Jarle Aarbakke

Human thiopurine methyltransferase pharmacogenetics: Gene sequence polymorphisms

Thiopurine methyltransferase (TPMT) catalyzes the S‐methylation of thiopurine drugs. TPMT activity is regulated by a common genetic polymorphism that is associated with large individual variations in thiopurine toxicity and efficacy. We previously cloned the functional gene for human TPMT and reported a common variant allele for low enzyme activity, TPMT*3A, that contains point mutations at cDNA nucleotides 460 and 719. In the present study, we set out to determine the number, types, and frequencies of TPMT variant alleles associated with low enzyme activity in clinical laboratory samples in the United States and to compare those results with data obtained from two different ethnic groups. We identified a total of six different variant alleles for low TPMT activity in the 283 clinical laboratory samples studied. The most common variant was *3A; the second most frequent variant allele, *3C, contained only the nucleotide 719 polymorphism; and four other variant alleles were detected. TPMT*3A also appeared to be the most common variant allele in a Norwegian white population sample, but it was not found in a population sample of Korean children. However, *3C was present in samples from the Korean children, as was a novel allele, *6. Characterization of variant alleles for low TPMT enzyme activity will help make it possible to assess the potential clinical utility of deoxyribonucleic acid‐based diagnostic tests for determining TPMT genotype.

Interethnic difference in thiopurine methyltransferase activity

A number of metabolic pathways are subject to both genetic polymorphism and interethnic differences. A catabolic pathway of 6‐mercaptopurine, red blood cell (RBC) thiopurine methyltransferase (TPMT) activity showed genetic polymorphism in Caucasians, but variation according to ethnicity has not been studied. We investigated if red blood cell thiopurine methyltransferase was subject to interethnic variation in a Saami (Lappish; n = 36) and a Caucasian population (n = 50). The Saami population sample had 29% higher thiopurine methyltransferase activity, 17.0 ±3.3 U/ml red blood cell compared with the Caucasian population sample, 13.1 ± 2.9 U/ml red blood cell (p < 0.001). Probit plots and frequency distribution histograms supported bimodality consistent with genetic polymorphism in both study populations. Differences in chronic diseases, drug consumption, age, or gender could not explain the interethnic difference in red blood cell thiopurine methyltransferase activity. The higher red blood cell thiopurine methyltransferase activity in the Saami population group indicates that these subjects may require higher dosages of thiopurine drugs than Caucasians.

Inhibition of human thiopurine methyltransferase by furosemide, bendroflumethiazide and trichlormethiazide

AbstractResults: Incubation in vitro of human recombinant and erythrocyte (RBC) thiopurine methyl transferase (TPMT) with furosemide, bendroflumethiazide and trichlormethiazide demonstrated inhibition of both enzyme preparations, with IC50 values of 170 μM, 360 μM and 1 mM, respectively. Kinetic studies revealed that the inhibition was mixed or non-competitive with regard both to the thiopurine substrate 6-mercaptopurine (6-MP) and the methyl donor S-adenosyl-L-methionine. Conclusion: Since S-methylation is a major pathway in the metabolism of thiopurines, our data point to the possibility of a clinically significant diuretic-thiopurine interaction in patients treated simultaneously with these drugs.

Thiopurine methyltransferase activity in a Korean population sample of children

Thiopurine methyltransferase (TPMT) is a cytoplasmic enzyme that catalyzes the S‐methylation of the cytotoxic drugs 6‐mercaptopurine and azathioprine. Red blood cell (RBC) TPMT activity is subject to genetic polymorphism, and we have previously demonstrated an interethnic difference in TPMT activity. To investigate whether there was a race‐related difference in RBC TPMT activity, TPMT was measured in a Korean population sample of 309 healthy children. Mean TPMT activity in healthy Korean children was 12.4 ± 2.4 units/ml RBC, which is similar to the earlier reported TPMT activities in white populations. In contrast to the bimodal or trimodal frequency distributions of RBC TPMT activity in most other population samples, the frequency distribution histogram, the probit plot, and the Shapiro‐Wilk test supported a normal distribution of TPMT activity in this Korean population sample of healthy children. Mean RBC TPMT activity showed a tendency to decrease with age, but it was not statistically significant. No gender‐related difference in RBC TPMT activity was found.

Clinical Pharmacokinetics of Phenylbutazone

More than 25 years after Phenylbutazone was introduced as a non-steroidal anti-inflammatory agent, basic knowledge is still accumulating on its pharmacokinetics in man. Phenylbutazone is almost completely absorbed after oral administration. A large fraction of the drug in plasma is bound to proteins, and the drug has a small volume of dislribution. Phenylbutazone is eliminated by metabolism, only 1% being excreted unchanged in the urine. Approximately 10% of a single dose of Phenylbutazone is excreted in bile as metabolites. About 60% of the urinary metabolites have been identified. A novel type of drug metabolite in man, the C-glucuronide, is formed by direct coupling of the pyrazolidine ring of Phenylbutazone to glucuronic acid via a C-C bond. Phenylbutazone is oxidised in a phenyl ring or in the side chain to hydroxylated metabolites, which may undergo subsequent O-glucuronidation. After a single dose, C-glucuronidation seems to be the dominant reaction, while oxidation becomes increasingly important after repeated administration. Due to different pharmacokinetic properties of the metabolites, the C-glucuronides are detected in highest concentrations in the urine, while the pharmacologically active compounds oxyphenbutazone and y-hydroxyphenbutazone predominate in plasma.The biological (elimination) half-life of Phenylbutazone in man is long, with a mean of about 70 hours, and exhibits large interindividual and intraindividual variation. The interindividual variation is largely due to genetic factors. The intraindividual variation is dose and time dependent. In an individual there may be several critical dose levels where a change in the elimination kinetics takes place.Since there is no correlation between the plasma level and the clinical or toxic effects of Phenylbutazone, there is at present no need for routine monitoring of plasma concentrations of the drug.

