Search

Assessment of thiopurine S-methyltransferase (TPMT) status is required before commencing thiopurine treatment to reduce the potential for adverse drug reactions. Our laboratory has provided a natio...

The common marmoset (Callithrix jacchus) is a New World monkey that is increasingly used in pharmacological and toxicological studies. Thiopurine S-methyltransferase (TPMT) plays roles in the metabolism of widely used anticancer and anti-inflammatory drugs. Here, we report the isolation and molecular characterization of marmoset TPMT cDNA, which was found to contain an open-reading frame of 245 amino acids that was approximately 92% identical to its human ortholog. Marmoset TPMT was phylogenetically closer to other primate orthologs than to its pig, dog, rabbit, or rodent orthologs. Among the five marmoset tissue types analyzed, marmoset TPMT mRNA was most abundant in kidney and liver, just as human TPMT is. These results suggest that marmoset and human TPMT are similar at the molecular level.

Up to 1/5 of patients with wildtype thiopurine- S -methyltransferase (TPMT) activity prescribed azathioprine (AZA) or mercaptopurine (MP) demonstrate a skewed drug metabolism in which MP is preferentially methylated to yield methylmercaptopurine (MeMP). This is known as thiopurine hypermethylation and is associated with drug toxicity and treatment non-response. Co-prescription of allopurinol with low dose AZA/MP (25–33%) circumvents this phenotype and leads to a dramatic reduction in methylated metabolites; however, the biochemical mechanism remains unclear. Using intact and lysate red cell models we propose a novel pathway of allopurinol mediated TPMT inhibition, through the production of thioxanthine (TX, 2-hydroxymercaptopurine). In red blood cells pre-incubated with 250 μM MP for 2 h prior to the addition of 250 μM TX or an equivalent volume of Earle's balanced salt solution, there was a significant reduction in the concentration of MeMP detected at 4 h and 6 h in cells exposed to TX (4 h, 1.68, p = 0.0005, t -test). TX acts as a direct TPMT inhibitor with an apparent Ki of 0.329 mM. In addition we have confirmed that the mechanism is relevant to in vivo metabolism by demonstrating raised urinary TX levels in patients receiving combination therapy. We conclude that the formation of TX in patients receiving combination therapy with AZA/MP and allopurinol, likely explains the significant reduction of methylated metabolites due to direct TPMT inhibition.

Background and Aims Thiopurine S-methyltransferase (TPMT) is responsible for inactivation of thiopurine drugs which are commonly used in leukemia, organ transplantation and autoimmune diseases. The gene encoding TPMT is polymorphic, and both phenotyping and genotyping studies have shown ethnic variations in gene sequence and enzyme activity worldwide. The aim of this study is to identify the most common genetic polymorphisms of TPMT in healthy Jordanian volunteers and patients with rheumatoid arthritis (RA). Methods A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was used to identify the frequency of TPMT ( *2, *3A, *3B, and *3C ) polymorphisms in 250 healthy Jordanian volunteers and 110 RA patients. Results Only four healthy subjects (1.6%) and one RA patient (0.9%) with variant alleles were identified in this study. Two healthy subjects had the TPMT*3A allele and the other two had the TPMT*3B allele, whereas the one RA patient had the TPMT*3A allele. No homozygous polymorphisms were detected and all genotypes detected were heterozygous ( *1/*3A ) ( *1/*3B ). None of the subjects had TPMT*2 or TPMT*3C variant alleles. Conclusions Mutant alleles identified in this study have a low frequency. TPMT ( *3A and *3B ) were the only detected heterozygous alleles. No homozygous variant allele was detected. Further studies are necessary to identify other variant alleles that might uniquely occur in Jordanians.

Thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) plays a pivotal role in thiopurine treatment outcomes. However, little has been known about its intracellular regulation. Here, we describe the effect of fluctuations in physiological levels of S-adenosyl-L-methionine (SAM) and related metabolites on TPMT activity levels in cell lines and erythrocytes from healthy donors. We determined higher TPMT activity in wild-type TPMT*1/*1 individuals with high SAM concentrations (n=96) compared to the low SAM level group (n=19; P

Aim Azathioprine, 6-Mercaptopurine and 6-Thioguanine are commonly used in the management of acute leukemia, autoimmune diseases and as the immunosuppressants in organ transplantation. These drugs belong to the class of thiopurine which can inhibit DNA synthesis and cell proliferation of fast growing lymphocytes by suppressing de novo purine synthesis. Thioguanine nucleotides (TGNs) are the active metabolites in which accumulation of high level cytotoxic TGNs can lead to myelosuppression. It has been demonstrated that Thiopurine S-methyltransferase (TPMT) is the key enzyme responsible for catalytic conversion of TGNs to non-cytotoxic methyl-thioguanine. The TPMT gene is polymorphic and TPMT protein variants with reduced enzyme activity are associated with inefficient removal of TGNs. In this study, we aim to investigate the allele frequency of the most common TPMT variants (TPMT*2 and *3C) in Hong Kong population. Methods Genomic DNA from whole blood was extracted. The presence of TPMT*2 and *3C mutant alleles were determined using Amplification-Refractory Mutation System (ARMS) PCR method. Beta-2-microglobulin was used as the internal control. All mutant alleles were confirmed by Sanger’s sequencing. Hong Kong healthy individuals (n = 300) were included in this study. Rare TPMT alleles were not determined. Results TPMT*1/3C genotype was detected in 9 out of 300 healthy subjects while TPMT*2 allele was absent. The allele frequency of TPMT*3C in Hong Kong population is 1.5%. Conclusions This study provides the first analysis of the allele frequency of TPMT variants in Hong Kong population and our finding is in accordance with the reported frequency range in Chinese population. TPMT*3C is the most common TPMT variant in Hong Kong subjects. The establishment of TPMT genotyping assay for patient requires thiopurine therapy will provide additional information to guide the reduction of drug dose to minimize the risk of myelosuppression.

The prevalence of thiopurine S-methyltransferase (TPMT) polymorphism and its association with clinical and hematological toxicities was retrospectively analyzed in 71 Indian children with acute lymphoblastic leukemia (ALL). Only heterozygous TPMT alleles were observed (10%, 7/71) with relative frequencies being *1/*3C (4.2%), *1/*2 (4.2%) and *1/*3A (1.4%). The median 6-mercaptopurine dose administered during the maintenance therapy was 31% lower among patients with heterozygous TPMT alleles versus the rest (32.1 mg/m 2 /day and 46.2 mg/m 2 /day, p = 0.005), though the myelosuppression and toxicities were similar in both the groups. Identification of TPMT genotype appears to be important in making the ALL treatment more effective and less toxic.

6-mercaptopurine, a key drug for the treatment of acute lymphoblastic leukaemia in children, is a prodrug metabolized into 6-thioguanine (6-TGN) which are the active compounds and into methylated metabolites, primary by thiopurine S-methyltransferase enzyme (TPMT). This enzyme displays important inter subject variability linked to a genetic polymorphism: when treated with standard doses of thiopurine, TPMT-deficient and heterozygous patients are at great risk for developing severe and potentially life-threatening toxicity (hematopoietic, hepatic, mucositis...) but show a better survival rate while patients with high TPMT activity (wild type) present lower peripheral red blood cells 6-TGN concentrations and a higher risk of leukemia relapse. Genotyping remains crucial before 6-MP administration at diagnosis to identify patients with homozygous mutant TPMT genotype and therefore prevent severe and life-threatening toxicity, and to individualize therapy according to TMPT genotype. Follow-up of ALL treatment should preferentially be based on repeated determinations of intracellular active metabolites (6-thioguanine nucleotides) and methylated metabolites in addition to haematological surveillance.

A novel human thiopurine S-methyltransferase (TPMT) variant allele, (319 TG, 107TyrAsp, *27), was identified in a Thai renal transplantation recipient with reduced erythrocyte TPMT activity. The TPMT*27 variant allozyme showed a striking decrease in both immunoreactive protein level and enzyme activity after transient expression in a mammalian cell line. We set out to explore the mechanism(s) responsible for decreased expression of this novel variant of an important drug-metabolizing enzyme. We observed accelerated degradation of TPMT*27 protein in a rabbit reticulocyte lysate. TPMT*27 degradation was slowed by proteasome inhibition and involved chaperone proteins-similar to observations with regard to the degradation of the common TPMT*3A variant allozyme. TPMT*27 aggresome formation was also observed in transfected mammalian cells after proteasome inhibition. Inhibition of autophagy also decreased TPMT*27 degradation. Finally, structural analysis and molecular dynamics simulation indicated that TPMT*27 was less stable than was the wild type TPMT allozyme. In summary, TPMT*27 serves to illustrate the potential importance of protein degradation - both proteasome and autophagy-mediated degradation - for the pharmacogenetic effects of nonsynonymous SNPs.

