TERMINALLY UNSATURATED VANILLOIDS AND MEDICINAL APPLICATION THEREOF

FIELD OF THE INVENTION

The present invention refers to terminally unsaturated vanilloids and their use for the manufacturing of composition to treat a capsaicin-responsive condition. BACKGROUND OF THE INVENTION

Itch originating in the skin is considered pruritoceptive and can be induced by a variety of stimuli, including mechanical, chemical, thermal, and electrical stimulation. The primary afferent neurons responsible for histamine induced itch are unmyelinated C-fibers. In human C-fiber nociceptors, two major classes exist: mechano-responsive nociceptors and mechano-insensitive nociceptors.

Mechano-responsive nociceptors have been shown in studies to respond to mostly pain, whilst mechano-insensitive receptors respond mostly to itch induced by histamine. However it does not explain mechanically-induced itch or when itch is produced without a flare reaction which involves no histamine. It is also possible that pruritoceptive nerve fibers have different classes of fibers.

Itch receptors are only found on the top two skin layers, the epidermis and the epidermal/dermal transition layers. Shelley and Arthur had verified the depth by injecting individual itch powder spicules (Muchuna puriens) and found that maximal sensitivity was found at the basal cell layer or the innermost layer of the epidermis. Surgical removal of those skin layers removed the ability for a patient to perceive itch. Itch is never felt in muscle, joints, or inner organs, which show that deep tissue does not contain itch signaling apparatuses.

Sensitivity to puritic stimuli is not even across the skin and has a random spot distribution with similar density to that of pain. The same substances that elicit itch upon intracutaneous injection (injection within the skin) elicit only pain when injected subcutaneously (beneath the skin). Itch is readily abolished in skin areas treated with nociceptor excitotoxin capsaicin but remains unchanged in skin areas rendered touch-insensitive when pretreated with saponins, an anti-inflammatory agent. Although experimentally induced itch can still be perceived under a complete

A-fiber conduction block, it is significantly diminished. Overall, itch sensation is mediated by A-delta and C nociceptors located in the uppermost layer of the skin.

Neuropathic itch can originate at any point along the afferent pathway as a result of damage of the nervous system. They could include diseases or disorders in the central nervous system or peripheral nervous system. Examples of neuropathic itch in origin are nostalgia paresthetica, brachioradial pruritus, brain tumors, multiple sclerosis, peripheral neuropathy, and nerve irritation.

Neurogenic itch is induced centrally but with no neural damage, being often associated with accumulation of endogenous opioids and possibly synethetic opioids.

Itch is also associated with some psychiatric disorders such as delusions of parasitosis or related obsessive-compulsive disorders, e.g. neurotic scratching.

The sensation of itch can be reduced by many painful sensations, such as the most commonly known one which is scratching. Many recent studies have shown that itch can be inhibited by other forms of painful stimuli, such as noxious heat, physical rubbing/scratching, chemically induced, electrical stimulation, and genetic differences. Any stimuli that caused pain will effectively show that itch was inhibited in that occurrence.

Inflammatory mediators such as bradykinin, serotonin (5-HT) and prostaglandins, released during a painful or pruritic inflammatory condition not only activates pruriceptors but also causes acute sensitization of the nociceptors. In addition, expression of neuro growth factors (NGF) can cause structural changes in of nociceptors such as sprouting. NGF is high in injured or inflamed tissue. Increased NGF is also found in atopic dermatitis, a hereditary and non-contagious skin disease with chronic inflammation.

NGF is known to up-regulate neuropeptides, especially substance P. Substance P has been found to have an important role in inducing pain however there is no confirmation that substance P directly causes acute sensitization. Instead substance P may contribute to itch by increasing neuronal sensitization and may the affect release of mast cells, which contain many granules rich in histamine, during long-term interaction.

Capsaicin is a pungent compound derived from Capsicum spp., of the Solanaceae family. It has long been used because of its selective action on the small diameter afferent nerve fibers C-fibers and A-delta fibers responsible for the pain signals.

Capsaicin, 8-methyl-6(E)-nonenoic acid vanillylamide, is synonym of capsaicinoids since it represents the entity recognized by the FDA, being one of the most potent beside being the predominant chemical entity in natural capsaicinoids.

Capsacin is effective in managing painful conditions such as rheuma/osteoarthritis, diabetic neuropathy, postherpetic neuralgia, postmastectomy pain syndrome, cluster headache, reflex sympathetic dystrophy, anal pruritus, vulvar vestibulitis syndrome.

