JPWO2009148169A1 - Tumor therapy using liposomes encapsulating ammine platinum complex at high concentration - Google Patents

Abstract

The present invention provides a therapeutic agent containing an ammine platinum complex (especially cisplatin) that has few side effects and acts specifically on tumors and cancer, and uses thereof. The present invention provides a composition for treating cancer or tumor. The composition includes cis-diamine dichloroplatinum (II), and the liposome includes a targeting agent on the surface of the liposome. In one embodiment, the targeting substance is selected from the group consisting of an antibody and a sugar chain. In a preferred embodiment, the targeting agent is sialyl Lewis X or an anti-E-selectin antibody. In one embodiment, the cancer or tumor is selected from the group consisting of colon cancer, lung cancer, squamous cell carcinoma, breast cancer and ovarian cancer.

Description

  The present invention relates to a composition comprising a liposome encapsulating an ammine platinum complex at a high concentration and a method for using the composition. Specifically, the present invention relates to a composition containing liposomes encapsulating cisplatin at a high concentration and a method for using the composition.

  Liposomes have a hollow spherical lipid bilayer structure. Therefore, a water-soluble drug can be encapsulated inside the lipid bilayer membrane. For this reason, liposomes have been extensively studied and used in fields such as pharmaceuticals, agricultural chemicals, and cosmetics. In particular, with regard to liposomes having a ligand bound to the outer membrane surface, many studies have been made as DDS materials for selectively delivering drugs or genes to target sites such as cancer and tumor.

  However, many of the drugs used for cancer / tumor treatment are poorly water-soluble, and the amount of drug that can be encapsulated in liposomes is very small. For example, in the case of cisplatin, since it does not dissolve in anything other than aqueous systems, and there is only a saturated concentration of about 2 mg / ml at room temperature even in aqueous systems, in order to administer an amount of a drug effective for cancer / tumor treatment to patients, There is an inconvenience that the amount of liposomes administered is necessarily high.

  Patent Document 1 describes a liposome preparation containing an anticancer agent such as cisplatin and carboplatin, an antipyretic agent such as aspirin, acetaminophen, and indomethacin, and a hormone such as cortisone acetate. However, the liposome preparation described in Patent Document 1 requires a heating step in its production process. In general, it is known that liposomes become unstable by heating. Therefore, the liposome produced by the method described in Patent Document 1 is unstable. In Patent Document 1, the amount of cisplatin encapsulated in liposomes is limited to 8.9 μg / mg lipid. Therefore, the liposome produced by the method described in Patent Document 1 could encapsulate cisplatin only in a small amount.

  Non-Patent Document 1 describes a liposome encapsulating cisplatin. However, this liposome could encapsulate the liposome only as much as 14.0 μg / mg lipid.

  Cisplatin is an anticancer drug that utilizes the reactivity of metal complexes. Cisplatin, which is a platinum metal complex, passes through the cancer cell membrane in an uncharged state and moves into the cell. Although the chloride ion concentration in blood is 103 mmol / l, it drops to 4 mmol / l in the cell. Therefore, the chloride ion of cisplatin is replaced with water molecules, and the equilibrium as shown in FIG. Is in a state.

  Cisplatin in this state binds to intracellular DNA in the manner shown in FIG. 1B, and exhibits anticancer activity by inhibiting DNA replication. However, since cisplatin has strong side effects such as nephrotoxicity and hearing loss, it has been attempted to develop various cisplatin derivatives and dosage forms as well as administration methods in order to maintain the anticancer action while reducing these side effects. It was.

  However, since drugs such as cisplatin with low water solubility cannot be encapsulated in liposomes at high concentrations, it has not been successful to deliver a sufficient amount of drug to the target site.

  Patent Document 2 describes a method for producing liposomes containing cisplatin complexed with lipids. However, the liposome of Citation 2 only incorporates cisplatin in an amount of 43 μg per mg of lipid.

  Patent Document 3 describes a method for producing liposomes containing cisplatin complexed with lipids and administering a lipid composition containing cisplatin using a nebulizer. However, the liposome of Reference 3 only incorporates cisplatin in an amount of 43 μg per mg of lipid.

  In Patent Documents 2 and 3, it is considered that most of the cisplatin taken up in the liposome is embedded on the surface of the liposome membrane or is present between the lipid membranes and hardly contained in the inner aqueous phase of the liposome. Accordingly, there is no description of liposomes that encapsulate cisplatin in a high concentration in the internal aqueous phase of the liposomes.

  Therefore, there is a demand for developing and providing a technique for delivering a large amount of poorly water-soluble drug to a target site.

Japanese Patent Laid-Open No. 5-255070 JP-T-2006-502233 International Publication No. 2005/084448 Pamphlet

Mary S. Newman, Gail T .; Colbern, Peter K. et al. Working et al., "Comparative pharmacokinetics, tissue distribution, and therapeutic effectiveness of cisplatin encapsulated in long-circulating, pegylated liposome (SPI-077) in tumor-bearing mice", Cancer Chemother Pharmacol (1999) 43: 1-7, pp.

  An object of the present invention is to provide a therapeutic agent containing an ammine platinum complex (particularly, cisplatin) that has few side effects and acts specifically on tumors and cancer, and a method for using the same.

  In order to solve the above-mentioned problems, the present inventors have conducted intensive research to include cisplatin by including target directivity on the surface of a liposome encapsulating a poorly water-soluble platinum complex (for example, cisplatin) at a high concentration. Succeeded to act specifically on tumors and cancer. Accordingly, the therapeutic agent comprising a liposome encapsulating the poorly water-soluble ammine platinum complex of the present invention at a high concentration makes it possible to minimize the side effects on the individual while at least maintaining the drug efficacy against the target. It has come to be solved.

In order to achieve the above object, the present invention provides, for example, the following means.
(Item 1) A composition for treating cancer or tumor, the composition comprising a liposome encapsulating cis-diamine dichloroplatinum (II), and the liposome is targeted to the surface of the liposome A composition comprising a directional substance.
(Item 2) A composition for treating cancer or tumor, the composition comprising liposomes containing more than 43 μg of cis-diamine dichloroplatinum (II) per 1 mg of lipid, A composition comprising a targeting substance on the surface of the liposome.
(Item 3) The composition according to the above item, wherein the cis-diamine dichloroplatinum (II) is contained in an amount of 200 µg or less per 1 mg of lipid.
(Item 4) The composition according to the above item, wherein the cancer or tumor is selected from the group consisting of colorectal cancer, lung cancer, squamous cell carcinoma, breast cancer and ovarian cancer.
(Item 5) The composition according to the above item, wherein the target-directing substance is selected from the group consisting of an antibody and a sugar chain.
(Item 6) The composition according to the above item, wherein the target-directing substance is sialyl Lewis X or an anti-E-selectin antibody.
(Item 7) The composition according to the above item, wherein the cis-diamine dichloroplatinum (II) is contained in an amount of more than 43 μg per 1 mg of lipid.
(Item 8) The composition according to the above item, wherein the cis-diamine dichloroplatinum (II) is formulated so as to be at least 500 mg / m ² .
(Item 9) Use of a liposome encapsulating cis-diamine dichloroplatinum (II) for the manufacture of a medicament for treating cancer or tumor, wherein the liposome is targeted to the surface of the liposome. Use, including substances.
(Item 10) The use according to item 9, wherein the target-directing substance is selected from the group consisting of an antibody and a sugar chain.
(Item 11) The use according to item 9 or 10, wherein the cis-diamine dichloroplatinum (II) is contained in an amount of more than 43 μg per 1 mg of lipid.
(Item 12) A liposome encapsulating cis-diamine dichloroplatinum (II) for treating cancer or tumor, wherein the liposome contains a targeting substance on the surface of the liposome.
(Item 13) The liposome according to item 12, wherein the target-directing substance is selected from the group consisting of an antibody and a sugar chain.
(Item 14) The liposome according to item 12 or 13, wherein the cis-diamine dichloroplatinum (II) is contained in an amount of more than 43 μg per 1 mg of lipid.
(Item 15) A method for treating cancer or tumor, comprising the step of administering a liposome encapsulating cis-diamine dichloroplatinum (II) to a subject in need of the treatment, The method, wherein the liposome comprises a targeting substance on the surface of the liposome.
(Item 16) The method according to item 15, wherein the target-directing substance is selected from the group consisting of an antibody and a sugar chain.
(Item 17) The method according to any one of Items 15 to 16, wherein the cis-diamine dichloroplatinum (II) is contained in an amount of more than 43 μg per 1 mg of lipid.
(Item 18) The method according to any one of Items 15 to 17, wherein the cis-diamine dichloroplatinum (II) is administered so as to be at least 500 mg / m ² .
It will be understood that these uses, liposomes, methods may further or alternatively include one or more of the features described above and are within the scope of the present invention.

  Accordingly, these and other advantages of the present invention will be apparent upon reading the following detailed description with reference to the accompanying drawings.

Until now, it was possible to encapsulate a poorly water-soluble ammine platinum complex (for example, cisplatin), which could not be encapsulated in liposomes, or to be efficiently encapsulated in liposomes. A platinum complex (eg, cisplatin) could be encapsulated in the liposome. In this liposome, most of the incorporated cisplatin is considered to exist in the internal aqueous phase of the liposome (confirmed by ¹⁹⁵ Pt-NMR). Thus, since cisplatin exists in the internal aqueous phase of the liposome, it is considered that cisplatin is not released until the liposome is taken into the cells at the target site and the liposome membrane is destroyed. Furthermore, the liposome can have the desired targeting. Therefore, by employing such liposomes, the composition of the present invention can be administered to a target at least to the same extent as before by administering a liposome preparation containing a poorly water-soluble ammine platinum complex at a much lower dose than the prior art. It was possible to reduce side effects on individuals while maintaining the efficacy (for example, anti-cancer effect).

  Preferred embodiments of the present invention will be shown below, but those skilled in the art can appropriately implement the embodiments and the like from the description of the present invention and well-known and common techniques in the field, and the effects and effects of the present invention can be easily achieved. It should be recognized to understand.

  Hereinafter, embodiments of the present invention will be described in detail.

FIG. 1A is a schematic diagram showing the behavior of cisplatin in cells. Cisplatin is present in a cisplatin state in plasma because the Ci ⁻ ion concentration is 103 mmol, but in the cell, the Cl ⁻ ion concentration is 4 mmol, so the Cl ⁻ ion of cisplatin is dissociated, It takes an equilibrium state with the structure coordinated by water molecules. FIG. 1B is a schematic diagram showing how cisplatin binds to DNA. A: A single bond (monofunctional bound) is a single bond with a guanine base. B: Interstrand crosslink is a state in which cisplatin is bound between two DNA strands. C: Intrastrand crosslink is a state in which cisplatin is single-bonded to DNA and protein is single-bonded. D: DNA-protein crosslink is a single bond with a guanine base, while it is bound to a protein. FIG. 1C shows the structures of cis-diamine dinitratoplatinum (II) (CDDP-3) and cis-diamine dichloroplatinum (II) (CDDP). FIG. 1D is a diagram showing a biosynthetic pathway of ganglio gangliosides. FIG. 2A shows the measurement result of the particle size of the cis-diamine dichloroplatinum (II) -encapsulating liposome prepared in Example 1 as a particle size distribution by strength. FIG. 2B shows the measurement result of the particle size of the cis-diamine dichloroplatinum (II) -encapsulating liposomes to which sugar chains are bound, prepared in Example 3, as a particle size distribution by strength. FIG. 3 is a diagram schematically illustrating the preparation of cisplatin-encapsulating liposomes. FIG. 4A shows that cisdiamine dinitratoplatinum (II) 5 mg / ml was dissolved in TAPS buffer (pH 8.4) containing 150 mM sodium chloride, and left at 25 ° C. (0 hour, 5 minutes, 10 minutes, The UV spectrum is shown after 15 minutes, 30 minutes and 45 minutes). 1: 0 hours, 2: 5 minutes later, 3:10 minutes later, 4:15 minutes later, 5:30 minutes later, 6:45 minutes later. FIG. 4B shows a UV spectrum when 1 mg of cis-diamine dichloroplatinum (II) was dissolved in 1 ml of TAPS buffer as a control. FIG. 5 shows the ¹⁹⁵ Pt NMR spectrum of cisplatin. In cisplatin, a chemical shift was observed at -2160 ppm. FIG. 5b shows the ¹⁹⁵ PtNMR spectrum of the nitric acid body (cis-diammine dinitratoplatinum (II)). In the nitric acid body (cis-diammine dinitratoplatinum (II)), a chemical shift was observed at -1620 ppm. FIG. 5 c shows a ¹⁹⁵ PtNMR spectrum of a nitric acid (cis-diammine dinitratoplatinum (II))-encapsulating liposome (NaCl ⁺ ). The chemical shift of nitric acid (cis-diammine dinitratoplatinum (II)) encapsulated in the liposome was observed at −2160 ppm, which is the same as cisplatin when NaCl was contained in the external liquid. FIG. 5d shows a ¹⁹⁵ Pt-NMR spectrum when cis-diammine dinitratoplatinum (II) encapsulated in liposomes was not changed to cis-diamine dichloroplatinum (II) (4.75 mM, heavy water). FIG. 6 shows the measurement result of the particle size of cis-diamine dichloroplatinum (II) -encapsulating liposomes to which the antibody prepared in Example 5 was bound, as a particle size distribution by strength. FIG. 7 is a photograph of cis-diamine dichloroplatinum (II) -encapsulating liposome I observed with a transmission electron microscope. Liposome I after cis-diammine dinitratoplatinum (II) was encapsulated in liposome I and then replaced with a TAPS buffer containing 150 mM sodium chloride was observed with H-7100S (HITACHI). Bar indicates 100 nm. FIG. 8 shows the ¹⁹⁵ Pt NMR spectrum of cisplatin. For the measurement, INOVA-600 (Varian) was used. FIG. 8a shows the ¹⁹⁵ Pt NMR spectrum of cis-diamine dichloroplatinum (II) (6.6 mM, heavy water). FIG. 8b shows the cis-diammine dinitratoplatinum (II) (13.7 mM, heavy water) ¹⁹⁵ PtNMR spectrum. FIG. 8c shows that after encapsulating cis-diammine dinitratoplatinum (II) in liposomes, the external solution was exchanged with a buffer containing 150 mM sodium chloride, and cis-diammine dinitratoplatinum (II) was replaced with cis-diamine dichloroplatinum (II). ¹⁹⁵ PtNMR spectrum when converted to (II) (4.75 mM, heavy water) is shown. FIG. 8d shows a ¹⁹⁵ Pt-NMR spectrum when cis-diammine dinitratoplatinum (II) encapsulated in liposomes was not changed to cis-diamine dichloroplatinum (II) (4.75 mM, heavy water). FIG. 9 shows in vitro cytostatic activity of SKBr3: human breast cancer cells by cisplatin-encapsulated liposomes. The vertical axis represents the cell viability (%), and the horizontal axis represents the added cisplatin concentration (CDDP concentration; μM). The cell viability (%) indicates the cell viability (%) of the section to which cisplatin was added, assuming that the control viability was 100%. The cisplatin concentration indicates the final concentration of cisplatin added to the cells. Black circle: cisplatin-encapsulated unmodified liposome, black triangle: cisplatin-encapsulated SLX-modified liposome, black square: cisplatin-encapsulated antibody-modified liposome FIG. 10 shows the in vitro cell growth inhibitory activity of cis29-encapsulated liposomes against HT29: human colon cancer cells. The vertical axis represents the cell viability (%), and the horizontal axis represents the added cisplatin concentration (CDDP concentration; μM). The cell viability (%) indicates the cell viability (%) of the section to which cisplatin was added, assuming that the control viability was 100%. The cisplatin concentration indicates the final concentration of cisplatin added to the cells. Black circle: cisplatin-encapsulated unmodified liposome, black triangle: cisplatin-encapsulated SLX-modified liposome, black square: cisplatin-encapsulated antibody-modified liposome FIG. 11 shows in vitro cytostatic activity of A549: human lung cancer cells by cisplatin-encapsulated liposomes. The vertical axis represents the cell viability (%), and the horizontal axis represents the added cisplatin concentration (CDDP concentration; μM). The cell viability (%) indicates the cell viability (%) of the section to which cisplatin was added, assuming that the control viability was 100%. The cisplatin concentration indicates the final concentration of cisplatin added to the cells. Black circle: cisplatin-encapsulated unmodified liposome, black triangle: cisplatin-encapsulated SLX-modified liposome, black square: cisplatin-encapsulated antibody-modified liposome FIG. 12 shows in vitro cytostatic activity of LLC: mouse lung cancer cells by cisplatin-encapsulated liposomes. The vertical axis represents the cell viability (%), and the horizontal axis represents the added cisplatin concentration (CDDP concentration; μM). The cell viability (%) indicates the cell viability (%) of the section to which cisplatin was added, assuming that the control viability was 100%. The cisplatin concentration indicates the final concentration of cisplatin added to the cells. Black circle: cisplatin-encapsulated unmodified liposome, black triangle: cisplatin-encapsulated SLX-modified liposome, black square: cisplatin-encapsulated antibody-modified liposome FIG. 13 shows in vitro cell growth inhibitory activity of A431: human squamous cell carcinoma cells by cisplatin-encapsulated liposomes. The vertical axis represents the cell viability (%), and the horizontal axis represents the added cisplatin concentration (CDDP concentration; μM). The cell viability (%) indicates the cell viability (%) of the section to which cisplatin was added, assuming that the control viability was 100%. The cisplatin concentration indicates the final concentration of cisplatin added to the cells. Black circle: cisplatin-encapsulated unmodified liposome, black triangle: cisplatin-encapsulated SLX-modified liposome, black square: cisplatin-encapsulated antibody-modified liposome FIG. 14 shows in vitro cytostatic activity by cisplatin (CDDP). The vertical axis represents the cell viability (%), and the horizontal axis represents the added cisplatin concentration (μM). The cell viability (%) indicates the cell viability (%) of the section to which cisplatin was added, assuming that the control viability was 100%. The cisplatin concentration indicates the final concentration of cisplatin added to the cells. There was a tendency for the cell viability to decrease depending on the cisplatin concentration. Differences in sensitivity to cisplatin were observed depending on the cell line, but all cells were sensitive to cisplatin. White circle: HT29 (human colon cancer cell), black diamond: A549 (human lung cancer cell), black circle: A431 (human squamous cell carcinoma cell), cross (x): LLC (mouse lung cancer cell), black square: SKOV3 (human ovary) Cancer cells) FIG. 15 is a graph showing a relationship between an acute toxicity test of CDDP-SLX-Lip and body weight fluctuation. CDDP-SLX-Lip (18 mg CDDP equivalent / Kg, 25 mg CDDP equivalent / Kg, 50 mg CDDP equivalent / Kg body weight) and CDDP (18 mg / Kg body weight, 25 mg / Kg body weight) from the tail vein of normal mice Balb / c (female, 8 weeks old) ) Was administered once, and the survival rate for 14 days was examined. The vertical axis indicates the survival rate (%), and the horizontal axis indicates the number of days (day) after administration. Black triangle: CDDP (25 mg / Kg), black circle: CDDP (18 mg / Kg), white square: CDDP-SLX-Lip (50 mg / Kg), white triangle: CDDP-SLX-Lip (25 mg / Kg), white circle: CDDP -SLX-Lip (18 mg / Kg) FIG. 16 is a graph showing changes in body weight when the body weight before administration is taken as 100%. The weight of the surviving mice was measured. The vertical axis represents weight fluctuation (%), and the horizontal axis represents the number of days (day) after administration. Black circle + dotted line: CDDP (25 mg / Kg), black triangle + dotted line: CDDP (18 mg / Kg), white square: CDDP-SLX-Lip (50 mg / Kg), white circle: CDDP-SLX-Lip (25 mg / Kg), White triangle: CDDP-SLX-Lip (18 mg / Kg), black circle + solid line: physiological saline, black triangle + solid line: HEPES buffer, black square + solid line: liposome FIG. 17a shows the results of HE staining of mouse kidney administered with CDDP-SLX-Lip equivalent to 25 mg CDDP per kg body weight of the mouse. FIG. 17b shows the results of Tunel immunohistochemical staining of kidneys administered with CDDP-SLX-Lip equivalent to 25 mg CDDP per kg of mouse body weight. FIG. 17 c shows the results of H · E staining of kidney administered with 25 mg CDDP per kg of mouse body weight. The arrow indicates the expansion site of the proximal tubule. FIG. 17d shows the results of Tunel immunohistochemical staining of kidney administered 25 mg CDDP per kg body weight of the mouse. Arrows indicate Tunel positive cells. FIG. 17e shows the result of HE staining of the spleen administered with CDDP-SLX-Lip equivalent to 25 mg CDDP per kg body weight of the mouse. FIG. 17 f shows the results of Tunel immunohistochemical staining of the spleen administered with CDDP-SLX-Lip equivalent to 25 mg CDDP per kg of mouse body weight. Black arrows indicate Tunel positive cells. FIG. 17g shows the results of HE staining of the spleen administered with 25 mg CDDP per kg of mouse body weight. A: Red spleen, B: Follicle FIG. 17h shows the results of Tunel immunohistochemical staining of the spleen administered with CDDP per kg of mouse body weight. Black arrows indicate Tunel positive cells. FIG. 17i shows the results of HE staining of the liver administered with CDDP-SLX-Lip equivalent to 25 mg CDDP per kg of mouse body weight. The arrow indicates the small granuloma site. FIG. 17j shows the results of Tunel immunohistochemical staining of the liver administered with CDDP-SLX-Lip equivalent to 25 mg CDDP per kg body weight of the mouse. FIG. 17k shows the results of H · E staining of liver administered with 25 mg CDDP per kg of mouse body weight. FIG. 17l shows the results of Tunel immunohistochemical staining of liver administered with 25 mg CDDP per kg body weight of the mouse. FIG. 18 shows the antitumor effect of CDDP-Lip and CDDP-SLX-Lip on A549 cell transplanted mice. Bar: mean ± SD, n = 4, white triangle indicates the administration date. The vertical axis is the tumor volume (mm ³ ), and the horizontal axis is the number of days after cell transplantation. Black circle: physiological saline, black triangle: CDDP-SLX-Lip, black square: CDDP-Lip FIG. 19 is a graph showing the toxicity and antitumor activity of sialyl Lewis X-conjugated cisplatin liposomes. Black circles indicate A549 cells, black triangles indicate SKBr3 cells, black squares indicate A431 cells, white circles indicate LLC cells, and white triangles indicate HT29 cells. The vertical axis represents the survival rate (%), and the horizontal axis represents the added CDDP concentration (μM). (Bar: mean ± SD, n = 3) FIG. 20 is a graph showing the accumulation property of sialyl Lewis X-bound cisplatin liposomes on a cancer site. CDDP-Lip is an unmodified liposome encapsulating CDDP, and CDDP-SLX-Lip is a sialyl Lewis X-modified liposome encapsulating CDDP. The vertical axis represents the amount of platinum (ng / g tumor). (Bar: mean ± SD, n = 3)

  The present invention will be described below. Throughout this specification, singular articles (eg, “a”, “an”, “the”, etc. in the English language) are understood to include the concept of the plural unless specifically stated otherwise. Should be. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. In case of conflict, the present specification, including definitions, will control.

(Definition of terms)
Listed below are definitions of terms particularly used in the present specification.

  As used herein, “ammine platinum complex” refers to a platinum complex having ammonia. As an ammine platinum complex, for example, cis-diamminedichloroplatinum (II) <“cisplatin” is known. >, Cis-diammine dinitratoplatinum (II), cis-diammine-1,1-cyclobutane-dicarboxylatoplatinum (II) <known as carboplatin. >, Cis-diammine (glycolato) platinum (II) <known as nedaplatin. >, (1R, 2R-diamminecyclohexane) oxalatoplatinum (II) <known as oxaliplatin. >, Cis-diamminedibromoplatinum (II) <having anticancer activity. >, Μ-hydroxoplatinum (II) <having anticancer activity. >, But is not limited thereto. Among these, as the poorly water-soluble ammine platinum complex, for example, cis-diamminedichloroplatinum (II), cis-diammine-1,1-cyclobutane-dicarboxylatoplatinum (II), cis-diamminedibromoplatinum (II) Etc.

  In the present specification, “cis-diamminedichloroplatinum (II) (CDDP)” is also referred to as cisplatin. This has the following structure:

Cisplatin has an effect on the treatment of various cancers and tumors. Examples of cancer and tumor include, for example, testicular tumor, bladder cancer, renal pelvis / ureteral tumor, prostate cancer, ovarian cancer, head and neck cancer, non-small cell lung cancer, esophageal cancer, cervical cancer , Neuroblastoma, gastric cancer, small cell lung cancer, osteosarcoma, germ cell tumor and the like. Here, cancer includes all neoplastic diseases such as tumor and leukemia.

  In the present specification, the “water-soluble form of cis-diamine dichloroplatinum (II)” refers to cisplatin in a water-soluble state. For example, the water-soluble form of cis-diamine dichloroplatinum (II) includes cis-diammine dinitratoplatinum (II) (CDDP-3).

  In the present specification, “cis-diammine dinitratoplatinum (II) (CDDP-3)” is also referred to as cisplatin nitrate. This has the following structure:

In the present specification, “cis-diammine-1,1-cyclobutane-dicarboxylatoplatinum (II)” is also referred to as carboplatin. This has the following structure:

Carboplatin has an effect on the treatment of various cancers and tumors. Examples of cancer and tumor include, for example, testicular tumor, bladder cancer, renal pelvis / ureteral tumor, prostate cancer, ovarian cancer, head and neck cancer, non-small cell lung cancer, esophageal cancer, cervical cancer , Neuroblastoma, gastric cancer, small cell lung cancer, osteosarcoma, germ cell tumor and the like. Here, cancer includes all neoplastic diseases such as tumor and leukemia.

  In the present specification, “cis-diammine (glycolato) platinum (II)” is also referred to as nedaplatin. This has the following structure:

Nedaplatin has an effect on the treatment of various cancers and tumors. Examples of cancer and tumor include, for example, testicular tumor, bladder cancer, renal pelvis / ureteral tumor, prostate cancer, ovarian cancer, head and neck cancer, non-small cell lung cancer, esophageal cancer, cervical cancer , Neuroblastoma, gastric cancer, small cell lung cancer, osteosarcoma, germ cell tumor and the like. Here, cancer includes all neoplastic diseases such as tumor and leukemia.

  In the present specification, “1R, 2R-diamminecyclohexane) oxalatoplatinum (II)” is also referred to as oxaliplatin. this is,:

Oxaliplatin has an effect on the treatment of various cancers and tumors. Examples of cancer and tumor include, for example, testicular tumor, bladder cancer, renal pelvis / ureteral tumor, prostate cancer, ovarian cancer, head and neck cancer, non-small cell lung cancer, esophageal cancer, cervical cancer , Neuroblastoma, gastric cancer, small cell lung cancer, osteosarcoma, germ cell tumor and the like. Here, cancer includes all neoplastic diseases such as tumor and leukemia.

  As used herein, “cis-diamminedibromoplatinum (II)” has the following structure:

Cis-diammine dibromoplatinum (II) has anticancer activity.

  In the present specification, the “μ-hydroxoplatinum (II) binuclear complex” includes, for example, a compound having the following structure:

The μ-hydroxoplatinum (II) binuclear complex has anticancer activity.

  In these platinum complexes, the bond between Pt and the ammine ligand is strong. Therefore, the platinum complex is present in an aqueous solution in a state where the bond part of Pt and O is dissociated.

  In the present invention, it is considered important that the water-soluble platinum complex is present in contact with the liposome when it is present as the liposome. Therefore, it is understood that the effects of the present invention can be achieved even if a platinum complex other than cisplatin can be once made into a water-soluble form and then form a poorly water-soluble salt. In this field, ionic species that form such water-soluble and poorly water-soluble salts for the above platinum complexes or other platinum complexes are known or can be appropriately selected. It will be understood that the present invention may be practiced even if it exists. And as a standard of selection of water solubility, 0.5g / 100g water-10g / 100g water (for example, 0.5g / 100g water, 1.0g / 100g water, 1.5g / 100g water, 2.0g / 100 g water, 2.5 g / 100 g water, 3.0 g / 100 g water, 3.5 g / 100 g water, 4.0 g / 100 g water, 4.5 g / 100 g water, 5.0 g / 100 g water, 5.5 g / 100 g Water, 6.0 g / 100 g water, 6.5 g / 100 g water, 7.0 g / 100 g water, 7.5 g / 100 g water, 8.0 g / 100 g water, 8.5 g / 100 g water, 9.0 g / 100 g water 9.5 g / 100 g water, 10 g / 100 g water or more (above, upper limit), 10 g / 100 g water, 9.5 g / 100 g water, 9.0 g / 100 g water, 8.5 g / 100 g water, 8.0 g / 100g water, 7.5g / 00 g water, 7.0 g / 100 g water, 6.5 g / 100 g water, 6.0 g / 100 g water, 5.5 g / 100 g water, 5.0 g / 100 g water, 4.5 g / 100 g water, 4.0 g / 100 g Water, 3.5 g / 100 g water, 3.0 g / 100 g water, 2.5 g / 100 g water, 2.0 g / 100 g water, 1.5 g / 100 g water, 1.0 g / 100 g water or less (above, lower limit) Any range of possible combinations) (solubility of water-soluble ones), and poorly water-soluble selection criteria include 0.0 g / 100 g water to 0.5 g / 100 g water (eg, 0.0 g / 100 g water, 0.05 g / 100 g water, 0.1 g / 100 g water, 0.15 g / 100 g water, 0.2 g / 100 g water, 0.25 g / 100 g water, 0.3 g / 100 g water, 0.35 g / 100 g water, 0 .4 g / 100 g water, 0. 5 g / 100 g water, 0.5 or more (above, upper limit), 0.5 g / 100 g water, 0.45 g / 100 g water, 0.4 g / 100 g water, 0.35 g / 100 g water, 0.25 g / 100 g Water, 0.2 g / 100 g water, 0.15 g / 100 g water, 0.1 g / 100 g water, 0.05 g / 100 g water or less (above, lower limit possible combinations in any range) Solubility) can be expressed as solubility.

In the present specification, the “platinum complex raw material” refers to a raw material capable of forming a water-soluble ammine platinum complex when mixed with a buffer solution. Therefore, any material may be used as long as it can form a water-soluble ammine platinum complex by mixing, and a plurality of raw materials may be used. Examples of the platinum complex material that can be used in the present specification include cis- [Pt (NH ₃ ) ₂ I ₂ ], cis- [Pt (NH ₃ ) ₂ Cl ₂ ], and cis- [Pt (NH ₃ ) _2. Br ₂ ], K ₂ PtCl _4, but are not limited thereto. For example, cis- [Pt (NH ₃ ) ₂ I ₂ ], cis- [Pt (NH ₃ ) ₂ Cl ₂ ], cis- [Pt (NH ₃ ) ₂ Br ₂ ], K ₂ PtCl ₄ is silver nitrate (AgNO ₃ ) Add nitric acid by adding 2 equivalents (1.98 equivalents).

