Photosensitive substance-containing lipoprotein, photodynamic therapy preparation, drug delivery method, and posterior ocular neovascular occlusion method

Abstract

To provide a photodynamic therapy preparation in which the delivery site selectivity of a sensitive substance (drug) is improved.SOLUTION: The present invention relates to a photosensitive-substance-containing lipoprotein, which contains a photosensitive substance and a modified high-density lipoprotein containing an apolipoprotein and a phospholipid, wherein the photosensitive-substance-containing lipoprotein contains one or more molecules of the photosensitive substance per particle of the modified high-density lipoprotein, and the apolipoprotein is obtained by bonding a vascular endothelial cell-targeting peptide or an aptamer to a cytophilic peptide.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a photosensitive substance-containing lipoprotein, a preparation for photodynamic therapy, a drug delivery method, and a method for blocking neovascularization in the posterior segment of the eye. [Background technology]

[0002] Age-related macular degeneration (AMD) is a disease that progresses due to the development of new blood vessels in the choroid, located on the outer side of the retina, leading to a state of reduced vision. One of the treatments for age-related macular degeneration is photodynamic therapy (PDT). In photodynamic therapy, photosensitive substances such as verteporfin are delivered to the affected area, and the photogenerated reactive oxygen from the photosensitive substance is used to block the new blood vessels, thereby halting the progression of the disease.

[0003] For example, Patent Document 1 discloses a method for treating wet age-related macular degeneration (wAMD), in which a patient is treated by PDT using a photosensitizer. It is disclosed that verteporfin is administered as a photosensitizer. Patent Document 2 discloses a method for treating ocular neovascular diseases, which includes administering at least one vascular occlusion agent to ocular neovascular tissue, including at least one photosensitizer that absorbs light in the range of about 380 nm to about 720 nm; and irradiating the eye with light having a wavelength or waveband that matches the excitation wavelength or waveband of the photosensitizer, activating the photosensitizer, and occluding one or more vessels of ocular neovascular tissue for a sufficient occlusion period, and porphyrin, purpurin, and verteporfin are listed as photosensitizers.

[0004] Non-Patent Document 1 discloses that photodynamic therapy is commonly used as a method for treating age-related macular degeneration, and that verteporfin (the component name of Visudyne (registered trademark), 15 mg for intravenous injection, Novartis Pharma KK) is used as a photosensitizer for PDT. Visudyne is a liposome preparation and is an age-related macular degeneration treatment agent that has been on the market since 2004. The liposomes that make up the liposome preparation function as a drug carrier.

[0005] The inventors have reported that modified high-density lipoproteins, including apolipoproteins and phospholipids bound to vascular endothelial cell targeting peptides or aptamers and cell affinity peptides, function as drug delivery carriers (Patent Document 3), but there is no specific disclosure in this document about their application in photodynamic therapy in combination with photosensitive substances. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 2017-537117 [Patent Document 2] Special Publication No. 2008-524224 [Patent Document 3] JP 2021-138629 A [Non-patent literature]

[0007] [Non-Patent Document 1] Ophthalmology PDT Study Group "Guidelines for Photodynamic Therapy for Age-Related Macular Degeneration" Journal of the Japan Ophthalmology Society, Vol. 108, No. 4 Summary of the Invention [Problem to be solved by the invention]

[0008] In photodynamic therapy, a photosensitive substance (drug) that can generate active oxygen at the lesion site is encapsulated in liposomes and delivered to the lesion site, and then laser light is irradiated only to the lesion site. However, in conventional age-related macular degeneration treatments, the selectivity of the drug delivery site is not high enough, and as a result, the drug may reach a site other than the lesion site. If the drug reaches a site other than the lesion site, and laser light is mistakenly irradiated to such a site, it will damage normal tissue and cause a decrease in vision, which is a problem. In addition, there is a time constraint that the laser light must be irradiated 15 minutes after intravenous administration of the drug. For this reason, in Japan today, in order to perform photodynamic therapy for age-related macular degeneration, it is recommended to attend a seminar co-sponsored by the Japanese Retina and Vitreous Society / Ophthalmology PDT Research Society and obtain a certified doctor's qualification, and high expertise is required for the treatment of age-related macular degeneration.

[0009] If the selectivity of the delivery site of the drug in the age-related macular degeneration treatment agent is increased, the delivery of the drug to sites other than the lesion site is suppressed, so that even if the entire eyeball is irradiated with the laser light used in age-related macular degeneration treatment, damage to normal tissues will not occur. Even if the entire eyeball is not irradiated with laser light, the treatment of age-related macular degeneration is significantly simplified by irradiating the laser light to a wide area rather than a pinpoint. Therefore, in order to solve the problems in conventional age-related macular degeneration treatment, the present inventors have conducted research with the aim of increasing the selectivity of the delivery site of the photosensitizer (drug) in age-related macular degeneration treatment and increasing the retention of the photosensitizer (drug) in the diseased site. Another object of the present invention is to provide a photodynamic therapy preparation with improved selectivity of the delivery site and retention of the photosensitizer (drug) in the diseased site. [Means for solving the problem]

[0010] Examples of specific embodiments of the present invention are given below.

[0011] [1] A photosensitizer-containing lipoprotein comprising a modified high density lipoprotein containing an apolipoprotein and a phospholipid, and a photosensitizer, The photosensitizer-containing lipoprotein contains one or more photosensitizer molecules per modified high density lipoprotein particle, The apolipoprotein is a photosensitive substance-containing lipoprotein to which a vascular endothelial cell targeting peptide or aptamer and a cell affinity peptide are bound. [2] The photosensitive substance-containing lipoprotein according to [1], wherein the photosensitive substance is one or more selected from the group consisting of porphyrin compounds, chlorin compounds, bacteriochlorin compounds, phthalocyanine compounds and cyanine dyes. [3] The photosensitive substance-containing lipoprotein according to [1] or [2], wherein the photosensitive substance is a porphyrin compound. [4] The photosensitive substance-containing lipoprotein according to [3], wherein the porphyrin compound has a porphyrin ring and a hydrophilic group. [5] The photosensitive substance-containing lipoprotein according to [3] or [4], wherein the porphyrin compound is verteporfin. [6] The photosensitizer-containing lipoprotein according to any one of [1] to [5], which has a volume average particle diameter of 140 nm or less. [7] The photosensitive substance-containing lipoprotein according to any one of [1] to [6], wherein the vascular endothelial cell targeting peptide or aptamer is a neovascular endothelial cell targeting peptide or aptamer. [8] The photosensitive substance-containing lipoprotein described in [7], wherein the neovascular endothelial cell targeting peptide is one or more selected from the group consisting of an NGR-containing peptide, an RGD-containing peptide, and a CPRECES-containing peptide. [9] The photosensitive substance-containing lipoprotein according to any one of [1] to [8], wherein the cell affinity peptide is one or more selected from the group consisting of TAT peptide, penetratin (PEN), polyarginine (R8), LL-37, transportan, Pep-1 and MTS.

[10] A formulation for photodynamic therapy comprising a photosensitizer-containing lipoprotein according to any one of [1] to [9].

[11] A photodynamic therapy formulation described in

[10] for use in treating age-related macular degeneration.

[12] A photodynamic therapy formulation described in

[10] or

[11] , wherein the photodynamic therapy comprises administering the photosensitive substance-containing lipoprotein to a subject suffering from age-related macular degeneration and irradiating the site of posterior ocular neovascularization with light.

[13] A method for blocking neovascularization in the posterior segment of the eye (excluding methods for treating humans), comprising administering a photosensitive substance-containing lipoprotein described in any one of [1] to [9] to a subject suffering from age-related macular degeneration, and irradiating the site of neovascularization in the posterior segment of the eye with light.

[14] A photodynamic therapy formulation described in

[10] for use in treating malignant tumors.

[15] A preparation for photodynamic therapy described in

[14] , wherein the photodynamic therapy comprises administering the photosensitive substance-containing lipoprotein to a subject having a malignant tumor and irradiating the site of neovascularization in the malignant tumor with light.

[16] A method for blocking neovascularization at a malignant tumor site (excluding methods for treating humans), comprising administering a photosensitive substance-containing lipoprotein described in any one of [1] to [9] to a subject having a malignant tumor, and irradiating the site of neovascularization at the malignant tumor site with light.

[0012] [A] A photosensitizer-containing lipoprotein according to any one of [1] to [9] for use in photodynamic therapy. [B] Use of a photosensitizer-containing lipoprotein according to any one of [1] to [9] for the manufacture of a preparation for photodynamic therapy. [C] A photosensitive substance-containing lipoprotein according to any one of [1] to [9] for use in treating age-related macular degeneration. [D] Use of a photosensitizer-containing lipoprotein according to any one of [1] to [9] for the manufacture of a preparation for treating age-related macular degeneration. [E] A photosensitizer-containing lipoprotein according to any one of [1] to [9] for use in treating a malignant tumor. [F] Use of the photosensitizer-containing lipoprotein according to any one of [1] to [9] for the manufacture of a preparation for treating malignant tumors. [G] A photosensitizer-containing lipoprotein according to any one of [1] to [9] for use in a method for blocking posterior segment neovascularization. [F] A photosensitizer-containing lipoprotein according to any one of [1] to [9] for use in a method for blocking neovascularization at a malignant tumor site. Effect of the Invention

[0013] According to the present invention, it is possible to improve the selectivity of the delivery site of a photosensitizer (drug) in the treatment of age-related macular degeneration. Also, according to the present invention, it is possible to provide a preparation for photodynamic therapy in which the selectivity of the delivery site of a photosensitizer (drug) is improved. [Brief description of the drawings]

