Pharmaceutical composition for stimulating stem cell division and suppressing bacterial virulence

Abstract

Scope: The invention relates to organic and bioorganic combinatorial chemistry and pharmacia, namely to new combinatorial library of dipiridamol derivative and supramolecular structures based on them, which when being used not separated in individual components, have high bioactivity as a means of stem cell fission encouragement as pharmaceutical compositions combined with phosphodiesterase inhibitors and histone deacetylase inhibitors, as well as pharmaceutically acceptable excipients. The composition can also be used to struggle with resistant microorganisms by establishing their sensitivity to antibiotics.

Claims

1. A pharmaceutical composition, including histone deacetylase inhibitors, dipyridamole and other phosphodiesterase inhibitors, as well as pharmaceutically acceptable excipients, wherein it also contains unseparated mixture of dipyridamole combinatorial derivatives obtained by means of simultaneous modification by at least two covalent modifying agents selected from succinic anhydride, monochloroacetic acid, malic anhydride, acetic anhydride, propionic anhydride, butane anhydride, acetic-propionic anhydride, acetic-butane anhydride, glutaric anhydride, phthalic anhydride, cis-aconitic anhydride, trans-aconitic anhydride, citric anhydride, isolemic anhydride, acetyl chloride, acetyl fluoride, propionyl chloride, butyroyl chloride, and ethoxyoxalyl monochloride.

2. The pharmaceutical composition according to claim 1, wherein it additionally contains ascorbic acid.

3. The pharmaceutical composition according to claim 1, wherein it additionally contains bendazole.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1. Scheme of combinatorial dipiridamol derivative synthesis (IV) in combinatorial reaction of dipiridamol (I) with two modifiers (II, III).

(2) FIG. 2. Thin-layer chromatogram of combinatorial dipiridamol derivative (IV), initial dipiridamol (I), completely acylated dipiridamol (Ib) and completely succinylated dipiridamol (Ic).

