RU2483739C1 - Use of liophilised preparation of allofibroblasts for treating herpes virus diseases - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine and veterinary science, and is intended for treating herpes virus diseases. A lyophilised preparation of allofibroblasts is used as an antiviral agent.

EFFECT: invention is effective for treating herpes virus of human and vertebrate animals.

6 tbl, 4 ex

Description

The invention relates to the field of medicine and veterinary medicine, namely to the creation of funds for the treatment of humans and animals from diseases caused by herpes viruses.

The herpes virus family, which now numbers more than 100, includes viruses not only in humans, but also in various vertebrates (monkeys, horses, cattle, pigs, rabbits, cats, dogs, mice, etc.). Animal herpes viruses are not pathogenic for humans, with the exception of herpes virus In monkeys. Human herpes viruses have 8 herpes viruses, the most common of which are the herpes simplex virus type 1 (HSV-1) and the herpes simplex virus type 2 (HSV-2). The result of human infection with HSV-1 are, as a rule, various lesions of the skin and mucous membranes. HSV-2 causes damage to the genital organs, in particular genital herpes, severe diseases of the newborn. According to some estimates, up to 90% of the world's population is affected by the herpes simplex virus.

With herpetic lesions of the mucous membranes and skin integument, not only epithelial and dermal cells, but also the regional lymphatic system are involved in the infectious process. The disease is accompanied by a pronounced local inflammatory reaction - in the dermis there is swelling of the papillary layer and infiltrates, consisting mainly of lymphocytes and neutrophils, acidophilic inclusions are found in the nuclei of degenerated cells. An immunological response develops both for a viral antigen and for cellular antigens, which often leads to the formation of autoimmune diseases of the skin and mucous membranes [V.A. Isakov, E.I. Arkhipova, D.V. Isakov. Human herpes virus infection. - SPb .: 2006; M.A.Samgin, A.A. Khaldin. Herpes simplex (dermatological aspects). M .: 2002].

The clinical picture of herpetic eye diseases in 61.7% of cases is complicated by concomitant infection, in 55.4% - metabolic damage to eye tissue, increased intraocular pressure and secondary glaucoma - in 16.8% of cases, impaired lens transparency and cataract development - in 12, 7%.

The basis of the defeat of the ocular stroma, with herpetic eye diseases, followed by its destruction and scarring, is the inadequate response of the immune system to the viral and corneal antigen [Maychuk Yu.F. Herpes virus diseases of the eyes. - M .: 2000].

The drugs currently used to treat herpesvirus infections based on the particular pathogenesis of the disease are divided into 2 groups: a complex of antiviral drugs based on acyclovir and immunotropic drugs (myelopid, tactivin, immunoglobulin, interferons, etc.).

Acyclovir (zovirax, ganciclovir) are a synthetic analogue of pure nucleoside with high activity against human herpes viruses. The drug blocks the reproduction of the virus during its replication in the cell. The external use of acyclovir-based drugs also requires long-term use - for 7-12 days 5 times a day - which is associated with the low penetration of acyclovir into the cornea and skin. With its prolonged use, variants of the virus that are insensitive to the action of acyclovir may occur.

An indication for the appointment of immunotropic drugs is immunological deficiency, manifested by a decrease in one of the indicators of the T-link and / or monocytic-macrophage link, as well as a decrease in the functional activity of phagocytic cells. It is advisable to combine immunotropic drugs with antiviral therapy to prevent the appearance of acyclovir-resistant variants of the virus. Interferon group preparations (reaferon, он-interferon, roferon, leukinferon, viferon) are used for herpetic diseases of any localization.

The disadvantages of known drugs include addiction and side effects, such as a burning sensation, pain, discomfort, peeling from ointments.

For example, an antiviral agent in the form of a gel is known containing in 1 g of a mixture: concentrated interferon 5000-10000 ME, nipagin 0.002-0.004 g, 2% solution of styrene-maleic anhydride copolymer - the rest [Patent RU 2302881, IPC A61K 38/21 , A61K 9/06, A61P 31/12, 2007].

Also known is a pharmaceutical composition for the treatment and prevention of genital herpes, chronic papillomovirus infection and the prevention of cervical cancer, containing in 1 g: interferon 100000-1000000 ME; C-aminocaproic acid - 0.1-0.5; polyvinylpyrrolidone - 0.005-0.15; aluminum oxide - 0.5-1.5; lanolin - up to 100.0 [Patent RU 2180593, IPC А61К 38/19, А61К 38/21, А61К 45/00, А61К 31/197, А61К 9/02, А61К 9/06, А61Р 31/22, А61Р 35 / 00, 2002].

