WO2017059786A1 - 一种肿瘤靶向的新型多肽 - Google Patents

一种肿瘤靶向的新型多肽 Download PDF

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WO2017059786A1
WO2017059786A1 PCT/CN2016/100989 CN2016100989W WO2017059786A1 WO 2017059786 A1 WO2017059786 A1 WO 2017059786A1 CN 2016100989 W CN2016100989 W CN 2016100989W WO 2017059786 A1 WO2017059786 A1 WO 2017059786A1
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nrgd
tumor
polypeptide
group
peptide
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PCT/CN2016/100989
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English (en)
French (fr)
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张志荣
龚涛
张彦
宋旭
陈体佳
符垚
孙逊
白小清
张贝
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四川大学
重庆药友制药有限责任公司
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Priority to US15/767,146 priority Critical patent/US10428114B2/en
Priority to EP16853091.3A priority patent/EP3360889A4/en
Publication of WO2017059786A1 publication Critical patent/WO2017059786A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • 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
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a novel polypeptide having tumor targeting, in particular to an alanine-alanine-asparagine (AAN)-containing sequence linked to an RGD-containing peptide, thereby obtaining a not only targeting tumor cells and blood vessels, but also A tandem polypeptide (nRGD) that targets tumor-associated macrophages to regulate the tumor microenvironment to enhance antitumor effects belongs to the field of medicine.
  • AAN alanine-alanine-asparagine
  • nRGD tandem polypeptide
  • tumor microenvironment at the tumor site which provides the necessary conditions for tumor development and metastasis, such as maintaining growth-promoting signals, maintaining neovascularization, resistance to apoptosis and growth inhibition signals, and tumor cell metastatic ability. And infinite proliferation, genomic instability and susceptibility, energy metabolism reforming, inflammation that promotes tumor growth, and evasion of the identification and killing of the immune system. (Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011, 144, 647-674.)
  • Integrin is a kind of cell adhesion receptor which exists on the cell membrane and belongs to the tumor penetrating peptide. Its main function is to mediate the adhesion between cells and cells, cells and extracellular matrix. Integrins form heterodimers by the non-covalent bond of alpha and beta subunits. In vertebrates, 18 alpha subunits and 8 beta subunits make up 24 different heterodimeric structures. Among them, ⁇ v ⁇ 3 integration It is highly expressed on the surface of various tumor cells and tumor-associated vascular endothelial cells such as ovarian cancer, melanoma, breast cancer and glioma, and is closely related to tumor neovascularization and metastasis.
  • a tripeptide sequence containing arginine-glycine-aspartate can specifically recognize and bind to integrin.
  • the commonly used tumor penetrating peptide iRGD has the characteristics of simultaneously targeting tumor neovascularization and tumor cells, and is highly integrated with the integrin receptor proteins ⁇ v ⁇ 3 and ⁇ v ⁇ 5 highly expressed on the membrane of neovascular endothelial cells to achieve tumor targeting of anticancer drugs. transfer. (Sugahara KN, Teesalu T, Karmali PP, Kotamraju VR, Agemy L, Greenwald DR, Ruoslahti E. Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs. Science 2010 May 21; 328 (5981): 1031-5.)
  • the prior art discloses that during the process of developing a malignant tumor cell into a tumor, the tumor cell utilizes its own high mutation in the process of interacting with the body, and down-regulates the expression of the protein molecule associated with the immune recognition and attack to generate an immune escape.
  • abnormal or overexpressing immunosuppressive-related proteins directly inhibit tumor immune responses or induce immunosuppressive cell differentiation and infiltration.
  • tumor cells not only overcome the body's immune system to recognize and kill it, but also establish a tumor immune microenvironment that provides sufficient nutrition and suitable for its rapid growth (Dunn GP, Bruce AT, Ikeda H, Old LJ, Schreiber RD. Cancer immunoediting: from immunosurveillance to tumor escape. Nat Immunol. 2002 Nov; 3 (11): 991-8).
  • the RGD peptide targets tumor blood vessels and tumor cells, thereby enhancing the therapeutic effect of the drug on the tumor
  • the biological treatment of the tumor is also an important aspect that cannot be ignored. Because tumor cells establish an immune barrier for themselves, the body cannot recognize and kill tumor cells. As long as there are residual tumor cells in the tumor environment, the tumor may have a recurrence. Moreover, the ideal therapeutic effect cannot be achieved by a single tumor treatment.
  • the present invention provides a more effective tumor targeting novel polypeptide.
  • One of the objects of the present invention is to provide a tumor-targeting novel multifunctional polypeptide which binds an alanine-alanine-asparagine (AAN)-containing sequence to an RGD-containing peptide, preferably by covalent
  • AAN alanine-alanine-asparagine
  • RGD a tandem polypeptide
  • nRGD tumor-associated macrophage-targeted AANs
  • Dox doxorubicin
  • One of the objects of the present invention is to provide a novel polypeptide (nRGD) having tumor targeting which can be directly used in combination with a drug or a carrier.
  • One of the objects of the present invention is to provide a novel polypeptide (nRGD) having tumor targeting capable of modifying an antitumor drug to obtain a prodrug of the antitumor drug; and also capable of modifying a drug delivery carrier.
  • nRGD novel polypeptide
  • One of the objects of the present invention is to provide a novel polypeptide having tumor targeting which targets tumor-associated macrophages via the peptide-alanine-alanine-asparagine (AAN).
  • AAN peptide-alanine-alanine-asparagine
  • One of the objects of the present invention is to provide a novel polypeptide having tumor targeting that targets tumor blood vessels and tumor cells via RGD polypeptides.
  • One of the objects of the present invention is to provide a novel polypeptide having tumor targeting, wherein the AAN-containing sequence is preferably covalently linked to the RGD-containing peptide; or preferably via a peptide bond or an amino acid peptide chain; or an AAN-containing sequence
  • AAN-containing and RGD-containing peptides may be linked by amino acid analogs or peptide analogs, and that more than one atom may be located between the bonding atoms, such as by gamma-aminobutyric acid.
  • new multifunctional polypeptide of the present invention has the same meaning as “new polypeptide”, “multifunctional polypeptide”, “nRGD”, “nRGD polypeptide”, and “polypeptide nRGD”.
  • the "RGD-containing peptide” described in the present invention has the same meaning as “RGD-containing peptide”, “RGD-containing peptide chain”, and “RGD-containing peptide chain”.
  • the AAN of the present invention is a substrate for an aspartic endoprotein (legumain). This hair
  • aspartic endonuclease and most of the integrin receptors such as ⁇ v ⁇ 3 co-localized to form a complex, high expression in tumor cells and tumor vascular endothelial cell membrane.
  • integrin receptors such as ⁇ v ⁇ 3
  • tumors such as breast cancer, colon cancer, lung cancer, liver cancer and the like all overexpress aspartic endopeptidase.
  • AAN alanine-alanine-asparagine
  • the target of AAN according to the present invention is aspartate endoproteinase (Legumain).
  • Legumain is not only highly expressed in tumor cells, but is associated with the tumor microenvironment. Tumor-associated macrophages are also highly expressed.
  • Reisfeld, RA The tumor microenvironment: a target for combination therapy of breast cancer. Crit. Rev. Oncog. 18, 115–133 (2013). Lin, Y. et al. Selective ablation of tumor-associated macrophages suppresses metastasis and angiogenesis. Cancer Sci. 104, 1217–1225 (2013).
  • Patent CN201310048238 states that the AAN tripeptide modified doxorubicin prodrug has the same antitumor efficacy as the original drug.
  • the literature also shows that AAN directly modified liposomes did not improve the anti-tumor efficacy of the drug.
  • the AAN tripeptide-modified anticancer drug or carrier cannot successfully enhance the antitumor effect of the anticancer drug or carrier.
  • TAM tumor-associated macrophages
  • TAM is an important inflammatory cell that exists around the blood vessels in the tumor site and has no blood vessel necrotic areas. TAM It is a key component of the tumor site, which secretes a large number of tumor growth promoting factors. It has also been found in mouse models to regulate angiogenesis, lymphangiogenesis, immunosuppression and tumor metastasis. (Seth B. Coffelt, Russell Hughes, Claire E. Lewis. Associated macrophages: Effects of angiogenesis and tumor progression. Biochimica et Biophysica Acta. 1796 (2009) 11–18) along with other bone marrow-associated cells expressed at the tumor site TAM is already a very attractive target for new biologic therapies for cancer.
  • the novel polypeptide of the present invention targets tumor blood vessels and cells to kill tumor blood vessels and cells, and also targets tumor-associated macrophages, thereby significantly enhancing the therapeutic effect by changing the tumor microenvironment.
  • novel multifunctional polypeptide of the present invention can not only be combined with other treatment methods for anti-tumor, but also can be used as an anti-tumor biological treatment method alone.
  • the AAN-containing sequence and the RGD-containing peptide also pass through a molecule similar to the peptide chain but not linked by the native peptide chain.
  • AAN does not affect its tumor targeting and tumor vascular targeting function, regardless of whether it is broken from the RGD peptide chain.
  • One of the objects of the present invention is to provide a pharmaceutical composition in which the nRGD polypeptide is mixed with a pharmaceutically active ingredient or the nRGD polypeptide is mixed with a delivery vehicle.
  • One of the objects of the present invention is to provide a pharmaceutical composition in which the nRGD polypeptide is covalently linked or non-covalently associated with a pharmaceutically active ingredient, or the nRGD polypeptide is covalently linked or non-covalently associated with a delivery vehicle.
