WO2006046584A1

WO2006046584A1 - Remedy for visual cell injuries

Info

Publication number: WO2006046584A1
Application number: PCT/JP2005/019654
Authority: WO
Inventors: Noriyuki Kuno; Fumihiko Takamatsu
Original assignee: Santen Pharmaceutical Co., Ltd.
Priority date: 2004-10-26
Filing date: 2005-10-26
Publication date: 2006-05-04

Abstract

It is intended to search for a remedy which is efficacious against intractable diseases causing visual cell injuries such as retinal pigment degeneration, cone dystrophy, age-related macular degeneration, age-related macula, macular edema, retinal detachment and so on and provide a material which is useful in regenerating visual cells or functions thereof. Sustained-release compositions, wherein hepatocyte growth factor (HGF), brain-derived neurotrophic factor (BDNF) or pigment epithelium-derived factor (PEDF) is impregnated with a crosslinked gelatin hydrogel, show each an excellent effect of suppressing retinal degeneration. When such a composition is combined with fat-derived interstitial cells and subretinally administered, visual cells or the functions thereof can be regenerated and the effect of suppressing retinal degeneration can be strengthened and prolonged.

Description

Specification

Photocell disorder treatment

Technical field

[0001] The present invention relates to a sustained-release composition comprising a crosslinked gelatin hydrogel impregnated with hepatocyte growth factor (HGF), brain-derived neurotrophic factor (BDNF) or reticulopigment epithelium-derived neurotrophic factor (PEDF). The therapeutic agent of the disease which causes the photoreceptor cell disorder which consists of. The present invention also provides a sustained release composition in which a crosslinked gelatin hydrogel is impregnated with HGF, BDNF or PEDF, and a visual cell comprising a photoreceptor cell or a cell having a role of regenerating or complementing the function, or the function thereof. It is related with the material for making it.

Background art

[0002] A photoreceptor cell is a cell that converts a light stimulus to the retina into an electrical signal, and controls an important function related to optical sensation and vision. Have serious consequences. Examples of diseases that cause photoreceptor cell damage include retinal pigment degeneration, cone dystrophy, age-related macular degeneration, macular edema, retinal detachment, cancer-related retinopathy, retinal vein occlusion, and retinal pigment epithelial detachment. These diseases are refractory and are not easily cured even by the administration of existing drugs, and once denatured, the function of the detached photoreceptor cells cannot be restored. Furthermore, it is desired to regenerate the function of photoreceptor cells. As a new treatment method, regenerative medicine in which cells and tissues of own and other families are transplanted to replace and regenerate dysfunctional cells, tissues and organs has been attempted.

[0003] By the way, hepatocyte growth factor (HGF) is a strong growth-promoting factor for mature hepatocytes, and as a liver regeneration factor, it only acts on liver repair 'regeneration and angiogenesis. (Patent Document 1, Non-Patent Document 1). Patent Document 2 describes an invention related to an HGF sustained-release preparation, and an HGF sustained-release preparation obtained by combining HGF and a gelatin hydrogel having specific physical properties can be used as an angiogenesis-promoting agent or an arterial disease therapeutic agent. Is stated.

[0004] Moon-derived neurotrophic factor (BDNF) is a photoreceptor disorder Patent Document 3 describes that ophthalmic solutions or eye ointments containing BDNF are effective in treating age-related macular degeneration and retinal pigment degeneration. Yes. Patent Document 4 describes that retinal pigment epithelium-derived neurotrophic factor (PEDF) is effective in treating retinal diseases. Patent Document 5 describes an invention relating to a material for cell transplantation, such as hydrogel, and that a hydrogel containing a cell growth factor such as hepatocyte growth factor (HGF) is effective as a therapeutic agent for heart failure. Is stated. Patent Document 6 describes an invention relating to a therapeutic agent for regenerating a tissue organ composed of cells and a cell growth factor, and a therapeutic agent combining a precursor cell with bFGF (basic fibroblast growth factor). It has been reported that adipose tissue regenerates when implanted under the skin.

[0005] However, there has been no report on the pharmacological action against photoreceptor cell damage regarding a sustained release composition in which a crosslinked gelatin hydrogel is impregnated with HGF, BDNF or PEDF.

Patent Document 1: Japanese Patent Publication No. 4-72840

Patent Document 2: International Publication No. 03Z007982 Pamphlet

Patent Document 3: Japanese Patent Laid-Open No. 2003-48851

Patent Document 4: U.S. Patent No. 6451763

Patent Document 5: JP 2002-145797

Patent Document 6: Japanese Patent Laid-Open No. 2001-316285

Non-patent literature l: Proc. Natl. Acad. Sci., 90, 1937-1941 (1993)

Disclosure of the invention

Problems to be solved by the invention

[0006] It is important to find an effective therapeutic agent for refractory diseases that cause visual cell disorders such as retinitis pigmentosa, cone dystrophy, age-related macular degeneration, age-related macular disease, macular edema, and retinal detachment It is a problem. In addition, if a material useful for regenerating photoreceptor cells or their functions can be provided, it can greatly contribute to the fundamental treatment of intractable retinal diseases that cause photoreceptor damage.

