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Dr B. E. Sands, MGH Crohn's and Colitis Center, 100 Charles River Plaza, 9th Floor, Cambridge Street, Boston, MA 02114, USA. E-mail: firstname.lastname@example.org
Azathioprine, mercaptopurine, methotrexate, ciclosporin and tacrolimus all have their respective niches in the treatment of inflammatory bowel disease. These immunomodulators are potent and effective medications; however, they potentially have serious toxicity. To maximize benefit and minimize risk, clinicians must understand the mechanism of action, appropriate indications, range of toxicity and proper dosing of these medications. Furthermore, once initiating therapy, patients need to be monitored appropriately for evidence of efficacy and toxicity.
This review includes the rationale behind recommendations for the management and monitoring of patients using immunomodulators. For the purine antagonists – azathioprine and mercaptopurine – the evidence for utility of thiopurine methyltransferase testing and mercaptopurine metabolite monitoring is addressed. The roles of liver biopsy and screening for methylenetetrahydrofolate reductase mutations in patients taking methotrexate are reviewed. With appropriate monitoring, the calcineurin inhibitors – ciclosporin and tacrolimus – can be used safely and effectively.
Immunomodulators are important agents for the treatment of Crohn's disease and ulcerative colitis, and prescribing clinicians should be comfortable recognizing both their value and their limitations.
Immunomodulators are integral to the treatment of inflammatory bowel disease (IBD).1 The goals of using this class of medication are to control active inflammation, allow for the withdrawal of steroids, and ultimately to maintain long-term remission of Crohn's disease and ulcerative colitis (UC). Various immunomodulators that have been found to be efficacious in IBD include azathioprine (AZA), mercaptopurine (MP), methotrexate, ciclosporin and tacrolimus. These effective and potent medications can have significant toxicity, even when used carefully. When prescribing these medications, it is imperative to understand their potential toxicity and how to appropriately monitor patients to ensure safety.
To identify articles for this review, a PubMed search was conducted using the following key words individually and in various combinations with Boolean operators: AZA, MP, methotrexate, ciclosporin, tacrolimus, Crohn's disease, UC, monitoring, metabolites, adverse and toxicity. Search of reference lists from pertinent articles identified additional publications. Relevant abstracts from the most recent American Gastroenterological Association and American College of Gastroenterology conferences were also included.
Mechanism of action and evidence for efficacy
Azathioprine and MP are purine analogues which have been shown to be effective in inducing and maintaining remission in patients with Crohn's disease and UC. AZA is a prodrug that is converted to MP and then metabolized to an active metabolite, thioguanine (thioguanine [TG]; Figure 1). TG is incorporated into ribonucleotides, thereby exerting an antiproliferative effect on mitotically active lymphocyte populations. AZA and MP may also possess direct anti-inflammatory properties by inhibiting cytotoxic T cell and natural killer cell function2 and inducing apoptosis of T cells through Rac1 target gene modulation.3 Although it has been speculated that AZA may possess immunosuppressive and metabolic benefits beyond that of MP,4, 5 these drugs are used interchangeably in clinical practice.
AZA has been studied for use in Crohn's disease in multiple randomized-controlled trials dating to 1971.6, 7 A meta-analysis in 1995 showed an estimated common odds ratio of 3.09 (95% CI: 2.45–3.91) for the treatment of active Crohn's disease and 2.27 (95% CI: 1.76–2.93) in maintaining remission.8 Extrapolation from Crohn's data, and controlled trials in both active disease9 and maintenance10 have led to widespread use in UC as well. To avoid potential toxicity associated with long-term use, there is a temptation to stop AZA/MP after a certain period of disease quiescence. However, drug-withdrawal studies in both Crohn's disease and UC have similarly shown an acceleration of relapse after stopping the previously effective medications.10–15
Rare, but potentially serious and even life-threatening adverse effects have been reported with AZA and MP. A report of experience with MP in 396 patients16 with IBD had evidence of direct toxicity including pancreatitis (3.3%), bone marrow suppression (2%), allergic reactions (2%) and drug hepatitis (0.3%). Importantly, all of these events were reversible upon stopping the offending medication. The incidence of these and other adverse effects is relatively constant in other series8, 17–22 that looked at long-term toxicity (Table 1). In a large series, Connell et al. reported on 739 IBD patients who were treated with AZA. Although the overall rate of leucopoenia was comparable (3.8% of patients with WBC <3.0 × 109/L), two patients died as a result of pancytopenia and sepsis.22
Table 1. Patients experiencing toxicity from AZA/MP
* Meta-analysis, included some patients from Present et al.16
† This is an updated report from the Present et al.16 series.
n/a, not available; LFT, liver function test; AZA, azathioprine; MP, mercaptopurine.
Number of patients
Stopped drug due to adverse effect (%)
Allergic reaction (%)
Hepatitis/abnormal LFTs (%)
Death related to leucopoenia (%)
Infection (significant) (%)
Death due to lymphoma (%)
Blood dyscrasia (%)
Death due to blood dyscrasia (%)
In an updated report from one case series,21 the incidence of allergic reactions was 3.9%. In five of the patients with allergic reactions, the authors attempted desensitization and were successful in four. They also noted that although desensitization can work, it is rarely successful in those who had an allergic mediated pancreatitis. Re-challenge with the same or alternative drug (i.e. AZA→MP) may be reasonable with cautious desensitization, if the adverse effect was not severe (e.g. nausea, non-severe infection) or if there is another plausible explanation (concomitant viral infection or drug interaction) for the event. Re-challenge is contraindicated in pancreatitis or cases of life-threatening toxicity (e.g. sepsis secondary to neutropenia).
