RU2401121C1 - Pharmaceutical composition based on herbal extracts for treatment and prevention of viral infections and syphilis and method of treatment and prevention of viral infections and syphilis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: pharmaceutical composition for treating syphilis and viral diseases specified from influenza, human immunodeficiency virus, cytomegalovirus infections, viral hepatitis type A, D, C and herpes infections, containing an amount of polyphenol compounds of sea buckthorn leaves, including at least 60 % of halloellagotannines, flacoside, a pharmacologically acceptable carrier and a substance of licorice extract, or glycyrrhizic acid, or its pharmaceutically acceptable salt, acridonoacetic acid or its pharmaceutically acceptable salt, birch bark extract or betulin taken in therapeutically effective amounts. A method of treating syphilis and viral diseases, including the introduction of the declared pharmaceutical composition by 2-3 doses 3 times a day.

EFFECT: composition exhibits an evident antiviral action with respect to the infections stated above.

22 cl, 1 dwg, 12 tbl, 7 ex

Description

The present invention relates to the field of medicine and pharmaceuticals, in particular to a pharmaceutical composition based on herbal extracts intended for the treatment and / or prevention of syphilis and viral diseases selected from influenza, HIV infection, neuroinfection, cytomegalovirus infection, viral hepatitis A, B , C, secondary immunodeficiency conditions against a background of chronic bacterial infections and herpes virus infection, as well as to a method for treating these diseases.

The problem of treatment and prevention of viral infections, including sexually transmitted infections, including HIV and hepatitis B and C, is becoming increasingly important every year. With the growing number of diseases of viral etiology, the relevance of creating effective drugs not only for their treatment, but also for prevention (today, several million people die every year from AIDS and viral hepatitis B and C in the world).

The relevance of creating a new tool for the treatment and prevention of syphilis is due to the complexity and inconvenience for the patient of existing treatment regimens, the need for long courses of therapy.

In addition, due to the danger of a pandemic of certain forms of influenza (avian influenza A caused by the H5N1 virus), it is important to develop means for the prevention and treatment of this viral disease.

Known medicinal products based on herbal extracts approved for medical use with antiviral effects, such as flacoside, hyporamine or glycyrrhizic acid contained in licorice root extract; glycyrrhizic acid is also used as sodium glycyrrhizinate.

Hyporamine, which is the sum of polyphenolic compounds from the leaves of buckthorn buckthorn, containing at least 60% galloellagotannins, is approved for medical use in adults and children as an antiviral agent. It has a wide range of antiviral activity against herpes simplex viruses, cytomegalovirus, Varicella Zoster, human immunodeficiency viruses (HIV infection), and others. Hydrolyzable tannins are biologically active components. (New domestic medicines Consolidated release, M., 2000. State. Register, t.1, 2001). On the basis of hyporamine, preparations were created against the human immunodeficiency virus (USSR patent No. 1805967), for the treatment and prevention of viral diseases (RF patent No. 2118163).

Flacoside is approved for medical use in adults and children as an antiviral agent for acute viral hepatitis A and B, primary and recurrent forms of herpes simplex extragenital and genital localization, herpes zoster and other infections. It is a derivative of flavones, obtained from the leaves of Amur velvet (Mashkovsky M.D. Medicines, v.2, 2000, State. Register of medicines, 2001, v.1). The use of flacoside for the treatment of herpes is disclosed in USSR copyright certificate No. 491387.

The closest technical solution to the claimed invention, in our opinion, is RF patent No. 2200571, which discloses the composition (complex) of natural compounds of plant origin, active against viral infections and other sexually transmitted infections, including hyporamine, characterized in that the complex additionally contains flacoside and helepin or helepin D (extracts of Canadian desmodium herb), in the ratio 1: (2.0-4.0) :( 2.0-2.5), respectively. It has been shown that the complex of hyporamine, flacoside and helepin is effective against the most relevant viral infections to date - HIV, herpes virus infection and viral hepatitis.

At the same time, one of the most important areas in the treatment of viral infections of various etiologies (HIV infection, herpes virus infection, viral hepatitis, etc.) is the use of endogenous interferon synthesis inducers.

Endogenous interferon inducers represent a new class of antiviral, antitumor and immunomodulating drugs. The most promising in this class are low molecular weight interferonogens - derivatives of 10-carboxymethylene-9-acridanone (acridonoacetic acid), which have a number of pharmacological properties (low toxicity, the absence of allergenic, mutagenic and embryotoxic effects on the body) necessary to create an "ideal" endogenous inducer interferon. An important pharmacological property of acridonoacetic acid is the lack of metabolic breakdown in the liver and accumulation in the body.

Acridonoacetic acid was first obtained by German researchers in 1923 by thermal cyclization of the amino acid substituted N-phenylglycine.

According to its chemical structure, acridonic acetic acid is a planar tricyclic heteroaromatic system with an N-carboxymethyl substituent; in its structure, a fragment of N, N-disubstituted pharmacologically active aminoacetic acid, glycine, is easily detected.

This compound simultaneously combines high lipophilicity due to a flat heteroaromatic ring and hydrophilicity due to the ring keto group and the side residue of acetic acid at the nitrogen atom. This unique chemical structure of acridonoacetic acid suggests that it has a high biological activity, due to both easy penetration into the organs and tissues of the body, and a possible effect on the receptor apparatus of the cell and the effect on metabolic reactions in the body.

The antiviral activity of acridonoacetic acid and its derivatives was shown by American researchers in 1972 - the sodium salt of acridonoacetic acid showed a high level of protection against viruses of the Semlica forest, western and eastern encephalitis, influenza and herpes. Later studies by various authors found that the antiviral activity of acridonoacetic acid, as well as its pharmaceutically acceptable salts, against a wide range of viruses is due to the induction in the body of high titers of endogenous interferon.

