piperidines and pirodavir

Adults frequently develop eustachian tube dysfunction and middle ear pressure (MEP) abnormalities during natural and experimental influenza and human rhinovirus (HRV) infections. Oral rimantadine treatment did not reduce the otologic manifestations of experimental influenza in adults or natural influenza in children. However, intranasal zanamivir and oral oseltamivir significantly reduced MEP abnormalities during experimental influenza in adults, and oseltamivir treatment appears to reduce the likelihood of otitis media in children with acute influenza. Investigational anti-HRV agents, including intranasal tremacamra, intranasal AG7088, and oral pleconaril, warrant study in this regard. Depending on the virus, early antiviral therapy has the potential to impact the risk of otitis media following respiratory tract infections.

Topics: Acetamides; Adult; Anti-Bacterial Agents; Antiviral Agents; Child; Child, Preschool; Clinical Trials as Topic; Eustachian Tube; Guanidines; Humans; Infant; Influenza A virus; Influenza B virus; Influenza, Human; Interferon-beta; Oseltamivir; Otitis Media; Otitis Media with Effusion; Picornaviridae Infections; Piperidines; Pressure; Pyrans; Pyridazines; Respiratory Tract Infections; Rhinovirus; Rimantadine; Sialic Acids; Superinfection; Virus Replication; Zanamivir

A randomized, double-blind, placebo-controlled trial assessed the therapeutic efficacy of intranasal pirodavir in naturally occurring rhinovirus colds. Adults with symptoms of < or = 2 days' duration were randomly assigned to intranasal sprays of pirodavir (2 mg per treatment) or placebo six times daily for 5 days. In people with laboratory-documented rhinovirus colds (53 in the pirodavir group, 55 in the placebo group), no significant differences in the resolution of respiratory symptoms were apparent between the groups. The median duration of illness was 7 days in each group. Similarly, scores for individual symptoms found no differences in favor of pirodavir during or after treatment. In contrast, reduced frequencies of rhinovirus shedding were observed in the pirodavir group on day 3 (70 versus 23%; P < 0.001) and day 5 (38 versus 12%; P = 0.002) but not after the cessation of treatment, on day 7 (19 versus 21%). No pirodavir-resistant viruses were recovered from treated individuals. The pirodavir group had higher rates of nasal dryness, blood in mucus, or unpleasant taste on several study days. In summary, intranasal sprays of pirodavir were associated with significant antiviral effects but no clinical benefit in treating naturally occurring rhinovirus colds.

Topics: Administration, Intranasal; Adolescent; Adult; Aged; Antiviral Agents; Common Cold; Double-Blind Method; Female; Humans; Male; Middle Aged; Patient Compliance; Piperidines; Pyridazines; Rhinovirus

Pirodavir (R77975) is a capsid-binding, antipicornaviral agent with in vitro activity against most rhinovirus (RV) serotypes. We conducted four double-blind, controlled trials to assess the efficacy of intranasal pirodavir in experimentally induced RV infection of susceptible volunteers. Intranasal pirodavir (2 mg per dose) or the hydroxypropyl-beta-cyclodextrin vehicle as a placebo was given by metered pump spray. In three prophylaxis trials, subjects were inoculated with RV within 10 min of the second and third doses. When sprays were given six times per day for a total of 25 doses, infection, detected by either virus shedding or seroconversion, developed in 100% of the 13 placebo-treated subjects and 58% of the 12 pirodavir-treated subjects (P = 0.015). Clinical colds developed in 54% of placebo-treated subjects and 8% of pirodavir-treated subjects during drug administration (efficacy = 85%, P = 0.03), although late-developing colds developed in several subjects in both groups. Significant reductions in morning symptom scores and in the frequency of abnormal middle-ear pressures were also found in the pirodavir group. In contrast, in two prophylaxis studies using three doses daily, no significant antiviral or clinical benefits were observed. When frequent sprays were initiated at 24 h after RV challenge, significant reductions in virus shedding but no clinical benefits were found. Intranasal pirodavir was generally well tolerated but was associated with an excess rate of transient unpleasant taste. The findings indicated that frequent intranasal sprays of pirodavir were effective in preventing experimentally induced RV illness.

