FIELD OF THE INVENTION The present invention relates to a novel use of nutritional/pharmaceutical compositions comprising as active ingredients resveratrol and cysteine and derivatives thereof, preferably the combination resveratrol and NAC (N-acetyl-1-cysteine).

More specifically, the invention relates to the use of such nutritional/pharmaceutical compositions for treating and/or preventing a hyaluronidase-related disorder such as skin senescence or j oint arthritis, in particular in a postmenopausal woman.

BACKGROUND OF THE INVENTION

Hyaluronic acid ("hyaluronan") is found in almost all vertebrate organs, but most abundantly in the extracellular matrix of soft connective tissues, such as in human skin wherein it constitutes a high fraction of the viscoelastic connective net- work of dermis.

Under normal physiological conditions, hyaluronan is one of the major components of synovial fluid. The molecular weight of hyaluronan in osteoarthritic or inflamed joints can decline as a result of the presence of proinflammatory cytokines, free radicals, and lytic enzymes, with a change in the viscoelasticity of synovial fluid leading to joint dysfunction. The hyaluronan retentive capacity on moisture is particularly evident in skin, thereby it contributes to make a clear difference between a young (hydrated) complexion and an aged (dry) skin. The level of hyaluronan can descrease by the hyaluronidase upregulation. The enzyme, hyaluronidase actually depolymerizes hyaluronan by degrading the original polymer into low molecular oligomers of D-glucuronic acid and N-acetyl-D-glucosamine. Inhibitors of hyaluronidase can, therefore, play a key role to preserve the hyaluronan structure and function within the connective tissue, to prevent or treat the related disorders. A recent overview on hyaluronidase inhibitors Botzki, A. "Structure-based design of hyaluronidase inhibitors", Dissertation 2004, University of Regensburg; pointed out that quite differentiated structural moieties (e.g. ascorbyl palmitate) can inhibit hyaluronidase. Hyaluronidase inhibitors were identified in plant polyphenols, e.g. apigenin and kaempferol (Kakegawa H; Planta Med 1988, 54, 385-389); saponins and sapogenis (Carini M; Arch Pharm, 1995, 328, 720-724); phenols from Areca catechu (Lee KK, Int J Cosm Science, 2001, 23(6), 341-346); Sorghum polyphenols (Bralley E, J Med Food, 2008, l l(2):307-312).

Resveratrol (3,5,4' -trihydroxystilbene) is a polyphenol produced by stilbene synthase being endowed of an array of beneficial properties such as anti-inflammatory, imunomodulatory, chemopreventive, neuroprotective, and cardioprotective actions.

To our knowledge no one has reported so far any activity if resveratrol on hyaluronidase.

Resveratrol was instead subject of intense researches on its classic anti-aging properties. For examples resveratrol-based is a skin care formulation seems to afford a 17 times greater antioxidant activity than idebenone (Baxter RA. J Cosmet Dermatol. 2008; 7(l):2-7). US2005107338 and US2006257502 disclose nutritional supplements to enhance the mitochondrial function comprising, inter alia, resveratrol and NAC.

WO2006127759 discloses an antioxidant nutritional supplements to enhance the resistance to the oxidative damages comprising, inter alia, resveratrol and NAC.

JP2001058916 and related application (US6147121, EP0953346 etc.) discloses the use of hydroxystilbenes, and specifically resveratrol, as effective collagen-synthesizing, fibroblast- proliferating, protease expression-inhibiting, and finally skin tonicization agent by its topical application thereof.

Instead, we have found out that resveratrol alone may be beneficial in collagenase inhibition but can be detrimental as mitotic agent (unpublished results) if used alone. Therefore, an optimized use of this valuable phytoalexin was still on demand.

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SUMMARY OF THE INVENTION

The present invention is based on the fact that a combination of resveratrol and cysteine or derivative thereof is surprisingly effective in the inhibition of the hyaluronidase activity. Accordingly, the present invention provides a composition suitable for medicinal use which comprises resveratrol and at least a cysteine or a cysteine derivative.

In one aspect, the composition of invention can be administered to a subject whose hyaluronidase-related disorder is skin senescence.

In one aspect, the composition of invention can be administered to a subject whose hyaluronidase-related disorder is joint arthritis. In another aspect, the composition of invention is administered to a postmenopausal women having said hyaluronidase-related disorders.

