Inflammation suppression by peripheral nerve agonists

Abstract

To provide methods and compositions for treating conditions associated with activation or over-activation of mast cells, that is, acute or chronic urticaria, mastocytosis or any subtype of mastocytosis, pseudo-allergy, any form of dermatitis such as contact or atopic dermatitis, wound healing, rosacea, acne, or psoriasis, food allergy, or irritable bowel syndrome.SOLUTION: Provided herein is a method of treating a patient having a condition associated with activation or over-activation of mast cells, comprising administering to the patient an amount of MRGPRD agonist effective to down-regulate mast cell degranulation in the patient. The MRGPRD agonist is: beta alanine; gamma-aminobutyric acid; beta aminoisobutyric acid; 5-oxoeicosatetraenoic acid; or any combination thereof. Also topical, enteric, delayed-release or aerosol formulations comprising β-alanine are provided.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Federal Grant Statement This invention was made with government support awarded by the National Institutes of Health under Grant No. AR067187. The United States government has certain rights in this invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 893,433, filed August 29, 2019.

[0003] Provided herein are methods and compositions for the suppression of inflammation in the skin and mucous membranes, which are useful for treating conditions caused by mast cell activation, including, but not limited to, urticaria, mastocytosis, allergies, eczema, allergic contact dermatitis, irritable bowel syndrome, and food allergies.

[0004] Mast cells have important immunoregulatory functions, for example, at the skin and mucosal boundaries between the body and the environment. Mast cells are an important part of the innate immune system and are abundant in barrier organs such as the skin. Mast cells express many activating receptors, including immunoglobulin (Ig) E and the Fc region of certain classes of IgG, which allow antibodies to bind to their cell surface. Upon antigen entry, granule-containing mast cells release many inflammatory mediators, such as histamine and serotonin (mast cell activation). Mast cells also express surface Mas-related G protein receptors. These receptors bind specific ligands, including the neuropeptide substance P, the antimicrobial peptide LL37, and exogenous factors such as certain drugs and components of house dust mites. This receptor binding results in the release of several biologically active molecules, including cytokines and proteases, which are important in allergic reactions, anaphylactic-type responses, and several inflammatory diseases. These responses can be attenuated by the use of drugs that inhibit the release of mast cell mediators (called mast cell stabilizers) or that block the action of the mediators (such as antihistamines).

[0005] Several conditions (e.g., pathologies or diseases) have been associated with mast cell activation, including, but not limited to, urticaria, mastocytosis (e.g., cutaneous or systemic), allergies, eczema, allergic contact dermatitis, irritable bowel syndrome, and food allergies.

[0006] Mast cells are an important part of the innate immune system and are abundant in barrier organs such as the skin. Although mast cells are primarily known for inducing allergic responses, many other biological functions have now been described for these cells. Several studies have shown that during wound repair, mast cells enhance acute inflammation, stimulate re-epithelialization and angiogenesis, and promote scarring. Mast cells have also been associated with abnormal sensations, and large numbers of MCs are found in chronic wounds, hypertrophic scars, and keloids. Summary of the Invention

[0007] Therapeutic agents effective in downregulating, e.g., reducing or ameliorating, mast cell activation or its effects are desirable, as such therapeutic agents and their uses are desirable in the treatment of pathologies resulting from mast cell activation.

[0008] In a first aspect or embodiment of the present invention, there is provided a method of treating a patient having a condition associated with mast cell activation or hyperactivation, comprising administering to the patient an amount of an MRGPRD agonist effective to downregulate mast cell degranulation in the patient.

[0009] In a second aspect or embodiment of the invention, there is provided a method of the first aspect or embodiment, wherein the MRGPRD agonist is beta-alanine (β-ala) or a pharmaceutically acceptable salt thereof.

[0010] In a third aspect or embodiment of the present invention, there is provided a method of the first aspect or embodiment, wherein the MRGPRD agonist is gamma aminobutyric acid (GABA) or a pharmaceutically acceptable salt thereof.

[0011] In a fourth aspect or embodiment of the present invention, there is provided a method of the first aspect or embodiment, wherein the MRGPRD agonist is β-aminoisobutyric acid (β-AIBA) or a pharmaceutically acceptable salt thereof, or 5-oxoeicosatetraenoic acid (5-oxoETE) or a pharmaceutically acceptable salt thereof.

[0012] In a fifth aspect or embodiment of the present invention, there is provided a method of any of the first to fourth aspects or embodiments, wherein the condition associated with mast cell activation or hyperactivation is uticaria, and the MRGPRD agonist is administered locally or systemically to the patient.

[0013] In a sixth aspect or embodiment of the present invention, there is provided a method of any of the first to fourth aspects or embodiments, wherein the condition associated with mast cell activation or hyperactivation is mastocytosis, and the MRGPRD agonist is administered locally or systemically to the patient.

[0014] In a seventh aspect or embodiment of the present invention, there is provided a method of any of the first to fourth aspects or embodiments, wherein the condition associated with mast cell activation or hyperactivation is food allergy, and the MRGPRD agonist is administered to the patient enterally (to or for release in the patient's intestine), such as in an enteric-coated or delayed-release oral dosage form.

[0015] In an eighth aspect or embodiment of the present invention, there is provided a method of any of the first to fourth aspects or embodiments, wherein the condition associated with mast cell activation or hyperactivation is acute or chronic urticaria, mastocytosis or any subtype of mastocytosis, pseudoallergy, any form of dermatitis such as contact or atopic dermatitis, wound healing, rosacea, acne, or psoriasis, and the method comprises administering to the patient an amount of an MRGPRD agonist effective to inhibit or reduce inflammation in the patient, for example, an amount effective to inhibit or reduce mast cell activation in the patient.