Six-mercaptopurine (6-MP) is a pro-drug widely used in treatment of various diseases, including acute lymphoblastic leukaemia (ALL). Side-effects of thiopurine therapy have been correlated with thiopurine methyltransferase (TPMT) activity. We propose a novel TPMT-mediated mechanism of S-adenosylmethionine (SAM)-specific effects on 6-mercaptopurine (6-MP) induced cytotoxicity in a model cell line for acute lymphoblastic leukemia (MOLT). Our results show that exogenous SAM (10-50microM) rescues cells from the toxic effects of 6-MP (5microM) by delaying the onset of apoptosis. We prove that the extent of methylthioinosine monophosphate (MeTIMP) induced inhibition of de novo purine synthesis (DNPS) determines the concentrations of intracellular ATP, and consequently SAM, which acts as a positive modulator of TPMT activity. This leads to a greater conversion of 6-MP to inactive 6-methylmercaptopurine, and thus lower availability of thioinosine monophosphate for the biotransformation to cytotoxic thioguanine nucleotides (TGNs) and MeTIMP. We further show that the addition of exogenous SAM to 6-MP treated cells maintains intracellular SAM levels, TPMT activity and protein levels, all of which are diminished in cells incubated with 6-MP. Since TPMT mRNA levels remained unaltered, the effect of SAM appears to be restricted to protein stabilisation rather than an increase of TPMT expression. We thus propose that SAM reverses the extent of 6-MP cytotoxicity, by acting as a TPMT-stabilizing factor. This study provides new insights into the pharmacogenetics of thiopurine drugs. Identification of SAM as critical modulator of TPMT activity and consequently thiopurine toxicity may set novel grounds for the rationalization of thiopurine therapy.

Thiopurine S-methyltransferase (TPMT) metabolizes cytotoxic thiopurine drugs used in the treatment of leukemia and inflammatory bowel disease. TPMT is a major pharmacogenomic target with 23 alleles identified to date. Several of these alleles cause rapid protein degradation and/or aggregation, making it extremely difficult to study the structural impact of the TPMT polymorphisms experimentally. We, therefore, have performed multiple molecular dynamics simulations of the four most common alleles [TPMT*2 (A80P), *3A (A154T/Y240C), *3B (A154T) and *3C (Y240C)] to investigate the molecular mechanism of TPMT inactivation at an atomic level. The A80P polymorphism in TPMT*2 disrupts helix alpha3 bordering the active site, which breaks several salt-bridge interactions and opens up a large cleft in the protein. The A154T polymorphism is located within the co-substrate binding site. The larger threonine alters the packing of substrate-binding residues (P68, L69, Y166), increasing the solvent exposure of the polymorphic site. This packing rearrangement may account for the complete lack of activity in the A154T mutant. The Y240C polymorphism is located in beta-strand 9, distant from the active site. Side-chain contacts between residue 240 and helix alpha8 are lost in TPMT*3C. Residues 154 and 240 in TPMT*3A are connected through a hydrogen-bonding network. The dual polymorphisms result in a flattened, slightly distorted protein structure and an increase in the thiopurine-binding site solvent accessibility. The two variants that undergo the most rapid degradation in vivo, TPMT*2 and *3A, are also the most deformed in the simulations.

