Cancer-targeting conjugate, and composition for preventing or treating cancer comprising same

Abstract

The present invention relates to a cancer-targeting conjugate which exhibits a higher accumulation rate in cancer cells compared to conventional anticancer agents, and is efficiently degraded within cancer cells to exert a rapid anticancer effect, thereby promptly alleviating symptoms in acute situations, minimizing long-term damage through rapid cancer treatment, and effectively managing cancer through swift evaluation of the efficacy of the corresponding drug.

Description

Cancer targeting complex, and a composition for cancer prevention or treatment comprising the same.

[0001] The present invention relates to a cancer-targeting complex, and more specifically, to a complex that targets cancer and is efficiently absorbed by cancer cells to effectively prevent or treat cancer, and a composition for preventing, treating, or improving cancer containing the same.

[0002] Cancer remains one of the leading causes of morbidity and mortality worldwide. Methods for treating cancer include chemotherapy, targeted therapies, and immunotherapy. Chemotherapy attacks not only cancer cells but also normal cells, leading to serious side effects such as loss of appetite, nausea, and weakened immune function due to bone marrow suppression.

[0003] To address the aforementioned problems, various targeted therapies have recently been developed. Targeted therapies are drugs that selectively inhibit the process of cancer cell development or growth. Since targeted therapies act by targeting specific proteins or genes that are overexpressed or mutated within cancer cells, rather than the cancer cells themselves, they have the advantage of minimizing impact on normal cells, resulting in fewer side effects, and providing efficient therapeutic effects. However, a limitation exists in that the therapeutic response to targeted therapies varies significantly depending on individual characteristics, as the genetic and molecular properties of cancer tissue differ from patient to patient. Currently, there are no suitable alternatives to evaluating treatment potential other than genetic analysis of cancer tissue or directly administering the drug to confirm the response. Consequently, despite the high cost of targeted therapies, the difficulty in predicting therapeutic response remains a clinical challenge. For example, while the anti-EGFR drug cetuximab (Erbitux®) is well known as an effective first-line treatment for cancer, only about 20% of patients actually respond to anti-EGFR therapy, and it is difficult to accurately predict drug responsiveness in advance.

[0004] To overcome the limitations of such targeted therapies, PROTAC (PROteolysis TArgeting Chimera) compounds, an innovative technology that induces specific degradation of protein targets, have recently been developed. PROTACs are attracting attention because they do not require direct access to the active site of the target protein, allowing them to effectively act on "undruggable" proteins that are difficult to target with conventional drug designs. However, PROTACs have limitations in terms of drug stability and in vivo efficacy due to their small molecule structure and high hydrophobicity. In fact, while some PROTAC compounds targeting BCL-XL (e.g., ABT263) have demonstrated potent effects, serious side effects such as thrombocytopenia have been reported, posing restrictions on their clinical application. In other words, there is currently a lack of suitable drugs for the treatment of cancer, and the development of more innovative and stable platform technologies is urgently required to address this issue.

[0005] [Prior Art Literature]

[0006] Patent Document 1. Republic of Korea Registered Patent Publication No. 10-1930399

[0007] The present invention aims to propose a drug with a novel structure utilizing a novel linker for the binding of a PROTAC substance and a maleimide functional group in the degradation of a target protein in cancer, in order to develop a new drug based on PROTAC technology.

[0008] The inventors designed a drug structure by using a linker represented by Chemical Formula 1 to combine a maleimide functional group with a PROTAC compound, so that when the existing PROTAC substance is administered in vivo, it forms a complex with albumin present in the body. As a result, they confirmed that not only the accumulation rate in cancer cells but also the degradation rate within cancer cells increases, thereby allowing for the expectation of a superior anticancer effect compared to existing anticancer drugs, and thus completed the present invention.

[0009] The objective of the present invention is to provide a cancer targeting complex having an excellent anticancer effect and its uses.

[0010] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer comprising the above-mentioned cancer-targeting complex as an active ingredient.

[0011] Another objective of the present invention is to provide a food composition for preventing or improving cancer as described above.

[0012] Another objective of the present invention is to provide a method for treating a disease, comprising the step of administering the cancer-targeting complex to a subject who requires it.

[0013] Another objective of the present invention is to provide a therapeutic use of a host cell comprising or transformed with the cancer-targeting complex.

[0014] To achieve the above objective, the present invention provides a cancer targeting complex comprising a maleimide group substituted or unsubstituted with R3, which is bonded to the side chain of PROTAC through a linker represented by the following chemical formula 1.

[0015] [Chemical Formula 1]

[0016]

[0017] In the above chemical formula 1, R1 is a single bond, or is selected from N, O, S, P, an alkylene group having 1 to 20 carbon atoms substituted or unsubstituted with at least one R2, or a heteroalkylene group having 1 to 20 carbon atoms substituted or unsubstituted with at least one R2, and

[0018] R2 is independently selected from -F, -Cl, -Br, -I, hydroxyl group, cyano group, nitro group, alkyl group having 1 to 20 carbon atoms, and carbonyl group.

[0019] R3 is any one selected from alkylamine groups having 1 to 20 carbon atoms.

[0020] In the above chemical formula 1, R1 may be any one selected from an alkylene group having 1 to 10 carbon atoms substituted or unsubstituted with at least one R2, or a heteroalkylene group having 1 to 10 carbon atoms substituted or unsubstituted with at least one R2.

[0021] In the above chemical formula 1, R1 may be any one selected from alkylene groups having 1 to 6 carbon atoms that are substituted or unsubstituted with at least one R2.

[0022] In the above chemical formula 1, R1 may be any one selected from alkylene groups having 1 to 6 carbon atoms substituted with at least one carbonyl group.

[0023] The above chemical formula 1 may be any one selected from the following chemical formulas 1-1 to 1-10.

[0024] 상기 프로탁(PROTAC)은 CDK9(Cyclin-dependent kinase 9) / CDK family, BRD4(Bromodomain-containing protein 4) / BET(Bromodomain and extra-terminal) family, SHP-2(Src homology region 2 domain-containing phosphatase-2), AR(Androgen receptor), ER(Estrogen receptor), RAR(Retinoic acid receptor), ERRα(Estrogen-related receptor alpha), BTK(Bruton's tyrosine kinase), ALK(Anaplastic lymphoma kinase), RIPK(Receptor-interacting serine / threonine-protein kinase), c-Met(MET proto-oncogene / receptor tyrosine kinase), FAK(Focal adhesion kinase), IRAK4(Interleukin-1 receptor-associated kinase 4), p38 MAPK(p38 mitogen-activated protein kinases), SGK(Serum and glucocorticoid-regulated kinase), TBK1(TANK-binding kinase 1), KRAS(Kirsten rat sarcoma virus protein), B-Raf(B-Raf proto-oncogene serine / threonine kinase), β-catenin, FKBP(FK506 binding protein), IDO(Indoleamine 2,3-dioxygenase), PD-1 / PD-L1(Programmed death protein 1 / programmed death-ligand 1), PARP1(Poly [ADP-ribose] polymerase 1),PRC2 (Polycomb repressive complex 2), EGFR (Epidermal growth factor receptor), Sirt2 (NAD-dependent deacetylase sirtuin 2), HER2 / ERBB2 (Human epidermal growth factor receptor 2), FRS2 (Fibroblast growth factor receptor substrate 2), BCL-XL (B-cell lymphoma-extra large), SMARCA (SWI / SNF related matrix associated actin dependent regulator of chromatin subfamily A), HDM2 (Human double minute 2 homolog), HDAC (Histone deacetylase) family, BCR-ABL (Breakpoint cluster region protein- Tyrosine-protein kinase ABL1), MCL1 (Modulator Of VRAC Current 1), FLT-3 (Fms Related Receptor Tyrosine Kinase 3), STAT3 (Transcription factor STAT3), Myc family and BAF One or more proteins selected from the group consisting of complex (Brg / Brahma-associated factors) It could be the target.

[0025] The above anticancer agent for cancer targeting may be represented by the following chemical formula 2.

[0026] [Chemical Formula 2]

[0027]

[0028] To achieve the above additional objective, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising a cancer-targeting complex as an active ingredient.

[0029] The above cancers include thyroid cancer, oral cancer, oropharyngeal cancer, ovarian cancer, male genital tumors, male breast cancer, brainstem tumors, cerebral lymphoma, brain tumors, pituitary meningioma, gallbladder cancer, biliary tract cancer, colorectal cancer, intracranial tumors, head and neck tumors, bladder cancer, leukemia, abdominal tumors, nasal cavity cancer, non-small cell lung cancer, nasopharyngeal cancer, esophageal cancer, breast cancer, stomach cancer, liver cancer, pancreatic cancer, ependymoma, adenoma, small cell lung cancer, small intestine cancer, kidney cancer, malignant astrocytoma, benign astrocytoma, female genital tumors, female external genital cancer, female urethral cancer, penile cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, mediastinal tumors, vaginal cancer, salivary gland cancer, skin cancer, malignant lymphoma, multiple myeloma, aplastic anemia, oligodendroglioma, hypopharyngeal cancer, anal cancer, hematological cancer, laryngeal cancer. It may be one or more selected from the group consisting of thoracic tumors and thymic cancers.

[0030] To achieve the above additional objective, the present invention provides a food composition for cancer prevention or improvement comprising a cancer-targeting complex as an active ingredient.

[0031] In addition, the present invention provides a method for preventing or treating cancer, comprising the step of administering a cancer-targeting complex to an individual.

[0032] In addition, the present invention provides a use of a cancer targeting complex for the manufacture of a drug for the prevention or treatment of cancer.

[0033] The cancer targeting complex according to the present invention exhibits the effect of inhibiting the expression of BRD4 and PD-L1 proteins in cancer cells.

[0034] The cancer targeting complex according to the present invention spontaneously binds to albumin in vivo without a separate carrier to form a stabilized inactive form. This minimizes side effects caused by the non-specific action of the drug and specifically accumulates in cancer tissue to provide enhanced therapeutic, preventive, and improvement effects. In particular, the cancer targeting complex according to the present invention acts as a unique platform that simultaneously increases cancer cell targeting and drug delivery efficiency, possessing excellent potential to overcome the limitations of existing therapeutic agents.

[0035] The cancer-targeting complex according to the present invention exhibits a superior accumulation rate in cancer cells compared to conventional anticancer drugs and is efficiently degraded within cancer cells to demonstrate a rapid anticancer effect. Consequently, it can rapidly alleviate symptoms in acute situations, facilitate rapid treatment of cancer to reduce long-term damage, enable rapid evaluation of the drug's efficacy to manage cancer, and is advantageous for integration with surgery or radiation therapy.

[0036] FIG. 1 is a schematic diagram illustrating the structure of a cancer targeting complex according to the present invention.

[0037] Figure 2a is the HPLC spectrum of the intermediate product prepared from Example 1.

[0038] FIG. 2b is the LC-MS spectrum for the intermediate product prepared from Example 1 ([M] + H + = 1086 m / z).

[0039] Figure 3a is the HPLC spectrum of Mal-PROTAC prepared from Example 1.

[0040] Figure 3b is the LC-MS spectrum of Mal-PROTAC prepared from Example 1 ([M] + H + = 604 m / z, 1208 m / z).

[0041] Figure 4a is the HPLC spectrum of ECMal-PROTAC prepared from Comparative Example 1.

[0042] Figure 4b is the LC-MS spectrum of ECMal-PROTAC prepared from Comparative Example 1 ([M] + H + = 1354 m / z).

