New pharmaceutical use

Abstract

The present invention provides a peptide having the sequence: Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Tyr-Lys, or a salt thereof, for use as a medicament. The peptide is particularly useful for treating conditions characterised by inflammation, including wounds, burns, haemorrhoids, psoriasis, acne, atopic dermatitis, COPD, ulcerative colitis and IPF.

Description

Technical Field

[0001] This invention relates to novel uses of known compounds in human medicine, and to pharmaceutical compositions comprising known compounds. In particular, this invention relates to the use of said compounds and those compositions in the treatment of inflammation.

[0002] Background Technology and Existing Technology

[0003] Inflammation is typically characterized by a local tissue response to the invasion of, for example, microorganisms, certain antigens, damaged cells, or physical and / or chemical agents. The inflammatory response is generally a protective mechanism used to eliminate, dilute, or isolate both the damaging agents and the damaged tissue, and to initiate tissue healing.

[0004] Inflammation can originate from physical trauma, infection, certain chronic diseases (such as psoriasis and autoimmune diseases such as rheumatoid arthritis), and / or chemical and / or physiological responses to external stimuli (e.g., as part of an allergic reaction). It may involve a complex series of events in which inflammatory mediators increase blood flow and cause local vasodilation, leading to redness and heat, causing fluid secretion, which often results in local swelling, allowing white blood cells to migrate into the inflamed area, and causing pain.

[0005] Many symptoms / conditions are characterized by abnormal tissue-damaging inflammation, and / or are caused by abnormal tissue-damaging inflammation. Such symptoms are typically characterized by the activation of immune defense mechanisms, with detrimental effects on the host outweighing any benefits, and are usually associated with: varying degrees of tissue redness or congestion, swelling, high fever, pain, itching, cell death, tissue destruction, cell proliferation, and / or loss of function. Examples include inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, psoriasis, glomerulonephritis, and transplant rejection.

[0006] Typically, a complex series of events produces inflammatory changes, such as increased blood flow through local vasodilation, leading to redness and heat; extravasation of white blood cells and plasma, which usually results in local swelling; activation of sensory nerves (leading to pain in some tissues); and loss of function. These inflammatory changes are triggered by a cascade of cellular and biochemical events involving cells such as neutrophils, monocytes, macrophages, and lymphocytes, along with inflammatory mediators such as vasoactive amines, cytokines, complement factors, and reactive oxygen species.

[0007] Inflammation plays a crucial role, especially in the wound healing process. Wounds and burns can therefore be classified as inflammation-related conditions. The conventional wisdom in the art is that anti-inflammatory drugs should not be applied directly to open wounds, as this would be detrimental to wound healing.

[0008] Mussel adhesive protein (MAP), also known as mytilus edulis foot protein (mefp), is a protein secreted by marine mollusks such as the purple mussel (Mytilus edulis), the thick-shelled mussel (Mytilus coruscus), and the green mussel (Perna viridis). The adhesive protein is secreted by mussels from their byssal glands, where it is produced and stored. When secreted on the surface of solids (such as rocks) and other solid objects (such as metal, wood, glass, etc.), it forms a waterproof bond that secures the mussel to the solid object. Mussels typically attach themselves in groups to coastal reefs or to the bottom of boats. This bond is extremely strong and can withstand the impact of waves in coastal waters.

[0009] Studies of the purple mussel (Mytilus edulis), Mediterranean mussel (Mytilus galloprovincialis), California mussel (Mytilus californias), and green mussel (Perna viridis) have identified 11 separate mussel-derived adhesion protein subtypes to date: mfp-1 (sometimes referred to as "mefp-1", which can be used interchangeably below), mfp-2 / mefp-2, mfp-3 / mefp-3, mfp-4 / mefp-4, mfp-5 / mefp-5, mfp-6 / mefp-6; collagen pre-COL-P, pre-COL-D, and pre-COL-NG; and mussel byssal matrix proteins PTMP (proximal filament matrix protein) and DTMP (distal proximal filament matrix protein). See, for example, Zhu et al., Advances in Marine Science, 32, 560 (2014) and Gao et al., Journal of Anhui Agr.Sci., 39, 19860 (2011)).

[0010] All mussel adhesive proteins (including their subtypes) have two structural features, which include: (1) lysine, which enables the protein to carry a high positive charge (due to the NH2 terminus); and (2) 3,4-dihydroxyphenylalanine (DOPA, dopamine), whose catechol portion is responsible for forming strong covalent bonds, thereby giving the mussel adhesive protein the ability to bind to solid surfaces.

[0011] Products based on mussel adhesive proteins are currently used in a limited number of applications (including as tissue adhesives for microporous bonding and for treating wounds and burns). Commercial products are either used directly as solutions of mussel adhesive proteins or stored as lyophilized powders to be dissolved before use.

[0012] A key portion of mefp-1 consists of 70 to 90 tandem repeats of the following decapeptide: Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-DOPA-Lys (see Waite, Int. J. Adhesion and Adhesives, 7, 9 (1987)). This decapeptide sequence can be isolated as a low molecular weight derivative of naturally occurring MAP, or synthesized, for example, according to Yamamoto's description in J. Chem. Soc., Perkin Trans. 1, 613 (1987). See also Dalsin et al., J. Am. Chem. Soc., 125, 4253 (2003).

[0013] DOPA residues are considered essential for the activity of MAP, such as mefp-1. There is no suggestion in the art that complete substitution of MAP with different amino acids (such as tyrosine) would result in the preservation of the physicochemical or biological properties of MAP.

[0014] There is certainly no suggestion in the art that isolated decapeptides lacking DOPA residues will or even possibly possess the same or even similar properties as MAP; these DOPA-free decapeptides are disclosed only as model compounds, for example, in the following literature: Kanyalkar et al., Biomaterials, 389 (2002) and Belli et al., Dental Materials, 26, e125 (2010). Surprisingly, we have found that isolated decapeptides in mefp-1 with structures different from repeating decapeptide units can be used to treat inflammation. See also US 5,616,311 and WO 96 / 39128. Summary of the Invention

[0015] According to the present invention, a (separated) peptide compound is provided, comprising or preferably consisting of the following sequence:

[0016] Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Tyr-Lys,

[0017] Its regioisomers, stereoisomers, or salts are used in medicine and / or as drugs, for example, for the treatment of inflammation, inflammatory conditions, and / or conditions characterized by inflammation.

[0018] The present invention further provides (isolated) peptide compounds of the above-described sequence for use in veterinary science. The compounds (including regioisomers and stereoisomers) and their salts disclosed herein for the uses mentioned herein are collectively referred to as "compounds of the present invention".

[0019] The compounds of the present invention, whether in salt form or other forms, include regioisomers of certain amino acids (e.g., Hyp and Tyr) in the above-described structures, as well as mixtures of these regioisomers. For example, Tyr includes not only tyrosine (4-hydroxyphenylalanine), but also 2-hydroxyphenylalanine and 3-hydroxyphenylalanine, and Hyp includes 4-hydroxyproline, 3-hydroxyproline, and 5-hydroxyproline. The Hyp residue adjacent to the Tyr residue in the compounds of the present invention may be 3-hydroxyproline, and the Hyp residue adjacent to the Thr residue in the compounds of the present invention may be 4-hydroxyproline. More preferably, both Hyp residues are 4-hydroxyproline.

[0020] Therefore, compounds of the present invention that are not in salt form may have the following specific chemical structures:

[0021]

[0022] Furthermore, in addition to the standard central carbon atom of the amino acids in the above sequence (with no exception in the L-configuration), some amino acids in the sequence further contain other chiral carbon atoms. All these stereoisomers and mixtures thereof (including racemic mixtures) are included within the scope of this invention. Hyp, by definition, includes trans-4-hydroxy-L-proline, cis-4-hydroxy-L-proline, trans-3-hydroxy-L-proline, cis-3-hydroxy-L-proline, trans-5-hydroxy-L-proline, and cis-5-hydroxy-L-proline, but we preferably use 4-hydroxy-L-proline for the compounds of this invention.

[0023] The compounds of this invention can be in the form of salts. Salts that may be mentioned include pharmaceutically acceptable salts, such as pharmaceutically acceptable acid addition salts and base addition salts. Such salts can be formed by conventional means, for example by reacting a free peptide with an equivalent or more of a suitable acid or base, optionally in a solvent or in a medium in which the salt is insoluble, followed by removal of the solvent or medium using standard techniques (e.g., in a vacuum, by lyophilization or filtration). Salts can also be prepared, for example, by using a suitable ion exchange resin to exchange an antiion of the active ingredient in salt form with another antiion.

[0024] Preferred salts include, for example, hydrochloride, hydrogen sulfate, maleate, methanesulfonate, toluenesulfonate, alkaline earth metal salts such as calcium and magnesium salts, or alkali metal salts such as sodium and potassium salts.

[0025] The compounds of the present invention are useful because they possess pharmacological activity. Therefore, the compounds of the present invention can be used in human and animal medicine. They are thus suitable as pharmaceuticals (and / or for use in veterinary science), although they can also be used as part of medical devices.

[0026] Although the compounds of the present invention may themselves possess pharmacological activity, certain pharmaceutically acceptable (e.g., "protected") derivatives of the compounds of the present invention may exist or be prepared, which may not possess such activity, but may be administered and then metabolized or chemically transformed to form the compounds of the present invention. Such compounds (which may possess some pharmacological activity, provided that such activity is significantly lower than that of the active compound to which the compound is metabolized / transformed) can therefore be described as "prodrugs" of the compounds of the present invention.

[0027] As used in this application, the prodrugs mentioned will include the following compounds, which, upon administration, form experimentally detectable amounts of the compounds of the present invention within a predetermined time period. All prodrugs of the compounds of the present invention are included within the scope of the present invention.

[0028] The compounds of this invention can be used specifically to treat inflammation.

[0029] "Treating inflammation" includes treating inflammation in any organ of the body (including soft tissues, joints, nerves, vascular system, internal organs, especially mucous membrane surfaces, particularly the skin) regardless of the cause, and also includes all such inflammatory conditions or symptoms, and / or conditions or symptoms characterized by inflammation (e.g., as symptoms).

[0030] Inflammatory symptoms can (and usually) involve the activation of immune defense mechanisms, with harmful effects on the host outweighing any benefits. Such symptoms are typically associated with: varying degrees of tissue redness or congestion, swelling, edema, high fever, pain (including aches), fluid exudation, itching (pruritus), cell death and tissue destruction, cell proliferation and / or loss of function.