Despite the fact that the ability to induce endogenous interferon under the influence of various "biological" (viruses, bacteria, toxins, etc.) and "chemical" (salts of heavy metals, polymers, heteroaromatic compounds) inducers have almost all types of body cells, it was found that only immunocompetent cells of the body are capable of inducing interferon under the action of AUC salts: monocytes, macrophages, lymphocytes and Kupffer liver cells. The optimal interferon-inducing concentration of AUC in cultures of immunocompetent cells is 6 μg / ml.

Acridonoacetic acid penetrates well through the placental barrier and induces high titers of interferon in the tissues of the fetus, without affecting its viability, which was established when studying the induction of interferon in pregnant female mice. This biological effect with the absence of teratogenic properties in acridonoacetic acid makes it possible to treat a number of diseases in obstetric and pediatric practice.

The high level of α / β induced interferon produced by the sodium salt of acridonoacetic acid has an inhibitory effect on the development of various tumors; the antiproliferative effect of the interferon inducer is significantly expressed in the model of a weakly metastatic tumor, and not in the model of a fast metastatic lymphosarcoma tumor.

Upon preliminary administration of corynebacterium (Corynebacterium parvum) to animals, followed by the induction of acridonoacetic acid salt, the antitumor effect of the drug increased significantly. It is typical that the introduction of a natural inducer of interferon, Newcastle virus in place of acridonoacetic acid salt in a parallel experiment, did not lead to an increase in the antitumor effect on mouse lymphoma models, which indicates various biological effects of interferons synthesized in response to the effects of a “chemical” and “biological” inducer.

Considering that the pharmacological effects of acridonoacetic acid go beyond what can be explained only by the induction of interferon, it is obvious that the antiviral, antitumor and antiproliferative effect of the drug can be associated not only with the effects of induced interferon, but with dose-dependent specific inhibition of the key intracellular enzyme cyclo-AMP- phosphodiesterase, which leads to a decrease in the content in the cell of the universal messenger cyclo-AMP and, accordingly, increases the sensitivity of cells to the antigen and mitogenic action.

In addition to the effects associated with the induction of interferon, a “direct” antiviral and antitumor effect of acridonoacetic acid is possible, mediation through inhibition of the functions of the key mitochondrial respiratory chain enzymes of the cell ubiquinone, or inhibition of ATP binding to the mitochondrial ADP / ATP-dependent transport protein by specific covalent conjugation coupling acids on the peptide bond lysine - cysteine.

A number of preparations were obtained on the basis of acridonoacetic acid and its derivatives, in particular, the Polisan Scientific and Production Company (Russia, St. Petersburg) developed Cycloferon (a salt of acridonoacetic acid with N-methylglucamine, a 12.5% solution for injection), successfully used in the clinic for HIV infection (AIDS, stage IIA-IIIB), neuroinfection (encephalitis, Lyme disease, neuroSPID), viral hepatitis, herpetic, cytomegalovirus and chlamydial infections, secondary immunodeficiency against chronic bacterial infections, rheumatic fever Sgiach and systemic connective tissue diseases (rheumatoid arthritis, systemic lupus erythematosus), degenerative diseases of the joints, including deforming osteoarthrosis; in children older than 4 years (as part of complex therapy) with viral hepatitis (A, B, C), herpetic and HIV infection.

NTFF "Polisan" obtained patents for Cycloferon in the Russian Federation, in Japan, the USA and the EU.

More detailed information on the properties and antiviral activity of acridonoacetic acid and its derivatives, including Cycloferon, is available (as of March 18, 2009) on the Internet at (WWW): http: //www.ama.dp.ua/Info/library/CycloClin /AUK.asp

Also, earlier in the prior art, the possibility of using licorice root extract (or its active principle, glycyrrhizic acid or a pharmaceutically acceptable salt of glycyrrhizic acid, in particular sodium glycyrrhizinate), and birch bark extract, or its active principle, was shown for the treatment and prevention of viral hepatitis. - betulin.

Betulin is a crystalline organic substance, insoluble in water, filling in the form of fine powder cork cells of birch bark and imparting a white color to birch bark. The molecular formula is C ₃₆ H ₆₀ O ₃ . Betulin is one of the first substances extracted from plant materials (1778).

The main source of natural betulin is birch. The white part of the birch bark in individual species of birch (Betula verrueosa, Betula papyfera) contains up to 30% or more betulin (Hayek, E.W. et al. (1989) A Bicentennial of Betulin, Phytochemistry, 28 (9) p. 229-2242).

Betulin is a pentacyclic triterpene with a carbon skeleton of a number of lupans, which is also called betulinol, trochotone, birch camphor and (cinnamon) resigul. A characteristic feature of the lupane series is a ring with five carbon atoms in the pentacyclic system, which at position C-19 has an isopentenyl group. Betulin is characterized by high thermal stability, its melting point is between 250 and 261 ° C, and even higher values are achieved after sublimation of the recrystallized product. Its molecular weight is 442.7, it is soluble in pyridine and tetrahydrofuran, but slightly soluble in dichloromethane, chloroform and cold organic solvents, and its solubility increases significantly with increasing temperature. Betulin is practically insoluble in water and cold petroleum ether. In addition, kinetic studies have shown a very low reactivity of betulin hydroxyl groups.

In 1899, the antiseptic properties of betulin were shown and therefore it was used to sterilize dressings and plasters (Wheeler J., (1899), Pharm. J., Die Darstellung des Betulin durch Sublimation, 494, Ref. Chem. Centr. 1900 I , p. 353). In naturopathy and traditional medicine, birch bark decoctions have been received and are still being used, which are used to treat malaria, dropsy, gout and skin diseases, as well as in the form of tinctures for compresses in the treatment of abscesses.

A large number of studies are devoted to the hepatoprotective activity of betulin. It was found that betulin reduces the activity of alanine amigutransferase, alkaline phosphatase, the content of maleic dialdehyde, prevents the inflammatory process in the liver (Yu.K. Vasilenko et al. "Pharmacological properties of birch bark triterpenoids" Experimental and Clinical Pharmacology, 1993, v. 54 No. 4 pp. .53-55).

