Anticancer composition comprising transdermal permeation peptide for transdermal delivery

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer, and preparation method therefor, the composition comprising a complex of a transdermal permeation peptide and a saccharide, and a lectin. It has been identified that, when the composition comprising the complex of the transdermal permeation peptide and the saccharide, and the lectin is used for cancer treatment, skin permeation of anticancer substances is improved, anti-tumor activity and anti-metastasis properties are exhibited, and hepatotoxicity is not exhibited, and thus effective use as a composition for anticancer treatment is possible.

Description

Anticancer composition for transdermal delivery containing a transdermal penetrating peptide

[0001] The present invention relates to an anticancer composition for transdermal delivery comprising a transdermal penetrating peptide.

[0002] Surgical management is the most common method for treating various types of cancer, including skin cancer. However, this approach has limitations in elderly patients, anatomically difficult tumor locations, extensive lesion areas, and cases involving unclear regions. In such situations, alternative treatments such as immunotherapy, radiation therapy, cryotherapy, and photodynamic therapy can be used. Additionally, imiquimod, one of the representative chemotherapy drugs for topical treatment, can serve as a simple and cost-effective alternative or adjuvant therapy to surgical resection. However, treatment with imiquimod carries the potential to cause side effects, including inflammatory diseases associated with its immunomodulatory effects. Furthermore, imiquimod achieved histological clearance in only 645 out of 1,803 lesions (35.8%).

[0003] In developing enhanced topical treatment strategies for various cancers, including skin cancer, excellent target affinity and selectivity are required. Based on this, transdermal delivery of protein therapeutics with proven high anticancer efficacy can be a promising approach. However, protein drugs, including antibodies, can be limited due to high costs and low thermal stability, which may hinder their widespread application. Furthermore, transporting high molecular weight hydrophilic proteins across the stratum corneum presents a significant challenge.

[0004] Various methodologies such as skin-penetrating antibiotics, micro / nanocarriers, microneedles, ion technology, electroporation, jet injection, and thermal scavenging are being studied to improve the transdermal delivery of proteins. Nevertheless, these approaches have the disadvantage of increasing cost and complexity.

[0005] Meanwhile, concanavalin A (ConA) has been proven to have antitumor efficacy across various cancer types, including hepatocellular carcinoma, melanoma, breast cancer, cervical cancer, and glioma. Since ConA is typically extracted from jack beans and purified using a relatively simple method, it can be produced very cost-effectively compared to other protein therapeutics manufactured through biotechnological methods. However, the use of ConA is significantly limited due to its hepatotoxicity. Because ConA has severe side effects, severe enough to be used to induce hepatitis in research for treatment, studies using ConA as a cancer treatment have decreased significantly in recent years.

[0006] Against this backdrop, the inventors developed a transdermal delivery strategy for ConA that can reduce side effects compared to injection, minimize liver exposure, and further enable its use as a topical therapeutic agent for various cancers, including skin cancer.

[0007] However, it is not easy to transport hydrophilic proteins through the skin barrier. The inventors used a skin-penetrating peptide (CPPecp) derived from human eosinophil cation protein and, considering the binding characteristics of ConA and sugars, developed a peptide drone (PD), a complex of mannose and CPPecp, for the transdermal delivery of ConA. The present invention was completed by confirming that when the complex of PD and ConA (CPC) is used in cancer treatment, it not only improves skin penetration but also possesses anticancer and antimetastatic properties and has no hepatotoxic side effects.

[0008] The object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer comprising a complex of a transdermal penetrating peptide and a sugar; and a lectin.

[0009] Another object of the present invention is to provide a method for preparing the above pharmaceutical composition comprising the following steps: i) preparing a complex of a transdermal penetrating peptide and a sugar; and ii) combining the sugar of the complex with a lectin.

[0010] The advantages and features of the present invention and the methods for achieving them will become clear by referring to the embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below but may be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the present invention, and the present invention is defined only by the scope of the claims.

[0011] The terms used in this specification are for describing embodiments and are not intended to limit the invention. In this specification, the singular form includes the plural form unless specifically stated otherwise in the text. The terms "comprises" and / or "comprising" used in this specification do not exclude the presence or addition of one or more other components in addition to the components mentioned. Throughout the specification, the same reference numerals refer to the same components, and "and / or" includes each of the mentioned components and all combinations of one or more. Although terms such as "first," "second," etc., are used to describe various components, these components are not limited by these terms. These terms are used merely to distinguish one component from another. Therefore, the first component mentioned below may be the second component within the technical scope of the invention.

[0012] Unless otherwise defined, all terms used herein (including technical and scientific terms) may be used in a meaning commonly understood by those skilled in the art to which the present invention pertains. Additionally, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless explicitly and specifically defined otherwise.

[0013] The present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising a complex of a transdermal penetrating peptide and a sugar; and a lectin.

[0014] The transdermal penetrating peptide of the present invention may include an amino acid sequence represented by SEQ ID NO. 1.

[0015] The above sequence number 1 may be the amino acid sequence of NYRWRCKNQN.

[0016] The sugars of the present invention may include, but are not limited to, mannose, glucose, N-acetylglucosamine, N-acetylneuraminic acid, mannose-6-phosphate, N-glycan, maltotriose, mannopentaose, galactose, N-acetylgalactosamine, or galacturonic acid.

[0017] The lectin of the present invention may include concanavalin A (ConA), wheat germ agglutinin (WGA), soybean agglutinin (SBA), lysine (Ricin), peanut agglutinin (PNA), phythemagglutinin (PHA), aloe lectin (ALA), seaweed lectins, or lupin lectin (LAL), and specifically may be ConA, but is not limited thereto.

