Influence of virus strain, challenge dose, and time of therapy initiation on the in vivo influenza inhibitory effects of RWJ-270201.

The influenza virus neuraminidase inhibitor RWJ-270201 (cyclopentane carboxylic acid, 3-[cis-1-(acetylamino)-2-ethylbutyl]-4[(aminoiminomethyl)amino]-2-hydroxy-[cis, 2S, 3R, 4R]) was significantly inhibitory to an infection in mice induced by influenza A/NWS/33 (H1N1) virus when oral gavage (p.o.) treatment with 10 mg/kg per day was delayed at least 60 h after virus exposure. Treatment was 5 mg/kg twice daily for 5 days. Viral challenge doses of influenza A/Shangdong/09/93 (H3N2) virus ranging from the LD(70) to the LD(100) did not affect the marked antiviral efficacy of 12.5 mg/kg of RWJ-270201 administered p.o. twice daily for 5 days beginning 4 h pre-virus exposure; infection by an approximate 2 LD(100) dose (10(8) cell culture infectious doses/ml) was only weakly inhibited by the same treatment as seen by significant increase in mean day to death. Murine infections induced by influenza A/Bayern/57/93 (H1N1) and B/Lee/40 viruses were significantly inhibited by 100, 10, and 1 mg/kg per day of RWJ-270201 using the above treatment regimen; influenza A/PR/8/34 (H1N1) virus infections in mice were only moderately inhibited, the antiviral effects using this virus being lessening of arterial oxygen decline, reduced lung consolidation, and inhibition of lung virus titers primarily at the higher dosages.

Cyclopentane neuraminidase inhibitors with potent in vitro anti-influenza virus activities.

A novel series of cyclopentane derivatives have been found to exhibit potent and selective inhibitory effects on influenza virus neuraminidase. These compounds, designated RWJ-270201, BCX-1827, BCX-1898, and BCX-1923, were tested in parallel with zanamivir and oseltamivir carboxylate against a spectrum of influenza A (H1N1, H3N2, and H5N1) and influenza B viruses in MDCK cells. Inhibition of viral cytopathic effect ascertained visually and by neutral red dye uptake was used, with 50% effective (virus-inhibitory) concentrations (EC(50)) determined. Against the H1N1 viruses A/Bayern/07/95, A/Beijing/262/95, A/PR/8/34, and A/Texas/36/91, EC(50)s (determined by neutral red assay) of the novel compounds were < or =1.5 microM. Twelve strains of H3N2 and two strains of avian H5N1 viruses were inhibited at <0.3 microM. Influenza B/Beijing/184/93 and B/Harbin/07/94 viruses were inhibited at <0.2 microM, with three other B virus strains inhibited at 0.8 to 8 microM. The novel inhibitors were comparable in potency to (or slightly more potent than) zanamivir and oseltamivir carboxylate. No cytotoxicity was seen with the compounds at concentrations of < or =1 mM in cell proliferation assays. The antiviral activity of RWJ-270201, chosen for clinical development, was studied in greater detail. Its potency and that of oseltamivir carboxylate decreased with increasing multiplicity of virus infection. Time-of-addition studies indicated that treatment with either compound needed to begin 0 to 12 h after virus exposure for optimal activity. Exposure of cells to RWJ-270201 caused most of the virus to remain cell associated, with extracellular virus decreasing in a concentration-dependent manner. This is consistent with its effect as a neuraminidase inhibitor. RWJ-270201 shows promise in the treatment of human influenza virus infections.

In vivo influenza virus-inhibitory effects of the cyclopentane neuraminidase inhibitor RJW-270201.

