Inflammatory signal-sensing GSK3 beta inhibitory peptide with separable cell-penetrating peptide

Abstract

The present invention relates to an inflammatory signal-sensing GSK3β inhibitory peptide with a separable cell-penetrating peptide (CTP), which has the structure of CTP-DEAD-IAGIP where caspase-1 cleavable sequence (DEAD) is inserted between the cell-penetrating peptide (CTP) and the inflammatory signal-sensing GSK3β inhibitory peptide (IAGIP). When the CTP-DEAD-IAGIP reaches inflamed cells, activated inflammasome activates caspase-1 which, in turn, cleaves CTP from IAGIP. The separation of CTP increases the binding affinity of IAGIP to GSK3β and restricts the extracellular migration of IAGIP, thereby increasing its intracellular concentration to improve the inhibitory efficacy against GSK3β, which potentially enhances therapeutic efficacy for inflammatory bowel disease.

Description

Inflammatory signaling GSK3 beta inhibitory peptide with a separate cell-penetrating peptide

[0001] The present invention relates to an inflammatory signal-recognizing GSK3β inhibitory peptide having a separate cell-penetrating peptide and its use.

[0002] Inflammatory bowel disease (IBD) is a chronic and refractory inflammatory disorder that primarily affects the colon, the distal portion of the gastrointestinal (GI) tract. The main types of IBD are ulcerative colitis (UC) and Crohn's disease (CD). While ulcerative colitis begins in the colon and is limited to the innermost mucosal layer, Crohn's disease can involve the entire GI tract, affecting the deeper barrier, even the muscularis propria. Although the exact etiology of IBD remains unknown, pharmacological treatments have been developed to alleviate and maintain IBD.

[0003] Current anti-inflammatory bowel disease agents, including aminosalicylates, glucocorticoids, and immunosuppressants, fail to provide satisfactory clinical outcomes for a significant number of patients with IBD due to low anti-inflammatory activity, drug resistance, and serious side effects with long-term use. Recently, biologic agents, particularly TNF inhibitors, have been introduced into clinical practice and have significantly contributed to the treatment of IBD. However, a significant number of patients with IBD do not respond to biologic agents, and resistance develops in a majority of patients with long-term use. Furthermore, the side effects associated with the action of biologic agents are significant. Therefore, there is a significant unmet medical need for the development of agents with superior anti-inflammatory bowel disease treatment effects and improved side effects.

[0004] Glycogen synthase kinase-3 (GSK-3) is a constitutively active serine / threonine protein kinase with two isoforms, GSK3α and GSK3β, which are encoded by multiple genes and are highly homologous. GSK-3 appears to play a critical role in numerous cellular processes, including glycogen metabolism, membrane-to-nucleus signaling, gene transcription, and survival.

[0005] Recently, GSK3β has been identified as a key regulator of multiple toll-like receptor (TLR) signaling pathways that regulate inflammatory responses. Agonist-induced TLR activation induces proinflammatory responses, primarily via the NFκB pathway. Simultaneously, TLR stimulation activates the PI-3K pathway, leading to Akt activation and GSK3β inhibition, which in turn liberates CREB and effectively suppresses NFκB activity. Furthermore, it promotes CREB-dependent production of the anti-inflammatory cytokine IL-10.

[0006] Recent studies have shown increasing evidence that selective activation of pro-inflammatory TLR signaling by gut bacterial antigens can perpetuate chronic enteritis. Based on the pathophysiological characteristics of chronic enteritis, GSK3β, a key regulator of the TLR signaling pathway, has been considered a promising drug target for inflammatory bowel disease. Inhibition of GSK3β is thought to be highly effective in suppressing inflammation in inflammatory bowel disease by increasing anti-inflammatory cytokine production while simultaneously suppressing pro-inflammatory cytokine production. Indeed, GSK3β inhibitors have been shown to have beneficial effects in experimental colitis.

[0007] Although many small-molecule GSK3β inhibitors, including SB216763 and SB415286, are already known, none have been approved for clinical use. This is likely because these inhibitors interfere with the normal function of GSK3β, a multifunctional kinase, and can cause adverse effects if they act off-target. Therefore, the development of more effective and safer GSK3β inhibitors is urgently needed.

[0008] The present invention relates to an inflammatory signal-recognizing GSK3β inhibitory peptide having a separate cell-penetrating peptide, and provides a CTP separate cell-penetrating GSK3β inhibitory peptide in which a cell-penetrating peptide (Cytoplasmic transduction peptide; CTP) consisting of an amino acid sequence represented by SEQ ID NO: 1, a Caspase-1 cleavable sequence (DXXD) peptide consisting of an amino acid sequence represented by SEQ ID NO: 2, and an IKK-activated GSK3β inhibitory peptide (IAGIP) consisting of an amino acid sequence represented by SEQ ID NO: 3 are fused; and a composition for preventing, improving, or treating an inflammatory disease comprising the CTP separate cell-penetrating GSK3β inhibitory peptide as an active ingredient.

