mercaptopurine and thioinosinic-acid

Methotrexate is widely administered with mercaptopurine, a prodrug requiring activation into thioguanine nucleotides (TGN) to exert antileukemic effects. In vitro, methotrexate enhances TGN formation, but in vivo, such enhancement has yet to be demonstrated. We investigated whether TGN concentrations were related to methotrexate concentrations in children with acute lymphoblastic leukemia who received a weekly intravenous methotrexate (40 mg/m(2)) dose combined with daily mercaptopurine (75 mg/m(2)). A total of 141 erythrocyte TGN concentrations were measured with erythrocyte methotrexate polyglutamates (MTX-PG) concentrations in 87 patients. Average TGN concentrations ranged from 137 to 958 pmol/8 x 10(8) cells (median 389), average total MTX-PG concentrations (MTX- PG(1-7)) from 0.60 to 97.7 pmol/10(9)cells (median 29), and average long chain polyglutamate concentrations (MTX-PG(5-7)) from 0 to 8.35 pmol/10(9) cells (median 2.43). Higher TGN concentrations correlated with higher MTX-PG(5-7) concentrations (P = 0.002). These data support the practice of administering methotrexate with mercaptopurine during continuation therapy of acute lymphoblastic leukemia.

Topics: Algorithms; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Biotransformation; Child; Drug Administration Schedule; Erythrocytes; Humans; Hypoxanthine Phosphoribosyltransferase; Inactivation, Metabolic; Injections, Intravenous; Inosine Monophosphate; Mercaptopurine; Methotrexate; Methyltransferases; Neoplasm Proteins; Nucleotides; Polyglutamic Acid; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Prodrugs; Thioguanine; Thionucleotides; Xanthine Oxidase

The effects of prolonged exposure to 2 and 10 microM 6-mercaptopurine (6MP) in the human lymphoblastic T-cell line MOLT-4 were studied with respect to cell-kinetic parameters, phosphoribosyl pyrophosphate (PRPP) and purine ribonucleotide levels, formation of 6MP-nucleotides, especially methyl-thio-IMP (Me-tIMP), DNA and RNA synthesis ([32P] incorporation), and [8-14C]6MP incorporation into newly synthesized DNA and RNA. The results provided new insights into the complex mechanism of action of 6MP in human malignant lymphoblasts. Exposure to 2 microM 6MP resulted in a rapid inhibition of purine de novo synthesis (PDNS) by increased levels of Me-tIMP, resulting in increased PRPP levels and decreased purine ribonucleotides, affecting cell growth and clonal growth, and less cell death. DNA synthesis decreased, associated with an increasing delay of cells in S phase. Incorporation of thioguanine nucleotides into newly synthesized DNA resulted in an increasing arrest of cells in G2 + M phase. RNA synthesis, initially decreased, recovered partially, associated with a recovery of purine ribonucleotides. New formation of 6MP-nucleotides (tIMP) was only detected within the first 24 hr, and 6MP levels in the culture medium were already undetectable after 6 hr of exposure to 2 microM, indicating a high rate of incorporation and complete conversion of 6MP within this period. Incorporation of 6MP-nucleotides into DNA was 5 times as high as incorporation into RNA. Exposure to 10 microM 6MP resulted in early cytotoxicity at 24 hr, associated with a complete inhibition of PDNS by a large pool of Me-tIMP and lower levels of purine ribonucleotides as compared to 2 microM 6MP. A more severe delay of cells in S phase was associated with an inhibition of DNA synthesis to 14% of control within the first 24 hr, and an arrest in G2 + M phase. Further increasing levels of Me-tIMP caused an arrest of cells and late cytotoxicity in S phase at 48 hr, preventing further progression into G2 + M phase. Our data suggest that inhibition of PDNS due to Me-tIMP is a crucial event in the mechanism of 6MP cytotoxicity. It is responsible for decreased RNA synthesis and decreased availability of natural deoxyribonucleotides, causing a delay of DNA synthesis in S phase. This enhances incorporation of 6MP as thioguanine nucleotides into DNA in the S phase and subsequent late cytotoxicity in the G2 phase. However, with high concentrations of 6MP, the large pool of Me-tIMP causes severe reduction o

