Ranking of prophylactic efficacy of poly(ICLC) against Rift Valley fever virus infection in mice by incremental relative risk of death.

The prophylactic efficacy of poly(ICLC) (stabilized, synthetic, double-stranded polyriboinosinic-polyribocytidylic acid) against Rift Valley fever virus infection in Swiss-Webster mice was dependent on the treatment schedule. The treatment schedule was optimized by ranking the results of various treatments by the Cox proportional-hazard model based on the incremental relative risk of death. With this ranking procedure, the schedule of choice was three doses of 20 micrograms each given 5 days apart. This regimen yielded a 90% survival rate. Additional parameters were determined, including the timing of the first and second drug dose, the temporal relationship of these treatments to the day of challenge, and the minimal effective dose (1 microgram per mouse).

Enhanced therapeutic efficacy of poly(ICLC) and ribavirin combinations against Rift Valley fever virus infection in mice.

The therapeutic efficacy of polyriboinosinic-polyribocytidylic acid stabilized with poly-L-lysine and carboxymethyl cellulose [poly(ICLC)] given alone or in combination with ribavirin was evaluated in Swiss Webster mice infected with Rift Valley fever virus. Four or more 20-micrograms doses of poly(ICLC) given at various intervals beginning 24 h after infection protected all mice against death. On the other hand, a treatment regimen consisting of only three doses of poly(ICLC) given 24 h postinfection resulted in a 50% survival rate. When initiated 48 h postinfection, an extended treatment regimen with the same dose was required to yield 40% survivors. Lower doses (5 micrograms) of poly(ICLC) per mouse were only marginally effective even when six injections were given between days 1 and 9 postinfection. The combined administration of ribavirin and poly(ICLC) initiated as late as 48 h postinfection was effective even when treatment consisted of doses that were ineffective when either drug was used alone.

Tolerance and antiviral effect of ribavirin in patients with Argentine hemorrhagic fever.

Tolerance and antiviral effect of ribavirin was studied in 6 patients with Argentine hemorrhagic fever (AHF) of more than 8 days of evolution. Administration of ribavirin resulted in a neutralization of viremia and a drop of endogenous interferon titers. The average time of death was delayed. A reversible anemia was the only adverse effect observed. From these results, we conclude that ribavirin has an antiviral effect in advanced cases of AHF, and that anemia, the only secondary reaction observed, can be easily managed. The possible beneficial effect of ribavirin during the initial days of AHF is discussed.

Effect of ribavirin and tributylribavirin on argentine hemorrhagic fever (Junin virus) in guinea pigs.

Subcutaneous injections of ribavirin into guinea pigs infected intraperitoneally or intracerebrally with Junin virus significantly increased the mean time to death but did not enhance survival of the animals. We found similar results with tributylribavirin. Virus replication was delayed, but not prevented, in ribavirin-treated infected guinea pigs. The animals usually died with high virus titers in their brains and frequently were paralyzed.

Synthesis and biological activity of 5-thiobredinin and certain related 5-substituted imidazole-4-carboxamide ribonucleosides.

A number of 5-substituted imidazole-4-carboxamide ribonucleosides were prepared and tested for their biological activity. Treatment of 5-chloro-1-beta-D-ribofuranosylimidazole-4-carboxamide (2) with methanethiol provided 5-(methylthio)-1-beta-D-ribofuranosylimidazole-4-carboxamide (3a). Similar treatment of 2 with ethanethiol or benzenemethanethiol gave the corresponding 5-ethylthio and 5-benzylthio derivatives 3b and 3c. Oxidation of 3a and 3b with m-chloroperoxybenzoic acid furnished the corresponding sulfonyl derivatives 4a and 4b. Reductive cleavage of 3c with sodium naphthalene or Na/NH3 gave 5-mercapto-1-beta-D-ribofuranosylimidazole-4-carboxamide (5-thiobredinin, 5). Direct treatment of 2 with sodium hydrosulfide provided an alternate route to 5, the structure of which was established by single-crystal X-ray analysis. 5-Thiobredinin has a zwitterionic structure similar to that of bredinin. Glycosylation of persilylated ethyl 5(4)-methylimidazole-4(5)-carboxylate (6) with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose in the presence of SnCl4 provided a quantitative yield of the corresponding tri-O-benzoyl nucleoside 7. Debenzoylation of 7 with MeOH/NH3 at ambient temperature gave ethyl 5-methyl-1-beta-D-ribofuranosylimidazole-4-carboxylate (8). Further ammonolysis of 8 or 7 at elevated temperature and pressure gave 5-methyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (9). All of these ribonucleosides were tested in Vero cell cultures and in mice against certain viruses. Compounds 3a and 3c exhibited significant activity against vaccinia virus in vitro, whereas 4a was effective against Rift Valley fever virus in mice. 5-Thiobredinin failed to exhibit appreciable antiviral or cytostatic activity (against L1210 and P388) in cell culture.

