WO2013097707A1

WO2013097707A1 - Use of integrin blockers polypeptide ap25 in preparation of drugs for treating tumours

Info

Publication number: WO2013097707A1
Application number: PCT/CN2012/087450
Authority: WO
Inventors: 徐寒梅; 杨永晶; 胡加亮
Original assignee: Xu Hanmei
Priority date: 2011-12-27
Filing date: 2012-12-25
Publication date: 2013-07-04
Also published as: CN102488890A

Abstract

Provided in the present invention is the use of integrin blocker polypeptide AP25 having a sequence of A1la-Cys-Asp-Cys-Arg-G1ly-Asp-Cys-Phe-Cys-G1ly-G1ly-G1ly-G1ly-I1le-Va1l-Arg-Arg-A1la-Asp-Arg-A1la-A1la-Va1l-Pro in the preparation of drugs for treating tumours, wherein the tumours are the primary and secondary cancers and sarcomas deriving from the human head and neck, brain, thyroid, pancreas, lung, liver, esophagus, stomach, breast, kidney, gall bladder, colon, rectum, ovary, uterus, cervix, prostate, bladder and testis.

Description

FIELD OF THE INVENTION The present invention relates to the field of medicine, and in particular to an integrin blocker polypeptide AP25 having tumor angiogenesis, integrin affinity and binding ability. The blocker is a polypeptide, and the integrin blocker polypeptide can be used for the treatment of solid tumors. BACKGROUND OF THE INVENTION Malignant tumors are the leading killer of human health. Although traditional tumor therapy has certain curative effect, due to the non-specific cytotoxicity of chemical drugs, it has great toxic and side effects on patients, and is also prone to drug resistance, which brings great pain to cancer patients. Since Folkman proposed the theory of tumor angiogenesis in 1972, reports on the treatment of tumors by inhibiting tumor angiogenesis have emerged in great success and have achieved great success. Tumor angiogenesis is the morphological basis for tumor growth and metastasis. Most malignant solid tumors such as ovarian cancer, liver cancer, cervical cancer and breast cancer are vascular-dependent tumors. Neovascularization provides nutrients and oxygen for tumor growth, and on the other hand is an important pathway for tumor metastasis. Therefore, inhibition of tumor neovascularization can effectively inhibit the occurrence, development and metastasis of tumors.

Tumor neovascularization is regulated by many cytokines, and one of the important tumor vascular endothelial cell molecules associated with tumor angiogenesis is a member of the integrin family. Integrin is an important adhesion molecule on the cell surface. It is a receptor for a variety of extracellular matrix components. It is widely present on the cell surface and is a fairly large family of receptors. Integrin mainly mediates the adhesion between cells and cells and cells and extracellular matrix, plays an important role in regulating cell proliferation, differentiation, migration and apoptosis, and induces neovascularization and tumor invasion and metastasis in tumors. Play an important role. Integrins are composed of an alpha chain and a chain, both of which play a role in binding to the ligand. The combination of different alpha chains and chains determines the specificity of the ligand. So far, 15 cc chains and 9 chains have been discovered. In tumor cells, complex components of integrin have undergone complex changes. In general, the number of integrins involved in tissue structure has decreased, while the number of integrins associated with cell migration has increased. Integrin a5W, ανβ5, avW, etc. are all related to angiogenesis and cell migration, and the role of avW is particularly important. a ^3 Transmembrane heterodimeric glycoprotein, also known as the VN receptor, formed by the av subunit (CD51, 150 kD) and the ^ subunit (CD61, 105 kD). avW recognizes the arg-gly-asp (RGD) sequence in the ligand molecule. Such ligands include FN, VN TSP-1 and vWF, and the like. ^^3 can be expressed in a variety of cell types and binds to multiple ligands during multicellular activity, and is involved in physiological and pathological processes such as tumor angiogenesis, invasion and metastasis, inflammation, wound healing, and coagulation. For example, co^3 can bind to metalloproteinases and degrade the extracellular matrix, thereby facilitating invasion. In addition, two processes affected by avW are apoptosis and angiogenesis, and the expression of ccvW is also increased on capillary endothelial cells involved in angiogenesis. In tumor vascular endothelial cells, angiogenic factors are closely related to the expression of integrin, such as Both VEGF and FGF can up-regulate the expression of ανβ3. Integrin ανβ3 is highly expressed on the surface of many malignant tumor cells and neovascular endothelial cells in tumor tissues, but not in mature vascular endothelial cells and most normal organ systems, in order to design ανβ3 ligand small molecule antagonist peptides containing RGD sequences and The monoclonal antibody of ανβ3 provides a theoretical basis for the treatment of tumors. Currently, internationally developed inhibitors against integrins are mainly specific antibodies, such as Vitaxine; or small molecule ligand inhibitors such as cilengitide. There are also some integrin blockers that have entered phase II clinical trials, and no similar or similar products have entered the market in China. Therefore, it is very necessary to develop such drugs with independent intellectual property rights in China. ZL200510040378.5 Highly effective inhibition of angiogenic polypeptides and preparation methods and applications thereof, introducing several integrin inhibitors, one of which is an integrin blocker polypeptide sequence: Ala-Cys-Asp-Cys-Arg-Gly-Asp-Cy s-Phe-Cys-Gly-Gly-Gly-Gly-Ile- Val- Arg- Arg-

Ala-Asp-Arg-Ala-Ala-Val-Pro (the sequence contains two pairs of disulfide bonds, paired in 1-4, 2-3) (designated AP25, see Seq. NO. l), the sequence contains Integrin Ligand Sequence (Ala-Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys-Gly

-Gly-Gly-Gly) (see Seq.NO.2) and neovascularization sequence (Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val

-Pro ) (see Seq. N0.3 ), in which the integrin ligand sequence contains the RGD-4C sequence ( Ala-Cys- Asp-Cys- Arg-Gly

-Asp-Cys-Phe-Cys-Gly) (see Seq. N0.4), such that the polypeptide sequence can efficiently bind to a tumor-specific expression of the integrin isoform, and the sequence contains a neovascular inhibitory sequence, capable of Inhibition of tumor neovascularization, thereby achieving the effect of inhibiting tumor growth and metastasis. In the prior patent, only melanoma has been studied. The present invention has further studied the sequence and found that it has therapeutic effects on various tumors, increases its applicability, and expands its social value and economic value. OBJECT OF THE INVENTION The present invention is directed to the sequence Ala-Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys-Gly-Gly-Gly-Gly-Ile-Val-Arg-Arg-Ala-Asp-Arg- Ala-Ala-Val-Pro (the sequence contains two pairs of disulfide bonds, paired in 1-4, 2-3) was further studied and found to have a therapeutic effect on a variety of solid tumors, increasing its indications. The use of the integrin blocker polypeptide AP25 for the preparation of a medicament for treating tumors, wherein the sequence of the integrin blocker is Ala-Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys- Gly-Gly-Gly-Gly-Ile-Val- Arg- Arg-Ala-Asp-Arg- Ala-Ala-Val-Pro (The sequence contains two pairs of disulfide bonds, paired in 1-4, 2-3) (designated AP25, see Seq. NO. l), characterized in that the tumor originates from the head and neck, brain, thyroid, pancreas, lung, liver, esophagus, stomach, breast, kidney, gallbladder, colon Or primary or secondary to the rectum, ovary, uterus, cervix, prostate, bladder, testicles Cancer and sarcoma. The beneficial effects of the study found that the sequence Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro is the 60th to 70th amino acids of endostatin, which has a good inhibitory effect on tumor angiogenesis in vitro. Its activity is even higher than that of endostatin itself. The RGD C sequence is a heavy ligand of integrin. Therefore, the polypeptide Ala-Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys-Gly-Gly-Gly-Gly containing the RGD-4C sequence can specifically recognize integrins. The integrin blocker polypeptide of the present invention has the sequence of Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala "Ala-Val-I ro" having an inhibitory effect on angiogenesis, and has an N-terminal linkage with an integration zero family. Affinity and binding ability of RGD-4C sequence Ala-Cys-Asp-Cys-Arg-piy-Asp-Cys-Phe-|Cys-Gly-Gly-Gly-Gly, constructed a high affinity with integrin Polypeptides of sexual and binding ability. The integrin blocker polypeptide sequence is: Ala-Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys-Gly-Gly-Gly-Gly-Ile-Val- Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro, which contains 25 amino acids, has an integrin affinity and binding ability in the RGD-4C sequence. Studies have shown that this integrin blocker polypeptide is mainly The target is the integrin ccv, subunit, and the sequence contains a neovascular inhibitory sequence, thereby inhibiting tumor neovascularization, thereby achieving the effect of inhibiting tumor growth and metastasis. Although the sequence is disclosed in the prior patent ZL200510040378.5 It has a therapeutic effect on melanoma, but it has not been disclosed to have therapeutic effects on other tumors. A large number of studies at home and abroad have shown that even the same tissue type, Tumors of the same degree may have different sensitivities to the same drug. Therefore, anti-tumor screening is required for a certain molecule in drug development. Because some drugs are only effective for specific tumor cells, they are ineffective or ineffective for other tumors. Therefore, experiments are needed to explore and verify the therapeutic effect. The inventors have learned through a large number of experiments that the integrin blocker has a clear target and can significantly inhibit the migration, proliferation and tubular structure of human umbilical vein endothelial cells (HUVEC) in vitro. It also inhibits the proliferation of some human tumor cells. The in vivo experiment has obvious anti-tumor effect, and has fewer side effects, less dosage and lower cost. The integrin blocker designed by the present invention is scientific, reasonable and feasible. It is effective as a therapeutic drug for the treatment of human solid tumors, greatly expanding the therapeutic spectrum of the integrin blocker, providing new ideas and prospects for future drug development, with significant social and market value. The integrin blocker polypeptide refers to the integrin blocker polypeptide AP25. BRIEF DESCRIPTION person 1 (of HUVEC) cell adhesion assay determines umbilical vein endothelial cells polypeptide targets; Figure 2 integrin antagonist polypeptide inhibits the proliferation of human umbilical vein endothelial cells (HUVEC); and Figure 3 Integrin blocker polypeptide inhibits the proliferation of human gastric cancer MGC-803 cells;

