WO2014043144A1

WO2014043144A1 - Methods for treating inflammation and pain

Info

Publication number: WO2014043144A1
Application number: PCT/US2013/059094
Authority: WO
Inventors: Joseph P. St. Laurent; Gerald S. Jones; David M. Bresse
Original assignee: Olatec Industries Llc
Priority date: 2012-09-11
Filing date: 2013-09-10
Publication date: 2014-03-20

Abstract

The present invention is directed to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an ω-(methanesulfonyl)alkenylnitrile compound, or a pharmaceutically acceptable salt thereof. The present invention is also directed to a method for treating inflammation, inflammatory-related disorders, or pain, by orally administering an ω-(methanesulfonyl)alkenylnitrile compound or a pharmaceutically acceptable salt or solvate thereof to a subject in need thereof.

Description

METHODS FOR TREATING INFLAMMATION AND PAIN

This application claims priority to U. S. Provisional Application No. 61/699,720, filed September 11, 2012; which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a pharmaceutical composition comprising a

pharmaceutically acceptable carrier and an ro-(methanesulfonyl)alkenylnitrile compound, or its pharmaceutically acceptable salts. The present invention also relates to methods of using the compound for treating inflammation or inflammatory-related disorders and pain.

BACKGROUND OF THE INVENTION

Inflammation is a process by which microbes or tissue injury induce the release of cytokines and chemokines from various cell types producing increased blood vessel permeability, upregulation of endothelial receptors, and thus increased egress of various cells of the innate and adaptive immune system which enter surrounding tissue and grossly produce the classical picture of inflammation, i.e. redness, swelling, heat and pain.

Inflammation is a localized reaction of live tissue due to an injury, which may be caused by various endogenous and exogenous factors. The exogenous factors include physical, chemical, and biological factors. The endogenous factors include inflammatory mediators, antigens, and antibodies. Endogenous factors often develop under the influence of an exogenous damage. An inflammatory reaction is often followed by an altered structure and penetrability of the cellular membrane. Endogenous factors, such as mediators and antigens define the nature and type of an inflammatory reaction, especially its course in the zone of injury. In the case where tissue damage is limited to the creation of mediators, an acute form of inflammation develops. If immunologic reactions are also involved in the process, through the interaction of antigens, antibodies, and autoantigens, a long-term inflammatory process will develop. Various exogenous agents, for example, infection, injury, radiation, also provide the course of

inflammatory process on a molecular level by damaging cellular membranes which initiate biochemical reactions.

Based on the physical causes, pain can be divided into three types: nociceptive, neuropathic, and mix -type.

Nociceptive pain is the term for pain that is detected by specialized sensory nerves called nociceptors. These nerves are located throughout the soft tissues, such as muscles and skin, as well as the internal organs. There are two types of nociceptive pain: somatic pain and visceral pain. Visceral pain comes from the internal organs. Deep somatic pain is initiated by stimulation of nociceptors in ligaments, tendons, bones, blood vessels, fasciae and muscles, and is dull, aching, poorly localized pain. Examples include sprains and broken bones. Superficial pain is initiated by activation of nociceptors in the skin or other superficial tissue, and is sharp, well- defined and clearly located. Examples of injuries that produce superficial somatic pain include minor wounds and minor (first degree) burns. Nociceptive pain is usually short in duration and end when the damage recovers. Examples of nociceptive pain include postoperative pain, sprains, bone fractures, burns, bumps, bruises, and inflammatory pain.

Neuropathic pain is pain caused by damage or disease that affects the somatosensory system. Neuropathic pain is originated from spontaneous ectopic neuron discharge in the nervous system either in central or in peripheral. Because the underlying etiologies are usually irreversible, most neuropathic pain are chronic pain. Most people describe neuropathic pain as shooting, burning, tingling, lancinating, electric shock qualities, numbness, and persistent allodynia. The nomenclature of neuropathic pain is based on the site of initiating nervous system with the etiology; for examples, central post-stroke pain, diabetes peripheral neuropathy, post- herpetic (or post-shingles) neuralgia, terminal cancer pain, phantom limb pain.

Mix -type pain is featured by the coexistence of both nociceptive and neuropathic pain. For example, muscle pain trigger central or peripheral neuron sensitization leading to chronic low back pain, migraine, and myofacial pain.

Connective tissues are subjected to a constant barrage of stress and injury. Acute or chronic impacts and the natural progression of various degenerative diseases all produce painful inflammation in joint regions, such as the neck, back, arms, hips, ankles and feet. These afflictions are common and often debilitating.

Current therapy is directed to some or all of the pathogenetic components of

inflammation. For example, corticosteroids have a broad spectrum of activities and NSAIDS are more specifically anti-prostaglandin and analgesic. All current therapies have relatively high rates of adverse effects and adverse effects are severe and serious. There is a need for a composition and a method for treating inflammation, inflammatory- related disorders, and pain. The composition should be economic and easy to manufacture, and the method should be effective and have no significant side effects. SUMMARY OF THE INVENTION

The present invention is directed to a pharmaceutical composition comprising a

pharmaceutically acceptable carrier and an ro-(methanesulfonyl)alkenylnitrile compound or a pharmaceutically acceptable salt or solvate thereof. The compound is at least 90% pure (w/w).

