METHOD OF PRODUCING OF PHARMACOLOGICALLY ACTIVE LIPOSOMAL COMPOSITION CONTAINING CYTOCHROME C, AND LIPOSOMAL COMPOSITION OBTAINED BY THIS METHOD

Abstract

This invention relates to pharmaceutics and a method of producing of liposomal composition containing cytochrome c and the pharmacologically active liposomal composition obtained by this method, which can be used as a means of polyfunctional pharmacotherapy, especially for ophthalmology, hematology and cardiology.

A method of producing of liposomal composition containing cytochrome c, the method includes preparing mixture of solutions of lipids in an organic solvents, drying in vacuum the mixture and emulsifying it in aqueous medium containing cytochrome c, adding of cryoprotectant, homogenization of the emulsion, filtration and freeze-drying, and according to the invention the lipids are egg or soybean phosphatidylcholine with one or two of lipids from the group consisting of dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol or dioleoyloxypropy trimethylammonium, whereas the ratio of phosphatidylcholine:to other lipids is 0.3-2.0:1, thereby to prepare the mixture of solutions of lipids, phosphatidylcholine is dissolved in ethyl alcohol, and other lipidsin chloroform and a volume ratio in a mixture of solutions of ethyl alcohol:chloroform is 1:1.5-2.5, emulsification is performed at a weight ratio of cytochrome c to lipids of 1:11.4-18.5 adding a cryoprotectant solution to the aqueous medium, and the cryoprotectant is selected from lactose, trehalose, sucrose oligosaccharides, wherein the said solution contains 60-80% of total cryoprotectant, and homogenization with step-by-step increasing pressure from 300 to 800 atm, after its completion a solution of selected cryoprotectant is added to emulsion, wherein solution contains 40-20% of total cryoprotectant, and the weight ratio of lipids mixture to cryoprotectant is 1:5.5-7.2.

It is claimed a liposomal composition containing cytochrome c, lipids and cryoprotectant, which is performed according to the method above, and the composition includes egg or soybean phosphatidylcholine in mixture with one or two of lipids from the group consisting of dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol or dioleoyloxypropy trimethylammonium, and cryoprotectant is selected from lactose, trehalose, sucrose oligosaccharides, wherein the weight ratio of cytochrome c:phosphatidylcholine:other lipids:cryoprotectant is 1:2.9-8.6:4.3-15.7:78.6-102.8 and percentage content ratio is (0.81-1.06)%:(3.03-8.26)%:(4.13-9.88)%:(78.67-83.30)% respectively.

Claims

1. A method of producing of a pharmacologically active liposomal composition by forming a mixture of lipids solutions in organic solvents, drying it in a vacuum and emulsifying the composition in an aqueous medium containing cytochrome c, dispersing of an emulsion with the addition of a cryoprotectant, followed by filtration, sterile filtration and freeze-drying, wherein to the medium for emulsification a solution of cryoprotectant is introduced, the said solution contains 60-80% wt. of its total amount, and dispersion is carried out at step-by-step increasing pressure from 300 to 800 atm controlling the size of an emulsion particles; after dispersion, a solution of cryoprotectant is added, the said solution contains 40-20% wt. of total cryoprotectant amount.

2. The method of claim 1, wherein phosphatidylcholine selected from egg phosphatidylcholine or soybean phosphatidylcholine, with a mixture of one or two different lipids selected from the group consisting of dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol or dioleoyloxypropy trimethylammonium, is selected as lipids.

3. The method of claim 1, wherein in order to form a mixture of lipids solutions, phosphatidylcholine is dissolved in ethyl alcohol, and dissolution of other lipids are carried out in chloroform with following merge of the said solutions at volumetric ratio of ethyl alcohol:chloroform in a range of 1:1.5-1:2.5.

4. The method of claim 1, wherein a weight ratio of cytochrome c:lipids mixture in a range of 1:11.4-1:18.5 is used.

5. The method of claim 4, wherein for a mixture of lipids a weight ratio of other lipids:phosphatidylcholine in a range of 1:0.3-1:2.0 is used.

6. The method of claim 1, wherein oligosaccharide selected from the group consisting of lactose, trehalose or sucrose is used as cryoprotectant.

7. The method of claim 6, wherein a weight ratio of lipids mixture:cryoprotectant is in a range of 1:5.5-1:7.2.

8. Pharmacologically active liposomal composition containing cytochrome c, a mixture of lipids and a cryoprotectant, wherein a mixture of lipids comprising egg phosphatidylcholine or soybean phosphatidylcholine and at most two different lipids selected from the group consisting of dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol or dioleoyloxypropy trimethylammonium, whereas a weight ration of cytochrome c:phosphatidylcholine:other lipids:cryoprotectant is in a range of (0.81-1.06)%:(3.03-8.26)%:(4.13-9.88)%:(78.67-83.30)%.

9. The composition of claim 8, wherein the composition includes oligosaccharide as a cryoprotectant, which is selected from the group consisting of lactose, trehalose or sucrose.

