Trend towards dose reduction of azathioprine as monotherapy in inflammatory bowel disease patients: what about for combination therapy?

Despite controversial data, efficacy of azathioprine for the maintenance of remission was confirmed in two recent update meta-analyses, both in Crohn’s disease (CD) and ulcerative colitis (UC) [Chande et al. 2015; Timmer et al. 2016]. Azathioprine is a prodrug that must be activated to form active metabolites [Tiede et al. 2003] including 6-thioguanine nucleotides (6-TGNs) [Neurath et al. 2005]. Metabolism of azathioprine is well known and involves three pathways including one dependent on xanthine oxidase (60% of metabolism) which produces inactive xanthic derivatives [Chouchana et al. 2014]. There are two other competitive pathways, related to thiopurine methyltransferase (TPMT) activity, which exhibits genetic polymorphism. TPMT induces the production of methyl derivatives, including 6-methylmercaptopurin (6-MMP), which have hepatic [Dubinsky et al. 2000] or hematological toxicities [Hindorf et al. 2006]. Another of these competitive pathways produces 6-TGNs which is an active metabolite with dose-dependent hematological toxicities. Thus, the greater the TPMT activity, the more the metabolism of azathioprine is turned to the production of methyl derivatives, resulting in an increase of the 6-MMP/6-TGN ratio. In contrast, if the TPMT activity is low or null, 6-TGN concentrations are very high and the 6-MMP/6-TGN ratio decreases.

The correlation between azathioprine dose and 6-TGN concentration is low. Indeed, 10% of patients have a heterozygous mutation of the TPMT gene, resulting in low enzyme activity and increased 6-TGN levels. A homozygous mutation is reported in 0.1% of patients, which induces a null TPMT activity and extremely high levels of 6-TGN. In these conditions, patients are exposed to a major risk of aplasia, even with standard doses of azathioprine. In contrast, 15% of patients have a very high TPMT activity, resulting in a very high methyl-derivatives/6-TGN ratio (>20) and inefficiency of the drug [Cuffari et al. 2004].

In the American Gastroenterological Association and the European Crohn’s and Colitis Organization guidelines, the recommended dose of azathioprine ranges from 2–2.5 mg/kg/day. However, international guidelines do not mention the possible use of TPMT genotyping and phenotyping and therapeutic drug monitoring before and during thiopurine therapy in inflammatory bowel disease (IBD). In a worldwide survey of experts [Roblin et al. 2011], thiopurine testing was relatively underutilized by IBD gastroenterologists. The availability and reimbursement of TPMT status and azathioprine metabolites strongly influenced the management of IBD patients treated with thiopurines.

When azathioprine is used as monotherapy, 6-TGN levels associated with clinical response, range from 235–270 pmoles. In a recent meta-analysis, Moreau and colleagues included 17 studies reporting 6-TGN levels more than 230–260 pmoles, with a consequent doubling in the rate of response [odds ratio (OR): 2.16 (1.76–2.61)] [Moreau et al. 2014]. No heterogeneity regarding geographical origin of studies was reported. Thus, rather than using a standard dose for all patients, a dose adjustment could be done individually, based on 6-TGN levels, maintaining the same efficacy of the drug.

For this purpose, Haines and colleagues suggested a personalized algorithm of treatment, by taking into account 6-TGN and 6-MMP levels [Haines et al. 2011]. In a large cohort of IBD patients, Shi and colleagues reported no difference in terms of clinical response in patients under normal or low doses of azathioprine. Despite these interesting outcomes, no extrapolation on non-Asian populations can be done. No measure of 6-TGN was performed in the Asian population study of Shi and colleagues [Shi et al. 2016]. However, authors argue that 6-TGN levels would be probably over the effective threshold value, with low doses of azathioprine. This point would be due to the polymorphism of the Nudix hydrolase gene (NUDT15), previously reported in the Asian population. In fact, NUDT15 R139C was strongly associated with thiopurine-induced leukopenia but not with 6-TGN levels or clinical activity. In a recent study [Kakuta et al. 2016], the authors performed an association study to investigate and replicate the association of R139C with adverse events of thiopurines in Japanese patients. A total of 142 Japanese patients with IBD previously treated with thiopurine, were examined. The association of R139C with early (<8 weeks) leukopenia [white blood cells (WBCs) < 3000/mm³],was reported in this Japanese IBD cohort (p = 1.92 × 10^–16, OR = 28.4), regardless of manipulating the doses, rates of thiopurine continuation were similar between the groups. In another study [Asada et al. 2016], the authors investigated the association of NUDT15 R139C with 6-TGN levels and thiopurine-induced leukocytopenia in Japanese patients with IBD. A total of 264 participants (103 healthy volunteers and 161 IBD patients treated with thiopurines) were enrolled. Of the 161 IBD patients treated with thiopurine, there was no significant difference in 6-TGN levels among the NUDT15 genotypes. Overall, 45 patients (27.9%) developed leukocytopenia [WBCs < 3000/μl, and the C/T and T/T genotypes were significantly associated with this hematological toxicity (p = 1.7 × 10^–5)]. These results suggest that NUDT15 R139C-related thiopurine-induced leukocytopenia is mediated by a 6-TGN-independent mechanism.