Figure 4 Integrin blocker polypeptide inhibits the proliferation of human cervical cancer HeLa cells;

Figure 5 Integrin blocker polypeptide inhibits the proliferation of human colon cancer HCT 116 cells;

Figure 6 Integrin blocker polypeptide inhibits the proliferation of human glioma U87 cells;

Figure 7 Integrin blocker polypeptide inhibits the proliferation of human breast cancer MDA-MB-231 cells;

Figure 8 Integrin blocker polypeptide inhibits migration of human umbilical vein endothelial cells (HUVEC);

Figure 9: Integrin blocker polypeptide inhibits tubular structure formation of human umbilical vein endothelial cells (HUVEC); Figure 10 Inhibitory effect of integrin blocker polypeptide on human nasopharyngeal carcinoma CE nude mouse xenograft tumor growth; 11 Integrin blocker polypeptide inhibits growth of human glioma U87 xenograft tumor in nude mice; Figure 12 Inhibitory effect of integrin blocker polypeptide on human thyroid carcinoma SW-579 xenograft xenograft tumor; 13 Integrin blocker polypeptide inhibits growth of human pancreatic cancer SW-1990 xenograft tumor in nude mice; Figure 14 Inhibitory effect of integrin blocker polypeptide on human lung cancer H460 xenograft xenograft tumor;

Figure 15: Inhibitory effect of integrin blocker polypeptide on human liver cancer Bel-7402 xenograft tumor in nude mice; Figure 16 Inhibitory effect of integrin blocker polypeptide on human esophageal cancer Ecl09 xenograft tumor in nude mice; Inhibitory effect of integrin blocker polypeptide on human gastric cancer MGC-803 xenograft tumor in nude mice; Figure 18 Inhibitory effect of integrin blocker polypeptide on human breast cancer MDA-MB-231 xenograft tumor in nude mice; 19 Integrin blocker polypeptide inhibits growth of human kidney cancer A498 xenograft tumor in nude mice;

Figure 20: Inhibitory effect of integrin blocker polypeptide on human gallbladder carcinoma GBC-SD xenograft tumor in nude mice; Figure 21 Inhibitory effect of integrin blocker polypeptide on human colon cancer HT-29 xenograft xenograft tumor; Figure 22: Inhibitory effect of integrin blocker polypeptide on human ovarian cancer SK-OV-3 xenograft tumor in nude mice; Figure 23 Integrin blocker polypeptide inhibits growth of human endometrial carcinoma HHUA nude mouse xenograft tumor Figure 24: Inhibitory effect of integrin blocker polypeptide on human cervical cancer HeLa nude mouse xenograft tumor growth; Figure 25 Inhibitory effect of integrin blocker polypeptide on human prostate cancer DU-145 xenograft xenograft tumor; Figure 26: Inhibitory effect of integrin blocker polypeptide on human bladder cancer HT1376 xenograft tumor in nude mice; Figure 27 Inhibitory effect of integrin blocker polypeptide on human testicular cancer 5637 xenograft xenograft tumor; Figure 28 Inhibitory effect of a peptide blocker polypeptide on sarcoma HT-1080 xenograft tumor growth in nude mice; DETAILED DESCRIPTION OF THE INVENTION Example 1 Integrin Blocker Polypeptide Target Analysis Human umbilical vein endothelial cell (HUVEC) adhesion assay was used to determine integrin resistance The target of the action of the polypeptide. That is, a 96-well plate was firstly plated with a certain concentration of polypeptide, and HUVEC cells were incubated with different integrin subunit antibodies and subjected to adhesion experiments. Compared to cells incubated with no antibody, if the adhesion of the cells incubated with an antibody to the polypeptide is significantly reduced, the polypeptide is said to interact with HUVEC via the integrin subunit. The specific operation is as follows: The polypeptide is prepared into a solution of 150 μ _§ /ιη1 in PBS, and the 96-well plate is placed in a ΙΟΟμΙ per well, and placed at 4 ° C for 16 hours, and then 1% BSA is blocked in a 22 ° C water bath for 4 to 5 hours.

HUVEC cells (only 2nd to 5th generation) were incubated with serum-free endothelial cell culture medium for 4 hours when cultured at 37 ° C in a 5% CO ₂ incubator to a confluence of 90% or more. The cells were collected by trypsinization, resuspended in serum-free endothelial cell culture medium, and counted, and the cell concentration was adjusted to ⁵ × 10 ⁵ /ml. The cells were incubated with αν, β3, αν+β3, α5, β1, cd+ ^ integrin subunit antibody at 4 ° C for 2 hours, and the cell suspension was shaken at intervals to allow the cells to fully contact the antibody. The BSA in the 96-well plate was discarded, and the cells were plated, and the cell suspension was pulverized per well (5 replicate wells per group) and adhered at 37 ° C for 90 minutes. Unattached cells were gently washed away with PBS and stained with methanol-Coomassie Brilliant Blue Mix and fixed for 40 minutes. The 96-well plate was rinsed with a decolorizing solution to remove the Coomassie blue background. 1% SDS was added to a 96-well plate at 100 μl per well, and the absorbance was measured at 620 nm. Results: As shown in Figure 1, the number of cell adhesions in the group with αν, αν+β3, ο5, a5+ W integrin subunit antibody was significantly different from that in the group without integrin subunit antibody; integration with β3 The number of cell adhesions in the subunit antibody-incubated group was significantly different from that in the group without the integrin subunit antibody; the number of cell adhesions in the group incubated with the βΐ integrin subunit antibody and the integrin subunit antibody There was no significant difference in the incubation group. From this, it can be judged that this integrin inhibitor polypeptide mainly interacts with HUVEC cells through two integrin subunits αν and α5. Example 2 Inhibition of proliferation of human umbilical vein endothelial cells (HUVEC) by integrin blocker polypeptide The activity of integrin blocker polypeptide to inhibit endothelial cell growth was examined by MTT assay, and endothelial cells were only used for passages 2-6. HUVEC cells were cultured to a concentration of 90% or more in a 37 ° C, 5% CO ₂ incubator and collected by trypsinization. The cells were resuspended in the culture medium and counted under a microscope to adjust the cell concentration to 3 χ 10 ^{4 .} The cells were inoculated into a 96-well plate, ΙΟΟμΙ/well, and cultured overnight at 37 ° C in a 5% CO ₂ incubator. The integrin blocker polypeptide is diluted with the culture solution to each predetermined concentration. Endo is diluted to the final concentration with the culture solution. After the cells were fully attached, each dilution was separately added to a 96-well plate (ΙΟΟμΙ/well). As the administration group, the integrin blocker polypeptide dilution was added, and Endo and Paclitaxel were added as the positive control group, and the culture medium without any drug was used as the negative control group. Incubate for 72 h at 37 ° C in a 5% CO ₂ incubator. 5 mg/ml of MTT was added to a 96-well plate at 20 μl per well, and incubation was continued for 4 hours. The medium was aspirated, dissolved in ΙΟΟμΙ DMSO per well, and gently mixed by shaking for 10 minutes. The absorbance was measured with a microplate reader at a measurement wavelength of 570 nm and a reference wavelength of 630 nm, and the growth inhibition (PI) was calculated. The formula is as follows:

PI (%) = 1 - administration group / negative group The results obtained by the experiment were expressed as mean ± SD, and a statistical T test was performed, *P < 0.05 was a significant difference, and **P < 0.01 was a very significant difference. Table 1. Inhibitory effect of integrin blocker polypeptide on proliferation of human umbilical vein endothelial cells (HUVEC)

Group

( /ml ) A570nm detection inhibition rate (%)