A novel gene delivery system termed artificial viral particles (AVPs) containing a plasmid coding for a recombinant fusion protein of enhanced green fluorescent protein (EGFP) with thiopurine-S-methyltransferase (TPMT) was designed for transfection of selected cell lines to establish stable clones which express recombinant EGFP-TPMT protein for further in vitro investigation of toxic effect of thiopurine drugs. Various AVPs based on a complex of the cationic polymer polyethylenimine (PEI) and anionic liposomes were formulated and transfection conditions were adapted in order to transfect the human Jurkat, HepG2 and HEK 293 cell lines. An adequate transfection rate was achieved with AVP containing branched low molecular weight PEI at a PEI:DNA charge ratio of 4.5:1 and liposomes composed of DOPS, DLPE, cholesterol and an activated N-glutaryl-DOPE membrane anchor. Stably transfected clones were successfully established and expression of recombinant EGFP-TPMT in homogeneous cell populations was demonstrated by flow cytometry, fluorescence microscopy and immunoblotting. The level of the expressed protein in stable clones was highest in HEK 293, followed by HepG2 and Jurkat. The enzymatic activity of the TPMT moiety was demonstrated by decreased sensitivity to 6-thioguanine and increased sensitivity to 6-mercaptopurine in HEK 293 cells expressing EGFP-TPMT. Formulation of AVP as transfection vector succeeded in establishing human cell lines stably expressing EGFP-TPMT, thereby proving a successful delivery system and providing an initial step to enable investigation of the role of the clinically important drug metabolizing enzyme TPMT.

Thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) is the key enzyme in the metabolism of thiopurine drugs. Determination of TPMT activity has been used for the individualization of thiopurine dose. We developed HPLC-UV assay for the determination of TPMT activity in human erythrocytes using 6-mercaptopurine as a substrate. Various extraction and chromatographic conditions were compared. In-house developed extraction with acetonitrile provided the lowest limit of quantification. TPMT activity was determined in 99 previously genotyped healthy Estonians. TPMT activity was expressed as the formation of 6-methylmercaptopurine ng/ml/h and normalized either to haemoglobin, haematocrit, erythrocyte count or protein content. The receiver-operating characteristic curve analysis revealed similar accuracy values for TPMT activity in predicting heterozygous and wild type individuals for each method of calculation. In healthy Estonians, TPMT activity varied from 21.5 to 129.6 ng/ml/h. For heterozygous individuals (n = 18), TPMT activity was 48.1 +/- 11.7 ng/ml/h. Wild type individuals (n = 81) revealed significantly higher TPMT activity 79.3 +/- 20.7 ng/ml/h (P0.001). This sensitive HPLC assay for quantitative determination of TPMT activity could easily be used in clinical settings. Under constant experimental conditions for haemolysate preparation no normalization is required.

Background Thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs. Several mutations in the TPMT gene have been identified which correlate with a low activity phenotype. The molecular basis for TPMT deficiency is not well defined in minority Chinese. We investigated differences in the activity of TPMT and the frequencies of mutant TPMT alleles in 4 ethnic groups of Chinese. Method The frequency of 4 common TPMT mutant alleles, TPMT*2, TPMT*3A, TPMT*3B and TPMT*3C, were determined in healthy subjects from Han (n = 312), Jing (n = 103), Yao (n = 126) and Uygur Chinese (n = 160) by allele-specific PCR and PCR-restriction RFLP analysis. TPMT activity in erythrocytes was determined by HPLC. Results There was no significant difference in the mean TPMT activity between all ethnic groups studied and no subject with TPMT deficiency was found in all populations studied. TPMT*3C was found in 2.2% of Han and 1.9% of Jing Chinese. TPMT*2, TPMT*3B and TPMT*3A alleles were not detected in any of the Han or Jing Chinese tested. In contrast, 3.7% of Uygur Chinese had TPMT*3C and TPMT*3A alleles. Neither allele was detected in Yao Chinese. The overall frequencies of variant TPMT allele in Uygur were higher than in Han or Jing Chinese. However, neither the overall frequency of mutant TPMT alleles nor the genotype frequencies were significantly different between Han, Jing, Yao and Uygur Chinese. Conclusions The TPMT*3C was the most prevalent allele in Han, Jing and Uygur Chinese, while TPMT*3A is a rare allele in Uygur Chinese who belong to Caucasian. Ethnicity may be an important factor affecting the variability in response to thiopurine chemotherapy.