PHARMACEUTICAL COMPOSITIONS

(3) Different methods can be used to introduce the supramolecular combinatorial dipiridamol derivative (SCDD). SCDD composition can be administered orally or as intravascular, subcutaneous, intraperitoneal injection, in aerosol form, via eye, in bladder, locally, etc. For example, inhaled administration methods are well known in this technical field. Dose of pharmaceutical composition will vary over a wide range depending on the specific administered SCDD, nature of disease, frequency of administration, administration method, clearance of the used agent from host organism and the like. Initial dose can be higher with the following lower maintaining doses. The dose can be administered with a frequency of one time per week or every other week, or can be divided on smaller doses and administered once or several times a day, twice a week and so on for maintaining the dose efficacy level. In many cases, higher dose will be required for oral administration than for intravascular administration. Within this invention, SCDD can be included to many compositions for therapeutic administration. In detail, within this invention SCDD can be included to pharmaceutical compositions combined with suitable pharmaceutically acceptable carriers or diluents and can be included to preparations with solid, semisolid, liquid or gaseous forms such as capsules, powders, granules, balms, creams, foams, solutions, suppositories, injections, forms for inhalation, gels, microspheres, lotions and aerosols. SCDD, as it is, can be administered in different ways, including oral, buccal, rectal, parenteral, intraperitoneal, subcutaneous, percutaneous, intratracheal administration and so on. According to invention, SCDD, after being administered, can be distributed on the system level or can be localized using implant or another composition holding down the active dose in the place of implantation. According to this invention, SCDD can be administered independently, combined with each other or they can be used combined with other known compounds (for example, ascorbic acid, bendazol, anti-inflammatory agents, etc.). SCDD can be administered to pharmaceutical dosage forms as their pharmaceutically acceptable salts. The following methods and excipients are mentioned only as examples and are not restricting by any means. For preparations with oral administration method, compounds can be used independently or combined with suitable additives for manufacture of pills, powders, granules or capsules, for example, with ordinary additives such as lactose, mannitol, corn starch or potato starch; with linking agents such as crystalline cellulose, cellulose derivatives, gum acacia, corn starch or gelatines; with disintegrators such as corn starch, potato starch orsodium carboxymethyl cellulose; with lubricating agents such as talc or magnesium stearate; and, if desired, with dilutants, buffering agents, wetting agents, preserving agents and correctives. SCDD can be included to compositions for injections via their dilution, suspending or emulsionizing inaqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetical glycerides of aliphatic acids, ethers of higher aliphatic acids or propylene glycol; and, if desired, with ordinary additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives. SCDD can be used in aerosol composition for inhaled administration. According to this invention, SCDD can be included to acceptable propellants under pressure such as dichlorodifluoromethane, propane, nitrogen, etc. Besides, compounds can be included in suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. According to this invention, SCDD can be administered rectally using suppository. Suppository can contain fillers such as cacao shell butter, carbowaxes and polyethyleneglycols melting at body temperature, though solid ones—at room temperature. Standard dosage forms can be manufactured for oral or rectal administration such as syrups, elixirs and suspensions, where each dose unit, for example, teaspoon, tablespoon, pill or suppository, contains predefined quantity of composition containing one or more compounds within this invention. Similarly, standard dosage forms for injections or intravascular administration can contain SCDD as per this invention in composition in the form of sterile water solution, normal physiological salt solution or other pharmaceutically acceptable carrier. Implants for long-term release of compositions are well known in this technical field. Implants are produced in the form of microspheres, bars, etc. with biodegradable or non-biodegradable polymers. For example, lactic and/or glycolic acid polymers form degradable polymer well tolerated by host. Implant containing SCDD as per invention shall be placed close to trauma focus so that local concentration of active agent is higher compared to remaining body regions. The term “standard dosage form” used here refers to physically discrete units suitable for usage as single doses for human and animal subjects; by this, each unit contains predefined quantity of compounds as per this invention sufficient, according to calculations, for desired effect, together with pharmaceutically acceptable diluent, carrier and filler. Descriptions of standard dosage forms as per this invention depend on the specific SCDD being used, as well as on the effect to be achieved, and on the pharmacodynamics of used compound in the host. Pharmaceutically acceptable excipients such as fillers, adjuvants, carriers or diluents, are generally accessible. Besides, pharmaceutically acceptable auxiliary substances are generally accessible, such as pH controlling agents and buffering agents, tonicity controlling agents, stabilizers, wetting agents, etc. Typical doses for system administration vary from 0.1 pg to 100 milligram per kg subject body mass for one administration. Typical dose can be one pill for administration from two to six times a day or one capsule or pill with long-term release for intake once a day with proportionally higher content of active ingredient. Long-term release effect can be caused by materials a capsule is made from, dissolving at different pH values, capsules providing slow release affected by osmotic pressure or any other method of controlled release. It will be clear to the specialists in this technical field that dose levels can vary depending on the specific compound, severity of symptoms and subject's liability to side effects. Preferred SCDD doses can be easily determined by specialists in this technical field by a variety of ways. The preferred method is measurement of SCDD bioactivity. One of the methods of interest is usage of liposomes as a filler for delivery. Liposomes merge with cells of target area and provide delivery on liposome content inside the cells. Contact of liposomes with cells shall be maintained for a time sufficient for merging, using different methods to maintain the contact such as extraction, linking agents and the like. In one aspect of invention, liposomes are developed to obtain aerosol for pulmonary administration. Liposomes can be made with purified proteins or peptides mediating the merging of membranes such as Sendai virus or influenza virus, etc. Lipids can represent any useful combination of known lipids forming liposomes, including cationic or zwiterionic lipids such as phosphatidylcholine. The remaining lipids will be normally neutral or acidic lipids such as cholesterin, phosphatidylserine, phosphatidylglycerol and the like. In order to obtain liposomes, we can use method described by Kato et al. (1991) J. Biol. Chem. 266:3361. In short, lipids are mixed with SCDD-containing composition for presenting to liposomes in the suitable water medium, in salt medium properly, where total solids will be within a range of 110 wt. %. After vigorous mixing within short periods of time, about 5-60 sec., glass tube is placed in warm water bath at about 25-40° C. and this cycle is repeated about 5-10 times. Then the composition is processed with ultrasound within suitable period of time, normally about 1-10 sec., and, possibly, is additionally mixed with vortex mixer. Then the volume is increased by adding water medium, normally about 1-2 times, with further shaking and cooling. This method allows including supramolecular structures with high cumulative molecular weight to liposomes.

(4) Compositions with Other Active Agents

(5) According to formula, the composition may include such phosphodiesterase inhibitors as vinpocetine, nicardipine, nimodipine, lixazinone, cyclostamide, milrinone, cilostazol, dihydropyridazinone, rolipram, denbufillin, cilomilast, roflumilast, sildenafil, ariflo, vardenafil, tadalafil, zaprinast, thiadiazole, papaverine, but the composition is not limited by the specified substances. The composition may also include histone deacetylase inhibitors: valproic acid, valproic-hydroxamic acid, cinnamichydroxamic acid, phenyl butyrate, tubacin, vorinostat, depsipeptide, butyrate, but the composition is not limited by the specified substances. For application in methods under consideration, according to invention SCDD may be included into compositions with other pharmaceutically active agents, in particular antimicrobial agents, immunomodulators, antiviral agents, and antiviral substances. Other agents of interest include a wide range of antibiotics known in this technical field. Antibiotic classes include penicillins, for example penicillin G, penicillin V, methicillin, oxacillin, carbenicillin, nafcillin, ampicillin and so on; penicillins in combination with betalactamase inhibitors; cephalosporins, For example cefaclor, cefazolin, cefuroxime, moxalactam, etc.; carbapenems; monobactams; aminoglycosides; tetracyclines; macrolides; lincomycins; polymyxins; sulfonamides; quinolones; chloramphenicol; metronidazole; spectinomycin; trimethoprim; vancomycin; and so on. Antifungal agents are also useful, including polyenes, for example amphotericin B, nystatin, flucosin; and azoles, for example miconazole, ketoconazole, itraconazole and fluconazol. Tuberculosis drugs include isoniazid, ethambutol, streptomycin and rifampin. Other agents of interest in terms of creating new compositions include a wide range of mononucleotide derivatives and other RNA polymerase inhibitors known in this technical field. Classes of viral inactivating agents include interferons, lamivudine, ribavirin and so on; amantadine; remantadine, For example zinamivir, oseltavimir and so on; acyclovir, valacyclovir, valganciclovir; and so on. Other groups of viral inactivating agents include adefovir, vbacavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir, efavirenz, nevirapine, indinavir, lopinavir and ritonavir, nelfinavir, ritonavir, sakinavir, daclatasvir, sovofbuvir. According to invention SCDD composition may also cytokines, for example interferon gamma, tumor necrosis factor alpha, interleukin 12, etc. The present invention is further described by the following examples which should not be considered as limiting the scope of the invention.