  For example, in the case of cisplatin, the solubility of cisplatin in water is 2 mg / ml (room temperature). Cisplatin nitrate is 20 mg / ml. That is, it can be used with a platinum complex having a solubility of cisplatin (room temperature) or higher. In the art, ionic species that form such water-soluble and sparingly water-soluble salts are known or can be appropriately selected for the above-mentioned cisplatin or other platinum complexes. However, as with cisplatin, it can be appropriately selected by considering its solubility. At this time, it can implement similarly by adjusting suitably the equivalent number of silver nitrate to add. Further, the bromide ion concentration and the iodide ion concentration are the same as those of the chloride ion and can be carried out without any problem. Since bromide ions and iodide ions are stronger in binding than chloride ions, even under the same conditions, poorly water-soluble substances are considered to be formed. Moreover, since it is thought that there is no difference in the liposome membrane permeability | transmittance of each ion, it can implement in consideration of these factors.

  In this specification, the “ammine group” is a name given when an ammonia molecule is bonded to another group.

  In the present specification, the “liposome” usually means a closed vesicle composed of a lipid layer assembled in a membrane shape and an inner aqueous layer. In addition to phospholipids typically used, cholesterol, glycolipids and the like can also be incorporated. Since liposomes are closed vesicles containing water inside, it is possible to retain water-soluble drugs and the like in the vesicles. Therefore, such liposomes are used to deliver drugs and genes that cannot pass through the cell membrane into the cell. Moreover, since biocompatibility is good, the expectation as a nanoparticulate carrier material for DDS is great. In the present invention, the liposome is a structural unit having a functional group that imparts an ester bond (eg, glycolipid, ganglioside, phosphatidylglycerol, etc.) or a structural unit that has a functional group that imparts a peptide bond in order to attach a modifying group. (Eg, phosphatidylethanolamine).

  The liposome used in the present invention may be prepared by any production method as long as it can pass a water-soluble ammine platinum complex. In the present invention, the liposome can be used by mixing liposomes prepared by a plurality of production methods (for example, modified cholate method and freeze-dried method). This is because it is sufficient that the water-soluble ammine platinum complex can be transferred and maintained in the liposome through the liposome membrane. Such liposomes can be used by those skilled in the art by examining the amount of water-soluble platinum complex encapsulated in the liposome combined with the constituent lipid components, the efficiency of encapsulation, leakage after encapsulation, and the stability of the liposome. It can be determined as appropriate. And the judgment criteria are as follows.

  The encapsulated amount may be any amount of platinum according to its use by measuring the amount of platinum in the liposome by the atomic absorption height method (FAAS).

  The encapsulation efficiency can be calculated as a ratio of the amount of platinum encapsulated in the liposome with respect to the initial amount of platinum complex. Although it depends on the application, for example, 0.5% or more of a platinum complex may be included.

  Leakage after encapsulation can be evaluated by quantifying the amount of platinum in the liposome over time by atomic absorption spectrophotometry (FAAS) and comparing it with the amount of platinum immediately after encapsulation.

  The stability of the liposome can be confirmed by measuring the particle size distribution over time.

  If the liposome membrane is too hard, it will not allow the substance to permeate. If it is too soft, the liposome itself will become unstable. The firmness of the liposome membrane is determined by the type and mixing ratio of the liposome constituent components. The firmness and fluidity of the liposome membrane affects the barrier ability of the liposome.

  The reason why the water-soluble substance remains in the aqueous phase in the liposome is that the lipid bilayer works as a barrier to the water-soluble substance. The fluidity and permeability of the membrane are completely different between the gel phase and the liquid crystal phase. In general, the liquid crystal phase contains more lipids having a low phase transition temperature, and the fluidity increases as the temperature increases. The substance encapsulated in the inner aqueous phase of the liposome varies in leakage depending on the phase transition and fluidity of the membrane.

  Liposomes composed of saturated lipids exhibit high barrier ability (difficult to permeate but difficult to leak) in the gel phase. Liposomes composed of saturated lipids lose their barrier ability above the phase transition temperature, but the barrier ability is restored by adding cholesterol or a small amount of unsaturated fatty acids to the saturated lipid in the liquid crystal phase.

  Liposomes composed of unsaturated fatty acids have a barrier ability in the liquid crystal phase, but their fluidity and low-molecular membrane permeability increase with increasing temperature. Even in unsaturated fatty acid membranes, the addition of cholesterol increases the barrier ability of the membrane. That is, as the cholesterol content increases, the permeability decreases, but the substance in the inner aqueous phase can remain stable.

  When cholesterol is contained in the membrane component, the state of phase transition and the fluidity of the membrane change greatly. Cholesterol increases the interaction between lipid molecules in unsaturated lipid membranes, causing a decrease in fluidity and membrane permeability. On the other hand, when added to a saturated lipid, the phase transition disappears and a film having fluidity is obtained even at the temperature of the gel phase.

  These effects are thought to occur because cholesterol forms a complex with lipid molecules. In the presence of cholesterol, the dramatic fluidity change at the phase transition disappears, resulting in a decrease in fluidity above the phase transition temperature, while an increase in fluidity is observed below the phase transition temperature.

  Liposomes used in the present invention, when cholesterol is contained, have a ratio sufficient to achieve hardness (for example, cholesterol to phospholipids) when the encapsulated substance passes through the membrane and remains in the liposome. For example, about 30 to 50 mol%, about 30 mol% or more, about 35 mol% or more, about 40 mol% or more, about 45 mol% or more, about 50 mol% or less, about 45 mol% or less, about 40 mol% or less, may be about 35 mol% or less, may be about 50 mol% or more, or about 30 mol%, any possible combination thereof).

  The liposome used in the present invention may be anything as long as the water-soluble ammine platinum complex can pass through the liposome membrane and enter the liposome. This is because the water-soluble ammine platinum complex only has to pass through the provided liposome or the membrane of the formed liposome and migrate into the liposome and remain there. As the liposome, commercially available liposomes may be used, for example, commercially available from NOF Corporation. Liposomes prepared by a plurality of production methods (for example, modified cholate method and lyophilized method) can also be used in combination.

  The preparation and characterization of general liposomes is known in the art and requires only routine experimentation and technical knowledge in the art. For example, it may be prepared by sonication method, ethanol injection method, French press method, ether injection method, cholic acid method, lyophilization method, reverse phase evaporation method (for example, “New Development of Liposome Application-Artificial Toward the development of cells-supervision Kazunari Akiyoshi / Kaoru Sakurai NTS p33-45 (2005) "," Liposome Nojima Shochichi p21-40 Nanedo (1988) ".

  For example, among them, a method using a cholic acid dialysis method is exemplified. In the cholate dialysis method, production is carried out by a) preparation of mixed micelles of lipid and surfactant, and b) dialysis of mixed micelles. Next, in a preferred embodiment of the sugar chain liposome of the present invention, it is preferable to use a protein as a linker, and coupling of a glycoprotein having a sugar chain bound to the protein to the liposome can be performed by the following two-step reaction. it can. a) periodate oxidation of the ganglioside moiety on the liposome membrane, and b) coupling of the glycoprotein to the oxidized liposome by reductive amination reaction. By such a technique, a glycoprotein containing a desired sugar chain can be bound to the liposome, and a wide variety of glycoprotein / liposome conjugates having the desired sugar chain can be obtained. It is very important to examine the particle size distribution to see the purity and stability of the liposomes. As the method, gel filtration chromatography (GPC), scanning electron microscope (SEM), dynamic light scattering (DLS), or the like can be used. As an example, dipalmitoyl phosphatidylcholine, cholesterol, ganglioside, dicetyl phosphate, dipalmitoyl phosphatidylethanolamine and sodium cholate can be used to produce liposomes of the type 35: 40: 15: 5: 5: 167.

  Liposomes can also be prepared by lyophilization. The lyophilization method is described in, for example, H.H. Kikuchi, N .; Suzuki et al, Biopharm. Drug Dispos. 17, 589-605 (1999). For example, it can be prepared by the following method. The liposome solution is frozen at −40 to −50 ° C. and lyophilized. Add the inclusion substance solution to the lyophilized powder and rehydrate. Remove the unencapsulated material by ultrafiltration or dialysis. If necessary, sugar or the like is added to the initial liposome solution. Further, depending on the purpose, the particle size is adjusted by a French press method or a membrane filter method.

  As used herein, the term “liposome source” refers to a lipid capable of forming liposomes when mixed with a buffer. These include, for example, micellar suspensions used in the examples, but can form liposomes when contacted with a water-soluble ammine platinum complex, allowing the water-soluble ammine platinum complex to pass through. Other lipids may be used as long as they can be used. The liposome raw material used in this production method depends on the use of the liposome to be produced. The amount of the water-soluble platinum complex encapsulated in the liposome combined with the constituent lipid components, the encapsulation efficiency, the leakage after encapsulation, and the stability of the liposome Those skilled in the art can appropriately determine the above.

  As used herein, the term “lipid” refers to a long chain aliphatic hydrocarbon or derivative thereof. “Lipid” is a general term for compounds composed of, for example, fatty acids, alcohols, amines, aminoalcohols, aldehydes and the like. Examples of the lipid having liposome-forming ability used in the present invention or the lipid constituting the liposome include, for example, phosphatidylcholines, phosphatidylethanolamines, phosphatidic acids, long-chain alkyl phosphates, glycolipids (gangliosides, etc.), Phosphatidylglycerols, sphingomyelins, cholesterols, etc.

  Examples of the phosphatidylcholines include dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, and the like.

  Examples of the phosphatidylethanolamines include dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, and the like.

  Examples of the phosphatidic acids include dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, and distearoyl phosphatidic acid. Examples of long-chain alkyl phosphates include dicetyl phosphate.

  Examples of glycolipids include galactosylceramide, glucosylceramide, lactosylceramide, phosphnatide, globoside, gangliosides and the like. Examples of gangliosides include ganglioside GM1 (Galβ1,3GalNAcβ1,4 (NeuAα2,3) Galβ1,4Glcβ1,1′Cer), ganglioside GDla, ganglioside GTlb, and the like.

  As the phosphatidylglycerols, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol and the like are preferable.

  Of these, phosphatidic acids, long-chain alkyl phosphates, glycolipids, and cholesterol have the effect of increasing the stability of the liposomes, so it is desirable to add them as constituent lipids. For example, as the lipid constituting the liposome used in the present invention, phosphatidylcholines (molar ratio 0 to 70%), phosphatidylethanolamines (molar ratio 0 to 30%), phosphatidic acids, and long-chain alkyl phosphates are used. One or more lipids selected from the group consisting of (1 to 30% molar ratio), one or more lipids selected from the group consisting of glycolipids, phosphatidylglycerols and sphingomyelin (molar ratio 0 to 40%) ), And cholesterol (molar ratio 0 to 70%). Preferably it contains gangliosides, glycolipids or phosphatidylglycerols. This is because a linker such as albumin is easily bonded.

  Examples of the lipid or liposome material constituting the liposome used in the present invention include dipalmitoyl phosphatidylcholine, cholesterol, ganglioside, dicetyl phosphate, dipalmitoyl phosphatidylethanolamine, sodium cholate, dicetylphosphatidylethanolamine-poly Glycerin 8G, Dimyristoylphosphatidylcholine, Distearoylphosphatidylcholine, Dioleoylphosphatidylcholine, Dimyristoylphosphatidylserine, Dipalmitoylphosphatidylserine, Distearoylphosphatidylserine, Dioleoylphosphatidylserine, Dimyristoylphosphatidylinositol I Cytol, dioleoylphosphatidylinositol, dimyristoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, distearoylphosphatidic acid, dioleoylphosphatidic acid, galactosylceramide , Glucosylceramide, lactosylceramide, phosphtide, globotide, GM1 (Galβ1,3GalNAcβ1,4 (NeuAα2,3) Galβ1,4Glcβ1,1′Cer), ganglioside GD1a, ganglioside GD1b, dimyristoylphosphatidylphosphine Stearoyl phosphatidyl glyce Lumpur, but dioleoylphosphatidylglycerol like, without limitation. Preferably, dipalmitoyl phosphatidylcholine, cholesterol, ganglioside, dicetyl phosphate, dipalmitoyl phosphatidylethanolamine, sodium cholate, dicetylphosphatidylethanolamine-polyglycerin 8G, dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylglycerol may be used.

  In the present specification, the “ammine platinum complex-encapsulating liposome” refers to a liposome encapsulating an ammine platinum complex. The “ammine platinum complex-encapsulating liposome” and the “ammine platinum complex-containing liposome” may be used interchangeably herein.

(Production method)
The ammine platinum complex-encapsulating liposome used in the present invention can be produced by the following method. Specifically, the method comprises: A) providing a water-soluble ammine platinum complex, a platinum complex raw material or a combination thereof; B) providing a liposome, a liposome raw material or a combination thereof; and C) the water-soluble ammine platinum. A step of preparing a mixture containing a complex, a platinum complex raw material or a combination thereof, and the liposome, the liposome raw material or a combination thereof, and subjecting the mixture to a liposome formation maintaining condition, provided that the platinum complex is present when the liposome is present. A step of subjecting the salt formed to a condition in which the salt is in a water-soluble state. The production method used in the present invention may further include a step of D) providing the mixture obtained in the step C) in the presence of a solution containing an ion in which the salt formed by the platinum complex is sparingly water-soluble. .

  Preferably, in step D), i) a step of hydrophilizing the formed liposome; ii) a step of binding a target-directing substance to the liposome; iii) a liposome having the modified target-directing substance bound thereto It may include a step of hydrophilizing, and iv) filtering the solution containing the hydrophilized liposome.

  In one embodiment, in this production method, in the step C), the water-soluble ammine platinum complex, platinum complex raw material or a combination thereof, and the liposome, liposome raw material or a combination thereof are 1: 9 to 9: 1. Can be mixed in a ratio within the range of

  In one embodiment, the platinum complex suspension and lipid suspension may be mixed in a ratio within the range of 1: 9 to 9: 1.

  In a preferred embodiment, the platinum complex suspension and lipid suspension may be mixed in a ratio of 7: 3.

  In another embodiment, the platinum complex solution and the liposome can be mixed in a ratio within the range of 1: 9 to 9: 1 (preferably 7: 3).

In one embodiment, in the present production method, in the step C), the mixture substantially does not contain ions in which a salt to be formed is poorly water-soluble. The ions that form the poorly water-soluble salt include, for example, chloride ions (Cl ⁻ ), bromide ions (Br ⁻ ), iodide ions (I ⁻ ), thiocyanate ions (SCN ⁻ ), cyanide ions ( CN ^-) may be like. Preferably, the ion that forms the poorly water-soluble salt may be a chloride ion (Cl ⁻ ). In one embodiment, the mixture may contain chloride ions (Cl ⁻ ) in the range of 0-4 mM. The mixture should not contain ions that are poorly water-soluble, or even contain enough ions to form a poorly water-soluble salt.

  In one embodiment, the mixture comprises N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4), carbonate buffer (pH 8.5), phosphate buffer (pH 8.0). 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES) buffer (pH 7.2), tris (hydroxy) aminomethane buffer, 3- (N-morpholino) propanesulfonic acid Buffer, N-tris (hydroxymethyl) 1-2-aminoethanesulfonic acid buffer, N-2-hydroxyethylpiperazine-N′-2 ethanesulfonic acid buffer, N-tris (hydroxymethyl) methyl-2- Hydroxy-3-aminopropanesulfonic acid buffer, piperazine-N, N′-bis (2-hydroxypropanesulfonic acid) buffer, N 2-hydroxyethylpiperazine-N′-2-hydroxypropane-3-propanesulfonic acid buffer, tris (hydroxymethylmethylglycine) buffer, N, N-bis (2-hydroxyethyl) glycine buffer, 2- ( (Cyclohexylamino) ethanesulfonic acid buffer, 3-N-cyclohexylamino-2-hydroxypropanesulfonic acid buffer, 3-cyclohexylaminopropanesulfonic acid buffer, etc. (preferably, N-tris (hydroxymethyl) -3-amino Propanesulfonic acid buffer (pH 8.4)).

  Liposomes, liposome raw materials or combinations thereof, platinum complexes, platinum complex raw materials or combinations thereof, and if necessary, buffers can be mixed in any order. For example, these orders are 1) the order in which the liposome raw material and the platinum complex are mixed and the buffer solution is added, 2) the order in which the platinum raw material and the buffer solution are mixed, and then the platinum complex is added, and 3) the platinum complex. However, the present invention is not limited to this. This is because, as long as the conditions under which the liposome is formed and maintained are maintained, the present invention can be achieved as long as the water-soluble ammine platinum complex can come into contact with the liposome at the time when the liposome is present, regardless of the order of mixing. Because it can be done. When using the platinum complex which shows strong acidity in aqueous solution, the range of pH 6-10 is preferable for a liquid mixture.

  In one embodiment, the targeting substance that can be used in the production method may be, for example, an antibody, a sugar chain, a lectin, a complementary nucleic acid, a receptor, a ligand, an aptamer, or an antigen. Preferably, the targeting substance may be an antibody or a sugar chain, but is not limited thereto. This is because the target directing substance may be any substance that imparts target directivity to the liposome without destroying the liposome. A person skilled in the art can appropriately determine the target-directing substance to be bound to the liposome according to the target.

  In the present invention, the water-soluble ammine platinum complex preferably has two ammine groups, and more preferably can be cis-diammine dinitratoplatinum (II).

  A preferable production method includes the following steps: A) a step of dissolving the water-soluble ammine platinum complex in a first buffer solution to prepare a platinum complex solution; B) a step of providing the liposome raw material; C) the platinum A step of mixing the complex solution and the lipid suspension and subjecting them to the liposome formation maintaining conditions may be included. In this method, the first buffer solution and the second buffer solution may be those that do not contain ions that are sparingly soluble in the salt formed by the platinum complex.

  In one embodiment, the step B) of the present production method comprises the steps of: B-i) suspending a lipid having liposome-forming ability in a methanol / chloroform solution and stirring the solution; evaporating the stirred solution; Obtaining a lipid membrane by vacuum drying; and B-ii) suspending the lipid membrane in a second buffer to form a lipid suspension.

In a more preferred embodiment, the production method used in the present invention comprises (A1) cis-diammine dinitratoplatinum (II) and N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer as step A). A step of preparing a solution containing cis-diammine dinitratoplatinum (II) dissolved in a liquid, wherein the N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer solution contains chloride ions (Cl ⁻ ) Or a chloride ion (Cl ⁻ ) in the range of 0 to 4 mM; (A2) pH of the solution containing the cis-diammine dinitratoplatinum (II) is adjusted to pH 6 to 10 (preferably Adjusting the pH to 8.4).

Further, as step B), (B1) dipalmitoyl phosphatidylcholine, cholesterol, ganglioside, dicetyl phosphate, dipalmitoyl phosphatidylethanolamine and sodium cholate (preferably, for example, 35: 40: 15: 5: 5: 167 (B2) The lipid is suspended in a methanol / chloroform (preferably 1: 1) solution and stirred, the stirred solution is evaporated, and the precipitate is evacuated. A step of obtaining a lipid membrane by drying; and (B3) the lipid membrane is suspended in N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4) to obtain a lipid suspension. The N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer Liquor, chloride ion (Cl ^-) or do not contain, or chloride ion (Cl ^-) and comprises in the range of 0 to 4 mM,; and (B4) lipid suspension at 30 ° C. to 40 ° C. The Stirring, purging with nitrogen, and sonicating may be included.

  Furthermore, as step C), (C1) the cis-diammine dinitratoplatinum (II) solution whose pH has been adjusted and a sonicated lipid suspension are used in a ratio of 1: 9 to 9: 1 (preferably, 7: 3) and may be subjected to ultrafiltration with a molecular weight cut-off of 500 to 300,000 (preferably 10,000).

  As another preferable production method, A) a step of dissolving the water-soluble ammine platinum complex in a first buffer solution to prepare a platinum complex solution; B) a step of providing the liposome; and C) the platinum complex solution And the liposome, and a method including a step of subjecting to the liposome formation and maintenance conditions. In this method, as the first buffer solution, a salt formed by the platinum complex that does not contain ions that are poorly water-soluble can be used. The liposome may have a composition sufficient, for example, for a water-soluble ammine platinum complex to migrate through and stay in the liposome membrane. The liposome formation and maintenance conditions can be, for example, sufficient conditions for maintaining the liposomes without destroying and at the same time allowing the water-soluble ammine platinum complex to pass through the liposome membrane and remain in the liposomes.

In another more preferred embodiment, step A) of the production method used in the present invention comprises (A1) cis-diammine dinitratoplatinum (II) and N-tris (hydroxymethyl) -3-aminopropanesulfonic acid. A step of preparing a solution containing cis-diammine dinitratoplatinum (II) dissolved in a buffer solution, wherein the N-tris (hydroxymethyl) -3-aminopropanesulfonate buffer solution contains chloride ions (Cl ^-) contain no, or chloride ions (Cl ^- a) comprises in the range of 0 to 4 mM, step; (A2) the pH of the solution containing the cis- diammine Min-ji nits Ratn platinum (II) pH 6 to 10 (preferably Can include adjusting to pH 8.4).

  The step B) of the production method used in the present invention comprises (B1) a step of providing a liposome; (C1) the cis-diammine dinitratoplatinum (II) solution adjusted in pH and the liposome; Mixing at a ratio in the range of 9-9: 1 and subjecting to ultrafiltration with a molecular weight cut off of 500-300,000 (preferably 10,000).

  In one embodiment, the liposome encapsulating the ammine platinum complex according to the present invention can be produced by the following method. Specifically, the method comprises: A) providing a solution containing a water-soluble platinum complex having two ammine groups; B) a solution containing the water-soluble platinum complex and a lipid suspension having liposome-forming ability A step of preparing a mixed solution by mixing with a liquid; C) a step of subjecting the mixed solution to conditions for forming liposomes; and D) a solution of an ion containing a salt that forms a poorly water-soluble salt. Subjecting to presence. Preferably, the water-soluble platinum complex can be cis-diammine dinitratoplatinum (II).

  In one embodiment, the lipid suspension in step B) is prepared by i) suspending the lipid in a methanol / chloroform solution and stirring, evaporating the stirred solution, and drying the precipitate in vacuum. Obtaining a lipid membrane; and ii) suspending the lipid membrane in a suspension buffer.

  In one embodiment, the methanol / chloroform solution may include methanol and chloroform in a 1: 1 ratio.

  In one embodiment, the method may further include a step of sonicating the lipid suspension following the step B). Preferably, the ultrasonic treatment step may be a step of stirring the lipid solution at 30 ° C. to 40 ° C., substituting with nitrogen, and performing ultrasonic treatment.

  In one embodiment, in the step C), the mixed solution may be subjected to conditions for forming the liposome for a time sufficient for the liposome to form. Preferably, the conditions for the liposome to form the mixed solution may be, for example, subjecting the mixed solution to ultrafiltration, or allowing the mixed solution to stand overnight. More preferably, the condition for the liposome to form the mixed solution may be that the mixed solution is subjected to ultrafiltration with a molecular weight cut-off of 10,000.

  In one embodiment, the step D) can be performed after confirming the formation of liposomes in the step C).

A more preferable production method includes the following steps: A-1) preparing a solution containing a water-soluble platinum complex having two ammine groups; A-2) adjusting the pH of the solution containing the water-soluble platinum complex. adjusting the pH within the range of 6 to 10; B-1) The cis-diammine dinitratoplatinum (II) solution with adjusted pH and the sonicated lipid suspension are 1: 9 to 9: Mixing at a ratio within the range of 1 to produce a mixed solution; and C-1) subjecting the mixed solution to ultrafiltration; and D-1) after ultrafiltration, the mixed solution is converted to chloride ions. And a step of subjecting to the presence of (Cl ⁻ ).

The liposome encapsulating cis-diamine dichloroplatinum (II) according to the present invention can be produced by the following method. Specifically, this method comprises (A1) cis-diammine dinitratoplatinum (II) and N-tris (hydroxymethyl) -3-aminopropanesulfonic acid substantially free from chloride ion (Cl ⁻ ). Dissolving in a buffer and preparing a solution containing cis-diammine dinitratoplatinum (II); (A2) adjusting the pH of the solution containing cis-diammine dinitratoplatinum (II) to pH 8.4; (B1) a step of preparing a lipid by mixing dipalmitoyl phosphatidylcholine, cholesterol, ganglioside, dicetyl phosphate, dipalmitoyl phosphatidylethanolamine and sodium cholate in a molar ratio of 35: 40: 15: 5: 5: 167; (B2) The lipid is suspended in a methanol / chloroform (1: 1) solution and stirred. , Evaporated the stirring solution, obtaining a lipid membrane by vacuum drying the precipitate; a and (B3) lipid membrane, chloride ion (Cl ^-) substantially free N- tris (hydroxymethyl A step of preparing a lipid suspension by suspending in methyl) -3-aminopropanesulfonic acid buffer (pH 8.4), and (B4) stirring the lipid suspension at 30 ° C. to 40 ° C., followed by nitrogen substitution And (B5) mixing the cis-diammine dinitratoplatinum (II) solution adjusted in pH and the sonicated lipid suspension in a ratio within a range of 7: 3. (C1) subjecting the mixed solution to ultrafiltration at a molecular weight cut-off of 10,000; and (D1) after ultrafiltration, the mixed solution is treated with N-tris (hydroxymethyl). ) -3-Aminopropanesulfone Including exposing to at least one buffer selected from the group consisting of a buffer (pH 8.4), carbonate buffer (pH 8.5), PBS (pH 8.0) and HEPES buffer (pH 7.2). .

  As used herein, the term “mixture” refers to a water-soluble ammine platinum complex, a platinum complex raw material or a combination thereof (also referred to herein as a water-soluble ammine platinum complex and the like), a liposome, This refers to a mixture containing the liposome raw materials or a combination thereof (also referred to herein as liposomes).

  As used herein, the term “mixed solution” refers to a solution prepared by mixing a solution containing a water-soluble platinum complex and a lipid suspension having liposome-forming ability.

  Liposomes, liposome raw materials or combinations thereof, platinum complexes, platinum complex raw materials or combinations thereof, and if necessary, buffers can be mixed in any order. For example, these orders are 1) the order in which the liposome raw material and the platinum complex are mixed and the buffer solution is added, 2) the order in which the platinum raw material and the buffer solution are mixed, and then the platinum complex is added, and 3) the platinum complex. However, the present invention is not limited to this. This is because, as long as the conditions under which the liposome is formed and maintained are maintained, the present invention can be achieved as long as the water-soluble ammine platinum complex can come into contact with the liposome at the time when the liposome is present, regardless of the order of mixing. Because it can be done. When using the platinum complex which shows strong acidity in aqueous solution, the range of pH 6-10 is preferable for a liquid mixture.

  That is, in the present invention, it is understood that there is no problem as long as the liposome “formation / maintenance” condition is maintained.

  While not wishing to be bound by theory, strongly acidic platinum complexes (including cisplatin nitrate) in aqueous solutions can be resuspended with alkaline buffer when the mixture of lipid components and cisplatin nitrate is redissolved in aqueous solution. These conditions need to be taken into account because the lipids must be degraded during acidification. As a necessary level of alkalinity, for example, when 80 mg cisplatin nitrate is dissolved in 4 ml of TAPS (pH 8.4), about 350 μl of 1N NaOH can be used.

  In the production method used in the present invention, this mixture is a step of subjecting to a liposome formation maintaining condition, provided that the salt formed by the platinum complex at the time when the liposome is present is in a water-soluble state. A liposome encapsulating the ammine platinum complex according to the present invention can be prepared. This mixture need only be in the range of pH 6-10 (preferably pH 8.4). This is because, if the pH is near neutral, it is possible to form liposomes and / or maintain liposomes.

  As used herein, the term “liposome formation maintenance conditions” refers to conditions under which liposomes form and / or are maintained. Liposome formation maintenance conditions can include subjecting the mixture of the platinum complex solution and the lipid suspension to ultrafiltration and allowing the solution to stand overnight. Subjecting the mixture of the platinum complex solution and the lipid suspension to ultrafiltration and allowing the mixture to stand overnight may be carried out continuously. The condition for maintaining the formation of liposomes may be sufficient for maintaining the liposomes without destroying them, and at the same time, allowing the water-soluble ammine platinum complex to pass through the liposome membrane and remain in the liposomes.

  As used herein, the term “liposome-forming” conditions refers to conditions for liposomes to form in this mixture (eg, a mixed solution). The conditions for forming liposomes can be, for example, subjecting the mixture to ultrafiltration, or allowing the mixture to stand overnight. More preferably, the conditions for liposome formation may be that the mixture is subjected to ultrafiltration at a molecular weight cut-off of 500 to 300,000 (preferably 10,000).

As used herein, the term “liposome-maintained” conditions refers to conditions under which liposomes are maintained in this mixture (eg, a mixed solution). The conditions under which the liposomes are maintained are not desired to be bound by theory, for example, but the liposomes are not broken and maintained at pH 6.0 or below. Since it breaks when a surfactant is added, it is not maintained. It is not maintained because it breaks due to physicochemical forces such as ultrasound. It is not maintained because it breaks when the temperature is higher than 60 ° C. When Cl ⁻ , Br ⁻ , I ⁻ , SCN ⁻ , CN ⁻ or a nucleobase (guanine, cytosine) is contained in the external liquid, it does not exist in the state of a water-soluble platinum complex or is inactivated. (Because it is inactivated when it binds to the nitrogen at the N7 position of the guanine base, these can be taken into account.

As used herein, the term “conditions in which the salt formed by the platinum complex is in a water-soluble state” refers to conditions under which the salt formed by the platinum complex is in a water-soluble state in the mixture. For example, such a condition means that the mixed solution is placed under the condition of a solution that does not contain ions in which the salt formed by the platinum complex is sparingly water-soluble. The ions that form a poorly water-soluble salt include, for example, chloride ions (Cl ⁻ ), bromide ions (Br ⁻ ), iodide ions (I ⁻ ), thiocyanate ions (SCN ⁻ ), and cyanide ions (CN). ^-) and the like (preferably, chloride ion (Cl ^-) may be). This solution may not contain ions that are poorly water-soluble or may not contain so much that the platinum complex forms a poorly water-soluble salt. The ion which the salt which this platinum complex forms is sparingly water-soluble can be contained in the range of 0 to 4 mM, for example, if it is a chloride ion (Cl ⁻ ). This is because in the case of chloride ions (Cl ⁻ ), this concentration of 0 to 4 mM is less than the concentration existing in the body, so it is considered that it does not precipitate as a poorly water-soluble salt.

