RU2304443C1 - Agent possessing antiviral activity - Google Patents

Abstract

FIELD: medicine, virology, pharmaceutical industry, pharmacy.

SUBSTANCE: invention proposes using drug fucoidan as an agent possessing antiviral activity with respect to Hantaan virus representing pathogen of hemorrhagic fever with renal syndrome. Fucoidan possesses high antiviral activity with respect to Hantaan virus representing a pathogen of hemorrhagic fever with renal syndrome. Invention can be used as an agent against Hantaan virus representing a pathogen of hemorrhagic fever with renal syndrome.

EFFECT: valuable property of agent.

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Description

The invention relates to medicine and relates to agents having antiviral activity against the Hantaan virus, a causative agent of hemorrhagic fever with renal syndrome (HFRS).

Hemorrhagic fever with renal syndrome is one of the first among natural focal infections in our country, as well as in several other countries, and is characterized by the development of immunocomplex generalized destructive panvasculitis with the subsequent occurrence of disseminated intravascular coagulation (DIC) and impaired microcirculation. An important role in the development of the disease belongs to violations of the mechanisms of cellular and humoral immunity [Ivanis V.A. Immunopathogenesis, clinic, immunocorrective therapy of hemorrhagic fever with renal syndrome (HFRS) in the region of circulation of different hantavirus serotypes. Abstract of a doctoral dissertation. 2004, 52 p .; Sirotin B.Z. Hemorrhagic fever with renal syndrome. Khabarovsk 1994, 302 p.]. Therefore, the search for new drugs for treating this disease is relevant.

Until now, the treatment of patients with HFRS is a difficult task. The use of antiviral drugs, in particular ribavirin, has not been widely used in practice and, according to the literature, remains a controversial issue [Huggins JW., Et.al. Ribavirin therapy for Hantaan virus infection in suckling mice. J. Infect. Dis. 1986, 153 (3): 489-497. Mertz C. J. et al. Placebo-controlled, double-blind trial of intravenous ribavirin for the treatment of hantavirus cardiopulmonary syndrome in North America. Clin. Infect Dis. 2004, 1; 39 (9): 1307-13].

Due to the fact that the severity of the course of the disease is largely determined by immune disorders, immunocorrective therapy began to be used in complex treatment. To date, specific immunoglobulin and α-interferon have been used in medical practice; recently, data have appeared on the use of roncoleukin in the complex treatment of HFRS [Ivanis V.A. Immunopathogenesis, clinic, immunocorrective therapy of hemorrhagic fever with renal syndrome (HFRS) in the region of circulation of different serotypes of hantavirus. Abstract of a doctoral dissertation. 2004, 52 p.].

The use of the antiviral drug amixin in the treatment of HFRS is known [RU 2196576 C1, 2003.01.20]. Amiksin refers to low molecular weight synthetic compounds of florenones and is an oral inducer of endogenous interferon. It primarily affects T cells, as well as enterocytes, hepatocytes and granulocytes and stimulates the synthesis of types 1 and 2 interferons in them.

The use of the non-steroidal anti-inflammatory drug iodantipyrine has been shown against the background of conventional therapy for HFRS [RU 2181046 C2, 2002.04.10]. Iodantipyrine belongs to the group of pyrazonol derivatives, has anti-inflammatory, antiviral, immunostimulating and interferonogenic properties. The drug is an inducer of interferons, significantly increases the activity of fibroplasts, delays the penetration of the virus into the cell due to the stabilizing effect on biological membranes, and significantly stimulates the production of antibodies. Despite the fact that no side effects were observed with the appointment of interferon inducers, it should be noted the possibility of exacerbation of diseases of the gastrointestinal tract and the development of allergic reactions.

It is known that one of the starting stages in the development of viral infections is the attachment of the virus to target cells. The study of the mechanism of viral replication showed that some substances can act selectively without affecting the host cell [F. Ershov Antiviral drugs. M., 1998, 192 p.]. Therefore, it is relevant to search for new, highly effective non-toxic biologically active drugs to interrupt or completely block the adsorption of the Hantaan virus.

