Cytosine Nucleoside Analogues Research Articles

Inhibition of replication of hepatitis B virus by cytallene in vitro.

The acyclic cytosine nucleoside analog cytallene [1-(4'-hydroxy-1',2'-butadienyl)cytosine], which has both (+)- and (-)-enantiomers, was evaluated for its anti-hepatitis B virus (HBV) activity in 2.2.15 cells and was found to have potent activity against HBV DNA synthesis. The R-(-)-enantiomer was found to be the more active of the cytallene enantiomers, with a 50% inhibition concentration against HBV synthesis (HBIC50) of 0.08 microM. Its antiviral activity could be reversed by deoxycytidine (dC) and less efficiently by cytidine. Upon removal of the R-(-)-enantiomer from culture medium, the synthesis of HBV DNA could reinitiate, which suggested that the antiviral action is reversible. The R-(-)-enantiomer was also found to be more cytotoxic than the S-(+)-enantiomer. The degree of cytotoxicity varied among the cell lines, with a 50% inhibition of cell growth at greater than 10 microM. The R-(-)-enantiomer had no effect on HBV RNA synthesis and mitochondrial DNA synthesis at a concentration of 10 times or more than the HBIC50. The two enantiomers cannot be deaminated by dC deaminase, and they can be phosphorylated by cytoplasmic dC kinase. The R-(-)-enantiomer of cytallene is the first acyclic cytosine analog with potent inhibitory activity against HBV similar to those of other L-(-)-ddC analogs.

Modulation of the metabolism of beta-L-(-)-2',3'-dideoxy-3'-thiacytidine by thymidine, fludarabine, and nitrobenzylthioinosine.

beta-L-(-)-2',3'-Dideoxy-3'-thiacytidine (3TC) is a cytosine nucleoside analog that potently inhibits the replication of human and duck hepatitis B viruses and human immunodeficiency virus through the activity of its 5'-triphosphate ester metabolite. The present study examined the intracellular decay of 3TC 5'-phosphates and tested strategies for modulating the cellular content of those nucleotides in primary cultures of duck hepatocytes and in human hepatoma 2.2.15 cells and CCRF-CEM T lymphoblasts. Inhibition by deoxycytidine of the 5'-phosphorylation of 3TC in duck hepatocytes confirmed that, as in mammalian cells, deoxycytidine kinase catalyzed 3TC activation. The 5'-mono, 5'-di-, and 5'-triphosphates of 3TC underwent monoexponential elimination from duck hepatocytes and 2.2.15 cells (half-lives, 3.6 to 8.0 h). Thymidine and fludarabine, which are agents that enhance the activity of deoxycytidine kinase, were tested in strategies for increasing the cellular content of 3TC 5'-phosphates. Coordinate treatment of cells with 3TC and thymidine (50 microM) increased the content of 3TC 5'-monophosphate in duck hepatocytes and the content of 3TC 5'-di- and 5'-triphosphates in 2.2.15 cells, but enhancement of 3TC 5'-phosphate levels in CCRF-CEM cells required a higher thymidine concentration (100 microM). Fludarabine (5 microM) did not affect the contents of 3TC 5'-di- and 5'-triphosphates in duck hepatocytes, but modestly increased the contents of those nucleotides in 2.2.15 cells and CCRF-CEM cells. Nitrobenzylthioinosine (NBMPR), an inhibitor of the es facilitated diffusion nucleoside transporter, reduced the level of entry of 3TC into 2.2.15 cells and abolished inward fluxes of thymidine, adenosine, and deoxycytidine. In 2.2.15 cells and CCRF-CEM cells, NBMPR reduced the formation of 3TC 5'-di- and 5'-triphosphates and reversed the thymidine- and fludarabine-induced increases in the formation of those nucleotides. NBMPR protected against the cytotoxicity of 3TC in CCRF-CEM cells, whereas thymidine potentiated that toxicity, apparently by enhancing the formation of 3TC 5'-triphosphate. Taken together, these results indicate that deoxycytidine kinase and the es nucleoside transporter are targets for manipulation of the metabolism and activity of 3TC.

Pharmacological approach for optimization of the dose schedule of 5-Aza-2'-deoxycytidine (Decitabine) for the therapy of leukemia.

5-Aza-2'-deoxycytidine (5-Aza-CdR; Decitabine) is an active antineoplastic agent in patients with leukemia. Since 5-Aza-CdR is an S phase specific agent and has a short plasma half-life, its antileukemic activity is dose schedule-dependent. Leukemia patients who are candidates for 5-Aza-CdR therapy following relapse after therapy with cytosine arabinoside are at greater risk for the problem of drug resistance since these cytosine nucleoside analogues are metabolized by the same enzymes. Due to its unique mechanism of action of demethylating DNA, 5-Aza-CdR has the potential to activate tumor (growth) suppressor and differentiation genes that have been accidentally silenced by DNA methylation in leukemic cells. All these factors should be taken into account in the design of the optimal dose schedule of this analogue. The optimal dose schedule of 5-Aza-CdR should be based on the kinetic parameters of deoxycytidine kinase, its pharmacokinetics, its effects on DNA methylation and the cell cycle parameters of the leukemic cells and the normal hematopoietic stem cells. Since granulocytopenia is the major toxic effect produced by 5-Aza-CdR, the use of hematopoietic growth factors to shorten the duration of leukopenia should be investigated. Another approach which we are investigating is to use the methods of gene therapy to insert the cytidine deaminase gene into normal hematopoietic progenitor cells so as to make them drug resistant to 5-Aza-CdR. The use of other agents that can induce the differentiation of leukemic cells in combination with 5-Aza-CdR may have the potential to increase the clinical effectiveness of this analogue for the therapy of leukemia.

