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

Abstract

The present invention addresses the problem of providing a photodynamic therapy preparation in which the selectivity of a sensitive substance (drug) to a delivery site is enhanced. 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.

Description

Lipoprotein containing photosensitizer, preparation for photodynamic therapy, drug delivery method, and method for occluding neovascularization in the posterior segment of the eye

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

[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, resulting in a state of visual impairment. One treatment for AMD is photodynamic therapy (PDT). In PDT, a photosensitive substance such as verteporfin is delivered to the lesion site, and the photo-generated reactive oxygen species occlude 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 with PDT using a photosensitizer. It discloses administering verteporfin as the photosensitizer. Furthermore, Patent Document 2 discloses a method for treating ocular neovascular diseases, including administering at least one vaso-occlusive substance to neovascular tissue of the eye, the vaso-occlusive substance 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 to activate the photosensitizer and occlude one or more vessels in the neovascular tissue of the eye for a sufficient occlusion period, and lists porphyrin, purpurin, and verteporfin as photosensitizers.

[0004] Non-Patent Document 1 discloses that photodynamic therapy is commonly used as a treatment method for 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 liposomal preparation and is a treatment for age-related macular degeneration that has been on the market since 2004. The liposomes that make up the liposomal 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 applying them in combination with photosensitizers to photodynamic therapy.

[0006] JP 2017-537117 A JP 2008-524224 A JP 2021-138629 A

[0007] Ophthalmology PDT Study Group "Guidelines for Photodynamic Therapy for Age-Related Macular Degeneration" Journal of the Japan Ophthalmology Association, Vol. 108, No. 4

[0008] In photodynamic therapy, a photosensitizer (drug) capable of generating reactive oxygen species at the lesion site is encapsulated in liposomes and delivered to the lesion site, followed by laser irradiation limited to the lesion site. However, conventional age-related macular degeneration treatments lack sufficient selectivity for the drug delivery site, resulting in the drug reaching areas other than the lesion site. If the drug reaches the lesion site, accidental laser irradiation of such areas can damage normal tissue and cause vision loss, creating problems. Furthermore, there is a time constraint of laser irradiation, requiring 15 minutes after intravenous drug administration. Therefore, in Japan, in order to perform photodynamic therapy for age-related macular degeneration, it is currently recommended to attend a training course cosponsored by the Japan Retina and Vitreous Society / Ophthalmology Photodynamic Therapy Study Group and obtain certified physician qualifications. High levels of expertise are required for the treatment of age-related macular degeneration.

[0009] If the selectivity of the drug delivery site for age-related macular degeneration treatment agents is improved, delivery of the drug to sites other than the lesion site is suppressed, so that even if the laser light used in age-related macular degeneration treatment is irradiated onto the entire eyeball, damage to normal tissues will not occur. Furthermore, even if the entire eyeball is not irradiated with laser light, the treatment of age-related macular degeneration can be significantly simplified by irradiating the laser light over a wide area rather than a pinpoint. Therefore, in order to solve the problems of conventional age-related macular degeneration treatments, the inventors have conducted research with the aim of improving the selectivity of the delivery site of photosensitizers (drugs) in age-related macular degeneration treatments and increasing the retention of the photosensitizers (drugs) at the diseased site. Another object of the present invention is to provide a photodynamic therapy formulation with improved selectivity of the delivery site and retention of the photosensitizers (drugs) at the diseased site.

[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, wherein the photosensitizer-containing lipoprotein contains one or more photosensitizer molecules per modified high-density lipoprotein particle, and the apolipoprotein is bound to a vascular endothelial cell-targeting peptide or aptamer and a cell affinity peptide. [2] The photosensitizer-containing lipoprotein according to [1], wherein the photosensitizer is one or more selected from the group consisting of a porphyrin compound, a chlorin compound, a bacteriochlorin compound, a phthalocyanine compound, and a cyanine dye. [3] The photosensitizer-containing lipoprotein according to [1] or [2], wherein the photosensitizer is a porphyrin compound. [4] The photosensitizer-containing lipoprotein according to [3], wherein the porphyrin compound has a porphyrin ring and a hydrophilic group. [5] The photosensitizer-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], wherein the volume average particle diameter is 140 nm or less. [7] The photosensitizer-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 photosensitizer-containing lipoprotein according to [7], wherein the neovascular endothelial cell targeting peptide is one or more peptides selected from the group consisting of NGR-containing peptides, RGD-containing peptides, and CPRECES-containing peptides. [9] The photosensitizer-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 preparation for photodynamic therapy, comprising the photosensitizer-containing lipoprotein according to any one of [1] to [9].

[11] The preparation for photodynamic therapy according to

[10] , which is used for treating age-related macular degeneration.

[12] The preparation for photodynamic therapy according to

[10] or

[11] , wherein the photodynamic therapy comprises administering the photosensitizer-containing lipoprotein to a subject suffering from age-related macular degeneration and irradiating a site where neovascularization has occurred in the posterior segment of the eye with light.

[13] A method for occluding neovascularization in the posterior segment of the eye, comprising administering the photosensitizer-containing lipoprotein according to any of [1] to [9] to a subject suffering from age-related macular degeneration and irradiating a site where neovascularization has occurred in the posterior segment of the eye with light.

[14] The preparation for photodynamic therapy according to

[10] , which is used for treating malignant tumors.

[15] The preparation for photodynamic therapy according to

[14] , wherein the photodynamic therapy comprises administering the photosensitizer-containing lipoprotein to a subject suffering from malignant tumors and irradiating a site where neovascularization has occurred in the malignant tumor with light.

[16] A method for blocking neovascularization at a malignant tumor site, comprising administering the photosensitizer-containing lipoprotein according to 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] The photosensitizer-containing lipoprotein according to any one of [1] to [9] for use in photodynamic therapy. [B] Use of the photosensitizer-containing lipoprotein according to any one of [1] to [9] for the manufacture of a preparation for photodynamic therapy. [C] The photosensitizer-containing lipoprotein according to any one of [1] to [9] for use in the treatment of age-related macular degeneration. [D] Use of the photosensitizer-containing lipoprotein according to any one of [1] to [9] for the manufacture of a preparation for the treatment of age-related macular degeneration. [E] The photosensitizer-containing lipoprotein according to any one of [1] to [9] for use in the treatment of malignant tumors. [F] Use of the photosensitizer-containing lipoprotein according to any one of [1] to [9] for the manufacture of a preparation for the treatment of malignant tumors. [G] The photosensitizer-containing lipoprotein according to any one of [1] to [9] for use in a method for blocking neovascularization in the posterior segment of the eye. [F] The photosensitizer-containing lipoprotein according to any one of [1] to [9] for use in a method for blocking neovascularization at the site of a malignant tumor.