As used herein, the term “a salt that forms does not contain ions that are sparingly water-soluble” means that the platinum complex is substantially free of ions that can form sparingly water-soluble salts in the solution. That means. Examples of the ions having poorly water-soluble salts include chloride ions (Cl ⁻ ), bromide ions (Br ⁻ ), iodide ions (I ⁻ ), thiocyanate ions (SCN ⁻ ), and cyanide ions (CN). ^- ) Etc. This solution may not contain ions that are poorly water-soluble or may not contain so much that the platinum complex forms a poorly water-soluble salt.

As used herein, “substantially free of” an ion that can form a poorly water-soluble salt means that the ion can be contained at a concentration that does not form a hardly water-soluble salt. For example, if the ion that can form the poorly water-soluble salt is chloride ion (CL ⁻ ), the concentration can be said to be a concentration (about 0 to 4 mM) that is lower than the concentration existing in the body. The concentration at which a sparingly water-soluble salt can be formed can be determined experimentally by a method comprising the following steps:
1) A step of preparing an aqueous solution (6 mM) containing an ammine platinum complex;
2) preparing a solution (final concentration of 0 to 200 mM) containing ions capable of forming a slightly water-soluble salt at various concentrations;
3) adding a solution containing ions that can form the poorly water-soluble salt of step 2) to the aqueous solution containing the ammine platinum complex of step 1) until the ions form a poorly water-soluble salt; and 4) A step of calculating a concentration capable of forming a poorly water-soluble salt from the amount of the solution containing the ions added.

  Concentrations that do not form poorly water-soluble salts are defined similarly for other salts.

  As used herein, the term “water-soluble” refers to the property of being soluble in water. The ammine platinum complex encapsulated in the liposome by the method for producing an ammine platinum complex-encapsulating liposome according to the present invention is preferably poorly water-soluble. As used herein, the term “poorly water-soluble” refers to the property of being insoluble or hardly soluble in water.

  In the present specification, the “first buffer solution” refers to a buffer solution for dissolving a water-soluble ammine platinum complex, a material of the platinum complex, or a combination thereof.

In the production method used in the present invention, as the first buffer solution, a solution containing no ion capable of forming a sparingly water-soluble salt in the platinum complex in the buffer solution can be used. This first buffer solution does not need to contain a salt that forms a poorly water-soluble salt, even if it does not contain ions that are poorly water-soluble. Examples of the ion that forms a poorly water-soluble salt include chloride ions (Cl ⁻ ), bromide ions (Br ⁻ ), iodide ions (I ⁻ ), thiocyanate ions (SCN ⁻ ), cyanide ions ( CN ^-) may be like. This first buffer includes, for example, N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4), carbonate buffer (pH 8.5), phosphate buffer (pH 8.0), 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES) buffer (pH 7.2), tris (hydroxy) aminomethane buffer, 3- (N-morpholino) propanesulfonic acid buffer Agent, N-tris (hydroxymethyl) 1-2-aminoethanesulfonic acid buffer, N-2-hydroxyethylpiperazine-N′-2 ethanesulfonic acid buffer, N-tris (hydroxymethyl) methyl-2-hydroxy -3-aminopropanesulfonic acid buffer, piperazine-N, N'-bis (2-hydroxypropanesulfonic acid) buffer, N-2-hydroxy Ethyl piperazine-N'-2-hydroxypropane-3-propanesulfonic acid buffer, tris (hydroxymethylmethylglycine) buffer, N, N-bis (2-hydroxyethyl) glycine buffer, 2- (cyclohexylamino) Buffers containing ethanesulfonic acid buffer, 3-N-cyclohexylamino-2-hydroxypropanesulfonic acid buffer, 3-cyclohexylaminopropanesulfonic acid buffer, and the like may be used. Preferably, the first buffer may be a buffer containing N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (preferably pH 8.4). The first buffer can be adjusted within the range of pH 6-10 (preferably 8.4). In the present invention, a buffer solution other than the above can also be used as the first buffer solution. This is because if the salt formed is a buffer solution that does not contain ions that are poorly water-soluble, or if the platinum complex does not contain enough water to form a poorly water-soluble salt, the platinum complex will contain a poorly water-soluble salt. It is because it does not form.

  In the present specification, the “platinum complex solution” refers to a solution containing a water-soluble platinum complex prepared by dissolving a water-soluble ammine platinum complex in a first buffer solution. For example, if the platinum complex solution is cisplatin nitrate, the range can be set based on the cisplatin nitrate (molecular weight 353) based on the solubility range of 15 mM to 300 mM. Solutions containing such water-soluble platinum complexes are known in the art and require only routine experimentation and common technical knowledge in the art. Therefore, the preparation of such a solution is within the skill of the artisan.

  As used herein, the term “solution containing a water-soluble platinum complex” may be a solution containing a platinum complex in a water-soluble state. The solution containing the water-soluble platinum complex can be adjusted within a range of pH 6 to 10 (preferably 8.4). The solution containing the water-soluble platinum complex may be a solution that does not contain ions in which the salt that is formed is sparingly water-soluble.

  The solution containing the water-soluble platinum complex used in the present invention is, for example, N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4), carbonate buffer (pH 8.5), phosphorus Acid buffer (pH 8.0), 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES) buffer (pH 7.2), tris (hydroxy) aminomethane buffer, 3- (N-morpholino) propane sulfonic acid buffer, N-tris (hydroxymethyl) 1-2-aminoethanesulfonic acid buffer, N-2-hydroxyethylpiperazine-N′-2 ethanesulfonic acid buffer, N-tris (Hydroxymethyl) methyl-2-hydroxy-3-aminopropanesulfonic acid buffer, piperazine-N, N′-bis (2-hydroxyprop Sulfonic acid) buffer, N-2-hydroxyethylpiperazine-N′-2-hydroxypropane-3-propanesulfonic acid buffer, tris (hydroxymethylmethylglycine) buffer, N, N-bis (2-hydroxyethyl) ) Glycine buffer, 2- (cyclohexylamino) ethanesulfonic acid buffer, 3-N-cyclohexylamino-2-hydroxypropanesulfonic acid buffer, 3-cyclohexylaminopropanesulfonic acid buffer, and the like. Preferably, the solution containing the water-soluble platinum complex may contain N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4). The “platinum complex solution” or “solution containing a water-soluble platinum complex” may contain a buffering agent other than those described above. This is because it is sufficient that the ammine platinum complex can be kept in a water-soluble state at the time when the liposome is present. Such a buffer has a buffer capacity at pH 6 to 10, and can maintain liposomes and maintain a water-soluble platinum complex if it does not contain an anion having binding properties with a platinum complex.

  As used herein, the term “lipid suspension” refers to a suspension of lipids having the ability to form liposomes, and the lipids that form liposomes when suspended under conditions for forming liposomes are suspended. Refers to any liquid. When suspended in a membrane state, it is also referred to as “lipid membrane suspension”. Further, when the lipid is dissolved in the solvent, it is also referred to as “lipid solution”. In a broad sense, the lipid solution may include a lipid suspension.

As described below, the composition of such a “suspension of lipid having the ability to form liposomes” can be appropriately determined by those skilled in the art, and the range can be clearly determined. The description is as follows: Lipid suspensions that can be used in the present invention can be made with lipids commonly used in the production of liposomes. Examples of lipids generally used for preparing liposomes include phosphatidylcholine and cholesterol. Lipids that can alternatively be used in liposome production include phosphatidylethanolamine, ganglioside, sodium cholate, dicetyl phosphate, phosphatidylserine, phosphatidylinositol, diphosphatidylglycerol, cardiolipin, sulfoxyribosyl diglyceride, digalactosyl diglyceride, galactosyl Examples include, but are not limited to, diglycerides. Preferably, the lipid suspension that may be used in the present invention may have the following range of compositions:
Phosphatidylcholine: 0-50 mM
Phosphatidylethanolamine: 0 to 3.3 mM
Cholesterol: 0 to 26.1 mM
Ganglioside: 0 to 9.3 mM
Sodium cholate: 0-108.9 mM
Dicetyl phosphate: 0 to 3.29 mM.

  The lipid suspension having the liposome-forming ability is prepared by suspending the lipid as a raw material for posome in a methanol / chloroform solution (preferably 1: 1) and evaporating the stirred solution. Step of obtaining a lipid membrane by drying the precipitate in vacuum; B-ii) It can be prepared by suspending the lipid membrane in a second buffer solution (suspension buffer solution). This lipid suspension may be sonicated. This lipid suspension comprises dipalmitoyl phosphatidylcholine, cholesterol, ganglioside, dicetyl phosphate, dipalmitoyl phosphatidylethanolamine and sodium cholate (preferably in a molar ratio of 35: 40: 15: 5: 5: 167). ). In the present method, the solution containing the water-soluble ammine platinum complex and the lipid suspension can be mixed in a ratio of 1: 9 to 9: 1 (preferably 7: 3).

  In the present specification, the “second buffer solution” refers to a buffer solution for dissolving a lipid suspension. This second buffer is also referred to herein as a “suspension buffer”.

In the production method used in the present invention, as the second buffer solution, one containing no ion capable of forming a sparingly water-soluble salt in the platinum complex in the buffer solution can be used. This suspension buffer should not contain ions that are sparingly water-soluble or, if included, not so much that the platinum complex forms sparingly water-soluble salts. Examples of the ion having a poorly water-soluble salt include chloride ion (Cl ⁻ ), bromide ion (Br ⁻ ), iodide ion (I ⁻ ), thiocyanate ion (SCN ⁻ ), cyanide ion ( CN ^-) may be like. This second buffer includes, for example, N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4), carbonate buffer (pH 8.5), phosphate buffer (pH 8.0), 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES) buffer (pH 7.2), tris (hydroxy) aminomethane buffer, 3- (N-morpholino) propanesulfonic acid buffer Agent, N-tris (hydroxymethyl) 1-2-aminoethanesulfonic acid buffer, N-2-hydroxyethylpiperazine-N′-2 ethanesulfonic acid buffer, N-tris (hydroxymethyl) methyl-2-hydroxy -3-aminopropanesulfonic acid buffer, piperazine-N, N'-bis (2-hydroxypropanesulfonic acid) buffer, N-2-hydroxy Ethyl piperazine-N'-2-hydroxypropane-3-propanesulfonic acid buffer, tris (hydroxymethylmethylglycine) buffer, N, N-bis (2-hydroxyethyl) glycine buffer, 2- (cyclohexylamino) Buffers containing ethanesulfonic acid buffer, 3-N-cyclohexylamino-2-hydroxypropanesulfonic acid buffer, 3-cyclohexylaminopropanesulfonic acid buffer, and the like may be used. Preferably, the first buffer may be a buffer containing N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (preferably pH 8.4). The second buffer can be adjusted within the range of pH 6-10 (preferably 8.4).

  The “second buffer solution” or “suspension buffer solution” may contain a buffering agent other than those described above. This is because it is sufficient that the ammine platinum complex can be kept in a water-soluble state at the time when the liposome is present. Such a buffer has a buffer capacity at pH 6 to 10, and can maintain liposomes and maintain a water-soluble platinum complex if it does not contain an anion having binding properties with a platinum complex.

  In the present specification, the first buffer solution and the second buffer solution may have the same composition or different compositions. This is because these buffers may be any buffer as long as the platinum complex can be kept in a water-soluble state when the liposome is present.

As used herein, the term “in the presence of a solution containing an ion in which the salt formed by the platinum complex is poorly water soluble” means that an ion in which the salt formed by the platinum complex is poorly water soluble is provided. It means to be in the environment to obtain. In a preferred embodiment, the presence of a solution containing ions in which the salt formed by the platinum complex is poorly water-soluble can be in the presence of chloride ions (Cl ⁻ ).

In the present specification, the “ion whose salt to be formed is sparingly water-soluble” of the platinum complex is, for example, chloride ion (Cl ⁻ ), bromide ion (Br ⁻ ), iodide ion (I ⁻ ), thiocyanic acid. Ions (SCN ⁻ ), cyanide ions (CN ⁻ ), and the like. This first buffer includes, for example, N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4), carbonate buffer (pH 8.5), phosphate buffer (pH 8.0), 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES) buffer (pH 7.2), tris (hydroxy) aminomethane buffer, 3- (N-morpholino) propanesulfonic acid buffer Agent, N-tris (hydroxymethyl) 1-2-aminoethanesulfonic acid buffer, N-2-hydroxyethylpiperazine-N′-2 ethanesulfonic acid buffer, N-tris (hydroxymethyl) methyl-2-hydroxy -3-aminopropanesulfonic acid buffer, piperazine-N, N'-bis (2-hydroxypropanesulfonic acid) buffer, N-2-hydroxy Ethyl piperazine-N'-2-hydroxypropane-3-propanesulfonic acid buffer, tris (hydroxymethylmethylglycine) buffer, N, N-bis (2-hydroxyethyl) glycine buffer, 2- (cyclohexylamino) Buffers including, but not limited to, ethanesulfonic acid buffer, 3-N-cyclohexylamino-2-hydroxypropanesulfonic acid buffer, 3-cyclohexylaminopropanesulfonic acid buffer, and the like may be used. Preferably, the second buffer is a buffer containing N-tris (hydroxymethyl) -3-aminopropane sulfonic acid buffer (preferably pH 8.4). In the present invention, a buffer solution other than the above can also be used as the second buffer solution. This is because if the buffer solution does not contain ions that are poorly water-soluble or does not contain so much that the platinum complex forms a poorly water-soluble salt, the platinum complex will be a poorly water-soluble salt. It is because it does not form.

For example, the following can be given as examples of solubility.
1. Two equivalents of chloride ion (Cl ⁻ ), bromide ion (Br ⁻ ), iodide ion (I ⁻ ), thiocyanate ion (SCN) with respect to a water-soluble platinum complex (in a state where two water molecules are coordinated) ^When- ) is present, two molecules of coordinated water of the water-soluble platinum complex are substituted with these ions to form a hardly soluble salt. When containing one equivalent of ions, one water molecule is replaced by these ions. When it does not contain ions, it takes a structure in which two water molecules are coordinated.
2. In the case of chloride ion (Cl ⁻ ), it is said that the intracellular chloride ion concentration is 0 to 4 mM and the water molecule is replaced, and it is said that it binds to DNA. From this, the range of chloride ion is expressed in mM. I was allowed to. Of the bromide ions (Br ⁻ ), iodide ions (I ⁻ ), and thiocyanate ions (SCN ⁻ ), the complexes in which Br ⁻ and I ⁻ are coordinated have different activity strengths. It is said that there is. Therefore, in the intracellular environment, it is considered to take a structure in which water molecules are coordinated.

It is known that poorly water-soluble substances are Cl ⁻ , Br ⁻ and I ⁻ and have anticancer activity. It is said that the bondability to the platinum atom is in the order of Cl ⁻ <Br ⁻ <I ⁻ . Br ⁻ and I ⁻ are more likely to bind to platinum than Cl ⁻ and form a hardly soluble substance. Therefore, it is considered that it can be made a poorly water-soluble substance under the same conditions as Cl ⁻ . The present invention can be implemented in consideration of these conditions.

  Further, in the present invention, if it is poorly water-soluble, it is contemplated that once the liposome of the present invention is formed, it becomes difficult for the platinum complex to come out of the liposome after the formation, thereby increasing the delivery efficiency.

In this production method, an ion (for example, chloride ion (Cl ⁻ )) in which the salt formed by the platinum complex is poorly water-soluble is, for example, N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer ( pH 8.4), carbonate buffer (pH 8.5), PBS (pH 8.0) or HEPES buffer (pH 7.2), tris (hydroxy) aminomethane buffer, 3- (N-morpholino) propanesulfonic acid buffer Solution, N-tris (hydroxymethyl) 1-2-aminoethanesulfonic acid buffer, N-2-hydroxyethylpiperazine-N′-2 ethanesulfonic acid buffer, N-tris (hydroxymethyl) methyl-2-hydroxy -3-aminopropanesulfonic acid buffer, piperazine-N, N'-bis (2-hydroxypropanesulfonic acid) buffer, N-2-H Droxyethylpiperazine-N′-2-hydroxypropane-3-propanesulfonic acid buffer, Tris (hydroxymethylmethylglycine) buffer, N, N-bis (2-hydroxyethyl) glycine buffer, 2- (cyclohexyl) Amino) ethanesulfonic acid buffer, 3-N-cyclohexylamino-2-hydroxypropanesulfonic acid buffer, 3-cyclohexylaminopropanesulfonic acid buffer and the like may be provided, but are not limited thereto. This is because the liposome can be maintained and the water-soluble platinum complex can be maintained as long as it does not contain an anion having a buffer capacity at pH 6 to 10 and binding ability to the platinum complex. The preparation and characterization of common buffers is known in the art and requires only routine experimentation and technical knowledge in the art. Accordingly, in view of the target poorly water-soluble salt of an ammine platinum complex and the above-mentioned pH, ions that the buffer solution can contain, and the like, those skilled in the art will know that the buffer solution contains an ion from which the platinum complex forms a slightly water-soluble salt Can be selected as appropriate.

In the present method, chloride ions (Cl ⁻ ) can be provided by NaCl, HCl, CaCl _2, but are not limited thereto. Preferably, chloride ion (Cl ⁻ ) can be provided by NaCl.

  In a preferred embodiment, the liposome according to the present invention contains ganglioside, glycolipid or phosphatidylglycerol, and is linked to a linker such as a peptide, and is directed to a targeting substance (eg, antibody, sugar chain, lectin, complementary nucleic acid, receptor). , Ligands, aptamers, antibodies, etc.).

  As used herein, the term “targeting substance” refers to a substance that specifically recognizes a disease state (particularly a tumor). Examples of the “targeting substance” used in the present invention include, but are not limited to, antibodies, sugar chains, lectins, complementary nucleic acids, receptors, ligands, aptamers, antibodies and the like. This is because the target directing substance may be any substance that imparts target directivity to the liposome without destroying the liposome. A person skilled in the art can appropriately determine the target-directing substance to be bound to the liposome according to the target. Any substance can be modified on the surface of the liposome membrane by using an appropriate cross-linking agent.

  As used herein, the term “antibody” refers to an immunoglobulin molecule having a specific amino acid sequence elicited by an antigen that is an immunogen. Antibodies are produced by B cells and are present in blood and body fluids. An antibody has the characteristic of reacting specifically with an antigen. The antibody may be present naturally rather than as a result of stimulation by presentation of the antigen. Basically, the molecular structure of an antibody is formed by two light chains and a heavy chain, but can also exist as a dimer, trimer, or pentamer. Examples of these include, but are not limited to, IgA, IgE, IgM, IgG, and the like.

  In the present specification, the “sugar chain” refers to a compound formed by connecting one or more unit sugars (monosaccharide and / or a derivative thereof). When two or more unit sugars are connected, each unit sugar is bound by dehydration condensation by a glycosidic bond. Examples of such sugar chains include polysaccharides (glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, sialic acid, and complexes and derivatives thereof) contained in the living body. Other examples include, but are not limited to, a wide range of sugar chains that are decomposed or derived from complex biomolecules such as degraded polysaccharides, glycoproteins, proteoglycans, glycosaminoglycans, glycolipids, and the like. Therefore, in the present specification, the sugar chain can be used interchangeably with “polysaccharide”, “carbohydrate”, and “carbohydrate”. Further, unless otherwise specified, the “sugar chain” in the present specification may include both sugar chains and sugar chain-containing substances. Typically, a substance in which about 20 types of monosaccharides (glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, sialic acid, and complexes and derivatives thereof) are connected in a chain. It is attached to proteins and lipids inside and outside the cells of the body. The functions differ depending on the sequence of monosaccharides, and they are usually branched in a complex manner. The human body is expected to have several hundreds of sugar chains with various structures, and there are tens of thousands of useful structures in the human body. It is believed that there are more than types. Although it is considered to be related to higher-order functions that proteins and lipids perform in vivo, such as cell-to-cell molecular and cell recognition functions, there are many unexplained mechanisms. It is attracting attention in current life science as the third life chain after nucleic acid and protein. In particular, the function of a sugar chain as a ligand (information molecule) in cell recognition is expected, and its application to the development of highly functional materials is being studied.

  As used herein, “sugar” or “monosaccharide” refers to polyhydroxyaldehyde or polyhydroxyketone containing at least one hydroxyl group and at least one aldehyde group or ketone group, and constitutes the basic unit of a sugar chain. As used herein, sugars are also referred to as carbohydrates, and both are used interchangeably. As used herein, when specifically mentioned, a sugar chain refers to a chain or a sequence in which one or more sugars are linked, and when a sugar or a monosaccharide is referred to, it refers to one unit constituting the sugar chain. . Here, those in which n = 2, 3, 4, 5, 6, 7, 8, 9 and 10 are referred to as diose, triose, tetrose, pentose, hexose, heptose, octose, nonose and decourse, respectively. Generally, it corresponds to an aldehyde or ketone of a chain polyhydric alcohol. The former is called aldose and the latter is called ketose. In the present invention, any type can be used.

  As used herein, the term “lectin” refers to a substance capable of binding to the sugar chains of cell membrane complex carbohydrates (glycoproteins and glycolipids), cell aggregation, mitogenesis, functional activation, cell It has the ability to exert effects such as obstacles. If sugar chains are information molecules transmitted by cells, lectins can be said to be receiving molecules. Cells or tissues with certain properties have a corresponding lectin pattern. Lectins achieve infection, biological defense, immunity, fertilization, targeting to target cells, cell differentiation, cell-cell adhesion, quality control of nascent glycoproteins and intracellular sorting and transport. Lectins have extensive sugar chain binding properties and are strictly controlled by their inherent physicochemical properties of rapid association and dissociation. Also called sugar chain recognition protein. Research on plant lectins is old, and about 300 kinds are already known. Recently, active research has been conducted on animal lectins, and the discovery of new lectins continues. Various sugar chain recognition functions based on lectins (about 100 types) of major lectin families present on animal cell membranes have been studied. In particular, the function as a receptor (information receiving protein or target molecule) for receiving structural information of sugar chain ligands having various structures has been attracting attention.

  As used herein, the term “complementary nucleic acid” is defined as the broadest meaning used in the art, and refers to nucleic acids that can form base pairs with each other by the base pairing rules of nucleic acids. Examples of these include, but are not limited to, DNA and RNA.

  As used herein, the term “receptor” is also referred to as “receptor” and refers to a substance that exists in a cell and has a structure for recognizing and transmitting an external stimulus. Examples of these include, but are not limited to, cell surface receptors and intracellular receptors.

  As used herein, the term “ligand” refers to a molecule that itself is very strongly adsorbed to a substance. Examples of these include, but are not limited to, proteins, nucleic acids, chemical substances, and the like.

  As used herein, the term “aptamer” refers to a nucleic acid having a relatively low molecular weight that can recognize and bind to the structures of various substances (proteins, chemical substances, etc.). Examples of these include, but are not limited to, RNA aptamers and DNA aptamers.

  As used herein, the term “antigen” refers to a substance that has a function of promoting antibody production. Examples include, but are not limited to, macromolecular sugars, proteins, complexes thereof, viruses, cells, and the like.

  As used herein, “hydrophilization” refers to binding of a hydrophilic compound to the liposome surface. Examples of the compound used for hydrophilization include a low molecular weight hydrophilic compound, preferably a low molecular weight hydrophilic compound having at least one OH group, and more preferably a low molecular weight hydrophilic compound having at least two OH groups. Can be mentioned. Further, a low molecular weight hydrophilic compound having at least one amino group, that is, a hydrophilic compound having at least one OH group and at least one amino group in the molecule can be mentioned. Examples of such hydrophilic compounds include amino alcohols such as tris (hydroxyalkyl) aminoalkanes including tris (hydroxymethyl) aminomethane, and more specifically, tris (hydroxymethinore) aminoethane. , Tris (hydroxyethyl) aminoethane, tris (hydroxypropyl) aminoethane, tris (hydroxymethyl) aminomethane, tris (hydroxyethyl) aminomethane, tris (hydroxypropyl) aminomethane, tris (hydroxymethyl) aminopropane, tris (hydroxy) And ethyl) aminopropane and tris (hydroxypropyl) aminopropane.

(Ammine platinum complex encapsulated liposome)
The ammine platinum complex-encapsulating liposome can be obtained by mixing a water-soluble ammine platinum complex raw material and a liposome raw material and subjecting them to liposome formation maintaining conditions.

  In one embodiment, the ammine platinum complex-encapsulating liposome used in the present invention contains ammonia having an ammine platinum complex (eg, cis-diamine dichloroplatinum (II)) in an amount of more than 43 μg per 1 mg lipid, for example, 43. 1, 43.2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44, 44.5, 45, 45.5, 46, 46. 5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 62.5, 65, 67. 5, 70, 72.5, 75, 77.5, 80, 82.5, 85, 87.5, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or more Preferably, more than 70 [mu] g, more preferably more than 85 [mu] g, most preferably, may contain at least 100 [mu] g. In one embodiment, the ammine platinum complex-encapsulating liposome used in the present invention may contain ammine platinum complex having ammonia at 200 μg or less per 1 mg lipid. The ammine platinum complex-encapsulating liposome contains an ammonia-containing ammine platinum complex (for example, cis-diamine dichloroplatinum (II)) of 200 μg or less per mg lipid, for example, 200, 190, 180, 170, 160, 150, 140, 130. 120, 110, 100, 95, 90, 87.5, 85, 82.5, 80, 77.5, 75, 72.5, 70, 67.5, 65, 62.5, 60, 59, 58 57, 56, 55, 54, 53, 52, 51, 50, 49.5, 49, 48.5, 48, 47.5, 47, 46.5, 46, 45.5, 45, 44.5 44, 43.9, 43.8, 43.7, 43.6, 43.5, 43.4, 43.3, 43.2, 43.1 or less, preferably 70 μg or less, more preferably Is 85 μg Lower, and most preferably, it can include at 100μg or less.

  In one embodiment, the ammine platinum complex-encapsulating liposome used in the present invention contains ammonia-containing ammine platinum complex (eg, cis-diamine dichloroplatinum (II)), eg, more than 249.4 per mg phospholipid. For example, 249.5, 249.6, 249.7, 249.8, 249.9, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 310, 320, 330 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580 590, 600, 650, 700, 750, 800, 850, 900, 950 1000,1100,1160 or more, preferably, 406Myug, more preferably, 493Myug, most preferably, may include at least 580Myug. In one embodiment, the ammine platinum complex-encapsulating liposome used in the present invention may contain 1160 μg or less of ammine platinum complex having ammonia per 1 mg phospholipid. The ammine platinum complex-encapsulating liposome is an ammonia-containing ammine platinum complex (for example, cis-diamine dichloroplatinum (II)) of 1160 μg or less, for example, 1100, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 590, 580, 570, 560, 550, 540, 530, 520, 510, 500, 490, 480, 470, 460, 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 295, 290, 285, 280, 275, 270, 265, 260, 255, 250, 249.9, 249.8, 249.7, 249. 6, 249.5, 249.4 or less, preferably , 580Myug less, more preferably, 493Myug less, and most preferably, can include the following 406Myug.

  In one embodiment, the ammine platinum complex-encapsulating liposome used in the present invention contains an ammonia-containing ammine platinum complex (for example, cis-diamine dichloroplatinum (II)), for example, 43.1, 43. 2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 62.5, 65, 67.5, 70, 72.5, 75, 77.5, 80, 82.5, 85, 87.5, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500 or more Moreover, preferably, more than 210Myug, more preferably, more than 255Myug, most preferably, may include at 300μg higher. In one embodiment, the ammine platinum complex-encapsulating liposome used in the present invention may contain an ammonia-containing ammine platinum complex at 500 μg or less per 1 mg of lipid. The ammine platinum complex-encapsulating liposome is an ammonia-containing ammine platinum complex (for example, cis-diamine dichloroplatinum (II)) of 500 μg or less, for example, 500, 450, 400, 350, 300, 250, 200, 150, per ml of liposome. 140, 130, 120, 110, 100, 95, 90, 87.5, 85, 82.5, 80, 77.5, 75, 72.5, 70, 67.5, 65, 62.5, 60 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49.5, 49, 48.5, 48, 47.5, 47, 46.5, 46, 45.5, 45 44.5, 44, 43.9, 43.8, 43.7, 43.6, 43.5, 43.4, 43.3, 43.2, 43.1 or less, preferably 210 μg or less, More preferable May be included at 255 μg or less, most preferably 300 μg or less.

