Modulation of cell-mediated immunity with 5-azapyrimidine nucleosides.

5-azacytidine [5-AzCR] and 5-aza-2'-deoxycytidine [5-AzCdR] can produce an immunosuppressive or an immunostimulatory effect on the graft-versus-host or host-versus-graft reactions in the regional popliteal lymph node of the mouse. The result depends on the dose of the drug and the time between its administration and induction of the cell-mediated immune response. Syngeneic spleen cells are capable of inducing regional lymphadenomegaly if injected into the footpads of recipients treated 1 day previously or concurrently with 5-AzCR or 5-AzCdR. Spleen cells from allogeneic and syngeneic donors treated with 5-AzCR or 5-AzCdR in vivo or in vitro can elicit greater regional lymphadenomegaly than the untreated cells. Both drugs not only seem to influence the proliferative activity of effector and regulatory populations of immunocompetent cells, but they probably also change their recognition ability and immunogenic properties.

Depression of experimental gastric ulcers and acute pancreatitis in rats treated by 5-azacytosine, 5-azacytidine and their N-methyl derivatives.

5-Azacytosine, 1-methyl-5-azacytosine and 5-azacytidine administered to rats with a ligated pylorus block gastric secretion, gastric acidity, the extent of hemorrhage and the number and size of gastric defects. The same drugs also depress the development of experimental acute pancreatitis mediated in rats by interstitial administration of 7.5% natrium cholate into the pancreas in vivo. The drugs affected the amount of abdominal fluid and 6 h after the treatment the pathological changes were significantly decreased.

Azapyrimidine nucleosides: metabolism and inhibitory mechanisms.

Triazine nucleosides represent highly active compounds affecting different cellular processes. While 6-azauridine displays a rather selective inhibitory effect, biological action of 5-azacytidine reflects the polyvalent inhibitory mechanism of the drug (interaction with pyrimidine synthesis de novo, incorporation into RNA and DNA, depressed maturation of ribosomal RNA, inhibition of RNA and DNA methylation, etc.) and the analog displays pronounced cytostatic and immunosuppressive activity. 5-Aza-2'-deoxycytidine action is directed against DNA synthesis similar to that of 5-azacytosine arabinoside. N4-Substituted derivatives of 5-azacytidine affect gastric secretion and together with 5-azacytosine and 5-azacytidine represent a new type of drugs with antiulcer activity. 6-Amino-5-azacytosine nucleosides interfere with the metabolism of purines rather than pyrimidines as evidenced by the character of their inhibitory mechanism and measurement of conformation. 6-Azauridine (as 2',3',5'-triacetate) and 5-azacytidine were used with certain success in human chemotherapy, the first one as a drug affecting recalcitrant psoriasis, the second one for the treatment of different forms of leukemia. The inhibitory mechanisms of individual azapyrimidine nucleosides are discussed in relation to their known biological effects.

Effects of N4-methyl and N4,N4-dimethyl derivatives of 5-azacytidine on liver RNA synthesis and gastric secretion in rats.

Of different N-alkyl substituted derivatives of 5-azacytidine tested for their action on gastric secretion in rats with ligated pylorus the most effective beside 5-azacytidine were N4-methyl and N4,N4-dimethyl derivatives. The drugs blocked gastric secretion, gastric acidity, the extent of hemorrhage and the number and size of gastric defects. Simultaneously a higher synthesis of RNA in the liver of drug-treated animals was observed.

Inhibition of phytohemagglutinin-induced DNA synthesis and of adenosine deamination by 5-azacytidine and 5-aza-2'-deoxycytidine in intact human cells.

5-Azacytosine nucleosides block phytohemagglutinin-induced thymidine incorporation into DNA in intact human blood lymphocytes in a dose-dependent manner at 1-100 micrograms/ml. Maximal inhibition was observed when mitogen and 5-azacytidine or 5-aza-2'-deoxycytidine were added to the culture in admixture. Both analogues also inhibit deamination of exogenous adenosine in stimulated cells without affecting the synthesis of nucleotides from adenosine, adenine or hypoxanthine. Similar inhibitory effect of 5-aza-2'-deoxycytidine on the metabolism of adenosine was observed in normal (WI-38) and SV40 virus-transformed (VA-13) human fibroblasts.

Inhibition of bovine heart adenosine deaminase activity by some pyrimidine analogues.

