JP4791067B2 - Method for producing liposome preparation - Google Patents

Description

  The present invention relates to a method for producing a liposome preparation useful for a drug delivery system.

In recent years, drug delivery systems (DDS) have been actively studied. Its purpose is sustained release of drugs (sustained release), extended life of drugs with a short half-life in the body, promotion of drug absorption at various lesion sites, or delivery of drugs only to target tissues and cells To do. As the method, use of closed vesicles such as liposomes, emulsions, lipid microspheres, and nanoparticles can be considered. However, there are various problems to be overcome in the practical application, and in particular, avoidance from the foreign body recognition mechanism on the living body side and control of pharmacokinetics are important. In other words, in order to deliver closed vesicles with high selectivity to the target site, the uptake of reticuloendothelial tissue (RES) such as the liver and spleen is avoided and interaction with opsonin and plasma proteins in the blood is avoided. It is necessary to increase the stability in blood by preventing aggregation due to action (adsorption).
As a method for solving this problem, membrane modification with a hydrophilic polymer is known. Closed vesicles modified with hydrophilic polymers, especially liposomes, have high retention in the blood, so that the liposomes passively accumulate in tissues with increased vascular permeability, such as tumor tissue and inflammatory sites. Is being put to practical use (see Patent Documents 1 to 3 and Non-Patent Documents 1 to 3). Among them, Doxil (registered trademark) already marketed in Europe and America is well known for liposomes modified with PEG using polyethylene glycol (hereinafter sometimes referred to as “PEG”) as a hydrophilic polymer. .
Japanese National Patent Publication No. 5-505173 Japanese Patent Publication No. 7-20857 Japanese Patent No. 2667051 Cancer Lett., 1997, 118 (2), p.153 Br.J.Cancer., 1997, 76 (1), p.83 `` LIPOSOMES from Physics to Applications '' by DD Lasic, Elsevier, 1993

In general, the production method is very important for the practical application of liposome preparations. A general method for producing liposomes includes a step of homogenizing liposome membrane constituents (hereinafter also referred to as a homogenization step), a liposome formation step of controlling the particle diameter and the like by mixing membrane lipids and an aqueous phase to form liposomes ( Hereinafter, it is also referred to as a liposome formation step), then a step of removing unencapsulated drug required in the case of drug-encapsulated liposomes, or an external liquid replacement step in the case of liposomes not containing a drug (in this specification, these are generically referred to In general, the flow may be simply referred to as an unencapsulated drug removal step), and finally the final purification or aseptic filtration step (hereinafter referred to as the sterilization step).
In these production processes, it is essential for liposomes to ensure physicochemical stability such as particle diameter.

However, the methods disclosed so far may be possible for small-scale production at the laboratory level, but are not practical for practical use, such as adsorption to production equipment and aggregation between liposomes at intermediate stages. -It was difficult to apply to large-scale production, and depending on the combination of processes, it was not possible to produce a preparation having the desired physicochemical properties such as aggregation being promoted.
For example, in the liposome formation process, a liposome formation method in the presence of alcohol for the purpose of improving lipid solubility, lowering the viscosity of the liposome dispersion, and suppressing the growth of microorganisms is disclosed (Non-patent Document 4). When the temperature is lowered after granulation, lipid precipitation and aggregation between liposomes occur immediately, followed by adsorption to the membrane and aggregation between liposomes in the unencapsulated drug removal process using a hollow fiber membrane or reverse osmosis membrane. Furthermore, further production became impossible due to adsorption to a sterile filtration membrane and occurrence of aggregation in the final sterilization step, and a liposome preparation having the desired physicochemical properties could not be produced.
G. Gregoriadis "Liposome Technology Liposome Preparation and Related Techniques" 2ndedition, Vol. I-III, CRC Press

In general, the production of hydrophilic polymer-modified liposomes homogenizes lipid membrane constituents including hydrophilic polymers in the step of homogenizing the liposome membrane constituents. Subsequent processes are manufactured in the same process as the above process (Patent Document 4). However, in this method, the hydrophilic polymer is oriented not only in the liposome external surface that is originally required but also in the interior, not only reducing the volume of the internal aqueous phase and lowering the drug encapsulation rate, but also during production. It was not a desirable method in terms of both formulation design and production because it would use extra hydrophilic polymer and affect production costs. In addition, there is a report that hydrophilic polymers increase the permeability of the liposome membrane, and the leakage of the encapsulated drug increases as the content of the hydrophilic polymer increases (Non-Patent Document 5, Non-Patent Document). 6).
For these reasons, it is desirable that the amount of the hydrophilic polymer to be modified in the liposome membrane is the minimum necessary amount, and the modification method is a method of modifying the liposome surface with a necessary amount of the hydrophilic polymer after liposome formation. Is desirable.
From these viewpoints, a method of adding a hydrophilic polymer after liposome formation is disclosed (Patent Document 5). In this method, the thin film method is used for the homogenization step, and liposome formation is performed in the absence of an organic solvent. Therefore, the stability of the liposome is higher than that in the presence of the organic solvent.
In general, it is known that the stability of liposomes is remarkably reduced in the presence of an organic solvent. Therefore, the conditions for ensuring the physicochemical stability of liposomes in the presence of an organic solvent are more severe.
US Pat. No. 5,013,556 Biochem. Biophys. Acta. 1304 (1996) 120-128 Biochem. Biophys. Acta. 1463 (2000) 167-178 Japanese Patent Publication No. 7-20857

When considering application to medium- and large-scale production for practical application, it is common to use an organic solvent in the homogenization step and perform the liposome formation step in the presence of the organic solvent. However, it is generally known that liposomes are destabilized in the presence of an organic solvent. In fact, after performing the liposome formation step in the presence of an organic solvent, it was difficult to ensure physicochemical stability such as particle size. A production method for ensuring physicochemical stability after the liposome formation step in the presence of an organic solvent is an important issue in medium- and large-scale production.

  As described above, none of the conventional methods for producing hydrophilic polymer-modified liposomes has a problem on an industrial scale and cannot be said to be satisfactory. The object of the present invention is a simple production method that can be satisfactorily satisfied from the viewpoint of production cost and liposome stability in a production method at an industrial level of a liposome preparation in which a hydrophilic polymer is introduced to the outer surface. In addition, in the liposome formation process, the unencapsulated drug removal process and the sterilization process, while adsorbing to production equipment etc. The object is to provide a production process that can be processed to obtain a liposomal formulation having the desired physicochemical properties.

  In view of the above points, as a result of studying to solve the above problems, a hydrophilic polymer modification step was set immediately after the liposome formation step, and in that step, the outer surface of the liposome was modified with a hydrophilic polymer, In the purification process (unencapsulated drug removal process and sterilization process) or the external solution replacement process of liposomes while ensuring the physicochemical stability such as the particle size, manufacturing cost, liposome stability From the above point of view, it was found that the method could be fully satisfied, and the following method for producing a liposome preparation according to the present invention was completed. In the present invention, when an organic solvent is used in the liposome formation step, the hydrophilic polymer introduction efficiency in the hydrophilic polymer addition step is dramatically increased, and a new finding is obtained that can be made more suitable for industrial level production. I was able to. That is, in this step using an organic solvent in the liposome forming step, the introduction of the hydrophilic polymer is simple, and the hydrophilic polymer can be introduced in a shorter time than when no organic solvent is present.

  The present invention is based on an external force that impairs the physical stability of the liposome in the liposome formation step, the liposome non-encapsulation step, the liposome external liquid replacement step, the sterilization step, etc., regardless of whether the drug is encapsulated or not. By setting the hydrophilic polymer modification step immediately before the liposome formation step, in other words, immediately after the liposome formation step, the physical properties such as the particle size in the subsequent purification step (unencapsulated drug removal step and sterilization step) It is a method for producing a liposome preparation, characterized in that the liposome is stably treated while ensuring chemical stability.

