PHARMACEUTICAL FORMULATION FOR THE ELIMINATION OF CAUSATIVE AGENTS OF HERPESVIRAL INFECTIONS FROM THE TISSUES OF A MACROORGANISM

Abstract

A pharmaceutical composition is proposed for the elimination of the causative agents of herpes virus infections from macroorganism tissues, including histone deacetylase inhibitors, acyclovir derivatives, epigallocatechin gallate and glycyrrhizin, characterized in that it additionally contains a supramolecular structure of a combination of gator epigalocatechin gallate and galactate epigalocatechol at least two covalent modifying agents.

Also, the composition may include such substances as chorlecalciferol, toll receptor activators acridonoacetic acid, tilorone, zymosan, imidazoquinoline, imiquimod. As modifiers of the structures of epigalocatechin gallate and glycyrrhizin can be used: succinic anhydride, acetic anhydride, propionic anhydride, butane anhydride, acetic-propionic anhydride, acetic-buttanic anhydride, glutaric anhydride, phthalic anhydride, citric anhydride, citric anhydride citric anhydride, isolimonic anhydride, acetyl chloride, acetyl fluoride, propionyl chloride, butyroyl chloride, ethoxyoxalyl monochloride, monochloroacetic acid.

Claims

1. A pharmaceutical composition designed to eliminate the causative agents of herpes virus infections from the tissues of a macroorganism, comprising histone deacetylase inhibitors, acyclovir derivatives, epigallocatechin gallate and glycyrrhizin, wherein it additionally contains a supramolecular structure from a combined mixture of combinatorial derivatives of gallate and epigallocatechin gallate at least two covalent modifying agents.

2. The pharmaceutical composition according to claim 1, wherein it further comprises a supramolecular structure from an undivided mixture of combinatorial derivatives of glycyrrhizin obtained by simultaneous modification of glycyrrhizin with at least two covalent modifying agents.

3. The pharmaceutical composition according to claim 1, wherein succinic anhydride and monochloroacetic acid are used as covalent modifiers.

4. The pharmaceutical composition according to claim 1, wherein maleic anhydride and succinic anhydride are used as covalent modifiers.

5. The pharmaceutical composition according to claim 1, wherein maleic anhydride and monochloroacetic acid are used as covalent modifiers.

6. The pharmaceutical composition according to claim 1, wherein any two modifiers from the list can be used as covalent modifiers: acetic anhydride, propionic anhydride, butane anhydride, acetic-propionic anhydride, acetic-butanic anhydride, glutaric anhydride, phthalic anhydride, cis-aconitic anhydride, trans-aconitic anhydride, citric anhydride, isolimonic anhydride, acetyl chloride, acetyl fluoride, propionyl chloride, butyroyl chloride, ethoxyoxalyl monochloride.

7. The pharmaceutical composition according to claim 1, wherein it further comprises not limited to those presented here, but including one or more low molecular weight activators of TOLL receptors: acridonoacetic acid, tilorone, zymosan, imidazoquinoline, imiquimod.

8. The pharmaceutical composition according to claim 1, wherein it further comprises cholecalciferol.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] FIG. 1. Scheme for the chemical synthesis of combinatorially modified epigalocatechin gallate derivatives.

[0051] FIG. 2. Scheme for the chemical synthesis of combinatorially modified derivatives of glycyrrhizic acid.

[0052] FIG. 3. HPLC (Milichrom A-02) of the combinatorial derivative of epigalocatechin gallate (2), its octasuccinyl (1) and octaacetyl (3) derivatives and control—unmodified epigalocatechin gallate (4), solution A gradient: 0.5 M lithium perchlorate/0.1 M chloric acid, solution B: acetonitrile (B from 5% to 100%)

[0053] FIG. 4. HPLC (Milichrom A-02) of the combinatorial derivative of glycyrrhizin (2), its octasuccinyl (1) and octaacetyl (3) derivatives and control—unmodified glycyrrhizin (4), solution A gradient: 0.5 M lithium perchlorate/0.1 M perchloric acid, solution B: acetonitrile (B from 5% to 100%)

PHARMACEUTICAL COMPOSITIONS

[0054] Various methods of preparing a patentable pharmaceutical composition (PFC) can be used. The PFC composition can be given orally or can be administered by intravascular, subcutaneous, intraperitoneal injection, in the form of an aerosol, by ocular route of administration, into the bladder, topically, and so on. For example, inhalation methods are well known in the art. The dose of the therapeutic composition will vary widely depending on the specific antiviral PFC administered, the nature of the disease, frequency of administration, route of administration, clearance of the agent used from the host, and the like. The initial dose may be higher with subsequent lower maintenance doses.