[0043] Figure 5 is the time-dependent HPLC spectrum of a mixture of BSA and a cancer targeting complex (Example 1).

[0044] Figure 6 is the MALDI-TOF spectrum for a mixture of BSA and a cancer targeting complex (Example 1).

[0045] Figure 7a shows the DLS analysis results for albumin (BSA) and albumin-complex (Alb-Mal-PROTAC), and Figure 7b shows the DLS analysis results for albumin (BSA) and the cancer targeting complex (ECMal-PROTAC) of Comparative Example 1.

[0046] Figure 8a shows the results of analyzing the degree of degradation of the albumin complex (Alb-Mal-PROTAC) over time in the presence of esterase using RP-HPLC.

[0047] Figure 8b is the result of analyzing the newly occurring peak in Figure 8a using MASS.

[0048] Figure 9a is a graph showing the cytotoxicity of the cancer targeting complex (Mal-PROTAC) of Example 1 on CT26 cells.

[0049] Figure 9b is a graph of the results of HPLC analysis after treating 4T1 cells with the cancer targeting complex (Mal-PROTAC) of Example 1 and performing cell lysis after 24 hours.

[0050] Figure 10a is a confocal microscope image of 4T1 cancer cells treated with ARV-771 or Alb-Mal-PROTAC.

[0051] FIG. 10b is a confocal microscope image of 4T1 cancer cells treated with ARV-771 or ECMal-PROTAC prepared from Comparative Example 1. Cy5.5 represents ARV-771.

[0052] Figure 11 is a Western blot result analyzing the expression levels of BRD4 protein and PD-L1 protein over time after treating CT26 cancer cells with a PD-L1 antibody, ARV-771, or the cancer targeting complex (Mal-PROTAC) of Example 1. Figure 12 is a graph showing the quantitative analysis of the results of Figure 11.

[0053] Figure 13 is a Western blot result analyzing the expression level of BRD4 protein after treating 4T1 cancer cells with ARV-771 or the cancer targeting complex (ECMal-PROTAC) of Comparative Example 1 at different concentrations.

[0054] Figure 14 shows NIRF (non-invasive near-infrared fluorescence) images of the control group (con), comparison group (Cy5.5-ARV-771 intravenous administration), and experimental group (Cy5.5-Alb-Mal-PROTAC intravenous administration) over time, and Figure 15 shows the quantification of only the fluorescence signal of the cancer tissue from the results of Figure 14.

[0055] Figure 16 is a fluorescence image of each tissue after collecting major organs (liver, lung, spleen, kidney, heart) and cancer tissues from the control group (intravenous administration of Cy5.5-ARV-771) and the experimental group (intravenous administration of Cy5.5-Alb-Mal-PROTAC), and Figure 17 is a fluorescence intensity graph quantifying Figure 16.

[0056] Figure 18 is an enlarged fluorescent image of a cross-section of cancer tissue taken from the control group (intravenous administration of Cy5.5-ARV-771) and the experimental group (intravenous administration of Cy5.5-Alb-ECMal-PROTAC).

[0057] Figure 19 is a graph showing the change in tumor tissue size according to the treatment period of the control group, comparison group, and experimental group, and at the bottom is the tumor tissue excised on the last day of treatment (15 days after drug administration).

[0058] Figure 20 is a graph showing the change in body weight according to the treatment period of the control group, comparison group, and experimental group.

[0059] Figure 21 is a graph showing the survival rate (%) according to the treatment period of the control group, comparison group, and experimental group.

[0060] Figure 22 shows the results of the histological evaluation of cancer tissues extracted from the control group, comparison group, and experimental group.

[0061] Figure 23 shows the results of analyzing the expression levels of BRD4 and PD-L1 in cancer tissues extracted from the control group, comparison group, and experimental group, and Figure 24 is a graph showing the quantitative results of Figure 23.

[0062] Figure 25 shows the results of immunofluorescence staining on cancer tissues extracted from the control group, comparison group, and experimental group.

[0063] Figure 26 shows the results of histological evaluation of liver, lung, spleen, kidney, and heart tissues extracted from the control group, comparison group, and experimental group.

[0064]

[0065] Below, various aspects and embodiments of the present invention will be examined in more detail.

[0066]

[0067] The objects, other objects, features, and advantages of the present invention will be readily understood through the following preferred embodiments associated with the accompanying drawings. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided to ensure that the disclosed content is thorough and complete, and to ensure that the spirit of the invention is sufficiently conveyed to a person skilled in the art.

[0068] In this specification, terms such as “comprising” or “having” are intended to specify the existence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should not be understood as precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0069] In this specification, where a range is described for a variable, it will be understood that the variable includes all values ​​within the described range, including the described endpoints of the range. For example, the range “5 to 10” will be understood to include not only the values ​​5, 6, 7, 8, 9, and 10, but also any sub-ranges such as 6 to 10, 7 to 10, 6 to 9, 7 to 9, etc., and any values ​​between integers valid for the category of the described range, such as 5.5, 6.5, 7.5, 5.5 to 8.5, and 6.5 to 9. Also, for example, the range “10% to 30%” will be understood to include all integers including values ​​such as 10%, 11%, 12%, 13%, etc. and up to 30%, as well as any sub-range such as 10% to 15%, 12% to 18%, 20% to 30%, etc., and any value between valid integers within the stated range category such as 10.5%, 15.5%, 25.5%, etc.

[0070] In the chemical formulas or structural formulas within this specification, * or indicates the joint location.

[0071] All technical and scientific terms used herein have the same meaning as commonly understood by a skilled expert in the art to which this invention pertains, unless otherwise defined. In general, the nomenclature used herein is well known and commonly used in the art.

[0072]

[0073] One aspect of the present invention relates to a cancer targeting complex comprising a maleimide group substituted or unsubstituted with R3, which is bonded to the side chain of PROTAC through a linker represented by the following chemical formula 1.

[0074] [Chemical Formula 1]

[0075]

[0076] In the above formula 1, R1 is a single bond, or is selected from N, O, S, P, an alkylene group having 1 to 20 carbon atoms substituted or unsubstituted by at least one R2, or a heteroalkylene group having 1 to 20 carbon atoms substituted or unsubstituted by at least one R2, R2 is independently selected from -F, -Cl, -Br, -I, a hydroxyl group, a cyano group, a nitro group, an alkyl group having 1 to 20 carbon atoms, and a carbonyl group, and R3 is selected from an alkylamine group having 1 to 20 carbon atoms.

[0077] The linker represented by the above chemical formula 1 is a linker that connects PROTAC and maleimide, and in the above chemical formula 1, R1 may form a covalent bond with the maleimide group.

[0078] In the present invention, an alkylene group refers to a divalent group derived from an alkyl group. Specifically, the number of carbon atoms in the alkylene group is not particularly limited, but may typically be in the range of 1 to 20, and preferably may have 1 to 16 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms. Examples include methylene groups, ethylene groups, n-propylene groups, isopropylene groups, n-butylene groups, sec-butylene groups, tert-butylene groups, pentylene groups, and hexylene groups. The alkylene group may be substituted or unsubstituted with at least one R2.

[0079] The above heteroalkylene group may be a heteroalkyl group connecting two or more other groups. Specifically, it refers to an alkylene group of any suitable length having 1 to 5 heteroatoms of N, O, S, and P. Here, 'alkylene group' is as defined above.

[0080] In the present invention, R2 may be any one selected independently from -F, -Cl, -Br, -I, hydroxyl group, cyano group, nitro group, alkyl group having 1 to 20 carbon atoms, alkyl carbonyl group having 1 to 20 carbon atoms, and carbonyl group.

[0081] In the present invention, "alkyl group" refers to a monovalent straight-chain or broken-chain saturated hydrocarbon radical composed only of carbon and hydrogen atoms. Typically, it may be in the range of 1 to 20, and preferably may have 1 to 16 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms. The alkyl group includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, hexyl, ethylhexyl, etc.

[0082] In the present invention, the "alkyl carbonyl group" is a -C(=O)-alkyl radical, where 'alkyl' is defined as above. Specific examples may include, but are not limited to, methyl carbonyl (acetyl, -C(=O)CH3), ethyl carbonyl, n-propyl carbonyl, i-propyl carbonyl, n-butyl carbonyl, sec-butyl carbonyl, i-butyl carbonyl, tert-butyl carbonyl, n-octyl carbonyl, etc.

[0083] In the present invention, "nitro group" means -NO2, "cyano" means -CN, "amine group" means -NH2, and "carbonyl group" means -C(=O)H.

[0084] In the present invention, "alkylamine group" is a -NH-alkyl radical, where 'alkyl' is defined as above. Specific examples include, but are not limited to, methylamine group, dimethylamine group, ethylamine group, diethylamine group, n-propylamine group, isopropylamine group, n-butylamine group, isobutylamine group, tert-butylamine group, n-pentylamine group, isopentylamine group, neopentylamine group, n-hexylamine group, etc.

[0085] Preferably, in the above formula 1, R1 may be any one selected from an alkylene group having 1 to 10 carbon atoms substituted or unsubstituted with at least one R2, or a heteroalkylene group having 1 to 10 carbon atoms substituted or unsubstituted with at least one R2.

[0086] More preferably, in the above formula 1, R1 may be any one selected from alkylene groups having 1 to 6 carbon atoms that are substituted or unsubstituted with at least one R2.

[0087] More preferably, in the above formula 1, R1 may be any one selected from alkylene groups having 1 to 6 carbon atoms substituted with at least one carbonyl group, and the above formula 1 may be represented by any one selected from the following formulas 1-1 to 1-10.

[0088] One end of the linker represented by Chemical Formula 1 may be covalently bonded to the amine group of the maleimide group that is substituted or unsubstituted with R3, and the linker of the structure described above and the maleimide group may form a carboxamide bond.

[0089] [Chemical Formula 1-1]

[0090] *-(CH2C(=O))-*

[0091] [Chemical Formula 1-2]

[0092] *-(CH2C(=O)CH2)-*

[0093] [Chemical Formula 1-3]

[0094] *-(CH2(=O)CH2CH2)-*

[0095] [Chemical Formula 1-4]

[0096] *-(CH2CH2C(=O))-*

[0097] [Chemical Formula 1-5]

[0098] *-(CH2CH2C(=O)CH2)-*

[0099] [Chemical Formula 1-6]

[0100] *-(CH2CH2CH2C(=O))-*

[0101] [Chemical Formula 1-7]

[0102] *-(CH2CH2CH2C(=O)CH2)-*

[0103] [Chemical Formula 1-8]

[0104] *-(CH2CH2CH2CH2C(=O))-*

[0105] [Chemical Formula 1-9]

[0106] *-(CH2CH2CH2CH2C(=O)CH2)-*

[0107] [Chemical Formula 1-10]

[0108] *-(CH2CH2CH2CH2CH2C(=O))-*

[0109] The cancer targeting complex according to the present invention has a relatively small molecular weight of 1 kDa to 15 kDa, but is not rapidly eliminated through the kidneys and has the characteristic of selectively accumulating in cancer tissue. Due to this characteristic, the cancer targeting complex of the present invention reaches a maximum concentration within approximately 9 hours after administration, which enables efficient drug delivery to cancer tissue and rapid and excellent therapeutic effects.

[0110] The term "PROTAC (proteolysis-targeting chimera)" used in the present invention is a platform technology composed of an E3 ligase binding module and a linkage target protein binding module, which can induce in vivo degradation of disease-causing target proteins through ubiquitination and can inhibit most disease-causing proteins that are difficult to inhibit with active site-directed drugs derived from conventional drug development methods by degrading them.

