Decay rates of anti-HIV dideoxynucleotides in tissue culture systems: a simple correction for the effect of cell replication.

Measurement of intracellular drug levels in cell culture systems can be of predictive value in establishing rational clinical dosage schedules. Such in vitro measurements carried out with anti-HIV agents of the 2',3'-dideoxynucleoside (ddN) class have shown that many of the pharmacologically active ddNTP metabolites of these agents have relatively long intracellular half-lives and little or no host-cell cytotoxicity. As a consequence, replication of drug-exposed cells continues at an unperturbed rate so that a systematic dilution error occurs in the measurement of ddNTP decay half-times. The aim of this study is to present a simple general formulation for the correction of measured t1/2-values for ddNTPs and for other agents with similar intracellular pharmacokinetic properties. Two factors of practical interest emerge: first, the error is greater for agents with slow intracellular clearance rates than for agents with rapid rates; and second, for cell lines with long doubling times, the measured t1/2-values approach more closely to the true t1/2-values, until with the extreme case (quiescent or "G(o)" cells), the observed and true decay times are identical. The greatest dilution errors are seen with adenodine-based agents such as ddATP and 2'-F-ddATP, while the smallest errors are seen with rapidly cleared agents of the dideoxythymidine class.

2',3'-Didehydro-3'-deoxythymidine: regulation of its metabolic activation by modulators of thymidine-5'-triphosphate biosynthesis.

The anti-human immunodeficiency virus (anti-HIV) agent 2',3'-didehydro-3'-deoxythymidine (D4T), like other 2',3'-dideoxynucleosides, requires conversion to its 5'-triphosphate to exert its pharmacological effect. Although D4T-triphosphate is unusually potent as an inhibitor of HIV-1 reverse transcriptase, the phosphorylation of the drug at low dose levels is inefficient because of its low affinity as an alternate substrate for the initial phosphorylation enzyme thymidine kinase. Because thymidine kinase is under feedback regulatory control by the physiological deoxynucleoside-5'-triphosphate dTTP, we examined the effect on D4T phosphorylation and thus, potentially, on its antiviral activity, of a variety of agents that lower intracellular dTTP pools. We found that agents that inhibit the de novo pyrimidine biosynthetic pathway have the ability to increase D4T phosphorylation, the most effective being two inhibitors of thymidylate formation, methotrexate and 5-fluoro-2'-deoxyuridine, compounds that block the enzymes dihydrofolate reductase and thymidylate synthetase, respectively. Because HIV itself lacks the capacity to synthesize dTTP and the other deoxynucleoside triphosphates essential for viral replication, combinations of D4T with modulatory agents that deplete host-cell dTTP, unlike conventional anti-HIV drug monotherapy directed solely at viral enzymes, have the ability to inhibit replication of mutant HIV strains as well as of wild-type virus.

Studies on the mechanism of action of 1-beta-D-arabinofuranosyl-5-azacytosine (fazarabine) in mammalian lymphoblasts.

Fazarabine has shown activity in the panel of 60 cultured human tumor lines of the National Cancer Institute. COMPARE analyses relating correlation coefficients of other anticancer drugs with those of fazarabine suggest that this agent operates through a similar mode of action to that of cytarabine. Studies have been carried out both in culture and in vivo to examine the mechanism of action of fazarabine in P388 murine and Molt-4 human lymphoblasts. Authentic fazarabine nucleotide standards were prepared by chemical and enzymatic methods and characterized on HPLC by comparison to related pyrimidine nucleoside-5'-phosphates as well as by enzymatic digestion. Fazarabine inhibited the incorporation of labeled thymidine into DNA without influencing the synthesis of RNA or protein. Deoxycytidine overcomes this inhibition of DNA synthesis and also prevents the cytotoxicity of the drug to lymphoblasts, probably by competing for fazarabine uptake and metabolism. Fazarabine was rapidly phosphorylated in both cell lines; in P388 cells it was incorporated into DNA, where it continued to undergo the same type of ring opening and degradation as the free nucleoside. Alkaline elution studies demonstrated that exposure to the agent resulted in the formation of alkaline labile sites. Fazarabine also inhibited the methylation of deoxycytidine residues in DNA, but this effect was less pronounced than that produced by 5-azacytidine. Taken together, these studies suggest that fazarabine probably acts by arresting the synthesis and/or altering the structural integrity or functional competence of DNA.

