Title

USE OF CHAVICOL FOR THE TREATMENT OF SUPERINFECTED OILY

SKIN AND SCALP

Description The invention relates to the use of chavicol (p-allylpenol) for the manufacture of a composition indicated for the treatment of superinfected oily skin and scalp.

The invention also relates to a nontherapeutic regimen or a method to treat superinfected oily skin and scalp which comprises topically applying or orally administering a composition comprising chavicol.

BACKGROUND OF THE INVENTION

The impairment in steroid metabolism plays an important role in the pathogenesis of several skin disorders. In fact lipid metabolism is primarily regulated by the 5(X- reductase steroid enzymes converting testosterone into dihydrotestosterone (DHT), this being a potent modulator of keratin formation, sebum production and hair growth cycles.

The lipid metabolism imbalance by the over-reactivity of 5α-reductases ("5(X-TR") contributes to the etiology of common disorders including acne and androgenetic alopecia. Oily skin and scalp are a perfect growth medium of lipophilic, generally nonpathogenic ubiquitous anaerobic micro-organisms. In fact, Propionibacterium acnes infect oily skin, whilst Pityrosporum ovale flourish in seborrheic scalp thereby enhancing the loss of hair. Hence, P. ovale and P. acnes aggravate the course of the androgenetic impairment. The international patent application WO03/082233 discloses the use of a large class of allylphenols for the treatment of androgenic disorders by the inhibition of 5(X-TR inhibition.

Subsequently most of the thereby claimed allylphenols have been found to either elicit an estrogen-like response, or to have reduced 5α-reductase inhibition, or even to have an opposite pro-testosterone activity, or to have sensitizing properties

(unpublished).

At present, no single topical therapeutic agent capable of ameliorating all factors involved in the etiopathogenesis of superinfected oily skin and scalp has yet emerged.

The treatment includes various topical and systemic therapies and is guided by the type of clinical manifestations. Successful management of such skin disorders requires also careful patient evaluation followed by consideration of several factors related to the patient age, skin type, coexisting conditions, lifestyle, menstrual regularity, and so on.

Topical therapy is often preferred and include comedolytic agents such as tretinoin, adapalene, azelaic acid, tazarotene and salicylic acid; antimicrobial agents such as benzoyl peroxide; antibiotics such as clindamycin, erythromycin and tetracycline; and anti-inflammatory agents such as sodium sulfacetamide. Oral antibiotics or other systemic treatments including estrogens, antiandrogens, and isotretinoin are often added to the treatment regimen when the condition does not respond satisfactorily to topical therapy.

The ideal agent would target all the pathogenic factors without producing adverse effects.

SUMMARY OF THE INVENTION In the investigation of activity/toxicology behaviour of allylphenols it has surprisingly been found out that several such compounds hold side effects which have been not detected hitherto and render them unsuitable for cosmetic/dermatologic applications.

In fact, several compounds disclosed in WO03/082233 were found estrogenic, or have reduced 5α-TR inhibition and even pro-testosterone activity, or are sensitizing agents.

Conversely, it has been found out that chavicol inhibits 5(X-TR without significant estrogenic or sensitizing potentials, thereby coupled with significant biocidal activity, which all together are excellent promises in superinfected oily skin and scalp treatment.

In other words, it has been therein discovered the chavicol has dual-action pattern, i.e. reduce sebum over-production and, concomitantly, kill or eradicate certain anaerobic micro-organisms, which both contribute to oily skin and scalp disorders.

An object of this invention is the use of chavicol for the manufacture of a composition to treat superinfected oily skin and scalp without significant estrogenic or sensitizing effects.

Another object of this invention is a cosmetic/dermatologic composition comprising chavicol intended for the treatment of superinfected oily skin and scalp as well as for combating ad/or preventing hair loss. Another object of this invention is a nutritional/pharmaceutical composition comprising chavicol intended for the treatment of superinfected oily skin and scalp.

A further object of this invention is a cosmetic/dermatologic method or regimen for the treatment of superinfected oily skin and scalp in subjects in need thereof.

