Antitumor Activity of Cytidine Dialdehyde and Its Effects on Nucleotide and Nucleic Acid Synthesis1

[CANCER RESEARCH 40, 598-603, March 1980] 0008-5472/80/0040-0000502.00 Antitumor Activity of Cytidine Dialdehyde and Its Effects on Nucleotide and Nucleic Acid Synthesis1

James J. Kinahan, Zlatko P. Pavebic, Robert J. Leonard, Alexander Bboch, and Gerald B. Grindey2

Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Memorial Institute, Buffalo, New York 14263

ABSTRACT have been proven effective in preventing the growth of Li 210 and Ehrlich ascites tumors in mice, while being inactive against Cytidine dialdehyde inhibited the growth of leukemia Li 210 a variety of other implanted ascites and solid tumors (1, 12, cells in culture at a 50% inhibitory concentration of 3.5 x 1O@ 13, 24). The peniodate oxidation products of inosine, inosine M and, when administered i.p. at 200 mg/kg daily for 5 days, dialdehyde, has produced objective responses in several pa increased the mean survival of Li 210 tumor-bearing mice by tients with neoplasms (22). As a consequence, the biochemical up to 171%. Given by the s.c. or i.v. routes, the compound action of these nucleoside analogs has received increasing was ineffective. The ethanol adduct of cytidine dialdehyde, attention. The dialdehyde derivatives of different nucleosides although inactive in cell culture, increased the mean survival of vary in their effect upon a number of enzymes, and thus they Li 210 tumor-bearing mice by up to 225% when administered may have unique sites of primary action. For example, the i.p. but was inactive upon s.c. administration. dialdehyde derivative of 6-methybthiopunineniboside has been Exposure of Li 210 cells in culture for 25 hr to cytidine shown to inhibit RNA polymenase (28, 32), whereas inosine dialdehyde at the 50% inhibitory concentration increased the diabdehyde has no effect on this enzyme (10). This study nibonucleoside di- and triphosphate pools, slightly increased addressed itself to the possible mechanism of action of cytidine deoxyadenosmnetniphosphate, deoxythymidine tniphosphate, dialdehyde, a compound that had been evaluated previously and deoxyguanosine tniphosphate pools, and caused a pro (i 2) for its effect upon the growth of some experimental tumors. nounced increase in the deoxycytidine tniphosphate pool. As Since the effectiveness of nucleoside diabdehydes appears to determined by the rate of pynimidine precursor incorporation depend largely upon the route of their administration, further into nucleic acids, this concentration of drug showed no effect evaluation of this parameten of drug action was carried out. on RNA synthesis but caused a reduction in DNA synthesis to Additionally, the diabdehyde groups were modified in order to 53% of control. Exposure of Li 2i 0 cells for 3 to 6 hr to 1O@ determine the contribution that these groups make towards M cytidine diabdehyde, a concentration which inhibits growth antitumor activity. completely, effected an increase in the nibonucleoside di- and tniphosphate pools and a rapid decrease of the deoxythymidine MATERIALS AND METHODS tniphosphate pool. The deoxycytidine tniphosphate and deox yguanosine tniphosphate pools decreased more slowly, and the Nonbabeled nucleosides and nucleotides were purchased deoxyadenosine triphosphate pool remained slightly elevated. from Sigma Chemical Co., St. Louis, Mo. Radiolabeled nucleo Analysis of the rate of substrate incorporation into nucleic sides and nucleotides were obtained from Schwarz/Mann, acids showed that this concentration of drug produced an 80% Orangeburg, N. Y., and from Amersham Corp., Arlington decrease in RNA synthesis and a 75% decrease in DNA syn Heights, III. The heteropobyrnersused in the enzymatic assay thesis 3 hr after drug exposure. These results suggest that the of dNTP@poolswere purchased from P. L. Biochemicabs, Inc., mechanism of action of cytidine diabdehyde may be due to its Milwaukee, Wis. , the Micrococcus luteus DNA pobymenase was initial