In one embodiment, the ammine platinum complex-encapsulating liposome used in the present invention contains ammonia-containing ammine platinum complex (for example, cis-diamine dichloroplatinum (II)), for example, 43.1 × 10 6 per liposome. ^-11 , 43.2 × 10 ⁻¹¹ , 43.3 × 10 ⁻¹¹ , 43.4 × 10 ⁻¹¹ , 43.5 × 10 ⁻¹¹ , 43.6 × 10 ⁻¹¹ , 43.7 × 10 ⁻¹¹ 43.8 × 10 ⁻¹¹ , 43.9 × 10 ⁻¹¹ , 44 × 10 ⁻¹¹ , 44.5 × 10 ⁻¹¹ , 45 × 10 ⁻¹¹ , 45.5 × 10 ⁻¹¹ , 46 × 10 ⁻¹¹ 46.5 × 10 ⁻¹¹ , 47 × 10 ⁻¹¹ , 47.5 × 10 ⁻¹¹ , 48 × 10 ⁻¹¹ , 48.5 × 10 ⁻¹¹ , 49 × 10 ⁻¹¹ , 49.5 × 10 ⁻¹¹ , 50 × 10 ⁻¹¹ , 51 × 10 ⁻¹¹ , 52 × 10 ⁻¹¹ , 53 × 10 ⁻¹¹ , 54 × 10 ⁻¹¹ , 55 × 10 ⁻¹¹ , 56 × 10 ⁻¹¹ , 57 × 10 ⁻¹¹ , 58 × 10 ⁻¹¹ , 59 × 10 ⁻¹¹ , 60 × 10 ⁻¹¹ , 62.5 × 10 ⁻¹¹ , 65 × 10 ⁻¹¹ , 67.5 × 10 ⁻¹¹ , 70 × 10 ⁻¹¹ , 72.5 × 10 ⁻¹¹ , 75 × 10 ^{− 11} , 77.5 × 10 ⁻¹¹ , 80 × 10 ⁻¹¹ , 82.5 × 10 ⁻¹¹ , 85 × 10 ⁻¹¹ , 87.5 × 10 ⁻¹¹ , 90 × 10 ⁻¹¹ , 95 × 10 ⁻¹¹ , 100 × 10 ⁻¹¹ , 110 × 10 ⁻¹¹ , 120 × 10 ⁻¹¹ , 130 × 10 ⁻¹¹ , 140 × 10 ⁻¹¹ , 150 × 10 ⁻¹¹ , 160 × 10 ⁻¹¹ , 170 × 10 ⁻¹¹ , 180 × ^{10 -11, 190 × 10 -11,} 200 10 ^-11 [mu] g or more, preferably, 70 × ^{10 -11} [mu] g, more preferably, 85 × ^{10 -11} [mu] g, most preferably, may include at 100 × ^{10 -11 μg} or more. In one embodiment, the ammine platinum complex-encapsulating liposome used in the present invention may contain ammine platinum complex having ammonia at 200 μg or less per liposome. The ammine platinum complex-encapsulating liposome contains an ammonia-containing ammine platinum complex (for example, cis-diamine dichloroplatinum (II)) at 200 × 10 ⁻¹¹ μg or less, for example, 200 × 10 ⁻¹¹ , 190 × 10 6 per liposome. ⁻¹¹ , 180 × 10 ⁻¹¹ , 170 × 10 ⁻¹¹ , 160 × 10 ⁻¹¹ , 150 × 10 ⁻¹¹ , 140 × 10 ⁻¹¹ , 130 × 10 ⁻¹¹ , 120 × 10 ⁻¹¹ , 110 × 10 ⁻¹¹ 100 × 10 ⁻¹¹ , 95 × 10 ⁻¹¹ , 90 × 10 ⁻¹¹ , 87.5 × 10 ⁻¹¹ , 85 × 10 ⁻¹¹ , 82.5 × 10 ⁻¹¹ , 80 × 10 ⁻¹¹ , 77.5 ^{× 10 -11, 75 × 10 -11} , 72.5 × 10 -11, 70 × 10 -11, 67.5 × 10 -11, 65 × 10 -11, 62.5 × 10 -1 ^{, 60 × 10 -11, 59 ×} 10 -11, 58 × 10 -11, 57 × 10 -11, 56 × 10 -11, 55 × 10 -11, 54 × 10 -11, 53 × 10 -11, 52 × 10 ⁻¹¹ , 51 × 10 ⁻¹¹ , 50 × 10 ⁻¹¹ , 49.5 × 10 ⁻¹¹ , 49 × 10 ⁻¹¹ , 48.5 × 10 ⁻¹¹ , 48 × 10 ⁻¹¹ , 47.5 × 10 ⁻¹¹ , 47 × 10 ⁻¹¹ , 46.5 × 10 ⁻¹¹ , 46 × 10 ⁻¹¹ , 45.5 × 10 ⁻¹¹ , 45 × 10 ⁻¹¹ , 44.5 × 10 ⁻¹¹ , 44 × 10 ⁻¹¹ 43.9 × 10 ⁻¹¹ , 43.8 × 10 ⁻¹¹ , 43.7 × 10 ⁻¹¹ , 43.6 × 10 ⁻¹¹ , 43.5 × 10 ⁻¹¹ , 43.4 × 10 ⁻¹¹ , 43 ^{.3 × 10 -11, 43.2 × 10} -11, 43.1 × 10 -11 or It is less, preferably, 70 [mu] g × ^{10 -11} or less, more preferably, 85 [mu] g × ^{10 -11} or less, and most preferably, may include at 100 [mu] g × ^{10 -11} or less.

  In one embodiment, the ammine platinum complex-encapsulating liposome used in the present invention may have two ammonia.

  In another embodiment, the ammine platinum complex-encapsulating liposome used in the present invention may be a water-soluble form of cis-diamine dichloroplatinum (II).

  In another embodiment, the ammine platinum complex-encapsulating liposome used in the present invention may be cis-diamine dichloroplatinum (II).

  In another embodiment, the liposome encapsulating the ammine platinum complex of the present invention contains, as lipids, dipalmitoyl phosphatidylcholine, cholesterol, ganglioside, dicetyl phosphate, dipalmitoyl phosphatidylethanolamine, sodium cholate, dicetylphosphatidylethanolamine. Polyglycerin 8G, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine, distearoylphosphatidylserine, dioleoylphosphatidylserine, dimyristoylphosphatidylinositol, dipalmitoylphosphatidyldisulfitol Stearoyl phosphatidyl boar Dioleoylphosphatidylinositol, dimyristoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, distearoylphosphatidic acid, dioleoylphosphatidic acid, galactosyl Ceramide, Glucosylceramide, Lactosylceramide, Phosphnatide, Globotide, GM1 (Galβ1,3GalNAcβ1,4 (NeuAα2,3) Galβ1,4Glcβ1,1'Cer), Ganglioside GD1a, Ganglioside GD1b, Dimyristoylphosphatidylglycerol, Dipalmitoylglycerol Distearoylphosphatidylglycero , It can include dioleoyl phosphatidylglycerol, and the like. Preferably, dipalmitoyl phosphatidylcholine, cholesterol, ganglioside, dicetyl phosphate, dipalmitoyl phosphatidylethanolamine, sodium cholate, dicetylphosphatidylethanolamine-polyglycerin 8G, dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylglycerol may be used. This is because even liposomes composed of lipids other than the above can contain cis-diamminedichloroplatinum (II) and / or a water-soluble form thereof in the liposome, and they can be retained in the liposome. This is because it may be composed of lipids that can be stored. Preferably, the liposome encapsulating the ammine platinum complex contains dipalmitoyl phosphatidylcholine, cholesterol, ganglioside, dicetyl phosphate, dipalmitoyl phosphatidylethanolamine and sodium cholate in a molar ratio of 35: 40: 15: 5: 5: 167. Can be included.

  In the present invention, the liposome can be used by mixing liposomes prepared by a plurality of production methods (for example, modified cholate method and freeze-dried method).

  In one embodiment, the liposome encapsulating the ammine platinum complex used in the present invention may further contain a targeting substance. Examples of the target-directing substance include, but are not limited to, antibodies, sugar chains, lectins, complementary nucleic acids, receptors, ligands, aptamers, and antigens (preferably antibodies, sugar chains). This is because any substance can be modified on the surface of the liposome membrane by using an appropriate crosslinking agent.

  Here, the liposome encapsulating the ammine platinum complex of the present invention can be produced by any method described in the above (Manufacturing method).

(Description of Preferred Embodiment)
Hereinafter, preferred embodiments of the present invention will be described. The embodiments provided below are provided for a better understanding of the present invention, and the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification.

(Composition)
In one aspect, the present invention provides a composition for treating cancer or tumor. The composition includes cis-diamine dichloroplatinum (II), and the liposome may include a targeting agent on the surface of the liposome.

  In one embodiment, the present invention provides a composition for treating cancer or tumor. The composition may comprise liposomes containing more than 43 μg of cis-diamine dichloroplatinum (II) (cisplatin) per mg lipid, and the liposomes may comprise a targeting substance on the surface of the liposomes.

In the cisplatin-encapsulated liposome used in the present invention, it is considered that most of the incorporated cisplatin is present in the inner aqueous phase of the liposome (confirmed by ¹⁹⁵ Pt-NMR). Thus, since cisplatin exists in the internal aqueous phase of the liposome, it is considered that cisplatin is not released until the liposome is taken into the cells at the target site and the liposome membrane is destroyed. Furthermore, the liposome can have a desired target directivity. Moreover, since the surface of the liposome used in the present invention is negatively charged, the binding to nonspecific blood vessel walls and blood components is reduced, and the liposome has more specific target directivity. Furthermore, by making the liposome membrane hydrophilic, it is possible to prevent phagocytosis by phagocytic cells such as macrophages and increase the retention in blood. Therefore, by adopting such liposomes, the composition of the present invention can be administered to a target at least as much as conventional drugs (eg, anticancer activity) even when administered at a much lower dose than the prior art. ), While reducing the side effects on the individual.

  In one embodiment, the cis-diamine dichloroplatinum (II) -encapsulated liposome according to the present invention contains more than 43 μg of cis-diamine dichloroplatinum (II) per mg lipid, for example, 43.1, 43.2, 43. 3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 62.5, 65, 67.5, 70, 72.5, 75, 77.5, 80, 82.5, 85, 87.5, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or more, preferably 70 μg or more, more preferably May be included at 85 μg or more, most preferably at 100 μg or more. In one embodiment, the liposome used in the present invention may contain, for example, cis-diamine dichloroplatinum (II) at 500 μg or less per mg lipid. The cis-diamine dichloroplatinum (II) -encapsulating liposome contains cis-diamine dichloroplatinum (II) in an amount of 200 μg or less per 1 mg lipid, for example, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 87.5, 85, 82.5, 80, 77.5, 75, 72.5, 70, 67.5, 65, 62.5, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49.5, 49, 48.5, 48, 47.5, 47, 46.5, 46, 45.5, 45, 44.5, 44, 43. 9, 43.8, 43.7, 43.6, 43.5, 43.4, 43.3, 43.2, 43.1 or less, preferably 70 μg or less, more preferably 85 μg or less, most Preferably, 100μ It may include the following.

  In another embodiment, the liposomes used in the present invention contain, for example, cis-diamine dichloroplatinum (II) greater than 249.4 per mg phospholipid, such as 249.5, 249.6, 249.7. 249.8, 249.9, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850 , 900, 950, 1000, 1100, 1160 or more, preferably 406μ , More preferably, 493Myug, most preferably, may include at least 580Myug. In one embodiment, the liposome used in the present invention may contain, for example, cis-diamine dichloroplatinum (II) at 1160 μg or less per 1 mg phospholipid. The cis-diamine dichloroplatinum (II) -encapsulating liposome contains cis-diamine dichloroplatinum (II) in an amount of 1160 μg or less per 1 mg phospholipid, for example, 1100, 1000, 950, 900, 850, 800, 750, 700, 650, 600. 590, 580, 570, 560, 550, 540, 530, 520, 510, 500, 490, 480, 470, 460, 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350 340, 330, 320, 310, 300, 295, 290, 285, 280, 275, 270, 265, 260, 255, 250, 249.9, 249.8, 249.7, 249.6, 249.5 249.4 or less, preferably 580 μg or less, more preferably Or 493 μg or less, most preferably 406 μg or less.

  In another embodiment, the liposome used in the present invention contains, for example, cis-diamine dichloroplatinum (II) at 43.1, 43.2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49. 5, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 62.5, 65, 67.5, 70, 72.5, 75, 77.5, 80, 82. 5, 85, 87.5, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500 or more, preferably 210 μg, more preferably 255 μg, most preferred May be included at 300 μg or more. In one embodiment, the liposome used in the present invention may contain, for example, cis-diamine dichloroplatinum (II) at 500 μg or less per ml of liposome. The cis-diamine dichloroplatinum (II) -encapsulating liposome contains cis-diamine dichloroplatinum (II) in an amount of 500 μg or less per ml of liposome, for example, 500, 450, 400, 350, 300, 250, 200, 150, 140, 130, 120, 110, 100, 95, 90, 87.5, 85, 82.5, 80, 77.5, 75, 72.5, 70, 67.5, 65, 62.5, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49.5, 49, 48.5, 48, 47.5, 47, 46.5, 46, 45.5, 45, 44.5, 44, 43.9, 43.8, 43.7, 43.6, 43.5, 43.4, 43.3, 43.2, 43.1 or less, preferably 210 μg or less, more preferably 255 μg The most favorable Details, may include at 300μg or less.

In another embodiment, the liposome used in the present invention contains, for example, cis-diamine dichloroplatinum (II) at 43.1 × 10 ⁻¹¹ , 43.2 × 10 ⁻¹¹ , 43.3 per liposome. × 10 ⁻¹¹ , 43.4 × 10 ⁻¹¹ , 43.5 × 10 ⁻¹¹ , 43.6 × 10 ⁻¹¹ , 43.7 × 10 ⁻¹¹ , 43.8 × 10 ⁻¹¹ , 43.9 × 10 ⁻¹¹ , 44 × 10 ⁻¹¹ , 44.5 × 10 ⁻¹¹ , 45 × 10 ⁻¹¹ , 45.5 × 10 ⁻¹¹ , 46 × 10 ⁻¹¹ , 46.5 × 10 ⁻¹¹ , 47 × 10 ⁻¹¹ 47.5 × 10 ⁻¹¹ , 48 × 10 ⁻¹¹ , 48.5 × 10 ⁻¹¹ , 49 × 10 ⁻¹¹ , 49.5 × 10 ⁻¹¹ , 50 × 10 ⁻¹¹ , 51 × 10 ⁻¹¹ , 52 × 10 ⁻¹¹ , 53 × 10 ⁻¹¹ , 54 × 10 ^{− 11} , 55 × 10 ⁻¹¹ , 56 × 10 ⁻¹¹ , 57 × 10 ⁻¹¹ , 58 × 10 ⁻¹¹ , 59 × 10 ⁻¹¹ , 60 × 10 ⁻¹¹ , 62.5 × 10 ⁻¹¹ , 65 × 10 ^{− 11} , 67.5 × 10 ⁻¹¹ , 70 × 10 ⁻¹¹ , 72.5 × 10 ⁻¹¹ , 75 × 10 ⁻¹¹ , 77.5 × 10 ⁻¹¹ , 80 × 10 ⁻¹¹ , 82.5 × 10 ^{− 11} , 85 × 10 ⁻¹¹ , 87.5 × 10 ⁻¹¹ , 90 × 10 ⁻¹¹ , 95 × 10 ⁻¹¹ , 100 × 10 ⁻¹¹ , 110 × 10 ⁻¹¹ , 120 × 10 ⁻¹¹ , 130 × 10 ^{− 11} , 140 × 10 ⁻¹¹ , 150 × 10 ⁻¹¹ , 160 × 10 ⁻¹¹ , 170 × 10 ⁻¹¹ , 180 × 10 ⁻¹¹ , 190 × 10 ⁻¹¹ , 200 × 10 ⁻¹¹ μg or more, preferably , 70 × ^{10 -11} μg, Ri is preferably 85 × ^{10 -11} [mu] g, most preferably, may include at 100 × ^{10 -11} μg or more. In one embodiment, the liposome used in the present invention may contain, for example, cis-diamine dichloroplatinum (II) at 200 μg or less per liposome. The cis-diamine dichloroplatinum (II) -encapsulating liposome contains cis-diamine dichloroplatinum (II) in an amount of 200 × 10 ⁻¹¹ μg or less, for example, 200 × 10 ⁻¹¹ , 190 × 10 ⁻¹¹ , 180 ×, per liposome. ^{10 -11, 170 × 10 -11,} 160 × 10 -11, 150 × 10 -11, 140 × 10 -11, 130 × 10 -11, 120 × 10 -11, 110 × 10 -11, 100 × 10 - ¹¹ , 95 × 10 ⁻¹¹ , 90 × 10 ⁻¹¹ , 87.5 × 10 ⁻¹¹ , 85 × 10 ⁻¹¹ , 82.5 × 10 ⁻¹¹ , 80 × 10 ⁻¹¹ , 77.5 × 10 ⁻¹¹ , 75 × 10 ⁻¹¹ , 72.5 × 10 ⁻¹¹ , 70 × 10 ⁻¹¹ , 67.5 × 10 ⁻¹¹ , 65 × 10 ⁻¹¹ , 62.5 × 10 ⁻¹¹ , 60 × 10 ⁻¹¹ , 59 × 10 ⁻¹¹ , 58 × 10 ⁻¹¹ , 57 × 10 ⁻¹¹ , 56 × 10 ⁻¹¹ , 55 × 10 ⁻¹¹ , 54 × 10 ⁻¹¹ , 53 × 10 ⁻¹¹ , 52 × 10 ⁻¹¹ , 51 × 10 ⁻¹¹ , 50 × 10 ⁻¹¹ , 49.5 × 10 ⁻¹¹ , 49 × 10 ⁻¹¹ , 48.5 × 10 ⁻¹¹ , 48 × 10 ⁻¹¹ , 47.5 × 10 ⁻¹¹ , 47 × 10 ⁻¹¹ 46.5 × 10 ⁻¹¹ , 46 × 10 ⁻¹¹ , 45.5 × 10 ⁻¹¹ , 45 × 10 ⁻¹¹ , 44.5 × 10 ⁻¹¹ , 44 × 10 ⁻¹¹ , 43.9 × 10 ⁻¹¹ 43.8 × 10 ⁻¹¹ , 43.7 × 10 ⁻¹¹ , 43.6 × 10 ⁻¹¹ , 43.5 × 10 ⁻¹¹ , 43.4 × 10 ⁻¹¹ , 43.3 × 10 ⁻¹¹ , 43 ^{.2 × 10 -11, 43.1 × 10} -11 or less, preferably, 70 g × ^{10 -11} or less, more preferably, 85 [mu] g × ^{10 -11} or less, and most preferably, may include at 100 [mu] g × ^{10 -11} or less.

In another embodiment, the compositions of the present invention, the cis- diamminedichloroplatinum (II) can be formulated to be administered to be at least 500 mg / ^{m 2.} The cis-diamine dichloroplatinum (II) is, for example, about 500 to about 2000 mg / m ² , preferably about 500 to about 1800 mg / m ² , more preferably about 500 to about 1400 mg / m ² , most preferably About 500 to about 1000 mg / m ² , but is not limited thereto. The compositions of the invention can be administered, for example, by intravenous administration. Since cisplatin has high side effects, the dosage is limited, and in order to reduce the side effects, it is necessary to repeatedly administer small doses. As a result, cancer and tumor cells become resistant to cisplatin and become ineffective, which is a clinical problem. Although it is not desired to be bound by theory in this regard, since the composition of the present invention has few side effects, it is possible to administer a large amount of cisplatin at a time and kill cancer / tumor cells all at once. It is thought that you can. Furthermore, the composition of the present invention can contain a targeting substance, and can target cancer / tumor cells pinpoint. In addition, since the antitumor effect of cancer / tumor cells is actually confirmed, the composition of the present invention suppresses side effects by releasing cisplatin after accumulating around the cancer / tumor tissue. However, it is thought that the antitumor effect is exhibited.

  In one embodiment, in the composition of the present invention, the cancer or tumor is testicular tumor, bladder cancer, renal pelvis / ureteral tumor, prostate cancer, ovarian cancer, head and neck cancer, non-small cell lung cancer. , Esophageal cancer, cervical cancer, neuroblastoma, stomach cancer, small cell lung cancer, osteosarcoma, germ cell tumor, and the like. Here, cancer includes all neoplastic diseases such as tumor and leukemia. Preferably, the cancer or tumor can be colon cancer, lung cancer, squamous cell carcinoma, breast cancer, ovarian cancer.

  In one embodiment, the targeting substance that can be included in the composition of the present invention can be, for example, an antibody, sugar chain, lectin, complementary nucleic acid, receptor, ligand, aptamer, antigen. Preferably, the targeting substance may be an antibody (eg, anti-E-selectin antibody) or a sugar chain (eg, sialyl Lewis X (SLX)), but is not limited thereto. This is because the target directing substance may be any substance that imparts target directivity to the liposome without destroying the liposome. A person skilled in the art can appropriately determine the target-directing substance to be bound to the liposome according to the target.

  The composition of the present invention can specifically accumulate cis-diamine dichloroplatinum (II) -encapsulating liposomes at a target site such as cancer or tumor by including a target-directing substance. In particular, liposomes modified with sialyl Lewis X or anti-E-selectin antibody are prominently accumulated in cancers and tumors that actively undergo angiogenesis, and thus are thought to suppress the growth and proliferation of cancer cells and angiogenesis. It is done. As a result, a composition containing cis-diamine dichloroplatinum (II) -encapsulating liposomes at a dose much lower than that of the prior art is administered, and at least as much drug efficacy (eg, anticancer effect) as that of the prior art is applied to the target. While maintaining, it became possible to reduce the side effects on the individual.

  In one embodiment, in the composition of the present invention, the cis-diamine dichloroplatinum (II) may be in a water-soluble form.

  In another embodiment, liposomes encapsulating cis-diamine dichloroplatinum (II) that can be used in the composition of the present invention include, as lipids, dipalmitoyl phosphatidylcholine, cholesterol, ganglioside, dicetyl phosphate, dipalmitoyl phosphatidylethanolamine, Sodium cholate Sodium cholate, Dicetylphosphatidylethanolamine-polyglycerin 8G, Dimyristoyl phosphatidylcholine, Distearoyl phosphatidylcholine, Dioleoylphosphatidylcholine, Dimyristoylphosphatidylserine, Dipalmitoylphosphatidylserine, Distearoylphosphatidylserine, Dioleoyl Phosphatidylserine, dimyristoylphosphatidylinositol, dipalmitoy Phosphatidylinositol, distearoylphosphatidylinositol, dioleoylphosphatidylinositol, dimyristoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, distearoylphosphatidic acid Oil phosphatidic acid, galactosylceramide, glucosylceramide, lactosylceramide, phosphnatide, globotide, GM1 (Galβ1,3GalNAcβ1,4 (NeuAα2,3) Galβ1,4Glcβ1,1′Cer), ganglioside GD1a, ganglioside GD1b, dimyristoyl Zipalmitoilho Phosphatidyl glycerol, distearoyl phosphatidyl glycerol, can include dioleoyl phosphatidylglycerol, and the like. Preferably, dipalmitoyl phosphatidylcholine, cholesterol, ganglioside, dicetyl phosphate, dipalmitoyl phosphatidylethanolamine, sodium cholate, dicetylphosphatidylethanolamine-polyglycerin 8G, dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylglycerol may be used. This is because even liposomes composed of lipids other than the above can contain cis-diamminedichloroplatinum (II) and / or a water-soluble form thereof in the liposome, and they can be retained in the liposome. This is because it may be composed of lipids that can be stored.

  The liposome used in the composition of the present invention is any one as long as the water-soluble form of cis-diamminedichloroplatinum (II) can enter the liposome through the liposome membrane. It may be. This is because the water-soluble form of cis-diamminedichloroplatinum (II) only needs to pass through the provided liposomes or the membrane of the formed liposomes and migrate into the liposomes and remain there. As the liposome, commercially available liposomes may be used, for example, commercially available from NOF Corporation. Liposomes prepared by a plurality of production methods (for example, modified cholate method and lyophilized method) can also be used in combination.

  The preparation and characterization of general liposomes is known in the art and requires only routine experimentation and technical knowledge in the art. For example, it may be prepared by sonication method, ethanol injection method, French press method, ether injection method, cholic acid method, lyophilization method, reverse phase evaporation method (for example, “New Development of Liposome Application-Artificial Toward the development of cells-supervision Kazunari Akiyoshi / Kaoru Sakurai NTS p33-45 (2005) "," Liposome Nojima Shochichi p21-40 Nanedo (1988) ".

  Here, as the liposome encapsulating cis-diamine dichloroplatinum (II) used in the present invention, any form described in the above (Production method) and (Ammine platinum complex encapsulating liposome) can be used.

  The composition, medicament, and preparation of the present invention may contain a suitable formulation material or a pharmaceutically acceptable carrier as required. Suitable formulation materials or pharmaceutically acceptable carriers include antioxidants, preservatives, colorants, fluorescent dyes, flavoring agents, diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffering agents. , Delivery vehicles and / or pharmaceutical adjuvants. Typically, the composition of the present invention is in the form of a composition comprising an ammine platinum complex, and optionally other active ingredients, together with at least one physiologically acceptable carrier, excipient or diluent. Is administered. For example, suitable vehicles can be micelles, injection solutions, physiological solutions, or artificial cerebrospinal fluid, which are supplemented with other substances commonly used in compositions for parenteral delivery. It is possible.

  Acceptable carriers, excipients or stabilizers used herein are preferably non-toxic to recipients and preferably inert at the dosages and concentrations used. Preferably, for example, phosphate, citrate, or other organic acid; ascorbic acid, α-tocopherol; low molecular weight polypeptide; protein (eg, serum albumin, gelatin or immunoglobulin); hydrophilic polymer (eg, Amino acids (eg, glycine, glutamine, asparagine, arginine or lysine); monosaccharides, disaccharides and other carbohydrates (glucose, mannose, or dextrin); chelating agents (eg, EDTA); sugar alcohols (eg, EDTA); Mannitol or sorbitol ); Salt-forming counterions (such as sodium); and / or non-ionic surface-active agents (e.g., Tween, Pluronic (pluronic) or polyethylene glycol (PEG)), and the like.

  Exemplary suitable carriers include neutral buffered saline or saline mixed with serum albumin. Preferably, the product is formulated as a lyophilizer using a suitable excipient (eg, sucrose). Other standard carriers, diluents and excipients may be included as desired. Other exemplary compositions include a Tris buffer with a pH of about 7.0-8.5 or an acetate buffer with a pH of about 4.0-5.5, which further includes sorbitol or a suitable replacement thereof. Can contain things.

  The general preparation method of the composition in this invention is shown below. It should be noted that veterinary drug compositions, quasi-drugs, marine drug compositions, cosmetic compositions, and the like can also be produced by known preparation methods.

  The composition of the present invention can be blended with a pharmaceutically acceptable carrier as required, and can be administered parenterally, for example, as a liquid preparation such as an injection, suspension, solution, spray or the like. . Examples of pharmaceutically acceptable carriers include excipients, lubricants, binders, disintegrants, disintegration inhibitors, absorption enhancers, absorbents, wetting agents, solvents, solubilizers, suspending agents, Examples include isotonic agents, buffering agents, soothing agents and the like. In addition, formulation additives such as preservatives, antioxidants, colorants, sweeteners and the like can be used as necessary. Moreover, it is also possible to mix | blend substances other than sialyl Lewis X, a lipid, etc. with the composition and pharmaceutical of this invention. Examples of parenteral administration routes include, but are not limited to, intravenous, intramuscular, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, intravaginal administration, topical administration, and dermal administration. When administered systemically, the medicament used in the present invention may be in the form of a pharmaceutically acceptable aqueous solution free of pyrogens. It is within the skill of the artisan to consider pH, isotonicity, stability, etc. for the preparation of such pharmaceutically acceptable compositions.

  Preferable examples of the solvent in the liquid preparation include injection solutions, alcohol, propylene glycol, macrogol, sesame oil and corn oil.

  Preferable examples of the solubilizer in the liquid preparation include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate and sodium citrate. It is not limited to them.

  Suitable examples of the suspending agent in the liquid preparation include, for example, surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate, Examples thereof include hydrophilic polymers such as polyvinyl alcohol, polyvinyl pyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxyethyl cellulose, and hydroxypropyl cellulose.

  Preferable examples of the isotonic agent in the liquid preparation include, but are not limited to, sodium chloride, glycerin, D-mannitol and the like.

  Preferable examples of the buffer in the liquid preparation include, but are not limited to, phosphate, acetate, carbonate, citrate and the like.

  Preferable examples of the soothing agent in the liquid preparation include, but are not limited to, benzyl alcohol, benzalkonium chloride, procaine hydrochloride and the like.

  Preferable examples of the preservative in the liquid preparation include, but are not limited to, paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, 2-phenylethyl alcohol, dehydroacetic acid, sorbic acid and the like.

  Preferable examples of the antioxidant in the liquid preparation include, but are not limited to, sulfite, ascorbic acid, α-tocopherol and cysteine.

  When prepared as injections, solutions and suspensions are preferably sterilized and isotonic with solvents in the injection site for blood or other purposes. Usually, these are sterilized by filtration using a bacteria retention filter or the like, blending with a bactericide, or irradiation. Furthermore, after these treatments, solidify by freeze-drying or other methods, and add sterile water or sterile diluent for injection (lidocaine hydrochloride aqueous solution, physiological saline, aqueous glucose solution, ethanol, or a mixed solution thereof) just before use. May be.

  Furthermore, the composition in this invention may contain the coloring agent, the preservative, the fragrance | flavor, the flavoring agent, the sweetener, etc., and another chemical | medical agent.

  The amount of the substance, composition, etc. used in the present invention takes into consideration the purpose of use, the target disease (type, severity, etc.), the age, weight, sex, past history, cell morphology or type of the subject. Thus, it can be easily determined by those skilled in the art. The frequency with which the method of the present invention is applied to a subject also takes into account the purpose of use, the target disease (type, severity, etc.), the subject's age, weight, gender, medical history, treatment course, etc. A person skilled in the art can easily determine. Examples of the frequency include administration every day to once every several months (for example, once a week to once a month). It is preferable to administer once a week to once a month while observing the course. The amount to be administered can be determined by estimating the amount required by the site to be treated.

  As used herein, “subject” refers to an organism to which the treatment of the present invention is applied, and is also referred to as a “patient”. The patient or subject may be, for example, a bird, a mammal, and the like. Preferably, such animals are mammals (eg, single pores, marsupials, rodents, wings, wings, carnivorous, carnivorous, long-nosed, odd-hoofed, cloven-hoofed) , Rodents, scales, sea cattle, cetaceans, primates, rodents, rabbits, etc.). Exemplary subjects include, but are not limited to, animals such as humans, cows, pigs, horses, chickens, cats, dogs and the like. More preferably, it may be a human.

  As used herein, an “effective amount to treat” is a term that is well recognized by those skilled in the art to produce the intended pharmacological result (eg, prevention, treatment, prevention of recurrence, etc.). Refers to the amount of an effective drug. Thus, a therapeutically effective amount is an amount sufficient to reduce the symptoms of the disease to be treated. One useful assay to ascertain an effective amount (eg, a therapeutically effective amount) for a given application is to measure the extent of recovery of the target disease. The amount actually administered will depend on the individual to whom the treatment is to be applied, and is preferably an amount optimized to achieve the desired effect without significant side effects. The determination of an effective dose is well within the ability of those skilled in the art.

  Therapeutically effective amount, prophylactically effective amount, etc. and toxicity are standard pharmaceutical procedures in cell culture or laboratory animals (eg ED50, therapeutically effective dose in 50% of the population; and LD50, 50% of the population). Doses that are fatal). The dose ratio between therapeutic and toxic effects is the therapeutic index and it can be expressed as the ratio ED50 / LD50. Drug delivery vehicles that exhibit large therapeutic indices are preferred. Data obtained from cell culture assays and animal experiments are used to formulate a range of quantities for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. This dose will vary within this range depending on the mode of administration used, the sensitivity of the patient, and the route of administration. As an example, the dose is appropriately selected depending on the age and other patient conditions, the type of disease, the type of cells used, and the like.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range from the description of specific preferred embodiments of the present invention based on the description of the present specification and the common general technical knowledge. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  Hereinafter, although an example explains the composition of the present invention in detail, the present invention is not limited to this. The reagents used in the following were commercially available unless otherwise specified.

