Emulsion of perfluoroorganic compounds of medicinal indication and method for its obtaining

FIELD: medicine.

SUBSTANCE: the suggested emulsion contains quickly excreting perfluoroorganic compounds as perfluorodecalin and perfluorooctylbromide, perfluoroorganic additive and phospholipids in the form of dispersion prepared due to homogenization at pressure of not less than 100 atm. in water-saline medium. Perfluoroorganic additive is being the mixture of perfluorated tertiary amines - perfluorotripropylamine and its co-products: cis- and trans-isomers of perfluoro-1-propyl-3.4-dimethylpyrrolidone and perfluoro-1-propyl-4-methylpiperidine. The method to obtain the emulsion deals with obtaining the dispersion of phospholipids due to homogenization at pressure of not less than 100 atm. in water-saline medium followed by thermal sterilization, then comes homogenization at pressure of the mentioned perfluoroorganic compounds in dispersion of phospholipids and thermal sterilization of the ready-to-use emulsion. The latter is indicated to treat blood losses, hypoxic and ischemic states, improve oxygen supply by blood and keep isolated perfused organs and tissues. In accordance to the present innovation stability of emulsion has been increased and its qualities have been improved. Storage period of emulsion in its unfrozen state at +4 C corresponds to 12 mo, not less, moreover, biocompatibility of emulsion with biological medium (blood, plasma or serum)has been kept.

EFFECT: higher efficiency of application.

20 cl, 13 ex, 21 tbl

 

The invention relates to the field of Biophysics and medicine, in particular to medicines intended for the treatment of hemorrhage, hypoxic and ischemic conditions improve oxygen delivery by blood, save isolated perfuziruemah organs and tissues.

The LIST of SYMBOLS, SYMBOLS, UNITS AND TERMS

PAVsurfactant
P-268, F-268proxanol is chosen 268, pluronic-268
PFDperpendicular
PFMSPperformancecriteria
Co-products PFMSPisomers performatives (Methylcyclopentane) amine, CIS/TRANS-performatives (4-methylcyclohexyl) Amin, performativity- (4 - methylcyclohexyl) amine, CIS/TRANS-PERFLUORO-N-(4 - methylcyclohexyl)-2-methylpyrrolidine
PFOBperforative
PFOperformancesee liquid, is a mixture of performapply and its coproducts: CIS - and TRANS-PERFLUORO-1-propyl-3,4 - dimethylpyridine and PERFLUORO-1-propyl-4-methylpiperidin.
An SFC PFCperfluorocarbons, perfluorinated compounds
DFTBA performability
PPTP (PAF-3)performapply
SFLthe phospholipids of soybean
FLphospholipids
AFLthe phospholipids of egg yolk
nwave Exhibitor
WithVvolumetric content of fluorocarbon phase PFC emulsions (ml/DL)
andthe average particle diameter
λwavelength
Ipindex reactogenicity

The success of the development of infusion media, including emulsion performancesin compounds (PFC) is largely dependent on the physicochemical properties of the selected SFC, physico-chemical properties of emulsions based on them and the technology of producing emulsions SFC.

SFC intended for creation on their basis of medical drugs that are fully fluorinated organic compounds of different classes. In appearance it is a transparent colourless liquid odourless, high density is about 2 times heavier than water. The extraordinary strength of connection C-F (485,6 kJ/mol) leads to the fact that the intermolecular forces in these compounds is very weak. A manifestation of weak intermolecular forces in PFCs is their ANO is real high ability to dissolve gases, including blood gases.

Thanks to the bond strength of C-F, SFC are characterized by chemical inertness. SFC not dissolve in N2Oh and do not undergo metabolism in the body. Chemical inertness SFC does not imply biological inertness. PFCs, injected in the form of emulsions, trapped in organs and tissues, and stay in the organs depends on the nature of the SFC and the dose of the emulsion.

The study of the biological properties of the SFC different classes showed that the rate of removal of organs depends on a number of interrelated physical and chemical parameters of these compounds: structure and molecular weight, boiling point and the elasticity of steam, and the critical temperature of dissolution of PFCs in hexane (Tkr). TCR.is the temperature at which blend together equal amounts of the compounds and hexane. Tkrconsidered as a measure of the relative solubility of PFCs in lipids, which characterizes the speed of their passage through the membrane. Than below Tkrthe better the compound is soluble in lipids and the faster it is excreted from the body. In table. 1 shows the values of physico-chemical parameters that are used as selection criteria for PFCs for medical purposes.

The table is 1 and

The values of the critical temperature of dissolution in hexane (Tkr), the elasticity of the pair (P) and half of the agencies (t1/2for different PPS [1].
Perfluorinated compoundsMMTkrP, mm Hg (37°)t1/2the day
Bicyclo[4.3.0]nonan41213334
Decalin46222127
Decapitalisation5242427
N-(4-methylcyclohexylamine)59538160(90)
1-propyl-2 methylpiperidin483351924
Tripropylamine521431765
Tributylamine671611900
Directroy ether652592500

According to the information provided, there was clear correlation between Tkrand t1/2(half-life). For the elasticity of the pair of such dependence is not detected. To a greater degree the Yeni linked T krand MM SFC. The optimal range MM for PFCs is the interval 460-520. In General, all the proposed criteria for the selection of the SFC for the biological goals do not contradict each other, but are qualitative in nature. Currently, the attention of researchers involved in the development and study of emulsions SFC, concentrated on a relatively small number of connections. In table 2 and 3 shows the structural formula and the main physico-chemical characteristics of the most famous SFC.

Table 2.

Structural formulas of the most well-known and promising PFCs.
Perpendicular (PFD)

< / br>
MM 462
Performapply (PPTP)

< / br>
MM 521
Performability (PFTB)

< / br>
MM 671
Performfiltering-nonan (Ptmn)

< / br>
MM 562
Performatively (Pfma)

< / br>
MM 495
Performancecriteria (PFMRP)

< / br>
MM 595
Perflurocarbon (PFOB)

CF3-(CF2)6-CF2Br

MM 499
bis-perfo butylate (F-44E)

With4F9-CH=CH-C4F9< / br>
MM 464
bis-perhexiline (F-66E)

C6F13-CH=CH-C6F13< / br>
MM 664

During the study the primary biological properties of various SFC issued an important requirement is the absence of unidentifiable impurities. The presence of impurities with unknown properties can distort the true picture of the behavior of the basic substance when administered intravenously to animals (delay in organs, toxicity, effect on various systems of the body).

Table 3.

Physico-chemical properties of the SFC that form the basis of medical drugs [2].
PropertiesPFDPPTPPFMSPPFOBPFDB
Chemical formulaC10F18With9F21NC12F23NC8F17BrC10F21Br
Molecular mass, g/mol462521595499599
Boiling point, °142131168143180
Vapor pressure, MRTT, (37°)a 12.718,02,010,51,5
The critical temperature of dissolution, Tkr°224440-207
The solubility of oxygen ml/100 ml (% vol), (37°)40454053-
Half-life, t1/276590(60)440
Note: PFD/PPTP form the basis of drug Fluor-YES (Fluosol DA); PFD/PFMSP - drug Perftoran; PFOB/PFDB are the basis of different types of drug Oxygent (Oxygent).

Liquid SFC are poor solvents for the various water-soluble biologically active substances. Therefore, to use the SFC as kislorodprovodyashchikh their environments is dispersed in an aqueous solution of the emulsifier to the formation of finely dispersed emulsions.

The ability to exchange for emulsions SFC is determined by the total amount of oxygen contained in the emulsion, the concentration of which obeys Henry's law: directly proportional to the partial pressure of oxygen. The physical principle of the dissolution of gases in the SFC applies to emulsions SFC. The number Of dissolved in the emulsion depends on the content of the fluorocarbon phase and does not depend on the particle size, i.e. the amount dissolved in the PFC emulsions of oxygen close to the calculated values when adding the value of the content of this gas in each phase separately (the amount of dissolved oxygen in the aqueous phase plus the amount of dissolved oxygen in the SFC). The content of inert gases in mixtures of emulsion SFC/plasma also subject to the rule of addition of gas volumes in each phase. Thus, the amount of any gas contained in the emulsion, PFC, may be calculated in accordance with the physical laws of their solubility, based on the partial pressure of gas and the volume ratio of the fractions SFC/N2O. This means that the oxygen content in the PFC emulsions will be higher, the higher is its partial pressure or tension (pO2) and the proportion of fluorocarbon phase.

Specific (functional) the action of any drug when introduced into the organism is determined by its portability, which is characterized by the value of LD50and the lack of side effects, which is mainly manifested in the form of reactogenicity. The value of LD50for PFC emulsions depends largely on particle size. The average particle diameter of PFC emulsions should not exceed 0.2 μm. While the proportion of large frequent the C (with an average diameter excess of 0.4 μm) from 3% to 10% LD50for these emulsions is reduced more than in 2 times. Identification of possible reactogenicity of PFC emulsions is one of the most difficult issues that must be addressed in the design of dosage forms for intravascular injection. With the introduction of reactive drug person can develop different degree of "allergic" reaction from a slight reddening of the skin until anaphylactoid reactions with respiratory arrest and heart.

Most researchers believe that the reactogenicity mainly depends on the nature of the emulsifier used for dispersion of fluorocarbon fundamentals of emulsion and forming adsorption (surface) layer around the particles. Prevalent idea that in the emulsions of the first generation cause of reactogenicity was nonionic surfactant block copolymer of polyoxyethylene and polyoxypropylene Pluronic F 68 (F-68) and his replacement by natural phospholipids completely eliminates the problem of reactogenicity. This view is not entirely correct, since the fat emulsion in spite of the fact that they stabilizirovannyi natural phospholipids, still have the reactogenicity. Reactogenicity emulsions SFC cannot be eliminated simply by using phospholipids as emulsifier and stabilizat the RA SFC. Indeed, it turned out that the reactogenicity emulsions SFC is primarily determined by the surface properties of emulsified particles, i.e. the state of stabilising particle layer of emulsifier. However, along with the chemical structure of the nature of the surfactant molecules, the key parameters which determine how the stability of dispersed systems, and the possibility of side reactions are: the strength of the surfactant with the oil "core" particles of the emulsion, the location of the surfactant molecules on the surface, the density of their packing, the severity of the adsorption properties with respect to proteins and other biologically active molecules in the bloodstream, and finally, the particle size of the emulsion. About the last option should say especially: the reduction in the average particle size of the emulsion in the preparation of Perftoran, stabilized only the block copolymer of polyoxyethylene and polycaproamide - proxanol is chosen 268, which is the closest analogue F-68, contributed to the sharp decrease in the frequency of adverse reactions. Hence, it is clear that surface phenomena (i.e. the interaction of two heterogeneous systems - emulsion and blood or plasma) in the behavior of PFC emulsions with intravenous play a crucial role in the formation of the formulation and techniques for obtaining emulsions SFC. And here you should experiment to find how the composition of the oil is the nucleus and interacts with surfactant, so to test the suitability of the adopted technology of producing emulsions SFC.

In the development presented in this invention the emulsion SFC medical devices and method of reception we explored each recipe and element technologies for biological effect using animated models. It is known that rabbits are one of the equivalents of reactive response to the introduction emulsions SFC is a sharp decrease in the content of neutrophilic leukocytes in the peripheral blood. In the experiment when evaluating potential reactogenicity emulsions SFC uses a metric called the index of reactogenicity Ip, which is determined by the following formula: Ip=SC/SB, where Sa and the number of neutrophils in interest to the original level in control and experimental groups of animals. If the value of the Ip after 5 and 20 minutes after administration of the drug does not exceed 3, then the probability of reactogenicity minimum [3].

There are various ways to obtain emulsions SFC. Emulsion oil-water, which include emulsion SFC and fluorocarbon base, is an oil phase, is obtained by the expenditure of energy. Grinding oil phase is carried out with the help of ultrasound or mechanical means.