Specifically, the present invention is represented by the following (1) to (11).
(1) A liposome forming step for obtaining a liposome dispersion, a hydrophilic polymer modifying step for modifying a hydrophilic polymer on the outer surface of the liposome, and a load comprising an external force that impairs the physical stability of the liposome A method for producing a liposome preparation having a liposome dispersion treatment step, wherein the hydrophilic polymer modification step is carried out while maintaining a temperature equal to or higher than the treatment temperature in the liposome formation step after completion of the liposome formation step. A method for producing a liposome preparation.
(2) The method for producing a liposome preparation according to the above (1), wherein the dispersion treatment step is one or more of an unencapsulated drug removal step, a sterilization step, and a liposome external solution replacement step.
(3) The method for producing a liposome preparation according to the above (1) or (2), wherein the liposome dispersion liquid obtained in the liposome forming step contains an organic solvent.
(4) The said organic solvent is a manufacturing method of the liposome formulation as described in said (3) derived from the solvent used in the homogenization process which mixes and homogenizes the liposomal lipid prior to a liposome formation process in a solvent.
(5) The method for producing a liposome preparation according to the above (3) or (4), wherein the organic solvent is alcohol.
(6) The method for producing a liposome preparation according to any one of the host machines (1) to (5), wherein the temperature in the hydrophilic polymer addition step is a temperature equal to or higher than a lipid membrane phase transition temperature.
(7) The said hydrophilic polymer addition process is a manufacturing method of the liposome formulation in any one of said (1)-(6) implemented within 180 minutes immediately after the said liposome formation process.
(8) The hydrophilic polymer introduction rate in the hydrophilic polymer addition step is 0.01 to 20 mol with respect to the total lipid constituting the liposome dispersed in the liposome dispersion obtained in the liposome formation step. % The manufacturing method of the liposome formulation in any one of said (1)-(7).
(9) The method for producing a liposome preparation according to any one of (1) to (8), wherein the hydrophilic polymer is polyethylene glycol.
(10) The method for producing a liposome preparation according to (9), wherein the hydrophilic polymer is polyethylene glycol having a molecular weight of 500 to 15,000 daltons.
(11) The method for producing a liposome preparation according to any one of (1) to (10), wherein the liposome has an average particle size of 50 to 250 nm.

  The production method of the present invention, which is considered to be applied to medium and large-scale production for practical use, has its physical chemistry without denaturation due to aggregation / adsorption in the steps after the liposome formation step including particle size control. The liposome can be stably treated while ensuring the mechanical stability, which is sufficiently satisfactory from the viewpoint of production cost and liposome stability.

Hereinafter, the present invention will be described in more detail. A liposome is a closed vesicle composed of a phospholipid bilayer membrane, and includes an aqueous phase (inner aqueous phase) in the vesicle space. Liposome preparations are prepared by using the liposome as a carrier and carrying a drug thereon. Liposomes have a membrane structure such as single lamella vesicles (Small Unilamellar Vesicle, SUV) and multilamellar vesicles (MLV) consisting of multiple lipid bilayers. Are known. In order to suppress the leakage of the drug encapsulated in the liposome, there is also a proposal that the membrane structure of the liposome is an MLV membrane (see Patent Document 6).
International Publication No. 01/000173 Pamphlet

  A general production process of a liposome preparation is in the order of a homogenization process, a liposome formation process, an unencapsulated drug removal process, and a sterilization process. In the present invention, the order of these steps is not particularly limited. Moreover, in the method using an ion gradient, etc., there are those in which an unencapsulated drug removal step including an outer solution replacement step is performed twice. Moreover, although a freeze-drying process may enter in a manufacturing method, this invention is not influenced by the duplication and addition of these processes.

  A homogenization process shows the process of melt | dissolving a liposome membrane structural component using an organic solvent etc., and homogenizing. In general, a plurality of lipid components such as phospholipid and cholesterol are often present as liposome components. When a plurality of lipid components are present, it is essential to take a homogenization step in order to avoid lipid heterogeneity during liposome membrane formation. When a single lipid is used, a homogenization step is not necessarily essential, but when assuming production aimed at practical use, it is desirable to take the following homogenization step. As a method for homogenization, a thin film method is well known in which chloroform and the like are completely dissolved and vacuum-dried for homogenization. As a production method aiming at practical use, a method of homogenizing by completely dissolving in an organic solvent such as ethanol and using the lipid-organic solvent solution in the subsequent liposome forming step is widely used.

A liposome formation process shows the process of forming a liposome using the homogenized lipid. As a general production method of the liposome forming step, a method using a hydration method (Bangham method), a sonication method, a reverse phase evaporation method or the like has already been reported. Moreover, as a manufacturing method aimed at practical use, there are a heating method (Patent Document 7) and a lipid dissolution method (Patent Document 8). In addition, DRV method (Dehydrated / Rehydrated Vesicles) and freeze-thaw method have been reported as methods for increasing the amount of drug retained in the inner aqueous phase. The liposome forming step includes a step of controlling the particle size after forming the liposome. The particle size control process can be controlled using various known techniques including membrane emulsification and shear force persistence. More specifically, there are a membrane emulsification method in which a filter is forced to pass a plurality of times, a high pressure emulsification method in which high pressure discharge is performed by a high pressure discharge type emulsifier. All of the production methods are known in G. Gregoriadis edited “Liposome Technology Liposome Preparation and Related Techniques” 2ndedition, Vol. I-III, CRC Press, and this description is incorporated herein by reference. . In recent years, as a new production method, a method of performing shear emulsification by jet flow under a liquid phase (jet flow emulsification) (Patent Document 9) using a speed conversion from compression by ultra high pressure, or supercritical carbon dioxide is used. There is a liposome preparation technique (Non-patent Document 7) that is used. In recent years, an improved ethanol injection method that simplifies the particle size control process has appeared (Non-Patent Document 8). These also correspond to the liposome forming step of the present invention.
JP 60-7933 JP 60-12127 A JP2005-2055 Pharm Tech Japan Vol.19 No.5 (2003) 91 (819) J. Liposome Research 11 (1), 115-125 (2001)

The liposome obtained by the production method of the present invention is preferably provided with a particle size control step in the liposome formation step. When the liposome takes a spherical shape or a form close thereto, the diameter is not particularly limited, but the diameter is 20 nm to 2000 nm, preferably 30 nm to 400 nm, and more preferably 50 nm to 250 nm.

The liposome dispersion liquid treatment process means various treatment processes performed after the liposome formation process for a known liposome dispersion, such as an unencapsulated drug removal process, a liposome external liquid replacement process, and a sterilization process.
The unencapsulated drug removal step indicates a step aimed at removing the unencapsulated drug when the liposome formation step is performed with a drug solution. In addition, the liposome external liquid replacement step refers to a step aimed at external liquid replacement when the liposome formation step is performed with a solution containing no drug. The purpose of the external solution replacement is to remove the organic solvent brought into the liposome formation step through the homogenization step, and to form an ion gradient inside and outside the liposome. The unencapsulated drug removal step and the external liquid replacement step are also useful as a step of removing the unbound hydrophilic polymer. In this step, among the hydrophilic polymers added in the hydrophilic polymer modification step, those not bound to the liposome can be removed. As such methods, dialysis, ultracentrifugation, gel filtration, and the like are known. All the manufacturing methods are known in G. Gregoriadis edited “Liposome Technology Liposome Preparation and Related Techniques” 2ndedition, Vol. I-III, CRC Press, and are described in this specification with reference to this description. . In particular, examples of a production method for practical use include a method using a hollow fiber such as a dialyzer, a tangential flow using a ultrafiltration membrane, diafiltration, and the like.

