RU2264819C2 - Pharmaceutical anti-herpetic composition and method for obtaining medicinal form upon its basis - Google Patents

Abstract

FIELD: immunology, pharmacology.

SUBSTANCE: the suggested composition includes virion anti-herpetic preparation that contains viruses of herpes simplex of 1 or 2 serotypes inactivated either with formalin or γ-radiation, moreover, the composition mentioned is supplemented with immunomodulator polyoxidonium; composition in question additionally contains valine and lysine and, also, the combination consisting of not less than 2 amino acids chosen out of the group of: phenylalanine, leucine, alanine, threonine, histidine, arginine, methionine at a certain ratio of components. The composition may additionally contain albumin and some vitamins. Another objective of the present innovation is the method to prepare a suppository based upon this pharmaceutical composition that includes mixing active components and cacao oil according to standard technique, moreover, as an active substance one should add the above-mentioned pharmaceutical composition and either one or several microelements chosen out of the following group: zinc, chromium, selenium and nickel. The innovation enables to keep antigenicity and stability of specific activity of the preparation mentioned along with enhanced antiviral properties; moreover, body resistance is increased and prolonged both in case of acute and chronic infection. New medicinal form of the composition suggested is easy for application.

EFFECT: higher efficiency of application.

6 cl, 4 ex, 2 tbl

Description

The invention relates to medicine, in particular to the creation of a new pharmaceutical antiherpetic composition and the preparation of a dosage form based on it.

Currently, the following anti-herpes virus drugs are known: acyclovir, virazole, foscarnet, vidarabine, etc. [1].

However, chemotherapeutic agents are not always effective enough, because they do not have a specific effect on only the herpes virus, in principle, are polyactive and can be used for other viral diseases.

The corresponding vaccines have specific activity against the herpes virus. A number of antiherpetic vaccines, including those based on inactivated virions, have been developed both in Russia and abroad, for example, they are described in RF patents No. 2014084, 1994, No. 2085582, 1997, No. 2085583, 1997, U.S. Patent Nos. 4,816,250, 1989, No. 5215745, 1993, No. 5602023, 1997.

Although the vaccines listed in these works are specific and are based on inactivated and attenuated herpes virus strains, not all of them are suitable for treating humans. To successfully fight the virus in a highly organized organism, it is necessary to take into account humoral and cellular factors, the state of the immune system, the degree of virus damage, the stage of the disease (latent infection, exacerbation).

At one time, experiments with the herpes simplex virus (HSV-1), belonging to the subfamily Alphaherpesvirinae, revealed its immunosuppressive activity. Inhibition of T cell proliferation may be combined with a defect in the humoral response to a heterologous antigen. The complex nature of the interaction of herpes simplex virus, serotype 2 (HSV-2) with the immune system of mice, demonstrates the fact that the host is more sensitive to infection with the Coxsackie B virus while maintaining a normal response to sheep erythrocytes. Infection of alveolar or peritoneal macrophages leads to a suppression of their ability to function as effectors of antibody-dependent cytotoxicity. At different times after administration, the virus suppresses the phagocytic activity of macrophages of the abdominal cavity of mice. In the early stages of infection in alveolar macrophages, a temporary increase in phagocytic activity was determined, followed by suppression of this function. The immunosuppressive activity of the virus was investigated, simulating this effect in a culture of human peripheral mononuclear cells. After infection and appropriate incubation, natural killer cells (ECs) were isolated and their cytotoxicity was determined. In the course of work with the described system, it was found that the virus suppresses the cytotoxic function of EC only when monocytes are in the culture of peripheral mononuclear cells. Removing the latter from the system cancels the development of virus-induced suppression; after the introduction of monocytes, the immunological defect is re-formed. It was also found that the development of one of the types of suppression due to herpes viruses is realized through monocytes. There is evidence of the direct action of herpes virus on regulatory T cells, which leads to impaired synthesis and the ability to interact with interleukin-2 [2].

