WO2023121153A1 - Composition pour le traitement de la spondylarthrite - Google Patents

Composition pour le traitement de la spondylarthrite Download PDF

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WO2023121153A1
WO2023121153A1 PCT/KR2022/020592 KR2022020592W WO2023121153A1 WO 2023121153 A1 WO2023121153 A1 WO 2023121153A1 KR 2022020592 W KR2022020592 W KR 2022020592W WO 2023121153 A1 WO2023121153 A1 WO 2023121153A1
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spondyloarthritis
peptide
present
amino acid
pharmaceutical composition
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PCT/KR2022/020592
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Korean (ko)
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민병무
박성환
조미라
최정원
이선영
나현식
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서울대학교산학협력단
가톨릭대학교 산학협력단
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Publication of WO2023121153A1 publication Critical patent/WO2023121153A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis

Definitions

  • the present invention relates to a use of a functional peptide derived from vitronectin for the treatment, prevention or improvement of spondyloarthritis.
  • Spondyloarthritis is an inflammatory arthritis affecting approximately 0.2 to 1.61% of the population.
  • Spondyloarthritis can be divided into two subtypes: axial SpA (axSpA) and peripheral spondyloarthritis (SpA).
  • Axial spondyloarthritis induces axial joint inflammation and eventually leads to new bone formation at the vertebral edges, also referred to as syndesmophyte formation.
  • Treatment of axial spondyloarthritis (axSpA) aims to reduce the inflammatory response and inhibit abnormal bone bridging of the axial joint.
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • Interleukin (IL)-17 mediates inflammation and abnormal hyperosteosis in axial spondyloarthritis, and various immune cells in patients with axial spondyloarthritis express IL-17 (J.A. Smith, R.A.
  • Th17 cells type 17 helper T cells
  • Th17 cells are It is one of the major IL-17 expressing cells.
  • Th17 cells are upregulated in the peripheral blood of patients with axial spondyloarthritis, and circulating Th17 levels and regulatory T cell (Treg) abundance are positively and negatively correlated with axial spondyloarthritis disease activity, respectively.
  • Th17/Treg imbalance may have therapeutic potential for axial spondyloarthritis.
  • SpA spondyloarthritic
  • Curdlan-induced SKG mouse is one of them.
  • SKG mice harbor the ZAP-70 W163C mutation, and curdlan immunity induces several spondyloarthritic features, including spinal stiffness, intestinal inflammation, uveitis, and psoriasis-like skin lesions. Since the ZAP-70 W163C mutation plays an important role in thymic T cell selection, this mouse model is suitable for evaluating T cell differentiation in the development of spondyloarthritic arthritis as well as structural damage in axial joints.
  • Vitronectin-derived functional peptide VnP-16 is an active peptide recently developed as a treatment for osteoporosis.
  • VnP-16 enhances osteoblast differentiation through ⁇ 1 integrin-FAK signaling and inhibits osteoclast differentiation and resorption activity through JNK-c-Fos-NFATc1 and ⁇ v ⁇ 3 integrin-c-Src-PYK2 signaling, respectively.
  • JNK-c-Fos-NFATc1 and ⁇ v ⁇ 3 integrin-c-Src-PYK2 signaling respectively.
  • anti-inflammatory effects of VnP-16 in spondyloarthritic (SpA) animal models have not been reported.
  • physiologically active peptides derived from vitronectin act to regulate the levels of Th17 cells and Treg cells in an animal model of spondyloarthritis (SpA).
  • SpA spondyloarthritis
  • an object of the present invention is to provide a pharmaceutical composition for treating or preventing spondyloarthritis comprising the peptide as an active ingredient.
  • a peptide consisting of a sequence of 12 to 173 consecutive amino acids including the amino acid sequence of SEQ ID NO: 1 (RVYFFKGKQYWE) in the amino acid sequence of SEQ ID NO: 2, encoding the peptide
  • a pharmaceutical composition for treating or preventing spondyloarthritis comprising a polynucleotide or a recombinant vector containing the polynucleotide as an active ingredient.
  • a peptide comprising the amino acid sequence of SEQ ID NO: 1, a polynucleotide encoding the peptide, or a backbone comprising a recombinant vector comprising the polynucleotide as an active ingredient
  • a pharmaceutical composition for treating or preventing arthritis is provided.
