RU2482866C2 - Method of treating papilloma viral infections implemented through interleukin-18 induction - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to dermatovenerology and may be used to treat the infections caused by human papilloma viruses, implemented through interleukin-18 induction. For this purpose, the inducer is presented by medicines of the peptide alloferon using the mechanical removal of any type of papilloma formation. The inducer is presented by injections of the preparation 'Allokin-alpha lyophilisate for solution for subcutaneous administration'; the therapeutic course is 3 injections every second day, and the course may be repeated. The inducter is presented by alloferon suppositories. The inducers are presented by the ointments containing alloferon.

EFFECT: method provides the effective treatment ensured by the stimulation of natural immunity through interleukin-18 induction.

4 cl, 3 tbl, 4 ex

Description

The invention relates to medicine and can be used in dermatology, venereology, oncology and other fields related to the stimulation of immunity by induction of interleukin-18. The invention relates to methods of immunocorrective therapy.

Known (Chernysh S.I., Kim Soo In, Becker G.P., Makhaldiani N., Hoffmann J., Bule F. RF Patent No. 2172322) a group of antiviral peptides isolated from insects with the common name alloferons. Based on one of the peptides of this group of alloferon-1, the drug Allokin-alpha lyophilisate was developed for the preparation of a solution for subcutaneous administration, which has been available since 2003 as a medicine against chronic recurrent herpes type 1 and 2 and acute hepatitis B.

Alloferon is a selective specific stimulator of the functional activity of natural killers (NK), which is associated with a direct increase in the cytotoxic activity of these cells, and not with an increase in their number (Ershov F.I., Kubanova A.A., Pinegin B.V. et al. Influence allokina-alpha for relapses of chronic genital herpes // Materia Medica. - 2003. - No. 4 (40). - P.103-111). Alloferon in its pharmacological action is close to interferon-alpha. The difference between alloferon is that it not only induces the synthesis of endogenous interferons, but also activates the natural killer (NK) system (Chernysh SI, Kim SI, Bekker G. et al. Antiviral and antitumor peptides from insects // Proceedings of National academy of science of USA. - 2002. - Vol.9. - No. 20. - P.12628-12632, Chernysh S.I. Allokins (cytokine-like insect peptides) as modulators of the immune response of humans and other mammals // Russian Journal of Immunology. - 2004 .-- Vol. 9 - S.1. - P.36).

NKs recognize certain structures of high molecular weight glycoproteins that are expressed on the membrane of virus-infected cells. HKs are not antigen-specific cells, cause lysis of target cells independent of antibodies and complement, recognize their target cells without the participation of histocompatibility antigens and are factors of natural resistance. The cell responds to stimuli to which there are receptors on the membrane. They have receptors for cytokines (at least for IL-2, 4, 10, 12, IFN-gamma), for MHC-1 antigens, CD16 (Fc-gammaRIIIA). The latter receptor is responsible for the activation of the HK cytotoxic apparatus and the implementation of the antibody-dependent cellular cytotoxicity (ADCCT) reaction. As a result, the contents of intracellular HK granules are ejected into the extracellular space. The main role here belongs to perforin (cytolysin). Perforin is embedded in the membrane of the target cell and forms transmembrane pores, which leads to cell death, since the contents of the cell flow through these pores (Cerwenka N. 2001. NATURAL KILLER CELLS, VIRUSES AND CANCER).

HK proliferation and activation is controlled by the cytokines EL-2, IL-15, IL-18. Significant synergy is observed between the effects of IL-12 and IL-18 (Papamichail M, Perez SA, Gritzapis AD, Baxevanis CN. Natural killer lymphocytes: biology, development, and function. Cancer Immunol Immunother. 2004; 53 (3): 176-86 , Baxevanis CN, Gritzapis AD, Papamichail M. In vivo antitumor activity of NKT cells activated by the combination of IL-12 and IL-18. J Immunol. 2003; 171: 2953-2959).

