RU2393849C2 - Medical emulsion of perfluororganic compounds, method of preparation and method of application - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: declared invention refers to chemical-pharmaceutical industry, and concerns an emulsion of perfluororganic compounds (PFOC) for medicine and biology, containing mixed perfluorocarbons (PFC) with a main ingredient being perfluorodecahydronaphthalene (PFD) and mixed perfluorochemical tertiary amines (PFTA) with a main ingredient being perfluoro-N-(4-methylcyclohexyl)-piperidine (PFMCP), having smaller excretion rates than PFD, a stabilising agent and a physiologically acceptable water-salt solution with energy metabolism substrata, differing that all the ingredients of PFOS have critical hexane solution temperatures (T_crH), differing no more than by 2-4°C, and the stabilising agent represents mixed block copolymers from the group of block copolymers polyoxyethylene-polyoxypropylene with an average weight content of polyoxypropylene 20%. The invention also concerns a method for making said emulsion, and a method of treating with applying the declared emulsion.

EFFECT: prepared emulsions show high stability and are non-toxic.

21 cl, 5 tbl, 17 ex, 2 dwg

Description

The claimed group of inventions relates to the field of biomedicine, transfusiology, pharmacology, biophysics, in particular to medicines based on an emulsion of perfluororganic compounds (PFOS), intended for the treatment of systemic and local disorders of the blood flow, hypoxic and ischemic conditions, improving the mass transfer of gases and metabolites between blood and tissues, maintaining the function of isolated organs and tissues, reducing the phenomena of inflammation, conducting infusion-transfusion therapy in shock and blood loss.

LIST OF CONVENTIONS, SYMBOLS, UNITS AND TERMS USED IN THIS APPLICATION

US Saturated affinity separation CA Surface-active substance P-168, P-268, Block copolymer of polyoxyethylene and polyoxypropylene P1F-268 Proxanol 168, Proxanol 268, Pluronic F68 POP Polyoxypropylene Proxanol Block POE Proxyanol Polyoxyethylene Block PFD Perfluorodecalin PFDE Perfluorodihexyl ether PFMTSP Perfluoromethylcyclohexylpiperidine PFOB Perfluorooctyl bromide PFOS Organofluorine compounds PFTA Perfluorinated Tertiary Amines PFBA Perfluorotributylamine PFTPA Perfluorotripropylamine PFU Perfluorocarbons PFE Perfluorinated ethers RES Reticulo-endothelial system CA Stabilizing agent T _crg Critical Dissolution Temperature in Hexane FL Phospholipids ⌀ Average particle diameter I _R Reactogenicity Index

PFOS used in biology and medicine are completely fluorinated organic compounds of various classes, including perfluorocarbons (PFCs) - compounds containing only fluorine and carbon atoms, perfluorinated tertiary amines (PFTA), in which, along with fluorine and carbon atoms, there is a nitrogen atom, and also perfluorinated ethers, in the structure of which there are oxygen atoms. In recent years, bromine-containing PFOS have also been used. Liquid PFOS is transparent, colorless, odorless, about 2 times heavier than water. The high bond strength C-F (485.6 kJ / mol) ensures the chemical stability of PFOS and weak intermolecular interaction forces (1, 2). The latter property provides an increased ability of PFOS to dissolve various gases.

PFOS practically does not dissolve in H ₂ O, but some PFOS are slightly soluble in lipids - lipophilic PFOS. All types of PFOS are not subject to metabolic transformations in humans and animals. The issue of stability and the removal of a bromine atom and, therefore, the possibility of decay of a molecule left without bromine has not been discussed in the literature. Lipophilic PFOS, and among them the most lipophilic is perfluorooctyl bromide (PFOB), due to physical interaction with biological membranes and hydrophobic sections of protein molecules, can have a pronounced effect on the metabolism and many functions of cells and body tissues in general (3, 4, 5, 6). According to the data of L. Clark (7), later confirmed by A.N. Sklifas et al. (8), the inclusion of other lipophilic PFOS in the emulsion composition along with lipophilic PFD can lead to the death of animals several months after the intravenous administration of such emulsions.

It is known that to eliminate this fatal effect, it is proposed to add a lipophobic organo perfluorine compound, for example, perfluoro-N- (4-methicyclohexyl) piperidine to lipophilic organofluorine compounds (8, 9). However, this raises the problem of selecting an emulsifier, since lipophilic organo perfluoro compounds are stabilized by phospholipids, and lipophobic organo perfluoro compounds are stabilized by block copolymers of oxyethylene and oxypropylene (Pluronic F-68 according to the western classification; proxanol 268 according to the Russian nomenclature). In addition, the use of a mixture of substances that differ sharply in their physicochemical properties (lipophilic and lipophobic) significantly reduces the stability of the emulsion during storage (freezing, thawing, or long-term storage in an unfrozen state) and when the emulsion enters the bloodstream. Calculations and model experiments (10) give reason to believe that this is due to the clustering of an insufficiently homogeneous mixture of substances inside the particles of the emulsion and the constant turbulent motion of the fluorocarbon phase near the adsorption layer of the surface-active component, the stabilizing agent.

Introduced intravenously in the form of emulsions PFOS quite quickly removed from the bloodstream. The bulk of PFOS is excreted through the lungs. Therefore, those PFOS that have a relatively high vapor pressure (they, as a rule, are more lipophilic) can cause emphysema up to the rupture of individual alveoli and impaired gas exchange and blood flow in the lung tissue. Up to 30% of PFOS emulsions introduced into the body are captured by the cells of the reticuloendothelial system (RES) - mainly in the liver, spleen, bone marrow, lymph nodes, and are also partially dissolved in adipose tissue and membrane phospholipids of almost all cells. The PFOS accumulated in this way is retained in organs and tissues for a period of several days (PFOB, PFD) to several years (PFBA), depending on the physicochemical properties of PFOS and the dose of the emulsion introduced. The rate of PFOS removal from organs depends on a number of physicochemical parameters interconnected: structure, molecular weight (MM), boiling point, vapor elasticity, and mainly on a parameter defined as the critical temperature for the dissolution of PFOS in hexane (T _crH ). T _crH is the temperature at which equal volumes of the test compound and hexane are mixed with each other. T _{crH is} considered as a measure of the relative solubility of PFOS in lipids, which characterizes both the degree of their dissolution in the lipids of the membranes, the speed of their passage through the membranes, and the degree of affinity for hydrophobic portions of protein molecules.

The lower T _crH , the lower MM and the higher the vapor _pressure , the better this PFOS is soluble in lipids, diffuses faster, and the more easily and quickly it is excreted from the body. A good correlation was observed between the indicated parameters and the half-life (t _1/2 ) of PFOS from RES cells. Currently, the attention of researchers involved in the development and study of PFOS emulsions is focused on a relatively small number of compounds (Table 1).

Table 1 The values of the critical temperature of dissolution in hexane (T _crg ), vapor _pressure (P) and half-life from organs (t _1/2 ) for various PFOS [1] Type of PFOS mm T _crG , ° C P, mmHg (37 °) t _1/2 , day F-octyl bromide 493 -twenty 10.5 four F-Bicyclo [4.3.0] nonane 412 13 33 four F-Decalin 462 22 12 7 F-decahydroacenaphthene 524 24 2 7 F-1-propyl-2 methylpiperidine 483 35 19 24 F-tripropylamine 521 43 17 65 F-N- (4-methylcyclohexylpiperidine) 595 40 one 90 F-tributylamine 671 61 one 900 F-Digexyl ether 652 59 2 500

In the manufacture of emulsions for intravascular administration, most specialists prefer rapidly excreting lipophilic PFOS (2, 10, 11, 12, 13, 14), which have a relatively small T _{crH value} , including high lipophilic PFOB. However, the composition of "Fluosol DA", produced in 70-80 years. Green Cross Corporation (Japan), along with rapidly excreted PFD, also included relatively slowly excreted PFPA (12). The Oxygent developed by Alliance Therapeutic (USA) includes high lipophilic PFOB and PFDB (13, 14). The commercial pharmacopoeial drug “Perftoran”, developed in the USSR and produced in the pharmacy network by the company NPF “Perftoran”, along with rapidly excreted PFD contains PFMTSP relatively long-delayed in the cells of RES (6).

It should be noted that the PFOS emulsions themselves, despite the chemical and metabolic inertness of their constituent components, also have pronounced biological activity, which is determined not only by the properties of the components themselves, but largely depends on the phase boundary formed by the surface-active substance (SA) , the presence of SA or its dispersion in the aqueous phase, on the stability of the adsorption layer, as well as on the degree of dispersion of the emulsion, which can play the role of a huge sorption and exchange catalysis nical active surface. The emulsion introduced into the body actually introduces a huge hydrophobic phase into the bloodstream and into membranes, which can act as a medium for various reactions between compounds having increased solubility in the hydrophobic phase. In other words, the PFOS emulsion can stimulate microcatalysis phenomena occurring in the hydrophobic phase or at the phase boundary.

Since liquid PFOS does not dissolve in water and polar compounds that play a crucial role in the homeostasis of biological objects do not dissolve in them, so far PFOS can be introduced into the bloodstream only in the form of a finely dispersed emulsion. The ability of PFOS and PFOS emulsions to participate in gas exchange is determined by the physical dissolution of gases, and according to Henry's law, the amount of dissolved gas is directly proportional to its partial pressure. The amount of gas dissolved in the PFOS emulsion depends on the volumetric content of the fluorocarbon phase and does not depend on the particle size. Thus, the amount of experimentally determined gas dissolved in PFOS emulsions is almost equal to the calculated values obtained by adding up the contents of this gas in each phase separately (the amount of dissolved oxygen in the aqueous phase plus the amount of dissolved gas in PFOS) for a given gas partial pressure. Thus, the amount of any gas contained in the PFOS emulsion can be calculated in accordance with the physical laws of their solubility, based on the partial pressure of the gas and the volume ratio of the PFOS / N ₂ O fractions. Due to the submicron size of the PFOS emulsions, when introduced into the bloodstream, more than an order of magnitude increases the surface area involved in gas exchange. Together with the acceleration of the processes of oxygen saturation and release, which is almost ten times higher than the hemoglobin in erythrocytes, this contributes to a significant acceleration of the mass transfer of oxygen and carbon dioxide, even if the dose of an emulsion containing only 10 vol.% PFOS introduced into the bloodstream does not exceed 1 -5% of the volume of circulating blood.

The therapeutic efficacy of PFOS emulsions described in experimental and clinical studies performed with Fluosol-DA (12) and Perftoran (6, 15) is limited by their possible side effects. The following factors limit the therapeutic use of PFOS emulsions as drugs. First, back in 1971, Fujita (12) found that when the particle size of the emulsion is more than 0.1 μm, their toxicity increases sharply, which is probably due to thromboembolic complications. With an increase in the proportion of large particles (with an average diameter exceeding 0.4 μm) from 3% to 10%, the LD ₅₀ for PFOS emulsions decreases by more than 2 times. The significance of particle size for the tolerance of emulsions was shown by S.I. Vorobiev (16), who established that an increase in particle size, even if their fraction is only 1-2%, leads to reactogenicity, which manifests itself in the development of anaphylactoid reactions after the introduction of the first drops of emulsion into the bloodstream. Manifestations of anaphylactoid reactions can be different in severity - mild, moderate and severe: from redness of the skin and mild allergic manifestations up to respiratory arrest.