[0014] [Figure 1] 1 is a schematic diagram illustrating the structure of a photosensitizer-containing lipoprotein according to this embodiment (center diagram). The left diagram is a schematic diagram illustrating the structure of a conventional modified high-density lipoprotein, and the right diagram is a schematic diagram illustrating the structure of an existing preparation (liposome preparation). [Diagram 2] 1 shows the absorption spectrum of verteporfin (VP) for each nanoparticle loaded with VP. [Diagram 3] 1 is a graph showing the generation ability of singlet oxygen (1O2). [Figure 4] 1 shows images of the amount of nanoparticles taken up by HUVEC cells and a graph showing the intracellular VP weight. [Diagram 5] 1 is a graph showing cytotoxicity (phototherapeutic effect) when HUVEC cells incorporating each nanoparticle were irradiated with light. [Figure 6] 1 is a graph showing the residence time of each nanoparticle in the blood after intravenous administration of each nanoparticle to a mouse. [Figure 7] These images show the accumulation of nanoparticles in choroidal neovascularization (CNV) in the posterior part of the eye after intravenous administration of each nanoparticle to mice. [Figure 8] FIG. 8 is a graph showing CNV residence time. [Figure 9] 1 shows images of the CNV area after each nanoparticle was administered intravenously to a mouse and laser light was irradiated, and a graph showing the area of ​​the CNV area. [Figure 10] The nanoparticles were administered intravenously to a mouse, and the images were taken of the condition of the entire eyeball (left) and the retina near the optic disc (right) after laser light irradiation. [Figure 11] 1 is a graph showing the NBD fluorescence intensity of HUVEC cells that have taken up each nanoparticle in the presence of a CD13 inhibitor. [Figure 12] 1 is a graph showing the VP fluorescence intensity of HUVEC cells that have taken up each nanoparticle in the presence of a CD13 inhibitor. [Figure 13] 1 shows graphs analyzing the expression levels of various transporter mRNAs in HUVEC cells that had taken up each nanoparticle. [Figure 14] 1 is a graph showing an analysis of the rate of increase in tumor volume when photodynamic therapy was applied to cancer model mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention will be described in detail below. The following description may be based on representative embodiments or specific examples, but the present invention is not limited to such embodiments. In this specification, a numerical range expressed using "~" means a range including the numerical values ​​before and after "~" as the lower and upper limits.

[0016] (Photosensitizer-containing lipoprotein) The present embodiment relates to a modified high-density lipoprotein containing an apolipoprotein and a phospholipid, and a photosensitive substance-containing lipoprotein containing a photosensitive substance, wherein the photosensitive substance-containing lipoprotein contains one or more molecules of the photosensitive substance per particle of the modified high-density lipoprotein, and the apolipoprotein is bound to a vascular endothelial cell targeting peptide or aptamer and a cell affinity peptide.

[0017] High density lipoprotein (HDL) constituting the photosensitive substance-containing lipoprotein contains apolipoprotein AI (apoA-I) and phospholipids. High density lipoprotein (HDL) may be either high density lipoprotein derived from natural plasma or reconstituted (i.e., artificial) high density lipoprotein (rHDL) produced by chemical synthesis or genetic engineering of apolipoprotein or its genetically modified apolipoprotein and phospholipids. High density lipoprotein (HDL) used in this embodiment contains apolipoprotein and phospholipid as main components. When high density lipoprotein (HDL) is artificially prepared, tens to hundreds of moles of phospholipid can be used per mole of apolipoprotein.

[0018] The density of high density lipoprotein (HDL) is preferably in the range of about 1.063 to 1.210 g / mL in the case of naturally derived HDL (see Antonio M. Gotto, Jr. et al. Methods Enzymol. 1986; 128: 3-41). On the other hand, in the case of rHDL, the size conforms to that of naturally derived HDL, but the density can be adjusted to a desired value during production, and the density is preferably 1.063 g / mL or more.

[0019] Apolipoproteins include, but are not limited to, apolipoproteins generally known to be contained in natural lipoproteins and genetically modified apolipoproteins thereof. Among them, apolipoproteins are preferably apolipoproteins known to be contained in high density lipoproteins (HDL) and genetically modified apolipoproteins thereof. Examples of apolipoproteins include proteins belonging to the groups of apolipoproteins A to F, H, J, LI, M and O, and are preferably one or more selected from the group consisting of apolipoprotein AI (apoA-I), apolipoprotein A-II (apoA-II), apolipoprotein C (apoC), apolipoprotein E (apoE) and genetically modified apolipoproteins thereof, and more preferably one or two selected from apolipoprotein AI (apoA-I) and genetically modified apoA-I thereof. Genetically modified apolipoproteins refer to mutants or analogs having a function equivalent to that of apolipoproteins (e.g., lipid binding function) (i.e., functional equivalents). Examples of genetically modified apolipoproteins include partial fragments of apolipoproteins and apolipoproteins that are combinations of these. An example of a genetically modified apolipoprotein of apolipoprotein AI is apoA-I, which lacks 44 amino acids at the N-terminus.

[0020] In this embodiment, the apolipoprotein is bound to a vascular endothelial cell targeting peptide or aptamer and a cell affinity peptide. In this specification, the apolipoprotein bound to the vascular endothelial cell targeting peptide or aptamer and the cell affinity peptide is also referred to as a modified apolipoprotein or a modified apolipoprotein.

[0021] Vascular endothelial cell targeting peptide or aptamer (hereinafter sometimes abbreviated as VTP) refers to a peptide or aptamer that can bind to vascular endothelial cells and target blood vessels. VTP is preferably a "neovascular endothelial cell targeting peptide or aptamer" that is a peptide or aptamer that can bind to neovascular endothelial cells and target vascular endothelial cells, and is particularly preferably a neovascular endothelial cell targeting peptide. For example, α v It is known that β3 integrin, aminopeptidase A, aminopeptidase N (APN; also called CD13), vascular endothelial growth factor (VEGF) receptor, etc. are expressed (J. Lahdenranta et al. (2007) The FASEB Journal; 21: 3272-3278). Therefore, the peptides and aptamers targeting neovascular endothelial cells include antibodies, fragments thereof, and aptamers that specifically bind to these molecules.

[0022] The neovascular endothelial cell targeting peptide may be, for example, one or more peptides selected from the group consisting of "asparagine-glycine-arginine (NGR)-containing peptides", RGD-containing peptides such as ACDCRGDCFC (SEQ ID NO: 2), and CPRECES-containing peptides such as CPKVCPRECESNC (SEQ ID NO: 3). These peptides target the CD13 receptor, α v It is a molecule found as a tumor neovascularization targeting peptide that binds to β3 integrin or aminopeptidase A, and has been reported to also bind to neovascularization in the retina (J. Lahdenranta et al. (2007) supra). These peptides may have any amino acid added before or after them as long as it does not impair the ability to bind to neovascularization. For example, an NGR-containing peptide may be an X-linked peptide that contains only NGR or has other amino acids attached to either or both of the front and back of the NGR. m NGRY nHere, X is any amino acid, m is an integer of 1 to 10, and when m is 2 or more, X is a combination of the same or different amino acids. Y is any amino acid, n is an integer of 1 to 10, and when n is 2 or more, Y is a combination of the same or different amino acids. X m NGRY n An example of a peptide represented by the formula (I) is CNGRCGG (SEQ ID NO: 4). By having cysteines (C) before and after NGR, an S-S bond is formed by oxidation, and the NGR portion is cyclized, thereby enhancing the receptor binding activity.

[0023] NGR-containing peptides are also expected to increase drug retention in CD13-positive cells, which could increase the retention of photosensitizer-containing lipoproteins at disease sites and alleviate time limitations in photodynamic therapy.

[0024] An example of an antibody that specifically binds to a molecule selected from VEGF receptors is bevacizumab (anti-VEGF humanized monoclonal antibody). In addition, the antibody may include a variable domain of heavy chain of heavy chain antibody (VHH), and an example of an antibody fragment may include Fab, F(ab')2, Fab', diabody, single chain antibody (e.g., scFv, dsFv), etc.

[0025] The vascular endothelial cell targeting aptamer is preferably, for example, a DNA aptamer or an RNA aptamer. For example, the aptamer that binds to the VEGF receptor is AptA, described in NUCLEIC ACID THERAPEUTICS, Volume 25, Number 5, pages 227-234, 2015. 80mer , Apt M80mer and APTs Divalent (See Table 1 below.) Apt 80mer The 5 in the base sequence represents a benzyl-containing base (BndU). M80mer is shown in SEQ ID NO:5.

[0026] [Table 1]

[0027] A cytophilic peptide (hereinafter sometimes abbreviated as CP) refers to a peptide that exhibits cytophilicity and is capable of binding to a cell membrane and then being transferred into the cell. In this specification, cytophilicity includes, but is not limited to, cell membrane permeability, cell adhesiveness, vascular endothelial cell accumulation, endosomal escape, cell nucleus accumulation, and mitochondrial accumulation.

[0028] CPs include natural peptides derived from natural proteins and synthetic peptides (e.g., chimeric peptides) artificially produced. CPs are classified into basic cell affinity peptides, amphipathic cell affinity peptides, and hydrophobic cell affinity peptides according to the chemical properties of their amino acid sequences. For example, basic cell affinity peptides include the TAT peptide derived from the TAT protein (Trans-activator of transcription protein) of the HIV-1 virus, penetratin (PEN) derived from Drosophila melanogaster, polyarginine (R8), and LL-37 derived from an antibacterial peptide, while amphipathic cell affinity peptides include the chimeric peptides transportan and Pep-1, and PEN sequence modifications or shuffling bodies, while hydrophobic cell affinity peptides include, but are not limited to, Pep-1, a synthetic peptide obtained by phage display, and MTS, a peptide derived from the signal peptide of a membrane protein. Among them, the CP used in this embodiment is preferably one or more selected from the group consisting of TAT peptide, penetratin (PEN), polyarginine (R8), LL-37, transportan, Pep-1, and MTS. In particular, the CP used in this embodiment is preferably a basic cell affinity peptide, preferably one or more selected from the group consisting of AT peptide, penetratin (PEN), polyarginine (R8), and LL-37, and particularly preferably penetratin (PEN).

[0029] VTP and CP bind to apolipoproteins to form VTP- and CP-associated apolipoproteins, which are sometimes referred to herein as vc-apo.