Means for solving the problem

[0007] The present inventors have developed hepatocyte growth factor (HGF), brain-derived neurotrophic factor (BDNF). In addition, as a result of intensive research aimed at exploring the pharmaceutical use of a sustained-release composition in which cross-linked gelatin hydrogel is impregnated with neurotrophic factor derived from retinal pigment epithelium (PEDF), spontaneously developed retinal pigment degeneration model rats ( In pharmacological studies using RCS rats, all sustained-release compositions obtained by impregnating cross-linked gelatin hydrogel with HGF, BDNF, or PEDF exhibit an excellent inhibitory effect on photoreceptor cell degeneration. When the release composition and the adipose-derived stromal cells were combined, it was found that photoreceptor cells or their functions regenerate and the effect of suppressing photoreceptor cell degeneration is enhanced and sustained, leading to the present invention.

That is, the present invention

(1) A therapeutic agent for diseases causing visual cell damage, comprising a sustained release composition comprising a crosslinked gelatin hydrogel impregnated with at least one of the following components 1) to 3):

1) Hepatocyte growth factor (HGF)

2) Brain-derived neurotrophic factor (BDNF)

3) Neurotrophic factor derived from retinal pigment epithelium (PEDF)

(2) A therapeutic agent for diseases causing visual cell damage, comprising a sustained release composition in which at least one of the following components 1) to 3) is impregnated in a crosslinked gelatin hydrogel and the following component 4):

1) Hepatocyte growth factor (HGF)

2) Brain-derived neurotrophic factor (BDNF)

3) Neurotrophic factor derived from retinal pigment epithelium (PEDF)

4) Cells that play a role in regenerating or complementing photoreceptor cells or their functions

(3) The therapeutic agent according to (1) or (2) above, wherein the disease causing photoreceptor cell damage is a retinal degenerative disease,

(4) Before the retinal degenerative disease is retinitis pigmentosa, cone dystrophy, age-related macular degeneration, age-related macular degeneration, macular edema, retinal detachment, cancer-related retinopathy, retinal vein occlusion or retinal pigment epithelial detachment (1) to the therapeutic agent according to (3),

(5) The therapeutic agent according to (2), wherein the photoreceptor cell or the cell having a role of regenerating or complementing its function is a stromal cell,

(6) The therapeutic agent according to (5), wherein the stromal cells are adipose-derived stromal cells, and

(7) Sustained release with at least one of the following components 1) to 3) impregnated in a crosslinked gelatin hydrogel A visual cell comprising the composition and the following component 4) or a material for regenerating the function thereof:

1) Hepatocyte growth factor (HGF)

2) Brain-derived neurotrophic factor (BDNF)

3) Neurotrophic factor derived from retinal pigment epithelium (PEDF)

4) cells having a role of regenerating or complementing photoreceptor cells or their functions,

About.

[0009] The first invention is a method wherein a crosslinked gelatin hydrogel is impregnated with hepatocyte growth factor (HGF), brain-derived neurotrophic factor (BDNF) or retinal pigment epithelium-derived neurotrophic factor (PEDF). It is a therapeutic agent for diseases that cause photoreceptor cell damage that also has the ability to release composition. As is clear from the results of the pharmacological test described below, the HGF sustained release composition, the BDNF sustained release composition, and the PEDF sustained release composition all stabilize HGF, BDNF, and PEDF as well as long-term. It is effective for sustained release of these drugs while maintaining activity, and when administered to the site of photoreceptor cell damage or its surrounding tissues (subretinal, intraretinal, intravitreous, etc.), it can be administered for a long time with a single administration. Sustains the effect of suppressing retinal degeneration over time.

[0010] Hepatocyte growth factor (HGF), brain-derived neurotrophic factor (BDNF), and retinal pigment epithelium-derived neurotrophic factor (PEDF) in the present invention are all purified to such a degree that they can be used as pharmaceuticals. It is not particularly limited as long as it is a thing, and may be a natural type or a recombinant type.

[0011] Examples of natural HGF include primary culture of cultured mesenchymal cells such as liver, lung, kidney, spleen, plasma, platelet force extract, fibroblasts, vascular endothelial cells, macrophages, and cell cultures. Examples include supernatants and those derived from these cell extracts. In addition, the thread-replaceable HGF may be one in which one or more amino acids in the amino acid sequence of the thread-replaceable HGF are substituted, deleted, or added as long as it has substantially the same action as the natural HGF. Alternatively, one or more sugar chains in the amino acid sequence of recombinant HGF may be substituted, deleted, or added. Furthermore, HGF may be a natural HGF or a modified form of recombinant HGF (for example, a complex with a polymer material such as polyethylene glycol or lecithin).

