Method of obtaining a pharmacologically active liposomal cytochrome c and nitric oxide complex

Abstract

The invention relates to pharmaceutical industry and discloses a method of obtaining a new pharmacologically active liposomal agent containing substances that exhibit specific pharmacological activity on peripheral vessels and cavernous bodies of a mammal. More particularly, the invention relates to a method of obtaining a pharmacologically active liposomal cytochrome c containing nitric oxide. The new liposomal agent acts as a donor of the key biologically active substancenitric oxide (NO). A method of obtaining a pharmacologically active liposomal cytochrome c and nitric oxide complex comprises the treatment of the liposomal cytochrome c emulsion with gaseous nitric oxide (NO) until liposomal cytochrome c is completely reconstituted and the addition of an S-nitroso compound to the liposomal cytochrome c emulsion.

Claims

1. A method of obtaining a pharmacologically active liposomal cytochrome c and nitric oxide complex, the method comprising treating a liposomal cytochrome c emulsion with gaseous nitric oxide (NO) until liposomal cytochrome c is completely reconstituted and adding an S-nitroso compound to the liposomal cytochrome c emulsion.

2. The method according to claim 1, wherein the liposomal cytochrome c emulsion is treated by supplying gaseous nitric oxide (NO) using an inert carrier gas.

3. The method according to claim 2, wherein the inert carrier gas is argon.

4. The method according to claim 2, wherein the inert carrier gas is pre-filtered to a purity of at least 99.995%.

5. The method according to claim 2, wherein the inert carrier gas is purified from salt-forming admixtures of nitric oxide (NO) after contact with gaseous nitric oxide (NO).

6. The method according to claim 2, wherein the liposomal cytochrome c emulsion is obtained by high-pressure homogenization followed by lyophilic drying to produce a lyophilizate.

7. The method according to claim 6, wherein the liposomal cytochrome c emulsion is reconstituted from the lyophilizate.

8. The method according to claim 1, wherein liposomal cytochrome c having the form of emulsion is pre-filtered using hydrophilic membranes before gaseous nitric oxide (NO) is supplied.

9. The method according to claim 8, wherein prefiltration is performed through at least two successively positioned hydrophilic membranes with a gradually decreasing pore diameter.

10. The method according to claim 1, wherein liposomal cytochrome c having the form of emulsion is treated with gaseous nitric oxide (NO) at a room temperature.

11. The method according to claim 1, wherein the emulsion is subject to additional filtration after the reconstitution of liposomal cytochrome.

12. The method according to claim 11, wherein, after the additional filtration, the emulsion is frozen at a temperature of minus 35 C. followed by lyophilic drying.

13. The method according to claim 1, wherein an S-nitroso compound is added to the emulsion to obtain a concentration in the range of 0.01 to 0.1 M in the emulsion.

14. The method according to claim 1, wherein an S-nitroso compound is added to the emulsion before the treatment with gaseous nitric oxide (NO) and/or during the treatment with gaseous nitric oxide (NO) and/or after the treatment with gaseous nitric oxide (NO).

15. The method according to claim 1, wherein an S-nitroso compound is selected from a group of S-nitrosothiols consisting of nitroso-N-acetylpenicillamine, S-nitrosoglutathione (GS-NO), S-nitrosocysteine (Cys-NO), and any mixture thereof.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a diagram illustrating the contractile activity of intact and deendothelized rat thoracic aorta segments pre-activated by phenylephrine (10.sup.6 mol/l) exposed to a sample of the ex-tempore liposomal cytochrome c and nitric oxide complex (Lipoxide).

(2) FIG. 2 is a diagram illustrating the comparative efficacy of liposomes acting as an element of the liposomal cytochrome c and nitric oxide complex (Lipoxide) and a sample of the ex-tempore liposomal cytochrome c and nitric oxide complex (Lipoxide) in terms of the contractile activity of rat thoracic aorta segments pre-activated by phenylephrine (10.sup.6 mol/l).

(3) FIG. 3 is a diagram illustrating the contractile activity of intact and deendothelized rat thoracic aorta segments pre-activated by phenylephrine (10.sup.6 mol/l) exposed to liposomes.