[0009] To solve the above problem, the present invention provides a CTP-separated cell-permeable GSK3β inhibitory peptide fused with a cytoplasmic transduction peptide (CTP) comprising an amino acid sequence represented by SEQ ID NO: 1, a caspase-1 cleavable sequence (DXXD) peptide comprising an amino acid sequence represented by SEQ ID NO: 2, and an IKK-activated GSK3β inhibitory peptide (IAGIP) comprising an amino acid sequence represented by SEQ ID NO: 3.

[0010] In addition, the present invention provides a polynucleotide encoding the CTP-separable cell-permeable GSK3β inhibitory peptide, a recombinant vector comprising the polynucleotide, and a transformant transformed with the recombinant vector.

[0011] In addition, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, which comprises the CTP-separated cell-permeable GSK3β inhibitory peptide as an active ingredient.

[0012] In addition, the present invention provides a health functional food composition for preventing or improving inflammatory diseases, which comprises the CTP-separated cell-permeable GSK3β inhibitory peptide as an active ingredient.

[0013] The present invention relates to an inflammatory signaling GSK3β inhibitory peptide having a split cell-penetrating peptide, and has a structure of CTP-DEAD-IAGIP in which a caspase-1 cleavable sequence (DEAD) is inserted between a cell-penetrating peptide (CTP) and an inflammatory signaling GSK3β inhibitory peptide (IAGIP). When CTP-DEAD-IAGIP reaches a cell in an inflammatory state, CTP is split from IAGIP by caspase-1 activated by an activated inflammasome. The splitting of CTP increases the binding affinity of IAGIP to GSK3β and limits the extracellular movement of IAGIP, thereby increasing its intracellular concentration and enhancing its inhibitory efficacy against GSK3β, thereby enhancing its potential therapeutic efficacy for inflammatory bowel disease.

[0014] Figure 1 shows the results of a comparative test on the GSK3β inhibitory efficacy of CTP-dead GSK3β inhibitory peptide (CTP-DEAD-IAGIP) and CTP-IAGIP in a mouse macrophage RAW 264.7 cell line.

[0015] Figure 2 shows the results of a comparative test on the treatment and improvement of colitis when CTP-isolated GSK3β inhibitory peptide (CTP-DEAD-IAGIP) and CTP-IAGIP were administered into the rectum.

[0016] Figure 3 shows a colon-specific capsule used for oral administration of a peptide drug.

[0017] Figure 4 shows the results of the colon tissue protection effect when CTP-DEAD-IAGIP and CTP-IAGIP were filled into colon-targeting capsules and administered orally.

[0018] Figure 5 shows the results of the colon inflammation relief effect when CTP-DEAD-IAGIP and CTP-IAGIP were filled into colon-targeting capsules and administered orally.

[0019] Figure 6 shows the experimental process for investigating whether CTP-dead GSK3β inhibitory peptide (CTP-DEAD-IAGIP) accumulates in inflammatory cells.

[0020] Figure 7 shows the results of comparing the accumulation effects in RAW264.7 cells under inflammatory conditions (LPS / ATP) treated with CTP-DEAD-IAGIP-FITC / CTP-DEAA-IAGIP-FITC.

[0021] Figure 8 shows a schematic diagram of the mechanism of action of CTP-DEAD-IAGIP and CTP-DEAA-IAGIP.

[0022] In a prior invention, the present inventors designed a GSK3β inhibitory peptide that is specifically activated in an intestinal inflammatory state, that is, a GSK3β inhibitory peptide (IKK-activated GSK3β inhibitory peptide; IAGIP) that is activated only when phosphorylated by IKK, a kinase activated in an inflammatory state, and evaluated its therapeutic activity in a TNBS-induced rat colitis model. In order to manufacture an orally administrable IAGIP, a cell-penetrating peptide (CTP) and a colon-targeted drug delivery technology were applied to IAGIP to invent a colon-targeted CTP-IAGIP, which was patented under Korean Patent No. 10-1562029.

[0023] The CTP of the prior art CTP-IAGIP is a part that is necessary for IAGIP, a GSK3β inhibitory peptide, to reach and bind to its cytoplasmic target GSK3β by imparting cell permeability, but is not necessary for IAGIP's target binding. In addition, since CTP can interfere with the binding of IAGIP activated by IKK to GSK3β, it is ideally removed after performing its role as a CTP (after delivery into the cell). Removal of the cell-penetrating peptide in CTP-IAGIP is expected to not only increase the target binding affinity of IAGIP, but also limit the extracellular movement of IAGIP that has been translocated into the cell, thereby causing intracellular accumulation and increasing its concentration. Accordingly, the present invention relates to a GSK3β inhibitory peptide (CTP-DEAD-IAGIP) that recognizes inflammation signals and is cleaved into a cell-penetrating peptide under an inflammatory condition by introducing a caspase-1 cleavable sequence between CTP and IAGIP of the prior art CTP-IAGIP. CTP-DEAD-IAGIP is a GSK3β inhibitory peptide that has cell permeability and inflammation recognition activation functions, and at the same time, CTP-DEAD is cleaved and converted into IAGIP by caspase-1 activated by inflammasomes activated under an inflammatory condition. The present inventors confirmed that CTP-DEAD-IAGIP exhibits superior GSK3β inhibitory activity than CTP-IAGIP and simultaneously exhibits superior anti-colitis effects in cell experiments and DNBS-induced colitis animal models, thereby completing the present invention.