  • the nRGD polypeptide of the present invention is in a mode of mixing with the composition, without covalent attachment or non-covalent association; covalently linked or non-covalently associated with the composition; the novel polypeptide precedes or Administration to the composition; attachment to the composition; using one or a combination of the above.
  • the nRGD polypeptide of the present invention can be used in combination with one or more accessory molecules.
  • the combined mode is association; at least one of the accessory molecules does not overlap with the nRGD polypeptide; at least one of the accessory molecules overlaps with the nRGD polypeptide.
  • the accessory molecule comprises an independent homing molecule, a targeting molecule, an affinity ligand, a cell penetrating peptide, an in vivo escape molecule, a subcellular targeting molecule, a nuclear targeting molecule, or a combination and mixture thereof.
  • homing molecule refers to a body that preferentially homing to a tumor or other specific tissue in preference to normal tissue;
  • conjugate refers to a substance formed by chemical bonds between molecules.
  • Drugs suitable for use in the present invention include, but are not limited to, therapeutic effects of therapies: therapeutic proteins, therapeutic compounds, therapeutic compositions, cancer chemotherapeutics, toxins, cytotoxic agents, anti-inflammatory agents, growth factors, cytokines, trends Compounds, compounds, antibodies, nucleic acids, nucleic acid analogs, cells, viruses, bacteriophage, viral particles, phage particles, viral capsids, phage capsids, virus-like particles, liposomes, micro-modulates that regulate one or more signaling pathways Balls, nanoparticles, micelles, emulsions, microemulsions, dendrimers, microparticles, chemotherapeutic agents, anti-angiogenic agents, or combinations and mixtures thereof.
  • Tumors suitable for use in the present invention include, but are not limited to, benign or malignant tumors, including benign or malignant tumors of the epithelial tissue; benign or malignant tumors of the mesenchymal tissue; benign or malignant tumors of the lymphoid hematopoietic tissue; benign or malignant tumors of the nervous tissue; gonad or embryo related Benign or malignant tumors; other tumors include pigmented nevi, hydatidiform mole, melanoma, chorionic epithelial cancer, seminoma, dysplasia, and embryonal carcinoma.
  • the RGD peptide chain contained in the nRGD peptide of the present invention includes, but is not limited to, an RGD peptide, a cyclic c (RGDfK), iRGD or a derivative thereof, preferably iRGD.
  • the nRGD peptide of the present invention can pass a cysteine residue Covalently linked to a pharmaceutically active ingredient or a carrier, the preferred nRGD peptide has the structure of formula 1:
  • nRGD peptide of the present invention may not contain the above cysteine residue when it is non-covalently associated or mixed with a pharmaceutically active ingredient or a drug delivery carrier.
  • the AAN of the novel targeting polypeptide comprises a derivative thereof, such as a polypeptide substrate R-AAN-sequence sensitive to aspartic endopeptidase, wherein the R group refers to hydrogen. Atom (H), acetyl (Ac), alanine (A), phenylalanine (F), glycine (G) or a combination thereof.
  • R is preferably H
  • the sequence of the nRGD is CCRGDK (NAA) GPDC, wherein the second cysteine is linked to the 10th cysteine to form a loop; or R is preferably H.
  • the sequence of the nRGD is CRGDK (NAA) GPDC, wherein two cysteines are joined to form a loop.
  • the drug to be used is preferably a chemotherapeutic drug, wherein the chemotherapeutic drug is preferably doxorubicin (Dox).
  • Dox doxorubicin
  • the pharmaceutical dosage form used in the present invention is preferably a PEGylated liposome.
  • the mode of use described in the present invention is covalent attachment and direct mixing.
  • the tumor model used in the present invention is a breast cancer ectopic tumor.
  • One of the objects of the present invention is to provide the use of the polypeptide of the present invention, nRGD, or a pharmaceutical composition thereof, for the preparation of an antitumor drug.
  • One of the objects of the present invention is to provide the use of the polypeptide nRGD of the present invention or a pharmaceutical composition thereof for the preparation of a pharmaceutically acceptable pharmaceutical preparation.
  • the addition of the multifunctional polypeptide of the present invention significantly increases the antitumor effect of the doxorubicin drug and the doxorubicin liposome. Moreover, the anti-tumor effect of nRGD and iRGD was also significantly increased. At the same time, the addition of multifunctional peptides also significantly increased the survival of mice, and found that most of the mice in the nRGD group survived for 90 days, and some of the tumors disappeared. This effect is amazing. It is also a significant effect not reported in previous anti-tumor drug treatments. Although only a simple mix, the survival of some mice in the PEGylated liposome and nRGD mixed groups was extended to 90 days. The iRGD group of mice can only live for 2 months. The addition of multifunctional peptides significantly reduces the toxicity of the drug due to enhanced targeting.
  • the present invention through creative research, covalently attaches an alanine-alanine-asparagine (AAN) sequence to an RGD-containing peptide to obtain a tumor cell and a blood vessel and a tumor-associated macrophage to thereby modulate the tumor.
  • a tandem polypeptide (nRGD) that enhances the anti-tumor effect in a microenvironment.
  • polypeptides of the present invention significantly enhance the effectiveness of anti-tumor components while also altering the single treatment modality of anti-tumor components. Has a good application prospects.
  • the multifunctional polypeptide nRGD of the present invention significantly enhances the antitumor effect of the antitumor component.
  • the multifunctional polypeptide nRGD of the present invention enhances the antitumor effect of the antitumor component by targeting tumor-associated macrophages, regulating the tumor microenvironment, and producing a biological therapeutic effect.
  • the multifunctional polypeptide nRGD of the present invention alters the single treatment mode of the anti-tumor component.
  • the multifunctional polypeptide of the present invention reduces the toxicity of the antitumor component while increasing the targeting property.
  • the multifunctional polypeptide of the present invention is simple to use and is expected to be a key component in the future development of tumor drugs.
  • Figure 1 Schematic and electron micrograph of doxorubicin liposomes.
  • Figure 1-1 shows a schematic diagram of the doxorubicin liposome;
  • Figure 1-2 shows an electron micrograph of the doxorubicin liposome.
  • Figure 2 shows that in the 4T1 tumor model, the nRGD group can significantly improve the efficacy of doxorubicin.
  • Figure 2-1 shows the tumor growth curve.
  • Figure 2-2 shows a tumor picture.
  • Figures 2-3 show tumor weight.
  • Figures 2-4 show the tumor inhibition rate.
  • Figure 2-5 shows changes in body weight.
  • Figure 2-6 shows the growth curve. *P ⁇ 0.05, ***P ⁇ 0.01.
  • Figure 3 shows tumor HE staining, Ki-67 and HER2 immunohistochemical staining evaluation.
  • Figure 3-1 shows HE staining, yellow arrow indicates active area of tumor growth;
  • Figure 3-2 shows Ki-67 immunohistochemical staining;
  • Figure 3-3 shows HER2 immunohistochemical staining.
  • Figure 3-4 shows the Ki-67 and HER2 optical density measurements.
  • Figure 4 shows that the nRGD group recognizes tumor blood vessels, increases tumor penetration, and targets tumor-associated macrophages.
  • Figure 4-1, Figure 4-2, and Figure 4-3 show that nRGD-Lipo-Dox can target CoCl2-treated 4T1 and M2 macrophages. *P ⁇ 0.05, ***P ⁇ 0.01.
  • Figure 5 shows the cytokine levels of tumor sites after treatment.
  • Figure 5-1 shows the ELISA assay for TGF- ⁇ 1. *P ⁇ 0.05, ***P ⁇ 0.01.
  • Figure 5-2 shows TGF- ⁇ 1
  • Figure 5-3 shows CCl 2
  • Figure 5-4 shows IL-10
  • Figure 5-5 shows IL-6
  • Figure 6 shows that the nRGD group can target TAMs to modulate the tumor microenvironment.
  • Figure 7 shows that the nRGD group is relatively low in toxicity.
  • Figure 7-1 shows different organ tissue sections (200X except bone 100X);
  • Figure 7-2 shows splenomegaly in each group;
  • Figure 7-3 shows serum cytokine IL-6 assay;
  • Figure 7-4 shows serum cells Factor IL-12 assay. *P ⁇ 0.05, ***P ⁇ 0.01.
  • Figure 8 shows that in the 4T1 tumor model, the nRGD group can significantly improve the efficacy of paclitaxel.
  • Tumor growth curve (Fig. 8-1), tumor picture (Fig. 8-2), tumor weight (Fig. 8-3), tumor inhibition rate (Fig. 8-4), and body weight change (Fig. 8-5).
  • Figure 9 shows that in the 4T1 tumor model, the nRGD group can significantly increase the efficacy of the lycopene.
  • Tumor growth curve (Fig. 9-1), tumor picture (Fig. 9-2), tumor weight (Fig. 9-3), tumor inhibition rate (Fig. 9-4), and body weight change (Fig. 9-5). *P ⁇ 0.05, ***P ⁇ 0.01.
  • Figure 10 shows that in the 4T1 tumor model, the nRGD group can significantly improve the efficacy of docetaxel.
  • Tumor growth curve (Figure 10-1), tumor picture ( Figure 10-2), tumor weight (Figure 10-3), tumor inhibition rate (Figure 10-4), body weight change (Figure 10-5). *P ⁇ 0.05, ***P ⁇ 0.01.