A still further object of this invention is a nutraceutical method or regimen to treat superinfected oily skin and scalp in subjects in need thereof.

DETAILED DESCRIPTION OF THE INVENTION

According to one aspect, the present invention refers to the use of chavicol for the manufacture of a composition to treat superinfected oily skin and scalp. As used herein "superinfected oily skin and/or scalp" means an skin or scalp undergoing a lipid over-production, often accompanied with continuously sheds dead skin cells for the outer most portion of the epidermis, associated with over-growth of anaerobic and lipophilic micro-organism such as yeast/fungi of the genus Malassezia or Pityrosporum and/or bacteria of the genus Propionibacterium. In particular, it includes skin and/or scalp affected by androgenic disorders such as alopecia, acne, seborrhea and/or dandruff.

As used herein, chavicol is defined to include derivatives or prodrugs thereof. A "derivative or prodrug" means any pharmaceutically acceptable salt, ester, salt of an ester, or other derivatives which capable of releasing chavicol. Particularly favoured derivatives and prodrugs are those that increase the bioavailability of chavicol when

administered to a subject in need thereof, e.g. enhance the delivery of chavicol into the skin layers or allow the orally administered compound to be more readily absorbed into the blood.

Chavicol, alias 4-(2-propenyl)-phenol, or p-hydroxy-allylbenzene, or 4-allylphenol, with MW 134.18 (CAS 501 -92-8) is a colourless low-melting product with m.p. of 16 ⁰ C.

According to another aspect, the present invention refers to the use of chavicol as active ingredient in the manufacturing of a composition suitable for the treatment of superinfected oily skin and/or scalp, wherein the composition is characterized in that: a) it reduces sebum over-production; b) it kills superinfecting anaerobic micro-organisms; c) it possesses no significant estrogenic potential; d) it possesses no significant sensitizing potential. chavicol suitable for purpose of the invention can be obtained from chavicol- containing plant distillates, or by chemical synthesis with known methods, provided that the final purity of the material is not less than 95% w/w of the active ingredient.

A convenient method is the conversion of Estragole (l-methoxy-4-(2- propenyl)benzene), which is readily available starting material obtained in large quantity as aroma chemicals from estragon oil, into chavicol by demethylation (methyl-ether cleavage), e.g. with EtMgBr, MeMgI, HBr/AcOH, EtSNa/DMF, TMSI, pyridine.HCl, BBr ₃ and the like.

Although the chavicol suitable for the inventive purpose may be from any natural source and/or synthetic method, we herein provide a convenient process to manufacture chavicol. The process entails the demethylation of Estragole with a Lewis acid followed a 2- step extractions in alkaline and acid-neutral media. In a preferred embodiment, this reaction is carried out under mild conditions with a Lewis acid such as boron alogenide (BX ₃ ) associated with a quaternarium ammonium-iodine salt, preferably the former is BCl ₃ and the latter 77-Bu ₄ NI, preferably in 1:1 ratio at around 1.5 eq., wherein the reaction is followed by at least two solvent-water extractions to afford

pure chavicol.

According to the invention, chavicol can be incorporated into a variety of formulations suitable for topical delivery of active ingredients. The topical formulations suitable for topical treatment of treat superinfected oily skin and scalp are creams, lotions, mousses, sprays, emulsions, gels, shampoos and the like, which are manufactured according to methods commonly known in the art (see, for instance: Topical Formulations: Design and Development-Bozena Michniak/Paperback/CRC Press, LLC/February 1999; Remington: The Science and Practice of Pharmacy 20 ^th - Alfonso L. Gennaro, Alfonso R. (Ed. ) Gennaro; Publisher: Lippincott Williams & Wilkins, December 2000,20th Ed.; Encyclopedia of Pharmaceutical Technology- James Swarbrick (Editor), James C. Boylan(Editor)/Hardcover/Marcel Dekker/May 1997).