[0030] In modified apolipoprotein (vc-apo), at least one VTP and at least one CP are bound to the apolipoprotein. When one VTP and one CP are bound, it is preferable that the VTP is bound to the N-terminus of the apolipoprotein, and the CP is bound to the C-terminus of the apolipoprotein. The VTP can be bound to the C-terminus of the apolipoprotein, and the CP can be bound to the N-terminus of the apolipoprotein. Alternatively, both the VTP and the CP can be bound to either the N-terminus or the C-terminus of the apolipoprotein. When two or more VTPs are bound, two or more VTPs can be bound consecutively, and when two or more CPs are bound, two or more CPs can be bound consecutively. Alternatively, the VTP and the CP can be bound to the N-terminus and the C-terminus of the apolipoprotein, respectively. By binding two or more VTPs and / or CPs to one apolipoprotein, it may be possible to enhance vascular targeting ability and / or cell affinity. A modified high density lipoprotein may contain two or more molecules of apolipoprotein, and each apolipoprotein may contain two or more molecules of VTP and CP.

[0031] Here, the method of binding the VTP and CP to the apolipoprotein can be a chemical synthesis method (e.g., a coupling method) or a biological method (e.g., a genetic engineering method). When the VTP and CP are artificially bound to the apolipoprotein by creating a transforming (fusion) gene using a genetic engineering method, the VTP and CP are bound to the apolipoprotein by "fusion". The functional group and position on the apolipoprotein to be fused can be appropriately modified depending on the VTP and CP to be fused.

[0032] The content of the modified apolipoprotein contained in the modified high density lipoprotein is preferably 0.01 μmol / L or more, more preferably 0.05 μmol / L or more, and preferably 0.5 μmol / L or less, more preferably 0.1 μmol / L or less.

[0033] In addition, the modified apolipoprotein preferably contains a VTP and a CP fused to the apolipoprotein in an equal molar ratio, although two or more molecules of VTP and CP may be bound to one molecule of apolipoprotein, in which case the modified apolipoprotein contains a VTP and / or a CP fused to the apolipoprotein in a molar ratio of two or more times the amount.

[0034] The modified high density lipoprotein includes a phospholipid. The phospholipid means a lipid having one or more phosphate ester moieties. The phospholipid in the present specification includes a phospholipid generally known to be contained in a natural lipoprotein. The phospholipid known to be contained in a high density lipoprotein (HDL) is preferable, but is not limited thereto. For example, sphingolipids having sphingosine as a backbone are included, and the number of carbon atoms of the acyl group is preferably 12 to 18, and more preferably 14 to 18. For example, phosphatidylglycerol, phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine are included, and phosphatidylcholine is preferable. Specific examples include, but are not limited to, dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), egg phosphatidylcholine (PC), 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC), and hydrogenated soy phosphatidylcholine (HSPC). Note that the hydrogenated soy phosphatidylcholine (HSPC) may contain about 85% distearoyl phosphatidylcholine (DSPC). The phospholipid may be one to which a saturated fatty acid is bound, or one or more selected from the group consisting of dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC) and hydrogenated soy phosphatidylcholine (HSPC), or one or more selected from distearoyl phosphatidylcholine (DSPC) and hydrogenated soy phosphatidylcholine (HSPC). For example, it is considered that the use of a phospholipid to which a saturated fatty acid is bound can more effectively increase the ability to generate active oxygen at the lesion site. The phospholipid may be used alone or in combination of two or more.

[0035] Phospholipids, for example, can easily adjust the hardness of the lipid membrane and can form a stabilized complex. Furthermore, phospholipids play a role in contributing to the inclusion of compounds such as drugs. In addition, cholesterol can be added to the phospholipids for use. By including cholesterol, the hardness of the lipid membrane formed by the phospholipids can be adjusted. By adding cholesterol to the phospholipids, it is possible to interact with active ingredients such as drugs and adjust the efficiency of inclusion in the lipid membrane. The amount of cholesterol added to the phospholipid is preferably, for example, in the range of 0.01 to 30% by mass.

[0036] The content of phospholipids contained in the modified high density lipoprotein is preferably 0.5 μmol / L or more, more preferably 1.0 μmol / L or more, even more preferably 1.5 μmol / L or more, even more preferably 2.0 μmol / L or more, and particularly preferably 2.5 μmol / L or more. The content of phospholipids contained in the modified high density lipoprotein is preferably 250 μmol / L or less, more preferably 200 μmol / L or less, even more preferably 150 μmol / L or less, even more preferably 100 μmol / L or less, even more preferably 50 μmol / L or less, and particularly preferably 20 μmol / L or less. The content of phospholipids contained in the modified high density lipoprotein is preferably 0.1 mg / mL or more, more preferably 1 mg / mL or more, and even more preferably 2 mg / mL or more per 1 mg / mL of the modified apolipoprotein. The content of phospholipids contained in the modified high density lipoprotein is preferably 100 mg / mL or less, more preferably 50 mg / mL or less, and even more preferably 20 mg / mL or less per 1 mg / mL of the modified apolipoprotein.

[0037] The photosensitizer-containing lipoprotein of this embodiment contains one or more molecules of the photosensitizer per particle of modified high density lipoprotein. The photosensitizer generates active oxygen and heat when irradiated with light. In the treatment of age-related macular degeneration, the active oxygen and heat generated from the photosensitizer can block new blood vessels that have developed in the choroid located on the outer side of the retina, thereby stopping the progression of the disease. In particular, the photosensitizer used in this embodiment is preferably one that photogenerates active oxygen.

[0038] The photosensitizer preferably absorbs light having a wavelength in the range of 380 to 850 nm. For example, the absorption wavelength of verteporfin is about 690 nm, that of indocyanine green (ICG) is about 800 nm, and that of porfimer sodium is about 630 nm.

[0039] Examples of photosensitive substances include porphyrin compounds, chlorin compounds, bacteriochlorin compounds, phthalocyanine compounds, cyanine dyes, etc. More specifically, examples of photosensitive substances include hematoporphyrin derivatives, porfimer sodium, talaporfin sodium, protoporphyrin IX (PpIX), pheophorbidea, indocyanine green, aluminum phthalocyanine tetrasulfate (AlPcS4), tin etiopurpurin (SnET2), zinc(II) octadecylphthalocyanine (ZnOPPc), purpurinimide, azachlorin, zinc etiopurpurin (ZnET2), phthalocyanine (Pc), cadmium texaphyrin (CdTX), texaphyrin, zinc tetraphyptaloporphyrin (ZnTNP), verdein, ... Examples of the photosensitizer include benzoporphyrin derivative mono acid ring A (BPDMA), purpurin, zinc tetrasulfonated phthalocyanine (ZnTSPc), gallium phthalocyanine (Ga-Pc), indium phthalocyanine (In-Pc), benzoporphyrin derivative (BPD), calcium sulfonated phthalocyanine (Ca-SPc), zinc phthalocyanine derivative (ZnPc), aluminum sulfonated phthalocyanine (AlSPc), benzoporphyrin derivative, N-aspartyl chlorin e6 (Npe6), methylene blue, verteporfin, rhodamine, temoporphyrin, porphycene, hypersine, and IR-700. These photosensitizers may be included as part of the complex, or may form a complex by binding the photosensitizer to an antibody or the like. Among them, the photosensitive substance is preferably a porphyrin compound or a cyanine dye, more preferably one or more selected from the group consisting of porfimer sodium, indocyanine green, verteporfin, N-aspartyl chlorin e6 (Npe6) and IR-700, and particularly preferably verteporfin.

[0040] The porphyrin compound preferably has a hydrophobic porphyrin ring and a hydrophilic group. The photosensitive substance has a hydrophobic porphyrin ring and a hydrophilic group, so that the photosensitive substance is easily stably encapsulated in the phospholipid. Examples of the hydrophilic group that the porphyrin compound may have include a carboxylic acid group, a sulfonic acid group, a phosphoric acid group, a phosphonic acid group, and a hydroxyl group.

[0041] The content of the photosensitizer in the photosensitizer-containing lipoprotein is preferably 0.001 mol or more, more preferably 0.005 mol or more, and even more preferably 0.01 mol or more, relative to 1 mol of phospholipid. The upper limit of the content of the photosensitizer in the photosensitizer-containing lipoprotein is not particularly limited, but is preferably 5 mol or less, more preferably 3 mol or less, and even more preferably 1 mol or less, relative to 1 mol of phospholipid.

[0042] The content of the photosensitizer contained in the photosensitizer-containing lipoprotein is preferably 0.1 mol or more, more preferably 0.5 mol or more, and even more preferably 1 mol or more, relative to 1 mol of the modified apolipoprotein. The upper limit of the content of the photosensitizer in the photosensitizer-containing lipoprotein is not particularly limited, but is preferably 500 mol or less, more preferably 300 mol or less, and even more preferably 100 mol or less, relative to 1 mol of the modified apolipoprotein. The amount of the photosensitizer contained in the photosensitizer-containing lipoprotein can be determined from the integrated fluorescence intensity in the fluorescence spectrum of the photosensitizer.

[0043] In a preferred embodiment, for example, the photosensitizer-containing lipoprotein contains about 5 mg / mL of phospholipid and about 0.05 mg (50 μg) / mL of photosensitizer per 1 mg / mL of modified apolipoprotein. In a preferred embodiment, the photosensitizer-containing lipoprotein contains 50 to 200 mol of phospholipid and 1 to 50 mol of photosensitizer per 1 mol of modified apolipoprotein.

[0044] The photosensitizer-containing lipoprotein of this embodiment may contain, in addition to the photosensitizer, other drugs, fluorescently labeled substances, bioactive substances, etc. In this case, the content of the other drugs, fluorescently labeled substances, or bioactive substances may be, for example, 0.01 to 25 μmol / L, or 0.05 to 2 μmol / L.

