Treatment of skin dysesthesia and skin inflammation

Abstract

NOT VISIBLE DUE TO STATUS OF PATENT

Description

TITLE OF THE INVENTIONTreatment of Skin Dysesthesia and Skin InflammationCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefits of US Patent Application No. 18 / 445,766 filed on January 25, 2024 and CN Patent Application No. 202410762256X filed on June 13, 2024, the contents of which are incorporated herein by reference in their entireties.TECHNICAL OF INVENTION

[0002] One aspect of the present invention is a method to treat inflammation of the skin. The preferred embodiment, FEMA 4557, is an example of a topically applied molecule that can be used to achieve these therapeutic goals. Treatment of the skin reduces pain, itch, swelling and promotes rapid wound healing. Another aspect of the invention is treatment of canine dermatitis and skin irritation.BACKGROUND OF THE INVENTION

[0003] Inflammation is the reaction of vascularized living tissue to local injury (Cotran et al., Pathologic Basis of Disease, 1989, 4th Edition). The cardinal signs of inflammation were described -2000 years ago by Celsus and by Galen. The expression of inflamed tissues was redness (rubor), and heat (calor), due to increased blood flow, swelling (tumor) due to leakage of blood contents into tissues, and pain (dolor) due to activation of nerve endings in the injured tissue. The loss of function (functio laesa) resulted from tissue dysfunction. Much of the injury caused by inflammation is due to changes in the microcirculation, primarily an increase in vascular permeability. Leakage is due to substances released by injured tissues called inflammatory mediators, such as histamine, that enlarge gaps in postcapillary venules. This release is called the inflammatory cascade. Excess fluids and solutes entering the interstitial spaces may block gas exchange and tissue nutrition. In severeinjuries, vascular leakage may persist for hours or days until thrombosis occurs, or the vessels rejuvenate. Inflammation is a dynamic process as the injured tissue seeks to defend, repair, and heal itself. In some instances, dysregulated inflammation itself becomes a source of further tissue injury, especially if the immune system is activated.

[0004] Anti-inflammatory drugs were in the clinic before mechanisms of drug action became understood. For example, non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin for pain relief, and corticosteroids, were used in rheumatoid arthritis before the cellular actions of prostaglandins and phosphorylases were known. More recent pharmacological strategies focus on anti-TNFa therapies and inhibitors of cytokines that may be pivotal for a given local pathology. The inhibitors of tyrosine kinases in the Janus family of enzymes is another promising source of new antiinflammatory drugs.

[0005] Anti-inflammatory drugs generally work as specific agonists, antagonists, enzyme inhibitors, or scavengers of inflammatory mediators. By design, each drug works one-on-one against substances that promote inflammation. However, the efficacy of a single antagonist may be limited if more than one mediator acts during tissue injury. For example, an antihistamine works against the histamine released in seasonal allergic rhinitis but is less effective against vasomotor rhinitis wherein histamine is one of many inflammatory mediators. Another problem of an antagonist is drug action at a non-target site. For example, TRPV1 antagonist may reduce the perception of thermal heat, but they also increase the risk of a scald when drinking hot coffee. An agonist, a term used to describe a chemical that activates biological events, is more effective than an antagonist if it can suppress convergent processes initiated by more than one inflammatory mediator. An example of a widely effective agonist is epinephrine for treating anaphylactic shock. Here, multiple actions of histamine are traversed by the adrenergic receptor agonist.

[0006] Treatment of acute inflammation is especially useful. Events happening in tissues within -three hours after injury are critical for survival. Currently, the best treatment for the injury of skin is application of ice or cold water, wherein the treatment goal is lowering of tissue temperatures to reduce pain and swelling.

[0007] The treatment for skin inflammation in animals is also important. The domesticated dog (Canis familiaris) has about 200 breeds recognized by Kennel Clubs. The human-dog bond is firm and eternal because the dog is such a good companion. However, care for the dog's health requires resources. The dog's skin, like human skin, is its largest organ, about 12 to 24% of body weight. In most breeds, hair covers the skin except on the footpads and nose. The density of canine hair is about 15x that of the human scalp, and each hair follicle has a sebaceous gland. But sweat glands are virtually absent on the dog's skin. Skin disorders most frequently initiate a dog owner's visit to the veterinarian.

[0008] The dog's skin is close to the ground, exposed to dirt, hard to keep clean, attacked by insects and microorganisms, and has an immune system that vigorously reacts to allergens. Common disorders in dogs are bites from fleas and mites, infections of the hair follicles by fungi and bacteria, and allergies. In addition, the genetics of the breed influence skin sensitivities. For example, the West Highland Terrier and Labrador Retriever are susceptible to atopic dermatitis. Spaniels, because of droopy ears, are susceptible to infections of the pinna, and Bulldogs are to irritation of the facial skin folds.

[0009] Hair-coated animals cannot describe their skin sensations in words. However, they point to the source of skin discomfort and irritation by licking, biting, and scratching. The general term “dysesthesia” encompasses reactions to noxious stimuli such as irritation, burning sensations, itch, and pain. Licking and rubbing may be a form of cleansing the site of dysesthesia. Itch is the sensation that makes us want to scratch and may be particularly annoying to the dog because of its coat. That is, the dog cannot “get to” the site of irritation. Biting an itchy site to cause pain may be preferable to the itch. However, biting and scratching exacerbate skin injury. A vicious “itch-scratch cycle” prevents the skin from healing. If scratching diminishes, the skin heals faster. Cooling gels in humans reduce itch but have not been used in dogs because the dog's hair impedes the access of the coolant to the skin receptors.

[0010] The canine sites of dysesthesia with thinner skin and less hair are on the belly, armpit (axillary), groin (inguinal), and raw or "hot spots" on the hindlimbs, the back, andat the base of the tail. Areas with thicker skin are the ear canal, neck area, and the skin between the toes (interdigital). Skin sensations at all sites are disrupted by disorders such as atopic dermatitis, atopic-like dermatitis, allergens, fungi, bacteria (folliculitis, pyoderma), and acute and chronic reactions to bites from insects and parasites. The cutaneous inflammatory response to injury is as important as the injury itself. For example, for bites from insects or arachnids, it is not the withdrawal of blood by the flea or the mite's consumption of cellular debris that causes injury. Instead, it is the host's allergic reactions to the invading arthropod's saliva, eggs, larvae, and feces.

[0011] Current drug treatments for veterinary skin disorders are sophisticated, a multibillion dollar business, and require accurate diagnosis and targeted therapy. The drug management strategy can be specific or general. For example, to treat scabies and other mites, use a topical scabicide such as permethrin, malathion, or lindane. For fleas or ticks, an orally ingested neurotoxin, afoxolaner (e.g., Nexgard), will poison the flea or tick when it drinks the animal’s blood. A topical or systemic antibiotic is used for bacterial or fungal infections of hair follicles (e.g., hot spots or pyoderma traumatic) or skin. Prescription drugs with a generalized effect on the immune system are for allergies. For example, an effective immunosuppressant in atopic or atopic-like dermatitis is the specific JANUS kinase inhibitor called oclacitinib (Apoquel). Other immunosuppressants are cyclosporine or anti-inflammatory steroids. Immunosuppressants, however, have a powerful effect on the body, with a variable onset, and long-term use may lead to undesirable side effects. Topical antiinflammatory steroids for dermatoses in humans have limited efficacy in the dog and have an additional risk of super-infection.

[0012] The owner of pets is most concerned when the animal repeatedly licks, bites, and scratches the skin disorder site. A dog's scratch with its claws, alternating contraction and relaxation of the limb muscles, arching of the spine, angulating the head, and targeting the site to scratch, is vigorous and will damage tissues. And so will the bite. The skin damage perpetuates the skin disorder in the "itch-scratch cycle." Interruption of the itch-scratch cycle will be an effective new method of treatment. In humans, l-menthol is sometimes used in anti-itch remedies. However, menthol isirritating and cannot be used for canine skin. Currently, no topical cooling drugs exist to relieve canine skin dysesthesia or interrupt the itch-scratch cycle.BRIEF SUMMARY OF THE INVENTION

[0013] This application describes chemicals that may be prototypes of a new class of anti-inflammatory agonists.

[0014] The applicant has investigated cooling agents, that is, chemicals that produce cooling sensations without a change in tissue temperatures. Early in 2012, he noticed that the isopropyl esters of p-menthane glycine and p-menthane D-alanine had prolonged cooling activity when applied to the ocular margins (Wei, 2012). Subsequently, he discovered that diisopropyl analogs of phosphoryl alkanes produced refreshing coolness Yang et al. (2017). The isopropyl group appears to have specific anchoring sites on the TRPM8 receptor, which mediates sensations of coolness. Significant activity is lost if the isopropyl groups of phosphoryl alkanes change to secbutyl. The studies of Xu et al. (2020) describe robust coupling energy between the isopropyl group on the cyclohexane ring of l-menthol and the L843 (leucine) and I846 (isoleucine) residues of TRPM8. This "pocket" site for the "isopropyl legs" may be essential for prolonged TRPM8 receptor activation.

[0015] FEMA 3804 (WS-23), a GRAS (generally recognized as safe) food additive used as a cooling agent, has two isopropyl groups. However, when applied to the oral cavity or skin, it is short-acting with less than 30 min of cooling.

[0016] FEMA 4557, an additive like WS-23 with two isopropyl groups, has yet to be thoroughly examined for activities on the skin or the nasal membranes. As of 2014, 30+ GRAS / FEMA cooling agents are listed (Leffingwell, 2014). One aspect of the discovery here is that the preferred embodiment, FEMA 4557, has a prolonged duration of cooling without irritancy on skin, and, more surprisingly, FEMA 4557 is a potent inhibitor of acute inflammation.

[0017] The two major classes of anti-inflammatory agents, gluco corti costeroids, exemplified by cortisone, and cyclooxygenase inhibitors, exemplified by aspirin, areused to treat chronic inflammatory conditions such as rheumatoid arthritis and asthma. These drugs also have short-term anti-inflammatory effects, such as on mosquito bites and headaches. Epinephrine and antihistamines can treat the acute inflammatory effects of histamine on anaphylaxis and urticaria. However, there is no category of drugs called topical "acute anti-inflammatories." Physical lowering of temperature, for example, cooling with cold water or ice, is known to have potent acute antiinflammatory properties. When applied to injured tissues, ice or an ice pack will stop the pain and swelling of traumatized or burned tissues. The mechanisms for how physical cooling stops acute inflammation are not known. If a chemical can mimic the effects of ice on acute inflammation, it will most likely act on a convergent point of the inflammatory cascade (Table 1). Imagine the injured tissue as a parched grassland on fire (inflamed). Dousing the field with water is a general anti-inflammatory action. Making the field wet may also dampen the chances of added conflagration.