According to the results of the study of fat emulsions and Fluosol DA, most researchers believe that reactogenicity is determined by the nature of the stabilizing agent - CA, used to disperse PFOS and form an adsorption layer around the particles. It is believed that the non-ionic CA block copolymer of polyoxyethylene (POE) and polyoxypropylene (POP) Pluronic F 68, formally analogous to proxanol 268, is responsible for the reactogenicity, and that its replacement with natural phospholipids can completely eliminate the problems of reactogenicity. However, it is known that fat emulsions stabilized by natural phospholipids are mostly reactogenic. Therefore, the reactogenicity of PFOS emulsions cannot be eliminated only through the use of phospholipids as an emulsifier and stabilizer of PFOS particles. According to the patent (17), the reactogenicity of PFOS emulsions, in addition to the degree of dispersion, is determined, first of all, by the surface properties of emulsified particles, i.e., by the state of the emulsifier layer stabilizing the particles. The leading parameters in this case are the bond strength of the CA with the oil fluorocarbon "core" of the emulsion particles, the nature of the location of the CA molecules on the surface, the density of their packing, the stability of adsorption properties with respect to proteins and other biologically active molecules in the bloodstream. To obtain the desired correspondence, the said patent empirically proposes to select the composition of the oil “core”, the composition of PFOS and the CA interacting with it. However, the empirical selection and the results of the combination of PFOS and SA in any case leave an indefinite range of variations in the stability of PFOS emulsions due to the variability of chemical technologies for producing PFOS and SA, as well as seemingly insignificant deviations in the dispersion technology.

The main causes of variations in the composition of SA are the very nature of polymer and phospholipid CAs. An important destabilizing factor is also the presence of constant turbulent flows inside the PFOS oil phase that arise when mixing dissimilar PSFs included in the oil “core”: from a mixture of lipophilic and lipophobic PFOS that differ significantly in T _crG .

In order to increase stability and reduce the reactogenicity of PFOS emulsions according to RF patent No. 2206319 (18), which was selected as a prototype, it was proposed to increase the uniformity of the “oil” core by using PFOS additives that are close to the main components of PFD and PFMCP in structure and somewhat reduce differences between PFD and PFMTSP in terms of T _crG . However, the range of differences in the values of T _KRG between PFOS used in the composition according to the patent of the Russian Federation No. 2206319 remains more than 10 ° C - quite large. An experimental analysis performed using a thermal imaging highly sensitive matrix detector showed that with such a gap in terms of T _crg in the mixture of PFOS used in the emulsion according to RF patent No. 2206319, very intense turbulent movements in the fluorocarbon phase are observed.

The objective of the claimed group of inventions related by a single inventive concept is to create a stable PFOS emulsion primarily by reducing the differences in T _{crH values} inside fluorocarbon self-assembly due to adsorption separation, in accordance with the type of PFOS used in the core of the emulsion particles and changing the composition of the stabilizing agent. The solution of this problem is achieved using the original method of obtaining emulsion.

The essence of the invention in terms of the emulsion of perfluororganic compounds (PFOS) for biological and medical purposes is that it contains a mixture of perfluorocarbons (PFCs), the main component of which is perfluorodecalin (PFD) and a mixture of perfluorinated tertiary amines (PFTA), the main component of which is perfluorine -N- (4-methylcyclohexyl) piperidine (PFMTSP), having lower than PFD, the rate of excretion from the body, a stabilizing agent and a physiologically acceptable water-salt solution with substrates of energy metabolism, p Moreover, all PFOS components have critical dissolution temperatures in hexane (T _crH ), which differ by no more than 2-4 ° C, and the stabilizing agent is a mixture of block copolymers from the group of block copolymers of polyoxyethylene-polyoxypropylene with an average mass content of polyoxypropylene 20 %, while the PFD mixture contains only fully fluorinated PFC impurities obtained by the synthesis of PFD, and the PFMTSP mixture contains only fully fluorinated PFTA impurities, as well as at least one component from the group: rtripropilamina (PFTPA) only with fully fluorinated impurities PPTA mixture of perfluorotributylamine (PFTBA) with only fully fluorinated impurities PPTA mixture perftordetsilovogo ester (PFDE) only with fully fluorinated impurities perfluorinated ethers (PPE).

In special cases, the implementation of the emulsion as perfluorocarbons contains a mixture of perfluorinated co-products of perfluorodecalin having an average half-life of at least 12 days and a range of T _crH in the range of 22-29 ° C, purified from hydrogen-containing and unsaturated impurities, it contains perfluorinated tertiary amines a mixture of all perfluorotripropylamine co-products having an average elimination half-life of at least 60 days and a range of T _crH in the range of 41-46 ° C, purified from hydrogen-containing and unsaturated at Mesi, or as perforated tertiary amines, it contains a mixture of co-products of perfluoro-N- (4-methylcyclohexyl) piperidine having an average half-life of at least 80 days and a range of T _crH in the range of 32-40 ° C, purified from hydrogen-containing and unsaturated impurities, as a slowly excreted PFOS, it contains a mixture of co-products of perfluorodecyl ether (PFDE) with an average half-life of at least 500 days and a _Tg range in the range of 49-53 ° С, purified from hydrogen-containing and unsaturated impurities, in as perfluorinated tertiary amines, it may contain a mixture of perfluorotributylamine co-products having an average half-life of at least 900 days, and a _Tg range in the range of 56-60 ° C, purified from hydrogen-containing and unsaturated impurities, the ratio of rapidly excreted and slowly excreted in it PFOS ranges from 5: 1 to 1: 5; as a stabilizing agent, it contains a mixture of copolymers of polyoxyethylene (POE) -polyoxypropylene (POP) selected from the molecules of saturation affinity separation (HAC) with a mass of 5–13 thousand. Yes, as a stabilizing agent, it contains a mixture of POE and POP copolymers selected by the NAS method with a POE: POP ratio in the range from 78:22 to 82:18, the content of the stabilizing agent in it is 1-5%, to provide a viscosity of 2-5 cP, and a physiologically acceptable aqueous salt solution includes NaCl, KCl, MgCl ₂ , NaHCO ₃ , NaH ₂ PO ₄ , Na ₂ HPO ₄ D-glucose to ensure isosmosis in the final dosage form, while maintaining the ratio Na and K ions at a level of 10: 1-30: 1 and a pH in the range of 6.9-8.0, the water-salt portion is ready the final dosage form contains 110-150 mM NaCl, 4-5 mM KCl, 1-3 mM MgCl ₂ 6-20 mM NaHCO _3, 2.1 mM NaH ₂ PO _4, 2.1 mM Na ₂ HPO ₄ and 10-15 mm D-glucose, and for use as an anti-ischemic protector, the water-salt part of the preparation contains 4-15 mm KCl, salts of tri-, di- and monocarboxylic acids, including 1-7 mm sodium citrate, 1-7 mm sodium isocitrate , 1-7 mm sodium succinate, 1-7 mm sodium sodium ketoglutarate, 1-7 mm sodium pyruvate, 1-7 mm sodium sodium hydroxybutyrate, 1-7 mm sodium glutamate, 1-7 mm sodium aspartate.

The invention in terms of a method for preparing an emulsion of PFOS (in accordance with the above set of characteristics of an emulsion) for biological and medical purposes includes mixing and dispersing a mixture of liquid PFOS with an aqueous solution of a stabilizing agent, followed by grinding in a high pressure homogenizer to a submicron particle size of the emulsion, and:

- a mixture of PFOS having differences in T _{crg of} not more than 2-3 ° C, is poured into a container containing a solution of a mixture of CA in a ratio of 10-30 volume parts of PFOS to 90-70 volumes of a solution of a mixture of CA,

- the obtained heterogeneous mixture is subjected to the NAS procedure at a temperature of 50-60 ° C, including obtaining a coarse dispersion of PFOS in a CA solution by stirring on a high-speed mixer at a rotor speed of 1000-3000 rpm, the obtained coarse dispersion is extruded in a homogenizer under pressure of 100-300 kg / cm ² in an atmosphere that does not contain oxygen, and the coarse dispersion of PFOS is separated from the aqueous solution of CA, the resulting precipitate of coarse dispersion is mixed with a fresh portion of the solution of CA from such a calculation that for PFOS was 10-30%,

- while the described operation US is repeated at least 2 times,

- after which the emulsion is passed repeatedly through a homogenizer at a pressure of 300-700 kg / cm ² ,

- the resulting submicron emulsion is diluted with water-salt solution so that the final concentration of PFOS is 4-12 vol.% and the emulsion has an osmotic pressure in the range of 270-330 mOsm.

In particular cases of the method implementation, mixtures of liquid PFCs and PFTA or PFE are obtained in a ratio of 5: 1 to 1: 5, subjected to washing after mixing with pyrogen-free water-alcohol solutions and water, and mixtures of denser liquid PFOS are separated from the water-alcohol solution and water , filtered under pressure of an oxygen-free gas, poured and sealed into vials for filling and autoclaving, CA is prepared in the form of a 10-30% aqueous solution of copolymers of polyoxyethylene-polyoxypropylene - proxanol 168 and proxanol 268 taken in from a ratio of 10: 1 to 1:10, depending on the composition of the composition of the PFOS mixture, they clean the CA solution by passing through a carbon sorbent and a sterilizing filter under the pressure of an oxygen-free gas, warm the CA solution for 8-24 hours and cool to 16- 20 ° C to completely restore the transparency of the solution, the aqueous solution of a mixture of copolymers of polyoxyethylene-polyoxypropylene is heated at a temperature that is selected in the range of ± 2 ° C from the cloud point established experimentally for the obtained mixture of CA.

The essence of the invention in terms of a method for treating diseases of the circulatory system is that they introduce into the bloodstream or lymphatic duct fast-releasing organofluorine compounds, characterized in that PFOS emulsion is introduced into the bloodstream as fast-releasing perfluororganic compounds (in accordance with the above set of emulsion characteristics).

In special cases, the implementation of the method for the treatment of the phenomena of hypoxia, ischemia, edema, inflammation in shock, blood loss, thrombo-embolic lesions, functional and organic disorders of vascular patency, tissue damage is carried out by intravenous administration of PFOS emulsion in a dose of 0.5-30 ml per 1 kg body weight, to suppress secondary alteration in inflammation and wound healing, as well as to accelerate the healing of traumatic, trophic and surgical wounds, intravenous administration of PFOS emulsion is carried out in doses of 0.3 - 6 ml per kg of body weight ate and / or locally in 0.5-50 ml portions subcutaneously, intramuscularly, in depth around the damaged area or in the cavity or biological fluids surrounding the inflamed and injured tissues, in particular intrapleural, intraperitoneal, intralumbal, into the common lymphatic duct at a dose of 1- 100 ml, as well as a cleaning or application fluid for treating open wound surfaces, and to improve systemic and local blood flow, regulate systemic blood pressure and perfusion regional pressure, lighten eniya and acceleration of mass transfer of gases, substrates and metabolites carried intravascular or topical administration PFOC emulsion in tissue, including with perfusion and besperfuzionnoy protection isolated or disconnected from the flow of organs and tissues.

Thus, the reduction of the main process of destruction of PFOS emulsions caused by molecular diffusion is achieved in the claimed composition in the same way as in other patents (12, 17, 18) by introducing a component with low vapor elasticity into the fluorocarbon base (for example, PFMTSP or PFTAA) and less soluble in water, having a higher boiling point and slowing down this process. But at the same time, in contrast to the prototype, in the present invention, the “windows” of the gap between the physicochemical characteristics of various PFOS included in the core of the emulsion droplets are eliminated as much as possible, and the adsorption layer of the emulsion particles is formed from the SA mixture by self-assembly - by the method of saturation adsorption separation (US).

The PFOS emulsion for biological and medical purposes contains a mixture of rapidly eliminating perfluorocarbons (PFCs) with the main component of PFD and a mixture of perfluorinated tertiary amines (PFTA) with the main component of perfluoro-N- (4-methylcyclohexyl) piperidine (PFMCP), which has lower rates of excretion from organism, a stabilizing agent and a physiologically acceptable water-salt solution with energy exchange substrates, PFOS additionally contain all fully fluorinated PFC impurities obtained in the synthesis of PFD in a mixture of PFD in a mixture of PFMCP - all completely fluorinated impurities of PFTA, and / or additionally contains perfluorotripropylamine (PFTPA) with all its completely fluorinated PFTA impurities, and / or additionally contains perfluorotributylamine (PFTA) with all its completely fluorinated impurities PFTA, and / or additionally contains perfluorodihexyl ether (PFDE) with all its perfluorinated ethers (PFE) inherent in it upon receipt of fully fluorinated impurities and a stabilizing agent in the form of a mixture of block porimers, with the possibility zhnosti formation of selected mixtures of PFOS homogenous structure inside the emulsion droplets, due to the fact that PFOS mixture components have small differences in the critical solution temperature in hexane _(AWG T) at intervals of no more than 2-4 ° C.