[0111] 상기 프로탁(PROTAC)은 CDK9(Cyclin-dependent kinase 9) / CDK family, BRD4(Bromodomain-containing protein 4) / BET(Bromodomain and extra-terminal) family, SHP-2(Src homology region 2 domain-containing phosphatase-2), AR(Androgen receptor), ER(Estrogen receptor), RAR(Retinoic acid receptor), ERRα(Estrogen-related receptor alpha), BTK(Bruton's tyrosine kinase), ALK(Anaplastic lymphoma kinase), RIPK(Receptor-interacting serine / threonine-protein kinase), c-Met(MET proto-oncogene / receptor tyrosine kinase), FAK(Focal adhesion kinase), IRAK4(Interleukin-1 receptor-associated kinase 4), p38 MAPK(p38 mitogen-activated protein kinases), SGK(Serum and glucocorticoid-regulated kinase), TBK1(TANK-binding kinase 1), KRAS(Kirsten rat sarcoma virus protein), B-Raf(B-Raf proto-oncogene serine / threonine kinase), β-catenin, FKBP(FK506 binding protein), IDO(Indoleamine 2,3-dioxygenase), PD-1 / PD-L1(Programmed death protein 1 / programmed death-ligand 1), PARP1(Poly [ADP-ribose] polymerase 1),PRC2 (Polycomb repressive complex 2), EGFR (Epidermal growth factor receptor), Sirt2 (NAD-dependent deacetylase sirtuin 2), HER2 / ERBB2 (Human epidermal growth factor receptor 2), FRS2 (Fibroblast growth factor receptor substrate 2), BCL-XL (B-cell lymphoma-extra large), SMARCA (SWI / SNF related matrix associated actin dependent regulator of chromatin subfamily A), HDM2 (Human double minute 2 homolog), HDAC (Histone deacetylase) family, BCR-ABL (Breakpoint cluster region protein- Tyrosine-protein kinase ABL1), MCL1 (Modulator Of VRAC Current 1), FLT-3 (Fms Related Receptor Tyrosine Kinase 3), STAT3 (Transcription factor STAT3), Myc family and BAF One or more proteins selected from the group consisting of complex (Brg / Brahma-associated factors) It could be the target.

[0112] The E3 ligase on which the above-mentioned PROTAC acts may exhibit a proteolytic effect through an E3 ligase selected from the group consisting of CRBN (Cereblon), VHL (Von Hippel-Lindau tumor suppressor), cIAP (Cellular inhibitor of apoptosis protein), Keap1 (Kelch-like ECH-associated protein 1), HDM2 (Human double minute 2 homolog), βTRCP (Beta-transducin repeat containing E3 ubiquitin protein ligase), CHIP (Carboxyl terminus of HSC70-interacting protein), and UHRF1 (ubiquitin like with PHD and ring finger domains 1). Specifically, it may exhibit a proteolytic effect through an E3 ligase selected from the group consisting of CRBN (Cereblon) and VHL (Von Hippel-Lindau tumor suppressor).

[0113] The cancer targeting complex of the present invention forms a single platform that binds to albumin in vivo by combining a PROTAC with a maleimide group, thereby enabling effective action regardless of the structure of the PROTAC as long as it includes a linker represented by Chemical Formula 1. Specifically, the linker according to the present invention is represented by Chemical Formula 1, and the terminal end of this linker is combined with the hydroxyl group of the PROTAC to form a carbonyl group. This allows for the formation of a bond without restriction to the general structure of the PROTAC, and due to the characteristics of this ester bond, it is selectively hydrolyzed by esterase within cancer cells to release the active PROTAC. Therefore, the cancer targeting complex of the present invention is not particularly limited to PROTAC compounds; in particular, it can be sufficiently understood from the disclosed examples that any PROTAC compound containing a hydroxyl group can be combined with a maleimide group by the linker represented by Chemical Formula 1 without separate experiments.

[0114] In one embodiment of the present invention, the PROTAC may be ARV-771. ARV-771 is composed of a ligand for a BRD4 (bromodomain 4) inhibitor (JQ1) and a VHL (Von Hippel-Lindau) E3 ligase, and is one of the BRD4-degrading PROTACs that treats cancer by degrading the BET (bromodomain and extra-terminal domain) protein and the bromodomain. The BET protein is known to be overexpressed in cancer cells.

[0115] In one embodiment of the present invention, the cancer targeting complex according to the present invention may be represented by the following chemical formula 2.

[0116] [Chemical Formula 2]

[0117]

[0118] Even if the cancer targeting complex according to the present invention is injected into the body through various administration routes, it spontaneously forms a binding with in vivo albumin and moves through blood vessels, thereby allowing it to specifically accumulate and be activated in cancer tissues or cancer cells. The cancer targeting complex according to the present invention specifically binds to plasma albumin through a thiol-maleated reaction. The cancer targeting complex bound to albumin is selectively accumulated in cancer tissues through the EPR effect. The cancer targeting complex accumulated within cancer cells releases free ARV-771 (PROTAC) as its ester bond is cleaved by esterases present within the cancer cells. The released free ARV-771 simultaneously binds to both BRD4 and VHL E3 ligases, inducing polyubiquitination of BRD4. Polyubiquitinated BRD4 is accepted by the proteasome, degraded into an oligopeptide, and inhibits PD-L1 protein expression. Dissociated ARV-771 is recycled and participates in the degradation of other BRD4s and the reduction of PD-L1, thereby having an anticancer effect.

[0119] As previously described, the cancer-targeting complex according to the present invention is cleaved by esterase. Since esterase is present in excess in cancer cells, it is a target substance suitable for the structural characteristics of the present invention. Most conventional prodrug anticancer agents target caspases, but if caspases are targeted due to the structural characteristics of the present invention, a problem may arise where the release of PROTAC is not easily achieved unless the cancer cells are stimulated by anticancer agents or the like to increase the caspase concentration.

[0120] The cancer-targeting complex according to the present invention binds to endogenous albumin present in the body's blood vessels via a thiol-maleimide reaction upon injection, and thus remains in the body in an inactive state until it enters cancer cells. Therefore, even without including a separate carrier, unlike conventional anticancer agents, it has a very low potential for side effects and high targeting characteristics and accumulation rates for tumor tissue, thereby providing excellent effects for cancer treatment, prevention, and improvement.

[0121] The inventors designed a drug structure by using a linker represented by Chemical Formula 1 to bind a maleimide group to a PROTAC compound, so that when the existing PROTAC substance is administered in vivo, it forms a complex with albumin present in the body. As a result, the cancer-targeting complex according to the present invention has a superior accumulation rate in cancer cells compared to conventional anticancer drugs and is efficiently degraded within cancer cells to exhibit a rapid anticancer effect. Consequently, it has been confirmed in various ways that it can rapidly alleviate symptoms in acute situations, rapidly treat cancer to reduce long-term damage, facilitate the rapid evaluation of the drug's efficacy to manage cancer, and is advantageous for integration with surgery or radiation therapy.

[0122]

[0123] Another aspect of the present invention relates to a pharmaceutical composition for the prevention or treatment of cancer comprising a cancer-targeting complex as an active ingredient.

[0124] The above cancer targeting complex binds to albumin present in the body to form a stabilized and inactivated form, specifically accumulates in cancer cells, and is specifically degraded by esterase present in cancer cells to release PROTAC, thereby inducing apoptosis in cancer cells.

[0125] The term "cancer" as used in this invention refers collectively to diseases caused by cells having aggressive characteristics, such as dividing and growing while disregarding normal growth limits; invasive characteristics, such as infiltrating surrounding tissues; and metastatic characteristics, such as spreading to other parts of the body.

[0126] In the present invention, the cancer is not particularly limited in its type, and preferably, the applicable cancer types may vary depending on the PROTAC. For example, the cancer may be selected as a BRD4-overexpressing cancer, a CDK9-overexpressing cancer, a BET-related cancer, etc., and more preferably, the cancer may be selected as thyroid cancer, oral cancer, oropharyngeal cancer, ovarian cancer, male reproductive tumor, male breast cancer, brainstem tumor, cerebral lymphoma, brain tumor, pituitary meningioma, gallbladder cancer, biliary tract cancer, colorectal cancer, intracranial tumor, head and neck tumor, bladder cancer, leukemia, abdominal tumor, nasal cavity cancer, non-small cell lung cancer, nasopharyngeal cancer, esophageal cancer, breast cancer, gastric cancer, liver cancer, pancreatic cancer, ependymoma, adenoma, small cell lung cancer, small intestine cancer, kidney cancer, malignant astrocytoma, benign astrocytoma, female reproductive tumor, female external genital cancer, female urethral cancer, penile cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, mediastinal tumor, vaginal cancer, salivary gland cancer, skin cancer, malignant lymphoma, It may be one or more selected from multiple myeloma, aplastic anemia, oligodendroglioma, hypopharyngeal cancer, anal cancer, blood cancer, laryngeal cancer, thoracic tumor and thymic cancer.

[0127] As used in the present invention, the term "prevention" refers to any act of suppressing cancer or delaying its onset by administering a pharmaceutical composition according to the present invention.

[0128] The term "treatment" as used in this invention refers to any act in which symptoms caused by cancer are improved or beneficially altered by the administration of a pharmaceutical composition according to this invention.

[0129] The inventors confirmed through specific embodiments that the cancer targeting complex according to the present invention can enhance the in vivo retention time of PROTAC, the cancer cell influx rate, and the anticancer effect.

[0130] The above "cancer targeting complex," etc., may be within the aforementioned range.

[0131] In the present invention, the term “administration” refers to introducing the pharmaceutical composition of the present invention to a subject by any appropriate method, and the route of administration may be administered via various oral or parenteral routes as long as it can reach the target tissue. Examples of parenteral administration routes may include subcutaneous, intraperitoneal, pulmonary, nasal, intramuscular, intravenous, or intra-arterial, and preferably intravenous administration.

[0132] The pharmaceutical composition of the present invention may further comprise a suitable carrier, excipient, or diluent commonly used in the manufacture of pharmaceutical compositions. A composition comprising a pharmaceutically acceptable carrier may be in various formulations for oral or parenteral administration. When formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Formulations for oral administration may include tablets, pills, powders, granules, capsules, suspensions, liquid formulations, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized formulations, etc., and such formulations may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. with one or more compounds.

[0133] In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients. Liquid preparations for oral administration include suspensions, oral liquids, emulsions, and syrups; in addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as humectants, sweeteners, flavorings, and preservatives, may be included. Preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Propylene glycol, polyethylene glycol, and vegetable oils such as olive oil may be used as non-aqueous solvents and suspension solvents. Witepsol, Macrogol, Tween 61, cocoa dough, laurin dough, and glycerogelatin may be used as bases for suppositories.

[0134] In addition, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" in the present invention means an amount sufficient to inhibit or alleviate cancer at a reasonable benefit / risk ratio applicable to medical use, and the effective dose level may be determined based on factors including individual type and severity, age, body weight, diet, gender, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0135] The pharmaceutical composition of the present invention may be administered once a day, divided into several doses, administered every other day, or administered once a week. Specifically, the pharmaceutical composition may be administered at a dose of 0.001 to 1000 mg / kg / day, more specifically at a dose of 0.1 to 100 mg / kg / day.