[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. Furthermore, according to the present invention, it is possible to provide a formulation for photodynamic therapy in which the selectivity of the delivery site of a photosensitizer (drug) is improved.

[0014] Schematic diagram illustrating the structure of the photosensitizer-containing lipoprotein of 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 formulation (liposome formulation). The absorption spectrum of verteporfin (VP) for each nanoparticle loaded with VP is shown. Singlet oxygen ( １ O ２ 7 is a graph showing the production ability of nanoparticles. It is an image of the amount of nanoparticles taken up by HUVEC cells and a graph showing the weight of intracellular VP. It is a graph showing the cytotoxicity (phototherapeutic effect) when HUVEC cells that have taken up each nanoparticle are irradiated with light. It is a graph showing the blood residence time of each nanoparticle after intravenous administration of each nanoparticle to mice. It is an image of the accumulation of each nanoparticle in choroidal neovascularization (CNV) in the posterior segment of the eye after intravenous administration of each nanoparticle to mice. It is a graph showing the CNV residence time in FIG. 7. It is an observation image showing the CNV area after intravenous administration of each nanoparticle to mice and irradiation with laser light, and a graph showing the area of ​​the CNV area. It is an image of the condition of the entire eyeball (left) and the retina near the optic disc (right) after intravenous administration of each nanoparticle to mice and irradiation with laser light. It is a graph showing the NBD fluorescence intensity of HUVEC cells that have taken up each nanoparticle in the presence of a CD13 inhibitor. 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. 2 is a graph analyzing the expression levels of various transporter mRNAs in HUVEC cells that have taken up each nanoparticle. 3 is a graph analyzing the tumor volume increase rate when photodynamic therapy is applied to cancer model mice.

[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 "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0016] (Photosensitive substance-containing lipoprotein) This 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), which constitutes the photosensitizer-containing lipoprotein, contains apolipoprotein AI (apoA-I) and phospholipids. The high density lipoprotein (HDL) may be either a high density lipoprotein derived from natural plasma, or a reconstituted (i.e., artificial) high density lipoprotein (rHDL) produced by chemical synthesis or genetic engineering of apolipoproteins or genetically modified apolipoproteins and phospholipids. The high density lipoprotein (HDL) used in this embodiment contains apolipoproteins and phospholipids as its main components. When artificially preparing high density lipoprotein (HDL), phospholipids can be used in an amount of several tens to several hundreds of moles 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 occurring 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 occurring HDL, but although the density can be adjusted to a desired value during production, the density is preferably 1.063 g / mL or higher.

[0019] Apolipoproteins include, but are not limited to, apolipoproteins generally known to be contained in natural lipoproteins and genetically modified apolipoproteins thereof. Among these, preferred apolipoproteins are apolipoproteins known to be contained in high-density lipoprotein (HDL) and genetically modified apolipoproteins thereof. Examples of apolipoproteins include proteins belonging to the groups of apolipoproteins A to F, H, J, L-I, M, and O. Preferred are one or more apolipoproteins selected from the group consisting of apolipoprotein A-I (apoA-I), apolipoprotein A-II (apoA-II), apolipoprotein C (apoC), apolipoprotein E (apoE), and genetically modified apolipoproteins thereof, and more preferred are one or two apolipoproteins selected from apolipoprotein A-I (apoA-I) and genetically modified apolipoproteins thereof. Genetically modified apolipoproteins refer to mutants or analogs (i.e., functional equivalents) that have the same function as an apolipoprotein (e.g., lipid-binding function). Genetically modified apolipoproteins include, for example, partial fragments of apolipoproteins and apolipoproteins that are combinations of these fragments. An example of a genetically modified apolipoprotein of apolipoprotein AI is apoA-I lacking 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 a vascular endothelial cell-targeting peptide or aptamer and a cell affinity peptide is also referred to as a modified apolipoprotein or a modified apolipoprotein.

[0021] A 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. The 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 β ３ It is known that integrins, aminopeptidase A, aminopeptidase N (APN; also known as CD13), vascular endothelial growth factor (VEGF) receptors, etc. are expressed in neovascular endothelial cells (J. Lahdenranta et al. (2007) The FASEB Journal; 21: 3272-3278). Therefore, peptides and aptamers targeting neovascular endothelial cells include antibodies, fragments thereof, and aptamers that specifically bind to these molecules.

[0022] Examples of the neovascular endothelial cell targeting peptide include 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 and α receptor, respectively, on tumor vascular endothelial cells. ｖ β ３ These molecules were discovered as tumor neovascularization targeting peptides that bind to integrin or aminopeptidase A, and have 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 their ability to bind to neovascularization. For example, NGR-containing peptides may contain only NGR or X-type peptides with other amino acids attached either before or after NGR, or both. ｍ NGRY ｎHere, X is any amino acid, m is an integer from 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 from 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). The presence of cysteines (C) before and after NGR allows for the formation of S—S bonds upon oxidation, resulting in cyclization of the NGR moiety, thereby enhancing receptor binding activity.

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

[0024] Examples of antibodies that specifically bind to a molecule selected from VEGF receptors include bevacizumab (anti-VEGF humanized monoclonal antibody), etc. Furthermore, antibodies can include variable domain of heavy chain (VHH) antibodies, and examples of antibody fragments can 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 may be the Apt. aptamer described in NUCLEIC ACID THERAPEUTICS, Volume 25, Number 5, pages 227-234, 2015. ８０ｍｅｒ , Apt Ｍ８０ｍｅｒ and APT Ｄｉｖａｌｅｎｔ (See Table 1 below). ８０ｍｅｒ The number 5 in the base sequence represents a benzyl-containing base (BndU). Ｍ８０ｍｅｒ is shown in SEQ ID NO:5.