A sterilization process shows the process of sterilizing after a liposome formation process. The sterilization method is not particularly limited. For example, filtration sterilization, high-pressure steam sterilization method, dry heat sterilization method, ethylene oxide gas sterilization method, radiation (eg, electron beam, X-ray, γ-ray, etc.) sterilization method, ozone water A sterilization method or a sterilization method using hydrogen peroxide water can be used. Further, depending on the manufacturing method, this sterilization step may not be set. As the sterilization step in the present invention, filtration sterilization is most preferable.
In the filtration sterilization method, liposomes permeate, but Brevundimonas diminuta (size, approximately 0.3 × 0.8 μm) used as an indicator bacterium is required not to be filtered, so the particles are sufficiently smaller than Brevundimonas diminuta. It is necessary to be. The liposome having a particle size of around 100 nm is also important for ensuring the filtration sterilization step. The filter used for filtration sterilization preferably has a pore size of 0.45 μm or less. For example, a filter sterilization filter having a pore size of 0.2 μm is used.

The production method in the present invention solves the problems in the production method of a liposome preparation having a homogenization step, a liposome formation step, an unencapsulated drug removal step, and a sterilization step for practical use. However, it is not limited to the manufacturing method aiming at practical use as described above.
The feature of the present invention is that a hydrophilic polymer modification step is set immediately after the liposome formation step in order to ensure physicochemical stability such as particle diameter in the production step, and the hydrophilic polymer is attached to the outer surface of the liposome in that step. It is to be modified. Modification of the hydrophilic polymer on the outer surface of the liposome is carried out by bringing the liposome and the hydrophilic polymer into contact with each other and immobilizing the hydrophilic polymer on the outer surface of the liposome. A portion capable of immobilizing the hydrophilic polymer may be provided on the surface, or a portion capable of immobilizing the liposome outer surface may be provided on the hydrophilic polymer. In the present invention, not only the hydrophilic polymer itself but also those having a portion that can be immobilized on the outer surface of the liposome as described above are collectively referred to as a hydrophilic polymer.
The method for modifying the hydrophilic polymer in the hydrophilic polymer modifying step is not particularly limited, but it is preferably used as a solution instead of powder in order to ensure uniformity and operability. The solvent for dissolving the hydrophilic polymer is not particularly limited, but considering the necessity of mixing with water, water, alcohols, DMF, THF, DMSO and the like are desirable, and water is most preferable.

The solution concentration of the hydrophilic polymer in the hydrophilic polymer addition step depends on the solubility of the hydrophilic polymer, but considering that the amount of solution brought into the liposome formation step is small, it is desirable that the amount of solution brought in is small . When the hydrophilic polymer is a liquid, it is most preferable to add it as it is. Although it depends on the hydrophilic polymer introduction rate (mol%) to the formed liposome, it is considered that the solution concentration is generally 0.01 mg / mL to 10 g / mL, but is not particularly limited.
“Hydrophilic polymer introduction rate (mol%)” indicates the ratio of the hydrophilic polymer to the liposome membrane constituents containing the hydrophilic polymer.

  The amount of hydrophilic polymer added to ensure physicochemical stability such as particle size depends on the amount of solvent other than water brought from the homogenization step to the liposome formation step. Specifically, the solvent other than water is an organic solvent. Further, the solvent other than water may be any one that is uniformly mixed with water, and examples thereof include alcohols such as ethanol, and aqueous nonaqueous solvents such as DMF, THF, and DMSO. The greater the amount of solvent other than water that is brought in, the greater the amount of hydrophilic polymer added. Moreover, since the minimum addition amount changes with kinds of solvents other than the water brought in, it needs to be careful. When the solvent to be brought in is only water, the amount of hydrophilic polymer added may be the smallest. However, even in the case of water alone, it is desirable to add a hydrophilic polymer to ensure physicochemical stability. The hydrophilic polymer introduction rate (mol%) modified by the addition is formed in the liposome formation process. 0.1 to 20 mol% is desirable with respect to the total lipid constituting the prepared liposome, but is not limited thereto.

  It is essential that the temperature condition for adding the hydrophilic polymer in the hydrophilic polymer adding step is higher than the phase transition temperature of the main membrane material. The phase transition temperature of the main membrane material of the liposome depends on the lipid structure, and it is common to use a phospholipid that is higher than the in vivo temperature (35 to 37 ° C.). Specifically, it is one of the preferred embodiments that the phase transition temperature of the main film material is 50 ° C. or higher. In this case, it should be noted that it is essential that the hydrophilic polymer is added at 50 ° C. or higher.

The liposome after the liposome forming step is destabilized such as aggregation depending on temperature and time. Since this destabilization differs depending on the lipid composition of the liposome, it is known that the temperature and time differ for each lipid composition. In order to avoid this destabilization that varies depending on the lipid composition, the timing of adding the hydrophilic polymer in the hydrophilic polymer modification step is preferably as close as possible immediately after the liposome formation step. Specifically, if it is within 180 minutes, it is preferable because the influence of heat on the membrane component and the inclusion is small, more preferably within 120 minutes, still more preferably within 45 minutes, immediately after the liposome formation step. Most desirable. More specifically, the liposome dispersion liquid after the liposome formation step may be directly added to the hydrophilic polymer solution, and vice versa, the method of adding the hydrophilic polymer solution to the liposome dispersion liquid after the liposome formation step. But it ’s okay. Alternatively, the liposome dispersion and the hydrophilic polymer solution may be simultaneously transferred to another container and mixed. At that time, it is desirable to take a step of stirring with a stirrer or the like from the viewpoint of concentration uniformity and temperature uniformity.
After the addition of the hydrophilic polymer in the hydrophilic polymer modification step, it is desirable to carry out heating and stirring for a predetermined time above the phase transition temperature. The time for heating and stirring is 0 to 120 minutes, preferably 0 to 60 minutes, more preferably 0 to 45 minutes.

In the present invention, it has been found that the presence of alcohol in the hydrophilic polymer modification step can significantly reduce the introduction time of the hydrophilic polymer and lower the introduction temperature condition. From the viewpoint of lipid stability, the liposome after the hydrophilic polymer modification step is desired to have a temperature condition as low as possible and a heating and stirring time as short as possible. The existence is suitable from these viewpoints.
In the present invention, as a method for bringing alcohol into the hydrophilic polymer addition step, it is most desirable to use alcohol in the homogenization step. This is because it is most convenient to completely solubilize with alcohol for the homogenization of the desired lipid in the homogenization step.

  It is desirable that the liposome after the hydrophilic polymer addition step is quickly cooled from the viewpoint of lipid stability. As a simpler cooling method, ice cooling is preferable. The hydrophilic polymer that has not been bound in the hydrophilic polymer addition step can be removed in the next unencapsulated drug removal step. Therefore, it is desirable that there is an unencapsulated drug removal step after the hydrophilic polymer addition step.

  The homogenization step, liposome formation step, and hydrophilic polymer addition step are steps that utilize the deformation of the liposome and the liquidation of the lipid membrane, so that the temperature is higher than the phase transition temperature of the main membrane material. However, the liposome after the hydrophilic polymer addition process, on the other hand, does not need to change the particle size due to the deformation of the liposome, so the unencapsulated drug removal process and the sterilization process are the phase transition of the main membrane material. It is desirable to carry out below the temperature. For example, when the phase transition temperature of the main film material is around 50 ° C., it is preferably about 0 to 40 ° C., and more specifically, it is preferably manufactured at about 5 to 30 ° C.

