WO2013097709A1 - 整合素阻断剂多肽及其应用 - Google Patents

整合素阻断剂多肽及其应用 Download PDF

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WO2013097709A1
WO2013097709A1 PCT/CN2012/087465 CN2012087465W WO2013097709A1 WO 2013097709 A1 WO2013097709 A1 WO 2013097709A1 CN 2012087465 W CN2012087465 W CN 2012087465W WO 2013097709 A1 WO2013097709 A1 WO 2013097709A1
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tumor
peptide
gly
polypeptide
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PCT/CN2012/087465
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English (en)
French (fr)
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徐寒梅
浦春艳
康志安
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Xu Hanmei
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Priority to AU2012361902A priority Critical patent/AU2012361902B2/en
Priority to PL12863907T priority patent/PL2799445T3/pl
Priority to EP12863907.7A priority patent/EP2799445B1/en
Priority to KR1020187026109A priority patent/KR102003422B1/ko
Priority to US14/368,960 priority patent/US9458203B2/en
Priority to KR1020147021078A priority patent/KR101966762B1/ko
Publication of WO2013097709A1 publication Critical patent/WO2013097709A1/zh
Priority to US15/245,487 priority patent/US9879052B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the field of medicine, and in particular to an integrin blocker having an inhibitory angiogenesis, an integrin affinity and a binding ability, the blocker comprising two polypeptides, the integrin blocker polypeptide can be used for solid tumors and Treatment of rheumatoid arthritis.
  • tumor growth depends on angiogenesis.
  • Tumor angiogenesis is the morphological basis of tumor growth and metastasis. It not only provides nutrition to tumors, but also exports a large number of tumor cells to the host to cause tumor growth and metastasis.
  • 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, it is an important pathway for tumor metastasis. Therefore, inhibition of tumor angiogenesis is an important anticancer measure.
  • Integrins are a class of receptors that are widely distributed on the cell surface and mediate the adhesion of vascular endothelial cells and tumor cells. They participate in angiogenesis and tumor metastasis by linking the interaction between intracellular cytoskeletal proteins and extracellular matrix molecules. At present, at least eight integrins ⁇ 1 ⁇ 1, ⁇ 2 ⁇ 1, ⁇ 3 ⁇ 1, ⁇ 6 ⁇ 1, ⁇ 6 ⁇ 4, ⁇ 5 ⁇ 1, ⁇ 3, ⁇ 5) are involved in tumor angiogenesis, and avW plays an important role. avW is a transmembrane heterodimeric glycoprotein formed by the av subunit (CD51, 150 kD) and the 3 subunit (CD61, 105 kD), also known as the VN receptor.
  • ccvW recognizes the arg-gly-asp (RGD) sequence in the ligand molecule.
  • avW can be expressed in a variety of cell types and binds to multiple ligands during multicellular activity, participating in tumor angiogenesis, invasion and metastasis.
  • the polypeptide containing the RGD sequence has an integrin antagonist action, can reduce the expression of cell surface adhesion molecules, regulate intracellular signal transduction, inhibit tumor angiogenesis and inhibit tumor growth and metastasis. Therefore, integrin-targeted integrin blockers can block intracellular signaling downstream of integrin, effectively inhibit tumor angiogenesis, thereby inhibiting tumor growth and metastasis, and have broad applications in cancer therapy. prospect.
  • polypeptide sequence polypeptides and peptides III are integrin blockers with targeted binding and affinity, and have been found to have therapeutic effects on a variety of tumors, increasing the applicability, expanding their social and economic value. .
  • RA Rheumatoid arthritis
  • RA is one of the most common inflammatory joint diseases and major disabling factors in the clinic. In the world, about 0.5%-1.0%, the incidence of RA is about 0.4% in China. RA can occur at any age, and as the age increases, the incidence increases. The high age of women is 45-55 years old. Gender is closely related to the incidence of RA. The ratio of male to female is about 1:3.
  • RA is a chronic systemic inflammatory disease whose etiology is not known. It is a chronic clinical manifestation of chronic, symmetrical, multiple synovitis and extra-articular lesions. It is an autoimmune inflammatory disease.
  • vasculitis and synovitis turn off In the cerebral synovial vasculature, vasospasm is formed, resulting in thickening of the synovial membrane, increased exudation, secretion of various cytokines, invasion of cartilage and bone damage. It can also erode the muscle cavity, ligament, tendon sheath and muscles around it, which affects the stability of the joint, and is prone to joint malformation and dysfunction. Vasculitis can also invade all organs of the body and form systemic diseases.
  • Non-organic anti-inflammatory drugs usually called first-line drugs
  • first-line drugs have a wide variety of such drugs, and there are dozens of domestic markets.
  • second, steroid hormones, hormones are a very good analgesic and anti-inflammatory drugs, but long-term use alone can not improve the condition, but it brings many side effects, hormones as a transitional use before the second-line slow acting drug is effective, but the dosage should be Small, time should not be too long.
  • short-term treatment shocks and combined with second-line medication must be necessary.
  • slow-acting anti-rheumatic drugs commonly known as second-line drugs, so-called slow-acting drugs include antimalarials, gold salts, penicillamine and guanidinium amide, they have a slow onset of treatment, long-term effects on RA Relieving effect, it is also called disease improvement medicine.
  • immunosuppressive agents commonly used are methotrexate, cyclophosphamide, azathioprine, tripterygium, blue vine and so on.
  • Neovascularization is accompanied by synovial hyperplasia and inflammatory cell infiltration. It is the basis of vasospasm formation and joint destruction in RA.
  • the articular cartilage which should have no blood vessels, has formed a new blood vessel due to some abnormal changes, causing the cartilage to be eroded, causing joint deformation or pain.
  • Neovascularization causes abnormal changes in synovial tissue in patients with rheumatoid arthritis.
  • the normal synovial lining of normal people consists of only 1-2 layers of cells, while the synovial lining of RA patients usually has 4-10 layers of cells (sometimes even more than 20 floors ;).
  • RA synovium a large number of inflammatory cells infiltrating into the RA synovium, such as T cells, B cells, and monocytes.
  • Neovascularization under normal physiological conditions, is highly regulated and is an essential process in reproductive, embryonic development, tissue repair and wound healing.
  • Angiogenesis also occurs under a variety of pathological conditions including: tumor growth and metastasis; inflammatory disorders such as rheumatoid arthritis, psoriasis, osteoarthritis, inflammatory bowel disease, Crohn's disease, Ulcerative colons and other inflammatory disorders.
  • Integrin is ⁇ that recognizes the arg-gly-asp (ROD) in the ligand molecule and participates in tumor angiogenesis, invasion, metastasis, and binding to multiple ligands during multicellular activity. Physiological and pathological processes such as inflammation, wound healing and coagulation. Therefore, a polypeptide containing an RGD sequence has an integrin antagonist action, and the RGD sequence can be used as a carrier for targeted transport to the neovascular endothelium, thereby achieving a more efficient treatment for neovascular diseases.
  • ROD arg-gly-asp
  • the inhibition of angiogenesis by the vasopressor polypeptide not only prevents the delivery of oxygen and nutrients to the synovial membrane, but also directly causes the blood vessel to degenerate, thereby possibly inhibiting the synovial proliferation of the RA.
  • Inhibition of neovascularization is the key to the treatment of these diseases, and proliferation and migration of endothelial cells is a critical step in neovascularization.
  • Peptide II and Peptide III contain the RGD sequence (Arg-Gly-Asp), which allows the polypeptide sequence to efficiently bind to integrin and inhibit cell and extracellular by inhibiting the interaction between intracellular cytoskeletal proteins and extracellular matrix molecules.
  • the matrix and the adhesion of cells to cells inhibit the signal transduction between cells and extracellular mediators and between cells and cells, thereby inhibiting angiogenesis.
  • the sequence also contains a neonatal angiogenesis sequence, which has been found to have a high activity in inhibiting neovascularization, particularly for rheumatoid arthritis.
  • the targeting of vasopressor polypeptides to inhibit angiogenesis in the treatment of rheumatoid arthritis provides a new research direction for the development of new rheumatoid arthritis drugs.
  • the newly designed polypeptide sequence polypeptides and peptides III are peptides with targeted binding and affinity for integrins.
  • Previous studies have found that peptides and peptides III can inhibit endothelial cell proliferation, migration, and tubule formation, and use flow cytometry.
  • the target of peptide ⁇ and peptide III was analyzed as integrin ⁇ 3.
  • Later studies found that it can also inhibit the formation of the tubular structure of rat arterial rings, and confirmed by cell adhesion experiments that the target of peptide II and peptide III is integrin co ⁇ 3, and found that it has rheumatoid arthritis
  • the therapeutic effect increases the applicability and expands its social and economic value. Summary of the invention
  • the present invention further studies on polypeptide I, polypeptide II and polypeptide III, and finds that it has therapeutic effects on various solid tumors and rheumatoid arthritis, and increases its indications.
  • sequence of X is a deletion, Arg-Gly-Asp, Arg-Gly-Asp-Gly-Gly-Gly-Gly Ala-Cys-Asp-Cys-Arg-Gly-
  • the sequence of Y is deletion, Arg-Gly-Asp, Arg- Gly-Asp-Gly-Gly-Gly-Gly, Ala-Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys or Ala-Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe- Cys-Gly-Gly-Gly-Gly-Gly-Gly.
  • Peptide I Phe-Gln-Pro-Val-Leu-His-Leu-Val- Ala-Leu- Asn-Ser-Pro-Leu-Ser-Gly-Gly-
  • Peptide III Phe-Gln-Pro-Val-Leu-His-Leu-Val-Ala-Leu-Asn-Ser-Pro-Leu-Ser-Gly-Gly-Met-Arg-Gly-Ile-Arg-Gly-Ala -Asp-Phe-Gln-Cys-Phe-Gln-Gln-Ala-Arg-Ala-Val-Gly-Leu-Ala-Gly- m m, ⁇ , ⁇ , ⁇ , ⁇ , hu, ⁇ , now a, ⁇ , ⁇ » »i -mm 'm ⁇ Y-im ⁇ m ⁇
  • the adjuvant is attached, and the adjuvant is bovine serum albumin, human serum albumin or polyethylene glycol.
  • an integrin blocker according to claim 4 for the preparation of a medicament for the treatment of tumors, characterized in that said pharmaceutical composition can treat primary or secondary cancer, melanoma and sarcoma by various modes of administration, including subcutaneous Or intramuscular injection, intravenous or intravenous drip, oral administration such as pills, capsules, etc., nasal spray.
  • Peptide II and Peptide III are new amino acid sequences designed and synthesized by our laboratory. They have not been applied for invention patents at home and abroad.
  • the amino acid numbers of EDSM-1 and EDSM-2 reported in the previous patent ZL200610039298.2 are 47 and 55, respectively.
  • peptide II and peptide III added 4 Gly between the integrin ligand sequence RGD and the angiogenesis inhibitory sequence, which increased the flexibility based on EDSM-1, making the integrin ligand sequence RGD easier to interact with.
  • peptide I has the effect of inhibiting tumor angiogenesis and treating rheumatoid arthritis.
  • the arginine-glycine-aspartate (RGD) sequence is an important ligand for integrin. Therefore, the Gly-Gly-Gly-Gly-Arg-Gly-Asp polypeptide containing the RGD sequence can also specifically recognize and integrate Prime.
  • the integrin blocker polypeptide of the present invention is in the order of Phe-Gln-Pro-Val-Leu-His-Leu-Val-Ala-Leu-Asn-Ser-Pro-Leu-Ser-Gly- which inhibits angiogenesis.
  • the Gly-Gly-Gly-Gly-Gly-Arg-Gly-Asp sequence of (RGD) constructs polypeptides II and III which have affinity and binding ability to integrin.
  • the integrin blocker polypeptide II (Arg-Gly-Asp-Gly-Gly-Gly-Gly-Phe-Gln-Pro-Val-Leu-His-Leu-Val-Ala-Leu-Asn-Ser-Pro- Leu-Ser-Gly-Gly-Met-Arg-Gly-Ile-Arg-Gly-Ala-Asp-P he-Gln-Cys-Phe-Gln-Gln-Ala-Arg-Ala-Val-Gly-Leu-Ala -Gly-Thr-Phe-Arg-Ala) and peptide III
  • the RGD sequence in the molecule has integrin affinity and binding ability, and the research indicates that the target is mainly integrin ⁇ ⁇ 3, which has an integrin antagonist action and can reduce Expression of cell surface adhesion molecules regulates intracellular signal transduction.
  • the sequence contains a neovascularization inhibitory sequence, thereby inhibiting tumor neovascularization, thereby achieving the effect of inhibiting tumor growth and metastasis.
  • a neovascularization inhibitory sequence thereby inhibiting tumor neovascularization, thereby achieving the effect of inhibiting tumor growth and metastasis.
  • the inventors have learned through a large number of experiments that the target of the integrin blocker is clear, and can inhibit the migration, proliferation and tubular structure of human umbilical vein endothelial cells (HUVEC) in vitro, and inhibit the proliferation of some human tumor cells. effect. In vivo experiments have significant anti-tumor effects with small side effects, low dosage, and low cost.
  • the integrin blocker polypeptide designed by the invention is scientific, reasonable, feasible and effective, and can be used as a therapeutic drug for treating human solid tumors, greatly expanding the therapeutic spectrum of the integrin blocker, and providing a new drug development in the future.
  • the ideas and prospects have significant social and market value.
  • the polypeptide ⁇ and polypeptide III of the present invention contain a targeted RGD sequence, and the polypeptide can be targeted to the neovascular endothelium during vasospasm formation in RA, inhibiting neovascularization, and thereby preventing or treating rheumatoid arthritis. effect.
  • the inventors have learned through a large number of experiments that polypeptide II and peptide III can inhibit the development of adjuvant rheumatoid arthritis in rats and collagen-type rheumatoid arthritis in DBA/1 mice. In vivo experiments have demonstrated significant treatment for rheumatoid arthritis. The effect, and fewer side effects, the cost is reduced with less usage.
  • the polypeptide designed by the invention is scientific, reasonable, feasible and effective, and can be used as a medicine for treating or preventing rheumatoid arthritis, which greatly expands the scope of treatment of the polypeptide, and provides new ideas and prospects for drug development in the future, and has significant Social value and market value.
  • FIG. 1 Western blotting detection of integrin ⁇ and integrin ⁇ 3 expression in Bel-7402 cells
  • Figure 2 Flow cytometry assay for binding of integrin blocker polypeptide II to a target
  • Figure 3 Flow cytometry assay to detect binding of integrin blocker polypeptide III to a target
  • Example 1 Preparation and assay of integrin blocker polypeptide synthesis peptides
  • Polypeptides I, II and III were synthesized by solid phase synthesis, purified by high performance liquid chromatography, and the molecular weight of the polypeptide was determined by MS. The peptide was determined by RP-HPLC. Purity.
  • the polypeptide phase I solid phase synthesis method is based on Fmoc-Phe ( Otbu) -wang resin or Fmoc- Arg (Otbu)-CTC resin.
  • the solid phase synthesis of peptide II is Fmoc-Arg ( Otbu) -wang resin Or Fmoc- Arg (Otbu)-CTC resin as the starting material
  • the multi-month too III solid phase synthesis method is Fmoc-Phe ( Otbu ) -wang resin or Fmoc- Phe (Otbu)-CTC resin is the starting material, and then the dipeptide is sequentially connected to the forty-three/fifty-fifty peptide with the protective amino acid.
  • ⁇ / peptide III crude.
  • the crude product was dissolved, purified twice by preparative high-performance liquid phase, and finally concentrated and lyophilized to obtain a pure product. This method not only ensures the efficiency of synthesis but also improves the purity.
  • Peptide I Weigh the appropriate amount of Fmoc-Phe (Otbu)-wang resin or Fmoc-Arg (Otbu)-CTC resin, pour it into the glass sand core reaction column, and add CH 2 C1 2 to make the resin fully expand.
  • Peptide II Weigh the appropriate amount of Fmoc- Arg (Otbu)-wang resin or Fmoc- Arg (Otbu)-CTC resin, pour it into the glass sand core reaction column, and add CH 2 C1 2 to make the resin fully expand.
  • Peptide III Weigh the appropriate amount of Fmoc-Phe (Otbu)-wang resin or Fmoc-Phe (Otbu)-CTC resin, pour it into the glass sand core reaction column, and add CH 2 C1 2 to make the resin fully expand.
  • Uncapping Add the appropriate amount of the dehydrogenating solution of the hexahydropyridine/DMF. After the reaction for a period of time, drain the capping solution, wash it with DMF in the middle, and then add a proper amount of the capping solution to remove the Fmoc protecting group.
  • the dried resin was placed in a round bottom flask, and the cleaved peptide intermediate was sufficiently cleaved by adding a cutting solution, and the resin was separated from the polypeptide by a sand core funnel.
  • the post-treatment steps are as follows: First, add anhydrous ether to the cutting solution to precipitate the polypeptide, then centrifuge, pour off the supernatant, then wash the polypeptide with anhydrous ether and drain to obtain the crude peptide.
  • Preparation 1
  • One-time purification The column was prepared by rinsing with 30%-40% acetonitrile and 60%-70% buffer solution at a flow rate of 50 ml/min-100 ml/min for 10 min-20 min. Load with an infusion pump, collect a baseline, and collect a solution with an absorbance of more than 200 mv at an ultraviolet wavelength of 220 nm to check for samples. With a gradient elution, the initial percentage of acetonitrile is 30%-40%, and the percentage of acetonitrile after 80-50 min is 80%-90%.
  • a solution having an ultraviolet wavelength of 220 nm and an absorption value greater than 200 mV is collected, and a purity of more than 95% is detected as a peak top, which is to be subjected to secondary separation and purification.
  • 2 secondary purification will The peak received at one time was rotated to evaporate the organic solvent and then loaded with an infusion pump using 30%-40% acetonitrile and 60%-70% buffer solution. The flow rate was 50-100 ml/min, and a baseline was collected to collect a solution that absorbed more than 200 mv at an ultraviolet wavelength of 220 nm to detect whether a sample was flushed out.
  • the initial percentage of acetonitrile is 30%-40%, and the percentage of acetonitrile after 30-50 min is 80%-90%.
  • a solution that absorbs more than 200 mv at an ultraviolet wavelength of 220 nm is charged, and is tested as having a purity greater than 95%.
  • the purified product after lyophilization was collected, and the purity of the peptide was analyzed by reverse phase liquid phase analysis.
  • solid phase synthesis was successfully used to synthesize integrin blocker peptide I, peptide II and peptide III.
  • This method has high reproducibility, high operability and less pollution.
  • Two kinds of resins can be used to synthesize peptides: wang Resin or CTC resin; experimental wang resin, relatively stable compared to other resins, less side reactions, better peak product type, higher relative yield of purification, so the cost is relatively low; experimental CTC resin reaction is less affected by temperature, reaction The rate is fast; and the peptide is purified by reversed-phase high-performance liquid phase method.
  • the gradient elution is better than the isocratic elution.
  • the separation process is appropriate, the retention time is high, the production efficiency is high, and the purity is high.
  • the purity of III was 95.39%, 98.41%, and the purity of 96.40% was more than 95%, which met the design requirements.
  • the logarithmic growth phase cell Bel-7402 was digested with 0.25% trypsin, and the cells were collected by centrifugation at 800 rpm for 5 min; count, add 20 ⁇ l/1 ⁇ 10 5 cells to the protein extract, and blow up the cells, 4 ° Place C for 30 min to lyse the cells; add 1/4 volume of 5 ⁇ protein loading buffer and boil for 5-10 min.
  • Cell Bel-7402 was cultured in a cell bottle to 80% confluence, digested and collected, washed twice with ice-cold PBS, and resuspended in PBS containing 1% BSA for 30 min; with 2 ⁇ murine anti-human ⁇ 3 function Blocking monoclonal antibodies ( ⁇ . ⁇ ⁇ ⁇ / ⁇ 1, 1 :200) and 2 ⁇ murine anti-human ⁇ 5 ⁇ 1 function blocking monoclonal antibodies (1.0 ⁇ 8/ ⁇ 1, 1 :200) with cell suspension at 4 ° C Incubate for 1.5 h ; collect the cells and wash them twice with ice-cold PBS, then incubate with 100 l FITC-labeled modified peptide II and peptide 111 (2 mg/ml) with the cell suspension for 1.5 h at 4 °C; The cells were then collected and washed twice with ice-cold PBS, then resuspended in 400 l PBS, analyzed by flow cytometry, and
  • RESULTS The target of cell expression, integrin ⁇ 3, was determined as shown in Fig. 1.
  • FITC-labeled integrin blocker polypeptide II is incubated in cells without antibody incubation. 80.4%, the fluorescence intensity decreased to 13.4% after addition of the integrin ⁇ 3 antibody, and the fluorescence intensity decreased to 33.2% after the addition of the integrin ⁇ 5 ⁇ 1 antibody; the FITC-labeled integrin blocker polypeptide III was incubated in the cells without antibody addition. The fluorescence intensity was approximately 82.4.
  • HUVEC cells cultured in logarithmic growth phase were digested with trypsin digest, collected, washed twice with PBS and resuspended in blank HUVECs-specific medium. The cells were counted under a microscope and the cell concentration was adjusted to lx10 5 /ml.
  • the test solutions of each group were prepared and diluted to 100 ⁇ with a blank HUVECs-specific medium. The cells were seeded into a transwell chamber, each well ⁇ ⁇ , and each set of test solution was added to the chamber.
  • Integrin blocker peptide II The effect of integrin blocker peptide II on HUVEC cell migration assay is shown in Table 2. Integrin blocker peptide ⁇ inhibited migration of 5% fetal bovine serum and 1% ECGS-induced HUVEC compared to the negative control. And it showed a dose-dependent effect at low and medium doses. At high, low, and low doses, the inhibition rate of peptide migration on cell migration was significantly different from that of the negative control (** ⁇ 0.01), and the ability to inhibit endothelial cell migration at high doses decreased compared with the medium dose. .
  • the inhibition rates were above 50% at doses of 4 g/ml, 8 g/ml, and 16 g/ml, which were 54.10%, 69.87%, 60.27%, and 57.24%, respectively.
  • the inhibition rate of cell migration reached a maximum of 69.87%.
  • integrin blocker peptide III The effect of integrin blocker peptide III on HUVEC cell proliferation assay is shown in Table 3. Compared to the negative control, integrin blocker peptide III inhibited migration of 5% fetal bovine serum and 1% ECGS-induced HUVEC. At a moderate to low dose, it showed a dose-dependent effect. At high, low and low doses, the ability to inhibit endothelial cell migration at high doses decreased compared with the medium dose. The inhibition rate of peptide III on cell migration at each dose was significantly different from that of the negative control (* ⁇ 0.01), and the inhibition rate was reached at 1 g/ml, 2 ⁇ ⁇ /ml, 4 g/ml. More than 50%, respectively, were 52.04%, 76.78% and 62.64%. When the dose of polypeptide polypeptide III was 2 g/ml, the inhibition rate of cell migration reached a maximum of 76.78%.
  • integrin blocker polypeptide on human umbilical vein endothelial cells 10 mg/ml Matrigel (BD, USA) frozen at -20 °C was placed in a 4 refrigerator overnight, diluted 1:1 with HUVEC medium, and coated at 30 ⁇ in 96-well plates (Greiner, USA) ), polymerization in a 37 ° C incubator for 1 h.
  • HUVEC cells cultured to logarithmic growth phase were digested with 0.2% EDTA, collected, washed twice with PBS and resuspended in blank HUVEC medium. The cells were counted under a microscope, and the cell concentration was adjusted to lxlO 5 /ml.
  • test solutions of each group were prepared and diluted to 100 ⁇ l with a blank HUVECs-specific medium.
  • the cells were seeded in 96-well plates at 100 ⁇ l per well, and each set of test solutions was added to the air and incubated at 5% CO 2 at 37 °C.
  • 5 fields were randomly selected for each dose and counted.
  • the number of differentiated tubes at each concentration was counted and analyzed for peptide II and peptide III to HUVEC. The effect of cell differentiation into tube capacity. Calculate the tubelet inhibition rate according to the formula:
  • N test is the number of small tubes in the test group
  • ⁇ is the number of small tubes in the blank control group.
  • peptide II inhibited the differentiation of HUVECs into small tubes at different doses, and reached more than 50% at medium and low doses, and the dose and inhibition rate were A certain dose dependency.
  • the peptide dose was 2 ⁇ ⁇ / ⁇ 1, 4 ⁇ ⁇ / ⁇ 8 g/ml, the number of tubules generated was the least and the inhibition rate was the highest.
  • the polypeptide II doses of 2 g/ml, 4 g/ml, and 8 ⁇ /! ⁇ were 74.65%, 78.87%, and 71.83%, respectively.
  • the peptide II dose was 2 g/ml, 4 g/
  • the inhibition rates of ml and 8 g/ml reached 80.77%, 81.68% and 76.19%, respectively.
  • Peptide II and endothelial cells were significantly different from the negative group at doses of 0.5 g/ml, lg/ml, 2 g/ml, 4 g/ml, and 8 g/ml for 6 h.
  • the effect of peptide III on the differentiation of HUVECs is shown in Table 5.
  • the dendritic capillary-like tubular structure was differentiated within 6 h to 60 h.
  • the tubules were formed at 6 h, and the number of tubules was the highest at 6 h and 12 h. Reduced, basically disappeared by 60 h.
  • the number of tubules formed at each dose of peptide III was less than that of negative. It can be seen that peptide III inhibited the differentiation of HUVECs into small tubes at different doses, and reached more than 60% at medium and low doses, and the dose and inhibition rate were A certain dose dependency.
  • Tumor tissues from growing period milled under sterile conditions to prepare a lx 10 7 cells / ml cell suspension to 0.1 ml was inoculated subcutaneously in the right axilla of mice.
  • the transplanted tumors were used to measure the diameter of the transplanted tumors.
  • the animals were randomly divided into groups.
  • the inhibitory effect of the integrin blocker polypeptide on the tumor of the test animal was dynamically observed using a method for measuring the tumor diameter.
  • the number of measurements of the diameter of the tumor is once every 2 days, and the weight of the mouse is also weighed for each measurement.
  • the polypeptide was injected subcutaneously into the left ankle, and the negative group was given the same amount of normal saline for 14 days.
  • the cyclophosphamide was administered subcutaneously every other day, purple.
  • the cedar group was administered subcutaneously every 3 days, and the low dose of the polypeptide was administered twice a day in two groups, and the other groups were administered once a day.
  • the mice were sacrificed and the tumor pieces were surgically removed for weighing.
  • the formula for calculating the tumor volume (TV) is:
  • RTV V t /V Q .
  • V Q is the tumor volume measured at the time of sub-cage administration (i.e., d Q )
  • V t the tumor volume at each measurement.
  • the evaluation index of antitumor activity is the relative tumor growth rate T/C (%), and the calculation formula is as follows:
  • TRTV treatment group RTV
  • C RTV negative control group RTV
  • Peptide II is 0.75 15.3 ⁇ 0. .9 8 22.4 ⁇ 1 .6 7 1.79 ⁇ 1.03** 55. .22% Dosing twice a day
  • the inhibitory effect of peptide I on the growth of melanoma B16F10 C57BL/6 black mice was shown in Table 6.
  • the paclitaxel group 10 mg/kg had a tumor inhibition rate of 70.80% for melanoma B16F10 C57BL/6 black mice. It has a significant effect on the body weight of the experimental animals, and the body weight of the animals in the negative group and the polypeptide-administered group is lighter, and the side effects are more obvious; the high-, low-dose and low-dose of the polypeptide I are administered twice a day to the melanoma B16F10 C57BL/6.
  • the tumor inhibition rate of the transplanted tumor of the black mouse was 37.26%, 32.17%.
  • the inhibitory effect of peptide II on tumor growth of melanoma B16F10 C57BL/6 black mice is shown in Table 7.
  • the inhibition rate of melanoma B16F10 C57BL/6 black mice transplanted tumor was 75.49%, but the experiment was performed.
  • the body weight of the animals has a significant effect, compared with the body weight of the negative group and the polypeptide-administered group, the toxic side effects are more obvious; the anti-tumor rate of the melanoma B16F10 C57BL/6 black mouse transplanted tumor is 15 mg/kg in the cyclophosphamide group. It was 72.89% but had a significant effect on the body weight of the experimental animals.
  • the body weight was lighter and the side effects were more obvious.
  • the peptides were high, medium, low dose and low dose twice a day.
  • the tumor inhibition rate of melanoma B16F 10 C57BL/6 black mouse transplanted tumor was 40.80%, 34.17%, 59.06%, 55.22%.
  • the negative control group there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the inhibitory effect of peptide III on tumor growth of melanoma B16F10 C57BL/6 black mice was shown in Table 8.
  • the high, medium and low doses of peptide III and low doses were administered twice a day to melanoma B16F10 C57BL/6 black mice transplanted tumors.
  • the tumor inhibition rate was 63.07%, 59.82%, 68.67%, and 69.21%.
  • the tumor volume in the middle-dose group, the low-dose group, and the low-dose two-dose group was significantly different from the negative group tumor volume.
  • the peptide ⁇ 0.09375 mg/kg was administered twice a day to the melanoma B16F10 C57BL/6 black mice had the best inhibitory effect on the growth of transplanted tumors; compared with the negative control group, there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • Tumor tissues from growing period milled under sterile conditions to prepare a lx 10 7 cells / ml cell suspension to 0.1 ml was inoculated subcutaneously in the right axilla of mice.
  • the transplanted tumors were measured with a vernier caliper and the animals were randomly divided into groups after the tumors were grown to 100-200 mm 3 .
  • the inhibitory effect of the integrin blocker polypeptide on the tumor of the test animal was dynamically observed using a method for measuring the tumor diameter.
  • the number of measurements of tumor diameter is once every 2 days, and the number of starting animals is also measured every time while weighing.
  • the peptide was injected into the tail vein, and the negative group was given the same amount of normal saline for 14 days.
  • the positive group was Avastin.
  • Avastin was administered once every 3 days in the tail vein, and the other group was administered once a day in the tail vein.
  • One week after the administration the mice were sacrificed 21 days later, and the tumor pieces were surgically removed and weighed.
  • the formula for calculating tumor volume (TV) is:
  • RTV V t /V Q .
  • V Q is the tumor volume measured at the time of sub-cage administration (i.e., d Q )
  • V t the tumor volume at each measurement.
  • the evaluation index of antitumor activity is the relative tumor growth rate T/C (%), and the calculation formula is as follows:
  • TRTV treatment group RTV
  • C RTV negative control group RTV
  • Peptide II high 3 20.38 ⁇ 0.39 8 23.13 ⁇ 0.67 8 0.43 ⁇ 0.12* 63.80% Peptone 1.5 1.5 1.16 ⁇ 0.45 8 22.86 ⁇ 0.65 8 0.38 ⁇ 0.12** 67.82%
  • Peptide II is 0.75 20.31 ⁇ 0.50 8 23.03 ⁇ 0.65 8 0.50 ⁇ 0.17* 57.83%
  • Table 11 Inhibition of polypeptide III on xenograft tumor growth in human breast cancer MDA-MB-231 nude mice
  • Avastin group, human breast cancer MDA-MB-231 The tumor inhibition rate of nude mice xenograft tumors was 78.53%, which had no significant effect on the body weight of experimental animals.
  • the high, medium and low doses of peptide I inhibited the tumor xenograft of human breast cancer MDA-MB-231 nude mice.
  • the rate was 52.95%, 58.82%, and 51.26%.
  • There was a significant difference between the low dose and the negative group and the tumor volume in the middle dose group was significantly different from the tumor volume in the negative group.
  • the negative control group there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the inhibitory effect of peptide ⁇ on the growth of human breast cancer MDA-MB-231 nude mice xenograft tumors is shown in Table 10.
  • Avastin group the tumor inhibition rate of human breast cancer MDA-MB-231 nude mice xenograft tumors 78.53%, no significant effect on the body weight of experimental animals; high, medium and low doses of peptide II inhibited human breast cancer MDA-MB-231 xenograft tumor in nude mice
  • the tumor rate was 63.80%, 67.82%, and 57.83%.
  • the tumor volume in the middle-dose group was significantly different from that in the negative group. Compared with the negative control group, there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the inhibitory effect of peptide III on the growth of human breast cancer MDA-MB-231 nude mice xenograft tumors is shown in Table 11.
  • Avastin group the tumor inhibition rate of human breast cancer MDA-MB-231 nude mice xenograft tumors 68.95% had no significant effect on the body weight of experimental animals; the tumor inhibition rate of human breast cancer MDA-MB-231 nude mice xenograft tumors was high, medium and low doses of 58.82%, 72.86%, 61.91%.
