JP2005002055A - Method for producing liposome containing physiologically active substance and liposome containing physiologically active substance obtained by the method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a liposome containing a biologically active substance, suitable for scale up required when the mass production is carried out industrially. <P>SOLUTION: The method for producing the liposome containing the biologically active substance involves extruding the biologically active substance and a liposome-forming lipid from (B) a slit-shaped structure of an extruding part having (A) a structure selected from each rotating helical structure and screw-shaped structure, and (B) the slit structure, at ≥9.0 L/sec extrusion flow rate and at ≥9.4 m/sec extrusion flow velocity. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a production method suitable for scale-up required when industrially mass-producing liposomes containing a physiologically active substance. The present invention also relates to a liposome containing a physiologically active substance obtained by the production method. The liposome has a desired average particle diameter and is suitable for use in the medical field because the contained physiologically active substance is not thermally denatured.
[0002]
[Prior art]
Many attempts have been made to apply bioactive substances and drugs in the medical field as a drug delivery system by microencapsulating them with liposomes. As a general liposome production method, a thin film method, a reverse phase method, a dialysis method, a membrane emulsification method, a high pressure discharge method, a high speed stirring method, or the like is used. Although these methods can be used to make liposomes of aqueous solutions of drugs with low concentrations and viscosities and physiologically active substances and protein solutions that do not easily cause heat denaturation, it is difficult to increase the drug content in liposomes. In order to increase the drug content, it is necessary to make the content liposomal at a high concentration. However, it has been difficult to make a physiologically active substance having a high viscosity and susceptible to thermal denaturation into a liposome.
[0003]
Patent Document 1 describes a method of forming a high-viscosity and high-concentration hemoglobin aqueous solution into a liposome using a high-speed stirring emulsifier. However, with this method and the above-mentioned method for producing liposomes that are generally used, it is possible to prepare liposomes containing a physiologically active substance on the scale of about several hundred mL. In order to scale up from L to several hundred L, it is necessary to examine manufacturing parameters at each scale. Therefore, there are problems associated with large waste and risk associated with the scale-up of liposome production, and further problems such as mechanical strength and durability, making it difficult to design and manufacture large devices. Moreover, the method of patent document 1 uses the shearing force by a cutter-shaped or comb-shaped stirring blade as the energy of emulsification. For this reason, it has been pointed out that a highly viscous mixture does not cause a sufficient flow in the container, and the temperature difference becomes large in the stirring container. High heat is generated at the tip of the stirring blade, and the temperature range of the mixture needs to be lowered in order not to cause denaturation of the physiologically active substance by heat. Furthermore, there is a problem that the progress of liposome formation is slow due to the temperature adjustment, and it takes a long processing time.
[0004]
In order to industrially produce medical liposomes containing physiologically active substances in Japan, the production amount is several tens of thousands to several hundred thousand L per year. In this case, it is necessary to produce several hundreds of liposomes as a production amount per day. In order to shorten the time required for the emulsification step in production due to productivity problems, a processing speed of about several tens to several hundreds L / hr is required. In a method using a conventional high-speed stirring type emulsifier, an emulsification apparatus having a capacity of about several tens to 100 L is necessary for sufficient emulsification. However, in order to scale up from a small experimental machine to a large machine, it is difficult to obtain a necessary parameter because it cannot be predicted with a simple similarity based on the peripheral speed and shape of the stirring blades. For this reason, it is necessary to consider manufacturing on an actual scale, which involves great waste and risk associated with scale-up.
[0005]
[Patent Document 1]
Japanese Patent Laid-Open No. 3-173813
[0006]
[Problems to be solved by the invention]
An object of this invention is to provide the manufacturing method of the liposome containing the bioactive substance which can be scaled up in order to satisfy | fill said request. Another object of the present invention is to provide a liposome containing a physiologically active substance obtained by this production method.
[0007]
[Means for Solving the Problems]
In order to solve the above object, the present invention has the following configurations (1) to (10).
(1) From the (B) slit-like structure of the discharge part having the following (A) structure and (B) slit-like structure provided in the sealed container, 9.0 L of a physiologically active substance and a liposome-forming lipid A method for producing a liposome containing a physiologically active substance, wherein the liposome is discharged at a discharge flow rate of at least / sec and a discharge flow rate of at least 9.4 m / sec.
(A) a structure that rotates about at least one axis selected from the group consisting of a helical structure and a screw-like structure;
(B) A slit-like structure that rotates about an axis.
Here, the (B) slit-like structure is disposed to face the (A) structure, and the (B) slit-like structure is forward or reverse with respect to the rotation direction of the (A) structure. Rotate in the direction.
[0008]
(2) The rotational speed of the structure (A) is 500 rpm or more and less than 20,000 rpm,
(B) The method for producing a liposome containing a physiologically active substance according to (1), wherein the rotational speed of the slit-like structure is 500 rpm or more and less than 20,000 rpm.
[0009]
(3) The method for producing a liposome containing a physiologically active substance according to (1) or (2), wherein the slit width in the slit-like structure is 0.5 mm to 5 mm.
[0010]
(4) The physiologically active substance according to any one of (1) to (3), wherein the shaft sealing mechanism of the rotating shaft of the structure (A) and the structure (B) is a mechanical seal A method for producing liposomes containing
[0011]
(5) The sealed container further incorporates a heat exchanger, and the inside of the sealed container is cooled using the heat exchanger, whereby the physiologically active substance and the liposome-forming lipid are discharged. A method for producing a liposome containing a physiologically active substance according to any one of (1) to (4), wherein the temperature in the sealed container is maintained at 5 ° C to 60 ° C.
[0012]
(6) When the physiologically active substance and liposome-forming lipid are ejected from the slit-like structure of the ejection part, the inside of the sealed container is pressurized at 0.1 atm or more and 2 atm or less (1) Or a method for producing a liposome containing the physiologically active substance of (5).
[0013]
(7) The method for producing a liposome containing the physiologically active substance according to any one of (1) to (6), wherein the physiologically active substance is hemoglobin.
[0014]
(8) The physiologically active substance is supplied to the sealed container as a solution having a concentration of 30 wt% to 60 wt%, and a liposome containing the physiologically active substance of any one of (1) to (7) is produced how to.
[0015]
(9) A liposome containing a physiologically active substance obtained by any one of the methods (1) to (8).
[0016]
(10) The liposome containing a physiologically active substance according to (9), wherein the average particle size is 150 nm to 220 nm.
[0017]
DETAILED DESCRIPTION OF THE INVENTION
The present invention will be further described below with reference to the drawings. However, the drawings are for illustrative purposes, and the present invention is not limited thereto.
FIG. 1 is a conceptual diagram of an example of an apparatus used in the method of the present invention, and a sealed container 2 is indicated by a broken line for easy understanding of the internal configuration.
