Dynamics of the antiviral activity of N-methanocarbathymidine against herpes simplex virus type 1 in cell culture.

N-Methanocarbathymidine [(N)-MCT], a thymidine analogue, exhibits potent activity in cell culture against herpes simplex virus1 (HSV-1). (N)-MCT showed higher antiviral activity than ganciclovir (GCV). Continuous treatment of Vero cells with (N)-MCT immediately or 10 h post-infection (p.i.) fully prevented the development of viral infection. However, when infected cells were treated with (N)-MCT at 12 h p.i., there was only a partial inhibition (ca. 50%). Additionally, continuous treatment of infected cells with (N)-MCT for about 48 h was sufficient to achieve full prevention of viral infection without further treatment. These findings suggest the complete loss of herpes simplex thymidine kinase (HSV-tk) activity occurs after 48 h of treatment with (N)-MCT. This study helps to understand the mechanism and dynamics of antiHSV activity of (N)-MCT, which is necessary for its future development as an antiviral drug.

Antitumor activity and metabolic activation of N-methanocarbathymidine, a novel thymidine analogue with a pseudosugar rigidly fixed in the northern conformation, in murine colon cancer cells expressing herpes simplex thymidine kinase.

N-Methanocarbathymidine [(N)-MCT], a thymidine analogue incorporating a pseudosugar with a fixed Northern conformation, exhibits antiherpetic activity against both herpes simplex virus (HSV) HSV-1 and HSV-2, with a potency greater than that of the reference standard, ganciclovir (GCV). In the present study, we have assessed the cytotoxic activity in vitro of (N)-MCT in wild-type murine colon cancer cells (MC38) and in cells expressing the herpes simplex thymidine kinase gene (MC38/HSV-tk), and the antitumor activity of (N)-MCT in vivo against HSV-tk transduced and nontransduced MC38 murine tumors. In vitro, when assessed over a 48-h period, the growth-inhibitory activity (IC50) of (N)-MCT toward MC38/HSV-tk cells was 2.9 microM. In parallel studies, the cytostatic activity of the reference compound GCV in these tumor lines was 3.0 microM. In studies in vivo, both (N)-MCT and GCV (100 mg/kg) given twice daily for 7 days completely inhibited the growth of HSV-tk-transduced MC38 tumors while exhibiting no effect on nontransduced MC38 tumors in mice. In nontransduced cells both in vitro and in vivo, only low levels of (N)-MCT and its monophosphate could be detected after administration of the parent drug, whereas in HSV-tk-transduced cells (N)-MCT was phosphorylated to its respective mono-, di-, and triphosphates. Furthermore, data showed that (N)-MCT incorporated in high levels into cellular DNA whereas trace levels were measured into RNA. These observations indicate that (N)-MCT may be a useful candidate prodrug for HSV-tk suicide gene therapy of cancer.

Pharmacokinetics and organ distribution of N-methanocarbathymidine, a novel thymidine analog, in mice bearing tumors transduced with the herpes simplex thymidine kinase gene.

PURPOSE: The conformationally rigid nucleoside, N-methanocarbathymidine [(N)-MCT] exerts a potent antiproliferative effect both in vitro and in vivo against murine colon cancer cells (MC38) expressing the herpes simplex virus thymidine kinase gene (MC38/HSV-tk). Metabolic studies have revealed that high levels of (N)-MCT triphosphate accumulate in transduced cells and are incorporated into DNA, resulting in cell death. The objective of the present study was to assess the pharmacokinetic profile of (N)-MCT in C57BL/6 mice bearing nontransduced MC38 and MC38/HSV-tk tumors. METHODS: Male black C57BL/6 mice bearing subcutaneous tumors derived from wildtype and HSV-tk-transduced MC38 murine colon cancer cells in the left and right flank, respectively, were treated i.p. with radiolabeled (N)-MCT (100 mg/kg). Mice were killed at each of the predetermined times after drug administration. Blood, urine, tumors and various organs and tissues were obtained for measurement of drug levels. RESULTS: Plasma and tissue concentrations of (N)-MCT peaked at 0.25-0.5 h. The major pharmacokinetic parameters calculated for (N)-MCT in plasma were: T(1/2)beta 4.7 h, AUC 147 micro g.h/ml, CL 0.69 l/kg per h. The penetration of (N)-MCT into brain and testes was slow. Between 4 and 24 h after drug administration, the levels of (N)-MCT measured in HSV-tk-expressing tumors were significantly higher than in wildtype tumors. HPLC analysis of methanolic extracts of plasma and urine obtained at various times after drug administration revealed no (N)-MCT metabolites in the plasma, and the compound was secreted unchanged in the urine. CONCLUSIONS: After i.p. injection into mice, (N)-MCT was rapidly absorbed and distributed in all organs examined. No drug metabolites were detectable in plasma and the compound was secreted unchanged in urine. These results are essential for the future development and in postulating the most efficient use of (N)-MCT in the HSV-tk enzyme prodrug system for gene therapy approaches for the treatment of cancer.

