Synthesis and biological evaluation of Ribo 7-N/O/S pyrimidine 9-deaza C-nucleoside analogs as new antiviral agents for inhibiting HCV RNA-dependent RNA polymerases.

Hepatitis C infection is caused by the bloodborne pathogen hepatitis C virus (HCV) and can lead to serious liver diseases and, ultimately, death if the treatment is ineffective. This work reports the synthesis and preclinical evaluation of 7 novel 9-O/N/S pyrimidine nucleosides, including compound 12, the triphosphate of known compound 7b. The nucleosides are 9-deaza modifications of adenosine and guanosine with ß-2'-C-methyl substituent on the ribose. Within this series of compounds, a 9-deaza furopyrimidine analog of adenosine, compound 7b, showed high anti-HCV activity in vitro, good stability, low toxicity, and low genotoxicity when administrated in low doses, and an adequate pharmacokinetics profile. An improved synthesis of compound 7b compared to a previous study is also reported. Compound 12 was synthesized as a control to verify phosphorylation of 7b occurred in vivo.

Discovery and optimization of orally bioavailable and potent plasma Kallikrein inhibitors bearing a quaternary carbon.

Hereditary angioedema (HAE) is a rare and potentially life-threatening disease that affects an estimated 1 in 50,000 individuals worldwide. Berotralstat (BCX7353) is the only small molecule approved by the US Food and Drug Administration (FDA) for the prophylactic treatment of HAE attacks in patients 12 years and older. During the discovery of BCX7353, we also identified a novel series of small molecules containing a quaternary carbon as potent and orally bioavailable Plasma Kallikrein (PKal) inhibitors. Lead compound was identified as a potent inhibitor following a detailed lead optimization process that balanced the lipophilic efficiency (LipE) and pharmacokinetic (PK) profile.

Scaffold hopping via ring opening enables identification of acyclic compounds as new complement Factor D inhibitors.

The three complement pathways comprising the early phase of the complement system (the classical, lectin, and alternative pathways) act together with the innate and adaptive immune systems to protect against foreign entities and maintain tissue homeostasis. While these systems are normally under tight regulatory control, several diseases have been reported to correlate with uncontrolled activation and amplification of the alternative pathway, including paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, C3 glomerulopathy, and age-related macular degeneration. Complement FactorD (CFD), a serine protease, is the rate-limiting enzyme for the activity of alternative pathway. CFD activates the alternative pathway by cleaving Complement Factor B complexed to C3b (C3bB) to generate alternative pathway C3 convertase (C3bBb). In our search for novel CFD inhibitors with therapeutic potential, we employed a hot-spot analysis of an ensemble of apo and holo CFD structures. This analysis identified potential pharmacophore features that aided in the design of a series of compounds based on an l-proline core. While these compounds inhibited CFD in an esterolytic assay (for example, a proline-based compound, IC50 = 161 nM), the pharmacokinetic (PK) properties were poor. A strategy of scaffold hopping via ring opening led to a novel series of acyclic compounds, with subsequent structure-based ligand design and lead optimization producing several novel CFD inhibitors. One of these inhibitors, 1-(2-((2-(3-chloro-2-fluorobenzylamino)-2-oxoethyl)(cyclopropyl)amino)-2-oxoethyl)-5-(3-methyl-3-(1-methylpiperidin-4-yl)ureido)-1H-indazole-3-carboxamide, showed good potency with IC50s of 37 nM in the esterolytic assay and 30 nM in a hemolytic assay and PK assessments following oral administration to rats revealed a Cmax of 113 ng/mL and an AUC0-24h of 257 hr.ng/mL.

An update on the progress of galidesivir (BCX4430), a broad-spectrum antiviral.

Galidesivir (BCX4430) is an adenosine nucleoside analog that is broadly active in cell culture against several RNA viruses of various families. This activity has also been shown in animal models of viral disease associated with Ebola, Marburg, yellow fever, Zika, and Rift Valley fever viruses. In many cases, the compound is more efficacious in animal models than cell culture activity would predict. Based on favorable data from in vivo animal studies, galidesivir has recently undergone evaluation in several phase I clinical trials, including against severe acute respiratory syndrome coronavirus 2, and as a medical countermeasure for the treatment of Marburg virus disease.