Enhanced efficacy of liposome-encapsulated ribavirin against Rift Valley fever virus infection in mice.

Administration of liposome-encapsulated ribavirin to mice led to ribavirin concentrations in the liver, the primary site of Rift Valley fever virus proliferation, that were fivefold greater than those attained with the same doses of free ribavirin. Liposomal ribavirin given at a dose of either 25 or 50 mg of drug per kg of body weight protected mice against a rapidly lethal high-titer challenge with Rift Valley fever virus, whereas similar doses of free drug or empty liposomes had no detectable benefit. Hence, tissue targeting of ribavirin with liposomes substantially increased the therapeutic index by increasing the efficacy of the treatment. By using liposomes as drug carriers, a nontoxic, low-dose regimen of ribavirin had a therapeutic effect that was comparable to that achieved with higher but potentially more toxic doses of free ribavirin.

Synthesis and antiviral/antitumor activities of certain 3-deazaguanine nucleosides and nucleotides.

A new procedure for the preparation of the antiviral and antitumor agent 3-deazaguanine (1) and its metabolite 3-deazaguanosine (2) has been developed by reacting methyl 5(4)-(cyanomethyl) imidazole-4(5)-carboxylate (4) and 5-(cyanomethyl)-1- (2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)imidazole-4-carboxylate (6), respectively, with hydrazine. The 3-deazaguanosine 3',5'-cyclic phosphate (13) was prepared from 5-(cyanomethyl)-1-beta-D-ribofuranosyl-imidazole-4-carboxamide 5'-phosphate. Glycosylation of the trimethylsilyl 4 with 1-O-methyl-2-deoxy-3,5-di-O-p-toluoyl-D-ribofuranose in the presence of trimethylsilyl trifluoromethanesulfonate gave the corresponding N-1 and N-3 glycosyl derivatives with alpha-configuration (18 and 20) as the major products, along with minor amounts of the beta-anomers (19 and 21). However, glycosylation of the sodium salt of 4 with 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-alpha-D-erythro-pentofurano se (17) gave exclusively the beta-anomers (19 and 21) in good yield. Base-catalyzed ring closure of these imidazole nucleosides gave 2'-deoxy-3-deazaguanosine (29), the alpha-anomer 28, and the corresponding N-3 positional isomers 27 and 26. The site of glycosylation and the anomeric configuration of these nucleosides have been assigned on the basis of 1' NMR and UV spectral characteristics and by single-crystal X-ray analysis for 27-29. In a preliminary screening, several of these compounds have demonstrated significant broad-spectrum antiviral activity against certain DNA and RNA viruses in vitro, as well as moderate activity against L1210 and P388 leukemia in cell culture.

Synergistic antiviral effects of ribavirin and the C-nucleoside analogs tiazofurin and selenazofurin against togaviruses, bunyaviruses, and arenaviruses.

