JP3443939B2 - Lipid endoplasmic reticulum and sustained-release preparations - Google Patents

Description

Detailed Description of the Invention

[0001]

The present invention relates to lipid vesicles using <br/> lipid vesicles forming material, mainly Liposomes - lipid vesicles for arm, the drug was contained in the inner aqueous phase particularly slowly The present invention relates to a lipid vesicle as a sustained release preparation that can be released.

[0002]

2. Description of the Related Art Sustained-release preparations are intended to reduce the side effects of drugs having severe side effects and to maintain the effects of drugs having a short residence time in blood over a long period of time. Conventionally, in order to encapsulate a drug and enhance its effectiveness, microcapsules [new edition "microcapsules-the manufacturing method / property / application-", Sankyo Publishing Co., Ltd., Nos. 42-6
Encapsulation using a synthetic polymer as a membrane material, as represented by page 3], has been performed, but due to the difficulty of controlling the particle size of the capsule,
It has problems such as biodegradability and controlled release, and is not suitable for medical use.

Further, JP-A-60-215622 and JP-A-6-215622.
As disclosed in gazettes such as 3-22516,
Sustained-release preparations in which a drug is contained in a polymer such as polylactic acid are known as drug carriers that are excellent in controlling dispersion time and sustained-release properties, but these are difficult to microparticulate and do not undergo modification. There is a problem in that it retains a water-soluble drug, and it is still not suitable for medical use.

Recently, it has been found that lipid vesicles composed of natural phospholipids are effective for encapsulating drugs and the like. For example, JP-A-64-9931, NOF Corporation and NOF Liposo- "NEW LIP" issued by Mu Co., Ltd.
IDS "vol. 1 (1990), JP-A-2-300
Many reports have been made in Japanese Patent Laid-Open No. 120, Japanese Patent Laid-Open No. 3-163031, and the like.

[0005] These lipid vesicles are called, as vesicles constituting the liposomes, phospholipid sequence molecule interface hydrophilic group an inner aqueous phase (phospholipid polar group) is located,
By forming a bilayer membrane with a phospholipid-arranged molecule in which a hydrophilic group (phospholipid polar group) is located at the interface with the outer aqueous phase, the water-soluble drug in the inner aqueous phase taken up inside this membrane is formed. The drug is gradually released to the outside through this film so that the drug effect is continuously exerted.

[0006] However, lipid vesicles composed of natural phospholipids have poor storage stability during freezing and long-term storage, and are not stable in the body. There is a problem that it is difficult to exert performance,
In addition, the drug's efficacy disappeared within a short period of time, and it was not sufficiently satisfactory in terms of sustained release.

On the other hand, JP-A-60-67489 and 62-62
-205092, 63-54384, 63-5
Publications such as 4385 propose that a phospholipid having a polymerizable group introduced therein is used to carry out a polymerization treatment after the formation of lipid vesicles. In this case, since the strength of the film is increased by the polymerization treatment, the storage stability can be expected to be improved, but the drug effect is unlikely to be exhibited to the contrary, and the sustained release property is still unsatisfactory. There is also a problem that the biodegradability deteriorates and the endoplasmic reticulum accumulates in the body.

[0008] Further, in Japanese Patent Laid-Open No. 62-104844, a lipid vesicle is formed from a mixture of a polymerizable group-introduced phospholipid and a non-polymerizable phospholipid in the hope of improving the sustained release property. After that, it has been proposed to remove non-polymerizable phospholipids to form lipid vesicles with small pores, but the removal operation for forming small pores is troublesome, and it is not practical. Japanese Unexamined Patent Publication No. 4-202123 proposes to use degradable peptide lipid vesicles. However, in addition to requiring a complicated polymerization operation, there are toxicity problems and antigenicity due to the use of heterologous peptides in vivo. Inevitable problems such as.

[0009]

Thus, at present, as a practical sustained-release preparation, it has excellent stability such as storage stability and excellent biodegradability, and accumulates in the body. There is almost no such drug that has excellent drug release and sustained release.

