WO2011037252A1 - Liposome preparation containing spicamycin derivative - Google Patents

Abstract

Disclosed is a liposome preparation in which a poorly water-soluble spicamycin derivative (KRN5500) can be retained stably, and which can exhibit excellent accumulation in blood when the constitutional proportions of a phospholipid and other lipids, which are membrane-constituting components, and the length of an acyl chain in the phospholipid are controlled properly and can also release KRN5500 with high efficiency in an affected part. The liposome preparation comprises a liposome and a spicamycin derivative represented by chemical formula (I) and carried on the liposome, wherein the liposome comprises 0 to 50 mol% of cholesterol and 100 to 50 mol% of a phospholipid having an acyl chain that is an acyl chain contained in a saturated C₁₄-C₁₈ fatty acid. (In the formula, R₁ represents H; R₂ represents OH; and R represents a linear or branched alkyl group having 9 to 15 carbon atoms or a linear alkenyl group having 10 to 17 carbon atoms.)

Description

Liposome preparation containing spicamycin derivative

The present invention relates to a liposome preparation containing a poorly water-soluble spicamycin derivative as a drug.

Anticancer agents used for cancer treatment are classified into DNA, RNA, and protein synthesis inhibitors based on the pattern of polymer synthesis inhibitory activity against cancer cells. Mainly, the development of anticancer agents acting on DNA and RNA synthesis is the mainstream. In this case, there are many cases where toxicity to limited organs such as myelosuppression becomes dose limiting toxicity (DLT). On the other hand, protein synthesis inhibitors are often toxic to organs that synthesize important proteins necessary for the maintenance of the body, including the liver. In many cases, clinical development is discontinued due to expression. For this reason, protein synthesis inhibitors are required to have higher selective toxicity between tumor cells and normal cells.

A spicamycin derivative represented by the formula (I) [wherein R ₁ = H, R ₂ = OH, and R is a linear or branched alkyl having 9 to 15 carbon atoms or a carbon having 10 to 17 carbon atoms] Straight-chain alkenyl] is

It is an antitumor substance derived from actinomycetes and was found as a protein synthesis inhibitor having an action of inducing differentiation of HL60 cells (see Patent Document 1). A peculiar aminoheptose (hereinafter abbreviated as spicamine) binds to the amino group at the 6-position of purine, glycine binds to the amino group at the 4-position of the spicamine, and a fatty acid is attached to the amino group of the glycine. It has an amide-bonded structure. For clinical use of the spicamycin as an anti-tumor agent, the fatty acid side chain moieties differ for the purpose of providing a less toxic, higher therapeutic index, componentally singulated spicamycin compound Various spicamycin derivatives have been developed. As a result, it has been found that the structure of the fatty acid moiety in these various derivatives contributes most to the antitumor activity, and spicamycin X (WO 90/15811 publication) (patent application PCT / JP90 / 00781). As well as KRN5500 (6- [4-deoxy-4- (2E, 4E) -tetradecadienoylglycyl] -amino-L-glycero-b-mannoheptopyranosyl) amino-9H-purine may be suitable for its purpose. It is reported that it is (refer patent document 2).

Of these, in particular the formula (II)

KRN5500 is a spicamycin derivative having the largest therapeutic index, and its effect is exerted by inhibiting protein synthesis of cancer cells. Therefore, it has been developed as a novel anticancer agent having a new antitumor mechanism. (Non-Patent Document 1). Since KRN5500 has strong interaction between the fatty acid side chain moiety and the cell, internalization easily occurs and is taken into the cell. When taken into cells, this fatty acid side chain is hydrolyzed only by the fatty acid moiety by an enzyme present in the cell membrane or the like to produce the active body SAN-Gly (4-N-glycylpicamicin aminoside) (Non-patent Document 2). ). The conversion activity of KRN5500 in each cell to SAN-Gly and the cell-killing effect of KRN5500 on that cell have been found to correlate. In normal cells, the conversion activity to SAN-Gly is low. It is high in cancer cell lines showing a strong cell killing effect, and it has been reported that KRN5500 has relatively cancer cell selectivity (Non-patent Document 3). Furthermore, KRN5500 shows the same cytocidal effect on adriamycin, vincristine, and mitomycin C resistant cells as the parent strain, and is effective against multidrug resistant cells by avoiding the drug excretion mechanism by most P-glycoproteins. It has also been found.

Thus, KRN5500 has a new antitumor mechanism of protein synthesis inhibition and is expected to be highly effective against cancers that have acquired the resistance of existing anticancer agents. Clinical trials were conducted both in Japan and in the United States (Non-Patent Document 4, Non-Patent Document 5, Non-Patent Document 6). However, in phase I clinical trials in Japan and the United States, pulmonary disorders and liver disorders appeared, respectively.
One of the problems associated with the side effects of KRN5500 is its poor water solubility, and the use of organic solvents or polyoxyethylene castor oil (Cremophor EL) is unavoidable due to its extremely low solubility in water. Therefore, in addition to the toxicity of the drug itself of KRN5500, there is a risk of developing side effects due to solvent toxicity, and it has been reported that these side effects are also involved in the side effects observed in clinical trials. In addition, since the pH of the preparation is as high as 10-11 due to the use of an organic solvent, it has been inevitably administered from the central vein in clinical trials, so the administration time is also long and very difficult to handle. Has been. Thus, KRN5500 has a problem of toxicity due to a solubilizer in addition to the toxicity of the drug itself, and there are still many problems to be solved.

Generally, studies have been made on DDS for poorly water-soluble drugs in order to avoid such side effects due to solvent toxicity. For example, paclitaxel is mentioned as a poorly water-soluble drug. Currently, Cremophor EL (polyoxyethylene castor oil) and ethanol are used as solvents for administration as an injection solution. This Cremophor EL is known to cause hypersensitivity reactions, causing anaphylactic reactions, and manifestation of dyspnea, facial flushing, generalized rash, chest pain, tachycardia, hypotension, angioedema, urticaria, etc. There is. Therefore, in order to prevent this severe hypersensitivity, pre-administration drugs such as steroids and antihistamines are required at the time of administration of paclitaxel, and it is currently difficult to use (Non-patent Document 7).

Therefore, in order to solve these problems, attempts have been made to make DCL of paclitaxel. NK105 (nanocarrier), which is currently under development, and Abraxane that is already in clinical use fall under these categories.
NK105 is a polymeric micelle preparation produced by mixing paclitaxel with a block copolymer in which a hydrophilic polyethylene glycol (PEG) chain and a polyaspartic acid chain that is a hydrophobic group are bonded in a chain form, Since the solubility of paclitaxel can be drastically improved without using Cremophor EL, it is possible to provide an injection solution that avoids solvent toxicity. In the non-clinical study of NK105, excellent retention in blood, antitumor effect, and reduction of neurotoxicity were observed, and a clinical phase II study is currently being conducted (Non-patent Document 8). Abraxane, which is also a paclitaxel DDS formulation, is a uniform nanoparticle formulation with human serum albumin as an additive. Like NK105, Cremophor EL and ethanol are not used as solvents, so the risk of solvent toxicity can be avoided, and a high dose of paclitaxel can be administered in a short time without the need for pre-administration. Patent Document 9). Thus, since a poorly water-soluble drug can be freed from the risk of side effects due to solvent toxicity by being converted to DDS, the safety range of the preparation can be expanded. Furthermore, the DDS preparation is expected to maintain or enhance the antitumor effect while reducing the toxicity inherent in the drug due to its characteristics.

Also in the above KRN5500, it is required to enhance the effect while reducing the side effects due to solvent toxicity, and further, the toxicity of KRN5500 itself. Therefore, in order to solve these problems, studies have been made on micellization of KRN5500 (Patent Document 3, Non-Patent Document 10, Non-Patent Document 11) and liposome formation. The micelleization of KRN5500 is achieved by converting a block copolymer having a hydrophilic polymer segment and a hydrophobic polymer segment (PEG-P (C ₁₆ , BLA)) and KRN5500 into N, N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO), respectively. It is obtained by dissolving, mixing and stirring them, and then performing dialysis and sonication (Non-patent Document 12). The toxicity and antitumor effect of this KRN5500 micelle are evaluated, and the toxicity is reported to be reduced compared with the single substance in pulmonary toxicity in the bleomycin (BLM) model in rats. On the other hand, the antitumor effect is almost the same as that of KRN5500 alone in a system in which human gastric cancer cell line MKN-45 cells are transplanted into nude mice, and an effect exceeding that of the single body is not obtained. Further, the particle size of these micelles has two peaks detected according to the scattering intensity, and it is difficult to control the particle size. Further, the particle size distribution is as wide as 81 nm to 390 nm, which is not preferable as a practical injection solution. Furthermore, it is not a production method suitable for scale-up, such as using an organic solvent or using a production method that requires dialysis or ultrasonic treatment.
As described above, KRN5500 micelles still have many problems such as medicinal efficacy, production method, particle size, etc., and have not reached the level that can be clinically endured. Therefore, in order to obtain the effectiveness of KRN5500, it is desired to develop a preparation that can be solubilized without using an organic solvent or Cremophor EL and can reduce the original toxicity of KRN5500 and side effects due to solvent toxicity.