[0031] Inflammatory conditions that can be mentioned include arteritis, diabetes, metabolic syndrome, rosacea, asthma and allergic reactions, ankylosing spondylitis, chronic obstructive pulmonary disease (COPD), gouty arthritis, inflammatory bowel diseases (such as Crohn's disease and ulcerative colitis), multiple sclerosis, osteoarthritis, pancreatitis, prostatitis, psoriatic arthritis, rheumatoid arthritis, tendinitis, bursitis, Sjogren's syndrome, systemic lupus erythematosus, uveitis, urticaria, vasculitis, mastocytosis, diabetic vascular complications, migraines, atherosclerosis, and related cardiovascular diseases. A characteristic feature of the inflammatory disease state is chronic obstructive pulmonary disease (COPD). Another characteristic feature is the inflammatory bowel disease, including Crohn's disease, especially ulcerative colitis.

[0032] Inflammatory conditions that may be mentioned more specifically include inflammation of the skin or mucous membranes (including the oral mucosa, nasal mucosa, ocular mucosa, vaginal mucosa, cervical mucosa, and / or anorectal mucosa, especially the oral or nasal mucosa), such as inflammation due to infection (such as viral and / or bacterial infections), or allergic / atopic conditions (such as rhinitis, pharyngitis, periodontitis, gingivitis, dry eye, conjunctivitis, dermatitis, urticaria (hives), and food allergies); and other inflammatory conditions such as herpes, drug eruption, polymorphic light eruption, sunburn, early signs of skin cancer (erythematous skin lesions), pathological alopecia (including after skin grafts), chemotherapy rash, psoriasis, erythema multiforme, folliculitis, eczema, and otitis externa.

[0033] More specifically, the compound can be used to treat conditions characterized by inflammation and / or certain conditions associated with inflammation. Such conditions can include wounds (including abrasions (scratches), incisions (including surgical incisions), lacerations, punctures, avulsions, bruises, and scar formation) and burns (including inflammation resulting from post-burn surgery such as skin grafts) and other conditions such as hemorrhoids.

[0034] Wounds on the skin or mucous membranes can arise from or be caused by physical damage to the membrane surface, either internally or externally (i.e., as a symptom of an underlying physiological disorder).

[0035] Physical (e.g., "open") wounds can be caused by: sharp instruments (cuts, incisions, punctures) or blunt / mechanical forces (lacerations, abrasions, avulsions), physical impacts (bruises), heat or chemicals (burns and blisters), UV light (sunburn), and cold (frostbite). Wounds can be superficial (damaging only the epidermis and / or dermis) or full-thickness wounds (damage below the epidermis and / or dermis). In severe cases, subcutaneous and / or submucosal tissues such as muscles, bones, joints, and even internal organs may be damaged.

[0036] The compounds of this invention can be used to relieve pain (including aches) associated with inflammation and / or wounds. In particular, the compounds of this invention can be used to relieve surgical pain and / or non-surgical pain. Those skilled in the art will understand that the term "surgical pain" (i.e., procedural pain) refers to acute pain associated with medical research and treatment performed for healthcare purposes. The term "non-surgical (pain)" refers to general pain associated with inflammation and / or wounds (e.g., pain associated with oral ulcers, burns, and / or scars) and is not the result of a specific medical intervention.

[0037] The compounds of this invention can be used not only to treat inflammation, pain (including aches) and / or itching (itching) associated with the wound itself and the healing process, but also to prevent fluid leakage from the wound, the risk of infection, and to prevent physiological reactions such as scar formation and melanin deposition arising from inflammation and / or the wound healing process.

[0038] Scar formation is a result of inflammation and / or wound healing, and is a general term for the formation of fibrotic tissue as a result of such inflammation / healing.

[0039] The compounds of this invention can also be used to inhibit the production of melanin that may lead to self-inflammatory / wound healing. The compounds of this invention can also be used to inhibit conditions associated with melanin deposition, such as melasma, freckles, melanosis, facial rashes and other pigmentation, skin cancer with melanoma, and pigmentation or skin diseases such as acne caused by sun exposure.

[0040] Wounds can also result from diseases or conditions. Such wounds can include the formation of blisters and / or ulcers on the skin and mucous membranes. These are common conditions that are often chronic and difficult to treat. Skin tissue can typically be damaged, ablated, liquefied, infected, and / or necrotic. Ulcers can lead to secondary health consequences, especially if they become infected, making them difficult to heal and causing significant damage. They can also cause significant psychological stress and financial burden on patients, impacting both overall well-being and quality of life.

[0041] In alternative embodiments, the compounds of the present invention may be used specifically for inflammatory skin conditions or diseases including psoriasis, acne, eczema and dermatitis, especially allergic / atopic dermatitis, as well as for the treatment of rhinitis (especially allergic rhinitis), hemorrhoids and chronic obstructive pulmonary disease.

[0042] Psoriasis is a chronic inflammatory skin disease that is prone to relapse (some patients never recover throughout their lives). The clinical manifestations of psoriasis primarily include erythema and scaling. It can occur all over the body, but is more commonly observed on the scalp and extremities.

[0043] Acne is a chronic inflammatory skin disease of the hair follicles (pilosebaceous units), which occurs in close association with the following major factors: excessive sebum production, occluded sebaceous ducts (including closed and open comedones), bacterial infection, and inflammatory response. It often occurs in adolescence and is characterized by polymorphic skin lesions on the face. The term acne therefore includes acne vulgaris and rosacea (i.e., brown nose).

[0044] Eczema is an inflammatory skin reaction characterized by intense itching, caused by a variety of internal and external factors. It has three phases: acute, subacute, and chronic. In the acute phase, exudation is often present, while the chronic phase includes infiltration and hypertrophy. Skin lesions are typically itchy and prone to recurrence.

[0045] Dermatitis is a common skin condition characterized by roughness, redness, itching, eczema, and dryness. Small bumps, refractory ulcers, and pigmented patches caused by dermatitis, if left untreated, can develop into basal cell carcinoma, squamous cell carcinoma, and malignant melanoma. Dermatitis can be caused by a variety of internal and external infectious or non-infectious factors, including substances (contact dermatitis) or allergic reactions (anaphylactic / atopic dermatitis). It also includes seborrheic dermatitis (seborrheic eczema) and all forms of steroid-dependent dermatitis (including photosensitive seborrheic dermatitis, perioral dermatitis, rosacea-like dermatitis, steroid rosacea, steroid-induced rosacea, iatrogenic rosacea, steroid-like rosacea, topical corticosteroid-induced rosacea-like dermatitis, and more particularly facial corticosteroid addictive dermatitis (FCAD) or facial corticosteroid-dependent dermatitis (facial corticosteroid addictive dermatitis). Corticosteroid-dependent dermatitis (FCDD) is characterized by facial flushing, erythema, telangiectasia, atrophy, papules and / or pustules in the facial area following prolonged treatment with topical corticosteroids (including uncontrolled, abused or misused); see, for example, Xiao et al., J. Dermatol., 42, 697 (2015) and Lu et al., Clin. Exp. Dermatol., 35, 618 (2009)).

[0046] Rhinitis is an irritation and inflammation of the nasal mucosa. Common symptoms of rhinitis include nasal congestion, runny nose, sneezing, and postnasal drip. The most common type of rhinitis is allergic rhinitis, which is caused by allergens such as pollen, dust, mold, or certain animal dander / scales. The compounds of this invention can relieve itchy eyes even when administered intranasally (i.e., through the nasal mucosa).

[0047] Hemorrhoids are swelling caused by inflammation of numerous blood vessels found inside or around the rectum and anus. Symptoms include bleeding after stool passes (i.e., wound), prolapsed hemorrhoids, mucus discharge and itching, pain, redness, and swelling in the anal area. Hemorrhoids are believed to result from increased abdominal pressure, for example, as a consequence of constipation or diarrhea.

[0048] Chronic obstructive pulmonary disease (COPD) is a name for a group of lung conditions that cause shortness of breath, including emphysema (damage to the alveoli) and chronic bronchitis (long-term inflammation of the airways). COPD occurs when the lungs become inflamed, damaged, and narrowed. The damage to the lungs is usually irreversible and results in impaired airflow into and out of the lungs. Symptoms of COPD include shortness of breath, expectorant cough, frequent chest infections, and persistent wheezing. The most common cause of this disease is smoking, but other risk factors include high levels of air pollution and occupational exposure to dust, chemicals, and fumes.

[0049] The compounds of the present invention can have a positive effect in alleviating erythema, redness and swelling, edema, blisters and bullous pemphigoid caused by various conditions (including those mentioned in general and specific in this application), and can inhibit the exudation of subcutaneous tissue fluid and suppress itching and pain caused by such inflammatory conditions.

[0050] Other inflammatory symptoms that may be mentioned include:

[0051] (a) Mucosal inflammation, such as oral mucositis, oral ulcers, otitis media, laryngitis, tracheitis, esophagitis, gastritis, enteritis and enterocolitis (including bacterial dysentery, chronic amoebic dysentery, schistosomiasis, nonspecific ulcerative colitis and segmental enteritis), cervicitis and cervical endometritis, endometritis, inflammation caused by inhalation injury, and inflammation associated with cancer and infection (e.g. viral infection, such as the common cold or influenza) that affects mucosal surfaces, such as the mucosal surfaces of the mouth, nasopharynx, ears, throat, trachea, gastrointestinal tract, cervix, etc.

[0052] (b) Orthopedic inflammation associated with, for example, fractures, purulent infections of bones and joints, caused by: rheumatic bone disease, and purulent osteomyelitis (acute, chronic, localized, sclerotic, post-traumatic), purulent arthritis; bone tumors (osteoma, osteoid osteoma, chondroma), bone cysts, osteoclastoma, primary osteosarcoma (osteosarcoma, chondrosarcoma, osteofibrosarcoma, Ewing's sarcoma, non-Hodgkin's lymphoma, myeloma, chordoma), bone metastases, tumor-like lesions of bone (bone cysts, aneurysmal bone cysts, eosinophilic granulomas, fibrous dysplasia of bone); and rheumatoid arthritis.

[0053] (c) Neurological inflammation, such as polyneuritis, facial neuritis, peripheral neuritis, subcutaneous neuritis, ulnar neuritis, intercostal neuritis, etc.

