New drug use

Abstract

Provided is a peptide of the sequence Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Tyr-Lys, or a salt thereof, for use as a pharmaceutical. The peptide is particularly useful in the treatment of conditions characterized by inflammation, including wounds, burns, hemorrhoids, psoriasis, acne, atopic dermatitis, COPD (chronic obstructive pulmonary disease), ulcerative colitis, and IPF. [Selected Figure] Figure 1

Description

[Technical Field]

[0001] The present invention relates to new uses of known compounds in human medicine, and to pharmaceutical compositions containing same. In particular, the present invention relates to the use of compounds and compositions thereof in the treatment of inflammation. [Background technology]

[0002] Inflammation is typically characterized as a localized tissue response to invasion by, for example, microorganisms, specific antigens, damaged cells, or physical and / or chemical agents. The inflammatory response is usually a protective mechanism that destroys, weakens, or sequesters both the injurious agent and the damaged tissue, and initiates tissue healing.

[0003] Inflammation can result from physical trauma, infection, certain chronic diseases (e.g., autoimmune diseases such as psoriasis and rheumatoid arthritis), and / or a chemical and / or physiological response to an external stimulus (e.g., part of an allergic reaction). A complex series of events can be involved in which inflammatory mediators increase blood flow and local vascular dilation, resulting in redness and heat, exudation of fluid, often resulting in local swelling, leukocyte migration to the inflamed area, and pain.

[0004] Many pathologies / disorders are characterized by and / or caused by abnormal tissue damage inflammation.These pathologies are typically characterized by the activation of immune defense mechanisms, resulting in more harmful effects than beneficial effects for the host, and are generally associated with varying degrees of tissue redness or hyperemia, swelling, hyperthermia, 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.

[0005] Typically, a complex series of events leads to inflammatory changes, including increased blood flow due to local vascular dilation resulting in redness and heat, extravasation of leukocytes and plasma often resulting in local swelling, activation of sensory nerves (resulting in pain in some tissues), and loss of function. These inflammatory changes are caused 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.

[0006] In particular, inflammation plays an important role in the wound healing process.Therefore, wounds and burns can be classified as inflammation-related pathologies.The conventional thinking in the art is that anti-inflammatory drugs should not be applied directly to open wounds, because it is harmful to the progress of wound healing.

[0007] Mussel adhesive protein (MAP), also known as Mytilus edulis foot protein (mefp), is a protein secreted by marine shellfish such as Mytilus edulis, Mytilus coruscus, and Perna viridis. The adhesive protein is secreted by mussels from their byssus glands, where it is produced and stored. When secreted onto solid surfaces such as rocks, as well as other solid objects such as metal, wood, and glass, it forms a waterproof bond that secures the mussel to the solid object. Mussels typically adhere in groups to coastal coral reefs or the bottom of ships. The bond is incredibly strong, allowing them to resist wave damage in coastal waters.

[0008] Studies of Mytilus edulis, Mytilus galloprovincialis, Mytilus californias, and Perna viridis have thus far identified 11 distinct adhesive protein subtypes from mussels: mfp-1 (sometimes referred to as "mefp-1" and used interchangeably below), mfp-2 / mefp-2, mfp-3 / mefp-3, mfp-4 / mefp-4, mfp-5 / mefp-5, and mfp-6 / mefp-6; collagens pre-COL-P, pre-COL-D, and pre-COL-NG; and the mussel foot matrix proteins PTMP (proximal filum matrix protein) and DTMP (distal-proximal filum 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).

[0009] All mussel adhesive proteins, including their subtypes, share two structural features in that they contain (1) lysine, which allows the protein to carry a high positive charge load (due to its NH2 terminus), and (2) 3,4-dihydroxyphenylalanine (DOPA, dopamine), whose catechol moiety is responsible for the formation of strong covalent bonds and, consequently, the ability of mussel adhesive proteins to bind to solid surfaces.

[0010] Products based on mussel adhesive protein products are currently used in a limited number of areas (including microcellular adhesion, as tissue adhesives, and wound and burn treatment). Commercially available products are either used directly as a solution of mussel adhesive protein or stored as a lyophilized powder for dissolution before use.

[0011] The majority of mefp-1 consists of 70-90 tandem repeats of the 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 derived and / or isolated as a low molecular weight derivative of naturally occurring MAP, or can be synthesized as described by Yamamoto in J. Chem. Soc., Perkin Trans. 1, 613 (1987). See also Dalsin et al., J. Am. Chem. Soc., 125, 4253 (2003).

[0012] The DOPA residue is considered essential for the activity of MAPs such as mefp-1, and there is no suggestion in the art that its complete replacement with a different amino acid, such as tyrosine, would result in the retention of the physicochemical or biological properties of the MAPs.

[0013] Indeed, there is no suggestion in the art that isolated decapeptides that do not contain DOPA residues, such as those disclosed only as model compounds by Kanyalkar et al. in Biomaterials, 389 (2002) and Belli et al. in Dental Materials, 26, e125 (2010), have or may have the same or even similar properties as MAP. Surprisingly, we have found that isolated decapeptides with structures different from the repeating decapeptide units of mefp-1 are useful in treating inflammation. See also US 5,616,311 and WO 96 / 39128. Summary of the Invention

[0014] According to the present invention, there is provided a method for the treatment of inflammation, inflammatory disorders, and / or disorders characterized by inflammation, comprising administering to a subject the sequence Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Tyr-Lys, an (isolated) peptidic compound comprising, or preferably consisting of, the Stereoisomers or salts are provided.

[0015] Further provided are (isolated) peptide compounds of the above sequences for use in veterinary medicine and / or as cosmetics. Compounds disclosed herein for the uses mentioned herein ( Stereoisomers included) and salts thereof are hereinafter collectively referred to as "compounds of the invention."

[0017] Thus, compounds of the present invention, not in the form of a salt, may have the following specific chemical structure:

[0018] [ka]

[0019] In addition to the standard central carbon atom of the amino acids in the above sequences (without exception of the L-configuration), certain amino acids in the sequences contain an additional chiral carbon atom. All such stereoisomers and mixtures thereof (including racemic mixtures) are included within the scope of the present invention. In comparison, the definition of Hyp 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, although it is preferred that Hyp used in the compounds of the present invention is 4-hydroxy-L-proline.

[0020] The compound of the present invention may be in the form of salt.The salts that can be mentioned include pharmaceutically acceptable salts and / or cosmetically acceptable salts, such as pharmaceutically and / or cosmetically acceptable acid addition salts and base addition salts.Such salts can be formed by conventional means, for example, by reacting free peptide with one or more equivalents of suitable acid or base in a solvent or medium in which the salt is insoluble, and then removing the solvent or medium using standard techniques (for example, under vacuum, by lyophilization, or by filtration).Salts can also be prepared by exchanging the counterion of the active ingredient in the form of salt with another counterion, for example, by using a suitable ion exchange resin.

[0021] Preferred salts include, for example, hydrochlorides, bisulfates, maleates, mesylates, tosylates, alkaline earth metal salts such as calcium and magnesium, or alkali metal salts such as sodium and potassium salts.

[0022] The compounds of the present invention are useful because they have pharmacological activity.Therefore, the compounds of the present invention are useful as human and animal medicines.Therefore, they are indicated as pharmaceuticals (and / or veterinary medicine), but can also be used as cosmetics and / or as part of medical devices.

[0023] While the compounds of the present invention may have pharmacological activity themselves, certain pharmaceutically acceptable (e.g., "protected") derivatives of the compounds of the present invention exist or may be prepared, which may have no such activity but can be orally administered and subsequently metabolized or chemically converted in the body to form the compounds of the present invention. Such compounds (which may have some pharmacological activity, provided that such activity is appreciably less than the activity of the active compound to which they are metabolized / converted) may therefore be described as "prodrugs" of the compounds of the present invention.

[0024] As used herein, reference to a prodrug will include a compound that forms an experimentally detectable amount of a compound of the invention within a given time period after administration. All prodrugs of the compounds of the invention are included within the scope of the invention.

[0025] The compounds of the present invention are particularly useful in the treatment of inflammation.

[0026] "Treatment of inflammation" includes treatment of inflammation in any organ of the body (including soft tissues, joints, nerves, vasculature, internal organs, particularly mucosal surfaces, and specifically skin), regardless of cause, and also includes all such inflammatory disorders or conditions and / or disorders or conditions characterized (e.g., as a symptom) by inflammation.

[0027] Inflammatory conditions can be (and typically are) characterized by the activation of immune defense mechanisms, resulting in effects that are more harmful than beneficial to the host. Such conditions are generally associated with varying degrees of tissue redness or hyperemia, swelling, edema, hyperthermia, pain (including soreness), fluid exudation, itching (pruritus), cell death, and tissue destruction, cell proliferation, and / or loss of function.

[0028] Inflammatory conditions that may be mentioned include arteritis, diabetes, metabolic syndrome, rosacea, asthma and allergy, ankylosing spondylitis, chronic obstructive pulmonary disease, gouty arthritis, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), multiple sclerosis, osteoarthritis, pancreatitis, prostatitis, psoriatic arthritis, rheumatoid arthritis, tendonitis, bursitis, Sjogren's syndrome, systemic lupus erythematosus, uveitis, urticaria, vasculitis, mastocytosis, diabetic vascular complications, migraine, atherosclerosis and related cardiovascular disorders.Another condition characterized by inflammation that may be mentioned is chronic obstructive pulmonary disease (COPD).Another condition characterized by inflammation that may be mentioned is inflammatory bowel disease, including Crohn's disease and especially ulcerative colitis.

[0029] Further inflammatory conditions that may be mentioned in particular include inflammation of the skin or mucous membranes (including the oral, nasal, ocular, vaginal, cervical, and / or anorectal mucosa, more particularly the oral or nasal mucosa), such as inflammation resulting from infection (such as viral and / or bacterial infection) or allergic / atopic conditions (such as rhinitis, pharyngitis, periodontitis, gingivitis, xerophthalmia, conjunctivitis, dermatitis, urticaria (hives), and food allergies), as well as other inflammatory conditions such as herpes, drug eruptions, polymorphous light eruption, sunburn, early symptoms of skin cancer (erythematous skin lesions), pathological alopecia (including after skin grafts), chemical rash, psoriasis, erythema multiforme, folliculitis, eczema, and otitis externa.