JP 59-161396 A Japanese Patent Laid-Open No. 5-186494 JP-A-11-335267

The poorly water-soluble spicamycin derivative (KRN5500) can be solubilized without using an organic solvent or Cremophor EL, thereby avoiding the risk of side effects due to solvent toxicity, and increasing the drug concentration in the blood for a long time after administration. An object of the present invention is to provide a KRN5500 formulation that can be maintained and can maintain or enhance the effect while reducing the toxicity of KRN5500 itself. Moreover, it is desired that this KRN5500 preparation is very excellent in stability in a vial.

The above problems are solved by the present invention described below.
The present inventor has studied to achieve the above-mentioned object, and as a result, the poorly water-soluble spicamycin derivative (KRN5500) can be made into a liposome by controlling the lipid composition ratio of phospholipid and cholesterol, which are the lipid membrane components of the liposome. I found it. Moreover, while examining the antitumor effect of this KRN5500 liposome preparation, it was found that the antitumor effect can be enhanced by using a phospholipid having a short acyl chain length (myristoyl, C ₁₄ ) (in this case, however) About half of the cholesterol as a lipid membrane component). Since KRN5500 is a fat-soluble drug, it is considered that it is encapsulated in the lipid membrane due to its characteristics, but by shortening the chain length of the acyl chain of phospholipid, the chain length of the acyl chain and the fatty acid side chain of KRN5500 This is probably because KRN5500 can be efficiently released in the affected area while maintaining the drug concentration for a long time in the blood. In addition, it was found that the antitumor effect is also enhanced in a membrane formulation not containing cholesterol as a lipid membrane constituent. From these results, it can be seen that, in order to obtain a high antitumor effect, the balance between stability in blood and release of KRN5500 in the affected area is very important. It is presumed that there is a hydrophobic interaction between the chain length of the phospholipid acyl chain, which is a lipid membrane component, and the fatty acid side chain of KRN5500. If this hydrophobic interaction is too strong, the fluidity of the lipid membrane is suppressed, so that the release of KRN5500 in the affected area is suppressed, and as a result, it is presumed that the expected antitumor effect cannot be obtained. On the other hand, if this hydrophobic interaction is too weak, the stability in the blood is reduced and disappears immediately, so that the antitumor effect expected in the same manner cannot be obtained. Therefore, there is a demand for a liposome preparation that can be efficiently released in the affected area while stably maintaining KRN5500 in the blood, and for that purpose, the higher the membrane fluidity (however, when cholesterol is included), the above objective can be achieved. It became clear. In addition, it became clear that a liposome preparation containing a relatively short chain length of PEG (preferably PEG ₂₀₀₀ -DSPE) on the lipid membrane surface can reduce side effects while maintaining a strong antitumor action. . From these, the following present invention is provided to solve the above-mentioned problems.

(1) A liposome containing 100 to 50 mol% of phospholipid which is an acyl chain of a saturated fatty acid having a cholesterol chain length of 0 to 50 mol% and an acyl chain of C ₁₄ to C ₁₈ is represented by the chemical formula (I) Liposome preparation carrying a spicamycin derivative:

(Wherein R ₁ = H, R ₂ = OH, and R is straight-chain or branched alkyl having 9 to 15 carbon atoms or straight-chain alkenyl having 10 to 17 carbon atoms).
(2) The liposome preparation according to (1), wherein the lipid membrane of the liposome is any of the following 1) to 3):
1) The lipid membrane of the liposome is a lipid membrane containing a phospholipid whose acyl chain is an acyl chain of a saturated fatty acid having an average chain length of more than 16 to 18 as a main constituent, and the acyl chain is distearoyl, palmitoyl stearoyl or stearoyl Palmitoyl or myristoyl stearoyl or stearoyl myristoyl is preferred.
2) The lipid membrane of the liposome contains phospholipids whose acyl chains are acyl chains of saturated fatty acids having an average chain length of more than 14 to 16 and cholesterol as main components, and the molar ratio of both components is 80: 20-50 Preferably, the acyl chain is dipalmitoyl or palmitoyl myristoyl or myristoyl palmitoyl.
3) The lipid membrane of the liposome contains a phospholipid whose acyl chain is an acyl chain of a saturated fatty acid having an average chain length of 14 and cholesterol as main components, and the molar ratio of both components is 60:40 to 50:50 It is preferred that the acyl chain is dimyristoyl.
The content of the main constituent component with respect to the total lipid of the lipid membrane of the liposome is 50% by mass or more, preferably 80% by mass or more.
Here, the chain length of the acyl chain means the carbon number of the acyl chain. The average chain length means the carbon number obtained by averaging the carbon numbers of the fatty acids constituting the phospholipid. The constituent fatty acids may be the same or different.
(3) The liposome preparation according to (1), wherein the liposome lipid membrane of the liposome preparation is any of the following 1) to 3):
1) The lipid membrane of the liposome contains, as a main component, a phospholipid whose acyl chain is an acyl chain of a saturated fatty acid having a chain length of 18.
2) The lipid membrane of the liposome contains a phospholipid whose acyl chain is an acyl chain of a saturated fatty acid having a chain length of 16 and cholesterol as main components, and the molar ratio of both components is 80:20 to 50:50 is there.
3) The lipid membrane of the liposome contains a phospholipid whose acyl chain is an acyl chain of a saturated fatty acid having a chain length of 14 and cholesterol as main components, and the molar ratio of both components is 60:40 to 50:50 is there.
The content of the main constituent component with respect to the total lipid of the lipid membrane of the liposome is 50% by mass or more, preferably 80% by mass or more.
(4) The liposome preparation according to any one of (1) to (3), wherein the phospholipid is phosphatidylcholine.
(5) The group R = CH ₃ (CH ₂ ) ₈ CH═CHCH═CH—, wherein the compound of the formula (I) is (6- [4-deoxy-4- (2E, 4E) -tetradecadenyloylglycyl]- The liposomal preparation according to any one of (1) to (4), which is amino-L-glycero-b-L-mannnohetopypyranosyl) amino-9H-purine) (referred to as KRN5500).
(6) The liposome preparation according to any one of (1) to (5), wherein the liposome preparation contains a hydrophilic polymer.
(7) Any one of (1) to (6), wherein the AUC (plasma concentration-area under the time curve) of the spicamycin derivative represented by chemical formula (I) is 45 times or more that of the spicamycin derivative alone The liposome preparation described in 1.

The above-described liposome preparation according to the present invention can stably retain, for example, a poorly water-soluble spicamycin derivative (KRN5500), the composition ratio of phospholipids and other lipids, which are membrane constituents, and acyl of phospholipids. By controlling the chain length, it is possible to efficiently release KRN5500 in the affected area while being excellent in retention in blood, and can further enhance the original antitumor effect of KRN5500, and side effects due to drug toxicity Can be reduced. In particular, by controlling the PEG chain length (molecular weight of polyethylene glycol) and the amount of PEG modification, side effects can be further reduced while maintaining a strong antitumor action. In the course of reaching the above invention, it was revealed that the KRN5500 preparation of the present invention has good stability in a vial.

FIG. 1 is a graph showing the amount of liposome drug loaded relative to the amount of drug charged. FIG. 2 is a graph showing the efficiency of drug encapsulation in liposomes with respect to the amount of drug charged. FIG. 3 is a graph in which a liposome preparation or the like was administered to a rat, and the KRN5500 concentration in plasma after the lapse of a predetermined time after the administration was quantified by absorbance measurement. FIG. 4 is a graph showing the in vitro cell-killing effect (Colo205) of KRN5500 liposome preparations having different lipid compositions. FIG. 5 is a graph showing the tumor volume over the elapsed days after administration of a liposome preparation or the like to mice (human colon cancer cells (CoL-1)). FIG. 6 is a graph showing the tumor volume when a liposome preparation or the like is repeatedly administered to mice (human lung cancer cell line PC-9). FIG. 7 is a graph showing the weight change rate of mice when a liposome preparation or the like is repeatedly administered to mice (human lung cancer cell line PC-9). FIG. 8 is a graph in which liposome preparations having different PEG modification amounts and PEG chain lengths were administered to rats, and the KRN5500 concentration in plasma after lapse of a predetermined time after the administration was quantified by absorbance measurement. FIG. 9 is a graph showing the in vitro cell killing effect (Colo205) of a KRN5500 liposome formulation containing PEG ₂₀₀₀ -DSPE. FIG. 10 is a graph showing the in vitro cell killing effect (HT-29) of a KRN5500 liposome preparation containing PEG ₂₀₀₀ -DSPE. FIG. 11 is a graph showing the tumor volume over the elapsed days after administration of a liposome preparation or the like to mice (human colon cancer cells (CoL-1)). FIG. 12 is a graph showing the relative tumor volume with respect to the Control group in the number of days after administration of a liposome preparation or the like to mice (human colon cancer cells (CoL-1)). FIG. 13 is a graph showing changes in the body weight of mice after administration of a liposome preparation or the like to mice (human colon cancer cells (CoL-1)). FIG. 14 is a graph showing the tumor volume over the elapsed days when a liposome preparation or the like was administered to mice (human colon cancer cells (CoL-1)) three times in total at 4-day intervals. FIG. 15 is a graph showing the relative tumor volume with respect to the Control group in the elapsed days when a liposome preparation or the like was administered to mice (human colorectal cancer cells (CoL-1)) three times at intervals of 4 days. FIG. 16 is a graph showing the change in body weight of mice over the elapsed days when a liposome preparation or the like was administered to mice (human colon cancer cells (CoL-1)) three times in total at 4-day intervals. FIG. 17 is a graph showing the tumor volume over the elapsed days when a liposome preparation or the like was administered to mice (human colon cancer cells (CoL-1)) twice at a total interval of 6 days. FIG. 18 is a graph showing the relative tumor volume with respect to the Control group in the elapsed days when a liposome preparation or the like was administered to mice (human colon cancer cells (CoL-1)) twice at a total interval of 6 days. FIG. 19 is a graph showing changes in the body weight of mice over the elapsed days when a liposome preparation or the like was administered to mice (human colon cancer cells (CoL-1)) twice at a 6-day interval.