[0018] The acute anti-inflammatory molecule identified here is FEMA 4557, a generally recognized safe (GRAS) flavoring agent approved as a food additive (Smith, 2009). The structure of the preferred embodiment, FEMA 4557 is shown as below.

[0019] N-alkyl-diisopropyl-butanamides have a total mass of about 200 Daltons. A synonym for FEMA 4557 is N-ethyl-2,2-diisopropyl butanamide (CAS No. 51115-70-9). In studies described here, FEMA 4557 exerts a robust anti-inflammatory activity at topical concentrations of ≥5% wt / vol and a total dose of several mg. FEMA 4557 stops pain and itch and exhibits unusual healing properties on injured tissues. FEMA 4557 may be a prototype of a new class of anti-inflammatory agonists for acute inflammation. The unique characteristics of FEMA 4557 are not shared by existing cooling agentsused as food additives, and the discovery of its anti-inflammatory suppressing properties in this invention is surprising and unexpected.

[0020] The mechanism for anti-inflammatory action is complex. The vascular tree is a network of vessels designed to maintain, at its outermost subdivisions, a surface area between blood and tissues for exchanging gases and nutrients and for the drainage of waste products. During the early stages of inflammation, the sensitive mechanisms regulating microvascular perfusion disrupt so that vascular patency diminishes, blood contents leak into tissues, and hemostasis may develop. After injury, tissue interstitial fluid pressure may turn negative and suck fluids from the vascular compartment into the extracellular matrix, thus creating edema. Modulating acute inflammation may lead to new drugs with clinical benefits.

[0021] About three decades ago, Wei et al. (1993) and Wei and Thomas (1993) reviewed the idea of using a drug agonist to inhibit a convergent pathway for modulating acute inflammation. The hypothalamic hormone, corticotropin-releasing factor, was identified as having potent peripheral anti-inflammatory actions and was later shown to be effective for reducing peritumoral edema in human subjects with brain metastases (Robert et al., 2020). However, no lead candidates were pinpointed for topical treatment. These intriguing observations persist and have not faded. FEMA 4557 unexpectedly provides new clues.

[0022] In this application, we accidentally found that FEMA 4557 had immediate antiinflammatory effects. These included:- inhibition of the skin's response to insect bites (fleas and a bumble bee sting),- reducing the signs of skin irritation in a case of canine skin allergy,- reducing itch in severe urticaria, and- reducing pain and heat-induced swelling from a cigar lighter burn on the hand.

[0023] The preferred embodiment, FEMA 4557, exerted an immediate, profound, and unexpected anti-inflammatory drug action not seen with related substances. FEMA4557 rendered the immediate injury reactions of pain, irritation, and burning sensations, less imperative. The subject could cope with the injury. Swelling and redness went down, and healing was rapid. This rapid tissue response to local injury was unusual and occurred after topical application.

[0024] Cooling sensations are generally associated with activating the integral membrane receptor called TRPM8. However, the anti-inflammatory action of FEMA 4557 did not correlate well to agonist potency at the TRPM8 receptor. FEMA 4557 is a weak TRPM8 agonist, having less potency than l-menthol. A potent TRPM8 agonist such as WS-12 did not affect inflammation, but FEMA 4557 was active. The antiinflammatory effect of FEMA 4557 may act on a downstream, convergent pathway of inflammation such as interstitial fluid pressure or Pif (Table 1 ), but its precise molecular site of action is not yet known. If FEMA 4557 preserves tissue architecture during acute injury, it can reduce pain and accelerate healing. The dynamic events of Table 1 occur within minutes after tissue injury. The actions of FEMA 4557 suggest that it may have utility for drug treatment of acute inflammation.

[0025] Traditionally, pharmacologists focus on chronic inflammation because of its role in persistent clinical conditions such as arthritis, asthma, and related diseases. The immediate stages of inflammation, for example, within three hours of injury, were studied here. In this application, we used bioassays for screening immediate antiinflammatory effects. These included a) visual observation of the skin’s response toinsect bites, b) assessment of pain and heat-induced swelling from an accidental burn from a torch, c) the inflammatory response seen in canine skin allergy and woundhealing, and d) the inflammatory response of skin inflammation in a subject with urticaria. In all instances, the preferred embodiment exerted an immediate and dramatic anti-inflammatory drug action. The immediate sensory reactions of pain, irritation, and burning sensations were no longer imperative. The subject could cope with the injury. Swelling and redness went down, and healing was rapid. This rapid organismic response to local tissue injury was unusual and occurred after topical application of the preferred embodiment.

[0026] The primary structures of the active agonists in the preferred embodiments share the features of a butanamide attached to a branched C2. A total mass of about 200 Daltons may be sufficient for bioactivity. The anti-inflammatory activity in vivo occurs at topical concentrations of >5% wt / vol and a total dose of several mg. The examples suggest that the preferred embodiments activate convergent, unitary events to inhibit acute inflammation. The anti-inflammatory action was non-selective against insect bites, heat, allergy, and skin disease stimuli. These multiple actions indicate an inhibitory effect on downstream pathways of inflammation.

[0027] In adapting this knowledge for experimental study of canine dysesthesia, these objectives were necessary: a) define the molecular target and mechanism for a drug, b) state the target's anatomic location, and c) develop methods for formulating and delivering the drug to the target. For experiments, the investigator selects the class of chemicals and the bioassay. A milestone occurs when a chemical entity is identified that a) has a rapid onset of action, b) can be produced, formulated, and delivered at reasonable costs, c) is free of adverse side effects, d) has a sufficient duration of action, and e) reduce dysesthesia, inhibits scratching, and progression of the itch-scratch cycle. Overall, the treatment should accelerate skin healing.

[0028] In one aspect of the present invention a pharmacological composition for the treatment of the inflammation of a dermatological disorder in a mammal in need of treatment thereof, comprisesa liquid or semi-liquid composition having a therapeutically effective amount of one or more compounds having Formula 1Formula 1 wherein R is ethyl, n-propyl, isopropyl, cyclopropyl, or acetyl.

[0029] In another aspect of the present invention, a therapeutic method for the treatment of the itch of a dermatological disorder in a mammal in need of treatment thereof, comprises topically applying a liquid or semi-liquid composition to the mammal’s skin, the composition comprising a therapeutically effective amount of one or more compounds having Formula 2wherein R1 is ethyl, isopropyl, or n-propyl, and R2 is ethyl, isopropyl, n-propyl, cyclopropyl, or acetyl.

[0030] The preferred embodiments of Formula 1 are cooling agents that presumably act as an agonist on a TRP channel protein called TRPM8 located on neuronal membranes. More precisely, the preferred embodiment is FEMA 4557, also known as N-ethyl-2,2- diisopropylbutanamide [CAS No. 511 15-70-9], The agent activates TRPM8 and generates a signal to the brain which suppresses dysesthetic sensations. The surprising finding was that the preferred embodiments can be applied at high concentrations on the skin from 5 to 60% wt / vol in liquid or semi-liquid form, and these formulations do not cause irritation and are effective anti-inflammatories. Examples of such formulation and case histories of successful treatment are described.

[0031] One disorder of interest is canine dermatitis and the skin’s reaction to injury, but the invention applies to other hair-coated mammals such as cats and horses. For the dog, the treatment goal is to stop the dog's scratching and to abort the irritant-scratch cycle so the inflamed tissues have time to heal. Other aspects and embodiments of the present invention are further clarified below.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0032] Fig. 1. Topical Application of a cooling DIPA1 -7 gel on skin of healthy volunteers inhibits itch induced by various pruritogens. n=30 subjects per trial. Data from Prof. Hyeone Kim, Sacred Hospital, Hallym University, Seoul, Korea. Itch intensity was selfrated on a scale of 0 to 10 for 10 min (maximum score = 100). (Lee, S., Wei, E. T., et al. Histamine- and pruritogen-induced itch is inhibited by a TRPM8 agonist: a randomized vehicle-controlled human trial. Brit. J. Dermatol. 190(6), 885-894, 2024. https: / / doi.org / 10.1093 / bjd / ljae054). DIPA1 -7 gel (Intrinsic IB, Dong Wha Pharmaceuticals, Seoul, Korea) significantly inhibited itching induced by all five test pruritogens.

[0033] Fig. 2. Cooling intensity of agents tested in the oral cavity at 100 ppm in 5% sucrose. Cooling intensity was self-rated on a scale of 0 to 9 units. Data from Fig. 4 of Sonya Johnson et al., 2017. The rank order of potency of WS-5> WS-3 ~ I- menthol>WS-12>WS23 agrees with compilations published by Leffingwell (2014) and by Erman (2007).

[0034] Fig. 3. Median Effective Dose (EC50) of various cooling agents in TRPM8 in vitro assay. The consensus is that icilin and WS-12 are the more potent agonists, followed by WS-5 > WS-3 ~ l-menthol. The acyclic amides WS-23 and FEMA 4557 are significantly less active than l-menthol. The applicant provided the test substances used in the publications by McKemy et al., Bodding et al., and Sherkheli et al. Further experiments by the applicant confirmed the weak activity of FEMA 4557 on TRPM8 (10% the activity of l-menthol) and the absence of activity on TRPA1 .

[0035] Fig. 4. Bioassay of FEMA 4557 (labeled as BUDA) in TRPM8 and TRPA1 . Data generated in the laboratory of Prof. Seok-Yong Lee at Duke University. The EC50 of FEMA 4557 was estimated at 0.41 mM or about 1 / 30x that of l-menthol. FEMA 4557 was not active in cells transfected with TRPA1 , although a positive TRPA1 control AITC (allyl isothiocyanate) was active on TRPA1 .

[0036] Fig. 5. Cooling agents, prepared as an 8% wt. / vol. gel, was tested topically on the philtrum skin assay. The duration of cooling was recorded (min on y-axis). The startling activity of FEMA 4557, cooling for - 3 hr, was unexpected and surprising. FEMA 3784 is also known as Cool 10 or menthoxypropanediol.

[0037] Fig. 6. A pen-like delivery system for punctate delivery of active ingredient to lesion sites. The tip of the pen has a solid foam-like matrix. The active ingredient is dissolved in a liquid.

[0038] Fig. 7. Example of skin's inflammatory response to flea bites and treatment with topical FEMA 4557. Half an hour after the bite (left panel), the skin is red, swollen, and intensely itchy. Within 10 min after application of FEMA 4557, 32% solution, with pen swelling and redness, is reduced at 12 hr. Surprisingly, itching disappears with a singleapplication of FEMA 4557. After 24 hr, the skin inflammation heals. A single application of FEMA 4557 is sufficient to counteract inflammation.