The cyclopentane influenza virus neuraminidase inhibitor RWJ-270201 was evaluated against influenza A/NWS/33 (H1N1), A/Shangdong/09/93 (H3N2), A/Victoria/3/75 (H3N2), and B/Hong Kong/05/72 virus infections in mice. Treatment was by oral gavage twice daily for 5 days beginning 4 h pre-virus exposure. The influenza virus inhibitor oseltamivir was run in parallel, and ribavirin was included in studies with the A/Shangdong and B/Hong Kong viruses. RWJ-270201 was inhibitory to all infections using doses as low as 1 mg/kg/day. Oseltamivir was generally up to 10-fold less effective than RWJ-270201. Ribavirin was also inhibitory but was less tolerated by the mice at the 75-mg/kg/day dose used. Disease-inhibitory effects included prevention of death, lessening of decline of arterial oxygen saturation, inhibition of lung consolidation, and reduction in lung virus titers. RWJ-270201 and oseltamivir, at doses of 10 and 1 mg/kg/day each, were compared with regard to their effects on daily lung parameters in influenza A/Shangdong/09/93 virus-infected mice. Maximum virus titer inhibition was seen on day 1, with RWJ-270201 exhibiting the greater inhibitory effect, a titer reduction of >10(4) cell culture 50% infective doses (CCID(50))/g. By day 8, the lung virus titers in mice treated with RWJ-270201 had declined to 10(1.2) CCID(50)/g, whereas titers from oseltamivir-treated animals were >10(3) CCID(50)/g. Mean lung consolidation was also higher in the oseltamivir-treated animals on day 8. Both neuraminidase inhibitors were well tolerated by the mice. RWJ-270201 was nontoxic at doses as high as 1,000 mg/kg/day. These data indicate potential for the oral use of RWJ-270201 in the treatment of influenza virus infections in humans.

Immunological effects of the orally administered neuraminidase inhibitor oseltamivir in influenza virus-infected and uninfected mice.

Oseltamivir (GS4104), the ethyl ester prodrug of the carbocyclic transition state sialic acid analog GS4071, has been reported to be a striking inhibitor of influenza A and B virus infections in mice and ferrets. Multiple studies indicate this material to also be active against the disease in humans, and it has recently been approved for human use. The effect of oral gavage (p.o.) therapy of oseltamivir on various immune factors considered to be of importance in primary influenza virus infection was studied in mice. Both uninfected animals and those infected with influenza A/NWS/33 (H1N1) virus were used. Doses of 100 mg kg(-1) day(-1) were administered twice daily for 5 days beginning 16 h pre-virus exposure. Two hours after end of treatment, the mice were killed and their spleens assayed for cytotoxic T lymphocyte (CTL) and natural killer (NK) cell activity. Subpopulations of splenic T, T-helper, T-cytotoxic and B lymphocytes as well as macrophages were determined using flow cytometry. Similar significant (P<0.01) increases in CTL activity were seen at effector:target cell ratios of 60:1 and 30:1 in the infected mice treated with oseltamivir or with placebo. NK cell activity was greater in the infected mice than in uninfected mice; the levels in all animals were not significantly affected by treatment with oseltamivir. Macrophage, T, T-helper, T-cytotoxic and B lymphocyte populations were similar in both treated and untreated animals. These data indicate treatment with oseltamivir does not adversely affect the primary in vivo cellular immune responses to influenza virus infection assayed in this study. The experiment was repeated to show that treatment with this compound significantly prevented the development of the infection and inhibited virus titers in the lung. Surviving treated mice on day 21 had mean neutralizing antibody titers of 1:208, and withstood rechallenge with the virus at this time, indicating the initial virus-inhibitory effect also did not prevent the animals from developing an adequate humoral immunity to the virus.

Spectrum of virus inhibition by consensus interferon YM643.

The spectrum of viruses inhibited by a genetically engineered consensus interferon (IFN) YM643 (interferon alfacon-1) was evaluated using a cytopathic effect inhibition assay or plaque inhibition assay for five DNA viruses and 12 RNA viruses. This activity was compared to that of natural IFN-alpha derived from Namalwa lymphoblastoid cell line [IFN-alpha (Namalwa)]. The viruses inhibited by both IFNs were herpesvirus types 1 and 2, human cytomegalovirus, varicella-zoster virus, vesicular stomatitis virus, yellow fever virus, bovine viral diarrhoea virus, Semliki Forest virus, western equine encephalitis virus, encephalomyocarditis virus, rhinovirus type A, respiratory syncytial virus, Newcastle disease virus and influenza virus type A (H1N1). Neither IFN inhibited coxsackie virus B1, reovirus type 3 or vaccinia virus in the experimental conditions used. The specific activity of YM643 in human cells generally ranged from 3.6x10(7) to 2.1x10(9) IU/mg, which was greater than that of IFN-alpha (Namalwa), which ranged from 3.1x10(6) to 4.6x10(8) IU/mg against all sensitive viruses, except human cytomegalovirus and rhinovirus type 1A, which displayed approximately equal sensitivity to both IFNs. Significantly, the potency of YM643 against bovine viral diarrhoea virus and yellow fever virus, which were selected to serve as surrogates of hepatitis C virus, equalled or exceeded that of IFN-alpha (Namalwa). These results suggest that the genetically engineered YM643 is more potent than natural IFN-alpha.