[0026]

[0027] A cytotropic peptide (hereinafter sometimes abbreviated as CP) refers to a peptide that exhibits cytotropy, capable of binding to a cell membrane and then being transported into cells. In this specification, cytotropy 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 artificially produced synthetic peptides (e.g., chimeric peptides). CPs are classified into basic cytotropic peptides, amphipathic cytotropic peptides, and hydrophobic cytotropic peptides based on their chemical properties defined by their amino acid sequences. Examples of basic cytotropic 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, polyarginine (R8), and LL-37 derived from an antibacterial peptide. Examples of amphipathic cytotropic peptides include chimeric peptides such as transportan and Pep-1, as well as modified or shuffled PEN sequences. Examples of hydrophobic cytotropic 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 these, 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, and is preferably one or more selected from the group consisting of AT peptide, penetratin (PEN), polyarginine (R8), and LL-37, with penetratin (PEN) being particularly preferred.

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

[0030] In modified apolipoproteins (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 be bound to the N-terminus of the apolipoprotein and the CP be bound to the C-terminus of the apolipoprotein. Alternatively, the VTP may be bound to the C-terminus of the apolipoprotein and the CP may be bound to the N-terminus of the apolipoprotein. Alternatively, both the VTP and the CP may 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 may be bound consecutively, and when two or more CPs are bound, two or more CPs may be bound consecutively. Alternatively, the VTP and the CP may be bound to the N-terminus and the C-terminus of the apolipoprotein, respectively. Binding two or more VTPs and / or CPs to one apolipoprotein may enhance vascular targeting ability and / or cell affinity. The 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, VTP and CP can be bound to apolipoproteins by chemical synthesis (e.g., coupling methods) or biological methods (e.g., genetic engineering methods). When VTP and CP are artificially bound to apolipoproteins by genetic engineering methods using a transforming (fusion) gene, VTP and CP are bound to apolipoproteins by "fusion." The functional groups and positions on the apolipoprotein to be fused can be modified appropriately 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] The modified apolipoprotein preferably contains VTP and CP fused to the apolipoprotein at 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 VTP and / or CP fused to the apolipoprotein at a molar ratio of at least twice the amount.

[0034] The modified high-density lipoprotein includes a phospholipid. Phospholipid means a lipid having one or more phosphate ester moieties. In this specification, phospholipids include phospholipids generally known to be contained in natural lipoproteins. Phospholipids known to be contained in high-density lipoproteins (HDL) are preferred, but are not limited to these. For example, sphingolipids with a sphingosine skeleton are included, and the number of carbon atoms in the acyl group is preferably 12 to 18, and more preferably 14 to 18. For example, phosphatidylglycerol, phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine are included, with phosphatidylcholine being preferred. Specific examples include, but are not limited to, dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), egg phosphatidylcholine (PC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), and hydrogenated soy phosphatidylcholine (HSPC). Note that the hydrogenated soy phosphatidylcholine (HSPC) may contain approximately 85% distearoylphosphatidylcholine (DSPC). The phospholipid may be one to which a saturated fatty acid is bound, or may be one or more selected from the group consisting of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), and hydrogenated soybean phosphatidylcholine (HSPC), or may be one or more selected from distearoylphosphatidylcholine (DSPC) and hydrogenated soybean phosphatidylcholine (HSPC). For example, it is believed that the use of a phospholipid to which a saturated fatty acid is bound can more effectively enhance the ability to generate active oxygen at the lesion site. Note that the phospholipid may be used alone or in combination of two or more types.

[0035] Phospholipids, for example, allow easy adjustment of the hardness of the lipid membrane and can form a stabilized complex. Furthermore, phospholipids play a role in contributing to the encapsulation of compounds such as drugs. Furthermore, cholesterol can be added to phospholipids for use. By incorporating cholesterol, the hardness of the lipid membrane formed by the phospholipids can be adjusted. Adding cholesterol to phospholipids can also interact with active ingredients such as drugs, thereby adjusting the encapsulation efficiency 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 phospholipid content 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 phospholipid content 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 phospholipid content 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 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 modified apolipoprotein.

[0037] The photosensitizer-containing lipoprotein of this embodiment contains one or more molecules of the photosensitizer per modified high-density lipoprotein particle. The photosensitizer generates reactive oxygen species and heat upon light irradiation. In the treatment of age-related macular degeneration, the reactive oxygen species and heat generated from the photosensitizer can block neovascularization that has developed in the choroid, located outside the retina, thereby halting the progression of the disease. In particular, the photosensitizer used in this embodiment is preferably one that photogenerates reactive oxygen species.

[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 approximately 690 nm, that of indocyanine green (ICG) is approximately 800 nm, and that of porfimer sodium is approximately 630 nm.