The present invention is also directed to a method for treating inflammation,

inflammatory-related disorders, and pain. The method comprises the step of administering an ω- (methanesulfonyl)alkenylnitrile compound or a pharmaceutically acceptable salt thereof to a subject in need thereof. The pharmaceutical composition comprising the active compound can be applied by any accepted mode of administration including oral and parenteral (such as intravenous, intramuscular, subcutaneous or rectal). Oral administration is preferred.

DETAILED DESCRIPTION OF THE INVENTION

Definition

"Alkyl" refers to groups of from 1 to 12 carbon atoms, either straight chained or branched, preferably from 1 to 8 carbon atoms, and more preferably 1 to 6 carbon atoms.

"Arylalkyl" refers to aryl -alkyl- groups preferably having from 1 to 6 carbon atoms in the alkyl moiety and from 6 to 10 carbon atoms in the aryl moiety. Such arylalkyl groups are exemplified by benzyl, phenethyl and the like.

"Cycloalkyl" refers to cyclic alkyl groups of from 3 to 12 carbon atoms having a single cyclic ring or multiple condensed rings which can be optionally substituted with from 1 to 3 alkyl groups. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl, 2- methylcyclooctyl, and the like, or multiple ring structures such as adamantyl, and the like.

"Pharmaceutically acceptable salts," as used herein, are salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects.

Pharmaceutically acceptable salt forms include various crystalline polymorphs as well as the amorphous form of the different salts. The pharmaceutically acceptable salts can be formed with metal or organic counterions and include, but are not limited to, alkali metal salts such as sodium or potassium; alkaline earth metal salts such as magnesium or calcium; and ammonium or tetraalkyl ammonium salts, i.e., NX₄+ (wherein X is C_1-4).

"Solvates," as used herein, are addition complexes in which the compound is combined with an acceptable co-solvent in some fixed proportion. Co-solvents include, but are not limited to, ethyl acetate, lauryl lactate, myristyl lactate, cetyl lactate, isopropyl myristate,

ethanol, 1-propanol, isopropanol, 1-butanol, isobutanol, tert-butanol, acetone, methyl ethyl ketone, and diethyl ether. The inventors have discovered that ro-(methanesulfonyl)alkenylnitriles are effective for treating inflammation, inflammatory-related disorders, and pain. ro-(Methanesulfonyl)alkenylnitriles

ro-(Methanesulfonyl)alkenylnitriles (methanesulfonylalkanenitriles) useful for the present invention are compounds of formula I (both E- and Z-isomers), or a pharmaceutically acceptably salt or solvate thereof:

Formula I

wherein Ri and R₂ are independently selected from the group consisting of H, straight-chain alkyl, branched alkyl, cycloalkyl, and arylalkyl, and halogen; and

n= 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; m= 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; and n + m = 0-9 . Preferably n + m = 0 - 4, more preferably n = 0 - 4 and m = 0.

The structures of (¾⁾-methanesulfonylacrylonitrile (1; n= m = 0; Ri = R₂ =H ; molecular weight MW= 131.15 ), (2 methanesulfonylacrylonitrile (2; n= m = 0; Ri = R₂ =H ; MW= 131.15 ), 2, 3-dichloro-3-methanesulfonylacrylonitrile (3; n=m=0; Ri = R₂ =C1 , MW=200.04), 4- methanesulfonylbut-2-enenitrile (4; n= 1, m = 0; Ri = R₂ =H, MW=145.18), 5- methanesulfonylpent-2-enenitrile (5; n= 2, m = 0; Ri = R₂ =H, MW=159.21), 6- methanesulfonylhex-2-enenitrile (6; n= 3, m = 0; Ri = R₂ =H, MW=173.23), and 7- methanesulfonylhept-2-enenitrile (7; n= 4, m = 0; Ri = R₂ =H, MW= 187.26) are shown below.

(¾⁾-Methanesulfonylacrylonitrile (1) can be prepared according to U.S. Patent No.

3,541,119. For example, the compound can be prepared by alkylating sodium methanesulfinate with 2,3-dichloropropionitrile in the presence of a base.

f¾⁾-Methanesulfonylacrylonitrile (2) can be prepared by modifying the procedure of Asscher and Vofsi (J. Chem. Soc. 1964, 4962-4971). For example, the compound can be prepared by a copper-catalyzed addition of methanesulfonyl chloride to acrylonitrile.

2, 3-dichloro -3-methanesulfonylacrylonitrile (3) can be prepared according to U.S. Patent No. 4,424,167. For example, the compound can be prepared from 3-methylsulfonylpriopionitrile by a series of chlorination/dehydrohalogenation reactions using chlorine gas in the presence of a basic catalyst.

ro-Methanesulfonylalk-2-enenitriles (e.g. Compounds 4, 5, 6, and 7) can be prepared by alkylating sodium methanesulfinate with requisite ro-halolalk-2-enenitriles, or by alkylating sodium thiomethoxide with the requisite ro-halolalk-2-enenitriles, followed by oxidation of the products with hydrogen peroxide or other appropriate oxidizing agent such as urea hydrogen peroxide.