[0016] In a ninth aspect or embodiment of the present invention, there is provided the method of any of the first to third aspects or embodiments, wherein 5 mg to 5000 mg or 1 mg to 10 g of the MRGPRD agonist is administered to the patient.

[0017] In a tenth aspect or embodiment of the present invention, there is provided a method of any of the first to third aspects or embodiments, wherein the MRGPRD agonist is administered to the patient for at least one week.

[0018] In an eleventh aspect or embodiment of the present invention, there is provided a method of treating a wound in a patient, the method comprising administering to the wound an amount of an MRGPRD agonist effective to enhance wound healing in the patient.

[0019] In a twelfth aspect or embodiment of the invention, there is provided a method of the eleventh aspect or embodiment, wherein the MRGPRD agonist is beta-alanine (β-ala) or a pharmaceutically acceptable salt thereof; gamma aminobutyric acid (GABA) or a pharmaceutically acceptable salt thereof; beta aminoisobutyric acid (β-AIBA) or a pharmaceutically acceptable salt thereof; 5-oxoeicosatetraenoic acid (5-oxoETE) or a pharmaceutically acceptable salt thereof, or a combination of any of the above.

[0020] In a thirteenth aspect or embodiment of the present invention, there is provided the method of the eleventh or twelfth aspect or embodiment, wherein the MRGPRD agonist is formulated as a topical pharmaceutical composition.

[0021] In a fourteenth aspect or embodiment of the present invention, there is provided the method of any of the eleventh to thirteenth aspects or embodiments, wherein the wound is a non-healing wound.

[0022] In a fifteenth aspect or embodiment of the present invention, there is provided a method of any of the eleventh to fourteenth aspects or embodiments, wherein the patient has diabetes.

[0023] In a sixteenth aspect or embodiment of the present invention, there is provided the method of the fifteenth aspect or embodiment, wherein the wound is a diabetic foot ulcer.

[0024] In a seventeenth aspect or embodiment, there is provided a topical composition comprising β-alanine, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier.

[0025] In an eighteenth aspect or embodiment of the invention, there is provided a composition of the seventeenth aspect or embodiment in an amount effective to treat a condition associated with mast cell activation or hyperactivation, for example acute or chronic urticaria, mastocytosis or any variant of mastocytosis, pseudoallergy, any form of dermatitis such as contact or atopic dermatitis, wound healing, rosacea, acne, or psoriasis.

[0026] In a nineteenth aspect or embodiment of the present invention, there is provided the composition of the eighteenth aspect or embodiment, which is an ointment, cream, lotion, spray, gel, or wound dressing.

[0027] In a twentieth aspect or embodiment, there is provided an aerosol or spray pharmaceutical formulation or device comprising β-alanine, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier.

[0028] In a twenty-first aspect or embodiment of the present invention, there is provided an aerosol or spray pharmaceutical formulation or device of the twentieth aspect or embodiment in the form of a nebulizer.

[0029] In a twenty-second aspect or embodiment of the present invention, there is provided an aerosol or spray pharmaceutical formulation or device of the twentieth aspect or embodiment in the form of a nasal sprayer.

[0030] In a twenty-third aspect or embodiment of the present invention, there is provided an aerosol or spray pharmaceutical formulation or device of the twentieth aspect or embodiment in the form of a metered dose inhaler or metered dose sprayer.

[0031] In a twenty-fourth aspect or embodiment of the present invention, there is provided a delayed release or enteric formulation comprising β-alanine or a pharmaceutically acceptable salt thereof in a delayed release and / or enteric composition and / or in a delayed release and / or enteric coating for enteric release of β-alanine in a patient (i.e., the drug is released primarily in the patient's intestine).

[0032] In a 25th aspect or embodiment of the present invention, there is provided a method of preventing, alleviating or treating pain or pruritus in a patient, e.g., neurogenic inflammation, pain, or pruritus in a patient, comprising administering to the patient an MRGPRD agonist in an amount effective to prevent, alleviate or treat pain or pruritus in the patient, e.g., an amount effective to inhibit or reduce mast cell activation in the patient. [Brief explanation of the drawings]

[0033] [Figure 1] FIG. 1 is a graph showing that β-alanine inhibits 48 / 80-mediated mast cell degranulation, as described in Example 2. [Figure 2] 2 is a graph showing that β-alanine inhibits DNFB contact hypersensitivity in vivo, as described in Example 3. Error bars + / - SEM. **p<0.01; *p<0.05. [Figure 3] Figure 3 is a graph showing that 5% topical β-alanine inhibits mast cell degranulation in mice, as described in Example 4. Values ​​represent the amount of Evans blue dye in the dermis in μg per gram of tissue. **p<0.01. Each symbol represents data from an individual animal. [Figure 4] FIG. 4 is a graph showing that β-alanine induces wound healing-related transcriptome changes, as described in Example 5. DETAILED DESCRIPTION OF THE INVENTION

[0034] The use of numerical values ​​in the various ranges set forth herein, unless otherwise specified, is described as an approximation, as if the word "about" were to precede both the minimum and maximum values ​​of the stated range. In this manner, slight variations above and below the stated ranges can be used to achieve substantially the same results as values ​​within the range. Also, unless otherwise specified, the disclosure of these ranges is intended as a continuous range, including all values ​​between the minimum and maximum values.

[0035] As used herein, the term "comprising" and variations thereof are meant to be open-ended. The terms "a" and "an" are intended to refer to one or more.

[0036] A "pathological condition," in the context of this disclosure, is a pathology, disease, disorder, sequelae, or any other abnormal or undesirable effect resulting from the activation or hyperactivation of mast cells.

[0037] As used herein, a "patient" may be an animal such as a mammal, including, but not limited to, a primate (e.g., a human, a non-human primate, e.g., a monkey, and a chimpanzee), a non-primate (e.g., a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or a goose).