Infection is a relatively common indirect toxicity and was seen in 7.4% of patients (1.8% of these were severe) in the series by Present et al.16 In other series, the incidence of significant infections ranges from 0.3 to 7.1% (Table 1).8, 17–21 In addition to bacterial infections, viral infections are associated with use of AZA/MP. The herpes viruses, specifically Epstein–Barr virus (EBV), cytomegalovirus (CMV), varicella zoster virus (VZV) and herpes simplex virus (HSV) have all been reported to cause some rare, but serious complications in IBD patients receiving AZA/MP. Most herpes virus infections are probably unrecognized and manifest as self-limited viral syndromes, but life-threatening complications such as disseminated varicella zoster,23, 24 CMV pneumonitis25 and viral-mediated haemophagocytic syndrome26, 27 have been reported.
Twelve neoplasms were seen (3.1%) in the Present et al.16 series, however only one (histiocytic lymphoma of the brain) had a probable association with MP use.16 Two further lymphomas were reported in the updated report. Both of those patients were treated successfully and were well after long-term follow-up.21 Although some have estimated either no or a very small increased risk of lymphoma associated with the use of AZA/MP,28, 29 other reports describe a threefold relative risk compared with Crohn's patients not taking these medications.30 Part of this risk is likely related to the role of EBV-mediated lymphoma, which is well described in IBD patients on AZA/MP31 and may be dose-related. With conflicting data, and controversy over an inherent risk of lymphoma in Crohn's disease itself, the true risk of lymphoma related to AZA/MP use is not entirely clear.
In addition to lymphoma, there has been concern that AZA may be related to other malignancies, as well. Fraser et al. reported on 626 patients with IBD, and found no increased risk of lymphoma, colorectal cancer or extra-intestinal malignancies.28 Connell et al. evaluated the cancer risk in IBD patients taking AZA and had 6975 patient-years of follow-up.32 They concluded that AZA use does not substantially increase the risk of colorectal, other gastrointestinal or extra-intestinal cancers in IBD. However, there was a slight, but non-significant, increase in cervical cancer, which has also been reported in the immunosuppressed transplant population.33, 34 An increased risk of non-melanoma skin cancer is well recognized in the immunosuppressed transplant population as well,35 and has also been reported in IBD.36 Our practice is to recommend routine cervical cancer screening and added caution with sun exposure to IBD patients taking immunomodulators.
Thiopurine methyltransferase testing
Thiopurine methyltransferase (TPMT) enzymatically converts MP to 6-methyl-mercaptopurine (6-MMP), diverting metabolism away from TG (Figure 1). There is an inverse relationship between expression of TPMT and level of TG. Therefore, lower TPMT activity yielding higher levels of TG has been associated both with an increased likelihood of clinical response and bone marrow suppression.37, 38 The wild type of TPMT, found in 89% of Caucasians is TPMT*1/TPMT*1. The most common variant allele is TPMT*3A, however a number of others have been described.39 About 11% of the population are heterozygotes carrying one wild type and one variant allele.40 Approximately one of 300 people are homozygous recessive and have no TPMT enzymatic activity, strongly favouring TG production. These individuals are at high risk for severe bone marrow toxicity.40 Although rare, identifying these patients who are homozygous recessive before initiating treatment can prevent significant morbidity and potential mortality. If we assume that homozygous recessive individuals would not receive AZA/MP and that identified heterozygotes (intermediate metabolizers) would receive a reduced initial dose, the number needed to screen for TPMT genotype to avoid one adverse event is 100. A decision analysis has shown this strategy to be cost-effective.41 This approach could help avoid preventable, profound bone marrow suppression early in treatment, however, patients with TPMT mutations only account for 27% of myelotoxicity.42 The remaining nearly three-fourths of patients developed sporadic neutropenia for unidentified reasons. Consequently, while TPMT testing is helpful in avoiding early, profound bone marrow suppression, it should not take the place of careful monitoring of full blood counts (FBC) throughout the duration of treatment on AZA/MP.
As an alternative to TPMT genotyping, TPMT enzymatic activity could be measured to determine phenotypic expression. Low (or no) TPMT enzyme activity corresponds to the homozygous recessive genotype, intermediate TPMT enzyme activity to heterozygotes, and normal or high TPMT enzyme activity with the homozygous normal wild type.43 Patients with intermediate TPMT enzyme activity may successfully respond to AZA/MP treatment more frequently, while those with the highest TPMT activity levels have been shown to be resistant to standard doses of therapy.44
Further research is needed to determine if TPMT testing truly provides benefit beyond close follow-up of blood counts. Although TPMT testing will not eliminate the risk of myelotoxicity, the potential to avoid even a few life-threatening adverse effects is certainly appealing. With the currently available data, we recommend TPMT genetic or enzyme activity testing prior to initiating AZA/MP (Table 2).