The ultrasonic impact dispersion occurs due to the tearing of the efforts of abrupt pressure changes, is the quiet arise due to two reasons. First, the alternation of local contractions and expansions in the liquid during the passage of waves; second, the impact of cavitary, i.e. education and the decay cavities filled dissolved in the liquid gas. The energy and power of ultrasound exposure necessary for obtaining submicron emulsion so great that along with the dispersion lead to loss of connection, C-F. In the aqueous phase of the emulsion appears highly toxic concentrations of ions F-of the order of 3-5 mm. The emulsion containing such F-not suitable for any compensation of blood loss or to preserve perfuziruemah bodies, it should be clean and free from excessive amounts of F-by passing through the ion exchange resin. A second disadvantage of emulsions SFC obtained using an ultrasonic dispersion is very wide size dispersion with an average particle size of 0.1 μm can meet a significant proportion of particles with a size of more than 0.4 μm and particles with a size less than 0.01 μm.

Mechanical dispersion by shaking or vigorous mixing allow only coarse emulsion SFC inappropriate for biomedical use size: more mm To obtain finely dispersed emulsions used method of extrusion of the substance of the dispersed phase through the thin resp is rste in the dispersion medium under high pressure (extrusion method), which leads to the rupture of the driving liquid jet into droplets. Dispersion is caused by the pressure gradient and hydraulic forces of friction. Usually the production of emulsions SFC carried out on the high-pressure homogenizers. The consolidating the progress achieved on the homogenizers grinding particles, i.e. stabilization of emulsions is achieved by using surfactants or emulsifiers. Their stabilizing effect is due to two reasons: firstly, the reduction of excess interfacial surface energy or surface tension decrease; secondly, the formation of structural-mechanical barrier (adsorption layer), ensuring the stability of the particles and preventing their contact or adhesion.

Among a large number of surfactants only a few meet the requirements of the possibility of their use to obtain drugs intended for intravenous administration (table. 4).

Table 4

The main surface-active substances used for producing emulsions PFC
NameThe structural formulaMain features
Proxanol is chosen-268 (Pluronic F-68)Synthetic block copolymer, MM˜13000 (P-268) and ˜9000 (F-68), x is the number of frame link guides the EB block CPE*, the number of links of the PDP. It is soluble in water.
PhospholipidsNatural compound. R1and R2- various chain fatty acids.
Lecithin (egg yolk)R3=N(CH3)3MM - 760-870. Practically insoluble in water.
* CPE - polices ethylene; PDP - polices propylene.

Up to the present time are mainly two emulsifier to obtain emulsions SFC - proxanol is chosen-268 (pluronic-F-68) and phospholipids of natural raw materials (egg yolk, soybean and others).

Structure proxanol is chosen does not match the characteristics of the molecules of water-soluble surfactants that have a polar head (hydrophilic) and nonpolar tail (hydrophobic part). The proxanol is chosen two chain policie ethylene (CPE) cause the hydrophilic nature of the molecules, forming hydrogen bonds with molecules of H2O. Metal group block policie propylene (PDP) determine the lipophilic properties of the molecule. The ratio of blocks CPE: SOCA for F-68 and P-268 on average equally and is 80:20. The stabilizing effect of these emulsifiers is mainly due to the steric effect of the protective film formed by the surfactant molecules around the particles PFCs. Thus n is the number of surfactant, related in the adsorption layer, a significant portion of the surfactant molecules form various micellar structures in aqueous phase, including those available from the SFC. Between the molecules of the surfactant in the adsorption layer and the micelles in the aqueous phase, it is the dynamic balance required to stabilize the adsorption layer on the one hand, and on the other, violating the packing density of surfactant molecules in the adsorption layer during long-term storage.

Phospholipids (PL) are a mixture of compounds of natural origin, the General structure of which is shown in the table. 4. Fluorescence is not soluble in water and at the same time are not a good lipophilic agent in relation to various SFC, although the bilayer particles of phosphatidylcholine (PC) partially soluble PFD and PPTP. The interaction of the individual and the SFC in the aqueous phase is dual in nature. Perhaps the conclusion of the SFC in the lamellar structure of the individual and (or) the formation of monolayers FL irreversibly associated with the surface of the particles. The possible existence of heterogeneous particles in emulsions SFC/vial, i.e. particles covered with a protective layer FL and free vials. This heterogeneity may be related to features of the process and/or with an excess of fluorescence relative to the fluorocarbon phase.

For finely dispersed emulsions SFC determining mechanism to reduce the degree of dispersion (the criminal code of opinie particles) is isothermal or molecular distillation of substances dispersed phase of small particles into larger particles by diffusion of molecules SFC through a dispersive medium. This process is also called ripening of the emulsion according to the Ostwald or reconcretion. The driving force of this process is more saturated vapor pressure over the particles of smaller sizes are relatively larger. In this case, an important parameter is the degree of solubility of the SFC in the aquatic environment. Prevention of precondensation can be crucial to maintain aggregate stability of PFC emulsions, i.e. the preservation of their dispersion and individuality of the particles. The main ways destabilization of the molecular diffusion and the smaller the significance of flocculation or coagulation is typical for relatively dilute emulsions PFCs, in which the content of the fluorocarbon phase does not exceed 20% (by volume), and for a more concentrated emulsion PFCs, in which the volume fraction of the fluorocarbon phase is 50%.

Known ways of stabilizing emulsions PFCs. The General principle of stabilization of colloidal systems - prevention mechanisms of their destruction. Introduction in the emulsion SFC/FL sugars and negatively charged aemulatio (minor components PL) process prevents flocculation of the particles by changing the spatial interactions of the surfactant molecules in the adsorbed layer, and also by increasing the electrostatic repulsive forces between particles.

The reduction of the principal processorsare emulsions SFC, caused by molecular diffusion, reach typically the introduction in fluorocarbon basis of the second component (additional perfluorinated compounds are less soluble in water, having a higher boiling point and slow down this process.

This principle of stabilization used in the development of drugs flusol-YES, perftoran and oxygen. Table 5 presents summary data on the composition and physicochemical properties of these drugs.

Table 5.

Summary data on the composition of drugs Fluor-YES (Japan), Perftoran (Russia), Oxygen (USA) /2/.
Components medicationsConcentration (% weight/volume)
Fluor

YES
PerftoranExigent
AF0104AF0143AF0144
PFD1413---
PPTP6----
PMCP-6,5---
PFOB--907 58
PFDB---32
Pluronic F-68 (proxanol is chosen-268)2,724---
Phospholipids0,4-4of 5.43,6
Potassium oleateto 0.032----
The buffer
Divalent cations++---

In the first two drugs to the PFD, which is the most oil phase, additives introduced perfluorinated compounds PPTP and PFMSP, more storable and less soluble in water. As the emulsifier used is a water-soluble pluronic F-68 with additives FL (flusol YES) or equivalent proxanol is chosen-268 (perftoran). Physico-chemical parameters of the drugs are not very different from each other. They belong to the first-generation drugs, a General lack of which is to you the minute storage in the frozen state due to insufficient stability. In the case of drug exigent to its fluorocarbon based PFOB added PFDB, which has a higher boiling point and less soluble in water. The advantage of the drug exigent related to the second generation, is determined by the ability to store it in is not frozen. In addition, PFOB comprising fluorocarbon based drug rapidly excreted from the body, at about the same speed as the PFD (t1/2˜ 4 and 7 days, respectively).

Drug Oxygent is a commercial name of infusion media, slightly different in composition.

The role of emulsifier in obtaining emulsions is not limited to the reduction of the interphase surface tension in the H2O/SFC necessary to reduce dispersion. The changing nature of the emulsifier may affect the speed of the process of molecular diffusion. Promising recognized fluorinated surfactant-FPAV containing in its molecule a hydrophobic fluorinated and hydrophilic non-fluorinated part. Great success on the synthesis of FPAV PFCs have been made in recent years by the French school of chemistry [4]. The General structure of synthesized FPAV represents a combination of polyfluorinated chain and a polar head. As the connecting link of these elements is taken hydrocarbon chain. The polar head is selected from nature is the breaking of the substances or their derivatives. FPAV, containing a polar head alcohols or derivatives of sugars, show synergism with pluronic F-68. Use as the polar heads of phospholipids, sugars or phosphates of phosphatidylcholine in the structure of FPAV increases the stability of PFC emulsions containing natural fluorescence as an emulsifier. Also we propose a new class of mixed FPAV to stabilize PFC [4]. Molecules of this class of FPAV represent a block of two linear components such as hydrocarbon and perfluorinated. The General formulas of these compounds are written as follows:

CnF2n+1CmH2m+1ornF2n+1SN=SNAmH2m+1

The authors call these molecules "dowel", which means literally "key" or otherwise connecting element, clip.

It is believed that the molecules of FPAV General linear structure RH-RF fulfill the role of a fixing element, the hydrocarbon end of which is included in the lipid film surrounding the particles PFCs, and other fluorinated, included in the oil phase PFC, i.e. molecule type RH-RF improve the adhesive properties of the surface layer of surfactant.

To date PFD and PFOB are the most appropriate connections when creating emulsions biomedical purposes because they are most easily excreted from the body than the other perfluorinated compounds.

Known patents [5, 6], in which the proposed composition of the blood, fluorocarbon based on a mixture of two SFC (PFD/PFMSP or PFD/PTBA or PFOB/PFMRP); mixture of three SFC (PFOB/PFD/PFMSP or PFOB/PFD/PFTB) and even a mixture of four SFC (PFOB/PFD/PFMP/PFTB)taken in different ratios. Dispersed the compounds with water-soluble emulsifier of proxanol is chosen P-268. The use of this emulsifier is not possible to store the emulsion of the aforementioned compositions at positive temperature. In addition, after thawing emulsions of this type have a limited shelf life at +4°With (no more than 1 month). This is their main drawback.

Patented emulsion using fluorinated surfactant FPAV). Microemulsions SFC containing FPAV [7], up to the present time have not found practical application as an infusion medium, most likely due to insufficient stability in vivo. Know of any other composition of the emulsions SFC obtained using mixed FPAV containing in the molecule forevernow and lipophilic parts [8]. These emulsions although maintains the average diameter of the particles at positive temperatures of storage, but only for 3 months.

Known patent [9], in which the source vial to obtain emulsions SFC is fat emulsion Liposyn 10%. As fluorocarbon OS is Ovi emulsions patented three groups SFC. The first group includes perftorsilanami or perftorpolietilena (including PFD, Pretilachlor, Pipelinerouten and others). The second group includes performancemanagement heterocyclic compounds. The third group consists of perfluorinated tertiary amines - PPTP, PFTB, etc. Included in the number of SFC and PFOB. However, with the help of the above emulsion Liposyn 10% failed to obtain a stable emulsion PFD. The maximum shelf life was 25 days.

In the patent [10] used fluorescence egg yolk to obtain emulsions SFC. The content of the fluorocarbon phase in these environments varies widely from 10 to 50 vol.%, and the contents of the vial from 0.5 to 7 wt.%. As the oil phase in the patent uses any one of an SFC selected from a wide class of compounds. In particular, the group of performdragoperation with the number of fluorine atoms from 1 to 24, as well as PFD; perfluorinated amines; PTB; performancerelated and performanceoriented.

The focus in the above two patents is given to methods of preservation of various organs and systems using the obtained emulsions SFC. Before physiological experiments developed emulsion is mixed with crystallinity solutions and/or oncotic agents (albumin, gidroxiatilkrahmal). The proposed emulsion though regarding what are the emulsions of the second generation, but have a major drawback. In both patents are missing observations for the stability of the proposed emulsions, i.e. the conservation of particle size during long term storage (over one month). Patents [9, 10] we considered as analogues.

Closer in essence to the claimed emulsion SFC emulsion is proposed in [11]. This emulsion is used as a prototype, is among the emulsions of the second generation and contains quickly displayed perfluorinated compound in an amount of from 40 to 50% vol. and the perforated Supplement more high-boiling compounds in an amount of 5 to 10 vol.%. As quickly displayed SFC use PFD or PFOB (main component), and as an additive - PFMSP. The emulsifier are used vials of egg yolk or soy.