Pharmaceutical Compositions

The present invention provides pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers and an active compound (an ω-

(methanesulfonyl)alkenylnitrile), or a pharmaceutically acceptable salt, or solvate thereof. The active compound or its pharmaceutically acceptable salt or solvate in the pharmaceutical compositions in general is about 0.1-5% for an injectable formulation, about 1-90% for a tablet formulation, and 1-100% for a capsule formulation.

In one embodiment, the pharmaceutical composition can be in a dosage form such as tablets, capsules, granules, fine granules, powders, solutions, suppositories, injectable solutions, or the like. The above pharmaceutical composition can be prepared by conventional methods.

Pharmaceutically acceptable carriers, which are inactive ingredients, can be selected by those skilled in the art using conventional criteria. The pharmaceutically acceptable carriers may contain ingredients that include, but are not limited to, saline and aqueous electrolyte solutions; ionic and nonionic osmotic agents such as sodium chloride, potassium chloride, glycerol, and dextrose; pH adjusters and buffers such as salts of hydroxide, phosphate, citrate, acetate, borate; and trolamine; antioxidants such as salts, acids and/or bases of bisulfite, sulfite, metabisulfite, thiosulfite, ascorbic acid, acetyl cysteine, cystein, glutathione, butylated hydroxyanisole, butylated hydroxytoluene, tocopherols, and ascorbyl palmitate; surfactants such as lecithin, phospholipids, including but not limited to phosphatidylcholine, phosphatidylethanolamine and phosphatidyl inositiol; poloxamers and ploxamines, polysorbates such as polysorbate 80, polysorbate 60, and polysorbate 20, polyethers such as polyethylene glycols and polypropylene glycols; polyvinyls such as polyvinyl alcohol and povidone; cellulose derivatives such as methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose and hydroxypropyl methylcellulose and their salts; petroleum derivatives such as mineral oil and white petrolatum; fats such as lanolin, peanut oil, palm oil, soybean oil; mono-, di-, and triglycerides; polysaccharides such as dextrans and glycosaminoglycans such as sodium hyaluronate. Such pharmaceutically acceptable carriers may be preserved against bacterial contamination using well-known preservatives, these include, but are not limited to,

benzalkonium chloride, ethylene diaminetetraacetic acid and its salts, benzethonium chloride, chlorhexidine, chlorobutanol, methylparaben, thimerosal, and phenylethyl alcohol, or may be formulated as a non-preserved formulation for either single or multiple use.

For example, a tablet formulation or a capsule formulation of the active compound may contain other excipients that have no bioactivity and no reaction with the active compound.

Excipients of a tablet or a capsule may include fillers, binders, lubricants and glidants,

disintegrators, wetting agents, and release rate modifiers. Binders promote the adhesion of particles of the formulation and are important for a tablet formulation. Examples of excipients of a tablet or a capsule include, but not limited to, carboxymethylcellulose, cellulose, ethylcellulose,

hydroxypropylmethylcellulose, methylcellulose, karaya gum, starch, tragacanth gum, gelatin, magnesium stearate, titanium dioxide , poly(acrylic acid), and polyvinylpyrrolidone. For example, a tablet formulation may contain inactive ingredients such as colloidal silicon dioxide,

crospovidone, hypromellose, magnesium stearate, microcrystalline cellulose, polyethylene glycol, sodium starch glycolate, and titanium dioxide. A capsule formulation may contain inactive ingredients such as gelatin, magnesium stearate, and titanium dioxide. Method of Use

Inflammation is a process and a state of tissue pathology resulting from activation and continuation of activity of the innate and acquired components of the immune system. The arachidonic acid cascade and cytokine production and action in cell to cell interactions are critical components of immune activation and response, which lead to inflammation.

Arachidonic acid is a component of membrane phospholipids. After it is freed from

phospholipids, arachidonic acid acts as a precursor to many of the known eicosinoids including prostaglandins and leucotrienes, which are known pro-inflammatory entities.

The active compounds are effective in inhibiting pro-inflammatory cytokine release (e.g., IL-Ιβ, IL-6, TNFa, IL-4 and IFNy) from human peripheral blood mononuclear cells in vitro. The active compound is anti-inflammatory when administered orally in a mouse ear swelling model, in which the inflammation is induced by arachidonic acid.

The present invention is directed to a method of treating inflammation and/or pain. The active compound can be used as is, or it can be administered in the form of a pharmaceutical composition that additionally contains a pharmaceutically acceptable carrier. The method comprises the steps of first identifying a subject suffering from inflammation and/or pain, and administering to the subject the active compound, in an amount effective to treat inflammation and/or pain. "An effective amount," as used herein, is the amount effective to treat a disease by ameliorating the pathological condition or reducing the symptoms of the disease.

In one embodiment, the method reduces or alleviates the symptoms associated with inflammation. The present invention provides a method to treat localized manifestations of inflammation characterized by acute or chronic swelling, pain, redness, increased temperature, or loss of function in some cases.