10. The composition of claim 8 having anticataract effect.

11. The composition of claim 8 having ability to restore the hemostatic coagulation system in acute massive blood loss.

12. The composition of claim 8 having anti-hipoxia and antitoxic effect.

Description

EXAMPLE 1

[0029] Claimed invention. The accurately weighed 6.0 g of (e)PC (in terms of 100% substance [for example, 14]) is dissolved in 100 ml of ethyl alcohol while stirring. The accurately weighed 5.0 g of DPPG (in terms of 100% substance [14]) is dissolved in 150 ml of chloroform while stirring, and after combining the resulted solutions with the (e)PC alcoholic solution (volumetric ratio of ethyl alcohol to chloroform is 1:1.5). A mixture of lipid solutions is filtered through a membrane with a pore diameter of 0.22 m, transferred to a rotary evaporator, and the solvents are removed by drying in a vacuum at a temperature of 40-45 C. until a thin film is obtained. Upon completion of the drying process, an inert gas is passed to the evaporator flask within 25-45 minutes. The accurately weighed 0.70 g of cytochrome c (in terms of 100% substance [for example, 15-16]) is dissolved in 80 ml of sterile phosphate buffered solution of pH 6.7-7.1, and filtered through a membrane with a pore diameter of 0.22 m. The accurately weighed 60.0 g of lactose (pharmacopeial milk sugar) in terms of 100% substance at a temperature of 50-60 C. is dissolved, while stirring, in 250 ml of sterile phosphate buffered solution of pH 67-7,1 and filtered through a membrane with a pore diameter of 0.22 m.

[0030] After drying, the resulting lipid film is quantitatively removed from the walls of the evaporator's flask using a mixture of 620 ml of sterile phosphate buffered solution of pH 6.7-7.1, 80 ml of cytochrome solution containing 0.7 g of cytochrome c, and 240 ml of lactose solution in a phosphate buffer containing 48.0 g of lactose, while stirring for 60 minutes at 100-120 rpm (for example, IKA, Germany) until a homogeneous emulsion is obtained.

[0031] The emulsion is transferred to a reactor of high pressure homogenizer (for example, M 110R Microfluidizer Processor, Microfluidics) and is dispersed at a temperature of 38-45 C. with a step-by-step increasing pressure from 300 atm to 800 atm for 1-3 cycles. The size of the emulsion particles (for example, Malvern Zetasizer Nano S) at the end of the dispersion process does not exceed 160 nm.

[0032] After completion of homogenization, 60 ml of a sterile lactose solution in a buffer solution of pH 6,7-7,1 containing 12.0 g of lactose is added to the emulsion and is mixed together. The volume of the emulsion is 1000 ml. The resulting emulsion is filtered through a membrane with a pore diameter of 0.22 m, and it is then a subject for sterile filtration, after that it is dosed in an aseptic manner in glass vials.

[0033] The vials with the emulsion are subjected to intensive freezing and freeze-drying (e.g., Martin Christ-2-6-D, USA). After drying, the vials with the lyophilized product are sealed in an inert gas atmosphere under aseptic conditions.

[0034] In the Examples 2-12, the claimed method is carried out in accordance with the Example 1. Changes in the parameters of the process implementation are reflected in Tables No. 1-3.

[0035] The targeted product is a light amorphous mass of yellow color with a characteristic smell.

EXAMPLE 13

[0036] Prototype according to [13]. PC in the amount of 3.6 g and DPPG in the amount of 1.0 g (in terms of 100% substance) is dissolved in 150 ml of a mixture of chloroform and ethyl alcohol (ratio of 4:1), and stirred. A lipid solution is filtered through a membrane with a pore diameter of 0.22 m, transferred to a rotary evaporator and evaporated at a temperature of 41-45 C. until a thin film is obtained. The film is treated with nitrogen gas for 15-20 min. The accurately weighed 0.135 g of cytochrome c (in terms of 100% substance) is dissolved in 135 ml of sterile phosphate buffered solution of pH 6.5-6.8 (cytochrome c concentration is 1 mg/ml) and filtered through a membrane with a pore diameter of 0.22 m. The accurately weighed 14.0 g of lactose, in terms of 100% substance, is dissolved in 35 ml of sterile phosphate buffered solution of pH 6.5-6.8 (lactose concentration 400 mg/ml) and filtered through a membrane with a pore diameter of 0.22 m.

[0037] The resulting lipid film is quantitatively removed from the walls of the evaporator flask with a mixture of 135 ml of cytochrome c solution (concentration of 1 mg/ml) and 30 ml of sterile phosphate buffered solution of pH 6.5-6.8. The content of the flask is stirred for 2 hours until a homogeneous emulsion is obtained.

[0038] The emulsion is transferred to a high pressure homogenizer, and dispersed at a temperature of 38-44 C. under pressure of 900 atm to obtain a particles size that does not exceed 200 nm. After achieving this particle size, 35 ml of lactose solution (containing 14 g of lactose) is added to the emulsion. The dispersion is continued until particles size is not more than 130-150 nm.

[0039] The resulting emulsion is filtered through a cascade of filters, wherein terminal filter has pores size of 0.22 m, and then sterile filtered, bottled into glass vials, freeze-dried and sealed under nitrogen atmosphere.

[0040] Based on the results of implementation of the prototype method, the product is in the form of a light amorphous mass of yellow color with a characteristic smell.

[0041] When identifying and establishing the quality parameters of a liposomal compositions obtained according to the proposed and prototype method, the compositions were used per se and in the form of an emulsion, reconstituted by adding in a vial with a lyophilized product of a sterile isotonic solution corresponding to the form of their potential pharmacotherapeutic application.

