Cancer cell-targeting drug delivery carrier comprising camptothecin-irgd conjugate, and manufacturing method therefor

Abstract

The present invention relates to a cancer cell-targeting drug delivery carrier comprising a Camptothecin-iRGD conjugate, and a manufacturing method therefor. The conjugate exhibited excellent anticancer activity against colon cancer compared to the combined administration of CPT and iRGD and administration of CPT alone, and the conjugate has effectively increased intracellular penetrability and intracellular dispersibility and thus has increased intracellular drug delivery efficiency, thereby being able to be usefully applied in the pharmaceutical field.

Description

Cancer cell-targeting drug delivery system comprising camptothecin-IRGD conjugate and method for preparing the same

[0001] The present invention relates to a cancer cell-targeting drug delivery system comprising a camptothecin-iRGD conjugate and a method for producing the same.

[0002]

[0003] Camptothecin (CPT) is a pentacyclic quinoline alkaloid isolated from the trunk and bark of Camptotheca (Happy tree), a tree native to China. Camptothecin exhibits significant antitumor activity by inhibiting topoisomerase I, an enzyme overexpressed in various tumor cell lines and essential for DNA synthesis. Due to its broad antitumor activity and unique mechanism of action, significant efforts have been made to develop clinical analogues of camptothecin. However, most camptothecin and its derivatives are poorly soluble and inactive under physiological conditions, which has hindered the clinical development of suitable camptothecin analogues.

[0004] Meanwhile, antibody-drug conjugates (ADCs), a new type of targeted drug, generally comprise three components: an antibody or antibody-like ligand, a small molecule drug, and a linker that couples the drug to the linker ligand. ADCs utilize the antibody's specific recognition of an antigen to transport the drug molecule to the vicinity of target cells and effectively release it, achieving a therapeutic goal. In August 2011, the U.S. Food and Drug Administration (FDA) approved the listing of Adecteis™, a novel ADC drug developed by Seattle Genetics for the treatment of Hodgkin lymphoma and recurrent degenerative large cell lymphoma (ALCL), and its clinical applications have demonstrated the safety and efficacy of this type of drug.

[0005] The utility of most clinically used anticancer agents has been limited by their toxicity to all proliferating cells and / or their inability to exert activity against all tumor cells. While novel agents with unique mechanisms of action, intended to provide enhanced targeting, continue to be developed, most of these compounds still lack absolute tumor selectivity, and off-target effects continue to limit their therapeutic utility. Antibody-drug conjugates (ADCs) are designed to bind to specific epitopes on the surface of tumor cells and have provided an alternative method of targeting tumor cells in an effort to reduce associated toxicity. Despite their high selectivity, very few ADCs achieve adequate cellular uptake and limited apoptotic activity, necessitating the development of more effective tumor-targeting platforms.

[0006] Accordingly, the inventors of the present invention have completed the present invention with the aim of providing a drug delivery technology that can effectively deliver drugs to tumor cells, and developing a drug delivery system having a treatment or prevention effect on colon cancer using a camptothecin-iRGD conjugate.

[0007]

[0008] The purpose of the present invention is to provide a camptothecin-iRGD conjugate represented by the following chemical formula 1.

[0009] [Chemical Formula 1]

[0010] .

[0011] Another object of the present invention is to provide a method for producing a camptothecin-iRGD conjugate, comprising the steps of: producing a camptothecin compound by reacting a camptothecin derivative and the heterobifunctional cross-linking agent; and cross-linking the camptothecin compound with iRGD comprising the amino acid sequence of SEQ ID NO: 1.

[0012] Another object of the present invention is to provide a drug delivery system comprising the camptothecin-iRGD conjugate.

[0013] Another object of the present invention is to provide a method for producing a drug delivery system, comprising the steps of: producing a drug compound by reacting a target drug and the heterobifunctional cross-linking agent; and cross-linking the drug compound with iRGD comprising the amino acid sequence of SEQ ID NO: 1.

[0014] Another object of the present invention is to provide a pharmaceutical composition for preventing and treating cancer comprising the camptothecin-iRGD conjugate.

[0015] Another object of the present invention is to provide an anticancer adjuvant comprising the camptothecin-iRGD conjugate.

[0016]

[0017] To achieve the above purpose,

[0018] The present invention provides a camptothecin-iRGD conjugate represented by the following chemical formula 1.

[0019] [Chemical Formula 1]

[0020] .

[0021] In addition, the present invention provides a method for producing a camptothecin-iRGD conjugate, comprising the steps of: producing a camptothecin compound by reacting a camptothecin derivative and the heterobifunctional cross-linking agent; and cross-linking the camptothecin compound with iRGD comprising the amino acid sequence of SEQ ID NO: 1.

[0022] In addition, the present invention provides a drug delivery system comprising the camptothecin-iRGD conjugate.

[0023] In addition, the present invention provides a method for producing a drug delivery system, comprising: a step of producing a drug compound by reacting a target drug and the heterobifunctional cross-linking agent; and a step of cross-linking the drug compound with iRGD comprising the amino acid sequence of SEQ ID NO: 1.

[0024] In addition, the present invention provides a pharmaceutical composition for preventing and treating cancer comprising the camptothecin-iRGD conjugate.

[0025] In addition, an anticancer adjuvant comprising the camptothecin-iRGD conjugate is provided.

[0026]

[0027] The camptothecin-iRGD conjugate of the present invention exhibited superior anticancer activity against colon cancer compared to a mixture of camptothecin and iRGD and administration of camptothecin alone. The conjugate effectively increases intracellular penetration and intracellular dispersion, thereby increasing intracellular drug delivery efficiency, and thus can be usefully used as a drug delivery vehicle or anticancer agent.

[0028]

[0029] Figure 1 is a schematic diagram showing the process of targeting cancer cells with an iRGD-based camptothecin conjugate.

[0030] Figure 2 is a diagram showing a synthetic scheme of a camptothecin-iRGD conjugate.

[0031] Figure 3 is a diagram of compound 1 according to manufacturing example 1-1. 1 This diagram shows the H NMR spectrum.

[0032] Figure 4 is a diagram of compound 1 according to manufacturing example 1-1. 13 This diagram shows the C NMR spectrum.

[0033] Figure 5 is a diagram showing the ESI-MS spectrum of compound 1 according to Manufacturing Example 1-1.