PFMRP is the stabilizer of the emulsion, reducing the speed of the process of molecular diffusion (precondensation) the main component PFD or PFOB and is used to obtain emulsions with different content, in particular drug Perftoran. The main drawback is claimed in [11] the emulsion SFC is a relatively large average particle diameter is more than 0.2 μm.

The present invention is to increase the stability of the emulsion and improve its quality, i.e. the preservation of biocompatibility with biological medium (blood, plasma or serum) is stored in a frozen state for at least 12 months.

For this proposed emulsion SFC medical devices containing quickly displayed connection PFD and PFOB, performances (perfluorinated) additive and phospholipids, characterized in that it contains phospholipids in the form of a dispersion prepared by homogenization under pressure of at least 100 atmospheres in the water-salt medium, and performancesee additive is a mixture of perforated tertiary amines - performapply and its coproducts: CIS - and TRANS-PERFLUORO-1-propyl-3,dimethylpyrimidine and PERFLUORO-1-propyl-4-methylpiperidine.

The emulsion is characterized by the fact that the total concentration of SFC in the emulsion is in the range from 2 to 40 vol.%;

The emulsion is characterized in that the composition quickly displayed SFC contains PFD and PFOB in a ratio of from 10:1 to 1:10, performancesee additive is from 1% to 50% of the total content of the SFC in the emulsion.

The emulsion is characterized by the fact that performancesee additive in the form of these perfluorinated amines further comprises PERFLUORO-N-methylcyclohexylamine and his coproduct: isomers performatives (Methylcyclopentane) amine, CIS/TRANS-performatives (4-methylcyclohexyl) Amin, performativity-(4 - methylcyclohexyl) amine, CIS/TRANS-PERFLUORO-N-(4-methylcyclohexyl)-2 - methylpyrrolidine.

The emulsion is characterized by Thu which contains the variance of the individual egg yolk or dispersion FL soy or their mixture in the water-salt medium at a concentration of from 0.2 to 5 wt.%.

Emulsion, characterized by the fact that the variance of the vial in the water-salt medium contains an adjuvant in an amount of from 1 to 15% of the total contents of the vial; adjuvant is vegetable oils: soybean, sunflower or castor.

The emulsion is characterized as adjuvant comprises a mixture of vegetable oils selected from the group including soy, sunflower and castor oil in the effective value.

The emulsion is characterized by the fact that the water-salt medium contains sodium and potassium salts of chlorides and phosphates and monosaccharide mannitol in water for injection and the concentration of the components of the water-salt medium is the osmotic pressure in the range from 100 to 350 momola per litre.

The emulsion is characterized by the fact that the average particle diameter not greater than 0.2 μm and is in the range 0.06 to 0.2 μm.

The emulsion is characterized by the fact that the shelf life in frozen form at + 4°With not less than 12 months.

The method of obtaining the claimed emulsion performancesin compounds, including the production of dispersions of phospholipids by homogenization under pressure of at least 100 atmospheres in the water-salt medium with subsequent heat sterilization, homogenization under pressure specified performancesin compounds in dispersion of phospholipids and heat sterilization of the finished emulsion.

The method of obtaining C is revealed emulsion SFC is characterized by that homogenize the SFC in dispersion FL under pressure from 300 to 650 bar.

The method of obtaining the claimed emulsion SFC characterized by the fact that the variance of the vial is sterilized at a temperature of 100°C.

The method of obtaining the claimed emulsion SFC characterized by the fact that the emulsion SFC sterilized at a temperature of 100°C.

As stated above, the object of the invention is to increase the stability of the emulsion and improve its quality, i.e. the preservation of biocompatibility with biological medium (blood, plasma or serum) when stored in a frozen condition for at least 6-12 months. The term "biocompatibility" is not uniquely defined in the scientific literature and includes different values and needs to be clarified in relation to the emulsions SFC. In the quotation above patents [8-11] the term biocompatibility use the following views is a relatively high rate of removal of organs selected SFC; the ability to maintain tissues and organs through the blood vessels which perpusilla emulsion SFC; relatively low toxicity to animals (at least two of the volume of circulating blood). These views are not mutually exclusive, but does not reflect the first stage of interaction between dispersed particles SFC with plasma and blood when hit emulsion SFC in the bloodstream. We consider the phenomenon bioso the compatibility, since the severity of the interaction (response) emulsion SFC with the biological medium (blood, plasma or serum). The result of this interaction can be assessed not only in vivo, but first of all in vitro experiments on the degree of stability of the emulsion when the impact of a number of factors, simulating damage adsorption layer during storage and penetration of the emulsion into the bloodstream.

The quality and stability of emulsions SFC generally be characterised on the basis of particle size: average particle diameter in the PFC emulsions should not exceed 0.2-0.3 microns. This approach is insufficient to disperse drugs medical-biological purposes, administered intravenously. This is due to the fact that the particles PFCs as foreign material, when injected into the bloodstream interact with proteins and molecules of other compounds present in the plasma, and blood cells. The General nature of the interaction depends on the surface properties of the particles. Functional activity (gas transport function) of PFC emulsions also depends largely on the compatibility of the surface of the emulsified particles of blood and plasma, as in the case of, for example, activation of the complement system on the alien surface, it starts a cascade of reactions that cause vasospasm and impaired regional blood flow. It should also be noted that the Stabi is inost emulsions in vitro is largely determined by the properties of the surfactant adsorption layer around the particles (strength, the surface topography and other). In light of the above question concerning the stability of emulsions PFCs cannot be solved only by using conventional colloidal-chemical methods for the study of particle size without assessing the peculiarities of the structure. It is highly important development for this purpose is quite simple methods and approaches to obtain information about the particle size and the integrity of their structure. It should also clarify the notion of structure as applied to the PFC emulsions.

Progress in the study of the stability of emulsions PFC in vitro and in vivo is associated with the expansion and deepening of the concept of patterns of PFC emulsions and methods for its study. The notion of stability of any drug or substance is determined by the stability of its properties. The parameters used to characterize the properties of PFC emulsions are not exhaustive characteristics of their stability. Experiments expanded the understanding of the criteria of stability of emulsions SFC with consideration of the peculiarities of their structure.

The stability of PFC emulsions are usually estimated on the basis of changes in particle size during storage. Such pure colloid-chemical approach is not sufficient. For emulsions PFCs, which is the basis of drugs intended for intravenous becomes very important information about their stability n is only in experiments in vitro, but the ability to predict their stability during circulation in the bloodstream. Obtaining such information can be achieved, if more clearly outline view of the structure of PFC emulsions. Particles of PFC emulsions have a two-layer structure of a ball in the center of which is PFCs (core particles), and on the surface layer of emulsifier (shell) [12]. The shell thickness of the emulsifier is small and is not more than 5-10% of the particle diameter. However, the behavior of PFC emulsions in the bloodstream (interaction with plasma proteins and blood cells, the rate of excretion and others), as well as stability during prolonged storage will largely depend on the strength and condition of surfactants around the particle. Therefore, it is necessary to obtain information about the particle size and structural changes taking place in the studied environments under certain influences.

For theoretical description and analysis of changes in the structure of emulsions SFC, as the basis of infusion media, it is proposed to consider the following representation [13].

1) "General structure" of PFC emulsions and its changes. This aspect is characterized by a mean diameter and distribution of particle sizes.

2) "the Microstructure, which is determined by the state of emulsifier in the shell, the degree of its interaction with the PFC, the mutual location of the Molek the surfactant, their ordering, packing density, degree of oxidation, the phase state of structured molecules.

So far, all the researchers were limited to the analysis of the "General structure"that is not enough, because the stability of emulsions, biocompatibility, and in particular the surface properties of the particles and their adsorption capacity, determined by the "microstructure".

In the present invention the emulsion SFC compared with the prototype and studied, first, on the parameters characterizing the change of the overall structure in different periods of storage of the obtained emulsions.

Secondly, simulated the effects of damaging the emulsion factors under conditions that allow to assess the condition of the "microstructure" of the PFC emulsion. In particular, we used "stress effect" in the form of dilution of the emulsion with water and determined the change of parameters compared with native emulsion. Breeding of PFC emulsions water disturbs the existing equilibrium between the adsorption layer surfactant (shell) and the molecules of the surfactant in the dispersion medium. Therefore, it has a certain predictive power in relation to the stability of the metastable system (emulsion SFC) or its destruction.

In addition, the observed microstructure changes and compatibility emulsions SFC in contact with the serum to the JVI as a model system to study the biocompatibility of emulsions SFC in experiments in vitro). The interaction of two heterogeneous disperse systems - serum and emulsion PFC - characterizes the change in the surface properties of the particles when injected into the bloodstream and microstructure changes of PFC emulsions during storage. Observation of the dynamics of the overall structure and microstructure conducted at the same time, within 12 months of storage of the emulsions PFCs.

To detect differences in these parameters during storage of the emulsions SFC required methods and approaches that did not contribute additional perturbations in the studied system while making the necessary measurements. As such have been selected, tested and developed optical methods of control.

To assess the overall structure was selected method spectrodensitometer or turbidity spectrum method (CM) [14]. It also used to estimate the distribution of particle sizes in the studied emulsions after centrifugation and fractionation. The change of microstructure of emulsions or surface properties of the particles, caused by the changing relationship of the surfactant molecules in the adsorption layer around the SFC, was estimated using the indirect method of finding the index of interaction (Kτ) studied emulsions with serum in relation to the physiological solution: the relative turbidity Toτ12whereτ 1and τ2- turbidity mixtures "serum/emulsion and serum/saline" with appropriate changes in the ratios of components of mixtures [15]. Additionally, to confirm the immutability of nature emulsified particles PFCs (lack of free FL) was performed to compare the experimental and calculated values τ: τest.=ΣNi·τi(ΣNi=1), where τiand Ni- turbidity and percentage of selected fractions; τexp.the turbidity of the same sample of emulsion fractionation.

I. the Following is the specific formulations of the proposed emulsion SFC in accordance with the present invention.

Composition 1. The emulsion contains 40% vol. fluorocarbon phase (Cvin the form of PFD and PFOB taken in the ratio 1:1, with perfluorinated additive in the form of a mixture of performapply and its coproducts: CIS - and TRANS-PERFLUORO-1-propyl-3,4-dimethylpyridine and PERFLUORO-1-propyl-4-methylpiperidin in the amount of 50% of the total content of the main SFC, stable emulsified state 5% dispersion vial containing AFL and adjuvant in the form of castor oil, taken at a concentration of 15% from the total content AFL, in the water-salt medium of the following composition: 2 mm (115 mg liter) of sodium chloride, 2 mm potassium phosphate one-deputizing (310 mg anhydrous with the and per liter), 7.5 mm sodium phosphate disubstituted (460 mg of anhydrous salt per liter), 318 mm mannitol (58 g of mannitol per liter) in water for injection. The osmotic pressure of the emulsion is 310 of momola per litre. The average particle size of the emulsion of € 0.195 μm.

Part 2. The emulsion according to example (composition) 1, characterized in that the emulsion contains 20 vol.% fluorocarbon phase (Cvin the form of PFD and PFOB taken in the ratio of 10:1, with perfluorinated additive mixture of performapply and its coproducts: CIS - and TRANS-PERFLUORO-1-propyl-3,4-dimethylpyridine and PERFLUORO-1-propyl-4-methylpiperidin, optionally containing PERFLUORO-N-methylcyclopentadiene and his coproduct isomers performatives (Methylcyclopentane) amine, CIS/TRANS-performatives (4-methylcyclohexyl) Amin, performativity-(4-methylcyclohexyl) amine, CIS/TRANS-PERFLUORO-N-(4-methylcyclohexyl)-2-methylpyrrolidine in total amount of 25% of the content of the main SFC, stable emulsified state, 2.5% of the variance vial containing SFL and adjuvant in the form of soybean oil, taken at a concentration of 10% from the total content AFL, in the water-salt medium of the following composition: 2 mm sodium phosphate one-deputizing (276 mg of anhydrous salt per liter), 7.5 mm sodium phosphate disubstituted (460 mg of anhydrous salt per liter), 278 mm mannitol (50 g of mannitol per liter) in the de for injection. The osmotic pressure of the emulsion is 270 momola per litre. The average particle size of the emulsion is 0.1 ám.