Thus, the described mechanisms of the action of the herpetic virus on immunocompetent cells and immune status in general suggest the need for inclusion in antiviral compounds of drugs that specifically affect the immune system, as well as drugs that normalize cellular metabolism when the body is affected by the herpes simplex virus, especially with sluggish, recurrent and poorly responding therapy, with a tendency to chronic diseases, which include chronic viral infection. It is advisable to include immunocompetent substances, such as interferons and antioxidants, in the complex therapy, as recommended in our previous developments (see, for example, RF patent 2142816, 1999), which also shows a more convenient dosage form for the patient (in the form of a suppository). This development is considered the closest analogue of the present invention.

Despite the significant advantages of the aforementioned vaccine preparation, it does not take into account some important aspects of the patient's condition affected by the herpes virus. In patients with chronic forms, there is a violation of cellular and humoral immunity, there is an increased secretion of glucocorticoid hormones, a sharp violation of the exchange of cells and tissues affected by the virus.

The technical task of the present invention is the creation of a new, more effective and progressive complex preparation, which would not only specifically act directly on the herpes simplex virus, but also provide an intensive immunomodulating effect on the body as a whole and normalize the metabolism in cells and tissues affected by the virus.

The problem is solved in that a virion vaccine antiherpetic drug, in which herpes simplex viruses of serotypes 1 or 2, inactivated by formalin or γ-radiation, additionally contains a high-tech immunomodulator polyoxidonium, as well as valine and lysine, in addition, a combination consisting of at least 2 amino acids selected from the group: phenylalanine, leucine, alanine, threonine, histidine, arginine, methionine.

The pharmaceutical composition has the following ratio of components:

antiherpetic drug - 10 ⁶ -10 ⁷ plaque-forming units / ml suspension polyoxidonium - 0.03-0.06 g valine - 0.18-0.25 g lysine - 0.15-0.30 g combination of 2 metabolic amino acids - 0.12-0.27 g physiological fluid - up to 100 ml

As a physiological medium, for example, Eagle's vaccine preparation medium can be used.

The composition may additionally contain human albumin as a stabilizer in an amount of 0.22-0.24 g per 100 ml, as well as a combination of 2-3 water and fat-soluble vitamins selected from the group: thiamine, riboflavin, nicotinamide, pyridoxine, ascorbic acid, retinol, tocopherol, or a mixture thereof in the total amount in the composition from 0.05 to 3.5%.

Such a drug is suitable in the treatment of herpes simplex types 1 and 2, for this, a combination of amino acids and vitamins is appropriate for each type of herpes, and the dose and frequency of use, depending on the patient's condition.

Domestic immunomodulator of the latest generation - polyoxidonium (PO) - is a very effective immunocompetent agent. PO is a copolymer of 1,4-ethylene piperazine N-oxide and (N-carboxyethyl) -1,4-ethylene piperazinium bromide; it is a lyophilized porous mass with a yellowish tint, highly soluble in water, molecular weight from 60,000 to 100,000. In addition to immunomodulating, PO It has a pronounced detoxifying, antioxidant and membrane stabilizing effect [3].

Recently, we have undertaken a number of experiments using software simultaneously with the use of antiherpetic vaccines when used together by parenteral administration. These experiments gave positive results on model infections in laboratory animals (keratitis in rabbits, genital herpes in guinea pigs, herpetic meningoencephalitis in mice) [4], the results of these experiments are shown in Table 1.

