CP2C-targeting peptide-based anticancer agents

Abstract

The present invention relates to an anticancer agent based on a CP2c targeting peptide. The CP2c targeting peptide according to the present invention binds to the transcription factor CP2c so as to inhibit the formation of a transcription factor complex (CP2c homotetramer and CP2c / CP2b / PIAS1 heterohexamer) containing CP2c, thereby inducing cancer cell-specific cell death, and a fatty acid is bound to the peptide to ensure stability that enables it to be maintained in vivo for a long time.

Description

Technical Field

[0001] The present invention relates to anticancer agents based on CP2c targeting peptides. Background Art

[0002] Although many protein / peptide anticancer agents and anticancer agents based on low molecular weight compounds have been developed and used, they do not selectively act only on cancer cells in vivo, but also produce serious side effects on normal cells, and their effects are sometimes not significant due to the emergence of drug-resistant cancer cells. In addition, many protein / peptide anticancer agents under development have short half-lives in vivo, so multiple studies are being conducted to overcome this problem. Similarly, although the world is competing to develop anticancer drugs, anticancer drugs with no problems in terms of in vivo stability, safety and drug resistance have not yet been developed. Therefore, it is highly beneficial to find drugs that can ensure long-term stability in vivo, selectively remove only various types of cancer cells, and act on drug-resistant cancer cells, which will be highly beneficial worldwide. Summary of the invention

[0003] Technical issues to be resolved

[0004] The present invention has been made in view of the above problems, wherein an emphasis is placed on providing an anticancer agent based on a CP2c targeting peptide that can ensure long-term stability in vivo, selectively remove only various types of cancer cells, and also act on drug-resistant cancer cells.

[0005] Technical Solution

[0006] According to the present invention, the CP2c targeting peptide refers to a peptide that binds to the transcription factor CP2c to inhibit the formation of a transcription factor complex (CP2c homotetramer and CP2c / CP2b / PIAS1 heterohexamer) containing CP2c, thereby inducing cancer cell-specific cell death. The CP2c targeting peptide corresponds to Tyr-Pro-Gln-Arg (SEQ ID NO: 1), which is a peptide of the smallest size consisting only of amino acids necessary for anticancer effects. Therefore, a peptide that substantially comprises these four amino acids and exhibits an anticancer effect by interacting with the CP2c protein can be used as a CP2c targeting peptide according to the present invention.

[0007] The CP2c targeting peptide according to the present invention may be a peptide consisting of 4 to 20 amino acids comprising the amino acid sequence of SEQ ID NO: 1. Preferably, the CP2c targeting peptide according to the present invention may be a peptide consisting of 6 amino acids (6aa)'NYPQRP (Asn-Try-Pro-Gln-Arg-Pro, SEQ ID NO: 2)' (ACP52).

[0008] The CP2c targeting peptide according to the present invention can be combined with a cell-penetrating peptide (CPP) to enhance the cell-penetrating activity.

[0009] For example, the CP2c targeting peptide according to the present invention may be a peptide comprising a peptide (ACP52) consisting of SEQ ID NO: 2; and an internalizing RGD (iRGD) peptide consisting of 9 amino acids (9aa) 'CRGDKGPDC (Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys, SEQ ID NO: 3)' as a cell penetrating peptide (CPP). As an example of a CP2c targeting peptide according to the present invention, 'ACP52C' is a peptide in which an acetyl group (Ac) is bound to the N-terminus of ACP52, iRGD is bound to the C-terminal Pro, and an amide group (NH2) is bound to the C-terminus of iRGD (see Figure 7 ).

[0010] In addition, for the purpose of ensuring the in vivo stability of the previously developed CP2c targeting peptide, the present invention prepares a CP2c targeting peptide-fatty acid conjugate bound to a fatty acid. When the fatty acid is bound to the CP2c targeting peptide, the resulting conjugate binds to albumin in the blood, thereby improving in vivo stability and providing a high therapeutic effect.

[0011] The fatty acids may be (but are not necessarily limited to) C 12 To C 20 In one embodiment of the present invention, the fatty acid may be C 16 Fatty acids, such as palmitoleic acid (also represented by 'pal'). In addition, in one embodiment of the present invention, the fatty acid may be a modified fatty acid bound to glutamic acid (Glu, E) or an amino acid sequence shown as EGLFG as a target sequence for the proteolytic enzyme cathepsin B. In particular, the modified fatty acid may be formed by a peptide bond between the carboxyl group of the fatty acid and the amino group of glutamic acid or the amino group of glycine in the amino acid sequence shown as EGLFG. In the present specification, palmitoleic acid coupled to glutamic acid may be referred to as "E-pal", and palmitoleic acid coupled to the amino acid sequence shown as EGLFG may be referred to as "EGLFG-pal".

[0012] In addition, according to the present invention, the CP2c targeting peptide-fatty acid conjugate may include a CP2c targeting peptide (e.g., ACP52) for CP2c targeting and a linker peptide for connecting CPP (e.g., iRGD) and / or fatty acids to each other. The linker peptide may be an amino acid or a peptide composed of a combination thereof known in the art, without specific limitations. In particular, the linker peptide may include glycine (Gly, G), such as G n(wherein n is an integer from 1 to 6). In another embodiment of the present invention, the linker peptide may be composed of G n KG m (wherein n and m are each independently an integer from 0 to 6). For example, when n or m is 0, lysine (Lys, K) may be located at the N-terminus or C-terminus of the linker peptide, and when n and m are not 0, lysine (Lys, K) may be located between glycines. For conjugation between fatty acids and peptides, lysine (Lys, K) is included in the linker peptide. The terminal functional group (-HN2) of lysine enables the binding of additional amino acids.