Chavicol may optionally be associated with other components which have auxiliary action in the treatment and/or the disinfection of superinfected oily skin and scalp or may provide skin benefits. Examples of said additional components are, for instance, active against the proliferation of Propionibacterium acnes and/or Pityrosporum ovale: e. g. antibiotics such as erythromycin, clindamycin and tetracyclines, antimicrobials such as chlorexidine and benzoylperoxide, cedrene, caryophyllene, longifolene andthujopsene comedolytic agents such as tretinoin, adapalene, azelaic acid, tazarotene, salicylic acid and derivatives thereof, antinfiammatory agents such as NSAID (i.e. acetylsalicylic acid, ibuprofen, naproxen, sulfacetamide), steroidal antinfiammatory agents (i.e. hydrocortisone), vitamins (e. g. retinoic acid and derivatives thereof), oil or sebum control agents (i.e. clay silicones), and skin healing agents. The amount of chavicol that may be used according to the invention obviously depends on the desired effect and must be in an amount that is effective for the treatment of oily skin and hair and for combating and/or preventing hair loss.

In general the amount of chavicol in the topical composition for treating and/or disinfecting oily skin and hair and for combating hair loss according to this invention may range from about 0.1% (w/w) to about 10% (w/w). The topical compositions

useful for delivery of chavicol may contain the usual acceptable excipients, including carriers and vehicles, preservative agents, surfactants, moisturizing agents, thickeners, perfumes, chelating agents, water, alcohols, antioxidants, antiseptics, colorants and adsorbents. Depending on the mode of administration, the composition according to the invention may be in any dermatological form normally used, particularly in cosmetology.

According to another aspect, a subject of the invention is a topically or orally administrable composition comprising chavicol and at least another active agent selected from the group consisting of: i) antiseborrheic agents, for instance progesterone; ii) antidandruff agents, for instance octopirox or zinc pyrithione; iii) antiacne agents, for instance retinoic acid, azelaic acid or benzoyl peroxide; iv) anti-alopecia agents, for instance minoxidil or finasteride. One embodiment of the invention thus features a nontherapeutic regimen or method to treat superinfected oily skin and scalp comprising topically applying chavicol in association with a cosmetically/dermatologically acceptable excipients.

Chavicol may be also administered by the oral route in a variety of oral dosage forms including tablets, capsules, sugar or film coated tablets, liquid solutions or suspensions.

The solid oral forms may further contain diluents, e.g. lactose, dextrose, saccharose, sucrose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate. and/or polyethylene glycols; binding agents, e.g. starches, arabic gum, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. a starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents such as lecithin, polysorbates, and, in general, food-grade substances. Said compositions may be manufactured in known manner, e.g., by mixing, granulating, tabletting, sugar- coating, or film-coating processes. The liquid dispersions for oral administration may be e.g. syrups, emulsions and suspensions. Syrups may contain as carrier, e.g.,

saccharose or saccharose with glycerine and/or mannitol and/or sorbitol. Suspensions and emulsions may contain as carrier, e.g agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or PVA.

Another embodiment of the invention features a nontherapeutic regimen or method to treat superinfected oily skin and scalp comprising orally administering chavicol in association with a nutricionally/pharmaceutically acceptable excipients.

The dosage level for oral administration depends on the patient age, weight and skin conditions may therefore range from 10 to 500 mg pro dose from 1 to 5 times daily.

Example 1 - Test on 5-α-reductase inhibition activity

The inhibition of 5α-reductase activity is tested by micro -radioassay as per Watanabe et all. in IFSCC Magazine 4(2):83-87,2001.

The conversion rate of testosterone expressed as 50% inhibitory concentration (IC50) indicate the anti-androgenic activity of chavicol an IC50 at around 10 ^"1 g/100. Comparative Example 1 - Test on 5-α-reductase inhibition activity

The same method of Example 2 has been carried out on 4-(2-methylene-hexyl)- phenol provide a IC50 of around 1 g/100g.