Is the anomeric effect an important factor in the rate of adenosine deaminase catalyzed hydrolysis of purine nucleosides? A direct comparison of nucleoside analogues constructed on ribose and carbocyclic templates with equivalent heterocyclic bases selected to promote hydration.

The aglycone of (North)-methanocarbadeoxyadenosine [(N)-MCdA, (5)], a relatively weak substrate for adenosine deaminase (ADA)-relative rate of deamination ca. 100 times lower than adenosine-was modified with substitutions at positions 6 (6-fluoro, compound 6) and 8 (8-aza, compound 7) with the intent to improve the level of hydration and hence hydrolysis by ADA. In these substrates the fused cyclopropane moiety constrains the cyclopentane ring to mimic the conformation of a furanose sugar in the North hemisphere of the pseudorotational cycle, which matches the conformation of the ribose ring of adenosine in complex with ADA. The order of susceptibility to ADA hydrolysis was adenosine>>(N)-MCdA (5) approximately equal to(N)-6F-MCdP (6)>(N)-8-aza-MCdA (7). Despite the known fact that 8-azaadenosine is hydrolyzed twice as fast as adenosine, the corresponding carbocyclic analogue 7 was hydrolyzed at approximately half the rate of the parent 5. These results argue in favor of the critical role of the O(4') oxygen atom and its associated anomeric effect in assisting hydrolysis by ADA.

Metabolic pathways of N-methanocarbathymidine, a novel antiviral agent, in native and herpes simplex virus type 1 infected Vero cells.

N-methanocarbathymidine ((N)-MCT), a thymidine analog incorporating a pseudosugar with a fixed Northern conformation, exhibits potent antiherpetic activity against herpes simplex virus types 1 (HSV-1) and 2 (HSV-2). This study contrasts the metabolic pathway of (N)-MCT and the well-known antiherpetic agent ganciclovir (GCV) in HSV-1-infected and uninfected Vero cells. Treatment of HSV-1 infected Vero cells immediately after viral infection with (N)-MCT profoundly inhibited the development of HSV-1 infection. Using standard plaque reduction assay to measure viral infection, (N)-MCT showed a potency greater than that of ganciclovir (GCV), the IC50s were 0.02 and 0.25 microM for (N)-MCT and GCV, respectively. (N)-MCT showed no cytotoxic effect on uninfected Vero cells (CC50>100 microM). Dose and time dependence studies showed high levels of (N)-MCT-triphosphate ((N)-MCT-TP), and GCV-triphosphate (GCV-TP) in HSV-1-infected cells incubated with (N)-MCT or GCV, respectively. In contrast, uninfected cells incubated with (N)-MCT showed elevated levels of (N)-MCT-monophosphate only, while low levels of mono, di- and triphosphates of GCV were found following incubation with GCV. Although the accumulation rate of (N)-MCT and GCV phosphates in HSV-1-infected cells were similar, the decay rate of (N)-MCT-TP was slower than that of GCV-TP. These results suggest that: (1) the antiviral activity of (N)-MCT against herpes viruses is mediated through its triphosphate metabolite; (2) in contrast to GCV, the diphosphorylation of (N)-MCT in HSV-1- infected cells is the rate limiting step; (3) (N)-MCT-TP accumulates rapidly and has a long half-life in HSV-1-infected cells; and (4) HSV-tk catalyzed the mono, and diphosphorylation of (N)-MCT while monophosphorylating GCV only. These results provide a biochemical rational for the highly selective and effective inhibition of HSV-1 by (N)-MCT.