WO2007088952A1

WO2007088952A1 - Liposome preparation comprising substance having anti-tumor activity

Info

Publication number: WO2007088952A1
Application number: PCT/JP2007/051741
Authority: WO
Inventors: Naoto Oku
Original assignee: Taiho Pharmaceutical Co., Ltd.
Priority date: 2006-01-31
Filing date: 2007-01-26
Publication date: 2007-08-09
Also published as: JP4881327B2; JPWO2007088952A1

Abstract

Disclosed is a liposome preparation which includes a substance having an anti-tumor activity, wherein at least two peptides each having an affinity to a tumor tissue are bound to the liposome surface.

Description

Description Liposome formulation of antitumor active substance Technical Field

The present invention relates to a liposome preparation including a medically useful antitumor active substance. Background art

To date, many drugs have been developed for cancer treatment and used in medicine. However, until now, no antitumor active substance has been developed that exhibits a selective cell killing effect only on tumor tissues. When an antitumor active substance is administered as it is into a blood vessel, it may disappear quickly from the blood or be distributed to organs other than the target, and it does not always effectively accumulate in cancer tissue. For this reason, many antitumor active substances cannot fully exert their antitumor effects (main effects) on force-sensitive tissues, and often have undesirable effects on normal tissues (side effects), resulting in serious toxicity. Is causing. Enhancement of the main effects of antitumor active substances and reduction of side effects are important issues in current cancer chemotherapy, and drug delivery systems that efficiently accumulate drugs in cancer tissues and cells (DDS, There is a strong demand for the development of Drug Delivery System.

Liposomes are closed vesicles mainly composed of phospholipids derived from biological components, and are characterized by low toxicity and antigenicity when administered to living bodies. It has also been shown that encapsulating a drug in a ribosome can improve the reach to the target tissue by changing the blood qualitative and biodistribution of the drug (for example, Non-patent Document 1, Patent Document 1). ) In addition, the blood vessel walls of new blood vessels that are abundant in cancer tissues are more permeable than those of existing blood vessels, and vesicles such as ribosomes are known to accumulate easily in cancer tissues (for example, non- Patent Document 2). Therefore, the antitumor active substance is enclosed in the ribosome. The added formulation is one of the DDS that is expected to enhance main effects and reduce side effects.

However, when a liposomal preparation containing an antitumor active substance is used for actual cancer treatment, the normal liposome composition has insufficient selective reachability to the cancer tissue, and thus has an antitumor effect. In many cases, it is not fully demonstrated. In addition, the occurrence of side effects due to the large amount of ribosomes distributed in organs other than the target is also a problem. The ribosomal lipid membrane is physically or chemically modified with a peptide (hereinafter referred to as a tumor tissue affinity peptide), protein, or antibody that selectively collects in cancer tissue, and then transferred to cancer tissue. Attempts have been made to increase sex.

As a tumor tissue affinity peptide, it is rare in normal tissues, and is expressed specifically in white blood vessels in tumors. Integrins, vasculargrowthfactors, matrime talloproteases ^ high—molecular—weightproteoglycan, etc. Those that migrate and join are well known. A typical example is a peptide containing an RGD sequence, which has been reported to selectively bind to integrin α- ν β-3 and _α — V; 3-5 in tumor neovasculature (for example, non-patent Reference 3). Peptides containing PRP sequences have been confirmed to accumulate specifically in new blood vessels, and are one of the vascular endothelial growth factor (VEGF) receptors fms— 1 iketyrosinekinase— 1 (f 1 t- 1) It has been suggested that it has an affinity (for example, Non-Patent Document 4, Patent Document 2). In addition, it has been confirmed that a peptad containing a PLPL sequence has an affinity for membranetype-1 matrix meloproteases (MT1-MMP) (for example, Non-Patent Document 5). In addition, it has been confirmed that a peptide containing an NGR sequence has an affinity for aminopeptidase N in tumor neovasculature (for example, Non-Patent Document 6). [Patent Document 1] WO 9 5/24 2 0 1

[Patent Document 2] Republished WO 0 0/2 34 7 6 [Non-Patent Document 1] J ournalof L ipos ome Reserch) 4, 667 (1994)

[Non-Patent Document 2] Dru g D e l i v e r y Sy s te m, 14, 4 ^ ΰ (1999)

[Non-Patent Document 3] B i o te c hno o g y, 12, 265 (1 995) [Non-Patent Document 4] On c o g e ne e, 21, 2662 (2002)

[Non-Patent Document 5] I NTERNAT I ONAL J OURNAL OF CA NCER, 108 301 (2004)

[Non-patent document 6] CANCER RESEARCH 60, 722 (2000) However, when these peptides are modified to ribosomal lipid membranes alone, selective migration to cancer tissues and an increase in antitumor effects are observed. It was often insufficient. In many cases, animal species and cancer types that exhibit antitumor effects were limited. Even if the type of peptide and the amount of modification were varied, there was a limit to the degree of increase in the antitumor effect, and it was thought that the hurdle to make a preparation that could be put to practical use in the clinical field was high.

