KR102465431B1 - Cell Penetrating Peptide-linker-anti-cancer drug and Pharmaceutical composition for prevention or treatment of colorectal cancer containing the same - Google Patents

Abstract

The present invention relates to a cell-penetrating peptide-linker-anticancer agent conjugate for the treatment of colorectal cancer and a pharmaceutical composition for preventing or treating colorectal cancer comprising the same, specifically, in a form in which octaarginine (R8) CPP is combined with oxaliplatin through a heterogeneous linker As a binding agent, it is effective in the treatment of colorectal cancer since it has a strong cytotoxic effect on colorectal cancer cells and efficient cellular uptake.

Description

Cell penetrating peptide-linker-anticancer drug conjugate and a pharmaceutical composition for preventing or treating colorectal cancer comprising the same

The present invention relates to a cell-penetrating peptide-linker-anticancer agent conjugate for the treatment of colorectal cancer and a pharmaceutical composition for preventing or treating colorectal cancer comprising the same, specifically, in a form in which octaarginine (R8) CPP is combined with oxaliplatin through a heterogeneous linker As a binding agent, it is effective in the treatment of colorectal cancer since it has a strong cytotoxic effect on colorectal cancer cells and efficient cellular uptake.

Colorectal cancer is the second most common cancer in Korea after gastric cancer and the third most common cancer worldwide. The most common sites of metastasis are the liver, lungs, peritoneum, and bone in that order. Despite such a poor prognosis, there is no clear treatment other than surgery. Recently, intraperitoneal chemotherapy has been helping to improve the survival rate, and currently used anticancer drugs are Mitomycin-C and Oxaliplatin. However, various side effects (leukopenia, anemia, thrombocytopenia, cardiac, lung, and renal toxicity) have been reported with such intraperitoneal chemotherapy, and some studies have reported that it does not lead to significant survival rates. For this reason, peritoneal metastasis is the reason that the permeability is low due to the peritoneal-vascular barrier, and therefore it is important to develop an effective drug delivery system to solve this problem.

In this regard, cell penetrating peptides have been widely used in drug delivery systems. CPPs are short amino acid sequences usually composed of 30 amino acids, which can be derived from natural peptides or proteins (eg, tart peptide, HIV-1 protein) or obtained from organic synthesis (eg, polyarginine).

Meanwhile, Oxaliplatin is a third-generation Pt-based drug approved by the FDA in 2002. Oxaliplatin is the third and second most common cause of death in men and women worldwide, and is widely used in chemotherapy mainly for colorectal cancer (CRC), a major cause of death. Despite its widespread clinical use, oxaliplatin has potential side effects such as neurotoxicity, diarrhea, nausea, and nephrotoxicity. The half-life of oxaliplatin in plasma was only 14.1 minutes, indicating unfavorable pharmacokinetics. In addition, the intracellular uptake of oxaliplatin is very low because oxaliplatin has low cell membrane permeability and is a substrate for the efflux pump present in the cell membrane. As a result, it is necessary to administer oxaliplatin for a long time, which may result in drug resistance to cancer and adverse drug reactions in patients. Therefore, there is a need to develop a new strategy for delivering oxaliplatin into cells.

Under this background, the present inventors use a cell-penetrating peptide (CPP), a nanomaterial, for the treatment of colorectal cancer that can lower toxicity and maximize the effect, and a cell-penetrating peptide-linker-anticancer agent conjugate comprising the same, and colorectal cancer prevention or An attempt was made to develop a therapeutic pharmaceutical composition.

Korean Patent Registration 10-1759261 (2017.07.12) Korean Patent Publication 10-2019-0083478 (2020.01.07)

The present inventors synthesized an oxaliplatin conjugate (CPP-Oxal) based on octaarginine (R8) CPP. Specifically, as conjugates of octaarginine (R8) CPP combined with oxaliplatin through a heterogeneous linker, the present inventors have efficiently It was experimentally confirmed that the cell uptake and cytotoxic effect on colon cancer cells were strong, and the intracellular uptake mechanism and intracellular localization of the conjugate were also confirmed. By doing so, it was found that the conjugate according to the present invention is effective for the treatment of colorectal cancer, and thus the present invention has been completed.

Accordingly, an object of the present invention is to provide a cell-penetrating peptide-linker-anticancer agent conjugate for the treatment of colorectal cancer and a pharmaceutical composition for preventing or treating colorectal cancer comprising the same.

In order to solve the above object, the present invention for use in the prevention or treatment of colorectal cancer, (a) octaarginine-K- mercaptoacetyl (RRRRRRRR-K-mercaptoacetyl); Or octaarginine-K-mercaptoacetyl (RRRRRRRR-K-mercaptoacetyl) labeled with FITC, a fluorescent substance; any one cell penetrating peptide (CPP) selected from; (b) a linker represented by the following formula (I) for binding to the cell-penetrating peptide; And (c) provides a cell-penetrating peptide-linker-anticancer agent conjugates comprising an anticancer agent binding to the linker.

[Formula I]

In addition, the present invention comprises the steps of (i) adding an anticancer agent to the linker represented by Formula I; and (ii) octaarginine-K-mercaptoacetyl (RRRRRRRR-K-mercaptoacetyl) labeled with octaarginine-K-mercaptoacetyl (RRRRRRRR-K-mercaptoacetyl) or FITC in the product of step (i) It provides a method for preparing cell-penetrating peptide-anticancer agent conjugates, comprising the step of adding and synthesizing one cell-penetrating peptide (CPP).

In addition, the present invention provides a pharmaceutical composition for preventing or treating colorectal cancer comprising the conjugate.

The conjugate according to the present invention can be used as a new drug carrier-anticancer agent to replace the existing anticancer agent, and is a promising material for the treatment of colorectal cancer, which can increase the survival rate and improve the quality of life from colorectal cancer. In addition, the drug delivery system as in the present invention is a high value-added industry, recording double-digit or more growth rates every year, and can contribute to securing original technology and developing new biopharmaceuticals.

