tiazofurin 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

A rapid and sensitive HPLC-RP method for simultaneous determination of tiazofurin, its 5'-O acetyl and benzoyl esters and their active metabolite thiazole-4-carboxamide adenine dinucleotide was developed and validated. The method allowed determination and quantification of nanomolar quantities of these substances in cell extracts of treated cells, and was also used in kinetic studies of cellular uptake of tiazofurin and its esters from the cultivation medium. Separation of the analyzed substances from unidentified peaks from both biological materials was achieved by gradient elution, thus reducing the possibility of interference. The mobile phase consisted of a 0.1 M sodium-hydrogen phosphate, pH 5.1 and methanol. Run time was 22 min, with 5 min equilibration time.

Topics: Adenine Nucleotides; Animals; Chromatography, High Pressure Liquid; Esters; Rats; Ribavirin; Tumor Cells, Cultured

Thiazole-4-carboxamide adenine dinucleotide (TAD) analogue 7 containing a fluorine atom at the C2' arabino configuration of the adenine nucleoside moiety was found to be a potent inducer of differentiation of K562 erythroid leukemia cells. This finding prompted us to synthesize its hydrolysis-resistant methylenebis(phosphonate) and difluoromethylenebis(phosphonate) analogues 8 and 9, respectively. Since both TAD and benzamide adenine dinucleotide (BAD) are potent inhibitors of inosine monophosphate dehydrogenase (IMPDH), the corresponding fluorine-substituted methylenebis(phosphonate) analogue 12 of BAD was also synthesized. Thus, 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine (13) was converted in five steps into the corresponding methylenebis(phosphonate) analogue 18. Dehydration of 18 with DCC led to the formation of the bicyclic trisanhydride intermediate 19a, which upon reaction with 2',3'-O-isopropylidenetiazofurin (20) or -benzamide riboside (21) followed by hydrolysis and deprotection afforded the desired methylene-bridged dinucleotides 8 and 12, respectively. The similar displacement of the 5'-mesyl function of 2',3'-O-isopropylidene-5'-O-mesyltiazofurin (24) with the difluoromethylenebis(phosphonic acid) derivative gave the phosphonate 25 which was coupled with 13 to afford 26. The desired difluoromethylenebis(phosphonate) analogue 9 was obtained by deprotection with Dowex 50/H+. This compound as well as beta-CF2-TAD (4) showed improved differentiation-inducing activity over beta-CH2-TAD (3), whereas analogues containing the -CH2-linkage (8 and 12) were inactive.

Topics: Adenine Nucleotides; Animals; Antimetabolites, Antineoplastic; Benzamides; Cell Differentiation; Chromatography, High Pressure Liquid; Enzyme Inhibitors; Fluorine; Humans; IMP Dehydrogenase; Tumor Cells, Cultured

Treatment of 3-(2,3-O-isopropylidene-beta-D-ribofuranosyl)benzamide (6) with POCl3 in (EtO)3-PO afforded only little phosphorylation product (8, 5%), but the major product was 5'-chlorobenzamide riboside (7, 85%). Reaction of 6 with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite followed by 2-cyanoethanol/tetrazole treatment and oxidation with tert-butyl peroxide gave a 1:1 mixture of the desired 5'-O-bis(2-cyanoethyl) phosphate 9 and the chloro derivative 7. This mixture was treated with methanolic ammonia and partitioned between CHCl3 and water. The 2',3'-O-isopropylidenebenzamide mononucleotide (8) was obtained in 21.2% overall yield from the aqueous layer. Compound 8 was then converted into the corresponding imidazolide 11b which, upon coupling with 2',3'-O-acetonide of AMP, afforded the acetonide of benzamide adenine dinucleotide (15) in 94% yield together with small amounts of symmetrical pyrophosphates P1,P2-bis(2',3'-O-isopropylideneadenosin-5'-yl)pyrophosphate (13, 3%) and P1,P2-bis(2',3'-O-isopropylidene-3-(carbamoylphenyl)-5'-ribosyl)py rophosphate (14, 2%). Deprotection of 15 with Dowex 50/H+ in water afforded the desired benzamide adenine dinucleotide (BAD) in 93% yield. BAD inhibits inosine monophosphate dehydrogenase type I (IC50 = 0.78 microM) and type II (IC50 = 0.88 microM) with same degree of potency.