In the publication Sun IC, Shen JK, Wang HK, Cosentino LM, Lee KH. Synthesis and anti - HIV activity of betulin derivaties, Bioorg Med Chem Lett. 1998 May 19, 8 (10), p.1267-1272 indicates that in vitro 3-substituted glutaryl derivatives of betulin exhibited cytotoxicity against lymphocytes transformed with HIV; anti-HIV activity is also indicated for a number of betulin derivatives (see: Hashimoto F, Kashiwada Y, Cosentino LM, Chen CH, Garrett PE, Lee KH. Anti-AIDS agents-XXVII. Synthesis and anti-HIV activity of betulinic acid and dihydrobetulinic acid derivatives. Bioorg Med Chem. 1997 Dec; 5 (12): pp. 2133-43).

It was later shown that birch bark extract or betulin can be successfully used both in treatment (RF patent No. 2244554) and in the prevention of viral hepatitis C (RF patent No. 2264820), however, to obtain combined formulations for the treatment of viral hepatitis and other viral infections of betulin not previously applied.

Surprisingly, it has been found within the framework of the present invention that a composition comprising hyporamine and flacoside in therapeutically effective amounts, further containing a therapeutically effective amount of acridioacetic acid (preferably as a pharmaceutically acceptable salt, more preferably as a salt with N-methylglucamine), as well as a therapeutically effective the amount of licorice root extract, or glycyrrhizic acid, or a pharmaceutically acceptable salt of glycyrrhizic acid (preferably gly irrizinata sodium) and a therapeutically effective amount of the extract of bark or betulin possesses significantly more pronounced antiviral activity against virus infections mentioned above in comparison with prior art antiviral activity and compared to the antiviral activity of each component alone; the effectiveness of the pharmaceutical composition of the invention in the treatment of avian influenza A caused by the H5N1 virus has also been unexpectedly shown.

Thus, in the framework of the present invention, the following group of inventions is claimed:

- A pharmaceutical composition for the treatment of syphilis and viral diseases selected from influenza, HIV infection, neuroinfection, cytomegalovirus infection, viral hepatitis, secondary immunodeficiency conditions against a background of chronic bacterial infections and herpes virus infection, made on the basis of plant extracts and containing therapeutically effective amounts of the amount polyphenolic compounds from buckthorn buckthorn leaves, including at least 60% galloellagotannins, flacoside and pharmaceutically acceptable my carrier, characterized in that it further comprises a therapeutically effective amount akridonouksusnoy acid or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of the extract of licorice or glycyrrhizic acid or pharmaceutically acceptable salts of glycyrrhizic acid and a therapeutically effective amount of the extract of bark or betulin.

- A method for the treatment of syphilis and viral diseases selected from influenza, HIV infection, cytomegalovirus infection, viral hepatitis, secondary immunodeficiency conditions against chronic bacterial infections and herpes virus infection, comprising administering a pharmaceutical composition of the invention to a patient in need of 2-3 doses 3 once a day.

In the framework of the present invention, the term "HIV infection" also includes AIDS, stages IIA-IIIB; the term "neuroinfection" includes neuro-AIDS, viral encephalitis, Lyme disease.

Within the framework of the present invention, the composition may be in the form of any convenient form for use, such as hard or soft gelatin capsules, pellets, tablets, including modified release tablets, sachets, using the manufacturing techniques and excipients traditional for these dosage forms.

It was found empirically that the following therapeutically effective amounts of the active ingredients of the claimed composition are optimal, although not limiting the scope of claims in accordance with this application: from 10 to 100 mg of hyporamine, from 100 to 200 mg of flacoside, from 250 to 1000 mg of acridonoacetic acid or its pharmaceutically acceptable salt (preferably salts with N-methylglucamine), from 30 to 300 mg of glycyrrhizic acid, and from 100 to 350 mg of betulin.

An unexpected technical result consists in a qualitative expansion of the spectrum and in a quantitative increase (in comparison with the prototype) of the antisyphilitic (antireponematous) and antiviral activity of the claimed pharmaceutical composition with respect to influenza, HIV infections, neuroinfections, cytomegalovirus infection, viral hepatitis, herpes virus infection, and secondary immunodeficiencies against the background of chronic bacterial infections.

It should be understood that the data presented in the examples below are provided only to illustrate the claimed invention, and not to limit the scope of claims, and confirm the presence of unexpectedly more pronounced antisyphilitic (anitreponematous) and antiviral activity of the claimed composition both in comparison with the composition of the prototype, and in comparison with individual components.

Example 1. The activity of the claimed composition against Treponema pallidum

Studies were carried out on the effect of the composition according to the invention, the plant complex in accordance with the prototype (hyporamine, flacoside and helepin taken in a ratio of 1: 2.0-4.0: 2.0-2.5), hyporamine and flacoside, on pale treponema - causative agent of syphilis. The quantitative composition of the composition according to the invention was selected so that it contained an equivalent amount of hyporamine and flacoside to the composition of the prototype. A suspension of pale treponemas was obtained from rabbit orchitis on the 7th day after infection with syphilis. Treponema was observed in a dark-field microscope - eyepiece x40, lens x7. A suspension of pale treponemas was maintained in sterile saline. For the experiments, a suspension with a content of about 100 pale treponemas in the field of view was used. Solutions of active compounds were prepared at the rate of 20 mg of the substance in 1 ml of liquid: for water-soluble compounds (e.g. hyporamine) - in 1 ml of distilled water, for water-insoluble (flacoside) - 5% ethyl alcohol solution + 0.9 ml of distilled water.

Treponemocidal effect was observed by direct contact in a drop. One drop (0.05 ml) of each solution was placed on a glass slide, mixed with an equal volume of terponium suspension in an isotonic NaCl solution, covered with a coverslip and examined under a microscope.

The results are presented in table 1.

Similar data were obtained for several compositions within the framework of this invention, differing in the quantitative ratio of the components, the results are summarized in table 2.