  • the tumor volume in the middle-dose group was significantly different from that in the negative group. Compared with the negative control group, there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the positive control drug oxaliplatin was administered once every 4 days in the tail vein, and the sirroin was administered once every two days by the intragastric administration.
  • the other group was administered once per day in the tail vein.
  • Multi-month too II high 3 19.95 ⁇ 1.21 10 19.60 ⁇ 1.26 10 0.48 ⁇ 0.13 38.28%
  • the polypeptide ⁇ is 1.5 20.15 ⁇ 0.97 10 19.67 ⁇ 1.41 10 0.16 ⁇ 0.10** 79.72%
  • Table 14 polypeptide III is small for human gastric cancer MGC-803 Inhibition of murine xenograft tumor growth group dose starting weight (g) terminal body weight (g) tumor weight (g) tumor inhibition rate
  • the tumor inhibition rate of human gastric cancer MGC-803 nude mice xenograft tumor was 38.28%, 38.46%, 35.90%. Compared with the negative control group, there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the inhibitory effect of peptide ⁇ on the growth of human gastric cancer MGC-803 nude mice xenograft tumors is shown in Table 13.
  • the combination of chemical drugs and drug groups has a tumor inhibition rate of 51.76% for human gastric cancer MGC-803 nude mice xenograft tumors, but The weight of the experimental animals had a significant effect, compared with the body weight of the negative group and the polypeptide-administered group, and the side effects were more obvious; the high, medium and low dose groups of the polypeptide II inhibited the xenograft tumor of human gastric cancer MGC-803 nude mice.
  • the tumor rate was 38.28%, 79.72%, and 70.67%.
  • the inhibitory effect of peptide III on the growth of human gastric cancer MGC-803 nude mice xenograft tumors is shown in Table 14.
  • the combination of chemical drugs and drug groups has a tumor inhibition rate of 51.76% for human gastric cancer MGC-803 nude mice xenograft tumors, but The body weight of the experimental animals had a significant effect, compared with the body weight of the negative group and the polypeptide-administered group, and the side effects were more obvious; the high, medium and low dose groups of the polypeptide III inhibited the xenograft tumor of human gastric cancer MGC-803 nude mice.
  • the tumor rate was 57.65%, 66.03%, and 70.08%.
  • Paclitaxel 10 20.13 ⁇ 0.57 8 17.80 ⁇ 0.63 7 0.28 ⁇ 0.07** 68.21% Peptide I high 3 19.94 ⁇ 0.65 8 22.96 ⁇ 0.69 8 0.47 ⁇ 0.17* 46.60% Number of starting animals Number of starting animals
  • polypeptide II on human lung cancer H460 nude mice xenograft Inhibition of tumor growth, group dose, initial body weight, terminal body weight, tumor weight (g), tumor inhibition rate
  • Peptide I is naked to human lung cancer H460
  • the inhibitory effect of mouse xenograft tumor growth is shown in Table 15, the chemical drug paclitaxel
  • the tumor inhibition rate of human lung cancer H460 nude mice xenograft tumor was 68.21%, but had significant effect on the body weight of the experimental animals.
  • the body weight was lighter and the side effects were more obvious.
  • the inhibition rate of I high-, medium-, and low-dose groups in human lung cancer H460 nude mice xenograft tumors was 46.60%, 54.54%, 42.05%.
  • the tumor volume in the middle-dose group was significantly different from that in the negative group.
  • the negative control group there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the inhibitory effect of peptide II on the growth of human lung cancer H460 nude mice xenograft tumors is shown in Table 16.
  • the chemotherapy drug paclitaxel group has a tumor inhibition rate of 68.21% in human lung cancer H460 nude mice xenograft tumors, but the body weight of experimental animals has Significantly, the body weight of the animals in the negative group and the peptide administration group was lighter, and the side effects were more obvious; the number of human lung starting animals in the high, medium and low dose groups of peptide II
  • the tumor inhibition rate of xenografted tumors of H460 nude mice was 57.68%, 65.37%, and 53.49%. There was a significant difference between the high-dose group and the low-dose group and the negative-sex group. The tumor volume in the middle-dose group was significantly different from that in the negative group. Compared with the negative control group, there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the inhibitory effect of peptide III on the growth of human lung cancer H460 nude mice xenograft tumors is shown in Table 17.
  • the inhibition rate of human lung cancer H460 nude mice xenograft tumors was 68.21%, but the final number of animals was tested.
  • the body weight of the animals had a significant effect, compared with the body weight of the negative group and the polypeptide-administered group, and the side effects were more obvious.
  • the tumor inhibition rate of the high, medium and low dose groups of polypeptide III on human lung cancer H460 nude mice xenograft tumors 68.05%, 74.42%, 69.23%. There were significant differences between the high-dose, middle-dose, low-dose, and negative groups. Compared with the negative control group, there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • Example 6 For the tumor inoculation and test evaluation methods, see Example 6.
  • the positive control drug paclitaxel group was administered once every 3 days, and the other groups were administered once per night.
  • Negative control one 19.83 ⁇ 0.61 12 23.26 ⁇ 0.55 12 1.27 ⁇ 0.23
  • Peptide II high 3 19.87 ⁇ 0.66 8 22.90 ⁇ 0.59 8 0.39 ⁇ 0.05** 68.75% number of starting animals
  • polypeptide 1.5 1.5 20.15 ⁇ 0.56 8 22.93 ⁇ 0.60 8 0.31 ⁇ 0.07** 75.12% polypeptide II low 0.75 20.10 ⁇ 0.39 8 23.21 ⁇ 0.60 8 0.36 ⁇ 0.05** 71.54%
  • the inhibition rate of human hepatocellular carcinoma SMMC-7721 nude mice xenograft tumors in the drug paclitaxel group It was 78.10%, but had a significant effect on the body weight of the experimental animals. Compared with the negative group and the polypeptide-administered group, the body weight was lighter and the side effects were more obvious.
  • the high, medium and low dose groups of the polypeptide I were naked to the human liver cancer SMMC-7721.
  • the tumor inhibition rate of murine xenograft tumors was 56.70%, 63.00%, and 58.26%. There was a significant difference between the middle dose group and the negative group, and there was a significant difference between the high dose group and the low dose group. Compared with the negative control group, there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the inhibitory effect of peptide II on the growth of human liver cancer SMMC-7721 nude mice xenograft tumors is shown in Table 19.
  • the inhibition rate of human hepatocellular carcinoma SMMC-7721 nude mice xenograft tumor was 78.10%, but the experiment was performed.
  • the body weight of the animals has a significant effect, compared with the body weight of the negative group and the polypeptide-administered group, the toxic side effects are more obvious; the high, medium and low doses of the polypeptide ⁇ inhibit the tumor of human liver cancer SMMC-7721 nude mice xenograft tumor
  • the rate is 68.75%, 75.12%, and 71.54%. There were significant differences between the high-dose, middle-dose, low-dose, and negative groups. Compared to the negative control group, the body of the experimental animal There was no effect on the weight, and no obvious side effects were observed.
  • the inhibitory effect of peptide III on the growth of xenograft tumor of human liver cancer SMMC-7721 nude mice is shown in Table 20.
  • the inhibition rate of human hepatocellular carcinoma SMMC-7721 nude mice xenograft tumor was 78.10%, but the experiment was performed.
  • the body weight of the animals has a significant effect, compared with the body weight of the negative group and the polypeptide-administered group, the toxic side effects are more obvious; the high, medium and low dose groups of the polypeptide III inhibit the tumor of the human liver cancer SMMC-7721 nude mouse xenograft tumor
  • the rate is 77.55%, 82.55%, and 71.85%.
  • Compared with the negative control group there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • Example 6 For the tumor inoculation and test evaluation methods, see Example 6.
  • the positive control drug cisplatin group was administered once every 3 days, and the other groups were administered once per night.
  • Negative control 20.05 ⁇ 0.58 12 23.12 ⁇ 0.57 12 1.40 ⁇ 0.37 cisplatin 10 20.16 ⁇ 0.54 8 17.28 ⁇ 0.56 7 0.38 ⁇ 0.10** 73.13% ⁇ 2.5 19.84 ⁇ 0.55 8 23.14 ⁇ 0.54 8 0.80 ⁇ 0.20 43.08% Polypeptide III height 0.75 19.76 ⁇ 0.43 8 22.88 ⁇ 0.66 8 0.50 ⁇ 0.15** 64.35% Polypeptide III 0.375 20.19 ⁇ 0.60 8 23.17 ⁇ 0.58 8 0.39 ⁇ 0.08** 72.41% Number of starting animals
  • Peptide III was 0.1875 19.98 ⁇ 0.65 8 23.10 ⁇ 0.56 8 0.44 ⁇ 0.11** 68.68% Results: The inhibitory effect of peptide I on the growth of human cervical cancer HeLa nude mice xenograft tumors is shown in Table 21. Chemical cisplatin group, for human The tumor inhibition rate of cervical cancer HeLa nude mice xenograft tumor was 73.13%, but it had a significant effect on the body weight of the experimental animals.
  • the body weight was lighter and the side effects were more obvious;
  • the tumor inhibition rate of the middle and low dose groups on human cervical cancer HeLa nude mice xenograft tumors was 62.85%, and the final number of animals was 70.71%, 65.00%.
  • the tumor volume in the high-dose, medium-dose, and low-dose groups was significantly different from that in the negative group.
  • the negative control group there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the inhibitory effect of peptide II on the growth of human cervical cancer HeLa nude mice xenograft tumors is shown in Table 22.
  • the inhibition rate of human cisplatin group on human cervical cancer HeLa nude mice xenograft tumor was 73.13%, but for experimental animals.
  • the body weight has a significant effect, compared with the body weight of the negative group and the polypeptide-administered group, the toxic side effects are more obvious; the high, medium and low dose groups of the polypeptide II have the tumor inhibition rate of human cervical cancer HeLa nude mice xenograft tumor 55.82%, 68.19%, 64.45%.
  • the tumor volume in the high-dose, medium-dose, and low-dose groups was significantly different from that in the negative group. Compared with the negative control group, there was no effect on the body weight of the test animals, and no obvious side effects were observed.
  • the inhibitory effect of peptide III on the growth of human cervical cancer HeLa nude mice xenograft tumors is shown in Table 23.
  • the inhibition rate of human cisplatin group on human cervical cancer HeLa nude mice xenograft tumor was 73.13%, but for experimental animals.
  • the body weight has a significant effect, compared with the body weight of the negative group and the polypeptide-administered group, the toxic side effects are more obvious; the high, medium and low dose groups of the polypeptide III have the tumor inhibition rate of human cervical cancer HeLa nude mice xenograft tumor 64.35%, 72.41%, 68.68%.
  • the tumor volume in the high-dose, medium-dose, and low-dose groups was significantly different from that in the negative group. Compared with the negative control group, there was no effect on the body weight of the test animals, and no obvious side effects were observed.
  • Example 6 For the tumor inoculation and test evaluation methods, see Example 6.
  • the positive control drug paclitaxel group was administered once every 3 days in the tail vein. His group was administered once a day in the tail vein.
  • Peptide I high 3 19.82 ⁇ 0.60 8 23.14 ⁇ 0.48 8 0.60 ⁇ 0.14 47.36% Number of starting animals Number of starting animals
  • polypeptide I 1.5 19.96 ⁇ 0.58 8 22.93 ⁇ 0.56 8 0.51 ⁇ 0.10* 55.30% polypeptide I was 0.75 19.87 ⁇ 0.58 8 22.81 ⁇ 0.60 8 0.52 ⁇ 0.12* 54.39% Table 25 Peptide II on human endometrial cancer HHUA nude mice Inhibition of xenograft tumor growth
  • Paclitaxel 10 20. .94 ⁇ 0. .58 8 18. .88 ⁇ 0.68 7 0.23 ⁇ 0.07** 79. .82% Enrity 2.5 19. .88 ⁇ 0. .79 8 22. .89 ⁇ 0.51 8 0.65 ⁇ 0.15 43. .00% Peptide III height 0.75 19. .89 ⁇ 0. .53 8 23. .07 ⁇ 0.56 8 0.38 ⁇ 0.10** 66. .67% Peptide III 0.375 20. .31 ⁇ 0. 51 8 23.18 ⁇ 0.57 8 0.30 ⁇ 0.06** 73. .21% Peptide III is 0.1875 19.74 ⁇ 0. .55 8 22.92 ⁇ 0.57 8 0.33 ⁇ 0.07** 70.
  • the inhibitory effect of peptide I on the growth of human endometrial carcinoma HHUA nude mice xenograft tumors is shown in Table 24, Chemicals In the paclitaxel group, the tumor inhibition rate of human endometrial cancer HHUA nude mice xenograft tumors was 79.82%, but it had a significant effect on the body weight of the experimental animals, and the body weight of the animals in the negative group and the polypeptide administration group was lighter.
  • the tumor inhibitory rate of high, medium and low doses of peptide I on human endometrial cancer HHUA nude mice xenograft tumors was 47.36%, 55.30%, 54.39%. There was a significant difference between the tumor volume in the middle dose group and the low dose group compared with the tumor volume in the negative group. Compared with the negative control group, there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the inhibitory effect of peptide II on the growth of human endometrial carcinoma HHUA nude mice xenograft tumors is shown in Table 25.
  • the tumor inhibition rate of human endometrial cancer HHUA nude mice xenograft tumors was 79.82%, but It has a significant effect on the body weight of the experimental animals, and the body weight of the animals in the negative group and the polypeptide-administered group is lighter, and the side effects are more obvious; the number of high, medium and starting animals of the polypeptide II
  • the inhibition rate of low-dose group on human endometrial cancer HHUA nude mouse xenograft tumor was 59.65%, 67.54%, 64.03%. There was a significant difference in tumor volume between the high-dose group, the middle-dose group, and the low-dose group compared with the negative group. Compared with the negative control group, there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the tumor inhibition rate of human endometrial cancer HHUA nude mouse xenograft tumor was 73.13%, but it had a significant effect on the body weight of the experimental animals, compared with the body weight of the negative group and the polypeptide-administered group.
  • the high, medium and low dose groups of peptide III inhibited the tumor xenograft tumor of human endometrial cancer HHUA nude mice by 66.67%, 73.21%, 70.53%.
  • the negative control group there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • Example 6 For the tumor inoculation and test evaluation methods, see Example 6.
  • the positive control drug cisplatin group was administered once every 3 days, and the other groups were administered once per night.
  • Negative control one 19.77 ⁇ 0.61 12 23.04 ⁇ 0.61 12 1.43 ⁇ 0.34
  • polypeptide 1.5 1.5 20.08 ⁇ 0.46 8 23.04 ⁇ 0.48 8 0.46 ⁇ 0.10** 67.83%
  • Polypeptide II is 0.75 20.31 ⁇ 0.60 8 23.10 ⁇ 0.48 8 0.53 ⁇ 0.12** 62.92%
  • Table 29 Peptide III is naked to human prostate cancer DU-145 Inhibition of mouse xenograft tumor growth, initial body weight (g), terminal body weight, tumor weight (g), tumor inhibition rate, end animal count
  • the inhibitory effect of peptide ⁇ on the growth of human prostate cancer DU-145 nude mice xenograft tumors is shown in Table 28.
  • the inhibition rate of human cisplatin group to human prostate cancer DU-145 nude mice xenograft tumor was 73.84%. However, it has a significant effect on the body weight of the experimental animals, and the body weight of the animals in the negative group and the polypeptide-administered group is lighter, and the side effects are more obvious; the high, medium and low dose groups of the polypeptide II are human xenografts of human prostate cancer DU-145 nude mice.
  • the tumor inhibition rate was 54.25%, 67.83%, and 62.92%.
  • the inhibitory effect of peptide III on the growth of human prostate cancer DU-145 nude mice xenograft tumors is shown in Table 29.
  • the inhibition rate of human prostate cancer cisplatin group to human prostate cancer DU-145 nude mice xenograft tumor was 73.84%. However, it has a significant effect on the body weight of the experimental animals, and the body weight of the animals in the negative group and the polypeptide-administered group is lighter, and the side effects are more obvious; the high, medium and low dose groups of the polypeptide III are human xenografts of human prostate cancer DU-145 nude mice.
  • the tumor inhibition rate was 60.73%, 69.90%, and 64.08%.
  • Integrin Blocker Polypeptide I, Polypeptide II and Peptide III for Human Testicular Cancer 5637 Nude Mouse Xenograft Tumor Growth Inhibition Test For tumor inoculation and test evaluation methods, see Example 6.
  • the positive control drug 5-fluorouracil group was administered once every 3 days, and the other group was administered once per night.
  • polypeptide III on human testicular cancer 5637 nude mice Xenograft tumor growth inhibitors starting weight (g) terminal weight tumor weight (g) tumor inhibition rate
  • the tumor inhibition rate of human testicular cancer 5637 nude mice xenograft tumor was 77.80%, but it had a significant effect on the body weight of the experimental animals, compared with the weight of the negative group and the polypeptide-administered group.
  • the toxic side effects were more obvious; the inhibition rate of the high, medium and low doses of peptide I on human testicular cancer 5637 nude mice xenograft tumors was 54.76%, 63.49%, 56.34%.
  • the tumor volume in the high-dose group and the low-dose group was significantly different from that in the negative group.
  • the tumor volume in the middle-dose group was significantly different from that in the negative group. Compared with the negative control group, there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the inhibitory effect of peptide II on the growth of human testicular carcinoma 5637 nude mice xenograft tumors is shown in Table 31.
  • the 5-Fu group of human testicular cancer 5637 nude mice xenograft tumors showed 77.80% inhibition rate, but the experiment
  • the body weight of the animals has a significant effect, compared with the body weight of the negative group and the polypeptide-administered group, the toxic side effects are more obvious;
  • the tumor suppressor rate of the high-, medium-, and low-dose peptide II on human testicular cancer 5637 nude mice xenograft tumor It is 52.49%, 64.32%, 60.96%.
  • the inhibitory effect of peptide III on the growth of human testicular carcinoma 5637 nude mice xenograft tumors is shown in Table 32.
  • the inhibitory rate of xenograft tumors in human testicular cancer 5637 nude mice was 73.84%, but the experiment was performed.
  • the body weight of the animals had a significant effect, compared with the body weight of the negative group and the polypeptide-administered group, and the side effects were more obvious.
  • the tumor inhibition rate of the high-, medium-, and low-dose group of peptide III on human testicular cancer 5637 nude mice xenograft tumors It is 60.51%, 65.11%, and 62.63%.
  • the animal's weight was not affected, and no obvious side effects were observed.
  • Example 6 For the tumor inoculation and test evaluation methods, see Example 6.
  • the positive control drug Avastin was administered once every 2 days in the tail vein, and the other groups were administered once per day in the tail vein.
  • Peptide II is 0.75 20.01 ⁇ 0.51 8 22.76 ⁇ 0.51 8 0.46 ⁇ 0.11* 62.00%
  • Table 35 Inhibition of polypeptide III on xenograft tumor growth in human gallbladder carcinoma GBC-SD nude mice
  • the tumor inhibition rate of transplanted tumors was 58.33%, 65.00%, and 56.67%.
  • the tumor volume in the high-dose group and the low-dose group was significantly different from that in the negative group.
  • the tumor volume in the middle-dose group was significantly different from that in the negative group. Compared with the negative control group, there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the inhibitory effect of peptide II on the growth of human gallbladder carcinoma GBC-SD nude mice xenograft tumors is shown in Table 34.
  • the tumor inhibition rate of human gallbladder carcinoma GBC-SD nude mice xenograft tumors was 78.85%, Compared with the negative control group, there was no effect on the body weight of the experimental animals; the inhibition rate of the high, medium and low doses of the polypeptide II on the human gallbladder carcinoma GBC-SD nude mice xenograft tumor was 60.48%, 69.19%, 62.00%.
  • the tumor volume in the high-dose group and the low-dose group was significantly different from that in the negative group.
  • the tumor volume in the middle dose group was significantly different from the tumor volume in the negative group. Compared with the negative control group, there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the inhibitory effect of peptide III on the growth of human gallbladder carcinoma GBC-SD nude mice xenograft tumors is shown in Table 35.
  • the tumor inhibition rate of human gallbladder carcinoma GBC-SD nude mice xenograft tumors was 76.79%.
  • the high, medium and low dose groups of the polypeptide III had a tumor inhibition rate of 63.73%, 74.62%, and 64.55% in the human gallbladder carcinoma GBC-SD nude mice xenograft tumor.
  • the tumor volume in the middle-dose group was significantly different from that in the negative group.
  • the negative control group there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • Avastin 10 19.83 ⁇ 0.58 8 20.94 ⁇ 0.47 0.24 ⁇ 0.06** 79.60% Entitude 2.5 20.15 ⁇ 0.52 8 22.99 ⁇ 0.62 0.73 ⁇ 0.16 42.06%
  • Peptide I high 3 19.84 ⁇ 0.55 8 23.14 ⁇ 0.54 0.64 ⁇ 0.13 49.20%
  • Peptide I 19.1.5 19.76 ⁇ 0.43 8 22.88 ⁇ 0.66 0.55 ⁇ 0.09* 56.34%
  • Peptide I is 0.75 20.19 ⁇ 0.60 8 23.17 ⁇ 0.58 0.59 ⁇ 0.16 53.17%
  • Peptide II inhibits the growth of human bladder cancer HT1376 nude mice xenograft tumor effect
  • Peptide II is low 0.75 20.00 ⁇ 0.67 8 22.86 ⁇ 0.63 8 0.39 ⁇ 0.10** 67.62%
  • Table 38 Inhibition of polypeptide III on human bladder cancer HT1376 nude mouse xenograft tumor growth
  • Avastin group, human bladder cancer HT1376 nude mice xenograft tumor The tumor inhibition rate was 79.60%, which had no effect on the body weight of the experimental animals compared with the negative control group.
  • the inhibition rate of the high, medium and low dose groups of peptide I on human bladder cancer HT1376 nude mice xenograft tumors was 49.20%. , 56.34%, 53.17%.
  • the tumor volume in the middle dose group was significantly different from the tumor volume in the negative group. Compared with the negative control group, there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the inhibitory effect of peptide II on the growth of human bladder cancer HT1376 nude mice xenograft tumors is shown in Table 37.
  • the tumor inhibition rate of human bladder cancer HT1376 nude mice xenograft tumors was 79.60%, which was compared with the negative control group. Comparison The weight of the material had no effect; the tumor inhibition rate of human bladder cancer HT1376 nude mice xenograft tumors was 60.97%, 71.10%, 67.72% in the high, medium and low dose groups of peptide II.
  • the tumor volume in the high-dose group was significantly different from that in the negative group.
  • the tumor volume in the middle-dose group and the low-dose group was significantly different from that in the negative group.
  • Compared with the negative control group there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the inhibitory effect of peptide III on the growth of human bladder cancer HT1376 nude mice xenograft tumors is shown in Table 38.
  • the tumor inhibition rate of human bladder cancer HT1376 nude mice xenograft tumors was 79.60%, which was compared with the negative control group.
  • the tumor inhibition rate of human bladder cancer HT1376 nude mice xenograft tumor was 67.92%, 76.94%, 72.55% in high, medium and low dose groups.
  • Example 6 For the tumor inoculation and test evaluation methods, see Example 6.
  • the positive control drug Avastin was administered once every 3 days in the tail vein, and the other groups were administered once per day in the tail vein.
  • Body weight had no effect; the tumor inhibitory rate of high, medium and low doses of peptide I on human pancreatic cancer SW-1990 nude mice xenograft tumors was 55.04%, 63.57%, 60.47%.
  • the tumor volume in the high-dose, medium-dose, and low-dose groups was significantly different from that in the negative group. Compared with the negative control group, there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the inhibitory effect of peptide II on the growth of human pancreatic cancer SW-1990 nude mice xenograft tumors is shown in Table 40.
  • the tumor inhibition rate of human pancreatic cancer SW-1990 nude mice xenograft tumors was 79.07%
  • the negative control group there was no effect on the body weight of the experimental animals
  • the high, medium and low dose groups of the polypeptide II had a tumor inhibition rate of 65.05%, 72.97%, and 70.09% of the human pancreatic cancer SW-1990 nude mouse xenograft tumor.
  • the tumor volume in the high-dose, medium-dose, and low-dose groups was significantly different from that in the negative group.
  • the negative control group there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the inhibitory effect of peptide III on the growth of human pancreatic cancer SW-1990 nude mice xenograft tumors is shown in Table 41.
  • the tumor inhibition rate of human pancreatic cancer SW-1990 nude mice xenograft tumors was 79.07%
  • the negative control group there was no effect on the body weight of the experimental animals
  • the inhibition rate of the high, medium and low dose groups of the polypeptide III on human pancreatic cancer SW-1990 nude mice xenograft tumors was 71.01%, 78.64%, 74.68%.
  • Tumor volume and negative group tumor mass in high dose group, middle dose group and low dose group There is a very significant difference in the product comparison.
  • the negative control group there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • Integrin Blocker Peptide I, Peptide II and Peptide III for Human Esophageal Carcinoma Ecl09 Nude Mouse Xenograft Tumor Growth Inhibition Test For tumor inoculation and test evaluation methods, see Example 6.
  • the positive control drug Avastin was administered once every 3 days in the tail vein, and the other groups were administered once per day in the tail vein.
  • the tumor inhibition rate of human esophageal cancer Ecl09 nude mice xenograft tumor was 69.00%, which had no effect on the body weight of the experimental animals compared with the negative control group;
  • the inhibition rate of I high-, medium-, and low-dose groups in human esophageal cancer Ecl09 nude mice xenograft tumors was 64.71%, 69.00%, 65.36%.
  • the inhibitory effect of peptide II on xenograft tumor growth in human esophageal cancer Ecl09 nude mice is shown in Table 43.
  • the tumor inhibition rate of human esophageal cancer Ecl09 nude mice xenograft tumor was 69.00%, which was compared with the negative control group.
  • the tumor inhibition rate of the high-, medium-, and low-dose peptides in the human esophageal cancer Ecl09 nude mice was 53.34%, 63.88%, and 59.65%.
  • the tumor volume in the middle-dose group was significantly different from that in the negative group.
  • the negative control group there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the inhibitory effect of peptide III on xenograft tumor growth of human esophageal cancer Ecl09 nude mice is shown in Table 44.
  • the tumor inhibition rate of human esophageal cancer Ecl09 nude mice xenograft tumor was 69.00%, which was compared with the negative control group.
  • the tumor inhibitory rate of human high-, medium-, and low-dose peptides in human esophageal cancer Ecl09 nude mice was 59.75%, 67.13%, and 65.63%.
  • the tumor volume in the medium-dose group and the low-dose group was significantly different from that in the negative group.
  • the negative control group there was no effect on the body weight of the test animals, and no obvious side effects were observed.
  • the positive control drug cisplatin group was administered once every 3 days, and the other groups were administered once per night.
  • Cisplatin 10 20.17 ⁇ 0.56 8 17.75 ⁇ 0.53 0.30 ⁇ 0.05** 79.17% End of animal number
  • Table 45 The inhibitory effect of peptide I on the growth of human colon cancer HT-29 nude mice xenograft tumors is shown in Table 45.
  • the inhibitory effect of peptide II on the growth of human colon cancer HT-29 nude mice xenograft tumors is shown in Table 46.
  • the tumor inhibition rate of human colon cancer HT-29 nude mice xenograft tumors was 79.17%, but It has a significant effect on the body weight of the experimental animals, and the body weight of the animals in the negative group and the polypeptide-administered group is lighter, and the side effects are more obvious; the high, medium and low dose groups of the polypeptide II are human xenograft tumors of human colon cancer HT-29 nude mice.
  • the tumor inhibition rate was 72.93%, 81.96%, and 78.32%.
  • the tumor volume in the high-dose, medium-dose, and low-dose groups was significantly different from that in the negative group. Compared with the negative control group, there was no effect on the body weight of the test animals, and no obvious side effects were observed.
  • the inhibitory effect of peptide III on the growth of human colon cancer HT-29 nude mice xenograft tumors is shown in Table 47.
  • the tumor inhibition rate of human colon cancer HT-29 nude mice xenograft tumors was 79.17%, but It has a significant effect on the body weight of the experimental animals, and the body weight of the animals in the negative group and the polypeptide-administered group is lighter, and the side effects are more obvious; the high, medium and low dose groups of the polypeptide III are human xenograft tumors of human colon cancer HT-29 nude mice.
  • the tumor inhibition rate was 79.11%, 85.62%, and 83.70%. High dose group, medium dose end animal number
  • Example 6 For the tumor inoculation and test evaluation methods, see Example 6.
  • the positive control drug cisplatin group was administered once every 3 days, and the other groups were administered once per night.
  • the inhibitory effect of peptide II on the growth of xenograft tumor of human thyroid carcinoma SW-579 nude mice is shown in Table 49.
  • the tumor inhibition rate of human thyroid cancer SW-579 nude mice xenograft tumor was 74.09%. However, it has a significant effect on the body weight of the experimental animals, and the body weight of the animals in the negative group and the polypeptide-administered group is lighter, and the side effects are more obvious; the high, medium and low dose groups of the polypeptide II are human xenografts of human thyroid cancer SW-579 nude mice.
  • the tumor inhibition rate was 56.59%, 66.94%, and 60.97%. There was a significant difference between the tumor volume in the high-dose group and the tumor volume in the negative group.
  • the tumor volume in the middle-dose group and the low-dose group was negative in the negative group. There was a significant difference in tumor volume compared to the tumor volume. Compared with the negative control group, there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the inhibitory effect of peptide III on the growth of xenograft tumor of human thyroid carcinoma SW-579 nude mice is shown in Table 50.
  • the tumor inhibition rate of human thyroid cancer SW-579 nude mice xenograft tumor was 74.09%. However, it has a significant effect on the body weight of the experimental animals, and the body weight of the animals in the negative group and the polypeptide-administered group is lighter, and the side effects are more obvious; the high, medium and low dose groups of the polypeptide III are human xenografts of human thyroid cancer SW-579 nude mice.
  • the tumor inhibition rate was 63.38%, 70.58%, and 66.40%.
  • Example 6 For the tumor inoculation and test evaluation methods, see Example 6.
  • the positive control drug cisplatin group was administered once every 3 days, and the other groups were administered once per night.
  • Cisplatin 10 20.13 ⁇ 0.57 8 17.80 ⁇ 0.63 0.25 ⁇ 0.06** 79.83% Entitude 2.5 19.94 ⁇ 0.65 8 22.96 ⁇ 0.69 8 0.70 ⁇ 0.14 43.20% Peptide I high 3 20.21 ⁇ 0.57 8 22.59 ⁇ 0.53 8 0.57 ⁇ 0.15* 53.65 % polypeptide I 1.5 2.06 ⁇ 0.47 8 23.26 ⁇ 0.40 8 0.49 ⁇ 0.10** 60.16% polypeptide I low 0.75 20.02 ⁇ 0.51 8 22.87 ⁇ 0.40 8 0.51 ⁇ 0.13 * 58.53% Table 52 peptide II on human kidney cancer A498 nude mice Inhibition of xenograft tumor growth
  • Cis molybdenum 10 20.13 ⁇ 0.57 8 17. 80 ⁇ 0. .63 7 0.25 ⁇ 0.06** 79.83% Entitude 2.5 19.94 ⁇ 0.65 8 22. 96 ⁇ 0. .69 8 0.70 ⁇ 0.14 43 .20% Peptide II High 3 20.05 ⁇ 0.52 8 22. 93 ⁇ 0. .51 8 0.43 ⁇ 0.11** 64 .90% Peptide II 1.5 19.91 ⁇ 0.57 8 23. 13 ⁇ 0. .58 8 0.35 ⁇ 0.09** 71 .41% Peptide II is 0.75 19.94 ⁇ 0.56 8 23.14 ⁇ 0.58 8 0.41 ⁇ 0.11** 67.
  • Peptide III is 0.75 19.68 ⁇ 0.59 8 23.18 ⁇ 0.50 8 0.41 ⁇ 0.12** 66.36% Polypeptide III 0.375 19.83 ⁇ 0.65 8 22.92 ⁇ 0.59 8 0.30 ⁇ 0.07** 75.36% Peptide III is low 0.1875 19.97 ⁇ 0.66 8 22.82 ⁇ 0.69 8 0.36 ⁇ 0.10** 70.74% Results: The inhibitory effect of peptide I on the growth of human kidney cancer ⁇ 498 nude mice xenograft tumors is shown in Table 51. The cisplatin group of the drug, the tumor suppressor of human kidney cancer ⁇ 498 nude mice xenograft tumor The rate was 79.83%, but the final number of animals had a significant effect on the body weight of the experimental animals.