Topics: Administration, Intranasal; Adolescent; Adult; Common Cold; Double-Blind Method; Humans; Middle Aged; Piperidines; Pyridazines

Enterovirus 71 (EV71) is a huge threat to the worldwide public health and there is no approved antiviral drug for EV71-induced disease therapy. Corydaline exists antiallergic and antinociceptive activities, but the anti-EV71 activity of corydaline is still not reported. In this study, corydaline could suppress the expression of viral structural and non-structural proteins. Furthermore, corydaline inhibits EV71 replication by suppressing the COX-2 expression and the phosphorylation of JNK MAPK and P38 MAPK but not ERK MAPK in vitro. Based on these findings, corydaline could be a potential lead or supplement for the development of new anti-EV71 agents in the future.

Topics: Antiviral Agents; Berberine Alkaloids; Cyclooxygenase 2 Inhibitors; Enterovirus A, Human; Humans; JNK Mitogen-Activated Protein Kinases; Molecular Structure; p38 Mitogen-Activated Protein Kinases; Piperidines; Pyridazines; Signal Transduction; Virus Replication

Compounds were evaluated for antiviral activity in rhabdomyosarcoma (RD) cells against a recent 2014 clinical isolate of enterovirus D68 (EV-D68), a 1962 strain of EV-68D, rhinovirus 87 (RV-87, serologically the same as EV-D68), and enterovirus 71 (EV-71). Test substances included known-active antipicornavirus agents (enviroxime, guanidine HCl, pirodavir, pleconaril, and rupintrivir), nucleobase/nucleoside analogs (3-deazaguanine and ribavirin), and three novel epidithiodiketopiperazines (KCN-2,2'-epi-19, KCN-19, and KCN-21). Of these, rupintrivir was the most potent, with 50% inhibition of viral cytopathic effect (EC50) and 90% inhibition (EC90) of virus yield at 0.0022-0.0053 μM against EV-D68. Enviroxime, pleconaril and the KCN compounds showed efficacy at 0.01-0.3 μM; 3-deazaguanine and pirodavir inhibited EV-D68 at 7-13 μM, and guanidine HCl and ribavirin were inhibitory at 80-135 μM. Pirodavir was active against EV-71 (EC50 of 0.78 μM) but not against RV-87 or EV-D68, and all other compounds were less effective against EV-71 than against RV-87 and EV-D68. The most promising compound inhibiting both virus infections at low concentrations was rupintrivir. Antiviral activity was confirmed for the ten compounds in virus yield reduction (VYR) assays in RD cells, and for enviroxime, guanidine HCl, and pirodavir by cytopathic effect (CPE) assays in A549, HeLa-Ohio-1, and RD cells. These studies may serve as a basis for further pre-clinical discovery of anti-enterovirus inhibitors. Furthermore, the antiviral profiles and growth characteristics observed herein support the assertion that EV-D68 should be classified together with RV-87.

Topics: A549 Cells; Antimetabolites; Antiviral Agents; Benzimidazoles; Enterovirus A, Human; Enterovirus D, Human; Guanine; HeLa Cells; Humans; Oxadiazoles; Oxazoles; Oximes; Picornaviridae; Piperazines; Piperidines; Pyridazines; Rhabdomyosarcoma; Rhinovirus; Ribavirin; Sulfonamides