[0012] Examples of natural BDNF include extracts of brain, spinal cord, heart, lung, and skeletal muscle strength. It is. Recombinant BDNF may be one in which one or more amino acids in the amino acid sequence of recombinant BDNF are substituted, deleted, or added as long as it has substantially the same action as natural BDNF. In addition, one in which one or more sugar chains in the amino acid sequence of recombinant BDNF are substituted, deleted, or added may be used. Further, BDNF may be natural BDNF or a modified form of recombinant BDNF (for example, a complex with a polymer material such as polyethylene glycol or lecithin).

[0013] Examples of natural PEDF include extracts from the retina, primary culture cells of retinal pigment epithelial cells and culture supernatants of cell lines, and cell extracts thereof. Recombinant PEDF may be one in which one or more amino acids in the amino acid sequence of recombinant PEDF are substituted, deleted, or added as long as it has substantially the same action as natural PEDF. Alternatively, one or more sugar chains in the amino acid sequence of recombinant PEDF may be substituted, deleted, or added. Furthermore, PDEDF may be a natural PEDF or a modified form of recombinant PEDF (for example, a complex with a polymer material such as polyethylene glycol or lecithin).

In the present invention, “photocytopathy” refers to a condition in which the photocell is damaged by various factors. Examples of the “disease causing photocytopathy” include retinal pigment degeneration. And retinal degenerative diseases such as cone dystrophy, age-related macular degeneration, age-related macular disease, macular edema, retinal detachment, cancer-related retinopathies, retinal vein occlusion, and retinal pigment epithelial detachment.

In the present invention, the “crosslinked gelatin hydrogel” refers to a hydrogel obtained by crosslinking gelatin, and is not particularly limited as long as it is a hydrogel obtained by crosslinking gelatin. Include those having at least one of the following physical properties 1) to 4).

[0016] 1) Gelatin constituting the crosslinked gelatin hydrogel is acidic gelatin or basic gelatin.

[0017] 2) Gelatin constituting the crosslinked gelatin hydrogel has a molecular weight of 0.5 to 200,000 danoleton under the non-reducing conditions of SDS-PAGE.

[0018] 3) Gelatin constituting the crosslinked gelatin hydrogel has an isoelectric point of 4.5 to 9.5.

[0019] 4) Water content power of crosslinked gelatin hydrogel is 0 to 99%.

[0020] HGF sustained-release composition, BDNF sustained-release composition or PEDF sustained-release composition of the present invention HGF, BDNF or PEDF and a cross-linked gelatin hydrogel form a complex. When administered in vivo, the hydrogel is hydrolyzed by proteolytic enzymes, and the gelatin gradually becomes water-soluble. This is thought to contribute to the sustained release of BDNF or PEDF.

[0021] Gelatin is not particularly limited, and can be obtained, for example, by alkaline hydrolysis treatment or acid hydrolysis treatment from skin, tendon or collagen of animals such as cattle. When using HGF or BDNF, acidic gelatin obtained by alkali treatment of type I collagen derived from urchin bone is preferred, for example, S-4 manufactured by Nitta Gelatin Co., Ltd. Can be mentioned. In addition, when using PEDF, a basic gelatin obtained by acid treatment of type I collagen derived from pig skin is preferred, for example, AP-150 manufactured by Nitta Gelatin Co., Ltd. It is done.

[0022] The gelatin of the present invention preferably has a molecular weight of 0.5 to 200,000 daltons under non-reducing conditions of SDS-PAGE.

[0023] The isoelectric point of the gelatin of the present invention is preferably 4.5 to 9.5. When BDNF is used, the isoelectric point of 4.5 to 5.5 is more preferable. Furthermore, when PEDF is used, 7.0 to 9.5 is more preferable. The isoelectric point is obtained by preparing a 1% gelatin aqueous solution, filtering the solution through a cation column or an anion column, and measuring the pH of the filtered aqueous solution.

[0024] The crosslinking method for obtaining the crosslinked gelatin hydrogel of the present invention is not particularly limited. For example, gelatin may be crosslinked by thermal dehydration, ultraviolet irradiation, electron beam irradiation, chemical crosslinking, or a combination thereof. it can. Preferred methods are thermal dehydration without using a crosslinking agent, ultraviolet irradiation or electron beam irradiation.

[0025] The shape of the crosslinked gelatin hydrogel of the present invention is not particularly limited, and examples thereof include particles, sheets, disks, columns, and prisms. When the sustained-release composition of the present invention is administered as an injection, a particulate form is preferred, and when it is transplanted or embedded in a wide range of modified sites, a sheet form is preferred.

[0026] The preparation of the particulate crosslinked gelatin hydrogel is preferably carried out as follows. For example, put olive oil in a three-necked round bottom flask equipped with a stirring motor and a propeller. Stir at a rotational speed of about 600 rpm, add gelatin aqueous solution dropwise to this to prepare a WZO emulsion, cool, add acetone, ethyl acetate, etc., stir, and centrifuge to collect gelatin particles. The collected gelatin particles are further washed with acetone, ethanol, etc. and dried. The dried gelatin particles are put into a constant-temperature vacuum dryer or the like and subjected to thermal dehydration crosslinking. Furthermore, this is put into an ultraviolet irradiation device and irradiated with ultraviolet rays to complete the crosslinking. A particulate dry cross-linked gelatin hydrogel is thus obtained. When these dry-crosslinked gelatin hydrogel particles are impregnated with aqueous solutions of HGF, BDNF, and PEDF, the particulate HGF sustained-release composition, BDNF sustained-release composition, or PEDF sustained-release composition (drug-containing crosslinked gelatin hydrogel) Microspheres) are obtained.

