KR100274172B1 - A synthetic peptide derived from TIMP-2 inhibiting the activity of type Ⅳ collagenase - Google Patents

Abstract

PURPOSE: Provided is a synthetic peptide having some amino acids of TIMP-2 which inhibits the activity of IV collagenase to prevent cancer growth and metastasis caused by vascularization. CONSTITUTION: The synthetic peptide is obtained by the next steps of: i) adding 0.25mol of parahydroxy methylphenyloxymethyl polystyrene resin to a tube and synthesizing Fmoc-amino acid, carboxyl terminal of peptide, in the tube; ii) deprotecting using 20% piperidine for 21 minutes; iii) washing and coupling the peptides with NMP for 9 minutes and with NMP for 71 minutes, respectively; iv) separating synthesized peptides with TFA from a solid support; v) adding resin attached with synthesized peptides to a flask and cooling the peptides; vi) washing the peptides with DCM and adding diethylether; vii) centrifuging the mixture to obtain sediments and eliminating supernatant; and viii) purifying the remaining sediments.

Description

A synthetic peptide derived from TIMP-2 inhibiting the activity of type IV collagenase}

The present invention relates to a synthetic peptide having a cancer suppression and angiogenesis inhibitory activity, and a method for producing the same, which are prepared by modifying a peptide derived from TMP-2 (Tissue Inhibitor of Metalloproteinase-2), and these peptides are metastatic inhibitors. Used as More specifically, the synthetic peptides of the present invention obtain peptides comprising amino acid sequences 6 to 15 of wild type TIMP-2 and modify them or add amino acid sequences 108 to 126 of TIMP-2. It can be used as a transfer inhibitor by inhibiting the activity of type IV collagenase.

Angiogenesis of cancer cells plays an important role in the growth and metastasis of cancer cells. If cancer cells fail to angiate or enter and exit blood vessels, most cancers will not grow to more than 1 to 2 mm in diameter and remain localized in the primary position (Folkman and Tyler; Cancer Invasion and metastasis: Biologic mechanisms and Therapy (SB Day ed.) Raven press, New York, 1977, PP: 94-103). Angiogenesis of cancer occurs through a process in which capillaries extend from existing blood vessels toward solid cancer, which can be seen by electron microscopy, and a pattern is observed in response to angiogenesis stimulation from cancer. Blood vessels, particularly the basement membrane around the lavage after the capillary vessels, are broken down, and then vascular endothelial cells exit from the vessels and travel towards cancer. At this time, the cells following the DNA synthesis and cell differentiation, once immature new blood vessels are extended to form branches and loops of blood vessels, forming a complete vascular network through the process of tubular formation. Finally, the capillaries are surrounded by the basement membrane or perivascular cell layer to form mature capillary images (Ausprunk and Folkman; Microvasc Res 14, 53-65 (1977); Burger, Chandler and Kintworth, Lab. Invest 48, 169-). 180 (1983).

Cancer cells cause angiogenesis by releasing angiogenic factors such as Fibroblast Growth Factor (FGF), Vascular Endothelial Growth Factor (VEGF), Angiogenin, and TGF-α. Angiogenesis plays two important roles in tumors: firstly, it provides the nutrition and oxygen needed for the growth and proliferation of primary and metastasized secondary tumors; It gives them a chance to get into and spread cancer cells all over the body to become metastasis.

The main cause of death of cancer patients is metastasis and metastasis is the growth of cancer cells isolated from primary tumor masses and implanted into anatomically distant tissues or organs [Sugarbaker, Weingard and Roseman; Cancer Invasion and Metastasis (L.A. Liotta and I. R. Hart ed.) Boston, Nijhoff. 1982. PP 427-465. Currently, the clinic uses chemotherapy or immunotherapy to improve the therapeutic performance of cancer. Although these treatments have some effects in some tumors, they do not contribute to increasing the survival rate of cancer patients. Because of the transition.

Riotta et al suggest a series of biochemical phenomena in which metastasis occurs: First, cancer cells attach to substrates such as laminin in the basement membrane or fibronectin in lipids through surface receptors of cancer cells. Cancer cells fixed in the second stage secrete hydrolytic enzymes or cause host cells to secrete enzymes to degrade the substrate. The third step is for cancer cells to migrate through parts of the hydrolyzed substrate. Subsequent invasion of the substrate occurs by repeating these three steps (Liotta, Kleinerman, Catanzara and Rynbrandt; J. Natl Cancer Inst 58, 1427-1439 (1977)). In order for cancer cells to metastasize to distant organs, cancer cells must first invade the surrounding tissues. Normal epithelial cells are surrounded by a basement membrane and isolated from the surrounding epilepsy, and the basement membrane outside the vascular endothelial cells is a barrier to cancer cells from penetrating into the tissue parenchyma, which is the primary barrier to cancer cell infiltration. Thus, basement membrane collapse indicates that metastasis is occurring.