[0013] In the CP2c targeting peptide-fatty acid conjugate according to the present invention, the conjugation of the CP2c targeting peptide and the fatty acid can be achieved through a linker peptide. In particular, the conjugation can be achieved by the binding between the glutamic acid of the peptide bonded to the modified fatty acid and the lysine of the linker peptide. More particularly, the binding between the glutamic acid of the modified fatty acid and the lysine of the linker peptide can be formed by a peptide bond between the functional group (NH2) of lysine and the carboxyl group of glutamic acid.

[0014] Meanwhile, the N-terminus and / or C-terminus of the peptide of the CP2c targeting peptide-fatty acid conjugate used in the present invention may be modified so as to obtain improved stability, enhanced pharmacological properties (half-life, absorption, efficacy, potency, etc.), altered specificity (e.g., broad-spectrum biological activity) and reduced antigenicity of the peptide used. Preferably, the modification may be in the form of an acetyl group, a fluorenylmethoxycarbonyl group, an amide group, a formyl group, a myristyl group, a stearoyl group or a polyethylene glycol (PEG) bonded to the N-terminus and / or the C-terminus of the peptide, but may include but is not limited to components capable of improving the modification of the peptide, particularly the stability of the peptide. As used in the present invention, the term "stability" means not only the in vivo stability that protects the peptide of the present invention from in vivo proteolytic enzyme attack, but also storage stability (e.g., room temperature storage stability). In the CP2c targeting peptide-fatty acid conjugate according to the present invention, the N-terminus and the C-terminus of the peptide may be modified with an acetyl group and an amide group, respectively.

[0015] The CP2c targeting peptide-fatty acid conjugate according to an embodiment of the present invention is as follows:

[0016] 1) Ac-K(E-pal)-GG- is bonded to the N-terminus of ACP52C in which Ac is absent (C16-ACP52Cn, ACP52CG),

[0017] 2) Ac-K(EGLFG-pal)-GG- is bonded to the N-terminus of ACP52C in which Ac is absent (A16-GFLG-ACP52Cn, ACP52CK),

[0018] 3) The functional group of K in the GG-K-GG linker peptide between ACP52 and iRGD is bonded to the α-carbon carboxyl group of glutamic acid of E-Pal (C-16-ACP52Cm, ACP52GK)

[0019] 4) The functional group of K in the GG-K-GG linker peptide between ACP52 and iRGD is bonded to the carboxyl group of the γ carbon of glutamic acid of E-Pal (γC16-ACP52Cm, ACP52CGK).

[0020] Delete a picture

[0021] For example, the structures of ACP52GK and ACP52CGK in the CP2c targeting peptide-fatty acid conjugate according to the present invention are as follows:

[0022]

[0023] In one example of the present invention, 4 kinds of synthetic CP2c targeting peptide-fatty acid conjugates were used to analyze the in vivo stability, anticancer effect, tumor metastasis inhibition and physiological toxicity in a mouse model with hepatoma cell (Hep3B) or breast cancer cell (MDA-MB-231) xenografts. As a result, it was confirmed that the in vivo stability, tumor inhibition and tumor metastasis inhibition were excellent, and the conjugate was a safe substance without toxicity to the body.

[0024] In addition, it was confirmed that in one example of the present invention, cells resistant to the CP2c targeting peptide-fatty acid conjugates were generated during treatment with 4 synthetic CP2c targeting peptide-fatty acid conjugates to study anticancer effects. Therefore, it was confirmed that the mechanism and reason for the generation of resistance was due to the reduction of cellular MDM2p90 levels in cells treated with the CP2c targeting peptide-fatty acid conjugates. In addition, it was confirmed that cancer cells resistant to the CP2c targeting peptide-fatty acid conjugates could be killed by co-treatment of the CP2c targeting peptide-fatty acid conjugates with a caspase 2 inhibitor capable of inhibiting the degradation of MDM2p90.

[0025] The present invention provides a pharmaceutical composition for preventing, improving and treating cancer, which comprises a CP2c targeting peptide-fatty acid conjugate as an active ingredient.

[0026] As another aspect of the present invention, the present invention also provides a method for treating an individual, comprising the step of administering a CP2c targeting peptide-fatty acid conjugate or a pharmaceutical composition comprising the same in a pharmaceutically effective amount to an individual in need of treatment.

[0027] In the present invention, cancer may also include resistant cancers that show resistance to anticancer agents, particularly anticancer agents based on the CP2c targeting peptide-fatty acid conjugates according to the present invention.

[0028] In the present invention, the term "treatment" refers to any action to improve or beneficially change the symptoms of cancer by administering the peptide according to the present invention or a pharmaceutical composition comprising the peptide.

[0029] In the present invention, the term "administering" refers to introducing a predetermined substance into a subject by any suitable method, i.e., a derivative of a peptide according to the present invention or a pharmaceutical composition comprising the same. The route of administration can be any general route, as long as the drug can reach the target tissue. For example, the route of administration may include, but is not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration. However, since the peptide is digested when administered orally, it is preferred to prepare an oral composition coated with an active agent or exempted from degradation in the stomach. Preferably, the peptide will be administered in the form of an injection. In addition, the pharmaceutical composition according to the present invention can be administered by any device capable of transporting the active substance to the target cell.

