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There is insufficient information about combination therapy with approved anti-influenza agents. We tested combinations that paired a neuraminidase (NA) inhibitor (zanamivir, oseltamivir carboxylate, or peramivir) with rimantadine against infection of MDCK cells with H1N1 and H3N2 subtypes of influenza A virus and characterized their mode of interaction. When reduction of extracellular virus was analyzed by individual regression models and three-dimensional representations of the data, all three combinations showed additive and synergistic effects with no cytotoxicity. Maximum synergy against A/New Caledonia/20/99 (H1N1) virus infection was observed with <2.5 microM rimantadine paired with low concentrations of NA inhibitors. All combinations reduced the extracellular yield of A/Panama/2007/99 (H3N2) influenza virus synergistically. However, our findings were different for the cell-associated virus yield. At some drug concentrations, the yield of cell-associated virus was inhibited antagonistically. Therefore, the method of analysis can be a crucial factor in evaluating the interactions of drugs with different mechanisms. We hypothesize that assays based on cell-associated virus yield may underestimate the efficacies of drug combinations that include an NA inhibitor. Taken together, our results suggest that regimens that combine NA inhibitors and rimantadine exert synergistic anti-influenza effects in vitro. These findings provide baseline information for therapeutic testing of the drug combinations in vivo.

Background: Antiviral agents could play a significant role in the response to a future influenza pandemic, especially if an effective vaccine is unavailable. There is, however, a limited availability of antiviral drugs, as was the case during the A/Fujian(H3N2) epidemic in the 2003-2004 season. There are major differences among the available antiviral agents in terms of clinical pharmacology, adverse effects and resistance profiles, all of which must be considered when selecting agents for pandemic use and stockpiling. The logistic issues involved in delivering the drug to large populations must also be considered. The M2 ion channel inhibitors amantadine and rimantadine are partially effective for chemoprophylaxis of pandemic influenza, and when used for early treatment they provide some symptom relief. Interpandemic studies demonstrate that the neuraminidase inhibitors would be effective for both prevention and treatment of influenza., Conclusion: Given the limited supply and the previously demonstrated inability of the manufacturing sector to meet surging needs, an effective antiviral response could not currently be launched in the United States. Antiviral agents could be important in managing and treating pandemic influenza and reduce lower respiratory complications and hospitalizations.

Effective antiviral agents are urgently needed to combat the possible return of severe acute respiratory syndrome (SARS). Commercial antiviral agents and pure chemical compounds extracted from traditional Chinese medicinal herbs were screened against 10 clinical isolates of SARS coronavirus by neutralisation tests with confirmation by plaque reduction assays. Interferon-beta-1a, leukocytic interferon-alpha, ribavirin, lopinavir, rimantadine, baicalin and glycyrrhizin showed antiviral activity. The two interferons were only active if the cell lines were pre-incubated with the drugs 16 h before viral inoculation. Results were confirmed by plaque reduction assays. Antiviral activity varied with the use of different cell lines. Checkerboard assays for synergy were performed showing combinations of interferon beta-1a or leukocytic interferon-alpha with ribavirin are synergistic. Since the clinical and toxicity profiles of these agents are well known, they should be considered either singly or in combination for prophylaxis or treatment of SARS in randomised placebo controlled trials in future epidemics.

The embryonated hen's egg can be infected with influenza A virus in laboratory experiments leading to death of chick embryos within 8 days post infection. This model can be used for rapid and reliable in vivo evaluation of potential anti-influenza inhibitors. It offers a realistic alternative to experiments with small laboratory rodents. By an example of treatment with the antiviral drugs amantadine (CAS 665-66-7), rimantadine (CAS 1501-84-4) and zanamivir (CAS 139110-80-8), it could be demonstrated that chick embryos survive a lethal influenza A virus infection when these drugs are administered immediately before or after the infective agent is inoculated. In conclusion, the use of influenza virus-infected chick embryos for evaluation of new antiviral substances can lead to a considerable reduction in the number of small laboratory rodents required.

2-(1-Adamantyl)pyrrolidines 6, 7, 2-(1-adamantyl)piperidines 10, 12a-c, 15a,b and 2-(1-adamantyl)hexahydroazepines 19, 21, 22 were synthesized and tested for their antiviral activity against influenza A, B viruses and the human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2). The synthetic procedure followed for the preparation of the parent piperidine 10 represents a general method for the synthesis of 2-alkyl- or cycloalkyl-substituted piperidine alkaloids. Parent aminoadamantanes 6, 10 and 19 contain the 1-aminoethyl pharmacophore group of rimantadine drug 2, extended into a saturated nitrogen heterocycle: pyrrolidine, piperidine and hexahydroazepine, respectively. The ring size effect in anti-influenza A activity was investigated. Rimantadine analogues 6 and 10 were, respectively, 6- and 4-fold more active than the drug Rimantadine 2, whereas the hexahydroazepine derivative 19 was inactive. Thus, enlargement from a 5-(pyrrolidine)- or 6-(piperidine)- to a 7-(hexahydroazepine)- membered heterocyclic ring dramatically reduced the anti-influenza virus A activity. Substitution of piperidine 10 with a dialkyaminoethyl group led to the active compounds 15a and 15b: compound 15a was active against influenza A virus whereas both 15a and 15b were active against HIV-1.

