HPLC/Tandem Ion Trap Mass Detector Methods for Determination of Inosine Monophosphate Dehydrogenase (IMPDH) and Thiopurine Methyltransferase (TPMT)

The efficiency of Mycophenolate mofetil (MMF) and Azathioprine (AZA) as immunosuppressive agents depends on the activity of 2 enzymes, inosine monophosphate dehydrogenase (IMPDH) and thiopurine methyltransferase (TPMT) respectively. We present preliminary evaluation of nonradioactive methods that apply HPLC with ion-trap mass detection to measure the activities of IMPDH in peripheral blood mononuclear cells (PBMC) and TPMT in the erythrocytes (RBC). We found IMPDH activity of 0.9 ± 0.2 nmol/hour/10⁶ PBMC and TPMT activity of 19.9 ± 4.7 nmol/hour/ml RBC in healthy subjects. These methods, following its further validation, could be useful for monitoring the activity in a clinical and experimental setting.     

Simultaneous determination of azathioprine and 6-mercaptopurine by high-performance liquid chromatography

A specific, sensitive, single-step solid-phase extraction and reversed-phase high-performance liquid chromatographic method for the simultaneous determination of plasma 6-mercaptopurine and azathioprine concentrations is reported. Following solid-phase extraction, analytes are separated on a C₁₈ column with mobile phase consisting of 0.8% acetonitrile in 1 mM triethylamine, pH 3.2, run on a gradient system. Quantitation limits were 5 ng/ml and 2 ng/ml for azathioprine and 6-mercaptopurine, respectively. Peak heights correlated linearly to known extracted standards for 6-mercaptopurine and azathioprine (r = 0.999) over a range of 2–200 ng/ml. No chromatographic interferences were detected.     

High-performance liquid chromatographic assay for the measurement of azathioprine in human serum samples

A quantitative, highly sensitive HPLC-based method for the direct measurement of azathioprine is described, introducing a newly synthesized 9-methyl derivative of this immunosuppressant as internal standard in combination with isocartic HPLC and UV-absorbance measurement at 285 nm. Analysis was performed on a RP18 select B column with acetonitrile-0.01 M potassium phosphate buffer (12:88, v/v) at pH 2.3 as mobile phase. Results of precision analysis from serum samples spiked with 3.125, 12.5, and 25 ng azathioprine, respectively, were (mean±S.D.): 3.148±0.259 ng (8.22%), 12.594±0.571 ng (4.53%), and 25.016±0.658 ng (2.63%) with C.V. values in parentheses for n=5. The accuracy of the assay ranged from −7.6 to 0.7% (expressed as % bias) tested on five consecutive days. The limit of quantification was at 2.5±0.256 ng (C.V. 10.25%), thus allowing drug monitoring in long-term patients. The method can also be used to evaluate individual pharmacokinetic parameters of a single patient, as well as for drug monitoring of a cohort of patients who suffer from azathioprine-induced symptoms of toxicity. An example of the pharmacokinetic behaviour in an individual is given in this paper.     

激光照射、免疫抑制剂及二者配合应用对大鼠全胰十二指肠移植的影响Ⅱ移植胰解剖组织学变化

以Ｗｉｓｔａｒ糖尿病模型大鼠作为受体,进行同佐间全胰十二指肠移植。用激光照射、免疫抑制剂及二者配合应用进行抗移植排斥反应试验,于术后７天及出现排斥反应相对,进行病理解剖与病理组织学观察,结果表明,日剂量为３９．７２４５Ｊ／ｃｍ￣２的激光照射,可推迟排斥反应的发生时间、降低排斥反应的发生程度;激光照射与８—５—３ｍｇ／ｋｇ／ｄａｙ的硫唑嘌呤（Ａｚａ）配合,上述作用显著,且与环孢霉素Ａ（ＣｓＡ）的效果接近。     

Effects of 4-nitro- and 4-sulpho-7-substituted benzofurazans on nucleic acid and protein synthesis of a malignant fibrosarcoma cell line in combination with mild hyperthermia

The inhibitory effects of substituted nitro- and sulphobenzofurazans on DNA, RNA and protein synthesis were compared in a new malignant fibrosarcoma cell line at 37°C and 41°C. The effects of these drugs with and without mild hyperthermia were evaluated by determining the % inhibition of incorporation of ³H-precursors into DNA, RNA and protein. None of the sulphobenzofurazan derivatives (Sbf) were effective inhibitors of nucleic acid and protein synthesis at 37°C nor did they enhance the inhibitory effect of hyperthermia alone. The nitrobenzofurazan derivatives (Nbf) at concentrations 10% that used for the Sbf derivatives strongly inhibited biopolymer synthesis in a dose related manner; 4-chloro-7-nitrobenzofurazan (Nbf-Cl) being the most potent inhibitor. Hyperthermia amplified the effect of all the Nbf compounds tested on RNA and protein synthesis but did not further affect DNA synthesis. This selective synergistic effect was most pronounced when the lowest concentrations of Nbf compounds were studied. The synergism however, did not follow a uniform pattern. 6-Mercaptopurine and 6-(1-methyl-4-nitro-5-imidazoyl)thiopurine (Azathioprine) (100 μM) had marginal effects on nucleic acid and protein synthesis when the cells were exposed to these two thiopurines for 1 h at both 37°C and 41°C and they had only a moderate inhibitory effect after exposure for 15 h.     