Table 1 Comparative characteristics of the treponemocidal effect of the composition according to the invention, the prototype and individual preparations of hyporamine and flacoside Treponemocidal effect (%), through: 2-5 min 10 min 20 minutes 30 minutes 40 min 60 min Prototype 70 80 85 90 90 90 Hyporamine 70 75 80 85 85 90 Flacoside twenty twenty 25 thirty 35 40 Control (isotonia, NaCl solution) 0 0 0 0 0 0 The composition according to the invention 80 87 90 one hundred one hundred one hundred

Table 2. Comparative characteristics of treponemocidal effect of various compositions according to the invention No. p / p Hyporamine: flacoside: glycyrrhizinate: cycloferon: betulin (mg) Treponemocidal effect after 30 minutes,% one 50: 100: 100: 250: 100 96% 2 70: 150: 200: 400: 150 98% 3 70: 200: 200: 300: 300 99% four 100: 200: 200: 250: 200 one hundred% 5 100: 170: 150: 450: 200 one hundred%

Example 2. Antiviral activity of the claimed composition against viral hepatitis C

We examined 74 patients (54 men, 20 women) aged 16 to 60 years old, with diagnosed chronic hepatitis C, without signs of cirrhosis. All patients were divided into 3 groups: I group (27 people, 17 of them men, 10 women) received 2 times a day an oral composition in accordance with the prototype (hyporamine + flacoside + helepin), II group (27 people, 15 of them men, 12 women) received 2 times a day orally the composition according to the invention, based on the equivalent amount of hyporamine and flacoside, group III (control, 20 people, 15 men, 5 women) received 2 times a day basic therapy (enzymatic and symptomatic preparations , vitamins of groups A and E and ribavirin).

Every day, a general examination of all patients was performed, including questioning the patient, measuring body temperature, pulse, blood pressure, determining the increase in the liver and spleen. In addition, daily laboratory monitoring of treatment effectiveness was carried out, which included:

- detection of specific markers of viral hepatitis A (anti-HAV IgM), B (HbsAg, anti-Bc IgM), C (anti-HCV, HCV RNA) and D (anti-HD);

- a biochemical blood test to determine the level of bilirubin, serum AlAT (alanine aminotransferase) and AcAT (aspartate aminotransferase), GGTP (gamma-glutamyl transpeptidase), sublimate and thymol samples, prothrombin index, total protein and albumin, the study of autoantibody antibodies (circulating autoantibodies );

- study of viral load and genotyping of HCV;

- the study of indicators of interferon status of patients.

The study was conducted before prescribing drugs and after 10, 20, 30 days from the start of therapy, at the end of treatment, and then after 3 and 6 months from the start of treatment and later (in a number of patients) for 1 year from the start of the drug.

As criteria for assessing the therapeutic efficacy of the claimed composition was used:

A. According to a general examination of patients - symptoms of liver failure, such as: headache, weakness, decreased appetite, nausea, pain in the right hypochondrium.

Comparative results for groups I-III are presented in table 3 (see below). From them it can be seen that in patients who received therapy in accordance with the prototype (hyporamine + flacoside + helation), but in comparison with the control group, some leveling of symptoms of liver failure was observed, however, in patients of group II who received therapy in accordance with the present invention, significantly more pronounced improvement; in addition, in patients of the control group, the symptoms of the disease often reappeared some time after the cancellation of the therapy. In addition, by the 30th day of treatment, patients receiving the composition in accordance with the invention, there was a significantly more distinct normalization of liver size compared with patients receiving the drug according to the prototype, and even more so compared with patients in the control group.

B. According to laboratory tests, there is a time frame for normalizing the level of AlAT and AsAT, as well as the dynamics of virological examination indicators (reduction of viral load, elimination of HCV from the blood of patients) and interferon-inducing properties of the composition. During the study, the possibility of adverse reactions was taken into account.

Statistical processing of the material was carried out using variational analysis and determination of the arithmetic mean (M), the average error of the arithmetic mean (m), followed by establishing the significance of the differences between the average indices of the groups (p). Comparative results for groups I-III are presented in table 4 (see below). From the presented data it can be seen that the dynamics of changes in the level of bilirubin, gamma-gtr, total protein and albumin in all three groups was approximately the same.

At the same time, both in group I and group II, a significant decrease in the level of AlAT and AsAT was observed (in the control group, the level of these enzymes did not change significantly), while in group II, which received the composition in accordance with the invention, this decrease was significantly more pronounced (the differences were statistically significant at p <0.05), and these indicators of AlAT and AsAT in the group of patients receiving the composition according to the invention remained stable even after 6 months from the start of treatment. For the values of AlAT and AsAT on the 60th day (immediately after the end of the course of therapy), the differences between groups I and II were statistically significant at p <0.01.

In patients receiving the composition in accordance with the invention, in comparison with patients receiving the composition according to the prototype, there was a clear improvement in interferon status (increased serum interferon, interferon alpha) 10 and 20 days after the start of therapy.

In groups I and II during treatment, a significant decrease in HCV RNA was detected (by the time the course was completed, the detection rate of HCV RNA in groups I and II was 22.5% and 13.0%, respectively) compared with the control group, where the detection frequency of HCV RNA by the time the course was completed, it was 90%, but after 1 month. after the course of treatment, HCV RNA was detected in 25.0% of group I patients and only in 5.0% of group II patients (in the control group, the detection rate of HCV RNA increased to 97%); after 6 months from the start of therapy, HCV RNA was detected in 30% of patients receiving the composition of the prototype, and only 13.0% of patients receiving the composition in accordance with the invention.