New water-soluble antiviral chemical agents, containing 10 to 30% of adamantane derivatives (amino-, aminopropyl-adamantane-, aminomethyl- and rimantadine), which were conjugated with polycarboxylic matrixes of the divinyl ether and maleic anhydride copolymers (DIVEMA), were developed. The polymeric drugs exhibited a low cytotoxicity (4 to 10 times less than rimantadine) and a wide spectrum of antiviral activity against influenza viruses, including both the remantadine-resistant strains of A/PR/8/34 (H1N1) and the B/Saint-Petersburg strain/71/77 as well as against herpes viruses of type 1, parainfluenza viruses of types 1 and 3 and RS-virus. A reduction of the viral infection titer in their reproduction in sensitive cells' cultures was more than 2.0 Ig ID50. Complete inhibition of viral-specific syntheses, registered by immune-enzyme assay (IEA) and by hemagglutination test was observed at low infection doses ranging from 1 to 100 ID50. The efficiency of the antiviral effect depends on a drug's molecular weight and a structure of chemical bonds between the adamantane nucleus and the polymeric matrix.

There is a lack of information in the medical chemistry literature concerning the anti-influenza A activity of the drug rimantadine's 2-isomer (2-rimantadine). We now present results showing that, although 2-adamantanamine (2-amantadine) 3 is only moderately active, some 2-rimantadine analogues are effective anti-influenza A virus agents in vitro. The 2-rimantadine analogues and their spirocyclobutane and spirocyclopentane congeners were synthesized through interesting routes. The 2-rimantadine analogues were 2-4 times more potent than rimantadine 2 against influenza virus A H2N2 strain; their spirocyclobutane congeners proved equally active to rimantadine 2. Two compounds exhibited a similar activity and one of the compounds was was fourfold more potent than rimantadine 2 against H3N2 strain.

The combined protective effect of a novel naturally glycosylated Cu/Zn-containing superoxide dismutase, produced by the fungus Humicula lutea (HL-SOD) strain 103, and the selective anti-influenza drug rimantadine hydrochloride (Rim) was evaluated in experimental virus infection in mice, induced with influenza virus A/Aichi/2/68 (H3N2). A combined application of HL-SOD and Rim in doses, which by themselves did not protect significantly mice against the infection, resulted in a synergistically increased protection, determined on the basis of protective indices. Lung virus titers, lung weights and consolidation and mortality rates were all decreased significantly, while survival times were prolonged.

The 1918 influenza pandemic caused more than 20 million deaths worldwide. Thus, the potential impact of a re-emergent 1918 or 1918-like influenza virus, whether through natural means or as a result of bioterrorism, is of significant concern. The genetic determinants of the virulence of the 1918 virus have not been defined yet, nor have specific clinical prophylaxis and/or treatment interventions that would be effective against a re-emergent 1918 or 1918-like virus been identified. Based on the reported nucleotide sequences, we have reconstructed the hemagglutinin (HA), neuraminidase (NA), and matrix (M) genes of the 1918 virus. Under biosafety level 3 (agricultural) conditions, we have generated recombinant influenza viruses bearing the 1918 HA, NA, or M segments. Strikingly, recombinant viruses possessing both the 1918 HA and 1918 NA were virulent in mice. In contrast, a control virus with the HA and NA from a more recent human isolate was unable to kill mice at any dose tested. The recombinant viruses were also tested for their sensitivity to U.S. Food and Drug Administration-approved antiinfluenza virus drugs in vitro and in vivo. Recombinant viruses possessing the 1918 NA or both the 1918 HA and 1918 NA were inhibited effectively in both tissue culture and mice by the NA inhibitors, zanamivir and oseltamivir. A recombinant virus possessing the 1918 M segment was inhibited effectively both in tissue culture and in vivo by the M2 ion-channel inhibitors amantadine and rimantadine. These data suggest that current antiviral strategies would be effective in curbing the dangers of a re-emergent 1918 or 1918-like virus.

Traditional methods used to monitor influenza infection typically require 2-5 days to perform, prompting a need for more rapid and quantitative methods for monitoring viral infection in 96-well formats. Such assays would find application in high-throughput screening for novel antiviral agents. A new method, based on branched DNA (bDNA) technology, is described for the specific detection of negative strand RNA of influenza A strains using a set of oligonucleotides designed for the A/PR/8/34 nucleoprotein (NP) transcript. By detecting the genomic strand, this signal amplification assay is appropriate for monitoring the kinetics of viral replication. Assay performance was monitored following infection of MDCK cells. The assay exhibited high reproducibility, good sensitivity over a range of multiplicity of infection and has a lower limit of detection of approximately 5 x 10 (5) RNA copies. Designed to quantitate the H1N1 strain A/PR/8/34, the assay also detects other influenza A subtypes, but not the evolutionarily more distant strain B/Yamagata/16/88. Validation as an antiviral assay was demonstrated with two influenza antivirals, zanamivir and rimantadine. The EC(50) values calculated following bDNA detection for zanamivir (265 nM) and rimantadine (9.4 microg/ml) in A/PR/8/34 infection correlate closely to data previously reported from visual CPE determinations, neutral red dye uptake and plaque assays, respectively. The advantages over the more time-consuming traditional assays suggest that the influenza bDNA assay is applicable to rapid screening of compound collections for antiviral activity.

