it remains the authors’ opinion that relying on either total leukocyte counts or mean corpuscular volume as the sole measure of dosing adequacy should be used with caution [54].
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