[0039] Fig. 8. Results of a canine model of skin allergy and treatment of inflammation with FEMA 4557. A 6-yr old long-haired dachshund had a skin allergy for at least eight months. Standard treatments were not effective. On the left panel, the white bar marks the inflamed sites in the right groin and belly area (with excoriations and hair loss). FEMA 4557 8%, topically applied, immediately stopped the licking, biting, and scratching of lesion sites. After three days of gel, skin healing became self-evident, and on day seven, the skin inflammation disappeared, with tissue healing and hair growth. The skin lesions have not recurred.

[0040] Fig. 9. Examples of canine irritated sites for topical treatment: (clockwise) belly and groin, interdigital skin folds, pinna and ear canal, and back. Photos from www.robbinsroadanimalclinic.com.

[0041] Fig. 10. A 53-year-old female subject developed recurrent episodes of urticaria six months after receiving a Covid-19 vaccination. Each episode lasted ~2 weeks, and there were 10+ episodes. The itch of urticaria was on all parts of the body. Inspection of the nape showed evidence of scratching (lighter shade skin where the epithelium is scratched). The dark patches are rash. Applying FEMA 4557 gel or solution controlled the itching, allowing the subject to rest and sleep. The subject is now controlling her itch with daily doses of fexofenadine (Allegra / Hives) and using the FEMA 4557 pen on an "as-needed basis."

[0042] Fig. 11. The upper figure shows the burn site from the cigar lighter. The white bars show rapid development of swelling. There was pain after the burn, together with redness and swelling. A 20% solution of FEMA 4557 was applied with a cotton-tipped applicator to the burn site 15 min after the burn. The pain diminished and was not noticeable after 5 hr. FEMA 4557 was only applied once. The redness, swelling, and pain disappeared after 24 hr, and the wound site formed an eschar which resolved within three weeks.

[0043] Fig. 12. Dose-response relationship of FEMA 4557 to cooling on the philtrum skin. FEMA 4557 was dissolved in a polymer gel (Carbopol® 980) and applied to the philtrum skin with a cotton-tipped applicator. Cooling intensity area-under-curve (AUC) duration was recorded on a standardized philtrum skin assay.

[0044] Fig. 13. Inhibition of pain and swelling from a bumble bee sting with FEMA 4557. The subject’s distal phalanx of the middle finger was stung by a bumble bee when he tried to grab the bee with tissue paper. The volume of the distal phalanx increased from 7.0 g to 9.0 g within 30 min, as measured by the fluid displacement method over a toploading balance. The swelling was accompanied by redness and pain. The left panel shows the normal middle finger of the contralateral hand. Half an hour after the bite (middle panel), the distal phalanx is swollen, and painful. The glistening FEMA 4557 32% solution on the finger is shown in the right panel. Surprisingly, the pain in the middle finger was converted to an intense itch within several minutes after FEMA 4557. This conversion of pain to itch is unexpected, and more tolerable to the subject. The itching lasted several hours.DETAILED DESCRIPTION OF THE INVENTIONBackground on Topical Cooling Agents and its Metamorphosis

[0045] To discover a molecule that suppresses skin dysesthesia and inflammation, after topical application, it is essential to identify the best chemical ingredient using an appropriate bioassay.

[0046] Drug discovery usually begins with herbal remedies, but when Friedrich Serturner crystallized morphine from opium resin in 1804, modern pharmacology emerged. Now, we think of the active ingredient as a molecule of defined chemistry, not a botanical mixture. Once isolated and characterized, the active ingredient in a herbal remedy becomes a seed for a slow metamorphosis, a glacial evolution, into an improved or better entity. Witness salicylic acid in willow bark become aspirin, then ibuprofen. After Serturner crystallized morphine from opium resin, the metamorphosis spawned codeine, heroin, oxycodone, methadone, and fentanyl.

[0047] The efforts to find better analogs from a prototype always entail extensive synthesis and bioassay, a common yardstick of synthesis being 1000+ analogs. Paul Ehrlich, the father of modern pharmacology, called his anti-syphilitic salvarsan 606 because it was among 606 analogs! Ehrlich then made an improved neosalvarsan after 900+ analogs and assays. Another example of synthetic efforts at "finding the right needle in a haystack" was Gerhard Domagk, who tested thousands of sulfonamides before finding Prontosil rubrum (which then progressed to sulfanilamide). Adams and Nicholson also screened thousands of compounds before finding ibuprofen. The prototype before ibuprofen was aspirin, which in turn is an analog of salicylic acid, first discovered as an analgesic in willow bark by Reverend Edward Stone in 1763. Drug discovery requires luck and effort. The discovery statistics are such that one would be fortunate to find ten candidates (1%) after making a thousand and refining one out of 10 (0.1%) to a lead candidate. And that lead candidate will likely fail in four out of five clinical trials!TRPM8 Agonists. Clockwise: l-menthol, Ax-8, iciiin, Cryosim-3.

[0048] The four major classes of TRPM8 agonist are monoterpenoids (represented by menthol), p-menthane carboxamides (represented by WS-12 and Ax-8), icilin, and 1- dialkylphosphorylalkane (DIPA-1-7, cryosims). With the exception of DIPA-1-7 (Cryosim-1), none will work for the scratching caused by canine dermatitis. Menthol is a skin irritant. Icilin will not dissolve and can’t be delivered. The large class of p- menthanecarboxamides are not easily soluble and have limited penetration of skin.DIPA-1 -7 will work on itch in humans, but expensive for use as a drug on the skin of fur- coated animals.

[0049] For menthol, Wilkinson Sword (WS) scientists tried to improve menthol 50+ years ago and made 1200+ analogs. Watson et al. (1978) summarized the results in a classical paper. The WS effort ended in 1980. Leffingwell and Rowsell re-visited and updated this subject in 2014; by then, thirty-two cooling agents were in the FEMA (Flavors, Extracts, Manufacturer's Association) database. FEMA-designated WS agents are GRAS (generally recognized as safe) and preferred for screening because FEMA has collected safety data. Such WS agents are assigned a FEMA number, periodically reviewed, and information posted online. Thus, the preference is for the FEMA label over the WS label.

[0050] The first intended use of WS analogs was for after-shave lotions (skin applications), but this shifted to cooling the oral cavity. Today, metric tons per year of WS-3, WS-5, WS-12, and WS-23, together with menthol, are used as cooling ingredients in toothpaste (~45%), chewing gum and confectionery (—45%), and skin cosmetics and toiletries (~ 10%). Recently, WS-5, WS-23, and FEMA 4557 (in China) entered the market as cooling substances in liquids for vaporization in e-cigarettes. However, no WS compounds ever became a therapeutic agent for skin dysesthesia management. FEMA 4549 and FEMA 3784 (menthoxypropanediol) were tested in clinical itch trials but did not enter development. The "WS" designations for WS-3, WS-5, WS-12, and WS-23 are familiar from past usage. But the preferred term is now the FEMA number, which has an extensive library online.

[0005] ] Sensory activity in the oral cavity was the first screen because the intended uses were for the oral cavity. The goal was to identify candidates producing ideal cooling sensations in the mouth. Test agents, prepared as 15 mg of white crystals, were placed on the dorsal surface of the tongue. The chemical structures and identifying characteristics are in Table 5. Surprisingly, FEMA 4557 (N-ethyl-2,2- diisopropylbutanamide) stood out as a potent agent producing stinging, freezing, and painful cold on the throat. Related cooling agents were not freezing cold, except for WS- 23, but this analog was too short-lived and lacked potency for skin treatment.Dermatological Applications of Cooling Agents and FEMA 4557

[0052] Scratching and skin dysesthesia are common manifestations of dermatoses of humans and other mammals. Susceptible fur-coated animals that candidates for treatment with topical FEMA 4557 include, for example, canines (e.g., a dog), felines (e.g., a cat), equines (e.g., a horse), primates, simians (e.g., a monkey or ape), or humans. Effective, inexpensive, topical treatment of canine itch is a large and unmet need. Herbal oils containing monoterpenoids such menthol, camphor, and eucalyptol are frequently used as topical agents for skin dysesthesia. But these agents are irritating to mucous membranes such as the eyelids, pungent, and short-lived. Sometimes, agents such as l-menthol may be present in remedies at concentrations as high as 32% wt / volume. But these oils are dangerous to use, especially in children.

[0053] Physical abstraction of heat by ice or cold packs reduces skin dysesthesia but is not sustainable because freezing damages tissues. Cooling can also trigger rashes in susceptible individuals with “cold urticaria.” Cold weather can precipitate itching episodes, either due a low humidity in air, or because of exposure to indoor heating.FEMA 4557

[0054] After comparing various agents in the oral cavity screen, FEMA 4557 was formulated into a topical gel and applied to the skin surface. This 4557-gel effectively treated the skin dysesthesia in subjects with insect bites, scalp dermatitis, and dogs with canine eczema. Related FEMA agents were not effective under similar test conditions. The effective doses for FEMA 4557’s topical activity on the skin were much higher than doses used commercially in the oral cavity, but no tissue irritation or inflammation was detected on the skin, despite concentrations of >5% wt / vol. The average use levels(ppm) / average maximum use levels (ppm) for FEMA 4557 at which the FEMA Expert Panel based its judgments that the substances are generally recognized as safe (GRAS) is 3000 / 6000 ppm (or 0.3 to 0.6%) for FEMA 4557 in chewing gum, and 1000 / 1500 for confectionery frostings and hard candy. The tests on skin confirmed and revealed the therapeutic value of FEMA 4557 as a topical medicament. There was an effective drug action, but adverse side effects were absent at therapeutic concentrations.

[0055] An example of a topical gel formulation of FEMA 4557 consisted of 8% wt / vol of FEMA 4557, 2% ethanol (or 1 ,2-propanediol) as a solvent, 0.5% carbomer as a gelling agent, and 1 % l-methanol as a dispersing agent and initiator. This gel applied topically, will stop the discomforts of an insect sting for 3+ hours with an onset of less than 3 min. A plastic reservoir container with a small rounded tip is ideal for dispensing the gel for human or canine skin. In certain fur-coated mammals such as dogs, l-menthol is not used in the topical formulation because licking and oral ingestion of the topical formulation can cause vomiting, diarrhea, and general discomfort. In summary, a known food additive was found to be an effective pharmaceutical ingredient for skin dysesthesia.Chemistry of Cooling Agents and Terminology

[0056] The prototype cooling agent for the oral cavity is menthol, found in plant species of the Mentha family. Today, the active ingredient used in commerce is the l-menthol enantiomer. The applicant described the properties of new cooling agents icilin and 1 - dialkylphosphorylalkanes, represented by 1 -diisopropylphosphorylheptane (synonyms: Cryosim-1 , DIPA-1 -7: CAS No.).