Chemical synthesis of 3-substituted analogues of remantadine is described. Derivatives IIIb and IIIc when compared with remantadine had not only potent activity against ethalon herpes simplex type 1 virus strain but also were active against herpes virus resistant to aciclovir. Compound IIIc demonstrated virulecidal effect. Combination of IIIc + aciclovir had additive effect against ethalon herpes simplex type 1 virus strain. Investigated 3-substituted analogues demonstrated low activity in the model system of influenzae virus. No antiviral activity was demonstrated in the model system of Syndbys virus (though compounds were evaluated in subtoxic concentrations).

Influenza virus infection accounts for significant morbidity, mortality, and healthcare expenditures among persons worldwide. Approximately 20,000 to 40,000 people in the US die each year as a result of influenza. Individuals most susceptible to adverse outcomes include the elderly and those with asthma, chronic obstructive pulmonary disease (COPD), heart disease, renal failure, malignancy, or immunosuppression. Prior to the AIDS epidemic, underlying respiratory disease was the greatest risk factor for influenza-related hospitalization ranking third to heart disease and malignancy for risk of mortality. Although the influenza vaccine can help prevent pneumonia and hospitalization, it is limited by less than ideal immunization rates and the possibility of viral antigenic shifts that render the vaccine ineffective. Pharmacologic interventions play an important role in the management of influenza virus infection by shortening the duration of symptoms. The advent of the neuraminidase inhibitors (NAIs) zanamivir and oseltamivir has significantly affected the treatment of influenza. Unlike NAIs, the older therapeutic options amantadine and rimantadine may cause significant central nervous system adverse effects. In addition, amantadine and rimantadine are not active against influenza B viruses, whereas NAIs are active against both influenza A and B. Post-marketing surveillance of the NAIs has revealed that bronchospasm may occur in patients with underlying respiratory disease treated with the NAI zanamivir. Recent data suggest zanamivir is effective in patients with underlying respiratory disease, but the data are insufficient to elucidate the true risk of bronchospasm. Based on post-marketing reports, zanamivir should be used with caution in patients with asthma or COPD. Although oseltamivir has not been associated with any significant respiratory adverse effects, no data exist on the safety and efficacy of this NAI in patients with underlying respiratory disease.

Antiviral agents could potentially play a major role in the initial response to pandemic influenza, particularly with the likelihood that an effective vaccine is unavailable, by reducing morbidity and mortality. The M2 inhibitors are partially effective for chemoprophylaxis of pandemic influenza and evidence from studies of interpandemic influenza indicate that the neuraminidase inhibitors would be effective in prevention. In addition to the symptom benefit observed with M2 inhibitor treatment, early therapeutic use of neuraminidase inhibitors has been shown to reduce the risk of lower respiratory complications. Clinical pharmacology and adverse drug effect profiles indicate that the neuraminidase inhibitors and rimantadine are preferable to amantadine with regard to the need for individual prescribing and tolerance monitoring. Transmission of drug-resistant virus could substantially limit the effectiveness of M2 inhibitors and the possibility exists for primary M2 inhibitor resistance in a pandemic strain. The frequency of resistance emergence is lower with neuraminidase inhibitors and mathematical modelling studies indicate that the reduced transmissibility of drug-resistant virus observed with neuraminidase inhibitor-resistant variants would lead to negligible community spread of such variants. Thus, there are antiviral drugs currently available that hold considerable promise for response to pandemic influenza before a vaccine is available, although considerable work remains in realizing this potential. Markedly increasing the quantity of available antiviral agents through mechanisms such as stockpiling, educating health care providers and the public and developing effective means of rapid distribution to those in need are essential in developing an effective response, but remain currently unresolved problems.

Influenza virus A/Hong Kong/1774/99, isolated from a young child with mild influenza, was shown to be similar in its antigenic and genetic characteristics to H3N2 viruses circulating in pigs in Europe during the 1990s and in particular to be closely related to viruses isolated from two children in the Netherlands in 1993. Similar viruses had previously not been identified outside Europe. Although there is little evidence as to how the child contracted the infection, it appears likely that pigs in southern China were the source of infection. Characteristics shared with the European swine viruses include resistance to the anti-influenza drugs amantadine and rimantadine. Thus not only does this incident once again highlight the potential of pigs as a source of novel human influenza viruses, but also indicates the potential for emergence of amantadine-resistant human viruses.

The viral ion channel protein M2 supports the transit of influenza virus and its glycoproteins through acidic compartments of the cell. M2 conducts endosomal protons into the virion to initiate uncoating and, by equilibrating the pH at trans-Golgi membranes, preserves the native conformation of acid-sensitive viral hemagglutinin. The exceptionally low conductance of the M2 channel thwarted resolution of single channels by electrophysiological techniques. Assays of liposome-reconstituted M2 yielded the average unitary channel current of the M2 tetramer--1.2 aA (1.2 x 10(-18) A) at neutral pH and 2.7 to 4.1 aA at pH 5.7--which activates the channel. Extrapolation to physiological temperature predicts 4.8 and 40 aA, respectively, and a unitary conductance of 0.03 versus 0.4 fS. This minute activity, below previous estimates, appears sufficient for virus reproduction, but low enough to avert abortive cytotoxicity. The unitary permeability of M2 was within the range reported for other proton channels. To address the ion selectivity of M2, we exploited the coupling of ionic influx and efflux in sealed liposomes. Metal ion fluxes were monitored by proton counterflow, employing a pH probe 1,000 times more sensitive than available Na+ or K+ probes. Even low-pH-activated M2 did not conduct Na+ and K+. The proton selectivity of M2 was estimated to be at least 3 x 10(6) (over sodium or potassium ions), in agreement with electrophysiological studies. The stringent proton selectivity of M2 suggests that the cytopathology of influenza virus does not involve direct perturbation of cellular sodium or potassium gradients.