[0027] Preparation of a sheet-like, disc-like, cylindrical, and prismatic crosslinked gelatin hydrogel is preferably carried out as follows. For example, a sheet-like dried crosslinked gelatin hydrogel can be obtained by extruding an aqueous gelatin solution into a sheet and drying it followed by thermal dehydration crosslinking or ultraviolet crosslinking. In addition, after injection molding using a mold having an appropriate shape, the molded product is dried and subjected to thermal dehydration crosslinking or UV crosslinking to obtain a dry crosslinked gelatin hydrogel having a disk shape, a cylindrical shape, or a prism shape. it can. When these dry cross-linked gelatin hydrogels are impregnated with aqueous solutions of HGF, BDNF, and PEDF, a sheet-like, disc-like, cylindrical, and prismatic HGF sustained-release composition, BDNF sustained-release composition, or PEDF sustained-release composition A sexual composition is obtained.

[0028] The particle size of the particulate crosslinked gelatin hydrogel can be appropriately selected depending on the purpose of use, but is preferably 500 µm or less, more preferably 200 µm or less. In the case of a sheet-like cross-linked gelatin hydrogel, the film thickness can be appropriately selected according to the purpose of use, but it is preferably 200 μm or less, more preferably 100 μm or less.

[0029] The water content of the crosslinked gelatin hydrogel of the present invention can be appropriately selected depending on the purpose of use, but it is preferably 30 to 99 wZw%, more preferably 40 to 98 wZw%.

[0030] The weight ratio of HGF, BDNF or PEDF and gelatin relative to the HGF sustained-release composition, BDNF sustained-release composition or PEDF sustained-release composition of the present invention is not particularly limited, but a preferred weight ratio is HGF. (BDNF, PEDF) Z gelatin = lZO.2 ~: LZ 10000, more preferably HGF (BDNF, PEDF) Z gelatin = lZl ~: LZlOOO. [0031] The second invention is a method in which a crosslinked gelatin hydrogel is impregnated with hepatocyte growth factor (HGF), brain-derived neurotrophic factor (BDNF) or retinal pigment epithelium-derived neurotrophic factor (PEDF). A releasable composition, a therapeutic agent for a disease causing a photoreceptor damage, comprising a cell having a role of regenerating or complementing a photoreceptor cell or a function thereof, and a material for regenerating the photoreceptor cell or a function thereof. As is clear from the results of the pharmacological test described below, an HGF sustained-release composition, a BDNF sustained-release composition, or a PEDF sustained-release composition, and cells that play a role in regenerating or complementing photoreceptor cells or their functions. In combination, when these are injected into the site of photoreceptor damage or the surrounding tissue (subretinal, intraretinal, intravitreal, etc.), the visual cell or its function is regenerated and the effect of suppressing retinal degeneration is enhanced.

[0032] The photoreceptor cell used in the present invention or a cell having a role of regenerating or complementing the function thereof is not particularly limited. For example, a stromal cell such as an adipose-derived stromal cell or a bone marrow stromal cell, an undifferentiated cell Leaf cells, retinal stem (progenitor) cells, retinal pigment epithelial cells, iris pigmented epithelial cells, neural stem cells, hematopoietic stem cells, skin stem cells, embryonic stem cells (ES cells), embryonic germ stem cells (EG cells), and embryos Examples thereof include the above-mentioned cells induced to differentiate from sex stem cells, embryonic germ stem cells, etc., preferably stromal cells, more preferably adipose-derived stromal cells. It should be noted that the cells that play a role to regenerate or complement the photoreceptor cells or their functions may be collected from the tissue strength of the patient who has a disorder in the photoreceptor cells, or from the tissue strength other than the patient. Well ...

[0033] For example, a human adipose tissue mass collected by adipose tissue is crushed with a scissors and scissors, and the resulting strips are shaken with a collagenase solution, filtered through a filter, and centrifuged to obtain cell groups. Collect. Next, only the collected cell group strength adherent cells are collected, cultured in an incubator, and proliferated to obtain adipose-derived stromal cells.

[0034] In the present invention, the adipose-derived stromal cells include those into which a photoreceptor cell-specific homeobox gene and a gene encoding a factor exhibiting a neuroprotective action or a degeneration inhibitory action are introduced.

[0035] A photoreceptor-specific homeobox gene is a gene or differentiation trait that has a region-specific expression pattern of a photoreceptor cell and controls region-specific morphogenesis in the process of photoreceptor cell development. There are genes involved in the expression of, such as Crx, Otx2, Nrl, Chx 10 etc. are mentioned.