Part 3. The emulsion of example 1, characterized in that the emulsion contains 15 vol.% fluorocarbon phase (Cvin the form of PFD and PFOB taken in the ratio 1:10, with perfluorinated additive mixture of performapply and its coproducts: CIS - and TRANS-PERFLUORO-1-propyl-3,4-dimethylpyridine and PERFLUORO-1-propyl-4-methylpiperidin, optionally containing PERFLUORO-N-methylcyclohexylamine in the total amount of 5% of the content of the main SFC, stable emulsified state 2% dispersion vial containing a mixture of AFL and SFL, as well as adjuvant in the form of sunflower oil, taken to a concentration of 5% of the total contents of the vial in the water-salt medium of the following composition: 1 mm sodium phosphate one-deputizing (138 mg of anhydrous salt per liter), 3.7 mm sodium phosphate disubstituted (230 mg anhydrous salt per liter), 100 mm mannitol (18 g of mannitol per liter) in water for injection. The osmotic pressure of the emulsion is 105 momola per litre. The average particle size of the emulsion of 0.08 μm.

Part 4. The emulsion of example 1, characterized in that the emulsion contains 10 vol.% fluorocarbon phase (Cvin the form of PFD and PFOB taken in the ratio 2:1, with perfluorinated additive mixture of performapply and his coproduct is in: CIS - and TRANS-isomers of PERFLUORO-1-propyl-3,4-dimethylpyridine and PERFLUORO-1-propyl-4-methylpiperidin, optionally containing PERFLUORO-N-methylcyclohexylamine in the total amount of 0.2% of the content of the main SFC, stable emulsified state 2% dispersion vial containing AFL, as well as adjuvant in the form of a mixture of sunflower and castor oil, taken at a concentration of 2% from the total content AFL, in the water-salt medium of the following composition: 1 mm sodium phosphate one-deputizing (138 mg of anhydrous salt per liter), 3.7 mm sodium phosphate disubstituted (230 mg anhydrous salt per liter), 90 mm mannitol (13 g of mannitol per liter) in water for injection. The osmotic pressure of the emulsion is 100 momola per litre. The average particle size of the emulsion of 0.07 μm.

Part 5. The emulsion of example 1, characterized in that the emulsion contains 2% vol. fluorocarbon phase (Cvin the form of PFD and PFOB taken in the ratio 1:2, with perfluorinated additive mixture of performapply and its coproducts: CIS - and TRANS-PERFLUORO-1-propyl-3,4-dimethylpyridine and PERFLUORO-1-propyl-4-methylpiperidin, optionally containing PERFLUORO-N-methyl-cyclohexylpiperidine in total amount of 10% of the content of the main SFC, stabilized emulsified 0.2% dispersion vial containing SFL, as well as adjuvant in the form of a mixture of soybean and castor oil, taken to a concentration of 5% of the total content SFL, water is alevai medium of the following composition: 2 mm (115 mg per liter) of sodium chloride, 2 mm sodium phosphate one-deputizing (276 mg of anhydrous salt per liter), 7.5 mm sodium phosphate disubstituted (460 mg of anhydrous salt per liter), 318 mm mannitol (58 g of mannitol per liter) in water for injection. The osmotic pressure of the emulsion is 350 momola per litre. The average particle size of the emulsion 0.06 micron.

Part 6. The emulsion of example 1, characterized in that the emulsion contains 10 vol.% fluorocarbon phase (Cvin the form of PFD and PFOB taken in the ratio of 4:1, with perfluorinated additive mixture of performapply and its coproducts: CIS - and TRANS-PERFLUORO-1-propyl-3,4-dimethylpyridine and PERFLUORO-1-propyl-4-methylpiperidin in the total amount of 4% of the content of the main SFC, stable emulsified state 2% dispersion vial containing SFL, as well as adjuvant in the form of a mixture of sunflower, soybean and castor oil, taken at a concentration of 4% from the total content SFL, in the water-salt medium of the following composition: 2 mm sodium phosphate one-deputizing (276 mg of anhydrous salt per liter), 7.5 mm sodium phosphate disubstituted (460 mg of anhydrous salt per liter), 200 mm mannitol (36 g of mannitol per liter) in water for injection. The osmotic pressure of the emulsion is 225 of momola per litre. The average particle size of the emulsion 0,09 ám.

Table 6 shows the compositions of the emulsions in accordance with izaberete is receiving in examples 1-6.

Table 6

The composition of the emulsions SFC in examples 1-6.
# exampleCvvol.%, PFD/ PTBSFC Supplement relates. content.The dispersion of the fluorescence (wt.)%Adjuvant treat. contentOsmolarityParticle sizeThe aqueous phase composition in mm
No. 140% vol 1:1PFO

50%
AFL 5%The castor. 15%310 mOsmof € 0.195 μm2l

2 NaH2P

7,5 Na2HP

318 mannitol
No. 2

20 about% 10:1

PFO PFMSP*

25%
SFL 2,5%

Soy

10%

270 mOsm

0,10 µm

2 NaH2P

7,5 Na2HP

278 mannitol
No. 325 vol% 1:10PFO PFMSP*

5%
AFL SFL

2%
Podsols. 10%110 mOsm0,08 µm1 NaH2P

3,7 Na2HP

100 mannitol
No. 4Ob%

2:1
PFO PFMSP*

0,2%
AFL

2%
Podsols. the castor.

2%
100 mOsm of 0.07 μm1 NaH2P

3,7 Na2HP

95 mannitol
No. 52 on% 1:2PFO PFMSP*

10%
SFL

0,2%
Soy castor.

5%
350 mOsm0.06 micron2 NaCl

2 NaH2P

7,5 Na2HP

318 mannitol
No. 610 about% 4:1PFO

4%
SFL

2%
Podsols. soy castor.

2%
225 mOsm0,09 ám2 NaH2P

7,5 Na2HP

200 mannitol
* PFMSP here and below is performancecriteria and his coproduct: isomers performatives (Methylcyclopentane) amine, CIS/TRANS-performatives (4-methylcyclohexyl) Amin, performativity-(4 - methylcyclohexyl) amine, CIS/TRANS-PERFLUORO-N-(4-methylcyclohexyl)-2-methylpyrrolidine

II. The following are specific examples of the method of producing emulsions SFC claimed composition and physico-chemical parameters.

Example 1. The emulsion was prepared under aseptic conditions.

1.1. For preparation of 1 l of emulsion containing 10 vol.% SFC, prepared 1% of the variance of the individual.

1.2. The first stage of the preparation of dispersion vials: sterile round bottom flask was filled with 100 ml of 10%alcoholic solution AFL and is chased alcohol on a rotary evaporator, added 1 g of castor oil (adjuvant concentration of 10% of the content AFL) and poured into 900 ml of water-salt solution.

1.3. For the preparation of water-salt solution is also used pyrogen-free water. Powder one-deputizing sodium phosphate, powder disubstituted phosphate of sodium and crystalline mannitol was dried in a dry-heat Cabinet at a temperature of 110°C for 2 hours. Then the sample was 0.138 g of anhydrous one-deputizing sodium phosphate, 0,523 g anhydrous disubstituted phosphate of sodium and 50.0 g of mannitol was dissolved in aseptic conditions in a laminar box in 1 l of pyrogen-free water. The resulting aqueous salt composition was passed through a sterile filter company Millipor with a pore diameter of 0.4 µm.

1.4. A mixture of phospholipids, vegetable oil and water-salt solution is mechanically stirred in the flask before the formation of a homogeneous suspension milky yellowish color. The resulting suspension vial was transferred into a sterile container homogenizer high pressure.

1.5. The homogenizer is pre-sterilized by passing superheated water vapor transmission of rubbing alcohol in a volume of 500 ml and washing with 500 ml of hot pyrogen-free water.

1.6. Suspension vial was dispersively in the homogenizer at a pressure of 100 ATM 4x transmission to the formation of the translucent is a homogeneous liquid - dispersion vial, which was filled into vials. Through the vials missed a sterile inert gas (nitrogen, argon or a mixture of nitrogen and carbon dioxide) within 2-4 minutes.

1.7. The vials were closed with rubber stoppers and cavalcaselle under running aluminum caps. Then the vials were sterilized by heat treatment at 100°C for 1 hour. The vials were kept at room temperature before the next acquisition phase of the emulsion SFC.

1.8. The next step was to process the SFC. Mixed 72 ml PFD with 8 ml PFOB. To 80 ml of this composition SFC was added 20 ml PFOS. The resulting composition is a mixture of PFD and PFOB with SFC additive was mixed with an equal volume of alcohol medical. Perfluorocarbon phase as the heavier separated from the alcohol in a separating funnel. Separated from the alcohol mixture SFC was mixed with 3-fold volume of pyrogen-free water, were shaken and separated in a separating funnel (specific gravity SFC almost twice the weight of water).

1.9. To further benefit from the emulsion. In the working capacity of the homogenizer was made 900 ml of the dispersion vials and 100 ml of the treated mixture SFC (composition PFD/PFOB=9/1 + PFO - 20%), all contents of the working capacity mechanically stirred and subjected to the dispersion under a pressure of 500 atmospheres, passing the entire volume 8 times through a high pressure chamber, before the formation of translucent opalescens the th yellowish fluid - submicron emulsion SFC. The emulsion was poured into 100 ml flasks were closed with rubber stoppers and cavalcaselle aluminum caps.

1.10. Vials of emulsion SFC sterilized by heating at 100°C for 1 hour, cooled and kept at 4°during the year.

The composition of the obtained emulsion: the content of the fluorocarbon phase (Cvis 10 vol.%, the ratio of the PFD/PFOB equal to 9/1, the relative amount of PFOS in a mixture of SFC is 20%, the concentration of APL - 1 wt.%, the concentration of castor oil is 0.1% (relative quantity of castor oil as adjuvant suspension is 10% of the total content AFL). Series 1.

Determined the viscosity of this series in the viscometer VPI-2. Its value amounted to 0,953 SDR. For comparison, the viscosity of the drug Perftoran with the same content of the fluorocarbon phase is 2.5 SDRs.

Example 2. Emulsion SFC was prepared as described in example 1, the identical composition. Only as an adjuvant to AFL took a mixture of two oils: castor and soybean oil at a ratio of 1/1. The composition ofv- 10 vol.%, the ratio of the PFD/PFOB equal to 9/1, the relative content of PFOS is 20%, the concentration of APL - 1 wt.%, the relative content of the adjuvant is a mixture of two oils (castor oil/soybean oil = 1/1) - 10%. Series 2.

Example 3. Emulsion SFC was prepared as described in example 1, but in a volume of 800 ml andkeeping 20% vol. SFC, identical to example 1 composition. In a round-bottom flask was introduced 200 ml of 10%alcohol solution of soybean FL (SFL). Alcohol drove on a rotary evaporator, was added into the flask adjuvant is a mixture of soybean oil/kastornoe oil in the ratio1/2- in the total amount of 3 g, which was 15% of the total number SFL. Water-salt solution contained 0,276 g anhydrous one-deputizing sodium phosphate, at 1,046 g anhydrous disubstituted phosphate of sodium and 10.0 g of mannitol. Into the flask containing SFL and adjuvant, was introduced 1 liter of water-salt solution, was dissolved, was transferred to a homogenizer, was dispersively, poured into vials were sterilized as in example 1. Preparing fluorocarbon phase. To 160 ml PFD was added 40 ml PFOB, selected 160 ml of this composition and mixed it with 40 ml PFOS. 200 ml of the mixture obtained after SFC purification dug in the homogenizer, which was 800 ml of the dispersion SFL. The resulting emulsion was poured into vials and sterilized.

The composition of the emulsion: Withv=20 vol.%, the ratio of the PFD/PFOB = 8/2; the relative content of PFOS - 20%; the concentration of SFL equal to 2 wt.%, the relative content adjuvant (mixture of two oils - soybean oil/castor oil in the ratio1/2) is 15%. Series 3.