Topics: Adenine Nucleotides; Antimetabolites, Antineoplastic; Benzamides; Chromatography, High Pressure Liquid; Enzyme Inhibitors; Humans; IMP Dehydrogenase; Isoenzymes; Molecular Structure; Neoplasm Proteins; Structure-Activity Relationship

Tiazofurin exhibits antitumor activity in murine and human tumor cells. In a recent phase I/II trial in patients with end-stage leukemia, tiazofurin showed good response; however, repeated treatment resulted in clinical resistance to the drug. To elucidate the mechanisms of resistance in human leukemic cells, two variants of human myelogenous leukemia K652 cells resistant to tiazofurin were developed by drug-selection pressure. Compared to a concentration producing 50% cell proliferation reduction that was 9.1 microM in sensitive cells, the resistant variants displayed concentrations producing 50% cell proliferation reductions of 12 and 16 mM. The activity of the target enzyme, IMP dehydrogenase, was not altered in the resistant cells. Studies on tiazofurin metabolism revealed that resistant variants formed < 10% of the active metabolite, thiazole-4-carboxamide adenine dinucleotide. This correlated with the activity of NAD pyrophosphorylase, the enzyme that synthesizes thiazole-4-carboxamide adenine dinucleotide, which was reduced to 10% in the resistant lines. Concurrently, the activity of thiazole-4-carboxamide adenine dinucleotide phosphodiesterase was elevated in the refractory cells. Compared to the sensitive counterpart, the levels of GMP and NAD were lower in the resistant lines. Guanine salvage activity was decreased in the resistant cells. Basal dGTP and dATP concentrations were elevated in the resistant line; nevertheless, tiazofurin incubation decreased dGTP levels in only the sensitive cells. Although there was no difference in the Km of tiazofurin transport or efflux, the Vmax of uptake of the drug was reduced in the resistant lines. Sensitive and resistant cells exhibit similar cytotoxicity to agents which do not share the mechanism of action of tiazofurin, suggesting that refractory cells are still sensitive to other standard antileukemic drugs.

Topics: Adenine Nucleotides; Antineoplastic Agents; Biological Transport; Carbon Radioisotopes; Deoxyribonucleotides; Drug Resistance; Drug Screening Assays, Antitumor; Guanine; Humans; IMP Dehydrogenase; Leukemia, Myeloid; Nicotinamide-Nucleotide Adenylyltransferase; Nucleic Acids; Phosphoric Diester Hydrolases; Phosphorylation; Purine Nucleotides; Ribavirin; Tumor Cells, Cultured

Ab initio computations (RHF/6-31G* parallel 3-21G*) were performed on the thiazole-4-carboxamide group found in the antitumor drug tiazofurin and its dehydrogenase-binding anabolite thiazole-4-carboxamide adenine dinucleotide (TAD). Results indicate that the carboxamide group is constrained in the conformation in which the amino group is cis-planar to the ring nitrogen. This finding is consistent with carboxamide conformations observed in crystal structures of the thiazole nucleosides. In contrast, ab initio computations on the nicotinamide and dihydronicotinamide rings found in the cofactors NAD+ and NADH indicate two stable conformations for the carboxamide group. This finding confirms previous computational studies and is consistent with results from a survey of the Cambridge Structural Database. Natural bond orbital analysis indicates that the low-energy carboxamide conformers of all three heterocycles are stabilized by a combination of electrostatic and charge transfer interactions. A survey of the Protein Data Bank indicates that the carboxamide group conformation in TAD is constrained to that favored by dehydrogenase-bound NAD(P)(H).

Topics: Adenine Nucleotides; Amides; Antineoplastic Agents; Crystallography; Enzyme Inhibitors; IMP Dehydrogenase; Molecular Conformation; NADP; Niacinamide; Ribavirin; Stereoisomerism; Structure-Activity Relationship

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

2-beta-D-Arabinofuranosylthiazole-4-carboxamide and 2-beta-D-xylofuranosyl-thiazole-4-carboxamide are sugar modified analogues of tiazofurin, a C-glycosyl nucleoside which after anabolism to the dinucleotide, TAD (thiazole-4-carboxamide adenine dinucleotide), exhibits antitumor activity. However, ara-T and xylo-T did not exhibit cytotoxicity. Compared to tiazofurin, only 12.5% of the ara-T and 8.8% of the xylo-T were metabolized to TAD derivatives by human myelogenous leukemia K562 cells. This was reflected in the finding that guanylate pools were not depressed after treatment with either tiazofurin derivative. These results provide evidence that the ribose moiety is essential for the metabolism and cytotoxicity of tiazofurin. This investigation should be helpful in the design of new analogues of tiazofurin for future clinical trials.