An object of the present invention is to provide a ribosome preparation that includes a substance having an anti-malignant tumor activity, has a high cancer tissue reachability, and has an enhanced anti-serum activity. Disclosure of the invention

The present invention relates to the following inventions.

1. A ribosome preparation that contains an anti-S heavy tumor active substance and has two or more tumor tissue affinity peptides bound to the liposome surface.

2. Two peptides selected from the group consisting of peptides containing an RGD sequence, peptides containing a PRP sequence, peptides containing a PLPL sequence, and peptides containing an NGR sequence as S-hepatic tissue affinity peptides A ribosome preparation comprising at least.

3. A ribosome preparation comprising at least a peptide containing an RGD sequence and a peptide containing a PRP sequence as a tumor tissue affinity peptide. 4. The tumor tissue affinity peptide comprises two or more peptides selected from the group consisting of a peptide containing an RGD sequence, a peptide containing a PRP sequence, a peptide containing a PLPL sequence and a peptide containing an NGR sequence. Some liposome preparations.

5. A liposome preparation in which the tumor tissue affinity peptide is a peptide containing RGD sequence and a peptide containing PRP sequence.

6. A liposome preparation in which the tumor or tissue affinity peptide is a peptide containing an RGD sequence, a peptide containing a PRP sequence or a peptide containing an NGR sequence.

7. A ribosome preparation in which each tumor tissue affinity peptide contains 3 to 15 amino acids.

8. A liposome preparation in which at least one lipid component is dipalmitoyl phosphatidylcholine or distearoyl phosphatidylcholine.

9. Use of the above liposomal formulation for the treatment of tumors.

10. A method for treating a tumor, wherein an effective amount of an antitumor active substance contained in the ribosome preparation is administered to a mammal.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have synergized the ability to migrate to tumor tissue and affinity by using a combination of multiple types of tumor tissue affinity peptides. I devised the possibility to increase. In fact, by modifying the membrane surface of liposomes containing compounds with anti-malignant tumor activity with multiple types of tumor tissue affinity peptides, these peptides have synergistic effects on heavy tumor tissue affinity. Antitumor activity when administered intravascularly is significantly increased compared to ribosomes that are not peptide-modified and ribosomes that are modified with a single tumor tissue affinity peptide, and increased toxicity Was found not to accompany, and the present invention was completed.

The liposome used in the liposome preparation of the present invention is a closed vesicle having an inner aqueous phase part surrounded by a lipid bilayer formed by dispersing phospholipids constituting a cell membrane in water as described above. Multi-phase ribosome (Mu 1 ti 1 ame 1 1 ar Ve sicle: MLV), large unilamellar ribosome (Large Un il ame llar Ve sicl) e: LUV) and small unilamellar ribosome (Sma 1 1 Un i 1 ame 1 1 ar vesicle: SUV). Any type of liposome can be used in the present invention. The liposome of the present invention forms a stable ribosome structure before and after in vivo administration.

Since the fluidity and membrane permeability of the bilayer membrane constituting the liposome increase greatly with the phase transition temperature as a boundary, it is generally preferable to use a phospholipid having a phase transition temperature of 37 ° C or higher. Examples of such phospholipids include hydrogenated purified egg yolk phosphatidylcholine (phase transition temperature 50-60 ° C, hereinafter referred to as HEPC), hydrogenated refined soybean phosphatidylcholine (phase transition temperature approximately 55 ° C, hereinafter referred to as HSPC) Dipalmitoylphosphatidylcholine (phase transition temperature about 41 ° C, hereinafter referred to as DPPC) and distearoylphosphatidylcholine (phase transition temperature about 58 ° C, hereinafter referred to as DSPC), more preferably DS PC And DP PC. These can be used alone or in combination. In addition to these phospholipids, the liposomes used in the present invention are preferably used in admixture with stabilizers such as cholesterol derivatives that have been reported to improve the stability of ribosomes. The molar ratio of cholesterol derivative to phospholipid is preferably 1: 0.3 to 3, more preferably 1: 1 to 2.5.

Further, as an isotonic agent, for example, glycerin, glucose, sodium chloride and the like can be added. Furthermore, preservatives such as parabens, chlorobutanol, benzyl alcohol, propylene glycol may be added.

The tumor tissue affinity peptide used in the present invention is not particularly limited as long as it is a peptide that exhibits directivity to the tumor tissue when bound to the surface of a liposome, but a peptide containing an RGD sequence, a PRP sequence And peptides containing PLPL sequences, peptides containing NPL sequences, and peptides containing PLPL sequences. In addition, peptides other than the above may be bound. Furthermore, the tumor tissue affinity peptide used in the present invention includes a peptide containing two or more sequences selected from the group consisting of RGD sequence, PRP sequence, PL PL sequence and NGR sequence in one peptide. A peptide can be illustrated.

The peptide containing the RGD sequence used in the present invention may be any peptide containing the amino acid sequence of arginine monoglycine-spartamate, and can be prepared by a known method in the field of peptide synthesis. The peptide may be linear or cyclic. For example, GRGDS, GRGDL, GRGDR, GR GD F can be exemplified.