[0136] Furthermore, the composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents. When administered in combination with other therapeutic agents, the composition of the present invention and the other therapeutic agents may be administered sequentially or simultaneously. The other therapeutic agents may be drugs, such as compounds or proteins, that promote cancer regression or additionally prevent cancer proliferation or growth, and also include all other anticancer therapies other than drug therapy, such as radiation therapy.

[0137]

[0138] Another aspect of the present invention relates to a food composition for the prevention or improvement of cancer comprising the cancer-targeting complex as an active ingredient.

[0139] The above "cancer targeting complex," "PROTAC," "cancer," "prevention," etc. may be within the aforementioned scope.

[0140] The food composition of the present invention has excellent effects in preventing or improving cancer.

[0141] The term "improvement" above may refer to any action that at least reduces parameters related to the condition being treated, such as the severity of symptoms. In this case, the food composition may be used for the prevention or improvement of cancer, either simultaneously with or separately from a therapeutic agent, either before or after the onset of the disease.

[0142] In the above food composition, the active ingredient may be added directly to the food or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. The amount of the active ingredient may be appropriately determined according to its purpose of use (for prevention or improvement). Generally, when manufacturing food or beverages, the above food composition may be added in an amount of about 15% by weight or less, specifically about 10% by weight or less, with respect to the raw materials. However, in the case of long-term consumption for the purpose of health and hygiene or health control, the above amount may be less than the above range.

[0143] The above food composition may further include one or more of a carrier, a diluent, an excipient, and an additive, and may be formulated into one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations. Foods to which a compound according to one aspect may be added include various types of food, powders, granules, tablets, capsules, syrups, beverages, chewing gum, tea, vitamin complexes, health functional foods, etc.

[0144] Specific examples of the above carrier, excipient, diluent, and additive may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0145] In addition to containing the active ingredient, the above food composition may contain other ingredients as essential components without special limitations. For example, it may contain various flavoring agents or natural carbohydrates as additional ingredients, such as in conventional beverages. Examples of the above-mentioned natural carbohydrates may be monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, etc.; polysaccharides, e.g., dextrin, cyclodextrin, etc., and conventional sugars, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used. The proportion of the above-mentioned natural carbohydrates may be appropriately determined by the choice of a person skilled in the art.

[0146] In addition to the above, the food composition of the present invention may contain various nutritional agents, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These ingredients may be used independently or in combination, and the proportion of these additives may also be appropriately selected by those skilled in the art.

[0147] The above food composition can be used as a health functional food for the prevention or improvement of cancer.

[0148] The above food composition may be provided by mixing it with a conventionally known health functional food for the prevention or improvement of cancer or a newly developed health functional food for the prevention or improvement of cancer.

[0149] If the above-mentioned health functional food further includes a health functional food for the prevention or improvement of cancer, it is important that an amount is mixed such that the maximum effect can be obtained with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

[0150]

[0151] Another aspect of the present invention may provide a method for treating a disease comprising the step of administering the cancer-targeting complex to a subject who requires it, and more specifically, may provide a method for preventing or treating cancer comprising the step of administering the cancer-targeting complex to an individual.

[0152] The above terms, such as "cancer targeting complex," "PROTAC," "administration," "cancer," "prevention," and "treatment," may fall within the aforementioned scope.

[0153] The term "individual" in the present invention refers to any animal, including humans, that has the potential to develop or has developed the cancer. By administering the composition of the present invention to an individual, cancer can be alleviated or treated.

[0154] The above method may involve administering the composition in conjunction with a known composition or other pharmaceutical composition having a preventive or therapeutic effect against cancer, or administering it simultaneously, separately, or sequentially, and may involve single or multiple administrations. It is important to consider all of the above factors to administer an amount that obtains maximum effect with a minimum amount without side effects, and this can be easily determined by a person skilled in the art.

[0155]

[0156] Another aspect of the present invention provides a use of a cancer-targeting complex for the manufacture of a drug for the prevention or treatment of cancer.

[0157] The above "cancer," "prevention," "treatment," "cancer targeting complex," "PROTAC," etc. may be within the aforementioned scope.

[0158]

[0159] The present invention is to be explained in more detail below through examples, etc.; however, the scope and content of the present invention shall not be interpreted as being narrowed or limited by the examples, etc. below. Furthermore, based on the disclosure of the present invention including the examples below, it is evident that a person skilled in the art can easily practice the present invention even without specific experimental results presented, and it is natural that such variations and modifications fall within the scope of the appended claims.

[0160] Furthermore, the experimental results presented below describe only the representative experimental results of the above examples and comparative examples, and the respective effects of various embodiments of the present invention not explicitly presented below will be described in detail in the relevant sections.

[0161]

[0162] Experimental materials

[0163] ARV-771 (99.02%) was obtained from Chemscene (Monmouth Junction, NJ, USA). DMF (N,N-dimethylformamide; 99.8%), EDC-HCl (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride), DMAP (4-dimethylaminopyridine; 99%), esterase obtained from porcine liver, and TEA (triethylamine; 99.5%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 5-Azidovaleric acid (98%), 4-maleimidobutyric acid (98%), and 2-mercaptoethanol (98%) were commercially obtained from Tokyo Chemical Industry (TCI; Toshima, Tokyo, Japan). BCN-PEG2-Mal (Bicyclononyne-polyethylene glycol-maleimide; 95%) was purchased from Axispharm (San Diego, CA, USA), and acetonitrile (ACN) was purchased from Honeywell (Charlotte, NC, USA). DMSO (Dimethyl sulfoxide; 99%), BSA (bovine serum albumin; 98%), and TWEEN 20 were purchased from Biosesang (Ansan, Gyeonggi, Republic of Korea). Cy5.5-NHS(Cyanine 5.5-N-hydroxysuccinimide ester) and Cy5.5-COOH(cyanine 5.5.5-carboxylic acid was purchased from Lumiprobe (Hunt Valley, MD, USA), and anti-BRD4, anti-PD-L1, and anti-β-actin were purchased from Abcam (Discovery Drive, Cambridge, United Kingdom). CCK-8 (Cell counting kit-8) was purchased from Vitascientific (Beltsville, MD, USA), and RPMI (Roswell Park Memorial Institute) 1640 medium, FBS (fetal bovine serum), penicillin, and streptomycin were purchased from WELGENE Inc (Daegu, Republic of Korea). The 4T1 (mouse breast cancer cells) cell line was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). All reagents were used exactly as received.

[0164]

[0165] Experimental equipment

[0166] Reverse-phase high-performance liquid chromatography (RP-HPLC; 1200 series, Agilent Technologies, Palo Alto, CA, USA) equipped with a C18 column (Eclipse XDB-C18, 4.6 x 150 mm, particle size = 5 μm, Agilent Technologies) was used. In all synthesis procedures, the molar mass of the products and their m / z (mass-to-charge ratios) were determined by liquid chromatography-mass spectrometry (LC-MS; Q-TOF 5600, AB SCIEX, Framingham, MA, USA). The hydrodynamic size of albumin was measured via dynamic light scattering (DLS; Zetasizer Nano ZS, Malvern Instruments, Malvern, UK). Chemical conjugation of FITC or Cy 5.5 dyes to ARV-771 or BSA was verified using a fluorescence spectrophotometer (FL; Hitachi F-7000, Hitachi High-Tech, Hitachinaka, Ibaraki, Japan). Observations were made using a confocal laser scanning microscope (CLSM; Leica TCS SP8, Leica Microsystems GmbH, Wetzlar, Germany) and a microplate reader (VERSAmax™, Molecular Devices Corp., Sunnyvale, CA, USA). To investigate the in vivo and in vitro distribution of the cancer targeting complex (Mal-PROTAC), an IVIS Lumina Series III system (PerkinElmer, Waltham, MA, USA) was used in conjunction with Living Image software (PerkinElmer).

[0167]

[0168] Preparation Examples 1 and 2. Preparation of samples labeled with fluorescent dyes

[0169] Specifically, ARV-771 (5 mg, 5.07 μmol), Cy5.5-COOH (5.8 mg, 7.60 μmol), and DMAP (3.1 mg, 25.34 μmol) were placed in a 4 mL vial, and 1 mL of anhydrous DMF was added to dissolve them. The EDC-HCl (3.9 mg, 25.34 μmol) was added to the vial. This activates the carboxylic acid group of Cy5.5-COOH. The reaction was carried out at room temperature for 12 hours to obtain a crude product. The crude product was then subjected to C using ACN / H2O (40 / 60) as the eluent. 18 Pure Cy5.5-linked ARV-771 (Cy5.5-ARV-771) was recovered by passing it through a column.

[0170] To obtain BSA labeled with Cy5.5 (Cy5.5-Alb), unlabeled BSA (66.5 mg, 1.00 μmol) and Cy5.5-NHS (0.1 mg, 0.10 μmol) were mixed and dissolved in 10 mL of anhydrous DMF. The reaction was carried out at room temperature for 12 hours to obtain the reaction product. After the reaction was completed, the reaction product was recovered, diluted with H2O, dialyzed using a dialysis membrane (MWCO = 10,000 Da), and freeze-dried to produce Cy5.5-Alb with a molar ratio of 1:10 between Cy5.5 and BSA.

[0171] The fluorescence intensity of Cy5.5-ARV-771 and Cy5.5-Alb was quantified as FL, and Cy5.5-ARV-771 was further blended with native ARV-771 to make the molal ratio of Cy5.5 to ARV-771 1:10.

[0172]

[0173] Example 1. Preparation of Cancer Targeting Complex (Mal-PROTAC)

[0174] PROTACs are drugs utilizing UPS, and various PROTAC substances targeting proteins that cause cancer resistance have been developed. In this invention, to enhance the drug delivery efficiency and pharmacological effects of PROTAC compounds, an albumin-PROTAC complex was designed by conjugating a maleimide compound to a PROTAC compound to form a bond with albumin present in vivo. During the design process, it was confirmed that the structure binding the PROTAC compound and the maleimide compound has an important correlation with the pharmacological properties of the complex. Accordingly, a cancer-targeting complex of Formula 2 was selected and synthesized based on cell permeability and pharmacological effects.

[0175] [Reaction Equation 1]

[0176]

[0177]

[0178] [Chemical Formula 2]

[0179]

[0180] ARV-771 (20.0 mg, 20.27 μmol) was placed in a 3 ml 1-neck round-bottom flask equipped with a magnetic stirrer bar, and 0.5 ml of anhydrous DMF was added to dissolve it.

[0181] 2,5-hexanedione (7.25 mg, 50.68 μmol), DMAP (12.38 mg, 101.35 μmol), DCM / THF (3:1 (v / v), 5 mL), and EDC-HCl (15.73 mg, 101.35 μmol) were added to a flask and reacted for 12 hours under an inert atmosphere (N2) at room temperature with slow mixing. The product was concentrated under low pressure and synthesized dicarbonyl-substituted PROTAC intermediates by C18 column chromatography (Sep-Pak® C18 cartridges 5 g; Waters Corporation, Milford, MA, USA) using an eluent of ACN / H2O (70 / 30, v / v). The product was freeze-dried to remove residual solvent (Fig. 2a, b) (intermediate: yield 88.7%, purity 98.9%).

