Kjell Stenberg

Inhibition of Influenza Virus Ribonucleic Acid Polymerase by Ribavirin Triphosphate

Ribavirin 5′-triphosphate (RTP), derived from the broad-spectrum antiviral compound ribavirin (Virazole), can selectively inhibit influenza virus ribonucleic acid polymerase in a cell-free assay. Ribavirin and its 5′-monophosphate have no effect on the polymerase. The inhibition is competitive with respect to adenosine 5′-triphosphate and guanosine 5′-triphosphate. RTP also inhibits ApG- and GpC-stimulated influenza virus ribonucleic acid polymerase. Since ribavirin is phosphorylated in the cell, the inhibition of influenza multiplication in the cell may also be caused by RTP.

Mode of action, toxicity, pharmacokinetics, and efficacy of some new antiherpesvirus guanosine analogs related to buciclovir.

9-[4-Hydroxy-3-(hydroxymethyl)butyl]guanine (3HM-HBG), (RS)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine ([+/-]2HM-HBG), and cis-9-(4-hydroxy-2-butenyl)guanine (2EN-HBG), new acyclic guanosine analogs structurally related to buciclovir (BCV [(R)-9-(3,4-dihydroxybutyl)guanine]), were evaluated in parallel with buciclovir as anti-herpes simplex virus (HSV) agents. In cell cultures, replication of different strains of HSV type 1 (HSV-1) and HSV-2 was inhibited at nontoxic drug concentrations. The concentrations giving 50% inhibition of plaque formation were, however, dependent on virus strain and cell type. In most cell types, the order of activity against HSV-1 strains was 3HM-HBG greater than (+/-)2HM-HBG greater than BCV greater than 2EN-HBG, whereas the drugs showed an approximately equivalent activity against HSV-2 strains in different cells. The cytotoxic effects of the drugs were also cell type dependent, the order of activity being BCV greater than 3HM-HBG = (+/-)2HM-HBG greater than 2EN-HBG. At growth-inhibitory concentrations, the guanosine analogs BCV, 3HM-HBG, and (+/-)2HM-HBG showed clastogenic effects in human lymphocytes, mainly because of the induction of chromatid breaks. When evaluated for their anti-HSV effects in systemic HSV-1 infections in mice, the order of activity was BCV = 3HM-HBG greater than (+/-)2HM-HBG greater than 2EN-HBG, and in mice infected systemically with HSV-2, only BCV and 3HM-HBG showed efficacy. The differences between efficacy in vitro and in vivo could be explained in part by differences in kinetics of the drugs in mouse plasma, as the more efficacious drugs, BCV and 3HM-HBG, showed lower clearances and longer half-lives than the less efficacious ones, (+/-)2HM-HBG and 2EN-HBG. When used topically against a cutaneous HSV-1 infection in guinea pigs, 3HM-HBG showed an effect equivalent to that of BCV, whereas (+/-)2HM-HBG and 2EN-HBG were inactive. Mechanistically, the guanosine analogs were characterized by a high affinity for the viral thymidine kinase and a low affinity fo a cellular thymidine kinase and by their inhibition of viral DNA synthesis in infected cells.

Critical determinants of antiherpes efficacy of buciclovir and related acyclic guanosine analogs

Buciclovir is an example of an antiherpes, acyclic guanosine analog activated by the viral thymidine kinase and inhibiting viral DNA synthesis in infected cells. An investigation of closely related buciclovir-analogs with similar antiherpes activities in cell cultures and similar, or identical, modes of action but with disparate effects in vivo, revealed the following critical determinants of antiherpes efficacy. (1) The accumulation of guanosine analog-triphosphates in infected cells, which is cell-type-specific and analog-dependent. (2) The potencies of the triphosphates as inhibitors of the viral DNA polymerase. (3) The plasma kinetics of the analogs, which are widely different despite the similar structures. (4) The penetration into nervous tissue relative to penetration into non-nervous tissues, of importance in connection with the neurotropic behavior of the virus. (5) The concentration of the antagonist thymidine in certain tissues. (6) The difference in pathogenesis between primary infections and recurrent infections, exemplified in the different efficacies of topically applied drugs in cutaneous and genital HSV-2 infections in guinea pigs.