  • Figure 11 shows that in the glioma model, the nRGD group has a better antitumor effect than the iRGD group.
  • the sequence is CCRGDK (NAA) GPDC, in which the second cysteine is linked to the 10th cysteine and looped out and synthesized by Jill Biochemical (Shanghai) Co., Ltd.
  • the prepared nRGD peptide was 85% pure.
  • the nRGD peptide When non-covalently associated or mixed with a pharmaceutically active ingredient or a carrier, the nRGD peptide may not contain the first cysteine as a linking group, and the corresponding sequence is CRGDK (NAA) GPDC, two of which Cysteine is linked to form a ring with a purity of 92%.
  • NAA CRGDK
  • the doxorubicin liposome was prepared by a film dispersion method and an ammonium sulfate gradient method. 56 parts of phospholipids, 34 parts of cholesterol, 8 parts of PEG 2000 -DSPE (purchased from Lipoid, Germany) and 2 parts of Mal-PEG 2000 -DSPE (PEGylated liposomes without Mal-PEG 2000 -DSPE, ie prescription 56 Separate phospholipids, 34 parts of cholesterol, 10 parts of PEG 2000 -DSPE) (purchased from Lipoid, Germany) were dissolved in 5 mL of chloroform. The organic solvent was removed by rotary evaporation, and a solution of 123 mMol ammonium sulfate was hydrated.
  • mice Female Balb/c mice were inoculated with 5 ⁇ 10 5 4T1 cells and randomly divided into 7 groups.
  • Saline group (NS) Dox, PEGylated liposome (PEG-Lipo-Dox), iRGD liposome (iRGD-Lipo-Dox), nRGD liposome (nRGD-Lipo-Dox), mixed with Dox and nRGD
  • the administration group Dox+nRGD
  • the PEGylated liposome and the nRGD mixed administration group PEG-Lipo-Dox+nRGD.
  • Dox+nRGD the PEGylated liposome
  • PEG-Lipo-Dox+nRGD On day 8, 12, 5 mg/kg Dox equivalent of the drug or various formulations were injected.
  • TGI (1 - (the average tumor weight of the treatment group / (the average weight of the control tumor)) X 100%.
  • HE immunohistochemistry, RT-PCR, immunofluorescence and ELISA Anti-tumor effects and mechanisms were studied. Plasma samples were collected for ELISA. Tissue samples were evaluated for toxicity by HE staining. The remaining mice were subjected to survival studies.
  • nRGD group improved the anti-tumor effect on the surface of the liposome whether it was directly mixed with Dox or liposome or modified.
  • the 4T1 tumor rapidly increased to 2161.7 ⁇ 422.6 mg and the volume was 1200 mm 3 in 20 days without treatment. Tumors in the treatment group were all reduced. Both PEG-Lipo-Dox+nRGD and nRGD-Lipo-Dox exhibited significant antitumor effects compared to the iRGD-Lipo-Dox group. Compared with the non-peptide group, the anti-tumor effect of the peptide-added group was also improved. The tumor growth inhibition rate is also consistent with the tumor growth curve. The weight of nRGD-Lipo-Dox tumor was 49.8 ⁇ 28.6 mg, and the inhibition rate was as high as 97.7%.
  • the tumor weights of Dox and PEG-Lipo-Dox were 1592.0 ⁇ 98.0 mg and 942.0 ⁇ 295.0 mg, and the inhibition rates were only 26.3% and 56.4%.
  • the tumors of the Dox+nRGD and PEG-Lipo-Dox+nRGD groups were only 462.0 ⁇ 43.2 mg and 195.0 ⁇ 116.1 mg, and the inhibition rates were 77.5% and 90.1%.
  • the nRGD group prolonged the survival of tumor mice. For nRGD-Lipo-Dox, 44.4% of the mice survived after 90 days and the tumors in the mice were completely cured.
  • the survival time of the mice in the nRGD mixed group was also prolonged compared with the control group.
  • the iRGD-Lipo-Dox group only lived for 65 days. It can be seen from the above experiments that the anti-tumor effect of the nRGD group is very remarkable.
  • nRGD antitumor effect of nRGD
  • the inventors performed HE and immunohistochemical studies on tumors, as shown in FIG. HE staining showed that the nRGD group significantly inhibited tumor growth, and the tumors in the nRGD-Lipo-Dox group were almost necrotic. Ki-67 immunohistochemistry is the evaluation of proliferating cells, and HER2 is an evaluation of oncogenic transforming factors and tumor growth in tumors. Both methods also confirmed that the nRGD group performed better. All results are consistent with the anti-tumor effect.
  • nRGD group can significantly improve the efficacy regardless of whether it is directly mixed with doxorubicin and its liposomes, or modified on the surface of liposomes. It is worth noting that the addition of nRGD, because the effect is very significant, reduces the number of administrations and achieves excellent therapeutic effects.
  • the nRGD group targets tumor blood vessels and cells while targeting TAMs, as shown in Figure 4 and Table 2.
  • nRGD-Lipo-Dox selectively targets CoCl2-stimulated 4T1 cells that activate legumain and M2-type macrophages after IL-4 stimulation .
  • nRGD significantly reduced uptake compared to iRGD.
  • Table 2 the IC50 values of each group were consistent with the cellular uptake results.
  • nRGD-Lipo-Dox had lower IC50 in CoCl2-stimulated 4T1 cells and IL-4-stimulated M2 macrophages, indicating that cells with high expression of legumain More lethal.
  • the inventors also evaluated the targeting of the nRGD group by in vivo. As shown in Figure 4-4, co-administration or modification of nRGD increased the accumulation of Dox at the tumor site, indicating that it increased the permeability of the drug at the tumor site. The inventors in the section saw that due to the killing effect of Dox and liposome on tumor cells, there was a void in the tumor with more permeation, and only the connective tissue of the tumor interstitial remained. The inventors evaluated the anti-angiogenic effect and found that the blood vessels of the tumor in the nRGD group and the iRGD-Lipo-Dox group gradually decreased with increasing time points.
  • Tumor-associated blood vessels were significantly reduced in the nRGD group and the iRGD–Lipo-Dox group compared to the saline group, Dox, and PEG–Lipo-Dox.
  • anti-angiogenesis and direct drug killing of tumors often result in tumor-associated macrophage aggregation at the tumor site.
  • Previous studies have shown that the nRGD and iRGD–Lipo-Dox groups cause rapid destruction of blood vessels and tumor cells at the tumor site. As shown in Figure 4-5, it was found that TAMs gradually increased over time in the iRGD–Lipo-Dox tumor site.
  • nRGD group as described by the inventor Like, with M2 type macrophage killing effect, did not lead to an increase in TAMs.
  • nRGD has the function of targeting tumor blood vessels and tumor cells, while having the effect of targeting TAMs.
  • the nRGD group targets TAMs to regulate the microenvironment of the tumor, as shown in Figures 5 and 6.
  • nRGD-targeted TAMs are of great significance for the regulation of the tumor microenvironment, and the inventors have studied changes in the tumor microenvironment.
  • TAMs are reported to be cells that highly express TGF ⁇ 1, CCl2 and IL-10.
  • the expression levels of all three in the nRGD group were reduced.
  • the expression levels of IL-6 and TNF- ⁇ in the nRGD group increased, and the increase in both helped to inhibit the effect of effector T cells and thereby suppress the immune response at the tumor site.
  • the results indicate that changes in cytokines in the tumor site of the nRGD group contribute to the improvement of its role in the treatment of tumors.
  • vascular endothelial growth factor VEGF
  • CD 34-labeled tumor blood vessels CD 34-labeled tumor blood vessels
  • CD 105-labeled tumor neovascularization As shown in Figures 6-1, 6-2 and 6-3, the nRGD group had a relatively low expression of VEGF, while the blood vessels and neovascularization at the tumor site were reduced. In the iRGD–Lipo-Dox group, although the number of blood vessels was reduced, the expression of VEGF and neovascularization was observed to increase. This indicates that nRGD still maintains tumor vascular normalization after treatment of TAMs, thereby inhibiting tumor angiogenesis and tumors. relapse.
  • VEGF vascular endothelial growth factor
  • TAMs are also associated with immune escape and inhibition at the tumor site.
  • the inventors studied the number of immune cells of tumor cells. As shown in Figure 6-4, there is no obvious rule for CD8+ T cell changes. As shown in Figures 6-5 and 6-6, both regulatory T cells and MDSCs showed an increase in the number of tumor sites in the Dox, PEG-Lipo-Dox and iRGD–Lipo-Dox groups, whereas there was no change in the nRGD group. These results indicate that the nRGD group targets TAMs, thereby reducing the number of regulatory T cells and MDSCs, and suppressing the effects of these cells on tumor growth and tumor immune escape.
  • nRGD neuropeptide kinase
  • the cytokine IL-6 and IL-12 assays also found that serum levels of IL-6 and IL-12 were reduced in the nRGD group.
  • the nRGD group achieved superior anti-tumor effects by targeting tumor blood vessels and tumor cells and targeting TAMs to regulate the tumor microenvironment, and showed lower toxicity.
  • mice Female Balb/c mice were inoculated with 5 ⁇ 10 5 4T1 cells and randomly divided into 5 groups. Saline group (NS), PTX, paclitaxel albumin nanoparticles (PTX-BSA), PTX and nRGD mixed administration group (PTX+nRGD) and paclitaxel albumin nanoparticles and nRGD mixed administration group (PTX-BSA+nRGD) ).