Influenza virus infection is associated with development of oxidative stress in lung and blood plasma, viz. increase of primary and secondary lipid peroxidation products. It was established that rimantadine treatment led to a decrease of the products of lipid peroxidation in tissues of mice experimentally infected with influenza virus A/Aichi/2/68 (H3N2). The effect is strongest in blood plasma (a decrease of about 50%) and weaker in the lung (about 20%). To elucidate the mechanism of this action of rimantadine, experiments were carried out with some model systems. The capability of rimantadine to scavenge superoxide radicals (scavenging properties) was studied in a system of xanthine-xanthine oxidase to generate superoxide. The amount of superoxide was measured spectrophotometrically by the NBT-test and chemiluminesce. Rimantadine does not show scavenging properties and its antioxidant effect observed in vivo, is not a result of its direct action on the processes of lipid peroxidation and/or interaction with antioxidant enzymes. The antioxidant properties of rimantadine were investigated by measurement of induced lipid peroxidation in a Fe2+ and (Fe2+ - EDTA) system with an egg liposomal suspension. Our findings with model systems do not prove an antioxidant or prooxidant effect of the drug on the processes of lipid peroxidation. Apparently, the observed antioxidant effect of rimantadine in vivo is not connected directly with free radical processes in the organism.

Amantadine, a drug known to inhibit influenza A viral matrix (M2) protein function, was reported to be an effective treatment in some patients with chronic hepatitis C virus (HCV) infection. Sequence comparison shows no homology between M2 and any of the HCV proteins. The effects of amantadine and a related analogue, rimantadine, on viral protease, helicase, ATPase, RNA-dependent RNA polymerase, and HCV internal ribosomal entry site (IRES) translation were tested by established in vitro biochemical assays. No inhibition (>15%) of HCV protease, helicase, ATPase, and polymerase was observed with concentrations up to 400 microgram/mL. IRES-specific inhibition was not observed at clinically relevant concentrations, but both cap and IRES reporter genes were suppressed at higher levels, suggesting nonspecific translation inhibition. In conclusion, amantadine and rimantadine have no direct and specific inhibitory effects against HCV protease, helicase, ATPase, polymerase, and IRES in vitro.

The spread of drug-resistant influenza viruses type A to close contacts in families, schools, and nursing homes has been well documented. To investigate whether drug-resistant influenza viruses circulate in the general population, 2017 isolates collected in 43 countries and territories during a 4-year period were tested for drug susceptibility in a bioassay. Drug resistance was confirmed by detection of specific mutations on the M2 gene that have been shown to confer resistance to amantadine or rimantadine. Sixteen viruses (0.8%) were found to be drug-resistant. Only 2 of these resistant viruses were isolated from individuals who received amantadine or rimantadine treatment at the time the specimens were collected. For 12 individuals use of amantadine or rimantadine could be excluded, and from the remaining 2 patients information about medication was unavailable. These results indicate that the circulation of drug-resistant influenza viruses is a rare event, but surveillance for drug resistance should be continued.

A variant obtained by a single passage of the initial FPV in the presence of 25 micrograms/ml rimantadine is more resistant to the drug than the parental virus. Sensitivity to rimantadine is higher in a variant obtained similarly by cell culture infection with rimantadine-resistant FPV. Potentiation of the antiviral effect of a combination of ribavirin (12-25 micrograms/ml) and rimantadine (0.1-6 micrograms/ml) is decreased or null if rimantadine dose is increased to 12-25 micrograms/ml; this phenomenon does not depend on the sensitivity of infective virus to rimantadine. Antiviral effect of drug combination with a high concentration of rimantadine is due to ribavirin alone.

We report here that bloodstream forms of the African trypanosome, Trypanosoma brucei, are sensitive to the anti-influenza virus drug rimantadine (50% inhibitory concentration of 1.26 micrograms ml-1 at pH 7.4). The activity is pH dependent and is consistent with a mechanism involving inhibition of the ability to regulate internal pH. Rimantadine is also toxic to the trypanosomatid parasites Trypanosoma cruzi and Leishmania major.

FPV/Rostock obtained in the presence of remantadine (25 micrograms/ml) is studied. This variant of the virus is characterized by a decreased amount of M1-protein. Its buoyant density in urograffin density gradient is 1.20 gamma/cm3 vs. 1.18 and 1.175 g/cm3 for initial FPV and remantadine-resistant FPV, respectively. Purification and concentration decreases the hemagglutinating activity of the virus 5-10 times more intensely than that of initial and remantadine-resistant FPV. On the other hand, the production of virus-specific proteins and intracellular distribution of hemagglutinin in this virus variant were not changed. The changes associated with a decrease of hemagglutinin and M1-protein content in virions are believed to manifest at a later stage preceding virion release from the cell.