Reversible effects on cellular metabolism and proliferation by trisodium phosphonoformate.

The antiviral compound trisodium phosphonoformate (PFA), which inhibits herpesvirus multiplication by 50% at a concentration of 10 μM, did not show any effects on macromolecular synthesis and cell proliferation in HeLa and human lung cells at this concentration. At the high concentration of 2 mM, PFA reduced DNA synthesis to 50% after 1 h of treatment, whereas no effects could be seen on RNA and protein synthesis. Treatment for 24 h with 1 mM PFA inhibited both DNA synthesis and cell proliferation to 50%. The inhibition of DNA synthesis and cell proliferation at 10 mM PFA was rapidly reversed by removing the drug from the cells.

Reversible Inhibition of Cellular Metabolism by Ribavirin

The broad spectrum antiviral drug ribavirin (Virazole, 1-β-d-ribofuranosyl-1,2,4-triazole-3-carboxamide) inhibits cellular macromolecular synthesis as well as cell division in eucaryotic cells. The concentration and time dependence have been studied. One-hour treatment with 25 μM ribavirin or 18 h with 2 μM inhibited the deoxyribonucleic acid synthesis to 50%. Higher concentrations of ribavirin were required to obtain a similar inhibition of ribonucleic acid and protein synthesis. This effect on cell metabolism and cell division can be reversed by removing the drug from the cells.

Concentration-dependent effects of foscarnet on the cell cycle.

The mechanism of toxicity of foscarnet was studied by monitoring its effects on the cell cycle of exponentially growing, semisynchronous human embryo cells in culture. The effects of foscarnet on the cell cycle were dependent on the concentration of drug used. At 1 mM, cell division was reduced by 50%, whereas the cell flow was mainly reduced in the G2 phase of the cell cycle, leading to an increase in the proportion of G2+M cells. The minor reduction of thymidine incorporation in S phase cells provided additional evidence that 1 mM foscarnet did not specifically inhibit DNA synthesis. Cell division was greatly reduced at 2.5 mM foscarnet, and the G2 phase was markedly affected, whereas S cell flow was less reduced. S cell flow was 10% per h and thymidine incorporation was 25% that of control cells, while a block in the G2+M phase was evident. On the other hand, at a concentration of 5 mM foscarnet, the cell flow was greatly reduced in the G1 and S phases, with less reduction of G2 cell flow and cells accumulated in the S phase. The effects of foscarnet on the cell cycle were more pronounced with increasing times up to 72 h, which could not be explained by the slow penetration of foscarnet which required only 4 to 8 h to achieve constant levels. At 2.5 and 5 mM foscarnet, there was the additional effect of the cell membranes becoming more leaky as a result of foscarnet toxicity which might contribute to the toxic effects of the drug at high concentrations. When foscarnet was removed from the medium, the effects on the cell cycle were rapidly reversed, in the time needed for foscarnet to diffuse out from the cells, which indicates the reversible nature of the toxic effects of foscarnet.

Incorporation into nucleic acids of the antiherpes guanosine analog buciclovir, and effects on DNA and protein synthesis.

Using cells expressing herpes simplex virus (HSV) thymidine kinase, we investigated the metabolism of the acyclic antiherpes guanosine analog buciclovir, in relation to the effects of the drug on viral DNA and protein synthesis. In these cells the predominant metabolite of buciclovir was its triphosphate, as in the HSV-1 infected Vero cells investigated in parallel. Further metabolism of buciclovir led to incorporation into RNA and DNA. Buciclovir inhibited DNA synthesis, not RNA synthesis, and prevented an increase in the size of newly synthesized DNA. To study the relative effects of BCV on cellular and viral DNA synthesis, human TK-cells transformed to a TK+ phenotype with HSV-2 DNA, were infected with HSV-1. In these HSV-1 infected cells buciclovir-triphosphate caused a preferential inhibition of viral DNA synthesis. Despite incorporation of buciclovir into RNA, and the presence of buciclovir-triphosphate from the time of infection onwards, no effect was observed on the synthesis of the beta proteins ICP-6 and ICP-8. Presumably as a consequence of inhibition of viral DNA synthesis, the synthesis of a beta gamma protein (gD) and a gamma protein (gC) were inhibited, and synthesis of the beta proteins (ICP-6 and ICP-8) was not shut-off. Glycosylation of gC that was still synthesized, was not inhibited. Thus, the biological effects of buciclovir can be explained by its inhibition of DNA synthesis.