Binary combinations of the N-nucleoside ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) and the C-nucleoside analog selenazofurin (2-beta-D-ribofuranosylselenazole-4-carboxamide) or tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) were tested in vitro for activity against Venezuelan equine encephalomyelitis, Japanese encephalitis, yellow fever, Rift Valley fever, Korean hemorrhagic fever, and Pichinde viruses. The 50% effective dose for each compound alone or in a series of combinations was determined with a plaque reduction assay. Combinations of ribavirin and selenazofurin were synergistic against Venezuelan equine encephalomyelitis, Japanese encephalitis, yellow fever, and Pichinde viruses, with fractional inhibitory concentrations of 0.1, 0.2, 0.4, 0.4, respectively, but showed additive effects against Korean hemorrhagic fever and Rift Valley fever viruses. Combinations of ribavirin and tiazofurin were synergistic against yellow fever and Japanese encephalitis (fractional inhibitory concentrations, 0.41 and 0.48, respectively) but showed additive effects against Korean hemorrhagic fever virus. Combinations of selenazofurin and tiazofurin had additive effects against Japanese encephalitis, yellow fever, and Korean hemorrhagic fever viruses. The effect of combinations on cell toxicity was additive, both in monolayers of nondividing cells incubated under agar for the same period as the plaque assay and for rapidly dividing cells given short-term exposure (4 h), followed by determination of the proportion of surviving cells with a colony forming assay.

In-vivo activity of antivirals against exotic RNA viral infections.

Infection of humans by viruses belonging to the families of Toga-, Bunya-, and Arenaviridae constitutes a major health problem worldwide and certain of the viruses have the potential to cause widespread epidemics. In the search for effective chemotherapy against these viruses several hundred nucleoside and nucleotide analogues have been screened for antiviral activity. Of the compounds tested, ribavirin, has been shown in laboratory animal models to have significant inhibitory effects against Rift Valley Fever virus, the bunyavirus Punta Toro, Hantaan virus and arenaviruses such as Pichinde, Junin, Machupo and Lassa Fever. Clinical studies with ribavirin in persons infected with Lassa Fever virus are in progress. Ribavirin or certain analogues have no detectable in vivo activity against arboviral encephalitic infections caused by Venezuelan or Western Equine Encephalitis or Semliki Forest Viruses. Other compounds including a series of triazolo derivatives, and the thiazole carboxamide nucleoside Tiazofurin appear to have in-vivo activity against certain of these exotic RNA viruses.

Effects of ribavirin on red blood cells.

Man and rhesus monkey may develop anemia during treatment with the broad-spectrum antiviral ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide). To assess whether the anemia is due to decreased production of erythrocytes, increased destruction, or a combination of both factors, the transport of ribavirin into erythrocytes and the drug's effect on the osmotic fragility, deformability, and half-life of erythrocytes were evaluated. The rate of uptake of ribavirin by erythrocytes was species and concentration dependent. Monkey cells accumulated the largest concentration of drug followed by human and rat cells. Monkey and human red cells, pretreated in vitro with ribavirin, retained 77 and 45% of the drug, respectively, when reincubated for 2 hr in drug-free medium. Rat red cells retained only 20% of their initial ribavirin content. Neither osmotic fragility nor deformability was altered by exposure of red cells to ribavirin in vitro. The half-life of 1,3-[3H]diisopropyl fluorophosphate (DFP)-labeled erythrocytes was measured in rhesus monkeys treated intramuscularly (im) for 10 days with either 15 or 60 mg/kg of ribavirin. A dose-related decrease in red cell survival was observed from Day 0 to 28. Thereafter, red cell half-lives were comparable to control values. These data indicate that ribavirin at a dose as low as 15 mg/kg decreases the half-life of red cells. This effect was reversible upon discontinuation of the drug. At 60 mg/kg, ribavirin also inhibited the release of red cells from the bone marrow. Termination of treatment was followed by release of red cells from the bone marrow as indicated by a drop in the specific activity of [3H]DFP-labeled red cells and marked reticulocytosis. No inhibition of red cell release from the bone marrow was seen in the low-dose group. These data suggest that ribavirin can decrease red cell survival as well as inhibit red cell release from the bone marrow. Both effects appear fully reversible when treatment is withdrawn.