[0039] Examples of photosensitizers include porphyrin compounds, chlorin compounds, bacteriochlorin compounds, phthalocyanine compounds, cyanine dyes, etc. More specifically, photosensitizers include hematoporphyrin derivatives, porfimer sodium, talaporfin sodium, protoporphyrin IX (PpIX), pheophorbide, indocyanine green, aluminum phthalocyanine tetrasulfate (AlPcS), etc. ４ ), tin etiopurpurin (SnET ２ ), zinc(II) octadecylphthalocyanine (ZnOPPc), purpurinimide, azachlorin, zinc etiopurpurin (ZnET ２), phthalocyanine (Pc), cadmium texaphyrin (CdTX), texaphyrin, zinc tetraphyptaloporphyrin (ZnTNP), verdein, 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, IR-700, and the like. These photosensitizers may be contained as part of a complex, and may form a complex by, for example, binding of the photosensitizer with an antibody, etc. Among these, the photosensitizer 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. When the photosensitizer has a hydrophobic porphyrin ring and a hydrophilic group, the photosensitizer is easily stably encapsulated in the phospholipid. Examples of the hydrophilic group that the porphyrin compound can have include a carboxylic acid group, a sulfonic acid group, a phosphoric acid group, a phosphonic acid group, and a hydroxy 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 calculated 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. Also, 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 lipoproteins (hereinafter sometimes abbreviated as vcHDL) containing modified apolipoproteins and phospholipids, the apolipoproteins contain VTP in addition to CP, which allows vcHDL to act as a drug delivery carrier in the posterior segment of the eye and target neovascularization. This allows vcHDL to have excellent delivery capabilities to neovascularization in the posterior segment of the eye, which is an area affected by age-related macular degeneration, which is believed to be caused by abnormal angiogenesis. Therefore, by loading a photosensitizer, a drug for treating age-related macular degeneration, onto vcHDL, the drug (photosensitizer) can be selectively delivered to areas affected by age-related macular degeneration. The photosensitizer-containing lipoprotein of this embodiment is the modified high-density lipoprotein (vcHDL) loaded with a photosensitizer, allowing the drug to be selectively delivered to areas affected by age-related macular degeneration. This allows for increased drug (photosensitizer) delivery efficiency. In this specification, the delivery efficiency of a drug (photosensitizer) refers to the proportion of the administered drug (photosensitizer) that reaches the neovascularization in the posterior segment of the eye, which is the site affected by age-related macular degeneration. Increasing the delivery efficiency of a drug (photosensitizer) makes it possible to increase the amount of drug (photosensitizer) that reaches the neovascularization in the posterior segment of the eye, thereby reducing the light irradiation intensity and further improving the safety of treatment. Furthermore, by selectively delivering the drug to the affected area, the occurrence of phototoxicity (side effects) can be suppressed even when light is irradiated to areas other than the lesion. This allows for a wider range of light irradiation sites, significantly simplifying the treatment of age-related macular degeneration. Furthermore, increasing the retention of the drug at the diseased site not only alleviates the time limit (15 minutes) between intravenous administration and laser light irradiation, but also promotes the clearance of the photosensitizer from normal tissue, thereby reducing 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. The smaller the size of the photosensitizer-containing lipoprotein, the better its ability to reach the posterior segment of the eye is expected. 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 diameter of the photosensitizer-containing lipoprotein is small, on the nanometer order, and therefore, in this specification, the photosensitizer-containing lipoprotein is sometimes referred to as a nanoparticle.

[0047] The volume-average particle size and surface charge of the photosensitizer-containing lipoprotein 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 capacity 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 capacity of the photosensitizer-containing lipoprotein is not particularly limited, but is preferably 3000% or less, for example.

[0049]

[0050] The ability to generate reactive oxygen (singlet oxygen) is １ O ２Detection was performed using Singlet Oxygen Sensor Green Reagent (SOSG; Invitrogen Thermo Fisher Scientific Inc., USA), which selectively reacts and emits fluorescence. Specifically, a solution containing a photosensitizer-containing lipoprotein and SOSG was prepared, and this solution was 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 using 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 determined as F ０ , the integrated fluorescence intensity after light irradiation is F, and from the above formula １ O ２ The productivity (%) is calculated.

[0051] In this embodiment, for example, by using a phospholipid in which saturated fatty acids are bonded, the ability to generate active oxygen (singlet oxygen) can be more effectively increased, and the ability to generate active oxygen at the lesion site can be more effectively increased. If the ability to generate active oxygen (singlet oxygen) can be increased, it becomes possible to reduce the amount of drug loaded or administered.

[0052] (Method for Producing Photosensitizer-Containing Lipoprotein) This embodiment relates to a method for producing a photosensitizer-containing lipoprotein, comprising the following steps i) to v): i) fusing VTP and CP to an apolipoprotein by genetic engineering to obtain a fusion protein (modified apolipoprotein); ii) mixing the obtained fusion protein with liposomes containing phospholipids to produce crude vcHDL (spontaneous interaction method); or alternatively, producing crude vcHDL by mixing with cholic acid micelles without going through liposome formation (cholate-dialysis method); iii) purifying the crude vcHDL by removing unreacted phospholipids and apolipoprotein; and iv) mixing the photosensitizer with vcHDL 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), genetic engineering techniques can be used. Examples of genetic engineering techniques include DNA editing using methylase or DNA polymerase I / DNA ligase, and polymerase chain reaction (PCR). Specifically, a penetratin (PEN) peptide gene with a Pst I recognition sequence at the 5' end and an Xba I recognition sequence at the 3' end is inserted into the Pst I and Xba I restriction enzyme recognition sites of the E. coli expression vector pCOLD I using DNA ligase. Next, a gene with a Kpn I restriction enzyme recognition sequence at the 5' end and a Pst I restriction enzyme recognition sequence at the 3' end of the N-terminal 44 amino acid deleted apoA-I gene is prepared by PCR, and inserted between the Kpn I and Pst I recognition sites of the pCOLD I using DNA ligase. By the above procedures, 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-binding apolipoprotein (modified apolipoprotein), the fusion protein gene is prepared by introducing the VTP gene and CP gene in equimolar ratios 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, several tens to several hundred times, for example, about 5 to about 2,000 times, about 20 to about 500 times, 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 described above can be adjusted by adjusting the molar ratio of the fusion protein to the phospholipid and 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 1 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 1 mol of phospholipid.

[0058] The temperature during mixing in step iv) is preferably 30 to 70°C, and more preferably 40 to 60°C. The mixing time is preferably 1 to 100 minutes, and 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 also be shortened. This allows for increased production efficiency of 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 Preparation) This embodiment relates to a photodynamic therapy preparation containing a photosensitizer-containing lipoprotein. Photodynamic therapy involves administering the 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, whereby the photosensitizer-containing lipoprotein is selectively delivered to the lesion (e.g., tumor tissue or neovascularization in the posterior segment of the eye, which is the site of age-related macular degeneration). The lesion to which the photosensitizer-containing lipoprotein has reached is then irradiated with light (laser light). This generates active oxygen and heat from the photosensitizer contained in the photosensitizer-containing lipoprotein, damaging the lesion and destroying the diseased tissue. Thus, photodynamic therapy is a treatment method that combines the delivery of a lipoprotein containing a photosensitizer to the lesion site with the irradiation of the lesion site with light (laser light).

[0061] When the photodynamic therapy preparation is used, for example, for 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 neovascularization, and the neovascularization includes α ｖ β ３ Since integrins, aminopeptidase A, aminopeptidase N (APN; also known as CD13), or vascular endothelial growth factor (VEGF) receptors are expressed in these cells, the peptides and aptamers targeting neovascular endothelial cells contained in the photosensitive substance-containing lipoproteins specifically bind to these molecules.

