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Abstract Background Patients with hypertension continue to have less than optimal blood pressure control, with nearly one in five Canadian adults having hypertension. Pharmacist prescribing is gaining favor as a potential clinically efficacious and cost-effective means to improve both access and quality of care. With Alberta being the first province in Canada to have independent prescribing by pharmacists, it offers a unique opportunity to evaluate outcomes in patients who are prescribed antihypertensive therapy by pharmacists. Methods The study is a randomized controlled trial of enhanced pharmacist care, with the unit of randomization being the patient. Participants will be randomized to enhanced pharmacist care (patient identification, assessment, education, close follow-up, and prescribing/titration of antihypertensive medications) or usual care. Participants are patients in rural Alberta with undiagnosed/uncontrolled blood pressure, as defined by the Canadian Hypertension Education Program. The primary outcome is the change in systolic blood pressure between baseline and 24 weeks in the enhanced-care versus usual-care arms. There are also three substudies running in conjunction with the project examining different remuneration models, investigating patient knowledge, and assessing health-resource utilization amongst patients in each group. Discussion To date, one-third of the required sample size has been recruited. There are 15 communities and 17 pharmacists actively screening, recruiting, and following patients. This study will provide high-level evidence regarding pharmacist prescribing. Trial Registration Clinicaltrials.gov NCT00878566.

A method is described for the preparation of ganciclovir triphosphate (GCV-TP) using murine colon cancer cells (MC38) transduced with the herpes simplex virus-thymidine kinase (MC38/HSV-tk). Murine cells transduced with viral-tk contain required viral and host enzymes needed for complete cellular synthesis of this potent antiviral metabolite. Dose response studies showed optimal intracellular levels of GCV-TP occurred after exposure of MC38/HSV-tk cells to 300 microM ganciclovir for 24 h producing 7.5 nmol GCV-TP/10(6) cells. This reflects cellular accumulation of GCV-TP to levels 25-fold greater than the medium concentration of parent drug. A simple isolation scheme included methanolic extraction and anion-exchange chromatography to recover the target triphosphate. Mass spectral analysis and selective enzyme degradation provided structural confirmation of the purified product. Biological activity of the purified GCV-TP was demonstrated by competitive inhibition experiments using human DNA polymerase alpha and HSV DNA polymerase that showed substantially greater sensitivity for the viral polymerase in agreement with previous reports. The GCV-TP obtained was further used to enzymatically prepare GCV mono- and diphosphate in high yield. This method provides an easily scalable means of preparing milligram amounts of the triphosphates of pharmacologically active acyclic nucleosides like ganciclovir.

Inosine 5 -monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme for the synthesis of GTP and dGTP. Two isoforms of IMPDH have been identified. IMPDH Type I is ubiquitous and predominantly present in normal cells, whereas IMPDH Type II is predominant in malignant cells. IMPDH plays an important role in the expression of cellular genes, such as p53, c-myc and Ki-ras. IMPDH activity is transformation and progression linked in cancer cells. IMPDH inhibitors, tiazofurin, selenazofurin, and benzamide riboside share similar mechanism of action and are metabolized to their respective NAD analogues to exert antitumor activity. Tiazofurin exhibits clinical responses in patients with acute myeloid leukemia and chronic myeloid leukemia in blast crisis. These responses relate to the level of the NAD analogue formed in the leukemic cells. Resistance to tiazofurin and related IMPDH inhibitors relate mainly to a decrease in NMN adenylyltransferase activity. IMPDH inhbitors induce apoptosis. IMPDH inhitors are valuable probes for examining biochemical functions of GTP as they selectively reduce guanylate concentration. Incomplete depletion of cellular GTP level seems to down-regulate G-protein function, thereby inhibit cell growth or induce apoptosis. Inosine 5'-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) catalyzes the dehydrogenation of IMP to XMP utilizing NAD as the proton acceptor. Studies have demonstrated that IMPDH is a rate-limiting step in the de novo synthesis of guanylates, including GTP and dGTP. The importance of IMPDH is central because dGTP is required for the DNA synthesis and GTP plays a major role not only for the cellular activity but also for cellular regulation. Two isoforms of IMPDH have been demonstrated. IMPDH Type I is ubiquitous and predominately present in normal cells, whereas the IMPDH Type II enzyme is predominant in malignant cells. Although guanylates could be salvaged from guanine by the enzyme hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8), the level of circulating guanine is low in dividing cells and this route is probably insufficient to satisfy the needs of guanylates in the cells.

Three carbocylic analogues of the potent cytidine deaminase inhibitor (CDA) zebularine [1-(beta-D-ribofuranosyl)-1, 2-dihydropyrimidin-2-one, 1a] were synthesized. The selected pseudosugar templates correspond, respectively, to the cyclopentenyl moiety of neplanocin A (compound 4), the cyclopentyl moiety of aristeromycin (compound 5), and a newly designed, rigid bicyclo[3.1. 0]hexane moiety (compound 6). These three carba-nucleoside versions of zebularine were fashioned to overcome the inherent instability of the parent drug. Each target compound was approached differently using either convergent or linear approaches. The immediate precursor to the cyclopentenyl analogue 4 was obtained by a Mitsunobu coupling of pseudosugar 7 with 2-hydroxypyrimidine. The cyclopentyl analogue 5 was linearly constructed from carbocyclic amine 17, and the final target 6 was similarly constructed from the carbobicyclic amine 27. Of the three target compounds, only 5 showed a significant level of inhibition against human CDA, but it was 16 times less potent than zebularine (Ki = 38 microM vs Ki(apparent) = 2.3 microM). Although these carbocyclic analogues appeared to be more stable than zebularine, replacement of the electronegative CO4' oxygen for the less electronegative carbon in 4-6 presumably reduces the capacity of the pyrimidin-2(1H)-one ring to form a covalent hydrate, a step considered crucial for the compound to function as a transition-state inhibitor of the enzyme.