[0038] As used herein, the term "treatment" can refer to a beneficial or specific result, such as the amelioration of one or more functions or symptoms of a condition, e.g., in the context of this disclosure, a condition resulting from mast cell activation or overactivation. The term "treatment" can also include, but is not limited to, the alleviation or amelioration of one or more symptoms of a condition resulting from mast cell activation or overactivation. "Treatment" can also mean the prolongation of survival compared to expected survival in the absence of treatment.

[0039] "Lower" in the context of a marker or symptom associated with a condition, e.g., in the context of the present disclosure, a condition related to mast cell activation or hyperactivation, can refer to a clinically meaningful and / or statistically significant reduction in such level. The reduction can be, for example, at least 10%, at least 20%, at least 30%, at least 40%, or more, to a level considered within the normal range for individuals without such a condition, or to a level below the detection level of the assay. In certain embodiments, the reduction can be to a level considered within the normal range for individuals without such a condition, which can also be referred to as normalization of the level. In certain embodiments, the decrease can be normalization of the level of a sign or symptom of the condition, i.e., a narrowing of the difference between the target level of the sign of the condition and the normal level of the sign of the condition (e.g., the upper limit of normal, if the target value must be reduced to reach a normal value, or the lower limit of normal, if the target value must be increased to reach a normal level). These methods can include clinically meaningful inhibition of mast cell degranulation as demonstrated by a clinically meaningful outcome after treating a subject with an MRGPRD agonist as described herein.

[0040] A "therapeutically effective amount," as used herein, can include an amount of an MRGPRD agonist as described herein that, when administered to a patient in need thereof, e.g., a patient having a condition resulting from mast cell activation or overactivation, can be sufficient to effect treatment of the condition (e.g., by resolving, alleviating, or maintaining an existing condition or one or more symptoms of the condition). A "therapeutically effective amount" can vary depending on the MRGPRD agonist, how the agent is administered, the condition and severity, and the medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment, if any, and other individual characteristics of the patient being treated.

[0041] A "therapeutically effective amount" can also include that amount of an agent that produces a local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The MRGPRD agonists used in the methods and compositions described herein can be administered in amounts sufficient to obtain a reasonable benefit / risk ratio applicable to such treatment.

[0042] For patient treatment, therapeutic compounds, for example, MRGPRD agonists such as β-alanine (β-ala), γ-aminobutyric acid (GABA), and / or β-aminoisobutyric acid (β-AIBA), can be administered by any effective route of administration, including, but not limited to, parenteral administration, such as intravenous, intraperitoneal, intraorgan, e.g., hepatic delivery, or intramuscular injection; inhalation, for example, in a spray or aerosol metered-dose inhaler; topical administration, such as dermal, transdermal, intraaural, or intraocular delivery; transmucosal administration, such as vaginal or oral administration; or oral administration. The composition may be administered in individual doses or in multiple doses over time to maintain reduced mast cell activation. Mucosal delivery includes, for example, but is not limited to, delivery to the mucosa of the respiratory system, gastrointestinal system, and vagina. The MRGPRD agonist can be delivered in a manner that targets specific tissues, such as the skin, rectum, intestine, or nasal cavity.

[0043] The MRGPRD agonist may be formulated or otherwise prepared as a suitable composition, such as a pharmaceutical dosage form or medicament, in which the compound is the active ingredient. The medicament may comprise a pharmaceutically acceptable carrier or excipient. The therapeutic agent / pharmaceutical composition may Remington: The Science and Practice of Pharmacy, 21st Edition, edited by Paul Beringer et al., Lippincott, Williams & Wilkins, Baltimore, MD, Easton, Pa. (2005). As is well known in the pharmaceutical arts, depending on the route of delivery, dosage forms may comprise additional carriers or excipients, such as water, saline (e.g., normal saline), or phosphate buffered saline. The composition may comprise a pharmaceutically acceptable carrier or excipient. An excipient is an inert substance used as a carrier for the active ingredient of a drug. An "inert" excipient may facilitate and assist in the delivery, stability, or bioavailability of the active ingredient in a pharmaceutical product. Non-limiting examples of useful excipients include anti-caking agents, binders, rheology modifiers, coating agents, disintegrants, emulsifiers, oils, buffers, salts, acids, bases, fillers, diluents, solvents, flavors, penetration enhancers, colorants, glidants, lubricants, preservatives, antioxidants, absorbents, vitamins, sweeteners, and the like, as may be utilized in the pharmaceutical / formulation arts.

[0044] Topical formulations or pharmaceutical agents can be used to deliver MRGPRD agonists as described herein. Topical delivery includes delivery through the skin or mucosa, such as dermal, oral, nasal, ocular, otic, or vaginal delivery routes. Suitable topical delivery formulations and considerations for formulating such topical formulations are widely known. (See, e.g., Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Lippincott, Williams, & Wilkins, Philadelphia, Pa., eds., 21st ed. (2005), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compositions, particularly Chapters 65 ("Optical Drugs"), 43 ("Ophthalmic Preparations"), and 44 ("Medical Topicals"); Ueda, CT et al. "Topical and Transdermal Drug Products" Pharmacopeial Forum 35(3) [May–June 2009]; and Benson, Heather AE et al. "Topical and Transdermal Drug Delivery: From Simple Potions to Smart Technologies." Current drug delivery vol. 16,5 (2019): 444-460.)

[0045] Alternatively, enteric or delayed release oral formulations or pharmaceutical preparations can be used to deliver the MRGPRD agonist as described herein.In enteric or delayed release formulations, dosage form is an oral dosage form that comprises delayed release coating, such as enteric coating, that surrounds therapeutic agent, so as to delay the release of therapeutic agent until it reaches target, such as small intestine.Dosage form can be provided as a unit dosage form, for example, with lipid particles packed in syringe or ampoule for single or multiple use.