interference with DNA synthesis followed by a general purchased from Miles Laboratories, Elkhart, bnd. ized inhibition of DNA, RNA, and protein synthesis at cytotoxic Preparation of the Periodate Oxidation Products of Cytl concentrations. dine and Their Derivatives. The peniodateoxidationproducts of cytidine and of [5-3H]cytidine were prepared according to INTRODUCTION the method described by Dvonch et a!. (i 2) based on the procedures of Jackson and Hudson (20) and Khym and Cohn The peniodate oxidation products of nucleosides, commonly (23). Briefly, the nucleosides were oxidized with periodic acid referred to as nucleoside dialdehydes, are equilibrium mixtures for 30 mm at room temperature in the dank. lodate was removed of various cyclic and acyclic hemiacetals and hydrates (i 4, i 6, by chromatography of the reaction mixture on Dowex 1-X8- i 7, 21, 23). Since they react chemically as do typical aIde acetate, and the eluate was lyophilized to a white powder. No hydes, the equilibrium forms are likely to have an effect only starting material was discernible on paper chromatography. on the nate at which the reactive species is regenerated upon Reduction of the diabdehyde was carried out by the proce cross-linking with proteins either by Schiff base or carbinola dune of Khym and Cohn (23) using sodium borohydnide at mine formation (6, 9, 27, 29). alkaline conditions, followed by chromatography of the acidi The peniodate oxidation products of several nucbeosides fied solution on Dowex [email protected] and ethyl adducts of cytidine diabdehyde were prepared by the method of Dvonch and Album (11) involving the dissolution of the peniodate ,ThisinvestigationwassupportedbyUSPHSResearchGrantsCA-i7156 and CA-i 2585 from the National Cancer Institute. 2 To whom requests for reprints should be addressed. 3 The abbreviatIons used are: dNTP, deoxyribonucleoside tniphosphates; lC@, Received August i 7, 1979; accepted November 28, 1979. 50% inhIbitory concentration.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1980 American Association for Cancer Research. Evaluation of Cytidine Dialdehyde Activity oxidized nucleoside in ethanol on methanol and precipitating radioactive thymidine as a control. The contribution of labeled the adducts with ether at 5Â°C.Inthis procedure, the alcohol is uridine to the UTP and CTP pools was negligible up to 25 mm presumed to add to the aldehyde groups to ultimately form a of exposure to label; thus, nonradioactive unidinecontrols were cyclic structure. unnecessary. At various times up to 25 mm, cell cultures were Survival Studies of Tumor-bearing Mice. Female DBA/2J rapidly cooled to 4Â°in a dry ice-ethanol bath, the cells were mice(1 8 to 21 g)were purchased from The Jackson Laboratory centrifuged and dissolved in 1 ml of 0.1 % sodium dodecyl (Bar Harbor, Maine). Female DBA/2Ha mice were supplied by sulfate with 5 @gofcalf thyrnus DNA as carrier for analysis of the breeding colony at this institute. The characteristics of acid-insoluble radioactivity or extracted with 1 ml 1.0 M HCIO4 leukemia LI 210 (26), SMT-F (19) and the procedure used for and neutralized with 8 N KOH for analysis of nucleoside tn continuous infusion of mice (31) have been described previ phosphates. Their rate of incorporation into DNA was calcu ously. Mean survival was counted from the day of tumor inoc bated from the acid-insoluble radioactivity by the method of ulation, considered Day 0, and drug administration was begun Henshkoet a!. (18). The acid-soluble UTP and CTP pools were 24 hr following inoculation. separated and quantitated by high-pressure liquid chnomatog Hlstopathology. Animalswere killed by cervicaldislocation. raphy, and radioactivity (of UTP, CTP, and dTTP) was quanti Tissues taken for histological study were fixed in Bouin's tated by a sample collection of the appropriate ebuted peaks solution, sectioned at 5 @srn,andstained with hematoxybinand (or of the material ebuted at the known dTTP retention time) eosin. into scintillation