Although angiogenesis and metastasis occur differently, the two processes have a common function in common, which means that the two processes can only occur when the basement membrane is broken down. The basement membrane serves as an important barrier when metastasis and angiogenesis occur. These base membranes are insoluble and continuous and have a soft structure that large proteins cannot pass through, and the main components of the base membrane are type IV collagen, laminin, and heparan sulfate proteoglycans (Vracko Am. J. Pathol 77, 313 -317 (1974)).

Type IV collagenase is an enzyme that increases activity in metastatic cancer cells. This type IV collagenase is an enzyme belonging to the matrix metalloproteinase (hereinafter, abbreviated as "MMP") system and is an enzyme that specifically breaks down type IV collagen which forms the main skeleton of the basement membrane. Several studies have reported a direct relationship with cancer metastasis (Garbisa et al .; Cancer Res 47, 1523-1528 (1987), Ostrowski et al .; Mol carcinogenesis 1, 13-19 (1988), Liotta et al. Nature 284, 67-68 (1980)). Overexpression or high activity of these enzymes is associated with several pathological conditions, such as malignancy and rheumatoid arthritis (Okada, et al .; J. Biol. Chem. 261, 14245-14255 (1986), Kalebic et. al .; Science 221, 281-283 (1983), Liotta et al .; Nature 284, 64-68 (1980).

Type IV collagenase is known to have two subtypes. The latent type IV collagenase having a molecular weight of 72 KDa is called MMP-2, and the type IV collagenase having a molecular weight of 92 KDa is called MMP-9. All of the enzymes belonging to these MMP systems are produced and secreted in an inactive manner, and are activated by treating an enzyme such as trypsin or plasmin or by exposure to an organic mercury compound. At this time, about 80 amino acid residues are removed from the amino terminus. Is activated.

However, even if MMP is activated, the presence of TIMP does not react with the substrate.

TIMP (Tissure Inhibitor of Metalloproteinase) is a natural MMP inhibitor present in the living body, and three types of TIMP-1,2,3 have been reported so far, and 12 cysteines form disulfide bonds to form 6 loops and Form two domains. It is reported that only three loops at the amino acid terminus can inhibit MMP activity, and three loops at the carboxy terminus are thought to play an important role in binding the latent type of MMP-9 (Murphy et. al .; Biochemistry 30, 8097-8102, 1991). It is also reported that the portion of TIMP-1 inhibiting MMP is discontinuous and will include amino acid sequences from two other loops of at least six loops (O'shea et al .; Biochem 31, 10146-10152, 1992). In addition, disulfide bonds (Cys ¹³ -Cys ¹²⁴ , Cys ¹²⁷ -Cys ¹⁷⁴ ) have been reported as an important part of TIMP-1 inhibition of MMP-1 (Bodden et al .; J. Biol. Chem. 269, 18943-18952 (1994).

TIMP-2 is 40% consistent with TIMP-1 and the amino acid sequence and binds to both the pro and active forms of MMP-2, and has been reported to inhibit all active MMPs. Some hydrolysis of TIMP-2 with trypsin has been reported to have the ability to bind the 13.5 kDa fragment near the amino acid end to the MMP-1 active form (DeClerck et al., Biochem J. 289, 65-69 (1993) ). Therefore, it is speculated that the part including the first three disulfide bonds among the six disulfide bonds will be an important part for forming a complex with MMP-2. Thus, since TIMP-2 has the activity which suppresses type IV collagenase activity, TIMP-2 can be used as an anticancer agent.

When developing TIMP-2 as an anticancer agent, the production of peptides containing only the portion of the structure of TIMP-2 that inhibits the activity of type IV collagenase results in lower molecular weight, so that it is absorbed when developed as a drug compared to full-size TIMP-2. It has the advantage of fast delivery and less susceptible to degradation enzymes, so that the concentration can be sustained.

Therefore, the present inventor has disclosed and patented the sequence of the peptide of the wild type TIMP-2 amino acid sequence that plays an important role in binding to type IV collagenase (Korean Patent Application No. 94-23476), and continued the research The peptides derived from wild type TIMP-2 were modified to obtain even more active peptides to complete the present invention.