[0036] In addition, specific examples of a gene encoding a factor that exhibits a neuroprotective action or a degeneration inhibitory effect include brain-derived neurotrophic factor (BDNF), retinal pigment epithelium-derived neurotrophic factor (PEDF), liver parenchyma. Examples include genes encoding factors such as cell growth factor (HGF)

[0037] A method for introducing a gene encoding a factor that exhibits a degeneration-inhibiting action into a stromal cell having a photoreceptor-specific homeobox gene or a neuroprotective action is not particularly limited. For example, the lipofussion method Physics and physics introduction methods such as the Electo mouth position method, calcium phosphate method, and gene gun method, adenovirus vector, adeno-associated virus vector, retrovirus vector, Sendai virus vector, Sendai virus envelope vector, etc. The method using is mentioned.

[0038] HGF sustained-release composition, BDNF sustained-release composition or PEDF sustained-release composition and fat-derived stromal cells obtained by the above method are mixed to regenerate photoreceptor cells or their functions. A material is obtained.

[0039] The HGF sustained-release composition, the BDNF sustained-release composition or the PEDF sustained-release composition of the present invention comprises a pharmaceutically acceptable stabilizer, preservative, solubilizer, and pH adjustment as necessary. May contain additives such as agents, thickeners, suspending agents, buffering agents, tonicity agents, preservatives, soothing agents, antioxidants, etc. it can. In order to adjust the sustained release period of these sustained release compositions, sugars or lipids having amino acids, amino groups, phosphate groups, sulfate groups, SH groups, carboxyl groups, etc. may be added. it can.

[0040] The present invention also provides a visual cell disorder comprising administering to a patient a therapeutically effective amount of a sustained release composition in which a crosslinked gelatin hydrogel is impregnated with at least one of HGF, BDNF and PEDF. A method for treating an induced disease, a combination of a sustained-release composition impregnated with a crosslinked gelatin hydrogel with at least one of HGF, BDNF, and PEDF and a cell that plays a role to regenerate or complement its function. A method for treating diseases causing photoreceptor cell damage comprising administering to a patient in an effective amount, and a sustained-release composition and photoreceptor cells in which at least one of HGF, BDNF and PEDF is impregnated in a crosslinked gelatin hydrogel Or a combination with a cell that plays a role in regenerating or complementing its function is effective for treatment The present invention also relates to a method for regenerating a photoreceptor cell or a function thereof, which comprises administering a suitable amount to a patient.

[0041] The dosage of the therapeutic agent for diseases causing photoreceptor cell damage and the material for regenerating photoreceptor cells or their functions according to the present invention takes into consideration the disease site, the degree of disease, the age and weight of the patient, and the like. and can be appropriately adjusted, HGF (BDNF, PEDF) is preferably from 0. OOl ^ lOOO ^ g range preferred tool as in the range of 0.01 to 500 _8. As the administration form, for example, it may be injected as an injection into the site of photoreceptor damage or the surrounding tissue (subretinal, intraretinal, intravitreal, etc.), and the site of photoreceptor damage or the surrounding tissue (subretinal, Transplanted into the retina, vitreous, etc.).

The invention's effect

[0042] As is clear from the results of a retinal degeneration inhibition test using spontaneous retinal degeneration model rats (RCS rats) described later, cross-linked gelatin hydrogen microspheres impregnated with drugs (HGF, BDNF, PEDF) When administered, it exerts an excellent retinal degeneration inhibitory effect, and when combined with drug-impregnated crosslinked gelatin hydrogel microspheres and adipose-derived stromal cells, the photoreceptor cells or their functions are regenerated. The effect of inhibiting retinal degeneration is further enhanced and sustained as compared with the case where drug-impregnated crosslinked gelatin hydrogel microspheres are administered subretinal.

[0043] Therefore, the HGF sustained-release composition, BDNF sustained-release composition, PEDF sustained-release composition of the present invention, and these sustained-release compositions and the photoreceptors or their functions are regenerated or complemented. Combinations with cells that have a role include: retinitis pigmentosa, cone dystrophy, age-related macular degeneration, age-related macular disease, macular edema, retinal detachment, cancer-related retinopathy, retinal vein occlusion, retinal chromatic epithelial detachment It is useful as a therapeutic agent for diseases that cause photoreceptor cell disorders such as, and is an excellent material for regenerating photoreceptor cells or their functions.

BEST MODE FOR CARRYING OUT THE INVENTION

[0044] The results of the pharmacological test and formulation examples are shown below, but these examples are for better understanding of the present invention and do not limit the scope of the present invention.

