The broad-spectrum anti-DNA virus agent cidofovir inhibits lung metastasis of virus-independent, FGF2-driven tumors.

The FDA-approved anti-DNA virus agent cidofovir (CDV) is being evaluated in phase II/III clinical trials for the treatment of human papillomavirus (HPV)-associated tumors. However, previous observations had shown that CDV also inhibits the growth of vascular tumors induced by fibroblast growth factor-2 (FGF2)-transformed FGF2-T-MAE cells. Here, we demonstrate that CDV inhibits metastasis induced by FGF2-driven, virus-independent tumor cells. Pre-treatment of luciferase-expressing FGF2-T-MAE cells with CDV reduced single cell survival and anchorage-independent growth in vitro and lung metastasis formation upon intravenous inoculation into SCID mice. This occurred in the absence of any effect on homing of FGF2-T-MAE cells to the lungs and on the growth of subconfluent cell cultures or subcutaneous tumors in mice. Accordingly, CDV protected against lung metastasis when given systemically after tumor cell injection. Lung metastases in CDV-treated mice showed reduced Ki67 expression and increased nuclear accumulation of p53, indicating that CDV inhibits metastasis by affecting single cell survival properties. The anti-metastatic potential of CDV was confirmed on B16-F10 melanoma cells, both in zebrafish embryos and mice. These findings suggest that CDV may have therapeutic potential as an anti-metastatic agent and warrants further study to select those tumor types that are most likely to benefit from CDV therapy.

The thymidine phosphorylase inhibitor 5'-O-tritylinosine (KIN59) is an antiangiogenic multitarget fibroblast growth factor-2 antagonist.

5'-O-Tritylinosine (KIN59) is an allosteric inhibitor of the angiogenic enzyme thymidine phosphorylase. Previous observations showed the capacity of KIN59 to abrogate thymidine phosphorylase-induced as well as developmental angiogenesis in the chicken chorioallantoic membrane (CAM) assay. Here, we show that KIN59 also inhibits the angiogenic response triggered by fibroblast growth factor-2 (FGF2) but not by VEGF in the CAM assay. Immunohistochemical and reverse transcriptase PCR analyses revealed that the expression of laminin, the major proteoglycan of the basement membrane of blood vessels, is downregulated by KIN59 administration in control as well as in thymidine phosphorylase- or FGF2-treated CAMs, but not in CAMs treated with VEGF. Also, KIN59 abrogated FGF2-induced endothelial cell proliferation, FGF receptor activation, and Akt signaling in vitro with no effect on VEGF-stimulated biologic responses. Accordingly, KIN59 inhibited the binding of FGF2 to FGF receptor-1 (FGFR1), thus preventing the formation of productive heparan sulphate proteoglycan/FGF2/FGFR1 ternary complexes, without affecting heparin interaction. In keeping with these observations, systemic administration of KIN59 inhibited the growth and neovascularization of subcutaneous tumors induced by FGF2-transformed endothelial cells injected in immunodeficient nude mice. Taken together, the data indicate that the thymidine phosphorylase inhibitor KIN59 is endowed with a significant FGF2 antagonist activity, thus representing a promising lead compound for the design of multitargeted antiangiogenic cancer drugs.

Preclinical development of bicyclic nucleoside analogues as potent and selective inhibitors of varicella zoster virus.

OBJECTIVES: To progress the anti-varicella-zoster-virus (VZV) aryl bicyclic nucleoside analogues (BCNAs) to the point of Phase 1 clinical trial for herpes zoster. METHODS: A new chromatography-free synthetic access to the lead anti-VZV aryl BCNAs is reported. The anti-VZV activity of lead Cf1743 was evaluated in monolayer cell cultures and organotypic epithelial raft cultures of primary human keratinocytes. Oral dosing in rodents and preliminary pharmacokinetics assessment was made, followed by an exploration of alternative formulations and the preparation of pro-drugs. We also studied uptake into cells of both parent drug and pro-drug using fluorescent microscopy and biological assays. RESULTS: Cf1743 proved to be significantly more potent than all reference anti-VZV compounds as measured either by inhibition of infectious virus particles and/or by viral DNA load. However, the very low water solubility of this compound gave poor oral bioavailability (approximately 14%). A Captisol admixture and the 5'-monophosphate pro-drug of Cf1743 greatly boosted water solubility but did not significantly improve oral bioavailability. The most promising pro-drug to emerge was the HCl salt of the 5'-valyl ester, designated as FV-100. Its uptake into cells studied using fluorescent microscopy and biological assays indicated that the compound is taken up by the cells after a short period of incubation and limited exposure to drug in vivo may have beneficial effects. CONCLUSIONS: On the basis of its favourable antiviral and pharmacokinetic properties, FV-100 is now being pursued as the clinical BCNA candidate for the treatment of VZV shingles.

Susceptibilities of several clinical varicella-zoster virus (VZV) isolates and drug-resistant VZV strains to bicyclic furano pyrimidine nucleosides.