TIMP-2-derived synthetic peptides of the present invention can prevent cancer growth and metastasis by inhibiting MMPs, which play a crucial role in cancer angiogenesis and metastasis.

It is an object of the present invention to provide a peptide derived from and modified from wild type TIMP-2. The synthetic peptide is derived from a peptide comprising an amino acid sequence of amino acids 6 to 15 of TIMP-2, in which TIMP-2 plays an important role in inhibiting MMP, or the sequence is modified or amino acid 108 of TIMP-2 To amino acids 126 and peptides to which this amino acid sequence has been added. These synthetic peptides inhibit MMP-2, an enzyme that plays a crucial role in cancer angiogenesis and metastasis, thereby preventing angiogenesis, preventing cancer growth and inhibiting cancer metastasis.

1 is a diagram confirming the activity of type IV collagenase (MMP-2, 9) isolated from the cancer cell culture medium (zymogram).

Figure 2 shows the results of experiments to inhibit the MMP-2 activity of the peptide.

Figure 3 measures the effect of inhibiting the transition according to the concentration of rTIMP-2.

4 shows the inhibition of MMP-2 activity of HB 703 derivatives.

Figure 5 is a test of the survival extension of mice that caused tumors with B16BL6.

Figure 6 is a test of the survival extension of the tumor-induced mice with B16BL6.

7 is an experiment showing the effect on the tube formation of HUVE cells by rTIMP-2.

8 is an experiment showing the effect on the tube formation of HUVE cells by the peptide.

9 shows the results of animal testing of inhibition of angiogenesis by rTIMP-2 and peptide.

Figure 10 is a drug efficacy test for human gastric cancer in nude mice.

Figure 11 measures the tumor weight of human gastric cancer grown in nude mice treated with peptides.

FIG. 12 measures 44 days of gastric cancer growth in nude mice with prolonged survival by peptides.

FIG. 13 shows survival extension experiments for human stomach cancer in nude mice.

Hereinafter, the present invention will be described in detail.

The present invention is to prepare a synthetic peptide derived from wild type TIMP-2, to prepare a wild type TIMP-2 by genetic engineering method in order to compare the MMP-2 inhibitory effect with wild type TIMP-2. The present inventors have applied for a patent for a method for manufacturing TIMP-2 (Korean Patent Application No. 94-23475), and according to the method, TIMP-2 can be easily obtained in high yield.

Hereinafter, the manufacturing method of TIMP-2 is described.

<Production Example> Preparation of Recombinant TIMP-2

CDNA library made from mRNA isolated from liver cancer tissue of liver cancer patient is used as a template, and the 5 'primer inserts some base sequence having complementarity with N-terminal region of TIMP-2 and recognition region of Nde I restriction enzyme. In the 3'- primer, polymerase chain reaction was carried out using two primers in which the nucleotide sequence of complementary to the C-terminal region of TIMP-2 and the recognition site of Hind III restriction enzyme were inserted. And amplified and purified TIMP-2 gene. DNA of the above polymerase chain reaction product was cut with restriction enzymes of Nde I and Hind III and inserted into pGEMEX-1 (Promega) vector which is widely used as an expression vector. This expression plasmid was named pGMX-TP, and using this, E. coli JM109 (DE3) was transformed. When transformed Escherichia coli was cultured in LB (Luria-Broth) medium under appropriate culture conditions and induced protein expression with isopropyl thio-β-D-galactoside (IPTG), the protein of TIMP-2 was expressed in the cytoplasm of E. coli. Produced as a clone body, the inclusion body was dissolved in protein denaturation and folded with native TIMP-2, and then purified and prepared using an anion exchange resin.

In addition, the inventors of the present invention prepared the peptides that are considered to be an important part of the amino acid sequence of TIMP-2 produced by the above-mentioned preparation for binding to type IV collagenase, the molecular weight is reduced to full-size TIMP-2 Compared with TIMP-2 prepared in Preparation Example, TIMP-2 amino acid sequence No. 1 was developed because it has the advantage of fast delivery and less degradation of enzymes to sustain the concentration when developed as a drug. Peptides, including up to 15, were found to be an important part of the inhibitory action of type IV collagenase, and peptides containing them have been synthesized (Korean Patent Application No. 94-23476).