It is also known immunocorrecting antiviral drug containing 1 g: interferon human recombinant alpha and / or beta and / or gamma - 100 IU-20 million ME, tocopherol acetate or tocopherol derivatives - 0.010-0.07 g, sodium or calcium ascorbate or ascorbyl palmitate - 0.010-0.05 g, calcium pantothenate - 0.010-0.3 g, antiviral component - 0.01-0.4 g, formative and excipients - the rest [Patent RU 2411039, IPC A61K 38/21, A61K 31/355, A61K 31/475, A61K 31/522, A61K 47/00, A61K 9/02, A61K 9/06, A61K 9/08, A61P 31/12, 2011].

For the treatment of herpetic diseases, compositions from plant materials are also used. For example, the antiviral drug "Alpizarin" is known, obtained from the grass of Alpine copepod and yellowing penny or from mango leaves, and produced in the form of tablets and ointments [M.D. Mashkovsky. Medicines - M .: Medicine, 1993, vol. II, p.401]. In addition, it is known alcohol tincture of flowers and leaves of hawthorn blood red and young leaves of the maple tree with a ratio of 2-4: 1-2 and a ratio of raw materials and alcohol 1: 3 [Patent RU 2196598, IPC A61K 35/78, A61K 9 / 06, 2003].

A disadvantage of known preparations from plant materials is the lack of antiviral activity.

Other complex preparations based on various active substances are known, such as, for example, fullerene-polyhydropolyaminocaproic acid [Patent RU 2333752, IPC A61K 31/195, A61K 33/44, A61K 9/06, A61P 31/12, 2008], hydrochlorides (3-R-1-adamantyl) -1-ethylamines [Patent RU 2247714, IPC С07С 215/14, А61К 31/14, А61Р 31/16, А61Р 31/22, 2005] and based on other substances that have a side effect action and do not have a sufficient regenerating effect on cells damaged by the virus.

Treatment of animals for herpetic diseases is also an important task.

For the treatment of bovine vulvovaginitis, for example, vaccines are prepared based on the herpes virus strain causing the disease [Patent RU 2279474, IPC C12N 7/00, A61K 39/265, C12R 1/93, 2006; Patent RU 2111011, IPC A61K 39/295, A61K 39/15, A61K 39/155, A61K 39/265, 1998]. In addition, it is known the use of intrauterine devices, for example, suppositories containing one suppository, g: vivaton - 1.0; lanolin, hch - 1.0; ASD-2f - 0.5; cocoa butter - 8.0 [Patent RU 2375067, A61K 35/12, A61K 35/32, A61K 35/34, A61K 36/00, A61P 15/00, A61P 15/02, 2009].

Most of the listed known agents and formulations against herpes virus infection have side effects. In addition, almost all known drugs do not cause the regeneration of cells damaged by the virus.

The purpose of the present invention is the expansion of the range of effective immunobiological preparations for the treatment of human and animal diseases caused by the herpes virus, with high regenerating properties and the absence of side effects.

The problem is solved in that for the treatment of herpesvirus diseases in humans and animals it is proposed to use a lyophilized preparation from allofibroblasts, namely, cells of strain LEC-4 (81).

Allofibroblasts are allogeneic (cultured) fibroblasts.

Fibroblasts (from lat. Fibra - fiber and Greek. Blastos - germ, germ) - the main cellular form of the connective tissue of the body of vertebrates and humans. Fibroblasts are formed during embryogenesis from stem cells of mesenchymal origin.

Fibroblasts produce fibers and the main substance of connective tissue. For example, it is known that fibroblasts can produce type I and II collagens and extracellular matrix components: laminin, nidogen, tinascin, chondroitin-4-sulfate, proteoglycan, fibronectin, as well as cytokines, which are understood as a variety of growth factors, interferons, chemokines and interleukins . Cytokines mainly play a regulatory role in intercellular interactions, activating or inhibiting the activity of certain cells, including cells involved in inflammation, regeneration and the development of the immune response. The locality of the action of cytokines is associated with their ability to bind to a large number of receptors on the surface of cells.

Currently, allofibroblast cultures are widely used, as a rule, for the treatment of superficial lesions of the skin and mucous membranes, in particular severe burns, trophic ulcers and other superficial lesions of the skin and mucous membranes. Cellular systems form the central element of cellular therapeutic technologies used in cardiology, neurology, hematology, hepatology, dentistry, endocrinology and for revitalization purposes.

It is known to use allofibroblasts, namely, cells of strain LEC-4 (81), as a cell culture for replacement therapy, which is used as a suspension or as cells on a synthetic substrate for the treatment of burn patients, patients with injuries of the musculoskeletal system, during restoration and correction of the functions of damaged tissues and organs [Patent RU 2213775, IPC C12N 5/08, A61K 35/12, C12N 5/08, C12R 1:91, 2003].