[0057] The WS compounds most widely used in commerce are the p- menthanecarboxamides. Note the p-menthane moiety contains a compact cyclohexane entity. Cyclohexane makes these compounds hydrophobic and difficult to dissolve in aqueous solvents. WS scientists also discovered "acyclic amides" that exerted cooling actions. For the acyclic amide, WS-23 and WS-24 were commercialized. These compounds are "acyclic" because they do not contain the cyclohexane ring.

[0058] Here are comments on grades assigned to specific classes of cooling agents (Table 2). The lower potency of FEMA 4557 does not influence costs because it can be synthesized at low costs. Icilin cannot be formulated for skin delivery: it does notdissolve in any standard dermatological solvent. The p-menthane carboxamides cover a wide range of compounds, but they are difficult for skin formulation to achieve a long duration of action. Menthol is short-acting and irritating, especially when it is near the ocular surface. The 1 -dialkylphosphoryl-alkane known as DIPA1 -7 (Cryosim-1 , 1 - dialkylphosphorylheptane) is an excellent skin agent, but costs are high for dermatological applications. It can be seen that FEMA 4557 has many favorable attributes.Both WS-23 and FEMA 4557 have diisopropyl "legs."

[0059] The diisopropyl legs are essential for receptor occupancy and activation. In this discovery, we find that FEMA 4557 has an unexpected and surprisingly long duration of action. The methyl group on FEMA 4557, marked as (***), and which is not on WS-23 or WS-24, may act as a "hook" or "coat-hanger" to trap the molecule at the receptor pocket and prolong occupancy and duration of action.

[0060] Fig. 5 shows the relative activity of FEMA 4557 to other compounds. FEMA 4557's potency on the philtrum test is exceptional. The dose-response relationship of FEMA 4557 on the philtrum skin is shown in Fig. 12. In the data for Fig. 12, the activityof FEMA 4557 was somewhat less potent than the data in Fig. 5. This lowered potency is because a higher concentration of ethanol was used in the excipient.

[0061] The cooling monoterpenoids (menthol group), DAPA compounds, icilin analogs, p-menthanecarboxamides, and acyclic amides can generate infinite analogs. But which will work in the clinic? In practice, one cannot test everything. After making a thousand compounds, the first step is to reduce the selection to ten candidates, or 1 %. One or two of these ten candidates might have a chance of success in the clinic.

[0062] For example, within the DAPA series, only two DIPA candidates, DIPA-1 -7 and DIPA-1 -8, have a chance of becoming a drug for human dermatological applications. However, with the right choice, clinical success is remarkable. In the clinic, DIPA-1 -7 inhibits itching in patients with urticaria. It also inhibits scalp itch, which is helpful for seborrheic dermatitis and psoriasis. DIPA-1 -7 is approved for such uses in S. Korea. Recent studies (Lee, S., Wei, E. T., et al. Histamine- and pruritogen-induced itch is inhibited by a TRPM8 agonist: a randomized vehicle-controlled human trial. Brit. J. Dermatol. 190(6), 885-894, 2024. https: / / doi.org / 10.1093 / bjd / ljae054) show DIPA1 -7 as a versatile agent to stop itching. Pretreatment with DIPA-1 -7 prevents itching elicited by five pruritogens that act by different mechanisms of action (Fig. 1 ). DIPA-1 -7 is ideal for short paroxysmal itch episodes, such as atopic eczema and urticaria. But for long-term use, DIPA-1 -7 has limitations. It is too cold and can be irritating on broken skin surfaces. To treat itch from atopic eczema, the itch of the mature patient, or vulvar itch, it would be ideal to have gentler, soothing, and long-acting agents acting on the skin. The limitations of DIPA-1 -7 led to a search for better alternatives. The applicant looked in the p-menthane carboxamides and the acyclic amides series.

[0063] Most data on structure-activity relationships of p-menthane carboxamides is for cooling intensity in the oral cavity (Table 5). Wei US Patent 6,599,524, 2014, identified some lead candidates for skin in the p-menthane carboxamide series, but these did not enter development.Bioassay Based on TRPM8 Receptor Potency, the EC50

[0064] In 2002, groups led by David Julius and Ardem Patapoutian (Nobel Laureates of 2021) showed that menthol and icilin, both purported cooling agents, acted on a neuronal membrane protein subsequently called the TRPM8 receptor. A receptor assay is a standard tool and can be done quickly. For example, in a 384-plate assay, you can easily test 10 compounds and get dose-response data. For a chemical agonist, an in vitro TRPM8 assay generates an EC50 (median effective concentration). But the quantitative correlation between the EC50 on TRPM8 and cooling sensation is not always clear. That is, the scientific measure was “roughly" predictive, but there was considerable uncertainty for each agonist. The EC50 values have scientific panache, but do not give information on the duration of action or the quality of the sensation. Table 3 summarizes results on TRPM8 EC50 (see Fig. 3).

[0065] Fig. 3. Median Effective Dose (EC50) of various cooling agents in TRPM8 in vitro assay. The consensus is that icilin and WS-12 are the more potent agonists, followed by WS-5 > WS-3 ~l-menthol. The acyclic amides WS-23 and FEMA 4557 are significantly less active than l-menthol. The applicant provided the test substances used in the publications by McKemy et al., Bodding et al., and Sherkheli et al. Further experiments by the applicant confirmed the weak activity of FEMA 4557 on TRPM8 (10% the activity of l-menthol) and the absence of activity on TRPA1 .Reference # in Table 31 . Behrendt, H-J. et al., Characterization of the mouse cold-menthol receptor TRPM8 and vanilloid receptor type-1 VR1 using a fluorometric imaging plate reader (FLIPR) assay. Brit. J. Pharmacol. (2004) 1 -9.2. Bodding M, et al.. Characterisation of TRPM8 as a pharmacophore receptor. Cell Calcium. 2007;42(6):618-28.3. Johnson S, et al., Trigeminal Receptor Study of High-Intensity Cooling Agents. J Agric Food Chem. 2018;66(10):2319-23.4. Leffingwell J, Rowsell D. Wilkinson Sword cooling compounds: from the beginning to now. Perfume & Flavorist 39: 34-44, 2014.5. McKemy DD, Neuhausser WM, Julius D. Identification of a cold receptor reveals a general role for TRP channels in thermosensation. Nature. 2002;416(6876):52-8.6. Sherkheli MA, et al., Characterization of Selective TRPM8 Ligands and their Structure. 2010;13(2):242-53.

[0066] The results of Johnson et al. (Ref #3) have the greatest discrepancy from the data of others. Closer inspection and analysis of Johnson's data show the numbers do not fit the Hill Equation criteria for an EC50 measurement. In a Sigmoid Curve, it is essential to have data points above and below the median value, but Johnson’s data doesn’t have it. Overall, one can conclude that the acyclic amide coolants, WS-23 and FEMA 4557 have potencies substantially lower (e.g., 1 / 5 to 1 / 10) than that of menthol. Thus, the surprising potency of FEMA 4557 against skin dysesthesia and inflammation is unexpected. Fig.4 shows the data on FEMA 4557 from Prof Lee's laboratory at Duke University and confirms its weak activity on TRPM8. The EC50 of FEMA 4557 (labeled as BUDA) was estimated at 0.41 mM or about 1 / 30x that of l-menthol. FEMA 4557 was not active in cells transfected with TRPA1 , although a positive TRPA1 control AITC (allyl isothiocyanate) was active on TRPA1. Prof. Seok-Yong Lee at Duke University is the world’s leading expert on TRPM8.

[0067] Fig. 2 and Table 4 show the cooling intensity of agents tested in the oral cavity at 100 ppm in 5% sucrose. Cooling intensity was self-rated on a scale of 0 to 9 units.Data are from Fig. 4 of Sonya Johnson et al., 2017. The rank order of potency of WS-5> WS-3 ~ l-menthol> WS-12> WS23 agrees with compilations published by Leffingwell (2014) and by Erman (2007). The data from the oral cavity do not predict the potent activity of FEMA 4557 on the skin.

[0068] The sensory neurons of the skin, studied in isolated dorsal root ganglia neurons, have TRPM8 as the primary protein for detecting cold. However, in about 20% of such labeled neurons, the TRPM8 co-exists with TRPV1 in the same neuron. Co-activation of such TRPM8 / TRPV1 neurons may give the sensation of icy, stinging, painful cold. Painful cold may also help stop skin dysesthesia. TRPA1 is another TRP channel present in sensory neurons and co-exist with TRPM8 and TRPV1 . It has been reported that FEMA 4557 activates TRPA1 (Johnson et al. 2017) and is active on TRPM8. But we were not able to repeat the TRPA1 results. The use FEMA 4557 for the treatment of skin dysesthesia and inflammation may be based on both pain and cold.Structure Activity and Isopropyl Legs

[0069] The applicant has noticed in his studies of cooling agents that the isopropyl groups confer greater potency in biological activity. The pocket for the isopropyl occupancy has not been identified with precision. Xu et al. (Molecular mechanisms underlying menthol binding and activation of TRPM8 ion channel. Nat Commun.] 2020. http: / / dx.doi.org / 10.1038 / s41467-020-17582-x) have conducted a thorough analysis of the binding of l-menthol to the mouse TRPM8 receptor. They noted the robust coupling energy between the isopropyl group on the cyclohexane ring and the channel based on the Kd values. For the critical binding amino acid residues, they stated that the strongest coupling energy values for the "isopropyl leg" were at L843 (leucine) and I846 (isoleucine). In contrast, the V775 (valine) residue outside the binding pocket exhibited smaller coupling energy with the isopropyl legs. Y745 (tyrosine) and F839 (phenylalanine) were within the binding pocket, but apart from the isopropyl legs, they exhibited smaller coupling energy than I846 and L843. These results suggested that the isopropyl legs point downward as predicted in the docking experiments, where they stand on residues L843 and I846 to facilitate channel activation.

[0070] The activity of FEMA 4557 relative to the other compounds tested are shown in Table 3 and Fig 3. In previous studies, Wei and Co-workers have found that 1 - diisopropylalkylphosphorylalkanes are potent stimulators of the TRPM8 receptor and inhibit itch. One entity, called DIPA-1 -7 (Cryosim-1 ), was exceptionally active and stopped the itch of urticarial rashes and scalp itch in human subjects (see refs). DIPA- 1 -7, at less than 2 mg on the tongue, produces a frigid, stinging, icy, and painful cold, like FEMA 4557. FEMA 4557 and FEMA 3804 are similar in structure (Table 5). Both have prominent isopropyl legs in close juxtaposition to the hydrogen bonding oxygen atom. FEMA 4557 produced potent stinging cold, but not FEMA 3804. The N-ethyl group or the extra methyl group in C-2 of the FEMA 4557 butanamide may fit better into the receptor pocket. Increasing the N-alkyl substituent to n-propyl or iPr may further alter the duration of action. Thus, the selection of FEMA 4557 as an ideal agonist based on its diisopropyl legs has a rational basis in structure-activity relationships.Philtrum Bioassay for Identifying Topical BioactivityJohn Gaddum, concept of Bioassay and EC50.