[0030] In the present invention, the term "as an active ingredient is included" means an amount sufficient to treat a disease under a reasonable risk-benefit ratio applicable to drug therapy. The effective dose level can be determined according to the patient's disease type, severity, drug activity, drug sensitivity, administration time, administration route, excretion rate, treatment duration, factors including concomitant drugs, and other factors known in the medical field. The peptide according to the present invention or a pharmaceutical composition comprising it can be used as a separate therapeutic agent or can be used in combination with other therapeutic agents, can be used sequentially or simultaneously with conventional therapeutic agents, and can be used once or multiple times. Taking all of the above factors into account, it is important to administer the minimum amount that can obtain the maximum effect and no side effects, which can be easily determined by those with medical expertise. The dosage and frequency of the pharmaceutical composition of the present invention are based on the type of drug as the active ingredient, together with several related factors such as disease type, administration route, age, sex and weight of the patient and the severity of the disease.

[0031] The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease under a reasonable risk-benefit ratio applicable to drug therapy. The appropriate dosage of the pharmaceutical composition of the present invention varies according to factors such as formulation method, administration method, age, weight and sex of the patient, severity of the disease, food, administration time and route, administration route, discharge rate and response sensitivity, and for the desired treatment, an ordinary skilled physician can easily determine and prescribe an effective dosage.

[0032] The term "individual" of the present invention used herein includes animals, such as horses, sheep, pigs, goats, camels, antelopes, dogs or humans, whose symptoms can be improved by administering the therapeutic composition according to the present invention. Diseases can be effectively prevented and treated by administering the pharmaceutical composition according to the present invention to individuals. The treatment method according to the present invention can be a method for treating animals other than humans, but is not limited thereto. In other words, considering that humans may suffer from diseases whose symptoms can be improved by administering the composition according to the present invention, the composition according to the present invention can be fully used for human treatment.

[0033] Therefore, the pharmaceutical composition according to the present invention may include a variety of pharmaceutically acceptable carriers, as long as the peptide according to the present invention is included as an active ingredient. Pharmaceutically acceptable carriers may include adhesives, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants and flavoring agents for oral administration. Injections can be used by mixing buffers, preservatives, analgesics, solubilizers, isotonic agents or stabilizers. For topical administration, a matrix, excipient, lubricant or preservative can be used. The preparation of the pharmaceutical composition of the present invention can be prepared in a variety of ways by mixing with a pharmaceutically acceptable carrier as described above. For example, it can be prepared in the form of tablets, lozenges, capsules, elixirs, suspensions, syrups or wafers for oral administration, and it can be prepared in the form of single-dose ampoules or multiple doses for injection. It can also be prepared in the form of other solutions, suspensions, tablets, pills, capsules or sustained release formulations.

[0034] Meanwhile, some examples of carriers, excipients and diluents suitable for preparations include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil. In addition, fillers, anti-agglomeration agents, lubricants, wetting agents, flavoring agents or preservatives can also be used.

[0035] Hereinafter, the present invention will be described in more detail with reference to the following examples. It should be understood that the included examples are only used to specifically illustrate the spirit of the present invention and are not intended to be any form of limitation of the present invention.

[0036] Effects of the Invention

[0037] To identify the final candidate for improved in vivo stability of the transcription factor CP2c targeting peptide ACP52C, which showed action as a universal anticancer agent, C 16The fatty acid was linked to the CP2c targeting peptide lead substance ACP52C, and the conjugate was confirmed to show in vivo stability and anticancer effect in a cancer cell xenograft mouse model. In addition, the cancer cell-specific anticancer effect of the final anticancer candidate was confirmed in a variety of cancer cells and normal cells. The anticancer effect of the final candidate was shown to be caspase 2-dependent, and its therapeutic effect on cancer cells resistant to anticancer drugs was confirmed by treatment with a combination of a caspase 2 inhibitor and the final candidate. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Shown is the GI depending on ACP52C treatment in various cancer cell lines 50 value.

[0039] Figure 2a to d show that ACP52C treatment induces G2 / M cell cycle arrest and cell death (sub G1 cells) as revealed by FACS.

[0040] Figure 3a Figures c to c show that ACP52C treatment causes an increase in the expression of p53, which regulates cell cycle and apoptosis.

[0041] Figure 4a to c show apoptosis induced by ACP52C treatment.

[0042] Figure 5 Shown are the time-dependent subcellular movement, localization, and amount of ACP52C in the MDA-MB-231 cell line.

[0043] Figure 6a h to 4 show the results of in vivo half-life analysis of the ACP52C peptide.

[0044] Figure 7 The construction of a C16 fatty acid binding peptide for improved in vivo stability is shown.

[0045] Figure 8a to d shows that it depends on the C 16 Results of cell growth analysis of fatty acid binding peptides (ACP52C; ACP52CG; ACP52CK) treated in various cancer cell lines.

[0046] Figure 9a to c shows that the 16 Results of cell growth assays of fatty acid binding peptides (ACP52C; ACP52CG; ACP52CK) treated in various p53 mutant or null cancer cell lines.