(Example 1: Preparation of liposome encapsulating cis-diamminedichloroplatinum (II))
(Synthesis of cis-diamine dinitratoplatinum (II) (cisplatin-nitrate))
After completely dissolving 4.15 g (10.0 mmol) of potassium platinum tetrachloride (K ₂ PtCl ₄ ) and 6.64 g (40 mmol) of potassium iodide in 50 ml of distilled water from which oxygen has been sufficiently removed, A precipitate was obtained by adding 1.35 ml (22 mmol) of 28% aqueous ammonia and stirring. This precipitate was washed twice with water and ethanol, respectively, and then dried at room temperature under vacuum (˜20 mmHg) to obtain 4.49 g of ammine-platinum intermediate cis- [Pt (NH ₃ ) ₂ I ₂ ]. . After 2.41 g (5.0 mmol) of this ammine-platinum intermediate cis- [Pt (NH ₃ ) ₂ I ₂ ] was suspended in 30 ml of distilled water, 1.68 g (9.9 mmol) of silver nitrate was added. The mixture was stirred for 24 hours in the dark. The produced silver iodide was filtered off, and the filtrate was concentrated with an evaporator to obtain white crystals. The white crystals were washed twice with cold distilled water and cold ethanol and dried to obtain 1.01 g of colorless cis- [Pt (NH ₃ ) ₂ (NO ₃ ) ₂ ] as a cisplatin-nitrate.

(Preparation of liposome encapsulating cis-diamine dinitratoplatinum (II))
Dipalmitoyl phosphatidylcholine (DPPC), cholesterol, ganglioside, dicetyl phosphate (DCP), dipalmitoyl phosphatidylethanolamine (DPPE) in a molar ratio of 35: 40: 15: 5: 5 to a total lipid content of 45.6 mg Then, 46.9 mg of sodium cholate was added and dissolved in 3 ml of a methanol / chloroform solution (1: 1). The mixture was stirred at 37 ° C. for 1 hour, and methanol / chloroform was evaporated by a rotary evaporator and vacuum-dried. The obtained lipid membrane was suspended in 3 ml of N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4) to which NaCl was not added, and stirred at 37 ° C. for 1 hour. The solution was then purged with nitrogen and sonicated to obtain a clear micelle suspension. Next, 54.1 mg of cis-diamine dinitratoplatinum (II) was weighed and dissolved in 7 ml of N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4) to which NaCl was not added. Ultrafiltration using PM10 (Amicon Co., USA) and N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4) without addition of NaCl (mixed with micelle suspension) Fractionated molecular weight: 10,000) to prepare 10 ml of uniform liposome (35.5 mg lipid amount).

(Confirmation that cis-diamine dinitratoplatinum (II) was encapsulated in the liposome)
It was confirmed by flameless atomic absorption spectrophotometry (FAAS) that the liposomes prepared in this example contained cis-diamine dinitratoplatinum (II).

  AA-6700 Atomic Absorption Flame Emission Spectrophotometer (Shimadzu) was used for flameless atomic absorption spectrophotometry (FAAS). Under the conditions of a wavelength of 265.9 nm, a slit width of 0.5, and a lamp current of 14 mA, 120 ° C. for 30 seconds, 250 ° C. for 10 seconds, 700 ° C. for 20 seconds, 700 ° C. for 5 seconds, 2600 ° C. for 3 seconds Processed sequentially. Prepare a standard curve by diluting a platinum standard solution (1 mg Pt / ml, 1000 ppm) with purified water to prepare solutions of 12.5 ng / ml, 25 ng / ml, 50 ng / ml, 100 ng / ml, and 200 ng / ml. did. The liposome solution was 10000 times with purified water to prepare a test sample.

(result)
In the liposome prepared in this example, cis-diamine dinitratoplatinum (II) was encapsulated in 323.33 μg / 1 ml (91.08 μg / mg lipid). The lipid content of the obtained liposome was 35.5 mg. The encapsulation rate of the cis-diamine dinitratoplatinum (II) in the liposome was 5.9%. The solution containing cis-diamine dinitratoplatinum (II) -encapsulating liposomes was colorless.

(Conversion of cis-diamine dinitratoplatinum (II) to cis-diamine dichloroplatinum (II))
Liposomes confirmed to contain cis-diamine dinitratoplatinum (II) were mixed with N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4), carbonate buffer (pH 8.5). ), PBS (pH 8.0), or HEPES buffer (pH 7.2) at 25 ° C. for 96 hours. Each of these buffers contains NaCl at a final concentration of 150 mM.

As a result, it is confirmed that the colorless solution turns pale yellow. This indicates that cis-diamine dinitratoplatinum (II) has been converted to cis-diamine dichloroplatinum (II). Further, this change can be detected by absorptiometry, IR method, and ¹⁹⁵ Pt-NMR method.

(Absorptiometric method)
(1) The NaCl of cis-diamine dinitratoplatinum (II) -encapsulated liposomes that have been subjected to a buffer containing NaCl at a final concentration of 150 mM for 96 hours is removed by buffer exchange and concentrated 10 times. (2) After heating at 100 ° C. for 30 minutes (breakdown of liposomes), solvent extraction is performed with chloroform. (3) Collect the aqueous layer. (4) An absorption spectrum with a wavelength of 200 to 400 nm is measured for the recovered aqueous layer. (5) The operations of (2) to (4) are also performed on the liposome immediately after encapsulating cis-diamine dinitratoplatinum (II) (before being subjected to 150 mM NaCl). (6) By comparing absorption spectra of cis-diamine dinitratoplatinum (II) liposomes subjected to buffer for 96 hours and liposomes immediately after inclusion of cis-diamine dinitratoplatinum (II), cis-diamine dinitrato The conversion from platinum (II) to cis-diamine dichloroplatinum (II) can be confirmed.

(IR method)
Solvent extraction is performed as in the spectrophotometric method. After collecting the aqueous layer, the water is evaporated using a rotary evaporator. Immediately after encapsulating cis-diamine dinitratoplatinum (II) -encapsulated liposomes and IR-spectrum measured by Nujol method and subjected to a buffer containing NaCl at a final concentration of 150 mM for 96 hours, and cis-diamine dinitratoplatinum (II) Compare the spectra of the liposomes (before subjecting to 150 mM NaCl).

In the case of cis-diamine dinitratoplatinum (II), a spectrum of Pt—O—H bending vibration appears in a region of 950 to 1100 cm ⁻¹ . In the case of cis-diamine dichloroplatinum (II), since there is only a Pt—Cl bond, no band is observed in the region of 950 to 1100 cm ⁻¹ . Therefore, conversion from cis-diamine dinitratoplatinum (II) to cis-diamine dichloroplatinum (II) can be confirmed.

  For details of the IR method for platinum complexes, see, for example, R.A. Fraggiani, B.M. Lippert, C.I. J. et al. Lock and B.M. Rosenberg, J. et al. Am. Chem. Soc. 99, 777 (1977), F.M. D. Rochon, A .; Morneau and R.M. Melason, Inorg. Chem. 27, 10 (1988).

(Conversion of nitrate contained in liposome to cisplatin)
(Analysis by ¹⁹⁵ PtNMR spectrum)
Conversion of nitric acid encapsulated in liposomes to cisplatin is analyzed by a ¹⁹⁵ Pt NMR spectrum. The sensitivity of the ¹⁹⁵ Pt NMR spectrum is 20 times greater than that of the natural ^13C- NMR spectrum. Moreover, the natural abundance ratio of ¹⁹⁵ Pt is 33.8%, and the relative sensitivity of naturally occurring platinum to ¹ H is relatively high at 3.4 × 10 ⁻³ . Furthermore, since the chemical shift is observed in a very wide frequency range of 1500 ppm, it is extremely useful for examining the structure and bonding properties.

(Sample preparation)
Samples of liposomes encapsulating cisplatin, nitrate (cis-diammine dinitratoplatinum (II)) and nitrate (cis-diammine dinitratoplatinum (II)) are prepared as follows.

Cisplatin: 2 mg of cisplatin (MW: 300) is dissolved in ₁ ml of D ₂ O to prepare a sample. (6.6 mM)
Nitric acid body (cis-diammine dinitratoplatinum (II)): 5 mg of nitric acid body (MW 365) is dissolved in ₁ ml of D ₂ O to prepare a sample. (13.7 mM)
Nitrate (cis-diammine dinitratoplatinum (II))-encapsulated liposome (NaCl ⁺ ): Concentrated to a Pt concentration of 4.75 mM.

(Measuring method)
An external standard solution is prepared by dissolving Na ₂ PtCl ₆ (sodium hexachloroplatinate (IV)) in D ₂ O. The NMR apparatus uses INOVA-600 (Varian). A sample is put in a 5 mm measuring tube and measurement is performed.

In this measuring method, a chemical shift is observed at -2160 ppm for cisplatin and -1620 ppm for nitric acid (cis-diammine dinitratoplatinum (II)) as controls. Therefore, the chemical shift of nitric acid (cis-diammine dinitratoplatinum (II)) encapsulated in the target liposome is observed at −2160 ppm, which is the same as cisplatin when NaCl is contained in the external liquid. Whether the encapsulated nitrate (cis-diammine dinitratoplatinum (II)) is substituted by sodium chloride-derived Cl ⁻ ions in the buffer solution and converted to cisplatin by measuring these chemical shifts, etc. You can check whether.

(Comparative example 1. Effect of sodium chloride ion)
DPPC, cholesterol, ganglioside, DCP, DPPE are mixed in a molar ratio of 35: 40: 15: 5: 5 so that the total lipid amount is 45.6 mg, 46.9 mg of sodium cholate is added, and methanol is added. -It was dissolved in 3 ml of a chloroform solution (1: 1) solution. The mixture was stirred at 37 ° C. for 1 hour, and methanol / chloroform was evaporated by a rotary evaporator and vacuum-dried. The obtained lipid membrane was suspended in 3 ml of N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4) to which NaCl was added and stirred at 37 ° C. for 1 hour. The solution was then purged with nitrogen and sonicated to obtain a clear micelle suspension. Next, 54.1 mg of cis-diamine dinitratoplatinum (II) was weighed and dissolved in 7 ml of N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4) to which NaCl was added. , Ultrafiltration using N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4) mixed with micelle suspension and supplemented with PM10 (Amicon Co., USA) and NaCl ( Fractionated molecular weight: 10,000), and 10 ml of uniform liposomes were prepared.

  When N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4) to which NaCl was added was used, cis-diamine dinitratoplatinum (II) was not encapsulated in liposomes. After dissolution in N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS) buffer, precipitation occurred in several tens of minutes. These were confirmed by the absorbance method. For the measurement, a UV 2500PC (Shimadzu) absorptiometer was used.

  Dissolve cis-diamine dinitratoplatinum (II) 5 mg / ml in TAPS buffer solution (pH 8.4) containing 150 mM sodium chloride, leave it at 25 ° C., and change with time (0 hour, 5 minutes, 10 minutes later). , 15 minutes, 30 minutes and 45 minutes later). As a control, 1 mg of cis-diamine dichloroplatinum (II) was dissolved in 1 ml of sodium chloride-containing TAPS buffer, and its UV spectrum was measured. As shown in FIGS. 4A and 4B, the absorption characteristics of the UV spectrum after 45 minutes were the same as those of cisplatin. Even after 45 minutes, no change was observed in the UV spectrum. Therefore, when an aqueous solution of cis-diamine dinitratoplatinum (II) is dissolved in TAPS containing 150 mM NaCl and allowed to stand at 25 ° C. for 45 minutes, cis-diamine dinitratoplatinum (II) is converted into cis-diamine dinitratoplatinum (II). ) Was found to change. It is considered that this is because cis-diamine dinitratoplatinum (II) is precipitated as cis-diamine dichloroplatinum (II), which is hardly soluble in water, due to the presence of NaCl.

(Example 2. Quantitative analysis of liposome)
(I. Lipid determination)
The amount of lipid constituting the liposome was calculated by quantifying the amount of cholesterol. Determinator TC555 kit (catalog number UCC / EAN128) (KYOWA Co. LTD) was used for lipid quantification. 50 mg / ml attached to the kit as a standard substance
Cholesterol was used.

As a standard solution, a standard substance (50 mg / ml: cholesterol) was diluted with a PBS buffer solution to prepare solutions of 0, 0.1, 0.25, 0.5, 0.75, 1, 5, 10 μg / 20 μl. . Cis-diamine dinitratoplatinum (II) -encapsulated liposomes were diluted 5-fold with PBS buffer to prepare a sample solution. 20 μl of each of the standard solution and the sample solution was dispensed into a test tube. To each test tube, 17 μl of Triton X-100 (10% solution) was added and stirred, and then allowed to stand at 37 ° C. for 40 minutes. 300 μl of the enzyme reagent of Determiner TC555 kit was added and stirred, and then allowed to stand at 37 ° C. for 20 minutes. Absorbance was measured at 540 nm, a calibration curve was prepared from the standard solution, the amount of cholesterol in the liposome was measured, and the amount of lipid was determined.
Conversion formula for determining lipid content from cholesterol content Lipid content (μg / 50 μl) = Cholesterol content (μg / 50 μl) × 4.51 (conversion factor)
The lipid content of the obtained liposome was 3.55 mg / ml.

(II. Particle size distribution and particle size)
Liposome particles were diluted 50 times with purified water and measured using Zetasizer Nano (Nan-ZS: MALVERN Co. LTD).

  The average particle size of the liposome particles was 159 nm (FIG. 2A).

(Example 3. Preparation of cis-diamine dinitratoplatinum (II) -encapsulated liposomes to which sugar chains are bound)
A cis-diamine dinitratoplatinum (II) -encapsulated liposome was prepared in the same manner as in “Preparation of cis-diaminedinitratoplatinum (II) -encapsulated liposome” in Example 1. In the liposome prepared in this example, cis-diamine dinitratoplatinum (II) was encapsulated in 323.33 μg / 1 ml (91.08 μg / mg lipid). The lipid content of the obtained liposome was 35.5 mg. The encapsulation rate of the cis-diamine dinitratoplatinum (II) in the liposome was 5.9%. The solution containing cis-diamine dinitratoplatinum (II) -encapsulating liposomes was colorless.

(Hydrophilic treatment on the liposomal lipid membrane surface)
10 ml of cis-diamine dinitratoplatinum (II) -encapsulated liposome solution was subjected to ultrafiltration (fractionated molecular weight: 300,000) using XM300 membrane (Amicon Co., USA) and carbonate buffer (pH 8.5). The pH of the solution was 8.5. Next, 10 mg of a crosslinking reagent bis (sulfosuccinimidyl) suberate (BS ³ ; Pierce Co., USA) was added, and the mixture was stirred at room temperature for 2 hours. Thereafter, the mixture was further stirred overnight under refrigeration to complete the chemical binding reaction between the lipid dipalmitoylphosphatidylethanolamine and BS ³ on the liposome membrane. Then, this liposome solution was subjected to ultrafiltration (fractionated molecular weight: 300,000) with an XM300 membrane and a carbonate buffer (pH 8.5). Next, 40 mg of tris (hydroxymethyl) aminomethane dissolved in 1 ml of carbonate buffer (pH 8.5) was added to 10 ml of liposome solution. The solution was then stirred at room temperature for 2 hours, then stirred overnight under refrigeration, ultrafiltered with a molecular weight cut off of 300,000 to remove free tris (hydroxymethyl) aminomethane, and the carbonate buffer Was replaced with N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4), and the chemical bonding reaction between BS ³ bound to the lipid on the liposome membrane and tris (hydroxymethyl) aminomethane was performed. Completed. As a result, the hydroxyl group of tris (hydroxymethyl) aminomethane was coordinated on the lipid dipalmitoylphosphatidylethanolamine of the liposome membrane to make it hydrated and hydrophilic.

(Binding of human serum albumin (HSA) on the liposome membrane surface)
The binding of human serum albumin (HSA) onto the surface of the liposome membrane was performed according to a previously reported method (Yamazaki, N., Kodama, M. and Gabis, H.-J. (1994) Methods Enzymol. 242, 56-65). The coupling reaction method was used. That is, this reaction is performed by a two-step chemical reaction. First, 1 ml of N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer solution was prepared by replacing ganglioside present on the surface of 10 ml of the liposome membrane obtained in this example. 43 mg of sodium metaperiodate dissolved in (pH 8.4) was added and periodate oxidized by stirring overnight under refrigeration. Free filtration of sodium periodate was removed by ultrafiltration (fractional molecular weight: 300,000) with XM300 membrane and PBS buffer (pH 8.0), and N-tris (hydroxymethyl) -3-amino was removed. The propanesulfonic acid buffer was exchanged with PBS buffer (pH 8.0) to obtain 10 ml of oxidized liposomes. To this liposome solution, 20 mg of human serum albumin (HSA) / PBS buffer (pH 8.0) was added and reacted at room temperature for 2 hours, and then 100 μl of 2M NaBH ₃ CN / PBS buffer (pH 8.0). The mixture was stirred at room temperature for 2 hours and further overnight under refrigeration, and HSA was bound by a coupling reaction between ganglioside on the liposome and HSA. Then, ultrafiltration (fractionated molecular weight: 300,000) was performed to remove free sodium cyanoborate and human serum albumin, and the buffer of this solution was replaced with carbonate buffer (pH 8.5). 10 ml of an HSA-bound liposome solution was obtained.

(Preparation of sugar chain)
Sialyl Lewis X was used as the sugar chain.

  The mass of each sugar chain was measured and pretreated for use in the following.

(Binding of sugar chain on liposome membrane surface-bound human serum albumin (HSA))
2 mg of each sugar chain prepared in this example was dissolved in purified water, added to a 0.5 ml aqueous solution in which 0.25 g of NH ₄ HCO ₃ was dissolved, stirred at 37 ° C. for 3 days, and then filtered with a 0.45 μm filter. Filtration was completed to complete the amination reaction at the reducing end of the sugar chain to obtain 4 mg / ml of the glycosylamine compound of each sugar chain (aminated sugar chain solution). Next, 10 mg of a crosslinking reagent 3,3′-dithiobis (sulfosuccinimidylpropionate) (DTSSP; Pierce Co., USA) was added to 10 ml of a part of the liposome solution obtained in this example, and the mixture was stirred for 2 hours at room temperature. Subsequently, the mixture was stirred overnight under refrigeration and ultrafiltered (fractionated molecular weight: 300,000) with an XM300 membrane and carbonate buffer (pH 8.5) to remove free DTSSP. 10 ml of liposomes bound to HSA. Next, the above glycosylamine compound (aminated sugar chain solution) 12.5, 37.5, 125, 250, 500, 1250, 2500 μl is added to this liposome solution, and the mixture is reacted at room temperature for 2 hours. Methyl) aminomethane / carbonate buffer (pH 8.5) was added and then stirred overnight under refrigeration to allow the glycosylated amine compound to bind to DTSSP on liposome membrane bound human serum albumin. Free sugar chains and tris (hydroxymethyl) aminomethane were removed by ultrafiltration (fraction molecular weight: 300,000) with XM300 membrane and HEPES buffer (pH 7.2). As a result, 10 ml of each liposome in which sugar chains, human serum albumin and liposomes were bound was obtained.

(Hydrophilic treatment on liposomal membrane-bound human serum albumin (HSA))
About the liposome with which the sugar chain couple | bonded with the present Example was couple | bonded, the hydrophilization process of the HSA protein surface on a liposome was performed separately with the following procedures, respectively. Separately, 26.4 mg of tris (hydroxymethyl) aminomethane was added to 10 ml of the liposomes, and the mixture was stirred at room temperature for 2 hours and then overnight under refrigeration, followed by XM300 membrane and HEPES buffer (pH 7.2). Ultrafiltration (fraction molecular weight: 300,000) was performed to remove unreacted substances. Filtration through a 0.45 μm filter yields 10 ml each of the liposome complex to which the hydrophilized sugar chain is bound as a final product (total lipid amount 15.2 mg, total protein amount 1100 μg, average particle size 171 nm). It was.

(Confirmation that cis-diamine dichloroplatinum (II) is encapsulated in liposomes)
The solution containing liposomes with sugar chains prepared according to this example changed from colorless to pale yellow. This shows that cis-diamine dinitratoplatinum (II) encapsulated in liposomes was converted to cis-diamine dichloroplatinum (II). Further, this change can be detected by absorptiometry, IR method, and ¹⁹⁵ Pt-NMR method.

(Conversion of nitrate contained in liposome to cisplatin)
(Analysis by ¹⁹⁵ PtNMR spectrum)
The conversion of nitrate encapsulated in liposomes to cisplatin was analyzed by ¹⁹⁵ Pt NMR spectrum.

(Sample preparation)
Samples of liposomes encapsulating cisplatin, nitrate (cis-diammine dinitratoplatinum (II)) and nitrate (cis-diammine dinitratoplatinum (II)) were prepared as follows.

Cisplatin: 2 mg of cisplatin (MW: 300) was dissolved in ₁ ml of D ₂ O to prepare a sample. (6.6 mM)
Nitric acid body (cis-diammine dinitratoplatinum (II)): 5 mg of nitric acid body (MW 365) was dissolved in ₁ ml of D ₂ O to prepare a sample. (13.7 mM)
Nitrate (cis-diammine dinitratoplatinum (II))-encapsulated liposomes (NaCl ⁺ ): Concentrated to a Pt concentration of 4.75 mM.

(Measuring method)
Na ₂ PtCl ₆ (sodium hexachloroplatinate (IV)) was dissolved in D ₂ O to prepare an external standard solution. As the NMR apparatus, INOVA-600 (Varian) was used. Measurement was performed by placing 600 μl of a sample in a measuring tube having a diameter of 5 mm.

(result)
As a control, a chemical shift was observed at -2160 ppm for cisplatin and -1620 ppm for nitric acid (cis-diammine dinitratoplatinum (II)) (FIGS. 5a and 5b). The chemical shift of nitric acid (cis-diammine dinitratoplatinum (II)) encapsulated in the target liposome was observed at −2160 ppm as in the case of cisplatin when NaCl was contained in the external liquid (FIG. 5 c). From this, it was confirmed that the encapsulated nitric acid (cis-diammine dinitratoplatinum (II)) was replaced by Cl ⁻ ions derived from sodium chloride in the buffer solution, and most of it was converted to cisplatin. It was. Moreover, when cis-diammine dinitratoplatinum (II) encapsulated in liposomes is not changed to cis-diamine dichloroplatinum (II), a peak of -1620 ppm which is a chemical shift value of cis-diamminedinitratoplatinum (II) Furthermore, -2160 ppm, which is the chemical shift value of cis-diamine dichloroplatinum (II), was not observed (FIG. 5d).

  In the liposome prepared in this example, cis-diamine dichloroplatinum (II) was encapsulated in 253.98 μg / 1 ml (78.38 μg / mg lipid). The lipid content of the obtained liposome was 32.4 mg. The encapsulation rate of the cis-diamine dichloroplatinum (II) in the liposome was 4.59%.

  The amount of cisplatin encapsulated in the liposome produced by the method of Patent Document 1 is 8.9 μg / mg lipid, and the lipid amount of the obtained liposome is 200 mg. Therefore, the amount of cisplatin encapsulated per liposome produced by this method is about 9 times as much as that of the liposome of Patent Document 1. Further, in the method of this example, 54.1 mg of cis-diamine dinitratoplatinum (II) (46.0 mg in terms of cisplatin) is encapsulated in the liposome, whereas in Patent Document 1, it is about twice as large. An amount of 100 mg of cisplatin is encapsulated in the liposomes. Therefore, when the encapsulation efficiency per unit lipid amount is converted from the amount of cisplatin encapsulated and the amount of cisplatin used for the encapsulation, it can be said that the liposome produced by the method of the present invention is about 18 times as excellent. Furthermore, the method of the present invention does not include a heating step, and it can be said that the prepared liposome is stable.

  In addition, the liposome described in Non-Patent Document 1 contains 14.0 μg / mg lipid cisplatin. Therefore, the amount of cisplatin encapsulated per liposome produced by this method is about 5.6 times as much as that of the liposome of Non-Patent Document 1.

  Therefore, it was confirmed that the cisplatin-encapsulated liposome prepared by this method can encapsulate much more cisplatin than the liposome produced by the conventional method.

(Example 4. Quantitative analysis of liposome)
In this example, the quantitative analysis of the cis-diamine dinitratoplatinum (II) -encapsulated liposomes to which the sugar chain was prepared prepared in Example 3 was performed.

(I. Protein quantification)
The amount of HSA encapsulated in the liposome and the total protein amount of HSA coupled to the liposome surface were measured by the BCA method.

Micro BCA ^™ Protein Assay Reagent kit (Cat. No. 23235BN) (PIERCE Co. LTD) was used for the measurement of protein amount. As a standard substance, 2 mg / ml albumin (BSA) attached to the kit was used.

  As a standard solution, a standard substance (2 mg / ml: albumin) was diluted with a PBS buffer solution to prepare solutions of 0, 0.25, 0.5, 1, 2, 3, 4, 5 μg / 50 μl. Cis-diamine dinitratoplatinum (II) -encapsulating liposomes were diluted 20-fold with PBS buffer to prepare a sample solution. 50 μl of each of the standard solution and the sample solution was dispensed into a test tube. 100 μl of 3% sodium lauryl sulfate solution (SDS solution) was added to each test tube. Reagents A, B, and C attached to the kit were mixed so that reagent A: reagent B: reagent C = 48: 2: 50, and 150 μl was added to each test tube. The test tube was allowed to stand at 60 ° C. for 1 hour. After returning to room temperature, the absorbance at 540 nm was measured, a calibration curve was prepared with the standard solution, and the amount of protein in the liposome was measured. The protein amount of the liposome was 110 μg / ml.

(II. Lipid quantification)
The amount of lipid constituting the liposome was calculated by quantifying the amount of cholesterol. Determinator TC555 kit (catalog number UCC / EAN128) (KYOWA Co. LTD) was used for lipid quantification. 50 mg / ml attached to the kit as a standard substance
Cholesterol was used.

As a standard solution, a standard substance (50 mg / ml: cholesterol) was diluted with a PBS buffer solution to prepare solutions of 0, 0.1, 0.25, 0.5, 0.75, 1, 5, 10 μg / 20 μl. . The cis-diamine dinitratoplatinum (II) -encapsulated liposomes to which sugar chains were bound prepared in Example 3 were diluted 5-fold with a PBS buffer solution to prepare a sample solution. 20 μl of each of the standard solution and the sample solution was dispensed into a test tube. To each test tube, 17 μl of Triton X-100 (10% solution) was added and stirred, and then allowed to stand at 37 ° C. for 40 minutes. 300 μl of the enzyme reagent of Determiner TC555 kit was added and stirred, and then allowed to stand at 37 ° C. for 20 minutes. Absorbance was measured at 540 nm, a calibration curve was prepared from the standard solution, the amount of cholesterol in the liposome was measured, and the amount of lipid was determined.
Conversion formula for determining lipid content from cholesterol content Lipid content (μg / 50 μl) = Cholesterol content (μg / 50 μl) × 4.51 (conversion factor)
The lipid content of the obtained liposome was 3.24 mg / ml.

(III. Particle size distribution and particle size)
Liposome particles were diluted 50 times with purified water and measured using Zetasizer Nano (Nan-ZS: MALVERN Co. LTD).

  The average particle diameter of the liposome particles was 171 nm (FIG. 2B).

(Example 5: Preparation of liposomes encapsulating cis-diamine dichloroplatinum (II) conjugated with an antibody)
A cis-diamine dinitratoplatinum (II) -encapsulated liposome was prepared in the same manner as in “Preparation of cis-diaminedinitratoplatinum (II) -encapsulated liposome” in Example 1. The solution containing the cis-diamine dinitratoplatinum (II) -encapsulating liposome is colorless.

(Hydrophilic treatment on the liposomal lipid membrane surface)
In the same manner as in Example 1, the liposome lipid membrane surface is subjected to a hydrophilic treatment.

(Preparation of target-directed substances)
As a target-directing substance, an anti-E-selectin antibody was used as an antibody specific to the tumor. This antibody was prepared as follows. Hybridoma cells (Number: CRL-2515 CL3) producing anti-human E-selectin mouse monoclonal antibody were purchased from ATCC and purified. Cells RPMI 1640 (GIBCO code.11875) / 10 % FBS / penicillin 100 unit / ml · streptomycin 100μg / ml (Antibiotic Antimycotic solution, stabiklized SIGMA A5955) in (fetal bovine serum SIGMA F0926), 37 ℃, 5 % CO 2 environment Cultured under. After pristane (500 μl / mouse) was administered twice (5 day intervals) to mouse Balb / c (Japan SLC female, 6 weeks old), 5 × 10 ⁶ hybridoma cells were suspended in PBS and 1 ml each was intraperitoneally injected. Injected. On day 14 after injection, the abdomen was opened and ascites was collected. Ammonium sulfate (0.4 saturation) was added to ascites and centrifuged at 10,000 rpm × 30 minutes (4 ° C.). The precipitate was dissolved in physiological saline and dialyzed (3 days, 4 ° C.). Centrifugation is performed at 10,000 rpm × 30 minutes (4 ° C.), and 2 volumes of 0.06 M acetate buffer (pH 4.8) is added to the supernatant, so that the final concentration of n-Capric acid is 6.8%. And stirred at room temperature for 30 minutes. Centrifugation was performed at 10,000 rpm × 30 minutes (0 to 4 ° C.), and dialysis (3 days, 4 ° C.) was performed. Centrifugation was performed at 10,000 rpm × 30 minutes (0 to 4 ° C.), and the supernatant was collected. A commercially available antibody can also be used as the antibody specific to the tumor (for example, an anti-E-selectin antibody (AF575) manufactured by R & D systems (MN, USA)).

(Binding of antibody on liposome membrane-bound human serum albumin (HSA))
To 10 ml of a part of the liposome liquid obtained in this example, 10 mg of a crosslinking reagent 3,3′-dithiobis (sulfosuccinimidylpropionate) (DTSSP; Pierce Co., USA) was added, and then at room temperature for 2 hours, Stir overnight under refrigeration, ultrafiltration (fraction molecular weight: 300,000) with XM300 membrane and carbonate buffer (pH 8.5) to remove free DTSSP, and DTSSP becomes HSA on liposomes. 10 ml of bound liposome was obtained. Next, 3.26 ml (15 mg antibody) of 4.6 mg / ml anti-E-selectin antibody prepared in this example was added to this liposome solution and reacted at 25 ° C. for 2 hours. (Hydroxymethyl) aminomethane / carbonate buffer (pH 8.5) was added and then stirred overnight under refrigeration to bind anti-E-selectin antibody to DTSSP on liposome membrane-bound human serum albumin. It was. The free anti-E-selectin antibody and tris (hydroxymethyl) aminomethane were removed by ultrafiltration (fraction molecular weight: 300,000) with XM300 membrane and HEPES buffer (pH 7.2). As a result, 10 ml of each liposome in which anti-E-selectin antibody, human serum albumin and liposome were bound was obtained.

(Hydrophilic treatment on liposomal membrane-bound human serum albumin (HSA))
The liposome to which the anti-E-selectin antibody prepared in this example was bound was subjected to a hydrophilic treatment by the same method as in Example 3.

