KR100450578B1 - Compositions comprising Nm23 protein for the use of matrix metalloproteinase inhibitor and angiogenesis inhibitor - Google Patents

Abstract

The present invention provides a composition for inhibiting MMP activity and inhibiting angiogenesis, containing Nm23 protein as an active ingredient.

The composition containing the Nm23 protein of the present invention as an active ingredient can be used as a prophylactic and therapeutic agent for a wide range of diseases mediated by excessive activity of MMP and various diseases caused by angiogenesis.

Description

Compositions comprising Nm23 protein for the use of matrix metalloproteinase inhibitor and angiogenesis inhibitor}

The present invention relates to a composition for inhibiting matrix metalloproteinase (hereinafter referred to as 'MMP') activity and a composition for inhibiting angiogenesis.

Angiogenesis is the process by which blood vessels grow out of the endothelial cells of existing vessels and are strictly regulated only when the corpus luteum, which is the development of embryonic development, tissue regeneration and wound healing, and changes in the female genital system periodically. Folkman and Cotran, Relation of vascular proliferation to tumor growth, Int Rev Exp Pathol 16, 207-248 (1976).

However, there are diseases caused by abnormal growth of angiogenesis that is not controlled autonomously. Diseases related to angiogenesis in pathological conditions include hemangioma, angiofibroma, angioplasty and cardiovascular diseases such as atherosclerosis, angiogenesis, and edema sclerosis. Eye diseases caused by angiogenesis include corneal graft angiogenesis and angiogenesis. Glaucoma, diabetic retinopathy, neovascularized corneal disease, spot degeneration, pterygium, retinal degeneration, posterior capsular fibrosis, granulomatous conjunctivitis, etc. There are dermatological diseases such as dermatitis and acne, and cancer growth and metastasis depends on angiogenesis [D'Amato RJ and Adamis AP, Ophthalmol 102, 1261-1262, 1995; Arbiser JL, J Am Acad Derm 34 (3): 486-497, 1996; O'Brien KD et al. Circulation 93 (4): 672-682, 1996; Hanahan D and Folkman J, Cell 86: 353-364, (1996).

Especially in cancer, neovascularization of cancer cells plays an important role in the growth and metastasis of cancer cells. Angiogenesis not only provides the nutrition and oxygen needed for tumor growth and proliferation, but also provides the opportunity for cancer cells to metastasize to the blood circulation by the new capillary blood vessels that have penetrated the tumor, causing the cancer cells to metastasize throughout the body. Polverini PJ, Critical Reviews in Oral Biology , Vol. 6, No. 3, 230-247 (1995).

Diseases caused by abnormal angiogenesis are deeply involved in the development or progression of the various diseases, and studies are actively being conducted to find substances that inhibit angiogenesis for the prevention or treatment of these diseases.

In order for angiogenesis to occur, the most important process is that the basement membrane must first be decomposed by enzymes, and through the decomposed basement membrane, blood vessels are reconstituted by vascular endothelial cell migration, proliferation, and vascular endothelial cell differentiation. Consists of being generated. At this time, enzymes that play an important role in decomposing type IV collagen, which is the main component of the basement membrane, are enzymes belonging to the MMP group, and more than 20 MMPs have been found to date.

MMP is a zinc-containing endopeptidase that is involved in the breakdown of connective tissues and basement membranes. New classes (MMP-3, etc.) and collagenases (MMP-14, etc.) present in the membrane. Of these, gelatinases are known to be involved in osteoarthritis, whereas gelatinases are primarily involved in cancer growth and metastasis by breaking down the basement membrane [Howell. D.S .; Pelaetier, J.P. In Arthritis and Allied Conditions; McCarthy, D. J .; Koopman, W. J., Eds; Lea and Febiger; Philadelphia, 1993; 12th Edition Vol. 2 pp. 1723].

MMPs are associated with many other diseases. Diseases in which excessive MMP activity is observed include restenosis, MMP-mediated osteopenia, inflammatory diseases of the central nervous system, skin aging, wrinkles, dermatitis, rheumatoid arthritis, sepsis arthritis, corneal ulcers, abnormal wound union, bone disease, proteinuria , Aortic aneurysm, Degenerative cartilage loss due to traumatic joint injury, Myelin deprivation disease, Liver cirrhosis, Renal glomerular disease, Immature rupture of the embryonic membrane, Inflammatory bowel disease, Periodontal membrane disease, Macular degeneration associated with aging, Diabetic retinopathy, Proliferation Sexual vitreoretinopathy, immature retinopathy, ophthalmic inflammation, cone cornea, Sjogren's syndrome, myopia, ocular tumor, and corneal rejection.

As such, MMP plays a very important role in angiogenesis and cancer metastasis through the decomposition of the basement membrane, and thus, various mediated diseases are required to develop inhibitors capable of inhibiting it.