In another embodiment, the present invention provides a method to alleviate the symptoms of pain regardless of the cause of the pain. The general term "pain" treatable by the present method includes traumatic pain, neuropathic pain, organ pain, and pain associated with diseases. Traumatic pain includes pain resulting from injury, post-surgical pain and

inflammatory pain. Neuropathic pain includes neuropathic and idiopathic pain syndromes, and pain associated with neuropathy such as diabetic neuropathy, causalgia, brachial plexus avulsion, occipital neuralgia, fibromyalgia, gout, and other forms of neuralgia. Organ pain includes ocular, corneal, bone, heart, skin/burn, visceral (kidney, gall bladder, etc.), joint, and muscle pain. Pain associated with diseases includes pain associated with cancer, AIDS, arthritis, herpes and migraine. The present invention reduces pain of varying severity, i.e. mild, moderate and severe pain; acute and chronic pain. The present invention is effective in treating joint pain, muscle pain, tendon pain, and burn pain.

In preferred embodiments, the present invention is useful in treating inflammation and/or pain associated in a musculoskeletal system or on the skin. The highly innervated, musculoskeletal and skin systems have a high capacity for demonstration of pain. In addition, the musculoskeletal system has a high capacity for tissue swelling, and the skin has a high capacity for redness, swelling, and heat. In musculoskeletal and skin systems, the degree of tissue damage is frequently magnified out of proportion to the resulting inflammatory response. In the skin for example, merely firm stroking will cause release of the cytokines, IL-1 and TNF.

The present invention provides a method for treating inflammation and/or pain associated with inflammatory skeletal or muscular diseases or conditions. The method comprises the steps of identifying a subject in need thereof, and administering to the subject the active compound, in an amount effective to treat inflammation and/or pain. The skeletal or muscular diseases or conditions include musculoskeletal sprains, musculoskeletal strains, tendonopathy, peripheral radiculopathy, rheumatoid arthritis, polymyalgia rheumatica, juvenile arthritis, gout, ankylosing spondylitis, psoriatic arthritis, systemic lupus erythematosus, costochondritis, tendonitis, bursitis, such as the common lateral epicondylitis (tennis elbow), medial epicondylitis (pitchers elbow) and trochanteric bursitis, temporomandibular joint syndrome, and fibromyalgia.

The present invention provides a method for treating inflammation and/or pain associated with inflammatory skin diseases such as dermatitis, psoriasis, and acne. The method comprises the steps of identifying a subject in need thereof, and administering to the subject the active compound, in an amount effective to treat inflammation and/or pain.

ro-(Methanesulfonyl)alkenylnitriles, which are effective in inhibiting arachidonic acid induced inflammation and in inhibiting the release of pro-inflammatory cytokine, are effective to treat inflammation and/or pain associated with psoriasis, acne, and dermatitis, particularly contact dermatitis, and atopic dermatitis.

The pharmaceutical composition of the present invention can be applied by systemic administration such as oral, parenteral (such as intravenous, intramuscular, subcutaneous or rectal), and other systemic routes of administration. In systemic administration, the active compound first reaches plasma and then distributes into target tissues. Oral administration is a preferred route of administration for the present invention.

Dosing of the composition can vary based on the extent of the injury and each patient's individual response. For systemic administration, plasma concentrations of active compounds delivered can vary; but are generally Ixl0^~10-lxl0^~4 moles/liter, and preferably Ixl0^~8-lxl0^~5 moles/liter.

In one embodiment, the pharmaceutical composition is administrated orally to the subject. The dosage for oral administration is generally 1-50, and preferably 1-10, or 1-5 mg/kg/day. For example, the active compound can be applied orally to an adult human at 100-800

mg/dosage, or 200-600 mg/dosage, 1-4 times a day, depends on the patient's condition.

In one embodiment, the pharmaceutical composition is administrated subcutaneously to the subject. The dosage for subcutaneous administration is generally 0.3-20, and preferably 0.3-3 mg/kg/day.

Those of skill in the art will recognize that a wide variety of delivery mechanisms are also suitable for the present invention.

The present invention is useful in treating a mammal subject, such as humans, horses, and dogs. The present invention is particularly useful in treating humans.

The following examples further illustrate the present invention. These examples are intended merely to be illustrative of the present invention and are not to be construed as being limiting. EXAMPLES

Example 1. Preparation of 3-(methylsulfonyl)acrylonitrile

A methanolic solution of 2,3-dichloropropionitrile (60 mmol, a) was added dropwise to a mixture of sodium methanesulfmate (1 equivalent) in aqueous methanol containing sodium acetate (2 equivalent). The reaction was monitored by TLC. After being stirred for about 1.5 hours, the reaction mixture was diluted with water and extracted with CHC1₃. The CHCI₃ extract was washed with water, treated with charcoal and concentrated. The residue was crystallized from hot ethanol. The ield of 3-(methylsulfonyl)acrylonitrile (b) was 1.12 g (14%).