[0024]

[0025] The present invention provides a CTP-separated cell-permeable GSK3β inhibitory peptide fused with a cytoplasmic transduction peptide (CTP) comprising an amino acid sequence represented by SEQ ID NO: 1, a caspase-1 cleavable sequence (DXXD) peptide comprising an amino acid sequence represented by SEQ ID NO: 2, and an IKK-activated GSK3β inhibitory peptide (IAGIP) comprising an amino acid sequence represented by SEQ ID NO: 3.

[0026] Preferably, the DXXD peptide may be a peptide consisting of an amino acid sequence represented by SEQ ID NO: 4, but is not limited thereto.

[0027] Preferably, the CTP-separable cell-permeable GSK3β inhibitory peptide may be a peptide consisting of an amino acid sequence represented by SEQ ID NO: 5, but is not limited thereto.

[0028]

[0029] The peptide of the present invention can be readily prepared by chemical synthesis methods known in the art. Representative methods include, but are not limited to, liquid or solid phase synthesis, fragment condensation, and F-MOC or T-BOC chemistry.

[0030] Furthermore, the peptide of the present invention can be produced by genetic engineering methods. First, a DNA sequence encoding the peptide is synthesized according to a conventional method. The DNA sequence can be synthesized by PCR amplification using appropriate primers. Alternatively, the DNA sequence can be synthesized by a standard method known in the art, for example, using an automatic DNA synthesizer (e.g., sold by Biosearch or Applied Biosystems). The produced DNA sequence is inserted into a vector containing one or more expression control sequences (e.g., promoter, enhancer, etc.) that are operatively linked to the DNA sequence and control the expression of the DNA sequence, and a host cell is transformed with the recombinant expression vector formed thereby. The resulting transformant is cultured in an appropriate medium and under conditions to allow the DNA sequence to be expressed, and a substantially pure peptide encoded by the DNA sequence is recovered from the culture. The recovery can be performed using a method known in the art (e.g., chromatography). The term “substantially pure peptide” as used above means that the peptide according to the present invention does not substantially contain any other protein derived from the host.

[0031]

[0032] In addition, the present invention provides a polynucleotide encoding the CTP-separable cell-permeable GSK3β inhibitory peptide.

[0033]

[0034] The above “polynucleotide” is a polymer of deoxyribonucleotides or ribonucleotides existing in single-stranded or double-stranded form. It encompasses RNA genome sequences, DNA (gDNA and cDNA), and RNA sequences transcribed therefrom, and includes analogs of natural polynucleotides unless otherwise specified.

[0035] The polynucleotide comprises not only a nucleotide sequence encoding the peptide, but also a complementary sequence to the sequence. The complementary sequence includes not only a perfectly complementary sequence but also a substantially complementary sequence.

[0036] Additionally, the polynucleotide may be modified. Such modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides. A polynucleotide encoding the amino acid sequence is also interpreted to include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence. Such substantial identity may be a sequence that exhibits at least 80% homology, at least 90% homology, or at least 95% homology when the nucleotide sequence and any other sequence are aligned to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0037]

[0038] In addition, the present invention provides a recombinant vector comprising the polynucleotide.

[0039] In addition, the present invention provides a transformant (excluding humans) transformed with the recombinant vector.

[0040]

[0041] In the present invention, “vector” means a self-replicating DNA molecule used to carry a clone gene (or other piece of clone DNA).

[0042]

[0043] In the present invention, “recombinant vector” refers to a plasmid, viral vector or other vector known in the art capable of expressing an inserted nucleic acid in a host cell, and may be a polynucleotide encoding a peptide of the present invention operably linked to a conventional expression vector known in the art. The recombinant vector may generally include a replication origin capable of proliferating in a host cell, one or more expression control sequences (e.g., promoter, enhancer, etc.) that regulate expression, a selective marker, and a polynucleotide encoding a peptide of the present invention operably linked to the expression control sequence. The transformant may be one transformed by the recombinant vector.

[0044] Preferably, the transformant can be obtained by introducing a recombinant vector containing a polynucleotide encoding the peptide of the present invention into a host cell by a method known in the art, for example, but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun, and other known methods for introducing nucleic acids into cells.

[0045]

[0046] In addition, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, which comprises the CTP-separated cell-permeable GSK3β inhibitory peptide as an active ingredient.

[0047] Preferably, the CTP-separable cell-permeable GSK3β inhibitory peptide can separate CTP and IAGIP from each other by Caspase-1 in an inflammatory state in which inflammasome is activated.

[0048] Preferably, the inflammatory disease may be, but is not limited to, colitis or inflammatory bowel disease (IBD).

[0049]

[0050] The pharmaceutical composition of the present invention can be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the active ingredient, and the adjuvants may include solubilizers such as excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, glidants, or flavoring agents. The pharmaceutical composition of the present invention can be preferably formulated as a pharmaceutical composition by additionally including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. In the composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and may include saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders and lubricants can be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions and emulsions, pills, capsules, granules or tablets.