Comparison of the DNA incorporation in human MOLT-4 cells of two 2'-beta-fluoronucleosides, 2'-beta-fluoro-2',3'-dideoxyadenosine and fialuridine.

Incorporation of 2'-beta-fluoro-2',3'-dideoxyadenosine (F-ddA), a recently developed anti-HIV agent, into the cellular DNA of human MOLT-4 cells has been compared with the DNA incorporation seen with fialuridine (FIAU; 1-[2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl]-5-iodouracil), a potent anti-hepatitis B (anti-HBV) nucleoside analogue recently found to cause severe hepatic toxicity in human subjects. At equimolar concentrations (10 microM), incorporation of F-ddA was less than 1% of that for FIAU, a difference attributable to the lack of a 3'-hydroxyl group in the former compound and a consequent inability of F-ddA, unlike FIAU, to form DNA internucleotide linkages.

Enhancement by 2'-deoxycoformycin of the 5'-phosphorylation and anti-human immunodeficiency virus activity of 2',3'-dideoxyadenosine and 2'-beta-fluoro-2',3'-dideoxyadenosine.

The anti-human immunodeficiency virus agents 2',3'-dideoxyadenosine (ddAdo) and 2'-beta-fluoro-2',3'-dideoxyadenosine (2'-beta-F-ddAdo) are rapidly converted, both in vitro and in vivo, to the corresponding inosine analogs by the widely distributed enzyme adenosine deaminase (EC 3.5.4.4). We have determined the effects of the potent adenosine deaminase inhibitor 2'-deoxycoformycin (2'-dCF) on ddAdo and 2'-beta-F-ddAdo metabolism in MOLT-4 cells and on ddAdo antiviral activity in the ATH8 test system. At levels as low as 5 nM in the incubation medium, 2'-dCF effectively blocks the extracellular deamination of both agents, thus permitting their rapid cellular uptake as the unchanged parent compounds, rather than as the less lipid-soluble 2',3'-dideoxyinosine or 2'-beta-fluoro-2',3'-dideoxyinosine. The result is a significant increase in intracellular levels of the pharmacologically active forms 2',3'-dideoxyadenosine-5'-triphosphate and 2'-beta-fluoro-2',3'-dideoxyadenosine-5'-triphosphate. The effect becomes maximal over the range of 50-250 nM 2'-dCF and declines to control levels when extracellular 2'-dCF levels exceed 1 microM. This decrease in ddAdo and 2'-beta-F-ddAdo phosphorylation with higher levels of the inhibitor appears to result from intracellular penetration of 2'-dCF and consequent inhibition of intracellular deamination, a critical step in the activation of both agents through the 5'-nucleotidase pathway. In anti-human immunodeficiency virus assays, a 2.2-fold increase in ddAdo antiviral potency was seen at 2'-dCF levels of 20 and 50 nM.

Comparison of detection methods for adenovirus from enteric clinical specimens.

Fecal samples submitted for virus examination over July 1990 to June 1991 from children < 3 years of age were examined by electron microscopy (EM), virus culture (VC), and enzyme immunoassay [EIA, group-reactive and adenovirus (Ad) 40/41 specific; Cambridge BioScience] to compare the detection rate of adenovirus from pediatric fecal specimens. Ad isolates of serotypes 1-7 grown in HEp-2 or primary rhesus monkey kidney cells were identified by neutralization. Graham 293 cell cultures were used only when specimens were found to be positive for Ad by EM, type-specific Ad40/41 EIA, and for isolates not identified by neutralization. Ads grown in 293 cells were identified by DNA restriction endonuclease analysis. Of the 1187 specimens examined, 105 (9%) were found to be positive for Ad. VC detected 93, while 12 additional positives were detected by EM or EIA. The relative sensitivity of VC, EIA, and EM for the 105 specimens was 89% (93), 45% (47), and 35% (37), respectively. Among the 105 positive specimens, enteric Ad, nonenteric Ad, and untypeable Ad were 28% (29), 65% (68), and 7% (8), respectively. Of 37 EM positives, 62% (23) were enteric Ad; 27% (10) were nonenteric including serotypes 2, 3, 4, 5, 12, and 31, with 4, 1, 1, 2, 1, and 1 isolates of each type positive, respectively; and 11% (4) were detectable only by EM. Five isolates were identified as variant of Ad 2(3), Ad 3(1) and Ad 31(1). Over a 1-year period, a single Ad41 variant strain was the most frequently detected enteric Ad in Winnipeg, Manitoba, Canada.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced stimulation by ribavirin of the 5'-phosphorylation and anti-human immunodeficiency virus activity of purine 2'-beta-fluoro-2',3'-dideoxynucleosides.