  • a 10 mg/kg PTX equivalent of the drug or formulation was injected on days 8, 12, and 16.
  • the single administration of the mixed administration group was two needles, one needle was 10 mg/kg PTX equivalent of the drug or preparation, and the other needle was injected with 4.8 mg/kg of nRGD. Volume and body weight were measured every 2 days. Part of the mice were sacrificed at 20 days.
  • RESULTS As shown in Figure 8, the nRGD group improved the anti-tumor effect of PTX and PTX-BSA, and the body weight No reduction, no increase in toxicity.
  • mice Female Balb/c mice were inoculated with 5 ⁇ 10 5 4T1 cells and randomly divided into 5 groups.
  • a 12 mg/kg IBT equivalent of the drug or formulation was injected on days 8, 9, and 10.
  • the single administration of the mixed administration group was two needles, one needle was 12 mg/kg IBT equivalent of the drug or preparation, and the other needle was injected with 4.8 mg/kg of nRGD.
  • TGI (1 - (the average tumor weight of the treatment group / (the average weight of the control tumor)) X 100%.
  • nRGD group increased the anti-tumor effect of PTX and PTX-BSA, and did not decrease body weight and did not increase toxicity.
  • mice Female Balb/c mice were inoculated with 5 ⁇ 10 5 4T1 cells and randomly divided into 5 groups. Saline group (NS), TXT, docetaxel micelles (TXT-micells), docetaxel and nRGD mixed dose group (TXT+nRGD) and docetaxel micelles and nRGD mixed dose group ( TXT-micells+nRGD).
  • a 15 mg/kg TXT equivalent of the drug or formulation was injected on days 8, 10, and 12.
  • the single administration of the mixed administration group was two needles, one needle was 15 mg/kg TXT equivalent of the drug or preparation, and the other needle was injected with 4.8 mg/kg of nRGD. Volume and body weight were measured every 2 days. Part of the mice were sacrificed at 20 days.
  • nRGD group increased the anti-tumor effect of TXT and TXT-micells, and did not decrease body weight and did not increase toxicity.
  • the nRGD group had a better anti-tumor effect than the iRGD group.
  • Glioblastoma mice were randomly divided into 9 groups, 20 in each group.
  • Saline group (NS) Teniposide, free lycopene and octreotide (free IBT & OCT), oxidized lycopene and octreotide PEGylated liposome (PEG-Liposome), liposome mixed with iRGD Administration group (PEG-Liposome+iRGD), liposome and nRGD mixed administration group (PEG-Liposome+nRGD), iRGD modified lycopene and octreotide PEGylated liposome (iRGD-Liposome), nRGD modification The oxidized lysine and octreotide PEGylated liposome (nRGD-Liposome) and the blank liposome and nRGD mixed administration group (blank).
  • 10 mg/kg IBT and 200 ⁇ g/kg OCT equivalent of the drug or preparation were injected on the 5th, 7th, 9th, 11th, and 12th day.
  • 10 mg/kg Teniposide was injected as a positive control.
  • the mixed administration group was administered in two doses, one for 10 mg/kg IBT and 200 ⁇ g/kg OCT equivalent for the drug or preparation, and the other for 5 mg/kg of iRGD or nRGD.
  • the survival of the mice was recorded daily and the survival curve was plotted.
  • the nRGD group had a longer life cycle than the iRGD group, either mixed or modified on the liposome surface.
  • the nRGD group had a better antitumor effect than the iRGD group, whether it was mixed administration or modified on the surface of the liposome.
  • the inventors screened the use of nRGD with doxorubicin as a model drug.
  • mice Female Balb/c mice were inoculated with 5 ⁇ 10 5 4T1 cells and randomly divided into 3 groups. Doxorubicin was administered in combination with nRGD, which was administered prior to doxorubicin or after doxorubicin.
  • the inventors screened the concentration of nRGD using doxorubicin as a model drug.
  • mice Female Balb/c mice were inoculated with 5 ⁇ 10 5 4T1 cells and randomly divided into 5 groups.
  • the doxorubicin dose was 5 mg/kg, and the nRGD concentrations were 1, 2, 4, 8, 10 mg/kg, respectively.
  • RESULTS The concentration of nRGD was positively correlated with Dox anti-tumor.
  • mice Female Balb/c mice were inoculated with 5 ⁇ 10 5 4T1 cells and randomly divided into 3 groups.
  • the doxorubicin dose was 5 mg/kg, and the other two groups were given the above polypeptides on the basis of doxorubicin.
  • the polypeptide sequences RGD and c (RGDfK) targeting the tumor angiogenic endothelial cell integrin receptor are respectively linked to AAN via -NHCH 2 CH 2 CH 2 CO-.
  • the sequences are CRGD-4Abu-NAA and c(RGDfK)-4Abu-AAN.
  • the effects of evaluating the two peptides are as follows:
  • mice Female Balb/c mice were inoculated with 5 ⁇ 10 5 4T1 cells and randomly divided into 3 groups.
  • the doxorubicin dose was 5 mg/kg, and the other two groups were given the above polypeptides on the basis of doxorubicin.
  • the nRGD of the present invention significantly increases the antitumor drug effect, has lower toxicity, and has wide applicability, which has not been reported in previous literatures and materials.
  • the inventors have rationally speculated that the present invention is useful for a variety of anti-tumor compositions and also for anti-tumor helper molecules to enhance their therapeutic effects on malignant or benign tumors.

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Abstract

提供一种由丙氨酸-丙氨酸-天冬酰胺（AAN）和含精氨酸-甘氨酸-天冬氨酸（RGD）多肽连接而成的多肽nRGD；所述多肽nRGD可靶向肿瘤血管、肿瘤细胞和肿瘤相关巨噬细胞，介导肿瘤靶向传递。

Description

一种肿瘤靶向的新型多肽
本申请要求于2015年10月10日提交中国专利局、申请号为201510650649.2、发明名称为“一种肿瘤靶向的新型多肽”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及一种具有肿瘤靶向的新型多肽，具体涉及含丙氨酸-丙氨酸-天冬酰胺(AAN)序列连接于含RGD肽，从而得到一种不仅靶向肿瘤细胞和血管，而且靶向肿瘤相关巨噬细胞，从而调节肿瘤微环境增强抗肿瘤的效果的串联型多肽(nRGD)，属于医药领域。
背景技术
肿瘤部位存在特殊的肿瘤的微环境，为肿瘤的发展和转移提供了所必需的条件，如保持促生长信号，保持新生血管生成，对细胞凋亡和生长抑制信号的抵抗，肿瘤细胞的转移能力和无限增殖，基因组的不稳定和易突变性，能量代谢重整，促肿瘤生长的炎症及逃避免疫系统的识别和杀伤等。(Hanahan D,Weinberg RA.Hallmarks of cancer:the next generation.Cell2011,144,647-674.)
随着生物医学技术的高速发展，肿瘤的靶向治疗已成为当前肿瘤治疗的主要发展方向。递药载体技术的发展为抗肿瘤药物的特异性、靶向性传递提供了可能。利用特异性的肿瘤靶向配体修饰抗肿瘤药物或递药载体，有效提高药物或递药载体在肿瘤部位的分布和蓄积，降低药物在非靶器官和组织的分布，从而实现提高疗效、降低毒副作用的目的。
整合素(Integrin)是一类存在于细胞膜上的细胞黏附受体，属于肿瘤穿透肽，其主要功能是介导细胞与细胞、细胞与细胞外基质间的黏附作用。整合素由α和β亚基通过非共价键结合而形成异二聚体。脊椎动物体内，18种α亚基和8种β亚基组成24种不同的异二聚体结构。其中，αvβ3整合 素在卵巢癌、黑色素瘤、乳腺癌、神经胶质瘤等多种肿瘤细胞及肿瘤相关血管内皮细胞的表面高度表达，与肿瘤的新生血管生成和转移密切相关。
已有研究证实，含有精氨酸-甘氨酸-天冬氨酸(RGD)的三肽序列能够特异性的识别并结合整合素。目前常用的肿瘤穿膜肽iRGD具有同时靶向肿瘤新生血管和肿瘤细胞的特性，与肿瘤新生血管内皮细胞膜上高表达的整合素受体蛋白αvβ3和αvβ5高度结合，实现抗癌药物的肿瘤靶向传递。(Sugahara KN,Teesalu T,Karmali PP,Kotamraju VR,Agemy L,Greenwald DR,Ruoslahti E.Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs.Science 2010May 21；328(5981):1031-5.)
