Composition to be delivered to posterior ocular part

Abstract

To provide an effective DDS for a posterior ocular part which realizes posterior ocular disease treatment by instillation.SOLUTION: A liposome for delivering a drug to a posterior ocular part by instillation which is a preparation having an average particle diameter of 600nm or less contains the following (A) to (D): (A) one or more kinds phospholipid selected from the group consisting of distearoyl phosphatidylcholine, dimyristolyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, egg-yolk lecithin and hydrogenated product thereof; (B) a compound having one or more kinds of steroid skeleton selected from the group consisting of cholesterol, cholesterol ester, cholestanol and dehydro cholesterol; (C) one kinds of charged substance selected from the group consisting of stearylamine, didodecyl dimethylammonium bromide and dicetyl phosphate; and (D) surface modifier selected from the group consisting of folic acid derivative, octaarginine, polylysine and intracellular penetrating peptide.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a composition for efficiently delivering a drug such as a nucleic acid to the posterior segment of the eye without the intervention of medical technology, a pharmaceutical composition (particularly an eye drop) comprising said composition and a drug, and the treatment and prevention of a posterior segment disease using said pharmaceutical composition. [Background technology]

[0002] Known intractable posterior segment diseases include age-related macular degeneration, retinal vein occlusion, retinitis pigmentosa, proliferative vitreoretinopathy, diabetic retinopathy, etc. Medical technology interventions such as laser photocoagulation (PDT) and vitreous surgery have traditionally been used to treat these diseases.

[0003] Regarding drug therapy, anti-inflammatory drugs and steroids have been used. In recent years, anti-vascular endothelial growth factor (VEGF) drugs such as pegaptanib (Macugen (registered trademark)), ranibizumab (Lucentis (registered trademark)), bevacizumab (Avastin (registered trademark)), and aflibercept (Eylea (registered trademark)) have been developed and their effectiveness has been demonstrated. However, even if these macromolecular bioactive substances are administered by eye drop, they are not delivered to the posterior segment of the eye where the retina is located due to barrier mechanisms such as the tear film and aqueous humor excretion. Therefore, at present, the administration method of these drugs is limited to intravitreal injection, which is highly invasive. Although embedded steroid-containing preparations (implants, etc.) have also been developed, they still require medical technology intervention, and the problem of invasiveness has not been solved.

[0004] Liposomal preparations are widely used as carriers for delivering macromolecules, such as antibodies and other proteins and nucleic acids, to target cells, and they have also been investigated as drug delivery systems (DDS) for the posterior segment of the eye (Non-Patent Documents 1 and 2). However, their effectiveness is not sufficient, and no preparations capable of delivering drugs to the posterior segment by instillation have yet been commercially available. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Int. J. Mol. Sci., 18: 2076 (2017) [Non-Patent Document 2] Yakugaku Zasshi, 132(12): 1365-1370 (2012) Summary of the Invention [Problem to be solved by the invention]

[0006] As mentioned above, any treatment for posterior segment disease: 1) It is highly invasive and there is concern about reduced compliance; 2) Drug therapy by intravitreal injection places a heavy burden on patients due to the fear of direct injection into the eye, the need for frequent administration, and the possibility of side effects such as endophthalmitis and retinal detachment; 3) current treatments do not restore retinal function or vision, and cannot restore lost nerve cells; 4) Conventional long-acting injections and implants require medical technology intervention and are expensive, raising concerns about medical economics; Due to these problems, there remains an unmet medical need for drugs to treat posterior segment diseases. Therefore, an object of the present invention is to provide an effective DDS for the posterior segment of the eye, which enables treatment of posterior segment diseases by instillation. [Means for solving the problem]

[0007] In order to achieve the above object, the present inventors have used a model polymer FITC-dextran (FD-10) with a molecular weight of about 10,000 to intensively study a liposome formulation capable of delivering the drug to the posterior segment of the eye by instillation. As a result, it has been revealed that by optimizing the lipid composition and particle size, the drug can be efficiently delivered to the posterior segment of the eye by instillation. In addition, by modifying the surface of the liposome with a cationic polymer, a cell membrane-permeable peptide, a folate-binding lipid derivative, etc., it has been successfully improved the delivery to the retinal site. In particular, it has been confirmed that the persistence of nucleic acid delivery is improved by applying the surface modification. Furthermore, it has been revealed that the characteristics of the liposome particles and the nucleic acid need to be matched when applying the actual nucleic acid. In addition, it has been revealed that the liposome formulation actually suppresses retinal edema using a mouse model of posterior segment pathology (RVO). Based on these findings, the present inventors conducted further research and have completed the present invention.

[0008] That is, the present invention is as follows. [1] (A) to (D) below: (A) one or more phospholipids selected from the group consisting of distearoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, egg yolk lecithin, and hydrogenated products thereof; (B) a compound having one or more steroid skeletons selected from the group consisting of cholesterol, cholesterol ester, cholestanol, and dehydrocholesterol; (C) one charged substance selected from the group consisting of stearylamine, didodecyldimethylammonium bromide, and dicetyl phosphate; and (D) a surface modifier selected from the group consisting of folic acid derivatives, octaarginine, polylysine, and cell-penetrating peptides; A liposome preparation for delivering a drug to the posterior segment of the eye by instillation, comprising the component (a) above, wherein the preparation has an average particle size of 600 nm or less. [2] The liposome preparation according to [1], having an average particle size of 200 nm or less. [3] The liposome formulation according to [1] or [2], wherein component (D) is a folic acid derivative, octaarginine or a cell-penetrating peptide. [4] The liposome preparation according to any one of [1] to [3], which has a surface potential of +20 to +60 mV. [5] The liposome formulation according to any one of [1] to [4], wherein the drug is a nucleic acid. [6] A preventive or therapeutic agent for a posterior segment disease, comprising the liposome preparation according to any one of [1] to [4] encapsulating a polymer effective for preventing or treating a posterior segment disease. [7] The agent according to [6], wherein the polymer is a nucleic acid. [8] The agent described in [7], wherein the nucleic acid is an siRNA, a single-stranded nucleic acid or a modified nucleic acid. [9] The agent according to any one of [6] to [8], which is an eye drop. Effect of the Invention

