JP2008031142A - Fat tissue-targeting peptide and liposome having the peptide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a substance having a specific migration ability to skeletal muscles or fat tissues, and to provide a drug delivery system using the substance. <P>SOLUTION: This heptapeptide comprising 3 to 6 basic amino acid residues and at least one hydrophobic amino acid residue or at least one hydrophilic amino acid residue except the basic amino acid residues and having a specific migration ability to fat tissues or skeletal muscles. A liposome bound to the heptapeptide and having a specific migration ability to fat tissues and the like. The peptide has a specific migration ability to fat tissues, and has an ability for targeting a liposome to whose lipid membrane the peptide is bound, on the fat tissue. Since having an escaping ability from endosome to cytoplasm, the liposome can encapsulate a remedy for type II diabetes to give a drug delivery system effective for the therapy of the type II diabetes. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a peptide having specific migration ability for adipose tissue, and a liposome having the peptide.

  Diabetes is broadly divided into type I diabetes and type II diabetes. Type I diabetes, also called insulin-requiring diabetes (IDDM), is a diabetes that develops when the pancreatic islets of Langerhans are inflamed and the insulin secretion by β-cells is markedly reduced, and cannot survive without supplementation with insulin Presents with insulin-dependent pathology. On the other hand, type II diabetes is also called non-insulin dependent diabetes mellitus (NIDDM), and is associated with environmental factors such as obesity, overeating, lack of exercise, and stress, and is likely to occur in middle-aged and older elderly people .

  Non-insulin resistance in type II diabetes is attributed to the fact that skeletal muscle and adipose tissue, which are inherently insulin-responsive peripheral tissues, acquire insulin resistance. Therefore, in the treatment of diseases such as type II diabetes treatment, it is an important issue to construct a delivery system that delivers drugs, genes, and the like to skeletal muscles and adipose tissues that are greatly involved in sugar metabolism.

  Virus vector systems such as retroviruses and adeno-associated viruses have been developed as one of systems for delivering substances such as proteins, drugs or genes to target sites of living organisms. While this viral vector system is generally excellent in specificity for target cells, problems such as antigenicity and particularly toxicity have been pointed out.

  Liposome vectors are known as delivery systems that replace viruses. Liposomes are vectors based on artificially prepared lipid bilayers, and have the ability to encapsulate various substances and deliver them into target cells. However, the specificity with respect to a target cell is scarce, and the delivery efficiency to a target cell of a drug etc. is not high. Therefore, various methods for imparting specificity to target cells to liposomes have been studied and reported.

  For example, there has been reported a method for improving uptake into tumor cells by binding a hydrophilic polymer to the surface of a lipid membrane constituting a liposome and enhancing the retention in blood (Patent Document 1). In addition, as a method for increasing the specificity for specific cells, a liposome in which a protein existing on the surface of a target cell, for example, a receptor protein or a specific ligand for an antigen is bound to the surface of a lipid membrane has been reported (Patent Document 2). ).

However, a delivery system specifically excellent for skeletal muscle or adipose tissue for delivering drugs or genes to skeletal muscle or adipose tissue that is useful for treating type II diabetes has not been constructed.
JP-A-1-249717 JP-A-4-346918

  The present invention relates to a ligand having specificity for skeletal muscle or adipose tissue, and a delivery system useful for the treatment of type II diabetes using the ligand to deliver drugs, genes, etc. to skeletal muscle or adipose tissue I will provide a.

  The present inventors conducted a phage display method using an individual animal, tried to search for peptide ligands having specificity for skeletal muscle or adipose tissue, and a specific oligopeptide had high specificity for adipose tissue. The following inventions have been completed.

(1) 3 to 6 basic amino acid residues, and at least one hydrophilic amino acid residue other than a hydrophobic amino acid residue or a basic amino acid, and a migration ability specific to adipose tissue or skeletal muscle A heptapeptide having

(2) The heptapeptide according to (1), wherein the hydrophobic amino acid is leucine, isoleucine or proline.

(3) The heptapeptide according to (1), wherein the hydrophilic amino acid is glutamine, threonine, methionine, serine or asparagine.

(4) The heptapeptide according to (1), wherein the amino acid sequence is IRQRRRR.

(5) A liposome to which the heptapeptide according to any one of (1) to (4) is bound.

(6) The liposome according to (5), which has a migration ability specific to adipose tissue.

(7) The liposome according to (5), which has an ability to migrate into cells via caveolae.

(8) The liposome according to (5), comprising a lipid to which a heptapeptide is bound.

(9) The liposome according to (8), wherein the heptapeptide comprises a lipid bound via a cysteine residue added to the heptapeptide and a maleimidized hydrophilic polymer. The liposome according to (8) or (9), comprising a lipid bound to a non-hydrophilic polymer.

