Oxygen carrier pharmaceutical composition containing perfluorocarbon and casein, and preparation method therefor

Abstract

The present invention provides a pharmaceutical composition in which perfluorocarbon is formulated in the form of a nanoemulsion using casein or a salt thereof, and a preparation method therefor. In the pharmaceutical composition of the present invention, perfluorocarbon may be present in the form of stable liquid particles in the nanoemulsion. Therefore, the pharmaceutical composition of the present invention can avoid side effects such as thrombus formation or vascular occlusion, and thus can be useful as an artificial oxygen carrier, i.e., a blood substitute.

Description

Oxygen carrier pharmaceutical composition containing perfluorocarbon and casein and method for producing the same

[0001] The present invention relates to a pharmaceutical composition containing perfluorocarbon as an oxygen carrier and a method for preparing the same. More specifically, the present invention relates to a pharmaceutical composition for use as an artificial oxygen carrier, wherein perfluorocarbon is formulated in the form of a nanoemulsion using casein or a salt thereof, and a method for preparing the same.

[0002] Red blood cells are essential components of blood, supplying oxygen to the body's tissues and organs and removing carbon dioxide. Therefore, when severe bleeding occurs due to accidents, trauma, surgery, or acute coagulation disorders, leading to hemorrhagic shock, death can occur if a new blood supply is not available. The oldest, most common, and most effective method for addressing these situations is homologous blood transfusion. However, transfusions of homologous blood incur significant costs, including the process of separating, testing, diagnosing, and storing the collected blood. Furthermore, they can lead to complications such as infection and immunological side effects due to different blood types.

[0003] One potential solution to these problems is artificial blood. Research on artificial blood is currently underway in areas such as the development of artificial red blood cells (RBCs) that carry oxygen, artificial platelets that aid in blood clotting, and artificial immune cells that prevent infection and clotting factors. Among these, active research is being conducted on artificial RBCs, or RBC substitutes. These RBCs function as a substitute for human RBCs, transporting oxygen to various tissues through the blood vessels.

[0004] Artificial oxygen carriers pursue oxygen-carrying capacity, maintaining a constant colloidal osmotic pressure, long-term blood retention, low toxicity of their components, ease of excretion, and ease of long-term storage. They can be transfused regardless of blood type, making them crucial for national health and security. To address the global blood shortage, various blood substitutes are being researched. Research and development is underway to produce universal artificial blood, including cell-free and chemically modified hemoglobins and oxygen-compatible polymers, to supply oxygen carriers that can replace the function of red blood cells. However, no successful examples have yet been reported.

[0005] Perfluorocarbons (PFCs) are chemically and biologically inert, and have a high dissolving capacity for oxygen and carbon dioxide, with a solubility in oxygen approximately 20 times that of plasma, which physically increases the dissolution of oxygen in arterial blood. Because PFCs do not mix well with water, they are used in emulsion form. Because emulsions contain particulate matter, they must be administered in low doses to avoid overload and subsequent dysfunction of reticuloendothelial system phagocytes.

[0006] Perfluorocarbon-containing preparations include Fluosol-DA and Oxygent. TMFluosol-DA (Green Cross Corp., Osaka, Japan) is an emulsion formulation containing perfluorodecalin, perfluorotripropylamine, Pluronic F-68, and phospholipids. However, Fluosol-DA was discontinued due to serious side effects such as bleeding, carbon monoxide poisoning, cerebral hypoxia, anemia, angioplasty, microvascular thrombosis and occlusion, and pulmonary toxicity. Oxygent TM (Alliance Pharm, USA) is an emulsion formulation containing perflubron (perfluorooctyl bromide) and phospholipids. However, Oxygent TM (Alliance Pharm, USA) Side effects such as stroke, flu-like symptoms, blood clots, and vascular lung damage have also been reported.

[0007] Therefore, there is a need in the art for the development of a formulation containing perfluorocarbons useful as oxygen carriers, which can avoid side effects such as thrombosis and vascular occlusion.

[0008] The present inventors have conducted extensive research to develop improved formulations containing perfluorocarbons. In particular, they have conducted extensive research to develop a perfluorocarbon-containing emulsion formulation that can avoid adverse effects such as thrombosis and vascular occlusion. The present inventors have discovered that when perfluorocarbons are formulated in a nanoemulsion form using casein or its salt, which has been proven to be safe, the perfluorocarbons can exist in the form of stable liquid particles within the nanoemulsion.

[0009] Accordingly, the present invention aims to provide a pharmaceutical composition in the form of a nanoemulsion comprising a perfluorocarbon and casein or a salt thereof, which functions as an artificial oxygen carrier.