Example 1. Obtainment of Supramolecular Combinatorial Dipiridamol Mixture (CD)

(6) Dilute 222 μM of dipirimadol (I) (CAS N 58-32-2, Mr=504.636 g/mol, n=4) in 50 ml of dioxane in mixture with 50 ml of glacial acetic acid, add 60 μM of succinic anhydride (III) and 61 μM of acetic anhydride (II), stir the solution and warm with backflow condenser within 5-50 minutes. Pour the solution into vials and lyophilize to remove solvent and acetic acid. Combinatorial mixture (IV) is used to obtain pharmaceutical compositions, to study structures, to determine bioactivity (CD). FIG. 4 shows the scheme of combinatorial dipiridamol derivatives synthesis. Instead of carboxylic acid anhydride modifiers, you can use halides of carboxylic and polycarboxylic acids, such as succinic, maleic, fumaric, lactic, propionic, other halogen derivatives, such as chloromethane, bromoethane, chloropropane, cyclic alkylating compounds like oxirane, propiolactone.

(7) FIG. 1.

(8) One parent molecule of diprimadol (I) contains 4 residual hydroxyl groups available for modification (n=4). The amino groups as a part of residual morpholine and the pyridmidine nucleus—protonated and protected against modification under the given reaction conditions.

(9) Calculations of the number of modifier moles are carried out according to the combinatorics formulas:
m=4×(3×2.sup.n-2−1);
k=n×(2.sup.n−1),
where m—number of different molecule derivatives in the combinatorial mixture and the number of dipyridamole moles for reaction; n—number of hydroxyl groups available for modification in the structure of dipyridamole (n=4); k—number of moles of each modifier. Thus, having only one parent dipyridamole molecule and two modifiers after combinatorial synthesis, we obtain 12 combinatorial derivatives with different degrees of substitution, different positions of the substituents and different shufflings of the modifier residues, not just as a mixture, but as difficultly separated supramolecular mixture. Due to the presence of both substituted and non-substituted hydroxyl groups in different derivatives, supramolecular structures are formed through both hydrogen and ionic bonds, including with tertiary amino groups of heterocycles. Modifiers—succinic anhydride or acetic anhydride can be introduced both simultaneously and sequentially—or first introduce succinic anhydride, warm the mixture with backflow condenser, and then introduce acetic anhydride and reheat the mixture. Similarly, in this reaction, maleic anhydride, aconitic anhydride, glutaric, phthalic anhydride and acetic anhydride, ethyl formic acid, monochloroacetic acid, propiolactone, ethylene oxide and other low-molecular alkylating agents (methyl chloride, ethyl chloride, propyl chloride) can be used instead of succinic anhydride as one of the modifiers.

(10) NMR C.sup.13 (carbon-13-nuclear magnetic resonance): C: 96,1; 161,8; 170,0; 157,8; CH.sub.2: 58,9; 61,7; 58,1; 61,4; 29,2; 29,1; CO: 173,1; 174,7; 170,2; CH.sub.2 (in morpholine cycle) 52,7; 25,4; 25,5C.sup.13 NMR data of the combinatorial derivative confirm the presence of both ethyl groups of succinic acid residues in its structure and acetyl residues—reaction products with acetic anhydride.

(11) For HPLC we used Milichrom A-02 microcolumn chromatograph in the gradient of acetonitrile (5-100%)/0.1 M chloric acid+0.5 Mlithium perchlorate. The combinatorial derivative in the chromatogram gave one clear broadened peak and was not separated into components, although the retention time differed from both the starting dipyridamole 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 12 of them). This combinatorial derivative (CD) also behaves similarly when separated in a thin layer (acetonitrile: water, UV detection) and gives only one band that does not coincide with any of the obtained derivatives.

(12) FIG. 2.

(13) As FIG. 2 shows, TLC, combinatorial mixture (IV) is less mobile, while the initial unmodified dipyridamol (I) is the lightest. Completely acylated dipyridamol (Ib) and succinylated dipyridamol (Ic) are intermediate between native dipyridamole and combinatorial one. The combinatorial dipyridamole band is not separated either by two-dimensional TLC or by HPLC (not shown).

(14) Next, a study was conducted of the inhibition of cAMP-phosphodiesterase from supramolecular combinatorial dipyridamole derivatives obtained in the reaction with different molar ratios of modifiers according to the final concentration of AMP by ELISA method. The reaction was stopped by the addition of a double volume of 1% TCA.