The peptide containing the PRP sequence used in the present invention may be any peptide containing the amino acid sequence of proline-arginine-proline, and can be prepared by a known method in the field of peptide synthesis. For example, APRPG, GPRPL, GP RPR can be exemplified.

The peptide containing the PLP L sequence used in the present invention may be a peptide containing the amino acid sequence of proline-synthetic-synaptic-synthetic-sin, and can be prepared by a known method in the peptide synthesis field. For example, GPLPLR, HP LPLS, PVPLPL, PLP LVR can be exemplified as follows:

The peptide containing the NGR sequence used in the present invention may be any peptide containing the amino acid sequence of asparagine, glycine and arginine, and can be prepared by a known method in the field of peptide synthesis. For example, CNGRC and CNGRG can be exemplified.

As described in Examples below, by introducing two or more types of tumor tissue affinity peptides into the ribosome membrane, it is possible to produce a ribosome preparation with improved tumor tissue affinity and reduced side effects. it can. In the ribosome preparation of the present invention, it is more preferable to introduce 2 to 4 kinds of tumor tissue affinity peptides. The combination of two kinds of tumor tissue affinity peptides in the liposome preparation of the present invention includes a peptide containing RGD sequence and a peptide containing PRP sequence; a peptide containing RGD sequence and a peptide containing PLPL sequence. A peptide containing an RGD sequence and an NGR sequence Peptides containing PRP sequences and peptides containing PLPL sequences; Peptides containing PRP sequences and peptides containing NGR sequences; Peptides containing PLPL sequences and peptides containing NGR sequences it can. Preferred are peptides containing RGD sequences and peptides containing PRP sequences.

The molar ratio of the two tumor tissue affinity peptides in the liposome preparation of the present invention can be arbitrarily set depending on the drug to be encapsulated and the cancer to be treated, but for example, a peptide containing an RGD sequence and a peptide containing a PRP sequence. When peptide is used, peptide containing RGD sequence: peptide containing PRP sequence = 1: 0.1 to 10 is preferable, 1: 0.3 to 3 is more preferable, and 1: 1 is more preferable.

The combination of the three tumor tissue affinity peptides in the liposome preparation of the present invention includes a peptide containing an RGD sequence, a peptide containing a PRP sequence, and a peptide containing a PLPL sequence; a peptide containing an RGD sequence, Peptide containing PRP sequence and peptide containing NGR sequence; Peptide containing RGD sequence, Peptide containing PLPL sequence and Peptide containing NGR sequence; Peptide containing PRP sequence, P LP L sequence included Peptides containing peptides and NGR sequences can be exemplified. Preference is given to peptides comprising an RGD sequence, peptides comprising a PRP sequence and peptides comprising an NGR sequence.

The molar ratio of the three tumor tissue-affinity peptides in the liposome preparation of the present invention can be arbitrarily set depending on the drug to be encapsulated and the cancer to be treated. For example, a peptide containing an RGD sequence, a peptide containing a PRP sequence When peptides containing NGR sequences are used, peptides containing RGD sequences: peptides containing PRP sequences: peptides containing NGR sequences = 1: 0. 1 to: 1 0: 0.:! To 10 is preferable, 1: 0.3 to 3: 3: 0.3 to 3 is more preferable, and 1: 1 to 1 is more preferable.

Furthermore, the combination of the four types of tumor tissue affinity peptides in the liposome preparation of the invention includes peptides containing RGD sequences, peptides containing PRP sequences, peptides containing PL PL sequences and peptides containing NGR sequences. It can be illustrated. The molar ratio of the four tumor tissue affinity peptides in the liposome preparation of the present invention can be arbitrarily set depending on the drug to be encapsulated and the cancer to be treated. For example, a peptide containing an RGD sequence, a peptide containing a PRP sequence, P When using peptides containing LP L sequences and peptides containing NGR sequences, peptides containing RGD sequences: peptides containing PRP sequences: peptides containing PLPL sequences: peptides containing NGR sequences == 1: 0. 1-10: 0.1-1-10: 0.1-10 is preferable, 1: 0.3-3: 0.3-3-3: 0.3-3 more preferable, and 1: 1: 1: 1 is more preferable.

The number of constituent amino acids of the tumor tissue affinity peptide used in the present invention is preferably 3 to 15 because of its low antigenicity, ease of modification to ribosomes and low production costs. 12 is more preferable, and 5 to 10 is particularly preferable. Specific examples of amino acids constituting the tumor tissue affinity peptide used in the present invention include arginine, glycine, aspartic acid, proline, leucine, phenenolealanine, serine, histidine, asparagine, cysteine and the like. it can.

The molar ratio between the phospholipid used in the present invention and the total amount of each peptide having affinity for tumor tissue is preferably from 1: 0.01 to 0.3, more preferably from 1: 0.05 to 0.2 force S, 1 : 0.1 is particularly preferred.