(Confirmation that cis-diamine dichloroplatinum (II) is encapsulated in liposomes)
The solution containing liposomes bound with anti-E-selectin antibody prepared according to this example changed from colorless to pale yellow. This shows that cis-diamine dinitratoplatinum (II) encapsulated in liposomes was converted to cis-diamine dichloroplatinum (II). Further, this change can be detected by the spectrophotometric method, IR method and ¹⁹⁵ Pt-NMR method using the same method as in Example 1.

(Quantitative analysis of liposomes)
Quantitative analysis (protein quantification, lipid quantification, particle size distribution and particle size) of Cis-diamine dinitratoplatinum (II) -encapsulated liposomes bound with anti-E-selectin antibody prepared in this example was the same as in Example 1. The method was used (FIG. 6).

(Example 6A: Examination of efficacy for cancer / tumor treatment)
In this example, cis-diamine dichloroplatinum (II) -encapsulated liposomes prepared according to Example 1 (liposome without sugar chain), Example 3 (sugar chain-modified liposome) and Example 5 (antibody-modified liposome) are cancerous. -The purpose is to confirm that it is effective in the treatment of tumors.

(Evaluation of anticancer activity of cis-diamine dichloroplatinum (II) -encapsulated liposomes)
(1. Accumulation in cancer-bearing site: evaluation of the amount of cis-diamine dichloroplatinum (II) accumulated in the cancer site)
The cis-diamine dichloroplatinum (II) -encapsulated liposome prepared in Example 3 (Sialyl Lewis X-modified liposome) was used.

1 × 10 ⁶ cells of Ehrlich ascites tumor cells (EAT cells) (ATCC Number: CCL-77TM E) subcutaneously in the right thigh of 6 week old mice (Balb / c, female) (one per group). Ehrlich-Letter assets)) was used for experiments 10 days after transplantation. Sialyl Lewis X-modified liposomes encapsulating cis-diamine dichloroplatinum (II), and cis-diamine dichloroplatinum (II) alone as a control at 150 mg to the mouse at 1.7 mg / kg body weight (226 μg / ml), tail vein Administered. Tumors were removed 48 and 96 hours after administration.

  Of the excised tumor tissue (0.2 to 1.0 g), 1 mL of concentrated nitric acid was added to 0.1 g and stirred for 5 minutes. After leaving still for 1 hour, it heated in the 60 degreeC water bath for 1 hour, and returned to room temperature. 300 μl of distilled water was added to 100 μl of the solution treated with nitric acid. The amount of platinum was measured by flameless atomic absorption spectrophotometry (FAAS: AA-6700 Atomic Absorption Flame Emission Spectrophotometer SIMAZU), and the amount of platinum per gram of tumor was compared. (Wavelength 265.9 nm, slit width 0.5, lamp current 14 mA, 120 ° C. 30 seconds, 250 ° C. 10 seconds, 700 ° C. 5 seconds, 2600 ° C. 3 seconds: calibration curve is 1 mg / ml platinum standard solution (NAKARAI) Diluted with distilled water to make solutions of 12.5 ng / ml, 25 ng / ml, 50 ng / ml, 100 ng / ml, 200 ng / ml). The results are shown in the table below.

Platinum concentration: Amount of platinum present per gram of tissue (ng)
Time after administration: time when each liposome was administered and the concentration of platinum present in the tissue was measured Cisplatin alone: cis-diamine dichloroplatinum (II) only SLX modification: sialyl Lewis X containing cis-diamine dichloroplatinum (II) Modified liposomes.

(Discussion)
48 hours after liposome administration, it was observed that in mice administered with sialyl Lewis X modified liposomes, liposomes were significantly accumulated at the cancer site compared to mice administered only with cis-diamine dichloroplatinum (II). It was done. In addition, when only cis-diamine dichloroplatinum (II) was administered 96 hours after administration, accumulation at the cancer site was significantly reduced. In sialyl Lewis X-modified liposomes, it was confirmed that cis-diamine dichloroplatinum (II) remained at the tumor site 2.5 times or more than when cis-diamine dichloroplatinum (II) alone was administered (Table). 1).

(2. Confirmation of activity to suppress cell growth)
In this example, cis-diamine dichloroplatinum (II) -encapsulating liposomes prepared in Example 1 (liposome without sugar chain), Example 3 (sugar chain-modified liposome) and Example 5 (antibody-modified liposome) were used.

Ehrlich ascites tumor cell (EAT cell) (ATCC Number: CCL-77TM)
E (Ehrlich-Letter assets)) was seeded at 1 × 10 ³ cells / 100 μl / well (Falcon 3072), and the sample was added after 24 hours of culture. Samples were cis-diamine dichloroplatinum (II) encapsulated unmodified liposome, cis-diamine dichloroplatinum (II) encapsulated sialyl Lewis X liposome, cis-diamine dichloroplatinum (II) encapsulated anti-E-selectin antibody-modified liposome, DMEM (Dulbecco) Modified Eagle Liquid Medium Low Glucose (SIGMA D6046) / 10% FBS (Fetal Bovine Serum SIGMA F0926) / Penicillin 100 unit / ml Streptomycin 100 μg / ml II) The amount was adjusted to 10 μg / ml. As a control, cis-diamine dichloroplatinum (II) alone was dissolved in DMEM / 10% FBS and adjusted to 10 μg / ml (cisplatin alone) was used. Next, this sample was added to the cultured cells so that the final concentrations were 8.3 μM and 16.6 μM, and cultured in a 37 ° C., 5% CO ₂ incubator for 48 hours. The number of cells 48 hours after the addition was evaluated by performing an MTT assay using the amount of formazan as an index.

  Furthermore, an MTT assay was also performed on an object containing no sample, to which only DMEM / 10% FBS was added.

20 μl of WST-8 for cell number measurement (Kishida Chemical) was added to each well, and the absorbance at 450 nm was measured after 2 hours. For the measurement of absorbance, GE Healthcare Ultraspec-6300 was used. Experiments were performed in 4 sections per sample; n = 3. The results are shown below.
(Table 2. Inhibition of cell proliferation by each sample)

Sample: Final concentration of added sample (μM)
No sample: Average cisplatin alone of the absorbance (450 nm) of the group to which no sample (cis-diamine dichloroplatinum (II)) was added: cis-diamine dichloroplatinum (II) alone Unmodified: cis-diamine dichloroplatinum (II) An unmodified liposomal SLX modification encapsulating cis-diamine dichloroplatinum (II) encapsulated, sialyl Lewis X modified liposome antibody modification: cis-diamine dichloroplatinum (II) encapsulating an anti-E-selectin antibody Modified liposome average: Absorbance (450 nm) average SD after administration of each sample: standard deviation.

(Discussion)
In the group to which no sample was added, the absorbance (450 nm) was 0.480 (Table 2, no sample).

  The group to which unmodified liposomes, SLX modified liposomes and antibody modified liposomes were administered was almost the same as that of cis-diamine dichloroplatinum (II) alone when the final sample concentration was 8.3 μM or 16.6 μM. It was shown to be effective. From this result, it was shown that even when cis-diamine dichloroplatinum (II) was encapsulated in liposomes, the anticancer effect was not lost. It was confirmed that cisplatin was released from the liposome in the cell and interacted with the nucleic acid.

  According to this example, it was confirmed that cis-diamine dichloroplatinum (II) encapsulated in liposomes exhibited growth inhibitory activity on cells in all of unmodified liposomes, SLX modified liposomes and antibody modified liposomes.

(3. Confirmation of tumor growth inhibitory effect)
1 × 10 ⁶ cells of Ehrlich ascites tumor cells (EAT cells) (ATCC Number: CCL-77TM E (Ehrlich-)) subcutaneously in the right thigh of 6-week-old mice (Balb / c female) (3 mice in each group) Letter assays)). At 5 days, 12 days, and 19 days after transplantation, cis-diamine dichloroplatinum (II) -encapsulated sialyl Lewis X-modified liposomes and anti-E-selectin antibody-modified liposomes were respectively administered at 1.5 mg / kg (200 μg / ml) to mice. Was administered into the tail vein. As a control, physiological saline and cis-diamine dichloroplatinum (II) were dissolved in physiological saline so as to be 200 μg / ml. This dissolved solution was administered similarly.

The size of the tumor was confirmed by measuring the major axis (Amm) and bearing diameter (Bmm) using digital calipers (Mitutoyo CD-S15C) 12 days, 19 days, and 29 days after tumor implantation. Tumor volume (mm ³ ) was calculated using the formula (A × B ² ) × 0.4.

  In this animal experiment, the liposome for encapsulation uses a sugar chain (SLX), which is known to be directed to cancer, and an antibody, to improve the anticancer effect against conventional cisplatin. Observed. The results are shown below.

Tumor volume (mm ³ )
Days: Time when tumor size was measured after tumor transplantation Saline: saline only cisplatin alone: cis-diamine dichloroplatinum (II) only SLX modification: sialyl Lewis containing cis-diamine dichloroplatinum (II) Liposome antibody modification modified with X: liposomes modified with anti-E-selectin antibody encapsulating cis-diamine dichloroplatinum (II) Average: average SD of tumor volume (mm ³ ) after administration of each sample: standard deviation .

(Results and Discussion)
From this result, when sialyl Lewis X-modified liposome or anti-E-selectin antibody-modified liposome encapsulating cis-diamine dichloroplatinum (II) was administered, the size of the tumor was 29 days after tumor cell transplantation. -Significantly smaller than those administered diaminedichloroplatinum (II) alone and unmodified liposomes.

  This is because liposomes encapsulating cis-diamine dichloroplatinum (II) were modified with sialyl Lewis X or anti-E-selectin antibody, so that the accumulation of liposomes in tumor tissue increased, and cis-diamine was added to the tumor. This is thought to be the result of the concentrated effects of dichloroplatinum (II).

  In this example, the effect of modification of sialyl Lewis X and anti-E-selectin antibody was confirmed. From this result, liposomes encapsulating cis-diamine dichloroplatinum (II) and modified with sialyl Lewis X or anti-E-selectin antibody are significantly less than cis-diamine dichloroplatinum (II) administered alone. Was confirmed to suppress tumor growth. This indicates that liposomes modified with sialyl Lewis X or anti-E-selectin antibody are effective for the treatment of tumors.

  It was also confirmed that the amount of cisplatin encapsulation per lipid or phospholipid can be significantly increased as compared with the conventional encapsulation method, and as a result, a remarkable therapeutic effect is achieved.

  This is, for example, a conventional anti-cancer agent-encapsulated liposome (Cancer Chemother Pharmacol (1999) 43: 1-7 Competitive pharmacological pharmacetic drugs, and therapeutic efficacy of citrate phosphocetic sensitivities.) It is understood that it is more prominent than that achieved in bearin rice). The SPI077 liposome described in this document is a liposome clinically used in the United States. In FIG. 1A of this document, data of SPI077 liposome is described, but its dosage is much larger than that of the liposome of this example. Therefore, it can be said that the present invention achieves the same or better effect while reducing the total dose. Depending on the type of cancer, probe-labeled liposomes (SPI077) have anti-cancer activity similar to this paper, so liposomes using sugar chains and antibodies as recognition probes can be used at this cisplatin dose. The effect is expected.

(Example 6B. Acute toxicity test)
In this example, cis-diamine dichloroplatinum (II) -encapsulating liposomes prepared by the same method as in Example 3 (sugar chain-modified liposome) were used.

(Method)
Balb / c Female 8-week-old normal mice (4 mice per group) were administered cisplatin (CDDP) and cisplatin-encapsulated liposomes (sialyl Lewis X-modified) at 25 mg / Kg and 18 mg / Kg via the tail vein. After administration, the survival rate up to 2 weeks was confirmed.

(result)
At a dose of cisplatin of 25 mg / Kg, the survival rate of mice administered cisplatin alone was 0% 5 days after administration. In cisplatin-encapsulated liposomes (sialyl Lewis X modification), the survival rate was 75%.

  When the dose of cisplatin was 18 mg / Kg, the survival rate of mice administered cisplatin alone was 25% after 5 days from the administration. In cisplatin-encapsulated liposomes (modified with sialyl Lewis X), 75% of mice survived.

(Discussion)
Cisplatin encapsulated liposomes (sialyl Lewis X modified) have been demonstrated to have very low acute toxicity. In other words, even if the dose of cisplatin alone is toxic, cisplatin-encapsulated liposomes may be administered without acute toxicity to the subject.

(Example 6C. In vitro cell growth inhibitory activity)
(Method)
(Cell culture)
HT29: human colon cancer cells (ATCC No. THB38), A549: human lung cancer cells (ATCC No. CCL-185), A431: human squamous cell carcinoma cells (ATCC No. CRL-1555), LLC: mouse lung cancer Cells (RIKEN: RCB0098), SKBr3: human breast cancer cells (RIKEN: No. TKG0592) were purchased and used for the following experiments.

Each cell was seeded in a 96-well microplate (Falcon 3072) at 1 × 10 ³ cells / 50 μL / well and cultured at 37 ° C. under 5% CO _{2 for} 24 hours. The cell culture medium used was DMEM (SIGMA D6046) supplemented with 10% FBS (Biowest Code. No. S1823) and 1% penicillin / streptomycin solution (SIGMA P0781).

(Preparation of cisplatin-encapsulated liposome-Hepes solution)
In this example, cis-diamine dichloroplatinum (II) -encapsulated liposomes prepared by the same method as Example 1 (liposome without sugar chain), Example 3 (sugar chain-modified liposome) and Example 5 (antibody-modified liposome). (Cisplatin-encapsulating liposomes) were used.

  The cisplatin-encapsulated unmodified liposome was diluted with 20 mM Hepes buffer (pH 7.2) to a concentration of 0.4 mM cisplatin to prepare a cisplatin-encapsulated unmodified liposome-Hepes solution.

  For the cisplatin-encapsulated SLX-modified liposome, sialyl Lewis X (SLX) was used as a sugar chain. The cisplatin-encapsulated SLX-modified liposome was diluted with 20 mM Hepes buffer (pH 7.2) to a concentration of 0.4 mM cisplatin to prepare a cisplatin-encapsulated SLX-modified liposome-Hepes solution.

Anti-E-selectin antibody specific for tumor was used as the cisplatin-encapsulating antibody-modified liposome. The anti-E-selectin antibody used in this example was prepared using the same method as in Example 5: Anti-E-selectin producing hybridoma (CL-3) ATCC No. CRL-2515 was purchased and purified. Mice Balb / c (Japan SLC female 6 weeks old) was administered pristane twice at 0.5 mL / mouse (5 day intervals). Hybridomas were prepared in RPMI 1640 medium (GIBCO code. 11875) / 10% FBS / penicillin 100 units / ml streptomycin 100 μg / ml (Antibiotic Antimicrobial solution, stabilized SIGMA A 5955) (5 fetal bovine serum CO ₂ F 9 SIG MA) Incubated at 0 ° C. Cells were suspended in PBS and administered intraperitoneally at 5 × 10 ⁶ cells / mouse. Ascites was collected about 2 weeks later. Ammonium sulfate was precipitated, the precipitate was dissolved in PBS, and dialyzed against PBS buffer (3 days, 4 ° C.). The dialyzed solution was passed through a protein G column (HiTrap Protein G column 1 mL). After washing the column, the monoclonal antibody bound to protein G was eluted with 0.1 M glycine buffer (pH 2.7), and the eluate was neutralized with 1 M Tris buffer (pH 9.0). A commercially available antibody can also be used as the antibody specific to the tumor (for example, an anti-E-selectin antibody (AF575) manufactured by R & D systems (MN, USA)).

  The cisplatin-encapsulated antibody-modified liposome was diluted with 20 mM Hepes buffer (pH 7.2) to a concentration of 0.4 mM cisplatin to prepare a cisplatin-encapsulated antibody-modified liposome-Hepes solution.

(Addition of cisplatin-encapsulating liposome solution)
Each cisplatin-encapsulating liposome-Hepes solution was added to the cell culture so that the final concentration of cisplatin was 10 μM, 50 μM, 100 μM, or 200 μM. More specifically, when the final concentration was 200 μM, 50 μL of the cisplatin-encapsulating liposome-Hepes solution was added as it was. When final concentrations of 10 μM, 50 μM, and 100 μM were added, the cisplatin-encapsulated liposome-Hepes solution was further diluted with the above cell culture medium to make 50 μL.

  As a control, 50 μL of 20 mM Hepes buffer (pH 7.2) was used instead of the cisplatin-encapsulating liposome-Hepes solution. This control is intended to take into consideration the influence of the Hepes buffer used for diluting the cisplatin-encapsulating liposomes on the cell growth ability. The maximum amount of Hepes buffer added per well, 50 μL, was used as a control.

72 hours after the addition of cisplatin-encapsulated liposome-Hepes solution or control, 10 μL of WST-8 (reagent for cell proliferation measurement: Kida Chemical Code No. 260-96165) was added to each well, and 5% CO was added for 1 hour. ₂ and cultured at 37 ° C. The number of cells was evaluated by taking advantage of the proportional relationship between the absorbance and the number of living cells. Specifically, the absorbance was measured at 450 nm using a BioRAD Model 680 microplate reader absorbance meter, and the number of cells was calculated. A 96-well microplate reader added with WST-8 and cultured at 37 ° C. under 5% CO _{2 for} 1 hour was placed on the microplate reader, and the absorbance was measured. The sensitivity of cisplatin was assessed by the number of survivors (%) relative to this control.

(result)
Cell viability decreased in all cell lines to which cisplatin-encapsulated unmodified liposomes, cisplatin-encapsulated SLX-modified liposomes, and cisplatin-encapsulated antibody-modified liposomes were added (FIGS. 9 to 13). That is, it can be said that all cell lines showed sensitivity to all cisplatin-encapsulating liposomes (FIGS. 9 to 13). Further, no difference in cell viability (sensitivity) was observed between these cisplatin-encapsulated liposomes. From this result, it was confirmed that the modification of liposome with sialyl Lewis X or anti-E-selectin antibody does not affect the effect of cisplatin encapsulated in liposome.

(Comparative Example 6C. In Vitro Cell Growth Inhibitory Activity with Cisplatin Alone) (Cell Culture)
In this comparative example, HT29: human colon cancer cells (ATCC No. THB38), A549: human lung cancer cells (ATCC No. CCL-185), A431: human squamous cell carcinoma cells (ATCC No. CRL-1555), LLC: Mouse lung cancer cells (RIKEN: RCB0098), SKOV3: human ovarian cancer cells (ATCC: Code. No. HTB-77) were used. Each cell was seeded in a 96-well microplate (Falcon 3072) at 1 × 10 ³ cells / 50 μL / well and cultured at 37 ° C. under 5% CO _{2 for} 24 hours. As the cell culture medium, DMEM (SIGMA D6046) supplemented with 10% FBS (Biowest Code. No. S1823) and 1% penicillin / streptomycin solution (SIGMA P0781) was used.

(Preparation of cis-diamine dichloroplatinum (II) (cisplatin) -Hepes solution)
P4394 purchased from SIGMA was used as cisplatin. As the cisplatin used in this example, those purchased from Bristol Pharmaceutical Co., Ltd., Nippon Kayaku, Marco, Yakult, Merck, Pfizer, Kyowa Hakko, etc. can be used. In addition, 1.98 equivalents of sodium chloride or potassium chloride are added to the cisplatin nitrate aqueous solution, and the mixture is stirred for 24 hours under light shielding. It is also possible to synthesize by collecting the resulting precipitate by filtration and washing twice with cold water.

  Cisplatin was diluted with 20 mM Hepes buffer (pH 7.2) to a concentration of 0.4 mM cisplatin to prepare a cisplatin-Hepes solution.

(Addition of cisplatin-Hepes solution)
The cisplatin-Hepes solution was further diluted with the cell culture medium so that the final concentration of cisplatin was 2 μM, 10 μM, 20 μM, or 40 μM, and 50 μL was added to the cell culture.

  As a control, 10 μL of 20 mM Hepes buffer (pH 7.2) was used in place of the cisplatin-Hepes solution. This was diluted to a total of 50 μL with the cell culture medium and added to the cell culture. The amount of Hepes of 10 μL is the same as the amount of Hepes buffer added per well when adding cisplatin solution at 40 μM. This control is for taking into consideration the effect of the Hepes buffer used for diluting cisplatin on the cell growth ability.

72 hours after the addition of cisplatin-Hepes solution or control, 10 μL of WST-8 (reagent for cell proliferation measurement: Kida Chemical Code 260-96165) was added to each well, and the reaction was performed for 1 hour under 5% CO ₂ at 37 ° C. Incubated at 0 ° C. The number of cells was evaluated by taking advantage of the proportional relationship between the absorbance and the number of living cells. Specifically, the absorbance was measured at 450 nm using a BioRAD Model 680 microplate reader absorbance meter, and the number of cells was calculated. Add 96-well microplate reader cultured at 37 ° C. under 5% CO _{2 for} 1 hour by adding WST-8 (reagent for cell proliferation measurement: Kida Chemical Code No. 260-96165) on the microplate reader The absorbance was measured. The sensitivity of cisplatin was assessed by the number of survivors (%) relative to this control.

(result)
Viability of all cells was reduced by cisplatin (FIG. 14). That is, all cells were sensitive to cisplatin. In Comparative Example 6C, since all cells were killed when the final concentration of cisplatin was 40 μM or higher, no experiment was conducted at a concentration higher than 40 μM.

(Summary of Example 6C and Comparative Example 6C)
In this example, all cisplatin-encapsulated liposomes decreased cell viability regardless of the presence or absence of the targeting substance (FIGS. 9 to 13). That is, it is considered that the modification with the targeting substance does not affect the effect of the encapsulated cisplatin.

  In addition, compared to cisplatin not encapsulated in liposomes, cisplatin encapsulated in liposomes had a high cell survival rate even when a high concentration was added. In cisplatin-encapsulated liposomes, it is thought that cisplatin is released into the cell by breaking the liposome after the liposome is taken into the cell, so that a sufficient amount of cisplatin from the liposome to reduce cell viability is in the cytoplasm. There is a possibility that a certain amount of time is required before being released into the water. The results of this example are data after 72 hours from the addition of cisplatin-encapsulated liposomes. Therefore, by extending the period from the addition of cisplatin-encapsulated liposomes to evaluation, it does not show the same sensitivity as cisplatin. It is thought. In addition, with cisplatin-encapsulated liposomes, there is a possibility that cisplatin can be released to cells over a long period of time.

(Example 6D: Examination of efficacy for treatment of other cancer / tumor)
The cis-diamine dichloroplatinum (II) -encapsulated liposomes prepared according to Example 1 (unmodified liposome), Example 3 (sugar chain-modified liposome) and Example 5 (antibody-modified liposome) are testis tumor, bladder cancer, Renal fistula / ureteral tumor, prostate cancer, ovarian cancer, head and neck cancer, non-small cell lung cancer, esophageal cancer, cervical cancer, neuroblastoma, stomach cancer, small cell lung cancer, osteosarcoma, germ cell tumor It is confirmed whether it is effective for the treatment.

Testicular tumor, bladder cancer, renal pelvis / ureteral tumor, prostate cancer, ovarian cancer, head and neck cancer, non-small cell lung cancer, esophageal cancer, cervical cancer in female Balb / c mouse subcutaneously Tumor, neuroblastoma, gastric cancer, small cell lung cancer, osteosarcoma, germ cell tumor cells (approximately 2 × 10 ⁷ cells) transplanted and cancer tissue developed to 0.3-0.6 g Used in this experiment as a cancer mouse.

  Each tumor-bearing mouse is administered with each cis-diamine dichloroplatinum (II) -encapsulating liposome (200 μl) by either oral administration or tail vein administration.

  In all cancer-bearing mice administered with cis-diamine dichloroplatinum (II) -encapsulating liposomes, the volume of cancer / tumor was measured using the same method and criteria as in Example 6A, and It can be determined whether the tumor / cancer disappears.

  Therefore, it is possible to examine whether or not the cis-diamine dichloroplatinum (II) -encapsulated liposome produced by this method is effective for cancer / tumor treatment.

(Example 7: Experiments with other water-soluble ammine platinum complexes)
(Preparation of liposome encapsulating cisplatin-sulfate)
(Preparation of cisplatin-sulfate)
After completely dissolving 4.15 g (10.0 mmol) of potassium platinum tetrachloride (K ₂ PtCl ₄ ) and 6.64 g (40 mmol) of potassium iodide in 50 ml of distilled water from which oxygen has been sufficiently removed, A precipitate is obtained by adding 1.35 ml (22 mmol) of 28% aqueous ammonia and stirring. The precipitate is washed twice with water and ethanol, respectively, and then dried at room temperature in vacuum (˜20 mmHg) to obtain ammine-platinum intermediate cis- [Pt (NH ₃ ) ₂ I ₂ ]. 1.45 g (3.0 mmol) of this ammine-platinum intermediate cis- [Pt (NH ₃ ) ₂ I ₂ ] was suspended in 300 ml of distilled water, and then 0.933 g (3.0 mmol) of silver sulfate was added. Then, the mixture is reacted for 4 hours with stirring, and the produced silver iodide is separated by filtration to obtain colorless cis- [Pt (NH ₃ ) ₂ SO ₄ ] as a cisplatin-sulfate.

(Preparation of liposome encapsulating cisplatin-sulfate)
DPPC, cholesterol, ganglioside, DCP, DPPE are mixed in a molar ratio of 35: 40: 15: 5: 5 so that the total lipid amount is 45.6 mg, 46.9 mg of sodium cholate is added, and methanol is added. -Dissolve in 3 ml of chloroform solution (1: 1). The mixture is stirred at 37 ° C. for 1 hour, and methanol / chloroform is evaporated by a rotary evaporator and vacuum-dried. The obtained lipid membrane is suspended in 3 ml of N-tris (hydroxymethyl) -3-aminomethane (TAPS) buffer solution (pH 8.4) not added with NaCl, and stirred at 37 ° C. for 1 hour. The solution is then purged with nitrogen and sonicated to obtain a clear micelle suspension. Next, 50 mg of cisplatin-sulfate is weighed and dissolved in 7 ml of a TAPS buffer (pH 8.4) not added with NaCl, mixed with the micelle suspension, PM10 (Amicon Co., USA) and NaCl not added. 10 ml of uniform liposomes are prepared by ultrafiltration (fraction molecular weight: 10,000) using added TAPS buffer (pH 8.4).

(Confirmation that cisplatin-sulfate was encapsulated in the liposome)
Using the same method as in Example 1, it is confirmed by flameless atomic absorption spectrophotometry (FAAS) that the liposome prepared in this example contains cisplatin-sulfate.

  As a result, it can be confirmed whether or not cisplatin-sulfate is encapsulated in the liposome.

(Conversion of cisplatin-sulfate to cis-diamine dichloroplatinum (II))
(1. Conversion of cisplatin-sulfate to cis-diamine dichloroplatinum (II) by buffer)
Liposomes confirmed to contain cisplatin-sulfate were mixed with N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4), carbonate buffer (pH 8.5), PBS (pH 8). 0.0) or HEPES buffer (pH 7.2) at 25 ° C. for 96 hours. Each of these buffers contains NaCl at a final concentration of 150 mM.

As a result, it is confirmed that the colorless solution turns pale yellow. This indicates that the cisplatin-sulfate was converted to cis-diamine dichloroplatinum (II). Further, this change can be detected by the spectrophotometric method, IR method and ¹⁹⁵ Pt-NMR method using the same method as in Example 1.

(2. Preparation of liposome encapsulating cisplatin-sulfuric acid linked with sugar chain)
Using the same method as in Example 2, liposomes encapsulating cis-diamine dichloroplatinum (II) bound with a sugar chain are prepared from liposomes that have been confirmed to encapsulate cisplatin-sulfate. In the obtained liposome, the conversion of cisplatin-sulfate into cis-diamine dichloroplatinum (II) was detected by the same method as in Example 1 using the spectrophotometric method, IR method, and ¹⁹⁵ Pt-NMR method. can do.

(3. Preparation of liposome encapsulating cis-diamine dichloroplatinum (II) bound with antibody)
Using the same method as in Example 6, liposomes encapsulating cis-diamine dichloroplatinum (II) bound with an antibody were prepared from each solution of liposomes that were confirmed to encapsulate cisplatin-sulfate. To do. In the obtained liposome, the conversion of cisplatin-sulfate into cis-diamine dichloroplatinum (II) was detected by the same method as in Example 1 using the spectrophotometric method, IR method, and ¹⁹⁵ Pt-NMR method. can do.

(Quantitative analysis of liposomes)
For each of the liposomes prepared in the above section (Conversion of cisplatin-sulfate to cis-diamine dichloroplatinum (II)), quantitative analysis (protein quantification, lipid quantification, particle size distribution and particle size) was carried out as in Example 1. The same method is used.

(Investigation of efficacy of cancer / tumor treatment)
Whether or not each of the liposomes prepared in the above section (Conversion of cisplatin-sulfate to cis-diamine dichloroplatinum (II)) by the same method as in Example 5 is effective in treating tumors and cancers. Check.

  As a result, in all cancer-bearing mice administered with liposomes encapsulating these poorly water-soluble drugs, the volume of cancer / tumor was measured, and the change in volume and whether the tumor / cancer disappeared. Can be considered.

  Therefore, it can be seen whether the liposome encapsulating cis-diamine dichloroplatinum (II) produced by this method is effective for the treatment of cancer / tumor.

(Example 8: Experiments with other platinum complexes)
Using the same method as in Example 1, instead of cis-diamminedichloroplatinum (II), cis-diammine-1,1-cyclobutane-dicarboxylatoplatinum (II), cis-diammine (glycolato) platinum (II ), (1R, 2R-diamminecyclohexane) oxalatoplatinum (II) and cis-diamminedibromoplatinum (II) are prepared.

(Confirmation that drug is encapsulated in liposome)
Using the same method as in Example 1, it can be confirmed by flameless atomic absorption spectrophotometry (FAAS) whether these drugs are encapsulated in liposomes as a water-soluble form.

(Conversion from water-soluble drugs to poorly water-soluble drugs)
(1. Conversion from water-soluble drug to poorly water-soluble drug by buffer solution)
Using the same method as in Example 1, each solution of liposomes confirmed to contain a water-soluble drug is subjected to a solution containing ions in which the salt formed is sparingly water-soluble. As a result, it was shown that the water-soluble drug was converted to a poorly water-soluble drug. Further, this change can be detected by the spectrophotometric method, IR method and ¹⁹⁵ Pt-NMR method using the same method as in Example 1.