[0034] Figure 6 is a diagram of compound 3 according to manufacturing example 1-3. 1 This diagram shows the H NMR spectrum.

[0035] Figure 7 is a diagram of compound 3 according to manufacturing example 1-3. 13 This diagram shows the C NMR spectrum.

[0036] Figure 8 is a diagram showing the ESI-MS spectrum of compound 3 according to Manufacturing Example 1-3.

[0037] Figure 9 is a diagram showing the HPLC spectrum of a camptothecin-iRGD conjugate according to Manufacturing Example 1-4.

[0038] Figure 10 is a diagram showing the MALDI-TOF MS spectrum of a camptothecin-iRGD conjugate according to Manufacturing Example 1-4.

[0039] Figures 11 to 13 are diagrams showing the results of evaluating the cell absorption capacity of a camptothecin-iRGD conjugate according to Manufacturing Example 1.

[0040] Figures 14 to 16 are diagrams showing the fluorescence spectrum of camptothecin alone according to Comparative Example 1.

[0041] Figures 17 and 18 are diagrams showing the results of toxicity evaluation of the camptothecin-iRGD conjugate according to Manufacturing Example 1 on HT29 and SW480 cells.

[0042] Figure 19 is a diagram showing the stability effect of a camptothecin-iRGD conjugate according to Manufacturing Example 1 in human plasma.

[0043] Figures 20 to 13 are diagrams showing the in vivo antitumor effect of the camptothecin-iRGD conjugate according to Manufacturing Example 1 on the SW480 xenograft mouse model.

[0044] Figures 24 and 25 are diagrams showing immunohistochemical staining for Ki67 of a camptothecin-iRGD conjugate according to Manufacturing Example 1.

[0045] Figures 26 and 27 are diagrams showing the tissue distribution pattern after the camptothecin-iRGD conjugate according to Manufacturing Example 1 and the camptothecin according to Comparative Example 1 were injected into mice.

[0046] Figures 28 and 29 are diagrams showing the tissue distribution pattern through intravenous injection and subcutaneous injection of the camptothecin-iRGD conjugate according to Manufacturing Example 1.

[0047]

[0048] Conditions associated with the reaction, such as solvent, base, amount of compound used, reaction temperature, and reaction time, are not limited to the description below. The compounds of the present invention can also be conveniently prepared by any combination of various synthetic methods described herein or known in the art, and such combinations can be readily determined by those skilled in the art.

[0049]

[0050] The present invention provides a camptothecin-iRGD conjugate represented by the following chemical formula 1.

[0051] [Chemical Formula 1]

[0052] .

[0053]

[0054] The "camptothecin (CPT)" of the present invention is a topoisomerase inhibitor discovered in the bark and stem of Camptotheca (Happy tree), and exhibits excellent anticancer effects in the preclinical stage.

[0055] The "iRGD" or "iRGD peptide" of the present invention refers to a 9-amino acid cyclic peptide having the sequence (sequence: CRGDKGPDC) or a variant thereof.

[0056] The iRGD peptide can target and deplete immunosuppressive regulatory T cells in a tumor-specific manner. Tumor-infiltrating regulatory T cells (Tregs) are abundant in immunotherapy-refractory tumors such as pancreatic ductal adenocarcinoma (PDAC) and contribute to the immunosuppressive tumor microenvironment. Treatments that induce systemic depletion of Tregs are undesirable due to the inflammatory and autoimmune side effects associated with nonspecific eradication. Therefore, because iRGD receptors are present only in tumors, the use of peptides that specifically target these receptors can enable the expansion of effector CD8 T cells within tumors and prevent the autoimmune toxicity caused by systemic regulatory T cell depletion.

[0057] The 9-amino acid cyclic peptide, iRGD, binds to αv integrin and promotes tumor-specific cell and tissue penetration of the linked drug / protein. iRGD therapy sensitizes PDAC tumors to both chemotherapy and immune checkpoint blockade, significantly reducing tumor burden and prolonging survival in animal models. Notably, this technology can also be applied to other peritoneal tumors, as iRGD receptors are abundant on tumor-infiltrating regulatory T cells in many tumor types. Thus, the iRGD peptide can significantly improve patient outcomes and overall survival in cancers refractory to various immunotherapies. Furthermore, this treatment synergizes with existing cancer therapies, leading to tumor reduction and improved survival in pancreatic cancer animal models.

[0058] According to an embodiment of the present invention, the camptothecin may include (S)-(+)-camptothecin.

[0059] The (S)-(+)-camptothecin of the present invention is a fluorescent hydrophobic compound composed of five rings, including one lactone-containing ring (E-ring) with a single chiral center at carbon 20, as shown in the following chemical formula 2. The 20-(S) isomer is known to inhibit Topo-I 10 to 100 times more potently than the 20-(R) isomer. Some CPT-based studies have covalently linked CPT to macromolecules through acylation or alkylation, taking advantage of the property that the carbon 20 hydroxyl group of the E-ring does not significantly inhibit the efficacy of CPT. The E-ring modification can provide highly effective CPT derivatives with increased solubility, cytotoxicity, and lactone stability.

[0060] [Chemical Formula 2]

[0061] .

[0062] According to an embodiment of the present invention, the iRGD may comprise a sequence (SEQ ID NO: 1: Ac-CCRGDKGPDC-NH2(Cys-iRGD), which is an iRGD peptide linked by a single internal disulfide bond (C2-C10).

[0063] According to an embodiment of the present invention, the conjugate may include a heterobifunctional crosslinker.

[0064] The term "heterobifunctional" in the present invention refers to a compound comprising two different chemically reactive functional groups.

[0065] According to an embodiment of the present invention, the heterobifunctional cross-linking agent may be a linker connecting the camptothecin and the iRGD.

[0066] The term "linker" of the present invention refers to a moiety that links two groups, such as a cell binding agent and a cytotoxic compound. Typically, the linker is substantially inert under the conditions under which the two groups it links are linked. A bifunctional cross-linker may comprise two reactive groups, one at each end of the linker moiety, such that one reactive group can first react with the cytotoxic compound to provide a compound containing the linker moiety, and then the second reactive group can react with the cell binding agent. Alternatively, one end of the bifunctional cross-linker can first react with the cell binding agent to provide a cell binding agent containing the linker moiety, and then the second reactive group can react with the cytotoxic compound. The linking moiety may contain a chemical bond that allows for release of the cytotoxic moiety at a specific site. Such chemical bonds may include a disulfide bond, a thioether bond, an acid-labile bond, a photo-labile bond, a peptidase-labile bond, and an esterase-labile bond.