(4) FIG. 4 is a diagram showing the comparative efficacy of samples of the ex-tempore liposomal cytochrome c and nitricoxide complex (Lipoxide) and lyophilized liposomal cytochrome c and nitric oxide complex (Lipoxide) in terms of the contractile activity of rat thoracic aorta segments pre-activated by phenylephrine (10.sup.6 mol/l).

(5) FIG. 5 is a diagram illustrating the comparative efficacy of samples of lyophilized liposomal cytochrome c and nitric oxide complex (Lipoxide) and liposomal cytochrome c and nitric oxide complex with the addition of S-nitrosoglutathione (Lipoxide GSNO) in terms of the contractile activity of rat thoracic aorta segments pre-activated by phenylephrine (10.sup.6 mol/l).

(6) FIG. 6 is a diagram illustrating the relaxing effect of the ex-tempore liposomal cytochrome c and nitric oxide complex (Lipoxide) on cavernous bodies taken from a rat penis pre-stimulated with phenylephrine 10.sup.6 M.

(7) FIG. 7 is a diagram illustrating the comparative effect of liposomes on cavernous bodies taken from a rat penis pre-stimulated with phenylephrine 10.sup.6 M.

(8) FIG. 8 is a diagram illustrating the relaxing effect of the lyophilized cytochrome c and nitric oxide complex (Lipoxide) on cavernous bodies taken from a rat penis pre-stimulated with phenylephrine 10.sup.6 M.

(9) FIG. 9 is a diagram illustrating the comparative effect of liposomes and liposomal cytochrome c and nitric oxide complexes (Lipoxide) on cavernous bodies taken from a rat penis pre-stimulated with phenylephrine 10.sup.6 M.

(10) FIG. 10 is a diagram illustrating the original record of the experiment to study the effects of the liposomal cytochrome c and nitric oxide complex with the addition of S-nitrosoglutathione (Lipoxide GSNO) on strips of cavernous bodies taken from a human penis pre-stimulated with phenylephrine 10.sup.6 M.

DETAILED DESCRIPTION

(11) The invention will now be explained in greater detail with the reference to embodiments of the method of obtaining a pharmacologically active liposomal cytochrome c and nitric oxide complex and experimental data gathered as part of the study of target products and with the reference to the accompanying drawings.

(12) The examples and figurative materials are in no way intended to limit the embodiments of the invention but to explain the essence of the invention and illustrate the possibility of achieving the claimed result.

(13) The method according to the invention is implemented as follows.

(14) The pharmacologically active liposomal cytochrome c and nitric oxide complex is obtained by S-nitrosylation of nitric oxide (NO) and nitroso compounds (nitrosothiols) of the protein cytochrome c, incorporated in the liposomal product Lipochrome, Lyophilizate for Preparation of Emulsion for Injection, with purified gas. For S-nitrosylation, a semi-finished product (a half product) of the medicinal product Lipochrome, Lyophilizate for Preparation of Emulsion for Injection is used. It is an emulsion obtained by high-pressure homogenization followed by lyophilic drying. The emulsion is filtered successively through hydrophilic membranes with a pore diameter of 0.45 m and 0.2 m and then, at a room temperature, nitric oxide gas is passed through the emulsion pre-purified through aqueous sodium hydroxide solution 5 M to remove salt-forming admixtures of nitric oxide. Nitric oxide is supplied to the emulsion using an inert gashigh-purity argon (at least 99.995%). S-nitrosylation with nitric oxide is carried out until the emulsion changes its color from light brown to a stable bright pink. In this case, an absorbance spectrophotometer will show maxima at the wavelength of 528 nm and 560 nm. Such change in physical and chemical properties of the emulsion of the semi-finished product (half product) of the medicinal product Lipochrome, Lyophilizate for Preparation of Emulsion for Injection is associated with the addition of nitric oxide (NO) to the cytochrome c hemocomplex and its conversion into the reconstituted form.

(15) S-nitrosothiols (Examples 3-6): S-nitroso-N-acetylpenicillamine (SNAP) (Examples 7-10), S-nitrosoglutathione (GS-NO) (Examples 1-6), and S-nitrosocysteine (Cys-NO) (Examples 11-14) are added to a semi-finished product (a half product) before and after the treatment with nitric oxide (NO) to obtain concentrations in the range of 0.01 to 0.1 M in the emulsion.