The inventors further continued to find that the peptides modified from the 6 to 15 sequences among the TIMP-2 amino acid sequences were superior to the type IV collagenase. Peptides having an amino acid sequence of the wild type TIMP-2 amino acid sequence 6 to 15 had no inhibitory effect on MMP-2, but the peptide sequence was modified or amino acid sequences 108 to 126 The added peptides inhibited MMP-2 activity. Peptides that inhibit MMP-2 activity showed excellent results in metastasis inhibition and survival extension experiments using B16BL6 melanoma, and strongly inhibited angiogenesis in angiogenesis experiments. In addition, measurement of the effect on human gastric cancer with nude mice inhibited tumor growth and prolonged survival of nude mice.

In order to use the peptide of the present invention as a therapeutic agent, it may be prepared by a known method in the pharmaceutical field, and may be prepared by itself or a pharmaceutically acceptable carrier, excipient, forming agent, diluent, and the like. The mixture may be prepared and used in the form of powder, granules, tablets, capsules or injections.

In addition, the pharmaceutical composition comprising the peptide of the present invention as an active ingredient may be administered by dividing 1-100 ml once or several times at a concentration of 0.2-10 mg / kg.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples illustrate the invention, but the scope of the invention is not limited by the examples.

Example 1 Preparation of TIMP-2 Derived Peptides

The peptides of the present invention are peptides obtained by modifying a part of an amino acid sequence including three loops from an amino acid terminal, which is a part of the structure of TIMP-2, which is considered to bind MMP-2. Biosystems Model 431A) was used to synthesize the solid phase (solid phase) peptide synthesis. 0.25 mmol parahydroxy methylphenyloxymethyl polystyrene (HMP) resin was added to a standard reaction vessel (38 ml), and the synthesis was started by adding the Fmoc-amino acid at the carboxyl terminal of the peptide to be synthesized.

Peptide synthesis was carried out according to the standard scale Fmoc coupling protocol developed by ABI, before editing the parameters and following the autosynthesis menu (see ABI User's Manual. Jan, 1992). When using standard scale Fmoc, deprotection was performed for 21 minutes with 20% piperidine diluted with N-methyl pyrrolidine (NMP), washed for 9 minutes with NMP and coupling for 71 minutes. Was carried out. Coupling used 1-hydroxy-benzotriazole (HOBT) and a system for 7 minutes washing with NMP.

Example 2 Isolation and Purification of Peptides

After the synthesis is complete, the peptide is separated from the solid support using trifluoroacetic acid (TFA), and the peptide is removed by referring to the ABI manual (Introduction to Cleavage Techniques, P6-19 (1990)). Separated. In other words, after the synthesis is completed, the resin with the peptide is placed in a round flask and cooled, and then 0.75 g crystalline phenol, 0.25 ml 1,2-ethanedithiol (EDT), 0.5 ml thioanisole, 0.5 ml distilled water and 10 ml TFA are added to the lid. Close and react for 1-2 hours at room temperature. After the reaction, the resin and the reaction solution were filtered through low vacuum through a sintered glass funnel to separate the resin and the peptide solution. The flask and glass funnel were washed with 5-10 ml dichloromethane (DCM) to allow the solution to mix with the peptide solution, and then 50 ml or more cold diethyl ether was added to obtain a precipitate of the peptide. The precipitate was centrifuged at about 3000 rpm to remove the supernatant and freeze-dried. The peptide thus obtained was purified by High Performance Liquid Chromatography (HPLC). The column was a C ₄ Semi-prep column (Vydac), buffer A was equilibrated with distilled water and 0.1% TFA, and B was eluted with 80% acetonitrile and 0.1% TFA. As a result, the purified peptide was obtained with high purity and the synthetic yield was about 60 ± 5%.

Thus, the peptide containing amino acid sequence 6 to 15 of the wild type TIMP-2 of the synthetic purified peptide was named HB 700, amino acid sequence 6 to 15 of the TIMP-2 and 108 to 126 Peptides made by modifying the amino acid sequence up to the names HB 701, 703, 705, 707, 710, 711, 714. The amino acid sequence from the amino terminus of the synthesized peptide is as follows.

HB 700: VHPQQAFCNA

HB 701: AFCNEHA

HB 703: VHPQQAFCNEHA

HB 705: LNHRYQMGCA

HB 707: KCLNHRYQMGCA

HB 710: DTLSTTQKKSLNHRYQMGCA

HB 711: PVHPQQAFCNPGCQYRHNLSHKQTTSL

HB 714:

As a result of mass spectrometry and amino acid sequencing of the synthesized peptides, the synthesized peptides were synthesized in the order of magnitude.