[0009] According to the liposome preparation of the present invention, a macromolecular physiologically active substance such as a nucleic acid can be efficiently delivered to the posterior segment of the eye by instillation, and therefore a non-invasive and effective therapeutic agent for posterior segment diseases can be provided. [Brief description of the drawings]

[0010] [Figure 1] Figure 1 shows the delivery of FD-10 to the inner plexiform layer of mice by instillation of FD-10-encapsulated PLA-modified liposomes (mean ± SEM, n = 7-9). * P < 0.05 versus untreated, †† P < 0.01 versus FD-10 solution. [Diagram 2] Figure 2 shows the retinal delivery of siRNA by eye drop administration of siRNA-encapsulated unmodified liposomes and folic acid (FA)-modified liposomes. Figure 2A shows an epifluorescence microscopy image of the retina 30 minutes after eye drop administration. Figure 2B shows the relative fluorescence intensity in the inner plexiform layer 30 minutes after eye drop administration. [Diagram 3]Figure 3 shows the retinal delivery of Bonac nucleic acid by instillation of unmodified (SA-containing) liposomes encapsulating Bonac nucleic acid into mice. Figure 3A shows an epifluorescence microscopic image of the retina 30 minutes after instillation (scale bar: 50 μm). Figure 3B shows the relative fluorescence intensity in the inner plexiform layer 30 minutes after instillation (mean ± SEM, n = 7-8). ** P < 0.01 versus siRNA solution. [Figure 4] Figure 4 shows the retinal delivery of Bonac nucleic acid by instillation of Bonac nucleic acid-encapsulated R8-modified liposomes into mice. Figure 4A shows a fluorescent microscopic image of the retina 30 minutes after instillation (scale bar: 50 μm). Figure 4B shows the relative fluorescence intensity in the inner plexiform layer 30 minutes after instillation (mean ± SEM, n = 7-8). [Diagram 5] Figure 5 shows a fluorescent microscopy image of the retina 30 minutes after instillation of Bonac nucleic acid-encapsulated unmodified (SA-containing) liposomes and R8-modified liposomes into a retinal vein occlusion (RVO) model mouse, showing the retinal delivery of Bonac nucleic acid (scale bar: 50 μm). [Figure 6] Figure 6 shows the therapeutic effect of eye drop administration of modified liposomes encapsulating nucleic acids with anti-VEGF activity to RVO mice. The group administered VEGF siRNA-encapsulated folic acid (FA)-modified liposomes suppressed thickening of the inner granular layer compared to the group administered vehicle solution (n=6-9) (Figure 6A). In addition, the group administered Bonac nucleic acid-encapsulated R8-modified liposomes suppressed thickening of the inner granular layer compared to the group administered vehicle solution (Figure 6B). Lipid composition: egg yolk lecithin (EPC) / Chol.=7 / 3, lipid concentration: 20.4 mM, R8 concentration: 2.5 mol% DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention will be described in detail below. In addition, the terms used in this specification can be used in the sense generally used in the technical field unless otherwise specified.

[0012] 1. Liposomal preparation of the present invention The present invention provides a liposome preparation for delivering a drug to the posterior segment of the eye by instillation (hereinafter, may be referred to as the "liposome preparation of the present invention.") The liposome preparation of the present invention contains the following components (A) to (D):

[0013] (A) Phospholipids The phospholipid used in the liposome preparation of the present invention is not particularly limited as long as it is medicamentously acceptable, and examples thereof include phosphatidylcholine (e.g., dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, etc.), phosphatidylglycerol (e.g., dioleoylphosphatidylglycerol, dilauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, etc.), and the like. oleyl phosphatidylethanolamine (e.g., dioleoyl phosphatidylethanolamine, dilauroyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dioleoyl glycerophosphoethanolamine, etc.), phosphatidylserine, phosphatidylinositol, phosphatidic acid, cardiolipin, egg yolk, soybean and other natural lipids derived from animals and plants (e.g., egg yolk lecithin, soybean lecithin, etc.), or hydrogenated products thereof. Preferred examples include phosphatidylcholine (e.g., distearoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, etc.), egg yolk lecithin, or hydrogenated products thereof. Only one of these phospholipids may be used, or two or more of them may be used in combination.

[0014] (B) Sterol The compound (sterol) having a steroid skeleton used in the liposome preparation of the present invention is not particularly limited as long as it is medicamentously acceptable, and examples thereof include cholesterol, its fatty acid ester (cholesterol ester), cholestanol (dihydrocholesterol), dehydrocholesterol, cholesterol succinate, lanosterol, dihydrolanosterol, desmosterol, and other animal-derived sterols, stigmasterol, sitosterol, campesterol, brassicasterol, and other plant-derived sterols (phytosterols), zymosterol, ergosterol, and other microorganism-derived sterols. Preferred examples include cholesterol, cholesterol ester, cholestanol, and dehydrocholesterol. These sterols may be used alone or in combination of two or more.

[0015] The blending ratio (molar ratio) of component (A) to component (B) in the liposome preparation of the present invention is not particularly limited as long as it does not impair the drug deliverability of the liposome preparation of the present invention to the posterior segment of the eye. For example, when component (A) is a phospholipid composed of a saturated fatty acid, (A) / (B) is, for example, less than 8, preferably less than 5, more preferably 2 to 4, even more preferably 2 to 3, and particularly preferably 2 to 2.5.