  • the body weight was lighter and the side effects were more obvious.
  • the peptide I high, medium and low dose groups were on the human kidney.
  • the tumor inhibition rate of xenograft tumors in cancer ⁇ 499 nude mice was 53.65%, 60.16%, 58.53%.
  • the negative control group there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the inhibitory effect of peptide ⁇ on the growth of human kidney cancer ⁇ 498 nude mice xenograft tumors is shown in Table 52.
  • the inhibition rate of human cisplatin group on human kidney cancer ⁇ 498 nude mice xenograft tumor was 79.83%, but for experimental animals.
  • the body weight has a significant effect, compared with the body weight of the negative group and the polypeptide-administered group, the toxic side effects are more obvious; the tumor inhibitory rate of the high, medium and low doses of the polypeptide II on the xenograft tumor of human kidney cancer ⁇ 498 nude mice 64.90%, 71.41%, 67.03%.
  • the tumor volume in the high-dose, medium-dose, and low-dose groups was significantly different from that in the negative group. Compared with the negative control group, there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the inhibitory effect of peptide III on the growth of human kidney cancer ⁇ 498 nude mice xenograft tumors is shown in Table 53, the cisplatin group, the tumor inhibition rate of human kidney cancer ⁇ 498 nude mice xenograft tumors was 79.83%, but for experimental animals
  • the body weight has a significant effect, compared with the body weight of the negative group and the polypeptide-administered group, the toxic side effects are more obvious; the inhibition rate of the high, medium and low dose groups of the polypeptide III on the xenograft tumor of human kidney cancer ⁇ 498 nude mice 66.36%, 75.36%, 70.74%.
  • the tumor volume in the high-dose, medium-dose, and low-dose groups was significantly different from that in the negative group. Compared with the negative control group, there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • Example 6 For the tumor inoculation and test evaluation methods, see Example 6.
  • the positive control drug cisplatin group was administered once every 3 days, and the other groups were administered once per night.
  • the inhibition rate of human ovarian cancer SK-OV-3 nude mice xenograft tumor was 78.36%, but the weight of experimental animals. Significantly, the body weight of the animals in the negative group and the peptide administration group was lighter, and the side effects were more obvious; the high, medium and low dose groups of the polypeptide I inhibited the tumor xenograft tumor of human ovarian cancer SK-OV-3 nude mice. The rate was 67.13%, 70.89%, and 69.40%. There was a significant difference in tumor volume between the high-dose group, the middle-dose group, and the low-dose group compared with the negative group. Compared with the negative control group, there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the tumor inhibition rate of human ovarian cancer SK-OV-3 nude mice xenograft tumor was 78.36%, but it had a significant effect on the body weight of the experimental animals. Compared with the negative group and the polypeptide administration group, the body weight was lighter, and the side effects were more serious. Obviously; the high, medium and low doses of peptide II inhibited tumor xenograft tumors in human ovarian cancer SK-OV-3 nude mice by 63.25%, 72.76%, and 68.36%. The tumor volume in the high-dose, medium-dose, and low-dose groups was significantly different from that in the negative group. Compared with the negative control group, the final number of animals
  • the inhibitory effect of peptide III on the growth of human ovarian cancer SK-OV-3 nude mice xenograft tumors is shown in Table 56.
  • the tumor inhibition rate of human ovarian cancer SK-OV-3 nude mice xenograft tumors It was 78.36%, but had a significant effect on the body weight of the experimental animals. Compared with the negative group and the polypeptide-administered group, the body weight was lighter and the side effects were more obvious.
  • the high, medium and low dose groups of the polypeptide III were applied to human ovarian cancer SK-OV- 3
  • the tumor inhibition rate of nude mice xenograft tumors was 69.98%, 76.63%, 73.87%.
  • the tumor volume in the high-dose, medium-dose, and low-dose groups was significantly different from that in the negative group. Compared with the negative control group, there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • Example 6 For the tumor inoculation and test evaluation methods, see Example 6.
  • the positive control drug 5-fluorouracil group was administered once every 3 days, and the other group was administered once per night.
  • the 5-Fu group has a tumor inhibition rate of 78.10% for sarcoma HT-1080 nude mice, but has a significant effect on the body weight of experimental animals.
  • the body weight was lighter and the side effects were more obvious;
  • the high, medium and low dose groups of the peptide I had a tumor inhibition rate of sarcoma HT-1080 nude mice xenograft tumors of 64.23%, 73.72%. , 69.34%.
  • the negative control group there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the inhibitory effect of peptide II on the xenograft tumor growth of sarcoma HT-1080 nude mice is shown in Table 58.
  • the 5-Fu group has a tumor inhibition rate of 78.10% for sarcoma HT-1080 nude mice xenograft tumors, but the experiment
  • the body weight of the animals has a significant effect, compared with the body weight of the negative group and the polypeptide-administered group, the toxic side effects are more obvious; the high, medium and low dose groups of the polypeptide II are sarcoma
  • the tumor inhibition rate of HT-1080 nude mice xenograft tumors was 67.06%, 74.43%, 70.60%.
  • the tumor volume in the high-dose group was significantly different from that in the negative group.
  • the tumor volume in the middle-dose group and the low-dose group was significantly different from that in the negative group.
  • the inhibitory effect of peptide III on xenograft tumor growth in sarcoma HT-1080 nude mice is shown in Table 59.
  • the tumor inhibition rate of sarcoma HT-1080 nude mice xenograft tumor was 78.10%, but the experiment The body weight of the animals had a significant effect, compared with the body weight of the negative group and the polypeptide-administered group, and the side effects were more obvious.
  • the tumor inhibition rate of the high, medium and low dose groups of the polypeptide III on the xenograft tumor of sarcoma HT-1080 nude mice It is 73.29%, 79.90%, and 75.87%. There was a significant difference in tumor volume between the high-dose group, the middle-dose group, and the low-dose group compared with the negative group. Compared with the negative control group, the number of animals that started the experiment
  • the weight of the substance had no effect and no obvious side effects were observed.
  • Example 6 For the tumor inoculation and test evaluation methods, see Example 6.
  • the positive control drug Avastin group was administered once every 3 days in the tail vein.
  • the other groups were administered once a day in the tail vein.
  • Peptide III is naked to human laryngeal carcinoma Hep-2 Inhibition of mouse xenograft tumor growth
  • Negative control A 20.00 ⁇ 0.52 12 23.33 ⁇ 0.44 12 1.12 ⁇ 0.20
  • a Avastin 10 20.09 ⁇ 0.70 8 20.47 ⁇ 0.70 6 0.26 ⁇ 0.06 76.79% Number of starting animals
  • the inhibitory effect of peptide II on the growth of human laryngeal carcinoma Hep-2 nude mice xenograft tumors is shown in Table 61.
  • the tumor inhibition rate of human laryngeal carcinoma Hep-2 nude mice xenograft tumors was 76.79%.
  • the body weight of the experimental animals had no significant effect; the tumor inhibitory rate of the high, medium and low doses of peptide II on human laryngeal carcinoma Hep-2 nude mice xenograft tumors was 60.76%, 70.41%, 64.98%.
  • the tumor volume in the high-dose group was significantly different from that in the negative group.
  • the tumor volume in the middle-dose group and the low-dose group was significantly different from that in the negative group.
  • Compared with the negative control group there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the inhibitory effect of peptide III on xenograft tumor growth in human laryngeal carcinoma Hep-2 nude mice is shown in Table 62.
  • the tumor inhibition rate of human laryngeal carcinoma Hep-2 nude mice xenograft tumor was 76.79%.
  • the body weight of the experimental animals had no significant effect; the inhibition rate of the high, medium and low doses of peptide III on human laryngeal carcinoma Hep-2 nude mice xenograft tumors was 62.81%, 75.13%, 67.29%.
  • the tumor volume in the high-dose group was significantly different from that in the negative group.
  • the tumor volume in the middle-dose group and the low-dose group was significantly different from that in the negative group.
  • Compared with the negative control group there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • Example 24 Integrin Blocker Polypeptide I, Polypeptide II and Polypeptide III for Human Brain Tumor SF763 Nude Mouse Xenograft Tumor Growth Inhibition Test Tumor inoculation and detection evaluation methods are shown in Example 6.
  • the positive control drug paclitaxel group was administered once every 3 days, and the other groups were administered once per night.
  • Peptide I for human brain The inhibitory effect of tumor SF763 xenograft tumor growth in nude mice is shown in Table 63, the chemical drug paclitaxel The tumor inhibition rate of human brain tumor SF763 nude mice xenograft tumor was 76.29%, but it had a significant effect on the body weight of the experimental animals.
  • the body weight was lighter and the side effects were more obvious;
  • the tumor inhibitory rate of high, medium and low doses of peptide I on human brain tumor SF763 nude mice xenograft tumors was 60.16%, 71.19%, 67.80%.
  • Compared with the negative control group there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the inhibitory effect of peptide II on the growth of xenograft tumors in human brain tumor SF763 nude mice is shown in Table 64.
  • the inhibition rate of xenograft tumors in human brain tumor SF763 nude mice was 76.29%, but for experimental animals.
  • the body weight has a significant effect, compared with the body weight of the negative group and the polypeptide-administered group, the toxic side effects are more obvious; the high, medium and low dose groups of the polypeptide II have a tumor inhibition rate of 65.48 on human brain tumor SF763 nude mouse xenograft tumor. %, 75.02%, 70.61%.
  • Compared with the negative control group there was no effect on the body weight of the experimental animals, and no obvious side
  • the inhibitory effect of peptide III on the growth of human brain tumor SF763 nude mice xenograft tumors is shown in Table 65.
  • the tumor inhibition rate of human brain tumor SF763 nude mice xenograft tumors was 76.29%, but for experimental animals.
  • the body weight has a significant effect, compared with the body weight of the negative group and the polypeptide-administered group, the toxic side effects are more obvious; the final, middle and low dose groups of the polypeptide III high-end animal group inhibit the xenograft tumor of human brain tumor SF763 nude mice.
  • the tumor rate was 74.06%, 79.76%, and 72.82%. There were significant differences between the high-dose, middle-dose, low-dose, and negative groups. Compared with the negative control group, there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • Example 6 For the tumor inoculation and test evaluation methods, see Example 6.
  • the positive control drug paclitaxel group was administered once every 3 days, and the other group was administered once per night.
  • Negative control 20.09 ⁇ 0.63 12 23.22 ⁇ 0.50 12 1.25 ⁇ 0.31 - Paclitaxel 10 19.96 ⁇ 0.59 8 17.08 ⁇ 0.57 7 0.29 ⁇ 0.07** 76.62% Entitude 2.5 20.05 ⁇ 0.70 8 23.05 ⁇ 0.67 8 0.68 ⁇ 0.28 45.41% Many months too I high 3 19.94 ⁇ 0.59 8 20.99 ⁇ 0.66 7 0.51 ⁇ 0.09** 59.20% Many months too I 1.5 1.88 ⁇ 0.50 8 22.87 ⁇ 0.57 7 0.44 ⁇ 0.05** 64.80% Multi-month too I low 0.75 20.21 ⁇ 0.64 8 23.11 ⁇ 0.61 8 0.52 ⁇ 0.08** 58.40% Table 67 Inhibition of polypeptide II on xenograft tumor growth in human rectal cancer Colo 320 nude mice
  • Negative control 20.09 ⁇ 0.63 12 23.22 ⁇ 0.50 12 1.25 ⁇ 0.31
  • One paclitaxel 10 19.96 ⁇ 0.59 8 17.08 ⁇ 0.57 7 0.29 ⁇ 0.07** 76.62% Entitude 2.5 20.05 ⁇ 0.70 8 23.05 ⁇ 0.67 8 0.68 ⁇ 0.28 45.41% Number of starting animals
  • Peptide III is small for human rectal cancer Colo 320 Inhibition of murine xenograft tumor growth dose starting weight (g) end-of-body end-of-border animal number final tumor weight (g) tumor inhibition rate (mg/k (g) end animal number
  • the chemical drug paclitaxel group, human colon cancer Colo 320 nude mice xenograft tumor The tumor inhibition rate was 76.62%, but it had a significant effect on the body weight of the experimental animals. Compared with the negative group and the polypeptide-administered group, the body weight was lighter and the side effects were more obvious.
  • the high, medium and low dose groups of the polypeptide I were used for human rectal cancer.
  • the tumor inhibition rate of Colo 320 nude mice xenograft tumors was 59.20%, 64.80%, 58.40%. There were significant differences between the high-dose, middle-dose, low-dose, and negative groups. Compared with the negative control group, there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the inhibitory effect of peptide ⁇ on the growth of human rectal cancer Colo 320 nude mice xenograft tumors is shown in Table 67.
  • the inhibitory rate of the drug paclitaxel group on human rectal cancer Colo 320 nude mice xenograft tumor was 76.62%, but the experiment The body weight of the animals has a significant effect, compared with the body weight of the negative group and the polypeptide-administered group, the toxic side effects are more obvious; the high, medium and low dose groups of the polypeptide II inhibit the tumor of the human rectal cancer Colo 320 nude mouse xenograft tumor The rate was 67.41%, 75.42%, and 72.26%. High dose group, There was a significant difference between the middle dose group, the low dose group and the negative group. Compared with the negative control group, there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • the inhibitory effect of peptide III on the growth of human rectal cancer Colo 320 nude mice xenograft tumors is shown in Table 68.
  • the inhibitory rate of the drug-type paclitaxel group on human rectal cancer Colo 320 nude mice xenograft tumors was 76.62%, but the experiment The body weight of the animals has a significant effect, compared with the body weight of the negative group and the polypeptide-administered group, the toxic side effects are more obvious; the high, medium and low dose groups of the polypeptide III inhibit the tumor of the human rectal cancer Colo 320 nude mouse xenograft tumor The rate was 73.51%, 80.89%, and 70.34%. There were significant differences between the high-dose, middle-dose, low-dose, and negative groups. Compared with the negative control group, there was no effect on the body weight of the experimental animals, and no obvious side effects were observed.
  • a collagen mouse arthritis animal model was constructed to study the therapeutic effect of the polypeptide on collagen induced arthritis (CIA) in mice.
  • Mice were used as test animals, SPF DBA/1 mice (supplied by Sino-British SIPPR Lab. Animal Ltd), animal production license number: SCXK (Shanghai) 2008 -0016), male, 7-8 weeks old, weighing 18-22 g, randomized, normal control group, model control group, peptide I low, medium and high dose groups (0.2, 0.4, 0.8 mg/kg) , peptide II low, medium and high dose groups (0.2, 0.4, 0.8 mg / kg), peptide III low, medium and high dose groups (0.1, 0.2, 0.4 mg / kg) and positive drug control group (methotrexate 1 mg /kg).
  • the experimental group established a mouse CIA model by dissolving chicken cartilage type III collagen (cIII) with O. l mol/1 acetic acid into a 4 mg/ml solution and overnight at 4 ° C in the refrigerator. .
  • type III collagen was fully emulsified with a full Freund's adjuvant (CF A) containing 4 mg/ml Myeobaeterium tuberculosis strain H37Rv. After anesthesia in DBA/1 mice, each emulsifier was injected into the tail skin.
  • CF A full Freund's adjuvant
  • Sensitization was carried out at 50 ⁇ , and after 21 days, 4 mg/ml of type III collagen (cIII) and incomplete Freund's adjuvant (IFA) were fully emulsified, and then re-immunized at the tail with the same dose of emulsifier.
  • cIII type III collagen
  • IFA incomplete Freund's adjuvant
  • Subcutaneous injection from the 30th day of modeling peptide I low, medium and high dose groups (0.2, 0.4, 0.8 mg / kg), peptide II low, medium and high dose groups (0.2, 0.4, 0.8 mg / kg), peptide III low, medium and high dose groups (0.1, 0.2, 0.4 mg/kg): twice daily for 10 consecutive days; positive drug control group (methotrexate 1 mg/kg): once every five days, 3 times in succession Normal control group and model control group (saline): 10 consecutive days.
  • the body weight and joint score were measured every 3 days from the 21st day to the 70th day after modeling, and the diameters of the foot and ankle were measured to observe the effect of the drug on collagen-induced arthritis in mice. On the 70th day, the mice were sacrificed by dislocation.
  • the arthritis evaluation indicators are as follows:
  • the diameters and ankle thicknesses of the left and right ankle of the left and right ankles were measured by vernier calipers every 3 days before and after the modeling and from the 21st to the 70th day after the modeling, and the results were recorded.
  • the measurement data of the test results were expressed by the mathematical mean plus or minus standard deviation (mean ⁇ SD).
  • the t test was performed between each drug-administered group and the control group using SPSS 11.0 software.
  • the table * indicates ⁇ ⁇ 0.05, ** Indicates ⁇ ⁇ 0.01.
  • mice were injected intradermally with inactivated M. tuberculosis complete Freund's adjuvant and collagen in an equal volume of emulsifier. After 21 days, the tail was injected intradermally. Completely mixed with emulsifier of Freund's adjuvant and collagen in the left, on the 27th day after immunization, the paws of CIA mice were swollen and the index of arthritis index was increased. The model group was the swelling peak on the 45th to 60th day. At 35 days, the weight did not increase at all, and there was a slight decrease in the later period.
  • Peptide I can exert immunoprotective effects in collagen-induced mouse arthritis animal models.
  • the results are shown in Table 69: Positive control group, peptide I high, medium and low dose group paw swelling degree compared with the model group, are extremely significant sexual differences ( ⁇ ⁇ 0.01)
  • the experimental results were statistically significant.
  • the low, medium and high dose groups of the peptide I scored in the limbs.
  • Peptide II can exert immunoprotection in vivo in collagen-induced mouse arthritis animal model.
  • the results are shown in Table 70:
  • the positive control group and the high, medium and low dose groups of the peptides have a significant degree of paw swelling compared with the model group.
  • Sexual differences ⁇ ⁇ 0.01
  • the experimental results were statistically significant.
  • the positive control group and the high, medium and low dose groups of the peptide II had significant difference ( ⁇ ⁇ 0.01), and the experimental results were statistically significant.