[0047] Fig.10aFigures 4 to 5 show the results of cell growth analysis depending on the treatment with ACP52CGK in normal cell lines (BEAS2B, hMSC) and cancer cell lines (Hep3B, Hs746T, Caov-3, MDA-MB-231, U343, HCT116, PANC1, PC3, A549, THP-1).

[0048] Fig.11a Figures 1 to 4 show the results of the analysis of the anticancer effects of ACP52CG and ACP52CK in the Hep3B cell line-xenograft mouse model.

[0049] Fig.12a Figures 1 to 2 show the results of analyzing the anticancer effect of ACP52CK in a liver cancer mouse model induced by DEN treatment.

[0050] Fig.13a Figures 1 to 2 show the results of analysis of the anticancer effects of ACP52CG and ACP52CK in a liver cancer mouse model induced by DEN treatment.

[0051] Fig.14a Figures 1 to 4 show the results of the analysis of the anticancer effects of ACP52CG and ACP52CK in the MDA-MB-231 (LM1) cell line-xenograft mouse model.

[0052] Fig.15a Figures 1 to 5 show the results of analysis of the anticancer effect (tumor size and weight, body weight, and hematological analysis) and metastasis inhibition in the Hep3B cell line-xenograft mouse model when treated with ACP52CGK at three different doses at 3-day intervals.

[0053] Fig.16a Figures 1 to 4 show the results of analysis of the anticancer effect (tumor size and weight, body weight, and hematological analysis) and metastasis inhibition in the Hep3B cell line-xenograft mouse model when treated with ACP52CGK at three different doses at 5-day intervals.

[0054] Fig.17a Figures 1 to 4 show the results of analysis of the anticancer effect (tumor size and weight, body weight, and hematological analysis) and metastasis inhibition in the MDA-MB-231 (LM1) cell line-xenograft mouse model when treated with ACP52CGK at three different doses at 3-day intervals.

[0055] Fig.18aFigures 1 to 4 show the results of analysis of the anticancer effect (tumor size and weight, body weight, and hematological analysis) and metastasis inhibition in the MDA-MB-231 (LM1) cell line-xenograft mouse model when treated with ACP52CGK at three different doses at 5-day intervals.

[0056] Fig.19a a to b show the results of in vivo half-life analysis using fluorescently labeled ACP52CGK.

[0057] Fig.20a b to b show the analysis results of the migration pathway, intracellular localization and intracellular residence time of ACP52CGK in the MDA-MB-231 cell line over time.

[0058] Fig.21 The results of analysis by ELISA to investigate whether antibodies against ACP52C, ACP52CG, ACP52CK, and ACP52CGK are formed are shown.

[0059] Fig.22a Figures 4 to 5 show the results of sudden and repeated in vivo toxicity tests of ACP52C.

[0060] Fig.23 The results of replicate toxicity testing of ACP52CGK are shown.

[0061] Fig.24 Shown are the results of histological analysis of major organs from repeated toxicity tests with ACP52CGK.

[0062] Fig.25a Figures 4 to 5 show the results of efficacy analysis of ACP52CGK in culture of cells derived from tumor tissues of breast cancer patients.

[0063] Fig.26a Figures 4 to 5 show the results of the efficacy analysis of ACP52CGK in culture of cells derived from cryopreserved breast cancer patient cancer tissues.

[0064] Fig.27a Figures 4 to 5 show the results of comparative analysis of the efficacy of ACP52CGK in cells of a specific generation cultured from PDX tumor tissues.

[0065] Fig.28a b to b show the results of efficacy and resistance analysis of ACP52CGK in cells derived from patient tumor tissues.

[0066] Fig.29 Shown are the results of expression analysis of CP2c transcriptional activity-independent pathway proteins in lung cancer cell lines showing resistance to ACP52C.

[0067] Fig.30a Figures 1 to 4 show the results of ACP52C treatment-dependent expression analysis of CP2c transcriptional activity-independent pathway proteins in lung cancer cell lines (A549, PC9, KCL22).

[0068] Fig.31a b show the results of MDM2 overexpression-dependent expression analysis of CP2c transcriptional activity-independent pathway proteins in lung cancer cell lines (A549, PC9).

[0069] Fig.32a to d show the results of ACP52C treatment-dependent cell growth analysis of the A549 cell line in which MDM2 overexpression was induced for a long time.

[0070] Fig.33a to d show the results of MDM2p60 expression model and lung cancer cell line-specific alternative splicing and SNP analysis.

[0071] Fig.34a Figures 4 to 5 show the results of efficacy analysis depending on the combined treatment of caspase 2 inhibitor and ACP52C in ACP52C-resistant cells.

[0072] Fig.35 Shown are changes in cell morphology depending on combined treatment of a caspase 2 inhibitor and ACP52C in ACP52C-resistant cells. DETAILED DESCRIPTION

[0073] Hereinafter, the present invention will be described in more detail with reference to the following examples. It should be understood that the included examples are only used to specifically illustrate the spirit of the present invention and are not intended to be any form of limitation of the present invention.