[0067] According to an embodiment of the present invention, the heterobifunctional crosslinking agent may include tertiary-butoxycarbonyl-N-amido-polyethylene glycol-carboxylic acid (t-Boc-N-amido-PEG3-CH2CO2H) and N-β-maleimidopropyloxysuccinimide ester (N-β-Maleimidopropyloxysuccinimide ester; BMPS).

[0068]

[0069] In addition, the present invention provides a method for producing a camptothecin-iRGD conjugate, comprising the steps of: producing a camptothecin compound by reacting a camptothecin derivative and the heterobifunctional cross-linking agent; and cross-linking the camptothecin compound with iRGD comprising the amino acid sequence of SEQ ID NO: 1.

[0070] According to an embodiment of the present invention, the camptothecin derivative may include (S)-(+)-camptothecin.

[0071]

[0072] In addition, the present invention provides a drug delivery system comprising the camptothecin-iRGD conjugate.

[0073] The drug delivery system of the present invention is a 'topical preparation', which means that the topical preparation is a fibrin clot-like structure that is intended to be locally applied to a specific area of ​​a subject, typically formed on a tissue or wound surface.

[0074] The drug delivery system according to the present invention not only targets cancer cells by binding the heterobifunctional cross-linking agent to camptothecin having antitumor properties and then covalently bonding an iRGD peptide having tumor-targeting properties, but also activates the tumor transport system to control the tumor tissue penetration of the compound, so that it can be usefully used as a drug delivery system.

[0075]

[0076] In addition, the present invention provides a method for producing a drug delivery system, comprising: a step of producing a drug compound by reacting a target drug and the heterobifunctional cross-linking agent; and a step of cross-linking the drug compound with iRGD comprising the amino acid sequence of SEQ ID NO: 1.

[0077] According to an embodiment of the present invention, the drug is camptothecin, insulin, antirheumatic agent, prednisolone 21-acetate, paclitaxel, doxorubicin, retinoic acid, cisplatin, Fluorouracil (5-FU), docetaxel, tamoxifen, anasterozole, carboplatin, topotecan, belotecan, irinotecan, gleevec, vincristine, aspirin, salicylates, ibuprofen, naproxen, fenoprofen, Indomethacin, phenylbutazone, methotrexate, cyclophosphamide, mechlorethamine, dexamethasone, prednisolone, celecoxib, valdecoxib, nimesulide, cortisone, corticosteroid, glabridin, Boswellin CG, Alma extract, Alpha lipoate, Sabi white, Symwhite 377, Rucinol, curcuminoids, safflower extract, silkworm cocoon extract, retinoids, vitamin A, vitamin C, Betulinic acid, TGF (Transforming growth factor), coenzyme Q10,Ascorbic acid, Ononin, protein derived from animal placenta, centigrass extract, lotus root extract, chlorella extract, α-lipoic acid, α-tocopherol, retinol, glutathione, genistein, quercetin, propyl gallate, epigallocatechin gallate, gallocatechin gallate, silybin, diosmetin, kaempferol, epicatechin, and galangin can be used alone or in combination of two or more.

[0078] According to an embodiment of the present invention, the drug may be camptothecin.

[0079] According to an embodiment of the present invention, the camptothecin may include (S)-(+)-camptothecin.

[0080]

[0081] In addition, the present invention provides a pharmaceutical composition for preventing and treating cancer comprising the camptothecin-iRGD conjugate.

[0082] The term "cancer" (and "cancerous") in the present invention refers to or describes a physiological condition or disorder in mammals that is typically characterized by uncontrolled cell growth. A "tumor" comprises one or more cancer cells.

[0083] The term “prevention” as used in the present invention means any act of suppressing symptoms or delaying progression of a specific disease by administering the composition of the present invention.

[0084] The term "treatment" as used in the present invention means any act of improving or beneficially altering the symptoms of a specific disease by administering the composition of the present invention.

[0085] The camptothecin conjugates of the present invention may be useful for inhibiting the proliferation of abnormal cells, tumor cells, or cancer cells, thereby inducing apoptosis of tumor or cancer cells, or for treating cancer in a patient. Accordingly, provided herein is a method for treating cancer in a subject in need thereof, which method may comprise administering to the subject one or more camptothecin conjugates described herein.

[0086] Cancers that can be treated with the camptothecin conjugate include, but are not limited to, colorectal cancers such as colon cancer, rectal cancer, and appendicitis. Examples of hematopoietic cancers include follicular lymphoma, anaplastic large cell lymphoma, mantle cell lymphoma, acute myeloblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and multiple myeloma. Examples of solid tumors include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, hemangiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, stomach cancer, oral cancer, nasal cancer, pharyngeal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, choriocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, small cell lung carcinoma, bladder carcinoma, lung cancer, epithelial These may include carcinomas, gliomas, glioblastoma multiforme, astrocytomas, medulloblastomas, craniopharyngiomas, ependymomas, pinealomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, skin cancers, melanomas, neuroblastomas, and retinoblastomas.

[0087] The pharmaceutical composition of the present invention may further include an adjuvant in addition to the active ingredient. Any adjuvant known in the art may be used without limitation. However, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to enhance its effectiveness.

[0088] The pharmaceutical composition according to the present invention can be prepared in a form in which the active ingredient is mixed with a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0089] The pharmaceutical composition of the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions, each according to a conventional method.

[0090] When formulated, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and such solid preparations can be prepared by mixing the active ingredient with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0091] The pharmaceutical composition according to the present invention can be administered to a subject via various routes. All modes of administration are contemplated, including oral, intravenous, intramuscular, subcutaneous, and intraperitoneal injection.

[0092] The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, physical condition, etc. of the subject. It is obvious that the concentration of the active ingredient included in the pharmaceutical composition can be selected in various ways depending on the subject, and it is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg / ml. If the concentration is less than 0.01 μg / ml, pharmaceutical activity may not be observed, and if it exceeds 5,000 μg / ml, it may be toxic to the human body.