<Example 3> MMP-2 activity inhibition experiment

(Step 1) Preparation of MMP-2

To obtain MMP-2, HT-1080 (ATCC CCL121) cells or T98G (ATCC CRL-1690), which are metastatic human fibrosarcoma cells, were cultured in DMEM 10% FBS medium, and then the medium was removed and serum-free DMEM medium was used. The culture medium was exchanged and cultured for 24-72 hours to recover a supernatant containing MMP-2. Precipitate with ammonium sulfate up to 0-70% and dissolve in 20mM Tris (pH 7.4) and then dialysis in equilibration buffer (50mM Tris, pH 7.6, 0.5M NaCl, 5mM CaCl ₂ , 0.05% Brij 35, 0.02% NaN ₃ ) ) And then loaded into gelatin-sepharose and eluted with an equilibration buffer mixed with 1M NaCl and 5% DMSO to obtain purified 72KDa MMP-2. The activity of MMP-2 was confirmed by zymogram (see FIG. 1 ). However, some of MMP-2 purified from cancer cell cultures are combined with TIMP-2 and are difficult to be separated by gelatin-sepharose column. Therefore, the baculovirus system is obtained to obtain pure MMP-2 only. Expression in Sf9 insect cells. MMP-2 cDNA was first cloned back into pBS-KS and named pBS-GEL. Xho I and Not I fragments containing 1.9 Kb MMP-2 cDNA of pBS-GEL were transferred to pBacPAK 9 migration vector, and the migration vector and BacPAK6 virus DNA were infected with Sf9 cells by calcium phosphate method. Sf9 cells are grown at 27 ° C. in TNM-FH medium with 10% fetal bovine serum, centrifuged for infection and resuspended in serum free medium to a concentration of 10 ⁷ cells / ml and at room temperature with virus. Incubate for 1 hour. Infected Sf9 cells were grown for 96 hours, and the medium was collected and purified according to the MMP-2 purification method. MMP-2 obtained from baculovirus showed a tendency to cut MMP-2 due to better autolysis than MMP-2 obtained from cancer cell culture medium.

(Step 2) MMP-2 activity inhibition experiment

In order to quantitatively analyze the inhibitory activity of MMP-2, the peptides of the present invention, these peptides were incubated with MMP-2 at room temperature for 20 minutes, followed by collagenase buffer (50 mM) using ³ H collagen I as a substrate. Incubate at 37 ° C. with Tris, pH 7.5, 200 mM NaCl, 10 mM CaCl ₂ , 0.05% Brij 35, 0.02% NaN ₃ ). At this time, 1 mM APMA is added to activate MMP-2. The reaction was stopped with 250 mM EDTA, 10% TCA / 0.5% tannic acid was added, centrifuged at 13000 rpm for 10 minutes, the supernatant was carefully taken, and the amount of radioactivity was measured by a scintillation measuring instrument. In the experiment of inhibiting MMP-2 activity with the peptides synthesized in the present invention, peptide HB 700 including amino acid sequence 6 to 15 of wild type TIMP-2 had no effect of inhibiting MMP-2 activity but HB 703, modified 700, strongly inhibited MMP-2 activity. Screening peptides that are more effective than wild type rTIMP-2 with synthesized peptides, HB 703, HB 710, HB 711, HB 714 have the effect of inhibiting MMP-2 activity as shown in FIG . Had a similar or better effect.

Example 4 Animal Experiments to Measure Metastasis (Experimental metastasis model)

C57BL / 6 male mice, 6-7 weeks of age, were divided into 10 rats per group and 5 x 10 ⁴ B16 melanoma cells per head were intravenously injected into the tail and rTIMP was determined first based on rTIMP-2 from the day of injection. The metastasis inhibitory effect of wild-type rTIMP-2 was measured by intraperitoneal injection of -2 at 0.2 mg per animal three times, intraperitoneal injection at 0.5 mg twice or once at 1.0 mg. Two weeks later, the necropsy was counted and the number of colonies transferred to the lungs was examined using a microscope. Intraperitoneal injection of rTIMP-2 at 0.2 mg per head three times, the number of colonies in which B16 melanoma cells had metastasized to the lung was suppressed by 39% compared to the control group. Once intravenous, 82% inhibition (see Figure 2 ). Based on the above experiments, intraperitoneal injections of peptides effective in MMP-2 activity inhibition experiments 0.2mg three times (total 0.6mg / head) and measured the number of colonies metastasized to the lung two weeks later were measured for rTIMP-2 and metastasis inhibition. Compare the effects. When rTIMP-2 inhibited metastasis by 39%, three injections of the same dose of 0.2 mg, peptide HB 703 showed the best metastasis inhibitory effect with 66%, followed by HB 714 with 45% inhibition. It showed an effect. In addition, HB 710 and HB 711, which were effective in MMP-2 activity suppression experiments, had almost no colonies in the lungs, which had no inhibitory effect. Also showed no effect (see Table 1).