Example 4. The emulsion prepared as in example 1 except that the ratio of components in the composition PFD/ PFOB was also the same and equal to 8/2. To 170 ml of this composition was added 30 ml PFMSP, mixed by shaking, was purified in a standard way and dug in the homogenizer, where there were 800 ml of the dispersion SFL (obtained as in example 3)containing the same adjuvant is a mixture of two oils: soybean oil/castor oil in the ratio1/2in the amount of 15% of the content SFL. The emulsion was dispersively under pressure of 400 ATM.

The composition of the emulsion: Withv=20 vol.%; the ratio of the PFD/PFOB = 8/2; the relative content PFMSP - 15%; the concentration of SFL equal to 2 wt.%, the relative content adjuvant (a mixture of two oils: soybean oil/castor oil in the ratio1/2in the dispersion SFL is 15%. Series 4.

Example 5. The emulsion was obtained as in example 1, except that used a different number of AFL to obtain a dispersion of the individual. 50 ml of alcohol solution AFL was placed in a round-bottom flask was distilled alcohol on a rotary evaporator, placed there by 0.6 g of sunflower oil added 0,95 l salt solution was mixed by shaking and homogenized at a pressure of 150 ATM. The composition of the PFD/PFOB in the ratio of 5/5 was prepared by mixing 25 ml PFD with 25 ml PFOB. a 49.5 ml of this mixed with 0.5 ml PFOS. Prepared with 50 ml of a mixture of SFC, after cleaning dug in the homogenizer, which was previously filled 0,95 l suspension AFL. Homogenization preliminary rough var is rsii carried out at a pressure of 350 ATM. The filling and sterilization of finely dispersed emulsion was performed according to the specified rules.

The composition of the obtained emulsion: Withv=5 vol.%; the ratio of the PFD/PFOB=5/5; the relative content of PFOS is 1%; the concentration of AFL equal to 0.5 wt.%; the relative content of sunflower oil, taken as adjuvant, is 12%. Series 5.

Example 6. 50 ml of 10%alcohol solution SFL was placed in a round-bottom flask was distilled alcohol according to the described method, put 0.6 g of soybean oil was added to 950 ml of saline solution, after stirring moved in a homogenizer to obtain a dispersion at a pressure of 180 atmospheres. After sterilization, it was used to obtain the emulsion.

The composition of the PFD/PFOB ratio (5/5) was obtained by mixing 25 ml PFD with 25 ml PFOB. To 49.5 ml of this composition was added 0.5 ml PFMRP. After cleaning with alcohol 50 ml of the mixture was given her in the homogenizer, which was 950 ml of the dispersion SFL. Homogenization was performed as described previously, in two stages. First, at a pressure of 200 ATM, and then at a pressure of 500 ATM.

The composition of the obtained emulsion: Cv=5 vol.%; the ratio of the PFD/PFOB=5/5; the relative content PFMRP is 1%; the concentration of SFL equal to 0.5 wt.%; the relative content of soybean oil, taken as adjuvant, is 12%. Series 6.

Example 7. Prepared suspension with con what entrala SFL, equal to 0.2 wt.%. For this purpose, 20 ml of alcohol solution SFL was placed in a rotary evaporator, drove the alcohol, put 0.02 g adjuvant mixture of two oils of sunflower and soybean in the ratio of 1/1. Added into the flask 980 ml of saline solution. Dispersion and sterilization was carried out as described in example 6.

The composition of the PFD/PFOB obtained by mixing 4 ml PFD and 16 ml PFOB (the ratio of the components 2/8). To 19 ml of the mixture SFC added 1 ml PFMRP. 20 ml of the resulting 3-component mixture dug in the homogenizer, which was previously filled 980 ml suspension SFL. The dispersion process was performed as indicated in the previous example, the filling and sterilization of the resulting emulsion was carried out by a standard method.

The composition of the obtained emulsion: Cv=2%; the ratio of the PFD/PFOB is 2/8; the relative content PFMRP is 5%; the concentration of SFL is 0.2 wt.%; the relative amount of the adjuvant is a mixture of sunflower and soybean oils in a ratio of 1/1 is equal to 1%. Series 7.

Example 8. To obtain an emulsion containing 40% vol. prepared suspension AFL at a concentration of 5 wt.%. For this, 500 ml of alcohol solution AFL was placed in a round bottom flask, kept the alcohol, making it 2.5 g of castor oil as adjuvant, poured it 600 ml of saline solution was stirred and dis is were argiroupoli on the homogenizer at 200 atmospheres until a homogeneous medium yellowish-white color. Sterilized it as mentioned previously.

The composition of the SFC was prepared by mixing 40 ml PFD with 360 ml PFOB (the ratio of the components 1/9). To 360 ml of this composition was added 40 ml PFMRP. Received 400 ml of 3-component mixture SFC dug in the homogenizer, where there were 600 ml of previously obtained suspension AFL. Homogenization was carried out in two stages: first at a pressure of 250 atmospheres, the second at a pressure of 600 atmospheres. Bottled and sterilized emulsion in accordance with the adopted methodology.

The composition of the obtained emulsion: Cv=40%vol.; the ratio of the PFD/PFOB=1/9; the relative amount of additive PFMRP is 10%; the concentration of AFL in emulsion (suspension) is 5 wt.%; the relative content of adjuvant - castor oil-equal to 5%. Series 8.

Example 9. Fluorocarbon phase emulsion was prepared by mixing 40 ml PFD with 360 ml PFOB. To 320 ml of this composition was added 80 ml of a mixture containing 40 ml PFMRP and 40 ml PFOS. Suspension of the emulsifier contained 4.2 wt.% AFL, 4.2 wt.% SFL and adjuvant is a mixture of castor and sunflower oils in a ratio of 9/1 in the amount of 4.2 g, i.e 5% of the total content AFL.

To obtain emulsion homogenizer was filled with 600 ml of the resulting suspension and dug into the homogenizer 400 ml 3-component mixture SFC of the above composition. The homogenization process, filling and sterilization was carried out as in PR the previous example.

The composition of the emulsion: Cv=40%vol.; the ratio of the PFD/PFOB=1/9; the relative content of the mixture PFMSP with PFO is 20%; the concentration of fluorescence (a mixture of AFL and SFL 1:1) emulsion 5 wt.%; the content of the adjuvant - 0.25% (mixture of two oils of castor and sunflower in a ratio of 9/1). Series 9.

Table 7

The composition of the obtained emulsions SFC, various series (examples 1-9)
the number of seriesCv about.

%
The ratio of the PFD/PTBSFC Supplement relates. content.FL,

(weight.) %
View adjuvantAdjuvant refers. content, %
No. 1109/1PFO 20%AFL

1%
the castor.10
No. 2109/1PFO 20%AFL

1%
the castor./Soi 1/110
No. 3208/2PFO 20%SFL

2%
castor/Soi 2/115
No. 4208/2PFMSP* 15%SFL

2%
the castor./Soi 2/115
No. 555/5PFO 1%AFL

0,5%
podsol.12
No. 655/5PFMSP* 1%SFL

0,5%
soy12
No. 722/8PFMSP* 5%SFL

0,2%
soy/podsol.

1/1
1
No. 8401/9PFMSP* 10%AFL

5%
the castor./podsol. 9/15
No. 9401/9PFO 10% PFMSP* 10%AFL

2.5% of SFL 2,5%
the castor./podsol 9/15
* PFMSP here and below is performancecriteria and his coproduct: isomers performatives (Methylcyclopentane) amine, CIS/TRANS-performatives (4-methylcyclohexyl) Amin, performativity-(4-methylcyclohexyl) amine, CIS/TRANS-PERFLUORO-N-(4-methylcyclohexyl)-2-methylpyrrolidine

Table 7 shows the composition of all series of emulsions.

Later in tabular form (table. 8) shows the results of observation of the change in the mean diameter of the particles for native (undiluted) and diluted with water emulsion in different SFC in different periods of storage.

with 3.27
Table 8.

Testing the value of the wave exponent and the mean diameter of the particles for both the original and diluted with water samples of the emulsion PFC, obtained according to the examples 1, 3, 4, 5, 8, 9.
the number of seriesStorage time, monthsnand, um
ex.RSV:2EX.times. 1:2
1-0103,403,200,1140,13
13,333,330,1190,119
33,233,200,1280,13
6with 3.273,230,1240,128
93,133,300,1360,121
123,053,140,143is 0.135
1-030with 3.273,330,1250,119
13,333,330,1190,119
33,133,200,1360,13
63,20with 3.270.1300,124
123,203,130,1300,136
1-0403,203,130,130,136
13,173,00,1320,148
32,873,100,1650,14
6of 3.073,100,141was 0.138
93,10of 3.07was 0.1380,141
122,942,850,1550,17
1-0503,20with 3.270,130,124
13,13of 3.070,1360,14
33,03of 3.070,1460,14
6of 3.073,030,1410,145
122,973,100,148was 0.138
1-0803,330,1130,124
13,233,260,1280,126
33,103,200,1390,13
63,033,110,140,14
92,883,100,1640138
122,872,90,1650,160
1-0903,163,200,1340,13
13,03,130,1480,137
33,0of 3.070,1480,141
62,863,02of 0.182of) 0.157
122,7of 3.07of € 0.1950,14

Calculations n was performed using the method of least squares. The RMS error in the determination of n was equal to 0,01-0,03. Hence the error in the determination of n is 0.3-1%. The parameter n is the characteristic function is y used method of turbidity spectrum and is calculated is not less than 3-5 points. For finely dispersed emulsions SFC n are positively correlated with the average diameter of the particles [14].

The results show that the averaged parameters n and practically did not change during 12 months of storage. Dilution as stress exposure had little impact on the size of the particles. Several large was the increase in the values of and for emulsions containing SFL, long-term follow up: 9-12 months. The change interval of the wave of the Exhibitor within 1 year of storage for all series of emulsions PFC/dispersion of the fluorescence was in the range of 3.4 and 2.7. This is consistent with the increase in the average particle diameter of from 0.11 to 0.15-of € 0.195 μm.

To monitor the change in the distribution of particle sizes used the fractionation of the studied environments. The emulsion was centrifuged at mild conditions (1500 rpm) and defined (exactly) 3 faction: top 20%, middle 60%, bottom 20% of the total sample (Fig. 1). As shown in Fig. 1, the emulsion SFC, taken as a prototype, in addition to the three fractions, differing in the average particle size of the emulsion of an SFC has a top light fraction containing free phospholipids (PLC), which shows a weak connection adsorption layer with performancesee oil phase and not related to the adsorption layer of surfactant. For each faction, find the values of n and A. The values of the mentioned parameters the ditch for fractionated emulsions of the claimed composition when stored within 1-12 months are presented in table. 9. It turned out that n and for the upper and middle fractions remained practically unchanged within 1-12 months of storage. At the bottom fraction slight increase in particle size with increasing storage time. This has led to an increase in the width of the distribution of particle sizes. The maximum width of the distribution was in the range of 0.06 to 0.19 μm.

The results showed that within 12 months of storage, the average particle diameter for native emulsion and dilution with water (stress-impact) increased slightly, while remaining within the acceptable range: less than 0,20 mm.

3,6
Table 9.