[0051] The pharmaceutical formulation form of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions, and sustained-release formulations of the active compound, etc. The pharmaceutical composition of the present invention may be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. The effective amount of the active ingredient of the pharmaceutical composition of the present invention refers to the amount required for the prevention or treatment of a disease. Therefore, it can be adjusted according to various factors including the type of disease, the severity of the disease, the types and contents of the active ingredient and other ingredients contained in the composition, the type of formulation, and the patient's age, body weight, general health condition, sex and diet, administration time, administration route and secretion rate of the composition, treatment period, and concurrently used drugs. Although not limited thereto, for example, in the case of adults, the composition of the present invention may be administered once to several times a day, and in the case of a compound, the composition may be administered at a dose of 0.1 ng / kg to 10 g / kg.

[0052]

[0053] In addition, the present invention provides a health functional food composition for preventing or improving inflammatory diseases, which comprises the CTP-separated cell-permeable GSK3β inhibitory peptide as an active ingredient.

[0054]

[0055] The health functional food composition of the present invention may be provided in the form of a powder, granules, tablets, capsules, syrup, or beverage. The health functional food composition may be used in combination with other foods or food additives in addition to the active ingredient, and may be appropriately used according to conventional methods. The amount of the active ingredient mixed may be appropriately determined depending on the intended use, for example, for preventive, health, or therapeutic treatment.

[0056] The effective dosage of the active ingredient contained in the above health functional food composition may be used in accordance with the effective dosage of the above pharmaceutical composition, but in the case of long-term intake for the purpose of health and hygiene or health control, it may be below the above range. It is certain that the active ingredient may be used in an amount exceeding the above range because there is no problem in terms of safety.

[0057] There are no special restrictions on the types of the above health foods, and examples include beverages, meat, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes.

[0058] Hereinafter, to aid understanding of the present invention, examples will be given to describe it in detail. However, the following examples are provided to more fully explain the present invention to those of average skill in the art and are merely illustrative of the content of the present invention. Therefore, the scope of the present invention is not limited to the examples below.

[0059]

[0060] <Preparation>

[0061] 1. Photide

[0062] A cell-permeable GSK3β inhibitory peptide (CTP-IAGIP YGRRARRRARR-EPVPPPPTPRSSRHDSGLDSMKD) activated by IKK and a split cell-permeable peptide designed by introducing a caspase-1 cleavable sequence, DEAD (SEQ ID NO: 4), between the cell-permeable peptide (CTP, YGRRARRRARR; SEQ ID NO: 1) and the IKK-activated GSK3β inhibitory peptide (IAGIP, EPVPPPPTPRSSRHDSGLDSMKD; SEQ ID NO: 3) were prepared with a purity of 95% (Anigen, Gwangju, Korea). A mutant peptide refractory to IKK-mediated activation (CTP-mIAGIP, YGRRARRRARR-EPVPPPPTPRSSRHDAGLDSMKD), in which the first serine in the IKK recognition motif was changed to alanine, and a GSK3β inhibitory peptide (CTP-DEAA-IAGIP, YGRRARRRARR-DEAA-EPVPPPPTPRSSRHDSGLDSMKD), in which the caspase-1 cleavable sequence DEAD was changed to DEAA, which is activated by IKK but not caspase-1, were also prepared (Anygen, Gwangju, Korea). To investigate peptide accumulation within cells, CTP-DEAD-IAGIP-FITC and CTP-DEAA-IAGIP-FITC, in which the fluorescent probe FITC was attached to the C terminus of CTP-DEAD-IAGIP and CTP-DEAA-IAGIP, were prepared (Anygen, Gwangju, Korea).

[0063]

[0064] 2. Cells and reagents

[0065] Human colon cancer cell line HCT116 and mouse macrophage RAW 264.7 (ATCC, Manassas, VA) cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium; HyClone) supplemented with 10% fetal bovine serum (Hyclone, Logan, Utah) and penicillin / streptomycin. Lipopolysaccharide (LPS) was purchased from Sigma Chemical Co. (St. Louis, MO). Antibodies were purchased from Cell Signaling (Beverly, MA) and Santa Cruz Biotechnology (Santa Cruz, CA). All ELISA assay kits were obtained from R & D systems. DNBS (2,4-dinitrobenzene sulfonic acid hydrate) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Adenosine triphosphate (ATP) was purchased from Sigma Chemical Co. (St. Louis, MO). (St. Louis, MO). All other compounds were reagent grade and commercially available.

[0066]

[0067] 3. Laboratory animals

[0068] Seven-week-old male Sprague-Dawley (SD) rats were purchased from Samtako Bio Korea (Gyeong-gi-do, South Korea) and housed in the Pusan ​​National University Animal Care Facility under controlled temperature, humidity, and light / dark cycles. The animal protocol used in the present invention was reviewed and approved by the Pusan ​​National University Institutional Animal Care and Use Committee (PNU-IACUC) for ethical procedures and scientific management (Approval Number: PNU-2022-0229).