[0045] In modified high density lipoprotein (hereinafter, sometimes abbreviated as vcHDL) containing modified apolipoprotein and phospholipid, the apolipoprotein contains VTP in addition to CP, so that vcHDL becomes a drug delivery carrier in the posterior segment of the eye and can target neovascularization, and therefore has excellent delivery ability to neovascularization in the posterior segment of the eye, which is an affected area of ​​age-related macular degeneration caused by abnormal angiogenesis. Therefore, by loading a photosensitizer, which is a drug for treating age-related macular degeneration, on vcHDL, the drug (photosensitizer) can be selectively delivered to the affected area of ​​age-related macular degeneration. The photosensitizer-containing lipoprotein of this embodiment is the modified high density lipoprotein (vcHDL) loaded with a photosensitizer, so that the drug can be selectively delivered to the affected area of ​​age-related macular degeneration. This can increase the delivery efficiency of the drug (photosensitizer). In this specification, the delivery efficiency of the drug (photosensitizer) indicates the ratio of the drug (photosensitizer) that reaches the neovascularization in the posterior segment of the eye, which is an affected area of ​​age-related macular degeneration, to the administered drug (photosensitizer). By increasing the delivery efficiency of the drug (photosensitive substance), it is possible to increase the amount of drug (photosensitive substance) that reaches the neovascularization of the posterior segment of the eye, and as a result, the light irradiation intensity can be reduced, and the safety of the treatment can be further improved. Furthermore, by selectively delivering the drug to the affected area, it is possible to suppress the occurrence of phototoxicity (side effects) even when light is irradiated to areas other than the affected area. This allows the light irradiation area to be made wider, making it possible to significantly simplify the treatment of age-related macular degeneration. Furthermore, by increasing the retention of the drug at the affected area, not only is the time limit (15 minutes) between intravenous administration and laser light irradiation relaxed, but the clearance of the photosensitive compound from normal tissue is also promoted, leading to a reduction in side effects.

[0046] The volume average particle diameter of the photosensitizer-containing lipoprotein of this embodiment is preferably 140 nm or less, more preferably 100 nm or less, even more preferably 75 nm or less, even more preferably 50 nm or less, and particularly preferably 30 nm or less. The lower limit of the volume average particle diameter of the photosensitizer-containing lipoprotein is not particularly limited, but is preferably 5 nm or more, for example. It is expected that the smaller the size of the photosensitizer-containing lipoprotein, the higher the delivery ability to the posterior segment of the eye. Therefore, by setting the volume average particle diameter of the photosensitizer-containing lipoprotein within the above range, the delivery efficiency of the drug (photosensitizer) can be more effectively improved. As described above, the volume average particle size of the photosensitizer-containing lipoprotein is small, on the order of nanometers, and therefore, in this specification, the photosensitizer-containing lipoprotein is sometimes referred to as nanoparticles.

[0047] The volume mean particle size and surface charge of the photosensitizer-containing lipoproteins can be measured using a Nanotrac UPA-UT151 particle size analyzer (MicrotracBEL Corp., Tokyo, Japan) and a ZETASIZER NANO Z (Malvern, Worcestershire, UK), respectively.

[0048] The photosensitizer-containing lipoprotein of this embodiment has an active oxygen (singlet oxygen) generating ability calculated using the following formula, which is preferably 400% or more, more preferably 450% or more, and even more preferably 500% or more. The upper limit of the active oxygen (singlet oxygen) generating ability of the photosensitizer-containing lipoprotein is not particularly limited, but is preferably, for example, 3000% or less.

[0049]

number

[0050] Reactive oxygen (singlet oxygen) generation ability is1 Detection is performed using Singlet Oxygen Sensor Green Reagent (SOSG; Invitrogen Thermo Fisher Scientific Inc., USA), which selectively reacts with O2 and emits fluorescence. Specifically, a solution containing photosensitive substance-containing lipoprotein and SOSG is prepared, and this solution is irradiated with light (10 mW, 5 min) using a 689 nm xenon lamp (Asahi Spectra, Tokyo, Japan). The fluorescence spectrum of the solution before and after light irradiation is measured with a plate reader (TECAN) (Ex. 480 nm / Em. 500-560 nm), and the integrated fluorescence intensity is calculated. The integrated fluorescence intensity before light irradiation is F0, and the integrated fluorescence intensity after light irradiation is F, and from the above formula, 1 Calculate O2 production capacity (%).

[0051] In this embodiment, for example, by using a phospholipid in which saturated fatty acid is bound, the active oxygen (singlet oxygen) generating ability can be more effectively increased, and the active oxygen generating ability at the lesion site can be more effectively increased. If the active oxygen (singlet oxygen) generating ability can be increased, it is also possible to reduce the loading amount and administration amount of the drug.

[0052] (Method of producing lipoprotein containing photosensitive substance) The present embodiment relates to a method for producing a photosensitizer-containing lipoprotein, comprising the following steps i) to v): i) The VTP and CP are fused to an apolipoprotein by genetic engineering techniques to obtain a fusion protein (modified apolipoprotein). ii) Crude vcHDL is produced by mixing the obtained fusion protein with liposomes containing phospholipids (spontaneous interaction method); alternatively, crude vcHDL is produced by mixing with cholate micelles without going through liposome formation (cholate-dialysis method). iii) The crude vcHDL is purified by removing unreacted phospholipids and apolipoproteins. iv) A photosensitizer and vcHDL are mixed to produce a photosensitizer-containing lipoprotein containing one or more photosensitizer molecules per vcHDL particle. v) Purifying the photosensitizer-containing lipoproteins by removing unreacted photosensitizer.

[0053] In step i), a genetic engineering technique can be used. Examples of the genetic engineering technique include DNA editing using methylase or DNA polymerase I / DNA ligase, and polymerase chain reaction (PCR). Specifically, a penetratin (PEN) peptide gene with Pst I and Xba I recognition sequences added to the 5'-end and 3'-end is inserted into the restriction enzyme Pst I and Xba-I recognition sites of the E. coli expression vector pCOLD I using DNA ligase. Next, a gene with the restriction enzyme Kpn I recognition sequence added to the 5'-end of the N-terminal 44 amino acid deleted apoA-I gene and the restriction enzyme Pst I recognition sequence added to the 3'-end is prepared by PCR, and inserted between the Kpn I and Pst I recognition sites of the pCOLD I using DNA ligase. Through the above operations, a polypeptide gene can be prepared in which the PEN peptide gene is fused to the C-terminus of the N-terminal 44 amino acid deleted apoA-I and the NGR peptide gene is fused to the N-terminus. When producing a VTP- and CP-bound apolipoprotein (modified apolipoprotein), a fusion protein gene is prepared by introducing an equimolar ratio of the VTP gene and the CP gene to the apolipoprotein gene before fusion.

[0054] In step ii), a modified high density lipoprotein (vcHDL) is prepared by adding several to several thousand times the molar amount, several tens to several hundreds of times the molar amount, for example, about 5 to about 2000 times the molar amount, about 20 to about 500 times the molar amount, or about 30 to about 200 times the molar amount, of phospholipid relative to 1 mole of the fusion protein.

[0055] The particle size of the modified high density lipoprotein (vcHDL) produced as above can be adjusted by adjusting the molar ratio of the fusion protein and phospholipid and further by obtaining a desired density fraction after density gradient ultracentrifugation, but is not limited to these methods.

[0056] In step iii), unreacted phospholipids and apolipoproteins are removed by gel filtration or ultracentrifugation, and the crude vcHDL is purified to obtain vcHDL.

[0057] In step iv), the photosensitizer and vcHDL are mixed. The amount of the photosensitizer is preferably 0.001 mol or more, more preferably 0.01 mol or more, and even more preferably 0.03 mol per mol of phospholipid. The amount of the photosensitizer is preferably 100 mol or less, more preferably 50 mol or less, and even more preferably 25 mol or less per mol of phospholipid.

[0058] The temperature during mixing in step iv) is preferably 30 to 70°C, more preferably 40 to 60°C. The mixing time is preferably 1 to 100 minutes, more preferably 1 to 60 minutes. In this embodiment, by using a compound having a porphyrin ring and a hydrophilic group as the photosensitizer, the affinity between vcHDL and the photosensitizer can be increased, and as a result, the mixing temperature in step iv) can be lowered and the mixing time can be shortened. This can increase the production efficiency of the photosensitizer-containing lipoprotein.

[0059] In step v), unreacted photosensitizer can be removed by gel filtration or ultracentrifugation to purify the photosensitizer-containing lipoprotein.

[0060] (Photodynamic Therapy Preparations) The present embodiment relates to a photodynamic therapy preparation containing a photosensitizer-containing lipoprotein. Photodynamic therapy includes administering a photosensitizer-containing lipoprotein to a subject suffering from, for example, a malignant tumor or age-related macular degeneration, and irradiating the lesion with light. In photodynamic therapy, the photosensitizer-containing lipoprotein is administered to a subject suffering from a malignant tumor or age-related macular degeneration, and the photosensitizer-containing lipoprotein is selectively delivered to the lesion (for example, tumor tissue or neovascularization in the posterior eye, which is the affected area of ​​age-related macular degeneration). Then, light irradiation (laser light irradiation) is performed on the lesion where the photosensitizer-containing lipoprotein has reached. This causes active oxygen and heat to be generated from the photosensitizer contained in the photosensitizer-containing lipoprotein, which can damage the lesion and destroy the diseased tissue. In this way, photodynamic therapy is a treatment method that combines delivery of a photosensitizer-containing lipoprotein to a lesion and light irradiation (laser light irradiation) on the lesion.

[0061] When the photodynamic therapy preparation is used, for example, in the treatment of malignant tumors, the type of malignant tumor is not particularly limited, and examples include brain / nerve tumors, skin cancer, stomach cancer, lung cancer, liver cancer, lymphoma / leukemia, colon cancer, pancreatic cancer, anal / rectal cancer, esophageal cancer, uterine cancer, breast cancer, adrenal cancer, kidney cancer, renal pelvis / ureter cancer, bladder cancer, prostate cancer, urethral cancer, penile cancer, testicular cancer, bone / osteosarcoma, leiomyoma, rhabdomyoma, mesothelioma, etc. Cancer cells have new blood vessels, and the new blood vessels include α v Because these cells express β3 integrin, aminopeptidase A, aminopeptidase N (APN; also known as CD13), or vascular endothelial growth factor (VEGF) receptors, the peptides and aptamers targeting neovascular endothelial cells contained in the photosensitive substance-containing lipoproteins specifically bind to these molecules.