The purine dideoxynucleosides 2'-beta-fluoro-2',3'-dideoxyadenosine (2'-beta-F-ddAdo), 2'-beta-fluoro-2',3'-dideoxyinosine, and 2'-beta-fluoro-2',3'-dideoxyguanosine (2'-beta-F-ddGuo) are active inhibitors of the replication of the human immunodeficiency virus (HIV) in the ATH8 assay system, with 2'-beta-F-ddAdo and 2'-beta-fluoro-2',3'- dideoxyinosine showing activity and potency equivalent to those of their respective parent compounds, 2',3'-dideoxyadenosine (ddAdo) and 2',3'-dideoxyinosine. Because inhibitors of IMP dehydrogenase such as ribavirin and tiazofurin stimulate the 5'-phosphorylation and consequently the anti-HIV activity of the three nonfluorinated parent compounds (ddAdo, 2',3'-dideoxyinosine, and 2',3'-dideoxyguanosine), we have undertaken a study in MOLT-4 cells to determine whether a similar stimulatory effect is observed with their 2'-beta-fluorinated analogs. The 5'-phosphorylation of all the fluoro compounds was found to be greatly enhanced by low levels (10 microM) of either ribavirin or tiazofurin, with the greatest increase being seen with 2'-beta-F-ddAdo, where stimulation of the formation of the 5'-mono-, di-, and triphosphorylated nucleotides was approximately 20-fold, 6-fold, and 5-fold, respectively. These increases were approximately 3-fold greater than the increases seen with the nonfluorinated parent compound ddAdo. In the case of 2'-beta-F-ddGuo, the greatest stimulation (8-fold) was seen in the formation of the 5'-diphosphate. In parallel with the increased phosphorylation of 2'-beta-F-ddAdo and 2'-beta-F-ddGuo, the anti-HIV potency of these two compounds at the 5 microM level was approximately doubled in the presence of ribavirin (5 microM).

Cellular pharmacology of cyclopentenyl cytosine in Molt-4 lymphoblasts.

