I. A. Leneva, I. T. Fedyakina, T. A. Guskova, R. G. Glushkov
The Shemyankin and Ovchinikov Institute of Bio-organic Chemistry, Moscow
The sensitivity of various influenza virus strains to arbidol. The influence of arbidol
in combination with other antiviral drugs on reproduction of influenza virus A.
Aim. To study the antiviral activity of arbidol in relation to various antigen subtypes of
influenza virus; to study the effectiveness of arbidol when used in combination with
adamantine-type antiviral drugs, ribavirin and ribamidil in inhibiting influenza virus
reproduction in cell culture.
Material and Methods. The activity of the drugs against viral reproduction was assessed
according to the expression of viral antigens in virus-infected cells, determined by
Results. Arbidol is not inferior to adamantine drugs, neuraminidase inhibitors, ribavirin
and ribamidil in its inhibiting effect on influenza virus A and B. Arbidol inhibits
reproduction of human IVA antigen strains H1N1, H2N2, H3N2 and rimantadine-
sensitive as well as rimantidine-resistant strains of the influenza virus. Arbidol inhibits
the reproduction of strains of avian influenza, pathogenic to humans, H5N1 and H9N2,
and H6N1 and H9N2, which have genes in common with H5N1 and H9N2. Arbidol’s
inhibitory effect on viral reproduction in cell culture was enhanced when it was used in
combination with amantidine, rimantadine, ribavirin and ribamidil.
Conclusion. Arbidol has broad-spectrum antiviral action, and inhibits reproduction of
various antigenic subtypes and rimantadine-resistant human IVA, avian viruses H5N1
and H9N2, and influenza viruses B and C.
Key words: arbidol, influenza viruses A and B, combined chemotherapy of influenza
At the present time, along with its campaign of vaccination as a main defense
against influenza, the World Health Organization (WHO) is recommendingadministration of etiotropic drugs, that is, drugs which target a specific point in the viralreproductive cycle. Included in the first generation of such drugs are theadamantadine types, amantadine and rimantadine. They block the ion channels in thetransmembrane of the virus protein M2, and impede the proton transport which lowersthe pH of the virion, a necessary condition for the release of virus ribonucleoproteinsfrom protein M1 and the beginning of the virus genome transcription. Amantadine andrimantadine molecules, having a diameter corresponding to these ion channels, inhibitproton transport, raising the pH level in the endosome, hindering release from M1, andstopping the reproduction of the virus . However, the adamantadine drugs are noteffective against influenza B, have serious side effects, and also cause a rapid andwidespread growth of resistant strains of the virus .
The second generation of drugs includes neuraminidase inhibitors, which are
taken in the form of inhalers or aerosol sprays (zanamivir) and tablets or capsules(oseltamivir). These preparations inhibit the function of viral neuraminidase, whichblocks the release of new viral particles from the cells and further spread of the viruswithin the organism . Not only are these drugs very expensive, zanamivir can causeirritation of the nasal passages, and oseltamivir can cause nausea and vomiting . From
the foregoing, it is clear that these existing remedies are lass than ideal; thus, there is aneed for creation of a new drug which works through a different pathway. Lately, therehas been widespread use in Russia of the drug arbidol, confirmed by the RussianFederation Pharmicological Committee for the prevention and treatment of influenzagroups A and B in adults and children. The high therapeutic effectiveness of this newrepresentative of the indole group is a result of its broad-spectrum biological action, andis based on its immunomodulator, interferon inductor, antioxidant and virus-specificqualities . Clinical trials among more than 10,000 patients, and also 15 years of use inmedical practice, have shown arbidol’s safety, and its lack of side effects .
The present work gives the results of studies of the effectiveness of arbidol
against various strains of the influenza virus which are pathogenic in humans, and also itseffectiveness against viral reproduction when used in combination with other drugs.
Materials and Methods
Viruses and cells.