Figure 1 shows (a) the structure of oxaliplatin with a bidentate ligand, DACH is stable and essential for providing cytotoxicity, oxalate is relatively unstable and can be replaced with other similar compounds, (b) Intrastrand Pt It shows the formation structure of -GG diadduct, and DACH is involved in Pt-DNA adduct.
Figure 2 shows the results of MTT analysis, showing the cytotoxic effects of CPP-Oxal and oxaliplatin in (a) SW480, (b) SW620 and (c) HCT116 cell lines (the curves in each figure use polynomial regression analysis); (d) Intracellular Pt content (ng per million cells) of CPP-Oxal and oxaliplatin was measured in HCT116 cells using ICP-MS.
3 is a result of cell uptake and colocalization analysis of F-CPP-Oxal (10 μM) in HCT116 cells. (a) green fluorescence of F-CPP-Oxal, (b) red fluorescence of mitotracker, (c) merged images ((a), (b) and Hoechst 33342 (blue, 8.11 µM) from Pearson's r is 0.953 (scale bar: 20 μm)), and (d) is a scatter plot consisting of the green and red pixel intensities of each fluorescence image. The green intensity in (a) represents the x-coordinate, (b) the red intensity of the y-coordinate, the frequencies of pixels appearing in the scatterplot are distinguished by different colors, and (d) the numbers next to the bars represent the frequency levels.
4 shows (a) Mitotracker (10 μM) (b) Lysotracker (10 μM) and (c) Rhodamine B-dextran (10 μM). Pearson's r is in each merged image, (d), (e) and (f) the relative intensity of each fluorescence signal along the arrows, Hoechst 33342's fluorescence is omitted for clarity (scale bar: 20 μm).
5 is a result of investigating the cellular uptake mechanism of F-CPP-Oxal using an endocytic inhibitor, (a) confocal microscopy images of HCT116 cells: (i) control and (ii) clathrin-mediated endocytosis, ( Showing inhibition of iii) caveola-mediated endocytosis and (iv) macrocytosis, nuclei were stained using Hoechst 33342 (scale bar: 20 μm), and (b) F-CPP- under different inhibitory conditions. The relative fluorescence intensity of Oxal was compared.
6 shows (a) the percentage change in body weight of each group, (b) the percentage change in tumor volume, and (c) the tumor inhibition rate (TIR) of each group calculated and shown (d) on days 0, 10 and 20 An image of a mouse with a tumor is shown. The tumor can be seen in the flank of the mouse, and (e) shows an image of a tumor excised after sacrificing the mouse on day 20.

Hereinafter, the present invention will be described in more detail through an embodiment.

The present inventors have experimentally confirmed that, in the case of a conjugate in which octaarginine (R8) CPP is combined with oxaliplatin through a heterogeneous linker, efficient cellular uptake and strong cytotoxic effect on colorectal cancer cells, the intracellular uptake mechanism of the conjugate And intracellular localization was also confirmed, and by confirming that the conjugate had an anticancer effect when administered to mice with tumors, it was found that the conjugate according to the present invention is effective in treating colorectal cancer, thereby completing the present invention.

Cell penetrating peptide (CPP) is a short chain consisting of 8 to 30 amino acids and is relatively simple, easy to synthesize, and has a good advantage as a drug delivery system because of its low toxicity. Therefore, the development of a linker that binds oxaliplatin and CPP used for the treatment of colorectal cancer peritoneal metastasis is of great significance.

Accordingly, the present invention provides (a) octaarginine-K-mercaptoacetyl (RRRRRRRR-K-mercaptoacetyl); Or octaarginine-K-mercaptoacetyl (RRRRRRRR-K-mercaptoacetyl) labeled with FITC, a fluorescent substance; any one cell penetrating peptide (CPP) selected from; (b) a linker represented by the following formula (I) for binding to the cell-penetrating peptide; And (c) provides a cell-penetrating peptide-linker-anticancer agent conjugates comprising an anticancer agent binding to the linker.

[Formula I]

The FITC-labeled octaarginine (R8) is a FITC-amh-octaarginine-K-mercaptoacetyl group (FITC-amh-RRRRRRRR-K-mercaptoacetyl). The anticancer agent is oxaliplatin. The binder according to the present invention may be represented by the following Chemical Formula II.

[Formula II]

The octaarginine may improve intracellular permeability by binding to the negatively charged material of the cell membrane.

In addition, the present invention provides ((i) adding an anticancer agent to the linker represented by Formula I; and (ii) octaarginine-K-mercaptoacetyl (RRRRRRRR-K-mercaptoacetyl) to the product of step (i). Or FITC-labeled octaarginine-K-mercaptoacetyl (RRRRRRRR-K-mercaptoacetyl) comprising the step of synthesizing by adding any one cell penetrating peptide (CPP) selected from, cell penetrating peptide-anticancer agent conjugates (conjugates) It provides a manufacturing method of

In addition, there is provided a pharmaceutical composition for preventing or treating colorectal cancer comprising the conjugate according to the present invention.

As used herein, the term “prevention” refers to any action that suppresses or delays the progression of an allergic disease by administration of the composition of the present invention.

As used herein, the term “treatment” refers to any action in which symptoms of allergic diseases are improved or beneficially changed by administration of the composition of the present invention.

The composition of the present invention may further include pharmaceutically acceptable additives, and pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, Mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, Sucrose, dextrose, sorbitol and talc and the like may be used. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

In addition, the composition of the present invention may be administered in various oral and parenteral formulations during actual clinical administration. When formulated, commonly used fillers, extenders, binders, wetting agents, disintegrants, diluents or excipients such as surfactants It can be prepared using

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and may be prepared by mixing excipients such as starch, calcium carbonate, sucrose, lactose or gelatin. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. Liquid formulations for oral use include suspensions, solutions, emulsions and syrups, and various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. in addition to commonly used simple diluents such as water and liquid paraffin. .

