WO2010147109A1

WO2010147109A1 - Angiogenesis inducer comprising genetically modified gelatin and basic fibroblast growth factor

Info

Publication number: WO2010147109A1
Application number: PCT/JP2010/060101
Authority: WO
Inventors: 一隆荻原; 乃梨子石川; 章二大屋; 哲男平等; 健太郎中村
Original assignee: 富士フイルム株式会社
Priority date: 2009-06-15
Filing date: 2010-06-15
Publication date: 2010-12-23

Abstract

Disclosed is an angiogenesis inducer which is safe for living organisms, has excellent bioadhesive properties, and can reduce the quantity of bFGF to be used. The angiogenesis inducer comprises, as active ingredients, a genetically modified gelatin having an amino acid sequence derived from a partial amino acid sequence for collagen and a basic fibroblast growth factor.

Description

Angiogenesis inducer containing genetically modified gelatin and basic fibroblast growth factor

The present invention relates to an angiogenesis inducer using genetically modified gelatin and basic fibroblast growth factor.

Angiogenesis mainly represents a phenomenon in which new blood vessels are formed from existing blood vessels. As normal physiological angiogenesis, there are angiogenesis related to angiogenesis in the embryonic period, endometrium and corpus luteum formation, wound healing and the like. Utilizing an angiogenic event for treatment is practiced as an angiogenic therapy. The importance of angiogenesis has been clarified as a therapeutic method for wound healing, ischemic diseases, etc., and in treatments widely called regenerative medicine such as organ regeneration, cell transplantation, and natural healing effects. Because of the therapeutic effect of angiogenesis itself, or because angiogenesis enhances the therapeutic effect, therapeutic agents targeting angiogenesis have been developed.

Among these, basic fibroblast growth factor (bFGF) is widely used. bFGF was found as a protein that strongly stimulates the proliferation of fibroblasts from the bovine pituitary gland, and subsequent research has been vigorously conducted, and not only fibroblasts but also vascular endothelial cells, vascular smooth muscle cells, corneal endothelial cells It has been shown to stimulate cell proliferation on many types of cells such as osteoblasts and chondrocytes.

However, when bFGF is used as an angiogenic agent, the growth factor is expensive, and since the risk of canceration is expected because it actively induces blood vessels, the growth factor is pointed out. It is preferable that the effect is exerted in as little amount as possible, and therefore, it is required to apply bFGF to a site where angiogenesis is required efficiently.

On the other hand, biopolymers such as gelatin have been widely used as medical materials. Due to recent advances in genetic engineering techniques, protein synthesis has been carried out by introducing genes into E. coli and yeast. By this technique, various recombinant collagen-like proteins have been synthesized (for example, Patent Documents 1 and 2), which are superior in non-infectivity and uniform compared to natural gelatin, and have been sequenced. It is said that it has advantages such as being able to precisely design strength and decomposability. However, the use of genetically modified gelatin that has been proposed so far does not exceed the range of replacement of natural gelatin, and of course, its use as an angiogenic carrier has not been known.

Patent Document 3 discloses a bFGF preparation using a naturally-derived cross-linked gelatin gel such as pig or bovine, but gelatin itself is a carrier for containing bFGF and reduces the amount of bFGF used. It is not used. In Patent Document 3, gelatin is cross-linked, and bFGF is contained in the gelatin gel to regenerate blood vessels. However, Patent Document 3 relies on bFGF only for angiogenesis, and gelatin is used only as a base material for sustained release. Therefore, there is a drawback that a large amount of bFGF is required for angiogenesis.

US Pat. No. 6,992,172 WO2008 / 103041 specification WO1994 / 027630 specification

The present invention has been made to solve the problem of providing an angiogenesis-inducing agent that is safe for a living body and excellent in bioadhesiveness and that can reduce the amount of bFGF used.

As a result of intensive studies to solve the above problems, the present inventors have induced vascular endothelial cells with bFGF by using genetically modified gelatin having high affinity with vascular endothelial cells, and the vascular endothelial cells with genetically modified gelatin. It was found that angiogenesis can be induced with a smaller amount of bFGF used by positively adhering to the present invention, and the present invention has been completed.

That is, according to the present invention, there is provided an angiogenesis-inducing agent comprising genetically modified gelatin having an amino acid sequence derived from a partial amino acid sequence of collagen and basic fibroblast growth factor as active ingredients.
Preferably, the genetically modified gelatin has a repetition of a sequence represented by Gly-XY, which is characteristic of collagen (X and Y each independently represents one of amino acids) (a plurality of Gly-XY are the same) However, the molecular weight is 2 KDa or more and 100 KDa or less.
Preferably, the genetically modified gelatin has a repetition of a sequence represented by Gly-XY, which is characteristic of collagen (X and Y each independently represents one of amino acids) (a plurality of Gly-XY are the same) However, the molecular weight is 10 KDa or more and 90 KDa or less.