[0074] [Preparation Example 1] Preparation of CP2c targeting peptide conjugated with cell penetrating peptide

[0075] It is known that the transcription factor CP2c is overexpressed in many cancers. According to a study by a US research group, it was reported that inhibiting the expression of CP2c in liver cancer cell lines inhibited cell growth, while overexpression of CP2c led to cancer progression and metastasis [Grant et al., Antiproliferative small-molecule inhibitors of transcription factor LSF reveal oncogene addiction to LSF in hepatocellular carcinoma, Proc. Natl. Acad. Sci. 2012; 109(12): 4503-4508].

[0076] The present inventors identified peptides that bind to transcription factor CP2c (also known as Tfcp2, LSF, LBP1, UBP1, etc.) by phage display [Kang et al., Identification and characterization of four novel peptide motifs that recognize distinct regions of the transcription factor CP2, FEBS Journal 2005; 272: 1265-1277], selected a type of peptide derived from a peptide that interferes with DNA binding of CP2c among the identified peptides (CP2c targeting peptide, SEQ ID NO: 2), and synthesized an ACP52C lead substance by combining an internalizing RGD (iRGD) peptide consisting of 9 amino acids (9aa)′CRGDKGPDC (Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys, SEQ ID NO: 3) as a cell penetrating peptide (CPP) with the CP2c-targeting peptide in order to improve cell permeability ( Figure 7 ).

[0077] [Example 1] Determination of the anticancer effect of ACP52C

[0078] The selected peptide binds to CP2c and inhibits the formation of transcription factor complexes containing CP2c (CP2c homotetramers and CP2c / CP2b / PIAS1 heterohexamers), thereby indirectly interfering with the DNA binding of CP2c. As a result of analyzing the anticancer effect of targeted synthetic ACP52C in various cancers, it was confirmed that it showed cancer cell-specific growth inhibition and cell death efficacy ( Figure 1 ).

[0079] The growth inhibition and cell death inducing effects of ACP52C were confirmed by FACS analysis performed by treating cells with ACP52C after synchronizing the cell cycle to the G1 / S phase by the double thymidine blocking method. As a result, it was confirmed that polyploidy was formed when the cell cycle was arrested in the G2 / M phase. On the other hand, it has been confirmed that when ACP52C is applied to a cell line synchronized to the G2 / M phase by thymidine / nocodazole treatment, the cell cycle is arrested in the sub-G1 phase and cell death is induced ( Figure 2a to d).

[0080] It has been demonstrated that ACP52C-induced G2 / M arrest is caused by increased expression of CHK1 / 2 proteins and decreased expression of Cdc25c, CDK1, and cyclin B proteins, whereas cell death induction is caused by increased expression of pro-apoptotic proteins and decreased expression of anti-apoptotic marker proteins, as well as apoptosis through caspase activation ( Figure 3a To c, Figure 4a to c).

[0081] The subcellular movement, localization, and stability of ACP52C in cells treated with Cy5-labeled peptides (Cy5-ACP52C) for 30 minutes were analyzed over time by confocal microscopy. Most peptides passed through the cytoplasm, were located in the nucleus from 4 hours on, moved back to the cytoplasm at 8 hours, and degraded in the cytoplasm at 16 hours. It was confirmed that the Cy5-ACP52C peptide colocalized with CP2c in the nucleus from 1 hour after treatment, and CP2c also tended to be distributed in the cytoplasm with Cy5-ACP52C 8 hours after treatment ( Figure 5 ).

[0082] After ACP52C was incubated multiple times in a solution containing 10% serum and then removed serum proteins with Centricon (Mw10,000) before HPLC analysis, the half-life of ACP52C in a solution containing serum was analyzed by HPLC (model: UHPLC DIONEX Ultimate 3000; flow rate = 1.000 ml / min). As a result of the experiment, ACP52C was not completely degraded until 24 hours. At the same time, after ACP52C was injected into mice through the tail vein, blood samples were extracted over time, and the degree of ACP52C degradation in the blood samples was analyzed by HPLC. As a result, EC50 (the time to retain 50% of the intact ACP52C) was about 2 hours. In addition, when the residual fluorescence intensity in the mouse body was measured over time by a real-time imaging process in mice injected with Cy5-labeled peptide (Cy5-ACP52C) via the tail vein, although the total fluorescence intensity in the mouse body was halved 7.95 hours after the treatment, ACP52C ( Figure 6a to h).

[0083] [Preparation Example 2] Synthesis of CP2c Targeting Peptide-Fatty Acid Conjugate

[0084] Our data suggest that although ACP52C exhibits promising anticancer activity, ACP52C may be unstable in vivo. As an effort to improve the in vivo stability of ACP52C, four types of albumin affinity C were synthesized. 16 Fatty acid (palmitoyl acid) conjugated peptide ( Figure 7Each peptide was synthesized with modifications at the N-terminus and the C-terminus, and then the C 16 Fatty acids were conjugated to each peptide.

[0085] [Example 2] Determination of the in vitro anticancer effect of CP2c targeting peptide-fatty acid conjugate

[0086] The anticancer effects of the CP2c targeting peptide-fatty acid conjugate synthesized in Preparation Example 2 were analyzed in various cancer cells. As a result, all three types of C 16 The fatty acid binding peptides (ACP52CG, ACP52CK, and ACP52CGK) all induced cancer cell-specific cell death similar to that of the control group (ACP52C), and the GI calculated 48 h after treatment was 50 GI values ​​and control 50 GI values ​​similar to or better than the control 50 Value is more effective ( Figure 8a To d, Fig.10a In addition, cell survival curves and GI were calculated by MTT assay after various cancer cell lines with different p53 mutations were treated with ACP52CG and ACP52CK. 50 As a result, although the GI of each cell line 50 The values ​​showed deviations, but they were 10 mM or less, which was significantly different from the results of normal cell lines (about 1,000 mM) ( Figure 9a To sum up, the anticancer effects of ACP52CG, ACP52CK, and ACP52CGK were similar to those of ACP52C, and the fatty acids bound to the complexes did not show any negative effects on either cancer cells or normal cells.