[0062] It is particularly preferable that the photodynamic therapy preparation of this embodiment is used for the treatment of age-related macular degeneration. That is, in a preferred embodiment, the photodynamic therapy preparation of this embodiment is a preparation for the treatment of age-related macular degeneration. In this case, in photodynamic therapy, a photosensitizer-containing lipoprotein is administered to a subject suffering from age-related macular degeneration, and the photosensitizer-containing lipoprotein is selectively delivered to neovascularization in the posterior eye, which is the affected area of ​​age-related macular degeneration. Then, light irradiation (laser light irradiation) is performed on the neovascularization site where the photosensitizer-containing lipoprotein has reached. This generates active oxygen from the photosensitizer contained in the photosensitizer-containing lipoprotein, which can damage the neovascularization and block the blood vessels.

[0063] The photodynamic therapy preparation of this embodiment can also be used to treat malignant tumors. In a preferred embodiment, the photodynamic therapy preparation of this embodiment is a preparation for treating malignant tumors. In this case, in photodynamic therapy, a photosensitizer-containing lipoprotein is administered to a subject having a malignant tumor, and the photosensitizer-containing lipoprotein is selectively delivered to neovascularization caused by the malignant tumor. After that, the site of neovascularization where the photosensitizer-containing lipoprotein has reached is irradiated with light (laser light irradiation). This generates active oxygen from the photosensitizer contained in the photosensitizer-containing lipoprotein, which can damage the neovascularization and block the blood vessels.

[0064] The photodynamic therapy preparation may further contain a pharma- ceutically acceptable additive in addition to the above-mentioned photosensitizer-containing lipoprotein. As the pharma-ceutically acceptable additive, various active ingredients or medicinal ingredients (including pharmacologically active ingredients and physiologically active ingredients) and additives according to various applications can be mentioned, so long as they do not impede the effects of the present invention. As the additive, for example, a buffering agent (e.g., sodium phosphate), an isotonicity agent (e.g., sodium chloride), a pH adjuster (e.g., boric acid), an antiseptic / preservative (e.g., benzalkonium chloride), a stabilizer (e.g., mannitol), a thickener (e.g., sodium alginate), a chelating agent (e.g., sodium edetate), a surfactant (e.g., polyoxyethylene sorbitan monooleate), an absorption enhancer (e.g., sodium oleate), a flavoring agent (e.g., menthol), etc. can be mentioned.

[0065] Furthermore, the photodynamic therapy preparation may contain, in addition to the above-mentioned photosensitizer-containing lipoprotein, a diagnostic, preventive or therapeutic agent for malignant tumors or age-related macular degeneration. For example, vascular endothelial growth factor (VEGF) inhibitors (e.g., VEGF antibodies, VEGF aptamers, siRNA), steroid preparations (e.g., dexamethasone, betamethasone, fluorometholone, prednisolone), non-steroid preparations (e.g., indomethacin, bromfenac, diclofenac sodium, etc.), prostaglandin preparations (e.g., latanoprost, tafluprost), pirenoxine, glutathione, memantine, epinephrine, pilocarpine hydrochloride, carbachol, dorzolamine hydrochloride, etc. These include acetazolamide, timolol maleate, carteolol hydrochloride, betaxolol hydrochloride, bunazosin hydrochloride, isopropyl unoprostone, pranoprafen, aspirin, SRPIN803 (6-(4-hydroxy-3-methoxybenzylidene)-5-imino-2-(trifluoromethyl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7(6H)-one), and pazopanib.

[0066] The preparation for photodynamic therapy of this embodiment can be used in the form of eye drops, eye ointment, nasal drops, ear drops, injections, and the like, but is preferably an injection.

[0067] The photodynamic therapy preparation of this embodiment is preferably an injection, and the photodynamic therapy preparation is preferably injected intravenously. When the photodynamic therapy preparation is injected intravenously, the photosensitizer-containing lipoprotein contained in the photodynamic therapy preparation selectively accumulates at the lesion. Then, by irradiating the lesion where the photosensitizer has accumulated with a laser beam, active oxygen and heat are generated from the photosensitizer, which damages the lesion. This can stop the progression of the disease. For example, when the photodynamic therapy preparation is used for the treatment of age-related macular degeneration, when the photodynamic therapy preparation is injected intravenously, the photosensitizer-containing lipoprotein contained in the photodynamic therapy preparation selectively accumulates in the neovascularization of the posterior eye, which is the lesion site of age-related macular degeneration, and then, by irradiating the lesion where the photosensitizer has accumulated with a laser beam, active oxygen and heat are generated from the photosensitizer, which damages the neovascularization and blocks the blood vessels. Furthermore, when a photodynamic therapy preparation is used to treat malignant tumors, when the photodynamic therapy preparation is injected intravenously, the photosensitive substance-containing lipoprotein contained in the photodynamic therapy preparation selectively accumulates in neovascularization caused by the malignant tumor, and then, by irradiating the lesion where the photosensitive substance has accumulated with laser light, active oxygen and heat are generated from the photosensitive substance, which damage the neovascularization and block the blood vessels.

[0068] The photosensitizer-containing lipoprotein contained in the photodynamic therapy formulation of this embodiment can be highly selectively accumulated in neovascularization of the posterior eye, which is an affected area of ​​age-related macular degeneration, and neovascularization caused by malignant tumors. This can increase the delivery efficiency of the photosensitizer contained in the photosensitizer-containing lipoprotein. Furthermore, by increasing the selectivity of the delivery site of the photodynamic therapy formulation, the occurrence of phototoxicity (side effects) can be suppressed even when light is irradiated to areas other than the lesion site. Furthermore, the photodynamic therapy formulation of this embodiment has a longer retention time at the diseased site compared to conventional drugs, so that a longer time period during which light can be irradiated can be secured. This makes it possible, for example, to administer the photodynamic therapy formulation outside the laser light irradiation room, thereby reducing the burden on medical staff performing photodynamic therapy, and further increasing the therapeutic efficiency of photodynamic therapy.

[0069] Drug Delivery Methods The present embodiment may relate to a method for drug delivery to neovascularization in the posterior segment of the eye using the above-mentioned photosensitizer-containing lipoprotein. Also, the present embodiment relates to a method for drug delivery to neovascularization in the posterior segment of the eye using the above-mentioned preparation for photodynamic therapy.

[0070] Furthermore, the present embodiment may relate to a method for drug delivery to neovasculature at a malignant tumor site using the above-mentioned photosensitizer-containing lipoprotein. Also, the present embodiment relates to a method for drug delivery to neovasculature at a malignant tumor site using the above-mentioned preparation for photodynamic therapy.

[0071] As described above, modified high density lipoprotein (vcHDL) containing modified apolipoprotein and phospholipids can selectively deliver photosensitive substances to neovascularization by apolipoprotein containing VTP in addition to CP, and can be a drug delivery carrier for the posterior segment of the eye or a drug delivery carrier for malignant tumors. Therefore, by applying the drug delivery method or by administering the above-mentioned photodynamic therapy preparation to a patient suffering from or at risk of suffering from age-related macular degeneration, it becomes possible to diagnose, prevent or treat age-related macular degeneration. In addition, by applying the drug delivery method or by administering the above-mentioned photodynamic therapy preparation to a patient with a malignant tumor, it becomes possible to treat cancer, such as shrinking, disappearing or suppressing metastasis of the malignant tumor.

[0072] In the drug delivery method of the present embodiment, by using modified high density lipoprotein (vcHDL) containing modified apolipoprotein and phospholipid as a drug delivery carrier, it is possible to selectively deliver a photosensitive substance (drug) to neovascularization in the posterior segment of the eye, which is an affected area of ​​age-related macular degeneration, which is believed to be caused by abnormal angiogenesis. In addition, in the drug delivery method of the present embodiment, it is possible to selectively deliver a photosensitive substance (drug) to neovascularization caused by malignant tumors. This can increase the delivery efficiency of the photosensitive substance (drug). Furthermore, by increasing the selectivity of the drug delivery site, it is possible to suppress the occurrence of phototoxicity (side effects) even when light is irradiated to areas other than the lesion site. This allows the light irradiation area to be made wider, which makes it possible to significantly simplify age-related macular degeneration treatment and cancer treatment. Furthermore, by increasing the retention of the drug in the diseased area, not only is the time limit (15 minutes) from intravenous administration to laser light irradiation relaxed, but it also leads to the reduction of side effects by promoting the clearance of the photosensitive compound from normal tissues.

[0073] In this embodiment, the modified high density lipoprotein (vcHDL) itself, which does not contain a photosensitive substance (drug), can exert an anti-inflammatory effect and angiogenesis inhibitory effect. Therefore, the above-mentioned drug delivery method itself can be used as a method for treating or preventing age-related macular degeneration or cancer.

[0074] (Posterior segment neovascular occlusion method) The present embodiment relates to a method for blocking neovascularization in the posterior segment of the eye, which includes administering the above-mentioned photosensitive substance-containing lipoprotein to a subject suffering from age-related macular degeneration and irradiating a site where neovascularization in the posterior segment of the eye is developed with light. The method for blocking neovascularization in the posterior segment of the eye can be applied to humans or mammals other than humans, but may not be applied to human treatment methods. In the method for blocking neovascularization in the posterior segment of the eye, the above-mentioned photosensitive substance-containing lipoprotein or photodynamic therapy preparation is, for example, intravenously injected, so that the photosensitive substance-containing lipoprotein reaches the site where neovascularization in the posterior segment of the eye is developed. Since the photosensitive substance-containing lipoprotein contains modified apolipoproteins including VTP and CP, the photosensitive substance-containing lipoprotein is selectively delivered to neovascularization in the posterior segment of the eye, which is the affected site of age-related macular degeneration. Thereafter, the site where neovascularization has developed to which the photosensitive substance-containing lipoprotein has reached is irradiated with light (laser light irradiation). This generates active oxygen from the photosensitive substance contained in the photosensitive substance-containing lipoprotein, which damages the neovascularization and blocks the blood vessels.