Preferably, the genetically modified gelatin has a repetition of a sequence represented by Gly-XY, which is characteristic of collagen (X and Y each independently represents one of amino acids) (a plurality of Gly-XYs are the same) Or may be different), including two or more cell adhesion signals per molecule.
Preferably, the cell adhesion signal is an amino acid sequence represented by Arg-Gly-Asp.

Preferably, the amino acid sequence of the recombinant gelatin does not include serine and threonine.
Preferably, the amino acid sequence of the genetically modified gelatin does not include serine, threonine, asparagine, tyrosine, and cysteine.
Preferably, the amino acid sequence of the recombinant gelatin does not include the amino acid sequence represented by Asp-Arg-Gly-Asp.

Preferably, the genetically modified gelatin is
Formula: A-[(Gly-XY) _n ] _m -B
(In the formula, A represents an arbitrary amino acid or amino acid sequence, B represents an arbitrary amino acid or amino acid sequence, n Xs independently represent any of the amino acids, and n Ys each independently represent an amino acid. N represents an integer of 3 to 100, and m represents an integer of 2 to 10. Note that n Gly-XY may be the same or different.

Preferably, the genetically modified gelatin is
Formula: Gly-Ala-Pro-[(Gly-XY) ₆₃ ] ₃ -Gly
(In the formula, 63 X's each independently represent any amino acid, and 63 Y's each independently represent any amino acid. The n Gly-XY may be the same or different. Good.)
Indicated by
Preferably, the genetically modified gelatin is (1) an amino acid sequence represented by SEQ ID NO: 1, or (2) an amino acid sequence having an angiogenic action having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 1. Have

Preferably, the genetically modified gelatin is cross-linked.
Preferably, crosslinking is performed with aldehydes, condensing agents, or enzymes.
Preferably, the angiogenesis inducer of the present invention induces angiogenesis by accumulating at an angiogenesis site.

According to the present invention, the angiogenesis further comprises administering a recombinant gelatin having an amino acid sequence derived from a partial amino acid sequence of collagen and a basic fibroblast growth factor to a subject in need of induction of angiogenesis. A method of inducing is provided.

According to the present invention, there is further provided use of a recombinant gelatin having an amino acid sequence derived from a partial amino acid sequence of collagen and a basic fibroblast growth factor for the production of an angiogenesis inducer.

Since the angiogenesis-inducing agent of the present invention is characterized by containing genetically modified gelatin, it has no risk of canceration, is safe for the living body, and has excellent bioadhesiveness. Moreover, according to the angiogenesis inducer of the present invention, it is possible to reduce the amount of bFGF used.

FIG. 1 shows the results of measuring the amount of hemoglobin contained in blood in order to quantify the angiogenic effect. FIG. 2 shows the results of the HUVEC cell adhesion test. FIG. 3 shows the results of the HUVEC cell adhesion test. FIG. 4 shows HUVEC cell photographs on plates coated with various proteins. FIG. 5 shows a comparison of the area of one HUVEC cell on a plate coated with various proteins. FIG. 6 shows inhibition of HUVEC cell adhesion by anti-αV antibody.

Hereinafter, embodiments of the present invention will be described in detail.
As the genetically modified gelatin used in the present invention, a genetically modified gelatin having an amino acid sequence derived from a partial amino acid sequence of collagen can be used, for example, those described in EP1014176A2, US6992172, WO2004-85473, WO2008 / 103041, etc. However, it is not limited to these. Preferred as the genetically modified gelatin used in the present invention is the genetically modified gelatin of the following embodiment.

The genetically modified gelatin used in the present invention is excellent in biocompatibility due to the inherent performance of natural gelatin, and is not naturally derived. In addition, since the genetically modified gelatin used in the present invention is more uniform than natural ones and the sequence is determined, the strength and degradability can be precisely designed with less blur due to cross-linking described later. It is.

The molecular weight of the genetically modified gelatin used in the present invention is preferably from 2 to 100 KDa. More preferably, it is 2.5 to 95 KDa. More preferably, it is 5 to KDa. Most preferably, it is 10 KDa or more and 90 KDa or less.

The genetically modified gelatin used in the present invention preferably has a repeating sequence represented by Gly-XY characteristic of collagen. Here, the plurality of Gly-X-Ys may be the same or different. In Gly-XY, Gly represents glycine, and X and Y represent any amino acid (preferably any amino acid other than glycine). The GXY sequence characteristic of collagen is a very specific partial structure in the amino acid composition and sequence of gelatin / collagen compared to other proteins. In this part, glycine accounts for about one third of the whole, and in the amino acid sequence, it is one in three repeats. Glycine is the simplest amino acid, has few constraints on the arrangement of molecular chains, and greatly contributes to the regeneration of the helix structure upon gelation. The amino acids represented by X and Y are rich in imino acids (proline, oxyproline), and preferably account for 10% to 45% of the total. Preferably, 80% or more of the sequence, more preferably 95% or more, and most preferably 99% or more of the amino acids are GXY repeating structures.