vials and subsequent counting in aqueous Cell Culture and Extracts. The beukemic Li 21 0 cells were counting solution. Acid extracts of the cultures containing grown at 37Â°and in a 5% CO2 atmosphere in Roswebl Park nonradioactive thymidine were used for the enzymatic quanti Memorial Institute Tissue Culture Medium 1640, supplemented tation of dTTP. with 10% dialyzed fetal calf serum (Grand Island Biological Effect of Cytidine Diabdehyde on Total Cellular RNA, DNA, Co., Grand Island, N. V.) and 20 m@imorpholinopropanesul and Protein Content. The Schrnidt-Thannhauser procedure tonic acid buffer. The doubling time of control cultures was 11 (30) was used to measure the effect of the drug on cellular hr. The cells were routinely checked by Microbiological Asso macromolecubecontent. ciates, Walkensville, Md., for the absence of Mycoplasma con tamination. RESULTS Determination of Ribonucleotide Pools and Detection of Derivatives of Cytidine Dialdehyde. Equal numbers of control Effects of Cytidine Dialdehyde on Tumor-bearing Mice. and drug-treated cells were harvested at the indicated times The effect of cytidine diabdehyde administered to leukemia by centrifuging an appropriate volume of the cultures at 1800 Li 210-bearing mice by the i.p., s.c., or i.v. routes is shown in rpm for 10 mm at 4Â°in a Beckman Model J-21 centrifuge. Table 1. Daily administration of the agent, at 50 or 100 mg/kg Analysis of nucleotide pools or radiolabeled cytidine diabde i.p. for 5 days, provided an increase in survival over controls of hyde metabolites was carried out by twice extracting 2.25 x up to 123%. Doses of 200 or 300 mg/kg administered i.p. on 106 or 2.25 x 1O@cells with 1 ml 1 M HCIO4, followed by Days 3, 6, and 9 after tumor inoculation increased the life span neutralization with 8 N KOH. Precipitated KCIO4was removed percentage of the mice by up to 171%. Administration of by centritugation, and the supennatant solution was analyzed cytidine dialdehyde by the s.c. or i.v. bobus routes or by at once or stored at â€”20Â°andanalyzed within 2 weeks. continuous i.v. infusion, however, produced only marginal on The separationand quantitationof acid-solublenibonucleo no antitumor effect. The administration of 300 mg/kg/day for side mono-, di-, and tniphosphates was carried out on a DuPont 4 days was toxic as indicated by mice dying before untreated model H 830 high-pressure liquid chromatograph using a Per controls (Table 1). maphase ABX column. The mobile phase consisted of a linear On the assumption that, bikeother nucleoside diabdehydes gradient of 2 mM KH2PO4,pH 3.0, to 0.5 M KH2PO4,pH 4.6, at (9, 10, 24, 32, 33), cytidine dialdehyde may react with plasma a mixing rate of 3%/mm to a final composition of 66% of the proteins upon s.c. or i.v. administration, derivatives were made higher phosphate concentration. Conditions of flow rate, pres which offered the potential of being more stable. Doses of 50 sure, and quantitation have been described (25). Analysis for to 200 mg of the methanol and ethanol adducts of cytidine radioactive materials was carried out by the collection of cob dialdehyde per kg increased the survival of Li 210 tumor umn effluents into scintillation vials. Aqueous-counting solution bearing mice by up to 203% when administered i.p. once daily (Amersham Corp., Arlington Heights, III.) was added, and ra for 4 or 5 days (Table 2). Larger doses of both drugs were dioactivity was measured with a Mark Ill liquid scintillation toxic when administered by either the i.v. or s.c. route (Table spectrometer (Searbe,Des Plaines, III.). 2). These adducts were inactive against Li 2i 0 cells in culture, Assay of dNTP Concentrations. Quantitation of the dNTP although 50% of the growth of these cells is inhibited by concentrations present in the Li 210 cell extracts was per cytidine dialdehyde at a concentration of 3.5 x 1O@M. Table formed using a DNA pobyrnenaseassayas described previously 2 also shows that reduction ofthe oxidation products of cytidine