The Enterovirus genus of the Picornaviridae is represented by several viral pathogens that are associated with human disease, namely Poliovirus 1, Enterovirus 71 and Rhinoviruses. Enterovirus 71 has been associated with encephalitis, while Rhinoviruses are a major cause of asthma exacerbations and chronic obstructive pulmonary disease. Based on the structure of both pleconaril and pirodavir, we previously synthesized some original compounds as potential inhibitors of Rhinovirus replication.. These compounds were explored for in vitro antiviral potential on other human pathogenic Enteroviruses, namely Enterovirus 71 on rhabdo-myosarcoma cells, Coxsackievirus B3 on Vero cells, Poliovirus 1 and Echovirus 11 on BGM cells.. Activity was confirmed for compound against Rhinovirus 14. Furthermore, few compounds showed a cell-protective effect on Enterovirus 71, presented a marked improvement as compared to the reference drug pleconaril for inhibitory activity on both Enterovirus 71 and Poliovirus 1. The most striking observation was the clear cell protective effect for the set of analogues in a virus-cell-based assay for Echovirus 11 with an effective concentration (EC50) as low as 0.3 µM (Selectivity index or SI = 483), and selectivity indexes greater than 857 (EC50 = 0.6 µM) and 1524 (EC50 = 0.33 µM).. Some of the evaluated compounds showed potent and selective antiviral activity against several enterovirus species, such as Enterovirus 71 (EV-A), Echovirus 11 (EV-B), and Poliovirus 1 (EV-C). This could be used as a starting point for the development of other pleconaril/pirodavir-like enterovirus inhibitors with broad-spectrum activity and improved effects as compared to the reference drugs.

Topics: Antiviral Agents; Dose-Response Relationship, Drug; Enterovirus; Microbial Sensitivity Tests; Oxadiazoles; Oxazoles; Piperidines; Pyridazines; Structure-Activity Relationship

Rhinovirus infections do not only cause common colds, but may also trigger severe exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Even though rhinoviruses have been the focus of extensive drug development efforts in the past, an anti-rhinoviral drug still has to make it to the market. In the past, the viral capsid protein VP1 has been shown to be an important target for the development of antiviral molecules. Furthermore, many different chemical scaffolds appear to possess the properties that are required to inhibit virus replication by this mechanism of action. I-6602, an analogue of the rhinovirus inhibitor pirodavir, was previously identified as a potent inhibitor of rhinovirus infection. Here, we describe the antiviral activity of its analogue ca603, a molecule with a modified linker structure, and corroborate its mechanism of action as a capsid binder.. The molecule ca603 shows antiviral activity against a panel of rhino-and enteroviruses. Cross-resistance is observed against viruses with mutations that render them resistant to the inhibitory effect of the capsid binder pleconaril and thermostability assays demonstrate that the compound binds and stabilizes the viral capsid. Binding of the molecule to the VP1 protein is corroborated by in silico modeling.. It is confirmed that ca603 inhibits rhinovirus replication by interaction with the VP1 protein and, by this, allows to further expand the chemical diversity of capsid-binding molecules.

Topics: Antiviral Agents; Capsid Proteins; Humans; Microbial Sensitivity Tests; Oxadiazoles; Oxazoles; Piperidines; Protein Binding; Pyridazines; Rhinovirus

Human rhinovirus type C (HRV-C) is a newly discovered enterovirus species frequently associated with exacerbation of asthma and other acute respiratory conditions. Until recently, HRV-C could not be propagated in vitro, hampering in-depth characterization of the virus replication cycle and preventing efficient testing of antiviral agents. Herein we describe several subgenomic RNA replicon systems and a cell culture infectious model for HRV-C that can be used for antiviral screening. The replicon constructs consist of genome sequences from HRVc15, HRVc11, HRVc24, and HRVc25 strains, with the P1 capsid region replaced by a Renilla luciferase coding sequence. Following transfection of the replicon RNA into HeLa cells, the constructs produced time-dependent increases in luciferase signal that can be inhibited in a dose-dependent manner by known inhibitors of HRV replication, including the 3C protease inhibitor rupintrivir, the nucleoside analog inhibitor MK-0608, and the phosphatidylinositol 4-kinase IIIβ (PI4K-IIIβ) kinase inhibitor PIK93. Furthermore, with the exception of pleconaril and pirodavir, the other tested classes of HRV inhibitors blocked the replication of full-length HRVc15 and HRVc11 in human airway epithelial cells (HAEs) that were differentiated in the air-liquid interface, exhibiting antiviral activities similar to those observed with HRV-16. In summary, this study is the first comprehensive profiling of multiple classes of antivirals against HRV-C, and the set of newly developed quantitative HRV-C antiviral assays represent indispensable tools for the identification and evaluation of novel panserotype HRV inhibitors.