[0069]

[0070] 4. Western blot

[0071] Cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.7% Na-deoxycholate, 1% NP-40, 150 mM NaCl, 0.3 μM aprotinin, 1 μM pepstatin, and 1 mM PMSF) and incubated on ice for 30 min. The supernatant was collected by centrifugation at 10,000 g for 10 min at 4°C. To prepare tissue lysates, the inflamed colonic distal end was removed, and 3 mL of ice-cold RIPA buffer was mixed per 1 g of tissue. The tissues were finely chopped and homogenized using a homogenizer. After incubation on ice for 30 min, the samples were centrifuged at 10,000 g for 10 min at 4°C. The protein concentration of the supernatant was measured by the BCA method. Cell or tissue extracts were separated by electrophoresis using 7.5 or 10% SDS-PAGE gels. The proteins were transferred to nitrocellulose membranes (Protran, Schleicher & Schuell, Keene, NH). Phosphorylated glycogen synthase (p-GS) was detected in whole cell extracts using a monoclonal anti-phospho-glycogen synthase antibody (Cell Signaling), and the signal intensity of glycogen synthase (GS) (Santa Cruz Biotechnology) detected in the same manner was used for normalization. COX-2 and iNOS proteins in cell lysates and tissues were detected using monoclonal anti-COX-2, iNOS (NOS-2) antibodies (Santa Cruz Biotechnology). Signals were visualized using Supersignal chemiluminescence substrate (Thermo Fisher Scientific, Waltham, MA, USA).The experiment was repeated twice and normalized with an antibody against α-tubulin (Santa Cruz Biotechnology).

[0072]

[0073] <Experimental Example>

[0074] The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

[0075]

[0076] 1. Comparison of the GSK3β inhibitory efficacy of CTP-isolated GSK3β inhibitory peptide (CTP-DEAD-IAGIP) and CTP-IAGIP.

[0077] A comparative experiment on the GSK3β inhibitory efficacy of CTP-DEAD-IAGIP and CTP-IAGIP was conducted in the mouse macrophage RAW 264.7 cell line of Preparation Example 2 above. The experimental conditions shown in Fig. 1A were carried out to confirm the GSK3β inhibitory efficacy of CTP-DEAD-IAGIP and CTP-IAGIP under conditions in which only IKK was activated. After pretreatment with each peptide at a concentration of 200 uM for 1 hour, LPS was treated and cultured for 7 hours. The concentrations of intracellular p-GS and GS were measured by the method described in Preparation Example 4 (Western blot) above. The experimental conditions shown in Fig. 1B were conducted to test whether CTP-DEAD-IAGIP, which has a DEAD sequence that is cleaved by Caspase-1 and thus a cell-penetrating peptide is separated, exhibits a superior GSK3β inhibitory effect than CTP-IAGIP, which does not separate a cell-penetrating peptide, by co-administering ATP / LPS, which activate Caspase-1. Each peptide was pretreated for 1 hour at a concentration of 200 uM, and then ATP was treated 5 hours after LPS treatment and incubated for 2 hours. The concentrations of intracellular p-GS and GS were measured by the method described in Preparation Example 4 (Western blot).

[0078]

[0079] 2. Induction of colitis in mice by 2,4-dinitrobenzenesulfonicacid hydrate (DNBS)

[0080] Experimental colitis was induced in the SD rats of Preparation Example 3. Before inducing colitis, the SD rats (250 to 260 g) were deprived of food except drinking water for 24 hours. Colitis induction was performed under anesthesia with isoflurane (Hana Pharm, Hwaseong, South Korea) using a Small Animal O2 Single Flow Anesthesia System (LMS, Pyeongtaek, South Korea). The concentration of isoflurane was 3% for induction and 2.5% for maintenance, with 1 L / min of oxygen.

[0081] When the SD rats above did not respond to physical stimulation under anesthesia, a rubber cannula (2 mm, OD) was inserted from the colon to the rectum, with the tip positioned at the splenic flexure, 8 cm proximal to the anus. Then, DNBS (48.0 mg) dissolved in 0.4 mL of 50% aqueous ethanol was injected into the colon through the rubber cannula.

[0082]

[0083] 3. Comparison of the anti-colitis effects of CTP-isolated GSK3β inhibitory peptide (CTP-DEAD-IAGIP) and CTP-IAGIP in a DNBS-induced rat colitis model.

[0084] To evaluate and compare the anti-colitis effects of CTP-DEAD-IAGIP and CTP-IAGIP administered orally or rectally, the male SD rats of Preparation Example 3 were divided into 6 (oral administration) or 5 (rectal administration) groups (n = 5 per group) and treated as follows.

[0085]

[0086] - Workplace administration group

[0087] Group 1 (normal group): 0.5 mL of PBS administered rectally;

[0088] Group 2 (peptide-free DNBS group): 0.5 mL of PBS administered rectally;

[0089] Group 3 (peptide-treated DNBS group): rectal administration of CTP-mIAGIP (200 uM / 500 uL) in 0.5 mL of PBS;

[0090] Group 4 (peptide-treated DNBS group): rectal administration of CTP-IAGIP (200 uM / 500 uL) in 0.5 mL of PBS;

[0091] Group 5 (peptide-treated DNBS group): Rectal administration of CTP-DEAD-IAGIP (200 uM / 500 uL) in 0.5 mL of PPBS.