(25). dialdehyde leads to their inactivation. It further demonstrates Effect of Cytidine Dlaldehyde on the Rate of Nucleoside that when given i.p. daily for 5 days, unidine dialdehyde in Triphosphate Incorporation Into Nucleic Acids. At the mdi creases the mean survival of the mice to an extent similar to cated times, 2.25 x 106 cells from control and drug-treated that provided by cytidine diabdehyde. The antitumor activities cultures were added to 125-mbflasks kept in a 37Â°water bath of these compounds are similar to those originally reported by under 5% CO2atmosphere and containing [3H]unidine(19 Ci/ Dvonch et a!. (12) upon i.p. administration to mice bearing mmol;0.1 6 @LM),[3H]thymidine(55 Ci/mmob;0.01 @tM)ornon leukemia Li 210.

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The effect of cytidine dialdehyde on a fast-growing mammary our studies on the mechanism by which cytidine diabdehyde carcinoma in mice is shown in Table 3. At a dose of 200 mg/ inhibits Li 210 cell growth, the effect of the drug on nucleotide kg, administered by the schedule outlined, the agent was only pools and nucleic acid synthesis in cultured Li 2i 0 cells was marginally effective. examined. Table 4 shows the changes occurring in the nibo Histopathobogy. The histopathobogical effects of the drug nucleoside di- and tniphosphate pools of Li 2i 0 cells grown in were evaluated in mice treated by continuous infusion of cyti the presence of cytidine diabdehyde. Cells exposed to the IC50 dine diabdehyde (300 mg/kg/day) for 3 days. All mice were concentration of cytidmnediabdehydegrew logarithmically at a sacrificed 24 hr after termination of treatment, and the major steady 68% of control growth rate. Continuous ICsodrug ex organs were taken. At autopsy, pallor of kidneys and liver, posunefor 25 hr resulted in an increase in the cellular nibonu reduction of lymphoid tissue, and watery intestinal content cleoside di- and tniphosphate concentrations, ranging from 32 were seen. Microscopic study revealed hydropic degeneration, to 66% above control. Exposure of cells to 1O@ M cytidine necrosis, and cystic dilation of the tubules in the outer cortex dialdehyde stopped cell growth within 3 hr and led to an of the kidneys. In addition to these renal lesions observed in increase of these pools as well. After a 6-hr exposure to this most animals, there was a depletion of lymphocytes in thymus, concentration of drug, the cell number was decreased, but the lymph nodes, and spleen, and depletion of all types of herna nibonucleoside di- and tniphosphate pools remained elevated. topoietic cells in bone marrow, slight necrotizing entenitis, and Table 5 shows the effects of cytidine diabdehydeon the dNTP fatty change around the portal tracts in the liven. pools. The IC50concentration of the drug increased the dNTP Mechanism of Action of Cytidine Diabdehyde.As part of pools slightly, the dCTP pool being increased to a substantially

Table 1 1210Route Effect of cytidine dialdehyde onDBA/2J micebearing leukemiaL of survivalDrug drug ad ministra of drug ad of increase in spani.p.50,tionScheduleDose (mg/kg) ministrationNo. miceMean treatedControla% life 123i.p.1 100 Daily for 5 days5/group1 5.2, 20.59.265, 171iv.50, 00, 200. 300 3 injections 3 days 5.5, 23, 238.582, 171, apart5/group1 0iv.12.5, 100, 3 injections 3 days 8.5, 8.8, 5, 9, 200, 300 apart5/group7.8, 7.88.10, 14iv.100, 25, 50, Continuous infusion 7.8, 9, 97.96, 0, 14, 100 for 3 days5/group8.4, 0s.c.50, 200, 300 Continuous infusion 8.2, 48.60. 0, for 4 days5/group8, 5a 100, 3 injections 3 days 8, 9.2, 0, 14, 200, 300 apart5/group8.5, 8.58.15,

Each group of controls consisted of 10 mice.