Table 2. Recommendations for the safe and effective use of azathioprine and mercaptopurine
TPMT, thiopurine methyltransferase; AZA, azathioprine; MP, mercaptopurine; FBC, full blood count; LFTs, liver function tests; WBC, white blood cell; 6-MMP, 6-methyl-mercaptopurine; TG, tioguanine; PO, per oral.
1. Consider TPMT genetic or enzyme activity testing prior to initiating therapy
• Do not treat if TPMT homozygous recessive or low enzyme activity
2. Initiate therapy at 50 mg PO daily (for both AZA/MP) for normal genotype or enzyme activity, 25 mg PO daily for heterozygote variants or intermediate enzyme activity
3. If normal TPMT genotype, measure FBC, LFTs and amylase biweekly for 3 months. If no significant abnormalities (or trend towards leucopoenia or transaminitis), measure FBC and LFTs every 3 months for the duration of therapy. If TPMT testing is not performed, follow labs weekly for the first month
4. After 2 weeks on therapy, as long as WBC is not <4.0 × 109 and has not decreased by >50% of starting WBC, and LFTs and amylase are not rising, escalate to the goal dose of 1.5 mg/kg for MP or 2.5 mg/kg for azathioprine. For heterozygotes, the goal dose is 0.75 mg/kg for MP or 1.25 mg/kg for azathioprine
5. Hold AZA/MP for WBC <3.5 × 109/L, recheck labs in 1 week. If WBC persistently <3.5 × 109/L on full weight-based dose, consider decreasing daily dose by 25–50 mg
6. For any signs of infection (viral or bacterial), patients should have a clinical evaluation and measurement of FBC and LFTs. If clinically significant infection, hold AZA/MP until symptoms resolve
7. If no significant clinical response at 3–4 months, consider measuring 6-MMP and TG metabolites to assess for selective shunting towards 6-MMP or non-compliance
• In this setting, 6-MMP: TG ratio >10:1 represents unfavourable metabolism unlikely to result in therapeutic efficacy and consider switching to a different agent
AZA is 55% of MP by molecular weight, and 88% of AZA is converted to MP.45 If changing from MP to AZA, a conversion factor of 2.07 can be used.46 The goal dose for MP is 1.5 mg/kg and for AZA 2.5 mg/kg. It is worth noting that strictly speaking, 1.5 mg/kg of MP multiplied by 2.07 is just over 3 mg/kg of AZA. Although lower doses had been favoured in early clinical trials, a meta-analysis demonstrated greater efficacy of higher doses.8 A study looking retrospectively at AZA dose escalation noted that increasing AZA to 2.5 mg/kg did appear beneficial in patients not responding to 2.0 mg/kg, however, further increases were less likely to add benefit and were associated with an increase in adverse effects.47 The optimal weight-based dose for AZA/MP has not been established in prospective randomized trials, however, such a dose-ranging study is about to be underway (B. E. Sands, personal communication).
Now that both generic and branded AZA/MP are available, questions arise with regard to equivalent bioavailability. One study showed that the branded AZA or MP led to higher levels of the active metabolite TG than generic AZA,48 but the clinical significance of this observation is uncertain. Until further data become available, it is worth considering that changing between generic and branded drugs may result in a subtle (but potentially significant) shift in metabolism.
Strategies for initiating treatment with AZA/MP vary, and range from slow titration based on WBC to immediately starting at the full weight-based dose. Although relying on the WBC as a surrogate marker for efficacy has been observed to be beneficial,49 when evaluated in a prospective manner, leucopoenia did not correlate with either efficacy of treatment50 or TG levels.38, 51 Furthermore, there is some concern that persistently depressed WBC may be a risk factor for developing neoplasms.52 A rationale behind slow titration is to carefully monitor for clinical signs of toxicity. While idiosyncratic reactions, such as pancreatitis, fever, rash, nausea/vomiting, diarrhoea and arthralgias may be minimized by this approach, dose-dependent toxicities (hepatitis, thrombocytopenia, delayed leucopoenia) are unlikely until a significant cumulative dose has been given.46 In addition, slow titration may further delay an already lengthy period before therapeutic effects are seen. As long as the TPMT genotype is normal, starting at a low dose (50 mg for both AZA/MP) for 2 weeks with monitoring of clinical side-effects, biweekly FBC and liver function tests (LFTs) before escalating to a full weight-based dose is a reasonable approach. Doctors and patients both need to understand that these medications are slow in onset. In general, 3–4 months may be needed before beneficial effects are seen, however, onset of effect is very variable. Although some patients will not see effects for 6 to even 12 months, 20% of patients may attain clinical remission by 2 weeks.53
Close follow-up of FBC and LFTs is necessary in all patients taking AZA/MP. The exact frequency of testing has not been examined systematically, however many practitioners measure FBC biweekly for 3 months and then every 3 months for the entire duration of treatment. As previously noted, three-fourths of neutropenia associated with these medications occur sporadically many months after starting therapy.42 These events are not accounted for by variations in TPMT, so TPMT testing should not take the place of regular monitoring of FBC. Practically, to monitor for drug-induced hepatitis, LFTs can be obtained at the same time intervals as FBC, however, measuring LFTs can probably be done less frequently. Pancreatitis usually occurs in the first 8 weeks after starting treatment with AZA/MP. This can be predicted and potentially prevented by noting a preclinical rise in serum amylase.54 Obtaining amylase with biweekly labs for the 8 weeks could pick up those patients at risk and avoid the few potential cases of drug-induced pancreatitis, however, this practice has not been prospectively evaluated. Measuring C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) are not necessary for monitoring of drug safety, however, if elevated at baseline, these may be helpful as markers of response to therapy.