[0062] The photodynamic therapy formulation of this embodiment is particularly preferably used for treating age-related macular degeneration. That is, in a preferred embodiment, the photodynamic therapy formulation of this embodiment is a formulation for treating 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 segment of the eye, which is the affected area of ​​age-related macular degeneration. The site of neovascularization reached by the photosensitizer-containing lipoprotein is then irradiated with light (laser light). This generates active oxygen from the photosensitizer contained in the photosensitizer-containing lipoprotein, which damages the neovascularization and causes vascular occlusion.

[0063] The photodynamic therapy formulation of this embodiment can also be used to treat malignant tumors. In a preferred embodiment, the photodynamic therapy formulation of this embodiment is a formulation for treating malignant tumors. In this case, in photodynamic therapy, a photosensitizer-containing lipoprotein is administered to a subject with a malignant tumor, and the photosensitizer-containing lipoprotein is selectively delivered to neovascularization caused by the malignant tumor. The site of neovascularization to which the photosensitizer-containing lipoprotein has reached is then irradiated with light (laser light irradiation). This generates active oxygen from the photosensitizer contained in the photosensitizer-containing lipoprotein, which damages the neovascularization and causes vascular occlusion.

[0064] In addition to the photosensitizer-containing lipoprotein, the photodynamic therapy preparation may further contain a pharmaceutically acceptable additive. Pharmaceutically acceptable additives include various active ingredients or medicinal ingredients (including pharmacologically active ingredients and physiologically active ingredients) and additives depending on the intended use, as long as they do not impair the effects of the present invention. Examples of additives include buffers (e.g., sodium phosphate), isotonicity agents (e.g., sodium chloride), pH adjusters (e.g., boric acid), preservatives (e.g., benzalkonium chloride), stabilizers (e.g., mannitol), thickeners (e.g., sodium alginate), chelating agents (e.g., sodium edetate), surfactants (e.g., polyoxyethylene sorbitan monooleate), absorption enhancers (e.g., sodium oleate), and flavorings (e.g., menthol).

[0065] Furthermore, the photodynamic therapy preparation may contain, in addition to the 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. 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), pazopanib, and the like.

[0066] The photodynamic therapy preparation 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 formulation of this embodiment is preferably an injectable formulation, and the photodynamic therapy formulation is preferably administered intravenously. When the photodynamic therapy formulation is administered intravenously, the photosensitizer-containing lipoprotein contained in the photodynamic therapy formulation selectively accumulates at the lesion. Laser light is then irradiated onto the lesion where the photosensitizer has accumulated, causing the photosensitizer to generate reactive oxygen and heat, which damage the lesion. This can halt the progression of the disease. For example, when the photodynamic therapy formulation is used to treat age-related macular degeneration, when the photodynamic therapy formulation is administered intravenously, the photosensitizer-containing lipoprotein contained in the photodynamic therapy formulation selectively accumulates in neovascularization in the posterior segment of the eye, which is the lesion site of age-related macular degeneration. Laser light is then irradiated onto the lesion where the photosensitizer has accumulated, causing the photosensitizer to generate reactive oxygen and heat, which damage the neovascularization and occlude the blood vessels. Furthermore, when a photodynamic therapy preparation is used to treat malignant tumors, when the photodynamic therapy preparation is injected intravenously, the photosensitizer-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 photosensitizer has accumulated with laser light, active oxygen and heat are generated from the photosensitizer, which damage the neovascularization and block the blood vessels.

[0068] The photosensitizer-containing lipoprotein contained in the photodynamic therapy formulation of this embodiment can accumulate with high selectivity, particularly in neovascularization in the posterior segment of the eye, which is an area affected by age-related macular degeneration, or in neovascularization caused by malignant tumors. This increases 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. Furthermore, the photodynamic therapy formulation of this embodiment has a longer retention time at the diseased site compared to conventional drugs, thereby ensuring a longer time period during which light can be irradiated. This allows, for example, the photodynamic therapy formulation to be administered outside of a laser light irradiation room, thereby reducing the burden on medical professionals performing photodynamic therapy and further increasing the therapeutic efficiency of photodynamic therapy.

[0069] (Drug Delivery Method) This embodiment may relate to a drug delivery method to neovascularization in the posterior segment of the eye using the above-described photosensitizer-containing lipoprotein. Also, this embodiment relates to a drug delivery method to neovascularization in the posterior segment of the eye using the above-described formulation for photodynamic therapy.

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

[0071] As described above, modified high-density lipoprotein (vcHDL), which contains modified apolipoproteins and phospholipids, can act as a drug delivery carrier for the posterior segment of the eye or for malignant tumors, since the apolipoprotein contains VTP in addition to CP, and can selectively deliver photosensitizers to neovascularization. Therefore, by applying the drug delivery method or administering the above-mentioned photodynamic therapy preparation to patients suffering from or at risk of suffering from age-related macular degeneration, it becomes possible to diagnose, prevent, or treat age-related macular degeneration. Furthermore, by applying the drug delivery method or administering the above-mentioned photodynamic therapy preparation to patients with malignant tumors, cancer treatments such as shrinkage, elimination, or suppression of metastasis of malignant tumors become possible.

[0072] The drug delivery method of this embodiment uses modified high-density lipoprotein (vcHDL), which contains modified apolipoproteins and phospholipids, as a drug delivery carrier, thereby enabling selective delivery of a photosensitizer (drug) to neovascularization in the posterior segment of the eye, an area affected by age-related macular degeneration (AMD), which is believed to be caused by abnormal angiogenesis. Furthermore, the drug delivery method of this embodiment allows selective delivery of a photosensitizer (drug) to neovascularization sites resulting from malignant tumors. This increases the delivery efficiency of the photosensitizer (drug). Furthermore, by increasing the selectivity of the drug delivery site, phototoxicity (side effects) can be suppressed even when light is irradiated to areas other than the lesion site. This allows for a wider range of light irradiation sites, significantly simplifying AMD and cancer treatments. Furthermore, increasing the retention of the drug at the diseased site not only alleviates the time limit (15 minutes) between intravenous administration and laser light irradiation, but also promotes clearance of the photosensitizer from normal tissue, thereby reducing side effects.

[0073] In this embodiment, the modified high-density lipoprotein (vcHDL) itself, which does not contain a photosensitizer (drug), can exert anti-inflammatory and anti-angiogenic effects, and therefore the drug delivery method itself can be used to treat or prevent age-related macular degeneration and cancer.