In the experiments we used single-layer cultures of embryonic
dog kidney cells (MDCK). The viruses studied were human influenza group A strainsA/PR/8/34 (H1N1), A/WSN/33 (H1N1), A/Singapore/1/57 (H2N2), A/Japan/305/57(H2N2), A/Krasnodar/101/59 (H2N2), A/Taiwan/79 (H3N2), A/Mississipi/85 (H3N2);influenza group B strains B/Lee/40, B/Singapore/222/79; and group C strains C/SSSR/and C/Leningrad, received from the Institute of Virology named after D. I. IvanovskyRAMN. The viruses A/Hong Kong/156/97 (H5N1) and A/Hong Kong/1074/99 (H9N2)were isolated from humans during the Hong Kong influenza outbreak. VirusesA/guineafowl/Hong Kong/G1/97 (H9N2) and A/duck/Hong Kong/W312/97 (H6N1)were isolated from birds in Hong Kong. Viruses A/Hong Kong/196/57, A/HongKong/1074/99, A/guineafowl/Hong Kong/G1/97, and A/guineafowl/Hong Kong/G1/97(P3) are the property of St. Jude Children’s Research Hospital, Memphis, TN. Allexperiments were conducted in Biosafety Level 3 facilities.
The study of antiviral actions of drugs in MDCK cells using the enzyme
MDCK cells were placed in 96-well plates (from Costar) with
an average density of 35,000 cells per well and cultured in Eagle medium with 5% fetal
calf serum and 10 mM glutamine to produce one full layer. The drugs or drug
combinations under study were added to the cells in two-round concentration in 100 mcl
Eagle medium. We added 100 mcl of this same medium to the virus control, and 200 mcl
to the cell control. In the experiments with human influenza virus strains, the virus
compounds and divisions under study were done in Eagle medium with added TPCK
trypsin (2.5 mcg/ml). To study the combined effect of preparations on viral reproduction,
each plate included wells with control cells (cells not infected by the virus); wells with
control virus (cells infected by the virus); wells infected by the virus in the presence of
the two preparations under study; and “control wells,” infected by the virus, with one or
the other of the studied preparations individually added. For the study of the combined
effectiveness of the drugs, we added 50 mcl of the drug solution to the cells in 4-stage
concentrations. To the “control wells” we added 50 mcl of one drug in 4-stage
concentrations, along with 50 mcl of medium with trypsin (2.5 mcg/ml).
After incubating the cells with the given preparations for 30 min. at 37 degrees
Celsius, we added 100 mcl of allantoic virus in Eagle medium (multiplicity of infection
0.1-1 MOI; in studies on combined preparations, 1-5 MOI) to the wells, except for thecell control wells. Then the plates were incubated for 17 hours in a 5% CO2 environmentat 37 degrees; then the cells were fixed with 80% acetone in PBS for 20 min., andthoroughly dried, after which point we determined the expression of viral antigens byenzyme immunoassay, as earlier written . The percent inhibition of the studiedcombinations was determined according to the formula: % inhibition = 100 – [OD490(experiment) – OD490(cell control)]/ [OD490(virus control without the combination) –OD490(cell control)]. The drug concentration which reduced the OD490 by 50% was takenas the minimal inhibitory concentration 50 (MIC50).
Results and Discussion
It is well-known that in humans, influenza can display antigens of type A, B, or C.
In the human population, the main subtypes of group A which circulate are H1N1, H2N2and H3N2, which in turn undergo continual changes in their antigen structure . Forthis reason, the most important characteristic of an anti-influenza preparation must be abroad-spectrum effect across strains. Clinical experiments showing the high prophylacticand therapeutic effects of arbidol have been conducted during influenza outbreaks causedby A/H2N2 and H3N1, B/369, and associated variants . However, laboratorydiagnostics and identification of the influenza strains causing illness in the specificpeople in the experiments were not completed. Thus, direct data of arbidol’s sensitivityto various strains of influenza, which cause illness in humans, has been lacking.
Table 1: The influence of antiviral drugs on viral reproduction of various strains of human
influenza virus A, B and C in MDCK cell culture
Note: Drug concentrations were the following: arbidol, virazol, ribamidil, zanamivir, oseltamivir– 10 mcg/ml; amantadine, rimantadine – 5 mcg/ml in experiments with group A and 20 mcg/mlin experiments with groups B and C. “—“ indicates reactions not studied; “n/a” = not active.
In our studies comparing arbidol’s effects to the effects of other anti-influenza
drugs on strains of influenza virus A and B, arbidol inhibited viral reproduction of all thetested group A strains at approximately the same level, that is, 80%; arbidol did no worsethan, and in fact, often surpassed the effects of other anti-influenza preparations (Table1). In contrast to amantadine and rimantadine, which had practically no influence on thereproduction of viral group B, arbidol, like the neuraminidase inhibitors, decreased viralreproduction by 60%. Inhibition of virus group C was weaker, at 20%.