Table 2 Effect of derivatives of cytidine dialydehyde on OBA/2J mice bearing leukemia L 1210 Route of survivalCon drug ad increaseCompoundtionDoseministra Schedule of drugNo. ofMean % spanMethanol-cytidinei.p.50Daily (mg/kg)administrationmiceDrug treatedtrolsaIn life 88125dialdehyde for 4 days51 adducti.p. 100, for 5 days 23, 6, 203, 200, 400 2.8 0, 0 s.c.50, 50, 100, Daily for 5 days5/group5/groupi4.6, 8.8, 8.8, 8.892, 0, 0 0,0Ethanol-cytidinei.p.50, 200,400Daily 6.2,2.27.6 185,dialdehyde 100,Daily for 5 days5/group14.2, 23.4,8.273, adducts.c.200, 400 2.4 0 50, iOO, for 5 days5/group7.2, 8.8, 8.6, 16, 13, 8, 0Unidine 200, 400Daily 8.2, 2.47.60, 93Reduceddialdehydei.p.50, 100Daily for 5 days5/group14.7, 15.4884, cytidine dialdehydei.p.50, 100, for 5 days6/group7.5, 8, 8.3, 13, i7,28a 200,300Daily 9.17.16,

Each group of controls consisted of i 0 mice.

Table 3 miceCompoundRoute Effect of cytidine diaIdehyde on SMT-F in DBA/2Ha

mor diame ter (50% of sur final size) of drug ad (mg/ of drug adminis of vival reached on DayControl ministrationDose kg)Schedule trationNo. miceAv. (days)i5.8-mmtu (0.9% NaCI solution) Cytidine dialde i.p.i 00 3, 6, 8, i 0, 13, i 5, 6/group22.3 28.7 12 hydei.p. 200Day 17,20,22,24,27,29, 23.211 16 316

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Table 4 Effect of cytidine dialdehyde on the ribonucleoside di- and triphosphate pools of leukemia L 12 10 cells Values given are averages of 3 separate determinations. Actual nibonucleoside tniphosphate concentration, pmol/ 10Â°cells,for control cultures were: CTP, 502 Â±44; UTP, 863 Â±65; ATP, 2097 Â±119; and GTP, 668 Â±36. controlCytidine Length of of drug % of expo growth% dialde surerelativeADP +UDP controlCDPUDPGDPCTPUTPATPGTPhexoses3.5hyde (M)(hrs)to x i0@ iOâ€”@25103.1i0â€”@[email protected] 28bi39.6 112.9150.6122.5144.4107.3132.81i3.2149.312i.i166.4145.8156.7130.3150.1

a Steadystategrowth. b Cell number stationary.

C Cell number decreasing.