The use of allofibroblasts in the surgical treatment of periodontitis and in dental implantation is known [Patent RU 2204332, IPC АВВ 17/00, А61К 35/48, С12Т 7/00, 2003; “The Use of Allofibroblasts in Dental Implantation” and “New Biotechnologies in the Surgical Treatment of Periodontitis”, authors - S.N. Fedotov, S.G. Shunevich, N.A. Soloviev, Materials of the VII All-Russian Scientific Forum with international participation “Dentistry 2005”, Moscow-2005, p. 279-281]. The use of allofibroblasts in the surgery of facial skull injuries is also known [“Cellular cultures in the surgery for facial skull injuries”, M.V. Turuntsev, N.L. Kuznetsova, S.A. Maltsev, www.medline.ru, volume 8, Surgery, May 2007].

According to the present invention, the cell preparations of strain LEC-4 (81) in lyophilized form are proposed for use in the treatment of herpetic infection in animals and humans.

Studies of the Yekaterinburg Research Institute of Viral Infections (JIIIVI) showed that a lyophilized preparation from allofibroblasts (AFB), namely cells of the LEC-4 strain (81), allows to reduce the treatment time and severity of the course of herpes virus disease not only in humans, but also in vertebrate mammals. Thus, it was found that APB affect not only human herpes viruses (HSV-1 and HSV-2), but also on herpes viruses pathogenic to animals. When using the claimed drug side effects were not detected.

The strain LEC-4 (81) is a strain of diploid cells, obtained by YNIIVI (AS USSR No. 1147748), certified by the Ministry of Health of Russia (registration certificate P No. 001402/01 2002), registered as original and stored in a low-temperature museum bank developer (applicant).

The cell culture from strain LEC-4 (81) is a morphologically homogeneous population of cells with a limited life span of a certain tissue origin, preserving a stable karyotype (2n at least 75% of cells), free of foreign agents, oncogenically safe, cultivated on artificial nutrient media in standard blood substitute bottles.

Cells of strain LEC-4 (81) lack histocompatibility antigens, which allows their use, including for the treatment of animals.

The results of the JINIVI studies showed that LEC-4 allofibroblasts (81) in addition to the previously known regenerating, immunomodelling and antibacterial effects, showed new properties - antiviral effect in the treatment of skin and mucous membranes affected by the herpes virus.

The antiviral effect of the claimed drug may be associated with an increase in the number of cytokines in the lyophilized preparation LEC-4 (81), which was found as a result of the JINR studies.

The lyophilized form of the strain LEC-4 (81) is obtained by the method described in patent RU 2398873, 2009. Cryopreserved diploid cell cultures are used as feedstock according to the regulatory documentation (“Isolation, cultivation and control of diploid cell strains.” Guidelines. Ministry of Health USSR. - M., 1979, p.16-22). As a raw material, cell cultures obtained from mammalian embryonic tissue, mainly pig, sheep embryonic tissue, can be used. For carrying out the claimed method, the most preferred are embryonic tissues of the first half of the gestational age (4-6 weeks). Cells obtained from embryonic tissue of such an early age are less differentiated, which positively affects the quality and quantity of the final product. As a source of embryonic tissue, embryos of the second half of pregnancy (10-12 weeks) can be used, however, in this case, the quality of the cells deteriorates, and the time of effective cultivation is significantly reduced.

The preparation of cell culture involves the use of pre-ground mammalian embryonic tissue washed with an enzyme solution at a temperature of 25-37 ° C, followed by trypsinization of the tissue by repeated processing of tissue pieces with stirring with a trypsin solution, the drained cells are treated with nutrient medium and centrifuged, the precipitate formed is resuspended and cultured in a monolayer in a growth medium based on the Eagle medium, incubated, followed by passage for at least five times h, the target product is treated with an enzyme solution to remove from the surface of the mattress, the suspension obtained is centrifuged, and the resulting cell mass is used to obtain the drug after cryopreservation.

For cultivation in a monolayer, a growth medium based on Eagle’s medium is used, which medium can be used, which consists preferably of equal amounts of MEM’s growth medium and 0.5% lactalbumin hydrolyzate (GLA) solution with 10% calf fetal serum (ETS) or a mixture of MEM Needle growth medium and 199 medium, or other known growth media containing a Needle growth medium.

The use of these media ensures the preservation of the viability of cell cultures and their maximum yield.

Cell harvesting is carried out with a mixture of solutions of 0.25% trypsin and 0.02% Versen followed by the addition of Eagle or GLA medium. The mode of cell harvesting ensures the maximum quantity and quality of the removed cell suspension, and subsequent centrifugation - high-quality deposition of the cell mass.

