In Vitro Antiviral Activity of a New Indol-3-carboxylic Acid Derivative Against SARS-CoV-2.

The coronavirus disease (COVID-19) pandemic has brought into sharp relief the threat posed by coronaviruses and laid the foundation for a fundamental analysis of this viral family, as well as a search for effective anti-COVID drugs. Work is underway to update existent vaccines against COVID-19, and screening for low-molecular-weight anti-COVID drug candidates for outpatient medicine continues. The opportunities and ways to accelerate the development of antiviral drugs against other pathogens are being discussed in the context of preparing for the next pandemic. In 2012-2015, Tsyshkova et al. synthesized a group of water-soluble low-molecular-weight compounds exhibiting an antiviral activity, whose chemical structure was similar to that of arbidol. Among those, there were a number of water-soluble compounds based on 5-methoxyindole-3-carboxylic acid aminoalkyl esters. Only one member of this rather extensive group of compounds, dihydrochloride of 6-bromo-5-methoxy-1-methyl-2-(1-piperidinomethyl)-3-(2-diethylaminoethoxy) carbonylindole, exhibited a reliable antiviral effect against SARS-CoV-2 in vitro. At a concentration of 52.0 µM, this compound completely inhibited the replication of the SARS-CoV-2 virus with an infectious activity of 106 TCID50/mL. The concentration curves of the analyzed compound indicate the specificity of its action. Interferon-inducing activity, as well as suppression of syncytium formation induced by the spike protein (S-glycoprotein) of SARS-CoV-2 by 89%, were also revealed. In view of its synthetic accessibility - high activity (IC50 = 1.06 µg/mL) and high selectivity index (SI = 78.6) - this compound appears to meets the requirements for the development of antiviral drugs for COVID-19 prevention and treatment.

Human Diploid Fibroblast Cell Lines - a Model System for Studying Immunodulatory Properties of Modern Immunobiological Drugs and Viruses.

The immunomodulatory properties of immunobiological drugs Glutoxim and Phosprenyl we well as vesicular stomatitis virus and inactivated tick-borne encephalitis vaccine virus were studied using human diploid fibroblast cell line from the collection of M. P. Chumakov Federal Research Center for Research and Development of Immunobiological Products. All tested preparations exhibited immunomodulatory activity in human diploid fibroblast cell line. Glutoxim in doses of 0.1 and 0.25 µg/ml stimulated production of IL-6 and IL-10 during 24-48 h of culturing, but did not stimulate production of IL-1ß. Phosprenyl, on the contrary, increased production of IL-1ß and the levels of IL-6 and IL-10. Vesicular stomatitis virus stimulated the production of IL-1ß, IL-6, and IL-10, while inactivated tick-borne encephalitis vaccine virus stimulated the production of cytokines IL-8 and IL-18. Immunomodulatory activity of inactivated tick-borne encephalitis vaccine virus was first demonstrated in the in vitro system.

Selection of Domestic Cell Lines for the Creation of Diagnostic and Preventive Preparations against Enterovirus 71.

We studied the sensitivity of domestic proprietary human and animal cell lines from the collection of M. P. Chumakov Federal Scientific Center for Research and Development of Immuneand-Biological Products to infection with different enterovirus 71 strains. A cell system based on domestic proprietary permanent cell line 4647 was for the first time used for reproduction of four enterovirus 71 strains (BrCr, 42266, 42934, and 43374). It was shown that strain 4647 is the optimal cell substrate for enterovirus 71 reproduction. The titers of enterovirus 71 for all four strains considerably (by 2 lgTCID50/ml and more) increased during sequential passages in permanent cell line 4647. The prospects of using permanent cell line 4647 for creation of diagnostic and preventive preparations against 71 was demonstrated.

Polyprenyl Phosphates Induce a High Humoral and Cellular Response to Immunization with Recombinant Proteins of the Replicative Complex of the Hepatitis C Virus.