  • peptide refers to a polymer compound in which two or more amino acids are linked by peptide bonds.
  • the peptide used for the treatment or prevention of spondyloarthritis is a physiologically active peptide derived from vitronectin, comprising 12 to 12 consecutive amino acid sequences including the amino acid sequence of SEQ ID NO: 1 (RVYFFKGKQYWE) within the amino acid sequence of SEQ ID NO: 2. It is a peptide consisting of 173 amino acid sequences.
  • the peptide consisting of the amino acid sequence of SEQ ID NO: 2 is an active peptide derived from vitronectin, and is a peptide derived from the biologically active peptide motif consisting of the amino acid sequence of SEQ ID NO: 1 of the present invention, and includes the peptide motif. It equally retains the activity of a peptide comprising the amino acid sequence of SEQ ID NO: 1 (RVYFFKGKQYWE) of the present invention.
  • the peptide used for the treatment or prevention of spondyloarthritis is a physiologically active peptide derived from vitronectin, and is a peptide containing the amino acid sequence of SEQ ID NO: 1 "RVYFFKGKQYWE”.
  • the peptide may include a mutant peptide having a different sequence by deletion, insertion, substitution, or a combination of amino acid residues within a range that does not affect the activity of the peptide, or a protein fragment having the same function.
  • amino acid modifications at the protein and peptide level including the amino acid sequence of SEQ ID NO: 1 but not changing its activity as a whole, are known in the art, and in some cases phosphorylation, sulfation , acrylation, glycosylation, methylation, and farnesylation. Accordingly, the peptide includes not only the amino acid sequence of SEQ ID NO: 1, but also a peptide having an amino acid sequence substantially identical thereto or a variant thereof.
  • the peptide having the substantially identical amino acid sequence is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more of the amino acid sequence of SEQ ID NO: 1 , It may be a peptide comprising an amino acid sequence having 98% or more, 99% or more, or 99.5% or more homology, but is not limited thereto, and a sequence having 90% or more amino acid sequence homology with the amino acid sequence of SEQ ID NO: 1 Any peptide having the same activity while including a is included in the scope of the present invention.
  • the peptide of the present invention may include the amino acid sequence of SEQ ID NO: 1, and may consist of 20 or less amino acids exhibiting activities for treating, preventing, and improving spondyloarthritis. Specifically, the peptide may be composed of 20 or less, 18 or less, 15 or less, or 12 amino acids.
  • the peptide may be a peptide consisting of the amino acid sequence of SEQ ID NO: 1.
  • the peptide consisting of the amino acid sequence of SEQ ID NO: 1 is also referred to as "VnP-16 peptide" and described.
  • the peptides of the present invention can be obtained by various methods well known in the art. As an example, it can be produced using a polynucleotide recombination and protein expression system, or synthesized in vitro through chemical synthesis such as peptide synthesis, and cell-free protein synthesis, but is not limited by its production method no.
  • the N-terminus or A protecting group may be bound to the C-terminus.
  • the protecting group may be an acetyl group, a fluorenyl methoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, or polyethylene glycol (PEG), but modification of the peptide, in particular, stability of the peptide may be improved. If there is a component, it can be included without limitation.
  • the 'stability' refers to storage stability (eg, storage stability at room temperature) as well as in vivo stability that protects the peptide of the present invention from attack by proteolytic enzymes in vivo.
  • the peptide of the present invention has an activity to inhibit the progression of spondyloarthritis, it can be used for prevention or treatment of spondyloarthritis.
  • the spondyloarthritis may be axial spondyloarthritis or peripheral spondyloarthritis, specifically axial spondyloarthritis.
  • spondyloarthritis which is a disease to be treated, may also be referred to as "ankylosing spondylitis”.
  • the peptides of the present invention are selected from the group consisting of IL-1 ⁇ , IL-6, TNF- ⁇ and IL-17, as demonstrated by the experimental results of the specific examples herein, of one or more inflammatory cytokines. expression can be inhibited.
  • the peptides of the present invention can down-regulate the level of type 17 helper T cells, as demonstrated by the experimental results of the specific examples herein.