[0055] The dose can be administered once a week or once every two weeks, or divided into smaller doses and administered once or several times a day, twice a week, and so on to maintain an effective dose level. In many cases, a higher dose will be needed for oral administration than for intravenous administration, PFCs can be included in many therapeutic compositions. More specifically, the PFCs of the present invention can be incorporated into pharmaceutical compositions in combination with suitable pharmaceutically acceptable carriers or diluents, and can be incorporated into preparations in solid, semi-solid, liquid or gaseous forms, such as capsules, powders, granules, ointments, creams, foams, solutions, suppositories, injections, forms for inhalation use, gels, microspheres, lotions and aerosols.

[0056] As such, the administration of the compounds can be carried out in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intratracheal administration and so on. The PFCs of the invention can be distributed systemically after administration or can be localized using an implant or other composition that holds the active dose at the site of implantation. The PFCs of the present invention can be administered alone, in combination with each other, or they can be used in combination with other known compounds (e.g. clopidogrel, anti-inflammatory agents, and so on).

[0057] In pharmaceutical dosage forms, the compounds may be administered in the form of their pharmaceutically acceptable salts. The following methods and excipients are given as examples only and are in no way limiting. For oral preparations, the compounds can be used alone or in combination with suitable additives for the manufacture of tablets, powders, granules or capsules. For example, with conventional additives such as lactose, mannitol, corn starch or potato starch; with binding agents such as crystalline cellulose, cellulose derivatives, gum arabic, corn starch or gelatins; with disintegrants such as corn starch, potato starch or sodium carboxymethyl cellulose; with lubricants such as talc or magnesium stearate; and, if desired with diluents, buffering agents, moisturizing agents, preservatives and flavoring agents.

[0058] PFCs should be included in injectable compositions by dissolving, suspending or emulsifying them in an aqueous or non-aqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and, if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives.

[0059] PFCs can be used in an aerosol composition for inhalation administration. The compounds of the present invention can be incorporated into suitable pressure propellants such as dichlorodifluoromethane, propane, nitrogen and the like. In addition, PFCs can be incorporated into suppositories by mixing with a variety of bases, such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally using a suppository. A suppository may contain excipients, such as cocoa butter, carboax, and polyethylene glycols, which melt at body temperature but are solid at room temperature.

[0060] Standard dosage forms for oral or rectal administration, such as syrups, elixirs and suspensions, where each unit dose, for example, a teaspoon, tablespoon, tablet or suppository, may contain a predetermined amount of a composition containing one or more compounds of the present invention . Similarly, unit dosage forms for injections or intravenous administration may contain the compound of the present invention in the composition in the form of a solution in sterile water, normal saline or another pharmaceutically acceptable carrier. Implants for the sustained release of compositions are well known in the art.

[0061] Implants are made in the form of microspheres, plates, and so on with biodegradable or non-biodegradable polymers. For example, lactic and/or glycolic acid polymers form a degradable polymer that is well tolerated by the host. An implant containing the PFC according to the invention is positioned close to the focus of the pathology, so that the local concentration of the active agent is increased compared to other areas of the body. As used here, the term “Unit dosage form” refers to physically discrete units suitable for use as single doses for human and animal subjects, each unit containing a predetermined number of compounds of the present invention, which, according to calculations, is sufficient to provide the desired effect, together with a pharmaceutically an acceptable diluent, carrier or excipient.

[0062] The descriptions of the unit dosage forms of the present invention depend on the particular compound used, and the effect to be achieved, as well as the pharmacodynamics of the compound used in the host. Pharmaceutically acceptable excipients, such as fillers, adjuvants, carriers or diluents, are generally available. In addition, pharmaceutically acceptable excipients are generally available, such as pH adjusting agents and buffering agents, tonicity agents, stabilizers, wetting agents and the like.

[0063] Typical doses for systemic administration range from 0.1 μg to 1000 milligrams per kg of subject body weight per administration. A typical dose may be a single tablet for administration from two to six times a day, or one capsule or sustained release tablet for administration once a day with a proportionally higher content of the active ingredient. The effect of prolonged release may be due to the materials of which the capsule is made, dissolving at different pH values, capsules providing a slow release under the influence of osmotic pressure or any other known controlled release method.