[0093] According to one embodiment of the present invention, the cancer is characterized in that at least one selected from the group consisting of colon cancer, colorectal cancer, CTH producing tumor, acute lymphocytic or lymphoblastic leukemia, acute or chronic lymphocytic leukemia, acute nonlymphocytic leukemia, bladder cancer, brain tumor, breast cancer, cervical cancer, chronic myelogenous leukemia, intestinal cancer, T-zone lymphoma, endometriosis, esophageal cancer, gall bladder cancer, Ewing's sarcoma, head and neck cancer, tongue cancer, Hopkins lymphoma, Kaposi's sarcoma, kidney cancer, liver cancer, lung cancer, mesothelioma, multiple myeloma, neuroblastoma, non-Hodkin's lymphoma, osteosarcoma, ovarian cancer, neuroblastoma, mammary cancer, cervical cancer, prostate cancer, pancreatic cancer, penile cancer, retinoblastoma, skin cancer, stomach cancer, thyroid cancer, uterine cancer, testicular cancer, Wilms' tumor, and trophoblastoma. A therapeutic pharmaceutical composition can be provided.

[0094]

[0095] In addition, the present invention provides an anticancer adjuvant comprising the camptothecin-iRGD conjugate.

[0096] The anticancer adjuvant of the present invention refers to a formulation that can improve, enhance, or augment the anticancer effect of an anticancer drug by being administered in combination with the anticancer drug. Depending on the treatment concentration, the anticancer adjuvant can be used as either an anticancer drug or an anticancer adjuvant, and can also enhance the sensitivity of the anticancer drug.

[0097] The above anticancer adjuvant may be administered simultaneously, separately, or sequentially with the anticancer drug. The order of administration of the anticancer adjuvant, i.e., whether the anticancer drug and the anticancer adjuvant are administered simultaneously, separately, or sequentially, at what point in time, can be determined by a physician or specialist. This order of administration may vary depending on many factors. The above anticancer adjuvant may be administered in combination with a known compound that has the effect of preventing, improving, or treating cancer. In this case, the known compound may be administered simultaneously or sequentially.

[0098]

[0099] In addition, the present invention provides a method for preventing or treating cancer, comprising administering to a subject a pharmaceutically effective amount of the camptothecin-iRGD conjugate or a composition comprising the same.

[0100] The pharmaceutical composition of the present invention is administered in a therapeutically effective amount or a pharmaceutically effective amount.

[0101] The term "pharmaceutically effective amount" as used in the present invention means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined based on factors including the type and severity of the individual, age, sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0102]

[0103] The advantages and features of the present invention, and the methods for achieving them, will become clearer with reference to the detailed examples described below. The present invention will now be described in detail through examples. However, these examples are intended to specifically illustrate the present invention and are not intended to limit the scope of the present invention.

[0104]

[0105] Manufacturing Example 1. Manufacturing of camptothecin-iRGD conjugate

[0106] Manufacturing Example 1-1. Synthesis of Compound 1

[0107] The method for preparing the camptothecin-iRGD conjugate of the present invention is shown in FIG. 2. Specifically, starting with compound 1 of FIG. 2, camptothecin-iRGD was finally synthesized. To synthesize compound 1, 60 mg (0.17 mmol) of (S)-(+)-camptothecin was dissolved in 3 mL of anhydrous DMF. To the camptothecin solution, 100 mg (0.32 mmol) of t-Boc-N-amido-TEG3-CH2CO2H, 66 mg (0.34 mmol) of EDC.HCl (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), and 10.4 mg (85 μmol) of DMAP (4-Dimethylaminopyridine) were added under nitrogen. The reaction was stopped after stirring at room temperature for 24 hours. The reaction mixture was quenched with 20 mL of water and extracted three times with EtOAc. The combined organic layers were washed sequentially with 0.1 M hydrochloric acid and distilled water. The separated organic layer was dried over anhydrous MgSO4, and the solvent was evaporated using a rotary evaporator. Compound 1, which was purified through a silica gel column in 95% CH2Cl2 and 5% MeOH using MPLC, was obtained in 95.7% yield.

[0108] [Compound 1]

[0109]

[0110] Compound 1 of the above manufacturing example 1-1 1 The H NMR spectrum is shown in Figure 3.

[0111] Compound 1 of the above manufacturing example 1-1 1 The C NMR spectrum is shown in Fig. 4.

[0112] The ESI-MS spectrum of compound 1 of the above manufacturing example 1-1 is shown in Figure 5.

[0113]

[0114] Manufacturing Example 1-2. Preparation of Compound 2 by removing the butoxycarbonyl protecting group (Boc-protected) of Compound 1.

[0115] 100 mg (0.156 mmol) of compound 1 prepared in the above Preparation Example 1-1 was dissolved in 4 mL of 1 M HCl / EtOAc and stirred at room temperature for 4 hours. The solution was reduced in pressure to evaporate the solvent, and the residue was triturated using 20 mL of diethyl ether. The triturated product was filtered, washed with 20 mL of CH2Cl2, and dried in a vacuum oven to obtain the following compound 2 in a yield of 97.2%.

[0116] [Compound 2]

[0117]

[0118]

[0119] Manufacturing Example 1-3. Synthesis of Compound 3

[0120] 100 mg (0.18 mmol) of compound 2 prepared in the above manufacturing example 1-2 was added to anhydrous CH2Cl2 After dissolving in 3 mL, 32 mg (0.31 mmol) of N-methylmorpholine (NMM) was added under nitrogen. 50 mg (0.18 mmol) of N-β-maleimidopropyloxysuccinimide ester (BMPS) was added at 0°C, and the mixture was stirred at room temperature for 24 hours under nitrogen. After completion of the reaction, the mixture was cooled to 5°C, and 1 N HCl was added dropwise to adjust the pH to 3 to 4. The aqueous layer was extracted three times with CH2Cl2. The separated organic layer was dried over anhydrous MgSO4, and the solvent was evaporated to obtain a dark brown sticky liquid. Compound 3 was purified through a silica gel column in 90% CH2Cl2 and 10% MeOH using MPLC, and the following compound 3 was obtained in 85% yield.

[0121] [Compound 3]

[0122]

[0123]

[0124] Compound 3 of the above manufacturing example 1-3 1 The H NMR spectrum is shown in Figure 6.

[0125] Compound 3 of the above manufacturing example 1-3 1 The C NMR spectrum is shown in Figure 7.