  • the Th17 cells may be CD4+ IL-17+ cells or CD4+ IL-22+ IL-17+ cells.
  • the peptides of the present invention can upregulate the level of Treg cells (regulatory T cells).
  • the Treg cells may be CD4+ CD25+ Foxp3+ cells.
  • the peptides of the present invention can inhibit pSTAT3 s727 expression.
  • composition of the present invention may further contain a non-steroidal anti-inflammatory agent in addition to the above-described peptide as an active ingredient.
  • the NSAIDs include drugs that block the production of cyclooxygenase (COX) or inhibit its activity.
  • drugs include, for example, salicylic acid, aspirin, ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flubiprofen, oxaprozin, loxoprofen, indomethacin , tolmetin, sulidac, etodolac, ketorolac, diclofenac, aceclofenac, or celecoxib, but are not limited thereto.
  • composition of the present invention may include, as an active ingredient, not only the peptide, but also a polynucleotide encoding the peptide, or a recombinant vector containing the polynucleotide.
  • polynucleotide is a polymer of nucleotides in which nucleotide monomers are linked in a chain shape by covalent bonds, and is a DNA or RNA nucleic acid molecule of a certain length or longer, which encodes (encodes) the peptide of the present invention. ) means a polynucleotide.
  • nucleic acid bases may be mutated by substitution, deletion, insertion, or a combination thereof, and these are also included in the scope of the present invention.
  • the recombinant vector of the present invention refers to an expression construct of a means for stably expressing the peptide of the present invention by introducing it into a cell, for example, a plasmid vector, a cosmid vector, a bacteriophage vector, a viral vector, etc. vectors, and such recombinant vectors can be easily prepared by those skilled in the art according to any known method using DNA recombinant technology.
  • the pharmaceutical composition of the present invention comprises (i) a therapeutically effective amount of the above-described peptide of the present invention, a polynucleotide encoding the peptide, or a recombinant vector containing the polynucleotide; and (ii) a pharmaceutically acceptable carrier.
  • terapéuticaally effective amount means an amount sufficient for the composition of the present invention to achieve a therapeutic or preventive effect on spondyloarthritis.
  • the pharmaceutically acceptable carrier is one commonly used in formulation, and may include lactose, dextrose, sucrose, sorbitol, mannitol, 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, and the like. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
  • the pharmaceutical composition of the present invention can be administered by any route suitable for treating spondyloarthritis, for example, it can be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular administration It can be administered by injection, intraperitoneal injection, or transdermal administration.
  • the suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, medical condition, food, administration time, administration route, excretion rate and reaction sensitivity, A ordinarily skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis.
  • the daily dosage of the pharmaceutical composition of the present invention is 0.001-10,000 mg/kg.
  • the pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art. or it may be prepared by incorporating into a multi-dose container.
  • the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally contain a dispersing agent or stabilizer.
  • prevention means suppression or delay of the occurrence of spondyloarthritis or symptoms caused by this disease or symptoms due to complications of this disease
  • treatment means spondyloarthritis or this disease. means the alleviation, amelioration, beneficial alteration, or complete elimination of symptoms resulting from complications of
  • a peptide consisting of a sequence of 12 to 173 consecutive amino acids including the amino acid sequence of SEQ ID NO: 1 (RVYFFKGKQYWE) in the amino acid sequence of SEQ ID NO: 2, a polynucleotide encoding the peptide, or the above
  • a method for treating or preventing spondyloarthritis comprising administering a therapeutically effective amount of a recombinant vector containing a polynucleotide to a subject in need of spondyloarthritis treatment.
  • the subject may include a human or a non-human mammal.
  • a subject in need of treatment for spondyloarthritis provides a method for treating or preventing spondyloarthritis comprising the step of administering to subject).
  • the subject may include a human or a non-human mammal.
  • a peptide consisting of a sequence of 12 to 173 consecutive amino acids including the amino acid sequence of SEQ ID NO: 1 (RVYFFKGKQYWE) in the amino acid sequence of SEQ ID NO: 2, a polynucleotide encoding the peptide, or
  • the use of the recombinant vector containing the polynucleotide for the treatment or prevention of spondyloarthritis is provided.