In view of the above circumstances, the present invention provides a lipid vesicle composed of a phospholipid, which is excellent in stability such as storage stability and biodegradability, and which is used as a liposomal vesicle for liposomes. With the aim of providing a lipid vesicle which is excellent in sustained release of a substance contained in an aqueous phase, particularly when the above substance is a drug, which is suitable as a sustained release preparation having a long-lasting medicinal effect. There is.

[0011]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies on the above-mentioned object, and as a result, as a lipid vesicle-forming material, a non-polymerizable phospholipid and a specific polymerizable phospholipid in a specific ratio were used. By using a mixture, it is excellent in stability and biodegradability, and also as a lipid vesicle for liposome, excellent in sustained release of the substance contained in the inner aqueous phase,
In particular, when the above substance was a drug, it was found that a lipid vesicle suitable for a sustained-release preparation having excellent sustained efficacy can be obtained, and the present invention was completed.

That is, the present invention comprises A) a non-polymerizable phospholipid and B) the following formula (1); (In the formula, ^one of R ¹ and R ² is a polymerizable acyl group, the other is a non-polymerizable acyl group, and X is a phospholipid polar group). A lipid vesicle-forming material containing a lipid, wherein the ratio of the polymerizable phospholipid of the component B to 0.1 mol of the non-polymerizable phospholipid of the component A is 0.1 to 10 mol. The present invention relates to lipid vesicles for liposomes and the like formed by a conventional method, particularly to lipid vesicles obtained by polymerizing the above-mentioned polymerizable phospholipid of component B after membrane formation.

[0013]

[Constitution and Action of the Invention] As the non-polymerizable phospholipid of the component A in the present invention, natural or synthetic phospholipids can be widely used. Specifically, common phospholipids such as egg yolk lecithin, soybean lecithin, hydrogenated egg yolk lecithin, hydrogenated soybean lecithin, and natural or synthetic phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine. , Phosphatidylinositol, phosphatidyglycerol, and the like, and one or more of them are used.

The component B polymerizable phospholipid in the present invention is a phosphatidyl ester represented by the formula (1), wherein X is a phosphorylcholine group, a phosphoryldiethanolamine group, a phosphorylserine group, ^One of R ¹ and R ² is a polymerizable acyl group and the other is a non-polymerizable acyl group, and a phospholipid polar group such as a phosphorylinositol group and a phosphorylglycerol group.

Those in which both R ¹ and R ² are non-polymerizable acyl groups are included in the non-polymerizable phospholipid of the component A.
Further, a compound in which both R ¹ and R ² are a polymerizable acyl group does not exhibit the effects of the present invention even when used in combination with the component A. That is, in the present invention, there is a great feature in mixing and using the polymerizable phospholipid having the above-mentioned specific composition and the non-polymerizable phospholipid of the component A.

The polymerizable acyl group constituting R ¹ and R ² is a residue of a polymerizable fatty acid such as a fatty acid having a conjugated double bond, a fatty acid having a triple bond and a fatty acid having a terminal double bond, For example, 2,4-octadecadienoic acid, 8,10,12-octadecatrienoic acid, 9- (p
-Vinylbenzoyl) nonanilic acid, 12-methacryloyl oxide decanoic acid, 10-undecic acid, 16-heptadecic acid, 22-tricosic acid, Tricosa-10,1
2-diynoic acid, tricosa-2,4-diynoic acid, nonadeca-2,4-diynoic acid, 11,13-tetradecadienoic acid, 11- (2,4-hexadienyloxy) undecanoic acid, etc. Mention may be made of residues of fatty acids.

The non-polymerizable acyl group which constitutes R ¹ and R ² is a residue of a non-polymerizable fatty acid such as a natural or synthetic saturated fatty acid or unsaturated fatty acid, for example, capric acid,
Examples thereof include residues of various fatty acids such as lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, elaidic acid, linoleic acid, linolenic acid, and eleostearic acid.

The polymerizable phospholipid as the component B is obtained by, for example, hydrolyzing a natural or synthetic phosphatidyl ester such as lecithin with phospholipase A to give a lysophosphatidyl ester such as lysolecithin. It can be synthesized by a method such as subjecting the polymerizable fatty acid to an esterification reaction.

