RU2070033C1 - Method of preparing perfluorocarbon emulsion for medicinal aims - Google Patents

Abstract

FIELD: chemical technology. SUBSTANCE: invention relates to preparing emulsions which are able to transfer oxygen and used as blood-substituents, roentgencontrasting agents and for isolated organs storage. Mixture of perfluoroorganic compounds is dropped through an aqueous solution of emulsifying agent, then prepared preemulsion is passed by turns through the basic and additional homogenizer circulatory contour increasing pressure in additional contour by 1.1-1.2-fold as compared with pressure in basic contour. To prepare emulsions a mixture of perfluorodecaline or perfluorooctyl bromide and perfluoromethylcyclohexyl-piperidine is taken at ratio 2:1. Copolymer of polyoxyethylene-polyoxypropylene is used as emulsifying agent. Average particle size is from 0.06 to 0.11 mcm, particle part of size below 0.10 mcm is 85%. Emulsion is stored at t = +20 C for one month and for 3 months in frozen state. EFFECT: simplified technology, improved sterility of preparing monodispersed emulsion. 2 cl, 9 tbl, 1 dwg

Description

 The invention relates to the medical industry and relates to a method for producing perfluorocarbon emulsions capable of transporting oxygen and intended primarily for use in medicine as blood substitutes, radiopaque agents, and also media for preserving isolated organs.

 It is known that the quality of emulsions is to a large extent determined by the size and distribution of particles. Thus, the presence of large particles larger than 0.2 0.3 μm, as shown in some works, is the reason for the toxicity of emulsions (Mitsuno Tokao, Kokuritsu Kobe, Medical Institute, Japan, "Practical use of artificial blood," Shizen, 1981, 36 (9), p. 62 69). An important indicator is also the stability of the emulsion during storage, which depends mainly on the properties of the used organofluorine compounds (PFOS) and emulsifying agent.

To obtain perfluorocarbon emulsions, as a rule, two types of perfluororganic compounds (PFOS) are used simultaneously, as a rule. One of them is selected from the group (C ₈ C ₁₀ ) containing, for example, perfluorodecalin (PFD) or perfluorooctyl bromide (PFOB), the second from the group (C ₁₁ C ₁₂ ) containing, for example, perfluorotripropylamine (PFPA), perfluoromethylcyclohexylpiperidine (PFMTSP) or perfluorotributylamine (PFBA). Compounds of the first type are quickly eliminated from the body (within a month), but do not provide sufficient stability for their emulsions, while compounds of the second type, on the contrary, give the emulsion high stability, allowing them to be stored without freezing, but for a long time (up to 2 years) ) are not excreted from the body.

 The most common emulsion technology of this kind is based on methods using ultrasound or high pressure homogenization. To obtain emulsions on an industrial scale, the second method is preferable, because it makes it possible to obtain emulsions in large quantities and with better physicochemical characteristics, for example, better particle distribution (Jean G. Riess and Maurice Le Blanc. Preparation of perfluorochemical emulsions for biochemical use: principles, materials and methods. Ellis Horwood Series in Biomedicine, VCH, Blood Substituts, Preparation, Physiology and Medical Applications. 1991. Ch. 5, pp 113 115).

A known method for producing emulsions containing, for example, perfluorodecalin (PFD), perfluorotripropylamine (PFPA), emulsifying agents, for example, a polyoxyethylene-polyoxypropylene copolymer (Pluronic F-68, domestic analogue - proxanol), egg yolk phospholipids or soybean phospholipids and water ( USSR N 797546 company Green Cross Corporation, publ. In the bulletin "Discoveries, inventions." N 2, 1981). In accordance with this method, the initial emulsion is prepared by mixing the components in a physiologically acceptable aqueous medium using a homogeneous mixer or a propeller mixer, then the initial emulsion is emulsified by injection in a high-pressure homogenizer like Manton Gaulin at a pressure of from 100 to 500 kg / cm ² and a temperature of + 20 to +55 ^o C. The mixture is passed through the slot of the homogenizer up to 12 times.

 As follows from the table. 1, the proportion of particles larger than 0.3 μm in some cases reaches 60%. This is explained by the use of phospholipids as an emulsifying agent, as well as by the fact that at high temperatures of the process of emulsification and sterilization coarsening of the particles of the emulsion occurs. In addition, due to the use of mixers such as a propeller mixer, a sufficient sterility of the process is not ensured. All this makes it necessary to filter the fraction of large particles and sterilize the emulsion. These emulsions were intended for use in animal experiments.

 The average particle size of an improved medical emulsion Fluosol-DA 20% of the same company, prepared in a similar way based on PFD and PFPA is 0.118 μm, the proportion of particles ranging in size from 0.3 to 0.5 μm is 1.9% (Mitsuno T. et al. "Intake and retension of perfluorochemical substance of Fluosol-DA in res human", Proceedings of the 5. Int. Sympos. on Oxygen-Carring Colloidal Blood Substituts, Meinz, March, 1981, p. 220). The drug is stored frozen, as after 8-12 hours of storage at room temperature, the emulsion particles coarsen.

