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A sensitive, specific and rapid HPLC-MS/MS method has been developed and validated for the simultaneous determination of cytarabine and valcytarabine (valyl prodrug of cytarabine) in rat plasma in the present study. The analytes were separated on a C18 column (50 mm x 2.1 mm, 1.7 microm) and a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source was applied for detection. Cation exchange solid-phase extraction cartridge was employed to extract the analytes from rat plasma, with high recovery of cytarabine (>85%). The method was linear over the concentration ranges of 10-20,000 ng/mL for cytarabine and 25-1000 ng/mL for valcytarabine. The lower limit of quantitation (LLOQ) of cytarabine and valcytarabine was 10 and 25 ng/mL, respectively. The intra-day and inter-day relative standard deviation (RSD) were less than 15% and the relative error (RE) were all within 15%. Finally, the method was successfully applied to support the prodrug pharmacokinetic study after valcytarabine and cytarabine were orally administrated to the Sprague-Dawley rat, respectively.

We established a facile regioselectively chemo-enzymatic synthesis procedure for the preparation of mutual derivatives of nucleosides and pyrimidines by sequential Markovnikov addition and acylation. Firstly, pyrimidine derivatives containing vinyl ester group were synthesized from pyrimidines and divinyl esters through Markovnikov addition catalyzed by K(2)CO(3) in DMSO at 80 degrees C, and the yields were ranged from 50% to 87%. Then regioselective acylation of ribavirin and cytarabine with pyrimidine vinyl ester was catalyzed by CAL-B (immobilized lipase from Candida antarctica) in anhydrous acetone. Reaction conditions of enzymatic acylation including enzyme resource and solvents were optimized. A series of mutual derivatives of nucleosides and pyrimidines were synthesized successfully and characterized with NMR, IR, and HRMS. This chemo-enzymatic protocol involving sequential Markovnikov addition and acylation provided a novel way of synthesizing complicated functional compounds regioselectively which was hard to be achieved either by chemical or by enzymatic methods.

Poly(2-ethyl-2-oxazoline) (POZ) was synthesized by living cationic ring-opening polymerization under microwave irradiation yielding polymers with low polydispersity indices (PDI, 1.15). The polymerization was quenched with sodium carbonate yielding a hydroxyl end-group with a high degree of functionality. The hydroxyl group was converted to carboxylate and the polymer was purified by ionic exchange chromatography. Following activation to succinimidyl ester, POZ-conjugates to high and low molecular weight biomolecules, trypsin and Ara-C, were obtained. The properties of the conjugates were compared to those of the corresponding conjugates with poly(ethylene glycol) (PEG) of similar size. The coupling of POZ to trypsin did not affect the enzymatic activity towards low mass substrates but, on the contrary, reduced the activity on the higher mass ones. Furthermore, the POZ-protein conjugates showed hydrodynamic volumes and protein rejecting properties similar to those of PEG-conjugates. Similarly, the POZ-Ara-C conjugate revealed a drug release profile, stability towards the degrading enzyme cytidine deaminase and in vitro cytotoxicity comparable to what has already been described for the PEG derivative. These data support the potential of POZ as a versatile alternative to the well-known and widely used PEG for protein and drug conjugation and delivery.

We have previously demonstrated the ability of poly(glycerol adipate) backbone (PGA) and PGA polymer backbone substituted with varying amounts of pendant C(18) chain length acyl groups to yield Dexamethasone phosphate DXMP loaded nanoparticles. The aim of this study was to obtain a deeper understanding of the underlying principles responsible for good drug incorporation and controlled release of drugs from poly (glycerol adipate) (PGA) nanoparticles. We compared the incorporation of the water soluble drugs DXMP and Cytosine arabinoside (CYT-ARA) in both unmodified and substituted PGA polymers. We investigated the effect of change in acyl group chain length and the degree of substitution on the physicochemical properties, drug loading and release of DXMP and CYT-ARA. Nanoparticles were prepared by the interfacial deposition technique and the simultaneous emulsification method. Amongst the nanoparticles prepared using acylated polymers with varying chain lengths (C(2) to C(10)) for DXMP incorporation, polymers with acyl group chain lengths containing 8 carbon atoms (C(8)) showed maximum drug incorporation. Amongst the C(8) series, polymers with 100% acylation provided both good drug incorporation and a controlled release for DXMP while for CYT-ARA it was the unsubstituted polymer backbone that had maximum drug loading and slower release. A number of inter-related factors are responsible for producing particles with particular size, zeta potential, drug loading and release characteristics. Drug loading and release from nanoparticles are primarily influenced by the nature of interactions between the drug and polymers which in turn depend upon the type of drug used and the physical chemistry of the polymer.

4-Seleno analogue of 1-beta-arabinofuranosyl cytosine (ara-C) was synthesized via 4'-selenouridine as a key intermediate, which was easily prepared from D-ribose. The arabino configuration was achieved by chemoselective ring opening of the 2,2'-anhydro-4'-selenouridine. The synthesized 4'-seleno-ara-C showed potent antitumor activity (IC(50) = 1.5 microM) against stomach cancer cells (SNU638).

This study assessed the antiproliferative activity of sapacitabine (CYC682, CS-682) in a panel of 10 human cancer cell lines with varying degrees of resistance or sensitivity to the commonly used nucleoside analogues ara-C and gemcitabine. Growth inhibition studies using sapacitabine and CNDAC were performed in the panel of cell lines and compared with both nucleoside analogues and other anticancer compounds including oxaliplatin, doxorubicin, docetaxel and seliciclib. Sapacitabine displayed antiproliferative activity across a range of concentrations in a variety of cell lines, including those shown to be resistant to several anticancer drugs. Sapacitabine is biotransformed by plasma, gut and liver amidases into CNDAC and causes cell cycle arrest predominantly in the G(2)/M phase. No clear correlation was observed between sensitivity to sapacitabine and the expression of critical factors involved in resistance to nucleoside analogues such as deoxycytidine kinase (dCK), human equilibrative nucleoside transporter 1, cytosolic 5'-nucleotidase and DNA polymerase-alpha. However, sapacitabine showed cytotoxic activity against dCK-deficient L1210 cells indicating that in some cells, a dCK-independent mechanism of action may be involved. In addition, sapacitabine showed a synergistic effect when combined with gemcitabine and sequence-specific synergy with doxorubicin and oxaliplatin. Sapacitabine is therefore a good candidate for further evaluation in combination with currently used anticancer agents in tumour types with unmet needs.