Interleukin-18 (IL-18, IL-18), also known as IFNγ-inducing factor (IGIF), was primarily characterized as a potential inducer of the synthesis of IFNγ T and NK cells. IL-18 is a non-glycosylated polypeptide that does not have a classical signal sequence. IL-18 is synthesized as an inactive propeptide with a mass of 24 kDa. After proteolytic cleavage under the influence of caspases, a mature active peptide with a molecular mass of 18 kDa is formed. Regardless of IL-12, IL-18 affects the secretion of IFNγ and quickly activates the cells of the monocyte / macrophage system, which leads to the activation of many antibacterial, antitumor and antiviral responses. IL-18 itself is induced by stress signals (neurogenic or bacterial origin). It is believed that stress-induced release of IL-18 can lead to an increase in the IFNγ / IL-18 cycle: following the first wave of IFNγ formation by lymphocytes induced by IL-18, the newly synthesized IFNγ, in turn, stimulates monocytes / macrophages, which leads to an increase their ICE activity. An increase in ICE activity, in particular, leads to the formation of IL-18. IL-18 not only stimulates the synthesis of IFNγ, but also modulates its functional activity. It was shown that expression of the Fas ligand by CD4 + - Th 1 and NK cells also occurs under the influence of IL-18. On the other hand, it was shown that IFNγ is involved in the activation of expression of Fas itself. Thus, it can be concluded that IL-18 alone (FasL) or through IFNγ (Fas) stimulates the initialization of apoptosis processes (Hirata J, Kotani J, Aoyama M, Kashiwamura S, Ueda H, Kuroda Y, Usami M, Okamura H, Marukawa SA role for IL-18 in human neutrophil apoptosis. Shock. 2008 Dec; 30 (6): 628-33).

Papillomavirus infection is one of the most common viral infections. More than 50% of the sexually active population of the world is infected with human papillomavirus (HPV) during their lifetime. Despite the fact that in recent years effective antiviral therapy has been developed for many viral infections, the number of virus-associated diseases remains high. Cervical cancer associated with persistent human papillomavirus (HPV) infection is one of the most common and dangerous types of malignant neoplasms in women around the world. According to the WHO, every year around 500,000 new cases of cervical cancer are diagnosed in the world, which is second in the world as the cause of cancer death among women. Today, at least 700 million people are carriers of HPV infection, and the number of deaths associated with HPV infection reaches 280 thousand a year (Kiselev V.I., Kiselev O.I. Human papillomaviruses in development cancer of the cervix uteri. St. Petersburg-M.: Rose of the World, 2003).

The result of the infection are diseases of the anogenital region: cancer of the cervix, vulva, vagina, perianal region and genital warts. It was established that the malignancy of HPV-infected epithelial cells is due to the synthesis of two oncoproteins - E6 and E7, encoded by the HPV genome. In this case, the main role in the induction of tumor transformation belongs to the oncoprotein E7 (Baxevanis CN, Gritzapis AD, Papamichail M. In vivo antitumor activity of NKT cells activated by the combination of IL-12 and IL-18. J Immunol. 2003; 171: 2953- 2959).

Protein E6 HPV types 16 and 18 can cause malignancy of epithelial cells of the mammary gland. Proteins E6 and E7 work cooperatively, causing uncontrolled proliferation of primary human fibroblasts and keratinocytes (Young-Sik Cho, Jeong-Woo Kang, MinChul Cho et al. Down modulation of EL-18 expession by human papillomavirus type 16 E6 oncogene via binding to IL- 18. FEBS Letters 2001; 501: 139-45).

The E6 protein of highly oncogenic HPV types is classified as a high-risk protein, interacts with the P53 protein and forms a stable complex with it. This leads to rapid proteolytic degradation of P53 in the ubiquitin-dependent pathway. Currently, a clear correlation has been established between the carriage of genital HPV, malignancy of human keratinocytes, proteolytic destabilization of P53 and the association of certain types (types 16 and 18) of HPV with human tumors (Phelps WC, Barnes JA and Loba DC. Human papillomavirus: molecular targets and targets for antiviral therapy. Internal Antiviral News 1999; 7: 4-80).

Moreover, it is E6 protein that binds to interleukin-18, which is the main inducer of gamma-interferon, which leads to blockade of cellular cytotoxic immunity reactions. The negative effect of E6 / E7 proteins on the expression of the gene encoding IL-18 was established, which indicates the presence of a specific immunosuppression mechanism in HPV that suppresses the production of IL-18 (Cho YS, Kang JW, Cho MC et al. Down modulation of EL-18 expession by human papillomavirus type 16 E6 oncogene via binding to IL-18. FEBS Letters 2001; 501: 139-45).

It was also shown that proteins E6 and E7 bind to interleukin-18 in the E42A region and neutralize its action (Lee CA, Cho KJ, Kim SH, Shim JH, Lim JS, Cho DH, Song MS, Dinarello CA, Yoon DY. IL -18 E42A mutant is resistant to the inhibitory effects of HPV-16 E6 and E7 oncogenes on the IL-18-mediated immune response. Cancer Lett. 2005; 229 (2): 261-70).