The parameters n and a, characterizing the width of the distribution of particle sizes for PFC emulsions according to examples 1, 3, 4, 5, 8, 9 within 12 months of storage (top., est., bottom, the fraction determined after centrifugation)
the number of seriest, monthsbreedingnand, um
the upper classes.core.min.the upper classes.core.min.
1-010b/p3,50with 3.27with 3.270,1050,119
1:2a 3.873,392,930,050,114of) 0.157
1b/p3,47with 3.273,00,1070,1240,148
1:23,573,433,130,0950,1110,136
3b/p3,433,332,930,110,119of) 0.157
1:23,833,303,230,0620,1210,128
6b/p3,47with 3.273,00,1060,1240,148
1:2a 3.93,43,130,0440,1140,136
12b/p3,47with 3.272,780,1070,1240,185
1:23,332,930,090,1190,156
1-030b/p3,372,872,83amount of 0.1180,1650,175
1:23,473,473,17to 0.108to 0.108of 0.133
1b/p3,4is 3.082,80,1140,1410,183
1:23,63,263,200,090,1250,13
3b/p3,333,232,870,1190,1280,165
1:23,503,333,030.104 g0,1190,146
6b/p3,533,333,100,1010,1190,139
1:23,53,333,130.104 g 0,1190,136
12b/p3,53with 3.272,930,100,124of) 0.157
1:23,63,43,00,090,1140,148
1-040b/p3,33of 3.07of 3.070,1190,1410,141
1:23,33,173,130,1210,1310,136
1b/p3,23,132,930,130,136of) 0.157
1:23,333,233,030,1190,1280,145
3b/pwith 3.273,032,740,1240,146of € 0.195
1:23,33,22,80,1210,13of 0.182
6 b/p3,333,17was 2.760,1190,132of € 0.195
1:23,503,202,930.104 g0,13of) 0.157
12b/p3,17of 3.072,80,1320,141of 0.182
1:23,333,202,930,1190,13of) 0.157

td align="center"> b/p
Continuation of table 9
the number of seriest, monthsbreedingnand, um
the upper classes.core.min.the upper classes.core.min.
1-050b/p3,03,232,930,1480,128of) 0.157
1:23,603,333,060,090,1190,141
13,333,172,90,1190,132rate £ 0.162
1:23,47with 3.27of 3.070,1060,1240,141
3b/p3,233,132,87to 0.1270,1360,166
1:23,233,293,03to 0.1270,1220,146
6b/p3,373,172,730,1160,132of € 0.195
1:23,533,232,970,1010,1280,151
12b/p3,23of 3.072,720,1280,1410,198
1:23,573,22,930,0950,13of) 0.157
1-080b/p3,403,37with 3.27 0,1140,1160,124
1:23,403,403,200,1140,1140,13
1b/p3,373,303,170,1160,1220,132
1:23,303,202,900,1220,1300,161
3b/p3,333,172,930,1190,1320,158
1:23,473,402,830,1060,1140,175
6b/p3,203,062,810,130,1510,181
1:23,373,142,730,1240,1650,196
12b/pis 3.082,972,720,140,150,198
Ȋ 1:23,523,162,990,100,130,15
1-090b/pwith 3.273,132,870,1240,1360,167
1:2with 3.273,302,830,1250,1220,175
1b/p3,033,02,770,1460,1490,188
1:23,303,032,930,1220,139of) 0.157
3b/p3,13of 3.072,730,1360,1410,196
1:23,173,103,00,132was 0.1380,148
6b/p3,113,012,780,1470,1560,185
1:23,40 3,172,340,1240,136rate £ 0.162
12b/pis 3.082,912,780,140,160,185
1:23,333,152,810,12is 0.1350,18

The detected increase was associated with an increase in the width of the distribution of particle sizes due to the appearance in the emulsions of relatively large particles. So fractionation of the emulsions after 12 months of storage the size of the particles in the bottom fraction was increased from 0.12 to 0,198 μm. In General, these results are consistent with the basic mechanism of destruction of the emulsions according to the Ostwald (or molecular distillation). However, the share of such relatively large particles was so small (˜10%)that this circumstance did not affect the increase in the average diameter of the particles. It should be emphasized that fractionation of the emulsions was observed only uniform settling of particles that talked about the lack of free emulsions fluorescence even after 1 year of storage, i.e. the distribution of particles in the emulsion did not change, remaining modal. The results obtained indicate the conservation of the overall structure of the obtained emulsions PFCs in the tech is of 1-12 months of storage.

In table. 10 shows the index values of the interaction, the particles of the investigated emulsions with serum, modified by the addition of a 5%solution of albumin in a ratio of 1/1.

The indices of the interaction of PFC emulsions with serum Kτthat characterize their microstructure, for emulsions PFC/AFL during the 12-month period monitoring fluctuated in a fairly narrow range (the ratio of serum:emulsion = 1:0.05 and 1:0,1). When increasing the ratio of serum:the emulsion to 1:0.10 interval oscillation indexτand Kτ/nwas increased. For emulsions PFC/SFL narrow range of fluctuations Toτremained only until the 6-month retention period. As already noted, the established variations Toτrelated, most likely, to the fact that standardize the mixture of sera from experience to experience at different periods of storage of the emulsions is very difficult. However, the saving interval oscillation index interaction emulsions with serum and in certain rather narrow limits for each series indicates that the surface properties of the particles for long periods of time (6-9 months) little change. Jump Toτby the end of shelf life in the absence of the emulsions free vials may be associated with the emergence of additional collaboration is s between the particles and macromolecules serum. To test this assumption were calculated experimental and calculated values of turbidity τ - additional independent parameter estimation integrity patterns of PFC emulsions (table. 11) [13].

Table 10.

Index values of interaction with serum PFC emulsions different periods of storage at +4°
the number of seriesShelf lifeτToτ
The ratio of serum:emulsion
1:0,051:0,11:0,051:0,1
1-0200,8±0,11,33±0,033,8±0,56,2±0,2
10,93±0,061,4±0,14,9±0,37,2±0,5
30,97±0,021,99±0,0033,50±0,075,01±0,02
60,97±0,011,63±0,033,5±0,43,4±0,1
91,23&x000B1; 0,022,09±0,033,7±0,27,0±0,1
121,72,32±0,017,2±0,3to 10.09±0,1
1-0300,77±0,071,1±0,13,3±0,34,8±0,4
10,84±0,051,29±0,064,0±0,36,6±03
31,0±0,11,46±0,053,6±0,65,28±0
60,92±01,53±03,3±0,43,19±0,03
121,33±01,92±04,0±0,26,4±0
1-0401,00±0,061,25±0,085,4±0,35,4±0,4
11,1±0,21,67±0,035,3±0,98,9±0,2
31,15±01,84±0,064,4±0,33,83±0,06
61,18±0,021,97±0,03/td> 5,7±0,810,7±0,2
91,83±0,041,76±0,024,2±0,44,40±0,04
121,30±0,042,22±0,077,7±0,513,9±0,8
1-0500,98±0,051,56±0,085,1±0,28,0±0,4
11,1±0,11,76±0,024,3±0,67,0±0,1
31,1±0,31,90±0,064,5±0,88,3±0,2
61,18±0,022,05±0,075,7±0,811,1±0,4
121,60±0,012,91±0,053,6±0,37,2±0,1
1-0800,77±0,051,15±03,4±0,45,4±0,7
10,95±0,021,43±0,034,0±1,06,4±0,3
31,10±0,11,57±0,028,7±0,5
61,4±0,11,94±0,033,3±0,65,4±0,5
91,52±0,032,68±0,0510±219±1
121,38±0,082,30±0,0311±221,3±0,9
1-0900,81±0,021,48±0,035,6±0,614,5±0,3
11,2±0,11,8±06±14,79±0,07
31,2±01,4±0,46,0±0,57±3
61,2±0,22,25±0,032,8±0,56,4±0,5
91,9±0,13,24±0,0314±224±2
121,52±0,032,68±012±125,3±0,7

In a physical sense τ for disperse systems is the sum of the power losses of the light beam on a separate cha is the tics in the absence of cooperative effects and multiple scattering. The coincidence of the experimental and calculated values τ for both native and diluted with water emulsions testified that the interaction between particles and macromolecules serum is practically unchanged even after 9-12 months of storage at +4°C. Jump Toτis likely to be associated with the appearance in the aqueous dispersion medium additional supramolecular structures PFC/vial, and the ratio of them PFCs, and FL is the same as in the emulsions.

Table 11.

Experimental and calculated values of turbidity for emulsions with different retention periods
No. series (volumetric content of UPF)Shelf life, months.τ500
Sourcediluted 1:2
exp.calculationexp.Calculation
1-0109,910,04,6the 4.7
(10 vol.%)111,7the 11.63,603,75
312,2to 12.03,91as 4.02
613,813,14,13,86
913,814,04,294,37
1216,114,1to 4.984,94
1-0308,239,063,60the 3.65
(20 vol.%)19,6610,2of 3.073,11
312,411,33,33,39
613,311,553,573,57
1213,112,4as 4.02as 4.02
1-04012,311,44,054,0
(20 vol.%)113,813,24,64,5
321,015,4to 4.985,0
616,115,55,55,5
917,217,25,6 6,2
1220,9to 19.96,86,5

continuation of table. 11
No. series (volumetric content of UPF)Shelf life, months.τ500
Sourcediluted 1:2
exp.calculationexp.Calculation
1-05012,8813,114,22of 4.38
(5 vol.%)114,0313,25to 4.684,71
319,116,95,65,56
616,816,155,35,3
1218,918,5of 5.44,5
1-0808,97a total of 8.742,912,81
(40 vol.%)111,7the 11.63,533,47
313,8 13,64,144,0
616,615,4to 4.684,59
921,818,95,65,6
12of 21.219,76,26,2
1-09014,0314,444,294,15
(40 vol.%)119,3of 17.5of 5.755,61
3KZT 19.0918,725,525,34
621,818,26,138,71
926,223,97,77,8
1226,925,54,914,3

Before talking about the benefits of the claimed composition and method of producing emulsions SFC, it should be emphasized that the main conditions of emulsions SFC gas transportation options during circulation in the bloodstream is the preservation of the corpuscular nature of the particles and the lack of reactogenicity. With POS is of colloid chemistry and Biophysics hit the PFC emulsion in the bloodstream can be considered as stress-impact, which should cause changes in the properties of the disperse medium. This impact can be reduced to the following. The dilution of the emulsion PFCs, and reducing the concentration of free emulsifier in the environment (fast stage). The result of this process is the weakening of the ties of surfactant molecules with the surface of the particles (slow phase). This change ties surfactant with PFC exacerbated by contact and interaction of particles with the plasma macromolecules, which can lead to changes in the composition of the adsorption layer or destruction of particles. The indicated sequence of processes is a somewhat simplified scheme.

In our experiments, the dilution of the emulsion with water simulates the first stage dilution of the emulsion and reduce the concentration of free emulsifier around the particles. The study of the interaction of the obtained emulsions with serum simulates the second stage - the influence of the contact of serum macromolecules on the surface properties of particles. It turned out that even after a year of storage particles of PFC emulsions remain "microstructure": dilution of the emulsion with water did not affect the particle size, indicating that the bond strength of the surfactant adsorption layer with the core particles SFC; did not change the index of particle interactions emulsions with serum (within measurement error), what evidence is olavo, on the preservation of the surface properties of the particles. Match the estimated value τ (after fractionation) with experimental (before fractionation), which confirms the preservation of the "nature" of the particles (their structure) and indicates the absence of free fluorescence in the emulsion after 1 year of storage.

Used methodological approaches significantly increase the accuracy of prediction of the stability of emulsions when injected into the bloodstream. Evidence of this are the results of parallel studies on the stability and index of reactogenicity (Ipseveral series of emulsions of identical composition. Ipfound by the method of [3].

Example 10. There were 4 series of the same type emulsion composition corresponding to example 2: the content of the fluorocarbon basis amounted to 9±1 vol.%, the ratio of the PFD/PFOB 9:1; addition of PFOS was 20%; the content of AFL was equal to 1 wt.%; the amount of adjuvant - castor oil and soybean oil at a ratio of 1:1 was 8% of the concentration of AFL.

Table 12 shows the values of n and for these series of different periods of storage.