There are two methods for introducing a tumor tissue affinity peptide into the liposome membrane: a method that uses chemical binding and a method that does not use chemical binding, but the latter method is selected to avoid denaturation of the ribosome membrane. It is desirable. Specific examples of the latter include a tumor tissue affinity peptide such as a stearoyl group, a acyl group (for example, Japanese Patent No. 2620729), a cholesteryl group (for example, Japanese Patent No. 2579730), a phospholipid (for example, Japanese Patent Laid-Open No. 04-099795), etc. By preparing a liposome by mixing the hydrophobic side chain of the tumor and mixing the lipophilic side chain with a tumor tissue affinity peptide with other lipid components, the hydrophobic side chain is attached to the lipid membrane part of the liposome. It is possible to produce peptide-modified liposomes that are coordinated and have a tumor tissue affinity peptide introduced on the surface of the liposome membrane. Even when multiple types of tumor tissue affinity peptides are introduced into the ribosomal membrane, as long as these peptides do not show adverse physicochemical interactions, It can be prepared by the method described above in the same manner as in the case of the affinity peptide. The binding direction of the hydrophobic side chain is arbitrary. For example, in the case of a peptide containing the RGD sequence, the hydrophobic side chain may be bound to either the R side or the D side, but preferably R On the side. The ribosome preparation of the present invention is prepared by a known method. Known preparation methods for ribosomes include, for example, the reverse phase evaporation method [Proceeding of the Nashonanore Academy 'Ob'Sciences'US'A (Proc. N at 1. USA, 75, 41 94 (1978), WO 97/48398], Freezing and thawing method [Arche's Biochemistry-And No Physics (Ar c h. B ioch em. B iop hy s), 2 12, 186 (1981)], pH gradient method Lehomicika 'Eto' pioff siege force 'actor (B ioch em. B iophy s. Ac ta), 8 1 6, 294 () 985) and JP-A-7-165560.

Among these, the pH gradient method is useful for encapsulating drugs with greatly different solubility depending on pH in the liposome at a high inclusion rate. As a method for preparing the liposomal preparation of the present invention by the pH gradient method, for example, a tumor tissue affinity peptide to which a lipid component and a hydrophobic side chain are bound is dissolved in a solvent such as chloroform, ether, ethanol, etc. Put in a mold flask and evaporate the solvent under reduced pressure to form a lipid film. Next, a weakly acidic buffer solution is added to the thin film and freeze-thawed to prepare large multiphase ribosomes (MLV) whose inner aqueous phase is weakly acidic. Furthermore, after making small single membrane ribosomes (SUV) by the extrusion method, etc., neutral buffer is added to neutralize the outer aqueous phase (the difference in pH between the inner aqueous phase and the outer aqueous phase is 3 It is desirable to be above. Add an aqueous solution of the drug here and incubate at a temperature about 10 ° C higher than the phase transition temperature of the lipid bilayer. By the above operation, the drug can be encapsulated at a high rate and quantitatively inside the ribosome.

Ribosome particle size has been reported to have a significant effect on the biodistribution of contained drugs and its delivery to tumor tissue. Biological and pharmacological Ph a rm aceutica 1 B ulletin), 1 7, 9 3 5 (1 9 9 4)]. Therefore, in the present invention, it is preferable to perform a sizing treatment in order to make the particle size of the drug-encapsulating ribosome appropriate and uniform, for example, using an Estatruder (manufactured by Lipex Biomembrane, etc.) It is desirable to adjust the average particle diameter to 50 to 200 nm, preferably 75 to 125 nm by passing it through a membrane filter having an appropriate pore size several times.

Moreover, by modifying the membrane surface of the ribosome with a ligand such as a polyethylene glycol derivative as necessary, the interaction with plasma proteins after administration of liposomes into the blood vessels is reduced, and reticules such as liver Kupffer cells. Incorporation into endothelial cells can be suppressed, and the transferability of liposomes to tumor tissue and the like can be further improved.

By the above operation, the ribosome preparation of the present invention is prepared, and if necessary, the ultracentrifugation treatment, the gel filtration treatment, the ultrafiltration treatment and the dialysis treatment can be carried out alone or in combination to appropriately encapsulate in the ribosome. Drugs that have not been done can be removed.

The ribosome preparation of the present invention obtained by the above method can be used as it is. Considering the storage period and storage conditions, add excipients such as mannitol, trehalose, lactose, glycine and freeze-dry. You can also Add cryopreservatives such as glycerin and store them frozen.