(15) TABLE-US-00001 TABLE 1 Inhibiting property regarding cAMP-phosphodiesterase (PDE) from supramolecular combinatorial derivatives of dipiridamol obtained in the reaction with different molar ratio of modifiers Item Reagent molecular ratio* ED.sub.50 as related to cAMP, No. m k1 k2 μg/ml, measuring error 10% 1 44   88***   88*** >500 2 -//- 70 70 100 3 -//- 61 60 0.01 4 -//- 30 30 5 5 -//- 15 15 10 6 -//-  7  7 60 7 -//-  3  3 115 8 -//-  2  2 210 9 -//-  1  1 300 10 -//-  0  0 300 *m-number of moles of dipiridamol in the combinatorial synthesis reaction; k1-number of moles of succinic anhydride in the reaction; k2-number of moles of acetic anhydride in the reaction; ** ED.sub.50 μg/ml of PDE inhibition was determined by diluting the initial concentration of the dipiridamol derivative; ***maximum molar ratio at which all groups in dipiridamol are replaced, exceeding this ratio leads to the fact that in the reaction unreacted modifiers-succinic anhydride and acetic anhydride remain in medium.

(16) As Table 1 shows, the smallest ED.sub.50 is observed precisely in the region with calculated molar ratios of modifiers (44:61:60). Thus, due to obtaining of combinatorial derivative of dipiridamol, the effective dose of dipiridamol can be reduced by 5 orders of magnitude to completely inhibit DPE.

(17) The following table 2 shows the formulations of the studied pharmaceutical compositions.

(18) TABLE-US-00002 TABLE 2 Formulation and ratio of ingredients of the pharmaceutical composition (FC CD) per capsule or pill Item No. Ingredient name % 1  2 3 1. CD 0.1-20.0 2. Papaverine 0.5-10.0 3. Ascorbic acid 0.2-10.0 4. Bendazole 0.5-10.0 5. Tadalafil  1-5.0 6. Sodium valproate   5-20.0 7. Excipients up to 100% As a control, the animals were applied the same composition with the same substances (in the form of Carbopol gel), but without CD (FC).

Example 2. Determination of FC and FCCD Compositions Impact on Tissue Regeneration

(19) The wound-healing properties of compositions were studied on male Vistar white rats. 38 animals previously anesthetized had a cut out of the skin area of 2 by 2 cm size on the dorsal side of the body, behind the right shield bone. The skin was taken by forceps and pulled back; the size of skin fragment was 2 cm, cut depth—2 mm, wound average area—4±1.0 cm.sup.2. The obtained polygonal shaped wounds bleeded intensively. Then, FC and FCCD were applied to the wound of the group 1 and 2 animals (10 in each). The wounds of group 3 rats were treated with “panthenol”, the 4th group of 8 animals was the control group; the wounds of these animals were not treated. The preparations were applied in such a way that the formed gels covered the entire surface of the wound and involved a small fragment around the wound. BF-6 glue was applied on top of the gel and dried; the animals were put back into the cells. 3, 6, 9, 11, and 13 days after the start of experiment (before the wound healing in animals of all groups) a planimetric study was carried out making it possible to judge the features of reparative processes. The wound area was measured as follows: the celluloid film was applied to the wound and wound contours were plotted on the film; after that the wound surface area was determined using graph paper. The results of the first series of experiments (Table 3) showed that under the impact of FCCD composition, wound healing at all study stages was significantly accelerated, while FC accelerated wound healing slightly. The effectiveness of the FCCD composition was statistically higher than that one of FC composition and Panthenol.

(20) TABLE-US-00003 TABLE 3 Indicators of cutaneous wound healing in rats under the impact of FCCD and FC compositions. Wound area* (S) during the monitoring period, cm.sup.2 (M ± m) Prepa- Composition 1-3 3-6 6-9 9-11 11-13 ration formulation n day day day day day FCCD CD, 10 4.1 ± 0.4 1.1 ± 0.1 0.1 ± 0.1 — — papaverine, ascorbic acid, bendazole, tadalafil, sodium valproate, carbopol and excipients FC papaverine, 10 4.3 ± 0.4 1.7 ± 0.2 0.7 ± 0.2 0.5 ± 0.1 — ascorbic acid, bendazole, tadalafil, sodium valproate, carbopol and excipients Panthenol Carbopol 10 4.0 ± 1.1 3.5 ± 0.3 2.6 ± 0.4 1.2 ± 0.3 0.3 ± 0.1 Control — 8 4.0 ± 0.6 3.6 ± 0.6 2.6 ± 0.6 1.5 ± 0.5 0.5 ± 0.2 * P ≤ 0.05 As Table 3 shows that in fact, wounds in animals treated with FCCD composition were healed 2 times faster (13 to 6 days), while the efficacy of control sample Panthenol did not differ from the control. Wound epithelization was initiated on the second day after composition application.