[0018] The above-mentioned lectin is a sugar-binding protein found in various plants, animals, microorganisms, etc., and has the characteristic of binding to specific sugar structures.

[0019] The above-mentioned concanavalin A is extracted from purple cannabali beans and can inhibit cancer cell growth and induce apoptosis. The above-mentioned wheat germ agglutinin is a lectin extracted from wheat germ and can inhibit cell proliferation by binding to sugar structures on the surface of cancer cells. The above-mentioned soybean agglutinin is extracted from soybeans and can inhibit cancer cells. The above-mentioned peanut agglutinin is extracted from peanuts and can inhibit cancer metastasis by interfering with the binding between cancer cells. The above-mentioned phythemagglutinin is extracted from kidney beans and can inhibit cancer cell growth. The above-mentioned aloe lectin is extracted from aloe and can inhibit cancer cell growth through the activation of the immune system. The above-mentioned seaweed lectin is extracted from various types of seaweed and can inhibit cancer cell growth and have antioxidant effects. The above-mentioned lupin lectin is a protein extracted from a plant called lupin and can induce apoptosis and inhibit cancer cell proliferation.

[0020] The composition of the present invention may include a molar ratio of the complex and the lectin of 3 to 5:1, specifically 4:1, but is not limited thereto.

[0021] In a specific embodiment of the present invention, it was confirmed that the molar ratio of the composite and ConA was 4:1. This indicates that a stable structure is maintained.

[0022] The cancer of the present invention may include, but is not limited to, skin cancer, liver cancer, breast cancer, colorectal cancer, stomach cancer, lung cancer, pancreatic cancer, kidney cancer, ovarian cancer, prostate cancer, or leukemia.

[0023] The above skin cancer may be a skin disease including melanoma, cutaneous lymphoma, cutaneous fibrosarcoma, squamous cell carcinoma, or basal cell carcinoma. The above skin disease may additionally include psoriasis, acne, eczema, or warts.

[0024] The composition of the present invention can increase anti-metastatic and anticancer activity.

[0025] In a specific embodiment of the present invention, it was confirmed that the composition effectively inhibits the migration of melanoma cells in a concentration-dependent manner through transwell migration analysis results to evaluate the effect on the migration and metastasis of cancer cells.

[0026] In another specific embodiment of the present invention, the composition is a positive control, Aldara TM It was confirmed that it inhibits tumor growth (approx. 54.7%) much more effectively than cream (approx. 19.0%).

[0027] The composition of the present invention can reduce side effects including skin inflammation or hepatotoxicity.

[0028] In a specific embodiment of the present invention, it was confirmed that a group of mice treated with the composition showed no signs of psoriasis or skin inflammation in the significantly reduced tumor sites, with no pustules.

[0029] In another specific embodiment of the present invention, it was confirmed that transdermal delivery of ConA through the transdermal permeable peptide did not cause hepatotoxicity in mice.

[0030] The composition of the present invention may be administered transdermally, and said composition may increase skin permeability.

[0031] In a specific embodiment of the present invention, it was confirmed that the transdermal permeable peptide facilitates enhanced transdermal delivery of ConA using various transdermal routes that can be used with hydrophilic substances.

[0032] The pharmaceutical composition of the present invention may include a complex of a transdermal penetrating peptide and a sugar; and an active substance comprising a lectin. In a specific embodiment of the present invention, the active substance may be ConA. In addition to the active substance, one or more active ingredients exhibiting the same or similar functions may be additionally included.

[0033] The term "pharmaceutical composition" of the present invention may be described as being used with "pharmaceutical composition."

[0034] The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives.

[0035] The above additive may further include suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions.

[0036] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is one that is commonly used in formulations and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0037] The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, or sterile injectable solutions, each according to conventional methods. Specifically, when formulating, it may be prepared using diluents or excipients such as fillers, weighting agents, binders, wetting agents, disintegrants, and surfactants that are commonly used.

[0038] The pharmaceutical composition of the present invention may additionally include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc., in addition to the above components.

[0039] The pharmaceutical composition of the present invention can be administered parenterally, intravenously, subcutaneously, intramuscularly, intraperitoneally, or transdermally, and specifically can be administered transdermally.

[0040] The appropriate dosage of the pharmaceutical composition of the present invention depends on factors such as the formulation method, mode of administration, age, body weight, sex, pathological condition, food, time of administration, route of administration, rate of excretion and response responsiveness, and the degree of cancer in the patient, and can be appropriately selected by a person skilled in the art.

[0041] The pharmaceutical composition of the present invention may be prepared in a unit volume form or contained in a multi-volume container by formulation using pharmaceutically acceptable carriers and / or excipients according to a method that can be easily carried out by a person skilled in the art to which the invention belongs. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0042] In addition, the present invention provides a method for treating cancer comprising the step of administering a pharmaceutical composition comprising a complex of a transdermal penetrating peptide and a sugar; and a lectin to a subject suspected of having cancer.

[0043] The subjects of administration of the present invention include, but are not limited to, humans, monkeys, cattle, horses, pigs, sheep, chickens, cats, dogs, mice, rabbits, etc.

[0044] In addition, the present invention provides a method for preparing the above pharmaceutical composition comprising the following steps. The method may include i) preparing a complex of a transdermal penetrating peptide and a sugar; and ii) combining the sugar of the complex with a lectin.

[0045] The complex of transdermal penetrating peptides and sugars of the present invention may be prepared through chemical reactions including SPAAC (Strain-Promoted Azide-Alkyne Cycloaddition), Staudinger Ligation, CuAAC (Copper-Catalyzed Azide-Alkyne Cycloaddition), Schiff Base Reaction, Michael Addition, or Diels-Alder Reaction, but is not limited thereto.