[0042] The efficacy of the claimed method with respect to the pharmaceutical quality of the produced liposomal composition is confirmed by the results of qualitative and quantitative identification of cytochrome c and lipid components (PC and other lipids), and the liposomal status of the targeted product is confirmed using a number of independent physical and chemical methods, namely: [0043] In spectrophotometric method, by the characteristic absorption spectrum in a range of 400-600 nm and an optical density of solution at wavelengths (4072) nm and (4902) nm, the solution is obtained after holding the targeted product in water at a temperature of 35 C. for 30 min, in comparison with such a solution of the standard sample of cytochrome c (identification and quantitative determination of cytochrome c respectively); [0044] In sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), by the electrophoregram of aqueous solution of the targeted product in comparison with such a solution of the standard sample of cytochrome c (identification of cytochrome c respectively); [0045] Spectrophotometric method by the characteristic absorption spectrum of solution in a range of 450-650 nm with maximum at wavelength of 522-526 nm and 559-561 nm, the solution is obtained after holding the targeted product in water at a temperature of 35 C. for 30 min, followed by passing of nitrogen(II) oxide until appearance of a bright pink color (identification of the iron-containing prosthetic group of cytochrome c); [0046] In thin-layer chromatography, by a chromatogram of the targeted product solution in a mixture of chloroform, methanol and water (volumetric ratio 73:23:3), the chromatogram contains spots of PC and other lipids at the level of the main spots on the chromatograms of solutions of standard samples of PC and other corresponding lipids (identification of PC and other lipids); [0047] In liquid chromatography technic with evaporative light scattering detector (ELSD), by a chromatogram of a solution of the targeted product in a mixture of chloroform, methanol and water (volumetric ratio 73:23:3) using for calibration and comparison a solution of standard samples of PC and corresponding lipids (quantitative determination of PC and other lipids); [0048] In liquid chromatography technic with spectrophotometric detection at a wavelength of 409 nm, by a chromatogram of solution obtained after holding the targeted product in a water at room temperature for 30 min, compared to the standard sample of the free cytochrome c (level of encapsulation of cytochrome c to liposomes); [0049] By measuring the size of the particles in emulsion of liposomal product by the method of dynamic light scattering (DLS); [0050] By index of oxidation of the targeted product lipid fraction (stability of liposomes); [0051] By parameters of formation time, separation stability and dispersed composition of the emulsion reconstituted from the freeze-dried targeted product (functional stability and distribution of liposomes by size); [0052] By pH and osmolality of the emulsion (determination of compliance with the requirements of functional use of parenteral and ophthalmic drugs).

[0053] According to the results of physical and chemical analysis (Table 4), the claimed method provides the pharmaceutical quality and stability of the targeted product as a liposomal composition containing cytochrome c, namely: [0054] With accordance to the methods of spectrophotometry, thin-layer chromatography and liquid chromatography, the targeted product keeps the native composition and nature of cytochrome c, PC and others selected lipids, which coincides with the corresponding standards of cytochrome c, PC and the corresponding lipids; [0055] Cytochrome c is quantitatively incorporated into liposomes with a level of encapsulation of more than 90%; [0056] The targeted product is characterized by quick formation of the emulsion from lyophilisate and emulsion separation stability; [0057] An emulsion of the targeted product has the stable liposome average size of 1473 nm accompanying by a practical monodispersity of their distribution by size (90-100%), including prolonged storage, and low oxidation index; [0058] Emulsion pH value is stable and it corresponds to the physiological norms for this factor in vivo; [0059] The osmolality index corresponds to pharmacopoeial requirements for ophthalmic drugs.

[0060] It should be noted that optimal pharmaceutical quality and stability is inherent to the targeted product produced by Examples 1-8, and the implementation of the claimed method, with excellent parameters (Examples 9-12), and of the prototype method (Example 13) causes a negative distortion of these parameters (Table 4), namely: [0061] decreasing of encapsulation level of cytochrome c into liposomes (by 2.8-7.0% in the examples 9-12 and 6.8-11.0% in the prototype); [0062] significant heterogeneity of liposomes dispersion by size (60-25-15%, 80-10-10% and 75-25% in examples 9,11 and 12 respectively, and 67-33% in the prototype), which increases during storage of the product, having tendency to increase the size of liposomes; [0063] relative index rise of oxidation of the liposomal product; [0064] increasing of time of emulsion formation from the freeze-dried product (in 1.5-2 times) simultaneously with decreasing of its separation stability (in 1.3 times); [0065] relative increasing of osmolality of the emulsion (for 10-40 mOsm/g until the upper limit of the pharmacopoeial norm of 380 mOsm/g for ophthalmic drugs).

[0066] The said important advantages of the pharmaceutical quality and stability of the composition produced by Examples 1-8 are the consequence and are coupled with optimized performance of the claimed method (Table 3) compared to the process parameters provided by Examples 9-12 and the prototype (Example 13), in particular: [0067] different dissolution in various solventsPC (in ethanol) and other lipids (in chloroform), followed by their merge at final ratio of ethyl alcohol:chloroform 1:1.5-2.5 (comp.:according to the prototype, analogical use of mixture of ethanol:chloroform 1:4 for dissolving in the Examples 9 and 12 resulted in deterioration of the pharmaceutical quality of the product); [0068] reduction of time in 1.5-2.6 for stirring after emulsification; [0069] dispersion within three successive cycles at a pressure in the range of 300-800 atm; [0070] two-stage introduction of cryoprotectant: at emulsification (60-80%) and after dispersion (40-20%) (compar.: introduction of all or part of cryoprotectant amount at the dispersion process in the Examples 9 and 11 resulted in deterioration of the pharmaceutical quality of the product); [0071] no filtration on cascade filters, therefore the total time of filtration is reduced in 2.1-2.9 times.

[0072] Thus, the claimed method which is performed according to the Examples 1-8, provides producing of a liposomal composition containing cytochrome c having competitive high pharmaceutical quality and stability, along with certain technological advantages of this method. It is important that this conclusion is true for the implementation when using all of the lipids and cryoprotectants of various nature selected in Table 1, which considerably extend the limits of method use.