[0054] (d) Subcutaneous and submucosal soft tissue inflammation, such as myositis, ligamentitis, tendinitis, panniculitis, bursitis, lymphadenitis, bubonadentitis, tonsillitis, synovitis, fasciitis, and soft tissue inflammation caused by injury, contusion or tear of muscle, ligament, fascia, tendon, synovium, fat, joint capsule and lymphatic tissue.

[0055] (e) Vascular inflammation, such as allergic leukocytoclastic small vessel vasculitis, allergic cutaneous vasculitis, polyarteritis nodosa, thrombotic vasculitis, granulomatous vasculitis, lymphocytic vasculitis, vasculitis with abnormal blood composition, and rheumatic vasculitis, as well as vascular inflammation associated with vascular cancer caused by: allergic leukocytoclastic small vessel vasculitis, polyarteritis nodosa, thrombotic vasculitis, granulomatous vasculitis, lymphocytic vasculitis, vasculitis with abnormal blood composition, and rheumatic vasculitis.

[0056] (f) Inflammation of internal organs (e.g., heart, stomach, intestines, lungs, liver, spleen, kidneys, pancreas, bladder, ovaries, and prostate), including but not limited to pericarditis, myocarditis, endocarditis, pneumonia, hepatitis, spleen inflammation, nephritis, pancreatitis, cystitis, oophoritis, prostatitis, and gastric ulcers.

[0057] (g) Inflammation of the eye and surrounding area, such as conjunctivitis, keratitis (e.g., acute epithelial keratitis, nummular keratitis, interstitial keratitis, discoid keratitis, neurotrophic keratitis, myxomatous keratitis, herpes simplex keratitis, herpes zoster keratitis, bacterial keratitis, fungal keratitis, Acanthamoeba keratitis, onchocerciasis keratitis, superficial punctate keratitis, ulcerative keratitis, exposure keratitis, photosensitive keratitis, and acute conjunctivitis from contact lenses), optic neuritis, etc.

[0058] (h) Inflammation of the gums and mouth, such as periodontitis, gingivitis, oral ulcers, etc.

[0059] (i) Inflammation associated with rheumatic diseases, such as rheumatic vasculitis, rheumatoid arthritis, rheumatic bone disease, ankylosing spondylitis, bursitis, Crohn's disease, gout, infectious arthritis, juvenile idiopathic arthritis, osteoarthritis, osteoporosis, polymyalgia rheumatica, polymyositis, psoriatic arthritis, scleroderma, Sjögren's syndrome, spondyloarthritis, systemic lupus erythematosus, tendinitis, etc.

[0060] The compounds of this invention can also be used to treat certain specific diseases of the respiratory system, such as cystic fibrosis, common interstitial pneumonia, allergic pneumonia, asbestosis, emphysema, cor pulmonale, and pulmonary embolism. One specific disease condition that can be mentioned is idiopathic pulmonary fibrosis.

[0061] Idiopathic pulmonary fibrosis (IPF) is a diffuse and fatal interstitial lung disease characterized by alveolar epithelial damage, massive proliferation of pulmonary fibroblasts, and excessive extracellular matrix deposition, ultimately leading to irreversible lung tissue damage. In the later stages of the disease, subjects with IPF experience respiratory failure and death. The compounds of this invention have been found to be useful for treating IPF and / or alleviating symptoms associated with the disease.

[0062] The compounds of this invention can further exhibit antioxidant activity by increasing SOD (superoxide dismutase) production and reducing lipid oxidation. Therefore, this compound and formulations comprising this compound can be considered to possess antioxidant properties.

[0063] The compounds of the present invention may also have antipyretic properties and can treat fever and / or relieve its symptoms; for example, by lowering the body temperature of the subject, thereby reducing fever. Therefore, the compounds of the present invention and formulations comprising the compounds of the present invention can be considered as antipyretics.

[0064] According to another aspect of the invention, a method for treating inflammation, inflammatory conditions and / or conditions / symptoms characterized by inflammation (e.g., as a symptom) is provided, the method comprising administering the compound of the invention to a patient in need of such treatment.

[0065] To avoid ambiguity, in the context of this invention, the terms “treatment,” “therapeutic method,” and “method of treatment” include therapeutic or alleviating treatment for patients in need, as well as preventive treatment and / or diagnosis for patients susceptible to inflammation and / or inflammatory conditions.

[0066] "Patients" include amphibians, birds, and mammals (especially humans).

[0067] According to the present invention, the compounds are preferably administered locally or systemically, such as orally, intravenously or intra-arterially (including via intravascular and other perivascular devices / dosage forms (e.g., stents)), intramuscularly, transdermally, subcutaneously, transmucosally (e.g., sublingual or oral), rectally, vaginally, transdermally, nasally, pulmonaryly (e.g., via the trachea or bronchi), preferably locally, or via any other non-enteric route, in the form of a pharmaceutically acceptable dosage form containing the compound. Inhalation (e.g., nasal) administration is particularly useful when the symptom to be treated is rhinitis or inflammation of a viral infection originating from the respiratory tract (e.g., the common cold, influenza). Pulmonary administration is particularly useful when the symptom to be treated is COPD or IPF. Local administration can be enhanced by producing a spray containing the active ingredient, for example by using powdered aerosols or by means of a water mist using suitable atomization techniques or devices such as nebulizers.

[0068] Preferred delivery modes of the compounds of the present invention include local delivery to sites of inflammation (e.g., mucous membranes, including the lungs, or more preferably the skin) using suitable (e.g., pharmaceutically and locally acceptable) media and / or commercially available formulations suitable for application to the skin and / or suitable mucosal surfaces, but may also include oral, intravenous, percutaneous or subcutaneous, nasal, intramuscular, intraperitoneal or pulmonary delivery.

[0069] The compounds of the present invention will typically be administered in the form of one or more pharmaceutical formulations, mixed with (e.g., pharmaceutically acceptable) adjuvants, diluents, or carriers, which may be selected appropriately taking into account the intended route of administration (e.g., topical administration to the relevant mucous membrane (including the lungs) or preferably the skin) and standard pharmaceutical or other practices. Such pharmaceutically acceptable carriers may be chemically inert to the active compound and may not have harmful side effects or toxicity under the conditions of use. Such pharmaceutically acceptable carriers may also impart immediate or modified release of the active ingredient.

[0070] Suitable pharmaceutical formulations can be commercially available or prepared in other ways according to techniques described in the literature, for example, Remington, The Science and Practice of Pharmacy, 22. nd edition, Pharmaceutical Press (2012) and Martindale–The Complete Drug Reference, 38 th Edition, Pharmaceutical Press (2014), and the documents mentioned therein, all relevant disclosures of which are incorporated herein by reference. Otherwise, the preparation of suitable formulations comprising the compounds of the present invention can be carried out by those skilled in the art using conventional techniques without inventive step.

[0071] The compounds of the present invention may be in the form of aqueous formulations such as emulsions, suspensions and / or solutions (e.g., (optionally) buffered aqueous formulations, such as formulations containing physiological saline, phosphate formulations (e.g., solutions, acetate formulations or borate formulations), or lyophilized powders.

[0072] The active ingredient can be further combined with appropriate excipients to prepare:

[0073] • Gel formulations (suitable gel matrix materials include cellulose derivatives, carbomer and alginate, tragacanth gum, gelatin, pectin, carrageenan, gellan gum, starch, xanthan gum, cationic guar gum, agar, non-cellulose polysaccharides, sugars such as glucose, glycerol, propylene glycol, vinyl polymers, acrylic resins, polyvinyl alcohol, carboxyvinyl polymers, and especially hyaluronic acid);

[0074] • Detergents (suitable matrix materials for which include cellulose derivatives, glycerol, non-cellulose polysaccharides, polyethylene glycol and propylene glycol of different molecular weights);

[0075] • Paste or ointment (suitable paste base materials include glycerin, vaseline, paraffin, polyethylene glycol with different molecular weights, etc.);

[0076] • Creams or foams (for which suitable excipients (e.g., foaming agents) include hydroxypropyl methylcellulose, gelatin, polyethylene glycol of different molecular weights, sodium lauryl sulfate, sodium fatty alcohol, polyoxyethylene ether sulfonate, corn gluten meal and acrylamide);

[0077] • Powdered aerosols (with suitable excipients including mannitol, glycine, dextrin, dextrose, sucrose, lactose, sorbitol, and polysorbates, such as dry powder inhalers); and / or

[0078] • Liquid (aerosol) spray for oral or inhalation use (suitable excipients include viscosity modifiers such as hyaluronic acid, sugars such as glucose and lactose, emulsifiers, buffers, alcohols, water, preservatives, sweeteners, flavorings, etc.).

[0079] Depending on the circumstances, the following substances may also be included in such formulations: humectants, such as glycerol, glycerin, polyethylene glycol, trehalose, petrolatum, paraffin oil, silicone oil, hyaluronic acid and its salts (e.g., sodium and potassium salts), caprylic / capric triglycerides, etc.; and / or antioxidants, such as vitamins and glutathione; and / or pH adjusters, such as acids, bases and pH buffers. In addition, the following substances may be included: surfactants / emulsifiers, such as cetyl alcohol, fatty acids (e.g., stearic acid), sodium lauryl sulfate, sorbitan esters (e.g., sorbitan stearate, sorbitan oleate, etc.), monoacylglycerol esters (e.g., glyceryl monostearate), polyethoxylated alcohols, polyvinyl alcohol, polyol esters, polyoxyethylene alkyl ethers (e.g., polyoxyethylene sorbitan monooleate), polyoxyethylene castor oil derivatives, ethoxylated fatty acid esters, polyoxyglycerol esters, lauryl dimethylamine oxide, bile salts (e.g., sodium deoxycholate, sodium cholate), phospholipids, N,N-dimethyldodecylamine-N-oxide, hexadecyltrimethylammonium bromide, poloxamer, lecithin, sterols (e.g., cholesterol), glycolipids, polysorbates, etc.; preservatives, such as phenoxyethanol, ethylhexylglycerin, etc.; and thickeners, such as acryloyl dimethyl taurate / VP copolymer. In particular, stearic acid, glyceryl monostearate, cetyl alcohol, sorbitan stearate, cetyl alcohol, caprylic / caprylic acid glyceryl ester, etc., may be specifically included in cream formulations.