Hereinafter, the present invention will be described in more detail.
The “poorly water-soluble spicamycin derivative” is a general term for compounds having a spicamycin skeleton that is insoluble or sparingly soluble in water. Specifically, it is represented by the following formula (I), wherein R ₁ = H, R ₂ = OH, and R is a linear or branched alkyl having 9 to 15 carbon atoms, or 10 to 10 carbon atoms. A derivative having 17 straight-chain alkenyl. In the compound represented by the formula (I), a derivative having R═CH ₃ (CH ₂ ) ₈ CH═CHCH═CH—, for example, a compound represented by the formula (II) (6- [4-deoxy-4- (2E, 4E) -tetradecadienoylglycyl] -amino-L-glycero-b-L-mannnohepttopyranosyl) amino-9H-purine) (KRN5500) or R = CH ₃ (CH ₂ ) 6- [4′-N- (N′-didecanoylglycyl) -spicaminyl-amino] purine) (referred to as SPM VIII or SPM X), more preferably (6- [4-deoxy- 4- (2E, 4E) -tetradecadienoylglycyl] -amino-L-glycero-b-L-mannnohetopy ranosyl) amino-9H-purine) (KRN5500).

The poorly water-soluble spicamycin derivative is a prodrug of the active form SAN-Gly (4-N-glycylspicamycinamine amineside). It is considered that the fatty acid side chain of the prodrug is necessary for permeation of the cell membrane of cancer cells and is hydrolyzed and cleaved by an enzyme between the fatty acid and glycine when permeating the cell membrane. The resulting SAN-Gly acts on the intracellular protein synthesis system and exhibits a cytocidal effect on cancer cells. It has been reported that an amide bond between glycine and a fatty acid is important in the process of synthesizing this prodrug derivative, and the antitumor activity disappears when this part is chemically converted.

In the present invention, a liposome preparation containing the poorly water-soluble spicamycin derivative (for example, KRN5500) is provided.
Liposomes are aqueous dispersions of closed vesicles composed of a phospholipid bilayer membrane and having a structure that forms a space separated from the outside by a membrane formed based on the polarity of the hydrophobic and hydrophilic groups of the lipid. . The aqueous phases inside and outside the closed vesicle across the membrane are called the inner aqueous phase and the outer aqueous phase, respectively. The liposome preparation refers to a preparation in which a liposome is used as a carrier and a drug is supported thereon. In the present invention, this liposome is loaded with a poorly water-soluble spicamycin derivative (for example, KRN5500) as a drug.

“Phospholipid”, which is one of the main components of the lipid membrane of liposomes carrying the drug, is a main component of biological membranes, and includes hydrophobic groups such as long-chain alkyl groups and phosphate groups in the molecule. It is an amphiphilic substance having a hydrophilic group. Phospholipids include phosphatidylcholine (= lecithin), phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin and other sphingophospholipids, cardiolipin And natural or synthetic phospholipids or derivatives thereof, derivatives bound with sugars (glycophospholipids), and hydrogenated phospholipids obtained by hydrogenating them according to a conventional method. Among these, hydrogenated soybean phosphatidylcholine, hydrogenated egg yolk phosphatidylcholine, distearoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, and dimyristoyl phosphatidylcholine are preferable. Liposomes may contain other membrane components together with the main constituent components. For example, lipids other than phospholipids or derivatives thereof (hereinafter sometimes referred to as other lipids) are included.
The “lipid other than phospholipid” is a lipid that has a hydrophobic group such as a long-chain alkyl group in the molecule and does not contain a phosphate group in the molecule, and is not particularly limited, but is not limited to glyceroglycolipid, sphingoglycolipid. And sterol derivatives such as cholesterol and derivatives such as hydrogenated products thereof. Cholesterol derivatives are sterols having a cyclopentanohydrophenanthrene ring, and specific examples include, but are not limited to, cholesterol.

The liposome in the present invention can be preferably formulated by controlling the lipid composition ratio of phospholipid and cholesterol, and also enhances the antitumor effect by shortening the chain length of the acyl chain of the phospholipid used. Can do.
When the average chain length of the acyl chain is more than 16 to 18, phospholipid 100 mol% or phospholipid 70 to 50 mol% and cholesterol 30 to 50 mol% are preferable. When the phospholipid acyl chain is a phospholipid selected from the group consisting of distearoyl, palmitoyl stearoyl, stearoyl palmitoyl, myristoyl stearoyl and stearoyl myristoyl, phospholipid 100 mol% or phospholipid 70-50 mol%, cholesterol 30- 50 mol% is preferable.
When the average chain length of the acyl chain is more than 14 to 16, phospholipids of 80 to 50 mol% and cholesterol of 20 to 50 mol% are preferable. When the phospholipid acyl chain is a phospholipid selected from the group consisting of dipalmitoyl, palmitoyl myristoyl and myristoyl palmitoyl, phospholipids of 80 to 50 mol% and cholesterol of 20 to 50 mol% are preferred. More preferred are phospholipid 70-50 mol% and cholesterol 30-50 mol%.
When the average chain length of the acyl chain is 14, phospholipids of 50 to 60 mol% and cholesterol of 50 to 40 mol% are preferable. When the acyl chain is dimyristoyl, phospholipids are preferably 50 to 60 mol% and cholesterol is 50 to 40 mol%. Within these ranges, the spicamycin derivative can be formulated into a liposome formulation by stabilizing the membrane while preventing a decrease in the drug loading.

In addition, the lipid membrane may be modified in order to change the physical properties of the lipid constituting the lipid membrane and impart desired properties to the lipid membrane. Specifically, a derivative in which a modifying group is linked to a compound main body having affinity for a lipid membrane can be included in the film, and the compound main body is usually a lipid. This lipid moiety may be either phospholipid or lipid other than phospholipid, or both, and is not particularly limited. For example, phospholipid, long chain aliphatic alcohol, sterol, polyoxypropylene alkyl, glycerin fatty acid ester and the like can be mentioned.
The modifying group is not particularly limited, and examples thereof include a charged group, a hydrophilic group such as a water-soluble polysaccharide, and a hydrophilic polymer chain, and one or a combination of two or more of these may be used.
The charged group is not particularly limited, and examples thereof include basic functional groups such as amino group, amidino group, and guanidino group, acidic functional groups, and the like, and a charged substance having these groups can be included in the film.
Examples of the charged substance having a basic functional group include DOTMA disclosed in JP-A No. 61-161246, DOTAP disclosed in JP-A-5-508626, and JP-A-2-292246. Transfectam, TMAG disclosed in JP-A-4-108391, 3,5-dipentadecyloxybenzamidine hydrochloride, DOSPA, TfxTM-50, DDAB disclosed in WO 97/42166, pamphlet , DC-CHOL, DMRIE and the like.
Examples of the charged substance having an acidic functional group include gangliosides having sialic acid such as ganglioside GM1 and ganglioside GM3, and acidic amino acid surfactants such as N-acyl-L-glutamic acid.
When the charged substance is a substance in which a compound having a basic functional group is bound to a lipid, it is called a cationized lipid. The basic functional group may be present on the lipid membrane surface of the liposome (on the outer membrane surface and / or on the inner membrane surface) as the lipid portion of the cationized lipid is inserted into the lipid bilayer of the liposome. it can. By modifying the lipid membrane with a cationized lipid, the adhesion between the lipid membrane of the liposome and the cells can be enhanced.

The water-soluble polysaccharide is not particularly limited, and examples thereof include water-soluble polysaccharides such as glucuronic acid, sialic acid, dextran, pullulan, amylose, amylopectin, chitosan, mannan, cyclodextrin, pectin, and carrageenan. Examples of water-soluble polysaccharide derivatives include glycolipids.
The membrane modification rate by the charged substance and the water-soluble polysaccharide can be appropriately set as necessary.
The hydrophilic polymer is not particularly limited, but polyethylene glycol, ficoll, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether-maleic anhydride alternating copolymer, polyvinyl pyrrolidone, polyvinyl methyl ether, polyvinyl methyl oxazoline. , Polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropyl methacrylate, polyhydroxyethyl acrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyaspartamide, synthetic polyamino acid, etc. Is mentioned.
The hydrophilic polymer preferably has a structure for modifying the lipid membrane of the liposome. In particular, the hydrophilic polymer chain preferably has this structure at one end. When the structure is a hydrophobic part such as a lipid, the hydrophilic polymer chain is immobilized so that the hydrophobic part protrudes from the outer surface of the liposome as the hydrophobic part is inserted into the lipid membrane. In the case of a reactive functional group capable of covalently bonding with a membrane component, the hydrophilic polymer chain is formed on the outer surface of the liposome by covalently bonding with a lipid membrane component such as phospholipid exposed on the outer surface of the liposome. It is fixed so that it may protrude from.