(11) The liposome according to any one of (8) to (10), wherein the lipid to which heptapeptide is bound is contained in an amount of 5 to 10 mol% per total lipid constituting the liposome.

(12) The liposome according to (10), comprising 5 to 10 mol% of a lipid bound to a hydrophilic polymer to which no heptapeptide is bound, per total lipid amount constituting the liposome.

(13) The liposome according to any one of (9) to (12), wherein the hydrophilic polymer is polyalkylene glycol.

(14) A heptapeptide containing 3 to 6 basic amino acid residues and at least one hydrophilic amino acid residue other than a hydrophobic amino acid residue or a basic amino acid is bound to a liposome and then passed through caveolae. A method for producing a liposome having the ability to move into a cell.

(15) The method according to (14), wherein the amino acid sequence of the heptapeptide is IRQRRRR.

  The peptide of the present invention has a specific ability to migrate to adipose tissue, and has the ability to target adipose tissue with liposomes bound to the surface of the lipid membrane. Moreover, since this liposome has the ability to escape from endosomes to the cytoplasm, it can be a drug delivery system effective for the treatment of type II diabetes by encapsulating a therapeutic agent for type II diabetes in the liposome. Furthermore, a heptapeptide consisting of the amino acid sequence IRQRRRR can be transferred to cells via caveolae by binding it to the liposome.

  The peptide of the present invention has 3 to 6 basic amino acid residues and at least one hydrophobic amino acid residue or a hydrophilic amino acid residue other than basic amino acid, and has a fat tissue-specific migration ability. It is a heptapeptide.

  These peptides are oligopeptides displayed by high-titer phages recovered from rat skeletal muscle and adipose tissue by the phage display method for rats.

  One of the features of the heptapeptide of the present invention is that it has 3 to 6 basic amino acid residues. The basic amino acid may be arginine (R, Arg) or lysine (K, Lys), but Arg is preferred.

  Another feature of the heptapeptide of the present invention is that it has at least one hydrophobic amino acid residue or hydrophilic amino acid residue. Preferred hydrophobic amino acids are leucine (L, Leu), isoleucine (I, Ile) or proline (P, Pro), but valine (V, Val), phenylalanine (F, Phe) and other hydrophobic amino acids. May be. The hydrophilic amino acids include glutamine (Q, Gln), threonine (T, Thr), methionine (M, Met), serine (S, Ser), asparagine (N, Asn) and the like.

  A preferred heptapeptide is a heptapeptide containing 4 or more basic amino acid residues, each containing one hydrophobic amino acid and one hydrophilic amino acid, and more preferably, the hydrophobic amino acid residue is isoleucine and hydrophilic. Sex amino acid residue is glutamine.

  In addition, 3 to 6 basic amino acid residues, at least one hydrophobic amino acid residue or hydrophilic amino acid residue may appear in any order on the amino acid sequence of the heptapeptide. Amino acid residues may appear continuously or discontinuously.

  In the present invention, a heptapeptide having high specificity for adipocytes is a heptapeptide whose amino acid sequence is IRQRRRR (hereinafter referred to as IRQ peptide). As will be shown later in Examples, the IRQ peptide exhibits a function of specifically inducing liposomes to which it is bound to lipid cells.

  In particular, as shown in the Examples, the IRQ peptide can impart a function of being incorporated into cells via caveolae to liposomes to which the peptide is bound. Caveolae are one of the membrane microdomains. They are present in the cell membrane and have a small dent structure (diameter ˜50 nm) made of specific lipids and proteins with a diameter of about several tens of nm. Caveolae are rich in glycolipids and cholesterol, and are formed by a membrane protein characteristic of this structure called caveolin, which binds to cholesterol and self-associates, and is one of the sites where endocytosis occurs, especially in vascular endothelial cells. It has been suggested that this is a useful mechanism for transporting substances from the blood vessel side to the basement membrane side via tosis. Therefore, the IRQ peptide of the present invention is extremely useful for the construction of a delivery system for delivering functional molecules such as genes and siRNA to normal tissue parenchymal cells.

  The heptapeptide referred to in the present invention contains 3 to 6 basic amino acid residues and at least one hydrophilic amino acid residue other than a hydrophobic amino acid residue or a basic amino acid, and is a fatty tissue or skeleton. As long as it has a migration ability specific to muscle, it also includes peptides in which one or several amino acid residues are further added or inserted into the heptapeptide.

  For example, as described later, when the heptapeptide of the present invention is bound to a liposome, an amino acid residue having a thiol group may be added or inserted into the heptapeptide, but such a peptide is still included in the heptapeptide of the present invention. It is.

  The heptapeptide of the present invention may be produced recombinantly using a general genetic recombination technique, or chemically synthesized using a device such as a peptide synthesizer that is widely commercially available and used. Good. It should be noted that for both recombinant production and chemical synthesis, a general method widely known by those skilled in the art may be used, and no special contrivance is required for the synthesis of the heptapeptide of the present invention.