From the above data it follows that an increase in the amount of IL-18 in the body should contribute to its fight against HPV. At the same time, drugs that induce the induction of IL-18 may be anti-HPV drugs.

Currently, drugs that selectively affect HPV do not exist. Most often, interferons (IFN) are used to treat HPV infection. They are a family of proteins that are produced by cells of the immune system in response to stimulation by viral antigens, mitogens. The recombinant preparation a-IFN, which has different trade names: “Etferon” (Egis), “Velferon” (Wellcome), “Reaferon”, “Interal” (Russia), “Realdiron” (Lithuania) and others (Silver, 2005). But in 60% of cases, even prolonged IFN therapy does not lead to clinical improvement. It was shown that the effectiveness of treatment directly depends on the level of synthesis of the oncoprotein E7. It was found that the E7 protein neutralizes the antiviral activity of a-IFN due to the fact that it selectively blocks most of the genes induced by IFN. In addition, E7 intracellularly inactivates IRF (IFN regulation factor), which involves the transcription of genes encoding the synthesis of antiviral proteins. This suggests that the E7 protein not only performs the functions of the main factor of malignancy, but also determines the resistance of cells to IFN therapy. Therefore, before IFN therapy, it is advisable to determine the level of oncoprotein E7. Its high values may indicate the inappropriateness of the use of IFN at the first stage of treatment of HPV-dependent pathology of the cervix. In this situation, the use of drugs based on the alloferon oligopeptide is a promising tool for blocking the development of human papillomavirus infection by inducing IL-18.

Thus, the prior art allows us to consider the effective use of drugs based on alloferon as an inducer of IL-18.

The present invention is a method of stimulating innate immunity through the induction of interleukin-18 using preparations based on the alloferon peptide, including the drug Allokin-alpha lyophilisate for the preparation of a solution for subcutaneous administration.

The ability to achieve the objectives of the invention is confirmed by the following examples.

Example 1. Induction of IL-18 by a single injection of the drug "Allokin-alpha"

Induction was carried out by administering Allokin-alpha subcutaneously once in a dose of 2 mg per kg of laboratory animal weight. For the experiment, BALB / C mice were used, females weighing 16-22 grams, age 14 weeks. The level of interleukin-18 (IL-18) in the blood serum was determined by enzyme-linked immunosorbent assay using specific antibodies. The amount of IL-18 in serum was determined at 1, 2, 3, 4, 5, 6, 8, 12, 16, 24, 32, 40, 48, 54 hours after administration of the drug. At the same time, the "background" amount of IL-18 present in the blood serum of the studied animals was studied. For this purpose, 14 mice were used as a control group, which received a subcutaneous injection of saline. In the experimental group, 42 animals were used. In the case of the control group, the confidence interval was calculated from the dilution measurements of the same sample. In the experimental group, the confidence interval was calculated relative to three samples from three animals for each measured point.

The test results of the drug are presented in Figure 1. 3-4 hours after administration, a small significant peak in the increase in IL-18 concentration is observed. After 32 hours, there is a second peak of a significant increase in the content of IL-18 in the blood serum of the experimental group of animals, which lasts about 20 hours. The presence of the first peak at 3-4 hours is probably due to nonspecific induction. A significant increase in IL-18 on the 2-3rd day of the experiment indicates the specific ability of the drug to induce the studied cytokine.

With subcutaneous administration of the drug "Allokin-alpha" after 3 hours, the induction of IL-18 is shown, which lasted up to 3 days. The maximum concentration of IL-18 is achieved 35 hours after a single injection of the drug.

Example 2. Induction of IL-18 by repeated administration of the drug "Allokin-alpha"

For this purpose, 2-, 3-, 6-, 8- and 10-fold administration of the drug was studied at intervals of 24, 48 and 72 hours.

For the experiment, BALB / C mice were used, females weighing 16-22 grams, age 14 weeks. The level of interleukin-18 (IL-18) in the blood serum was determined by enzyme-linked immunosorbent assay using specific antibodies. The amount of IL-18 in serum was determined at 24, 32, 40, 48, 54 hours after each administration of the drug. At the same time, the "background" amount of IL-18 present in the blood serum of the studied animals was studied. For this purpose, 11 mice were used as a control group, which received a subcutaneous injection of saline. In the experimental groups, a total of 150 animals were used. In the case of the control group, the confidence interval was calculated from the dilution measurements of the same sample. In the experimental group, the confidence interval was calculated relative to one sample from three animals for each measured point. The test results of the drug are presented in Table 1.