The apparatus 1 in FIG. 1 has a sealed container 2 that can store a liquid. The device 1 has a structure 3 in which one end exists in the sealed container 2 and is rotatable about an axis. A spiral structure or a screw-like structure 31 is provided at the end of the structure 3 in the sealed container 2. FIGS. 2A to 2C are partial enlarged views of an example of the structure 31, and the structure 31 is shown as a conical screw-like structure. 2 (a) is a side view of the structure 31, FIG. 2 (b) is a view of the structure 31 from below, and FIG. 2 (c) is a view of the structure 31 from above. FIG.
In FIG. 1, when the structure 3 is rotated around an axis, a spiral structure or a screw-like structure 31 rotates in the sealed container 2, and a liquid flow is generated in the sealed container 2. The opposite end 32 of the structure 3 is located outside the sealed container 2 and connected to a power device such as a motor (not shown) attached to the outside or the surface of the sealed container 2.
[0018]
The device 1 has a structure 4 having one end portion in the sealed container 2 and capable of rotating about an axis. A structure (slit-like structure) 41 having a slit 42 is provided at an end of the structure 4 in the sealed container 2, and an end 43 on the opposite side of the structure 4 is located outside the sealed container 2. The motor is connected to a power device (not shown) such as a motor attached to the outside or the surface of the sealed container 2. In FIG. 1, the slit 42 is partially omitted to show the structure 31, but the slit 42 is formed in the slit-like structure 41 over the entire circumference.
[0019]
In FIG. 1, the spiral structure or screw-like structure 31 and the slit-like structure 41 are arranged to face each other. More specifically, the slit-like structure 41 has a shape that wraps part or all of the spiral structure or screw-like structure 31 at a portion facing the spiral structure or screw-like structure 31. The structure 31 and the structure 41 are arranged in such a relationship to constitute the discharge unit of the apparatus 1.
[0020]
A shaft sealing mechanism 5 is provided in a portion where the structures 3 and 4 penetrate the sealed container 2.
Furthermore, the apparatus 1 includes a raw material supply port 6 for supplying liposome-forming lipids and physiologically active substances into the sealed container 2, a pressurizing tube 7 for pressurizing the sealed container 2 with gas or liquid, A heat exchanger 8 for cooling the sample and a sampling tube 9 for taking out the produced liposomes are provided.
[0021]
In order to carry out the method for producing the liposome of the present invention using the apparatus 1 of FIG. 1, first, the liposome-forming lipid and the physiologically active substance are supplied into the sealed container 2 through the raw material supply port 6.
[0022]
The liposome-forming lipid used in the present invention is a substance that forms a lipid membrane of liposome and encapsulates (encapsulates) a physiologically active substance therein. The liposome-forming lipid is not particularly limited as long as it can form liposomes, and natural or synthetic lipids can be used. Among these, phospholipids are particularly preferably used, and at least one phospholipid consisting of lecithin, phosphatidylethanolamine, phosphatidylcholine phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, sphingoemilin, and those hydrogenated according to a conventional method is used. can do. These phospholipids can be used alone or in combination of two or more.
[0023]
Furthermore, sterol membrane stabilizers such as cholesterol and charge-imparting substances (for example, stearic acid, oleic acid, myristic acid, linoleic acid, linolenic acid, etc. and those having a terminal group as a salt) are optionally added to the liposome-forming lipid You can also The above-mentioned liposome-forming lipid may be introduced into the closed container 2 in any form of powder or solution. The liposome-forming lipid may contain other additives. The additive is not particularly limited, and examples thereof include carriers, excipients, preservatives, stabilizers, binders, antioxidants, swelling agents, isotonic agents, solubilizers, preservatives, buffers, and diluents. It is done.
[0024]
The physiologically active substance contained in the liposome is not particularly limited as long as the formation of the liposome is not impaired and the method of the present invention is applicable. The method of the present invention is suitably used for producing liposomes having a high viscosity in a solution state and encapsulating biologically or pharmacologically active substances that are susceptible to thermal denaturation. Specific examples of such physiologically active substances include proteins. Specific examples of the protein contained in the liposome include colony stimulating factors such as hemoglobin, interferons, interleukins, tumor necrosis factor, granulocyte macrophage colony stimulating factor, granulocyte colony stimulating factor, macrophage colony stimulating factor, etc. , Stem cell factor (SCF), transforming growth factor, hepatocyte growth factor, vascular endothelial cell growth factor and other cytokines, growth factor, erythropoietin (EPO), vaccine, antibody and the like. These proteins are preferably human proteins or proteins that can be administered to humans.
[0025]
Among these physiologically active substances, the method of the present invention is more preferably used for containing in a liposome a high-concentration protein that has high viscosity and is easily heat-denatured. Here, the high concentration protein means a protein concentrated to 30% (w / w) or more.
[0026]
These physiologically active substances may contain only one kind of physiologically active substance in the liposome or two or more kinds of physiologically active substances in the liposome according to the purpose of use.
The physiologically active substance can be introduced into the closed container 2 in the form of powder, solution or gel.
[0027]
As specific examples of these physiologically active substances, hemoglobin used as an artificial oxygen carrier in the medical field is preferably exemplified, and high concentration hemoglobin of 30% (w / w) or more is particularly preferable. High concentration hemoglobin may be contained alone in the liposome, or may be contained in the liposome together with other physiologically active substances. High-concentration hemoglobin can be obtained by hemolyzing erythrocytes by a conventional method and removing the erythrocyte membrane using a membrane having a fractional molecular weight of 100,000 (Japanese Patent Laid-Open No. 2002-161047). Then, what is concentrated to 30% (w / w) or more by ultrafiltration is used as high-concentration hemoglobin (Japanese Patent Laid-Open No. 63-212230). Alternatively, recombinant hemoglobin (Nature, (USA), March 1992, 356 (19), No. 19, p. 258-260) produced in E. coli, yeast, etc. by genetic manipulation is subjected to ultraconcentration, etc. It is also possible to use what is concentrated to 30% (w / w) or more as high-concentration hemoglobin.
[0028]
The physiologically active substance is supplied into the sealed container 2 in the form of an aqueous solution, and its concentration is preferably 30 to 60% (w / w), and particularly preferably 40 to 50% (w / w). In particular, when the physiologically active substance is high-concentration hemoglobin, it is preferable to supply at the above concentration. The amount of oxygen transport to peripheral tissues, which is a function of the artificial oxygen carrier, increases depending on the hemoglobin concentration in the liposome. If hemoglobin is introduced at the above-mentioned concentration, the hemoglobin concentration can be maximized within a range in which the concentration of hemoglobin and the inclusion of hemoglobin in the liposome are compatible.
[0029]
For the purpose of efficiently incorporating a physiologically active substance such as hemoglobin into the liposome, it is an important technical problem to contain as much physiologically active substance as possible with the smallest amount of liposome-forming lipid. In both relations, it is known that the ratio (L / H ratio) between the weight (L) of the liposome-forming lipid and the weight (H) of the physiologically active substance contained in the liposome is preferably as small as possible. . For example, in Japanese Patent Laid-Open No. 2-86841, the L / H ratio is set to 0.40 to 1.67. In the present invention, the L / H ratio is preferably 0.4 to 1.67. If the L / H ratio is in the above range, the liposome has sufficient strength, there is no risk of leakage of the physiologically active substance, and the body is loaded with a lipid component more than necessary per dose of the physiologically active substance. There is no risk of giving it to.