[0030] More specifically, the compounds can be used to treat certain conditions characterized by and / or associated with inflammation, which may include wounds (including abrasions (scratches), incisions (including surgical incisions), lacerations, punctures, avulsions, contusions, and scars), burns (including inflammation resulting from post-burn surgery, such as skin grafts), and other conditions such as hemorrhoids.

[0031] Skin or mucosal wounds can result from or be caused by internal or external physical injury to the membrane surface (ie, are a symptom of an underlying physiological disorder).

[0032] Physical (e.g., "open") wounds can be caused by sharp objects (cuts, incisions, punctures) or blunt objects / mechanical forces (lacerations, abrasions, avulsions), physical blows (contusions), heat or chemicals (burns and blisters), ultraviolet light (sunburn), cold (chills or frostbite). Wounds can be superficial (damage to the epidermis and / or dermis only) 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.

[0033] The compounds of the present invention can be used to alleviate pain (including pain) associated with inflammation and / or wounds. Specifically, the compounds of the present invention can be used to alleviate procedural pain and / or non-procedural pain. Those skilled in the art will understand that the term "procedural pain" (i.e., surgical pain) refers to acute pain associated with medical investigation and treatment carried out for medical purposes. The term "non-procedural" refers to general pain associated with inflammation and / or wounds (e.g., pain associated with dental ulcers, burns, and / or scars), and is not the result of a specific medical intervention.

[0034] The compounds of the invention can be used to treat inflammation, pain (including pain), and / or pruritus (itching) associated with the wound itself and the healing process, as well as to prevent exudation of fluid from the wound, the risk of infection, and physiological responses resulting from inflammation and / or the wound healing process, such as scarring and melanin pigmentation.

[0035] Scarring is the result of inflammation and / or wound healing and is a general term for the formation of fibrous tissue that is the result of such inflammation / healing.

[0036] The compound of the present invention can also be useful for suppressing the production of melanin pigmentation, which may be caused by inflammation and / or wound healing.The compound of the present invention can also be useful for suppressing the pigmentation caused by melanin pigmentation-related disorders, such as melasma, freckles, melanin pigmentation, butterfly rash and other pigmentation, skin cancer including melanoma, and skin diseases such as sun exposure or acne.

[0037] Wounds can also occur as a result of disease or injury. Such wounds can include blisters and / or ulcers of the skin and mucous membranes. These are common conditions that are often long-lasting and difficult to treat. Skin tissue can often be damaged, removed, liquefied, infected, and / or necrotic. Ulcers, especially if infected, can be difficult to heal, costly, and have secondary health consequences. They can also cause significant psychological stress and economic loss to patients, affecting both their overall well-being and quality of life.

[0038] Alternatively, inflammatory skin conditions or diseases in which the compounds of the invention find particular utility include the treatment of psoriasis, acne, eczema and dermatitis, particularly allergic / atopic dermatitis, as well as rhinitis, particularly allergic rhinitis, hemorrhoids, and chronic obstructive pulmonary disease.

[0039] Psoriasis is a chronic inflammatory skin disease that tends to recur (some patients never recover). Clinical symptoms of psoriasis mainly include erythema and scaling. It can occur all over the body, but is more commonly observed on the scalp and hands and feet.

[0040] Acne is a chronic inflammatory skin disease of the follicular (pilosebaceous) system, the development of which is closely related to factors such as excessive sebum, obstruction of the pilosebaceous ducts (including closed and open comedones), bacterial infection, and inflammatory reactions. It tends to occur at a young age and is characterized by polymorphic skin lesions on the face. Therefore, the term acne includes both ordinary acne and acne rosacea (i.e., red nose).

[0041] Eczema is an intensely itchy inflammatory reaction of the skin caused by various internal and external factors. It has three stages: acute, subacute, and chronic. The acute stage tends to produce exudate, while the chronic stage involves infiltration and hypertrophy. Skin lesions are often itchy and easily recur.

[0042] Dermatitis is a common skin disorder characterized by roughness, redness, itchiness, eczema, and dryness. The small lumps, intractable ulcers, and pigmented patches caused by dermatitis can develop into basal cell carcinoma, squamous cell carcinoma, and malignant melanoma if not treated promptly. Dermatitis can be caused by a variety of internal and external infectious or non-infectious factors, including substances (contact dermatitis) or allergies (allergic / atopic dermatitis). Also included are 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, iatrosacea, steroid-dermatitis-like rosacea, topical corticosteroid-induced rosacea-like dermatitis, and more specifically, facial corticosteroid-addicted dermatitis (FCAD) or facial corticosteroid-dependent dermatitis (FCDD), characterized by flushing, erythema, telangiectasias, atrophy, papules, and / or pustules of the facial area after long-term treatment (including uncontrolled use, abuse, or misuse) with topical corticosteroids (see, e.g., Xiao et al., J. Dermatol., 42, 697 (2015), and Lu et al., Clin. Exp. Dermatol., 35, 618 (2009)).

[0043] Rhinitis is the irritation and inflammation of the mucous membrane inside the nose.The common symptoms of rhinitis include stuffy nose, 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 skin flakes.The compound of the present invention can also relieve itchy eyes when administered intranasally (i.e., to the nasal mucous membrane).

[0044] Hemorrhoids are swellings caused by large inflammations of the hemorrhoidal blood vessels found in or around the rectum and anus. Symptoms include bleeding (i.e., wounds) after passing stool, prolapsed hemorrhoids, mucus secretion, and itching, burning, redness, and swelling in the anal area. Hemorrhoids are thought to be the result of increased abdominal pressure, for example, as a result of constipation or diarrhea.

[0045] Chronic obstructive pulmonary disease (COPD) is the name for a group of lung conditions that cause difficulty breathing, including emphysema (damage to the alveoli) and chronic bronchitis (long-term airway inflammation). COPD occurs when the lungs become inflamed, damaged, and narrowed. The damage to the lungs is usually irreversible, resulting in impaired airflow in and out of the lungs. Symptoms of COPD include shortness of breath, a productive cough, frequent chest infections, and persistent wheezing. The most common cause of the disease is smoking, but other risk factors include high levels of air pollution and occupational exposure to dust, chemicals, and smoke.

[0046] The compounds of the present invention may have a positive effect on the relief of erythema, redness and swelling, edema, blisters, and bullous pemphigoid caused by various conditions, including those generally and specifically mentioned herein, and may inhibit the exudation of subcutaneous tissue fluid and suppress the itching and pain caused by such inflammatory conditions.

[0047] Other inflammatory conditions that may be mentioned include: (a) Mucosal inflammation such as inflammation caused by oral mucositis, aphthous ulcers, otitis media, laryngitis, tracheitis, esophagitis, gastritis, enteritis, and enterocolitis (including bacterial dysentery, chronic amebic dysentery, schistosomiasis, nonspecific ulcerative colitis, and regional enteritis), cervicitis and endometritis, endometritis, inhalation injuries, and the like, as well as inflammation of the mucosa associated with cancer and infections (e.g., viral infections such as the common cold or influenza) affecting mucosal surfaces such as the oral cavity, nasopharynx, ears, throat, trachea, gastrointestinal tract, and cervix.

[0048] (b) For example, fractures, pyogenic infections of bones and joints, inflammation due to rheumatic bone disease, as well as pyogenic osteomyelitis (acute, chronic, localized, sclerosing, post-traumatic), septic arthritis, bone tumors (osteoma, osteoid osteoma, chondroma), bone cysts, osteoclastoma, primary bone sarcomas (osteosarcoma, chondrosarcoma, osteofibrosarcoma, Ewing's sarcoma, non-Hodgkin's lymphoma, myeloma, chordoma), metastatic bone tumors, tumor-like lesions of bone (bone cysts, aneurysmal bone cysts, eosinophilic granuloma, fibrous dysplasia), and orthopedic inflammation associated with rheumatoid arthritis.

[0049] (c) Nerve inflammation such as peripheral polyneuritis, facial neuritis, peripheral neuritis, subcutaneous neuritis, ulnar neuritis, and intercostal neuritis.

[0050] (d) Inflammation of subcutaneous and submucosal soft tissues, such as myositis, dendritis, tendonitis, cystitis, lymphadenitis, cryptitis, tonsillitis, synovitis, and fasciitis, as well as inflammation of soft tissues caused by injury, contusion, or tear of muscle, ligament, fascia, tendon, synovium, fat, joint capsule, and lymphatic tissue.

[0051] (e) Vascular inflammation such as allergic leukocytoclastic vasculitis, allergic cutaneous vasculitis, polyarteritis nodosa, thromboangiitis, granulomatous vasculitis, lymphocytic vasculitis, vasculitis with abnormal blood composition, and rheumatoid vasculitis, as well as vascular inflammation associated with vascular cancer caused by allergic leukocytoclastic vasculitis, polyarteritis nodosa, thromboangiitis, granulomatous vasculitis, lymphocytic vasculitis, vasculitis with abnormal blood composition, and rheumatoid vasculitis.

[0052] (f) Inflammation of internal organs such as the heart, stomach, intestines, lungs, liver, spleen, kidneys, pancreas, bladder, ovaries, and prostate, including, but not limited to, the treatment of pericarditis, myocarditis, endocarditis, pneumonia, hepatitis, splenitis, nephritis, pancreatitis, cystitis, oophoritis, prostatitis, and gastric ulcers.

[0053] (g) Inflammation of the eye and surrounding area, such as conjunctivitis, keratitis (e.g., acute superficial keratitis, nummular keratitis, interstitial keratitis, disciform keratitis, neurotrophic keratitis, mucoplaque keratitis, herpes simplex keratitis, herpes zoster keratitis, bacterial keratitis, fungal keratitis, acanthamoeba keratitis, Onchocerca volvulus keratitis, superficial punctate keratitis, ulcerative keratitis, lagophthalmos keratitis, photokeratitis, and acute hyperemia during contact lens wear), and optic neuritis.

[0054] (h) Inflammation of the gums and oral cavity, such as periodontitis, gingivitis, and dental ulcers.