0.06 0.1728±0.0085 7.08

0.13 0.1652±0.0093** 11.22

0.25 0.1664±0.0092** 10.57

0.5 0.1634±0.0072** 12.20

1 0.1480±0.0068** 16.01 Peptide 2 0.1332±0.0051** 24.50

4 0.1040±0.0086** 41.25

8 0.0720±0.0088** 59.61

16 0.0536±0.0079** 70.17

32 0.0680±0.0026** 61.90

64 0.0173±0.0015** 91.02 Endo (ES) 8 0.1702±0.0074** 8.50 Taxol 10 0.0040±0.0007** 98.68 Negative group (Control) ― 0.1858±0.00736** ― Result: See Table 1 and Figure 2 Compared with the negative control, the integrin blocker polypeptide significantly inhibited the proliferation of HUVEC and showed a significant dose-dependent relationship. Example 3 Inhibition of Proliferation of Human Gastric Cancer MGC-803 Cells by Integrin Blocker Polypeptide The activity of integrin blocker polypeptide for inhibiting the growth of human gastric cancer MGC-803 cells was examined by MTT assay. Tumor cells were cultured in a 37 ° C, 5% CO ₂ incubator to a confluence of more than 90%, collected by trypsin digestion, and resuspended in culture medium. The cells were counted under a microscope, and the cell concentration was adjusted to 2 x 10 ⁴ /ml. The cell suspension was inoculated into a 96-well plate, ΙΟΟμΙ/well, and cultured overnight at 37 ° C in a 5% CO ₂ incubator. The integrin blocker polypeptide is diluted with the culture solution to each predetermined concentration. Endo is diluted to the final concentration with the culture solution. After the cells were fully attached, each dilution was separately added to a 96-well plate (ΙΟΟμΙ/well). As the administration group, the integrin blocker polypeptide dilution was added, and Endo and Paclitaxel were added as the positive control group, and the culture medium without any drug was used as the negative control group. Incubate for 48 h at 37 ° C in a 5% CO ₂ incubator. 5 mg/ml of MTT was added to a 96-well plate at 20 μl per well, and incubation was continued for 4 hours. The medium was aspirated, dissolved in 150 μl of DMSO per well, and gently mixed by shaking for 10 minutes. The absorbance was measured with a microplate reader at a measurement wavelength of 570 nm and a reference wavelength of 630 nm, and the growth inhibition (PI) was calculated. The formula is as follows:

PI (%) = 1 - administration group / negative group The results obtained by the experiment are expressed as mean ± SD, and a statistical T test is performed, *P < 0.05 is a significant difference, **P <

0.01 is a very significant difference.

Table 2. Integrin blocker polypeptide inhibits proliferation of human gastric cancer MGC-803 cells group dose g / ml ) A570nm detection inhibition rate (%)

1 0.8802±0.1119* 34.87%

2 0.8336±0.0980* 38.32%

4 0.9118±0.0765* 32.53%

8 1.0386±0.1028 23.15%

Peptide 16 0.8806±0.0631* 34.84%

32 0.6704±0.0732* 50.39%

64 0.4264±0.0315** 68.45%

128 0.1626±0.0316** 87.97%

256 0.0980±0.0247** 92.75%

Endo (ES) 50 0.8818±0.1321** 34.75%

Taxol 10 0.0622±0.0066** 95.40%

Negative group (Control) - 1.3514 ± 0.1192 0.00% Result: See Table 2 and Figure 3, compared with the negative control, integrin blocker peptide can significantly inhibit the proliferation of human gastric cancer MGC-803 cells in vitro, and it is obvious Dose dependence. Example 4 Inhibition of Proliferation Inhibition of Human Cervical Carcinoma HeLa Cells by Integrin Blocker Polypeptide The activity of integrin blocker polypeptide for inhibiting the growth of human cervical cancer HeLa cells was examined by MTT assay. See Example 3 for a detailed implementation.

Table 3. Inhibitory effect of integrin blocker polypeptide on proliferation of human cervical cancer HeLa cells Group dose (g/ml) A570nm detection inhibition rate (%)

1 1.0346±0.1671 29.12%

2 1.1972±0.0646 17.98%

4 1.0570±0.1257 27.59%

8 1.1642±0.0678 20.24%

Peptide 16 0.9804±0.0928* 32.84%

32 0.7822±0.0716* 46.41%

64 0.5618±0.0805** 61.51%

128 0.3596±0.0437** 75.36%

256 0.2720±0.0617** 81.37%

Endo (ES) 50 1.1334±0.159* 22.35%

Taxol 10 0.3500±0.0641 ** 76.02%

Negative group (Control) - 1.4597 ± 0.1937 0.00% Result: See Table 3 and Figure 4. Compared with the negative control, integrin blocker peptide can significantly inhibit the proliferation of human cervical cancer HeLa cells in vitro, and exhibits a significant dose. Dependencies. Example 5 Inhibition of proliferation of human colon cancer HCT 116 cells by integrin blocker polypeptide The activity of integrin blocker polypeptide for inhibiting the growth of human colon cancer HCT 116 cells was examined by MTT assay. See Example 3 for a specific implementation.

Table 4. Inhibitor blocker polypeptide inhibits proliferation of human colon cancer HCT 116 cells Group dose g / ml ) A570nm detection inhibition rate (%)

1 0.6214±0.0906 1.83%

2 0.6184±0.0710 2.31%

4 0.4882±0.0412* 22.88%

8 0.4657±0.0842* 26.43%

Peptide 16 0.3702±0.0596* 41.51%

32 0.3833±0.0947* 39.44%

64 0.3450±0.0505* 45.50%

128 0.2875±0.0766** 54.58%

256 0.1887±0.0589** 70.19%

Endo (ES) 50 0.4586±0.0521 * 27.55%

Taxol 10 0.1094±0.0414** 82.72%

Negative group (Control) - 0.6330 ± 0.0422 0.00% Result: See Table 4 and Figure 5, integrin blocker peptide significantly inhibits human colon cancer in vitro compared to the negative control HCT 116 cells proliferated and showed a significant dose-dependent relationship. Example 6 Inhibition of Proliferation of Human Glioma U87 Cells by Integrin Blocker Polypeptide The activity of integrin blocker polypeptide to inhibit the growth of human glioma U87 cells was examined by MTT assay. See Example 3 for a specific embodiment. Table 5. Inhibitory effect of integrin blocker polypeptide on proliferation of human glioma U87 cells

Group dose g /ml ) A570nm detection inhibition rate (%)

1 0.7536±0.0586 3.06%

2 0.6866±0.0457 11.68%

4 0.6086±0.0457 21.71%

8 0.5368±0.0382* 30.95%

Peptide 16 0.3940±0.0187** 49.32%

32 0.2492±0.0257** 67.94%

64 0.1530±0.0456** 80.32%

128 0.1344±0.0124** 82.71%

256 0.0812±0.0067** 89.55%

Endo (ES) 50 0.7108±0.0410 8.57%

Taxol 10 0.1306±0.0105** 83.20%

Negative group (Control) - 0.7774±0.0562 0.00% Result: See Table 5 and Figure 6. Integrin blocker peptide significantly inhibited the proliferation of human glioma U87 cells in vitro compared with the negative control. The dose dependency. Example 7 Inhibition of proliferation of human breast cancer MDA-MB-231 cells by integrin blocker polypeptide The activity of integrin blocker polypeptide for inhibiting the growth of human breast cancer MDA-MB-231 cells was examined by MTT assay. See Example 3 for a specific embodiment.

Table 6. Inhibitory effect of integrin blocker polypeptide on proliferation of human breast cancer MDA-MB-231 cells

Έ3\ dose (; /ml) A570nm detection inhibition rate (%)

1 0.9103±0.0240 4.46%

2 0.8652±0.0330** 9.21%

4 0.8650±0.0397* 9.23%

8 0.8613±0.0341 * 9.62% _

Peptide 16 0.7802±0 0270** 18.16%

32 0.7163±0 0412** 24.89%

64 0.4840±0 0242** 49.36%

128 0.3343±0 0480** 65.13%

256 0.2262±0 0305** 76.51%

Endo (ES) 50 0.8546±0 0356* 10.32%

Taxol 10 0.0538±0 0034** 94.67%

Negative group (Control) 0.9526±0 0233 0.00% Result: See Table 6 and Figure 7, integrin blocker polypeptide significantly inhibited the proliferation of human breast cancer MDA-MB-231 cells in vitro compared to the negative control, and Significant dose dependence is presented. Example 8 Inhibition inhibition assay of integrin blocker polypeptide on human umbilical vein endothelial cells (HUVEC) 10 mg/ml Matrigel (BD, USA) was diluted 1:3 with serum-free endothelial cell culture medium and applied to Transwell chamber (Greiner, USA) membrane, air dried at room temperature. HUVEC cells cultured in logarithmic growth phase were trypsinized, collected, resuspended in serum-free endothelial cell culture medium, counted under a microscope, and the cell concentration was adjusted to I x 10 ⁵ /ml. Each group of test liquids was prepared and grouped as follows: blank control group: serum-free endothelial cell culture solution without drug; Endo group: 5 mg/ml Endo liquid storage solution with drug-free serum-free endothelial cell culture solution Dilution to a predetermined concentration; integrin blocker polypeptide group: The integrin blocker polypeptide was diluted to each predetermined concentration with a drug-free serum-free endothelial cell culture solution. The cells were seeded into a Transwell chamber at 100 μl per well, and each set of test solution was added to the chamber. Into a 24-well plate, 0.6 ml of endothelial cell culture medium containing 5% fetal bovine serum and 1% endothelial cell growth factor (ECGS) was added to stimulate cell migration, and incubated at 5% CO ₂ at 37 ° C for 24 hours. Discard the medium in the well, fix it with absolute ethanol at room temperature for 30 minutes, 0.1% crystal violet for 10 minutes at room temperature, rinse with water, gently wipe off the unsprayed cells in the upper layer with a cotton swab, observe under the microscope and randomly select four fields to take pictures. Calculate the migration inhibition (MI) according to the formula:

MI (%) = ( 1 -Ntest/Ncontrol) 100% where Ntest is the number of cell migrations in the test group and Ncontrol is the number of cell migrations in the blank control group. The results obtained by the experiment were expressed as mean ± SD, and a statistical T test was performed. *P < 0.05 was a significant difference, and **P < 0.01 was a very significant difference. Table 7. Inhibition of migration of human umbilical vein endothelial cells (HUVEC) by integrin blocker polypeptides. Group dose (μ _§ /ιη1) migration cell number migration inhibition rate (%)

0.25 2191.5±121.8 16.11

0.5 842.5±39.2** 67.75 -„ —, , , , , , , , ―, ―^

""^" ^""""""""" ϊ Ϊ64Γ3ΪΪ35 ** 37Τ ~~~~TM~TM~TM" En (ES) 8 1749.0±136.1 * 33.5

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ The number of migrated endothelial cells was significantly reduced. The integrin blocker polypeptide inhibited the migration of HUVEC induced by 5% fetal bovine serum and 1% ECGS compared to the blank control group. At 0.5μ _§ /ιη1 and 1μ _§ /ιη1, the inhibitory effect of integrin blocker polypeptide on cell migration was significantly different from that of the blank control, when the integrin blocker peptide dose was 0.5. When μ _§ /ιη1, the inhibition rate of HUVEC cell migration was maximized. Example 9 Integrin Blocker Polypeptide Inhibits Tubular Structure Formation Inhibition Test of Human Umbilical Vein Endothelial Cells (HUVEC)

10 mg/ml Matrigel (BD, USA) was diluted 1:1 with serum-free endothelial cell culture medium containing 1% ECGS, 30 μl per well was plated in 96-well plates (Greiner, USA), and cultured at 37 °C. Aggregate lh in the box. The HUVEC cells in the logarithmic growth phase were digested with 0.25% trypsin, collected, resuspended in serum-free endothelial cell culture medium containing 1% ECGS, and counted under a microscope to adjust the cell concentration to 1.5 χ 10 ⁵ /ml. The test solutions of each group were prepared as follows: blank control group: 1% ECGS serum-free endothelial cell culture medium without drug; Endo group: 5 mg/ml with serum-free endothelial cell culture medium containing 1% ECGS The Endo stock solution was diluted to a predetermined concentration; Paclitaxel group: The 6 mg/ml paclitaxel stock solution was diluted to a predetermined concentration with a serum-free endothelial cell culture medium containing 1% ECGS. Integrin Blocker Polypeptide Set: The 10 mg/ml polypeptide stock solution was diluted to a predetermined concentration with serum-free endothelial cell culture medium containing 1% ECGS. The cells were seeded into 96-well plates, each well ΙΟΟμΙ, and each group of test solutions was added to the wells, 100 μl per well, incubated at 5% C0 ₂ , 37 °C. They were observed under an inverted microscope at 6 h, 12 h, 24 h, 36 h, and 48 h, photographed and counted.

Results: As shown in Figure 9, HUVEC cells differentiated into irregular luminal structures within 6h~24h, and the 36h luminal structure decreased significantly, and disappeared at 48h. The inhibitory effect of integrin blocker polypeptide on the tubular structure formation of HUVEC cells at 6h, 12h, 24h and 36h was dose-dependent. Moreover, the polypeptide inhibited the formation of tubular structures of HUVEC cells at three doses of 0.5μ _§ /ιη1 , 1μ _§ /ιη1 and 2μ _§ /ιη1. Example 10 Integrin Blocker Polypeptide for Human Nasopharyngeal Carcinoma CE Nude Mouse Xenograft Tumor Growth Inhibition Test Tumor cells in logarithmic growth phase were prepared as 5×10 ⁷ /ml cell suspension under aseptic conditions to 0.1 Ml was inoculated subcutaneously into the right axilla of nude mice. The diameter of the transplanted tumor of nude mice was measured with a vernier caliper, and the animals were randomly divided into groups after the tumor grew to 100-200 mm ³ . The antitumor effect of the test polypeptide was dynamically observed using a method for measuring the tumor diameter. The measurement of the diameter of the tumor is once every 2 days, and the weight of the mouse is also weighed for each measurement. The administration methods were all injected by tail vein. Negative control group injected with the same amount of physiology Saline, once a day; cisplatin group 10mg/kg, once a week; Endo group 2.5mg/kg, once a day; peptide high, medium and low groups were 20mg/kg, 10mg/kg, 5mg/ Kg, administered once a day. Tumor volume calculation formula:

TV = 0.52 X aX b ² where a and b represent the length and width, respectively. The relative tumor volume was calculated based on the measured results. The evaluation index of antitumor activity is the relative tumor growth rate T/C (%;), and the calculation formula is as follows:

T/C (%) = TRTV / CRT _V X 100%

T _RTV : treatment group RTV; CRTV: negative control group RTV Table 8. Inhibition of polypeptide on human nasopharyngeal carcinoma CNE nude mice xenograft tumor growth

Group dose Starting weight (g) Starting End body weight (g) Tumor weight (g) Tumor inhibition rate

(mg/kg) starting material

counting

Negative control a 22.87 12 21.56 12 1.102 a cisplatin 10 23.04 8 20.33 7 0.326** 70.38% Endo 2.5 22.48 8 22.03 8 0.647 41.25% Peptide height 20 22.31 8 22.42 8 End of action 0.384** 65.18%

10 23.22 8 23.37 8 0.525* 52.36% Peptide lower 5 22.98 8 23.54 8 0.664 39.77% Result: See Table 8 and Figure 10, the inhibition rate of cisplatin 10 mg/kg group on human nasopharyngeal carcinoma CE nude mice xenografts 70.38%; Endostar 2.5mg/kg group inhibited the tumor growth rate of human nasopharyngeal carcinoma CE nude mice xenografts 41.25%; high, medium and low doses of peptides inhibited human nasopharyngeal carcinoma CE nude mice xenografts The rates were 65.18%, 52.36%, and 39.77%, respectively. However, cisplatin was more toxic, and the body weight of the animal decreased significantly. The animals died during the experiment. The peptide had no significant effect on the body weight of the experimental animals. Therefore, the results of the polypeptide growth inhibition test on human nasopharyngeal carcinoma CE nude mice showed that the polypeptide 20 mg/kg group had a significant inhibitory effect on the growth of human nasopharyngeal carcinoma CE xenografts compared with the negative control group. The peptide 10 mg/kg group had a significant inhibitory effect on the growth of human nasopharyngeal carcinoma CE xenografts. Compared with the positive control cisplatin, the polypeptide had no significant effect on the body weight of the experimental animals, and no obvious side effects were observed. Example 11 Inhibition Test of Integrin Blocker Polypeptide on Human Gliomas U87 Nude Mouse Xenograft Tumor Growth See Example 10 for a specific embodiment. The dosage regimen was as follows: Negative control group was injected with the same amount of normal saline once a day; Paclitaxel group 10 mg/kg, once a week; Endo group 2.5 mg/kg, once a day; Peptide high, medium and low groups respectively Take 20 mg/kg, 10 mg/kg, 5 mg/kg, administered once a day. Table 9. Inhibition of polypeptide on human glioma U87 xenograft xenograft tumor growth

Group dose Starting weight (g) Terminal weight (g) Tumor weight (g) Tumor inhibition rate

(mg/kg) Negative control - 22.57 12 22.78 11 1.517 One paclitaxel 10 23.97 8 21.02 6 0.407** 73.11% Enteol 2.5 22.02 8 22.34 8 0.933 38.47% Polypeptide height 20 22.88 8 22.91 8 78.19% Peptide 10 21.55 8 21.87 8 76.21% polypeptide low 5 22.69 8 22.83 8 0.459** 69.73 % start

Primer

Results: See Table 9 and Figure 11. The inhibition rate of paclitaxel 10 mg/kg group on human glioma U87 nude mice xenografts was 73.11%; Endemic 2.5 mg/kg group was transplanted into human glioma U87 nude mice. The tumor inhibition rate of tumor was 38.47%; the tumor inhibition rate of human glioma U87 nude mice was 78.19%, 76.21%, 69.73% in the high, medium and low dose groups. However, paclitaxel is more toxic, the animal loses weight, and the animal has died during the experiment. The peptide had no significant effect on the body weight of nude mice.

End

Therefore, the results of the polypeptide growth inhibition test on human glioma U87 nude mice xenografts showed that the polypeptides of human glioma U87 were compared with the negative control group, peptides 20 mg/kg, 10 mg/kg and 5 mg/kg. The growth of transplanted tumors has a very significant inhibition of p

effect. Compared with paclitaxel in the positive control group, the peptide had no significant effect on the body weight of the experimental animals, and no obvious toxic and side effects were observed.