Topics: Adenine Nucleotides; Antimetabolites, Antineoplastic; Arabinonucleosides; Cell Line; Cell Survival; Dose-Response Relationship, Drug; Humans; IMP Dehydrogenase; Kinetics; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Nucleosides; Ribavirin

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

Thiazole-4-carboxamide adenine dinucleotide (TAD) is the active anabolite of the new antitumor agent tiazofurin (NSC 286193). TAD is an analogue of NAD in which the nicotinamide ring has been replaced by a thiazole-4-carboxamide heterocycle. TAD putatively acts by inhibition of inosine monophosphate dehydrogenase (IMPd). In this study it is shown that TAD is a competitive inhibitor, with respect to NAD, of mammalian glutamate, alcohol, lactate, and malate dehydrogenases. TAD binds to these enzymes with 1-2 orders of magnitude less affinity than it binds to IMPd. Computer modeling studies suggest that dehydrogenase binding by TAD occurs at the regular cofactor site, the thiazole-4-carboxamide group mimicking the steric and hydrogen-bonding properties of the nicotinamide ring. Noncompetitive kinetics of TAD inhibition of the target enzyme IMPd are potentially due to a reverse order of addition of substrate and cofactor from that observed in the dehydrogenases studied here. The weaker binding of TAD to these dehydrogenases may be due to their inability to preserve a close sulfur-oxygen contact in the bound inhibitor.

Topics: Adenine Nucleotides; Antineoplastic Agents; Binding, Competitive; Computer Simulation; IMP Dehydrogenase; Ketone Oxidoreductases; Kinetics; Models, Chemical; Oxidoreductases; Ribavirin; Ribonucleosides

Tiazofurin was demonstrated to be an effective inhibitor of the growth of human cultured blast cells, and the high specific activities of IMP dehydrogenase (EC 1.1.1.205) were observed in all the cell extracts tested. IMP dehydrogenase has been purified to homogeneity from MOLT 4F human T-lymphoblast, and the Km values for IMP and NAD were 29 and 54 microM, respectively. The inhibitory mechanisms of thiazole-4-carboxamide adenine dinucleotide (TAD) and ribavirin 5'-monophosphate (RMP), the active forms of the antimetabolites tiazofurin and ribavirin, were investigated on the purified enzyme. RMP inhibits competitively with respect to IMP as well as XMP, and the inhibition by TAD was similar to that by NADH, which was uncompetitive with NAD. However, the Ki values of RMP (0.58 microM) and TAD (0.075 microM) were several orders of magnitude lower than those of XMP (85 microM) and NADH (94 microM). Thus, the drugs interact with the two distinct sites of IMP dehydrogenase with much higher affinities than the natural substrates and products. Preincubation of the purified enzyme with RMP enhanced its inhibitory effect in a time-dependent manner, and the enhancement was further increased by the addition of TAD. The combination of tiazofurin and ribavirin exerted a synergistic effect on the growth inhibition in MOLT 4F cells.

Topics: Adenine Nucleotides; Antimetabolites, Antineoplastic; Cell Division; Cell Line; Humans; IMP Dehydrogenase; Inosine Monophosphate; Ketone Oxidoreductases; Kinetics; Leukemia; NAD; Ribavirin; Ribonucleosides

Tiazofurin through its active metabolite thiazole-4-carboxamide adenine dinucleotide (TAD) inhibits IMP dehydrogenase, the rate-limiting enzyme of GTP biosynthesis. IMP dehydrogenase activity in human leukemic cell extracts (33.4 +/- 0.1 nmol/h/mg protein) was increased 11-fold compared to normal leukocytes (3.1 +/- 0.5). Km values for IMP and NAD+ of leukemic IMP dehydrogenase were 22.7 and 44.0 microM, respectively. XMP inhibited competitively with IMP and noncompetitively with NAD+. NADH exerted mixed type inhibition with respect to both IMP and NAD+. The inhibitory pattern of TAD was quite similar to that of NADH; however, the affinity of TAD to leukemic IMP dehydrogenase (Ki = 0.1 microM) was three orders of magnitude higher than the natural product NADH (Ki = 150 microM). These results contribute to an understanding of the mechanism of action of tiazofurin in the treatment of leukemia.