[0055] (i) Rheumatoid inflammation such as rheumatoid 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, Sjogren's syndrome, spondyloarthropathy, systemic lupus erythematosus, and tendonitis.

[0056] The compounds of the present 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, pulmonary embolism, etc. A specific condition that may be mentioned is idiopathic pulmonary fibrosis.

[0057] Idiopathic pulmonary fibrosis is a diffuse and fatal pulmonary interstitial disease, with pathological characteristics including alveolar epithelial damage, extensive proliferation of pulmonary fibroblasts, and excessive deposition of extracellular matrix, which ultimately leads to irreversible lung tissue damage.In the later stage of the disease, the subject with idiopathic pulmonary fibrosis experiences respiratory failure and death.It has been found that the compound of the present invention can be useful in treating idiopathic pulmonary fibrosis and / or alleviating the symptoms associated with this disease.

[0058] The compounds of the present invention may further have antioxidant effects by increasing SOD (superoxide dismutase) production and reducing lipid oxidation. Therefore, such compounds and formulations containing them may be considered to have antioxidant properties.

[0059] The compounds of the present invention may also have antipyretic properties, allowing for the treatment of fever and / or alleviating its symptoms, for example, by reducing the body temperature of a subject (which results in a reduction in fever). Thus, the compounds of the present invention and formulations containing them may be considered to be antipyretics.

[0060] According to a further aspect of the present invention, there is provided a method for treating inflammation, inflammatory disorders, and / or disorders / conditions characterized by inflammation (e.g., as a symptom), comprising administering a compound of the present invention to a patient in need of such treatment.

[0061] For the avoidance of doubt, in the context of the present invention, the terms "treatment", "therapy" and "treatment" include therapeutic or palliative treatment of patients in need of treatment, as well as prophylactic treatment and / or diagnosis of patients susceptible to inflammation and / or inflammatory disorders.

[0062] "Patient" includes reptilian, avian, and mammalian (especially human) patients.

[0063] According to the present invention, the compounds of the present invention are administered in the form of a pharmaceutical preparation containing the compound(s) in a pharmaceutically acceptable dosage form(s), preferably locally or systemically, for example, orally, intravenously, or intra-arterially (including intravascular and other perivascular devices / dosage forms (e.g., stents)), intramuscularly, cutaneously, subcutaneously, transmucosally (e.g., sublingually or bucally), rectally, intravaginally, transdermally, intranasally, pulmonary (e.g., tracheal or bronchial), preferably topically, or by any other parenteral route. Administration by inhalation (e.g., intranasal) is particularly useful when the condition being treated is inflammation caused by rhinitis or a viral infection of the respiratory tract (e.g., cold, influenza). Pulmonary administration is particularly useful when the condition being treated is COPD or IPF. Local administration forms can be enhanced by creating a spray containing the active ingredient, for example, by using a powder aerosol or an aqueous mist using appropriate atomization technology or devices, such as a nebulizer.

[0064] Preferred modes of delivery of the compounds of the invention include local delivery to the site of inflammation (e.g., lung or more preferably mucous membranes, including skin) in a suitable (e.g., pharmaceutically and topically acceptable) vehicle suitable for application to the skin and / or suitable mucosal surfaces, and / or in a commercially available formulation, but may also include oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal, or pulmonary delivery.

[0065] The compounds of the present invention will generally be administered in the form of one or more, e.g., pharmaceutical formulations, mixed with (e.g., pharmaceutically acceptable) adjuvants, diluents, or carriers, which may be selected with due consideration of the intended route of administration (e.g., topical administration to relevant mucous membranes (including the lungs) or preferably to the skin) and standard pharmaceutical or other (e.g., cosmetic) practice. Such pharmaceutically acceptable carriers may be chemically inert to the active compounds and may be free of adverse side effects or toxicity under the conditions of use. Such pharmaceutically acceptable carriers may also provide immediate or modified release of the active ingredient.

[0066] Suitable pharmaceutical formulations are commercially available or can be found 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), as well as documents referenced therein, the relevant disclosures of all of which documents are incorporated herein by reference. Otherwise, the preparation of suitable formulations containing the compounds of the present invention can be achieved in ways not of the present invention by those skilled in the art using conventional techniques.

[0067] 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 saline-, phosphate-, acetate-, or borate-containing formulations), or lyophilized powders.

[0068] The active ingredient may be further combined with suitable excipients to prepare the following: gel formulations (for which suitable gel matrix materials include cellulose derivatives, carbomers and alginates, tragacanth gum, gelatin, pectin, carrageenan, gellan gum, starch, xanthan gum, cationic guar gum, agar, non-cellulose polysaccharides, sugars such as glucose, glycerin, propanediol, vinyl polymers, acrylic resins, polyvinyl alcohol, carboxyvinyl polymers, and, in particular, hyaluronic acid); lotions (suitable matrix materials for which include cellulose derivatives, glycerin, non-cellulose polysaccharides, polyethylene glycols of different molecular weights, and propanediol); pastes or ointments (for which suitable paste matrix materials include glycerin, petrolatum, paraffin, polyethylene glycols of different molecular weights, etc.); creams or foams (for which suitable excipients (e.g., foaming agents) include hydroxypropyl methylcellulose, gelatin, polyethylene glycols of different molecular weights, sodium dodecyl sulfate, sodium fatty alcohol polyoxyethylene ether sulfonate, corn gluten flour, and acrylamide); powder aerosols (suitable excipients for which include mannitol, glycine, dextrin, dextrose, sucrose, lactose, sorbitol, and polysorbates, e.g., dry powder inhalants); and / or Liquid (aerosol) sprays for oral administration or inhalation (suitable excipients of which include viscosity adjusting agents such as hyaluronic acid, sugars such as glucose and lactose, emulsifiers, buffers, alcohol, water, preservatives, sweeteners, flavors, etc.).

[0069] Moisturizing agents such as glycerol, glycerin, polyethylene glycol, trehalose, glycerol, petrolatum, paraffin oil, silicone oil, hyaluronic acid and its salts (e.g., sodium and potassium salts), octanoic / capric triglyceride, and / or antioxidants such as vitamins and glutathione, and / or pH adjusters such as acids, bases, and pH buffers may also be included in such formulations as needed. In addition, hexadecanol (cetyl alcohol), fatty acids (e.g., stearic acid), sodium dodecyl sulfate (sodium lauryl sulfate), sorbitan esters (e.g., sorbitan stearate, sorbitan oleate, etc.), monoacylglycerides (e.g., glyceryl monostearate), polyethoxylated alcohols, polyvinyl alcohols, polyol esters, polyoxyethylene alkyl ethers (e.g., polyoxyethylene sorbitan monooleate), polyoxyethylene castor oil derivatives, ethoxylated fatty acid esters Other examples of surfactants / emulsifiers include polyoxylglycerides, lauryldimethylamine oxide, bile salts (e.g., sodium deoxycholate, sodium cholate), phospholipids, N,N-dimethyldodecylamine-N-oxide, hexadecyltrimethylammonium bromide, poloxamers, lecithin, sterols (e.g., cholesterol), sugar esters, polysorbates, preservatives such as phenoxyethanol and ethylhexyglycerin, and thickeners such as acryloyldimethyltaurate / VP copolymer. Specifically, stearic acid, glyceryl monostearate, hexadecanol, sorbitan stearate, cetyl alcohol, and octanoic / capric glycerides may be included, particularly in cream formulations.

[0070] The compounds of the present invention and (for example, the above-mentioned solutions, gels, creams, ointments, lotions, foams, pastes, and / or dry powders, including those mentioned above) can be further combined with a suitable matrix material to prepare bandages or therapeutic patches for application to biological surfaces, such as skin or mucosal surfaces. Thus, such formulations can be used to impregnate matrix materials, such as gauze, nonwoven fabrics, or silk paper. Alternatively, the therapeutic patch can be, for example, a bandage, a face mask, an eye mask, a hand mask, a foot mask, etc.

[0071] Although petrolatum may be used for use in applying such dressings to wounds, we have also found that a PEG (e.g., PEG 400) based ointment can be combined with a matrix material to prepare a dressing without the need for petrolatum.

[0072] The compounds of the present invention may also be combined in treatment with one or more growth factors selected from platelet-type growth factors (including platelet-derived growth factor, PDGF), osteosarcoma-derived growth factor (ODGF), epidermal growth factor (EGF), transforming growth factors (TGFα and TGFβ), fibroblast growth factors (αFGF, βFGF), insulin-like growth factors (IGF-I, IGF-II), nerve growth factor (NGF), interleukin-type growth factors (IL-1, IL-1, IL-3), erythropoietin (EPO), and colony-stimulating factors (CSF).

[0073] According to a further aspect of the present invention, there is provided a (e.g., pharmaceutical) composition comprising a compound of the present invention and one or more pharmaceutically acceptable excipients, such as an adjuvant, diluent, or carrier. Preferred formulations are suitable for topical application, for example, to mucous membranes (including the lungs), or more preferably to the skin, and therefore include a topically acceptable adjuvant, diluent, or carrier.

[0074] Thus, formulations comprising compounds of the invention may be suitable, adapted for, and / or packaged and presented for topical administration (e.g., to the lungs, mucosa, or skin) to treat, for example, disorders involving inflammation, inflammatory disorders, and / or conditions characterized by inflammation (as a symptom) by direct local administration of the formulation (e.g., to the lungs, mucosa, or preferably to the skin).

[0075] For the avoidance of doubt with respect to this aspect of the invention, the topical formulations of the invention may be used in any and all conditions described herein, including the treatment of inflammation, in the treatment of any and all inflammatory disorder(s), and / or in the treatment of any and all condition(s) characterised by inflammation as mentioned, defined or described hereinbefore.

[0076] The topical (e.g., liquid or (e.g., aqueous) solution-based) formulations of the present invention may be particularly useful in wound healing and may alleviate pain (including soreness) and pruritus / itch, particularly associated with the wound itself and the wound healing process. Such topical formulations of the present invention may be particularly useful in preventing and / or inhibiting exudation of fluid from a wound, particularly during the acute inflammatory phase, e.g., the first 48 hours, after a burn or wound has been sustained. This prevents the risk of infection and other physiological reactions. Such topical formulations of the present invention may also be particularly useful in preventing and / or inhibiting scarring and melanin pigmentation (see above), whether or not associated with a wound.