  Another embodiment of the present invention is a liposome having a migration ability specific to adipose tissue to which the heptapeptide described above is bound. A preferred embodiment is a liposome containing a lipid to which a heptapeptide is bound, and a more preferred embodiment is a liposome comprising a cysteine residue added to the C-terminus of the heptapeptide and a lipid bound via maleimide polyethylene glycol. It is.

  Liposomes are vector particles based on an artificially prepared lipid membrane. When forming this lipid membrane, drugs, proteins, DNA, antisense RNA, siRNA and other physiologically active substances can be encapsulated in the membrane. The liposome encapsulating the physiologically active substance in this manner can be incorporated into the endosome mainly by endocytosis of the cell, so that the encapsulated physiologically active substance can be incorporated into the cell. One of the important properties in the principle of this liposome is that it is specifically taken up by the target cell, and the other is that it is taken up by the endosome and then from the endosome to the cytoplasm or nucleus, mitochondria and other organelles. To move specifically.

  Liposomes to which the heptapeptides of the present invention, particularly IRQ peptides are bound, have a migration ability specific to adipose tissue, and also have the ability to move into the cytoplasm after being taken into cells by endocytosis. It became clear. This endocytosis takes place via caveolae. Such liposomes contain various drugs, proteins, DNA, antisense RNA, siRNA and other physiologically active substances that are effective in the treatment of diabetes, particularly type II diabetes. It can be used as an excellent delivery system capable of reaching an active substance. In particular, since liposomes bound with IRQ peptides are delivered via caveolae, they are extremely useful as delivery systems for delivering functional molecules such as genes and siRNA to normal tissue parenchymal cells.

  Liposomes of the present invention are lipids (phospholipids, glycolipids, sterols, saturated or unsaturated fatty acids, etc.) that are essential components constituting the lipid layer, and membrane stabilization that is an optional component other than the heptapeptide described above. It is comprised from conventionally well-known components which comprise a general liposome, such as an agent, an antioxidant, a charged substance, and a membrane protein.

  Phospholipids include dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, phosphatidylcholine such as distearoylphosphatidylcholine, dioleoylphosphatidylglycerol, dilauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylglycerol, Phosphatidylglycerols such as distearoyl phosphatidylglycerol, dioleoyl phosphatidylethanolamine, dilauroyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidie ethanol Phosphatidylethanolamine such as an amine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, cardiolipin, sphingomyelin, mention may be made of egg yolk lecithin, soybean lecithin or hydrogenation products thereof and the like.

  Examples of glycolipids include glyceroglycolipids such as sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride and glycosyl diglyceride, and sphingoglycolipids such as galactosyl cerebroside, lactosyl cerebroside and ganglioside. Examples of sterols include animal-derived sterols such as cholesterol, cholesterol succinic acid, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol, plant-derived sterols such as phytosterol, stigmasterol, sitosterol, campesterol, and brassicasterol. Examples include sterols derived from microorganisms such as timosterol and ergosterol.

  Examples of the saturated or unsaturated fatty acid include saturated or unsaturated fatty acids having 12 to 20 carbon atoms such as palmitic acid, oleic acid, stearic acid, arachidonic acid, and myristic acid.

  The amount of lipid blended is 50 to 100% (molar ratio), preferably 70 to 100% (molar ratio), more preferably 85 to 100% (molar ratio) with respect to the total amount of components constituting the lipid layer of the liposome. ).

  Further, by incorporating an anionic lipid in the lipid layer of the liposome of the present invention, it is also possible to impart nuclear translocation ability to the liposome. In particular, the use of an anionic lipid selected from cholesteryl hemisuccinate, phosphatidylic acid or cardiolipin is preferred. When an anionic lipid is contained, it is preferably added so as to be an amount corresponding to 20 to 80 mol% of the total amount of lipid contained in the lipid layer.

  The membrane stabilizer is an optional component that is added to physically or chemically stabilize the lipid membrane or adjust the fluidity of the lipid membrane. The blending amount can be appropriately adjusted within a range of 0 to 50% (molar ratio) with respect to the total amount of components constituting the lipid layer of the liposome. Suitable membrane stabilizers are glycerin or triolein, trioctanoin, etc. and fatty acid esters of glycerin.

  Antioxidants are optional ingredients added to prevent lipid membrane oxidation. The blending amount can be appropriately adjusted within a range of 0 to 10% (molar ratio) with respect to the total amount of components constituting the lipid layer of the liposome. Examples of the antioxidant include tocopherol, propyl gallate, ascorbyl palmitate, butylated hydroxytoluene and the like.

  The charged substance is an optional component added to impart a positive or negative charge to the lipid membrane. The blending amount can be appropriately adjusted within a range of 0 to 95% (molar ratio) with respect to the total amount of components constituting the lipid layer of the liposome.