[0016] (C) Charged substances The liposome preparation of the present invention is characterized by further containing a charged substance. The charged substance has an opposite charge to that of the drug to be encapsulated, and thus enhances the interaction between the drug and the liposome through electrostatic interaction, thereby improving the encapsulation rate of the drug. For example, when the drug is a nucleic acid, a positively charged charged substance can be added since the nucleic acid is negatively charged. Examples of the positively charged substance include fatty acid amides such as stearylamine and oleylamine, didodecyldimethylammonium bromide, dioctadecyldimethylammonium chloride, N-(2,3-oleyloxy)propyl-N,N,N-trimethylammonium, didodecylammonium bromide, 1,2-dioleyloxy-3-trimethylammonium propane, 3β-N-(N',N'-dimethylaminoethane)carbamol cholesterol, 1,2-dimyristoyloxypropyl-3-dimethylhydroxyethylammonium, 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propane ammonium trifluoroacetate, and other cationic lipids. Preferred are stearylamine and didodecyldimethylammonium bromide. On the other hand, when the drug has a positive charge, a negatively charged substance can be added. Examples of the negatively charged substance include dicetyl phosphate.

[0017] The blending ratio (molar ratio) of component (C) in the liposome preparation of the present invention is not particularly limited as long as it does not impair the drug deliverability of the liposome preparation of the present invention to the posterior segment of the eye. For example, the ratio of the sum of components (A) and (B) to component (C) is 10:1 to 30:1, preferably 15:1 to 25:1, more preferably 18:1 to 25:1, and particularly preferably 18:1 to 22:1.

[0018] (D) Surface Modifier The liposome preparation of the present invention is characterized by further containing a surface modifier. Examples of the surface modifier include substances having affinity with molecules (e.g., folate receptors, heparan sulfate proteoglycans, etc.) expressed on the surface of target cells in the posterior segment of the eye, such as the retina, choroid, sclera, and optic nerve. Specific examples of the surface modifier include folic acid derivatives, polymers of basic amino acids such as polyarginine and polylysine, and cell-penetrating peptides. Examples of the folic acid derivatives include, but are not limited to, FA-PEGylation reagents in which the end of a linker such as polyethylene glycol (e.g., molecular weight 1000 to 20000) is modified with folic acid. Polylysine and polyarginine are not particularly limited as long as they have a molecular weight of 500 or more, and in the case of polylysine, examples include polylysines having a molecular weight distribution of 500 to 2000, 5000 to 15000, 15000 to 30000, and 30000 to 70000, and preferably polylysines having a molecular weight distribution of 5000 to 15000, 15000 to 30000, and 30000 to 70000. Polyarginine having a similar molecular weight can also be used, and polyarginine having a molecular weight of 500 to 2000, such as octaarginine, can also be preferably used.Examples of cell-penetrating peptides include AntP derived from Drosophila, TAT derived from HIV (Frankel, A. et al., Cell 55, 1189-93 (1988); Green, M. & Loewenstein, PM Cell 55, 1179-88 (1988)), Penetratin (Derossi, D. et al., J. Biol. Chem. 269, 10444-50 (1994)), Buforin II (Park, CB et al., Proc. Natl Acad. Sci. USA 97, 8245-50 (2000)), Transportan (Pooga, M. et al., FASEB J. 12, 67-77 (1998)), and MAP (model amphipathic peptide) (Oehlke, J. et al., Biochim. Biophys. Acta. 1414, 127-39(1998)), K-FGF(Lin, YZ etal. J. Biol. Chem. 270, 14255-14258(1995)), Ku70(Sawada, M. et al. Nature CellBiol. 5, 352-7(2003)), Prion(Lundberg, P. et al. Biochem. Biophys. Res. Commun. 299, 85-90(2002)), pVEC (Elmquist, A. et al. Exp. Cell Res. 269, 237-44(2001)), Pep-1 (Morris, MC et al. Nature Biotechnol. 19, 1173-6(2001)), Pep-7 (Gao, C. et al. Bioorg. Med. Chem. 10, 4057-65(2002)), SynBl (Rousselle, C. et al. MoI. Pharmacol. 57, 679-86(2000)), HN-I (Hong, FD & Clayman, G L. Cancer Res. 60, 6551-6(2000)), and those using the cell passage domain of proteins such as VP22 derived from HSV, but are not limited to these.These surface modifiers may be used alone or in combination of two or more. The modification of liposomes with a surface modifier can be carried out by chemically binding the above-mentioned surface modifier to the surface of prepared liposomes by a method known per se, or by mixing the surface modifier previously bound to a lipid with the liposomes.

[0019] The blending ratio (molar ratio) of component (D) in the liposome preparation of the present invention is not particularly limited as long as it does not impair the drug delivery of the liposome preparation of the present invention to the posterior segment of the eye, and is, for example, 0.01 to 10 mol %, preferably 0.1 to 1 mol %, as a molar fraction relative to the entire preparation. Alternatively, the blending amount of component (D) in the liposome preparation of the present invention can be adjusted so that the surface potential of the liposome after surface modification is +20 to 60 mV.

[0020] The liposome preparation of the present invention may further contain, in addition to the components (A) to (D), for example, glycolipids (e.g., glyceroglycolipids such as sphingomyelin, sulfoxyribosylglyceride, diglycosyldiglyceride, digalactosyldiglyceride, galactosyldiglyceride, and glycosyldiglyceride, sphingoglycolipids such as galactosylcerebroside, lactosylcerebroside, and ganglioside), long-chain fatty acids or long-chain aliphatic alcohols (e.g., fatty acids having 10 to 20 carbon atoms or alcohols thereof (e.g., palmitic acid, stearic acid, lauric acid, may contain saturated fatty acids such as myristic acid, pentadecylic acid, arachidic acid, margaric acid, and tuberculin stearic acid; unsaturated fatty acids such as palmitoleic acid, oleic acid, arachidonic acid, vaccenic acid, linoleic acid, linolenic acid, arachidonic acid, and eleostearic acid; oleyl alcohol, stearyl alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol, and linolyl alcohol; and glycerin fatty acid esters (e.g., monoacylglycerides, diacylglycerides, and triacylglycerides).