Topics: Carbon Radioisotopes; Cell Cycle; DNA; Humans; Inosine Monophosphate; Leukemia, Lymphoid; Mercaptopurine; Phosphoribosyl Pyrophosphate; Phosphorus Radioisotopes; Ribonucleotides; RNA; Stem Cells; T-Lymphocytes; Thionucleotides

The chemicals 6-mercaptopurine (6-MP) and methotrexate (MTX) are the cornerstones in the maintenance treatment of acute lymphoblastic leukemia. The intracellular metabolism of 6-MP to 6-thioguanosine nucleotides (TGN) via 6-thioinosine 5'-monophosphate (TIMP) is crucial for its cytotoxic effect. MTX inhibits purine de novo synthesis and thereby increases the intracellular PRPP being a substrate for the phosphoribosylation of 6-MP to TIMP. Hypoxanthine has been shown to inhibit the uptake of 6-MP over the cell membrane and the phosphoribosylation of 6-MP to TIMP. We have previously shown that the conversion of TIMP to TGN decreases at 6-MP concentrations above 5 microM in vitro. The aim of the present study was therefore to investigate the effect of MTX increasing the PRPP and TIMP concentrations and of hypoxanthine decreasing the TIMP concentration on the formation of TGN from TIMP. Murine myelomonocytic leukemia cells (WEHI-3b) were treated with 6-MP in vitro. The drug concentration was kept constant by continuous addition of 6-MP during the experiment. With this technique, the concentration of TGN begins to decrease already at 6-MP concentrations above 2 microM. The addition of 0.2 microM MTX 6 h before 6-MP strongly inhibited the purine de novo synthesis, decreased the ATP, and increased the PRPP concentration 4-fold. The intracellular concentrations of TIMP and to a lesser extent TXMP also increased. The concentrations of the TGN were, however, basically unaffected by the preincubation with MTX. Simultaneous addition of 20-50 microM hypoxanthine and 6-MP decreased the accumulation of all cellular 6-MP metabolites. It is concluded that the synergistic cytotoxic effect of the combination of 6-MP and MTX is not based on biochemical modulation of the 6-MP metabolism by MTX.

Topics: Animals; Drug Interactions; Extracellular Space; Hypoxanthine; Hypoxanthines; Inosine Monophosphate; Kinetics; Leukemia, Experimental; Leukemia, Myelomonocytic, Acute; Mercaptopurine; Methotrexate; Mice; Phosphoribosyl Pyrophosphate; Purines; Thionucleotides; Tumor Cells, Cultured; Xanthines

A reversed-phase high-performance liquid chromatographic assay was developed to quantify intracellular metabolites of the cytotoxic drug 6-mercaptopurine in the human red blood cell. The 6-thioguanine nucleotides, 6-thioinosinic acid and 6-methylmercaptopurine metabolites are measured in a single sample. A similar assay measures the parent thiopurine compounds. The limit of quantitation of the assay is 0.03, 0.03 and 0.12 nmol per 8 x 10(8) red blood cells for the 6-thioguanine nucleotides, 6-thioinosinic acid and the 6-methylmercaptopurine metabolites, respectively.

Topics: Chromatography, High Pressure Liquid; Erythrocytes; Humans; Hydrolysis; Inosine Monophosphate; Mercaptopurine; Methylthioinosine; Thionucleotides

Co-incubation of human leukemia cell lines with naturally occurring nucleobases (hypoxanthine or adenine) significantly prevented the cytotoxic activity of 6-thiopurines. Extracellular hypoxanthine decreased the transport of 6-mercaptopurine into cells, but adenine had no significant effect. However, intracellular thioinosine monophosphate accumulation in the presence of 10 microM, 6-mercaptopurine was reduced to below 1% or 10% of that of the controls when 50 microM hypoxanthine or adenine was added, respectively. Finally, in adenine phosphoribosyl transferase deficient mutants, adenine provided no protective effect against 6-thiopurines, whereas hypoxanthine retained its modulating activity. These data suggest that the nucleobases compete with 6-thiopurines for the ribose-phosphate donor, 5'-phosphoribosyl-1-pyrophosphate, thus preventing the formation of active metabolites of 6-thiopurines.