  • a peptide containing the amino acid sequence of SEQ ID NO: 1 (RVYFFKGKQYWE), a polynucleotide encoding the peptide, or a recombinant vector containing the polynucleotide are used to treat or prevent spondyloarthritis provide use.
  • consecutive 12 to 173 amino acids comprising the amino acid sequence of SEQ ID NO: 1 (RVYFFKGKQYWE) in the amino acid sequence of SEQ ID NO: 2 for the preparation of a medicament for the treatment of spondyloarthritis
  • RVYFFKGKQYWE amino acid sequence of SEQ ID NO: 1
  • a peptide comprising the amino acid sequence of SEQ ID NO: 1 (RVYFFKGKQYWE) for the preparation of a medicament for the treatment of spondyloarthritis, a polynucleotide encoding the peptide, or the polynucleotide It provides the use of the recombinant vector comprising.
  • the peptides of the present invention have an activity to inhibit the progression of spondyloarthritis. Specifically, the peptides of the present invention inhibit the production of inflammatory cytokines IL-1 ⁇ , IL-6, TNF- ⁇ , and IL-17A, down-regulate the level of Th17 cells (type 17 helper T cells), and regulate T The level of cells (Treg) is up-regulated, and the expression of pSTAT3 s727 is suppressed.
  • the peptide of the present invention having such an activity can be developed as a therapeutic agent for spondyloarthritis.
  • Figure 1 shows the anti-arthritic effect of vitronectin-derived physiologically active peptide VnP-16 in spondyloarthritic (SpA) mice.
  • Panel B is a hematoxylin and eosin (H&E)/safranin O staining image of an isolated ankle joint from SpA mice 12 weeks after Curdlan injection.
  • the bar graph shows the arthritis score.
  • Data are the mean ⁇ standard error of the mean (SEM) of three independent experiments. *P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001. P values were obtained compared to the vehicle group.
  • Figure 2 shows the results that VnP-16 reduced the spondylitis score of SpA mice.
  • spinal tissues were obtained from vehicle-treated group, celecoxib-treated group, VnP-16 single-treated group, and VnP-16 + celecoxib co-treated group, and H&E and safranin It was stained with O (safranin O).
  • Black arrows indicate inflammatory cell infiltration.
  • the bar graph shows the spondylitis score. *P ⁇ 0.05. P values were obtained compared to the vehicle group.
  • Figure 3 shows the results of VnP-16 reducing the expression of inflammatory cytokines in the nucleus pulposus of mice with spondyloarthritis (SpA).
  • Cells expressing IL-1 ⁇ , IL-6, TNF- ⁇ , and IL-17A were counted in the nucleus pulposus by immunohistochemical staining.
  • P values were obtained compared to the vehicle group.
  • Figure 4 shows the results of VnP-16 reducing the expression of inflammatory cytokines in cartilaginous end plates of mice with spondyloarthritis (SpA). Immunohistochemical staining results of IL-1 ⁇ , IL-6, TNF- ⁇ and IL-17A in cartilage endplates. Data are means ⁇ SEM. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001. P values were obtained compared to the vehicle group.
  • 5a to 5c show the results of VnP-16 regulating Th17 helper T cells and regulatory T cells (Treg) populations in the annulus fibrosus of mice with spondyloarthritis (SpA).
  • Teg Th17 helper T cells and regulatory T cells
  • SpA spondyloarthritis
  • FIG. 5A shows Spinal tissues were stained with CD4-FITC, IL-17-PE, CD25-APC, and Foxp3-PE to evaluate the Th17 population (FIG. 5A), the IL-22+ Th17 population (FIG. 5B), and the Treg population (FIG. 5C), respectively. did Double positive cells are indicated on the bar graph. Data are mean ⁇ SEM. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001. P values were obtained compared to the vehicle-administered group.
  • FIG. 6 shows the results of VnP-16 regulating Th17 (type 17 helper T cell) and regulatory T cell (Treg) differentiation in the spleen of spondyloarthritis (SpA) mice.
  • Spleen tissues were subjected to flow cytometry using antibodies against IL-17A, CD4, CD25 and Foxp3 to determine Th17 and Treg populations.
  • Data are mean ⁇ SEM. **P ⁇ 0.01. P values were obtained compared to the vehicle group.
