WO2012053484A1

WO2012053484A1 - Process for production of uni-lamellar liposome by two-stage emulsification technique which involves adding water-soluble lipid to inner aqueous phase, and uni-lamellar liposome produced by the process

Info

Publication number: WO2012053484A1
Application number: PCT/JP2011/073847
Authority: WO
Inventors: 武寿磯田
Original assignee: コニカミノルタホールディングス株式会社
Priority date: 2010-10-19
Filing date: 2011-10-17
Publication date: 2012-04-26
Also published as: JP5838970B2; JPWO2012053484A1

Abstract

The present invention provides: a uni-lamellar liposome having an improved water-soluble medicinal agent encapsulation rate; and a process for producing the uni-lamellar liposome. This uni-lamellar liposome is characterized in that an inner aqueous phase is composed of an aqueous solvent (W1) having, dissolved therein, a water-soluble lipid component (Fw) composed of a lipid having a ClogP value smaller than 11. This process for producing the uni-lamellar liposome is characterized by comprising: (1) a primary emulsification step of dissolving a water-soluble lipid component (Fw) in an aqueous solvent (W1), dissolving a mixed lipid component (F1) that does not contain the water-soluble lipid (Fw) in an organic solvent (O), and mixing/emulsifying these solutions with each other, thereby preparing W1/O emulsion; (2) a secondary emulsification step of mixing/emulsifying the W1/O emulsion produced in step (1) with an aqueous solvent (W2), thereby preparing a W1/O/W2 emulsion; and (3) a solvent removal step of removing the organic solvent from the W1/O/W2 emulsion produced in step (2), thereby preparing an aqueous liposome suspension.

Description

Method for producing single-cell liposome by two-stage emulsification method in which water-soluble lipid is added to the inner aqueous phase, and single-cell liposome obtained by the production method

The present invention relates to a production method using a two-stage emulsification method of single cell liposomes that can be used as a drug carrier for pharmaceuticals, foods, and cosmetics, and a single cell liposome obtained by the production method.

Liposomes are closed vesicles consisting of a lipid bilayer composed mainly of phospholipids. Since it has a structure and function similar to a cell membrane, it is difficult to stimulate the immune system and is highly safe as a material. In addition, the water-soluble drug can be retained in the internal aqueous phase surrounded by the lipid bilayer membrane, and the fat-soluble drug can be retained in the bilayer membrane. Can be included.

Regarding such liposomes that can be used in fields such as pharmaceuticals and cosmetics and methods for producing the same, in particular, regarding means for improving the encapsulation rate of water-soluble substances (drugs) to be encapsulated in liposomes, for example, the following documents are available: It has been published.

In Non-Patent Document 1, when preparing a W / O / W emulsion by a microchannel emulsification method using a W / O emulsion as a dispersed phase and a Tris-HCl buffer as an outer aqueous phase, sodium caseinate is used as an emulsifier in the outer aqueous phase. As a result, it was described that the inclusion rate of calcein in the liposome (lipid capsule) could be increased to about 80%. However, in this non-patent document 1, no attention is paid to the additive to the inner aqueous phase. In addition, calcein is only exemplified as the inclusion substance, the versatility of the drug is not shown, and practical verification considering the production scale has not been made.

Patent Document 1 discloses that “it is composed of an inner membrane composed of a lipid containing one or two or more functional lipids and a lipid containing or not containing one or two or more functional lipids. A lipid bilayer comprising an outer membrane, and at least for any one type of functional lipid contained in the inner membrane, the amount in the inner membrane exceeds the amount in the outer membrane Is disclosed, characterized by satisfying the following conditions. Further, as one of the above functional lipids, “water-soluble lipid” such as “a compound in which a polyoxyalkylene group (— (CH ₂ CH ₂ O) _n H) is introduced into cholesterol” is mentioned. "By introducing a polyoxyalkylene group into the membrane lipid, the surface of the liposome becomes hydrophilic, improving the stability of the liposome (improving its disintegration and aggregation properties) and recognizing it as a foreign substance in the body. It is described that an effect such as “cannot be done” is obtained. Furthermore, it is described that a microencapsulation method, a membrane emulsification method, a supercritical carbon dioxide method and the like can be applied as a method for producing the liposome of the present invention.

However, what is specifically mentioned as the “water-soluble lipid” in the invention according to the above-mentioned Patent Document 1 includes a part that enters the inner membrane and constitutes it, such as cholesterol into which a polyoxyalkylene group is introduced. Is a compound. In addition, regarding the present invention, “liposomes having different constituent components (amount of functional lipid) in the inner and outer membranes, such as containing more functional lipids that chemically interact with compounds such as drugs in the inner membrane than in the outer membrane. Is superior to liposomes containing the same amount of such functional lipids in the inner and outer membranes, and is superior in drug encapsulation, dispersion stability, and controlled release. As a more specific embodiment of such an invention, “the charged lipid contributing to the retention of the drug is contained only in the inner membrane, while the water-soluble lipid involved in the stability of the liposome and the body movement is contained only in the outer membrane” And the amount of the charged lipid exceeds the amount of the water-soluble lipid ”. That is, the production method in which a water-soluble lipid that does not necessarily form a part of the inner membrane is added to the inner aqueous phase and the liposomes obtained thereby are not specifically disclosed by the above-mentioned Patent Document 1.

In order to make liposomes administered into the body difficult to be recognized by the immune system (stealth), to make it easier to collect in specific organs, to increase hydrophilicity and to improve blood stability, it is applied to the outer surface of the liposome. A technique for introducing a polyethylene glycol (PEG) chain is also known (for example, Non-Patent Document 2). However, for such a technique, for example, a compound such as PEGylated cholesterol is used as a constituent lipid component of the outer membrane of the liposome. The addition of such compounds is not described or suggested by such techniques.

International Publication No. 2008/140081 Pamphlet

The present invention is capable of realizing a high encapsulation rate of a water-soluble drug in a liposome, improving the temporal stability of a W / O / W emulsion, and maintaining the encapsulation rate in the process. It is an object of the present invention to provide a method for producing a single liposome having a high encapsulation rate of a water-soluble drug obtained by such a production method.

The present inventors have found that the above problem can be solved by adding “water-soluble lipid” to the inner aqueous phase when producing liposomes by a two-stage emulsification method, and have completed the present invention. .

That is, the present invention provides a single cell liposome characterized in that an aqueous solvent (W1) in which a water-soluble lipid component (Fw) comprising a lipid having a ClogP value of less than 11 is dissolved is used as an inner aqueous phase.

The water-soluble lipid component (Fw) preferably contains at least one selected from the group consisting of lysolipids, short-chain phospholipids, PEG lipids, and chemically synthesized lipids having a hydrophilic group.

It is preferable that a water-soluble drug is further dissolved in the aqueous solvent (W1).

The present invention also provides a dry powder of the above-mentioned single cell liposome, an aqueous suspension containing the above single cell liposome, or obtained by adding the above dry powder to an aqueous solvent.

In addition, the present invention provides a method for producing single-cell liposomes, which comprises the following steps (1) to (3):
(1) The water-soluble lipid component (Fw) is dissolved in the aqueous solvent (W1), the mixed lipid component (F1) other than the water-soluble lipid (Fw) is dissolved in the organic solvent (O), and these are mixed and emulsified. A primary emulsification step to prepare a W1 / O emulsion by
(2) A secondary emulsification step of preparing a W1 / O / W2 emulsion by mixing and emulsifying the W1 / O emulsion obtained through the step (1) and the aqueous solvent (W2);
(3) A solvent removal step of preparing an aqueous suspension of liposomes by removing the organic solvent contained in the W1 / O / W2 emulsion obtained through the step (2).

The primary emulsification step (1) is preferably performed by further adding a water-soluble drug.

The solvent removal step (3) is preferably performed while stirring the W1 / O / W2 emulsion.

The present invention also provides single cell liposomes produced by any of the above production methods, aqueous suspensions thereof, or dry powders thereof.

In the present invention, “monocystic liposome” (ULV, synonymous with mononuclear liposome) refers to a liposome structure having a single inner aqueous phase, and usually has a volume average particle size in the range of about 20 to 500 nm. On the other hand, “multivesicular liposome” (MVL: リポソーム multivesicular liposomes) refers to a liposome structure comprising a lipid membrane surrounding a plurality of non-concentric inner aqueous phases, and also referred to as “multilamellar liposome” (MLV ) Refers to a liposome structure having a plurality of concentric membranes, such as “onion skin”, with a shell-like concentric aqueous compartment in between. The characteristics of multivesicular liposomes and multilamellar liposomes are that the volume average particle diameter is in the micrometer range, usually 0.5 to 25 μm.

The stability of the inner membrane side of the liposome is improved by adding a predetermined water-soluble lipid to the inner aqueous phase, and the step includes removing the O phase (oil phase) by stirring or leaving the W / O / W emulsion. Even when liposomes are produced by such a method, it is possible to suppress a decrease in the encapsulation rate of the water-soluble drug. Conventionally, it is known that the preparation of a W / O / W emulsion easily proceeds in such a liposome production method when the O phase is an oil having a high boiling point such as olive oil, decane, or hexadecane, On the other hand, when an organic solvent having a boiling point lower than that of water is used for the O phase, it is not easy to prepare a W / O / W emulsion. This is because the surface tension of the organic solvent is low, so that the ability to maintain the spherical shape of the particles is sufficient. It was interpreted as not. According to the present invention, even when an organic solvent having a boiling point lower than that of water is used for the O phase, preparation of a W / O / W emulsion is facilitated, and liposomes having a relatively high water-soluble drug encapsulation rate are easily obtained.

Although the principle that the above effect is produced is not necessarily clarified, for example, the water-soluble lipid accumulates in the vicinity of the inner wall of the liposome membrane, or the membrane is strengthened, or depending on the type of drug. Is partly mixed in the liposome membrane and disturbs the membrane, which may reduce the strength of the membrane. It is considered that such contamination can be prevented. It is also possible that the water-soluble lipid has a function like an emulsifier and improves the emulsification behavior, or the water-soluble lipid forms reverse micelles and acts on the drug to control its solubility. .