Capsaicin and the structurally related vanilloids thought to exert their actions via the vanilloid receptor which is coupled to a non-specific cation channel. Capsaicin itself, the primary pungent principle of capsicums, is selectively stimulating sensory nerve endings to cause pain, and evoking an inflammatory reaction. Capsaicin activates the vanilloid receptor- 1 (VRl, or transient receptor potential vanilloid- 1 receptor, TRPVl). TRPVl was originally described on sensory neurons as a central integrator of various nociceptive stimuli.

However, several human skin cell populations are also now recognized to express TRPVl. Cultured human keratinocytes also expressed functional TRPVl, whose stimulation inhibited proliferation, induced apoptosis, elevated intracellular Ca concentration, up-regulated interleukin-lβ, transforming growth factor-β ₂ .

This support the physio-logical importance of TRPVl in human skin beyond nociception, and identify TRPVl as a promising novel target for manipulations of epithelial growth disorders.

Recent studies (Bodό et all, Am. J. Path., 2005, 166, 4, 985-998) highlights that TRPVl signaling is a major, newly recognized player in epithelial biology in general, both by its direct effect on epithelial tissues (eg, epidermal and keratinocytes) and its complex indirect effects on neuroectodermal-mesenchymal interaction e.g., via the modification of neuropeptide release from sensory skin nerves and of skin mast cell activation. This invites to systemically explore how antiproliferative TRPVl signalling can be manipulated by endogenous and exogenous ligands to treat

hyperproliferative epithelial growth disorders of the skin, e.g. psoriasis, actinic keratosis, keratoacanthoma, and squamous cell carcinoma, and elsewhere.

WO 92/09287 by Procter & Gamble discloses a very wide family of vanilloid compounds to treat or prevent herpes lesions and pain associated therewith. At least the vanilamide derivatives of these compounds are provided with highly pungent properties (high irritating effect) that make these compounds scarcely suitable for use especially for repeated use, this may be the reason for the fact that this application was abandoned.

Legin, Pharmaceutical Chemistry Journal, vol. 30, 1, 1996, pages 60-68, describes 10-undecylenic acid vanillamide together with its use to treat different capsaicin responsive conditions. This document too, discloses that the active compound has a very high pungent property, i.e. a high irritating ability, or effect.

There is therefore the need for capsaicin compounds that are as active as the known natural compounds (and analogues thereof) and that are also non irritating, i.e. they are free of the pungent properties of said compounds of the known art.

It is the aim of the present invention to solve the above mentioned problem.

SUMMARY

This aim is reached by means of the present invention that provides terminally unsaturated vanilloids according to claim 1.

The present invention also provides a composition according to claim 4.

The use of said compounds according to claim 10 is a further object of the invention.

Therefore, an object of the present invention are terminally unsaturated vanilloids.

Another object is a medicinal composition comprising a terminally unsaturated vanilloids to treat a capsaicin-responsive condition.

Another object is a medicinal composition to treat a skin disorder characterized by itching.

Another object is a medicinal composition to treat a skin disorder characterized by a benign hyperproliferative condition.

Another object is a medicinal composition to treat a skin disorder characterized by

an oncologic hyperproliferative condition.

No prior art has mentioned ra-unsaturated vanilloids, i.e. derived from terminally unsaturated carboxilyc acid for the use in medicinal compositions, particularly in compositions comprising thereof.

Terminally unsaturated fatty acid are non-natural oleochemicals artifacts, sometimes found in trace amounts in non-vegetal metabolism. For istance, undecylenic acid has been detected in human sweat, while 9-decylenic acid (caproleic acid) has been identified in cow milk and in royal jelly.

Terminally unsaturated fatty acids are also available by metathetical ethenolysis of unsaturated fatty acid methyl esters of oleic and others usaturate fatty acids (Warwel S et all, Fat. Sci. TechnoL; 1992; 94, 323; ibid, Chemosphere; 2000, 43, 39).

New analgesic and pro-apoptotic vanilloids are suitable for the treatment of a variety of capsaicin-responsive conditions.

These vanilloids are in fact intended for the treatment of acute or chronic pain and itching (pruritus) and in hyperproliferative disorders, said treatment devoided of the discomfort associated with the use of natural vanilloid. DETAILED DESCRIPTION OF THE INVENTION

The active ingredients of the present invention are compound of formula (I) :

wherein:

R denotes lower alkyl or H; n is an integer from 2 to 8.

-X- denotes -O-CO-; -CH ₂ -NH-CO-; and -NH-CO-; with the provisio that when -X- is -NH-CO-, then n is < 6. As used herein "lower alkyl" are alkyl groups containing from 1 to 5 carbon atoms such as, e.g., methyl, ethyl, propyl, butyl, isopropyl, isobutyl, pentyl, isopentyl, etc.