[0073] All characteristics of the pharmaceutical quality of the product of claimed method are established according to validated methods. The composition content is calculated basing upon reliable data on the content of cytochrome c, PC and other lipids in the product which was obtained by the claimed method and the prototype method, by weight ratio and percentage content of the components (Table 5). While calculating the percentage composition content (for 100%), an experimentally determined index of weight loss during drying was taken into account.

[0074] In accordance with the object of the invention, the quality of the produced liposomal composition of cytochrome c is evaluated by pharmacological activity factors in preclinical studies at various pathological conditions and methods of administration.

[0075] The selected specificity of the studies of the produced composition corresponds to the object of the invention, with reference to the proof of the production of the targeted product with polyfunctional pharmacological activity.

[0076] A comparison of the specific pharmacological effect of a liposomal composition containing cytochrome c obtained by the claimed method and prototype method was carried out in experimental models of the following pathologies:

[0077] 1) a model of irradiation cataract caused by chronic general irradiation of animals (rabbits) with polychromatic light in a range of 350-1150 nm for 26 weeks, which according to clinical features corresponds to hard nuclear cataract in humans [17].

[0078] The composition in question was administered in a form of drop emulsion (0.675 mg/ml of cytochrome c). Instillations (three times a day) were began on the 21st week after the created cataract model, they were continued for 6 weeks.

[0079] As an analogue of functional activity in the evaluation of the pharmacological effect of the liposomal composition, the cytochrome c drug Oftan-Katachrom (Santen Oy, Finland) was used. An aqueous solution containing nicotinamide, adenosine and benzalkonium chloride (20, 2 and 0.20 mg/ml respectively), which is similar to the solvent of the Otan-Katachrom, was used to correctly compare the effects of the products to create the emulsion of the liposomal composition. Animals of the control group received instillations of the same solvent in appropriate doses.

[0080] Evaluation of the pharmacological effect of a liposomal composition containing cytochrome c, which is obtained by the claimed and prototype method in the cataract model, was performed according to standards for assessing the state of the eye lens (5 stages of cataract: no changesstage 0 and intense cataract lesionstage 5) [18]. The biochemical parameters were also determined (enzymes and lipid peroxidation products (LOPs)) in aqueous humour in chamber and lens.

[0081] 2) a model of hemostatic system damage due to acute massive blood loss (MBL) in volume of 30% of circulating blood of animals (sexually mature white rats), which leads to disseminated intravascular coagulation of blood with metabolic acidosis [19, 20].

[0082] The composition in question was administered 30 minutes after MBL, intravenously, in the form of an emulsion in a saline solution (1 mg/kg cytochrome c) in volume corresponding to the volume of lost blood (isovolumic replenishment).

[0083] The evaluation of the pharmacological effect of the liposomal composition containing cytochrome c, which is obtained by the claimed and the prototype method in the MBL model, was performed according to the parameters of the coagulation system, acidbase homeostasis balance state and gas exchange of arterial blood of animals.

[0084] 3) models of normobaric, gemic and histotoxic (tissue) hypoxia, which are used to assess the anti-hypoxic effect of potential drugs [21].

[0085] Normobaric hypoxia was caused by placing experimental animals in a hermetic chamber with a closed volume of living space of 0.5 l. Gemic hypoxia was caused by the introduction of methemoglobin-forming substance to the animals, i.e. sodium nitrite at a dose of 200 mg/kg. Tissue hypoxia was modeled by the introduction of nitroprusside sodium according to the following scheme: 1st-4th day1 mg/kg (once a day), 5th day25 mg/kg. In all models of hypoxia in experiments, adult white mice of both sexes are used.

[0086] The products in question were administered as intraperitoneal injection at a dose of 2 mg/kg (basing on cytochrome c content) according to the following scheme: 1st-4th dayonce a day, 5th dayin 30 min before hipoxia modelling.

[0087] The criterion for estimating the anti-hypoxic effect of a liposomal composition containing cytochrome c, which is obtained by the claimed and prototype method, was the lifetime of the experimental animals.

[0088] 4) a model of narcotic intoxication [21] caused by administration of thiopental sodium in a dose of 45 mg/kg to adult white rats of both sexes.

[0089] The products in question were administered as intraperitoneal injections in 30 min before the introduction of xenobiotic at a dose of 1 mg/kg (basing on cytochrome c content).

[0090] The criterion for estimating the antitoxic effect of a liposomal composition containing cytochrome c, which is obtained by the claimed and prototype method, was the time of narcotic sleep.

[0091] For a correct assessment of the pharmacological effect in the models of MBL, hypoxia and narcotic intoxication cytochrome c solution in saline solution was used as an analogue of the liposomal composition with respect to the functional activity (for example, Farmasino Pharm. Co., Ltd, China) in equimolar amounts based on cytochrome c content. To animals of the control group saline solution in appropriate doses were injected.

[0092] According to the current requirements for the pharmacological products considered as potential medicinal products, as well as to confirm the correctness of the dose regimen and method of administration, the harmlessness of the liposomal composition containing cytochrome c, which is obtained by the claimed method and the prototype method, with respect to the provided methods of its administration (injections and eye drops), was assessed.

[0093] The applied products in the form of an emulsion in a saline solution with single intravenous administration and repeated intraperitoneal injections (14 days) did not cause death of experimental animals (white rats), local irritation, negative influence on body weight and weight coefficients of internal organs, changes in blood formulas and basic biochemical parameters of blood serum.