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

On the other hand, injections may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives.

In addition, the therapeutic composition of the present invention may contain any physiologically acceptable carrier, excipient or stabilizer (Remington: The Science and Practice of Pharmacy, 19th Edition, Alfonso, R., ed, Mack Publishing Co. (Easton, PA). : 1995)) may be additionally included. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphoric acid, citric acid and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG).

The dosage for the human body of the pharmaceutical composition of the present invention may vary depending on the subject's age, weight, sex, dosage form, health status and disease level, and is generally 0.01 - 100 mg/kg/day, preferably 0.1 to 20 mg/kg/day, and more preferably 1 to 5 mg/kg/day, but is not limited thereto. Administration may be administered once a day or may be administered in several divided doses, thereby not limiting the scope of the present invention.

In the present invention, a CPP oxaliplatin conjugate (CPP-Oxal) was prepared to overcome the problems associated with the systemic drug delivery of oxaliplatin, and the linker according to the present invention was optimized to bind CPP to the drug product. The in vitro cytotoxicity of CPP-Oxal against various colon cancer cells was confirmed using the MTT assay, and the IC50 values were significantly lower than the parent drug, indicating enhanced cellular uptake of oxaliplatin through CPP. Efficient cell uptake of CPP-Oxal was confirmed based on confocal microscopy and ICP-MS studies, and the cellular localization and internalization mechanism of CPP-Oxal were further investigated. In addition, tumor growth inhibition was more pronounced with CPP-Oxal than with oxaliplatin, confirming that CPP-Oxal exhibited enhanced antitumor effect in vivo on xenografts.

Hereinafter, preferred examples are presented to help the understanding of the present invention, but the following examples are only illustrative of the present invention and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Experimental example>

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. General synthesis method

Di-tert-butylmalonate, sodium hydride (NaH), N-(3-bromopropyl)phthalimide, hydrazine monohydrate, N-methylmorpholine (NMM), trifluoroacetic acid (TFA), tri Ethylsilane, dichloro(1,2-diaminocyclohexane)platinum(II), phosphate buffered saline and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were purchased from Sigma-Aldrich Korea (Yongin, Korea). N-β-maleimidopropyloxysuccinimide ester (BMPS) and oxaliplatin were purchased from Thermo Fisher Scientific (Rockford, Illinois, USA) and Alfa Aesar (Korea Fisher Science Co., Ltd.), respectively. Octaarginine (R8) CPP, RRRRRRRR-K-mercaptoacetyl (RRRRRRRR-K-mercaptoacetyl), FITC-amh-RRRRRRRR-K-mercaptoacetyl (FITC-amh-RRRRRRRR-K-mercaptoacetyl), where amh is 6 -Aminohexanoic acid) was synthesized by Peptron (Daejeon, Korea). A lysine residue was added to CPP for attachment of the mercaptoacetyl group. Mass spectra were obtained using the high-resolution fast atomic bombardment (HR-FAB-MS) or high-resolution electrospray ionization (HR-ESI-MS) methods of the Korea Basic Science Institute (KBSI) (Ochang, Korea). For the analysis of high molecular weight compounds, matrix-assisted laser desorption/ionization mass spectroscopy (MALDI-TOF MS) data were obtained from BIONEER (Daejeon, Korea).

Medium pressure liquid chromatography (MPLC) was performed using a CombiFlash RF+ Lumen instrument (MA, USA) with integrated UV and ELS detectors.

Analytical reversed-phase high-performance liquid chromatography (RP-HPLC) and preparative RP-HPLC were performed using a Shimadzu SPD-20A UV detector and a Shimadzu LC-20AR series pumping system (Shimadzu Corporation, Kyoto, Japan). The Vivaspin 2 Hydrosart® (HY) ultrafiltration column (MWCO = 2 kDa) was manufactured by Sartorius Stedim Lab Ltd. (Stonehouse GL10 3UT, UK).

2. Synthetic Characteristics

2-1. di-tert-butyl 2-(3-aminopropyl)malonate [ Di-tert-butyl 2-(3-aminopropyl)malonate (1) ]

Compound 1 is described in Bioconjugate Chem. 20 (2009) 1869-1878 was synthesized similarly to the method described. Briefly, di-tert-butylmalonate was first conjugated to 3-bromopropyl phthalimide to give di-tert-butyl 2- (3-phthalimidopropyl-malonate). Subsequently, compound 1 was obtained by deprotection of di-tert-butyl 2-(3-phthalimidopropyl-malonate) using hydrazine monohydrate (25 mmol) in absolute ethanol. A quantitative yield was obtained. ¹ H NMR (CDCl ₃ )δ (ppm): 3.17 (t, 1H, R—CH), 2.71 (t, 2H, NH ₂ CH ₂ ), 1.85 (m, 2H, CH ₂ CH), 1.62 (m, 2H, CH ₂ CH ₂ CH ₂ ), 1.48(s, 18H, t-Bu); ¹³ CNMR(CDCl ₃ )δ (ppm): 168.52 (C(O)-t-Bu), 81.81 (C quaternary, t-Bu), 53.41 (CH ₂ NH), 39.29 (R-CH, malonate), 29.70 (NH CH ₂ CH ₂ ), 27.78 (CH ₃ ,t-Bu), 25.69 (CH ₂ CH). HR-FAB-MS:exact m/z calculated for C ₁₄ H ₂₇ NO ₄ 273.1916; found 274.2016[M+H] ⁺ .