Amantadine and rimantadine hydrochloride were tested for stability after storage at different temperatures and under different conditions for extended periods of time. Both compounds were quite stable after storage for at least 25 years at ambient temperature; they both retained full antiviral activity after long-term storage or after boiling and holding at 65-85 degrees C for several days. Thus, amantadine and rimantadine could be synthesized in large quantities and stored for at least one generation without loss of activity in preparation for the next influenza A pandemic in humans.

The protective effect of an immunostimulatory bacterial preparation, cytoplasmic membranes of Escherichia coli WF stable protoplast type L-forms (CM) alone and in combination with the selective antiviral drug rimantadine was evaluated in experimental influenza A/Aichi/2/68 (H3N2) virus infection in mice. In sublethal infection, CM administered intraperitoneally (i.p.) 7 days before virus exposure in a single dose of 25 mg/kg did not reduce significantly the virus lung titers. In lethal infection, CM applied in the same way weakly reduced the mortality rate. The combined application of CM with rimantadine resulted in synergistically increased protection, determined on the basis of virus lung titers, lung consolidation, mortality rates, protective indices, and survival times.

1. The M2 protein of influenza A virus is implicated in transmembrane pH regulation during infection. Whole-cell patch clamp of mouse erythroleukaemia cells expressing the M2 protein in the surface membrane showed a conductance due to M2 which was specifically blocked by the anti-influenza drug rimantadine. 2. The ion selectivity of the rimantadine-sensitive current through M2 was determined. Reversal potentials were close to equilibrium potentials for transmembrane pH gradients and not to those for Na+, K+ or Cl- concentration gradients. M2 permeability to Na+ relative to H+ was estimated to be less than 6 x 10(-7). 3. The M2 conductance increased as external pH decreased below 8.5 and approached saturation at an external pH of 4, effects attributable to increased permeability due to increased driving potential and to activation by low external pH. Both activation and permeation could be described by interaction of protons with sites on M2, with apparent dissociation constants of approximately 0.1 microM and 1 microM, respectively, under physiological conditions. 4. The M2 protein can transfer protons selectively across membranes with the H+ electrochemical gradient, properties consistent with its role in modifying virion and trans-Golgi pH during virus infection.

Amide of 1-adamantane carboxylic acid (AACA), the same as remantadine, was shown to have no antiviral action and any influence on the efficiency of the Sindbis virus adsorption. However, it significantly inhibited the virus specific synthesis of RNA and protein. The main difference between AACA and remantadine was in the fact that the efficiency of the inhibition of the Sindbis virus reproduction and synthesis of the virus specific RNA in the presence of AACA did not markedly decrease even when AACA was added to the experimental system 7 hours after the adsorption whereas for the inhibitory effect of remantadine it was to be added to the system not later than 2 hours after the adsorption.

The recognition of viruses as causes of pneumonia in both immunocompetent and immunocompromised hosts has expanded dramatically. The number of therapeutic agents available for treatment of these illness also has increased in the last decade. Each of these agents has demonstrated a limited therapeutic indication for treatment of viral pneumonia. Many of these agents inhibit viral DNA synthesis through actions as nucleoside analogs (such as acyclovir and ganciclovir). However, a variety of alternative mechanisms of inhibition of viral replication are used. Ribavirin, while being a nucleoside analogue, also appears to exert broad antiviral activity by a variety of enzymatic inhibitory mechanisms. Foscarnet, an inorganic pyrophosphate analogue, offers additional treatment options for herpesviruses by acting as a direct virus DNA polymerase inhibitor. The tricyclic amines amantadine and rimantadine inhibit influenza A replication by interfering with viral uncoating after cell penetration. Thus, these two agents are largely effective as prophylaxis. The search for novel antiviral drugs, such as neuraminadases inhibitors with selective influenza activity, is currently in progress.