[0080] The compounds of the present invention and (e.g., solutions, gels, creams, ointments, lotions, foams, pastes, and / or dry powders comprising them as described above) can be further combined with suitable matrix materials to prepare dressings or therapeutic patches for administration onto biological surfaces such as skin or mucous membranes. Such formulations can therefore be used to impregnate matrix materials such as gauze, nonwoven fabric, or silk paper. The therapeutic patch may also be, for example, a band-aid, face mask, eye mask, hand mask, foot mask, etc.

[0081] Vaseline can be used to apply such dressings to wounds, but we have also found that PEG-based ointments (such as PEG400) can be combined with matrix materials to prepare dressings without the need for Vaseline.

[0082] The compounds of the present invention can also be used in combination with one or more growth factors selected from the following for treatment: platelet-type growth factors (including platelet-derived growth factor, PDGF); osteosarcoma-derived growth factor (ODGF), epidermal growth factor (EGF), transforming growth factor (TGFα and TGFβ), fibroblast growth factor (αFGF, βFGF), insulin-like growth factor (IGF-I, IGF-II), nerve growth factor (NGF), interleukin-type growth factors (IL-1, IL-3), erythropoietin (EPO), and colony-stimulating factor (CSF).

[0083] According to another aspect of the invention, compositions (e.g., pharmaceuticals) are provided comprising the compounds of the invention and one or more pharmaceutically acceptable excipients, such as adjuvants, diluents, or carriers. Preferred formulations are suitable for topical application, for example, to mucous membranes (including the lungs), or more preferably to the skin, and thus comprise topically acceptable adjuvants, diluents, or carriers.

[0084] Therefore, formulations containing the compounds of the present invention are suitable for, adapted for and / or packaged for local administration (e.g., administration to the lungs, mucous membranes or skin) by direct local administration of the formulation (e.g. administration to the lungs, mucous membranes or skin), for example for the treatment of conditions, including inflammation, inflammatory conditions and / or symptoms characterized by inflammation (e.g. as symptoms).

[0085] In this respect, to avoid doubt, the topical formulation of the present invention can be used for any and all conditions described in this application, including the treatment of inflammation, the treatment of any and all inflammatory conditions, and / or the treatment of any and all conditions characterized by inflammation, as previously mentioned, defined or described.

[0086] The topical (e.g., liquid-based or solution-based) formulations of the present invention are particularly useful for wound healing and can relieve pain (including aches and pains), especially itching / itching associated with the wound itself and the wound healing process. Such topical formulations of the present invention are particularly useful for preventing and / or inhibiting fluid exudation from the wound, especially during the acute inflammatory phase, such as during the first 48 hours after a burn or wound infection. This prevents the risk of infection and other physiological reactions. Such topical formulations of the present invention can also be particularly useful for preventing and / or inhibiting scarring and hyperpigmentation (see above), whether or not related to the wound.

[0087] The active ingredient can be administered continuously or intermittently. The dosing pattern can also be determined by the timing and frequency of administration, but in the case of therapeutic treatment of inflammation, it also depends on the severity of the symptoms.

[0088] Depending on the condition to be treated, the patient, and the route of administration, the compounds of the present invention can be administered to patients in need at different therapeutically effective doses.

[0089] Similarly, the amount of active ingredient in the formulation will depend on the severity of the condition and on the patient to be treated, but can be determined by those skilled in the art.

[0090] In any case, depending on the severity of the condition and the route of administration, a medical practitioner or other skilled worker will be able to determine the actual dose most appropriate for the individual patient in a conventional manner. The doses mentioned in this application are examples of average cases; of course, there may be individual cases in which higher or lower dose ranges are advantageous, and these are also within the scope of this invention.

[0091] The dosage can be administered once or four times a day.

[0092] The appropriate concentration of the compounds of the present invention in aqueous products can be from about 0.01 (e.g., about 0.1) to about 15.0 mg / mL, in all cases calculated as free (non-salt) peptides.

[0093] The appropriate local dose of the compounds of the present invention is from about 0.05 to about 50 μg / cm². 2 The treatment area, such as about 0.1 (e.g., about 0.5) to about 20 μg / cm². 2 The treatment area includes approximately 1 to approximately 10 μg / cm². 2 The treatment area, such as approximately 5 μg / cm² 2 The treatment area is calculated as a free (non-salt) peptide in all cases.

[0094] We preferably have formulations containing the compounds of the present invention with a pH value in the range of about 1.0 to about 9.0 (e.g., about 3.0 to about 8.0). However, we have found that the compounds of the present invention are significantly more stable at all pH values ​​(including neutral and alkaline pH) than, for example, MAP and isolated compounds consisting of the mefp-1 decapeptide sequence.

[0095] In any event, the dosage administered to mammals, particularly humans, within the scope of this invention should be sufficient to produce a therapeutic response in mammals within a reasonable timeframe (as described above). Those skilled in the art will recognize that the selection of precise dosages and compositions, as well as the most appropriate delivery methods, will also be particularly influenced by: the pharmacological properties of the formulation, the nature and severity of the condition to be treated, the recipient's physical condition and mental susceptibility, and the age, condition, weight, sex, and response of the patient to be treated, the stage / severity of the disease, and genetic differences between patients.

[0096] In the uses and methods described in this application, the compounds of the present invention may also be combined with one or more active ingredients (another anti-inflammatory agent) for treating inflammation and / or inflammatory conditions. Such patients may therefore also (and / or have) received therapy based on the administration of one or more of such another active ingredient, meaning receiving a prescribed dose of one or more of those active ingredients mentioned in this application before, in addition to, and / or after treatment with the compounds of the present invention.

[0097] Such anti-inflammatory agents that can be used in combination with the compounds of the present invention in the treatment of inflammation include therapeutic agents for treating inflammation and / or diseases characterized by inflammation as one of their symptoms. Depending on the condition to be treated, such anti-inflammatory agents may also include NSAIDs, leukotriene receptor antagonists (e.g., montelukast, as described below), corticosteroids, analgesics, and certain enzymes, such as trypsin, as described below. The compounds of the present invention may also be combined with leukotriene B4 (LTB4).

[0098] In this document, the compounds of the present invention can also be combined with one or more mussel adhesion proteins (MAPs) for the treatment of inflammation, comprising any adhesion protein that may be derived from mussel species, such as the blue mussel (Mytilus edulis), including full-length proteins, including all subtypes derived from or potentially derived from mussels, such as collagen pre-COL-P, pre-COL-D, and pre-COL-NG, mussel byssal matrix proteins PTMP and DTMP, and more preferably mfps or mefps, such as mefp-2, mefp-3, mefp-4, mefp-5, mefp-6, and especially mefp-1, and including mixtures or combinations of any of these proteins, such as mefps. While mixtures / combinations of the above-described MAP subtypes may be provided according to the present invention, it is preferred that the purity of the dominant MAP subtype (e.g., mefp-1) is at least 25% by weight of the total amount of any such mixture.

[0099] Naturally occurring MAP can be prepared, for example, by mixed adsorption chromatography (see Chinese Patent No. ZL200710179491.0), by carboxymethyl ion exchange chromatography (see Chinese Patent No. ZL200710179492.5), and / or by salting out and dialysis (Chinese Patent No. ZL200910087567.6). Commercial sources of MAP include USUN BioCo. (China; as MAP Medical). Available for sale by BD Biosciences (USA), Kollodis (South Korea), and Biopolymer (Sweden). MAP can also be prepared using known recombinant DNA methods.

[0100] Pharmaceutically acceptable derivatives of MAP can also be combined with the compounds of the present invention, and include compounds having molecular weights ranging from about 500 Da to about 2000 Da (e.g., about 1500, such as about 1200, including about 800) Da, which allow for easier penetration of biological membranes, such as skin barriers or mucosal surfaces. Such derivatives can also include other compounds containing amino acid sequences that are identical to or (e.g., minor) variants of sequences already identified in naturally occurring MAP, and which can be synthesized by chemical and / or biological methods (e.g., chemical modification of naturally occurring MAP, or direct synthesis). We use "(e.g., minor) variants of amino acid sequences identified in naturally occurring MAP" to indicate variations in those sequences that do not negatively affect the essential properties of naturally occurring MAP to a measurable degree.

[0101] For example, as described above, the isolated decapeptide compound having the following sequence: Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-DOPA-Lys(mefp-1 decapeptide) is a pharmaceutically acceptable low molecular weight MAP derivative that can be combined with the compounds of the present invention.

[0102] This MAP derivative can be used alone in the combined product according to the invention, or in combination with one or more other such derivatives and / or one or more of the aforementioned full-length MAPs.

[0103] Suitable concentrations of MAP and its derivatives in aqueous topical formulations can be from about 0.01 (e.g., about 0.1) to about 15.0 (e.g., about 1.5) mg / mL, and suitable pH values ​​can be in the range of about 1.0 to about 7.0 (e.g., about 3.0 to about 6.5), regardless of whether the formulation used is a combination formulation or a kit of parts as described above. Suitable commercial sources of such aqueous solutions include USUN Bio Co., Jiangyin, Jiangsu Province, China.

[0104] The appropriate local dose of MAP and its derivatives is in the range of approximately 0.1 to approximately 50 μg / cm³. 2 The treatment area, such as approximately 1 to approximately 20 μg / cm² 2 The treatment area includes approximately 2 to approximately 10 μg / cm². 2The therapeutic domain, such as approximately 5 μg / cm 2 The treatment area.

[0105] The combination of the compounds of the present invention and mefp-1 has been found to be particularly useful in the treatment of inflammatory bowel diseases such as ulcerative colitis, especially in stopping bleeding in patients with this disease.

[0106] Other preferred agents that can be combined with the compounds of the present invention include LTB4 (for treating wounds and burns), montelukast (generally used to treat inflammation), and trypsin (for treating mucosal inflammation associated with, for example, viral infections).

[0107] The compounds of the present invention can also be combined with other therapeutic agents, which are known to cause inflammation as a side effect when administered.

[0108] When the compounds of the present invention can be "combined" with other therapeutic agents in this manner, the active ingredients can be administered co-administered in the same formulation or separately (simultaneously or sequentially) in different formulations.

[0109] Such combination products are provided to administer the compounds of the present invention with other therapeutic agents, and thus such combination products can be embodied as individual formulations, wherein at least one of those formulations contains the compounds of the present invention and at least one contains other therapeutic agents, or can be embodied (i.e. formulated) as combination formulations (i.e. embodied as a single formulation containing the compounds of the present invention and other therapeutic agents).