[0030]
The physiologically active substance and the liposome-forming lipid are introduced into the closed container 2 without mixing or as a mixture. In the present specification, these non-mixed or mixed physiologically active substances and liposome-forming lipids may be referred to as “raw materials” hereinafter. Specifically, the physiologically active substance and the liposome-forming lipid may be separately introduced into one sealed container 2, or the physiologically active substance and the liposome-forming lipid are mixed partially or completely in advance, Then, this mixture may be introduced into the sealed container 2.
[0031]
When the physiologically active substance and the liposome-forming lipid are not in a liquid state, the physiologically active substance and the liposome-forming lipid may be appropriately dissolved in a solvent or mixed in the solvent and supplied into the sealed container 2. Examples of the solvent used for this purpose include aqueous solvents (water, purified water, physiological saline, buffer solution, etc.), and organic solvents (methanol, ethanol, butanol, etc.).
[0032]
The pH of the physiologically active substance and liposome-forming lipid before being mixed, or the mixture of a partly or wholly mixed physiologically active substance and liposome-forming lipid is not particularly limited as long as it is in a physiological range. In particular, it is preferably in the pH range in blood, specifically in the range of pH 4.0 to 10.0, more preferably in the range of pH 6.0 to 8.0.
[0033]
After the raw material is introduced into the container 2, the container 2 is sealed in a state filled with the liquid. Since liposomes containing a physiologically active substance are mainly used for medical purposes, it is preferable that the raw material is introduced into the container 2 in a sterile state, and is sealed as it is, and is manufactured in a sealed state.
[0034]
The inner shape of the sealed container 2 is not particularly limited as long as it can be maintained filled with a liquid and sealed, and the raw material can be discharged from the slit 42 of the discharge unit and mixed therein, and can be mixed. The shape can be appropriately selected from a cube, a rectangular parallelepiped, an ellipsoid, a sphere, and a combination thereof.
[0035]
It is preferable that the capacity | capacitance of the airtight container 2 is about 1L-100L, More preferably, it is about 10L-50L. If the capacity | capacitance of the airtight container 2 is about 1L-100L, the discharge part which has sufficient discharge amount can be accommodated in the airtight container 2, and sufficient discharge amount can be obtained with realistic energy. .
[0036]
In the method for producing the liposome of the present invention, the inside of the container 2 of the apparatus of FIG. 1 is filled with the raw material and sealed, and then the structures 3 and 4 are rotated around the axis. Thereby, the spiral structure or screw-like structure 31 and the slit-like structure 41 in the sealed container 2 rotate. When the spiral structure or the screw-like structure 31 is rotated, the material 31 enters the space where the structure 31 and the structure 41 face each other, that is, in the space filled with the material between the structure 31 and the structure 41. Is formed. Part or all of the raw material flow produced by the rotation of the structure 31 collides with the structure 41. Therefore, the structure 31 may be other than a spiral structure or a screw structure as long as the structure 31 forms a flow of the raw material in the direction of the structure 41 when rotated. However, a spiral structure or a screw-like structure is preferable, and in particular, a structure in which all of the raw material flow formed by the structure 31 passes through the slits 42 of the structure 41 is preferable. Specific examples of such a structure include a conical screw structure shown in FIGS. 2A to 2C and a propeller-like structure.
[0037]
The flow of the raw material colliding with the structure 41 passes through a slit 42 provided in the structure 41 to form a discharge flow. The discharge flow that has passed through the slit 42 rapidly loses speed in the sealed container 2. As a result, a liquid-liquid shearing force acts at the flow interface, this shearing force acts as emulsification energy, the physiologically active substance and the liposome-forming lipid are mixed, the raw material is emulsified, and the liposome is formed (of the physiologically active substance). Encapsulation proceeds. Therefore, the speed difference at the interface of the discharge flow is an important factor for liposome formation.
[0038]
The method of the present invention is characterized in that the discharge flow rate of the discharge flow passing through the slit 42 is 9.0 L / sec or more and the discharge flow rate is 9.4 m / sec. Efficient liposomes can be formed by discharging the raw material from the slits 42 under the conditions of the discharge flow parameters.
[0039]
The discharge flow rate of the discharge flow is preferably 9.9 L / sec to 1000 L / sec, more preferably 9.9 L / sec to 500 L / sec, and even more preferably 9.9 L / sec to 200 L / sec. 9.9 L / sec to 100 L / sec is more preferable, and 50 L / sec to 100 L / sec is particularly preferable. The discharge flow rate of the discharge flow is preferably 9.4 m / sec to 30 m / sec, more preferably 12 m / sec to 20 m / sec. When the flow rate of the discharge flow is less than 9.4 m / sec, the encapsulation does not proceed sufficiently, and thus liposomes having an optimum particle size cannot be obtained. On the other hand, when the flow rate is 50 m / sec or more, although it depends on the capacity of the heat exchanger 8 to be used, excessive heat generation may occur and cooling may be difficult.
[0040]
The discharge flow rate of the discharge flow can be changed according to the capacity of the sealed container 2 as long as the above conditions are satisfied. The ratio between the discharge flow rate (L / sec) of the discharge flow and the capacity (L) of the sealed container 2 is not particularly limited, but is preferably 5 (1 / sec) or more, more preferably 5 (1 / sec). ) To 10 (1 / sec). When the ratio between the discharge flow rate (L / sec) of the discharge flow and the capacity (L) of the closed container 2 is in the above range, the stirring of the raw material in the closed container 2 by the discharge flow is sufficient, and the inside of the container 2 This eliminates the possibility of heat non-uniformity in the sealed container 2 due to insufficient stirring of the raw material. In addition, the size of the discharge section necessary to obtain the ratio between the desired flow rate (L / sec) of the discharge flow and the container capacity (L) is a realistic size with respect to the capacity of the sealed container 2. By selecting the ratio between the discharge flow rate and the capacity of the sealed container 2, the heat generated by the shearing force described above is quickly and uniformly distributed in the sealed container 2, and the temperature distribution in the sealed container 2. Can be reduced.
[0041]
In the apparatus 1 shown in FIG. 1, the distance between the structure 31 and the structure 41 depends on the shape of the structure 31 and the structure 41 and the opening area of the slit 42 provided in the structure 41, but the structure 31 rotates. As long as a part or all of the flow of the raw material produced by the collision with the structure 41 exhibits the effect of the present invention, it is not particularly limited. However, in order for the flow of the raw material generated by the rotation of the structure 31 to be efficiently discharged from the slits 42 provided in the structure 41, the distance between the structure 31 and the structure 41 is structural. It is desirable that it is as small as possible as long as it does not touch the surface. The distance between the structure 31 and the structure 41 is preferably 0.1 mm to 5 mm, more preferably 0.1 mm to 2 mm.
