巯嘌呤甲基转移酶基因多态性联合硫鸟嘌呤核苷酸血药浓度监测在硫唑嘌呤治疗炎症性肠病治疗中的临床应用

目的：探讨巯嘌呤甲基转移酶（TPMT）基因多态性联合红细胞中硫鸟嘌呤核苷酸（6-TGNs）血药浓度监测在硫唑嘌呤治疗炎症性肠病中的临床应用,为实施个体化治疗方案提供参考。方法：选取临床确诊为炎症性肠病的患者,采用PCR扩增、焦磷酸基因测序法技术检测其TPMT基因第7外显子G460A和第10外显子A719G的2个多态性位点;应用高效液相色谱法测定患者红细胞（RBC）中6-TGNs浓度。结果：15例患者TPMT＊3基因均为野生型,未发现突变;首次检测的6-TGNs浓度为 147.31～875.26 pmol/L（8×10^8）RBC,服用相同剂量的患者个体差异较大。结论：TPMT基因多态性检测有助于预测硫唑嘌呤治疗炎症性肠病的骨髓毒副作用,6 TGNs浓度测定有助于药物剂量的调整,两者联合应用可为接受硫唑嘌呤治疗的炎症性肠病患者的个体化治疗提供参考依据。     

HPLC测定炎症性肠病患者红细胞中6-硫鸟嘌呤核苷酸及6-甲巯基嘌呤的浓度

摘要：目的:建立HPLC法同时测定炎症性肠病（IBD）患者红细胞中硫唑嘌呤代谢物6-硫鸟嘌呤核苷酸（6-TGNs）及6-甲巯基嘌呤（6-MMP）浓度,为硫唑嘌呤（AZA）个体化治疗提供依据。方法:红细胞经高氯酸沉淀,6-TGNs在酸性条件下加热水解生成6-硫鸟嘌呤（6-TG）,6-MMP水解生成4-氨基-5-（甲硫基）羰基咪唑（AMTCI）,以5-溴尿嘧啶（5-BU）为内标,采用HPLC法测定其含量。色谱柱:Kromasil 100-5-C18（150 mm×4.6 mm,5μm）,流动相:乙腈-20 mmol·L1磷酸二氢钾溶液（6∶94）,流速:0.8 ml·min-1,柱温:30℃;6-TG、6-MMP及5-BU检测波长分别为340,303,280 nm。10例IBD患者,口服AZA 50～100 mg·d-11个月以上,检测其6-TGNs及6-MMP浓度,焦磷酸测序检测TPMT基因型。结果:6-TG在8～800pmol﹒（8×108）-1RBC浓度范围内线性关系良好（r=0.994 0）;低、中、高3个浓度的绝对回收率为63.24%～69.09%（n=5）。AMTCI在250～16 000 pmol·（8×108）-1RBC浓度范围内线性关系良好（r=0.997 8）;低、中、高3个浓度的绝对回收率为85.17%～92.28%（n=5）。10例患者TPMT均为野生型。1例患者6-TGNs浓度偏高,白细胞减少;2例患者6-MMP浓度较高,碱性磷酸酶轻度升高。结论:本方法专属性、精密度及稳定性较好,灵敏度高,适用于同时测定炎症性肠病患者红细胞中6-TGNs及6-MMP的浓度。     

嘌呤类药物代谢酶和代谢物与药物反应关联及影响因素研究进展

临床上嘌呤类药物作为免疫抑制剂广泛应用于治疗实体器官移植、自身免疫性疾病和炎症性肠病、儿童急性淋巴细胞性白血病等疾病,但该类药物体内处置个体间差异大,治疗窗窄,且所致不良反应（如肾毒性和肝毒性）发生率较高。本文综述近年来嘌呤类药物代谢酶（TPMT、NDPK、ITPA、GST、XO/XDH、HGPRT、IMPDH和NUDT15）和代谢物（6-MMPR、6-TGNs、6-TGTP）对该类药物临床疗效和不良反应的影响,以及患者服药剂量和疗程、性别、年龄、体质量、遗传等多因素对代谢酶和代谢物影响的研究进展。研究表明该类药物临床疗效和不良反应与其体内代谢酶活性和代谢物浓度密切相关,但结论并不完全一致,因此有待大规模临床研究进一步验证以上关联性及影响因素。     