2-2. di-tert-butyl 2-(3-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)propyl)malonate [ Di-tert-butyl 2-(3-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido) propyl)malonate (2) ]

NMM (0.34 mmol) was added to a solution of compound 1 (0.47 mmol) in dry CH ₂ Cl ₂ under nitrogen gas. After the reaction mixture was cooled to 0° C., BMPS (0.22 mmol) was added at the same temperature. The reaction mixture was left overnight with stirring under a nitrogen atmosphere. After completion the reaction mixture was cooled to 5 °C in a water bath and the pH was lowered to 3-4 pH level by addition of 1N HCl. The layers were separated and the aqueous layer was extracted 3 times with CH ₂ Cl ₂ . The organic layer was dried over MgSO ₄ and evaporated to give the desired. The final product was purified by MPLC on a simple silica gel column using a gradient system (95% CH ₂ Cl ₂ /5% MeOH) with an integrated ELS and UV detector. A quantitative yield was obtained. ¹ H NMR (CDCl ₃ )δ (ppm): 6.71 (s, 2H, CHCH), 3.84 (t, 2H, NCH2), 3.22(q,2H,NHCH2), 3.1(t,1H,R-CH), 2.51(t, 2H, NCH2CH2), 1.80 (m, 2H , CH2CH), 1.44( _m , 2H , _CH2CH2CH2 ), 1.48(s,18H, _{t -} Bu); ¹³ CNMR(CDCl ₃ )δ (ppm): 172 (CH ₂ C ONH), 170 ( C ON C O), 169 ( C OCH C O), 135 ( C H C H), 81.81 (C quaternary, t- Bu), 53.41 (CO C HCO), 39 (NCH ₂ ), 35 (NHCH ₂ ), 34 (N C H ₂ CH ₂ ), 27.78 (CH ₃ ,t-Bu), 26.75 (NHCH ₂ ), 25.69 ( CH C H ₂ ). HR-FAB-MS: exact m/z calculated for C ₂₁ H ₃₂ NO ₂ 424.2290; found 425.2290 [M+H] ⁺ .

2-3. 2-(3-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)propyl) malonic acid [ 2-(3-(3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)propyl)malonic acid (3) ]

Compound 2 (0.14 mmol) was acid decomposed by stirring in trifluoroacetic acid (2.4 mmol) and dry CH ₂ Cl ₂ in the presence of triethylsilane (1.0 mmol). After reacting at room temperature for 4 hours, the solvent was removed by evaporation, and the crude product was washed with Et ₂ O and used further without purification. A quantitative yield was obtained. ¹ H NMR (CDCl ₃ )δ (ppm): 6.86 (s, 2H, CHCH), 3.8 (t, 2H, NCH ₂ ), 3.3(m,3H,NHC H ₂ ,RC H ), 2.5(t,2H) ,NCH ₂ C H ₂ ), 1.77 (s, 2H, CHC H ₂ ), 1.46(m,2H,NHCH ₂ C H ₂ ); ¹³ CNMR (CDCl ₃ )δ (ppm): 173.4 (NHCO), 172.5 ( C ON C O), 172.5 ( C OCH C O), 134.4 ( C H C H), 39 (CO C HCO), 34.8 (NCH ₂ ), 34.5 (NHCH ₂ ), 26.8 (NCH ₂ CH ₂ ), 26.6 ( C HCH ₂ ), 26.23 (NHCH ₂ CH ₂ ). HR-FAB-MS: exact m/z calculated for C ₁₃ H ₁₆ N ₂ O ₇ 312.1033; found 313.1033 [M+H] ⁺ .

2-4. cis-Cyclohexane-1,2-diaminoplatinum(II)-[2-(3-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1yl)propane-mido) ) propyl) malonate cis-Cyclohexane-1,2-diaminoplatinum(II)-[2-(3-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1yl)propane-mido)propyl)malonate] ( 4)

After reacting for 4 hours, the solvent is removed by evaporation and the product is used as Et2O and further used without purification. A quantitative yield was shown. Initially, [DACHPt(OH ₂ ) ₂ ] ²⁺ ·2NO ^3- is Chemother. Res. Pract. 2012 (2012) 1-10 was synthesized according to the literature. [DACHPt/(Cl) ₂ ] was suspended in AgNO ₃ aqueous solution and stirred at room temperature in the dark for 24 hours. After stirring, the AgCl precipitate was filtered through a 0.22 μm filter by centrifugation at 3000 rpm for 15 min. [DACHPt(H ₂ O) ₂ ] ²⁺ ·2NO ^3- was stirred in deionized water and heated to 50°C. The solution was cooled to room temperature and then compound 3 was added. The reaction mixture was adjusted to pH 5.5 with 0.1 M Na ₂ CO ₃ solution. The reaction was carried out in the dark for 4 h, the reaction mixture was freeze-dried and min using H ₂ O/MeOH (80:20) mobile phase on a C18 column (220 nm, 2 ml min-1, tR = 1.50 min). Purification by preparative RP-HPLC gave compound 4 as a sticky solid. A quantitative yield was obtained. ¹ H NMR (D ₂ O)δ (ppm): 6.7 (s, 2H, CHCH), 3.6 (t, 2H, NCH ₂ ), 3.4(m,3H,NHC H ₂ ,RC H ), 2.5 (m, 2H,NCH ₂ C H ₂ ), 1.77(s,2H,CHC H ₂ ), 1.46(m,2H,NHCH ₂ C H ₂ ). HR ESI-MS: exact m/z calculated for C ₁₉ H ₂₈ N ₄ O ₇ NaPt 642.1503; found 642.1511. ¹ H NMR (D ₂ O)δ (ppm): 6.7 (s, 2H, CHCH), 3.6 (t, 2H, NCH ₂ ), 3.4(m,3H,NHC H ₂ ,RC H ), 2.5 (m, 2H,NCH ₂ C H ₂ ), 1.77(s,2H,CHC H ₂ ), 1.46(m,2H,NHCH ₂ C H ₂ ).