[0042]
In the method of the present invention, both structure 31 and structure 41 are rotated. Here, the rotation direction of the structure 31 and the structure 41 may be the same direction or may be opposite directions. Even when the structure 31 and the structure 41 are rotated in the same direction, rotating the structure 41 changes the flow direction of the discharge flow, thereby increasing the velocity vector of the discharge flow and increasing the emulsification energy. The effect can be expected.
However, in the method of the present invention, the rotation direction of the structure 31 and the structure 41 is preferably opposite. If the structure 31 and the structure 41 are rotated in the opposite directions, in addition to the above effects, the opening time of the slit 42 is shortened, thereby increasing the discharge flow rate of the discharge flow. This can also be expected to increase the emulsification energy by increasing the velocity vector of the discharge flow.
[0043]
The number of rotations (rpm) of the structure 31 is preferably 500 rpm or more and less than 20,000 rpm, more preferably 1, although it depends on the diameter of the structure 31 and the shape of the part forming the fluid or the capacity of the sealed container 2. 000 rpm to 10,000 rpm, more preferably 4,000 rpm to 7,000 rpm. By rotating the structure 41 at a rotation speed described later and rotating the structure 31 at a rotation speed of 500 rpm or more and less than 20,000 rpm, the capacity of the closed container 2 is not so limited, and the closed container 2 with a wide range of capacities is used. The discharge flow rate of the discharge flow passing through the slit 42 of the structure 41 is 9.0 L / sec or more, and the discharge flow rate is 9.4 m / sec or more. If the rotational speed of the structure 31 is 20,000 rpm or more, the bearing portion of the structure 31 may be mechanically damaged, and heat is generated in the bearing portion, so that the physiologically active substance is thermally transformed during the production of liposomes. There is a risk.
[0044]
The size of the structure 31 is determined by the capacity of the sealed container 2, but is usually 20 mm to 300 mm in diameter. However, in order to obtain an optimum rotational speed for setting the discharge flow rate of the discharge flow to 9.0 L / sec or more, the diameter is more preferably 50 mm to 300 mm. The material of the structure 31 is not particularly limited, but a steel alloy, an aluminum alloy, a titanium alloy, a ceramic, or the like is optimally used.
[0045]
The number of rotations (rpm) of the structure 41 is preferably 500 rpm or more and less than 20,000 rpm, more preferably, depending on the diameter of the structure 41, the length of the part forming the fluid, the shape, or the capacity of the container. 1,000 rpm to 10,000 rpm, more preferably 4,000 rpm to 7,000 rpm. By rotating the structure 31 at the above-described rotation speed and rotating the structure 41 at a rotation speed of 500 rpm or more and less than 20,000 rpm, the capacity of the closed container 2 is not so limited, and the closed container 2 with a wide range of capacities can be used. The discharge flow rate of the discharge flow passing through the slit 42 of the structure 41 is 9.0 L / sec or more, and the discharge flow rate is 9.4 m / sec or more. If the rotation speed of the structure 41 is 20,000 rpm or more, the bearing portion of the structure 41 may be mechanically damaged, and heat is generated in the bearing portion, so that the physiologically active substance is thermally transformed during the production of liposomes. There is a risk.
[0046]
As described above, the structure 41 has the slit 42 and has a shape that wraps part or all of the structure 31 at the facing portion. The structure 41 is preferably cylindrical or conical with an opening that can accommodate the structure 31 inside. The slit 42 is formed on the side surface or the bottom surface of the structure 41 when the structure 41 is cylindrical, and at the bottom surface when the structure 41 is conical. Although the size of the structure 41 depends on the size of the structure 31 and the closed container 2, when the shape is cylindrical or conical, the diameter is usually 20 mm to 300 mm, more preferably 50 mm to 300 mm.
[0047]
In the structure 41, the shape of each slit 42, the number of the slits 42, and the like are not particularly limited. However, when the structure 31 and the structure 41 are rotated under the above-described conditions, the discharge flow rate of the discharge flow passing through the slit 42 is 9 The total opening area is 0.0 L / sec or more and the discharge flow rate is at least 9.4 m / sec or more. Thereby, as described above, a liquid-liquid shearing force acts at the interface of the flow, and this shearing force acts as an emulsifying energy, and liposome formation proceeds.
[0048]
Here, if the width of the individual slits 42 is too wide in the structure 41, the speed difference becomes small at the center of the discharge flow, and the liquid-liquid shear force necessary for liposome formation does not work sufficiently. For this reason, the width of the slit 42 is preferably in the range of about 0.5 mm to 5 mm, and more preferably in the range of about 1 mm to 3 mm. When the width of the slit 42 is within the above range, the difference in velocity at the central portion of the discharge flow is sufficient, which is preferable for promoting liposome formation. In addition, even when the physiologically active substance or the liposome-forming lipid, or a mixture thereof is highly viscous, a sufficient discharge amount can be obtained, and the entire mixture can be sufficiently stirred, so that the liposome formation is entirely performed. proceed. The length of the slit 42 in the axial direction of the structure 41 is not particularly limited, but is preferably in the range of 10 mm to 300 mm. The shape of each slit 42 is not particularly limited, and may be a rectangle, a trapezoid, a rhombus, a triangle, an ellipse, or the like. Furthermore, although the number of the slits 42 depends on the diameter or shape of the structure 41, it is preferably 10 to 100, more preferably 20 to 50.
The material of the structure 41 is not particularly limited, but a steel alloy, an aluminum alloy, a titanium alloy, a ceramic, or the like is optimally used.
[0049]
Further, the time required for emulsifying the raw material by discharging the raw material from the slit 42 in the closed container 2 is not particularly limited as long as the above-described object can be achieved, but it is preferably 1 minute to 60 minutes, more preferably. Is from 3 minutes to 30 minutes.
[0050]
As shown in FIG. 1, the method of the present invention is preferably carried out using an apparatus 1 in which a heat exchanger 8 is built in a sealed container 2. By incorporating the heat exchanger 8 in the hermetic container 2, the inside of the hermetic container 2 can be cooled when the raw material is discharged from the slit 42 and emulsified. Thereby, the temperature in the airtight container 2 is maintained at a suitable temperature. Here, the preferable temperature in the closed container 2 is a temperature at which the physiologically active substance is not likely to be thermally denatured when the liposome is produced using the method of the present invention, and specifically, 5 ° C. to 60 ° C. It is preferable, More preferably, it is 5 to 40 degreeC.
[0051]
The place where the heat exchanger 8 is disposed is not particularly limited as long as the purpose is achieved, and may be any place in the sealed container 2, but heat is generated in the flow path of the discharge flow in the sealed container 2. It is preferable to arrange the exchanger 8. With this arrangement, the heat exchange efficiency in the heat exchanger 8 can be increased. By increasing the heat exchange rate in the container 2, liposomes can be encapsulated without causing thermal denaturation of the physiologically active substance, and the time required for the encapsulation process can be greatly shortened.