TG/6-MMP metabolite monitoring
The utility of measuring the MP metabolites TG and 6-MMP has been debated in the literature and even referred to as the ‘metabolite controversy’.55 In 2000, Dubinsky et al. showed that in children, higher TG levels corresponded with a higher frequency of response. In fact, 65% of patients with TG levels in the therapeutic range (>235 on their assay) had a beneficial response as opposed to 27% with suboptimal levels.38 Similar results have been reported by others,56, 57 but this has not been consistent among all groups. In one study of 170 adults, there was no correlation between TG levels and IBDQ scores.58 A similarly negative result was found in 71 patients with Crohn's disease where metabolite monitoring had no correlation with clinical status based on Harvey-Bradshaw index.59 More recently in 2004, Wright et al. concluded that lower TG levels did correspond with increased disease activity, however the variability of metabolites over time makes single measurements inadequate for directing dose titration.60 Variability of TPMT enzyme activity likely plays a role in the inconsistency of TG levels, influenced by concomitant medications such as 5-aminosalicylates (5-ASAs) and other environmental exposures. The different patient populations, metabolite assays and disease activity indices used in these studies make it difficult to draw conclusions from the previously reported literature. The wide range and broad overlap of TG levels among patients responding or resistant to therapy also complicates this issue. Furthermore, in trials where IBD patients were treated with TG directly, serum TG levels were up to ten-fold higher (range: 6299–20 441) than when treated with AZA/MP, without associated leucopoenia.61 These facts argue that although TG is an important metabolite associated with both efficacy and toxicity of AZA/MP, other metabolites are likely also to play an essential role. At the current time, the data are insufficient to support routine monitoring of MP metabolites. However, it does appear that in a subset of patients who are not responding to treatment, obtaining TG and 6-MMP levels can be very helpful to identify non-compliant patients and those who preferentially metabolize away from TG favouring 6-MMP production.62, 63 A further use of metabolite testing may be to confirm adequate absorption in patients with short-gut syndrome.64 A prospective trial evaluating the utility of TG and 6-MMP levels is currently underway (S. B. Hanauer, personal communication), and will hopefully lead to a more clear understanding of the role of metabolite testing.
Despite therapeutic efficacy and normal LFTs, clinicians will oftentimes continue to follow metabolite levels. Other than the expense, the pitfalls of this practice may be inappropriate dose adjustments to put patients into the ‘therapeutic range’ for TG, and fear of pushing 6-MMP levels above the ‘toxic’ threshold of 5700. Although patients with hepatotoxicity have higher median 6-MMP levels than those without,38, 63 most patients with elevated 6-MMP levels have normal LFTs and no evidence of hepatotoxicity. Furthermore, some experience hepatotoxicity with normal 6-MMP levels. In a recent publication, 12% of patients had 6-MMP level >5700, and no hepatotoxicity was seen.62 Elevated 6-MMP appears to have a low predictive value for abnormal liver tests.
Based on the above data, it would seem reasonable to recommend checking TG/6-MMP metabolites when patients are not achieving therapeutic efficacy despite adequate weight-based dosing to ascertain non-compliance or metabolism favouring 6-MMP (Table 2). A 6-MMP:TG ratio >10 has been suggested as a threshold representing unfavourable metabolism which is unlikely to result in therapeutic efficacy.63 In such individuals, further dose escalation may be fruitless. Incidentally found high 6-MMP levels warrant continued close follow-up of LFTs; however, as long as LFTs remain normal, dose-reduction is unnecessary.
Inosine triphosphate pyrophosphatase (ITPA) is another enzyme involved in the metabolism of MP (Figure 1). ITPA-deficient patients accumulate 6-thio-ITP, a potentially toxic metabolite. In a study of 62 patients with IBD suffering adverse effects while taking AZA, a deficiency-associated allele was significantly associated with adverse drug reactions when compared with controls.65 Specifically, associations were found with flu-like symptoms (OR: 4.7, 95% CI: 1.2–18.1), rash (OR: 10.3, 95% CI: 4.7–62.9) and pancreatitis (OR: 6.2, 95% CI: 1.1–32.6). A subsequent report from the same investigators confirmed that ITPA polymorphisms were associated with flu-like symptoms, however, an increase in overall toxicity (including pancreatitis) was not seen.66 Others also found no association between ITPA mutations and pancreatitis, but did see a significant correlation between ITPA mutations and leucopoenia (OR: 2.647, 95% CI: 1.214–5.773, P = 0.013).67 Until further studies confirm the utility of ITPA testing, we do not recommend use in clinical practice.
As with any medication, drug interactions can quickly change metabolism leading to either loss of efficacy or toxicity. Debate on the influence of 5-ASA on AZA/MP metabolite level and the clinical significance of this effect continues. In vitro studies have confirmed that 5-ASAs are non-competitive inhibitors of TPMT, leading to potentially toxic intracellular levels of TG.68 Clinically, higher TG levels have been seen in patients concomitantly taking certain 5-ASAs along with AZA/MP.58, 69 This has not been observed in all studies,63, 70 and although there is some selective shunting towards TG based on TPMT inhibition, this is likely of small clinical importance. Nonetheless, patients oftentimes ask about stopping 5-ASAs once finally achieving remission with AZA/MP. Although in most cases this can be successful, caution needs to be taken, as the TPMT inhibition provided by the 5-ASA may in some cases be the key factor in maintenance over the fine line of efficacy.