Influence of treatment schedule and viral challenge dose on the in vivo influenza virus-inhibitory effects of the orally administered neuraminidase inhibitor GS 4104.

Experiments were done to determine how an alteration of the treatment schedule of 5 or 32 mg/kg/day per os (p.o.) doses of GS 4104 [the ethyl ester prodrug of the neuraminidase inhibitor (3R, 4R,5S)-4-acetamido-5-amino-3-(1-ethylpropoxy)-1-cylohexene-1 -carboxylic acid (GS 4071)] would affect influenza A (H1N1) virus infection in mice. Treatments with a low dose, one, two, three or four times daily, were highly inhibitory, unless therapy was terminated relatively early in the infection (days 2-3), in which case efficacy was curtailed. Single administrations at various times relative to virus exposure had essentially no effect. The 32 mg/kg/day dose was significantly inhibitory using all treatment schedules. These data indicated a requirement for the compound to be in the host when lung virus titres were reaching maximal levels and, for minimally effective doses, that at least continued daily therapy was needed to maintain adequate serum levels to achieve an appropriate antiviral effect. Twice daily p.o. treatment for 5 days with 20 mg/kg/day of GS 4104 totally prevented deaths in mice receiving high viral challenge doses that were sufficient to kill placebo-treated controls in less than 5 days. Other parameters of antiviral efficacy (lung consolidation, arterial oxygen saturation, lung virus titres) were also markedly inhibited regardless of viral challenge doses. These data provide further insights into how the maximum therapeutic benefit can be derived from use of this orally effective influenza virus neuraminidase inhibitor.

Antiviral activities of biflavonoids.

Biflavonoids such as amentoflavone (1), agathisflavone (2), robustaflavone (3), hinokiflavone (4), volkensiflavone (5), rhusflavanone (7), succedaneflavanone (9), all isolated from Rhus succedanea and Garcinia multiflora, as well as their methyl ethers and acetates, volkensiflavone hexamethyl ether (6), rhusflavanone hexaacetate (8), and succedaneflavanone hexaacetate (10) were evaluated for their antiviral activities. The inhibitory activities against a number of viruses including respiratory viruses (influenza A, influenza B, respiratory syncytial, parainfluenza type 3, adenovirus type 5, and measles) and herpes viruses (HSV-1, HSV-2, HCMV, and VZV) were investigated. The results indicated that robustaflavone exhibited strong inhibitory effects against influenza A and influenza B viruses with EC50 values of 2.0 micrograms/ml and 0.2 microgram/ml, respectively, and selectivity index values (SI) of 16 and 454, respectively. Amentoflavone and agathisflavone also demonstrated significant activity against influenza A and B viruses. Amentoflavone and robustaflavone exhibited moderate anti-HSV-1 anti-HSV-2 activities with EC50 values of 17.9 micrograms/ml (HSV-1) and 48.0 micrograms/ml (HSV-2) and SI values of > 5.6 (HSV-1) and > 2.1 (HSV-2) for amentoflavone; EC50 values of 8.6 micrograms/ml (HSV-1) and 8.5 micrograms/ml (HSV-2), and SI values of > 11.6 (HSV-1) and > 11.8 (HSV-2) for robustaflavone. Rhusflavanone demonstrated inhibitory activities against influenza B, measles, and HSV-2 viruses with SI values of 9.3, 8 and > 6.4, respectively. Succedaneaflavanone exhibited inhibitory activities against influenza B virus and VZV with SI values of 15 and < 3.0, respectively.

Inhibition of influenza virus infections in mice by GS4104, an orally effective influenza virus neuraminidase inhibitor.