[0074] (Method for Occlusion of Posterior Segment Neovascularization) This embodiment relates to a method for occlusion of posterior segment neovascularization, which includes administering the above-described photosensitizer-containing lipoprotein to a subject suffering from age-related macular degeneration and irradiating a site of posterior segment neovascularization with light. The method for occlusion of posterior segment neovascularization can be applied to humans or mammals other than humans, but may exclude treatment methods for humans. In the method for occlusion of posterior segment neovascularization, the above-described photosensitizer-containing lipoprotein or photodynamic therapy formulation is, for example, intravenously injected, so that the photosensitizer-containing lipoprotein reaches the site of posterior segment neovascularization. Because the photosensitizer-containing lipoprotein contains a modified apolipoprotein containing VTP and CP, the photosensitizer-containing lipoprotein is selectively delivered to the neovascularization site in the posterior segment of the eye, which is the site affected by age-related macular degeneration. Light (laser light) is then irradiated to the site of neovascularization where the photosensitizer-containing lipoprotein has reached. This causes the generation of active oxygen from the photosensitive substance contained in the lipoprotein, which damages the newly formed blood vessels and blocks them.

[0075] This embodiment also relates to a method for blocking neovascularization at a malignant tumor site, which includes administering the photosensitizer-containing lipoprotein described above to a subject with a malignant tumor and irradiating the site of neovascularization at the 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 exclude human treatment methods. In the method for blocking neovascularization at a malignant tumor site, the photosensitizer-containing lipoprotein or photodynamic therapy formulation described above is delivered to the site of neovascularization at the malignant tumor site by, for example, intravenous injection. Because the photosensitizer-containing lipoprotein contains modified apolipoproteins including VTP and CP, the photosensitizer-containing lipoprotein is selectively delivered to neovascularization caused by the malignant tumor. The site of neovascularization at which the photosensitizer-containing lipoprotein has reached is then irradiated with light (laser light). This generates reactive oxygen from the photosensitizer contained in the photosensitizer-containing lipoprotein, damaging the neovascularization and resulting in vascular occlusion. This makes it possible to treat cancer by shrinking or eliminating malignant tumors or inhibiting metastasis.

[0076] The light to be irradiated may be light emitted by, for example, a semiconductor laser, a dye laser, etc. Specifically, Nd-YAG (yttrium aluminum garnet) laser, CO ２ Examples of lasers include laser, argon dye laser, and excimer laser. For example, light irradiation (laser light irradiation) can be performed in accordance with the treatment guidelines disclosed in Non-Patent Document 1, etc. 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 (83 seconds).

[0077] When the photosensitizer-containing lipoprotein or photodynamic therapy formulation of the present embodiment is intravenously injected, its blood residence time 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 photodynamic therapy formulation. When a conventional photodynamic therapy formulation 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 formulation. Therefore, by using the photosensitizer-containing lipoprotein or photodynamic therapy formulation 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 one or more times.

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

[0080] The features of the present invention will be explained in more detail below with reference to examples and comparative examples. The materials, amounts used, ratios, treatment contents, treatment procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed 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 N-terminal 44 amino acid-deleted human apoA-I. Furthermore, RQIKIWFQNRRMKWKKK (SEQ ID NO: 1), in which a K was added to the C-terminus of PEN, was ligated to the C-terminus of the N-terminal 44 amino acid-deleted human apoA-I. The molecular weight of the NGR and PEN-fused apoA-I was approximately 29,319 Da. Furthermore, human apoA-I lacking 44 amino acids at the N-terminus was used as a template, and RQIKIWFQNRRMKWKKK (SEQ ID NO: 1), in which PEN has a K added to the C-terminus, was ligated to the C-terminus of the N-terminal 44 amino acid-deleted human apoA-I 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 variants were expressed in BL21(DE3) (Novagen, Addison, WI, USA) and purified under guanidine-denaturing conditions on Ni Sepharose High-Performance resin packed in a Tricorn 10 / 100 column on an AKTAstart chromatography system (Cytiva, Tokyo, Japan).

[0083] Preparation of Modified High-Density Lipoproteins Distearoylphosphatidylcholine (DSPC) was mixed with sodium cholate at a molar ratio of 1:3.6, and the DSPC was dispersed by bath sonication at 65°C for 10 min (Elma Schmidbauer GmbH, Mannheim, Germany). Subsequently, each apoA-I variant was dissolved in PBS containing 4 M urea, mixed with a DSPC dispersion (DSPC:apoA-I variant = 80:1 (mol)), and allowed to react at 55°C for 30 min. After the reaction, the mixture was dialyzed 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. Dialysis of the mixture of DSPC, NGR, and PEN-fused apoA-I was initiated at 55°C and then allowed to cool to room temperature. The reaction solution inside the dialysis membrane was transferred to a microtube and centrifuged (20,000 x g, 24°C, 30 min). The supernatant was collected and purified by size exclusion chromatography using an HPLC (Shimadzu Corporation, Kyoto, Japan) connected to a Superose 6prep grade packed column (Cytiva). Modified high-density lipoprotein (vcHDL) containing apoA-I variants (hereinafter, the HDL variants using PEN-fused apoA-I are referred to as eLP1, and the HDL variants using NGR and PEN-fused apoA-I are referred to as eLP2) was eluted 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 at room temperature. The eluate of the modified HDL fraction (50-75 min) was collected and dialyzed overnight at room temperature against 3 L of PBS 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 minutes), and the supernatant was stored in a low protein-binding tube.

[0084] Loading of verteporfin (VP) onto modified high-density lipoproteins. Modified HDL was mixed with VP dissolved in DMSO at a VP:lipid molar ratio of 1:10 and incubated at 55°C for 30 min. Purification was performed by size-exclusion chromatography equilibrated with PBS. VP-loaded modified high-density lipoproteins (VP@eLP1 and 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. The ethanol was evaporated under reduced pressure at 35°C using a rotary evaporator R-300 (Nihon Buchi, Tokyo, Japan) to form a lipid film. The resulting lipid film 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 film, and the mixture was subjected to bath sonication 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 Vis (Figure 1).

[0086] (Evaluation) Characterization of VP-loaded modified high-density lipoprotein and Vis The protein concentration in VP-loaded 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, 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 concentrations in VP-loaded modified high-density lipoproteins and Vis were 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 little difference in the absorption peaks of VP in the VP-loaded modified high-density lipoprotein and Vis.

