Thiopurine S-methyltransferase (TPMT; Sadenosyl-L-methionine:thiopurine S-methyltransferase, EC 2.1.1.67) activity exhibits genetic polymorphism, with -0.33% of Caucasians and African-Americans inheriting TPMT deficiency as an autosomal recessive trait. To determine the molecular genetic basis for this polymorphism, we cloned the TPMT cDNA from a TPMT-deficient patient who had developed severe hematopoietic toxicity during mercaptopurine therapy. Northern blot analysis of RNA isolated from leukocytes of the deficient patient demonstrated the presence of TPMT mRNAs of comparable size to that in subjects with high TPMT activity. Sequencing of the mutant TPMT cDNA revealed a single point mutation (G238 C), leading to an amino acid substitution at codon 80 (Ala80 Pro). When assessed in a yeast heterologous expression system, this mutation led to a 100-fold reduction in TPMT catalytic activity relative to the wild-type cDNA, despite a comparable level of mRNA expression. A mutation-specific PCR amplification method was developed and used to detect the G238 -> C mutation in genomic DNA of the propositus and her mother. This inactivating mutation in the human TPMT gene provides insights into the genetic basis for this inherited polymorphism in drug metabolism.Thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) is a cytoplasmic enzyme that preferentially catalyzes the Smethylation of aromatic and heterocyclic sulfhydryl compounds, including the anticancer agents 6-mercaptopurine and 6-thioguanine. TPMT activity exhibits genetic polymorphism, with -89% of Caucasians and African-Americans having high TPMT activity, -11% intermediate activity (presumed heterozygotes), and -1 in 300 inheriting TPMT deficiency as an autosomal recessive trait (1, 2). TPMT activity is typically measured in erythrocytes, as the level of TPMT activity in human liver, kidney, and normal lymphocytes has been shown to correlate with that in erythrocytes (3, 4).As part of multiagent chemotherapy for the treatment of acute lymphoblastic leukemia, mercaptopurine typically comprises a large percentage of therapy and is often administered daily throughout 2.5 years of continuation treatment. Mercaptopurine is a prodrug with no intrinsic anticancer activity, requiring intracellular conversion to thioguanine nucleotides, with subsequent incorporation into DNA, as one mechanism of its antiproliferative effects (5). Alternatively, mercaptopurine is metabolized to 6-methylmercaptopurine by TPMT or to 6-thiouric acid by xanthine oxidase. 6-Thiouric acid is an inactive metabolite, whereas 6-methylmercaptopurine nucleotide inhibits phosphoribosyl diphosphate amidotransferase, an enzyme catalyzing the first step in de novo purine synthesis (6, 7). It is not clear whether mercaptopurine's principal mechanism of cytotoxicity is via incorporation of thioguanine nucleotides into DNA and RNA, or via 6-methylmercaptopurine nucleotide inhibition of de novo purine synthesis, or a combination of these effects.Clinical studies in children with acute lymphoblastic l...
SUMMARYCellular accumulation of methotrexate polyglutamates (MTXPGs) is recognized as an important determinant of the cytotoxicity and selectivity of methotrexate in acute lymphoblastic leukemia (ALL). We identified a significantly lower cellular accumulation of MTXPGs in T-lineage versus B-lineage lymphoblasts in children with ALL, which is consistent with the worse prognosis of T-lineage ALL when treated with conventional antimetabolite-based therapy. Maximum MTXPG accumulation in leukemic blasts in vivo was 3-fold greater in lymphoblasts of children with B-lineage ALL (129 children) compared with those with T-lineage ALL (20 children) ( p Ͻ 0.01) and was characterized by a saturable (E max ) model in both groups. The human leukemia cell lines NALM6 (B-lineage) and CCRF/CEM (T-lineage) were used to assess potential mechanisms for these lineage differences in MTX accumulation, revealing i) greater total and long-chain MTXPG accumulation in NALM6 over a wide range of methotrexate concentrations (0.2-100 M), ii) saturation of MTXPG accumulation in both cell lines, with a higher maximum (E max ) in NALM6, iii) 3-fold higher constitutive FPGS mRNA expression and enzyme activity in NALM6 cells, iv) 2-fold lower levels of DHFR mRNA and protein in NALM6 cells, and v) 4 -6 fold lower extracellular MTX concentration and 2-fold lower intracellular MTXPG concentration to produce equivalent cytotoxicity (LC 50 ) in NALM6 versus CEM. There was a significant relationship between FPGS mRNA and enzyme activity in lymphoblasts from children with newly diagnosed ALL, and blast FPGS mRNA and activity increased after methotrexate treatment. These data indicate higher FPGS and lower DHFR levels as potential mechanisms contributing to greater MTXPG accumulation and cytotoxicity in B-lineage lymphoblasts.
Several anticancer drugs display characteristics that make them suitable candidates for therapeutic drug monitoring (TDM), including substantial pharmacokinetic variability and a narrow therapeutic index. However, concentration-effect relationships (pharmacodynamics) of most antineoplastic agents have not been well defined, thus limiting the widespread clinical application of TDM for cancer chemotherapy. Strategic incorporation of pharmacokinetic studies during phase I-III clinical trials should facilitate the identification of concentration-effect relationships and the definition of clinically useful levels of treatment intensity. We review representative clinical studies that have defined pharmacodynamic relationships for methotrexate, teniposide, etoposide, carboplatin, and mercaptopurine. Given that TDM has impacted positively on the clinical use of many drugs belonging to other therapeutic classes, and that pharmacodynamic correlations have been identified in several recent studies of anticancer drugs, we consider implementation of TDM a rational strategy for optimizing the use of selected antineoplastics.
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