The carbocyclic transition state sialic acid analog GS4071 ([3R,4R,5S]-4-acetamido-5-amino-3-[1-ethylpropoxy]-1-cyclohexane-1 -carboxylic acid), a potent influenza virus neuraminidase inhibitor, was highly inhibitory to influenza A/NWS/33 (H1N1), A/Victoria/3/75 (H3N2), A/Shangdong/09/93 (H3N2) and B/Hong Kong/5/72 viruses in Madin Darby canine kidney (MDCK) cells. The 50% effective concentrations in these experiments ranged from 1.8 to 59.5 microM, with no cytotoxicity evident at 1000 microM, using inhibition of viral cytopathic effect determined visually and by neutral red dye uptake. The ethyl ester prodrug of GS4071, GS4104, administered by oral gavage (p.o.), had significant inhibitory effects on infections in mice induced by these viruses. Antiviral effects were seen as prevention of death, increase in mean day to death, inhibition of decline of arterial oxygen saturation, lessened lung consolidation and inhibition of infectious virus recovered from the lungs. No toxicity was seen in dosages up to 100 mg/kg/day (highest evaluated). Comparison experiments run versus the influenza A (H1N1) virus-induced infection using GS4104, GS4071 and the neuraminidase inhibitor zanamivir (GG167, 4-guanidino-Neu5Ac2en), all administered p.o., indicated a 10-fold or greater potency for inhibiting the infection by GS4104. The minimum effective dosage for GS4104 was 0.1 mg/kg/day, with the compound administered twice daily for 5 days beginning 4 h pre-virus exposure. Oral therapy with GS4104 could be delayed from 48 to at least 60 h after exposure of mice to influenza A (H1N1) virus and still render a significant antiviral effect, the time of delay being dependent on the viral challenge dose. Intranasal instillation of GS4071 and GG167 to mice infected with influenza virus was highly inhibitory to the infection, the minimum effective dosages to significantly prevent death being 0.01 mg/kg/day for GS4071 and 0.1 mg/kg/day for GG167. Caging of infected mice treated with 10 mg/kg/day of GS4104 with infected saline-treated animals did not transfer any influenza-inhibitory effect to the latter animals. These data provide strong evidence of the potential of orally administered GS4104 for treatment of influenza A and B virus infections in humans.

Oral administration of a prodrug of the influenza virus neuraminidase inhibitor GS 4071 protects mice and ferrets against influenza infection.

We have recently described GS 4071, a carbocyclic transition-state analog inhibitor of the influenza virus neuraminidase, which has potent inhibitory activity comparable to that of 4-guanidino-Neu5Ac2en (GG167; zanamivir) when tested against influenza A virus replication and neuraminidase activity in vitro. We now report that GS 4071 is active against several strains of influenza A and B viruses in vitro and that oral GS 4104, an ethyl ester prodrug which is converted to GS 4071 in vivo, is active in the mouse and ferret models of influenza virus infection. Oral administration of 10 mg of GS 4104 per kg of body weight per day caused a 100-fold reduction in lung homogenate viral titers and enhanced survival in mice infected with influenza A or B viruses. In ferrets, a 25-mg/kg dose of GS 4104 given twice daily reduced peak viral titers in nasal washings and eliminated constitutional responses to influenza virus infection including fever, increased nasal signs (sneezing, nasal discharge, mouth breathing), and decreased activity. Consistent with our demonstration that the parent compound is highly specific for influenza virus neuraminidases, no significant drug-related toxicity was observed after the administration of oral dosages of GS 4104 of up to 800 mg/kg/day for 14 days in nonclinical toxicology studies with rats. These results indicate that GS 4104 is a novel, orally active antiviral agent with the potential to be used for the prophylaxis and treatment of influenza A and B virus infections.

Thiazolo[4,5-d]pyrimidines. Part II. Synthesis and anti-human cytomegalovirus activity in vitro of certain acyclonucleosides and acyclonucleotides derived from the guanine analogue 5-aminothiazolo[4,5-d]pyrimidine-2,7(3H,6H)-dione.

The synthesis and in vitro antiviral activity of certain hydroxyalkoxymethyl, hydroxyalkyl, hydroxyalkenyl and phosphonoalkenyl derivatives of the guanine congener 5-aminothiazolo[4,5-d]pyrimidine-2,7(3H,6H)-dione are reported. The compounds of this study were selected for their structural similarity to acyclonucleosides with known anti-herpesvirus activity. 5-Amino-3-[(Z)-4-hydroxy-2-buten-1-yl]thiazolo[4,5-d]pyrimidine-2, 7(3H,6H)- dione was the only member of the series to display significant in vitro activity against human cytomegalovirus (HCMV); however, this compound did not inhibit other herpesviruses, human immunodeficiency virus type 1 or murine cytomegalovirus. It was found to have a cytotoxicity profile similar to that of ganciclovir (DHPG). The antiviral effect was found to be sensitive to the initial viral input and the time of addition during the virus replication cycle. Significantly, the compound was found to have equal anti-HCMV activity, against standard virus strains, to DHPG, but also showed potent activity against DHPG-resistant virus strains, except for a strain mutated in the UL97 (phosphotransferase) gene.