BACKGROUND & AIMS: The use of azathioprine (AZA) in inflammatory bowel disease (IBD) patients is limited by toxicity, which occurs in up to 20% of treated patients. Mutations in the thiopurine S-methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) genes have been associated with the occurrence of AZA-related toxicity. The aim of our study was to determine the relative contribution of ITPA and TPMT mutations to the development of toxicity induced by AZA treatment in IBD patients. METHODS: ITPA(94C>A, IVS2+21A>C) and TPMT (238G>C, 460G>A, and 719A>G) genotypes were assessed in 262 IBD patients (159 females, 103 males; 67 patients with ulcerative colitis, 195 patients with Crohn's disease) treated with AZA and were correlated with the development of leukopenia and hepatotoxicity. RESULTS: Leukopenia (leukocyte count, A and TPMT alleles were significantly higher in the leukopenic population compared with patients without leukopenia (16.7% and 5.4%, respectively, for ITPA 94C>A, and 20.8% and 4%, respectively, for TPMT). Moreover, the ITPA 94C>A and TPMT mutations predicted leukopenia: ITPA 94C>A odds ratio, 3.504; 95% confidence interval, 1.119-10.971 (P = .046); TPMT odds ratio, 6.316; 95% confidence interval, 2.141-18.634 (P = .004). Neither TPMT nor ITPA genotype predicted hepatotoxicity. CONCLUSIONS: ITPA 94C>A and TPMT polymorphisms are associated with AZA-related leukopenia in IBD patients

Human thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs. TPMT is genetically polymorphic and is associated with large interindividual variations in thiopurine drug toxicity and therapeutic efficacy. During routine genotyping of patients with Crohn's disease, one novel missense mutation, 365A>C (TPMT*19, Lys(122)Thr), and a recently described missense mutation, 488G>A (TPMT*16, Arg(163)His), were identified in a Caucasian and a Moroccan patient, respectively. Using a heterologous yeast expression system, kinetic parameters (K(m) and V(max)) of the two variants with respect to 6-thioguanine S-methylation were determined and compared with those obtained with the wild-type enzyme. The Lys(122)Thr exchange did not significantly decrease the intrinsic clearance value (V(max)/K(m)) of the variant enzyme. In contrast, the Arg(163)His substitution significantly decreased the intrinsic clearance value by three-fold. The Arg(163) is located in a highly conserved region of the human TPMT protein and, as such, the Arg(163)His substitution is expected to result in a marked reduction of enzyme activity, as confirmed by the in vitro data. Phenotyping by measurement of red blood cell TPMT activity indicated that the patient heterozygous for the Lys(122)Thr mutation had normal TPMT activity, whereas the patient heterozygous for the Arg(163)His mutation was an intermediate methylator, which demonstrated a positive correlation between TPMT phenotyping and the in vitro data. The identification of a novel non-functional allele of the TPMT gene improves our knowledge of the genetic basis of interindividual variability in TPMT activity. These data further enhance the efficiency of genotyping methods to predict patients at risk of an inadequate response to thiopurine therapy.

Background: Thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs that are commonly used to treat a wide range of conditions. It is now well established that interpatient variation in sensitivity to these drugs is due to point mutations in the TPMT gene. The mutant alleles TPMT*2 (238G>C), TPMT*3A (460G>A, 719A>G), TPMT*3B (460G>A), and TPMT*3C (719A>G) account for 80–95% of TPMT deficiency observed in Caucasian populations. In this paper, we describe a novel, multiplex, allele-specific polymerase chain reaction (PCR) method that detects the 238G>C, 460G>A, and 719A>G mutations, allowing for the simultaneous identification of TPMT*2 and TPMT*3 alleles. The assay is internally controlled, robust, and does not require the use of restriction endonucleases. Therefore, the assay is not prone to erroneous readings due to incomplete restriction digestion, as documented for existing PCR restriction fragment length polymorphism (RFLP) assays of TPMT.

A non-extraction high-performance liquid chromatographic (HPLC) method has been developed for the determination of 6-methylthioguanine (6-MTG), as part of the determination of thiopurine S -methyltransferase activity (TPMT) in erythrocytes. Erythrocyte lysate is added to a glass vial containing substrates and incubation buffer, which is then sealed for the rest of the analysis. Enzyme incubation, sample preparation, and analysis are then undertaken without further sample-handling steps. The need for a solvent extraction step has been overcome by heating the incubate to 85 °C to stop the enzyme reaction. The heat inactivation step precipitates protein which upon centrifugation forms a thin film in the bottom of the glass vial enabling the supernatant to be injected directly onto the HPLC system. The assay shows excellent precision and recovery with a within-batch imprecision giving a co-efficient of variation of 2.9% (mean=41.5 nmol 6-MTG/g Hb/h, n =10) and 5.1% (mean=12.6 nmol 6-MTG/g Hb/h, n =10). The between-batch imprecision gives a co-efficient of variation of 8.2% (mean=11.1 nmol 6-MTG/g Hb/h, n =11) and 7.3% (mean=41.0 nmol 6-MTG/g Hb/h, n =16). Determination of the TPMT activity in 120 people shows a range of enzyme activity of 11.3–63.8 nmol 6-MTG/g Hb/h with a mean and median activity of 34.8 and 34.2 nmol 6-MTG/g Hb/h, respectively. TPMT is increasingly used in clinical practice to ensure optimisation of treatment with thioguanine drugs. This direct HPLC method minimises sample-handling, reduces inherent imprecision, the possibility of laboratory error and with the potential for further automation, makes it ideal for use in a regional referral laboratory.