[0126] The ESI-MS spectrum of compound 3 of the above manufacturing example 1-3 is shown in Figure 8.

[0127]

[0128] Manufacturing Example 1-4. Synthesis of camptothecin-iRGD conjugate

[0129] 15 mg (21 μmol) of compound 3 prepared in the above Preparation Example 1-3 was dissolved in 3 mL of PBS buffer containing 26 mM TCEP (tris(2-carboxyethyl)phosphine) and stirred for 10 minutes. 4 mg (3 μmol) of iRGD (SEQ ID NO: 1: Ac-CCRGDKGPDC-NH2(Cys-iRGD), an iRGD peptide linked by a single internal disulfide bond (C2-C10)) was added to the solution and stirred at room temperature for 6 hours. The mixture was purified using an ultrafiltration column (Vivaspin 2 Hydrosart® (HD)) for 30 minutes, purified again with DI H2O at 2500 rpm for 30 minutes, and finally lyophilized to obtain a camptothecin-iRGD conjugate represented by the following chemical formula 1 in a yield of 84.6%.

[0130] [Chemical Formula 1]

[0131]

[0132] The HPLC spectrum of the camptothecin-iRGD conjugate of the above manufacturing example 1-4 is shown in Figure 9.

[0133] The MALDI-TOF MS spectrum of the camptothecin-iRGD conjugate of the above manufacturing example 1-4 is shown in Figure 10.

[0134]

[0135] Comparative Example 1. Preparation of camptothecin sample

[0136] In order to confirm the cancer cell targeting function of the camptothecin-iRGD conjugate of the present invention, a camptothecin-only compound was prepared, and specifically, 1 mg of camptothecin was added to 95.7 mL of cell culture medium to prepare a concentration of 30 μM.

[0137]

[0138] Comparative Example 2. Preparation of a mixture of camptothecin and iRGD

[0139] In order to confirm the cancer cell targeting function of the camptothecin-iRGD conjugate of the present invention, a mixture of camptothecin and iRGD was prepared, and specifically, 1 mg of camptothecin and 2.7 mg of iRGD were mixed and added to 95.7 mL of cell culture medium to prepare a concentration of 30 μM.

[0140]

[0141] Experimental Example 1. Preparation of Reagents and Devices

[0142] 4-Dimethylaminopyridine (DMAP) 99%, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) 98%, N-methylmorpholine (NMM), phosphate-buffered saline (PBS), tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and t-Boc-N-amido-TEG3-CH2CO2H were purchased from Sigma-Aldrich (Yongin, Korea). N-β-Maleimidopropyloxysuccinimide ester (BMPS) crosslinker and ethyl acetate were dissolved in 1 M hydrogen chloride solution (1 M HCl / EtOAc) was purchased from Thermo Fisher Scientific (Rockford, IL, USA). (S)-(+)-camptothecin was purchased from Tokyo Chemical Industry (TCI), Japan. Ac-CCRGDKGPDC-NH2 (Cys-iRGD), an iRGD peptide linked by a single internal disulfide bond (C2-C10), was synthesized by Anygen (Gwangju, Korea).

[0143] All anhydrous reactions of the present invention were performed using oven-dried glassware apparatus under an inert atmosphere of dry nitrogen. Medium-pressure liquid chromatography (MPLC) was performed using a CombiFlash RF+ Lumen instrument (MA, USA) with integrated UV and ELS detectors. 1 H and 13 C NMR spectra were recorded on a Jeol resonance spectrometer (400 MHz). Mass spectra were recorded using high-resolution electrospray ionization (HR-ESI-MS) at the Korea Basic Science Institute (Ochang, Korea). Matrix-assisted laser desorption / ionization-time-of-flight mass spectrometry (MALDI-TOF MS) was performed at BIONEER (Daejeon, Korea) to analyze high-molecular-weight compounds. A Vivaspin 2 Hydrosart® (HY) ultrafiltration column (MWCO = 2 kDa) was purchased from Sartorius Stedim Lab Ltd. (Stonehouse GL10 3UT, UK).

[0144]

[0145] Experimental Example 2. Preparation of Bioassay Samples

[0146] Dulbecco's phosphate-buffered saline (DPBS, pH 7.4), Roswell Park Memorial Institute (RPMI) 1640, fetal bovine serum (FBS), and trypsin / EDTA were purchased from Gibco, Thermo Fisher Scientific (Loughborough, UK). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and lyophilized human citrated plasma were purchased from Sigma-Aldrich, Korea. The specific organelle marker DRAQ5, a red-infrared fluorescent dye specific for nuclei and DNA quantification, was obtained from Thermo Fisher Scientific (Invitrogen, OR, USA). Primary Ki67 antibody (1:200 dilution) and secondary goat anti-rabbit antibody (1:1000 dilution) for Ki67 protein staining were purchased from Abcam (Cambridge, USA) and Invitrogen (Carlsbad, Germany), respectively. All aqueous solutions were prepared using Milli-Q purified water (18.2 MΩ).

[0147]

[0148] Experimental Example 3. SW480 and HT29 cell culture

[0149] Human colon cancer cells, SW480 and HT29, were provided by the Korea Cell Line Bank (Seoul, Korea). The cells and penicillin were added to Dulbecco's Modified Eagle Medium (DMEM) containing 10% (v / v) FBS and cultured at 37°C in a humidified 5% CO2 atmosphere.

[0150]

[0151] Experimental Example 4. Cell Absorption Analysis

[0152] SW480 cells cultured from Experimental Example 3 were seeded at 1X10 per well in a 4-well chamber cell culture slide. 5 After seeding cells individually and culturing for 24 h, the existing medium was replaced with serum-free RPMI 1640, and the cells were pretreated with 30 μM each of camptothecin-iRGD conjugates, a mixture of camptothecin and iRGD, and camptothecin alone for 1 h at 37°C. The cells were rinsed three times with serum-free medium to remove any uninternalized samples, and then 5 μM of DRAQ5 fluorophore was added and incubated for 10 min. The fluorescence intensity of the intracellular compounds was evaluated and quantified using a confocal laser scanning microscope (Carl Zeiss LSM 710) equipped with an oil immersion lens (NA 1.30, 100x).