[0182] The previously synthesized intermediate (20.0 mg, 17.99 μmol) was placed in a 3 ml 1-neck round-bottom flask and dissolved in 0.5 ml DMSO, then 1-(2-Aminoethyl)maleimide hydrochloride (12.8 mg, 26.98 μmol), HATU (10.2 mg, 26.98 μmol), and DIPEA (5.81 mg, 44.9 μmol) were added, and the mixture was reacted for 3 hours under an inert atmosphere (N2) at room temperature. The reaction mixture was purified using reverse-phase high-performance liquid chromatography (RP-HPLC; 1200 series, Agilent Technologies, Palo Alto, CA, USA) equipped with a C18 column (Eclipse XDB-C18, 4.6 x 150 mm, particle size = 5 μm, Agilent Technologies) and lyophilized to synthesize a cancer targeting complex (Mal-PROTAC) in powder form. The purification process was performed using a concentration gradient method in which the ACN / H2O eluent was varied from 20:80 (v / v) to 80:20 (v / v). The fractions obtained at each concentration were analyzed by RP-HPLC and LC-MS and are shown in Figures 3a and 3b.

[0183] As shown in Figures 3a, b, and c, it was confirmed that a high-purity cancer-targeting complex (Mal-PROTAC) can be obtained through the synthesis process of the present invention (Mal-PROTAC: 62.9% yield and 98.2% purity) (Figure 3). Since a single spectral peak was observed in the HPLC results, it can be seen that the cancer-targeting complex (Mal-PROTAC) was synthesized with high purity without impurities or mixtures through the synthesis method of the present invention. The LC-MS spectrum results also showed that the measured molecular weight was similar to the theoretical value ([M + H] + = 604 m / z, 1208 m / z), it can be confirmed that the cancer targeting complex (Mal-PROTAC) was synthesized in a structurally accurate and high-purity state.

[0184]

[0185] Comparative Example 1. ECMal-PROTAC Synthesis

[0186] [Reaction Equation 2]

[0187]

[0188] ARV-771 (20.0 mg, 20.27 μmol) was placed in a 3 ml 1-neck round-bottom flask equipped with a magnetic stirrer bar, and dissolved by adding 0.5 ml of anhydrous DMF. After successfully adding 5-azidovaleric acid (7.25 mg, 50.68 μmol), DMAP (12.38 mg, 101.35 μmol), and EDC-HCl (15.73 mg, 101.35 μmol) to the flask, the mixture was reacted for 12 hours at room temperature while slowly mixing. The resulting product was concentrated under low pressure conditions and purified and separated by C18 column chromatography (Sep-Pak® C18 cartridges 5 g; Waters Corporation, Milford, MA, USA) using an ACN / H2O (70 / 30, v / v) concentration gradient eluent to obtain N3-PROTAC. N3-PROTAC in powder form was recovered by freeze-drying to remove residual solvent.

[0189] The above N3-PROTAC (20.0 mg, 17.99 μmol) was placed in a 3 ml single-neck round-bottom flask and dissolved in 0.5 ml of DMSO. BCN-PEG2-Mal (12.8 mg, 26.98 μmol) was added to the solution and reacted at room temperature for 2 hours. Afterward, the solution was purified using reverse-phase high-performance liquid chromatography (RP-HPLC; 1200 series, Agilent Technologies, Palo Alto, CA, USA) equipped with a C18 column (Eclipse XDB-C18, 4.6 x 150 mm, particle size = 5 μm, Agilent Technologies) and freeze-dried to synthesize ECMal-PROTAC in powder form. The purification process was performed using a concentration gradient method in which the ACN / H2O eluent was changed from 20:80 (v / v) to 80:20 (v / v). The fractions obtained at each concentration were analyzed using RP-HPLC and LC-MS analysis and are shown in Figures 4a and 4b.

[0190] As shown in Figures 4a and 4b, it was confirmed that the ECMal-PROTAC of Comparative Example 1 was successfully synthesized with high purity (N3-PROTAC: yield 88.7%, purity 98.9%, ECmal-PROTAC: yield 62.9% and purity 98.2%).

[0191] The ECMal-PROTAC of Comparative Example 1 was also synthesized with high purity without impurities or mixtures, but it can be confirmed that there is a clear difference in molecular weight compared to the cancer targeting complex (Mal-PROTAC) of Example 1. Through this, it can be seen that there are differences in chemical structure and ionization characteristics between the complex of Example 1 and the complex of Comparative Example 1.

[0192]

[0193] Experimental Example 1. Analysis of whether the cancer-targeting complex efficiently forms a binding with albumin

[0194] In order for the cancer targeting complex according to the present invention to selectively and efficiently accumulate within cancer cells in vivo and to induce apoptosis in cancer cells, it is necessary to form an immediate binding with albumin in vivo when administered via various routes. To verify whether the cancer targeting complex having the structure designed in the present invention achieves these characteristics, experiments were conducted as follows.

[0195] An aqueous solution of a cancer-targeting complex (Example 1) at a concentration of 140 μM was prepared and mixed with a BSA solution (200 μM) diluted with PBS in a 1:1 volume ratio for co-incubation. Samples were then collected at time intervals (0 min, 5 min, 30 min, 60 min). The collected samples were quenched with mercaptoethanol, and RP-HPLC analysis was performed using an eluent concentration gradient (ACN / H2O, 20 / 80 (v / v) to 80 / 20 (v / v), 30 min) (0 min, 5 min, 30 min, 60 min in Fig. 5).

[0196] As control groups, only the cancer targeting complex (Example 1) was used (Mal-PROTAC in Fig. 5), and only BSA was used (Albumin in Fig. 5).

[0197] As a control group, 1 ml of PBS solution containing thiol-blocked albumin (BSA) at a concentration of 1% (w / v) was prepared, mixed with an aqueous solution of a cancer targeting complex (Example 1) at a concentration of 140 μM at room temperature in a 1:1 volume ratio, reacted for 30 minutes, and then RP-HPLC analysis was performed (Thiol Blocked in Fig. 5). Thiol-blocked BSA is prepared by mixing 4-maleimidobutyric acid and BSA for 1 hour.

[0198] In addition, to verify the albumin binding ability of the cancer targeting complex of Example 1 (Example 1), a sample (Alb-Mal-PROTAC) co-cultured for 30 minutes was analyzed by LC-MS spectrum and compared with the LC-MS spectrum of free BSA (Albumin) (Fig. 6).

[0199]

[0200] Figure 5 shows the time-dependent HPLC spectrum of a mixture of BSA and the cancer targeting complex (Example 1). According to this, it can be seen that the cancer targeting complex (Mal-PROTAC) prepared from Example 1 of the present invention rapidly forms a binding with albumin through a thiol-maleimide click chemical reaction. It can be observed that as the incubation time with albumin increases, the peak of the cancer targeting complex (Mal-PROTAC) of Example 1 decreases sharply, and the peak of BSA shifts to the right. In other words, the cancer targeting complex (Mal-PROTAC) of the present invention successfully forms an albumin-complex within 30 minutes of being injected into the body and coming into contact with albumin.

[0201]

[0202] Figure 6 is the LC-MS spectrum for a mixture of BSA and a cancer-targeting complex (Example 1). The LC-MS results for albumin (BSA) alone as a control are also shown.

[0203] As shown in Figure 6, the change in molecular weight of the cancer targeting complex (Mal-PROTAC) according to Example 1 and Albumin was confirmed to be 1,200 Da. This can be confirmed to be consistent with the change in molecular weight of the cancer targeting complex (Mal-PROTAC) according to Example 1. In other words, it can be seen that the cancer targeting complex (Mal-PROTAC) according to Example 1 was safely bound to albumin without causing chemical or physical changes when forming a complex.

[0204]

[0205] Experimental Example 2. Analysis of Physicochemical Properties of Cancer Targeting Complex (Mal-PROTAC)

[0206] A BSA solution (200 μM) diluted with PBS was mixed in a 1:1 volume ratio with an aqueous solution (140 μM) of the cancer targeting complex (Mal-PROTAC) of Example 1 or the cancer targeting complex (ECMal-PROTAC) of Comparative Example 1, and then co-incubated for 30 minutes, after which the albumin-complex (Alb-Mal-PROTAC or Alb-ECMal-PROTAC) was recovered. Their hydrodynamic size was measured by dynamic light scattering (DLS; Zetasizer Nano ZS, Malvern Instruments, Malvern, UK).

[0207]

[0208] Figure 7a shows the DLS analysis results for albumin (BSA) and albumin-complex (Alb-Mal-PROTAC), and Figure 7b shows the DLS analysis results for albumin (BSA) and the cancer targeting complex (ECMal-PROTAC) of Comparative Example 1.

[0209] As shown in Fig. 7, the particle size of ECMal-PROTAC of Comparative Example 1 increased by 1.25 to 1.3 times (5.43 ± 1.42 -> 6.99 ± 1.38 nm) when combined with albumin, but no significant change in size was observed in Mal-PROTAC of Example 1 of the present invention even when combined with albumin to form an albumin-complex (Alb-Mal-PROTAC), indicating that Mal-PROTAC according to the present invention does not have any physical effect on the change in size of albumin when injected into the body.

[0210]

[0211] Experimental Example 3. Degradation of the cancer targeting complex (Mal-PROTAC)

[0212] 15 units of esterase were added to the albumin-complex (Alb-Mal-PROTAC) prepared in Experimental Example 2, and the samples were recovered at reaction times of 0, 3, 6, and 12 hours and analyzed using reverse-phase high-performance liquid chromatography (RP-HPLC; 1200 series, Agilent Technologies, Palo Alto, CA, USA) equipped with a C18 column (Eclipse XDB-C18, 4.6 x 150 mm, particle size = 5 μm, Agilent Technologies). Since the actual cancer targeting complex (Mal-PROTAC) is provided in vivo in the form of an albumin-complex, the experiment was performed using the albumin-complex (Alb-Mal-PROTAC) rather than the cancer targeting complex (Mal-PROTAC).

[0213]

[0214] Figure 8a shows the results of analyzing the degree of degradation of the albumin complex (Alb-Mal-PROTAC) over time in the presence of esterase using RP-HPLC, and Figure 8b shows the results of analyzing the newly occurring peak in Figure 8a using MASS.

[0215] As shown in Fig. 8, it was confirmed that the ester bond of the albumin-complex (Alb-Mal-PROTAC) according to the present invention was successfully cleaved by esterase present in cancer cells, and that almost all of ARV-771 (PROTAC) was released within 12 hours. The albumin-complex (Alb-Mal-PROTAC) was degraded within 6 to 12 hours and released ARV-771.

[0216] In previous experiments, it was confirmed that the albumin-complex (Alb-ECMal-PROTAC) of Comparative Example 1 gradually released 63.2% of ARV-771 within 48 hours. That is, it can be seen that the albumin-complex (Alb-Mal-PROTAC) according to the present invention has a faster degradation rate compared to the cancer targeting complex of Comparative Example 1.

[0217] In Fig. 8a, a peak (10-11 min) of the albumin-complex (Alb-Mal-PROTAC) according to the present invention is present, and upon esterase treatment, a new peak (13 min) occurred. As a result of analyzing the MASS for this (Fig. 8b), it was confirmed that it is ARV-771 ([M + Na] + = 1008 m / z). That is, it can be seen that the albumin-complex (Alb-Mal-PROTAC) according to the present invention is rapidly and successfully completely degraded into ARV-771 by the esterase of tumor cells.

[0218]

[0219] Experimental Example 4. Cancer cell death effect of cancer targeting complex (Mal-PROTAC)

[0220] The cancer cell killing effect of the PROTAC compound ARV-771 and the cancer targeting complex (Mal-PROTAC) of Example 1 was analyzed using the CCK-8 (cell counting kit-8) method.