Potent inhibition of respiratory syncytial virus by polyoxometalates of several structural classes.

A series of polyoxometalates (POM) were synthesized and evaluated for anti-respiratory syncytial virus (RSV) activity. POM containing zirconium, tungsten, silicon, platinum, niobium or germanium of a variety of structural types have been evaluated. Sixteen of the compounds had very striking anti-RSV activity against a clinical isolate, Utah 89, with median effective concentration (EC50) values < or = 3 microM and selective indices > 80 as determined by viral cytopathic inhibition effect, neutral red uptake and virus yield reduction assays. The EC50 values for all three assays correlated very well (Pearson correlation coefficients > 0.90). POM containing sodium cations were totally inactive. Germanium-, niobium-, tin- and zirconium-containing compounds were found to be highly potent and selective. The antiviral activity was not cell line-dependent. The median cytotoxic concentration (IC50) values were generally greater than 100 microM. The compounds were also comparably active against a known laboratory RSV strain, A2, as well as other RSV strains. To detect any virus strain-specific inhibitory activity, seven POM were tested against other RSV strains; all were nearly equally inhibitory to the human virus strains, suggesting no strain specificity. Timing studies suggested that these compounds were most inhibitory during virus adsorption and penetration, although RSV was still significantly inhibited when the compound was added 3 h post-infection; which is considered well into the eclipse period. These data suggest that these potent, non-toxic compounds should be further studied as potential chemotherapeutic agents for treating RSV infections.

In vivo anti-influenza virus activity of a zinc finger peptide.

Matrix protein (M1) is a major structural protein of influenza virus, and it inhibits its own polymerase. A 19-amino-acid peptide, corresponding to a zinc finger region of the M1 sequence of influenza virus strain A/PR/8/34 (H1N1), centered around amino acids 148 to 166, was synthesized. This peptide, designated peptide 6, represents a zinc finger which includes a 7-amino-acid loop or finger and a 4-amino-acid tail at the carboxyl terminus, in addition to the 8 amino acids involved in the coordination of Zn. Three experiments were run to evaluate the activity of peptide 6 on infections induced in mice by influenza A/PR/8/34 and A/Victoria/3/75 (H3N2) viruses. Intranasal (i.n.) treatment of the H1N1 virus infection with 30 or 60 mg/kg of body weight/day, three times daily for 5 days, beginning 4 h pre-or 8 h post-virus exposure, was effective in preventing death, reducing the arterial oxygen decline, and inhibiting lung consolidation. Virus titers in the lungs determined on day 5 were reduced by up to 1.5 log10 in treated groups, but considerable variation in the titers of the recovered virus was seen. The H3N2 virus infection was treated i.n. with 30, 60, or 120 mg of peptide 6/kg/day by using the above-mentioned delayed initiation treatment schedule, and similar protection was seen, although lung virus titers were not reduced in the day-5 assay. Peptide 6 was well tolerated at doses up to 60 mg/kg/day. This zinc finger peptide may provide a new class of antivirals effective against influenza virus.

Inhibitory effects of recombinant manganese superoxide dismutase on influenza virus infections in mice.