Aim: To evaluate the pharmacokinetic characteristics of L-valyl-ara-C, a peptidomimetic prodrug of ara-C., Methods: After the synthesis of L-valyl-ara-C, the in vitro stability of L-valyl-ara-C was examined in various biological media. Plasma pharmacokinetic profiles of ara-C and L-valyl-ara-C were also evaluated in rats., Results: The degradation of L-valyl-ara-C was negligible in fresh plasma and also in the presence of plasmin over a 2 h incubation period. Furthermore, L-valyl-ara-C appeared to be stable in the leukemia cell homogenates, and subsequently, it was far less cytotoxic than the parent, ara-C in AML2 and L1210 cells. The chemical hydrolysis of L-valyl-ara-C was rather accelerated in acidic pH. Following an oral administration of L-valyl-ara-C, the appearance of ara-C was observed in plasma although the systemic exposure of the prodrug was much higher than that of ara-C. The bioavailability of ara-C was about 4% via prodrug administration., Conclusion: The amide bond of L-valyl-ara-C was stable against the enzymatic hydrolysis, and the utility of L-valyl-ara-C as an oral delivery system of ara-C appeared to be limited by its low metabolic conversion to ara-C in rats.

Various amphiphilic heterodinucleoside phosphates containing 1-beta-D-arabinofuranosylcytosine (ara-C) and 5- fluorodeoxyuridine (5-FdUrd) have recently been synthesized in order to increase the efficacy of ara-C and 5-FdUrd. Employing growth inhibition and growth recovery assays, we evaluated the in vitro effects of four of these dimers (No. 2, 2A, 3, 10) in L1210 and P388D1 murine leukemia cells. Although ara-C and 5-FdUrd appeared equimolar in all dimers, their contribution to the cytotoxicity of these agents was different. Thus, the liberation of ara-C and 5-FdUrd from their dimeric origin and their subsequent metabolic activation had a different course. In another set of experiments, we examined the in vivo effects of these agents in mice. The dimer with the highest cytotoxicity in vitro exerted the lowest acute toxicity and yielded the lowest therapeutic effect in vivo. The obtained data indicate that dimers with slower liberation of ara-C and 5-FdUrd were less cytotoxic, but prolonged liberation of both antimetabolites protected them from inactivation and extended the time period of therapeutic action. Some of the dimers exceeded the synergistic effects yielded by simultaneous application of both ara-C and 5-FdUrd. The significantly higher therapeutic potential of these new antitumor agents indicates that further studies are warranted.

ErbB2 overexpressing breast tumors have a poor prognosis and a high risk to develop chemoresistance to therapeutic treatment. "Chemoresistance" is a response of cells to toxic stress, and, although it is a common phenomenon, it is still poorly defined. However, a detailed understanding is required to target desensitized pathways and mechanisms for successful reactivation as part of a tailored therapy. To gain insight, which malfunctions contribute to chemoresistance, two mechanisms relevant for tissue homeostasis, the regulation of the cell cycle and of apoptosis, were investigated. Maternal MCF-7- and ErbB2-overexpressing MCF-7(erbB2) breast cancer cells were long term pretreated with 2'-deoxy-5-fluorodeoxyuridine (5-FdUrd) or 1-beta-d-arabinofuranosylcytosine (AraC) and the acquisition of drug-insensitivity was analyzed. A phosphate-conjugated heterodinucleoside consisting of one 5-FdUrd- and one AraC-moiety (5-fluoro-2'-desoxyuridylyl-(3'-->5')-Arabinocytidine) was utilized as a tool to assess the type of acquired resistances. ErbB2-overexpression disrupted proper cell cycle regulation and furthermore facilitated the development of an apoptosis-refractory phenotype upon exposure to 5-FdUrd. Experiments with dimer 5-FdUrd-araC in ErbB2-overexpressing MCF-7(erbB2) cells, and also with nucleoside 5-FdUrd in maternal MCF-7 cells, evidenced that the phenotypes of resistance to cell cycle inhibition and to apoptosis induction were differently affected. The expression profile of cyclin D1 (but not that of p53, p21, or p27) correlated with the proliferative phenotypes and nuclear accumulation of apoptosis inducing factor (but not activation of caspase 7) with apoptotic phenotypes. Dimer 5-FdUrd-araC overrode acquired chemoresistances, whereas combined application of 5-FdUrd and AraC exhibited significantly less activity. Dimer 5-FdUrd-araC remained active in MCF-7 clones most likely by circumventing the prerequisite of first-step phosphorylation. The acquisition of chemoresistance encompassed the affection of apoptosis- and cell-cycle regulation to, respectively, different extents. Thus, drug-induced cell cycle arrest and apoptosis induction are independent of each other.

A comparative study was made of Novozym 435-catalyzed regioselective acylation of 1-beta-D-arabinofuranosylcytosine with vinyl propionate for the preparation of the 5'-O-monoester in eleven co-solvent mixtures and three pure polar solvents. Novozym 435 displayed low or no acylation activity toward 1-beta-D-arabinofuranosylcytosine in pure polar solvents, although those solvents can dissolve the nucleosides well. When a hexane-pyridine co-solvent system was adopted, both the initial rate and the substrate conversion were enhanced markedly. The polarity of co-solvent mixtures had significant effect on the reaction. Among the solvent mixtures investigated, the higher the polarity of the solvent mixture, the lower the initial reaction rate and the substrate conversion. It was also found that the acylation was dependent on the hydrophobic solvent content, the water activity and the reaction temperature. The most suitable co-solvent, initial water activity, and reaction temperature were hexane-pyridine (28:72, v/v), 0.07, and 50 degrees C, respectively. Under these conditions, the initial rate, the substrate conversion and the regioselectivity were as high as 91.1 mM h(-1), >97% and >98%, respectively, after a reaction time of 6 h. Among the reaction mediums examined, the lowest apparent activation energy was achieved with hexane-pyridine (28:72, v/v), in which Novozym 435 also exhibited good thermal stability.

This study aimed to investigate the gastrointestinal stability and the cellular uptake characteristics of L-valyl-ara-C, a peptidomimetic prodrug of ara-C (cytarabine). After the synthesis of L-valyl-ara-C via the incorporation of L-valine into the N4-amino group of the cytosine ring in araC, the gastrointestinal stability of L-valyl-ara-C was examined using artificial gastric juice and artificial intestinal fluids. The cellular uptake characteristics of L-valyl-ara-C were also examined in Caco-2 cells. The disappearance half-life of L-valyl-ara-C was 2.2 h in artificial gastric juice, while the degradation of L-valyl-ara-C was negligible in artificial intestinal fluid and also in the supernatant above the Caco-2 cell monolayer during the 2-h incubation. The cellular accumulation of L-valyl-ara-C was 5-fold higher than that of ara-C in Caco-2 cells. Furthermore, the cellular uptake of L-valyl-ara-C did not increase proportionally to the increase in drug concentration. The cellular accumulation of L-valyl-ara-C was significantly reduced in the presence of uridine, p-aminohippurate, tetraethylammonium and small dipeptides, while it was not changed in the presence of L-valine and benzoic acid, suggesting that L-valyl-ara-C could interact with multiple uptake transporters, including peptide transporters, organic anion and cation transporters and nucleoside transporters, but might not interact with amino acid transporters. In conclusion, L-valyl-ara-C could be effective to improve the oral absorption of ara-C via the carrier-mediated transport pathway.