Table 1 №№
p / p Herpesvirus vaccines used Test virus titration results in Ig TDC
50 / ml Dose of Polyoxidonium in mg per injection The concentration of virus-neutralizing antibodies in the neutralization index Killed vaccine - 2,3 1 against the virus 6.5 0.1 3.75 ** herpes simplex type 1 0.2 3.3 * Killed vaccine - 2.0 2 against the virus 5.5 0.1 3.0 * herpes simplex 0.2 2,5 1 type Killed vaccine - 2.0 3 against 5,0 0.1 3.0 ** cytomegalovirus 0.2 2,5 of man Note: 1. The significance of differences in the concentration of virus neutralizing antibodies in vaccinated animals without the use of Polyoxidonium and with its use. ** P≤0.01; * P≤0.05

As studies show, the choice of an immunomodulatory drug and the regimen for its use in patients are determined by the immunologist depending on the severity and type of underlying disease, concomitant pathology, as well as the detected (sometimes hidden) immunological defect.

When the cells of the monocytic-macrophage system are affected, the following are used: polyoxidonium, lycopid. In the most severe forms of lesion in the monocyte-macrophage system, granulocyte-macrophage colony-stimulating factors are used: milgramostim (leukomax), filgrastim (neupogen).

For defects in the cellular component of immunity, one of the following drugs is used: polyoxidonium, tactivin, thymoptin, thymogen, thymoline. If the synthesis of antibodies is disturbed, B-lymphocytes use: myelopid, polyoxidonium.

Among the drugs with immunostimulating properties, three main groups can be distinguished that are used in practical health care: preparations of microbial origin (Pyrogenal, Prodigiosan, Ribomunil, Sodium Nucleinate and others), drugs of endogenous origin: preparations of thymus (T-activin, Timalin, Timoptin, Timaktid, Timostimulin, etc.), medications of bone marrow origin (Myelopid), cytokines (Molgramostin, Reaferon, etc.), chemically pure, synthetic drugs: therapeutic drugs with identified immunomodulatory and properties (e.g., diutsifon et al.), analogues of endogenous substances (Likopid, Timogen et al.), the actual synthetic drugs (polioksidony et al.).

Our studies have shown that the best effect for subjects with various forms of herpes has a high-tech synthetic drug polyoxidonium.

In vivo, software has a more complex and multifaceted effect on the immune system. Since the development of any immune response begins with cells of the monocyte-macrophage system, and since the cytokines produced by monocytes / macrophages have a pleiotropic effect, an increase in their functional activity under the influence of PO leads to the activation of both cellular and humoral immunity. So, in particular, with the introduction of PO together with low doses of antigen, the synthesis of antibodies to this antigen is increased by 5-10 times compared to the control. It is important to note that such an increase can be observed in animals with a genetically determined weak reaction to this antigen. Thus, the software has the ability to set in motion all the factors protecting the body from foreign antigenic agents and this movement spreads naturally, as it does with the development of the immune response in the body. These observations allowed us to choose among many modern immunomodulators, namely polyoxidonium for its successful use in complex pharmaceutical antiherpetic compositions. Empirically, we were able to establish that the very effective components in the composition of the composition are the essential amino acids valine and lysine, as well as some other amino acids that lead to accelerated epithelization of skin tissue affected by the virus, such a composition, especially with the addition of trace elements, which enhances the immune response to the introduction of the drug allows it to be used for a more effective fight against viral lesions of herpes serotypes 1 and 2.

The following mechanisms of action of trace elements (ME) in the immune system exist:

1. Effect on specific receptors.

To receptors localized on the cytoplasmic membrane: HLA system, MHC system (Ni, Cr, Hg).

Adhesins: selectins and integrins (Mn, Hg).

Transferrin receptors (Al, Ga).

Receptors involved in EC lysis (Zn).

Cytokine receptors (Zn).

T - cell receptor (Zn, Hg).

Receptors for calcium and magnesium ions (Zn, Mn, Be, Cd, Hg, etc.)

Receptor for immunoglobulins (Zn).

For receptors localized in intracellular compartments: Mitochondria (Fe, Zn), Cytoskeleton LIM proteins (Zn, Se, Li).

Intracellular calcium receptors on mitochondria, endoplasmic reticulum (Cd, Zn).