Table 1 The concentration of IL-18 (PG / ml) in the blood of mice after varying the frequency of administration of the drug "Allokin-alpha" Interval between injections, hours Multiplicity of introduction 2 3 6 8 10 24 380 ± 15 560 ± 24 1050 ± 41 459 ± 19 498 ± 14 48 402 ± 22 592 ± 21 800 ± 27 600 ± 23 744 ± 27 72 520 ± 14 740 ± 32 1444 ± 49 1200 ± 35 924 ± 31 The control 200 ± 12

Table 1 presents the maximum concentrations of IL-18 after the last injection of the drug.

The maximum level of interleukin-18 (1444 pg / ml) was achieved in the group where 6-time administration of the drug was used with an interval of 72 hours. The average background level in the group of control animals was 200 pg per ml. The level of induced IL-18 from a single injection was about 400 pg / ml. When administered after 48 hours, the maximum level was 800 pg / ml. When administered after 24 hours, 1050 pg / ml (six-time administration schedule). Exceeding the frequency of administration by more than six times caused a decline in the induction of IL-18.

Example 3

Patients, 24 people with diagnosed HPV infection were divided into 2 groups: the 1st group included 15 people, the 2nd - 9 people. In both groups, electroexcision of the site of papillomatosis was also performed. Patients of the 1st group took Allokin-alpha for the purpose of immunocorrection twice (the first and second series of injections), with an interval of 1 month, according to the scheme: 1 mg subcutaneously (3 injections) every other day. Patients of the 2nd group underwent only electroexcision.

Observation groups were representative by gender and age of patients. The results of treatment of the underlying disease were evaluated 3, 4, and 6 weeks after the end of the course of therapy. The level of interleukin-18 (IL-18) in the blood serum of patients was determined by enzyme-linked immunosorbent assay using specific antibodies. The amount of IL-18 in serum was determined at 40 hours after the last injection. In parallel, the "background" amount of IL-18 present in the blood serum of patients of the 2nd group was studied. The measurement results are presented in Table 2.

table 2 The concentration of IL-18 (PG / ml) in the blood of patients after administration of the drug "Allokin-alpha" and patients who did not receive immunotherapy. Patient group Serum IL-18 concentration after the first series of injections The concentration of IL-18 in serum after the second series of injections 1st group, therapy with Allokin-alpha 88 ± 13 134 ± 25 2nd group, control, only electro-excision was performed 34 ± 11 37 ± 14

When diagnosed by PCR, HPV was detected in the 1st group in three patients (16.7%), in the 2nd - in six (66.6%) patients.

The results of the study show the relationship between the increase in the concentration of IL-18 in the serum of patients using the drug Allokina-alpha and the concomitant treatment of HPV infection.

Example 4. Induction of IL-18 using a preparation of alloferon, administered as part of suppositories and ointments.

Suppositories based on cocoa butter or Vitebsol weighing 1.0 g and containing 1.0 mg of the alloferon peptide were used. The ointment used in the experiment had the following composition: Alloferon 0.1 g; liquid paraffin 13.0 g; petrolatum 4.0 g; glycerol 3.0; PEG sorbitan (monostearate) 4.0; sorbitan monostearate 3.0; stearyl alcohol 3.0; cetyl alcohol 2.0; diethylene glycol monoethyl ether 2.0; methyl parahydroxybenzoate (nipagin) 0.5; propyl parahydroxybenzoate 0.2; water up to 100.0 g.

For the experiment, BALB / C mice were used, females weighing 16-22 grams, age 14 weeks. The level of interleukin-18 (IL-18) in the blood serum was determined by enzyme-linked immunosorbent assay using specific antibodies. The amount of IL-18 in serum was determined at 24, 32, 40 hours after each administration of the drug. At the same time, the "background" amount of IL-18 present in the blood serum of the studied animals was studied. For this purpose, 5 mice were used as a control group, which received a subcutaneous injection of saline. In each experimental group (1st group - candles, 2nd group - ointment) 15 animals were used. For vaginal administration to laboratory animals, 1/50 of the suppository was used. The ointment was administered using an impregnated cotton swab (50 mg ointment). A single administration was performed.

In the case of the control group, the confidence interval was calculated from the dilution measurements of the same sample. In the experimental group, the confidence interval was calculated relative to one sample from three animals for each measured point. The test results of the drug are presented in Table 3.