The product had a white, crystalline appearance. The melting point was 106.5-107.5°C. The results of FTIR-ATR, 1H NMR, ¹³C NMR analyses were consistent the structure of b. The elemental analysis found C 36.57, H 3.84, and N 10.70. The GC-MS study showed m/z of 132. Example 2. In Vitro Inhibition of Cytokine Activities by 3-(methylsulfonyl)acrylonitrile Objective: Test 3-(methylsulfonyl)acrylonitrile for inhibition of cytokine secretion in blood mononuclear cells (PBMCs) stimulated with lipopolysaccharide (LPS).

Cytokines: IL-Ιβ, IL-6, TNFa

Test Compound Dose Concentrations: 50, 10, 5, 1, 0.5, 0.1 μΜ

Controls

Vehicle: DMSO, 0.1% v/v

LPS control: LPS + Vehicle

Cells negative control : Vehicle only / No LPS

Positive control: Dexamethasone (100 nM, n=2)

Protocols:

1. Microtiter plates were seeded with lx 10⁴ PBMCs/well in 150μί culture medium (RPMI 1640, 10% FBS, 1% Pen/Strep, 2 mM L-Alanyl-L-glutamine) 2. The plates were incubated at 37°C in 5% C0₂ for 1 hour.

3. 10 of test compound (at different concentrations), dexamethasone, and vehicle controls were added to appropriate wells, and the plates were incubated at 37°C in 5% C0₂ for 1 hour.

4. 40μΙ, of LPS (50 pg/mL final) were added and the plates were incubated at 37°C in 5% C0₂ for 1 hour.

5. The plates were centrifuged at 1000 rpm for 10 minutes and supernatants were collected.

6. The supernatants were assayed for IL-Ι β, IL-6, TNFa levels using the Luminex Bead kit.

7. EC50 values were determined. Results:

The EC50's of 3-(methylsulfonyl)acrylonitrile for inhibiting IL-Ι β, IL-6, and TNFa were 0.16 μΜ, 0.19 μΜ, and 0.30 μΜ respectively. The results demonstrate that the active compound has an inhibitory effect on cytokines involved in the inflammatory process. Example 3. Anti-inflammatory Activity of Oral Administration of 3- (methylsulfonyl)acrylonitrile in Mice

3-(Methylsulfonyl)acrylonitrile was suspended in vehicle (1% Tween 80 in water) to 0.1-3 mg/mL. The test compound, dexamethasone (positive control), and vehicle were orally administered to mice and evaluated for anti-inflammatory activity in the topical arachidonic acid induced ear swelling model in mice.

Male ICR derived mice weighing 22 ± 2 g were used in this experiment. 5 mice were used for each group (active compound, positive control, and vehicle). All animals were maintained in a controlled temperature (22-24°C) and humidity (60% - 70%) environment with 12-hour light/dark cycles for at least one week prior to use.

Arachidonic acid (0.5 mg in 20 μΐ_^ acetone) was applied topically onto the anterior and posterior surfaces of the right ear of test animals to induce inflammation. Active compound of different concentrations in vehicle (10 mL/kg) and vehicle (10 mL/kg) were orally administered by gavage 1 hour before arachidonic acid, whereas dexamethasone (0.3 mg/kg) was orally administered by gavage 3 hour before arachidonic acid challenge. At 60 minutes after arachidonic acid induction of ear edema, the thickness of the right ear and the left ear was measured and the difference calculated as an indication of the inflammation in the right ear. Significant activity was defined as a statistically significant inhibition (p-value determined by t- test was < 0.05) in arachidonic acid induced ear swelling relative to the vehicle-treated group. The results are shown in Table 1. Oral administration of 3-(methylsulfonyl)acrylonitrile at dosage of 10 mg/kg caused significant inhibition in arachidonic acid induced ear swelling at 60 minutes.

Table 1

Example 4. Inhibition of Cytokine Activities (Prophetic Example)

Active compounds ro-(methanesulfonyl)alkenylnitriles are tested for their inhibitory effects on in vitro cytokine release from human peripheral blood mononuclear cells (PBMCs). Secretion of cytokines by PBMCs plays a significant role in the inflammatory response.

Each active compound is added to cultures of fresh human PBMCs at 162 μΜ (22 μg/mL) in duplicate. One hour later, PBMCs are stimulated to secrete cytokines using the mitogens lipopolysaccharide and concanavalin A (ConA). Lipopolysaccharide at 50pg/mL is used to stimulate the release of interleukin IL-Ιβ, IL-6 and tumor necrosis factor TNFa. ConA at 20 μg/mL is used to stimulate the release of IL-4 and ConA at 5 μg/mL is used to stimulate interferon IFNy. The corticosteroid dexamethasone (100 nM) is used as a positive control. After 24 hours of incubation, the supernatants are assayed for the cytokines using the Luminex Bead kit. The percent inhibition of IL-Ιβ, IL-6, TNFa, IL-4 and IFNy by the active compounds and the positive compound are calculated. Example 5. Anti-inflammatory Activity of Oral Administration of ω- (methanesulfonyl)alkenylnitriles in Mice (Prophetic Example)

Active compounds of ro-(methanesulfonyl)alkenylnitrile are suspended in vehicle (1% Tween 80 in water) to 0.5-10 mg/mL. The test compounds, dexamethasone (positive control), and vehicle are orally administered to mice and evaluated for anti-inflammatory activity in the arachidonic acid induced ear swelling model in mice.