[0021] The liposome of the present invention may be a single-layer membrane or a multi-layer membrane, but is preferably a single-layer membrane. The average particle size of the liposome preparation of the present invention is not particularly limited as long as it does not impair the drug delivery of the liposome preparation of the present invention to the posterior segment of the eye, but is, for example, less than 1 μm, preferably 600 nm or less, more preferably 400 nm or less, and even more preferably 200 nm or less. The lower limit of the average particle size is also not particularly limited, but is, for example, 10 nm or more, preferably 50 nm or more, and more preferably 100 nm or more. Therefore, the preferred range of the average particle size of the liposome preparation of the present invention is, for example, 10 to 600 nm, more preferably 50 to 400 nm, even more preferably 50 to 200 nm, and particularly preferably 100 to 200 nm.

[0022] The dispersion of the liposome preparation of the present invention can be prepared, for example, by mixing components (A) to (C) and dispersing them in an aqueous solution by a conventional method. An ultrasonic dispersion device, an emulsification dispersion device, or the like can be appropriately used for dispersion. The obtained dispersion can be passed through a filter having an appropriate pore size using, for example, an extruder, to adjust the average particle size of the liposomes to a desired value.

[0023] 2. Pharmaceutical Compositions The present invention also provides an agent for preventing or treating posterior ocular diseases (hereinafter also referred to as "pharmaceutical composition of the present invention"), which comprises the liposome preparation of the present invention encapsulating a polymer effective for preventing or treating posterior ocular diseases. In the present specification, when referring to "encapsulation", the nucleic acid molecule of the present invention may be retained in the liposome internal phase or in the lipid bilayer. In the present specification, "prevention" includes not only inhibition of onset but also delay of onset, and "treatment" is used as a concept including not only complete cure but also alleviation of symptoms and inhibition of progression.

[0024] The "posterior ocular disease", which is a target disease of the pharmaceutical composition of the present invention, generally means a disease in the vitreous body, retina, choroid, sclera, or optic nerve, and examples thereof include, but are not limited to, age-related macular degeneration, diabetic retinopathy, diabetic macular edema, retinal vein occlusion, uveitis, etc.

[0025] The active ingredient contained in the pharmaceutical composition of the present invention is not particularly limited as long as it is a polymeric bioactive substance effective for the prevention or treatment of posterior ocular diseases, but since many posterior ocular diseases are deeply related to the expression of neovascularization, examples of the polymeric bioactive substance include polymers having anti-VEGF activity. Examples of polymers having anti-VDGF activity include nucleic acids that suppress the expression of VFGF, such as siRNA, shRNA, and miRNA against VDGF, anti-VEGF antibodies or fragments thereof, VEGF antagonists containing the VFGF-binding domain of the VEGF receptor, and proteins or nucleic acids that suppress the function of VEGF, such as aptamers that bind to VEGF or its receptor. In a preferred embodiment, the drug encapsulated in the liposome preparation of the present invention is a nucleic acid, more specifically, a nucleic acid molecule having 10 to 100 nucleotides in one molecule or a derivative thereof, which exerts a physiologically active effect in cells of the posterior segment of the eye, such as cells of the retina (e.g., retinal pigment epithelial cells), and inhibits the function of the target DNA, RNA, or proteins that are its expression products. Preferred examples of such a structure include oligonucleic acids, which are single-stranded nucleic acid molecules, or nucleic acid molecules containing double or multiple strands in one molecule, or combinations thereof (e.g., double-stranded nucleic acid molecules formed from two single-stranded nucleic acid molecules).

[0026] The nucleotides constituting the oligonucleic acid used in the present invention may be DNA, RNA, DNA / RNA chimera, or derivatives or analogs thereof. Specific examples of oligonucleic acids include, but are not limited to, siRNA, shRNA, microRNA, antisense nucleic acid, ribozyme, and aptamer.

[0027] In another preferred embodiment, the oligonucleic acid includes, but is not limited to, "single-stranded nucleic acid molecules comprising an expression suppression sequence that suppresses the expression of a target gene" as described in, for example, WO 2012 / 017919, WO 2013 / 103146, WO 2012 / 005368, WO 2012 / 077446, WO 2013 / 133393, etc.

[0028] The oligonucleic acid used in the present invention is not limited to the natural type, and at least a part of the sugar or phosphate backbone constituting the nucleotide may be modified in order to increase stability in the body, such as nuclease resistance. When modified, desirable modifications include modification of the 2'-position of the sugar, modification of other parts of the sugar, modification of the phosphate backbone of the oligonucleic acid, etc. Examples of modifications of the 2'-position of the sugar include substituents such as OR, R, R'OR, SH, SR, NH2, NHR, NR2, N3, CN, F, Cl, Br, and I. Here, R represents an alkyl or aryl, preferably an alkyl group having 1 to 6 carbon atoms, and R' represents an alkylene, preferably an alkylene having 1 to 6 carbon atoms. Examples of modifications of other parts of the sugar include 4'thio bodies. Examples of modifications of the phosphate backbone of the oligonucleic acid include phosphorothioates, phosphorodithioates, alkylphosphonates, and phosphoramidates.

[0029] Oligonucleic acids can be synthesized in solid phase or liquid phase by the well-known phosphoramidite method or triester method. The most common mode is solid phase synthesis by the phosphoramidite method, which can be synthesized by an automatic nucleic acid synthesizer or manually. After the solid phase synthesis is completed, the nucleic acid is removed from the solid phase, the protecting group is deprotected, and the target product is purified. By purification, nucleic acids with a purity of 90% or more, preferably 95% or more, can be obtained.

[0030] The pharmaceutical composition of the present invention can be prepared by forming a complex between the above-mentioned nucleic acid, preferably an oligonucleic acid, and the above-mentioned liposome of the present invention.