[0075] The present embodiment also relates to a method for blocking neovascularization at a malignant tumor site, which includes administering the above-mentioned photosensitizer-containing lipoprotein to a subject having a malignant tumor and irradiating a neovascularization site at a malignant tumor site with light. The method for blocking neovascularization at a malignant tumor site can be applied to humans or mammals other than humans, but may not be applied to human treatment methods. In the method for blocking neovascularization at a malignant tumor site, the above-mentioned photosensitizer-containing lipoprotein or photodynamic therapy preparation is, for example, intravenously injected, so that the photosensitizer-containing lipoprotein reaches the neovascularization site at a malignant tumor site. Since the photosensitizer-containing lipoprotein contains modified apolipoproteins containing VTP and CP, the photosensitizer-containing lipoprotein is selectively delivered to neovascularization caused by a malignant tumor. Thereafter, the neovascularization site to which the photosensitizer-containing lipoprotein has reached is irradiated with light (laser light irradiation). This generates active oxygen from the photosensitizer contained in the photosensitizer-containing lipoprotein, which damages the neovascularization and blocks the blood vessels. This makes it possible to treat cancer by shrinking or eliminating malignant tumors or inhibiting metastasis.

[0076] Examples of the light to be irradiated include light emitted by semiconductor lasers, dye lasers, etc. Specific examples include Nd-YAG (yttrium aluminum garnet) lasers, CO2 lasers, argon dye lasers, excimer lasers, etc. For example, light irradiation (laser light irradiation) can be performed in accordance with the treatment guidelines disclosed in Non-Patent Document 1 and the like. In this case, the wavelength of the laser light is 689±3 (mean±standard deviation) nm, and the amount of light irradiation energy is 50 J / (light irradiation output 600 mW / cm 2 for 83 seconds).

[0077] In addition, when the photosensitizer-containing lipoprotein or the photodynamic therapy preparation of the present embodiment is intravenously injected, the blood residence time of the photosensitizer-containing lipoprotein or the photodynamic therapy preparation is relatively long. Therefore, the time period for light irradiation (laser light irradiation) can be set to, for example, about 5 to 80 minutes after the start of intravenous injection of the photosensitizer-containing lipoprotein or the photodynamic therapy preparation. When a conventional photodynamic therapy preparation is used, the time period for light irradiation (laser light irradiation) is about 5 to 20 minutes after the start of intravenous injection of the photodynamic therapy preparation. Therefore, by using the photosensitizer-containing lipoprotein or the photodynamic therapy preparation of the present embodiment, the time period for light irradiation (laser light irradiation) can be significantly extended.

[0078] When treating age-related macular degeneration or cancer, the posterior segment neovascularization occlusion method is preferably performed once or multiple times.

[0079] Other examples of this embodiment include the following: The treatment method may be applied to humans or mammals other than humans, but may exclude the treatment method for humans. [A] A photosensitizer-containing lipoprotein as described above for use in photodynamic therapy. [B] Use of the above-mentioned photosensitizer-containing lipoprotein for the manufacture of a preparation for photodynamic therapy. [C] A photosensitizer-containing lipoprotein as described above for use in treating age-related macular degeneration. [D] Use of the above-mentioned photosensitizer-containing lipoprotein for the manufacture of a preparation for treating age-related macular degeneration. [E] A photosensitizer-containing lipoprotein as described above for use in treating malignant tumors. [F] Use of the above-mentioned photosensitizer-containing lipoprotein for the manufacture of a preparation for treating malignant tumors. [G] The above-mentioned photosensitizer-containing lipoprotein for use in a method for blocking neovascularization in the posterior segment of the eye. [F] The above-mentioned lipoprotein containing a sensitizer for use in a method for blocking neovascularization at a malignant tumor site. EXAMPLES

[0080] The features of the present invention will be described in more detail below with reference to examples and comparative examples. The materials, amounts used, ratios, processing contents, processing procedures, etc. shown in the following examples can be appropriately changed without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be interpreted as being limited by the specific examples shown below.

[0081] (Example 1) Preparation of NGR and PEN fused apoA-I and PEN fused apoA-I The structure of NGR and penetratin (PEN) fused apolipoprotein AI (apoA-I) is as follows: Human apoA-I lacking 44 amino acids at the N-terminus was used as a template, and the NGR (CNGRCGG) moiety was ligated to the N-terminus of the human apoA-I lacking 44 amino acids at the N-terminus. Furthermore, RQIKIWFQNRRMKWKKK (SEQ ID NO: 1), in which K was added to the C-terminus of PEN, was ligated to the C-terminus of the human apoA-I lacking 44 amino acids at the N-terminus. The molecular weight of the NGR and PEN fused apoA-I was approximately 29,319 Da. In addition, human apoA-I lacking 44 amino acids at the N-terminus was used as a template, and RQIKIWFQNRRMKWKKK (sequence number 1), in which a K was added to the C-terminus of PEN, was linked to the C-terminus of human apoA-I lacking 44 amino acids at the N-terminus to obtain PEN-fused apoA-I. Hereinafter, NGR and PEN-fused apoA-I and PEN-fused apoA-I will also be collectively referred to as apoA-I variants.

[0082] ApoA-I mutants were expressed in BL21(DE3) (Novagen, Dallas, WI, USA) and purified under guanidine denaturing conditions using Ni Sepharose High-Performance resin packed in a Tricorn 10 / 100 column of an AKTAstart chromatography system (Cytiva, Tokyo, Japan).

[0083] Preparation of modified high density lipoproteins Distearoylphosphatidylcholine (DSPC) was mixed with sodium cholate in a molar ratio of 1:3.6, and DSPC was dispersed by bath sonication (Elma Schmidbauer GmbH, Mannheim, Germany) at 65°C for 10 min. Then, each apoA-I variant was dissolved in PBS containing 4M urea, mixed with the DSPC dispersion (DSPC:apoA-I variant = 80:1 (mol)), and reacted at 55°C for 30 min. After the reaction, dialysis was performed overnight at room temperature against 3 L of PBS. A dialysis membrane with a molecular weight cutoff of 50 kDa (Spectrum Chemical Manufacturing Corp.) was used for dialysis. Note that only the mixture of DSPC and NGR- and PEN-fused apoA-I was allowed to cool to room temperature after starting dialysis at 55°C. The reaction solution inside the dialysis membrane was transferred to a microtube and centrifuged (20,000×g, 24°C, 30 min). The supernatant was collected and purified by size-exclusion chromatography using a HPLC system (Shimadzu Corporation, Kyoto, Japan) equipped with a Superose 6prep grade packed column (Cytiva). Modified high-density lipoproteins (vcHDLs) containing apoA-I variants (hereafter, HDL variants using PEN-fused apoA-I are referred to as eLP1, and HDL variants using NGR and PEN-fused apoA-I are referred to as eLP2) were eluted at room temperature with tris(hydroxymethyl)aminomethane buffer (150 mM NaCl, 50 mM Tris·HCl (pH 7.4), 2 mM ethylenediaminetetraacetic acid, 0.5 mM phenylmethylsulfonyl fluoride) at a flow rate of 1 mL / min. The eluate of the modified HDL fraction (50-75 min) was collected and dialyzed against 3 L of PBS overnight at room temperature using a dialysis membrane with a molecular weight cutoff of 50 kDa. The modified HDL inside the dialysis membrane was transferred to a microtube and centrifuged (20,000×g, 24° C., 15 min), and the supernatant was stored in a low-protein binding tube.

[0084] Loading of verteporfin (VP) into modified high density lipoproteins The modified HDL was mixed with VP dissolved in DMSO at a VP to lipid molar ratio of 1:10 and incubated at 55°C for 30 min. Purification was performed by size exclusion chromatography equilibrated with PBS. The VP-loaded modified high density lipoproteins (VP@eLP1, VP@eLP2) were stored in low protein binding tubes (Figure 1).

[0085] (Example 2) Preparation of Visudyne®-like nanoparticles (Vis) DMPC, EggPG, and VP dissolved in ethanol were mixed in a molar ratio of 40:24:5, and the ethanol was evaporated under reduced pressure at 35°C using a rotary evaporator R-300 (Nihon BUCHI, Tokyo, Japan) to form a lipid membrane. The lipid membrane was then freeze-dried for 2 h using a freeze dryer FDS-1000 (TOKYO RIKAKIKAI Co., Ltd., Tokyo, Japan). A 9.9% lactose solution was added to the lipid membrane, and bath sonication was performed at room temperature for 10 min. After sonication, the lipid suspension was passed through a polycarbonate filter (Avanti Polar Lipids) with a pore size of 400 nm using a mini-extruder (Avanti Polar Lipids, Alabama, USA) to form a viscoelastic membrane (Figure 1).

[0086] (evaluation) Characterization of VP-loaded modified high density lipoproteins and Vis The protein concentration in the VP-modified high density lipoprotein was quantified using bovine serum albumin (Thermo Fisher Scientific) as a standard solution in the presence of 1% Triton X-100, using a DC protein assay kit (Bio-Rad Laboratories, Inc.) and a Synergy HTX Multi-Mode Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA). The phosphatidylcholine concentration in the VP-modified high density lipoprotein and Vis was quantified using a Phospholipid C assay kit (FUJIFILM Wako Pure Chemical Corporation). The volume-average particle size and surface charge of each nanoparticle were measured using a Nanotrac UPA-UT151 particle size analyzer (MicrotracBEL Corp., Tokyo, Japan) and a ZETASIZER NANO Z (Malvern, Worcestershire, UK), respectively. The VP concentration in each nanoparticle was calculated from the integrated fluorescence intensity in the fluorescence spectrum using a plate reader (TECAN Group Ltd., Mannedorf, Switzerland). The absorption spectrum of each nanoparticle was measured using a UV-3600 Plus ultraviolet-visible near-infrared spectrophotometer (Shimadzu Corporation, Kyoto, Japan) (Figure 2). As shown in Figure 2, there was almost no difference in the absorption peak of VP in the VP-loaded modified high density lipoprotein and Vis.