Next, the hydrophobic compound used for bonding with the hydrophilic polymer chain to form the hydrophilic polymer-hydrophobic polymer compound will be described below.
The hydrophobic compound is not particularly limited. For example, the compound (hydrophobic compound) which has a hydrophobic area | region can be mentioned. Examples of the hydrophobic compound include other lipids such as phospholipids and sterols, long-chain aliphatic alcohols, glycerin fatty acid esters, and the like. Among them, phospholipid is one of the preferred embodiments. Moreover, these hydrophobic compounds may have a reactive functional group. The bond formed by the reactive functional group is preferably a covalent bond, and specific examples include an amide bond, an ester bond, an ether bond, a sulfide bond, and a disulfide bond, but are not particularly limited.
The acyl chain of the phospholipid is preferably a saturated fatty acid acyl chain. The chain length of the acyl chain is preferably C ₁₄ to C ₂₀ , and more preferably C ₁₄ to C ₁₈ . Examples of the acyl chain include dimyristoyl, dipalmitoyl, distearoyl, and palmitoyl stearoyl.
The phospholipid is not particularly limited. As the phospholipid, for example, one having a functional group capable of reacting with the hydrophilic polymer can be used. Specific examples of the phospholipid having a functional group capable of reacting with such a hydrophilic polymer include phosphatidylethanolamine having an amino group, phosphatidylglycerol having a hydroxy group, and phosphatidylserine having a carboxy group. Is mentioned. One preferred mode is to use the above phosphatidylethanolamine.
The lipid derivative of the hydrophilic polymer comprises the above hydrophilic polymer and the above lipid. The combination of the hydrophilic polymer and the lipid is not particularly limited. What was combined suitably according to the objective can be used. For example, at least one selected from other lipids such as phospholipids and sterols, long chain fatty alcohols, glycerin fatty acid esters, and at least selected from PEG, polyglycerol (PG), and polypropylene glycol (PPG). One example is a derivative of a hydrophilic polymer bonded to one. Specific examples include polyoxypropylene alkyl, and in particular, when the hydrophilic polymer is polyethylene glycol (PEG), it is one of preferred embodiments to select phospholipid and cholesterol as the lipid. Examples of the PEG lipid derivative by such a combination include PEG phospholipid derivatives and PEG cholesterol derivatives.

The lipid derivative of the hydrophilic polymer can be positively charged, negatively charged, or neutral depending on the choice of lipid. For example, when DSPE is selected as the lipid, it becomes a lipid derivative that shows a negative charge due to the influence of the phosphate group, and when cholesterol is selected as the lipid, it becomes a neutral lipid derivative. Lipids can be selected according to the purpose.
The molecular weight of PEG is not particularly limited, but is usually 500 to 10,000 daltons, preferably 1,000 to 7,000 daltons, more preferably 1,500 to 5,500 daltons, and more preferably 1,500 daltons. ~ 5,000 Daltons. More preferably, it is 1,500 to 2,500 daltons.
The molecular weight of PG is not particularly limited, but is usually 100 to 10,000 daltons, preferably 200 to 7,000 daltons, more preferably 400 to 5,000 daltons.
The molecular weight of PPG is not particularly limited, but is usually 100 to 10,000 daltons, preferably 200 to 7,000 daltons, more preferably 10,000 to 5,000 daltons.
Among these, phospholipid derivatives of PEG are mentioned as one of preferable embodiments. Examples of the phospholipid derivative of PEG include polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE). PEG-DSPE is preferred because it is a general-purpose compound and is easily available.

Liposomes whose surface has been modified using lipid derivatives of such hydrophilic polymers prevent blood opsonin protein and the like from adsorbing to the surface of the liposomes, increasing the blood stability of the liposomes, In addition, it is possible to avoid capture by reticuloendothelial tissue (RES) such as spleen, and to improve delivery to a target tissue or cell. That is, high blood retention is obtained. As a result, it is possible to passively accumulate in the tissue in which the vascular permeability of the tumor tissue is enhanced.
In the present invention, “retention in blood” means a property in which a drug carried in a liposome preparation is present in blood in a host administered with the liposome preparation carrying the drug. If the drug is released from the liposome before delivery to the target tissue or cell, it quickly disappears from the blood. When the retention in blood is good, it is possible to improve the delivery to a target tissue or cell even if a small amount of drug is administered.
The modification rate by the hydrophilic polymer lipid derivative is usually 0.1 to 20 mol%, preferably 0.1 to 10 mol%, more preferably 0.5 to 10 mol%, as a ratio to the total lipid. Furthermore, it is 0.5 to 4 mol%.
In the present invention, “total lipid” refers to a lipid obtained by removing a hydrophilic polymer lipid derivative from lipids constituting a lipid membrane.

In the present invention, the amount of the poorly water-soluble spicamycin derivative supported on the liposome as described above, that is, the drug / total lipid molar ratio of the liposome preparation is usually 0.1 or less from the viewpoint of liposome stability and encapsulation efficiency. Yes, preferably 0.001 to 0.03.
The drug loading in the liposome or the drug content in the liposome formulation means a state in which the drug is supported and retained in the liposome formulation (dispersion), and the drug not retained in the liposome, that is, dispersed. This means that the outer aqueous phase of the liquid is essentially free of any drug that is freely present regardless of liposomes. In the liposome preparation of the present invention using a poorly water-soluble spicamycin derivative as a drug, it is considered that at least a part of the drug is substantially contained in the lipid film and is supported on the membrane. The preparation is not required to have a free drug in the outer aqueous phase, and the loading state of the drug is not limited, and may be supported on the lipid membrane of the liposome and / or encapsulated in the inner aqueous phase.

The liposome preparation of the present invention may further contain a pharmaceutically acceptable stabilizer and / or antioxidant depending on the administration route. These are collectively referred to as pharmaceutically auxiliaries. Stabilizers include, but are not limited to, saccharides such as glycerol or sucrose. Examples of the antioxidant include, but are not limited to, ascorbic acid, uric acid, and tocophenol analogues such as vitamin E. Tocophenol has four isomers, α, β, γ, and δ, and any of them can be used in the present invention.
The liposome preparation of the present invention may further contain a pharmaceutically acceptable additive depending on the administration route. Examples of such additives include water, saline, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethyl cellulose, sodium polyacrylate, sodium alginate, water-soluble Dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, human serum albumin (HSA), mannitol, Sorbitol, lactose, PBS, biodegradable polymer, serum-free medium, acceptable as a pharmaceutical additive, and stability of liposome preparation Surfactants do not affect the concentration, and the concentrations that do not affect the stability refers to 15 mass% or less of the total formulation. Alternatively, a buffer solution of physiological pH acceptable in vivo can be used. The additive to be used is selected appropriately or in combination from the above depending on the dosage form, but is not limited thereto.

In this invention, the liposome formulation of the aspect containing these additives and / or pharmaceutically-added adjuvant can be provided as a pharmaceutical composition. The pharmaceutical composition of the present invention can be stored by refrigeration at room temperature (generally 21 ° C. to 25 ° C.), preferably 0 to 8 ° C.
As the above-mentioned liposome preparation, known liposome preparation methods can be widely used as long as the medicine can be stably supported.
In the present invention, the method for preparing the liposome suspension includes hydration method (Bangham method), sonication method, reverse phase evaporation vesicles, heating method, lipid dissolution method, DRV method (Dehydrated / Rehydrated Vesicles), freeze-thaw method, ethanol injection method, thin film method, extrusion method, high-pressure emulsification method using high-pressure discharge type emulsifier ("Liposome in Life Science" edited by Terada, Yoshimura et al .; Springer Fairlark Tokyo (1992) Various known techniques such as) can be employed.
In addition, recently developed technologies include the jet flow emulsification method that uses shear rate emulsification by jet flow under a liquid phase using the speed conversion from compression by ultra high pressure, the liposome preparation technology using supercritical carbon dioxide, There is also an improved ethanol injection method that simplifies the sizing process.
In the present invention, a typical liposome preparation step includes the steps (i) of producing a liposome encapsulating a drug to obtain a coarse liposome suspension, the granulating step (ii) of the coarse liposome suspension, and an external solution. It includes a step (iv) of obtaining a liposome suspension by substitution (removal of unencapsulated drug), and preferably a step (iii) of surface modification with a hydrophilic polymer is further included between steps (ii) and (iv).

In performing these steps, a preparation method as described above can be appropriately employed as necessary. Further, not only one method but also two or more methods can be selected, and the same or different methods can be duplicated or added.
Since liposomes generally have a phase transition point, each step before the external liquid replacement step (unencapsulated drug removal step) (iii) for liposome preparation is performed at a temperature above the phase transition point of the main membrane material. It is preferable.