[0010] In addition, the present invention aims to provide a method for producing a pharmaceutical composition in the form of a nanoemulsion.

[0011] According to the present invention, a pharmaceutical composition in the form of a nanoemulsion for use as an artificial oxygen carrier is provided, comprising a dispersed phase comprising a perfluorocarbon, a surfactant, and casein or a salt thereof in an aqueous continuous phase.

[0012] The above aqueous continuous phase may be water, a phosphate buffer solution, or a saline solution.

[0013] The perfluorocarbon may be selected from the group consisting of perfluorooctyl bromide (PFOB), perfluorodecalin (PFD), and perfluorohexane (PFH), and preferably may be perfluorooctyl bromide (PFOB). The perfluorocarbon may be present in a concentration of 10 to 70 w / w%.

[0014] The surfactant may be selected from the group consisting of phospholipids, glycerin fatty acid esters, sorbitan fatty acid esters, polyoxyethylene fatty acid esters, propylene glycol fatty acid esters, tocopherol polyethylene glycol succinate (TPGS), macrogol 15 hydrostearate, poloxamer, polyoxyl hydrogenate castor oil, polyoxyl castor oil, polyoxyl cetosteryl ester, and cholesterol. The surfactant may be selected from the group consisting of phospholipids, for example, phosphatidylcholine, phosphatidylethanolamine, and lecithin. The surfactant may be present at a concentration of 1 to 10 w / w%.

[0015] The above casein or its salt may be present at a concentration of 1 to 10 w / w%.

[0016] The pharmaceutical composition of the present invention may further comprise at least one viscosity modifier selected from the group consisting of glycerin, mannitol, lactose, trehalose, polyvinyl alcohol, propylene glycol, dextrin, and povidone. The viscosity modifier may be present at a concentration of 1 to 5 w / w%.

[0017] In the pharmaceutical composition of the present invention, the dispersed phase may have a diameter of 300 nm or less.

[0018] In addition, according to the present invention, a method for preparing a pharmaceutical composition is provided, comprising the steps of (a) adding casein or a salt thereof, a surfactant, and optionally a viscosity modifier, and perfluorocarbon to an aqueous continuous phase and homogenizing the same, and (b) dispersing the dispersion obtained in step (a) using a high-pressure disperser to form a nanoemulsion.

[0019] The present invention has revealed that when perfluorocarbon is formulated in the form of a nanoemulsion using casein or its salt, the perfluorocarbon can exist in the form of stable liquid particles within the nanoemulsion. Therefore, the pharmaceutical composition in the form of a nanoemulsion according to the present invention can avoid the adverse effects of thrombosis or vascular occlusion, and thus can be usefully used as an artificial oxygen carrier, i.e., a blood substitute.

[0020] Figure 1 is a Cryo-TEM image of a nanoemulsion (No. 12 of Example 2) containing casein at a concentration of 2.5 w / w%.

[0021] Figure 2 is a cryo-TEM image of a nanoemulsion (No. 17 of Example 2) that does not contain casein.

[0022] The present invention provides a pharmaceutical composition in the form of a nanoemulsion for use as an artificial oxygen carrier, comprising a dispersed phase comprising a perfluorocarbon, a surfactant, and casein or a salt thereof in an aqueous continuous phase.

[0023] In the pharmaceutical composition of the present invention, the aqueous continuous phase may be water (e.g., water for injection, etc.), phosphate buffer, physiological saline, etc. If necessary, the aqueous continuous phase may additionally contain a viscosity modifier, such as glycerin.

[0024] The perfluorocarbon may include various perfluorocarbons known as artificial oxygen carriers. For example, the perfluorocarbon may be selected from the group consisting of perfluorooctyl bromide (PFOB) (also known as perflubron), perfluorodecalin (PFD), and perfluorohexane (PFH). In one embodiment, the perfluorocarbon may be perfluorooctyl bromide (PFOB). The perfluorocarbon may be used in a concentration sufficient to exhibit effectiveness as an artificial oxygen carrier. For example, the perfluorocarbon may be present in a concentration of 10 to 70 w / w%, preferably 20 to 60 w / w%.