The type of drug encapsulated in the ribosome preparation of the present invention is not particularly limited, but the liposomal preparation of the present invention has an excellent affinity for tumor tissue, so long as it has activity in the tumor tissue, antitumor activity Substances are preferred. Specific antitumor active substances are not particularly limited. For example, alkylic drugs, various antimetabolites, antitumor antibiotics, other antitumor agents, antitumor plant components, BRM (biological response) Sex control substances), angiogenesis inhibitors, cell adhesion inhibitors, matrix / metalloprotease inhibitors or hormones. More specifically, alkyl alkylating agents include, for example, nitrogen mustard, nitrogen mustard N-oxide, ifosfamide, menolephalan, chlorophosphamide, chloroethylamine alkylating agents such as kulamumbucil, For example, aziridin-based alkylating agents such as carbocone and thiotepa; for example, epoxide-based alkylating agents such as dib mouth momannitol and dipromodalcitol; And chlorozotocin, ranimustine and other nitrosolean-type anorequinolei chiral lj; busu ^ / fan, tosinoreic acid improsulfan, pisnolevane and other sulfonic acid estenoles; dacarbazine;

Examples of various antagonists include, for example, 6-mercaptopurine, azathioprine, 6-thioguanine, thioinosin and other purine antimetabolites; fluorouracil, tegafur, tegafur uracil, tegafur, gimeracil oteracyl potassium combined lj, canolemofur, And pyrimidine antimetabolite such as doxyfluridine, proxuridine, cytarabine, and enocytabine; antifolate antimetabolite such as methotrexate, trimetrexate, etc., and salts or complexes thereof.

Antitumor antibiotics include, for example, anthracyclines such as daunorubicin, aclarubicin, doxorubicin, pirarubicin, and epilubicin; actinomycins such as actinomycin D; chromomycins such as chromomycin A 3; Tomycins; bleomycins such as bleomycin and peplomycin; and their salts or complexes. Other anti-tumor agents include, for example, cisplatin, carboplatin, oxalibratin, TAS-1103, tamoxifen, L-parasinease, asperaton, schizophyllan, picibanil, ubenimex, krestin, and the like. A salt or a complex may be mentioned.

Examples of the antitumor plant component include plant altoroids such as camptothecin, vindesine, vintalistin, and vinblastine; epipodophyllotoxins such as etoposide and teniposide; and salts or complexes thereof. Also, Other examples include piperoproman, neocalcinostatin, and hydroxyurea.

Examples of BRM include tumor lobe death factor, indomethacin, and salts or complexes thereof.

Examples of the angiogenesis inhibitor include fumagillol derivatives, and salts or complexes thereof.

Examples of the cell adhesion inhibitor include substances having an RGD sequence, and salts or complexes thereof.

Examples of matrix 'meta-oral protease inhibitors include marimastat, batimastat and the like, and salts or complexes thereof.

Hormones include, for example, hydroconorethone, dexamethasone, methylprednisolone, prednisolone, plasterone, betamethasone, triamcinolone, oximetron, nandrolone, methenolone, phosfestrone, ethininorestradiol, chlormadinone, salt, Or a composite is mentioned.

Examples of the form of the drug include low molecular weight compounds, peptides, proteins, antibodies, siRNA V, and genes.

The ribosome preparation of the present invention is generally used as an injection (intravenous, intramuscular, subcutaneous administration) by suspending or diluting with a physiologically acceptable aqueous solution at the time of use. It can also be used as nasal drops, inhalants, suppositories, transdermal absorbents, transmucosal absorbents, and the like. Final preparations include malignant tumors (lung cancers of mammals such as humans, extirpator cancers (esophageal cancer, stomach cancer, rectal cancer, colon cancer, etc.), breast cancer, head and neck cancer, liver cancer, gallbladder. , S spleen cancer, gynecological cancer (uterine cancer, cervical cancer, ovarian cancer, etc.), urinary cancer (renal cancer, bladder cancer, prostate cancer, etc.), brain tumor, leukemia, It may be designed so that an effective dose for treatment and remission of melanoma, malignant lymphoma, etc.) is administered. For example, an antitumor active substance may be contained as an intravenously administered ribosome preparation in an amount of 0 · 01 to 100 mg. Ribosome preparation of the present invention The dosage of the active ingredient contained in is preferably from 0.01 to 1 000 mg / day. Brief Description of Drawings

FIG. 1 is a graph showing the antitumor effect of Test Example 3.

FIG. 2 is a graph showing the change in body weight of the mouse of Test Example 3.

FIG. 3 is a draaf showing the antitumor effect of Test Example 4.

FIG. 4 is a graph showing changes in body weight of the mice of Test Example 4. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail with reference to Examples and Test Examples, but the present invention is not limited thereto.

Example 1 (TAS— 1 0 3 liposome formulation modified with multiple peptides)

DS PC40 ₁ amo K Cholesterol monole 20 μηιο 1, Steer mouth inlay Ρ Peptide containing RP sequence (C 1 8—APRPG) 4 μm ο 1 and Peptide containing stearoyl RGD sequence (C 1 8—GRGDS ) Put 4 m ο 1 in eggplant-shaped flask (lipid composition: DSPC / cholesterol / C 1 8 -APRPG / C 1 8—GRGD S = 10Ζ5Ζ0.5 ノ 0.5) added. The vacuum form was distilled off under reduced pressure with an evaporator to form an oily thin film on the inner wall surface of the eggplant type flask. Furthermore, it was vacuum-dried for 1 hour in a desiccator. Next, after hydration with 0.3 mL of 0.3 M citrate solution (pH 4.0), freeze-thaw was performed three times. After sonication for 10 minutes using a bath sonicator, the average particle size of ribosome was passed through a polycarbonate membrane plano letterer (Nuclepore) with a pore size of 100 nm three times using an etas truder (manufactured by Lipetux Biomembrane). The diameter was adjusted to about 100 nm. Next, add 0.5 mL of 0.5 M sodium carbonate solution 0.76 pH 7.