-Production raw materials-
・ Water-soluble lipid component (Fw)
In the present invention, the “water-soluble lipid component” refers to a component composed of a lipid having a ClogP value of less than 11. “ClogP” is a coefficient indicating the affinity of an organic compound for water and 1-octanol, and can be calculated by molecular structure analysis and physical property calculation tool ChemBioDraw Ultra of CambridgeSoft. This is a value obtained by calculating the “partition coefficient” expressed by the ratio of water and 1-octanol dissolved in an experiment. When ClogP is large, it is relatively easy to dissolve in 1-octanol, and when ClogP is small, it is relatively easy to dissolve in water.

The water-soluble lipid that can be used in the present invention is not particularly limited as long as this “ClogP” shows a value smaller than 11, among which lysolipid, short-chain phospholipid, PEG lipid and chemical synthesis having a hydrophilic group. Lipids are preferred. Any one of these water-soluble lipids may be used alone, or two or more thereof may be used in combination. The amount of the water-soluble lipid component (Fw) added to the aqueous solvent (W1) can be adjusted within an appropriate range in consideration of the effects of the invention and the amount of the water-soluble lipid component (Fw) dissolved in water. it can.

A lysolipid is a kind of glycerophospholipid and is a monoacylglycerophospholipid from which either one of the fatty acids bonded to the 1-position or 2-position of glycerol has been removed. A phosphatidylglycerol is mentioned.

The short-chain phospholipid is a glycerophospholipid or sphingophospholipid having a small number of bound fatty acids (for example, 2 to 10), such as didecanoylphosphatidylcholine.

The PEG lipid is a derivative in which a PEG (polyethylene glycol) chain is introduced into the lipid, while the chemically synthesized lipid having a hydrophilic group is a derivative in which a hydrophilic group other than the PEG chain is introduced into the lipid. Examples of the lipid include phospholipid and cholesterol. Examples of hydrophilic groups other than PEG chains include sugar chains, alcohol groups, amino groups, thiol groups and carboxylic acid groups, and amino acid chains combining these. In particular, details of chemically synthesized lipids (synthetic sterols) having a sugar chain as a hydrophilic group are disclosed in, for example, International Publication WO2008 / 081686.

・ Mixed lipid component (F1) ・ (F2)
The mixed lipid component (F1) dissolved in the organic solvent (O) in the primary emulsification step mainly constitutes the inner membrane of the lipid bilayer of the liposome, and possibly contributes to the outer membrane. On the other hand, the mixed lipid component (F2) added in the secondary emulsification step as necessary mainly constitutes the outer membrane of the liposome. The mixed lipid components (F1) and (F2) may have the same composition or different compositions.

The blending composition of these mixed lipid components is not particularly limited, and can be in accordance with the blending composition of known liposomes. In general, phospholipids (lecithins derived from animals and plants; phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid or their fatty acid esters, glycerophospholipids; sphingophospholipids; derivatives thereof, etc.), Consists mainly of sterols that contribute to stabilization (cholesterol, phytosterol, ergosterol, derivatives thereof, etc.), and also glycolipids, glycols, aliphatic amines, long chain fatty acids (oleic acid, stearic acid, palmitic acid, etc.) ) And other compounds that impart various functions may be blended. Further, F2 can be blended with lipids for modifying the liposome surface and imparting various functions such as PEGylated phospholipid. The compounding ratio of these compounds in the mixed lipid component may be appropriately adjusted according to the use while taking into consideration properties such as the stability of the lipid membrane and the behavior of the liposome in vivo.

Aqueous solvent (W1) / (W2), organic solvent (O)
As the aqueous solvents (W1) and (W2) and the organic solvent (O), general solvents such as those used in known liposome production methods can be used. The aqueous solvent (W1) and the organic solvent (O) used in the primary emulsification step form an aqueous phase and an oil phase of the W1 / O emulsion, respectively. The aqueous solvent (W2) used in the secondary emulsification step is W1 / O. / The outer water phase of the W2 emulsion is formed. Examples of the aqueous solvent include pure water containing other solvents mixed with water as necessary, salts / sugars for adjusting osmotic pressure, buffers for adjusting pH, and the like. Examples of organic solvents include hexane (n-hexane), chloroform, cyclohexane, 1,2-dichloroethene, dichloromethane, 1,2-dimethoxyethane, 1,1,2-trichloroethene, t-butylmethyl ether, ethyl acetate. Water-insoluble organic solvents such as diethyl ether, ethyl formate, isopropyl acetate, methyl acetate, methyl ethyl ketone, and pentane. In addition, water-soluble organic solvents such as acetonitrile, methanol, acetone, ethanol, 2-propanol, and ethers, hydrocarbons, halogenated hydrocarbons, halogenated ethers, and esters other than those described above can be used. Of these, chloroform, cyclohexane, dichloromethane, hexane, t-butyl methyl ether, ethyl acetate, diethyl ether, ethyl formate, isopropyl acetate, methyl acetate, methyl ethyl ketone, pentane, acetonitrile, methanol, acetone, ethanol, 2-propanol and the like are preferable. Considering the solvent removal step, the organic solvent is preferably a volatile compound, and a compound having a boiling point lower than that of water is preferable. These compounds may be used alone or in combination of two or more. For example, since the resulting nano-sized W1 / O emulsion has good monodispersity, an organic solvent containing hexane as a main component (50% by volume or more), preferably an organic solvent containing 60% by volume or more of hexane. It is also preferable that the solvent is an organic solvent (O).

・ Water-soluble drugs (water-soluble substances encapsulated in liposomes)
In the present invention, the water-soluble substance (referred to as “water-soluble drug”) encapsulated in the liposome is not particularly limited, and is known in the fields of pharmaceuticals, cosmetics, foods and the like depending on the use of the liposome. Various substances can be used. However, such water-soluble drugs do not include the water-soluble lipid component described above.

Among water-soluble drugs, those used for medical liposomes include, for example, contrast agents (nonionic iodo compounds for X-ray contrast, complexes composed of gadolinium and chelating agents for MRI contrast, etc.), Anticancer drugs (adriamycin, viralrubicin, vincristine, taxol, cisplatin, mitomycin, 5-fluorouracil, irinotecan, estrasite, epirubicin, carboplatin, intron, gemzar, methotrexate, cytarabine isoborine, tegafur, cisplatin, etoposide tine , Cyclophosphamide, melphalan, ifosfamide, tespamine, nimustine, ranimustine, dacarbatin, enocitabine, fludarabine, pentostatin, cladrivi , Daunomycin, aclarubicin, ibirubicin, amrubicin, actinomycin, taxotere, trastuzumab, rituximab, gemtuzumab, lentinan, schizophyllan, interferon, interleukin, asparaginase, phosfestol, busulfan, bortezomib, alimta, bevacizumab, Agents (macrolide antibiotics, ketolide antibiotics, cephalosporin antibiotics, oxacephem antibiotics, penicillin antibiotics, beta-lactamases, aminoglycoside antibiotics, tetracycline antibiotics, fosfomycin antibiotics , Carbapenem antibiotics, penem antibiotics), MRSA / VRE / PRSP infection treatment, polyene antifungal, pyrimidine antifungal Agent, azole antifungal agent, candin antifungal agent, new quinolone synthetic antibacterial agent, antioxidant agent, anti-inflammatory agent, blood circulation promoter, whitening agent, skin roughening agent, anti-aging agent, hair growth promoting agent , Moisturizers, hormonal agents, vitamins, nucleic acids (sense or antisense strands of DNA or RNA, plasmids, vectors, mRNA, siRNA, etc.), proteins (enzymes, antibodies, peptides, etc.), vaccine preparations (toxoids such as tetanus) Antigens such as diphtheria, Japanese encephalitis, polio, rubella, mumps, hepatitis, etc. Antigens such as DNA or RNA vaccines, dyes / fluorescent dyes, chelation And pharmaceutical auxiliaries such as agents, stabilizers and preservatives.

The purpose of encapsulating the water-soluble drug is achieved by dissolving the water-soluble drug in the inner aqueous phase (W1). Accordingly, if the drug having high water solubility is dissolved in the inner aqueous phase (W1) at a high concentration, the absolute amount contained can be increased. On the other hand, the amount of the inner aqueous phase (W1) can be changed as appropriate, and the amount (number) of lipids required for it can be calculated if particles (W1 / O) having a predetermined particle size are to be prepared. For example, when a 100 nm W1 / O emulsion (particle volume: 0.0005 μm ³ ) is formed, it is calculated that 2.0 × 10 ¹⁵ W1 / O particles are produced if 1.0 mL of W1 phase (inner water phase) is produced. On the other hand, a 100 nm W1 / O nanoemulsion (particle surface area 2500 nm ² ) is calculated to be composed of 0.4 × 10 ⁵ phospholipid molecules (lecithin surface area 0.7 nm ² ). Therefore, the amount of lipid necessary for the primary emulsification of 1.0 mL of the drug solution is 2.0 × 10 ¹⁵ particles × 0.4 × 10 ⁵ particles = 0.8 × 10 ²⁰ cells, that is, 0.132 mmol. Since lipid molecules other than lecithin can be approximately 0.7 nm ^{2 in} surface area, the total amount of lipid is considered to be the minimum amount necessary to produce a W1 / O nanoemulsion with 0.132 mmol as 100 nm. To make liposomes, 0.264 mmol of that is required, and 193 mg is required in terms of molecular weight in DPPC of a typical phospholipid.