Example 3. Rise of Level of Cells-Precursors in Mice (Pluripotent)

(21) TABLE-US-00004 TABLE 5 Absolute count of cells-precursors in 1 ml of blood Methylcellulose culture CFU-GM BFU-E CFU-GEMM Control 188.2 17 18 FCCD: 10 mg/kg 831.7 122.7 80.9 FCCD: 5 mg/kg 611.5 93.3 71.7 FCCD: 2.5 mg/kg 689.7 99.9 77.2 FCCD: 1 mg/kg 426 62 27.7 Precursors Methylcellulose culture Time GM BFU-E CFU-GEMM 15″ 2.88 2.85 3.87 30″ 6.74 4.28 4.67  2′ 2.90 2.16 1.93

(22) (pluripotent)

(23) The impact on subcutaneous (s.c.) administration of FCCD to C3H/H3 J mice for a number of granulocyte macrophage (CFU-GM), erythroid (BFU-E), and polypotent (KOEGEMM) cells-precursors in 1 ml of blood was evaluated. For in vitro colony formation, the precursors were stimulated with a combination of 1 U/ml rhu Epo, 50 ng/ml SLE, conditioned spleen murine cell medium containing 5% v/v. pituitary mitogen (PWMSCM), and 0.1 mM of hemin Plates were counted after 7 days of incubation.

(24) The number of precursor cells of mobilized FCCD was observed versus the time with 5 mg/kg single subcutaneous injection and the results are given in Table 4.

(25) TABLE-US-00005 TABLE 4 Absolute count of cells-precursors in 1 ml of blood Methylcellulose culture CFU-GM BFU-E CFU-GEMM Control 290.2 48.3 26.1 FCCD: 15″ 793.7 129.4 92.3 FCCD: 33″ 1803.3 210.1 116.7 FCCD: 120″ 830.6 103.2 50.3

(26) To evaluate the dose-dependent effects, FCCD was administered at a concentration of 1, 2.5, 5 and 10 mg/kg by a single subcutaneous injection and the number of precursors in 1 ml of blood was determined 1 hour after administration, the results are given in Table 5. The maximum mobilization of precursor cells when using a dose of 2.5-10 mg/kg FCCD achieved approximately 0.5-1 hour after injection, as shown in the Table 6.

(27) Mobilization of Mouse Precursors-Cells when Combined with MIP-1α and G-CSF

(28) The ability of FCCD combined with the macrophage mouse inflammation protein (mu) (MIP-1a), to mobilize precursor cells with or without prior administration of rhu G-CSF was studied. It has been previously demonstrated that MIP-1α promotes the mobilization of precursor cells in mice and humans (Broxmeyer, H. E. et al. Blood Cells, Molecules and Diseases (1998) 24 (2): 14-30). Groups of mice were randomized for subcutaneous injection of a control diluent (physiological salt solution) or G-CSF at a dose of 2.5 μg per mouse twice a day for two days. Eleven hours after the last injection of physiological salt solution or G-CSF, the mice were divided into groups that received MIP-1α administered intravenously at a total dose of 5 μg, FCCD administered subcutaneously at a dose of 5 mg/kg, or a combination of MIP-1α and FCCD at the same doses. In an hour, the mice were sacrificed and the number of precursor cells in 1 ml of blood was determined. FCCD acted more efficiently than the additive method in mobilizing precursor cells when used combined with macrophage mouse inflammation protein (mu) (MIP)-1α, each administered 11 hours after administration of rhu G-CSF or control diluent (physiological salt solution) and 1 hour before taking blood.

(29) Clinical Rise of Level of Cells-Precursors

(30) The study was carried out on five healthy volunteers (P1-P5) with an initial number of white blood cells from 4500 to 7500 cells/mm3. Each patient received a single subcutaneous (s.c.) injection of 80 μg/kg FCCD in 0.9% physiological salt solution from stock solution of 10 mg/ml FCCD in saline under sterile conditions. Blood samples were taken using a catheter prior to dosing and at various time periods up to 24 hours after administration of preparation. Blood samples were evaluated relative to the total count of white blood cells, CD34-positive precursor cells (using FACS analysis) as CD34-positive precursor cells (using FACS analysis) as a percentage of the total count of white blood cells, as well as the absolute count in 1 ml and circulatory status of granulocyte-macrophage (CFU-GM), erythroid (BFU-E) and pluripotent (KOEGEMM) precursor cells. As Tables 4 and 5 show, FCCD administration caused rise of the count of white blood cells and CD34-positive precursor cells in volunteers, which turned out to be maximum 6 hours after administration.

(31) TABLE-US-00006 TABLE 6 FCCD-induced mobilization of white blood cells in different volunteers (x10.sup.3 WBC) Test- Initial Treatment, hours ID ing data 0.5 1 2 4 6 9 12 P1 6.9 6.5 8.15 14.9 21.9 23.9 28.7 22.53 7.15 P2 6.12 5.7 6.77 8.97 16.7 19.0 19.9 21.6 9.12 P3 4.52 5.5 7.63 9.44 17.9 18.15 19.9 19.94 5.12 P4 5.17 5.29 4.25 7.73 12.7 16.13 16.8 18.5 5.16 P5 4.55 5.16 6.18 8.64 10.9 16.83 19.32 19.14 4.93

(32) TABLE-US-00007 TABLE 7 FCCD-induced mobilization of CD34- positive cells expressed as a percentage of the total count of WBC in different volunteers Treatment, hours ID Initial data 1 3 6 9 2 P1 .06 .03 .09 .12 .12 .09 P2 .07 .08 .09 .13 .12 .12 P3 .06 .16 .07 .08 .13 .09 P4 .06 .09 .09 .11 .10 .10 P5 .12 .12 .13 .2 .2 .16

(33) The blood was also studied for the mentioned FCCD activated precursors.