[0221] First, mouse breast cancer cells (4T1) were placed in a 96-well culture plate at a rate of 3 × 10⁶ per well. 3Cells were seeded and cultured in RPMI 1640 (Roswell Park Memorial Institute 1640) medium at 37°C under a 5% carbon dioxide atmosphere. After 24 hours, each cell was treated with ARV-771 or the cancer-targeting complex (Mal-PROTAC) of Example 1 at various concentrations (0, 0.01, 0.1, 1, 7, 10, 25, 50 μM) and cultured for 48 hours. After treating each well with culture medium containing 10% CCK-8 and incubating for 30 minutes, the absorbance of each well was analyzed using a microplate reader. A VERSAmax™, Molecular Devices Corp., Sunnyvale, CA microplate reader was used, and absorbance was measured at 450 nm. Changes in cell viability were calculated using the IC50 of GraphPad Prism 8 software (GraphPad Software, San Diego, CA, USA). 50 The (haf-maximal inhibitory concentration) value was calculated and presented.

[0222]

[0223] Figure 9a is a graph showing the cytotoxicity of the cancer targeting complex (Mal-PROTAC) of Example 1 measured against 4T1 cells. As shown in Figure 9a, it can be confirmed that the cancer targeting complex (Mal-PROTAC) of Example 1 exhibits excellent toxicity against cancer cells. Specifically, the cancer targeting complex (Mal-PROTAC) of Example 1 [shows] IC [indicating] in cancer cells 50 It was confirmed that [it] appears at very low concentrations. In other words, the cancer targeting complex according to the present invention has a notable anticancer effect against cancer cells rich in BRD4.

[0224]

[0225] Figure 9b is a graph of the results of HPLC analysis after treating 4T1 cells with the cancer targeting complex (Mal-PROTAC) of Example 1 and performing cell lysis after 24 hours.

[0226] As shown in Fig. 9b, it was confirmed that Free ARV-771 (PROTAC) was detected within cancer cells. In other words, it was confirmed that the cancer targeting complex (Mal-PROTAC) of Example 1 was rapidly and successfully introduced into the cell and normally released the drug. Thus, it can be clearly confirmed that the anticancer effect in Fig. 9a is the anticancer effect caused by the cancer targeting complex according to the present invention.

[0227]

[0228] Experimental Example 5. Analysis of the cancer cell healing effect of the cancer targeting complex (Mal-PROTAC).

[0229] The cancer targeting complex (Mal-PROTAC) of Example 1 was designed with improved chemical structure to ensure rapid absorption into cancer cells, excellent accumulation rate, and enhanced degradation rate within the cell. To verify these effects, an aqueous solution (140 μM) of the cancer targeting complex (Mal-PROTAC) of Example 1 was mixed with a BSA solution (200 μM) in a 1:1 volume ratio and reacted for 30 minutes to prepare an albumin-complex (Alb-Mal-PROTAC). ARV-771 was used as a control. ECMal-PROTAC prepared from Comparative Example 1 was used as a comparative group.

[0230] Prepare 4T1 mouse breast cancer cell lines, and 1 × 10 5Cells were seeded into a glass-bottom confocal dish (35 mm diameter) and cultured in RPMI 1640 (Roswell Park Memorial Institute 1640) medium for 24 hours. Next, the cells were treated with ARV-771 (5 μM) or Alb-Mal-PROTAC (5 μM) and cultured at 37°C for 1, 3, and 6 hours. After washing three times with PBS, the cells were treated with a 4% formaldehyde aqueous solution for 15 minutes, and then the cell nuclei were stained with DAPI (4,6-diamidino-2-phenylindole) for 10 minutes. The DAPI and DOX fluorescence signals in the 4T1 cells were measured and analyzed using CLSM. Based on the analysis results, the cellular uptake of ARV-771 and Alb-Mal-PROTAC over time was evaluated.

[0231]

[0232] Fig. 10a is a confocal microscope image of 4T1 cancer cells treated with ARV-771 or Alb-Mal-PROTAC. Fig. 10b is a confocal microscope image of 4T1 cancer cells treated with ARV-771 or ECMal-PROTAC prepared from Comparative Example 1. Cy5.5 represents ARV-771.

[0233] As shown in Figure 10, ARV-771 and Alb-Mal-PROTAC were both absorbed into cancer cells within 6 hours, and were also successfully rapidly degraded within the cancer cells, so that at 12 hours, a large amount of DOX was found near the nucleus of the cancer cells.

[0234] On the other hand, it was confirmed that the amount of ECMal-PROTAC prepared from Comparative Example 1 absorbed into cancer cells was lower than that of ARV-771 at 6 hours, and that it was fully absorbed into cancer cells only after 12 hours. In other words, it can be seen that the cancer targeting complex (Mal-PROTAC) according to the present invention is significantly faster (approximately 1.5 to 2 times) in terms of the time required for accumulation and drug release compared to ECMal-PROTAC prepared from Comparative Example 1.

[0235] In particular, since DOX was detected in the cell starting from 1 hour for the cancer targeting complex (Mal-PROTAC), it can be seen that the intracellular absorption and degradation are significantly superior compared to when ARV-771 was treated alone.

[0236] That is, the cancer targeting complex (Mal-PROTAC) according to the present invention has excellent absorption and accumulation in cancer cells, and its degradation rate is fast, so it is degraded as it is absorbed into the cell, and the presence of DOX was confirmed in the nucleus of the cancer cell from 1 hour. As a result, cancer cell death was observed more quickly with the cancer targeting complex (Mal-PROTAC) according to the present invention than with the conventional PROTAC compound (ARV-771).

[0237] Since the cancer targeting complex (ECMal-PROTAC) of Comparative Example 1 is absorbed into 4T1 cells at a somewhat slower rate than the PROTAC compound (ARV-771), it can be seen that the cancer targeting complex (Mal-PROTAC) according to the present invention is absorbed faster than conventional PROTAC complexes and induces a faster therapeutic effect on cancer cells.

[0238]

[0239] Experimental Example 6. Therapeutic effect of ECMal-PROTAC of Example 1 in vitro

[0240] BRD4 (Bromodomain-containing protein 4) is a protein that plays a crucial role in the growth, survival, and metastasis of cancer cells. Since it regulates the expression of cancer-related genes such as myc and bcl2, the silencing of BRD4 in cancer cells implies the inhibition of cancer cell proliferation and survival. PD-L1 (Programmed Death-Ligand 1) is an important immunomodulatory protein that induces immune evasion, helping cancer cells avoid immune attacks by T cells. A decrease in PD-L1 in cancer cells means that the cells are better recognized by the immune system, thereby increasing the therapeutic effect of anticancer drugs.

[0241] In this experiment, we aimed to confirm the cancer therapeutic ability by evaluating the removal of BRD4 and PD-L1 when cancer cells were treated with the cancer-targeting complex (ECMal-PROTAC) of Example 1. First, 1 × 10 54T1 cells were seeded into each well of a 6-well culture plate and cultured for 24 hours in RPIM1640 medium containing MSA (mouse serum albumin) at 37°C and under a 5% carbon dioxide atmosphere. PD-L1 antibody (1 μM), ARV-771 (1 μM), or the cancer targeting complex (Mal-PROTAC) of Example 1 (1 μM) were added to the cell culture medium, and the cells were cultured at 37°C for various time intervals (0h, 1h, 3h, 6h, 24h, 48h). After the culture was completed, the cells were washed three times with PBS and lysed with a lysis buffer containing 1% protease. Cell debris from the recovered lysate was removed by centrifugation at 12,000 rpm for 25 minutes, and the supernatant was collected and the protein content was measured using a BCA kit. The supernatant was loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel, subjected to electrophoresis (SDS-PAGE), and then blotted onto a polyvinylidene difluoride (PVDF) membrane. After blotting, to inhibit the indiscriminate binding of IgG (immunoglobulin G), the PVDF membrane was treated with 5% BSA-containing tris buffered saline containing 0.1% Tween 20 (TBS-T) for 2 hours, and then immediately exposed to rabbit anti-mouse primary antibodies (BRD4 (1:1000, anti-mouse IgG), PD-L1 (1:200, anti-mouse IgG)). After treatment at 4°C for 24 hours, the PVDF membrane was washed three times with TBS-T and then incubated with HRP-conjugated mouse anti-rabbit IgG antibodies for an additional 1 hour at room temperature. The immunoreactive bands of the PVDF membrane were analyzed using an enhanced chemiluminescence (ECL) system.

[0242]

[0243] Figure 11 is a Western blot result analyzing the expression levels of BRD4 protein and PD-L1 protein over time after treating 4T1 cancer cells with a PD-L1 antibody, ARV-771, or the cancer targeting complex (Mal-PROTAC) of Example 1. Figure 12 is a graph showing the quantitative analysis of the results of Figure 11.

[0244] As shown in Figures 11 and 12, it was confirmed that the cancer targeting complex (Mal-PROTAC) of Example 1 binds to MSA (mouse serum albumin) in cell culture medium, is rapidly and efficiently internalized into 4T1 cells, and that the overexpressed BRD4 protein is degraded to less than 50% within 6 hours and completely degraded to less than 25% after 24 hours. Furthermore, it was confirmed that the expression of PD-L1 protein was also reduced to less than 50% after 6 hours and completely reduced to less than 20% after 24 hours.

[0245]

[0246] Previous experiments confirmed that 24 hours is the maximum reaction time that significantly reduces BRD4, even with conventional PROTAC compounds. Accordingly, the BRD4 protein expression level was analyzed by Western blot in the same manner as in the previous experiments, except that the cancer targeting complex of Comparative Example 1 (ECMal-PROTAC) was treated at different concentrations (0 μM, 0.1 μM, 0.5 μM, 1 μM, 5 μM) and cultured for 24 hours instead of the cancer targeting complex of Example 1. For accurate control, ARV-771 was used as a control.

[0247] Figure 13 is a Western blot result analyzing the expression level of BRD4 protein after treating 4T1 cancer cells with ARV-771 or the cancer targeting complex (ECMal-PROTAC) of Comparative Example 1 at different concentrations.

[0248] As shown in Figure 13, it was confirmed that the cancer targeting complex (ECMal-PROTAC) of Comparative Example 1 inhibited the BRD4 protein by 55% at the same reaction time (24 hours) and concentration (1 μM) as the cancer targeting complex (Mal-PROTAC) of Example 1, and inhibited the BRD4 protein by 50% at a concentration 5 times higher (5 μM).

[0249] That is, it was confirmed that the cancer targeting complex (Mal-PROTAC) of Example 1 induces rapid and strong (2-fold) inhibition of BRD4 due to having a chemical structure that is differentiated from the cancer targeting complex (ECMal-PROTAC) of Comparative Example 1. In addition, the cancer targeting complex (Mal-PROTAC) of Example 1 also showed excellent effects on PD-L1 protein inhibition.

[0250] Synthesizing the results described above, it was confirmed that the cancer targeting complex (Mal-PROTAC) of Example 1 is absorbed more rapidly into cancer cells than conventional PROTAC compounds through structural improvement, and after absorption, the linker is rapidly cleaved by esterase present in the cancer cells to release the PROTAC compound, thereby inducing a significantly faster and stronger apoptotic effect on cancer cells.