Male ICR derived mice are used in this experiment. 5 mice are used for each group (active compounds, positive control, and vehicle). All animals are maintained in a controlled temperature (22-24°C) and humidity (60% - 70%) environment with 12-hour light/dark cycles for at least one week prior to use.

Arachidonic acid (0.5 mg in 20 acetone) is applied topically onto the anterior and posterior surfaces of the right ear of test animals to induce inflammation. Each active compound in vehicle (10 mL/kg) and vehicle (10 mL/kg) is orally administered by gavage 1 hour before arachidonic acid, whereas dexamethasone (0.3 mg/kg) is orally administered by gavage 3 hour before arachidonic acid challenge. At 60 minutes after arachidonic acid induction of ear edema, the thickness of the right ear and the left ear of each mouse is measured and the difference calculated as an indication of the inflammation in the right ear. Significant activity is defined as a statistically significant inhibition (p-value determined by t-test was < 0.05) in arachidonic acid induced ear swelling relative to the vehicle-treated group.

Example 6. Systemic Exposure of Oral and Subcutaneous Administration of ω- (methanesulfonyl)alkenylnitrile Formulations in Rats (Prophetic Example)

This study is done to determine the systemic (plasma) exposure of ω- (methanesulfonyl)alkenylnitriles after administration by the oral and subcutaneous routes to rats. ro-(Methanesulfonyl)alkenylnitriles are prepared in water containing Tween 80 for oral administration and in saline for subcutaneous administration. Rats are used in the study. Rats are given a single dose at 1-25 mg/kg by either oral or subcutaneous route. The blood is drawn from each rat at 0.25, 1, 2, 3, 4, 6, 12, 24, and 48 hours and measured for ω- (methanesulfonyl)alkenylnitrile concentrations by GC/MS/MS.

The average maximum plasma concentrations measured (Cmax) after oral dosing and after subcutaneous are determined. The above results demonstrate that significant bioavailability of ro-(methanesulfonyl)alkenylnitriles after both the oral and subcutaneous routes.

Example 7. Anti-inflammatory and Analgesic Activity of the Active Compound by Oral Administration in a Carrageenan Model (Prophetic Example)

Active compounds of ro-(methanesulfonyl)alkenylnitrile such as 3-

(methylsulfonyl)acrylonitrile are suspended in vehicle (1% Tween 80 in water) to 0.5-10 mg/mL. Active compounds, indomethacin (positive control), and vehicle (1% Tween 80 in water) are orally administered and evaluated for anti-inflammatory and analgesic activity in the rat carrageenan-induced paw inflammation model.

Rats are used in the experiment. Carrageenan (0.1 mL of a 1% suspension) is injected subcutaneously into the left hind paw to induce inflammation. Active compounds in vehicle (10 mL/kg) and vehicle are orally administered 1-2 hours before the carrageenan administration. Indomethacin is given orally at 5 mg/kg, 1 hour prior to carrageenan administration. The degree of inflammation (edema, or swelling) is determined using a plethysmograph to measure paw volume. Analgesia is determined by measuring paw withdrawal to a mechanical stimulus using von Frey filaments. Inflammation and analgesia are measured 4 hours after carrageenan administration. ro-(methanesulfonyl)alkenylnitriles are expected to have anti-inflammatory and/or analgesic properties as measured by a significant decrease in paw volume and/or a significant increase in mechanical pressure needed to elicit paw withdrawal, respectively, as compared to the vehicle control.

Example 8. Analgesic activity of the Active Compound by Oral Administration in a Hot Plate Model (Prophetic Example)

Active compounds of ro-(methanesulfonyl)alkenylnitrile such as 3- (methylsulfonyl)acrylonitrile are suspended in vehicle (1% Tween 80 in water) to 0.5-10 mg/mL. Active compound, morphine (positive control), and vehicle (1% Tween 80 in water) are orally administered and evaluated for analgesic activity in the rat hot plate model.

Rats are used in the experiment. Active compounds in vehicle (10 mL/kg) and vehicle (1% Tween 80 in water) are orally administered 1-2 hours before the rat is placed on a 55°C hot plate, and the time to lick the paw is measured. The positive control, morphine, is given orally at 30 mg/kg, 1 hour prior to hot plate testing. ro-(Methanesulfonyl)alkenylnitriles are expected to have analgesic properties as measured by a significant increase in time to licking as compared to the vehicle control (t-test, p<0.05).

Example 9. Analgesic Activity of Active Compounds by Oral Administration in CFA- Induced Thermal Hyperalgesia (Prophetic Example)

CFA (Complete Freund's Adjuvant) is known to induce inflammatory pain. (Walker, et al. JPET. 304: 56-62, 2003.)