[0045] 1. Pharmacological test

(1) Preparation of drug-impregnated crosslinked gelatin hydrogel microspheres Using acidic gelatin derived from cattle (isoelectric point; 5.0, molecular weight; approx. 100,000 daltons, manufactured by Nitta Gelatin Co., Ltd.), prepare 0.2 ml of 10wZv% gelatin aqueous solution and add 5 ml of olive oil dropwise. And emulsified using a vortex mixer. The emulsion was cooled until the aqueous phase solidified. To this emulsion was added 1.5 ml of cold acetone, and the solidified gelatin droplets were dehydrated to obtain gelatin microspheres. This was centrifuged under cooling to remove the supernatant, the residue was redispersed with acetone, and further centrifuged. This operation was performed three times to wash the gelatin microspheres, and then the gelatin microspheres were dried at 4 ° C for 24 hours. The dried gelatin microspheres were placed in a thermostatic vacuum dryer and subjected to thermal dehydration crosslinking at 160 ° C for 72 hours. Furthermore, this was put into an ultraviolet irradiation device and irradiated with ultraviolet rays for 30 minutes to crosslink the gelatin microsphere. The crosslinked gelatin microspheres were sieved to obtain dry crosslinked gelatin hydrogel microspheres having an average particle size of 10 / ζ πι and a water content of 66%.

[0046] This dry crosslinked gelatin hydrogel microsphere 2.5 mg human recombinant HGF (1

gZ 1) Aqueous solution 10 μ 1 (manufactured by PeproTech) was impregnated and allowed to stand at 4 ° C. for 24 hours. To this, 90 L of phosphate buffered saline was added to prepare an HGF-impregnated crosslinked gelatin hydrogel microsphere suspension (HGF ^ / z gZmg microsphere concentration: 2.5 wZv%).

[0047] Next, in the above preparation method, instead of “Human recombinant HGF (1 μgZ 1) aqueous solution 10 1 (manufactured by eproTech)”, “Human recombinant BDNF (1 μ & μ \) aqueous solution” BDNF-impregnated crosslinked gelatin hydrogel microsphere suspension (BDNF; 4 gZmg, microsphere concentration: 2.5 wZv%) in the same manner as above except that “10 μ 1 (PeproTech)” is used. Was prepared. Furthermore, instead of “bovine-derived acidic gelatin (isoelectric point; 5.0, molecular weight; approximately 100,000 Dalton, manufactured by Nitta Gelatin)”, “pig-derived basic gelatin (isoelectric point; 9.0, "Molecular weight: approx. 80,000 Dalton, manufactured by Nitta Gelatin Co., Ltd.)", and "human recombinant PEDF (manufactured by PeproTech)" instead of "human recombinant HGF (1 μgZ μ 1) aqueous solution 10 μ 1 PEDF impregnated crosslinked gelatin hydrogel microsphere suspension (PEDF; 4 μgZmg, microsphere concentration: 2) in the same manner as above except that 1 μ & / μ \) aqueous solution 10 1 (manufactured by Upstate) is used. 5 w / v%) was prepared. [0048] (2) Isolation and culture of rat adipose-derived stromal cells

Green fluorescent protein transgenic rats (3 weeks old) were euthanized by over anesthesia with jetyl ether, and subcutaneous fat was collected. The collected subcutaneous fat was shredded with scissors in phosphate buffered saline (5 ml), and 5 ml of collagenase solution (2 mgZml) was added and shaken at 37 ° C for 15-20 minutes. After shaking, 10 ml of Medium 99 containing 10% urine fetal serum (10 ml, manufactured by Invitrogen) was added to form a suspension. The suspension was filtered through a sterile 200 m filter to remove cell clumps. The filtrate is centrifuged (2000 rpm, 5 minutes), the supernatant is removed, the residue is suspended in Medium 99 (10 ml) containing 10% ushi fetal serum, and this suspension is centrifuged again (1000 rpm, 10 minutes). Minutes). After removal of the supernatant, the residue is suspended in 5 ml of Medium 99 containing 10% urine fetal serum, seeded in a culture flask, and maintained at 37 ° C and 5% CO.

2 cultured under. 12-24 hours after seeding, medium change (mediu ml99 containing urchin fetal serum (10%) and basic fibroblast growth factor (lOOngZml)) to remove non-adherent cells, and adherent cells Incubate at 37 ° C, 5% CO until confluent

2

Experimental fat-derived stromal cells were obtained.

[0049] (3) Retinal degeneration suppression test

In accordance with the method of Jiang LQ et al. (Invest. Ophthalmol. Vis. Sci 35 (13): 4300-4309 (1994)), a retinal degeneration suppression test was performed. In the retinal degeneration suppression test, RCS rats (Royal College of Surgeons Rat) were used as spontaneous retinal degeneration model rats, and the b-wave amplitude (before administration and 4 weeks after administration) was measured on the electroretinogram. Evaluation of denaturation inhibitory effect

[0050] i) Inhibition test of retinal degeneration by subretinal administration of drug-impregnated crosslinked gelatin hydrogel microspheres

(experimental method)

The HGF-impregnated crosslinked gelatin hydrogel microsphere suspension, BDNF-impregnated crosslinked gelatin hydrogel microsphere suspension and PEDF-impregnated crosslinked gelatin hydrogel microsphere suspension were suspended under the retina of both eyes of 3-week-old RCS rats. Each suspension was injected with 5 μ 1 (HGF, BDNF, PEDF; 0.5 g, microspheres; 125 g) / eye. Phosphate buffered saline was used as a control. [0051] At 4 weeks after subretinal administration, an electroretinogram was taken and the amplitude of the b wave was measured. The residual rate of b wave at 4 weeks after subretinal administration was calculated based on the b wave amplitude before administration (100%). The test results are shown in Fig. 1 [HGF], Fig. 2 [BDNF] and Fig. 3 [PEDF]. (The effect of suppressing retinal degeneration is greater as the residual rate of b-wave amplitude is higher.) ₀ Yes, the survival rate is the average value.