Preferred R are CH ₃ and H.

The composition of the invention comprises at least a compound of formula (I) in a suitable ingredients are contained in a carrier in a concentration within the range of between about 0.01% and about 25% by weight of a compound of formula (I).

The selected concentration of active ingredient may depend on patient's condition, age, body weight, and mode of administration.

The preparation of pharmaceutical compositions is well known in the art and is been described in many articles and textbooks, e.g. Remington's Pharmaceutical Sciences, Gennaro, A. R. ed., Mack Publishing Company, Easton, Pa., 1990, and especially pp. 1521-1712 therein, or U.S. Pat. Nos. 5,736,519, 5,733,877, 5,554,378, 5,439,688, 5,418,219, 5,354,900, 5,298,246, and 5,164,372.

The compositions of the present invention are preferably produced by mixing the active ingredients with excipient, carrier, diluent, and optionally, a preservative or the like, to constitute a pharmacologically acceptable vehicle as known in the art.

Examples of excipients include glucose, mannitol, inositol, sucrose, lactose, fructose, starch, corn starch, microcrystalline cellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl pyrrolidone and the like.

Optionally, a thickener may be added, such as a natural gum, a cellulose derivative, an acrylic or vinyl polymer, or the like. The pharmaceutical composition may be provided in liquid or semi-solid form. The liquid preparation is provided preferably as oil suspension, fat emulsion or microcapsule composition. A semi-solid composition is provided preferably as a hydrous or oily gel, or an ointment. An oil suspension may be prepared by suspending or emulsifying capsaicin in an oleaginous base, such as sesame oil, olive oil, corn oil, soybean oil, cottonseed oil, peanut oil, lanolin, petroleum jelly, paraffin, Isopar, silicone oil, fatty acids of 6 to 30 carbon atoms or the corresponding glycerol or alcohol esters.

Emulsions may be prepared by adding to a fat or oil about 0.1 to 10% by weight of an emulsifier, and the ingredients are mixed with heating. Water and an isotonic agent are added, and optionally the pH is adjusted. The mixture is then homogenized. Preferably, such emulsions will contain an electric charge adjusting agent, such as

phospholipids, fatty acids, bilic acids, and salts thereof.

The composition may also be prepared as a hydrous gel. A hydrous gel base is dissolved or dispersed in aqueous solution containing a buffer and the active agents, and the solution is warmed or cooled to give a stable gel. As indicated above, the active ingredients may also be contained in oil droplets, particularly useful for aerosol or spray preparations, or in liposomes, microcapsules and/or nanoparticles.

The carrier may be water-based and forms an aqueous solution containing the other ingredients. An oil-based carrier solution containing the ingredients is an alternative to the aqueous carrier solution. Either aqueous or oil-based solutions further contain thickening agents to provide the composition with the viscosity of a liniment, cream, ointment, gel, or the like. Alternative embodiments of the present invention can also use a solid carrier containing the active ingredients. This enables the alternative embodiments to be applied via a stick applicator, patch or suppository. The solid carrier further contains thickening agents.

The present invention also refers to method to treat acute or chronic pain, skin disorders, by the administration of compounds of formula (I) without the discomfort normally associated with capsicum extract or the natural capsacinoids in pure form.

The composition of invention may be applied to an area on the surface of skin or mucosa to treat a capsaicin-responsive condition.

In one embodiment is the capsaicin-responsive condition is neuropathic or nociceptive pain or, more in general, itching. hi another embodiment, the capsaicin-responsive condition is a skin disorder with benign hypeproliferative including psoriasis, common warts, keratoacanthoma, seborrhoeic keratosis, seborrhea and ichthyosis.

In another embodiment, the capsaicin-responsive condition is a skin disorder with benign hypeproliferative including actinic keratosis and squamous cell carcinoma.

In one embodiment, the invention provides a method of treating a capsaicin- responsive condition in a subject by administration of a composition that contains a vanilloid of formula (I) to deliver at least about 4 nmoles active to skin as measured in a mouse skin absorption assay.

δ

Compounds of invention can be administered in a variety of ways including local, oral, enteral, parenteral, subcutaneous, transdermal, transmucosal, intravenous, intramuscular, intraperitoneal, intranasal, subdural, rectal, vaginal, etc.

The present invention is illustrated by the following examples that should not be considered limiting.