Table 3 The concentration of IL-18 (PG / ml) in the blood of mice after administration of the drug alloferon in the form of suppositories. Laboratory
animals Measurement of IL-18 concentration after administration
drug watch 24 32 40 Vaginal administration of suppositories 153 ± 15 171 ± 13 263 ± 19 Vaginal administration of ointments 161 ± 14 201 ± 17 280 ± 16 Control group 164 ± 12

Terms used in the present description:

Cytokines are hormone-like molecules of a protein-peptide nature that are produced by body cells. Cytokines carry out short-range regulation of intercellular and intersystem interactions, determine cell survival, stimulation or inhibition of their growth, as well as differentiation, functional activation and apoptosis of cells. Cytokines can be divided into 4 following groups:

1. Hematopoietic factors (CSF-G, -M, -GM, IL-3 and IL-7, erythropoietin) - stimulators of growth and maturation of immature hematopoietic cells.

2. Regulators of natural immunity - pro-inflammatory cytokines (IFNα, β, IL-1 and IL-6, TNFα, chemokines - IL-8, MCP-1, RANTES, etc.). They participate in the nonspecific defense of the body against bacterial and viral infections. Their main targets are phagocyte cells - macrophages and granulocytes.

3. Cytokines that regulate specific immune responses (IL-2 and IL-4, transforming growth factor (TGFβ), etc.). These proteins are involved in the activation, growth and differentiation of mature lymphocytes.

4. Cytokines that regulate inflammatory reactions that develop in the process of a specific immune response (INFγ, lymphotoxin, IL-5, IL-10, IL-18, etc.). Their main function is the activation of non-specific effector cells: cytotoxic macrophages and natural killers.

Interferons are glycoproteins, representatives of the cytokine group. Depending on the origin and, accordingly, the structure of the molecule, human interferons are divided into 3 main types: IFNα, the producers of which are mainly macrophages and B cells; IFNβ produced by fibroblasts, and IFNγ, which is synthesized mainly by activated T-lymphocytes and natural killer cells. The molecular weight of interferons varies from 17-45 kDa for IFNα and IFNβ and from 20 to 80 kDa for IFNγ. It is believed that IFNα and IFNβ have predominantly antiviral, and IFNγ - immunomodulatory activity.

Interleukins are a large group of cytokines synthesized mainly by T-lymphocytes, but in some cases also by natural killer cells or other tissue cells. Interleukins have various functions, but most of them stimulate other cells for division or differentiation, while each of them acts on a separate, limited group of cells expressing receptors specific for a given interleukin. These are soluble peptides, strong local immunoregulators; activate T cells.

Natural (natural) killers (NKs) are lymphocytes involved in the innate immunity system. NKs were discovered by their ability to kill tumor cells, from where they got their name. The main function of activated NKs is the recognition and destruction of the body's own cells in which there are disorders - tumor cells and cells infected with the pathogen. NKs have a characteristic morphology: the main part of the abundant cytoplasm contains several mitochondria, free ribosomes with individual elements of a rough endoplasmic reticulum, the Golgi apparatus, and characteristic electron-dense granules associated with the membrane. On the surface of NK there are many receptors for activating the cells themselves with the help of cytokines, as well as for recognizing ligands on the surface of tumor and infected cells.

Human papillomavirus (HPV) is an infection that causes cervical cancer due to the development of cervical intraepithelial neoplasia, which is caused by a violation of the control of cell proliferation. Enhanced proliferation of HPV-infected suprabasal cells of the cervical epithelium is accompanied by the expression of viral oncogenes - proteins E6 and E7. The ability to persist for a long time in the patient’s body is determined by the peculiarities of the life strategy of these viruses, based on blocking the mechanisms of innate and adaptive antiviral immunity. The risk of malignant degeneration is associated with several types of papillomavirus infection. They are referred to as high risk oncogenic risk viruses. These are types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. There are 5 types of low-risk viruses - 6, 11, 42, 43, 44.

Claims (4)

1. A method for the treatment of infections caused by human papillomaviruses, implemented through the induction of interleukin-18, which consists in the use of drugs based on the alloferon peptide using mechanical removal of papillomaviruses of any type. 2. The method according to claim 1, characterized in that, as an inducer, injections of the drug "Allokin-alpha lyophilisate for preparing a solution for subcutaneous administration" are used, a course of 3 injections with an interval of 1 day between injections, and the course can be repeated. 3. The method according to claim 1, characterized in that suppositories based on alloferon are used as an inductor. 4. The method according to claim 1, characterized in that as an inducer use ointments containing alloferon.

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