The PFOS emulsion is characterized in that a mixture of all perfluorinated perfluorodecalin coproducts having an average half-life of at least 12 days and a range of T _{crH of} 22-29 ° C, purified from hydrogen-containing and unsaturated impurities, is used as rapidly excreting PFCs.

The PFOS emulsion is characterized in that as PFTA it contains a mixture of all PFPA co-products having an average elimination half-life of at least 60 days and a range of T _{crH of} 41-46 ° C, purified from hydrogen-containing and unsaturated impurities.

The PFOS emulsion is characterized in that PFTA contains a mixture of PFMCP co-products having an average elimination half-life of at least 80 days and a _Tg range of 32-40 ° C, purified from hydrogen-containing and unsaturated impurities.

The PFOS emulsion is characterized by the fact that, as a slowly excreted PFOS, it contains a mixture of PFDE co-products having an average elimination half-life of at least 500 days and a _Tg range of 49-53 ° C, purified of hydrogen-containing and unsaturated impurities.

The PFOS emulsion is characterized in that, as PFTA, it contains a mixture of perfluorotributylamine co-products (PFBA) with an average half-life of at least 900 days and a range of T _crG 56-60 ° C, purified from hydrogen-containing and unsaturated impurities.

The PFOS emulsion is characterized in that the ratio of rapidly excreted and slowly excreted PFOS is 5: 1 or 1: 5.

The PFOS emulsion is characterized in that as a stabilizing agent it contains a mixture of copolymers of polyoxyethylene (POE) -polyoxypropylene (POP) with a molecular weight of 5-13 thousand Da, formed (selected) by saturation affinity separation (NAS).

The PFOS emulsion is characterized in that it contains a mixture of POE and POP copolymers selected by the NAS method as a stabilizing agent with a POE: POP weight ratio in the range from 78:22 to 82:18.

The PFOS emulsion is characterized in that the content of the stabilizing agent in the aqueous phase is 1-5% to provide a viscosity of 2-5 cPoise.

The PFOS emulsion is characterized in that a physiologically acceptable aqueous salt solution includes NaCl, KCl, MgCl ₂ , NaHCO ₃ , NaH ₂ PO ₄ , Na ₂ HPO ₄ and D-glucose to ensure isosmosis in the final dosage form, while maintaining the ratio of Na ions and K at a level of 8: 1-30: 1 and a pH in the range of 6.9-8.0.

The PFOS emulsion is characterized in that the water-salt portion of the finished final dosage form contains 110-150 mM NaCl, 4-5 mM KCl, 1-3 mM MgCl ₂ , 6-20 mM NaHCO ₃ , 1-2 mM NaH ₂ PO ₄ , 1-2 mM Na ₂ HPO ₄ and 10-15 mM D-glucose.

The PFOS emulsion is characterized in that when used as an anti-ischemic protector, the water-salt portion contains 4-15 mm KCl, 10-20 mm D-glucose and salts of tri-, di- and monocarboxylic acids, including 1-7 mm citrate sodium, 1-7 mm sodium isocitrate, 1-7 mm sodium succinate, 1-7 mm sodium sodium ketoglutarate, 1-7 mm sodium pyruvate, 1-7 mm sodium β-hydroxybutyrate, 1-7 mm sodium glutamate, 1- 7 mM sodium aspartate.

The method of preparing the PFOS emulsion for biological and medical purposes is implemented by mixing and dispersing a mixture of liquid PFOS with an aqueous solution of a stabilizing agent, followed by grinding in a high pressure homogenizer to a submicron particle size of the emulsion, and:

- a mixture of PFOS containing components differing in T _{crg by} no more than 4 ° C is poured into a container containing a solution of a mixture of CA in a ratio of 10-30 volume parts of PFOS to 90-70 volumes of a solution of a mixture of CA,

- the resulting heterogeneous mixture is subjected to the NAS procedure, carried out at a temperature of 50-60 ° C, including obtaining a coarse dispersion of PFOS in a solution of a mixture of CA by mixing on a high-speed mixer at a rotor speed of 1000-3000 rpm; the obtained coarse dispersion is extruded in a homogenizer under a pressure of 100-300 kg cm ² in an atmosphere not containing oxygen, and the coarse dispersion of PFOS is separated from the aqueous solution of CA; the obtained precipitate of a coarse dispersion is mixed with a fresh portion of a CA solution from such a calculation that the volume fraction of PFOS is 10-30%,

- while the described operation US is repeated at least 2 times,

- after which the emulsion is passed repeatedly through a homogenizer at a pressure of 300-700 kg / cm ² ,

- the resulting submicron emulsion is diluted with water-salt solution so that the final concentration of PFOS is 4-12 vol.% and the emulsion has an osmotic pressure in the range of 270-330 mOsm per liter.

The method for preparing the PFOS emulsion is implemented in such a way that mixtures of liquid PFCs and PFTA or PFE are obtained in a ratio of 5: 1 to 1: 5, subjected to washing after mixing with pyrogen-free aqueous-alcoholic solutions and water, and mixtures of denser liquid PFOS are separated from aqueous alcohol solution and water, filtered under pressure of a gas that does not contain oxygen, poured and sealed in bottles for rolling and carry out autoclaving.

The method of preparing the PFOS emulsion is implemented in such a way that the CA mixture is prepared in the form of a 10-30% aqueous solution of polyoxyethylene-polyoxypropylene block copolymers - a mixture of proxanol 168 and proxanol 268, taken in a ratio of 10: 1 to 1:10 depending on the composition of the composition of the PFOS mixture , the CA solution is purified by passing through a carbon sorbent and a sterilizing filter under pressure of an oxygen-free gas, the CA solution is heated for 8-24 hours and cooled to 16-20 ° C to completely restore the transparency of the solution.

The method of preparing the PFOS emulsion is implemented in such a way that an aqueous solution of a mixture of block copolymers of polyoxyethylene-polyoxypropylene is heated at a temperature that is selected in the range of ± 2 ° C from the cloud point established experimentally for the obtained mixture of CA.

The method of using PFOS emulsion to reduce the effects of hypoxia, ischemia, edema, inflammation in shock, blood loss, thrombotic-embolic lesions, functional and organic disorders of vascular patency, tissue damage is realized by intravenous administration at a dose of 0.5-30 ml per 1 kg of body weight .

The method of using PFOS emulsion to suppress secondary alteration in inflammation and wound healing, as well as to accelerate the healing of traumatic, trophic and surgical wounds, is implemented by intravenous administration in doses of 0.3-6 ml per kg of body weight and / or topically in portions of 0.5-50 ml subcutaneously, intramuscularly, in depth around the damaged area or in the surrounding inflamed and injured tissues of the cavity or biological fluids, in particular intrapleural, intraperitoneal, intralumbal, into the common lymphatic duct in doses 1-100 ml, and as the cleaning liquid or the processing applicative open wound surfaces.

The method of applying the PFOS emulsion to improve systemic and local blood flow, regulate systemic blood pressure and regional perfusion pressure, facilitate and accelerate mass transfer through gases, substrates, and metabolites is realized by intravascular or local injection into tissues, as well as by perfusion and perfusion-free protection of those isolated or disconnected from blood flow of organs and tissues.

A decrease in the main process of destruction of PFOS emulsions caused by molecular diffusion is usually achieved by introducing into the fluorocarbon base a component of PFOS with low vapor elasticity (for example, PFMTSP or PFBA) and less soluble in water, which has a higher boiling point and slows down this process. However, this leads to the appearance of inhomogeneities in the fluorocarbon phase, which reduce the stability of the emulsions. In order to increase the stability of PFOS emulsions, the present invention uses such PFOS mixtures that significantly increase the uniformity of the “oil phase” and contribute to the formation of an adsorption layer from a CA mixture by saturation affinity separation, when physical self-assembly and enrichment of the adsorption layer of emulsion particles are most suitable for this PFOS compositions with CA molecules contained in the emulsion of the general CA mixture repeatedly updated during the preparation of the emulsion.

Judging by the T _{crG diagrams} for PFOS according to RF patent No. 2206319, there is a gap of more than 10 ° C between them, which indicates the presence of quite significant differences in the physicochemical properties of the components of the “oil” core, and therefore it is not physically homogeneous , which is manifested in the clustering of the fluorocarbon phase and in the constant turbulent movement of clusters inside this phase. In order to increase the uniformity of the fluorocarbon phase in our invention, PFOS compositions are used, which practically form an almost continuous series of T _crG values and thereby significantly increase the uniformity of the “oil” phase, and increase the stability of PFOS emulsions. At the same time, the rates of removal of PFOS from RES cells and lipids change somewhat: the half-life of PFD when introduced as part of the proposed mixtures increases by an average of 10-15%, that is, it is not 12-13 days (typical for PFD outside these mixtures), but 14-15 days, while, on the contrary, the initial excretion rate of PFTA and PFDE increases by 10-15%, and therefore the half-life decreases by 5-7%.

Increasing the uniformity of the oil phase is achieved at the stage of selection of PFOS. As you know, obtaining individual PFOS on an industrial scale is the most expensive step in the process of converting organic compounds to PFOS. Studies conducted by V.A. Arkhipov and K.N. Makarov showed that the toxicity of PFOS for human cells depends only on impurities of hydrogen-containing and unsaturated compounds, i.e., on the presence of incompletely fluorinated substances, even if only one hydrogen atom or one unsatisfactory connection. Examples of formulas of such compounds found in insufficiently purified PFOS impurities are given below in the form of a structural diagram of some toxic impurities of under-fluorinated PFOS molecules and their radicals.

The most important point from the point of view of safety is that with the variation of impurities of completely fluorinated substances in the PFD, the toxicity of the drug does not change if it did not have underfluorinated PFOS-mixture remains completely safe for animal cells (table 2).

table 2 The effect of purified PFOS and PFOS containing impurities of 1ppm under-fluorinated fluorocarbon compounds Cultivation Conditions of Transformed Raji Human Lymphoid Cells Initial cell density Cell growth after cultivation Benchmark Comparison RPMI 1640 environment 70 thousand / ml 290 thousand / ml Accepted as 100% RPMI1640 medium + purified PFD 65 thousand / ml 300 thousand / ml 103.4% RPMI 1640 + H-PFD medium (mixed with 1 ppm under-fluorinated compounds) 69 thousand / ml 170 thousand / ml 58.6%

Similar studies were performed for other types of PFOS, in particular for PFTA and PFE, and the same conclusions were drawn. This led to the proposal not to isolate the individual compounds PFD, PFMTSP, PFTPA, PFTA, PFDE and their co-products (close homologues, isomers and impurities), but to obtain these PFOS surrounded by minor impurities synthesized with them, isomers and homologs close to T _crH , but at the same time it is tough to ensure and control the release of the resulting substances from under-fluorinated impurities so that the level of under-fluorinated impurities does not exceed 1 part per million (1 ppm). In such a situation, the production efficiency of PFOS for the yield of a useful product also increases sharply, and accordingly, the cost of the process for obtaining PFOS falls without affecting their biological safety. Moreover, close homologues, isomers, and impurities (let's call them co-products) form an expanded field around the main components in terms of T _{crg values} , which allows the formation of PFOS compositions more uniform in physicochemical properties with a given field width T _crg . At the same time, the stability of emulsions increases (Table 3).