[0052]
In the method of the present invention, when the structure 31 and the structure 41 are rotated to form a discharge flow passing through the slit 42, it is preferable to apply a low pressure to the hermetic container 2. When the structure 31 is rotated at a high speed while the inside of the sealed container 2 is at normal pressure, a vacuum bubble called cavitation is generated behind the rotating structure 31 and it may be difficult to control emulsification. Specifically, due to the generation of cavitation, the rotational energy of the structure 31 is not sufficiently transmitted to the discharge flow, the shock wave generated when this cavitation bubble collapses excessively emulsifies emulsification, and the emulsion contains bubbles. May be mixed in. By applying a low pressure to the hermetic container 2, excessive generation of cavitation is suppressed, and emulsification can be easily controlled.
[0053]
In the present invention, the “low pressure” means a pressure that can control the problems associated with cavitation without causing excessive cavitation. Therefore, the “low pressure” here is not particularly limited as long as it is a pressure that does not cause the above-mentioned problem, but is preferably 0.1 to 2 atm, more preferably 0.3 to 1 atm. . In order to pressurize the inside of the sealed container 2 with the above-mentioned pressure, the inside of the sealed container 2 may be pressurized with gas or liquid through the pressurizing tube 7 after supplying the raw material into the sealed container 2.
[0054]
As shown in FIG. 1, a shaft sealing structure for sealing the rotating shafts of the structures 3 and 4 is provided in the through portions of the structures 3 and 4 of the sealed container 2 in order to keep the inside of the container 2 in a sealed state. 5 is provided. As such a shaft seal structure 5, a shaft seal structure using a ring-shaped seal such as a rotating shaft and a resin in contact with the rotation shaft is generally used. However, when a ring-shaped seal made of resin or the like is used for the shaft seal structure 5, the contact portion between the rotating shaft and the ring-shaped seal slides at a high speed, so that the seal becomes high temperature. If this heat is transferred into the sealed container 2, it may cause thermal denaturation of the physiologically active substance. Thermal denaturation of a physiologically active substance is a problem in itself for producing liposomes containing the physiologically active substance. However, when thermal alteration of the physiologically active substance occurs, there is a risk that the thermal metabolite will stick to the shaft seal structure. is there. When the heat-transformed product adheres to the shaft seal structure, the ring-shaped seal is broken, and it becomes difficult to maintain the hermeticity of the container 2 for a long period of time.
[0055]
In the method of the present invention, it is preferable that the shaft seal structure 5 does not cause the above problem. Specific examples of such a shaft seal structure 5 include a mechanical seal. Here, the mechanical seal is a sealed structure in which one of upper and lower smooth metal plates or ceramic plates is fixed to the sealed container 2 and the other is fixed to the rotating shaft of the structure 3 or structure 4 and these are in contact with each other. is there. A liquid film is formed with water or the like in a gap of several μm to several tens of μm between the two upper and lower plates, and lubrication between the shaft seal and the seal is performed with this liquid film. By using a mechanical seal for the shaft seal structure 5, heat generation in the shaft seal structure is prevented, and the fear of the above-mentioned problems associated with thermal transformation of the physiologically active substance during liposome production is further eliminated.
[0056]
As mentioned above, although the method of this invention was demonstrated with reference to drawings, the method of this invention is not limited to the aspect shown in figure. For example, in the apparatus 1 shown in FIG. 1, a single combination of the structure 31 and the structure 41 is shown in the sealed container 2, but the number of the combinations is not particularly limited, and may be plural. Further, in the combination of the structure 31 and the structure 41, the structures 31 and 41 are not limited to the combination including one each, and one or a plurality of both may be included in the combination.
Further, in the apparatus shown in FIG. 1, the structure 31 and the structure 41 are arranged so that their rotation axes are on the same straight line, but the rotation axes of the structure 31 and the structure 41 are not on the same straight line, The other may be arranged eccentric to the other.
[0057]
The present invention also provides a liposome containing a physiologically active substance obtained by the aforementioned method. The liposome of the present invention contains a physiologically active substance having physiological activity or pharmacological activity represented by the above-mentioned hemoglobin, and can be used for medical purposes. Although the average particle diameter of the obtained liposome is not particularly limited, it is preferably 150 nm to 220 nm, more preferably 150 nm to 200 nm. When the average particle size of the liposome is less than 150 nm, the weight (L) of the liposome-forming lipid increases and the above-mentioned L / H ratio becomes large, which is not preferable. Moreover, when the average particle diameter of the liposome is more than 300 nm, there is a possibility that embolization is caused in capillaries and the like. In order to obtain liposomes having an average particle diameter of about 150 nm to 220 nm, which is the optimum average particle diameter in the present invention, the discharge flow rate of the discharge flow from the slit 42 is 9.0 L / sec or more, and the discharge flow rate is 9.4 m / second. It must be at least sec. Here, the discharge flow rate is more preferably 12 m / sec or more. On the other hand, the discharge flow rate is more preferably 9.9 L / sec or more, and further preferably 14.8 L / sec or more. However, if the discharge flow rate or the discharge flow rate of the discharge flow is extremely large, the heat generation in the sealed container 2 is abruptly generated and the bioactive substance may be thermally denatured. It is desirable to do it in a range. For this purpose, the discharge flow rate of the discharge flow is preferably 30 m / sec or less, more preferably 20 m / sec or less. The discharge flow rate of the discharge flow is preferably 1000 L / sec or less, more preferably 500 L / sec or less, still more preferably 200 L / sec or less, and particularly preferably 100 L / sec or less.
[0058]
From the obtained liposome containing the physiologically active substance, the physiologically active substance that has not been encapsulated is separated and removed according to a conventional method, and collected by filtration, ultracentrifugation or the like. An antiaggregation agent may be added to the prepared liposome. In this case, the aggregation inhibitor is used in combination with a long-chain aliphatic alcohol, sterol, polyoxyproline alkyl, or a hydrophobic compound such as glycerin fatty acid ester or phospholipid. The lipid derivative of the aggregation inhibitor is not particularly limited as long as it does not impair the structural stability of the liposome, and examples thereof include polyethylene glycol and dextran. Of these, polyethylene glycol (PEG) and polyethylene glycol lipid derivatives are preferred.
[0059]
The liposome of the present invention can be systemically or locally administered to a patient parenterally. Examples of the host to be administered include mammals, preferably humans, monkeys, mice, livestock and the like. As a parenteral administration route, for example, intravenous injection (intravenous injection) such as infusion, intramuscular injection, intraperitoneal injection, and subcutaneous injection can be selected, and an appropriate administration method is selected depending on the age and symptoms of the patient. be able to. Liposomes are administered to patients already suffering from the disease in an amount sufficient to cure or at least partially block the symptoms of the disease. For example, the effective dose of the physiologically active substance encapsulated in the liposome is selected in the range of 0.01 mg to 2000 mg per kg body weight per day. The effective dose of the physiologically active substance is more preferably 0.01 mg to 100 mg. However, the liposome of the present invention is not limited to these doses. The administration time may be administered after the disease has occurred, or may be administered prophylactically to relieve symptoms at the time of onset when the onset of the disease is predicted. The administration period can be appropriately selected depending on the age and symptoms of the patient.