Objective: To provide a review of rimantadine, including its antiviral activity, pharmacokinetics, efficacy, adverse effects, drug interactions, and dosage and administration. Information on influenza A virus and clinical features of influenza disease are presented. Comparative data on rimantadine and amantadine are described., Data Sources: A MEDLINE search restricted to English-language literature published from 1966 through 1994 and an extensive review of journals was conducted., Data Extraction: The data on antiviral activity, pharmacokinetics, adverse effects, and drug interactions were obtained from various articles on rimantadine in open and controlled studies. Controlled double-blind studies were evaluated to assess the efficacy of rimantadine in prophylaxis and treatment of influenza A infection., Data Synthesis: Over 90% of a rimantadine dose was absorbed in 3-6 hours in healthy adults. Steady-state plasma concentrations have ranged from 0.10 to 2.60 micrograms/mL at doses of 3 mg/kg/d in infants to 100 mg twice daily in the elderly. Nasal fluid concentrations of rimantadine at steady-state were 1.5 times higher than plasma concentrations, which may explain the effectiveness of rimantadine despite a low plasma concentration. Over 75% of a rimantadine dose was metabolized in the liver, and the parent compound and metabolites were almost completely eliminated by the kidneys. The elimination half-life ranged from 24.8 to 36.5 hours, which allows once-daily dosing. Dosage adjustment is recommended for patients with severe renal impairment (creatinine clearance < or = 0.17 mL/s), severe hepatic dysfunction, or elderly nursing home patients. Drug-resistant strains of influenza A virus to rimantadine occurred in several studies with children and/or adults. Clinical significance of drug-resistant strains has not been established. Rimantadine appeared to be effective in 85-90% of individuals for prevention of influenza A illness and in 50-65% for prevention of influenza A infection. Rimantadine reduced the time to a 50% reduction in symptoms by 1-3 days versus placebo. Differences in symptom reduction between rimantadine and placebo after the first 3 days of treatment was not generally clinically significant. The most common adverse effects of rimantadine administration were associated with the central nervous system (CNS) and the gastrointestinal (GI) tract. CNS-related adverse effects occurred in 3.2% of children younger than 10 years of age and 8.4% of adults. In elderly patients, the incidence of CNS-related adverse effects ranged from 4.9% at 100 mg/d to 12.5% at 200 mg/d. GI adverse effects occurred in 8.4% of children younger than 10 years of age, 3.1% of adults, and 2.9% at 100 mg/d and 17.0% at 200 mg/d in the elderly., Conclusions: Rimantadine offers some desirable features for the treatment and prophylaxis of influenza A infection. It appears to be an attractive choice in elderly patients with a history of CNS adverse effects from amantadine and in patients with mild or moderate renal impairment. Although approved for twice-daily dosing, rimantadine has a pharmacokinetic profile that would allow once-daily dosing. It is effective for prophylaxis (not postexposure prophylaxis) and treatment of influenza A virus. It also has a low incidence of adverse effects.

Amphetamine, caffeine, sydnocarb, meclofenoxate, adapromine, midantan, and nomifensine were studied for their effects on bioelectrical activity and Fourier EEG power spectra of the sensomotor cortex, dorsal hippocamp and lateral hypothalamus of freely behaving awake rats. The drop in the absolute power of all frequency ranges with the enhanced power of fast beta 1,2-ranges was common to the action of psychostimulants. In addition to the common properties, specific features of their action were revealed. Amphetamine, meclofenoxate, and nomifensine were found to increase the amplitude of the dominant peak in the theta-range and amphetamine shifts the frequency of the dominant peak to the region of faster ranges. The agents-induced electrophysiological changes correspond to the varying degrees of activation of the central nervous system, causing the optimization of behavioral functions, abolition of fatigue and drowsiness and enhancing physical and mental working capacity.

The mechanisms responsible for the formation of resistance of influenza A virus isolates during the natural circulation of the influenza viruses in the environment were studied. The influenza viruses H1N1 and H3N2 resistant to remantadine, adapromine, and deitiforine have been isolated in the USSR and Mongolia since 1982. The majority of natural resistant isolates appeared to be atypical both in antigenic properties and genomic structure as compared to the isolates prevalent in the common epidemic process. The nucleotide sequences of the M2 gene of some resistant strains and virus A/PR8/34 used in our country as an attenuation donor for preparation of killed recombinant vaccines. The electrophoretic mobility of genomic RNA of two resistant isolates is similar to that of the vaccine strain X-54 based on the virus A/PR/8/34. In this connection, the appearance of resistant strains in the environment may be due not only to spontaneous mutagenesis or selective drug actions, but also to the involvement into the circulation of vaccinal strains.

The influenza virus M2 protein was expressed from a recombinant baculovirus in Spodoptera frugiperda Sf9 cells, purified and reconstituted into artificial membrane vesicles. The specific inhibitor amantadine overcame the toxic activity of the protein and boosted the rate of M2 synthesis by a factor of 10, allowing yields of about 1 mg of purified M2 protein per g of Sf9 cells. M2 protein expressed in this system was phosphorylated and palmitoylated and displayed properties similar to the authentic virus protein. Purified wild-type M2 protein and an amantadine-resistant mutant M2 (M2 delta) with a deletion in the trans-membrane domain (amino acids 28 to 31) were incorporated into lipid vesicles, which were loaded with the fluorescent pH indicator pyranine. On imposition of an ionic gradient, M2 caused a decrease in intravesicular pH, which was susceptible to inhibition by 0.1 to 1 microM-rimantadine or N-ethyl-rimantadine. M2 delta behaved similarly but exhibited the expected drug resistance. These experiments indicate that isolated M2 functions as an ion channel and demonstrates in vitro M2-mediated proton translocation.

The anti-influenza activity of the neuraminidase inhibitor 4-guanidino-Neu5Ac2en (4-G-NAc) was determined in Madin-Darby canine kidney (MDCK) cells by yield reduction and ELISA and in explants of human respiratory epithelium by yield reduction. In MDCK cells, 50% inhibitory concentrations (EC50) averaged 0.5 microgram/ml for influenza A/Virginia/88(H3N2) and 0.04 microgram/ml for A/Texas/36/91(H1N1) by ELISA, and < 0.01 microgram/ml for influenza A/Virginia by yield reduction. In human adenoid explants, concentrations causing at least 1.0 log10 TCID50/ml decrease in yield (EC90) at 48 h were < 0.01 microgram/ml for A(H1N1) and A(H3N2) viruses and 0.25 microgram/ml for B/Hong Kong/5/72. 100 micrograms/ml 4-G-NAc did not inhibit outgrowth of human adenoid epithelium at 6 days, whereas ribavirin 10 micrograms/ml reduced outgrowth by > 50%. 4-G-NAc is a potent and selective inhibitor of clinical isolates of influenza A and B viruses in human respiratory epithelium.