The aim of this study was to describe a competition between cytarabine (araC) and aspirin (ASA) in binding with bovine serum albumin (BSA). High-affinity binding sites for both drugs were determined using a spectrofluorimetric method. Cytarabine as well as aspirin binds in the IIA hydrophobic subdomain of the transporting protein. Binding constants for araC-BSA and ASA-BSA were calculated by the Scatchard method. Analysis of ultraviolet (UV) difference spectra showed that araC, which has a higher affinity to BSA in comparison to ASA [Ka(araC) > Ka(ASA)] displaces ASA in high-affinity binding sites. The competition between drugs in low-affinity binding sites was investigated using (1)H nuclear magnetic resonance (NMR) and 13C-NMR spectra. We concluded that in the low-affinity binding sites cytarabine decreases the affinity of albumin toward aspirin. However, the interaction between araC and BSA becomes more difficult in the presence of aspirin., (Copyright 2006 Wiley Periodicals, Inc.)

Chemoresistance is a biological response of cells to survive toxic stress. During cancer treatment the development of chemoresistance is a major problem. The mechanisms how cells become insensitive, and which downstream pathways are affected are not completely understood. Since it has not been well analysed which and how many regulative disorders are subsummised under the term "chemoresistance", we examined and measured arabinosylcytosine (AraC)-mediated desensitation of two mechanisms relevant for tissue homeostasis, cell cycle inhibition and apoptosis induction. MCF-7 cells harbouring ectopic mutated p53 were suitable for this investigation because they activated these mechanisms subsequently and became insensitive to AraC with regard to cell cycle inhibition and apoptosis induction. The major causal mechanism of acquired resistance against AraC was most likely through the inhibition of the first step of AraC phosphorylation within the cell, which is rate limiting for its activation. With regard to cell cycle inhibition AraC-resistant cells were also resistant against 5-fluorodeoxyuridine (5-FdUrd), but fully responsive to 5-FdUrd-induced apoptosis, evidencing that cell cycle and apoptosis are independent of each other. Apoptosis correlated with AIF-activation and was independent of Caspase 7, whereas cell cycle inhibition correlated with cyclinD1 expression but not with induction of p21 or p27. The phosphate conjugated 5-FdUrd-araC heterodimer (5-Fluoro-2'-desoxyuridylyl-(3'-->5')-Arabinocytidine), which is a prodrug of AraC-monophosphate, reactivated AIF and down-regulated cyclin D1 in AraC-resistant cells and circumvented resistance to apoptosis and to cell cycle inhibition. Also, cells which were resistant to 5-FdUrd or doxorubicin were sensitive to 5-FdUrd-araC. This investigation demonstrates that chemoresistance affects apoptosis induction and cell cycle inhibition independently and that detailed knowledge about the affected downstream pathways would enable the design of targeted intervention with small molecules to restore chemosensitivity.

Selectivity of enzymatic and chemical methods for transesterifications of cytarabine with divinyl dicarboxylates was described. Catalysis by lipase acrylic resin from Candida antarctica (CAL-B) in acetone facilitated the single step synthesis of polymerizable 5'-O-acyl cytarabine derivatives in high yields, while the use of alkaline protease from Bacillus subtilis (subtilisin) in pyridine afforded the mixture products of 5'-O-acyl and 4-N-acyl cytarabine derivatives. Interestingly, polymerizable 4-N-acyl cytarabine vinyl derivatives can be selectively prepared by chemical transesterification in dioxane. The obtained series of cytarabine derivatives would be useful for a significant monomer for a polymeric anticancer drug.

Various amphiphilic heterodinucleoside phosphates have recently been synthesized in order to overcome drug resistance. These agents contain 5-fluorodeoxyuridine (5-FdUrd) and arabinofuranosylcytosine (Ara-C). We now investigated the action of two of these novel dimers (#2 and #10) in sensitive and 5-FdUrd/Ara-C cross-resistant H9 human lymphoma cells. The dimers were compared with 5-FdUrd and Ara-C for growth inhibition, apoptosis induction, and cell-cycle effects. No significant difference in the cytotoxicity of dimer #2 could be observed between sensitive and 5-FdUrd/Ara-C cross-resistant H9 cells (IC50 values of 220 nM and 200 nM, respectively), indicating that further studies with this compound are warranted.

The Institut Gustave-Roussy (IGR) Department of Clinical Pharmacy (DCP) ensures the annual preparation of about 30 000 therapeutic batches of anti-neoplastic agents. High performance thin-layer chromatography (HPTLC) allows postproduction quality control of these batches. Although the centralized chemotherapy manufacturing unit has been recently ISO 9001:2000 certified, it was considered to improve the quality level of manufactured batches even further. The viability of micro-organisms (bacteria and fungi) in appropriate sterile media containing various anti-neoplastic agents at therapeutic concentration was assessed to demonstrate the lack of contamination during our manufacturing process in the isolator. After 14 days of incubation in these media, the results show the absence of contamination of the manufactured batches. This leads us to conclude that using sterile drugs and sterile medical devices in a sterile isolator allows the manufacture of sterile therapeutic batches with excellent confidence.

Amidox (3,4-dihydroxybenzamidoxime), a new polyhydroxy-substituted benzoic acid derivative, is a potent inhibitor of the enzyme ribonucleotide reductase (RR), which catalyses the de novo synthesis of DNA. RR is considered to be an excellent target for cancer chemotherapy. In the present study we investigated the antineoplastic effects of Amidox alone and in combination with Arabinofuranosylcytosine (Ara-C) in HL-60 human promyelocytic leukemia cells. In growth inhibition experiments Amidox yielded an IC50 of 30 microM, colony formation was inhibited at an IC50 of 20 microM as determined by a soft agar assay. Exposure of the cells to 75 and 100 microM Amidox for 24 hours was shown to significantly decrease intracellular dCTP, dGTP and dATP pools, whereas dTTP concentration increased, as determined by HPLC. The combination of Amidox with Ara-C yielded more than additive cytotoxic effects both in growth inhibition assays and in soft agar assays. We could show that--after preincubating the cells with 75 and 100 microM Amidox and subsequent exposure to Ara-C--intracellular Ara-CTP levels increased by 576% and 1143%, respectively. In conclusion, Amidox might offer an additional option for the treatment of leukemia and thus be further investigated in vitro and in vivo.