[0060]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples using hemoglobin as a physiologically active substance, but the present invention is not limited to these examples.
Comparative Example 1
Hydrogenated soybean lecithin (HSPC) (manufactured by NOF Corporation), cholesterol (Chol) (manufactured by NOF Corporation) and stearic acid (SA) (manufactured by NOF Corporation), HSPC / Chol / SA = 1/1/1 ( mol) was dissolved in 200 mL of t-BuOH (manufactured by Wako Pure Chemical Industries, Ltd.) and then lyophilized to remove t-BuOH. To this lipid mixture, 60 g of purified water was added and heated at 80 ° C. for 30 minutes to be fully hydrated. A high-speed stirrer (stirring capacity 500 mL, cutter-type stirring blade diameter 33 mm, Waring) was obtained by adding 400 mL of a hemoglobin (45 wt%) solution (disclosed in JP-A No. 2002-161047) to this lipid hydrate and inverting and mixing. The product was supplied to a sealable container equipped with a Waring blender manufactured by Kogyo Co., Ltd., and the container was sealed. From this state, while the cooling jacket provided outside the container is cooled with a −10 ° C. refrigerant, the container is pressurized with air from a pressure tube provided in the container in a temperature range of 5 ° C. to 10 ° C. While applying a pressure of 0.3 atm, the rotation speed of the stirring blade was changed (10000 rpm (circumferential speed 17.2 m / sec), 12000 rpm (circumferential speed 20.7 m / sec), 15000 rpm (circumferential speed 25.9 m). / Sec) for 3 min, 30 times) and emulsified by stirring using a conventional high-speed stirring method. After emulsification, 30 g of a hemoglobin-lipid mixture treated under each emulsification condition was collected, 30 mL of physiological saline was added, and centrifugal washing was performed 3 times at 40,000 G, 60 min to remove hemoglobin that was not encapsulated. Thereafter, it is dispersed in 20 mL of physiological saline, and polyethyleneglycol (PEG) -linked phospholipid (manufactured by NOF Corporation) is added to an amount of 0.1 wt% as an aggregation inhibitor to obtain hemoglobin-containing liposomes surface-modified with PEG. It was. The same operation was repeated 3 times.
[0061]
The particle size of each of the hemoglobin-containing liposomes surface-modified with PEG thus prepared was measured with a light scattering diffractometer (LS230, manufactured by COULTER), and the standard for the particle size of the liposomes obtained by three operations. Deviation (SD) was determined.
[0062]
Comparative Example 2
Hydrogenated soybean lecithin (HSPC), mixing ratio consisting of cholesterol (Chol) and stearic acid (SA), 180 g of a mixture of HSPC / Chol / SA = 1/1/1 (mol) was dissolved in 600 mL of t-BuOH and then frozen. It was prepared by drying and removing t-BuOH. To this lipid mixture, 180 g of purified water was added and heated at 80 ° C. for 30 minutes to be fully hydrated. A hermetic container equipped with a high-speed stirrer (stirring capacity 1500 mL, cutter-type stirring blade diameter 90 mm, manufactured by Tokushu Kika Kogyo Co., Ltd., Ajihomomixer) obtained by adding 1200 mL of hemoglobin (45 wt%) solution to the lipid hydrate and mixing by inverting And the vessel was sealed. From this state, while the cooling jacket provided outside the container is cooled with a −10 ° C. refrigerant, the container is pressurized with air from a pressure tube provided in the container in a temperature range of 5 ° C. to 10 ° C. While the pressure of 0.3 atm was applied, the rotation speed of the stirring blade was changed (3600 rpm (circumferential speed 17.0 m / sec), 4400 rpm (circumferential speed 20.9 m / sec), 5400 rpm (circumferential speed 25.4 m) / Sec) for 3 min, 30 times) and emulsified with stirring. After emulsification, 30 g of the hemoglobin-lipid mixture treated under each emulsification condition was collected, 30 mL of physiological saline was added, and centrifugal washing was performed 3 times at 40,000 G, 60 min to remove hemoglobin that was not encapsulated. Thereafter, the resultant was dispersed in 20 mL of physiological saline, to which polyethylene glycol (PEG) -bound phospholipid was added to a concentration of 0.1 wt% as an anti-aggregation agent, and hemoglobin-containing liposomes surface-modified with PEG were obtained. The same operation was repeated 3 times.
The particle size of each of the hemoglobin-containing liposomes surface-modified with PEG thus prepared was measured with a light scattering diffractometer (LS230, manufactured by COULTER), and the standard for the particle size of the liposomes obtained by three operations. Deviation (SD) was determined.
[0063]
The particle diameters of the hemoglobin-containing liposomes obtained in Comparative Examples 1 and 2 are shown in FIG. 3 with the peripheral speed of the cutter-type stirring blade as the treatment condition as a parameter.
From the results of Comparative Example 1 and Comparative Example 2 (FIG. 3), when a high-speed stirrer having a similar cutter-type stirring blade is used, the hemoglobin-containing liposome particles are used when the stirring blade peripheral speed is used as an emulsification parameter. The effect on the diameter is different, and it is clear that it is difficult to predict the effect of emulsification conditions and the design of the stirrer on the emulsification when the scale is further scaled up. .
[0064]
Example 1
Hydrogenated soybean lecithin (HSPC), mixing ratio consisting of cholesterol (Chol) and stearic acid (SA), 180 g of a mixture of HSPC / Chol / SA = 1/1/1 (mol) was dissolved in 600 mL of t-BuOH and then frozen. It was prepared by drying and removing t-BuOH. To this lipid mixture, 180 g of purified water was added and heated at 80 ° C. for 30 minutes to be fully hydrated. This lipid hydrate was added with 1200 mL of hemoglobin (45 wt%) solution and mixed by inversion. The apparatus 1 shown in FIG. 1 (stirring capacity 1500 mL, spiral structure (spiral screw structure) 31 maximum diameter 80 mm, 1 rotation) Supplied from the raw material supply port 6 into the sealed container 2 having a discharge amount of 148 mL, a slit width of 1.5 mm, a slit length of 22 mm, a number of slits of 32, made by M Technique, CLEARMIX CLM-5.5 / 11W) The container 2 was sealed. The pressurized tube 7 is pressurized with air, a pressure of 0.3 atm is applied to the sealed container 2, the heat exchanger 8 provided in the sealed container 2 is cooled with a −10 ° C. refrigerant, and the sealed container 2 While maintaining the liquid temperature in the temperature range of 5 ° C. or more and 60 ° C. or less, the spiral structure 31 and the slit structure 41 are rotated under the following conditions to discharge the raw material from the slit 42 of the slit structure 41. And emulsified. The discharge was carried out 5 times for 3 minutes under each discharge condition.