硫嘌呤甲基转移酶遗传多态性检测在6-MP个体化治疗中的意义

目的：研究硫嘌呤甲基转移酶活性和基因型检测对6-巯基嘌呤（6-MP）的个体化治疗的意义。方法-94例口服6-MP维持治疗的白血病患者，19例口服6-Mp后出现血液系统危象的为观察组，同时服用6-MP没有出现血液系统损害的75患者为对照组，采用高效液相色谱法测定硫嘌呤甲基转移酶（TPMT）活性，等位基因特异性PCR和限制性片段长度多态性（RFLP）的方法检测TPMT＊2、TPMT＊3A、TPMT＊3B和TPMT＊3C的等位基因频率。通过临床白细胞记数和骨髓象检查及临床表现判定巯嘌呤的疗效和不良反应。结果：观察组TPMT活性是（6．1±2．1）U·mL^-1 pRBCs显著低于对照组（15．3±2．3）U·mL^-1 pRBCs，而观察组的基因突变率10．7％高于对照组1．2％。结论：TPMT活性低下和／或TPMT基因突变型个体，在服用标准剂量的巯嘌呤后，发生不良反应的危险较高，应及时发现此类患者并积极调整剂量实现安全有效的治疗。     

环孢素、硝苯地平等5种肾移植受者常用药物对人体红细胞硫嘌呤甲基转移酶活性的影响

目的:研究肾移植受者中常与硫唑嘌呤(AZA)同时服用的药物如环孢素、泼尼松的活性代谢物氢化可的松、硝苯地平、卡托普利以及别嘌呤醇对硫嘌呤甲基转移酶(TPMT)活性的影响。方法:应用高效液相色谱法(HPLC)测定健康受试者红细胞TPMT活性,其中中等活性和正常活性受试者各8例,计算药物各浓度对应的平均TPMT活性抑制率及有抑制作用药物的平均IC_(50)值。结果:环孢素、氢化可的松、卡托普利以及别嘌呤醇对TPMT活性抑制作用较弱。硝苯地平能明显抑制TPMT的活性,中等活性组的平均IC_(50)值为(24±17)μg/mL,正常活性组的平均,IC_(50)值为(12±10)μg/mL。结论:硝苯地平能明显抑制TPMT活性,当硝笨地平与嘌呤类药物同时服用时,应警惕可能发生不良相互作用。     

侧柏炭黄酮类成分群对干酵母致血热复合出血模型大鼠的止血作用研究

目的：观察侧柏炭黄酮类成分群对干酵母致血热复合出血模型大鼠的止血作用。方法：对照组和模型组灌胃CMC-Na,以云南白药为阳性药,阳性组、剔除前组、剔除后组、成分群低剂量组和成分群高剂量组分别灌胃相应的治疗药物,连续给药8 d,于第7天复制出血热复合出血模型,以活化部分凝血活酶时间（APTT）、血浆凝血酶时间（TT）、凝血酶原时间（PT）、纤维蛋白原的量（FIB）、血小板计数（PLT）、血小板分布宽度（PDW）、平均血小板体积（MPV）、ADP诱导的血小板聚集率、血栓素（TXB₂）、6-酮前列腺素（6-keto-PGF_1α）、红细胞计数（RBC）、红细胞压积（HCT）、血红蛋白（HGB）的量、全血黏度、环氧合酶2（COX-2）等评价指标为导向,考察侧柏炭乙酸乙酯部位成分群的止血作用。结果：与对照组比较,模型组大鼠APTT,TT显著延长（P<0.01,P<0.05）,PT显著缩短（P<0.05）,FIB的量明显增加（P<0.01）;PLT,PDW,MPV显著增加（P<0.01,P<0.05）,ADP诱导的血小板聚集率显著降低（P<0.05）,6-keto-PGF_1α显著降低（P<0.01）,TXB₂显著升高（P<0.01）;各切变率下全血黏度明显增加（P<0.01）,RBC,PLT和HGB明显升高（P<0.01）,C0X-2的表达量显著升高（P<0.01）。黄酮类成分群可明显缩短APTT,TT（P<0.01,P<0.05）,明显降低FIB的量（P<0.05）;明显降低PDW和PLT（P<0.01,P<0.05）,显著提高ADP诱导的血小板聚集率（P<0.05）,6-keto-PGF_1α显著升高（P<0.01,P<0.05）,TXB₂显著降低（P<0.01）;明显降低模型大鼠RBC、HCT、HGB及全血低切黏度（P<0.01,P<0.05）,C0X-2的表达量显著降低（P<0.01）,改善肺组织病理性出血现象。剔除后组对模型大鼠各环节的作用较弱。结论：侧柏炭黄酮类成分群主要通过改善内源性凝血功能及促进血小板聚集功能、降低全血低切黏度等发挥其凉血止血作用,是侧柏炭止血有效部位。     