Table 5 Table 6 Effects of cytidine dialdehyde on dNTP pools in leukemia L 12 10 cells Effect of cytidine dialdehyde on UTPplus CTP incorporation into RNA and Valuesgivenare averagesof2 separatedeterminations.ActualpmoldNTP dTTP incorporation into DNA per i0@cells for control cultures were: dATP, 5.6 Â±0.3; dTTP, 17.9 Â±0.9; Total radioactivity after cell exposure to [3H]unidinefor a 15-mm period ranged dGTP, 1.5 Â±0.3; and dCTP, 5.2 Â±0.4. from 1 265 cpm (CTP) to 20,1 14 cpm (UTP) in [email protected] activity controlhyde Length % of of @ of drug growth [3H]UTP [3H]CTP Cytidine dialde expo relative to% UTP CTP (hr)controldATPdTTPdGTPdCTP3.5(M)sure [email protected]@328141.724.682.073.7io-@620136.813.368.157.9forradioactivityranged 106 cells ranged from 306 to 347 dpm/pmol. Incorporated (25hr. from 2,839 (3 hr, 1O@M cytidine dialdehyde treatment) to 39,4i 9 to[3H]thymidinecontrol cells) dpm/1 0Â°cells/mm. Total radioactivity after cell exposure 50-@tI for a i 5-mm period ranged from 4,1 28 to 5,313 (dTTP) cpm in 2,129andsamples. Specific activity of dTTP, [3H]dTTP/dTTP, for 106 cells was and2,89621,934 dpm/pmol for drug-treated cells in Parts 1 and 2, respectively, greater extent than were the dATP, dTTP, on the dGTP pools. was1 and 4,848 dpm/pmol for control cells. Incorporated radioactivity Exposure of the cells to i O@M cytidine dialdehyde decreased 04,921 and 104,826 dpm/i 0Â°cells/mmcultures.Incorporation for the respective control the dTTP pool most markedly and the dGTP and dCTP pools IncorporationUTPof to a lesser extent, while the dATP pool remained elevated over +intoRNA CTP into of dTTP DNALength the period of drug exposure. Table 6 shows the effects of cytidine diabdehyde on the pmol/of % of pmol/Cytidine drug growth 1o@ incorporation of nucleoside triphosphates into RNA and DNA. ofhydedialde- expo- relative celbs/ % of 106 % Specific activities of the nucleoside tniphosphates were con controli. (M) sure (hr) to control mm control cells stant after the first 2 mm of exposure of cells to label; hence, Control49.33.5 a 129 cell extracts were analyzed at time points between 2 and 25 52.72. x i0@ 25 48a 1i8 91.5 26.0 mm. The use of [3H]unidinegave rise to label in both UTP and Control 44.4 21.6 CTP pools, and the sum of the specific activities of these 2 i0-4 3 28b 8.6 19.4 5.2 24.4

triphosphate precursors was therefore used in determining the a Steady state growth. rates of substrate incorporation into RNA. Results similar to b Cell number stationary. those seen with [3Hlunidmnewere obtained with [3H]guanosine, which gave rise to a single-labeled nibonucleoside tniphosphate i , metabolites of cytidine dialdehyde were found in the fraction pool. At the ICseconcentration of cytidine diabdehyde,the rate which eluted in the region where nucleoside di- and tniphos of incorporation of the tniphosphate precursors into RNA was phates commonly ebute. Although this observation does not unaffected, whereas the rate of dTTP incorporation into DNA constitute proof that phosphorylation does, indeed, take place, was reduced to 53% of control. At a concentration of cytidine such an event has been described to occur with inosine dialde dialdehyde (1O@M), which inhibits growth completely, labeled hyde (i 3). nucleoside tniphosphate incorporation into RNA and DNA was decreased by approximately 80% after 3 hr of exposure to the DISCUSSION drug. The effect of i O@ M cytidine diabdehyde on total cellular The inhibition of cell growth by the peniodate oxidation prod RNA, DNA, and protein was also measured, using the Schmidt ucts of inosine, adenosine, and methybthioinosine has been Thannhauser (30) procedure (data not presented). At 6 hr after found to be associated with the ability of these agents to bind drug exposure, total DNA content of the treated cells remained enzymes as diverse as thymidylate kinase (24), DNA polymer at the initial level, whereas it increased by 77% in nontreated ase (24), RNA pobymenase(28, 32), pancreatic RNase (33), cells. In contrast, the total RNA content of control cells was and nibonucleotide reductase (2â€”6,8). Thus, the further dis only slightly higher than that of drug-treated cells. Protein section of the metabolic changes that occur in drug-treated synthesis was also inhibited. cells is of value in assigning the primary site of action of such To gain some idea concerning the metabolic disposition of nucleoside derivatives. the drug, Li 2i 0 cells were treated with [3H]cytidine dialdehyde, Since, at the IC50concentration, cytidine diabdehydecauses and the acid-soluble fraction was extracted. As shown in Chart an increase in both the rNTP and dNTP pools above control