General gelatin has 1: 1 polar amino acids, both charged and uncharged. Here, the polar amino acid specifically means cysteine, aspartic acid, glutamic acid, histidine, lysine, asparagine, glutamine, serine, threonine, tyrosine, arginine, and among these polar uncharged amino acids are cysteine, asparagine, glutamine, serine. , Threonine, tyrosine. In the genetically modified gelatin used in the present invention, the proportion of polar amino acids is 10 to 40%, preferably 20 to 30%, of all the constituent amino acids. And it is preferable that the ratio of the uncharged amino acid in the polar amino acid is 5% or more and less than 20%, preferably less than 10%. Furthermore, it is preferable that any one amino acid among serine, threonine, asparagine, tyrosine and cysteine is not included in the sequence, preferably two or more amino acids.

Generally, the minimum amino acid sequence that acts as a cell adhesion signal in a polypeptide is known (for example, “Pathophysiology”, Vol. 9, No. 7 (1990), page 527, published by Nagai Publishing Co., Ltd.). The genetically modified gelatin used in the present invention preferably has two or more of these cell adhesion signals in one molecule. As specific sequences, RGD sequences, LDV sequences, REDV sequences, YIGSR sequences, PDSGR sequences, RYVVLPR sequences, LGITIPG sequences, RNIAEIIKDI sequences, which are expressed in one-letter amino acid notation in that there are many types of cells that adhere. , IKVAV sequence, LRE sequence, DGEA sequence, and HAV sequence are preferable, RGD sequence, YIGSR sequence, PDSGR sequence, LGTIPG sequence, IKVAV sequence, and HAV sequence, and particularly preferably RGD sequence. Of the RGD sequences, an ERGD sequence is preferred.

As the arrangement of RGD sequences in the genetically modified gelatin used in the present invention, it is preferable that the number of amino acids between RGDs is not uniform between 0 and 100, preferably between 25 and 60.

The content of the minimum amino acid sequence is preferably 3 to 50, more preferably 4 to 30, and particularly preferably 5 to 20 per protein molecule from the viewpoint of cell adhesion and proliferation. Most preferably, it is 12.

In the genetically modified gelatin used in the present invention, the ratio of the RGD motif to the total number of amino acids is preferably at least 0.4%. When the genetically modified gelatin contains 350 or more amino acids, each stretch of 350 amino acids has at least 1 stretch. Preferably it contains two RGD motifs. The ratio of RGD motif to the total number of amino acids is more preferably at least 0.6%, more preferably at least 0.8%, more preferably at least 1.0%, more preferably at least 1.2%. And most preferably at least 1.5%. The number of RGD motifs in the genetically modified gelatin is preferably at least 4, more preferably 6, more preferably 8, more preferably 12 or more and 16 or less per 250 amino acids. A ratio of 0.4% of the RGD motif corresponds to at least one RGD sequence per 250 amino acids. Since the number of RGD motifs is an integer, a gelatin of 251 amino acids must contain at least two RGD sequences to meet the 0.4% feature. Preferably, the recombinant gelatin of the present invention comprises at least 2 RGD sequences per 250 amino acids, more preferably comprises at least 3 RGD sequences per 250 amino acids, more preferably at least 4 per 250 amino acids. Contains one RGD sequence. As a further aspect of the genetically modified gelatin of the present invention, it contains at least 4 RGD motifs, preferably 6, more preferably 8, more preferably 12 or more and 16 or less.
Further, the genetically modified gelatin may be partially hydrolyzed.

The genetically modified gelatin used in the present invention preferably has a repeating structure of A-[(Gly-XY) _n ] _m -B. m is preferably 2 to 10, and preferably 3 to 5. n is preferably 3 to 100, more preferably 15 to 70, and most preferably 50 to 65.

It is preferable to bind a plurality of naturally occurring collagen sequence units to the repeating unit. The naturally occurring collagen referred to here may be any naturally occurring collagen, but is preferably type I, type II, type III, type IV, and type V. More preferred are type I, type II and type III. According to another form, the collagen origin is preferably human, cow, pig, mouse, rat. More preferably, it is a human.

The isoelectric point of the genetically modified gelatin used in the present invention is preferably 5 to 10, more preferably 6 to 10, and further preferably 7 to 9.5.

Preferably, the genetically modified gelatin is not deaminated.
Preferably, the genetically modified gelatin does not have procollagen and procollagen.
Preferably, the genetically modified gelatin has no telopeptide.
Preferably, the genetically modified gelatin is a substantially pure collagen material prepared with a nucleic acid encoding natural collagen.