Example 2 - Test on estrogenic potential The method is adapted from Gould JC, et all, MoI Cell Endocrinol 142:203-214

Serial dilutions of Estradiol are prepared in 10 mm Tris (pH 7.6), 0.3 m KCl, 5 mm dithiothreitol, and 1 mg/ml BSA. 100 microl of 17β-Estradiol and chavicol in concentration from 10 ^"5 -10 ^"10 M is transferred to a polystyrene tube. ^[3H] Estradiol (Amersham Pharmacia Biotech, IL, USA) is added to each tube at 5 nM. Recombinant human ERa (PanVera Corp., Madison, WI) are added at 8 pmol/ml to each tube. After overnight incubation at 4 ⁰ C, 100 ml of a 6% hydroxyapatite (HAP) slurry in dilution buffer (10 ml Tris (pH 7.6) and 5 mm dithiothreitol) are added to each tube. Tubes are then incubated at 4 ⁰ C for 30 min and spun at 1000 3 g for 10 min. HAP pellets are washed x 4 in dilution buffer containing 1% Triton X-100. Pellets are resuspended in 1 ml dilution buffer and transferred to scintillation vials.

Radioactivity is measured on a Packard Tri-Carb 460 scintillation counter (Packard Instruments, CT, USA).

Results expressed as relative estrogenic potential, i.e. taking 17β-Estradiol as reference (value 1): > 10 ^~9 for chavicol, hence it has negligible, if any, estrogenic potential.

Comparative Example 2 - Test on estrogenic potential

The same method of Example 2 carried out on 4-(2-Methylene-hexyl)-phenol at 10 ^"

⁵ -10 ^"8 M with 17β-Estradiol as reference (value 1) provided an estrogenic potential of

10 ^"6 , which compares 10 ^"3 found for 4-n-Nonylphenols and 0.5x10 ⁵ for 4-n- Octylphenol (Tanamoto et all, Abstract of 2 ^nd IVA World Water Congress, Berlin

2001, 15-19 Oct).

Example 3 - Test on anaerobic bacteria Determination of the efficacy of chavicol on strains of Propionibacterium acnes and Pityrosporum ovale. For this purpose, samples are taken from the patients' skin or scalp are isolated, purified on agar and stored in peptone/dextrose slant agar tubes. The strains were applied, undiluted or diluted 1:100, to an RPMI 1640 agar (Gibco/BRL, Life Technologies GmbH, Eggenstein, Germany) containing about 1.0 [mu]g/ml Propionibacterium acnes and Pityrosporum ovale strains. The activity of chavicol is determined by the microtiter dilution technique in RPMI 1640 medium. The growth medium RPMI 1640 buffered with 0.165 M morpholinopropanesulfonic acid pH 7.0 is introduced into 96-well microtiter plates. Serial dilutions by a factor of 2 are prepared to result in final concentrations of 256 to 0.002 [mu]g/ml of chavicol. The microtiter plates prepared in this way are incubated with the test strains. The initial cell count is 1-5x10 ³ colony- forming units per ml of growth medium. The microtiter plates are incubated at 35 ⁰ C for 48 hours. As determined by photometry at 510 nm, chavicol MIC50 is ~ 2.5 mmol on Propionibacterium acnes, and ~ 0.5 mmol on Pityrosporum ovale.

Example 4 - Assessment of efficacy and safety

A 2% cream is made by melting 2 g of chavicol at 75 ⁰ C in 10 g Emulvama™ AGC (premix of: glyceril stearate, cetearyl alcohol, stearic acid, sodium cocoyl glutamate) from VaMa Farmacosmetica (Zibido (MI), Italy) in water at 75 °C under high stirring. Ten adult female volunteers, mean age of 43 years (range 25-56) with oily facial skin enrolled in the study are free of dermatosis and not receiving any prescribed drugs. Patients are instructed to apply the lotion twice daily to forehead and cheeks, by applying - 1.5 mg/cm ² but in the 24 hr-period preceding the clinic visits after 4 and 8 weeks. Sebum excretion levels are assessed on the central forehead and nasolabial region of the cheek (sebum-test sites) at baseline and after 4 and 8 weeks of treatment by Sebutape ^® (Pagnoni et all, J Soc Cosmet. Chem. 1994; 45: 221-225). Influence of chavicol 2% on sebum excretion rate is expressed as % area occupied by sebum spots. Each line represents an individual study volunteer (Skin Oiliness Reference: Low: 4%>; Normal: 4-6.5%; Oily: 6.5-9%; Very Oily: >9%). Safety evaluations are based on detection of potential skin irritation or other signs of adverse reaction to the treatment.