As the lipid bilayer structure of liposomes, there are known membrane structures such as Unilamellar Vesicle (SUV, Large Unilamellar Vesicle, LUV) and multilamellar vesicle (MLV) composed of a plurality of sheets.
The liposome according to the present invention may have any membrane structure, but multilamellar vesicle liposomes are preferred from the viewpoint of encapsulation efficiency.
For example, when carrying out the sizing step by the above-mentioned membrane emulsification method, it can be made into a mono-lamellar by forcibly passing a membrane filter made of a commercially available polycarbonate or the like several times using an extruder. Can be controlled. For example, when the particle size is adjusted to 100 nm, the particle size can be adjusted stepwise by combining membrane filters of 400 nm, 200 nm, 100 nm and the like.
In the granulating step, if the temperature of the liposome coarse suspension is equal to or higher than the phase transition point of the main membrane material, the particle size can be easily controlled.
The size of the liposome after sizing is not particularly limited, but it can take a spherical shape or a form close thereto, and its particle diameter (diameter of particle outer diameter) is not particularly limited, but is usually 0.02 to 2 μm, The thickness is preferably 0.03 to 0.4 μm, more preferably 0.05 to 0.25 μm. This particle diameter is measured as a mean diameter value of all particles by a dynamic light scattering method using a Zetasizer (Malvern Instruments. 3000HS, Zatasizer Nano ZS90).

The tumor to be treated with the liposome preparation of the present invention is not particularly limited, but is a solid tumor, specifically, esophageal cancer, stomach cancer, colon cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer, laryngeal cancer. Lung cancer, prostate cancer, bladder cancer, breast cancer, uterine cancer or ovarian cancer. Target sites include tumor cells, tissues, organs or organs and their interiors. Therefore, in the present invention, the disease means the above-mentioned tumor, and the drug is expected to show an antitumor effect against them.
In the present invention, “exposure” means that the drug released to the outside of the liposome acts on the external environment. Specifically, the released spicamycin derivative is close to the target site, and when it penetrates the cell membrane of cancer cells, the fatty acid side chain is enzymatically hydrolyzed between the fatty acid and glycine, and the active form SAN-Gly. This SAN-Gly acts on the intracellular protein synthesis system and exhibits an antitumor effect. In order to show such an effect, it is necessary to maintain a balance between the drug release from the liposome preparation and the blood retention of the liposome preparation.
In the present invention, “release” means that the drug contained in the liposome preparation is released from the liposome.
It is important to control the release of the drug contained in the liposome preparation of the present invention because it exhibits strong antitumor activity when exposed to a target site at a high concentration for a long time in plasma.

The liposome preparation of the present invention can maintain the drug concentration in plasma at a high concentration. Since conventional liposome preparations disappear quickly from blood, it is difficult to expect a sufficient effect because the exposure time at the target site is short. Further, rapid disappearance from the blood is not preferable because the drug is exposed to organs such as liver and spleen, which are metabolic organs, at a high concentration, leading to side effects at the site. Since the liposome preparation of the present invention can maintain the drug concentration in plasma at a high concentration, it can reduce the exposure of the drug to organs such as the liver and spleen. It is preferable because it can be exposed and side effects can be reduced.
In the present invention, the drug concentration in plasma 1 hour after administration is 10% or more of the initial value, preferably 15% or more. This “initial value” is the theoretical concentration immediately after administration of the liposome preparation of the present invention, and is generally assumed that the total plasma volume calculated from the body weight of the host is used to dilute with the dose volume. Calculated. The plasma concentration at each time point can be expressed as a ratio to the initial value, and is generally expressed as “% dose”.
In the present invention, the drug is used for long exposure to the desired target site. Therefore, in the present invention, for the prevention and / or treatment of host diseases, an effective amount of a drug is released in the host by administering to the host a liposome preparation carrying an effective amount of the drug, and the target site is prolonged. For exposure at high concentrations over time, it can be parenterally administered systemically or locally to the host (patient). Examples of the host to be administered include mammals, preferably humans, monkeys, mice, livestock and the like.

As the parenteral route of administration, for example, intravenous injection (intravenous injection) such as infusion, intramuscular injection, intraperitoneal injection, and subcutaneous injection can be selected, and an appropriate administration method should be selected depending on the age and symptoms of the patient. Can do. The carrier of the invention is administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially block the symptoms of the disease. For example, the effective dose of the drug enclosed in the carrier is selected in the range of 0.01 mg to 100 mg per kg body weight per day. However, the carrier of the present invention is not limited to these dosages. The administration time may be administered after the disease has occurred, or may be administered prophylactically to relieve symptoms at the time of onset when the onset of the disease is predicted. The administration period can be appropriately selected depending on the age and symptoms of the patient.
As a specific administration method, the liposome preparation can be administered by syringe or infusion. Further, a catheter is inserted into the body of a patient or host, for example, into a lumen, for example, into a blood vessel, and the tip thereof is guided to the vicinity of the target site, and the blood flow to the desired target site, the vicinity thereof, or the target site through the catheter. It is also possible to administer from the site where is expected.

EXAMPLES Next, although an Example is given and this invention is demonstrated in more detail, this invention should not be limited to these Examples.
Each concentration and particle diameter of the drug-encapsulated liposome prepared in each example were determined as follows.
Phospholipid concentration (mg / mL): Phospholipid concentration in the liposome suspension quantified using high performance liquid chromatography.
Cholesterol concentration (mg / mL): Cholesterol concentration in a liposome suspension quantified using high performance liquid chromatography.
Total lipid concentration (mol / L): Total molar concentration (mM) of lipid as a membrane component calculated from the above phospholipid concentration and cholesterol concentration. This total lipid does not include lipids in PEG derivatives for introducing PEG (in the example, DSPE in PEG-DSPE).
Drug concentration (mg / mL): About the solution obtained by diluting the preparation obtained above with RO water (reverse osmosis membrane water) so that the phospholipid concentration is about 20 mg / mL, and further diluting 20 times with methanol, Absorbance at 264 nm was quantified by high performance liquid chromatography using an ultraviolet absorptiometer. The concentration of the enclosed KRN5500 is shown as drug amount (mg) / total preparation amount (mL).
High performance liquid chromatography test conditions:
Column: A stainless steel tube having an inner diameter of 6 mm and a length of 15 cm is filled with fully porous spherical silica gel (Nacalai Tesque COSMOSIL 5C18-ARII).
Column temperature: around 30 ° C. Mobile phase: 400 mL of liquid chromatogram methanol is added to and mixed with 100 mL of citrate buffer at pH 6.0.
Flow rate: 1.5mL / min
Drug loading (drug / total lipid molar ratio): The concentration of KRN5500 encapsulated in liposomes is shown as the drug / total lipid molar ratio from the ratio of the drug concentration to the total lipid concentration.
Particle size (nm): average particle size measured with a Zatasizer 3000HS (Malvern Instruments) or Zatasizer Nano ZS90 after diluting 20 μL of the liposome dispersion in 3 mL of physiological saline.

The abbreviations and molecular weights of the components used are shown below.
DSPC: Distearoyl phosphatidylcholine (molecular weight 790.2, manufactured by NOF Corporation)
HSPC: hydrogenated soybean phosphatidylcholine (molecular weight 790, Lipoid SPC3)
HEPC: Hydrogen egg yolk soybean phosphatidylcholine (molecular weight 777, manufactured by NOF Corporation)
DPPC: dipalmitoylphosphatidylcholine (molecular weight 734.0, manufactured by NOF Corporation)
DMPC: Dimyristoylphosphatidylcholine (molecular weight 677.9, manufactured by NOF Corporation)
Chol: cholesterol (molecular weight 388.66, manufactured by Solvay)
PEG ₅₀₀₀ -DSPE: Polyethylene glycol (molecular weight 5000) -phosphatidylethanolamine (molecular weight 6081, manufactured by NOF Corporation)
PEG ₂₀₀₀ -DSPE: Polyethylene glycol (molecular weight 2000) -phosphatidylethanolamine (molecular weight 2927, manufactured by NOF Corporation)
KRN5500 (molecular weight 589.7, manufactured by Kyowa Hakko Kirin Co., Ltd.)

Here, the relationship between the notation used in the drawings and the preparation examples in the following examples will be described.
HSPC / Chol-Lipo: Preparation Example 1, HEPC / Chol-Lipo: Preparation Example 2,
DPPC / Chol-Lipo: Preparation Example 3, DMPC / Chol-Lipo: Preparation Example 4,
HSPC (PEG 0.75 mol%)-Lipo: Preparation Example 5,
HSPC (PEG 2.0 mol%)-Lipo: Preparation Example 6, DMPC / Chol-Lipo: Preparation Example 17,
HEPC / Chol-Lipo: Preparation Example 18, DMPC / Chol (PEG 0.75 mol%)-Lipo: Preparation Example 19,
DMPC / Chol (PEG 1.5 mol%)-lipo: Preparation Example 20,
DMPC / Chol (PEG 2.0 mol%)-lipo: Preparation Example 21,
DMPC / Chol (PEG 1.5 mol%)-L lipo: Preparation Example 22,
DMPC / Chol (PEG2000-4.0 mol%)-lipo: Preparation Example 23, HEPC-lipo: Preparation Example 24.