Therefore, considering the case where a drug is dissolved in 1.0 μmL and a 100 nm nm liposome is prepared, cytarabine is dissolved in an amount of 0.1 to 1.0 μg as described in the pharmacy method, so the drug weight ratio is 0.1 μg / 0.193 to 1.0 μg / 0.193. g, iohexol (contrast agent) dissolves 1.0 g or more, so the drug weight ratio is 1.0 g / 0.193 g or more. This means that the amount of lipid can be reduced and the drug can be efficiently encapsulated, and the amount of lipid can be reduced. This is clinically significant, and this method can achieve a drug weight ratio of 0.5 to 5. Furthermore, when more drug is dissolved, the viscosity generally increases as it approaches saturation. By this method, the internal water phase can be included up to 10 mPa · s.

Also, when producing particles larger than 100 nm, the required amount of lipid is smaller, which means that it is more efficient.

-Liposomes and aqueous suspensions thereof The liposomes of the present invention, typically the liposomes obtained by the production method of the present invention as described below, contain an aqueous solvent (W1) in which a water-soluble lipid component (Fw) is dissolved. A higher drug encapsulation rate can be achieved as compared with the case where an aqueous solvent in which the water-soluble lipid component is not dissolved is used as the inner aqueous phase.

The liposome of the present invention is typically obtained in a state suspended in an aqueous solvent (W2: outer aqueous phase) by the production method of the present invention as described below. Such aqueous suspensions of liposomes are used for various applications. Until they are used, they can be stored as powdered liposomes, for example, by freeze-drying. In use, the powdered liposomes may be added to an aqueous solvent and suspended again.

The size of the liposome of the present invention is not particularly limited, but can be adjusted, for example, so that the volume average particle diameter is 50 to 1,000 nm. In particular, liposomes with a volume average particle size of 50 to 200 nm have almost no risk of occluding capillaries and can pass through gaps formed in blood vessels in the vicinity of cancer tissues. This is convenient.

In the present invention, the volume average particle diameter of the liposome and the emulsion is measured by a dynamic light scattering method. For example, a dynamic light scattering nanotrack particle size analyzer (UPA-EX150, Nikkiso Co., Ltd.) Can be used to calculate. In the case of liposomes, an aqueous suspension of liposomes can be diluted 10 times with, for example, an isotonic PBS solution, and the particle size distribution and volume average particle size can be calculated using a dynamic light scattering nanotrack particle size analyzer. In the case of an emulsion, the emulsion (for example, W1 / O emulsion) is diluted 10 times with, for example, a chloroform / hexane mixed solvent, and the particle size distribution and volume average particle size are measured using a dynamic light scattering nanotrack particle size analyzer. The diameter can be calculated.

-Manufacturing method of liposome-
The liposome production method of the present invention includes at least the following steps (1) to (3), and may further include other steps as necessary.

(1) Primary emulsification step In the primary emulsification step, the water-soluble lipid component (Fw) is dissolved in the aqueous solvent (W1), and the mixed lipid component (F1) other than the water-soluble lipid (Fw) is dissolved in the organic solvent (O). It is a step of preparing a W1 / O emulsion by dissolving and mixing and emulsifying these.

The method for preparing the W1 / O emulsion is not particularly limited, and a method that is also used in the conventional method for producing liposomes can be employed. For example, an emulsification method using an apparatus such as an ultrasonic emulsifier, a stirring emulsifier, a membrane emulsifier, or a high-pressure homogenizer can be mentioned. The average particle diameter can be controlled in a wide range, and the obtained W1 / O emulsion is monodispersed. Such a method is preferable.

The pH of the aqueous solvent (W1) is usually adjusted in the range of 3 to 10, and for example, when oleic acid is used for the mixed lipid component, the pH is preferably 6 to 8.5. In order to adjust the pH, an appropriate buffer may be used.

Other conditions in the primary emulsification step, such as the amount of the mixed lipid component (F1) (ratio to the organic solvent (O)), the volume ratio of the organic solvent (O) and the aqueous solvent (W1), and the average particle size of the W1 / O emulsion The diameter and the like can be appropriately adjusted according to a known liposome production method (primary emulsification step), taking into consideration the conditions of the subsequent secondary emulsification step, the form of the liposome to be finally prepared, and the like. Usually, the amount of the mixed lipid component (F1) is 1 to 50% by mass with respect to the organic solvent (O), and the volume ratio of the organic solvent (O) to the aqueous solvent (W1) is 100: 1 to 1: 2. The volume average particle size of the W1 / O emulsion is preferably 50 to 1,000 nm, more preferably 50 to 200 nm.

In the present invention, in order to encapsulate the water-soluble drug in the liposome, (i) the water-soluble drug is added during the primary emulsification step, the water-soluble drug is dissolved in the aqueous solvent (W1), and the end of the secondary emulsification step And (ii) after obtaining a (empty) liposome that does not contain a water-soluble drug, an aqueous solvent in which the liposome is dispersed or a lyophilized product of the liposome. Any method of adding a water-soluble drug to the re-dispersed aqueous solvent and stirring it to incorporate it into the liposome can be used. In the production method of the present invention, even when the method (i) is used, the encapsulation rate is relatively high, and the water-soluble drug can be efficiently encapsulated in the liposome.

If necessary, a fat-soluble substance (fat-soluble drug) can be encapsulated in the lipid membrane of the liposome of the present invention. In that case, the fat-soluble drug is added during the primary emulsification step as in (i) above and dissolved in the organic solvent (O), or empty liposomes are obtained as in (ii) above. It may be added later.

(2) Secondary emulsification step The secondary emulsification step is a step of preparing a W1 / O / W2 emulsion by mixing and emulsifying the W1 / O emulsion obtained in the above step (1) and the aqueous solvent (W2). It is.

The method for preparing the W1 / O / W2 emulsion is not particularly limited, and a method that is also used in the conventional method for producing a W1 / O / W2 emulsion can be employed.

For example, it is preferable to use a microchannel emulsification method that does not require a large mechanical shearing force for the emulsification treatment in order to suppress the collapse of the droplets during the emulsification operation and the leakage of the inclusion substance from the droplets. In the microchannel emulsification method, for example, a microchannel emulsification device module composed of a silicon microchannel substrate and a glass plate covering the upper portion of the substrate is used. The outlet side of the groove-type microchannel formed by the substrate and the glass plate, or the outlet side of the through-type microchannel processed on the substrate is filled with an aqueous solvent (W2) that forms an outer aqueous phase, A W1 / O / W2 emulsion can be formed by press-fitting a W1 / O emulsion from the inlet side of the microchannel. As the substrate, various types of substrates such as a dead end type, a cross flow type and a through hole type can be used.

Alternatively, a membrane emulsification method can be used in which a W1 / O / W2 emulsion is prepared by passing a W1 / O emulsion through an emulsion membrane and dispersing it as droplets in an aqueous solvent (W2). In particular, a membrane emulsification method using an emulsified membrane formed of SPG (Shirasu Porous Glass) having fine pores with a diameter of about 0.1 to 5.0 μm is suitable, and the cost is low. Therefore, it can be an industrially advantageous method.

In addition, in order to improve the monodispersity of the average particle diameter of the W1 / O / W2 emulsion, after obtaining the W1 / O / W2 emulsion by membrane emulsification by the above-described method or other methods, once more or once Multiple times, the membrane treatment with the W1 / O / W2 emulsion may be performed on the same membrane as the membrane used in the membrane emulsification or a different membrane. In particular, when membrane treatment is performed using a membrane having a pore size smaller than the membrane used for membrane emulsification, when preparing a W1 / O / W2 emulsion by one membrane emulsification without membrane treatment. Compared to membrane emulsification and membrane treatment, the load on each membrane (pressure required to pass the emulsion through the membrane) can be reduced, thereby increasing the membrane life and processing time required for the secondary emulsification process. This is advantageous for improving the productivity of the liposome and reducing the cost.

Furthermore, a W1 / O / W2 emulsion can also be obtained by stirring emulsification that may cause mechanical shearing force. Also in this invention, the various methods and apparatuses used in order to mix two or more fluids in the well-known stirring emulsification method can be used. For example, there are various types of stirrers, and many of them simply rotate a bar, plate, or propeller-shaped stirrer in a tank at a constant speed in one direction. There is also a case to let you. In special circumstances, various devices such as arranging a plurality of stirrers in reverse and alternately rotating or attaching a protrusion or plate combined with a stirrer on the tank side to increase the shear stress generated by the stirrer are made. . There are various ways to transmit power to the stirrer, and most of them rotate the stirrer via a rotating shaft. However, a stirrer coated with magnet and coated with Teflon (registered trademark) is rotated from the outside of the container. There is also a magnetic stirrer that transmits power with a magnetic field. Conditions such as the size (radius) of the stirrer and the number of revolutions per minute may be adjusted within an appropriate range in consideration of the effects of the present invention and the conditions in the known stirring emulsification method.

Other conditions in the secondary emulsification step, such as the volume ratio of the W1 / O emulsion to the aqueous solvent (W2), the average particle size of the W1 / O / W2 emulsion, etc., are known liposome production methods (secondary emulsification step). According to the above, it can be adjusted as appropriate in consideration of the use of the liposome to be finally prepared.

Further, in the secondary emulsification step, the mixed lipid component (F2) and the mixed lipid component (F2) are mixed in the aqueous solvent (W2) and the W1 / O emulsion as necessary. (F1) may be added, or a water-soluble emulsifier that does not destroy the liposome lipid membrane may be added. Many emulsifiers are known in the field of surface chemistry. Typically, proteins, polysaccharides, ionic surfactants and nonionic surfactants are used as water-soluble emulsifiers in emulsification and dispersion processes. ing.

Examples of the protein include gelatin (a soluble protein obtained by denaturing collagen by heating), albumin and trypsin. Gelatin usually has a molecular weight distribution of several thousand to several million, but preferably has a weight average molecular weight of 1,000 to 100,000, for example. Gelatin commercially available for medical use or food use can be used. Albumin includes egg albumin (molecular weight about 45,000), serum albumin (molecular weight about 66,000 ... bovine serum albumin), milk albumin (molecular weight about 14,000 ... α-lactalbumin), etc. A dry desugared egg white is preferred.