(16) The emulsion is then re-filtered through hydrophilic membranes with a pore diameter of 0.45 m and 0.2 m, dosed in vials and is subject to lyophilic drying.

(17) The following examples illustrate methods of obtaining a pharmacologically active liposomal cytochrome and nitric oxide (NO) complex (cyt c3+-NO) according to the invention and obtaining a stable form of the said complex.

EXAMPLE 1

(18) An emulsion of a half-product of the medicinal product Lipochrome, Lyophilizate for Preparation of Emulsion for Injection is bubbled (purged) for 30 minutes with argon, an inert gas, pre-filtered through a hydrophobic filter with a pore diameter of 0.1 m to remove atmospheric air. After bubbling of the emulsion, an argon supply line is switched to supply nitric oxide (NO) from a solution tank where metallic copper reacts with 30% nitric acid solution with the release of gaseous nitric oxide (NO). After the reaction vessel, argon, a carrier gas, passes through a vessel with a solution of 5 M sodium hydroxide to remove salt-forming admixtures of nitric oxide (NO) and enters a vessel with the emulsion. Treatment with nitric oxide (NO) is performed until complete reconstitution of liposomal cytochrome c and formation of the liposomal cytochrome c and nitric oxide complex (cyt c3+-NO). A change in the color of the emulsion from light brown to bright pink and spectrophotometric maxima at the wavelength of 528 and 560 nm suggest that the reaction is complete. Next, the emulsion is filtered through a membrane filter with a pore diameter of 0.2 m, dosed in 3 ml vials, exposed to intense freezing at a temperature of minus 35 C. and is then subject to lyophilic drying in a lyophilizer (e.g. Martin Christ 2-6D, Germany).

EXAMPLE 2

(19) The finished medicinal product Lipochrome, Lyophilizate for Preparation of Emulsion for Injection in vials is dissolved in water to obtain an emulsion with cytochrome c at a concentration of 0.675 mg/ml. Contents of vials are combined in one vessel to obtain 100 ml of the emulsion and are bubbled (purged) for 30 minutes with argon, an inert gas, pre-filtered through a hydrophobic filter with a pore diameter of 0.1 m to remove atmospheric air. After bubbling of the emulsion, an argon supply line is switched to supply nitric oxide (NO) from a solution tank where metallic copper reacts with 30% nitric acid solution with the release of gaseous nitric oxide (NO). After the reaction vessel, argon, a carrier gas, passes through a vessel with a solution of 5 M sodium hydroxide to remove salt-forming admixtures of nitric oxide (NO) and enters a vessel with the emulsion. Treatment with nitric oxide (NO) is performed until complete reconstitution of liposomal cytochrome c and formation of the liposomal cytochrome c and nitric oxide complex (cyt c3+-NO). A change in the color of the emulsion from light brown to bright pink and spectrophotometric maxima at the wavelength of 528 and 560 nm suggest that the reaction is complete. Next, the emulsion is filtered through a membrane filter with a pore diameter of 0.2 m, dosed in 3 ml vials, exposed to intense freezing at a temperature of minus 35 C. and is then again subject to lyophilic drying in a lyophilizer (e.g. Martin Christ 2-6D, Germany).

EXAMPLE 3

(20) The nitroso compound S-nitrosoglutathione (GS-NO) is added to 100 ml of the emulsion of the half-product of the medicinal product Lipochrome, Lyophilizate for Preparation of Emulsion for Injection to obtain a concentration of 0.01 M and is bubbled (purged) for 30 minutes with argon, an inert gas, pre-filtered through a hydrophobic filter with a pore diameter of 0.1 m to remove atmospheric air. After bubbling of the emulsion, an argon supply line is switched to supply nitric oxide (NO) from a solution tank where metallic copper reacts with 30% nitric acid solution with the release of gaseous nitric oxide (NO). After the reaction vessel, argon, a carrier gas, passes through a vessel with a solution of 5 M sodium hydroxide to remove salt-forming admixtures of nitric oxide (NO) and enters a vessel with the emulsion. Treatment with nitric oxide (NO) is performed until complete reconstitution of liposomal cytochrome c and formation of the liposomal cytochrome c and nitric oxide complex (cyt c3+-NO). A change in the color of the emulsion from light brown to bright pink and spectrophotometric maxima at the wavelength of 528 and 560 nm suggest that the reaction is complete. Next, the emulsion is filtered through a membrane filter with a pore diameter of 0.2 m, dosed in 3 ml vials, exposed to intense freezing at a temperature of minus 35 C. and is then subject to lyophilic drying in a lyophilizer (e.g. Martin Christ 2-6D, Germany).