Studies of the molecular aspects of resistance of influenza virus A to drugs (rimantadine, deytiforine, amantadine) allow a purposeful design of new compounds with a broad spectrum of antiviral activity and evoking no resistance. In this work the nucleotide sequence of rimantadine- and deytiforine-resistant influenza A strain Leningrad/156/83 (H3N2) was compared with that of A/Victoria/35/72. The influence of aminoacid substitutions in the M2 protein on its secondary structure in the membrane and its role in resistance development was shown.

Using 14 monoclonal antibodies (MoAbs) in solid-phase ELISA it was found that influenza virus A/Hong Kong/1/68 (H3N2) mutants resistant to the antiviral compound mopyridone as compared to the mopyridone-sensitive mutant manifested significant changes in the antigenic structure (sites 1A, 2 and 3) of M1 protein. No differences in M1 were found between rimantadine-resistant and rimantadine-sensitive mutants of influenza virus A(H3N2).

Clinical isolates of influenza A viruses identified during outbreaks in two winters were tested for their rimantadine susceptibilities by an enzyme-linked immunosorbent assay modified from that described previously by Belshe et al. (R. B. Belshe, B. Burk, F. Newman, R. L. Cerruti, and I. S. Sim, J. Virol. 62:1508-1512, 1988). The infectivity titer and the 50% inhibitory concentration of rimantadine were calculated for each virus. Of 105 influenza virus A isolates tested, 28 influenza A/H1N1 isolates from the 1988 and 1989 outbreak and 77 influenza A/H3N2 isolates from the outbreak in following year, were susceptible to the antiviral action of rimantadine.

The results of the study on the combined antiviral activity of the SHS-174 preparation (a lyophylized infusion from three higher plants) and three amantadine derivatives (rimantadine, amantadine glucuronide and its derivative) are presented. The antiviral effect of the drugs on the reproduction of influenza virus strains A/H1N1 and A/H3N2 in vitro was studied. The combined antiviral effect was evaluated on the basis of viral yields and in many cases a synergism was found. The most synergistic effect was shown for the combination of SHS-174 with the derivative of amantadine glucuronide.

Rimantadine and its structural analogs, i. e. amide of 1-adamantane carboxylic acid (AACA) and 1-adamantane acetic acid amide, were shown to be able to inhibit reproduction of Sindbis virus in culture Vero cells. AACA had the maximum antiviral activity. Subcultures of the initial sensitive population of Sindbis virus in the presence of AACA led to formation of mutants resistant to AACA as well as to rimantadine, adamantane acetic acid amide and ammonium chloride. The Sindbis virus population was heterogenous in sensitivity to AACA, which was evident from isolation of separate clones with various levels of sensitivity to the above mentioned compounds from the population. It was found that reproduction of the AACA sensitive and resistant strains of Sindbis virus differed: the latent period of the resistant strain was 2 hours longer than that of the sensitive strain. The same effect was observed in the comparative study on synthesis of the virus-specific RNA.

The susceptibility of H7 influenza A viruses to the M2-mediated alteration in HA resulting from treatment with amantadine or rimantadine depends both on the pH stability of HA and the pH encountered during transport to the plasma membrane of the particular virus-infected cell. pH stabilities of a range of virus mutant HAs exhibited an inverse correlation with drug sensitivity and the proportion of low-pH HA expressed on the surface of infected cells in the absence of drug. The lowest pH encountered during transport was thus estimated from the proportion of HA expressed in its native conformation and its pH stability profile. Lower drug sensitivity and improved HA maturation of MDCK cells compared to that in CEF reflect the higher pH within the appropriate compartment of the former. Differences in apparent pH resulting from infection with two closely related virus strains, Rostock or Weybridge, e.g., approximately 6.0 and 5.4, respectively, in CEF, were abrogated by rimantadine treatment (pH approximately 5.2 in CEF) and were attributable to intrinsic properties of their respective M2 proteins. The greater activity of the Rostock M2, which was estimated to be capable of increasing vesicular pH by as much as 0.8 pH units, correlates with the lower pH stability of the HA. This emphasizes the essential relationship between the characteristics of the two virus proteins as well as the subtle role of M2 in regulating the pH of the transport pathway to protect the structural integrity of the hemagglutinin glycoprotein.

A study was made of the influence of adapromine on bioelectrical activity of the brain, sensorimotor cortex, dorsal hippocamp and lateral hypothalamus in freely moving wakeful rats. Adapromine was established to evoke a decrease of the amplitude of the dominant peak and dominant theta-activity in power spectra of the EEG in the cortex and hippocamp, with an increase of rapid wave activity in the beta 2 range in the right cortex and hippocamp. These changes attained maximum after 1 to 1.5 hour and lasted up to 4-5 hours after adapromine administration. These changes can be viewed as activation of the cortex and hippocamp, which may attest to the presence of antidepressive and psychostimulant effects in the action spectrum of adapromine. The specific influence of adapromine on catecholaminergic processes of the brain may lie at the basis of the above effects.