[0064] To the professionals in the field of this art it will be clear, that dose levels may vary depending on the particular compound, the severity of the symptoms, and the subject's predisposition to side effects. Some of the specific compounds are more potent than others. Preferred doses of this compound can be readily determined by specialists who are skilled in this art in a variety of ways. The preferred method is to measure the physiological activity of PFC.

[0065] One of the methods of interest is the use of liposomes as a vehicle for delivery. Liposomes fuse with the cells of the target region and provide delivery of liposome contents to the cells. The contact of the liposomes with the cells is maintained for a time sufficient for fusion using various methods of maintaining contact, such as isolation, binding agents and the like. In one aspect of the invention, liposomes are designed to produce an aerosol for pulmonary administration. Liposomes can be made with purified proteins or peptides that mediate membrane fusion, such as Sendai virus or influenza virus and so on.

[0066] Lipids can be any useful combination of known liposome forming lipids, including cationic or zwitterionic lipids, such as phosphatidylcholine. The remaining lipids will usually be neutral or acidic lipids, such as cholesterol, phosphatidylserine, phosphatidylglycerol and the like. To obtain liposomes, the method described by Kato et al. (1991) J. Biol. Chem. 266: 3361. is utilized.

[0067] Briefly, lipids and a composition for incorporation into liposomes containing a patented composition are mixed in a suitable aqueous medium, suitably in a salt medium, where the total solids content will be in the range of about 110 wt. %. After vigorous stirring for short periods of approximately 5-60 seconds, the tube is placed in a warm water bath at approximately 25-40° C. and this cycle is repeated approximately 5-10 times.

[0068] The composition is then sonicated for a suitable period of time, typically approximately 1-10 seconds, and optionally further mixed with a vortex mixer. Then the volume is increased by adding an aqueous medium, usually increasing the volume by about 1-2 times, followed by agitation and cooling. This method allows the inlusion of supramolecular structures with high total molecular weight in liposomes.

Compositions With Other Active Agents

[0069] For use in the methods under consideration, the PFCs of the invention can be formulated with other pharmaceutically active agents, in particular other antiviral, immunomodulatory and antimicrobial agents known in the art. Classes of drugs for the treatment of herpes virus infections and their complications are presented in standardized protocols for the treatment of these pathologies and can be combined with patented PFCs, for example, ribavirin, iododeoxyuridine, ganciclovir, valganciclovir, valaciclovir, penciclovir.

[0070] Cytokines, for example, interferon alpha, interferon gamma, interferon-beta, tumor necrosis factor alpha, interleukin 12, interferon inducers cycloferon, tilorone, other activators of TOLL receptors: zymosan, imidazoquinoline, imiquimod, inducer of completion can also be included in the PFC composition of the invention. phagocytosis of cholecalciferol. The present invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

EXAMPLE 1

Obtaining a Combinatorial Mixture of Derivatives of Epigallocatechin Gallate (KEGG)

[0071] 764 μM epigallocatechin gallate (I) is dissolved in 10 ml of dioxane, 2040 μM succinic anhydride (III) and 2040 μM acetic anhydride are added, the solution is stirred and heated under reflux for 20 minutes. The solution is then poured into ampoules and lyophilized to remove solvent and acetic acid. The combinatorial mixture is used to obtain pharmaceutical compositions, study the structure, and determine the biological activity. FIG. 1 shows the synthesis scheme for combinatorial derivatives of I. One initial molecule (I) contains 8 phenolic hydroxyl groups available for modification.

[0072] Calculations of the number of moles of modifiers are carried out according to the combinatorics formulas:

[0073] m=4×(3×2.sup.n−2−1); k=n×(2.sup.n−1), where m is the number of different derivatives of molecules in the combinatorial mixture and the number of moles (I) for the reaction; n is the number of phenolic hydroxides available for modification in structure I (n=8); k is the number of moles of each modifier.

[0074] Thus, having only one initial molecule (I) and two modifiers after combinatorial synthesis, we obtain 764 combinatorial derivatives (IV ad) with different degrees of substitution, different positions of substituents and different permutations of the modifier residues, not just in the form of a mixture, but in the form of it is difficult to separate supramolecular mixture. Modifiers—succinic anhydride or acetic anhydride can be entered both simultaneously and sequentially—or first introduce succinic anhydride, warm the mixture under reflux for 20 minutes, then add acetic anhydride and also warm the mixture for another 20 minutes.