2. Effect on enzyme activity.

Many essential MEs are a component of the catalytic center of a number of enzymes. For example, Mn is the essential part of the superoxide dismutase (SOD) of immunocytes, Se is part of the catalytic center of glutathione peroxidase (isoenzyme VI), Zn is the most important part of numerous finger proteins that regulate the level of transcription of other intracellular proteins. There are also ways of effecting ME on enzyme activity, which include competitive inhibition or allosteric activation of metalloenzymes. For example, Zn is a competitive inhibitor of Ca ^{+ 2} , Mg ²⁺ - dependent endonuclease. This action of Zn has determined its leading role in the immune system as an anti-apoptotic factor.

3. The effect on the activity of hormones.

- ME as an integral part of hormones.

Zn is a key component of thymosin, a hormone that implements effects: thymus on the T-cell unit of the immune system.

ME and hormone deposition.

Zn, Cr - are involved in the deposition and stabilization of the insulin molecule, which has a multimodulating effect on all insulin-dependent cells of the body, which include immunocytes. Zinc provides intracellular deposition and stabilization of the hormones of the neurohypophysis.

Participation in the degradation and elimination of hormones.

The angiotensin converting enzyme is known to be Zn-dependent.

Participation in the mechanism of action of hormones.

4. Effect on carrier proteins.

Albumins

Metallothioneins, which are synthesized in mononuclear cells of the reticuloendotemial system of the body.

Stress proteins, as universal proteins synthesized in cells in response to stressful effects (heat shock, hunger, UV radiation, exposure to heavy metals, chronic infection).

5. Physico-chemical effect of ME on immunocyte membranes.

It was found that, for example, selenium can have an antioxidant effect, acting as a cofactor of glutathione peroxidase, which provides the inactivation of free oxygen species, the birth of which in the immune system ensures both the destruction or elimination of a foreign agent (parasite, bacterium), and with excess production of singlet O ₂ , H ₂ O ₂ , OH causes damage to the membrane apparatus of the immunocytes themselves. The occurrence of O ₂ , H ₂ O ₂ , OH is associated with the induction of the Gaber-Weiss and Fenton reactions under the influence of transition metals (Cu, Zn, Mn, Fe).

Thus, MEs are able, through the mediation of enzymatic and non-enzymatic mechanisms of lipid peroxidation (LPO), as well as activation of antioxidant mechanisms to regulate the physicochemical properties of cell membranes, including the property of semi-permeability to various biological substrates (antigens, infectious agents, etc. .).

6. Impact on presentation, intracellular processing and degradation of antigens (see paragraph 1).

7. Impact on the formation of immunological memory, as well as, probably, in the long-term existence of memory cells, anti-apoptotic MEs play a role (Zn, Se, etc.)

8. Impact on the production of immunoglobulins (Zn, Be).

9. Influence on the processes of chemotaxis, adhesion and phagocytosis [5, 6].

Numerous tests on laboratory animals made it possible to choose the most effective microelements for inclusion in the composition of the developed dosage form (suppository): 2-3 microelements selected from the group: zinc, chromium, selenium and nickel. The presence of these trace elements in the dosage form enhances the effect of the immunomodulator chosen by us by 25-30% in comparison with the control (without ME).

Table 2 below shows the level of immunological parameters before and after the introduction of a composition containing amino acids, polyoxidonium.

The table shows the effect of PO on immunological parameters, where cellular immunity and the level of immunocompetent proteins significantly increase. It should be noted that the introduction of amino acids into the composition (valine and lysine, in addition, a combination consisting of at least 2 amino acids selected from the group: phenylalanine, leucine, alanine, threonine, histidine, arginine, methionine) our combinations accelerated the re-epithelization of the affected tissues (from 15 to 30% compared with the prototype, see above), depending on the type of virus. The introduction of polyoxidonium together with amino acids not only activated immunity, which significantly prolonged the period of remission, accelerated the process of re-epithelialization and healing of the skin, and in some cases practically cured chronic forms of the disease in individual animals (12% of all subjects).