[0046] The above SPAAC reaction is a click chemical reaction in which azide and cyclooctyne react to form a triazole ring without a copper catalyst, which can avoid the problem of cytotoxicity caused by copper and is therefore suitable for in vivo application.

[0047] The above Staudinger ligation reaction is a reaction in which an azide reacts with a phosphine compound to form a stable bond, and it can be primarily used for labeling reactions in vivo.

[0048] The above CuAAC reaction is a reaction in which an azide and an alkyne combine under a copper catalyst to form a triazole ring, and is a representative example of click chemistry.

[0049] The above Schiff Base Reaction is a reaction in which an amine combines with an aldehyde or ketone to form an imine bond, and it can also be used when combining proteins or peptides with sugars.

[0050] The above Michael Addition reaction is a reaction in which an α,β-unsaturated carbonyl compound and a nucleophyll are combined, and can be used for the combination between a specific amino acid of a peptide (e.g., cysteine) and a variant of mannose.

[0051] The above Diels-Alder Reaction is a reaction in which a conjugated diene and a dienefil combine to form a ring structure, and can be used in a modified form for the binding between a peptide and a mannose structure.

[0052] In a specific embodiment of the present invention, a complex of a transdermal penetrating peptide and a sugar can be prepared due to a click reaction between the azide functional group of mannose and the dibenzocyclooctyne (DBCO) functional group of the peptide.

[0053] The transdermal penetrating peptide of the present invention may include an amino acid sequence represented by SEQ ID NO. 1, and SEQ ID NO. 1 may be NYRWRCKNQN.

[0054] The sugars of the present invention may include, but are not limited to, mannose, glucose, N-acetylglucosamine, N-acetylneuraminic acid, mannose-6-phosphate, N-glycan, maltotriose, mannopentaose, galactose, N-acetylgalactosamine, or galacturonic acid.

[0055] The lectins of the present invention may include, but are not limited to, concanavalin A (ConA), wheat germ agglutinin (WGA), soybean agglutinin (SBA), lysine (Ricin), peanut agglutinin (PNA), phythemagglutinin (PHA), aloe lectin (ALA), seaweed lectins, or lupin lectin (LAL).

[0056] The present invention relates to a pharmaceutical composition for the prevention or treatment of cancer comprising a complex of a transdermal penetrating peptide and a sugar; and a lectin, and a method for preparing the same. When the composition comprising the complex of the transdermal penetrating peptide and a sugar; and the lectin is used for cancer treatment, it is confirmed that the skin penetration of an anticancer substance is improved, it has antitumor activity and anti-metastatic properties, and does not exhibit hepatotoxicity, thereby making it useful as a composition for anticancer treatment.

[0057] Figure 1 is a schematic diagram illustrating a method of transdermal delivery of an anticancer protein (ConA) using a skin-penetrating peptide-based peptide drone for the treatment of melanoma.

[0058] Figure 2 shows the formation of a ConA-PD complex based on ConA-mannose interactions, Figure 2a shows the chemical structure and sequence of PD, Figure 2b shows a schematic FRET study showing the interaction between fluorescein-labeled PD (PD-Fl) and rhodamine-labeled concanavalin A (ConA-Rho), Figure 2c shows a FRET study with varying ratios of ConA-Rho and PD-Fl using an emission filter 580 / 20 nm and an excitation filter 480 / 30 nm, and Figure 2d shows a CD study of a complex containing ConA (black) and PD (blue) or CPPecp peptide (red) in a 1:5 molar ratio.

[0059] Figure 3 shows the in vitro antitumor activity of ConA and CPC, and the relative survival rates of B16F10 cells treated with ConA in Figure 3a and CPC in Figure 3c, and NIH3T3 cells treated with ConA in Figure 3b and CPC in Figure 3d were evaluated using the MTT assay. Figure 3e shows the caspase-3 activity of B16F10 cells treated with PD (green), ConA (red), and CPC (blue), measured using a colorimetric analysis kit and expressed as a multiple increase compared to the control group.

[0060] Figure 4 shows the anti-metastatic and skin penetration activities of PD, Figure 4a shows a schematic description of the transwell migration assay, in which B16F10 cells on the top surface of a transwell plate were treated with PD at the indicated concentration and cultured at 37°C for 24 hours, Figure 4b shows the number of migrated cells on the bottom surface stained with DAPI and analyzed using ImageJ software, Figure 4c shows a representative image (100x magnification) of the migrated cells on the bottom surface stained with DAPI, Figure 4d shows the evaluation of CPC penetration ability using an ex vivo human skin model kit topically treated with ConA-Rho (0.5 mg / ml), and Figure 4e shows the evaluation of CPC skin penetration ability using an ex vivo human skin model kit topically treated with CPC containing 0.5 mg / ml (4.808 μM) of ConA-Rho containing 48.08 μM of PD.

[0061] Figure 5 shows the in vivo antitumor efficacy of CPC, Figure 5a shows the experimental schedule of the in vivo study, and Figure 5b shows the non-treatment group (black), Aldara for 12 days. TMFigure 5 shows the mean tumor growth curves of the cream (imiquimod 12.5 mg) treatment group (light green) and the CPC (2.5 mg) treatment group (blue); Figure 5c shows the TUNEL staining of tumor tissues of the untreated group (left) and the CPC treatment group (right); and Figure 5d shows CPC (left) and Aldara on day 6 of the experiment. TM Figure 5 shows the skin condition of a mouse treated with the cream (right), Figure 5e shows the serum AST levels measured at the end of the experiment using a colorimeter kit after ConA intravenous injection (ConA IV) or topical CPC treatment, Figure 5f shows the serum ALT levels measured at the end of the experiment using a colorimeter kit after ConA intravenous injection (ConA IV) or topical CPC treatment, and Figure 5g shows the H&E staining of liver tissue in the ConA IV and CPC groups, where the white arrow indicates the cell division pattern and the black arrow indicates single cell necrosis.

