1 Rega Institute for Medical Research, K. U. Leuven, B-3000 Leuven, Belgium; 2 Welsh School of Pharmacy, University of Wales Cardiff, Cardiff CF1 3XF, UK
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Abstract |
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Antiviral activity of bicyclic pyrimidine nucleoside analogues |
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Resistance development of BCNAs against VZV |
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Molecular basis for the anti-VZV specificity of BCNAs |
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Interestingly, when human erythrocyte NDP kinase was added to the reaction mixture containing BVDU or BCNAs and VZV TK, a considerable amount of BVDU-triphosphate was formed, whereas no trace of BCNA-triphosphate could be detected.7 Although we cannot exclude the possibility that BCNA-diphosphate can be recognized by other NDP kinase isoenzymes or other cellular enzymes, our observations may suggest that the mechanism of anti-VZV activity of the BCNAs does not occur through the 5'-triphosphate derivatives, but through the 5'-monophosphate or 5'-diphosphate derivative, virtually excluding viral DNA polymerase as the eventual target for anti-VZV activity. The mechanism of antiviral action of the BCNAs is currently under further investigation in our laboratories.
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Catabolic properties of BCNAs |
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Pyrimidine nucleoside analogues are often prone to hydrolysis to their free base by the pyrimidine nucleoside catabolic enzyme uridine phosphorylase (UPase) or thymidine phosphorylase (TPase). The free bases are, as a rule, devoid of any therapeutic activity. Thus, the UPase and TPase enzymes may inactivate the antiviral pyrimidine nucleoside analogues. BVDU is a well-known example of an antiherpes virus drug that is highly susceptible to hydrolytic cleavage by both human and Escherichia coli TPase.8,9 Interestingly, the BCNAs were found to be entirely resistant to the phosphorolytic cleavage by TPase, and thus are expected to be relatively stable in biological fluids.10 Human plasma does indeed not catabolize BCNAs to their free bases. When modelled in the active site of E. coli TPase,11,12 it became clear why the BCNA Cf 1743 did not function as a substrate for TPase. First of all, the BCNAs have no free carbonyl at the corresponding position (C-4) in the pyrimidine ring, but, more importantly, the BCNA base has no hydrogen available on the original N-3 position of the pyrimidine ring that forms a hydrogen bond with Ser-186 in the active site of TPase. Moreover, the presence of a bulky 6-alkylphenyl side chain on the fused bicyclic pyrimidine ring clashes with the main chain residues (176 and 177) of E. coli TPase, which must prevent proper positioning of Cf 1743 in the active site of the enzyme. Our observation that Cf 2002, which represents a BCNA without the 6-substituent on the fused pyrimidine ring, also lacks substrate activity for human and E. coli TPase indicates that lacking one hydrogen bond between the BCNA and the enzyme active site may be sufficient to annihilate substrate activity of the BCNAs for TPase.
Role of dihydropyrimidine dehydrogenase
The free nucleobase of BVDU, (E)-5-(2-bromovinyl)uracil (BVU), and related compounds such as 5-propynyl-uracil have proven to be efficient inhibitors of human dihydropyrimidine dehydrogenase (DPD).13 DPD is a key catabolic enzyme in the degradation of natural pyrimidines and pyrimidine analogues such as 5-fluorouracil (5-FU). It has been shown that the combination of BVDU (upon conversion to BVU by TPase) with 5-FU results in a marked potentiation of the toxicity and/or antitumour activity of 5-FU in a variety of cell cultures and animal models.1416 Indeed, co-administration of oral sorivudine (BVaraU) with 5-FU in cancer patients suffering from a VZV infection has led to the appearance of severe toxicity of 5-FU, resulting in a number of deaths.1618 Presumably, degradation of sorivudine by intestinal prokaryotic TPase(s) had released BVU from sorivudine,17 which then blocked the catabolic inactivation of 5-FU by DPD resulting in unacceptably high plasma levels of 5-FU.18 Further clinical trials with sorivudine were suspended. Given the potential above-mentioned complications of co-administration of BVU (as BVDU or BVaraU) or BVU analogues and 5-FU to patients, it is of clinical importance to reveal whether the free base of the BCNAs also inhibits DPD. Indeed, in marked contrast to BVU, which inhibited human liver DPD activity at an IC50 of c. 10 µM,10 several free BCNA bases were found to be completely ineffective in inhibiting the DPD enzyme at 100250 µM. In addition, when 5-FU was administered to mice in combination with free BCNA bases or with BCNA Cf 1368, no effect of BCNAs on 5-FU plasma levels was observed under the experimental conditions at which BVDU markedly raised and prolonged the plasma levels of 5-FU.10 Thus, it could be concluded that the BCNAs may be expected not to affect 5-FU plasma levels in patients treated with 5-FU for cancer that would concomitantly be treated with the BCNAs for a concurrent VZV infection.
Pharmacokinetics of BCNAs in vivo
It is important that antiviral drugs have favourable pharmacokinetics and a good oral bioavailability. The BCNAs with optimal antiviral activity have a pronounced lipophilicity with optimal calculated octanol:water log P values ranking between 2.5 and 3.5. The high log P values correlate with a low water solubility (<1 mg/L) of the BCNAs. This property presents a challenge for an appropriate oral formulation. We made a reasonable solution of 2 mg/mL Cf 1368 in 11% dimethyl sulphoxide, 22% cremophore and 67% phosphate-buffered saline and administered the drug (in 200 µL) to adult NMRI mice by oral gavage. Drug plasma levels were determined at a variety of time points after drug administration and were compared with the plasma levels of drug given by intravenous route. Preliminary data revealed a very high oral bioavailability (>50%) of the BCNA Cf 1368 in mice (R. Sienaert, L. Naesens and J. Balzarini, unpublished results), which is a strong beneficial property from a clinical viewpoint.
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Conclusions |
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Footnotes |
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References |
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