[0153] For quantitative analysis, a calibration curve of the compound was constructed using a reverse-phase HPLC (Shimadzu Corp., Japan) equipped with a fluorescence detector. An aliquot of the compound (2% DMSO in H2O) was injected and passed through a C18 column (Shimpack GIS ODS) at a flow rate of 1.0 ml / min (80% MeOH in H2O, 0.1% TFA). A standard curve was constructed by calculating the area under the curve (AUC) of the fluorescence peak and the corresponding concentration of the compound. The SW480 cells were cultured with the compound, trypsinized, and centrifuged. The resulting cell pellet was suspended in an aqueous HNO3 (30% in H2O) / H2O2 (70% in H2O) solution (8:1 ratio), sonicated, and then prepared in the same manner as the standard curve.

[0154]

[0155] Experimental Example 5. Stability Test of Camptothecin-iRGD Conjugates

[0156] 500 μg of camptothecin-iRGD conjugate was dissolved in 2 mL H2O and added to human plasma, incubated at 37°C to obtain a homogeneous solution (70.22 μM CPT, 1 mL). Aliquots of the solution were analyzed by HPLC at various time intervals (0, 0.25, 0.5, 0.75, 1, 1.5, 2.0, 2.5, 5, 9, 12, 15, and 18 h). The mobile phase consisted of MeOH (80% in H2O) and 0.1% TFA and was applied in gradient mode at a flow rate of 1.0 mL / min. The UV absorption of the conjugate was measured at 370 nm using an analytical column (Shimpack Prep-ODS(H)KIT), and the percentage of conjugate residue at each time interval was calculated based on the initial concentration of the conjugate.

[0157]

[0158] Experimental Example 6. MTT Assay

[0159] After seeding SW480 and HT29 cells in 96-well plates, the medium was replaced with serum-free RPMI 1640 within 24 hours, and the cells were treated with camptothecin-iRGD conjugates, a mixture of camptothecin and iRGD, and camptothecin alone at concentrations of 0 to 50 μM each for 24 hours, incubated for 48 hours, and rinsed with cold DPBS. After 4 hours, the medium was replaced with DMSO, and the dissolved formazan dye was quantified using a microplate reader to measure the absorbance at 540 nm. The group that was not treated with cells in the above process was used as a control group. The cell viability (%) was calculated as the percentage of fluorescence intensity of the experimental wells compared to the fluorescence intensity of the control wells.

[0160]

[0161] Experimental Example 7. In vivo mouse study

[0162] All animal experiments were conducted at the Soonchunhyang University Laboratory Animal Center, and the research protocol was approved by the Soonchunhyang University Animal Ethics Committee in compliance with relevant laws and regulations (SCH19-0057).

[0163] Fifteen 6-week-old female BALB / c nude mice, weighing 17–21 g, were provided by Korea Orient Bio (Seongnam, Korea). To establish xenografts, 2X107 SW480 cells / 200 μL were injected subcutaneously into the flank of each mouse. Tumor sizes were 100 mm. 3 When the mice grew to , they were randomly divided into three groups (n = 5), and drug solutions (each 20 μL) were administered through the tail vein of the mice. Each mouse was injected with 0.1 mg / kg of camptothecin-iRGD conjugate, 0.019 mg / kg of camptothecin, or PBS once every two days for a total of 9 times (18 days), and then the rate of change in tumor volume was observed, and the tumor burden percentage and tumor inhibition rate (TIR) ​​were calculated using the following equation 1.

[0164] [Formula 1]

[0165] TIR(%) = 100 Х(average tumor weight of control group - average tumor weight of experimental group) / average tumor weight of control group

[0166] To determine the tissue distribution of the compounds in vivo, 22.23 mg / kg camptothecin-iRGD and 2.5 mg / kg camptothecin were prepared in sterile distilled water (4.68 mM), and 100 μL of the solution was administered three times via tail vein or subcutaneous injection to each of three groups of mice. After 24 hours, the mice were euthanized, and the heart, liver, lung, spleen, kidney, and tumor tissues were collected and weighed. The amount of camptothecin internalized in the tissues was measured using HPLC equipped with a fluorescence detector. In addition, each tissue was dissolved in 400 μL of acidic solution, sonicated at 35°C for 1 hour, and the resulting homogenate solution was diluted with distilled water, filtered, and then injected into the HPLC. The operating conditions of the HPLC were the same as those used in the calibration curve above. The AUC of each sample was obtained in triplicate, and the average value was converted to the mass of camptothecin.

[0167]

[0168] Experimental Example 8. Immunochemical Study

[0169] SW480 xenograft mouse models (BALB / c) were injected intra-tail vein with 56 μM each of camptothecin-iRGD conjugates, a mixture of camptothecin and iRGD, and PBS every other day for a total of 9 times. The mouse models were sacrificed, and the tumor tissues obtained were fixed in 10% formaldehyde dissolved in PBS at 37°C for 2 hours, embedded in paraffin, and sectioned to 4 μm thickness. The sectioned tissues were heated in 10 mM citrate buffer (pH 6.0) for 15 minutes to perform antigen retrieval before staining, and the tissues were incubated with Ki67 antibody at 4°C for 24 hours, then with goat anti-rabbit antibody at room temperature for 1 hour, and then treated with 100 mL of 3'-3-diaminobenzide (Dako). The average number of stained cells was calculated in three randomly selected fixed square areas (600 mm Х 450 mm) from each tumor tissue.

[0170]

[0171] Experimental Example 9. Statistical Analysis

[0172] The biological activities of the compounds prepared from Manufacturing Example 1, Comparative Example 1, and Comparative Example 2 were compared using Student's t-test. Statistical analysis was performed using SPSS 19.0 for Windows OS (SPSS, Chicago, IL, USA), and P < 0.05 was considered significant. The error bars in the figure represent the standard deviation of the mean.

[0173]

[0174] Example 1. Cell absorption analysis

[0175] To confirm the cellular uptake of the camptothecin-iRGD conjugate prepared from Preparation Example 1, SW480 cells were treated with 30 μM of the camptothecin-iRGD conjugate, the camptothecin / iRGD mixture, or camptothecin alone, respectively, and cultured for 1 hour. The cells were washed three times with fresh medium and then cultured again with DRAQ5 for 10 minutes, and then the internalization of the compounds was analyzed. Since camptothecin exhibits blue fluorescence under 405 nm illumination, the degree of cellular uptake of the conjugate, the mixture, and camptothecin alone was evaluated by blue fluorescence intensity using a confocal microscope (Carl Zeiss LSM 710, NA 1.30, 100X).