Particularly preferably as the genetically modified gelatin used in the present invention,
(1) the amino acid sequence of SEQ ID NO: 1; or (2) 80% or more (more preferably 90% or more, most preferably 95% or more) of homology with the amino acid sequence of SEQ ID NO: 1, An amino acid sequence having an angiogenic effect;
A genetically modified gelatin having

The genetically modified gelatin used in the present invention can be produced by a genetic recombination technique known to those skilled in the art, for example, according to the method described in EP1014176A2, US6992172, WO2004-85473, WO2008 / 103041, and the like. Specifically, a gene encoding the amino acid sequence of a predetermined recombinant gelatin is obtained, and this is incorporated into an expression vector to produce a recombinant expression vector, which is then introduced into a suitable host to produce a transformant. To do. By culturing the obtained transformant in an appropriate medium, genetically modified gelatin is produced. The genetically modified gelatin used in the present invention is prepared by recovering the genetically modified gelatin produced from the culture. be able to.

If the performance of the genetically modified gelatin alone is insufficient, it may be mixed with other materials or combined. For example, different types of genetically modified gelatin, other biopolymers or synthetic polymers may be mixed. Examples of the biopolymer include polysaccharides, polypeptides, proteins, nucleic acids, antibodies and the like. Preferred are polysaccharides, polypeptides, and proteins. Examples of polysaccharides, polypeptides, and proteins include collagen, gelatin, albumin, fibroin, and casein. Furthermore, these may be partially chemically modified as necessary. For example, hyaluronic acid ethyl ester may be used. Examples of the polysaccharide include glycosaminoglycans represented by hyaluronic acid and heparin, chitin, and chitosan. Furthermore, examples of polyamino acids include poly-γ-glutamic acid.

The genetically modified gelatin used in the present invention can be chemically modified depending on the application. Chemical modifications include the introduction of low molecular weight compounds or various polymers (biopolymers (sugars, proteins), synthetic polymers, polyamides) into the carboxyl group or amino group of the side chain of genetically modified gelatin, Examples include cross-linking between gelatins. Examples of the introduction of the low molecular weight compound into the genetically modified gelatin include a carbodiimide-based condensing agent.

The crosslinking agent used in the present invention is not particularly limited as long as the present invention can be carried out, and may be a chemical crosslinking agent or an enzyme. Examples of the chemical cross-linking agent include formaldehyde, glutaraldehyde, carbodiimide, cyanamide and the like. Preferred are formaldehyde and glutaraldehyde. Furthermore, examples of cross-linking of genetically modified gelatin include light irradiation on gelatin into which a photoreactive group has been introduced, or light irradiation in the presence of a photosensitizer. Examples of the photoreactive group include a cinnamyl group, a coumarin group, a dithiocarbamyl group, a xanthene dye, and camphorquinone.

When performing cross-linking by an enzyme, the enzyme is not particularly limited as long as it has a cross-linking action between genetically modified gelatin chains. Preferably, trans-glutaminase and laccase, most preferably trans-glutaminase is used for cross-linking. it can. A specific example of a protein that is enzymatically cross-linked with transglutaminase is not particularly limited as long as it has a lysine residue and a glutamine residue. The transglutaminase may be derived from a mammal or may be derived from a microorganism. Specifically, transglutaminase derived from a mammal that has been marketed as an Ajinomoto Co., Ltd. activa series, for example, Human-derived blood coagulation factors (Factor XIIIa, Haematologic Technologies, Inc.), including guinea pig liver-derived transglutaminase, goat-derived transglutaminase, and rabbit-derived transglutaminase manufactured by Oriental Yeast Co., Ltd., Upstate USA Inc., Biodesign International, etc. Etc.).

The cross-linking of genetically modified gelatin has two processes: a process of mixing a genetically modified gelatin solution and a cross-linking agent and a process of reacting these uniform solutions.

In the present invention, the mixing temperature when the genetically modified gelatin is treated with the crosslinking agent is not particularly limited as long as the solution can be uniformly stirred, but is preferably 0 ° C. to 40 ° C., more preferably 0 ° C. to 30 ° C. More preferably, it is 3 ° C to 25 ° C, more preferably 3 ° C to 15 ° C, still more preferably 3 ° C to 10 ° C, and particularly preferably 3 ° C to 7 ° C.

After stirring the genetically modified gelatin and the crosslinking agent, the temperature can be raised. The reaction temperature is not particularly limited as long as the crosslinking proceeds, but is substantially 0 ° C. to 60 ° C., more preferably 0 ° C. to 40 ° C. in view of denaturation and degradation of the recombinant gelatin. The temperature is preferably 3 ° C to 25 ° C, more preferably 3 ° C to 15 ° C, still more preferably 3 ° C to 10 ° C, and particularly preferably 3 ° C to 7 ° C.

In the present invention, basic fibroblast growth factor (bFGF) is used in combination with genetically modified gelatin. The form of bFGF used in the present invention is not particularly limited, and may be naturally derived bFGF or genetically modified bFGF. As the genetically modified bFGF, for example, those commercially available as Trafermin (Fiblast) (Kaken Pharmaceutical Co., Ltd.) may be used.