Physiologic deoxynucleotides are required for an error-proof DNA replication, repair and synthesis. Any inaccuracy in this process results in a block in DNA synthesis until the error is corrected. If the cell enzymes are unable to correct the error, a signal for apoptosis is generated. This mechanism is the main target for anticancer nucleoside analogs. They also interact with the metabolism of physiological nucleosides, and consequently, have a large number of intracellular targets to induce cytotoxicity. In addition, it is now reported that some analogs may interfere directly with RNA synthesis. A great deal of synthesized nucleoside analogs provide the opportunity to understand the structure-based differences in their metabolism and mechanisms of action as well as to identify the specific intracellular targets and diseases, in which each of these newer nucleoside analogs acts most efficiently. This paper summarizes developments in the area of new nucleoside analogs undergoing clinical evaluation for the treatment of solid tumors, namely tezacitabine, troxacitabine, DMDC, CNDAC, ECyD, clofarabine, and decitabine.

The electrochemical oxidation and reduction behaviour of adsorbed species of antimetabolic antineoplastic agent Tarabine PFS (Cytosar-U) in Sorensen buffer solution of different pH values at an in situ-mercury film electrode (MFE) is studied using cyclic voltammetry (CV) and Osteryoung square-wave stripping voltammetry (OSWSV). Optimal experimental and operational parameters have been selected for the drug preconcentration and determination in aqueous medium. Based on the adsorption and accumulation of Tarabine PFS using Osteryoung square-wave anodic stripping voltammetry (OSWASV) at MFE, the drug is easily detected as 0.134 ng/ml (5.51 x 10(-10) M). Calibration plots have been constructed at different accumulation times. The standard deviation (n=10) at a concentration level of 6 x 10(-8) M Tarabine PFS is 0.062. The interaction of ssDNA with the drug under the optimal conditions at pH 7.7 has been studied. The formal potentials E degrees and E degrees ' and the equilibrium constants K(1) and K(2) have been calculated for the free form of Tarabine PFS and the bonded form with ssDNA, respectively. It was found that K(2) value for the bonded oxidized form is 298 times than that of K(1) for the bonded reduced form. Therefore, ssDNA has been found to interact strongly with the oxidized form of the drug. The method has been used for the nanogram determination of ssDNA with 1.9% variation coefficient. Detection limit of 3 ng/ml ssDNA has been achieved. Possible interfering organic compounds, cations and anions have been tested. The method has been applied for the drug determination in urine samples, down to 0.23 ng/ml could be easily achieved in such samples.

Cytarabine (ara-C) was included in albumin microspheres and these microspheres were immersed in a poly(lactide-co-glycolide) (PLGA) film to constitute a comatrix system to develop a prolonged form of release. Cytarabine-loaded albumin microspheres were synthesized by emulsion, and 25 or 50 mg of drug were included in the disperse phase. Thus, microspheres with 46+/-4 microg drug/mg microspheres and 50+/-5 microg drug/mg microspheres were obtained, which means a percentage of incorporation efficiency of 42+/-4% and 25+/-2%, respectively. These cytarabine-loaded microspheres were used to prepare PLGA-comatrices. Kinetic release studies indicated that total cytarabine release only takes place in the presence of protease, probably due to the fact that glutaraldehyde establishes covalent links with the amine side group of the drug and cross-links it with the protein matrix. A slower kinetic release of the drug was obtained from PLGA-comatrices, although only 80% of the included cytarabine was released on day 7. The comatrices were subcutaneously implanted in the back of rats and in both cases the ara-C administered dose was 36 mg of ara-C per kg of body weight. The drug was detected in plasma 10 days. The mean residence time (MRT) of the drug administered by these comatrices was 87-91 times larger when compared to the value obtained when the drug was administered in solution by intraperitoneal injection. The histological studies show that a degradative process of the comatrices takes place. The comatrices do not damage surrounding tissue; a normal regeneration of the implanted zone was observed.

The objective of the study was to optimize the formulation parameters of cytarabine liposomes by using artificial neural networks (ANN) and multiple regression analysis using 3(3) factorial design (FD). As model formulations, 27 formulations were prepared. The formulation variables, drug (cytarabine)/lipid (phosphatidyl choline [PC] and cholesterol [Chol]) molar ratio (X1), PC/Chol in percentage ratio of total lipids (X2), and the volume of hydration medium (X3) were selected as the independent variables; and the percentage drug entrapment (PDE) was selected as the dependent variable. A set of causal factors was used as tutorial data for ANN and fed into a computer. The optimization was performed by minimizing the generalized distance between the predicted values of each response and the optimized one that was obtained individually. In case of 3(3) factorial design, a second-order full-model polynomial equation and a reduced model were established by subjecting the transformed values of independent variables to multiple regression analysis, and contour plots were drawn using the equation. The optimization methods developed by both ANN and FD were validated by preparing another 5 liposomal formulations. The predetermined PDE and the experimental data were compared with predicted data by paired t test, no statistically significant difference was observed. ANN showed less error compared with multiple regression analysis. These findings demonstrate that ANN provides more accurate prediction and is quite useful in the optimization of pharmaceutical formulations when compared with the multiple regression analysis method.

1-beta-D-arabinofuranosylcytosine (ara-C) conjugates of phospholipid were shown to be highly antineoplastic against various tumor cells. In this study, we report that these conjugates are potent activators of protein kinase C (PKC, EC) in vitro. Although required Ca2+, PKC activation by the conjugates occurred even in the absence of phospholipid and diacylglycerol. Among the conjugates, 1-beta-D-arabino-furanosylcytosine 5'-diphosphate-rac-1-O-octadecyl-2-O-palmitoylglycerol [(ara-CDP-DL-PBA); ara-C conjugate of ether phospholipid], was employed to investigate its mode of activation, since ether phospholipid has been reported to be a regulator of the PKC. When PKC was activated by ara-CDP-DL-PBA, diacylglycerol enhanced its activity with 3-fold reduction of an apparent Ka value for ara-CDP-DL-PBA and no change in the Vmax. During the PKC activation by phosphatidylserine, ara-CDP-DL-PBA exhibited a synergistic effect on the activation. Studies on the relationship between the structures of ara-CDP-DL-PBA and their effects on PKC activity showed that phosphate group of ether lipid was important for its activation of PKC, and that conjugation of ara-C and ether lipid further enhanced the enzyme activity. These results suggest that the ara-C conjugate of phospholipid activates PKC in a co-operative manner with diacylglycerol and/or phosphatidylserine, however, the exact mechanism of the antineoplastic effect of ara-CDP-DL-PBA through PKC activation still remains speculative.