[0087] [Example 3] Determination of tumor growth inhibition and general physiological toxicity depending on treatment with CP2c targeting peptide-fatty acid conjugates in a mouse model transplanted with various cancer cell lines

[0088] [3-1] Determination of the tumor growth inhibitory effects and general physiological toxicity of ACP52CG, ACP52CK, and ACP52GK in mouse models transplanted with hepatocellular carcinoma and breast cancer cell lines

[0089] In order to analyze the anticancer efficacy of the CP2c targeting peptide-fatty acid conjugate synthesized in Preparation Example 2 in an animal model, ACP52CG and ACP52CK were injected into Hep3B xenograft mice 5 times at intervals of 3 days via the tail vein. As a result, both ACP52CG and ACP52CK showed efficacy similar to that of sorafenib, but their efficacy was less than that of ACP52C. However, no specific abnormalities were found in normal tissues and blood levels, and no toxicity was observed ( Fig.11a to e).

[0090] In addition, mice after 22 weeks of DEN treatment were divided into 3 groups (7 to 8 mice / group), such as a control group (mock) treated with a vehicle alone, a sorafenib group (approved drug for liver cancer treatment), and an ACP52CG-GFLGE (ACP52CK) group. The drug was injected 12 times at a concentration of 5 mg / kg through the tail vein at intervals of 3 days. As a result of the analysis, the sorafenib-treated group showed a significant anticancer effect compared with the control group (p = 0.04), and the ACP52CG-GFLGE (ACP52CK)-treated group showed an anticancer effect similar to that of the sorafenib-treated group (p = 0.001) ( Fig.12a In addition, the efficacy analysis results of ACP52CG and ACP52CK in mice 15 weeks after DEN treatment were also superior to another control FQI1, but did not show any superior efficacy compared to ACP52C ( Fig.13a to d).

[0091] When ACP52CG and ACP52CK were injected into MDA-MB-231 (LM1) xenograft mice 5 times at 3-day intervals via the tail vein, both ACP52CG and ACP52CK showed tumor regression efficacy, but the anticancer effect was not significant. No specific abnormalities were found in the blood levels, so no toxicity was observed ( Fig.14a to d).

[0092] When ACP52GK was injected into Hep3B xenograft mice five times at 3-day intervals via the tail vein and then the efficacy was analyzed, it was confirmed that the efficacy of ACP52GK was lower than that of ACP52C, but the efficacy was better than that of the control FQI1 group.

[0093] [3-2] Determination of the tumor growth inhibitory effect, metastasis inhibitory effect, and general physiological toxicity of ACP52CGK in mouse models transplanted with hepatocellular carcinoma and breast cancer cell lines

[0094] ACP52CGK was injected into Hep3B xenograft mice 5 times at 3 different concentrations (1.39, 2.77 or 5.54 mg / kg) via the tail vein at 3-day intervals. Tumor volume was measured every 3 days after drug injection. Tumors and major tissues were excised from mice sacrificed on the 24th day after tumor cell injection. The tumors were weighed and fixed with 4% formaldehyde together with major organs. Hematoxylin / eosin staining was performed after tissue slides were made by paraffin sectioning. Basic CBC analysis was performed on the collected blood samples using a Coulter LH 750 hematology analyzer. The data obtained were statistically processed using the Excel program.

[0095] As a result, ACP52CGK exhibited tumor suppressor effects similar to those of ACP52C, and no abnormalities were found in normal tissue and blood levels ( Fig.15a to d and 15g to j). In addition, according to Fig.15e to f, show the mean and standard deviation of the tumor area metastasized to the lung, confirming that ACP52CGK is superior to ACP52C in tumor metastasis inhibition.

[0096] ACP52CGK was injected into Hep3B xenograft mice 5 times at 5-day intervals via the tail vein at 3 different concentrations (1.39, 2.77, 5.54 mg / kg), and the anticancer effect was evaluated in the same manner as above. As a result, all three concentrations of ACP52CGK showed tumor inhibition and metastasis inhibition effects superior to those of the sorafenib and ACP52C treatment groups, and the inhibition was concentration-dependent ( Fig.16a In addition, ACP52CGK injected at 5-day intervals showed more effective tumor suppression and metastasis suppression than ACP52CGK injected at 3-day intervals (compare Fig.15a to j and Fig.16a As a result of comprehensive analysis of all the results, 2.77 mg / kg was the optimal concentration. In addition, no specific abnormality was found in the blood level, so no toxicity was observed ( Figure 16h to k).

[0097] The anticancer effect of ACP52CGK was analyzed in MDA-MB-231 (LM1) xenograft mice in the same manner as in the Hep3B xenograft mouse model experiment. Similarly, the tumor inhibition and metastasis inhibition effects of ACP52CGK at all three concentrations (1.39, 2.77, 5.54 mg / kg) were superior to those of the sorafenib and ACP52C treatment groups ( Fig.17a When the injections were performed at 5-day intervals instead of 3-day intervals, a better anticancer effect was observed, and 2.77 mg / kg was the optimal concentration. In addition, no specific abnormalities were found in the blood levels, so no toxicity was observed ( Figure 17g to j and 18h ​​to k).