[0046] The oral dosage form may comprise a delayed-release coating to delay release of the therapeutic agent until it reaches the small intestine. The dosage form may be provided, for example, as a unit dosage form with the therapeutic agent packed in suitable packaging for single or multiple use. The dosage form may also be a suitable gastrointestinal dosage form, for example, as a suppository, enema, or via a feeding tube for delivery to the patient's intestine.

[0047] A "delayed-release coating" on a pharmaceutical dosage form is a coating or barrier applied to an oral dosage form that delays the release of the active ingredient of the dosage form, e.g., a therapeutic agent. A delayed-release coating may be an "enteric coating" that remains intact in the stomach and survives gastric pH and enzymatic processing, but dissolves in the small intestinal environment. In the context of the pharmaceutical compositions, medicaments, or dosage forms described herein, the goal of this delivery may be to delay the release of the therapeutic agent until it passes into the small intestine. Because the rate of stomach passage varies significantly depending on stomach contents and individual differences, an enteric-release coating or barrier may be preferable to a delayed-release barrier that relies on transit time rather than passage into the intestinal environment.

[0048] Polymers useful for enteric coatings can remain intact at low pH, but as the pH rises in the small intestine, the polymers swell and become soluble in intestinal fluids. Non-limiting examples of materials used in enteric coatings include cellulose acetate phthalate (CAP), poly(methacrylic acid-co-methyl methacrylate), cellulose acetate trimellitate (CAT), poly(vinyl acetate phthalate) (PVAP), and hydroxypropyl methylcellulose phthalate (HPMCP), fatty acids, waxes, shellac, plastics, and plant fibers. Delayed-release coatings also include polymer coatings, such as, but not limited to, hydroxypropyl methylcellulose (HPMC), carboxymethylcellulose (CMC), or other polymer compounds that can be coated onto immediate-release cores comprising a therapeutic agent. The thickness of the coating can be varied to produce the desired delay in the release of the therapeutic agent. Similarly, particles comprising a therapeutic agent can be embedded in HPMC, CMC, or other polymers useful for controlling the release of therapeutic agents. Delayed-release coatings can be coated with an enteric coating to further control the release of the therapeutic agent. Those skilled in the art of formulation will be able to optimize the overall dosage form structure and release pattern in the dosage forms described herein for any particular therapeutic agent.

[0049] The MRGPRD agonist can be delivered intranasally or by inhalation as an aerosol to treat conditions affecting the respiratory system, such as for systemic treatment or for delivery of the MRGPRG agonist to the nasal cavity, nasopharynx, trachea, bronchi, and / or lungs. Non-limiting examples of aerosols include dry powders, droplets generated by nebulizers or atomizers, aerosols generated by metered-dose aerosol inhalers, and droplets generated by nasal inhalers. In one example, the MRGPRD agonist is delivered by a dry or liquid metered-dose inhaler that can be configured for delivery by inhalation from the oral cavity or nasal cavity (see, for example, Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Lippincott, Williams, & Wilkins, Philadelphia, Pa., ed., 21st Edition (2005), e.g., Chapter 50 ("Aerosols"). The metered-dose inhaler can include a propellant and an oral adapter for oral inhalation and general pulmonary delivery. For nasal and nasopharyngeal delivery, the dosage form can be a spray device such as a pump sprayer equipped with a nasal adapter for spraying into the nasal cavity. The composition in the nasal sprayer can comprise a thickening agent to aid retention in the nasal cavity and nasopharynx.

[0050] The choice of delivery route may depend on the condition being treated. While systemic delivery may be effective, focused delivery to affected tissues may be preferable. Skin conditions such as urtucaria, contact dermatitis, cutaneous mastocytosis, eczema, or wounds can be treated locally, e.g., on the skin, using topical creams or ointments. Respiratory conditions such as nasal allergies, asthma, or anaphylaxis can be treated by aerosol delivery to the nasal and oral cavities. Intestinal conditions can be treated with delayed-release / enteric-coated medications or suppositories. Systemic conditions such as systemic mastocytosis may be treated by any effective route, such as oral administration or inhalation of an aerosol.

[0051] In addition to its use in downregulating mast cell degranulation, β-ala has been found to induce wound healing-related transcriptome changes.As a result, β-ala may be useful for promoting wound healing, for example, in the treatment of wounds and diabetic wounds, such as diabetic foot ulcers, which are slow to heal or do not heal.In one example, β-ala is administered topically to treat diabetic wounds, such as diabetic foot ulcers.

[0052] In one aspect or embodiment, the MRGPRD agonist is β-ala, which may be preferred due to its superior MRGPRD activation compared to GABA or β-AIBA. β-ala is widely available and inexpensive. β-ala has been taken orally as a functional food to improve athletic performance and overall physical performance in elderly people. Provided herein is a novel use of β-ala and related MRGPRD agonists for the treatment of conditions associated with mast cell activation or overactivation. While β-ala, currently available as a powder or capsule, can be effective for systemic conditions, targeted local and intestinal delivery has not previously been of interest.

[0053] Thus, topical and intestinal anti-inflammatory pharmaceutical compositions and medicaments are described herein for targeted delivery of β-ala. Thus, pharmaceutical compositions containing β-ala (including pharmaceutically acceptable salts thereof) and a pharmaceutically acceptable carrier are provided. Pharmaceutical compositions containing the recognition reagent β-ala are useful for treating conditions resulting from mast cell activation or hyperactivation, as described herein.