Some pyrimidine analogues--especially 6-azauridine and 5-azacytidine at a final concentration of 1 mmol dm-3--significantly depressed adenosine deaminase activity isolated and purified from bovine heart. Both the above mentioned azapyrimidine nucleosides were effective competitive inhibitors of this enzyme. The inhibitory effect of some clinically used pyrimidine analogues may be of importance for explanation of their mechanisms of action on the cell metabolism.

Inhibitory effects of 5-azapyrimidine nucleosides on cellular immunity.

The effect of 5-azacytidine (5-AzCR) and 5-aza-deoxycytidine (5-AzCdR) on the survival of skin grafts in mice and rats, the action of these drugs on regional GVH reaction, as well as the formation of haemopoietic colonies (CFU-5) in the spleen were studied. Both drugs prolonged the life span of skin grafts when administered 24 hr before transplantation, or on the 4th post-transplantation day. However, they were little effective when injected 24 hr after skin grafting, or after induction of the regional GVHR. Following intraperitoneal administration, they inhibited CFU-5 formation. Two-hour incubation in vitro of cells with 5-AzCR significantly reduced their GVH reactivity and capacity to form CFU-5; 5-AzCdR under the same conditions was ineffective.

The role of putrescine in modulation of DNA synthesis in regenerating liver of rats pretreated with 5-azacytidine.

The relationship between polyamine metabolism and DNA synthesis in regenerating liver was investigated using normal and 5-azacytidine-treated rats. The increase of DNA synthesis in regenerating rat liver was completely inhibited by administration of the analogue 1 h after partial hepatectomy. On the contrary, early and enhanced increase (modulation) of DNA synthesis was observed when the analogue was injected 24 h before partial hepatectomy. Changes in ornithine decarboxylase [EC 4.1.1.17] activity and the tissue levels of polyamines in liver remnants showed similar patterns in normal and 5-azacytidine-pretreated rats, except that the putrescine level in removed liver and the spermidine level in the S-phase were significantly higher in the drug-pretreated rats. On the other hand, a single injection of the analogue into intact rats evoked marked increase in hepatic ornithine decarboxylase activity followed by increase in the tissue level of putrescine, but not of spermidine or spermine. No similar increase in the enzyme activity was detected after injection of 5-aza-2'-deoxycytidine, which did not cause modulation of DNA synthesis. No modulation of DNA synthesis in regenerating liver of rats pretreated with 5-azacytidine was observed when partial hepatectomy was carried out before increase of the hepatic level of putrescine. In addition, a positive and highly significant correlation was observed between the level of putrescine in the liver removed at partial hepatectomy and the extent of modulation of DNA synthesis in regenerating liver of rats pretreated with 5-azacytidine. These results suggest that putrescine plays an important role in modulation of DNA synthesis and support our previous proposal that putrescine may have at least two roles in DNA synthesis.

Tryptophan-mediated induction of hepatic enzymes stimulated by 5-azacytidine pretreatment.

The effect of 5-azacytidine on tryptophan-mediated induction of various enzymes in rat livers was analyzed. Pretreatment of animals with 5-azacytidine results in a different response of tryptophan oxygenase to tryptophan and cortisone administration. While the hormonal induction of this enzyme was not affected by the drug, the tryptophan-mediated induction was stimulated. Pretreatment of rats with 5-azacytidine resulted also in the enhancement of liver tyrosine aminotransferase activity. There was no difference between the effect of tryptophan and cortisone. Tryptophan affects also the level of thymidine kinase at various stages of liver regeneration. 5-azacytidine given 16-40 hours prior to partial hepatectomy resulted in the enhancement of thymidine kinase activity at 24 hours of regeneration.

Synthesis and biological activity of 2'-deoxy-6-methyl-5-azacytidine and its alpha-D-anomer.

The stannic chloride catalyzed glycosylation of bis-tri-methylsilyl-6-methyl-5-azacytosine 2 with the halogenose 3 leading to the protected anomeric nucleosides 4a and 4b was investigated. Methanolysis of 4a and 4b afforded the corresponding free nucleosides 1a and 1b. Compounds 4a and 4b were also prepared by the isocyanate method via acetylamidinourea derivatives 6. Antileukemic activity in vitro and inhibition of growth of E. coli by the title compounds are reported.

Effect of 5-aza-2'-deoxycytidine on antibody formation and DNA synthesis in rat spleen.