Table 3. Comparative characteristics of the severity of disease symptoms and their duration (days) in patients with chronic viral hepatitis C (groups I-III) Symptoms of the disease Group I (27 people) Group II (27 people) Group III (20 people) ABS. % Lasts. symptoms (days) ABS % Lasts. symptoms (days) ABS % Lasts. symptoms (days) Headache 5 18.5 3.22 ± 0.66 one 3,7 3.0 ± 0.1 8 40,0 3.78 ± 0.92 Weakness 22 81.4 27.1 ± 1.43 10 37.0 15.2 ± 0.7 eighteen 90.0 35.3 ± 1.78 Decreased appetite fifteen 55.6 13.1 ± 0.8 8 29.6 7.2 ± 0.9 19 95.0 17.2 ± 3.11 Nausea 7 25.9 2.1 ± 0.5 3 11.1 1.0 ± 0.2 13 65.0 9.3 ± 1.7 Pain in the right hypochondrium 10 37.0 6.7 ± 2.1 5 18.5 3.2 ± 0.7 twenty 100.0 30.3 ± 5.5

Table 4. Data of a biochemical analysis of blood in patients with chronic viral hepatitis C (groups I-III) Indicator Group Before treatment On the 30th day of therapy By the end. therapy (day 60) In a month after the end. treatment After 6 months from the start of therapy AlAT (U / L) I 133.2 ± 22.1 65.1 ± 13.2 148.1 ± 9.6 49.8 ± 8.2 55.3 ± 8.4 II 116.8 ± 19.9 49.9 ± 10.0 30.0 ± 9.2 32.0 ± 8.4 35.9 ± 7.3 III 122.1 ± 20.8 113.9 ± 15 103.1 ± 10 90.0 ± 4.8 99.7 ± 8.1 AsAT (U / L) I 83.7 ± 20.5 42.3 ± 13.9 49.5 ± 10.2 55.3 ± 11.2 55.7 ± 12.2 II 78.5 ± 16.7 35.2 ± 11.0 31.0 ± 9.7 35.9 ± 12.1 33.0 ± 11.4 III 85.2 ± 11.9 88.1 ± 12.3 75.4 ± 12.5 77.7 ± 13.5 89.7 ± 15.8 GGTP (U / L) I 99.2 ± 15.2 59.1 ± 11.7 48.1 ± 12.0 53.2 ± 10.0 42.0 ± 11.1 II 95.8 ± 11.5 63.2 ± 15.1 47.0 ± 11.9 50.0 ± 11.2 41.1 ± 12.5 III 99.4 ± 17.3 60.0 ± 14.2 49.0 ± 13.5 52.1 ± 11.8 43.1 ± 15.0 Bilirubin (μmol / L) I 25.7 ± 5.2 15.5 ± 2.0 15.8 ± 1.4 17.1 ± 3.1 19.2 ± 4.2 II 29.7 ± 4.6 16.7 ± 3.0 16.4 ± 1.9 16.5 ± 1.4 16.9 ± 2.2 III 22.3 ± 2.9 19.9 ± 3.6 18.0 ± 1.2 20.2 ± 3.2 17.1 ± 1.0 Total protein (g / l) I 77.2 ± 8.9 N / A 75.3 ± 7.6 N / A 78.1 ± 4.9 II 79.9 ± 11.3 N / A 77.9 ± 9.0 N / A 79.2 ± 5.6 III 75.4 ± 9.2 N / A 77.1 ± 12.2 N / A 77.5 ± 10.0 Albumin (g / l) I 50.7 ± 1.9 N / A 49.4 ± 1.8 N / A 48.7 ± 2.4 II 48.4 ± 2.8 N / A 50.5 ± 2.2 N / A 49.5 ± 3.5 III 49.7 ± 1.3 N / A 53.5 ± 1.9 N / A 49.9 ± 4.1 PHKHCV (%) I 100.0 49.2 22.5 25.0 30,0 II 100.0 30.1 13.0 5,0 13.0 III 100.0 95.0 90.0 97.0 100.0 * N / A - RESEARCH NOT PERFORMED

In addition, in the framework of the present invention, it was shown that the composition in accordance with the invention did not have adverse side effects in the treatment of chronic viral hepatitis C. The composition according to the invention was well tolerated by patients, normalized their emotional state; no patients had allergic reactions.

The composition according to the invention, in comparison with the prototype, showed unexpectedly high antiviral activity, which resulted in a statistically significant decrease in the symptoms of liver failure and a reduction in the time of their disappearance, as well as in a significantly faster rate (in comparison with the prototype) of the decrease in the levels of AlAT, AsAT and alkaline phosphatase, and in a statistically significant decrease (in some cases, the disappearance) of HCV RNA and in the improvement of indicators of ititerferon status and production of endogenous alpha-interferon.

Example 3. Antiviral activity of the claimed composition against viral hepatitis B.

The study of the effectiveness of the claimed composition in the treatment of viral hepatitis B involved 60 people (38 men, 22 women), aged 22 to 58 years, suffering from acute viral hepatitis B.

One third of the patients (20 people) received the composition three times a day orally (in capsule form) in accordance with the prototype (hyporamine + flacoside + helepin) and symptomatic therapy (comparison group), another third of the patients (20 people) received three times a day orally (in in the form of capsules) a composition in accordance with the invention containing an equivalent amount of hyporamine and flacoside (experimental group); a third of patients (20 people) received a placebo preparation three times a day (in the form of capsules that did not seem to differ from the studied compositions) (control group), while the patients were distributed into groups randomly; There were no significant differences between the groups by age, weight, gender, timing and severity of the disease.

All patients underwent the acute phase of the disease, while not a single patient took interferon drugs, immunostimulants, or steroids for 2-3 months before the study and during it.

The effectiveness of the claimed composition and composition in accordance with the prototype was evaluated both by the duration of preservation of the clinical symptoms of the disease (weakness, dyspeptic syndrome, pain in the right hypochondrium, jaundice, skin itching), and by the change in the biochemical parameters of blood serum (total bilirubin, alanine aminotransferase (AlAT) , aspartate aminotransferase (AsAT)), as well as the changes observed in the interferon system observed during treatment in patients of the “experimental” group, the control group and the comparison group (receiving com position according to the prototype) and the presence of HBV replication markers in patients by the time of treatment, namely the disappearance of HBV DNA, the HBeAg seroconversion in HBeAB (it is believed that HBeAg seroconversion in HBeAB with viral hepatitis B means the termination of virus replication and a favorable outcome diseases), by the presence of HBsAg, as well as by the presence in patients of antibodies (IgM) to the “cow” antigen (anti-HBc IgM).