For example, Table 3 shows a comparison of the contribution of decreasing _{ΔТ crg} to the stability of PFOS emulsions with the contribution of reducing the vapor pressure of PFOS. In the case when a mixture of PFBA and PFD was used, the stability of the PFOS emulsion is achieved due to 10 times less vapor pressure of PFTA (compared to PFD), since PFBA diffuses weakly and prevents the diffusion of PFDA (example 4, Table 3), despite a large value of ΔT _crg . If in this situation the ΔT _crG value is _reduced by adding other PFTA having co-product impurities (according to the claimed invention), then, despite the loss in vapor elasticity (which is several times higher for PFMCP and PFTPA than for PFTA), there is an additional increase in the stability of PFOS emulsions (example 5, table 3). In Example 5, despite the replacement of a portion of PFTA with PFTA with higher vapor pressure, instead of the expected decrease in the stability of emulsions, a significant increase in the stability of the new composition was observed due to a decrease in _{ΔТ crg} inside the PFOS core.

Figure 1 shows diagrams of critical dissolution temperatures of various PFOS in hexane.

The use of such PFOS with extended boundaries of T _crg led to an unexpected biological effect: the rate of excretion of individual components from RES cells changed. So, if an individual PFD had a T / 2 of 12–13 days, then in the presence of PFMTSP and PFTA, this value for PFD extended to 14–15 days. On the contrary, if an individual PFMCP was removed from T / 2 of the order of 90 days, then in the presence of PFD, the T / 2 value for PFMCP decreased to 80 days, which allows one to understand the previously obtained data for T / 2 variations for PFMCP, previously given in the scientific literature without taking into account the fact that the data were obtained in the case of T / 2 equal to 90 days for an individual substance, and T / 2 of the order of 65 days (according to our data, this is an extremely low estimate) was obtained for the PFD / PFMTSP mixture with a component ratio of 2: 1.

The dispersion and stability of the PFOS emulsions obtained according to the previous patents (Example 1 according to the RF patent No. 2070033) and the prototype (Example 2 according to the RF patent No. 2206319) were compared with the PFOS emulsions obtained according to the claimed invention (groups 3, 5 - table 3). Stability was checked during storage, freezing-thawing and when mixed with dextran solutions (polyglucin) with an average molecular weight of 60,000 Da. The results are shown in table 3.

Table 3 Group number, type of PFOS in the composition of emulsions, the meaning of the example. The average particle size at the original size of 0.08 microns The effect of a solution of dextran on the optical density of the emulsion PFOS when mixed in a ratio of 1: 1 After 45 days of storage at 4 ° C After 5-fold freeze-thaw No dextran, after a week of storage + 3% dextran (after 30 minutes) + 6% dextran (after 30 minutes) 1) PFD and PFMTSP (patent No. 2070033) in a ratio of 2: 1. PFOS of the core with _{ΔТ crG} ~ 20 ° С. 0.14 ± 0.01 0.15 ± 0.02 0.10 ± 0.01 0.36 ± 0.02 0.90 ± 0.71 2) PFD and PFMTSP (prototype No. 2206319) in the ratio of 2: 1. PFOS core with ΔT _crg > 6 ° C 0.12 ± 0.01 0.11 ± 0.01
P ₁ <0.05 0.10 ± 0.01 0.19 ± 0.015
P ₁ <0.01 0.62 ± 0.03
P ₁ <0.01 3) PFD and PFMTSP (according to the invention) in a ratio of 2: 1 with impurities, PFOS core with ΔT _crg <4 ° C 0.10 ± 0.01
P ₁ <0.05 0.11 ± 0.01
P ₁ <0.05 0.08 ± 0.009 0.16 ± 0.01
P ₁ <0.01 0.50 ± 0.02
P <0.01 P ₂ <0.05 4) PFD and PFBA in a ratio of 2: 1. An example of stabilization of the emulsion due to PFBA at ΔT _crG > 30 ° C. 0.11 ± 0.01 0.12 ± 0.01 0.09 ± 0.01 0.16 ± 0.01
P ₁ <0.01 0.53 ± 0.02 P ₁ <0.001
P ₁ <0.05 5) PFD, PFTA, PFTPA, PFMTSP, (according to the proposed invention), the ratio of 2: 0.8: 0.1: 0.1, ΔT _crG <4 ° C. 0.10 ± 0.01
P ₁ <0.05 0.11 ± 0.01
P ₁ <0.05 0.08 ± 0.009 0.13 ± 0.01
P ₁ <0.01 P ₂ <0.05 0.43 ± 0.02
P _1.2 <0.01 P _3.4 <0.05

P _i shows the level of significance of differences when compared with the group corresponding to the index number at (n = 9-10).

Matching the type of stabilizing agent (CA) to the PFOS composition plays a key role in dispersing PFOS and maintaining the stability and biological activity of PFOS emulsions.

To obtain PFOS emulsions, mainly two types of SA are used:

1) non-ionic block copolymer of polyoxyethylene (POE) with polyoxypropylene (POP) in the form of Proxanol-268 or its analog Pluronic F-68, as well as phospholipids (PL) isolated from egg yolk or soy, etc.

For proxanol 268, a centrally located hydrophobic POP chain is connected at its ends with two POE chains. According to the accepted nomenclature of the names of block copolymers, the number 8 in the name of proxanol 268 corresponds to the fact that the average total mass of POE blocks in the molecule is 80%, and the number 26 means that the average MM of POP, which is on average 20% of the mass of the proxanol molecule, equal to 2600 Yes. Thus, the average MM of the proxanol 268 block copolymer as a whole is 13,000 Da, with a PEO / POP block ratio of 80% and 20% on average, respectively. Recall that in the synthesis of a block copolymer, as in the synthesis of any polymer, far from identical molecular masses and the ratio of individual molecule blocks are obtained. Therefore, they are characterized by average MM, the presence of a molecular weight distribution that varies even in one batch and the average ratio of POP to POE blocks.

Proxanol interacts with the hydrophobic part, that is, through POP with PFOS, and with water - with the hydrophilic part - with the POE block. The stabilizing effect of proxanol and Pluronic is due to the steric effect of the protective film formed by CA molecules around PFOS particles. At the same time, along with SA bound in the adsorption layer, a significant part (up to 50%) of SA molecules form various micellar structures in the aqueous phase that are free of PFOS. Between the CA molecules located in the adsorption layer of the emulsion particles and in the aqueous phase in the micelles, the dynamic equilibrium is established, which is necessary to stabilize the adsorption layer, on the one hand, and, on the other hand, violating the packing density of the CA molecules in the adsorption layer during long-term storage. Due to differences in the compositions of SA bound in the adsorption layer of PFOS particles and free micellar CA, substitution as a result of the exchange of molecules of the adsorption layer to molecules from micelles of the aqueous phase is unfavorable to maintain the stability of the adsorption layer. In the presence of sharp differences, the achievement of an effective (maintaining a stationary level and type of SA in the adsorption layer of particles) dynamic equilibrium between CA molecules in the particles and in the aqueous phase is practically unattainable.

Note that when using natural PL as an emulsion stabilizer of PFOS, there are also problems of heterogeneity of the substance used both in composition and quality (the presence of polar components, free fatty acids, lysoforms).

When choosing and determining the quality of block copolymers taken as SA, we proceeded from an assessment of two criteria: the stability of PFOS emulsions and the degree of damage to biological membranes. It turned out that proxanol 268 provides, with the same composition, the PFOS selected by us, significantly greater emulsion stability than proxanol 168, apparently due to the more pronounced detergent (amphiphilic) properties of proxanol 268: on average, an emulsion from a mixture of PFD with PFMCP with coproducts of both components taken in a 2: 1 ratio, stabilized with proxanol 268 without freezing, stored longer than in the case of proxanol 168: average doubling period of the average particle size (at 0.07 μm in the original emulsion) avil 45 ± 4 days proxanol 268 and only 30 ± 3 days proxanol 168 (p <0.02). However, proxanol 168 does not damage biological membranes due to its weak detergent properties, in particular, it does not cause a separation of oxidative phosphorylation reactions when added to isolated mitochondria, a drop in the transmembrane electrochemical potential of hydrogen ions on the inner membrane, and suppression of the phosphorylated respiration of liver mitochondria. In contrast, proxanol 268 causes a marked decrease in parameters as oxidative phosphorylation when added to isolated mitochondria and a decrease in the rate of phosphorylating respiration is evidence of membrane damage. The damaging activity against biological membranes of Pluronic F68 is even more pronounced, despite the fact that formally it should simply be an analogue of proxanol 268.

Figure 2 shows the effect of various additives (50 μl) to rat liver mitochondria on the safety of phosphorylating respiration during oxidation of succinate (5 mM). Additives: non-fluorinated PDF (N-PFD), purified from non-fluorinated PDF impurities with co-products (PDF) and 4% aqueous solutions of Pluronic F68 (Pl F68), proxanol 268 (Pr 268) and proxanol 168 (Pr 168). The incubation medium was 250 mM sasarose, 3 mM KH ₂ PO ₄ (pH 7.4), 1 mM MgCl ₂ , 15 mM KCl, 200 μm ADP. The concentration of MX 3 mg protein per ml. The cell volume is 2 ml. The incubation temperature is 26 ° C.

Since in the synthesis of proxanols, the ratio of POE and POP blocks is only 80:20 on average, and in industrial batches of raw materials this ratio can differ in one direction or another, we conducted a study in which we determined the effect of variations of this ratio on the stability of emulsions and damage to biological membranes . It turned out that an increase in the proportion of POP above 18% is absolutely necessary to obtain more stable emulsions, a decrease in the proportion of POP below 18% reduces the retention time of emulsions with proxanol 268 by one third, and with sposanol 168 by 2 times. An increase in the proportion of POP block above 22% is unacceptable due to increased damage to biological membranes, and then both proxanol approach in their damaging properties to Pluronic F68. Thus, the range of permissible variations in the selected types of proxanol was determined. In order to combine a sufficiently high stability of emulsions with a reduced damaging ability with respect to biological objects represented by membrane structures at most levels of body organization, we used a mixture of proxanols 268 and 168. It was found that emulsions containing PFTA along with PFD and other PFOS can stabilize without a significant loss in stability with an increase in the proportion of proxanol 168, and these emulsions have a reduced damaging ability with respect to biological objects, such as isolated perfused organs: heart and kidney, compared to emulsions with a high content of proxanol 268. But these emulsions can hardly be introduced into the body, since PFBA has too long a delay period in RES cells (T / 2 is 900 days). For PFOS emulsions containing more lipophilic than PFTA components, proxanol 268 is more acceptable as a CA, however, as indicated, damage to biological membranes increases. Ultimately, we came to the conclusion that for each PFOS composition, it is necessary to select its own mixture of proxanols 168 and 268, which is very laborious, since it requires additional tests to select a mixture of proxanols each time. The way out of this difficulty is achieved by using the self-assembly procedure based on the use of physicochemical affinity between the PFOS composition taken and the proxanol mixture 168 and 268. In the process of repeated self-assembly, the adsorption layer of PFOS dispersion particles is sequentially saturated with the composition of SA that has the highest affinity for the taken PFOS composition, it is only important to speed up the saturation process, which is achieved by raising the temperature in the mixer to 50-60 ° C (depending on the proxanol mixture taken and its rate turbidity), as well as the repetition of the procedure with updating the aqueous phase of SA, which did not react with particles of the PFOS dispersion.