  • Lower limb scores in low, medium and high dose groups of peptide II Significantly lower than the model control group (p ⁇ 0.01), compared with the model control group, the results were statistically significant.
  • Table 70 Immunoprotection of polypeptide II in collagen-type mouse arthritis animal model
  • Peptide III can exert immunoprotective effects in collagen-induced mouse arthritis animal models.
  • the results are shown in Table 71: Positive control group, peptide III high, medium and low dose group paw swelling degree is extremely significant compared with the model group. Sexual differences ( ⁇ ⁇ 0.01) The experimental results were statistically significant. Compared with the model group, the positive control group and the high, medium and low dose groups of the peptide III had significant difference ( ⁇ ⁇ 0.01), and the experimental results were statistically significant. The limbs scores of the low, medium and high doses of peptide III were significantly lower than the model control group ( ⁇ ⁇ 0.01), which was significantly different from the model control group. The experimental results were statistically significant. Table 71 Immunoprotection of peptide III against collagen-type mouse arthritis animal model
  • Peptide I, Peptide II and Peptide III have therapeutic effects on collagen-induced arthritis in mice.
  • the rats in the experimental group established the AA model on the 0th day by injecting the inactivated Mycobacterium tuberculosis (H37RA, 10 mg/ml) completely in the left hind foot of the rat. 0.08 ml of the agent caused a rat adjuvant arthritis model. Subcutaneous injection was given on the 10th day of modeling: peptide I was low, medium and high in three dose groups (0.4, 0.8, 1.6 mg/kg), and peptide II was low, medium and high in three dose groups (0.4,
  • the arthritis evaluation indicators are as follows:
  • Diameter and ankle thickness of the left and right ankle of the rat were measured with vernier calipers before and after modeling and on 8, 8, 14, 20, 23 and 26 days after modeling, and the results were recorded.
  • the measurement data of the test results were expressed by the mathematical mean plus or minus standard deviation (mean ⁇ SD).
  • the t test was performed between each drug-administered group and the control group using SPSS 11.0 software.
  • the table * indicates ⁇ ⁇ 0.05, ** Indicates ⁇ ⁇ 0.01.
  • Peptide I can exert immunoprotective effects in collagen-induced rat arthritis animal model.
  • the results are shown in Table 72: Rat positive control group, peptide I middle dose group, left hind foot diameter compared with model group, there is a significant difference (p ⁇ 0.01); Integrin blocker peptide I low and high dose group left hind foot diameter compared with the model group, there were significant differences ( ⁇ ⁇ 0.05), the experimental results were statistically significant.
  • the diameter of the right hind paw was lower in the low, middle and high dose groups of the peptide I compared with the model group, and there was a significant difference ⁇ 0.05).
  • the limbs scores of the low, medium and high doses of peptide I were significantly lower than the model control group ⁇ 0.05). The differences were statistically significant compared with the model control group.
  • Peptide II can exert immunoprotective effects in collagen-induced rat arthritis animal models.
  • the results are shown in Table 73: Rat positive control group, peptide II dose group, left hind foot diameter compared with model group, there is a significant difference (p ⁇ 0.01); The degree of swelling of the left foot and paw in the low- and high-dose groups of the peptides was significantly different from that in the model group ( ⁇ ⁇ 0.05), and the experimental results were statistically significant. There was a significant difference between the positive control group and the low, medium and high dose groups of peptide II in the right foot and paw group compared with the model group ( ⁇ ⁇ 0.05). The limbs scores of the low, medium and high doses of peptide II were significantly lower than those of the model control group ( ⁇ ⁇ 0.05), and the differences were statistically significant compared with the model control group.
  • Polypeptide II has a therapeutic effect on arthritis in AA rats.
  • Peptide III can exert immunoprotective effects in collagen-induced rat arthritis animal models.
  • the results are shown in Table 74: Rat positive control group, peptide III medium dose group left hind foot diameter compared with model group, there is a significant difference (p ⁇ 0.01); integration Compared with the model group, the low and high dose groups of the peptide blocker III had significant differences ( ⁇ ⁇ 0.05), and the experimental results were statistically significant.
  • the diameter of the right hind paw was lower in the rat positive control group and the low, medium and high dose groups of the peptide III compared with the model group, and there was a significant difference ⁇ 0.05).
  • the scores of the limbs in the low, medium and high dose groups of peptide III were significantly lower than those in the model control group ( ⁇ 0.05), and the differences were statistically significant compared with the model control group.

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Abstract

提供了一种整合素阻断剂，其包含经修饰的内皮抑素第6-48位氨基酸，还可以进一步在N端或C端连接含有RGD的序列，该整合素阻断剂对整合素具有亲和性和结合能力，可用于实体肿瘤和类风湿性关节炎的预防和治疗。所述的肿瘤为起源于人的头颈部、脑部、甲状腺、食管、胰腺、肺脏、肝脏、胃、乳腺、肾脏、胆囊、结肠或直肠、卵巢、子宫颈、子宫、前列腺、膀胱、睾丸的原发或继发的癌、黑色素瘤以及肉瘤。

Description

说 明 书
整合素阻断剂多肽及其应用
技术领域
本发明涉及药物领域， 具体涉及具有抑制血管生成、 具有整合素亲和性和结合能力的整 合素阻断剂， 此阻断剂包括两种多肽， 该整合素阻断剂多肽可用于实体肿瘤和类风湿性关节 炎的治疗。
背景技术
近年我国肿瘤的发病率和病死率不断增长。 无限制生长、 侵袭和转移是肿瘤的恶性标志 和特征， 也是导致治疗失败和死亡的主要原因。 因此， 控制肿瘤的生长、 侵袭和转移是改善 预后， 提高生存率的主要措施。 1971年 Folkman 首先提出肿瘤生长依赖血管新生的理论， 肿 瘤血管生成是肿瘤生长和转移的形态学基础， 它不仅向肿瘤提供营养， 也向宿主输出大量的 肿瘤细胞导致肿瘤的生长和转移。 绝大多数恶性实体肿瘤如卵巢癌、 肝癌、 宫颈癌和乳腺癌 等都是血管依赖性肿瘤。 新生血管一方面为肿瘤生长提供营养和氧气， 另一方面还是肿瘤转 移的重要途径。 因此， 抑制肿瘤血管形成是重要的抗癌措施。
整合素是一类广泛分布于细胞表面的受体， 能介导血管内皮细胞和肿瘤细胞的黏附， 它 们通过连接胞内细胞骨架蛋白和细胞外基质分子的相互作用参与血管生成和肿瘤转移。 目前 至少有 8种的整合素 α1β1、 α2β1、 α3β1、 α6β1、 α6β4、 α5β1、 ανβ3、 ανβ5) 参与肿瘤血管生 成， 其中 avW发挥着重要作用。 avW由 av亚基 （CD51， 150kD ) 和 3亚基 （CD61， 105kD ) 形成的跨膜异二聚体糖蛋白， 又名 VN受体。 ccvW能识别配体分子中的精-甘-天冬序列 （arg- gly-asp , RGD)。 avW可以表达于多种细胞类型， 并与多细胞活动过程中的多种配体结合， 参 与肿瘤的血管生成， 侵袭转移过程。 含有 RGD序列的多肽具有整合素拮抗剂作用， 能减少细 胞表面黏附分子的表达， 调节细胞内信号转导， 抑制肿瘤血管生成从而抑制肿瘤生长和转移。 因此， 以整合素为靶点的整合素阻断剂能够阻断整合素的下游的胞内信号传导， 可有效地抑 制肿瘤血管生成， 从而抑制肿瘤生长和转移， 在肿瘤治疗中具有广阔的应用前景。
目前， 国际上开发出的整合素阻断剂已进入 Π期临床， 而我国尚未有相似或同类产品进 入市场， 非常有必要开发我国自主知识产权的此类药物。先前的专利 ZL200610039298.2血管 生成抑制多肽及其制备方法和应用， 主要是通过对整合素第 6-49个氨基酸进行改造和修饰， 修饰改造后的多肽具有比内皮抑素更好的体内活性和肿瘤靶向性。 其介绍了几种整合素抑制 齐 U， 其中有两个为 Arg-Gly-Asp-Phe-Gln-Pro-Val-Leu-His-Leu-Val- Ala-Leu- Asn-Ser-Pro-Leu-
Ser-Gly-Gly-Met-Arg-Gly-Ile-Arg-Gly-Ala-Asp-Phe-Gln-Cys-Phe-Gln-Gln-Ala-Arg-Ala-Val-Gly-
Leu-Ala-Gly-Thr-Phe-Arg-Ala-Phe (简称 EDSM-l ,见 Seq NO.4 ) Phe-Gln-Pro-Val-Leu-His-Leu-
Val- Ala- Leu-Asn-Ser-Pro-Leu-Ser-Gly-Gly-Met-Arg-Gly-Ile-Arg-Gly-Ala-Asp-Phe-Gln-Cys-Phe -Gln-Gln-Ala-Arg-Ala-Val-Gly-Leu-Ala-Gly-Thr-Phe-Arg-Ala-Phe-Gly-Gly-Gly-Gly-Ala-Cys-Arg -Gly-Asp-Cys-Phe-Cys(简称 EDSM-2, 见 Seq N0.5)。 这两个序列包含了整合素配体序列 Arg- Gly-Asp和 Gly-Gly-Gly-Gly- Ala-Cys-Arg-Gly-Asp-Cys-Phe-Cys (见 Seq NO.6)和新生血管抑 制序列（ Phe-Gln-Pro-Val-Leu- His-Leu-Val-Ala-Leu-Asn-Ser-Pro-Leu-Ser-Gly-Gly-Met-Arg-Gly- Ile-Arg-Gly-Ala-Asp-Phe-Gln-Cys-Phe-Gln-Gln-Ala-Arg-Ala-Val-Gly-Leu-Ala-Gly- Thr-Phe -Arg-Ala-Phe, 见 Seq N0.7)。 先前专利中只是对多肽 EDSM-1和 EDSM-2的克隆、 原核表达 载体的构建和 EDSM-1治疗肝癌和胃癌进行了初步研究。 本发明针对内皮抑素序列做了进一 步研究， 发现整合素第 6-48个氨基酸具有更好的抑制血管生成的作用， 并对其进行了修饰， 修饰后的多肽是在血管生成抑制序列 Phe-Gln-Pro-Val- Leu-His-Leu-Val-Ala- Leu-Asn-Ser-Pro -Leu-Ser-Gly-Gly-Met-Arg-Gly-Ile-Arg-Gly-Ala-Asp-Phe-Gln-Cys-Phe-Gln-Gln-Ala-Arg-Ala-Val- Gly-Leu-Ala-Gly-Thr-Phe-Arg-Ala (见 Seq NO.1 ) 的 N端 /C端加上了整合素配体序列
( Arg-Gly- Asp-Gly-Gly-Gly-Gly ) 构建了两种整合素阻断剂即多肽 II ( Arg-Gly- Asp-Gly -Gly-Gly-Gly-Phe-Gln-Pro-Val-Leu-His-Leu-Val-Ala-Leu-Asn-Ser-Pro-Leu-Ser-Gly-Gly-Met-Arg- Gly-Ile-Arg-Gly-Ala-Asp-Phe-Gln-Cys-Phe-Gln-Gln-Ala-Arg-Ala-Val-Gly-Leu-Ala-Gly-Thr-Phe- Arg-Ala (见 Seq NO.2)) 和多肽 III (Phe-Gln-Pro-Val-Leu-His-Leu-Val-Ala-Leu-Asn-Ser-Pro- Leu-Ser-Gly-Gly-Met-Arg-Gly-Ile-Arg-Gly-Ala-Asp-Phe-Gln-Cys-Phe-Gln-Gln-Ala-Arg-Ala-Val- Gly-Leu-Ala-Gly-Thr-Phe-Arg-Ala-Gly-Gly-Gly-Gly-Arg-Gly-Asp (见 Seq N0.3 ))， 其中整合素 配体序列中含有 RGD序列（Arg-Gly- Asp), 使该多肽序列可以有效地结合于肿瘤特异性表达 的整合素亚型， 并且该序列中含有新生血管抑制序列， 能够抑制肿瘤新生血管形成， 进而达 到抑制肿瘤生长和转移的效果。 新设计的多肽序列多肽 Π和多肽 III是具有靶向结合作用和亲 和性的整合素阻断剂， 并发现其对多种肿瘤有治疗作用， 增加了适用症， 拓展了其社会价值 和经济价值。
类风湿关节炎 （rheumatoid arthritis, RA)是临床最常见的炎性关节病和主要致残因素之 一。 在全世界约为 0.5%-1.0%， RA的发病在我国约为 0.4%。 RA可发生于任何年龄， 随着年 龄的增长， 发病率也随之增高。 女性高发年龄为 45-55岁， 性别与 RA发病关系密切， 男女 之比约 1 :3。 RA是一种病因尚未明了的慢性全身性炎症性疾病， 以慢性、 对称性、 多滑膜关 节炎和关节外病变为主要临床表现， 属于自身免疫炎性疾病。 患者常以手部或腕部疼痛及肿 胀 (特别是腕背部的肿胀)为首发症状， 症状持续不缓解， 普通的对症治疗虽可以缓解症状， 但常常由于用药不规则或不足量而导致症状反复。 病情进展时可以出现明显的晨僵， 通常可 达 1小时以上， 并不断加重； 同时出现一定的关节功能障碍。
类风湿关节炎的病因和发病机制尚未完全明了， 其基本病理特点是血管炎和滑膜炎。 关 节内滑膜血管增生， 形成血管翳， 导致滑膜增厚， 渗出增多， 分泌多种细胞因子， 侵犯软骨 并引起骨质损害。 对其周围的肌腔、 韧带、 腱鞘以及肌肉等组织也均可侵蚀， 从而影响关节 的稳定， 容易发生关节畸形而出现功能障碍。 血管炎亦可侵犯周身各脏器组织， 形成系统性 疾病。
目前治疗 RA的药物分为两大类： 控制症状药和控制疾病药。 控制症状的抗风湿药分为 四类： 一、 非 体类抗炎药， 通常称为一线药， 这类药物种类繁多， 国内市场已多达数十种。 二、 类固醇激素， 激素是一个非常好的止痛抗炎药， 但长期单独使用不能改善病情， 反而带 来许多副作用， 激素作为二线慢作用药起效前的过渡性用是可以的， 但用量要小， 时间不宜 过长。 在病情较重， 伴有关节外表现的患者， 短期治疗冲击， 并联合二线药治疗时必须的。 三、 慢作用抗风湿药， 通常称为二线药， 所谓慢作用药包括抗疟药、 金盐、 青霉胺和柳氮碘 胺吡啶， 它们治疗 RA起效慢， 长期作用对 RA病情有一定缓解作用， 故也称病情改善药。 四、 免疫抑制剂： 常用有甲氨喋吟、 环磷酰胺、 硫唑嘌吟、 雷公藤、 青风藤等。
RA的病理过程中， 血管新生是一种标志性的组织学改变， 新生血管形成伴随滑膜增生和 炎细胞浸润， 是 RA中血管翳形成及关节破坏的基础。 原来应该没有血管存在的关节软骨因 某种异常变化， 而形成新的血管， 使得软骨受到侵蚀， 造成关节变形或疼痛。 新生血管引起 类风湿关节炎患者滑膜组织发生异常变化，正常人的关节滑膜内层仅 1-2层细胞组成，而 RA 患者的滑膜内层通常有 4-10层细胞 (有时甚至超过 20层；)。 这些细胞不仅在数量上异常增多， 而且在功能上处于异常活跃的状态， 它们可以分泌大量的细胞因子、 信号分子和蛋白酶， 加 速关节破坏的进程。 另外， RA滑膜中还有大量炎性细胞的浸润， 如 T细胞、 B细胞和单核 细胞。
新生血管生成， 在正常生理条件下， 受到高度调节， 在生殖、 胚胎发育、 组织修复和创 伤愈合中是必不可少的过程。 血管生成在多种病理条件下也会发生， 所述病理条件包括： 肿 瘤生长和转移； 炎性障碍， 例如类风湿性关节炎、 银屑病、 骨关节炎、 炎性肠病、 克隆病、 溃疡性结肠类以及其它炎性障碍。
整合素是 αφ能识别配体分子中的精-甘-天冬序列 （arg-gly-asp, ROD) 并与多细胞活 动过程中的多种配体结合参与肿瘤的血管生成、 侵袭、 转移、 炎症、 伤口愈合和凝血等 生理和病理过程。 因此， 含有 RGD序列的多肽具有整合素拮抗剂作用， RGD序列可以作为 一种载体， 靶向运输到新生血管内皮， 从而对新生血管性疾病达到更高效率的治疗。 因此， 血管抑制多肽通过抑制血管生成不仅可阻止向滑膜输送氧气和营养物质， 且直接导致血管退 化， 从而可能抑制 RA滑膜增生。 抑制新生血管的形成是治疗这类疾病的关键， 而内皮细胞 的增殖和迁移是新生血管形成的关键步骤。 多肽 II和多肽 III含有 RGD序列（Arg-Gly-Asp)，使该多肽序列可以有效地结合于整合素， 并通过抑制胞内细胞骨架蛋白和细胞外基质分子的相互作用， 抑制细胞与细胞外基质及细胞 与细胞之间的相互黏附， 来抑制细胞与细胞外介质及细胞与细胞间的信号传导， 从而抑制血 管生成。 此外该序列中还含有新生血管抑制序列， 研究发现其具有高效抑制新生血管生成的 活性， 特别是对类风湿性关节炎类疾病有很好的作用。 血管抑制多肽针对抑制血管新生治疗 类风湿关节炎的作用靶点， 为开发新型类风湿关节炎药物提供新的研究方向。
新设计的多肽序列多肽 Π和多肽 III是对整合素具有靶向结合作用和亲和性的多肽， 前期 研究发现多肽 Π和多肽 III能够抑制内皮细胞的增殖、 迁移、 小管生成， 并用流式细胞仪分析 了多肽 Π和多肽 III的作用靶点为整合素 ανβ3。 后研究发现其还能抑制大鼠动脉环的管状结构 形成， 并通过细胞粘附实验验证了多肽 II和多肽 III的作用靶点为整合素 co^3， 并发现其对类 风湿性关节炎有治疗作用， 增加了适用症， 拓展了其社会价值和经济价值。 发明内容
发明目的
本发明针对多肽 I、 多肽 II和多肽 III做了进一步研究， 发现其对多种实体肿瘤和类风湿 性关节炎有治疗作用， 增加了其适应症。
技术方案
整合素阻断剂多肽， 其特征在于其氨基酸序列为：
X-Phe-Gln-Pro-Val-Leu-His-Leu-Val-Ala-Leu-Asn-Ser-Pro-Leu-Ser-Gly-Gly-Met- Arg- Gly-Ile-A rg-Gly-Ala-Asp-Phe-Gln-Cys-Phe-Gln-Gln-Ala-Arg-Ala-Val-Gly-Leu-Ala-Gly-Thr-Phe-Arg-Ala-Y
， X的序列为缺失、 Arg-Gly-Asp、 Arg-Gly- Asp-Gly-Gly-Gly-Gly Ala-Cys-Asp-Cys-Arg-Gly-
Asp-Cys-Phe-Cys或 Ala-Cys- Asp-Cys- Arg-Gly- Asp-Cys-Phe-Cys-Gly-Gly-Gly-Gly; Y的序列为 缺失、 Arg-Gly-Asp、Arg-Gly-Asp-Gly-Gly-Gly-Gly、Ala-Cys- Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys 或 Ala-Cys- Asp-Cys- Arg-Gly- Asp-Cys-Phe-Cys-Gly-Gly-Gly-Gly。
所述的整合素阻断剂多肽， 其特征在于所述的氨基酸序列为：
多肽 I： Phe-Gln-Pro-Val-Leu-His-Leu-Val- Ala-Leu- Asn-Ser-Pro-Leu-Ser-Gly-Gly-
Met-Arg-Gly-Ile-Arg-Gly-Ala-Asp-Phe-Gln-Cys-Phe-Gln-Gln-Ala-Arg-Ala-Val-Gly
-Leu-Ala-Gly-Thr-Phe-Arg-Ala;
多月太 II : Arg-Gly-Asp-Gly-Gly-Gly-Gly-Phe-Gln-Pro-Val-Leu-His-Leu-Val-Ala-Leu-
Asn-Ser-Pro-Leu-Ser-Gly-Gly-Met-Arg-Gly-Ile-Arg-Gly-Ala-Asp-Phe-Gln-Cys-Phe-Gln-Gln-Ala-
Arg-Ala-Val-Gly-Leu-Ala-Gly-Thr-Phe-Arg-Ala;
多肽 III： Phe-Gln-Pro-Val-Leu-His-Leu-Val-Ala-Leu-Asn-Ser-Pro-Leu-Ser-Gly-Gly- Met-Arg-Gly-Ile-Arg-Gly-Ala-Asp-Phe-Gln-Cys-Phe-Gln-Gln-Ala-Arg-Ala-Val-Gly-Leu-Ala-Gly- m
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根据权利要求 4整合素阻断剂在制备治疗肿瘤药物中的应用， 其特征在于所述的药物组合物 可通过多种给药方式治疗原发或继发的癌、 黑色素瘤及肉瘤， 包括皮下或肌肉注射， 静脉注 射或者静脉滴注， 口服给药如药丸、 胶囊等， 鼻喷剂。
有益效果
1、先前专利 ZL200610039298.2报道了内皮抑素第 6-49个氨基酸具有较好的抗肿瘤作用， 本发明对此做了大量研究， 发现内皮抑素第 6-48个氨基酸 （多肽 I， 命名为 EDSM, 见 Seq NO.1 ) 具有更好的抗肿瘤作用， 同时还发现多肽 I能有效治疗类风湿性关节炎， 增强了适应 性， 增加了适用症， 同时减少一个氨基酸， 合成成本更低， 拓展了其社会价值和经济价值。
多肽 II和多肽 III是本实验室设计合成的新的氨基酸序列， 尚未在国内外申请发明专利授 权， 先前专利 ZL200610039298.2所报道的 EDSM-1和 EDSM-2氨基酸数目分别是 47和 55， 相比之前的序列， 多肽 II和多肽 III在整合素配体序列 RGD和新生血管抑制序列之间加入了 4 个 Gly， 在 EDSM-1的基础上增加了柔性， 使得整合素配体序列 RGD更易与靶点结合， 活性 更高； 在 EDSM-2的基础上减少了氨基酸数目， 但依然保持了较高的抗肿瘤活性， 大大降低 了成本； 同时还发现多肽 Π和多肽 III具有较好的治疗类风湿性关节炎的作用， 增强了适应性， 增加了适用症， 拓展了其社会价值和经济价值。
2、 药理作用机理介绍：
研究发现， 多肽 I具有抑制肿瘤血管生成和治疗类风湿性关节炎的作用。 精氨酸-甘氨酸 -天冬氨酸 (RGD)序列是整合素的一个重要配体，因此，含有 RGD序列的多肽 Gly-Gly-Gly-Gly -Arg-Gly-Asp也能够特异性的识别整合素。 本发明的整合素阻断剂多肽是在具有抑制血管生 成作用的序 Phe-Gln-Pro-Val-Leu-His-Leu-Val-Ala-Leu-Asn-Ser-Pro-Leu-Ser-Gly-Gly-Met-Arg- Gly-Ile-Arg-Gly-Ala-Asp-Phe-Gln-Cys-Phe-Gln-Gln-Ala-Arg-Ala-Val-Gly-Leu-Ala-Gly-Thr-Phe- Arg-Ala的 N/C端连接上与整合素家族具有亲和性和结合能力的含有精氨酸 -甘氨酸-天冬氨酸
(RGD) 的 Gly-Gly-Gly-Gly- Arg-Gly-Asp序列， 构建了与整合素有亲和性和结合能力的多肽 II和多肽 III。 该整合素阻断剂： 多肽 II (Arg-Gly-Asp-Gly-Gly-Gly-Gly-Phe-Gln-Pro- Val-Leu-His-Leu-Val- Ala-Leu- Asn-Ser-Pro-Leu-Ser-Gly-Gly-Met-Arg-Gly-Ile-Arg-Gly-Ala-Asp-P he-Gln-Cys-Phe-Gln-Gln-Ala-Arg-Ala-Val-Gly-Leu-Ala-Gly-Thr-Phe- Arg-Ala) 和多肽 III
(Phe-Gln- Pro-Val-Leu-His-Leu-Val-Ala-Leu-Asn-Ser-Pro-Leu-Ser-Gly-Gly-Met-Arg-Gly-Ile- Arg-Gly-Ala-Asp-Phe-Gln-Cys-Phe-Gln-Gln-Ala-Arg-Ala-Val-Gly-Leu-Ala-Gly-Thr-Phe-Arg-Ala- Gly-Gly-Gly-Gly- Arg-Gly-Asp ) , 为含有 50个氨基酸的多肽， 分子中 RGD序列具有整合素亲 和性和结合能力， 研究表明其作用靶点主要为整合素 α ^3， 具有整合素拮抗剂作用， 能减少 细胞表面黏附分子的表达， 调节细胞内信号转导。 并且该序列中含有新生血管抑制序列， 从 而抑制肿瘤新生血管形成， 进而达到抑制肿瘤生长和转移的效果。 国内外大量的研究表明即使是同一组织类型、 分化程度相同的肿瘤， 对同一药物的敏感 性也有可能不同， 因此， 药物开发中对某一分子需要进行抗肿瘤谱的筛选。 因为有些药物只 对特定肿瘤细胞有效， 对其他肿瘤是无效或低效， 因此需要实验去摸索和验证其治疗效果。 发明人经过大量实验获知该整合素阻断剂靶点明确， 在体外能够明显抑制人脐静脉内皮细胞 (HUVEC ) 的迁移、 增殖及管状结构的形成， 对人的某些肿瘤细胞的增殖也有抑制作用。 体 内实验具有明显的抗肿瘤效果， 且副作用小、 用量少、 成本低。 说明本发明设计的整合素阻 断剂多肽科学、 合理、 可行有效， 能作为制备治疗人类实体肿瘤的治疗药物， 极大拓展了该 整合素阻断剂的治疗谱， 为将来药物开发提供了新的思路和前景， 具有显著的社会价值和市 场价值。
本发明的多肽 Π和多肽 III含有由于具有靶向性的 RGD序列，多肽可以靶向到 RA中血管 翳形成过程中新生血管内皮， 抑制新生血管形成， 进而达到预防或治疗类风湿性关节炎的效 果。 发明人经过大量实验获知多肽 II、 多肽 III能够抑制佐剂型大鼠类风湿关节炎和 DBA/1小 鼠胶原型类风湿性关节炎的发展， 体内实验证明具有显著的治疗类风湿型关节炎的效果， 并 且副作用少， 用量少成本降低。 说明本发明设计的多肽科学、 合理、 可行有效， 能作为治疗 或预防类风湿性关节炎药物， 极大拓展了该多肽的疾病治疗范围， 为将来药物开发提供了新 的思路和前景， 具有显著的社会价值和市场价值。
附图说明
附图 1 Western blotting检测 integrin ν和 integrin β3在 Bel-7402细胞中的表达；
附图 2 流式细胞实验检测整合素阻断剂多肽 II与靶点的结合；
附图 3 流式细胞实验检测整合素阻断剂多肽 III与靶点的结合；
具体实施方式
实施例 1 整合素阻断剂多肽合成肽的制备及检验 多肽 I、 II、 III均采用固相合成法合成， 采用高效液相色谱法纯化， 并采用 MS测定多 肽的分子量， RP-HPLC测定多肽的纯度。
多肽多肽 I固相合成法是以 Fmoc-Phe ( Otbu) -wang resin或 Fmoc- Arg (Otbu)-CTC resin 为起始原料， 多肽 II固相合成法是以 Fmoc-Arg ( Otbu) -wang resin或 Fmoc- Arg (Otbu)-CTC resin 为起始原料， 多月太 III固相合成法是以 Fmoc-Phe ( Otbu ) -wang resin 或 Fmoc- Phe (Otbu)-CTC resin 为起始原料， 然后用保护氨基酸依次接二肽至四十三 /五十肽， 接肽工作完 成后充分洗涤， 然后切肽、 后处理即得整合素阻断剂多肽 Π/多肽 III粗品。 将粗品溶解， 用制 备型高效液相经过两次纯化， 最后浓缩冻干得到纯品。 该方法不仅保证了合成的效率， 又提 高了纯度。
1. 接肽 （包括接二肽至四十三 /五十肽） 的步骤如下：
多肽 I： 称取 Fmoc-Phe ( Otbu) -wang resin或 Fmoc- Arg (Otbu)-CTC resin适量， 倒入玻 璃砂芯反应柱中， 加入 CH2C12 适量使树脂充分膨胀。
多肽 II： 称取 Fmoc- Arg (Otbu) -wang resin或 Fmoc- Arg (Otbu)-CTC resin适量， 倒入玻 璃砂芯反应柱中， 加入 CH2C12 适量使树脂充分膨胀。
多肽 III： 称取 Fmoc-Phe (Otbu) -wang resin或 Fmoc- Phe (Otbu)-CTC resin适量， 倒入玻 璃砂芯反应柱中， 加入 CH2C12 适量使树脂充分膨胀。
a、 脱帽：加入六氢吡啶 / DMF的脱帽液适量， 反应一段时间后将脱帽液抽干，中间用 DMF 洗涤一次， 然后再加入一次脱帽液适量反应， 脱去 Fmoc保护基。
b、 洗涤： 将脱帽液抽干， 用 DMF洗涤树脂几次， 充分洗净副产物。
c、 缩合： 将用于接肽的保护氨基酸和激活剂溶于 DMF和缩合剂中， 摇匀， 并充分反应。 d、 洗涤： 将反应液抽干， 用 DMF充分洗涤树脂， 洗净副产物。
所述切肽的步骤如下：
把抽干的树脂装入圆底烧瓶中， 加入切割液充分裂解合好的五十肽中间体， 用砂芯漏斗 将树脂与多肽分离，所述的裂解液的体积组成为：三氟乙酸：苯酚：水：苯甲硫醚： EDT=88-92: 2.5-3.5： 2.5-3.5： 1.5-2.5： 1.5-2.5。
2. 后处理步骤如下： 先加无水乙醚到切割液中将多肽析出， 然后离心， 把清液倒掉,然后再用 无水乙醚洗涤多肽， 抽干得多肽粗品。
3. 纯化的步骤如下：
a、 溶解： 称取粗品配制成 5-20 g/1的溶液， 用 0.45 μιη的混合滤膜过滤。
制备： ①一次纯化： 用 30%-40%的乙腈和 60%-70%的缓冲溶液， 流速为 50 ml/min-100 ml/min下冲洗 10 min-20 min平衡制备柱。用输液泵上样， 采集基线， 收集紫外波长 220 nm吸收值大于 200 mv 的溶液， 检验是否有样品。 用梯度洗脱， 乙腈的起始百分数为 30%-40%, 30-50 min后乙腈的百分数为 80%-90%。收集紫外波长 220 nm吸收值大于 200 mv的溶液， 检测纯度大于 95%的合并作为峰顶， 待做二次分离纯化。 ②二次纯化： 将 一次收到的峰顶旋转蒸发掉有机溶剂后用输液泵上样， 用 30%-40%的乙腈和 60%-70% 的缓冲溶液。流速 50-100 ml/min,并采集基线，收取在紫外波长 220 nm吸收大于 200 mv 的溶液， 检测是否有样品冲出。 用梯度洗脱， 乙腈的起始百分数为 30%-40%， 30-50 min 后乙腈的百分数为 80%-90%。收取在紫外波长 220 nm吸收大于 200 mv的溶液， 通过检 测纯度大于 95%的作为合格。
b、 浓缩、 过滤、 冻干： 将合格溶液用旋转蒸发仪 37°C减压浓缩， 除去残留溶剂和注射 用水。 最后用 0.22 μιη滤膜过滤， 滤液装入冻干盘中， 用冷冻干燥机进行冷冻干燥。 4. 纯度检测及分子量检测
收集冻干后的纯化产物， 通过反向液相检测多肽纯度分析纯度。 本实验成功采用固相合成法合成了整合素阻断剂多肽 I、 多肽 II和多肽 III， 此方法重复 性高， 可操作性强， 污染少； 实验可采用两种树脂来合成多肽即： wang resin或 CTC resin; 实验用 wang resin,相对其它树脂比较稳定，副反应少，工艺粗品峰型较好，纯化相对收率高， 所以成本相对较低； 实验用 CTC resin反应受温度影响小， 反应速率快； 并使用反相高效液相 法纯化多肽， 使用梯度洗脱相对于等度洗脱来说， 分离效果更好， 分离过程中， 保留时间合 适， 生产效率高， 纯度高。
结果： 合成的多肽经反向液相色谱分析进行纯度鉴定结果如下： 多肽 I、 多肽 II、 多肽
III纯度分别为 95.39%， 98.41%, 96.40%纯度均大于 95%， 符合设计要求。
实施例 2
整合素阻断剂多肽 II和多肽 III靶点分析。
( 1 ) Western Blotting分析细胞表达的靶点 integrin ανβ3
用 0. 25%胰蛋白酶消化对数生长期细胞 Bel-7402， 800 rpm离心 5 min收集细胞； 计数， 按 20 μ1/1 χ 105个细胞加入蛋白质提取液， 吹吸打散细胞， 4 °C放置 30 min使细胞裂解； 加入 1/4体积 5χ蛋白质上样缓冲液， 沸水浴 5-10 min。 取 20 μΐ蛋白质样品， 进行 10% SDS-PAGE 电泳； 半干式电转移法将凝胶上的蛋白质转移到 PVDF膜上， 恒流 l mA/cm2， 3 h; 将 PVDF 膜在丽春红中染色 30 s， dH20脱色至有清晰条带， 将右上角剪去以区分蛋白面； 将 PVDF膜 用封闭液室温封闭 1.5 h; PBST洗膜 2-3次， 每次 5-10 min。 加入一抗， 4°C过夜或 37°C孵育 1.5 h; PBST洗膜 3-5次， 每次 5-10 min。 加入二抗， 室温孵育 1 h; PBST洗膜 3-5次， 每次 5-10 min。 在暗室 （允许红色光源） 中， 将 PVDF膜蛋白面朝上放在保鲜膜上， 向表面均匀 滴加发光液， 反应 5 min; 将 PVDF膜包裹在保鲜膜中， 剪下一块等大的 X光胶片， 放置夹 片盒曝光 0.5-5 min (视条带光强度而定）； 取出 X光胶片， 在显影剂中漂至条带出现， 放到 停影液 （5%冰醋酸） 中停影 1 min， 用流水冲洗 1 min后在定影剂中漂至背景部分透明， 最 后流水冲洗 20 min固定影像。
(2) 流式细胞实验分析整合素阻断剂多肽 II和多肽 III与靶点的结合
细胞 Bel-7402在细胞瓶中培养至 80%的融合后， 消化收集， 用冰预冷的 PBS洗 2次， 将细胞用含 1% BSA 的 PBS 重悬 30min; 用 2 μΐ 鼠抗人 ανβ3 功能阻断单克隆抗体 (Ι .Ο μ§/μ1, 1 :200) 和 2 μΐ鼠抗人 α5β1功能阻断单克隆抗体 (；1.0 μ8/μ1, 1 :200)与细胞悬液在 4°C孵育 1.5 h; 将细胞收集， 并用冰预冷的 PBS洗涤 2次， 之后用 100 l FITC标记的修饰多肽 II和 多肽 111(2 mg/ml)与细胞悬液在 4°C孵育 1.5 h;标记后将细胞收集并用冰预冷的 PBS洗涤 2次， 之后 400 l PBS重悬， 用流式细胞仪分析， FITC荧光用 FL1通道检测荧光强度。
结果：细胞表达的靶点 integrin ανβ3的测定，如图 1所示， Bel-7402细胞表面表达 integrin ανβ3, 可以作为后期靶点结合试验的对象。
流式细胞试验分析整合素阻断剂多肽 Π和多肽 III与靶点的结合，结果如图 2、3所示: FITC 标记的整合素阻断剂多肽 II在细胞不加入抗体孵育是荧光强度大约是 80.4%， 加入 integrin ανβ3抗体之后荧光强度减弱至 13.4%， 加入 integrin α5β1抗体之后荧光强度减弱至 33.2%; FITC标记的整合素阻断剂多肽 III在细胞不加入抗体孵育是荧光强度大约是 82.4%，加入 α 抗体之后荧光强度减弱至 11.3%， 加入 integrin cd^抗体之后荧光强度减弱至 25.9%。经流式 细胞仪检测分析发现， 多肽 II和多肽 III能够同时和 integrin avW和 integrin α5βΙ结合， 但主 要的结合靶点仍为 integrin α ^3。
实施例 3
整合素阻断剂多肽对人脐静脉内皮细胞 （HUVEC) 的迁移抑制试验
将 lO mg/ml Matrigel (BD公司， USA)用 HUVEC专用培养基以 1 :2稀释， 涂布于 transwell 小室膜上， 室温风干。 将培养到对数生长期的 HUVEC细胞用胰蛋白酶消化液消化， 收集， 用 PBS洗涤两次后用空白 HUVECs专用培养基重悬。 在显微镜下计数， 将细胞浓度调整到 l x lO5 个 /ml。配制各组的试验用液，用空白 HUVECs专用培养基稀释到 100 μΐ。将细胞接种到 transwell 小室中， 每孔 ΙΟΟ μΙ, 并且将各组试验用液加入小室中。 24孔板中加入 0.6 ml含 5%胎牛血清和 1% ECGS的内皮细胞培养液剌激细胞迁移，于 5% C02， 37 °C培养 24 h。弃去孔中培液，用 90% 酒精常温固定 30 min， 0.1%结晶紫常温染色 10 min， 清水漂净， 用棉签轻轻擦掉上层未迁移 细胞，显微镜下观察并选择四个视野拍照计数。按照公式计算迁移抑制率（migration inhibition, MI):
Ntest
MI (%) = 1 100% 其中 Ntest为测试组的细胞迁移数， N∞ntTOl为空白对照组的细胞迁移数。
试验独立重复 3次， 试验得到的结果计算 mean ±SD， 并进行统计 t检验， *P<0.05为显著 性差异， **P<0.01为极显著性差异。
表 1 整合素阻断剂多 Jft I对人脐静脉内皮细胞 （HUVEC) 迁移抑制作用 组别 Cn=8;> 剂量 ^g/ml) 迁移细胞数 Ml (%)
Mean ± SD
多肽 I 0.5 979.8±75.3** 18.69%
1 956.0±78.2** 20.66%
2 961.3±89.5** 20.23%
4 903.5±68.7** 25.02%
8 815.5±62.32** 32.32%
16 706.3±48.7** 41.39%
32 871.8±67.9** 27.66%
64 929.5±99.7** 22.86%
ES 20 911.7±33.5** 24.34% control 0 1204.8±29.3 -
0.05, **P<0M.
表 2 整合素阻断剂多肽 II对人脐静脉内皮细胞 （HUVEC) 迁移抑制作用 组别 (n=8) 剂量 (^g/ml) 迁移细胞数 Ml (%)
Mean ± SD
多肽 II 0.13
875.0±35.3** 24.44%
0.25
800.7±64.8** 30.85%
0.5
685.7±32.5** 40.78%
1
611.0±74.1** 47.24%
2
600.3±39.3** 48.15%
4
531.5±72.6** 54.10%
8
348.8±39.9** 69.87%
16
460.1±97.3** 60.27%
20 880.2±33.3** 23.99%
_ 1158.0±54.5** __ a: * <0.05, **P<0M.
表 3 整合素阻断剂多肽 III对人脐静脉内皮细胞 （HUVEC) 迁移抑制作用
Group (n=8) Dose (^g/ml) Migration cell number Ml (%)
Mean士 SD
多肽 III 0.13
787.1±46.3** 23.08%
0.25
595.7±29.3** 41.78%
0.5
490.7±17.1** 52.04%
1
237.6±19.0** 76.78%
2
384.1±43.0** 62.46%
4
482.3±19.2** 52.86%
ES 20 866.0±27.2 15.37% control - 1023.2±11.1 ― a: * <0.05, **P<0M.