Benzamide riboside (BR) exhibits potent antitumor activity in a variety of cultured human tumor cells. The drug is metabolized to benzamide adenine dinucleotide (BAD), which in turn functions as a selective inhibitor of IMP dehydrogenase (IMPDH) activity with a Ki of 0.118 microM. In vitro, BR is a more potent antitumor inhibitor of IMPDH than tiazofurin, another IMPDH inhibitor which has shown significant oncolytic activity in adult patients with end-stage leukemia. To elucidate the mechanism of resistance, a variant of human myelogenous leukemia K562 cells was developed by subculturing sensitive cells in sublethal concentrations of BR over 60 generations. The BR resistant line that emerged exhibited an IC50 (a concentration producing 50% reduction in cell proliferation) of 148 microM, compared to the sensitive line which had an IC50 of 1.6 microM. The activity of the target enzyme, IMPDH, was increased 3-fold in the resistant variant. Studies on BR metabolism revealed that resistant cells formed only 18% of the active metabolite, BAD, compared to sensitive cells. This finding, in turn, correlated with the specific activity of NAD pyrophosphorylase (the enzyme responsible for the synthesis of BAD) which was reduced to undetectable levels in the resistant variant. The basal levels of NAD and guanylates were also significantly decreased to 41% and 48%, respectively, in the resistant line compared to the parent line. Additionally, after treatment with BR a decrease in guanylate level was observed only in the sensitive cells. Sensitive and resistant cells exhibit comparable cytotoxicity to agents outside the tiazofurin family, suggesting that a multidrug resistance was unlikely to explain the resistance to BR. Moreover, BR resistant cells exhibit collatoral sensitivity to 6-aminopurine, cytarabine and 5-fluorouracil, which have different mechanisms of action. In conclusion, these studies establish that the primary mechanism of resistance to BR involves an increase in IMPDH (target enzyme) activity with a concurrent decrease in NAD pyrophosphorylase (BAD synthetic enzyme) activity.

A novel inhibitor of dihydroorotate dehydrogenase (DHO-DH) has been discovered using data from the National Cancer Institute's in vitro drug screen. Upon analysis of cytotoxicity results from the sixty tumor cell lines used in this screen, the COMPARE program predicted that NSC 665564 was likely to have the same mechanism of inhibition as brequinar, a known potent inhibitor of DHO-DH. We validated this prediction experimentally using MOLT-4 lymphoblast and found the IC50 of brequinar (0.5 microM) and NSC 665564 (0.3 microM) were comparable and that this induced cytotoxicity was reversed by either uridine or cytidine. The enzyme target of NSC 665564 was shown to be identical to that of brequinar when incubation with each drug followed by a 1 h pulse with [14C] sodium bicarbonate resulted in cellular accumulation of [14C]N-carbamyl-L-aspartic acid and [14C]L-dihydroorotic acid, with concurrent marked depletion of CTP and UTP. The Ki's for NSC 665564 and brequinar were 0.14 and 0.24 microM, respectively, when partially purified MOLT-4 mitochondria (the site of DHO-DH) were used. These results show that mechanistic predictions obtained using correlations from the COMPARE algorithm are independent of structure since the structure of NSC 665564 is dissimilar to that of other established DHO-DH inhibitors.

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.

The computer algorithm COMPARE provides information regarding the biological mechanism of action of a compound. In this study, excellent correlations were obtained for 2,2'-[3,3'-dimethoxy[1,1'-biphenyl]-4,4'-diyl)diimino]bis- benzoic acid (redoxal) and 1-(p-bromophenyl)-2-methyl-1H- naphth[2,3-d]imidazole-4,9-dione (BNID) and two well-studied dihydroorotate dehydrogenase (DHOD) inhibitors, dichloroallyl lawsone and brequinar, in terms of antiproliferative activity against tumor cell lines in vitro. When redoxal and BNID were incubated with MOLT-4 cells for 72 hr, 50% growth inhibition was achieved at 0.7 and 3.5 microM, respectively. After 24 hr of incubation, pyrimidine triphosphate pools were shown to be decreased by 50% by redoxal (1 microM) and BNID (0.25 microM). Addition of either uridine (50 microM) or cytidine (100 microM) antagonized the cellular cytotoxicity caused by either drug; uridine corrected the UTP and CTP deficit, whereas cytidine corrected only the CTP deficit. Exposure of MOLT-4 cells to a 1 microM concentration of either drug for 18 hr followed by a 1-hr exposure to [14C]bicarbonate showed a 97% decrease of incorporation of [14C] into pyrimidine triphosphates accompanied by a 91- and 82-fold increase in radioactive incorporation into L-dihydroorotate and N-carbamyl-L-aspartate, respectively. By direct exposure of DHOD prepared from MOLT-4 cell mitochondria to a range of concentrations of the two drugs, apparent Ki values of 0.33 microM (redoxal) and 0.53 microM (BNID) were determined. These data provide direct evidence for inhibition of DHOD by redoxal and BNID in MOLT-4 lymphoblasts.

The 2'-deoxy (2a) and 2'-ara-fluoro (3a) derivatives of zebularine [1-(beta-D-ribofuranosyl)-dihydropyrimidin-2-one, 1a] were phosphorylated in high yield to the 5'-nucleotides 2b and 3b, respectively, and characterized by HPLC, enzyme degradation, 1H, 13C and 31P NMR, and high resolution mass spectral analysis. Their inhibitory activity against partially purified MOLT-4 deoxycytidylate deaminase (dCMPD) in the presence of the allosteric effector deoxycytidine triphosphate (dCTP) and Mg+2 ion was examined. Compounds 2b and 3b inhibited dCMPD with Ki values of 2.1 x 10(-8) M and 1.2 x 10(-8) M, respectively. The parent nucleotide, zebularine monophosphate 1b was ineffective at concentrations > 100 mumol. The effect of the nucleosides, 1a-3a, as well as tetrahydrouridine (THU) and 2'-deoxy THU (dTHU), on the cellular production of DNA precursors was examined in human MOLT-4 peripheral lymphoblasts. It was shown that 1a, 2a and 3a all elevated intracellular dCTP and TTP levels in whole cells with the most powerful effect elicited by 1a. The 2'-fluoro derivative 3a was chemically phosphorylated much more cleanly and higher yield than 2a, without the formation of diphosphorylated by-products. This compound was found to be infinitely less sensitive to acid-catalyzed degradation than 2a. Since the substitution of fluorine for hydrogen had a slight potentiating effect on the dCMPD inhibitory activity while stabilizing the compound toward acid-catalyzed and enzymatic depyrimidination, compound 3b emerges as a very attractive tool for the pharmacological modulation of pyrimidine deaminase activity.