[0094] While studding acute and chronic (28 days) ophthalmological toxicity of the liposomal composition containing cytochrome C, and the composition is in the form of drops in the saline solution, no negative manifestations were found regarding the structures of the anterior part of the eye and the optic disc, as well as the locally irritating and allergic effect (on rabbits).

[0095] Thus, according to the characteristics of acute and chronic toxicity, including ophthalmological harmlessness, that do not differ for the liposomal composition containing cytochrome c, which is obtained by the claimed method and prototype method, they should be attributed to virtually harmlessness means, which makes possible the potential pharmacotherapeutic use.

[0096] In order to comply with the bioethical regulations governing the rules of humane treatment of animals and minimizing animals number in preclinical studies [22], in highly invasive experimental models (acute massive blood loss, cataract with lens removal), separate indicative examples of a liposomal composition containing cytochrome c obtained by the claimed method, are used. For optimal balance between adherence to bioethical regulations and the reliability of estimation of pharmacological activity in models of different pathologies, examples of compositions have been selected on a cross-sectional basis, and in the models of hypoxia (less invasive), all samples of the composition with the highest pharmaceutical quality (Examples 1-8) were analyzed, and for comparisonalso examples 9 and 10. To be noted, the pharmacological effect of the product produced by the prototype method was analyzed in all experimental models (Example 13).

[0097] Tables 6-8 show the results of establishing the pharmacological effect of a liposomal composition containing cytochrome c, which is produced by the claimed method and prototype method. In all models of pathologies, the products have pharmacological activity, but the very implementation of the claimed method provides producing of a composition with a high polyfunctional pharmacological effect.

[0098] In the model of irradiation without treatment (negative control) in animals, severe 3 and 4 stages of damages caused by cataract (50% of eyes with cataract of each stage correspondingly) are achieved, which is accompanied by pathological changes in the biochemical parameters of the lens (accumulation in 1.7 times of products of LOP and imbalance of the enzymes activity in 1.4-1.7 times). At the same time the claimed liposomal composition of cytochrome c shows high effect against cataract (Table 6): [0099] prevents cataract development reducing the lesion of the lens to the initial stages 1 and 2 (12.5-25% and 87.5-75% of eyes amount, correspondingly), in the absence of manifestations of cataract with severe irreversible stages 3 and 4; [0100] virtually normalizes an enzymes activity and the content of LPO products in the lens (89-95% of the norm).

[0101] In the MBL model, lesions of the hemostasis system are manifested in reducing of parameters of blood coagulation for 15-30% and distortion of the acidbase homeostasis state and gas exchange in blood (in particular, a decreasing of pH for 0.243 units to non-physiological values and reducing for 10% to critical range of oxygen saturation of blood).

[0102] The liposomal composition of cytochrome c, which is produced by the claimed method, has the ability to restore the major components of the hemostasis system which were affected by the MBL, causing (Table 7): [0103] restoration of parameters of the functional state of the blood coagulation system (up to 88-99% of normal) with a clear tendency to support coagulation hemostasis (thrombocytopeniathe level of APTT, PT, TT and fibrinolysisthe level of fibrinogen); [0104] practical elimination of decompensated metabolic acidosis according to the acid-base homeostasis state (increasing of pH and BB to physiological normal state) and gas exchange of arterial blood, including normalization of important parameter of oxygen blood saturation, O.sub.2Sat (up to 99-100% of normal value).

[0105] In the models of hypoxia of different etiologies (normobaric, hemic, tissue), the liposomal composition with cytochrome c provides increasing of life time of animals by 47-68%, that is, it has a universal anti-hypoxic activity. The composition has an antitoxic effect, reducing the duration of narcotic sleep by 33-40% in contrast to narcotic intoxication (Table 8).

[0106] By the level of confirmed pharmacological activity in experimental pathologies of different genesis, the liposomal composition produced by the claimed method is competitive both in respect of the product of prototype method (effectiveness of which in the models of different pathologies is less in 1.2-1.8 times) and with respect to analogue drugs (the effectiveness of which in models of different pathologies less in 1.3-2.4 times).

[0107] In general, the highest quality is inherent to the liposomal composition with cytochrome c, which is produced by the claimed method with accordance to the parameters set forth in Examples 1-8, and which are different from those provided for producing of the composition by the prototype method, namely: as lipids the following is used: phosphatidylcholine selected from (e)PC or (s)PC in a mixture with one or two lipids selected from the group of DPPG, DPPC, DSPC, DPG, PG, PI or DOTA at weight ratio of phosphatidylcholine:to other lipids of 0.3-2.0:1; dissolution of PC is carried out in ethyl alcohol and dissolution of other lipidsin chloroform with their following merge, wherein the volumetric ratio of ethyl alcohol:chloroform is 1:1.5-2.5; emulsification is performed at a weight ratio of 1:11.4-18.5 of cytochrome c:lipids respectively

adding a cryoprotectant solution, wherein a cryoprotectant is selected from lactose, trehalose, sucrose oligosaccharides, whereas the said solution contains 60-80% of total cryoprotectant; and homogenization with step-by-step increasing pressure from 300 to 800 atm monitoring the size of emulsion particles, after its completion a solution of selected cryoprotectant is added to emulsion, wherein solution contains 40-20% of total cryoprotectant, and the weight ratio of lipids:cryoprotectant is 1:5.5-7.2.

