Carol L. Szumlanski

Human catechol-O-methyltransferase pharmacogenetics: Description of a functional polymorphism and its potential application to neuropsychiatric disorders

Catechol-O-methyltransferase (COMT) inactivates catecholamines and catechol drugs such as L-DOPA. A common genetic polymorphism in humans is associated with a three-to-four-fold variation in COMT enzyme activity and is also associated with individual variation in COMT thermal instability. We now show that this is due to G-->A transition at codon 158 of the COMT gene that results in a valine to methionine substitution. The two alleles can be identified with a PCR-based restriction fragment length polymorphism analysis using the restriction enzyme Nla III. The identification of a gentic marker associated with significant alterations in enzyme activity will facilitate the analysis of a possible role for the COMT gene in neuropsychiatric conditions in which abnormalities in catecholamine neurotransmission are believed to occur, including mood disorders, schizophrenia, obsessive compulsive disorder, alcohol and substance abuse, and attention deficit hyperactivity disorder. In addition, this polymorphism may have pharmacogenetic significance in that it will help make it possible to identify patients who display altered metabolism of catechol drugs.

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.

Human liver catechol‐O‐methyltransferase pharmacogenetics

Catechol‐O‐methyltransferase activity and thermal stability in the human red blood cell are controlled by a common genetic polymorphism. Approximately 25% to 30% of a randomly selected population sample is homozygous for the traits of low catechol‐O‐methyltransferase activity and thermolabile enzyme in the red blood cell. We tested the hypothesis that the catechol‐O‐methyltransferase genetic polymorphism might also control those same characteristics of the enzyme in an important human drug‐metabolizing organ, the liver. Catechol‐O‐methyltransferase enzyme activity and thermal stability were measured in 99 hepatic biopsy samples obtained during clinically indicated surgery. The frequency distribution of heated/control ratios, a measure of enzyme thermal stability, was bimodal, with 28% of samples included in a subgroup with thermolabile enzyme. There were no sex‐related differences in hepatic catechol‐O‐methyltransferase thermal stability. However, catechol‐O‐methyltransferase enzyme activity in hepatic tissue from male subjects was significantly higher than that in samples from female subjects: 61.3 ± 20.2 units/mg protein (mean ± SD; n = 50) versus 46.6 ± 22.2 units/mg protein (n = 49; p = 0.0002). There was a significant correlation of hepatic catechol‐O‐methyltransferase activity and thermal stability in samples from both female (rs = 0.698; p = 0.0001) and male subjects (rs = 0.429; p = 0.002). Finally, when both red blood cell catechol‐O‐methyltransferase activity and thermal stability were measured in blood samples from 34 of these patients, there was a significant correlation between catechol‐O‐methyltransferase heated/control ratios and levels of enzyme activity in hepatic tissue and in red blood cell lysates. These findings indicate that the genetic polymorphism that controls catechol‐O‐methyltransferase activity level and thermal stability in red blood cells also controls those same properties of the enzyme in the human liver.

Immunosuppressive CD14+HLA-DRlow/− Monocytes in Prostate Cancer

To determine if the levels of circulating myeloid‐derived suppressor cells increase with progression of prostate cancer (PCa); to determine if such cells could contribute to the relative inefficiency of PCa immunotherapy.

Human liver thiopurine methyltransferase pharmacogenetics: Biochemical properties, liver-erythrocyte correlation and presence of isozymes

Thiopurine methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs such as 6-mercaptopurine (6-MP). TPMT activity in the human red blood cell (RBC) is controlled by a common genetic polymorphism. Gene frequencies for this polymorphism are such that approximately one in 300 subjects is homozygous for the allele for low activity and lacks RBC TPMT activity, 11% of subjects are heterozygous and have intermediate levels of enzyme activity and 89% are homozygous for the allele for high activity. Our experiments were performed to determine whether the properties of TPMT in an important human drug metabolizing organ, the liver, were similar to those of RBC TPMT and to test the hypothesis that the genetic polymorphism which controls TPMT activity in the human RBC might also regulate the level of this enzyme activity in hepatic tissue. Human liver TPMT is a cytoplasmic enzyme and the Km values for 6-MP and S-adenosyl-L-methionine, cosubstrates for the reaction, were 580 microM and 2.7 microM, respectively. These properties, as well as the sensitivity of human liver TPMT to a panel of methyltransferase inhibitors, were similar to those of RBC TPMT. The enzyme activity was then measured in 119 surgical biopsy samples of hepatic tissue. Average hepatic TPMT activity was 13.6% higher in samples from male than in those from female patients. Frequency distribution histograms demonstrated the presence of a subgroup with intermediate enzyme activity that included 8.4% of samples. In addition, when TPMT activity was measured in both RBCs and hepatic tissue for 35 patients, those with inherited intermediate levels of RBC TPMT activity also had intermediate hepatic enzyme activity. Finally, ion exchange chromatography demonstrated the presence of two isozymes of TPMT in human hepatic tissue, but the isozymes did not appear to explain the molecular mechanism responsible for the genetic polymorphism. These results were compatible with the conclusion that the genetic polymorphism which controls TPMT activity in the RBC also controls levels of this important enzyme activity in a major human drug metabolizing organ, the liver.