结果： 整合素阻断剂多肽 I对 HUVEC细胞迁移实验的影响见表 1， 与阴性对照相比， 整 合素阻断剂多肽 I能抑制 5%的胎牛血清及 1%ECGS诱导的 HUVEC的迁移作用， 并在中低剂 量下呈现一定的剂量依赖性。 在高中低各剂量下， 多肽 I对细胞迁移的抑制率与阴性对照相 比有极显著性的差异（**Ρ<0.01) ， 在高剂量下抑制内皮细胞迁移的能力相对中剂量有所下 降。 当多肽 I的剂量为 6μ§/ιη1时， 对细胞迁移的抑制率达到最大值 41.39%。
整合素阻断剂多肽 II对 HUVEC细胞迁移实验的影响见表 2， 与阴性对照相比， 整合素阻 断剂多肽 Π能抑制 5%的胎牛血清及 1%ECGS诱导的 HUVEC的迁移作用， 并在中低剂量下呈 现一定的剂量依赖性。 在高中低各剂量下， 多肽 Π对细胞迁移的抑制率与阴性对照相比有极 显著性的差异 （**Ρ<0.01) ， 在高剂量下抑制内皮细胞迁移的能力相对中剂量有所下降。 在 剂量 4 g/ml， 8 g/ml， 16 g/ml时抑制率都达到了 50%以上， 分别为 54.10%， 69.87%, 60.27% 和 57.24%。 当多肽 II的剂量为 8 g/ml时， 对细胞迁移的抑制率达到最大值 69.87%。
整合素阻断剂多肽 III对 HUVEC细胞增殖实验的影响见表 3， 与阴性对照相比， 整合素阻 断剂多肽 III能抑制 5%的胎牛血清及 1%ECGS诱导的 HUVEC的迁移作用， 并在中低剂量下呈 现一定的剂量依赖性， 在高中低各剂量下， 在高剂量下抑制内皮细胞迁移的能力相对中剂量 有所下降。 多肽 III在各剂量下对细胞迁移的抑制率与阴性对照相比有极显著性的差异 (* < 0.01) ， 在 1 g/ml， 2 μβ /ml ， 4 g/ml时抑制率都达到了 50%以上， 分别为 52.04%， 76.78% 和 62.64%， 当多肽多肽 III的剂量为 2 g/ml时， 对细胞迁移的抑制率达到最大值 76.78%。
实施例 4
整合素阻断剂多肽对人脐静脉内皮细胞 （HUVEC) 的小管生成抑制试验 将 -20 °C冻存的 10 mg/ml Matrigel (BD公司， USA) 放于 4 冰箱过夜化开， 用 HUVEC 培养基以 1 : 1稀释， 30 μΐ涂布于 96孔板 （Greiner公司， USA) 上， 37 °C温箱中聚合 1 h。 将培 养到对数生长期的 HUVEC细胞用 0.2% EDTA消化， 收集，用 PBS洗涤两次用空白 HUVEC培养 基重悬。 在显微镜下计数， 将细胞浓度调整到 lxlO5个 /ml。 配制各组的试验用液， 用空白 HUVECs专用培养基稀释到 100 μ1。 将细胞接种到 96孔板中， 每孔 100 μ1， 并且将各组试验用 液加入小空中， 于 5%C02， 37 °C孵育。 于 6 h， 12 h, 24 h, 36 h， 48 h， 60 h每个剂量随机选 5个视野进行拍照并计数， 统计每个浓度下分化成管的数量并分析多肽 II和多肽 III对 HUVEC 细胞分化成管能力的影响。 按照公式计算小管生成抑制率：
Ntest
小管生成抑制率 （％) 1 - 100%
Ncontrol
其中 Ntest为测试组的小管数目， ^^^^为空白对照组的小管数目。
试验独立重复 3次， 试验得到的结果计算 mean ± SD， 并进行统计 t检验， *P < 0.05为显著 性差异， **P < 0.01为极显著性差异。
表 4 整合素阻断剂多肽 II对人脐静脉内皮细胞 （HUVEC) 小管生成的抑制作用 时间 6 h 12h
小管生 小管生成 剂量 小管数目 剂量 小管数目
组别 成抑制 抑制率
(n=5) ( g/ml) Mean ± SD ( g/ml) Mean ± SD
率 （％) (%)
0.25 26.0±4.3 8.45% 0.25 14.0±3.3 * 23.08%
0.5 20.0±0.8** 29.58% 0.5 11.4±1.6** 37.36%
1 18.5±5.0** 34.86% 1 10.7±1.2** 40.93% 多肽 II
2 7.2±0.8** 74.65% 2 3.5±0.5 ** 80.77%
4 6.0±1.0** 78.87% 4 3.3±2.5 ** 81.68%
8 8.0±1.4** 71.83% 8 4.3±0.5 ** 76.19%
ES 20 17.0±3.6** 40.14% 20 10.3±2.3 ** 43.22% taxol 10 0.0±0.0** 100.00% 10 0.0±0.0** 100.00% control ― 28.4±3.0 ― ― 18.2±1.7 ―
表 5 整合素阻断剂多肽 III对人脐静脉内皮细胞 （HUVEC) 小管生成的抑制作用
Time 6 h 12h
小管生 小管生成 剂量 小管数目 剂剂量量 小管数目
抑制率
Figure imgf000014_0001
(%) 0.25 26.4±1.9** 19.02% 0.25 16.4±1.5 ** 18.81%
0.5 20.2±2.3 ** 37.88% 0.5 14.6±2.0** 27.39% 多肽 III
1 11.0±1.0** 66.26% 1 8.6±1.5 ** 57.10%
2 10.6±2.8** 67.28% 2 9.0±1.0** 55.45%
ES 20 22.0±2.5 ** 32.52% 20 18.0±2.1 ** 10.89% taxol 10 0.0±0.0** 100% 10 0.0±0.0** 100% control 32.60±1.34 20.20±1.92 ― 结果： 多肽 II对 HUVECs分化的影响见表 4: 在 6 h至 60 h内分化成毛细血管样管状结构， 6 h时小管已经形成， 6 h和 12 h时小管数目最多， 12 h后小管渐渐减少， 至 60 h时基本消失。 多肽 Π各剂量下形成小管的数目均比阴性少， 可见各剂量下多肽 II对 HUVECs分化成小管均 有抑制作用， 在中低剂量时都能达到 50%以上， 且给药剂量和抑制率呈一定的剂量依赖关系。 多肽 Π剂量 2 μ§/ιη1、 4 μ§/ιηΚ 8 g/ml时生成的小管数目最少， 抑制率最高。在 6 h时多肽 II剂 量 2 g/ml、 4 g/ml、 8 §/!^抑制率分别达到74.65%、 78.87%、 71.83%, 在 12 h时多肽 II剂量 2 g/ml、 4 g/ml、 8 g/ml抑制率分别达到 80.77%、 81.68%、 76.19%。 多肽 II与内皮细胞作用 6 h后在剂量为 0.5 g/ml、 l g/ml、 2 g/ml、 4 g/ml、 8 g/ml时与阴性组具有极显著性差异。
多肽 III对 HUVECs分化的影响见表 5: 在 6 h至 60 h内分化成分枝状毛细血管样管状结 构， 6 h时小管已经形成， 6 h和 12 h时小管数目最多， 12 h后小管渐渐减少， 至 60 h时基本 消失。 多肽 III各剂量下形成小管的数目均比阴性少， 可见各剂量下多肽 III对 HUVECs分化成 小管均有抑制作用， 在中低剂量时都能达到 60%以上， 且给药剂量和抑制率呈一定的剂量依 赖关系。 多肽 III剂量 1 g/ml 、 2 g/ml时生成的小管数目最少， 抑制率最高。 在 6h时多肽 III剂量 1 g/ml 、 2 g/ml抑制率分别达到 66.26%、 67.28%。 在 12 h时多肽 III剂量 1 g/ml、 2 g/ml抑制率分别达到 57.10%、 55.45%。 多肽 III与内皮细胞作用 6 h后在所有剂量： 0.25 μ§/ιη1 、 0.5 μ§/ιηΚ 1 μ§/ιηΚ 2 g/ml下与阴性组均具有极显著性差异。
实施例 5
整合素阻断剂多肽 I、 多肽 II和多肽 III对黑色素瘤 B16F10 C57BL/6黑鼠移植肿瘤生长抑制 试验
取生长旺盛期的瘤组织在无菌条件下碾磨， 制备成 l x 107个 /ml细胞悬液， 以 0.1 ml接 种于小鼠右侧腋部皮下。小鼠移植瘤用游标卡尺测量移植瘤直径，待肿瘤生长至 100-200 mm3 后动物随机分组。 使用测量瘤径的方法， 动态观察整合素阻断剂多肽对受试动物肿瘤的抑制 效果。 肿瘤直径的测量次数为每 2天 1次， 每次测量同时还需称量鼠重。 实验组左侧腋部皮 下注射多肽， 阴性组同时给等量生理盐水， 给药 14 d， 其中环磷酰胺隔天皮下给药一次， 紫 杉醇组每 3天皮下给药一次， 多肽低剂量一天给药两次组一天给两次， 其它各组一天给药一 次。治疗 14 d后， 小鼠处死， 手术剥取瘤块称重。肿瘤体积 (tumor volume , TV)的计算公式为：
TV = l/2xaxb2
其中 a、 b分别表示长宽。
根据测量的结果计算出相对肿瘤体积（relative tumor volume, RTV),计算公式为： RTV = Vt/VQ。 其中 VQ为分笼给药时 (即 dQ)测量所得肿瘤体积， Vt为每一次测量时的肿瘤体积。 抗 肿瘤活性的评价指标为相对肿瘤增殖率 T/C (%)， 计算公式如下：
TRTV
T/C (%) = ΐοο%
起动起动
始物始物
Figure imgf000016_0001
TRTV: 治疗组 RTV ； CRTV: 阴性对照组 RTV。
试验独立重复 3次， 试验得到的结果计算 mean ± SD， 并进行统计 t检验， *P < 0.05为显著 性差异， **P < 0.01为极显著性差异。
表 6 多肽 I对黑色素瘤 B16F10 C57BL/6黑鼠移植肿瘤动动终终生长的抑制作用
末物末物
剂量
组别 （mg/kg/ 起始体重 （g) 终末体重（g) 瘤重 （g ) 抑瘤率 次）
2.65 ±1.22 - 阴性对照 一 16.7 ±1.0 12 21.9 ±1.4
0.77 ±0.62** 70.84% 紫杉醇 10 16.5 ±0.7 10 16.4 ±2.8
1.66 ±1.35 37.26% 多肽 I高 3 17.1 ±0.8 10 22.9 ±1.2
1.80±0.78 32.17% 多肽 I低 0.75 16.9 ±1.0 10 23.6 ±2.0 表 7 多肽 II对黑色素瘤 B16F10 C57BL/6黑鼠移植肿瘤生长的抑制作用 组别 剂量 起始体重 （g) 终末体重（g) 瘤重 （g) 抑瘤率
( mg/kg/
次）
阴性对照 15 .8 ±1. .0 12 22.9 ±1 .4 12 3.99±1.03 紫杉醇 10 15 .3 ±0. .7 8 16.8 ±2 .8 6 0.98±0.30** 75. .49% 环磷酰胺 15 15 .2 ±0. .9 8 17.3 ±1 .5 8 1.40±0.06** 64. .95% 多肽 II高 3 15 .7 ±0. .8 8 21.9 ±1 .2 6 2.36±0.38 40. .80% 多肽 II中 1.5 15 .1 ±0. .5 8 22.6 ±0 .6 7 2.63±0.69 34. .17% 多肽 II低 0.75 15 .3 ±1. .0 8 21.6 ±2 .0 6 1.63±0.53** 59. .06% 多肽 II低 0.75 15 .3 ±0. .9 8 22.4 ±1 .6 7 1.79±1.03** 55. .22% 天给药两次
表 8 多肽 III对黑色素瘤 B16F10 C57BL/6黑鼠移植肿瘤生长的抑制作用
(mg/kg/ 动物 动物
次）
阴性对照 一 15.8 ±1.0 12 22.9 ±1.4 12 3.99±1.03 一 紫杉醇 10 15.3 ±0.7 8 16.8 ±2.8 6 0.98±0.30** 75.49% 环磷酰胺 15 15.2 ±0.9 8 17.3 ±1.5 8 1.40±0.06** 64.95% 多肽 III高 0.375 14.7 ±0.6 8 22.2 ±1.8 7 1.47 ±0.42* 63.07% 多肽 III中 0.1875 15.4±0.5 8 21.2 ±1.5 7 1.60±1.09** 59.82% 多肽 III低 0.09375 15.5±0.6 8 21.5 ±0.8 6 1.25±0.47** 68.67% 多肽 III低一
0.09375 15.3±0.9 8 22.4 ±1.6 7 1.23±0.27** 69.21% 天给药两次
结果： 多肽 I对黑色素瘤 B16F10 C57BL/6黑鼠移植肿瘤生长的抑制作用见表 6， 紫杉醇 组 10 mg/kg, 对黑色素瘤 B16F10 C57BL/6黑鼠移植肿瘤的抑瘤率为 70.80%， 但对实验动物 的体重有显著影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 I高、 低剂量及低剂量一天给药两次组对黑色素瘤 B16F10 C57BL/6 黑鼠移植肿瘤的抑瘤率为 37.26%, 32.17%。 多肽 I对黑色素瘤 B16F10 C57BL/6黑鼠移植肿瘤生长抑制试验结果表明， 与阴性对照组相比， 多肽 I对黑色素瘤 B16F10 C57BL/6黑鼠移植肿瘤的生长有较好的抑制 作用； 与阴性对照组相比， 对实验动物的体重没有影响， 未见明显的毒副反应。
多肽 II对黑色素瘤 B16F10 C57BL/6黑鼠移植肿瘤生长的抑制作用见表 7， 紫杉醇组 10 mg/kg,对黑色素瘤 B16F10 C57BL/6黑鼠移植肿瘤的抑瘤率为 75.49%，但对实验动物的体重 有显著影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 环磷酰胺组 15 mg/kg,对黑色素瘤 B16F10 C57BL/6黑鼠移植肿瘤的抑瘤率为 72.89%但对实验动物的体重有 显著影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 Π高、 中、 低 剂量及低剂量一天给药两次组对黑色素瘤 B16F 10 C57BL/6黑鼠移植肿瘤的抑瘤率为 40.80%， 34.17%, 59.06%, 55.22%。 低剂量组和低剂量一天给药两次组肿瘤体积与阴性组肿瘤体积相 比有极显著性差异。 与阴性对照组相比， 对实验动物的体重没有影响， 未见明显的毒副反应。
多肽 III对黑色素瘤 B16F10 C57BL/6黑鼠移植肿瘤生长的抑制作用见表 8，多肽 III高、中、 低剂量及低剂量一天给药两次组对黑色素瘤 B16F10 C57BL/6 黑鼠移植肿瘤的抑瘤率为 63.07%, 59.82%, 68.67%, 69.21%。 高剂量组肿瘤体积与阴性组肿瘤体积相比具有显著性差 异， 中剂量组、 低剂量组和低剂量一天给药两次组肿瘤体积与阴性组肿瘤体积相比有极显著 性差异。 与阴性对照组相比， 多肽 ΠΙ0.09375 mg/kg 每天给药两次组对黑色素瘤 B16F10 C57BL/6黑鼠移植肿瘤的生长有最好的抑制作用； 与阴性对照组相比， 对实验动物的体重没 有影响， 未见明显的毒副反应。
实施例 6
整合素阻断剂多肽 I、多肽 II和多肽 III对人乳腺癌 MDA-MB-231裸小鼠异种移植肿瘤生长抑 制试验
取生长旺盛期的瘤组织在无菌条件下碾磨， 制备成 l x 107个 /ml细胞悬液， 以 0.1 ml接种 于小鼠右侧腋部皮下。 小鼠移植瘤用游标卡尺测量移植瘤直径， 待肿瘤生长至 100-200 mm3 后动物随机分组。 使用测量瘤径的方法， 动态观察整合素阻断剂多肽对受试动物肿瘤的抑制 效果。 肿瘤直径的测量次数为每 2天 1次，起始动物数每次测量同时还需称量鼠重。 实验组尾静脉注射 多肽， 阴性组同时给等量生理盐水， 给药 14 d。 阳性组为阿瓦斯汀。 阿瓦斯汀每 3天尾静脉 给药一次， 其他组一天尾静脉给药一次。 给药后休息一周， 21 d后， 小鼠处死， 手术剥取瘤 块称重。 肿瘤体积 (tumor volume ,TV)的计算公式为：
TV = l/2xaxb2
末动物数终
其中 a、 b分别表示长宽。
根据测量的结果计算出相对肿瘤体积（relative tumor volume, RTV),计算公式为： RTV = Vt/VQ。 其中 VQ为分笼给药时 (即 dQ)测量所得肿瘤体积， Vt为每一次测量时的肿瘤体积。 抗 肿瘤活性的评价指标为相对肿瘤增殖率 T/C (%)， 计算公式如下：
TRTV
T/C (%) = ΐοο%
Figure imgf000018_0001
TRTV: 治疗组 RTV ； CRTV: 阴性对照组 RTV。
试验独立重复 3次， 试验得到的结果计算 mean ± SD， 并进行统计 t检验， *P < 0.05为显著 性差异， **P < 0.01为极显著性差异。
表 9 多肽 I对人乳腺癌 MDA-MB-231裸小鼠异种移植肿瘤生长的抑制作用
组别 剂量 起始体重（g) 终末体重 瘤重 （g) 抑瘤率
( mg/kg/ (g)
次）
20.19 ±1.24 12 22.18 ±1.88 1.19±0.31
20.50 ±0.77 10 17.56 ±1.65 0.26±0.08**
19.97 ±1.19 10 19.67 ±1.36 0.62±0.21
20.35 ±0.95 10 19.34 ±1.13 0.56 ±0.10 多月太 I中 1.5 20.50 ±1.21 10 20.21±1.97 10 0.49 ±0.13** 58.82% 多月太 I低 0.75 20.69 ±1.20 10 22.28 ±1.75 12 0.58 ±0.22* 51.26% 表 10 多肽 II对人乳腺癌 MDA-MB-231裸小鼠异种移植肿瘤生长的抑制作用 组别 剂量 起始体重（g) 终末体重 瘤重 （g) 抑瘤率
( mg/kg/ (g)
次） 阴性对照 一 20.11±0.60 12 23.10±0.61 12 1.19±0.31 一 阿瓦斯汀 10 20.25±0.64 8 21.23±0.47 6 0.26±0.08** 78.53% 恩度 2.5 20.00±0.67 起始动物数 8 22.94±0.64 7 0.62±0.21 47.71% 起始动物数
多肽 II高 3 20.38±0.39 8 23.13±0.67 8 0.43±0.12* 63.80% 多肽 Π中 1.5 20.16±0.45 8 22.86±0.65 8 0.38±0.12** 67.82% 多肽 II低 0.75 20.31±0.50 8 23.03±0.65 8 0.50±0.17* 57.83% 表 11 多肽 III对人乳腺癌 MDA-MB-231裸小鼠异种移植肿瘤生长的抑制作用
末动物数终
起始体重（g) 终末体重 瘤重 （g) 抑瘤率 末动物数终
( mg/k (g)
g/次） 阴性对照 一 20.11±0.60 12 23.10±0.61 12 1.19±0.31 一 阿瓦斯汀 10 20.25±0.64 8 21.23±0.47 6 0.26±0.08** 78.53% 恩度 2.5 20.00±0.67 8 22.94±0.64 7 0.62±0.21 47.71% 多肽 III高 0.75 20.09±0.56 8 23.06±0.64 8 0.49±0.17* 58.82% 多肽 III中 0.375 20.00±0.58 8 22.63±0.57 8 0.32±0.09** 72.86% 多肽 III低 0.1875 20.03±0.44 8 23.10±0.42 8 0.45±0.15* 61.91% 结果： 多肽 I对人乳腺癌 MDA-MB-231裸小鼠异种移植肿瘤生长的抑制作用见表 9， 阿 瓦斯汀组， 对人乳腺癌 MDA-MB-231裸小鼠异种移植肿瘤的抑瘤率为 78.53%， 对实验动物 的体重无显著影响； 多肽 I高、 中、低剂量组对人乳腺癌 MDA-MB-231裸小鼠异种移植肿瘤 的抑瘤率为 52.95%， 58.82%, 51.26%。 低剂量与阴性组具有显著性差异， 中剂量组肿瘤体积 与阴性组肿瘤体积相比有极显著性差异。 与阴性对照组相比， 对实验动物的体重没有影响， 未见明显的毒副反应。
多肽 Π对人乳腺癌 MDA-MB-231裸小鼠异种移植肿瘤生长的抑制作用见表 10， 阿瓦斯 汀组， 对人乳腺癌 MDA-MB-231裸小鼠异种移植肿瘤的抑瘤率为 78.53%， 对实验动物的体 重无显著影响； 多肽 II高、 中、低剂量组对人乳腺癌 MDA-MB-231裸小鼠异种移植肿瘤的抑 瘤率为 63.80%， 67.82%, 57.83%。 高剂量组、 低剂量与阴性组具有显著性差异， 中剂量组肿 瘤体积与阴性组肿瘤体积相比有极显著性差异。 与阴性对照组相比， 对实验动物的体重没有 影响， 未见明显的毒副反应。
多肽 III对人乳腺癌 MDA-MB-231裸小鼠异种移植肿瘤生长的抑制作用见表 11， 阿瓦斯 汀组， 对人乳腺癌 MDA-MB-231裸小鼠异种移植肿瘤的抑瘤率为 68.95%， 对实验动物的体 重无显著影响； 多肽 III高、 中、低剂量组对人乳腺癌 MDA-MB-231裸小鼠异种移植肿瘤的抑 瘤率为 58.82%， 72.86%, 61.91%。 高剂量组、 低剂量与阴性组具有显著性差异， 中剂量组肿 瘤体积与阴性组肿瘤体积相比有极显著性差异。 与阴性对照组相比， 对实验动物的体重没有 影响， 未见明显的毒副反应。
实施例 7
整合素阻断剂多肽 I、多肽 II和多肽 III对人胃癌 MGC-803裸小鼠异种移植肿瘤生长抑制试验 肿瘤接种及检测评价方法见实施例 6。 阳性对照药奥沙利铂每 4天尾静脉给药一次， 希罗 达灌胃给药每两天给药尾静脉给药一次， 其他组每天尾静脉给药一次。
表 12 多肽 I对人胃癌
剂量
组别 （mg/kg/ 起始体重（g) 终末体重（g) 瘤重 （g ) 抑瘤率 次）
Figure imgf000020_0001
阴性对照 20.00±0.52 12 23.33±0.44 12 0.78 ±0.22
奥沙利铂 20.09±0.70 8 20.47±0.70 6
奥沙利铂 3.3 mg/kg
0.32 ±0.13* 51.76% +希罗达 希罗达 135
mg/kg
多肽 I高 3 20.18±0.61 23.23±0.67 0.48 ±0.13 38.28% 多肽 I中 1.5 20.02±0.52 22.66±0.61 8 0.42 ±0.10 38.46% 多肽 I低 0.75 20.14±0.52 22.98±0.60 8 0.50 ±0.13 35.90% 表 13多肽 II对人胃癌 MGC-803裸小鼠异种移植肿瘤生长的抑制作用
剂量
组别 （mg/kg/ 起始体重（g) 终末体重（g) 瘤重 （g ) 抑瘤率 次）
阴性对照 一 20.29 ±1.29 12 22.08 ±1.88 12 0.78 ±0.22 ― 奥沙利铂
奥 ίΦ禾 Ι|ί白 ^ m /
i m ' 20.20 ±0.75 10 17.50 ±1.95 9 0.32 ±0.13* 51.76%
+希罗达 希罗达 135
mg/kg
多月太 II高 3 19.95 ±1.21 10 19.60 ±1.26 10 0.48 ±0.13 38.28% 多肽 π中 1.5 20.15 ±0.97 10 19.67 ±1.41 10 0.16 ±0.10** 79.72% 多肽 π低 0.75 20.00 ±1.22 10 20.40 ±1.26 10 0.23 ±0.13** 70.67% 表 14多肽 III对人胃癌 MGC-803裸小鼠异种移植肿瘤生长的抑制作用 组别 剂量 起始体重 (g) 终末体重（g) 瘤重 (g) 抑瘤率
( mg/kg/
次）
阴性对照 20.29 ±1.29 12 22.08 ±1.88 12 0.78 ±0.22 一 奥沙利铂
奥沙利铂 +希 3.3 mg/kg
20.20 ±0.75 10 17.50 ±1.95 9 0.32 ±0.13* 51.76% 罗达 希罗达 135
mg/kg
多肽 III高 0. 75 19.75 ±0.65 1 ^0 20.88 ±0.85 10 0.33 ±0.09 57.65% 多肽 III中 0.375 19.63 ±0.75 10 20.13 ±1.03 10 0.27 ±0.06* 66.03% 多肽 III低 0.1875 20.25 ±0.65 10 22.25 ±1.71 10 0.23 ±0.05** 70.08% 结果： 多肽 I对人胃癌 MGC-803裸小鼠异种移植肿瘤生长的抑制作用见表 12， 化药联 合用药组， 对人胃癌 MGC-803裸小鼠异种移植肿瘤的抑瘤率为 51.76%， 但对实验动物的体 重有显著影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 I高、 中、
¾
低剂量组对人胃癌 MGC-803裸小鼠异种移植肿瘤的抑瘤率为 38.28%， 38.46%, 35.90%。 与 阴性对照组相比， 对实验动物的体重没有影响， 未见明显的毒副反应。
多肽 Π对人胃癌 MGC-803裸小鼠异种移植肿瘤生长的抑制作用见表 13， 化药联合用药 组， 对人胃癌 MGC-803裸小鼠异种移植肿瘤的抑瘤率为 51.76%， 但对实验动物的体重有显 著影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 II高、 中、 低剂 量组对人胃癌 MGC-803裸小鼠异种移植肿瘤的抑瘤率为 38.28%， 79.72%, 70.67%。 中剂量 组和低剂量组肿瘤体积与阴性组肿瘤体积相比有极显著性差异。 与阴性对照组相比， 对实验 动物的体重没有影响， 未见明显的毒副反应。
多肽 III对人胃癌 MGC-803裸小鼠异种移植肿瘤生长的抑制作用见表 14， 化药联合用药 组， 对人胃癌 MGC-803裸小鼠异种移植肿瘤的抑瘤率为 51.76%， 但对实验动物的体重有显 著影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 III高、 中、 低剂 量组对人胃癌 MGC-803裸小鼠异种移植肿瘤的抑瘤率为 57.65%， 66.03%, 70.08%。 中剂量 组肿瘤体积与阴性组肿瘤体积相比有显著性差异， 低剂量组肿瘤体积与阴性组肿瘤体积相比 有极显著性差异。 与阴性对照组相比， 对实验动物的体重没有影响， 未见明显的毒副反应。
实施例 8
整合素阻断剂多肽 I、 多肽 II和多肽 III对人肺癌 H460裸小鼠异种移植肿瘤生长抑制试验 肿瘤接种及检测评价方法见实施例 6。 阳性对照药紫杉醇组每 3天尾静脉给药一次， 其 他组每天尾静脉给药一次。
表 15多肽 I对人肺癌 H460裸小鼠异种移植肿瘤生长的抑制作用 组别 剂量 起始体重 终末体重 瘤重 （g) 抑瘤率
( mg/kg/次） （g) (g)
阴性对照 19.99±0.72 1 0.88±0.23 一
2
紫杉醇 10 20.13±0.57 8 17.80±0.63 7 0.28±0.07** 68.21% 多肽 I高 3 19.94±0.65 8 22.96±0.69 8 0.47±0.17* 46.60% 起始动物数起始动物数
多肽 I中 1.5 20.05±0.52 8 22.93±0.51 8 0.40±0.05** 54.54% 多肽 I低 0.75 19.91±0.57 8 23.13±0.58 8 0.51±0.17* 42.05% 表 16多肽 II对人肺癌 H460裸小鼠异种移植肿瘤生长的抑制作用 组别 剂量 起始体重 终末体重 瘤重 （g) 抑瘤率
( mg/kg/次） （g) (g) 末动物数末动物数终终
阴性对照 一 20.13±0.61 12 23.07±0.53 1 0.88±0.23
2
紫杉醇 10 20.05±0.68 8 18.09±0.55 7 0.28±0.07** 68.21% 多肽 II高 3 20.21±0.57 8 22.59±0.53 8 0.37±0.17* 57.68% 多肽 Π中 1.5 20.06±0.47 8 23.26±0.40 8 0.30±0.05** 65.37% 多肽 II低 0.75 20.02±0.51 8 22.87±0.40 8 0.41±0.17* 53.49% 表 17 多肽 III对人肺癌 H460裸小鼠异种移植肿瘤生长的抑制作用
组别 剂量 起始体重 (g) 起 终末体重（g) 终 瘤重 (g) 抑瘤率
( mg/kg/ 始 末
次） 动 动
物 物
数 数
阴性对照 一 20.13±0.61 12 23.07±0.53 12 0.88±0.23 一 紫杉醇 10 20.05±0.68 8 18.09±0.55 7 0.28±0.07** 68.21% 多肽 III高 0. 75 20.14±0.64 8 23.14±0.51 8 0.28±0.11** 68.05% 多肽 III中 0.375 19.89±0.62 8 23.12±0.53 8 0.23±0.06** 74.42% 多肽 III低 0.1875 20.36±0.68 8 23.15±0.49 8 0.27±0.05** 69.23% 结果： 多肽 I对人肺癌 H460裸小鼠异种移植肿瘤生长的抑制作用见表 15， 化药紫杉醇 组， 对人肺癌 H460裸小鼠异种移植肿瘤的抑瘤率为 68.21%， 但对实验动物的体重有显著影 响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 I高、 中、 低剂量组 对人肺癌 H460裸小鼠异种移植肿瘤的抑瘤率为 46.60%， 54.54%, 42.05%。 高剂量组、 低齐 U 量与阴性组有显著性差异， 中剂量组肿瘤体积与阴性组肿瘤体积相比有极显著性差异。 与阴 性对照组相比， 对实验动物的体重没有影响， 未见明显的毒副反应。
多肽 II对人肺癌 H460裸小鼠异种移植肿瘤生长的抑制作用见表 16， 化药紫杉醇组， 对 人肺癌 H460裸小鼠异种移植肿瘤的抑瘤率为 68.21%， 但对实验动物的体重有显著影响， 与 阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 II高、 中、 低剂量组对人肺 起始动物数
癌 H460裸小鼠异种移植肿瘤的抑瘤率为 57.68%， 65.37%, 53.49%。 高剂量组、 低剂量与阴 性组有显著性差异， 中剂量组肿瘤体积与阴性组肿瘤体积相比有极显著性差异。 与阴性对照 组相比， 对实验动物的体重没有影响， 未见明显的毒副反应。
多肽 III对人肺癌 H460裸小鼠异种移植肿瘤生长的抑制作用见表 17， 化药紫杉醇组， 对 人肺癌 H460裸小鼠异种移植肿瘤的抑瘤率为 68.21%， 但对末动物数终实验动物的体重有显著影响， 与 阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 III高、 中、 低剂量组对人肺 癌 H460裸小鼠异种移植肿瘤的抑瘤率为 68.05%， 74.42%, 69.23%。 高剂量组、 中剂量组、 低剂量与阴性组均有极显著性差异。 与阴性对照组相比， 对实验动物的体重没有影响， 未见 明显的毒副反应。
实施例 9
整合素阻断剂多肽 I、 多肽 II和多肽 III对人肝癌 SMMC-7721 裸小鼠异种移植肿瘤生长抑制 试验
肿瘤接种及检测评价方法见实施例 6。 阳性对照药紫杉醇组每 3天尾静脉给药一次， 其他 组每天尾静脉给药一次。
表 18多肽 I对人肝癌 SMMC-7721裸小鼠异种移植肿瘤生长的抑制作用
起始体重（g) 终末体重 瘤重 （g) 抑瘤率
(mg/k (g)
g/次） 阴性对照 一 19.77±0.61 12 23.04±0.61 12 1.27±0.23
紫杉醇 10 20.11±0.52 8 17.22±0.51 7 0.28±0.05** 78.10% 恩度 2.5 19.83±0.49 8 23.04±0.68 8 0.79±0.29 36.96% 多肽 I高 3 20.18±0.62 8 22.84±0.55 8 0.55±0.12* 56.70% 多肽 I中 1.5 20.23±0.67 8 22.99±0.55 8 0.47±0.14** 63.00% 多月太 I低 0.75 19.93±0.54 8 23.17±0.63 8 0. 53±0.04* 58.26% 表 19 多肽 II对人肝癌 SMMC-7721裸小鼠异种移植肿瘤生长的抑制作用 组别 剂量 起始体重 (g) 终末体重 瘤重 （g) 抑瘤率
( mg/kg/ (g)
次） 阴性对照 一 19.83±0.61 12 23.26±0.55 12 1.27±0.23 一 紫杉醇 10 19.81±0.54 8 16.90±0.51 7 0.28±0.05** 78.10% 恩度 2.5 19.73±0.50 8 23.02±0.62 8 0.79±0.29 36.96% 起始动物数
多肽 II高 3 19.87±0.66 8 22.90±0.59 8 0.39±0.05** 68.75% 起始动物数
多肽 Π中 1.5 20.15±0.56 8 22.93±0.60 8 0.31±0.07** 75.12% 多肽 II低 0.75 20.10±0.39 8 23.21±0.60 8 0.36±0.05** 71.54% 表 20多肽 III对人肝癌 SMMC-7721裸小鼠异种移植肿瘤生长的抑制作用
剂量 起始体重（g) 终末体重 瘤重 （g) 抑瘤率 ( mg/k 末动物数终
(g)
g/次） 末动物数终
阴性对照 一 19.83±0.61 12 23.26±0.55 12 1.27±0.23 一 紫杉醇 10 19.81±0.54 8 16.90±0.51 7 0.28±0.05** 78.10% 恩度 2.5 19.73±0.50 8 23.02±0.62 8 0.79±0.29 36.96% 多肽 III高 0.75 19.89±0.72 8 22.63±0.39 8 0.28±0.03** 77.55% 多肽 III中 0.375 19.97±0.50 8 23.25±0.54 8 0.22±0.03** 82.55% 多肽 III低 0.1875 19.91±0.46 8 22.70±0.57 8 0.35±0.04** 71.85% 结果： 多肽 I对人肝癌 SMMC-7721裸小鼠异种移植肿瘤生长的抑制作用见表 18， 化药 紫杉醇组， 对人肝癌 SMMC-7721裸小鼠异种移植肿瘤的抑瘤率为 78.10%， 但对实验动物的 体重有显著影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 I高、 中、低剂量组对人肝癌 SMMC-7721裸小鼠异种移植肿瘤的抑瘤率为 56.70%， 63.00%， 58.26%。 中剂量组与阴性组有极显著性差异， 高剂量组、 低剂量与阴性组有显著性差异。 与阴性对照 组相比， 对实验动物的体重没有影响， 未见明显的毒副反应。