Topics: Antiviral Agents; Common Cold; Dose-Response Relationship, Drug; HeLa Cells; Humans; In Vitro Techniques; Isoxazoles; Oxadiazoles; Oxazoles; Phenylalanine; Piperidines; Pyridazines; Pyrrolidinones; Replicon; Rhinovirus; RNA, Viral; Tubercidin; Valine; Virus Replication

The human common cold, which is a benign disease caused by the Rhinoviruses, generally receives palliative symptomatic treatments, since no specific therapy against any of these viruses currently exists. In this work, some original synthetic compounds were produced and tested, in order to find non-toxic substances with an improved protection index (PI) for infected cells, as compared to reference drugs such as Pirodavir. We designed a series of novel molecules with a double oxygen in the central hydrocarbon chain and some modifications of the lateral methylisoxazole and propoxybenzoate moieties of lead compound 6602 (ethyl 4-{3-[2-(3-methyl-1,2-isoxazol-5-yl)ethoxy]propoxy}benzoate). It was found that most of these substances were actually less toxic than Pirodavir; in addition, the new molecule indicated as 8c was more than 30 times less toxic than Pirodavir, about twice as active on the group A strain of Rhinovirus HRV14, and even four times more effective on the group B strain HRV39, as compared to Pirodavir's PI.

Topics: Antiviral Agents; Benzoic Acid; Benzoxazoles; Cell Survival; Drug Design; HeLa Cells; Humans; Piperidines; Pyridazines; Rhinovirus; Serotyping; Structure-Activity Relationship

A novel small molecule, H1PVAT, was identified as a potent and selective inhibitor of the in vitro replication of all three poliovirus serotypes, whereas no activity was observed against other enteroviruses. Time-of-drug-addition studies revealed that the compound interfered with an early stage of virus replication. Four independently-selected H1PVAT-resistant virus variants uniformly carried the single amino acid substitution I194F in the VP1 capsid protein. Poliovirus type 1 strain Sabin, reverse-engineered to contain this substitution, proved to be completely insensitive to the antiviral effect of H1PVAT and was cross-resistant to the capsid-binding inhibitors V-073 and pirodavir. The VP1 I194F mutant had a smaller plaque phenotype than wild-type virus, and the amino acid substitution rendered the virus more susceptible to heat inactivation. Both for the wild-type and VP1 I194F mutant virus, the presence of H1PVAT increased the temperature at which the virus was inactivated, providing evidence that the compound interacts with the viral capsid, and that capsid stabilization and antiviral activity are not necessarily correlated. Molecular modeling suggested that H1PVAT binds with high affinity in the pocket underneath the floor of the canyon that is involved in receptor binding. Introduction of the I194F substitution in the model of VP1 induced a slight concerted rearrangement of the core β-barrel in this pocket, which disfavors binding of the compound. Taken together, the compound scaffold, to which H1PVAT belongs, may represent another promising class of poliovirus capsid-binding inhibitors next to V-073 and pirodavir. Potent antivirals against poliovirus will be essential in the poliovirus eradication end-game.