[0075] Similarly, in this reaction, instead of succinic and/or acetic anhydrides, the following modifiers can be used: maleic anhydride, aconitic anhydride, glutaric, phthalic anhydride and acetic anhydride, formic acid ethyl ester, monochloroacetic acid, propiolactone alkylene glycol ethylene oxide , ethyl chloride, propyl chloride). C13 NMR spectra were determined on a Brucker spectrometer.

[0076] C.sub.13 NMR spectra for the combinatorial derivative of epigalocatechin (IVa-d): 157.8, 94.8, 95.3, 157.2, 99.4, 24.8, 157.3, 68.6, 82.7, 130.9, 129.9, 112.0, 145.3, 144.6, 146.3, 169.0, 2-.3, 165.9 , 121.2, 108.6, 109.6, 146.1, 140.3, 133/4, 154.3, 171.1, 28.8, 29.1, 174.7

[0077] For the HPLC, a Milichrom A-02 microcolumn chromatograph in a gradient of acetonitrile (5-100%)/0.1 M perchloric acid +0.5 M lithium perchlorate was used. The combinatorial derivative in the chromatogram (FIG. 4) gave one clear broadened peak and was not divided into components, although the retention time differed from both epigallocatechin and its completely substituted derivatives. This indicated that complex supramolecular structures were formed between different combinatorial derivatives (in our case there were 764 of them), which were not separated chromatographically.

[0078] This combinatorial derivative (KEGG) also behaves similarly when separated in a thin layer (acetonitrile: water) and gives only one band, which does not coincide with any of the obtained derivatives.

[0079] Table 1 shows the screening of derivatives (I) with different ratios of modifiers as histone deacetylase inhibitors.

[0080] HAT and HDAC. Nuclear extract of HeLa cells (NE) was obtained as previously described [Yoon H G, Choi Y, Cole P A, Wong J Mol Cell Biol 2005, 25: 324-35.]. SIRT1 and HDAC-3 activity analysis was determined using a commercially available kit (Biovision Biotechnology) according to the manufacturer's instructions. SIRT1 deacetylase activity was analyzed using the SIRT1/Sir2 Deacetylase Fluorometric Assay (CycLex) kit. The table shows the data on the inhibition of histone deacetylases SIRT1 and HDAC-3 with epigalocatechin derivatives with different ratios of modifiers.

TABLE-US-00001 TABLE 1 The inhibitory ability against SIRT1 and HDAC-3 from the side of supramolecular combinatorial derivatives (I) obtained in the reaction with different molar ratio of modifiers The molar No ratio of reagents * K.sub.i (mM) p/p m k1 k2 HDAC-3 SIRT 1 764 8160*** 8160*** >4 >5 2 -//- 4080   4080   >4 >5 3 -//- 2040   2040   0.10 ± 0.02 0.24 ± 0.03 4 -//- 1020   1020   0.60 ± 0.03 0.80 ± 0.05 5 -//- 510  39  >4 >5 6 -//- 255  19  >4 >5 7 -//- 127  10  >4 >5 8 -//- 63  5 >4 >5 9 -//- 31  2 >4 >5 10 -//- 15  1 >4 >5 13 -//- 0 0 >4 >5 14 -//- 8160*** 0 >4 >5 16 -//- 4080   0 >4 >5 17 -//- 2040   0 >4 >5 18 -//- 1020   0 >4 >5 19 -//- 510  0 >4 >5 20 -//- 255  0 >4 >5 21 -//- 127  0 >4 >5 22 -//- 63  0 >4 >5 23 -//- 31  0 >4 >5 24 -//- 15  0 >4 >5 25 -//- 0 1 >4 >5 26 -//- 1 15  >4 >5 27 -//- 0 31  >4 >5 28 -//- 0 63  >4 >5 29 -//- 0 127  >4 >5 30 -//- 0 255  >4 >5 31 -//- 0 510  >4 >5 32 -//- 0 1020   >4 >5 33 -//- 0 2040   >4 >5 34 -//- 0 4080   >4 >5 35 -//- 0 8160   >4 >5 36 -//- 8160*** 1 >4 >5 37 -//- 4080   15  >4 >5 38 -//- 2040   31  >4 >5 39 -//- 1020   63  >4 >5 40 -//- 510  127  >4 >5 41 -//- 255  255  >4 >5 42 -//- 127  510  >4 >5 43 -//- 63  1020   >4 >5 44 -//- 31  2040   >4 >5 45 -//- 15  4080   >4 >5 46 -//- 0 8160   >4 >5 * m is the number of moles (1) in the combinatorial synthesis reaction; K1 is the number of moles of succinic anhydride in the reaction; K2 is the number of moles of acetic anhydride in the reaction; ** Ki (mM)—The release concentrations of the fluorescent diacetylated product of different substrate concentrations were used for Lineweaver-Burk calculations. Average data from two independent experiments. ***the maximum molar ratio at which all groups in (1) are replaced, the excess of this ratio leads to the fact that unreacted modifiers remain in the reaction medium—succinic anhydride and acetic anhydride.