[0087] [Table 2]

[0088] Singlet oxygen generation capacity Singlet oxygen ( 1 O2) 1Detection was performed using Singlet Oxygen Sensor Green Reagent (SOSG; Invitrogen Thermo Fisher Scientific Inc., USA), which selectively reacts with O2 and emits fluorescence. A solution containing 10 μg / mL VP-containing nanoparticles (VP@eLP1, VP@eLP2, or Vis) and 10 mM SOSG was prepared. A dispersion of each nanoparticle was placed in a square cell and irradiated with light (10 mW, 5 min) using a 689 nm xenon lamp (Asahi Spectra, Tokyo, Japan). The fluorescence spectrum of the solution before and after light irradiation was measured with a plate reader (TECAN) (Ex. 480 nm / Em. 500-560 nm), and the integrated fluorescence intensity was calculated. The integrated fluorescence intensity before light irradiation was F0, and the integrated fluorescence intensity after light irradiation was F, and the integrated fluorescence intensity was calculated from the following equation: 1 The O2 generating capacity was calculated. 1 The calculation results of O2 generating capacity are shown in Figure 3. 1 The reason why the O2 generating ability is smaller than that of VP@eLP1 and VP@eLP2 is that the lipids that make up Vis contain unsaturated fatty acids, and these unsaturated fatty acids 1 This is thought to be due to oxidation through reaction with O2.

[0089]

number

[0090] cell culture HUVECs were cultured in a 100-mm dish in a medium containing endothelial cell growth basal medium-2 (EBM2), 2% fetal bovine serum, and 1% antibiotic antimycotic mixed stock solution (Nacalai Tesque, Inc.) in a HERAcell 150 (Heraeus Holding GmbH, Hanau, Germany) incubator (37°C, 5% CO2). Medium was changed every 2 days. Cells were subcultured when they reached 70-90% confluence. Cells were detached by treatment with TrypLE Express (Thermo Fisher Scientific). The detached cells were centrifuged (200 × g, 24°C, 3 min), the supernatant was removed, and the cells were suspended in new medium and transferred to a new dish for subculture.

[0091] Analysis of interactions between each nanoparticle and vascular endothelial cells HUVECs were used as a model of normal vascular endothelial cells and were cultured in EBM2 containing 2% FBS. Basic fibroblast growth factor (bFGF) was added to a final concentration of 50 ng / mL to enhance CD13 expression. HUVECs were cultured in triple-well glass base dishes at 1 × 10 5 The cells were seeded at a density of 10000000000 cells / mL and cultured in a CO2 incubator at 37°C for 24 hours. The adherent cells were treated with each nanoparticle (eLP1, eLP2, Vis containing 1μg / mL VP), and after 15 and 60 minutes, the cells were washed with D-PBS containing heparin and intracellular VP fluorescence was observed using a confocal fluorescence microscope LSM800 with Airyscan (Carl Zeiss Microscopy GmbH, Jena, Germany). A 40x oil immersion objective lens (Plan-Apochromat 40x / 1.4oil DIC M27) ​​was used for observation. To quantify the amount of intracellular VP, cells were seeded in a 12-well plate (1 × 10 5HUVECs (1000×g, ...

[0092] Phototherapy effect of each nanoparticle The photocytotoxicity test of each nanoparticle was performed using the Cytotoxicity LDH Assay Kit-WST (DOJINDO LABORATORIES, Kumamoto, Japan). HUVECs were placed in a 96-well plate at 1 × 10 5 The cells were seeded at a density of 100 cells / mL and stimulated with bFGF in the same manner as above, after which each nanoparticle containing 100 ng / mL of VP was applied. After 60 min, the cells were washed with D-PBS containing heparin, new medium was added, and light irradiation (31 mW, 1 min) was performed using a xenon lamp. After light irradiation, the medium was replaced and the cells were left to stand in a CO2 incubator at 37°C for 24 hours. Cytotoxicity was evaluated according to the reagent manufacturer's protocol (Figure 5). As shown in Figure 5, P@eLP2 showed the highest cytotoxicity (= phototherapy effect).

[0093] Blood retention of each nanoparticle The animal experiments using mice in this study were conducted in accordance with the ethical guidelines set out by the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, the Kyoto University Animal Experiment Guidelines, and the Toyama Prefectural University Animal Experiment Ethics Committee. C57BL / 6JJmsSlc mice (male, 8-10 weeks old) were purchased from Sankyo Labo Service Corporation, INC. (Toyama, Japan). Each nanoparticle was administered to the tail vein of the mouse at a dose of 2 mgVP / kg of body weight. Blood was collected 0.16, 1, 4, 7, and 24 hours after administration and mixed with a tube containing 3.8% sodium citrate (Iwaki Seiyaku Co., Ltd., Tokyo, Japan). After centrifugation (3000 rpm, 4°C, 5 min) to separate plasma components and blood cell components, the supernatant component, plasma, was collected, pre-frozen at -80°C, and then freeze-dried. Methanol was added to all freeze-dried plasma samples, vortex-mixed, bath sonicated for 2 min, and vortex-mixed again. The samples were then mixed using a tube rotator (4°C, 1 h). After bath sonication for 2 min, the samples were vortex-mixed again. After centrifugation (20,000×g, 4°C, 5 min), the supernatant was analyzed by reverse phase high performance liquid chromatography (RP-HPLC) to quantify VP. VP was eluted using an HPLC LC-20Ai (Shimadzu Corporation) equipped with a liquid delivery unit (LC-20AD), an autosampler (SIL-20A), a column oven (CTO-20A), a photodiode array detector (SPD-M20A), and a spectrofluorometric detector (RF-20A xs). A COSMOSIL 5C18-MS-II column was used, and a gradient mobile phase consisting of (A) 0.05 mM NaH2PO4 solution and (B) MeOH was used for elution.The gradient program was set as follows: 0-2 min, 15% mobile phase B; 2-5 min, 15%-85% mobile phase B; 5-10 min, 5%-100% mobile phase B. The flow rate was 1.1 mL / min, and the column oven was set at 35 °C (Figure 6). As shown in Figure 6, the vis. 1 / 2 was 5.2±1.8h, and the t 1 / 2 is 4.8±0.5h, and the t 1 / 2 The mean blood retention time was 5.1±1.6 hours, and no significant difference was observed.

[0094] Evaluation of CNV accumulation of VP loaded on each nanoparticle C57BL / 6JJmsSlc mice (male, 8-10 weeks old) were used. The mice were anesthetized with 10 mg / kg xylazine and 80 mg / kg ketamine. The pupils were sufficiently dilated with 5% phenylephrine hydrochloride / 0.8% tropicamide (Sandol P, Rhoto Nitten Co., Ltd, Nagoya, Japan). Cover glasses were attached to the mouse corneas and used as contact lenses. Laser photocoagulation was performed using a photocoagulator (DC-3000; Nidek, Gamagori, Japan) set to 75 lm, 0.05 seconds, and 150 mW, and CNV was induced by irradiating four locations 4-5 disk diameters away from the optic disc with laser light. Seven days after laser light irradiation, each nanoparticle (Vis and VP@eLP2) was administered to the mice via the tail vein at a dose of 10 mg / kg in terms of VP. The eyes were removed 15 and 60 min after administration. After removal, the tissue was fixed with 4% PFA, blocked, and immunostained using rat anti-CD102 antibody (1:200, BD Pharmingen, San Diego, CA, USA) and Alexa 488-labeled anti-rat IgG antibody (1:500, Invitrogen, Eugene, OR, USA) as primary and secondary antibodies, respectively. The RPE / choroid complex was then flattened and radially incised to obtain flat mounts. Fluorescence from the CNV (Ex. 488 nm / Em. 495-530 nm) and fluorescence from the VP in the CNV (Ex. 405 nm / Em. 610-700 nm) were observed by confocal microscopy (Figure 7). A 10x dry objective lens (Plan-Apochromat 20x / 0.8) was used for observation. The fluorescence intensity of the VP in the CNV was quantitatively analyzed using ImageJ (Figure 8). As shown in Figure 7, VP@eLP2 showed higher choroidal neovascularization (CNV) accumulation than Vis. Furthermore, as shown in Figure 8, VP@eLP2 also showed significantly higher CNV retention than Vis.

[0095] Effect of photodynamic therapy (PDT) on CNV mice CNV was generated in the same manner as in the evaluation of CNV accumulation. Seven days after laser light irradiation, Vis and VP@eLP2 were administered to mice via the tail vein at 2 mg / kg in terms of VP. As a control, PBS was administered to mice via the tail vein. Laser light irradiation was performed 60 min after administration. Laser light irradiation was performed using a Vislas PDT system 690S (Carl Zeiss) with a 7000 μm 2 , 60s, 600mW / cm 2 The entire eyeball was irradiated with laser light. Seven days later, the eyeball was enucleated, fixed with 4% PFA, blocked, and immunostained using rat anti-CD102 antibody and Alexa 488-labeled anti-rat IgG antibody as the primary and secondary antibodies, respectively. The RPE / choroid complex was then flattened, radially incised, and flat-mounted. CNV-derived fluorescence was observed using a confocal microscope. A 20x dry objective lens (Plan-Apochromat 20x / 0.8) was used for observation. CNV area values ​​were quantitatively analyzed (Figure 9). As shown in Figure 9, the CNV area was reduced by laser light irradiation in mice administered with VP@eLP2 compared to mice administered with Vis, and a high phototherapy effect was obtained. In addition, a reduction in CNV area was observed in mice administered with VP@eLP2 even without laser light irradiation, which is thought to be due to the anti-inflammatory and angiogenesis inhibitory effects of VP@eLP2 itself.

[0096] statistical analysis Statistical analyses were performed using Microsoft Excel. Differences between means of two or more groups were determined by Welch's t-test. Differences between means of three or more groups were determined by Tukey or Tukey-Kramer tests. In all statistical analyses, p<0.05 was considered statistically significant. All data were graphed as mean ± standard deviation.

[0097] Consideration As shown by the above results, VP@eLP2 showed higher CNV selectivity and significantly higher CNV retention than Vis. The high CNV selectivity of VP@eLP2 allows for the reduction of side effects (phototoxicity). In addition, the high CNV retention of VP@eLP2 allows for the extension of the PDT window. These effects will lead to the development of photodynamic therapy that is less burdensome for both medical professionals and patients.