[0071] The rationale of using cooling to gate the perception of noxious stimuli is described in Fig.1 . Here, the bioassays for identifying the best agent are discussed. In the search for a molecule that will suppress skin dysesthesia and inflammation after topical application, the correct bioassay must be used. The importance of bioassay was emphasized by John Henry Gaddum, FRS (1900-1965). Gaddum, a physiologist, became a pharmacologist when he trained with Trevan at Wellcome Research Labs. Trevan standardized the mathematics of the bioassay of digitalis leaf which eventually led to the identification of digoxin, an essential drug for patients with heart failure. The concepts of the dose-response curve and median effective doses emanate from these scientists. Gaddum was fond of pithy sayings. Some memorable quotes are:- A branch of science comes of age when it becomes quantitative.- On research. “Enthusiasm is not enough."- “The pharmacologist has been a ‘jack of all trades’, borrowing from physiology, biochemistry, pathology, microbiology, and statistics - but he has developed one technique of his own and that is the technique of bioassay.’’

[0072] Trevan and Gaddum recognized that a normal dose-response curve on linear axes was a rectangular hyperbola. The hyperbola became a sigmoid (S)-shaped curve when the x-axis was logarithmic. Further transformation of the y-axis to probit units gave a straight line and a facilitated readout of the median effective dose (EC50). This type of pharmacologic analysis is now computerized and routine for in vivo and in vitro assays, such receptor activation potencies.

[0073] Threshold for Cooling on the Surface of the Tongue. Wilkinson Sword in their program for finding better menthol analogs, tested substances by impregnating them on filter paper and applying the filter paper to the tongue of volunteers. The threshold for cooling was first rated as “+++” signs to signify potency and later quantified as “pg” on the tongue. The data were “semi-quantitative” and there is overlap in the potency of many analogs

[0074] Intensity of Cooling in Oral Cavity. As WS compounds became widely used in the 1990s in toothpaste, chewing gum, mouthwash, and confectionery, there was a need to re-design the bioassay for the oral cavity. The filter paper on the tongue method was replaced by the “hold and spit" method. In this assay, a test substance is suspended or dissolved in a liquid, and the liquid held for 30 sec in the mouth and then expectorated. Sometimes, sucrose is added to the test liquid to mask tastes. The sensory effects are recorded by the subject and “cooling intensity” is rated on a scale of 0 to 9. No attempt was made to relate oral cavity cooling to effects on skin. The consensus was that WS-5 gave the most intense cool, followed by WS-3 and l-mentho I , and then WS-12. WS-23 and WS-24 were considered less potent.

[0075] TRPM8 Receptor Assay. In 2002, groups led by David Julius and Ardem Patapoutian (Nobel Laureates of 2021 ) showed that menthol and icilin, both purported cooling agents, acted on a neuronal membrane protein called the TRPM8 receptor. A receptor assay is a standard tool, and can be done quickly. For example, in a 384-plate assay, 10 compounds can be tested to generate an in vitro TRPM8 EC50 (median effective concentration) value. The EC50 was “roughly" predictive, but there was considerable uncertainty for each agonist. The EC50 do not give information on the duration of action or the quality of the sensation.

[0076] Philtrum (Skin) Assay. For cooling sensations on the skin, it is necessary to test the substance on human skin. Wei proposed the use of the skin on the philtrum as a test site for cooling activity of chemicals. The test substance is dissolved in a carrier, such as solvent, gel, or ointment and applied to the philtrum, a surface area of ~10 to 12 cm2.

[0077] The subject is given a form to record observations and instructions to mark sensations on the philtrum and environs as:0 = no changes in sensation1 = a slight feeling of coolness, cold, or tingling2 = a clearcut feeling of coolness or cold3 = a robust feeling of coolness or cold

[0078] The intervals for measurements are usually 0, 1 , 3, 5 min after application, and at then 5 min or longer intervals thereafter until two successive zeros are obtained.

[0079] The philtrum is the midline groove above the upper lip border and vermilion and is the drug application site. The gel is placed on the philtrum with a cotton-tipped applicator up to the nasolabial folds and sometimes, but not deliberately, on the lower nostrils (subnasale). Cold receptors densely innervate the philtrum skin, second only to the surfaces of the eyeball and anogenitalia. The lips also have a high density of "cold spots."

[0080] Hensel et al. mapped the cold spots on the human face (see Dhaka et al., 2008. TRPM8-expressing sensory neurons and their projections. J Neurosci. 2008 Jan 16: 28(3):566-75). The mystacial pad (nose pad with vibrissae) is abundantly labeled with TRPM8-fibers. Other receptors present are TRPV1 and TRPA1 . "Cold Points" linked to single-unit neuronal discharges can be measured in the brain of experimental animals (e.g., see Poree LR, Dickenson AH, Wei ET. Corticotropin-releasing factor inhibits the response of trigeminal neurons to noxious heat. Brain Res. 1989 Nov 20: 502(2) :349-55.

[0081] In practice, test substances are applied to the philtrum with a cotton-tipped applicator (Puritan Cotton-tipped applicators, or Q-Tips) after dipping the tip in a reservoir bottle containing the liquid or semi-liquid (gel) test substance. The composition adhering to the cotton (about 0.2 mL) is put on the skin. A subject can practice the method in front of a mirror with a high-intensity light source. The results are described in Table 7.

[0082] Fig. 5. shows results for cooling agents, prepared as an 8% wt. / vol. gel, when tested on the philtrum skin assay. The duration of cooling was recorded (min on y-axis). The startling long activity of FEMA 4557, cooling for ~ 3 hr, was unexpected and surprising. FEMA 3784 is also known as Cool 10 or menthoxypropanediol.The Preferred Embodiments are shown in Formula 1.wherein R is ethyl, isopropyl, n-propyl, cyclopropyl, or acetyl

[0083] where R = ethyl, the compound Is FEMA 4557. The isopropyl, n-propyl cyclopropyl, or acetyl analogs have not been synthesized and tested. There is reason to believe that these analogs may be more potent than the ethyl analog. The extra methyl group forming the “hook” or “coat hanger” on FEMA 4557 is critical for the practice of this invention. The hook is ethyl in FEMA 4557, but can be modified to n- propyl, or i-propyl to increase activity, as shown in Formula 2. Permeation through the stratum corneum is not an essential factor for success because FEMA 4557 is much more active than WS-23, despite having an extra methyl group.Structures of FEMA 4557 analogs. wherein R1 is ethyl, isopropyl, or n-propyl, and R2 is ethyl, isopropyl, n-propyl, cyclopropyl, or acetyl.Formulation, Excipients, and Initiators

[0084] The formulation of skin care products is usually a carefully guarded secret. Success may depend on slight changes in the selection or levels of ingredients without an obvious explanation for differences. Here, the choice of the carbomer polymer for gelling is straightforward, and varying the concentration will determine the appropriate gel viscosity.

[0085] The dispersion of FEMA 4557, the bulk ingredient, is facilitated by ethanol and, surprisingly, by about an added 1 % by weight of l-menthol. The menthol helps FEMA 4557 to be homogeneous and dispersed in the gel. Ethanol, as a solvent, inhibits TRPM8 agonist binding, but a concentration of ethanol < 1 % wt / vol improves solubility and dispersion. Another ingredient for the formulation is an initiator of cooling. An 8% wt / vol gel of FEMA 4557 alone has an onset of 15 to 20 min for producing cool and cold. An initiator added to the formulation immediately makes the cold within 1 to 3 min and sustains the anti-itch effect. The initiator enhances the patient's appreciation of the antiitch impact. Table 7 lists effective initiators and their active concentrations in the gel. Wei in US Patent 10,195,217 2019 describes the chemistry of these compounds. The use of initiators is part of the discovery process. Other compounds to extend the duration of action, such as hyaluronic acid, may be considered as an excipient.Therapeutic Applications, Different Forms of Inflammation

[0086] In the Examples below, FEMA 4557 compositions inhibit the inflammation of flea bites, thermal injury, skin allergy, and urticaria. The surprising finding is that FEMA 4557 has a prolonged anti-inflammatory effect on all five cardinal signs of inflammation after a single application. Intense physical cooling with ice has a comparable effect, but this is not sustainable because the ice will damage tissue metabolism. A potent topical glucocorticoid has anti-inflammatory actions, but its onset is slower and will not work for thermal injury. A strong cooling agent such as Cryosim-1 (also known as DI PA-1 -7, 1 - diisopropylphosphorylheptane) is antipruritic and analgesic but does not affect the redness and the diameter of the wheal produced by histamine (Lee et al., 2024, vide supra). Thus, the anti-inflammatory effects of FEMA 4557 are unusual, unique for its chemical class, and not previously described.Study 1. Insect Bites

[0087] Fig. 7. Shows an example of skin's inflammatory response to flea bites and treatment with topical FEMA 4557. An elderly male gardener was bitten on the thigh. The row of inflamed red spots is typical of flea bites. Half an hour after the bites, the skin is red, swollen, and intensely itchy (left panel). Within 10 min after application of FEMA 4557, 32% solution, with a pen swelling and redness becomes reduced (middle panel at 12 hr). Surprisingly, itching disappears within 10 min application and a single application of FEMA 4557 is sufficient to suppress itch. After 24 hr, the skin inflammation heals. The rapid onset of the anti-itch effect, and effectiveness of a single application of FEMA 4557 to counteract inflammation is surprising and not expected. Subsequently, the ability of FEMA 4557 to counteract the inflammation of insect bites have been confirmed in more than 12 cases.

[0088] This incident illustrates how FEMA 4557 is used. A furry yellow-black bumble bee, about 1.5 cm long, was buzzing behind the windowpane in the kitchen. Instead of swatting the bee, the applicant tried to trap the bee with a piece of tissue in order to release him outside into the garden. The applicat was stung on the distal phalanx of the middle finger (Fig. 13), which became swollen and sharply painful. The volume of the distal phalanx increased from 7.0 g to 9.0 g within 30 min, as measured by the fluid displacement method over a top-loading balance (middle panel). The applied 32% wt / vol FEMA 4557 solution glistens on the fingertip (right panel). Surprisingly, the sharp pain transforms into an intense itch within a few minutes and the pain is reduced. This itch persisted for at least three hours.