[0077] The administration of the active ingredient can be continuous or intermittent. The mode of administration can also be determined by the timing and frequency of administration, and in the case of therapeutic treatment of inflammation, by the severity of the condition.

[0078] Depending on the disorder and patient being treated, as well as the route of administration, the compounds of the present invention may be administered to a patient in need of treatment in different therapeutically effective doses.

[0079] Similarly, the amount of active ingredient in the formulation will depend on the severity of the condition being treated and the patient, but may be determined by one skilled in the art.

[0080] In any event, a practitioner or other skilled artisan will be able to routinely determine the actual dosage that will be most suitable for an individual patient, depending on the severity of the condition and the route of administration. The dosages mentioned herein are exemplary of the average case, and there can, of course, be individual instances in which higher or lower dosage ranges are merited, and these are within the scope of this invention.

[0081] The dose can be administered once to four times daily.

[0082] Suitable concentrations of the compounds of the invention in the aqueous solution product can be from about 0.01 (eg, about 0.1) to about 15.0 mg / mL, in all cases calculated as the free (non-salt) peptide.

[0083] A suitable topical dose of the compounds of the invention is about 5 μg / cm, calculated in all cases as the free (non-salt) peptide. 2 Approximately 1 to 10 μg / cm of the treatment area 2 ) treatment area, about 0.1 (e.g., about 0.5) to about 20 μg / cm 2 Approximately 0.05 to approximately 50 μg / cm of treatment area 2 within the therapeutic range of

[0084] The pH of formulations containing the compounds of the present invention is preferably within 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 basic pH, than, for example, MAP and isolated compounds consisting of the mefp-1 decapeptide sequence.

[0085] In any event, the dose administered to a mammal, particularly a human, in the context of the present invention must be sufficient to effect a therapeutic response in the mammal over a reasonable time frame (as described above). Those skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by, among other things, the pharmacological properties of the formulation, the nature and severity of the condition being treated, the physical and mental state of the recipient, and the age, condition, weight, sex, and response of the patient being treated, the stage / severity of the disease, and genetic differences between patients.

[0086] In the uses and methods described herein, the compounds of the present invention may be combined with one or more active ingredients (other anti-inflammatory agents) useful in the treatment of inflammation and / or inflammatory disorders. Accordingly, such patients may also (and / or already) be undergoing a therapy based on the administration of one or more of such other active ingredients, by which is meant receiving prescribed doses of one or more of the active ingredients mentioned herein before, in addition to, and / or after treatment with the compounds of the present invention.

[0087] The anti-inflammatory agent that can be used in combination with the compound of the present invention in the treatment of inflammation includes the therapeutic agent that is useful for treating inflammation and / or the disease characterized by inflammation as one of its symptoms.Depending on the pathological condition to be treated, such anti-inflammatory agent can also include NSAIDs, leukotriene receptor antagonists (for example, montelukast, as described below), corticosteroids, analgesics, and certain enzymes, such as trypsin, as described below.The compound of the present invention can also be combined with leukotriene B4 (LTB4).

[0088] In this context, the compounds of the present invention can also be combined with one or more mussel adhesive proteins (MAPs) for therapeutic use, including collagens pre-COL-P, pre-COL-D, and pre-COL-NG, mussel foot matrix proteins PTMP and DTMP, and any adhesive protein that may be derived from a mussel species, such as Mytilus edulis (blue mussel), including full-length proteins including all subtypes that are or may be derived from mussels, such as mfp or mefp (e.g., mefp-2, mefp-3, mefp-4, mefp-5, mefp-6, and especially mefp-1), including mixtures or combinations of any of these proteins, such as mefp. While mixtures / combinations of the above-mentioned MAP subtypes can be provided in accordance with the present invention, it is preferred that the purity of the predominant MAP subtype (e.g., mefp-1) be at least 25% by weight of the total amount of any such mixture.

[0089] 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 Bio Co. (China, sold as MAP Medical Device®), BD Biosciences (USA), Kollodis (Korea), and Biopolymer (Sweden). Alternatively, MAP may be produced using known recombinant DNA methods.

[0090] Derivatives (e.g., pharmaceutically acceptable derivatives) of MAPs can also be combined with the compounds of the invention, including, for example, compounds having molecular weights in the range of about 500 to about 2000 (e.g., about 1,500, such as about 1,200, including about 800 Da), which can allow for easier penetration through biological membranes, such as the skin barrier or mucosal surfaces. Such derivatives can also include other compounds containing amino acid sequences that are the same as or (e.g., minor) variants of sequences identified in naturally occurring MAPs, which can be synthesized by chemical and / or biological processes (e.g., chemical modification of naturally occurring MAPs or direct synthesis). A "(e.g., minor) variant of an amino acid sequence identified in a naturally occurring MAP" refers to a variation in the sequence that does not measurably adversely affect the desired properties of the related naturally occurring MAP.

[0091] For example, as discussed above, the isolated decapeptide compound of the sequence: Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-DOPA-Lys (mefp-1 decapeptide) is a pharmaceutically acceptable low molecular weight derivative of MAP that can be combined with the compounds of the present invention.

[0092] Such derivatives of MAPs may be used in combination products according to the invention either alone or in combination with one or more other such derivatives, and / or with one or more of the full-length MAPs mentioned above.

[0093] Whether the formulation used is a combination preparation or a kit-of-parts as described above, a suitable concentration of MAP and its derivatives in the aqueous topical formulation product may be about 0.01 (e.g., about 0.1) to about 15.0 (e.g., about 1.5) mg / mL, and a suitable pH value may be within the range of about 1.0 to about 7.0 (e.g., about 3.0 to about 6.5). Suitable commercial sources of such aqueous solutions include USUN Bio Co., Jiangyin, Jiangsu Province, China.

[0094] A suitable topical dose of MAP and its derivatives is approximately 5 μg / cm 2 Approximately 2 to 10 μg / cm of the treatment area 2 Approximately 1 to approximately 20 μg / cm, including the treatment area 2 Approximately 0.1 to approximately 50 μg / cm of the treatment area 2 within the therapeutic range of

[0095] Combinations of the compounds of the present invention with mefp-1 have been found to be particularly useful in the treatment of inflammatory bowel diseases such as ulcerative colitis, and particularly in stopping bleeding in patients with such diseases.

[0096] Other preferred agents that can be combined with the compounds of the invention include LTB4 (for treating wounds and burns), montelukast (for treating inflammation generally), and trypsin (for treating inflammation of the mucous membranes associated with, for example, viral infections).

[0097] The compounds of the present invention may also be combined with other therapeutic agents that are known to cause inflammation as a side effect when administered.

[0098] When the compounds of the invention are "combined" with other therapeutic agents in this manner, the active ingredients may be administered together in the same formulation or separately (simultaneously or sequentially) in different formulations.

[0099] Such combination products provide for the administration of a compound of the invention in combination with another therapeutic agent, and therefore may be presented as separate formulations (at least one of which contains a compound of the invention and at least one of which contains the other therapeutic agent) or may be presented (i.e., formulated) as a combined preparation (i.e., presented as a single formulation containing a compound of the invention and the other therapeutic agent).

[0100] therefore, (1) A pharmaceutical formulation comprising a 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 (this formulation is hereinafter referred to as a "combination preparation"); and (2) A parts kit comprising: (A) a pharmaceutical formulation comprising a compound of the present invention in admixture with a pharmaceutically acceptable adjuvant, diluent, or carrier; (B) a pharmaceutical formulation containing another anti-inflammatory agent, or an agent known to cause inflammation as a side effect, mixed with a pharmaceutically acceptable adjuvant, diluent, or carrier; The components (A) and (B) are each provided in a form suitable for administration in combination with the other.

[0101] According to a further aspect of the present invention there is provided a method of making a kit of parts as defined above, comprising associating component (A) as defined above with component (B) as defined above, thus making the two components suitable for administration in combination with each other.

[0102] By "associating" two components with each other, we are saying that components (A) and (B) of a kit of parts are: (i) provided as separate formulations (i.e., independently of each other) and then brought together for use in combination with each other in a combination therapy; (ii) They may be packaged and presented together as separate components of a "combination pack" for use in combination with each other in combination therapy.

[0103] therefore, (I) one of components (A) and (B) as defined herein, (II) A kit of parts is further provided that includes the component together with instructions for use in combination with the other of the two components.

[0104] The kits of parts described herein may contain two or more formulations containing suitable amounts / doses of a compound of the invention and / or two or more formulations containing suitable amounts / doses of another anti-inflammatory agent to provide for repeated administration. When two or more formulations (containing any of the active compounds) are present, such formulations may be the same or different with respect to the dose, chemical composition(s), and / or physical form(s) of any of the compounds.

[0105] With respect to the kits of parts described herein, by "administration in combination with," we include sequential, separate, and / or simultaneous administration of respective formulations comprising a compound of the invention and another anti-inflammatory agent over the course of treatment of the relevant condition.

[0106] Thus, with respect to a combination product according to the invention, the term "administration in combination with" includes administration of the two components of the combination product (a compound of the invention and another anti-inflammatory agent) either together or sufficiently closely in time (optionally repeatedly) so as to permit a greater beneficial effect to the patient over the course of treatment of the relevant condition than would occur if, over the same course of treatment, either a formulation comprising the compound of the invention or a formulation comprising the other agent were administered alone (optionally repeatedly) without the other component. Determining whether a combination provides a greater beneficial effect with respect to the treatment of a particular condition, and over the course of treatment, will depend on the condition being treated or prevented, but can be routinely accomplished by one of ordinary skill in the art.

[0107] Furthermore, in the context of the kit-of-parts according to the invention, the term "in combination with" includes that one or the other of the two formulations may be administered before, after, and / or simultaneously (optionally repeatedly) with the administration of the other component. When used in this context, the terms "co-administered" and "administered simultaneously with" include administration of individual doses of the relevant compound of the invention and the other anti-inflammatory agent within 48 hours (e.g., 24 hours) of each other.

[0108] In a further aspect of the present invention, there is provided a process for preparing a combination preparation as defined above, which comprises associating a compound of the present invention, another anti-inflammatory agent, or an agent known to cause inflammation as a side effect, with at least one (e.g., pharmaceutically acceptable) excipient.