The Clinical Impact of Thiopurine Methyltransferase Polymorphisms on Thiopurine Treatment

Acute lymphoblastic leukaemia (ALL) is the most common malignancy of childhood. Although current treatment results in long term survival in over 70% of cases there is evidence that as many as 50% could have been cured using a less complex regimen with a lower incidence of long term side effects. In previous studies it has been found that thiopurines given as part of continuing therapy are key agents in preventing relapse. However, optimal administration during continuing therapy is often not achieved. Variation in the level of thiopurine methyltransferase (TPMT) activity appears to be a major molecular determinant of the extent of thiopurine metabolism. TPMT activity shows a trimodal distribution pattern. A lack of activity is found in approximately one in 300 Caucasians; approximately 11% have intermediate activity and the remaining 89% high activity. Congenital loss of activity is associated with grossly elevated levels of active drug and profound myelosuppression on exposure to thiopurines. This loss of activity has been attributed to single nucleotide polymorphisms (SNPs) within the TPMT gene. The frequency of SNPs is related to ethnicity, with the most common in Caucasians being TPMT*3A which is characterized by a G to A transition at position 460 with a substitution of alanine for tyrosine at amino acid 154 (A154Y) and a transition of A to G at nucleotide 719 resulting in a change of tyrosine to cysteine at position 240 (Y240C). Polymorphisms have also been identified within the 5′ flanking promoter region of the TPMT gene due to a variable number of tandem repeats (VNTR*3–*8). An overview of the polymorphisms identified to date, their implication on the metabolism of the thiopurine drugs and therapeutic importance will be discussed.     

Allele Frequency of Inosine Triphosphate Pyrophosphatase Gene Polymorphisms in a Japanese Population

The enzyme inosine triphosphate pyrophosphatase (ITPase) catalyses the pyrophosphohydrolysis of ITP to IMP. ITPase deficiency is a clinically benign autosomal recessive condition characterised by the abnormal accumulation of ITP in erythrocytes. A deficiency of ITPase may predict adverse reactions to therapy with the thiopurine drug 6‐mercaptopurine and its prodrug azathioprine. In this study, we examine the frequencies of ITPA polymorphisms in 100 healthy Japanese individuals. The allele frequency of the 94C > A variant in the Japanese sample was 0.135 (Caucasian allele frequency 0.06). The IV2 + 21A > C polymorphism was not found in Japanese (Caucasian allele frequency 0.130). Allele frequencies of the 138G > A, 561G > A and 708G > A polymorphisms were 0.57, 0.18 and 0.06 respectively in the Japanese population, and with the exception of the 138G > A polymorphism, similar to allele frequencies in Caucasians.     

Genetic Determinants of the Pre‐ and Post‐Azathioprine Therapy Thiopurine Methyltransferase Activity Phenotype

Thiopurine drug therapy has been reported to lead to a variable increase in red cell TPMT activity that may alter effective dose and therapeutic outcome. The aim of this study was to correlate Variable Number Tandem Repeat (VNTR) in the promoter region of the TPMT gene with induction of red cell TPMT activity in patients treated with azathioprine (AZA). In 58 patients, TPMT activity measured at 3 months was not significantly induced on average above pre‐therapy levels. Individual patients showed variation in TPMT activity pre‐ and post‐AZA therapy, however changes in TPMT activity were not predicted by VNTR configuration. In conclusion, TPMT promoter VNTRs are unlikely to play a significant role in changes in TPMT activity in response to AZA therapy.     

Mutation in the ITPA Gene Predicts Intolerance to Azathioprine

Inosine triphosphate pyrophosphatase (ITPase) deficiency occurs with polymorphic frequencies in Caucasians and results in the benign accumulation of the inosine nucleotide ITP. In 62 patients treated with azathioprine for inflammatory bowel disease, the ITPA 94C > A deficiency‐associated allele was significantly associated with adverse drug reactions (OR 4.2, 95% CI 1.6–11.5, p = 0.0034). Significant associations were found for flu‐like symptoms (OR 4.7, 95% CI 1.2–18.1, p = 0.0308), rash (OR 10.3, 95% CI 4.7–62.9, p = 0.0213) and pancreatitis (OR 6.2,CI 1.1–32.6, p = 0.0485). Polymorphism in the ITPA gene thus predicts AZA intolerance. Alternative immunosuppressive drugs, particularly 6‐thioguanine, should be considered for AZA‐intolerant patients with ITPase deficiency.     

激光照射、免疫抑制剂及二者配合应用对大鼠全胰十二指肠移植的影响Ⅲ、对大鼠全胰十二指肠移植物功能存活的影响

用健康雄性Ｗｉｓｔａｒ大鼠制备糖尿病模型,以成功的模型大鼠作为受体,健康雄性大鼠为供体,行全胰十二指肠移植术。用移植成功大鼠进行大剂量激光照射、免疫抑制剂及二者配合抗移植排斥反应的实验,于术后隔天监测血糖、尿糖的变化,于术后７天及出现血糖、尿糖持续升高时采取移植胰脏,进行病理组织学观察,以了解排斥反应的发生与程度。结果表明,日剂量为３９．７２４５Ｊ／ｃｍ￣２的激光照射,可推迟排斥反应的发生时间、降低排斥反应的发生程度及延长大鼠全胰十二脂肠移植物的存活时间。３９．７２４５Ｊ／ｃｍ￣２的激光照射与８－５－３ｍｇ／ｋｇ／ｄａｙ的硫唑嘌呤（Ａｚａ）配合,上述作用更为显著,超过各单一使用的效果,且与环隐霉素（Ａ（ＣｓＡ）的作用效果接近。并证实２５－２０－１５ｍｇ／ｋｇ／ｄａｙ的ＣｓＡ及１０－８－５ｍｇ／ｋｇ／ｄａｙ的Ａｚａ抗全胰十二指肠移植抗排斥反应效果明显。