The preparation of a dry preparation is carried out by lyophilization of a pre-treated cell culture. The processing of cell culture is as follows. The reconstituted cell culture is cultured in a monolayer in the above-mentioned growth medium based on Eagle's medium, preferably for 4-5 days, after which the cell culture is removed and separated from the conditioned growth medium. The latter is taken into vials, and the removed cell layer is washed by centrifugation, the resulting cell suspension is brought to the desired concentration and poured into vials. Sterility samples are taken from vials with conditioned growth medium and from vials with cell suspension. Vials with conditioned growth medium and vials with cell suspension are first frozen to a temperature not lower than -20 ° C, and then thawed at a temperature of + 20 ° C - + 25 ° C. Frozen vials with cell suspension and conditioned growth medium are stored until bacteriological analyzes, but not more than 3 weeks. After thawing, the contents of the vials are sterilely poured into pharmaceutical vials, then frozen in open vials in sterile freeze drying cassettes at a temperature not exceeding minus 50 ° C, kept frozen for at least 48 hours, followed by lyophilization in two stages, at the first stages carry out desorption for at least 20 hours at a temperature of minus 50 ° С ÷ 0 ° С, and in the second stage, sublimation for at least 20 hours at a temperature of 0 ° С ÷ 30 ° С at a pressure of no higher than 15 Pa. dry bottles target pro uktom aseptically sealed.

The resulting preparation is stable when stored at a temperature of + 4 ° C for at least 12 months.

As examples of specific performance, we present the results of studies on the effects of allofibroblasts on human organisms, rats, and cows.

Example 1. Investigation of the response of outbred white mice to the administration of a lyophilized preparation of LEC-4 diploid cells (81), namely, the effect of the drug on the indicators of humoral immunity of white mice.

Studies were conducted on outbred white mice of both sexes, weighing 18.0-22.0 g (648 individuals). Mice were kept under standard vivarium conditions with the same diet. Work with mice was carried out in accordance with the "Rules for the use of experimental animals", approved by order of the Ministry of Health of the USSR No. 75 of 08/12/1977

The effect of the drug on immunogenesis was studied in vivo. The test drug was administered to animals intraperitoneally in an amount of 0.5 ml in various dilutions in physiological saline (0.9% NaCl solution). According to the experimental conditions, the animals were divided into the following groups:

Group I - animals that were injected with 0.5 μg / kg;

Group II - animals that were injected with 5.0 μg / kg;

Group III - animals that were injected with 50.0 μg / kg;

Group IV - animals that were injected with 500.0 μg / kg;

Group V - animals that were injected with 5000.0 mcg / kg

Group VI (control) - animals that were injected with saline in an amount equal to the volume of the study drug.

Studies of humoral immunity were performed 6 and 24 hours after exposure to the solutions of the test drug by evaluating the following quantitative blood counts:

- titer of immunoglobulins M (IgM), mg · cm ^-3 ;

- titer of immunoglobulins G (IgG), mg · cm ^-3 ;

- titer of immunoglobulins A (IgA), mg · cm ^-3 ;

- titer of immunoglobulins E (IgE), mg · cm ^-3 ;

- titer of γ-interferon (γ-IFN), pg · cm ^-3 ;

- titer of the tumor necrosis factor α-factor (α-TNF), pg · cm ^-3 ;

- titer of interleukin-1 (IL-1), pg · cm ^-3 ;

- titer of interleukin-2 (IL-2), PG · cm ^-3 ;

- titer of interleukin-6 (IL-6), PG · cm ^-3 .

The quantitative determination of titers of immunoglobulins and cytokines in blood serum was carried out using test systems based on the classic solid-state two-step “sandwich” enzyme-linked immunosorbent assay on microplates (Table 1).

The state of the formed peripheral blood elements in experimental mice was recorded on a Digicell-500+ hematology analyzer (Austria) 24 hours after drug administration (Table 2).

Statistical processing of the obtained experimental data was carried out according to generally accepted methods with the calculation of the arithmetic mean value (M) and confidence interval (m). Assessment of the significance of differences was determined by Student's criterion.

According to the results of the studies, the following was reliably established.

The inventive drug has a pronounced immunomodelling effect. A stimulating effect was noted for a dose of 500 μg / kg and above with an intraperitoneal route of administration. First of all, the production of α-TNF, IL-1 and IL-2 is activated. The suppression of the synthesis of IL-6 is expressed for a short time (observation period of 6 hours) and levels after 24 hours (Table 1).

The inventive preparation does not have a sensitizing effect on the macroorganism, because IgE level in all groups of experimental animals remained close to the values in the control group of animals.

An increase in the level of IgM and IgG concentrations in doses exceeding 500 μg / kg is apparently the result of a nonspecific inflammatory reaction with the intraperitoneal administration of a test drug containing xenogenic protein.

The indices of the quantitative composition of the formed peripheral blood elements (Table 2) indicate that, when administered intraperitoneally, the drug does not affect the hematopoiesis of experimental animals - after 24 hours, the indicators of the formed blood elements remained similar to those obtained in mice of the control group with physiological saline.

Example 2. The study of the effects of the claimed drug on a model of ophthalmic herpes.

The model is reproduced in rats by infection on a scarified cornea.

The purpose of the experiment is to determine the protective properties of the claimed drug with a localized focus of infection.

The drug was diluted with injection water (5 ml per vial). In 0.025 ml of the solution contains 5 thousand cells (when calculating lyophilization).