Topics: Amino Acid Substitution; Animals; Antiviral Agents; Base Sequence; Binding Sites; Capsid; Capsid Proteins; Cell Line, Tumor; Chlorocebus aethiops; Drug Resistance, Viral; HeLa Cells; Humans; Models, Molecular; Piperidines; Poliomyelitis; Poliovirus; Pyrazoles; Pyridazines; Pyrimidines; RNA, Viral; Sequence Analysis, RNA; Virus Replication

Antivirals against enterovirus 71 (EV71) are urgently needed. We demonstrate that the novel enteroviral protease inhibitor (PI) SG85 and capsid binder (CB) vapendavir efficiently inhibit the in vitro replication of 21 EV71 strains/isolates that are representative of the different genogroups A, B, and C. The PI rupintrivir, the CB pirodavir, and the host-targeting compound enviroxime, which were included as reference compounds, also inhibited the replication of all isolates. Remarkably, the CB compound pleconaril was devoid of any anti-EV71 activity. An in silico docking study revealed that pleconaril-unlike vapendavir and pirodavir-lacks essential binding interactions with the viral capsid. Vapendavir and SG85 (or analogues) should be further explored for the treatment of EV71 infections. The data presented here may serve as a reference when developing yet-novel inhibitors.

Topics: Antiviral Agents; Benzimidazoles; Capsid; Capsid Proteins; Drug Resistance, Viral; Enterovirus A, Human; Enterovirus Infections; Isoxazoles; Molecular Docking Simulation; Oximes; Phenylalanine; Piperidines; Protease Inhibitors; Protein Binding; Pyridazines; Pyrrolidinones; Sulfonamides; Valine; Virus Replication

A series of novel N-phenylbenzamide derivatives were synthesized and their anti-EV 71 activities were assayed in vitro. Among the compounds tested, 3-amino-N-(4-bromophenyl)-4-methoxybenzamide (1e) was active against the EV 71 strains tested at low micromolar concentrations, with IC50 values ranging from 5.7 ± 0.8-12 ± 1.2 μM, and its cytotoxicity to Vero cells (TC50 = 620 ± 0.0 μM) was far lower than that of pirodavir (TC50 = 31 ± 2.2 μM). Based on these results, compound 1e is a promising lead compound for the development of anti-EV 71 drugs.

Topics: Animals; Antiviral Agents; Benzamides; Chlorocebus aethiops; Drug Evaluation, Preclinical; Enterovirus; Inhibitory Concentration 50; Piperidines; Pyridazines; Structure-Activity Relationship; Vero Cells

Picornaviridae represent a very large family of small RNA viruses, some of which are the cause of important human and animal diseases. Since no specific therapy against any of these viruses currently exists, palliative symptomatic treatments are employed. The early steps of the picornavirus replicative cycle seem to be privileged targets for some antiviral compounds like disoxaril and pirodavir. Pirodavir's main weakness is its cytotoxicity on cell cultures at relatively low doses. In this work some original synthetic compounds were tested, in order to find less toxic compounds with an improved protection index (PI) on infected cells. Using an amino group to substitute the oxygen atom in the central chain, such as that in the control molecule pirodavir, resulted in decreased activity against Rhinoviruses and Polioviruses. The presence of an -ethoxy-propoxy- group in the central chain (as in compound I-6602) resulted in decreased cell toxicity and in improved anti-Rhinovirus activity. This compound actually showed a PI >700 on HRV14, while pirodavir had a PI of 250. These results demonstrate that modification of pirodavir's central hydrocarbon chain can lead to the production of novel derivatives with low cytotoxicity and improved PI against some strains of Rhinoviruses.

Topics: Antiviral Agents; Cell Line, Tumor; Cell Survival; HeLa Cells; Humans; Piperidines; Pyridazines; Rhinovirus

Human rhinoviruses (HRVs) are the causative pathogens in more than half of viral upper respiratory tract infections. Currently, no antiviral agents that are active against HRVs are available for clinical use. Because only higher primates are susceptible to HRVs, the screening of new drug is most commonly based on the cell line model. In this study, isolated rabbit airway smooth muscles (ASM) tissue model has been established, and the airway responsiveness with different treatment has been examined. Relative to control tissues, the maximal constrictor (Tmax) response to ACh increased significantly 150% in ASM inoculated with HRV, and relaxation to isoproterenol has been attenuated to 63%. And the abnormal responsiveness can be inhibited in presence of pretreatment with several new compounds which have been exhibited effective anti-HRV activity on cell lines. The results demonstrate that the established ASM model will be applied to screening the anti-HRVs drugs.