[0054] A suitable dose of β-ala is about 0.001 milligrams (mg) per kilogram (kg -1 ) in the range of about 1,000 mg / kilogram of recipient body weight, e.g., about 1 to 50 mg / kg -1Unit doses and topical (local) treatments can range, for example, from about 0.001 mg to about 10,000 mg, or from 5 mg to 5,000 mg, and topical compositions such as ointments or creams can range, for example, from 1 nanogram (ng) per milliliter (ml -1 ) ~ 10mg ml ‑1 or a concentration in the range of 1% w / v (weight / volume) to 10% w / v, for example, 5% w / v. Repeated dose regimens can involve administering a therapeutic amount of an MRGPRD agonist periodically, such as multiple times daily, every other day, or once a year, or as needed. After an initial treatment regimen, treatment can be administered less frequently. Certain factors, including but not limited to, the severity of the condition, previous treatments, the health status and / or age of the subject, and other diseases present, can affect the dose and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount can include a single treatment or a series of treatments.

[0055] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, etc. may be used. The composition may be an ointment in which the MRGPRD agonist is mixed in a carrier comprising petrolatum. Suitable topical formulations also include, but are not limited to, those in which the MRGPRD agonist is mixed with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprylate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or C 1-20Examples of suitable MRGPRD agonist delivery agents include alkyl esters (e.g., isopropyl myristate), monoglycerides, or diglycerides, or pharmaceutically acceptable salts thereof.Those skilled in the pharmaceutical and formulation fields can prepare suitable formulations for delivery of MRGPRD agonists, as described herein.

[0056] Non-limiting examples of MRGPRD agonists include β-ala, GABA, and β-AIBA.Other MRGPRD agonists are also known or can be developed.Such other MRGPRD agonists are also expected to be effective in treating conditions associated with mast cell activation or hyperactivation.The effectiveness of MRGPRD agonists can be tested, for example, by other assays that can be used to determine the ability of a compound to act as an MRGPRD agonist and to determine its effectiveness in treating conditions associated with mast cell activation or hyperactivation, as described herein.

[0057] B-alanine (β-ala) is also known as 3-aminopropanoic acid, for example, having the following structure: [ka]

[0058] β-ala or a pharmaceutically acceptable salt thereof is useful in the methods and compositions provided herein.

[0059] Non-limiting examples of pharmaceutically acceptable salts of β-ala include, but are not limited to, β-ala hydrochloride or acid addition salts such as malate, fumarate, or citrate as disclosed in U.S. Pat. No. 7,956,218 B2, having the following structure: [ka] (wherein n is, for example, 1, 2, or 3, and A is a pharmaceutically acceptable counterion.) For example, n is 2 when A is malate or fumarate, and 3 when A is citrate.

[0060] Gamma-aminobutyric acid (GABA, or 4-aminobutanoic acid) and its pharmaceutically acceptable salts are also useful in the methods and compositions provided herein. The structures of GABA (left) and exemplary acid addition salts thereof (right) are as follows: [ka] (wherein n is, for example, 1, 2, or 3, and A is a pharmaceutically acceptable counterion.) For example, n is 2 when A is malate or fumarate, and 3 when A is citrate.

[0061] β-aminoisobutyric acid (β-AIBA, or 3-aminoisobutyric acid) and its pharmaceutically acceptable salts are also useful in the methods and compositions provided herein. The structures of β-AIBA (left) and exemplary acid addition salts thereof (right) are as follows: [ka] (wherein n is, for example, 1, 2, or 3, and A is a pharmaceutically acceptable counterion.) For example, n is 2 when A is malate or fumarate, and 3 when A is citrate.

[0062] 5-oxoeicosatetraenoic acid (5-oxoETE) or a pharmaceutically acceptable salt thereof is also useful in the methods and compositions provided herein. The structures of 5-oxoETE (top) and an exemplary base addition salt thereof (bottom) are as follows: [ka] (wherein n is, for example, 1, 2, or 3, and A is a pharmaceutically acceptable counterion.) For example, n is 2 when A is malate or fumarate, and 3 when A is citrate.

[0063] Any pharmaceutically acceptable salt of any compound described herein can be used in the methods described herein.The pharmaceutically acceptable salt forms of the compounds described herein can be prepared by conventional methods known in the pharmaceutical field, including the preparation of certain veterinary acceptable salts.For example, but not limited to, if a compound comprises a carboxylic acid group, its suitable salt can be formed by reacting the compound with a suitable base to obtain the corresponding base addition salt.Non-limiting examples include alkali metal hydroxides such as potassium hydroxide, sodium hydroxide and lithium hydroxide; alkaline earth metal hydroxides such as barium hydroxide and calcium hydroxide; alkali metal alkoxides such as potassium ethanolate and sodium propanolate; and various organic bases such as piperidine, diethanolamine and N-methylglutamine.

[0064] Acid addition salts and base addition salts can be prepared by contacting the free base form with a sufficient amount of the desired acid or base to produce the salt by methods known in the art. The free base can be regenerated by contacting the salt form with a base or acid (depending on the nature of the salt) and isolating the free base. The free base forms differ slightly from their respective salt forms in certain physical properties, such as solubility in polar solvents, but these salts are otherwise identical to their respective free base forms for purposes described herein.

[0065] The compound comprising a basic nitrogen group is C 1-4 Alkyl halides, such as methyl, ethyl, isopropyl and tert-butyl chlorides, bromides and iodides; C 1-4 Alkyl sulfates, for example, dimethyl, diethyl and diamyl sulfate; C 10-18 Alkyl halides, such as decyl, dodecyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; and aryl-C 1-4 They may be quaternized with agents such as alkyl halides, e.g., benzyl chloride and phenethyl bromide. Salts allow for the preparation of both water- and oil-soluble compounds.