Thereby, 6-TGN levels in the Asian population are not higher than those from other race groups. We think that, conversely to what suggested by Shi and colleagues [Shi et al. 2016], it seems premature to recommend low dose azathioprine, based on this hypothesis. Indeed, several studies showed insufficient evidence of the effectiveness of low dose of azathioprine therapy or TPMT measurement in IBD patients [Booth et al. 2011; Dassopoulos et al. 2014; González-Lama et al. 2011; Reinshagen et al. 2007]. Moreover, in this Asian cohort, almost all patients used 5-aminosalicylates in combination with thiopurine. Even if 6-TGN levels are higher in patients treated with a combination of 5-aminosalicylic acid (5-ASA) and azathioprine, particularly in those who have heterozygous mutations of TPMT, no study has demonstrated yet an increase in the efficacy of azathioprine by this combination therapy. However, in clinical practice, thiopurines are frequently associated with 5-ASA. Combination of treatments should increase the chemopreventive effect of 5-ASA in IBD patients. In a large cohort of IBD patients, van Schaik and colleagues [van Schaik et al. 2012] showed that the use of thiopurines protected IBD patients against the development of advanced neoplasia. In this study, no patient under combination therapy presented advanced neoplasia, however, the number of patients were too low to conclude anything significant.

Recent studies on CD and UC revealed that combination therapy with azathioprine plus infliximab (IFX) is more effective than monotherapy in the treatment of active CD or UC [Colombel et al. 2010; Panaccione et al. 2011]. In both studies, concomitant immunomodulator use (azathioprine, mercaptopurine) increased the concentration of serum monoclonal antibodies (mAbs). One mechanism is the reduction of antidrug antibody (ADA) formation (0.9% in patients receiving combination therapy versus 14.6% in patients receiving IFX monotherapy in the SONIC trial) [Colombel et al. 2010]. In a post-hoc analysis of the SONIC trial [Bouguen et al. 2015] the authors investigated the relationship between delta mean corpuscular volume (mCV) value and CD outcomes. Changes in erythrocyte mCVs are correlated with 6-TGN concentrations [Jobson et al. 2001]. In the combination therapy group, at week 26, patients with a delta mCV of at least 7 achieved more frequent mucosal healing compared with those with delta mCV < 7 (75% versus 47.1%, p = 0.0172), while the percentage of patients presenting steroid-free remission was comparable in both groups (77.9% versus 64.4%, p = 0.015).

Importantly, the percentage of patients with a trough level of IFX (TRI) > 3 µg/ml was significantly higher in patients with a delta mCV > 7 (68.4% versus 38.8% in patients with delta CMV < 7; p = 0.0032) [Jobson et al. 2001]. Yarur and colleagues (Yarur et al. 2015) assessed the correlation between 6-TGN levels and TRI using a nonradiolabeled homogeneous mobility shift assay or antibodies to IFX (ATI). Levels of 6-TGN were correlated with those of IFX (rho: 0.53; p < 0.0001). In this study, there was no difference regarding TRI between patients on IFX monotherapy and those on combination therapy when 6-TGN levels were <125 pmol/8 × 10⁸ red blood cells (RBCs). By contrast, TRIs were significantly decreased in IBD patients treated with monotherapy compared with those on combination therapy when 6-TGN levels were >125 pmol/8 × 10⁸ RBCs (p < 0.0001).

Furthermore, patients with detectable ATI (antibodies-to-IFX) had lower levels of 6-TGN when compared with those with no ATI. Patients with 6-TGN levels <125 pmol/8 × 10⁸ RBCs had a greater risk of developing detectable ATI [OR: 13; 95% confidence interval (CI): 2.3 –72.7; p < 0.01]. The best cutoff that predicted a significantly higher level of IFX was 125 pmol/8 × 10⁸ RBCs [receiver operating characteristic (ROC): 0.86; p < 0.001].