Patients are often treated concomitantly with AZA/MP and infliximab. A small study looked at levels of TG and WBC prior to treatment with infliximab, at 1–3 weeks after infusion and 3 months later.71 Results showed that TG levels were significantly higher and WBC significantly lower within 1–3 weeks after infliximab infusion. Furthermore, higher levels of TG corresponded with better efficacy and tolerance of infliximab. These intriguing results warrant further investigation.
Allopurinol, by inhibiting xanthine oxidase (Figure 1), dramatically affects the metabolism of AZA/MP by allowing a significantly higher proportion of medication to continue down the path towards TG. If possible, avoiding the co-administration of allopurinol and AZA/MP is the safest option, but if both are necessary medications, cutting AZA/MP by half is recommended along with very close follow-up of the FBC. Medications with known immunosuppressant properties should be avoided, and other medications with possible interactions including warfarin [decreased International Normalized Ratio (INR) possibly because of an increased synthesis of prothrombin], furosemide (increased bone marrow suppression by TPMT inhibition)72 and angiotensin converting enzyme (ACE) inhibitors (increased bone marrow suppression by unknown mechanism) should be used with caution. At every office visit, a complete medication list should be carefully reviewed with patients to monitor for important drug–drug interactions.
Mechanism of action and evidence of efficacy
Methotrexate was initially used for the treatment of leukaemia in children, at which time it was noticed that those with concomitant psoriasis or rheumatoid arthritis (RA) showed improvement in these conditions.73 Subsequent studies confirmed efficacy in psoriasis and RA, which led to trials in IBD. Methotrexate is a folic acid structural analogue that competitively inhibits the binding of dihydrofolic acid to the enzyme dihydrofolate reductase, involved in purine and pyrimidine synthesis.74 In addition to this impaired DNA synthesis, a variety of effects of methotrexate have been reported, including generation of adenosine, decreased expression of interleukin (IL)-1, and induction of apoptosis.75 The relevant mechanism of action in IBD, however, has not been fully elucidated. Methotrexate has been shown to be effective in Crohn's disease for both treating active disease76 and maintaining remission.77 A few small studies looking at the efficacy of methotrexate in UC have had mixed results.78–82 Although there are no randomized-controlled data, if patients are intolerant to, or failing AZA/MP, methotrexate could be considered for patients with refractory or steroid-dependent UC.
Liver toxicity related to methotrexate is seen frequently in patients with psoriasis. In a study from 1996, a series of 104 patients with psoriasis were treated with methotrexate over a period of 3.4 years.83 Nearly a quarter of these patients had either active hepatitis or cirrhosis on follow-up liver biopsy. Patients with psoriasis have a higher prevalence of risk factors for liver disease including obesity, alcohol overuse and diabetes than those with IBD. The incidence of hepatotoxicity from methotrexate in patients with IBD has not been studied fully, but is thought to be significantly lower than in psoriasis. A collaborative study from Te et al. at the University of Chicago and the Mount Sinai Hospital in New York reported on liver biopsies in 20 IBD patients who had received a mean dose of 2633 mg of methotrexate over 131.7 weeks.84 The majority (95%) had Roenigk grade I scoring85 (normal-to-mild steatosis or inflammation) and one patient had Roenigk grade IIIb (moderate-to-severe fibrosis) histological changes. This patient also had diabetes, was obese and was on multiple other potentially hepatotoxic medications in addition to methotrexate. Although methotrexate-induced hepatotoxicity can occur in patients with IBD, in properly selected patients (avoiding use in patients who are obese, regular alcohol users, have fatty liver or other pre-existing liver disease), this risk is likely low.
Hypersensitivity pneumonitis has been reported in about 1% of patients, and risk factors include older age, diabetes and rheumatoid lung disease.86 Rare cases have been reported in patients with IBD.82, 87 Pre-treatment chest X-ray or pulmonary function tests are not routinely ordered, but a high clinical suspicion is essential if pulmonary symptoms begin during treatment. Nausea is a common complaint of patients taking methotrexate. This can usually be minimized by changing the time of dosing (before bedtime), ensuring adequate intake of folic acid (minimum of 1 mg PO daily, possibly some added benefit with 2 mg daily), and if needed, adding antiemetics around the time of the weekly dose. As methotrexate is both teratogenic and a known abortifacient, ensuring adequate birth control is absolutely essential. In addition, methotrexate may be toxic to sperm,88, 89 and men should stop methotrexate at least for 3 months before trying to conceive.