In addition, since such inhibitors usually need to be administered to patients for a long time, there is a need for the development of drugs with low toxicity.

Nm23 protein is an endogenous protein and has been reported to have NDP (Nucleoside diphosphate) -kinase enzymatic activity [Biggs et al., A Drosphila gene that is homologous to a mammalian gene associated with tumor metastasis codes for a nucleoside diphosphate kinase. Cell 63, 933-940 (1990)], also known as transcription factor and I factor [Postel et al., Human c-myc transcription factor PuF identified as Nm23-H2 nucleoside diphosphate kinase , a candidate suppressor of tumor metastasis. Science 261, 478-480 (1993); Okabe-Kado et al., Inhibitory action of Nm23 proteins on induction of erythroid differentiation of human leukemia cells. Biochim. Biophys. Acta 1267, 101-106 (1995).

Two genes, Nm23-H1 and Nm23-H2, have been found in human genes, and both are known to be involved in inhibiting cancer metastasis [Rosengard et al. Reduced Nm23 / Awd protein in tumor metastasis and aberrant Drosophila development. Nature 342, 177-180 (1989); Charpin C. et al., Automated and quantitative immuocytochemical assays of Nm23 / NDPK protein in breast carcinomas, Int. J. Cancer, 74, 416-420 (1997).

However, although some of the above effects have been found, the specific mechanism of action of Nm23 protein is not known and its usefulness is extremely limited. Therefore, by specifically identifying the mechanism of action of Nm23 protein, it is useful for various diseases. The need to provide has emerged. There is also an urgent need for the development of effective prophylactic and therapeutic agents for a number of diseases caused by overexpression of MMPs and abnormal angiogenesis.

Therefore, the present inventors will attempt to provide a composition for effectively preventing and treating various diseases caused by overexpression of MMP and abnormal angiogenesis by identifying the relationship between Nm23 protein and MMP activity and angiogenesis.

1 is a diagram showing the effect of inhibiting MMP activity by Nm23-H1 and Nm23-H2.

Figure 2 is a diagram showing the formation of umbilical cord endothelial cells human.

Figure 3 is a diagram showing the effect of Nm23-H1 protein inhibits the tube formation of vascular endothelial cells cultured on Matrigel.

4 is a diagram showing the effect of Nm23-H2 protein inhibits the tube formation of vascular endothelial cells cultured on Matrigel.

5 is a diagram showing the effects of Nm23-H1 protein and Nm23-H2 protein in inhibiting angiogenesis in the mouse Matrigel model.

The present invention provides a composition for inhibiting MMP activity comprising Nm23 protein as an active ingredient.

In addition, the present invention is a NMP23 protein as an active ingredient MMP-mediated diseases, ie restenosis, MMP-mediated osteopenia, inflammatory diseases of the central nervous system, skin aging, wrinkles, dermatitis, rheumatoid arthritis, septic arthritis, corneal ulcer, Aberrant wound union, bone disease, proteinuria, aortic aneurysm, degenerative cartilage loss due to traumatic joint injury, demyelination of the nervous system, liver cirrhosis, renal glomerular disease, immature rupture of the embryonic membrane, inflammatory bowel disease, fascia membrane disease, aging It provides a composition for the prevention and treatment of macular degeneration, diabetic retinopathy, proliferative vitreoretinopathy, immature retinopathy, ocular inflammation, cone cornea, Sjogren's syndrome, myopia, ophthalmic tumor, corneal rejection.

In another aspect, the present invention provides a composition for inhibiting angiogenesis comprising the Nm23 protein as an active ingredient.

In addition, the present invention is an Nm23 protein as an active ingredient, angiogenic diseases, that is, hemangioma, hemangiofibroma, diabetic retinopathy, retinopathy of premature infants, neovascular glaucoma, corneal disease caused by neovascularization, degenerative plaque, degeneration of pneumonia, pterygium It provides a composition for the prevention and treatment of retinal degeneration, posterior capsular fibrosis, granular conjunctivitis, psoriasis, capillary dilatation, purulent granulomas, seborrheic dermatitis, obesity, acne and arthritis.

The Nm23 protein used in the present invention includes an Nm23 protein, a derivative thereof, and a fragment thereof which exhibit the same function as a protein containing an amino acid sequence encoded by a human Nm23 gene (US Patent No. 6,329,198).

The Nm23 protein used in the present invention includes an Nm23 protein, derivatives thereof and fragments thereof which exhibit the same functions as proteins encoded by human Nm23-H1 gene and human Nm23-H2 gene.

The Nm23 protein used in the present invention is not limited to either natural extracts, those produced by typical peptide chemistry using peptide synthesizers, or those produced by genetic engineering methods. Nm23 protein used in the present invention can be produced in large quantities using E. coli, and in this case, it is preferable to use the purified by ATP-Sepharose column chromatography.