Cytarabine, an analog of deoxycytidine, is an important agent in the treatment of ovarian carcinoma, acute myeloid and lymphoblastic leukemia. Its mechanism of action has been attributed to an interference with DNA replication. The plasma membrane has received increasing attention as a possible target of antitumor drugs, where the drugs may act as growth factor antagonists and receptor blockers, interfere with mitogenic signal transduction or exert direct cytotoxic effects. Furthermore, it has been reported that drugs that exert their antiproliferative effect by interacting with DNA generally cause structural and functional membrane alterations which may be essential for growth inhibition by these agents. This paper describes the studies undertaken to determine the structural effects induced by cytarabine to cell membranes. The results showed that cytarabine, at a concentration about one thousand times higher than that found in plasma when it is therapeutically administered, did not induce significant structural perturbation in any of these systems. Therefore, it can be unambiguously concluded that this widely used anticancer drug does not interact at all with erythrocyte membranes.

As an approach to the development of specific drug delivery systems, a new class of low macromolecular carriers called 'telomers' endowed with an antitumour agent, such as arabinofuranosylcytosine (Ara-C), RGDSK peptidic sequences, as tumour targeting moieties, and tyrosine groups labelled with 125I atoms allowing the in vivo scintigraphic follow up, were synthesized. Their tumour targeting ability was assessed in vivo in mice bearing a murine B16 melanoma. The biological results showed that the presence of RGDSK sequences onto the macromolecules leads to the selective targeting and the accumulation of telomers within the vascularized zone of the tumour. Moreover, such compounds exhibited in vitro a better IC(50) (0.015 muM) than pure Ara-C and in vivo an oncostatic index higher than 160%.

alpha-Phosphono lactone derivatives of the nucleosides cytidine and cytosine arabinoside have been prepared from the corresponding nucleoside aldehydes. The stereochemical outcome of allylation reactions with these aldehydes was found to be dependent upon both the choice of protecting groups for the 2'- and 3'-hydroxyl groups and, to some extent, the nature of the Lewis acid catalyst employed. Ultimately, conditions were found that favored either the 5'R or 5'S diastereomer from different cytidine aldehydes, and gave some stereoselectivity in additions to an aldehyde derivative of ara-C. The resulting homoallylic alcohols were used as substrates in attempted Knovenagel and Horner-Wadsworth-Emmons condensations, but elimination was found to predominate over lactone formation under the conditions employed. The desired alpha-phosphono lactones could be prepared through a reaction sequence that included ring-closing metathesis on acrylate esters of the homoallylic alcohols, followed by reduction of the resulting alpha,beta-unsaturated lactones and carbon-phosphorus bond formation on enolates generated from the saturated lactones.

The 5'-amino-5'-phosphono derivatives of cytidine, cytosine arabinoside (ara-C), and uridine have been prepared via the corresponding nucleoside aldehydes. Phosphite addition to imines derived from the nucleoside aldehydes and p-methoxybenzylamine was efficient, and use of this amine allowed cleavage of the products to the parent amino phosphonic acids. The phosphite additions proved to be diastereoselective, with the cytidine and uridine derivatives favoring the 5'S stereochemistry and the ara-C derivative favoring the 5'R isomer. The stereochemistry of one cytidine derivative was established by single-crystal diffraction analysis, and detailed comparisons of the (13)C NMR data allowed assignments of the other amino phosphonates.

1-beta-d-Arabinofuranosylcytosine (Ara-C) is a potent antineoplastic drug used in the treatment of acute leukemia. Previous biochemical studies indicated the incorporation of Ara-C into DNA reduced the catalytic activity of human topoisomerase I by decreasing the rate of single DNA strand religation by the enzyme by 2-3-fold. We present the 3.1 A crystal structure of human topoisomerase I in covalent complex with an oligonucleotide containing Ara-C at the +1 position of the non-scissile DNA strand. The structure reveals that a hydrogen bond formed between the 2'-hydroxyl of Ara-C and the O4' of the adjacent -1 base 5' to the damage site stabilizes a C3'-endo pucker in the Ara-C arabinose ring. The structural distortions at the site of damage are translated across the DNA double helix to the active site of human topoisomerase I. The free sulfhydryl at the 5'-end of the nicked DNA strand in this trapped covalent complex is shifted out of alignment with the 3'-phosphotyrosine linkage at the catalytic tyrosine 723 residue, producing a geometry not optimal for religation. The subtle structural changes caused by the presence of Ara-C in the DNA duplex may contribute to the cytotoxicity of this leukemia drug by prolonging the lifetime of the covalent human topoisomerase I-DNA complex.

Both the (R)- and (S)-5'-hydroxy 5'-phosphonate derivatives of cytidine and cytosine arabinoside (ara-C) have been prepared via phosphite addition or a Lewis acid mediated hydrophosphonylation of appropriately protected 5'-nucleoside aldehydes. Phosphite addition to a cytosine aldehyde protected as the 2',3'-acetonide gave predominately the 5'R isomer, while phosphite addition to the corresponding 2',3'-bis TBS derivative favored the 5'S stereochemistry. In contrast, phosphite addition to the 2',3'-bis TBS protected aldehyde derived from ara-C gave only the 5'R adduct. However, TiCl(4)-mediated hydrophosphonylation of the same ara-C aldehyde favored the 5'S stereoisomer by a 2:1 ratio. Once all four of the diastereomers were in hand, the stereochemistry of these compounds could be assigned based on their spectral data or that obtained from their O-methyl mandelate derivatives. After hydrolysis of the phosphonate esters and various protecting groups, the four alpha-hydroxy phosphonic acids were tested for their ability to serve as substrates for the enzyme nucleoside monophosphate kinase and for their toxicity to K562 cells.

pH-sensitive niosomal and liposomal formulations bearing alkylated N-isopropylacrylamide (NIPAM) copolymers were characterized with regard to vesicle-polymer interaction, pH-responsiveness and stability in human serum. The interactions between the pH-sensitive NIPAM copolymer and the vesicles were studied by spectrofluorimetry, using covalently-attached pyrene as a probe. In contrast to liposomes, where complexation of copolymer to the lipid bilayer is essentially mediated by hydrophobic interactions, the binding between niosomes and PNIPAM was mainly driven by hydrogen bonding. Both formulations were found to rapidly release their contents under mildly acidic conditions. However, the niosomes lost their pH-sensitivity after incubation in serum, whereas liposomes maintained their ability to respond to pH only when complexed with a copolymer containing a high proportion of hydrophobic anchor. The ability of pH-sensitive liposome/polymer complexes to enhance the cytotoxicity of cytosine arabinofuranoside (ara-C) was evaluated in vitro using macrophage-like J774 cells. Ara-C encapsulated in pH-sensitive liposomes exhibited a higher cytotoxicity than the control formulation. This study showed that both niosomes and liposomes can be rendered pH-sensitive by anchoring a randomly-alkylated NIPAM copolymer to their surface. The interactions that take place between the polymer and the vesicles strongly depend on the vesicle nature. pH-sensitive PNIPAM-based liposomes can improve the in vitro efficiency of ara-C.