[0098] [Example 4] Stability and safety evaluation of CP2c targeting peptide-fatty acid conjugate

[0099] To analyze the in vivo half-life of ACP52CGK, Cy5-labeled ACP52CGK was injected into the tail vein of mice, and then the remaining fluorescence intensity in the mice was measured over time by real-time imaging. As a result, the half-life was about 20.2 hours ( Fig.19a to b). These results indicate that conjugation to fatty acids results in a significantly improved in vivo stability compared to the lead substance ACP52C which exhibited a half-life of 7.95 hours.

[0100] In order to analyze the subcellular movement, localization and stability of ACP52CGK in cultured cells, Cy5-labeled ACP52CGK was treated in cell culture medium for 30 minutes, and then the subcellular movement of the peptide was tracked over time. In addition, subcellular movement was analyzed by confocal microscopy to see whether Cy5-labeled ACP52CGK also migrated to mitochondria (Hsp60) and lysosomes (LC3). As a result, ACP52CGK passed through the cytoplasm from 4 hours and was mainly located in the nucleus, and came out of the cytoplasm after 8 hours; some of them were in mitochondria, but eventually migrated to lysosomes and degraded at 16 hours. These subcellular movement, localization and stability phenomena of ACP52CGK are no different from those in ACP52C ( Fig.20a To b).

[0101] At the same time, it is speculated that ACP52C does not show immunogenicity because it consists of 15 amino acids. In order to verify this, the immunogenicity of ACP52CG and ACP52CK was directly tested in the process of obtaining the final new drug candidate. Since the final candidate substance can be a C-16 palmitoleic acid-conjugated ACP52C peptide that is indistinguishable from ACP52CG and ACP52CK in terms of chemical composition, it was decided to analyze whether antibodies are formed on ACP52CG and ACP52CK. As a result of ELISA analysis of sera isolated from rabbits injected three times with ACP52CG and ACP52CK, neither rabbit serum induced an immune response on ACP52CG, ACP52CK and ACP52CGK, including ACP52C. Therefore, it is concluded that the final candidate substance (ACP52CGK) also did not show immunogenicity in humans ( Fig.21 ).

[0102] Repeated toxicity tests of ACP52CGK in vivo administration were performed with female and male mice. As a preliminary experiment, when 100mg / kg and 1000mg / kg of ACP52C were repeatedly administered twice, all mice survived and no hepatotoxicity or adverse reactions to major organs were observed. As a comprehensive experiment, after ACP52CGK was intravenously infused into solvent groups and high-dose groups (100mg / Kg) of 6 male and female mice at intervals of 3 days for 28 days, weight changes, water intake, and major histological analysis of organs, organ weights, hematological tests, and blood biochemical tests were analyzed. In short, no abnormal findings were detected ( Fig.22a to e, 23 and 24, and Tables 1, 2, 3 and 4).

[0103]

Table 1

[0104] Table 1. Absolute organ weights (g) of mice treated or not treated with ACP52CGK

[0105]

[0106]

Table 2

[0107] Table 2. Relative organ weights (%) of mice treated or not treated with ACP52CGK

[0108]

[0109]

Table 3

[0110] Table 3. Hematology of mice treated or not treated with ACP52CGK

[0111]

[0112]

Table 4

[0113] Table 4. Serum biochemistry of mice treated or not treated with ACP52CGK

[0114]

[0115] In order to investigate whether the efficacy of ACP52CGK tested on cancer cell lines and xenograft mouse models can be equally applied to actual clinical practice, the efficacy of ACP52CGK was evaluated in cells cultured from tumor tissues in the fresh state of breast cancer patients. As a preliminary result, it was confirmed that ACP52CGK induced cell death at GI50: about 2 μM, which is similar to those in cancer cell line experiments ( Fig.25a To d). Therefore, the frozen cancer tissue of breast cancer patients was thawed and primary cultured. When the effect of ACP52CGK was analyzed for cells obtained therefrom, it was also confirmed that cell death was induced at GI50: about 2 μM ( Fig.26a to d).

[0116] [Example 5] Analysis of the cause of resistance of cells showing resistance to CP2c targeting peptide-fatty acid conjugate

[0117] As a result of analyzing the efficacy of ACP52CGK in each generation of cells primary cultured from PDX (patient-derived xenograft) tumor tissues, it was confirmed that there was no significant difference in sensitivity to ACP52CGK among cells derived from the same origin ( Fig.27aHowever, primary cultured cells from some PDX tumor tissues showed resistance to ACP52CGK ( Fig.28a To b).

[0118] Cells (mainly lung cancer cell lines) that show resistance to ACP52CGK tend to show low expression levels of MDM2p90 and relatively high expression of MDM2p60. In fact, it was confirmed by immunoblotting that the expression of YY1 was reduced in three lung cancer cell lines (A549, PC9, KCL22) treated with ACP52C, but the expression of p53, p63 and p73 did not show any changes. The expression of MDM2 (p90 and p60) was not reduced in these cell lines. Since MDM2p60 is known to be a protein in which the C-terminal region of MDM2p90 containing the sites phosphorylated by ATM (S386, S395 and S407) is deleted, it is speculated that MDM2 degradation is not properly regulated by ACP52C treatment ( Fig.29 , Fig.30a to c).