The results are presented in tables 5-7. So, table 5 shows the duration of the clinical symptoms of the disease in the experimental group, the control group and the comparison group; table 6 shows the data of biochemical analysis of blood serum in patients with acute viral hepatitis B in the experimental group, the control group and the comparison group; Table 7 shows the data on the presence of HBV and anti-HBc IgM replication markers in the experimental group, the control group, and the control group at the end of the treatment) in the experimental group, the comparison group, and the control group.

Table 5. The duration of the clinical symptoms of the disease in the experimental group, the control group and the comparison group, day Symptoms Experimental group (0) (n = 20) Comparison Group (C) (n = 20) Control group (K) (n = 20) Weakness 2.4 ± 1.3 5.7 ± 2.2 6.9 ± 1.2 Dyspeptic syndrome 3.1 ± 0.9 5.2 ± 1.5 6.5 ± 1.7 Pain in the right hypochondrium 2.2 ± 0.7 3.4 ± 1.9 4.6 ± 2.1 Jaundice 4,5 ± 1,0 6.2 ± 1.4 10.9 ± 1.6 Itchy skin 3.2 ± 1.5 4.8 ± 2.4 9.3 ± 1.7

Table 6. Biochemical parameters of blood serum in patients with acute viral hepatitis B in the experimental group, the control group and the comparison group. Before starting treatment Mid-course of treatment At the end of treatment About ¹ C ² K ³ About ¹ C ² K ³ About ¹ C ² K ³ Total bilirubin ⁴ , mmol / l 209.3 ± 11.2 173.4 ± 15.9 185.7 ± 13.8 64.3 ± 7.7 109.2 ± 11.0 127.9 ± 12.2 29.8 ± 7.9 57.3 ± 10.1 85.4 ± 13.0 AlAT ⁵ , nmol / (s · l) 2178.1 ± 113.9 1812.5 ± 209.3 1956.4 ± 195.6 752.9 ± 19.2 849.1 ± 13.5 979.1 ± 15.8 719.4 ± 12.2 799.4 ± 10.1 858.4 ± 23.5 AsAT ⁶ , nmol / (s · l) 1783.5 ± 123.4 1844.1 ± 142.5 1629.3 ± 142.1 793.2 ± 13.4 867.1 ± 16.6 989.4 ± 19.7 694.6 ± 13.8 768.2 ± 11.4 809.3 ± 12.9 ¹ Experimental group (n = 20) (patients receiving the composition according to the invention)
² comparison group (n = 20) (patients receiving the composition in accordance with the prototype)
³ Control group (n = 20) (patients receiving placebo)
⁴ Normal - 3.4-17.1 μmol / L
⁵ Normal - up to 666 nmol / (s · l)
⁶ Normal - up to 666 nmol / (s · l)

Table 7. HBV replication markers and the presence of anti-HBc IgM in the experimental group, the control group and the comparison group. Patient group Experimental group ⁷ (n = 20) Comparison Group ⁸ (n = 20) Control group ⁹ (n = 20) HBV DNA (-) 48.90% 31.50% 12.0% Seroconversion (HBeAg => HBeAB) 62.50% 46.50% 15.0% HBsAg (-) 78.0% 51.5% 10.0% anti HBc IgM 13.5% 41.5% 82.50% ⁷ Patients receiving the composition of the invention (n = 20) ⁸ Patients receiving a complex of substances in accordance with the prototype (according to RU 2200571) (n = 20) ⁹ Patients ^on Placebo (n = 20)

Example 4. Antiviral activity of the claimed composition against HIV infection

The study of the antiviral activity of the claimed pharmaceutical composition (in comparison with the prototype) was carried out in vitro in a transplanted mixed culture of human T-lymphocytes H9 / IIIB and H9 infected with HIV-1 (strain HTLV-III). A cell culture mixture (1: 1 ratio) was prepared immediately before the experiment and 2 ml was poured into each well of the Coster plate. At the same time, the test composition in accordance with the present invention was added to 1/3 of the wells (with a final concentration (according to hyporamine) of 30, 10, 5, and 1 μg / ml (experimental preparation, “O”); in accordance with the prototype (with obtaining a similar final concentration for hyporamine) (comparison drug, "C"); another 1/3 of the holes were used as control (control, "K").

The plate was incubated at a temperature of 37 ° C at 5% CO ₂ for 7 days. Cell viability was assessed daily using an inverted microscope. The results of the experiment were taken into account on the 4th day by indirect immunofluorescence using serum of an HIV-infected person at a 1:40 dilution, determining the number of HIV ⁺ cells in the experimental group, in the comparison group and in the control group (cell culture not treated with antiviral compounds). Each experiment (study of each concentration of the experimental drug and the comparison drug) was repeated three times.

In the control group, the number of HIV ⁺ cells amounted to 24.9 ± 2.4% by the 4th day; this value was subsequently taken as 100%.

The results of the study were summarized in table 8 (indicated% in relation to the number of HIV ⁺ cells in the control).