Human thiopurine S-methyltransferase (TPMT) is an enzyme responsible for the detoxification of widely used thiopurine drugs such as azathioprine (Aza). Its activity is inversely related to the risk of developing severe hematopoietic toxicity in certain patients treated with standard doses of thiopurines. DNA samples from four leucopenic patients treated with Aza were screened by PCR-SSCP analysis for mutations in the 10 exons of the TPMT gene. Four missense mutations comprising two novel mutations, A83T (TPMT*13, Glu(28)Val) and C374T (TPMT*12, Ser(125)Leu), and two previously described mutations, G430C (TPMT*10, Gly(144)Arg) and T681G (TPMT*7, His(227)Gln) were identified. Using a recombinant yeast expression system, kinetic parameters (K(m) and V(max)) of 6-thioguanine S-methylation of the four TPMT variants were determined and compared to those obtained with wild-type TPMT. This functional analysis suggests that these rare allelic variants are defective TPMT alleles. The His(227)Gln variant retained only 10% of the intrinsic clearance value (V(max)/K(m) ratio) of the wild-type enzyme. The Ser(125)Leu and Gly(144)Arg variants were associated with a significant decrease in intrinsic clearance values, retaining about 30% of the wild-type enzyme, whereas the Glu(28)Val variant produced a more modest decrease (57% of the wild-type enzyme). The data suggest that the sporadic contribution of the rare Glu(28)Val, Ser(125)Leu, Gly(144)Arg, and His(227)Gln variants may account for the occurrence of altered metabolism of TPMT substrates. These findings improve our knowledge of the genetic basis of interindividual variability in TPMT activity and would enhance the efficiency of genotyping methods to predict patients at risk of inadequate responses to thiopurine therapy.

Background/Aims : Toxicity and efficacy of azathioprine is governed partly by the activity of thiopurine methyltransferase (TPMT). Azathioprine has been used for many years, with corticosteroids or alone, for the treatment of autoimmune hepatitis (AIH) but no studies of TPMT phenotype and genotype in relation to response to the drug in AIH have been published. Methods : Erythrocyte TPMT activities were measured by a radioincorporation assay in 72 consecutive outpatients with AIH, 53 of whom were genotyped for the commonest defective alleles in Europeans ( TPMT*3A , *3B and *3C ) by restriction fragment length polymorphism analysis. Results : TPMT activities were significantly lower in patients intolerant of azathioprine (group I, n =15) than in those who sustained remission on azathioprine alone (group II, n =28; P =0.003) and those who tolerated azathioprine but continued to require corticosteroids (group III, n =29; P P =0.034). Ten patients with defective alleles (all heterozygotes) had significantly lower TPMT activities ( P =0.002). However, in 25% there was discordance between phenotype and/or genotype and response to azathioprine. Conclusions : TPMT phenotyping or genotyping may be advisable before institution of azathioprine therapy in AIH but neither approach invariably predicts response to the drug.

Background: Thiopurine S-methyltransferase (TPMT), which exhibits autosomal codominant polymorphism, plays an important role in the metabolism of thiopurine drugs such as mercaptopurine, thioguanine and azathioprine. Decreased activity of TPMT is associated with severe hematopoietic toxicity after administration of standard doses of these drugs. Methods: We developed a specific high-performance liquid chromatographic (HPLC) assay for measuring 6-methylmercaptopurine (6-MMP) formed from 6-mercaptopurine (6-MP) in red blood cells (RBC) lysates. The assay was used to study the distribution of TPMT activities in 44 healthy Japanese subjects with different TPMT genotypes. Results: The TPMT activities in the subjects ranged from 17.9 to 37.1 pmol/h/mgHb. The TPMT activity of one subject with TPMT*1/*3C (17.9 pmol/h/mgHb) was 40% lower than the mean value of TPMT activities in 43 subjects with TPMT*1/*1 (29.6±4.3 pmol/h/mgHb). Conclusions: This sensitive and reproducible HPLC assay for determination of TPMT activity in RBC clinical studies has been designed to optimize dosage regimens of thiopurine drugs.