Closest to the claimed tool according to the mechanism of action on hantavirus is lactoferrin [Kariwa H. at. al. In vitro antiviral activity of lactoferrin and ribavirin upon hantavirus. Arch Virol. 2000, 145 (8): 1571-82]. Lactoferrin, an iron-binding glycoprotein with a high molecular weight, is found in colostrum, saliva, and tears, is produced by cells of the glandular epithelium, is a marker of the inflammatory process and one of the factors of nonspecific defense of the body, has an antioxidant effect, and is involved in the development of the immune response [Sukharev A.E. and other Questions of medical chemistry, 1990, 3: 81-83]. It has been established that bovine lactoferrin inhibits the adsorption of hantaviruses on cell culture, inhibiting their infection both during processing of the virus and cells. Contacting viral particles, lactoferrin prevents their penetration into cells, thereby preventing or attenuating the development of the disease. At the same time, washing the cells treated with lactoferrin with phosphate-buffered saline reduces the inhibitory effect, which indicates weak adhesion of the drug to the cells [Kariwa H. at. al. In vitro antiviral activity of lactoferrin and ribavirin upon hantavirus. Arch Virol. 2000,145 (8): 1571-82].

The objective of the invention is the expansion of the arsenal of funds with activity against the Hantaan virus.

The problem was solved by using fucoidan as an agent with antiviral activity due to inhibition of adsorption on target cells of the Hantaan virus, the causative agent of hemorrhagic fever with renal syndrome.

It is known to use fucoidan from Fucus evanescens as an agent having anticoagulant and immunotropic effects [RU 2247574 C2 A61K 35/80; Kuznetsova TA, Zaporozhets TS, Besednova et al. Immunostimulating and anticoagulant activity of fucoidan from the brown alga of the Sea of Okhotsk Fucus evanescens. Antibiotics and chemotherapy. 2003, 48 (4): 11-13], the use of fucoidan from Laminariajaponica to inhibit HIV infection [RU 2019186 C1, 1994.09.15].

Fucoidan preparations from Laminaria cichorioides, Laminariajaponica, Fucus evanescens act on the compliment system [Tatiana N. Zvyagintseva, Natalia M. Shevchenko, Irina V. Nazarova et al. "Inhibition of complement activation by water-soluble polysaccharides of some far-eastern brown seaweeds." CBP Part C. 126. 2000, 209-215]. Fucoidan from Laminaria japonica stimulates the phagocytic and bactericidal activity of neutrophils [Zaporozhets TS, Makarenkova ID et al. Activation of neutrophil effector functions by biopolymers of marine hydrobionts. Medical immunology, 2003, 5 (1-2): 149-156].

With respect to fucoidans isolated from other algae species, it was found that fucoidan from Leathesia difformis reduces the penetration into the cell of herpes simplex virus type I and II and cytomegalovirus [Feldman S.C., et al. Antiviral properties of fucoidan fractions from Leathesia difformis. Phytomedicine. 1999, 6 (5): 335-40.], Fucoidan from Sargassum horneri in vitro inhibits the attachment and penetration of herpes simplex viruses I, cytomegalovirus and human immunodeficiency virus type I [Hoshino T. et al. An antivirally active sulfated polysaccharide from Sargassum homeri (TURNER) C. AGARDH. Biol. Pharm. Bull. 1998, 21 (7): 730-4.], Fucoidan from Macrocystis pyrifera inhibits the action of the vesicular stomatitis virus [Mayer A.MS. et al. Biological activity in Macrocystis pyrifera from Argentina: sodium alginate, fucoidan and laminaran. I. Antitumor, cytotoxicity and humoral response. Hydrobiologia 1987, 151: 483-489], sulfated polysaccharides from the alga Sargassum patents inhibit the adsorption and replication of herpes simplex virus type 2 [Zhu W., et al. Antiviral property and mode of action of a sulphated polysaccharide from Sargassum patents against herpes simplex virus type 2. Int. J. Antimicrob. Agents. 2004.24 (3), 279-83].