[Discharge condition 1]
Rotation speed of the spiral structure 31: 4000 rpm
(Discharge rate 9.9L / sec, discharge flow rate 9.4m / sec)
Rotation direction of slit-like structure 41: reverse direction to spiral-like structure 31
Rotation speed of slit-like structure 41: 4000 rpm
[Discharge condition 2]
Rotation speed of spiral structure 31: 5000 rpm
(Discharge rate 12.4L / sec, discharge flow rate 11.7m / sec)
Rotation direction of slit-like structure 41: reverse direction to spiral-like structure 31
Rotation speed of slit-like structure 41: 4000 rpm
[Discharge condition 3]
Rotation speed of spiral structure 31: 6000 rpm
(Discharge rate 14.8L / sec, discharge flow rate 14.1m / sec)
Rotation direction of slit-like structure 41: reverse direction to spiral-like structure 31
Number of revolutions of slit-like structure 41: 6000 rpm
[0065]
After emulsification, 30 g of the hemoglobin-lipid mixture treated under the respective emulsification conditions was collected, 30 mL of physiological saline was added, and centrifugal washing was performed 3 times at 40,000 G, 60 min to remove unencapsulated hemoglobin. Thereafter, the resultant was dispersed in 20 mL of physiological saline, to which polyethylene glycol (PEG) -bound phospholipid was added to a concentration of 0.1 wt% as an anti-aggregation agent, and hemoglobin-containing liposomes surface-modified with PEG were obtained. The same operation was repeated 3 times.
[0066]
The particle size of each of the hemoglobin-containing liposomes surface-modified with PEG thus prepared was measured with a light scattering diffractometer (LS230, manufactured by COULTER), and the standard for the particle size of the liposomes obtained by three operations. Deviation (SD) was determined.
[0067]
Comparative Example 3
Hydrogenated soybean lecithin (HSPC), mixing ratio of cholesterol (Chol) and stearic acid (SA), 60 g of a mixture of HSPC / Chol / SA = 1/1/1 (mol) was dissolved in 200 mL of t-BuOH and then frozen. It was prepared by drying and removing t-BuOH. To this lipid mixture, 60 g of purified water was added and heated at 80 ° C. for 30 minutes to be fully hydrated. The lipid hydrate was added with 400 mL of a hemoglobin (45 wt%) solution and mixed by inversion, and the apparatus 1 shown in FIG. 1 (stirring capacity 500 mL, spiral structure (spiral screw structure) 31 maximum diameter 33 mm, one rotation) Supplied from the raw material supply port 6 into the sealed container 2 having a discharge amount of 12.8 mL, a slit width of 1.15 mm, a slit length of 11 mm, a number of slits of 32, manufactured by M Technique Co., Ltd., CLEARMIX CLM-1.5 / 22W) The container 2 was sealed. The pressurized tube 7 is pressurized with air, a pressure of 0.3 atm is applied to the sealed container 2, the heat exchanger 8 provided in the sealed container 2 is cooled with a −10 ° C. refrigerant, and the sealed container 2 While maintaining the liquid temperature in the temperature range of 5 ° C. or more and 60 ° C. or less, the spiral structure 31 and the slit structure 41 are rotated under the following conditions to discharge the raw material from the slit 42 of the slit structure 41. And emulsified. The discharge was carried out 5 times for 3 minutes under each discharge condition.
[Discharge condition 1]
Rotation speed of the spiral structure 31: 10,000 rpm
(Discharge rate 2.1L / sec, discharge flow rate 5.2m / sec)
Rotation direction of slit-like structure 41: reverse direction to spiral-like structure 31
Number of rotations of slit-like structure 41: 10,000 rpm
[Discharge condition 2]
Rotation speed of spiral structure 31: 12000 rpm
(Discharge rate 2.6L / sec, discharge flow rate 6.3m / sec)
Rotation direction of slit-like structure 41: reverse direction to spiral-like structure 31
Rotation speed of slit-like structure 41: 12000 rpm
[Discharge condition 3]
Number of revolutions of spiral structure 31: 15000 rpm
(Discharge rate 3.2L / sec, discharge flow rate 7.9m / sec)
Rotation direction of slit-like structure 41: reverse direction to spiral-like structure 31
Rotation speed of slit-like structure 41: 15000 rpm
[0068]
After emulsification, 30 g of a hemoglobin-lipid mixture treated under the respective emulsification conditions was collected, 30 mL of physiological saline was added, and centrifugal washing was performed 3 times at 40,000 G, 60 min to remove unencapsulated hemoglobin. Thereafter, the resultant was dispersed in 20 mL of physiological saline, to which polyethylene glycol (PEG) -bound phospholipid was added to a concentration of 0.1 wt% as an anti-aggregation agent, and hemoglobin-containing liposomes surface-modified with PEG were obtained. The same operation was repeated 3 times.
The particle size of each of the hemoglobin-containing liposomes surface-modified with PEG thus prepared was measured with a light scattering diffraction measurement device (LS230, manufactured by COULTER), and the standard for the particle size of the liposomes obtained by three operations. Deviation (SD) was determined.
[0069]
Comparative Example 4
Hydrogenated soybean lecithin (HSPC), a mixture ratio of cholesterol (Chol) and stearic acid (SA), 60 g of a mixture of HSPC / Chol / SA = 1/1/1 (mol) dissolved in t-BuOH, 200 mL, It was prepared by lyophilization and removal of t-BuOH. To this lipid mixture, 60 g of purified water was added and heated at 80 ° C. for 30 minutes to be fully hydrated. A solution obtained by adding 400 mL of a hemoglobin (45 wt%) solution to this lipid hydrate and tumbling and mixing was used to prepare a device 1 (stirring capacity 500 mL, spiral structure (spiral screw structure) 31 having a maximum diameter of 33 mm, 1 From the raw material supply port 6 into the hermetic container 2 having a rotary discharge amount of 12.8 mL, a slit width of 1.15 mm, a slit length of 11 mm, a number of slits of 32, manufactured by M Technique, CLEARMIX CLM-1.5 / 22W) The container 2 was sealed. The pressurized tube 7 is pressurized with air, a pressure of 0.3 atm is applied to the sealed container 2, the heat exchanger 8 provided in the sealed container 2 is cooled with a −10 ° C. refrigerant, and the sealed container 2 While maintaining the liquid temperature in the temperature range of 5 ° C. or more and 60 ° C. or less, the spiral structure 31 and the slit structure 41 are rotated under the following conditions to discharge the raw material from the slit 42 of the slit structure 41. And emulsified. In addition, discharge was implemented 3 minutes and 5 times on the following conditions. Number of rotations of the spiral structure 31: 20000 rpm
(Discharge rate 3.4L / sec, discharge flow rate 9.2m / sec)
Slit structure 41: fixed (not rotated)
[0070]
After emulsification, 30 g of a hemoglobin-lipid mixture was collected, 30 mL of physiological saline was added, and hemoglobin that was not encapsulated 3 times at 40,000 G, 60 min was centrifuged and washed. Thereafter, the resultant was dispersed in 20 mL of physiological saline, to which polyethylene glycol (PEG) -bound phospholipid was added to a concentration of 0.1 wt% as an anti-aggregation agent, and hemoglobin-containing liposomes surface-modified with PEG were obtained. The same operation was repeated 3 times.