In the angiogenesis inducer of the present invention, a drug can be encapsulated as desired. The drug is a physiologically active ingredient. Specific examples include transdermal absorption agents, topical therapeutic agents, oral therapeutic agents, cosmetic ingredients, and supplement ingredients. Specific examples of the drug include anti-inflammatory agents, antibacterial agents, antibiotic agents, immunosuppressive agents, antioxidant agents, anticancer agents, vitamins, nucleic acids, and antibodies. Particularly preferred are anti-inflammatory agents. As the anti-inflammatory agent, either steroidal or non-steroidal may be used. Examples of anti-inflammatory agents include, for example, aspirin, acetaminophen, phenachicene, indomethacin, diclofenac sodium, piroxicam, fenoprofen calcium, ibuprofen, chlorpheniramine maleate, diflunisal, dexamethasone sodium phosphate, paclitaxel, docetaxel, 5 -Fluorouracil, topotensin, cisplatin, rapamycin, tacrolimus, cyclosporine. As vitamins, both water-soluble and fat-soluble are used. Specific examples of the vitamin include vitamin A, vitamin B group, vitamin C, vitamin D group, vitamin E, and vitamin K, for example. Specific drugs have been listed above, but as long as the genetically modified gelatin used in the present invention is used, the present invention is not limited to the drugs listed above.

In the present invention, an angiogenesis is achieved by administering a genetically modified gelatin having an amino acid sequence derived from the above-described partial amino acid sequence of collagen to a subject (for example, a mammal such as a human) in need of angiogenesis induction. Can be induced.

The angiogenesis-inducing agent of the present invention can be appropriately determined in dosage, usage, and dosage form according to the intended use. For example, the angiogenesis-inducing agent of the present invention may be directly administered to a target site in a living body, or distilled water for injection, physiological saline for injection, pH 5-8 buffer (phosphate system, citric acid) It may be suspended in a liquid excipient such as an aqueous solvent such as a system and administered by injection, coating, or the like. Further, it may be applied after mixing with an appropriate excipient to form an ointment, gel or cream. That is, the administration form of the angiogenesis inducer of the present invention may be oral or parenteral (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, etc.). In addition, for the treatment of coronary artery disease, obstructive peripheral arteriosclerosis, angiogenesis failure, etc., direct injection into the heart muscle from the intraventricular lumen using a catheter, or catheter in the stenosis or occlusion part in the coronary artery It is also possible to release or apply them locally using. Examples of the dosage form include orally administered drugs such as tablets, powders, capsules, granules, extracts and syrups, or injections (for example, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections). Etc.) and the like. For example, when administered locally, the form of the angiogenesis-inducing agent of the present invention is not particularly defined, and examples thereof include sponges, films, nonwoven fabrics, fibers (tubes), particles, meshes and the like.

The formulation of the angiogenesis-inducing agent of the present invention can be performed according to methods known to those skilled in the art. For example, when the pharmaceutical carrier is a liquid, it can be dissolved or dispersed, and when the pharmaceutical carrier is a powder, it can be mixed or adsorbed. Further, if necessary, pharmaceutically acceptable additives (for example, preservatives, stabilizers, antioxidants, excipients, binders, disintegrants, wetting agents, lubricants, coloring agents, fragrances) , Flavoring agents, skin coats, suspending agents, emulsifiers, solubilizers, buffering agents, tonicity agents, plasticizers, surfactants or soothing agents, and the like.

The dose of the genetically modified gelatin is not particularly limited, but is, for example, 1 to 100 mg, preferably 1 to 50 mg per 1 cm ² of the surface area of the organism to be administered.

The dose of bFGF is not particularly limited, and is, for example, 1 μg to 1 mg, preferably 1 μg to 100 μg, per 1 cm ² of the surface area of the organism to be administered.

Examples of the target diseases of the angiogenesis-inducing agent of the present invention include ischemic diseases, cell / tissue regeneration therapy, cell transplantation treatment, diabetic skin ulcer, hearing loss, heart disease, acute coronary syndrome, acute myocardial infarction, unstable angina Disease, promotion of fracture healing, pseudo joint, bone union failure, nerve regeneration treatment, and the like.

The following examples further illustrate the present invention, but the present invention is not limited to the examples.

CBE3 described below was prepared as a genetically modified gelatin (described in WO2008-103041).
CBE3
Molecular weight: 51.6kD
Structure: GAP [(GXY) 63] 3G
Number of amino acids: 571 RGD sequence: 12 Imino acid content: 33%
Almost 100% of amino acids are GXY repeating structures.
The amino acid sequence of CBE3 does not include serine, threonine, asparagine, tyrosine and cysteine.
CBE3 has an ERGD sequence.
Isoelectric point: 9.34

Amino acid sequence (SEQ ID NO: 1 in the sequence listing) (same as SEQ ID NO: 3 in WO2008 / 103041, except that X at the end is corrected to “P”)
GAP (GAPGLQGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGPAGAPGAPGLQGMPGERGAAGLPGAPG

In the following examples, unless otherwise specified, the genetically modified gelatin represented as CBE3 is described as R-Gel.