  A phospholipid is generally an amphiphile having a hydrophobic group composed of a long-chain alkyl group and a hydrophilic group composed of a phosphate group in the molecule. Examples of phospholipids include glycerophospholipids such as phosphatidylcholine (= lecithin), phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol; sphingomyelin ( Sphingophosphorines such as Sphingomyelin, SM; natural or synthetic diphosphatidyl phospholipids such as cardiolipin and their derivatives; those hydrogenated according to conventional methods (eg, hydrogenated soybean phosphatidylcholine) (HSPC)). Hereinafter, these phospholipids may be referred to as “phospholipids”. Among these, hydrogenated phospholipids such as hydrogenated soybean phosphatidylcholine, sphingomyelin, and the like are preferable.

In addition, it is preferable to use a phospholipid having a phase transition temperature higher than the in-vivo temperature (35 to 37 ° C.) as a main membrane material. This is because by using such phospholipid, it becomes possible to prevent the drug enclosed in the liposome from leaking easily from the liposome to the outside during storage or in a living body such as blood. is there. These liposomes are preferably produced at or above the phase transition temperature of the main membrane material. This is because it is difficult to control the particle size at a temperature lower than the phase transition temperature of the main film material. For example, when the phase transition temperature of the main film material is around 50 ° C., it is preferably about 50 to 80 ° C., more specifically, it is preferably produced at about 60 to 70 ° C. Liposomes can contain a single type of phospholipid or multiple types of phospholipids as the main membrane material.
The amount of phospholipid which is a main constituent component is usually 20 to 100 mol%, preferably 40 to 100 mol% in the whole membrane lipid. The amount of lipids other than phospholipids is usually 0 to 80 mol%, preferably 0 to 60 mol% in the whole membrane lipid.

  In the present invention, in the lipid bilayer membrane as described above, the hydrophilic polymer is selectively modified only on the outside of the lipid bilayer membrane in the hydrophilic polymer modification step. The hydrophilic polymer to be modified is not particularly limited, but polyethylene glycol, Ficoll, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether-maleic anhydride alternating copolymer, polyvinyl pyrrolidone, polyvinyl methyl ether, Polyvinylmethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropyl methacrylate, polyhydroxyethyl acrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyaspartamide, synthesis Examples include polyamino acids.

Among these, it is effective to have a high retention in blood of the formulation, polyethylene glycols, polyglycerols, polypropylene glycols are preferable, polyethylene glycol (PEG), polyglycerine (PG), polypropylene glycol (PPG) is more preferable. In addition, such hydrophilic polymer is preferably one in which one end is alkoxylated (for example, methoxylation, ethoxylation, propoxylation). This is because the storage stability is excellent. Among these, polyethylene glycol (PEG) is the most general purpose, and has the effect of improving the blood retention, and is preferable.

In the present invention, “retention in blood” means, for example, the property that a drug is present in blood in a state where the drug is encapsulated in the drug carrier in a host to which the drug carrier is administered. Once released from the drug carrier, the drug quickly disappears from the blood and is exposed. When the retention in the blood is good, it is possible to administer the drug in a smaller amount.
In the present invention, “exposure” means that the drug released to the outside of the drug carrier acts on the external environment. Specifically, the released drug approaches the target site and exerts its action (for example, an antitumor effect) by contacting. When the drug acts on the target site, it acts locally on the cells in the cell cycle where DNA synthesis of the target site is performed, and shows the expected effect.

    The hydrophilic polymer preferably has a structure for modifying the liposome. In particular, the hydrophilic polymer chain preferably has this structure at one end. That is, the hydrophilic polymer used for modification is preferably composed of a main body portion of the hydrophilic polymer and a structure portion for modifying the liposome. When the structure is a hydrophobic part such as a lipid, the hydrophilic polymer main body part is immobilized so as to protrude from the outer surface of the liposome in such a manner that the hydrophobic part is inserted into the liposome membrane. Is a reactive functional group that can be covalently bonded to the liposome membrane component, the hydrophilic polymer main body portion is formed by covalently bonding to the liposome membrane component such as phospholipid exposed on the outer surface of the liposome. Immobilized so as to protrude from the outer surface of the liposome.

Next, the hydrophobic compound used for bonding with the hydrophilic polymer main body to form a hydrophilic polymer-hydrophobic polymer compound will be described below.
The hydrophobic compound is not particularly limited. For example, the compound (hydrophobic compound) which has a hydrophobic area | region can be mentioned. Examples of the hydrophobic compound include phospholipids constituting mixed lipids described later, other lipids such as sterols, long-chain aliphatic alcohols, glycerin fatty acid esters, and the like. Among them, phospholipid is one of the preferred embodiments. Moreover, these hydrophobic compounds may have a reactive functional group. The bond formed by the reactive functional group is preferably a covalent bond, and specific examples include an amide bond, an ester bond, an ether bond, a sulfide bond, and a disulfide bond, but are not particularly limited.

The acyl chain contained in the phospholipid is desirably a saturated fatty acid. The chain length of the acyl chain is preferably C ₁₄ -C ₂₀ , and more preferably C ₁₆ -C ₁₈ . Examples of the acyl chain include dipalmitoyl, distearoyl, and palmitoyl stearoyl.
The phospholipid is not particularly limited. As the phospholipid, for example, one having a functional group capable of reacting with the above hydrophilic polymer can be used. Specific examples of the phospholipid having a functional group capable of reacting with such a hydrophilic polymer include phosphatidylethanolamine having an amino group, phosphatidylglycerol having a hydroxy group, and phosphatidylserine having a carboxy group. Is mentioned. One preferred embodiment is to use the above phosphatidylethanolamine.

  The lipid derivative of the hydrophilic polymer comprises the above hydrophilic polymer and the above lipid. The combination of the hydrophilic polymer and the lipid is not particularly limited. What was combined suitably according to the objective can be used. For example, at least one selected from phospholipids, other lipids such as sterols, long-chain fatty alcohols, and glycerin fatty acid esters, and at least one selected from PEG, PG, and PPG are bonded with high hydrophilicity. And molecular derivatives. Specific examples include polyoxypropylene alkyl, and in particular, when the hydrophilic polymer is polyethylene glycol (PEG), it is one of preferred embodiments to select phospholipid and cholesterol as the lipid. Examples of the PEG lipid derivative by such a combination include PEG phospholipid derivatives and PEG cholesterol derivatives.

  The lipid derivative of the hydrophilic polymer can be positively charged, negatively charged, or neutral depending on the choice of lipid. For example, when DSPE is selected as the lipid, it becomes a lipid derivative that shows a negative charge due to the influence of the phosphate group, and when cholesterol is selected as the lipid, it becomes a neutral lipid derivative. The lipid can be selected according to the purpose.

The molecular weight of PEG is not particularly limited. The molecular weight of PEG is usually 500 to 10,000 daltons, preferably 1,000 to 7,000 daltons, more preferably 2,000 to 5,000 daltons.
The molecular weight of PG is not particularly limited. The molecular weight of PG is usually 100 to 10,000 daltons, preferably 200 to 7000 daltons, more preferably 400 to 5000 daltons.
The molecular weight of PPG is not particularly limited. The molecular weight of PPG is usually 100 to 10,000 daltons, preferably 200 to 7,000 daltons, and more preferably 1,000 to 5,000 daltons.