In a further aspect, the composition of invention also comprise one or more phytoestrogen, or a carotenoid such as astaxanthin.

DETAILED DESCRIPTION OF THE INVENTION

It has now been found surprisingly that a composition containing a combination of the following as its characterising components: resveratrol and cysteine and/or derivatives of cysteine, or a pharmacologically acceptable salt thereof, is extremely effective in the prevention and/or therapeutic treatment of disorders caused by the hyaluronidase up- regulation, as a result of the potent effect exerted by its components. Noteworthy, the inventive combination also add useful ancillary activities such as collagenase inhibition and fibroblast proliferation.

Therefore, the present invention relates to the use of resveratrol and cysteine and/or derivatives thereof, preferably N-acetyl-cysteine (NAC) to prepare a pharmaceutical product in which the two components are admixed together or separately packaged as a kit-of-parts for the prevention and treatment of disease forms related to hyaluronidase-related disorders.

The two active ingredients can be admixed together or separately packaged is for the "coordinated use" of said ingredients; in any case, the active components are resveratrol and cysteine and/or derivatives of cysteine, or a pharmacologically acceptable salt thereof.

The term "resveratrol" as used herein more precisely define trans-resveratrol. i.e. the compound of formula (I):

Trans-resveratrol is preferably extracted from natural products containing thereof, such as Vitis vinifera, Vitis rotundifolia, Vitis lambrusca and other Vitis spp. or from the roots of the Polygonum genus, e.g. P. cuspidatum and P. multiflorum. Preferably a pure 99+% resveratrol obtained from P. cuspidatum is used, e.g. supplied by Chromadex (Irvine, CA, USA). The term "cysteine and derivatives thereof herein comprises compounds of formula (II):

RNH ₂ -CH(COOR')-CH ₂ -SR" (II) wherein:

R denotes the optionally substituted C2-C22 acyl group; R' denotes an optionally substituted C2-C22 alkyl group; and R" denotes an optionally substituted C2-C22 o alkyl or carboxyalkyl group.

Preferred substances of formula (II) preferred 1-cysteine (R ₅ R', R" = H) and N-acetyl-1- cystine, or NAC (R = COCH ₃ , R', R" = H).

Other suitable substances of formula (II) include S-carboxymethyl-1-cysteine (R" = CH ₂ COOH, R, R' = H) and 1-cysteine ethyl ester (R' = CH ₂ CH ₃ , R, R" = H).

The expression "cysteine and/or cystein derivatives" more commonly include the physiologically acceptable salt thereof, i.e. any salt which is medicinally acceptable and does not greatly reduce the inhibitory activity on hyaluronidase.

Suitable examples include acid addition salts, with an organic or inorganic acid such as acetic acid, tartaric acid, trifluoroacetic acid, lactic acid, maleic acid, fumaric acid, citric acid, methanesulfonic acid, sulfuric acid, phosphoric acid, nitric acid, or hydrochloric acid. It is to be further understood that cysteine and cysteine derivatives, or derivatives thereof, can be used in hydrated forms of these compounds as well as the anhydrous forms.

The invention also relates to nutritional/pharmaceutical compositions according to claim 13. The nutritional/pharmaceutical compositions of the present invention contain resveratrol in an amount sufficient to provide to a human adult (weighing about 70 kg) a dosage from about 0.5 mg/day to about 3000 mg/day, preferably from about 25 mg/day to about 100 mg/day. Thus, if the nutritional/pharmaceutical composition is a food or beverage the amount of resveratrol contained therein is suitably in the range from about 10 mg to about 100 mg per serving. If the composition is a medicinal formulation it may contain from about 5 mg to about 150 mg per solid dosage unit, e.g., per capsule or tablet, or from about 5 mg per daily dose to about 300 mg per daily dose of a liquid formulation.

The composition preferably comprises resveratrol and cysteine and derivatives thereof in a weight ratio of 1:1 to 1:100, preferably from 1:2 to 1:50, more preferably from 1:2.5 to 1:10. The composition preferably comprises resveratrol and NAC (as cysteine derivative) in the afore mentioned weight ratios. The term "nutritional/pharmaceutical" as used herein denotes usefulness in both the nutritional and pharmaceutical field of application. Thus, the novel nutritional/pharmaceutical compositions can find use as supplement to food and beverages, dietary supplement and as pharmaceutical formulations for enteral or parenteral application which may be solid formulations such as capsules or tablets, or liquid formulations, such as solutions or suspensions. As will be evident from the foregoing, the term nutritional/pharmaceutical composition also comprises food and beverages containing the above-specified active ingredients.