多肽 II对人肝癌 SMMC-7721裸小鼠异种移植肿瘤生长的抑制作用见表 19， 化药紫杉醇 组， 对人肝癌 SMMC-7721裸小鼠异种移植肿瘤的抑瘤率为 78.10%， 但对实验动物的体重有 显著影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 Π高、 中、 低 剂量组对人肝癌 SMMC-7721裸小鼠异种移植肿瘤的抑瘤率为 68.75%， 75.12%, 71.54%。 高 剂量组、 中剂量组、 低剂量与阴性组有极显著性差异。 与阴性对照组相比， 对实验动物的体 重没有影响， 未见明显的毒副反应。
多肽 III对人肝癌 SMMC-7721裸小鼠异种移植肿瘤生长的抑制作用见表 20， 化药紫杉醇 组， 对人肝癌 SMMC-7721裸小鼠异种移植肿瘤的抑瘤率为 78.10%， 但对实验动物的体重有 显著影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 III高、 中、 低 剂量组对人肝癌 SMMC-7721裸小鼠异种移植肿瘤的抑瘤率为 77.55%， 82.55%, 71. 85%。 高 剂量组、 中剂量组、 低剂量与阴性组有极显著性差异。 与阴性对照组相比， 对实验动物的体 重没有影响， 未见明显的毒副反应。
实施例 10
整合素阻断剂多肽 I、 多肽 II和多肽 III对人宫颈癌 HeLa裸小鼠异种移植肿瘤生长抑制试验 起始动物数
起始动物数
肿瘤接种及检测评价方法见实施例 6。 阳性对照药顺铂组每 3天尾静脉给药一次， 其他 组每天尾静脉给药一次。
表 21多肽 I对人宫颈癌 HeLa裸小鼠异种移植肿瘤生长的抑制作用 组别 剂量 起始体重 (g) 终末体重 瘤重 （g) 抑瘤率
(mg/kg/ 末动物数终
(g)
末动物数终
次） 阴性对照 一 20.00±0.52 12 23.33±0.44 12 1.40±0.37 一 顺铂 10 20.09±0.70 8 20.47±0.70 6 0.38±0.10** 73.13% 恩度 2.5 20.18±0.61 8 23.23±0.67 7 0.80±0.20 43.08% 多肽 I高 3 20.07±0.53 8 23.18±0.51 8 0.52±0.05** 62.85% 多肽 I中 1.5 20.17±0.48 8 22.96±0.57 8 0.41±0.07** 70.71% 多肽 I低 0.75 19.62±0.52 8 22.94±0.46 8 0.49±0.05** 65.00% 表 22 多肽 II对人宫颈癌 HeLa裸小鼠异种移植肿瘤生长的抑制作用 组别 剂量 起始体重 终末体重 瘤重 （g) 抑瘤率
(mg/kg/ (g) (g)
次） 阴性对照 一 20. 05±0. .58 12 23.12±0. 57 12 1.40±0.37 一 顺铂 10 20. 16±0. .54 8 17.28±0. 56 7 0.38±0.10** 73.13% 恩度 2.5 19. 84±0. .55 8 23.14±0. 54 8 0.80±0.20 43.08% 多肽 Π高 3 19. 92±0. .75 8 22.99±0. 52 8 0.62±0.18* 55.82% 多肽 Π中 1.5 20. 12±0. .63 8 22.88±0. 55 8 0.45±0.11** 68.19% 多肽 II低 0.75 20. 03±0. .57 8 23.14±0. 55 8 0.50±0.12** 64.45% 表 23 多肽 III对人宫颈癌 HeLa裸小鼠异种移植肿瘤生长的抑制作用 起始体重（g) 终末体重 瘤重 （g) 抑瘤率
(mg/k (g)
g/次） 阴性对照 一 20.05±0.58 12 23.12±0.57 12 1.40±0.37 一 顺铂 10 20.16±0.54 8 17.28±0.56 7 0.38±0.10** 73.13% 恩度 2.5 19.84±0.55 8 23.14±0.54 8 0.80±0.20 43.08% 多肽 III高 0.75 19.76±0.43 8 22.88±0.66 8 0.50±0.15** 64.35% 多肽 III中 0.375 20.19±0.60 8 23.17±0.58 8 0.39±0.08** 72.41% 起始动物数
多肽 III低 0.1875 19.98±0.65 8 23.10±0.56 8 0.44±0.11** 68.68% 结果： 多肽 I对人宫颈癌 HeLa裸小鼠异种移植肿瘤生长的抑制作用见表 21， 化药顺铂 组， 对人宫颈癌 HeLa裸小鼠异种移植肿瘤的抑瘤率为 73.13%， 但对实验动物的体重有显著 影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 I高、 中、 低剂量 组对人宫颈癌 HeLa裸小鼠异种移植肿瘤的抑瘤率为 62.85%末动物数终， 70.71%, 65.00%。 高剂量组、 中剂量组、低剂量组肿瘤体积与阴性组肿瘤体积相比均有极显著性差异。与阴性对照组相比， 对实验动物的体重没有影响， 未见明显的毒副反应。
多肽 II对人宫颈癌 HeLa裸小鼠异种移植肿瘤生长的抑制作用见表 22， 化药顺铂组， 对 人宫颈癌 HeLa裸小鼠异种移植肿瘤的抑瘤率为 73.13%， 但对实验动物的体重有显著影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 II高、 中、 低剂量组对人 宫颈癌 HeLa裸小鼠异种移植肿瘤的抑瘤率为 55.82%， 68.19%, 64.45%。 高剂量组、 中剂量 组、 低剂量组肿瘤体积与阴性组肿瘤体积相比均有极显著性差异。 与阴性对照组相比， 对实 验动物的体重没有影响， 未见明显的毒副反应。
多肽 III对人宫颈癌 HeLa裸小鼠异种移植肿瘤生长的抑制作用见表 23， 化药顺铂组， 对 人宫颈癌 HeLa裸小鼠异种移植肿瘤的抑瘤率为 73.13%， 但对实验动物的体重有显著影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 III高、 中、 低剂量组对人 宫颈癌 HeLa裸小鼠异种移植肿瘤的抑瘤率为 64.35%， 72.41%, 68.68%。 高剂量组、 中剂量 组、 低剂量组肿瘤体积与阴性组肿瘤体积相比均有极显著性差异。 与阴性对照组相比， 对实 验动物的体重没有影响， 未见明显毒副作用。
实施例 11
整合素阻断剂多肽 I、多肽 II和多肽 III对人子宫内膜癌 HHUA裸小鼠异种移植肿瘤生长抑制 试验
肿瘤接种及检测评价方法见实施例 6。 阳性对照药紫杉醇组每 3天尾静脉给药一次， 其 他组每天尾静脉给药一次。
表 24多肽 I对人子宫内膜癌 HHUA裸小鼠异种移植肿瘤生长的抑制作用
组别 剂量 起始体重 终末体重 瘤重 （g) 抑瘤率
( mg/kg/ (g) (g)
次） 阴性对照 一 20.09±0.66 12 23.01±0.48 12 1.14±0.35 一 紫杉醇 10 20.94±0.58 8 18.88±0.68 7 0.23±0.07** 79.82% 恩度 2.5 20.88±0.79 8 22.89±0.51 8 0.65±0.15 43.00% 起始动物数
多肽 I高 3 19.82±0.60 8 23.14±0.48 8 0.60±0.14 47.36% 起始动物数起始动物数
多肽 I中 1.5 19.96±0.58 8 22.93±0.56 8 0.51±0.10* 55.30% 多肽 I低 0.75 19.87±0.58 8 22.81±0.60 8 0.52±0.12* 54.39% 表 25 多肽 II对人子宫内膜癌 HHUA裸小鼠异种移植肿瘤生长的抑制作用
组别 剂量 起始体重（g) 终末体重 瘤重 （g) 抑瘤率
( mg/kg/ 末动物数终
(g)
次） 末动物数终末动物数终 阴性对照 一 20.09±0.66 12 23.01±0.48 12 1.14±0.35 一 紫杉醇 10 20.94±0.58 8 18.88±0.68 7 0.23±0.07** 79.82% 恩度 2.5 20.88±0.79 8 22.89±0.51 8 0.65±0.15 43.00% 多肽 II高 3 20.08±0.59 8 23.04±0.55 8 0.46±0.11** 59.65% 多肽 Π中 1.5 19.70±0.52 8 22.95±0.46 8 0.37±0.07** 67.54% 多肽 II低 0.75 20.28±0.61 8 22.93±0.46 8 0.41±0.11** 64.03% 表 26 多肽 III对人子宫内膜癌 HHUA裸小鼠异种移植肿瘤生长的抑制作用 组别 剂量 起始体重 （g) 终末体重 瘤重 （g) 抑瘤率
( mg/k (g)
g/次） 阴性对照 一 20. .09±0. .66 12 23. .01±0.48 12 1.14±0.35
紫杉醇 10 20. .94±0. .58 8 18. .88±0.68 7 0.23±0.07** 79. .82% 恩度 2.5 19. .88±0. .79 8 22. .89±0.51 8 0.65±0.15 43. .00% 多肽 III高 0.75 19. .89±0. .53 8 23. .07±0.56 8 0.38±0.10** 66. .67% 多肽 III中 0.375 20. .31±0. .51 8 23. .18±0.57 8 0.30±0.06** 73. .21% 多肽 III低 0.1875 19. .74±0. .55 8 22. .92±0.57 8 0.33±0.07** 70. .53% 结果： 多肽 I对人子宫内膜癌 HHUA裸小鼠异种移植肿瘤生长的抑制作用见表 24，化药 紫杉醇组， 对人子宫内膜癌 HHUA裸小鼠异种移植肿瘤的抑瘤率为 79.82%， 但对实验动物 的体重有显著影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 I高、 中、 低剂量组对人子宫内膜癌 HHUA裸小鼠异种移植肿瘤的抑瘤率为 47.36%， 55.30%, 54.39%。 中剂量组、 低剂量组肿瘤体积与阴性组肿瘤体积相比均有显著性差异。 与阴性对照 组相比， 对实验动物的体重没有影响， 未见明显的毒副反应。
多肽 II对人子宫内膜癌 HHUA裸小鼠异种移植肿瘤生长的抑制作用见表 25，化药紫杉醇 组， 对人子宫内膜癌 HHUA裸小鼠异种移植肿瘤的抑瘤率为 79.82%， 但对实验动物的体重 有显著影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 II高、 中、 起始动物数
低剂量组对人子宫内膜癌 HHUA裸小鼠异种移植肿瘤的抑瘤率为 59.65%， 67.54%, 64.03%。 高剂量组、 中剂量组、 低剂量组肿瘤体积与阴性组肿瘤体积相比均有极显著性差异。 与阴性 对照组相比， 对实验动物的体重没有影响， 未见明显的毒副反应。
多肽 III对人子宫内膜癌 HHUA裸小鼠异种移植肿瘤生长的抑制作用见表 26，化药紫杉醇 末动物数终
组， 对人子宫内膜癌 HHUA裸小鼠异种移植肿瘤的抑瘤率为 73.13%， 但对实验动物的体重 有显著影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 III高、 中、 低剂量组对人子宫内膜癌 HHUA裸小鼠异种移植肿瘤的抑瘤率为 66.67%， 73.21%, 70.53%。 高剂量组、 中剂量组、 低剂量组肿瘤体积与阴性组肿瘤体积相比均有极显著性差异。 与阴性 对照组相比， 对实验动物的体重没有影响， 未见明显毒副作用。
实施例 12
整合素阻断剂多肽 I、 多肽 II和多肽 III对人前列腺癌 DU-145裸小鼠异种移植肿瘤生长抑制 试验
肿瘤接种及检测评价方法见实施例 6。 阳性对照药顺铂组每 3天尾静脉给药一次， 其他 组每天尾静脉给药一次。
表 27 多肽 I对人前列腺癌 DU-145裸小鼠异种移植肿瘤生长的抑制作用 组别 剂量 起始体重 （g) 终末体重 瘤重 （g) 抑瘤率
( mg/k (g)
g/次）
19.77±0.61 23.04±0.61 12 1.43±0.34 一 20.11±0.52 17.22±0.51 7 0.37±0.10** 73.84% 19.83±0.49 23.04±0.68 8 0.86±0.16 38.46% 19.88±0.58 22.99±0.37 8 0.48±0.10** 66.43% 19.97±0.66 23.10±0.61 8 0.40±0.16** 72.02% 多月太 I低 0.75 20.07±0.54 8 22.85±0.61 8 0.46±0.14** 67.83% 表 28多肽 II对人前列腺癌 DU-145裸小鼠异种移植肿瘤生长的抑制作用
组别 剂量 起始体重 终末体重 瘤重 （g) 抑瘤率
( mg/kg/ (g) (g)
次） 阴性对照 一 19.77±0.61 12 23.04±0.61 12 1.43±0.34 一 顺铂 10 20.11±0.52 8 17.22±0.51 7 0.37±0.10** 73.84% 恩度 2.5 19.83±0.49 8 23.04±0.68 8 0.86±0.16 38.46% 起始动物数
多肽 Π高 3 19.63±0.49 8 22.99±0.55 8 0.65±0.14* 54.25% 起始动物数
多肽 Π中 1.5 20.08±0.46 8 23.04±0.48 8 0.46±0.10** 67.83% 多肽 II低 0.75 20.31±0.60 8 23.10±0.48 8 0.53±0.12** 62.92% 表 29 多肽 III对人前列腺癌 DU-145裸小鼠异种移植肿瘤生长的抑制作用 起始体重（g) 终末体重 瘤重 （g) 抑瘤率 末动物数终
( mg/k (g)
g/次） 末动物数终
阴性对照 10 19.77±0.61 12 23.04±0.61 12 1.43±0.34 一
5 20.11±0.52 8 17.22±0.51 7 0.37±0.10** 73.84%
2.5 19.83±0.49 8 23.04±0.68 8 0.86±0.16 38.46% 多肽 III高 0.75 20.18±0.62 8 22.84±0.55 8 0.56±0.14** 60.73% 多肽 III中 0.375 20.23±0.67 8 22.99±0.55 8 0.43±0.13** 69.90% 多肽 III低 0.1875 19.93±0.54 8 23.17±0.63 8 0.51±0.15** 64.08% 结果： 多肽 I对人前列腺癌 DU-145裸小鼠异种移植肿瘤生长的抑制作用见表 27， 化药 顺铂组， 对人前列腺癌 DU-145裸小鼠异种移植肿瘤的抑瘤率为 73.84%， 但对实验动物的体 重有显著影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 I高、 中、 低剂量组对人前列腺癌 DU-145裸小鼠异种移植肿瘤的抑瘤率为 66.43%， 72.02%, 67.83%。 高剂量组、 中剂量组、 低剂量组肿瘤体积与阴性组肿瘤体积相比均有极显著性差异。 与阴性 对照组相比， 对实验动物的体重没有影响， 未见明显毒副作用。
多肽 Π对人前列腺癌 DU-145裸小鼠异种移植肿瘤生长的抑制作用见表 28，化药顺铂组， 对人前列腺癌 DU-145裸小鼠异种移植肿瘤的抑瘤率为 73.84%， 但对实验动物的体重有显著 影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 II高、 中、 低剂量 组对人前列腺癌 DU-145裸小鼠异种移植肿瘤的抑瘤率为 54.25%， 67.83%, 62.92%。 高剂量 组肿瘤体积与阴性组肿瘤体积相比具有显著性差异， 中剂量组、 低剂量组肿瘤体积与阴性组 肿瘤体积相比均有极显著性差异。 与阴性对照组相比， 对实验动物的体重没有影响， 未见明 显的毒副反应。
多肽 III对人前列腺癌 DU-145裸小鼠异种移植肿瘤生长的抑制作用见表 29，化药顺铂组， 对人前列腺癌 DU-145裸小鼠异种移植肿瘤的抑瘤率为 73.84%， 但对实验动物的体重有显著 影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 III高、 中、 低剂量 组对人前列腺癌 DU-145裸小鼠异种移植肿瘤的抑瘤率为 60.73%， 69.90%, 64.08%。 高剂量 组、 中剂量组、 低剂量组肿瘤体积与阴性组肿瘤体积相比均有极显著性差异。 与阴性对照组 相比， 对实验动物的体重没有影响， 未见明起始动物数起始动物数显毒副作用。
实施例 13
整合素阻断剂多肽 I、 多肽 II和多肽 III对人睾丸癌 5637裸小鼠异种移植肿瘤生长抑制试验 肿瘤接种及检测评价方法见实施例 6。 阳性对照药 5-氟尿嘧啶组每 3天尾静脉给药一次， 其他组每天尾静脉给药一次。
末动物数末动物数终终
表 30多肽 I对人睾丸癌 5637裸小鼠异种移植肿瘤生长的抑制作用
组别 剂量 起始体重（g) 终末体重 瘤重 （g) 抑瘤率
(mg/kg/ (g)
次） 阴性对照 一 20.09±0.65 12 23.11±0.61 12 1.26±0.29 一
5-Fu 10 19.90±0.48 8 17.03±0.53 7 0.28±0.06** 77.80% 恩度 2.5 20.00±0.70 8 22.98±0.72 8 0.76±0.21 39.49% 多肽 I高 3 19.83±0.49 8 23.04±0.68 8 0.57±0.11* 54.76% 多肽 I中 1.5 19.63±0.49 8 22.99±0.55 8 0.46±0.10** 63.49% 多肽 I低 0.75 20.08±0.46 8 23.04±0.48 8 0.55±0.11* 56.34% 表 31多肽 II对人睾丸癌 5637裸小鼠异种移植肿瘤生长的抑制作用
组别 剂量 起始体重（g) 终末体重 瘤重 （g) 抑瘤率
(mg/kg/ (g)
次）
20.09±0.65 23.11±0.61 12 1.26±0.29 ― 19.90±0.48 17.03±0.53 7 0.28±0.06** 77.80%
20.00±0.70 22.98±0.72 8 0.76±0.21 39.49%
20.03±0.61 23.16±0.51 8 0.60±0.16* 52.49% 多肽 II中 1.5 20.05±0.58 8 22.96±0.59 8 0.45±0.10** 64.32% 多肽 II低 0.75 19.97±0.62 8 23.09±0.59 8 0.49±0.11** 60.96% 表 32多肽 III对人睾丸癌 5637裸小鼠异种移植肿瘤生长的抑制作用 剂 起始体重 （g) 终末体重 瘤重 （g) 抑瘤率
(g)
(mg/
kg/次） 阴性对照 一 20.09±0.65 12 23.11±0.61 12 1.26±0.29 一
5-Fu 10 19.90±0.48 8 17.03±0.53 7 0.28±0.06** 77.80% 起始动物数
恩度 2.5 20.00±0.70 8 22.98±0.72 8 0.76±0.21 39.49% 多肽 III高 0.75 19.96±0.57 8 23.12±0.73 8 0.50±0.10** 60.51% 多肽 III中 0.375 20.03±0.60 8 23.35±0.50 8 0.44±0.12** 65.11% 多肽 III低 0.1875 19.89±0.57 8 23.02±0.64 8 0.47±0.12** 62.63% 结果： 多肽 I对人睾丸癌 5637裸小鼠异种移植肿瘤生末动物数终长的抑制作用见表 30， 化药 5-Fu 组， 对人睾丸癌 5637裸小鼠异种移植肿瘤的抑瘤率为 77.80%， 但对实验动物的体重有显著 影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 I高、 中、 低剂量 组对人睾丸癌 5637裸小鼠异种移植肿瘤的抑瘤率为 54.76%， 63.49%, 56.34%。 高剂量组、 低剂量组肿瘤体积与阴性组肿瘤体积相比具有显著性差异， 中剂量组肿瘤体积与阴性组肿瘤 体积相比均有极显著性差异。 与阴性对照组相比， 对实验动物的体重没有影响， 未见明显的 毒副反应。
多肽 II对人睾丸癌 5637裸小鼠异种移植肿瘤生长的抑制作用见表 31， 化药 5-Fu组， 对 人睾丸癌 5637裸小鼠异种移植肿瘤的抑瘤率为 77.80%， 但对实验动物的体重有显著影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 II高、 中、 低剂量组对人 睾丸癌 5637裸小鼠异种移植肿瘤的抑瘤率为 52.49%， 64.32%, 60.96%。 高剂量组肿瘤体积 与阴性组肿瘤体积相比具有显著性差异， 中剂量组、 低剂量组肿瘤体积与阴性组肿瘤体积相 比均有极显著性差异。 与阴性对照组相比， 对实验动物的体重没有影响， 未见明显的毒副反 应。
多肽 III对人睾丸癌 5637裸小鼠异种移植肿瘤生长的抑制作用见表 32， 化药 5-Fu组， 对 人睾丸癌 5637裸小鼠异种移植肿瘤的抑瘤率为 73.84%， 但对实验动物的体重有显著影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 III高、 中、 低剂量组对人 睾丸癌 5637裸小鼠异种移植肿瘤的抑瘤率为 60.51%， 65.11%, 62.63%。 高剂量组、 中剂量 组、 低剂量组肿瘤体积与阴性组肿瘤体积相比均有极显著性差异。 与阴性对照组相比， 对实 验动物的体重没有影响， 未见明显毒副作用。
实施例 14
整合素阻断剂多肽 I、 多肽 II和多肽 III对人胆囊癌 GBC-SD裸小鼠异种移植肿瘤生长抑制试 验
肿瘤接种及检测评价方法见实施例 6。 阳性对照药阿瓦斯汀组每 2天尾静脉给药一次， 其他组每天尾静脉给药一次。
表 33多肽 I对人胆囊癌 GBC-SD裸小鼠异种移植肿瘤生长的抑制作用
组别 剂量 起始体重（g) 终末体重 瘤重 （g) 抑瘤率
( mg/kg/ 起始动物数
(g)
次） 起始动物数起始动物数 阴性对照 一 20.09±0.34 12 23.11±0.70 12 1.20±0.30 一 阿瓦斯汀 10 20.13±0.65 8 20.89±0.61 7 0.25±0.06** 78.85% 末动物数终
恩度 2.5 20.11±0.59 8 23.13±0.38 7 0.71±0.15 40.83% 末动物数末动物数终终
多肽 I高 3 20.88±0.79 8 22.89±0.51 8 0.50±0.11* 58.33% 多肽 I中 1.5 20.08±0.59 8 23.04±0.55 8 0.42±0.10** 65.00% 多肽 I低 0.75 19.70±0.52 8 22.95±0.46 8 0.52±0.11* 56.67% 表 34 多肽 II对人胆囊癌 GBC-SD裸小鼠异种移植肿瘤生长的抑制作用
组别 剂量 起始体重（g) 终末体重 瘤重 （g) 抑瘤率
( mg/kg/ (g)
次） 阴性对照 一 20.09±0.34 12 23.11±0.70 12 1.20±0.30 一 阿瓦斯汀 10 20.13±0.65 8 20.89±0.61 7 0.25±0.06** 78.85% 恩度 2.5 20.11±0.59 8 23.13±0.38 7 0.71±0.15 40.83% 多肽 II高 3 20.06±0.63 8 23.10±0.64 8 0.47±0.11* 60.48% 多肽 Π中 1.5 19.80±0.39 8 22.99±0.51 8 0.37±0.10** 69.19% 多肽 II低 0.75 20.01±0.51 8 22.76±0.51 8 0.46±0.11* 62.00% 表 35 多肽 III对人胆囊癌 GBC-SD裸小鼠异种移植肿瘤生长的抑制作用
起始体重（g) 终末体重 瘤重 （g) 抑瘤率
( mg/k (g)
g/次） 阴性对照 一 20.09±0.34 12 23.11±0.70 12 1.20±0.30 阿瓦斯汀 10 20.13±0.65 8 20.89±0.61 0.25±0.06** 78.85% 2.5 20.11±0.59 8 23.13±0.38 0.71±0.15 40.83% 多肽 III高 0.75 20.22±0.40 8 23.10±0.56 8 0.44±0.11* 63.73% 多肽 III中 0.375 20.10±0.56 8 22.85±0.57 8 0.30±0.09** 74.62% 多肽 III低 0.1875 20.05±0.52 8 22.94±0.51 8 0.43±0.09* 64.55% 结果： 多肽 I对人胆囊癌 GBC-SD裸小鼠异种移植肿瘤生长的抑制作用见表 313种移植 肿瘤的抑瘤率为 58.33%， 65.00%, 56.67%。 高剂量组、 低剂量组肿瘤体积与阴性组肿瘤体积 相比具有显著性差异， 中剂量组肿瘤体积与阴性组肿瘤体积相比有极显著性差异。 与阴性对 照组相比， 对实验动物的体重没有影响， 未见明显的毒副反应。
起始动物数
多肽 II对人胆囊癌 GBC-SD裸小鼠异种移植肿瘤生长的抑制作用见表 34， 阿瓦斯汀组， 对人胆囊癌 GBC-SD裸小鼠异种移植肿瘤的抑瘤率为 78.85%， 与阴性对照组相比， 对实验动 物的体重没有影响； 多肽 II高、 中、 低剂量组对人胆囊癌 GBC-SD裸小鼠异种移植肿瘤的抑 瘤率为 60.48%， 69.19%, 62.00%。 高剂量组、 低剂量组肿瘤体积与阴性组肿瘤体积相比具有 显著性差异， 中剂量组肿瘤体积与阴性组肿瘤体积相比有极显著末动物数终性差异。与阴性对照组相比， 对实验动物的体重没有影响， 未见明显的毒副反应。
多肽 III对人胆囊癌 GBC-SD裸小鼠异种移植肿瘤生长的抑制作用见表 35， 阿瓦斯汀组， 对人胆囊癌 GBC-SD裸小鼠异种移植肿瘤的抑瘤率为 76.79%， 与阴性对照组相比， 对实验动 物的体重没有影响； 多肽 III高、 中、 低剂量组对人胆囊癌 GBC-SD裸小鼠异种移植肿瘤的抑 瘤率为 63.73%， 74.62%, 64.55%。 高剂量组、 低剂量组肿瘤体积与阴性组肿瘤体积相比具有 显著性差异， 中剂量组肿瘤体积与阴性组肿瘤体积相比有极显著性差异。与阴性对照组相比， 对实验动物的体重没有影响， 未见明显的毒副反应。
实施例 15
整合素阻断剂多肽 I、 多肽 II和多肽 III对人膀胱癌 HT1376裸小鼠异种移植肿瘤生长抑制试 肿瘤接种及检测评价方法见实施例 6。 阳性对照药阿瓦斯汀组每 3天尾静脉给药 其他组每天尾静脉给药一次。
表 36 多肽 I对人膀胱癌 HT1376裸小鼠异种移植肿瘤生长的抑制作用
组别 剂量 起始体重（g) 终末体重 瘤重 （g) 抑瘤率
( mg/kg/ (g)
次） 阴性对照 一 20.21±0.60 12 23.03±0.43 12 1.26±0.32 阿瓦斯汀 10 19.83±0.58 8 20.94±0.47 0.24±0.06** 79.60% 恩度 2.5 20.15±0.52 8 22.99±0.62 0.73±0.16 42.06% 多肽 I高 3 19.84±0.55 8 23.14±0.54 0.64±0.13 49.20% 多肽 I中 1.5 19.76±0.43 8 22.88±0.66 0.55±0.09* 56.34% 多肽 I低 0.75 20.19±0.60 8 23.17±0.58 0.59±0.16 53.17% 表 37 多肽 II对人膀胱癌 HT1376裸小鼠异种移植肿瘤生长的抑制作用
组别 剂量 起始体重（g) 终末体重 瘤重 （g) 抑瘤率
( mg/kg/ (g)
次）
起始动物数
起始动物数
阴性对照 一 20.21±0.60 12 23.03±0.43 12 1.26±0.32 一 阿瓦斯汀 10 19.83±0.58 8 20.94±0.47 7 0.24±0.06** 79.60% 恩度 2.5 20.15±0.52 8 22.99±0.62 7 0.73±0.16 42.06% 多肽 II高 3 19.85±0.55 8 23.21±0.62末动物数终 8 0.47±0.12* 60.97% 多肽 Π中 1.5 20.01±0.66 8 22.88±0.63 8 0.35±0.09** 71.10% 末动物数终
多肽 II低 0.75 20.00±0.67 8 22.86±0.63 8 0.39±0.10** 67.62% 表 38 多肽 III对人膀胱癌 HT1376裸小鼠异种移植肿瘤生长的抑制作用
起始体重（g) 终末体重 瘤重 （g) 抑瘤率
( mg/k (g)
g/次） 阴性对照 一 20.21±0.60 12 23.03±0.43 12 1.26±0.32 一 阿瓦斯汀 10 19.83±0.58 8 20.94±0.47 7 0.24±0.06** 79.60% 恩度 2.5 20.15±0.52 8 22.99±0.62 7 0.73±0.16 42.06% 多肽 III高 0.75 20.12±0.70 8 22.90±0.69 8 0.38±0.10** 67.92% 多肽 III中 0.375 20.13±0.67 8 22.85±0.46 8 0.28±0.08** 76.94% 多肽 III低 0.1875 19.89±0.62 8 23.20±0.61 8 0.33±0.09** 72.55% 结果： 多肽 I对人膀胱癌 HT1376裸小鼠异种移植肿瘤生长的抑制作用见表 36， 阿瓦斯 汀组， 对人膀胱癌 HT1376裸小鼠异种移植肿瘤的抑瘤率为 79.60%， 与阴性对照组相比， 对 实验动物的体重没有影响； 多肽 I高、 中、 低剂量组对人膀胱癌 HT1376裸小鼠异种移植肿 瘤的抑瘤率为 49.20%， 56.34%, 53.17%。 中剂量组肿瘤体积与阴性组肿瘤体积相比极显著性 差异。 与阴性对照组相比， 对实验动物的体重没有影响， 未见明显的毒副反应。
多肽 II对人膀胱癌 HT1376裸小鼠异种移植肿瘤生长的抑制作用见表 37， 阿瓦斯汀组， 对人膀胱癌 HT1376裸小鼠异种移植肿瘤的抑瘤率为 79.60%， 与阴性对照组相比， 对实验动 物的体重没有影响； 多肽 II高、 中、 低剂量组对人膀胱癌 HT1376裸小鼠异种移植肿瘤的抑 瘤率为 60.97%， 71.10%, 67.72%。高剂量组肿瘤体积与阴性组肿瘤体积相比具有显著性差异， 中剂量组、 低剂量组肿瘤体积与阴性组肿瘤体积相比有极显著性差异。 与阴性对照组相比， 对实验动物的体重没有影响， 未见明显的毒副反应。
多肽 III对人膀胱癌 HT1376裸小鼠异种移植肿瘤生长的抑制作用见表 38， 阿瓦斯汀组， 对人膀胱癌 HT1376裸小鼠异种移植肿瘤的抑瘤率为 79.60%， 与阴性对照组相比， 对实验动 物的体重没有影响； 多肽 III高、 中、 低剂量组对人膀胱癌 HT1376裸小鼠异种移植肿瘤的抑 瘤率为 67.92%， 76.94%, 72.55%。 高剂量组、 低剂量组、 中剂量组肿瘤体积与阴性组肿瘤体 起始动物数
积相比均有极显著性差异。 与阴性对照组相起始动物数比， 对实验动物的体重没有影响， 未见明显的毒 副反应。
实施例 16
整合素阻断剂多肽 I、 多肽 II和多肽 III对人胰腺癌 SW-1990裸小鼠异种移植肿瘤生长抑制试 末动物数终
验
末动物数终
肿瘤接种及检测评价方法见实施例 6。 阳性对照药阿瓦斯汀组每 3天尾静脉给药一次， 其他组每天尾静脉给药一次。
表 39 多肽 I对人胰腺癌 SW-1990裸小鼠异种移植肿瘤生长的抑制作用
起始体重（g) 终末体重 瘤重 （g) 抑瘤率
( mg/k (g)
g/次） 阴性对照 一 19.87±0.41 12 23.08±0.59 12 1.29±0.32 一 阿瓦斯汀 10 19.94±0.59 8 20.99±0.66 7 0.27±0.07** 79.07% 恩度 2.5 19.88±0.50 8 22.87±0.57 7 0.65±0.14 49.66% 多肽 I高 3 20.16±0.45 8 22.86±0.65 8 0.58±0.10* 55.04% 多肽 I中 1.5 20.31±0.50 8 23.03±0.65 8 0.47±0.08** 63.57% 多肽 I低 0.75 20.09±0.56 8 23.06±0.64 8 0.51±0.09** 60.47% 表 40 多肽 II对人胰腺癌 SW-1990裸小鼠异种移植肿瘤生长的抑制作用
组别 剂量 起始体重（g) 终末体重 瘤重 （g) 抑瘤率
( mg/kg/ (g)
次） 阴性对照 一 19.87±0.41 12 23.08±0.59 12 1.29±0.32 阿瓦斯汀 10 19.94±0.59 8 20.99±0.66 0.27±0.07** 79.07% 恩度 2.5 19.88±0.50 8 22.87±0.57 0.65±0.14 49.66% 多肽 II高 3 20.21±0.64 8 23.11±0.61 0.45±0.10** 65.05% 多肽 Π中 1.5 19.76±0.55 8 22.8±0.57 0.35±0.09** 72.97% 多肽 II低 0.75 19.91±0.58 8 22.80±0.57 0.39±0.10** 70.09% 表 41多肽 III对人胰腺癌 SW-1990裸小鼠异种移植肿瘤生长的抑制作用 剂量 起始体重（g) 终末体重 瘤重 （g) 抑瘤率 ( mg/k (g)
g/次）
起始动物数 阴性对照 一 19.87±0.41 12 23.08±0.59 12 1.29±0.32 一 阿瓦斯汀 10 19.94±0.59 8 20.99±0.66 7 0.27±0.07** 79.07% 恩度 2.5 19.88±0.50 8 22.87±0.57 7 0.65±0.14 49.66% 多肽 III高 0.75 20.28±0.53 8 23.00±0.39 8 0.37±0.08** 71.01% 末动物数终