Background & Aims: Myelosuppression in patients with Crohn's disease (CD) treated with azathioprine has been attributed to low activity of thiopurine S -methyltransferase (TPMT). Allelic variants of the TPMT gene responsible for changes in the enzyme activity have been characterized. We investigated the distribution of mutant alleles associated with TPMT deficiency in patients with CD and myelosuppression during azathioprine/6-mercaptopurine therapy. Methods: Forty-one patients with CD were included. They developed leukopenia or thrombocytopenia during azathioprine or 6-mercaptopurine treatment. Polymerase chain reaction–based methods were used to search for mutations associated with TPMT deficiency. Results: Four patients (10%) had 2 mutant alleles associated with TPMT deficiency, 7 (17%) had 1 mutant allele, and 30 (73%) had no known TPMT mutation. The delay between administration of the drug and occurrence of bone marrow toxicity was less than 1.5 months in the 4 patients with 2 mutant alleles, and ranged from 1 to 18 months in patients with 1 mutant allele and from 0.5 to 87 months in patients with normal genotype. Conclusions: Twenty-seven percent of patients with CD and myelosuppression during azathioprine therapy had mutant alleles of the TPMT gene associated with enzyme deficiency. Myelosuppression is more often caused by other factors. Continued monitoring of blood cell counts remains mandatory in patients treated with azathioprine. GASTROENTEROLOGY 2000;118:1025-1030

The risk of azathioprine-induced myelosuppression can be predicted by detecting patients with intermediate or low thiopurine methyltransferase (TPMT) activity. Population studies have shown that 89% of whites have high TPMT activity, 11% have intermediate TPMT activity, and 0.3% have low TPMT activity. Three specific mutations in the TPMT gene that cause decreased TPMT activity have recently been identified. Patients homozygous for the TPMT mutations have low TPMT activity, and patients heterozygous for TPMT mutations have intermediate TPMT activity. This has led to the development of a technique for TPMT genotype analysis that will identify patients at risk of azathioprine-induced myelosuppression. We report a case of a patient with bullous pemphigoid who experienced azathioprine-induced myelosuppression and who was later found to be homozygous for TPMT mutant alleles. Using the cost of treatment required for this patient and the estimated population prevalence of TPMT mutations, we examined the cost impact of screening for TPMT mutations in all patients being considered for azathioprine therapy. We calculated that screening is cost-neutral assuming patients homozygous for TPMT mutations experience myelosuppression, and that it is cost-beneficial assuming patients heterozygous for TPMT mutations also experience myelosuppression while receiving azathioprine. Screening patients for TPMT mutations will reduce the risk of azathioprine-induced myelosuppression, and our study suggests that it may also be a cost-attractive strategy. (J Am Acad Dermatol 2000;42:628-32.)

A new liquid–liquid extraction is described for thiopurine methyl transferase (TPMT, EC 2.1.1.67) activity determination: the use of a pH 9.5 NH 4 Cl buffer solution, before adding the solvent mixture, allows more rapid extraction, avoiding a centrifugation step, and reduces the global cost of analysis. After the extraction step, 6-methylmercaptopurine, synthesised during the enzymatic reaction, is determined by a liquid chromatographic assay. Analytical performance of the assay was tested on spiked erythrocyte lysates. The linear concentration range was 5–250 ng ml −1 ( r ≥0.997, slope=1.497, intercept=−0.367). The recoveries were 82.8, 89.9 and 82.2% for 75, 125 and 225 ng ml −1 , respectively. The coefficients of variation were ≤6.1% for within-day assay ( n =6) and ≤9.5% for between-day assay precision ( n =6; 14 days). TPMT activity was determined in a French adult Caucasian population ( n =70). The results ranged from 7.8 to 27.8 nmol h −1 ml −1 packed red blood cells and the frequency distribution histogram is similar to that previously published.