So, to improve the stability of the emulsion during storage in the claimed invention, a saturating affinity separation method (HAC) is used, which allows optimizing the composition of the adsorption layer of the particles of the emulsion in accordance with the composition of the PFOS - “oil” core of particles and reducing the differences between the SA located in the adsorption layer of particles emulsion, and micellar CA aqueous phase. NAS is performed in several successive sessions, including mechanical mixing of the PFOS composition taken for dispersion in a volume of 10-30 parts with 90-70 parts of a solution of 20-30% CA, which is a mixture of proxanol 168 (average MM of the order of 8000 Da) and proxanol 268 (average MM of the order 13,000 Yes). An inhomogeneous mixture having two layers of liquids: the heavy lower layer of liquid PFOS and the upper layer - micellar solution of CA, is mixed on a high-speed mixer at a rotor speed of 1-3 thousand rpm with immersion of a rotating blade, turbine or pestle of a magnetic stirrer in a dense phase PFOS followed by extrusion of the coarse dispersion using a homogenizer under a pressure of 100-300 kg / cm ² while blowing oxygen-free gas into the homogenizer. The resulting coarse dispersion of PFOS is precipitated by gravity or centrifugation. Then, on the separatory funnel and / or using a centrifugal separator, the PFOS dispersion itself is separated from the aqueous solution of a micellar composition of SA not bound to PFOS, which is subsequently discarded. The separated coarse dispersion of PFOS is mixed with a fresh portion of an aqueous solution of CA in such a way that the volume fraction of PFOS is again 10-30%. The process of dispersing and separating the dispersion of PFOS from an aqueous solution of CA is repeated again 1-3 times. The resulting pre-emulsion is passed repeatedly through a homogenizer at a pressure of 300-700 kg / cm ² to obtain an average particle size of the emulsion in the range from 0.05 to 0.1 μm when the emulsion leaves the plateau in particle size.

Thus, in the process of obtaining the emulsion, it is PFOS due to the physicochemical affinity that the SA molecules that have the highest affinity for them are selected from the solution of the mixture of POE-POP copolymers. Replacing the spent CA solution with a fresh solution allows not only optimizing the composition of the adsorption layer, but also minimizing the differences between the compositions of the CA in the adsorption layer and in the micellar aqueous part of the emulsion. Due to this, an additional stabilization of the adsorption layer and an increase in the stability of the PFOS emulsion as a whole is achieved in comparison with the prototype (Tables 3 and 4).

The obtained PFOS submicron emulsion is immediately mixed with a water-salt solution, the presence of salt ions in which contributes to the formation of a relative negative charge on the radicals of the block copolymer molecules that are turned into the aqueous phase, thereby providing additional stabilization of the adsorption layer, since this forms a kind of double an electric layer of dipoles of water molecules at the surface of the particles of the emulsion PFOS. As a result, the diffusion of nonpolar PFOS molecules through the charged adsorption layer is additionally slowed down and thereby the molecular distillation of the “oil” phase from small particles to larger particles (Ostwald ripening of emulsions) is reduced, and additional electrostatic forces repel particles from each other (electrical “spacer”) ), which prevents the adhesion of the particles of the emulsion.

The effect of the NAS procedure when using a mixture of proxanol 168 with proxanol 268 in various PFOS formulations for the time (day) of doubling the average particle size with an initial particle size of 0.06 ± 0.01 μm is illustrated in Table 4.

Table 4 The method of obtaining emulsions PFOS Composition of PFOS (PFOS ratio), time in days PFD and PFBA (2: 1) PFD and PFMTSP (2: 1) With a mixture of proxanol 168 and proxanol 268 without NAS, the prototype method 43 ± 4 (n = 6) 30 ± 3 (n = 5) With a mixture of Proxanol 168 and Proxanol 268 using NAS, the inventive method. 60 ± 5 (n = 6), t = 2.655, p <0.05 42 ± 4 (n = 5), t = 2.40, p <0.05

p - indicated when comparing with the prototype.

In the claimed invention, the water-salt solution contains 1.5-2 times higher concentrations of salts, compared with the prototype, so that after mixing the submicron emulsion with a water-salt solution, when the final concentration of PFOS is reduced to 4-12 vol.%, the aqueous part of the emulsion was isosmotic to the extracellular fluid, in particular blood plasma and lymph, and contained ions of sodium, potassium, bicarbonate, chlorine, phosphate and magnesium, as well as substrates necessary for maintaining energy and plastic metabolism. Salts of NaCl, KCl, MgCl ₂ , NaHCO ₃ , NaH ₂ PO ₄ , Na ₂ HPO ₄ , D-glucose, carboxylic acids and amino acids are used in the dosage form. The water-salt solution ensures the maintenance of the osmotic pressure at the level of 270-330 mOsm in the final dosage form, the ratio of Na and K ions at the level from 10: 1 to 30: 1 and the pH in the range from 6.9 to 8.0. The water-salt part of the finished final dosage form contains 110-150 mm NaCl, 4-5 mm KCl, 1-3 mm MgCl ₂ , 6-20 mm NaHCO ₃ , 1-2 mm NaH ₂ PO ₄ , 1-2 mm Na ₂ HPO ₄ and 10-15 mM D-glucose and can be supplemented with substrates that simultaneously play an anti-ischemic protective role, in particular salts of tri-, di- and monocarboxylic acids, including 1-7 mM sodium citrate, 1-7 mM isocitrate sodium, 1-7 mm sodium succinate, 1-7 mm sodium β-hydroxybutyrate; and also those capable of converting to succinate during hypoxia and ischemia - 1-7 mm sodium sodium ketoglutarate, 1-7 mm sodium pyruvate, 1-7 mm sodium malate, 1-7 mm sodium glutamate, 1-7 mm sodium aspartate.

The procedure for obtaining PFOS emulsion after NAS is completed by high pressure homogenization, which can lead to damage, destruction and peroxidation of the molecules of the stabilizing agent. Therefore, protective measures against “oxidative stress” must be taken. This is achieved by the fact that the process of preparing the components and dispersing - NAS and homogenization is carried out in an oxygen-free environment.

The proposed method allows, by updating the CA solution, to select from the mixture of CA having a wide molecular weight distribution and variations in the ratio of polyoxyethylene and polyoxypropylene blocks, CA molecules that have the highest affinity for the used PFOS composition, regardless of variations in the composition of the mixture of PFOS and CA, which increases stability, chemical uniformity and calibration of the resulting PFOS emulsions, and also reduces the likelihood of CA peroxides and thereby increases the safety of emulsions. The PFOS emulsion obtained by the proposed method is more stable during storage and when it enters the bloodstream, and also has a viscosity close to blood viscosity, which ensures shear stress in the parietal blood flow and normalizes the tone of blood vessels, which is necessary to improve mass transfer between intravascular fluid and tissues both for oxygen and carbon dioxide, and for substrates and metabolites. It is proposed to use PFOS emulsion as an agent for intravascular and interstitial administration with the aim of treating systemic and local disturbances in blood flow, hypoxic and ischemic conditions, improving the mass transfer of gases and metabolites between blood and tissues, maintaining the function of isolated organs and tissues, reducing inflammation, and conducting infusion transfusion therapy for shock and blood loss, as well as for hydrophobic anti-ischemic modification of membranes and maintenance of microcatalysis occurring in gy hydrophobicity phase and at the interface.

Just like the prototype, the resulting finished dosage form is stored in a frozen state to slow down molecular distillation, stabilize the adsorption layer and prevent particles from sticking together. After thawing, despite maintaining the particle size for 2–3 weeks, it is recommended that, in the case of intravascular administration, a thawed preparation stored at 2–4 ° C for 2–3 days is recommended. With interstitial or intracavitary administration, a thawed preparation can be used for 2-3 weeks.

More specific examples regarding the composition of the emulsion and the implementation of the method of its manufacture, as well as the method of application are given below.

Example 1

The emulsion consists of a mixture of PFD (fast-releasing PFOS) containing, along with PFD, all completely fluorinated impurities that accompany the main substance in the synthesis, and a mixture of slowly removing PFOS-PFTA or PFE, for example PFMTSP (No. 1 general ratio of components 2 parts of PFD: 1 part PFMTSP) or a mixture of PFMTSP and PFTPA (No. 2, the total ratio of components 2: 0.5: 0.5), or a mixture of PFMTSP, PFTPA and PFDE (No. 3, the general ratio of components 1: 1: 0.5), or a mixture of PFMTSP and PFDE (No. 4 general ratio of components 1: 1: 1), or PFMTSP and PFTA (No. 5 general ratio of components 1: 1: 2), with holding along with the main PFTA and PFE all fully fluorinated impurities accompanying the main substance in the synthesis. The prepared PFOS mixtures after sterilization and pyrogenation are mixed with SA, which is a mixture of copolymers of proxanol 268 and proxanol 168, taken in different ratios, in particular for composition No. 1 in a ratio of 5: 1, for No. 2 -5: 2, for No. 3 - 2 : 1, for No. 4 - 1: 1, for No. 5 - 1: 2. The dispersion is obtained by the NAS method with 4 repetitions of the NAS procedure and subsequent extrusion at a pressure of 400 kg per cm ² - 6 cycles, then 3 cycles at 700 kg per cm ² and then 2 cycles at 500 kg per cm ² . Emulsions are obtained with an average particle size of 0.6 to 0.8 μm.

Example 2

The PFOS emulsion described in Example 1 contains one part of a mixture of PFD (fast-releasing PFOS) and two parts of a mixture of 1 slowly removing PFOS No. 1, or No. 2, or No. 3, or No. 4 or No. 5, stabilized with a mixture of copolymers of proxanol 268 and proxanol 168, taken in different ratios, in particular for composition No. 1 in a ratio of 1: 1, for No. 2 - 1: 2, for No. 3 - 1: 4, for No. 4 1: 6, for No. 5 - 1:10. The dispersion is produced by the NAS method. The dispersion is obtained by the NAS method with 2 repetitions of the NAS procedure, including extrusion under a pressure of 100 kg per cm ² and then 200 kg per cm, followed by extrusion of the NAS product at a pressure of 400 kg per cm ² - 3 cycles, then 2 cycles at 600 kg each per cm ² and then 2 cycles at 500 kg per cm ² . Emulsions with an average particle size of 0.50 to 0.75 μm are obtained. The LD50 value of such emulsions reaches 165-200 ml per kg of mouse body weight with intraperitoneal administration.

Example 3

The emulsions described in Example 1 or 2 contain PFOS components having a half-life of 12 to 900 days from an animal’s RES cells after intravenous administration at a dose of 30 ml per kg of body weight. Moreover, the range ΔТ _crg for the components of the PFOS core in emulsions according to examples 1 and 2 is less than 2 ° C for No. 1 in the range from 22 ° C to 41 ° C and the half-life is about 85 days, for No. 2 ΔT _crG does not exceed 3 ° C in the range from 22 ° C to 47 ° C and an elimination half-life of about 74 days, for No. 3 and No. 4 _{ΔТ crG} does not exceed 4 ° C in the range from 22 ° C to 56 ° C and an elimination half-life of about 450 days, for No. 5, the maximum _{ΔТ crG value} is 4 ° С in the range from 22 ° С to 60 ° С and the half-life is about 850 days.

Example 4

The emulsion described in Example 1 contains fast-releasing PFOS and slowly releasing PFOS in a ratio of 5: 1 (sample A) or in a ratio of 1: 5 (sample B) and a stabilizing agent in which for sample A it has a POE to POP ratio of 78:22 and for Sample B - 82:18. The average particle size in emulsions is 0.08 μm, the duration of perfusion of an isolated rat heart according to Languedorf using 11 mM glucose as a substrate is without loss of the initial spontaneous frequency (at the level of 250 cuts per min) for sample A (with a high content of POP block in CA) 3 hours, and for sample B (with a low POP content) - 9 hours. Conclusion: to maintain an isolated heart, an emulsion is preferable, in which a mixture of proxanols with a low POP block content of up to 18% is used as a stabilizing agent.

Example 5

When diluting the PFOS emulsion according to examples 1,2,3,4 with a water-salt solution, the content of PFOS was 4, 10 or 12 vol.% Depending on the amount of added water-salt solution, while the content of the stabilizing agent in the aqueous phase was 1%, respectively . 4% and 5%, and the viscosity of the finished product based on the PFOS emulsion reached 1.5 cPoise, 2.6 cPoise and 5 cPoise, respectively. When a rat replaced 30% of the circulating (BCC) blood volume with these emulsions in non-anesthetized Wistar rats (animal weight 290-320 g) with implanted telemetry sensors, the following dynamics of mean arterial pressure was observed (Table 5).