Among these, phospholipid derivatives of PEG are mentioned as one of preferable embodiments. Examples of the phospholipid derivative of PEG include polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE). PEG-DSPE is preferred because it is a general-purpose compound and is easily available.
Said hydrophilic polymer can be used individually or in combination of 2 types or more, respectively.

Such a lipid derivative of a hydrophilic polymer can be produced by a conventionally known method. As a method for synthesizing a phospholipid derivative of PEG, which is an example of a lipid derivative of a hydrophilic polymer, for example, a method in which a phospholipid having a functional group capable of reacting with PEG is reacted with PEG using a catalyst Is mentioned. Examples of the catalyst include cyanuric chloride, carbodiimide, acid anhydride, and glutaraldehyde. Through such a reaction, the functional group and PEG can be covalently bonded to obtain a phospholipid derivative of PEG.
Liposomes whose surface has been modified using lipid derivatives of such hydrophilic polymers prevent the opsonin protein and the like in the plasma from adsorbing to the surface of the liposomes, thereby improving the blood stability of the liposomes. Can be avoided, and the deliverability of the drug to the target tissue or cell can be improved.

  The modification rate of the membrane lipid (total lipid) by the hydrophilic polymer lipid derivative is usually 0.1 to 20 mol%, preferably 0.1 to 5 mol%, more preferably 0.5 to 5 mol, as a ratio to the membrane lipid. %. The total lipid here is the total amount of all lipids constituting the membrane other than the hydrophilic polymer lipid derivative, and specifically includes phospholipids and other lipids, and other surface modifiers. Includes this surface modifier.

  Liposomes can contain other membrane constituents in addition to the above phospholipids and lipid derivatives of hydrophilic polymers. Examples of other membrane constituents include lipids other than phospholipids and derivatives thereof (hereinafter, these may be referred to as “other lipids”). Liposomes are preferably formed as a membrane of a mixed lipid containing other lipids together with the above-described phospholipid and hydrophilic polymer lipid derivative as a main membrane material. Thus, the liposome according to the present invention can maintain the above-mentioned membrane structure together with the lipid and the hydrophilic polymer, and other membrane components that can be included in the liposome do not impair the purpose of the present invention. Can be included in a range.

Various drugs can be carried on the liposome as described above. For example, therapeutic drugs include nucleic acids, polynucleotides, genes and analogs thereof, anticancer agents, antibiotics, enzyme agents, antioxidant agents, lipid uptake inhibitors, hormone agents, anti-inflammatory agents, steroid agents, blood vessels Expansion agent, angiotensin converting enzyme inhibitor, angiotensin receptor antagonist, smooth muscle cell proliferation / migration inhibitor, platelet aggregation inhibitor, anticoagulant, chemical mediator release inhibitor, vascular endothelial cell proliferation promoter or suppressor , Aldose reductase inhibitor, mesangial cell growth inhibitor, lipoxygenase inhibitor, immunosuppressive agent, immunostimulant, antiviral agent, Maillard reaction inhibitor, amyloidosis inhibitor, nitric oxide synthesis inhibitor, AGFs (Advanced glycation endproducts ) Inhibitors, radical scavengers, proteins, peptides, glycosaminoglycans And derivatives thereof, oligosaccharides and polysaccharides. Specifically, corticosteroids and their derivatives such as prednisolone, methylprednisolone and dexamethasone, non-steroidal anti-inflammatory agents such as aspirin, indomethacin, ibuprofen, mefenamic acid and phenylbutazone, and mesangial cell proliferation such as heparin and low molecular weight heparin. Inhibitors, immunosuppressants such as cyclosporine, ACE (angiotensin converting enzyme) inhibitors such as captopril, AGE (advanced glycation endoproduct) inhibitors such as methylguanidine, TGF-β antagonists such as biglycan and decorin, PKC (protein kinase) C) inhibitors, peripheral vasodilators such as prostaglandin preparations such as PGE ₁ and PGI ₂ , papaverine drugs, nicotinic acid drugs, tocopherol drugs, and Ca antagonists, phosphodiesterase inhibitors, Antithrombotic drugs such as ticlopidine and aspirin, wharf Anticoagulants such as gallin, heparin and antithrombin, thrombolytic agents such as urokinase, chemical mediator release inhibitors, antibiotics, antioxidants, enzymes, lipid uptake inhibitors, hormones, vitamin C, Examples include radical scavengers such as vitamin E and SOD, and antisense oligonucleotides, decoys or genes having a mesangial cell growth inhibitory action.
Examples of the diagnostic drug include in-vivo diagnostic agents such as an X-ray contrast agent, an ultrasonic diagnostic agent, a radioisotope-labeled nuclear medicine diagnostic agent, and a diagnostic agent for nuclear magnetic resonance diagnosis.

“Liposome preparation” is a general term for liposomes themselves or those in which liposomes are used as carriers and drugs are supported thereon. “Supported” as used herein means a state in which a drug is contained in a carrier. More specifically, the drug may be present in the inner aqueous phase of the liposome, or may be present in a state immobilized on the surface of the lipid layer, which is a component of the carrier, by electrostatic interaction or the like. It may be in a state where a part or all of the part is included in the layer. Examples of the place where the drug is carried include the liposome surface, the lipid membrane, and the inner aqueous phase. Among them, the inner aqueous phase of the liposome is preferable because it has a large volume and a large amount of the drug. The liposome preparation in the present invention is not particularly limited as long as the drug is supported on the liposome, and the liposome preparation may be in a state in which the liposome preparation is dispersed or suspended in the liposome external solution or lyophilized. The state is good.

  The liposome preparation of the present invention may further contain a pharmaceutically acceptable stabilizer and / or antioxidant and / or osmotic pressure adjusting agent and / or pH adjusting agent depending on the administration route.

  Examples of the stabilizer include, but are not particularly limited to, a saccharide such as glycerol, mannitol, sorbitol, lactose, or sucrose. Further, sterols such as cholesterol (Cholesterol) described above as other lipids of membrane constituents act as this stabilizer.

  Although it does not specifically limit as antioxidant, For example, ascorbic acid, uric acid, a tocopherol analog (for example, vitamin E) is mentioned. Tocopherol has four isomers, α, β, γ, and δ, and any of them can be used in the present invention. The stabilizer and / or antioxidant used is appropriately selected from the above depending on the dosage form, but is not limited thereto. Such stabilizers and antioxidants can be used alone or in combination of two or more. Further, from the viewpoint of preventing oxidation, it is desirable that the dispersion be a nitrogen-filled package. Further, it may be housed in a gas permeable container and hermetically packaged with a packaging material having gas barrier properties together with an oxygen scavenger.

Examples of the pH adjuster include sodium hydroxide, citric acid, acetic acid, triethanolamine, sodium hydrogen phosphate, sodium dihydrogen phosphate and the like.
The liposome preparation of the present invention may further contain an additive. Examples of such additives include pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch Pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, PBS, sodium chloride, saccharides, biodegradable polymer, serum-free medium, surface activity acceptable as a pharmaceutical additive Agents and buffers at physiological pH acceptable in vivo as described above.
The additive is appropriately selected from the above or a combination thereof, but is not limited thereto.

  In the production method of the present invention, a liposome preparation containing these additives can be provided as a pharmaceutical composition. The pharmaceutical composition containing the intermediate product produced by the production method of the present invention can be stored in a usual manner, for example, refrigeration at 0 to 8 ° C. or room temperature of 1 to 30 ° C.

  As a route for parenteral administration of the liposome preparation, for example, intravenous injection (intravenous injection) such as infusion, intramuscular injection, intraperitoneal injection, or subcutaneous injection can be selected, and an appropriate administration method depending on the age and symptoms of the patient Can be selected. As a specific administration method of the liposome preparation, the pharmaceutical composition can be administered by syringe or infusion. Further, the catheter is inserted into the body of the patient or host, for example, into a lumen, for example, into a blood vessel, and the tip thereof is guided to the vicinity of the target site. It is also possible to administer from the site where is expected.