  Examples of the charged substance that imparts a positive charge include saturated or unsaturated aliphatic amines such as stearylamine and oleylamine, and saturated or unsaturated cationic synthetic lipids such as dioleoyltrimethylammonium propane. Examples of the charged substance that imparts a negative charge include dicetyl phosphate, cholesteryl hemisuccinate, phosphatidylserine, phosphatidylinositol, and phosphatidic acid.

  Membrane protein is an optional component added to maintain the structure of the lipid membrane or to impart functionality to the lipid membrane. The blending amount is 0 to 10% (molar ratio), preferably 0 to 8% (molar ratio), more preferably 0 to 5% (molar ratio) based on the total amount of components constituting the lipid layer of the liposome. It is.

  The liposome of the present invention is a liposome containing a liposome constituent component to which the heptapeptide is bound, preferably a liposome containing the lipid exemplified above bound to a peptapeptide. More preferably, in a liposome having a structure in which the hydrophilic polymer is bonded to a part of a maleimidized hydrophilic polymer via a cysteine residue added to a heptapeptide, and the hydrophilic polymer is bonded to a component of the liposome, particularly a lipid. is there.

  The hydrophilic polymer and heptapeptide can be bonded by forming a covalent bond between the functional group of the hydrophilic polymer and the functional group of the heptapeptide. In general, combinations of functional groups capable of forming a covalent bond include, for example, amino group / carboxyl group, amino group / acyl halide group, amino group / N-hydroxysuccinimide ester group, amino group / benzotriazole carbonate group, Examples include amino group / aldehyde group, thiol group / maleimide group, thiol group / vinylsulfone group, etc., but in the case of the heptapeptide of the present invention, it is a peptide containing three or more basic amino acid residues. It is preferable to newly add an amino acid residue having a thiol group to the heptapeptide and bind to the hydrophilic polymer using this thiol group.

  A method of binding a peptide having a thiol group or a peptide to which an amino acid residue having a thiol group such as cysteine is added to a hydrophilic polymer, typically polyalkylene glycol, via such a thiol group, and such hydrophilicity The binding of a functional polymer and a lipid has been reported by Lu et al. (J. Controlled release, 2006, 110, 505-513). The liposome of the present invention can be prepared using the method of Lu et al., But is not limited to such a method. Addition of an amino acid residue having a thiol group may be on either the N-terminal side or the C-terminal side of the heptapeptide. Moreover, you may insert the amino acid residue which has a thiol group in parts other than the N terminal or C terminal of a heptapeptide. Therefore, the heptapeptide of the present invention comprises 3 to 6 basic amino acid residues and at least one hydrophilic amino acid residue other than a hydrophobic amino acid residue or a basic amino acid residue, or adipose tissue or skeletal muscle As long as it has a specific migration ability, it includes a heptapeptide in which one or several amino acid residues are added or inserted.

  The liposome of the present invention comprises a lipid to which heptapeptide is bound, preferably a lipid to which heptapeptide is bound via a hydrophilic polymer, in an amount of 0.1 to 15% (molar ratio), preferably 1 per total lipid amount constituting the liposome. It is preferable to contain -10% (molar ratio), more preferably 5-10% (molar ratio). As shown in the Examples below, even when the lipids to which heptapeptide is bound are contained in an amount of 10% (molar ratio) or more per total lipid constituting the liposome, no significant change is observed in the specific ability to migrate to adipose tissue.

  Moreover, it is preferable that the liposome of this invention further contains the lipid couple | bonded with the hydrophilic polymer which the heptapeptide has not couple | bonded. In general, it is known that the liposome contains a lipid bound to a hydrophilic polymer to which no peptide is bound, so that the blood retention capacity of the liposome is improved. By including in the liposome a lipid bound to a hydrophilic polymer to which no is bound, nonspecific migration of the liposome to tissues other than adipose tissue can be suppressed. In the present invention, the content of the lipid bound to the hydrophilic polymer to which the heptapeptide is not bound is preferably 5 to 10% (molar ratio) per total lipid constituting the liposome. In the content within this range, suppression of nonspecific migration of liposomes to tissues other than adipose tissue is observed.

  Any hydrophilic polymer used in a preferred embodiment of the present invention can be used as long as it is generally known to have a function of improving the blood retention of liposomes. Examples thereof include polyalkylene glycols such as polyethylene glycol, polypropylene glycol, polytetramethylene glycol, polyhexamethylene glycol, other dextran, pullulan, ficoll, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether. -Maleic anhydride alternating copolymer, amylose, amylopectin, chitosan, mannan, cyclodextrin, pectin, carrageenan and the like.

  A preferred hydrophilic polymer in the present invention is polyalkylene glycol, and polyethylene glycol is particularly preferred. The molecular weight of the polyalkylene glycol is preferably 300 to 10,000, more preferably 500 to 10,000, and particularly preferably 1,000 to 5,000.