The search for new adjuvants remains the critical task for the creation of hepatitis C vaccines due to the weak immunogenicity of biotechnological products. When immunizing mice with the recombinant proteins NS3 and NS5B of the hepatitis C virus (HCV), the adjuvant activity of three immunomodulators was compared. Phosprenyl® on the basis of polyprenyl phosphate (PPP), chemically synthesized analogue of the bacterial cell wall glucosaminyl muramyl dipeptide (GMDP), and IFN-α recombinant protein were tested. GMDP increased the activity of IgG1 antibodies 4-6 times but did not stimulate the production of IFN-γ; IFN-α has not shown any adjuvant properties. The introduction of recombinant HCV proteins together with PPP in low doses increased the activity of IgG2a isotype antibodies 4-7 times and increased IFN-γ secretion 3 times. Thus, it was first shown that PPP polarizes the immune response to Th1-type and is a promising adjuvant for the development of a vaccine against hepatitis C.

[Antiviral activity of phosprenyl and gamapren against infection caused by influenza a (H5N1) virus in cell culture].

The antiviral activity of Phosprenyl and Gamapren in vitro against highly pathogenic strain of avian influenza H5N1 virus was studied. Inoculation of the virus to the susceptible cell culture led to development of the cytopathogenic effect. Preliminary introduction of Phosprenyl and Gamapren an hour prior to infecting the cells with virus 10.0 TCID50 dose completely inhibited the cytopathogenic activity of the virus. At higher doses of virus (100.0 TCID50) significant inhibition of the infectious activity of the virus was observed: 70% of infected cells survived under the action of Phosprenyl, and 90% under the action of Gamapren. With the introduction of the preparations simultaneously with the infection of cells with virus at a dose of 10.0 TCID50 virtually 100% of infected cells survived, while in control cultures death of 100% of the cells occurred. After infection with the virus at a dose of 100.0 TCID50 Phosprenyl and Gamapren caused 50% protection of the cells. The antiviral effect of the drugs Phosprenyl and Gamapren may be associated not only with their virulicidal, but with anti-viral activity as well.

[Efficacy of polyprenyl phosphates in the experimental genital herpes model].

An experimental model of the primary genital herpes (herpes simplex type 2, HSV-2) in the female guinea pigs was suggested to study the infectious process activity of polyprenyl phosphates (PPP) and PPP+acyclovir (AC) complex treatment. The morphofunctional features of the guinea pig ovaries were studied in the control and experimental groups (the latter were inoculated with PPP and/or AC as a primary infection treatment) at the stage of the recurrent genital herpes aggravation. It was shown that in the case of combined PPP +AC use significant changes in the disease symptoms were observed, as well as a decrease in the infectious process activity and duration, and positive remote effect on the ovarian morphophysiology.

[TREATMENT OF PATIENTS WITH CHRONIC RECURRENT HERPES VIRUS INFECTION OF GENITAL LOCALIZATION: A CLINICAL STUDY OF FORTEPREN PREPARATION].