However, the new appointment of fucoidan, claimed as a means of inhibiting the adsorption of hantavirus, does not follow with obviousness from its known properties and was discovered by the authors for the first time.

Fucoidan preparations with activity against the Hantaan virus are obtained as a result of the method of complex processing of brown algae developed at the Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences [RU 2240816 C1, 11.27.2004]. The structural characteristics of fucoidan preparations are presented in the above information source.

The technical result provided by the invention consists in a more pronounced inhibitory effect of fucoidan on the adsorption of the Hantaan virus. It was found that when washing buffer with cells treated with fucoidan, the inhibitory effect of fucoidan on the adsorption of hantavirus does not decrease, unlike the prototype.

The technical result also consists in combining the inhibitory properties of fucoidan on the adsorption of the Hantaan virus, with its anticoagulant and immunostimulating effect in a wide range of doses. This is a very important advantage of the claimed drug in comparison with the known drugs used in the complex treatment of hemorrhagic fever with renal syndrome, because it is known that this disease is accompanied by severe violations of the mechanisms of cellular and humoral immunity and the occurrence of thrombolytic phenomena.

The inventive tool is non-toxic. The technology of its production is environmentally friendly, and the source of fucoidan is brown algae are widespread off the coast of the northern and eastern seas of Russia and are characterized by rapid renewal. All these advantages open up the possibility for the production of the claimed drug and its use in the complex treatment of HFRS with official therapeutic drugs.

Acute toxicity study of fucoidan.

Conducted on linear mice with intraperitoneal and oral methods of administration in doses of 0.5, 5, 25, 50 and 250 mg / kg, the observation period was 10 days. The studied indicators: survival, total body weight, general condition. Control animals received saline or distilled water. All mice that received fucoidan intraperitoneally (ip) or orally (p / o) remained alive during the entire observation period (table 1).

Table 1 The survival of mice with intraperitoneal and oral administration of fucoidan Dose (mg / kg), route of administration Duration of observation (day) one 2 3 four 5 6 7 8 9 10 0.5 in / b 10 10 10 10 10 10 10 10 10 10 5 b / w 10 10 10 10 10 10 10 10 10 10 25 b / w 10 10 10 10 10 10 10 10 10 10 50 b / w 10 10 10 10 10 10 10 10 10 10 250 b / w 10 10 10 10 10 10 10 10 10 10 50 p / o 10 10 10 10 10 10 10 10 10 10 250 p / o 10 10 10 10 10 10 10 10 10 10 The control 10 10 10 10 10 10 10 10 10 10

Observation in dynamics of the body weight of mice treated with fucoidan showed no differences compared with the control. Thus, it was found that fucoidan is not toxic in the studied dose range (from 0.5 to 250 mg / kg).

Study of the effect of fucoidan on the adsorption of hantavirus on a model of peritoneal macrophages.

Materials

1. Mice - white, outbred, weight 18-20 g.

2. Hantaan virus - strain 76-118, isolated from a suspension of lungs of field mouse Apodemus agrarius koreae. The experiment used a virus-containing culture fluid obtained by culturing the virus on Vero E6 cells (African green monkey kidney transplant line, C1106-CRL-1586 American Type Collection).

3. Fucoidan preparations from brown algae Laminaria japonica (L. japonica), Laminaria cichorioides (L. cichorioides), Fucus evanescens (F. evanescens).

4. Media: for the growth of Vero E6 cells and the propagation of hantavirus, MEM Needle medium was used with a double set of amino acids and vitamins, supplemented with L-glutamine (1 mg / ml), 7% SEC, penicillin (100 IU / ml) and streptomycin (50 mg / ml), as well as 3.5% serum of cow embryos (CEC). Nutrient medium 199 was used to isolate macrophages (production of GUP IPVE named after MP Chumakov RAMS, Moscow).

Research Methods.