[0089] The principal pain-inducing substance in bee sting venoms is a 26-residue peptide called melittin (Chen et al., 2016). Melittin affects membrane pores, has multiple actions of various nociceptive receptors, such as TRPV1 , and initiates cascades of phospholipid inflammatory mediators. The ability of FEMA 4557 to convert a painful melittin sting to itch may offer clues on the treatment of acute pain. If acute pain from tissue injury converts to an itch after FEMA 4557, the itch is more tolerable than pain.Study 2. Thermal Injury

[0090] Fig. 11 shows a 4-jet butane-fueled cigar lighter with a cigar punch. A cigar punch is used to make a hole on top of the cigar to facilitate airflow during smoking. When lit, the cigar lighter's flame is like a blowtorch. A subject accidentally burned his hand when trying to use the cigar punch. Fig. 11 shows the burn site from the cigar lighter at the juncture of the forefinger and thumb of the left hand. The white bars show the sites for rapid development of swelling. There was pain after the burn, together with redness and edema. A 20% solution of FEMA 4557 was applied with a cotton-tipped applicator to the burn site 15 min after the burn. The pain diminished and was not noticeable after 5 hr. FEMA 4557 was only applied once. The redness, swelling, and pain disappeared after 24 hr, and the wound site formed an eschar which resolved within three weeks. The rapid suppression of pain and healing without any side effects was surprising and indicated a potent anti-inflammatory effect on thermal injury.Study 3. Urticaria and Histamine Release

[0091] Urtica (Latin for sting) is a genus of flowering plants from the family Urticaceae. Many species have stinging nettles that contain histamine, serotonin, and acetylcholine. The mediators account for the signs and symptoms of urticaria, a human skin condition of histamine release triggered by IgE-antigen antibody type of allergy.

[0092] A 53-year-old female subject developed recurrent episodes of urticaria six months after receiving a Covid-19 vaccination (Fig. 10). The itch of urticaria was on all parts of the body. Each episode lasted ~2 weeks, and there were 10+ episodes in the post-vaccination period of one year. The Figure shows the urticarial lesions on the nape. There is evidence of scratching (lighter shade skin where the epithelium is removed) and rash (darker shade skin). Applying FEMA 4557 gel or solution controlled the itching within 10 min, allowing the subject to rest and sleep. The subject is now controlling her itch with daily doses of fexofenadine (Allegra / Hives) and using the FEMA 4557 pen on an "as-needed basis." As shown elsewhere in this application (Fig. 1 ), this anti-itch effect is not selective for histamine, but also works for other pruritogens on the human skin.Study 4. Skin Dysesthesia, Itch, and Inflammation

[0093] The compositions and formulations contemplated here have special utility in two types of human itch that are quite common: itch of the mature patient and the patient with the genitourinary syndrome of menopause. Everybody gets old and all females undergo menopause, so this is a large population.

[0094] The terminology to describe ageing and itch has evolved from senile pruritus, to itch of the elderly and now itch of the mature patient. The exact age of being elderly is between sixty to sixty-five years, but the risk of itch progresses as one becomes older so that about 50% of individuals over eighty have itch. The prevalence has been ascertained in general surveys and studies of the elderly in nursing homes. The exact causes of itching in the mature patient are varied and often not known, but include psychogenic and neurogenic triggers, inflammation, xerosis, and allergy.

[0095] Therapeutic Indications and Treatment Goals. The indications are itch of the mature patient, atopic dermatitis and vulvar itch. These skin sites are usually keratinized but may become more permeable because of inflammation and scratching. The number of epithelial cell layers is -8 to 15. The clinical conditions are chronic and not "curable." The treatment goals are to ameliorate symptoms and interrupt the itchscratch cycle. A composition containing the preferred embodiments has the advantages of immediate onset of drug action.

[0096] The three clinical conditions have these features in common:- The dermatitis is a dominant symptom that destroys the QoL for the patients.- The three indications are not "curable" but chronic and persistent.- The philtrum assay is essential for finding the active ingredient and its optimal formulation.- The dermatitis occurs on the skin with a stratum corneum of 10-15 cell layers, ~1 mm thick. The stratum corneum limits drug penetration to target receptors at the basal layer of the epidermis.- The proposed treatment is topical and repeated. An essential goal is to interrupt the itch-scratch cycle.- The preferred duration of action in the philtrum assay is > 3hr cooling, with another 3 hr residual cooling.- The formulation of FEMA 4557 should cool the philtrum for at least 3 hr and preferably longer, with residual cooling action.- In formulations with FEMA 4557, anti-inflammatory steroids, hormones, or emollients can be mixed.- The drug should be potent, cost-effective, and receptor-selective to avoid side effects.- The formulation for delivery should be easy to prepare.- The technology should be original and allow patentability for development.- The quality of sensation should be refreshingly cool or cold, without irritation or pain.- Identifying the ideal chemical class and molecule is a matter of continued and persistent experiment. When the criteria defined above, 95+% of known cooling molecules will fail! However, one N-alkyl, 2,2 diisopropyl butanamide structure, e.g., FEMA 4557, meets these criteria.Mechanisms of Anti-Inflammatory Actions of Preferred Embodiments

[0097] Inflammation is a dynamic clinical event that negatively impacts an individual's quality of life. An anti-inflammatory with a new mechanism of action can potentially treat many indications. Physical cooling of injured tissues relieves pain, but its benefits are not sustainable because of cell damage by heat abstraction. Chemical cooling agents, such as menthol, also relieve skin irritation but are not considered anti-inflammatory. Here, we show the preferred embodiment, a specific TRPM8 agonist delivered to its receptor, counteracts the inflammation caused by insect stings, heat, allergy, and skin disorders. Specific means the molecule activates only TRPM8 but not other TRP receptors. The term agonist means a molecule that activates a biological event. The agonist is not an antagonist, defined as a substance that blocks agonists at their receptors. For inflammation, the TRPM8 agonist is non-selective: it will inhibit a variety of inflammatory stimuli. Table 9 illustrates the pathways of inflammation. The exact mechanisms of the anti-inflammatory effect of the preferred embodiments are not unknown. The presence of TRPM8 on neuronal membranes may hint at the inhibition of neurogenic inflammation as a mechanism. However, TRPM8 receptors are also on the skin's keratinocytes, which may influence inflammatory response. One intriguing possibility is that TRPM8 activation inhibits the generation of negative interstitial fluid pressure (Pif) in the extracellular matrix. A negative Pif in the extracellular matrix creates a suction pressure on fluids from the blood compartment, resulting in edema and the disruption of tissue architecture. In previous studies with corticotropin-releasing factor (CRF), a neurohormone peptide, the applicant and others observed that CRF inhibits negative Pif and reduces brain edema in rats and humans. CRF also inhibits heat-induced edema in the rat.Therapeutic Applications, Different Forms of Dermatitis

[0098] The preferred embodiments, compositions, and formulations contemplated here have special utility in the treatment of dermatitis of the patient with diabetes, the mature patient, and individuals with atopic dermatitis.

[0099] Dermatitis and Itch in Patient with Type 2 Diabetes (T2DM). T2DM occurs in >10% of the world’s population and individuals become more overweight and have a sedentary lifestyle. T2DM is readily defined by lab tests on fasting plasma glucose levels (>126 mg / mL) or elevated glycated hemoglobin. The diabetic state is associated with dry skin (xerosis) and neuropathy and such T2DM patients have chronic dermatitis (-25%) which disrupts the quality of life. The preferred embodiment which reduces dermatitis and skin dysesthesia will have benefit in such patients.

[0100] itch of the Mature Patient. Everybody gets older. The skin, the body's largest organ, deteriorates with age. The skin becomes xerotic and is more fragile, and there are defects in wound healing. Dermatitis is a prominent symptom seen in geriatricdermatology. The medical terms to describe aging and dermatitis have evolved from senile pruritus to "itch of the elderly" and now “itch of the mature patient.” The age for being “elderly or mature" varies from 60 to 65 years, but at about 80 years, 30% of individuals are likely to have a chronic itch. "Chronic" means the itch is present for more than six weeks. The itch of the mature patient is a serious management problem in nursing homes. The causes of itching in older patients are varied and often challenging to diagnose. Triggers for itch include cutaneous defects and non-cutaneous causes such as psychogenic and neurogenic factors, medications, and allergies. The exact causes of itching in the mature patient are often not known, Treatments include creams and emollients, anti-inflammatory steroids, antihistamines, and psychotropic drugs. A topical gel, lotion, or cream to reduce itching in the mature patient would be valuable to physicians and patient.

[0101] Atopic dermatitis (AD), also known as atopic eczema, is a chronic inflammatory dermatologic condition that affects 12 to 13 % of children and approximately 7% of adults in the United States. The onset of many cases begins before age five, is more frequent in females, and 50% of children may have symptoms that persist into adulthood. The principal features of AD are severe itch and dry skin, associated with hyperreactivity to ordinary stimuli such as food allergens, irritants, and infection (comorbidities). The common skin lesions of the AD patient are red skin papules with oozing, crusting, excoriation, and scaling. AD imposes a substantial burden on the patient's quality of life, causing limits on social and functional engagements, psychological distress, and overall diminished satisfaction for the patient and family members. Itch, difficulty in sleep, and pain are the principal complaints of AD, together with concerns about cosmetic disfigurement. One Web study found that daily itch occurred in 91 % of AD respondents. In moderate-to-severe AD, 70% reported severe or unbearable itch in the past two weeks, 86% reported daily itch, and 63% reported itch lasting at least 12 hours per day. The numerical rating scale of itch intensity (on a scale of 0 to 10) correlates well with an overall ranking of mild, moderate, and severe disease. The QoL scale of a moderate and severe AD patient is worse than a patient with heart disease, diabetes, or high blood pressure. AD patients suffer most from a limited lifestyle, avoiding social interactions because of itching, skin appearance (dry-scaly skin,blisters or bumps, flushed inflamed skin, thickened skin, open sores or oozing), and because of pain and disturbed sleep. Disturbed QoL is conspicuous in patients with moderate to severe AD.

[0102] An excellent illustration of diabetic dermatitis is shown in Murao, S., & Murao, K. (2021 ). Multiple skin ulceration and itch-scratch cycle in a diabetic patient. In Journal of Diabetes Investigation (Vol. 12, Issue 11 , pp. 2102-2103). John Wiley and Sons Inc. https: / / doi.Org / 10.1 1 11 / jd i .13560. Reviews of the topics of “itch of the mature patient” and “atopic dermatitis can be found in these articles, which are incorporated herein by reference. Mature Patient: Chung BY et al., Pathophysiology and treatment of pruritus in elderly. Int J Mol Sci. 2021 ;22(1 ):1 — 12; Shevchenko A et al. Causes, pathophysiology, and treatment of pruritus in the mature patient. Clin Dermatol [Internet], 2018;36(2):140-51 . General Background: Kahremany S et al. Advances in understanding the initial steps of pruritoceptive itch: How the itch hits the switch. Int J Mol Sci. 2020;21 (14):1— 45. Smith RL et al. et al. GRAS flavoring substances 24. Food Technol. 2009;63(6):46-62.Acute Anti-Inflammatory Actions of FEMA 4557

[0103] Acute inflammation is a dynamic clinical event that accompanies tissue injury. A new anti-inflammatory mechanism of action can potentially treat many indications. Physical cooling by lowering tissue temperatures of injury relieves pain, but its benefits are not sustainable because cooling will cause cell damage by interference with metabolism. Chemical monoterpenoid cooling agents, such as l-menthol, are irritating and not considered anti-inflammatory. Here, we show FEMA 4557, the preferred embodiment, counteracts the acute inflammation caused by insect stings, heat, allergy, urticaria, and thermal injury.