  The liposome of the present invention may be in any form such as MLV (Multi Layer Vesicle), SUV (small unilamellar vesicle), LUV (large unilamellar vesicle), GUV (giant unilamellar vesicle) and the like. The size of the liposome of the present invention may be 30 to 100 nm in diameter, and preferably 50 to 300 nm in diameter, but is not particularly limited thereto.

  The liposome of the present invention is characterized by its constitution as described above, but the preparation can be performed based on a usual method. Examples of the usual method include simple hydration method, ultrasonic treatment method, ethanol injection method, ether injection method, reverse phase evaporation method, surfactant method, freezing / thawing method and the like. Also, conversion from multilamellar liposomes to single membrane liposomes or vice versa is possible.

  The target substance to be delivered into the cell can be encapsulated inside the liposome of the present invention. Examples of the target substance include drugs, nucleic acids, peptides, proteins, complexes thereof, and the like effective for treating diabetes, particularly type II diabetes. When the target substance is water-soluble, the target substance can be encapsulated in the aqueous phase inside the liposome by dissolving the target substance in an aqueous solvent used for hydrating the lipid membrane. When the target substance is fat-soluble, the target substance can be encapsulated in the lipid layer of the liposome by dissolving the target substance in an organic solvent used in the production of the liposome.

The liposome of the present invention can be used by being dispersed in an appropriate aqueous solvent such as physiological saline, phosphate buffer, citrate buffer, and acetate buffer. Additives such as saccharides, polyhydric alcohols, water-soluble polymers, nonionic surfactants, antioxidants, pH adjusters, hydration accelerators may be appropriately added to the dispersion. Further, the liposome of the present invention can be stored in a state where the above dispersion is dried. The liposome of the present invention can be administered orally, and parenteral such as vein, intraperitoneal, subcutaneous, nasal and the like. Administration is also possible.

  EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited to these examples.

Example 1 Determination of a peptide having a migration ability specific to adipose tissue 1) Phage display Synthetic DNA (21 nucleotides: equivalent to 7 amino acids) having an arbitrary base sequence is the 5 ′ end of DNA encoding the coat protein pIII of M13 phage The Ph.D. phage library 2 × 10 ¹¹ pfu manufactured by New England Biolabs, which was inserted in the above, was administered to the Balb / c mouse via the tail vein. This phage has a feature that an arbitrary peptide sequence corresponding to 7 amino acids encoded by the synthetic DNA inserted at the 5 ′ end is displayed outside the phage particle. Ten minutes after the administration, 100 ml of PBS / EDTA was perfused from the heart to remove the phage remaining in the blood vessel without binding to the tissue.

  Muscle tissue and adipose tissue were removed from the thigh and homogenized in homogenate buffer containing 20 mM Hepes, 250 mM Sucrose, 1 mM EDTA, 10 mg / mL aprotinin, 10 mg / mL Leupeptin, and 1 mM PMSF, respectively. . The titer of M13 phage in the supernatant after homogenization was measured. In addition, the phages grown by infecting the collected phages with 200 mL of the E. coli strain ER2738 culture solution were precipitated by PEG / HCl and collected. The above phage infection, propagation and purification procedures were in accordance with the package insert of the Ph.D. phage library kit manufactured by New England Biolabs.

  The collected phage was suspended in TBS solution containing 1.5 mL of 50 mM Tris-HCl; pH 7.5, 150 ml NaCl. Centrifugation was carried out at 15000 r.p.m (TOMY MX-200) for 10 minutes at 4 ° C., and the phage suspension was recovered in the supernatant.

  Add 1.5 mL of high-concentration CsCl solution (0.93 g / mL in TBS) to a new centrifuge tube, overlay 1.5 mL of the phage suspension above, and then add 1.5 mL of low-concentration CsCl solution (0.47 g / mL in TBS) 1.5 Layered with mL, filled the tube, and centrifuged at 265,000 × g, 15 ° C. for 15 hours.

  After centrifugation, the phage-derived band confirmed by UV irradiation was recovered using a syringe and dialyzed against TBS buffer to obtain the first recovered phage.

2 × 10 ¹¹ pfu of the first recovered phage was administered to the Balb / c mouse via the tail vein, and the above operation was repeated to obtain 2nd recovered phage. Further, 2 × 10 ¹¹ pfu of 2nd recovered phage was administered to Balb / c mice via the tail vein, and the above operation was repeated again to obtain 3rd recovered phage.

  When the titer of each recovered phage was measured, as shown in FIG. 1 (muscle tissue) and FIG. 2 (adipose tissue), the titer of the phage recovered from each of the muscle tissue and adipose tissue increased with each repetition. Confirmed to do.