Examples of the polysaccharide include dextran, starch, glycogen, agarose, pectin, chitosan, sodium carboxymethylcellulose, xanthan gum, locust bean gum, guar gum, maltotriose, amylose, pullulan, heparin, dextrin, and the like. Is preferably from 1,000 to 100,000.

Examples of the ionic surfactant include sodium cholate and sodium deoxycholate.

Examples of the nonionic surfactant include alkylglycosides such as octyl glucoside, polyalkylene oxide compounds such as “Tween 80” (Tokyo Chemical Industry Co., Ltd., polyoxyethylene sorbitan monooleate, molecular weight 1309.68) and “Pluronic F- 68 ”(BASF, polyoxyethylene (160) polyoxypropylene (30) glycol, number average molecular weight 9600), polyethylene glycol having a weight average molecular weight of 1000 to 100,000, and the like. Polyethylene glycol (PEG) products are “Unilube” (NOF Corporation), GL4-400NP, GL4-800NP (NOF Corporation), PEG200,000 (Wako Pure Chemical Industries), Macrogol (Sanyo Chemical Industries, Ltd.) ) And the like.

If the molecular weight is too small, it is likely to be mixed into the lipid membrane and inhibit the formation of liposomes. Conversely, if the molecular weight is too large, the dispersion of the W1 / O / W2 emulsion in the outer aqueous phase or the orientation to the interface may occur. The speed may be delayed, leading to liposome coalescence and multivesicular liposome formation. Therefore, the weight average molecular weight of the water-soluble emulsifier is preferably in the range of 1,000 to 100,000. Moreover, when the weight average molecular weight is in this range, the drug encapsulation rate of the liposome is good.

Various conditions when using the mixed lipid component (F2) and / or the water-soluble emulsifier as described above, for example, the amount thereof, and the mixing mode with the aqueous solvent (W2) and the W1 / O emulsion (addition order, etc.) ) Is not particularly limited, and may be appropriate according to known liposome production methods. For example, when F2 is mainly composed of a water-soluble lipid, such F2 and / or a water-soluble emulsifier can be added to W2 in advance, and a W1 / O emulsion can be added thereto for emulsification. On the other hand, when F2 is mainly composed of a fat-soluble lipid, (after preparation of the W1 / O emulsion) such F2 is added to the oil phase of the W1 / O emulsion in advance, and if necessary, a water-soluble emulsifier is added. The emulsification treatment can be performed by adding to the added W2.

(3) Solvent removal step The solvent removal step is a step of forming liposomes by removing the organic solvent phase (O) contained in the W1 / O / W2 emulsion obtained by the secondary emulsification step. That is, the organic solvent (O) contained in the W1 / O / W2 emulsion can be removed by evaporation by collecting the W1 / O / W2 emulsion, transferring it into an open container, and allowing it to stand or stir. Thereby, the lipid membrane which consists of a mixed lipid membrane component (F1) (and (F2) added as needed) is formed around an inner water phase, and a liposome can be obtained.

The removal of the solvent from the W1 / O / W2 emulsion by the method as described above may be performed using heating, decompression, and stirring as necessary according to a conventional method. Depending on the type of the compound contained in the organic solvent (O), a condition range in which the organic solvent does not bump suddenly is set. The temperature condition is preferably in the range of 0 to 60 ° C, more preferably 0 to 25 ° C. The decompression condition is preferably set within the range of the saturated vapor pressure of the solvent to atmospheric pressure, and more preferably within the range of + 1% to 10% of the saturated vapor pressure of the solvent. When different solvents are used in combination, conditions that match the solvent species having a higher saturated vapor pressure are preferred. These removal conditions may be combined within a range in which the solvent does not suddenly boil. For example, when a chemical that is weak against heat is used, it is preferable that the solvent is distilled off at a lower temperature and under reduced pressure. The solvent removal may not require stirring of the W1 / O / W2 emulsion, but the solvent removal proceeds more uniformly with stirring.

In addition, the liposome obtained by the production method as described above may contain a certain percentage of W / O / W emulsion-derived multivesicular liposomes in the production process, but in order to reduce this, stirring, It is effective to perform decompression or a combination thereof. For example, by reducing the pressure and stirring for longer than the time required for most of the solvent to escape, the hydration of the lipids that make up the liposome proceeds, and the multivesicular liposomes can be dissolved into a single-cell liposome state without causing inclusion leakage. It is possible to release. In addition, since the multivesicular liposome by-produced or remains as a by-product in this method has a structure containing many water droplets of about 50 to 200 nm derived from W / O, the pore diameter is slightly larger than the particle diameter of W / O. It is also possible to convert to single-cell liposomes of about 50 to 200 nm by passing through this filter. If there are multivesicular liposomes remaining after such operation, they can be removed by a filter.

<Calcein-encapsulating liposome>
[Example 1]
1. Primary emulsification process (preparation of W1 / O emulsion)
15 ml of hexane containing 0.3 g of egg yolk lecithin “COATSOME NC-50” (NOF Corporation) having a phosphatidylcholine content of 95%, 0.152 g of cholesterol (Chol) and 0.108 g of oleic acid (OA) ( O) and tris-hydrochloric acid buffer (pH 8, 50 mmol / L) containing calcein (0.4 mM) and M-LysoPC (lysophosphatidylcholine having a myristoyl group (lysolecithin), NOF Corporation, ClogP = 6.7) ) 5 mL was used as an aqueous dispersion phase (W1) for the inner aqueous phase. These mixed liquids were put into a 50 mL beaker, and ultrasonic waves were radiated at 25 ° C. for 15 minutes by an ultrasonic dispersion apparatus (UH-600S, SMT Co., Ltd.) with a 20 mm diameter probe set (output 5.5). The emulsification treatment was performed. When measured according to the above method, it was confirmed that the W1 / O emulsion obtained in this primary emulsification step was a monodispersed W / O emulsion having a volume average particle size of about 200 nm, and the CV value was 29%.

2. Secondary emulsification process (Preparation of W1 / O / W2 emulsion)
Subsequently, the W1 / O emulsion obtained by the primary emulsification step was used as a dispersed phase, and a W1 / O / W2 emulsion was produced by a microchannel emulsification method using an experimental dead-end type microchannel emulsifier module. It was.

The microchannel substrate of the module was made of silicon, and the terrace length, channel depth, and channel width of the microchannel substrate were about 60 μm, about 11 μm, and about 16 μm, respectively. A glass plate is pressure-bonded to the microchannel substrate to form a channel, and a tris-hydrochloric acid buffer containing 3% alkali-treated gelatin (an isoelectric point of about 5), which is an external aqueous phase solution (W2), on the outlet side of the channel The liquid (pH 8, 50 mmol / L) was filled, and the W1 / O emulsion was supplied from the inlet side of the channel to produce a W1 / O / W2 emulsion.

3. Solvent removal step (Preparation of aqueous suspension of liposome)
Next, the W1 / O / W2 emulsion was placed in a sealed sample bottle, allowed to stand at 20 ° C. for 15 minutes, then transferred to an open glass container without a lid, and stirred with a stir bar at room temperature for about 20 hours. Hexane was volatilized. A suspension of fine liposome particles was obtained, and it was confirmed that calcein was contained in the particles. The encapsulation rate of calcein was 71%.

[Example 2]
In the solvent removal step, the same procedure as in Example 1 was conducted except that the W1 / O / W2 emulsion was placed in a sealed sample bottle and allowed to stand at 20 ° C. for 360 minutes and then transferred to an open glass container without a lid. An aqueous suspension of liposomes was prepared. The encapsulation rate of calcein was 68%.

[Example 3]
In the solvent removal step, the same procedure as in Example 1 was conducted except that the W1 / O / W2 emulsion was placed in a sealed sample bottle, shaken at 20 ° C. for 360 minutes, and then transferred to an open glass container without a lid. An aqueous suspension of liposomes was prepared. The encapsulation rate of calcein was 61%.

[Comparative Example 1]
1. Primary emulsification process (preparation of W1 / O emulsion)
15 ml of hexane containing 0.3 g of egg yolk lecithin “COATSOME NC-50” (NOF Corporation) having a phosphatidylcholine content of 95%, 0.152 g of cholesterol (Chol) and 0.108 g of oleic acid (OA) ( O) and 5 mL of Tris-HCl buffer solution (pH 8, 50 mmol / L) containing calcein (0.4 mM) was used as the aqueous dispersion phase (W1) for the inner aqueous phase. These mixed liquids were put into a 50 mL beaker, and ultrasonic waves were radiated at 25 ° C. for 15 minutes by an ultrasonic dispersion apparatus (UH-600S, SMT Co., Ltd.) with a 20 mm diameter probe set (output 5.5). The emulsification treatment was performed. When measured according to the above method, it was confirmed that the W1 / O emulsion obtained in this primary emulsification step was a monodispersed W / O emulsion having a volume average particle size of about 200 nm, and the CV value was 29%.

3. Solvent removal step (Preparation of aqueous suspension of liposome)
Next, the W1 / O / W2 emulsion was placed in a sealed sample bottle, allowed to stand at 20 ° C. for 15 minutes, then transferred to an open glass container without a lid, and stirred with a stir bar at room temperature for about 20 hours. Hexane was volatilized. A suspension of fine liposome particles was obtained, and it was confirmed that calcein was contained in the particles. The encapsulation rate of calcein was 65%.

[Comparative Example 2]
In the solvent removal step, the same procedure as in Comparative Example 1 was conducted except that the W1 / O / W2 emulsion was placed in a sealed sample bottle and allowed to stand at 20 ° C. for 360 minutes and then transferred to an open glass container without a lid. An aqueous suspension of liposomes was prepared. The encapsulation rate of calcein was 48%.

[Comparative Example 3]
In the solvent removal step, the same procedure as in Comparative Example 1 was conducted except that the W1 / O / W2 emulsion was placed in a sealed sample bottle, shaken at 20 ° C. for 360 minutes, and then transferred to an open glass container without a lid. An aqueous suspension of liposomes was prepared. The encapsulation rate of calcein was 31%.