The drugs concerned by this review are cytarabine (ara-C), gemcitabine and fludarabine. Seventy-eighty per cent of a dose of ara-C are excreted under the form of ara-U (main metabolite). Plasma concentrations of ara-C are not related to drug pharmacodynamics (response to treatment) in contrast to intracellular levels of ara-CTP (active metabolite) which are associated with cytotoxic activity. Gemcitabine is able to autoactivate its own mechanism of action. Gemcitabine is characterized by a short half-life of elimination (15-20 min) and plasma pharmacokinetics of the drug are not linked to pharmacodynamics. Prolonged administration of gemcitabine is pharmacokinetically and pharmacologically justified and should deserve more intense clinical investigations. Total body clearance of F-ara-A (main circulating metabolite of fludarabine) is linked to creatinine clearance and drug-related neutropenia are more frequent in patients with creatinine clearance below 50 mL/min. So far there are no relationships between intracellular levels of F-ara-CTP and response to treatment.

We previously reported that liposomalized 5'-O-dipalmitoylphosphatidyl 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine (DPP-CNDAC), a hydrophobized derivative of the novel antitumor nucleoside CNDAC, is quite useful for cancer therapy. On the other hand, for anti-neovascular therapy, we recently isolated peptides homing to angiogenic vessels from a phage-displayed random peptide library, and observed that peptide-modified liposomal adriamycin strongly suppressed tumor growth, perhaps through damaging angiogenic endothelial cells. In the present study, we modified DPP-CNDAC-liposomes with one of the angiogenic homing peptides, APRPG, and examined their antitumor activity. Three doses of APRPG-modified DPP-CNDAC-liposomes (15 mg/kg as CNDAC) strongly inhibited tumor growth compared with the same number of doses of unmodified DPP-CNDAC-liposomes. The life span was increased 31.8%, with one completely cured mouse out of the six mice treated. Since the accumulation of liposomes in the tumor tissue was not so much different between APRPG-liposomes and non-modified liposomes, the enhanced therapeutic efficacy may be explained as the alteration of targets, i.e. APRPG-modified DPP-CNDAC-liposomes caused tumor growth suppression through damage of angiogenic endothelial cells. Anti-neovascular therapy promises no drug resistance, and should be effective against essentially any kind of solid tumor; and thus the present results demonstrate another benefit of the therapy, namely, high efficacy of cancer treatment.

A systematic study of N(4) amino PEG-prodrugs of ara-C (1) was conducted and provided a series of disubstituted amides, as well as a carbamate derivative. These conjugates showed hydrolysis half lives in rat plasma from about 1 h to 3 days, but were stable for >24 h in phosphate buffer, pH 7.4. In an LX-1 solid lung tumor model some of the PEG prodrugs exhibited superior activity to ara-C when compared on a molar basis. One problematic issue that was identified in this investigation was the need to increase the loading of ara-C onto PEG in order to avoid highly viscous solutions.

The synthesis of branched PEG (40,000) acids has been achieved using aspartic acid (Asp) and AspAsp dendrons. Complete conjugation of these dendritic acids with cytosine arabinoside (ara-C) was achieved by the use of spacers that allowed a greater separation of the branches to accommodate several large ara-C molecules in proximity to each other. The tetrameric and octameric PEG-ara-C amide prodrugs were much more effective in the treatment of solid and ascites tumors compared to the native drug. The greater loading of the PEG backbone appears to have achieved a minimum threshold concentration for the therapeutic delivery of ara-C.

Nucleoside analogues (NA) are essential components of AML induction therapy (cytosine arabinoside), effective treatments of lymphoproliferative disorders (fludarabine, cladribine) and are also used in the treatment of some solid tumors (gemcitabine). These important compounds share some general common characteristics, namely in terms of requiring transport by specific membrane transporters, metabolism and interaction with intracellular targets. However, these compounds differ in regard to the types of transporters that most efficiently transport a given compound, and their preferential interaction with certain targets which may explain why some compounds are more effective against rapidly proliferating tumors and others on neoplasia with a more protracted evolution. In this review, we analyze the available data concerning mechanisms of action of and resistance to NA, with particular emphasis on recent advances in the characterization of nucleoside transporters and on the potential role of activating or inactivating enzymes in the induction of clinical resistance to these compounds. We performed an extensive search of published in vitro and clinical data in which the levels of expression of nucleoside-activating or inactivating enzymes have been correlated with tumor response or patient outcome. Strategies aiming to increase the intracellular concentrations of active compounds are presented.

The 1'alpha-phenylselenouridine derivative 13 was successfully synthesized by enolization of the 3',5'-O-TIPDS-2'-ketouridine 8, and was subjected to a radical reaction with a vinylsilyl tether--an efficient procedure for preparing 1'alpha-branched-chain sugar pyrimidine nucleosides. Successive treatment of 8 with LiHMDS and PhSeCl in THF at < -70 degrees C gave the desired 1'-phenylseleno products in 85% yield as an anomeric mixture of the 1'alpha-product 11 and the 1'beta-product 12 (11/12= 2.5:1). Highly stereoselective reduction at the 2'-carbonyl of the 1'alpha-product 11 occurred from the beta-face by using NaBH4/CeCl3 in MeOH, and subsequent introduction of a dimethylvinylsilyl tether at the 2'-hydroxyl gave the radical reaction substrate 14. The photochemical radical atom-transfer reaction of 14 by using a high-pressure mercury lamp proceeded effectively in benzene to give the exo-cyclized PhSe-transferred product 18, in which (PhSe)2 proved to be essential as an additive for radical atom-transfer cyclization reactions. Subsequent phenylseleno-group elimination of 18 gave the sugar-protected 1'alpha-vinyluridine. With this procedure, 1'alpha-vinyluridine (22) and -cytidine (25), designed to be potential antitumor agents, were successfully synthesized. This study is the first example of functionalization at the anomeric 1'-position of a nucleoside by starting from a natural nucleoside to produce a ribo-type 1'-modified nucleoside.