多药耐药基因MDR1C1236T多态性与炎症性肠病患者的活性代谢物6-TGNs的相关性

目的考察多药耐药基因MDR1的单核苷酸多态性(SNP)C1236T与炎症性肠病(IBD)患者服用硫嘌呤类药物后的活性代谢产物6-硫鸟苷酸(6-TGNs)的相关性。方法有105名患者纳入研究,收集全血提取DNA,采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)法,进行MDR1C1236T基因型分析,采用HPLC方法检测红细胞内6-TGNs的浓度,并统计药物不良反应情况。卡方检验分析相关性。结果相同剂量下,6-TGNs浓度在1236CT/TT型携带者比CC型携带者高(P=0.048);同时,1236CT/TT型携带者服药后发生不良反应的风险显著高于1236CC型携带者(P=0.045)。结论 MDR1 1236 C>T突变与高6-TGNs浓度及高不良反应发生率密切相关。     

CYP1A2、CYP2D6和CYP2C19基因多态性与氯氮平及其代谢物血药浓度的相关性研究

目的：研究CYP1A2、CYP2D6和CYP2C19基因多态性与精神分裂症患者氯氮平（clozapine,CLZ）及其活性代谢物去甲氯氮平（N-desmethy clozapine,N-CLZ）血药浓度的相关性。方法：纳入156例经CLZ单药治疗1个月以上的精神分裂症患者,采集清晨服药前空腹血,采用液相色谱-串联质谱（LC-MS/MS）法测定CLZ及N-CLZ稳态谷浓度。通过Axiom基因芯片分析技术检测CYP1A2（*1C、*1F）、CYP2D6（*2、*10）、CYP2C19（*2、*3）等6个SNP位点的基因型,比较不同基因型患者CLZ及其代谢物的浓度剂量比（C/D）及代谢物与CLZ血药浓度比值（C_N-CLZ/C_CLZ）的差异。结果：CYP2D6*10基因多态性与N-CLZ C/D具有相关性（P<0.01）。CYP1A2*1C、CYP2D6（*2、*10）基因多态性与C_N-CLZ/C_CLZ具有相关性（P<0.05,P<0.01,P<0.01）。CYP1A2*1F、CYP2C19*2、CYP2C19*3基因多态性与C/D及C_N-CLZ/C_CLZ无相关性（P>0.05）。结论：CYP1A2*1C和CYP2D6（*2、*10）基因多态性对CLZ代谢存在影响,建议临床在使用CLZ治疗前检测患者CYP1A2*1C和CYP2D6（*2、*10）基因型,为CLZ个体化治疗提供参考。     

百事乐胶囊对抑郁模型大鼠海马GR/p-GR/SGK1表达的影响

目的：探讨百事乐胶囊对慢性应激抑郁模型大鼠学习记忆的保护作用。方法：将SD大鼠随机分为6组,即空白对照组、抑郁模型组、氟西汀组及百事乐胶囊高、中、低（2.88,1.44,0.72 g·kg^-1）剂量组,采用慢性温和不可预见性应激加孤养的方法建立抑郁模型,造模同时灌胃给药,连续21 d Morris水迷宫测定对大鼠行为学的影响,放射免疫法观察大鼠血浆CORT含量的变化,Western-blotting观察大鼠海马GR,p-GR,SGK1蛋白表达的变化。结果：与空白对照组比较,模型组大鼠逃避潜伏期（EL）、目标象限潜伏时间显著增长（P<0.01）;穿越目标象限的次数显著降低（P<0.05）;CORT含量显著增高（P<0.01）,海马GR、p-GR的表达下调（P<0.01）,SGK1的表达增加（P<0.01）。与模型组比较,百事乐胶囊高剂量能缩短模型大鼠的EL（P<0.05）,并能缩短目标象限潜伏时间（P<0.05）,增加目标象限的穿越次数（P<0.05）;同时高、中剂量组大鼠血浆CORT的含量显著下降（P<0.05或P<0.01）;高、中剂量组海马GR、p-GR的表达显著增加（P<0.05或P<0.01）,高剂量组SGK1的表达显著降低（P<0.05）。结论：百事乐胶囊可能通过GR/p-GR/SGK1信号级联增加应激抑郁模型大鼠海马学习记忆的功能,进而实现抗抑郁的作用。     