Another object of the invention is a method for preparing a suppository based on the aforementioned pharmaceutical composition using standard technology for the manufacture of candles, while the active ingredients of the above pharmaceutical composition according to standard technology and 2-3 microelements selected from the group are additionally added to the mass of candles based on cocoa butter: zinc, chromium, selenium and nickel. A set of trace elements in the form of soluble chelate forms is introduced into the suppository in an amount of 0.01-0.08% by weight.

The test results of the obtained suppositories in a group of 76 experimental animals showed that the use of the new drug not only boosted the immune system, but allowed to significantly extend the remission period, accelerate the re-epithelization and healing of the skin and mucous tissues, and in some cases, practically cure the disease in individual individuals ( 12% of all subjects with a chronic form of the disease).

In our opinion, the inclusion of vital ingredients that regulate metabolic processes at all levels, namely: PO, valine and lysine, vitamins of all groups, as well as ME: zinc, chromium, selenium and nickel, played an important role in this result.

Key performance indicators. The time to local recovery (complete re-epithelization) was reduced by 15-30%, the duration of remission increased on average to 6-7 months, the absence of the virus in the smear (PCR diagnosis) in 98.7% of cases, the activation of antiviral immunity, almost complete cure in 12% of animal observations.

Among the experimental animals (herpetic keratitis in rabbits, genital herpes in guinea pigs), 52 individuals were affected by the herpes simplex virus (serotypes 1 and 2) in acute form, and 24 by the chronic form of herpes simplex. The animals with the acute form were divided into three groups: in the first group, the herpetic vaccine described in the prototype was treated, in the second - a new pharmaceutical composition, in the control group the animals received chemotherapy in the form of ointments and solutions containing known anti-herpes drugs. A group of animals with a chronic form received new suppositories with a full range of amino acids, vitamins and trace elements.

Results. - In the 1st group, complete re-capitalization occurred on average on the 6th day of treatment, while in the 2nd group, full re-capitalization was observed on average already on the 4th-5th day of treatment. In the control group, complete local recovery was observed on average on the 7-10th day from the start of treatment. Moreover, the virus in control smears was not detected in 95.2% of the 1st group, in 97.8% of the 2nd group and in 92.5% of the control group. Duration of remission up to 4 months, took place in 92% of cases in the first group and up to 6-7 months in 85% of cases in the second group. At the same time, in the control group, the duration of remission up to 4 months was only 28%. A high-intensity immune response was recorded in the 1st group in 92.1% of cases, while in the 2nd group in 98.6% with a clear significance (p <0.01). So, in the blood serum of experimental groups of both groups (the second group is 11-18% higher) after treatment, there was a tendency to increase the percentage and absolute content of natural killer cells, cytotoxic T-lymphocytes of CD8 + phenotypes, and activation of virus-induced IF a was detected (index phagocytosis) mitogen-induced IF%.

The resulting pharmaceutical suspension, taken in an amount of 20 ml, is mixed with 0.01-0.03 ml of each of the solutions of salts of 2-3 ME and cocoa butter to a total mass of 100 g, from the obtained mass candles are made in a known manner weighing 0.15 -0.20 g each. The suppository contains ≤400 μg / g of protein, has a residual moisture content of ≤2.2% and a physiological pH of 7.3 ± 0.2, is non-toxic and can induce virus synthesis in rats with a neutralization index of 3.0 lg TCD ₅₀ / ml for HSV-1 and 2.0 lg TCD ₅₀ / ml for HSV-2, where TCD ₅₀ means the dose causing a cytopathic effect in 50% virus-infected tubes with a monolayer of cells.

Thanks to the creation of a new highly immunogenic composition, the antigenicity and stability of the specific activity of the drug are preserved, not only the antiviral properties are enhanced, but the body's resistance is increased and prolonged not only in acute, but also in chronic infections.