[0092]

[0093] - Oral administration group

[0094] Oral administration of 2.5 mg / kg of peptide filled in the colon-targeting capsule shown in Figure 3

[0095] Group 1 (normal group): Oral administration of colon-targeting capsules not filled with peptides;

[0096] Group 2 (peptide-free DNBS group): oral administration of colon-targeting capsules not filled with peptides;

[0097] Group 3 (peptide-treated DNBS group): CTP-mIAGIP (2.5 mg / kg) was filled into colon-targeting capsules and administered orally;

[0098] Group 4 (peptide-treated DNBS group): CTP-IAGIP (2.5 mg / kg) was filled into colon-targeting capsules and administered orally;

[0099] Group 5 (peptide-treated DNBS group): CTP-DEAA-IAGIP (2.5 mg / kg) was filled into colon-targeting capsules and administered orally;

[0100] Group 6 (peptide-treated DNBS group): CTP-DEAD-IAGIP (2.5 mg / kg) was filled into colon-targeting capsules and administered orally;

[0101]

[0102] Inflammation was induced, and after 3 days, each drug was administered orally or rectally once daily to the SD rats described above. After 6 days of administration, the anti-colitis effects were evaluated. The colonic damage score (CDS) was calculated using a modified version of a previously reported standard. The modified scoring system is as follows, and the CDS was assessed by four independent observers blinded to the experimental conditions.

[0103]

[0104] 0, normal appearance;

[0105] 1, local congestion but no ulceration;

[0106] 2, linear ulcers without severe inflammation;

[0107] 3, Inflammation and ulceration measuring 2 to 4 cm;

[0108] 4, adhesions to other organs, inflammation and ulcers measuring 2 to 4 cm;

[0109] 5, stricture, adhesions of the serosal membrane involving multiple intestinal loops, inflammation and ulceration greater than 4 cm in size.

[0110]

[0111] Additionally, myeloperoxidase (MPO) activity was measured in the distal colon (4 cm). Inflamed colon (0.2 g) was homogenized in a vial containing 2.0 mL of 5.0% hexadecyltrimethylammonium bromide (HTAB, pH 6.0) and incubated on ice for 20 min. The homogenates were centrifuged at 10,000 × g for 10 min at 4 °C, and 0.1 mL of the supernatant was mixed with 2.9 mL of 0.05 M phosphate buffer (pH 6.0) containing o-dianisidine (16.7 mg) and 30.0% H2O2 (1.7 mL). MPO units were measured at 460 nm for 5 min using a UV spectrophotometer (Shimadzu, Tokyo, Japan). One unit of MPO activity was defined as decomposing 1.0 μmol of peroxide per min at 25°C.

[0112]

[0113] 4. Measurement of CINC-3 using ELISA

[0114] The concentration of the inflammatory chemokine CINC-3 was measured in inflamed colons using an ELISA kit. Small pieces of colon were homogenized in potassium phosphate buffer (pH 6.0), and then centrifuged at 10,000 × g for 10 min at 4°C. CINC-3 in the supernatant was quantified using an ELISA kit. ELISA was performed according to the manufacturer's instructions.

[0115]

[0116] 5. Inflammatory cell accumulation effect of CTP-dead-IAGIP, a CTP-isolated GSK3β inhibitory peptide

[0117] A comparative experiment on the accumulation effect of CTP-DEAD-IAGIP in inflammatory cells was conducted in the mouse macrophage RAW 264.7 cell line of Preparation Example 2 above. The experimental conditions were carried out to confirm that CTP-DEAD-IAGIP accumulates in cells under conditions in which IKK and inflammasome are activated. RAW 264.7 cell lines pretreated with or without LPS / ATP for 8 hours were treated with CTP-DEAD-IAGIP-FITC and CTP-DEAA-IAGIP-FITC at a concentration of 200 uM for 1 hour, and then the medium was removed and the FITC-derived fluorescence intensity was measured using a confocal microscope. In addition, fresh medium was added, and after 30 minutes, the medium was removed and the FITC-derived fluorescence intensity was measured. The operation of replacing the medium was repeated once more (Fig. 6).

[0118]

[0119] 6. Data Analysis

[0120] Results were expressed as mean ± standard deviation (SD). To test for differences between groups, one-way analysis of variance (ANOVA) followed by Tukey's HSD test or Mann-Whitney U test (for CDS) was used. Differences with α or P < 0.05 were considered statistically significant.

[0121]

[0122] <Example 1> CTP-isolated GSK3β inhibitory peptide (CTP-DEAD-IAGIP) with superior GSK3β inhibitory efficacy compared to CTP-IAGIP

[0123] The same experiment was conducted under conditions where IKK alone or IKK and inflammasome were activated simultaneously by treating mouse macrophage Raw 264.7 cells with inflammatory factors, LPS or LPS / ATP.

[0124] When treated with the inflammatory factor LPS (only IKK is activated), CTP-DEAD-IAGIP and CTP-IAGIP had almost the same inhibitory efficacy on glycogen synthase (GS) phosphorylation (Fig. 1A). However, when treated with LPS / ATP, CTP-DEAD-IAGIP inhibited GS phosphorylation more than CTP-IAGIP. This suggests that under conditions where IKK and inflammasome are activated together, i.e., under conditions where cell-penetrating peptides are released, CTP-DEAD-IAGIP has a higher GSK3β inhibitory efficacy than CTP-IAGIP (Fig. 1B).