(1) Angiogenesis test using mice After adding 0.27 ml of glutaraldehyde (manufactured by Wako Pure Chemical Industries, Ltd.) aqueous solution (1%) to 2.43 ml of R-Gel aqueous solution (5.56%), 3cm x 3cm x 4mm Poured into a silicon mold and allowed to crosslink for 1 hour at room temperature and then at 24 ° C. for 24 hours. After completion of the reaction, the gel was removed from the template, the crosslinking reaction was stopped in 30 ml of 0.1 M glycine aqueous solution, and the obtained crosslinked R-Gel was washed with distilled water at room temperature for 1 hour for 3 hours. The resulting gel was quenched at −50 ° C. and lyophilized at −10 ° C. for 48 hours. The water content of the gel was measured from the change in the weight of the crosslinked R-Gel before and after the swelling treatment with PBS for 24 hours at 4 ° C., and was 95.4%. The obtained gel was cut out at 5 mg. To this gel, 50 μl of PBS containing 35 μg of bFGF was dropped, and left overnight at 4 ° C. to impregnate 35 μg of bFGF into the cross-linked R-Gel to prepare a bFGF-containing R-Gel preparation.

The 35 μg bFGF-containing crosslinked R-Gel preparation prepared above was implanted subcutaneously in the back of the mouse. Separately, as a control group, 50 μl of a PBS solution containing 35 μg of bFGF was subcutaneously administered. As another control group, 5 mg of bFGF-impregnated PI-5 gel (medgel) impregnated with 50 μl of 35 μg of bFGF in PBS was subcutaneously implanted. Three days after the administration, the skin of the mouse was peeled off, and the preparation embedding and the PBS solution administration site were observed. In the bFGF-containing PBS solution administration group, the state of the tissue around the administration site was the same as in the untreated group, and no gross change was observed. In the bFGF-impregnated PI-5 gel-administered group, almost no angiogenesis image was observed as in the untreated group. However, when a bFGF-containing cross-linked R-Gel preparation was embedded, the tissue around the preparation implantation site was also visually red, and an angiogenic effect that was clearly one of bFGF was confirmed.

From the above results, the bFGF-impregnated PI-5 gel administration group has not been able to efficiently adhere vascular endothelial cells induced with bFGF. In contrast, when the bFGF-containing crosslinked R-Gel preparation of the present invention is used, vascular endothelial cells induced with bFGF can be positively adhered to exert an angiogenic effect.

In order to quantify the angiogenic effect, the amount of hemoglobin contained in the blood was measured. A hemoglobin B-Test Wako kit (Wako Pure Chemical Industries) was used for the measurement.
5 mg of the above-prepared 35 μg bFGF-containing cross-linked R-Gel preparation was implanted subcutaneously in the back of the mouse. Separately, as a control group, 50 μl of PBS solution was subcutaneously administered. As another control group, 5 mg of bFGF-impregnated PI-5 gel (medgel) impregnated with 50 μl of 35 μg of bFGF in PBS was subcutaneously implanted. Two weeks after the administration, the skin of the mouse was peeled off, and the surrounding tissue of 1.5 cm × 1.5 cm was taken out mainly from the site where the preparation was embedded and the PBS solution was administered, and placed in a tube. The surrounding tissue was finely cut in a tube, and 300 μl of an extract (10 mM Tris, 1 mM EDTA, pH 7.8) was added. The mixture was stirred overnight at 4 ° C. with a rotator, and then the supernatant was separated by centrifugation. Using the supernatant, the amount of hemoglobin was measured according to the protocol of the kit.

The results are shown in FIG. As a result, the amount of hemoglobin in the bFGF-containing cross-linked R-Gel preparation administration group was clearly increased as compared with the PBS solution administration group and the bFGF-impregnated PI-5 gel administration group. In addition, as a result of t-testing the bFGF-containing cross-linked R-Gel preparation administration group with respect to each comparison group, p <0.01 was found, showing a significant difference, and confirming the angiogenic effect of R-Gel.

(2) Cell adhesion test (interaction with αVβ3 integrin)
In order to obtain the details of R-Gel's mechanism of neovascularization, R-Gel's cell adhesion test to vascular endothelial cells and experiments to investigate the interaction with αVβ3 integrin were conducted.
As a vascular endothelial cell used, HUVEC (normal human umbilical vein endothelial cell: Takara Bio Inc.) was used. It is known that the cells constantly express a number of αVβ3 integrin on the cell surface, and testing cell adhesion to the cells leads to vascular endothelial cells activated in new blood vessels. And the binding ability to αVβ3 integrin, which is reported to be highly expressed at the neovascular site.