[0251] The cancer targeting complex (Mal-PROTAC) of Example 1 is efficiently accumulated and degraded within cancer cells, thereby inhibiting not only BRD4 but also PD-L1, inducing apoptosis in cancer cells, and simultaneously reducing the immune evasion ability of cancer cells, which increases the activity of the anticancer agent. Since the cancer targeting complex (Mal-PROTAC) of Example 1 exists in the body in an inactive state by binding to albumin in vivo, it does not exhibit toxicity in normal cells, but exhibits a significant anticancer effect specifically in cancer cells. In particular, it has the advantage of exhibiting a faster anticancer effect (drug efficacy) compared to conventional PROTAC compounds and complexes containing them. Therefore, the cancer-targeting complex (Mal-PROTAC) according to the present invention simultaneously induces a reduction of BRD4 protein and PD-L1 protein, thereby reducing the immunosuppressive state in the tumor microenvironment and increasing the cancer cell death effect. Since it is rapidly degraded within cancer cells and exhibits rapid pharmacological effects, it can quickly alleviate symptoms in acute situations and allow for rapid treatment of cancer, thereby reducing long-term damage. Furthermore, it facilitates the management of cancer by rapidly evaluating the efficacy of the drug and is more advantageous for integration with surgery or radiation therapy.

[0252]

[0253] Experimental Example 7. Analysis of the accumulation rate of the cancer-targeting complex (Mal-PROTAC) in cancer cells in vivo

[0254] Cancer animal model

[0255] Five-week-old female Balb / c and Balb / c nu / nu mice were purchased from NaraBio, Inc. (Seoul, Republic of Korea). Prior to the experiment, the mice underwent a 2-week acclimatization period. During the experiment, the mice were free-fed in a room maintained at a temperature of 22 ± 2 ℃ and humidity of 40–60%, and the light-dark cycle was controlled at 12-hour intervals. All experiments were conducted in accordance with the regulations of the Institutional Animal Care and Use Committee (IACUC) of Ewha Womans University. The following 1 × 10⁶ were placed on the left thigh of each mouse. 6 Inoculated with 4T1 cells, tumor volume 150-200 mm³ 3 A female animal model was manufactured by rearing until it reached [the target].

[0256] Preparation of samples

[0257] Three animals were randomly distributed to each group of the above cancer animal model. To analyze the in vivo therapeutic effect of the cancer targeting complex (Mal-PROTAC) of Example 1, Cy5.5-BSA solution (Cy5.5-Alb, 200 μM) diluted with PBS was mixed with the Mal-PROTAC aqueous solution (140 μM) of Example 1 in a 1:1 volume ratio and co-incubated. The resulting Cy5.5-Alb-Mal-PROTAC, prepared by reacting for 30 minutes under pH 8.0 conditions, was used as the sample for the experimental group. Cy5.5-ARV-771 was used as the comparison group, and PBS solution was used as the control group.

[0258] Sample administration and analysis

[0259] The comparison group was administered a single dose of Cy5.5-ARV-771 (3 mg / kg, equivalent to ARV-771) via the tail vein. The experimental group was administered a single dose of Cy5.5-Alb-Mal-PROTAC (3 mg / kg, equivalent to ARV-771) via the tail vein. The control group was administered a single dose of saline (3 mg / kg) via the tail vein.

[0260] Whole-body fluorescence images were taken using the IVIS Lumina Series III system starting immediately after drug administration (0 hours) and at elapsed times (1 hour, 3 hours, 6 hours, 9 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours).

[0261]

[0262] Figure 14 shows NIRF (non-invasive near-infrared fluorescence) images of the control group (con), comparison group (Cy5.5-ARV-771 intravenous administration), and experimental group (Cy5.5-Alb-Mal-PROTAC intravenous administration) over time, and Figure 15 shows the quantification of only the fluorescence signal of the cancer tissue from the results of Figure 14.

[0263] As shown in FIGS. 14 and 15, the experimental group (Cy5.5-Alb-Mal-PROTAC) administered with the cancer-targeting anticancer agent (Mal-PROTAC) according to the present invention specifically accumulated in cancer tissue and reached its highest level after 9 hours. This is considered to be significantly faster than conventional anticancer agents or anticancer complexes. In addition, the experimental group accumulated in cancer cells at a significantly higher concentration than the control group (Cy5.5-ARV-771), and the fluorescence intensity was also confirmed to be 87.4 times stronger in the experimental group compared to the control group.

[0264]

[0265] Experimental Example 8. Analysis of the in vivo behavior of the cancer-targeting complex (Mal-PROTAC)

[0266] The comparison group, experimental group, and control group were all prepared in the same manner as in Experimental Example 7. In summary, the comparison group was administered a single dose of Cy5.5-ARV-771 (3 mg / kg, equivalent to ARV-771) via the tail vein. The experimental group was administered a single dose of Cy5.5-Alb-Mal-PROTAC (3 mg / kg, equivalent to ARV-771) via the tail vein. The control group was administered a single dose of saline (3 mg / kg) via the tail vein.

[0267] After 24 hours had passed since the drug was administered, the major organs (heart, liver, lungs, spleen, and kidneys) and cancerous tissues were removed. The biodistribution and histological analysis of each removed tissue were performed as follows. To analyze the distribution of samples in the removed cancerous tissues, the removed major organs and cancerous tissues were imaged using an IVIS Lumina Series III system (PerkinElmer, Waltham, MA, USA) and analyzed using Living Image software (PerkinElmer).

[0268] For the histological analysis of the excised cancer tissue, the isolated cancer tissue was immersed in a 4% formaldehyde solution, fixed at 4°C, and then 1-2 mm 3Sections were prepared by cutting into pieces of a specific size. Each section was shaken and incubated in TBST containing 3% BSA for 3 hours, after which FITC-conjugated anti-CD31 primary antibody (Biolegend, USA) was added and stained by incubating overnight at 4°C. The sections were washed three times with TBST, placed in DAPI mounting medium, placed on glass cover slides, and then photographed using a confocal laser scanning microscope (CLSM; Leica TCS SP8, Leica Microsystems GmbH, Wetzlar, Germany). The captured images were quantified using a microplate reader (VERSAmax™, Molecular Devices Corp., Sunnyvale, CA, USA).

[0269]

[0270] Figure 16 is a fluorescence image of each tissue after major organs (liver, lung, spleen, kidney, heart) and cancer tissues were collected from the control group (intravenous administration of Cy5.5-ARV-771) and the experimental group (intravenous administration of Cy5.5-Alb-Mal-PROTAC), and Figure 17 is a graph of fluorescence intensity quantified from Figure 16. Figure 18 is an enlarged fluorescence image of a cross-section of cancer tissue collected from the control group (intravenous administration of Cy5.5-ARV-771) and the experimental group (intravenous administration of Cy5.5-Alb-ECMal-PROTAC).

[0271] As shown in Figures 16 and 17, it was confirmed that the control group administered with Cy5.5-ARV-771 had a very strong fluorescence signal in kidney tissue, while having a very weak fluorescence signal in cancer tissue and other organ tissues. In other words, it is believed that simple PROTAC compounds, like the control group, do not accumulate in cancer cells in vivo but are eliminated directly through the kidneys, and thus fail to exert a sufficient anticancer effect.

[0272] In the experimental group administered with the anticancer agent for cancer targeting according to the present invention, the strongest fluorescence signal was observed in the cancer tissue, while the fluorescence signals in the liver tissue and kidney were only partial. In particular, the intensity of the fluorescence signal in the cancer tissue was 3 times higher than in the liver tissue and 1.5 times higher than in the kidney, confirming that the anticancer agent according to the present invention has a significantly superior potential for selective accumulation in the cancer tissue compared to the amount excreted through the kidney. This implies that the present invention has superior target specificity for cancer tissue, compared to conventional anticancer agents or anticancer complexes, which tend to accumulate at higher concentrations in the kidney than in the cancer tissue.

[0273] As shown in Fig. 18, in the experimental group administered with the anticancer drug for cancer targeting according to the present invention, the drug was uniformly distributed throughout the entire cancer tissue. In particular, it was confirmed that the drug was effectively delivered even to the center of the high-density cancer tissue, where it is difficult for conventional anticancer drugs to penetrate, thereby exhibiting an anticancer effect. Conventional anticancer drugs struggled to achieve complete eradication of cancer because they could not reach the center due to the high density within the cancer tissue, which necessitated the administration of excessive doses or long-term treatment. However, it was confirmed that the cancer targeting complex (Mal-PROTAC) according to the present invention effectively penetrates to the center of the cancer tissue at the same dose, thereby achieving superior pharmacological effects and high therapeutic efficiency that were unpredictable with conventional technology.

[0274]

[0275] Experimental Example 9. Analysis of the anticancer efficacy of a cancer-targeting complex (ECMal-PROTAC)

[0276] Cancer animal model

[0277] Five-week-old female Balb / c and Balb / c nu / nu mice were purchased from NaraBio, Inc. (Seoul, Republic of Korea). Prior to the experiment, the mice underwent a 2-week acclimatization period. During the experiment, the mice were free-fed in a room maintained at a temperature of 22 ± 2 ℃ and humidity of 40–60%, and the light-dark cycle was controlled at 12-hour intervals. All experiments were conducted in accordance with the regulations of the Institutional Animal Care and Use Committee (IACUC) of Ewha Womans University. The following 1 × 10⁶ were placed on the left thigh of each mouse. 6 Two 4T1 cells were inoculated, and the tumor volume was 50-80 mm³ 3 A female animal model was manufactured by rearing until it reached [the target].

[0278] Sample administration and analysis

[0279] In the previous Experimental Examples 7 and 8, samples labeled with fluorescent dyes were used to confirm the behavior of the anticancer drug targeting cancer in vivo, but in this experiment, the anticancer effect was analyzed, so the cancer targeting complex (Mal-PROTAC) of Example 1 was used directly as the drug without separate treatment.

[0280] The above-mentioned cancer animal models were randomly distributed with 5 animals per group. The control group was administered 10 mg / kg of physiological saline via the tail vein once every 3 days for 15 days (total 4 administrations). The comparison group was administered 10 mg / kg of ARV-771 via the tail vein once every 3 days for 15 days (total 4 administrations). The experimental group was administered 10 mg / kg of Mal-PROTAC (without substitution for ARV-771) from Example 1 via the tail vein once every 3 days for 15 days (total 4 administrations).

[0281]

[0282] The above control group, comparison group, and experimental group measured tumor size, body weight, and survival rate (%) once every two days. After a total of 15 days, the animals were euthanized, and major organs (heart, liver, lungs, spleen, kidneys) and tumor tissues were excised for Western blot, histological analysis (H&E, TUNEL), and immunofluorescence staining. In the case of the control group, the tumor size had already increased excessively by day 11, so the animals were culled in accordance with animal ethics guidelines; therefore, tissue from day 11 was used.

[0283] First, to determine the degree of apoptosis, major organ tissues and cancer tissues from each group (control group, comparison group, and experimental group) were fixed in a 10% buffered formaldehyde solution, embedded in paraffin, and then sliced ​​to prepare sections. The sections were stained with DAPI, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling), or H&E (hematoxylin & eosin) and analyzed using a BX51 optical microscope (Olympus, Tokyo, Japan) and a TCS SP8 confocal laser microscope.