The composition comprising the Nm23 protein of the present invention may include one or more components of Nm23 protein, derivatives thereof and fragments thereof having the same function as human Nm23 protein as an active ingredient.

The composition comprising the Nm23 protein of the present invention includes, as an active ingredient, one or more components of Nm23 protein, derivatives thereof, and fragments thereof having the same function as human Nm23 protein, and further include other active ingredients with these components. can do.

The composition comprising the Nm23 protein of the present invention can be preferably formulated into a pharmaceutical composition including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration.

Acceptable pharmaceutical carriers in compositions formulated as liquid solutions may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, If desired, other conventional additives such as antioxidants, buffers, bacteriostatics, and the like may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.

The compositions of the present invention can be administered in a conventional manner via the intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, nasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. Among these, parenteral administration includes intravenous, intramuscular, intraperitoneal, intrasternal, transdermal and intraarterial injection and infusion.

Specific effective amounts for a particular disease include the type of disease to be prevented or treated, the type and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the age, body weight, general health, sex and diet of the patient, and the time of administration. The range varies depending on various factors, including the route of administration and the rate of administration of the composition, the duration of treatment, and the drugs used concurrently (e.g., chemotherapeutic agents), but the daily dosage is divided between 0.05 mg and 2 g in one to several doses. It is preferable to administer.

It was confirmed that the composition of the present invention strongly inhibited the activity of MMP in an MMP activity inhibition experiment using a spectrofluorometer.

In addition, it was confirmed that the composition of the present invention strongly inhibited angiogenesis in tube formation experiments of vascular endothelial cells using gelled Matrigel and animal experiments using a mouse Matrigel model.

Hereinafter, preferred embodiments are illustrated to help understand the present invention. However, this is merely provided to understand the present invention more easily, but is not limited thereto.

Production Example : Preparation of Nm23-H1 Protein and Nm23-H2 Protein

Nm23-H1 protein and Nm23-H2 protein used in the present invention were overexpressed in E. coli as a recombinant protein, and then a protein having a purity of 99% or more was prepared using ATP-Sepharose column chromatography [ Kim et al., Rapid purification and characterization of NDP kinase using ATP-Sepharose affinity column chromatography, Mol. Cell 7, 630-634 (1997). The recombinant Nm23 protein thus obtained was used after confirming that it was structurally identical to Nm23 protein existing in nature.

Example 1 : MMP's activity inhibitory effect

The inhibitory effect of Nm23 protein on MMP activity was examined using a spectrofluorometer (Perkin-Elmer LS50B).

MMPs used at this time were genetically prepared in insect cells using a baculovirus system.

2,4-dinitrophenyl-Pro-Leu-Ala-Leu-Trp-Ala-Arg (SEQ ID NO: 1, CALBIOCHEM Cat.No. 444211) was used as the substrate, and MMP-2 and MMP-9 2,4-dinitrophenyl-Pro-Leu-Gly-Met-Trp-Ser-Arg (SEQ ID NO: 2, CALBIOCHEM Cat.No. 444215) and 2,4-dinitrophenyl-Pro-Tyr- for MMP-3 Ala-Tyr-Trp-Met-Arg (SEQ ID NO: 3, CALBIOCHEM Cat. No. 444220) was used.

Fluorescence intensities were measured at an excitation wavelength of 280 nm and an emission wavelength of 360 nm.

As a result, as shown in FIG. 1, the Nm23 protein was 2 μg / ml, and the Nm23-H1 protein was 63% MMP-1, 90% MMP-2, and 82% MMP-3 when compared with the control. Nm23-H2 protein inhibited MMP-1 by 40% and MMP-2 by 84%, respectively.

Example 2 : In Vitro Inhibitory Effect of Nm23 Protein

As an in vitro experiment to indirectly understand the effects on angiogenesis, the effect on the formation of capillary-like structures by human vascular endothelial cells was observed. Human Umbilical Vein Endothelial Cells (HUVECs) were isolated from cultured umbilical cords of fresh humans obtained by caesarean section, cultured, and confirmed to be vascular endothelial cells by cellular immunological staining using antibodies of factor VII. Was used. The vascular endothelial cells thus obtained were cultured on gelled Matrigel (Matrigel, commercially available from Collaborative Biomedical Products) at 37 ° C. for 16-18 hours.

Vascular endothelial cells cultured by the above method were used as controls, and the Nm23 protein was treated at 125 µg / ml to compare with vascular endothelial cells cultured on Matrigel.

Figure 2 is observed that the vascular endothelial cells cultured on Matrigel formed a reticular tubular structure can be seen that the reticular tubular structure is thought to be a process of angiogenesis.

FIG. 3 shows vascular endothelial cells cultured on Matrigel by treating Nm23-H1 protein at a concentration of 125 µg / ml.