EXAMPLE 4

(21) The nitroso compound S-nitrosoglutathione (GS-NO) is added to 100 ml of the emulsion of the half-product of the medicinal product Lipochrome, Lyophilizate for Preparation of Emulsion for Injection to obtain a concentration of 0.1 M. Then follow Example 3 for nitrosylation with nitric oxide (NO) and obtaining a pharmacologically active liposomal agent (target product).

EXAMPLE 5

(22) 100 ml of the emulsion of the half-product of the medicinal product Lipochrome, Lyophilizate for Preparation of Emulsion for Injection is bubbled (purged) for 30 minutes with argon, an inert gas, pre-filtered through a hydrophobic filter with a pore diameter of 0.1 m to remove atmospheric air. After bubbling of the emulsion, an argon supply line is switched to supply nitric oxide (NO) from a solution tank where metallic copper reacts with 30% nitric acid solution with the release of gaseous nitric oxide (NO). After the reaction vessel, argon, a carrier gas, passes through a vessel with a solution of 5 M sodium hydroxide to remove salt-forming admixtures of nitric oxide (NO) and enters a vessel with the emulsion. Treatment with nitric oxide (NO) is performed until complete reconstitution of liposomal cytochrome c and formation of the liposomal cytochrome c and nitric oxide complex (cyt c3+-NO). A change in the color of the emulsion from light brown to bright pink and spectrophotometric maxima at the wavelength of 528 and 560 nm suggest that the reaction is complete.

(23) Next, the nitroso compound S-nitrosoglutathione (GS-NO) is added to a concentration of 0.01 M to the emulsion and mixed. Then the emulsion is filtered through a membrane filter with a pore diameter of 0.2 m, dosed in 3 ml vials, exposed to intense freezing at a temperature of minus 35 C. and is then subject to lyophilic drying in a lyophilizer (e.g. Martin Christ 2-6D, Germany).

EXAMPLE 6

(24) A pharmacologically active liposomal agent (the target product) is obtained in accordance with Example 5 with the only modification: the nitroso compound S-nitrosoglutathione (GS-NO) is added to a concentration of 0.1 M.

(25) In Examples 7-10, the technological processes of the claimed method were performed with S-nitrosocysteine (Cys-NO) in accordance with Examples 3-6.

(26) In examples 11-14, the technological processes of the claimed method were performed with S-nitroso-N-acetylpenicillamine (SNAP) in accordance with Examples 3-6.

(27) In accordance with the object of the invention, the quality of the target productthe liposomal agent containing nitric oxide (NO) was evaluated for pharmaceutical activity in preclinical in vitro studies.

(28) The pharmacological action of the target products obtained by the claimed methods (Examples 1-6) was studied using the following experimental models:

(29) 1) contractile activity of rat thoracic aorta segments;

(30) 2) contractile activity of strips of cavernous bodies taken from a rat penis.

(31) The experimental results show that, starting with a concentration of 10.sup.8 mol/l, target products (Examples 1, 2) are able to induce dose-dependent relaxation of intact segments of aorta in rats, though the marked effect was induced by a sample at a concentration of 310.sup.8 mol/l.

(32) When exposed to the liposomal cytochrome c and nitric oxide complex (cyt c3+-NO) at a concentration of 10.sup.6 mol/l, the maximum amplitude of vasodilation was (87.82.2)% (n=11). The median effective concentration (EC50) of a sample, expressed as the logarithm of concentration (Log M), was (6.80.01), (n=11) (FIG. 1). The study of a sample on deendothelized ring aorta segments shows that the contractile activity of deendothelized aorta specimens did not change compared with intact vessels: the maximum amplitude of vasodilation was (91.42.7)%, (n=9), which did not significantly differ from that of control (p>0.05). The analysis of the results shows that the removal of endothelium did not significantly affect the sensitivity of effector elements of vessels exposed to the liposomal cytochrome c and nitric oxide complex (cyt c3+-NO): EC50=(6.80.03) (n=9; P>0.05) (FIG. 1).