Effects of foscarnet on the cell kinetics of Madin-Darby canine kidney cells

The antiherpes compound, foscarnet (trisodium phosphonoformate), showed concentration-dependent effects on the cell kinetics of Madin-Darby canine kidney cells. At 1 mM, only minor effects could be seen on cell proliferation and cell cycle distribution, as measured by flow cytometry DNA analysis. Treatment with 5 mM foscarnet resulted in an accumulation of cells in the S-phase although no complete cell cycle block was evident. At 10 mM foscarnet, cells accumulated earlier in the S phase, probably at the G1/S border. However, at both 5 and 10 mM foscarnet the block was not established until after 15 h incubation. Upon removing 10 mM foscarnet after 24 h incubation, G1 cells rapidly entered the S phase, whereas the progression through S and G2 + M was delayed considerably. The DNA synthesizing S phase seems, therefore, to be the main cell cycle phase affected by foscarnet.

Quantitative analysis of antiviral drug toxicity in proliferating cells.

The toxicity of most antiviral compounds was dependent on the type of cell used to assay toxicity. Ranking of compounds according to toxicity was, however, very similar (p < 0.01) in the three different cell types used in this study. The difference in toxicity observed for 9-β-D-arabinofuranosyladenine between Flow 5000 cells and CCRF-SB cells could not be accounted for by differences in the intracellular concentrations. On the other hand, the different toxicities observed for ribavirin and 2′-deoxy-5-triuorothymidine between Flow 5000 cells and CCRF-SB cells may be caused by the culture conditions (as shown for one cell type, HeLa S3, grown either as monolayer or in suspension) rather than by cell-specific differences. The growth-inhibitory effect of most antiviral compounds increased with treatment time, indicating an additive nature of toxicity. The ability of cells to recover from toxic treatment with drugs varied greatly from compound to compound (from undetectable regrowth to 140% growth compared to control cells). Coaddition of natural nucleosides could, at best, only partly protect cells from the toxic influences of antiviral nucleoside analogs. As a result of comparing antiviral effects and toxicity in vitro, the unselective compounds may be eliminated from further development at the screening level.

Toxic effects of some xanthine derivatives with special emphasis on adverse effects on rat testes.

Testicular toxicity and effects on thymus and body weights of 4 xanthine derivatives (D4026: 1,8-dimethyl-3-phenylxanthine, D4152: 8-methyl-3-phenylxanthine, D4160: 1,8-dimethyl-3-(2-methylbutyl)-xanthine, D4173: 8-methyl-3-(2-methylbutyl)-xanthine) were studied in Sprague-Dawley rats and cellular toxicity in human embryonal cells. The effect on toxicity by variation of substituent at positions 1 and 3 was tested. The compounds were administered orally to the rats once a day for 1 month. Mortalities were noted only with D4160. Dose related decreases in body weight gain were found for all substances, but only marginally with D4152. A significant decrease in thymus weight relative to control was observed with all substances, D4152 being the least potent. No effects on testes weights were found with any treatment but histological examination disclosed degeneration of germ producing epithelium of all rats given 100 mumol/kg of D4026 but not at 25 mumol/kg. One rat out of 5 showed testicular damage at 400 mumol/kg of D4173 or D4152. Plasma analysis for unchanged compounds showed significantly higher plasma concentrations at the high dose compared with the low dose with the exception for D4152 showing unexpectedly low levels. In the cellular toxicity test, D4160 was the most potent while D4152 was the least potent. D4026 had a steeper dose-response curve than the others but was less potent than D4160. The 1-methylated xanthine derivatives seemed to be more toxic than the two in position 1 unsubstituted analogues. Mechanisms for testicular toxicity of xanthine derivatives in the rat and clinical relevance of animal data are discussed.