[0104] The preferred embodiment of this discovery, FEMA 4557 is a non-selective inhibitor of a variety of inflammatory stimuli. The exact mechanisms of the antiinflammatory effect of FEMA 4557 are not unknown. The presence of TRP channels on neuronal membranes hints at the inhibition of neurogenic inflammation as a possible mechanism. One intriguing possibility is that TRP activation inhibits the generation ofnegative interstitial fluid pressure (Pif) in the extracellular matrix. Vascular leakage occurs when fluids pass from the blood compartment into the extracellular matrix. Inflammatory mediators such as histamine open gaps or endothelial pores (Baluk et al. 1998). A negative Pif in the extracellular matrix creates suction pressure on fluids from the blood compartment, resulting in edema and the disruption of tissue architecture (Wiig, 2011 ). In previous studies with hormones and peptides, the applicant has correlated negative Pif to tissue edema in experimental studies (Gjerde et al., 1998; Gjerde et al., 2000). FEMA 4557 may be a prototype of an agent to treat acute inflammation.Therapeutic Applications, Different Forms of Canine Dermatitis

[0105] The compositions and formulations contemplated here have utility in canine dermatitis conditions shown in Table 10.

[0106] Ectoparasites. A parasite is an organism that lives on or in a host and gets it food from the host. The ectoparasites that live on or in the skin of dogs belong to the arthropod phylum and include fleas, ticks, mites, and lice. For example, the mites that cause itch in dogs include Sarcoptidae and Decmodecidae. Sarcoptes scabiei cause sarcoptic mange in domestic ranging animals. The mange mite is similar to an arachnid and has four pairs of legs as an adult. By contrast, the flea is an insect with three pairs of legs. The dog flea belongs to the genus Ctenocephalides. Mites are barely visible at < 1 mm, whereas the flea at ~2 to 3 mm visibly hops around in the hair. The burrowing of the mite, its allergenic potential, and the flea bite with its saliva and secretions cause intense skin itching in the dog. Flea prevention with afoxolaner (Nexgard), a prescription drug, is a multi-billion dollar business. Like head lice in children, mites in dogs are treatable with topical scabicide solutions such as permethrin. The allergic reaction and scratching of the dog skin in response to mites and fleas can persist for weeks after removing the ectoparasites.

[0107] Fungal infestations of the skin are common in humans and other mammals. Witness the high incidence (~10-15%) of Athlete's foot (Tinea pedis) and flaky dandruff. In dogs some common fungi and yeast are Dermatophytosis (ringworm) and MalasseziaFungi grow well in warm and moist conditions and on the skin of dogs with thick hair. Fungi infections of the skin are irritating and cause intense scratching. Shampoos containing antifungal conazoles (ketoconazole and miconazole) kill fungi, but only if drug delivery succeeds. The 1 % conazole solutions sold as OTC products are less effective than 2% prescription solutions. And the latter are expensive. Users of the shampoos need instructions to soak the infected skin for about 15 min to get adequate drug exposure and to repeat the shampoo within several days to eradicate the fungi better. However, adherence to such procedures is variable, and treatment of fungi infection is erratic. Chlorhexidine is sometimes added to anti-fungal shampoos but it has more value if there is a concurrent bacterial infection.

[0108] Atopic dermatitis (AD) in dogs is a common (5 to 15%) chronic inflammatory skin disease with altered skin barrier function and hypersensitive reactions to external allergens. No specific diagnostic test is available for AD, and the primary signs are itchiness, scratching, and damaged skin. Affected areas are frequently the belly, behind the legs, and around the base of the tail. AD may be seasonal. Susceptible breeds are retrievers (Golden and Labrador) and terriers (Wheaton and West Highland), but all species are at risk. Atopic dermatitis is relentless and not curable, and signs manifest when the dog is young. Affected dogs have a diminished quality of life, and incessant scratching and secondary infections strain the bond between dog and owner.

[0109] An effective drug for AD is oclacitinib (Apoquel, Zoetis Corp.). Oclacitinib inhibits the function of pruritogenic and pro-inflammatory cytokines dependent on JAK1 or JAK3 enzyme activity but not the hematopoietic JAK2. Oclacitinib is not a corticosteroid or an antihistamine but a specific JANUS kinase inhibitor. Oclacitinib has a fast onset of action and is effective in 50 to 66% of treated subjects. The drug requires a prescription from a veterinarian and is priced (as of 2023) online at ~$75 per month. Adverse effects on the drug brochure include vomiting, lethargy, anorexia, diarrhea, elevated liver enzymes, dermatitis, seizures, polydipsia, and demodicosis. Nevertheless, the drug has a good profile, and JANUS kinase inhibitors are now at the forefront of pharmacology.

[0110] The three clinical conditions have these features in common:- The skin irritation and scratching is a dominant symptom that destroys the quality of life (QoL) for the dog.- The philtrum assay is ideal for finding the active ingredient with anti-irritant properties.- The preferred duration of action in the philtrum assay is > 3hr cooling, with another 3 hr residual cooling, and preferably longer.- The proposed treatment is topical and repeated. An essential goal is to reduce scratching behavior.- The formulation for delivery is easy to prepare and utilizes a “pen” for delivery (Fig.6).- The application of the drops does not cause avoidance behavior, indicating the absence of pain or discomfort.Comparison to JANUS Kinase inhibitors

[0111] For canine atopic dermatitis a significant advance has been the use of oclacitinib, a specific JANUS kinase inhibitor. This drug, introduced in 2013, is an oral tablet administered once or twice a day and may have anti -scratching effects as early as four hours after dosing. Oclacitinib requires a prescription from a veterinarian and is pricedat about $75 per month. It acts as an immunosuppressant and does not cure the underlying hyperactivity of the immune response. Dosing has to be maintained for the lifetime of the dog. Side-effects from oclacitinib listed on the prescribing information brochure include diarrhea, lethargy, abnormal white blood cell counts, and increase risks of infestations with mites. The use of FEMA 4557 for atopic dermatitis employs a different strategy and summarized in Table 1 1 .

[0112] In humans specific inhibitors of JANUS kinase are approved for AD, psoriasis and rheumatoid arthritis, but use of these drugs is carefully monitored because of potential serious side-effects such as cardiovascular risks. These drugs will not be useful for itch for insect bites, contact dermatitis, or other skin allergies. The use of a FEMA 4557 would be the preferred first choice in humans and dogs because cooling has a broad spectrum of actions and topical use has a good safety profile.CASE STUDY EXAMPLESStudy 1Dose-Response Study on Philtrum (see Fig. 12)

[0113] Canine skin is thinner than human skin by about 50%. The stratum corneum has about 47 cell layers and a thickness of 13 pm. The living epidermis has 3 to 6 cell layers and a thickness of 10 pm. In dogs, the hair may grow from compound hair follicles. These follicles are surrounded by 3 to 15 secondary hairs growing out of the same pore. Sebaceous glands within the skin lubricate the hair, keeping the coat shiny and water resistant. The thickness of the hair coat varies with the breed. The hair coat hinders the delivery of a topical drug to its receptors in the basal layer of the epidermis. Dogs do not have eccrine sweat glands like humans. Instead, there is some sweating from merocrine glands in the paw pads. Apocrine glands near hair follicles in the areas such as the armpit and groins secrete pheoromes which is not used for temperature regulation.

[0114] The test solutions were prepared using FEMA 4557 dissolved in a solvent containing racemic 1 ,2-propanediol , 4% ethanol, and 4% l-menthol. All percentages are given as weight / volume, so an 8% solution, for example, contains 8 g of FEMA 4557 IN 100 g of solvent. The solutions prepared were 0%, 4%, 8%, 16%, 32%, and 64%. The solution was applied to the philtrum with a cotton-tipped applicator. The volume offloaded per application, obtained by weighing the solution before and after use, ranged from 55 pl to 85 pl, averaging 60 pJ. For a 16% solution, the dose is equivalent to 160 mg / ml x 60 pl = 9.6 mg or about 10 mg. This delivered dose to a philtrum surface of 1to 2 cm2 is 5 to 10 mg / cm2. The recording of the intensity of sensations from the philtrum is as noted above. The results of Fig. 12 lead to these conclusions.

[0115] The solubility of FEMA 4557 (> 64% wt / vol in liquids) facilitate delivery to receptors. As shown in Fig. 6 and 12, FEMA 4557 is miscible with the solvent and forms a transparent solution at room temperature. This property enhances the delivery of the active agent to the receptive field, located in the epidermal layers of the organism's skin below the stratum corneum. For the canine skin, the barriers to drug delivery are the dense hair follicles, which may number several hundred thousand per cm2. Hair density for the dog, on average, is 15x greater than hair on the human scalp. A liquid will wick down the hair shaft via surface tension and distribute better than any solid delivery vehicle, such as a gel or cream, which will adhere to the surface of the hair shaft. A liquid is quickly delivered and distributes the active ingredient evenly onto the skin's surface. This delivery mechanism is impossible with other compounds such as WS-5, WS-12, or WS-30 because they are not as soluble as FEMA 4557.

[0116] Absence of adverse sensations of stinging cold. The tested FEMA 4557 solution at > 64% on the philtrum produced a pleasant and robust cool. No cold stinging sensations were felt. This is contrasted to DI PA-1 -7 (Cryosim-1), another agent with good skin penetrability but which produces a frigid cold and irritation. This safety feature of FEMA 4557 was unexpected and beneficial.

[0117] Sufficient duration of action. As can be seen from the graph, a single dose of the 16 to 32% will provide ~3 hr of cooling. This effect is accompanied by an "after effect" of another 3 hr cooling on the skin. A warm towel applied to the philtrum skin will reveal this aftereffect. That is, there is a memory, reservoir, or residual cooling that linger and supplement the drug action on dysesthesia. The total therapeutic duration is at least 6 hr at 16 to 32% and provides sufficient daily control of dysesthesia at a bid., t.i.d, or p.r.n. dosing. Other test agents did not have a similar duration of action.