Macrophages were obtained by introducing into the abdominal cavity of white mongrel mice 1 ml of sterile 10% peptone. Animals were opened on 3 days. To isolate macrophages, the abdominal cavity was washed with 199 heparin medium (5 U / ml). The tubes were pretreated with silicone (15-30 min) and kept on ice to prevent macrophages from sticking to the walls and bottom of the tubes. The resulting exudate was filtered and centrifuged for 10 min at 1000 rpm (2 times). Cells were counted in a Goryaev chamber (0.1 exudate + 4.9 ml of 3% acetic acid). The cell suspension was resuspended in medium 199 to a volume of 2 × 10 ⁶ [Laboratory methods for the diagnosis of hemorrhagic fever with renal syndrome. Guidelines. M., 1982].

In the neutralization reaction on the Vero E6 cell culture under the action of fucoidan (at a dose of 5 mg / kg) from Laminaria japonica, the decrease in the tissue infectious dose of the virus (TKID ₅₀ / 0.2) in relation to the control was 2.0 lg during pretreatment of cells and 3 , 0 lg when processing the virus. The action of fucoidan from Laminaria cichorioides was 2.0 and 2.5 lg, respectively. The fucoidan from Fucus evanescens had the least effect (a decrease of 1.5 and 2.0 lg).

Thus, the study showed that all studied fucoidan preparations interfere with the adsorption of hantavirus on Vero E6 cell culture. The decrease in the TKID of the virus indicates the effectiveness of the action of fucoidan, which is more pronounced during pre-treatment of the virus.

Taking into account that macrophages are target cells for hantavirus, and, taking into account the results obtained, we continued to study the effect of fucoidan on an in vitro model of mouse peritoneal macrophages.

To study the inhibitory effect of fucoidan on the adsorption of hantavirus, the Brilis method was used [V. Brilis. and other Methods of studying the adhesive process of microorganisms. Lab Case 1986, 4: 210-212.] in its own modification.

1. For pre-treatment of macrophages, 2 ml of cells and 0.2 μl of fucoidan in a final concentration of 5, 2.5 and 0.5 mg / ml were introduced into sterile paired vials with coverslips. The cell suspension was incubated at 37 ° C for 15, 30 min and 1 h. Then, 0.2 μl of virus-containing liquid (titer 10 ⁵ TKID 50 / ml) was added to the treated macrophages and left to interact, depending on the experimental conditions , for 15, 30 minutes and 1 hour at a temperature of 37 ° C.

2. For preliminary processing of hantavirus, 0.2 μl of virus-containing liquid and fucoidan in a final concentration of 5, 2.5 and 0.5 mg / ml were added to sterile paired vials with coverslips. Then they were incubated at a temperature of 37 ° C for 15, 30 min, and 1 h, and 2 ml of untreated macrophages were added. The cell suspension was left to interact for 15, 30 min and 1 h at a temperature of 37 ° C.

Intact macrophages and virus-containing fluid were used in the control. The interaction time was also 15, 30 min and 1 h at a temperature of 37 ° C.

3. To study the effect of fucoidan on further propagation of the Hantaan virus in macrophage culture after preliminary contact of the virus with fucoidan and macrophages with fucoidan for 1 hour, the culture fluid and non-attached macrophages were drained, the preparation was washed twice with MEM medium. Then, the MEM support medium containing 3.5% SEC was added to the vials and incubated for 2, 4, 6 hours at a temperature of 37 ° C, examining the drug for each time interval. Intact macrophages and virus-containing fluid were used in the control. The reaction time was 2, 4, and 6 hours at a temperature of 37 ° C.