Laboratory rats (18 individuals) weighing 180-200 g under ether anesthesia were infected by applying a suspension of HSV-1 (with an infectious titer of 4.5 lg TCD ₅₀ / ml) on a scarified eye cornea (in a volume of 0.025 ml).

After infection, rats were divided into 3 groups of 6 animals:

Group I - "Virus Control";

Group II - “Experiment”;

Group III - Acyclovir.

Six rats of the Experiment group, 24 hours after infection, performed a twofold application of a solution of the claimed preparation with a 3-hour interval between manipulations. The specified procedure was carried out within 3 days from the moment of infection. Six rats of the Virus Control group received no treatment.

Six rats of the Acyclovir group, 24 hours after infection, were twice applied with the Acyclovir Hexal eye ointment with a 3-hour interval between manipulations. The specified procedure was carried out for 5 days.

Six rats of the Virus Control group received no treatment.

Monitoring the development of the clinical process (table 3) was carried out by the following methods:

- Definition of a security indicator (PZ)

$$P3=\frac{\mathrm{one\; hundred}*\left(\mathrm{AND}E-\mathrm{one}\right)}{\mathrm{AND}E}$$

$$\mathrm{AND}E=\frac{P\mathrm{one}}{P2}$$

IE - performance index;

P1 - the indicator of fatal cases in the group "Virus Control" (in%);

P2 - the indicator of fatal cases in the group "Experiment" or "Acyclovir" (in%);

- Identification and comparison of the HSV-1 infectious titer between groups of experimental animals in rinses from the corneal mucosa (Table 4).

The results of the study:

The group "Virus Control" observed the development of herpetic conjunctivitis in the period of 2-5 days, which was accompanied by severe general intoxication. Against the background of conjunctivitis, 4 individuals developed herpetic stomatitis, 1 rat showed symptoms of central nervous system damage. By 12 days, 3 rats had clouding of the cornea of the eye (Table 3). In rats of the Experiment group, the development of herpetic eye lesions was observed in only 2 rats, while the infection process was mild, the duration of the disease was less than 3 days. Symptoms of intoxication and damage to the central nervous system were absent. In rats of the Acyclovir group, herpetic eye lesions were observed in 3 individuals. By the 15th day in 1 animal revealed clouding of the cornea and symptoms of intoxication.

The protection index (PZ) when using the “Lyophilized cell preparation AFB” in the treatment of experimental ophthalmic herpes in rats was 66.6%, which is comparable with highly active chemicals like Acyclovir. The protection rate when using the eye ointment "Acyclovir Hexal" in our study was 67.4%.

The use of swabs from the corneal mucosa of the eyes of rats infected with HSV-1 showed that the production of the virus is observed in the animals of the “Virus Control” group (Table 4). In the period from 1 to 7 days, an increase in the HSV infectious titer was noted, which indicated the development of the disease with the corresponding clinical picture (Table 1). The presence of infectious HSV in animals of this group was recorded up to 25 days inclusive.

In infected rats treated with Acyclovir Hexal, the maximum infectious titer of the virus was determined 3 days after infection and amounted to 1.33 lg TCD ₅₀ / ml. By the 20th day, the infectious active virus in washes from the corneal mucosa of the eyes could not be isolated.

In infected rats of the Experiment group, the maximum infectious titer of the virus was 1.0 lg TTC ₅₀ / ml was determined 1-3 days after infection, and by the 9th day the infectious active virus could not be isolated in washes from the corneal mucosa.

Preliminary findings on the use of the “Lyophilized Antibiotic Drug" in the treatment of experimental rat ophthalmic herpes.

The inventive drug has a multifunctional effect, which is expressed in total in selective inhibition of cellular antigens, which reduces the severity of the inflammatory reaction and accelerates the regeneration of the mucous membrane of the cornea of the eye, and also inhibits the reproduction of the virus.

1) The local effect of the cytokines contained in the claimed preparation activates the production of pro-inflammatory IL-1, IL-2 and Ф-TNF, inhibiting the synthesis of IL-6 (see example 1), which regulates the processes of inflammation of the eye tissues. The regeneration processes are due to both direct and inducing action of the components of the extracellular matrix, which is part of the claimed drug.

2) Suppression of virus reproduction in the tissues of the mucous membrane of the cornea of the eye occurs as a result of activation of transcription of INF-ά, INF-γ, INF-1λ in the cells of the monocyte-macrophage link by the components of the claimed preparation — mRNA INF-ά, INF-γ, INF-1λ, INF -1β, INF-2λ. The suppression of the reproduction of the virus is indicated by a decrease in the infectious titer and the absence of pathological changes in the tissues of the eye, which are formed as a result of the active infectious process.

Example 3. Treatment of cows with pustular vulvitis (synonyms: infectious rhinotracheitis, pustular vulvovaginitis) with the BAV-S cell preparation.

The causative agent is Herpesvirus bovis 1.

The study was conducted by employees of the Department of Animal Infectious Pathology of the GNU Mezenskoye, Beloyarsky District.