Specific examples of the composition:

Example 1. The composition contains the following components:

An antiherpetic drug, including 10 ⁶ -10 ⁷ plaque forming units / ml suspension

polyoxidonium - 0.03 g valine - 0.18 g lysine - 0.15 g combination of metabolic amino acids: phenylalanine, leucine 0.12 g saline - up to 100 ml

Example 2

The composition contains:

antiherpetic drug - 10 ⁶ -10 ⁷ plaque-forming units / ml suspension polyoxidonium - 0.06 g valine - 0.25 g lysine - 0.30 g metabolic amino acid combination: alanine, threonine, histidine - 0.27 g human albumin - 0.22 g saline - up to 100 ml

Example 3

The composition contains:

antiherpetic drug - 10 ⁶ -10 ⁷ plaque-forming units / ml suspension polyoxidonium - 0.05 g valine - 0.20 g lysine - 0.20 g metabolic amino acid combination: histidine, arginine, methionine - 0.25 g human albumin - 0.24 g vitamin C - 0.1 g saline - up to 100 ml

Example 4

The composition contains:

antiherpetic drug - 10 ⁶ -10 ⁷ plaque-forming units / ml suspension polyoxidonium - 0.03 g valine - 0.25 g lysine - 0.25 g combination of metabolic amino acids: phenylalanine, leucine, alanine, threonine, histidine, arginine, methionine, taken in equal parts - 0.25 g nicotinamide, pyridoxine - 0.03 g human albumin - 0.23 g saline - up to 100 ml

Literature

1. D.A. Kharkevich "Pharmacology", GEOTAR-MED, 2002, pp. 549-550.

2. Khaitov P.M. "Immunogenetics and immunology: resistance to infection", Tashkent, 1991.

3. Petrov R.V., Khaitov P.M. New generation vaccines based on synthetic polions: creation history, phenomenology and mechanisms of action, implementation. International journal on immunorehabiltation. - 1999, N11, p. 13-36.

4. I.F. Barinsky et al. "The ability of polyoxidonium to enhance the immunogenicity of herpes vaccines." Immunology, No. 2, 2001, pp. 17-20.

5. Skalny A.V. "Human microelementoses (diagnosis and treatment), Moscow, 1997.

6. Khaitov P.M. and other "Environmental immunology", Moscow, 1995.

Claims (6)

1. Pharmaceutical antiherpetic composition, comprising a virion vaccine antiherpetic drug, which contains herpes simplex viruses of serotypes 1 or 2, inactivated by formalin or γ-radiation, and an immunocompetent substance, characterized in that it contains an immunomodulator polyoxidonium, additionally contains valine and lysine, as well as a combination of at least 2 amino acids selected from the group of phenylalanine, leucine, alanine, threonine, histidine, arginine, methionine, in the following ratio of components, g: Antiherpetic drug 10 ⁶ -10 ⁷ plaque forming units / ml suspensions Polyoxidonium 0.03-0.06 Valine 0.18-0.25 Lysine 0.15-0.30 Combination of metabolic amino acids 0.12-0.27 Physiological fluid medium Up to 100 ml 2. The composition according to claim 1, characterized in that it further comprises human albumin in an amount of 0.22-0.24 g per 100 ml. 3. The composition according to claim 1, characterized in that it further comprises one or more water and fat-soluble vitamins selected from the group of thiamine, riboflavin, nicotinamide, pyridoxine, ascorbic acid, retinol, tocopherol, or a mixture thereof in a total amount of 0.05 to 3.5%. 4. The composition according to claim 1, characterized in that it can be made in the form of a suppository. 5. A method of preparing a suppository based on a pharmaceutical composition described in claims 1 to 4, comprising mixing according to standard technology the active components and cocoa butter, characterized in that the pharmaceutical composition according to claims 1 to 3 and one or more are introduced as an active principle trace elements (MEs) selected from the group zinc, chromium, selenium and nickel. 6. The method according to claim 5, characterized in that the ME is introduced in the form of soluble chelate forms in an amount of 0.01-0.08% of the total weight of the composition.