[0066] Non-limiting examples of pharmaceutically acceptable base salts include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, and zinc salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include, but are not limited to, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, chloroprocaine, choline, N,N'-dibenzylethylenediamine (benzathine), dicyclohexylamine, diethanolamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine. Examples of suitable anti-inflammatory agents include salts of methylparaben, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lidocaine, lysine, meglumine, N-methyl-D-glucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethanolamine, triethylamine, trimethylamine, tripropylamine, and tris-(hydroxymethyl)-methylamine (tromethamine).

[0067] Acid addition salts can be made by treating the compounds with pharmaceutically acceptable organic and inorganic acids, including, but not limited to, hydrohalides, e.g., hydrochloride, hydrobromide, hydroiodide; other inorganic acids and their corresponding salts, e.g., sulfate, nitrate, and phosphate; alkyl- and mono-arylsulfonates, e.g., ethanesulfonate, toluenesulfonate, and benzenesulfonate; and other organic acids and their corresponding salts, e.g., acetate, tartrate, maleate, succinate, citrate, benzoate, salicylate, and ascorbate.

[0068] Non-limiting examples of pharmaceutically acceptable acid salts include acetate, adipate, alginate, aspartate, benzoate, besylate (benzenesulfonate), bisulfate, bisulfite, bromide, butyrate, camphorate, camphorsulfonate, caprylate, chloride, chlorobenzoate, citrate, cyclopentanepropionate, digluconate, dihydrogenphosphate, dinitrobenzoate, dodecyl sulfate, ethanesulfonate, fumarate, galactarate, galacturonate, glucoheptanoate, gluconate, glutamate, and glycerophosphate. , hemisuccinate, hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, iodide, isethionate, isobutyrate, lactate, lactobionate, malate, maleate, malonate, mandelate, metaphosphate, methanesulfonate, methylbenzoate, monohydrogenphosphate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, oleate, pamoate, pectinate, persulfate, phenylacetate, 3-phenylpropionate, phosphate, phosphonate, and phthalate Examples include:

[0069] Multiple salt forms are also considered to be pharmaceutically acceptable salts. Common, non-limiting examples of multiple salt forms include bitartrate, biacetate, bifumarate, dimeglumine, biphosphate, disodium, and trihydrochloride.

[0070] Thus, "pharmaceutically acceptable salts," as used herein, is intended to mean active ingredients (drugs) comprising salt forms of any compounds as described herein, wherein the salt forms preferably impart improved and / or desirable pharmacokinetic / pharmacodynamic properties to the compounds described herein.

[0071] MRGPRD, as described in, for example, Gene ID: 116512, refers to "MAS-related GPR family member D." MRGPRD is also known as the β-alanine receptor. MRGPRD is a G protein-coupled receptor expressed in a specific subpopulation of sensory neurons. MRGPRD marks a distinct subset of sensory neurons that transmit polymodal nociceptive information from the skin epidermis to the substantia gelatinosa (SG, lamina II) of the spinal cord. Furthermore, Mrgprd-expressing (Mrgprd(+)) neurons are required for the full expression of mechanical nociceptive responses but not thermal nociceptive responses (Wang H, et al. Mrgprd-expressing polymodal nociceptive neurons innervate most known classes of substantia gelatinosa neurons. J Neurosci. 2009;29(42):13202-13209).

[0072] β-alanine reduces forskolin-stimulated cAMP production in cells expressing MRGPRD, suggesting that this receptor couples to the G proteins G(q) and G(i). Agonists for MRGPRD include β-ala, GABA, and β-AIBA (although GABA and β-AIBA are significantly less active as MRGPRD agonists than β-ala). It should be noted that β-ala, GABA, and β-AIBA are exemplary MRGPRD agonists, and other MRGPRD agonists may also function in downregulating mast cell activation (degranulation) in the manner described herein.

[0073] Provided herein are methods for treating patients with conditions associated with mast cell activation or hyperactivation. The methods comprise administering to the patient an amount of an MRGPRD agonist effective to downregulate mast cell degranulation in the patient. Examples of useful MRGPRD agonists include, but are not limited to, beta-alanine (β-ala) or a pharmaceutically acceptable salt thereof, gamma-aminobutyric acid (GABA) or a pharmaceutically acceptable salt thereof, and beta-aminoisobutyric acid (β-AIBA) or a pharmaceutically acceptable salt thereof. Combinations of any of the above compounds can also be used to treat patients. Examples include, but are not limited to, urticaria, mastocytosis, food allergies, anaphylaxis, and irritable bowel syndrome.

[0074] Also provided herein are topical, delayed-release, or enteric-coated pharmaceutical formulations comprising β-ala for targeting delivery of β-ala to the skin or mucosa to address specific conditions associated with mast cell activation or hyperactivation. The formulations may be in unit dosage form, spray devices, aerosol devices, metered-dose inhalers, spray or aerosol devices, or nebulizers. The formulations may be topical formulations such as lotions, creams, ointments, eye drops, ear drops, or bandages. Spray or aerosol devices may comprise propellants and / or rheology modifiers. Topical formulations may comprise penetration enhancers, rheology modifiers, antibiotics, anti-inflammatory agents, or anesthetics. Any formulation may also contain any useful additional active agent.

[0075] Non-limiting examples of antibiotics useful in the described formulations include acyclovir, afloxacin, ampicillin, amphotericin B, atovaquone, azithromycin, ciprofloxacin, clarithromycin, clindamycin, clofazimine, dapsone, diclazalil, doxycycline, erythromycin, ethambutol, fluconazole, fluoroquinolones, foscarnet, ganciclovir, gentamicin, iatroconazole, isoniazid, ketoconazole, levofloxacin, lincomycin, miconazole, neomycin, norfloxacin, ofloxacin, paromomycin, penicillin, pentamidine, polymyxin B, pyrazinamide, pyrimethamine, rifabutin, rifampin, sparfloxacin, streptomycin, sulfadiazine, tetracycline, tobramycin, trifluorouridine, trimethoprim sulfate, Zn-pyrithione, ciprofloxacin, norfloxacin, afloxacin, levofloxacin, gentamicin, tobramycin, neomycin, erythromycin, trimethoprim sulfate, polymyxin B, and silver salts such as chloride, bromide, iodide, and periodate.