MARCH 1980 601

of histopathobogical changes, including bone marrow depres sion and kidney degeneration, would indicate, however, that 1.0241 A B antitumor activity may not be achieveabbe even at such high 0.512 0.256 dose levels. 0256- FuN Scols A. cho@@g., â€¢120.4'â€”O.S4 â€”+â€”o sa ACKNOWLEDGMENTS @ 26 @ 24 The authors gratefully acknowledge Alice Atwood, Louise Tomei, and Gary Bullard for their competent assistance with the animal systems. 22 E 20 @ C 18 REFERENCES I') 16â€¢ @C,) 1. Bell, J. P., Faures, M. L., LePage, G. A., and Kimball, A. P. lmmunosup 14 w pressive and antitumor activity of the periodate oxidation product of $-D zC) 12 ribosyl-6-methylthiopurine. Cancer Res., 28: 782â€”787,1968. 10 2. Cory, J. G., and George, C. B. Irreversible inhibition of nibonucbeotide reductase from Ehrlich tumor cells by a modulator analog. Biochem. Bio 0 phys. Res. Commun., 52: 496â€”503,1973. U) 6@ 3. Cony, J. G., and Mansell, M. M. Studies on mammalian nibonucleotide 4@ reductase inhibition by pynidoxal phosphate and the dialdehyde derivatives of adenosine, adenosine 5'-monophosphate, and adenosine 5'-tnphosphate. Cancer Res., 35: 390â€”396,1975. 4. Cony, J. G., and Mansell, M. M. Ribonucleotide reductase as a site of 10 20 30 10 20 30 inhibition of DNA synthesis by the diabdehydederivative of punine nucleo MINUTES MINUTES sides. Cancer Left., 1: 133â€”138,1976. 5. Cony,J. G., Mansell, M. M., George, C. B., and Wilkinson, D. S. Inhibition of Chart 1. Elution profile of ribonucleotides and [3H]cytidine dialdehyde metab nucleic acid synthesis in Ehnlichtumor cells by peniodate-oxidizedadenosine olites extracted from leukemia Li 210 cells. A, cells were concentrated and and adenylic acid. Arch. Biochem. Biophys., 160: 495â€”503,1974. exposed to 102 M [3Hjcytidine diabdehyde(93 MCi/mg) for 6 hr and harvested 6. Cony,J. G., Mansell, M. M., and Whitford, T. W., Jr. Inhibition of nibonucleo @@@ as described in Materials and Methods. Manual changes in A sensitivity were side reductase activity and nucleic acid synthesis in tumor cells by the made during the gradient elution at the indicated points in order to facilitate dialdehyde derivatives of inosine (NSC 118994) and inosine acid. Cancer detection of the nibonucleotides. â€”â€”â€”â€”,radioactivecytidine dialdehyde and its Res., 36: 3166-31 70, 1976. metabolites eluted. B, cells were treated as in A but without the addition of labeled drug. 7. Cony, J. G., Parker, S. H., and Fox, C. S. Inhibition of RNA synthesis in Ehrlich tumor cells by the dialdehyde derivative of inosine (NSC 118994). Cancer Res., 38: 815â€”822,1978. levels, the effect of the agent is not likely due to its interference 8. Cony, J. G., and Whitford, T. W., Jr. Ribonucleotide reductase and DNA synthesis in Ehrlich ascites tumor cells. Cancer Res., 32: 1301 â€”1306,1972. with nibonucleotide reductase activity. Only when the cells are 9. Cysyk, R. L., and Adamson, R. H. Pharmacologic disposition of Inosine exposed to concentrations of the drug which inhibit growth diabdehyde(NSC 118994) in mice, rats, dogs and monkeys. Cancer Treat. Rep., 60: 555â€”562,1976. completely are the dNTP pools effectively decreased. It thus 10. Cysyk, R. L., and Adamson, R. H. Protein cross-linking properties of the appears that the inhibition of dNTP incorporation into DNA may antitumor agent inosine diabdehyde(NSC 1i 8994). Cancer Treat. Rep., 60: be the primary action of the drug in the Li 2i 0 cells. Whether 563â€”570,1976. i 1. Dvonch, W., and Album, H. E. Antitumor