[0081] As can be seen from table 1, only with the calculated ratio of components, when the maximum number of different derivatives (I) is formed, then a biologically active and effective supramolecular structure (derivative IV (a-d)) is formed. This supramolecular structure is able to inhibit both HDAC-3 and SIRT at a dose of 0.1 μM/L by 50%, which is 3 orders of magnitude less amount, than the initial dose of unmodified (I). Other derivatives either did not differ from unmodified (I) in their ability to inhibit HDAC-3 and SIRT, or were significantly less active.

[0082] This indicates that with the optimal ratio of modifiers when all possible derivatives are formed in the solution (764 variations of derivatives (I) with different permutations and arrangements in the substituents), a more complex supramolecular “quasi-lowering” structure with other properties and more than 3 orders of magnitude higher pharmacological activity is formed .

EXAMPLE 2

Obtaining a Fully Succinyl Epiglocatechin

[0083] 10 mM epigalocatechin (I) is dissolved in 10 ml of dioxane, 80 mM succinic anhydride (III) is added, the solution is stirred and heated under reflux for 20 minutes. The solution is poured into ampoules and lyophilized.

[0084] C.sub.13 NMR of octasuccinyl epigalocatechin: 174.7, 29.1, 28.8, 171.1, 149.3, 108.5, 149.6, 107.4, 155.3, 112.9, 25.0, 68.3, 82.1, 174.7, 29.1, 28.8, 133.1, 118.9, 118.9, 144.5, 133.5, 144.5, 165, 9, 123.4, 118.5, 146.0, 140.5, 146.0, 167.9

[0085] HPLC (Milichrom A-02; Gradient HClO4/LiClO4: AcCN 5-100%): 1 peak 18.0 min

EXAMPLE 3

Obtaining Fully Acetylated Epigalocatechin

[0086] 10 mM epigalocatechin (I) is dissolved in 10 ml of dioxane, 80 mM acetic anhydride (II) is added, the solution is stirred and heated under reflux for 20 minutes. The solution is poured into ampoules and lyophilized.

[0087] C.sub.13 NMR octaacetyl epigalocatechin: 20.3, 169.0, 149.3, 108.5, 20.3, 169.0, 107.4, 155.3, 112.9, 25.0, 68.3, 82.1, 133.1, 118.9, 118.9, 144.5, 133.5, 118.5, 123.4

[0088] HPLC (Milichrom A-02; Gradient HClO4/LiClO4: AcCN 5-100%): 1 peak 23.3 min

EXAMPLE 4

Obtaining a Combinatorial Mixture of Derivatives of Glycyrrhizin (CPG)

[0089] 92 μM glycyrrhizin (V) is dissolved in 10 ml of dioxane, 155 μM succinic anhydride (III) and 155 μM acetic anhydride are then added. After that the solution is stirred and heated under reflux for 20 minutes. The solution is then poured into ampoules and lyophilized to remove solvent and acetic acid. The combinatorial mixture is used to obtain pharmaceutical compositions, study the structure, and determine the biological activity. FIG. 2 shows the synthesis scheme for combinatorial derivatives of V. One of the original molecule (V) contains 5 hydroxyl groups available for modification in the glycoside residue.

[0090] Calculations of the number of moles of modifiers are carried out according to the combinatorics formulas:

[0091] m=4×(3×2.sup.n−2−1); k=n×(2.sup.n−1), where m is the number of different derivatives of molecules in the combinatorial mixture and the number of moles (I) for the reaction; n is the number of alcohol glycoside hydroxyls available for modification in structure I (n=5); k is the number of moles of each modifier. Thus, having only one initial molecule (V) and two modifiers after combinatorial synthesis, we obtain 92 combinatorial derivatives (VI ad) with different degrees of substitution, different positions of substituents and different permutations of the modifier residues, not just in the form of a mixture, but in the form of it is difficult to separate supramolecular mixture.

