Prospective Evaluation of the Pharmacogenetics of Azathioprine in the Treatment of Inflammatory Bowel Disease

A. Ansari; M. Arenas; S. M. Greenfield; D. Morris; J. Lindsay; K. Gilshenan; M. Smith; C. Lewis; A. Marinaki; J. Duley; J. Sanderson

Disclosures

Aliment Pharmacol Ther. 2008;28(8):973-983. 

In This Article

Results

A total of 215 patients were recruited into the study from 11 centres. Of these two patients broke the protocol, three had incomplete data, two had surgery before week 12 and one withdrew from the study leaving 207 for final analysis. The mean age was 40.3 years (range 18-80 years) and 115 (54%) were female. One hundred and seventeen patients had CD [65/117 (56%) female] of whom 25 (21%) had ileal disease, 52 (44%) ileocolonic, 31 (27%) colonic disease, nine (8%) disease limited to the proximal small bowel/upper gastrointestinal (GI) tract. Eleven (9%) had coexistent perianal or enterocutaneous fistulating disease which was not the primary indication for AZA use. Of the 90 patients [46/90 (51%) female] with UC, 50 (55%) had pancolitis, six (7%) subtotal colitis and 34 (38%) distal colitis.

In the majority of patients (85%), the indication for AZA was as steroid-sparing therapy. Induction of remission of active disease was the indication in six (3%), maintenance of remission in 26 (12%). The median starting dose of AZA was 1.95 mg/kg (range 1.88-2.38 mg/kg). The TPMT distribution conformed to the expected population distribution (data not shown). No patients with zero TPMT were detected and 19 of 219 (9%) had a heterozygous TPMT genotype. No significant induction of TPMT was observed from baseline to subsequent measurement. The correlation between TPMT genotype and phenotype was 100%, whereas between phenotype and genotype it was 99% (data not shown).

There was a high overall withdrawal rate of 83 (out of 215) patients (39%) due to adverse effects on an intention-to-treat analysis. A further 12 (6%) reported side effects but continued treatment over the 6 month period. The details of adverse effects leading to drug withdrawal are given in Table 1 . In particular, there was an unexpectedly high occurrence of gastric intolerance. One patient had asymptomatic amylasaemia which persisted despite withdrawal of AZA, and a diagnosis of macroamylasaemia was made.

Amongst the whole cohort of 207 patients, a complete clinical response to AZA occurred in 79 of 207 patients (38%). Clinical response amongst the 124 patients completing the 6 months required for inclusion in the analysis was 64% (79/124).

TPMT vs. Adverse Effects

A heterozygous TPMT genotype strongly predicted withdrawal of AZA due to adverse effects, such that 15 of the 19 (79%) heterozygotes did not tolerate 6 months of AZA, compared with 66 of 188 (35%) who were TPMT wild type (P= 0.000265). Gastric intolerance was the most frequent reason for withdrawal amongst heterozygotes and was significantly more common than in those with normal TPMT. Twenty patients had GI intolerance: seven (37%) were TPMT heterozygotes while only 13 (7%) were wild type (χ2 = 13.9, P< 0.001). Myelotoxicity was also more frequent in those with a heterozygous TPMT genotype (26% vs. 0.5%, P< 0.01). Importantly, gastric intolerance occurred early, within 6 weeks and only those remaining on AZA for longer went on to develop myelotoxicity (Figure 2).

Figure 2.

Timing of adverse drug reactions leading to withdrawal of azathioprine (AZA) amongst patients with heterozygous thiopurine S-methyltransferase (TPMT) deficiency. Patients all received 2 mg/kg of AZA as per study protocol. Overall, 15 of the 19 patients (79%) with heterozygous TPMT deficiency withdrew treatment due to ADRs during the 6 month study period. Of these, most (n = 11) withdrew treatment due to non-haematological ADRs (mainly gastrointestinal intolerance) with all but one case occurring within 6 weeks of starting AZA. Conversely, myelotoxicity (n = 4) did not occur until week 12 or later. Individuals with heterozygous TPMT deficiency are most likely to withdraw therapy due to gastric intolerance before they are able to develop myelotoxicity.