EXAMPLES Example 2 — 10-Undecylenic acid vanillyl ester

A solution of 4.05 g (20 mmol) of undecilenoyl chloride in 10 ml ethyl acetate was added dropwise into 5 mL of a pyridine solution of 2.47 g of vanillyl alcohol (15 mmol). The mixture has been stirred for 3 h, allowing it warm to room temperature. After, water and 2 N HCl were added, the mixture has been partitioned with 3x30 ml of ethyl acetate, the organic phases have been collected and washed with water, dried under Na ₂ SO ₄ and filtered. The filtrate is vacuum evaporated and chromatografated in silica gel to afford the compound of formula (II):

¹ H-NMR δ: 6.96 (s, IH, ArH), 6.93 (d, IH, ArH), 6.91 (d, 1η, ArH), 6.79 (s, 1η, OH), 5.82 (m, 1η, =CH), 5.03 (dd, 1η, (Z)HC=), 4.96 (s, 2η, OCH ₂ ), 4.93 (dd, 1η, (E)HC=), 3.78 (s, 3η, OCH ₃ ), 2.25 (t, 2η, CH ₂ CO), 2.04 (m, 2η, CH ₂ CH=CH ₂ ), 1.51 (q, 2H, CH ₂ CH ₂ CO), 1.33 (m, 1OH, CH ₂ ); liquid a room temperature. Example 2 - Enzymatic synthesis of 10-υndecylenic acid vanillyl ester

The compound of formula (II) is obtained from 50 mM of vanillyl alchohol and 1 M if undecylenic acid dissolved in 1 ml of acetone, in the presence of 20 mg of Novozym 435 and 50 mg of molecular sieves A 4 at 37°C, with the work-up as described by Watanabe and all. in Biosci Biotechnol Biochem, 66(2), 319-327, 2002. Example 3 - 4-AUylacetic acid vanillamide

3.15 g of vanillamine HCl have been suspended in 25 ml of DMF and added with 6.6 ml of a 5N solution of NaOH, and the mixture stirred for 15 minutes. The DMF

mixture has been chilled in an ice bath, and 2.4 g of undecylenoyl chloride dissolved in 5 ml of CH ₂ Cl ₂ added dropwise. The mixture has been stirred for 3 hours, allowing it warm to room temperature. After, it has been poured into 300 ml water, the layers separated, and the aqueous layer extracted with CH ₂ Cl ₂ . The organic phase has been washed with diluted HCl, sodium bicarbonate, water, and brine, dried and filtered. The crude product in purified on silica gel ethyl acetate/hexane (40:60) and recrystallized from methanol and water affording the product of formula (III):

¹ H-NMR δ: 7.59 (s, 2H, NH e OH), 6.70 (d, 1η, ArH), 6.62 (d, 1η, ArH), 6.60 (s, 1η, ArH), 5.82 (m, 1η, =CH), 5.02 (dd, 1η, (Z)H ₂ C=), 4.93 (dd, 1η, (E)H ₂ C=), 4.48 (s, 2η, NHCH ₂ ), 3.57 (s, 3η, OCH ₃ ), 2.16 (t, 2η, CH ₂ CO), 2.04 (m, 2η, CH ₂ CH=CH ₂ ); m.p. 48-50°C. Example 4 — 10-Undecylenic acid dopamide

A solution of undecylenic acid in acetonitrile at O ⁰ C have been added with triethylamine (1.4 :1 molar ratio) and isobuthylchloroformate (1.2 :1 molar ratio). After 30±40 min the mixture has been diluted with water and extracted with ethyl acetate. The resulting mixed anhydride has been treated with a solution of dopamine hydrochloride (1.1:1 molar ratio) in DMF containing triethylamine (1.1:1 molar ratio) for 18 h at 0 °C. The mixture diluted with water and extracted with ethyl acetate is washed with water, dried over Na ₂ SO ₄ and concentrated under vacuum. The product purified on silica gel by EtOAc/n-hexane afford a compound of formula (FV):

(IV)

¹ H NMR δ: 7.39 (s, 3H, 2 OH, NH), 6.76 (d, IH, ArH), 6.66 (d, 1η, ArH), 6.56 (s, 1η, ArH), 5.82 (m, 1η, =CH), 5.01 (dd, 1η, (Z)H ₂ C=), 4.93 (dd, 1η, (E)H ₂ C-), 3.44 (t, 2η, CH ₂ NH) ₅ 2.50 (t, 2H, ArCH ₂ ), 2.12 (t, 2η, COCH ₂ ), 2.04 (m, 2η, CH ₂ CH=CH ₂ ), 1.48 (q, 2H, COCH ₂ CH ₂ ), 1.31 (m, 10η, chain CH ₂ ). Example 5 — 10-Undecylenic acid homovanillamide