AIM: Selection of optimal dosage regimen, length of treatment course (frequency of administration), safety, tolerance and clinical effectiveness evaluation of the medical preparation fortepren in patients with chronical recurrent herpes virus infection of genital localization. MATERIALS AND METHODS: The medical product of antiviral and immune modulating effect--fortepren (sodium polyprenyl phosphate) as a 4 mg/ml solution for injections combined with the base course of acyclic nucleoside acyclovir, 400 mg tablets, held studies. 40 male and female patients participated in the study. After a 10-day acyclovir course (400 mg x 3 times a day) for removing the acute phase, 4 groups of 10 individuals were formed: 1--5 ml (20 mg) of fortepren i/m once at day 13 ± 2 after the start of the study after the completion of the treatment of the acute phase of the disease; 2--5 ml (20 mg) fortepren i/m 3 times at an interval of 21 days; 3--2 ml (8 mg) fortepren i/m 3 times at an interval of 21 days; 4 (control)--5 ml of placebo i/m at remission stage 3 times at an interval of 21 days. Increase of the duration of inter-recurrence period, decrease of the severity of the recurrences, state of skin and mucous damage elements, improvements of immunologic parameters were considered during effectiveness evaluation. RESULTS: Significant differences in the frequency of recurrences of genital herpes were shown for 3 months of observation in experimental and control groups. A significant reduction of genital herpes recurrence frequency from 3.52 ± 0.09 (before treatment) to 2.89 ± 0.08 (after treatment) was noted in patients of group 3 (p < 0.001). The frequency of recurrences in the control group was 3.84 ± 0.10, that was higher than the parameters in all the experimental groups. A significant reduction of the rash area was noted in group 3, moreover, a redution of frequency of detection of clinical manifestations of genital herpes in the form of vesicle elements after treatment in groups 2 (p = 0.02) and 3 (p = 0.005) was found. Evaluation of local symptoms has established that burning have caused minimal discomfort for patients of groups 3 and 4 and itch and soreness--of groups 1 and 3. The least pronounced exacerbations were noted in patients of group 3. Intramuscular administration of fortepren preparation was established to result in the increase of titers of leukocyte virus-induced interferon for the whole duration of treatment. CONCLUSION: An intramuscular dose of 2 ml (8 mg) at recurrence stage 3 times at an interval of 21 days after the completion of the 10-day base course of treatment of the acute phase of chronical recurrent herpes virus infection of genital localization using acyclovir was accepted as an optimal dosage regimen. Analysis of the obtained results has shown an acceptable safety profile and a good level of tolerance for fortepren preparation.

[Antiviral activity and possible mechanisms of action of moraprenil phosphates during experimental infection caused by herpes simplex type 1 virus].

AIM: Study of antiviral activity of moraprenil phosphates (MPP) against herpes simplex type 1 virus (HSV1) in vitro and during experimental infection caused by HSV1 in mice. MATERIALS AND METHODS: Activity of MPP in vitro was tested by the ability to suppress formation of symplasts in VERO cells infected with HSV1, strain VR-3. A series of MPP that suppress virus-induced symplast-formation by 30 times was selected for in vivo experiments. Anti-viral activity of MPP in vivo was studied in HSV-1 infected mice after administration of either prophylaxis or therapy regimens. RESULTS: MPP at the dose of 20 microg/mice during s/c administration exhibited a pronounced prophylactic-therapeutic effect. Effectiveness of MPP during clinically evident herpes against the background of developing neurologic symptoms was demonstrated for the first time. Visual observation of the mice, that had received MPP as the first clinical symptoms of the disease appeared, has shown that against the background of preparation injection the clinical signs have ceased after 2 - 3 days and did not registered at least for the whole duration of the observation period (14 days). CONCLUSION: Active herpes infection is accompanied by the increase of FoxP3 expression in-thymus was shown. Possible mechanisms of anti-viral effect of MPP are discussed.

[Adjuvants in modern medicine and veterinary].

The review is dedicated to immunologic adjuvants--various natural and synthetics substances that are added to vaccines for stimulation of specific immune response, but they do not induce specific response themselves. Critically important is the selection of the correct adjuvants, for which mechanisms of effect on immune system are studied the most. The majority of these mechanisms as well as physical-chemical and biological features of modern adjuvants are analyzed in the review. The problem of safety of adjuvants, types of immune response induced by adjuvants of various nature, excipients that are being verified or already in use in modern medicine and veterinary are also examined.

[Antiviral activity of polyprenylphosphates in experimental infection caused by hepatitis C virus in vitro].