Topics: 2-Aminopurine; Adenine; Adenine Phosphoribosyltransferase; Antineoplastic Agents; Biological Transport; Humans; Hypoxanthine; Hypoxanthine Phosphoribosyltransferase; Hypoxanthines; Inosine Monophosphate; Leukemia-Lymphoma, Adult T-Cell; Leukemia, Promyelocytic, Acute; Mercaptopurine; Thioguanine; Thionucleotides; Tumor Cells, Cultured

A highly sensitive reversed-phase high-performance liquid chromatographic assay, with ultraviolet detection, for 6-thioinosinic acid and the 6-thioguanine nucleotides (6TGNs) was developed. The 6TGNs are major red blood cell metabolites of the immunosuppressive agent azathioprine and the cytotoxic drugs 6-thioguanine and 6-mercaptopurine. The assay is based on the specific extraction, via phenyl mercury adduct formation, of the thiopurine released on acid hydrolysis of the thionucleotide metabolite. Red blood cell 6TGN concentrations in eighteen leukaemic children receiving chronic 6-mercaptopurine chemotherapy were measured and compared to a previously published spectrophotofluorometric assay. Linear regression analysis gave r = 0.991; P less than 0.001; y = 40 + 0.94x.

Topics: Chromatography, High Pressure Liquid; Drug Stability; Erythrocytes; Humans; Inosine Monophosphate; Inosine Nucleotides; Leukemia; Mercaptopurine; Nucleotides; Spectrometry, Fluorescence; Thioguanine; Thionucleotides

A sensitive and rapid assay for the quantitation of thioinosine monophosphate and thioguanosine monophosphate, the major intracellular active metabolites of mercaptopurine and thioguanine, respectively, has been developed. Neoplastic cells are extracted with trichloracetic acid, and the neutralized acid extracts are analyzed by ion-pairing high-performance liquid chromatography with dual-channel uv-wavelength detection. This technique provides a lower limit of sensitivity of 30 pmol of thioinosine monophosphate and 10 pmol of thioguanosine monophosphate. The number of cells assayed per sample was 2 X 10(7). This assay makes it possible to detect and quantitate low levels of thioinosine monophosphate and thioguanosine monophosphate present in neoplastic cells obtained directly from patients receiving mercaptopurine or thioguanine chemotherapy.

Topics: Bone Marrow; Cell Line; Chromatography, High Pressure Liquid; Guanine Nucleotides; Humans; Inosine Monophosphate; Inosine Nucleotides; Leukemia, Myeloid, Acute; Lymphoma; Mercaptopurine; Thioguanine; Thionucleotides

The activation of purine antimetabolites to their respective nucleotides is a step critical to their effectiveness as chemotherapeutic agents. Erythrocytes, with their relatively simple purine metabolism, are useful as a model for identifying mechanisms which enhance this 5-phosphoribosyl 1-pyrophosphate (P-Rib-PP)-dependent activation. We previously showed that pyrroline-5-carboxylate, a physiologic intermediate in the interconversions of proline, ornithine, and glutamate, markedly stimulated the pentose phosphate pathway, increased the formation of P-Rib-PP, and increased purine incorporation into nucleotides. We now report that the events initiated by pyrroline-5-carboxylate markedly increased the activation of 6-thiohypoxanthine, 6-thioguanine, and azathioprine to their respective nucleotides in intact human erythrocytes. The mechanism of this effect was directly demonstrated in studies using the conversion of hypoxanthine to inosine monophosphate as a model for pyrroline-5-carboxylate-mediated stimulation of P-Rib-PP-dependent nucleotide formation. Since the P-Rib-PP-dependent activation of these chemotherapeutic agents may be important to their clinical effectiveness, the events initiated by pyrroline-5-carboxylate may provide new insight into the nature of tumor sensitivity and resistance to these agents.