[0176] As a result, as shown in Figures 11 to 13, it was confirmed that the intensity of intracellular fluorescence was significantly increased in the camptothecin-iRGD conjugate of the present invention compared to the control group of camptothecin alone and the mixed use of camptothecin and iRGD, which confirms that the cancer cell penetration ability of the conjugate was significantly increased compared to the control group.

[0177] Additionally, to more quantitatively compare the cellular uptake of each sample, a linear calibration curve of pure camptothecin fluorescence according to concentration was generated by HPLC using a fluorescence detector. Since camptothecin can only fluoresce at a specific wavelength (maximum absorption wavelength 440 nm when excitation wavelength is 370 nm in H2O or MeOH), it is possible to determine the amount of camptothecin in drug-treated cells.

[0178] As a result, as shown in Figures 14 to 16, the camptothecin-iRGD conjugate exhibited an internalization amount 1.36 times higher than that of the camptothecin / iRGD mixture and 1.63 times higher than that of camptothecin alone, demonstrating the ability of iRGD as an excellent drug delivery vehicle. Therefore, it was confirmed that the camptothecin-iRGD conjugate could more efficiently penetrate the cell membrane and diffuse into the cytoplasm than the camptothecin / iRGD mixture and was much better than camptothecin alone.

[0179]

[0180] Example 2. Toxicity assay for SW480 and HT29 cells (in vitro assay)

[0181] To determine the toxicity of the camptothecin-iRGD conjugate prepared from Preparation Example 1 to HT29 and SW480 cells, SW480 and HT29 colon cancer cells were cultured with various concentrations (0-50 μL) of the camptothecin-iRGD conjugate, camptothecin / iRGD mixture, or camptothecin alone for 48 hours, and then the percentage of viable cells was determined using the MTT assay.

[0182] As a result, as shown in Figures 17 and 18, when the sample concentration was between 0 and 0.039 μM, there was little difference in the effect on cell viability among the samples, but at concentrations of 0.078 μM or higher, the cell viability of cells treated with the camptothecin-iRGD conjugate was dramatically reduced compared to cells treated with the camptothecin / iRGD mixture or camptothecin alone. Specifically, the cell viability of HT29 cells was 16.31% at 25 μM camptothecin-iRGD conjugate, 26.47% for the camptothecin / iRGD mixture, and 32.43% for camptothecin alone, and the cell viability of SW480 cells was 22.27% for 25 μM camptothecin-iRGD conjugate, 25.78% for the 25 μM camptothecin / iRGD mixture, and 29.51% for 25 μM camptothecin alone.

[0183] Additionally, as shown in Table 1 below, the IC of each sample for SW480 and HT29 cell lines at 48 hours 50 The values ​​are shown. IC of camptothecin-iRGD conjugate in SW480 cells 50 The IC value was 16-fold lower than that of the camptothecin / iRGD mixture and 24-fold lower than that of camptothecin alone, and the IC of the camptothecin-iRGD conjugate in HT29 cells 50 The values ​​were 4.4-fold lower than the camptothecin / iRGD mixture and 6.7-fold lower than camptothecin alone.

[0184] Therefore, compared to the control group of camptothecin alone and the use of a camptothecin / iRGD mixture, it was confirmed that the viability of cancer cells was significantly reduced in the camptothecin-iRGD conjugate of the present invention, which confirms that the cancer cell death induction ability of the conjugate was significantly increased compared to the control group.

[0185]

[0186] Additionally, the in vitro stability of iRGD-CPT in human plasma was investigated using HPLC. As shown in Figure 19, after 15 minutes of incubation of the camptothecin-iRGD conjugate in human plasma, only 1.4% of the initial concentration of the conjugate was hydrolyzed. After 5 hours of incubation, approximately 15.5% of the camptothecin-iRGD conjugate was hydrolyzed in plasma. The final reduction of the camptothecin-iRGD conjugate was measured after 18 hours of incubation, and the residual concentration in plasma was 76.4%.

[0187] Therefore, it can be confirmed that the camptothecin-iRGD conjugate has sufficient stability and long half-life in human plasma, so that it can reach the target site within a time period.

[0188]

[0189] Example 3. In vivo antitumor assay

[0190] To confirm the in vivo antitumor effect of the camptothecin-iRGD conjugate prepared from Manufacturing Example 1, 56 μM each of the camptothecin-iRGD conjugate, a mixture of camptothecin and iRGD, and PBS were injected through the tail vein of a SW480 xenograft mouse model (BALB / c) every other day for a total of 9 times, and the tumor volume and body weight were observed for up to 18 days.

[0191] As a result, as shown in Figures 20 to 23, compared to the use of camptothecin alone and the camptothecin / iRGD mixture as controls, the tumor volume was reduced by 39.7% and the tumor inhibition rate (TIR) ​​was increased by 75% in the camptothecin-iRGD conjugate of the present invention. In addition, as a result of comparing the actual tumor sizes of the mouse models, it was confirmed that the tumor size was reduced the most in the camptothecin-iRGD conjugate of the present invention, which confirms that the antitumor efficacy of the conjugate is significantly increased compared to the control group.

[0192]

[0193] Example 4. Immunohistochemistry (IHC) test

[0194] To confirm the immunohistochemical test of the camptothecin-iRGD conjugate prepared from Manufacturing Example 1, the camptothecin-iRGD conjugate, the mixture of camptothecin and iRGD, and PBS were dissolved in sterile distilled water at a concentration of 4.68 mM, and then injected intravenously and subcutaneously into each xenograft mouse model (BALB / c). After 24 hours, the drug-treated mice were sacrificed, and the tissues of major organs, including the heart, lungs, kidneys, liver, spleen, and tumors, were dissected, sonicated, and dissolved under acidic conditions. The solutions were aliquoted, and the amount of internalized compound was determined using a fluorescence detector and HPLC.

[0195] As a result, as shown in FIGS. 24 and 25, compared to the control group, a mixture of camptothecin and iRGD, and PBS, the average number of Ki67 protein expression and stained cells was reduced in the camptothecin-iRGD conjugate of the present invention, which confirms that the colon cancer cell death and tumor growth inhibition effects of the conjugate are significantly increased compared to the control group.