[Example 4]
Example 1 except that S-LysoPC (lysophosphatidylcholine having a stearoyl group, NOF Corporation, ClogP = 8.7) was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. Thus, an aqueous suspension of liposomes was prepared. The encapsulation rate of calcein was 73%.

[Example 5]
An aqueous liposome suspension was prepared in the same manner as in Example 2 except that S-LysoPC was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The encapsulation rate of calcein was 70%.

[Example 6]
An aqueous liposome suspension was prepared in the same manner as in Example 3 except that S-LysoPC was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The encapsulation rate of calcein was 65%.

[Example 7]
Example 1 except that DLPG-Na (dilauroylphosphatidylglycerol sodium salt, NOF Corporation, ClogP = 9.02) was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. Thus, an aqueous suspension of liposomes was prepared. The encapsulation rate of calcein was 82%.

[Example 8]
An aqueous suspension of liposomes was prepared in the same manner as in Example 2 except that DLPG-Na was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The encapsulation rate of calcein was 77%.

[Example 9]
An aqueous suspension of liposomes was prepared in the same manner as in Example 3 except that DLPG-Na was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The encapsulation rate of calcein was 71%.

[Example 10]
Example 1 except that in the primary emulsification step, short-chain phospholipid DDPC (didecanoylphosphatidylcholine, NOF Corporation, ClogP = 10) was dissolved in the inner aqueous phase (W1) instead of M-LysoPC. Thus, an aqueous suspension of liposomes was prepared. The encapsulation rate of calcein was 70%.

[Example 11]
An aqueous suspension of liposomes was prepared in the same manner as in Example 2 except that in the primary emulsification step, short-chain phospholipid DDPC was dissolved in the inner aqueous phase (W1) instead of M-LysoPC. The encapsulation rate of calcein was 70%.

[Example 12]
An aqueous suspension of liposomes was prepared in the same manner as in Example 3, except that in the primary emulsification step, short-chain phospholipid DDPC was dissolved in the inner aqueous phase (W1) instead of M-LysoPC. The encapsulation rate of calcein was 66%.

[Example 13]
In the primary emulsification step, instead of M-LysoPC, PEG phospholipid “PM020CN” (PEG phospholipid with a PEG chain (number average molecular weight of about 2000) in dimyristoylphosphatidylethanolamine, NOF Corporation, ClogP = 8) An aqueous liposome suspension was prepared in the same manner as in Example 1 except that it was dissolved in the inner aqueous phase (W1). The encapsulation rate of calcein was 81%.

[Example 14]
An aqueous suspension of liposomes was prepared in the same manner as in Example 2, except that in the primary emulsification step, PEG phospholipid “PM020CN” was dissolved in the inner aqueous phase (W1) instead of M-LysoPC. The encapsulation rate of calcein was 75%.

[Example 15]
An aqueous suspension of liposomes was prepared in the same manner as in Example 3, except that in the primary emulsification step, PEG phospholipid “PM020CN” was dissolved in the inner aqueous phase (W1) instead of M-LysoPC. The encapsulation rate of calcein was 77%.

[Example 16]
In the primary emulsification step, instead of M-LysoPC, a synthetic sterol man-3-chol prepared according to the method described in International Publication WO2008 / 081686 (see Synthesis Example 1) (a compound in which mannose is introduced at the 3-position of cholesterol, ClogP = 9.5) was dissolved in the inner aqueous phase (W1) in the same manner as in Example 1 to prepare an aqueous suspension of liposomes. The encapsulation rate of calcein was 74%.

[Example 17]
An aqueous suspension of liposomes was prepared in the same manner as in Example 2 except that man-3-chol was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The encapsulation rate of calcein was 70%.

[Example 18]
An aqueous suspension of liposomes was prepared in the same manner as in Example 3 except that man-3-chol was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The encapsulation rate of calcein was 70%.

[Comparative Example 4]
In the primary emulsification step, in the same manner as in Example 1 except that DLPE (dilauroylphosphatidylethanolamine, NOF Corporation, ClogP = 11.2) was dissolved in the inner aqueous phase (W1) instead of M-LysoPC, An aqueous suspension of liposomes was prepared. The encapsulation rate of calcein was 70%.

[Comparative Example 5]
An aqueous suspension of liposomes was prepared in the same manner as in Example 2 except that DLPE was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The encapsulation rate of calcein was 60%.

[Comparative Example 6]
An aqueous suspension of liposomes was prepared in the same manner as in Example 3, except that DLPE was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The encapsulation rate of calcein was 33%.

[Comparative Example 7]
In the primary emulsification step, the same procedure as in Example 1 was conducted except that DSPE (distearoylphosphatidylethanolamine, NOF Corporation, ClogP = 18.9) was dissolved in the inner aqueous phase (W1) instead of M-LysoPC. An aqueous suspension of liposomes was prepared. The encapsulation rate of calcein was 70%.

[Comparative Example 8]
An aqueous suspension of liposomes was prepared in the same manner as in Example 2 except that DSPE was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The encapsulation rate of calcein was 52%.

[Comparative Example 9]
An aqueous suspension of liposomes was prepared in the same manner as in Example 3 except that DSPE was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The encapsulation rate of calcein was 40%.

[Comparative Example 10]
In the primary emulsification step, in the same manner as in Example 1 except that DEPE (Diel Coil Phosphatidylethanolamine, NOF Corporation, ClogP = 24.5) was dissolved in the inner aqueous phase (W1) instead of M-LysoPC, An aqueous suspension of liposomes was prepared. The encapsulation rate of calcein was 71%.

[Comparative Example 11]
An aqueous suspension of liposomes was prepared in the same manner as in Example 2 except that DEPE was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The encapsulation rate of calcein was 51%.

[Comparative Example 12]
An aqueous suspension of liposomes was prepared in the same manner as in Example 3 except that DEPE was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The encapsulation rate of calcein was 39%.

[Comparative Example 13]
Example 1 except that DSPG-Na (distearoylphosphatidylglycerol sodium salt, NOF Corporation, ClogP = 16.7) was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. Thus, an aqueous suspension of liposomes was prepared. The encapsulation rate of calcein was 69%.

[Comparative Example 14]
An aqueous suspension of liposomes was prepared in the same manner as in Example 2 except that DSPG-Na was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The encapsulation rate of calcein was 56%.

[Comparative Example 15]
An aqueous suspension of liposomes was prepared in the same manner as in Example 3, except that DSPG-Na was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The encapsulation rate of calcein was 38%.

[Comparative Example 16]
Example 1 and Example 1 except that DEPG-Na (Dielcoyl phosphatidylethanolamine sodium salt, NOF Corporation, ClogP = 22.5) was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. Similarly, an aqueous suspension of liposomes was prepared. The encapsulation rate of calcein was 70%.

[Comparative Example 17]
An aqueous suspension of liposomes was prepared in the same manner as in Example 2 except that DEPE-Na was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The encapsulation rate of calcein was 55%.

[Comparative Example 18]
An aqueous suspension of liposomes was prepared in the same manner as in Example 3, except that DEPE-Na was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The encapsulation rate of calcein was 40%.

[Comparative Example 19]
In the primary emulsification step, in the same manner as in Example 1, except that DLPC (dilauroylphosphatidylcholine, NOF Corporation, ClogP = 12.2) was dissolved in the inner aqueous phase (W1) instead of M-LysoPC, An aqueous suspension was prepared. The encapsulation rate of calcein was 72%.

[Comparative Example 20]
An aqueous suspension of liposomes was prepared in the same manner as in Example 2 except that DLPC was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The encapsulation rate of calcein was 52%.

[Comparative Example 21]
An aqueous suspension of liposomes was prepared in the same manner as in Example 3 except that DLPC was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The encapsulation rate of calcein was 44%.

[Comparative Example 22]
In the primary emulsification step, in the same manner as in Example 1 except that DPPC (dipalmitoylphosphatidylcholine, NOF Corporation, ClogP = 17.8) was dissolved in the inner aqueous phase (W1) instead of M-LysoPC, An aqueous suspension was prepared. The encapsulation rate of calcein was 75%.

[Comparative Example 23]
An aqueous suspension of liposomes was prepared in the same manner as in Example 2 except that DPPC was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The encapsulation rate of calcein was 55%.

[Comparative Example 24]
An aqueous suspension of liposomes was prepared in the same manner as in Example 3, except that DPPC was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The encapsulation rate of calcein was 40%.

[Comparative Example 25]
In the primary emulsification step, instead of M-LysoPC, DSPE020 (PEG phospholipid in which a PEG chain (number average molecular weight of about 2000) was introduced into distearoylphosphatidylethanolamine, NOF Corporation, ClogP = 13.6) was used as the inner aqueous phase (W1 An aqueous suspension of liposomes was prepared in the same manner as in Example 1 except that it was dissolved in (1). The encapsulation rate of calcein was 80%.

[Comparative Example 26]
An aqueous suspension of liposomes was prepared in the same manner as in Example 2 except that DSPE020 was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The encapsulation rate of calcein was 60%.

[Comparative Example 27]
An aqueous suspension of liposomes was prepared in the same manner as in Example 3, except that DSPE020 was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The encapsulation rate of calcein was 38%.

[Comparative Example 28]
In the primary emulsification step, instead of M-LysoPC, a synthetic sterol man-6-chol prepared by the method described in International Publication WO2008 / 081686 (see Synthesis Example 13) (a compound in which mannose is introduced at the 6-position of cholesterol, ClogP = 11.1) was dissolved in the inner aqueous phase (W1) in the same manner as in Example 1 to prepare an aqueous suspension of liposomes. The encapsulation rate of calcein was 74%.

[Comparative Example 29]
An aqueous suspension of liposomes was prepared in the same manner as in Example 2 except that man-6-chol was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The encapsulation rate of calcein was 58%.