2-5. Synthesis of CPP Conjugates

To evaluate the biological activity, two types of conjugates were synthesized: CPP-oxaliplatin conjugate (CPP-Oxal) and FITC-labeled CPP-oxaliplatin conjugate (F-CPP-Oxal). Initially, compound 4 was gently dissolved in PBS buffer with TCEP (26 mM) and stirred for 10 min. Consequently, CPP, octaarginine were added and the reaction mixture was stirred at room temperature for 4 hours. The crude material was purified by an ultrafiltration column, Vivaspin 2 Hydrosart® (HD) for 30 minutes, re-purified with DI H ₂ O at 3500 rpm for an additional 30 minutes, then lyophilized to give a white solid. . A conjugate comprising FITC-CPP was also prepared by the same synthetic route. Both products were analyzed by MALDI-ToF mass spectrometer (FIG. 5). MALDI-TOF MS: CPP-Oxal = 2088.1600 by exact m/z ; found 2113.4631 [M+Na+H] ⁺ , and for F-CPP-Oxal = 2590.1600; It was calculated as found 2726.8453 [M+3-HPA matrix] ⁺ .

3. Biological analysis

3-1. ingredient

Dulbecco's Phosphate Buffered Saline (DPBS, pH 7.4), Roswell Park Memorial Institute (RPMI) 1640, High Glucose Dulbecco's Modified Eagle's Medium (DMEM), Fetal Bovine Serum (FBS), and Trypsin/EDTA from Gibco™ Thermo Fischer It was purchased from Scientific (Loughborough, UK). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was purchased from Sigma-Aldrich (Korea). Specific organelle markers, mitotracker™ Red CMXRos, lysotracker Red DND-99, Hoechst 33342, and rhodamine B-dextran (MW 10,000) were obtained from Thermo Fisher Scientific (Invitrogen, Oregon, USA). Imipramine hydrochloride, chlorpromazine hydrochloride and methyl-β-cyclodextrin were purchased from Sigma-Aldrich (Korea). All aqueous solutions were prepared using Milli-Q purified water (18.2 MΩ).

3-2. cell culture

HCT116, HeLa, SW480 and SW620 cells were purchased from the Korean Cell Line Bank (Seoul, Korea). Cells were cultured in DMEM at 37° C. in 10% (v/v) FBS with a humidified 5% CO ₂ environment and penicillin.

3-3. Cytotoxicity measurement using MTT assay

SW480, SW620 and HCT116 cells (4.5×10 ³ cells per well) were seeded in 96 well plates. The medium was changed to serum-free RPMI 1640 within 24 hours, and after an additional 24 hours, the cells were treated with different concentrations (0-40 μM) of oxaliplatin and CPP-Oxal. The treated cells were incubated for 48 h, washed with cold PBS, and then exposed to MTT together with the medium for 4 h. The medium was changed to DMSO and the dissolved formazan dye was quantified by measuring absorbance spectrophotometry at 540 nm using a plate reader. Untreated cells were used as controls. The percent viability of cells was calculated as the percentage of fluorescence intensity formed in the experimental wells compared to the control wells. Each experiment was performed three times.

3-4. Confocal microscopy studies of cellular uptake, localization and uptake mechanisms

HeLa and HCT116 cells (1 x 10 ⁵ cells per well) were seeded on 35 mm cover glass bottom dishes and 4-well chamber cell culture slides (SPL Ltd., Pocheon, Korea) and cultured for 24 hours. The old medium was removed and the cells were pretreated with serum-free RPMI 1640 containing 10 μM of F-CPP-Oxal at 37° C. for 30 min, followed by mitotracker (200 nM), lysotracker (150 nM), They were stained with specific organelle markers such as rhodamine B-dextran (1 mg/mL, 1 hour co-incubation time) and Hoechst 33342 (8.11 μM). Cells were washed three times with serum-free RPMI 1640 to remove uninternalized samples and incubated for 10 min with Hoechst33342 using a confocal laser scanning microscope (Carl Zeiss LSM 710) equipped with an oil immersion lens (NA 1.30, 100x). and analyzed. FITC was excited at 488 nm with an argon laser and fluorescence was observed at 500-530 nm using an emission band filter. Mitotracker, rhodamine B-dextran and lysotracker were excited at 543 nm with a HeNe laser and fluorescence was observed at 600-700 nm using an emission filter. The resulting fluorescence images were used to identify the degree of colocalization using the line intensity profile diagram and Pearson's correlation coefficient (Pearson'sr). The line intensity profile was analyzed using Zen software (Carl Zeiss, Germany). Pearson's r and scatterplots were obtained using ImageJ software (https://imagej.nih.gov/ij) installed with Just Another Colocalization Plugin (JACoP). The region of interest (ROI) was the entire area of the confocal microscopy image. To investigate the cellular uptake mechanism, HCT116 cells were pretreated with various membrane entry inhibitors such as chlorpromazine hydrochloride (30 μM), methyl-β-cyclodextrin (10 mM) and imipramine hydrochloride (5 μM) in serum-free RPMI 1640 medium. Post-treatment with F-CPP-Oxal (10 μM) for 30 min at 37 °C for an additional 30 min. Fluorescence intensity inside the cells was quantified using ImageJ software. Ten cells of similar size were selected for fluorescence intensity analysis. All analyzes were performed in triplicate.

3-5. Quantification of cell uptake by ICP-MS

The intracellular Pt metal content was measured by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) (Elan DRC II, PerkinElmer SCIEXTM) of the Korea Institute of Basic Science (Seoul, Korea). HCT116 cells were grown well in 6 well-plates and then treated with 20 mM CPP-Oxal and oxaliplatin for 1 hour. The old medium was removed, the cells were washed with PBS (x3), isolated with trypsin, and counted with a hemocytometer. Cells were centrifuged at 7000 rpm for 5 min to obtain a cell pellet. Cell pellet in HNO ₃ aqueous solution (70%): H ₂ O2 It was further suspended in (30%) (8: 1) solution, sonicated until a clear solution appeared, and then analyzed using ICP-MS. Cell uptake experiments were performed in triplicate and each experimental slot was used for ICP-MS analysis.