In clinical trials, the effective dose in treatment of active disease was 25 mg intramuscular (IM) weekly, and 15 mg IM weekly for maintenance of remission.76, 77 Trials with oral dosing of methotrexate in Crohn's disease have included a study using 12.5 mg PO weekly90 which showed no difference when compared with placebo, and another trial using dose titration up to 22.5 mg PO weekly which showed a non-significant trend towards improvement.91 Studies of the bioavailability of orally dosed methotrexate have been variable, with one study showing adequate methotrexate absorption, including among those with severe small bowel disease,92 and another demonstrating highly variable absorption with an average of 73% of that of subcutaneous administration.93 Pharmacokinetic studies in RA have shown that lower oral doses have more reliable absorption than higher doses,94 and this may be true in Crohn's disease as well.93 It is unclear if the lack of efficacy of oral dosing in the above trials was due to under-dosing, poor absorption, a type II error in those small trials, or a true lack of efficacy, but at this time oral dosing cannot be recommended. Subcutaneous dosing, on the other hand, is more easily tolerated than the proven IM regimen, and has been shown to be bioequivalent to IM injection in patients with RA.95 Our recommendation is for 25 mg subcutaneous weekly for treatment of active disease with dose-reduction to 15 mg subcutaneous weekly for maintenance of remission (Table 3). In Feagan's maintenance study, all patients at entry had received a minimum of 16 weeks at 25 mg subcutaneous weekly before decreasing their dose. As long as remission has been achieved, 16 weeks is a reasonable time to reduce the dose to 15 mg subcutaneous weekly. Co-administration of folic acid 1 mg PO daily helps prevent clinically significant folate deficiency and may diminish potential dose-related nausea and mouth sores. Time to onset of action is probably similar to AZA/MP. The only head-to-head study comparing methotrexate with AZA was not powered to address the question of rapidity of onset of action,96 but there was a suggestion that methotrexate may provide clinical benefit earlier than AZA/MP.73
Table 3. Recommendations for the safe and effective use of methotrexate
FBC, full blood count; LFTs, liver function tests; AST, aspartate aminotransferase; PO, per oral; WBC, white blood cell.
1. Avoid use in patients with known liver disease (including fatty liver), alcohol overuse, obesity, diabetes or in women trying to conceive
2. Start methotrexate at 25 mg subcutaneous weekly for active disease
3. Concomitantly begin folic acid 1 mg PO daily
4. Women required to use at least one form of highly effective birth control, men should avoid conceiving while on methotrexate and for 3 months after stopping
5. Monthly FBC and LFTs with albumin for 2 months, then every 4–8 weeks for the duration of therapy. Hold methotrexate for WBC <3.5 × 109/L, recheck labs in 1 week
6. At 16 weeks, if remission has been achieved, reduce dose to 15 mg subcutaneous weekly
7. For any signs of infection (viral or bacterial), patient should have a clinical evaluation and measurement of FBC and LFTs. If clinically significant infection, hold methotrexate until symptoms resolve
8. Perform liver biopsy only if there are persistent elevations of AST above the upper limit of normal in five of nine (or six of 12 if performed monthly) tests in a given year or a decrease in serum albumin below the normal range (in the setting of well-controlled inflammatory disease). Pre-treatment liver biopsies are not recommended unless there is concern about underlying liver disease
9. Stop methotrexate if liver biopsy reveals Roenigk grades IIIb or IV or if a patient has persistently abnormal LFTs and refuses liver biopsy
Laboratory values should be followed primarily to evaluate for pancytopenia and hepatotoxicity. Pancytopenia is uncommon and there were no severe episodes in either of Feagan et al.'s studies of methotrexate in Crohn's disease.76, 77 Nevertheless, this complication has been reported and can be life-threatening.97 A FBC and liver tests should be obtained monthly for the first 2 months and then performed every 4–8 weeks for the duration of therapy. The role of liver biopsy is discussed in detail below. Pharmacokinetic drug monitoring was examined in patients with RA, and found not to be useful.98
Role of liver biopsy
The initial high rate of hepatotoxicity seen in patients with psoriasis led to guidelines by the American Academy of Dermatology to obtain pre-treatment liver biopsies in addition to serial liver biopsies after each 1500 mg cumulative dose of methotrexate.85 The American College of Rheumatology, noting a much lower risk of hepatotoxicity in RA patients, recommends monitoring LFTs including albumin every 4–8 weeks. They advise to perform a liver biopsy only if there are persistent elevations of AST above the upper limit of normal in five of nine (or six of 12 if performed monthly) tests in a given year, or a decrease in albumin below the normal level in the setting of improving inflammatory disease.99 This guideline suggests that methotrexate should be stopped if liver biopsy reveals Roenigk grades IIIB or IV histology, or in persistently abnormal LFTs in a patient who refuses liver biopsy.99 Recently, a study reported on 66 patients with psoriasis followed for >5 years while receiving a median cumulative dose of methotrexate >3000 mg.100 The subjects underwent a total of 121 liver biopsies, and although some advanced fibrosis was reported, no cirrhosis was seen and not a single patient discontinued therapy based on liver biopsy findings. The previously mentioned study by Te et al.84 also supports the low risk of significant hepatotoxicity related to methotrexate. Thus far, formal guidelines for the treatment of IBD patients have not been stated by any gastroenterological association. Our recommendation is to follow those of the American Rheumatologic Association as outlined above (Table 3). However, as more safety data becomes available over time, the trend towards performing fewer liver biopsies is likely to continue.