In the present invention, one or more of the polymerizable phospholipids as the component B are used, and the amount used is 0.1 to 10 mol per 1 mol of the component A. It should be 0.1 to 8 moles.
If it is less than 0.1 mol, the storage stability and the stability in the body will be poor, and in the lipid vesicles for liposomes etc., the substance contained in the inner aqueous phase, for example, the drug, will be rapidly released, Good sustained release cannot be obtained. When the amount is more than 10 moles, the dense high molecular weight polymer domain of the polymerizable phospholipid formed in the membrane impairs the biodegradability and easily accumulates in the body, and the lipid is small for liposomes. In the endoplasmic reticulum, the release of substances, such as drugs, contained in the inner aqueous phase is impaired, and good sustained release cannot be obtained.

The lipid vesicle-forming material of the present invention contains the non-polymerizable phospholipid as the component A and the polymerizable phospholipid as the component B in the above proportions, and also as a constituent component other than the phospholipid, a known membrane stabilizing agent. Agents such as cholesterol and fatty acids can be included as necessary. The amount of cholesterol used is usually 1.5 per 1 mol of phospholipids of both A and B components.
It is preferable to use up to moles, and the amount of fatty acid used is A,
It is usually preferable to add up to 0.5 mol per 1 mol of the phospholipids of both components B. Further, as the other component, a known membrane-affinity amphipathic substance such as a surfactant can be used if necessary, as long as the effects of the present invention are not impaired.

In the present invention, the lipid vesicle-forming material described above is used to obtain a desired lipid vesicle depending on the usage form such as a liposome comprising a W / O / W emulsion and an O / W emulsion. Form the endoplasmic reticulum. The forming method is arbitrary and may be any known method. For example,
For lipid vesicles for liposomes, as described in detail in "Liposome", Nankodo Publishing (1988), thin film method, ultrasonic treatment method, ethanol injection method, French press method, extrusion method. It can be formed by a dialysis method, a freeze-thaw method, a reverse phase evaporation method, or the like, and it is particularly preferable that the particle diameter is formed to about 0.2 μm by an extrusion method.

After forming the membrane as a lipid vesicle in this way, by polymerizing the polymerizable phospholipid of the component B, and at that time by adjusting the degree of polymerization and the rate of polymerization, the storage stability can be improved. In terms of stability and sustained release of drugs, etc., the characteristics as designed can be obtained. Polymerization method is not particularly limited, initiator polymerization, ultraviolet polymerization, γ-ray polymerization,
Known methods such as X-ray polymerization and electron beam polymerization can be freely selected, and these may be appropriately combined. From the point that purification is not required after polymerization, UV polymerization, γ-ray polymerization, X
Line polymerization, electron beam polymerization and the like are preferable.

When forming lipid vesicles for liposomes, a water-soluble sustained-release substance can be included in the inner aqueous phase. Particularly when a sustained release preparation is to be obtained, a drug can be included as the above-mentioned substance, and the drug can be widely used as long as it is suitable for the endoplasmic reticulum preparation. For example, antipyretics such as aspirin and acetaminophenone, anti-inflammatory agents such as linderone and cerestamin, anticancer agents such as doxorubicin hydrochloride, cisplatin, carboplatin and bleomycin, immunosuppressive agents such as mizoribine,
Antimetabolites such as 5-fluorouracil, beta blockers such as propranolol, central nervous system drugs such as trifluoperazine and thioproperazine mesylate, lymphokines such as natural or recombinant interleukin 2, natural Type or genetically engineered interferon (α, β, γ), erythropoietin, which is a hematopoietic factor, and other vitamins such as nicotinic acid and cocarboxylase, and enzymes with various physiological actions, antibodies, hemoglobin, etc. There are various drugs including proteins.
Among these, anticancer agents such as doxorubicin hydrochloride are particularly preferably used in the present invention.