Male Sprague-Dawley rats weighing 180 ± 20 g are used. The animals, divided into groups of 8-10 each, receive a subplantar injection (0.1 ml) of CFA (0.1% solution) to the tested hindpaw at 24 hours prior to experimentation. Thermal hyperalgesia is tested by using the IITC Model-336G (IITC INC.USA) apparatus with a thermally regulated glass floors set at 30°C. Each rat is placed within a plastic box atop a glass floor. A light beam under the floor is aimed at the plantar surface of the right hind paw. The time is measured automatically when the paw is withdrawn away from the thermal stimulus. The intensity of the light is adjusted with average group baseline latency from 12 to 14 sec (pre-CFA) and a cut-off latency of 20 sec imposed. The latency to withdrawal is obtained for each rat and defined as the heat pain threshold. Twenty four hours after CFA injection, rats are pre-selected (with clear presence of thermal hyperalgesia) for experimentation only if the latency to withdrawal is less than 75% of baseline.

Active compounds of ro-(methanesulfonyl)alkenylnitrile such as 3- (methylsulfonyl)acrylonitrile are suspended in vehicle (1% Tween 80 in water) to 0.5-10 mg/mL. Active compound, morphine (positive control, 20 mg/kg), and vehicle (1%> Tween 80 in water) are orally administered and evaluated for analgesic activity in the rat hot plate model.

Test substance, positive control or vehicle is administered orally (10 mL/kg) to the rats at 60 minutes before the level of thermal hyperalgesia is again measured (post-treatment). Mean ± SEM of thermal paw withdrawal time is calculated. Unpaired Student's t test is applied for comparison the values of post-treatment between test substance treated group and vehicle control group. Positive activity is considered at P<0.05. Example 10. Analgesic Activity of Active Compounds by Oral Administration in a

Formalin Test (Prophetic Example)

Formalin test is a model of continuous pain resulting from formalin-induced tissue injury. The formalin model encompasses inflammatory, neurogenic, and central mechanism of nociception. The assay described below relates primarily to the late inflammatory algesic phase sensitive to both strong central analgesic as well as weaker analgesic/anti-inflammatory agents (Hunskaar, et al, J. Neuroscience Meth. 14: 69-76, 1985). The formalin test represents a suitable model for testing compounds for treating neuropathic pain (Benson, et al. Proceedings of Measuring Behavior, 2008, Eds. Spink, et al, 324-325).

Active compounds of ro-(methanesulfonyl)alkenylnitrile such as 3-

(methylsulfonyl)acrylonitrile are suspended in vehicle (1% Tween 80 in water) to 0.5-10 mg/mL. Active compound, morphine (positive control, 30 mg/kg), and vehicle (1% Tween 80 in water) are orally administered and evaluated for analgesic activity in the formalin model.

Test substance (10 mL/kg), positive control or vehicle is administered orally to groups of 8-10 CD-I derived male mice weighing 23±3 g one hour before subplantar injection of formalin (0.02 ml, 2% solution). Reduction of the induced hind paw licking time recorded during the following 10 to 30 minute period by 50% or more indicates analgesic activity. MS AN is expected to have analgesic properties as measured by a significant increase in time to licking as compared to the vehicle control (t-test, p<0.05).

Example 11. Analgesic Activity of Active Compounds by Oral Administration in Chronic Constriction Injury Model (Prophetic Example)

Peripheral nerve lesions may generate a syndrome comprising, in addition to spontaneous pain, exaggerated responses to light touch (tactile allodynia). Chronic constriction injury model is a neuropathic pain model.

Male Sprague Dawley rats weighing 180 ± 20 g are used. Under pentobarbital (50 mg/kg, 5 ml/kg, i.p.) anesthesia, the sciatic nerve is exposed at mid-thigh level. Four ligatures (4-0 chromic gut), about 1 mm apart, are loosely tied around the nerve. The animals are then housed individually in cages with soft bedding for 7 days before testing. Constriction of the sciatic nerve produces nerve injury and unilateral neuropathic pain.

On the day of experiments, the animals have no access to food overnight before testing. The rats are placed under inverted plexiglass cages on a wire mesh rack and allowed to acclimate for 20 to 30 minutes. Mechanic allodynia is evaluated by the Chaplan up/down method using von Frey filaments to the plantar surface of the left hind paw. See Chaplan, et al. J. Neuroscience Methods, 53: 55-63, 1994.

Rats are pre-selected for experimentation only if the pain threshold 7-14 days after nerve ligation (pre-treatment) is reduced by 10 grams of force relative to the response of the individual paw before nerve ligation (pre-ligation), namely, with clear presence of allodynia.

Active compounds of ro-(methanesulfonyl)alkenylnitrile such as 3- (methylsulfonyl)acrylonitrile are suspended in vehicle (1% Tween 80 in water) to 0.5-10 mg/mL. Active compound, morphine (positive control, 30 mg/kg), and vehicle (1% Tween 80 in water) are orally administered and evaluated for analgesic activity.

Test substance (10 mL/kg), positive control or vehicle is administered orally to rats. The mechanical allodynia test is performed 30 min before (pre-treatment) and 1 and 3 hours after a single dose of test substance or vehicle (post treatment). Paw withdraw thresholds of control and tested compounds are measured.