多肽 III中 0.375 20.05±0.60 8 22.93±0.56 8 0.28±0.06** 78.64% 多肽 III低 0.1875 20.27±0.67 8 23.21±0.61 8 0.33±0.08** 74.68% 结果： 多肽 I对人胰腺癌 SW-1990裸小鼠异种移植肿瘤生长的抑制作用见表 39， 阿瓦斯 汀组， 对人胰腺癌 SW-1990裸小鼠异种移植肿瘤的抑瘤率为 79.07%， 与阴性对照组相比， 对 实验动物的体重没有影响； 多肽 I高、 中、 低剂量组对人胰腺癌 SW-1990裸小鼠异种移植肿 瘤的抑瘤率为 55.04%， 63.57%, 60.47%。 高剂量组、 中剂量组、 低剂量组肿瘤体积与阴性组 肿瘤体积相比均具有极显著性差异。 与阴性对照组相比， 对实验动物的体重没有影响， 未见 明显的毒副反应。
多肽 II对人胰腺癌 SW-1990裸小鼠异种移植肿瘤生长的抑制作用见表 40， 阿瓦斯汀组， 对人胰腺癌 SW-1990裸小鼠异种移植肿瘤的抑瘤率为 79.07%， 与阴性对照组相比，对实验动 物的体重没有影响； 多肽 II高、 中、 低剂量组对人胰腺癌 SW-1990裸小鼠异种移植肿瘤的抑 瘤率为 65.05%， 72.97%, 70.09%。 高剂量组、 中剂量组、 低剂量组肿瘤体积与阴性组肿瘤体 积相比均具有极显著性差异。 与阴性对照组相比， 对实验动物的体重没有影响， 未见明显的 毒副反应。
多肽 III对人胰腺癌 SW-1990裸小鼠异种移植肿瘤生长的抑制作用见表 41， 阿瓦斯汀组， 对人胰腺癌 SW-1990裸小鼠异种移植肿瘤的抑瘤率为 79.07%， 与阴性对照组相比，对实验动 物的体重没有影响； 多肽 III高、 中、 低剂量组对人胰腺癌 SW-1990裸小鼠异种移植肿瘤的抑 瘤率为 71.01%， 78.64%, 74.68%。 高剂量组、 中剂量组、 低剂量组肿瘤体积与阴性组肿瘤体 积相比均具有极显著性差异。 与阴性对照组相比， 对实验动物的体重没有影响， 未见明显的 毒副反应。
实施例 17
整合素阻断剂多肽 I、 多肽 II和多肽 III对人食管癌 Ecl09裸小鼠异种移植肿瘤生长抑制试验 肿瘤接种及检测评价方法见实施例 6。 阳性对照药阿瓦斯汀组每 3天尾静脉给药一次， 其他组每天尾静脉给药一次。
表 42多肽 I对对人食管癌 Ecl09裸小鼠异种移植肿瘤生长的抑制作用
起始体重（g) 终末体重 瘤重 （g) 抑瘤率
(mg/k 起始动物数起始动物数
(g)
g/次） 起始动物数 阴性对照 一 20.19±0.57 12 22.82±0.63 12 1.53±0.38 一 阿瓦斯汀 10 20.04±0.55 8 20.93±0.51 7 0.47±0.10** 69.00% 末动物数末动物数终终
恩度 2.5 19.86±0.49 8 22.92±0.45 7 0.89±0.24 41.57% I高 3 20.00±0.67 8 22.94±0.64 7 末动物数终
多肽 0.54±0.08** 64.71% 多肽 I中 1.5 20.09±0.56 8 23.06±0.64 8 0.47±0.06** 69.00% 多肽 I低 0.75 20.00±0.58 8 22.63±0.57 8 0.53±0.08** 65.36% 表 43 多肽 II对人食管癌 Ecl09裸小鼠异种移植肿瘤生长的抑制作用
组别 剂量 起始体重（g) 终末体重 瘤重 （g) 抑瘤率
(mg/kg/ (g)
次） 阴性对照 一 20.19±0.57 12 22.82±0.63 12 1.53±0.38 一 阿瓦斯汀 10 20.04±0.55 8 20.93±0.51 7 0.47±0.10** 69.00% 恩度 2.5 19.86±0.49 8 22.92±0.45 7 0.89±0.24 41.57% 多肽 II高 3 19.93±0.64 8 22.90±0.59 8 0.71±0.15* 53.34% 多肽 Π中 1.5 20.06±0.56 8 23.08±0.58 8 0.55±0.12** 63.88% 多肽 II低 0.75 20.05±0.66 8 22.97±0.58 8 0.62±0.13* 59.65% 表 44 多肽 III对人食管癌 Ecl09裸小鼠异种移植肿瘤生长的抑制作用 剂量 起始体重（g) 终末体重 瘤重 （g) 抑瘤率 (mg/k (g)
g/次） 阴性对照 一 20.19±0.57 12 22.82±0.63 12 1.53±0.38 阿瓦斯汀 10 20.04±0.55 8 20.93±0.51 0.47±0.10** 69.00% 2.5 19.86±0.49 8 22.92±0.45 0.89±0.24 41.57% 多肽 III高 0.75 19.98±0.57 8 23.17±0.67 8 0.62±0.18* 59.75% 多肽 III中 0.375 20.18±0.53 8 23.31±0.73 8 0.50±0.12** 67.13% 多肽 III低 0.1875 20.06±0.44 8 23.14±0.52 8 0.53±0.14** 65.63% 结果： 多肽 I对人食管癌 Ecl09裸小鼠异种移植肿瘤生长的抑制作用见表 42， 阿瓦斯汀 组， 对人食管癌 Ecl09裸小鼠异种移植肿瘤的抑瘤率为 69.00%， 与阴性对照组相比， 对实验 动物的体重没有影响； 多肽 I高、 中、 低剂量组对人食管癌 Ecl09裸小鼠异种移植肿瘤的抑 瘤率为 64.71%， 69.00%, 65.36%。 高剂量组、 中剂量组、 低剂量组肿瘤体积与阴性组肿瘤体 起始动物
积相比有极显著性差异。 与阴性对照组相比， 对实验动物的体重没有影响， 未见明显的毒副 反应。
多肽 II对人食管癌 Ecl09裸小鼠异种移植肿瘤生长的抑制作用见表 43， 阿瓦斯汀组， 对 人食管癌 Ecl09裸小鼠异种移植肿瘤的抑瘤率为 69.00%， 与阴性对照组相比， 对实验动物的 体重没有影响； 多肽 Π高、 中、 低剂量组对人食管癌 Ecl09裸末动物终小鼠异种移植肿瘤的抑瘤率为 53.34%, 63.88%, 59.65%。 高剂量组、 低剂量与阴性组具有显著性差异， 中剂量组组肿瘤体 积与阴性组肿瘤体积相比有极显著性差异。与阴性对照组相比， 对实验动物的体重没有影响， 未见明显的毒副反应。
多肽 III对人食管癌 Ecl09裸小鼠异种移植肿瘤生长的抑制作用见表 44， 阿瓦斯汀组， 对 人食管癌 Ecl09裸小鼠异种移植肿瘤的抑瘤率为 69.00%， 与阴性对照组相比， 对实验动物的 体重没有影响； 多肽 III高、 中、 低剂量组对人食管癌 Ecl09裸小鼠异种移植肿瘤的抑瘤率为 59.75%, 67.13%, 65.63%。 高剂量组肿瘤体积与阴性组肿瘤体积相比具有显著性差异， 中剂 量组、 低剂量组肿瘤体积与阴性组肿瘤体积相比有极显著性差异。 与阴性对照组相比， 对实 验动物的体重没有影响， 未见明显的毒副反应。
实施例 18
整合素阻断剂多肽 I、 多肽 II和多肽 III对人结肠癌 HT-29裸小鼠异种移植肿瘤生长抑制试验 肿瘤接种及检测评价方法见实施例 6。 阳性对照药顺铂组每 3天尾静脉给药一次， 其他 组每天尾静脉给药一次。
表 45 多肽 I对人结肠癌 HT-29裸小鼠异种移植肿瘤生长的抑制作用
组别 剂量 起始体重（g) 终末体重 瘤重 （g) 抑瘤率
(mg/kg/ (g)
次） 阴性对照 一 20.09±0.64 12 22.78±0.62 12 1.44±0.36 一 顺铂 10 20.17±0.56 8 17.75±0.53 7 0.30±0.05** 79.17% 恩度 2.5 19.96±0.60 8 22.91±0.61 8 0.58±0.14 59.50% 多肽 I高 3 19.95 ±1.21 10 19.60 ±1.26 10 0.56±0.15** 61.11% 多肽 I中 1.5 20.15 ±0.97 10 19.67 ±1.41 10 0.49±0.12** 65.97% 多肽 I低 0.75 20.00 ±1.22 10 20.40 ±1.26 10 0.46±0.13** 68.05% 表 46 多肽 II对人结肠癌起始动物数 HT-29裸小鼠异种移植肿瘤生长的抑制作用
组别 剂量 起始体重（g) 起始动物数 终末体重 瘤重 （g) 抑瘤率
( mg/kg/ (g)
次） 阴性对照 20.09±0.64 12 22.78±0.62末动物数终 12 1.44±0.36
顺铂 10 20.17±0.56 8 17.75±0.53 0.30±0.05** 79.17% 末动物数终
恩度 2.5 19.96±0.60 8 22.91±0.61 8 0.58±0.14 59.50% 多肽 Π高 3 19.90±0.65 8 22.77±0.60 8 0.39±0.09** 72.93% 多肽 Π中 1.5 19.80±0.54 8 23.13±0.71 8 0.26±0.07** 81.96% 多肽 II低 0.75 19.89±0.61 8 22.81±0.71 8 0.31±0.07** 78.32% 表 47 多肽 III对人结肠癌 HT-29裸小鼠异种移植肿瘤生长的抑制作用 组别 剂量 起始体重（g) 终末体重 瘤重 （g) 抑瘤率
( mg/k (g)
g/次） 阴性对照 一 20.09±0.64 12 22.78±0.62 12 1.44±0.36 一 顺铂 10 20.17±0.56 8 17.75±0.53 7 0.30±0.05** 79.17% 恩度 2.5 19.96±0.60 8 22.91±0.61 8 0.58±0.14 59.50% 多肽 III高 0.75 20.12±0.67 8 22.90±0.52 8 0.30±0.09** 79.11% 多肽 III中 0.375 20.12±0.58 8 23.04±0.63 8 0.21±0.06** 85.62% 多肽 III低 0.1875 20.28±0.70 8 22.84±0.52 8 0.23±0.07** 83.70% 结果： 多肽 I对人结肠癌 HT-29裸小鼠异种移植肿瘤生长的抑制作用见表 45， 化药紫杉 醇组， 对人结肠癌 HT-29裸小鼠异种移植肿瘤的抑瘤率为 79.17%， 但对实验动物的体重有显 著影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 I高、 中、 低剂 量组对人结肠癌 HT-29裸小鼠异种移植肿瘤的抑瘤率为 61.11%， 65.97%, 68.05%。高剂量组、 中剂量组、低剂量组肿瘤体积与阴性组肿瘤体积相比均有极显著性差异。与阴性对照组相比， 对实验动物的体重没有影响， 未见明显的毒副反应。
多肽 II对人结肠癌 HT-29裸小鼠异种移植肿瘤生长的抑制作用见表 46， 化药紫杉醇组， 对人结肠癌 HT-29裸小鼠异种移植肿瘤的抑瘤率为 79.17%，但对实验动物的体重有显著影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 II高、 中、 低剂量组对人 结肠癌 HT-29裸小鼠异种移植肿瘤的抑瘤率为 72.93%， 81.96%, 78.32%。 高剂量组、 中剂量 组、 低剂量组肿瘤体积与阴性组肿瘤体积相比均有极显著性差异。 与阴性对照组相比， 对实 验动物的体重没有影响， 未见明显的毒副反应。
起始动物数
多肽 III对人结肠癌 HT-29裸小鼠异种移植肿瘤生长的抑制作用见表 47， 化药紫杉醇组， 对人结肠癌 HT-29裸小鼠异种移植肿瘤的抑瘤率为 79.17%，但对实验动物的体重有显著影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 III高、 中、 低剂量组对人 结肠癌 HT-29裸小鼠异种移植肿瘤的抑瘤率为 79.11%， 85.62%, 83.70%。 高剂量组、 中剂量 末动物数终
组、 低剂量组肿瘤体积与阴性组肿瘤体积相比均有极显著性差异。 与阴性对照组相比， 对实 验动物的体重没有影响， 未见明显的毒副反应。
实施例 19
整合素阻断剂多肽 I、 多肽 II和多肽 III对人甲状腺癌 SW-579裸小鼠异种移植肿瘤生长抑制 试验
肿瘤接种及检测评价方法见实施例 6。 阳性对照药顺铂组每 3天尾静脉给药一次， 其他 组每天尾静脉给药一次。
表 48 多肽 I对人甲状腺癌 SW-579裸小鼠异种移植肿瘤生长的抑制作用 起始体重（g) 终末体重 瘤重 （g) 抑瘤率
( mg/k (g)
g/次） 阴性对照 一 19.95±0.54 12 22.83±0.56 12 1.31±0.30 一 顺铂 10 20.15±0.66 8 18.25±0.65 7 0.34±0.07** 74.09% 恩度 2.5 20.07±0.61 8 22.70±0.54 8 0.88±0.19 32.82% 多肽 I高 3 19.75 ±0.65 10 20.88 ±0.85 10 0.55±0.12* 58.01% 多肽 I中 1.5 19.63 ±0.75 10 20.13 ±1.03 10 0.46±0.16** 64.89% 多肽 I低 0.75 20.25 ±0.65 10 22.25 ±1.71 10 0.47±0.14** 64.12% 表 49 多肽 II对人甲状腺癌 SW-579裸小鼠异种移植肿瘤生长的抑制作用 组别 剂量 起始体重（g) 终末体重 瘤重 （g) 抑瘤率
( mg/kg/ (g)
次） 阴性对照 一 19.95±0.54 12 22.83±0.56 12 1.31±0.30 ― 顺钼 10 20.15±0.66 8 18.25±0.65 7 0.34±0.07** 74.09% 恩度 2.5 20.07±0.61 8 22.70±0.54 8 0.88±0.19 32.82% 多肽 II高 3 19.82±0.39 8 23.15±0.67 8 0.57±0.15* 56.59% 多月太 II中 1.5 19.88±0.50 8 23.23±0.67 8 0.43±0.10** 66.94% 起始动物数
多月太 IU氐 0.75 19.64±0.44 8 22.92±0.67 8 0.51±0.13** 60.97% 起始动物数
表 50 多肽 III对人甲状腺癌 SW-579裸小鼠异种移植肿瘤生长的抑制作用 剂量 起始体重（g) 终末体重 瘤重 （g) 抑瘤率 ( mg/k (g)
g/次）
末动物数终
末动物数终
阴性对照 一 19.95±0.54 12 22.83±0.56 12 1.31±0.30 一 顺铂 10 20.15±0.66 8 18.25±0.65 7 0.34±0.07** 74.09% 恩度 2.5 20.07±0.61 8 22.70±0.54 8 0.88±0.19 32.82% 多肽 III高 0.75 19.76±0.53 8 22.56±0.55 8 0.48±0.14** 63.38% 多肽 III中 0.375 19.96±0.54 8 23.09±0.54 8 0.39±0.08** 70.58% 多肽 III低 0.1875 19.98±0.55 8 23.18±0.66 8 0.44±0.09** 66.40% 结果： 多肽 I对人甲状腺癌 SW-579裸小鼠异种移植肿瘤生长的抑制作用见表 48， 化药 顺铂组， 对人甲状腺癌 SW-579裸小鼠异种移植肿瘤的抑瘤率为 74.09%， 但对实验动物的体 重有显著影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 I高、 中、 低剂量组对人甲状腺癌 SW-579裸小鼠异种移植肿瘤的抑瘤率为 58.01%， 64.89%, 64.12%。 高剂量组肿瘤体积与阴性组肿瘤体积相比均有显著性差异； 中剂量组、 低剂量组肿瘤体积与 阴性组肿瘤体积相比均有极显著性差异。 与阴性对照组相比， 对实验动物的体重没有影响， 未见明显的毒副反应。
多肽 II对人甲状腺癌 SW-579裸小鼠异种移植肿瘤生长的抑制作用见表 49，化药顺铂组， 对人甲状腺癌 SW-579裸小鼠异种移植肿瘤的抑瘤率为 74.09%， 但对实验动物的体重有显著 影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 II高、 中、 低剂量 组对人甲状腺癌 SW-579裸小鼠异种移植肿瘤的抑瘤率为 56.59%， 66.94%, 60.97%。 高剂量 组肿瘤体积与阴性组肿瘤体积相比有显著性差异， 中剂量组、 低剂量组肿瘤体积与阴性组肿 瘤体积相比均有极显著性差异。 与阴性对照组相比， 对实验动物的体重没有影响， 未见明显 的毒副反应。
多肽 III对人甲状腺癌 SW-579裸小鼠异种移植肿瘤生长的抑制作用见表 50，化药顺铂组， 对人甲状腺癌 SW-579裸小鼠异种移植肿瘤的抑瘤率为 74.09%， 但对实验动物的体重有显著 影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 III高、 中、 低剂量 组对人甲状腺癌 SW-579裸小鼠异种移植肿瘤的抑瘤率为 63.38%， 70.58%, 66.40%。 高剂量 组、 中剂量组、 低剂量组肿瘤体积与阴性组肿瘤体积相比均有极显著性差异。 与阴性对照组 相比， 对实验动物的体重没有影响， 未见明显的毒副反应。
实起始动物数起始动物数施例 20
整合素阻断剂多肽 I、 多肽 II和多肽 III对人肾癌 A498裸小鼠异种移植肿瘤生长抑制试验
肿瘤接种及检测评价方法见实施例 6。 阳性对照药顺铂组每 3天尾静脉给药一次， 其他 组每天尾静脉给药一次。
表 51 多肽 I对人肾癌 A498裸小鼠异种移植肿瘤生长的抑制作用
末动物数末动物数终终
组别 剂量 起始体重（g) 终末体重 瘤重 （g) 抑瘤率
( mg/kg/ (g)
次） 阴性对照 19.99±0.72 12 23.13±0.51 12 1.23±0.26
顺铂 10 20.13±0.57 8 17.80±0.63 0.25±0.06** 79.83% 恩度 2.5 19.94±0.65 8 22.96±0.69 8 0.70±0.14 43.20% 多肽 I高 3 20.21±0.57 8 22.59±0.53 8 0.57±0.15* 53.65% 多肽 I中 1.5 20.06±0.47 8 23.26±0.40 8 0.49±0.10** 60.16% 多肽 I低 0.75 20.02±0.51 8 22.87±0.40 8 0.51±0.13* 58.53% 表 52多肽 II对人肾癌 A498裸小鼠异种移植肿瘤生长的抑制作用
组别 剂量 起始体重（g) 终末体重 瘤重 （g) 抑瘤率
( mg/kg/ (g)
次） 阴性对照 一 19.99±0.72 12 23. 13±0. .51 12 1.23±0.26
顺钼 10 20.13±0.57 8 17. 80±0. .63 7 0.25±0.06** 79 .83% 恩度 2.5 19.94±0.65 8 22. 96±0. .69 8 0.70±0.14 43 .20% 多肽 II高 3 20.05±0.52 8 22. 93±0. .51 8 0.43±0.11** 64 .90% 多肽 II中 1.5 19.91±0.57 8 23. 13±0. .58 8 0.35±0.09** 71 .41% 多肽 II低 0.75 19.94±0.56 8 23.14±0.58 8 0.41±0.11** 67.
表 53 多肽 III对人肾癌 Α498裸小鼠异种移植肿瘤生长的抑制作用 剂量 起始体重（g) 终末体重 瘤重 （g) 抑瘤率 ( mg/k (g)
g/次） 阴性对照 一 19.99±0.72 12 23.13±0.51 12 1.23±0.26 一 顺铂 10 20.13±0.57 8 17.80±0.63 7 0.25±0.06** 79.83% 恩度 2.5 19.94±0.65 8 22.96±0.69 8 0.70±0.14 43.20% 起始动物数
多肽 III高 0.75 19.68±0.59 8 23.18±0.50 8 0.41±0.12** 66.36% 多肽 III中 0.375 19.83±0.65 8 22.92±0.59 8 0.30±0.07** 75.36% 多肽 III低 0.1875 19.97±0.66 8 22.82±0.69 8 0.36±0.10** 70.74% 结果： 多肽 I对人肾癌 Α498裸小鼠异种移植肿瘤生长的抑制作用见表 51， 化药顺铂组， 对人肾癌 Α498裸小鼠异种移植肿瘤的抑瘤率为 79.83%， 但末动物数终对实验动物的体重有显著影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 I高、 中、 低剂量组对人 肾癌 Α498裸小鼠异种移植肿瘤的抑瘤率为 53.65%， 60.16%, 58.53%。 高剂量组、 低剂量组 肿瘤体积与阴性组肿瘤体积相比均有显著性差异； 中剂量组肿瘤体积与阴性组肿瘤体积相比 有极显著性差异。 与阴性对照组相比， 对实验动物的体重没有影响， 未见明显的毒副反应。
多肽 Π对人肾癌 Α498裸小鼠异种移植肿瘤生长的抑制作用见表 52， 化药顺铂组， 对人 肾癌 Α498裸小鼠异种移植肿瘤的抑瘤率为 79.83%， 但对实验动物的体重有显著影响， 与阴 性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 II高、 中、 低剂量组对人肾癌 Α498裸小鼠异种移植肿瘤的抑瘤率为 64.90%， 71.41%, 67.03%。 高剂量组、 中剂量组、 低 剂量组肿瘤体积与阴性组肿瘤体积相比均有极显著性差异。 与阴性对照组相比， 对实验动物 的体重没有影响， 未见明显的毒副反应。
多肽 III对人肾癌 Α498裸小鼠异种移植肿瘤生长的抑制作用见表 53， 化药顺铂组， 对人 肾癌 Α498裸小鼠异种移植肿瘤的抑瘤率为 79.83%， 但对实验动物的体重有显著影响， 与阴 性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 III高、 中、 低剂量组对人肾癌 Α498裸小鼠异种移植肿瘤的抑瘤率为 66.36%， 75.36%, 70.74%。 高剂量组、 中剂量组、 低 剂量组肿瘤体积与阴性组肿瘤体积相比均有极显著性差异。 与阴性对照组相比， 对实验动物 的体重没有影响， 未见明显毒副作用。
实施例 21
整合素阻断剂多肽 I、 多肽 II和多肽 III对人卵巢癌 SK-OV-3裸小鼠异种移植肿瘤生长抑制试 验
肿瘤接种及检测评价方法见实施例 6。 阳性对照药顺铂组每 3天尾静脉给药一次， 其他 组每天尾静脉给药一次。
表 54多肽 I对人卵巢癌 SK-0V-3裸小鼠异种移植肿瘤生长的抑制作用
起始体重（g) 终末体重 瘤重 （g) 抑瘤率
( mg/k (g)
g/次） 阴性对照 一 19.87±0.43 起始动物数起始动物数 12 23.03±0.59 12 1.34±0.27 一 顺铂 10 20.04±0.58 8 起始动物数 18.15±0.57 7 0.29±0.06** 78.36% 恩度 2.5 19.99±0.71 8 23.19±0.72 8 0.88±0.17 34.32% 多肽 I高 3 20.03±0.61 8 23.16±0.51 8 0.47±0.12** 67.13% 多肽 I中 1.5 20.05±0.58 8 22.96±0.59 8 0.39±0.07** 70.89% 多肽 I低 0.75 19.97±0.62 8 23.09±0.59 末动物数末动物数终终 8 0.41±0.10** 69.40% 末动物数终
表 55 多肽 II对人卵巢癌 SK-OV-3裸小鼠异种移植肿瘤生长的抑制作用
组别 剂量 起始体重 (g) 终末体重 瘤重 （g) 抑瘤率
( mg/kg/ (g)
次） 阴性对照 一 19.87±0.43 12 23.03±0.59 12 1.34±0.27 一 顺铂 10 20.04±0.58 8 18.15±0.57 7 0.29±0.06** 78.36% 恩度 2.5 19.99±0.71 8 23.19±0.72 8 0.88±0.17 34.32% 多肽 Π高 3 20.22±0.52 8 22.97±0.65 8 0.49±0.14** 63.25% 多肽 Π中 1.5 19.83±0.58 8 23.32±0.50 8 0.36±0.08** 72.76% 多肽 II低 0.75 19.91±0.61 8 23.07±0.50 8 0.42±0.10** 68.36% 表 56 多肽 III对人卵巢癌 SK-OV-3裸小鼠异种移植肿瘤生长的抑制作用
起始体重（g) 终末体重 瘤重 （g) 抑瘤率
( mg/k (g)
g/次） 阴性对照 一 19.87±0.43 23.03±0.59 12 1.34±0.27 — 顺铂 10 20.04±0.58 18.15±0.57 7 0.29±0.06** 78.36% 恩度 2.5 19.99±0.71 23.19±0.72 8 0.88±0.17 34.32% 多肽 III高 0.75 20.18±0.58 22.95±0.64 8 0.40±0.11 ** 69.98% 多肽 III中 0.375 19.91±0.55 8 22.92±0.70 8 0.31±0.07** 76.63% 多肽 III低 0.1875 19.55±0.57 8 22.83±0.50 8 0.35±0.09** 73.87% 结果： 多肽 I对人卵巢癌 SK-OV-3裸小鼠异种移植肿瘤生长的抑制作用见表 54，化药顺 铂组，对人卵巢癌 SK-OV-3裸小鼠异种移植肿瘤的抑瘤率为 78.36%，但对实验动物的体重有 显著影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 I高、 中、 低 剂量组对人卵巢癌 SK-OV-3裸小鼠异种移植肿瘤的抑瘤率为 67.13%， 70.89%, 69.40%。 高 剂量组、 中剂量组、 低剂量组肿瘤体积与阴性组肿瘤体积相比均有极显著性差异。 与阴性对 照组相比， 对实验动物的体重没有影响， 未见明显毒副作用。
多肽 II对人卵巢癌 SK-OV-3裸小鼠异种移植肿瘤生长的抑制作用见表 55， 化药顺铂组， 起始动物数
对人卵巢癌 SK-OV-3裸小鼠异种移植肿瘤的抑瘤率为 78.36%，但对实验动物的体重有显著影 响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 II高、 中、 低剂量组 对人卵巢癌 SK-OV-3裸小鼠异种移植肿瘤的抑瘤率为 63.25%， 72.76%, 68.36%。 高剂量组、 中剂量组、低剂量组肿瘤体积与阴性组肿瘤体积相比均有极显著性差异。与阴性对照组相比， 末动物数终
对实验动物的体重没有影响， 未见明显的毒副反应。
多肽 III对人卵巢癌 SK-OV-3裸小鼠异种移植肿瘤生长的抑制作用见表 56， 化药顺铂组， 对人卵巢癌 SK-OV-3裸小鼠异种移植肿瘤的抑瘤率为 78.36%，但对实验动物的体重有显著影 响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 III高、 中、 低剂量组 对人卵巢癌 SK-OV-3裸小鼠异种移植肿瘤的抑瘤率为 69.98%， 76.63%, 73.87%。 高剂量组、 中剂量组、低剂量组肿瘤体积与阴性组肿瘤体积相比均有极显著性差异。与阴性对照组相比， 对实验动物的体重没有影响， 未见明显毒副作用。
实施例 22
整合素阻断剂多肽 I、 多肽 II和多肽 III对肉瘤 HT-1080裸小鼠异种移植肿瘤生长抑制试验
肿瘤接种及检测评价方法见实施例 6。 阳性对照药 5-氟尿嘧啶组每 3天尾静脉给药一次， 其他组每天尾静脉给药一次。
表 57 多肽 I对肉瘤 HT-1080裸小鼠异种移植肿瘤生长的抑制作用
组别 剂量 起始体重 (g) 终末体重 瘤重 （g) 抑瘤率
( mg/kg/ (g)
次）
20.09±0.66 12 23.01±0.48 12 1.37±0.35
19.94±0.58 8 17.88±0.68 7 0.30±0.07**
19.88±0.79 8 22.89±0.51 8 0.69±0.15 多肽 I高 3 20.11±0.59 8 23.13±0.38 0.49±0.14** 64.23% 多肽 I中 1.5 20.06±0.63 8 23.10±0.64 8 0.36±0.08** 73.72% 多肽 I低 0.75 19.80±0.39 8 22.99±0.51 8 0.42±0.10** 69.34% 表 58 多肽 II对肉瘤 HT-1080裸小鼠异种移植肿瘤生长的抑制作用
组别 剂量 起始体重 终末体重 瘤重 （g) 抑瘤率
( mg/kg/ (g) (g)
次） 阴性对照 一 20.09±0.66 12 23.01±0.48 12 1.37±0.35 一
5-Fu 10 19.94±0.58起始动物数 8 17.88±0.68 7 0.30±0.07** 78.10%
2.5 19.88±0.79 起始动物数
恩度 8 22.89±0.51 8 0.69±0.15 49.72% 多肽 II高 3 20.08±0.59 8 23.04±0.55 8 0.45±0.11** 67.06% 多肽 Π中 1.5 19.70±0.52 8 22.95±0.46 8 0.35±0.07** 74.43% 多肽 II低 0.75 20.28±0.61 8 22.93±0.46 8 0.40±0.11** 70.60% 末动物数终
表 59 多肽 III对肉瘤 HT-1080裸小鼠异种移植肿瘤生长的抑制作用
末动物数终
剂 起始体重 （g) 终末体重 瘤重 （g) 抑瘤率
(g)
( mg/
kg/次） 阴性对照 一 20.09±0.66 12 23.01±0.48 12 1.37±0.35 一
5-Fu 10 19.94±0.58 8 17.88±0.68 7 0.30±0.07** 78.10% 恩度 2.5 19.88±0.79 8 22.89±0.51 8 0.69±0.15 49.72% 多肽 III高 0.75 19.89±0.53 8 23.07±0.56 8 0.37±0.10** 73.29% 多肽 III中 0.375 20.31±0.51 8 23.18±0.57 8 0.28±0.06** 79.90% 多肽 III低 0.1875 19.74±0.55 8 22.92±0.57 8 0.33±0.07** 75.87% 结果： 多肽 I对肉瘤 HT-1080裸小鼠异种移植肿瘤生长的抑制作用见表 57， 化药 5-Fu 组， 对肉瘤 HT-1080裸小鼠异种移植肿瘤的抑瘤率为 78.10%， 但对实验动物的体重有显著影 响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 I高、 中、 低剂量组 对肉瘤 HT-1080裸小鼠异种移植肿瘤的抑瘤率为 64.23%， 73.72%, 69.34%。 高剂量组、 中剂 量组、 低剂量组肿瘤体积与阴性组肿瘤体积相比均有极显著性差异。 与阴性对照组相比， 对 实验动物的体重没有影响， 未见明显的毒副反应。
多肽 II对肉瘤 HT-1080裸小鼠异种移植肿瘤生长的抑制作用见表 58， 化药 5-Fu组， 对 肉瘤 HT-1080裸小鼠异种移植肿瘤的抑瘤率为 78.10%， 但对实验动物的体重有显著影响， 与 阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 II高、 中、 低剂量组对肉瘤 HT-1080裸小鼠异种移植肿瘤的抑瘤率为 67.06%， 74.43%, 70.60%。 高剂量组肿瘤体积与阴 性组肿瘤体积相比具有显著性差异， 中剂量组、 低剂量组肿瘤体积与阴性组肿瘤体积相比均 有极显著性差异。 与阴性对照组相比， 对实验动物的体重没有影响， 未见明显的毒副反应。
多肽 III对肉瘤 HT-1080裸小鼠异种移植肿瘤生长的抑制作用见表 59， 化药 5-Fu组， 对 肉瘤 HT-1080裸小鼠异种移植肿瘤的抑瘤率为 78.10%， 但对实验动物的体重有显著影响， 与 阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 III高、 中、 低剂量组对肉瘤 HT-1080裸小鼠异种移植肿瘤的抑瘤率为 73.29%， 79.90%, 75.87%。 高剂量组、 中剂量组、 低剂量组肿瘤体积与阴性组肿瘤体积相比均有极显著性差异。 与阴性对照组相比， 对实验动 起始动物数
物的体重没有影响， 未见明显毒副作用。
起始动物数
实施例 23
整合素阻断剂多肽 I、 多肽 II和多肽 III对人喉癌 Hep-2裸小鼠异种移植肿瘤生长抑制试验
肿瘤接种及检测评价方法见实施例 6。 阳性对照药阿瓦斯汀组每 3天尾静脉给药一次， 末动物数终
其他组每天尾静脉给药一次。
表 60 多肽 I对人喉癌 Hep-2裸小鼠异种移植肿瘤生长末动物数终的抑制作用
剂 起始体重 （g) 终末体重 瘤重 （g) 抑瘤率
(g)
(mg/
kg/次） 阴性对照 一 20.00±0.52 12 23.33±0.44 12 1.12±0.20 一 阿瓦斯汀 10 20.09±0.70 8 20.47±0.70 6 0.26±0.06** 76.79% 恩度 2.5 20.18±0.61 8 23.23±0.67 7 0.59±0.10 46.96% 多肽 I高 3 20.15±0.52 8 22.99±0.62 8 0.50±0.10* 55.35% 多肽 I中 1.5 19.85±0.55 8 23.21±0.62 8 0.41±0.16** 63.39% 多肽 I低 0.75 20.01±0.66 8 22.88±0.63 8 0.43±0.07** 60.90% 表 61 多肽 II对人喉癌 Hep-2裸小鼠异种移植肿瘤生长的抑制作用 组别 剂量 起始体重（g) 终末体重 瘤重 （g) 抑瘤率
(mg/kg/ (g)
次）
20.00±0.52 23.33±0.44 12 1.12±0.20 ― 20.09±0.70 20.47±0.70 6 0.26±0.06** 76.79%
20.18±0.61 23.23±0.67 7 0.59±0.10 46.96%