The composition may be administered with a frequency of several times a day to once every two days, preferably daily. Treatment should be ongoing. In general, for administration to adults, an appropriate daily dosage is in the range of about 5 mg to about 500 mg, although the upper limit may be exceeded if expedient. The daily dosage can be administered as a single dosage or in divided dosages.

The present composition may be administered in any conventional form, preferably by oral route, for example, as tablets, coated tablets, dragees, troches, lozenges, aqueous or oily suspensions, liquid solutions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of medicinal compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide medicinally elegant and palatable preparations.

Tablets contain the active ingredient in admixture with non-toxic medicinally acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, dextrose, saccharose, cellulose, corn starch, potato starch, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, maize starch, alginic acid, alginates or sodium starch glycolate; binding agents, for example starch, gelatin or acacia; lubricating agents, for example silica, magnesium or calcium stearate, stearic acid or talc; effervescing mixtures; dyestuffs, sweeteners, wetting agents such as lecithin, polysorbates or lauryl sulphate. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. Such preparations may be manufactured in a known manner, for example by means of mixing, granulating, tableting, sugar coating or film coating processes. Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, e.g., calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is present as such, or mixed with water or an oil medium, e.g., peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, e.g., sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinylpyrrolidone gum tragacanth and gum acacia; dispersing or wetting agents may be naturally-occurring phosphatides, e.g. lecithin, or condensation products of an alkylene oxide with fatty acids, e.g. polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, e.g. heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate.

The said aqueous suspensions may also contain one or more preservatives, e.g. ethyl or n- propyl p-hydroxybenzoate, one or more colouring agents, and/or one or more sweetening agents such as sucrose, saccharin, glucose, sorbitol and mannitol.

Oily suspension may be formulated by suspending the active ingredient in a vegetable oil, e.g. arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.

Oily suspensions may contain a thickening agent, e.g. beeswax, hard paraffin or cetyl alcohol.

Sweetening agents, such as those set forth above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by this addition of an antioxidant such as ascorbic acid. Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, such as sweetening, flavouring and colouring agents, may also be present.

The medicinal compositions of the invention may also be in the form of O/W emulsions. The oily phase may be a vegetable oil, e.g. olive oil or arachis oils, or a mineral oil, e.g. liquid paraffin or mixtures of these.

Suitable emulsifying agents may be naturally-occurring gums, e.g. gum acacia or gum tragacanth, naturally occurring phospholipids, e.g. soy lecithin, and esters or partial esters derived from fatty acids an hexitol anhydrides, e.g. sorbitan mono-oleate, and condensation products thereof with ethylene oxide, e.g. polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavouring agents. Syrups and elixirs may be formulated with sweetening agents, e.g. glycerol, sorbitol or sucrose. In particular a syrup for diabetic patients can contain as carriers only products, e.g. sorbitol, which do not metabolise to glucose or which only metabolise a very small amount to glucose.

This suspension may be formulated according to the known art using those suitable dispersing of wetting agents and suspending agents which have been mentioned above.

In a preferred embodiment, the composition according to the present invention comprises also a phytosterol and/or a phytoestrogen from naturally sources by known means.

Useful phytosterols include campesterol, sitosterol, fucosterol, stigmasterol, stigmastanol, or stigmastadienone. A preferred phytosterol is β-sitosterol, but α-or γ-sitosterol are also useful phyitosterols. Other preferred phytosterols include derivatives or conjugates thereof, e.g. β- sitosterol-3-O-β-D-glucopyranoside, and mixture thereof.

Useful phytoestrogens include lignans, isoflavones, or coumestans, and derivatives or conjugates thereof.

Phytoestrogens include free (unconjugated) or conjugated forms, for example, sulfated, or sulfonated phytoestrogen conjugates, or glucoside, glucuronide, or sulfoglucuronide conjugates. A mixture of phytoestrogens can also be employed.