3-6. In vivo mouse study

Female BALB/c nude mice (6 weeks old) were purchased from Korea Orient Bio (Seongnam, Korea) and placed under standard living conditions. To generate xenografts, HCT116 colon cancer cells were injected subcutaneously into the flanks of female nude mice (2 x 10 ⁷ cells/200 μL). When the tumors grew and reached 100 mm ³ in size, mice were started with a series of injections and the first injection was considered day 0. Three groups of mice (5 mice per group) received subcutaneous injections on days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20. CPP-Oxal at 0.083 mg/mL, oxaliplatin and PBS were used as controls. Before the drug injection procedure, each mouse was weighed every other day. For the calculation of the tumor volume ratio (%), the tumor volume of each mouse on day 0 was set to 100%, and the rate of change in tumor volume every other day was calculated relative to the volume on day 0. Body weight ratio (%) was calculated in the same way. On day 20, mice were sacrificed by cervical dislocation and tumors were dissected, weighed and photographed. The Tumor Inhibition Ratio (TIR) was calculated using the following equation:

* TIR (%) = 100 × (mean tumor weight of control group - mean tumor weight of experimental group)/mean tumor weight of control group

All experiments on animals were performed at Soonchunhyang University Animal Experiment Center, and the research procedures were approved by the Animal Ethics Committee of Soonchunhyang University in accordance with the standards for handling and use of laboratory animals.

Experimental Example 1. Synthesis of oxaliplatin conjugate and its linker

The structure of oxaliplatin has a square plane with a central Pt atom to which two bidentate organic ligands are coordinated (Fig. 1a). One of the ligands 1,2-diaminocyclohexane (DACH) acts as a stable carrier ligand and is essential for the intrinsic cytotoxicity of oxaliplatin. Compared to the ammonia ligands of other Pt compounds such as cisplatin and carboplatin, bulky DACH ligands are more soluble and form larger distorted DNA adducts in cells (Fig. 1b). This results in severe DNA damage that cannot be repaired by DNA mismatch repair or nucleotide excision repair systems, whose activity greatly increases after Pt drug administration. Thus, DACH contributes to the enhancement of the cytotoxicity of oxaliplatin to cisplatin or carboplatin-resistant cells. In contrast, the oxalate ligand of oxaliplatin is highly unstable, so oxaliplatin can be hydrolyzed under biological conditions to generate active species. Since oxalate is not critical for the pharmacological activity of oxaliplatin, other moieties can be substituted for conjugation to the delivery vector while keeping DACH intact. In addition, CPP or amino acid residues cannot bind directly to the Pt atom like oxalate, indicating the need for a suitable linker for conjugating CPP and oxaliplatin simultaneously. Therefore, we attempted to design and synthesize a special linker that binds to CPP octaarginine (R8) at one end and an oxaliplatin derivative at the other end (Scheme 1).

[Scheme 1]

Scheme 1 is (a) BMPS, NMM, CH ₂ Cl ₂ (rt, overnight), (b) TFA, DCM, triethyl silane (4h, rt), (c) [DACHPt(OH ₂ ) ₂ ] ²⁺ In 2NO ^{3- ,} Na ₂ CO ₃ , H ₂ O (2h, rt), (d) CPP (R = R8KCOS or FITC-amh-R8KCOS), PBS buffer (pH = 7), TCEP (6h, rt) shows the reaction process.

Specifically, first, the compound 1 is described in the literature Bioconjugate Chem. 20 (2009) 1869-1878. Briefly, a higher yield of the product was obtained using a strong base (NaH) for the alkylation of malonate, followed by base hydrolysis to perform Gabriel synthesis to give malonate (1) and amino compounds.

Compound 2 was synthesized by coupling Compound 1 with a heterobifunctional linker, BMPS, an amine-to-sulfhydryl crosslinker containing NHS-ester and maleimide groups at opposite ends. . The tert-butyl protecting group on the malonate moiety of compound 2 was removed in CH ₂ Cl ₂ and triethylsilane with 25% TFA to give compound 3 in quantitative yield, which was used further without purification. Triethylsilane acts as a selective carbohydrate scavenger in strong acids such as TFA solutions for mild deprotection of tert-butyl esters. Triethylsilane made it possible to improve yield, reduce reaction time and simplify preparation. Subsequent synthesis is to react compound 3 with [DACHPt(H2O)2] ²⁺ -(NO ₃ ) ₂ to obtain compound 4. The oxaliplatin precursor [DACHPt(H ₂ O) ₂ ] ²⁺ -(NO ₃ ) ₂ was prepared as described in Scheme S1. The dicarboxylic acid group of compound 3 can form a bidentate chelate with Pt(II) through a coordination bond. In particular, the binding structure is similar to the oxalate ligand, so it can easily release the Pt drug inside the cell instead of a tight bond. However, hydrolysis of the maleimide group of compound 4 was observed under basic pH conditions. To prevent hydrolysis, conjugate formation was performed at pH 5.5-6.0. The BMPS crosslinking agent increased the hydrophilicity of compound 4, allowing the crude product to be purified using preparative RP-HPLC with a C18 column. All compounds could be fully analyzed using a variety of analytical techniques.

Experimental Example 2. Conjugation of CPP to Linker-Pt Derivative (4)

Compound 4 was gently mixed with octa-arginine in PBS to obtain two types of CPP conjugates: CPP-Oxal and FITC-labeled CPP-Oxal (F-CPP-Oxal). F-CPP-Oxal was used to investigate cellular uptake and localization using confocal microscopy. CPP has a thiol group at the C-terminus and can easily react with the maleimide group of compound 4. However, the thiol is prone to oxidation before being attached to the maleimide group, thereby reducing the reaction yield. To prevent disulfide formation, water-soluble TCEP was applied to rapidly and stoichiometrically reduce disulfide even under acidic conditions. To remove unreacted reagents, the conjugate was purified by centrifugation using an ultrafiltration column, Vivaspin 2 Hydrosart® (HD). The resultant was lyophilized and then analyzed using MALDI-TOF MS (FIG. 5).