[0052] (Result)

As evident from Figures 1-3, the HGF impregnated crosslinked gelatin hydrogel microsphere suspension, BDNF impregnated crosslinked gelatin hydrogel microsphere suspension, PEDF impregnated crosslinked gelatin hydrogel microsphere suspension When injected, V and shift were excellent in suppressing retinal degeneration.

[0053] ii) Inhibition of retinal degeneration by subretinal administration of drug-impregnated crosslinked gelatin hydrogel microspheres and adipose-derived stromal cells

(experimental method)

Add 4 x experimental fat-derived stromal cells to the HGF-impregnated crosslinked gelatin hydrogel microsphere suspension, BDNF-impregnated crosslinked gelatin hydrogel microsphere suspension, and PEDF-impregnated crosslinked gelatin hydrogel microsphere suspension, respectively. 10 ⁴ pieces // z 1 were dispersed. Next, a suspension of these experimental fat-derived stromal cells dispersed in 5 / zl (HGF, BDNF, PEDF; 0.5 / zg, microspheres; 125 g, adipose-derived stromal cells; 2 X 10 ⁵ ) / eye was injected into the retina of both eyes of 3-week-old RCS rats. Phosphate buffered saline was used as a control.

[0054] At 4 weeks after subretinal administration, an electroretinogram was taken and the amplitude of the b wave was measured. The residual rate of b wave at 4 weeks after subretinal administration was calculated based on the b wave amplitude before administration (100%). The test results are shown in Fig. 1 [HGF], Fig. 2 [BDNF] and Fig. 3 [PEDF]. The number of cases is 8 eyes each, and the survival rate is the average value.

[0055] (Result)

As is clear from Figs. 1 to 3, when a drug (HGF, BDNF, PEDF) -impregnated cross-linked gelatin hydrogel microspheres and adipose-derived stromal cells are combined and injected under the retina, V is also a photoreceptor cell or its function Regenerated and drug-impregnated crosslinked gelatin hydrogel micro The effect of suppressing retinal degeneration was further enhanced compared to the case of injecting sufuair under the retina.

[0056] 2. Formulation Examples

The typical formulation example of this invention is shown below.

[0057] Injection 1 (lml)

HGF impregnated crosslinked gelatin hydrogel microspheres

(HGF; 100 g) 25mg

Concentrated glycerin 26mg

Polysonole 80 80mg

Sterile purified ice 2ϋ_

lml

Injection 2 (lml)

BDNF-impregnated crosslinked gelatin hydrogen microspheres

(BDNFjlOO ^ g) 25mg

Concentrated glycerin 26mg

Polysorbate 80 4mg

my

lml

Injection 3 (lml)

HGF impregnated crosslinked gelatin hydrogel microspheres

(HGFjlOOMg) 25mg

Adipose-derived stromal cells 4X10 ⁷

Concentrated glycerin 26mg

Sterilized ice

lml

Injection 4 (lml)

PEDF impregnated crosslinked gelatin hydrogel microspheres

(PEDFjlOO ^ g) 25mg

Adipose-derived stromal cells 4X10 ⁷ Concentrated glycerin 2omg

Dredging

lml

Brief Description of Drawings

[FIG. 1] FIG. 1 shows a b wave at 4 weeks after subretinal administration in a retinal degeneration inhibition test using a suspension of HGF-impregnated crosslinked gelatin hydrogel microspheres and a mixture thereof with adipose-derived stromal cells. It is a graph which shows an amplitude residual ratio (average value).

[Figure 2] Figure 2 shows the b-wave amplitude at 4 weeks after subretinal administration in a retinal degeneration inhibition test using a BDNF-impregnated crosslinked gelatin hydrogel microsphere suspension and a mixture of it with adipose-derived stromal cells. It is a graph which shows a residual rate (average value).

[FIG. 3] FIG. 3 shows a b wave at 4 weeks after subretinal administration in a retinal degeneration suppression test using a PEDF-impregnated crosslinked gelatin hydrogel microsphere suspension and a mixture thereof with adipose-derived stromal cells. It is a graph which shows an amplitude residual ratio (average value).

Claims

The scope of the claims

[1] A therapeutic agent for diseases causing visual cell damage, comprising a sustained-release composition in which at least one of the following components 1) to 3) is impregnated in a crosslinked gelatin hydrogel.

1) Hepatocyte growth factor (HGF)

2) Brain-derived neurotrophic factor (BDNF)

3) Neurotrophic factor derived from retinal pigment epithelium (PEDF)

[2] A therapeutic agent for diseases causing visual cell damage, comprising a sustained release composition obtained by impregnating a crosslinked gelatin hydrogel with at least one of the following components 1) to 3) and the following component 4).