The oxygen free-radical scavenger recombinant human manganese superoxide dismutase (MnSOD) was studied for its effects on influenza virus infections in mice when used alone and in combination with ribavirin. Mice challenged with influenza A/NWS/33 (H1N1) virus were treated parenterally in doses of 25, 50, and 100 mg/kg of body weight per day every 8 h for 5 days beginning at 48 h post-virus exposure. An increase in mean day to death, lessened decline in arterial oxygen saturation, and reduced lung consolidation and lung virus titers occurred in the treated animals. To determine the influence of viral challenge, experiments were run in which mice were infected with a 100 or 75% lethal dose of virus and were treated intravenously once daily for 5 days beginning 96 h after virus exposure. Weak inhibition of the mortality rate was seen in mice receiving the high viral challenge, whereas significant inhibition occurred in the animals infected with the lower viral challenge, indicating that MnSOD effects are virus dose dependent. To determine if treatment with small-particle aerosol would render an antiviral effect, infected mice were treated by this route for 1 h daily for 5 days beginning 72 h after virus exposure. A dose-responsive disease inhibition was seen. An infection induced by influenza B/Hong Kong/5/72 virus in mice was mildly inhibited by intravenous MnSOD treatment as seen by increased mean day to death, lessened arterial oxygen saturation decline, and lowered lung consolidation. MnSOD was well tolerated in all experiments. A combination of MnSOD and ribavirin, each administered with small-particle aerosol, resulted in a generally mild improvement of the disease induced by the influenza A virus compared with use of either material alone.

Antiviral activities of tragacanthin polysaccharides on Punta Toro virus infections in mice.

Tragacanthin polysaccharides from Astragalus brachycentrus (AV208) and Astragalus echidnaeformis (AV212) plants, which are devoid of in vitro antiviral activity, were evaluated in a mouse model of Punta Toro virus (PTV) infection. The PTV (a phlebovirus member of the Bunyaviridae family of viruses) is a model for studying the treatment of Rift Valley fever and hantavirus infections. Single intraperitoneal treatments with 12.5-200 mg/kg/day doses of AV212 given 24 h before or 4 and 24 h after virus inoculation protected the majority of mice from mortality. Single treatments with AV208 were ineffective when given 24 h before the virus challenge; however, protection was afforded when treatments were administered at 4 and 24 h following virus inoculation. In a follow-up study, AV208 treatments of 1.6-50 mg/kg/day given 24 h subsequent to virus exposure caused reductions in mortality, liver infection scores, liver and spleen virus titers, and serum transaminases. The polysaccharides did not activate lymphocytes or natural killer cells, nor was interferon induced in treated mice. However, mice pretreated with fumed silica (a macrophage poison) and infected with the PTV were not protected by subsequent administration of AV208 or AV212 at 50 mg/kg, providing evidence that activation of peritoneal macrophages by the polysaccharides affords protection to infected animals. These compounds should be considered for the potential treatment of significant human infections induced by bunyaviruses and hantaviruses.

In vitro and in vivo sensitivity of a non-mouse-adapted influenza A (Beijing) virus infection to amantadine and ribavirin.

A clinically isolated non-mouse-adapted influenza A/Beijing/32/92 virus was assayed for sensitivity to amantadine and ribavirin in vitro and in mice. When multiple concentrations of each drug were assayed for ability to inhibit the virus-induced cytopathic effect in MDCK cells, the 50% effective (virus-inhibitory) concentration was 0.12 microgram/ml for amantadine and 1.9 micrograms/ml for ribavirin. The 50% cytotoxic concentrations were 25 and 100 micrograms/ml, respectively. It is known that intranasal challenge of mice with high concentrations of non-mouse-adapted influenza virus will induce a toxic pneumonitis in the absence of significant viral replication in the lung. Treatment of such virus-infected mice with approximately 1,250, approximately 625 and approximately 313 mg/kg/day of amantadine in the drinking water resulted in significant inhibition of lung scores and weights and a lessened decline in arterial oxygen saturation (SaO2) in the mice, but virus was in low titer or not recoverable from drug- or placebo-treated animals. Intraperitoneal treatment with 75, 37.5 and 18.8 mg/kg/day of ribavirin given twice daily for 5 days was effective only in preventing SaO2 decline, which contrasts with strong inhibition of infections induced by mouse-adapted viruses seen in other studies. These in vivo data indicate that when non-mouse-adapted influenza virus infections are used to evaluate potential antiviral drugs, false-negative results may be obtained.

Synthesis and anti-DNA viral activities in vitro of certain 2,4-disubstituted-7-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)pyrrolo[2,3-d d pyrimidine nucleosides.