[0062] Figure 6a shows the chemical structure and sequence of CPPecp, Figure 6b shows the chemical structure and sequence of peptide drone (PD), and Figure 6c shows the chemical structure and sequence of fluorescein-labeled PD.

[0063] Figure 7 shows a synthetic scheme for a peptide drone (PD), in which DBCO was conjugated to the peptide after a final deprotection step to expose the N-terminal amine group, 1.1 equivalents (44 μmol) of DBCO-acid and 1.1 equivalents (44 μmol) of HCTU were dissolved in 3 mL of DMSO, 2.2 equivalents (88 μmol) of DIPEA were added to the mixture and reacted for 4 hours, after which α-D-Mannopyranosylazide was added to CPPecp-DBCO in DMSO and the reaction tube was shaken at room temperature for 7 hours to promote the click chemical reaction between DBCO and the azide group.

[0064] Figure 8 shows the synthesis scheme of fluorescein-labeled PD (PD-Fl). Since mannose is connected to the N-terminus of the peptide, PD-Fl is synthesized using the amine group of the lysine (Lys, K) residue within the peptide sequence. PD-Fl is synthesized by reacting 100 nmol of 5(6)-carboxyfluorescein N-hydroxysuccinimide ester with 100 nmol of PD in 0.5 ml DMF for 12 hours. The synthesized PD-Fl is then ground with tert-butyl methyl ether and purified using reverse-phase high-performance liquid chromatography.

[0065] Figure 9a shows the results of the MALDI-TOF MS spectrum of CPPecp-NH2 (calculated molecular weight: 1380.56, observed molecular weight: 1381.74), Figure 9b shows the results of the MALDI-TOF MS spectrum of peptide drone (calculated molecular weight: 1902.07, observed molecular weight: 1901.59), and Figure 9c shows the results of the MALDI-TOF MS spectrum of fluorescein-labeled PD (calculated molecular weight: 2260.37, observed molecular weight: 2260.33).

[0066] Figure 10a shows the RP-HPLC chromatogram of the purified peptide of CPPecp-NH2, Figure 10b shows the RP-HPLC chromatogram of the purified peptide of peptide drone, and Figure 10c shows the RP-HPLC chromatogram of the purified peptide of fluorescein-labeled PD.

[0067] Figure 11 shows the CD spectra of PD (blue) and CPPecp (red) peptides.

[0068] Figure 12a shows the relative survival rate of B16F10 cells treated with PD peptide evaluated by MTT analysis, and Figure 12b shows the relative survival rate of NIH3T3 cells treated with PD peptide evaluated by MTT analysis.

[0069] Figure 13 is a figure showing a FRET study to confirm the maintenance of the CPC structure in a 50% glycerol solution.

[0070] Figure 14a shows the average tumor growth curves of the untreated group (black) and the ConA treatment group (red) for 12 days, and Figure 14b shows the body weight of mice during the experiment period.

[0071] Figure 15 shows the expected synergistic therapeutic effect across the entire skin when CPC treatment and intravenous administration treatment are combined.

[0072] The contents of the present invention will be explained in more detail below through the following examples and experimental examples. However, the scope of the present invention is not limited to the following examples and experimental examples, but includes variations of equivalent technical concepts.

[0073] Example 1. Preparation of materials

[0074] Fmoc-amino acid and coupling reagents were purchased from Novabiochem (Germany) and Anaspec (USA). All other reagents and general chemicals were purchased from Sigma-Aldrich (USA) and Merck (Germany).

[0075] Example 2. Peptide Synthesis

[0076] The peptide was synthesized using the fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS) protocol with Rink Amide MBHA resin LL (Novabiochem) as a solid support. All amino acids were protected by standard protecting groups.

[0077] The binding of each amino acid residue consists of 5 equivalents of Fmoc-protected amino acids, 4.5 equivalents of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HCTU), 4.5 equivalents of 1-hydroxybenzotriazole (HOBt), and 10 equivalents of N,N-diisopropylethylamine (DIPEA). This was achieved by dissolving in N,N-dimethylformamide (DMF) and reacting for 1.5 hours relative to the resin.

[0078] The Fmoc protecting group was removed during synthesis for 30 minutes by adding 20% ​​piperidine to DMF (v / v). After synthesis, the resin was treated for 3 hours with a cutting cocktail consisting of trifluoroacetic acid (TFA), 1,2-ethanedithiol, and thioanisole in a 95:2.5:2.5 ratio to remove protection from all protecting groups and to cut the resin. The solution was ground using an excess amount of tert-butyl methyl ether.

[0079] The cleaved peptides were purified by reverse phase high-performance liquid chromatography (RP-HPLC) using a concentration gradient of 0 to 50% acetonitrile in 0.1% TFA on a C18 column. The molecular weight of the peptides was verified using matrix-assisted laser desorption / ionization-time of flight mass spectrometry (MALDI-TOF MS) with an α-cyano-4-hydroxycinnamic acid (CHCA) matrix.

[0080] The peptide concentration was 6780 M for quantitative analysis. -1 cm -1 It was measured by spectrophotometry at 280 nm in 40% acetonitrile using the molar extinction coefficient.

[0081] Example 3. Transwell Migration Assay

[0082] To evaluate the effect of PD on cancer cell migration and metastasis, a Transwell migration assay was performed using Transwell plates with polycarbonate membranes having 8 μm pores. 600 μL of medium containing 10% fetal bovine serum (FBS) was added to each well of a 24-well plate. Approximately 200,000 B16F10 cells suspended in 200 μL of medium without FBS were applied to the upper chamber of the Transwell plate and cultured for 24 hours.