[0031] Since the liposome of the present invention has a charged substance (component (C)), by appropriately selecting the charged substance, it can easily form a complex with an active ingredient having an opposite charge. For example, when the active ingredient is a negatively charged nucleic acid, a positively charged substance can be blended as component (C). Thus, the pharmaceutical composition of the present invention can be produced by first dispersing components (A) to (C) in an aqueous solvent to form liposomes, then adding nucleic acid and dispersing again, or by dispersing components (A) to (C) and nucleic acid in an aqueous solvent, passing the resulting dispersion through a filter having an appropriate pore size as described above to form liposomes with a desired average particle size, and then surface-modifying with component (D) as described above. Alternatively, active ingredients (e.g., nucleic acids) can be encapsulated in liposomes by known methods such as the lipid film method (vortex method), reverse phase evaporation method, surfactant removal method, freeze-thaw method, and remote loading method.

[0032] Examples of the aqueous solvent used for forming the complex include water for injection, distilled water for injection, electrolyte solutions such as physiological saline, and sugar solutions such as glucose solution and maltose solution.

[0033] Examples of the dispersion treatment include homomixers, homogenizers, ultrasonic dispersers, ultrasonic homogenizers, high-pressure emulsifying dispersers, Microfluidizers (trade name), Nanomizers (trade name), Ultimizers (trade name), DeBEE2000 (trade name), and Manton-Gaulin high-pressure homogenizers. Treatment conditions, treatment time, treatment temperature, and the like can be appropriately selected. The dispersion treatment can also be carried out in several stages, such as through a coarse dispersion.

[0034] The pharmaceutical composition of the present invention can also be formulated with any carrier, for example, a pharma- ceutically acceptable carrier. Examples of pharma-ceutically acceptable carriers include, but are not limited to, excipients such as sucrose and starch, binders such as cellulose and methylcellulose, disintegrants such as starch and carboxymethylcellulose, lubricants such as magnesium stearate and aerosil, flavors such as citric acid and menthol, preservatives such as sodium benzoate and sodium hydrogen sulfite, stabilizers such as citric acid and sodium citrate, suspending agents such as methylcellulose and polyvinylpyrrolidone, dispersing agents such as surfactants, diluents such as water and physiological saline, and base waxes.

[0035] The pharmaceutical composition of the present invention can be administered to mammals locally through the eyes, and is particularly preferably administered by instillation into the eyes.

[0036] Examples of preparations suitable for topical administration to the eye include eye drops (aqueous eye drops, non-aqueous eye drops, suspension eye drops, emulsion eye drops, etc.), ointments, lotions, creams, etc. When the agent of the present invention is an eye drop, a suitable base material can be used. Examples of base materials used for eye drops include phosphate buffer, Hanks' buffer solution, physiological saline, irrigation solution, artificial tears, etc.

[0037] For example, buffering agents, isotonicity agents, solubilizing agents, preservatives, viscosity bases, chelating agents, cooling agents, pH adjusters, antioxidants and the like may be appropriately selected and added to the pharmaceutical composition of the present invention. Examples of the buffer include a phosphate buffer, a borate buffer, a citrate buffer, a tartrate buffer, an acetate buffer, and an amino acid. Examples of the isotonic agent include sugars such as sorbitol, glucose, and mannitol; polyhydric alcohols such as glycerin and propylene glycol; salts such as sodium chloride; and boric acid. Examples of solubilizing agents include nonionic surfactants such as polyoxyethylene sorbitan monooleate (eg, polysorbate 80), polyoxyethylene hydrogenated castor oil, tyloxapol, and Pluronic, and polyhydric alcohols such as glycerin and macrogol. Examples of preservatives include quaternary ammonium salts such as benzalkonium chloride, benzethonium chloride, and cetylpyridinium chloride; paraoxybenzoic acid esters such as methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, and butyl paraoxybenzoate; benzyl alcohol, sorbic acid and its salts (sodium salt, potassium salt, and the like); thimerosal (trade name), chlorobutanol, and sodium dehydroacetate. Examples of the viscous base include water-soluble polymers such as polyvinylpyrrolidone, polyethylene glycol, and polyvinyl alcohol; and celluloses such as hydroxyethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, and sodium carboxymethyl cellulose. Chelating agents include sodium edetate and citric acid. Cooling agents include l-menthol, borneol, camphor, eucalyptus oil, and the like. Examples of pH adjusters include sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, boric acid or its salts (borax), hydrochloric acid, citric acid or its salts (sodium citrate, sodium dihydrogen citrate, etc.), phosphoric acid or its salts (disodium hydrogen phosphate, potassium dihydrogen phosphate, etc.), acetic acid or its salts (sodium acetate, ammonium acetate, etc.), and tartaric acid or its salts (sodium tartrate, etc.). Antioxidants include, for example, sodium bisulfite, dry sodium sulfite, sodium pyrosulfite, concentrated mixed tocopherols, and the like.

[0038] The molar ratio of the active ingredient (e.g., nucleic acid) to the liposome constituents in the pharmaceutical composition of the present invention is usually 1 / 100,000 to 1 / 1,000. The amount of liposomes encapsulating the active ingredient contained in the liposome preparation is not particularly limited as long as it is an amount that does not cause the liposome particles to aggregate and can exert a sufficient medicinal effect, and is usually 10 to 100 mM.

[0039] The dosage of the pharmaceutical composition of the present invention will vary depending on the method of administration, the type and severity of the posterior ocular disease, and the condition of the recipient (sex, age, body weight, etc.); for example, when a nucleic acid having anti-VEGF activity is administered by eye drop administration to an adult, it is generally desirable to administer a single dose of 0.01 to 1000 μg, preferably 0.05 to 100 μg, and more preferably 0.1 to 50 μg of the nucleic acid once to 10 times a day, and preferably 5 to 10 times a day.