The toxicity, uptake, and metabolism of the oncolytic nucleoside cyclopentenyl cytosine (CPEC) have been examined in the Molt-4 line of human lymphoblasts. This compound is known to be converted to its 5'-triphosphate, which inhibits CTP synthetase and depletes the pools of cytidine nucleotides. In the Molt-4 system, the concentration of drug reducing proliferation by 50% in a 24-h incubation was between 50 and 100 nM. Cytidine, uridine, and nitrobenzylthioinosine almost fully prevented the cytotoxicity of CPEC when introduced shortly before or together with the drug, but only cytidine was effective as an antidote when added 12 h after 200 nM CPEC. Studies of the cellular entry of CPEC revealed that nitrobenzylthioinosine fully blocked this process over a 60-s interval and for as long as 2 h, suggesting that the initial interiorization was mediated by facilitated diffusion. In Molt-4 cells incubated with tritiated CPEC, 9 metabolites could be distinguished: prominent among these was cyclopentenyl uridine (CPEU), the deamination product of CPEC; other major metabolites included the 5'-mono-, di-, and triphosphates of CPEC, and of CPEU, along with two phosphodiesters provisionally identified as CPEC-diphosphate choline and CPEC-diphosphate ethanolamine. When the accumulation of CPEC-5'-triphosphate was measured as a function of concentration of the drug in the medium, the process was found not to be saturable by levels of CPEC up to 1000 nM. In cells incubated with 200 nM drug, CPEC-5'-triphosphate accumulated rapidly and linearly for approximately 4 h, the time for doubling of the concentration being 2 h. After a 16-h incubation with 100 nM CPEC, the concentration of CPEC-5'-triphosphate was 50-fold that of the parent drug in the medium and could be readily monitored spectrophotometrically in high-pressure liquid chromatography effluents without recourse to radiolabeled nucleoside. In 2-h incubations, the concentration of free CPEC required to reduce CTP by 50% was 150 nM; this corresponded to a CPEC-5'-triphosphate level of 750 nM. After washout of extracellular CPEC, CPEC-5'-triphosphate decayed with a half-life that ranged from 9 to 14 h. Twenty-four h after washout of 200 nM CPEC (the concentration of drug reducing proliferation by 80%), cells had not resumed proliferation, and CTP pools were still depressed by 90%. Cytidine, uridine, and nitrobenzylthioinosine all strongly repressed the anabolic phosphorylation of CPEC when added to Molt-4 cells along with the drug.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibitors of IMP dehydrogenase stimulate the phosphorylation of the anti-human immunodeficiency virus nucleosides 2',3'-dideoxyadenosine and 2',3'-dideoxyinosine.

2',3'-Dideoxyadenosine (ddAdo) and its deamination product 2',3'-dideoxyinosine (ddIno) (didanosine) inhibit the replication and infectivity of the human immunodeficiency virus (HIV) in a number of in vitro assay systems. Early clinical studies (phase I) have indicated a role for ddIno in the treatment of patients with severe HIV infection. In the present in vitro study, the formation in human T cells (MOLT-4, ATH8, and CCRF-CEM) of the pharmacologically active metabolite of ddIno and ddAdo, 2',3'-dideoxyadenosine-5'-triphosphate (ddATP), was found to be stimulated 2-4-fold by appropriate concentrations of inosinate dehydrogenase (IMPD) inhibitors such as ribavirin, tiazofurin, and mycophenolic acid. Concomitant with this increase in ddATP formation from ddIno was an increase in anti-HIV activity of this agent when it was combined with ribavirin in the ATH8 cell assay system and with tiazofurin in the MOLT-4 assay system. No change was noted in the intracellular concentration of the corresponding physiological deoxynucleoside-5'-triphosphate, dATP; positive correlation was observed, however, between the increase in ddATP formation from ddIno and the increase in intracellular IMP occurring as a consequence of IMPD inhibition. The results support the hypothesis that the stimulation of ddATP formation seen when ddIno is combined with ribavirin or other IMPD inhibitors is a consequence of an increased concentration of IMP, the major phosphate donor for the initial phosphorylation step in the anabolism of ddIno to ddATP, i.e., ddIno----ddIMP.

2'-Fluoro-2',3'-dideoxyarabinosyladenine: a metabolically stable analogue of the antiretroviral agent 2',3'-dideoxyadenosine.