Intracellular rebinding of transition-state analogues provides extended in vivo inhibition lifetimes on human purine nucleoside phosphorylase.

Purine nucleoside phosphorylase (PNP) is part of the human purine salvage pathway. Its deficiency triggers apoptosis of activated T-cells, making it a target for T-cell proliferative disorders. Transition-state analogues of PNP bind with picomolar (pm) dissociation constants. Tight-binding PNP inhibitors show exceptionally long lifetimes on the target enzyme. We solve the mechanism of the target residence time by comparing functional off-rates in vitro and in vivo We report in vitro PNP-inhibitor dissociation rates (t½) from 3 to 31 min for seven Immucillins with dissociation constants of 115 to 6 pm Treatment of human erythrocytes with DADMe-Immucillin-H (DADMe-ImmH, 22 pm) causes complete inhibition of PNP. Loss of [14C]DADMe-ImmH from erythrocytes during multiple washes is slow and biphasic, resulting from inhibitor release and rebinding to PNP catalytic sites. The slow phase gave a t½ of 84 h. Loss of [14C]DADMe-ImmH from erythrocytes in the presence of excess unlabeled DADMe-ImmH increased to a t½ of 1.6 h by preventing rebinding. Thus, in human erythrocytes, rebinding of DADMe-ImmH is 50-fold more likely than diffusional loss of the inhibitor from the erythrocyte. Humans treated with a single oral dose of DADMe-ImmH in phase 1 clinical trials exhibit regain of PNP activity with a t½ of 59 days, corresponding to the erythropoiesis rate in humans. Thus, the PNP catalytic site recapture of DADMe-ImmH is highly favored in vivo We conclude that transition-state analogues with picomolar dissociation constants exhibit long lifetimes on their targets in vivo because the probability of the target enzyme recapturing inhibitor molecules is greater than diffusional loss to the extracellular space.

Efficacy of the broad-spectrum antiviral compound BCX4430 against Zika virus in cell culture and in a mouse model.

Zika virus (ZIKV) is currently undergoing pandemic emergence. While disease is typically subclinical, severe neurologic manifestations in fetuses and newborns after congenital infection underscore an urgent need for antiviral interventions. The adenosine analog BCX4430 has broad-spectrum activity against a wide range of RNA viruses, including potent in vivo activity against yellow fever, Marburg and Ebola viruses. We tested this compound against African and Asian lineage ZIKV in cytopathic effect inhibition and virus yield reduction assays in various cell lines. To further evaluate the efficacy in a relevant animal model, we developed a mouse model of severe ZIKV infection, which recapitulates various human disease manifestations including peripheral virus replication, conjunctivitis, encephalitis and myelitis. Time-course quantification of viral RNA accumulation demonstrated robust viral replication in several relevant tissues, including high and persistent viral loads observed in the brain and testis. The presence of viral RNA in various tissues was confirmed by an infectious culture assay as well as immunohistochemical staining of tissue sections. Treatment of ZIKV-infected mice with BCX4430 significantly improved outcome even when treatment was initiated during the peak of viremia. The demonstration of potent activity of BCX4430 against ZIKV in a lethal mouse model warrant its continued clinical development.

Safety, pharmacokinetics, and pharmacodynamics of avoralstat, an oral plasma kallikrein inhibitor: phase 1 study.