[0087]

[0088] Singlet oxygen production ability Singlet oxygen ( １ O ２ )teeth １ O ２Detection was performed using Singlet Oxygen Sensor Green Reagent (SOSG; Invitrogen Thermo Fisher Scientific Inc., USA), which selectively reacts and emits fluorescence. A solution containing 10 μg / mL VP-containing nanoparticles (VP@eLP1, VP@eLP2, or Vis) and 10 mM SOSG was prepared. Each nanoparticle dispersion 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 spectra of the solution before and after irradiation were measured using a plate reader (TECAN) (Ex. 480 nm / Em. 500-560 nm), and the integrated fluorescence intensity was calculated. The integrated fluorescence intensity before irradiation was measured as F ０ , and the integrated fluorescence intensity after light irradiation is F, and from the following equation １ O ２ The production potential was calculated. １ O ２ The calculation results of the production potential are shown in Figure 3. １ O ２ The reason why Vis has a lower production capacity than VP@eLP1 and VP@eLP2 is that the lipids that make up Vis contain unsaturated fatty acids, and these unsaturated fatty acids １ O ２ This is thought to be due to oxidation by reaction with

[0089]

[0090] Cell Culture HUVECs were cultured in 100 mm dishes in 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% CO). ２) and cultured at 100°C for 30 min. The medium was changed every 2 days. Subculture was performed after confirming that each cell line had 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 fresh medium and transferred to a new dish for subculture.

[0091] Analysis of the interaction of each nanoparticle with vascular endothelial cells HUVECs were used as a normal vascular endothelial cell model and cultured in EBM2 containing 2% FBS. To enhance CD13 expression, basic fibroblast growth factor (bFGF) was added to a final concentration of 50 ng / mL. HUVECs were cultured in a triple-well glass base dish at a concentration of 1 × 10 ５ The cells were seeded at a density of 1000 cells / mL and incubated at 37°C in CO ２ The cells were cultured statically in an incubator for 24 hours. Each nanoparticle (eLP1, eLP2, Vis containing 1 μg / mL VP) was applied to the adherent cells. 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 / 1.4 oil DIC M27) ​​was used for observation. To quantify the amount of intracellular VP, 1 x 10 nanoparticles were seeded onto a 12-well plate. ５HUVECs (12,000 × g, 4 ° C, 10 min) were used. After treating the cells with each nanoparticle as described above and detaching them with a cell scraper, the resulting cell pellet was centrifuged (1,000 × g, 4 ° C, 5 min). The resulting cell pellet was dissolved in 400 μL of RIPA buffer (25 mM Tris HCl (pH 7.6), 0.1% SDS, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate) and subjected to two freeze-thaw cycles (-80 ° C; 30 min, 37 ° C; 30 min). The protein concentration in the supernatant obtained by centrifugation (12,000 × g, 4 ° C, 10 min) was measured using a BCA protein assay kit, and the VP concentration was quantified by fluorescence spectroscopy using a plate reader. The intracellular VP weight was calculated by normalizing the VP weight with the cellular protein weight (Figure 4). VP@eLP2 showed the same cellular uptake of VP as Vis.

[0092] Phototherapeutic effect of each nanoparticle Photocytotoxicity test of each nanoparticle was performed using Cytotoxicity LDH Assay Kit-WST (DOJINDO LABORATORIES, Kumamoto, Japan). HUVECs were plated at 1 × 10 in a 96-well plate. ５ The cells were seeded at a density of 100 ng / mL and stimulated with bFGF in the same manner as above, followed by treatment with each of the nanoparticles containing 100 ng / mL of VP. After 60 min, the cells were washed with D-PBS containing heparin, and fresh medium was added. Light irradiation was performed using a xenon lamp (31 mW, 1 min). After light irradiation, the medium was replaced and the cells were incubated at 37°C in CO. ２ The cells were cultured in an incubator 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 (i.e., phototherapeutic effect).

[0093] Blood retention of each nanoparticle: The animal experiments using mice conducted in this study were conducted in accordance with the ethical guidelines established 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 into the tail vein of the mice at a dose of 2 mgVP / kg body weight. Blood samples were collected 0.16, 1, 4, 7, and 24 hours after administration and mixed with tubes containing 3.8% sodium citrate (Iwaki Seiyaku Co., Ltd., Tokyo, Japan). After centrifugation (3000 rpm, 4°C, 5 min) to separate plasma and blood cell components, the supernatant plasma was collected, pre-frozen at -80°C, and then freeze-dried. Methanol was added to all freeze-dried plasma samples, followed by vortex mixing, bath sonication for 2 min, and then vortex mixing again. The samples were stirred in a tube rotator (4°C, 1 h). After bath sonication for 2 min, vortex mixing was again performed. 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 elution was performed with (A) 0.05 mM NaH ２ P.O. ４A gradient mobile phase consisting of (A) MeOH was used. 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 t １／２ is 5.2±1.8h, and the t １／２ is 4.8±0.5h, and the t １／２ The 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. Mice were anesthetized with 10 mg / kg xylazine and 80 mg / kg ketamine. Pupils were fully dilated with 5% phenylephrine hydrochloride / 0.8% tropicamide (Sandol P, Rhoto Nitten Co., Ltd., Nagoya, Japan). Cover glasses were placed on the mouse corneas and used as contact lenses. Laser photocoagulation was performed using a photocoagulator (DC-3000; Nidek, Gamagori, Japan) set at 75 lm, 0.05 seconds, and 150 mW. CNV was induced by laser irradiation at four locations 4-5 disk diameters away from the optic nerve head. Seven days after laser irradiation, each nanoparticle (Vis or VP@eLP2) was administered to mice via the tail vein at a dose of 10 mg / kg (equivalent to VP). The eyes were enucleated 15 and 60 min after administration. After enucleation, the eyes were fixed in 4% PFA and blocked. Immunostaining was performed 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 dissected to prepare flat mounts. Fluorescence from CNV (Ex. 488 nm / Em. 495-530 nm) and VP within CNV (Ex. 405 nm / Em. 610-700 nm) were observed using a confocal microscope (Figure 7). A 10x dry objective (Plan-Apochromat 20x / 0.8) was used for observation. The fluorescence intensity of VP within 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 exhibited significantly higher CNV retention than Vis.

