Monitoring Of Azathioprine Therapy In Chinese Han Patients With Inflammatory Bowel Disease

Inflammatory bowel disease(IBD),including Crohn's disease(CD)and ulcerative colitis(UC),is a chronic disorder of the gastrointestinal tract.The prevalence of IBD is about 1.0/1000 in Western countries.No large scale epidemiological investigation is available on the prevalence or incidence of IBD in China so far,but the diagnosed IBD in China has increased obviously in the past ten years.The thiopurine drugs azathioprine(AZA)and 6-mercaptopurine(6-MP)are well established in the treatment of inflammatory bowel disease.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.However,there is a wide inter- and intra-patient variation in the concentrations of active and toxic metabolites due to their complex metabolism and genetic polymorphisms in metabolizing enzymes.Serious drug toxicity leads to cessation of therapy in 9-28%of patients,and there is failure to achieve efficacy in approximately 15%of cases.Advances in the understanding of thiopurine drug metabolism have led to new genetic and metabolite tests to help clinicians monitor and optimize thiopurine use. Azathioprine and 6-MP are not therapeutically active.Azathioprine is a pro-drug that is converted to 6-MP via a nonenzymatic metabolism pathway.Then,6-MP may be activated via a multistep enzymatic pathway to produce the active metabolites,the 6-thioguanine nucleotides(6TGNs).6-TGNs is the predominant active metabolite that confers efficacy but is also associated with myelotoxicity.6-MP also metabolized by thiopurine methyltransferase(TPMT)to 6-methylmercaptopurine(6-MMP)or by xanthine oxidase(XO)to 6-thiouric acid(6-TU).TPMT is the key enzyme in the metabolism pathway,decreased TPMT activity correlated with myelotoxieity.Thiopurine methyltransterase enzyme activity is genetically determined.There is a trimodal distribution of TPMT in the general population:homozygous low activity occurs at a frequency of 0.3%;heterozygous or intermediate activity occurs at a frequency of 11.1%;and homozygous high or normal activity occurs at a frequency of 88.6%.This trimodal distribution is typical of monogenic inheritance.The TPMT gene is located on the short arm of chromosome 6.A total of 21 TPMT genetic polymorphisms(*2,*3A,*3B,*3C,*3D,*4-*19).have been identified which are associated with decreased levels of TPMT enzyme activity and/or thiopurine drug-induced toxicity.TPMT*2,*3A,*3B,*3C are the most prevalent mutant alleles.TPMT testing may also aid in dose individualization.Clearly,if a patient is TPMT-defieient,he will not tolerate standard thiopurine drug doses.It has been suggested that thiopurine drugs should be avoided in this subset of IBD patients due to the high risk of life threatening myelotoxicity.However,it has been shown that very small doses of a thiopudne can be used safely and effectively in a small series of TPMT-deficient patients,with close monitoring.Patients with intermediate TPMT activity should be commenced on half or one-third of the initial dose of thiopurine.Patients with very high TPMT activity will either be resistant to thiopurine drugs due to shunting of 6-MP down the 6-MMP pathway,or require a high dose to achieve efficacy,but at the risk of hepatotoxieity due to high 6-MMP(R) concentrations.As the incidence of IBD is increasing,what is the status of care for these patients? There are clear population differences of the TPMT genotype and phenotype, can TPMT phenotype or genotype predict the side effect or efficacy of thiopurine therapy in Chinese Hart IBD patients? To study the relationship of TPMT status with thiopurine therapy in Chinese Han IBD patients,we performed a four-stage study.Part 1 Quality of care for patients with inflammatory bowel disease Background and Aims:Clinical practice guidelines for management of inflammatory bowel disease are means of bringing the science of evidence-based medicine into clinical practice with the goal of improving patient care.Our aim to this study was to investigate the quality of care of IBD in a hospital based cohort of patients from eastern China according to the current practice guidelines.Methods:A retrospective review was conducted,involving 177 patients with IBD admitted to Sir Run Run Shaw Hospital,College of Medicine,Zhejiang University between June 2000 and June 2006.Data regarding demographic,clinical characteristics and medical therapy including use of oral aminosalisylates,topical therapy,coricosteroid agents,immunomodulatory agents(such as azathioprine)at the time of admission and outpatient clinic visit were analysed.Results:A total of 177 eligible patients were evaluated in this study,including 71 patients with Crohn's disease and 106 with ulcerative colitis.All were of Chinese Han population,and had active disease at baseline.27.8%of patients who had severe inflammatory bowel disease had not received oral or intravenous steroid therapy. Topical therapy was only used in 31(54.4%)patients with active distal or left-sided ulcerative colitis.Among the 51 patients in whom thiopurine were indicated,only 10 (19.6%)patients were receiving