亚洲炎症性肠病患者中NUDT15 R139C基因多态性与巯嘌呤类药物诱导的白细胞减少的Meta分析

目的：系统评价亚洲炎症性肠病患者中NUDT15 R139C基因多态性与巯嘌呤类药物诱导的白细胞减少的关系。方法：计算机检索PubMed、Embase、Web of Science、CNKI和万方等数据库中NUDT15 R139C基因多态性与巯嘌呤诱导的白细胞减少的相关研究,检索时间均为建库至2018年6月。由2位评价员独立筛选文献、提取资料并评价纳入研究的质量,采用Revman 5.3软件进行Meta分析。结果：共纳入8个研究,3 676例炎症性肠病患者,Meta分析结果表明：NUDT15 R139C纯合突变与杂合突变者发生白细胞减少的风险显著高于野生型患者,差异有显著性（[TT&CC：OR=73.24,95% CI（32.81,163.47）,P<0.000 01;TC&CC：OR=5.99,95% CI（4.97,7.22）,P<0.000 01;TT+TC&CC：OR=7.33 95% CI（6.12,8.78）,P<0.000 01]）;此外还发现,NUDT15 R139C纯合突变与杂合突变者发生早期白细胞减少的风险[TT&CC：OR=56.90,95% CI（25.40,127.49）,P<0.000 01;TC&CC：OR=2.20,95% CI（1.42,3.41）,P=0.000 2;TT+TC&CC：OR=6.19,95% CI（2.76,13.85）,P=0.002]、严重白细胞减少的风险[TT&CC：OR=34.32,95% CI（16.74,70.37）,P<0.000 01;TC&CC：OR=2.50,95% CI（1.54,4.05）,P=0.000 2;TT+TC&CC：OR=4.87 95% CI（3.13,7.59）,P<0.000 01]均较野生型患者高,差异有显著性。结论：在炎症性肠病患者中,NUDT15 R139C基因突变患者使用巯嘌呤药物发生白细胞减少、早期白细胞减少、严重白细胞减少的风险显著高于野生型,临床上NUDT 15R139C基因型的测定可对巯嘌呤类药物个体化治疗提供有力的帮助。     

Determination of 6-thioguanine and 6-methylmercaptopurine metabolites in renal transplantation recipients and patients with glomerulonephritis treated with azathioprine

The metabolism of azathioprine (AZA) was studied by monitoring the concentrations of red blood cell (RBC) 6-thioguanine nucleotides (6-TGN) and of 6-methylmercaptopurine metabolites (6-mMP) in 27 renal transplantation recipients and in 10 patient subjects with glomerulonephritis (GN). Concentrations of 6-TGNs and 6-mMP metabolites were measured using high-performance liquid chromatography (HPLC). Six patients from the group of renal transplantation recipients were also administered allopurinol. Median values of RBC 6-TGN and of 6-mMP metabolites concentrations in 21 renal transplantation recipients (without allopurinol) were 122 pmol/8x10(8) RBCs (range, <60-298) and 280 pmol/8x10(8) RBC (range, <150-1330), respectively; there was no correlation between concentrations of 6-TGN and of 6-mMP metabolites. The group of 21 renal transplantation recipients received different AZA doses (100 or 50 mg/d) related to clinical symptoms of AZA intolerance. The median values of 6-TGN concentrations in these subgroups were 131 and 122 pmol/8x10(8) RBCs and were not significantly different. Median values of 6-TGN concentrations in patients given allopurinol were significantly higher, despite AZA dose reduction, compared with the group without allopurinol and were equal to 363 and 122 pmol/8x10(8) RBC, p < 0.004, respectively. No significant differences were found between the concentrations of 6-mMP metabolites in either group. In the group of renal transplantation recipients, a significant correlation between white blood cell (WBC) count and 6-TGN concentration was established (r(s) = -0.59, p < 0.005). In the group of GN patients, the median values of 6-TGN and of 6-mMP metabolites concentrations were 108 pmol/8x10(8) RBCs (range, 0-297) and 420 pmol/8x10(8) RBC (range, 0-1440), respectively. There were no significant correlations between either the WBC count and 6-TGN concentrations or between 6-TGN concentrations and 6-mMP metabolites. We expect the results of our study to provide indications for better individualization of AZA therapy.     