Peptide Inhibition Experiment Intraperitoneal injection Number of colonies metastasized to the lung % Inhibition PBS (Control) 270 ± 114 0 Wild type rTIMP-2 166 ± 67 39 HB-703 93 ± 40 66 HB-707 243 ± 86 10 HB-710 255 ± 73 6 HB-711 264 ± 64 3 HB-714 148 ± 58 45

Example 5 Synthesis of Modified Peptide from Lead

A peptide (HB 715) in which HB 703 was used as a dimer was synthesized using the peptide HB 703 including amino acid sequences 6 to 15 of TIMP-2 as a leader. To maximize efficacy, an amino acid sequence known to inhibit cancer cell adhesion to HB 703 was added. That is, the RGD amino acid sequence which is a part of fibronectin or vitronectin recognized by cancer cell receptor (HB 716) or the YIGSR amino acid sequence which recognizes laminin (HB 717), or SIKVAV Peptides with amino acid sequence (HB 718) were synthesized and purified. In addition, HB 703 was re-inserted with histidine (HB 719, HB 723), which is thought to play an important role in MMP-2 inhibition, or a peptide (HB 722) with one amino acid converted to histidine was synthesized. Two fewer peptides were synthesized (HB 721) and histidine-inserted peptides were synthesized (HB 724). In addition, peptides (HB 726) connecting each of HB 703 and HB 710 by disulfide bonds were synthesized, and peptides (HB 727) connecting by disulfide bonds were synthesized and purified after successive synthesis of HB 703 and HB 710.

Synthesis of the branched peptide was performed using ABI's 431A autosynthesizer as described in Example 1 above. First, Fmoc-Gly (Advanced Chemtech.) Is added to parahydroxy methylphenyloxymethyl polystyrene (HMP) resin to add glycine, followed by Fmoc-Lys (fmoc) to which Fmoc is attached to the amino group at the end of lysine. Afterwards, since two amino groups are exposed per molecule after deprotection, two peptides of the same sequence exist in one molecule of the synthesized peptide. In this way, branched peptide HB 715 was synthesized from HB 703.

The amino acid sequence of the synthesized peptides is as follows.

HB 703: VHPQQAFCNEHA

HB 715:

HB 716: RGDVHPQQAFCNEHA

HB 717: YIGSRVHPQQAFCNEHA

HB 718: SIKVAVVHPQQAFCNEHA

HB 719: VHPQQHAFCNEHA

HB 721: PQQAFCNEHA

HB 722: VHPQQHFCNEHA

HB 723: VHPQQAFCHNEHA

HB 724: PQQHAFCNEHA

HB 726:

HB 727:

<Example 6> Measurement of the inhibitory effect of MMP-2 activity

As shown in Figure 4 , HB 718, HB 726 and HB 727 was very good MMP-2 inhibitory effect, followed by HB 715, HB 716, HB 722, HB 723, HB 724, HB 719, HB 717 Inhibitory effects were observed, and all showed an increased MMP-2 inhibitory effect compared to HB 703. However, animal experiments that inhibited the metastasis of B16BL6 cells injected intravenously in C57BL / 6 mice to the lung with these peptides showed that HB 718 had the best effect, but among other peptides HB 715, HB 719, HB 722 and HB 723. This showed a similar effect to HB 703.

<Example 7> Survival Extension Experiment

Subcutaneously injected 2 × 10 ⁵ B16BL6 melanoma cells suspended in 0.2 ml HBSS in C57BL / 6 mice, randomly mixed and divided into 6 animals per group, 0.2 mg per animal, rTIMP-2 and each peptide HB 703, HB 715, HB 719, HB 722, and HB 723 were intraperitoneally injected daily to measure survival rates for 30 days to select the best ones. That is, HB 703 was the most effective, and rTIMP-2 was the most effective, as compared with the change in the number of control groups and the number of groups injected with rTIMP-2 and peptides for one month. And HB 715 also had a prolonged survival effect. During the month after the cancer cell injection, the control group was reduced from 6 to 2 mice, whereas the HB 703, rTIMP-2 and HB 715 treatment groups had only one death and 5 remained (see FIG. 5 ). HB 719, HB 722 and HB 723 showed similar results as the control (see FIG. 6 ).