[0110] Therefore, the following further information is provided:

[0111] (1) A pharmaceutical preparation comprising: the compound of the present invention; another anti-inflammatory agent, or an agent known to cause inflammation as a side effect; and a pharmaceutically acceptable adjuvant, diluent, or carrier (the preparation is referred to below as a "combination preparation"); and

[0112] (2) A medicine box containing the following components:

[0113] (A) A pharmaceutical preparation comprising: a mixture of the compound of the present invention with a pharmaceutically acceptable adjuvant, diluent, or carrier; and

[0114] (B) A pharmaceutical preparation comprising: another anti-inflammatory agent, or an agent known to cause inflammation as a side effect, mixed with a pharmaceutically acceptable adjuvant, diluent, or carrier.

[0115] Components (A) and (B) are each provided in a form suitable for administration in combination with another.

[0116] According to another aspect of the invention, a method for preparing a kit as defined above is provided, the method comprising combining component (A) as defined above with component (B) as defined above, thereby making the two components suitable for co-administration.

[0117] By “combining” the two components with each other, the components (A) and (B) of the pillbox can be:

[0118] (i) provided as separate formulations (i.e., independently of each other), and subsequently combined together for use in combination therapy; or

[0119] (ii) Packaged and presented together as individual components of a “combination package” for use in combination therapy.

[0120] Therefore, a pillbox kit is further provided, which includes:

[0121] (I) One of components (A) and (B) as defined in this application; together with

[0122] (II) Instructions for using the component in combination with another component of the two components.

[0123] The kit described in this application may contain more than one formulation comprising an appropriate amount / dosage of the compound of the present invention, and / or more than one formulation comprising an appropriate amount / dosage of another anti-inflammatory agent to provide repeated doses. If more than one formulation (containing any one active compound) is provided, the formulation may be the same or may be different in terms of the dosage, chemical composition and / or physical form of any one compound.

[0124] Regarding the kit described in this application, for "combined administration with...", we include administering, sequentially, separately and / or simultaneously, various formulations containing the compound of the present invention and another anti-inflammatory agent throughout the course of treatment of the relevant condition.

[0125] Therefore, with respect to the combination product according to the invention, the term "combined administration" includes administering the two components of the combination product (the compound of the invention and another anti-inflammatory agent) together or at sufficiently close temporal intervals (optionally repeatedly) such that the beneficial effect on the patient during the treatment of the relevant condition is greater than the effect obtained by administering the formulation containing the compound of the invention or the formulation containing another agent alone (optionally repeatedly) during the same treatment in the absence of other components. Determining whether the combination provides a greater beneficial effect on a particular condition during the treatment of that condition will depend on the condition to be treated or prevented, but can be conventionally accomplished by those skilled in the art.

[0126] Furthermore, in the case of the kit according to the invention, the term "in combination with" includes the possibility that one or the other of the two formulations may be administered before, after, and / or concurrently with the administration of the other component (optionally repeatedly). When used in this application, the terms "concurrent administration" and "concurrent administration with" include individual doses of the relevant compound of the invention and another anti-inflammatory agent administered at intervals of 48 hours (e.g., 24 hours) between each other.

[0127] In another aspect of the invention, a method for preparing a combination formulation as defined above is provided, the method comprising combining the compound of the invention, another anti-inflammatory agent or an agent known to cause inflammation as a side effect, and at least one (e.g., pharmaceutically acceptable) excipient.

[0128] When the term “about” is used in this application, for example in the context of quantity, such as the concentration and / or dosage of the active ingredient, molecular weight, or pH, it should be understood that such variables are approximate values ​​and, in themselves, can vary within the range of: ±10% of the number specified in this application, for example ±5% of the number specified in this application, preferably ±2% (e.g., ±1%) of the number specified in this application. In this respect, the term “about 10%” means, for example, ±10% around the number 10, i.e., between 9% and 11%.

[0129] The advantage of the compounds of the present invention is that they are applicable to a variety of symptoms characterized by inflammation, whether the symptom itself is an organic inflammatory disease or is related to or characterized by inflammation (e.g., wounds, burns, or viral infections).

[0130] The compounds of the present invention also have the following advantages: compared with similar compounds known in the prior art such as MAP and isolated mefp-1 decapeptide, the compounds of the present invention have physicochemical stability to processes such as oxidation at various pH levels (including neutral and alkaline pH).

[0131] The uses and methods described in this application also have the following advantages: in the treatment of the aforementioned symptoms, compared with similar methods (treatments) known in the art for treating inflammatory conditions, the uses and methods described in this application are more convenient, more effective, less toxic, have a wider range of activities, are more potent, produce fewer side effects, or may have other useful pharmacological properties, whether used to treat inflammation, inflammatory conditions, or conditions characterized by inflammation as symptoms (including wounds) or for the treatment of other aspects.

[0132] The present invention is illustrated by the following embodiments, wherein, in a mouse wound model, Figure 1The results of ELISA assays using various test compounds to obtain various inflammatory markers from exudates from air pouches induced in mice, according to Example 1, are shown. Figure 2 It demonstrated the effect of various test compounds on the acute healing of wounds induced in mice; and Figures 3 to 5 The results of ELISA assays for inflammatory markers (Hyp, VEGF, and TNF-β1, respectively) obtained from samples taken from the wounds of those mice are shown. Example

[0133] Example 1

[0134] Synthesis of Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Tyr-Lys

[0135] Fmoc-Lys-Boc-Wang resin (0.3 mmol / g; GLS180322-41301, GL Biochem, Shanghai, China) was loaded into the reaction column.

[0136] Add 2 liters of dichloromethane (DCM; Shandong Jinling Chemical Industry Inc. Co., Shandong, China) to the column and allow the resin to soak for approximately half an hour. Then, remove the DCM and wash the column three times with 2 liters of N,N-dimethylformamide (DMF; Shandong Shitaifeng Fertilizer Industry Inc. Co., Shandong, China).

[0137] Mix 200 mL of piperidine (Shanghai Li Ming Industry and Trade Co., Ltd., China) with 1 L of DMF and use as the deprotection solution. Drain the liquid after 15 minutes and wash the column 6 times with DMF.

[0138] 69 g of Fmoc-Tyr(tBu)-OH (GLS170916-36901, GL Biochem) and 48 g of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyltetrafluoroborate ammonium (TBTU; GL Biochem) were dissolved in 300 mL of DMF and added to the reaction column. Then, 53 mL of N,N-diisopropylethylamine (DIPEA; Suzhou Highfine Biotech Co. Ltd, Jiangsu, China) was added. The reaction time was 1 hour.

[0139] Samples were taken, and ninhydrin (Shanghai Shanpu Chemical Co., Ltd., China) was used to determine when the reaction was complete. At this point, the liquid was drained and the residue was washed three times with DMF.

[0140] Repeat the above coupling steps to couple the same amount of the remaining amino acids: Fmoc-Thr(tBu)-OH, Fmoc-4-Hyp(tBu)-OH, Fmoc-4-Hyp(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Ala-OH.

[0141] At the end of the reaction sequence, 20% piperidine in DMF was added as a deprotection solution as described above. The liquid was then drained after 15 minutes, and the column was washed three times each with DMF, DCM, and methanol.

[0142] The liquid is drained to obtain the resin-bound polypeptide.

[0143] An appropriate amount of lysis buffer, consisting of 95% trifluoroacetic acid (TFA), 2.5% water, and 2.5% triisopropylsilane (Tis), was added to impregnate the resin-bound peptides. The mixture was placed on a shaker and incubated at 30°C to 35°C for 2 hours. The resin was then removed by filtration.

[0144] Add anhydrous ice-cold ether to the filtrate, centrifuge, and discard the supernatant. Wash the resulting product three times with anhydrous ice-cold ether. Dry the separated peptides using a desiccation agent to obtain 128 g of crude peptide. Desalt the crude compound using anion exchange resin, analyze, and freeze-dry. Approximately 70.7 g of purified peptide was obtained after purification and retested for confirmation.

[0145] 1 mg of the crude product was dissolved in 1 mL of a mixture of acetonitrile and water (1:3) and detected using a P3000A HPLC pump and an LC3000 semi-preparative apparatus (preparative column: GS-120-10-C18-AP 30 mm; Beijing Chuangxintongheng Science & Technology Co., Ltd., Beijing, China). An appropriate elution gradient was calculated, and the target peak was detected at 14.351 min using LCMS (analytical column: GS-120-5-C18-BIO, 4.6*250 mm; detection: UV at 220 nm; solvent A: 0.1% TFA in MeCN, solvent B: 0.1% TFA in water; flow rate: 1.0 mL / min; volume: 10 μL).

[0146] m / z 592.65 [M+2H] 2+ (97.89%).

[0147] Example 2

[0148] airbag model

[0149] Healthy adult male C57BL / 6 mice weighing between 20 and 30 g were provided by the Nanjing Biomedical Research Institute (NBRI), Nanjing University. Prior to any experiments, the mice were placed under standardized conditions (a constant temperature of 22 ± 2 °C, with alternating light and dark periods of 12 hours each) and fed a standard mouse diet with water for approximately one week.

[0150] General anesthesia was administered via intraperitoneal injection of 3% chloral hydrate (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China; 1 mL / 10 g body weight). Hair was shaved and removed from the entire back one day prior to the sterile air injection.

[0151] The air sac was generated as follows: sterile air (5 mL) was subcutaneously injected into the scapular region of mice. Three days later, a second injection of air (3 mL) was performed to maintain the air sac. To induce acute inflammation, three days after this final injection, the animals were injected with a sterile carrageenan solution (CP Kelco, Taixing, Jiangsu Province, China; 1%, 0.5 mL; prepared by adding 0.1 g of carrageenan powder to a beaker containing 10 mL of 0.9% saline solution and stirring). Mice were pretreated with test samples or media 1 hour before and 23 hours after the carrageenan injection into the subcutaneous air sac. The animals were sacrificed 24 hours after the carrageenan injection.

[0152] Skin biopsies were obtained from an air bag. A portion of the biopsy was fixed in formalin (prepared by adding ultrapure water to 50 mL of 40% formaldehyde solution (Nanchang Rain Dew Experimental Equipment Co., Ltd., Nanchang, Hubei Province, China) to a total volume of 500 mL) and analyzed by embedding the tissue in paraffin, sectioning, and staining.