derivatives of peniodate-oxidized this inhibition occurs as a result of the interference of the drug nucleosides. U.S. Patent 4,000,i37. chem. Abstr., 86: 498, 1977. with DNA polyrnerase activity or indicates impaired template i 2. Dvonch, W., Fletcher, H., III, Gregory, F. J., Healy, E-M., Warren, G. H., and function as has been observed previously with diabdehyde Album, H. E. Antitumor activity of peniodate-oxidation products of carbohy drates and their derivatives. Cancer Res., 26: 2386â€”2389,1966. derivatives of different nucleosides (7) remains to be estab 13. George, C. B., and Cony, J. G. Metabolism of the dialdehyde derivative of bished. inosine (NSC 118994): transport, distribution and incorporation into RNA of Ehrlich ascites tumor cells. Biochem. Pharmacol., 26: 1197â€”i204, 1977. The lack of a substantial therapeutic effect after i.v. on s.c. 14. Grant, A. J., and Lerner, L. M. Dialdehydes derived from adenine nucleosides administration of the same concentrations of cytidine dialde as substrates and inhibitors of adenosine aminohydrolase. Biochemistry, hyde that are active when given i.p. may be due to the reaction 18: 2838-2842, 1979. 15. Gnindey, G. B., and Nichol, C. A. Interaction of drugs inhibiting different of the diabdehydewith plasma proteins, as has been suggested steps in the synthesis of DNA. Cancer Res., 32: 527â€”531,1972. @ for other dialdehyde derivatives (9, 10, 24, 32, 33). Several 16. Guthnie, A. D. The â€˜dialdehydes'â€˜fromthe peniodate-oxidation of canbohy modifications of the analog were evaluated in an attempt to drates. Adv. Carbohydr. Chem. Biochem., 16: 105-i 58, 1961. 17. Hansske, F., and Cramer, F. Untersuchungen zur struktur perjodatoxydierter overcome this limitation. Reduction of the 2 aldehyde groups ribonucleoside and nibonucleotide. Carbohydr. Res., 54: 75â€”84,1977. abolished both therapeutic activity and in vitro cytotoxicity. 18. Hershko, A., Mamont, P., Shields, A., and Tomkins, G. M. Pleiotypic re sponse. Nat. New Biob.,232: 206-21 1, 1971. Formation of the methanol or ethanol adducts of cytidine di 19. Hosokawa, M., Orsini, F., and Mihich, E. Fast- and slow-growing transplant abdehyderesulted in derivatives which, unlike cytidine dialde able tumors derived from spontaneous mammary tumors of the DBA/2 Ha hyde, were inactive against Li 2i 0 cells in culture but were DD mouse. Cancer Res., 35: 2657â€”2662,1975. 20. Jackson, E. L., and Hudson, C. S. Studies on the cleavage of the carbon more active than the parent compound following i.p. adminis chain of glycosides by oxidation. A new method for determining ring struc tration to the tumor-bearing mice, signaling some protection tures and alpha and beta configurations of glycosides. J. Am. Chem. Soc., from inactivation. This stabilization did not, however, lead to 59: 994-1003, 1937. 2i . Jones, A. S., Markham, A. F., and Walker, R. T. A simple method for the significant antitumor activity upon s.c. administration of the preparation of ribonucleoside dialdehydes' and some comments on their adducts, suggesting that these agents may not reach the structure. J. Chem. Soc. 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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1980 American Association for Cancer Research. Antitumor Activity of Cytidine Dialdehyde and Its Effects on Nucleotide and Nucleic Acid Synthesis

James J. Kinahan, Zlatko P. Pavelic, Robert J. Leonard, et al.

Cancer Res 1980;40:598-603.

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