Table 5 The viscosity of the emulsion in SP and the content of PFOS in vol.% Initial BP * in mmHg HELL * (mmHg) after 30% bcc withdrawal HELL * (mmHg) after administration of PFOS emulsion in a volume of 30% bcc The retention time of average blood pressure after compensation for blood loss (hour) 1.5 cP, 4 vol% (n = 3) 130/80 80/40 120/85 2-3 2.6 cP, 10 vol% (n = 3) 125/65 60/35 125/75 4-6 5 cP, 12 vol% (n = 3) 128/70 60/40 120/75 7-8

BP * indicates the average of 3 animals.

As can be seen from the data presented in the table, an increase in the volume content of PFOS and the viscosity of the emulsion contributes to a longer retention of blood pressure after blood substitution.

Example 6

The finished product based on the PFOS emulsion according to examples 1-5 contains 110 mM NaCl, 5 mM KCl, 2 mM MgCl ₂ 8 mM NaHCO ₃ , 2 mM NaH ₂ PO ₄ , 1 mM Na ₂ HPO ₄ and 20 mM D-glucose 12 volume% PFOS. With massive hemorrhage, 55% of the circulating blood volume in dogs with emulsion No. 1 (5 animals) and No. 2 (6 animals) of Example 1, all dogs survive, despite the significant difference in the shift of buffer bases up to a deficit of 12, and an increase in the lactate ratio pyruvate in the blood up to 20.

Example 7

The finished product based on the emulsion PFOS according to examples 1-5 contains 120 mm NaCl, 5 mm KCl, 1 mm MgCl ₂ 6 mm NaHCO ₃ , 1.2 mm NaH ₂ PO ₄ , 1 mm Na ₂ HPO ₄ and 10 mm D-glucose with a content of 4 volume% PFOS. Emulsions with a low content of PFOS were administered intravenously to rats and their effect was tested at a dose of 1-2 ml on regional blood flow. It was shown that, despite small doses of PFOS administered, such doses of the emulsion are sufficient to activate blood flow in the event of edema or trauma in animals (see example 16 in detail). The increase in blood flow, determined by a laser Dopplerometer, averages from 30% to 90%, depending on the degree of the initial blood flow disturbance.

Example 8

The finished product based on the emulsion PFOS according to examples 1-5 contains 120 mm NaCl, 5 mm KCl, 3 mm MgCl ₂ 6 mm NaHCO ₃ , 1.2 mm NaH ₂ PO ₄ , 1 mm Na ₂ HPO ₄ and 20 mm D-glucose , 1 mM sodium citrate, 1 mM sodium isocitrate, 1 mM sodium succinate, 1 mM sodium α-ketoglutarate, 1 mM sodium pyruvate, 1 mM sodium β-hydroxybutyrate, 1 mM sodium glutamate, 1 mM sodium aspartate with 8 vol% PFOS . The resulting emulsion was used to preserve a Langendorff-perfused rabbit heart on a special perfusion bench: 3 hearts — comparison group (emulsion according to the prototype), 3 hearts — experience (emulsion No. 2 and No. 3 in Example 1 with the salt and substrate composition obtained in this example ), 3 hearts - control with water-salt solution with a composition of salts and substrates, as in this example. The retention time of the initial heart rate without stimulation in the control group was 1.5-2 hours, in the comparison group (according to the prototype) - 4-5 hours; in the experimental group according to the claimed invention - 6-7 hours.

Example 9

The finished product based on the PFOS emulsion according to examples 1-5 contains 150 mm NaCl, 15 mm KCl, 3 mm MgCl ₂ 12 mm NaHCO ₃ , 1.2 mm NaH ₂ PO ₄ 1 mm Na ₂ HPO ₄ and 20 mm D-glucose, 7 mM sodium citrate, 7 mM sodium isocitrate, 7 mM sodium succinate, 7 mM sodium α-ketoglutarate, 7 mM sodium pyruvate, 7 mM sodium β-hydroxybutyrate, 7 mM sodium glutamate, 7 mM sodium aspartate with 4 volume% PFOS. Used for cardioplegia of an isolated rat heart at a temperature of 18-20 ° C. It was shown that, in contrast to conventional water-salt and pharmacological cold cardioplegia, when an hour after cardiac arrest, electrical and contractile ability are restored in no more than 50% of cases and the magnitude of recovery of contractile activity does not exceed 75-80% (10 animals), when using emulsions PFOS composition No. 4 and No. 5 from example 1 with the indicated substrate-salt composition, the electrical and contractility are restored in more than 70% of cases and the recovery value of contractile activity is not lower than 9 0% (10 animals, p <0.02)

Example 10

The obtained emulsion No. 4 according to example 1 with a substrate-salt composition according to example 8 was used as a perfusion composition and according to example 9 was used as a cardioplegic composition on an isolated rabbit heart in the following mode: normothermic perfusion with an emulsion according to example 8 for 30 minutes at a temperature of 37 ° C (perfusion preservation procedure), then changing the perfusate to an emulsion of a cardioplegic composition with the substrate-salt composition according to Example 9 and perfusion at 20 ° C until cardiac arrest (cardioplegia procedure), then for 1 hour dice was disconnected from the bloodstream and placed in a bath with an emulsion of a cardioplegic composition, after an hour the heart was again perfused with an emulsion with a substrate-salt composition according to Example 8. In the comparison group, an emulsion of the prototype was used as a perfusion composition and a cardioplegic composition with a full substrate-salt composition of the prototype , supplementing in the latter case, the concentration of potassium ions up to 15 mm. The initial diastolic and systolic pressure, the degree of restoration of pulse pressure and the level of ATP and creatine phosphate in the heart tissue were measured 30 minutes after reconstructive perfusion at 37 ° C. In the experiment and in the comparison group of 5 experiments. It turned out that in the comparison group (prototype), diastolic pressure was higher by 20% (evidence of residual contracture), and systolic pressure was 10% lower (evidence of less recovery of myocardial contractility), respectively, the value of pulse pressure was lower by an average of 30 ± 10% ( p <0.05) than in the group with the claimed formulation of PFOS emulsions. The total content of adenyl nucleotides fell in the experimental group (the claimed formulation) by 30 ± 5%, and in the comparison group (according to the prototype) by 40 ± 6% (unreliable differences), while in the comparison group (prototype) the ratio of ATP / AMP = 4.2 ± 0.5, while in the group with the claimed composition the ratio of ATP / AMP = 6.5 ± 0.7 (p <0.05), respectively, in the group with the claimed compositions of the PFOS emulsion, the concentration of creatine phosphate in the heart tissue is 25 ± 7% (p <0.02). Thus, the claimed composition improves by 30% the restoration of the contractile function of the heart after cardioplegic arrest and helps preserve the energy status of the tissue judging by the ratio of ATP / AMP and the level of creatine phosphate.

Example 11 (a method of obtaining an emulsion of PFOS and the finished product). In a sterilized thermostatically controlled reactor (1) with a volume of 20 l with nitrogen injection while filling the reactor and with an integrated stirrer, pour with light stirring (200-300 rpm) 11 l of an aqueous sterile pyrogen-free solution of surface-active substances (CA) containing 10% proxanol 168 and 20% proxanol 268 (the total CA concentration is 30%) and 4 l of sterilized pyrogen-free mixture of PFOS purified from under-fluorinated impurities containing 2 l of perfluorodecalin (PFD) with its inherent set of perfluororganic impurities, 1.5 l of perfluoro-N - ( 4-methylcyclohexyl) -piperidine with its inherent set of perfluororganic impurities and 0.5 L mixture of perfluorinated tertiary amines containing up to 40% perfluoro-1-propyl-3,4-dimethylpyrrolidine and up to 16% perfluoro-1 as main substances -propyl-3-metipiperidine, up to 40% perfluorotripropylamine with their inherent upon receipt of a set of perfluororganic impurities. Next, the NAS procedure is carried out. The poured mixture of PFOS and a stabilizing agent is intensively mixed at a stirrer speed of about 1000 rpm, heated to a temperature of 50 ° C, and stirring is continued for 10 minutes. Then the mixer is stopped and allowed to settle - settle on the bottom of the coarse dispersion of PFOS. The supernatant in a volume of 9 l is pumped out of the reactor and 9 l of a fresh solution of the initial CA mixture is added. The stirrer is turned on again at a speed of up to 2000 rpm and the contents of the reactor are heated to 50 ° C, stirring is continued for 10 minutes. Then the mixer is again stopped, allowed to settle to the bottom of the coarse dispersion of PFOS. The supernatant in a volume of 6 liters is pumped out of the reactor and 6 liters of a fresh solution of the initial CA mixture are added. The intensive mixing cycle is again repeated with heating for 10 minutes, and the resulting dispersion is sent to the receiving container of the high-pressure homogenizer (2), where the extrusion is carried out, under pressure in the range of 100-200 kg / cm ² , maintaining the temperature in the extruder valve area not higher 60 ° C. The emulsion obtained upon exit from the homogenizer is separated in a centrifugal separator (3) from a solution of CA not bound to PFOS particles. The supernatant is drained, and the emulsion concentrate is again poured into thermostatic reactor 1 and then the volume of the liquid phase in the reactor is adjusted to 15 L with a fresh solution of the initial CA solution (the volume of the added solution is about 4 L). The resulting mixture of the preemulsion and the CA solution is intensively mixed at a stirrer speed of 3000 rpm at a temperature of 50 ° C and fed to the recirculation loop of the homogenizer, in which the extrusion is carried out under a pressure of 400-600 kg / cm ² , maintaining the temperature in the extruder valve area not higher than 60 ° C. The emulsion is passed through the extruder at least 6 times under the control of light transmission until it reaches a plateau. After the light transmission reaches a plateau, the emulsion goes through another homogenization cycle under a pressure of 400 kg / cm ² and an extruder temperature of not higher than 50 ° C. The resulting submicron emulsion is discharged into a sterile thermostatically controlled reactor (collection tank 4) with a stirrer, while 1 ml of the submicron emulsion is taken for operational control (5-7 min) using a laser nanosizer (type N5, Berlin Coulter) and to determine the volume of the fluorocarbon phase with using a hematocrit centrifuge. If the average particle size exceeds 100 nm, the emulsion from the reactor 4 is again subjected to 3-4 extrusion cycles until the average particle size in the freshly prepared submicron emulsion decreases to 60-80 nm. Then the submicron emulsion is poured into the collection tank 4 in a volume of about 14 liters. 14 l of submicron emulsion contains about 26.6 volume percent of the fluorocarbon phase. To the obtained submicron emulsion, 23.24 l of sterile pyrogen-free concentrate of the water-salt composition is added with gentle stirring (100 rpm) so that after the final dosage form, 110 mM NaCl, 5 mM KCl, 1 mM MgCl ₂ are contained in the aqueous phase of the preparation, 6 mM NaHO ₃ , 1.2 mM NaH ₂ PO ₄ , 1 mM Na ₂ HPO ₄ and 10 mM D-glucose. The resulting preparation is poured using an automatic machine under pressure through a filter with a pore size of 0.4 μm in a tangential flow into sterilized vials, sealed with rubber stoppers with caps for running in and left for 6-8 hours at a temperature of 2 ÷ 4 ° C. After measuring the average particle size (64 nm) and their distribution (see figure 2), the content of PFOS (10 vol%), pyrogenicity (total Δ <0.6 ° C in three rabbits for 3 hours) and osmoticity (300 mOsm ) the vials are labeled and placed in a freezer for storage at sub-zero temperatures of -17-20 ° C. The LD ₅₀ value for the PFOS emulsion obtained in this example was 165-170 ml per kg of mouse body weight. Under the same conditions of administration, the emulsion obtained by the prototype had an LD ₅₀ value of 140-150 ml per kg of mouse body weight. After intravenous administration to the rabbits of the PFOS emulsion obtained in this example at a dose of 20 ml per kg, including after 2–3 repeated administration with an interval of 2 weeks, all rabbits survived for 4 months of observation.