Suitable lignans include sesamin, justiciresinol, lariciresinol, isolariciresinol. secoisolariciresinol, 0-demethylsecoisolariciresinol, didemethylsecoisolariciresinol, demethoxysecoisolariciresinol, matairesinol, syringaresinol, episyringaresinol, diasyringaresinol, massoniresinol, lirioresinol, entrodiol, enterolactone, gomisin A, gomisin

C, gomisin D, nordihydroguaiaretic acid, 3'-O~methyl nordihydroguaiaretic acid, arctigenin, or 3'-O-demethylarctigenin, and derivatives or conjugates thereof. Examplary lignan conjugates include syringaresinol-3-D-glucoside, massoniresinol 4'-O-D-glucopyranoside, secoisolariciresinol diglycoside, and ramontoside, a butyrolactone lignan disaccharide.

Suitable isoflavones include genistein, daidzein, biochanin A, glycitein, zearalenone, β- zearalenol, formononetin, laburnetin, isoprunetin, O-desmethylangolensin, ipriflavone, phloretin, baicalein, alpinumisoflavone, hydroxyalpinumisoflavone, and derivatives or conjugates thereof. Exemplary phytoestrogen derivative is equol.

Suitable coumestan include coumestrol, wedelolactone, 4'-methoxycoumestrol, and plicadin, or derivatives or conjugates of these. In a preferred embodiment, the composition also comprise a carotenoid such as astaxanthin.

The following examples further illustrates the present invention.

EXAMPLES

Example 1 - Hyaluronidase inhibition

Hyaluronidase activity was determinated by a modified Morgan_Elson method as described in Arch. Pharm. (1995, 328:720-724). Briefly, Hyaluronidase (100 μl of 0.6 mg/ml hyluronidase from bovine testes in 0.05 M acetate buffer, pH 4 containing 0.15 M NaCl) was preincubated with samples dissolved in dimethyl sulphoxide (DMSO) or acetate buffer for 15 min a 37°C parallel to a control containing only DMSO or acetate buffer. After preincubation, the reaction was started by addiction of 0.35 ml of hyaluronic acid (1.43 mg/ml in acetate buffer) to a final volume of 0.5 ml. Incubation were carried out for 45' at 37°C. After that time enzyme reaction was stopped and the Morgan:Elson color reaction started by addiction of 100 μl of potassium tetraborate (0.8 M) and heating in a boiling water bath for 3'. After cooling to room temperature, 3 ml of p-dimethylaminobenzaldehyde (DMBA, 10 mg/ml in acetic acid containing 1.25 μl of HCl 10N) reagent was added and incubated at 37°C for 10'. The absorbance at 585 nm was measured against a blank test containing all reagents less hyaluronidase in a U V- Vis spectrophotometer Varian 50Bio. The percentage of inhibition was calculated as follow: Inhibition % = (Control OD 585 nm - Sample 585 nm) / (Control OD 585 nm) x 100 Then the linear regression curve of Inhibition % versus concentration of samples was built and IC ₅₀ or inhibition cone, calculated. The hyaluronidase activity is illustrated in Table I. TABLE I - Hyaluronidase inhibition of NAC (fixed cone.) and resveratrol (variable cone.)

% Inhibition Mean increment

NAC 0.3 mg/ml 50

NAC 0.3 mg/ml + Resveratrol 0.2 mg/ml 73.31 ± 2.07 +23.3%

NAC 0.3 mg/ml + Resveratrol 0.1 mg/ml 76.02 ± 3.98 +26.0%

NAC 0.3 mg/ml + Resveratrol 0.05 mg/ml 74.25 ± 2.93 +24.2%

NAC 0.3 mg/ml + Resveratrol 0.025 mg/ml 53.6 ± 1.25 +3.6%

Example 2 - Proliferative activity

The evaluation of the cell proliferation of human skin fibroblasts (ATCC-CRL-2703) exposed to treatments at different concentrations was measured by means of colorimetric assay (MTT). Culture exposition to test substances were kept for 24 and 48 hours. At the end of each experimental time cell proliferation was determined and 24 hours after cell treatment samples for collagenase activity were withdrawed. MTT-medium was prepared by adding 15 mg of MTT to 30 ml of culture medium. After exposure of cells to the test items, they were washed with 200 ml of PBS. After removal of the washing solution, 200 μl of MTT-medium were added to each well and then incubated for 4 hours at 37 ⁰ C and 5% CO ₂ . At the end of incubation period, the MTT medium is removed and 200 μl of MTT solution (10% Triton X- 100 plus 0.1 N HCl in isopropanol) were added. The plate was shaken on a rotatory plate for 20-30 minutes until homogeneous solution. The absorbance was measured at 570 nm on microplates with background at 690 nm. Results are expressed as % cell viability compared to an untreated control cell, as illustrated in Table II.