The clinical usefulness of antitumor chemotherapy has been strongly limited by the lack of specificity of most anticancer drugs, which act also against healthy cells. The aim of this work was to design, synthesize, and evaluate a macromolecular prodrug of Cytarabine, a known antitumor drug, which is a specific substrate for plasmin enzyme whose concentration is high in various kinds of tumor mass as a result of plasminogen activator secretion. alpha,beta-Poly(N-hydroxyethyl)-DL-aspartamide (PHEA), a known synthetic and biocompatible polyamino acid, was used as a drug carrier, and Cytarabine was linked to PHEA by D-Val-Leu-Lys spacer synthesized beginning from Cbz-D-Val-LeuOH dipeptide and N6-CbzLys methyl ester. The content of Cytarabine in the purified PHEA-D-Val-Leu-Lys-Cytarabine conjugate was equal to 3% w/w. In vitro experiments in the presence of plasmin evidenced the ability of this enzyme to strongly increase drug release from the macromolecular prodrug, as well as plasma incubation shows high stability of drug-polymer linkage. The direct linkage of Cytarabine to PHEA was also performed and, like PHEA-D-Val-Leu-Lys-Cytarabine conjugate, the obtained PHEA-Cytarabine conjugate showed high stability in plasma, but no release of Cytarabine was revealed in the presence of plasmin.

beta-l-Dioxolane-cytidine (l-OddC, BCH-4556, Troxacitabine) is a novel unnatural stereochemical nucleoside analog that is under phase II clinical study for cancer treatment. This nucleoside analog could be phosphorylated and subsequently incorporated into the 3' terminus of DNA. The cytotoxicity of l-OddC was correlated with the amount of l-OddCMP in DNA, which depends on the incorporation by DNA polymerases and the removal by exonucleases. Here we reported the purification and identification of the major enzyme that could preferentially remove l-OddCMP compared with dCMP from the 3' termini of DNA in human cells. Surprisingly, this enzyme was found to be apurinic/apyrimidinic endonuclease (APE1) (), a well characterized DNA base excision repair protein. APE1 preferred to remove l- over d-configuration nucleosides from 3' termini of DNA. The efficiency of removal of these deoxycytidine analogs were as follows: l-OddC > beta-l-2',3'-dideoxy-2', 3'-didehydro-5-fluorocytidine > beta-l-2',3'-dideoxycytidine > beta-l-2',3'-dideoxy-3'-thiocytidine > beta-d-2',3'-dideoxycytidine > beta-d-2',2'-difluorodeoxycytidine > beta-d-2'-deoxycytidine >/= beta-d-arabinofuranosylcytosine. This report is the first demonstration that an exonuclease can preferentially excise l-configuration nucleoside analogs. This discovery suggests that APE1 could be critical for the activity of l-OddC or other l-nucleoside analogs and may play additional important roles in cells that were not previously known.

Due to the capability of peptidyl derivatives of araC to behave as prodrugs of this antimetabolite, and because of the well known biological properties of peptide T and its analogues (in particular that of targeting CD4+ cells), new peptide T-araC conjugates were prepared and tested in vitro for antiproliferative activity. The aim was that of specifically delivering the antitumor drug to CD4+ cells. N4-(Acylpeptidyl)-derivatives of araC were synthesized by a new general approach involving solid-phase synthesis, which allows mild conditions, avoids the usually required protection of the glycoside portion of nucleosides and affords high yields of the final products. After the demonstration that peptide T-araC conjugates were able to activate chemotaxis by binding CD4 receptor on monocyte membranes, the antiproliferative activity was evaluated against a panel of leukemia lymphoma and carcinoma cell lines derived from human tumors, three CD4+ cell lines included. Title compounds resulted effective as antiproliferative agents at concentrations 4- to 10-fold higher than those of araC alone, did not preferentially inhibit CD4+ cells and proved stable not only in cell culture medium containing 20% FCS, but also in human plasma. All this suggests their potential utility in vivo.

4'-thio-beta-D-arabinofuranosylcytosine was synthesized by a facile route in high yields. It was evaluated for antitumor activity against a panel of human tumors, both in vitro and in vivo.

A convenient and mild synthesis of 5-bromo-N4-substituted-1-(beta-D-arabinofuranosyl)cytosine and 5-bromo-O4-methyl-1-(beta-D-arabinofuranosyl)pyrimidin-2(1H)-one derivatives by selective oxyfunctionalization of the corresponding 4-thionucleosides with 3,3-dimethyldioxirane is reported. The cytotoxicity and the antiviral activity against parainfluenza 1 (Sendai virus) of all new synthesized products are also reported.

Continuing our studies on ribonucleotide reductase (RNR) mechanism-based inhibitors, we have now prepared the diphosphates (DP) of 2'-O-allyl-1-beta-D-arabinofuranosyl-uracil and -cytosine and 2'-O-allyl-9-beta-D-arabinofuranosyl-adenine and evaluated their inhibitory activity against recombinant murine RNR. 2'-O-Allyl-araUDP proved to be inhibitory to RNR at an IC(50) of 100 microM, whereas 2'-O-allyl-araCDP was only marginally active (IC(50) 1 mM) and 2'-O-allyl-araADP was completely inactive. The susceptibility of the parent nucleosides to phosphorylation by thymidine kinase and 2'-deoxycytidine kinase was also investigated, and all nucleosides proved to be poor substrates for the above-cited kinases. Moreover, prodrugs of 2'-O-allyl-araU and -araC monophosphates, namely 2'-O-allyl-5'-(phenylethoxy-L-alanyl phosphate)-araU and -araC, were prepared and tested against tumor cell proliferation but proved to be inactive. A molecular modeling study has been conducted in order to explain our results. The data confirm that for both the natural and analogue nucleoside diphosphates, the principal determinant interaction with the active site of RNR is with the diphosphate group, which forms strong hydrogen bonds with Glu623, Thr624, Ser625, and Thr209. Our findings indicate that the poor phosphorylation may represent an explanation for the lack of marked in vitro cytostatic activity of the test compounds.