In a recent study [del Tedesco et al. 2016] IBD patients in deep remission under combination therapy with IFX–azathioprine (IFX maintenance regimen at a dose of 5 mg/kg every 8 weeks with baseline TRI > 2 µg/ml and stable doses of azathioprine 2–2.5 mg/kg/day) for at least 1 year were prospectively enrolled and separated in 3 distinct cohorts. In cohort A (n = 28), the dose of azathioprine in combination therapy was unchanged; in cohort B (n = 27), the dose of azathioprine was halved, with a minimum dose of 50 mg/day; in cohort C (n = 26), azathioprine was stopped. Overall, 5 patients (17.8%) in cohort A, 3 patients (11.5%) in cohort B, and 8 patients (30.7%) in cohort C experienced failure at 1 year (p = 0.1 across groups). Overall, 14.2% of patients in cohort A, 14.8% in cohort B and 42.3% in cohort C experienced unfavourable outcomes of IFX pharmacokinetics (a drop of TRI < 1 µg/ml or undetectable TRI with positive ATI). A threshold of 6-TGN <105 pmoles was associated with an unfavourable evolution of IFX pharmacokinetics with a sensitivity of 67%, specificity of 92%; and likelihood ratio of 7.67. A threshold of 6-TGN < 105 pmoles was associated with an unfavourable evolution of IFX pharmacokinetics.

Adverse events: in IBD patients, the use of combination therapy increases the risk of malignancy, especially lymphoma. In a large, French, prospective, observational study, patients receiving thiopurines for IBD had an increased risk of developing lymphoproliferative disorders [Beaugerie et al. 2009]. Similarly, past exposure to thiopurines increased the risk of myeloid disorders by 7-fold among patients with IBD [Lopez et al. 2014]. Moreover, the authors showed that ongoing and past exposure to thiopurines significantly increases the risk of nonmelanoma skin cancer (NMSC) in patients with IBD, even before the age of 50 years [Peyrin-Biroulet et al. 2011]. Finally, in the CESAME cohort, patients with IBD receiving thiopurines had an increased risk of urinary tract cancers [Bourrier et al. 2016]. Also, co-administration of immunomodulator therapy was associated with an increased risk of NMSC in adalimumab-treated patients [Osterman et al. 2014]. The risk of development of hepatosplenic T-cell lymphoma (HSTCL) is increased with tumor necrosis factor (TNF)-α inhibitor use in combination with thiopurines [Deepak et al. 2013].

It is unknown whether a low dose of thiopurines (6-TGN levels >125 pmol/8 × 10⁸ RBCs) may be a way to decrease cancer risk in IBD patients on combination therapy with thiopurine and anti-TNF therapy. However, studies conducted in other diseases reported an association between 6-TGN levels and cancer risk [Bo et al. 1999; Lennard et al. 1985; Schmiegelow et al. 2009]. Skin cancer in 108 renal transplantation recipients was associated with increased concentrations of 6-TGN (p = 0.005). The concentration of 6-TGN was 120–425 pmol/8 × 10⁸ RBCs (mean 276) in transplant patients who developed skin lesions and 54–203 pmol/8 × 10⁸ RBCs (mean 130) in a matched control group of renal transplant recipients (p = 0.005) [Lennard et al. 1985].

After childhood acute lymphoblastic leukemia, there is an increased risk of a second malignant cancer due to maintenance therapy with thiopurines. High 6-TGN levels were found to be risk factors in this patient population [Schmiegelow et al. 2009]. During 6-mercaptopurine (6-MMP) therapy in 439 children with acute lymphoblastic leukemia, 5 children later developed secondary myelodysplasia (sMDS) or acute myeloid leukemia (AML), associated particularly with those who had low TPMT activity. A TPMT cutoff of <14 U/ml RBCs was the strongest predictor of risk for developing sMDS (Cox regression; p = 0.02). The average 6-TGN or 6-MMP levels during maintenance therapy were above the 90th percentiles in four of the five secondary MDS/AML patients [Schmiegelow et al. 2009]. These results indicate that it is not only high 6-TGN levels that may lead to DNA damage and contribute to promoting carcinogenesis in these patients [Schmiegelow et al. 2009]. Hence, thiopurine dose reduction may be recommended for selected patients with low TPMT activity or high 6-TGN or 6-MMP levels. A recent population-based cohort study [Na et al. 2016] was conducted on 4131 adult Australian liver-, heart- and lung-transplant recipients (1984–2006). The authors ascertained non-Hodgkin’s lymphoma incidence by probabilistic record linkage between transplant registries and the Australian Cancer Database, and abstracted risk factor data at transplantation and at regular intervals thereafter from medical records. In final models, higher mean daily doses (1.2 mg/kg/day) of azathioprine were associated with increased risk of both early [hazard ratio (HR) 2.20, 95% CI: 1.21–4.01] and late non-Hodgkin’s lymphoma (HR 1.78, 95% CI: 1.12–2.84). All these studies are reported in distinct populations with different risks for development of neoplastic or hematological disorders than IBD patients.

In conclusion, the dose of azathioprine should be adjusted by considering its use as mono or combination therapy. Excluding patients with mutations of NUDT15 (increased risk of leukopenia) or TPMT genes, a dose of 2–2.5 mg/kg/day should be proposed. Determination of the phenotype of TPMT would lead to a reduced dose to the minimum efficient level. In patients under combination therapy, the lowest dose of azathioprine leading to 6-TGN levels >125 pmoles, seems sufficient to get a favorable pharmacokinetic of IFX.