It is known that one defect of anti-influenza drugs is the rapid mutation of
resistant strains to the drugs. This rapid mutation (within 1-2 trials) of drug-resistantstrains has been shown in many experiments in animals and cell cultures; resistant strainsin the human population can develop as soon as 2-4 days after treatment with these drugs.
The circulation of these strains (moreover, with a relatively high level of virulence) in thehuman population has become a reality [2, 3]. Therefore, conquering these infectionsrequires expedient use of drugs that work through a different mechanism. Our previousdata points to the difference between the antiviral actions of arbidol and rimantadine .
Because of this, the study of arbidol’s effect on strains of influenza which are resistant torimantadine is of undoubted interest. Studies of arbidol’s effects on the reproduction ofstrains sensitive to rimantadine and strains resistant to rimantadine have shown thatarbidol inhibits the reproduction of strains both sensitive and resistant to rimantadine(Table 2).
Table 2: Influence of antiviral drugs on viral reproduction of rimantadine-sensitive and
rimantadine-resistant strains of influenza A in MDCK cell culture
Viral avian illness – Weybridge strain (H7N7)
For most of the past century, it was widely accepted that only the antigens of
influenza virus groups B and C and H1N1, H2N2 and H3N2 of group A could causeinfections in humans. But in 1997, the avian virus H5N1 caused 18 cases of illness inHong Kong, 6 of them resulting in death . At the end of 2003 and beginning of 2004,a new outbreak of influenza caused by avian viruses appeared in various Asian countries,again with fatalities. The WHO recommended administration of anti-viral chemicalpreparations as one of the measures against the “bird flu.” Virus A/Hong Kong/157/97and A/guineafowl/Hong Kong/G1/97, causing infection in humans, and also the thirdavian virus A/duck/Hong Kong/W312/97 (H6N1) have six common genes which codeinternal proteins. Still another avian virus, A/chicken/Hong Kong/G9/97, has two genes(PB1 and PB2) in common with the abovementioned three viruses. These virusescontinue to circulate among bird populations, and we cannot rule out the possibility of
their crossing the barrier between species and entering the human population. At presentthey are considered candidates for future influenza virus epidemics .
For this reason, we decided to study arbidol’s effect on the reproduction of avian
viruses A/Hong Kong/157/97 (H5N1), A/guineafowl/Hong Kong/G1/97 (H9N2),A/chicken/Hong Kong/G9/9797 (H9N2), and A/duck/Hong Kong/W312/97 (H6N1) inMDCK cell culture. Arbidol inhibited reproduction in all of the studied avian viruses,although they varied in their sensitivity to it. The MIC50 for both H9N2 viruses was 15mcg/ml, at the same time that it was 30 and 25 mcg/ml, respectively, for A/HongKong/157/97 (H5N1) and A/duck/Hong Kong/W312/97 (H6N1). The MIC50 for virusA/Singapore/1/57, taken as a control, was 10 mcg/ml (Table 3).
Table 3: Antiviral activity of arbidol on avian influenza A viruses, having genes in common
with H5N1 which code for internal proteins, in MDCK cell culture
Thus, arbidol suppresses the reproduction of avian viruses H5N1 and H9N2,
which cause infection in humans, and also represses reproduction of other avian viruseswhich have genes in common with them – although arbidol is less effective on theseviruses than it is on H1N1, H2N2, and H3N2 antigen subgroups of the influenza viruscirculating in the human population.
Combining one anti-influenza chemical preparation with another preparation is
one way to increase the effectiveness of treatment. With this method, the best results areobtained by a combination of two drugs which work through different mechanisms, andoperate independently on different stages of the interaction of the virus with cells. Thestudy of arbidol’s effects on different stages of viral reproduction showed that it inhibitsthe fusion of the viral lipid coat with the endosome membrane of the cell, preventing theentrance of the virus into the cell and the subsequent release of the viral genome withinthe cell and start of transcription [10, 13]. That process is induced by the conformationalchanges in the influenza virus surface protein hemagglutinin that take place during alowered pH, allowing conditions facilitating membrane fusion. Experiments done withmonoclonal antibodies on the conformational changes of hemagglutinin have shown thatarbidol changes the conformation of hemagglutinin, preventing it from destabilizing to amore active form which would allow it to induce membrane fusion. These findings wereconfirmed through determination of nucleotide sequences from mutants resistant toarbidol, which were found to have mutations only at the gene which codes forhemagglutinin . Taking into account our earlier data on the mechanism of action ofarbidol, and the differences between this mechanism and the effective mechanism of
other anti-influenza drugs, we could study the combined effect of arbidol with other anti-viral preparations on viral reproduction in MDK cell cultures.