(34) The absolute count of undivided nuclear cells and low density nuclear cells in 1 ml of blood was determined (separation in Fico-hypaque), as well as the absolute count in 1 ml and the status in the circulation of granulocyte-macrophage (CFU-GM), erythroid (BFU-E) and polypotent (KOGHEMM) precursor cells in normal donors injected subcutaneously with FCCD. The above indicators were evaluated before administration and 1, 3, 6, 9 and 24 hours after FCCD administration. All results for precursor cells are given based on the rating of 3 culture plates per point analysis. The count of precursor cells and status in circulation, count of CFU-GM, BFU-E and KOEGEMM were studied in methyl cellulose cultures with cell stimulation of 1 U/ml of recombinant human (rhu) erythropoietin, 100 U/ml of rhu granulocyte-macrophage colony stimulating factor (GM-CSF), 100 U/ml rhu of Interleukin-3 (IL-3) and 50 ng/ml of rhu steel factor (SLF=stem cell factor (SCF)). CFU-GM was also evaluated in agar cultures after stimulation with 100 U/ml rhu GM-CSF and 50 ng/ml rhu SLE. In both types of studies, colonies were evaluated after 14 hours of incubation in a humidified atmosphere with 5% CO.sub.2 and reduced (5%) O.sub.2 pressure. The status of precursor cells in circulation was determined using a highly specific cytolytic method according to the activity of [3H]-thymidine as described previously (Broxmeyer, H. E. et al. Exp. Hematol. (1989) 17:455-459. The results were initially presented as mean total change in the absolute count of nuclear cells and precursors for 1, 3, 6, 9 and 24 hours compared with their count before injection (=Time (T) 0) for all five donors as given below in Tables 8-10: STD—Standard Deviation; STE—Standard Error; PBL-US—peripheral blood-undivided; PBL-LD—peripheral blood—low density (separation in Ficoll); P—Significance using 2-parametric t-test.

(35) TABLE-US-00008 TABLE 8 Total change compared to period of time = 0 (Average value from 5 clones) Content of nuclear cells PBL-US PBL-LD Average STD STE % CHG P Average STD STE % CHG P T = 0  1.00 0.00 0.00 0.0 1.00 0.00 0.00 0.0 T = 1  1.69 0.00 0.00 68.6 0.017 1.86 0.00 0.00 86.2 0.000 T = 3  2.80 0.51 0.23 180.2 0.000 2.86 0.28 0.12 185.6 0.000 T = 6  3.26 0.61 0.27 225.8 0.000 3.66 0.43 0.19 266.3 0.001 T = 9  3.09 0.69 0.31 209.4 0.000 3.64 1.18 0.53 264.3 0.001 T = 24 1.07 0.65 0.29 7.0 0.553 1.05 1.19 0.53 4.6 0.815 Methylcellulose culture CFU-GM BFU-E CFU- GEMM Average STD STE % CHG P Average STD STE % CHG P Average STD STE % CH T = 0  1.00 0.00 0.00 0.0 1.00 0.00 0.00 0.0 1.00 0.00 0.00 0.0 T = 1  4.77 0.00 0.00 376.7 0.001 1.99 0.00 0.00 98.9 0.002 2.32 0.00 0.00 131.8 T = 3  13.52 1.55 0.72 1242.5 0.001 3.21 0.50 0.22 221.3 0.004 4.33 0.44 0.20 332.5 T = 6  21.77 5.58 2.58 2079.6 0.000 6.01 1.25 0.56 500.5 0.006 10.07 0.59 0.27 907.2 T = 9  10.41 5.11 2.29 952.3 0.000 4.34 2.99 1.34 334.4 0.000 5.25 4.54 2.03 425.4 T = 24 1.48 3.11 1.34 55.5 0.005 1.26 1.02 0.45 26.3 0.194 1.53 3.04 1.36 53.2

(36) In this case, the results are presented as the total change from T=0 levels for each individual donor as shown in Tables 8-10.

(37) TABLE-US-00009 TABLE 9 Total change compared to period of time = 0 for each individual patient (P) Content of nuclear cells PBL-US PBL-LD P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 T = 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0 T = 2.54 1.38 1.38 1.36 1.76 2.07 1.99 1.48 1.66 2.10 1 T = 3.55 2.74 2.02 2.46 3.23 2.83 3.25 2.17 2.82 3.20 3 T = 3.97 2.94 2.74 2.60 4.04 4.07 3.90 2.27 2.78 5.30 6 T = 3.27 3.30 2.69 2.24 3.96 3.65 4.43 2.47 2.48 5.17 9 T = 1.21 1.43 0.96 0.77 0.99 1.01 1.71 0.79 0.60 1.12 24