Example 12

The method according to example 11, characterized in that the initial mixture of PFOS containing 2 parts of a mixture of PFD, 0.8 parts of a mixture of PFTA and 0.2 parts of a mixture of PFMTSP, after mixing, washed with a pyrogen-free aqueous-alcoholic solution (HRV): mix the mixture of PFOS and HRV in equal volumes, shaken for 5 minutes and then the HRV phase is separated. The remaining PFOS mixture is washed with pyrogen-free water: the mixture of PFOS and water is mixed in equal volumes, shaken for 5 minutes and then the aqueous phase is separated. Then, rinse with pyrogen-free water again. The washed PFOS mixture is filtered through a filter with a pore size of 0.4 μm under pressure of nitrogen not containing oxygen, and poured into glass vials. PFOS is poured into each bottle not more than 2/3 of the total volume of the bottle. Vials are sealed with rubber stoppers for running-in and sterilized by autoclaving at 120 ° C. The stabilizing agent for this composition of the PFOS mixture is prepared by mixing 1 part of proxanol 268 and 1 part of proxanol 268 in the form of a 20% solution in pyrogen-free water. The resulting solution of CA is passed through a column of activated granular carbon and then through an acetate-cellulose filter with a pore size of 1-2 μm at a pressure of nitrogen that does not contain oxygen. Using a turbidimeter, the cloud point of the resulting stabilizing agent solution is determined by heating from room temperature at a rate of 1 degree per minute. The registered cloud point in this case was 70 ° C. This temperature is used for heating in a reactor for a proxanol solution, where the accuracy of temperature retention is ± 2 ° C. The solution is heated for 8 hours, then slowly cooled to 18 ° C for 6 hours and check the degree of transparency on the nephelometer. A completely cooled solution should have the same light scattering as the initial solution before heating. Then carry out the NAS procedure, as described in example 10, despite the fact that the obtained submicron emulsion is divided into two portions. The first portion is diluted with a water-salt composition obtained according to example 8 in such a way that the PFOS content is 10 volume% (preparation for perfusion preservation of organs). The second portion of the emulsion is diluted with water-salt solution according to example 9 in such a way that the concentration of PFOS is 4% by volume (the drug for cardioplegia is to temporarily keep the heart disconnected from the bloodstream).

The resulting preparation based on PFOS emulsion is intended for the treatment of systemic and local blood flow disorders, hypoxic and ischemic conditions, improvement of mass transfer of gases and metabolites between blood and tissues, maintenance of the function of isolated organs and tissues, reduction of inflammation, and infusion-transfusion therapy in shock and blood loss .

PFOS emulsion is used to reduce the effects of hypoxia, ischemia, edema, inflammation in shock, blood loss, thrombotic-embolic lesions, functional and organic disorders of vascular patency, tissue damage by intravenous administration in a dose of 0.5-30 ml per 1 kg of body weight.

PFOS emulsion is used to suppress secondary alteration in inflammation and wound healing, as well as to accelerate the healing of traumatic, trophic and surgical wounds by intravenous administration in doses of 0.3-6 ml per kg body weight and / or topically in 0.5-50 ml portions subcutaneously. , intramuscularly, in depth around the damaged area or in the surrounding inflamed and injured tissues of the cavity or biological fluids, in particular intrapleural, intraperitoneal, intralumbal, into the common lymphatic duct at a dose of 1-100 ml, as well as honors applicative cleaning or processing liquid at the open wound surfaces.

PFOS emulsion is used to improve systemic and local blood flow, regulate systemic arterial pressure and regional perfusion pressure, facilitate and accelerate mass transfer through gases, substrates and metabolites by intravascular or local injection into tissues, as well as during perfusion and perfusion-free protection of organs isolated or disconnected from the bloodstream and tissues.

Example 13 (method of using emulsion for perfusion preservation of the kidneys)

PFOS emulsions of compositions No. 4 and No. 5 according to example 1 (the production method according to examples 11 and 12, respectively) and the emulsion obtained according to the prototype were used as perfusion compositions to maintain isolated dog kidneys in recirculation mode with normothermic perfusion with a change of perfusion solution every 10 hours. The experiment was performed on 6 dogs in each group, a total of 18 dogs weighing from 10 to 22 kg. As substrates in emulsion No. 4, 10 mM D-glucose, 1 mm sodium citrate, 1 mm sodium isocitrate, 1 mm sodium succinate, 1 mm sodium sodium ketoglutarate, 1 mm sodium pyruvate, 1 mm sodium sodium hydroxybutyrate, 7 mm monosodium glutamate, 7 mM sodium aspartate. The time of preservation of the kidney before the rise in perfusion pressure (before the development of tissue edema) was 38 ± 3 hours (emulsion No. 4) and 40 ± 3 hours (emulsion No. 5), whereas when using the PFOS emulsion according to the prototype it was 34 ± 2 hours. During perfusion, the emulsion was changed every 9 hours. The functioning of the preserved kidneys was tested in each case by replanting the perfused kidney at the moment of perfusion termination (30 minutes after the start of the exponential increase in the perfused pressure) to the recipient dog (under inhalation anesthesia with premedication with promedol and sodium gamma hydroxybutyrate) to the femoral artery and femur . The restoration of blood flow, the elasticity of the kidney, and the restoration of diuresis were recorded. In the experiment with the prototype, restoration of blood flow and diuresis was observed in 4 cases out of 6, with emulsion No. 4 in 5 dogs out of 6, with emulsion No. 5 in all 6 dogs, the transplant began to give urine immediately after restoration of blood flow.

Example 14

The use of the PFOS emulsion according to Example 12 for suppressing inflammation by intravenous administration in doses or locally in portions intramuscularly, in depth around the damaged area. The experiment was performed on 48 Wistar male rats anesthetized with thiopental and oxybutyrate, which, using a dental drill, applied a calibrated wound to the soft and bone tissues of the thigh. Then the bone wound was filled with bone crumb, and the soft muscle tissue, fascia and skin were sutured with tightening sutures. In the control group, animals were injected with saline in the tail vein at a dose of 1.5 ml per kg of body weight (group 1), or intramuscularly 0.3 ml above and below the wound (group 2); in groups 3 and 4, PFOS emulsion was administered, respectively in group 3 - intravenously, in group 4 - intramuscularly. In all groups of animals, 4 animals were slaughtered on days 3, 5 and 7, at the indicated times in each group. Thigh tissues were fixed in formalin and then in Canadian balsam, after which soft tissues and bone tissue were subjected to pathomorphological examination. In the control groups (administration of saline solution intravenously and locally) on day 3, pronounced leukocyte infiltration phenomena were observed, leukocyte infiltrates penetrated the soft layers with thick layers, including those surrounding the initially undamaged tissues, the lesion was increased due to secondary alteration with a large number of osteoclasts along the periphery bone wounds; leukocyte infiltration decreased significantly by 5 days, the first signs of connective tissue regeneration appeared and the number of osteoclasts decreased, the first osteoblastic elements appeared; on the 7th day, signs of lymphoid and epithelioid infiltration persisted, the number of osteoblasts and elements of the procollagenic framework of the connective tissue increased. In group 3 with intravenous administration of the emulsion, the effect of leukocyte infiltration, ubiquitous lymphoid infiltration, and secondary alteration phenomena are not pronounced already on day 3; on the 5th day in the soft and bone tissues signs of epithelioid infiltration and pronounced signs of the beginning of regeneration of connective tissue elements; on the 7th day, against the background of widespread regeneration of connective tissue elements, separate differentiating cells with structures characteristic of the original tissue appear. In group 4 with intramuscular injection of the PFOS emulsion, the pathomorphological picture is closer to that in group 3, despite the fact that the emulsion was injected not into the systemic circulation, but locally. Thus, the introduction of the PFOS emulsion has a pronounced anti-inflammatory effect, reduces leukocyte infiltration and practically eliminates the phenomena of secondary alteration in inflammation and wound healing, due to which the damage zone does not increase earlier and regenerative healing processes begin earlier. The indicated anti-inflammatory and wound healing processes are more pronounced with the intravenous administration of the PFOS emulsion, but these phenomena are unexpectedly bright in nature with the local administration of the PFOS emulsion.

Example 15. The treatment of fat and air embolism in rabbits.

The PFOS emulsion obtained in Example 11 was used to treat fatty and air embolism in rabbits, which are especially sensitive to impaired patency of the brain vessels. Rabbits weighing 3-4 kg were injected through the ear vein with 10 ml of a coarse emulsion of corn oil in water with an average particle size of 100-200 microns and visible air bubbles. After 3-5 minutes, the animals rolled their eyes, they fell on their sides, one rabbit showed a disturbance in the respiratory rhythm according to Chain-Stokes, and one animal developed apnea - breathing stopped. 20 minutes after the rabbit lost consciousness or 3-4 minutes after the appearance of Cheyne-Stokes breathing, or immediately after stopping breathing using a catheter previously fixed in the ear vein, rabbits were injected with PFOS emulsion at a dose of 4 ml per kg body weight. The therapeutic effect in the form of removing animals from a coma and a state of clinical death (as a result of respiratory arrest) developed almost immediately "on the needle": rhythmic breathing was restored, rabbits regained consciousness, opened their eyes, pupil reflexes were restored. In the control experiment with the introduction of physical. no solution of the therapeutic effect was observed: rabbits with Chain-Stokes breathing and with apnea died.

Example 16. Regulation of regional blood flow and improvement of blood flow in the affected area. In rats after a thigh injury in Example 13, tissue edema and a violation of local blood flow on the injured hip were observed on days 1-3. Emulsion PFOS or physical. the solution was administered intravenously at a dose of 2 ml per kg of body weight or locally 0.2 ml at 4 points around the wound, retreating 5-8 mm. After 30 minutes, the systemic blood flow in the region of large vessels proximal to the wound and local blood flow directly above the sutured wound, as well as in the opposite intact limb, were measured using an ultrasonic dopplerometer. Intravenous administration of emulsion PFOS. The experiment was performed on 40 Wistar male rats weighing 250-300 g. The rats were administered as controls, which were injected with 0.9% sodium chloride solution intravenously (group 3, n = 10) and intramuscularly (group 4, n = 10). In all rats, the state of blood flow in the initial state was additionally assessed prior to drug administration. Dopplerographic research was carried out on an ultrasonic diagnostic apparatus “Biomedical AU-5” (Italy) using a linear sensor with a frequency of 10-13 MHz according to the standard technique. Doppler mapping was performed based on the analysis of the direction and energy of the flow in the femoral arteries and in the tissues of the thigh before and after the introduction of the emulsion PFOS and physical. solution.

With the intravenous administration of PFOS emulsion, the maximum systolic blood flow velocity in the femoral artery exceeded the original more than 3 times (p <0.05), the minimum diastolic linear blood flow velocity along the vessel increased 2.5 times (p <0.05), the average speed - 4.5 times (p <0.05). At the same time, the resistance index decreased by 26.0% (p <0.05), which indicates a decrease in total peripheral vascular resistance. The pulse index significantly exceeded the background value by 2.02 times. When mapping, there was a significant increase in arterial and venous blood flow compared to the original in both the femoral artery and in the affected tissues of the thigh, while on the unaffected limb there was a clear tendency to decrease blood flow after intravenous administration of the PFOS emulsion. After intramuscular injection of PFOS emulsion, blood flow rates increased, but differences from control (saline solution) were not significant. When mapping, a significant increase in arterial and venous blood flow was visually observed compared with the initial in the femoral artery and in the tissues of the affected hip of rats. Energy Doppler mapping showed an increase in red blood cell mass both in the area of tissue infiltration with PFOS emulsion and with intravenous administration.

After intravenous administration physical. solution, the maximum systolic blood flow velocity in the femoral artery was 139.9%, the minimum diastolic linear blood flow velocity along the vessel was 161.1%, the average speed was 191.8% of the initial value (p <0.05). However, these changes in blood flow velocities were observed with a practically constant index of resistance, which indicates the preservation of spastic vascular tone in the microcirculation system. The pulse index characterizing the state of blood supply in the wound area exceeded the background value by 26.8% (p> 0.05). Differences in blood flow rates and indices of resistance and pulse with intramuscular injection of physical. solution in comparison with the initial background is not detected. When mapping, a slight increase in arterial blood flow was visually observed compared with the original in the femoral artery and in the affected tissues of the thigh. Energy Doppler mapping showed a slight increase in the mass of red blood cells in the area of tissue infiltration nat. solution and after its intravenous administration.