1) Hepatocyte growth factor (HGF)

2) Brain-derived neurotrophic factor (BDNF)

3) Neurotrophic factor derived from retinal pigment epithelium (PEDF)

[3] The therapeutic agent according to claim 1 or 2, wherein the disease causing photoreceptor cell damage is a retinal degenerative disease.

[4] The retinal degenerative disease is retinal pigment degeneration, cone dystrophy, age-related macular degeneration, age-related macular disease, macular edema, retinal detachment, cancer-related retinopathy, retinal vein occlusion or retinal pigment epithelial detachment 3. The therapeutic agent according to 3.

5. The therapeutic agent according to claim 2, wherein the cell having a role of regenerating or complementing a photoreceptor cell or its function is a stromal cell.

6. The therapeutic agent according to claim 5, wherein the stromal cells are adipose-derived stromal cells.

[7] A visual cell or a material for regenerating its function, comprising a sustained release composition obtained by impregnating a crosslinked gelatin hydrogel with at least one of the following components 1) to 3) and the following component 4).

1) Hepatocyte growth factor (HGF)

2) Brain-derived neurotrophic factor (BDNF)

3) Neurotrophic factor derived from retinal pigment epithelium (PEDF)

[8] Treatment of diseases causing visual cell damage, comprising administering to a patient a therapeutically effective amount of a sustained-release composition in which at least one of the following components 1) to 3) is impregnated in a crosslinked gelatin hydrogel: Method.

1) Hepatocyte growth factor (HGF)

2) Brain-derived neurotrophic factor (BDNF)

3) Neurotrophic factor derived from retinal pigment epithelium (PEDF)

[9] Visual cells comprising administering to a patient a therapeutically effective amount of a combination of a sustained release composition impregnated with a crosslinked gelatin hydrogel with at least one of the following components 1) to 3) and the following component 4) A method for treating a disorder causing a disorder.

1) Hepatocyte growth factor (HGF)

2) Brain-derived neurotrophic factor (BDNF)

3) Neurotrophic factor derived from retinal pigment epithelium (PEDF)

[10] The method according to claim 8 or 9, wherein the disease causing a photoreceptor cell disorder is a retinal degenerative disease.

[11] The retinal degenerative disease is retinal pigment degeneration, cone dystrophy, age-related macular degeneration, age-related macular disease, macular edema, retinal detachment, cancer-related retinopathy, retinal vein occlusion or retinal pigment epithelial detachment 10. The treatment method according to 10.

12. The treatment method according to claim 9, wherein the photoreceptor cell or the cell having a role of regenerating or complementing its function is a stromal cell.

13. The method according to claim 12, wherein the stromal cells are adipose-derived stromal cells.

[14] A photoreceptor cell comprising administering to a patient a therapeutically effective amount of a combination of a sustained release composition impregnated with at least one of the following components 1) to 3) in a crosslinked gelatin hydrogel and the following component 4): Or how to play the function.

1) Hepatocyte growth factor (HGF)

2) Brain-derived neurotrophic factor (BDNF)

3) Neurotrophic factor derived from retinal pigment epithelium (PEDF)

[15] Use of a sustained-release composition in which a crosslinked gelatin hydrogel is impregnated with at least one of the following components 1) to 3) for producing a therapeutic agent for a disease causing a photoreceptor cell disorder. 1) Hepatocyte growth factor (HGF)

2) Brain-derived neurotrophic factor (BDNF)

3) Neurotrophic factor derived from retinal pigment epithelium (PEDF)

[16] A sustained-release composition in which at least one of the following components 1) to 3) is impregnated in a crosslinked gelatin hydrogel for producing a therapeutic agent for diseases causing photoreceptor cell damage, and the following component 4): Use of unions.

1) Hepatocyte growth factor (HGF)

2) Brain-derived neurotrophic factor (BDNF)

3) Neurotrophic factor derived from retinal pigment epithelium (PEDF)

[17] The use according to claim 15 or 16, wherein the disease causing a photoreceptor cell disorder is a retinal degenerative disease.

[18] The retinal degenerative disease is retinitis pigmentosa, cone dystrophy, age-related macular degeneration, age-related macular disease, macular edema, retinal detachment, cancer-related retinopathy, retinal vein occlusion or retinal pigment epithelial detachment Use as described in 17.

[19] The use according to claim 16, wherein the cell having a role of regenerating or complementing a photoreceptor cell or its function is a stromal cell.

20. The use according to claim 19, wherein the stromal cells are adipose-derived stromal cells.

[21] The following components 1) to 3 for producing a material for regenerating photoreceptor cells or their functions

) And a sustained-release composition impregnated with a crosslinked gelatin hydrogel and the following component 4

Use in combination with).

1) Hepatocyte growth factor (HGF)

2) Brain-derived neurotrophic factor (BDNF)

3) Neurotrophic factor derived from retinal pigment epithelium (PEDF)