Topics: Acetylcholine; Animals; Antiviral Agents; Cytopathogenic Effect, Viral; HeLa Cells; Humans; Isoproterenol; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Piperidines; Pyridazines; Rabbits; Rhinovirus; Trachea

Topics: Deuterium Oxide; Drug Stability; Excipients; Humans; Piperidines; Pyridazines; Smallpox Vaccine

Picornaviruses (PV) include human rhinovirus (HRV), the primary cause of the common cold, and the enteroviruses (EV), which cause serious diseases such as poliomyelitis, meningoencephalitis, and systemic neonatal disease. Although no compounds for PV infections have been approved in the United States, pirodavir was one of the most promising capsid-binding compounds to show efficacy in human clinical trials for chemoprophylaxis of the common cold. Susceptibility to hydrolysis precluded its use as an oral agent. We have developed orally bioavailable pyridazinyl oxime ethers that are as potent as pirodavir. Compounds BTA39 and BTA188 inhibited a total of 56 HRV laboratory strains and three clinical isolates as determined by neutral red uptake assay. At concentrations of <100 nM, BTA39 inhibited 69% of the HRV serotypes and isolates evaluated, BTA188 inhibited 75%, and pirodavir inhibited 59% of the serotypes and isolates. The 50% inhibitory concentrations (IC(50)s) for the two compounds ranged from 0.5 nM to 6,701 nM. The compounds also inhibited EV, including coxsackie A and B viruses (IC(50) = 773 to 3,608 nM) and echoviruses (IC(50) = 193 to 5,155 nM). BTA39 only inhibited poliovirus strain WM-1 at 204 nM, and BTA188 only inhibited poliovirus strain Chat at 82 nM. EV 71 was inhibited by BTA39 and BTA188, with IC(50)s of 1 and 82 nM, respectively. Both compounds were relatively nontoxic in actively growing cells (50% cytotoxic doses, >/=4,588 nM). These data suggest that these oxime ethers warrant further investigation as potential agents for treating selected PV infections.

Topics: Animals; Antiviral Agents; Biological Availability; Capsid; Cell Line; Cytopathogenic Effect, Viral; Enterovirus; Enterovirus B, Human; Haplorhini; Humans; KB Cells; Neutral Red; Oximes; Picornaviridae; Piperidines; Pyridazines; Rhinovirus; Virus Replication

A series of capsid-binding compounds was screened against human rhinovirus (HRV) using a CPE based assay. The ethyl oxime ether 14 was found to have outstanding anti-HRV activity (median IC(50) 4.75 ng/mL), and unlike the equivalent ethyl ester compound 3 (Pirodavir), it has good oral bioavailability, making it a promising development candidate. Compound 14 illustrates that an oxime ether group can act as a metabolically stable bioisostere for an ester functionality.

Topics: Administration, Oral; Animals; Antiviral Agents; Biological Availability; Capsid; Cell Line; Ethers; Female; Humans; Male; Mice; Oximes; Piperidines; Protein Binding; Pyridazines; Rats; Rhinovirus; Structure-Activity Relationship

In a cell-free system derived from uninfected HeLa cells and programmed with poliovirus RNA, an unbalance between the different replication steps is observed. After programming, the vRNA is exclusively used as a template for viral translation. It takes hours before there is a switch from protein synthesis to RNA replication. This is probably the reason for the inefficient production of infectious virus (compared to the synthesis in infected cells). If, however, the cell-free system is programmed with vRNA and with a mRNA coding for the viral protein 3CD, an increase in vRNA synthesis is found early post-programming, resulting in a better balance of protein synthesis and RNA synthesis and an increased virus yield of at least 2 log10. These data show that a balance between translation, RNA replication and packaging is required to allow efficient viral proliferation. The virus yield could be increased by a further log10 by the addition of pirodavir (a capsid stabiliser) and 10% of rabbit reticulocyte lysate to the cell-free system.