With increasing awareness of the mitochondrial toxicity associated with certain 2',3'-dideoxynucleosides used in anti-human immunodeficiency virus therapy, procedures for quantitative analyses of drug effects on mitochondrial DNA (mtDNA) have assumed enhanced importance. For this reason we have developed a method to measure the copy numbers of mtDNA in cultured MOLT-4 cells. First a hybrid competitive DNA template was synthesized by conventional polymerase chain reaction (PCR), using two custom-synthesized 40-mer composite primers incorporating mitochondrial displacement loop sequences linked by a non-mitochondrial cDNA template (a 76-base pair sequence from the tat/rev region of human immunodeficiency virus cDNA). For the competitive assay, increasing known copy numbers of the hybrid competitive template were added as an internal control to samples containing total cellular DNA. With this approach, two competitive PCR products were generated, 1) a mitochondrial displacement loop-derived fragment (182 base pairs) and 2) a competitive DNA template-derived fragment (156 base pairs). Absolute quantitation was achieved by radiometric comparison of the relative amounts of the two products. To test the versatility of this method, varying amounts of competitive template (6.6 x 10(4) to 6.6 x 10(9) copies) were used with a fixed quantity of total cellular DNA taken from cells cultured for 9 days in the presence or absence of selected pyrimidine and purine dideoxynucleosides. The results showed that the copy number of cellular mtDNA is 823 +/- 71 copies/cell in MOLT-4 cells. Little selective depletion of mtDNA, compared with total cellular DNA, was seen with the purine dideoxynucleosides examined; however, when the cells were exposed to the pyrimidine dideoxynucleoside 2',3'-dideoxycytidine (50 nM) for 9 days, mtDNA content was specifically depleted, although total cellular DNA decreased by only 10%. Thus, in addition to the presently used methods of assessing mitochondrial impairment, i.e., Southern blot analysis and electron microscopy, the competitive PCR method provides a third and convenient assay, with particular applicability to determination of mtDNA in very small numbers of cells.

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.

The biochemical and cytotoxic activities of the IMP dehydrogenase (IMPDH) inhibitors benzamide riboside, tiazofurin, and selenazofurin were compared. These three C-nucleosides exert their cytotoxicity by forming an analogue of NAD, wherein nicotinamide is replaced by the C-nucleoside base. The antiproliferative activities of these three agents were compared in a panel of 60 human cancer cell lines. To examine the relationship of benzamide riboside and selenazofurin to tiazofurin, COMPARE computer analysis was performed, and correlation coefficients of 0.761 and 0.815 were obtained for benzamide riboside and selenazofurin, respectively. The biochemical activities of these agents were examined in human myelogenous leukemia K562 cells. Incubation of K562 cells for 4 hr with 10 microM each of benzamide riboside, selenazofurin and tiazofurin resulted in a 49, 71, and 26% decrease in IMPDH activity with a concurrent increase in intracellular IMP pools. As a consequence of IMPDH inhibition, GTP and dGTP concentrations were curtailed. These studies demonstrated that selenazofurin was the most potent of the three agents. To compare the cellular synthesis of NAD analogues of these agents, K562 cells were incubated with 10 microM each of benzamide riboside, tiazofurin and selenazofurin after prelabeling the cells with [2,8-3H]adenosine. The results demonstrated that benzamide riboside produced 2- and 3-fold more of NAD analogue (BAD) than tiazofurin and selenazofurin did. To elucidate the effects of the three compounds on other NAD-utilizing enzymes, the inhibitory activities of purified benzamide adenine dinucleotide (BAD), thiazole-4-carboxamide adenine dinucleotide (TAD) and selenazole-4-carboxamide adenine dinucleotide (SAD) were studied in commercially available purified preparations of lactate dehydrogenase, glutamate dehydrogenase and malate dehydrogenase. TAD and SAD did not inhibit these three dehydrogenases. Although BAD did not influence lactate and glutamate dehydrogenases, it selectively inhibited 50% of malate dehydrogenase activity at a 3.2 microM concentration. These studies demonstrate similarities and differences in the biochemical actions of the three C-nucleosides, even though they share similar mechanisms of action.

Megakaryocyte differentiation is a lengthy process with cells moving through a continuum delineated by the sequential expression of specific gene products. The limited number of primary cells available from marrow for analysis has brought attention to some leukemic cell lines which show enhanced megakaryocyte marker expression following incubation with inducing agents, the most common of which is phorbol myristate acetate (PMA). We developed an alternative induction protocol for the megakaryocytic leukemic cell line CMK, which involved incubation of the cells with IL-3 and the nucleoside analog, ribavirin, for 1-2 weeks. This treatment was neither toxic nor cytostatic and yielded increased levels of the surface glycoproteins GPIIb/IIIA and GPIb-IX. Levels of some megakaryocytic messages (GPIIIa, GPIX) showed a marked rise by 12 days of incubation in the inducer combination. This was due to a synergistic interaction between IL-3 and ribavirin which influenced both transcriptional and posttranscriptional events. Light and electron microscopy demonstrated the presence of large polyploid cells, with morphological features similar to those of megakaryocytes, in the induced cultures. Analysis of the heterogeneity of response in the cell population to the induction regimen after several days of treatment suggested that cells which failed to display surface markers had been stimulated by the inducers but did not have sufficient time to complete expression of that marker. The results were consistent with the view that the cells in the starting population were distributed along a temporal expression pathway, and those which were first to express the earliest marker would also lead in the expression of a later marker. The order of expression was the same as that during normal megakaryocyte development.