Leucopenia resulting from a drug interaction between azathioprine or 6-mercaptopurine and mesalamine, sulphasalazine, or balsalazide

AIM We evaluated the effect of coadministration of sulphasalazine, mesalamine, and balsalazide on the pharmacokinetics and pharmacodynamics of azathioprine and 6-mercaptopurine. METHODS Thirty four patients with Crohns disease receiving azathioprine or 6-mercaptopurine were enrolled in an eight week non-randomised parallel group drug interaction study and treated with mesalamine 4 g/day, sulphasalazine 4 g/day, or balsalazide 6.75 g/day. The primary outcome measure was the occurrence of clinically important leucopenia during the study, defined separately as total leucocyte counts <3.0 x 109/l and ⩽3.5×109/l. Whole blood 6-thioguanine nucleotide concentrations were determined. RESULTS Three patients could not be evaluated for the primary outcome measure. In the remaining 31 patients, the frequency of total leucocyte counts <3.0 and ⩽3.5 were: 1/10 and 5/10 in the mesalamine group; 1/11 and 6/11 in the sulphasalazine group; and 0/10 and 2/10 in the balsalazide group. There were significant increases in mean whole blood 6-thioguanine nucleotide concentrations from baseline at most time points in the mesalamine and sulphasalazine groups but not in the balsalazide group. CONCLUSIONS In patients with Crohns disease receiving azathioprine or 6-mercaptopurine, coadministration of mesalamine, sulphasalazine, and possibly balsalazide results in an increase in whole blood 6-thioguanine nucleotide concentrations and a high frequency of leucopenia.

Olsalazine and 6-mercaptopurine-related bone marrow suppression : A possible drug-drug interaction

A patient with refractory Crohns disease had two separate episodes of bone marrow suppression while receiving 50 to 75 mg 6‐mercaptopurine a day and 1000 to 1750 mg olsalazine a day. This adverse reaction necessitated dose reduction of 6‐mercaptopurine on the first occasion and withdrawal of 6‐mercaptopurine and olsalazine on the second occasion. The patients red blood cell thiopurine methyltransferase (TPMT) activity was 12.2 U per milliliter red blood cells (low normal range) and her TPMT genotype was wild‐type sequence for all known alleles of TPMT that result in low TPMT enzyme activity. In vitro enzyme kinetic studies confirmed the hypothesis that olsalazine and olsalazine‐O‐sulfate are potent noncompetitive inhibitors of recombinant human TPMT. We suggest that the patients relatively low baseline level of TPMT activity was inhibited by olsalazine and olsalazine‐O‐sulfate, leading to decreased clearance of 6‐mercaptopurine and its accumulation. This ultimately increased intracellular 6‐thiopurine nucleotide levels to toxic concentrations, which caused bone marrow suppression.

Thiopurine methyltransferase polymorphic tandem repeat: Genotype‐phenotype correlation analysis

Thiopurine methyltransferase (TPMT) is a genetically polymorphic enzyme that catalyzes the S‐methylation of thiopurine drugs such as 6‐mercaptopurine. Recently, a variable number tandem repeat (VNTR) within the TPMT promoter has been reported to “modulate” levels of this enzyme activity.

Human liver xanthine oxidase: Nature and extent of individual variation

Xanthine oxidase catalyzes the biotransformation of many drugs, including the thiopurines and methylxanthines. We used a sensitive radiochemical assay to determine optimal conditions for the assay of human liver xanthine oxidase activity. We then used those assay conditions to study the nature and extent of individual variation of xanthine oxidase activity in 189 samples of hepatic tissue from patients undergoing clinically indicated partial hepatectomy or open liver biopsy. The average hepatic xanthine oxidase activity was 21% higher in samples from male patients than in those from female patients (1.27 ± 0.43 [mean ± SD, n= 92] versus 1.05 ± 0.38 U/gm tissue [n= 97, p< 0.0001], respectively). Seventynine of these tissue samples had been obtained from patients with normal liver function studies and normal serum creatinine values. Average xanthine oxidase activity in these 79 samples remained approximately 20% higher for men than for women (1.35 ± 0.38 versus 1.12 ± 0.33 U/gm tissue, respectively). Probit analysis of the data for samples from patients with normal liver function studies and normal creatinine values suggested the presence of a subgroup of samples with relatively low xanthine oxidase activity in 21% (9 of 42) of male patients and 27% (10 of 37) of female patients. These observations may have implications with regard to individual variation in the biotransformation of drugs metabolized by xanthine oxidase.

Pharmacokinetics, dose adjustments, and 6-mercaptopurine/ methotrexate drug interactions in two patients with thiopurine methyltransferase deficiency

Two children with acute lymphoblastic leukaemia (ALL) were found to be thiopurine methyltransferase (TPMT)‐deficient by both genotype and phenotype. They were monitored with haematological parameters and red blood cell concentrations of 6‐thioguanine nucleotides (E‐6TGN) and methotrexate (E‐MTX, including MTX polyglutamates), in relation to the doses of 6‐mercaptopurine (6MP) and methotrexate (MTX), during their maintenance chemotherapy. Both patients developed severe pancytopenia at the standard protocol dose of 6MP. Even at 25% and 5%, respectively, of the protocol dose of 6MP, they achieved E‐6TGN values several‐fold above the population median, but without unacceptable bone‐marrow toxicity. Their high E‐6TGN values had only a minor influence on their E‐MTX values and their tolerance to oral MTX, but severe pancytopenia followed high‐dose MTX infusions. Due to the risk of fatal myelo‐suppression we recommend up‐front determination of TPMT activity in patients treated with 6MP or azathioprine.