 The objective of the invention is to increase the monodispersity of the emulsion by reducing in it a fraction of particles larger than 0.2 microns.

 Another objective of the invention is to improve the sterility of the process and simplify the technology for producing emulsions.

The problem is solved in that in the known method, including the preparation of a pre-emulsion by mixing organofluorine compounds with an emulsifying agent and subsequent repeated homogenization of the mixture in a closed high-pressure circuit, a pre-emulsion is obtained by dropping a mixture of perfluorodecalin or perfluorooctyl bromide in a ratio of 1: a solution of a copolymer of polyoxyethylene-polyoxypropylene in a closed loop of a homogenizer to obtain the required analyte ratio, then the mixture is passed alternately through the primary and secondary circuit homogenizer under pressure of 300 660 kg / cm ² to 12 times, increasing the pressure in the secondary circuit is 1.1 1.2 times as compared with the pressure in the main circuit.

 The invention is illustrated by a diagram of a device for implementing the method, which shows a container 1 for a mixture of PFOS, a pipe 2 connecting the tank 1 to the tank 3 of the main circuit, pipelines 4 and 6 connecting the tank 3 to the high-pressure homogenizer 5. Pipelines 7 and 9 and the container 8 form an additional circulation circuit, where the emulsion is pumped through the homogenizer 5 from the main circuit.

The method is as follows:
To obtain a pre-emulsion, the PFOS mixture is supplied dropwise from tank 1 through pipeline 2 to tank 3 with an emulsifier and is pumped together with the emulsifier in the main circuit through pipelines 4 and 6 through homogenizer 5 at a lower pressure threshold for 10 15 minutes. to obtain the desired ratio of components. Then the pre-emulsion is pumped through the pipe 7 into the container 8 of the additional circuit, increasing the pressure in the homogenizer by 1.1-1.2 times, while the container 3 is completely released and the so-called "stagnant zones" are eliminated, where the largest particles of the emulsion accumulate. After that, the pressure in the homogenizer 5 is reduced to the initial value and the emulsion is pumped through the pipeline 9 into the tank 3 of the main circuit, completely freeing the tank 8. Thus, the process of creating submicron emulsions in a two-circuit way consists in repeatedly (up to 12 times) alternating the passage of the emulsion through the circuits of the containers 3 and 8, during which the emulsion located in one working tank when changing the circuit is completely and evenly pumped to another working tank, which allows you to create almost one native, monodisperse in particle size composition.

 The experimentally selected mode of homogenization pressure made it possible to obtain an unexpected effect: eliminate repeated coarsening of emulsion particles, which is usually observed during homogenization at constant pressure, which helps to reduce the average particle size.

Thus, the proposed method allows to obtain a higher monodispersity and reduce the average particle size compared with the drug Fluosol-DA 20%
The emulsion can be stored without freezing for one month or more, because particle enlargement occurs within acceptable limits and does not affect the quality of the emulsion; moreover, with repeated freezing and thawing, the particle size of the emulsion does not change significantly.

 The table below 2 5, illustrating the change in the stability of the emulsion obtained by this method, according to the change in the average particle size.

 The described method allows to improve the sterility of the process and to abandon the sterilization of the emulsion using hard autoclaving, during which there is a significant enlargement of the particles of the emulsion (see tab. 6, 7, 8).

 As can be seen from the table. 7 and 8, the half-life of perfluorocarbons introduced into the emulsion obtained by this method from the bloodstream is 24 hours; the time for complete removal of perfluorocarbons from the body is 18 months.

 In the table. Figure 9 shows the comparative characteristics of 20% perfluorocarbon emulsions.

 Thus, the proposed method allows to obtain highly stable fully submicron emulsions that do not require additional sterilization, are stored at room temperature for a long time and have all the necessary qualities for medical use.

 Getting a 20% emulsion.

Example 1. In 900 ml of pyrogen-free water in a tank 3 was dissolved 40 g of a copolymer of polyoxyethylene and polyoxypropylene M.V. 7500 (proxanol-268). 100 ml of the PFD / PFMTSP mixture in a 2: 1 ratio containing 130 g PFD specific gravity 1.938 and 65 g PFMCP specific gravity 1.899 were passed dropwise from the container 1 through a pipe 2 through the resulting solution. At the same time, the resulting mixture was passed through homogenizer 5 at a pressure of 300 kg / cm ² for 10 min until the mixture was completely dripped with PFOS. The resulting pre-emulsion was then passed alternately through containers 3 and 8 and the homogenizer 5 twelve times, increasing the pressure through each cycle to 360 kg / cm ² . The temperature was maintained within 20 22 ^o C using water cooling. Thus, a 20% emulsion was obtained, the average particle size of which was 0.11 μm, the concentration F ^was 11 * 10 ^-6 M, the number of particles with a diameter of more than 0.2 μm was 1.4%. A sterile electrolyte solution supporting osmotic was added to the emulsion pressure, in quantity, g / l: sodium chloride 6.0; potassium chloride 0.39; magnesium chloride 0.19; calcium chloride 0.28; sodium hydrogen phosphate 0.2; sodium bicarbonate 1.3; glucose 2.0; pyrogen-free water up to 1000 ml, and was used as a blood substitute for intravenous administration to mice for a week according to standard methods. The LD ₅₀ value was 136 ml / kg.