After the interaction of the virus-containing liquid with macrophages, the cells fixed on the glasses were washed with physiological saline (3 sec), fixed in 96 ° alcohol (8-10 sec) and stained in the indirect method of fluorescent antibodies - NFMA [Besednova N.N. Experimental and clinical-immunological study of pseudotuberculosis infection - dis. doc honey. Sciences, 1980, 352 pp.]. Immune murine sera containing antibodies to the Hantaan virus (50 μl) were applied to fixed paired glass coverslips and left in a humid chamber at 37 ° C for 45 minutes. As secondary antibodies, we used a working dilution of anti-species prepared against mouse immunoglobulins, immunoglobulins labeled with fluorescein isothiocyanate - FITC (manufactured by the N.F. Gamalei Institute, Moscow) at a dose of 50 μl. To contrast the preparation, a solution of blue Evans was used on an FSB pH of 7.2, at a final concentration of 1: 100000. After application of immunoglobulin, the preparation was left for contact for 45 minutes in a moist chamber at a temperature of 37 ° C. After each contact, the preparation was washed three times for 3 minutes with phosphate-buffered saline pH 7.2, then twice with distilled water and dried at room temperature. The stained preparations were examined in a LUMAM-I2 luminescent microscope using an introduction-immersion lens (with magnification × 100). The presence of virus antigen was assessed by the presence of a bright green luminescent glow of cells. The results were calculated by the formula: the average number of luminous cells / 100 macrophages (CCM / 100 MF). The results are presented in table 2.

The effect of fucoidan on the adsorption of the Hantaan virus

table 2 Substances The control Macrophage processing 1 hour 1 hour Dose 5.0 mg / kg Abs. % Abs. % Fucoidan (L. japonica) 21.87 ± 2.67 one hundred 6.84 ± 1.55 ↓ to 31.27 Fucoidan (L. cichorioides) 22.44 ± 9.0 one hundred 6.47 ± 3.06 ↓ to 28.83 Fucoidan (F. evanescens) 22.44 ± 9.0 one hundred 9.66 ± 4.56 ↓ until 43.05 Substances The control Virus treatment 1 hour 1 hour Dose 5.0 mg / kg Abs. % Abs. % Fucoidan (L. japonica) 21.87 ± 2.67 one hundred 2.77 ± 0.82 ↓ to 12.66 Fucoidan (L. cichorioides) 33.74 ± 2.3 one hundred 5.27 ± 1.39 ↓ to 15.61 Fucoidan (F. evanescens) 33.74 ± 2.3 one hundred 9.70 ± 4.81 ↓ to 28.74

The experimental results showed that all preparations of fucoidan at a dose of 5 mg / kg inhibit the adsorption of the Hantaan virus. Fucoidan from L. japonica has the greatest effect. Therefore, in further experiments, we studied its effect on adsorption, depending on the dose and timing of the interaction. The results of the study are presented in table 3.

The effect of fucoidan (L. japonica) on the adsorption of the Hantaan virus in the model of peritoneal macrophages depending on the dose and time of interaction

Table 3 The control Fucoidan (L. japonica) 5 mg / kg 1 hour 1 hour (macrophages) 1 hour (virus) 21.22 ± 2.0 one hundred% 6.98 ± 1.1 ** ↓ up to 32.89% 3.15 ± 0.69 * ↓ up to 14.84% 30 minutes 30 minutes 27.97 ± 2.33 one hundred% 8.41 ± 1.51 ** ↓ up to 30.06% 3.34 ± 0.68 * ↓ to 11.94% 15 minutes 15 minutes 37.59 ± 5.13 one hundred% 9.05 ± 1.28 ** ↓ up to 24.07% 5.035 ± 1.02 ** ↓ to13.39% The control Fucoidan (L. japonica) 2.5 mg / kg 1 hour 1 hour (macrophages) 1 hour (virus) 21.22 ± 2.0 one hundred% 7.71 ± 1.28 ** ↓ up to 36.33% 4.56 ± 1.62 * ↓ up to 21.48% 30 minutes 30 minutes 27.97 ± 2.33 one hundred% 10.79 ± 1.86 ** ↓ up to 38.57% 5.015 ± 1.40 * ↓ up to 17.92% 15 minutes 15 minutes 37.59 ± 5.13 one hundred% 15.28 ± 2.56 ** ↓ up to 40.64% 5.79 ± 0.79 ** ↓ to15.40%