The effects of cytarabine on the structural and thermodynamic properties of an Okazaki fragment were investigated using UV hyperchromicity and 2D 1H NMR. Cytarabine significantly decreased the stability of this model Okazaki fragment, decreasing the melting temperature from 46.8 degrees C to 42.4 degrees C at 1.33 x 10(-5) M. Cytarabine also markedly increased the bend angle of the Okazaki fragment duplex from 20 degrees to 42 degrees. Changes to the structures and stabilities of Okazaki fragments may cause the biological effects of cytarabine.

Lipophilic derivatives of ara-C were developed with the aim to improve drug penetration and retention in solid tumors. Ara-C was esterified at the 5'-position with fatty acids (16-22 C-atoms, 0-3 double bonds). The derivatives were inactive in cell lines with various forms of ara-C and 2',2'-difluorodeoxycytidine (dFdC, gemcitabine) resistance, including deoxycytidine kinase (dCK) deficiency. The activity in the parent cell lines correlated negatively with chain length and positively with double bonds.

We investigate here the effects of the incorporation of the nucleoside analogs araC (1-beta-D-arabinofuranosylcytosine) and ganciclovir (9-[(1,3-dihydroxy-2-propoxy)methyl] guanine) into the DNA binding recognition sequence for the GATA-1 erythroid transcription factor. A 10-fold decrease in binding affinity was observed for the ganciclovir-substituted DNA complex in comparison to an unmodified DNA of the same sequence composition. AraC substitution did not result in any changes in binding affinity. 1H-15N HSQC and NOESY NMR experiments revealed a number of chemical shift changes in both DNA and protein in the ganciclovir-modified DNA-protein complex when compared to the unmodified DNA-protein complex. These changes in chemical shift and binding affinity suggest a change in the binding mode of the complex when ganciclovir is incorporated into the GATA DNA binding site.

Gemcitabine (2', 2'-difluorodeoxycytidine: dFdC) is a nucleoside analogue of deoxycytidine (dCyd). In the cells, dFdC is rapidly phosphorylated by dCyd kinase. dFdC inhibits ribonucleotide reductase and the subsequent decrease in cellular deoxynucleotides (particularly dCTP) is an important self-potentiating mechanism, resulting in decreased dFdC nucleotide incorporation into DNA. Other self-potentiating mechanisms of dFdC include increased formation of active dFdCDP and dFdC TP, and decreased elimination of dFdC nucleotides. After dFdC nucleotide is incorporated on the end of the DNA strand, one more deoxynucleotide is added and thereafter the DNA polymerases are unable to proceed (masked chain termination). dFdC is a better transport substrate compared with Ara-C, phosphorylated more efficiently and eliminated more slowly. These differences together with self-potentiation, masked chain termination and the inhibition of ribonucleotide reductase may explain why dFdC is active against human solid cancers compared with Ara-C. Schedule-dependent antitumor activities of dFdC were observed against various rodent tumors and human cancer xenograft systems.

5'-Chloro-5'-deoxy arabinosylcytosine (Cl-araC) is a more lipophilic analog of the clinically used drug--arabinosylcytosine (araC). The resistance toward the enzyme cytidine-deaminase action was described as an characteristic feature of this synthetic nucleoside. The kinetics of the Cl-araC transformation in acid and alkaline solutions was studied at various temperatures. When compared with parent compound araC, the chlorine atom at the 5' position of the nucleoside sugar moiety increases the Cl-araC stability. The chlorine atom stabilizing effect is higher in acidic conditions. Cl-araC increased stability, antileukemic activity accompanied by higher lipophilicity confirm the fact that Cl-araC belong among interesting compounds from the point of view of cancer chemotherapy.

The polymerization of nucleotide analogs into DNA is a common strategy used to inhibit DNA synthesis in rapidly dividing tumor cells and viruses. The mammalian DNA polymerases catalyze the insertion of the arabinofuranosyl analogs of dNTPs (aranucleotides) into DNA efficiently, but elongate from the 3' aranucleotides poorly. Slow elongation provides an opportunity for exonucleases to remove aranucleotides. The exonuclease activity associated with DNA polymerase delta removes araCMP from 3' termini with the same efficiency that it removes a paired 3' deoxycytosine suggesting that the proofreading exonucleases associated with DNA polymerases might remove aranucleotides inefficiently. A separate 30 kDa exonuclease has been purified from mammalian cells that removes araCMP from 3' termini. The activity of this enzyme in the cell could remove aranucleotides from 3' termini of DNA and decrease the efficacy of the analogs. Inhibition analysis of the purified exonuclease shows that this enzyme is inhibited by thioinosine monophosphate (TIMP) with a Ki = 17 microM. When high TIMP levels are generated in HL-60 cells, incorporation of araC in DNA is increased about 16-fold relative to total DNA synthesis. This increased araC in DNA is likely a result of exonuclease inhibition in the cell. Thus, exonucleases in cells might play an important role in removing aranucleotides inserted by DNA polymerases.

Matrices loaded with cytarabine were prepared by compression of the tailor made triblock copolymers C17E227C17 and C17E454C17 (where C=methylene and E=oxyethylene). Observations of the swelling characteristics of copolymer matrices on immersion in distilled water indicated an increase in the thickness of the gel layer around the matrices following ingress of water into the matrices. The in vitro release of cytarabine was characterised from matrices of different molar mass and with different known drug loadings. The release of cytarabine from the copolymer matrices was predominantly by a Fickian diffusion mechanism; the release rate was dependent on drug loading and independent of copolymer molar mass.

To investigate the stability of 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentafuranosylcytosine 3'-phosphoric acid, its thymidine ester was prepared via the phosphoramidite method using allyl protection for the phosphate function. This ester is stable under acidic conditions but extremely labile under basic conditions, decomposing with a cleavage of the internucleotide bond even at pH 7.3.

The recent cloning of human cytidine deaminase (CDD) revealed two variants with a nonconservative amino acid deviation (Gln<-->Lys) at codon 27 within a region of structural homology to a core domain of bacterial CDDs. We here confirm the occurrence of both CDD sequences by cDNA cloning and show that at cytarabine (ara-C) concentrations of 1x10(-8) to 2x10(-2) M, the recombinant enzyme corresponding to the Lys-carrying natural variant (CDD-2) exerts a 1.3- to 3.3-fold higher in vitro deamination rate of ara-C than the Gln-carrying enzyme (CDD-1). These results suggest that this genetic polymorphism contributes to the different deamination phenotypes of ara-C observed in vivo, and that investigation of CDD allelotype frequencies and their correlation with ara-C resistance in patients with acute leukemia may be warranted. In addition, our data may be relevant to recently considered CDD gene transfer strategies for the detoxification of hematopoietic stem cells during high-dose therapy with cytosine nucleoside analogs.