现有技术披露，恶性肿瘤细胞发展成为肿瘤的过程中，肿瘤细胞在与机体相互作用的过程中，利用自己的高突变性，一方面下调与免疫识别和攻击相关蛋白分子的表达而产生免疫逃逸；另一方面异常或过表达免疫抑制相关蛋白，直接抑制肿瘤免疫反应，或者诱导免疫抑制细胞分化和浸润。在这个过程中，肿瘤细胞不仅克服机体免疫系统对其识别和杀伤，而且建立起能为其提供充足营养和适合其快速生长的肿瘤免疫微环境(Dunn GP,Bruce AT,Ikeda H,Old LJ,Schreiber RD.Cancer immunoediting:from immunosurveillance to tumor escape.Nat Immunol.2002Nov；3(11):991-8)。
由此可见，RGD肽虽然靶向肿瘤血管和肿瘤细胞，从而增强药物对肿瘤的治疗作用，但是肿瘤的生物治疗也是不可忽视的重要方面。由于肿瘤细胞为自己建立起免疫屏障，机体无法识别和杀伤肿瘤细胞。只要肿瘤环境中有残存的肿瘤细胞，那么肿瘤就有复发的可能。而且，仅仅只是通过单一的肿瘤治疗方式不能达到理想的治疗效果。
为了解决上述提及的至少一个问题，本发明提供一种更加有效的肿瘤靶向新型多肽。
发明内容
本发明的目的之一，提供了一种肿瘤靶向新型多功能多肽，所述多肽将含丙氨酸-丙氨酸-天冬酰胺(AAN)序列连接于含RGD肽，优选通过共价 方式进行连接，从而获得能够靶向肿瘤细胞和血管和肿瘤相关巨噬细胞，从而调节肿瘤微环境增强抗肿瘤效果的串联型多肽(nRGD)。
研究中，本发明人将对肿瘤和肿瘤血管靶向作用非常突出的iRGD肽和肿瘤相关巨噬细胞靶向的AAN通过共价键连接起来获得nRGD，再以阿霉素(Dox)为模型药物，制备了含药脂质体。
在研究中意外地发现，将多肽nRGD与阿霉素(Dox)制备的含药脂质体同iRGD与阿霉素(Dox)制备的含药脂质体相比，取得了出乎意料的抗肿瘤效果。本发明人将该肽nRGD和Dox原药或Dox脂质体混合给药，也取得了显著的抗肿瘤效果。
本发明的目的之一，提供了一种具有肿瘤靶向的新型多肽(nRGD)，其能直接与药物或载体混合使用。
本发明的目的之一，提供了一种具有肿瘤靶向的新型多肽(nRGD)，其能够修饰抗肿瘤药物，得到所述抗肿瘤药物的前药；也能够修饰递药载体。
本发明的目的之一，提供了一种具有肿瘤靶向的新型多肽，其通过肽段—丙氨酸-丙氨酸-天冬酰胺(AAN)，靶向肿瘤相关巨噬细胞。
本发明的目的之一，提供了一种具有肿瘤靶向的新型多肽，通过RGD多肽，靶向肿瘤血管和肿瘤细胞。
本发明的目的之一，提供了一种具有肿瘤靶向的新型多肽，其中含AAN的序列与含RGD肽是优选共价连接；或者优选通过肽键或氨基酸肽链连接；或者含AAN的序列与含RGD肽通过以下方式连接：-CH2NH-，-CH2S-,-CH2-CH2-,-CH＝CH-,-COCH2-和-CH(OH)CH2-。应理解，含AAN和RGD肽可以由氨基酸类似物或者肽类似物连接，可以有多于一个原子位于键合原子之间，如通过γ-氨基丁酸连接。
本发明所述的“新型多功能多肽”，含义与“新型多肽”、“多功能多肽”、“nRGD”、“nRGD多肽”、“多肽nRGD”相同。
本发明所述的“含RGD肽”，含义与“含RGD的肽”、“含RGD肽链”、“含RGD的肽链”相同。
本发明所述的AAN，为天冬氨酸蛋白内切酶(legumain)的底物。本发 明所述的AAN的靶点为天冬氨酸蛋白内切酶(Legumain)。legumain广泛存在于肿瘤细胞和肿瘤微环境，在酸性(pH＝4.0-6.5)环境中激活，在中性环境中失活。
研究发现，天冬氨酸蛋白内切酶和大多数的整合素受体如αvβ3共定位形成复合物，在肿瘤细胞和肿瘤新生血管内皮细胞膜上均存在高表达。多种类型的肿瘤如乳腺癌、结肠癌、肺癌、肝癌等均过度表达天冬氨酸蛋白内切酶。
进一步研究发现，含有丙氨酸-丙氨酸-天冬酰胺(AAN)序列的三肽底物能被天冬氨酸蛋白内切酶特异性地识别并进行酶切。
更重要的是，本发明所述的AAN的靶点为天冬氨酸蛋白内切酶(Legumain)。在肿瘤环境中，legumain的表达位点可以从细胞质转移到细胞表面。(Liao,D.et al.Synthetic enzyme inhibitor:a novel targeting ligand for nanotherapeutic drug delivery inhibiting tumor growth without systemic toxicity.Nanomedicine.7,665–673(2011).)Legumain不仅在肿瘤细胞中高表达，与肿瘤微环境相关的肿瘤相关巨噬细胞也高表达。(Reisfeld,R.A.The tumor microenvironment:a target for combination therapy of breast cancer.Crit.Rev.Oncog.18,115–133(2013).Lin,Y.et al.Selective ablation of tumor-associated macrophages suppresses metastasis and angiogenesis.Cancer Sci.104,1217–1225(2013).)
专利CN201310048238中指出，AAN三肽修饰多柔比星前药与原药具有相同的抗肿瘤药效。文献也表明，AAN直接修饰脂质体没有提高药物的抗肿瘤药效。(Ze Liu,Min Xiong,Junbo Gong,Yan Zhang,Nan Bai,Yunping Luo,Luyuan Li,Yuquan Wei,Yanhua Liu,Xiaoyue Tan&Rong Xiang.Legumain protease-activated TAT-liposome cargo for targeting tumours and their microenvironment.Nat Commun.2014；5 4280.)由此可见，作为靶点，仅AAN三肽修饰抗癌药物或者载体不能成功地提高抗癌药物或载体的抗肿瘤效果。
本发明所述的新型多肽也能够靶向肿瘤相关巨噬细胞(TAM)。TAM是存在于肿瘤部位血管周围，肿瘤无血管的坏死区域的重要的炎症细胞。TAM 是肿瘤部位关键组成部分,其分泌大量的肿瘤生长促进因子。在小鼠模型中也发现，其调节血管生成，淋巴管生成，免疫抑制和肿瘤转移。(Seth B.Coffelt,Russell Hughes,Claire E.Lewis.Tumor-associated macrophages:Effectors of angiogenesis and tumor progression.Biochimica et Biophysica Acta.1796(2009)11–18)和其他的肿瘤部位表达的骨髓相关细胞一起，TAM已经是一个肿瘤新型生物治疗的非常有吸引力的靶点。
目前，通过TAM治疗肿瘤主要有以下三个方面：抑制单核细胞在肿瘤部位的蓄积，消除在肿瘤部位的巨噬细胞和中和TAM分泌的关键细胞因子。这些均为单一的通过TAM治疗肿瘤的方式。(Seth B.Coffelt,Russell Hughes,Claire E.Lewis.Tumor-associated macrophages:Effectors of angiogenesis and tumor progression.Biochimica et Biophysica Acta 1796(2009)11–18)。
本发明所述的新型多肽在靶向肿瘤血管和细胞以杀死肿瘤血管和细胞的同时，还靶向肿瘤相关巨噬细胞，从而通过改变肿瘤微环境，显著增强治疗作用。
本发明的新型多功能多肽不仅能够结合其他治疗方式来抗肿瘤，也可以单独作为抗肿瘤的生物治疗方式。
除了肽链，含AAN的序列与含RGD肽也通过与肽链类似，但非经天然肽链连接的分子。氨基酸或氨基酸类似物的连接包括但不限于-CH2NH-，-CH2S-,-CH2-CH2-,-CH＝CH-,-COCH2-和-CH(OH)CH2-。AAN不管是否从RGD肽链断裂，均不影响其肿瘤靶向和肿瘤血管靶向的功能。
本发明的目的之一，提供一种药物组合物，所述nRGD多肽与药物活性成分混合，或nRGD多肽与递药载体混合。
本发明的目的之一，提供一种药物组合物，所述nRGD多肽与药物活性成分共价连接或者非共价缔合，或nRGD多肽与递药载体共价连接或者非共价缔合。
本发明所述的nRGD多肽，其作用方式为与组合物混合，没有共价连接或者非共价缔合；与组合物之间共价连接或者非共价缔合；所述新型多肽先于或者后于组合物给药；连接于组合物；使用上述方式的一种或组合。