Several novel 2,4-disubstituted-7-(2-deoxy-2-fluoro-beta-D- arabinofuranosyl)pyrrolo[2,3-d]pyrimidines have been synthesized and evaluated for their anti-human cytomegalovirus (HCMV), anti-hepatitis B virus (HBV), and anti-herpes simplex virus (HSV) activities in vitro. These nucleosides were prepared starting from 2-amino-4-chloro-7-(2-deoxy-2-fluoro- 3,5-di-O-benzoyl-beta-D-arabinofuranosyl)pyrrolo[2,3-d]pyrimidine (3), which in turn was synthesized by direct glycosylation of the sodium salt of 2-amino-4-chloropyrrolo[2,3-d]pyrimidine (1) with 2-deoxy-2-fluoro-3,5-di-O-benzoyl-alpha-D-arabinofuranosyl bromide (2). Displacement of the 4-chloro group of 3 with OH, NH2, NHOH, SH, and SeH nucleophiles furnished the corresponding nucleosides 6-8, 12, and 14, respectively. The 3'-deoxygenation of 2-amino-4-chloro-7- (2-deoxy-2-fluoro-beta-D-arabinofuranosyl)pyrrolo[2,3-d]pyrimidine (4) and subsequent amination gave 2,4-diamino-2',3'-dideoxy derivative 19. Catalytic hydrogenation of 3 followed by debenzoylation afforded 2-aminopyrrolo[2,3-d]pyrimidine nucleoside 23. Among the compounds evaluated for their ability to inhibit the growth of HCMV (strain AD169) in MRC-5 cells using a plaque reduction assay, only 7 was significantly active in vitro with a 50% inhibitory concentration (IC50) of 3.7 micrograms/mL (TI > 125), whereas the IC50 value of ganciclovir (DHPG) was 3.2 micrograms/mL. Strain D16 of HCMV was more resistant to 7 (IC50 11 micrograms/mL) than the AD169 strain. When 7 was tested in combination with DHPG, the resultant anti-HCMV activity was found to be moderately synergistic with no evidence of antagonism. Nucleoside 7 also reduced episomal HBV replication in human hepatoblastoma 2.2.15 cells with an IC50 of 0.7 micrograms/mL (TI > 143). Development of cells harboring HBV which had become resistant to the drug was not observed with 7. Compound 7 also exhibited significant activity against herpes simplex virus types 1 and 2 (IC50 of 4.1 and 6.3 micrograms/mL, respectively) in Vero cells.

Effect of methionine enkephalin on natural killer cell and cytotoxic T lymphocyte activity in mice infected with influenza A virus.

Methionine enkephalin (Met-Enk) was evaluated for efficacy as an immune activator and potential therapeutic agent in influenza A/NWS/33 (H1N1) viral infections in female BALB/C mice. Influenza infection was induced intranasally with an approximate 90% lethal dose of virus and mice were treated intraperitoneally with doses of 10, 3 and 1 mg/kg/day, with treatments given 24 h pre-, 24 h post- and 72 h post-virus exposure. Splenocytes were assayed for natural killer cell (NK) and cytotoxic T lymphocyte (CTL) activity at time periods 76, 96 and 120 h post virus exposure. The 10 mg/kg dosage level significantly increased both CTL and NK activity at all time periods assayed. Other treatment schedules included single doses of 20, 10 and 3 mg/kg/day Met-Enk at either 24 h post- or 72 h post-virus exposure, with highly significant increases in NK and CTL activity noted after the latter treatment. The results of this study demonstrate the immunomodulatory effects of Met-Enk on NK and CTL in influenza infected mice and suggest a potential for therapeutic applications.

Antiviral activity of an immunomodulatory lipophilic desmuramyl dipeptide analog.

BCH-527, the lipophilic hydrochloride salt of octadecyl D-alanyl L-glutamine, was evaluated for efficacy against experimentally induced murine cytomegalovirus (MCMV), influenza A (H1N1) (IV-A), and Punta Toro virus (PTV) infections in mice. The compound was administered i.p. every other day for a total of 4 injections commencing 24 h previrus exposure. Doses ranged from 12.5 to 200 mg/kg per injection in the various experiments. The MCMV infection was significantly inhibited in two experiments by doses of 25-200 mg/kg, as manifested by increased numbers of survivors and decreased titers of virus recoverable from tissues. The IV-A infection was weakly inhibited, with antiviral activity seen in lowered lung scores and lung weights and less decline in arterial oxygen saturation values. The PTV infection was not inhibited. BCH-527 was stimulatory to cytotoxic T-cells, natural killer (NK) cells, macrophages, and splenic B-cells. The highest dose tested, 200 mg/kg, was inhibitory to cytotoxic T-cell activity and to some extent to NK cell and macrophage activity. These data suggest BCH-527 functions as an immune modulator in exerting the observed antiviral activity.