We have undertaken to characterize the role of cytoplasmic 5'-nucleotidase (EC 3.1.3.5) in the phosphorylation of the anti-herpes simplex virus and anti-human cytomegalovirus agent ganciclovir (GCV) in MOLT-4 cells, a human T cell line adapted to grow in suspension culture. The rate of formation of GCV triphosphate was found to be approximately doubled by preincubation of nontransfected MOLT-4 cells with agents that cause the accumulation of IMP, such as ribavirin (20 microM) and mycophenolic acid (1 microM), and the reaction rate was found to be unaffected by high levels of thymidine (100 microM). With herpes simplex virus-1 thymidine kinase (HStk) gene-transduced MOLT-4 cells, the rate of GCV phosphorylation was approximately 40-fold faster than that in uninfected cells and, in marked contrast to uninfected cells, the reaction was significantly inhibited both by IMP dehydrogenase inhibitors and by thymidine. These latter effects appear to be the result of 1) the accumulation of high levels of dTTP in IMP dehydrogenase inhibitor-treated cells, with consequent feedback inhibition of HStk, and 2) direct competitive substrate inhibition by thymidine of the HStk-catalyzed phosphorylation of GCV. Thus, agents that enhance 5'-nucleotidase-catalyzed phosphorylation of GCV in uninfected cells do not play a similar role in HStk-transfected cells, a consequence of the quantitative predominance of the viral thymidine kinase-catalyzed reaction over that attributable to endogenous cytoplasmic 5'-nucleotidase.

We have examined a series of tyrosine kinase inhibitors structurally related to erbstatin (tyrphostins) for inhibition of p210bcr-abl autokinase activity in vitro and for growth inhibition of chronic myelogenous leukemia (CML)K562 cells. Of the tyrphostins with IC50 for growth < 50 microM, AG814, AG946, AG952, AG896, AG953, AG956 and AG957 (structurally related to lavendustin A and piceatannol) completely inhibited p210bcr-abl kinase activity in an immune complex kinase assay. Another group of tyrphostins (AG807, AG568, AG763, AG1076, AG490, AG1318, AG556, AG1319, AG555 and AG1111) inhibits growth of K562 cells but not p210bcr-abl tyrosine kinase activity. Of the compounds which inhibit growth and p210bcr-abl tyrosine kinase activity, AG957 inhibits DNA synthesis as early as 2 h (60% inhibition at 20 microM of AG957), a time and concentration of drug where RNA and protein synthesis were not affected. AG957 inhibits p210bcr-abl tyrosine phosphorylation in living cells by 1 h without an inhibition of total protein phosphorylation. Growth inhibition by AG957 was reversible after 4 h of exposure, but irreversible after 24 h. AG957 can be considered as an important lead structure for the development of anti-bcr-abl tyrosine kinase antagonists. These data also raise the possibility that bcr-abl kinase activity is directly linked to maintenance of DNA synthesis in Philadelphia chromosome positive (Ph+) CML cells.

Benzamide riboside exhibits significant cytotoxicity against a variety of human tumor cells in culture. On the basis of metabolic studies, the primary target of this drug's action appears to be IMP dehydrogenase (IMPDH). Incubation of human myelogenous leukemia K562 cells with an IC50 concentration of benzamide riboside resulted in an expansion of IMP pools (5.9-fold), with a parallel reduction in the concentration of GMP (90%), GDP (63%), GTP (55%) and dGTP (40%). On kinetic grounds, it was deduced that benzamide riboside (whose Ki versus IMPDH is 6.4 mM, while that of its 5'-monophosphate is 3.9 mM) or its 5'-monophosphate were unlikely to be responsible for inhibition of this target enzyme, IMPDH, since only micromolar concentrations of benzamide riboside were needed to exert potent inhibition of tumor-cell growth. Studies on the metabolism of this C-nucleoside have revealed the presence of a new peak eluting in the nucleoside diphosphate area on HPLC. Treatment of this peak with venom phosphodiesterase degraded it and concurrently nullified its inhibitory activity versus IMPDH; alkaline phosphatase, on the other hand, totally failed to digest the anabolite. These results suggest that the metabolite in question is the phosphodiester, benzamide adenine dinucleotide (BAD). Evidence that the inhibitor was an analog of NAD, wherein the nicotinamide moiety has been replaced by benzamide, was provided by both NMR and mass spectrometric analysis and confirmed by enzymatic synthesis. Further insight into the nature of the active principle was obtained from kinetic studies, which established that BAD competitively inhibited NAD utilization by partially purified IMPDH from K562 cells with a Ki of 0.118 microM. In concert, these studies establish that benzamide riboside exhibits potent antiproliferative activity by inhibiting IMPDH through BAD.

Cyclopentenyl cytosine (CPEC) exhibits oncological activity in murine and human tumor cells and has now entered Phase I clinical trials. Its mode of action as an antitumor agent appears to be inhibition by its triphosphate (CPEC-TP) of CTP synthase, the enzyme which converts UTP to CTP. In an attempt to elucidate the mechanism of resistance to CPEC, a murine leukemia cell line resistant to CPEC (L1210/CPEC) was developed by N-methyl-N-nitro-N-nitrosoguanidine-induced mutagenesis and subsequent selection by cultivation of the L1210 cells in the presence of 2 microM CPEC. Resistant clones were maintained in CPEC-free medium for 6 generations before biochemical studies were performed. The resistant clone selected for further studies was approximately 13,000-fold less sensitive to growth inhibition by CPEC than the parental cells, and the concentration of CPEC required to deplete CTP in the resistant cells was 50-fold higher than in the sensitive cells. A comparison of the kinetic properties of CTP synthase from sensitive and resistant cells indicated alteration in the properties of the enzyme from the latter; the median inhibitory concentration for CPEC-TP increased from 2 to 14 microM, Km for UTP decreased from 126 to 50 microM, and Vmax increased 12-fold from 0.2 to 2.3 nmol/mg/min. Northern blot analyses of polyadenylated RNA from the resistant and sensitive cells indicated a 3-fold increase in transcripts of the CTP synthase gene in the resistant line. Consistent with these alterations in the properties of the enzyme, the resistant cells exhibited significantly expanded CTP and dCTP pools (4- 5-fold) when compared with the sensitive cells. No change was observed, however, in the properties of uridine-cytidine kinase, the enzyme responsible for the initial phosphorylation of CPEC; despite this, however, cellular uptake of CPEC was greatly decreased, and phosphorylation of CPEC and its incorporation into RNA were 10-fold less than in the parental cells. These latter observations are most readily explained by feedback inhibition by the increased CTP levels of the resistant cells of uridine-cytidine kinase and/or of the membrane transport process used for initial entry of CPEC.

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).