Then, 20 mM HEPES buffer (pH 7.5) was further added to make 2 mL, and the obtained ribosome solution was allowed to stand at 60 ° C. Next, the antitumor active substance TAS-103 (between scientific names: 6- [[2- (dimethylamino) ethyl] amino] 1-hydroxy-7 H-indeno [2, 1 c]] which has topoisomerase inhibitory activity Add 1 mL of quinolin-7-one dihydrochloride dissolved in 20 mM HE PES buffer to a concentration of 5 mg ZmL, and incubate TAS_103 in the ribosome by shaking at 60 ° C for 30 minutes. Sealed in. Furthermore, in order to remove TAS—103 not encapsulated, ultracentrifugation (90000 rpm, 15 min, 4 ° C.) was performed three times to remove the supernatant, and then phosphate buffer [PBS (— )] Was used to prepare a TAS-103-containing ribosome preparation (APRPG · GRGD S-lipo. TAS-103) modified with APRPG peptide and GRGD S peptide. The average particle size of the liposomes was 0.2 μm.

Comparative Example 1 (TAS-103 liposome preparation modified with a single peptide)

DS PC40, mo 1, cholesterol 20, umo 1, C 18-APRPG 4 ^ mo 1 are placed in eggplant type flask (lipid composition: DS PCZ cholesterol ZC 1 8-APRPG = 10X5 / 1), 5 ml Black mouth form was added. Then, according to the method of Example 1, a TAS-103-containing liposome preparation (APRPG-1 ipo. TAS-103) modified with an APRPG peptide was prepared. The average particle size of the ribosome was 0.2 m.

Comparative Example 2 (TAS-103 liposome preparation modified with a single peptide)

DS PC40 zmo 1, cholesterol 20 μπιο 1, C 1 8— GRGDS 4 μ ΐΊΐ ο 1 in eggplant type flask (lipid composition: DSPC / cholesterol ZC 1 8-GRGDS = 10/5/1), 5 m L black mouth form was added. Thereafter, according to the method of Example 1, a TAS-103-containing liposome preparation (GRGDS-1 ipo. TAS-103) modified with a GRGDS peptide was prepared. The average particle size of the ribosome was 0.2 μm.

Comparative Example 3 (TAS-103 liposome preparation not modified with peptide) W 200

15

D S P C 40 ^ mo 1 and cholesterol 20 μm o 1 were placed in an eggplant type flask (lipid composition: D S P CZ cholesterol = 10.0 / 5), and 5 mL of black mouth form was added. Hereinafter, according to the method of Example 1, a control T A S—10 03-containing ribosome preparation (c o t —1 i p o. TAS—10 3) not modified with a peptide was prepared. The average particle size of the ribosome was 0.2 μπι.

Test Example 1 (TAS — 1 0 3 encapsulation rate in ribosome)

A 10% diluted solution of the liposome solution prepared in Example 1 and Comparative Examples 1 to 3 in 10 μL and 10% Triton X—l OO reduced solution in 200 μL and 0.3 M citrate Buffer solution 1 7 90 0 L was added and mixed, and further sonication was performed to solubilize the liposome membrane. Using a fluorometer, measure the fluorescence intensity of the mixed solution under the conditions of an excitation wavelength of 4 68 nm and a fluorescence wavelength of 5 68 8 nm. From the calibration curve prepared in advance, TAS — 1 0 in the ribosome Three quantities were determined. As a result, it was confirmed that all ribosome preparations had a TAS-10 3 encapsulation rate of 90% or more in the liposome, and were encapsulated with high efficiency and efficiency.

Test Example 2 (TAS—10 3 liposome stability in serum)

The liposome solution prepared in Example 1 and Comparative Examples 1 to 3 was incubated with 50% non-immobilized serum serum PBS (—) at 37 ° C. for 3 hours, and then released. After removing 3 with a centrifuge, the amount of TAS-10 3 in the liposome was determined by the method of Test Example 1. As a result, it was confirmed that the encapsulation rate of 99% or more was maintained compared to that before incubation, and that it was stable in serum.