Example 8 Effect on Tubular Formation of HUVE Cells in Matrigel

As an in vitro experiment to indirectly know the effects on angiogenesis, the effect on the formation of capillary-like structures by human vascular endothelial cells was observed. In order to perform the Tube Formation experiment, the experiment was performed to obtain Human Umbilical Vein Endothelial (HUVE) cells. In other words, after the caesarean section, the endothelial cells of the veins were isolated from the umbilical cord of fresh human, and the cells obtained by culturing the vascular endothelial cells were successfully isolated by HUVE cells by cytoimmune staining using antibody of factor VIII. Vascular endothelial cells were cultured on Matrigel. After gelling the Matrigel and culturing the endothelial cells on the Matrigel, two-dimensional tubular formation occurs. The reticular tubular structure observed after 16-18 hours of incubation at 37 ° C can be thought of as a process of angiogenesis. The effect on angiogenesis was indirectly confirmed by observing the results of treatment with the same concentration of peptides as when TIMP-2 was treated at 100 ug / ml. When rTIMP-2 was treated, the tube formation was strongly inhibited, and the connection between the tubes was seen a lot, and when 300 ug / ml was added, the cells forming the tube did not have a proper shape (see FIG. 7 ). . HB 703, HB 715 and HB 718 were also weaker than rTIMP-2 at 100 ug / ml and 300 ug / ml concentrations, but inhibited the formation of HUVE cells and HB 718 strongly inhibited the tube formation at 300 ug / ml concentration. Agglomeration was observed (see FIG. 8 ).

<Example 9> Animal experiment for measuring angiogenesis (Mouse matrigel model)

The mouse matrigel model (Mouse matrigel) showed that the candidate substances HB 703, HB 715, and HB 718, which have the effect of inhibiting metastasis in animal experiments, were effective in survival extension experiments, and inhibited HUVE cell tube formation. model). Subcutaneous injection of 0.5 ml of Matrigel, 1 ng / ml of basic FGF and 20-40 units / ml of heparin in C57BL / 6 mice of 6-8 weeks results in strong angiogenesis. After 3-5 days, the epidermis was removed, the matrigel was carefully separated to recover the gel, and the amount of hemoglobin was measured using a Drabkin reagent. Peptide HB 703 and its derivatives and rTIMP-2, each of which was recognized as a metastasis inhibitory effect, were mixed with the above Matrigel. Good, followed by HB 705, HB 703, rTIMP-2 (see FIG. 9 ).

Example 10 Efficacy Test on Human Gastric Cancer in Nude Mice

KHH, a human gastric cancer cell line established by Yonsei Cancer Center, was cultured and subcutaneous 5 × 10 ⁶ gastric cancer cells per horse were fed to Balb / C / nu / nu 5-week-old male nude mice (purchased from B & K Universal Inc., USA). Xenografts were implanted and HB 703, HB 715 and HB 718 were injected daily into the abdominal cavity from day 1 of tumor injection at 0.2 mg per horse. The control group was injected with saline. The change in tumor size was measured daily from day 5, and at 21 days of treatment, the mice were sacrificed to remove the mass, skin was removed and the final size and weight were measured to determine the effect of the peptide. As shown in FIG. 10 , the group administered HB 703 had only 60% of the final tumor volume after 21 days compared with the control group. HB 703 was clearly effective in both tumor volume and weight, HB 715 was less effective than HB 703, but effective, HB 718 was not effective in tumor volume but effective in weight ( FIGS. 10 and 11). Reference).

Example 11 Survival Extension Test for Human Gastric Cancer in Nude Mice

Experiments to observe the prolonged survival by the peptide was carried out in the same manner as in the drug efficacy experiment of Example 10, but 0.2 mg per peptide were injected intraperitoneally every day from the day of xenograft gastric cancer cell line, treated until the death of the rat Proceeded. Analyzing the results up to 44 days after tumor injection, HB 703 was the most effective in this experiment, and it certainly had the effect of prolonging survival. Next came HB 715. In addition, there was a slight curve change when comparing the size of the tumor to the area 44 days after injecting the tumor. This may be due to the change in the size of the tumor when the mouse died. HB 703 inhibits the growth of the tumor compared to the control group. It is shown. This result is in agreement with the results of the survival extension test for B16BL6. HB 715 went into effect later and HB 718 had no effect (see FIGS . 12 and 13 ). During the drug efficacy test and survival test of the candidates, the mice did not lose weight and did not show any toxicity.