Hepatitis-B-virus resistance to lamivudine given for recurrent infection after orthotopic liver transplantation

Orthotopic liver transplantation for end-stage hepatitis-B-virus (HBV) infection is commonly complicated by recurrence of HBV. Lamivudine, a cytosine nucleoside analogue, has been shown to suppress HBV infection. We report the development of resistance to lamivudine in three patients who underwent transplantation for end-stage liver disease secondary to hepatitis B. Two of the patients received lamivudine for recurrent HBV infection after transplantation, whereas the third patient began treatment 1 month before transplantation in an attempt to prevent HBV recurrence after transplantation. The three patients initially responded well to treatment, but viral recurrence occurred after 9-10 months of treatment in all patients. HBV DNA was amplified from serum and sequenced through a conserved polymerase domain-the tyrosine, methionine, aspartate, aspartate (YMDD) locus. We assessed the susceptibility of HBV to lamivudine by infecting primary human hepatocytes with serum taken before the start of treatment and after recurrence in varying concentrations of lamivudine. DNA sequencing showed a common mutation within the YMDD locus of the HBV polymerase gene in all patients during lamivudine treatment. In hepatocyte cultures infected with pretreatment serum, HBV DNA concentrations were reduced to less than 6% of those in control cultures by addition of lamivudine in concentrations as low as 0.03 mumol/L. By contrast, in cultures treated with serum taken after recurrence, HBV DNA concentrations did not fall below 20% of control values, even with lamivudine at 30 mumol/L. Resistance to lamivudine has been reported in HIV patients with mutations in the YMDD locus of the polymerase gene. Our findings indicate a common mechanism of lamivudine resistance for HIV and HBV that involves similar point mutations in homologous domains of the viral polymerases.

Assignment of the human cytidine deaminase (CDA) gene to chromosome 1 band p35-p36.2.

The enzyme cytidine deaminase (EC 3.5.4.12; CDA) catalyzes the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. It can also catalyze the deamination of cytosine nucleoside analogues such as cytosine arabinoside and 5-azacytidine, which results in a loss of their cytotoxic and antitumor activity. Cytosine arabinoside is used in the treatment of acute myeloid leukemia, and the antileukemic activity of the drug is dependent on phosphorylation by deoxycytidine kinase. The occurrence of clinical cytosine arabinoside resistance is one of the main problems in the successful treatment of acute myeloid leukemia. Resistance to the drug has been ascribed to functional deoxycytidine kinase deficiency and to increased expression of the CDA gene. In this study, we report on the isolation of a CDA genomic fragment and its use as a probe for the chromosomal localization of the human CDA gene by in situ hybridization. 9 refs., 1 fig.

Synthesis and Biological Activity of Ara and 2′-Deoxycyclopentenyl Cytosine Nucleoside Analogues

Abstract The cytosine analogue of Neplanocin A, cyclopentenyl cytosine (CPE-C, 4), has significant antitumor and antiviral activity. Two closely related analogues modified at the 2′-position, ara-CPE-C (6) and 2′-deoxy-CPE-C (5), have been synthesized from the corresponding uracil derivative CPE-U. Both compounds were devoid of cytotoxicity against L1210 leukemia in vitro. Ara-CPE-C displayed antiviral activity against influenza type A2 but was not very potent.

Induction of cytidine deaminase in HL-60 myeloid leukemic cells by 5-AZA-2′-deoxycytidine

5-Aza-2′-deoxycytidine (5-AZA-CdR), a potent inhibitor of DNA methylation and an active antileukemic agent, produced an induction of cytidine deaminase activity in human HL-60 myeloid leukemic cells. This increase in enzyme activity correlated with the induction of differentiation of the HL-60 leukemic cells as shown by an increase in nitroblue tetrazolium reduction, loss of clonogenicity and decrease in DNA synthesis. The concentration of 5-AZA-CdR that produced these effects was in the range of 0.1–1.0 μM. The increase in cytidine deaminase activity became apparent starting at 48 h from the start of the 5-AZA-CdR treatment and at 72 h was about 6-fold greater than the non-treated cell. Since cytidine deaminase is the major enzyme involved in the catabolism of cytosine nucleoside analogues, these results may be relevant in the evaluation of the clinical response of leukemic patients treated with 5-AZA-CdR.

Acyclic pyrimidine nucleoside analogues: influence on growth of L5178y mouse lymphoma cells and antiherpes activity in KB cells.

Several uracil and cytosine nucleoside analogues with 2-hydroxyethoxymethyl, 2-aminoethoxymethyl or 1,3-dihydroxypropoxymethyl side chains were synthesized and evaluated for cytostatic (L5178y mouse lymphoma cells) and antiviral (herpes simplex virus type 1 infected KB cells) activity. Two compounds exhibited antiherpesvirus activity. These were 1-(2'-hydroxyethoxymethyl)-5-aza-cytosine and 1-(1',3'-dihydroxypropoxymethyl)-5-iodouracil. The MIC values were 25.8 and 325.7 micrograms/ml, respectively.

Mutagenicity of a cytosine-nucleoside analog, N4-aminocytidine, in Salmonella: A case for indirect mechanism

Mutagenicity of a cytosine-nucleoside analog, N4-aminocytidine, in Salmonella: A case for indirect mechanism