Table 8. A comparative study of the effect of the test drug and the reference drug on the expression of HIV-1 antigens by H9 culture cells on the 4th day after infection. A drug Concentration (hyporamine) The number of repetitions of the experiment The number of HIV ⁺ cells in culture,%, ± SD Test drug 30 mcg / ml 3 4.1 ± 0.5 Test drug 10 mcg / ml 3 10.5 ± 2.9 Test drug 5 mcg / ml 3 17.4 ± 5.2 Test drug 1 mcg / ml 3 28.3 ± 5.9 Comparison drug (according to RU2200571) 30 mcg / ml 3 24.3 ± 3.8 Comparison drug (according to RU2200571) 10 mcg / ml 3 31.4 ± 4.7 Comparison drug (according to RU2200571) 5 mcg / ml 3 47.4 ± 6.2 Comparison drug (according to RU2200571) 1 mcg / ml 3 64.8 ± 7.2

Example 5. Antiviral activity of the claimed composition against avian influenza A virus H5N1

A comparative study of the neutralizing activity of the claimed composition and complex of substances in accordance with the prototype (RU 2200571) was carried out in the chicken embryo system using the H5N1 avian influenza A virus. The virus-neutralizing effect of the claimed composition and complex of substances in accordance with the prototype was studied in in vitro contact experiments, as well as indication on chicken embryos. The composition in accordance with the invention (experimental preparation, “O”) or a complex of substances in accordance with the prototype (comparative preparation, “C”) was dissolved in a buffer solution with a pH of 7.2-7.4 and mixed with 1, 10, 100 and 1000 EID100 (embryonic infectious doses that cause infection of chicken embryos in 100% of cases), with the final concentration of solutions (hyporamine) being 100 or 50, or 25, or 10, or 5, or 2.5, or 1, or 0, 5, or 0.25, or 0.1 or 0.05 μg / ml. Mixtures of preparations with H5N1 avian influenza A virus were kept for 1 h at a temperature of + 4 ° С, then 9-10 intact chicken embryos were injected into the allantoic cavity in a volume of 0.2 ml. 48 hours after the introduction, the chicken embryos were opened and the presence of the influenza virus was determined in the allantoic fluid (hemagglutination method). Activity was expressed by the number of neutralized embryonic infectious doses of influenza virus.

Each measurement was repeated three times; the arithmetic mean and standard deviation were calculated from the results of three measurements.

The results of the study of the antiviral activity of the claimed composition in comparison with the preparation of the prototype are presented in table 9.

Table 9. A comparative study of the antiviral activity of the claimed composition against influenza A virus of birds H5N1. Hyporamine drug concentration, mcg / ml The number of neutralized EID ₁₀₀ virus (composition according to the invention, "O") The number of neutralized EID _{100 of the} virus (comparison drug according to RU 2200571, "C") one hundred 8000.0 ± 123.9 1520 ± 110.5 fifty 4040.0 ± 68.9 1028 ± 97.4 25 1787.0 ± 49.4 460.0 ± 72.2 10 1280.0 ± 38.8 320.0 ± 44.3 5 750.0 ± 24.7 170.0 ± 22.9 2.5 450.0 ± 24.3 100.0 ± 9.8 one 176.8 ± 14.2 53.8 ± 3.1 0.5 75 ± 13.6 43.1 ± 5.5 0.25 45.1 ± 12.9 23.2 ± 2.2 0.1 20.0 ± 10.2 14.2 ± 1.4 0.05 1.0 ± 1.4 1.0 ± 0.7

Example 6. Antiviral activity of the claimed composition against herpes virus infection (HSV1 virus) in an in vivo experiment

Experiment 1. A preclinical study was conducted of the comparative antiviral activity of the claimed pharmaceutical composition against herpes simplex virus type 1 (HSV-1 virus, strain SC16) in comparison with the pharmaceutical composition of the prototype.

The study was conducted on laboratory mice (female BALB / c and DBA / 2, Bantin and Kingman, UK). Animals in the control group (25 animals), the experimental group (25 animals) or the comparison group (25 animals) were infected by intraperitoneal administration of a suspension of HSV-1 SC16 virus at a dose of 5 × 10 ⁵ PFU (plaque-forming units) in 0.1 ml of phosphate buffer.

Antiviral treatment (oral administration of the claimed composition (in the experimental group) or a comparison drug known from RU 2200571 complex of substances (in the comparison group) containing an equivalent amount of hyporamine and flacoside) was started 24 hours after infection and continued for 120 hours from moment of infection. The experimental drug and the comparison drug were given in such a way as to provide a dose (for hyporamine) of about 100 mg / kg body weight per day.

Since it is known from the literature that the spleen and peritoneal exudate are the main reservoirs of herpes virus in the body and that the titer of the herpes virus (PFU / 0.1 ml) in peritoneal washes correlates well with the virus content in the spleen tissues, the titer of the virus was determined in the framework of this experiment in peritoneal swabs obtained from animals of the experimental group, the comparison group and the control group 24, 48, 72, 96 and 120 hours after infection.

Peritoneal washings were obtained by injecting 2 ml of ice-cold Eagle minimal nutrient medium containing 10% serum of newborn calves into the abdominal cavity of animals, gently massaging the abdomen in mice and then taking samples to study peritoneal fluid. The obtained samples were subjected to freezing / thawing three times, then the virus content (HSV-1 SC-16) was determined in each of the samples by plaque formation in a Vero cell culture (renal epithelial cells).

The results are presented in table 10 and in the drawing (as decimal logarithms of the values of PFU / 0.1 ml).

Table 10. The antiviral activity of the claimed composition and the reference drug against herpes simplex virus (HSV-1 SC-16) (with repeated administration). Time after infection, hour. 24 hours 48 hours 72 hours 96 hours 120 hours Experimental preparation, dose 100 mg / kg (for hyporamine), virus titer (Ig (PFU / 0.1 ml)) 0.7 ± 0.2 * 0.3 ± 0.1 * 0.2 ± 0.07 * 0.08 ± 0.02 * 0.04 ± 0.01 * Comparison drug (complex according to RU 2200571), dose 100 mg / kg (for hyporamine), virus titer (Ig (PFU / 0.1 ml)) 2.4 ± 0.3 * 1.5 ± 0.2 * 1.3 ± 0.3 * 1.4 ± 0.2 * 2.0 ± 0.4 * The control 2.7 ± 0.4 * 3.6 ± 0.3 * 3.8 ± 0.7 * 2.7 ± 0.8 * 2.2 ± 0.5 * * The differences are significant at p <0.01.