Cyclopentenyl cytosine 5'-triphosphate (CPEC-TP) is the active metabolite of the investigational drug cyclopentenyl cytosine (CPEC), a nucleoside analogue which exhibits noteworthy antineoplastic activity against several murine and human tumors in tissue culture, and which is now undergoing Phase I clinical trials. This study describes a method to measure the intracellular CPEC-TP levels in peripheral blood mononuclear cells (PBM cells) from patients treated with CPEC, without using radiolabeled drug. The method utilizes on-line multidimensional high-performance liquid chromatography (HPLC) with two columns of different retention mechanisms connected via an automated programmable switching valve. The elution fraction containing CPEC-TP is initially separated from cellular components using a gel sizing column (TSK-G2000-SW) and then rechromatographed by means of a reversed-phase column operated in an ion-pairing mode (YMC-A-312-ODS). The limit of quantitation of CPEC-TP by this method is 2.5 pmol per injection. When CPEC-TP levels were measured in PBM cells from 12 cancer patients after 20 h continuous infusion of CPEC at doses ranging from 3.5 to 5.9 mg/m2/h, the levels attained ranged from 1.4 to 13.4 microM (3.6 to 33.5 pmol/10(7) cells). However, wide variability in the concentrations of CPEC-TP achieved were evident at each dose and did not appear to correlate either with the CPEC dose or with CPEC plasma levels. This method was validated by a comparison of the quantitation of CPEC-TP in cultured PBM cells and Molt-4 cells (a human T-cell line adapted for growth in tissue culture) after incubation with both unlabeled and radiolabeled CPEC.

The rates of accumulation and decay of 2',3'-dideoxyadenosine-5'-triphosphate (ddATP) have been examined after incubation with the anti-human immunodeficiency virus (HIV) agents 2',3'-dideoxyinosine (ddIno) and 2',3'-dideoxyadenosine (ddAdo) in human T-cell systems frequently used for assay of anti-HIV agents (MOLT-4 and CEM). Formation of ddATP from ddIno or ddAdo was rapid and concentration-dependent, with no saturation of phosphorylation being observed up to extremely high levels (1 mM) of drug. Rates of removal of ddATP from MOLT-4 cells were slow (t1/2 = 25-40 hr) and appeared to be monophasic. These unusually long half-times for ddATP utilization are not a general property of purine dideoxypurine nucleosides: when the corresponding guanine analog (2',3'-dideoxyguanosine) was examined under the same conditions, the t1/2 of ddGTP removal was only 3-5 hr. Similar results were observed with the human T-cell line CCRF-CEM. Coadministration with ddIno of inosine monophosphate dehydrogenase inhibitors, such as ribavirin and tiazofurin, yielded higher levels of ddATP in MOLT-4 and CEM cells, but did not influence the slow removal of ddATP from T-cells. The long half-time for disappearance of ddATP from cells may permit the maintenance of pharmacologically effective levels of ddATP within cells with relatively infrequent administration of the parent drug (ddIno or ddAdo).

2',3'-Dideoxyguanosine (ddGuo) is a selective inhibitor of the replication of human immunodeficiency virus in vitro and the most active antihepadnavirus nucleoside analog known in vitro and in vivo, in a Peking duck model. However, the exact route by which this and related guanosine analogs are anabolized to their putative active metabolites in target cells is controversial. The anabolic pathway for the activation of ddGuo was investigated with the use of mutant human lymphoid CCRF-CEM and WI-L2 cell lines deficient in known nucleoside kinases. Uptake of ddGuo by human lymphoid cells and subsequent conversion to mono-, di-, and triphosphorylated metabolites is dose dependent and occurs proportionately to the exogenous concentration of drug. Studies with kinase-deficient CCRF-CEM and WI-L2 mutants revealed that at least two different routes of metabolism are operating in these cells to initiate the phosphorylation of ddGuo to its active dideoxynucleotides, one being deoxycytidine (dCyd) kinase and the other a cytosolic-5'-nucleotidase acting in the anabolic direction as a phosphotransferase. The evidence for this included 1) a lower but significant accumulation of drug anabolites in dCyd kinase-deficient mutants, 2) a lack of cross-resistance of the kinase-deficient mutants to growth inhibition by ddGuo, compared with that by the related analogs dideoxycytidine and arabinosylcytosine, known substrates for dCyd kinase, and 3) identification of different phosphorylation activities for ddGuo in extracts of wild-type cells and kinase-deficient mutants. Knowledge of the enzyme systems involved in anabolism of ddGuo analogs should be important for both new drug design and optimal therapeutic application.

Hep G2-derived hepatoblastoma cells (2.2.15), which actively produce hepatitis B virus (HBV), were cultured in the presence of 2',3'-dideoxyguanosine (ddG), 2',3'-dideoxyinosine, or 3'-azido-2',3'-dideoxythymidine (AZT). ddG was the most potent agent. It diminished viral replication by up to 95%, as assessed by the amount of episomal HBV DNA, without impairing cellular growth. AZT was the least effective against HBV. Northern blot analysis revealed no apparent difference in the pregenomic viral RNA profile, suggesting that these dideoxynucleosides suppress reverse transcription in the replicative cycle of HBV. The effect of varying the time of drug exposure showed that these agents can suppress HBV replication even when added late in culture. HBV replication in another 2.2.15 cell population of the same lineage was affected by ddG differently, which may enable the investigation of phenotypic or genetic alterations during culture. The present data suggest that some 2',3'-dideoxynucleosides can exert a potent antiviral activity against HBV in vitro, at least under certain circumstances, although the data do not prove that any of these agents have utility in patients with hepatitis.

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.

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)

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.

The inosinate dehydrogenase (IMPD) inhibitors ribavirin, tiazofurin and mycophenolic acid were found to stimulate by as much as 20-fold the anabolism of the anti-HIV agent 2' ,3'dideoxyguanosine to its 5'-diphosphate (ddGDP) in a human T-cell culture system (Molt-4 cells). Stimulation of the further conversion to ddGTP (the active form of the drug) was lesser in magnitude but still highly significant (up to 4-fold at appropriate concentrations of ribavirin or tiazofurin). In parallel with these increases, the inhibitors also produced increases of up to 35-fold in IMP levels. These results support the proposal that the initial phosphorylation of ddGuo is catalyzed by a phosphotransferase (5'-nucleotidase) which utilizes IMP as its phosphate donor (Johnson and Fridland, [1989] Molec. Pharmacol. 36, 291-295). Concomitant with this increase in 5'-phosphorylation of ddGuo, an increase in its anti-HIV activity of up to 6.5-fold was observed when this agent was combined with ribavirin (5 microM) in the H9 [corrected] cell assay system.