BACKGROUND: Avoralstat is a potent small-molecule oral plasma kallikrein inhibitor under development for treatment of hereditary angioedema (HAE). This first-in-human study evaluated the safety, tolerability, pharmacokinetics, and pharmacodynamics of avoralstat. METHODS: This double-blind, placebo-controlled, ascending-dose cohort trial evaluated avoralstat single doses of 50, 125, 250, 500, and 1000 mg and multiple doses up to 2400 mg daily (100, 200, 400, and 800 mg every 8 h [q8 h] up to 7 days). RESULTS: Avoralstat (n = 71) was generally well tolerated with no signals for a safety concern; there were no serious adverse events (AEs) or discontinuations due to AEs, and compared to placebo (n = 18), no notable difference in AEs. Four moderate severity AEs were reported in two subjects; syncope after a single 250 mg dose (one subject) and abdominal pain, back pain, and eczema after multiple doses of 800 mg avoralstat (one subject). For multiple-dose cohorts, the incidence of gastrointestinal AEs was highest at the 2400 mg/day dose. Elimination of avoralstat was bi-exponential with a terminal half-life of 12-31 h. Inhibition of plasma kallikrein was observed at all doses, and the degree of inhibition was highly correlated with avoralstat concentrations (R = 0.93). Mean avoralstat concentrations at doses ≥400 mg q8 h met or exceeded plasma kallikrein EC50 values throughout the dosing interval. CONCLUSION: Avoralstat was well tolerated, and drug exposure was sufficient to meet target levels for inhibition of plasma kallikrein. Based on these results, the 400 mg q8 h dose was selected for further evaluation in patients with HAE.

Combinations of favipiravir and peramivir for the treatment of pandemic influenza A/California/04/2009 (H1N1) virus infections in mice.

Favipiravir, an influenza virus RNA polymerase inhibitor, and peramivir, an influenza virus neuraminidase inhibitor, were evaluated alone and in combination against pandemic influenza A/California/04/2009 (H1N1) virus infections in mice. Infected mice were treated twice daily for 5 d starting 4 h after virus challenge. Favipiravir was 40%, 70%, and 100% protective at 20, 40, and 100 mg/kg/d. Peramivir was 30% protective at 0.5 mg/kg/d, but ineffective at lower doses when used as monotherapy. Combinations of favipiravir and peramivir increased the numbers of survivors by 10-50% when the 0.025, 0.05, and 0.1 mg/kg/d doses of peramivir were combined with 20 mg/kg/d favipiravir and when all doses of peramivir were combined with 40 mg/kg/d favipiravir. Three-dimensional analysis of drug interactions using the MacSynergy method indicates strong synergy for these drug combinations. In addition, an increase in lifespan for groups of mice treated with drug combinations, compared to the most effective monotherapy group, was observed for the 0.025, 0.05, and 0.1 mg/kg/d doses of peramivir combined with favipiravir at the 20 mg dose level. Therefore, the 20 mg/kg/d dose of favipiravir was selected for further combination studies. Increased survival was exhibited when this dose was combined with peramivir doses of 0.1, 0.25 and 0.5 mg/kg/d (1 mg/kg/d of peramivir alone was 100% protective in this experiment). Improved body weight relative to either compound alone was evident using 0.25, 0.5, and 1 mg/kg/d of peramivir. Significant reductions in lung hemorrhage score and lung weight were evident on day 6 post-infection. In addition, virus titers were reduced significantly on day 4 post-infection by combination therapy containing favipiravir combined with peramivir at 0.25 and 0.5 mg/kg/d. These data demonstrate that combinations of favipiravir and peramivir perform better than suboptimal doses of each compound alone for the treatment of influenza virus infections in mice.

Characterization of the binding affinities of peramivir and oseltamivir carboxylate to the neuraminidase enzyme.

With the continued threat of morbidity and mortality from influenza and the development of resistance to influenza antiviral drugs, there is increasing interest in new treatments, such as the investigational intravenous drug peramivir, and in combination treatments. In this study, we determined the impact of oseltamivir carboxylate on the binding affinity of peramivir/neuraminidase (NA) enzyme complex and vice versa. Influenza NA was incubated with peramivir and oseltamivir carboxylate alone and in combination. Dissociation rates of the enzyme-inhibitor complex measured in the presence of NA substrate for peramivir alone and the combination were similar, suggesting that peramivir competitively inhibits the neuraminidase enzyme and that oseltamivir carboxylate when added to peramivir does not impact the binding affinity of peramivir to the NA enzyme.

A single intramuscular injection of neuraminidase inhibitor peramivir demonstrates antiviral activity against novel pandemic A/California/04/2009 (H1N1) influenza virus infection in mice.