Synthetic peptides of the present invention are derived from a peptide having a sequence of amino acid sequence 6 to 15 of TIMP-2 that inhibits the activity of type IV collagenase or modified from 108 to 126 amino acid sequence of TIMP-2 As peptides containing or added to the amino acid sequence of, these peptides have the effect of inhibiting the activity of MMP-2, inhibiting angiogenesis, inhibiting cancer growth, and preventing metastasis, thereby prolonging life. It can be used for various purposes such as:

These peptides can be used as anti-cancer agents to inhibit the growth and metastasis of solid cancers and to prevent further growth and inhibit metastasis even if there is microtransition after surgery.

It can also be used as a treatment for diseases caused by excessive angiogenesis. Diseases caused by, for example, excessive MMP activity; Can be used for arthritis, diabetic retinopathy, hypertrophic scars, psoriasis, ulcers of mucous membranes and epithelial tissues, autoimmune inflammation, lung damage, granulomatous diseases, diseases associated with degradation of the basement membrane; It can also be used for lupus, autoimmune neurological disorders, myocyte destruction, ie myocardial infarction and glomerular abnormalities. In particular, it can be applied to lower intraocular pressure such as neovascular glaucoma and ocular blood vessel formation.

Claims (17)

A synthetic peptide derived from TIMP-2 that inhibits the activity of type IV collagenase, wherein the amino acid sequence from the amino terminus is VHPQQAFCNEHA. A synthetic peptide derived from TIMP-2 that inhibits the activity of type IV collagenase, wherein the amino acid sequence from the amino terminus is

Phosphorus Peptides. A synthetic peptide derived from TIMP-2 that inhibits the activity of type IV collagenase, wherein the amino acid sequence from the amino terminus is SIKVAVVHPQQAFCNEHA. A synthetic peptide derived from TIMP-2 that inhibits the activity of type IV collagenase, wherein the amino acid sequence from the amino terminus is RGDVHPQQAFCNEHA. A synthetic peptide derived from TIMP-2 that inhibits the activity of type IV collagenase, wherein the amino acid sequence from the amino terminus is YIGSRVHPQQAFCNEHA. A synthetic peptide derived from TIMP-2 that inhibits the activity of type IV collagenase, wherein the amino acid sequence from the amino terminus is VHPQQHAFCNEHA. A synthetic peptide derived from TIMP-2 that inhibits the activity of type IV collagenase, wherein the amino acid sequence from the amino terminus is PQQAFCNEHA. A synthetic peptide derived from TIMP-2 that inhibits the activity of type IV collagenase, wherein the amino acid sequence from the amino terminus is VHPQQHFCNEHA. A synthetic peptide derived from TIMP-2 that inhibits the activity of type IV collagenase, wherein the amino acid sequence from the amino terminus is VHPQQAFCHNEHA. A synthetic peptide derived from TIMP-2 that inhibits the activity of type IV collagenase, wherein the amino acid sequence from the amino terminus is PQQHAFCNEHA. A synthetic peptide derived from TIMP-2 that inhibits the activity of type IV collagenase, wherein the amino acid sequence from the amino terminus is

Phosphorus Peptides. A synthetic peptide derived from TIMP-2 that inhibits the activity of type IV collagenase, wherein the amino acid sequence from the amino terminus is

Phosphorus Peptides. A synthetic peptide derived from TIMP-2 that inhibits the activity of type IV collagenase, wherein the amino acid sequence from the amino terminus is DTLSTTQKKSLNHRYQMGCA. A synthetic peptide derived from TIMP-2 that inhibits the activity of type IV collagenase, wherein the amino acid sequence from the amino terminus is PVHPQQAFCNPGCQYRHNLSHKQTTSL. A synthetic peptide derived from TIMP-2 that inhibits the activity of type IV collagenase, wherein the amino acid sequence from the amino terminus is

Phosphorus Peptides. The peptide according to any one of claims 1 to 15, synthesized using L-amino acid. Arthritis, diabetic retinopathy, hypertrophic scar, psoriasis, mucous membrane and epithelial tissue ulcer, autoimmune inflammation, lung injury, granulomatous disease, which are diseases used as metastasis inhibitors, anticancer agents or due to excessive MMP activity; Diseases associated with degradation of the basement membrane, lupus, autoimmune neuropathy, myocyte destruction, ie myocardial infarction, glomerular abnormalities; The peptide of claim 1, wherein the peptide is used as a therapeutic agent for diseases caused by excessive angiogenesis such as ocular disease neovascular glaucoma, angiogenesis of the eye, and the like.

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