[0108] Deviating from the above parameters (Examples 9-12 and the prototypeExample 13), the implementation of the method is prolonged and somewhat complicated (Table 3increasing of time for stirring during emulsification, cascade filtration and increasing of the filtration time after dispersing) and desired pharmaceutical quality of the targeted product as a liposomal composition with cytochrome c is not achieved (Table 4).

[0109] Accordingly, the high pharmaceutical quality of the targeted product determines for liposomal composition polyfunctional pharmacological efficacy and a higher level of anti-catalytic, anti-hypoxic and antitoxic activity and the ability to restore the hemostatic blood coagulation system for MBL, the composition contains cytochrome c, a mixture of lipids, and a cryoprotectant, providing its components defined by Examples 1-8, and other than those for a prototype-produced composition, namely: a mixture of lipids includes phosphatidylcholine selected from (e)PC or (s)PC in a mixture with one or two lipids selected from the group of DPPG, DPPC, DSPC, DPG, PG, PI or DOTA, a cryoprotectant is a oligosaccharide selected from lactose, trehalose or sucrose, wherein in the composition a weight ratio of cytochrome c:phosphatidylcholine:other lipids:cryoprotectant is 1:2.9-8.6:4.3-15.7:78.6-102.8, and a percentage ratio of cytochrome c:phosphatidylcholine:other lipids:cryoprotectant is (0.81-1.06)%:(3.03-8.26)%:(4.13-9.88)%:(78.67-83.30)%.

[0110] Variation of these parameters of composition (Examples 9-12 and prototypeExample 13) reduces the level of its pharmacological effects.

[0111] The achieved optimal combination of favorable production effectiveness and operations for implementation of a method with a reliable pharmaceutical identification and positive pharmacological properties of the targeted product demonstrates the advantages of the claimed method for the object of the invention, that is, obtaining of a liposomal drug containing cytochrome c. Produced liposomal composition with cytochrome c and with identified individual composition, for which the polyfunctional pharmacological effects and harmlessness were first established, advantageously differs from the prototype-produced liposomal product containing cytochrome c.

[0112] This justifies the reasonability of using the claimed method for obtaining a stable and high-quality liposomal drug containing cytochrome c and introducing of this liposomal composition with cytochrome c as a potential effective drug with a multifunctional pharmacotherapeutic effect for use in ophthalmology, cardiology and hematology.

TABLE-US-00002 TABLE 1 Parameters for implementing the claimed method for obtaining a liposomal composition containing cytochrome c, and a prototype method with respect to nature of lipids and cryoprotectants that are used thereof .sup.1) Applied lipids Other lipids Example Phosphatidyl- Lipid name Weight ratio Cryo- No choline (conventional name) of other lipids protectant Proposed method 1 (e)PC DPPG Lactose 2 (s)PC DPPG Lactose 3 (e)PC DPPG + DPPC 1:1 Trehalose 4 (s)PC DSPC + DPG 2.5:1 Lactose 5 (e)PC DPPG + PG 1:1 Trehalose 6 (e)PC DPPG + PI 2:1 Sucrose 7 (e)PC DPPG + DOTA 2.5:1 Lactose 8 (e)PC DPPC + DOTA 0.5:1 Trehalose 9 (e)PC DPPG + DSPC 0.3:1 Lactose 10 (e)PC DPPC + DOTA 0.5:1 Trehalose 11 (s)PC DSPC + DPG 1:1 Lactose 12 (e)PC DPPG + PG 2:1 Lactose Prototype method 14 (e)PC .sup.2) DPPG Lactose .sup.1) in all Examples cytochrome c is an integral component of the process implementation. .sup.2) origin of the substance in the prototype is not identified.

TABLE-US-00003 TABLE 2 Parameters for implementing the claimed method for obtaining a liposomal composition containing cytochrome c, and a prototype method with respect to ratios of components of the produced composition, which are used thereof Weight ratio Example Cytochrome c: Lipids No lipids mixture * other lipids:PC mixture:cryoprotectant Claimed method 1 1:15.7 1:1.2 1:5.5 2 1:12.9 1:0.8 1:7.2 3 1:14.3 1:0.7 1:6.5 4 1:18.5 1:0.9 1:5.6 5 1:15.7 1:0.4 1:6.0 6 1:12.9 1:2.0 1:6.7 7 1:17.1 1:0.7 1:5.8 8 1:11.4 1:0.3 1:6.9 9 1:12.9 1:0.2 1:4.6 10 1:21.4 1:4.0 1:5.3 11 1:12.8 1:0.15 1:5.3 12 1:11.4 1:1.7 1:4.3 Prototype method 13 1:34.1 1:3.6 1:3.0 * a mixture of lipids is PC with other applied lipids indicated in Table 1.

TABLE-US-00004 TABLE 3 Parameters of the claimed method for obtaining a liposomal composition containing cytochrome c, and a prototype method with respect to technical data which are used thereof Example No Claimed method 13- 1 2 3 4 5 6 7 8 9 10 11 12 Prototype A medium for + + + + + + + + + + dissolution of + + + lipids: a) ethyl alcohol - PC, chloroform - other lipids, wherein ratio of ethyl alcohol:chloroform is 1: 1.5-2.5 (vol.) ) all lipids: mixture of ethyl alcohol and chloroform (1:4) Parameters for + + + + + + + + + + emulsification: + + + a) Medium: 60 70 55 45 60 45 65 80 112 85 90 95 120 mixture of 80 69 62 65 80 70 60 60 0 40 50 20 0 cytochrome c, cryoprotectant and buffered solution: mixture of cytochrome c and buffered solution: b) Stirring time, min c) amount of introduced cryoprotectant (% of total amount) Parameters 300 300 300 300 300 300 300 300 300 300 200 300 900 of 600 600 600 500 600 700 500 600 400 500 600 500 The dispersion: 800 800 800 800 800 800 800 800 800 900 800 500 whole a) Pressure process for cycles, <150 <145 <150 <145 <150 145 <150 <150 <160 <130 <130 <150 <145 at: 1st + + + 2nd + + + + + + + + + + 3rd 20 31 38 35 20 40 30 40 100 60 50 80 100 b) Particles size after dispersion, nm c) introduction of cryoprotectant solution: within dispersion process after dispersion c) amount of introduced cryoprotectant (% of total amount) Parameters + + for filtration + + + + + + + + + + + after 10 11 12 10 12 10 14 10 15 20 28 19 29 dispersion: a) filtration: cascade of filters filter of 0.22 m b) filtration average time (for 11). min