Inhibition of human cytomegalovirus in culture by alkenyl guanine analogs of the thiazolo[4,5-d]pyrimidine ring system.

A series of alkyl and alkenyl guanine analogs containing a thiazolo[4,5-d]pyrimidine ring system were prepared by reaction of the appropriate alkyl halide with the sodium salt of the heterocycle. In preliminary antiviral efficacy evaluations against laboratory strains of both human cytomegalovirus (HCMV) and herpes simplex virus types 1 and 2, it was determined that two of the compounds (T70072 and T01132) were more active and less toxic in stationary-phase cell monolayers than were the other derivatives tested. T01132 and T70072, which have 2-pentenyl and 3-methyl-2-butenyl moieties attached to position 3 of the 5-aminothiazolo[4,5-d]pyrimidine-2,7-dione, respectively, were then more extensively evaluated for anti-HCMV activity. The concentrations of T01132 and T70072 required to inhibit HCMV by 50% in plaque reduction assays were approximately 0.5 and 6.8 microM, respectively. These two compounds inhibited the growth of KB, MRC-5, or Vero cells at concentrations of 75 to 150 microM, depending upon the cell line. In bone marrow progenitor cells T01132 was slightly less toxic than ganciclovir (DHPG). The 50% inhibitory concentrations of T01132 against clinical isolates and DHPG-resistant strains of HCMV were approximately the same as those obtained for laboratory strains of HCMV (approximately 0.5 microM). When tested in combination with DHPG, the resultant antiviral activity was determined to be additive but not synergistic. Experiments performed using variations of the viral multiplicity of infection (MOI) demonstrated that T01132 was more active than DHPG at a low MOI (0.002 or 0.02). However, when a higher MOI (0.2 or 2.0) was used, DHPG was more efficacious than T01132. In experiments in which drug was added at various times post-viral infection, T01132 was most effective when added within the first 24 h post-HCMV infection while DHPG was able to protect cells in this assay system when added up to 48 h postinfection, indicating that T01132 is exerting its antiviral effect on events leading up to and possibly including viral DNA synthesis. The data presented in this report suggest that the antiviral activity of alkenyl-substituted thiazolopyrimidine derivatives may represent a mechanism of action against herpesviruses alternative to that of classical nucleoside analogs such as acyclovir or DHPG.

Antiviral and immunomodulating inhibitors of experimentally-induced Punta Toro virus infections.

A major component of a US Army Medical Research and Development Command-supported program to discover and develop new drugs for the treatment of Rift Valley fever, sandfly fever, and Crimean-Congo hemorrhagic fever has been to study candidate test materials against hepatotropic infections of C57BL/6 mice induced by the related but less biohazardous Punta Toro virus (PTV). The effects of 75 compounds, some of which were considered immunomodulators in their primary mechanism of activity, were studied in the PTV infection model. Of these, ribavirin, ribamidine, ribavirin 2',3',5'-triacetate, tiazofurin, tiazofurin-5'-monophosphate, tiazofurin-2',3',5'-triacetate, selenazofurin, pyrazofurin, 3-deazaguanine, and 3-deazaguanosine were considered significantly inhibitory, acting against the infection by a direct antiviral (non-immunomodulatory) fashion. These compounds had therapeutic indices (TI) ranging from > or = 5 to 65, using increased survivors as the evaluation parameter. Immunomodulators considered significantly inhibitory to this infection were poly (ICLC), ampligen, human recombinant interferon-alpha-A/D, MVE-1, MVE-2, AM-3, AM-5, mannozym, bropirimine, CL246,738, phenyleneamine, and 7-thia-8-oxoguanosine. Utilizing increased survivor numbers as measure of activity, these inhibitors had TI ranging from > or = 16 to 1000. Other antiviral effects exerted by the active compounds included reduction of hepatic icterus, lowered serum glutamic oxaloacetic and pyruvic acid transaminases, and inhibition of recoverable serum and liver virus titers. The active immunomodulators were significantly effective when therapy was initiated as late as 48 h after virus inoculation, at a time when clinical signs of the PTV disease were being manifested in the animal.