(38) TABLE-US-00010 TABLE 10 Precursors Methylcellulose culture CFU-GM BFU-E CFU-GEMM P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 P1 P2 P3 P4 P5 T = 0  1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 T = 1  5.09 5.33 3.70 6.87 2.84 2.58 1.46 2.30 1.46 2.13 2.07 2.26 2.22 1.96 3.07 T = 3  7.12 17.02 15.07 20.72 8.40 5.13 1.98 2.61 2.60 3.75 4.25 3.47 4.34 5.14 4.43 T = 6  14.66 23.96 20.99 28.54 20.39 9.14 3.67 4.54 3.34 9.35 7.47 9.35 6.52 9.10 17.92 T = 9  6.26 12.51 9.42 14.08 10.09 5.43 4.61 3.71 2.93 5.05 2.64 7.09 2.47 4.52 9.55 T = 24 1.10 1.91 1.43 1.51 1.83 1.06 1.88 1.14 0.79 1.44 1.12 2.62 0.69 0.98 2.25

(39) As the above tables show, the use of FCCD composition significantly increases the number of all studied classes of pluripotent cells in the blood of volunteers within 6 hours after the start of composition administration.

Example 4. —Reduction of P. aeruginosa Virulence Under FCCD Impact on the Example Depression of Adhesive Properties

(40) It is known that the adhesion of microorganisms is the first stage of colonization, the main and determining factor of their virulence and pathogenicity. Using adhesins, microbes recognize receptors on cell membranes, attach to them and colonize various surface structures of the cell wall. The ability of bacteria to adhesion and colonization of surfaces is fixed by natural selection. This function is necessary for bacteria with saprophytic existence. For example, Legionella are actively attached to the surface of cyanobacteria, cholera vibrios actively colonize zooplankton using their chitin as a source of nutrition and chitin also stimulates the multiplication of cholera vibrios. The study of the microorganisms adhesion is of particular importance for medical microbiology which has received clinical evidence: it was found that in the absence of adhesins, neither bacteria nor fungi can grow and form colonies, and if there is no colonization, then there is no infection and disease.

(41) Adhesion of bacterial pathogen can be carried out to the components of the extracellular matrix—fibronectin, collagen, laminin, etc. Matrix proteins have RGD sequence with which cell surface integrins interact. Thus, extracellular matrix proteins contribute to the adhesion of bacteria to the host target cells. The adhesion of bacteria to such proteins is specific and each pathogen implements this possibility in its own way. For the manifestation of the pathogenicity of some bacteria, their interaction with matrix proteins is critical.

(42) Most gram-negative bacteria attach to epithelial cells of humans and animals using adhesins which are special organelles. Many pathogenic microorganisms are able to penetrate into the host cells and proliferate actively in them. To penetrate into the cells of the bacteria, adhesive molecules called invasins are used. The most common mechanism involves the activation of signals in the host cell allowing the invasion of bacteria by triggering normal cellular reactions. Considering the presence of substances that can affect the manifestation of adhesion, it is possible to direct their action to prevent the development of infectious process. One of the ways to block the adhesion mechanisms is to use anti-infective preparations in low concentrations that inhibit the process of attaching pathogens in the primary infection zone. For this purpose, it is possible to use specific bacteriophages, as well as use in the development of vaccines.

(43) To determine the adhesive properties of microorganisms, the most convenient model in which human red blood cells are used as macroorganism cells. The increase in cell biomass under the influence of enhancers leads to a change in some biochemical tests. One of them is the adhesive properties of microorganisms (Table. 11)

(44) TABLE-US-00011 TABLE 11 Comparative adhesive properties (AI) of P. aeruginosa over FCCD Adhesion Index (AI) Pseudomonas Pseudomonas Pseudomonas aeruginosa aeruginosa aeruginosa FCCD, % ATCC 27853 ATCC 9027 12-76  0.01 ± 0.005 2.6 ± 0.3* 3.4 ± 0.2* 3.5 ± 0.3* 0.001 ± 0.0005 1.8 ± 0.2* 1.4 ± 0.2* 1.7 ± 0.4* Control 3.2 ± 0.3 3.1 ± 0.3 3.2 ± 0.3 Note: *the difference in indicators is statistically significant (p < 0.05)

(45) As shown in data on determination of adhesion degree as per AI during P. aeruginosa cultivation on media with FCCD, which are given in Table 11, adhesion indices differed from control indices. FCCD composition expressed in terms of combinatorial dipyridamole at a concentration of 0.001±0.0005% contributed to a decrease in adhesive activity of Pseudomonas aeruginosa strains to (1.4±0.3)-(1.8±0.4). Medium-adhesive strains under the influence of FCCD became low-adhesive. Adhesion index was (1.4-1.7) versus (3.1-3.2) when grown on medium without adding FCCD.

(46) As a result of processing the statistical data from Table 11, it was demonstrated that the differences between the adhesion indices of strains cultured on FCCD media at concentrations from 0.001±0.0005% to 0.01±0.005% and control are statistically significant. This indicates the effectiveness of using FCCD in a concentration from 0.001±0.0005% to 0.1±0.05% to reduce the adhesive activity of microorganisms.