[0153] The cavity was washed with 4 mL of sterile phosphate buffer solution (pH 7.4; prepared by dissolving 4 g of NaCl, 0.1 g of KCl, 1.749 g of Na2HPO4·12H2O and 0.1 g of KH2PO4 in ultrapure water, adjusting the pH to 7.4 with HCl and diluting with water to a total volume of 500 mL).

[0154] Dissolve the collected exudate and quantify its volume. Centrifuge the exudate at 3000 rpm at 4°C for 10 minutes, collect the supernatant and store it at -20°C for ELISA analysis of the following: tissue necrosis factor-α (TNF-α), interleukin-1-β (IL-1β), and interleukin-6 (IL-6) using a standard ELISA test kit (from Beijing 4ABiotech Co., Ltd. (Beijing)) and an ELISA reader (SH-1000Hitachi, Japan).

[0155] After conducting some preliminary experiments to validate the model, the following experiments were performed, in which mice were treated with test samples or media according to Table 1 below.

[0156] In Table 1, the MAP solutions were prepared as follows. Blue mussels were collected from the coastal area of ​​Shandong Province, China. The byssal threads were collected, cut into small pieces, and homogenized in an extraction buffer containing 5% acetic acid in 4 mol / L aqueous urea. The crude extract was collected after centrifugation and then purified by liquid chromatography. The purified protein (semi-finished product; concentration 8 mg / mL; purity 91.72% as determined by HPLC; pH 4.2) was stored at 0°C. The solutions used below were prepared by adding saline solution to the semi-finished product to obtain the concentrations shown in Table 1.

[0157] The isolated compound (hereinafter "Compound A"), consisting of the mefp-1 decapeptide sequence Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-DOPA-Lys, was purchased from Innovagen (Innovagen AB, Lund, Sweden). It was stored as a powder at -20°C and dissolved in saline solution to a concentration of 0.6 mg / mL at pH 5.5. 0.5 mL of the solution was injected.

[0158] The compound of this invention (compound B) was synthesized by GL Biochem (Shanghai) Ltd. according to Example 1 above, and was also stored as a powder at -20°C and dissolved in brine at a concentration of 0.6 mg / ml and pH 5.5. 0.5 mL of the solution was injected.

[0159] All substances listed in Table 1 are administered locally via direct injection into the balloon.

[0160] Table 1

[0161]

[0162] Histological specimens were analyzed and assessed as follows: inflammation score, activity score (i.e., the number and density of neutrophils shown in pathological slides, indicating the degree of inflammation and, in the case of open wounds and infectious diseases), edema score, and fibroblast proliferation score.

[0163] HE-stained sections were examined under an optical microscope and scored according to perceived inflammation levels (1, 2, or 3 points) (1 point for mild inflammation showing only a small number of inflammatory cells scattered throughout the area; 2 points for moderate inflammation showing many inflammatory cells; and 3 points for severe inflammation showing diffuse infiltration). A similar scoring system was used for edema levels after overall observation (3 points for most severe and 1 point for mild). Neutrophil scoring was performed using the same method as for inflammatory cells.

[0164] Table 2

[0165]

[0166] The ELISA results of TNF-α, IL-6 and IL-1β in the exudate are as follows: Figure 1 The results show that compound B has a very strong anti-inflammatory effect. (See diagram.)

[0167] Example 3

[0168] Acute wound model

[0169] Male C57BL / 6 mice aged 6-8 weeks were provided by Changzhou Cvens Experimental Animal Co., Ltd. (Changzhou, Jiangsu Province, China). Before any experiments, the mice were placed under standardized conditions (a constant temperature of 22±2℃, with alternating light and dark periods of 12 hours each) and fed a standard mouse diet with water for approximately one week.

[0170] General anesthesia was administered via intraperitoneal injection of 3% chloral hydrate (1 mL / 10 g body weight). Hair on the back was shaved with an infant razor and treated with hair removal cream. The skin area was wiped and disinfected twice with 75% alcohol.

[0171] A circular incision was made on the back using an 18mm diameter EMS skin sampler (Electron Microscopy Sciences, PO Box 550, 1560 Industry Road, Hatfield, PA, USA). Full-thickness skin was removed, with the incision reaching the fascia. The incision was left open and not sutured.

[0172] Different drugs were administered topically at 50 μL per wound, once daily from day 0 to day 12, as shown in Table 3 below. The control group did not have wounds treated. The model group received the same amount of saline. Each group contained 8 mice.

[0173] Recombinant human epidermal growth factor (rhEGF, Shanghai Haohai Biological Technology Co., Ltd., 23 / F, Shanghai, China) was purchased and prepared according to the manufacturer's instructions. Lyophilized rhEGF powder (100,000 IU / vial) was dissolved in 20 mL of physiological saline to prepare a solution with a concentration of 5000 IU / mL. The working dose of rhEGF in this experiment was 1285 IU / wound.

[0174] For this experiment, compound A was obtained from GL Biochem (Shanghai) Ltd. The peptide powder was stored at -20°C and dissolved in saline at concentrations of 61.8 μg / mL (compound A) and 61 μg / mL (compound B). 50 μL of the solution was applied to the wound surface.

[0175] Table 3

[0176] Group sample dose Comparison brine / Model Model + Salt Water / rhEGF Model + rhEGF 1285 IU / mouse Compound A Model + Compound A 3.09 μg / mouse Compound B Model + Compound B 3.05 μg / mouse

[0177] After medication administration, the wounds were covered with gauze and transparent dressings. Photographs were taken every other day starting from day 0. The photographs were scanned into a computer, and the wound area was calculated using ImageJ image analysis software (National Institutes of Health, China).

[0178] The area of ​​unhealed wounds is expressed as a percentage of the original wound area:

[0179] A t / A0×100%,

[0180] Where A0 and A t These refer to the initial area on day 0 and the wound area on the measurement date (time t), respectively.

[0181] Samples were collected on days 4 and 7 post-wound injury. Mice were euthanized and wound tissue was extracted using the same skin sampler used to create the wound. A 5 mm section of tissue was then excised from the center of the sample, preserved in 10% neutral buffered formalin (Nanchang Rain Dew Experimental Equipment Co., Ltd., Nanchang, Hubei Province, China), and analyzed by hematoxylin and eosin (HE) embedding in paraffin, sectioning, and staining.

[0182] Paraffin sections stained with hematoxylin and eosin (HE) and Masson's Law were analyzed under an optical microscope. Skin regeneration, fibroblast proliferation, collagen regeneration scores, and inflammation scores were assessed.

[0183] The remaining sample was stored at -80°C for further analysis. The tissue was cut into small pieces, and liquid nitrogen was added to increase its fragility. 9 mL of physiological saline was added to 1 g of tissue, and the tissue was then ground at 55 Hz for 60 seconds using a tissue homogenizer (Shanghai Jingxin Industrial Development Co., Ltd., Shanghai, China), followed by centrifugation at 8000 rpm at 4°C for 10 minutes.

[0184] The supernatant was collected, and the extracted proteins were used to determine vascular endothelial growth factor (VEGF), transforming growth factor β1 (TGF-β1), and hydroxyproline (Hyp) using a standard ELISA kit and ELISA reader (SH-1000 Hitachi, Japan). The ELISA kit was purchased from Beijing 4ABiotech Co., Ltd. (Beijing, China).

[0185] The effect of compound B on wound healing is shown in Table 4 and Figure 2 The figure shows the ratio of the remaining wound area of ​​the initial wound in different groups (±SD, in the case of Table 4).

[0186] Table 4

[0187]

[0188]

[0189] The data above show that low-dose compound B improved wound healing. The improvement rate was defined as (residual wound ratio in the treatment group / residual wound ratio in the model group) × 100%. On day 4, the improvement rate in the compound B group was approximately 10%.

[0190] The wound Hyp (μg / mg) content, as an indicator of collagen regeneration, is shown in Table 5 below. Figure 3 middle.

[0191] Table 5

[0192]

[0193] The results for VEGF content (pg / g) are listed in Table 6 below, and... Figure 4 The diagram in the middle is shown.

[0194] Table 6

[0195]

[0196] The results showed that, compared with the model group, compound B increased the production of VEGF in the injured tissue by approximately 56.7%.

[0197] The results of TGF-β1 content (pg / g) are listed in Table 7 below, and... Figure 5 The diagram in the middle is shown.

[0198] Table 7

[0199]

[0200] Example 4

[0201] Liquid spray for treating allergic dermatitis .

[0202] Compound B (see Example 1 above) was dissolved in water for injection (WFI; prepared by Yangzhou Tiancheng Water Treatment Devices & Engineering Co., Ltd., Yangzhou, China) at a concentration of 0.3 mg / mL.

[0203] The participants enrolled in this study had sensitive skin. Prior to treatment, patients experienced symptoms including redness, itching, and swelling. Participants were required to use a liquid spray in the morning and evening after cleansing their faces.

[0204] All participants reported relief from itching within 8 minutes of the first use. After one day of use, the symptoms of capillary congestion disappeared, and the patients' faces were no longer red and swollen.

[0205] The experiment showed that the liquid spray containing the compounds of the present invention rapidly relieved itching and reduced redness, swelling and other symptoms caused by allergies.

[0206] Example 5

[0207] Creams for treating closed comedones

[0208] Compound B (10 mg; see Example 1 above) was first dissolved in WFI (10 g).

[0209] Then a mixture containing sorbitan stearate (2g), cetyl alcohol (4g), caprylic / capric glyceride (6g) and glyceryl monostearate (3g) (all from Sinopharm Chemical Reagent Co. Ltd.) was prepared, stirred and heated to 85°C to completely melt the mixture.

[0210] Glycerin (5 g; Sinopharm Chemical Reagent Co., Ltd.) and ammonium acryloyldimethyl taurate / VP copolymer (0.13 g; Clariant Chemical (Guangzhou) Co., Ltd., China) were mixed with 68.86 g WFI. The mixture was stirred and heated to 85 °C to obtain a homogeneous colloidal suspension.

[0211] The copolymer / water mixture was added to the mixture containing sorbitol stearate, and then rapidly stirred for 5 minutes using an emulsifier. The resulting emulsion was cooled to room temperature.

[0212] The compound B solution was then added to the resulting mixture while stirring to obtain the final cream. The concentration of compound B in the final cream was 0.1 mg / g.