Example 2. An emulsion was prepared as described in example 1, but at a pressure of 500 to 550 550 kg / cm ² . The average particle size was 0.06 μm, the concentration of F ^was 12 * 10 ^-6 M, the number of particles with a diameter greater than 0.2 μm was 0.4%. After adding the electrolyte solution in accordance with the specified formulation, the emulsion was administered to mice, LD ₅₀ was 158 ml / kg

Example 3. An emulsion was prepared as described in example 1, but at a pressure of 600 660 kg / cm ² . The average particle size was 0.082 μm, the concentration of F ^was 12 * 10 ^-6 M, the number of particles with a diameter greater than 0.2 μm was 0.56%. After adding the electrolyte solution in accordance with the specified formulation, the emulsion was administered to mice, the LD ₅₀ value was 142 ml / kg

Example 4. Received an emulsion as described in example 1, but at a pressure of 500 to 550 kg / cm ² . An electrolyte solution was added to the emulsion in the amount, g / l: sodium chloride 8.0; potassium chloride 1.1; magnesium chloride - 0.12; calcium chloride 0.04; sodium hydrogen phosphate 0.2; sodium bicarbonate 1.3; glucose 2.0; pyrogen-free water up to 1000 ml. The emulsion was used as a cardioplegic composition for preserving the heart of a rabbit. Cardiac conservation was provided for 14 hours.

Example 5. Received an emulsion as described in example 1, but at a pressure of 500 to 550 kg / cm ² . An electrolyte solution was added to the emulsion in the amount, g / l: sodium chloride 8.0; potassium chloride 0.39; magnesium chloride - 0.19; calcium chloride 0.28; sodium hydrogen phosphate 0.2; sodium bicarbonate 1.3; glucose 2.0; pyrogen-free water up to 1000 ml. The emulsion was used to perfuse an isolated rabbit heart. The vital activity of the heart was maintained for 12 hours.

Example 6. An emulsion was prepared as described in Example 1, but perfluorodecalin was replaced with perfluorooctyl bromide of a specific gravity of 1.901 in the same amount and in the same ratio, the mixture was homogenized at a pressure of 500-550 kg / cm ² . An electrolyte solution was added to the resulting emulsion as described in Example 1. The emulsion was used as a radiopaque agent when administered intravenously to mice. The LD ₅₀ value was 152 ml / kg.

Getting 40% emulsion
Example 7. In 800 ml of pyrogen-free water in a tank 3 was dissolved 80 g of a copolymer of polyoxyethylene and polyoxypropylene M.V. 7500.

200 ml of a mixture of PFOB / PFMCP (2: 1) containing 266 g of PFOB specific gravity 1.901 and 133 g of PFMCP specific gravity 1.899 were passed through the obtained solution dropwise from tank 1 through pipeline 2, while the resulting mixture was passed through homogenizer 5 at a pressure of 500 kg / cm ² for 10 min until the mixture is completely dripped with PFOS. The resulting pre-emulsion was passed alternately through containers 3 and 8 and the homogenizer 5 twelve times, increasing the pressure through each cycle to 550 kg / cm ² . The temperature was maintained within 20 22 ^o C using water cooling. The average particle size of the emulsion was 0.07 μm, the concentration F ^was 12 * 10 ^-6 M, the number of particles with a diameter of more than 0.2 μm was 0.4%. A sterile electrolyte solution supporting the osmotic pressure was added to the emulsion, in g / l : sodium chloride 6.0; potassium chloride 0.39; magnesium chloride 0.19; sodium hydrogen phosphate 0.2; sodium bicarbonate 1.3, glucose 2.0; pyrogen-free water up to 1000 ml, and was used as a radiopaque agent for intravenous administration to mice. The LD ₅₀ value was 150 ml / kg.

Claims (2)

 1. A method of producing perfluorocarbon emulsions for medical purposes, comprising preparing a preemulsion by mixing the total amount of organofluorine compounds with an emulsifying agent and repeatedly homogenizing the mixture under high pressure in a closed circulation loop, characterized in that the mixture of perfluororganic compounds and emulsifying agent is alternately passed through the primary and secondary circuits homogenizer up to 12 times, increasing the pressure in the additional circuit by 1.1 - 1.2 times compared to the pressure we take in the main circuit, and the pre-emulsion is obtained by rolling a mixture of perfluororganic compounds through a container with an emulsifying agent and simultaneously passing the resulting pre-emulsion through the main circuit of the homogenizer.  2. The method according to p. 1, characterized in that for the preparation of the emulsion take a mixture of perfluorodecalin or perfluorooctyl bromide and perfluoromethylcyclohexylpiperidine in a ratio of 2: 1, and proxanol is used as an emulsifying agent.

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