*

should. * Example 12 Integrin blocker polypeptide against human thyroid cancer SW-579 nude mouse xenograft tumor growth inhibition test See Example 10 for a specific embodiment. The dosage regimen was as follows: Negative control group was given the same amount of normal saline once a day; 5-fluorouracil group 10 mg/kg, once a week; Endo group 2.5 mg/kg, once a day; Peptide high, medium and low The groups were administered once a day at 20 mg/kg, 10 mg/kg, and 5 mg/kg, respectively. Table 10. Inhibition of polypeptide on human thyroid carcinoma SW-579 xenograft tumor growth in nude mice

Group dose starting weight (g) initial terminal weight (g) terminal tumor weight (g) tumor inhibition rate

(mg/kg) animal animal negative control one 21.46 12 21.38 11 1.310 one

5-Fu 10 22.89 8 20.97 6 0.403** 69.26% Endo 2.5 21.76 8 20.42 7 1.017 22.37% Polypeptide height 20 22.33 8 22.19 8 0.382** 70.81% Peptide 10 22.45 8 22.13 8 0.493** 62.35% Peptide lower 5 21.35 8 21.24 8 0.550** 57.98 % Result: See table 10 and Fig. 12, the 5-Fu (5-fluorouracil) 10 mg/kg group inhibited the tumor growth rate of human thyroid cancer SW-579 nude mice xenografts by 69.26%; the Endo group 2.5 mg/kg, for human thyroid cancer SW- The tumor inhibition rate of 579 nude mice xenografts was 22.37%; the tumor inhibition rate of human thyroid cancer SW-579 nude mice was 70.81%, 62.35%, 57.98%, respectively. However, 5-Fu was more toxic, the animal lost weight, and the animals died during the experiment. The peptide had no significant effect on the body weight of nude mice. Therefore, the results of the polypeptide growth inhibition test on human thyroid cancer SW-579 nude mice showed that the phase contrast with the negative control group

Compared with the 20mg/kg, 10mg/kg and 5mg/kg peptides, the growth of human thyroid cancer SW-579 xenografts was significantly inhibited. Compared with the positive control group 5-Fu, the polypeptide had no significant effect on the body weight of the experimental animals, and no obvious side effects were observed. Example 13

End

Integrin Blocker Polypeptide Against Human Pancreatic Cancer SW-1990 Nude Mouse Xenograft Tumor Growth Inhibition End-Temperature Test See Example 10 for a specific embodiment. The dosage regimen was as follows: Negative control group was given the same amount of normal saline once a day; 5-fluorouracil group 10 mg/kg, once a week; Endo group 2.5 mg/kg, once a day; Peptide high, medium and low The groups were administered once a day at 20 mg/kg, 10 mg/kg, and 5 mg/kg, respectively. Table 11. Inhibition of polypeptide on human pancreatic cancer SW-1990 xenograft tumor growth in nude mice

(mg/kg) Negative control one 23.01 12 22.92 12 1.291 one

5-Fu 10 23.27 8 21.06 7 0.448** 65.26 % Enrity 2.5 22.33 8 21.97 8 0.855 33.76% Polypeptide height 20 22.45 8 22.21 8 0.399** 69.08% Peptide 10 23.11 8 23.02 8 0.596* 53.82% Peptide lower 5 22.78 8 22.55 8 0.706 45.31% Results: See Table 11 and Figure 13, 5-Fu 10mg/kg group on human pancreatic cancer SW-1990 nude mice xenograft tumor inhibition rate of 65.26%; Endemic 2.5mg / kg group to human The tumor inhibition rate of pancreatic cancer SW-1990 nude mice xenografts was 33.76%; peptide high, medium, The tumor inhibition rate of human pancreatic cancer SW-1990 xenografts in the low-dose group was 69.08%, 53.82%, and 45.31%, respectively. However, 5-Fu was more toxic, the animal lost weight, and the animals died during the experiment. The peptide had no significant effect on the body weight of nude mice. Therefore, the results of the polypeptide growth inhibition test on human pancreatic cancer SW-1990 nude mice xenograft showed that the polypeptide 20 mg/kg group had a significant inhibitory effect on the growth of human pancreatic cancer SW-1990 xenografts compared with the negative control group. The polypeptide 10 mg/kg group significantly inhibited the growth of human pancreatic cancer SW-1990 xenografts. Compared with the positive control group 5-Fu, the polypeptide had no significant effect on the body weight of the experimental animals, and no obvious side effects were observed. Example 14 Integrin Blocker Polypeptide Against Human Lung Cancer H460 Nude Mouse Xenograft Tumor Growth Inhibition Assay See Example 10 for a specific embodiment. The dosage regimen was initiated as follows: The negative control group was injected with the same amount of normal saline once a day;

Paclitaxel group 10mg/kg, once a week; Endo group 2.5mg/kg, once a day; Peptide high, medium and low groups were 20mg/kg, 10mg/kg, 5mg/kg, once a day .

Table 12. Inhibition of polypeptide on human lung cancer H460 xenograft tumor growth in nude mice

End

Group dose starting weight terminal weight (g) terminal tumor weight (g) tumor inhibition rate

(mg/kg) (g)

p

Negative control one 22.37 12 22.58 10 1.142 o * one

*

Paclitaxel 10 23.06 8 20.12 6 0.282** 75.32% Entitude 2.5 22.09 8 21.32 8 55.37% Polypeptide height 20 21.67 8 21.55 8 0.339** 70.29% Peptide 10 22.33 8 22.25 8 0.457 45.02% Peptide lower 5 21.38 8 21.17 8 0.656 42.58% Results: See Table 12 and Figure 14. The inhibition rate of paclitaxel 10 mg/kg group on human lung cancer H460 xenografts in nude mice was 75.32%, and the effect of enrollment 2.5 mg/kg group on human lung cancer H460 xenografts in nude mice The tumor rate was 55.37%. The tumor inhibition rate of human lung cancer H460 nude mice was 70.29%, 45.02%, 42.58% in the high, medium and low dose groups. However, paclitaxel was more toxic, the body weight decreased significantly, and there was death during the test, and the peptide had no significant effect on the body weight of nude mice. Therefore, the results of the polypeptide growth inhibition test on human lung cancer H460 xenografts in nude mice showed that the polypeptide 20 mg/kg group had a significant inhibitory effect on the growth of human lung cancer H460 xenografts compared with the negative control group. Compared with paclitaxel in the positive control group, the polypeptide had no significant effect on the body weight of the experimental animals, and no obvious side effects were observed. Example 15 Inhibition Test of Integrin Blocker Polypeptide on Human Liver Cancer Bel-7402 Nude Mouse Xenograft Tumor Growth See Example 10 for a specific embodiment. The dosage regimen was as follows: Negative control group was injected with the same amount of normal saline once a day; Paclitaxel group 10 mg/kg, once a week; Endo group 2.5 mg/kg, once a day; Peptide high, medium and low groups respectively The drug was administered once a day at 20 mg/kg, 10 mg/kg, and 5 mg/kg. Table 13. Inhibition of polypeptide on human hepatocellular carcinoma Bel-7402 xenograft tumor growth in nude mice

( mg/kg)

Negative control 22.38 10 22.56 9 1.378

From

Paclitaxel 10 23.67 8 Beginning 21.09 6 0.379** 72.47% Enrity 2.5 22.59 8 22.37 8 0.851 38.21% Polypeptide height 20 21.12 8 21.82 8 0.433** 68.55% Peptide 10 22.65 8 22.73 8 0.753 45.31% Peptide lower 22.05 8 22.24 8 0.919 33.33% final

Results: See Table 13 and Figure 15. Paclitaxel 10 mg/kg group for human liver cancer

72.47%; The degree of inhibition of human liver cancer Bel-7402 xenografts was 38.21% in the group of 2.5mg/kg; the tumor inhibition rate of human hepatocellular carcinoma Bel-7402 xenografts in high, medium and low dose groups of peptides They reached 68.55%, 45.31% and 33.33% respectively. However, paclitaxel was more toxic, the body weight decreased significantly, and there was death during the test. The peptide had no significant effect on the body weight of nude mice. Therefore, the results of the polypeptide growth inhibition test on human liver cancer Bel-7402 nude mice showed that the polypeptide 20 mg/kg group had a significant inhibitory effect on the growth of human liver cancer Bel-7402 xenografts compared with the negative control group. Compared with paclitaxel in the positive control group, the polypeptide had no significant effect on the body weight of the experimental animals, and no obvious side effects were observed. Example 16 Integrin Blocker Polypeptide Against Human Esophageal Cancer Ecl09 Xenograft Xenograft Tumor Growth Inhibition Test See Example 10 for a specific embodiment. The dosage regimen was as follows: Negative control group was injected with the same amount of normal saline once a day; Paclitaxel group 10 mg/kg, once a week; Endo group 2.5 mg/kg, once a day; Peptide high, medium and low groups respectively The drug was administered once a day at 20 mg/kg, 10 mg/kg, and 5 mg/kg. Table 14. Inhibition of polypeptide on human esophageal cancer Ecl09 xenograft tumor growth in nude mice Group dose starting weight (g) terminal body weight (g) tumor weight (g) tumor inhibition rate