Topics: Adenine Nucleotides; Antineoplastic Agents; Blast Crisis; Female; Humans; IMP Dehydrogenase; Ketone Oxidoreductases; Kinetics; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Middle Aged; NADP; Ribavirin; Ribonucleosides

Thiazole-4-carboxamide adenine dinucleotide (TAD), the active metabolite of the oncolytic C-nucleoside tiazofurin (TR), is susceptible to phosphodiesteratic breakdown by a unique phosphodiesterase present at high levels in TR-resistant tumors. Since accumulation of TAD, as regulated by its synthetic and degradative enzymes, appears to be an important determinant for sensitivity to the drug, a series of hydrolytically resistant phosphonate analogues of TAD were synthesized with the intent of producing more stable compounds with an ability to inhibit IMP dehydrogenase equivalent to TAD itself. Isosteric phosphonic acid analogues of TR and adenosine nucleotides were coupled with activated forms of AMP and TR monophosphate to give dinucleotides 2 and 4. Coupling of protected adenosine 5'-(alpha, beta-methylene)diphosphate with isopropylidene-TR in the presence of DCC afforded compound 3 after deprotection. These compounds are more resistant than TAD toward hydrolysis and still retain potent activity against IMP dehydrogenase in vitro. beta-Methylene-TAD (3), the most stable of the TAD phosphonate analogues, produced a depletion of guanine nucleotide pools in an experimentally induced TR-resistant P388 tumor variant that was superior to that obtained with TR in the corresponding sensitive line.

Topics: Adenine Nucleotides; Cell Line; Chemical Phenomena; Chemistry; Drug Resistance; Guanine Nucleotides; IMP Dehydrogenase; Neoplasms; Organophosphonates; Phosphoric Diester Hydrolases

In order to exert its antitumor effects, the C-nucleoside tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) is converted to the dinucleotide TAD (thiazole-4-carboxamide adenine dinucleotide), an inhibitor of IMP dehydrogenase (IMPD). With few exceptions, sensitive tumors (such as the P388 leukemia) have been found to accumulate substantially more of this inhibitory dinucleotide than resistant strains (exemplified by the colon 38 carcinoma). Previous studies have attributed this difference to a depressed capacity to synthesize TAD on the part of tumors refractory to tiazofurin. In the present study, a second contributory factor has been identified, viz. an enhanced ability to degrade preformed TAD. This degradation has been traced to a soluble phosphodiesterase present at high levels in tumors naturally resistant to tiazofurin. Using standard techniques, this TAD-phosphodiesterase has been purified 200-fold from the colon 38 carcinoma. The activity so purified readily hydrolyzed TAD and ADP-ribose, but exhibited a comparatively weak activity toward NAD and thymidine-5'-monophosphate-nitrophenyl ester. ADP-Ribose was also an excellent inhibitor of the hydrolysis of TAD. It is concluded, on the basis of these results, that TAD-phosphodiesterase plays an important role in the expression of the oncolytic activity of tiazofurin. The suggestion is also made that ADP-ribose may be the natural substrate for this enzyme.

Topics: Adenine Nucleotides; Animals; Chromatography, High Pressure Liquid; Colonic Neoplasms; IMP Dehydrogenase; Leukemia P388; Male; Mice; Phosphoric Diester Hydrolases; Ribavirin; Ribonucleosides

