guanosine-triphosphate and thiazole-4-carboxamide-adenine-dinucleotide

The increased activity in cancer cells of inosine 5'-monophosphate dehydrogenase (IMP DH, EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis, was suggested as a sensitive target for chemotherapy. Tiazofurin (NSC 286193), through its conversion to the active metabolite, thiazole-4-carboxamide adenine dinucleotide (TAD), is a strong inhibitor of IMP DH. In our clinical trial, tiazofurin caused return to the chronic phase in patients with chronic granulocytic leukemia in blast crisis (Tricot, G.; Jayaram, H.N.; Weber, G.; Hoffman, R. Tiazofurin: Biological effects and clinical uses. Int. J. Cell Cloning 8:161-170; 1990). In K562 human leukemic cells, tiazofurin down-regulated the expression of c-Ki-ras and c-myc oncogenes, which was followed by induced differentiation. We now report down-regulation by tiazofurin of the c-Ki-ras oncogene in a patient with chronic granulocytic leukemia in blast crisis. A single tiazofurin infusion (2,200 mg/m2) on days one and two decreased IMP dehydrogenase activity (the apparent t1/2 was 30 min), GTP concentration (the apparent t1/2 was 6 hr), and expression of ras (the apparent t1/2 was 8 hr) and c-myc (the apparent t1/2 was 38.5 hr) oncogenes in the leukemic cells. No further tiazofurin was given, because on days three and four the chemotherapeutic impact became evident in a tumor-lysis syndrome and the blast cells were cleared from the periphery by day five. The decrease in IMP DH activity, GTP concentration, and expression of c-Ki-ras oncogene were early markers of the successful chemotherapeutic impact of tiazofurin in a patient with chronic granulocytic leukemia in blast crisis.

Topics: Adenine Nucleotides; Antimetabolites, Antineoplastic; Blast Crisis; Blotting, Northern; Down-Regulation; Gene Expression Regulation; Genes, ras; Guanosine Triphosphate; Humans; Hypoxanthine; Hypoxanthines; IMP Dehydrogenase; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Pilot Projects; Ribavirin; RNA; Uric Acid

Tiazofurin is effective in treating end-stage leukemic patients (Tricot et al., Cancer Res 49:3696-3701, 1989). In sensitive tumors, the active metabolite of tiazofurin, TAD, potently inhibits IMP dehydrogenase activity, resulting in reduced guanylate pools. To elucidate tiazofurin activity in human solid tumors, we examined its activity in human colon carcinoma HT-29. Tiazofurin exhibited an LC50 of 35 microM in cultured HT-29 cells. Incubation of HT-29 cells with 100 microM tiazofurin for 2 h resulted in TAD formation (9.3 nmol/g cells) and in a 64% decrease in GTP pools. For biochemical and chemotherapy studies, athymic nude mice were transplanted s.c. with HT-29 cells. Twenty-four days later, mice were injected i.p. with tiazofurin (500 mg/kg); 6 h later, tumors were removed and analyzed. These tumors formed 17 nmol/g of TAD with decreased GTP pools (56%). To study oncolytic activity, transplanted mice were treated 24 h later with tiazofurin (500 mg/kg, once a day for 10 days). To examine the effectiveness of tiazofurin in established tumors, the drug was administered to mice 14 days after tumor implantation (500 mg/kg, once a day for 5 days, course repeated 4 times with a 10-day rest). Both treatment schedules resulted in significant antitumor activity. This study illustrates the potential usefulness of tiazofurin in treating human colon carcinoma.

Topics: Adenine Nucleotides; Animals; Antineoplastic Agents; Colonic Neoplasms; Drug Screening Assays, Antitumor; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Male; Mice; Mice, Nude; Ribavirin; Tumor Cells, Cultured

Tiazofurin is an oncolytic agent which has shown therapeutic activity in end-stage acute nonlymphocytic leukemia (ANLL) and blast crisis of chronic granulocytic leukemia (CGL-BC). Tiazofurin is anabolized to the active metabolite, thiazole-4-carboxamide adenine dinucleotide (TAD), which inhibits IMP dehydrogenase activity, leading to reduction of guanylate pools and cessation of cancer cell proliferation. The concentration of TAD in neoplastic cells of patients treated with tiazofurin should be a good indicator of sensitivity to the drug and also might herald the emergence of drug-resistant cells. Therefore, the precise quantitation of TAD in cancer cells during tiazofurin treatment is essential. In this paper we report a highly sensitive method for the determination of TAD in biological samples. With this technique, in addition to TAD, thirteen other biologically relevant adenine, guanine, cytosine and uridine nucleotides can be separated and quantitated accurately. TAD standard was separated on a Waters Partisil 10-SAX column in a RCM-10 module using an ammonium phosphate buffer system. TAD eluted at 21 min with a limit of detection of 15 pmol and linearity up to 3 nmol. The coefficient of variation was 0.6 +/- 0.1% for retention time and 2 +/- 0.3% for TAD concentration. Recovery of TAD was 96% with reproducibility of 98%. To examine the applicability of this method to a clinical setting, blood samples were obtained from a patient with CGL-BC and leukocytes were separated on a Ficoll-Hypaq gradient, extracted with trichloroacetic acid, and an aliquot was analyzed on HPLC. The TAD peak was identified by comparing the retention time and spectral analysis of the standard. After the patient was treated with a 2200 mg/m2 (12.7 mM) dose of tiazofurin, the TAD concentrations in the mononuclear cells at 2, 6, and 24 hr were 23.1, 13.6, and 0.8 microM. TAD levels at 2, 6, and 24 hr after a tiazofurin dose of 3300 mg/m2 (21.1 mM) were 42.8, 26.1, and 1.4 microM respectively.

Topics: Adenine Nucleotides; Antineoplastic Agents; Chromatography, High Pressure Liquid; Guanosine Triphosphate; Humans; Leukemia; Monocytes; Reproducibility of Results; Ribavirin; Sensitivity and Specificity

This study reports the selective sensitivity to tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide, NSC-286193) of human leukemic leukocytes as compared to normal ones in bone marrow and peripheral blood samples by comparing the production of the active metabolite, thiazole-4-carboxamide adenine dinucleotide (TAD), from labeled tiazofurin and the depression of GTP concentration. When labeled tiazofurin was incubated with leukocytes obtained from healthy volunteers or from leukemic patients (acute non-lymphocytic leukemia or acute lymphoblastic leukemia), the TAD production was 27.0 +/- 8.3, 551.3 +/- 71.8 and 755.9 +/- 94.1 pmoles/10(9) cells per hr, respectively. Thus, the leukemic cells produced over 20-fold higher concentrations of TAD than the normal leukocytes. Incubation with tiazofurin in leukemic leukocytes decreased the GTP pools (to 48-79%), whereas there was no change in the normal leukocytes. These results indicate a selectivity of response to tiazofurin in human normal and leukemic leukocytes. The procedure reported in this work may be suitable as a rapid predictive test for the sensitivity of leukemic leukocytes to tiazofurin. Such a diagnostic test should be helpful in identifying neoplastic cells sensitive to tiazofurin in the Phase II trials now being developed.

Topics: Adenine Nucleotides; Adult; Aged; Antineoplastic Agents; Female; Guanosine Triphosphate; Humans; Leukemia; Leukocytes; Male; Middle Aged; Ribavirin; Ribonucleosides