[0040] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these. EXAMPLES

[0041] Reference Example 1 Preparation of liposomes encapsulating a polymer model substance (FITC-labeled dextrin with a molecular weight of approximately 10,000; FD-10) and evaluation of surface modification and retinal delivery (1) Preparation of FD-10 (FD)-encapsulated liposomes FD-encapsulated multilamellar liposomes (MLVs) were prepared by the thin film hydration method. Distearoylphosphatidylcholine (DSPC, Nippon Oil & Fats) and cholesterol (Chol., Sigma) were weighed into a recovery flask, dissolved in an appropriate amount of chloroform, and the solvent was removed under reduced pressure in a 40°C water bath using a rotary evaporator to prepare a thin film. After drying overnight under reduced pressure, the thin film was hydrated using FD solution dissolved in Milli-Q in a 70°C water bath and incubated at 10°C for 30 minutes to prepare FD-encapsulated MLVs. The prepared FD-encapsulated MLV was passed through a filter (Nuclepore (registered trademark) Track-Etch Membrane, Whatman) with a pore size of 100 nm 41 times at a pressure of 150 to 200 kPa using an extruder (LiposoFastTM-Pneumatic, AVESTIN) to prepare liposomes (ssLip) finely divided into submicron sizes. The prepared ssLip was then frozen in a prefreezer at -60°C, thawed at 40°C, and subjected to ultrasonic treatment in four cycles. The FD-encapsulated ssLip was mixed with an equal amount of PLA Poly-L-arginine (PLA, Sigma) solution under stirring to prepare FD-encapsulated PLA-modified liposomes. The FD-encapsulated ssLip was mixed with an equal amount of HBSS-MES buffer (pH 6.0) that does not contain the polymer to prepare unmodified liposomes.

[0042] (2) The liposome suspension (ssLip) containing FD obtained in (1) above was administered to mice by eye dropwise, and the mouse eyeball was removed 30 minutes later. The removed eyeball was washed with a large excess of saline, immersed in 4% paraformaldehyde overnight to fix the tissue, and then immersed in 20% sucrose solution (in phosphate buffer) for 1 to 2 days. Then, the tissue was fixed in Cryomold No. 1 (Tissue-Tek (registered trademark) Cryomold, Sakura Finetech Japan Co., Ltd.) by adding a tissue freezing solution (Tissue-Tek (R) OCT Compound, Sakura Finetech Japan Co., Ltd.), and the eyeball was embedded by rapid freezing in liquid nitrogen. The embedded eyeball was horizontally cut using a Cryostat (Leica CM1850), and a 10 μm-thick slice was cut at the position where the optic nerve was exposed to prepare a section for observation. The sections were observed using an epifluorescence microscope (BX51, Olympus) with the focus on the retina at a position 500 μm from the optic nerve. The median density of a certain area (50 × 50 pixels) of the inner plexiform layer (IPL) in the obtained retinal images was quantified using ImageJ (National Institute of Mental Health). The mean fluorescence intensity was expressed as a value of 0-255, with the fluorescence intensity of the untreated eye being set to 1. The results are shown in Figure 1. In the FD-encapsulated ssLip administration group, fluorescence derived from FD-10 was observed in the retina. In addition, stronger fluorescence was confirmed in the PLA-modified ssLip administration group compared to the other groups.

[0043] Example 1 Preparation of liposomes encapsulating nucleic acids (siRNA or Bonac nucleic acid) and evaluation of their physical properties (1) Preparation of nucleic acid-encapsulating liposomes Folic acid-modified DY547-labeled siRNA-encapsulated liposomes were prepared by the thin film hydration method. DSPC, Chol., and stearylamine (SA) were weighed out in a recovery flask, dissolved in an appropriate amount of chloroform, and the solvent was removed under reduced pressure using a rotary evaporator on a 40°C water bath to prepare a thin film. After drying overnight under reduced pressure, the liposomes were hydrated in a 60°C water bath using a solution of DY547-labeled nucleic acid (vascular endothelial growth factor-small interfering RNA (VEGF-siRNA, Nippon Gene) or a single-stranded nucleic acid in which the guide strand and passenger strand of VEGF siRNA are linked by a non-nucleotide linker, a technology owned by Bonac Co., Ltd. (referred to as "Bonac nucleic acid" in this specification)) dissolved in Milli-Q, and incubated at 10°C for 30 minutes to prepare nucleic acid-encapsulated MLVs. The prepared nucleic acid-encapsulated MLVs were placed in an extruder (LiposoFast TM The liposomes were passed through a filter with a pore size of 100 nm (Nuclepore (registered trademark) Track-Etch Membrane, Whatman) 41 times at a pressure of 150 to 200 kPa using a suction pump (Fuji-Pneumatic, AVESTIN) to prepare submicron-sized liposomes (ssLip). The siRNA-encapsulated ssLip was prepared by mixing an equal amount of an aqueous dispersion of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-2000] (FA-PEG-DSPE, Avanti) with an aqueous dispersion of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-2000] (FA-PEG-DSPE, Avanti), and the Bonac nucleic acid-encapsulated ssLip was prepared by mixing an equal amount of an aqueous dispersion of Stearoyl-Octa-Arginine (St-R8, BEX Co., Ltd.) with an aqueous dispersion of 1,2-distearoyl-octa-arginine (St-R8, BEX Co., Ltd.), and incubating at 40°C for 1 hour to prepare siRNA-encapsulated folate-modified liposomes and Bonac nucleic acid-encapsulated R8-modified ssLip. The nucleic acid (siRNA or Bonac nucleic acid)-encapsulated ssLip was also mixed with an equal amount of HBSS-MES buffer (pH 6.0) or HBSS-MEPES buffer (pH 7.4) to prepare unmodified liposomes. (2) Evaluation of particle properties of siRNA-encapsulated liposomes The particle properties of the folic acid-modified DY547-siRNA-encapsulated liposomes are shown in Table 1. Liposomes were prepared with two different blend ratios (molar ratios) using DSPC / Chol. / SA as the lipid composition. siRNA was encapsulated in the liposomes by electrostatic interaction between the positive charge of SA and the negative charge of siRNA. The drug encapsulation rate was high at about 100% in both liposomes, and was improved compared to the case where FD was used as a model drug. This is thought to be because the interaction between the liposomes and siRNA was high due to electrostatic interaction. Therefore, it is suggested that the drug encapsulation rate when siRNA was used includes siRNA that was not encapsulated in the liposomes but interacted with the liposome surface and attached to it. The liposome composition with a DSPC / Chol. molar ratio of 7 / 3 had a smaller average particle size than the 8 / 1 liposome, and better particles were obtained. The increase in particle size is thought to be due to aggregation due to electrostatic interaction between the unencapsulated DY547-siRNA and the liposomes. It has been reported that phosphatidylcholine containing saturated fatty acids such as DSPC can eliminate the phase transition and increase the membrane fluidity of liposomes by adding Chol. In the case of a DSPC / Chol. molar ratio of 7 / 3, the increased Chol. content made it easier for DY547-siRNA to enter the inner aqueous phase of the liposome membrane, which is thought to be why the amount of unencapsulated DY547-siRNA decreased. In the case of folic acid-modified liposomes, the zeta potential decreased with modification with FA-PEG-DSPE compared to unmodified liposomes, confirming that folic acid was modified on the liposome surface. In addition, the drug encapsulation rate decreased with modification with folic acid. This is thought to be because folic acid is negatively charged, causing electrostatic repulsion with siRNA that was not encapsulated in the liposome and was attached to the liposome surface. From these results, it became clear that the liposome composition for this purpose should have a DSPC / Chol molar ratio of 7 / 3.