  The liposomal formulation of the present invention is administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially block the symptoms of the disease. For example, the effective dose of the drug encapsulated in the liposome preparation is usually selected in the range of 0.01 mg to 100 mg per kg body weight per day. However, the liposome preparation 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.

Next, the production method of the present invention is illustrated.
(Uniformization process)
For example, a liposome membrane component substance and a membrane stabilizer are dissolved by heating in a volatile organic solvent, preferably absolute ethanol, to prepare a lipid solution. The heating temperature is usually not lower than the phase transition temperature. For example, when hydrogenated soybean phosphatidylcholine is used, it is 50 ° C to 80 ° C, preferably 60 ° C to 70 ° C.

(Liposome formation process)
Next, an osmotic pressure adjusting agent, pH adjusting agent, encapsulating drug, etc. are dissolved in water for injection as necessary to prepare an aqueous solution. A crude liposome dispersion is obtained by mixing and stirring the pre-warmed aqueous solution and the lipid solution obtained in the homogenization step. The heating temperature is usually not lower than the phase transition temperature. For example, when hydrogenated soybean phosphatidylcholine is used, it is 50 ° C. or higher, preferably 60 ° C. to 70 ° C. In the stirring, it can be heated and stirred with a stirring device such as a propeller type stirrer, if necessary. Next, the average particle size of the liposome coarse dispersion can be controlled by a membrane filter made of polycarbonate. For example, when the particle size is adjusted to 100 nm, the particle size is often adjusted stepwise by combining membrane filters such as 400 nm, 200 nm, and 100 nm.

(Hydrophilic polymer modification process)
Next, a lipid derivative of a hydrophilic polymer is added to the sized liposome dispersion and heated and mixed. It is preferable that the liposome dispersion before the addition of the lipid derivative of the hydrophilic polymer is maintained in a heated state until the addition of the lipid derivative of the hydrophilic polymer, and the heating temperature is usually the temperature at which the granulation is performed. In the case of a liposome preparation using hydrogenated soybean phosphatidylcholine, the temperature is 50 ° C. or higher, preferably 60 ° C. to 70 ° C. The hydrophilic polymer is added as an aqueous solution as necessary. As the hydrophilic polymer, polyethylene glycol (PEG) is preferably used. The hydrophilic polymer can be added immediately after completion of the liposome formation step such as granulation, and is preferably added within 180 minutes immediately after the end of the previous step. The time until the addition is preferably as short as possible in the process. The addition method may be a method in which the liposome dispersion and the hydrophilic polymer solution are simultaneously mixed. The liposome dispersion is added to the hydrophilic polymer solution, or vice versa. The method of adding may be used. In addition, it is preferable to stir. In the stirring, it can be heated and stirred with a stirring device such as a propeller type stirrer, if necessary. In the hydrophilic polymer addition step, it is essential that the step is at least the phase transition temperature described above. In the present invention, when alcohol is present in the hydrophilic polymer addition step, it has been found that the hydrophilic polymer introduction time can be significantly shortened and the introduction temperature condition can be lowered. From the viewpoint of lipid stability, the liposome after the hydrophilic polymer addition step is desired to have a temperature condition as low as possible and a heating and stirring time as short as possible. The existence is suitable from these viewpoints. In the present invention, as a method for bringing alcohol into the hydrophilic polymer addition step, it is most desirable to use alcohol in the homogenization step. This is because it is most convenient to completely solubilize with alcohol for the homogenization of the desired lipid in the homogenization step. If necessary, the drug can be supported on the liposome by a remote loading method typified by a pH gradient method such as a citric acid method or an ammonium sulfate method after this step.

(Unencapsulated drug removal process)
The liposome dispersion having a uniform particle size thus obtained can be stably treated after this step, and the unencapsulated drug removal step, sterilization step, etc. should be carried out under generally known conditions. Can do. Since the homogenization step, liposome formation step, and hydrophilic polymer addition step are performed using the deformation of the liposome, it is essential that the temperature is equal to or higher than the phase transition temperature of the main membrane material. On the contrary, since it is difficult for the liposome after the process to undergo a particle size fluctuation due to the deformation of the liposome, it is desirable that the unencapsulated drug removal step and the sterilization step be performed below the phase transition temperature of the main membrane material. For example, when the phase transition temperature of the main film material is around 50 ° C., it is preferably about 0 to 40 ° C., and more specifically, it is preferably manufactured at about 5 to 30 ° C. The final preparation that has undergone the sterilization step can be stored at room temperature of 21 ° C. to 30 ° C., preferably refrigerated at 0 to 8 ° C., from the viewpoint of lipid stability and physicochemical stability such as particle size. it can. An outline of the production method of the present invention is shown in FIG.

EXAMPLES Next, although an Example and a test example are given and this invention is demonstrated in more detail, this invention should not be limited to these Examples and a test example.
The particle diameter (average particle diameter) of the liposome preparation in the present invention including the liposome preparation prepared in each example is an average particle diameter measured with a laser diffraction particle measuring apparatus (Beckman Coulter LS230). Moreover, each lipid component and the concentration (mg / mL) of the encapsulated drug were quantified by high performance liquid chromatography.
The abbreviations and molecular weights of the respective components used are shown below.
Hydrogenated soybean lecithin (HSPC, molecular weight 790)
Cholesterol (molecular weight 386.66)
Polyethylene glycol ₅₀₀₀ -phosphatidylethanolamine (PEG ₅₀₀₀ -DSPE, molecular weight 5938)

Example 1
Liposomes were produced in Preparation Example 1 and Comparative Example 1 in order to compare the production method of the present invention using alcohol in the homogenization step and the production method not using alcohol in the homogenization step.

(Preparation Example 1) Production method of the present invention using alcohol in the homogenization step (1) Homogenization step 4.9 g of hydrogenated soybean lecithin (HSPC) and 2.1 g of cholesterol are weighed, and absolute ethanol (10 mL) is added. Added and warmed to dissolve.
(2) Liposome formation step 90 mL of ammonium sulfate solution adjusted to 250 mM is heated to around 70 ° C. and added to 10 mL of the lipid ethanol solution obtained through the homogenization step, and the resulting dispersion is stirred to give coarse liposomes A dispersion was obtained. The obtained crude liposome dispersion was passed through a polycarbonate membrane filter having a pore size of 200 nm and 100 nm using an extruder heated to around 65 ° C. to obtain a liposome dispersion.

(Comparative Example 1) Production method not using alcohol in the homogenization step (1) Uniformation step 4.9 g of hydrogenated soybean lecithin (HSPC) and 2.1 g of cholesterol were weighed and heated to 60 ° C. Alcohol (200 mL) was added and dissolved by heating. After dissolution by heating, it was completely frozen by ice cooling. T-Butyl alcohol was completely removed by distillation under reduced pressure to obtain a mixed lipid.
(2) Liposome formation step 100 mL of ammonium sulfate solution adjusted to 250 mM is heated to around 70 ° C, added to the mixed lipid obtained through the homogenization step, and heated and swollen while stirring to give a crude liposome dispersion. Obtained. The obtained crude liposome dispersion was passed through a polycarbonate membrane filter having a pore size of 200 nm and 100 nm using an extruder heated to around 65 ° C. to obtain a liposome dispersion.