(33) Thus, the experimental results suggest that the removal of an endothelial layer of vessels does not affect the dilatory action of the ex-tempore complex (cyt c3+-NO). This allows us to claim that this sample is characterized by endothelium-independent action.

(34) To identify a possible contribution of liposomes to the vasodilatory effect of the ex-tempore liposomal cytochrome c and nitric oxide complex (cyt c3+-NO), liposomes were separately tested for their effect on the contractile activity of aorta smooth muscles (SM). The results show that liposomes are able to exhibit a weak dose-dependent vasodilator activity starting with a concentration of 10.sup.8 mol/l with a maximum effect (12.32.2%) seen at a concentration (10.sup.6 mol/l), which significantly differs from the maximum amplitude of the dilatory response achieved with the ex-tempore liposomal cytochrome c and nitric oxide complex (cyt c3+-NO) (n=8, P0.001) (FIG. 2). As shown on FIG. 2, the effect of low concentrations of liposomes does not differ from the overall efficacy of the complex. A significant difference in the efficacy was seen at a concentration of 10.sup.7 mol/l. At the same time, EC50 (7.50.07) was significantly different from that of the ex-tempore liposomal cytochrome c and nitric oxide complex (cyt c3+-NO) (n=9; P0.05) indicating an increased sensitivity of vascular tissue specimens to the action of liposomes.

(35) Thus, the study results show that liposomes make a significant contribution to the dilatory effect of low concentrations of the ex-tempore liposomal cytochrome c and nitric oxide complex (cyt c3+-NO). It is fair to assume that efficacy of the ex-tempore liposomal cytochrome c and nitric oxide complex (cyt c3+-NO) at low concentrations (up to 10.sup.7 mol/l) is due to the effect of liposomes. At the same time, liposomes exhibit an insignificant effect on the maximum vasodilation caused by the action of the ex-tempore liposomal cytochrome c and nitric oxide complex (cyt c3+-NO).

(36) The testing of liposomes using deendothelized ring aortic segments shows that efficacy of liposomes did not change compared with their effect on intact vessels: the maximum dilatory effect did not significantly differ from the response of intact vessels: (12.32.1)%, (n=6, p>0.05). The analysis of the results shows that the removal of the endothelium did not significantly affect sensitivity of effector elements of vessels to the action of the complex: EC50=(7.50.2) (n=6; P>0.05) (FIG. 3).

(37) A study was conducted to compare two samples of the liposomal cytochrome c and nitric oxide complex II (cyt c3+-NO) (Examples 1, 2): a sample prepared several hours before the experiment (ex tempore) and a lyophilized sample, a working solution of which was prepared 5 minutes before testing because previous studies suggest that a sample of the ex-tempore liposomal cytochrome c and nitric oxide (cyt c3+-NO) was characterized by physical and chemical parameters of an unstable compound capable of losing its vasodilatory properties within a rather short period of time under the influence of oxidation.

(38) The study results show that the lyophilized liposomal cytochrome c and nitric oxide complex (cyt c3+-NO) also induced an effective dose-dependent dilation of intact specimens of rat aorta smooth muscle (SM) with a maximum effect (89.93.8)% seen at a concentration of (10.sup.6 mol/l), which did not significantly differ from the maximum relaxation seen in response to a sample of the ex-tempore liposomal cytochrome c and nitric oxide (cyt c3+-NO) (n=7, P>0.05) (FIG. 4). However, the analysis of EC50 values revealed significant differences in the action of the lyophilized liposomal cytochrome c and nitric oxide complex (cyt c3+-NO): the latter had EC50 of (7.10.03) (n=7; P0.05), which was significantly different from that of the ex-tempore liposomal cytochrome c and nitric oxide (cyt c3+-NO) (FIG. 4). The left shift of the dose-effect curve that is characterized by EC50 is an indicator of increased sensitivity of vascular tissue specimens to the action of the lyophilized sample compared to the ex-tempore sample.