Experimental Example 3. Cytotoxicity test and quantification of the conjugate

The in vitro cytotoxic effects of synthetic CPP-Oxal and the parent drug were compared and analyzed. The cell lines tested were SW480, SW620 and HCT116, all established in primary adenocarcinoma of human colon tissue. All cell lines were incubated for 48 h with different concentrations of CPP-Oxal and oxaliplatin (0-40 μM) and then cell viability was measured using MTT assay. CPP-Oxal showed reduced cell viability in all cell lines compared to oxaliplatin, indicating that the conjugate provided significantly better drug efficacy against the selected cell lines ( FIGS. 2A , 2B , and 2C ).

[Table 1]

Table 1 shows the IC50 measurements of CPP-Oxal and oxaliplatin in different colon cancer cell lines, and each value is a result calculated based on the MTT analysis result. As shown in Table 1, the IC50 values of CPP-Oxal in SW480, SW620, and HCT116 cell lines were 6.8 μM, 8.1 μM, and 12.4 μM, respectively. For oxaliplatin, it was 18.0 μM , 24.3 μM and 21.5 μM, and in general, the IC50 values decreased by 1.7 to 3.0 fold with the use of the conjugate (Table 1). In addition, the total amount of oxaliplatin and CPP-Oxal inside the cell was compared by quantifying Pt using ICP-MS (Fig. 2d). HCT116 cells were incubated with oxaliplatin (20 μM) and CPP-Oxal (20 μM) for 1 h, then cells were washed and lysed. ICP-MS showed that the total Pt of CPP-Oxal treated cells was 154 ng per million cells. On the other hand, the cells treated with 5.3 ng of oxaliplatin per million cells suggest that the large difference in intracellular Pt content is based on the enhanced cellular internalization of CPP-Oxal.

These results show that the conjugate enters the cell more efficiently than oxaliplatin alone. Therefore, CPP aids in cellular uptake of drugs and enhances intracellular drug efficacy. Similarly, cytotoxicity assays were also performed for linker (2) and CPP alone. No cytotoxicity was observed in any cell line despite the high concentration (40 μM), suggesting that the cytotoxicity of the conjugate was essentially due only to the oxaliplatin moiety.

Experimental Example 4. Cell uptake and localization of the conjugate

Cellular uptake and intracellular distribution of F-CPP-Oxal were investigated using a confocal microscope (Carl Zeiss LSM 710, NA 1.30, 100x). First, HCT116 cells were initially treated with 10 μM of F-CPP-Oxal for 30 min and then treated with Mito Tracker (100 nM) for an additional 30 min at 37°C for mitochondrial staining. After that, the cell medium was discarded, rinsed 3 times with fresh medium and incubated with Hoechst 33342 for 10 min for DNA staining. F-CPP-Oxal was rapidly taken up by HCT116 cells within 1 h and the fluorescence of the conjugate was observed throughout the cytoplasm but to a lesser extent in the nucleus, suggesting that the zygote tends to diffuse in the cytoplasm first before entering the nucleus. (Fig. 3a). In addition, the merged image shows significant colocalization of the fluorescence signal between F-CPP-Oxal and mitotracker (Fig. 3c). The degree of colocalization was determined by calculating Pearson's r, which shows the intensity correlation between the two variable signals (green and red). Since Pearson's r ranged from -1 to 1, with a value of 1 indicating a perfect positive correlation, a value of 0.953 reflected the high colocalization between F-CPP-Oxal and mitotracker. Colocalization was further confirmed based on a scatterplot in which the green fluorescence signal intensity of F-CPP-Oxal was plotted against the red fluorescence signal of mitotracker in the pixels of each fluorescence image (Fig. 3d). The green and red pixels at the same spot in each figure all show similar intensities, indicating fairly good colocalization. All results show that the zygote is well internalized into the cell and that the cellular localization to the mitochondria is desirable.

Next, in order to understand the internalization process, the cellular localization of the zygote was further confirmed through staining of intracellular organelles. When F-CPP-Oxal was co-cultured with mitotracker (200 nM) in HeLa cells for 1 hour, significant colocalization between the two fluorescent signals was observed, confirming the localization of the conjugate in mitochondria (Fig. 4a).

FIG. 4d shows that Pearson's r was 0.983 and shows the relative intensity of fluorescence using arrows, confirming colocalization. Conversely, noticeable colocalization was observed when F-CPP-Oxal was co-cultured with Lysotracker (150 nM) and Rhodamine B-dextran (1 mg/mL; early and late endosome markers) in a similar manner. It didn't happen. The zygote and organelle markers meant that the zygote was not trapped inside the lysosome or intracellular vesicle ( FIGS. 4B and 4C ). These results were confirmed based on the low Pearson's r values (0.059 and 0.328, respectively) and intensity profiles using arrows (Figs. 4e and 4f). These results also suggest that the conjugate may not follow the receptor-mediated endocytosis pathway common to intracellular translocation.