New and emerging influenza virus strains, such as the pandemic influenza A (H1N1) virus require constant vigilance for antiviral drug sensitivity and resistance. Efficacy of intramuscularly (IM) administered neuraminidase (NA) inhibitor, peramivir, was evaluated in mice infected with recently isolated pandemic A/California/04/2009 (H1N1, swine origin, mouse adapted) influenza virus. A single IM injection of peramivir (four dose groups), given 1h prior to inoculation, significantly reduced weight loss (p < 0.001) and mortality (p < 0.05) in mice infected with LD90 dose of pandemic A/California/04/2009 (H1N1) influenza virus compared to vehicle group. There was 20% survival in the vehicle-treated group, whereas in the peramivir-treated groups, survival increased in a dose-dependent manner with 60, 60, 90 and 100% survivors for the 1, 3, 10, and 30 mg/kg doses, respectively. Weight loss on day 4 in the vehicle-treated group was 3.4 gm, and in the peramivir-treated groups was 2.1, 1.5, 1.8 and 1.8 g for the 1, 3, 10 and 30 mg/kg dose groups, respectively. In the treatment model, peramivir given 24h after infection as a single IM injection at 50mg/kg dose, showed significant protection against lethality and weight loss. There was 13% survival in the vehicle-treated group while in the peramivir-treated group at 24, 48, and 72 h post infection, survival was 100, 40, and 50%, respectively. Survival in the oseltamivir groups (10 mg/kg/d twice a day, orally for 5 days) was 90, 30 and 20% at 24, 48 and 72 h, respectively. These data demonstrate efficacy of parenterally administered peramivir against the recently isolated pandemic influenza virus in murine infection models.

Combination of peramivir and rimantadine demonstrate synergistic antiviral effects in sub-lethal influenza A (H3N2) virus mouse model.

Efficacy of combination of the intramuscularly administered neuraminidase (NA) inhibitor, peramivir, and the orally administered M2 ion channel blocker, rimantadine was evaluated in mouse influenza A/Victoria/3/75 (H3N2) model. Mice were challenged with a sub-lethal virus dose (0-40% mortality in placebo group) and changes in body weights were analyzed by three-dimensional effect analysis to assess mode of drug interactions. Compounds were administered in a 5-day treatment course starting 1h before viral inoculation. The peramivir and rimantadine doses ranged from 0.3-3 mg/kg/d and 5-30 mg/kg/d, respectively. The maximum mean weight loss of 5.19 g was observed in the vehicle-infected group on day 10. In the 1 and 3 mg/kg/d peramivir monotherapy groups, the weight losses were 4.3 and 3.55 g, respectively. In the rimantadine monotherapy group, the weight losses were 3.43, 2.1, and 1.64 g for the 5, 10, and 30 mg/kg/d groups, respectively. Combination of 1mg/kg/d peramivir with 5 and 10 mg/kg/d rimantadine produced weight losses of 1.69 and 0.69 (p<0.05 vs. vehicle and individual agent), respectively, whereas the combination of 3.0 mg/kg/d peramivir with 10 and 30 mg/kg/d rimantadine did not show any weight loss (p<0.05 vs. vehicle and individual agent). The three-dimensional analysis of the weight loss for the majority of the drug combinations of peramivir and rimantadine tested demonstrated synergistic antiviral effects.

Combinations of oseltamivir and peramivir for the treatment of influenza A (H1N1) virus infections in cell culture and in mice.

Oseltamivir and peramivir are being considered for combination treatment of serious influenza virus infections in humans. Both compounds are influenza virus neuraminidase inhibitors, and since peramivir binds tighter to the enzyme than oseltamivir carboxylate (the active form of oseltamivir), the possibility exists that antagonistic interactions might result when using the two compounds together. To study this possibility, combination chemotherapy experiments were conducted in vitro and in mice infected with influenza A/NWS/33 (H1N1) virus. Treatment of infected MDCK cells was performed with combinations of oseltamivir carboxylate and peramivir at 0.32-100µM for 3 days, followed by virus yield determinations. Additive drug interactions with a narrow region of synergy were found using the MacSynergy method. In a viral neuraminidase assay with combinations of inhibitors at 0.01-10nM, no significant antagonistic or synergistic interactions were observed across the range of concentrations. Infected mice were treated twice daily for 5 days starting 2h prior to virus challenge using drug doses of 0.05-0.4mg/kg/day. Consistent and statistically significant increases in the numbers of survivors were seen when twice daily oral oseltamivir (0.4mg/kg/day) was combined with twice daily intramuscular peramivir (0.1 and 0.2mg/kg/day) compared to single drug treatments. The data demonstrate that combinations of oseltamivir and peramivir perform better than suboptimal doses of each compound alone to treat influenza infections in mice. Treatment with these two compounds should be considered as an option.