( mg/kg)

Negative control 21.56 10 21.38 9 1.019

Paclitaxel 10 22.38 8 20.01 6 0.308** 69.76%

2.5 21.65 8 21.36 8 0.518* 49.21% Peptide height 20 22.17 8 21.98 8 0.360** 67.55% Peptide 10 22.83 8 22.67 8 0.509* 50.03% Peptide lower 21.87 8 21.06 8 0.610 40.12% Result: See Table 14 and Figure 16, The inhibition rate of paclitaxel 10mg/kg group on human esophageal cancer Ecl09 xenografts was 69.76%. The inhibition rate of human esophageal cancer Ecl09 xenografts was 49.21%. The peptides were high and medium. The low-dose group was 67.55%, 50.03%, and 40.21% of human esophageal cancer. However, paclitaxel was more toxic, the body weight decreased significantly, and there was death during the test. The peptide had no significant effect on the body weight of nude mice. Therefore, the results of the polypeptide growth inhibition test on human esophageal cancer Ecl09 xenografts showed that compared with the negative control group, the polypeptide 20 mg/kg group had a significant inhibitory effect on the growth of human esophageal cancer Ecl09 xenografts, peptide 10 mg/ The kg group had a significant inhibitory effect on the growth of human esophageal cancer Ecl09 xenografts. The peptide had no significant effect on the body weight of the experimental animals, and no obvious side effects were observed. Example 17 Integrin Blocker Polypeptide Against Human Gastric Cancer MGC-803 Nude Mouse Xenograft Tumor Growth Inhibition Test See Example 10 for a specific embodiment. The dosage regimen was as follows: Negative control group was injected with the same amount of normal saline once a day; Paclitaxel group 10 mg/kg, once a week; Endo group 2.5 mg/kg, once a day; Peptide high, medium and low groups respectively The drug was administered once a day at 20 mg/kg, 10 mg/kg, and 5 mg/kg. Table 15. Inhibition of polypeptide growth on human gastric cancer MGC-803 xenograft tumors in different groups Group dose Starting weight (g) Terminal weight (g) Tumor weight (g) Tumor inhibition rate

(mg/kg) Negative control 23.42 23.67 10 1.367 Paclitaxel 22.77 20.21 7 0.376** 72.48% Ente 23.16 23.98 8 0.749* 45.22% Peptide height 22.56 22.78 8 0.405** 70.35% Peptide 22.13 22.25 8 0.475** 65.26% Peptide lower 5 22.68 8 22.89 8 0.718* 47.45% Result: See Table 15 and Figure 17, the inhibition rate of paclitaxel 10mg/kg group on human gastric cancer MGC-803 nude mice xenografts was 72.48%; Endeavour 2.5mg/kg group The tumor inhibition rate of human gastric cancer MGC-803 xenografts in nude mice was 45.22%; the tumor inhibition rate of human gastric cancer MGC-803 nude mice was 70.35%, 65.26%, 47.45% in high, medium and low dose groups, respectively. . However, paclitaxel was more toxic, the body weight decreased significantly, and there was death during the test. The peptide had no significant effect on the body weight of nude mice. Therefore, the results of the polypeptide growth inhibition test on human gastric cancer MGC-803 xenografts showed that compared with the negative control group, the polypeptide 20 mg/kg group and the polypeptide 10 mg/kg group had extremely strong growth of human gastric cancer MGC-803 transplanted tumor. Significant inhibition, peptide 5mg / kg group significantly inhibited the growth of human gastric cancer MGC-803 transplanted tumor. Compared with paclitaxel in the positive control group, the polypeptide had no significant effect on the body weight of the experimental animals, and no obvious side effects were observed.

Start

Example of Initiating Example 18 Integrin Blocker Polypeptide Against Human Breast Cancer MDA-MB-231 Nude Mouse Xenograft Tumor Growth Inhibition Test See Example 10 for a specific embodiment. The dosage regimen was as follows: Negative control group was injected with the same amount of normal saline once a day; Paclitaxel group 10 mg/kg, once a week; Endo group 2.5 mg/kg, once a day; Peptide high, medium and low groups respectively The drug was administered once a day at 20 mg/kg, 10 mg/kg, and 5 mg/kg. End

End

Table 16. Inhibition of polypeptide on human breast cancer MDA-MB-231 xenograft tumor growth

(mg/kg) * * Negative control one 22.36 10 22.83 10 0.998 One paclitaxel 10 23.88 8 21.23 7 70.87% Enteol 2.5 22.16 8 21.98 8 0.507* 49.21% Peptide height 20 22.34 8 22.67 8 0.246** 75.36% Peptide 10 22.78 8 21.99 8 0.498* 50.09% Peptide lower 5 22.43 8 22.87 8 0.587 41.32% Result: See Table 16 and Figure 18, the inhibition rate of paclitaxel 10 mg/kg group on human breast cancer MDA-MB-231 xenografts in nude mice 70.87%; Endostar 2.5mg/kg group inhibited tumor growth rate of human breast cancer MDA-MB-231 xenografts in nude mice; 49.21%; high, medium and low doses of peptides on human breast cancer MDA-MB-231 nude mice The tumor inhibition rate of transplanted tumors was 75.36%, 50.09%, and 41.32%, respectively. However, paclitaxel was more toxic, the body weight decreased significantly, and there was death during the test. The peptide had no significant effect on the body weight of nude mice. Therefore, the results of the polypeptide growth inhibition test on human breast cancer MDA-MB-231 xenografts showed that compared with the negative control group, the polypeptide 20 mg/kg group had a significant growth on human breast cancer MDA-MB-231 xenografts. Sexual inhibition, peptide

The 10 mg/kg group had a significant inhibitory effect on the growth of human breast cancer MDA-MB-231 xenografts. Compared with the positive control group, paclitaxel had no significant effect on the body weight of the experimental animals, and no obvious side effects were observed. Example 19 Integrin Blocker Polypeptide Against Human Renal Carcinoma A498 Nude Mouse Xenograft Tumor Growth Inhibition Assay See Example 10 for a specific embodiment. The dosage regimen was as follows: Negative control group was injected with the same amount of normal saline once a day; Paclitaxel group 10 mg/kg, once a week; Endo group 2.5 mg/kg, once a day; Peptide high, medium and low groups respectively The drug was administered once a day at 20 mg/kg, 10 mg/kg, and 5 mg/kg. Table 17. Inhibition of polypeptide on human kidney cancer A498 xenograft tumor growth in nude mice

Group dose starting weight initial terminal weight (g) terminal tumor weight (g) tumor inhibition rate

(mg/kg) (g) Negative control for animal animals 21.53 10 21.07 9 1.117 paclitaxel 10 22.33 6 19.56 6 0.311 ** 72.19% Entitude 2.5 21.45 6 21.87 6 0.690 38.23% Peptide height 20 22.56 6 22.49 6 0.340** 69.55% Peptide 10 21.12 6 20.98 6 0.411 ** 60.21% Peptide lower 5 21.49 6 21.32 6 0.507** 54.58% Result: See Table 17 and Figure 19, paclitaxel 10 mg/kg group for human kidney cancer A498 nude mice xenografts The tumor inhibition rate was 72.19%; the tumor inhibition rate of human kidney cancer A498 nude mice was 38.23% in the group of 2.5mg/kg; the high, medium and low dose groups of peptide inhibited the transplanted tumor of human kidney cancer A498 nude mice. The tumor rate was 69.55%, 60.21%, and 54.58%, respectively. However, paclitaxel was more toxic and the animals lost weight during the test. The peptide had no significant effect on the body weight of nude mice. Therefore, the results of the polypeptide growth inhibition test on human kidney cancer A498 nude mice showed that compared with the negative control group, the polypeptides of 20mg/kg, 10mg/kg and 5mg/kg had a very strong growth of human kidney cancer A498 xenografts. Significant inhibition. Compared with paclitaxel in the positive control group, the polypeptide had no significant effect on the body weight of the experimental animals, and no obvious side effects were observed. Example 20 Integrin Blocker Polypeptide Against Human Gallbladder Carcinoma GBC-SD Nude Mouse Xenograft Tumor Growth Inhibition Test See Example 10 for a specific embodiment. The dosage regimen was as follows: The negative control group was injected with the same amount of normal saline once a day; Paclitaxel group 10mg/kg, once a week; Endo group 2.5mg/kg, once a day; Peptide high, medium and low groups were 20mg/kg, 10mg/kg, 5mg/kg, once a day . Table 18. Inhibition of polypeptide on human gallbladder carcinoma GBC-SD xenograft tumor growth in nude mice

(mg/kg) Negative control - 21.36 12 20.07 11 1.039 One paclitaxel 10 22.68 8 20.59 7 0.305** 70.66% Enteol 2.5 21.73 8 21.58 8 0.736 29.12% Polypeptide height 20 22.39 8 22.05 8 0.360** 65.35% Peptide 10 21.97 8 21.70 8 0.495* 52.33% start

Peptide low 5 21.29 8 Starting material 21.61 8 0.530* 48.97% Result: See Table 18 and Figure 20, paclitaxel 10mg/kg group on human gallbladder carcinoma GBC-SD xenograft tumor xenograft tumor rate is 70.66%; Enrity 2.5mg The tumor inhibition rate of human gallbladder carcinoma GBC-SD nude mice was 29.12%; the tumor inhibition rate of human gallbladder carcinoma GBC-SD xenografts was 65.35%, respectively. 52.33%, 48.97%. However, paclitaxel was more toxic, the body weight decreased significantly, and there was death during the test. The peptide had no significant effect on the body weight of the nude mice.