[0284] To perform a western blotting assay to confirm the expression of BRD4, cancer tissues from each group were lysed and treated with 40 μL of RIPA buffer containing a protease inhibitor for 30 minutes. The lysate was then collected on ice and centrifuged at 4°C and 15,000 rpm for 30 minutes to recover the protein-containing supernatant. The protein content in each supernatant was measured using a BCA kit, and 3 μg of protein was used per group. The supernatant was loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel, subjected to electrophoresis (SDS-PAGE), and then blotted onto a polyvinylidene difluoride (PVDF) membrane. After blotting, to inhibit the indiscriminate binding of IgG (immunoglobulin G), PVDF membranes were treated with 5% BSA-containing tris buffered saline containing 0.1% Tween 20 (TBS-T) for 2 hours and immediately exposed to rabbit anti-mouse primary antibodies (BRD4 (1:1000, anti-mouse IgG), PD-L1 (1:200, anti-mouse IgG)). After treatment at 4°C for 24 hours, the PVDF membranes were washed three times with TBS-T and then incubated for an additional 1 hour at room temperature with HRP-conjugated mouse anti-rabbit IgG antibodies. Immunoreactive bands on the PVDF membranes were analyzed using an enhanced chemiluminescence (ECL) system. The expression levels of BRD4 or PD-L1 in each group were calculated as a percentage (%), with the control group set at 100%.

[0285] Meanwhile, cancer tissues from each group were fixed in a 10% buffered formaldehyde solution, embedded in paraffin, and sliced ​​to prepare sections. The sections were treated with appropriate primary antibodies (BRD4 (1:1000, anti-mouse IgG), PD-L1 (1:200, anti-mouse IgG)), reacted at 4°C for 24 hours, washed three times with TSB-T, then cultured with green Alexa Fluor 488 (1:1000, Invitrogen) and red Alexa Fluor 568 (1:500, Invitrogen), respectively, reacted with DAPI for 5 minutes, and analyzed using a confocal laser fluorescence microscope.

[0286]

[0287] Figure 19 is a graph showing the change in tumor tissue size according to the treatment period of the control group, comparison group, and experimental group, and at the bottom is the tumor tissue excised on the last day of treatment (15 days after drug administration). Figure 20 is a graph showing the change in body weight according to the treatment period of the control group, comparison group, and experimental group, and Figure 21 is a graph showing the survival rate (%) according to the treatment period of the control group, comparison group, and experimental group.

[0288] As shown in Figure 19, it was confirmed that the tumor size of the experimental group administered the cancer targeting complex (Mal-PROTAC) of Example 1 was significantly reduced.

[0289] In addition, the control group and the comparison group showed a tendency for the size of the tumor to increase over time (1672.56 ± 74.49 mm, respectively). 3 (Day 11) and 1366.24 ± 346.71 mm 3 (Day 15)), in the experimental group administered the cancer-targeting complex (Mal-PROTAC) of Example 1, it can be confirmed that the tumor size showed a tendency to increase and then decrease again (112.17 ± 58.88 mm 3 (Day 15)).

[0290] As shown in FIGS. 20 and 21, the control group administered with the PROTAC compound showed an overall decrease in body weight. On the other hand, no significant difference in body weight or survival rate was observed between the control group and the experimental group administered with the cancer targeting complex (Mal-PROTAC) of Example 1, confirming that the cancer targeting complex of the present invention does not exhibit adverse effects in vivo.

[0291]

[0292] Figure 22 shows the results of the histological evaluation of cancer tissues extracted from the control group, comparison group, and experimental group.

[0293] As shown in Fig. 22, in the control group administered with the PROTAC compound, some cancer cell death was observed, but it did not reach a statistically significant level compared to the control group. On the other hand, in the experimental group administered with the cancer targeting complex (Mal-PROTAC) of Example 1, cancer cell death significantly increased, and a clear killing effect was observed, particularly in the center of the cancer tissue, which is difficult for conventional anticancer drugs to reach. These results indicate that the cancer targeting complex of the present invention can exhibit excellent therapeutic efficacy against cancer cells in vivo.

[0294]

[0295] Figure 23 shows the results of analyzing the expression levels of BRD4 and PD-L1 in cancer tissues extracted from the control group, comparison group, and experimental group, Figure 24 is a graph showing the quantitative results of Figure 23, and Figure 25 shows the results of immunofluorescence staining on cancer tissues extracted from the control group, comparison group, and experimental group.

[0296] As shown in FIGS. 23 and 24, it was confirmed that BRD4 and PD-L1 expression decreased in cancer cells in both the experimental group administered with the cancer targeting complex (Mal-PROTAC) of Example 1 and the control group administered with the PROTAC compound. However, through the results of immunofluorescence staining on the cancer tissue (Fig. 25), it was confirmed that BRD4 and PD-L1 expression decreased statistically significantly more in the experimental group administered with the cancer targeting complex (Mal-PROTAC) of Example 1 compared to the control group.

[0297] These results clearly confirm that the cancer-targeting complex of the present invention can exhibit superior therapeutic efficacy compared to existing anticancer drugs by effectively inhibiting the expression of BRD4 and PD-L1 in cancer cells in vivo.

[0298]

[0299] Figure 26 shows the results of histological evaluation of liver, lung, spleen, kidney, and heart tissues extracted from the control group, comparison group, and experimental group.

[0300] As shown in Figure 26, no significant damage was observed in the liver, lung, spleen, kidney, and heart tissues of the experimental group treated with the cancer targeting complex (Mal-PROTAC) of Example 1.

[0301] On the other hand, in the control group administered the PROTAC compound (ARV-771), slight changes in cell density were observed in the liver tissue, and slight damage was confirmed in the lung tissue structure and myocardial fibers. In other words, it can be seen that the cancer targeting complex (Mal-PROTAC) of Example 1 exhibits excellent safety by protecting normal tissues with low toxic side effects in vivo.

Claims

1. A cancer targeting complex comprising a maleimide group substituted or unsubstituted with R3 attached to the side chain of a PROTAC through a linker represented by the following chemical formula 1: [Chemical Formula 1] In the above chemical formula 1, R1 is a single bond, or is selected from N, O, S, P, an alkylene group having 1 to 20 carbon atoms substituted or unsubstituted with at least one R2, or a heteroalkylene group having 1 to 20 carbon atoms substituted or unsubstituted with at least one R2, and R2 is independently selected from -F, -Cl, -Br, -I, hydroxyl group, cyano group, nitro group, alkyl group having 1 to 20 carbon atoms, alkyl carbonyl group having 1 to 20 carbon atoms, and carbonyl group, and R3 is any one selected from alkylamine groups having 1 to 20 carbon atoms.

2. In Paragraph 1, A cancer targeting complex characterized in that, in the above chemical formula 1, R1 is selected from any one of an alkylene group having 1 to 10 carbon atoms substituted or unsubstituted with at least one R2, or a heteroalkylene group having 1 to 10 carbon atoms substituted or unsubstituted with at least one R2.

3. In Paragraph 1, A cancer targeting complex characterized in that, in the above chemical formula 1, R1 is selected from any one of an alkylene group having 1 to 6 carbon atoms that is substituted or unsubstituted with at least one R2.

4. In Paragraph 1, A cancer targeting complex characterized in that, in the above chemical formula 1, R1 is selected from any one of an alkylene group having 1 to 6 carbon atoms substituted with at least one carbonyl group.

5. In Paragraph 1, Cancer targeting complex characterized in that the above chemical formula 1 is any one selected from the following chemical formulas 1-1 to 1-10: [Chemical Formula 1-1] *-(CH2C(=O))-* [Chemical Formula 1-2] *-(CH2C(=O)CH2)-* [Chemical Formula 1-3] *-(CH2(=O)CH2CH2)-* [Chemical Formula 1-4] *-(CH2CH2C(=O))-* [Chemical Formula 1-5] *-(CH2CH2C(=O)CH2)-* [Chemical Formula 1-6] *-(CH2CH2CH2C(=O))-* [Chemical Formula 1-7] *-(CH2CH2CH2C(=O)CH2)-* [Chemical Formula 1-8] *-(CH2CH2CH2CH2C(=O))-* [Chemical Formula 1-9] *-(CH2CH2CH2CH2C(=O)CH2)-* [Chemical Formula 1-10] *-(CH2CH2CH2CH2CH2C(=O))-* 6. In Paragraph 1, 상기 프로탁(PROTAC)은 CDK9(Cyclin-dependent kinase 9) / CDK family, BRD4(Bromodomain-containing protein 4) / BET(Bromodomain and extra-terminal) family, SHP-2(Src homology region 2 domain-containing phosphatase-2), AR(Androgen receptor), ER(Estrogen receptor), RAR(Retinoic acid receptor), ERRα(Estrogen-related receptor alpha), BTK(Bruton's tyrosine kinase), ALK(Anaplastic lymphoma kinase), RIPK(Receptor-interacting serine / threonine-protein kinase), c-Met(MET proto-oncogene / receptor tyrosine kinase), FAK(Focal adhesion kinase), IRAK4(Interleukin-1 receptor-associated kinase 4), p38 MAPK(p38 mitogen-activated protein kinases), SGK(Serum and glucocorticoid-regulated kinase), TBK1(TANK-binding kinase 1), KRAS(Kirsten rat sarcoma virus protein), B-Raf(B-Raf proto-oncogene serine / threonine kinase), β-catenin, FKBP(FK506 binding protein), IDO(Indoleamine 2,3-dioxygenase), PD-1 / PD-L1(Programmed death protein 1 / programmed death-ligand 1), PARP1(Poly [ADP-ribose] polymerase 1),PRC2 (Polycomb repressive complex 2), EGFR (Epidermal growth factor receptor), Sirt2 (NAD-dependent deacetylase sirtuin 2), HER2 / ERBB2 (Human epidermal growth factor receptor 2), FRS2 (Fibroblast growth factor receptor substrate 2), BCL-XL (B-cell lymphoma-extra large), SMARCA (SWI / SNF related matrix associated actin dependent regulator of chromatin subfamily A), HDM2 (Human double minute 2 homolog), HDAC (Histone deacetylase) family, BCR-ABL (Breakpoint cluster region protein- Tyrosine-protein kinase ABL1), MCL1 (Modulator Of VRAC Current 1), FLT-3 (Fms Related Receptor Tyrosine Kinase 3), STAT3 (Transcription factor STAT3), Myc family and BAF One or more proteins selected from the group consisting of complex (Brg / Brahma-associated factors) Cancer targeting complex characterized by targeting.

7. In Paragraph 1, The cancer-targeting anticancer agent is a cancer-targeting complex characterized by being represented by the following chemical formula 2. [Chemical Formula 2] 8. A pharmaceutical composition for the prevention or treatment of cancer comprising a cancer-targeting complex according to claim 1 as an active ingredient.

9. In Paragraph 8, The above cancers include thyroid cancer, oral cancer, oropharyngeal cancer, ovarian cancer, male genital tumors, male breast cancer, brainstem tumors, cerebral lymphoma, brain tumors, pituitary meningioma, gallbladder cancer, biliary tract cancer, colorectal cancer, intracranial tumors, head and neck tumors, bladder cancer, leukemia, abdominal tumors, nasal cavity cancer, non-small cell lung cancer, nasopharyngeal cancer, esophageal cancer, breast cancer, stomach cancer, liver cancer, pancreatic cancer, ependymoma, adenoma, small cell lung cancer, small intestine cancer, kidney cancer, malignant astrocytoma, benign astrocytoma, female genital tumors, female external genital cancer, female urethral cancer, penile cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, mediastinal tumors, vaginal cancer, salivary gland cancer, skin cancer, malignant lymphoma, multiple myeloma, aplastic anemia, oligodendroglioma, hypopharyngeal cancer, anal cancer, hematological cancer, laryngeal cancer. A pharmaceutical composition for the prevention or treatment of cancer characterized by being one or more selected from thoracic tumors and thymic cancers.

10. A food composition for preventing or improving cancer comprising a cancer-targeting complex according to claim 1 as an active ingredient.

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