(Preparation Examples 1 to 6) Preparation of KRN5500 Liposome Formulation In order to confirm a membrane formulation capable of achieving a KRN5500 encapsulated liposome formulation, liposome formation was attempted using phospholipids having different chain lengths of acyl chains. We also tried membrane formulations with different phospholipid / Chol ratios. Preparation Examples 1, 5, and 6 were prepared on a 100 mL scale, and Preparation Examples 2, 3, and 4 were manufactured on a 10 mL scale. In addition, the following shows the preparation method in a 100 mL scale.
(1) Production of KRN5500 liposomes Each type of phospholipid, cholesterol, and KRN5500 were weighed so as to have a predetermined molar ratio shown in Table 1, and 10 mL of absolute ethanol was added and dissolved by heating.
To 10 mL of the obtained lipid / drug mixed ethanol solution, 90 mL of an inner aqueous phase (10 mM citric acid monohydrate / 0.9% sodium chloride solution at pH 6.5) heated to about 70 ° C. was added, and ultrasonic waves were added. A crude liposome suspension was prepared by stirring in an apparatus. This crude liposome suspension was filtered with a filter (pore size 0.2 μm × 3 times, 0.1 μm × 10 times, Whatman) attached to an extruder (The Extruder T.100, Lipexbiomembranes Inc.) heated to about 70 ° C. ) In order to prepare a liposome suspension.
(2) Surface modification While maintaining the above-mentioned liposome suspension in a heated state, an aqueous solution of PEG ₅₀₀₀ -DSPE (37.7 mg / mL) was immediately added so as to achieve the PEG introduction rate shown in Table 1, The membrane surface (outer surface) of the liposome was PEG-modified by warm stirring. The liposome suspension after the heating was quickly cooled with ice.
The PEG introduction rate (mol%) = (PEG ₅₀₀₀ −DSPE / total lipid) × 100
It is. The total lipid here does not include lipids in PEG derivatives (DSPE in PEG ₅₀₀₀ -DSPE).
(3) External solution replacement The ice-cooled liposome suspension after PEG modification is crossed with an external aqueous phase solution (10 mM citric acid monohydrate / 0.9% sodium chloride solution (pH 6.5)). External liquid replacement was performed by a flow filtration system (Vivaflow MW 100,000). In addition, it concentrated in order to obtain a high concentration KRN5500 liposome formulation (Preparation Examples 1, 5, and 6).
(4) Filtration sterilization Filtration sterilization was carried out using the above-mentioned liposome suspension after replacement with an external solution to obtain a final preparation. Phospholipid, Chol, and KRN5500 concentrations were quantified in the liposome suspension after filter sterilization using high performance liquid chromatography. The total of the phospholipid concentration and the Chol concentration was taken as the total lipid concentration, and the amount of encapsulated KRN5500 was determined.
Table 1 shows the membrane composition ratio and drug loading (drug concentration, drug / total lipid molar ratio) particle size of the KRN5500 liposome preparation obtained above.
As shown in Preparation Examples 1 to 4, the drug loading of the membrane formulation containing about half of the cholesterol is almost KRN5500 / total lipid = 0.01 (mol / mol) regardless of the chain length of the acyl chain of the phospholipid. showed that. On the other hand, the membrane formulation containing no cholesterol (Preparation Examples 5 and 6) can increase the amount of KRN5500 charged to about twice when preparing a lipid / drug mixed ethanol solution. The loading amount also increased by a factor of about 2.
Further, as a result of examining the influence of the lipid composition ratio in the formulation, as shown in Table 2, it became clear that there is a membrane formulation that is difficult to form into a liposome depending on the chain length of the acyl chain of the phospholipid and the membrane composition ratio. It was. If the chain length of the phospholipid acyl chain is long (distearoyl, palmitoyl stearoyl), it can be made into liposomes without cholesterol, but if it is short (dipalmitoyl, dimyristoyl), it contains no cholesterol. It became clear that it could not be formulated. In particular, when dimyristoyl (C ₁₄ ) was used, it was found that about half the amount of cholesterol was required for the phospholipid. This is thought to be due to the membrane stabilization effect of cholesterol. That is, if the chain length of the acyl chain is long, the hydrophobic interaction is strengthened. Therefore, it is presumed that KRN5500 can be stably encapsulated even if there is no membrane stabilization effect by cholesterol. On the other hand, when the chain length of the acyl chain is short, the hydrophobic interaction is weakened and the fluidity of the membrane is increased, so that KRN5500 cannot be stably encapsulated in the membrane, and as a result, aggregation of KRN5500 itself occurs. Presumed to gel.

In the liposome of the present invention, the preparation amount necessary to obtain the maximum amount of drug encapsulated was examined.
(Preparation Examples 7 to 16)
In the HSPC / Chol (molar ratio) = 54/46 membrane formulation, the amount of KRN5500 charged is 0.002, 0.003, 0.005, 0.007 in order of drug / total lipid (mol / mol) ratio. , 0.008, 0.009, 0.010, 0.012, 0.014, and 0.016 except that the amount was changed to an amount of 0.06, 0.016, and 0.016 to obtain a KRN5500 liposome preparation. Table 3 shows the drug / total lipid and particle size of the obtained KRN5500 liposome preparation. FIG. 1 shows a drug loading amount, and FIG. 2 shows a graph of encapsulation efficiency.
As a result of studying liposome formation by changing the amount of KRN5500 charged from drug / total lipid (mol / mol) = 0.002 to 0.016, as shown in FIG. 1, with the increase in the amount of KRN5500 charged, An increase in the amount enclosed was observed. If it is possible to further increase the amount of KRN5500 charged, it is expected that the encapsulated amount can be further increased. However, clogging may occur in the granulation process, or KRN5500 may not be dissolved in the lipid ethanol solution. For this reason, it is difficult to increase the charged amount further. Therefore, it was revealed that the maximum drug loading in the formulation was about 1 mol% with respect to the total lipid. This result was the same when other phospholipids were used. The encapsulation efficiency of KRN5500 showed a value of 60 to 80% regardless of the charged amount (FIG. 2).

[Test Example 1] Pharmacokinetics of liposome preparations with different lipid compositions (retention in blood)
The KRN5500 liposome preparation prepared in Preparation Examples 1, 2, 4, 5 and 6 and KRN5500 alone were administered to rats at a dose of 0.2 mg / kg as the amount of KRN5500. After administration, blood was collected after 0.25, 1, 2, 4, 6, 8, 12, 24, and 48 hours, and plasma was collected by centrifugation (5,000 rpm, 10 minutes, 4 ° C.). Subsequently, 200 μL of methanol was added to 40 μL of plasma, and the supernatant was collected by centrifugation (10,000 rpm, 10 minutes, 4 ° C.). The absorbance at 264 nm of the collected solution was quantified by high performance liquid chromatography using an ultraviolet absorptiometer, and the concentration of KRN5500 in each plasma was determined. The high-performance liquid chromatography test conditions are the same as the KRN5500 concentration conditions encapsulated in the liposomes. The results are shown in Table 4 and FIG.
In the case of KRN5500 alone, the KRN5500 concentration in plasma decreased rapidly after administration, and was detected only for 0.25 and 1 hour. On the other hand, all liposome preparations were detected up to 48 hours after administration, and a significant increase in residence time was observed compared to KRN5500 alone. In terms of “% dose”, KRN5500 alone was about 5-6% 1 hour after administration, whereas the liposome preparation was 70-80%. In addition, the area under the plasma concentration-time curve (AUC) was 60 to 100 times larger in the liposome preparation than in the KRN5500 alone. From these results, it was confirmed that the liposome preparation of the present invention can maintain the plasma KRN5500 concentration at a high concentration for a long time in any membrane formulation. However, the retention in blood of the HSPC 100 mol% prescription containing no cholesterol tends to be slightly lower than that of the prescription containing cholesterol, and when the modification rate of PEG ₅₀₀₀ -DSPE is 0.75 mol%, Immediate distribution, showing blood kinetics appearing again in the blood, and by increasing the modification rate of PEG ₅₀₀₀ -DSPE to 2.0 mol%, the initially observed distribution disappears, and the retention in blood is improved. Admitted.

[Test Example 2] Cytocidal effect of liposome preparation having different lipid composition For evaluation of cytocidal effect in vitro, the liposome preparations of Preparation Examples 1, 2, 3, 6 and Preparation Example 17 shown below were used. .
(Preparation Example 17)
DMPC, Chol, and KRN5500 were weighed so that the film formulation was DMPC / Chol (molar ratio) = 54/46, and KRN5500 liposome preparations were obtained in the same manner as in Preparation Examples 1 to 6 (100 mL scale). Table 5 shows the drug / total lipid and particle size of the obtained KRN5500 liposome preparation.

FIG. 4 shows the cell killing effect in vitro of the KRN5500 liposome preparation prepared in Preparation Examples 1, 2, 3, 6, and 17.
Human colon cancer strain Colo205 was plated on a 96-well plate in RPMI medium containing 10% fetal bovine serum so as to be 2000 cells / 90 micro L / well, and cultured at 37 ° C., 5% CO _{2 for} 5 hours. The test substance is DMSO for KRN5500, and 10 mM citric acid / 0.9% sodium chloride solution at pH 6.5 for KRN5500 liposome preparation, 200-fold dilution series of final concentration (10 concentrations from 0 to 10000 nM as KRN5500) A test substance solution (specimen) was prepared by diluting it 20 times with RPMI medium. The sample was added to a 96-well plate containing 10 microliters / well, cultured at 37 ° C. and 5% CO _{2 for} 72 hours, and then added with Cell Counting Kit (DOJINDO) 10 microliters / well. ° C., and cultured in 5% CO ₂ for about 20 minutes, the absorbance was measured (450 nm, reference wavelength 650 nm). Cell growth inhibitory action is 100% inhibition of cell (-), medium + test substance medium well absorbance (A), cell (+), medium + test substance medium well absorbance (B). With 0% inhibition, the cell growth inhibitory action by the sample was calculated by the cell killing effect (%) = 100 × (B−sample absorbance) / (BA).
The cell killing effect of the KRN5500 liposome preparation having a membrane composition of phospholipid / Chol ratio = 54/46 (molar ratio) was higher as the chain length of the phospholipid acyl chain was shorter (FIG. 4). This is probably because the shorter the chain length of the acyl chain of the phospholipid, the weaker the hydrophobic interaction between the chain length of the acyl chain and the fatty acid side chain of KRN5500, and KRN5500 is more likely to be released from the liposome. Further, even in the HSPC 100 mol% formulation not containing cholesterol, the same high activity as that of KRN5500 alone was obtained.