End

Therefore, the results of the test on the tumor inhibition rate of human gallbladder carcinoma GBC-SD mouse xenografts showed that the polypeptide 20mg/kg group had a significant effect on the growth of human gallbladder carcinoma GBC-SD xenografts compared with the negative control group. The inhibitory effect of the polypeptides 10 mg/kg and 5 mg/kg on the growth of human gallbladder carcinoma GBC-SD xenografts was significantly inhibited. Compared with paclitaxel in the positive control group, the polypeptide had no significant effect on the body weight of the experimental animals, and no obvious side effects were observed. Example 21 Inhibition test of integrin blocker polypeptide on human colon cancer HT-29 xenograft tumor growth in nude mice See Example 10 for a specific embodiment. The dosage regimen was as follows: Negative control group was injected with the same amount of normal saline once a day; Paclitaxel group 10 mg/kg, once a week; Endo group 2.5 mg/kg, once a day; Peptide high, medium and low groups respectively The drug was administered once a day at 20 mg/kg, 10 mg/kg, and 5 mg/kg. Table 19. Inhibition of polypeptide on human colon cancer HT-29 xenograft tumor growth in nude mice

Group dose starting weight initial terminal weight end stage tumor weight (g) tumor inhibition rate (%)

(mg/kg) (g) Animal (g) Animal negative control - 23.87 12 23.68 12 1.157 ― Paclitaxel 10 23.88 8 20.03 6 0.368** 68.23% Entitude 2.5 23.55 8 22.83 8 0.720 37.78% Polypeptide height 20 23.14 8 22.37 8 0.430** 62.88% Peptide 10 24.42 8 25.39 8 0.542* 53.13% Peptide lower 5 23.28 8 23.11 8 0.633 45.33% Results: See Table 19 and Figure 21. The inhibition rate of paclitaxel 10 mg/kg group on human colon cancer HT-29 xenografts in nude mice was 68.23%; Endemic 2.5 mg/kg group on human colon cancer HT- The tumor inhibition rate of nude mice xenografts was 37.78%. The tumor inhibition rates of human colon cancer HT-29 nude mice were 62.88%, 53.13% and 45.33%, respectively. However, paclitaxel was more toxic, the body weight decreased significantly, and there was death during the test. The peptide had no significant effect on the body weight of nude mice.

Start

Therefore, the results of the polypeptide growth inhibition test on human colon cancer HT-29 nude mice showed that the polypeptide 20mg/kg group had a significant effect on the growth of human colon cancer HT-29 xenografts compared with the negative control group. Inhibition, the peptide 10 mg/kg group significantly inhibited the growth of human colon cancer HT-29 xenografts. Compared with paclitaxel in the positive control group, the polypeptide had no significant effect on the body weight of the experimental animals, and no obvious side effects were observed. Example 22

End

Inhibition test of integrin blocker polypeptide on human ovarian cancer SK-OV-3 nude mouse xenograft tumor growth See Example 10 for a specific embodiment. The dosage regimen was as follows: Negative control group was injected with the same amount of normal saline once a day; Cisplatin group 10 mg/kg, once a week; Endo group 2.5 mg/kg, once a day; Peptide high, medium and low groups They were administered once a day at 20 mg/kg, 10 mg/kg, and 5 mg/kg, respectively. Table 20. Inhibition of polypeptide on human ovarian cancer SK-OV-3 xenograft tumor growth

Group dose Starting weight End-of-life weight Tumor weight (g) Tumor inhibition rate (%)

(mg/kg) (g) (g)

Negative control one 23.56 12 22.07 12 1.019 One cisplatin 10 22.93 8 20.21 6 0.323** 68.24% Entitude 2.5 22.30 8 22.07 8 0.661 35.14% Peptide height 20 22.68 8 22.43 8 0.304** 70.13% Peptide 10 21.53 8 21.30 8 0.409** 59.87% Peptide lower 5 2187 8 21.84 8 0.529* 48.08% Result: See Table 20 and Figure 22, the inhibition rate of cisplatin 10mg/kg group on human ovarian cancer SK-OV-3 nude mice xenografts is 68.24 %; Enta 2.5mg/kg group on human ovarian cancer SK-OV-3 nude mice xenograft tumor inhibition rate was 35.14%; peptide high, medium, The tumor inhibition rates of human ovarian cancer SK-OV-3 xenografts in the low-dose group were 70.13%, 59.87%, and 48.08%, respectively. However, cisplatin was more toxic, the body weight decreased significantly, and there was death during the test. The peptide had no significant effect on the body weight of nude mice. Therefore, the results of the polypeptide growth inhibition test on human ovarian cancer SK-OV-3 nude mice showed that compared with the negative control group, the polypeptides of 20 mg/kg and 10 mg/kg were implanted into human ovarian cancer SK-OV-3 tumors. Growth has a very significant inhibitory effect, and the polypeptide 5 mg/kg group has a significant inhibitory effect on the growth of human ovarian cancer SK-OV-3 xenografts. Compared with the positive control group, cisplatin had no significant effect on the body weight of the experimental animals, and no obvious side effects were observed. Example 23 Inhibition of integrin blocker polypeptides on human endometrial cancer HHUA nude mouse xenograft tumor growth The specific embodiment is shown in Example 10. The dosage regimen was as follows: Negative control group was injected with the same amount of normal saline once a day; Paclitaxel group 10 mg/kg, once a week; Endo group 2.5 mg/kg, once a day; Peptide high, medium and low groups respectively The drug was administered once a day at 20 mg/kg, 10 mg/kg, and 5 mg/kg. Table 21. Inhibition of polypeptide on human endometrial carcinoma HHUA nude mouse xenograft tumor growth

Group dose starting weight initial terminal weight end stage tumor weight (g) tumor inhibition rate (%;)

(mg/kg) (g) Animal (g) Animal negative control - 23.06 10 23.57 9 1.215 One paclitaxel 10 23.78 6 21.01 6 0.398** 67.21% Enteol 2.5 22.57 6 22.29 6 0.759 37.56% Polypeptide height 20 23.49 6 23.75 6 0.373** 69.34% Peptide 10 22.88 6 22.93 6 0.501 ** 58.77% Peptide lower 5 22.09 6 22.34 6 0.665* 45.25% Result: See Table 21 and Figure 23, paclitaxel 10 mg/kg group for human endometrial cancer HHUA nude The tumor inhibition rate of mouse xenografts was 67.21%; the degree of inhibition of human endometrial cancer HHUA nude mice xenografts was 37.56%; the high, medium and low doses of peptides were in human uterus. The tumor inhibition rate of HICA nude mice xenografts was 69.34% and 58.77%, respectively.

45.25 %. However, paclitaxel was more toxic and the body weight decreased significantly during the test. The peptide had no significant effect on the body weight of nude mice. Therefore, the results of the polypeptide growth inhibition test on human endometrial cancer HHUA nude mice showed that compared with the negative control group, the 20mg/kg group and the 10mg/kg group had a very strong growth of human endometrial cancer HHUA xenografts. Significant inhibition, peptide 5mg / kg group significantly inhibited the growth of human endometrial cancer HHUA transplanted tumor. Compared with paclitaxel in the positive control group, the polypeptide had no significant effect on the body weight of the experimental animals, and no obvious side effects were observed. Inhibition test of integrin blocker polypeptide on human cervical cancer HeLa nude mouse xenograft tumor growth is shown in Example 10. The dosage regimen was as follows: Negative control group was injected with the same amount of normal saline once a day; Paclitaxel group 10 mg/kg, once a week; Endo group 2.5 mg/kg, once a day; Peptide high, medium and low groups respectively The drug was administered once a day at 20 mg/kg, 10 mg/kg, and 5 mg/kg. Table 22. Inhibition of polypeptide on human cervical cancer HeLa nude mouse xenograft tumor growth

(mg/kg) (g) (g)

Negative control 22.87 23.05 8 1.005

Beginning

Paclitaxel 10 23.28 6 21.37 6 0.348** 65.38% Entity 2.5 23.54 6 23.29 6 0.604 39.88% Polypeptide height 20 23.78 6 23.09 6 0.318** 68.37% Peptide 10 22.74 6 22.41 6 0.436* 56.54% Peptide lower 23.09 6 22.97 6 0.602 40.01% of the end