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

The molecular correlation concept proposed that IMP dehydrogenase activity should be a sensitive target of chemotherapy. This hypothesis received support from an array of evidence. IMP dehydrogenase has the lowest activity in purine biosynthesis; it is the rate-limiting enzyme in GTP production; the enzymic activity is transformation-and progression-linked; it is elevated in all examined animal and human neoplastic cells. The activity of GMP synthetase and the concentrations of GMP and dGTP were increased in cancer cells. Whereas guanine salvage has a high potential activity, the low guanine content may well curtail actual salvage capacity. Ribonucleotide reductase activity was two orders of magnitude lower than that of IMP dehydrogenase. Tiazofurin, a C-nucleoside, had marked cytotoxicity on hepatoma cells in vitro and was the first drug that as a single agent profoundly inhibited the proliferation of the subcutaneously inoculated solid hepatoma 3924A in the rat. The impact of tiazofurin administration in hepatoma cells was revealed in a cascade of biochemical alterations involving primary, secondary and tertiary targets and markers of this drug action. The primary target was IMP dehydrogenase where the active metabolite of tiazofurin, TAD, was thought to be absorbed to the NADH site of the enzyme. As a consequence, the enzymic activity declined rapidly to about 30-40% and returned to normal range by 36 to 48 hr after injection. The secondary targets and markers are the profoundly decreased pools of guanylates (GMP, GDP, GTP). Concurrently, the concentrations of IMP and PRPP were increased 8- to 15-fold. The elevated IMP pools were attributed to the de-inhibition of the AMP deaminase activity subsequent to the decline in GTP concentration. The rise in PRPP pools was attributed to the selective inhibition of GPRT and HPRT activities by the high IMP pool which did not affect APRT activity. This interpretation is supported by the 6- to 8-fold increase in the concentrations of guanine and hypoxanthine and the lack of change in the adenine pools inthe hepatomas after tiazofurin administration. The marked drop in NAD concentration which was drug dose- and time-dependent is attributed to the competition for NAD pyrophosphorylase activity by the precursors of NAD and tiazofurin monophosphate. The tertiary targets were dominated by the profound alterations in the concentrations of the dNTPs. This was characterized by a rapid and persistent drop (for 3 days) of th

Topics: Adenine Nucleotides; Animals; Antineoplastic Agents; Guanine Nucleotides; IMP Dehydrogenase; Liver; Liver Neoplasms, Experimental; NAD; Phosphoribosyl Pyrophosphate; Rats; Rats, Inbred Strains; Ribavirin; Ribonucleosides

The antitumor activity of the antineoplastic agent, tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide), has previously been shown to require intracellular anabolism of the drug to a nicotinamide adenine dinucleotide (NAD) analog (2-beta-D-ribofuranosylthiazole-4-carboxamide adenine dinucleotide or "tiazofurin adenine dinucleotide"), which then acts as a potent inhibitor of the target enzyme inosine monophosphate (IMP) dehydrogenase. Inhibition of the latter enzyme in turn brings about a profound depletion of intracellular guanosine nucleotides essential for tumor cell growth and replication. In the present study, the cytotoxicity and metabolism of tiazofurin have been examined in six human lung cancer cell lines. At the pharmacologically attainable drug concentration of 100 microM, colony survival was less than 1.5% in three cell lines ("sensitive"), while survival in the remaining three was greater than 50% ("resistant"). The metabolism of tritiated tiazofurin was examined at concentrations ranging from 0.5 to 100 microM following both brief (6 h) and protracted (14 d) exposures. The sensitive lines accumulated concentrations of tiazofurin adenine dinucleotide that were approximately 10 times those achieved by the resistant lines at both time points. We also observed tendencies for the sensitive cell lines to exhibit: (a) higher specific activities of NAD pyrophosphorylase, the enzyme required for the synthesis of tiazofurin adenine dinucleotide, (b) significantly lower levels of a phosphodiesterase which degrades the latter dinucleotide, (c) greater inhibition of the target enzyme IMP dehydrogenase, and (d) greater depressions of guanosine nucleotide pools after drug treatment. By contrast, the basal levels of IMP dehydrogenase and purine nucleotides in these six lines did not correlate in any obvious way with their responsiveness or resistance. The accumulation and monophosphorylation of parent drug were also not prognostic variables. These studies thus suggest that the extent of accumulation of tiazofurin adenine dinucleotide, as regulated by its synthetic and degradative enzyme activities, is the single most predictive determinant of the responsiveness of cultured human lung tumor cells to tiazofurin.

Topics: Adenine Nucleotides; Antineoplastic Agents; Cell Line; Cells, Cultured; Cytotoxicity Tests, Immunologic; Humans; IMP Dehydrogenase; Lung Neoplasms; Purine Nucleotides; Pyrimidine Nucleotides; Ribavirin; Ribonucleosides; Sepharose; Tumor Stem Cell Assay