[0044] [Table 1]

[0045] (3) Evaluation of retinal delivery of nucleic acids using nucleic acid-encapsulated liposomes The nucleic acid-encapsulated liposome suspension (ssLip) obtained in (1) above was administered by eye drop to mice, and 30 minutes later, the mouse eyeballs were removed and observed under a fluorescent microscope and the fluorescence intensity was quantified in the same manner as in Reference Example 1 (2). Figure 2(A) shows the retina observed 30 minutes after the first eye drop administration of DY547-siRNA solution and DY547-siRNA-encapsulated liposome. No fluorescence was observed in the DY547-siRNA solution administration group, whereas fluorescence derived from DY547-siRNA was observed in the retina in the DY547-siRNA-encapsulated liposome administration group. Furthermore, stronger fluorescence was observed in the folate-modified liposome administration group compared to the other groups. Figure 2(B) shows the results of quantifying the fluorescence intensity of the retinal IPL. Compared to the DY547-siRNA solution administration group, the fluorescence intensity was significantly increased in the folate-modified DY547-siRNA encapsulated liposomes, indicating good retinal migration. One of the reasons for this is thought to be that the surface modification of the liposomes with folate conferred targeting to the folate receptors present in the retinal pigment epithelium cells.

[0046] The retinal migration of Bonac nucleic acid-encapsulated liposomes is shown in Figure 3. In the SA-containing liposome administration group, the fluorescence intensity increased significantly compared to the vehicle solution, indicating retinal migration. The retinal migration of Bonac nucleic acid-encapsulated R8-modified liposomes is shown in Figure 4. The R8-modified liposomes also showed a significant increase in fluorescence intensity compared to the vehicle solution and unmodified liposomes (SA-containing liposomes), indicating excellent retinal migration. These results demonstrated that by encapsulating nucleic acid drugs in liposomes modified with folic acid or R8, nucleic acids can be delivered to the retina with high efficiency even by eye drops.

[0047] Example 2 Evaluation of retinal delivery of nucleic acid by instillation of Bonac nucleic acid-encapsulated liposomes in a mouse model of retinal vein occlusion (RVO) (1) Creation of a mouse model of retinal vein occlusion The animals used were 8-week-old male ddY mice (Japan SLC Co., Ltd.). The mice were anesthetized by intramuscular administration of a combination of ketamine (120 mg / kg, Daiichi Sankyo Propharma) and xylazine (6 mg / kg, Bayer Healthcare), and the photosensitizer rose bengal (20 mg / kg, Wako Pure Chemical Industries, Ltd.) was administered into the tail vein. To dilate the pupil, 5 μL of Mydrin (registered trademark) P (0.5% tropicamide and 0.5% phenylephrine hydrochloride: tropicamide·phenylephrine hydrochloride, Santen Pharmaceutical Co., Ltd.) was administered in drops using a micropipette. The laser was a fundus photography device, Micron4 (Phoenix Research lab.), and a laser irradiation device (MERIDIAN AG, Bierigutstrasse) was attached. The retinal vein was occluded by irradiating the retinal vein located three papilla systems away from the optic disc of the right eye of the mouse with a laser (wavelength: 532 nm, spot size: 50 μm, irradiation time: 5 s, laser output: 50 mW). Three veins were occluded per eye, and the laser was irradiated 20 to 30 times per vein, and it was confirmed that the vein irradiated with the laser was completely occluded in the fundus image. In addition, to prevent the mouse's eyes from drying out, an appropriate amount of Hyalein (registered trademark) (purified sodium hyaluronate: hyalein, Santen Pharmaceutical Co., Ltd.) was administered by eye drop.

[0048] (2) Evaluation of retinal delivery of nucleic acids by instillation of Bonac nucleic acid-encapsulating liposomes The Bonac nucleic acid-encapsulated liposome suspension was administered by eye drop to the RVO model mice obtained in (1) above. Thirty minutes later, the mouse eyeballs were removed and observed under a fluorescent microscope in the same manner as in Reference Example 1 (2) to examine retinal migration. The results are shown in Figure 5. In the groups administered with liposomes encapsulating Bonac nucleic acid (SA-containing liposomes) and R8-modified liposomes, the fluorescence intensity increased significantly compared to untreated mice, indicating good retinal migration. The R8 modification further improved the retinal migration of Bonac nucleic acid.