  • FIG. 7 shows the results of VnP-16 suppressing the expression of pSTAT3 s727 in splenocytes.
  • Splenocytes collected from BALB/c mice were stimulated with vehicle or VnP-16, and expression of total STAT3, pSTAT3 s727, and GAPDH was measured by Western blotting. Data are mean ⁇ SEM. *P ⁇ 0.05. P values were obtained compared to the vehicle group.
  • mice with the ZAP-70 W163C mutation, 8-10 weeks of age were purchased from Saeronbio, Inc. (Uiwang, South Korea). Mice were housed under specific pathogen-free conditions and fed standard mouse chow (Ralston Purina, St. Louis, MO) and water ad libitum. The experiment was evaluated and approved by the Institutional Animal Care and Use Committee of the College of Medicine and the Animal Research Ethics Committee of the clergy University of Korea, and was conducted in accordance with the Laboratory Animal Welfare Act and Guidelines for Management and Use of Laboratory Animals (No. CUMC-2019-0298-02).
  • Curdlan (3 mg/kg) was injected intraperitoneally (IP) into 8-10 week old SKG mice.
  • the VnP-16 peptide was synthesized through a 9-fluorenylmethoxycarbonyl-based solid phase method using a C-terminal amide using a Pioneer Peptide synthesizer (Applied Biosystems, Foster City, CA, USA).
  • the synthesized peptides were purified by Peptron (Peptron, Daejeon, Korea) and analyzed for their properties. It was confirmed that the peptide used in the experiment had a purity of 95% or more using high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the NSAID, celecoxib was purchased from Hanlim Pharmaceutical.
  • Treatment started 1 week after Curdlan injection and continued for 11 weeks.
  • the clinical score was measured weekly for 12 weeks, as described in the previous study literature (M. Ruutu, G. et al., Arthritis and rheumatism 64(7) (2012) 2211-2222.), and the effect was The joint scores received were summed for each mouse.
  • Tissue samples obtained from peripheral joints and vertebrae were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at 7 ⁇ m thickness. Sections were dewaxed using xylene, dehydrated in an alcohol gradient, stained with hematoxylin and eosin (H&E) and stained with Safranin O. Inflammation was then evaluated and recorded as a score. The histological scores of the peripheral joints and spine were calculated as described in the literature (M. Ruutu, G. et al., Arthritis and rheumatism 64(7) (2012) 2211-2222.). Histopathological analysis was scored by three experimenters in a blinded manner, and stained tissues were observed under a microscope (Olympus, Tokyo, Japan, magnification: 40 ⁇ , 200 ⁇ ).
  • Immunohistochemical analysis was performed using the Dako REALTM EnvisionTM detection system kit (DAKO, Glostrup, Denmark, #5007). Tissues were first treated overnight at 4°C with primary antibodies against IL-1 ⁇ , IL-6, IL-17A and tumor necrosis factor (TNF)- ⁇ (all purchased from Abcam, Cambridge, UK), followed by Dako REAL It was incubated for 30 minutes with ⁇ Envision ⁇ /HRP. The final colored product was developed using a diaminobenzidine colorant. All histological scores were evaluated by three independent, blinded observers. Images were taken using a DP71 digital camera (Olympus, Center Valley, PA) attached to a BX41 microscope (Olympus). Positive cells were counted using Adobe Photoshop software (magnification 400x) and averaged in three randomly selected fields per tissue section.
  • the spines of the mice were removed, and to evaluate the differentiation of Th17 cells and Tregs, the spine tissues were stained with CD4-fluorescein isothiocyanate (FITC), IL-17-phycoerythrin (PE), CD25-allophycocyanin and forkhead boxes. It was reacted with an antibody against P3 (Foxp3)-PE (all purchased from eBioscience, San Diego, CA). Stained tissue sections were visualized using a confocal microscope (LSM 700 Meta; Carl Zeiss, Oberkochen, Germany). Double or triple positive cells were counted in three high magnification fields (magnification 400x) per section.