[0076] The formulation may include an analgesic or anesthetic agent, for example a local anesthetic. analgesics, including but not limited to acetaminophen, tramadol, or cannabinoids; nonsteroidal anti-inflammatory drugs (NSAIDs), including but not limited to bromfena, colchicine, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, indoprofen, ketoprofen, ketorolac, meclofenamate, mefenamic acid, meloxicam, nabumetone, naproxen, napafenac, oxaprozin, phenylbutazone, piroxicam, salicylamide, sulindac, and tolmetin; COX-2 inhibitors, including but not limited to celecoxib, rofecoxib, and etoricoxib; buprenorphine, butorphanol, codeine, hydrocodone, hydromorphone, levorphanol, meperidine, methadone, morphine, nalbuphine, oxycodone, oxymorphone, pentazolinone, benzodiazepine, benzocaine ... Narcotic analgesics (pain relievers) including cinnamyl, propoxyphene, tapentadol; as well as, but not limited to, ambucaine, amylocaine, articaine, benzonatate, bupivacaine, butacaine, butanilicaine, chloroprocaine, cinchocaine, cocaine, cuclomethylcaine, dimethocaine, diperodone, benzocaine, dibucaine, lidocaine, oxybuprocaine, butambene, pramoxine, propanediol, Topical analgesics or anesthetics include rakaine, proxymetacaine, tetracaine, meprylcaine, metabutoxycaine, nitracaine, orthocaine, oxetacaine, paraethoxycaine, phenacaine, piperocaine, pyridocaine, pramocaine, prilocaine, primacaine, procainamide, procaine, propoxycaine, pyrocaine, quinisocaine, ropivacaine, tricaine, trimecaine, tripacocaine, and capsaicin. [Example]

[0077] Example 1: Activation of MRGPRD neurons suppresses mast cell activation The skin contains various interval afferent neurons that are specialized to respond to specific stimuli, which are then interpreted as sensations such as pain or itch. We found that a specific subset of neurons in the epidermis of the skin and in the intestine (and other organs) is required to suppress mast cell activation. These neurons express the receptor Mas-related G protein receptor D (MRGPRD). Genetic ablation of MRGPRD-expressing neurons in mice results in increased mast cell activation. Furthermore, we found that the small molecule MRGPRD agonist, β-alanine, is sufficient to suppress cutaneous mast cell function in wild-type mice.

[0078] As described herein, small molecule agonists of MRGPRD can be applied to inhibit mast cell activation.

[0079] Potential uses include suppressing skin inflammation related to: Any form of acute or chronic urticaria, All types of urticarial diseases, Any subtype of mastocytosis, false allergies, All forms of dermatitis including contact and atopic dermatitis, wound healing, rosacea, acne and psoriasis.

[0080] Other uses in the skin include suppressing neurogenic inflammation, pain, and itching. Uses in other tissues include treating asthma, angioedema, and diarrhea. Additional uses include treating other diseases in which mast cells play a pathogenic role.

[0081] The MRGPRD agonist may be given topically, orally, or parenterally in any effective dose, dosage form, route of treatment, or treatment regimen.

[0082] Example 2: β-alanine inhibits 48 / 80-mediated mast cell degranulation C57BL / 6 mice were pretreated with an i.d. injection of either β-alanine or vehicle for two days, followed by an i.d. administration of compound 48 / 80. Compound 48 / 80, a condensation product of N-methyl-p-methoxyphenethylamine and formaldehyde, is a known chemical that induces rapid mast cell degranulation and associated edema. The degree of skin edema was measured by the amount of Evans blue dye in the skin. As shown in Figure 1, compound 48 / 80 increased Evans blue dye in vehicle-treated mice (compare columns 1 and 2), whereas pretreatment with β-alanine inhibited this effect (compare columns 3 and 4). In mice lacking MRGPRD neurons, β-alanine had no effect on the mast cell activation potential of 48 / 80 (compare columns 5 and 6). This serves as a specificity control, as neurons expressing receptors for β-alanine are absent.

[0083] Example 3: β-alanine (MRGPRD agonist) suppresses DNFB contact hypersensitivity in vivo (mouse model of allergic contact dermatitis) Referring to Figure 2, a cohort of C57BL / 6 mice (n = 5) was pretreated with either β-alanine (WT + β-ala) or vehicle (WT) via intraperitoneal intraperitoneal injection for 2 days, followed by sensitization with 0.2% DNFB (a chemosensitizer) at the same site. Five days later, 0.2% DNFB was applied to the ears. The increase in ear swelling on the indicated days was measured relative to ear thickness before DNFB application. Increased ear thickness was used as a proxy for measuring the amount of immune response to DNFB. As a specificity control, MRGPRD-DTR mice were treated with DT to ablate MRGPRD-expressing neurons (labeled MRGPRD in Figure DD) and similarly treated mice (MRGPRD and MRGPRD + β-ala). Ear thickness in these mice was generally increased, consistent with hyperactive mast cells. Importantly, β-alanine injection had no effect on MRGPRD-ablated mice, demonstrating that this effect of β-alanine requires the presence of MRGPRD-expressing neurons. In WT mice, β-alanine had a significant effect on ear swelling and therefore on the immune response.