[0213] Participants in the study had closed comedones (clogged pores) and were in the acute phase of acne. Symptoms included sebaceous glands blocked by keratinocytes, forming slightly hard bumps and protruding whiteheads.

[0214] After cleansing the face, apply the cream evenly both morning and evening. The bumps disappeared after one day of use, indicating that the cream containing the compound of this invention can be used to treat closed comedones.

[0215] Example 6

[0216] Determining the antioxidant capacity of the compounds of this invention.

[0217] The antioxidant capacity of compound B was determined using the 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl (DPPH) assay.

[0218] Compounds A and B were obtained as described in Example 3 above. The peptide powder was stored at -20°C and dissolved in brine at the concentrations shown below. DPPH was purchased from Sigma-Aldrich, China, Shanghai. Other reagents used and identified below were purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd., China.

[0219] The DPPH method is one of the most commonly used methods for determining antioxidant capacity. DPPH is a stable nitrogen-centered free radical, and its solution is deep purple with a maximum absorption peak at 517 nm. When a free radical scavenger is present in the reaction system, the free radical scavenger can pair with a single electron of DPPH·, and the absorption peak at 517 nm gradually disappears.

[0220] The degree of color change is stoichiometrically related to the number of paired electrons. Therefore, antioxidant activity can be measured based on changes in absorbance. The greater the inhibition rate, the stronger the antioxidant capacity.

[0221] Weigh 6 mg of DPPH and place it in a 100 mL volumetric flask. Dilute to volume with 80% ethanol to prepare a solution with a concentration of 0.06 mg / mL. Store in the dark at 4°C.

[0222] The peptide solution is prepared by dissolving 0.6g of peptide powder in 1mL of distilled water.

[0223] Add 200 μL of peptide solution to 3.8 mL of DPPH solution. Incubate the solution at room temperature in the dark for 1.5 hours. Measure the absorbance at 517 nm and express it as A1. The absorbance of the blank control sample is expressed as A0. Therefore, the percentage of remaining DPPH is calculated as follows: Clearance (%) = [A0 - A1] / A0 × 100

[0224] Compound A exhibited a DPPH scavenging rate of 52.11%. Compound B showed a much lower DPPH scavenging rate of 8.9%, indicating a significant improvement in antioxidant capacity.

[0225] Example 7

[0226] Stability of the compounds of this invention

[0227] The stability of compounds A and B (obtained as described in Examples 1 and 3 above) was tested as follows.

[0228] Dissolve 10 mg of each compound in 1 mL of distilled water to prepare a stock solution with a concentration of 10 mg / mL. Then, dilute 200 μL of the stock solution with 20 mM phosphate buffer (PB; Sigma-Aldrich China) with different pH values ​​as shown in Table 8 below.

[0229] Table 8

[0230]

[0231] After preparation, all the above samples were filtered through a 0.22 μm membrane (SLGV033RS; Sigma-Aldrich China) on a clean bench to remove potential bacteria in the solution.

[0232] All samples were stored at room temperature. After 3 days, they were transferred to a 45°C oven after preliminary HPLC analysis (column: Angilent ZORBAX Eclipse XDB-C18 (4.6×250mm; 5μm) (SN: USNH008244); buffer: A: 0.1% TFA in water; B: 0.1% TFA in ACN; gradient: 0–25 min: 5%–30% B; 25–30 min: 100% B; flow rate: 1 mL / min; detection wavelength: 220 nm; sample volume: 20 μL).

[0233] The stability of the above samples was studied by observing changes in color (Table 9), HPLC peak area (Table 10), and purity (Table 11). The pH value of all samples was stable during the testing period.

[0234] Table 9

[0235]

[0236] NO indicates that no color change was observed. + indicates that a slight color change was observed. ++ indicates that a significant color change was observed.

[0237] As shown in Table 9, the compound A solution became darker over time, especially at higher pH values. The colors of the other samples remained unchanged.

[0238] Table 10

[0239]

[0240] In Table 10 above, the peak area percentage representing the results for day 0 indicates peptide concentration. As shown in Table 10, the peak area of ​​the three samples of compound B showed almost no change. For compound B, the peak area at pH 5 remained unchanged, but the peak area decreased significantly at pH 7.2 and pH 8.1, indicating that the peptide concentration decreased over time.

[0241] Table 11

[0242]

[0243] When peptides are oxidized, the HPLC peak shifts and the purity of the peptide decreases. As shown in Table 11 above, the results indicate that most compound A is oxidized at pH 7.2 and pH 8.3.

[0244] The results above show that the compounds of the present invention are stable at all pH values, but compound A is stable only at pH 5.3.

[0245] Example 8

[0246] Compound B / Montaluscare Dressing

[0247] The ointment was prepared as follows: First, montelukast sodium (200 mg; Arromax Pharmatech Co., Ltd., Suzhou, China) was dissolved in polyethylene glycol 400 (20.0 g; Sinopharm Chemical Reagent Co., Ltd.) with stirring. Then, compound B (16 mg; see Example 1 above) was added to the solution.

[0248] Polyethylene glycol 3350 (21.3 g; Sinopharm Chemical Reagent Co., Ltd.) was dissolved in polyethylene glycol 400 (58.5 g) by heating to 60 °C with stirring. After the solution was cooled to 40-50 °C, a solution containing compound B and montelukast was added to it with stirring, and then mixed for 5 to 10 minutes. The mixture was cooled to room temperature to obtain the final product.

[0249] Apply the ointment evenly to the gauze using a flat plate. Cool to room temperature to obtain the final dressing.

[0250] Example 9

[0251] Compound B / trypsin spray

[0252] Compound B (30 mg; see Example 1 above) was dissolved in 10 mL of water. Trypsin (30 mg; SichuanDeebio Pharmacutical Co. Ltd., Guanhan, Sichuan, China) was dissolved in another 10 mL of water.

[0253] Calcium chloride (0.1 g), ethanol (0.5 g), water-soluble methanol (0.01 g), lactic acid (0.01 g), and glycerol (30 g) (all from Sinopharm Chemical Reagent Co. Ltd) were mixed together in 49.32 mL of water.

[0254] A solution containing compound B and trypsin is added to the mixture while stirring to provide the liquid for spraying.

[0255] Example 10

[0256] Compound B / trypsin aerosol for inhalation

[0257] Essentially as described in Example 9 above, an aerosol formulation was prepared from 20 mg of compound B and 30 mg of trypsin, without glycerol, and with a total of 99.32 mL of water.

[0258] Example 11

[0259] Common cold research

[0260] The participants in the study had a viral infection that they self-diagnosed as a common cold. They experienced symptoms, including cough, sore throat, and loss of voice, lasting for two days. They had not used any other cold medications prior to treatment.

[0261] Subjects were required to use the liquid spray of Example 10 described above in the morning and evening.

[0262] Each time you use the spray, pour 3 mL of the spray into the nebulizer reservoir, which is connected to the nebulizer. Then turn on the nebulizer and place the mask over the subject's mouth and nose.

[0263] Subjects reported relief from sore throat after the first use. After the second use, subjects no longer experienced sore throat and were able to speak, indicating that the liquid spray containing the compound of the present invention can relieve cough and sore throat symptoms caused by the common cold.

[0264] Example 12

[0265] Idiopathic pulmonary fibrosis (IPF) model

[0266] Experimental animals and grouping: Forty adult male Sprague Dawley rats were divided into four groups after 7 days of acclimatization: sham operation group, IPF model group (no treatment; model), experimental group (compound B), and positive control group.

[0267] Compound B was administered to rats at a dose of 130 μg / rat via nebulized inhalation. Pirfenidone was administered orally at a single-bolus dose of 120 mg / kg. Beijing Continent Pharmaceutical Co., Ltd. (Beijing, China) was used as a positive control.

[0268] A pulmonary fibrosis model was established by intratracheal instillation of bleomycin. Rats were anesthetized and placed on an operating table in a supine position to expose the trachea. Bleomycin (5 mg / kg) saline solution was injected into the trachea through the gap between the tracheal cartilage rings. The sham-operated group received an equal volume of physiological saline. Shortly after administration, the rats were lifted vertically and rotated to evenly distribute the drug. Once the rats recovered, approximately 5 days later, they were administered different drugs continuously for 28 days as planned. The experimental protocol is shown in Table 12.

[0269] Table 12

[0270] Group treat Dosage dose Sham brine Nebulized inhalation / Model brine Nebulized inhalation / Positive control Pirfenidone tube feeding 250mg / kg Compound B Compound B Nebulized inhalation 130 μg / rat

[0271] The exhaust volume of the nebulizer was 0.15 mL / min. The inhalation time for each rat was 1 minute.

[0272] On day 29 after drug administration, rats were anesthetized by intraperitoneal injection of chloral hydrate. Blood was collected from the orbital cavity, and the rats were euthanized. The thoracic cavity was rapidly opened, and the lungs were collected for further analysis. Lung wet weight was measured, and the lung coefficient (lung wet weight / rat weight × 1000) was calculated. The tissues were stored at -80°C for further use.

[0273] A lung tissue sample was accurately weighed and nine times its weight in saline solution was added. The tissue was then homogenized and centrifuged at 3000 rpm for 10 minutes. The homogenate was used to detect the expression of transforming growth factor-β1 (TGF-β1), tumor necrosis factor-α (TNF-α), and α-smooth muscle actin (α-SMA). Detection was performed using a standard ELISA method. The levels of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in the lung tissue were also measured.

[0274] The effects of compound B on the levels of TNF-α, α-SMA, and TGF-β1 in bleomycin-induced pulmonary fibrosis tissues of rats are shown in Table 13.

[0275] Table 13

[0276]

[0277]

[0278] The results showed that compound B inhibited the production of TNF-α, α-SMA, and TGF-β1 in bleomycin-induced pulmonary fibrosis lung tissue in rats. The inhibitory effect was almost comparable to that of the known IPF drug, pirfenidone.

[0279] The effects of compound B on lung coefficient, MDA and SOD content are shown in Table 14.

[0280] Table 14

[0281]

[0282] The results showed that the lung coefficient value of the compound B group was lower than that of the model group, indicating that compound B can reduce edema. Compared with the model group, the lower MDA value and higher SOD value in the compound B group indicate that compound B has an antioxidant effect by increasing SOD production and reducing lipid oxidation.

[0283] Example 13

[0284] Mouse cough suppression experiment - ammonia-induced cough method .