The 3rd recovered phages from both tissues were each infected with E. coli ER2738 spread on the plate, and 96 plaques were recovered to obtain 96 types of phages displaying the peptide on the outside of the phage particles. The obtained phage DNA was collected, and the amino acid sequence of the peptide displayed outside the phage particle was determined. These 96 types of peptides are shown in Table 1 (muscle tissue) and Table 2 (adipose tissue).

2) Sequence analysis of peptides
Select any one peptide from 96 types of peptides, search for the appearance frequency of amino acid residues with all other 95 types of peptides, and search for 3 or more of 7 amino acid residues in 95 types of peptides The number of peptides having the same amino acid residue was examined, and the number was defined as the Frequency Value for the arbitrary peptide. Regarding the evaluation of the appearance frequency of amino acid residues, the order of appearance was not taken into consideration, and it was accepted as long as three or more common amino acid residues exist continuously or at equal intervals. The frequency values of all 96 amino acids obtained according to this standard are shown in FIG. 3 (skeletal muscle) and FIG. 4 (adipose tissue). Among these, peptides having a frequency value of 20 or more were selected (Table 3: Skeletal muscle, Table 4: Adipose tissue).

  In addition to the above, any one peptide is selected from 96 types of peptides, and the appearance frequency of amino acid residues is searched for in total with all other 95 types of peptides. Among the residues, those having a peptide having 5 or more identical amino acid residues were selected. Regarding the evaluation of the appearance frequency of amino acid residues, the order of appearance was not taken into consideration, and it was accepted as long as five or more common amino acid residues existed continuously or at equal intervals.

Table 5 (skeletal muscle) and Table 6 (adipocyte) show peptides that meet this criterion and have the above Frequency Value of less than 20.

3) Selection of peptides Each of the phages shown in Table 2 and Table 5 was injected with 1 × ^{10 10} pfu / mouse from the rat tail vein, and 10 minutes after administration, 100 ml of PBS / EDTA was perfused from the heart. The muscle tissue was extracted and homogenized, and the phage titer in the supernatant was measured. In addition, as a control, a phage (non-selective phage) before selection of Ph.D. phage library was used.

  As a result, as shown in FIG. 5, accumulation in a high muscular tissue was confirmed with Clone 95, 52, 12, 30, 37, 8, and 47. In particular, Clone52 showed about 10 times higher accumulation than the control.

Example 2 Preparation of peptide-bonded liposome 1) Preparation of liposome
According to the method of Lu et al. (Described above), a peptide IRQRRRRC (hereinafter referred to as IRQ peptide) in which cysteine is added to the C-terminal of Clone 52, polyethylene glycol (Mal-PEG) to which a maleimide group is bonded, and Di Stearyl Phosphatidyl Ethanolamine (DSPE) ) And a conjugate (Mal-PEG-DSPE) were dissolved in pure water so that each concentration was 10 mM, and incubated at room temperature for 24 hours while stirring with a stirrer.

  Dilute the reaction mixture after incubation with 5 μl by adding 100 μl acetonitrile / 0.1% TFA, and then add 50 μL of an equal volume of matrix sinapinic acid (3,5-dimethoxy-4-hydroxy cinnamic acid) solution (10 mg / mL in acetonitrile / 0.1% TFA), and a sample with a small amount of NaCl added as an ionization accelerator is placed on a slide, dried at room temperature, and then subjected to MALDI-TOF MS measurement. The lipid IRQ-Mal-PEG-DSPE bound to -PEG-DSPE was confirmed.

From a lipid having a composition of phosphatidylcholine (EPC) / cholesterol (Chol) = 2: 1 to which Mal-PEG-DSPE corresponding to 5% of the total lipid is added, Mal--corresponding to 5% of the total lipid Liposomes were prepared by simple hydration methods from lipids in which part of PEG-DSPE was gradually replaced with IRQ-Mal-PEG-DSPE. In order to analyze the pharmacokinetics of liposomes, a tracer amount (30 μCi) of [ ³ H] cholesteryl hexadecyl ether (CHE) was added during liposome preparation to label the liposomes.

  The above liposomes were injected from the tail vein, and the remaining amount of each liposome in the blood was measured 24 hours later. As a result, a decrease in the blood remaining amount of the liposomes was observed with an increase in the substitution rate of IRQ-Mal-PEG-DSPE (FIG. 6).

On the other hand, as a result of observing the accumulation of liposomes in muscle tissue and adipose tissue, increase in the amount of liposome accumulation in muscle tissue was not observed even when the content of IRQ-Mal-PEG-DSPE increased (FIG. 7). Rather, the amount of liposomes accumulated in adipose tissue was increased (FIG. 8). In addition, even when the content of IRQ-Mal-PEP-DSPE with respect to the total amount of lipid was 10%, no significant increase in accumulation in adipose tissue and lung was observed (FIG. 9).
It was revealed that the liposome containing IRQ-Mal-PEG-DSPE also migrated to the lung depending on the content of the IRQ peptide under the above conditions (FIG. 10). Therefore, the content of IRQ-Mal-PEP-DSPE with respect to the total lipid amount was fixed at 5%, and liposomes added with Mal-PEG-DSPE were prepared, and their pharmacokinetics were analyzed. It was confirmed that as the content was increased to 5% and 10%, liposome accumulation in the lung could be suppressed (FIG. 11). Under the same conditions, the accumulation of liposomes in the adipose tissue hardly changed, indicating that the content of IRQ-Mal-PEP-DSPE is a determinant for the accumulation of liposomes in fat.