Monocyte/macrophages (M/M) are an important target cell for human immunodeficiency virus (HIV) infection in the body. The study of HIV infection in these cells, however, is rather complicated because they represent a variable population, and because HIV entry and replication in M/M may be markedly influenced by a number of factors. These must be considered in therapeutic approaches to HIV infection. In the present set of experiments, we studied the interaction between certain agents which increase the infection of monocyte/macrophages (M/M) by HIV and two groups of anti-HIV agents: dideoxynucleosides and specific inhibitors of gp120-CD4 binding. We found that the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), which markedly enhances HIV replication in M/M, does not affect the activity of recombinant soluble CD4 (sCD4) or OKT4A, two agents which block gp120-CD4 binding. However, it had varying effects on different dideoxynucleosides: GM-CSF increased the net anti-HIV activity of 3'-azido2',3'-dideoxythymidine (AZT), while at the same time it reduced the activity of 2',3'-dideoxycytidine (ddC) and 2',3'-dideoxyinosine (ddI). These effects probably represent an interplay between varying effects of GM-CSF on drug entry and phosphorylation. In additional experiments, we showed that very low concentrations of anti-HIV antibodies could enhance HIV infection of the U937 monocytoid cell line. Interestingly, while this effect has been hypothesized to occur through a CD4-independent mechanism, we found that the anti-HIV activities of both sCD4 and OKT4A were unchanged under conditions of enhancement.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

2',3'-Dideoxyuridine (ddUrd) exhibits poor if any anti-human immunodeficiency virus (HIV) activity in ATH8 and MT-4 cells. This is in agreement with the failure of ddUrd to be efficiently anabolized intracellularly to its 5'-triphosphate metabolite. However, 2',3'-dideoxyuridine-5'-triphosphate (ddUTP) proved to be a potent and selective inhibitor of the reverse transcriptase of HIV (Ki, 0.05 microM) and avian myeloblastosis virus (Ki, 1.0 microM). Bacterial DNA polymerase I, mammalian DNA polymerase alpha, terminal deoxyribonucleotidyl transferase, and Moloney murine leukemia virus reverse transcriptase were resistant to ddUTP. ddUTP is incorporated into the growing DNA chain principally at dTTP sites and inhibits further elongation. The potential of ddUTP as an anti-HIV therapeutic agent merits further investigation. However, to achieve this goal, it will be necessary to resort to techniques capable of delivering preformed phosphorylated ddUrd to the susceptible cells.

Of the dideoxynucleosides described to date, the purine analogues ddA and ddI have exhibited very favorable therapeutic ratios in vitro. ddI is presently undergoing extensive phase I-II clinical trials. Whereas the action of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) is usually to convert a given analogue of Ado to an inactive or less active form, ddI appears to retain the same biological activity as that of the parent ddA. An explanation for these observations was possible when we found that ddI (1) underwent only a slow cleavage to hypoxanthine through the action of PNP and (2) accumulated the same active antiviral metabolite (i.e., ddATP) as ddA in human lymphoid cells. The use of human lymphoid cells with deficiencies in cellular nucleoside kinases and of inhibitors of pathways of nucleotide metabolism have also revealed new aspects of dideoxypurine metabolism in human lymphoid cells, including the identification of a salvage pathway (phosphotransferase/5'-nucleotide pathway) by which ddA/ddI may be metabolized preferentially to the active nucleotide. The effectiveness of ddA and ddI as orally administered antiviral agents may be limited by their susceptibility to acid hydrolysis and the low efficiency for nucleotide conversion in human lymphoid cells. The presence of a fluorine atom in the arabinose configuration on C-2 confers resistance to solvolysis and renders the analogue less susceptible to enzymatic deamination and resistant to phosphorylytic cleavage by PNP. In addition, human lymphoid cells accumulated several fold higher levels of the putative active triphosphate, 2'-F-dd-ara-ATP, than those of ddA or ddI. This increased accumulation of the analogue triphosphate could be accounted for by a more direct conversion of 2'-F-dd-ara-A by a direct phosphorylation through dCyd kinase than ddA. Thus, a single substitution with fluorine at the 2' "up" position of the sugar moiety of ddA markedly improves several biochemical properties relating to dideoxynucleotide accumulation in human lymphoid cells. Whether there are significant alterations of other biochemical properties, such as the ability of the analogue triphosphate to interact with the target enzyme reverse transcriptase, has not yet been determined. Thus, a definitive resolution of the relative merit of ddA/ddI and its 2'-fluoro-arabinosyl analogue is not yet possible on the basis of the studies described here.

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.

[2-3H]Inosinic acid ([2-3H]IMP) has been biosynthesized in good yield from [2-3H]hypoxanthine and PRPP via the action of a partially purified preparation of hypoxanthine/guanine phosphoribosyl transferase from mouse brain. The product was purified in one step by ascending paper chromatography, and used to assess the activity of IMP dehydrogenase. To conduct the assay, tritiated substrate is admixed with enzyme in a final volume of 10 microliters; NAD is present to serve as cofactor for the reaction, and allopurinol to inhibit the oxidation of any hypoxanthine generated as a consequence of side reactions. After an appropriate period of incubation, the 3H2O arising from the oxidation of tritiated IMP via [3H]NAD is isolated by quantitative microdistillation. Performed as described, the assay is facile, sensitive, and accurate, with the capability of detecting the dehydrogenation of as little as 1 pmol of [3H]IMP. Using it, measurements have been made of IMP dehydrogenase in a comprehensive array of mouse organs. Of these, pancreas contained the enzyme at the highest specific activity.