作为本发明的具体实施方案之一，本发明所述的nRGD多肽能够与一个或多个辅助分子合用。
优选地，所述合用方式为缔合；至少一个所述辅助分子不与nRGD多肽重叠；至少一个所述辅助分子与nRGD多肽重叠。
优选地，所述辅助分子包括独立的归巢分子、靶向分子、亲和配体、细胞穿入肽，体内逃逸分子、亚细胞靶向分子、核靶向分子或它们的结合物和混合物。
其中，“归巢分子”是指体内优先于正常组织选择性地归巢于肿瘤或其他特定组织；“结合物”是指分子间以化学键相连形成的物质。
适用于本发明的药物包括但不限于具有肿瘤治疗作用的：治疗性蛋白质、治疗性化合物、治疗性组合物、癌症化疗剂、毒素、细胞毒性剂、抗炎剂、生长因子，细胞因子、趋化因子、调节一个或多个信号通路的化合物、抗体、核酸、核酸类似物、细胞、病毒、噬菌体、病毒颗粒、噬菌体颗粒、病毒衣壳、噬菌体衣壳、病毒样颗粒、脂质体、微球、纳米粒、胶束，乳剂，微乳，树突状大分子，微粒、化疗剂、抗血管生成剂或它们的结合物和混合物。
适用于本发明的肿瘤包括但不限于良性或恶性肿瘤，包括上皮组织良性或恶性肿瘤；间叶组织良性或恶性肿瘤；淋巴造血组织良性或恶性肿瘤；神经组织良性或恶性肿瘤；性腺或胚胎相关的良性或恶性肿瘤；其他肿瘤包括色素痣，葡萄胎，黑色素瘤，绒毛膜上皮癌，精原细胞癌，无性细胞瘤，胚胎性癌。
在具体实施例中，本发明所述的nRGD肽中所含的RGD肽链包含但不限于：RGD肽、环状c(RGDfK)、iRGD或它们的衍生物，优选为iRGD。
本发明所述的nRGD肽可通过半胱氨酸残基
Figure PCTCN2016100989-appb-000001
与药物活性成分或递药载体进行共价连接，优选的nRGD肽具有式1结构：
Figure PCTCN2016100989-appb-000002
在与药物活性成分或递药载体非共价缔合或者混合使用时，本发明所述的nRGD肽可不包含上述半胱氨酸残基。
作为优选的实施方案之一，所述的新型靶向多肽中AAN包括其衍生物，如对天冬氨酸蛋白内切酶敏感的多肽底物R-AAN-序列，其中R基团指代氢原子(H)、乙酰基(Ac)，丙氨酸(A)，苯丙氨酸(F)，甘氨酸(G)或它们的结合物。R-AAN-序列中，R优选为H，所述的nRGD的序列为CCRGDK(NAA)GPDC，其中第2个半胱氨酸与第10个半胱氨酸连接成环；或者R优选为H，所述的nRGD的序列为CRGDK(NAA)GPDC，其中两个半胱氨酸连接成环。
作为优选的实施方案之一，使用的药物优选为化疗药物，其中化疗药物优选为阿霉素(Dox)。
作为优选的实施方案之一，本发明使用的药物剂型优选为PEG化脂质体。
作为优选的实施方案之一，本发明所述的使用的方式为共价连接和直接混合。
作为优选的实施方案之一，本发明所述的使用的肿瘤模型为乳腺癌异位瘤。
本发明的目的之一，提供了本发明所述的多肽nRGD或其药物组合物在制备抗肿瘤的药物中的应用。
本发明的目的之一，提供了本发明所述的多肽nRGD或其药物组合物在制备药学上可接受的药物制剂中的应用。
本发明的多功能多肽的加入，显著增加了阿霉素原药和阿霉素脂质体的抗肿瘤效果。而且nRGD与iRGD抗肿瘤效果也显著增加。同时，多功能多肽的加入也显著增加了小鼠的生存期，并且发现nRGD组的小鼠大部分小鼠生存期都达到了90天，而且有部分小鼠肿瘤竟然消失了，这个效果是惊人的，也是之前抗肿瘤药物治疗中没有报道过的显著效果。尽管只是简单的混合，PEG化脂质体和nRGD混合用药组的部分小鼠生存期也延长了到了90天。而iRGD组小鼠只能活到2个月。多功能多肽的加入，由于靶向性的增强，显著降低了药物的毒性。
本发明通过创造性的研究，将含丙氨酸-丙氨酸-天冬酰胺(AAN)序列共价连接于含RGD肽，得到一种靶向肿瘤细胞和血管和肿瘤相关巨噬细胞从而调节肿瘤微环境增强抗肿瘤效果的串联型多肽(nRGD)。采用模型药物阿霉素及脂质体，氧化石蒜碱及纳米脂质载体，紫杉醇及白蛋白纳米粒和多西他赛及聚合物胶束为模型药物，通过混合或共价连接的方式，治疗小鼠乳腺癌异位瘤，均取得了显著的抗肿瘤效果。
因此，本发明所述的多肽显著提高了抗肿瘤组分的效果，同时也改变了抗肿瘤组分的单一治疗方式。具有良好的应用前景。
有益效果
(1)本发明的多功能多肽nRGD,显著提高了抗肿瘤组分的抗肿瘤效果。
(2)本发明的多功能多肽nRGD,提高抗肿瘤组分的抗肿瘤效果为靶向肿瘤相关巨噬细胞，调节肿瘤微环境，产生生物治疗的作用。
(3)本发明的多功能多肽nRGD,改变了抗肿瘤组分的单一治疗方式。
(4)本发明的多功能多肽，在增加靶向性的同时，降低了抗肿瘤组分的毒性。
(5)本发明的多功能多肽，使用方法简单，有望作为将来肿瘤药物研发中关键组分。
附图说明
如下，结合附图来详细说明本发明的实施方案，其中：
图1阿霉素脂质体的示意图与电镜图。图1-1表示阿霉素脂质体示意图；图1-2表示阿霉素脂质体电镜图。
图2表示在4T1肿瘤模型中，nRGD组可以显著提高阿霉素的药效。图2-1表示肿瘤生长曲线。图2-2表示肿瘤图片。图2-3表示肿瘤重量。图2-4表示抑瘤率。图2-5表示体重变化。图2-6表示生长曲线。*P<0.05,***P<0.01。
图3表示肿瘤HE染色，Ki-67和HER2免疫组化染色评价。图3-1表示HE染色,黄色箭头指示肿瘤生长活跃区域；图3-2表示Ki-67免疫组化染色；图3-3表示HER2免疫组化染色。图3-4表示Ki-67和HER2光密度值测量结果。
图4表示nRGD组可以识别肿瘤血管，增加肿瘤渗透以及靶向杀死肿瘤相关巨噬细胞。图4-1、图4-2、图4-3、表示nRGD-Lipo-Dox可以靶向CoCl2处理过的4T1以及M2型巨噬细胞。*P<0.05,***P<0.01。图4-4表示肿瘤切片表明nRGD组可以增加肿瘤部位的蓄积，也可以使肿瘤相关血管正常化。标尺＝200μm.绿色:Dox.红色:CD34。图4-5表示肿瘤切片表明nRGD组可以使肿瘤部位浸润的TAMs减少。标尺＝200μm.红色:CD206.蓝色:DAPI-细胞核。
图5表示治疗后肿瘤部位的细胞因子水平。图5-1表示ELISA测定TGF-β1。*P<0.05,***P<0.01。图5-2表示TGF-β1,图5-3表示CCl2,图5-4 表示IL-10,图5-5表示IL-6,图5-6表示TNF-α(n＝3)。
图6表示nRGD组可以靶向TAMs从而调节肿瘤微环境。图6-1表示VEGF免疫组化染色。放大倍数100X。血管生长抑制通过CD 34(图6-2)和CD 105(图6-3)(红色)免疫荧光染色评价。免疫微环境的改变通过CD8+T细胞(红)(图6-4),CD4+(绿)/Foxp3+(红)regulatory T细胞(黄)(图6-5)和CD11b+(绿)/Gr-1+(红)MDSCs(黄)(图6-6)评价。核通过DAPI染色为蓝色。标尺＝200μm。
图7表示nRGD组相对毒性低。图7-1表示不同器官组织切片(200X除了骨头100X)；图7-2表示各组脾肿大情况；图7-3表示血清中细胞因子IL-6测定；图7-4表示血清中细胞因子IL-12测定。*P<0.05,***P<0.01。
图8表示在4T1肿瘤模型中，nRGD组可以显著提高紫杉醇的药效。肿瘤生长曲线(图8-1)，肿瘤图片(图8-2),肿瘤重量(图8-3),抑瘤率(图8-4),体重变化(图8-5)。*P<0.05,***P<0.01。
图9表示在4T1肿瘤模型中，nRGD组可以显著提高氧化石蒜碱的药效。肿瘤生长曲线(图9-1)，肿瘤图片(图9-2),肿瘤重量(图9-3),抑瘤率(图9-4),体重变化(图9-5)。*P<0.05,***P<0.01。
图10表示在4T1肿瘤模型中，nRGD组可以显著提高多西他赛的药效。肿瘤生长曲线(图10-1)，肿瘤图片(图10-2),肿瘤重量(图10-3),抑瘤率(图10-4),体重变化(图10-5)。*P<0.05,***P<0.01。
图11表示在脑胶质瘤模型中，nRGD组较iRGD组具有更好的抗肿瘤效果。
具体实施方式
实施例1
nRGD肽的合成
通过固相合成法合成，序列为CCRGDK(NAA)GPDC，其中第2个半胱氨酸与第10个半胱氨酸连接成环，通过吉尔生化(上海)有限公司外包合成。所制备的nRGD肽纯度85％。
在与药物活性成分或递药载体非共价缔合或者混合使用时，该nRGD肽可不包含作为连接基团的第1个半胱氨酸，对应的序列为CRGDK(NAA)GPDC，其中两个半胱氨酸连接成环，纯度92％。
实施例2
阿霉素脂质体的制备和表征