The drug-containing lipid vesicles thus obtained have excellent stability and biodegradability, and in addition, they are remarkably excellent in sustained-release of the drug. It can be used as a carrier for sending blood into the body and can be used for a wide range of purposes such as DDS (Drach Delivery System), artificial blood, artificial vaccines, etc.
Even for industrial use, it can be used as a dispersion liquid for various applications such as biodevices and microcell reactors.

[0025]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, as the lipid vesicle-forming material, the non-polymerizable phospholipid and the specific polymerizable phospholipid are mixed and used at a specific ratio, whereby storage stability and in vivo It can form lipid vesicles with excellent stability and biodegradability. In addition, by controlling the amount of polymerizable phospholipids used and the degree of polymerization and degree of polymerization after membrane formation, the sustained release of water-soluble substances contained in the internal aqueous phase as lipid vesicles for liposomes can be freely controlled. In particular, when the above substance is a drug, it is possible to obtain a practical sustained-release preparation which can be freely released with the desired sustained-release effect.

[0026]

EXAMPLES Next, examples of the present invention will be described to more specifically describe. However, the present invention is not limited to the following examples.

Example 1 Commercially available hydrogenated egg yolk lecithin was treated by a known method with phospholipase A ₂ obtained from the venom of Nadiya Nadiya to give 1-acyl-L-3-glycerophosphorylcholine. It was Next, 2E, 4E-octadecadienoic acid is reacted with this by a known method using N, N′-carbodiimidazole to give 1-acyl-2- (2E, 4
E-octadecadienoyl) -L-3-glycerophosphorylcholine (hereinafter referred to as AODPC) was synthesized.

A polymerizable phospholipid composed of AODPC thus synthesized and a non-polymerizable phospholipid composed of commercially available L-α-dipalmitoylphosphatidylcholine (hereinafter referred to as DPPC) were used as the former: the latter mol. It was weighed so that the ratio was 8: 1 and the total amount was 2.4 g, added to an eggplant-shaped flask, and 15 ml of benzene was added and dissolved. This was frozen with liquid nitrogen and freeze-dried for two nights to prepare a lipid vesicle-forming material X ₁ consisting of a dry powder.

Example 2 Example 1 was repeated except that the ratio of the polymerizable phospholipid composed of AODPC to the non-polymerizable phospholipid composed of DPPC was changed so that the molar ratio of the former to the latter was 1: 1. Similarly to the above, a lipid vesicle-forming material X ₂ consisting of dry powder was prepared.

Example 3 Example 3 was carried out except that the ratio of the polymerizable phospholipid composed of AODPC and the non-polymerizable phospholipid composed of DPPC was changed so that the molar ratio of the former to the latter was 0.12: 1. In the same manner as in Example 1, the lipid vesicle-forming material X ₃ consisting of dry powder was used.
Was prepared.

Comparative Example 1 A lipid vesicle-forming material Y ₁ consisting of a dry powder was prepared by the same freeze-drying operation as in Example 1 using only the polymerizable phospholipid consisting of AODPC.

Comparative Example 2 As the polymerizable phospholipid, 1,2-di (2E, 4E-octadecadienoyl) -L-3-glycerophosphorylcholine (hereinafter
DODPC) was used and the non-polymerizable phospholipid consisting of DPPC was mixed in such a manner that the molar ratio of the former to the latter was 1: 1. The following lipid vesicle forming material Y ₂ was prepared.

Comparative Example 3 A lipid vesicle-forming material Y ₃ consisting of a dry powder was prepared by the same freeze-drying operation as in Example 1 using only non-polymerizable phospholipids consisting of DPPC.

Examples 4 to 6 10 mM HEPES buffer (pH 9.0) 100
After dissolving 100 mmol of 5 (6) -carboxyfluororescein (hereinafter referred to as CF) in ml and confirming the dissolution of CF, 1N NaOH aqueous solution was used to adjust the pH.
The pH was adjusted to 7.4 to prepare a CF-dissolved HEPES buffer solution.