[0092] Modifiers—succinic anhydride or acetic anhydride can be entered both simultaneously and sequentially—or first introduce succinic anhydride, warm the mixture under reflux for 20 minutes, then add acetic anhydride and also warm the mixture for another 20 minutes. Similarly, in this reaction, instead of succinic anhydride and/or acetic anhydride, the following can be used as modifiers: maleic anhydride, aconitic anhydride, glutaric, phthalic anhydride and acetic anhydride, formic acid ethyl ester, monochloroacetic acid, propiolactone, ethylene oxide and other low molecular weight alkylating substances (methyl chloride, ethyl chloride). C13 NMR spectra were determined on a Brucker spectrometer.

[0093] C.sub.13 NMR for the combinatorial derivative of glycyrrhizin (VI ad): 174.7, 29.1, 29.5, 173.1, 172.9, 68.0, 71.6, 84.2, 82.5, 78.5, 109.8, 68.5, 68.6, 78.8, 112.1, 77.6, 173.2, 67.9, 53.1, 39.0, 29.6, 23.5, 57.2, 36.6, 17.2, 69.0, 18.0, 43.1, 40.4, 18.7, 200.8, 123.0, 158.1, 47.1, 32.4, 33.1, 15.4, 39.1, 51.5, 25.0, 43.3, 42.2, 36.2, 19.9, 182.7

[0094] For the HPLC, a Milichrom A-02 microcolumn chromatograph in a gradient of acetonitrile (5-100%)/0.1 M perchloric acid +0.5 M lithium perchlorate was used. The combinatorial derivative in the chromatogram gave one clear broadened peak and was not divided into components, although the retention time differed from both glycyrrhizin and its completely substituted derivatives. This indicated that complex supramolecular structures that were not separated chromatographically formed between different combinatorial derivatives (in our case, there were 92 of them). This combinatorial derivative (CSPG) behaves similarly when separated in a thin layer (acetonitrile: water) and gives only one band, which does not coincide with any of the obtained derivatives.

EXAMPLE 5

Obtaining Fully Succinylated Glycyrrhizin

[0095] 10 mM glycyrrhizin (V) is dissolved in 10 ml of dioxane, 50 mM succinic anhydride (III) is added, the solution is then stirred and heated under reflux for 20 minutes. The solution is poured into ampoules and lyophilized.

[0096] C.sub.13 NMR octasuccinyl glycyrrhizin: 174.7, 29.1, 29.5, 173.1, 71.6, 84.2, 82.5, 78.5, 109.8, 68.5, 68.6, 78.8, 112.1, 77.6, 173.2, 67.9, 53.1, 39.0, 29.6, 23.5, 57.2, 36.6, 17.2 , 69.0, 18.0, 43.1, 40.4, 18.7, 200.8, 123.0, 158.1, 47.1, 32.4, 33.1, 15.4, 39.1, 51.5, 25.0, 43.3, 42.2, 36.2, 19.9, 182.7

[0097] HPLC (Milichrom A-02; Gradient HClO4/LiClO4: AcCN 5-100%): 1 19,6

EXAMPLE 6

Obtaining Fully Acetylated Glycyrrhizin

[0098] 10 mM glycyrrhizin (V) is dissolved in 10 ml of dioxane, 50 mM acetic anhydride (II) is added, the solution is then stirred and heated under reflux for 20 minutes. The solution is poured into ampoules and lyophilized.

[0099] C13 NMR octaacetyl glycyrrhizin: 172.9, 68.0, 71.6, 84.2, 82.5, 78.5, 109.8, 68.5, 68.6, 78.8, 112.1, 77.6, 173.2, 67.9, 53.1, 39.0, 29.6, 23.5, 57.2, 36.6, 17.2, 69.0, 18.0 , 43.1, 40.4, 18.7, 200.8, 123.0, 158.1, 47.1, 32.4, 33.1, 15.4, 39.1, 51.5, 25.0, 43.3, 42.2, 36.2, 19.9, 182.7

[0100] HPLC (Milichrom A-02; Gradient HClO4/LiClO4: AcCN 5-100%): 1 peak 22.1 min

[0101] FIG. 3 shows an integrated chromatogram of 4 substances: a combinatorial derivative of epigalocatechin gallate (2), its octasuccinyl (1) and octaacetyl (3) derivatives, and control -unmodified epigalocatechin gallate (4). As can be seen from the graphs, the retention time (volume) of the derivatives differs both from the original epigalocatechin gallate and from each other, which indicates that these are different compounds. Also, peak (2) was not divided into several small fragments, nor was it possible to separate it using different conditions of thin layer chromatography, which indicates the stable nature of the formed supramolecular structure from different derivatives. Fully succinylated and fully acetylated epigalocatechins did not exhibit biological activity.