阿霉素脂质体通过薄膜分散法和硫酸铵梯度法制备。56份磷脂，34份胆固醇，8份PEG2000-DSPE(购自德国Lipoid公司)和2份Mal-PEG2000-DSPE(PEG化脂质体为不加Mal-PEG2000-DSPE，即处方为56份磷脂，34份胆固醇，10份PEG2000-DSPE)(购自德国Lipoid公司)溶解于5mL的氯仿。旋蒸除去有机溶剂，123mMol硫酸铵溶液水化。探头超声后，用G75洗脱，与Dox孵育8h。去除未包载阿霉素后，得到未修饰的含有Mal末端的脂质体以及PEG化脂质体(PEG-Lipo-Dox)。通过与相应多肽(肽：MAL-PEG2000-DSPE摩尔比＝5:1)孵育4h，再通过Sepharose CL-4B柱去除未反应的多肽得到iRGD脂质体(iRGD-Lipo-Dox)和nRGD脂质体(nRGD-Lipo-Dox)。通过马尔文粒径仪测量粒径和电位，透射电镜进行形态表征，并通过超滤法测定包封率。
结果:如表1，所有的脂质体均为150nm左右，带负电。iRGD脂质体(iRGD-Lipo-Dox)和nRGD脂质体(nRGD-Lipo-Dox)与PEG化脂质体(PEG-Lipo-Dox)相比电位增加。包封率均大于90％。如图1，所得脂质体呈类圆形，且粒径均一。
表1：脂质体的性质(n＝3)
Figure PCTCN2016100989-appb-000003
实施例3
阿霉素和其脂质体药效和毒性评价
雌性Balb/c小鼠接种5X 105个4T1细胞，随机分成7组。生理盐水组(N.S),Dox,PEG化脂质体(PEG-Lipo-Dox),iRGD脂质体(iRGD-Lipo-Dox),nRGD脂质体(nRGD-Lipo-Dox),Dox与nRGD混合给药组(Dox+nRGD),PEG化脂质体与nRGD混合给药组(PEG-Lipo-Dox+nRGD)。在第8,12天注射5mg/kg Dox当量的药物或各种制剂。混合给药组的单次给药为两针，一针为5mg/kg Dox的当量的药物或制剂，同时注射另一针为4.8mg/kg的nRGD。每过2天量一次体积和体重。在20天的时候处死部分小鼠作为机制和毒性研究。肿瘤称量后计算平均抑瘤率:TGI＝(1-(治疗组的肿瘤平均重量/(对照组肿瘤平均重量))X 100％。通过HE，免疫组化，RT-PCR，免疫荧光和ELISA对抗肿瘤效果和机制进行研究。血浆样品收集起来进行ELISA检测。组织样品通过HE染色进行毒性评价。剩下的小鼠进行生存期研究。
结果：
1、nRGD组不管是直接和Dox及脂质体混合还是修饰在脂质体表面均提高抗肿瘤的效果。
如图2，4T1肿瘤在不治疗的情况下20天迅速增长至2161.7±422.6mg，体积为1200mm3。治疗组肿瘤均有所降低。与iRGD-Lipo-Dox组相比，PEG-Lipo-Dox+nRGD和nRGD-Lipo-Dox均表现出显著的抗肿瘤效果。而与不加肽组相比，加肽组的抗肿瘤效果也有所提高。肿瘤生长抑制率也与肿瘤生长曲线一致。nRGD-Lipo-Dox肿瘤重量为49.8±28.6mg，抑制率高达97.7％。Dox和PEG-Lipo-Dox的肿瘤重量为1592.0±98.0mg和942.0±295.0mg，抑制率仅为26.3％和56.4％。而相比较而言，Dox+nRGD和PEG-Lipo-Dox+nRGD组的肿瘤仅为462.0±43.2mg和195.0±116.1mg，抑制率为77.5％和90.1％。与抗肿瘤实验一致，nRGD组可以延长肿瘤小鼠的生存期。对于nRGD-Lipo-Dox,44.4％的小鼠在90天后仍然存活，而且小鼠的肿瘤完全治愈了。nRGD混合组的小鼠与对照组相比生存期也延长了。iRGD-Lipo-Dox组仅活到了65天。由以上实验可以看出，nRGD组抗肿瘤效果是非常卓越的。
基于上述结果，为了进一步证实nRGD的抗肿瘤效果，发明人对肿瘤进行了HE和免疫组化研究,如图3。HE染色表明，nRGD组显著抑制肿瘤的生长，nRGD-Lipo-Dox组的肿瘤基本上都坏死了。Ki-67免疫组化是评价繁殖活跃的细胞，HER2是评价肿瘤中致癌转化因子和肿瘤生长。两种方法也证实，nRGD组效果更佳。所有的结果与抗肿瘤的效果一致。
结论：nRGD组不管是直接和阿霉素及其脂质体混合使用，还是修饰在脂质体表面，均能显著提高药效。值得注意的是nRGD的加入，由于效果非常显著，降低了给药的次数，达到卓越的治疗效果。
2、nRGD组靶向肿瘤血管和细胞，同时靶向TAMs，如图4和表2。
发明人通过体外和体内实验研究了nRGD的靶向性。细胞摄取实验(图4-1、图4-2、图4-3)说明nRGD-Lipo-Dox选择性靶向表达激活legumain的CoCl2刺激的4T1细胞和IL-4刺激后的M2型巨噬细胞。同时对于表达legumain低的HUVEC和未处理的4T1细胞，nRGD与iRGD相比显著降低摄取。如表2，各组的IC50值与细胞摄取结果一致，nRGD-Lipo-Dox在CoCl2刺激的4T1细胞和IL-4刺激后的M2型巨噬细胞IC50更低,说明其对legumain高表达的细胞杀伤力更强。
本发明人也通过体内评价了nRGD组的靶向性。如图4-4,nRGD共给药或者修饰均增加Dox在肿瘤部位的蓄积，说明其提高药物在肿瘤部位的渗透性。切片中发明人看到由于Dox及脂质体对肿瘤细胞的杀伤作用，在渗透多的肿瘤出现了空洞，而且只剩下肿瘤间质的结缔组织。发明人对抗血管生成作用进行了评价，发现随着时间点的增加，肿瘤在nRGD组和iRGD–Lipo-Dox组的血管在逐渐减少。与生理盐水组，Dox以及PEG–Lipo-Dox相比，nRGD组和iRGD–Lipo-Dox组肿瘤相关血管急剧减少。然而，抗血管生成以及药物对肿瘤直接杀伤常常会导致肿瘤部位肿瘤相关巨噬细胞聚集。之前的研究表明nRGD组和iRGD–Lipo-Dox组会导致肿瘤部位血管和肿瘤细胞快速的摧毁。如图4-5，发现TAMs果然在iRGD–Lipo-Dox组肿瘤部位随时间逐渐增多。nRGD组正如发明人描述的一 样，具有M2型巨噬细胞杀伤作用，并没有导致TAMs的增加。
这些结果均证实nRGD具有靶向肿瘤血管和肿瘤细胞，同时具有靶向TAMs的作用。
表2：Dox及脂质体对4T1,HUVEC和Raw 264.7细胞24h的半数致死率(IC50)。(n＝3)
Figure PCTCN2016100989-appb-000004
3.nRGD组靶向TAMs从而调节肿瘤的微环境，如图5和图6。
正如发明内容中所述，nRGD靶向TAMs对于肿瘤微环境的调控是意义重大的，发明人对肿瘤微环境的变化进行了研究。
首先发明人对肿瘤部位的细胞因子水平进行了研究。据报道，TAMs是高表达TGFβ1,CCl2和IL-10的细胞。如图5，nRGD组三者的表达量均降低了。相反，nRGD组的IL-6和TNF-α表达量增加了，两者的增加有助于抑制效应T细胞的作用从而抑制肿瘤部位的免疫反应。结果表明nRGD组肿瘤部位细胞因子的改变有助于提高其治疗肿瘤的作用。
由于靶向TAMs有助于使肿瘤血管正常化从而提高肿瘤的治疗效果。发明人对血管内皮生长因子(VEGF),CD 34标记的肿瘤血管以及CD 105标记的肿瘤新生血管进行了研究。如图6-1,6-2和6-3，nRGD组具有相对较低的VEGF的表达，同时肿瘤部位的血管和新生血管减少。而iRGD–Lipo-Dox组，虽然血管数量减少，但是看到VEGF和新生血管表达增加。说明nRGD靶向TAMs治疗后仍然保持肿瘤血管正常化从而抑制肿瘤血管生成和肿瘤 复发。
TAMs也与肿瘤部位的免疫逃逸和抑制有关。发明人对肿瘤细胞的免疫细胞的数量进行了研究。如图6-4，CD8+T细胞变化没有明显的规律。如图6-5和6-6，调节性T细胞和MDSCs在Dox,PEG-Lipo-Dox和iRGD–Lipo-Dox组均出现了肿瘤部位数量增加，而nRGD组则没有变化。这些结果表明，nRGD组靶向TAMs,从而减低了调节性T细胞和MDSCs的数量,压制了这些细胞对肿瘤生长和肿瘤免疫逃逸的作用。
4.nRGD组表现出更低的毒性，如图7。
对于化疗药物来说，生物安全性是需要注意的不可忽略的一部分。发明人评价了给药后的毒性。发明人发现nRGD的加入并没有降低小鼠的体重。对脏器切片发现，nRGD组降低了对心脏，肾和肝的毒性，可能与其高靶向性和体内分布的改变有关。nRGD组降低系统毒性，数据显示，nRGD组具有更低的脾重量。细胞因子IL-6和IL-12测定也发现，nRGD组血清中IL-6和IL-12含量降低。
综上所述，nRGD组通过靶向肿瘤血管和肿瘤细胞且靶向TAMs调节肿瘤微环境，达到了卓越的抗肿瘤效果，并且表现出更低的毒性。
实施例4
紫杉醇(PTX)及其白蛋白纳米粒的药效评价