L-[alphaS,5S]-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (NSC-163501), an antibiotic elaborated by Streptomyces sviceus, has been shown to be a powerful inhibitor of many mammalian and bacterial reactions involving the transfer of nitrogen from the gamma-carboxamide of L-glutamine. Thus, the utilization of L-glutamine for the synthesis of carbamyl phosphate, L-asparagine, guanosine-5'-monophosphate, cytidine-5'-triphosphate, N-formylglycinamidine ribonucleotide, NAD, glucosamine-6-phosphate, and anthranilic acid is strongly or totally inhibited by a concentration of NSC-163501 of 1 X 10(-3) M. L-Glutamate synthetase of Escherichia coli is only modestly inhibited and 5-phosphoribosylamine synthesis in fetal rat liver is comparatively refractory to inhibition. NSC-163501 treatment of L1210 cells growing in a low L-glutamine culture medium produced arrest in G or early S phase. Of the amino acids tested, only L-glutamine antagonized such growth inhibition.

Variants of the Lewis lung carcinoma were selected for resistance to N-(phosphonacetyl)-L-aspartic acid (PALA) by treatment of tumor-bearing mice with repetitive subcurative doses of PALA. The specific activity of the target enzyme, L-aspartic acid transcarbamylase (ATCase), was measured in the four variants developed. Three had markedly elevated ATCase activities; however, the fourth line, LL/PALA-C, had an ATCase activity identical to that of the parent, PALA-sensitive line (LL/O). One high-ATCase variant, LL/PALA-J, and LL/PALA-C were compared with LL/O in subsequent biochemical studies on the mechanism of resistance to PALA. Enzyme activities in the salvage pathways which phosphorylate pyrimidine nucleosides and deoxynucleosides were found to be similar in all three lines. ATCase in these lines exhibits closely comparable kinetics with its natural substrates as well as with PALA. The time courses of restitution of ATCase after a single therapeutic dose of PALA show that both resistant variants recover full activity more rapidly than the parent. Additionally, inhibition of ATCase 24 hr following graded doses of PALA is lower in the resistant lines. The uptake of [14C]PALA in vitro into cell lines derived from the three Lewis lung carcinomas apparently occurs by passive diffusion and at comparable rates in both sensitive and resistant cells. Analysis of the nucleotide content of tumors reveals comparable spectrums of purine and pyrimidine nucleotide levels in the LL/O and LL/PALA-C lines, whereas the LL/PALA-J line has augmented nucleotide pools. In all three lines, 24 hr after treatment with PALA (400 mg/kg), uridine and cytidine nucleotide levels were substantially diminished (70 to 80%) while adenosine 5'-triphosphate and guanosine 5'-triphosphate levels were elevated (50 to 100%). Estimations of precursor flux through the de novo pyrimidine pathway by measuring orotate and orotidine levels in tumors of mice treated with pyrazofurin (an inhibitor of orotidine-5'-monophosphate decarboxylase) and either 0.9% NaCl solution or PALA shows that PALA treatment eliminates orotate and orotidine accumulation in LL/O but reduces it by only 75 and 50% in LL/PALA-C and LL/PALA-J, respectively. Similarly, PALA treatment (20 microM) of tumor lines in culture provokes a dramatic decrease in the incorporation of NaH14CO3 into pyrimidine intermediates and nucleotides in the LL/O cell line only. Determinations of specific activities of the other enzymes in this pathway reveal that the activity of carbamyl phosphate synthetase II, the rate-limiting step, is elevated 2- to 3-fold in both resistant lines. Since carbamyl phosphate synthetase II exists as a complex with ATCase, the suggestion is made that levels of carbamyl phosphate synthetase II are collaterally important determinants of PALA activity. An augmented pool of carbamyl phosphate in the resistant variants may serve to competitively displace PALA from ATCase, diminish enzyme inhibition, and allow pyrimidine biosynthesis to proceed despite therapy.

The embryolethal effects of phosphonacetyl-L-aspartic acid (PALA) are markedly gestational stage-specific in the Swiss albino mouse. Embryos are most sensitive to the lethal effects of PALA on days 7 and 8 of gestation, with embryonic LD50's of 9 and 8 mg/kg, respectively. In contrast, the embryonic LD50 on day 10 of gestation is 144 mg/kg. Following an IP dose of (acetyl-14C)-PALA to pregnant mice, approximately threefold higher levels of radioactivity were present in day 8 than in day 10 embryonic tissue, whereas the radioactive content of placentas from day 10 pregnant animals was significantly higher than in placentas from day 8 pregnant mice. Similarly, L-aspartate transcarbamylase (ATCase) activity was greater in maternal spleen, placentas, and embryos on day 8 than on day 10 of gestation, and PALA treatment produced a greater inhibition of ATCase in embryonic tissue on day 8 than on day 10; however, inhibition of placental ATCase activity was more pronounced on day 10 than on day 8. Neither single nor multiple doses of uridine (UR) given orally to pregnant mice on day 8 of gestation were effective in reducing day 8 PALA embryolethality. Carbamyl-L-aspartic acid, given in the drinking water of pregnant mice on days 7-9 of gestation, reduced the day 8 embryolethality of PALA from 100% to approximately 50%. In a similar experiment, the presence of UR in the drinking water of pregnant mice reduced PALA-induced embryolethality in day 10, but not day 8, embryos. These results indicate that the embryotoxic effects of PALA are the result of ATCase inhibition; furthermore, they suggest that the relative insensitivity of the day 10 embryo to the lethal effects of PALA may result either from a greater availability of UR to the day 10 (versus the day 8) embryo, or from an enhanced ability of the day 10 placenta to bind PALA and prevent its passage into the embryo.

A systematic search has been made for inhibitors of L-asparagine synthetase (L-glutamine hydrolyzing, EC 6.3.5.4) from leukemia 5178Y/AR, a rodent neoplasm resistant to the oncolytic enzyme L-asparaginase (EC 3.5.1.1), The classes of chemicals examined in this search included substrate and product analogs, agents capable of reacting with sulfhydryl functions, and a variety of modifiers whose mechanism of interaction with proteins is known. In general, antagonists of L-glutamine and thiol reagents proved to be the most effective inhibitors of L-asparagine synthetase from this tumor source. Within these groups, certain structural prerequisites to inhibition are reported. Attempts to correlate oncolytic potency with enzyme-inhibitory potency were unsuccesful.