[0109] Whenever the term "about" is used herein in the context of quantities such as concentration and / or dosage of an active ingredient, molecular weight, or pH, it will be understood that such variables are approximate and thus can vary from the numerical values ​​specified herein by ±10%, e.g., ±5%, and preferably ±2% (e.g., ±1%). In this regard, the term "about 10%" means, for example, ±10% for a numerical value of 10, i.e., 9% to 11%.

[0110] The compounds of the present invention have the advantage that they can be used in a variety of conditions characterized by inflammation, whether the condition itself is an organic inflammatory disease or is associated with or characterized by inflammation (e.g., wounds, burns, or viral infections).

[0111] The compounds of the present invention also have the advantage that they are physicochemically stable to processes such as oxidation at a variety of pHs, including neutral and basic pH, when compared to similar compounds known in the prior art, such as MAP and isolated mefp-1 decapeptide.

[0112] The uses and methods described herein may also have advantages over similar methods (treatments) known in the prior art in treating the above-mentioned conditions, whether for use in treating inflammation, inflammatory disorders, or disorders characterized by inflammation as a symptom (including wounds), or other methods, in that they may be more convenient for the physician and / or patient, more effective, less toxic, have a broad spectrum of activity, be more potent, cause fewer side effects, or have other useful pharmacological properties over similar methods (treatments) known in the prior art.

[0113] The present invention is illustrated by the following examples, in which FIG. 1 shows ELISA test results for various inflammatory markers tested with various test compounds obtained from exudates from air pouches induced in mice according to Example 1, FIG. 2 shows the effect on acute healing of wounds sustained by mice when treated with various test compounds, and FIGS. 3-5 show ELISA test results for inflammatory markers (Hyp, VEGF, and TNF-β1, respectively) obtained from samples taken from the wounds of each of the mice in a mouse wound model. [Brief explanation of the drawings]

[0114] [Figure 1] FIG. 1 shows the ELISA test results of various inflammatory markers tested with various test compounds obtained from exudates from air pouches induced in mice according to Example 1. [Figure 2] FIG. 2 shows the effect of treatment with various test compounds on acute healing of wounds inflicted on mice. [Figure 3] Figures 3 to 5 show the results of ELISA tests for inflammatory markers (Hyp, VEGF, and TNF-β1, respectively) obtained from samples taken from the wounds of each of these mice. [Figure 4] Figures 3 to 5 show the results of ELISA tests for inflammatory markers (Hyp, VEGF, and TNF-β1, respectively) obtained from samples taken from the wounds of each of these mice. [Figure 5]Figures 3 to 5 show the results of ELISA tests for inflammatory markers (Hyp, VEGF, and TNF-β1, respectively) obtained from samples taken from the wounds of each of these mice. [Example]

[0115] Example 1 Synthesis of Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Tyr-Lys Fmoc-Lys-Boc-Wang resin (0.3 mmol / g, GLS180322-41301, GL Biochem, Shanghai, China) was packed into the reaction column.

[0116] Two liters of methylene chloride (DCM, Shandong Jinling Chemical Industry Inc., Shandong, China) was added to the column, and the resin was allowed to soak for approximately 30 minutes. Then, the DCM was removed, and the column was washed three times with 2 liters of N,N-dimethylformamide (DMF, Shandong Shitaifeng Fertilizer Industry Inc. Co., Shandong, China).

[0117] 200 mL of piperidine (Shanghai Li Ming Industry and Trade Co., Ltd., China) was mixed with 1 L of DMF and used as the deprotection solution. The liquid was drained after 15 min, and the column was washed with DMF six times.

[0118] 69 g of Fmoc-Tyr(tBu)-OH (GLS170916-36901, GL Biochem) and 48 g of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU, GL Biochem) were dissolved in 300 mL of DMF and added to a reaction column. Next, 53 mL of N,N-diisopropylethylamine (DIPEA, Suzhou Highfine Biotech Co. Ltd., Jiangsu, China) was added. The reaction time was 1 h.

[0119] A sample was taken and ninhydrin (Shanghai Shanpu Chemical Co. Ltd, China) was used to detect when the reaction was complete, at which point the liquid was drained and the residue was washed three times with DMF.

[0120] The above coupling steps were repeated to couple the remaining amino acids in equal amounts: 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.

[0121] 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.

[0122] The liquid was drained to give the resin-bound polypeptide.

[0123] An appropriate amount of lysate consisting of 95% trifluoroacetic acid (TFA), 2.5% water, and 2.5% triisopropylsilane (TiS) was added to soak the resin-bound polypeptides. The mixture was placed on a shaker and incubated at 30–35°C for 2 hours. The resin was then removed by filtration.

[0124] Anhydrous glacial ether was added to the filtrate, which was then centrifuged and the supernatant was discarded. The resulting product was washed three times with anhydrous glacial ether. The isolated peptide was dried to obtain 128 g of crude polypeptide. The crude compound was desalted using an anion exchange resin, analyzed, and lyophilized. After purification, approximately 70.7 g of purified peptide was obtained, which was retested for confirmation.

[0125] 1 mg of the crude product was dissolved in 1 mL of a 1:3 mixture of acetonitrile and water and detected using a P3000A HPLC pump and an LC3000 semi-preparative instrument (preparative column model: GS-120-10-C18-AP 30 mm, Beijing Chuangxin Tonggheng Science & Technology Co., Ltd., Beijing, China). A suitable gradient was calculated for elution, and the target peak was detected by LCMS at 14:351 min (analytical column model: 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). m / z 592.65[M+2H] 2+ (97.89%).

[0126] Example 2 Air bladder model Healthy adult male C57BL / 6 mice weighing 20–30 g were provided by the Nanjing Biomedical Research Institute of Nanjing University (NBRI). Before any experiments, the mice were housed under standardized conditions (constant temperature of 22 ± 2°C, 12-h alternating light and dark cycles) and fed standard mouse chow with water for approximately 1 week.

[0127] General anesthesia was induced using intraperitoneal 3% chloral hydrate (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China, 1 mL / 10 g body weight). One day before sterile air injection, the entire back was shaved and depilated.

[0128] Air pouches were created by subcutaneously injecting 5 mL of sterile air into the intrascapular region of mice. Three days later, another 3 mL injection of air was administered to maintain the pouch. To induce acute inflammation, three days after this final injection, animals received an injection of sterile carrageenan solution (CP Kelco, Taixing, Jiangsu Province, China, prepared by adding 0.1 g of carrageenan powder to a beaker containing 10 mL of 0.9% saline and stirring). Mice were pretreated with the test sample or vehicle 1 hour before and 23 hours after carrageenan injection into the subcutaneous air pouch. Animals were sacrificed 24 hours after carrageenan injection.

[0129] Skin biopsies were taken from the air pouches. Portions of the biopsies were 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 histological embedding in paraffin wax, sectioning, and staining.

[0130] The cavity was washed with 4 mL of sterile phosphate buffer (pH 7.4, prepared by dissolving 4 g NaCl, 0.1 g KCl, 1.749 g Na2HPO4·12H2O, and 0.1 g KH2PO4 in ultrapure water, adjusting the pH to 7.4 with HCl, and diluting with water to a total volume of 500 mL).

[0131] The exudate was collected and its volume quantified. The exudate was centrifuged at 3000 rpm for 10 minutes at 4°C, and the supernatant was collected and stored at -20°C for ELISA analysis using standard ELISA test kits from Beijing4A Biotech Co., Ltd. (Beijing) and an ELISA reader (SH-1000Hitachi, Japan) for tissue necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6).

[0132] Several preliminary experiments were performed to validate this model, and then experiments were performed in which mice were treated by administering test samples or vehicle according to Table 1 below.

[0133] In Table 1, the MAP solution was prepared as follows. Mussels were collected from the coastal areas of Shandong Province, China. Mussel legs were collected, cut into small pieces, and homogenized in an extraction buffer containing 5% acetic acid in 4 mol / L urea water. The crude extract was collected after centrifugation and then purified by liquid chromatography. The purified protein (semi-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 to this semi-product to obtain the concentrations listed in Table 1.

[0134] An isolated compound consisting of the mefp-1 decapeptide sequence Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-DOPA-Lys (hereinafter "Compound A") was purchased from Innovagen (Innovagen AB, Lund, Sweden). It was stored as a powder at -20 °C and dissolved in saline at a concentration of 0.6 mg / ml, pH 5.5. 0.5 mL of the solution was injected.

[0135] The compound of the present 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. It was dissolved in saline at a concentration of 0.6 mg / ml, pH 5.5. 0.5 mL of the solution was injected.

[0136] All substances listed in Table 1 were administered locally by direct injection into the air pouch.

[0137] [Table 1]

[0138] Histological specimens were analyzed, and inflammation scores, activity scores (i.e., the number and density of neutrophils shown on the pathology slides, indicating the degree of inflammation and, in the case of open wounds and infectious diseases, the degree of infection), edema scores, and fibroproliferative scores were estimated as follows:

[0139] HE-stained sections were examined under a light microscope and scored (1, 2, or 3 points) according to the perceived level of inflammation (showing only a few scattered inflammatory cells within the area—1 point (mild); many inflammatory cells observed—2 points (moderate); and diffuse infiltration—3 points (severe)). After overall observation, a similar scoring system was used for the level of edema (3 points for most severe and 1 point for mild). Neutrophils were scored using the same methodology as for inflammatory cells.

[0140] [Table 2]

[0141] The ELISA test results of the exudates for TNF-α, IL-6, and IL-1β are shown graphically in Figure 1. The results showed that Compound B had very strong anti-inflammatory effects.

[0142] Example 3 Acute wound model Male C57BL / 6 mice, 6–8 weeks old, were supplied by Changzhou Cvens Experimental Animal Co. Ltd. (Changzhou, Jiangsu Province, China). Before any experiments, the mice were housed under standardized conditions (constant temperature of 22 ± 2°C, 12-hour alternating light and dark cycles) and fed standard mouse chow with water for approximately 1 week.

[0143] General anesthesia was induced using intraperitoneal 3% chloral hydrate (1 mL / 10 g body weight). The back was shaved with a baby shaver and depilated with cream. The skin area was wiped and sterilized twice with 75% alcohol.