雌性Balb/c小鼠接种5X 105个4T1细胞，随机分成5组。生理盐水组(N.S),PTX,紫杉醇白蛋白纳米粒(PTX-BSA),PTX与nRGD混合给药组(PTX+nRGD)和紫杉醇白蛋白纳米粒与nRGD混合给药组(PTX-BSA+nRGD)。在第8,12,16天注射10mg/kg PTX当量的药物或制剂。混合给药组单次给药为两针，一针为10mg/kg PTX当量的药物或制剂，同时注射另一针为4.8mg/kg的nRGD。每过2天量一次体积和体重。在20天的时候处死部分小鼠。肿瘤称量后计算平均抑瘤率:TGI＝(1-(治疗组的肿瘤平均重量/(对照组肿瘤平均重量))X 100％。
结果：如图8，nRGD组提高PTX和PTX-BSA抗肿瘤的效果,并且体重 没有降低，没有增加毒性。
实施例5
氧化石蒜碱(IBT)及其脂质纳米粒的药效评价
雌性Balb/c小鼠接种5X 105个4T1细胞，随机分成5组。生理盐水组(N.S),IBT,氧化石蒜碱脂质纳米粒(IBT-NLC),氧化石蒜碱与nRGD混合给药组(IBT+nRGD)和氧化石蒜碱脂质纳米粒与nRGD混合给药组(IBT-NLC+nRGD)。在第8,9,10天注射12mg/kg IBT当量的药物或制剂。混合给药组单次给药为两针，一针为12mg/kg IBT当量的药物或制剂，同时注射另一针为4.8mg/kg的nRGD。每过2天量一次体积和体重。在20天的时候处死部分小鼠。肿瘤称量后计算平均抑瘤率:TGI＝(1-(治疗组的肿瘤平均重量/(对照组肿瘤平均重量))X 100％。
结果：如图9，nRGD组提高PTX和PTX-BSA抗肿瘤的效果，并且体重没有降低，没有增加毒性。
实施例6
多西他赛(TXT)及其胶束的药效评价
雌性Balb/c小鼠接种5X 105个4T1细胞，随机分成5组。生理盐水组(N.S),TXT,多西他赛胶束(TXT-micells),多西他赛与nRGD混合给药组(TXT+nRGD)和多西他赛胶束与nRGD混合给药组(TXT-micells+nRGD)。在第8,10,12天注射15mg/kg TXT当量的药物或制剂。混合给药组单次给药为两针，一针为15mg/kg TXT当量的药物或制剂，同时注射另一针为4.8mg/kg的nRGD。每过2天量一次体积和体重。在20天的时候处死部分小鼠。肿瘤称量后计算平均抑瘤率:TGI＝(1-(治疗组的肿瘤平均重量/(对照组肿瘤平均重量))X 100％。
结果：如图10，nRGD组提高TXT和TXT-micells抗肿瘤的效果，并且体重没有降低，没有增加毒性。
实施例7
在脑胶质瘤模型中，nRGD组较iRGD组具有更好的抗肿瘤效果。
脑胶质瘤小鼠，随机分成9组，每组20只。生理盐水组(N.S),替尼泊苷(Teniposide),游离氧化石蒜碱和奥曲肽(free IBT&OCT),氧化石蒜碱和奥曲肽PEG化脂质体(PEG-Liposome),脂质体与iRGD混合给药组(PEG-Liposome+iRGD),脂质体与nRGD混合给药组(PEG-Liposome+nRGD),iRGD修饰的氧化石蒜碱和奥曲肽PEG化脂质体(iRGD-Liposome),nRGD修饰的氧化石蒜碱和奥曲肽PEG化脂质体(nRGD-Liposome)和空白脂质体和nRGD混合给药组(blank)。在第5,7,9,11,12天注射10mg/kg IBT和200μg/kg OCT当量的药物或制剂。在第5,7,9,11,12天注射10mg/kg Teniposide为阳性对照。混合给药组单次给药为两针，一针为10mg/kg IBT和200μg/kg OCT当量的药物或制剂，同时注射另一针为5mg/kg的iRGD或nRGD。每天记录小鼠生存情况，绘生存曲线。
结果：如图11，在脑胶质瘤模型中，不管是混合给药，还是修饰在脂质体表面，nRGD组小鼠较iRGD组具有更长的生存周期。因而，不管是混合给药，还是修饰在脂质体表面，nRGD组较iRGD组具有更好的抗肿瘤效果。
实施例8
发明人以阿霉素为模型药物对nRGD使用方式进行了筛选。
雌性Balb/c小鼠接种5X 105个4T1细胞，随机分成3组。分别将阿霉素与nRGD混合给药，nRGD先于阿霉素给药或后于阿霉素给药。
结果：不同的给药方式对抗肿瘤效果没有明显差异。
实施例9
发明人以阿霉素为模型药物对nRGD使用浓度进行了筛选。
雌性Balb/c小鼠接种5X 105个4T1细胞，随机分成5组。给阿霉素剂量均为5mg/kg,而给nRGD浓度分别为1,2,4,8,10mg/kg。
结果：nRGD浓度跟Dox抗肿瘤呈正相关。
实施例10
通过固相合成法合成(吉尔生化(上海)有限公司外包合成)。将靶向肿瘤新生血管内皮细胞整合素受体的多肽序列RGD及c(RGDfK)分别与AAN通过肽键相连。序列为CRGDNAA和c(RGDfK)AAN。评价这两个肽的效果如下所述：
雌性Balb/c小鼠接种5X 105个4T1细胞，随机分成3组。给阿霉素剂量均为5mg/kg,其余两组在阿霉素的基础上分别给上述多肽。
结果：CRGDNAA及c(RGDfK)AAN均可提高阿霉素的药效。
实施例11
通过固相合成法合成(吉尔生化(上海)有限公司外包合成)。将靶向肿瘤新生血管内皮细胞整合素受体的多肽序列RGD及c(RGDfK)分别与AAN通过-NHCH2CH2CH2CO-相连。序列为CRGD-4Abu-NAA和c(RGDfK)-4Abu-AAN。评价这两个肽的效果如下所述：
雌性Balb/c小鼠接种5X 105个4T1细胞，随机分成3组。给阿霉素剂量均为5mg/kg,其余两组在阿霉素的基础上分别给上述多肽。
结果：CRGD-4Abu-NAA和c(RGDfK)-4Abu-AAN均可提高阿霉素的药效。
综上所述，本发明的nRGD显著增加抗肿瘤药物的效果，并具有更低的毒性，而且具有广泛适用性，这是之前文献和资料都均未报道过的。发明人通过合理的推测，本发明用于多种抗肿瘤组合物，也和抗肿瘤辅助分子混用，提高其对恶性或良性肿瘤的治疗作用。

Claims (12)

  1. 一种多肽nRGD，其特征在于将含丙氨酸-丙氨酸-天冬酰胺(AAN)的序列连接于含RGD肽。
  2. 根据权利要求1所述的多肽nRGD，其特征在于nRGD肽中所述的含RGD肽选自：RGD肽、环状c(RGDfK)、iRGD或其衍生物。
  3. 根据权利要求1所述的多肽nRGD，其特征在于含丙氨酸-丙氨酸-天冬酰胺(AAN)的序列包括其衍生物，如对天冬氨酸蛋白内切酶敏感的多肽底物R-AAN-序列，其中R基团指代氢原子(H)、乙酰基(Ac)，丙氨酸(A)，苯丙氨酸(F)，甘氨酸(G)或它们的结合物；所述R-AAN-序列中R优选为H。
  4. 根据权利要求1-3任一项所述的多肽nRGD，其特征在于，所述含AAN的序列与含RGD肽优选通过共价方式连接；更优选是通过肽键或氨基酸肽链连接；进一步优选地，所述含AAN的序列与含RGD肽通过以下方式连接：-CH2NH-，-CH2S-,-CH2-CH2-,-CH＝CH-和-CH(OH)CH2-。
  5. 根据权利要求3所述的多肽nRGD，其特征在于，序列为CCRGDK(NAA)GPDC，其中第2个半胱氨酸与第10个半胱氨酸连接成环。
  6. 根据权利要求3所述的多肽nRGD，其特征在于，序列为CRGDK(NAA)GPDC，其中两个半胱氨酸连接成环。
  7. 一种药物组合物，其特征在于包含权利要求1-6任一项所述的多肽nRGD与药物活性成分，或包含权利要求1-6任一项所述的多肽nRGD与递药载体。
  8. 根据权利要求7所述的药物组合物，其特征在于所述多肽nRGD与药物活性成分通过共价连接或者非共价缔合，或所述多肽nRGD与递药载体通过共价连接或者非共价缔合。
  9. 根据权利要求7或8所述的药物组合物，其特征在于所述多肽nRGD能够与一个或多个辅助分子合用；优选地，所述辅助分子包括独立的归巢分子、靶向分子、亲和配体、细胞穿入肽，体内逃逸分子、亚细胞靶向分子、 核靶向分子或它们的结合物和混合物。
  10. 权利要求1-6任一项所述的多肽nRGD或权利要求7-9任一项所述的药物组合物在制备药物中的应用。
  11. 根据权利要求10所述的应用，其特征在于所述药物用于抗肿瘤。
  12. 根据权利要求11所述的应用，其特征在于所述肿瘤包括良性或恶性肿瘤，包括上皮组织良性或恶性肿瘤；间叶组织良性或恶性肿瘤；淋巴造血组织良性或恶性肿瘤；神经组织良性或恶性肿瘤；性腺或胚胎相关的良性或恶性肿瘤；色素痣，葡萄胎，黑色素瘤，绒毛膜上皮癌，精原细胞癌，无性细胞瘤，胚胎性癌。
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