(Test Example 1) Time dependency of PEG ₅₀₀₀ -DSPE introduction efficiency (%) PEG ₅₀₀₀ -DSPE solution (0.51 g / 20 mL (water for injection)) with respect to the liposome dispersions obtained in Preparation Example 1 and Comparative Example 1 ) Was immediately added, and the mixture was heated and stirred at 60 ° C. for a predetermined time to introduce PEG ₅₀₀₀ -DSPE into the liposome (introduction rate of PEG ₅₀₀₀ -DSPE (mol%) = 0.75). After the introduction, the liposomes were ice-cooled, and external solution replacement was performed using a gel column sufficiently substituted with a 10 mM histidine / 10% sucrose solution (pH 6.5) solution. Introduction efficiency of _PEG 5000 -DSPE (percent) was calculated based on the following equation for the external solution liposome dispersion after _replacement, the results of examining the time dependence of the PEG 5000 -DSPE transfer efficiency (%) in FIG. 2 Show. It was revealed that the introduction of PEG ₅₀₀₀ -DSPE was completed in a short time in the presence of ethanol compared to the absence of ethanol. As a result, it has been clarified that the presence of ethanol in the hydrophilic polymer addition step can shorten the stirring time in the hydrophilic polymer addition step.

Formula: PEG ₅₀₀₀ -DSPE introduction efficiency (%) = (PEG ₅₀₀₀ -DSPE introduction rate with respect to the total lipid amount in the final preparation (mol%)) / (PEG ₅₀₀₀ -DSPE introduction rate with respect to the total lipid amount in the charged amount (mol) %))

(Test Example 2) Temperature dependency of PEG ₅₀₀₀ -DSPE introduction efficiency (%) The PEG ₅₀₀₀ -DSPE solution (0.51 g / 20 mL (water for injection)) was rapidly added to the liposome dispersion obtained in Preparation Example 1. PEG ₅₀₀₀ -DSPE was introduced into the liposome by immediately adding and stirring at 40 to 70 ° C. for a predetermined time (PEG ₅₀₀₀ -DSPE introduction rate (mol%) = 0.75). After the introduction, the liposomes were ice-cooled, and external solution replacement was performed using a gel column sufficiently substituted with a 10 mM histidine / 10% sucrose solution (pH 6.5) solution. The PEG ₅₀₀₀ -DSPE introduction rate (mol%) was calculated for the liposome dispersion after replacement with the outer solution. The results of examining the temperature dependence of PEG ₅₀₀₀ -DSPE introduction efficiency (%) are shown in FIG. As a result, it has been clarified that the presence of ethanol in the hydrophilic polymer addition step can lower the stirring temperature in the hydrophilic polymer addition step.

(Example 2)
Liposomes were produced in Preparation Example 2 and Comparative Example 2 in order to compare the production method of the present invention with the conventional production method.

(Preparation Example 2) Production Method of the Present Invention (1) Homogenization Step 70.8 g of hydrogenated soybean lecithin (HSPC) and 29.1 g of cholesterol were weighed, and absolute ethanol (100 mL) was added and dissolved by heating.
(2) Liposome formation step 900 mL of ammonium sulfate solution adjusted to 250 mM is heated to around 70 ° C and added to 100 mL of the lipid ethanol solution obtained through the homogenization step, and the resulting dispersion is stirred to give coarse liposomes A dispersion was obtained. Using an extruder in which the obtained crude liposome dispersion was heated to around 65 ° C., a liposome dispersion was obtained by passing through a polycarbonate membrane filter having a pore size of 100 nm five times.
(3) Hydrophilic polymer addition step PEG _5000- DSPE solution (7.69 g / 200 mL (water for injection)) is immediately added and the mixture is heated and stirred while maintaining the obtained liposome dispersion in a heated state. Then, PEG ₅₀₀₀ -DSPE was introduced into the liposome (introduction rate of PEG ₅₀₀₀ -DSPE (mol%) = 0.75).

Comparative Example 2 Conventional Method (1) Homogenization Step 70.8 g of hydrogenated soybean lecithin (HSPC) and 29.1 g of cholesterol were weighed, and absolute ethanol (100 mL) was added and dissolved by heating.
(2) Liposome formation step 900 mL of ammonium sulfate solution adjusted to 250 mM is heated to around 70 ° C and added to 100 mL of the lipid ethanol solution obtained through the homogenization step, and the resulting dispersion is stirred to give coarse liposomes A dispersion was obtained. Using an extruder in which the obtained crude liposome dispersion was heated to around 65 ° C., a liposome dispersion was obtained by passing through a polycarbonate membrane filter having a pore size of 100 nm five times.

(Test Example 3) Liposome stability (temperature conditions)
The liposome dispersion prepared in Preparation Example 2 and Comparative Example 2 was ice-cooled immediately after preparation. Table 1 shows the results of measuring the average particle size of the liposomes after cooling. In Preparation Example 2 prepared by the production method of the present invention including a hydrophilic polymer addition step, the average particle size and the particle size distribution pattern did not change at all (FIG. 4). However, in Comparative Example 2 using the conventional method that does not include a hydrophilic polymer addition step, aggregation of liposomes proceeds by cooling with ice, and an increase in particle diameter and particle size distribution of multiple peaks instead of a single peak are observed. (FIG. 5). As a result, it became clear that it is desirable to cool the hydrophilic polymer after the hydrophilic polymer addition step, not immediately after the liposome formation step.

＊）複数ピークの平均粒子径として算出

*) Calculated as the average particle size of multiple peaks

(Test Example 4) Liposome stability (retention time)
The liposome dispersion liquid prepared in Preparation Example 2 and Comparative Example 2 was kept warmed at 65 ° C. for 60 minutes immediately after preparation. The results of measuring the average particle size of the liposomes during the retention time are shown in Table 2 and FIG. In Preparation Example 2 prepared by the production method of the present invention including the step of adding a hydrophilic polymer, the average particle size and the particle size distribution pattern did not change at all (FIG. 6). However, in Comparative Example 2 using the conventional method that does not include the hydrophilic polymer addition step, the aggregation of the liposome proceeds as the retention time becomes longer, and the particle diameter increases and the particle size distribution of a plurality of peaks instead of a single peak is observed. (FIG. 7). As a result, it was revealed that liposome aggregation can be suppressed by introducing a hydrophilic polymer addition step (FIG. 8). Moreover, in this prescription, it has become clear that the time from immediately after the liposome formation step to the transition to the hydrophilic polymer addition step is preferably within 45 minutes.

＊）複数ピークの平均粒子径として算出

*) Calculated as the average particle size of multiple peaks

(Example 3)
Liposomes were produced in Preparation Example 3 and Comparative Example 3 in order to compare the production method of the present invention with the conventional production method.

(Preparation example 3) Production method of the present invention (1) Homogenization step 70.8 g of hydrogenated soybean lecithin (HSPC) and 29.1 g of cholesterol were weighed, and absolute ethanol (100 mL) was added and dissolved by heating.
(2) Liposome formation step 900 mL of ammonium sulfate solution adjusted to 250 mM is heated to around 70 ° C and added to 100 mL of the lipid ethanol solution obtained through the homogenization step, and the resulting dispersion is stirred to give coarse liposomes A dispersion was obtained. Using an extruder in which the obtained crude liposome dispersion was heated to around 65 ° C., a liposome dispersion was obtained by passing through a polycarbonate membrane filter having a pore size of 100 nm five times.
(3) Hydrophilic polymer addition step While maintaining the obtained liposome dispersion in a heated state, immediately add a PEG ₅₀₀₀ -DSPE solution (7.69 g / 200 mL (water for injection)) and stir with warming. Then, PEG ₅₀₀₀ -DSPE was introduced into the liposome (introduction rate of PEG ₅₀₀₀ -DSPE (mol%) = 0.75). The liposome dispersion after completion of heating was quickly ice-cooled.
(4) Unencapsulated drug removal process (external liquid replacement process)
The cooled liposome dispersion is subjected to a tangential flow filtration method using an ultrafiltration membrane made of polyethersulfone having a molecular weight cut off of 300 K, and the external solution is obtained with a 10 mM histidine / 10% sucrose solution (pH 6.5). Replaced.
(5) Sterilization process Transfer the liposome dispersion after replacement of the external liquid to a pressurized tank, apply a pressure of about 1.5 kg / cm ² , pass through a 0.2 μm filter for filter sterilization, and perform filter sterilization. It was.