[0083] After washing with phosphate-buffered saline (PBS), the prepared PD peptide was added to the upper chamber, and the medium in the lower chamber was replaced and cultured for another 24 hours. Subsequently, the cells were washed with PBS, fixed with 4% paraformaldehyde for 15 minutes, and then washed again. Then, the cells were stained with 10 μg / mL of 4',6-diamidino-2-phenylindole (DAPI) in PBS for 15 minutes.

[0084] Non-migrating cells in the upper chamber were removed using a cotton swab. Then, the chamber was transferred to a slide glass, and migrating cells were observed using an Invitrogen EVOS M7000 imaging system. The number of migrating cells was quantified using ImageJ.

[0085] Example 4. Measurement of skin penetration activity

[0086] To evaluate the skin penetration ability of PD, an in vitro human skin model kit (NATIVESKIN®GenoSkin, France) composed of donated human skin was used. Rhodamine B-labeled Concanavalin A (ConA-Rho) was used to investigate whether the degree of penetration through the skin varied depending on the presence of PD.

[0087] For the in vitro human skin model, the sample was diluted with the provided skin culture medium, applied evenly to the skin, and incubated at 37°C for 24 hours. Afterward, excess sample was wiped off with a cotton swab and fixed in 10% neutral buffered formalin. The skin tissue was completely immersed in a 30% gum and sucrose solution at -4°C for 2 days.

[0088] Then, the tissue was frozen in Epredia™ Cryomatrix™ embedding resin inside a freezing mold at -30 ℃. The sample was subsequently sliced ​​into 8 μm thick slices using a CryoStar NX50. After cryopreservation, the slides were air-dried for 1 hour and immersed in distilled water for 10 minutes.

[0089] Staining was performed using DAPI, and slides were scanned at 200x magnification using an AxioScan.Z1 equipped with Rhodamine B and DAPI filters. The present invention was approved by the Inha University Institutional Review Board (230221-1A). Fluorescence measurements were outsourced to K20 in Korea.

[0090] Example 5. Animal experiment

[0091] Six-week-old female C57BL / 6 mice were purchased from Orient Bio (Korea). The mice were housed at the experimental animal facility of Inha University College of Medicine. All animal experiments were conducted in accordance with Inha University’s institutional guidelines on animal welfare and were approved by the Institutional Animal Care and Use Committee (IACUC). Through these procedures, the guidelines for the care and use of experimental animals were strictly adhered to.

[0092] Example 6. In vivo anticancer efficacy

[0093] To investigate the antitumor efficacy of CPC and compare it with Aldara™ cream (Donga ST), a melanoma mouse model was established using C57BL / 6 mice anesthetized by isoflurane inhalation anesthesia. After anesthesia, 100 μL of 3.5 x 10⁶ isoflurane was administered to each mouse. 4 B16F10 cells were inoculated into the skin.

[0094] Starting from the 9th day after cancer cell inoculation, Aldara™ cream and CPC were applied topically at intervals of 2 to 3 days for 2 weeks. Tumor growth was measured using calipers, and tumor volume was determined as π / 6 x width x length x height. Mouse body weight was also measured during the same period.

[0095] Example 7. Histological Analysis and TUNEL Staining

[0096] Harris hematoxylin-eosin staining was performed to visualize the tissue morphology. The tissue sections were first treated with xylene (GDCHEM) three times to remove paraffin, and then rehydrated using a graded ethanol series (100%, 95%, 80% ethanol; GDCHEM).

[0097] After rehydration, the sections were washed with tap water. The sections were stained with Harris hematoxylin (BBC), washed with tap water, and then differentiated with 1% hydrochloric acid-alcohol. Then, they were treated with 1% ammonia water and washed again. After eosin staining (Sigma Aldrich), the sections were briefly rinsed with tap water. The sections were dehydrated with 95% and 100% ethanol and cleared with xylene.

[0098] TUNEL staining was performed using the Millipore TUNEL Assay Kit (S7100) to visualize apoptotic cells in tumor tissue. Paraffin was removed from the tissue sections, and they were treated with 0.03% hydrogen peroxide for 15 minutes. After rinsing with distilled water, 150 μL of Proteinase K was applied for 10 minutes. Subsequently, the sections were washed with TBST. Then, 20 μL of equilibrium buffer was applied for 20 seconds, followed by 30 μL of reaction buffer.

[0099] The sections were covered with coverslips and incubated at a dilution ratio of 1:9 in a drying oven at 37°C for 1 hour. The reaction was stopped with stop buffer for 10 minutes, followed by washing with TBST. 30 μL of anti-digoxigenin antibody was applied, and the mixture was incubated for 30 minutes.

[0100] After washing with TBST, 150 μL of DAB was applied for 3 minutes. After rinsing the sections with running tap water, they were counterstained with Mayer's Hematoxylin for 3 minutes, immersed in 1% ammonia solution, and mounted. These experiments and measurements were outsourced to K20 in Korea.

[0101] Example 8. Measurement of hepatotoxicity in vivo

[0102] To evaluate the difference in liver damage between the ConA IV injection group and the CPC transdermal administration group, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) tests were performed. Serum samples were collected from each group 12 hours after the 15 mg / kg ConA IV injection. Serum ALT and AST activities were measured using an Abcam assay kit. Additionally, photographs were taken to document psoriatic adverse effects caused by Aldara™ cream.

[0103] Example 9. Fluorescence Resonance Energy Transfer

[0104] To evaluate whether Concanavalin A (ConA), known to have a specific affinity for mannose, can recognize and bind to the mannose portion attached to a skin-penetrating peptide, CPC formation was confirmed through experiments utilizing the Foster Resonance Energy Transfer (FRET) phenomenon. Since mannose is attached to the N-terminus of the peptide, the amine group of the lysine (Lys, K) residue within the peptide sequence was utilized.