AIM: Study antiviral effect of sodium polyprenylphosphate (PPP) in experimental infection model caused by hepatitis C virus (HCV) in cell culture. MATERIALS AND METHODS: Cytopathogenic variant of HCV isolated from blood serum of a chronically infected patient was used. HCV infectious dose was 10.0 TCD50. Highly sensitive to cytopathogenic effect of HCV continuous swine embryo kidney cells (SPEV) as 1 day monolayer grown in 24 well plastic plates on 199 medium with 10% calf serum with addition of L-glutamine and antibiotics (100 U/ml) were used. PPP was used in concentrations that do not have cytotoxic effect (from 60 to 7.5 microg in 50 microl); introduced into SPEV cell cultures immediately after infection, 24 hours before or 24 hours after the infection of cells with HCV. Infectious activity of HCV was evaluated by using Reed-Muench formula based on results of medium samples titration obtained 3 days after the infection of cells. RESULTS: PPP was shown to have antiviral properties when added into the cell cultures immediately after the infection with HCV. Under the effect of PPP HCV titers were established to decrease by 3.0 lg (PPV dose of 60 microg) and by 1.9 lg (PPV dose of 30 microg). Positive effect was also obtained for prophylactic use of PPP. When PPP at a dose of 60 microg was introduced 24 hours before the infection of SPEV with HCV, the titer of the virus decreased by 3.5 lg. Prophylactic administration of low doses of the preparation (7.5 and 15.0 microg) also showed evident antiviral effect (decrease of infectious activity of HCV by 3.2 - 2.3 lg, respectively). CONCLUSION: PPP at the doses tested has an ability to reduce concentration of HCV in SPEV cell cultures when added immediately after infection or 24 hours before.

[Multidirectional effect of MIF and sodium polyprenyl phosphate on the course of experimental flavivirus infection in mice].

AIM: Study of macrophage migration inhibiting factor (MIF) effect after intracerebral administration on the course of experimental infection induced in mice by tick borne encephalitis virus (TEV), and study of sodium polyprenyl phosphate (PPP) and/or antibodies against MIF on the course of this infection against the background of MIF administration. MATERIALS AND METHODS: Phosprenil preparation was used as a source of PPP. PPP was administered intracerebrally. MIF--human recombinant (R&D, USA), mice--Balb/c line. RESULTS: In the sera of mice infected with TEV, MIF production stimulation was detected at days 8 through 10 after the infection--against the background of clinical signs presentation of tick borne encephalitis (TE). Administration of PPP to infected mice, on the contrary, resulted in MIF production suppression at the specified period. After administration of 20 ng of MIF to mice, lethality increased by 40% and average life span decreased by 2.3 days. Thus, MIF at high doses caused an increase of infection course severity, induced by TEV in mice, and administration of 60 microg of PPP resulted in the protection from infection in 100% of cases. Intracerebral administrationto mice of antibodies against MIF resulted in a decrease of lethality indicator up to 26% as compared with control and an increase of averagelife span by 5.5 days. During simultaneous administration into the brain of infected mice of MIF, PPP and antibodies against MIF, prevention of MIF-induced increase of TE course severity was registered. CONCLUSION: The data obtained allow to conclude that MIF may serve as an indicator of TE course severity, and possible prognostic indicator of meningo-encephalitic form development in humans.

[Antiviral and immunomodulating activity of polyprenyl phosphates in viral infections].

Expressed antiviral activity of Fortepren (FP) and Gamapren (GP), polyprenyl phosphate (PPP)-contaning agents, was demonstrated in experiments on mice infected with the human herpes simplex virus, type 1 (HSV1) or the vernal encephalitis virus (VEV). Since both the viral infections are of great social significance, the PPP-containing agents should be considered prospective for the medical practice. The experimental data suggested that both the drugs considerably inhibited the VEV infectiousness in the susceptible cell culture. The quantity of protein E, the main immunogen of VEV, in the culture fluid of the VEV infected cells was shown to be markedly lowered under the effect of FP and GP. It was demonstrated for the first time that FP and GP significantly inhibited evolution of the VEV protein E in the cell culture J-96. The experiments with the infectious rhinotracheitis virus (IRTV) of the corned cattle revealed that FP and GP greatly retarded the HSV1 development in the susceptible cell culture. One of the mechanisms of the antiviral action of the PPP-containing agents was likely the effect on the evolution of the virus proteins in the cells. The impact of FP on production of some key cytokines (CT) was studied on mice with experimental vernal encephalitis (IFN-gamma, IL-4 and IL-12). The content of the above mentioned CT in blood of the mice was determined by the IFA test. Under the normal conditions and in the mice infected with VEV, production of IL-12 and IFN-gamma was shown to be stimulated during the first 3-5 days after the FP administration, whereas in the animals not exposed to FP there was observed stimulation of the IL-4 production during the first 3 days after the contamination, followed by increased production of IL-12 and IFN-gamma.