Probing the S2 site of factor VIIa to generate potent and selective inhibitors: the structure of BCX-3607 in complex with tissue factor-factor VIIa.

Factor VIIa (FVIIa) is a trypsin-like serine protease in the coagulation cascade. Its complex with tissue factor (TF) triggers the extrinsic pathway of the coagulation cascade, generating a blood clot. Research programs at several centers now recognize the important roles played by TF and FVIIa in both the thrombotic and inflammatory processes associated with cardiovascular diseases. Therefore, inhibition of the TF-FVIIa complex is seen as a promising target that is key to the development of clinical candidates for various cardiovascular applications. The crystal structure of the TF-FVIIa enzyme complex has been analyzed in order to design and synthesize small-molecule inhibitors. Using structure-based drug design (SBDD), a new series of inhibitors have been discovered that demonstrate high potency against the TF-FVIIa complex while maintaining substantial selectivity versus other closely related serine proteases such as trypsin, thrombin, factor Xa and plasmin.

Discovery of highly potent small molecule kallikrein inhibitors.

Uncontrolled kallikrein activation is involved in diseases such as hereditary angioedema, bacterial septic shock and procedures such as cardiopulmonary bypass. Here we report a series of small molecule compounds that potently inhibit kallikrein activity in vitro. Kinetic studies indicate that some of these compounds are slow binding inhibitors of kallikrein with Ki final less than a nanomolar. The ability of these compounds to inhibit the activity of kallikrein was further confirmed in a plasma model by quantitating the release of bradykinin, an endogenous cleavage product of plasma kallikrein. To understand the inhibitory mechanism of the selected compounds toward kallikrein, the interactions between the selected compounds and kallikrein was explored using molecular modeling based on the information of crystal structures of TF/FVIIa and kallikrein. The information presented in the current study provides an initial approach to develop more selective and therapeutically useful small molecule inhibitors.

Intravenous and oral pharmacokinetic study of BCX-1777, a novel purine nucleoside phosphorylase transition-state inhibitor. In vivo effects on blood 2'-deoxyguanosine in primates.

Administration of BCX-1777 to primates results in a rapid elevation of plasma 2'-deoxyguanosine (up to 0.4 microg/ml, 1.5 microM). Maximum 2'-deoxyguanosine C(max), 0.4 microg/ml, was achieved with the lowest IV dose of BCX-1777 and increasing the IV dose of BCX-1777 did not increase the 2'-deoxyguanosine C(max). However, plasma 2'-deoxyguanosine remained elevated longer as the dose of BCX-1777 increased. In contrast, increases in the oral dose of BCX-1777 did increase the plasma C(max) of 2'-deoxyguanosine. This was in spite of the observation that overall oral bioavailability of BCX-1777 was only 8.2%. This suggests that the BCX-1777 was absorbed slowly producing a sustained low concentration of BCX-1777, resulting in prolonged plasma concentrations of 2'-deoxyguanosine. After IV dosing, the BCX-1777 was cleared relatively quickly and the plasma 2'-deoxyguanosine tracked slightly behind the BCX-1777. IV administration of 5 mg/kg of BCX-1777 twice daily maintains the plasma 2'-deoxyguanosine concentrations at around 0.3 microg/ml (1.1 microM). These data indicate that oral and IV administration of BCX-1777 induce a rapid rise in 2'-deoxyguanosine and that oral dosing at 8.8 and 17.6 mg/kg are at least equivalent to 4.4 mg/kg IV in effecting the accumulation of 2'-deoxyguanosine. Finally, 2'-deoxyguanosine plasma concentration was maintained longer in the three highest oral doses in comparison to all IV doses.