5-Aza-2'-deoxycytidine which is preferentially taken up by the lymphatic tissue and is incorporated into DNA strongly affects the ability of the immune system to synthesize IgG antibodies against sheep red blood cells without affecting IgM antibody formation. A single dose of the drug results in a prolonged inhibition persisting 14 days after the secondary immunization. The inhibitory effect is dose-dependent and is maximal when 5-aza-2'-deoxycytidine is given 2 days after sheep erythrocytes. The drug affects the utilization of thymidine for the synthesis of DNA in the spleen; under certain conditions the enhancement of the rate of DNA synthesis in the spleen has been observed.

5-Aza-2'-deoxycytidine: preclinical studies in mice.

5-Aza-2'-deoxycytidine administered to normal mice repeatedly on daily schedule affects mainly the number of blood leukocytes and bone marrow myeloid cells. Using the cell-free extract from mouse spleen phosphorylated intermediates of 5-aza-2'-deoxycytidine-3H have been isolated and kinetics of their formation both in vitro and in vivo in mouse spleen cells have been studied. The administration of the drug affects the synthesis of DNA in mouse spleen resulting in its initial depression followed later by the enhancement of thymidine uptake.

Kinetics of 5-aza-2'-deoxycytidine phosphorylation in mouse spleen and L1210 leukemic cell extracts.

Out of different nucleosides and deoxynucleosides testes for their ability to block the phosphorylation of labeled 5-aza-2'-deoxycytidine in the presence of ATP and the cell-free extract from mouse spleen only deoxycytidine and cytosine arabinoside depressed the reaction significantly. With the same system the phosphate donor specificity for 5-aza-2'-deoxycytidine and deoxycytidine was determined. All of the 5'-triphosphates used were less efficient donors with respect to the analogue than to the natural substrate. The apparent Michaelis constants for the phosphorylation of 5-aza-2'-deoxycytidine and deoxycytidine were 2.9 and 2.5 x 10(-5) M, respectively. Using the extract from L1210 leukemic cells the Km constant for 5-aza-2'-deoxycytidine was 6.4 x 10(-5) M and that for deoxycytidine 2.7 x 10(-5) M. The Ki constants with the spleen extract were 1.2 x 10(-3) M for 5-aza-2'-deoxycytidine and 5.8 x 10(-6) M for deoxycytidine. Both compounds acted as competitive inhibitors of one another.

Depression of DNA synthesis in mouse spleen after treatment with 5-aza-2'-deoxycytidine.

5-Aza-2'-deoxycytidine is a highly effective cytostatic agent that preferentially affects the lymphatic system. Pretreatment of noninbred H mice with the drug markedly depressed the level of thymidine (dThd) incorporation into DNA in the spleen and also lowered the dThd and thymidylate kinase activities. Maximum effects were observed following administration of the analog in a single dose 24 hours before the mice were killed. Whereas cytidine and dThd did not reverse the inhibitory effect of 5-aza-2'-deoxycytidine, excessive doses of deoxycytidine partially reversed this inhibition. Similar to the depression of dThd incorporation, a depression in the incorporation of deoxycytidine and cytidine into spleen DNA was found after 24-hour pretreatment with 5-aza-2'-deoxycytidine. However, 7 days following 5-aza-2'-deoxycytidine treatment, the incorporation of dThd into DNA in the spleens of mice was significantly increased. [3H]5-aza-2'-deoxycytidine was rapidly incorporated into spleen DNA, whereas deoxycytidine interfered with the incorporation of [3H]5-aza-2'-deoxycytidine.

Synthesis of DNA in the liver and testes of cadmium-tested partially hepatectomized rats.

Cadmium affects the induction of thymidine and thymidylate kinases in regenerating rat liver. EDTA administered simultaneously with cadmium reverses its inhibitory action on enzyme synthesis, and prevents the depression of thymidine incorporation into DNA observed in cadmium-treated animals. Zinc does not abolish the inhibitory action of cadmium on the synthesis of DNA in regenerating liver, and the incorporation of thymidine into DNA in the testes was inhibited more by intraperitoneal injection of cadmium plus zinc than by injection of cadmium alone. Inhibition of thymidine incorporation into DNA in the liver and testes was proportional to the amount of cadmium administered up to about 2 mg CdCl2/kg body weight, but surprisingly, higher doses of cadmium caused less inhibition.