[0025] The surfactant acts as an emulsifier for forming a nanoemulsion. The surfactant includes, without limitation, emulsifiers used in perfluorocarbon-containing formulations. For example, the surfactant may be at least one selected from the group consisting of phospholipids, glycerin fatty acid esters, sorbitan fatty acid esters, polyoxyethylene fatty acid esters (e.g., polyoxyethylene 20 sorbitan monolaurate, polyoxyethylene 80 sorbitan monooleate, etc.), propylene glycol fatty acid esters, tocopherol polyethylene glycol succinate (TPGS), macrogol 15 hydrostearate, poloxamers, polyoxyl hydrogenate castor oil, polyoxyl castor oil, polyoxyl cetosteryl ester, and cholesterol. Preferably, the surfactant may be a phospholipid. In one embodiment, the phospholipid may be at least one selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, and lecithin. The above surfactant may be present at a concentration of 1 to 10 w / w%, preferably 1 to 5 w / w%.

[0026] In the pharmaceutical composition of the present invention, casein or a salt thereof (e.g., sodium caseinate) functions to enable perfluorocarbon to exist in the form of stable liquid particles in the nanoemulsion. It is presumed that casein or a salt thereof encapsulates perfluorocarbon liquid particles to form a core-shell structure or to modify the surface of the perfluorocarbon liquid particles. The casein or a salt thereof may be used in an amount that allows perfluorocarbon to exist in the form of stable liquid particles in the nanoemulsion. For example, the casein or a salt thereof may be present in a concentration of 1 to 10 w / w%, preferably 1 to 5 w / w%.

[0027] The pharmaceutical composition of the present invention may include pharmaceutically acceptable additives commonly used in perfluorocarbon-containing preparations. For example, the pharmaceutical composition of the present invention may further include one or more viscosity modifiers selected from the group consisting of glycerin, mannitol, lactose, trehalose, polyvinyl alcohol, propylene glycol, dextrin, and povidone. The viscosity modifier may be present at a concentration of 1 to 5 w / w%.

[0028] The pharmaceutical composition of the present invention is in the form of a nanoemulsion, and for example, the dispersed phase (i.e., perfluorocarbon liquid particles) may have a diameter of 300 nm or less, preferably 70 to 200 nm.

[0029] The present invention includes a method for preparing the pharmaceutical composition described above. For example, the method may include the steps of (a) adding casein or a salt thereof, a surfactant, and optionally a viscosity modifier and a perfluorocarbon to an aqueous continuous phase and homogenizing the mixture, and (b) dispersing the dispersion obtained in step (a) using a high-pressure disperser to form a nanoemulsion.

[0030] In the manufacturing method of the present invention, the aqueous continuous phase, perfluorocarbon, surfactant, viscosity modifier, etc. are as described in relation to the pharmaceutical composition. The homogenization in step (a) and the dispersion in step (b) can be performed using a homogenizer and a high pressure homogenizer according to conventional methods.

[0031] Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention and the present invention is not limited to these examples.

[0032] Example 1: Preparation and Evaluation of Nanoemulsions

[0033] An emulsion was prepared according to the ingredients and contents in Table 1 below. Specifically, sodium caseinate was dissolved in distilled water, followed by the addition of lecithin, glycerin, and perfluorooctyl bromide (PFOB), and homogenization. The resulting dispersion (in the form of a pre-emulsion) was dispersed using a high-pressure homogenizer to prepare a nanoemulsion.

[0034] The particle size, zeta potential, and oxygen release concentration of the obtained nanoemulsion were max ) were measured respectively. Particle size and zeta potential were measured using ELSZ-2000 equipment (Otsuka Electronics Korea).

[0035] The maximum oxygen release concentration was measured under the following conditions.

[0036] - Sample volume: 1.5 mL

[0037] - Flask volume: 50 mL

[0038] - Sample Oxygenation: 1 hour

[0039] - Medium: 50 mL of nitrogen-exchanged pH7.4 PBS buffer (oxygen concentration 0.01 mg / L)

[0040] - Stirring speed: 200 rpm

[0041] - Evaluation item: Dissolved oxygen (mg / L)

[0042] The sample was oxygenated by blocking air with a rubber cap and stirring at 200 rpm for 1 hour. 50 mL of pH 7.4 PBS buffer was blocked with air with a rubber cap and stirred at 200 rpm for 1 hour to replace with nitrogen so that the oxygen concentration was 0.01 mg / L or less. 1.5 mL of the sample was added to 50 mL of pH 7.4 PBS buffer and the oxygen release concentration was checked while stirring at 200 rpm.

[0043] As described above, the results of measuring particle size, zeta potential, and maximum oxygen release concentration are shown in Table 1 below.

[0044] [Table 1]

[0045]

[0046] As the casein concentration increased, particle size tended to decrease, while as the lecithin concentration increased, particle size also increased. The ratio of PFOB to glycerin did not significantly affect particle size.

[0047] As the casein concentration increased, the zeta potential tended to decrease. No significant effects were observed on zeta potential depending on the concentration of PFOB, lecithin, or glycerin.