[Comparative Example 30]
An aqueous suspension of liposomes was prepared in the same manner as in Example 3 except that man-6-chol was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The encapsulation rate of calcein was 45%.

<Cytarabine-encapsulated liposome>
[Example 19]
1. Primary emulsification process (preparation of W1 / O emulsion)
15 ml of hexane containing 0.3 g of egg yolk lecithin “COATSOME NC-50” (NOF Corporation) having a phosphatidylcholine content of 95%, 0.152 g of cholesterol (Chol) and 0.108 g of oleic acid (OA) ( 5) Tris-hydrochloric acid buffer (pH 8, 50 mmol / L) containing 0.010 g of cytarabine (80 mM) and M-LysoPC (lysophosphatidylcholine (lysolecithin) having a myristoyl group, NOF Corporation, ClogP = 6.7) Was an aqueous dispersion phase (W1) for the inner aqueous phase. These mixed liquids were put into a 50 mL beaker, and ultrasonic waves were radiated at 25 ° C. for 15 minutes by an ultrasonic dispersion apparatus (UH-600S, SMT Co., Ltd.) with a 20 mm diameter probe set (output 5.5). The emulsification treatment was performed. When measured according to the above method, it was confirmed that the W1 / O emulsion obtained in this primary emulsification step was a monodispersed W / O emulsion having a volume average particle size of about 200 nm, and the CV value was 29%.

3. Solvent removal step (Preparation of aqueous suspension of liposome)
Next, the W1 / O / W2 emulsion was placed in a sealed sample bottle, allowed to stand at 20 ° C. for 15 minutes, then transferred to an open glass container without a lid, and stirred with a stir bar at room temperature for about 20 hours. Hexane was volatilized. A suspension of fine liposome particles was obtained, and it was confirmed that calcein was contained in the particles. The inclusion rate of cytarabine was 47%.

[Example 20]
In the solvent removal step, except that the W1 / O / W2 emulsion was placed in a sealed sample bottle and allowed to stand at 20 ° C. for 360 minutes and then transferred to an open glass container without a lid, as in Example 19, An aqueous suspension of liposomes was prepared. The inclusion rate of cytarabine was 43%.

[Example 21]
In the solvent removal step, except that the W1 / O / W2 emulsion was placed in a sealed sample bottle, shaken at 20 ° C. for 360 minutes, and then transferred to an open glass container without a lid, as in Example 19, An aqueous suspension of liposomes was prepared. The inclusion rate of cytarabine was 40%.

[Example 22]
1. Primary emulsification process (preparation of W1 / O emulsion)
15 ml of hexane containing 0.3 g of egg yolk lecithin “COATSOME NC-50” (NOF Corporation) having a phosphatidylcholine content of 95%, 0.152 g of cholesterol (Chol) and 0.108 g of oleic acid (OA) ( 5) Tris-hydrochloric acid buffer (pH 8, 50 mmol / L) containing 0.010 g of cytarabine (80 mM) and M-LysoPC (lysophosphatidylcholine (lysolecithin) having a myristoyl group, NOF Corporation, ClogP = 6.7) Was an aqueous dispersion phase (W1) for the inner aqueous phase. These mixed liquids were put into a 50 mL beaker, and ultrasonic waves were radiated at 25 ° C. for 15 minutes by an ultrasonic dispersion apparatus (UH-600S, SMT Co., Ltd.) with a 20 mm diameter probe set (output 5.5). The emulsification treatment was performed. When measured according to the above method, it was confirmed that the W1 / O emulsion obtained in this primary emulsification step was a monodispersed W / O emulsion having a volume average particle size of about 200 nm, and the CV value was 29%.

2. Secondary emulsification process (Preparation of W1 / O / W2 emulsion)
Using the obtained W1 / O emulsion as a dispersed phase, a W1 / O / W2 emulsion was produced by an SPG membrane emulsification method. A cylindrical SPG membrane having a diameter of 10 mm, a length of 20 mm, and a pore diameter of 10 μm was used for an SPG membrane emulsifying device (trade name “external pressure micro kit” manufactured by SPG Techno Co., Ltd.). A tris-hydrochloric acid buffer solution (pH 7.4, 50 mM) containing a certain 3% alkali-treated gelatin (isoelectric point about 5) is filled, and the W1 / O emulsion is supplied from the inlet side of the apparatus. W1 / O / W2 emulsion was prepared so that the ratio was 1:40.

3. Solvent removal step (Preparation of aqueous suspension of liposome)
Next, the W1 / O / W2 emulsion was placed in a sealed sample bottle, allowed to stand at 20 ° C. for 15 minutes, then transferred to an open glass container without a lid, and stirred with a stir bar at room temperature for about 20 hours. Hexane was volatilized. A suspension of fine liposome particles was obtained, and it was confirmed that calcein was contained in the particles. The inclusion rate of cytarabine was 50%.

[Example 23]
In the solvent removal step, except that the W1 / O / W2 emulsion was placed in a sealed sample bottle and allowed to stand at 20 ° C. for 360 minutes and then transferred to an open glass container without a lid, as in Example 22, An aqueous suspension of liposomes was prepared. The inclusion rate of cytarabine was 46%.

[Example 24]
In the solvent removal step, except that the W1 / O / W2 emulsion was put in a sealed sample bottle and shaken at 20 ° C. for 360 minutes and then transferred to an open glass container without a lid, as in Example 22, An aqueous suspension of liposomes was prepared. The inclusion rate of cytarabine was 40%.

[Example 25]
1. Primary emulsification process (preparation of W1 / O emulsion)
15 ml of hexane containing 0.3 g of egg yolk lecithin “COATSOME NC-50” (NOF Corporation) having a phosphatidylcholine content of 95%, 0.152 g of cholesterol (Chol) and 0.108 g of oleic acid (OA) ( 5) Tris-hydrochloric acid buffer (pH 8, 50 mmol / L) containing 0.010 g of cytarabine (80 mM) and M-LysoPC (lysophosphatidylcholine (lysolecithin) having a myristoyl group, NOF Corporation, ClogP = 6.7) Was an aqueous dispersion phase (W1) for the inner aqueous phase. These mixed liquids were put into a 50 mL beaker, and ultrasonic waves were radiated at 25 ° C. for 15 minutes by an ultrasonic dispersion apparatus (UH-600S, SMT Co., Ltd.) with a 20 mm diameter probe set (output 5.5). The emulsification treatment was performed. When measured according to the above method, it was confirmed that the W1 / O emulsion obtained in this primary emulsification step was a monodispersed W / O emulsion having a volume average particle size of about 200 nm, and the CV value was 29%.

2. Secondary emulsification process (Preparation of W1 / O / W2 emulsion)
The obtained W1 / O emulsion was used as a dispersed phase, and an aqueous dispersed phase (W2) of Tris-HCl buffer (pH 7.4, 50 mM) containing 3% alkali-treated gelatin (isoelectric point about 5) was used as an outer aqueous phase. As described above, a W1 / O / W2 emulsion was produced by a stirring emulsification method. In the stirring emulsification, the W1 / O emulsion was supplied to a place where W2 was vigorously stirred with a stirrer, and a W1 / O / W2 emulsion was produced so that W1: W2 was 1:40.

[Example 26]
In the solvent removal step, except that the W1 / O / W2 emulsion was placed in a sealed sample bottle and allowed to stand at 20 ° C. for 360 minutes and then transferred to an open glass container without a lid, as in Example 25. An aqueous suspension of liposomes was prepared. The inclusion rate of cytarabine was 43%.

[Example 27]
In the solvent removal step, except that the W1 / O / W2 emulsion was placed in a sealed sample bottle, shaken at 20 ° C. for 360 minutes, and then transferred to an open glass container without a lid, as in Example 25, An aqueous suspension of liposomes was prepared. The inclusion rate of cytarabine was 39%.

[Example 28]
An aqueous suspension of liposomes was prepared in the same manner as in Example 22 except that DLPG-Na was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The inclusion rate of cytarabine was 49%.

[Example 29]
An aqueous suspension of liposomes was prepared in the same manner as in Example 23 except that DLPG-Na was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The inclusion rate of cytarabine was 48%.

[Example 30]
An aqueous suspension of liposomes was prepared in the same manner as in Example 24 except that DLPG-Na was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The inclusion rate of cytarabine was 45%.

[Example 31]
An aqueous suspension of liposomes was prepared in the same manner as in Example 25 except that PM020CN was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The inclusion rate of cytarabine was 53%.

[Example 32]
An aqueous suspension of liposomes was prepared in the same manner as in Example 26 except that PM020CN was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The inclusion rate of cytarabine was 51%.

[Example 33]
An aqueous suspension of liposomes was prepared in the same manner as in Example 27 except that PM020CN was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The inclusion rate of cytarabine was 53%.

[Example 34]
PM020CN was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step, and 0.1% instead of 3% alkali-treated gelatin (isoelectric point about 5) in the secondary emulsification step. Tris-HCl buffer containing Pluronic F-68 (polyoxyethylene (160) polyoxypropylene (30) glycol, number average molecular weight 9600, registered trademark, nonionic surfactant Pronon (# 188P, NOF Corporation)) An aqueous liposome suspension was prepared in the same manner as in Example 19 except that the liquid (pH 8, 50 mmol / L) was used. The inclusion rate of cytarabine was 60%.

[Example 35]
PM020CN was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step, and 0.1% instead of 3% alkali-treated gelatin (isoelectric point about 5) in the secondary emulsification step. An aqueous liposome suspension was prepared in the same manner as in Example 20 except that Tris-HCl buffer (pH 8, 50 mmol / L) containing Pluronic F-68 was used. The inclusion rate of cytarabine was 60%.

[Example 36]
PM020CN was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step, and 0.1% instead of 3% alkali-treated gelatin (isoelectric point about 5) in the secondary emulsification step. An aqueous liposome suspension was prepared in the same manner as in Example 21, except that Tris-HCl buffer (pH 8, 50 mmol / L) containing Pluronic F-68 was used. The inclusion rate of cytarabine was 52%.