Hematological and bone marrow effects of ribavirin in rhesus monkeys.

Ribavirin (Virazole, 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), a broad-spectrum antiviral compound, was evaluated for effects on blood and bone marrow of rhesus monkeys when administered by intramuscular injection for 10 days in doses of 30 or 100 mg/kg/day (four monkeys/group). Both groups developed a normochromic, normocytic anemia that was mild in the low-dose group and severe in the high-dose group. A dose-related erythroid hypoplasia occurred during the treatment period. Myeloid precursors were not affected. Differential counts of erythroid precursors showed a significant decrease in late erythroid forms while early erythroid forms were either unchanged or increased. Megakaryocyte numbers were increased in both groups. Qualitative changes in marrow cells included vacuolization of erythroid precursors and of occasional white cell precursors and megakaryocytes, and the appearance of bone marrow histiocytes containing red cells in various stages of disintegration. Thrombocytosis occurred in both treatment groups, with platelet counts returning to control values after drug withdrawal. Platelet function was not affected by treatment. No drug-related changes were seen during the treatment period for total and differential leukocyte counts, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration. Reticulocyte counts and mean corpuscular volume increased after treatment then returned to control values. Osmotic fragility of erythrocytes was not changed. These data show that in monkey, ribavirin causes a dose-related decrease in circulating red blood cell mass that is due in part to suppression of late erythroid precursors in bone marrow. These effects are reversible when treatment is discontinued and are not predictive of potentially serious or lasting untoward effects of ribavirin.

Morphological alterations in blood and bone marrow of ribavirin-treated monkeys.

The antiviral drug ribavirin (1, beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) produced significant hematologic effects when administered to rhesus monkeys by intramuscular injection over 10 days in doses of 30 or 100 mg/kg/day. The monkeys developed dose-related progressive anemia and thrombocytosis associated with marrow erythroid hypoplasia and megakaryocyte hyperplasia. In addition, bone marrow examination revealed phagocytosis of erythroid elements by histiocytes; vacuolization of erythroid precursors, and to a lesser extent precursors of other cell types; and occasional erythroid precursors with megaloblastoid appearance. The alterations were transient and disappeared on discontinuation of the drug.

Broad-spectrum antiviral activity of 2-beta-D-ribofuranosylselenazole-4-carboxamide, a new antiviral agent.

The relative in vitro antiviral activities of three related nucleoside carboxamides, ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide), and selenazole (2-beta-D-ribofuranosylselenazole-4-carboxamide), were studied against selected DNA and RNA viruses. Although the activity of selenazole against different viruses varied, it was significantly more potent than ribavirin and tiazofurin against all tested representatives of the families Paramyxoviridae (parainfluenza virus type 3, mumps virus, measles virus), Reoviridae (reovirus type 3), Poxviridae (vaccinia virus), Herpes-viridae (herpes simplex virus types 1 and 2), Togaviridae (Venezuelan equine encephalomyelitis virus, yellow fever virus, Japanese encephalitis virus), Bunyaviridae (Rift Valley fever virus, sandfly fever virus [strain Sicilian], Korean hemorrhagic fever virus), Arenaviridae (Pichinde virus), Picornaviridae (coxsackieviruses B1 and B4, echovirus type 6, encephalomyocarditis virus), Adenoviridae (adenovirus type 2), and Rhabdoviridae (vesicular stomatitis virus). The antiviral activity of selenazole was also cell line dependent, being greatest in HeLa, Vero-76, and Vero E6 cells. Selenazole was relatively nontoxic for Vero, Vero-76, Vero E6, and HeLa cells at concentrations of up to 1,000 micrograms/ml. The relative plating efficiency at that concentration was over 90%. The effects of selenazole on viral replication were greatest when this agent was present at the time of viral infection. The removal of selenazole from the medium of infected cells did not reverse the antiviral effect against vaccinia virus, but there was a gradual resumption of viral replication in cells infected with parainfluenza type 3 or herpes simplex virus type 1 (strain KOS). However, the antiviral activity of ribavirin against the same viruses was reversible when the drug was removed.