[0095] Photodynamic therapy (PDT) effect 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 a dose of 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 Visrus PDT system 690S (Carl Zeiss) with a 7000 μm ２ , 60s, 600mW / cm ２ The entire eyeball was irradiated with laser light at a setting of 0.05°C. Seven days later, the eyeballs were enucleated, fixed in 4% PFA, and blocked. Immunostaining was performed using rat anti-CD102 antibody and Alexa 488-labeled anti-rat IgG antibody as 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 was 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, demonstrating a high phototherapeutic effect. Furthermore, mice administered VP@eLP2 showed a reduction in the CNV area 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 analysis was performed using Microsoft Excel. Differences between the mean values ​​of two or more groups were determined by Welch's t-test. Differences between the mean values ​​of three or more groups were determined by the Tukey or Tukey-Kramer test. In all statistical analyses, p<0.05 was considered statistically significant. All data were graphed as mean ± standard deviation.

[0097] Discussion: As shown by the above results, VP@eLP2 exhibits higher CNV selectivity and significantly higher CNV retention than Vis. The high CNV selectivity of VP@eLP2 can reduce side effects (phototoxicity). Furthermore, the high CNV retention of VP@eLP2 can extend 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 effect) test The following test was conducted to confirm that phototoxicity (side effects) is reduced when VP@eLP2 is used. Vis and VP@eLP2 were administered via the tail vein to normal C57BL / 6JJmsSlc mice (male, 8-10 weeks old) at a VP equivalent of 2 mg / kg. As a control, PBS was administered to the mice via the tail vein. Laser light irradiation was performed 15 minutes after administration. Laser light irradiation was performed using a Bislas PDT System 690S (Carl Zeiss) at a 7000 μm ２ , 60s, 600mW / cm ２The laser was set to 0.05°C and the entire eyeball was irradiated with laser light. Seven days later, the eyeballs were enucleated, fixed in 4% paraformaldehyde (PFA, Nacalai Tesque, Inc.) (4°C, overnight), and dehydrated in 20% sucrose solution (4°C, 24 hours). The dehydrated eyeballs were embedded in OCT compound (Miles, Elkhart, IN, USA) and frozen in liquid nitrogen. The frozen eyeballs were 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 peripheral retina of 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. However, when mice administered Vis were irradiated with laser light, the entire retina disappeared and the eyeball became deformed. This is thought to be due to the low CNV selectivity of Vis, which caused retinal damage (phototoxicity) when irradiated with laser light.

[0099] VP@eLP2 uptake by HUVECs in the presence of CD13 inhibitors HUVECs were used as a normal vascular endothelial cell model. Culture was performed in EBM2 containing 2% FBS. To enhance CD13 expression, basic fibroblast growth factor (bFGF) was added to the medium to a final concentration of 50 ng / mL. Furthermore, 250 μM bestatin (Sigma-Aldrich, St. Louis, MO) was added as a CD13 inhibitor. HUVECs were cultured at 1 × 10 in a triple-well glass-based dish. ５ The cells were seeded at a density of 1000 cells / mL and incubated at 37°C in CO ２The cells were cultured in an incubator for 24 hours. VP@eLP2, containing 1 μg / mL VP and fluorescent lipid (NBD), was applied to the adherent cells. After 60 minutes, 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] Discussion: In the presence of bestatin, NBD fluorescence intensity did not change significantly. On the other hand, VP fluorescence intensity significantly increased. This difference may be explained by the function of CD13 in enhancing the expression of drug efflux transporters (Guo et al., Oncol. Res. 2020, 28, 533-540). Assuming that VP is a better substrate for drug efflux transporters than NBD, the inhibition of drug efflux transporter expression by bestatin may have enhanced the intracellular retention of VP beyond the inhibition of cellular uptake of VP@eLP2. In other words, the NGR peptide appears to have 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). HUVECs were seeded at a density of 1 x 10 in a 6-well plate. ５ After 24 hours, the culture supernatant was removed, and the cells were washed with 1 mL of D-PBS. 2 mL of VP@eLP2 (100 μM VP) containing 100 μM VP was added, and the cells were incubated for 24 hours at 20°C. ２The plate was incubated at 37°C for 1 hour in an incubator. The cells were then washed with 1 mL of D-PBS, and the plate was immediately cooled on ice. Total RNA was extracted from the cells 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 RNase H Plus], TaKaRa Bio, Inc.) and a Thermal Cycler Dice Real Time System (TaKaRa Bio, Inc.). -ΔΔＣｔ The relative changes in gene expression were analyzed using the ELISA 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] Treatment with VP@eLP2 significantly reduced MDR1 mRNA expression (Fig. 13), supporting the results above 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 in cancer model mice Human prostate cancer cell line PC-3 (RIKEN Cell Bank) was used. PC-3 was suspended in RPMI 1640 (4 × 10 ７ The tumor was then subcutaneously injected into the left and right dorsal regions of nude mice. 12 days later (day 0), the major and minor axes of the subcutaneous tumor were measured with a vernier caliper, and the tumor volume was calculated. VP@eLP2 or Vis (2 mg VP / kg) was then administered intravenously to the tail vein. One hour later, under isoflurane anesthesia, a 690 nm laser was irradiated (815 mW / cm) onto only one of the tumors on the left and right dorsal region. ２, φ5 mm, 2 min). 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) × minor axis (cm) × major axis (cm) / 2 The PDT effect was determined by normalizing the tumor volume on day 5 with the value on day 0 (Figure 14).

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

Claims

1. A photosensitive substance-containing lipoprotein comprising a modified high density lipoprotein containing an apolipoprotein and a phospholipid, and 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.

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

11. A photodynamic therapy preparation according to claim 10, for use in treating age-related macular degeneration.

12. A preparation for photodynamic therapy described in 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 ocular neovascularization with light.

13. A method for blocking neovascularization in the posterior segment of the eye, comprising administering the photosensitive substance-containing lipoprotein described in any one of claims 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. The photodynamic therapy formulation according to claim 10, for use in the treatment of malignant tumors.

15. A preparation for photodynamic therapy 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, comprising administering to a subject having a malignant tumor the photosensitive substance-containing lipoprotein described in any one of claims 1 to 9, and irradiating the site of neovascularization at the malignant tumor site with light.

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