A series of 4-(substituted aminosulfonyl)- and 4-(substituted hydrazinosulfonyl)-2-aminobutanoic acids, compounds structurally related to glutamine, was synthesized as potential inhibitors of L-asparagine synthetase and subjected to screening as antitumor agents. Target amino acids were obtained by condensation of a blocked reactive sulfonyl chloride with the appropriate amine or hydrazide, followed by deblocking with hydrogen--palladium or liquid hydrogen fluoride--anisole. Neither the target compounds nor their protected precursors inhibited the enzyme from L5178Y/AR or prolonged the life of mice with P-388 lymphocytic leukemia. However, DL-4,4'-dithiobis[2-(benzyloxycarbonylamino)butanoic acid], an intermediate in the synthesis of the target amino acids, exhibited 90% inhibition of L-asparagine synthetase at 10 mM.

2',3'-Dideoxycytidinene (ddeCyd), the 2',3'-unsaturated derivative of 2',3'-dideoxycytidine (ddCyd) is, like ddCyd itself, a potent and selective inhibitor of HTLV-III/LAV in vitro. This conclusion is based on the relatively high ratio of effective antiviral dose (0.3 microM) versus cell growth inhibitory concentration (20-35 microM) and the lack of any appreciable inhibitory activity against a series of non-oncogenic RNA and DNA viruses. Both compounds were considerably more inhibitory to human lymphoid cell lines than human nonlymphoid or murine cell lines. They were highly dependent on prior activation by deoxycytidine kinase to exert their anti-HTLV-III/LAV and cytostatic effects. In contrast with ddCyd, ddeCyd lost part of its anti-retrovirus effect upon prolonged incubation (10 days) with the virus-infected cells in culture.

The conjugate of L-alanosine [L-2-amino-3-(N-hydroxyN-nitrosamino)propionic acid] and 5-amino-4-imidazolecarboxylic acid ribonucleotide has been synthesized in good yield by enzymatic means, using partially purified chicken liver 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide synthetase (EC 6.3.2.6). The chromatographic behavior of this molecule was characterized, as was its ability to inhibit adenylosuccinate synthetase, an enzyme long considered to be the locus of action of the drug. The Ki of-L-alanosyl-5-amino-4-imidazolecarboxylic acid ribonucleotide versus a partially purified adenylosuccinate synthetase frm the L5178y/AR leukemia of C57BL X DBA/2 F1 (hereafter called BD2F1) mice was 0.228 microM, whereas the Ki of L-alanosine was 57.23 mM. Administration of 50 microCi of DL-[1-14C]alanosine along with unlabeled L-alanosine (500 mg/kg) to BD2F1 mice bearing s.c. nodules of Leukemia L5178Y/AR resulted in the accumulation in tumors of a material with properties compatible with those of L-alanosyl-5-amino-4-imidazolecarboxylic acid ribonucleotide. It coeluted with L-alanosyl-5-amino-r-imidazolecarboxylic acid ribonucleotide in the high-resolution chromatographic system used, was Bratton-Marshall positive, and inhibited adenylosuccinate synthetase strongly. In tumor nodules 2 hr after dosage, the concentration of this compound approximated 70 microM. Under the same circumstances, the intratumoral concentration of L-alanosine was found to be 440 microM. At this concentration, the antibiotic itself exerts only a marginal inhibition of leukemic adenylosuccinate synthetase. In ancillary studies, it was shown for the first time in vivo that the parenteral administration of L-alanosine reduces the specific activity of intratumoral adenylosuccinate synthetase by 70% and depresses the synthesis of DNA to an equivalent or greater extent; adenine but not hypoxanthine (both at 250 mg/kg) was able to reverse the latter inhibition. No effect on purine salvage enzymes was exerted by L-alanosine. Viewed in concert, these experiments establish that the adduct of L-alanosine with 5-amino-4-imidazolecarboxylic acid is formed by neoplastic cells in vivo and that this anabolite is most probably responsible for the inhibition of adneylosuccinate synthetase and, in turn, for the diminished synthesis of DNA seen after a therapeutic dose of L-alanosine.

The antiretroviral action of 2',3'-dideoxycytidine (ddCyd) depends on its intracellular conversion to the 5'-triphosphate metabolite ddCTP. The effect of natural pyrimidines and pyrimidine nucleosides, as well as of a number of inhibitors of pyrimidine nucleotide synthesis (i.e., N-(phosphonacetyl)-L-aspartate, 6-azauridine, pyrazofurin, 3-deazauridine, and hydroxyurea) on the metabolism of the potent anti-human immunodeficiency virus drug ddCyd has been investigated in human and murine cell lines. Deoxycytidine (dCyd) and cytidine (Cyd) effectively blocked the intracellular phosphorylation of ddCyd: dCyd by competition with ddCyd for 2'-deoxycytidine kinase, and Cyd probably by competition with the higher nucleoside mono- and diphosphate kinases. These conclusions are supported by the observations that (i) the cytostatic effects of ddCyd against human Molt/4F cells are significantly reversed by dCyd; (ii) the antiviral effects of ddCyd against hman immunodeficiency virus-infected human ATH8 cells are reversed by dCyd and Cyd; (iii) phosphorylated metabolites of ddCyd could not be detected in a 2'-deoxycytidine kinase-deficient murine leukemia (L1210)/araC cell line; and (iv) ddCyd lacked any cytostatic effect against this araC-resistant L1210 cell line. In contrast to dCyd and Cyd, thymidine (dThd) stimulated formation of phosphorylated ddCyd metabolites. The degree of this stimulation proved dependent on preincubation time and dThd concentration. There was a correlation between the increased ddCTP levels upon preincubation of the cells with dThd, and decreased dCyd-5'-triphosphate pools, presumably caused by inhibition of cytidine-5' -diphosphate reductase by dThd-5'-triphosphate. In an attempt to discover compounds other than dThd that are able to stimulate ddCTP formation, a number of inhibitors of pyrimidine nucleotide metabolism were also studied. Under our experimental conditions, 3-deazauridine and hydroxyurea proved equally as effective as dThd in stimulating ddCyd phosphorylation. Finally, we could demonstrate that dThd significantly enhanced the protective effect of ddCyd against human immunodeficiency virus-infected ATH8 cells.