The purpose of the study is to study the possibility of using the claimed drug for the treatment of genital forms of infectious rhinotracheitis of cows during pregnancy.

Clinical examination of cows was carried out in the second half of pregnancy. 3 groups of cows were formed (6 individuals in each group) with a pustular rash in the vulva with an intensity of at least 75%: 1 group (experimental) and 2 groups (control). Before treatment, all the cows of the groups were treated with the external surface of the labia with a warm weakly pink solution of potassium permanganate. The treatment in groups was carried out according to the schemes:

Group 1 (experimental): treatment with a solution of furatsilin, then liniment from thysol and the claimed drug. Processing was carried out 1 time in 3 days.

Group 2 (control): treatment with furatsilinom solution and tizol. Three times with an interval of 2-3 days.

Group 3 (control): treatment only with furatsilinom solution. Three times with an interval of 2-3 days.

The preparations were prepared ex tempore immediately before treatment of the animals. The drug tizol with hydrophilic properties was used as a tread.

The treatment results were recorded daily according to the results of a clinical examination and 2 days after the last treatment. On examination, the presence of pustular formations, hyperemia, and the amount of mucus on the surface of the vestibule vestibule were taken into account (Table 5). Based on the results of clinical observations, the application efficiency index (IE) was calculated:

$$\mathrm{AND}E=\frac{P\mathrm{one}}{P2}$$

P1 - an indicator of the severity of the clinical manifestations of pustular vulvitis in group 3 (in%);

P2 is an indicator of the severity of the clinical manifestations of pustular vulvitis in group 1 or group 2 (in%).

When analyzing the data obtained, it was found that the use of liniment from the insulator and the inventive preparation leads to the relief of the inflammatory process: in all cows of group 1, after triple treatment, swelling and hyperemia of the mucous membrane of the vestibule were reduced; a decrease in the intensity of pustular rash by 25-50% was observed, which indicates inhibition of virus reproduction. The efficacy index of the drug was 3.34.

In cows of the second (control) group, no pronounced changes were observed during treatment with tizol: in one cow, tissue infiltration of the vestibule of the vagina decreased and in two cows the number of pustules decreased by 25%. Index of the effectiveness of the drug -> 1.

In the third (control) group, after treatment with furatsilin expressed changes were not observed.

The effect of the claimed drug is expressed in total in selective inhibition of cellular antigens, which reduces the inflammatory response and accelerates the regeneration processes and, most importantly, enhances the direct antiviral effect due to the expression of cytokine genes.

Example 4. The use of the claimed drug for the treatment of patients with chronic genital herpes in the surface treatment of lesions of the skin and mucous membranes.

The study was conducted on the basis of the Sverdlovsk Regional Dermatovenerologic Dispensary.

The survey group included 17 men aged 20 to 43 years, who are registered in the dispensary. The duration of the disease was 2-5 years, the number of relapses - 3-5 per year. The main clinical manifestations during relapse are multiple small erosions and / or fused into larger lesions on the mucous membranes and skin of the genital organ, scrotum and perineum, accompanied by swelling and hyperemia of the genital, painful urination, burning, itching, low-grade fever, sleep disorder and general psychological depression. The clinical diagnosis of genital herpes was confirmed by laboratory tests by detecting HSV antigen in scrapings and smear impressions from the urethral canal and in urine sediment using fluorescent antibodies, as well as virus isolation on sensitive cell cultures (LEC and Vero) from blood, urine and scrapings from urethral canal.

1. Control group (9 people): the drug “Acyclovir” was used for treatment for 5 days, 200 mg 5 times a day per os and five-fold treatment of lesions with the antiviral drug “Acyclovir Hexa”;

2. Experimental group (8 people): during the relapse, patients took the drug “Acyclovir” per os for 5 days at 200 mg 5 times a day; for the treatment of herpetic erosion of the skin and mucous membranes of the penis, the claimed preparation was used at the rate of 50 thousand cells per 1 cm ^{2 of the} affected surface, 2 times a day for 5 days.

To obtain the greatest therapeutic effect, treatment was started no later than 72 hours from the onset of clinical manifestations.

Clinical examination of patients was performed on the 1st, 3rd, 7th and 10th day of treatment. Upon examination, the presence of: 1) vesicular and pustular elements, erosion and ulceration; 2) hyperemia and edema of tissues; 3) elevated temperature; 4) painful urination, burning, itching; 5) sleep disorders and general psychological depression. The intensity of clinical manifestations was expressed as a percentage (table 6).

The detection of HSV antigen in smears from the mucosa of the urogenital tract and erosive lesions was carried out in the reaction of indirect immunofluorescence on the HerpesFluoScreen test systems (NPO Niarmedik plus, Moscow) on the day of treatment, on days 3, 7, and 10 of treatment (Table 6 )

The onset of relapse was recorded upon the appearance of class M immunoglobulins (Ig M) against HSV in the blood serum, which were detected by ELISA on the Herpes-screen test systems manufactured by Bioservice CJSC (Moscow).