(2. Preparation of liposomes encapsulating a poorly water-soluble drug with sugar chains attached)
Using the same method as in Example 3, liposomes encapsulating a poorly water-soluble drug to which a sugar chain is bound are prepared from each solution of liposomes that have been confirmed to encapsulate a water-soluble drug. About this liposome, it can be detected that the encapsulated water-soluble drug is converted to a poorly water-soluble drug by the same method as in Example 1 by the spectrophotometric method, IR method, and ¹⁹⁵ Pt-NMR method. .

(3. Preparation of liposome encapsulating a poorly water-soluble drug to which an antibody is bound)
Using the same method as in Example 6, liposomes encapsulating a poorly water-soluble drug to which an antibody is bound are prepared from each solution of liposomes that have been confirmed to encapsulate a water-soluble drug. About this liposome, it can be detected that the encapsulated water-soluble drug is converted to a poorly water-soluble drug by the same method as in Example 1 by the spectrophotometric method, IR method, and ¹⁹⁵ Pt-NMR method. .

(Quantitative analysis of liposomes)
For each of the liposomes prepared in the above section (Conversion from water-soluble drug to poorly water-soluble drug), quantitative analysis (protein quantification, lipid quantification, particle size distribution and particle size) is performed in the same manner as in Example 1. .

(Investigation of efficacy of cancer / tumor treatment)
In the same manner as in Example 5, it is confirmed whether each of the liposomes prepared in the above section (Conversion from a water-soluble drug to a poorly water-soluble drug) is effective for tumor / cancer treatment.

  As a result, in all cancer-bearing mice administered with liposomes encapsulating these poorly water-soluble drugs, the volume of cancer / tumor was measured, and the change in volume and whether the tumor / cancer disappeared. Can be considered.

  Therefore, it can be seen whether the liposome encapsulating the poorly water-soluble drug produced by this method is effective for the treatment of cancer / tumor.

(Example 9. Encapsulation of cis-diammine dinitratoplatinum (II) in lyophilized empty liposome)
In this example, in order to confirm that the encapsulation technique of the present invention can be applied to any liposome, an improved cholate method (N. Yamazaki, M. kodama, HJ. Gabis, Methods Enzymol., 242). , 56-65 (1994); N. Yamazaki, J. Membr. Sci., 41, 249-267 (1989); and M. Hiroi, H. Minematsu, N Kondo, K Oie, K. Igarashi, N. Yamazaki. , BBRC., 353, 553-558 (2007)), a lyophilized empty liposome method (H. Kikuchi, N. Suzuki et al.).
al, Biopharm. Drug Dispos. , 17, 589-605 (1999)), cis-diammine dinitratoplatinum (II) was encapsulated.

(Synthesis of cis-diammine dinitratoplatinum (II) (CDDP-3))
The same method as in Example 1 was used. Cis-diammine dinitratoplatinum (II) was synthesized by the method of Dahara (SG Dhara, Indian J. Chem., 7, 335 (1970)).

After dissolving 4.15 g (10 mmol) of potassium chloroplatinate in distilled water, 6.64 g (40 mmol) of potassium iodide was added while nitrogen was blown in a light-shielded and ice-cooled state and stirred for 5 minutes. To this reaction solution, 1.35 mL (22 mmol) of 28% aqueous ammonia solution was added dropwise and stirred for 3 hours. The produced yellow crystals were collected by filtration, washed successively with distilled water and ethanol, and then dried under reduced pressure at 40 ° C. for 10 hours. Here, 4.49 g of ammine-platinum intermediate cis- [Pt (NH ₃ ) ₂ I ₂ ] (cis-diaminediodeplatinum (II) (CDDP-2)) was obtained. After suspending 2.41 g (5 mmol) of cis- [Pt (NH ₃ ) ₂ I ₂ ] in distilled water, 1.68 g (9.9 mmol) of silver nitrate was added, and the mixture was stirred for 24 hours under light shielding. The produced silver iodide was filtered off, and the filtrate was concentrated by an evaporator to obtain white crystals. The white crystals were washed successively with cold distilled water and ethanol and then dried under reduced pressure at 40 ° C. for 10 hours to obtain 1.01 g of cis-diammine dinitratoplatinum (II).

(Preparation of two types of liposomes and inclusion of cis-diamine dichloroplatinum (II))
(1. Improved cholate method (Liposome I))
Liposome I was prepared using a method similar to the improved cholate method of Example 1. DPPC, cholesterol, ganglioside, DCP, DPPE are mixed at a molar ratio of 35: 40: 15: 5: 5 so that the total lipid amount is 45.6 mg, and 46.9 mg of sodium cholate is added, and methanol is added. -It was dissolved in 3 mL of chloroform (1: 1) solution. After stirring at 37 ° C. for 1 hour, methanol and chloroform were evaporated with a rotary evaporator, and after vacuum drying, sodium chloride-free TAPS (N-tris (hydroxymethyl) -3-aminopropanesulfonic acid) buffer (pH 8.4) Suspended in 3 mL. After stirring at 37 ° C. for 1 hour, sonication was performed to obtain a transparent micelle suspension.

  Next, cis-diammine dinitratoplatinum (II) (108.2 mg) was completely dissolved in 7 ml of sodium chloride-free TAPS buffer (pH 8.4), adjusted to pH 8.4 with 1 M sodium hydroxide, Mixed with micelle suspension. 10 mL of liposomes were prepared by ultrafiltration (fractionation molecular weight: 10,000) using PM10 (AMICON) and a sodium chloride-free TAPS buffer (pH 8.4). The external solution of liposome was replaced with 150 mM sodium chloride-containing TAPS buffer (pH 8.4) by ultrafiltration using PM10 (Amicon), and cis-diammine dinitratoplatinum (II) encapsulated in the liposome was replaced with cis-diamine. Conversion into dichloroplatinum (II).

(2. Lyophilization method (Liposome II)
As the freeze-dried empty liposome, COASTOME EL series (EL-01-PA, NOF Corporation) was used. The composition of this COASTOME EL-01-PA (negatively charged liposome) was dicetylphosphatidylethanolamine-polyglycerin 8G: dipalmitoylphosphatidylcholine: cholesterol: dipalmitoylphosphatidylglycerol = 4.2: 11.4: 15.2. 11.4 (μMol / vial). Dissolve cis-diammine dinitratoplatinum (II) (40 mg) in 2 mL of sodium chloride-free TAPS buffer (pH 8.4), adjust to pH 8.4 with 1 M sodium hydroxide, and place it in the vial of lyophilized liposomes. 2 mL was added dropwise. The vial was mixed by inverting 5 times, and 2 mL of liposomes were prepared by ultrafiltration (fractionated molecular weight: 10,000) using PM10 (AMICON) and TAPS buffer solution (pH 8.4) containing no sodium chloride. Sodium chloride was added to the liposome solution so as to have a concentration of 150 mM, and the resultant was stirred at 4 ° C. for 96 hours to convert the encapsulated cis-diammine dinitratoplatinum (II) into cis-diamine dichloroplatinum (II).

(Comparative Example 2. Liposomes directly encapsulating cis-diamine dichloroplatinum (II) without using cis-diammine dinitratoplatinum (II))
As a comparative example, instead of encapsulating cis-diammine dinitratoplatinum (II) in a liposome and converting it to cis-diamine dichloroplatinum (II), a liposome was prepared in which cis-diamine dichloroplatinum (II) was encapsulated directly. .

(1. Improved cholate method)
The liposome directly encapsulating cis-diamine dichloroplatinum (II) was completely dissolved in 7 ml of TAPS buffer (pH 8.4) containing 150 mM sodium chloride in 28 mg of cis-diamine dichloroplatinum (II) (SIGMA). The pH was adjusted to 8.4 with sodium oxide and mixed with the micelle suspension. The inclusion of cis-diammine dinitratoplatinum (II) in this example except that cis-diamine dichloroplatinum (II) is used instead of cis-diammine dinitratoplatinum (II) and that the buffer contains sodium chloride The same method was used.

(2. Freeze-drying method)
COASTOME EL series (EL-01-PA, Nippon Oil & Fats) was used as the lyophilized empty liposome. Liposomes in which cis-diamine dichloroplatinum (II) was directly encapsulated were prepared as follows. COASTOME EL-01-PA was returned to room temperature. 4 mg of cisplatin was completely dissolved in 2 ml of 150 mM sodium chloride-containing TAPS buffer (pH 8.4), added to COASTOME EL-01-PA, and mixed by inversion three times. In order to remove free cisplatin, ultrafiltration was performed using a membrane with a molecular weight cut-off of 10,000.

(3. Production of cis-diamine dichloroplatinum (II) -encapsulated liposome I modified with a sugar chain)
Liposome I modified with a sugar chain was prepared in the same manner as in Example 3 using a part of cis-diamine dichloroplatinum (II) -encapsulating liposome I prepared by the improved cholic acid method of this example.

(Hydrophilic treatment on the liposomal lipid membrane surface)
Ultrafiltration using a cis-diamine dinitratoplatinum (II) -encapsulated liposome I using XM300 membrane (Amicon Co., USA) and carbonate buffer (pH 8.5) (fractionated molecular weight: 300,000) ) To bring the pH of the solution to 8.5. Next, 10 mg of a crosslinking reagent bis (sulfosuccinimidyl) suberate (BS ³ ; Pierce Co., USA) was added, and the mixture was stirred at room temperature for 2 hours. Thereafter, the mixture was further stirred overnight under refrigeration to complete the chemical binding reaction between the lipid dipalmitoylphosphatidylethanolamine and BS ³ on the liposome membrane. Then, this liposome solution was subjected to ultrafiltration (fractionated molecular weight: 300,000) with an XM300 membrane and a carbonate buffer (pH 8.5). Next, 40 mg of tris (hydroxymethyl) aminomethane dissolved in 1 ml of carbonate buffer (pH 8.5) was added to 10 ml of liposome solution. The solution was then stirred at room temperature for 2 hours, then stirred overnight under refrigeration, ultrafiltered with a molecular weight cut off of 300,000 to remove free tris (hydroxymethyl) aminomethane, and the carbonate buffer Was replaced with N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4), and the chemical bonding reaction between BS ³ bound to the lipid on the liposome membrane and tris (hydroxymethyl) aminomethane was performed. Completed. As a result, the hydroxyl group of tris (hydroxymethyl) aminomethane was coordinated on the lipid dipalmitoylphosphatidylethanolamine of the liposome membrane to make it hydrated and hydrophilic.

(Binding of human serum albumin (HSA) on the liposome membrane surface)
Metaperiodate in which ganglioside present on the membrane surface of 10 ml of liposome I obtained in this Example was dissolved in 1 ml of N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4). Sodium acid 43 mg was added and periodate oxidized by stirring overnight under refrigeration. Free filtration of sodium periodate was removed by ultrafiltration (fractional molecular weight: 300,000) with XM300 membrane and PBS buffer (pH 8.0), and N-tris (hydroxymethyl) -3-amino was removed. The propanesulfonic acid buffer was exchanged with PBS buffer (pH 8.0) to obtain 10 ml of oxidized liposomes. To this liposome solution, 20 mg of human serum albumin (HSA) / PBS buffer (pH 8.0) was added and reacted at room temperature for 2 hours, and then 100 μl of 2M NaBH ₃ CN / PBS buffer (pH 8.0). The mixture was stirred at room temperature for 2 hours and further overnight under refrigeration, and HSA was bound by a coupling reaction between ganglioside on the liposome and HSA. Then, ultrafiltration (fraction molecular weight: 300,000) was performed to remove free sodium cyanoborate and human serum albumin, and the buffer of this solution was replaced with carbonate buffer (pH 8.5). 10 ml of bound liposome solution was obtained.

(Preparation of sugar chain)
Sialyl Lewis X was used as the sugar chain.

  The mass of each sugar chain was measured and pretreated for use in the following.

(Binding of sugar chain on liposome membrane surface-bound human serum albumin (HSA))
2 mg of a sugar chain is dissolved in purified water, added to a 0.5 ml aqueous solution containing 0.25 g of NH ₄ HCO ₃ , stirred at 37 ° C. for 3 days, and then filtered through a 0.45 μm filter to reduce the sugar chain. The terminal amination reaction was completed to obtain 4 mg / ml glycosylamine compound (aminated sugar chain solution) of each sugar chain. Next, 10 mg of a crosslinking reagent 3,3′-dithiobis (sulfosuccinimidylpropionate) (DTSSP; Pierce Co., USA) was added to a part (10 ml) of the liposome I solution obtained in this example. Stir at room temperature for 2 hours, then overnight under refrigeration, ultrafiltered (fractionated molecular weight: 300,000) with XM300 membrane and carbonate buffer (pH 8.5) to remove free DTSSP, 10 ml of liposomes in which DTSSP was bound to HSA on the liposomes were obtained. Next, 37.5 μl of the above-mentioned glycosylamine compound (aminated sugar chain solution) is added to this liposome solution, and the mixture is reacted at room temperature for 2 hours. Tris (hydroxymethyl) aminomethane / carbonate buffer (pH 8.5) Then, the mixture was stirred overnight under refrigeration, and the glycosylated amine compound was bound to DTSSP on liposome membrane surface-bound human serum albumin. Free sugar chains and tris (hydroxymethyl) aminomethane were removed by ultrafiltration (fraction molecular weight: 300,000) with XM300 membrane and HEPES buffer (pH 7.2). As a result, 10 ml of each liposome in which sugar chains, human serum albumin and liposomes were bound was obtained.

(Hydrophilic treatment on liposomal membrane-bound human serum albumin (HSA))
About the liposome I with which the sugar chain prepared in the present Example was combined, the surface of the HSA protein on the liposome was hydrophilized. To this liposome (10 ml), tris (hydroxymethyl) aminomethane (26.4 mg) was added and stirred at room temperature for 2 hours and then overnight under refrigeration, followed by ultrafiltration with an XM300 membrane and HEPES buffer (pH 7.2). (Fractionation molecular weight: 300,000) and unreacted substances were removed. Filtration through a 0.45 μm filter yielded 10 ml each of the liposome complex to which the sugar chains that had been subjected to hydrophilic treatment as the final product were bound.

(Measurement of particle size and zeta potential)
Each liposome solution (without sugar chain) prepared in this example and comparative example was diluted 50-fold with pure water, and measured by a dynamic scattering method using Zetasizer Nano Nan-ZS (Malvern).

  The particle size distribution of cis-diamine dichloroplatinum (II) -encapsulated liposome I encapsulated with cis-diammine dinitratoplatinum (II) and then converted to cis-diamine dichloroplatinum (II) shows a uniform particle size distribution, The average particle size was 159 nm (Table 4). The particle size distribution of the cis-diamine dichloroplatinum (II) -encapsulated liposome II encapsulated with cis-diammine dinitratoplatinum (II) and then converted into cis-diamine dichloroplatinum (II) shows a uniform particle size distribution, The average particle size was 147 nm (Table 4).

As a result of confirmation of the particle sizes and shapes of the liposomes I and II, as shown in Table 4, the particle size of the cis-diamine dichloroplatinum (II) -encapsulating liposomes was about 150 nm in any liposome. In order to accumulate liposomes in tumor tissue or inflamed tissue, it is important to adjust the particle size of the liposomes. Since it has been clarified that there is a gap of 100 to 200 nm in the neovascularization of the cancer site and the blood vessel of the inflammation site, the size of the liposome I and liposome II particles is transferred from the inside of the blood vessel to the tissue. (A. A. Gabizon, Cancer Res., 52, 891-896 (1992); SK Huang, F. J. Martin, G. Jay et al.). , Am. J. Pathol., 143, 10-14 (1993); S. K. Huang, E. Mayhew, S. Gilani et al., Cancer Res., 52, 6774-6781 (1992); Wu, D. Da, TL Rudoll et al, Cancer Res., 53, 3765-3770 (1 93)).

(Observation with electron microscope)
Liposomes I encapsulating cis-diamine dichloroplatinum (II) prepared in this Example (no sugar chain) were negatively stained with 1% uranium acetate (JD Almeida, CM Brand, DC). Edwards and TD Heath, Lancet 2 (7941), 899-901 (1975) Specifically, one drop of the liposome solution was dropped onto a copper mesh, washed well with PBS buffer, and washed with 1% acetic acid. After immersing in uranium for several seconds, excess moisture was absorbed and dried, and then the shape and size of the particles were observed at 80000 times with a transmission electron microscope H-7100S (HITACHI).

  As a result, it was found that the liposome I encapsulating cis-diamine dichloroplatinum (II) was a uniform spherical liposome (FIG. 7). Moreover, the particle diameter of the liposome I observed with the electron microscope obtained the result which corresponds with the average particle diameter by a dynamic scattering method.

(Quantification of lipid content)
The same method as in Example 2 was used. Total cholesterol level was measured using Determiner TC555 kit (KYOWA) in the presence of 0.5% TitonX-100, and the total lipid level was calculated from the molar ratio of each lipid. Was 3.5 mg / mL for liposome I and 7.6 mg / mL for liposome II (Table 4).

(Quantification of cis-diammine dinitratoplatinum (II))
The cis-diammine dinitratoplatinum (II) encapsulated in the liposome was quantified by flameless atomic absorption spectrometry (FAAS) using AA-6700 Atomic Absorption Flame Emission Spectrophotometer (SHIMAZU)). Sequential treatment under conditions of wavelength 265.9nm, slit width 0.5, lamp current 14mA at 120 ° C for 30 seconds, 250 ° C for 10 seconds, 700 ° C for 20 seconds, 700 ° C for 5 seconds, 2600 ° C for 3 seconds Went. A 1 mg / mL platinum standard solution (NAKARAI) was diluted with purified water to prepare solutions of 12.5 ng / ml, 25 ng / ml, 50 ng / ml, 100 ng / ml, and 200 ng / ml, and prepared a calibration curve. The liposome solution was diluted 10,000 times with purified water to prepare a test sample.

  As a result of calculating the amount of cis-diamine dichloroplatinum (II) encapsulated in liposomes by measuring the amount of platinum by flameless atomic absorption spectrophotometry (FAAS), in the improved cholate method, cis-diammine dinitrate The cis-diamine dichloroplatinum (II) concentration in the liposome I solution encapsulated with platinum (II) and converted to cis-diamine dichloroplatinum (II) was 91.08 μg / mg lipid (323.3 μg / mL). (Table 4 left column). On the other hand, when cis-diamine dichloroplatinum (II) was encapsulated directly in the liposome, it was 0.306 μg / mg lipid (1.07 μg / mL) (left column in Table 4).

  In the freeze-drying method, the cis-diamine dichloroplatinum (II) concentration in the liposome II solution encapsulated in cis-diammine dinitratoplatinum (II) and converted into cis-diamine dichloroplatinum (II) was 117.5 μg / lipid. mg (893 μg / mL) (Table 4, right column). On the other hand, when cis-diamine dichloroplatinum (II) was directly encapsulated in the liposome, it was 17.7 μg / mg lipid (134.6 μg / mL) (right column in Table 4).

  The amount of inclusion per 1 mg of lipid when encapsulating cis-diammine dinitratoplatinum (II) and then converting to cis-diamine dichloroplatinum (II) is as follows. It was about 300 times the amount when directly encapsulated, and 6.6 times the amount for liposome II. As a result, it was confirmed that the difference in the amount of inclusion was particularly large in the case of liposome I produced by the improved cholate method in which a substance was encapsulated simultaneously with the formation of the liposome.

  Cis-diammine dinitratoplatinum (II) is about 10 times more water soluble than cis-diamine dichloroplatinum (II). Therefore, by encapsulating cis-diammine dinitratoplatinum (II) in liposomes and then converting it into cis-diamine dichloroplatinum (II), about 300 times the amount of cis-diamine dichloroplatinum (II) per 1 mg of lipid is converted into liposomes. It can be said that cis-diamine dichloroplatinum (II) was included at a much higher concentration than expected. Further, since the difference in liposome II was about 6 to 7 times the difference in the amount of encapsulation, this encapsulation technique is particularly effective with liposome I prepared by the improved cholate method. I can say that.

  In general, in the case of Liposome I produced by using the improved cholate method in which a substance is encapsulated simultaneously with the formation of the liposome, the amount of uncharged low molecular weight compound such as cis-diamine dichloroplatinum (II) is very large. Low. Therefore, in the method in which cis-diamine dichloroplatinum (II) is directly encapsulated in liposomes, liposome I can encapsulate only 0.306 μg / mg of lipid cis-diamine dichloroplatinum (II). However, when the encapsulation technique of the present invention was used, 91.1 μg / mg lipid cis-diamine dichloroplatinum (II) could be encapsulated. The reason why the difference in the amount of cis-diamine dichloroplatinum (II) encapsulated in the liposome I produced by the modified cholate method was larger than that in the liposome II produced by the freeze-dried empty liposome method was It is considered that the acid salt method can hardly contain an uncharged low molecular weight compound.

  In addition, liposome I and liposome II of this example are composed of a lipid component having a negative charge so that the charge on the surface of the liposome becomes negative in order to prevent binding with proteins in the blood, but those having a positive charge However, it is applicable, and those skilled in the art can change the design as appropriate. Since cis-diammine dinitratoplatinum (II) has a positive charge in an aqueous solution, it may be an advantageous molecular form for inclusion in a liposome using a lipid component having a negative charge. Most of the liposomes that target inflammation and cancer sites by the EPR effect (Y. Matsumura and H. Maeda, Cancer Res., 46, 6387-6392 (1986)) have a surface charge to improve blood retention. Since it is designed to be negative, the amount of the poorly water-soluble drug encapsulated in the liposome can be increased by using the encapsulation technology of the present invention in the same manner in many commonly used liposomes. it is conceivable that.

(Confirmation of conversion of cis-diammine dinitratoplatinum (II) to cis-diamine dichloroplatinum (II))
It was confirmed using the absorption spectrum method that the nitrate ion of cis-diammine dinitratoplatinum (II) was replaced by the chloride ion of 150 mM sodium chloride (Comparative Example 1 and FIGS. 4A and 4B).

  As shown in FIGS. 4A and 4B, the absorption spectrum of cis-diammine dinitratoplatinum (II) (FIG. 4A) changes over time immediately after the addition of chloride ions, and cis-diamine dichloroplatinum shown in FIG. 4B. Consistent with the absorption spectrum of (II). From this, it was confirmed that 99% or more of cis-diammine dinitratoplatinum (II) was converted to cis-diamine dichloroplatinum (II) in the presence of 150 mM sodium chloride.

Next, conversion of cis-diammine dinitratoplatinum (II) encapsulated in liposome I and liposome II to cis-diamine dichloroplatinum (II) in a 150 mM sodium chloride solution was confirmed by a ¹⁹⁵ Pt-NMR method. .

( ¹⁹⁵ Pt-NMR method)
The same method as in Example 3 was used. Liposome II prepared in this example was used as a liposome encapsulating cis-diammine dinitratoplatinum (II). Conversion of cis-diammine dinitratoplatinum (II) encapsulated in these liposomes into cis-diamine dichloroplatinum (II) was placed in a measuring tube having a diameter of 5 mm at 25 ° C. and INOVA-600 (Varian) was added. Analysis was performed by the ¹⁹⁵ Pt-NMR method used.

(Sample preparation)
Sodium hexachloroplatinate (IV) was dissolved in heavy water to 50 mM to prepare an external standard solution. Samples were prepared by dissolving cis-diamine dichloroplatinum (II) and cis-diammine dinitratoplatinum (II) in heavy water to a final concentration of 6.6 mM and 13.7 mM, respectively. Liposome samples with and without conversion of cis-diammine dinitratoplatinum (II) encapsulated in liposome II to cis-diamine dichloroplatinum (II) were lyophilized and then suspended in heavy water. Was also prepared so that the platinum concentration was 4.75 mM.

  As shown in FIGS. 8a and 8b, the chemical shift value when cis-diamine dichloroplatinum (II) is dissolved in heavy water is −2160 ppm, and cis-diammine dinitratoplatinum (II) is dissolved in heavy water. The chemical shift value when it was made to be -1620 ppm. This chemical shift value was consistent with literature values (B. Rosenberg, Biochimie., 60 859 (1978)). As shown in FIG. 8c, an external solution of liposome II encapsulating cis-diammine dinitratoplatinum (II) was used as a 150 mM sodium chloride-containing TAPS (pH 8.4) buffer, and the chemical shift after being allowed to stand at 25 ° C. for 96 hours. The value was −2160 ppm, which was consistent with the chemical shift value of cis-diamine dichloroplatinum (II) shown in FIG. From this, it was confirmed that cis-diammine dinitratoplatinum (II) encapsulated in liposome II was converted to cis-diamine dichloroplatinum (II). When cis-diammine dinitratoplatinum (II) is not changed to cis-diamine dichloroplatinum (II), the peak at -1620 ppm, which is the chemical shift value of cis-diammine dinitratoplatinum (II), is The chemical shift value of −2160 ppm of dichloroplatinum (II) was not observed (FIG. 8d).

  For liposome I, as shown in FIGS. 5a to 5d and Example 3, similar results were obtained, and cis-diammine dinitratoplatinum (II) encapsulated in the liposome was converted to cis-diaminedichloroplatinum (II). It was confirmed that

In the liposome ¹⁹⁵ (modified cholate method) and the liposome II (freeze drying method), the chemical shift of cis-diammine dinitratoplatinum (II) in ¹⁹⁵ Pt-NMR analysis was not recognized, It was confirmed at -2160 ppm which is the chemical shift position of cis-diamine dichloroplatinum (II) (FIGS. 5c and 8c). From this, it was confirmed that in any liposome, the structure of cis-diammine dinitratoplatinum (II) in the liposome was changed to cis-diamine dichloroplatinum (II).

  When cis-diammine dinitratoplatinum (II) encapsulated in liposomes was not converted to cis-diamine dichloroplatinum (II), as shown in FIGS. 5d and 8d, cis-diammine dinitratoplatinum (II ) And a chemical shift of cis-diamine dichloroplatinum (II) were not observed. This is because the encapsulated cis-diammine dinitratoplatinum (II) interacts with the membrane components of the liposome by the coordination of water molecules as shown in FIG. Therefore, it is considered that the chemical shift was not detected.

(Example 10)
The purpose of this example is to confirm the duration of the therapeutic effect of cisplatin-encapsulated liposomes prepared by the same method as in Example 1, Example 3 and Example 5, and conventional cisplatin-encapsulated liposomes.

  Cisplatin-encapsulated liposomes are prepared in the same manner as in Example 3 and Example 5. As conventional techniques, cisplatin-encapsulating liposomes are prepared according to the descriptions in Patent Documents 1 to 3 and Non-Patent Document 1.

(Effects on cells)
Various tumor cell lines are purchased and cultured. Each cell culture is administered at the same dose and the cell culture is observed over time. The period of time during which tumor cell growth is suppressed or tumor cell decrease is measured to determine how long the therapeutic effect of each cisplatin-encapsulated liposome lasts.

(In vivo effect)
As a subject, various tumor-bearing mice are prepared by transplanting various tumor cells subcutaneously into the thigh of the mouse. Cisplatin-encapsulated liposomes are administered to cancer-bearing mice at the same dose and the same number of times, and tumors are observed over time. The period during which tumor growth is suppressed or tumor volume is decreased is measured to examine how long the therapeutic effect of each cisplatin-encapsulating liposome lasts.

(Comparative example)
Cisplatin alone is used as a control. The same experiment as in this example is performed to examine the effect of cisplatin alone.

(Example 11)
The purpose of this example is to confirm the time when a therapeutic effect appears for the cisplatin-encapsulated liposomes prepared by the same method as in Example 1, Example 3 and Example 5, and the conventional cisplatin-encapsulated liposomes.

  Cisplatin-encapsulated liposomes are prepared in the same manner as in Example 3 and Example 5. As conventional techniques, cisplatin-encapsulating liposomes are prepared according to the descriptions in Patent Documents 1 to 3 and Non-Patent Document 1.

(Effects on cells)
Various tumor cell lines are purchased and cultured. Each cell culture is administered at the same dose and the cell culture is observed over time. The time when suppression of tumor cell growth begins or the time when the decrease in the number of tumor cells begins is compared for each cisplatin-encapsulated liposome.

(In vivo effect)
As a subject, various tumor-bearing mice are prepared by transplanting various tumor cells subcutaneously into the thigh of the mouse. Cisplatin-encapsulated liposomes are administered to cancer-bearing mice at the same dose and the same number of times, and tumors are observed over time. The time when tumor growth suppression starts or the time when tumor volume starts decreasing is compared for each cisplatin-encapsulated liposome.

(Comparative example)
Cisplatin alone is used as a control. The same experiment as in this example is performed to examine the effect of cisplatin alone.

(Example 12)
The purpose of this example is to confirm the therapeutic efficiency of cisplatin-encapsulated liposomes prepared by the same method as in Example 1, Example 3 and Example 5, and the prior art cisplatin-encapsulated liposomes.

  Cisplatin-encapsulated liposomes are prepared in the same manner as in Example 3 and Example 5. As conventional techniques, cisplatin-encapsulating liposomes are prepared according to the descriptions in Patent Documents 1 to 3 and Non-Patent Document 1.

(Effects on cells)
Various tumor cell lines are purchased and cultured. Each cell culture is administered at the same dose and the cell culture is observed over time. Measure the number of tumor cells and examine treatment efficiency.

(In vivo effect)
As a subject, various tumor-bearing mice are prepared by transplanting various tumor cells subcutaneously into the thigh of the mouse. Cisplatin-encapsulated liposomes are administered to cancer-bearing mice at the same dose and the same number of times, and tumors are observed over time. The tumor volume is measured, and the therapeutic effect when the same dose is administered is compared for each cisplatin-encapsulated liposome.

(Comparative example)
Cisplatin alone is used as a control. The same experiment as in this example is performed to examine the effect of cisplatin alone.

(Example 13)
The purpose of this example is to confirm the inhibitory effect on recurrence of cisplatin-encapsulated liposomes prepared by the same method as in Example 1, Example 3 and Example 5, and the prior art cisplatin-encapsulated liposomes.

  Cisplatin-encapsulated liposomes are prepared in the same manner as in Example 3 and Example 5. As conventional techniques, cisplatin-encapsulating liposomes are prepared according to the descriptions in Patent Documents 1 to 3 and Non-Patent Document 1.

(In vivo effect)
As a subject, various tumor-bearing mice are prepared by transplanting various tumor cells subcutaneously into the thigh of the mouse. Cisplatin-encapsulated liposomes are administered to tumor-bearing mice, and tumors are observed over time. Liposome administration is discontinued when the tumor disappears. Thereafter, it is observed whether the tumor will recur.

(Comparative example)
Cisplatin alone is used as a control. The same experiment as in this example is performed to examine the effect of cisplatin alone.

(Example 14)
This example examines the therapeutic effects on tumors and cancers that could not be treated with the prior art.

  In this example, cisplatin-encapsulated liposomes prepared by the same method as in Example 1, Example 3 and Example 5, and conventional cisplatin-encapsulated liposomes are used.