[0196]

[0197] Example 5. Analysis of tissue distribution patterns

[0198] In order to confirm the tissue distribution pattern of the camptothecin-iRGD conjugate prepared from Manufacturing Example 1, the conjugate and camptothecin alone were dissolved in sterile distilled water (4.68 mM each) and the solution (camptothecin-iRGD conjugate: 22.23 mg / kg, camptothecin alone: ​​2.5 mg / kg) was injected intravenously through the tail vein or subcutaneously into the same xenograft mouse model. After 24 hours, the drug-treated mice were sacrificed, and tissues of major organs such as the heart, lungs, kidneys, liver, spleen, and tumors were dissected, and pieces of each tissue were sonicated and dissolved under acidic conditions until a homogeneous solution was obtained. An aliquot of the solution containing the camptothecin-iRGD conjugate or camptothecin was analyzed by HPLC using a fluorescence detector to determine the amount of internalized camptothecin.

[0199] As a result, as shown in FIGS. 26 and 27, compared to the use of camptothecin alone and the camptothecin / iRGD mixture as controls, it was confirmed that the amount of the compound in the tissue significantly increased when the camptothecin-iRGD conjugate of the present invention was injected intravenously and subcutaneously. Specifically, in the case of intravenous injection, the camptothecin-iRGD conjugate was mainly found in the tumor, spleen, and heart, whereas camptothecin alone was found in the spleen, lung, and heart, and in the case of subcutaneous injection, the camptothecin-iRGD conjugate was mainly found in the tumor, lung, and heart, whereas camptothecin alone was found in the spleen, lung, and heart. Therefore, it can be confirmed that the camptothecin-iRGD conjugate has some tissue selectivity for tumors, whereas camptothecin alone has very low selectivity.

[0200] In addition, as shown in Figures 28 and 29, when the amount of intratumoral CPT was compared according to the drug administration method, the tumors deposited a much larger amount of camptothecin-iRGD conjugate than camptothecin alone in all of the above injection routes, and in particular, the amount of intratumoral camptothecin-iRGD conjugate significantly increased in the case of subcutaneous injection.

[0201] Therefore, it was confirmed that the cancer-targeting drug delivery system comprising the camptothecin-iRGD conjugate of the present invention not only increases intracellular drug delivery efficiency and cancer cell penetration ability compared to a mixture of camptothecin and iRGD and administration of camptothecin alone, but also increases colon cancer cell apoptosis induction ability and exhibits an anti-tumor effect of inhibiting tumor growth.

Claims

1. Camptothecin-iRGD conjugate represented by the following chemical formula 1: [Chemical Formula 1] .

2. A conjugate according to claim 1, wherein the camptothecin comprises (S)-(+)-camptothecin.

3. A conjugate according to claim 1, wherein the iRGD comprises sequence number 1.

4. A conjugate in the first paragraph, wherein the conjugate comprises a heterobifunctional crosslinker.

5. A conjugate in claim 4, wherein the heterobifunctional cross-linking agent is a linker connecting the camptothecin and iRGD.

6. In the fourth paragraph, the heterofunctional crosslinking agent is tertiary-butoxycarbonyl-N-amido-polyethylene glycol-carboxylic acid (t-Boc-N-amido-PEG) 3 -CH 2 CO 2 A conjugate comprising H) or N-β-maleimidopropyloxysuccinimide ester (BMPS).

7. A step of producing a camptothecin compound by reacting a camptothecin derivative and the heterobifunctional cross-linking agent; and A method for producing a camptothecin-iRGD conjugate, comprising the step of cross-linking the camptothecin compound with iRGD comprising the amino acid sequence of sequence number 1.

8. A method for producing a conjugate in claim 7, wherein the camptothecin derivative comprises (S)-(+)-camptothecin.

9. A drug delivery system comprising the camptothecin-iRGD conjugate of claim 1.

10. A step of producing a drug compound by reacting a target drug and the heterobifunctional cross-linking agent; and A method for producing a drug delivery system, comprising the step of cross-linking the drug compound with iRGD having an amino acid sequence of sequence number 1.

11. Alpha lipoate, Sabi white, Symwhite 377, Rucinol, curcuminoid, mothball extract, silkworm cocoon extract, retinoids, vitamin A, vitamin C, betulinic acid, TGF (transforming growth factor), coenzyme Q10, ascorbic acid, ononin, protein derived from animal placenta, centigrass extract, lotus root extract, chlorella extract, α-lipoic acid, α-tocopherol, retinol, glutathione, genistein, quercetin, propyl gallate, epigallocatechin A method for producing a drug delivery system, characterized in that at least one is selected from the group consisting of epigallocatechin gallate, gallocatechin gallate, silybin, diosmetin, kaempferol, epicatechin, and galangin.

12. A method for producing a drug delivery system, characterized in that the drug in claim 11 is camptothecin.

13. A method for producing a drug delivery system in claim 12, wherein the camptothecin comprises (S)-(+)-camptothecin.

14. A pharmaceutical composition for preventing and treating cancer, comprising the camptothecin-iRGD conjugate of claim 1.

15. In the 14th paragraph, the cancer is characterized in that at least one selected from the group consisting of colon cancer, CTH producing tumor, acute lymphocytic or lymphoblastic leukemia, acute or chronic lymphocytic leukemia, acute nonlymphocytic leukemia, bladder cancer, brain tumor, breast cancer, cervical cancer, chronic myelogenous leukemia, intestinal cancer, T-zone lymphoma, endometriosis, esophageal cancer, gall bladder cancer, Ewing's sarcoma, head and neck cancer, tongue cancer, Hopkins lymphoma, Kaposi's sarcoma, kidney cancer, liver cancer, lung cancer, mesothelioma, multiple myeloma, neuroblastoma, non-Hodkin's lymphoma, osteosarcoma, ovarian cancer, neuroblastoma, mammary cancer, cervical cancer, prostate cancer, pancreatic cancer, penile cancer, retinoblastoma, skin cancer, stomach cancer, thyroid cancer, uterine cancer, testicular cancer, Wilms' tumor, and trophoblastoma. and pharmaceutical compositions for therapeutic purposes.

16. An anticancer adjuvant comprising the camptothecin-iRGD conjugate of clause 1.

17. A method for preventing or treating cancer, comprising administering to a subject a pharmaceutically effective amount of the pharmaceutical composition of claim 14.

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