Example 5. Reduction of P. aeruginosa Virulence Under the Impact of FCCD on the Example of Recovery of the Bacteria Sensitivity to Antibiotics (Inhibition Zones on Solid Nutrient Medium)

(47) The studies are carried out in vitro by diffusion method on P. Aeruginosa strains indicated in previous experiments. Two rows of plates containing Müller-Hinton agar were prepared in parallel, with a 0.01-0.005% FCCD solution added to the second-row plates. After that, bacteria of the studied strains from a suspension containing 10.sup.8 μl/ml are inoculated on the surface of the agar. To do this, prepare a bacterial suspension corresponding to a concentration of 10.sup.9 μl/ml according to the optic turbidity standard of 10 units diluted with physiological salt solution 10 times (up to 10.sup.8 μl/m1). After the suspension is absorbed into agar, discs with polymyxin, ceftriaxone, levofloxacin, amikacin, imipinem are applied to its surface (cefazolin and amoxicillin were also added with additional passages, but they did not appear sensitive). Inoculations on plates with FCCD-free medium are used as controls, as well as media on which discs are not applied. Inoculations are incubated for 48 hours at 37° C. in one passage, then re-inoculation is carried out under similar conditions for the following passages. A total of 4 passages were carried out with controls. The results are taken into account by the diameter of the bacteria inhibition zones. On FCCD-free medium, there are no inhibition zones (an indicator of antibiotic resistance of bacteria), whereas on a medium containing 0.005-0.01% FCCD, a zone of bacterial growth inhibition from 25 to 40 mm in diameter forms around discs (appearance of sensitivity to antibiotic).

(48) As a result of studies, it was found that in FCCD-free medium there is no zone of bacterial growth inhibition around disks with polymyxin and amikacin. With administration of (0.005-0.01)% FCCD (plate 2) into medium around the disks with polymyxin and amikacin, zone of bacterial growth inhibition appears indicating an increase in the bacteria sensitivity to antibiotics. Growth inhibition zones were not observed for antibiotics Pseudomonas aeruginosa has genetically determined (initial) antibiotic resistance. Thus, FCCD composition is able to statistically significantly inhibit acquired antibiotic resistance in Pseudomonas aeruginosa.

(49) Similar studies were conducted for multi-resistant hospital strains K. pneumonia, A. bauiannii, S. aureus. In all cases, the sensitivity of bacteria to antibiotics was recovered at passage 3-4 and did not differ from similar ATCC strains.

Example 6. Reduction of P. aeruginosa Virulence Under the Impact of FCCD on the Example of Recovery of Bacterial Sensitivity to Antibiotics (Growth Inhibition in a Liquid Nutrient Medium, Determination of Changes in MIC)

(50) The study is carried out on P. aeruginosa strains given in the previous examples by in vitro serial dilution method using FCCD. Evaluate the decrease in resistance of Pseudomonas aeruginosa strains to amikacin and polymyxin. Prepare two rows of plates with Müller-Hinton nutrient medium: the first row contains various concentrations of the studied antibiotic (amikacin and polymyxin)—31, 62, 125, 250, 500 μg/ml of medium (double dilution). At the same time, prepare the same row of plates, but add FCCD additionally into the medium at a concentration of (0.0001-0.001) %. Inoculate 0.05 ml (drop) of each test strain on the plates from a bacterial suspension containing 10.sup.8 μl/ml according to the optic turbidity standard of 10 units. Müller-Hinton medium without antibiotic and Müller-Hinton medium containing 0.0001% of FCCD without antibiotic are used as controls. Inoculations are incubated at 37° C. for 48 hours. The results are considered by the value of MIC (minimum inhibitory concentration) with the obligatory growth of bacteria on control plates (see table 12). The table shows that when bacteria are exposed to FCCD, the causative agent of pseudomonosis reduces resistance to the studied antibiotics by 10 times.

(51) TABLE-US-00012 TABLE 12 Determination of the degree of sensitivity recovery of multiresistant nosocomial pathogens under the action of FCCD composition in different concentrations FCCD, Antimicrobial agents (MIC, μg/ml) Strains % Polymyxin Amikacin Ceftriaxone Levofloxacin Imipinem P. Control 250 >500 >500 >500 >500 aeruginosa (without IMI-2016 FCCD) FCCD 31 15 250 125 250 0.001 FCCD 125 125 500 250 125 0.001 A. Control >500 >500 >500 >500 >500 baumannii (without IMI-2016 FCCD) FCCD 31 15 250 125 250 0.001 FCCD 62 125 125 62 31 0.001 K. Control >500 >500 >500 >500 >500 pneumoniae (without IMI-2016 FCCD) FCCD 62 31 125 125 250 0,001 FCCD 125 62 250 250 125 0,001

(52) As Table 12 shows, sensitivity to antibiotics increased 33 times for amikacin and Pseudomonas aeruginosa and 16 times for polymyxin and Pseudomonas aeruginosa. A similar pattern was observed for acinetobacter and Klebsiella. Although sensitivity to other antibiotics also increased 2-4 times, but their concentration did not decrease to values probable for use in humans. Thus, the use of FCCD composition is promising for recovery of sensitivity to antibiotics in multiresistant strains of microorganisms.