From the presented data it can be seen that although in the group receiving a complex of substances of plant origin in accordance with the prototype (hyporamine, flacoside and helepin or helepin D, taken in a certain quantitative ratio), after 48 hours from the moment of infection there was a slight decrease in virus titer (in compared with the control, where the titer of the virus practically did not change in the first 3 days from the moment of infection), however, on the contrary, in the group of animals that received the composition in accordance with the claimed invention, containing equivalent The corresponding amount of hyporamine, the titer of the virus by 48 hours from the time of infection was close to the level of sensitivity of the method, and by the end of the experiment (120 hours from the time of infection), the virus was not detected at all in the peritoneal washes of animals of the experimental group.

Experiment 2. The effectiveness of the claimed composition against HSV-2 infection

Mice (60 Balb / c females, including 20 animals of the experimental group, 20 animals of the comparison group and 20 animals of the control group) were infected with HSV-2 herpes virus (Bry) (by inoculating a suspension of the virus in the earlobe) and the effect of the claimed pharmaceutical composition and preparation was studied comparisons (a complex of substances according to RU 2200571) for latent herpes virus infection. Treatment began on the 1st day after infection.

Table 11 shows the number of animals with latent HSV-1 infection in the trigeminal (TG) and cervical dorsal root (CDC) ganglia in each group (experience, control, comparison drug) 7 days after the start of treatment.

Table 11. The number of animals with latent HSV-2 infection in the trigeminal (TG) and cervical dorsal root (CDC) ganglia: Group The number of mice with latent HSV-1 infection % of animals with detected latent infection in at least the 1st ganglion Left TG Right TG Left CDK Right CDK Control (n = 20) ¹⁰ twenty twenty twenty twenty one hundred% Experienced (n = 20) ¹¹ 0 0 one one 10% Comparison drug (n = 20) ¹² 2 four four 5 75% ¹⁰ Animals not treated ¹¹ Animals receiving the drug in accordance with the invention ¹² Animals treated with a complex of substances in accordance with the prototype
(RU 2200571)

The presented data, in our opinion, convincingly indicate that the claimed composition in comparison with the prototype (a complex of substances according to the patent RU 2200571) has an unexpectedly higher quantitatively antitreponematic and antiviral activity and is characterized by a wide spectrum of antiviral activity, which is an unexpected technical result of the present invention.

Claims (22)

1. A pharmaceutical composition for the treatment of syphilis and viral diseases selected from influenza, HIV infection, neuroinfection, cytomegalovirus infection, viral hepatitis, secondary immunodeficiency conditions against a background of chronic bacterial infections and herpes virus infection, made on the basis of plant extracts and containing in therapeutically effective amounts the amount of polyphenolic compounds from the leaves of buckthorn buckthorn, including at least 60% galloellagotannins, flacoside and pharmaceutically when mlemy carrier, characterized in that it further comprises a therapeutically effective amount akridonouksusnoy acid or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of liquorice root extract or glycyrrhizic acid or pharmaceutically acceptable salts of glycyrrhizic acid and a therapeutically effective amount of the extract of bark or betulin. 2. The pharmaceutical composition according to claim 1, characterized in that the therapeutically effective amount of the sum of polyphenolic compounds from buckthorn buckthorn leaves is from 10 to 100 mg. 3. The pharmaceutical composition according to claim 1, characterized in that the therapeutically effective amount of flacoside is from 100 to 200 mg. 4. The pharmaceutical composition according to claim 1, characterized in that the pharmaceutically acceptable salt of acridonoacetic acid is a salt of acridonoacetic acid with N-methylglucamine. 5. The pharmaceutical composition according to claim 1, characterized in that the therapeutically effective amount of acridonoacetic acid or its pharmaceutically acceptable salt is from 250 to 1000 mg. 6. The pharmaceutical composition according to claim 1, characterized in that the pharmaceutically acceptable salt of glycyrrhizic acid is sodium glycyrrhizinate. 7. The pharmaceutical composition according to claim 1, characterized in that the therapeutically effective amount of glycyrrhizic acid or its pharmaceutically acceptable salt is from 30 to 300 mg. 8. The pharmaceutical composition according to claim 1, characterized in that the therapeutically effective amount of birch bark or betulin extract is from 100 to 350 mg. 9. The pharmaceutical composition according to claim 1, characterized in that the viral hepatitis is a viral hepatitis A, or viral hepatitis B, or viral hepatitis C. 10. The pharmaceutical composition according to claim 1, characterized in that the HIV infection is AIDS (stages IIA-IIIB). 11. The pharmaceutical composition according to claim 1, characterized in that the neuroinfection is neuro-AIDS, or viral encephalitis, or Lyme disease. 12. The pharmaceutical composition according to claim 1, wherein the flu is avian influenza A caused by the H5N1 virus. 13. The pharmaceutical composition according to any one of claims 1 to 12, characterized in that it is made in the form of hard or soft gelatin capsules. 14. The pharmaceutical composition according to any one of claims 1 to 12, characterized in that it is made in the form of pellets (s). 15. The pharmaceutical composition according to any one of claims 1 to 12, characterized in that it is made in the form of tablets. 16. The pharmaceutical composition according to p. 15, characterized in that it is made in the form of tablets with a modified release. 17. The pharmaceutical composition according to any one of claims 1 to 12, characterized in that it is made in the form of a sachet. 18. A method of treating syphilis and viral diseases selected from influenza, HIV infection (including AIDS, stages IIA-IIIB), neuroinfection, cytomegalovirus infection, viral hepatitis, secondary immunodeficiency conditions against a background of chronic bacterial infections and herpes virus infection, comprising administering a pharmaceutical composition according to any one of claims 1 to 17, in need of this patient, 2-3 doses 3 times a day. 19. The method according to p. 18, characterized in that the flu is an avian influenza A caused by the H5N1 virus. 20. The method according to p. 18, wherein the viral hepatitis is viral hepatitis A, or viral hepatitis B, or viral hepatitis C. 21. The method according to p. 18, wherein the neuroinfection is neuro-AIDS, or viral encephalitis, or Lyme disease. 22. The method according to p. 18, wherein the HIV infection is AIDS (stages IIA-IIIB).

Images