As previously mentioned, methotrexate is involved in disrupting folate metabolism. Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in folate metabolism and has been shown to have polymorphisms that affect enzyme activity. In a study involving Japanese patients with RA, the MTHFR C677C genotype was associated with lower effective doses of methotrexate while the C667T mutation appeared more frequently in patients experiencing drug toxicity.101 Concordant with this observation, among bone marrow transplant patients, the C677T genotype was associated with 30% lower MTHFR activity and coincided with an increased incidence in methotrexate-related toxicity.102 In a recent study in IBD patients, C677T and C1298C polymorphisms were examined and not found to be predictive of toxicity or efficacy of methotrexate treatment.103 More work is to be done before MTHFR polymorphisms can be considered useful in predicting methotrexate efficacy, resistance or drug toxicity, but these data suggest an exciting avenue of investigation.
In addition to modifying drug metabolism, the MTHFR C667T genotype is associated with elevated homocysteine levels in RA patients.104 As MTHFR mutations have also been associated with hypercoagulable states, a number of groups have looked into the possible relationship between MTHFR, homocysteine and thrombosis in IBD patients. Although elevated homocysteine levels have been seen more frequently in patients with IBD when compared with controls,105, 106 a clear association with MTHFR mutations and thrombosis has not been seen in these patients.105, 107–109 Elevated homocysteine levels may be a result of methotrexate use (particularly with concomitant sulfasalazine use),104 however, to our knowledge, a connection between methotrexate use and thrombosis has not been established in patients with IBD. Folic acid supplementation does decrease homocysteine levels,110 and although its clinical benefit in diminishing cardiovascular and cerebrovascular risk is controversial, this is another reason to recommend folic acid supplementation to all patients taking methotrexate.
Concomitant immunosuppressive drugs or medications that have potential to cause bone marrow toxicity (e.g. sulfonamides) should be used with caution. As adenosine may play a role in the immunosuppressive effects of methotrexate, avoiding adenosine receptor antagonists (caffeine, theophylline) has been recommended to prevent inhibiting therapeutic benefit.111 At the doses used in IBD (as opposed to chemotherapy), other significant drug–drug interactions are unusual.
Ciclosporin is a cyclic peptide, which forms a complex with cyclophilin within the cell. This complex inhibits calcineurin, a serine threonine phosphatase, which is responsible for activating proinflammatory transcription factors. By inhibiting nuclear factor of activated T cells, ciclosporin prevents production of IL-2, as well as interferon (IFN)-γ, tumour necrosis factor (TNF)-α, granulocyte–macrophage colony-stimulating factor and IL-4. Consequently, ciclosporin diminishes cytokine production and exerts an antiproliferative effect on lymphocytes.112
Although some data have suggested a beneficial effect of high-dose ciclosporin in active Crohn's disease,113 the benefit was not durable.114 Ciclosporin's main role has been in treating patients with severe steroid refractory UC. In the placebo-controlled trial by Lichtiger et al.115 intravenous (IV) ciclosporin showed an impressive 83% response rate in steroid refractory UC patients. At 6 months, after conversion to oral ciclosporin, 63% of patients were able to discontinue steroids and remain in remission while 37% had undergone colectomy.116 The addition of AZA/MP improves long-term response rates.117, 118 As a bridge to other maintenance therapy (i.e. AZA/MP), ciclosporin can be an effective treatment option.
Sandborn reviewed adverse effects of ciclosporin seen in 343 IBD patients across 27 different studies.119 This analysis showed the following: paresthesias (26%), hypertrichosis (13%), hypertension (11%), tremor (7%), nausea or vomiting (6%), renal insufficiency (6%), headache (5%), infection (3%), hepatotoxicity (3%), gingival hyperplasia (2%), seizure (1%) and anaphylaxis (0.3%). It was thought that ciclosporin caused a reversible reduction in glomerular filtration rate without significant histopathological changes.120 However, irreversible nephrotoxicity became a concern when a study in patients with autoimmune diseases (without renal disease) revealed evidence of nephropathy on renal biopsy in 21% of patients treated with oral ciclosporin.121 In that study, risk factors for nephropathy included higher dose of ciclosporin, greater rise in serum creatinine, and older age. Seizures are rare, but may result in significant morbidity. To decrease this risk, ciclosporin should be avoided in those with serum cholesterol values lower than 3.1 mm (120 mg/dL).122 Magnesium levels should be followed carefully123 as ciclosporin may lead to magnesium wasting,124 which may further lower the seizure threshold. Opportunistic infections may be severe and potentially fatal. Concomitant use of steroids or other immunomodulators (MP or AZA) likely compound this risk.
Successful trials with ciclosporin for UC used doses of 8–10 mg/kg/day orally or 2–4 mg/kg/day IV.115, 116, 125 The recent trial by Van Assche et al.125 concluded that 2 mg/kg/day IV was as efficacious as and had lower toxicity than 4 mg/kg/day dosing. An oral microemulsion formula (available as Neoral) has been shown to be effective in uncontrolled trials in UC patients (4.6–7.5 mg/day), but has not been studied in a placebo-controlled trial. Based on its more dependable bioavailability, early success in uncontrolled trials, and acceptance in the transplant field, the oral microemulsion formula may become more widely used and eventually replace IV administration.
Before treating with ciclosporin, we review surgical options with the patient and we make certain the patient has understood the risks and benefits of medical therapy vs. colectomy. Clinicians might consider obtaining informed consent before starting treatment. Kornbluth et al.'s ‘user's guide’,126 outlines precise logistics for giving the medication and recommendations for monitoring of patients on both IV and out-patient oral ciclosporin. These recommendations, based on their experience, are summarized below.