12 ml of this CF-dissolved HEPES buffer
And 0.6 g of each lipid vesicle-forming material X _{1 to} X ₃ of Examples 1 to 3 weighed in a beaker were mixed, and the mixture was stirred for 12 hours.
Three types of lipid dispersions were obtained by stirring and mixing with a laser. Subsequently, each of the lipid dispersions was used with an extruder [trade name of NOF Refosome Co., Ltd.]
It was sized to have a size of 0.2 μm by passing it through a filter made of polycarbonate.

Next, in order to remove CF which was not encapsulated, the sized dispersion liquid was subjected to Sephadex G-5.
Gel filtration was performed using a 0 (trade name, manufactured by Pharmacia) gel column. At that time, as a solvent for gel filtration, 150
10 mmol HEPE with the addition of mmol NaCl
S buffer (pH 7.4) was used. In this way, 3
A CF-encapsulated lipid vesicle dispersion was obtained.

Comparative Examples 4 to 6 Instead of the lipid vesicle-forming materials X _{1 to} X ₃ of Examples 1 to 3, the lipid vesicle-forming materials Y _{1 to} Y 3 of Comparative Examples 1 to ₃ were used. In the same manner as in Examples 4 to 6
An encapsulated lipid vesicle dispersion was obtained.

The CF-encapsulated lipid vesicle dispersions obtained in Examples 4 to 6 and Comparative Examples 4 to 6 were polymerized by γ-ray for 1 hour under irradiation conditions of 5 KGy / hour, and then stored as follows. A stability test and a sustained release test were performed.
The results of these tests are shown in Table 1 below. For reference, the table also shows the type of lipid vesicle-forming material used and the mixing molar ratio of the polymerizable phospholipid and the non-polymerizable phospholipid.

<Storage Stability Test> The CF-encapsulated lipid vesicle dispersion was instantaneously frozen using liquid nitrogen, and then thawed at room temperature to examine the particle size stability before and after freeze-thawing. The particle size is 15 for lipid vesicles before and after freezing and thawing.
10 mmol HEP with addition of 0 mmol NaCl
A certain amount was once dispersed in a cell filled with ES buffer (pH 7.4) and measured using NICOMP model 370 (trade name, manufactured by NOF Liposome Co., Ltd.). The particle size stability before and after freezing and thawing is shown as the ratio of particle size change by the ratio of particle size after freezing and thawing / particle size before freezing. Therefore, the closer the particle size change due to freeze-thawing is to 1, the smaller the particle size change and the more stable the film.

<Sustained release test> CF taken out from the dispersion liquid
A certain amount of the encapsulated lipid vesicle was once dispersed in a fluorescent cell at a temperature of 25 ° C filled with 10 mM HEPES buffer (pH 7.4) containing 150 mM NaCl, and λ _em was measured using a fluorometer. = 490 nm, λ _em = 52
The time-releasing property of CF was examined by measuring 0 nm after a fixed time. The total amount of encapsulated CF is the surfactant Tri
Ton-X-100 was added and ultrasonic irradiation was carried out to destroy the endoplasmic reticulum for measurement. The sustained release property was evaluated by comparing the CF release rate after 120 minutes. A preferred sustained release property is when the CF release rate after 120 minutes is in the range of about 10 to 90%.

[0041]

[Table 1]

Examples 7-9 Using ultrapure water, 10 mM HEPES buffer (p
H7.4) 100 ml was prepared. 12 ml of this HEPES buffer solution and each lipid vesicle-forming material X of Examples 1 to 3
0.6 g of _{1 to} X ₃ was mixed in a beaker and mixed with stirring for 12 hours using a stirrer to obtain three kinds of lipid dispersions.

Then, each of the lipid dispersions was applied using an extruder [trade name of NOF Refosome Co., Ltd.].
It was sized to have a size of 0.2 μm by passing it through a filter made of polycarbonate.
All of these operations were carried out on a clean bench installed in the clean room, and by using a sterilized instrument, three types of lipid vesicle dispersion liquids were obtained aseptically.

Comparative Examples 7 to 9 Except that the lipid vesicle forming materials Y _{1 to} Y 3 of Comparative Examples 1 to ₃ were used in place of the lipid vesicle forming materials X _{1 to} X ₃ of Examples 1 to 3. In the same manner as in Examples 7 to 9, three types of lipid vesicle dispersion liquids were aseptically obtained.