Topics: 3C Viral Proteases; Animals; Cell-Free System; Cysteine Endopeptidases; HeLa Cells; Humans; Piperidines; Poliovirus; Pyridazines; Reticulocytes; RNA, Viral; Time Factors; Viral Proteins; Virus Replication

The oral polio vaccine is the least stable vaccine of the common childhood vaccines. Two different inactivation mechanisms are responsible for the thermolability of the vaccine, i.e. denaturation of the viral capsid and degradation of the viral RNA within the capsid. Pirodavir, a capsid-binding compound, inhibits the viral capsid thermodenaturation. In this paper we show that deuterium oxide is able to stabilise the viral RNA against thermodegradation and that a combination of pirodavir and deuterium oxide leads to an additive effect indicating that both stabilisers work indeed on different inactivation mechanisms. Furthermore, it is shown that the variation in thermostability of the different vaccine strains is due to the different thermostability of their capsids.

Topics: Capsid; Child; Deuterium Oxide; Drug Stability; Drug Storage; Humans; In Vitro Techniques; Piperidines; Poliovirus; Poliovirus Vaccine, Oral; Preservatives, Pharmaceutical; Protein Denaturation; Pyridazines; RNA Stability; RNA, Viral; Species Specificity; Temperature

The Children's Vaccine Initiative established a Product Development Group in 1991 to help in the development of oral poliovaccines of improved thermostability. Under the auspices of this group, the effect of different stabilizers on the loss of infectivity of commercial monovalent type 3 live poliovaccine bulks at 37 degrees C, 42 degrees C and 45 degrees C has been examined. The only effective conditions were the maintenance of the pH at 6.5, and the use of deuterium oxide at a concentration of 90%. All other additives, including detergents, albumin, antiviral compounds, sucrose and magnesium chloride, alone or in combination either had no effect or increased the rate of loss of infectivity.

Topics: Antiviral Agents; Cells, Cultured; Drug Stability; Drug Storage; Humans; Hydrogen-Ion Concentration; Piperidines; Poliovirus; Poliovirus Vaccine, Oral; Polysorbates; Preservatives, Pharmaceutical; Pyridazines; Surface-Active Agents

Polioviral genes coding for P1, the precursor for the structural proteins, and 3CD, the viral protease, were cloned in a Saccharomyces cerevisiae inducible expression system. N-antigenic empty capsids could be isolated from the yeast cell extract provided that pirodavir, a capsid-binding compound and capsid stabilizer, was added during the induction period and during purification. Purification was by immunoaffinity chromatography. The purified empty capsids had the same immunogenicity as poliovirus virions. The techniques described might be useful for the production of new non-infectious vaccines.

Topics: 3C Viral Proteases; Antigens, Viral; Capsid; Chromatography, Affinity; Cloning, Molecular; Cysteine Endopeptidases; Genes, Viral; Piperidines; Poliovirus; Poliovirus Vaccine, Inactivated; Preservatives, Pharmaceutical; Pyridazines; Recombinant Proteins; Saccharomyces cerevisiae; Viral Proteins; Viral Structural Proteins

Two hundred and forty pyridazinamine derivates were assayed for their ability to stabilize the Oral Polio Vaccine (OPV). Of these, pirodavir (R 77975) was selected for vaccine potency tests. Although pirodavir turned out to be an effective stabilizer of OPV, the protection induced by pirodavir was not better, nor additive to the effect by MgCl2 1 M, the usual stabilizer. Both stabilizers were effective in preventing damage to the viral proteins, but could not prevent damage of viral RNA.