Amantadine- and rimantadine-resistant viruses have been recovered from approximately 30% of patients treated for acute H3N2 subtype influenza and less often from their close contacts receiving drug prophylaxis. The limited data suggest that resistant viruses can emerge rapidly during drug therapy, as early as 2-3 days into treatment. These viruses retain their resistance phenotype during multiple passages in the laboratory and appear to be genetically stable in this regard. Studies in families and in nursing homes indicate that resistant isolates appear to be transmissible from treated patients and cause typical influenza in contacts receiving drug prophylaxis. It is unknown whether resistant human viruses are capable of competing with wild-type ones during multiple cycles of infection in the absence of the drug. These viruses appear to be pathogenic, and no evidence indicates that they differ from wild-type strains. Thus, these viruses clearly possess the biologic properties that are associated with clinically important drug resistance. However, limited information is available to assess their actual impact. It is unknown what degree of selective drug pressure would be required to cause substantial transmission of resistant viruses during community outbreaks. Natural selection of antigenic variants and disappearance of previous variants may prevent the emergence of viruses that have been altered in the genes coding both for the surface glycoproteins and for the M2 protein. However, the emergence of drug-resistant influenza viruses appears to pose potential clinical problems in certain epidemiologic situations involving close contact with treated patients.

The influence of rimantadine-resistance mutations on the virulence of human H3N2 viruses in ferrets was examined. The similarities in virulence of the drug-resistant mutants with single amino acid substitutions at three different locations, 27, 30, and 31, within the M2 sequence and their corresponding sensitive wild-type isolates contrasted with differences in virulence between the three pairs of viruses. These data provide further evidence that rimantadine-resistant viruses that emerge during treatment of patients with the drug are unaltered both in their growth characteristics and virulence.

It was shown that amiloride, orthovanade and uabain induced almost a two-fold decrease in the rate of incubation medium oxidation by the chick embryo fibroblasts due to the Na+/H+ exchange and inhibited by more than 95 per cent the influenza virus activity. The following mechanism for inhibition of the influenza virus multiplication in the cells under the effect of the above mentioned substances was proposed: suppression of the cellular Na+/H+ exchange responsible for the decrease in pH value in the virus-carrying endosomes----prevention of the decrease in the intraendosomal pH value to the critical level----blocking of the acid dependent process of the virus uncoating----inhibition of the influenza infection as a whole.

The specific neurotropic action of rimantadine was demonstrated in mollusk neuron experiments. No specific neurotropic activity of dejtiphorin was found. In combined application, the latter blocked the neurotropic effect of rimantadine. In experiments in white mice and rats, high doses of both drugs decreased the emotional and motor activity of the animals. After the use of the combination of the two drugs the activity of the animals did not differ significantly from that in the controls. The antiviral effect of each drug alone and of combination thereof was similar.

A placebo-controlled, double-blind study to evaluate the safety and prophylactic efficacy of a low dose (100 mg) of rimantadine hydrochloride against naturally occurring influenza in adults was conducted at two sites. After the onset of the influenza season, volunteers (ages, 18 to 55 years) were assigned randomly to receive rimantadine or placebo daily. Subjects were monitored for adverse effects and evidence of influenza virus infection weekly for six weeks. Only 10 (8.7%) of 114 rimantadine recipients and 5 (4.4%) of 114 placebo control recipients reported one or more mild to moderate adverse symptoms, most of which were related to the gastrointestinal or central nervous system. Compared with placebo, low-dose rimantadine was highly effective in the prevention of influenza A virus infection (20 of 110 versus 7 of 112 participants; P less than 0.01) and influenza illness (7 of 110 versus 1 of 112 participants; P = 0.04). Influenza A/Leningrad/87-like (H3N2) virus was recovered from the nasopharynxes of only five placebo recipients. These findings indicate that low-dose rimantadine is well tolerated and highly effective for the prevention of influenza A illness in healthy adults.

The hydroxy metabolites of rimantadine (3-5) were synthesized and compared to amantadine (1) and rimantadine (2) for their ability to inhibit the replication of influenza viruses in vitro. All three metabolites were inhibitory to wild-type influenza A viruses (H3N2 and H1N1). In particular, 2-hydroxyrimantadine (3) showed similar activity to amantadine, but the 3- and 4-hydroxy metabolites (4 and 5, respectively), both of which are found in rimantadine-treated patients, showed only modest inhibitory activity. A rimantadine-resistant isolate of influenza A virus exhibited cross-resistance to amantadine and to each of the metabolites 3-5. None of the compounds were effective against influenza B virus.

The method of fluorescence dequenching was used to study the interaction between influenza virus A/Krasnodar/101/59 and Namalwa and Raji lymphoblastoid cells. Experiments with endocytosis inhibitors and fluorescence quenchers have shown that at pH = 5.0 the virus lipid envelopes are fused with the plasma membranes of the cells, and at pH = 7.4 the virus lipid envelopes are fused with the internal, presumably endosomal, membranes of the cells. Remantadine at a concentration of 50-1000 micrograms/ml did not influence the fusion of virus lipid envelopes with intracellular membranes at pH = 7.4 whereas at pH = 5.0 it inhibited, beginning from 25 micrograms/ml concentration, the fusion of virus lipid envelopes with the plasma membranes of cells.