The particle size of each of the hemoglobin-containing liposomes surface-modified with PEG thus prepared was measured with a light scattering diffraction measurement device (LS230, manufactured by COULTER), and the standard for the particle size of the liposomes obtained by three operations. Deviation (SD) was determined.
[0071]
The particle diameters of the hemoglobin-containing liposomes obtained in Example 1 and Comparative Examples 3 and 4 are shown in FIG. 4 with the discharge amount as each processing condition as a parameter. Furthermore, the particle diameters of the hemoglobin-containing liposomes obtained in Example 1 and Comparative Examples 3 and 4 are shown in FIG.
Comparing the results of Example 1 and Comparative Examples 3 and 4 (FIGS. 4 and 5), Comparative Example 3 implemented under a discharge condition lower than the discharge flow rate and discharge flow rate of the present invention was carried out without rotating the slit-like structure 41. In Comparative Example 4, the resulting liposome had a large variation in particle size, and the average particle size was 270 nm or more. In particular, in Comparative Example 4 carried out without rotating the slit-like structure, the average particle diameter of the obtained liposomes was higher than that in Example 1 although the discharge flow rate of the discharge flow was not so different from that in Example 1. It was remarkably large and about 370 nm. This is presumably because, in Example 1, both the spiral structure 31 and the slit-like structure 41 opposed to each other rotate to increase the velocity vector of the discharge flow, and the refinement of the liposome-type hemoglobin has progressed.
In Comparative Example 4, it was confirmed that the hemoglobin contained in the obtained liposome was partially denatured. This is considered to be caused by heat denaturation of hemoglobin due to heat generated by rotating the helical structure 31 at a rotational speed of 20000 rpm or more.
[0072]
4 and 5, the discharge rate of the high-concentration hemoglobin and liposome-forming lipid mixture discharged from the slit-like structure 41 is about 9 L / sec or more, and the discharge flow rate is 9.4 m / sec or more. It is clear that the particle size of hemoglobin-containing liposomes is small depending on the flow rate, and it is clear that the above conditions are necessary for scale-up production of liposomes having an average particle size of 150 to 220 nm. became. Therefore, in the liposome production method of the present invention, it was shown that the production of liposomes can be scaled up by paying attention to the discharge flow rate and the discharge flow rate as the liposome production parameters.
[0073]
【The invention's effect】
As described above in detail, the present invention provides a method for producing a liposome containing a physiologically active substance suitable for scale-up required for industrially mass-producing liposomes containing a physiologically active substance such as hemoglobin. To do. By adopting the method of the present invention, it becomes possible to design a production facility by predicting an optimal structure at the time of scale-up for mass production of liposomes containing a physiologically active substance on an industrial scale. According to the method of the present invention, heat generated by liposome formation can be efficiently absorbed without being localized. This makes it possible to produce liposomes incorporating a physiologically active substance that is unstable to heat, and can be applied in various fields in the future.
[Brief description of the drawings]
FIG. 1 is a conceptual diagram showing an example of an apparatus used in a method of the present invention.
FIGS. 2A to 2C are partial enlarged views of an example of the structure 31 of FIG. 1, FIG. 2A is a side view of the structure 31, and FIG. It is the figure seen, (c) is the figure which looked at the structure 31 from upper direction.
FIG. 3 is a graph showing the relationship between the stirring capacity and the peripheral speed of stirring blades and the particle size of hemoglobin-containing liposomes in Comparative Examples 1 and 2.
4 is a graph showing the relationship between the discharge amount and the particle size of hemoglobin-containing liposomes in Example 1 and Comparative Examples 3 and 4. FIG.
FIG. 5 is a graph showing the relationship between the discharge flow rate and the particle size of hemoglobin liposomes in Example 1 and Comparative Examples 3 and 4;
[Explanation of symbols]
1: Device
2: Airtight container (container)
3: Structure
31: Spiral structure or screw structure
32: End
4: Structure
41: slit-like structure
42: slit
43: End
5: Shaft seal structure
6: Raw material supply port
7: Pressurized tube
8: Heat exchanger
9: Sampling tube

Claims (10)

From the (B) slit-like structure of the discharge part having the following (A) structure and (B) slit-like structure provided in the sealed container, 9.0 L / sec or more of the physiologically active substance and the liposome-forming lipid are added. A liposome containing a physiologically active substance, wherein the liposome is discharged at a discharge flow rate of 9.4 m / sec or more.
(A) a structure that rotates about at least one axis selected from the group consisting of a helical structure and a screw-like structure;
(B) A slit-like structure that rotates about an axis.
Here, the (B) slit-like structure is disposed to face the (A) structure, and the (B) slit-like structure is forward or reverse with respect to the rotation direction of the (A) structure. Rotate in the direction. The rotational speed of the structure (A) is 500 rpm or more and less than 20,000 rpm,
The method for producing a liposome containing a physiologically active substance according to claim 1, wherein the rotational speed of the (B) slit-like structure is 500 rpm or more and less than 20,000 rpm. The method for producing a liposome containing a physiologically active substance according to claim 1 or 2, wherein a width of the slit in the slit-like structure is 0.5 mm to 5 mm. 4. The physiologically active substance according to claim 1, wherein the (A) structure and the (B) slit-like structure have a mechanical seal as a shaft sealing mechanism of a rotating shaft. 5. A method for producing liposomes. The sealed container further incorporates a heat exchanger, and the inside of the sealed container when the physiologically active substance and the liposome-forming lipid are discharged by cooling the sealed container using the heat exchanger. The method for producing a liposome containing a physiologically active substance according to any one of claims 1 to 4, wherein the temperature of 5 to 60 ° C is maintained. 6. The inside of the sealed container is pressurized at 0.1 atm or more and 2 atm or less when the physiologically active substance and liposome-forming lipid are ejected from the slit-like structure of the ejection part. A method for producing a liposome containing the physiologically active substance according to any one of the above. The method for producing a liposome containing a physiologically active substance according to any one of claims 1 to 6, wherein the physiologically active substance is hemoglobin. The method for producing a liposome containing a physiologically active substance according to any one of claims 1 to 7, wherein the physiologically active substance is supplied to the sealed container as a solution having a concentration of 30 wt% to 60 wt%. Liposomes containing a physiologically active substance obtained by the method according to any one of claims 1 to 8. The average particle diameter is 150 nm-220 nm, The liposome containing the physiologically active substance according to claim 9.

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