At present, anti-influenza chemotherapy widely incorporates amantadine and its
relative, rimantadine. Aside from the fact that these preparations are ineffective againstinfluenza group B, their usefulness is limited by the rapid growth of resistant strains tothem, and by the side effects which high doses of these drugs produce [2, 3]. Oftenribavirin (or its structural relative ribamidil) is used in the treatment of RSV. Thesepreparations exist in the form of inhalants, or are administered intravenously in hospitalsettings under a doctor’s direction, particularly to immunocompromised patients. Apartfrom the inconvenience of administration, these drugs also produce side effects [5, 15,16]. Apart from its specific action against the influenza virus, arbidol has animmunomodulating effect, has the capability to induce interferon, and is recommendedfor patients with compromised immune systems [6, 7, 17].
In order to reduce the likelihood of the development of resistant strains, or
increase the effectiveness of lower doses of amantadine, rimantadine, ribavirin andribamidil, we studied the effects of combining these drugs with arbidol. Not one of theconcentrations of arbidol we used weakened the effect of amantadine or rimantadine onviral reproduction. Adding amantadine 1 mcg/ml to arbidol did not have any significantinfluence on the inhibition degree of viral reproduction, greater than the effect of thesame concentrations given alone. The combinations of arbidol and amantadine (arbidol 1mcg/ml + amantadine 10 mcg/ml; arbidol 5 mcg/ml + amantadine in all studiedconcentrations; and arbidol 10 mcg/ml + amantadine in all studied concentrations)strengthened the suppressive effect of arbidol on viral reproduction, in comparison to theeffect shown by the same concentrations of arbidol and amantadine alone, but thestrongest inhibitory effect was observed for the combination 5 mcg/ml arbidol +amantadine in concentrations 0.3 mcg/ml or 1 mcg/ml. The data was similar for arbidol’seffect on viral reproduction in experiments in combination with rimantadine (Table 4).
Table 4: The effect of arbidol in combination with various anti-influenza drugs on viral
reproduction of influenza virus A/Singapore/1/57 in MDCK cell culture
The addition of ribavirin in concentrations of 1, 3 and 10 mcg/ml to all tested
concentrations of arbidol increased its inhibitory effect, but the rate of increase differedamong the different concentrations. The biggest increase in inhibitory effect of bothpreparations was observed under the following combinations: arbidol 3 mcg/ml +ribavirin 1 mcg/ml; arbidol 10 mcg/ml + ribavirin 10 mcg/ml; and arbidol 15 mcg/ml +ribavirin 10 mcg/ml. Adding ribamidil to arbidol in various concentrations also increasedthe inhibiting effect of arbidol on viral reproduction in every case; however, thecombination of arbidol and ribamidil which resulted in the biggest increase in inhibitionof viral reproduction differed somewhat from the result of the arbidol and ribavirincombination. The biggest increase in inhibiting effect of arbidol and ribamidil wasobserved under the following combinations: arbidol 3 mcg/ml + ribamidil 1 mcg/ml;arbidol 3 mcg/ml + ribamidil 3 mcg/ml; arbidol 10 mcg/ml + ribamidil 3 mcg/ml; andarbidol 10 mcg/ml + ribamidil 10 mcg/ml (Table 4).
Thus, in the study of the effects of arbidol in combination with other anti-
influenza drugs which have a different mechanism of action from it, nowhere wasobserved an antagonistic reaction between the drugs. The combinations of amantadine,rimantadine, ribavirin and ribamidil with arbidol increased the effectiveness of lowereddoses of these drugs, which lessens the chance of side effects from the drugs, anddecreases the likelihood of the development of resistant strains from use of theadamantadine drug group.
The data concerning arbidol’s lack of specificity in regards to influenza virus
groups A and B; its effectiveness against strains resistant to rimantadine, and againstavian influenza strains which are pathogenic in humans; and its heightened effectivenessin combination with other anti-influenza preparations in cell culture experiments – allprovide a foundation for widening the possibilities for arbidol in RSV chemotherapy, andincreasing arbidol’s use in clinical settings.
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