[0144] A single round wound was created on the back using an 18 mm diameter EMS skin biopsy punch (Electron Microscopy Sciences, PO Box 550, 1560 Industry Road, Hatfield, PA, USA). The full thickness of the skin was removed, and the wound was left open without sutures.

[0145] As shown in Table 3 below, different drugs were topically administered at 50 μL per wound once daily from day 0 to day 12. The control group received no wound. The model group received the same amount of saline. Each group contained eight mice.

[0146] Recombinant human epidermal growth factor (rhEGF, Shanghai Haohai Biological Technology Co., Ltd., 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 saline to produce a solution with a concentration of 5,000 IU / mL. The working dose of rhEGF in this experiment was 1,285 IU / wound.

[0147] In this experiment, Compound A was also obtained from GL Biochem (Shanghai) Ltd. The peptide powder was stored at -20°C and dissolved in saline at a concentration of 61.8 μg / mL (Compound A) and 61 μg / mL (Compound B). 50 μL of the solution was applied to the wound surface.

[0148] [Table 3]

[0149] The wounds were wrapped with gauze and transparent bandages after drug administration. Photographs of each wound 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).

[0150] The non-healed wound area was expressed as a percentage of the original wound area. A t / A0×100%, In the formula, A0 and A t refer to the initial area on day 0 and the wound area on the measurement day (time t), respectively.

[0151] After wounding, samples were collected on days 4 and 7. The mice were sacrificed, and wound tissue was collected using the same biopsy punch used to create the wound. Five-mm tissues were then excised from the center of the samples and preserved in 10% neutral buffered formalin (Nanchang Rain Dew Experimental Equipment Co., Ltd., Nanchang, Hubei Province, China), and analyzed by histological embedding in paraffin wax (HE), sectioning, and staining.

[0152] HE and Masson-stained paraffin sections were analyzed under a light microscope. Skin regeneration, fibroblast proliferation, collagen regeneration score, and inflammation score were estimated.

[0153] The remaining samples were stored at -80°C for further analysis. The tissues were cut into small pieces and liquid nitrogen was added to increase brittleness. 9 mL of saline was added to 1 g of tissue, and the tissues were ground at 55 Hz for 60 seconds using a Tissuelyser (Shanghai Jingxin Industrial Development Co., Ltd., Shanghai, China), followed by centrifugation at 8000 rpm for 10 minutes at 4°C.

[0154] The supernatant was collected and the extracted proteins were analyzed by ELISA using a standard ELISA test kit and an ELISA reader (SH-1000 Hitachi, Japan). Vascular endothelial growth factor (VEGF), transforming growth factor beta 1 (TGF-β1), and hydroxyproline (Hyp) were assayed. ELISA kits were purchased from Beijing4A Biotech Co., Ltd. (Beijing, China).

[0155] The effect of Compound B on wound healing is shown in Table 4 and Figure 2, which show the remaining wound area of ​​the initial wound in the different groups (±standard deviation in the case of Table 4).

[0156] [Table 4]

[0157] The above data show that low doses of Compound B improve wound healing. The improvement rate is defined as (remaining wound rate in treatment group / remaining wound rate in model group) × 100%. On the fourth day, the improvement rate of Compound B group was about 10%.

[0158] The wound Hyp (μg / mg) content in the samples, an indicator of collagen regeneration, is shown in Table 5 below and in FIG.

[0159] [Table 5]

[0160] The results for VEGF content (pg / g) are tabulated below in Table 6 and graphically depicted in FIG.

[0161] [Table 6]

[0162] The results showed that Compound B increased VEGF production in the wounded tissue by about 56.7% compared with that in the model group.

[0163] The results for TGF-β1 content (pg / g) are tabulated below in Table 7 and graphically depicted in FIG.

[0164] [Table 7]

[0165] Example 4 A liquid spray for treating allergic dermatitis. Compound B (see Example 1 above) was dissolved in water for injection (WFI, prepared from TC-RO-0.25 / h-2 Water Treatment System, Yangzhou Tiancheng Water Treatment Devices & Engineering Co., Ltd., Yangzhou, China) at a concentration of 0.3 mg / mL.

[0166] The subjects enrolled in this study have allergic skin.Before treatment, patients show symptoms including redness, itching and swelling of the skin.Subjects are required to use liquid spray in the morning and evening after washing their face.

[0167] All subjects experienced relief from itching within eight minutes of first use. Symptoms of capillary congestion disappeared, and after one day of use, patients' faces were no longer red or swollen.

[0168] This experiment demonstrated that a liquid spray containing the compounds of the present invention provided rapid relief of itching and reduced redness, swelling, and other symptoms caused by allergies.

[0169] Example 5 Cream for treating closed microcomedones Compound B (10 mg, see Example 1 above) was first dissolved in WFI (10 g).

[0170] Next, a mixture containing sorbitan stearate (2 g), cetyl alcohol (4 g), octanoic / capric acid glyceride (6 g), and glyceryl monostearate (3 g) (all from Sinopharm Chemical Reagent Co. Ltd.) was prepared, stirred, and heated to 85°C until the mixture was completely dissolved.

[0171] Glycerin (5 g, Sinopharm Chemical Reagent Co. Ltd.) and ammonium acryloyldimethyltaurate / VP copolymer (0.13 g, Clariant Chemical (Guangzhou) Co., Ltd., China) were mixed with 68.86 g of WFI. The mixture was stirred and heated to 85°C to provide a uniform colloidal suspension.

[0172] The copolymer / water mixture was added to the sorbitan stearate-containing mixture and then rapidly stirred for 5 minutes using an emulsifying device. The resulting emulsion was cooled to room temperature.

[0173] Next, the compound B solution was added to the resulting mixture with stirring to obtain the final cream, in which the concentration of compound B was 0.1 mg / g.

[0174] Subjects with closed microcomedones (blocked pores) and acute bouts of acne were enrolled in the study. Symptoms included sebaceous glands blocked by keratinocytes forming slightly hard lumps and raised whiteheads.

[0175] After washing the face, the cream was applied evenly in the morning and evening. The lumps disappeared after one day of use, indicating that the cream containing the compound of the present invention can be used to treat closed microcomedones.

[0176] Example 6 Determination of the antioxidant capacity of the compounds of the present invention The antioxidant capacity of compound B was determined using the 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl (DPPH) assay.

[0177] Compounds A and B were obtained as described in Example 3 above. The peptide powder was stored at -20°C and dissolved in saline 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.

[0178] The DPPH method is one of the most commonly used methods for determining antioxidant capacity. DPPH is a stable nitrogen-centered free radical that produces a deep purple solution and has a maximum absorption peak at 517 nm. If a free radical scavenger is present in the reaction system, it can pair with the single electron of DPPH, causing the absorption peak at 517 nm to gradually dissipate.

[0179] The degree of color change has a stoichiometric relationship with the number of paired electrons. Therefore, the antioxidant activity can be measured according to the change in absorbance. The greater the inhibition rate, the stronger the antioxidant ability.

[0180] 6 mg of DPPH was weighed into a 100 mL volumetric flask, and the volume was adjusted with 80% ethanol to prepare a solution with a concentration of 0.06 mg / mL. This was stored in the dark at 4°C.

[0181] A peptide solution was prepared by dissolving 0.6 g of peptide powder in 1 mL of distilled water.

[0182] 200 μL of peptide solution was added to 3.8 mL of DPPH solution. The solution was kept in the dark at room temperature for 1.5 hours. The absorbance at 517 nm was measured and designated as A1. The absorbance of the blank control sample was designated as A0. Therefore, the percentage of remaining DPPH was calculated as follows: Clearance rate (%) = [A0 - A1] / A0 × 100.

[0183] The DPPH clearance rate for Compound A was 52.11%. The DPPH clearance rate for Compound B was much lower at 8.9%, representing a significant improvement in antioxidant capacity.

[0184] Example 7 Stability of the compounds of the present invention The stability of Compounds A and B (obtained as described in Examples 1 and 3 above) was tested as follows.

[0185] 10 mg of each compound was dissolved in 1 mL of distilled water to prepare a stock solution with a concentration of 10 mg / mL. Then, 200 μL of the stock solution was diluted with 20 mM phosphate buffer (PB, Sigma-Aldrich China) with different pH values ​​as shown in Table 8 below.

[0186] [Table 8]

[0187] All of the above samples were filtered through a 0.22 μm membrane (SLGV033RS, Sigma-Aldrich China) to remove potential bacteria in the solution in a super clean bench after preparation.

[0188] All the above samples were stored at room temperature and transferred to a 45°C oven after 3 days of initial sample analysis by HPLC (Column: Angilent ZORBAX Eclipse XDB-C18 (4.6 x 250 mm, 5 μm) (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).

[0189] The stability of the above samples was studied by observing the color change (Table 9), HPLC peak area (Table 10), and purity (Table 11). The pH values ​​of all samples were measured to be stable over the test period.

[0190] [Table 9]

[0191] None means no color change was observed, + means a slight color change was observed, and ++ means a significant color change was observed.

[0192] As shown in Table 9, solutions of Compound A became darker over time, especially at higher pH. The color of the remaining samples remained unchanged.

[0193] [Table 10]

[0194] The peak areas, shown in Table 10 above as area % of the results on day 0, represent the peptide concentrations. As shown in Table 10, the peak areas of the three samples of Compound B remained nearly unchanged. For Compound B, the peak area remained unchanged at pH 5, but decreased significantly at pH 7.2 and pH 8.1, indicating that the concentration of the peptide decreased over time.

[0195] [Table 11]

[0196] Oxidation of the peptide results in a shift in the HPLC peak and a decrease in the purity of the peptide. As shown in Table 11 above, the results indicate that most of Compound A was oxidized at pH 7.2 and pH 8.3.

[0197] From the above results, it can be seen that the compounds of the present invention are stable at all pH values, whereas Compound A is only stable at pH 5.3.

[0198] Example 8 Compound B / Montelukast Bandage An ointment was prepared by first dissolving montelukast sodium (200 mg, Arromax Pharmatech Co., Ltd., Suzhou, China) in polyethylene glycol 400 (20.0 g, Sinopharm Chemical Reagent Co., Ltd.) with stirring. Compound B (16 mg, see Example 1 above) was then added to the solution.