ITPA Genotype and Adverse Effects

In the study group 202 were genotyped for the ITPA 94C>A and IVS2 + 21A>C polymorphisms. The allele frequencies were 0.089 for the ITPA 94C>A mutation and 0.093 for the ITPA IVS2 + 21A>C mutation. Overall, withdrawal due to adverse effects was not predicted by the ITPA genotype (ITPA 94C>A: P= 0.31 and ITPA IVS2 + 21A>C: P= 0.46). However, analysis by specific side effects and clusters of ITPA polymorphisms compared with patients without side effects revealed that the ITPA 94C>A mutation was strongly associated with flu-like symptoms (P= 0.014, 95% CI = 1.23-13.94, OR = 4.13). There was no association with any other side effect (gastric intolerance, P= 0.50 for ITPA 94C>A).

TPMT vs. clinical response

The activity of TPMT was strongly predictive of clinical response. There was a 43% (24/56) response in those with a red cell TPMT activity greater than 35 pmol/h/mg Hb, compared with 81% (55/68) in those with red cell TPMT activity below 35 pmol/h/mg Hb (Fisher's exact P< 0.001). Likewise, mean TPMT activity was significantly higher in non-responders vs. responders to AZA (38.6 pmol/h/mg Hb vs. 32 pmol/h/mg Hb respectively, P< 0.001). Dose of AZA (mg/kg) received did not differ either between responders and non-responders (1.95 mg/kg in both groups, t-test P= 0.65) or in each TPMT group (above and below 35 pmol/h/mg Hb) (1.94 mg/kg vs. 1.96 mg/kg, t-test P= 0.54).

TGN Measurements vs. Clinical Response

The TGN values took 4 weeks to reach steady state. TGN concentrations at 2 weeks were therefore not included in the analysis which comprised an average of the concentrations at 4, 12 and 24 weeks. All three measurements were available for 75 patients, at least two TGN concentrations in 112 patients and at least one measurement in 124 patients. Overall, there was no relationship between mean TGN level and clinical response (P= 0.14). However, an analysis comparing patients with TGN values above and below 100 pmol/8 × 108 RBC showed that there was a significant difference in clinical response between these two groups: 74% (59/80) response in those with TGN > 100 pmol/8 × 108 RBC vs. 46% (20/44) in those with TGN < 100 pmol/8 × 108 RBC (P= 0.0017). There was also a significant trend (P= 0.018) towards increasing the likelihood of successful clinical response with greater numbers of TGN values above 100 in any particular patient (Figure 3).

Figure 3.

Relationship between clinical response to azathioprine (AZA) and frequency of thioguanine nucleotide (TGN) concentrations above 100 pmol/8 × 108 RBC during study. Bar chart showing the relationship between clinical response to AZA and the numbers of TGN concentrations above 100 pmol/8 × 108 RBC. TGN concentrations taken during study at 4, 12 and 24 weeks. Clinical response assessed after 6 months of AZA 2 mg/kg. Patients with no TGN level over 100 pmol/8 × 108 RBC had a very low likelihood of successful clinical response whilst those with all three TGN values above this level had a very high likelihood of response. There was a significant trend towards increasing chances of clinical response with numbers of TGN values above 100 pmol/8 × 108 RBC (P= 0.018).

Haematological Parameters vs. Response

There was a significant relationship between lowest neutrophil count and complete response to AZA. The mean lowest neutrophil count was less in those patients who responded to AZA compared to those who had no response (4.1 vs. 5.6 respectively, P= 0.024). However, the lowest lymphocyte count did not correlate with clinical response. A strong relationship for MCV and clinical response was found (responders 87.2 fl vs. non-responders 82.6 fl, P= 0.001). Change in MCV from baseline to 6 months, however, did not correlate with clinical response.

Influence of 5-aminosalicylates

Co-prescription of 5-ASA did not influence TPMT activity (33.4 pmol/h/mg/Hb in those receiving 5-ASA vs. 33.7 pmol/h/mg Hb in those not on 5-ASA, t-test P= 0.79) and was not associated with any difference in clinical response to AZA (64% vs. 63%, on and off 5-ASA respectively, χ2 P= 0.93). Use of 5-ASA was, however, independently associated with a reduced risk of side effects (28% in those on 5-ASA vs. 48% in those not taking 5-ASA, χ2 P= 0.007). There was no effect of 5ASA on TGN concentrations [mean TGN on 5ASA 137 (n = 76) vs. 130 (n = 48) off 5ASA] (P= 0.49, Mann-Whitney U-test.).

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