TABLE-US-00005 TABLE 4 Identification and pharmaceutical quality parameters of a liposomal composition obtained by the claimed method and prototype method Example No Claimed method 13- 1 2 3 4 5 6 7 8 9 10 11 12 Prototype Identification: + + + + + + + + + + + + + cytochrome c + + + + + + + + + + + + + PC + + + + + + + + + + + + + other lipids Encapsulation 95.1 94.1 98.4 95.6 96.8 99.0 95.0 94.8 89.0 91.3 90.3 92.0 88.0 level of cytochrome c into liposomes (% of introduced amount) Liposomes 147/ 150/ 145/ 145/ 147/ 147/ 145/ 150/ 165/ 150/ 148/ 165/ 155/67 size 95 90 100 95 97 92 94 90 60 90 80 75 (nm)/% 132/5 138/ 135/5 140/3 135/8 130/6 140/ 130/ 132/ 125/ 150/ 62/33 liposomes 10 10 25 10 10 25 by the 100/ 100/ size: * 15 10 a) after 150/ 150/ 148/ 148/ 150/ 150/ 145/ 152/ 160/ 145/ 150/ 160/ 155/60 freeze- 95 90 100 95 95 92 95 90 55 90 75 80 60/25 drying 135/5 138/ 135/5 140/5 140/9 135/5 142/ 130/ 130/ 130/ 140/ 52/15 b) after 9 10 10 25 10 15 20 months 95/10 100/ storage 65/5 10 Oxidation 0.24 0.28 0.22 0.20 0.24 0.25 0.26 0.22 0.33 0.24 0.31 0.30 0.31 index, *** conv. unit Formation 1.2/ 1.5/ 1.5/ 1.0/ 1.2/ 1.2/ 1.0/ 1.0/ 2.0/ 1.0/ 1.8/ 2.0/ 1.8/ time/emulsion 115 110 120 110 112 120 100 105 80 90 100 95 95 *** stability (min)* pH of 6.56 6.50 6.56 6.70 6.55 6.61 6.50 6.50 6.62 6.48 6.75 6.58 6.74 emulsion* *** Emulsion 320 330 310 330 320 340 350 330 380 340 360 330 380 osmolality, mOsm/g**)*** Weight loss 5 5 5 5 4 4 5 4 6 5 5 3 6 within drying (%)*** Quantitative 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.70 0.67 0.70 0.70 0.70 0.67 content 6.0 4.0 4.0 6.0 3.0 6.0 2.0 5.0 9.0 3.0 8.0 5.0 18.0 (mg/ml)*: 5.0 5.0 6.0 7.0 8.0 3.0 6.0 7.0 2.0 12.0 1.0 3.0 5.0 cytochrome c PC other lipids (+) - positive identification *determined for an emulsion that is reconstructed by adding of 10 ml of the saline solution to the lyophilized product, the liposomal composition **in accordance with the requirements of the State Pharmacopoeia of Ukraine (2.2.35), for ophthalmologic drugs the index of osmolality should be within the range of 300-380 mOsm/g ***not regulated by the prototype

TABLE-US-00006 TABLE 5 Content of liposomal composition with cytochrome c obtained by claimed method and prototype method, by weight and percentage ratio of com- ponents determined by established parameters of pharmaceutical quality Weight ratio Ex- cytochrome c:PC: Content (% wt) ample :other lipids: Cytochrome Other Cryo- No :cryoprotectant * c PC lipids protectant Composition obtained by the claimed method 1 1:8.6:7.1:85.7 0.93 7.95 6.63 79.50 2 1:5.7:7.1:92.9 0.89 5.09 6.36 82.66 3 1:5.7:8.6:92.9 0.88 5.02 7.53 81.57 4 1:8.6:10.0:102.8 0.84 7.15 8.34 78.67 5 1:4.3:15.7:94.3 0.86 3.71 9.88 81.54 6 1:8.6:4.3:85.7 0.96 8.26 4.13 82.64 7 1:7.1:7.1:100.0 0.81 5.80 8.13 81.95 8 1:2.9:8.6:78.6 1.06 3.03 9.09 83.30 9 1:12.8:2.9:71.4 1.09 14.02 3.11 77.88 10 1:4.3:17.1:114.3 0.70 3.00 12.0 80.0 11 1:11.4:1.4:68.6 1.19 13.61 1.70 81.69 12 1:7.1:4.3:50 1.52 10.86 6.52 76.08 13 1:14.3:8.6:92.9 1.30 18.63 11.80 65.20 Composition obtained by the prototype method 14 1:26.7:7.4:103.7 0.72 19.22 5.33 74.72 * content of cryoprotectant (pharmaceutical excipient) is taken according to the introduced amount