Bedside monitoring should be performed every 15 min for the first hour of the infusion for signs of allergy or anaphylaxis. Blood pressure should be monitored every 4 h while awake. Daily clinical monitoring of disease activity and for side-effects such as headache, nausea or paresthesias is imperative. After initiating therapy at 2 or 4 mg/kg as a continuous IV infusion, ciclosporin levels should be obtained every 2 days. At the 4 mg/kg dose, the goal whole blood level of ciclosporin is 300–400 ng/mL, however, the literature on 2 mg/kg dosing suggests that lower levels (mean 237 ± 33 ng/mL) are as efficacious and potentially less toxic. If not within the therapeutic range, ciclosporin levels should be checked daily with every dose titration. Serum creatinine, potassium, magnesium and LFTs should be checked every 2 days, unless abnormal, in which case they should be checked daily. Serum cholesterol should be measured daily if ≤3.1–3.6 mm (120–140 mg/dL). Ciclosporin dose should be reduced if drug levels are >500 ng/mL for 2 consecutive days or if serum creatinine rises >30% over baseline, serum liver enzymes double, diastolic blood pressure exceeds 90 mmHg, or systolic blood pressure exceeds 150 mmHg despite antihypertensive treatment. If any of these occur, the dose should be decreased by a minimum of 25%. There are no clear guidelines with regard to Pneumocystis carinii pneumonia (PCP) prophylaxis. As PCP is entirely preventable, our general practice is to keep patients on trimethoprim/sulfamethoxazole 160/800 1 tab PO three times weekly while taking ciclosporin.
A clinical response should be noticed within 4–5 days. If clearly responding, ciclosporin infusion should be continued for a minimum of 7 days before conversion to oral ciclosporin. If there is not a significant improvement in clinical status after 10 days of IV therapy, patients should be referred for surgery.
Patients can be safely discharged home after 1–2 days of observation on oral ciclosporin. The total daily oral dose is twice the daily IV dose, divided and given every 12 h. While on oral ciclosporin, patients should be seen weekly for the first month, biweekly for the second month, and then every 3–4 weeks. Ciclosporin levels, serum chemistries, magnesium, FBC and ESR should be performed at the same interval as the clinic visits or weekly for any dose changes. Prednisone should be slowly tapered with a goal to be entirely weaned off by 6 months. AZA/MP should be started about 2–3 months after hospital discharge with plans to maintain remission on this medication after stopping ciclosporin. The ‘user's guide’ recommends discontinuation of ciclosporin at 6 months, by reducing the dose by 50% for 2 weeks followed by complete ciclosporin withdrawal. Others treat with ciclosporin for a shorter period of time, stopping by 3 months, in which case maintenance therapy will need to be initiated sooner than 2–3 months after discharge.
Tacrolimus is a macrolide antibiotic isolated from Streptomyces tsukibaensis. Although structurally different, its mechanism of action is similar to ciclosporin by acting as a calcineurin inhibitor, leading to decreased production of IL-2.127 Given its efficacy in transplant rejection and reliable oral absorption, clinical trials in Crohn's disease and UC have been performed. There has been one placebo-controlled trial showing efficacy of tacrolimus improving perianal fistulas in patients with Crohn's disease,128 and small open-label experiences showing benefit in active lumenal129, 130 and fistulizing Crohn's disease.131 In both adults and children, there is also open-label experience suggesting efficacy in UC.132, 133 Similar to ciclosporin, onset of action may be rapid with clinical improvement seen within 1–4 weeks. Duration of therapy has ranged from 3 months in the paediatric study132 to >2 years in adult Crohn's patients.130 Until further safety data become available, tacrolimus (like ciclosporin) should be used short-term (<6 months) as a therapeutic bridge to some other therapy.
Tacrolimus has an adverse effect profile similar to ciclosporin, however there is much less experience in IBD patients to guide recommendations to avoid toxicity. In the only controlled trial, in which 21 patients received tacrolimus for fistulizing Crohn's disease,128 the most commonly reported adverse events were paresthesias (57%), headache (48%), creatinine increase >30% (38%), insomnia (29%), nausea or vomiting (29%), leg cramps (24%), diarrhoea (19%) and pruritus (19%). Adverse effects were dose-related and patients were managed successfully with dose reduction.
Dosing should be 0.1–0.2 mg/kg/day if given orally or 0.01–0.02 mg/kg/day if given IV. As with ciclosporin, we recommend trimethoprim/sulfamethoxazole 160/800 1 tab PO three times weekly for PCP prophylaxis.
The target range for tacrolimus concentrations is 10–20 ng/mL.120 Whole blood tacrolimus levels, and electrolytes with renal function should be measured weekly for the first month, biweekly for the second month, and then monthly thereafter. Tacrolimus dose should be decreased if serum creatinine level rises >30% above baseline.
Azathioprine, MP, methotrexate, ciclosporin and tacrolimus all have their respective niches in the treatment of patients with IBD. Irreversible, severe toxicity is uncommon, however very rare but devastating outcomes can occur. With sufficient knowledge of the spectrum of adverse effects and appropriate monitoring, these medications can be used both safely and effectively. In the future, further understanding of the determinants of metabolism may lead to improved safety of these drugs and a more precise treatment strategy for patients with IBD.
Conflict of Interest
Dr Sands receives research support and honoraria from Prometheus Laboratories.