The metod of the Example 3 is applied with hamovanillylamine instead of dopamine HCl with similar molar ratio to afford compound of formula (V):

¹ H NMR δ: 7.61 (s, 2H, OH, NH), 6.85 (d, 1η, ArH), 6.69 (d, 1η, ArH), 6.55 (s, 1η, ArH), 5.82 (m, 1η, =CH), 5.02 (dd, 1η, (Z)H ₂ C=), 4.93 (dd, 1η, (E)H ₂ C=), 3.87 (s, 3η, CH ₅ O), 3.44 (t, 2η, CH ₂ NH), 2.30 (t, 2H, ArCH ₂ ), 2.12 (t, 2η, COCH ₂ ), 2.04 (m, 2η, CH ₂ CH=CH ₂ ), 1.48 (q, 2H, CH ₂ CH ₂ CO), 1.29 (m, 1OH, alkyl CH ₂ ). Biological Example 1 - Agonist activity on vanilloid receptors

The compound of invention have been tested for their agonist behaviour on human vanilloid receptor according to the procedure of Appendino G. et all., J. Med. Chem. 2002, 45, 3739-3745.

This method allows the compbined evaluation of potency as pECso, where EC ₅ 0 is the molar concentration needed to induce a relative half-maximal response for each compound; and efficacy, as percent of maximum possible effect ("PMPE") obtained with ionomycin. The results are illustrated in Table 1.

TABLE l

Test Compound PMPE pE50

4-Allylacetic acid vanillamide 64.5 7.5 ± 0.4

10-Undecylenic acid dopamide 59.4 8.6 ± 0.1

10-Undecylenic acid vanillyl ester 37.4 6.1 ± 0.2

10-Undecylenic acid homovanillamide 62.9 7.3 ± 0.1

Biological Example 2 - Assessment of stinging/burning

The compound of invention have been testes according to our previous application PCT/IB06/03577, Example 2, by Scoville Organolpetic Test according to ASTA Method 21.0 as revised by ASTA.

The results show that the compound of formula (I) as listed in Table 1 are at least 2 logs less pungent than capsaicin and also "unvanil" (see PCT/IB06/03577, Preparative Example 2).

The combined results indicate that the compound of invention are suitable for the treatment of nociceptive and hypeproliferative disorders without the discomfort associated with the classic capsaicinoids. Composition Example 1-2 - Ointments

Ingredient Quantity Quantity

4-Allylacetic acid vanillamide 0.1 g -

10-Undecylenic acid homovanillamide - 0.1 g

Liquid paraffin 2O g 20 g

White vaseline 52 g 52 g

Cetyl alcohol 9.9 g 9.9 g

Polyoxyetylene (60) hydrogenated castor oil 18 g 18 g

Composition Examples 3-4 - Gel

Ingredient Quantity Quantity

10-Undecylenic acid dopamide 0.25 g

4-Allylacetic acid vanillamide

Carbopol 1342 0.5 g 0.5 g

Triethanolamine 1.5 g 1.5 g

Polysorbate 20 0.2 g 0.2 g

Diazolidinyl urea 0.3 g 0.3 g

Disodium EDTA 0.02 g 0.02 g

Ethyl alcohol 2O g 2O g

Purified Water q. .b. to 100 g q.b. to 100 g

Composition Example 5 - Plaster for itching reeliei

According to the formula below, a styrene-isoprene-styrene block copolymer, an alicyclic saturated hydrocarbon resin, a synthetic rubber, and dibutylhydroxytoluene have been melted and kneaded together. To the kneaded product 10-undecylenic acid, methyl salicylate, and dl-camphor, have been added and the material has been further kneaded until uniform. The product is then spread over a liner to obtain a plaster.

Ingredient Quantity

10-Undecylenic acid vanillyl ester 5 g

Methyl salicylate 5.O g dl-Camphor 2.0 g Styrene-isoprene-styrene block copolymer 22.0 g

Alicyclic saturated hydrocarbon resin 41.5 g

Synthetic rubber 18.0 g

Dibutylhydroxytoluene 1.0 g Composition Examples 6-7 - Emulsions

Ingredient Quantity Quantity

Hydrogenated coconut oil 3.9 g 3.9 g

L-Arginine 1.0 g 1.0 g

4-Allylacetic acid vanillamide 0.5 g -

10-Undecylenic acid vanillyl ester - 1.5 g

Oleth-2 5.0 g 5.0 g

Quaternarium- 18-ectorite 0.5 g 0.5 g

MgSO ₄ JH ₂ O 0.3 g 0.3 g

Parfume, preservatives q.b q.b

Acqua qb to 100 g qb to 100 g