The term “pharmacogenetics” was coined in the 1950s to describe inherited differences in the disposition and effects of medications and other xenobiotics. Pharmacogenetic studies have focused largely on inherited variations in drug metabolism and polymorphisms of the genes encoding drug-metabolizing enzymes, to elucidate the molecular basis of these variations (Nebert 1997). Although genetic polymorphism in drug metabolism remains a primary focus, inherited differences in drug targets (e.g., receptors, genetic subsets of diseases, etc.) represent another facet of pharmacogenetics that can be of equal importance in determining the effects of some medications.

Thiopurine methyltransferase (TPMT) is an important enzyme in the metabolism of 6-mercaptopurine (6MP), which is used in the treatment of acute lymphoblastic leukemia (ALL). TPMT catalyzes the formation of methylthioinosine monophosphate (MetIMP), which is cytotoxic for cultured cell lines, and it plays a role in detoxification of 6MP. Population studies show a genetic polymorphism for TPMT with both high and low activity alleles. About 1 of 300 subjects is homozygous for the low activity. The function TPMT plays in detoxification or therapeutic efficacy of 6MP in vivo is not clear. In this article the genetic polymorphism of TPMT is reviewed and the contribution of TPMT to the cytotoxic action, or detoxification, of 6MP in children with ALL is discussed. Induction of TPMT activity has been described during the treatment for ALL. We performed a pilot study on the influence of high-dose 6MP infusions (1300 mg/m2 in 24 h) on TPMT activity of peripheral blood mononuclear cells (pMNC) of eleven patients with ALL. The TPMT activities were in, or, above the normal range. There was no statistically significant difference between the TPMT activities before and after the 6MP infusions. MetIMP levels in pMNC increased during successive courses. This might be explained by TPMT induction, but other explanations are plausible as well. Twenty five percent of the TPMT assays failed, because less than the necessary 5.10(6) pMNC could be isolated from the blood of leukopenic patients. Red blood cells can not be used for TPMT measurements, since transfusions are frequently required during the treatment with 6MP infusions. Therefore, the influence of high-dose 6MP infusions on TPMT activity can only be investigated further when a TPMT assay which requires less pMNC has been developed.

Azathioprine can cause severe myelosuppression. The inherited activity of the enzyme thiopurine methyltransferase has been recently recognised as a major factor in the susceptibility to myelosuppression. Thiopurine methyltransferase deficiency occurs at a frequency of one in 300 and is associated with profound myelosuppression after a short course of azathioprine. Very low thiopurine methyltransferase activity represents the TPMTL/TPMTL genotype, and can be detected before therapy with azathioprine is started. We describe the first documented case of azathioprine-induced severe myelosuppression due to thiopurine methyltransferase deficiency in autoimmune liver disease. The azathioprine dose was low (1 mg/kg) and pancytopenia occurred after 56 days therapy. It would be advisable to measure thiopurine methyltransferase activity before patients with autoimmune hepatitis are exposed to azathioprine.

6-mercaptopurine (6-MP) can be inactivated by S-methylation, which is catalysed by thiopurine methyltransferase (TPMT). An alternative metabolic route leads to the formation of cytotoxic 6-thioguanine nucleotides (6-TGN). To investigate whether these two pathways compete with each other to affect the therapeutic response to 6-MP, 6-TGN concentrations and TPMT enzymatic activity were measured in erythrocytes (RBC) from 95 children on long-term 6-MP therapy for lymphoblastic leukaemia (ALL). RBC TPMT activities were also measured in 130 control children and 104 long-term survivors of ALL no longer on treatment. The 95 children on 6-MP showed wide interindividual differences in RBC 6-TGN concentrations at the full protocol dose of 75 mg/m2, and RBC 6-TGN concentrations correlated negatively with RBC TPMT activity. Children with 6-TGN concentrations below the group median had higher TPMT activities and a higher subsequent relapse rate. 50 of the 104 long-term survivors had been treated with "gentle" low-dose protocols, and this subgroup contained an excess of children with lower TPMT activities compared with normal controls. These results indicate that genetically determined TPMT activity may be a substantial regulator of the cytotoxic effect of 6-MP, an effect which in turn could be important in influencing the outcome of therapy for childhood ALL.