  • FITC CD4-fluorescein isothiocyanate
  • PE IL-17-phycoerythrin
  • CD25-allophycocyanin CD25-allophycocyanin and forkhead boxes. It was reacted with an antibody against P3 (Foxp3)-PE (all purchased from eBioscience, San Diego, CA
  • Cell pellets were prepared from spleen tissue and controlled T using anti-mouse CD4-peridin chlorophyll protein (perCP) and anti-mouse CD25-allophycocyanin (APC) (eBioscience). After irradiation of cell populations, cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer set (Thermo Fisher Scientific) according to the manufacturer's instructions. For Th17 cell analysis, prior to FAC staining, cells were incubated with 25 ng/mL phosphomolybdic acid (Sigma-Aldrich), 250 ng/mL ionomycin (Sigma-Aldrich) and Golgi Stop (BD Biosciences, San Diego, CA, USA).
  • the protein levels of p-STAT3 (s727) (cat: #9134, 100 kDa, cell signaling, USA), total STAT3 (cat: #9189, 100 kDa, cell signaling, USA), and GAPDH (#ab181602, Abcam, UK) were analyzed by western It was measured using a blotting system (SNAP id Protein Detection System, Merck Millipore, Danvers, MD, USA). Splenocytes were collected from BALB/c mice, stimulated with VnP-16 (100 ⁇ g/mL) or vehicle for 2 hours, and then stimulated with IL-6 (10 ng/ml) for 1 hour. Whole cell lysates were then prepared.
  • Protein concentration was measured using the BCA assay method (#23235, thermo, USA), and samples were separated using 4-12% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel), followed by nitrocellulose membrane (Amersham Pharmacia, USA). moved to Uppsala, Sweden).
  • Primary antibodies against the following proteins [p-STAT3 s727 (cat: #9134, 100 kDa, cell signaling, USA), total STAT3 (cat: #9189, 100 kDa, cell signaling), GAPDH (cat: ab181602, 36 kDa, abcam, UK)] were diluted in 0.1% skim milk of Tris-buffered saline Tween-20 and incubated for 20 minutes at room temperature. The membrane was washed and incubated for 20 minutes at room temperature with horseradish peroxidase-conjugated secondary antibody. Band densities were calculated by image capture densitometry.
  • Example 9 VnP-16 inhibited the progression of spondyloarthritis (SpA).
  • Example 10 VnP-16 inhibited inflammatory cytokine expression.
  • the expression levels of IL-1 ⁇ , IL-6, TNF- ⁇ and IL-17 were evaluated by immunohistochemistry (IHC) in the nucleus pulposus (NP) and cartilaginous end plate (CEP) of the vertebral corner. .
  • IHC immunohistochemistry
  • the VnP-16 + celecoxib administration group significantly suppressed IL-1 ⁇ and TNF- ⁇ expression compared to the vehicle group.
  • the expression of IL-6 and IL-17A was significantly suppressed in the VnP-16 alone administration group and the VnP-16 + celecoxib administration group (FIG. 3).
  • VnP-16 showed an immunoregulatory role in helper T cell differentiation.
  • CD4+ IL-17+ cells and CD4+ IL-22+ IL- in the annulus fibrosus area in the VnP-16 alone administration group and VnP-16 + celecoxib administration group compared to the vehicle administration group 17+ cells were downregulated and CD4+CD25+Foxp3+ cells were upregulated (FIG. 5).
  • FIG. 6 shows that CD4+ CD25+ Foxp3+ T cells were up-regulated in the VnP-16 + celecoxib administration group compared to the vehicle administration group (FIG. 6).
  • the Th17 population (CD4+ IL-17+ T cells) tended to decrease in the VnP-16 + celecoxib administration group compared to the vehicle administration group.
  • pSTAT3 s727 expression was significantly inhibited by 100 ⁇ g/mL of VnP-16 (FIG. 7), which demonstrates the Th17/Treg regulatory mechanism of VnP-16.

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Abstract

La présente invention concerne l'utilisation d'un peptide physiologiquement actif dérivé de la vitronectine pour le traitement de la spondylarthrite. Le peptide de la présente invention a une activité pour inhiber la progression de la spondylarthrite et peut donc être développé en tant que principe actif d'un agent thérapeutique pour la spondylarthrite.
PCT/KR2022/020592 2021-12-20 2022-12-16 Composition pour le traitement de la spondylarthrite WO2023121153A1 (fr)

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