2) Analysis of intracellular kinetics of liposomes Whether or not the liposomes containing IRQ-Mal-PEP-DSPE prepared in the above 4) have endosome escape ability was evaluated using a confocal laser microscope. Liposomes containing IRQ-Mal-PEP-DSPE encapsulating 10 mM rhodamine were prepared according to the method described in 4) above. 75.7 μg of this liposome was added to 1 × 10 ⁵ NIH3T3 cells grown to confluence in DMEM medium and incubated for 1 hour or 3 hours. 30 minutes before observation with a confocal laser microscope, a lysosensor (Lysosenser, manufactured by Invitrogen) was added to 2 μM, and further incubated at 37 ^° C. for 30 minutes. After this, as a result of observing the intracellular localization of the liposome with a confocal laser microscope, dots were observed where the lysosensor (colored green) and the rhodamine signal (colored red) overlapped for 1 hour and 3 hours. However, many rhodamines alone (red only) were observed (FIG. 12). From this fact, it was shown that this peptide was transferred from the endosome to the cytoplasmic fraction.

3) Confirmation of liposome uptake route Lipid with the composition of phosphatidylcholine (EPC) / cholesterol (Chol) = 7: 3, equivalent to 5% of total lipid 4) Lipid IRQ-Mal-PEG-DSPE prepared in 4) (PEG is PEG2000) and Rho-DOPE (Avanti) corresponding to 1 mol% of the total lipid were added to prepare Rho-DOPE-labeled liposomes (IRQ-Lip) by the simple hydration method. In addition, as a comparative control, a peptide in which cysteine was added to the C-terminus of octaarginine (R8 peptide) instead of the IRQ peptide was used, and lipid R8-Mal-PEG-DSPE (PEG is PEG2000) according to the method described in 4) above In addition, R8-Mal-PEG-DSPE was used instead of the lipid IRQ-Mal-PEG-DSPE, and liposomes (R8-Lip) labeled with Rho-DOPE were prepared according to the method described above.

  In addition, liposomes (STR-IRQ-Lip) labeled with Rho-DOPE using IRQ peptide (STR-IRQ) with stearic acid added instead of lipid IRQ-Mal-PEG-DSPE were converted to lipid IRQ-Mal- Using R8 peptide (STR-R8-Lip) with stearic acid added instead of PEG-DSPE, liposomes labeled with Rho-DOPE (STR-R8-Lip) were prepared according to the above method.

Add 2 x 10 ⁵ NIH3T3 cells to 2 mL of DMEM medium / 3.5 cm plastic plate dish (3.5 cm) containing 10% fetal calf serum, let stand for one day, remove the medium, and add 1 mL of cells. Washed with PBS. The washed cells were suspended in serum-free DMEM medium, and 5 mM amiloride, 0.4 M sucrose, 1 μg / mL filipin III were added thereto, and the cells were incubated at 37 ° C. for 1 hour. The various liposomes shown above were added separately so that the final amount of Rho-DOPE was 0.05 mM / dish, and further incubated at 37 ° C. for 1 hour. The cells were washed once with PBS, three times with ice-cold PBS containing 20 units / mL heparin, and once with Krebs buffer. After staining the cell nuclei suspended in Krebs buffer with Syto-24 (final concentration 0.5 μM), the cells were observed with a confocal microscope.

  The results are shown in the table below. ++ indicates that significant inhibition of liposome uptake was observed, + indicates that weak uptake inhibition was observed, and − indicates that almost no inhibition of uptake was confirmed.

  Amiloride and sucrose are inhibitors of macropinocytosis and clathrin-mediated endocytosis, and filipin III is an inhibitor of endocytosis via caveolae. It became clear that the uptake to Caveola was done via Caveola.

Example 3 Preparation of liposomes encapsulating siRNA 1) Anti-luciferase siRNA capable of suppressing the expression of luciferase (McCaffrey et al., Nature, 2002, 418, pp. 38-39) at a concentration of 0.5 μg / μL The aqueous solution (18 μL) contained in 1 was gradually added dropwise to 2 μg / μL STR-R8 aqueous solution (9 μL) to prepare a siRNA core (N / P ratio was 2.9). A mixed solution of DOPE (Avanti) and CHEMS (Sigma) dissolved in chloroform at 9: 2 was placed in a glass tube, dried, and charged positively containing the lipid inclusion body (final lipid concentration 0.05 mM). 0.25 mL of water was added and sonicated for 1 minute to prepare IRQ-MEND1 (multi functional envelop type nano device consisting of DOPE and CHEMS containing siRNA and STR-IRQ). In addition, the above operation was performed on a mixed solution of PA (Sigma) and CHEMS dissolved in chloroform at 2: 7, and IRQ-MEND2 (multifunctional envelop type nano device consisting of PA and CHEMS including siRNA and STR-IRQ) ) Was produced.