[0119] Accordingly, by treating with doxycycline for 3 weeks, MDM2p90 was continuously overexpressed, and interestingly, by immunoblotting, the expression of p53 protein and YY1 in CP2c transcriptional activity-dependent and -independent marker proteins decreased week by week. At the same time, since cell death induced by ACP52C treatment was analyzed by MTT assay, it was confirmed that cell death was induced from the second week. Therefore, it was proved that the low expression of MDM2p90 in lung cancer cell lines was the cause of ACP52C resistance ( Fig.31a To b, Fig.32a to d).

[0120] Two models have been proposed that the production of MDM2p60 is due to caspase 2 cleavage or alternative splicing of MDM2p90. When RT-PCR was performed using a primer set capable of detecting alternative spliced ​​forms of MDM2 in a variety of cancer cell lines, no lung cancer cell-specific spliced ​​forms were found. In addition, when nucleotide sequence analysis of genomic DNA was tested after PCR cloning to determine whether MDM2p60 is produced from mRNA that is truncated due to the presence of SNPs in the MDM2 gene specifically for lung cancer cell lines, no lung cancer cell-specific SNPs were observed ( Fig.33a to d).

[0121] Meanwhile, when a caspase 2 inhibitor (AC-VDVAD-CHO) was treated to identify the phenomenon occurring due to the high activity of caspase 2 particularly in lung cancer cells, it was found by immunoblotting that the protein amount of MDM2p90 increased, while the protein amount of MDM2p60 decreased according to the treatment with the caspase 2 inhibitor. Therefore, we concluded that the overexpressed MDM2p60 form in lung cancer cells is the result of cleavage of MDM2p90 by caspase 2. Based on these results, the cell death induction effect was analyzed by treating cells (A549 and PDX cells, breast F0-JOS) that showed resistance to ACP52C with a combination of a caspase 2 inhibitor and ACP52C. As a result, efficacy ( ) was observed at around GI50 of about 1 μM in the group treated with a combination of a caspase 2 inhibitor and ACP52C. Fig.34a To d, Fig.35 ).

[0122] Therefore, the ACP52CGK peptide has improved in vivo stability compared to the existing ACP52C peptide, and shows growth inhibition / cell death as in ACP52C in all cancer cells, but has no significant effect on normal cells. In addition, it was confirmed that cancer cells that show resistance to ACP52C can be killed by combined treatment of ACP52C and caspase 2 inhibitors. <110> Industry-University Cooperation Foundation HanyangUniversity <120> Anticancer peptides targeting CP2c <130> X20U10C0057 <150> KR 10-2019-0038023 <151> 2019-04-01 <150> KR 10-2019-0085790 <151> 2019-07-16 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Peptides targeting CP2c <400> 1 Tyr Pro Gln Arg 1 <210> 2 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Peptides targeting CP2c <400> 2 Asn Tyr Pro Gln Arg Pro 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Cell Penetrating Peptides <400> 3 Cys Arg Gly Asp Lys Gly Pro Asp Cys 1 5

Claims

1. A CP2c targeting peptide-fatty acid conjugate, comprising: a transcription factor CP2c targeting peptide consisting of an amino acid sequence as shown in SEQ ID NO: 2; a linker peptide bonded to the C-terminus of the CP2c targeting peptide, wherein the linker peptide consists of an amino acid sequence as shown in GGKGG; a cell penetrating peptide (CPP) bonded to the C-terminus of a linker peptide bonded to the C-terminus of the CP2c targeting peptide, wherein the cell penetrating peptide (CPP) consists of an amino acid sequence as shown in SEQ ID NO: 3; and a C-terminus of a lysine (Lys, K) functional group conjugated to the linker peptide. 16 Palmitoleic acid, wherein the C 16 Palmitoylic acid is bonded to glutamic acid (Glu, E), wherein the gamma carbon carboxyl group of the glutamic acid is bonded to the functional group of lysine (Lys, K) of the linker peptide.

2. The CP2c targeting peptide-fatty acid conjugate according to claim 1, wherein the N-terminus and / or C-terminus of the peptide of the CP2c targeting peptide-fatty acid conjugate is modified to improve stability.

3. The CP2c targeting peptide-fatty acid conjugate according to claim 2, wherein the N-terminus of the peptide of the CP2c targeting peptide-fatty acid conjugate is modified with an acetyl group, and the C-terminus of the peptide is modified with an amide group.

4. Use of the CP2c targeting peptide-fatty acid conjugate according to any one of claims 1 to 3 for the manufacture of a medicament for treating cancer, wherein the cancer is selected from glioblastoma, breast cancer, leukemia, hepatocellular carcinoma, lymphoma, colon cancer, melanoma, pancreatic cancer, prostate cancer, gastric cancer and ovarian cancer.

5. Use of the CP2c targeting peptide-fatty acid conjugate and AC-VDVAD-CHO according to any one of claims 1 to 3 for the manufacture of a medicament for treating cancer, wherein the cancer is lung cancer, breast cancer, hepatocellular carcinoma or colon cancer that is resistant to the CP2c targeting peptide-fatty acid conjugate.

Images