[0118] Practice of invention. The potency of FEMA 4557, costs of production of the active ingredient, solubility in biocompatible solvents, ease of formulation into a liquid for delivery, absence of side effects, cooling efficacy, and duration of therapeutic action areunique and surprising. No other chemical tested by the applicant has all of these desirable properties.Study 2

[0119] A six-year-old long-haired Dachshund, over eight months, developed an inflamed area on the belly and lower right groin where it frequently licked and scratched. Upon examination, the site was red and swollen (Fig. 8). A visit to the veterinarian ruled out fleas and mites or fungal and bacteria growth. The veterinarian called it a non-specific allergy and recommended cetirizine hydrochloride liquid compounded for dogs. Taking cetirizine (Zyrtec, an antihistamine) for one week did not affect the red skin or the licking. The Applicant gave the dog's owner an airless pump dispenser containing 8% wt / vol of FEMA 4557 dispersed in 4 mg / mL of carbapol 980, formulated with 1 % ethanol and 1 % menthol, and instructed him to apply the gel to the skin of the dog's belly and groin. Within one day after the application of FEMA 4557, scratching and licking were reduced, and after three days of daily application, the redness and swelling disappeared. Treatment continued for one week, after which the skin appeared utterly normal. The inflammation and scratching have not come back.Study 3

[0120] A twelve-year-old West Highland Terrier had periodic episodes of seasonal skin allergy. She would scratch until the site (groin and hindquarters) lost its fur and was bare down to the skin. A visit to the vet concluded that fleas, mites, or pyoderma were not causing the skin irritation, but the condition may be due to fungus. The vet recommended an antifungal shampoo, but repeated bathing with a ketoconazole or miconazole shampoo did not make any difference. The owner of the dog, who had access to a pharmacy, applied nystatin ointment and a potent skin steroid cream containing triamcinolone acetate. The treated dog initially seemed to respond but then licked its inflamed skin and occasionally "chased its tail" as it tried to reduce discomfort by biting. However, the skin remained inflamed, looked fragile, and oozed liquid, and the paws became red from excessive licking.

[0121] An 8% FEMA 4557 gel spread on the bare spots of the groin, hindquarters, and perianal area stopped the scratching. Next, a 20% wt / vol FEMA 4557 solution, dissolved in propylene glycol, was applied to the interdigital folds of the paws with a cotton-tipped applicator. The dog stopped licking its front paws. All signs of skin discomfort disappeared after five days of treatment with FEMA 4557, and the bare spots began to grow hair.

[0122] In summary, a chemical y, FEMA 4557, was identified as an entity that a) has a rapid onset of action, b) can be produced, formulated, and delivered at reasonable costs, c) is free of adverse side effects, d) reduce dysesthesia, inhibits scratching, and progression of the itch-scratch cycle and e) has potent anti-inflammatory actions of insect bites and thermal injury. The treatment of canine skin disorders with this molecule accelerates skin healing.REFERENCES1 . Biro T, Ko MC, Bromm B, Wei ET, Bigliardi P, Siebenhaar F, et al. How best to fight that nasty itch - From new insights into the neuroimmunological, neuroendocrine, and neurophysiological bases of pruritus to novel therapeutic approaches. Exp Dermatol. 2005; 14(3).2. Cevikbas F, Lerner EA. Physiology and pathophysiology of itch. Physiol Rev. 2020; 100(3): 945-82.3. Dhaka et al., 2008. TRPM8-expressing sensory neurons and their projections. J Neurosci. 2008 Jan 16: 28(3):566-75.4. Erman, M.B. Cooling Agents and Skin Care Applications. Cosmetics and Toiletries Magazine, Vol. 120, pg. 105-118, 2005.5. Fokkens WJ et al;, European position paper on rhinosinusitis and nasal polyps. Rhinology Vol. 58, Supplement 29, Feb. 2020.6. Hill B. et al., Physiology and pathophysiology of human airway mucus. Physiol. Rev. 102: 1757-1836, 2022.7. Johnson S et al. Trigeminal Receptor Study of High-Intensity Cooling Agents. J Agric Food Chem. 2018;66(10):2319-23.8. Jung MJ, Wei ET. et al. A randomized, vehicle-controlled clinical trial of a synthetic TRPM8 agonist (DI PA-1 -7) gel for itch. J Am Acad Dermatol. 2021 ;84(3):869— 71 .9. Kang SY, Wei ET, et al. TRPM8 agonist (cryosim-1) gel for scalp itch: A randomized, vehicle controlled clinical trial. J Eur Acad Dermatology VenereoL 2022;1— 2.10. Lee, S., Wei, E. T., et al. Histamine- and pruritogen-induced itch is inhibited by a TRPM8 agonist: a randomized vehicle-controlled human trial. Brit. J. Dermatol. 190(6), 885-894, 2024. https: / / doi.Org / 10.1093 / bjd / ljae054.11. Leffingwell, J.C. Cooling ingredients and their mechanism of action. Handbook of cosmetic science and technology. Chpt. 65. Pp. 661 -675. Barel, Paye, and Maibach. (edited). Informa Health Care, New York, 2009.12. Rogers, D. Physiology of Airway Mucus Secretion and Pathophysiology of Hypersecretion. Conference Proceedings. Respiratory Care 52:1134-1149, 2007.13. Roswell DG et al. Acyclic carboxamides having a physiological cooling effect. US 4,296,255. October 20, 1981.14. Sherkheli MA et al. Characterization of Selective TRPM8 Ligands and their Structure. 2010; 13(2):242— 53.15. Smith RL et al. GRAS flavoring substances 24. Food Technol. 2009;63(6):46-62.16. Steinhoff M, ET Wei, Neurophysiological, neuroimmunological, and neuroendocrine basis of pruritus. J Invest Dermatol. 2006 Aug;126(8):1705-18.17. Wei, ET. 1 ,2,3,6-Tetrahydropyrimidine-2-one compositions and therapeutic methods therewith for pain and inflammation. US 6,743,801 . June 1 , 2004.18. Wei, ET. [((1 R,2S,5R)-2-lsopropyl-5-methyl-cyclohexanecarbonyl)-amino]-acetic acid isopropyl ester and related compounds and their use in therapy. US 8,426, 463. April 23, 2013.19. Wei, ET. A / -Alkylcarbonyl-amino acid ester compounds and their use for skin irritation, itch and pain. US Patent 6,599,524. Oct. 7, 2014.20. Widdicombe JH, Wine JJ. Airway gland structure and function. Physiol Rev.2015;95(4): 1241 — 319.21 . Wu ZH et al / . Acute and subacute inhalation toxicity assessment of WS-23 in Sprague- Dawley rats. J Appl Toxicol. 2021 ;41 (1 1):1826— 38.22. Yogeswaran S et al. The role of Synthetic Coolants, WS-3 and WS-23, in modulating E- cigarette-induced Reactive Oxygen Species (ROS) in lung epithelial cells. Toxicol Reports [Internet], 2022;9(June):1700-9. https: / / doi.Org / 10.1016 / i.toxrep.2022.08.007 .

Claims

Claims:1 A pharmacological composition for the topical treatment of inflammation in a mammal in need of treatment thereof, comprising: a liquid or semi-liquid composition having a therapeutically effective amount of one or more compounds having Formula 1wherein R is n-propyl, isopropyl, cyclopropyl, or acetyl2. The composition as in claim 1 wherein the Formula 1 compounds is N-(n- propyl)-2,2-diisopropyl butanamide and the therapeutically effective amount is from about 2 to 20 mg per dose.

3. The composition as in claim 1 wherein the Formula 1 compounds is N- (isopropyl)-2,2-diisopropyl butanamide, and the therapeutically effective amount is from about 2 to 50 mg per dose.

4. The composition as in claim 1 wherein the Formula 1 compounds is / V- (cyclopropyl)-2,2-diisopropyl butanamide.and the therapeutically effective amount is from about 2 to 50 mg per dose.

5. The composition as in claim 1 wherein the Formula 1 compounds is A / - (acetyl)-2,2-diisopropyl butanamide and the therapeutically effective amount is from about 2 to 50 mg per dose.

6. The composition as in claim 1 wherein the composition has from about 3% to 60% by weight of the Formula 1 compounds.

7. The composition as in claim 1 wherein the treatment of inflammation is the treatment of the reactions of vascularized tissues to localized injury.

8. The composition as in claim 1 wherein the inflammation being treated is dermatitis.

9. The composition as in claim 1 wherein the inflammation being treated are the symptoms of irritation, itch, soreness, burning sensations, and pain.

10. The composition as in claim 1 wherein the treatment of inflammation is treatment of swelling, redness, and loss of function of living tissues.11 . The composition as in claim 8 wherein the dermatitis is caused by diabetes mellitus.

12. The composition as in claim 8 wherein the dermatitis is caused by skin allergy.

13. The composition as in claim 8 wherein the dermatitis in need of treatment is the inflammatory response to insect bites.

14. The composition as in claim 1 wherein the inflammation in need of treatment is the dermatitis of acneiform eruptions, of insect stings, and of milaria rubra.

15. The composition as in claim 1 wherein the inflammation in need of treatment is the dermatitis of psoriasis, scalp psoriasis, an inflamed scalp, or the inflammation of seborrheic dermatitis.

16. The composition as in claim 1 wherein the inflammation in need of treatment is the inflammation of the itch scratch cycle with a plurality of doses being applied, wherein the Formula 1 compounds is N-methyl-2,2-diisopropyl- butanamide, N-meth-y2l,2-diisopropyl-butanamide, N-n-propyl-2, 2-diisopropyl- butanamide, or A / -isopropyl-2,2-diisopropyl-butanamide and the therapeutically effective amount is from about 10 to 50 mg per dose.

17. A therapeutic composition comprising a therapeutically effective amount of one or more compounds having Formula 2wherein R1 is ethyl, isopropyl, or n-propyl, and R2 is ethyl, isopropyl, n- propyl, cyclopropyl, or acetyl.

18. The composition as claimed in claim 17, further comprising 2 to 5% by weight of l-menthol.

19. A method for the treatment of the inflammation of a dermatological disorder in a mammal in need of treatment thereof, comprising: topically applying a liquid or semi-liquid composition to the mammal’s skin, the composition comprising a therapeutically effective amount of one or more compounds having Formula 1wherein R is methyl, ethyl, isopropyl, n-propyl,r cyclopropyl, or acetyl and a second cooling agent comprising menthol or a 1 -dialkylphosphorylalkane.

20. The method as claimed in claim 19, wherein the mammal is human.21 . The method as claimed in claim 19, wherein the mammal is canine.

22. The method as claimed in claim 21 , wherein the inflammation of a dermatological disorder is the inflammation of canine dermatitis.

23. The method as claimed in claim 21 , wherein the inflammation of a dermatological disorder is the inflammation of canine atopic dermatitis or canine atopic-like dermatitis.

24. Use of compound of formula 1 in preparation of a medicament for treating topical inflammation in a subject, said inflammation is the inflammation of skin,Formula 1 wherein R is methyl, ethyl, isopropyl, n-propyl, cyclopropyl, or acetyl.