[Test Example 3] Antitumor effect of liposome preparation having different lipid composition (single administration)
For the antitumor effect of a single dose in human colon cancer cells (CoL-1), the liposome preparations of Preparation Examples 1, 5, and 17 and Preparation Example 18 shown below were used.
(Preparation Example 18)
HEPC, Chol, and KRN5500 were weighed so that the membrane formulation was HEPC / Chol (molar ratio) = 54/46, and a KRN5500 liposome preparation was obtained in the same manner as in Preparation Examples 1 to 6 (prepared on a 100 mL scale). Table 6 shows the drug / total lipid and particle size of the obtained KRN5500 liposome preparation.

The human colon cancer strain CoL-1 tumor was inoculated subcutaneously into nude mice (BALB / C, nu / nu, pupa) and transplanted in normal breeding. For the test, a nude mouse was subcutaneously inoculated with the tumor fragment on the dorsal skin and reared normally, and when the tumor volume reached around 100 mm ³ , a group setting was performed with n = 4 so that the tumor volume became comparable ( Day 1).
The KRN5500 liposome preparation prepared in Preparation Examples 1, 5, 17, and 18 and KRN5500 alone were mixed with a medium [KRN5500 medium (N, N-dimethylacetamide 3.75%, polysorbate 80 3.0% , 2-aminoethanol 0.45%, physiological saline 92.8%), liposome preparation medium (pH 6.5, 10 mM citric acid / 0.9% sodium chloride solution)] and intravenous at 10 mg / kg It was administered internally.
The medium group of KRN5500 and the medium group of liposome preparation were used as the control group [Control (Liposome medium), Control (KRN5500 medium)].
The tumor volume was calculated as L × W × H / 2 mm ³ by measuring the major axis (Lmm), minor axis (Wmm), and thickness (Hmm) of the tumor at intervals of 2 to 3 days from Day 1 with calipers.
FIG. 5 shows the tumor volume in days elapsed after administration.
For human colon cancer, the liposome preparation and KRN5500 alone showed a significant tumor growth inhibitory effect compared to the control group. In addition, the liposome preparation showed a high sustained antitumor effect as compared with KRN5500 alone. In particular, the maximum antitumor effect of the liposome preparations of Preparation Examples 5 and 17 was similar to that of KRN5500 alone, and a remarkable sustained effect was recognized as compared with KRN5500 alone.

[Test Example 4] Antitumor effect of liposome preparation having different lipid composition (repeated administration)
Human lung cancer cell line PC-9 was cultured in RPMI medium containing 10% fetal bovine serum, washed with PBS, treated with 10% Tripsin-EDTA, washed twice with RPMI medium, and then nude mice (BALB / C, nu / nu, pupa) were inoculated subcutaneously in the dorsal part of the dorsal side so as to be 5 × 10 ⁶ cells / 100 micro L / individual and reared normally. Group setting was performed with n = 4 so that the tumor volume became comparable when the tumor volume reached around 100 mm ³ (Day 1).
7 times from Day 1 ( Day 1, 8, 15, 22) in total, the liposome preparation prepared in Preparation Examples 6 and 17 and KRN5500 alone were prepared to 1 mg / mL in each medium, and 10 mg / kg Was administered intravenously. The medium group of KRN5500 and the medium group of liposome preparation were used as the control group [Control (liposome medium), Control (KRN5500 medium)].
From Day 1, tumor volume was measured at intervals of 2 to 3 days according to Test Example 3, and mouse body weight (g) was calculated as body weight measurement value (g) −tumor volume (mm ³ ) / 1000. The weight change rate with a value of 1 was used as an index.
Estimated tumor volume and mouse body weight were determined 1, 3, 5, 8, 10, 12, 15, 17, 19, 22, 24 days after administration. The results are shown in Table 7 and FIGS.

Relative tumor volume (RTV) = Day X tumor volume / Day 1 tumor volume Tumor growth inhibition rate (TGIR, (%)) = (1−RTV of test substance administration group / RTV of Control group) × 100
For human lung cancer cells, both the liposome preparation and KRN5500 alone showed a significant tumor growth inhibitory effect compared to the control group. Furthermore, in any of the liposome preparations, a high antitumor effect was obtained as compared with KRN5500 alone, and a sustained effect was recognized. In particular, the liposome preparation of DMPC / Chol film formulation has the highest antitumor effect. In addition, the liposome preparation hardly affected the body weight of the mouse, whereas KRN5500 alone was observed to have a transient weight loss accompanying administration. From these results, it was clarified that the antitumor effect can be enhanced and the side effects can be reduced by making KRN5500 into liposomes. In addition, it became clear that the liposome preparation of other prescriptions has little influence on the body weight change of a mouse | mouth, and can reduce a side effect. In addition, as a result of the rat acute toxicity test, it was found that the acute death observed with KRN5500 alone can be avoided and the whitening of the liver can be reduced by liposome formation.

The KRN5500 liposome preparation shown in Table 8 was obtained in the same manner as in Preparation Examples 1-6. Preparation Examples 19 to 22 and 24 prepared PEG5000-DSPE liposome preparations with different modification amounts, and Preparation Example 23 prepared PEG2000-DSPE liposome preparations with different PEG molecular weights. In addition, a large liposome preparation of about 200 nm was obtained only in Preparation Example 22. The results are shown in Table 8.
It became clear that the encapsulated amount of KRN5500 increased and the drug / total lipid ratio improved as the particle size increased. On the other hand, it became clear that the molecular weight of PEG does not significantly affect the drug / total lipid ratio.

[Test Example 5] Pharmacokinetics of liposome preparation containing water-soluble polymer having different PEG modification amount and PEG chain (retention in blood)
The KRN5500 liposome preparation prepared in Preparation Examples 19 to 23 was administered to rats at a dose of 0.2 mg / kg as the amount of KRN5500. In the same manner as in Test Example 1, blood was collected after a predetermined time, and the KRN5500 concentration in plasma was determined by high performance liquid chromatography. The results are shown in FIG.
It was clarified that the liposome preparation having a large particle size (Preparation Example 22) has a short half-life compared to the others, and the retention in blood is slightly reduced. However, it was not a large decrease, but it was suggested that the blood retention was within an acceptable range in which the EPR effect can be expected. In all other liposome preparations, a significant increase in residence time was observed compared to KRN5500 alone.

Test Example 6 In Vitro Cell Killing Effect of KRN5000 Liposome Formulation Containing PEG2000-DSPE Human colon cancer strain Colo205 or HT-29 in RPMI or McCoy's 5A medium containing 10% fetal bovine serum in 2000 cells / 90 were plated in 96well plates at a micro L / well, 37 ℃, and incubated for 5 hours at 5% CO _2. KRN5500 and the KRN5500 liposome preparation prepared in Preparation Example 23 were used as test substances, KRN5500 was DMSO, and the liposome preparation was a final concentration of 10 mM citric acid / 0.9% sodium chloride solution at pH 6.5 (0 to 10,000 nM as KRN5500). A 200-fold dilution series of 10 concentrations was prepared, and a test substance solution (specimen) was prepared by diluting it 20 times with RPMI or McCoy's 5A.
After adding 10 microL / well of the sample to a 96-well plate in which cells were seeded and culturing at 37 ° C., 5% CO _{2 for} 72 hours, CellTiter-Glo Luminescent Cell Viability Assay (Promega) reagent was added at 50 microL / well. Wells were added and the fluorescence intensity was measured with a luminometer.
Cell growth inhibitory action is 100% inhibition of fluorescence intensity (A) of cells (−) and medium + test substance medium alone, and fluorescence intensity (B) of cells (+) and medium + test substance medium alone is 0%. The cell killing effect (%) = 100 × (B−analyte fluorescence intensity) / (BA) was calculated as inhibition. An empty liposome preparation (same as the liposome of Preparation Example 23 but does not carry KRN5500 and therefore carries liposome and medium) was used as a control group. The results are shown in FIG. 9 and FIG. In any cell type, no cell killing effect was observed with the empty liposome preparation, whereas the KRN5000 liposome preparation containing PEG2000-DSPE showed high activity. The reason why the IC50 value of the liposome preparation is higher than that of KRN5500 alone is thought to be that KRN5500 exhibits an inhibitory action after being released from the liposome.