Clinical blood analysis from patients was performed using a System 9020 hematology analyzer on the day of treatment and on the 10th day of treatment.

To assess the severity of the inflammatory process, we used the leukocyte intoxication index (LII) - according to the formula of Y. Y. Kalf-Kalifa, taking into account all the cellular elements of the leukogram:

$$L\mathrm{AND}\mathrm{AND}=\frac{\left(\mathrm{four}mc+3\mathrm{Yu}+2n+\mathrm{from}\right)\times \left(Pl+\mathrm{one}\right)}{\left(l+m\mathrm{about}\right)\times \left(\mathrm{uh}+\mathrm{one}\right)},$$

where in the percentage - mc - myelocytes, juvenile metamyelocytes, n - stab neutrophils, s - segmented neutrophils; pl - plasma cells, l - lymphocytes, mono - monocytes, e - eosinophils; 1, 2, 3, 4 - coefficients.

The level of pro-inflammatory (IL-1β, IL-6 and TNF-ά) and anti-inflammatory (IL-4) cytokines in the blood serum was determined by a standard method using the ProCon test systems (Protein circuit LLC St. Petersburg) per day treatment and on the 10th day of treatment (table 7).

Statistical data processing was performed using standard methods, the program "Statsoft Windows", version 4.0 (USA).

Research results

In patients on the day of treatment, clinical manifestations of recurrence of genital herpes virus infection were recorded in 100% of cases. In the blood serum of patients by ELISA, class M immunoglobulins (Ig M) were diagnosed for HSV, which confirms the onset of relapse. HSV antigen by IF was detected in the cells of the mucous membrane of the urogenital tract and / or erosive lesions in 100% of cases.

With a relapse of genital herpes in patients, leukocytosis (up to 17.5 ± 0.49 · 10 ⁹ / L), acceleration of ESR (up to 28.7 ± 1.53 mm / h) and a change in the percentage of blood cell elements in the leukocyte formula ( neutrophils up to 69.7 ± 3.41%, lymphocytes 24.18 ± 0.05%). The data obtained showed that in relapse in all 17 patients, LII increased almost 5 times compared with the same indicator in practically healthy patients (0.61 ± 0.09) and amounted to 3.01 ± 0.18, which indicates a pronounced inflammatory process.

The study of blood serum from patients on the day of treatment showed that the level of IL-1β and TNF-ά did not increase more than 2 times compared with normal values. At the same time, a progressive increase in the level of IL-6 was noted. The concentration of the anti-inflammatory cytokine IL-4 in the blood serum of all patients during the recurrence of HBV was 2.2 times higher than the level of normal values (table 6). One of the pathological effects of high concentrations of IL-6 (a mediator of local inflammation) is an increase in vascular endothelial permeability, which leads to the development of edema, which was observed in all patients. Moreover, unbalanced concentrations of the cytokines IL-1β, TNF-ά, and IL-4 are negative in themselves, damaging in nature, exacerbating pathological changes. Violation of the synthesis of IL-1β has an immunosuppressive effect on the functional activity of phagocytic cells, significantly reducing their ability to eliminate the virus from the body and contributing to the expansion of the focus of infection. As a result, not only epithelial cells, but cells of connective tissue and skin are involved in the infectious process (table 7).

In patients of group 1 who received only the antiviral drug Acyclovir Hexa, a decrease in the severity of the clinical manifestations of recurrence of genital herpes was observed on the 7th day in 33.3% of cases, epithelization of erosive lesions of the skin of the genital organ and perineum on the 10th day at 66 , 6% of cases. HSV antigen by IF method was detected in the cells of the mucous membrane of the urogenital tract and / or erosive lesions on the 10th day in 44.4%. An analysis of the results of studies of hematological parameters and the level of IL-1β, IL-6, TNF-ά, IL-4 cytokines in the blood serum revealed a positive dynamics of changes characteristic of the end of relapse stage.

In patients of group 2, by the 7th day, clinical manifestations of recurrence of genital herpes were observed in 50% of cases, epithelization of erosive lesions of the skin of the penis and perineum - on the 10th day in 75% of cases. HSV antigen by IF method was detected in the cells of the mucous membrane of the urogenital tract and / or erosive lesions on the 10th day in 37.5%. The severity of inflammatory reactions significantly decreased and was more pronounced than in patients of group 1. The results obtained corresponded to indicators of the stage of disease remission.

Thus, the use of the claimed drug in the complex treatment of recurrence of genital herpes showed that the "Lyophilized cellular preparation of APB" has a local anti-inflammatory and antiviral effect.