(39) Thus, according to the study results, the lyophilized liposomal cytochrome c and nitric oxide complex (cyt c3+-NO) (Examples 1, 2) has superior efficacy compared with that of the ex-tempore liposomal cytochrome c and nitric oxide complex (cyt c3+-NO), since it exhibits more pronounced efficacy at low concentrations, though the samples do not differ in terms of their maximum effect. It is fair to assume that the higher efficacy of the lyophilized complex is due to the fact that its working solution was prepared immediately before testing and, as such, was less exposed to oxidation processes.

(40) The liposomal cytochrome c and nitric oxide complex (cyt c3+-NO) was found to be an unstable substance capable of oxidizing quickly and losing its dilatory activity (Table 1). However, the lyophilized liposomal cytochrome c and nitric oxide complex (cyt c3+-NO) exhibited greater efficacy than the ex-tempore liposomal cytochrome c and nitric oxide complex (cyt c3+-NO). Therefore, the lyophilized liposomal cytochrome c and nitric oxide (cyt c3+-NO) were modified by synthesized glutathione to get a complex with stable properties (Examples 3-6). Studies have been conducted to determine its efficacy compared to the lyophilized liposomal cytochrome c and nitric oxide complex (cyt c3+-NO). The preliminary results suggest that the complex of liposomal cytochrome and nitric oxide with nitrosoglutathione (cyt c3+-NOGS-NO) is able to induce a dose-dependent dilatory response in vascular tissue specimens apparently seen at lower concentrations than vasodilation induced by unmodified lyophilized liposomal cytochrome c and nitric oxide complex (cyt c3+-NO) (FIG. 5). At the same time, the maximum vasodilation response did not change significantly: (89.35.7)%, (n=3, P>0.05).

(41) An additional study of the target products of the complex (Examples 1-6) used cavernous bodies taken from rats, i.e. syncytia of smooth muscle cells and endothelium located on the stroma of the connective tissue, as an object of research.

(42) After rats were euthanatized in accordance with the recommendations of the European Convention for the Protection of Animals, the penis was quickly dissected and immediately placed in a 20 ml preparative bath with the Krebs solution. Cavernous bodies were released from the connective tissue under microscopic control. Next, a few longitudinal strips of SM syncytia were taken from cavernous bodies and placed in the flow chamber between hooks of the strain gauge and the device for the preliminary (0.3-0.4 g) stretching. After preliminary treatment (for 30-40 minutes) and pumping with a solution with an increased (60 mM) KCl content, the strips were subject to pre-contraction with the phenylephrine solution (10.sup.6 M) and used in experiments with the test substances.

(43) Experimental data was recorded using the analog-digital converter LabTrax-4/16 and the software application LabScribe2. Given that stimulation with phenylephrine resulted in a significant autorhythmic activity, an adequate evaluation of the results was almost impossible. As a result of thorough digital filtration and the use of data-smoothing methods (using a moving average or the Savitzky-Golay filter based on polynomial approximation of peaks), the data became quite appropriate for further processing. The final data was downloaded to the OriginLab software for processing and graphic design.

(44) The results so obtained are presented in Table 3 below and on FIG. 6-10.

(45) TABLE-US-00003 TABLE 3 Relative relaxation of strips of cavernous bodies taken from a rat penis pre-stimulated with phenylephrine 10.sup.6 M induced by study substances. Liposomal cytochrome Liposomal cytochrome c (cyt c (cyt c3 + NO), emulsion Liposomes (control) c3 + NO), lyophilized Mean- Mean- Mean- Concentration, Relative square Relative square Relative square log (mol/l) contractility, % deviation contractility, % deviation contractility, % deviation PhE-6 100 100 100 Lipo-9 84.23 7.59 87.85 8.28 88.14 3.10 Lipo-8 63.73 12.14 78.77 12.75 73.52 6.45 Lipo-7 49.18 21.47 63.49 22.79 68.00 4.13 Lipo-6 34.41 19.68 48.91 31.97 36.04 10.94

(46) In recent studies, strips of cavernous bodies taken from a human penis were used as an object of research. Original curves (see, for example, FIG. 10) allow us to observe how the liposomal cytochrome c and nitric oxide complex with the addition of S-nitrosoglutathione (Lipoxide GSNO) is able to induce a pronounced relaxation of smooth muscles. At the same time, it should be noted that the relaxation begins with very low concentrations.

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