HUVEC was cultured using endothelial cell basic medium-2 (serum-free) (EBM ^TM- 2) and endothelial cell medium kit-2 (2% FBS) (EGM ^TM- 2 BulletKit ^TM ) (Takara Bio Inc.) . An EDTA-containing 0.25% trypsin solution was used at the time of passage and cell detachment. HUVEC grown to a sufficient amount in a T-75 flask was peeled from the bottom of the flask, and the supernatant was removed by centrifugation. Thereafter, the cells were washed with endothelial cell culture medium-2 containing the above endothelial cell culture medium kit-2, and the supernatant was again removed by centrifugation. 0.1% BSA was added to endothelial cell culture medium-2 without endothelial cell culture medium kit-2. The number of viable cells was counted using a cell counter, and the final cell concentration was adjusted to 500,000 cells / mL.

On the other hand, for cell adhesion test, various proteins (R-Gel, fibronectin, collagen manufactured by Fibrogen (hereinafter referred to as Fibrogen), pig skin-derived gelatin (hereinafter referred to as PSK), cow bone-derived gelatin (hereinafter referred to as A plate coated with G1917P was prepared, and R-Gel was dissolved in PBS (phosphate buffer) at a concentration of 1 mg / mL to prepare an R-Gel solution. Fibronectin was dissolved at a concentration of 1 mg / mL to prepare a fibronectin solution, and Fibrogen was dissolved at a concentration of 1 mg / mL in PBS (phosphate buffer) to prepare a fibrogen solution. PSK was dissolved at a concentration of 1 mg / mL to prepare a PSK solution.G1917P was dissolved at a concentration of 1 mg / mL in PBS (phosphate buffer solution) to prepare a G1917P solution. Dilute with PBS and use for plate addition.

¡Non-treated 96-well plate (IWAKI) was used for the plate. A solution obtained by diluting the above lysate with PBS was added to a non-treated 96-well plate at 50 μL / well so that the protein concentration was 0.02, 0.1, 0.2, 2.0 μg / well. Thereafter, incubation was performed at 37 ° C. for 2 hours, and after removing the solution, 100 μL of PBS was added to all wells and washed to remove PBS (washing step). The washing step was performed 3 times. This resulted in coating plates with different coating proteins and coating concentrations.

In this coating plate, 100 μL each of the HUVEC suspension (500,000 cells / mL) prepared above was seeded. After incubating at 37 ° C. for 1 hour, the medium was removed by aspiration, 100 μL of PBS was added for washing, and PBS was removed by aspiration (PBS washing). This PBS washing was performed 3 times to obtain a plate from which PBS was removed.

For assaying the number of cells on the obtained plate, DNA assay was used. In each well of the plate, 100 μL of SDS solution (20 mg of SDS dissolved in 100 mL of 1 × SSC solution: 1 × SSC solution is 17.999 g NaCl and 8.823 g Na ₃ Citrate 2 L ultrapure 2) and leave at 37 ° C. for 1 hour. Transfer the total amount of each individual solution to a 96-well black plate (Non-treated) and add 100 μL of Hoechst solution (20 μL of Hoechst 33258 and 20 mL of 1 × SSC solution) to all wells. The fluorescence intensity was measured with a reader. The plate reader used was Gemini EM (Molecular Devices), and the fluorescence intensity was measured at an excitation wavelength of 355 nm and a measurement wavelength of 460 nm. A calibration curve was prepared from a suspension of HUVEC cells with the number of cells adjusted.

The results of the obtained cell adhesion test (DNA assay) are shown in FIG. 2 and FIG. As a result, R-Gel showed better cell adhesion to HUVEC than fibronectin, fibrogen, PSK and G1917P. Moreover, the state of cell adhesion on the R-Gel coating plate, cell adhesion on the Fibrogen coating plate, cell adhesion on the PSK coating plate, and cell adhesion on the G1917P coating plate is shown in FIG. In the R-Gel coated plate, it can be visually confirmed that the number of adherent cells is large. At the same time, from this photograph, the area of one individually attached cell was determined with the software ImageJ. The result is shown in FIG. This indicates that R-Gel has a significantly larger cell area than Fibrogen, PSK, and G1917P, so there is a stronger bond between R-Gel and HUVEC than the others. I found out.

(3) αVβ3 integrin inhibition experiment by HUVEC adhesion Regarding the cell adhesion test of R-Gel performed in (2) above to confirm that the binding of R-Gel and HUVEC is mediated by αVβ3 integrin, αVβ3 An experiment was conducted to determine whether the adhesion was suppressed by blocking the integrin with an anti-αV antibody.

Details of the cell adhesion experiment were performed in the same manner as in (2) above. The coating concentration was 0.2 μg / well, and the experiment was conducted using an R-Gel coating plate and a fibronectin coating plate. The prepared HUVEC cells were incubated with a sufficient concentration of anti-human αV monoclonal antibody (MAB1980: CHEMICON) at 37 ° C for 30 minutes, and the same amount of PBS was added and incubated at 37 ° C for 30 minutes, respectively. It indicated as antibody-treated HUVEC and untreated HUVEC. Cell seeding was performed by adding a solution prepared so that the antibody-treated HUVEC or untreated HUVEC was 1 million cells / mL to the plate at 100 μL / well. The cell adhesion time was 1 hour at 37 ° C. as in (2) above. Quantification of the number of cells was also performed by DNA assay in the same manner as in (2) above.