In this report, we have compared the uptake, metabolism, and relevant enzymology of a novel anti-acquired immunodeficiency syndrome drug, 2'-fluoro-2',3'-dideoxyarabinosyladenine (2'-F-dd-ara-A) with the corresponding properties of its parent compound 2',3'-dideoxyadenosine (2',3'-ddAdo) in three human T cell lines, MOLT-4, ATH8, and CEM. In previous communications, we have reported that the primary route of metabolism of 2',3'-ddAdo in human T lymphoblasts is catabolic, i.e., deamination to 2',3'-dideoxyinosine (2',3'-ddlno). At this point, the metabolic pathway diverges, to result in either cleavage and inactivation of 2',3'-ddlno by purine nucleoside phosphorylase or in 5'-phosphorylation by a phosphotransferase, a reaction that generates 2',3'-inosine monophosphate and ultimately the putative active metabolite 2',3'-dideoxy-ATP. Studies with kinase-deficient mutant CEM lines indicate, however, that 2'-F-dd-ara-A favors a more direct anabolic route toward formation of 2'-fluoro-dideoxynucleotides, catalyzed initially by 2'-deoxycytidine kinase. In MOLT-4 cells, amounts of 2'-fluoro-dideoxyarabinosyladenine di- and triphosphate formed were approximately 20-fold and 5-fold greater than the respective accumulation of 2',3'-dideoxy-ADP and 2',3'-dideoxy-ATP over the same time of exposure. This metabolic profile was supported by enzymological studies, which revealed that 2'-F-dd-ara-A is deaminated 10 times less rapidly than ddAdo and that the resulting deaminated product is resistant to hydrolysis by purine nucleoside phosphorylase. Under similar conditions, ddAdo was rapidly degraded through cleavage of its deamination product ddlno. Like ddAdo, 2'-F-dd-ara-A was found to be transported by passive diffusion and does not enter cells via the purine nucleoside transport carrier system. However, the rate of entry of 2'-F-dd-ara-A was about half that of ddAdo (9.7 pmol/10(6) cells/min for 2'-F-dd-ara-A versus 18.4 pmol/10(6) cells/min for ddAdo). This investigation, therefore, demonstrates that, under the conditions studied, 2'-F-dd-ara-A and its deamination product 2'-fluoro-2',3'-dideoxyarabinosylhypoxanthine have metabolic properties that differ significantly from those of their parent compounds ddAdo and ddlno. These properties, combined with the previously reported resistance of the fluorinated nucleosides to acid degradation, make these compounds interesting candidates for further study as orally administered agents for the inhibition of human immunodeficiency virus replication in patients with acquired immunodeficiency syndrome.

Metabolism and action of amino acid analog anti-cancer agents.

The preclinical pharmacology, antitumor activity and toxicity of seven of the more important amino acid analogs, with antineoplastic activity, is discussed in this review. Three of these compounds are antagonists of L-glutamine: acivicin, DON and azaserine; and two are analogs of L-aspartic acid: PALA and L-alanosine. All five of these antimetabolites interrupt cellular nucleotide synthesis and thereby halt the formation of DNA and/or RNA in the tumor cell. The remaining two compounds, buthionine sulfoximine and difluoromethylornithine, are inhibitors of glutathione and polyamine synthesis, respectively, with limited intrinsic antitumor activity; however, because of their powerful biochemical actions and their low systemic toxicities, they are being evaluated as chemotherapeutic adjuncts to or modulators of other more toxic antineoplastic agents.

Comparison of cell culture and three enzyme-linked immunosorbent assays for the rapid diagnosis of respiratory syncytial virus from nasopharyngeal aspirate and tracheal secretion specimens.

A total of 211 specimens, including 144 nasopharyngeal aspirates and 67 tracheal secretions, were evaluated for the rapid detection of Respiratory Syncytial Virus (RSV) antigen by three commercial enzyme-linked immunosorbent assays (ELISA; Kallestad Diagnostic, Austin, TX; Ortho Diagnostics, Raritan, NJ, and Abbott Laboratories, North Chicago, IL) and by isolation of RSV in cell culture. Of the 61 RSV culture positive specimens, Kallestad ELISA, Ortho ELISA, and Abbott ELISA detected RSV antigen in 80%, 95%, and 92% of the specimens, respectively. An additional 28 specimens were found to be positive only by one or more RSV ELISA tests. A blocking assay confirmed the specificity of ELISA in 71% (20/28) of the RSV ELISA positive and the culture-negative specimens, and 29% were found to be false positive. Through the use of cell culture, with the resolution of ELISA positive and culture negative specimens by blocking assay, 81 specimens (61 + 20) were considered to be true positive. The sensitivity, specificity, and diagnostic accuracy were, for cell culture, 75%, 100%, and 91%; for Kallestad ELISA, 79%, 98%, and 91%; for Ortho ELISA, 95%, 99%, and 98%; and for Abbott ELISA, 93%, 96%, and 95% respectively. In our study, commercial ELISA tests have been shown to be rapid, reliable tests for the detection of RSV. Ortho ELISA and Abbott ELISA showed better sensitivity than the Kallestad ELISA for RSV detection directly in the clinical specimens.