[Test Example 7] Antitumor effect of liposome preparation containing water-soluble polymer having different PEG modification amount and PEG chain (CoL-1 repeated administration)
Similar to Test Example 3, human colon cancer strain CoL-1 tumors were inoculated subcutaneously into nude mice (BALB / C, nu / nu, rabbits) and were transplanted in normal breeding. For the test, a tumor fragment was subcutaneously inoculated subcutaneously on the dorsal side of nude mice and reared normally. When the tumor volume reached around 100 mm ³ , a group setting was performed with n = 5 so that the tumor volume became comparable ( Day 0).
The KRN5500 liposome preparation prepared in Preparation Examples 19, 20, 22, 23, and 24 and KRN5500 alone were prepared in a medium to 1 mg / mL and intravenously administered at 10 mg / kg. After 0, 2, 4, 6, 8, 10, 13, 15, 18, 21 days after administration, the tumor volume was measured according to Test Example 3, and the mouse body weight (g) was measured as the body weight measurement value (g). -Calculated by tumor volume (mm ³ ) / 1000, and the weight change rate with Day 0 as 1 was used as an index. The results are shown in FIG. 11, FIG. 12, and FIG. The control group was a vehicle group of liposome preparation [Control (Liposome medium)], and the relative tumor volume (T / C) relative to the Control group was calculated using the following formula.
Relative tumor volume (T / C) for the Control group = Tumor volume of Day X / Tumor volume of Control group of Day X

For human colon cancer, the liposome preparation and KRN5500 alone showed a significant tumor growth inhibitory effect compared to the control group. In addition, the liposome preparation had a high and sustained antitumor effect, and tumor regression was observed up to 1/2 volume or less at the start of administration. Furthermore, it became clear that the mouse body weight loss of the liposome preparation was greatly suppressed as compared with KRN5500 alone and the side effects were reduced. In particular, the KRN5500 liposome preparation containing PEG ₂₀₀₀ -DSPE shows tumor regression at an early stage after administration up to 1/2 volume or less at the start of administration, and weight loss while maintaining the regression for a long period of time. It became clear that it was relaxation as well. On the other hand, KRN5500 alone, although having a high antitumor effect, also showed an increase in weight loss (particularly 2 to 4 days after administration), suggesting a strong side effect.

[Test Example 8] Antitumor effect of KRN5500 liposome preparation containing PEG2000-DSPE (CoL-1 repeated administration)
From the results of Test Example 6, it was speculated that the liposome preparation that exhibits the strong antitumor effect and can reduce the side effects to the maximum is the preparation containing PEG2000-DSPE. The anti-tumor effect of repeated administration on human colon cancer cells (CoL-1) was tested.
The liposome preparation prepared in Preparation Example 23 was prepared to 1 mg / mL with a medium, and 3.3, 5.5, 10.0, and 16.5 mg / kg were each three times at intervals of 4 days from Day 0 ( Day 0, 4, 8) Intravenous administration. In addition, 5.5 and 16.5 mg / kg were intravenously administered twice at a 6-day interval from Day 0 (Day 0, 6), respectively. The vehicle group of the liposome preparation was used as a control group (Control).
After Day 0, 2, 4, 6, 8, 10, 13, 15, 18 days, the tumor volume was measured according to Test Example 3, and the mouse body weight (g) was determined. The results of the group administered three times at a 4-day interval from Day 0 (q4d × 3) are shown in FIG. 14, FIG. 15 and FIG. 16, and the group administered twice at a 6-day interval from Day 0 (q6d × 2) It is shown in FIG. 17, FIG. 18 and FIG.
In human colorectal cancer, tumor regression was observed at a dose of 5.5 mg / kg or more and ½ volume or less at the start of administration in a group in which the liposome preparation was administered three times at intervals of 4 days from Day 0. At 3.3 mg / kg, although the tumor growth inhibitory effect was recognized, it did not reach the regression to 1/2 volume or less. As for body weight loss, a transient body weight loss was observed after the second administration at 16.5 mg / kg, but other body weights at 3.3, 5.5, 10.0 mg / kg There was little effect.
On the other hand, in the group in which the liposome preparation was administered twice from Day 0 at an interval of 6 days, tumor regression was observed up to 1/2 volume or less at the start of administration in both 5.5 and 16.5 mg / kg. Moreover, there was almost no effect on the body weight of the mice at any dose. From this, it became clear that even at a high dose of 16.5 mg / kg, the weight loss after the second administration can be avoided by setting the administration interval to 6 days.
From the above, it was revealed that the KRN5500 liposome preparation containing PEG2000-DSPE can avoid side effects while maintaining a strong antitumor action. In addition, in q4d × 3 and q6d × 2, the antitumor activity up to around Day 15 was almost the same between 5.5 mg / kg and 16.5 mg / kg. It was suggested that there is a possibility that side effects can be avoided while maintaining a strong antitumor effect.

[Test Example 9] Rat rat intermittent administration toxicity test of KRN5000 liposome formulation containing PEG2000-DSPE The KRN5500 liposome formulation prepared in Preparation Example 23 was prepared to 0.3 and 1.0 mg / mL in a medium, and 3 And 10 mg / kg were administered intravenously to male Crl: CD (SD) rats twice at 6-day intervals, and hematologic examination, blood biochemical examination, autopsy and histopathology 2 days after the second administration Examination was carried out. In addition, as a control, a group for administering a liposome preparation medium and empty liposomes was provided. The number of animals was 5 in each group. The results are shown in Table 10 and Table 11.

Inhibition of body weight gain was observed after the second dose in the 3 mg / kg group and after the first and second doses in the 10 mg / kg group compared to the empty liposome dose group. Hematological examinations showed mild anemia and elevated platelet levels in the 10 mg / kg group compared to the empty liposome administration group. In the blood biochemical test, alanine aminotransferase was used in the groups of 3 and 10 mg / kg in which the low values of total protein and albumin, the high values of aspartate aminotransferase (AST) and leucine aminopeptidase (LAP) were compared with the empty liposome administration group. A high (ALT) value was observed in the 10 mg / kg group. The grades were all mild to mild. On gross examination at necropsy, slight fading (whitening) of the liver was observed in 4 of 5 cases in the 3 mg / kg group and in all 5 cases in the 10 mg / kg group. Histopathological examination revealed hepatocyte vacuolation in 3 of 5 cases in the 3 mg / kg group, all 5 cases in the 10 mg / kg group, and decreased splenic extramedullary hematopoiesis in 5 cases in the 10 mg / kg group. It was observed in 4 cases.
In a single intravenous administration of 3.1 to 25.0 mg / kg of KRN5500 alone (n = 5 in each group), hepatocellular vacuolation and extramedullary hematopoiesis of the spleen were confirmed by histopathological examination 2 days after administration. A decrease was observed in each group of 3.1 mg / kg or more. Cortical area atrophy of the thymus, decreased hematopoiesis of the bone marrow, and necrosis of the seminal vesicle epithelium were observed in each group of 3.1 mg / kg or more, and necrosis of the prostate epithelium was observed in each group of 6.3 mg / kg or more. The changes in hematological test values and blood biochemical test values were almost the same as those of the KRN5500 liposome preparation.

Based on the above, it was considered that the toxicity when the KRN5000 liposome preparation containing PEG2000-DSPE was administered twice at intervals of 6 days was milder than when the KRN5500 alone was administered once. Therefore, it was clarified that the KRN5500 liposome preparation of this formulation can further reduce the toxicity of KRN5500 while maintaining a high antitumor effect.

Claims (6)

1. A liposome containing 100 to 50 mol% of phospholipid having cholesterol of 0 to 50 mol% and an acyl chain of a saturated fatty acid having a chain length of C ₁₄ to C ₁₈ is loaded with a spicamycin derivative represented by the chemical formula (I). Liposome formulation held:
   

   

   
   (Wherein R ₁ = H, R ₂ = OH, and R is straight-chain or branched alkyl having 9 to 15 carbon atoms or straight-chain alkenyl having 10 to 17 carbon atoms).
2. 2. The liposome preparation according to claim 1, wherein the lipid membrane of the liposome of the liposome preparation is any of the following 1) to 3):
   1) The lipid membrane of the liposome contains, as a main component, a phospholipid whose acyl chain is an acyl chain of a saturated fatty acid having an average chain length of more than 16 to 18.
   2) The lipid membrane of the liposome contains a phospholipid whose acyl chain is an acyl chain of a saturated fatty acid having an average chain length of more than 14 to 16 and cholesterol as main components, and the molar ratio of both components is 80:20 to 50:50,
   3) The lipid membrane of the liposome contains a phospholipid whose acyl chain is an acyl chain of a saturated fatty acid having an average chain length of 14 and cholesterol as main components, and the molar ratio of both components is 60:40 to 50:50 is there.
3. The liposome preparation according to claim 1 or 2, wherein the phospholipid is phosphatidylcholine.
4. The group R = CH ₃ (CH ₂ ) ₈ CH═CHCH═CH—, wherein the compound of formula (I) is (6- [4-de
   4. Oxy-4- (2E, 4E) -tetradecadienoyllycyl] -amino-L-glycero-b-L-mannenohypertopynoylyl) amino-9H-purine) (referred to as KRN5500) Liposome formulation.
5. The liposome preparation according to any one of claims 1 to 4, wherein the liposome preparation contains a hydrophilic polymer.
6. The liposome preparation according to any one of claims 1 to 5, wherein the area under the plasma concentration-time curve of the spicamycin derivative represented by the chemical formula (I) is 45 times or more that of the spicamycin derivative alone.

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