Systematic structure-based design and stereoselective synthesis of novel multisubstituted cyclopentane derivatives with potent antiinfluenza activity.

The design and synthesis of novel, orally active, potent, and selective inhibitors of influenza neuraminidase differing structurally from existing neuraminidase inhibitors are described. X-ray crystal structures of complexes of neuraminidase with known five- and six-membered ring inhibitors revealed that potent inhibition of the enzyme is determined by the relative positions of the interacting inhibitor substituents (carboxylate, glycerol, acetamido, hydroxyl) rather than by the absolute position of the central ring. This led us to design potential neuraminidase inhibitors in which the cyclopentane ring served as a scaffold for substituents (carboxylate, guanidino, acetamido, alkyl) that would interact with the four binding pockets of the neuraminidase active site at least as effectively as those of the established six-membered ring inhibitors such as DANA (2), zanamivir (3), and oseltamivir (4). A mixture of the isomers was prepared initially. Protein crystallography of inhibitor-enzyme complexes was used to screen mixtures of isomers in order to identify the most active stereoisomer. A synthetic route to the identified candidate 50 was developed, which featured (3 + 2) cycloaddition of 2-ethylbutyronitrile oxide to methyl (1S,4R)-4[(tert-butoxycarbonyl)amino]cyclopent-2-ene-1-carboxylate (43). Structures of the synthetic compounds were verified by NMR spectroscopy using nuclear Overhauser effect methodology. Two new neuraminidase inhibitors discovered in this work, 50 and 54, have IC(50) values vs neuraminidase from influenza A and B of <1 and <10 nM, respectively. These IC(50) values are comparable or superior to those for zanamivir and oseltamivir, agents recently approved by the FDA for treatment of influenza. The synthetic route used to prepare 50 and 54 was refined so that synthesis of pure active isomer 54, which has five chiral centers, required only seven steps from readily available intermediates. Further manipulation was required to prepare deoxy derivative 50. Because the activities of the two compounds are comparable and 54 [RWJ-270201 (BCX-1812)] is the easier to synthesize, it was selected for further clinical evaluation.

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.

Comparison of the anti-influenza virus activity of RWJ-270201 with those of oseltamivir and zanamivir.

We have recently reported an influenza virus neuraminidase inhibitor, RWJ-270201 (BCX-1812), a novel cyclopentane derivative discovered through structure-based drug design. In this paper, we compare the potency of three compounds, RWJ-270201, oseltamivir, and zanamivir, against neuraminidase enzymes from various subtypes of influenza. RWJ-270201 effectively inhibited all tested influenza A and influenza B neuraminidases in vitro, with 50% inhibitory concentrations of 0.09 to 1.4 nM for influenza A neuraminidases and 0.6 to 11 nM for influenza B neuraminidases. These values were comparable to or lower than those for oseltamivir carboxylate (GS4071) and zanamivir (GG167). RWJ-270201 demonstrated excellent selectivity (>10,000-fold) for influenza virus neuraminidase over mammalian, bacterial, or other viral neuraminidases. Oral administration of a dosage of 1 mg/kg of body weight/day of RWJ-270201 for 5 days (beginning 4 h preinfection) showed efficacy in the murine model of influenza virus infection as determined by lethality and weight loss protection. RWJ-270201 administered intranasally at 0.01 mg/kg/day in the murine influenza model demonstrated complete protection against lethality, whereas oseltamivir carboxylate and zanamivir at the same dose demonstrated only partial protection. In the delayed-treatment murine influenza model, oral administration of a 10-mg/kg/day dose of RWJ-270201 or oseltamivir (GS4104, a prodrug of GS4071) at 24 h postinfection showed significant protection against lethality (P < 0.001 versus control). However, when the treatment was delayed for 48 h, no significant protection was observed in either drug group. No drug-related toxicity was observed in mice receiving 100 mg/kg/day of RWJ-270201 for 5 days. These efficacy and safety profiles justify further consideration of RWJ-270201 for the treatment and prevention of human influenza.

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.