Thiopurine S-methyltransferase phenotype-genotype correlation in hemodialyzed patients

Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme, catalyzing S-methylation of thiopurine drugs. TPMT exhibits autosomal codominant polymorphism. Patients carrying a variant genotype have low TPMT activity, and produce elevated levels of 6-thioguanine nucleotides (6-TGN) in their red blood cells (RBC). 6-TGN accumulation may result in azathioprine (AZA)-induced bone marrow myelosuppression in the course of treatment with the drug in a standard dosage regimen in patients following renal transplantation. In the current study, TPMT activity (phenotype) and genotype were determined in dialyzed patients, qualified for renal transplantation. TPMT activity was measured in RBC after dialysis by HPLC method. Patients were genotyped for TPMT *2, *3A and *3C variant alleles using PCR-RFLP and allele-specific PCR methods. TPMT activity ranged between 12.2 and 45.5 nmol 6-mMP/g Hb/h (median value 30.6). A significant correlation between TPMTphenotype and genotype was noted: the heterozygous patients (11.5%) demonstrated significantly lower mean TPMT activity as compared to the wild homozygotes (17 +/- 3.6 vs. 32.4 +/- 4.8 nmol 6-mMP/g Hb/h, p < 0.0003). No overlap in TPMT activity values between the group of heterozygous (range 12.2-20.6) and wild-type homozygous patients (range 22.7-45.5) was noted. TPMT activity, established after hemodialysis and TPMT genotyping results seem to be convergent in dialyzed patients, so both methods can be used for the identification of patients with lower TPMT activity. Such tests could be helpful in AZA dose individualization, and thus in reducing the risk of myelosuppression during AZA therapy following renal transplantation.     

MDR1 C3435T基因多态性与肾移植患者他克莫司血药浓度关系的Meta分析

目的:系统评价多药耐药基因1(MDR1)C3435T基因多态性与肾移植患者他克莫司(FK506)血药浓度的关系,为器官移植术后免疫抑制剂的精准化治疗提供循证参考。方法:计算机检索数据库Embase、Science Direct、Pubmed、CNKI、Wan fang、Conchrane,Clinicaltrials.gov,检索年限为1990年1月至2016年10月,检索语种为中文和英文,收集有关MDR1C3435T基因多态性与肾移植患者FK506血药浓度关系的研究。对纳入的研究进行资料提取与质量评价。用Cochrane提供Revman 5.3软件进行Meta分析。结果:有8项研究纳入此次Meta分析,共827例患者。Meta分析结果显示,MDR1C3435T基因型中CT型患者在肾移植术后1周[MD=-16.93,95%CI(-27.67,-6.19),P=0.002]、1月[MD=-18.09,95%CI(-26.41,-9.78),P<0.0001]、6月[MD=-15.52,95%CI(-25.18,-5.85),P=0.002]时,血药浓度高于同期CC型患者;TT型患者在肾移植术后1周[MD=-30.76,95%CI(-53.04,-8.48),P=0.007]、1月[MD=-25.92,95%CI(-48.34,-3.51),P=0.02]、3月[MD=-33.77,95%CI(-48.74,-18.80),P <0.00001]、6月[MD=-22.25,95%CI(-32.97,-11.53),P<0.0001]、12月[MD=-22.74,95%CI(-42.76,-2.72),P=0.03]时,血药浓度高于同期CC型患者;TT型患者在肾移植术后3月[MD=-18.81,95%CI(-34.30,-3.33),P=0.002]时,血药浓度高于同期CT型患者。结论:MDR1C3435T基因多态性与FK506血药浓度/剂量存在相关性,且血药浓度的关系是携带者(CT型或者TT型)>非携带者(CC型)。在肾移植术后不同时期内,根据MDR1C3435T基因多态性与FK506血药浓度/剂量存在的相关性,做基因检测可以在短时间内达到有效血药浓度。由于纳入研究数量较少、样本量不大、该结论有待大样本、高质量研究进一步证实。     