2) The final amount of diRNA of each MEND prepared in 1) is 0.4 μg for Hela cells stably expressing luciferase (5 × 10 ⁴ cells / (DMEM medium containing 10% fetal bovine serum)). In addition, the cells were transformed with lipofectamine 2000 (Invitrogen). After 3 hours, 1 mL of DMEM medium containing serum was added and cultured at 37 ° C. for 24 hours, and then the luciferase activity of each cell was measured (FIG. 13).

  Expression suppression (%) was confirmed when the luciferase activity in the control cells was taken as 100. As a result, 50% of IRQ-MEND and 30% of IRQ-MEND2 were suppressed. The lipid membrane of IRQ-MEND2 was replaced with a bilayer membrane according to the method of Nakamura et al. (Nakamura T et al., Biol Pharm. Bull., 2006, Vol. 29, No. 6, pp. 1290-1293). The suppression of expression increased to 52%.

The change in the titer of phage recovered from skeletal muscle by the phage display method is shown. The change in the titer of phage recovered from adipose tissue by the phage display method is shown. The Frequency Value for 96 types of peptides exhibited by phage recovered from skeletal muscle is shown. The Frequency Value for 96 types of peptides exhibited by phage recovered from adipose tissue is shown. The titer of the phage collect | recovered by the phage display method using the phage which displays the peptide of Table 3 is shown. The remaining amount of the liposome in the blood 24 hours after the injection of the liposome of the present invention from the tail vein is shown. The relationship between the content of IRQ-Mal-PEP-DSPE in the liposome of the present invention and the ability of the liposome to migrate to skeletal muscle is shown. The relationship between the content of IRQ-Mal-PEP-DSPE and the ability of liposomes to migrate to adipose tissue in the liposome of the present invention is shown. The relationship between the content of IRQ-Mal-PEP-DSPE and the ability of liposomes to migrate to adipose tissue in the liposome of the present invention is shown. The relationship between the content of IRQ-Mal-PEP-DSPE and the ability to migrate to lung tissue in the liposome of the present invention is shown. The relationship between the content of Mal-PEP-DSPE and the ability to migrate to lung tissue in the liposome of the present invention is shown. The color development of rhodamine and sisosensor in fibroblasts incorporating the liposome of the present invention is shown. It is a graph which shows the inhibitory effect of luciferase expression by the liposome which enclosed siRNA.

Claims (15)

Hepta having 3 to 6 basic amino acid residues and at least one hydrophilic amino acid residue other than a hydrophobic amino acid residue or a basic amino acid and having a migration ability specific to adipose tissue or skeletal muscle peptide. The heptapeptide according to claim 1, wherein the hydrophobic amino acid is leucine, isoleucine or proline. The heptapeptide according to claim 1, wherein the hydrophilic amino acid is glutamine, threonine, methionine, serine or asparagine. The heptapeptide according to claim 1, wherein the amino acid sequence is IRQRRRR. The liposome which the heptapeptide in any one of Claims 1-4 couple | bonded. The liposome according to claim 5, which has a migration ability specific to adipose tissue. The liposome according to claim 5, which has an ability to migrate into cells via caveolae. The liposome according to claim 5, comprising a lipid to which a heptapeptide is bound. The liposome according to claim 8, wherein the heptapeptide comprises a lipid bound via a cysteine residue added to the heptapeptide and a maleimidized hydrophilic polymer. Furthermore, the liposome of Claim 8 or 9 containing the lipid couple | bonded with the hydrophilic polymer which the heptapeptide has not couple | bonded. The liposome according to any one of claims 8 to 10, comprising 5 to 10 mol% of lipid to which heptapeptide is bound per total amount of lipid constituting the liposome. The liposome according to claim 10, comprising 5 to 10 mol% of a lipid bound to a hydrophilic polymer to which heptapeptide is not bound, based on the total amount of lipid constituting the liposome. The liposome according to any one of claims 9 to 12, wherein the hydrophilic polymer is polyalkylene glycol. Intracellular via caveolae by binding a heptapeptide containing 3 to 6 basic amino acid residues and a hydrophobic amino acid residue or at least one hydrophilic amino acid residue other than basic amino acid residues to liposomes A method for producing liposomes having migration ability. The method according to claim 14, wherein the amino acid sequence of the heptapeptide is IRQRRRR.