[Comparative Example 31]
An aqueous liposome suspension was prepared in the same manner as in Example 19 except that M-LysoPC was not dissolved in the inner aqueous phase (W1) in the primary emulsification step. The inclusion rate of cytarabine was 40%.

[Comparative Example 32]
An aqueous liposome suspension was prepared in the same manner as in Example 20, except that M-LysoPC was not dissolved in the inner aqueous phase (W1) in the primary emulsification step. The inclusion rate of cytarabine was 32%.

[Comparative Example 33]
An aqueous liposome suspension was prepared in the same manner as in Example 21, except that M-LysoPC was not dissolved in the inner aqueous phase (W1) in the primary emulsification step. The inclusion rate of cytarabine was 12%.

[Comparative Example 34]
An aqueous liposome suspension was prepared in the same manner as in Example 22 except that M-LysoPC was not dissolved in the inner aqueous phase (W1) in the primary emulsification step. The inclusion rate of cytarabine was 43%.

[Comparative Example 35]
An aqueous liposome suspension was prepared in the same manner as in Example 23 except that M-LysoPC was not dissolved in the inner aqueous phase (W1) in the primary emulsification step. The inclusion rate of cytarabine was 30%.

[Comparative Example 36]
An aqueous liposome suspension was prepared in the same manner as in Example 24 except that M-LysoPC was not dissolved in the inner aqueous phase (W1) in the primary emulsification step. The inclusion rate of cytarabine was 20%.

[Comparative Example 37]
An aqueous liposome suspension was prepared in the same manner as in Example 25 except that M-LysoPC was not dissolved in the inner aqueous phase (W1) in the primary emulsification step. The inclusion rate of cytarabine was 43%.

[Comparative Example 38]
An aqueous liposome suspension was prepared in the same manner as in Example 26 except that M-LysoPC was not dissolved in the inner aqueous phase (W1) in the primary emulsification step. The inclusion rate of cytarabine was 34%.

[Comparative Example 39]
An aqueous liposome suspension was prepared in the same manner as in Example 27, except that M-LysoPC was not dissolved in the inner aqueous phase (W1) in the primary emulsification step. The inclusion rate of cytarabine was 23%.

[Comparative Example 40]
An aqueous suspension of liposomes was prepared in the same manner as in Example 22 except that DSPG-Na was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The inclusion rate of cytarabine was 49%.

[Comparative Example 41]
An aqueous suspension of liposomes was prepared in the same manner as in Example 23 except that DSPG-Na was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The inclusion rate of cytarabine was 39%.

[Comparative Example 42]
An aqueous suspension of liposomes was prepared in the same manner as in Example 24 except that DSPG-Na was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The inclusion rate of cytarabine was 26%.

[Comparative Example 43]
An aqueous suspension of liposomes was prepared in the same manner as in Example 25 except that DSPE020 was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The inclusion rate of cytarabine was 51%.

[Comparative Example 44]
An aqueous suspension of liposomes was prepared in the same manner as in Example 26 except that DSPE020 was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The inclusion rate of cytarabine was 40%.

[Comparative Example 45]
An aqueous suspension of liposomes was prepared in the same manner as in Example 27 except that DSPE020 was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step. The inclusion rate of cytarabine was 32%.

[Comparative Example 46]
M-LysoPC was not dissolved in the inner aqueous phase (W1) in the primary emulsification step, and 0.1% Pluronic F was used in the secondary emulsification step instead of 3% alkali-treated gelatin (isoelectric point about 5). An aqueous liposome suspension was prepared in the same manner as in Example 19 except that Tris-HCl buffer solution (pH 8, 50 mmol / L) containing -68 was used. The inclusion rate of cytarabine was 44%.

[Comparative Example 47]
M-LysoPC was not dissolved in the inner aqueous phase (W1) in the primary emulsification step, and 0.1% Pluronic F was used in the secondary emulsification step instead of 3% alkali-treated gelatin (isoelectric point about 5). An aqueous liposome suspension was prepared in the same manner as in Example 20, except that Tris-HCl buffer (pH 8, 50 mmol / L) containing -68 was used. The inclusion rate of cytarabine was 30%.

[Comparative Example 48]
M-LysoPC was not dissolved in the inner aqueous phase (W1) in the primary emulsification step, and 0.1% Pluronic F was used in the secondary emulsification step instead of 3% alkali-treated gelatin (isoelectric point about 5). An aqueous liposome suspension was prepared in the same manner as in Example 21, except that Tris-HCl buffer (pH 8, 50 mmol / L) containing -68 was used. The inclusion rate of cytarabine was 22%.

[Comparative Example 49]
DSPE020 was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step, and 0.1% instead of 3% alkali-treated gelatin (isoelectric point about 5) in the secondary emulsification step. An aqueous liposome suspension was prepared in the same manner as in Example 19, except that Tris-HCl buffer (pH 8, 50 mmol / L) containing Pluronic F-68 was used. The inclusion rate of cytarabine was 58%.

[Comparative Example 50]
DSPE020 was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step, and 0.1% instead of 3% alkali-treated gelatin (isoelectric point about 5) in the secondary emulsification step. An aqueous liposome suspension was prepared in the same manner as in Example 20 except that Tris-HCl buffer (pH 8, 50 mmol / L) containing Pluronic F-68 was used. The inclusion rate of cytarabine was 44%.

[Comparative Example 51]
PM020CN was dissolved in the inner aqueous phase (W1) instead of M-LysoPC in the primary emulsification step, and 0.1% instead of 3% alkali-treated gelatin (isoelectric point about 5) in the secondary emulsification step. An aqueous liposome suspension was prepared in the same manner as in Example 21, except that Tris-HCl buffer (pH 8, 50 mmol / L) containing Pluronic F-68 was used. The inclusion rate of cytarabine was 32%.

<Contrast-encapsulated liposome>
[Example 37]
1. Primary emulsification process (preparation of W1 / O emulsion)
Omnipark 350 (iohexol content: 750 mg / mL, 10 mPa · s) dissolved in 0.25 mL of M-LysoPC (lysophosphatidylcholine (lysolecithin) having a myristoyl group, NOF Corporation, ClogP = 6.7) 0.010 g Mixed solvent containing 50 mg of DPPC (dipalmitoylphosphatidylcholine, “MC-6060”, NOF Corporation) and 10 mg of DPPG (dipalmitoylphosphatidylglycerol, “COATSOME MG-6060LA”, NOF Corporation) as phase (W1) 1.25 mL of (hexane / dichloromethane = 3/1) was used as the organic solvent phase (O). These mixed liquids are put into a 3.5 mL sample bottle and subjected to an emulsification treatment by irradiating ultrasonic waves at 20 ° C. for 15 minutes by an ultrasonic dispersion apparatus (UH-600S, SMT Co., Ltd.) equipped with a φ7 mm probe. It was.

2. Secondary emulsification process (Preparation of W1 / O / W2 emulsion)
The resulting W1 / O emulsion was added to 15 mL of Tris-HCl buffer (W2, pH 7.4, 50 mM) containing 0.1% Pluronic F68 under stirring with a stirrer, and stirred for 15 minutes. An O / W2 emulsion was prepared.

3. Solvent removal step (Preparation of aqueous suspension of liposome)
The obtained W1 / O / W2 emulsion was transferred to a closed container and stirred for about 8 hours under a reduced pressure condition of 20 ° C./500 mbar, and then stirred for about 8 hours under a reduced pressure condition of 20 ° C./180 mbar. The solvent was volatilized. The obtained liposome suspension was translucent yellow, and it was confirmed that the contrast agent iohexol was contained in the particles. The iohexol encapsulation rate of the liposome was 48%.

[Comparative Example 52]
An aqueous liposome suspension was prepared in the same manner as in Example 37 except that M-LysoPC was not dissolved in the inner aqueous phase (W1) in the primary emulsification step. The encapsulation rate of iohexol was 39%.

<SiRNA-encapsulated liposome>
[Example 38]
As an inner aqueous phase (W1), 10 mg of siRNA (random sequence) and M-LysoPC (lysophosphatidylcholine having a myristoyl group (lysolecithin), 0.010 g of NOF Corporation, ClogP = 6.7) are added to 0.25 mL of isotonic PBS solution. An aqueous liposome suspension was prepared in the same manner as in Example 37, except that the dissolved solution was used and an isotonic PBS solution containing 0.1% Pluronic F68 was used as the outer aqueous phase (W2). Prepared. The inclusion rate of siRNA was 57%.

[Comparative Example 53]
An aqueous liposome suspension was prepared in the same manner as in Example 38, except that M-LysoPC was not dissolved in the inner aqueous phase (W1) in the primary emulsification step. The encapsulation rate of siRNA was 48%.

[Example 39] (Scale up 20 times with SPG secondary emulsification)
(Production of W1 / O emulsion by primary emulsification process)
A mixed solvent (hexane: dichloromethane) containing 6.0 g of egg yolk lecithin “COATSOME NC-50” (manufactured by NOF Corporation) having a phosphatidylcholine content of 95%, 3.04 g of cholesterol (Chol) and 2.16 g of oleic acid (OA) = 8: 2) 300 mL as an organic solvent phase (O), cytarabine (MW 243.22, 20 mg / mL, 80 mM) and M-LysoPC (lysophosphatidylcholine having a myristoyl group (lysolecithin), NOF Corporation, ClogP = 6.7 ) 100 mL of Tris-HCl buffer solution (pH 7.4, 50 mmol / L) containing 0.20 g was used as an aqueous dispersion phase (W1) for the inner aqueous phase. The primary emulsification process was performed under a pressure condition of 100 MPa using a high-pressure homogenizer (Nanomizer, manufactured by Yoshida Kikai Kogyo Co., Ltd.). The average particle size of the obtained W1 / O emulsion at room temperature was 137 nm.