[0102] FIG. 4 shows an integrated chromatogram of 4 substances: the combinatorial derivative of glycyrrhizin (2), its octasuccinyl (1) and octaacetyl (3) derivatives, and the control is unmodified glycyrrhizin (4). As can be seen from the graphs, the retention time (volume) of the derivatives differs both from the initial glycyrrhizin and among themselves, which indicates that these are different compounds. Also, peak (2) was not divided into several small fragments, nor was it possible to separate it using different conditions of thin layer chromatography, which indicates the stable nature of the formed supramolecular structure from different derivatives. Fully succinylated and fully acetylated glycyrrhizin did not exhibit biological activity.

[0103] To check the biological (antiviral) activity of the synthesized derivatives with different ratios of components in the combinatorial synthesis reaction, the antiviral activity of the derivatives was studied by the screening method on Epstein-Barr virus models (strain X-2069b) in B-lymphoma culture plates by changing the number of copies of the virus genomes in ml PCR culture medium by detecting amplicon of LAT virus fragments (Synevo Lab). Substances were administered in 1/10 dose of the initial glycyrrhizic acid (final concentration of derivatives in the medium was 11 μm/ml), which initially had antiviral activity against Epstein-Barr virus in ED50=55 μm/ml. Cells were cultured in tablets in the environment of the Needle with the addition of donor blood plasma at a temperature of 37 0 C. The results of in vitro studies are shown in table 2.

TABLE-US-00002 TABLE 2 The ability of the supramolecular combinatorial derivative of glycyrrhizin CPG to eliminate Epstein-Barr virus from a lymphoma cell culture The molar The number of log No ratio of reagents * copies ** virus genomes p/p M k1 k2 in ml of culture fluid 1 92  930***  930*** >4.0 2 -//- 465  465  >4.0 3 -//- 155  155  0 4 -//- 77  77  2.0 5 -//- 39  39  >4.0 6 -//- 19  19  >4.0 7 -//- 10  10  >4.0 8 -//- 5 5 >4.0 9 -//- 2 2 >4.0 10 -//- 1 1 3.0 13 -//- 0 0 3.0 14 -//-  930*** 0 >4.0 16 -//- 465  0 >4.0 17 -//- 155  0 >4.0 18 -//- 77  0 >4.0 19 -//- 39  0 >4.0 20 -//- 19  0 >4.0 21 -//- 10  0 >4.0 22 -//- 5 0 >4.0 23 -//- 2 0 3.0 24 -//- 1 0 3.0 25 -//- 0 1860*** >4.0 26 -//- 1 930  >4.0 27 -//- 0 465  >4.0 28 -//- 0 155  >4.0 29 -//- 0 77  >4.0 30 -//- 0 39  >4.0 31 -//- 0 19  >4.0 32 -//- 0 10  >4.0 33 -//- 0 5 >4.0 34 -//- 0 2 2.0 35 -//- 0 1 3.0 36 -//- 1860*** 1 >4.0 37 -//- 930  1 >4.0 38 -//- 465  2 >4.0 39 -//- 155  5 >4.0 40 -//- 77  10  >4.0 41 -//- 39  19  >4.0 42 -//- 19  39  >4.0 43 -//- 10  77  >4.0 44 -//- 5 155  >4.0 45 -//- 2 465  >4.0 46 -//- 1 930  >4.0 * m is the number of moles of glycyrrhizin in the combinatorial synthesis reaction; K1 is the number of moles of succinic anhydride in the reaction; K2 is the number of moles of acetic anhydride in the reaction; ** Log means that the number in the column is the decimal logarithm of the DNA concentration, for example 3 in the column means 103 genomes/ml, the measurement error is 0.5 log/ml. ***the maximum molar ratio at which all groups in glycyrrhizin are replaced, an excess of this ratio leads to the fact that unreacted modifiers remain in the reaction medium—succinic anhydride and acetic anhydride.