【table 1】

Ribosome preparation based on pre-incubation

TAS—103 encapsulation rate after 3 hours (%) Example 1

APRPG-GRGDS- 1 i p o. TAS— 103 99.0 ± 0.0

Comparative Example 1

APRPG- 1 i p o. TAS— 103 99.0 ± 0. 0

Comparative Example 2

GRGDS-1 i po.TAS- 103 98. 9 ± 0. 1

Comparative Example 3

cont— 1 i po. TAS— 103 99.0 ± 0.0 Test Example 3 (Anti-tumor effect and toxicity evaluation of TAS-103 liposome) High lung metastatic tumor cell line used for evaluation of anti-tumor effect: Lewys Lung C arcinoma 5. Cultivation was carried out in 37 ° C 10% FBS (Fetal Bovine Serum, Fetal Bovine Serum) and DMEM (Dulbecco's Modified Eagle's Medium) in the presence of ₀ ° C ₀ ₂ and subcultured. A high lung metastatic tumor cell line, Lewis Lung Carcinoma, prepared to 5 X 10 ⁶ ce 11 sZmL, was administered 0.2 mL subcutaneously to the left ventral region of 6-week-old male C57 B LZ6 mice. Cancer-bearing mice were created. L ew is Lung Carcinoma transplantation 7, 11 1 and 15 days in total, 3 times in total, phosphate buffer [PBS (—)] as control and TAS prepared in Example 1 and Comparative Examples 1-3 — 103-encapsulated ribosome solution (20 mg / kg as TAS-103) was administered 0.2 mL Zbo dyZday into the tail vein of mice. The antitumor effect of the TAS-103-encapsulated liposome solution was examined by measuring the tumor volume 3 days after cancer transplantation. Tumor volume was calculated using the following formula.

B volume (Turn or Volume) = 0. 4 X a X b ²

(a: major axis of the tumor site, b: minor axis of the tumor site)

As a result, as shown in Figure 1, APRPG 'GRGDS— 1 i p o. TAS—

103 treatment group consists of A RPG— l i p o. TAS-103 treatment group, GRGD S—

1 1 p o. TAS-103 treatment group and cn t— 1 i p o. TAS—103 treatment group showed significant tumor growth inhibitory effect. C o n t— 1 i p o. TAS

It has been previously confirmed that the anti-tumor effect of -103 is higher than that of TAS-103 solution that has not been made into liposomes. In Figure 1, ** indicates that the result of the significant difference test is p <0.01, and * indicates that the result of the significant difference test is p <0.05. In addition, changes in body weight were examined as an index of side effects. As shown in Figure 2, weight loss was observed in all groups, but the least was observed in the APRPG.GRGD S—1 ipo. TAS—103 treatment group.

Example 2 (doxorubicin liposome preparation modified with multiple peptides)

DSPC20; zmol, cholesterol-containing 10, umo 1, peptide containing PRP sequence (C 18— APRPG) 2 mo 1 with stear mouth inlay and peptide C 18— GRGDS 2 imo containing RGD sequence with stearoyl 1 was placed in an eggplant-shaped flask (lipid composition: DSPC / cholesterol ZC 1 8— APRPGZC 18 — GRGD S = 10/5 / 0.5 5 0.5), and 5 mL of black mouth form was added. The vacuum form was distilled off under reduced pressure with an evaporator to form a lipid thin film on the inner wall surface of the eggplant type flask. Furthermore, it was vacuum-dried for 1 hour in a desiccator. Next, it was hydrated with 0.5 mL of 0.3 M citrate solution (pH 4.0), and then freeze-thawed three times. After sonication for 10 minutes using a bath sonicator, the average particle size of the ribosome was passed through a polycarbonate membrane filter (Nuclepore) with a pore size of 100 nm three times using an Estatruder (manufactured by Lipetks BioMenplan). Was adjusted to about 100 nm. Next, add 0.5 M sodium carbonate solution 0.211: << 7.5 and add 2 OmM HE PES buffer (ρ Η 7.5) to make the total volume 1 mL. The solution was left at 60 ° C.

Next, add 1 mL of an antitumor active substance, doxorubicin hydrochloride dissolved in 2 OmM HEPE S buffer at a concentration of 2 mg mL, and incubate at 60 for 30 minutes to encapsulate doxorubicin in the ribosome. . Furthermore, to remove the unencapsulated drug, ultracentrifugation (100,000 g, 10 min, 4 ° C) was performed three times to remove the supernatant, and then 0.3 M sucrose phosphate buffer (p.

H7.4), and the doxorubicin-containing ribosome preparation modified with APRPG and GRGDS peptides (APRPG · GRGDS— 1 i p o. D

OX) was prepared. The average particle size of the ribosome was 0.2 μπι.

Comparative Example 5— (Liposome formulation modified with a single peptide) Place DSPC 2 0 Ai mo 1 and cholesterol 1 0 μmo KC 1 8 -AP RP G 2 μπιο 1 into eggplant flask (lipid composition: DSP CZ cholesterol ZC 1 8 -APRPG = 1 0/5/1), 5 mL of black mouth form was added. A doxorubicin-containing liposome preparation (APRPG-1 ipo. DOX) modified with an APRPG peptide was prepared according to the method of Example 2 below. The average particle size of the ribosome was 0.2 μπι.

Comparative Example 6 (Liposome preparation modified with a single peptide)

Place DSPCSO / zmo 1 and cholesterol 1 10 μmo 1, C 1 8 -GRGD S 2 z mo 1 in eggplant flask (lipid composition: DSPC / cholesterol ZC 1 8 -GRGD S = l 0/5/1 ), 5 mL of black mouth form was added. Thereafter, a doxorubicin-containing liposome preparation (GRGD S-1 ipo. DOX) modified with GRGD S peptide was prepared according to the method of Example 2. The average particle size of the ribosome was 0.2 μm.