[0049] Example 3: Evaluation of efficacy of eye drops administered to RVO model mice containing nucleic acids (siRNA or Bonac nucleic acid) (1) Administration conditions The nucleic acid-encapsulated liposomes were administered by eye drop to RVO model mice prepared in the same manner as in Example 2(1) as follows. Two administration times were examined. 1) When examining both preventive and therapeutic effects, the compound was administered three times at 5-minute intervals to the right eye of mice 1, 3, 6, 12, 24, 36, and 48 hours before laser irradiation. The retinal veins of the mice were then blocked by laser irradiation, and the compound was administered three times at 5-minute intervals to the right eye of the mice 1, 3, and 6 hours later. 2) The drugs were administered repeatedly to the right eyes of mice 2, 3, 6, 12, 18, 24, 30, and 36 hours after laser irradiation. The drug was administered by instilling 3 μL of liposome suspension encapsulating various VEGF-siRNA into the right eyes using a micropipette. The group of RVO model mice not administered the drug was administered the same amount of HBSS-HEPES buffer and used as the vehicle group.

[0050] (2) Observation using optical coherence tomography (OCT) Mice were anesthetized 24 hours after laser irradiation for the eye drop schedule 1) and 48 hours after laser irradiation for the eye drop schedule 2) by intramuscular administration of a combination of ketamine (120 mg / kg, Daiichi Sankyo Propharma) and xylazine (6 mg / kg, Bayer Healthcare). 5 μL of Mydorin (registered trademark) P (0.5% tropicamide and 0.5% phenylephrine hydrochloride, Santen Pharmaceutical Co., Ltd.) was administered by eye drop, and OCT images of the right eye were observed (830 nm) using Micron4 (Phoenix Research lab.). In addition, to prevent the eyes from drying, To prevent this, an appropriate amount of Hyalein (registered trademark) (purified sodium hyaluronate: hyalein, Santen Pharmaceutical Co., Ltd.) was administered by instillation into the eyes.

[0051] (3) Preparation and evaluation of frozen tissue sections of the eye After OCT observation, the animals were euthanized by cervical dislocation and the eyeballs were removed. The removed eyeballs were washed with a large excess of saline and immersed in 4% paraformaldehyde for 2 days to fix the tissue, then immersed in 5, 10, 15, and 20% sucrose solutions (in phosphate buffer) for 2 hours each, and then immersed in 25% sucrose solution (in phosphate buffer) overnight. The tissue was then fixed in Cryomold No. 1 (Tissue-Tek® Cryomold, Sakura Finetech Japan Co., Ltd.) with the addition of tissue freezing solution (Tissue-Tek® OCT Compound, Sakura Finetech Japan Co., Ltd.), and the eyeballs were embedded by rapid freezing in liquid nitrogen. The embedded eyeballs were horizontally cut using a Cryostat (Leica CM1850), and 20 μm-thick slices were cut at the position where the optic nerve was exposed to prepare sections for observation. The sections were stained with hematoxylin and eosin, and the entire retina was observed using an all-in-one microscope (BZ-9000, KEYENCE). The thickness of the inner nuclear layer (INL) of the retina at positions 240 μm apart from the optic nerve in the obtained images was quantified using ImageJ (National Institute of Mental Health). The results are shown in Figure 6. In the VEGF-siRNA encapsulated folic acid modified liposome administration group, thickening of the inner nuclear layer (INL) of the retina was significantly suppressed compared to the vehicle solution administration group (Figure 6A). In addition, in the Bonac nucleic acid encapsulated R8 modified liposome administration group, thickening of the INL was significantly suppressed compared to the vehicle solution (Figure 6B). These results indicate that when a nucleic acid having anti-VEGF activity is encapsulated in a folic acid or octaarginine (R8) modified liposome and administered by ocular instillation, the nucleic acid is efficiently delivered to the retina and exerts a therapeutic effect against retinal diseases. [Industrial Applicability]

[0052] The liposome preparation of the present invention can efficiently deliver an encapsulated drug (preferably a nucleic acid) to the posterior segment of the eye by instillation, and is therefore expected to lead to the development of therapeutic drugs for posterior segment diseases as eye drops that have not yet been marketed. In other words, it is extremely useful because it enables non-invasive treatment of posterior segment diseases and meets unmet medical needs.

[0053] This application is based on Patent Application No. 2019-053908 filed in Japan on March 20, 2019, the contents of which are incorporated in their entirety by reference herein.

Claims

1. The following (A) to (D): (A) one or more phospholipids selected from the group consisting of distearoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, egg yolk lecithin, and hydrogenated products thereof; (B) a compound having one or more steroid skeletons selected from the group consisting of cholesterol, cholesterol ester, cholestanol, and dehydrocholesterol; (C) one charged substance selected from the group consisting of stearylamine, didodecyldimethylammonium bromide, and dicetyl phosphate; and (D) a surface modifier selected from the group consisting of folic acid derivatives, octaarginine, polylysine, and cell-penetrating peptides; A liposome preparation for delivering a drug to the posterior segment of the eye by instillation, comprising the component (a) above, wherein the preparation has an average particle size of 600 nm or less.

2. 2. The liposome preparation according to claim 1, having an average particle size of 200 nm or less.

3. The liposome preparation according to claim 1 or 2, wherein component (D) is a folic acid derivative, octaarginine or a cell-penetrating peptide.

4. The liposome preparation according to any one of claims 1 to 3, having a surface potential of +20 to +60 mV.

5. The liposome preparation according to any one of claims 1 to 4, wherein the drug is a nucleic acid.

6. A preventive or therapeutic agent for posterior segment diseases, comprising the liposome preparation according to any one of claims 1 to 4 encapsulating a polymer effective for preventing or treating posterior segment diseases.

7. The agent according to claim 6 , wherein the polymer is a nucleic acid.

8. The agent according to claim 7 , wherein the nucleic acid is an siRNA, a single-stranded nucleic acid or a modified nucleic acid.

9. The agent according to any one of claims 6 to 8, which is an eye drop.

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