Table 3 The dynamics of the development of clinical manifestations of experimental herpes virus infection in rats treated with the “Lyophilized drug APB” Group Clinical manifestations (number of individuals in%) The nature of eye damage (conjunctivitis, clouding of the cornea of the eye) The presence of a bacterial infection CNS lesions (paresis, paralysis, etc.) Lesions of the mucous membranes of the mouth, nose, genitals Asthenic syndrome, intoxication "Virus control" (6 rats infected with HSV-1, not receiving treatment) 100/50 67 16.7 67 83.3 "Experiment" (6 rats infected with HSV-1, treatment with the claimed drug) 33.3 / 0 - - 16.7 - "Acyclovir" (6 rats infected with HSV-1, treatment with the drug "Acyclovir Hexal") 33.3 / 16.7 16.7 - 16.7 16.7

Table 4 Isolation of Infectious HSV-1 in Rinses from Corneal Mucosa of Rats of Experimental Groups in Vero Cell Culture Group Day after HSV infection (virus infectious titer in lg / number of individuals in%) one 3 5 7 9 12 fifteen twenty 25 "Virus control" (6 rats infected with HSV-1, not receiving treatment) 1.0 / 33.3 1.66 / 67 2.0 / 83.3 2.0 / 100 1.66 / 83.3 1,5 / 50 1.0 / 50 0.66 / 50 0.5 / 16.7 "Experiment" (6 rats infected with HSV-1, treatment with the claimed drug) 1.0 / 50 1.0 / 33.3 0.6 / 16.7 0.33 / 16.7 - - - - - Acyclovir (6 rats infected with HSV-1, treatment with Acyclovir Hexal) 1.0 / 33.3 1.33 / 50 1.0 / 67 0.6 / 33.3 0.33 / 33.3 0.33 / 16.7 0.33 / 16.7 - -

Table 5 The results of clinical studies of cows with vulvovaginitis in the treatment of lyophilized preparation "APB" Groups of animals The severity of the clinical manifestations of vulvovaginitis (in%) Inspection Results Before Treatment After 1 use of drugs After 2 use of drugs After 3 use of drugs one 2 3 four 5 1 group Hyperemia of the mucous surface of the vestibule with severe infiltration, pustular rashes Hyperemia of the mucous surface of the vestibule, swelling, pustular rashes Hyperemia of the mucosal surface of the vestibule with a weak infiltrate, pustular rashes Slight hyperemia of the mucous surface of the vestibule, pustular rashes treatment: tizol + "APB n = 6 75-100% 75% 60-50% 25-30% 2 group Hyperemia of the mucous surface of the vestibule with severe infiltration, pustular rashes Hyperemia of the mucous surface of the vestibule with severe infiltration, pustular rashes Hyperemia and infiltrate in the tissues of the vestibule of the vagina, pustular rashes Hyperemia of the mucous surface of the vestibule with severe infiltration, pustular rashes treatment: tizol n = 6 75-100% 75% 75% 75% 3 group Hyperemia of the mucous surface of the vestibule with severe infiltration, pustular rashes Hyperemia of the mucous surface of the vestibule with severe infiltration, pustular rashes Hyperemia of the mucous surface of the vestibule with severe infiltration, pustular rashes Hyperemia of the mucous surface of the vestibule with severe infiltration, pustular rashes without treatment n = 6 75-100% 75% 75% 75%

Table 6 Manifestations of recurrent genital herpes in patients and detection of antigen HSV by IF method in clinical materials Indicator Group 1 (drug "Acyclovir") day after treatment (n = 9) Group 2 (drug APB) day after treatment (n = 8) 0 * 3rd 7th 10th 0 3rd 7th 10th Clinical manifestations of genital herpes (in%) one hundred 88.8 67.7 55.6 one hundred 75 fifty 25 Detection of HSV antigen (in cells of the urethral mucosa and erosive foci) (in%) one hundred 88.8 55.6 44,4 one hundred one hundred fifty 37.5 Note: "0 *" - the day of treatment of the patient; "Clinical manifestations" - the number of patients with symptoms of genital herpes.

Table 7 Changes in the inflammatory response in patients with genital herpes with different treatment regimens Indicator Group 1 (drug "Acyclovir") (n = 9) Group 2 (drug APB) (n = 8) before treatment after treatment before treatment after treatment LII 3.01 ± 0.14 2.24 ± 0.11 3.07 ± 0.14 1.57 ± 0.15 IL-1 (pg / ml) 73.8 ± 11.7 55.7 ± 6.3 75.2 ± 9.7 35.4 ± 5.1 IL-6 (pg / ml) 379.7 ± 22.1 327.1 ± 21.7 384.1 ± 21.8 231.4 ± 19.7 IL-4 (pg / ml) 71.8 ± 18.2 81.7 ± 20.9 70.4 ± 18.5 96.9 ± 23.7 ά-TNF (pg / ml) 61.5 ± 16.1 56.2 ± 11.6 62.9 ± 14.5 37.2 ± 10.9

Claims (1)

The use of a lyophilized preparation of allofibroblasts for the treatment of human and vertebrate diseases caused by the herpes virus.