Varicella-zoster virus (VZV) is responsible for primary infections as well as reactivations after latency in the dorsal root ganglia. The treatment of such infections is mandatory for immunocompromised patients and highly recommended for elderly patients with herpes zoster infections (also called zona or shingles). The treatment of choice is presently based on four molecules, acyclovir (ACV), valaciclovir, famciclovir, and (in Europe) brivudine (BVDU). We present here our data on the antiviral activity of a new class of potent and selective anti-VZV compounds, bicylic pyrimidine nucleoside analogues (BCNAs), against a broad variety of clinical isolates and different drug-resistant virus strains. The results show that the BCNAs are far more potent inhibitors than ACV and BVDU against clinical VZV isolates as well as the VZV reference strains Oka and YS. The BCNAs were not active against ACV- and BVDU-resistant VZV strains bearing mutations in the viral thymidine kinase gene but kept their inhibitory potential against virus strains with mutations in the VZV DNA polymerase gene. Mutant virus strains selected in the presence of the BCNAs were solely cross-resistant to drugs, such as ACV and BVDU, that depend for their antiviral action on metabolic activation by the viral thymidine kinase.

Specific recognition of the bicyclic pyrimidine nucleoside analogs, a new class of highly potent and selective inhibitors of varicella-zoster virus (VZV), by the VZV-encoded thymidine kinase.

Recently, an entirely new class of bicyclic nucleoside analogs (BCNAs) was found to display exquisite potency and selectivity as inhibitors of varicella-zoster virus (VZV) replication in cell culture. A striking difference in their ability to convert the BCNAs to their phosphorylated derivatives was observed between the VZV-encoded thymidine kinase (TK) and the very closely related herpes simplex virus type 1 (HSV-1) TK. Whereas VZV TK efficiently phosphorylated the BCNAs, HSV-1 TK was unable to do so. In addition, the thymidylate (dTMP) kinase activity of VZV TK further converted BCNA-5'-MP to BCNA-5'-DP. The BCNAs (or their phosphorylated derivatives) were not a substrate for cytosolic TK, mitochondrial TK, or cytosolic dTMP kinase. Human erythrocyte nucleoside diphosphate (NDP) kinase was unable to phosphorylate the BCNA 5'-diphosphates to BCNA 5'-triphosphates. Under the same experimental conditions, the anti-herpetic (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) derivative was efficiently converted to BVDU-MP and BVDU-DP by both VZV TK and HSV-1 TK and further, into BVDU-TP, by NDP kinase. Our observations may account for the unprecedented specificity of BCNAs as anti-VZV agents.

Lack of susceptibility of bicyclic nucleoside analogs, highly potent inhibitors of varicella-zoster virus, to the catabolic action of thymidine phosphorylase and dihydropyrimidine dehydrogenase.

The susceptibility of the bicyclic nucleoside analogs (BCNAs), highly potent and selective inhibitors of varicella-zoster virus (VZV), to the enzymes involved in nucleoside/nucleobase catabolism has been investigated in comparison with the established anti-VZV agent (E)-5-(2-bromovinyl)-2'-deoxyuridine [BVDU; brivudine (Zostex)]. Whereas human and bacterial thymidine phosphorylases (TPases) efficiently converted BVDU to its antivirally inactive free base (E)-5-(2-bromovinyl)uracil (BVU), BCNAs showed no evidence of conversion to the free base in the presence of these enzymes. The lack of substrate affinity of TPase for the BCNAs could be rationalized by computer-assisted molecular modeling of the BCNAs in the TPase active site. Moreover, in contrast with BVU, which is a potent and selective inhibitor of dihydropyrimidine dehydrogenase (DPD) (50% inhibitory concentration; 10 microM in the presence of a 25 microM concentration of the natural substrate thymine), the free base (Cf 1381; 6-octyl-2,3-dihydrofuro[2,3-d]pyrimidin-2-one) of BCNA (Cf 1368; 3-(2'-deoxy-beta-D-ribofuranosyl)-6-octyl-2,3-dihydrofuro[2,3-d]pyrimidin-2-one) and the free base Cf 2200 [6-(4-n-pentylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one] of BCNA (Cf 1743; 3-(2'-deoxy-beta-D-ribofuranosyl)-6-(4-n-pentylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one) did not inhibit the DPD-catalyzed catabolic reaction of pyrimidine bases (i.e., thymine) and pyrimidine base analogs [i.e., 5-fluorouracil (FU)] at a concentration of 250 microM. Consequently, whereas BVU caused a dramatic rise of FU levels in FU-treated mice, the BCNAs did not affect FU levels in such mice. From our data it is evident that BCNAs represent highly stable anti-VZV compounds that are not susceptible to breakdown by nucleoside/nucleobase catabolic enzymes and are not expected to interfere with cellular catabolic processes such as those involved in FU catabolism.