[0048] As the concentrations of casein and PFOB increased, the peak oxygen release concentration tended to increase. The ratio of lecithin and glycerin concentrations did not significantly affect the peak oxygen release concentration.

[0049] Example 2: Preparation and Evaluation of Nanoemulsions

[0050] To select the optimal composition based on the results obtained in Example 1, nanoemulsions were prepared according to the components and contents in Table 2 with a concentration of perfluorooctyl bromide (PFOB) of 40 w / w%. In addition, the particle size and zeta potential of each nanoemulsion were measured using the same method as in Example 1, and the results are shown in Table 2 below.

[0051] [Table 2]

[0052]

[0053] The results of measuring the particle structure using cryo-TEM for the nanoemulsion containing casein at a concentration of 2.5 w / w% (No. 12) and the nanoemulsion not containing casein (No. 17) are as shown in Figs. 1 and 2, respectively. The nanoemulsion containing casein showed a particle shape close to a circle (Fig. 1), whereas the nanoemulsion not containing casein showed a shape in which the particles were crushed or pressed together with very close interparticle spacing (Fig. 2). Therefore, it can be confirmed that the nanoemulsion containing casein exhibits excellent stability.

[0054] Example 3: Preparation of nanoemulsion

[0055] An emulsion was prepared according to the ingredients and contents in Table 3 below. Specifically, sodium caseinate was dissolved in distilled water, and then a surfactant and / or viscosity modifier (lecithin, TPGS, poloxamer 407, Kolliphor HS 15, glycerin, and / or mannitol) and perfluorocarbon [perfluorooctyl bromide (PFOB) and / or perfluorodecalin (PFD)] were added and homogenized. The obtained dispersion (dispersion in the form of a pre-emulsion) was dispersed using a high pressure homogenizer to prepare a nanoemulsion.

[0056] [Table 3]

[0057]

Claims

1. A pharmaceutical composition in the form of a nanoemulsion for use as an artificial oxygen carrier, comprising a dispersed phase comprising a perfluorocarbon, a surfactant, and casein or a salt thereof in an aqueous continuous phase.

2. A pharmaceutical composition according to claim 1, characterized in that the aqueous continuous phase is water, a phosphate buffer solution, or a physiological saline solution.

3. A pharmaceutical composition according to claim 1, characterized in that the perfluorocarbon is at least one selected from the group consisting of perfluorooctyl bromide (PFOB), perfluorodecalin (PFD), and perfluorohexane (PFH).

4. A pharmaceutical composition according to claim 3, characterized in that the perfluorocarbon is perfluorooctyl bromide (PFOB).

5. A pharmaceutical composition according to claim 1, characterized in that the perfluorocarbon is present at a concentration of 10 to 70 w / w%.

6. A pharmaceutical composition according to claim 1, characterized in that the surfactant is at least one selected from the group consisting of phospholipids, glycerin fatty acid esters, sorbitan fatty acid esters, polyoxyethylene fatty acid esters, propylene glycol fatty acid esters, tocopherol polyethylene glycol succinate (TPGS), macrogol 15 hydrostearate, poloxamer, polyoxyl hydrogenate castor oil, polyoxyl castor oil, polyoxyl cetosteryl ester, and cholesterol.

7. A pharmaceutical composition according to claim 6, characterized in that the surfactant is a phospholipid.

8. A pharmaceutical composition according to claim 7, characterized in that the phospholipid is at least one selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, and lecithin.

9. A pharmaceutical composition according to claim 1, characterized in that the surfactant is present at a concentration of 1 to 10 w / w%.

10. A pharmaceutical composition according to claim 1, characterized in that casein or a salt thereof is present at a concentration of 1 to 10 w / w%.

11. A pharmaceutical composition further comprising at least one viscosity modifier selected from the group consisting of glycerin, mannitol, lactose, trehalose, polyvinyl alcohol, propylene glycol, dextrin, and povidone, in the first paragraph.

12. A pharmaceutical composition according to claim 11, characterized in that the viscosity modifier is present at a concentration of 1 to 5 w / w%.

13. A pharmaceutical composition according to any one of claims 1 to 12, characterized in that the dispersed phase has a diameter of 300 nm or less. 14.(a) a step of adding casein or a salt thereof, a surfactant, and optionally a viscosity modifier, and perfluorocarbon to the aqueous continuous phase and homogenizing it, and (b) A step of forming a nanoemulsion by dispersing the dispersion obtained in step (a) using a high-pressure disperser. A method for producing a pharmaceutical composition according to any one of claims 1 to 12, comprising:

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