(Production of W1 / O / W2 emulsion by secondary emulsification process)
Using the W1 / O emulsion obtained by the primary emulsification step as a dispersed phase, a W1 / O / W2 emulsion was produced by the SPG emulsification method. A cylindrical SPG membrane having a diameter of 10 mm, a length of 125 mm, and a pore diameter of 1.0 μm was used for an SPG membrane emulsifier (trade name “High Speed Mini Kit KH-125” manufactured by SPG Techno Co.), and an external aqueous phase solution ( Filled with Tris-hydrochloric acid buffer solution (pH 7.4, 50 mmol / L) containing 3% alkali-treated gelatin (isoelectric point about 5), which is W2), and the above W1 / O emulsion was supplied from the apparatus inlet side. Thus, a W1 / O / W2 emulsion was produced. The pressure required for membrane emulsification was about 10 kPa.

(Production of liposomes by removal of organic solvent phase)
The W1 / O / W2 emulsion obtained by the secondary emulsification step was transferred to a closed container and stirred for about 4 hours under a reduced pressure room temperature condition of 500 mbar, and then stirred for about 18 hours under a reduced pressure room temperature condition of 180 mbar. Was volatilized. A suspension of fine liposome particles was obtained, and it was confirmed that cytarabine was contained in the particles. The resulting liposome had a cytarabine encapsulation rate of 47%.

(Discussion) SPG membrane emulsification took 90 minutes with increasing processing amount. Thereafter, the organic solvent was removed after standing at 20 ° C. for 270 minutes, so that the same standing as in Example 23 was carried out. In the scale-up experiment, the same cytarabine encapsulation rate as in Example 23 could be obtained.

[Example 40] (Scale up by 20 times by stirring secondary emulsification)
In the production of a W1 / O / W2 emulsion by the secondary emulsification step, a Tris-HCl buffer solution (pH 7.4, 50 mmol) containing 3% alkali-treated gelatin (isoelectric point about 5) as an outer aqueous phase solution (W2). / L) was used in the same manner as in Example 39, except that a W1 / O / W2 emulsion was produced by a stirring emulsification method. In the stirring emulsification, the W1 / O emulsion was supplied to a place where W2 was vigorously stirred with a stirrer, and a W1 / O / W2 emulsion was produced so that W1: W2 was 1:40. The resulting liposome had a cytarabine encapsulation rate of 47%.

(Discussion) The stirring time was set to 30 minutes with the increase of the processing amount. Thereafter, the organic solvent was removed after standing at 20 ° C. for 330 minutes, so that the same standing as in Example 26 was carried out. In the scale-up experiment, the same cytarabine encapsulation rate as in Example 26 could be obtained.

(Discussion 2) A liposome production method by stirring secondary emulsification is a microencapsulation method (for example, Ishii et al., J. Dispers. Sci. Technol .. vol.9, No.1, pp.1-15, 1988. However, depending on the drug to be included, a high encapsulation rate cannot be realized. It is considered that the soft W / O / W particles are broken by the shearing force and the encapsulated drug comes out, which may result in an encapsulation rate of several percent. In order to maintain the stirring efficiency on a large scale, it is necessary to stir with a large torque, and this tendency becomes remarkable because the shearing force locally increases near the stirring blade. However, the effect of strengthening the membrane by the addition of water-soluble lipids appears, and it is considered that reproducibility was obtained even on a large scale.

(Filter filtration)
When the liposome suspensions obtained in Examples 31, 32 and 33 and Comparative Examples 43, 44 and 45 were observed with a microscope, the presence of multivesicular liposomes was confirmed, and microparticles were also confirmed in the particle size distribution measurement results. By passing the obtained liposome suspension through a membrane filter DISMIC (0.2 μm cellulose acetate, Advantech Toyo) at 20 ° C., it can be confirmed from microscopic observation and particle size distribution measurement that multivesicular liposomes have disappeared, Moreover, it was confirmed that there was no change in the encapsulation rate before and after membrane filter filtration. In other examples and comparative examples, multivesicular liposomes were not observed.

(Measurement method of calcein inclusion rate)
The fluorescence intensity (Ftotal) of the entire liposome aqueous solution (3 mL) was measured with a spectrophotometer (U-3310, JASCO Corporation). Next, the fluorescence intensity (Fin) in the vesicle was measured by adding 30 μL of 0.01 M CoCl ₂ Tris-HCl buffer and quenching the fluorescence of calcein leaked into the outer aqueous phase with Co ²⁺ . Furthermore, vesicles were prepared under the same conditions as the sample without adding calcein, and the fluorescence (Fl) emitted by the lipids themselves was measured. The inclusion rate was calculated from the following formula;
Inclusion rate E (%) = (Fin−Fl) / (Ftotal−Fl) × 100
(Measuring method of particle size distribution)
The volume average particle diameters and CV values of the W1 / O emulsions obtained in the primary emulsification step of Examples and Comparative Examples were measured according to the following methods.

The W1 / O emulsion is diluted 10 times with a hexane / dichloromethane mixed solvent (volume ratio: 1/1), and the particle size distribution is measured using a dynamic light scattering nanotrack particle size analyzer (UPA-EX150, Nikkiso Co., Ltd.). The volume average particle diameter and the CV value (= (standard deviation / volume average particle diameter) × 100 [%]) were calculated based on the measurement. Moreover, the volume average particle diameter of the liposome was measured as it was for the prepared liposome suspension using the same apparatus.

(Method for measuring the inclusion rate of cytarabine)
Components of the suspension of liposome particles were separated using an ultracentrifuge under ultracentrifugation conditions. The amount of cytarabine contained in the solid content (liposome) and the supernatant solution was determined by HPLC (column: VarianPolaris C18-A (3 μm , 2 × 40 mm)). The amount of cytarabine encapsulated in the liposome, ie, the amount of cytarabine encapsulated in the liposome, and the amount of cytarabine not encapsulated in the liposome, The value obtained by dividing the amount of cytarabine is multiplied by 100 to calculate the inclusion rate (%) of cytarabine.

(Method of measuring the encapsulation rate of iohexol)
50 μL of a contrast agent compound (iohexol) -encapsulating liposome dispersion was collected, 950 μL of 1.8% physiological saline was added, and centrifugation (6,000 rpm, 20 minutes) was performed. The obtained supernatant and residue (liposomes) were completely separated, and then each was dissolved by adding alcohol to make 20 mL. The absorbance at a wavelength of about 245 nm is measured, and based on a calibration curve of the absorbance of the encapsulated contrast agent compound and the concentration of the contrast agent compound, the mass of the contrast agent compound encapsulated in the liposomes and the total contrast agent compound in the system The mass was calculated, and the encapsulation rate of the contrast agent compound was determined by the following formula. Here, the mass of the contrast agent compound encapsulated in the liposome is the mass of the contrast agent compound measured from the residue, and the total mass of the contrast agent compound is the sum of the contrast agent compound mass measured from the supernatant and the residue.

Inclusion rate of contrast agent compound (%)
= Compound mass for liposome-encapsulated contrast agent / Total mass of compound for contrast agent × 100
(Method for measuring the inclusion rate of siRNA)
The encapsulation rate of siRNA was determined by ultracentrifuging the liposome solution to separate the external solution and the liposome, and measuring the amount of each siRNA by HPLC.

(ClogP calculation method)
The partition coefficient can be obtained by calculation using a quantitative structure-activity relationship algorithm without actually measuring. In the fragment method, the distribution coefficient of a molecule is calculated by the sum of the distribution coefficients for each partial structure of the molecule. The ClogP value of the fragment was calculated with a calculation program installed in the software ChemDraw, and the value was calculated by adding the value according to the fragment-based ClogP value calculation developed by BioByte. For example, the structural formula of M-LysoPC can be divided into two fragments, and the ClogP of each is calculated to be 5.18 and 1.42. Therefore, the ClogP value of M-LysoPC was set to 6.7 by adding them.

Claims

Single cell liposome characterized in that an aqueous solvent (W1) in which a water-soluble lipid component (Fw) comprising a lipid having a ClogP value of less than 11 is dissolved is used as an inner aqueous phase.
The water-soluble lipid component (Fw) contains at least one selected from the group consisting of lysolipids, short-chain phospholipids, PEG lipids, and chemically synthesized lipids having a hydrophilic group. Single-vesicle liposomes.
The single cell liposome according to claim 1 or 2, wherein a water-soluble drug is further dissolved in the aqueous solvent (W1).
The dry powder of single vesicle liposome according to any one of claims 1 to 3.
An aqueous suspension containing the single cell liposome according to any one of claims 1 to 3 or obtained by adding the dry powder according to claim 4 to an aqueous solvent.
A method for producing a single cell liposome, comprising the following steps (1) to (3):
(1) The water-soluble lipid component (Fw) is dissolved in the aqueous solvent (W1), the mixed lipid component (F1) other than the water-soluble lipid (Fw) is dissolved in the organic solvent (O), and these are mixed and emulsified. A primary emulsification step to prepare a W1 / O emulsion by
(2) A secondary emulsification step of preparing a W1 / O / W2 emulsion by mixing and emulsifying the W1 / O emulsion obtained through the step (1) and the aqueous solvent (W2);
(3) A solvent removal step of preparing an aqueous suspension of liposomes by removing the organic solvent contained in the W1 / O / W2 emulsion obtained through the step (2).
The water-soluble lipid component (Fw) contains at least one selected from the group consisting of a lysolipid, a short-chain phospholipid, a PEG lipid, and a chemically synthesized lipid having a hydrophilic group. Manufacturing method.
The production method according to claim 7, wherein the primary emulsification step (1) is performed by further adding a water-soluble drug.
The production method according to claim 7 or 8, wherein the solvent removal step (3) is performed under stirring of a W1 / O / W2 emulsion.
A single vesicle liposome produced by the production method according to any one of claims 7 to 9, an aqueous suspension thereof, or a dry powder thereof.