Topics: Antibodies, Viral; Antigens, Viral; Capsid; Cells, Cultured; Cytopathogenic Effect, Viral; Drug Stability; Drug Synergism; Hot Temperature; Magnesium Chloride; Piperidines; Poliovirus; Poliovirus Vaccine, Oral; Preservatives, Pharmaceutical; Protein Binding; Protein Conformation; Protein Denaturation; Pyridazines; RNA, Viral

Heating the Sabin strains of poliovirus at 42 to 45 degrees C caused inactivation, loss of native antigen, and release of the viral RNA (vRNA). The loss of virion infectivity exceeded the loss of vRNA infectivity (as measured by transfection) by roughly 2 log10. Pirodavir inhibited the loss of native antigen and RNA release and reduced the loss of virion infectivity to the same level as the loss of vRNA infectivity. Thermoinactivation thus involves an RNA and a protein component, and pirodavir protected only against the latter.

Topics: Antiviral Agents; Cell Line; Centrifugation, Density Gradient; Hot Temperature; Humans; Kidney; Methionine; Piperidines; Poliovirus; Poliovirus Vaccine, Oral; Pyridazines; RNA, Viral; Transfection; Uridine; Vaccines, Inactivated; Viral Plaque Assay; Viral Proteins

Two hundred forty pyridazinamine derivatives were tested for the ability to stabilize the antigenicity and infectivity of oral poliovirus vaccine subjected to 45 degrees C for 2 h. Seven compounds stabilized the antigenicity of all three vaccine strains and neutralized the viral particles in a way that is reversible by dilution. Of these, R 77975 (pirodavir) was selected for vaccine potency tests. Sabin type 2 and type 3 strains were subjected to 4, 25, 42, and 45 degrees C for 1 week in the presence and absence of R 77975. Although R 77975 particularly stabilized the infectivity of the most thermolabile vaccine strain (Sabin type 3), the protection did not exceed that of 1 M MgCl2. When virus was inactivated in the absence of R 77975, the native or N antigenicity changed in H antigenicity. However, in the presence of the capsid-binding compound, N antigenicity was preserved in particles that had lost infectivity.

Topics: Antiviral Agents; Drug Stability; HeLa Cells; Humans; Piperidines; Poliovirus; Poliovirus Vaccine, Oral; Pyridazines

Pirodavir (R 77975) is the prototype of a novel class of broad-spectrum antipicornavirus compounds. Although its predecessor, R 61837, a substituted phenyl-pyridazinamine, was effective in inhibiting 80% of 100 serotypes tested (EC80) at concentrations above 32 micrograms/ml, pirodavir inhibits the same percentage of viruses at 0.064 micrograms/ml. Whereas R 61837 was active almost exclusively against rhinovirus serotypes of antiviral group B, pirodavir is broad spectrum in that it is highly active against both group A and group B rhinovirus serotypes. Pirodavir is also effective in inhibiting 16 enteroviruses, with an EC80 of 1.3 micrograms/ml. Susceptible rhinovirus serotypes were rendered noninfectious by direct contact with the antiviral compound. Their infectivity was not restored by dilution of virus-drug complexes, but was regained by organic solvent extraction of the compound for most serotypes. Neutralized viruses became stabilized to acid and heat, strongly suggesting a direct interaction of the compounds with viral capsid proteins. Mutants resistant to R 61837 (up to 85 times the MIC) were shown to bear some cross-resistance (up to 23 times the MIC) to the new compound, indicating that pirodavir also binds into the hydrophobic pocket beneath the canyon floor of rhinoviruses. Pirodavir acts at an early stage of the viral replication cycle (up to 40 min after infection) and reduces the yield of selected rhinoviruses 1,000- to 100,000-fold in a single round of replication. The mode of action appears to be serotype specific, since pirodavir was able to inhibit the adsorption of human rhinovirus 9 but not that of human rhinovirus 1A. Pirodavir is a novel capsid-binding antipicornavirus agent with potent in vitro activity against both group A and group B rhinovirus serotypes.

Topics: Antiviral Agents; Enterovirus; Microbial Sensitivity Tests; Piperidines; Pyridazines; Rhinovirus