No skin irritation are observed after 8 weeks of treatment. Neither changes in skin infrastructure, nor skin dryness, redness or peeling are noted during the 8-weeks period. In all volunteers, baseline sebum excretion levels on the forehead are higher than those detected on the nasolabial area of the cheek. Baseline SER measurements indicate oily to very-oily forehead skin and oily skin at the nasolabial region of the cheek (%areas covered by spots = 8.9 ± 1.0 and 6.5 ± 0.6, respectively). Mean sebum excretion rates after 4 weeks of chavicol 2% application, declined 35% (forehead) and 29% (cheek) and approached normal values in all participants. At 8 weeks of treatment, SER is 55% and 62% of the baseline at the forehead and cheek, respectively. It may be concluded that chavicol is a sebum regulator, being safe and useful in the application to skin and scalp.

Example 5 - Emulsion for acneic skin

100 g of emulsion contains

Laurylmethicone copolyol 2.O g

Liquid paraffine 1.0 g chavicol 2.O g

Lanolin 1.5 g

Soybean oil 10.0 g

Cyclomethycone 6.O g

Azelaic acid 1.5 g

Parfum, additives, preservatives q.b.

Demineralized water q.b. to 100 g

Sodium biphosphate q.b. to pH 3.8

Example 6 - Shampoo for oily scalp and falling hairs 100 g of detergent contains: Sodium laurylsulfate 5.O g

Triethanolamine laurylsulfate 5.O g

Betaine lauryldimethylaminoacetate 6.O g

Ethylene glycol distearate 2.O g

Octopirox 2.0 g chavicol 4.O g

Ethanol 2.O g

Parfum, additives, preservatives q.b.

Deionized water q.b. to 100 g

Example 7 - Gel to control armpit sweating and vellus 100 g of gel contains:

Aluminum oxychloride 1.2 g

Alantoin 0.1 g alfa-Bisabolol 0.1 g chavicol 2.O g

Acrylic gel of Carbopol™ q.b. to 100 g

Example 8 - Tablets to control hair loss or acneic skin

Each 350 g tablet contains: chavicol 120 mg,

Maize starch 30 mg

Lactose 115 mg

Carboxymethyl cellulose 15 mg

Microcrystalline cellulose 60 mg Magnesium stearate 10 mg.

Example 9 - Synthesis of chavicol in mild conditions

Reaction conditions Estragole (6.25 mmol; 1 eq.; 0.92 g) and 77-Bu ₄ NI (9.39 mmol; 1.5 eq.; 3.46 g) are stirred in dry CH ₂ Cl ₂ (18 ml) at -78 ⁰ C under N ₂ . A solution of BCl ₃ 9.38 ml, 1 M in CH ₂ Cl ₂ (9.38 mmol; 1.5 eq.) is added over 2 min. After 5 min, the solution is warmed to 0 ⁰ C and stirred for 2 h. The reaction solution is quenched with ice and H ₂ O, stirred for 30 min, and partially concentrated to remove CH ₂ CI ₂ .

Recovery The reaction mixture is extracted with Et ₂ O. The combined organic layers are separated and extracted with 2x10 ml of NaOH IN. This alkaline solution is washed with 10 ml CH ₂ Cl ₂ , then added with 7 ml of HCl 3N and extracted with CH ₂ Cl ₂ (2x15 ml), which is then separated, decolorizing (charcoal, 0.2 g) for 15 min, washed with brine, dried (Na ₂ SO ₄ ), filtered and vacuum evaporated affording around 0.5 g of chavicol as colourless products. Crystallization in ethanol at 10 ⁰ C afforded a single spot in silica gel TLC: CHCl ₃ /MeOH/AcOH 95:5:4 (Iodine); Rf= 0.25.