Comparative Example 3 Conventional Method (1) Homogenization Step 70.8 g of hydrogenated soybean lecithin (HSPC) and 29.1 g of cholesterol were weighed, absolute ethanol (100 mL) was added and dissolved by heating.
(2) Liposome formation step 900 mL of ammonium sulfate solution adjusted to 250 mM is heated to around 70 ° C and added to 100 mL of the lipid ethanol solution obtained through the homogenization step, and the resulting dispersion is stirred to give coarse liposomes A dispersion was obtained. Using an extruder in which the obtained crude liposome dispersion was heated to around 65 ° C., a liposome dispersion was obtained by passing through a polycarbonate membrane filter having a pore size of 100 nm five times.
(3) Unencapsulated drug removal process (external liquid replacement process)
The cooled liposome dispersion was subjected to a tangential flow filtration method using a polyethersulfone ultrafiltration membrane with a molecular weight cut off of 300 K, and the solution was externally added with a 10 mM histidine / 10% sucrose solution (pH 6.5). Liquid replacement was performed.
(4) Sterilization process Since aggregates were present in the liposome dispersion, the 0.2 μm filter sterilization filter was clogged, and filtration sterilization was not possible.

(Test Example 5) Liposome stability (sterilization process)
Table 3 shows the results of the liposome particle size in each step in Preparation Example 3 and Comparative Example 3. In the production method of the present invention, the final preparation could be produced through the sterilization step without increasing the particle size, but in the conventional method, the increase in the particle size in the unencapsulated drug removal step (external liquid replacement step). As a result, the 0.2 μm filter sterilization filter was clogged and could not be sterilized by filtration. Table 4 shows the physical property values of the final preparation obtained in Preparation Example 3. As a result, it was revealed that PEG ₅₀₀₀ -DSPE was introduced almost as charged. As a result of the above, it has been clarified that by using the production method of the present invention, liposomes having a sharp particle size distribution can be produced without impairing the physical stability during the process even if the particles are made into the nanometer range. .

Example 4
Liposomes encapsulating the drug were produced using the production method of the present invention.

(Preparation Example 4) Production of drug-encapsulated liposomes using the production method of the present invention (1) Homogenization step 7.1 g of hydrogenated soybean lecithin (HSPC) and 2.9 g of cholesterol were weighed and absolute ethanol (10 mL) Was added and dissolved by heating.
(2) Liposome formation step 6.78 g / 100 mL (PBS: physiological saline-phosphate buffer solution (pH 7.2)) 90 mL of the prepared prednisolone phosphate solution was heated to around 70 ° C., followed by a homogenization step. The dispersion obtained by adding to 10 mL of the obtained lipid ethanol solution was stirred to obtain a crude liposome dispersion. The obtained crude liposome dispersion was passed through a polycarbonate membrane filter having a pore size of 200 nm and 100 nm using an extruder heated to around 65 ° C. to obtain a liposome dispersion.
(3) Hydrophilic polymer addition step PEG ₅₀₀₀ -DSPE solution (0.77 g / 20 mL (water for injection)) is immediately added and the mixture is heated and stirred while maintaining the obtained liposome dispersion in a heated state. Then, PEG ₅₀₀₀ -DSPE was introduced into the liposome. The liposome dispersion after completion of heating was quickly ice-cooled.
(4) Unencapsulated drug removal process (external liquid replacement process)
After diluting the cooled liposome dispersion by adding a 5-fold amount of an external aqueous phase (PBS: physiological saline-phosphate buffer (pH 7.2)), an ultra-fine product made of polyethersulfone having a molecular weight cut off of 300K is used. Unencapsulated drug was removed by tangential flow filtration using a filtration membrane.
(5) Sterilization step After preparing the concentration of the liposome dispersion after removal of the unencapsulated drug and transferring it to a pressurized tank, apply a pressure of about 1.5 kg / cm ² and install a 0.2 μm filter for filter sterilization. Passed through and sterilized by filtration.

(Test Example 5) Physical property values of liposomes The physical property values of the liposomes obtained in Preparation Example 4 were analyzed. The results are shown in Table 5.

It is the schematic showing the outline of the manufacturing method of this invention. It is a diagram showing the time dependence of the introduced _{PEG 5000} -DSPE ethanol presence and absence of a homogenization process efficiency (%). It is a graph showing the temperature dependence of the introduction of ethanol present at _{PEG 5000} -DSPE homogenization process efficiency (%). It is a figure which shows the particle size distribution at the time of ice-cooling a PEG introduction | transduction liposome. It is a figure which shows the particle size distribution when the PEG non-introduced liposome is ice-cooled. It is a figure which shows the particle size distribution at the time of heating-maintaining a PEG introduction | transduction liposome for 60 minutes. It is a figure which shows a particle size distribution at the time of carrying out the heat retention of the PEG non-introduction liposome for 60 minutes. It is a figure shown about the relationship between holding time and a particle diameter.

Claims (10)

Liposome formation step for obtaining a liposome dispersion, hydrophilic polymer modification step for modifying a hydrophilic polymer on the outer surface of the liposome, and an external force that impairs physical stability can be applied to the liposome. A liposome dispersion treatment step, wherein the liposome dispersion contains an organic solvent, and the hydrophilic polymer modification step is performed in the liposome formation step after completion of the liposome formation step. A method for producing a liposome preparation, which is carried out while maintaining a temperature equal to or higher than the treatment temperature. 2. The method for producing a liposome preparation according to claim 1, wherein the dispersion treatment step is one or more of an unencapsulated drug removal step, a sterilization step, and a liposome external solution replacement step. The method for producing a liposome preparation according to claim 1 or 2 , wherein the organic solvent is derived from a solvent used in a homogenization step of mixing and homogenizing the liposome-constituting lipid in the solvent prior to the liposome formation step. The method for producing a liposome preparation according to any one of claims 1 to 3 , wherein the organic solvent is an alcohol. The method for producing a liposome preparation according to any one of claims 1 to 4 , wherein the temperature in the hydrophilic polymer addition step is a temperature equal to or higher than a lipid membrane phase transition temperature. The method for producing a liposome preparation according to any one of claims 1 to 5 , wherein the hydrophilic polymer addition step is performed within 180 minutes immediately after the liposome formation step. The hydrophilic polymer introduction rate in the hydrophilic polymer addition step is 0.01 to 20 mol% with respect to the total lipid constituting the liposomes dispersed in the liposome dispersion obtained in the liposome formation step. Item 7. A method for producing a liposome preparation according to any one of Items 1 to 6 . The method for producing a liposome preparation according to any one of claims 1 to 7 , wherein the hydrophilic polymer is polyethylene glycol. The method for producing a liposome preparation according to claim 8 , wherein the hydrophilic polymer is polyethylene glycol having a molecular weight of 500 to 15,000 daltons. The method for producing a liposome preparation according to any one of claims 1 to 9 , wherein the liposome has an average particle diameter of 50 to 250 nm.

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