Experimental Example 5. Conjugate internalization mechanism

To investigate the endocytosis behavior of drugs, the cellular uptake mechanism of F-CPP-Oxal was studied using various endocytosis inhibitors. This investigation focused on three major intracellular cellular pathways: clathrin-mediated endocytosis, caveola-mediated endocytosis, and macropinocytosis. Therefore, chlorpromazine hydrochloride (30 μM) was used to inhibit clathrin-mediated endocytosis, a representative internalization process of metabolites, hormones and proteins in response to their binding reaction with cell membrane receptors. Methyl-β-cyclodextrin extremely depletes the cellular cholesterol domain rich in caveolae, a flask-shaped protein. Therefore, methyl-β-cyclodextrin (10 mM) was used to inhibit caveola-mediated endocytosis internalizing sphingolipids, albumin, viruses and pathogenic bacteria. Reportedly, imipramine interferes with membrane wrinkling, an essential step for initiating macropinocytosis. Therefore, imipramine hydrochloride (5 μM) was used to inhibit macrotheliosis, which can absorb solutes or substances non-specifically from the extracellular environment. To inhibit each inner ear cell pathway, HCT116 cells were pretreated with each inhibitor for 30 min. The cells were then treated with F-CPP-Oxal (10 μM) and incubated again for 30 min. After washing the cells with fresh cell medium and Hoechst33342, the fluorescence of the conjugate was observed using a confocal microscope.

As can be seen in Figure 5a, clathrin and caveolae inhibitors did not affect the internalization process, and F-CPP-Oxal was internalized almost similarly to the control (no inhibitor). In contrast, cells treated with imipramine hydrochloride showed significantly reduced fluorescence inside the cells, indicating that the internalization of F-CPP-Oxal was clearly inhibited.

Thus, cellular internalization of the zygote appears to follow macrocytosis-mediated endocytosis. The result was also confirmed when comparing the relative fluorescence intensity inside the cell (Fig. 5b). The intensity of green fluorescence was quantified and compared using Imag J.

Experimental Example 6. In vivo antitumor activity of the conjugate

Since CPP-Oxal was more effective than the parent drug affecting the viability of colorectal cancer cells, the in vivo applicability of CPP-Oxal for the treatment of colorectal cancer was investigated. Xenografts, considered a CRC mouse model, were prepared by injecting HCT116 cancer cells (2 x 107 cells/200 μL) into nude mice. When the tumor size reached 100 mm3, mice were divided into 3 groups (n = 5 / group) based on the treated samples, and administration was started (day 0) and continued until day 20. Every other day, 200 μL of CPP-Oxal (40 μM) and oxaliplatin (40 μM) were injected subcutaneously near the tumor tissue of each mouse. In addition, PBS was injected as a control. The body weight and tumor volume of each mouse were monitored periodically until day 20 of injection.

As shown in FIG. 6A , initially all mice gained a slight increase in body weight. However, the weight of the mice started to decrease after 4 days, and the mice treated with oxaliplatin showed the greatest weight loss. In particular, the body weight of CPP-Oxal-treated mice started to increase after 16 days, and finally showed the highest value, which may be due to the high cytotoxicity of the conjugate to tumors, but also because of the small number of side effects. On the last day, mice treated with oxaliplatin, PBS and CPP-Oxal showed weight loss of approximately 17%, 9% and 4%, respectively.

Regarding tumor volume, all mice showed an increase in tumor volume. However, CPP-Oxal-treated mice had the slowest increase as shown in FIG. 6b . Compared with the tumor volume of the PBS-treated group on day 20, oxaliplatin-treated mice decreased by 30%, whereas CPP-Oxal-treated mice decreased by 60%, indicating tumor volume in the CPP-Oxal-treated group. The mouse group was significantly smaller than the other groups. Tumor inhibition ratio (TIR) based on tumor volume at day 20 was calculated as indicated in Figure 6c. The tumor inhibition ratio (TIR) of CPP-Oxal-treated mice was approximately 47% higher than that of oxaliplatin-treated mice (22%).

Tumors in the flank of each mouse were photographed on different days and shown in Fig. 6d. Tumor growth was more pronounced in mice injected with PBS or oxaliplatin. On day 20, mice in each group were sacrificed and all tumors were dissected to determine their actual size. Tumors treated with CPP-Oxal were the smallest and oxaliplatin treatment was less effective than the conjugates as shown in Figure 6e. Therefore, it can be seen that CPP-Oxal had a significant effect on inhibiting tumor growth.

Taken together, through the results of the above experimental examples, it was confirmed that the conjugate according to the present invention has a great potential to slow tumor progression in vivo and to slow down significant antitumor efficacy in xenografts in a CRC model.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (7)

Cell penetrating peptide-linker-anticancer agent conjugates, comprising:
(a) octaarginine-K-mercaptoacetyl (RRRRRRRR-K-mercaptoacetyl); Or octaarginine-K-mercaptoacetyl (RRRRRRRR-K-mercaptoacetyl) labeled with FITC, a fluorescent substance; any one cell penetrating peptide (CPP) selected from;
(b) a linker represented by the following formula (I) for binding to the cell-penetrating peptide; and
(c) an anticancer agent that binds to the linker.
[Formula I]

According to claim 1,
The FITC-labeled octaarginine (R8) is a FITC-amh-octaarginine-K-mercaptoacetyl group (FITC-amh-RRRRRRRR-K-mercaptoacetyl), the conjugate.
According to claim 1,
The conjugate, characterized in that the anticancer agent is oxaliplatin (oxaliplatin).
According to claim 1,
The conjugate is characterized in that it is represented by the following formula (II).
[Formula II]

According to claim 1,
Wherein the octaarginine binds to the negatively charged material of the cell membrane to improve intracellular permeability, the conjugate.
(i) adding an anticancer agent to the linker represented by Formula I; and
(ii) any one selected from octaarginine-K-mercaptoacetyl (RRRRRRRR-K-mercaptoacetyl) labeled with octaarginine-K-mercaptoacetyl (RRRRRRRR-K-mercaptoacetyl) or FITC in the product of step (i) Synthesis by adding a cell penetrating peptide (CPP) of;
A method for producing cell-penetrating peptide-anticancer agent conjugates (conjugates), comprising a.
[Formula I]

A pharmaceutical composition for preventing or treating colorectal cancer, comprising any one of the conjugates selected from claims 1 to 5.

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