The obtained results are shown in FIG. Thus, it was found that the cell adhesion of R-Gel and fibronectin to HUVEC was significantly suppressed by the anti-human αV antibody. Fibronectin is known to bind to HUVEC via αVβ3 integrin, and in this example, R-Gel was also shown to bind to HUVEC via αVβ3 integrin in the same manner as fibronectin. .

This indicates that R-Gel binds to αVβ3 integrin, and the results of (2) and (3) above show that R-Gel shows good binding to αVβ3 integrin, and that the binding is not related to other collagens. It is stronger than gelatin, and R-Gel shows better binding to vascular endothelial cells, and its binding is stronger than other collagen and gelatin, and has high binding power and specificity to new blood vessels.・ Results shown at the molecular level.

From the above experiments, it was shown that genetically modified gelatin showed good binding to vascular endothelial cells, and that, in combination with bFGF, angiogenesis was induced even at a dose at which natural gelatin was not effective.

Claims

An angiogenesis-inducing agent comprising genetically modified gelatin having an amino acid sequence derived from a partial amino acid sequence of collagen and basic fibroblast growth factor as active ingredients.
The genetically modified gelatin has a repetition of the sequence represented by Gly-XY, which is characteristic of collagen (X and Y each independently represents one of amino acids) (a plurality of Gly-XY are the same or different from each other) The angiogenesis-inducing agent according to claim 1, wherein the molecular weight is 2 to 100 KDa.
The genetically modified gelatin has a repetition of the sequence represented by Gly-XY, which is characteristic of collagen (X and Y each independently represents one of amino acids) (a plurality of Gly-XY are the same or different from each other) The angiogenesis-inducing agent according to claim 1 or 2, which has a molecular weight of 10 KDa or more and 90 KDa or less.
The genetically modified gelatin has a repetition of the sequence represented by Gly-XY, which is characteristic of collagen (X and Y each independently represents one of amino acids) (a plurality of Gly-XY are the same or different from each other) The angiogenesis-inducing agent according to any one of claims 1 to 3, wherein two or more cell adhesion signals are contained in one molecule.
The angiogenesis inducer according to claim 4, wherein the cell adhesion signal is an amino acid sequence represented by Arg-Gly-Asp.
The angiogenesis inducer according to any one of claims 1 to 5, wherein the amino acid sequence of the recombinant gelatin does not contain serine and threonine.
The angiogenesis inducer according to any one of claims 1 to 6, wherein the amino acid sequence of the genetically modified gelatin does not contain serine, threonine, asparagine, tyrosine, and cysteine.
The angiogenesis inducer according to any one of claims 1 to 7, wherein the amino acid sequence of the recombinant gelatin does not include the amino acid sequence represented by Asp-Arg-Gly-Asp.
Genetically modified gelatin
Formula: A-[(Gly-XY) n ] m -B
(In the formula, A represents an arbitrary amino acid or amino acid sequence, B represents an arbitrary amino acid or amino acid sequence, n Xs independently represent any of the amino acids, and n Ys each independently represent an amino acid. N represents an integer of 3 to 100, and m represents an integer of 2 to 10. Note that n Gly-XY may be the same or different. Item 9. The angiogenesis inducer according to any one of Items 1 to 8.
Genetically modified gelatin
Formula: Gly-Ala-Pro-[(Gly-XY) 63 ] 3 -Gly
(In the formula, 63 X's each independently represent any amino acid, and 63 Y's each independently represent any amino acid. The n Gly-XY may be the same or different. Good.)
The angiogenesis inducer according to any one of claims 1 to 9, which is represented by:
The genetically modified gelatin has (1) the amino acid sequence described in SEQ ID NO: 1 or (2) the amino acid sequence described in SEQ ID NO: 1 has an amino acid sequence having an angiogenic action having 80% or more homology. The angiogenesis inducer according to any one of claims 1 to 10.
The angiogenesis inducer according to any one of claims 1 to 11, wherein the genetically modified gelatin is crosslinked.
The angiogenesis inducer according to claim 12, wherein the crosslinking is performed by aldehydes, a condensing agent, or an enzyme.
The angiogenesis inducer according to any one of claims 1 to 13, which accumulates at an angiogenesis site and induces angiogenesis.
A method of inducing angiogenesis, comprising administering a recombinant gelatin having an amino acid sequence derived from a partial amino acid sequence of collagen and a basic fibroblast growth factor to a subject in need of angiogenesis induction.
Use of a recombinant gelatin having an amino acid sequence derived from a partial amino acid sequence of collagen and a basic fibroblast growth factor for the production of an angiogenesis inducer.