Table 12

The values of the wave exponent n and the mean diameter of the particles and emulsions of the same composition PFD/PTB/PPOWELL different periods of storage
No. the ' series Shelf life (months)nand, umnand, um
sourcediluted
5-0303,70±0,030,083,81±0,070,06(5)
13,62±0,030,093,83±0,050,06(5)
63,80±0,040,073,76±0,060,07(5)
5-0403,36±0,020,117to 3.58±0,010,09
13,30±0,040,1223,53±0,050,10(1)
63,37±0,050,117is 3.08±0,080,14
5-0503,35±0,050,1163,47±0,040,11
13,07±0,070,1413,24±0,090,12(7)
63,37±0,030,1173,13±0,020,13(6)
5-0603,32±0,020,123,35±0,050,11(6)
13,07±0,070,1413,09±0,080,139
63,45±0,04to 0.108/td> 3,26±0,090,12(5)
Table 13

The value of the wave exponent p and the mean diameter of the particles and characterizing the width of the distribution of particles by size fractionation by centrifuging for emulsions of the same composition PFD/PTB/PFO/AFL
the number of seriesShelf life (month)dilutionna mcm
topCore.NII.Top.Core.Min.
5-040native3,533,473,210,100,110,13
1/2of 3.773,63with 3.270,070,090,124
1native3,533,513,190,101 0.104 g0,132
1/23,853,663,260,060,0850,125
6native3,61of 3.463,150,0890,109is 0.135
1/23,613,122,90,0890,1360,160
5-050native3,713,433,170,0790,1100,132
1/23,88to 3.673,310,0550,0840,120
1native3,603,303,050,0940,1220,145
1/23,72 3,352,780,0780,1220,184
6nativeof 3.563,342,900,098amount of 0.118rate £ 0.162
1/23,533,152,710,101is 0.1350,20

According to the data obtained, the average particle diameter in all cases for native and diluted with water emulsion did not change during 6 months of storage, while remaining within 0.06 to 0.17 microns. The width of the distribution of particle sizes for native and diluted with water emulsions of the above mentioned composition was virtually unchanged in the specified observation period (table. 13). The index of the interaction of the obtained emulsion with modified serum Toτ taking into account the relative measurement error (±10%) fluctuated in a fairly narrow range (table. 14).

Table 14

Index value of interaction Toτ emulsions of the same composition PFD/PTB/PFO/AFL with modified serum in the presence of albumin (80%).
The series numberShelf life (months)The ratio of serum/emulsion
1:0,051:0,1
5-0302,283,37
12,343,70
62,604,00
5-0403,766,00
13,635,62
64,056,03
5-0504,05,56
14,33the 6.06
64,536,1
Table 15

Value index reactogenicity of emulsions of the same composition PFD/PTB/PFO/AFL (as control is taken dispersion vial).
The series numberStorage duration (months)
01 6
Dispersion FL1,4--
5-032,831,681,92
5-041,142,142,24
5-051,831,831,70
5-062,421,352,63

The results shows that the proposed emulsions and method of production thereof is possible to achieve high-quality microstructure of emulsions, are not damaged during storage in not frozen and subsequent stress in vitro (dilution, interaction with serum, enriched with albumin). The test results reactogenicity of the same samples of emulsions fully confirmed by the data model studies: none of the terms research index reactogenicity did not exceed a critical value equal to 3 (table. 15).

Example 11. The safety of the structure and the analysis of the level of reactogenicity emulsions with low volumetric content of the SFC at 5 vol.%. The composition of the emulsion PFD/PFOB 1:1, P is MCP 1%, SFL - 0,5%, adjuvant soybean oil - 12%. The width of the size distribution of the particles was in the range of 0.03 to 0.12 μm, the original index reactogenicity was 1,61. We investigated changes during storage medium particle size of native and diluted with water four similar emulsions (table. 16) and reactogenicity after 6 months of storage (table. 17). According to these data, the observed increment of the size of the particles when used in the formulation and the proposed method of obtaining provide the lowest level of the index reactogenicity.

Table 16

The value of the wave exponent n and the mean diameter of the particles and for native and diluted with water emulsions of the same composition PFD/PTB/PFMP/SFL
The series numberShelf life, monthsnand, umnand, um
sourcediluted with water 1:2
6-0213,27±0,040,1223,29±0,030,123
6is 3.08±0,09was 0.1383,18±0,020,132
6-0313,19±0,01 0,133,28±0,020.124
63,06±0,010,1453,11±0,03was 0.138
6-0513,31±0,010,123,48±0,010,105
63,24±0,010,1263,39±0,010,114
6-0613,11±0,030,1373,23±0,030,128

Table 17

Value index reactogenicity Ip for emulsions with low content of SFC after 6 months of storage in a frozen state
A series of emulsions6-026-036-056-06
Index reactogenicity1,872,001,361,8

Example 12. Long within 18 months of storage of the emulsion containing 10 vol.% SFC, with a ratio of PFD/PFOB 8:2, PFO 20%, AFL 2%, adjuvant castor oil 10%. The results of the study changes in the average size of particles during storage and dilution of the emulsion with water are presented in table. 18, the dynamics of the index of interaction emulsion with serum, enriched with albumin up to 50%-in table is. 19.

Table 18

The value of the wave exponent n and the mean diameter of the particles and for native and diluted with water emulsions PFD/PTB/PFO/AFL in different periods of storage in not frozen.
The series numberShelf life, monthsnand, umnand, um
sourcediluted with water 1:2
7-0303,70±0,030,083,81±0,070,065
13,62±0,030,093,83±0,050,065
63,80±0,040,073,76±0,060,07
183,81±0,040,065of 3.77±0,060,07

Table 19

Index value of interaction Toτ emulsion PFD/PTB/PFO/AFL with serum in the presence of albumin (50%) in different periods of storage.
The series numberShelf life, monthsItτ
The ratio of serum /emulsion
1:0,051:0,1
7-0301,742,37
11,682,25
61,482,74
181,221,78

As can be seen from the above data, the obtained emulsion was kept of the measured physico-chemical characteristics. Obviously, thanks in 18 months. store the index of the reactogenicity Ip emulsion PFD/PTB/PFO/AFL was only 1.5.

Example 13. Mapping quality emulsions SFC obtained on the prototype, the emulsion Exigent AF-0104 (firm Therapeutic Alliance, USA) and emulsion SFC received proposed in the present invention method. The comparison was conducted on the change of the wave of the Exhibitor and the mean diameter of the particles when diluted with water.

In the comparison emulsions at different absolute content SFC met is equal to the ratio of the components SFC/vial. These emulsions differ in the way of receiving. In the emulsion PFOB-2 (obtained by the present method) does not contain after centrifugation free phospholipid phase (Fig. 1B), whereas the emulsion Oxygen and emulsion prototype PFOB-1 are not associated in the adsorption layer of particles available phospholipids, easy sun is Livadia by centrifugation (Fig. 1A). That is why when breeding emulsions with water, when there is a destruction of aggregates fluorescence and particle emulsion was reduction of the average particle size of the emulsion in the preparation Oxygen from 0.35 to 0.15.

In the emulsion of the prototype (PFOB-1), obviously, there was no such coarse aggregates, but their presence is evidenced, in addition to the results of the centrifugation and separation of fraction-free vial, sharp differences between the calculated and experimentally defined parameters turbidity values defined by the rules of additivity for native and diluted with water emulsions. While drugs emulsions SFC received the claimed method, was observed almost complete according to parameter turbidity between the experimental and calculated values (table 21). It should be noted that physically defined parameter reflects the amount of power loss of the light beam to separate the particles in the absence of cooperative effects and multiple scattering. The discrepancy between experimental and calculated values of turbidity for drug Exigent and emulsion PFOB-1 (prototype) testified to the failure of additivity rule for these environments, i.e. whether named in disperse systems additional interactions between particles and luminous flux. These interactions are distinct is detected at a dilution water preparation Exigent and emulsions PFOB-1, reflecting the heterogeneity of the types of particles, in particular the presence along with the particles of the emulsion SFC different micel-lar structures vial. For emulsion PFOB-2 and emulsion PFD/PFOB series (5-03) interaction between particles and light flux obey the rule of additivity even after months of storage in a frozen state.

Fig. 1. The scheme of separation of the emulsion SFC on fractions depending on the method of obtaining: A - prototype (emulsion contains free FL); B - the proposed method, the emulsion contains only a different size particles). 1, 2, 3 - upper, main and lower fractions 1a - free fluorescence in the upper fractions

Table 20

The various emulsions of an SFC value of the wave exponent p and the mean diameter of the particles and for native and diluted with water emulsion (1:1).
The drug, type SFCCompositionnand, um
The SFC.

mass./ volume
FL, wt.%sourcebreeding N2About 1:1sourcebreeding

H2O 1:1
F0104 (PFOB)90 wt.% 45%vol.4 2,34±0,042,93±0,100,350,15
Emulsion PFOB-1 (prototype)45 wt.% 22%vol.22,91±0,092,82±0,100,160,18
The emulsion according to the proposed method (composition by prototype PFOB-2)20%

10 vol.%
13,38±0,023,33±0,050,1140,115
Table 21

The degree of compliance with experimental experience) turbidity values with the estimated values of this parameter (calculation)determined by the rule of additivity for different emulsions.
MedicationNativeDiluted with water 1:1
experiencecalculationexperiencecalculation
Exigent133,9±1,033,259,3±0,523
PFOB-1 (prototype)26,6±0,3 18,616,4±0,2to 5.21
PFOB-2 (the claimed method)9,310,02,352,31
PFD/PFOB 9:1 (inventive composition and method of example 10)12,911,23,243,17

Thus, the above examples demonstrate the whole range of benefits described composition and method of obtaining the claimed emulsion compared with the prototype and analogues. This is due to the following synergistically interacting factors.

1. PFD and PFOB taken as main components because these SFC tested for their biological and physico-chemical properties of a biologically acceptable, with a high rate of excretion from the body, i.e. from the cells of the reticulo-endothelial system, accumulating particles SFC.

2. Sharing PFD and PFOB in the effective ratio forms the mixed oil phase, the properties of which are changed graduale from the center to the periphery so that you can use in the recipe a little lipophilic and negitively perfluorinated tert is cnie amines, which have a substantially lower vapor pressure (see table. 1) and thereby reduce the speed of diffusion penetration of lipophilic molecules PFD and PFOB in the aqueous phase. This fact slows down the speed of the basic mechanism of destruction of emulsions - "maturation by Ostwald," and increases the stability of the selected composition of the emulsions SFC.

3. Introduction PFOB in the composition of the emulsions increases their oxygen capacity with the same content SFC and gives them additional radiopaque properties.

4. The presence in the composition of PFOB/PFD and a mixture of perfluorinated tertiary amines helps to reduce the viscosity of the final form due to the stronger binding of the surfactant adsorption layer around the particles, which eliminates the content of free and micellar forms of fluorescence in the aqueous phase of the emulsions.

5. Along with phospholipids with different physico-chemical characteristics of oils contributes to the formation of more dense membranophones adsorption layer around the particles with a smaller number of phospholipids, prevents the formation of micellar not containing SFC structures.

6. Features used water-salt environments provide conservation on the surface of the particles a negative charge that prevents them from sticking together during storage and transportation.

7. Along the technological methods providing vysokokachestvennoj (with a narrow distribution of particle sizes) of the emulsion, the above techniques weaken the process of molecular distillation SFC and also contribute to better preservation of emulsions.

8. The absence of aggregates of particles and micellar forms FL provides a reduction of the adsorption and complement activating properties of the emulsion when getting it into the bloodstream, which causes a low level of reactogenicity and contributes to the improvement of the biocompatibility of the emulsions of the proposed structure.