[0125]

[0126] <Example 2> CTP-isolated GSK3β inhibitory peptide (CTP-DEAD-IAGIP) showing superior anti-colitis effect compared to CTP-IAGIP when administered intrarectally in a DNBS-induced rat colitis model

[0127] To test whether CTP-DEAD-IAGIP has a therapeutic advantage over CTP-IAGIP in treating colitis, the anti-colitis effects of CTP-DEAD-IAGIP were compared with those of CTP-IAGIP. After inducing colitis in SD rats according to Experimental Example 3, the rats with colitis were treated with CTP-DEAD-IAGIP (200 uM / 500 uL), CTP-mIAGIP (200 uM / 500 uL), and CTP-IAGIP (200 uM / 500 uL) via rectal administration once daily for 6 days. After the final (6th) treatment, the rats with colitis were euthanized 24 hours later, and the anti-colitis therapeutic effects of each drug were evaluated.

[0128] As a result, according to Fig. 2A, DNBS showed a severely damaged colon corresponding to 4.5 in the colon damage score (CDS) evaluation shown in Experimental Example 3, and CTP-mIAGIP, which does not have GSK3β inhibitory activity, also showed a damaged state corresponding to 4.2, which was not much different from DNBS. On the other hand, the CTP-IAGIP treatment group showed an improved colon damage state of 1.75, and the CTP-DEAD-IAGIP treatment group showed a CDS evaluation of 1.1, showing a higher effect in improving colon damage than CTP-IAGIP. Fig. 2B shows that when the state of colon tissue recovery was confirmed by H&E staining, similar to the results of Fig. 2A, DNBS and CTP-mIAGIP showed severe tissue damage and almost no normal morphology, whereas CTP-DEAD-IAGIP and CTP-IAGIP significantly improved colon damage, and CTP-DEAD-IAGIP showed a better recovery effect on colon epithelial cells and mucosa than CTP-IAGIP. Fig. 2C shows the results of measuring MPO activity as shown in Experimental Example 4. In the case of DNBS and CTP-mIAGIP, inflammation was severe, resulting in high MPO levels, while CTP-DEAD-IAGIP and CTP-IAGIP showed low MPO levels, and CTP-DEAD-IAGIP was more effective than CTP-IAGIP in lowering MPO levels. Figure 2D shows the experimental results of the CINC-3 ELISA kit according to Experimental Example 5. As with Figure 2C, in the case of DNBS and CTP-mIAGIP, the levels of the inflammatory factor CINC-3 were almost the same and high, and both CTP-DEAD-IAGIP and CTP-IAGIP effectively lowered the concentration of CINC-3, and CTP-DEAD-IAGIP was more effective than CTP-IAGIP in lowering the concentration of CINC-3.Figure 2E shows that iNOS and COX-2, which are inflammatory factors, were confirmed by Western blot in Preparation Example 4. Similar to other results, DNBS and CTP-mIAGIP detected high levels of iNOS and COX-2 proteins in inflamed colon tissues, and CTP-IAGIP also lowered the amounts of iNOS and COX-2 proteins in inflamed tissues, but CTP-DEAD-IAGIP lowered the concentrations of iNOS and COX-2 proteins in inflamed tissues much more effectively. These results indicate that CTP-DEAD-IAGIP has a superior anti-colitis effect than CTP-IAGIP.

[0129]

[0130] <Example 3> CTP-isolated GSK3β inhibitory peptide (CTP-DEAD-IAGIP) showing superior anti-colitis effect compared to CTP-IAGIP when administered orally in a DNBS-induced rat colitis model

[0131] Experimental content: Colon-targeting capsules filled with CTP-isolated GSK3β inhibitory peptide (CTP-DEAD-IAGIP), CTP-IAGIP, and control peptides (CTP-mIAGIP and CTP-DEAA-IAGIP) were manufactured (Fig. 3), and the anti-colitis effect was investigated by orally administering the peptide drug at a dose of 2.5 mg / kg (once a day for 6 days) to rats with inflammatory inflammation (DNBS-induced rat colitis model).