The control Fucoidan (L. japonica) 0.5 mg / kg 1 hour 1 hour (macrophages) 1 hour (virus) 21.22 ± 2.0 one hundred% 9.62 ± 0.94 ** ↓ up to 45.33% 6.32 ± 1.97 * ↓ up to 29.78% 30 minutes 30 minutes 27.97 ± 2.33 one hundred% 12.53 ± 1.97 ** ↓ up to 44.79% 7.87 ± 0.57 * ↓ up to 28.13% 15 minutes 15 minutes 33.14 ± 4.45 one hundred% 15.635 ± 0.005 ** ↓ up to 47.17% 5.715 ± 0.005 * ↓ up to 17.24% Note * - p 0.01; ** - p <0.05

Studies have shown that fucoidan, during pretreatment of macrophages, at a dose of 0.5 mg / kg (for 15, 30 minutes and 1 hour) reduces the adsorption of hantavirus to an average of 46%. When processing the virus for 15 minutes - up to 17%, 30 minutes and 1 hour - up to 29% in relation to the control. The action of fucoidan in a dose of 2.5 mg / kg - up to 38% when processing macrophages and up to 18% - with preliminary contact with the virus. At a dose of 5 mg / kg, the adsorption of hantavirus on average decreased to 29% and to 13.5%, respectively. It was established that the inhibitory effect of fucoidan is more pronounced upon preliminary contact with the virus, nevertheless, its effect on macrophages was also effective.

When studying the effect of fucoidan on the reproduction of the Hantaan virus, it was found that pre-treatment with a drug (at a dose of 5 mg / kg) during subsequent interaction for 2, 4 and 6 hours prevents the virus from multiplying by blocking receptors and inhibiting adsorption on target cells (table four).

The effect of fucoidan (Z. japonica) on the reproduction of the Hantaan virus

Table 4 Interaction time Macrophages Virus 2 hours control 11.57 ± 0.62 one hundred% 11.57 ± 0.62 one hundred% Fucoidan 5 mcg / ml 2 hours 3.95 ± 0.81 ↓ up to 34.18% 3.86 ± 0.52 ↓ up to 33.40% 4 hours control 16.72 ± 3.69 one hundred% 16.72 ± 3.69 one hundred% Fucoidan 5 mcg / ml 4 hours 5.53 ± 0.46 ↓ up to 33.11% 5.42 ± 0.069 ↓ up to 32.41% 6 hours control 19.15 ± 0.71 one hundred% 19.15 ± 0.71 one hundred% Fucoidan 5 mcg / ml 6 hours 8.45 ± 0.12 ↓ up to 44.12% 7.28 ± 0.36 ↓ up to 38.01%

It is known that on the surface of the hantavirus numerous protrusions are located, which are glycoproteins G-1 and G-2 [Dantas JR. Jr., et. al. Characterization of glicoproteins of viruses causing hemorrhagic fever with renal syndrome (HFRS) using monoclonal antibodies. Virol 1986, 151: 379-384], which have hemagglutinating activity and induce a pH-dependent cellular interaction [Arikawa J. et. al. Characterization of Hantaan virus envelope glicoprotein antigenic determinants defined by monoclonal antybodies. J. Gen. Virol. 1989, 70: 615-624]. These functions play a crucial role in the attachment of the virus to the surface of sensitive cells when the virus is "undressed" at the initial stage of infection. We believe that when the virus is treated with fucoidan, the glycoproteins of the virus are blocked, which leads to inhibition of adsorption on target cells and consequently leads to a decrease in virus replication.

The mechanism of the antiviral action of fucoidan in the processing of macrophages is realized due to their binding to certain membrane receptors on the cell surface. Blocking receptors and changing the surface structures of cells is an obstacle to the adsorption and further penetration of the virus into the cells of a macroorganism.

Claims (1)

The use of fucoidan as an agent with antiviral activity against the Hantaan virus, a causative agent of hemorrhagic fever with renal syndrome.