Thus, intravenous administration of PFOS emulsion in small doses significantly improves systemic and local blood flow in the wound area, while even a slight decrease in local blood flow occurred on the unaffected limb, which is in good agreement with the hypothesis of O. Rafikova et al. (2004) on the role of the phenomena of hydrophobic NO-dependent microcatalysis, manifested in improving blood flow, and the possibility of decreasing blood flow during sequencing of NO by PFOS emulsion, which can cause a decrease in blood flow (as happened in our experiment on the healthy side). In this regard, the detection of a significant effect on the local blood flow after local administration of the PFOS emulsion turned out to be very indicative.

Example 17

Intracavitary administration of PFOS emulsion to reduce inflammation.

Male rats weighing 250-280 g under thiopental anesthesia performed laparotomy and subsequent suturing of the abdominal wound with a clean but not sterilized instrument - without observing aseptic rules (5 animals - control, 10 animals - experience, 5 for each emulsion PFOS). As a result, animals of the control group, which were intraperitoneally injected with 3 ml of physical. solution 24 and 48 hours after surgery, after 72 hours, pronounced infiltrative changes in the abdominal cavity and effusion in the form of neutrophilic activated leukocytes were observed. At the same time, the isolated leukocytes possessed a 3-5-fold increased ability to generate reactive oxygen species detected using a chemoluminometer in the presence of luminol after the addition of formyl myristyl acetate. When introduced into the abdominal cavity after 24 and 48 hours in 3 ml of PFOS emulsion obtained according to the claimed invention (samples No. 2 and No. 3 in example 1), the phenomena of inflammation in the abdominal cavity sharply decreased, the amount of effusion was so small that it was difficult it was possible to obtain a suspension of leukocytes, and the leukocytes obtained increased the production of reactive oxygen species after the addition of formyl myristyl acetate only 1.5-2 times. The pathomorphological analysis of organs in no case - neither control, nor in experience - did not reveal the appearance of septic foci. Thus, intracavitary administration of an emulsion contributed to a decrease in the inflammatory process in the abdominal cavity without the use of antibiotics, suppressed leukocyte hyperactivity, without causing any septic complications that could be expected in connection with the suppression of PFOS emulsion of leukocytes penetrating into the abdominal cavity. Subsequent healing proceeded in the control with the formation of a rather pronounced hypertrophic scar, while in animals that were injected with PFOS emulsion twice intraperitoneally, the suture was soft, smooth, without cicatricial changes.

As a result, a stable PFOS emulsion was created, an effective method for its preparation and an effective method for its use.

Information sources

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Claims (21)

1. An emulsion of perfluororganic compounds (PFOS) for biological and medical purposes, containing a mixture of perfluorocarbons (PFCs), the main component of which is perfluorodecalin (PFD) and a mixture of perfluorinated tertiary amines (PFTA), the main component of which is perfluoro-N- (4-methylcyclohexyl ) -piperidine (PFMTSP), having lower than PFD, the rate of excretion from the body, a stabilizing agent and a physiologically acceptable water-salt solution with energy exchange substrates, characterized in that all components of PFOS have the critical temperature of dissolution in hexane (T _crg ), differing by no more than 2-4 ° C, and the stabilizing agent is a mixture of block copolymers from the group of block copolymers of polyoxyethylene-polyoxypropylene, with an average mass content of polyoxypropylene of 20%, while PFD mixtures contain only completely fluorinated PFC impurities obtained by the synthesis of PFD, and PFMPP contains only fully fluorinated PFTA impurities, as well as at least one component from the group: perfluorotripropylamine (PFPA) mixtures only with fully fluorinated impurities of PFTA, a mixture of perfluorotributylamine (PFBA) only with fully fluorinated impurities of PFTA, a mixture of perfluorodecyl ether (PFDE) only with fully fluorinated impurities of perfluorinated ethers (PFE). 2. The emulsion PFOS according to claim 1, characterized in that as perfluorocarbons it contains a mixture of perfluorinated co-products of perfluorodecalin having an average half-life of at least 12 days and a range of T _crH in the range of 22-29 ° C, purified from hydrogen-containing and unsaturated impurities. 3. The PFOS emulsion according to claim 1, characterized in that, as perfluorinated tertiary amines, it contains a mixture of all perfluorotripropylamine co-products having an average elimination half-life of at least 60 days and a T _crH range of 41-46 ° C, purified from hydrogen-containing and unsaturated impurities. 4. The emulsion PFOS according to claim 1, characterized in that as the perforated tertiary amines it contains a mixture of co-products of perfluoro-N-4- (methylcyclohexyl) piperidine having an average half-life of at least 80 days and a range of T _crH within 32-40 ° C, purified from hydrogen-containing and unsaturated impurities. 5. The emulsion PFOS according to claim 1, characterized in that as slowly removing PFOS it contains a mixture of co-products of perfluorodecyl ether (PFDE) having an average half-life from the body of at least 500 days and a _Tg range in the range of 49-53 ° C, purified from hydrogen-containing and unsaturated impurities. 6. The PFOS emulsion according to claim 1, characterized in that as perfluorinated tertiary amines it contains a mixture of perfluorotributylamine co-products having an average elimination half-life of at least 900 days and a range of T _crH in the range of 56-60 ° C, purified from hydrogen-containing and unsaturated impurities. 7. The emulsion PFOS according to claim 1, characterized in that the ratio of the rapidly excreted and slowly excreted PFOS contained therein is from 5: 1 to 1: 5. 8. The PFOS emulsion according to claim 1, characterized in that it contains a mixture of polyoxyethylene (POE) -polyoxypropylene (POP) copolymers selected by saturation affinity separation (NAS) with a molecular weight of 5-13 thousand Da. 9. The PFOS emulsion according to any one of claims 1, 8, characterized in that it contains a mixture of POE and POP copolymers selected by the NAS method with a POE: POP ratio in the range from 78:22 to 82:18 as a stabilizing agent. 10. The emulsion PFOS according to any one of claims 1 to 8, characterized in that the content of the stabilizing agent in it is 1-5%, to ensure a viscosity of 2-5 cP. 11. The PFOS emulsion according to any one of claims 1 to 8, characterized in that the physiologically acceptable aqueous salt solution includes NaCl, KCl, MgCl ₂ , NaHCO ₃ , NaH ₂ PO ₄ , Na ₂ HPO ₄ , D-glucose to ensure isosmosis in the final dosage form, while maintaining the ratio of Na and K ions at a level of 10: 1-30: 1 and a pH in the range of 6.9-8.0. 12. The emulsion PFOS according to claim 1, characterized in that the water-salt part of the finished final dosage form contains 110-150 mm NaCl, 4-5 mm KCl, 1-3 mm MgCl ₂ 6-20 mm NaHCO ₃ , 1-2 mM NaH ₂ PO ₄ 1-2 mM Na ₂ HPO ₄ and 10-15 mM D-glucose. 13. The PFOS emulsion according to any one of claims 1, 12, characterized in that for use as an anti-ischemic protector, the water-salt portion of the preparation contains 4-15 mM KCl, salts of tri-, da- and monocarboxylic acids, including 1- 7 mm sodium citrate, 1-7 mm sodium isocitrate, 1-7 mm sodium succinate, 1-7 mm sodium sodium ketoglutarate, 1-7 mm sodium pyruvate, 1-7 mm sodium β-hydroxybutyrate, 1-7 mm sodium glutamate , 1-7 mm sodium aspartate. 14. The method of preparing the emulsion PFOS according to claim 1 for biological and medical purposes, comprising mixing and dispersing a mixture of liquid PFOS with an aqueous solution of a stabilizing agent, followed by grinding in a high pressure homogenizer to a submicron particle size of the emulsion, characterized in that
a mixture of PFOS having differences in T _{crg of} not more than 2-3 ° C is poured into a container containing a solution of a mixture of CA in a ratio of 10-30 volume parts of PFOS to 90-70 volumes of a solution of a mixture of CA,
the resulting heterogeneous mixture is subjected to the NAS procedure at a temperature of 50-60 ° C, including obtaining a coarse dispersion of PFOS in a CA solution by stirring on a high-speed mixer at a rotor speed of 1000-3000 rpm, the obtained coarse dispersion is extruded in a homogenizer under pressure of 100-300 kg / cm ² in an atmosphere containing no oxygen, and the coarse dispersion was separated from the aqueous solution PFOS CA resulting precipitate coarse dispersion is mixed with a fresh portion of solution CA from this calculation that the volume to I PFOS is 10-30%
while the described operation of the NAS is repeated at least 2 times,
after which the emulsion is passed repeatedly through a homogenizer at a pressure of 300-700 kg / cm ² ,
the resulting submicron emulsion is diluted with water-salt solution so that the final concentration of PFOS is 4-12 vol.% and the emulsion has an osmotic pressure in the range of 270-330 mOsm. 15. The method of preparing the emulsion PFOS according to claim 14, characterized in that mixtures of liquid PFCs and PFTA or PFE are obtained in a ratio of 5: 1 to 1: 5, subjected to washing after mixing with pyrogen-free aqueous-alcoholic solutions and water, the mixtures are separated by more dense liquid PFOS from a water-alcohol solution and water, filtered under pressure of a gas that does not contain oxygen, poured and sealed into bottles for rolling and carry out autoclaving. 16. The method of preparing the PFOS emulsion according to 14, characterized in that the CA mixture is prepared in the form of a 10-30% aqueous solution of copolymers of polyoxyethylene-polyoxypropylene - proxanol 168 and proxanol 268, taken in a ratio of from 10: 1 to 1:10 depending on the composition of the composition of the PFOS mixture, the CA solution is purified by passing through a carbon sorbent and a sterilizing filter under oxygen-free gas pressure, the CA solution is heated for 8-24 hours and cooled to 16-20 ° C to completely restore the transparency of the solution. 17. The method of preparing the PFOS emulsion according to claim 14, characterized in that the aqueous solution of the mixture of polyoxyethylene-polyoxypropylene copolymers is heated at a temperature that is selected in the range of ± 2 ° C from the cloud point established experimentally for the obtained CA mixture. 18. A method of treating diseases of the circulatory system, in which fast-releasing organofluorine compounds are introduced into the bloodstream or lymphatic duct, characterized in that the emulsion PFOS according to claim 1 is introduced into the bloodstream as fast-releasing organofluorine compounds. 19. The method according to p. 18, characterized in that for the treatment of the phenomena of hypoxia, ischemia, edema, inflammation in shock, blood loss, thrombotic-embolic lesions, functional and organic disorders of vascular patency, tissue damage is carried out by intravenous administration of PFOS emulsion in a dose of 0.5 -30 ml per 1 kg of body weight. 20. The method according to p. 18, characterized in that in order to suppress secondary alteration in inflammation and wound healing, as well as to accelerate the healing of traumatic, trophic and surgical wounds, the PFOS emulsion is administered intravenously in doses of 0.3-6 ml per kg body weight and / or locally in portions of 0.5-50 ml subcutaneously, intramuscularly, in depth around the damaged area, or in the surrounding inflamed and injured tissues of the cavity or biological fluids, in particular intrapleural, intraperitoneal, intralumbal, into the general lymphatic flow at a dose of 1-100 ml, as well as the cleaning liquid or the processing applicative open wound surfaces. 21. The method according to p. 18, characterized in that in order to improve systemic and local blood flow, regulate systemic blood pressure and regional perfusion pressure, facilitate and accelerate mass transfer through gases, substrates and metabolites, intravascular or local injection of PFOS emulsion into tissues is carried out, including including perfusion and non-perfusion protection of organs and tissues isolated or disconnected from the bloodstream.

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