III. Below is a biomedical experiments using the proposed emulsions.

Experiment 1. The use of emulsion SFC for massive cruiseline. Healthy rats of Wistar breed weighing 250-300 g (n=20) under Nembutal anesthesia was performed isovolumic cruiseline emulsion SFC, prepared according to example 1 (sec. II). Determined the survival of rats after massive cruiseline and preservation of liver mitochondria after compensation of blood loss (method see [16]). To ensure maintenance of oncotic pressure after massive cruisemate emulsion SFC mixed before infusion of the animal with a 20%solution of human albumin in a ratio of 1 part of a solution of 20% albumin and 6 parts of an emulsion SFC so that the final concentration of albumin was 3.5% (Uch is the fact that, 10 volume percent of the emulsion are SFC). During the operation of cruisemate rats breathed air, oxygen-enriched with value FiO20,5 supplied under a transparent cover made of plexiglass, which was covered head fixed on the back of the animal. From the venous sinus (right atrium) were taken with a syringe and 3.5 ml of blood was injected 3.5 ml emulsion SFC. After 10 minutes again took 3.5 ml of blood and was administered an equal amount of emulsion SFC. Then again repeated the procedure cruisemate so that the final amount of blood was on average not less than 3.5% of the body weight of the animal, for example, the animal has a mass of 250 g, the volume of the inflowing blood and the volume of injected emulsion SFC was of 8.8 ml Before and after cruisemate determined the content of hemoglobin in the peripheral blood, the partial pressure of oxygen and pH in arterial and venous blood. In this series of experiments, the concentration of hemoglobin was decreased after cruisemate on average 1.9 times. In the control group (n=20) animals instead emulsion SFC has introduced a 0.15 M sodium chloride solution containing 3.5% bovine serum albumin. Using the NMR spectrometer was determined in animals, the content of the SFC in the peripheral blood. After surgery cruisemate animals within 5 days he was kept in a special chamber with air, oxygen-enriched with FiO20,5.

In the experimental group (cruiseline emulsion SFC) all animals survived and the content of hemoglobin, erythrocytes and leukocytes recovered within 5 days. In the control group, 3 animals died. After 5 days all surviving animals were scored under Nembutal anesthesia and liver tissue was isolated mitochondria. Respiration of liver mitochondria was detected polarographically cooled in a closed cell at 27°C. it Was found that in the control group the rate of respiration in the active state (ATP synthesis) and the actual rate of ATP synthesis in the oxidation of NAD-dependent substrates 3-hydroxy-butyrate decrease on average 1.5 times, at 20% activation of the oxidation of succinate. These data testify to the transferred animals of moderate ischemic lesion of the liver tissue. Then, as in liver mitochondria isolated from animals 5 days after compensation of blood loss emulsion SFC was observed activation speed respiration and ATP synthesis by 25%on average, testified to the history of only moderate hypoxia.

Experiment 2. All procedures were performed as in the previous example, but used an emulsion containing 20% vol. The SFC, and the hemoglobin content was reduced in 3 times in comparison with the original, replacing the average 65-70% of circulating blood volume. The volume of blood and entered the shelter is alwaysgo composition was 12.25 ml for animal weighing 250 g As a result, in the experimental group survived all animals in the control killed 5 animals.

Experiment 3. All procedures were performed as in example 1, except that 5 animals in each group were scored after 6 hours, 1 day and 3 days after cruiseline. Was isolated mitochondria from liver and registered options fosforiliruyusciye breathing. Animals of the control group during all these periods, there was a sharp suppression of rates of respiration and phosphorylation in the oxidation of as NAD-dependent substrates, and succinate on average more than 50%, which is typical for severe ischemic damage to the mitochondria. In the experimental group after 6 hours after cruiseline was observed 40% activation fosforiliruyusciye breathing, which continued through the day and was not more than 25% after 3 days after cruisemate emulsion SFC. Such shifts are characteristic of intact liver mitochondria of animals undergoing hypoxia, but not ischemia.

Experiment 4. Keeping the kidneys in dogs undergoing hemorrhagic shock. The safety of the kidneys was determined by the recovery of kidney function after transplantation animal recipient with both remote kidneys (the study was conducted by special permission of the Ministry of health), as well as the results of the assessment of the level Danilovich nucleotides and lactate content, and PI is the Uvat in kidney tissue one hour after recovery of blood loss. The study was performed in 10 dogs, 5 dogs in each group.

The course of the study. Intubirovannah dogs weighing 20 kg, under General inhalation anesthesia with controlled breathing, from the femoral artery jet climbs 400 ml of blood, which is accompanied by a sharp drop in blood pressure (up to 50-60 mm Hg), a twofold increase in heart rate, increase in the concentration of lactate in the blood plasma of up to 20 mm. One hour after blood collection the animal is injected with a blood substitute in a dose exceeding the amount of blood loss by 15%in the experimental group - emulsion containing 10 vol.% SFC in example 1 with the addition of albumin to 3.5% (as in example 14)in the control group was administered plasmasphere poliglyukin. An hour later, animals were scored and took the two kidneys, one for transplantation after an hour the dog recipient, another for research of parameters of energy metabolism kidney tissue.

In the control group, the ratio of ATP/ADP decreased in 3 times, and the value of the energy charge ([ATP]+1/2[ADP])/([ATP]+[ADP]+[AMP]) - up to 0.45. In the experimental group (cruiseline emulsion SFC) the ratio of ATP/ADP was reduced to no more than 2 times, and the value of the energy charge decreased to 0.65 to 0.70. The ratio of lactate/pyruvate in kidney tissue in animals of the control group increased to 25-30, whereas in the experimental group did not exceed 6.

In all cases, at first the cages dogs-recipients of kidney, taken from dogs treated with emulsion SFC, immediately after connecting the kidneys to the blood flow began the urine. In the control group in 2 cases out of 5 after transplantation was observed in the development of reperfusion injury with a sharp tissue edema and complete cessation of blood flow (kidneys were killed). In 3 cases in the control group, the blood flow in the transplanted kidney was recovered, but the urine was started only a few hours.

The data presented indicate that the treatment of hemorrhagic shock in dogs using the obtained emulsion SFC provides significantly better protection of organs from ischemic and subsequent reperfusion damage.

Experiment 5. The use of emulsion SFC obtained in example 2, to save perfusing rabbit heart. Immediately prior to use (1-2 hours) emulsion SFC mixed with isotonic Krebs-Henseleit in the ratio 2:1:400 ml of emulsion and 200 ml of saline solution. To 600 ml of the mixture was added 80 ml of a 20%aqueous solution of serum albumin. The control composition for comparative studies contained 600 ml of saline solution with the addition of 7.2 g of mannitol and 80 ml of 20%aqueous solution of albumin. These compounds are used as the perfusion medium to save the rabbit heart. Perfusion in Langendor who have performed in the recirculating mode at a temperature of 37° C. Registered time to save the frequency and amplitude of contractions of the heart. In the control and in the experiment was taken at 8 hearts. When using perfusion solution based emulsion of PFOS contractility of the isolated rabbit heart was preserved for at least 6-8 hours, whereas perfusion of the control composition after 2 hours there was a sharp decrease in the frequency and amplitude of contractions up to full cardiac arrest.

In conclusion, it should be noted that the advantages over the prototype and analogues proposed emulsions are as follows.

Presents the formulation and method of producing emulsions SFC provide finely calibrated emulsion with a specified average particle size in the range from 0.06 to 0.195 μm, containing from 2 to 40 vol.% SFC stabilized phospholipid dispersion in a biologically acceptable water-salt composition. Shows high safety dispersion and microstructure" emulsions SFC during storage for up to 18 months in not frozen, allowing you to maintain high biocompatibility, which is expressed in low reactogenicity. Developed emulsion SFC suitable for biomedical use, in particular, for the reimbursement of massive blood loss, treatment of hemorrhagic shock, prevention of postischemic reperfusion p is uridine, preparation of organs for transplantation, perfusion preservation of isolated organs, have a fairly pronounced oxygen transport and rheological properties that ensure the prevention and elimination of ischemic damage oxygendependent functions of mitochondria and maintenance of aerobic energy metabolism in the tissues under conditions of cruiseline and treatment of hemorrhagic shock.

Sources of information

1. Journe. All-Union chemical society them. D., 1985, t, No. 4, s-394.

2. J.G. Riess et al.// Physiological activity of fluorine-containing compounds (experiment and clinic): Collection of scientific. the labor. - Pushchino, 1995. - s-90.

3. Mvers, Abstract. the dissertation. ... candles. the honey. of Sciences, Leningrad, 1991, 24 S.

4. J.G. Riess // Chem. Rev., 2001, v.101, no. 9, s-2914.

5. RF patent 2162692, CL And 61 To 31/02, 9/10, 1999.

6. RF patent 2199311, CL 7 And 61 9/107, 31/02, 2001.

7. The US patent No. 3778381, 1973.

8. The US patent No. 6113919, 2000.

9. The US patent No. 4866096; a 61 K 31/025, 1989.

10. The US patent No. 5374624 And 61 To 31/025, 1994.

11. RF patent №2088217,6 a 61 K 9/10, 31/02, 1997.

12. Biophysics, 1988, v.33, No.1, pagination 126-129.

13. Ingushetia. Abstract. the dissertation. ... Prof. Biol. of Sciences, St. Petersburg., 1999, 38 S.

14. Chemical and pharmaceutical journal, 1987, No. 12, s-1503.

15. Journal of physical chemistry, 1993, T. 67, No. 9, s-1888.

16. Eyisi. Abstract. the dissertation. ... Prof. the honey. of Sciences, Moscow, 1998, 36 S.

1. Emulsion perftoran hanicheskih compounds for medical purposes, containing bistabilities performancesee connection perpendicular and perforative, performances additive and phospholipids, characterized in that it contains phospholipids in the form of a dispersion prepared by homogenization under pressure of at least 100 atmospheres in the water-salt medium, and performancesee additive is a mixture of perfluorinated tertiary amines - performapply and its coproducts: CIS - and TRANS-PERFLUORO-1-propyl-3,4-dimethylpyridine and PERFLUORO-1-propyl-4-methylpiperidine.

2. The emulsion according to claim 1, characterized in that it contains 2-40% vol. performancesin connections.

3. The emulsion according to claim 1, characterized in that the composition bystrovytsia performancesin compounds contains perpendicular and perforative in the ratio of 10:1-1:10.

4. The emulsion according to claim 1, characterized in that performancesee additive is 1-50% of the total content of the composition bystrovytsia performancesin connections.

5. The emulsion according to claim 1, characterized in that the mixture of these perfluorinated tertiary amines further comprises PERFLUORO-N-methylcyclohexylamine and his coproduct.

6. The emulsion according to claim 1, characterized in that it contains a dispersion of phospholipids in water-salt medium at a concentration of 0.2-5 wt.%.

7. The emulsion according to claim 1, characterized in that todisperse phospholipids in water-salt medium contains phospholipids of egg yolk, or soybean phospholipids, or a mixture.

8. The emulsion according to claim 1, characterized in that the dispersion of phospholipids in water-salt medium contains an adjuvant in the form of vegetable oil in the amount of 1-15% of the total content of phospholipids.

9. Emulsion of claim 8, wherein the adjuvant is soybean oil.

10. Emulsion of claim 8, wherein the adjuvant is sunflower oil.

11. Emulsion of claim 8, wherein the adjuvant is castor oil.

12. Emulsion of claim 8, wherein the adjuvant comprises a mixture of vegetable oils selected from the group including soy, sunflower and castor oil, taken in the effective value.

13. The emulsion according to claim 1, characterized in that the water-salt medium contains sodium and potassium salts of chlorides and phosphates and monosaccharide mannitol in water for injection.

14. The emulsion according to claim 1, characterized in that the concentration of the components of the water-salt medium is the osmotic pressure in the range of 100-350 momola per litre.

15. The emulsion according to claim 1, characterized in that the average particle diameter not greater than 0.2 μm and is in the range 0.06 to 0.2 μm.

16. The emulsion according to claim 1, characterized in that the shelf life in frozen form at + 4°With not less than 12 months.

17. The production method of the emulsion performancesin compounds, Ohara is teresovannost in any one of claims 1 to 16, including the production of dispersions of phospholipids by homogenization under pressure of at least 100 atmospheres in the water-salt medium with subsequent heat sterilization, homogenization under pressure specified performancesin compounds in dispersion of phospholipids and heat sterilization of the finished emulsion.

18. The production method of the emulsion according to 17, characterized in that performancesee connection homogenized dispersion of phospholipids under pressure 300-650 atmospheres.

19. The production method of the emulsion according to 17, characterized in that the dispersion of phospholipids is sterilized at a temperature of 100°C.

20. The production method of the emulsion according to 17, characterized in that the emulsion performancesin compounds is sterilized at a temperature of 100°C.



 

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