[0105] PD-Fl molecules were synthesized by reacting 5(6)-carboxyfluorescein N-hydroxysuccinimide ester (Sigma-Aldrich) with CPPecp-mannose in DMF for 12 hours. For Concanavalin A (ConA), Rhodamine B-labeled Concanavalin A (NanoCS) was purchased and used.

[0106] To activate the sugar-binding ability of ConA, the reaction solvent was set to 0.1 M HEPES buffer containing 0.9 M NaCl, 1 mM MnCl2, and 1 mM CaCl2 (pH 7.4). The concentration of Rhodamine B-labeled ConA was set to 0.1 μM, and the concentration of PD-Fl was adjusted in multiples to investigate the induction of the FRET phenomenon at various ratios. Fluorescence intensity and FRET were measured and analyzed using a PerkinElmer Victor Nivo plate reader with a 480 / 30 nm excitation filter and a 580 / 20 nm emission filter.

[0107] Example 10. Circular Dichroism (CD)

[0108] CD spectra were recorded at 200 to 260 nm using a J-815 (Jasco) with a 1 mm path length cuvette. The concentration of ConA was 1 mg / mL, and the concentrations of PD and CPPecp binding to ConA were 48 μM, which is 5 times the molar amount of ConA. The concentration used for CD measurements of the peptides alone was 20 μM.

[0109] The measurement solvent was Cellove™ DPBS pH 7.3 containing 1 mM CaCl2 and 1 mM MnCl2. The scan was repeated 5 times, and the average value was recorded.

[0110] Example 11. Evaluation of cell viability

[0111] Mouse melanoma cell line B16F10 and mouse fibroblast cell line NIH3T3 cells were cultured in Dulbecco's Modified Eagle's Medium containing 4.5 g / L glucose, L-glutamine, and 25 mM HEPES (supplied by Gibco), supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Welgene).

[0112] Culture conditions were maintained at 37°C in an environment of 5% CO2 and 95% humidity. The cytotoxicity of ConA, PD, and CPC on B16F10 and NIH3T3 cells was analyzed using the MTT assay (Non-Radioactive Cell Proliferation Assay, Promega). One day prior to analysis, B16F10 and NIH3T3 cells were seeded into 96-well plates at a seeding density of 5,000 cells per well.

[0113] The next day, the medium was removed, and various concentrations of ConA, PD, and CPC were incubated for 1 hour in 0.1 M HEPES buffer containing 0.9 M NaCl, 1 mM MnCl2, and 1 mM CaCl2 to allow ConA and PD to bind, and then diluted with FBS(-) medium. Each sample was treated three times and incubated at 37 °C for 24 hours. Cell viability was evaluated using the ability of cells to convert the water-soluble tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) into an insoluble formazan product.

[0114] After treating the samples for 24 hours, 15 μL of MTT staining solution was added to each well and incubated for 4 hours. Subsequently, a solubilization solution / stopping mixture was added to dissolve the formazan product. Absorbance was measured at 570 nm using a PerkinElmer Victor Nivo plate reader and expressed as a normalized value relative to untreated control cells (100%).

[0115] Example 12. Caspase-3 Analysis

[0116] To determine whether CPC induces apoptosis in B16F10 cells in a caspase-dependent manner in conjunction with ConA, caspase-3 activity was evaluated using a colorimetric assay kit (Abcam). Each sample was incubated for 24 hours to obtain 1 x 10 6 After inducing apoptosis in more than [number] cells, the cells were lysed to extract proteins.

[0117] After incubating 120 μg of the extracted protein with the caspase-3 substrate DEVD-pNA at 37°C for 1 to 2 hours, the optical density was measured on a 405 / 10 nm filter using a Victor Nivo microplate reader (PerkinElmer).

[0118] Example 13. Statistical Analysis

[0119] For in vitro and in vivo experiments, results are expressed as mean ± standard deviation and mean ± standard error, respectively. All statistical data were processed using Sigmaplot. T-tests were used to analyze differences between two groups, and ANOVA was used to compare differences among multiple groups.

[0120] Experimental Example 1. Formation of ConA-PD Complex (CPC)

[0121] Considering the binding characteristics of ConA and sugars, the inventors developed a peptide drone (PD) containing CPPecp and mannose for the transdermal delivery of ConA (Fig. 1).

[0122] Specifically, to develop a PD for the transdermal delivery of ConA, mannose was conjugated to a CPPecp peptide (SEQ No. 1: NYRWRCKNQN) via a strain-promoted azide-alkyne click (SPAAC) reaction (Fig. 2a, Fig. 6, and Fig. 7). To determine whether the PD molecule forms an effectively regulated complex with ConA, the binding ratio between PD and ConA was determined using fluorescence resonance energy transfer (FRET) technology, considering that ConA forms a tetramer with four monosaccharide binding sites (Fig. 2b).

[0123] To this end, fluorescently labeled PD (PD-Fl) was synthesized by conjugating a fluorescent substance to the lysine side chain located at the center of the CPPecp sequence (Fig. 8). Additionally, rhodamine-labeled ConA (ConA-Rho) was prepared, and FRET studies were performed by increasing the ratio of ConA-Rho to PD-Fl from 1:0 to 1:20. As shown in Fig. 2c, the FRET signal increased with increasing ratio and remained stable at a ratio of 1:5 or higher, indicating that the peptide-mannose conjugate bound to the ConA tetramer at a 4:1 ratio.