FIG. 4 shows vascular endothelial cells cultured on Matrigel by treatment with 125 μg / ml Nm23-H2 protein. As shown in FIG.

When the area of the tube formed on the Matrigel was analyzed by Image-Pro Plus (Media Cybernetics), the Nm23-H1 protein and the Nm23-H2 protein were treated as shown in Table 1 below. It can be seen that the formation is inhibited to about 41% and 45%, respectively.

Pipe forming area Tube formation rate (%) Control 92 100 Nm23-H1 protein treatment group 54 59 Nm23-H2 protein treatment group 51 55

Example 3 : Inhibitory Effect of Nm23 Protein on Angiogenesis in Vivo

Angiogenesis inhibitory effects of Nm23-H1 protein and Nm23-H2 protein were measured using a mouse matrigel model, an animal experiment that can quantitatively inhibit angiogenesis.

Subcutaneous injection of 0.4 ml of Matrigel, 50 ng / ml of basic Fibroblast Growth Factor (FGF) and 50 units / ml of heparin in 6 to 8 week C57BL / 6 mice showed strong angiogenesis. After 3-5 days, the epidermis was removed, the matrigel was carefully separated to recover the gel, and then the amount of hemoglobin was measured using a Labin's reagent (commercially available from Sigma), which measures the total amount of hemoglobin in the blood. Measured and used as a control. In addition, the Nm23-H1 protein and Nm23-H2 protein were mixed with Matrigel at 49 μg per horse, respectively, and the hemoglobin content was measured and compared with the control group.

As a result, as shown in Table 2 and Figure 5, it can be seen that when the Nm23-H1 protein and Nm23-H2 protein containing the hemoglobin content is very low compared to the control. In other words, Nm23-H1 protein and Nm23-H2 protein can be seen that the angiogenesis inhibition rate of 74%, 77% respectively.

Hemoglobin content (g / ㎗) Angiogenesis inhibition rate (%) Control 390 ± 254 100 Nm23-H1 protein treatment group 102 ± 134 74 Nm23-H2 protein treatment group 91 ± 96 77

Since the composition comprising Nm23 of the present invention exhibits an action effect of inhibiting the activity of MMP, it is possible to prevent various symptoms and diseases mediated by MMP overexpression in advance or to reduce and eliminate the extent of symptoms and diseases already advanced. It has an effect.

In addition, since the composition comprising the Nm23 protein of the present invention has an action effect of inhibiting angiogenesis, it is possible to prevent various symptoms and diseases caused by abnormal angiogenesis in advance or to reduce the extent of symptoms and diseases already advanced and to eliminate them. It has a therapeutic effect.

<110> KIM, Min-Young KIM Min-Young; AngioLab, Inc. <120> Compositions comprising Nm23 protein for the use of matrix metalloproteinase inhibitor and angiogenesis inhibitor <130> 02p-36 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence < 220> No. 1 Pro is 2,4-dinitrophenyl Pro. <400> 1 Pro Leu Ala Leu Trp Ala Arg 1 5 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> No. 1 Pro is 2,4-dinitrophenyl Pro. <400> 2 Pro Leu Gly Met Trp Ser Arg 1 5 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> No. 1 Pro is 2,4-dinitrophenyl Pro. <400> 3 Pro Tyr Ala Tyr Trp Met Arg 1 5

Claims (10)

Restenosis, matrix metalloprotease-mediated osteopenia, inflammatory diseases of the central nervous system, skin aging, wrinkles, dermatitis, rheumatoid arthritis, septic arthritis, corneal ulcer, abnormal wound union, bone disease, Proteinuria, Aortic aneurysm, Degenerative cartilage loss due to traumatic joint injury, Myelin deprivation disease, Liver cirrhosis, Renal glomerular disease, Immature rupture of the embryonic membrane, Inflammatory bowel disease, Periodontal membrane disease, Macular degeneration associated with aging, Diabetic retinopathy Composition for the prophylaxis and treatment of matrix metalloprotease-mediated diseases selected from the group consisting of proliferative vitreoretinopathy, immature retinopathy, ophthalmic inflammation, cone cornea, Sjogren's syndrome, myopia, ophthalmic tumor and corneal rejection The composition for preventing and treating matrix metalloprotease-mediated diseases according to claim 1, wherein the Nm23 protein is Nm23-H1 protein. The composition for preventing and treating matrix metalloprotease-mediated diseases according to claim 1, wherein the Nm23 protein is Nm23-H2 protein. delete The composition for the prophylaxis and treatment of matrix metalloprotease-mediated diseases according to any one of claims 1 to 3, wherein the formulation is an injection, inhalant, spray, tablet, capsule, soft capsule, infusion, granule, pill or coating. delete delete delete delete delete