[0199] 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, the solution containing Compound B and montelukast was added to it with stirring, followed by mixing for 5-10 minutes. After cooling to room temperature, the final product was obtained.

[0200] The ointment was evenly coated onto the gauze using a flat plate and allowed to cool to room temperature to give the final dressing.

[0201] Example 9 Compound B / Trypsin Spray Compound B (30 mg, see Example 1 above) was dissolved in 10 mL of water. Trypsin (30 mg, Sichuan Deebio Pharmaceutical Co. Ltd., Guanhan, Sichuan, China) was dissolved in another 10 mL of water.

[0202] Calcium chloride (0.1 g), ethanol (0.5 g), water-soluble menthol (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.

[0203] A solution containing Compound B and trypsin was added to this mixture with stirring to provide a liquid for spraying.

[0204] Example 10 Compound B / trypsin aerosol for inhalation An aerosol formulation was prepared essentially as described above in Example 9 from 20 mg of Compound B and 30 mg of trypsin, this time without glycerol, in a total of 99.32 mL of water.

[0205] Example 11 Cold research Subjects enrolled in this study had a self-diagnosed viral infection, commonly referred to as the common cold. They presented with symptoms including cough, sore throat, and loss of voice for two days. No other cold medications were being used prior to treatment.

[0206] Subjects were required to use the liquid spray of Example 10 above in the morning and evening.

[0207] For each use, 3 mL of spray was poured into the atomizer reservoir attached to the atomizer. The atomizer was then opened and the mask was placed over the subject's mouth and nose.

[0208] After the first use, the subject felt that the pain in his throat was relieved.After the second use, the subject felt that the pain in his throat was gone and he was able to speak, which showed that the liquid spray containing the compound of the present invention can relieve the symptoms of cough and sore throat caused by colds.

[0209] Example 12 Idiopathic pulmonary fibrosis (IPF) model Experimental animals and group assignment: Forty adult male Sprague-Dawley rats were divided into four groups after 7 days of adaptive feeding: a sham-operated group (sham), an IPF model group (no treatment, model), a test group (compound B), and a positive control group (positive control).

[0210] Compound B was administered at a dose of 130 μg / rat in the study, which was administered by aerosol inhalation. Oral administration of pirfenidone (Etuary®, Beijing Continent Pharmaceutical Co., Ltd., Beijing, China) at a single bolus dose of 120 mg / kg served as a positive control.

[0211] A pulmonary fibrosis model was established by intratracheal instillation of bleomycin. Rats were anesthetized and placed supine on the operating table to expose the trachea. Bleomycin (5 mg / kg) in saline was injected into the trachea through the gap between the tracheal cartilage rings. The sham-operated group received an equal volume of saline. Immediately after administration, the rats were lifted vertically and rotated to ensure even distribution of the drug. After approximately 5 days of recovery, the rats were administered different drugs according to the schedule for 28 consecutive days. The experimental design is shown in Table 12.

[0212] [Table 12]

[0213] The exhaust volume of the nebulizer was 0.15 mL / min. The inhalation time for each rat was 1 min.

[0214] On the 29th day after drug administration, rats were anesthetized by intraperitoneal injection of chloral hydrate. After orbital blood collection, the rats were sacrificed. The chest cavity was quickly opened, and the lungs were collected for further analysis. The lung wet weight was measured, and the lung index was calculated (lung wet weight / rat weight × 1000). The tissues were stored in a refrigerator at -80°C for further use.

[0215] Lung tissue pieces were accurately weighed and 9 times their weight of saline 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 alpha-smooth muscle actin (α-SMA). Standard ELISA methods were used for detection. Malonaldehyde (MDA) content and superoxide dismutase (SOD) activity in the lung tissue were also detected.

[0216] The effects of Compound B on the contents of TNF-α, α-SMA, and TGF-β1 in tissues of bleomycin-induced pulmonary fibrosis in rats are shown in Table 13.

[0217] [Table 13]

[0218] The results show that Compound B inhibited the production of TNF-α, α-SMA, and TGF-β1 in lung tissue of rats with bleomycin-induced pulmonary fibrosis. The inhibitory effect was similar to that of pirfenidone, a known IPF drug.

[0219] The effects of Compound B on lung index, MDA, and SOD content are shown in Table 14.

[0220] [Table 14]

[0221] The results showed that the lung index value in the compound B group was lower than that in the model group, indicating that compound B could reduce edema. Compared with the model group, the lower MDA value and higher SOD value in the compound B group indicated that compound B has antioxidant effects by increasing SOD production and reducing lipid oxidation.

[0222] Example 13 Antitussive experiment - Ammonia-induced cough method in mice. Thirty-six ICR mice were randomly divided into three groups according to body weight, labeled as CMC-Na (negative) control group, dextromethorphan hydrobromide (positive) control group, and Compound B (B) group, with each group containing 12 mice, 6 males and 6 females.

[0223] Compound B was administered by nebulization using a YLS-8A multi-function cough-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 by intragastric administration at 10 mg / kg once daily for 5 days.

[0224] One, two, and four hours after the last drug administration, the mice were placed in an inverted beaker. One mL of ammonia water (25.0–28.0%) was placed in a boiling water bath and allowed to evaporate.

[0225] The duration of ammonia exposure for mice was determined by analyzing data immediately after each test or series of tests. The results of the previous analysis determined the method for conducting the next test or series of tests. In this way, the process of experiment-analysis-experiment proceeded sequentially: (a) The time it took for the previous mouse to cough determined the (shorter) time the next mouse was scheduled to receive the ammonia challenge, or (b) If the previous mouse did not cough, the subsequent mouse was stimulated for a longer period of time.

[0226] The difference in logarithm between two adjacent periods was 0.1.

[0227] A stethoscope was used to detect the number of coughs within one minute: if a mouse coughed more than three times per minute, this was labeled as a "cough," and if it coughed less than three times per minute, it was labeled as a "no cough."

[0228] EDT 50 was defined as the ammonia challenge time at which half of the mice developed a "cough" as calculated by the following formula: EDT 50 =lg -1 c / n (C is equal to the sum of r and x, where r is the number of animals in each stimulation time group, x is the logarithm of the stimulation time, and n is the number of animals in each group). lg is the base 10 logarithm. The results are shown in Table 15 below.

[0229] [Table 15]

[0230] In Table 15, R is the EDT concentration in the control group expressed as a percentage. 50 EDT in treatment group divided by 50 An R value of more than 130% means that the compound has an antitussive effect, and an R value of more than 150% indicates that it has a clear antitussive effect.

[0231] The results show that Compound B has a positive effect on cough relief at 1 and 2 hours.

[0232] Example 14 Liquid spray for treating colds Compound B was dissolved in water for injection (WFI, TC-RO-0.25T / h water treatment system, Yangzhou Tiancheng Water Treatment Devices & Engineering Co., Ltd., Yangzhou, China). The concentration was 0.5 mg / mL. 5 mL was filled into a plastic water spray bottle.

[0233] Subjects enrolled in the study had a cold for two days prior to treatment. No other cold treatments were used prior to treatment. Subjects who presented with cough, sore throat, and voice loss were required to use the liquid spray (3-5 pumps each morning and evening).

[0234] Subject noticed relief from sore throat after first use. After second use, subject no longer felt sore throat and was able to speak.

[0235] This experiment demonstrated that a liquid spray containing the compound of the present invention relieves the symptoms of cough and sore throat caused by the common cold.

[0236] Example 15 Aerosol inhalation to treat fever. The same formulation as described in Example 14 above was administered to subjects enrolled in the study who had a fever (temperature above 38.5°C) for 1 day. No other anti-pyretic medications were used prior to treatment.

[0237] Subjects were required to use the aerosol inhalation device in the morning and evening by pouring 3 mL of the formulation into the atomizer reservoir connected to the atomizer and mask and placing the mask over their mouth and nose.

[0238] The subject noticed that his body temperature had dropped to normal within two hours. After three uses, the fever symptoms did not recur.

[0239] This experiment demonstrates that inhalation of an aerosol containing a compound of the present invention reduces fever.

[0240] Example 16 A liquid spray for surgical pain relief during laser surgery. Enrolled subjects were required to use 1 mL of a liquid spray containing the same formulation as described in Example 14 above in a water spray bottle on half of their face before and after lattice laser surgery to remove facial melanin.

[0241] The subject reported that she experienced no pain during surgery on the half of her face that had the spray applied. Similarly, she experienced no pain after surgery. Conversely, she experienced pain during surgery on the half of her face that had not been sprayed. Furthermore, the pain lasted for approximately two hours after surgery.

[0242] This experiment demonstrates that a liquid spray containing a compound of the present invention reduces surgical pain during laser surgery.

[0243] Example 17 Combination Treatment for Ulcerative Colitis A gel containing Compound B (obtained as described above) and mefp-1 (USUN Bio Co., Jiangyin, China), both at a concentration of 1 mg / g, was prepared by combining the active ingredients with methylcellulose (2.5%), propanediol (11%), glycerol (11%), and acetic acid (pH control agent, up to 0.5 g) to yield a gel precursor with a pH of 5.5. All excipients were obtained from Sinopharm Chemical Reagent Co. Ltd. The gel was made up with water for injection. The gel was filled into a disposable anal-enteric drug delivery system.

[0244] Before treatment, the patient has more than 21 bowel movements per day, and shows severe bleeding from the colon and ulceration in the colon.The gel formulation is given by passing through the anus.After giving one dose on the first day, the patient only has three bowel movements on the second day.Then, the patient receives two more doses on the second day.By the third day, bleeding is controlled.

[0245] After 14 days of two doses per day, the patient's ulcerative colitis symptoms were alleviated and he had no further bleeding.

Claims

1. The following compounds Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Tyr-Lys, a stereoisomer thereof, or a pharmaceutically or cosmetically acceptable salt thereof; a pharmaceutically acceptable adjuvant, diluent, or carrier; 1. A topical pharmaceutical formulation for use in the treatment of inflammation, an inflammatory disorder, and / or a condition or disorder characterized by inflammation, comprising:

2. 2. The topical pharmaceutical formulation of claim 1, wherein the compound is: 【Chemical 1】

3. 3. The topical pharmaceutical formulation of claim 1 or claim 2, wherein the compound is not in the form of a salt.