Influence of xanthine oxidase on thiopurine metabolism in Crohn's disease

Background  The thiopurines, azathioprine (AZA) and mercaptopurine are extensively used in Crohn's disease (CD). Thiopurine bioactivation can be diverted by either thiopurine methyltransferase (TPMT), or by xanthine oxidase/dehydrogenase (XOD) which forms 6-thiouric acid (6TU).
Aim  To investigate whether chronic inflammation could influence small intestinal XOD activity using urinary excretion of 6TU as a surrogate marker of XOD activity.
Methods  6-Thiouric acid excretion was compared between 32 CD patients and nine dermatology patients (control group), on AZA. Six CD patients were interesting: five with low TPMT activity (one deficient, four intermediate), and one receiving AZA/allopurinol co-therapy.
Results  There was no statistical difference in 6TU excretion between the CD and control group. CD location, severity or surgery did not affect excretion. The TPMT-deficient patient excreted 89% of daily AZA dose as 6TU, but excretion by TPMT carriers was essentially normal. Concurrent 5-aminosalicylic acid therapy increased 6TU excretion significantly (median 32.9%), consistent with inhibiting TPMT. 6TU was undetectable in the patient on AZA/allopurinol co-therapy.
Conclusions  The results refuted our hypothesis, but fitted a model where most of an oral thiopurine dose effectively escapes first-pass metabolism by gut XOD, but is heavily catabolized by TPMT. Bioavailability of thiopurines may be competitively inhibited by dietary purines.     

Pharmacokinetics of 6-thiouric acid and 6-mercaptopurine in renal allograft recipients after oral administration of azathioprine

Summary The immunosuppressive activity of azathioprine (AZA) is unpredictable and depends on the formation of intracellular thiopurine ribonucleotides. However, the quantification of these active thiopurines presents difficult analytical problems. It has recently been postulated that plasma concentrations of 6-thiouric acid (6-TU) and 6-mercaptopurine (6-MP), metabolites of AZA, may provide more readily measurable indices of the pharmacologic activity of AZA. In order to evaluate the utility of 6-TU and 6-MP plasma concentrations in monitoring AZA therapy, we studied their pharmacokinetics in 6 renal transplant patients, and their in vitro immunosuppressive potency in a mixed lymphocyte proliferation assay.A peak plasma 6-TU concentration of 710.7 ng/ml was observed at 3.8 h after oral dosing. Good correlation was observed between the elimination t_1/2 of 6-TU and serum creatinine, and between AUC over 24 h and serum creatinine. However, we did not observe a second peak in plasma 6-TU concentration that could be attributed to the degradation of active AZA metabolites. 6-MP plasma concentrations in the patients were low (mean peak concentration 36.0 ng/ml) and rapidly disappeared within 8 h. In vitro immunosuppressive activity could not be demonstrated for 6-TU over a concentration range of 1.25 ng/ml to 0.25 mg/ml.We conclude that 6-TU is pharmacologically inert and is primarily eliminated by the kidneys. Our findings currently do not support the use of plasma concentrations of 6-TU or 6-MP to monitor AZA therapy. In order to optimize AZA therapy, analytical techniques that are technically feasible and that can directly quantify the active intracellular thiopurines are being explored.     

Assessment of thiopurine methyltransferase and metabolite formation during thiopurine therapy: results from a large Swedish patient population