  Cisplatin-encapsulated liposomes are prepared in the same manner as in Example 3 and Example 5. As conventional techniques, cisplatin-encapsulating liposomes are prepared according to the descriptions in Patent Documents 1 to 3 and Non-Patent Document 1.

(Effects on cells)
Purchase and culture tumor and cancer cell lines that could not be treated with conventional technology. Each cell culture is administered at the same dose and the cell culture is observed over time. The number of tumor cells is measured and the therapeutic effect is examined.

(In vivo effect)
As a subject, various tumor-bearing mice are prepared by transplanting tumor / cancer cells, which could not be treated by conventional techniques, into the subcutaneous thighs of mice. Cisplatin-encapsulated liposomes are administered to cancer-bearing mice and observed over time. Measure the volume of the tumor / cancer and compare the therapeutic effect of each cisplatin-encapsulated liposome.

(Comparative example)
Cisplatin alone is used as a control. The same experiment as in this example is performed to examine the effect of cisplatin alone.

(Example 15)
CDDP: Cisplatin CDDP-3: Cisplatin nitrate CDDP-SLX-Lip: Sialyl Lewis X modified liposome encapsulating CDDP CDDP-Lip: Unmodified liposome encapsulating CDDP In this example, highly toxic CDDP is encapsulated in a liposome. We examined whether the toxicity could be reduced by administration. In this example, CDDP, CDDP-3, CDDP-SLX-Lip and CDDP-Lip prepared by the following method were used. CDDP (18 mg or 25 mg CDDP equivalent / Kg body weight) and CDDP-SLX-Lip (25 mg or 50 mg CDDP equivalent / Kg body weight as the CDDP amount) were each administered to the normal mouse via tail vein, and survival was observed for 5 days after administration. . This will be described in detail below.

(Preparation of CDDP, CDDP-3, CDDP-SLX-Lip and CDDP-Lip)
CDDP: P4394 purchased from SIGMA was used for CDDP. Dissolved in HEPES (pH 7.2) to 2 mg / ml.

(Synthesis of CDDP-3)
The same method as in Example 1 was used for the synthesis of CDDP-3. Cis-diammine dinitratoplatinum (II) was synthesized by the method of Dahara (SG Dhara, Indian J. Chem., 7, 335 (1970)).

After dissolving 4.15 g (10 mmol) of potassium chloroplatinate in distilled water, 6.64 g (40 mmol) of potassium iodide was added while nitrogen was blown in a light-shielded and ice-cooled state and stirred for 5 minutes. To this reaction solution, 1.35 mL (22 mmol) of 28% aqueous ammonia solution was added dropwise and stirred for 3 hours. The produced yellow crystals were collected by filtration, washed successively with distilled water and ethanol, and then dried under reduced pressure at 40 ° C. for 10 hours. Here, 4.49 g of ammine-platinum intermediate cis- [Pt (NH ₃ ) ₂ I ₂ ] (cis-diaminediodeplatinum (II) (CDDP-2)) was obtained. After suspending 2.41 g (5 mmol) of cis- [Pt (NH ₃ ) ₂ I ₂ ] in distilled water, 1.68 g (9.9 mmol) of silver nitrate was added, and the mixture was stirred for 24 hours under light shielding. The produced silver iodide was filtered off, and the filtrate was concentrated by an evaporator to obtain white crystals. The white crystals were washed successively with cold distilled water and ethanol and then dried under reduced pressure at 40 ° C. for 10 hours to obtain 1.01 g of cis-diammine dinitratoplatinum (II).

(Preparation of CDDP-SLX-Lip and CDDP-Lip)
Liposome I modified with a sugar chain was prepared in the same manner as in Example 3 using a part of cis-diamine dichloroplatinum (II) -encapsulating liposome I prepared by the improved cholic acid method of this example.

(Hydrophilic treatment on the liposomal lipid membrane surface)
Ultrafiltration using a cis-diamine dinitratoplatinum (II) -encapsulated liposome I using XM300 membrane (Amicon Co., USA) and carbonate buffer (pH 8.5) (fractionated molecular weight: 300,000) ) To bring the pH of the solution to 8.5. Next, 10 mg of a crosslinking reagent bis (sulfosuccinimidyl) suberate (BS ³ ; Pierce Co., USA) was added, and the mixture was stirred at room temperature for 2 hours. Thereafter, the mixture was further stirred overnight under refrigeration to complete the chemical binding reaction between the lipid dipalmitoylphosphatidylethanolamine and BS ³ on the liposome membrane. Then, this liposome solution was subjected to ultrafiltration (fractionated molecular weight: 300,000) with an XM300 membrane and a carbonate buffer (pH 8.5). Next, 40 mg of tris (hydroxymethyl) aminomethane dissolved in 1 ml of carbonate buffer (pH 8.5) was added to 10 ml of liposome solution. The solution was then stirred at room temperature for 2 hours, then stirred overnight under refrigeration, ultrafiltered with a molecular weight cut off of 300,000 to remove free tris (hydroxymethyl) aminomethane, and the carbonate buffer Was replaced with N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4), and the chemical bonding reaction between BS ³ bound to the lipid on the liposome membrane and tris (hydroxymethyl) aminomethane was performed. Completed. As a result, the hydroxyl group of tris (hydroxymethyl) aminomethane was coordinated on the lipid dipalmitoylphosphatidylethanolamine of the liposome membrane to make it hydrated and hydrophilic.

(Binding of human serum albumin (HSA) on the liposome membrane surface)
Metaperiodate in which ganglioside present on the membrane surface of 10 ml of liposome I obtained in this Example was dissolved in 1 ml of N-tris (hydroxymethyl) -3-aminopropanesulfonic acid buffer (pH 8.4). Sodium acid 43 mg was added and periodate oxidized by stirring overnight under refrigeration. Free filtration of sodium periodate was removed by ultrafiltration (fractional molecular weight: 300,000) with XM300 membrane and PBS buffer (pH 8.0), and N-tris (hydroxymethyl) -3-amino was removed. The propanesulfonic acid buffer was exchanged with PBS buffer (pH 8.0) to obtain 10 ml of oxidized liposomes. To this liposome solution, 20 mg of human serum albumin (HSA) / PBS buffer (pH 8.0) was added and reacted at room temperature for 2 hours, and then 100 μl of 2M NaBH ₃ CN / PBS buffer (pH 8.0). The mixture was stirred at room temperature for 2 hours and further overnight under refrigeration, and HSA was bound by a coupling reaction between ganglioside on the liposome and HSA. Then, ultrafiltration (fraction molecular weight: 300,000) was performed to remove free sodium cyanoborate and human serum albumin, and the buffer of this solution was replaced with carbonate buffer (pH 8.5). 10 ml of bound liposome solution was obtained.

(Preparation of sugar chain)
Sialyl Lewis X was used as the sugar chain.

  The mass of each sugar chain was measured and pretreated for use in the following.

(Binding of sugar chain on liposome membrane surface-bound human serum albumin (HSA))
2 mg of sugar chain (SLX) is dissolved in purified water, added to 0.5 ml aqueous solution containing 0.25 g NH ₄ HCO ₃ , stirred at 37 ° C. for 3 days, filtered through a 0.45 μm filter, and sugar The amination reaction at the reducing end of the chain was completed to obtain 4 mg / ml of a glycosylamine compound of each sugar chain (aminated sugar chain solution). Next, 10 mg of a crosslinking reagent 3,3′-dithiobis (sulfosuccinimidylpropionate) (DTSSP; Pierce Co., USA) was added to a part (10 ml) of the liposome I solution obtained in this example. Stir at room temperature for 2 hours, then overnight under refrigeration, ultrafiltered (fractionated molecular weight: 300,000) with XM300 membrane and carbonate buffer (pH 8.5) to remove free DTSSP, 10 ml of liposomes in which DTSSP was bound to HSA on the liposomes were obtained. Next, 37.5 μl of the above-mentioned glycosylamine compound (aminated sugar chain solution) is added to this liposome solution, and the mixture is reacted at room temperature for 2 hours. Tris (hydroxymethyl) aminomethane / carbonate buffer (pH 8.5) Was added. Thereafter, the mixture was stirred overnight under refrigeration, and the glycosylated amine compound was bound to DTSSP on human serum albumin bound to the liposome membrane surface. The free sugar chain and tris (hydroxymethyl) aminomethane were removed by ultrafiltration (fraction molecular weight: 300,000) with an XM300 membrane and HEPES buffer (pH 7.2). As a result, 10 ml each of liposomes (CDDP-SLX-Lip) in which sugar chains, human serum albumin, and liposomes were bound was obtained.

Moreover, CDDP-Lip was produced using the method similar to a present Example except not adding sugar_chain | carbohydrate SLX. For the SLX-modified liposomes encapsulating CDDP and CDDP-3 produced in this example, the same method as in Examples 1 and 2 was used, and the lipid amount (mg / mL), average particle diameter (nm), The amount of CDDP per lipid (μg / mg) was measured. For the quantification of lipids, the total cholesterol amount was measured using the Determiner TC555 kit (catalog number UCC / EAN128) (KYOWA Co. LTD) in the presence of 0.5% Triton X-100, and the total lipid was determined from the molar ratio of each lipid. The amount was calculated. The average particle diameter was measured at 25 ° C. using a Zetasizer Nano (Nan-ZS: MALVERN Co. LTD) after diluting the liposome solution 50 times with purified water. The amount of CDDP was quantified by flameless atomic absorption spectrophotometry (FAAS) using AA-6700 (Shimadzu) by diluting the liposome solution 10,000 times with purified water to obtain a test sample. Measurement conditions: wavelength 265.9 nm, slit width 0.5, lamp current 14 mA, 120 ° C. 30 seconds, 250 ° C. 10 seconds, 700 ° C. 20 seconds, 700 ° C. 5 seconds, 2600 ° C. 3 seconds, were sequentially processed. CDDP amount is
Calculated by the formula: A × (300/195). A is the amount of platinum, 300 is the molecular weight of CDDP, and 195 is the molecular weight of platinum. The results are shown in the following table.

I II
Substance used for inclusion CDDP CDDP3
Lipid content (mg / mL) 3.5 3.5
Average particle diameter (nm) 158 159
CDDP / lipid (μg / mg) 0.2 60.3
I: CDDP liposome produced by directly encapsulating CDDP II: CDDP liposome produced by encapsulating CDDP3 and then substituting a buffer solution containing NaCl

(Acute toxicity test)
For the acute toxicity test, CDDP-SLX-Lip and CDDP-Lip concentrated 20 times by ultrafiltration (XM300; Amicon) were used. A HEPES buffer (pH 7.2) was used as an external solution for ultrafiltration. The CDDP concentration of CDDP-SLX-Lip was measured by flameless atomic absorption spectrophotometry (FAAS). The details of the quantification method are as described above. The CDDP concentration of CDDP-SLX-Lip at this time was 4 mg / ml (lipid amount 70 mg). A CDDP solution was prepared by dissolving CDDP in a HEPES buffer (pH 7.2) to give a 2 mg / ml solution.

  A normal mouse Balb / c (female, 8-week-old Japanese SLC) was added to a CDDP solution, CDDP-SLX-Lip, 200 μl of physiological saline, 200 μl of 20 mM HEPES buffer (pH 7.2) or SLX liposomes not encapsulating CDDP ( CDDP non-encapsulated SLX liposomes) were each administered once via the tail vein. Four individuals were used for each experiment. Each dose is shown in the table below. The survival rate for 14 days after administration was examined. In addition, the body weight for 5 days after administration was measured over time. The administered lipid amount of the CDDP non-encapsulated SLX liposome was adjusted to the lipid amount when CDDP-SLX-Lip was administered at 50 mg CDDP equivalent / Kg mouse body weight.

(result)
In the 25 mg administration group, the survival rate after 5 days of administration was 0%, but in the 20 mg and 50 mg administration groups of CDDP-SLX-Lip, the survival rate was 75%. By encapsulating in liposomes, considerable toxicity can be reduced. It became clear (FIG. 15). Further, the body weight of the mice continued to decrease over time in the CDDP 18 mg and 25 mg administration groups, and the CDDP 18 mg administration group showed a 20% decrease in body weight over 5 days and the CDDP 25 mg administration group over 25 days. On the other hand, in the groups administered with 18 mg, 25 mg, and 50 mg of CDDP-SLX-Lip, about 15% body weight loss was observed in all 3 days after administration, but the body weight increased thereafter, and the weight loss after 5 days after administration was It was less than 10% (FIG. 16). From the above results, it was found that CDDP-SLX-Lip has an excellent toxicity mitigating effect.

(Pathological observation)
CDDP or CDDP-SLX-Lip was administered to normal mice Balb / c (female, 8-week-old Japanese SLC) (4 mice in each group) in an amount corresponding to 25 mg CDDP per kg of mouse body weight, and 4 days later, kidney, spleen The liver was removed and fixed by immersion in a 10% neutral buffered formalin solution. After embedding in paraffin, it was sliced into 2 μm, and hematoxylin and eosin (HE) staining and Tunel immunohistochemical staining were performed. In situ apoptosis detection kit (TaKaRa MK500) was used for detection of apoptosis by Tunnel immunohistochemical staining.

(result)
CDDP (25 mg / Kg body weight) and CDDP-SLX-Lip (corresponding to CDDP 25 mg / Kg body weight) were each administered to the normal mouse via the tail vein, and the kidney, spleen, and liver were excised on the third day, and the tissue sections were isolated from H. pylori. E staining and Tunel immunostaining. As a result, in the kidney, no abnormal findings were observed in the CDDP-SLX-Lip administration group, and no Tunel positive cells were observed (FIGS. 17a and b). On the other hand, in the CDDP administration group, proximal tubule dilatation was observed, and tubular epithelial cells that caused Tunel-positive apoptosis were scattered (FIGS. 17c and d). In the spleen, no abnormal findings were observed in the CDDP-SLX-Lip administration group, and a very small number of Tunel positive cells were found (FIGS. 17e and f). However, in the CDDP-administered group, follicular atrophy accompanied by lymphocyte necrosis and a decrease in the number of red spleen cells were observed. In the follicle, necrotic lymphocytes were phagocytosed, and a Starry sky image was observed. Furthermore, many Tunel positive cells were observed in the follicle and red spleen. (FIGS. 17g and h). In the liver, a slight increase in benign microgranulomas was observed in the CDDP-SLX-Lip administration group, but no Tunnel positive cells were observed (FIGS. 17i and j). No abnormal findings were observed in the CDDP administration group, and no Tunel positive cells were observed (FIGS. 17k and 1).

(In vivo antitumor activity)
In this example, A549 cells (A549: human lung cancer cells (ATCC No. CCL-185)) having the lowest CDDP sensitivity were transplanted into mice in vitro, and the antitumor effects of CDDP-SLX-Lip and CDDP-Lip Specifically, it was performed as follows.

1 × 10 ⁷ A549 cells were subcutaneously administered to the right back of nude mice Balb / c Slc-nu / nu (female, 6 weeks old, 4 mice per group; Japan SLC). At 5 days, 12 days and 19 days after transplantation, 200 μl of physiological saline, 25 mg CDDP equivalent / kg body weight of CDDP-SLX-Lip or CDDP-Lip was administered from the tail vein. After 5 days, 12 days, 19 days, and 26 days after transplantation, the major axis (Amm) and minor axis (Bmm) of the tumor were measured using a digital caliper (Mitutoyo CD-20C), and the formula (A × B ² ) × Tumor volume (mm ³ ) was calculated by 0.5.

As shown in FIG. 18, at 12 days after cell transplantation, the tumor volumes (mm ³ ) of the CDDP-SLX-Lip administration group and the CDDP-Lip administration group were both small compared with the physiological saline administration group. There was no difference between the groups. However, 19 days after cell transplantation, a clear difference in the antitumor effect was observed between the CDDP-SLX-Lip administration group and the CDDP-Lip administration group. Specifically, the tumor volume of the CDDP-SLX-Lip administration group was the smallest, and a stronger antitumor effect was confirmed than CDDP-Lip not modified with a sugar chain (FIG. 18).

  Furthermore, on the 26th day of cell transplantation, the difference in tumor volume further increased between the CDDP-Lip administration group and the CDDP-SLX-Lip administration group. Further, in the CDDP-SLX-Lip administration group, it was found that a complete healing effect was observed in 1 out of 4 mice and the tumor disappeared.

(Cell culture)
Ehrlich ascites cancer cells (EAT), human colon cancer cells (HT29) and human squamous cell carcinoma cells (A431) were purchased from ATCC. Human lung cancer cells (A549), mouse lung cancer cells (LLC) and human breast cancer cells (SKBr3) were purchased from RIKEN. DMEM (SIGMA) supplemented with 10% FBS (Biowest) and 1% penicillin / streptomycin solution (SIGMA) was used for the culture of all cells. Culturing was performed at 37 ° C. with 5% CO ₂ .

(CDDP-SLX-Lip Cancer Cell Growth Inhibitory Activity)
Each cell of A549, HT29, A431, LLC and SKBr3 was seeded in a 96-well microplate at 1 × 10 ³ cells / 50 μl / well and cultured at 37 ° C. under 5% CO ₂ for 24 hours. CDDP-SLX-Lip was added to each well so that the final concentration of CDDP was 10 μM, 50 μM, 100 μM, and 200 μM. 72 hours after the addition, 10 μl of WST-8 (cell count measuring reagent Kishida Chemical) was added to each well and cultured at 37 ° C. for 1 hour under 5% CO ₂ . Absorbance at 450 nm was measured using a microplate reader (BioRAD), and the number of cells was estimated by using the correlation between the absorbance and the number of living cells. Specifically, the absorbance (%) at the time of addition relative to the absorbance at the time when liposome was not added (control) was calculated and used as the cell viability.

As shown in FIG. 19, CDDP-SLX-Lip was added to 5 types of cancer cells, and the IC ₅₀ after 72 hours was 200 μM for SKBr cells, 50 μM for TH29 cells, 175 μM for LLC cells, and 431 μM for A431 cells. 100 μM. For A549 cells, the cell viability was 65% at 200 μM. Although a difference was observed between cells, CDDP sensitivity was shown by adding CDDP-SLX-Lip in any cell.

(Accumulation of CDDP-SLX-Lip to cancer site)
5 × 10 ⁵ EAT cells were transplanted subcutaneously into the right thigh of a mouse (Balb / c, female, 6 weeks old: Japan SLC), and CDDP− corresponding to CDDP of 2.3 mg / Kg mouse 10 days later. SLX-Lip and CDDP-Lip were administered via the tail vein. 1 ml of concentrated nitric acid was added per 0.1 g of cancer removed 48 hours later, and the mixture was allowed to stand in a 60 ° C. water bath for 1 hour. After returning to room temperature, the solution was diluted 4-fold with distilled water, and the amount of platinum was quantified by FAAS to obtain the degree of liposome accumulation.

  SLX-modified liposomes or SLX-unmodified liposomes were administered to tumor-bearing mice transplanted with EAT cells, and the accumulation of the liposomes 48 hours later was examined by quantifying the amount of CDDP platinum encapsulated in the liposomes. As a result, as shown in FIG. 20, the accumulation of CDDP-SLX-Lip having SLX bound to the surface thereof at the cancer site was 5.7 times that of CDDP-Lip having no sugar chain bound thereto. This reveals that SLX affects the targeting properties of liposomes.

(Discussion)
In the examination of acute toxicity when administered at 25 mg / kg body weight of CDDP, the survival rate after 5 days from the administration was 0%. However, the survival rate of the CDDP-SLX-Lip25 and 50 mg administration groups was 75%, and the body weight was also recovered after the third day of administration in the case of CDDP-SLX-Lip (FIG. 19). Severe nephrotoxicity is known as one of the side effects of CDDP. At 25 mg CDDP, this nephrotoxicity was confirmed in the form of abnormal proximal tubules. On the other hand, in the case of CDDP-SLX-Lip, no toxicity to the kidney was observed. Presumably, CDDP-SLX-Lip is excreted from the urine in a form that does not show nephrotoxicity due to significant changes in pharmacokinetics. The effect of CDDP-SLX-Lip on the spleen was significantly milder than CDDP. Small granulomas observed in the liver did not show apoptosis in the TUNEL assay. Since an increase in small granulation species was also observed in CDDP non-encapsulated liposomes, this is considered to be a benign granulation species resulting from the phagocytosis of liposomes by macrophages in the liver (FIG. 20). Therefore, CDDP-SLX-Lip has extremely low toxicity compared to CDDP, and it was suggested that administration at 25 mg / Kg body weight that leads to lethality in CDDP is sufficiently possible in mice. Based on this finding, we will examine the dose of antineoplastic drugs to humans. The dosage of a human antineoplastic agent can be determined by body surface area. Body surface area can be calculated by the Dubois equation.

Body surface area ^{(BSA: m 2) = 0.007184} × Weight ^{(Kg) 0.425} × Height ^{(cm) 0.725}
With this, cisplatin is administered intravenously to humans in the range of about 30 to about 200 mg / m ² / day. For example, in a human of 170 cm and 60 kg, the body surface area is 1.9 m ² . Thus:
About 57 to about ^{380mg / 1.9m 2 / 60Kg / day} → about 0.95 to about 6.33mg / Kg / day
In this case, the mouse dose of CDDP is about 18 to about 50 mg / Kg body weight, and when converted to a dose of 170 cm and 60 Kg for humans, it is about 1080 to about 3000 mg / 60 Kg / 1.9 m ^2. . When this is converted into intravenous administration, it is about 568 to about 1578 mg / m ² .

Regarding the in vitro cell growth inhibitory activity against various cancer cells, the IC ₅₀ value varied somewhat depending on the cell line, but CDDP-SLX-Lip exhibited growth inhibitory activity against all cancer cells 72 hours after addition. (FIG. 19). Further, _{IC 50} values of CDDP-SLX-Lip is was 10-20 times higher compared to CDDP alone. In the case of CDDP-SLX-Lip, after being once taken into the cell as a liposome, CDDP is released into the cell by breaking the liposome, so that a sufficient amount of CDDP is sufficient to reduce the cell viability. It may take a certain amount of time to be released from the liposome into the cytoplasm. Since the data shown in FIG. 19 is the survival rate after 72 hours from the addition of CCDP-SLX-Lip, it is considered that the sensitivity from CDDP to the evaluation is increased by extending the time from the addition of liposome to the evaluation. Furthermore, CDDP-SLX-Lip may release CDDP into cells for a long time.

  The present inventors previously confirmed and reported that the accumulation of SLX-bound liposomes at the cancer site was highest 48 hours after administration by fluorescence imaging experiments using fluorescent substance Cy5.5-encapsulated SLX liposomes [ M.M. Hirai et al., BBRC. , 353, 553-558 (2007)]. Based on this finding, in this example, the amount of CDDP-SLX-Lip accumulated at the cancer site was examined 48 hours after administration. As a result, it was revealed from the measurement of the amount of platinum contained in the cancer tissue that CDDP-SLX-Lip accumulates about 6 times more in the cancer site than CDDP-Lip (FIG. 20). Here, the data of CDDP-Lip show the EPR effect of liposome [Y. Matsumura et al., Cancer Res,. 46, 6387-6392 (1986)].

  In vitro cell growth inhibition experiments revealed that A549 cells were less sensitive to CDDP-SLX-Lip than other cells (FIG. 19). Therefore, it was examined whether CDDP-SLX-Lip exhibits an antitumor effect in vivo on this low-sensitivity A549 cell. Specifically, the antitumor activity was examined when CDDP-SLX-Lip and CDDP-Lip were each administered at a CDDP equivalent of 25 mg / Kg body weight. This concentration of CDDP-SLX-Lip has been shown to have no side effects on mice, as shown in FIGS. After 5 days, 12 days and 19 days (every 7 days) after transplantation of A549 cells, liposomes were administered three times via the tail vein. On the 12th day after transplantation, the tumor volumes of the CDDP-SLX-Lip and CDDP-Lip administration groups were small compared to the physiological saline administration group, and a clear antitumor effect was observed. However, there was no difference between the two liposomes, and the effectiveness of SLX modified with liposomes was not recognized. However, at 19 and 26 days after transplantation, a clear difference was observed between CDDP-SLX-Lip and CDDP-Lip, confirming the effectiveness of SLX (FIG. 18). This is thought to be due to the sensitivity of A549 cells to CDDP. That is, although CDDP is accumulated at a high concentration in the cancer site using SLX-linked liposomes, A549 cells have low CDDP sensitivity (FIG. 19), and it did not take 19 days to exhibit antitumor activity. It is thought. By using cells that are highly sensitive to CDDP, it is highly possible that CDDP-SLX-Lip causes high concentration of CDDP / antitumor effect at an early stage. Moreover, in this example, evaluation was performed for 26 days after cancer transplantation, but since a CDDP-release effect from CDDP-SLX-Lip accumulated in cancer tissue can be expected, longer-term evaluation is performed. Thus, it is considered that the difference in the antitumor effect between CDDP-SLX-Lip and CDDP-Lip is increased.

(Example 16: Antitumor activity test)
In this example, CDDP, CDDP-3, CDDP-encapsulated sialyl Lewis X-modified liposomes and CDDP-encapsulated sugar chain-unmodified liposomes prepared by the same method as in Example 1 are used.

(Anti-tumor activity test I using SKBr3 cell line (human breast cancer cell))
1 × 10 ⁷ SKBr3 cell line (human breast cancer cell: SK-Br-3 Code) subcutaneously in the right thigh of a 5-week-old mouse (Balb / c Slc-nu / nu female) (5 mice per group) No. 04-030). The cells are cultured in DMEM medium (added with 2 mM glutamine and 10% fetal calf serum and no antibiotics) at 37 ° C. and 5% CO ₂ . 5 days, 12 days, and 19 days after transplantation, CDDP-encapsulated sialyl Lewis X-modified liposomes, CDDP-encapsulated sugar chain-unmodified liposomes, and CDDP are each administered to the tail vein of mice at a CDDP amount of 25 mg (200 μl / mouse) per kg of mouse body weight. . As a control, HEPES buffer (pH 7.2) is similarly administered at 200 μl / mouse.

  The size of the tumor can be measured over time by measuring the major axis (mm) and minor axis (mm) of the tumor using a digital caliper (Mitutoyo CD-S15C) (for example, 5 days, 12 days, 19 days and 26 days later).

Tumor volume (mm ³ ) is calculated by the following formula: minor axis (mm) × minor axis (mm) × major axis (mm) × 1/2. In addition, the measurement is performed on the tumor cell administration day (day 0) and the test substance administration day (for example, after 5 days, 12 days, 19 days, and 26 days). The weight can be measured using an electronic balance (A & D Corporation, HY-3000).

(Anti-tumor activity test II using SKBr3 cell line (human breast cancer cell))
1 × 10 ⁷ SKBr3 cell line (human breast cancer cell: SK-Br-3 Code. No. 04-) was subcutaneously injected into the right thigh of a 5-week-old mouse (Balb / c female) (5 mice in each group). 030). Cells are cultured in DMEM medium (2 mM glutamine and 10% fetal calf serum added, no antibiotics added) at 37 ° C. and 5% CO ₂ . After 5 days, 12 days, and 19 days after transplantation, in the liposome administration group, CDDP-encapsulated sialyl Lewis X-modified liposomes or CDDP-encapsulated sugar chain unmodified liposomes were administered at a CDDP amount of 1 mg or 4 mg (200 μl / mouse) per kg of mouse body weight. Each is administered from the tail vein of mice. In addition, in the cisplatin administration group, 1 mg or 4 mg (200 μl / mouse) of CDDP is administered from the tail vein of the mouse per kg body weight of the mouse. As controls, HEPES buffer (pH 7.2) and empty liposomes not containing CDDP are similarly administered at 200 μl / mouse.

The size of the tumor can be measured over time by measuring the major axis (mm) and minor axis (mm) of the tumor using a digital caliper (Mitutoyo CD-S15C) (for example, 5 days, 12 days, 19 days and 26 days later). The tumor volume (mm ³ ) can be calculated by the following formula: minor axis (mm) × minor axis (mm) × major axis (mm) × 1/2. Further, it is performed on the tumor cell administration day (day 0) and the test substance administration day (for example, 5 days, 12 days, 19 days and 26 days later). The weight can be measured using an electronic balance (A & D Co., Ltd. HY-3000).

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, it is understood that the scope of this invention should be construed only by the claims. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  INDUSTRIAL APPLICABILITY The present invention has the utility that liposomes can be efficiently encapsulated with poorly water-soluble ammine platinum complexes that cannot be encapsulated in liposomes or have a low encapsulation efficiency. Therefore, the usefulness of utilizing the poorly water-soluble ammine platinum complex which could not be administered orally as a liposome preparation is provided.

Claims (16)

A composition for treating cancer or tumor comprising a liposome encapsulating cis-diamine dichloroplatinum (II), and the liposome has a targeting substance on the surface of the liposome. A composition comprising. The composition according to claim 1, wherein the targeting substance is selected from the group consisting of an antibody and a sugar chain. The composition according to claim 1, wherein the targeting substance is sialyl Lewis X or an anti-E-selectin antibody. The composition according to claim 1, wherein the cancer or tumor is selected from the group consisting of colon cancer, lung cancer, squamous cell carcinoma, breast cancer and ovarian cancer. The composition according to claim 1, wherein the cis-diamine dichloroplatinum (II) is contained in an amount of more than 43 μg per mg lipid. The composition of claim 1, wherein the cis-diamine dichloroplatinum (II) is formulated to be administered to be at least 500 mg / m ² . Use of a liposome encapsulating cis-diamine dichloroplatinum (II) for the manufacture of a medicament for treating cancer or tumor, wherein the liposome comprises a targeting substance on the surface of the liposome, use. The use according to claim 7, wherein the targeting substance is selected from the group consisting of an antibody and a sugar chain. 8. Use according to claim 7, wherein the cis-diamine dichloroplatinum (II) is present in an amount of more than 43 [mu] g / mg lipid. A liposome encapsulating cis-diamine dichloroplatinum (II) for treating cancer or tumor, wherein the liposome contains a targeting substance on the surface of the liposome. The liposome according to claim 10, wherein the targeting substance is selected from the group consisting of an antibody and a sugar chain. The liposome according to claim 10, wherein the cis-diamine dichloroplatinum (II) is contained in an amount of more than 43 µg per 1 mg of lipid. A method for treating cancer or tumor comprising administering a liposome encapsulating cis-diamine dichloroplatinum (II) to a subject in need of the treatment, the liposome comprising: A method comprising a targeting substance on the surface of the liposome. The method according to claim 13, wherein the targeting substance is selected from the group consisting of an antibody and a sugar chain. The method according to claim 13, wherein the cis-diamine dichloroplatinum (II) is contained in an amount of more than 43 μg per 1 mg of lipid. The cis- diamminedichloroplatinum (II) are administered such that at least 500 mg / ^{m 2,} The method of claim 13.