[Experimental study of the phenomenon of antibody dependent tick-borne encephalitis virus infectivity enhancement in vitro].

Phenomenon of antibody dependent tick-borne encephalitis virus (TBEV) infectivity enhancement in the human monocytic cell culture J-96 has been studied. Three specific commercial sera that are generally used for therapy and prevention of TBE in humans were used. Results showed that enhancement of TBEV infectivity was markedly apparent following use of 2 out of 3 studied sera. Data obtained in the study prove that phenomenon of antibody dependent TBEV infectivity enhancement in monocytes cell culture may exist, but conditions for its realization in the organism are very unlikely due to the narrow range of antibodies' concentration and quantity of viral particles. A the same time, the fact of possible aggravation of TBE course after use of specific antisera for treatment and prevention of TBE in humans should not be ignored.

[Moraprenylphosphates suppress reproduction of Taylor murine encephalomyelitis virus and accumulation of VP3 viral protein in susceptible cell cultures BHK-21 and P388D1].

Influence of moraprenylphosphates (phosphorylated polyprenol of plant origin) upon the accumulation of Taylor murine encephalomyelitis virus VP3 protein in the susceptible cell cultures was studied. It has been shown that moraprenylphosphates inhibited the accumulation of VP3 at early stages of infectious process. Moraprenylphosphates were found to decrease infectivity of the virus as well as virus-induced cellular apoptosis. Mechanisms of immunomodulating and antiviral activity of moraprenylphosphates and prospects of their use as antiviral drugs have been discussed.

[Prolonged stimulation of hematopoiesis in mice with chemotactic peptide N-f-met-leu-phe-gly incorporated into liposomes].

The experimental study of macrophage-activating chemotactic peptide N-f-met-leu-phe-gly (chemotaxic peptide), as well as its liposomal form, on the proliferation and migration of colony-forming precursor cells in mice was carried out. The study revealed that the subcutaneous injection of chemotaxic peptide into mice in a dose of 20 Mg led to a pronounced, but short-term increase in the proliferation of such precursor cells in the marrow: as shown by the hydroxyurea "suicide" method, a day after the injection almost 50% of hematopoietic stem cells entered the S-phase of the cellular cycle; in addition, an increase in the content of colony-forming precursor cells in the peripheral blood and the spleen was observed. The injection of chemotaxic peptide, incapsulated into liposomes, led to a considerable increase in the duration of the stimulating effect, manifested by the maintenance of a stable proliferative state of the pool of hematopoietic stem cells during 4 weeks (the term of observation). This effect could be attributed to the formation of the liposomal "depot" and the gradual liberation of chemotaxic peptide from it.

[The correction action of Phosprenyl and Gamavit on the functional activity of mouse peritoneal macrophages in response to high doses of Staphylococcus aureus alpha-toxin].

The study of the functional activity of peritoneal macrophages of BALB/c mice at different stages of the toxic action caused by S. aureus alpha-toxin (ST) was carried out. The analysis of the dynamics of toxic reaction revealed the main critical points of triggering necrotic processes: the first hour and day 2. One hour after the injection of large doses of ST a sharp increase in the process of antigen binding with its subsequent sharp decrease. Simultaneously, a decrease in the activity of the lysosomal enzymes cathepsin D and acidic phosphatase was established, which was indicative of the destabilization of both lysosomal and cellular macrophage membranes. The increase of oxygen metabolism on day 2, together with the release of lysosomal proteases into the extracellular area, correlated with the maximum death rate of mice and served as the main index of the development of necrosis. The prophylactic and therapeutic use of the preparations Gamavit and Phosprenyl revealed their antitoxic activity and capacityfor stimulating the level of natural body resistance.

[The preparation phosprenyl suppresses diarrhea and cattle infectious rhinotracheitis virus multiplication in sensitive cell cultures]. / Preparat fosprenyl podavliaet razmnozhenie virusov diarei i infektsionnogo rinotrakheita krupnogo rogatogo skota v chuvstvitel'nykh kul'turakh kletok.