Comparative Example 7 (Liposome preparation not modified with peptide)

D S P C 20 m o 1 and Cholesterol Nore 10 μm o 1 were placed in an eggplant type flask (lipid composition: D S P CZ cholesterol = 1 0/5), and 5 mL of black mouth form was added. Then, according to the method of Example 1, a control doxorubicin-containing ribosome preparation (c ont-1 i po. DOX) not modified with a peptide was prepared. The average particle size of the liposome was 0.2 iz m.

Test Example 4 (Anti-tumor effect and toxicity evaluation of doxorubicin ribosome)

Mouse colon cancer cell line Cololon ₂ 6 NL 1 7 used for evaluation of antitumor effect was cultured in 3 7 ° C 10% FBS—DMEMZH am F 1 2 in the presence of 5% C0 ₂ and passaged did. Cololon 2 6 NL 17 prepared at 5 X 10 ⁶ ce 11 s / mL was administered 0.2 mL subcutaneously to the left ventral region of 6-week-old male B a 1 b / c mice. Created a kangan mouse. Co 1 0 n 2 6 NL 1 7 Transplantation 1 0, 1 3 and 16 days later, 3 times in total, 0.3 M sucrose phosphate buffer and doxorubicin prepared in Example 2 Encapsulated ribosome solution (5 mg as doxorubicin kg) was administered 0.2 mL / bod yZd ay into the tail vein of mice. The antitumor effect of doxorubicin-encapsulated ribosome solution was examined by measuring the tumor volume 8 days after cancer transplantation. The evaluation method is the same as Test Example 3.

As a result, as shown in Fig. 3, APRPG 'GRGDS— 1 ip o. DOX treatment group is divided into APRPG— 1 ip o. DOX treatment group, GRGDS— 1 ip o. DOX treatment group and c 0 nt— 1 ip. o. Compared to the DOX treatment group, the tumor growth was significantly suppressed. It has been previously confirmed that the antitumor effect of cn t-1 i p o.D0X is higher than that of the non-ribosomal doxorubicin hydrochloride solution. In Fig. 3, * indicates that the result of the significant difference test is p <0.05.

In addition, changes in body weight were examined as an index of side effects. As shown in FIG. 4, no weight loss was observed in the APRPG · GRGDS-1 ipo. DOX treatment group.

From these test results, anti-tumor active substance-encapsulated ribosome preparations modified with multiple tumor tissue affinity peptides are compared to non-ribosomal solutions, normal ribosome preparations, and liposomes modified with a single peptide. However, it was shown to significantly enhance antitumor activity and reduce toxicity. The reason for the enhancement of anticancer activity is that the reachability and affinity of the antitumor active substance to the tumor tissue have been significantly improved by the synergistic contribution of multiple peptides with affinity for different tumor molecules. Conceivable. INDUSTRIAL APPLICABILITY The liposomal preparation of the present invention has an effect of enhancing the effect of a substance having an antineoplastic activity contained therein and reducing its toxicity, which is clearly superior to conventional ribosome preparations. Therefore, in the actual treatment of malignant tumors, it is highly expected to suppress the progression of cancer while reducing side effects.

Claims

The scope of the claims

1. A ribosome preparation that contains an antitumor active substance and has two or more kinds of tumor tissue affinity peptides bound to the surface of the liposome.

2. A peptide containing RGD sequence as a tumor tissue affinity peptide, P R

The ribosome preparation according to claim 1, comprising at least two peptides selected from the group consisting of a peptide containing a P sequence, a peptide containing a PLP L sequence and a peptide containing an NGR sequence.

3. The liposome preparation according to any one of claims 1 and 2, comprising at least a peptide containing an RGD sequence and a peptide containing a PRP sequence as a tumor tissue affinity peptide.

4. Two or more peptides selected from the group consisting of a peptide containing an RGD sequence, a peptide containing a PRP sequence, a peptide containing a PLPL sequence, and a peptide containing an NGR sequence. The liposomal preparation according to claim 1, which is

5. The ribosome preparation according to any one of claims 1 and 4, wherein the tumor tissue affinity peptide is a peptide containing RGD sequence and a peptide containing PRP sequence.

6. The tumor tissue affinity peptide is a peptide comprising an RGD sequence, a peptide comprising a PRP sequence and a peptide comprising an NGR sequence.

5. The ribosome preparation according to any one of items 4.

7. The liposome preparation according to any one of claims 1 to 6, wherein the number of constituent amino acids of each tumor tissue affinity peptide is 3 to 15.

8. The liposome preparation according to any one of claims 1 to 7, wherein at least one lipid component is dipalmitoylphosphatidylcholine or distearoylphosphatidylcholine.

9. Encapsulates antitumor active substances and contains two or more tumor tissue affinity peptides. Use of a liposomal formulation bound to a posome surface for the treatment of tumors.

10. A method for treating tumors comprising administering to a mammal an effective amount of an antitumor active substance contained in a ribosome preparation containing an antitumor active substance and having two or more kinds of tumor tissue affinity peptides bound to the ribosome surface .