[0132] As a result, according to Fig. 4A, DNBS was in a state of severe colon damage corresponding to 4.2 in the colon damage score (CDS) evaluation shown in Experimental Example 3, and CTP-mIAGIP, which does not have GSK3β inhibitory activity, also had a state of damage corresponding to 4.1, which was not much different from DNBS. On the other hand, the CTP-IAGIP and CTP-DEAA-IAGIP treatment groups showed improved colon damage states of 2.3 each, and the CTP-DEAD-IAGIP treatment group showed a state of 1.1 in the CDS evaluation, showing a higher effect of improving colon damage than CTP-IAGIP or CTP-DEAA-IAGIP. Figure 4B shows that when the state of colon tissue recovery was confirmed by H&E staining, similar to the results of Figure 4A, DNBS and CTP-mIAGIP showed severe tissue damage and almost no normal morphology, whereas CTP-DEAD-IAGIP, CTP-DEAA-IAGIP, and CTP-IAGIP significantly improved colon tissue damage, and CTP-DEAD-IAGIP showed a superior recovery effect on colon epithelial cells and mucosa than CTP-DEAA-IAGIP or CTP-IAGIP. Figure 5C shows the results of measuring MPO activity in Experimental Example 3. In the case of DNBS and CTP-mIAGIP, the MPO level was high due to severe inflammation, while CTP-DEAD-IAGIP, CTP-DEAA-IAGIP, and CTP-IAGIP had low MPO levels, and CTP-DEAD-IAGIP was more effective than CTP-DEAA-IAGIP or CTP-IAGIP in lowering the MPO level.Figure 5D shows the experimental results of the CINC-3 ELISA kit according to Experimental Example 4. As with Figure 5C, in the case of DNBS and CTP-mIAGIP, the levels of the inflammatory factor CINC-3 were almost the same and high, and CTP-DEAD-IAGIP, CTP-DEAA-IAGIP, and CTP-IAGIP all effectively lowered the concentration of CINC-3, and CTP-DEAD-IAGIP was more effective in lowering the concentration of CINC-3 than CTP-DEAA-IAGIP or CTP-IAGIP. Figure 5E shows that iNOS and COX-2, which are inflammatory factors, were confirmed by Western blot in Preparation Example 4. Similar to other results, DNBS and CTP-mIAGIP detected high levels of iNOS and COX-2 proteins in inflamed colon tissues, and CTP-DEAA-IAGIP and CTP-IAGIP also lowered the amounts of iNOS and COX-2 proteins in inflamed tissues, but CTP-DEAD-IAGIP lowered the concentrations of iNOS and COX-2 proteins in inflamed tissues much more effectively. These results indicate that CTP-DEAD-IAGIP has a superior anti-colitis effect than CTP-IAGIP even when administered orally.

[0133]

[0134] <Example 4> Comparison of accumulation effects in RAW264.7 cells under inflammatory conditions (LPS / ATP) treated with CTP-DEAD-IAGIP-FITC / CTP-DEAA-IAGIP-FITC

[0135] RAW264.7 cells were divided into a group treated with LPS+ATP (inflammasome-activated inflammatory state) for 8 hours and an untreated group. After treating CTP-DEAD-IAGIP-FITC and CTP-DEAA-IAGIP-FITC for 1 hour each, the medium was removed to obtain cell samples (30×0), a sample (30×0) was added with new medium, incubated for an additional 30 minutes, and the medium was removed. A sample (30×1) was added with new medium, incubated for an additional 30 minutes, and the medium was removed. The intensity of FITC was measured using a confocal microscope (Experimental Example 5, Fig. 6).

[0136] In normal cells, the same FITC intensity was observed regardless of the treated peptide. In cells pretreated with LPS / ATP, when CTP-DEAD-IAGIP-FITC was added, much stronger fluorescence was measured than when CTP-DEAA-IAGIP-FITC, which does not release the cell-penetrating peptide (CTP), was added. When fluorescence was measured while replacing the medium, the decrease in fluorescence was significantly delayed in cells treated with CTP-DEAD-IAGIP-FITC. This result suggests that CTP-DEAD-IAGIP, which releases CTP due to the presence of DEAD, can selectively accumulate in inflammatory tissue cells, thereby increasing its efficacy (Fig. 7).

[0137]

[0138] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A CTP-separated cell-permeable GSK3β inhibitory peptide fused with a cytoplasmic transduction peptide (CTP) consisting of an amino acid sequence represented by sequence number 1, a caspase-1 cleavable sequence (DXXD) peptide consisting of an amino acid sequence represented by sequence number 2, and an IKK-activated GSK3β inhibitory peptide (IAGIP) consisting of an amino acid sequence represented by sequence number 3.

2. A CTP-separable cell-permeable GSK3β inhibitory peptide, characterized in that the DXXD peptide in the first paragraph is a peptide consisting of an amino acid sequence represented by sequence number 4.

3. In the first paragraph, the CTP-separable cell-permeable GSK3β inhibitory peptide is characterized in that it is a peptide consisting of an amino acid sequence represented by SEQ ID NO:

5.

4. A polynucleotide encoding a CTP-separable cell-permeable GSK3β inhibitory peptide according to any one of claims 1 to 3.

5. A recombinant vector containing the polynucleotide of paragraph 4.

6. Transformants transformed with the recombinant vector of Article 5 (excluding humans).

7. A pharmaceutical composition for preventing or treating inflammatory diseases, comprising a CTP-separated cell-permeable GSK3β inhibitory peptide according to any one of claims 1 to 3 as an active ingredient.

8. In the 7th paragraph, the CTP-separated cell-permeable GSK3β inhibitory peptide is a pharmaceutical composition for preventing or treating inflammatory diseases, characterized in that CTP and IAGIP are separated from each other by Caspase-1 in an inflammatory state in which inflammasome is activated.

9. A pharmaceutical composition for preventing or treating an inflammatory disease, characterized in that the inflammatory disease in paragraph 7 is colitis or inflammatory bowel disease (IBD).

10. A health functional food composition for preventing or improving inflammatory diseases, comprising a CTP-separated cell-permeable GSK3β inhibitory peptide according to any one of claims 1 to 3 as an active ingredient.

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