20.02±0.52 22.66±0.61 8 0.44±0.08* 60.76% 多肽 II中 1.5 20.14±0.52 8 22.98±0.60 8 0.33±0.07** 70.41% 多肽 II低 0.75 19.96±0.52 8 22.88±0.70 8 0.39±0.06** 64.98% 表 62 多肽 III对人喉癌 Hep-2裸小鼠异种移植肿瘤生长的抑制作用
剂量 起始体重（g) 终末体重 瘤重 （g) 抑瘤率 (mg/k (g)
g/次） 阴性对照 一 20.00±0.52 12 23.33±0.44 12 1.12±0.20 一 阿瓦斯汀 10 20.09±0.70 8 20.47±0.70 6 0.26±0.06 76.79% 起始动物数
恩度 2.5 20.18±0.61 8 23.23±0.67 7 0.59±0.10 46.96% 多肽 III高 0.75 20.07±0.53 8 23.18±0.51 8 0.42±0.10* 62.81% 多肽 III中 0.375 20.17±0.48 8 22.96±0.57 8 0.28±0.05** 75.13% 多肽 III低 0.1875 19.62±0.52 8 22.94±0.46 8 0.37±0.08** 67.29% '结呆:'多'肽 1对人喉擷 Hep- 裸小一鼠异'种'栘植肿瘤生长末动物数终的抑制作用见表 60，阿瓦斯〕组， 对人喉癌 Hep-2裸小鼠异种移植肿瘤的抑瘤率为 76.79%， 对实验动物的体重无显著影响； 多 肽 I高、 中、 低剂量组对人喉癌 Hep-2 裸小鼠异种移植肿瘤的抑瘤率为 55.35%， 63.39%, 60.90%。 高剂量组肿瘤体积与阴性组肿瘤体积相比具有显著性差异， 中剂量组、 低剂量组肿 瘤体积与阴性组肿瘤体积相比有极显著性差异。 与阴性对照组相比， 对实验动物的体重没有 影响， 未见明显的毒副反应。
多肽 II对人喉癌 Hep-2裸小鼠异种移植肿瘤生长的抑制作用见表 61， 阿瓦斯汀组， 对人 喉癌 Hep-2裸小鼠异种移植肿瘤的抑瘤率为 76.79%， 对实验动物的体重无显著影响； 多肽 II 高、中、低剂量组对人喉癌 Hep-2裸小鼠异种移植肿瘤的抑瘤率为 60.76%， 70.41%, 64.98%。 高剂量组肿瘤体积与阴性组肿瘤体积相比具有显著性差异， 中剂量组、 低剂量组肿瘤体积与 阴性组肿瘤体积相比有极显著性差异。 与阴性对照组相比， 对实验动物的体重没有影响， 未 见明显的毒副反应。
多肽 III对人喉癌 Hep-2裸小鼠异种移植肿瘤生长的抑制作用见表 62， 阿瓦斯汀组， 对人 喉癌 Hep-2裸小鼠异种移植肿瘤的抑瘤率为 76.79%， 对实验动物的体重无显著影响； 多肽 III 高、中、低剂量组对人喉癌 Hep-2裸小鼠异种移植肿瘤的抑瘤率为 62.81%， 75.13%, 67.29%。 高剂量组肿瘤体积与阴性组肿瘤体积相比具有显著性差异， 中剂量组、 低剂量组肿瘤体积与 阴性组肿瘤体积相比有极显著性差异。 与阴性对照组相比， 对实验动物的体重没有影响， 未 见明显的毒副反应。
实施例 24 整合素阻断剂多肽 I、 多肽 II和多肽 III对人脑瘤 SF763裸小鼠异种移植肿瘤生长抑制试验 肿瘤接种及检测评价方法见实施例 6。 阳性对照药紫杉醇组每 3天尾静脉给药一次， 其 他组每天尾静脉给药一次。
表 63 多肽 I对人脑瘤 SF763裸小鼠异种移植肿瘤生长的抑制作用 组别 剂量 起始体重（g) 终末体重 瘤重 （g) 抑瘤率
( mg/kg/ (g)
次） 阴性对照 19.89±0.64 12 23.03 ±0.81 12 1.18 ±0.33
紫杉醇 10 20.14 ±0.43 8 17.97 ±0.63 6 0.28 ±0.07** 76.29% 起始动物数起始动物数起始动物数
多肽 I高 3 19.83±0.58 8 20.94±0.47 8 0.47±0.08** 60.16% 多肽 I中 1.5 20.15±0.52 8 22.99±0.62 8 0.34±0.07** 71.19% 多肽 I低 0.75 20.12±0.70 8 22.90±0.69 8 0.38±0.06** 67.80% 表 64 多肽 II对人脑瘤 SF763裸小鼠异种移植肿瘤生长的抑制作用
组别 剂量 起始体重（g) 终末体重 末动物数末动物数终终末动物数终 瘤重 （g) 抑瘤率
( mg/kg/ (g)
次） 阴性对照 19.89±0.64 23.03 ±0.81 1.18 ±0.33
紫杉醇 10 20.14 ±0.43 17.97 ±0.63 0.28 ±0.07** 76.29% 多肽 II高 3 19.94 ±0.48 23.24 ±0.66 0.41 ±0.14* 65.48% 多肽 Π中 1.5 20.21 ±0.58 22.88 ±0.52 0.29 ±0.07** 75.02% 多肽 II低 0.75 20.01 ±0.78 22.95 ±0.52 0.35 ±0.13** 70.61% 表 65 多肽 III对人脑瘤 SF763裸小鼠异种移植肿瘤生长的抑制作用
组别 剂量 起始体重（g) 终末体重 瘤重 （g) 抑瘤率
( mg/kg/ (g)
次） 阴性对照 一 19.89±0.64 12 23.03 ±0.81 12 1.18 ±0.33 一 紫杉醇 10 20.14 ±0.43 8 17.97 ±0.63 7 0.28 ±0.07** 76.29% 多肽 III高 0. 75 19.86 ±0.48 8 23.09 ±0.53 8 0.31 ±0.09** 74.06% 多肽 III中 0.375 20.18 ±0.64 8 23.37 ±0.56 8 0.24 ±0.05** 79.76% 多肽 III低 0.1875 19.99 ±0.62 8 23.31 ±0.57 8 0.32 ±0.08** 72.82% 结果： 多肽 I对人脑瘤 SF763裸小鼠异种移植肿瘤生长的抑制作用见表 63， 化药紫杉醇 组， 对人脑瘤 SF763裸小鼠异种移植肿瘤的抑瘤率为 76.29%， 但对实验动物的体重有显著影 响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 I高、 中、 低剂量组 对人脑瘤 SF763裸小鼠异种移植肿瘤的抑瘤率为 60.16%， 71.19%, 67.80%。 高剂量组、 中齐 U 量、 低剂量组肿瘤体积与阴性组肿瘤体积相比有极显著性差异。 与阴性对照组相比， 对实验 动物的体重没有影响， 未见明显的毒副反应。
多肽 II对人脑瘤 SF763裸小鼠异种移植肿瘤生长的抑制作用见表 64， 化药紫杉醇组， 对 人脑瘤 SF763裸小鼠异种移植肿瘤的抑瘤率为 76.29%， 但对实验动物的体重有显著影响， 与 阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 II高、 中、 低剂量组对人脑 瘤 SF763裸小鼠异种移植肿瘤的抑瘤率为 65.48%， 75.02%, 70.61%。高剂量组肿瘤体积与阴 性组肿瘤体积相比有显著性差异， 中剂量、 低起始动物数剂量组肿瘤体积与阴性组肿瘤体积相比有极显 著性差异。 与阴性对照组相比， 对实验动物的体重没有影响， 未见明显的毒副反应。
多肽 III对人脑瘤 SF763裸小鼠异种移植肿瘤生长的抑制作用见表 65， 阿瓦斯汀组， 对人 脑瘤 SF763裸小鼠异种移植肿瘤的抑瘤率为 76.29%， 但对实验动物的体重有显著影响， 与阴 性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 III高末动物数终、 中、 低剂量组对人脑瘤 SF763裸小鼠异种移植肿瘤的抑瘤率为 74.06%， 79.76%, 72.82%。 高剂量组、 中剂量组、 低 剂量与阴性组有极显著性差异。 与阴性对照组相比， 对实验动物的体重没有影响， 未见明显 的毒副反应。
实施例 25
整合素阻断剂多肽 I、 多肽 II和多肽 III对人直肠癌 Colo 320裸小鼠异种移植肿瘤生长抑制试 验
肿瘤接种及检测评价方法见实施例 6。 阳性对照药紫杉醇组每 3天尾静脉给药一次， 其 他组每天尾静脉给药一次。
表 66 多肽 I对人直肠癌 Colo 320裸小鼠异种移植肿瘤生长的抑制作用
组别 剂量 起始体重（g) 终末体重 瘤重 （g) 抑瘤率
(mg/kg/ (g)
次） 阴性对照 一 20.09±0.63 12 23.22±0.50 12 1.25±0.31 ― 紫杉醇 10 19.96±0.59 8 17.08±0.57 7 0.29±0.07** 76.62% 恩度 2.5 20.05±0.70 8 23.05±0.67 8 0.68±0.28 45.41% 多月太 I高 3 19.94±0.59 8 20.99±0.66 7 0.51 ±0.09** 59.20% 多月太 I中 1.5 19.88±0.50 8 22.87±0.57 7 0.44 ±0.05** 64.80% 多月太 I低 0.75 20.21±0.64 8 23.11±0.61 8 0.52 ±0.08** 58.40% 表 67 多肽 II对人直肠癌 Colo 320裸小鼠异种移植肿瘤生长的抑制作用
组别 剂量 起始体重（g) 终末体重 瘤重 （g) 抑瘤率
(mg/kg/ (g)
次） 阴性对照 一 20.09±0.63 12 23.22±0.50 12 1.25±0.31 一 紫杉醇 10 19.96±0.59 8 17.08±0.57 7 0.29±0.07** 76.62% 恩度 2.5 20.05±0.70 8 23.05±0.67 8 0.68±0.28 45.41% 起始动物数
多肽 II高 3 19.88±0.58 8 22.99±0.37 8 0.41±0.09** 67.41% 起始动物数
多肽 Π中 1.5 19.97±0.66 8 23.10±0.61 8 0.31±0.03** 75.42% 多肽 II低 0.75 20.07±0.54 8 22.85±0.61 8 0.35±0.10** 72.26% 表 68 多肽 III对人直肠癌 Colo 320裸小鼠异种移植肿瘤生长的抑制作用 剂量 起始体重（g) 终末体重 末动物数终 瘤重 （g) 抑瘤率 (mg/k (g) 末动物数终
g/次） 阴性对照 一 20.09±0.63 12 23.22±0.50 12 1.25±0.31 一 紫杉醇 10 19.96±0.59 8 17.08±0.57 7 0.29±0.07** 76.62% 恩度 2.5 20.05±0.70 8 23.05±0.67 8 0.68±0.28 45.41% 多肽 III高 0.75 19.82±0.60 8 23.14±0.48 8 0.33±0.08** 73.51% 多肽 III中 0.375 19.96±0.58 8 22.93±0.56 8 0.24±0.05** 80.89% 多肽 III低 0.1875 19.87±0.58 8 22.81±0.60 8 0.37±0.07** 70.34% 结果： 多肽 I对人直肠癌 Colo 320裸小鼠异种移植肿瘤生长的抑制作用见表 66， 化药紫 杉醇组， 对人直肠癌 Colo 320裸小鼠异种移植肿瘤的抑瘤率为 76.62%， 但对实验动物的体重 有显著影响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 I高、 中、 低剂量组对人直肠癌 Colo 320裸小鼠异种移植肿瘤的抑瘤率为 59.20%， 64.80%, 58.40%。 高剂量组、 中剂量组、 低剂量与阴性组有极显著性差异。 与阴性对照组相比， 对实验动物的 体重没有影响， 未见明显的毒副反应。
多肽 Π对人直肠癌 Colo 320裸小鼠异种移植肿瘤生长的抑制作用见表 67，化药紫杉醇组， 对人直肠癌 Colo 320裸小鼠异种移植肿瘤的抑瘤率为 76.62%，但对实验动物的体重有显著影 响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 II高、 中、 低剂量组 对人直肠癌 Colo 320裸小鼠异种移植肿瘤的抑瘤率为 67.41%， 75.42%, 72.26%。 高剂量组、 中剂量组、 低剂量与阴性组有极显著性差异。 与阴性对照组相比， 对实验动物的体重没有影 响， 未见明显的毒副反应。
多肽 III对人直肠癌 Colo 320裸小鼠异种移植肿瘤生长的抑制作用见表 68，化药紫杉醇组， 对人直肠癌 Colo 320裸小鼠异种移植肿瘤的抑瘤率为 76.62%，但对实验动物的体重有显著影 响， 与阴性组和多肽给药组动物体重相比较轻， 毒副作用较明显； 多肽 III高、 中、 低剂量组 对人直肠癌 Colo 320裸小鼠异种移植肿瘤的抑瘤率为 73.51%， 80.89%, 70.34%。 高剂量组、 中剂量组、 低剂量与阴性组有极显著性差异。 与阴性对照组相比， 对实验动物的体重没有影 响， 未见明显的毒副反应。
实施例 26
多肽 I、 多肽 II和多肽 III在胶原诱导小鼠关节炎动物模型体内免疫保护作用
构建胶原型小鼠关节炎动物模型， 研究多肽对小鼠胶原诱导性关节炎 （collagen induced arthritis, CIA) 的治疗作用。 采用小鼠作为受试动物， SPF级 DBA/1小鼠 （由上海西普尔 -必凯 实验动物有限公司 （Sino-British SIPPR Lab. Animal Ltd) 提供， 动物生产许可证号码： SCXK (沪) 2008-0016)， 雄性， 7-8周龄， 体重为 18-22 g， 随机分组， 分别是正常对照组， 模型 对照组， 多肽 I低中高 3个剂量组（0.2， 0.4， 0.8 mg/kg)， 多肽 II低中高 3个剂量组（0.2， 0.4， 0.8 mg/kg),多肽 III低中高 3个剂量组（0.1， 0.2， 0.4 mg/kg)和阳性药对照组 (甲氨蝶吟 1 mg/kg)。 除正常组外， 第 0天各实验组建立小鼠 CIA模型， 方法是鸡软骨 III型胶原 (cIII)用 O. l mol/1醋酸 溶解成 4 mg/ml的溶液， 于 4°C冰箱过夜。 实验当天用 III型胶原与含 4 mg/ml Myeobaeterium tuberculosis strain H37Rv的完全弗氏佐剂 (CF A)等体积充分乳化， 待 DBA/1小鼠麻醉后， 于其 尾部皮内每只注射乳化剂 50 μΐ进行致敏， 21 d后用 4 mg/ml的 III型胶原 (cIII)与不完全弗氏佐剂 (IFA)等体积充分乳化后以相同剂量的乳化剂于尾部进行再次免疫。 造模第 30 d起皮下注射给 药： 多肽 I低中高 3个剂量组（0.2， 0.4， 0.8 mg/kg), 多肽 II低中高 3个剂量组（0.2， 0.4， 0.8 mg/kg), 多肽 III低中高 3个剂量组（0.1， 0.2， 0.4 mg/kg): 每日两次， 连续 10天； 阳性药对照 组 (甲氨蝶吟 1 mg/kg ): 每五天一次， 连续 3次； 正常对照组和模型对照组 （生理盐水）： 连续 10天。 分别于造模后第 21天至第 70天每 3天称量体重、 关节评分、 分别检测左右后足足踝直径 来观察药物对小鼠胶原型关节炎的影响。 在第 70 d， 脱臼处死小鼠。
关节炎评价指标如下：
1 )关节评分： 四肢： 按 0— 4五级评分： 0 = 无红斑或红肿； 1 = 轻微的红斑或肿胀， 其中的 一个前 /后趾关节有红斑或肿胀； 2 = 多于一只趾出现红斑或肿胀； 3 =踝或腕关节下的足爪 肿胀； 4 =包括踝关节在内的全部足爪肿胀。 小鼠四只足分别评分， 最高评分为 16分。 分别 于造模后第 21天至第 70天每 3天进行关节评分， 并记录结果。
2) 测量足踝直径
分别于造模前和造模后第 21天至第 70天每 3天用游标卡尺测量小鼠左、 右足踝内侧到 外侧的直径和足踝厚度， 并记录结果。
试验结果的计量资料以数学平均数加减标准差（ mean ± SD ) 表示， 用 SPSS 11.0软件进 行各给药组与对照组之间进行 t检验， 表中 *表示 ρ < 0. 05， **表示 ρ < 0. 01。
结果： 造模后小鼠与正常小鼠相比较， 在小鼠尾部皮内注射含灭活的结核分枝杆菌完全 弗氏佐剂和胶原等体积混合的乳化剂， 21天后尾部皮内注射不完全弗氏佐剂和胶原等体积混 合的乳化剂左后，免疫后第 27天， CIA小鼠足爪红肿，关节炎指数评分增高，模型组第 45-60 天为肿胀高峰， 模型组自第 35天开始体重几乎不增加， 后期还略微有下降。
多肽 I在胶原诱导小鼠关节炎动物模型都能发挥体内免疫保护作用， 结果见表 69: 阳性 对照组、 多肽 I高、 中、 低剂量组足爪肿胀度与模型组比较， 均有极显著性差异 （ρ <0.01 ) 实验结果具有统计学意义。 阳性对照组、 多肽 I高、 中、 低剂量组关节肿胀度与模型组比较， 均有极显著性差异（ρ <0.01 )， 实验结果具有统计学意义多肽 I低、 中、 高剂量组四肢评分显 著低于模型对照组 （ρ <0.01 )， 与模型对照组比较极显著性差异， 实验结果具有统计学意义。
表 69多肽 I对胶原型类小鼠关节炎动物模型体内免疫保护作用
剂量
左右足爪肿胀度 关节肿胀度
组别 (mg/kg 临床评分
(η) (mm) (mm)
)
正常对照组 10 ― 0.15±0.07** 0.14±0.04** 0.00±0.00** 模型对照组 10 ― 2.12±0.37 1.90±0.40 15.5±2.3 阳性对照组 10 1 0.90±0.19** 0.75±0.17** 8.3±1.2** 多肽 I高 10 0.8 1.31±0.26** 1.05±0.31** 11.4±1.6** 多肽 I中 10 0.4 0.94±0.18** 0.79±0.17** 9.1±1.4** 多肽 I低 10 0.2 1.21±0.22** 0.97±0.23** 9 7±ι 5**
*表示 p < 0. 05， **表示 p < 0. 01.
多肽 II在胶原诱导小鼠关节炎动物模型都能发挥体内免疫保护， 作用结果见表 70: 阳性 对照组、 多肽 Π高、 中、 低剂量组足爪肿胀度与模型组比较， 均有极显著性差异 （ρ <0.01 ) 实验结果具有统计学意义。 阳性对照组、 多肽 II高、 中、 低剂量组关节肿胀度与模型组比较， 均有极显著性差异 （ρ <0.01 )， 实验结果具有统计学意义。 多肽 II低、 中、 高剂量组四肢评分 显著低于模型对照组 (p <0.01 )，与模型对照组比较极显著性差异，实验结果具有统计学意义。 表 70 多肽 II对胶原型类小鼠关节炎动物模型体内免疫保护作用
剂量
左右足爪肿胀度 关节肿胀度
组别 (mg/kg 临床评分
？ if (mm) (mm) 正常对照组 10 ― 0.13±0.03** 0.13±0.03** 0.00±0.00** 模型对照组 10 ― 1.90±0.38 1.86±0.38 15.55±2.33 阳性对照组 10 1 0.90±0.18** 0.75±0.18** 8.01±1.21** 多肽 II高 10 0.8 1.44±0.29** 1.16±0.32** 10.73±1.65** 多肽 Π中 10 0.4 0.99±0.20** 0.83±0.21** 9.269±1.3** 多肽 II低 10 0.2 1.12±0.32** 0.95±0.24** 9.82±1.48**
*表示 p < 0. 05， **表示 p< 0. 01.
多肽 III在胶原诱导小鼠关节炎动物模型都能发挥体内免疫保护作用， 结果见表 71 : 阳性 对照组、 多肽 III高、 中、 低剂量组足爪肿胀度与模型组比较， 均有极显著性差异 （ρ <0.01 ) 实验结果具有统计学意义。 阳性对照组、 多肽 III高、 中、 低剂量组关节肿胀度与模型组比较， 均有极显著性差异 （ρ <0.01 )， 实验结果具有统计学意义。 多肽 III低、 中、 高剂量组四肢评分 显著低于模型对照组 (ρ <0.01 )，与模型对照组比较极显著性差异，实验结果具有统计学意义。 表 71多肽 III对胶原型类小鼠关节炎动物模型体内免疫保护作用
剂量
左右足爪肿胀度 关节肿胀度
组别 (mg/kg 临床评分
？ if (mm) (mm) 正常对照组 10 ― 0.13±0.03** 0.13±0.03** 0.00±0.00** 模型对照组 10 ― 1.90±0.38 1.86±0.38 15.55±2.33 阳性对照组 10 1 0.90±0.18** 0.75±0.18** 8.01±1.21** 多肽 III高 10 0.4 1.23±0.25** 1.05±0.26** 10.41±1.56** 多肽 III中 10 0.2 0.85±0.17** 0.68±0.19** 9.13±1.37** 多肽 III低 10 0.1 1.03±0.21** 0.92±0.27** 9.73±1.46**
*表示 p < 0. 05， **表示 p < 0. 01.
结论： 多肽 I、 多肽 II和多肽 III对小鼠胶原型关节炎具有治疗作用。
实施例 27
多肽 I、 多肽 II和多肽 III对佐剂型大鼠关节炎动物模型体内免疫保护作用
构建佐剂型大鼠关节炎动物模型， 研究多肽对佐剂性关节炎 （Adjuvant arthritis, AA) 大 鼠的治疗作用。 采用大鼠作为受试动物， SPF级 SD大鼠 （由上海西普尔 -必凯实验动物有限 公司（Sino-British SIPPR Lab. Animal Ltd)提供，动物生产许可证号码： SCXK (沪) 2008-0016)， 雄性， 体重为 140 g-160 g， 随机分组， 分别是正常对照组， 模型对照组， 多肽 I低中高 3个 剂量组 （0.4， 0.8， 1.6 mg/kg)， 多肽 II低中高 3个剂量组 （0.4， 0.8， 1.6 mg/kg)， 多肽 III低 中高 3个剂量组 （0.2， 0.4， 0.8 mg/kg) 和阳性药对照组 (甲氨蝶吟 1 mg/kg)。 除正常组外， 第 0天各实验组建立大鼠 AA模型， 方法是在大鼠的左侧后足足跎注射含灭活的结核分枝杆 菌 (H37RA, 10 mg/ml)完全弗氏佐剂 0.08 ml造成大鼠佐剂性关节炎模型。 造模第 10天起皮 下注射给药：多肽 I低中高 3个剂量组（0.4， 0.8， 1.6 mg/kg),多肽 II低中高 3个剂量组（0.4，
0.8， 1.6 mg/kg), 多肽 III低中高 3个剂量组 （0.2， 0.4， 0.8 mg/kg): 每日两次， 连续 10天； 阳性药对照组 (甲氨蝶吟 1 mg/kg ): 每五天一次， 连续 3次； 正常对照组和模型对照组（生理 盐水）： 连续 10天。 分别于造模后第 8， 11， 14， 17， 20， 23和 26天， 关节评分、 分别检测 左右后足足踝直径来观察药物对大鼠佐剂型关节炎的影响。
关节炎评价指标如下：
1 )关节评分四肢： 按 0— 4五级评分： 0 =无红斑或红肿； 1 = 轻微的红斑或肿胀， 其中的一 个前 /后趾关节有红斑或肿胀； 2 = 多于一只趾出现红斑或肿胀； 3 =踝或腕关节下的足爪肿 胀； 4 =包括踝关节在内的全部足爪肿胀。 大鼠四只足分别评分， 最高评分为 16分。
分别于造模后 8， 11， 14， 17， 20， 23和 26天进行关节评分， 并记录结果。
2) 测量足踝直径
分别于造模前和造模后 8， 11， 14， 17， 20， 23和 26天用游标卡尺测量大鼠左、 右足踝 内侧到外侧的直径和足踝厚度， 并记录结果。
试验结果的计量资料以数学平均数加减标准差（ mean ± SD ) 表示， 用 SPSS 11.0软件进 行各给药组与对照组之间进行 t检验， 表中 *表示 ρ < 0. 05， **表示 ρ < 0. 01。
结果： 造模后大鼠与正常大鼠相比较， 在大鼠左后足跎处注射含灭活的结核分枝杆菌完 全弗氏佐剂后， 左后足会迅速产生原发性关节炎， 出现明显的肿胀， 并伴有溃烂； 右后足大 约 10 d后开始出现继发性关节炎， 评分的分值逐渐增大； 同时耳部血管增生明显， 红肿明显； 尾部关节出现肿胀。
多肽 I在胶原诱导大鼠关节炎动物模型都能发挥体内免疫保护作用， 结果见表 72: 大鼠 阳性对照组、 多肽 I中剂量组左后足踝直径与模型组比较， 有极显著性差异（p <0.01 ); 整合 素阻断剂多肽 I低、 高剂量组左后足踝直径与模型组比较， 均有显著性差异（ρ <0.05 )， 实验 结果具有统计学意义。 大鼠阳性对照组、 多肽 I低、 中、 高剂量组右后足踝直径与模型组比 较，有显著性差异 <0.05 )。多肽 I低、中、高剂量组四肢评分显著低于模型对照组 <0.05 )， 与模型对照组比较差异均有统计学意义。
表 72多肽 I对佐剂型类大鼠关节炎动物模型体内免疫保护作用
剂量
左足爪肿胀度 右足爪肿胀度
组别 (mg/k 临床评分
(n) (mm) (mm)
g)
正常对照组 10 ― 0.79±0.18** 0.56±0.08** 0.0±0.0** 模型对照组 10 ― 7.11±1.4 3.38±0.94 13.5±2.6 阳性对照组 10 1 3.52±0.72** 0.63±0.19** 4.8±1.0* 多肽 I高 10 1.6 4.81±0.95* 1.35±0.30* 7.0±1.2* 多肽 I中 10 0.8 3.83±0.76** 0.94±0.18* 5.5±1.0* 多肽 I低 10 0.4 4.25±0.85* 1.36±0.31* 7.1±1.1*
*表示 p < 0. 05， **表示 p < 0. 01.
多肽 II在胶原诱导大鼠关节炎动物模型都能发挥体内免疫保护作用， 结果见表 73: 大鼠 阳性对照组、 多肽 II中剂量组左后足踝直径与模型组比较， 有极显著性差异（p <0.01 ); 多肽 Π低、 高剂量组左足足爪肿胀度与模型组比较， 均有显著性差异（ρ <0.05)， 实验结果具有统 计学意义。 大鼠阳性对照组、 多肽 II低、 中、 高剂量组右足足爪肿胀度与模型组比较， 有显 著性差异 （ρ <0.05)。 多肽 II低、 中、 高剂量组四肢评分显著低于模型对照组 （ρ <0.05)， 与 模型对照组比较差异均有统计学意义。
表 73多肽 II对佐剂型类大鼠关节炎动物模型体内免疫保护作用
Figure imgf000056_0001
正常对照组 10 0.90±0.17** 0.38±0.11** 0.00±0.00** 模型对照组 10 ― 7.01±1.42 3.21±0.69 13.11±2.62 阳性对照组 10 1 3.51±0.77** 0.59±0.17** 5.02±1.11* 多肽 II高 10 1.6 4.69±0.94* 1.39±0.36* 7.21±1.44* 多肽 Π中 10 0.8 3.88±0.75** 0.82±0.24* 5.57±1.25* 多肽 II低 10 0.4 4.42±0.86* 1.1±0.23* 6.34±1.22*
*表示 p < 0. 05， **表示 p < 0. 01.
结论： 多肽 II对 AA大鼠关节炎具有治疗作用。 多肽 III在胶原诱导大鼠关节炎动物模型都能发挥体内免疫保护作用， 结果见表 74: 大鼠 阳性对照组、 多肽 III中剂量组左后足踝直径与模型组比较， 有极显著性差异（p <0.01 ); 整合 素阻断剂多肽 III低、 高剂量组左后足踝直径与模型组比较， 均有显著性差异（ρ <0.05)， 实验 结果具有统计学意义。 大鼠阳性对照组、 多肽 III低、 中、 高剂量组右后足踝直径与模型组比 较，有显著性差异 <0.05)。多肽 III低、中、高剂量组四肢评分显著低于模型对照组 <0.05)， 与模型对照组比较差异均有统计学意义。
表 74多肽 III对佐剂型类大鼠关节炎动物模型体内免疫保护作用
剂量
左足爪肿胀度 右足爪肿胀度
组别 ？ if (mg/k 临床评分
(mm) (mm)
正常对照组 10 ― 0.90±0.17** 0.38±0.11** 0.00±0.00** 模型对照组 10 ― 7.01±1.42 3.21±0.69 13.11±2.62 阳性对照组 10 1 3.51±0.77** 0.59±0.17** 5.02±1.11* 多肽 III高 10 0.8 4.65±0.94* 1.17±0.26* 6.33±1.20* 多肽 III中 10 0.4 3.72±0.76** 0.73±0.21* 5.29±1.04* 多肽 III低 10 0.2 4.0±0.74* 1.09±0.28* 5.69±1.12*
*表示 p < 0. 05， **表示 p < 0. 01.
结论： 多肽 I、 多肽 Π和多肽 III对 AA大鼠关节炎具有治疗作用。

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^ 產 fi*
19 多肽 VII： Phe-Gln-Pro-Val-Leu-His-Leu-Val-Ala-Leu-Asn-Ser-Pro-Leu-Ser-Gly-Gly-
Met-Arg-Gly-Ile-Arg-Gly-Ala-Asp-Phe-Gln-Cys-Phe-Gln-Gln-Ala-Arg-Ala-Val-Gly-Leu-Ala-Gly-
Thr-Phe-Arg-Ala-Gly-Gly-Gly-Gly-Ala-Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys;
多肽環： Phe-Gln-Pro-Val-Leu-His-Leu-Val- Ala-Leu- Asn-Ser-Pro-Leu-Ser-Gly-Gly-
Met-Arg-Gly-Ile-Arg-Gly-Ala-Asp-Phe-Gln-Cys-Phe-Gln-Gln-Ala-Arg-Ala-Val-Gly-Leu-Ala-Gly-
Thr-Phe-Arg-Ala-Ala-Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys;
多月太 IX : Arg-Gly-Asp-Phe-Gln-Pro-Val-Leu-His-Leu-Val-Ala-Leu-Asn-Ser-Pro
-Leu-Ser-Gly-Gly-Met-Arg-Gly-Ile-Arg-Gly-Ala-Asp-Phe-Gln-Cys-Phe-Gln-Gln-Ala-Arg-Ala-Val-
Gly-Leu-Ala-Gly-Thr-Phe-Arg-Ala及其含有有效量上述多肽可接受的盐，或必要时药学上可接 受的载体或者赋形剂。
4. 根据权利要求 1、 2、 3任意一项所述的整合素阻断剂在制备治疗肿瘤药物中的应用， 其特 征在于所述的肿瘤为起源于人的头颈部、 脑部、 甲状腺、 食管、 胰腺、 肺脏、 肝脏、 胃、 乳 腺、 肾脏、 胆囊、 结肠或直肠、 卵巢、 子宫颈、 子宫、 前列腺、 膀胱或睾丸的原发或继发的 癌、 黑色素瘤以及肉瘤。
5. 根据权利要求根据权利要求 1、 2、 3任意一项所述的整合素阻断剂， 其特征在于所述的多 肽共价连接佐剂， 佐剂是牛血清白蛋白、 人血清白蛋白或聚乙二醇。
6. 根据权利要求 1、 2、 3任意一项所述的整合素阻断剂多肽在制备治疗或预防类风湿性关节 炎药物中的应用。
7. 根据权利要求 5整合素阻断剂在制备治疗肿瘤药物中的应用， 其特征在于所述的药物组合 物可通过多种给药方式治疗原发或继发的癌、 黑色素瘤及肉瘤， 包括皮下或肌肉注射， 静脉 注射或者静脉滴注， 口服给药如药丸、 胶囊等， 鼻喷剂。
8. 根据权利要求 1、 2、 3任意一项所述的整合素阻断剂的制备方法， 其特征在于所述的多肽 是通过合成方法或通过重组表达体的方法制备而得。
PCT/CN2012/087465 2011-12-27 2012-12-26 整合素阻断剂多肽及其应用 WO2013097709A1 (zh)

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PL12863907T PL2799445T3 (pl) 2011-12-27 2012-12-26 Polipeptyd blokujący integrynę do zastosowania w leczeniu reumatoidalnego zapalenia stawów
EP12863907.7A EP2799445B1 (en) 2011-12-27 2012-12-26 Integrin blocker polypeptide for use in the treatment of rheumatoid arthrits
KR1020187026109A KR102003422B1 (ko) 2011-12-27 2012-12-26 인테그린 차단제 폴리펩타이드 및 그의 응용
US14/368,960 US9458203B2 (en) 2011-12-27 2012-12-26 Integrin-blocking polypeptides and uses thereof
KR1020147021078A KR101966762B1 (ko) 2011-12-27 2012-12-26 인테그린 차단제 폴리펩타이드 및 그의 응용
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CN104327169B (zh) * 2014-10-08 2017-12-26 南京安吉生物科技有限公司 整合素阻断剂多肽及其在制备治疗新生血管性眼病药物中的应用
CN105218647A (zh) * 2015-10-28 2016-01-06 苏州普罗达生物科技有限公司 Vegfr2阻断剂多肽及其应用
CN105198965A (zh) * 2015-10-28 2015-12-30 苏州普罗达生物科技有限公司 一种vegfr2阻断剂多肽及其应用
CN105713095B (zh) * 2016-03-14 2021-05-07 南京安吉生物科技有限公司 一种多功能融合多肽及其制备方法和应用
CN108623692B (zh) * 2017-03-20 2020-11-17 徐寒梅 一种融合蛋白及其制备方法和其应用
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US20140316106A1 (en) 2014-10-23
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