[0124] Subsequently, circular dichroism (CD) analysis was performed to confirm complex formation through alternative methods. CD studies on ConA complexes containing ConA and PD or CPPecp peptides in a 1:5 molar ratio confirmed that PD reduced the intensity of the characteristic negative band of ConA at 220 nm, whereas the effect of CPPecp on the protein structure was negligible (Figs. 2d and 6).

[0125] These results indicate that monosaccharide binding relaxes the negative CD band of lectin protein in a concentration-dependent manner. Although the CD spectra of PD and CPPecp peptides showed minor differences (Fig. 11), they were not sufficient to have a significant effect on the CD spectrum of ConA, considering the difference in molecular weight between the peptides and proteins. This indicates that CPC is effectively organized based on ConA-mannose interactions.

[0126] Experimental Example 2. In vitro anticancer activity of ConA and CPC

[0127] Next, the inventors investigated the effect of PD binding on the anticancer activity of ConA. When melanoma cells (B16F10) and fibroblasts (NIH3T3) were treated with ConA, the protein exhibited selective and concentration-dependent cytotoxicity only in cancer cells (Figs. 3a and 3b). Considering that 10 μg / mL of ConA results in a cell viability of approximately 50%, the effect of PD on antitumor activity at the above concentration was evaluated.

[0128] As shown in Fig. 3c, ConA conjugated with PD at various molar ratios from 1:0 to 1:20 consistently inhibited the growth of B16F10 cells by approximately 50% while maintaining negligible cytotoxicity against NIH3T3 cells (Fig. 3d). ConA is known to induce apoptosis in cancer cells through a caspase-dependent mechanism. To observe the effect of PD on this process, caspase-3 activity was measured, and it was confirmed that both ConA and CPC significantly increased caspase-3 activity (Fig. 3e).

[0129] Although complex formation and PD alone were shown to slightly increase cancer cell toxicity and caspase-3 activity, B16F10 cells showed a cell viability of about 80% compared to NIH3T3 cells (Fig. 12). These in vitro experiments clearly confirmed that the binding of PD does not interfere with the intrinsic anticancer efficacy of ConA.

[0130] Experimental Example 3. Anti-metastatic and skin penetration activity of PD

[0131] CPPecp peptides are known to inhibit cancer cell metastasis through interactions with glycosaminoglycans such as heparin sulfate. Given that melanoma is highly metastatic, the anti-metastatic activity of CPPecp may exhibit a synergistic effect with the anticancer effect of ConA. Therefore, the inventors tested the anti-metastatic ability of CPPecp against melanoma using a 24-well transwell plate equipped with a filter membrane having 8 μm pores (Fig. 4a).

[0132] As shown in Figures 4b and 4c, PD effectively inhibited melanoma cell migration in a concentration-dependent manner, reaching approximately 36.1% at 1.92 μM. Therefore, based on the collective results, the ConA-PD molar ratio was determined to be 1:10 to ensure stable complex formation in subsequent experiments and to utilize the anti-metastatic properties of the excess peptide.

[0133] Next, we investigated whether PD helps deliver ConA across the skin barrier. As shown in Figure 4d, when applied to an in vitro human skin model kit without PD, ConA-Rho alone exhibited negligible skin penetration. In contrast, when ConA-Rho was co-administered with more than 10 times the amount of PD, it was confirmed that the fluorescence signal in the epidermis and beyond was significantly enhanced.

[0134] Generally, the following mechanisms are used to explain skin barrier penetration: i) intracellular pathways where a substance passes directly through cells, ii) intercellular pathways where a substance passes through the space between cells, and iii) pathways involving skin components such as hair follicles and sweat glands. Nanoparticles decorated with CPPecp exhibited greater transdermal permeability when they had a flexible structure than when they had a rigid structure, suggesting that the nanoparticles were transported through narrow pathways (mechanisms ii and iii).

[0135] In addition, CPPecp was found to enable the penetration of nanostructured cells through interaction with glycosaminoglycans (Mechanism i). Through this, it was hypothesized that PD could facilitate enhanced transdermal delivery of ConA by utilizing various transdermal pathways available for hydrophilic materials.

[0136] Experimental Example 4. In vivo antitumor efficacy of CPC

[0137] Based on proven in vitro anticancer activity and transdermal penetration capabilities, the inventors investigated the antitumor efficacy of the ConA-PD complex in a C57BL / 6 mouse model with B16F10 tumors. Treatment was performed according to the schedule shown in Fig. 5a. When the tumor reached approximately 50 mm³ (9 days after tumor cell inoculation), the animals were randomly divided into three groups and treated on days 0, 3, 5, 7, and 10. As a positive control, Aldara containing 12.5 mg of imiquimod was used. TM One pack of cream was applied per treatment.

[0138] On the other hand, CPC was dissolved in a 50% glycerol solution to prevent leakage without additional formulation optimization, which maintained its complex structure as confirmed in the FRET experiment (Fig. 13). To ensure cost-effectiveness and equity, this solution contained 2.5 mg of CPC, which is five times less than imiquimod.

[0139] Despite the quantitative difference, CPC is Aldara TM It inhibited tumor growth much more effectively (approx. 54.7%) than the cream (approx. 19.0%) (Fig. 5b). In cases without PD, treatment with ConA alone is Aldara TM Even at a level of 12.5 mg of imiquimod present in the cream, melanoma growth was not significantly inhibited (Fig. 14). This indicates that a transdermal penetration enhancer is needed.

[0140] In addition, as shown in Fig. 5c, it was confirmed through the TUNEL (transferase dUTP end labeling) assay that CPC inhibits cancer cell growth by inducing apoptosis. This was consistent with the results of the in vitro experiment mentioned above. The body weight of the mice was well maintained throughout the experiment period, which indicates low acute toxicity of the sample (Fig. 14).