Example 12. Treatment of Knee Pain (Prophetic Example)

Objectives: To investigate the efficacy of the active compound by oral administration to patients with mild to moderate knee pain associated with osteoarthritis following temporary cessation of standard NSAID therapy.

Formulation: Capsules or tablets each containing 100-600 mg of an active compound ω- (methanesulfonyl)alkenylnitrile such as 3-(methylsulfonyl)acrylonitrile are used in this example. Placebo capsules or tablets do not contain the active compound.

Methodology: A randomized, double -blind, placebo controlled, parallel treatment multicenter clinical activity study.

Patients with painful osteoarthritis of the knee, controlled by a stable dose of standard NSAID therapy for at least 2 months, discontinue use of the NSAIDs for a 7-day washout period. Patients are enrolled and treated for 7 days with follow-up at 8, 10, 14 and 21 days.

The capsules or tablets are orally administered to patients 1-4 times a day for 7 days. Patients are treated for 7 days and followed up for a further 14 days.

Criteria for Evaluation:

Safety:

Adverse Events (AEs) throughout the study.

• Physical examination at enrollment (-7 days, start of NSAID washout period), Baseline (Day 1, start of treatment), Day 10 and Day 21.

• Vital signs at enrollment (-7 days, start of NSAID washout period), Baseline (Day 1, start of treatment) and Days 2, 4, 8, 10, 14 and 21.

• Clinical laboratory measurements at Baseline (Day 1), Day 8 and Day 14.

Clinical Activity:

The primary clinical activity parameters are the measurement of pain at the site of application, as quantified by VAS and the Western Ontario and McMaster University (WOMAC) scale. The effect of treatment on swelling, tenderness and inflammation of the knee is recorded, also the time to reduction or eradication of pain after treatment is recorded.

Study Endpoints:

The primary clinical activity endpoints are:

• Change from Baseline (Day 1) to Day 8 in WOMAC functional disability index:

Pain (Scale 0 - 20).

Stiffness (Scale 0 - 8).

Physical function (Scale 0 - 68).

Change from Baseline (Day 1) to Day 8 in VAS pain scale (1 - 100). The secondary clinical activity endpoints are:

• Within-day change in VAS pain scale on Day 2 and Day 3 as measured by change from daily Baseline (Pre-Treatment 1) to 30 minutes Post Treatment 2.

• Change in investigator evaluation of swelling, tenderness and inflammation between Baseline (Day 1) and 30 minutes and 60 minutes after the first application on Day 1.

• Change in investigator evaluation of swelling, tenderness and inflammation between Baseline (Day 1) and Day 8.

• Time to reduction or eradication of pain subsequent to each application of active compound.

• Use of rescue medication (APAP).

The invention, and the manner and process of making and using it, are now described in such full, clear, concise and exact terms as to enable any person skilled in the art to which it pertains, to make and use the same. It is to be understood that the foregoing describes preferred embodiments of the present invention and that modifications may be made therein without departing from the scope of the present invention as set forth in the claims. To particularly point out and distinctly claim the subject matter regarded as invention, the following claims conclude the specification.

Claims

WHAT IS CLAIMED IS:

1. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of Formula I, or a pharmaceutically acceptable salt thereof;

Formula I

wherein Ri and R₂ are independently selected from the group consisting of H, straight-chain Ci_₆alkyl, branched Ci_₆alkyl, and halogen;

n= 0-4;

m= 0.

2. The pharmaceutical composition according to Claim 1, wherein the compound has at least 90% (w/w) purity, and the composition is in an oral form of tablets or capsules.

3. The pharmaceutical composition according to Claim 1, wherein Ri and R₂ are

independently H or CI.

4. The pharmaceutical composition according to Claim 1, wherein the compound is methanesulfonylacrylonitrile.

5. The pharmaceutical composition according to Claim 4, wherein the compound has at least 90% (w/w) purity, and the composition is in an oral form of tablets or capsules.

6. The pharmaceutical composition according to Claim 2 or 5, for treating inflammation or pain in a subject by oral administration.

7. The pharmaceutical composition according to Claim 6, for reducing or alleviating the symptoms of localized manifestations of inflammation characterized by acute or chronic swelling, pain, or redness.

8. The pharmaceutical composition according to Claim 7, wherein said inflammation and/or pain is associated with a skeletal or muscular disease or condition selected from the group consisting of: musculoskeletal sprains, musculoskeletal strains, tendonopathy, peripheral radiculopathy, osteoarthritis, degenerative joint disease, juvenile arthritis, gout, ankylosing spondylitis, psoriatic arthritis, system lupus erythematosus, costochondritis, tendonitis, bursitis, temporomandibular joint syndrome, and fibromyalgia.

9. The pharmaceutical composition according to Claim 7, wherein said inflammation and/or pain is associated with joints, ligaments, tendons, bone, muscles, or fascia.

10. The pharmaceutical composition according to Claim 7, wherein said inflammation and/or pain is associated with dermatitis or psoriasis.

11. The pharmaceutical composition according to Claim 13, wherein said dermatitis is atopic dermatitis or contact dermatitis.