immunomodulatory agents,and half of the 8 patients who received azathiopurine were suboptimal dosed without an attempt to increase dosage.Conclusion:There is room to improve the quality of care for IBD patients.There is inadequate use of topical mesalazine,failure to use corticosteroids in some severe patients,and most strikingly,the limited use of immunomodulatory agents. Part 2 Thiopurine methyitransferase genotype distribution in Chinese Han patients with inflammatory bowel diseaseAim:To determine the genotype distribution of thiopurine S-methyltransferase (TPMT)polymorphisms to evaluate the possible risk of the toxicity of azathioprine (AZA)in IBD patients of Chinese Han population.Methods:189 IBD patients(87 with CD,102 with UC)and 273 healthy Chinese Han volunteers were included,and allele specific polymerase chain reaction or PCR-restriction fragment length polymorphisms(PCR-RFLP)assays were used for TPMT genotyping.The subjects were genotyped for four TPMT single nucleotide polymorphisms(SNPs)as TPMT*2, TPMT*3A,TPMT*3B and TPMT*3C.Results:1,5 and 8 TPMT*1/*3C heterozygotes were found in CD,UC and healthy volunteers group,respectively. TPMT*2,TPMT*3A,TPMT*3B were not detected in the current study.The allele frequency of TPMT*3C in IBD group was 1.59%,which was similar to that of healthy volunteer group(1.47%).The allele frequency of TPMT*3C in UC group (2.45%)was higher than that in CD group(0.57%),though it did not reach statistically significant difference(p=0.146).Conclusions:The study suggested that TPMT*3C was the most prevalent mutant allele in Chinese Han population.Allelic frequency of TPMT*3C amongst patients with inflammatory bowel disease was similar to the distribution normally found in healthy Chinese Han population,but lower than that in Caucasians,suggesting that thiopurine therapy may be safer in Chinese Han IBD patients.Part 3 Pharmacogenetic association with adverse effects to thiopurine therapy in Chinese Han IBD patientsAim:To investigate the association of TPMT polymorphisms with the occurrence of adverse events during azathioprine therapy in Chinese Han IBD patients.Methods: Forty-three azathioprine treated IBD patients were assessed for four TPMT single nucleotide polymorphisms(SNPs)as TPMT*2,TPMT*3A,TPMT*3B and TPMT*3C and for adverse events.The relation between polymorphisms and adverse events was evaluated.Furthermore,we sequenced the exons of TPMT gene in patients who suffered AZA-related side effects.Results:Of all azathioprine treated patients,4 experienced myelotoxicity and 1 experienced hepatotoxieity.Of these 5 patients,no TPMT*2,TPMT*3A,TPMT*3B or TPMT*3C polymorphisms were detected.After direct sequencing of exons of TPMT gene,we found one synonymous single-nucleotide polymorphism(TPMT* 1S)in 3 patients,but which don't alter the encoded amino acid.Conclusions:Single-nueleotide polymorphisms of TPMT could't explain the azathioprine related adverse events in our study.Since the four TPMT single nucleotide polymorphisms were not detected in these patients,the possible association should be investigated further.Part 4 Thiopurine methyltransferase:early experience of use in clinical practiceAim:To measure the thiopurine methyltransferase(TPMT)activity of patients with inflammatory bowel disease,and investigate the correlation of TPMT activity with thiopurine intolerance.Methods:Thirteen patients with IBD on long term azathioprine therapy were involved.Erythroeyte TPMT activity was measured using reverse phase high performance liquid chromatography(HPLC)and correlated with azathioprine intolerance.Results:TPMT activity range of these 13 patients was 7.2-28.8 U/ml pRBC(mean 15.7±6.1 U/ml pRBC).Among the 5 patients who suffered from myelotoxicity,3 suffered in the early stage of AZA therapy.Of these 3 patients,TPMT level were lower than that of patients without myelotoxieity,which reached statistical significance(9.3±2.1 U/ml pRBC vs 18.0±6.2 U/ml pRBC; p=0.046).One patient who had higher TPMT activity(28.8 U/ml pRBC)suffered from hepatotoxieity during AZA therapy.3 of the 5 patients who had myelotoxicity tolerated AZA therapy after receiving a lower dose(50mg/d).Patients who achieved clinical response had significantly lower TPMT activity(13.7±3.5 U/ml pRBC vs 22.0±5.5 U/ml pRBC;p=0.009).Conlusions:The study suggested that TPMT activity associate with AZA-related toxicity.Measurement of TPMT activity is helpful to reduce the risk of AZA related toxicity,predict the therapy efficacy,and can be used to optimize the initial dose of AZA in IBD patients.In the four-stage study,we found that immulomodulatory drugs were inadequately used in IBD patients.Uncertainty regarding the risk for neutropenia and lack of predictive assays deters some physicians from using AZA at effective doses and longer treatment with it.In the study of TPMT polymorphisms,we found that single-nucleotide polymorphisms of TPMT could't explain the azathioprine related adverse events in our study,however,TPMT activity is helpful to reduce the risk of AZA related toxicity,predict the therapy efficacy,and may also be useful for determining the initial dose.