TABLE-US-00007 TABLE 6 The efficacy of the claimed method and prototype method in respect of the pharmacological activity of the liposomal composition with cytochrome c obtained thereof for the effect on the state of cataract development, and biochemical parameters of the eye lens in the model of irradiation cataract Parameters of enzymes activity of and content of lipid peroxidation products in the lens.sup.1) The number of eyes with Catalase, cataract (%) at stage: MDA, DK, kat/ LDH, AP, 1 2 3 4 nmol/ml nmol/ml ml kat/ml nkat/l Parameter 0 0 0 0 8.1 1.9 43.1 12.2 14.9 initial level (normal) Irradiation (26 weeks) without treatment (saline solution - 6 weeks) Negative 0 0 50 50 13.8 3.2 26.0 16.2 25.1 control Irradiation (26 weeks) + introduction of the next products (6 weeks): The proposed composition is obtained by the claimed method (examples)*.sup.)** 1 25.0 75.0 0 0 9.6 2.4 36.1 13.2 16.0 5 25.0 75.0 0 0 8.8 2.0 40.1 12.9 15.5 7 12.5 87.5 0 0 9.0 2.1 38.0 12.6 16.5 11 0 87.5 12.5 0 10.2 2.7 32.0 14.0 19.1 Composition obtained by the prototype method*.sup.)** 13 0 87.5 12.5 0 10.8 2.8 31.1 14.1 20.0 Prototype drug* Oftan 0 87.5 12.5 0 11.5 2.7 29.9 14.6 21.8 Katachrom .sup.1)MDAmalondialdehyde; DKdien conjugates; LDHlactate dehydrogenase; APacid phosphatase. *p < 0.05 comparing to the parameter initial level **p < 0.05 comparing to the control

TABLE-US-00008 TABLE 7 The efficacy of the claimed method and the prototype method for the pharmacological activity of the liposomal composition with cytochrome c obtained by these methods with respect to its effect on the hemostatic blood coagulation system in acute massive blood loss (MBL) Parameters of acid- Parameters of blood base homeostasis state and coagulation system.sup.1) gas exchange of blood.sup.2) APTT PT TT FG pH pCO.sub.2 HCO.sub.3.sup. O.sub.2Sat BB Initial value 39.4 41.0 47.2 2.07 7.465 38.62 22.52 99.29 46.9 (before MBL) MBL 29.0 28.1 43.2 1.67 7.222 34.64 17.37 90.40 38.8 (30%)* MBL + proposed composition obtained by the claimed method (examples)*.sup.)**.sup.) 1 33.7 39.6 48.0 1.99 7.442 36.64 24.21 99.34 49.34 3 34.6 39.2 47.0 1.98 7.415 36.85 24.0 99.20 47.95 4 33.8 40.0 48.0 2.00 7.450 37.44 23.65 99.0 46.02 8 37.9 40.5 48.2 2.02 7.460 37.70 24.10 99.61 48.30 9 32.5 36.5 46.3 1.89 7.341 36.0 21.75 97.20 45.26 MBL + composition according to the prototype method*.sup.)**.sup.) 14 32.4 36.6 46.8 1.90 7.345 36.0 21.80 96.40 44.49 MBL + analogue drug* Cytochrome c 30.1 31.1 45.8 1.81 7.301 35.38 18.47 92.06 41.27 .sup.1)APTTactivated partial thromboplastin time, s; PTprothrombin time, s; TTthrombin time, s; FGfibrinogen, g/L. .sup.2)pCO.sub.2partial pressure of CO.sub.2, mmHg; O.sub.2Satoxygen blood saturation, %; HCO.sub.3.sup.plasma bicarbonate, mmol/L BBtotal amount of anions in the blood, mmol/L. *p < 0.05 comparing to the parameter initial value *p < 0.05 comparing to the MBL group

TABLE-US-00009 TABLE 8 The efficacy of the claimed method and the prototype method for the pharmacological activity of the liposomal composition with cytochrome c obtained by these methods with respect to hypoxia of different etiology and narcotic intoxication Parameters of pharmacological activity in pathology models: Lifetime at hypoxia, min/% * Duration of sleep at Normobaric Gemic Tissue narcotic intoxication, hypoxia hypoxia hypoxia min/% * Composition obtained by the claimed method (Examples): 1 119.8/48.5 16.9/53.6 15.2/60.0 45.0/38.6 2 119.0/47.5 16.7/51.8 15.1/58.9 44.8/38.9 3 122.1/51.3 17.3/57.2 15.8/66.3 44.0/40.0 4 119.0/47.5 16.6/50.9 15.2/60.0 44.5/39.3 5 122.4/51.7 17.0/54.5 15.7/65.3 44.1/39.8 6 123.0/52.4 17.4/58.2 16.0/68.4 44.0/40.0 7 119.1/47.6 16.8/52.7 15.7/65.3 44.8/38.9 8 119.6/48.2 16.7/51.8 15.8/66.3 44.1/39.8 9 108.1/33.9 15.5/40.9 13.9/46.3 49.0/33.1 10 109.3/35.4 16.0/45.5 14.2/49.5 48.9/33.3 Composition obtained by the prototype method 13 106.5/31.9 15.3/40.1 13.9/46.3 55.3/24.5 Cytochrome 98.9/22.6 14.8/34.5 13.3/39.5 61.0/16.8 c-analogue Control- 80.7/ 11.0/ 9.5/ 73.3/ saline solution * min/%: minabsolute value of parameter in minutes; % parameter change as to control group in percent. p < 0.05 comparing to the control

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