[0098] Phototoxicity (side effects) testing In order to confirm that phototoxicity (side effects) is reduced when VP@eLP2 is used, the following test was carried out. Vis and VP@eLP2 were administered to normal C57BL / 6JJmsSlc mice (male, 8-10 weeks old) via the tail vein at a dose equivalent to 2 mg / kg of VP. As a control, PBS was administered to the mice via the tail vein. Laser light irradiation was performed 15 min after administration. Laser light irradiation was performed using a Vislas PDT system 690S (Carl Zeiss) with a 7000 μm 2 , 60s, 600mW / cm 2 The entire eyeball was irradiated with the laser light at a setting of 0.05°C. Seven days later, the eyeball was enucleated, fixed in 4% paraformaldehyde (PFA, Nacalai Tesque, Inc.) (4°C, O / N), and dehydrated in 20% sucrose solution (4°C, 24 h). After dehydration, the eyeball was embedded in OCT compound (Miles, Elkhart, IN, USA) and frozen in liquid nitrogen. The frozen eyeball was sectioned at 15 μm using a cryostat microtome (Leica Microsystems, Tokyo, Japan). The sections were stained with H&E and mounted in Aqua-Poly / Mount (Polysciences, Inc., USA). The retinal periphery of the H&E-stained sections was observed using an OLYMPUS IXplore Pro (Olympus Corporation, Tokyo, Japan). As shown in Figure 10, when mice administered PBS or VP@eLP2 were irradiated with laser light, no abnormalities were observed in the retina and no phototoxicity occurred, whereas when mice administered Vis were irradiated with laser light, the entire retina disappeared and the eyeball was deformed. This is thought to be due to the low CNV selectivity of Vis, which caused damage (phototoxicity) to the retina when irradiated with laser light.

[0099] VP@eLP2 uptake by HUVECs in the presence of CD13 inhibitors HUVECs were used as a model of normal vascular endothelial cells. They were cultured in EBM2 containing 2% FBS. To enhance CD13 expression, basic fibroblast growth factor (bFGF) was added to the medium at a final concentration of 50 ng / mL. In addition, 250 μM bestatin (Sigma-Aldrich, St. Louis, MO) was added as a CD13 inhibitor. HUVECs were cultured at a concentration of 1 × 10 5 The cells were seeded at a density of 1000 cells / mL and cultured for 24 hours in a CO2 incubator at 37°C. VP@eLP2 containing 1 μg / mL VP and fluorescent lipid (NBD) was applied to the adherent cells, and after 60 min, the cells were washed with D-PBS containing heparin and observed under a confocal fluorescence microscope LSM800 with Airyscan (Carl Zeiss Microscopy GmbH, Jena, Germany). The intracellular VP and NBD fluorescence intensities were evaluated using ImageJ (Figure 11 (NBD fluorescence intensity) and Figure 12 (VP fluorescence intensity)).

[0100] Consideration In the presence of Bestatin, NBD fluorescence intensity did not change significantly. On the other hand, VP fluorescence intensity increased significantly. This difference may be explained by the function of CD13 to enhance drug efflux transporter expression (Guo et al., Oncol.Res.2020,28,533-540). Assuming that VP is a better substrate for drug efflux transporters than NBD, the effect of inhibition of drug efflux transporter expression by Bestatin is thought to have enhanced the intracellular retention of VP more than the inhibition of cellular uptake of VP@eLP2. In other words, it is thought that NGR peptide has a greater effect on drug retention in CD13-positive cells than on drug targeting to CD13-positive cells.

[0101] Analysis of mRNA expression levels of various transporters by quantitative reverse transcription polymerase chain reaction (qRT-PCR) Seed HUVECs in 6-well plates at a density of 1 x 10 5 The plates were seeded at 2 mL at 100 cells / mL (medium composition: EBM2, 2% FBS, 50 μg / mL bFGF). After 24 hours, the culture supernatant was removed, washed with 1 mL of D-PBS, and then 2 mL of VP@eLP2 (100 μM VP) containing 100 μM VP was added and left to stand at 37°C for 1 hour in a CO2 incubator. The cells were then washed with 1 mL of D-PBS, and the plate was immediately cooled on ice. Total RNA extraction from the cells was performed using ISOGEN II (Nippon Gene Co., Ltd., Tokyo). To obtain cDNA, RNA was reverse transcribed using SuperScript VILO Master Mix. Real-time PCR was then performed using a master mix (TB Green Premix Ex TaqII [Tli RNaseH Plus], TaKaRa Bio, Inc.) and a Thermal Cycler Dice Real Time System (TaKaRa Bio, Inc.). 2 -ΔΔCt The relative changes in gene expression were analyzed using the method. Human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) was used as an endogenous normalization control. The primer sequences used in real-time PCR are shown below.

[0102] Consideration VP@eLP2 treatment significantly reduced MDR1 mRNA expression (Figure 13), supporting the above results and suggesting that MDR1 is the drug efflux transporter responsible for VP efflux in vascular endothelial cells.

[0103] hMDR1 forward, 5'-AAGCTTAGTACCAAAGAGGCTCTG-3' (SEQ ID NO: 6) reverse, 5'-GGCTAGAAACAATAGTGAAAACAA-3' (SEQ ID NO: 7) hMRP1 forward, 5'-AGTGACCTCTGGTCCTTAAACAAGG-3' (SEQ ID NO: 8) reverse, 5'-GAGGTAGAGAGCAAGGATGACTTGC-3' (SEQ ID NO: 9) hMRP4 forward, 5'-TCCTCCTCCATTTACAGTGACA-3' (SEQ ID NO: 10) reverse, 5'-TTATTCTCCTAAACACTGCAGCTC-3' (SEQ ID NO: 11) hMRP5 forward, 5'-TGAATCTGAAGTGATGGAGAATGG-3' (SEQ ID NO: 12) reverse, 5'-CCTATCGGAGCCTAGAACCG-3' (SEQ ID NO: 13) hBCRP forward, 5'-CCCAGTACGACTGTGACAATG-3' (SEQ ID NO: 14) reverse, 5'-CACAGTCTTCAAGGAGATCAGCTA-3' (SEQ ID NO: 15) hGAPDH forward, 5'-ACGGATTTGGTCGTATTGGG-3' (SEQ ID NO: 16) reverse, 5'-ACGGATTTGGTCGTATTGGG-3' (SEQ ID NO: 17)

[0104] PDT on cancer model mice Human prostate cancer cell line PC-3 (RIKEN Cell Bank) was used. A suspension of PC-3 in RPMI1640 (4 × 10 7 The tumor was incubated for 12 days at 37 °C for 1 hour at 4 °C for 1 hour (1 min) with 0.5% CO2 and 1.5% CO2. The tumor was incubated for 12 hours at 4 °C for 1 hour ... 2 , φ5mm, 2min). Five days later (day 5), the major and minor axes of the subcutaneous tumor were measured with a vernier caliper, and the tumor volume was calculated according to the following formula. Tumor volume (cm 3 ) = minor axis (cm) x minor axis (cm) x major axis (cm) / 2 The PDT effect was assessed by normalizing the tumor volume on day 5 by the value on day 0 (FIG. 14).

[0105] Consideration In all groups, the tumor volume increased on day 5 compared to day 0. However, the tumor volume increase rate in both the VP@eLP2+PDT group and the Vis+PDT group was smaller than that in the non-laser-irradiated group (VP@eLP2, Vis), suggesting that both samples show similar PDT effects in tumor-bearing mice.

Claims

1. A photosensitizer-containing lipoprotein comprising a modified high density lipoprotein containing an apolipoprotein and a phospholipid, and a photosensitizer, The photosensitizer-containing lipoprotein contains one or more photosensitizer molecules per modified high density lipoprotein particle, The apolipoprotein is a photosensitive substance-containing lipoprotein to which a vascular endothelial cell targeting peptide or aptamer and a cell affinity peptide are bound.

2. The photosensitive substance-containing lipoprotein according to claim 1, wherein the photosensitive substance is one or more selected from the group consisting of porphyrin compounds, chlorin compounds, bacteriochlorin compounds, phthalocyanine compounds and cyanine dyes.

3. The photosensitive substance-containing lipoprotein according to claim 1 , wherein the photosensitive substance is a porphyrin compound.

4. The photosensitive substance-containing lipoprotein according to claim 3 , wherein the porphyrin compound has a porphyrin ring and a hydrophilic group.

5. The photosensitive substance-containing lipoprotein according to claim 3 , wherein the porphyrin compound is verteporfin.

6. 2. The photosensitive substance-containing lipoprotein according to claim 1, having a volume average particle size of 140 nm or less.

7. The photosensitive substance-containing lipoprotein according to claim 1 , wherein the vascular endothelial cell targeting peptide or aptamer is a neovascular endothelial cell targeting peptide or aptamer.

8. The photosensitive substance-containing lipoprotein according to claim 7, wherein the neovascular endothelial cell targeting peptide is one or more selected from the group consisting of an NGR-containing peptide, an RGD-containing peptide and a CPRECES-containing peptide.

9. The photosensitive substance-containing lipoprotein according to claim 1, wherein the cell affinity peptide is one or more selected from the group consisting of TAT peptide, penetratin (PEN), polyarginine (R8), LL-37, transportan, Pep-1 and MTS.

10. A formulation for photodynamic therapy comprising the photosensitizer-containing lipoprotein according to any one of claims 1 to 9.

11. The photodynamic therapy formulation according to claim 10, which is used for treating age-related macular degeneration.

12. The photodynamic therapy formulation according to claim 10 or 11, wherein the photodynamic therapy comprises administering the photosensitive substance-containing lipoprotein to a subject suffering from age-related macular degeneration and irradiating the site of posterior segment neovascularization with light.

13. A method for blocking neovascularization in the posterior segment of the eye (excluding methods for treating humans), comprising administering the photosensitive substance-containing lipoprotein according to any one of claims 1 to 9 to a subject suffering from age-related macular degeneration, and irradiating a site where neovascularization in the posterior segment of the eye has occurred with light.

14. The photodynamic therapy formulation according to claim 10 for use in the treatment of malignant tumors.

15. The photodynamic therapy formulation according to claim 14, wherein the photodynamic therapy comprises administering the photosensitive substance-containing lipoprotein to a subject having a malignant tumor and irradiating the site of neovascularization in the malignant tumor with light.

16. A method for blocking neovascularization at a malignant tumor site (excluding methods for treating humans), comprising administering the photosensitive substance-containing lipoprotein according to any one of claims 1 to 9 to a subject having a malignant tumor, and irradiating the site of neovascularization at the malignant tumor site with light.

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