Fosprenil suppressed the multiplication of cattle diarrhea virus in calf coronary vessel cell culture. Added to the culture of infected cells in a dose of 200 mg, the drug decreased the virus titer 30-fold in comparison with infected control cultures. Antiviral activity of fosprenil towards infective rhinotracheitis virus multiplication was still higher: in a dose of 100 mg it decreased the virus titer in fetal calf lung culture 100-fold in comparison with the control. Moreover, the cytopathogenic effects of the viruses in infected cultures were 24-48 h delayed under the effect of fosprenil in comparison with infected control cultures. These results recommend fosprenil for the treatment of cattle viral diseases.

[Suppression of graft versus host reaction by depositing a magnet-controlled adriamycin dosage form in bone marrow allotransplantation in animal experiments]. / Podavlenie reaktsii transplantat protiv khoziaina s pomoshch'iu deponirovaniia magnitoupravliaemoi formy adriamitsina pri allotransplantatsii kostnogo mozga v ékperimente.

The main causes of failures during allogenous bone marrow transplantation is the development of graft versus host reactions. The methods of its prevention and treatment are the use of large doses of human toxic cytostatics and immunosuppressants aimed against donor immunocompetent cells. A way of preventing the adverse effects of cytostatics are targeted transport of long-acting cytostatic dosage forms to an organ or target cell through carriers, such as liposomes, microcapsules, microspheres and their conjugates with monoclonal antibodies. For this purpose, the study used gelatin and gum arabic microspheres containing the immunosuppressive cytostatic adriamycin. To enhance the efficiency of cytostatic depositing at the site of transplantation, the procedure of intraosseous transplantation of allogenous bone marrow transplantation with long-acting adriamycin depositing was developed. With this approach, the authors could not only deposit the agent, but could substantially increase the proportion of donor cells kept at the site of grafting as compared to the intravenous and intraosseous infusion of donor cells. The main advantage of the new technique of allogenous bone marrow transplantation in combination with a long-acting cytostatic dosage form is that acute and chronic graft versus host reactions can be inhibited long by using adriamycin in subtherapeutic dosages.

[Protective effect of a new antiviral preparation of phosprenyl in experimental tick-borne encephalitis]. / Zashchitnoe deistvie novogo protivovirusnogo preparata fosprenil pri éksperimental'nom kleshchevom éntsefalite.

Antiviral activity of phosprenyl was studied in BALB/c mice infected with tick-borne encephalitis (TBE) virus. Up to 60% animals infected with TBE virus survived after 1-3 intramuscular injections of phosprenyl. The mortality in the untreated group infected with the virus was 100%. Direct antiviral effect of phosprenyl was studied in sensitive SPEV cells infected with TBE virus. The titer of the virus decreased 10-fold in the cells treated with the drug vs. untreated control cells. Phosprenyl stimulates some interleukins: gamma-interferon, tumor necrosis factor-alpha, and interleukin-6. The stimulating effect of the drug manifests in intact animals and in those infected with TBE virus and treated with phosprenyl. The prospects of further trials of the drug as a therapeutic and prophylactic agent in TBE are discussed.

[Microorganism persistence in hematopoietic tissue: means for detecting it and its significance]. / Persistentsiia mirkoorganizmov v krovetvornoi tkani: puti vyiavleniia i znachimost'.

Many microorganisms are capable of prolonged persistence in the marrow. In this study, carried out by the method of negative selection based on the treatment of mouse marrow cells with specific antimicrobial sera and complement, Mycoplasma arthritidis and L-forms of group B streptococci were found to be capable of persisting in the marrow in close association with the late category of clonogenic precursor cells, CFU-7, as well as, to a lesser extent, with late erythroid precursors, CFUe. Early colony-forming cells, CFUs-12 and PFUe, as well as granulocyto-macrophagal precursors, CFUgm, did not practically express antigens to the given infective agents on their surface.