[0104] As can be seen from the table, only with the calculated ratio of the components, when the maximum number of different derivatives of glycyrrhizin is formed, a biological active and effective supramolecular structure (derivative No. 3 in the table or CPGH) is formed, capable of completely eliminating the Epstein-Barr virus pathogen at a dose of 11 μm/ml from cell culture (ED100) in the absence of any cytotoxic effect on the cells. Similar effects are exerted by derivative No. 3 from Table 1 in monkey kidney cultures latently infected with viruses such as type 1 human herpes virus, type 2 human herpes virus, Zoster herpes virus, human cytomegalovirus, type 6 human herpes virus. Latent infection of the culture was caused by pretreatment of the cell culture with a subeffective concentration of acyclovir. The addition of KEGG to CPGG additionally reduced the effective concentration of CPGG by 10 times to 5 μg/ml and reduced the period of determination of the viral genome in the culture medium to 3 days.

EXAMPLE 7

The study of the ANTIVIRAL ACTIVITY of CPG and KEGG in an Animal Experiment (Herpes Virus Kerato-Conjunctivitis/Encephalitis in Rabbits)

[0105] As mentioned earlier, the combination of an inhibitor of histone deacetylase CPG and an antiviral agent that is effective in the latent phase of viral replication of KEGG can either eliminate the pathogen from the body or significantly reduce its load on the body. Given the role of herpes viruses in the etiology of atherosclerosis, cancer, allergic autoimmune pathologies, arthrosis, allow more successful treatment of a group of pathologies associated with herpes viruses. For studies on animal models, a 5% aqueous solution of potassium salt of an equivalent mixture of KSPG and KEGG was used.

[0106] The features of the experimental system and its level of adequacy to a natural human disease undoubtedly play a decisive role in assessing the effect of antiviral substances on the course of infection. Herpetic experimental infection is of interest due to the fact that herpetic diseases are widespread and extremely variable in clinical manifestations. Models of experimental herpes in animals are finding wider application in the study of new antiviral substances.

[0107] As you know, one of the clinical forms of systemic herpes is herpetic encephalitis, which is reproduced in guinea pigs, hamsters, rats, mice, rabbits, dogs, and monkeys. Herpetic keratoconjunctivitis in rabbits, with an average weight of 3.5 kg, was obtained by applying infectious material (herpes simplex virus type 1 strain L-2) on a scarified cornea. The animal was fixed, anesthesia of the eye was performed with dikain (instilled into the eye). Eyelids were opened, then several scratches were applied to the cornea using a syringe needle. Then the virus-containing material was introduced and, closing the eyelids, rubbed it into the cornea in circular motions.

[0108] Dose of the virus: 0.05 ml. 16 rabbits were used in the experiment, ten of them were injected with a mixture of CPG and KEGG (daily from the second day of infection −14 days at a dose of 20 mg/kg, and six-placebo (0.9% sodium chloride). After infection of the rabbits by HSV1, initiated daily monitoring of cornea, the presence of keratoconjunctivitis, encephalic disorders, and the presence of virus genomes in the blood by PCR after infection. Before infection, in all animals, amplicons of the herpes virus were absent in the blood. On day 3 after infection, HSV1 was determined in the amount of 3 log/ml in the blood of all animals in the blood. In addition, two rabbits developed encephalic manifestations—convulsive syndrome, lack of appetite. On the 4th day after infection, the experimental group of rabbits was injected into the ear vein with a mixture of CPG and KEGG at a dose of 50 μg/kg body weight, and a 0.9% sodium chloride solution was introduced into the control group.

[0109] Every day for two weeks this procedure was repeated once a day. In the experimental group, all animals survived, and the HSV1 antigen in the blood was not determined on days 13-14. In addition, in the experimental group, encephal manifestations disappeared by the 5th day of drug administration, while in the control group 2 animals died. By the 14th day of treatment, two animals died in the experimental group, while in the control group 6 died. Accordingly, the efficacy index was 83.3%, which indicates the high therapeutic efficacy of CPG/KEGG in the model of herpetic keratoconjunctivitis/encephalitis in rabbits.

[0110] In addition, the rabbits in the experimental group gained weight and all animals showed no signs of keratoconjunctivitis. The chemotherapeutic index for rabbits for the drug CPG/KEGG was 1000, which indicates the promisingness of CPG/KEGG as a highly effective antiviral drug with a wide spectrum of action and low toxicity and the ability to completely eliminate the causative agent of herpes virus.