[0141] Imiquimod and Aldara TM The ingredients contained in the cream are known to cause inflammatory diseases associated with immunomodulatory effects, including skin inflammation such as psoriasis. These side effects are addressed in the present invention by Aldara TM This was also observed in mice treated with the cream (Fig. 5d). In contrast, the group treated with CPC showed clear skin without signs of pustules or psoriasis at significantly reduced tumor sites. However, ConA-associated hepatotoxicity remains a major obstacle to the application of CPC as an anticancer agent.

[0142] To determine whether a transdermal delivery method using PD could mitigate this problem, serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were evaluated (Figs. 5e and 5f). As a control group, one group was administered 15 mg / kg of ConA intravenously, which is a much lower dose than the transdermal dose and is commonly used to induce hepatitis.

[0143] The results indicated that transdermal delivery of ConA via PD maintained ALT and AST levels (46.3 U / L and 140.37 U / L, respectively) within the normal range (22 to 133 U / L and 46 to 221 U / L, respectively). In addition, hematoxylin and eosin (H&E) staining confirmed that transdermal delivery of ConA via PD did not cause hepatotoxicity in mice (Fig. 5g).

[0144] The results of the collective biological experiments show that CPC is Aldara TMIt has demonstrated the potential to exhibit excellent anticancer efficacy while addressing the side effects of creams and ConA. Furthermore, this agent is expected to produce synergistic effects when combined with other treatment methods, particularly intravenous therapy. As shown in Figure 15, CPC acts inward from the outer layer of the skin, whereas intravenous therapy diffuses outward from the deeper dermis, providing therapeutic efficacy across the entire skin area. Additionally, using this strategy allows for a reduction in the dosage of co-therapy drugs, such as antibody-based immune checkpoint inhibitors, compared to monotherapy, thereby enhancing therapeutic effects and mitigating drug resistance.

[0145] The inventors have developed a technology for treating cancer by transdermally delivering the anticancer protein ConA using a skin-penetrating peptide. Despite its proven anticancer efficacy, the use of ConA has been limited due to the significant hepatotoxicity of the protein. According to the present invention, transdermal administration of ConA is used in the treatment of Aldara TM It demonstrated superior anticancer efficacy compared to cream, and it was confirmed that liver toxicity was negligible. In addition, Aldara TM The absence of side effects, such as skin inflammation similar to psoriasis associated with the cream, indicated that effective treatment is possible. In the present invention, the peptide-protein complex was simply treated in a 50% glycerol solution, which suggests that formulation optimization can further enhance its function. Considering the confirmed anti-metastatic activity of the CPPecp sequence, optimizing the protein / peptide ratio may further increase anticancer efficacy. Therefore, the present invention provides an effective cancer treatment strategy as a promising therapy that can be used in conjunction with other treatments when surgical treatment is difficult.

[0146] Although embodiments of the present invention have been described above, the present invention is not limited to the above embodiments and can be manufactured in various different forms, and those skilled in the art will understand that the present invention can be implemented in other specific forms without changing the technical concept or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A complex of a transdermal penetrating peptide and a sugar; and a pharmaceutical composition for the prevention or treatment of cancer comprising a lectin.

2. In Paragraph 1, A pharmaceutical composition characterized in that the above-mentioned transdermal penetrating peptide comprises an amino acid sequence represented by SEQ ID NO.

1.

3. In Paragraph 1, A pharmaceutical composition characterized in that the above sugars comprise mannose, glucose, N-acetylglucosamine, N-acetylneuraminic acid, mannose-6-phosphate, N-glycan, maltotriose, mannopentoside, galactose, N-acetylgalactosamine, or galacturonic acid.

4. In Paragraph 1, A pharmaceutical composition characterized in that the above lectin comprises concanavalin A (ConA), wheat germ agglutinin (WGA), soybean agglutinin (SBA), lysine (Ricin), peanut agglutinin (PNA), phythemagglutinin (PHA), aloe lectin (ALA), seaweed lectins, or lupin lectin (LAL).

5. In Paragraph 1, A pharmaceutical composition characterized by the molar ratio of the complex and the lectin being 3 to 5:

1.

6. In Paragraph 1, A pharmaceutical composition characterized in that the above cancer includes skin cancer, liver cancer, breast cancer, colorectal cancer, stomach cancer, lung cancer, pancreatic cancer, kidney cancer, ovarian cancer, prostate cancer, or leukemia.

7. In Paragraph 6, A pharmaceutical composition characterized in that the above skin cancer is a skin disease including melanoma, cutaneous lymphoma, cutaneous fibrosarcoma, squamous cell carcinoma, or basal cell carcinoma.

8. In Paragraph 7, A pharmaceutical composition characterized by the above skin disease further comprising psoriasis, acne, eczema, or warts.

9. In Paragraph 1, The above composition is a pharmaceutical composition characterized by increasing anti-metastatic and anticancer activity.

10. In Paragraph 1, The above composition is a pharmaceutical composition characterized by reducing side effects including skin inflammation or hepatotoxicity.

11. In Paragraph 1, A pharmaceutical composition characterized by being administered transdermally.

12. A method for preparing the pharmaceutical composition of claim 1, comprising the following steps: i) a step of preparing a complex of a transdermal penetrating peptide and a sugar; and ii) A step of combining the sugars of the above complex with lectins.

13. In Paragraph 12, A method for manufacturing the above-mentioned complex, characterized by preparing it through a chemical reaction including SPAAC (Strain-Promoted Azide-Alkyne Cycloaddition), Staudinger Ligation, CuAAC (Copper-Catalyzed Azide-Alkyne Cycloaddition), Schiff Base Reaction, Michael Addition, or Diels-Alder Reaction.

Images