4. The following compounds 【Chemistry 2】 or a pharmaceutically or cosmetically acceptable salt thereof; a pharmaceutically acceptable adjuvant, diluent, or carrier; 1. A topical pharmaceutical formulation for use in the treatment of inflammation, an inflammatory disorder, and / or a condition or disorder characterized by inflammation, comprising:

5. The following compounds Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Tyr-Lys, a stereoisomer thereof, or a pharmaceutically or cosmetically acceptable salt thereof, or The following compounds 【Chemistry 3】 or a pharmaceutically or cosmetically acceptable salt thereof; Another anti-inflammatory agent A topical pharmaceutical formulation for use in the treatment of inflammation, an inflammatory disorder, and / or a condition or disorder characterized by inflammation, comprising a pharmaceutically acceptable adjuvant, diluent, or carrier.

6. 1. A kit of parts for use in the treatment of inflammation, inflammatory disorders, and / or conditions or disorders characterized by inflammation, comprising: (A) a topical pharmaceutical formulation according to claim 1; (B) a pharmaceutical formulation comprising another anti-inflammatory agent mixed with a pharmaceutically acceptable adjuvant, diluent, or carrier; A kit of parts, wherein the components (A) and (B) are each provided in a form suitable for administration in combination with the other.

7. 1. A kit of parts for use in the treatment of inflammation, inflammatory disorders, and / or conditions or disorders characterized by inflammation, comprising: (A) a topical pharmaceutical formulation according to claim 4; (B) a pharmaceutical formulation comprising another anti-inflammatory agent mixed with a pharmaceutically acceptable adjuvant, diluent, or carrier; A kit of parts, wherein the components (A) and (B) are each provided in a form suitable for administration in combination with the other.

8. For the manufacture of a medicament for the treatment of inflammation, inflammatory disorders, and / or conditions or disorders characterized by inflammation, The following compounds Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Tyr-Lys, 【Chemistry 4】 (The compound may or may not be in the form of a salt.) A topical pharmaceutical formulation according to any one of claims 1 to 5, or The kit of parts according to claim 6 or 7. Use of.

9. 4. The topical pharmaceutical formulation according to any one of claims 1 to 3, wherein the inflammatory disorder is selected from psoriasis, acne, eczema, and dermatitis, rhinitis, chronic obstructive pulmonary disease, and ulcerative colitis.

10. 10. The topical pharmaceutical formulation of claim 9, wherein the dermatitis is atopic dermatitis or steroid-dependent dermatitis.

11. 4. The topical pharmaceutical formulation of any one of claims 1 to 3, wherein the inflammatory disorder is selected from conjunctivitis, keratitis, acute superficial keratitis, nummular keratitis, interstitial keratitis, disciform keratitis, neurotrophic keratitis, mucoplaque keratitis, herpes simplex keratitis, herpes zoster keratitis, bacterial keratitis, fungal keratitis, acanthamoeba keratitis, Onchocerca volvulus keratitis, superficial punctate keratitis, ulcerative keratitis, lagophthalmos keratitis, photokeratitis, and acute hyperemia and optic neuritis during contact lens wear.

12. The topical pharmaceutical formulation of any one of claims 1 to 3 or 9 to 11, wherein the disorder characterized by inflammation is a wound or a burn.

13. 13. The topical pharmaceutical formulation of claim 12, wherein the wound is an abrasion, scratch, incision, laceration, skin puncture, laceration, contusion, scar or blister, or itching associated with any of the above.

14. 12. The topical pharmaceutical formulation of any one of claims 1 to 3 or 9 to 11, wherein said condition or disorder characterized by inflammation is hemorrhoids.

15. The topical pharmaceutical formulation of any one of claims 1 to 3 or 9 to 11, wherein the disorder characterized by inflammation is a viral infection.

16. 16. The topical pharmaceutical formulation of claim 15, wherein the viral infection is a cold.

17. A topical pharmaceutical formulation according to any one of claims 1 to 4 for use in the treatment of idiopathic pulmonary fibrosis.

18. 18. The topical pharmaceutical formulation of any one of claims 1 to 3 or 9 to 17, for direct topical administration to a mucosal surface.

19. 18. The topical pharmaceutical formulation of any one of claims 1 to 3 or 15 to 17, for direct topical administration to the lungs.

20. 15. The topical pharmaceutical formulation of any one of claims 1 to 3, 10 or 12 to 14, for direct topical administration to the skin.

21. 6. The topical pharmaceutical formulation of claim 5, wherein the additional anti-inflammatory agent is montelukast or a pharmaceutically acceptable salt thereof and the condition is a wound, a burn, or hemorrhoids.

22. 6. The topical pharmaceutical formulation of claim 5, wherein the additional anti-inflammatory agent is montelukast or a pharmaceutically acceptable salt thereof and the condition is chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis.

23. 6. The topical pharmaceutical formulation of claim 5, wherein said additional anti-inflammatory agent is mefp-1 and said condition is ulcerative colitis.

24. 6. The topical pharmaceutical formulation of claim 5, wherein said additional anti-inflammatory agent is trypsin and said condition characterized by inflammation is a viral infection.

25. 25. The topical pharmaceutical formulation of claim 24, wherein the viral infection is a cold.

26. 7. The kit of parts of claim 6, wherein the inflammatory disorder is selected from psoriasis, acne, eczema, and dermatitis, rhinitis, chronic obstructive pulmonary disease, and ulcerative colitis.

27. 27. The kit of parts according to claim 26, wherein the dermatitis is atopic dermatitis or steroid-dependent dermatitis.

28. 7. The kit of parts of claim 6, wherein the inflammatory disorder is selected from conjunctivitis, keratitis, acute superficial keratitis, nummular keratitis, interstitial keratitis, disciform keratitis, neurotrophic keratitis, mucoplaque keratitis, herpes simplex keratitis, herpes zoster keratitis, bacterial keratitis, fungal keratitis, acanthamoeba keratitis, Onchocerca volvulus keratitis, superficial punctate keratitis, ulcerative keratitis, lagophthalmos keratitis, photokeratitis, and acute hyperemia and optic neuritis during contact lens wear.

29. 7. The kit of parts of claim 6, wherein the disorder characterized by inflammation is a wound or a burn.

30. 30. The kit of parts of claim 29, wherein the wound is an abrasion, scratch, incision, laceration, skin puncture, laceration, contusion, scar or blister, or itching associated with any of the above.

31. 7. The kit of parts of claim 6, wherein the condition or disorder characterized by inflammation is hemorrhoids.

32. The kit of parts according to claim 6, wherein the disorder characterized by inflammation is a viral infection.

33. 33. The kit of parts of claim 32, wherein the viral infection is a cold.

34. For the manufacture of a drug for the treatment of idiopathic pulmonary fibrosis, The following compounds Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Tyr-Lys, 【Chemistry 5】 (The compound may or may not be in the form of a salt.) A topical pharmaceutical formulation according to any one of claims 1 to 5, or The kit of parts according to claim 6 or 7. Use of.

35. 34. The kit of parts according to any one of claims 6, 7 or 26 to 33, wherein said administration is direct topical administration to a mucosal surface.

36. 34. The kit of parts according to any one of claims 6, 7 or 26 to 33, wherein said administration is local administration directly to the lung.

37. 34. The kit of parts according to any one of claims 6, 7 or 26 to 33, wherein said administration is topical administration directly to the skin.

38. 38. The kit of parts of any one of claims 6, 7, 26-33, or 35-37, wherein the additional anti-inflammatory agent is montelukast or a pharmaceutically acceptable salt thereof, and the condition is a wound, a burn, or a hemorrhoid.

39. 38. The kit of parts of any one of claims 6, 7, 26-33, or 35-37, wherein the additional anti-inflammatory agent is montelukast or a pharmaceutically acceptable salt thereof, and the condition is chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis.

40. 38. The kit of parts of any one of claims 6, 7, 26-33 or 35-37, wherein the additional anti-inflammatory agent is mefp-1 and the condition is ulcerative colitis.

41. 38. The kit of parts of any one of claims 6, 7, 26-33 or 35-37, wherein the additional anti-inflammatory agent is trypsin and the condition characterized by inflammation is a viral infection.

42. 42. The kit of parts of claim 41, wherein the viral infection is a cold.

43. 43. A method for preparing the kit-of-parts of any one of claims 6, 7, 26-33 or 35-42, comprising providing components (A) and (B) as separate formulations.

44. 43. A method for preparing the kit-of-parts of any one of claims 6, 7, 26-33 or 35-42, comprising packaging components (A) and (B) together as separate components of a "combination pack" for use in combination with each other in combination therapy.

45. 9. The use according to claim 8, wherein the inflammatory disorder is selected from psoriasis, acne, eczema, and dermatitis, rhinitis, chronic obstructive pulmonary disease, and ulcerative colitis.

46. 46. ​​The use according to claim 45, wherein the dermatitis is atopic dermatitis or steroid-dependent dermatitis.

47. 9. The use of claim 8, wherein the inflammatory disorder is selected from conjunctivitis, keratitis, acute superficial keratitis, nummular keratitis, interstitial keratitis, disciform keratitis, neurotrophic keratitis, mucoplaque keratitis, herpes simplex keratitis, herpes zoster keratitis, bacterial keratitis, fungal keratitis, acanthamoeba keratitis, Onchocerca volvulus keratitis, superficial punctate keratitis, ulcerative keratitis, lagophthalmos keratitis, photokeratitis, and acute hyperemia and optic neuritis during contact lens wear.

48. 9. The use according to claim 8, wherein the disorder characterized by inflammation is a wound or a burn.

49. 49. The use of claim 48, wherein the wound is an abrasion, scratch, incision, laceration, skin puncture, laceration, contusion, scar or blister, or itching associated with any of the above.

50. 9. The use according to claim 8, wherein the condition or disorder characterized by inflammation is hemorrhoids.

51. 9. The use according to claim 8, wherein the disorder characterized by inflammation is a viral infection.

52. 52. The use of claim 51, wherein the viral infection is a cold.

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