End

RESULTS: See Table 22 and Figure 24. The inhibition rate of paclitaxel 10 mg/kg group on human cervical cancer HeLa nude mice xenografts was 65.38%; Endemic 2.5 mg/kg group inhibited human cervical carcinoma HeLa nude mice xenografts The rate of inhibition was 39.88%. The inhibition rates of high, medium and low dose groups of peptides in human cervical cancer HeLa nude mice were 68.37%, 56.54% and 40.01%, respectively. However, paclitaxel is more toxic and the body weight of the animal is significantly reduced. The peptide had no significant effect on the body weight of nude mice. Therefore, the results of the polypeptide growth inhibition test on human cervical cancer HeLa nude mice showed that compared with the negative control group, the polypeptide 20 mg/kg group had a significant inhibitory effect on the growth of human cervical cancer HeLa xenografts, peptide 10 mg/ The kg group had a significant inhibitory effect on the growth of human cervical cancer HeLa xenografts. Compared with paclitaxel in the positive control group, the polypeptide had no significant effect on the body weight of the experimental animals, and no obvious side effects were observed. Example 25 Inhibitory Test of Integrin Blocker Polypeptide on Human Prostate Cancer DU-145 Xenograft Xenograft Tumor Growth See Example 10 for a specific embodiment. The dosage regimen was as follows: Negative control group was injected with the same amount of normal saline once a day; Cisplatin group 10 mg/kg, once a week; Endo group 2.5 mg/kg, once a day; Peptide high, medium and low groups They were administered once a day at 20 mg/kg, 10 mg/kg, and 5 mg/kg, respectively. Table 23. Inhibition of polypeptide on human prostate cancer DU-145 xenograft tumor growth in nude mice Group dose, initial body weight, terminal body weight, tumor weight (g), tumor inhibition rate (%)

(mg/kg) (g) (g)

Negative control one 23.67 12 23.79 12 1.245 One cisplatin 10 23.51 8 21.88 6 0.356** 71.38% Entropy 2.5 22.37 8 22.89 8 0.900 27.68% Polypeptide height 20 22.13 8 22.57 8 0.307** 75.31% Peptide 10 22.09 8 22.48 8 0.552* 55.63% Peptide lower 5 23.26 8 23.64 8 0.644* 48.27% Result: See Table 23 and Figure 25. The inhibition rate of cisplatin 10 mg/kg group on human prostate cancer DU-145 nude mice xenografts is 71.38%; The tumor inhibition rate of DU-145 nude mice xenografts was 27.68%. The tumor inhibition rates of human prostate cancer DU-145 nude mice were 75.31%, 55.63% and 48.27%, respectively. However, cisplatin was more toxic, the body weight decreased significantly, and there was death during the test. The peptide had no significant effect on the body weight of nude mice. Therefore, the results of the polypeptide growth inhibition test on human prostate cancer DU-145 nude mice showed that the polypeptide 20 mg/kg group had a significant inhibitory effect on the growth of human prostate cancer DU-145 xenografts compared with the negative control group. , peptide 10mg/kg and 5mg/kg group for human prostate cancer

In comparison, the polypeptide had no significant effect on the body weight of the experimental animals, and no obvious side effects were observed. Example 26 Inhibition test of integrin blocker polypeptide on human bladder cancer HT1376 xenograft tumor growth in nude mice See Example 10 for a specific embodiment. The dosage regimen was as follows: Negative control group was injected with the same amount of normal saline once a day; Paclitaxel group 10 mg/kg, once a week; Endo group 2.5 mg/kg, once a day; Peptide high, medium and low groups respectively The drug was administered once a day at 20 mg/kg, 10 mg/kg, and 5 mg/kg. Table 24. Inhibition of polypeptide on human bladder cancer HT1376 xenograft tumor growth in nude mice

(mg/kg) (g) (g) Negative control 22.75 10 22.56 10 1.227

Paclitaxel 10 22.09 6 20.01 0.386** 68.55% Entitude 2.5 21.45 6 21.87 6 0.842 31.41% Peptide height 20 22.56 6 22.38 6 0.466** 62.03% Peptide 10 21.50 6 21.62 6 0.591 * 51.80% Peptide low 5 21.80 6 22.06 6 0.745 39.27% Results: See Table 24 and Figure 26, paclitaxel 10mg/kg group on human bladder cancer HT1376 nude mice xenograft tumor inhibition rate of 68.55%; Endemic 2.5mg / kg group to human The tumor inhibition rate of bladder cancer HT1376 xenografts was 31.41%. The tumor inhibition rates of human bladder cancer HT1376 xenografts were 62.03%, 51.80%, 39.27%, respectively. However, paclitaxel was more toxic, the body weight decreased significantly, and there was death during the test. The peptide had no significant effect on the body weight of nude mice. Therefore, the results of the polypeptide growth inhibition test on human bladder cancer HT1376 xenografts showed that compared with the negative control group, the polypeptide 20 mg/kg group had a significant inhibitory effect on the growth of human bladder cancer HT1376 xenografts, peptide 10 mg/ The kg group had a significant inhibitory effect on the growth of human bladder cancer HT1376 xenografts. Compared with paclitaxel in the positive control group, the polypeptide had no significant effect on the body weight of the experimental animals, and no obvious side effects were observed.

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Example 27 Inhibitory Test of Integrin Blocker Polypeptide on Human Testicular Cancer 5637 Nude Mouse Xenograft Tumor Growth The specific embodiment is shown in Example 10. The dosage regimen was as follows: Negative control group was injected with the same amount of normal saline once a day; Cisplatin group 10 mg/kg, once a week; Endo group 2.5 mg/kg, once a day; Peptide high, medium and low groups Separately

20 mg/kg, 10 mg/kg, 5 mg/kg, administered once a day. The final substance Table 25. Inhibition of polypeptide on human testicular cancer 5637 xenograft tumor growth

Group dose Starting weight End-of-life weight Tumor weight *(g) Tumor inhibition rate (%)

(mg/kg) (g) (g) Negative control one 23.04 10 23.61 10 1.572 One cisplatin 10 24.64 6 22.37 6 0.477** 69.64% Endpoint 2.5 23.88 6 24.09 6 1.111 29.33% Peptide height 20 22.57 6 22.88 6

71.92% Peptide 10 23.36 6 23.49 6 0.670** 57.36% Peptide Low 5 23.67 6 23.77 6 0.956 39.21% Result: See Table 25 and Figure 27, cisplatin 10 mg/kg group inhibits human testicular cancer 5637 nude mice xenografts The tumor rate was 69.64%, and the tumor inhibition rate of human testicular cancer 5637 nude mice was 29.33%. The high, medium and low dose groups of peptides inhibited the growth of human testicular carcinoma 5637 nude mice. The rates were 71.92%, 57.36%, and 39.21%, respectively. However, cisplatin is more toxic, and the body weight of the animal is significantly decreased, while the polypeptide has no significant effect on the body weight of nude mice. Therefore, the results of the polypeptide growth inhibition test on human testicular cancer 5637 nude mice showed that compared with the negative control group, The peptide 20 mg/kg group had a very significant inhibitory effect on the growth of human testicular cancer 5637 xenografts. The polypeptide 10 mg/kg group had a significant inhibitory effect on the growth of human testicular cancer 5637 xenografts. Compared with the positive control group, cisplatin had no significant effect on the body weight of the experimental animals, and no obvious side effects were observed. Example 28 Inhibition of integrin blocker polypeptides on sarcoma HT-1080 xenograft xenograft tumor growth The specific embodiment is shown in Example 10. The dosing regimen was as follows: The negative control group was injected with the same amount of normal saline once a day; the cyclophosphamide group was administered at 15 mg/kg once a week; and the polypeptide was administered once a day at 20 mg/kg. Table 26. Inhibition of polypeptide on xenograft tumor growth in sarcoma HT-1080 nude mice

(mg/kg) (g) Animal (g) Animal negative control - 22.15 8 22.76 8 1.533 monocyclic phosphoramide 15 23.18 8 21.90 8 0.526** 65.67% Peptide 20 23.47 8 23.69 8 0.439** 71.33% Result: See table 26 and Fig. 28, the inhibition rate of cyclophosphamide 15mg/kg group on sarcoma HT-1080 nude mice xenografts was 65.67%, and the tumor inhibition rate of sarcoma HT-1080 nude mice was 71.33% in peptide 20mg/kg group. . Compared with the negative control group, both the cyclophosphamide group and the polypeptide group had extremely significant differences. However, cyclophosphamide was more toxic and the body weight decreased significantly; while the peptide had no significant effect on the body weight of nude mice. Therefore, the results of the polypeptide growth inhibition test on sarcoma HT-1080 xenografts showed that the 20 mg/kg peptide group had a significant inhibitory effect on the growth of HT-1080 xenografts. Compared with the cyclophosphamide group, there was no significant effect on the body weight of the experimental animals, and no obvious side effects were observed.

Claims

Claim WO 2013/097707 PCT/CN2012/087450

1. The use of the integrin blocker polypeptide AP25 for the preparation of a medicament for treating a tumor, wherein the sequence of the integrin blocker is Ala-Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys- Gly-Gly-Gly-Gly-Ile-Val-Arg-Arg-Ala-Asp-Arg-

Ala-Ala-Val-Pro, characterized in that the tumor originates from the human head and neck, brain, thyroid, pancreas, lung, liver, esophagus, stomach, breast, kidney, gallbladder, colon, rectum, ovary, Uterus, cervix, prostate, bladder, primary testis, secondary cancer, and sarcoma.