[0084] Example 4: 5% topical β-alanine inhibits mast cell degranulation in mice As shown in Figure 3, mice were treated twice daily for 2 days with the indicated concentrations of a topical formulation of β-alanine ("b-ala," open squares) or vehicle alone ("con," closed circles) on the shaved flank skin. Mice were then given an intravenous dose of Evans blue dye, followed by an intradermal injection of Compound 48 / 80. Ten minutes later, the amount of Evans blue dye present in the skin (μg g) was measured as an index of the degree of mast cell degranulation. -1 Tissue) was determined by skin biopsy, enzymatic digestion, and colorimetric evaluation.

[0085] Example 5: B-alanine induces transcriptome changes associated with wound healing Wild-type C57BL / 6 mice were injected with 100 mM β-alanine or vehicle twice daily for two days. Skin samples were collected from the treatment sites, mRNA was obtained, and RNA sequencing was performed. Genes with increased expression in the β-alanine-treated group were analyzed by pathway analysis. The -log2p values ​​normalized to the number of genes are shown in Figure 4 (black bars). A similar analysis was performed using published RNA sequencing data (Naik S, et al. Nature 2017 Oct 26;550(7677):475-480. PMID: 29045388) obtained from mouse skin collected 6 hours after tape stripping or from sham controls (gray bars).

[0086] The strong similarity of the pathways activated after β-alanine administration and tape stripping strongly suggests that β-alanine activates wound healing pathways.

[0087] The present invention has been described with reference to certain exemplary embodiments, dispersible compositions, and uses thereof. However, it will be recognized by those skilled in the art that various substitutions, modifications, or combinations of any of these exemplary embodiments may be made without departing from the spirit and scope of the present invention. Accordingly, the present invention is not limited by the description of the exemplary embodiments.

Claims

1. A method for treating a patient having a condition associated with mast cell activation or hyperactivation, comprising administering to the patient an MRGPRD agonist in an amount effective to downregulate mast cell degranulation in the patient.

2. 2. The method of claim 1, wherein the MRGPRD agonist is beta-alanine (β-ala) or a pharmaceutically acceptable salt thereof; gamma-aminobutyric acid (GABA) or a pharmaceutically acceptable salt thereof; beta-aminoisobutyric acid (β-AIBA) or a pharmaceutically acceptable salt thereof; 5-oxoeicosatetraenoic acid (5-oxoETE) or a pharmaceutically acceptable salt thereof; or any combination thereof.

3. 2. The method of claim 1, wherein the condition associated with mast cell activation or hyperactivation is urticaria, and the MRGPRD agonist is administered locally or systemically to the patient.

4. 2. The method of claim 1, wherein the condition associated with mast cell activation or hyperactivation is mastocytosis, and the MRGPRD agonist is administered locally or systemically to the patient.

5. 10. The method of claim 1, wherein the condition associated with mast cell activation or hyperactivation is food allergy and the MRGPRD agonist is administered to the patient enterally, such as in an enteric-coated or delayed-release oral dosage form.

6. The method of claim 1, wherein the pathology associated with mast cell activation or hyperactivation is acute or chronic urticaria, mastocytosis or a subtype of mastocytosis, pseudoallergy, any form of dermatitis such as contact or atopic dermatitis, wound healing, rosacea, acne, or psoriasis, and the method comprises administering to the patient an amount of an MRGPRD agonist effective to inhibit or reduce inflammation in the patient, for example, an amount effective to inhibit or reduce mast cell activation in the patient.

7. 10. The method of claim 1, wherein 5 mg to 5,000 mg of the MRGPRD agonist is administered to the patient.

8. 10. The method of claim 1, wherein the MRGPRD agonist is administered to the patient for at least one week.

9. 10. A method of treating a wound in a patient, comprising administering to the wound an amount of an MRGPRD agonist effective to enhance wound healing in said patient.

10. 10. The method of claim 9, wherein the MRGPRD agonist is beta-alanine (β-ala) or a pharmaceutically acceptable salt thereof; gamma-aminobutyric acid (GABA) or a pharmaceutically acceptable salt thereof; beta-aminoisobutyric acid (β-AIBA) or a pharmaceutically acceptable salt thereof; 5-oxoeicosatetraenoic acid (5-oxoETE) or a pharmaceutically acceptable salt thereof; or any combination thereof.

11. 10. The method of claim 9, wherein the MRGPRD agonist is formulated as a topical pharmaceutical composition.

12. 10. The method of claim 9, wherein the wound is a non-healing wound.

13. 10. The method of claim 9, wherein the patient has diabetes and the wound is optionally a foot ulcer.

14. A topical composition comprising β-alanine or a pharmaceutically acceptable salt thereof in combination with a pharmaceutically acceptable carrier.

15. 15. The topical composition of claim 14 in an amount effective to treat a condition associated with mast cell activation or hyperactivation, such as acute or chronic urticaria, mastocytosis or any subtype of mastocytosis, pseudoallergy, any form of dermatitis such as contact or atopic dermatitis, wound healing, rosacea, acne, or psoriasis.

16. 15. The composition of claim 14, which is an ointment, cream, lotion, spray, gel, or wound dressing.

17. An aerosol or spray pharmaceutical formulation or device comprising β-alanine or a pharmaceutically acceptable salt thereof in combination with a pharmaceutically acceptable carrier.

18. 18. The pharmaceutical formulation or device of claim 17, in the form of a sprayer, such as a nasal spray or a metered dose sprayer or a metered dose inhaler.

19. A delayed release or enteric formulation comprising β-alanine or a pharmaceutically acceptable salt thereof in a delayed release and / or enteric composition and / or within a delayed release and / or enteric coating for the intestinal release of β-alanine in a patient.

20. A method for preventing, alleviating or treating pain or pruritus in a patient, e.g., neurogenic inflammation, pain, or pruritus in a patient, comprising administering to the patient an MRGPRD agonist in an amount effective to prevent, alleviate or treat pain or pruritus in the patient, e.g., in an amount effective to inhibit or reduce mast cell activation in the patient.

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