The lipid vesicle dispersions obtained in Examples 7 to 9 and Comparative Examples 7 to 9 were polymerized by γ-ray for 1 hour under irradiation conditions of 5 KGy / hour, and then bioaccumulated in the following manner. I conducted a sex test. The test results are shown in Table 2 below. For reference, the type of lipid vesicle-forming material used and the mixing molar ratio of the polymerizable phospholipid and the non-polymerizable phospholipid are also shown in the table.

<Bioaccumulation test> For experimental animals, Cr was used.
j: CD (SD) system male rats (Japan Charles River) were used. The lipid vesicle dispersion was taken into a syringe in an amount corresponding to 8.0 ml / Kg of rat body weight and administered once into the tail vein. As a control, physiological saline (Pharmacopoeia) was used, and the same amount as the above was administered by the same method.

These rats were bred, and 3 days and 120 days after administration, the blood was removed from the abdominal aorta under anesthesia with Nembutal (trade name) and sacrificed. The liver in which lipid vesicles had accumulated was removed and histologically examined. The liver was fixed in 10% neutral buffered formalin solution, and then paraffin sections were prepared by a conventional method. After preparing E-stained and Nile blue-stained specimens, they were observed under a microscope.

According to this observation, H. In E-stained specimens, macrophages (Kupffer cells) swollen by ingesting foamy substances were observed except for those administered with physiological saline. All of them were purple by Nile blue staining and were positive for lipid. The lipid endoplasmic reticulum itself is N
Since it showed a purple color on ile blue staining, it was judged that the phagocytic substance was lipid vesicles.

On the other hand, for the specimen 120 days after administration,
Only for lipid vesicles consisting only of AODPC and solids to which lipid vesicles using DODPC as a part of phospholipids were administered, the macrophore that was poor in stainability but ingested droplets of weakly basic substance -I admitted. These products exhibited a pale pink color even when stained with Nile blue, and were lipid-positive. By the above test, AODPC
It was determined that the lipid vesicles consisting of only the vesicles and the lipid vesicles using DODPC as a part of the phospholipid had a high bioaccumulation potential.

As a result of the above-mentioned test, with respect to the specimen 120 days after administration, positive reaction was observed by Nile blue staining, +, positive reaction was not observed by Nile blue staining, that is, lipid endoplasmic reticulum-like substance was detected. The results are shown in Table 2 with the result that accumulation was not recognized as-.
It was shown to.

[0051]

[Table 2]

As is clear from the above test results, the lipid vesicles obtained by polymerizing the lipid vesicle-forming material of the present invention after film formation are excellent in storage stability and sustained release of the encapsulated substance. Moreover, since it has the characteristic of low bioaccumulation, it can be widely used as a sustained-release preparation having high practicality.

Continued front page       (56) References Japanese Patent Laid-Open No. 63-139187 (JP, A)                 JP-A-61-129192 (JP, A)                 JP 61-91 (JP, A)                 JP 63-232841 (JP, A)                 JP-A-63-54384 (JP, A)                 JP 62-96431 (JP, A)

Claims (3)

(57) [Claims] 1. A) non-polymerizable phospholipid and B) the following formula (1); (In the formula, ^one of R ¹ and R ² is a polymerizable acyl group, the other is a non-polymerizable acyl group, and X is a phospholipid polar group). A lipid vesicle-forming material containing a lipid, wherein the ratio of the polymerizable phospholipid of the B component is 0.1 to 10 mol relative to 1 mol of the non-polymerizable phospholipid of the A component , and B after the membrane formation.
Characterized by polymerizing a polymerizable phospholipid as a component
Biodegradable lipid vesicles . 2. The drug according to claim 1, which is a lipid vesicle for liposome, wherein the drug is contained in the internal aqueous phase.
Lipid endoplasmic reticulum used as a carrier to be sent to . 3. A lipid endoplasmic reticulum for liposomes, comprising:
The lipid vesicle according to claim 1, wherein the aqueous phase contains a drug.
A sustained-release preparation consisting of