This study examined thiopurine methyltransferase (TPMT) and the relationship to thioguanine nucleotides (TGN) and methylthioinosine monophosphate (meTIMP) in a large Swedish patient population. The current hypothesis is that the cytotoxic effects of thiopurine drugs are mediated by the incorporation of TGN into DNA. The authors assayed the TPMT activity in red blood cells from 1151 subjects and the concentrations of TGN (n = 602) and meTIMP (n = 593) from patients treated with thiopurine drugs. The TPMT frequency distribution in both adults and children showed some differences from what had been found in unselected general populations. Children had lower median TPMT activity than adults (12.0 versus 12.9 U/mL RBC; P < 0.001). Relative differences in both TGN formation [medians: normal TPMT, 1.3; intermediate TPMT, 3.3; low TPMT, 47.9 pmol/8 x 10(8) RBC per mg azathioprine (AZA); P < 0.001] and meTIMP formation (medians: normal TPMT, 13; intermediate TPMT, 7.3; low TPMT, 0 pmol/8 x 10(8) RBC per mg AZA; P = 0.001) per 1 mg administered drug were noted among the 3 TPMT activity groups. Women formed higher concentrations of both TGN (1.5 versus 1.3 pmol/8 x 10(8) RBC per mg AZA; P = 0.01) and meTIMP (14.4 versus 10.7 pmol/8 x 10(8) RBC per mg AZA; P = 0.01) than men did. There was a significant correlation between the AZA dose and the meTIMP concentrations (r = 0.45; P < 0.001). Furthermore, dose alterations made in subjects with normal TPMT (n = 84) and intermediate TPMT (n = 22) activity resulted in more pronounced increases in TGN concentrations (170 versus 30 pmol/8 x 10(8) RBC; P < 0.001) in intermediate TPMT activity, whereas in normal TPMT activity changes in meTIMP concentrations were more pronounced (1.3 versus 0 nmol/8 x 10(8) RBC; P < 0.001). In normal TPMT activity both metabolites increased in a dose-dependent fashion, whereas in intermediate TPMT activity only TGN concentrations increased. The results of this study demonstrate the dynamic nature of thiopurine metabolism and its importance for thiopurine dosing.     

Long-term outcome of using allopurinol co-therapy as a strategy for overcoming thiopurine hepatotoxicity in treating inflammatory bowel disease

Background  Hepatotoxicity results in the withdrawal of thiopurines drugs, azathioprine (AZA) and mercaptopurine (MP), in up to 10% of patients with inflammatory bowel disease. Our group previously demonstrated that allopurinol with AZA/ciclosporin/steroid 'triple therapy' improved renal graft survival.
Aim  To confirm the hypothesis that allopurinol may alleviate thiopurine hepatotoxicity by similar mechanisms as proposed in our renal study.
Methods  Unselected patients with acute thiopurine hepatotoxicity were offered allopurinol co-therapy with low-dose AZA or MP. The starting AZA/MP dose was determined by thiopurine methyltransferase (TPMT) activity (two patients were intermediate TPMT); then this dose was reduced to 25% for allopurinol co-therapy. Response to treatment was assessed by clinical severity indices, endoscopy and blood tests.
Results  Of 11 patients (three Crohn's disease, eight ulcerative colitis) treated, nine (82%) remain in long-term remission (median 42 months) with normal liver tests. One patient also successfully bypassed flu-like symptoms. Two stopped: one nausea, one abnormal liver function (steatosis on biopsy). Leucopenia occurred in two cases and resolved with minor dose reductions.
Conclusions  Allopurinol co-therapy with low-dose AZA/MP can alleviate thiopurine hepatotoxicity. It appears safe and effective for long-term use, but requires monitoring for myelotoxicity. Assessing the TPMT activity helps tailor the AZA/MP doses.     

The impact of thiopurine s-methyltransferase polymorphism on azathioprine-induced myelotoxicity in renal transplant recipients

Thiopurine S-methyltransferase (TPMT) is an enzyme that catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathioprine. TPMT activity exhibits an interindividual variability, mainly as a result of genetic polymorphism. Patients with intermediate or deficient TMPT activity are at risk for toxicity after receiving standard doses of thiopurine drugs. It has previously been reported that 3 variant alleles: TPMT*2, *3A, and *3C are responsible for over 95% cases of low enzyme activity. The purpose of this study was to explore the association between these polymorphisms and the occurrence of azathioprine adverse effects in 112 renal transplant recipients undergoing triple immunosuppressive therapy including azathioprine, cyclosporine, and prednisone. TPMT genetic polymorphism was determined using PCR-RFLP and allele-specific PCR methods. Azathioprine dose, leukocyte, erythrocyte, and platelet counts, graft rejection episodes, as well as cyclosporine levels were analyzed throughout the first year after organ transplantation. We found the frequency of leukopenia episodes (WBC < 4.0 x 10(9)/L) significantly higher in heterozygous patients (53.8%) compared with those with TPMT wild-type genotype (23.5%). One patient, who was a compound homozygote (3A/*3C), experienced severe azathioprine-related myelotoxicity each time after receiving the standard drug dose. Our results suggest that polymorphisms in TPMT gene may be responsible for approximately 12.5% of all leukopenia episodes in renal transplant recipients treated with azathioprine. Genotyping for the major TPMT variant alleles may be a valuable tool in preventing AZA toxicity and optimization of immunosuppressive therapy.