The purpose of this investigation was to examine factors which regulate the reprogramming of gene expression in tumors responsible for resistance to tiazofurin. To study the resistance phenomenon drug-induced tumor lines were selected and examined for the mechanism of resistance. A comparison of the biochemical expression of resistance to tiazofurin in drug-induced resistant lines of hepatoma 3924A, leukemias L1210 and P388 revealed that the 3 lines expressed similar genetic alterations related to reduced TAD content, decreased NAD pyrophosphorylase activity and increased synthesis of guanylates from salvaging preformed guanine indicating that these 3 factors play an important role in the resistance to tiazofurin. Resistance was stable in the leukemia lines and did not require drug to maintain resistance. Hepatoma 3924A resistant line reverted to sensitive state in the absence of drug selection pressure. NAD pyrophosphorylase activity was substantially deleted in the tiazofurin resistant leukemia lines, but was only significantly decreased in the hepatoma resistant line. Extensive biochemical alterations including enhanced activity of IMP dehydrogenase, increased inosinate and guanylate pools, and reduced uptake of tiazofurin were found in the hepatoma line resistant to tiazofurin. To examine the applicability of these results to naturally sensitive and spontaneously resistant tumors, murine tumors were examined. In murine tumors, TAD accumulation, ratios of enzyme activities responsible for the synthesis and degradation of TAD, and the ratios of perturbation of inosinate and guanylate pools following tiazofurin challenge demonstrated significant correlation with the sensitive or resistant nature of the tumors. To extrapolate these observations to human tumor systems, cytotoxicity of tiazofurin and its metabolic effects were compared in 6 human lung cancer cell lines derived from cancer patients with small cell lung cancer (4 lines) and lung adenocarcinoma (2 lines). Cell lines exhibiting greater sensitivity to tiazofurin accumulated significantly larger amounts of TAD and showed significant reduction of guanylate pools following tiazofurin incubation. The activity of the enzyme responsible for the formation of TAD, NAD pyrophosphorylase, did not correlate with responsiveness to tiazofurin but the enzyme which hydrolyzes TAD, TADase, correlated positively with the status of resistance.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Adenine Nucleotides; Animals; Antineoplastic Agents; Cell Line; Cell Survival; Drug Resistance; Guanine Nucleotides; Humans; Inosine Nucleotides; Leukemia L1210; Leukemia P388; Liver Neoplasms, Experimental; Lung Neoplasms; Mice; Nicotinamide-Nucleotide Adenylyltransferase; Rats; Ribavirin; Ribonucleosides

The pharmacological effects and metabolism of tiazofurin have been compared in the six transplantable tumors comprising the NCI rodent tumor panel, viz. the P388 leukemia (S); the L1210 leukemia (S); the Lewis lung carcinoma (S); the B16 melanoma (R); the colon 38 carcinoma (R); and the M5076 sarcoma (R), where (S) denotes sensitivity and (R) resistance to tiazofurin. In addition, a variant of the P388 leukemia rendered resistant to the drug in vitro, and maintaining stable resistance in vivo, P388/TR, was also studied. Intraperitoneal administration of tiazofurin (100 mg/kg) resulted in a 3- to 30-fold greater accumulation of thiazole-4-carboxamide adenine dinucleotide (TAD), the proposed active metabolite of the drug in S versus R lines. In general, levels of TAD, percent inhibition of IMP dehydrogenase (mean 40% in S versus 10% in R), depression in the concentration of guanosine nucleotides, (50% in S versus 20% in R) and percent elevation of levels of IMP (500% in S versus 60% in R) correlated well with sensitivity or resistance. However, the B16 melanoma, although resistant to tiazofurin treatment, showed certain biochemical features characteristic of an S line. The sensitive and resistant tumors displayed comparable abilities to phosphorylate tiazofurin, but there was significant depression only in the R lines of the pyrophosphorylase which converts tiazofurin-5'-monophosphate to TAD (mean 78 nmoles/mg protein/hr in S versus 22 nmoles/mg protein/hr in R). The naturally resistant tumors were also found to exhibit a greater ability to degrade synthetic TAD than the sensitive lines (mean 102 nmoles/mg protein/hr in R versus 29 nmoles/mg protein/hr in S lines). The state of sensitivity or resistance could not be attributed to the basal levels of IMP dehydrogenase, to the specific activities of the enzymes of purine salvage, or to the basal concentration of purine and pyrimidine nucleotides. Moreover, treatment with tiazofurin did not influence the enzymes of TAD synthesis or of purine salvage.

Topics: Adenine Nucleotides; Animals; Antineoplastic Agents; Biotransformation; Dose-Response Relationship, Drug; Drug Resistance; Female; IMP Dehydrogenase; Male; Mice; Neoplasms, Experimental; Nucleotides; Ribavirin; Ribonucleosides