[0285] Thirty-six ICR mice were randomly divided into three groups based on their body weight: a CMC-Na (negative) control group, a dextromethorphan hydrobromide (positive) control group, and a compound B (B) group. Each group contained 12 mice, with 6 males and 6 females in each group.

[0286] Compound B was administered via nebulized inhalation using the YLS-8A multifunctional cough and sputum induction device (Jinan Yiyan Science and Technology Co., Ltd.) (0.15 mL / min) for 1 minute, once daily for 5 days. The positive control drug was administered via gavage at a dose of 10 mg / kg, once daily for 5 days.

[0287] Mice were placed in inverted beakers at 1, 2, and 4 hours after the last drug administration. 1 mL of ammonia (25.0% to 28.0%) was placed on top of a boiling water bath and evaporated into the beakers.

[0288] The duration of ammonia exposure in mice was determined by a method characterized by immediate data analysis following a single trial or batch of trials. The results of the previous analysis determined how to conduct the next trial or batch of trials. In this way, the experiment-analysis-experiment process proceeded sequentially as follows:

[0289] (a) The time taken for the previous mouse to cough determined the predetermined time (shorter) for the next mouse to receive ammonia stimulation; or

[0290] (b) If the previous mouse does not cough, the subsequent mouse will be stimulated for a longer period of time.

[0291] The difference between the logarithms of two adjacent time periods is 0.1.

[0292] The number of coughs per minute was detected using a stethoscope. A mouse was marked as "coughing" if it coughed three or more times in one minute, and marked as "no coughing" if it coughed less than three times in one minute.

[0293] EDT 50 The ammonia stimulation time, defined as the time at which half of the mice develop a "cough," is calculated using the following formula:

[0294] EDT 50 =lg -1 c / n

[0295] (c equals the sum of r and x, where r is the number of animals in each stimulus time group, x is the logarithm of the stimulus time, and n is the number of animals in each group). lg is the logarithmic value to the base 10. The results are shown in Table 15 below.

[0296] Table 15

[0297]

[0298] In Table 15, R represents the EDT in the treatment group. 50 Divided by the EDT in the control group 50 R value is expressed as a percentage. An R value > 130% indicates that the compound has an antitussive effect, and an R value > 150% indicates that the compound has a significant antitussive effect.

[0299] The results showed that compound B had a positive effect on cough relief at 1 hour and 2 hours.

[0300] Example 14

[0301] Liquid spray for treating the common cold

[0302] Compound B was dissolved in water for injection (WFI; from a TC-RO-0.25T / h-2 water treatment system, Yangzhou Tiancheng Water Treatment Devices & Engineering Co., Ltd., Yangzhou, China). The concentration was 0.5 mg / mL. 5 mL was then dispensed into a plastic water spray bottle.

[0303] Participants enrolled in the study had a common cold for 2 days prior to treatment. No other cold treatments were used prior to treatment. Patients experiencing cough, sore throat, and loss of voice were required to use a liquid spray (3 to 5 times each in the morning and evening).

[0304] The subjects reported relief from throat pain after the first use. After the second use, the subjects felt no more throat pain and were able to speak.

[0305] The experiment showed that the liquid spray containing the compounds of the present invention reduced symptoms of cough and sore throat caused by the common cold.

[0306] Example 15

[0307] aerosol inhalation for treating fever .

[0308] The same formulation described in Example 14 was administered to a study subject who had a fever (body temperature exceeding 38.5°C) for one day. No other antipyretics had been used prior to treatment.

[0309] Subjects were instructed to use the aerosol inhalation device in the morning and evening as follows: pour 3 mL of the formulation into the nebulizer reservoir connected to the nebulizer and mask, and wear the mask over the mouth and nose.

[0310] The subjects felt their body temperature return to normal within two hours. After three uses, no further fever symptoms occurred.

[0311] The experiment showed that inhalation of aerosols containing the compounds of the present invention reduced fever.

[0312] Example 16

[0313] Liquid spray to relieve pain during laser surgery .

[0314] Registered subjects were required to use a 1 mL liquid spray from a water spray bottle on half of their face before and after the lattice laser operation. The liquid spray contained the same formulation as described in Example 14 above to remove facial melanin.

[0315] Subjects reported no pain on the side of their face where the spray was applied during the procedure. Similarly, subjects reported no pain after the procedure. Conversely, they experienced pain on the side of their face where the spray was not applied during the procedure. Furthermore, the pain persisted for approximately 2 hours after the procedure.

[0316] The experiment showed that the liquid spray containing the compounds of the present invention reduced surgical pain during laser surgery.

[0317] Example 17

[0318] Combination therapy for ulcerative colitis

[0319] A gel containing compound B (obtained as described above) and mefp-1 (USUN Bio Co., Jiangyin, China) at a concentration of 1 mg / g was prepared as follows: the active ingredient was combined with methylcellulose (2.5%), propylene glycol (11%), glycerol (11%), and acetic acid (pH adjuster; up to 0.5 g) to obtain a gel precursor with a pH of 5.5. All excipients were obtained from Sinopharm Chemical Reagent Co., Ltd. The gel was prepared with water for injection. The gel was then loaded into a disposable anal-enteric drug delivery system.

[0320] Prior to treatment, the subject had more than 20 bowel movements per day and exhibited severe bleeding and ulceration in the colon. The gel preparation was administered rectally. After one dose on the first day, the patient had only 3 bowel movements on the second day. The subject then received two more doses on the second day. By the third day, the bleeding was under control.

[0321] After 14 days of administration of two doses daily, the patient's symptoms of ulcerative colitis had improved and there was no further bleeding. sequence list <110> Aetia (Shanghai) Pharmaceutical Co., Ltd. <120> New drug uses <130> TSACZ / P69389CN <140> 201980036092X <141> 2019-05-27 <150> PCT / CN2018 / 088701 <151> 2018-05-28 <160> 1 <210> 1 <211> 10 <212> PRT <213> Synthetic <220> <221> SITE <222> 6 <223> Xaa is 3- or 4-hydroxyproline. <220> <221> SITE <222> 7 <223> Xaa is 3- or 4-hydroxyproline. <400> 1 Ala Lys Pro Ser Tyr Xaa Xaa Thr Tyr Lys 1 5 10

Claims

1. Compound Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Tyr-Lys, The use of a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating inflammation, inflammatory conditions and / or conditions characterized by inflammation, wherein said conditions are selected from wounds, dermatitis, acne and idiopathic pulmonary fibrosis, and wherein said compound is administered topically in the form of a topical formulation.

2. The use as described in claim 1, wherein the compound is: 。 3. The use as described in claim 1 or claim 2, wherein the compound is not in the form of a salt.

4. Use of a pharmaceutical preparation in the manufacture of a medicament for treating inflammation, inflammatory conditions, and / or conditions characterized by inflammation, said pharmaceutical preparation comprising a compound as defined in any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant, diluent, or carrier, wherein said condition is selected from wounds, dermatitis, acne, and idiopathic pulmonary fibrosis, and wherein the compound or salt thereof provided in the preparation is administered topically as a topical preparation.

5. Use of a pharmaceutical preparation in the preparation of a medicament for treating inflammation, inflammatory conditions, and / or conditions characterized by inflammation, said pharmaceutical preparation comprising a compound as defined in any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof; another anti-inflammatory agent; and a pharmaceutically acceptable adjuvant, diluent, or carrier, wherein said condition is selected from wounds, dermatitis, acne, and idiopathic pulmonary fibrosis, and wherein the compound or salt thereof provided in the preparation is administered topically in the form of a topical preparation.

6. Use of a kit in the preparation of a medicament for treating inflammation, inflammatory conditions, and / or conditions characterized by inflammation, said kit comprising the following components: (A) A pharmaceutical preparation comprising a compound as defined in any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, and a mixture thereof with a pharmaceutically acceptable adjuvant, diluent, or carrier; and (B) A pharmaceutical preparation comprising a mixture of another anti-inflammatory agent and a pharmaceutically acceptable adjuvant, diluent, or carrier. Components (A) and (B) are each provided in a form suitable for co-administration with another component. The conditions described are selected from wounds, dermatitis, acne, and idiopathic pulmonary fibrosis, and the compounds or their salts provided in the kit are administered topically in the form of a topical preparation.

7. The use as described in any one of claims 1 to 6, wherein the inflammatory condition is psoriasis or eczema.

8. The use as described in any one of claims 1 to 6, wherein the dermatitis is atopic dermatitis or steroid-dependent dermatitis.

9. The use as described in any one of claims 1 to 6, wherein the inflammatory condition is a burn.

10. The use as described in any one of claims 1 to 6, wherein the wound is an abrasion, scratch, incision, tear, puncture wound, avulsion, bruise, scar, or blister, or itching associated with any of the foregoing.

11. The use as described in any one of claims 1 to 6, wherein the characteristic is that the inflammatory symptom or condition is hemorrhoids.

12. The use as described in any one of claims 1 to 6, wherein the administration is a direct local administration onto the mucosal surface.

13. The use as described in claim 12, wherein the administration is a direct local administration to the lungs.

14. The use as described in claim 12, wherein the administration is a direct topical application to the skin.

15. The use as described in claim 5 or claim 6, wherein the other anti-inflammatory agent is montelukast or a pharmaceutically acceptable salt thereof, and the condition is a wound, burn, or hemorrhoids.

16. The use as claimed in claim 5 or claim 6, wherein the other anti-inflammatory agent is montelukast or a pharmaceutically acceptable salt thereof, and the condition is idiopathic pulmonary fibrosis.

17. The use as described in claim 5 or claim 6, wherein the other anti-inflammatory agent is mefp-1.

18. The use as described in claim 5 or claim 6, wherein the other anti-inflammatory agent is trypsin.

19. A method for preparing a pharmaceutical formulation as defined in any one of claims 4 to 18, the method comprising combining a compound as defined in any one of claims 1 to 3 with one or more adjuvants, diluents or carriers.

20. A method for preparing a pillbox as defined in any one of claims 6 to 18, the method comprising combining component (A) of the pillbox with component (B) of the pillbox.

21. Use of the compound of any one of claims 1 to 6 or a pharmaceutically acceptable salt, formulation, or kit of the compound or the compound thereof in the preparation of a medicament for relieving pain associated with inflammation and / or wounds, wherein the pain is surgical pain, and wherein the compound or salt thereof provided in the formulation or kit is administered topically as a topical formulation.

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