Detection of one single mutation predicts thiopurine S-methyltransferase activity in a population of Saami in northern Norway

Thiopurine S‐methyltransferase (TPMT) activity exhibits genetic polymorphism. The purpose of this investigation was to identify TPMT mutant alleles in the Saami population as a basis of developing genotyping tests for prediction of TPMT activity. The most predominant allele in Saamis (n = 194) was the TPMT*3C allele (A719G mutation) representing 92% of the mutant alleles, with an estimated allelic frequency of 3.3%. The most frequent allele in Caucasians (n = 66) living in the same geographic area was the TPMT*3A (A719G and G460A mutations) representing 91% of the mutant alleles, with an estimated allelic frequency of 3.4%. A test for one mutation, A719G, may prospectively identify more than 90% of the Saami individuals who require reduction in thiopurine dose to avoid hematopoietic toxicity. In a Norwegian population, comprising both the major Caucasian population and a minor Saami population, the same genotyping tests (eg, tests for the A719G and G460A mutations) may be used.

Identification of factors regulating thiopurine methyltransferase activity in a Norwegian population

SummaryRed blood cell (RBC) thiopurine methyltransferase (TPMT), an inactivating pathway of 6-mercaptopurine, is controlled by genetic polymorphism and is subject to ethnic variation. RBC TPMT is a good predictor of clinical outcome in children with acute lymphoblastic leukemia. RBC TPMT activity was determined in 226 patients, 176 of them living in northern Norway (of which 123 were Saami (Lapps)). Demographic variables, use of drugs and presence of chronic diseases were evaluated as possible predictors of RBC TPMT activity by a multiple regression model.Men had higher RBC TPMT activity compared to women. Living in the northernmost county of Norway was associated with increased RBC TPMT activity irrespective of ethnicity. The use of diuretics was associated with increased RBC TPMT activity.The gender difference in RBC TPMT activity may indicate a need to treat male subjects more aggressively with thiopurine drugs compared to female subjects.

Methotrexate measurements in plasma: comparison of enzyme multiplied immunoassay technique, TDx fluorescence polarization immunoassay, and high pressure liquid chromatography.

One hundred nine patient plasma samples were examined for methotrexate (MTX) levels by enzyme multiplied immunoassay technique (EMIT), fluorescence polarization immunoassay (TDx), and high pressure liquid chromatography (HPLC). EMIT analysis was performed twice within a time span of 18 months. All three methods measure MTX with a high degree of specificity, sensitivity, and precision. There was no evidence of decay of MTX concentrations in samples stored at -20 degrees C for 1.5 years. EMIT, TDx, and HPLC are adequate methods for MTX quantification in the clinical laboratory.

High-dose 7-hydroxymethotrexate: Acute toxicity and lethality in a rat model

To elucidate mechanisms for methotrexate (MTX)-induced renal and hepatic toxicity, we investigated the acute effects of bolus plus continuous infusion of up to 0.4 g/kg 7-hydroxymethotrexate (7-OH-MTX) in the rat. We demonstrate for the first time in any species the occurrence of acute lethal toxicity within a few hours after 7-OH-MTX administration. Serum concentrations of 7-OH-MTX measured at the time of death were 1.4 mM (mean), about one-half of those achieved in some patients after infusion of high-dose MTX (HD-MTX) in the clinic. The data suggest an approximate LD50 (the dose lethal to 50% of the study population) of 0.3 g/kg and a steep dose/ lethality curve for 7-OH-MTX. Moreover, acute renal and hepatic toxicity occurred as evidenced by severe morphological findings and increased serum levels of creatinine and liver transaminases. In all rats subjected to continuous infusion of 7-OH-MTX, yellow microscopic precipitations were apparent in the kidney tubules. Crystallization was also seen in bile ducts of the liver in some of the rats. These results further support that the formation of 7-OH-MTX is disadvantageous and that reported attempts to prevent its formation during MTX treatment are warranted.

The interaction of 6-mercaptopurine (6-MP) and methotrexate (MTX).

The antimetabolites 6-mercaptopurine (6-MP) and methotrexate (MTX) are the cornerstones in the maintenance treatment of childrens acute lymphoblastic leukemia (ALL). The biochemical mechanisms underlying the increased therapeutic efficacy of the combination of these drugs have not yet been elucidated. However, both drugs interact with important pathways. such as purine de novo synthesis (PDNS), purine salvage, and methylation reactions. A review of the mechanistic aspects of the interactions between 6-MP and MTX is given.