Intracellular fate of phase I Coxiella burnetii in guniea pig peritoneal macrophages.

Cultivated guinea pig peritoneal macrophages were infected with radio-labeled phase I Coxiella burnetii in order to assess the intracellular distribution of ingested rickettsiae. Localization of organisms was determined by fractionation of macrophage homogenates by equilibrium density centrifugation on sucrose gradients. Macrophages isolated from either nonimmune or immune guinea pigs and infected with C burnetii opsonized with immune serum yielded equilibrium density distribution for rickettsiae similar to lysosomal enzymes, suggesting sequestration within macrophage lysosomes. To confirm these observations nonimmune or immune guinea pigs were injected with Triton WR-1339 prior to macrophage harvest to decrease the density of macrophage lysosomes. Triton-laden macrophages infected with opsonized rickettsiae resulted in equilibrium density distribution for lysosomal enzymes and organisms in less dense regions of the gradient. In contrast, when either nonimmune or immune macrophages were infected in the presence of normal guinea pig serum, the distribution of labeled rickettsiae in the gradient did not correspond with lysosomes. We conclude that in the absence of immune serum, ingested C burnetii are not sequestered within macrophage lysosomes. Phagolysomal fusion and subsequent degradation of rickettsiae within the lysosomes of the macrophages appear to occur only when C burnetii are opsonized with immune serum.

Antiviral efficacy of pyrazofurin against selected RNA viruses.

The antibiotic pyrazofurin, 3-(beta-D-ribofuranosyl)-4-hydroxypyrazole-5-carboxamide, markedly inhibited the in vitro replication of a number of RNA viruses including Rift Valley fever (RVF), Venezuelan equine encephalomyelitis (VEE), Sandfly, Pichinde, Lassa and LCM virus. Plaque formation was reduced by 80% or more with 2-10 micrograms/ml of pyrazofurin while 2 micrograms/ml reduced by 1000-fold the yield of Lassa and LCM virus in a yield reduction assay. In vivo, pyrazofurin failed to protect mice and guinea pigs against a lethal challenge with VEE and Pichinde virus, respectively. On the other hand, pyrazofurin caused a slight increase in the mean time to death of mice infected with RVF virus.

Synthesis and antiviral activity of certain carbamoylpyrrolopyrimidine and pyrazolopyrimidine nucleosides.

Following our recent discovery that 9-beta-D-ribofuranosylpurine-6-carboxamide (1) exhibits potent antiviral activity, we were prompted to synthesize certain pyrrolopyrimidine and pyrazolopyrimidine nucleosides containing a carbamoyl function (7a,b and 13). The key precursor, 7-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-4- carbonitrile (8a), required for the synthesis of 7a was prepared from the corresponding 4-chloro analogue (4a). Reaction of 4a with methanethiol, followed by oxidation, gave the 4-methylsulfonyl derivative (6a), which with NaCN in DMF gave 8a. Alkaline hydrolysis of 8a provided 7a. Similarly, 7b was prepared from 4-chloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)pyrazolo[3,4-d] pyrimidine (4b) via the carbonitrile intermediate 8b. Starting with thioformycin B or 7-chloro-3-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)pyrazolo[4,3-d]pyrimidine (10) and following the similar sequence of reactions, we obtained compound 13. The in vitro antiviral studies of these carbamoyl and certain related nucleosides indicated 7a to be a potent antiviral agent against vaccinia virus, whereas 13 was moderately active. 4-Chloro-7-beta-D-ribofuranosylpyrrolo[2,3-d]pyrimidine was found to be one of the most active compounds against RVF, PICH, YF, and SF viruses in culture.

Inhibition of RNA viruses in vitro and in Rift Valley fever-infected mice by didemnins A and B.

Didemnins, a new class of depsipeptides isolated from a Caribbean tunicate, have been shown to have potent antiviral activity against a broad range of RNA viruses in vitro. Didemnins A and B both protected mice against a lethal challenge of Rift Valley fever virus.