Studies on the mechanism of action of tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide). VI. Biochemical and pharmacological studies on the degradation of thiazole-4-carboxamide adenine dinucleotide (TAD).

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.

Arabinosyl-5-azacytosine: mechanisms of native and acquired resistance.

Factors influencing the activity of the nucleoside analogue arabinosyl-5-azacytosine (ara-AC) were studied in P388 murine lymphoblasts in vitro and in vivo, in variants of these cells with artificially acquired resistance, in the naturally resistant colon 38 carcinoma in vivo, and in a panel of six human tumors maintained in continuous culture. Differences were noted not only between the sensitive and artificially developed resistant variants of P388, but also between the naturally sensitive (P388) and naturally resistant (colon 38) tumors. The artificially developed resistant P388 cell lines showed an inhibited capacity to accumulate nucleotides derived from ara-AC and deoxycytidine, whereas the accumulation of cytidine nucleotides remained unchanged. Studies of the initial velocity of facilitated diffusion of ara-AC showed only minor differences between parental and resistant lines, while the nucleotide formation rates from both ara-AC and deoxycytidine were markedly depressed in the latter cells. It is concluded, therefore, that the failure of resistant P388 cells to accumulate these compounds results not from a transport deficit per se but rather from a failure to convert the nucleosides to nondiffusible (i.e., phosphorylated) species inside the cell. This failure was accompanied by a substantial reduction in the incorporation of a radiolabeled product derived from deoxycytidine into the nucleic acids of the resistant clones. The common factor responsible for the resistance of P388 variants toward ara-AC appears to be a markedly decreased level of deoxycytidine kinase activity. The naturally resistant colon 38 carcinoma, on the other hand, in addition to a decrease in the activity of its deoxycytidine kinase, showed a lower level of activity of all its purine and pyrimidine kinases, along with a notably elevated nucleoside triphosphatase activity (with ATP as substrate) when compared to P388. These differences were reflected in lower endogenous nucleoside triphosphate pool sizes in colon 38, and in a lower level of ara-AC-5'-triphosphate accumulation in colon 38 than in P388 after comparable drug exposure. In the six human tumor lines, a positive correlation was established between sensitivity to ara-AC (as determined by its median inhibitory concentration) and cellular content of deoxycytidine kinase. It is concluded that this latter enzyme is a generally important determinant of sensitivity to arabinosyl-5-azacytosine.

Thiazole-4-carboxamide adenine dinucleotide (TAD). Analogues stable to phosphodiesterase hydrolysis.

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.

Relationships between the cytotoxicity of tiazofurin and its metabolism by cultured human lung cancer cells.

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.

Ribavirin, tiazofurin, and selenazofurin: mononucleotides and nicotinamide adenine dinucleotide analogues. Synthesis, structure, and interactions with IMP dehydrogenase.

A series of dinucleotides, analogous to nicotinamide adenine dinucleotide but containing the five-membered base nucleosides tiazofurin (1a), selenazofurin (1b), ribavirin (2), and AICAR (3) in place of nicotinamide ribonucleoside, were prepared in greater than 50% yield by reacting the corresponding nucleotide imidazolidates (6a-d) with adenosine 5'-monophosphate (AMP). The symmetric dinucleotides of tiazofurin (TTD, 8e) and selenazofurin (SSD, 8f) were also prepared by a similar methodology. These dinucleotides were characterized by 1H NMR and fast atom bombardment MS and were evaluated for their inhibitory potency against a partially purified preparation of tumoral inosine monophosphate dehydrogenase (IMPD) from P388 cells. The order of potency found was SAD (8b) greater than TAD (8a) much greater than SSD (8f) congruent to TTD (8e) congruent to RAD (8c) much much greater than ZAD (8d). On kinetic analysis none of the dinucleotides produced competitive inhibition of IMPD with NAD as a variable substrate. In addition to their superior IMPD inhibitory activity, SAD and TAD appear to be the only dinucleotides, besides NAD, that are formed naturally by the NAD pyrophosphorylase from P388 lymphoblasts.