Topics: Azathioprine; Erythrocytes; Humans; Inosine Monophosphate; Inosine Nucleotides; Mercaptopurine; Oxidation-Reduction; Phosphates; Phosphoribosyl Pyrophosphate; Pyrroles; Thioguanine; Thionucleotides

1 A specific assay for the measurement of thioinosinic acid, in human lymphocytes, has been developed with a sensitivity of 50 ng of thioinosinic acid per 5 x 10(6) lymphocytes. 2 Thioinosinic acid is precipitated from purified lymphocytes as the lanthanum salt. Acid hydrolysis results in the formation of 6-mercaptopurine which, when converted into its phenyl mercury derivative, can be easily extracted into toluene. Back-extraction of the toluene layer with 0.1N HCl regenerates 6-mercaptopurine which is then assayed fluorometrically. 3 Blood samples were taken from renal transplant recipients 3 h after an oral dose of 50 mg azathioprine. The results from 5 patients gave a range of 54 to 173 ng of thioinosinic acid per 5 x 10(6) lymphocytes, with a mean of 110 ng. 4 In an in vitro incubation of azathioprine, 1mM with fresh human blood, 160 and 180 ng of thioinosinic acid per 5 x 10(6) lymphocytes was formed after 0.5 h and 5 h respectively. 5 The assay is suitable for the study of the kinetics of thioinosinic acid formation in lymphocytes of patients with kidney transplants. It could also prove useful in the study of thioinosinic acid formation in leukaemia patients undergoing 6-mercaptopurine treatment.

Topics: Azathioprine; Chemical Phenomena; Chemical Precipitation; Chemistry; Chromatography, Thin Layer; Humans; Inosine Monophosphate; Inosine Nucleotides; Lymphocytes; Mercaptopurine; Spectrometry, Fluorescence; Spectrophotometry; Thionucleotides

In male BALB/c mice, a combination of individually non-lethal doses of 6-mercaptopurine and endotoxin was significantly lethal. In contrast, mice treated with phenobarbital were resistant to this lethal effect. The high levels of thioinosinic acid in mice that were treated with endotoxin contrasted significantly with the levels in phenobarbital-treated mice. On the other hand, the concentration of hypoxanthine was increased by the administration of phenobarbital and decreased by the administration of endotoxin. The sleeping time and levels of pentobarbital hydroxylase found in endotoxin-treated mice were consistent with the lethality and levels of thioinosinic acid. After mice were treated with endotoxin, their sleeping time was prolonged, which agrees with the course of the stimulatory effects of 6-mercaptopurine anabolism. However, there were no significant differences in hypoxanthine-guanine phosphoribosyltransferase. Furthermore, contrary to expectation, there were significant increases in xanthine oxidase after treatment with endotoxin. Thus, the metabolism of 6-mercaptopurine might be modified by hepatic microsomal enzyme activity.

Topics: Animals; Drug Interactions; Endotoxins; Hypoxanthine Phosphoribosyltransferase; Hypoxanthines; Inosine Monophosphate; Liver; Male; Mercaptopurine; Mice; Mice, Inbred BALB C; Microsomes, Liver; Phenobarbital; Sleep; Thionucleotides; Time Factors; Xanthine Oxidase

Topics: Carbon Isotopes; Cell Fractionation; Cell-Free System; Chromatography, Ion Exchange; Chromatography, Paper; Drug Resistance; Female; Humans; Hypoxanthines; Inosine; Inosine Monophosphate; Inosine Nucleotides; Leukemia, Lymphoid; Leukocytes; Male; Mercaptopurine; Methods; Nucleotides; Spectrophotometry; Thionucleotides