TABLE II - Fibroblast proliferation of resveratrol (fixed cone.) and NAC (variable cone.)

24 h 48 h

Resveratrol 5 μM -5.75% + 4.07%

Resveratrol 5 μM + NAC 10 μM + 12.19% + 9.25% Resveratrol 5 μM + NAC 100 μM + 19.93% + 12.26%

Resveratrol 5 μM + NAC 1000 μM + 26.92% + 16.07%

Example 3 — Collagenase inhibition

A modification of the procedure of Mandl et al. (J Clin Invest. 1953, 32, 1323) was used in this experiment. Samples containing collagenase are incubated for 5 hours with native collagen. The extent of collagen breakdown is determined by the Moore & Stein (Biol Chem.. 1948, 176, 367) colorimetric ninhydrin method. Amino acids liberated are expressed as μmoles leucine per mg collagenase. One unit equals one μmole of 1-leucine equivalents from collagen in 5 hours at 37 ⁰ C and pH 7.5. For each sample a tube containing 5 mg of native collagen and 1 ml of TES 0.05M pH 7.5 was prepared. Tube was wormed at 37°C, 0.1 ml of sample added and incubated at 37°C for 5 hours. After incubation 0.2 ml of reaction mix (except entire collagen) were transferred into 1 ml of 2% ninhydrin solution and incubated in boiling water bath for 20 minutes. After cooling, mixex were diluted with 50% n-propanol, let stand for 15 minutes and then red at 570 nm. Collagenase activities in tested sample were calculated by interpolation of obtained reading on a standard curve with scalar collagenase units. Results are illustrated in Table III. TABLE III - Collagenase inhibition of resveratrol (fixed cone.) and NAC (variable cone.)

Collagenase Units % Inhibition

2 h 4 h 24 h

Resveratrol 5 μM 0.87 0.81 0.85 -22.47%

Resveratrol 5 μM + NAC 10 μM 0.83 0.83 0.81 -26.08%

Resveratrol 5 μM + NAC 100 μM 0.51 0.51 0.52 -48.08%

Resveratrol 5 μM + NAC 1000 μM 0.58 0.60 0.58 -42.33%

Composition Example 1 - Tablets with resveratrol, NAC and phytoestrogens

Ingredient Quantity per unit

Resveratrol mg 25

N- Acetyl-cysteine mg 100

Soy isoflavones mg 60 Linseed lignans mg 25 Eccipients for granulation q.b. to 1 g

Composition Example 2 - Tablets with resveratrol, NAC and astaxanthin Ingredient Quantity per unit

Resveratrol mg25

N- Acetyl-cysteine mglOO

Astaxanthin 5% mg80

Excipients for granulation q.b. to 0.7 g

Composition Examples 3-4 - Capsules with resveratrol and various cysteine derivatives Ingredient Quantity per unit

Resveratrol mg25 mg25

N- Acetyl-cysteine - mg250

1-Cysteine HCl H2O mgl25 -

S-Carboxymethyl-cysteine mgl25 -

Citric acid - mg30

Tocoferyl acetate mg20 mg20

Soy isoflavones mg60 mg60

Linseed lignans mg30 mg30 α-Lipoic acid mglO mg 10

Hard-capusle of gelatine of 0-side as primary package.

Composition Examples 5-6 - Sachets with resveratrol, cysteine derivatives and phytosterols Ingredient Quantity per unit

Resveratrol mg30 mg30

S-Carboxymethyl-cysteine mgl25

N-Acetyl-cysteine - mgl50

Soy isoflavones mg60 mg60 β-Sytosterol mg25 -

Cumestans mg30 mg30 Ascorbyl palmitate mg 100 mg 100 Excipients for free-flowing powder q.b. q.b.

Composition Examples 7-8 - Capsules with resveratrol, cysteine derivatives and other micro- nutrients

Ingredient Quantity per unit

Resveratrol mg25 mg25

S-Carboxymethyl-cysteine mglOO

N-Acetyl-cysteine - mglOO Phosphatidylcholine mg50 mg50

Phosphatidylserine mg50 mg50

Glycero-phosphorylcholine mg50 mg50

VitE mglO mglO

Selenium μglO μglO Magnesium chloride mg50 mg50

Zinc orotate mg30 ms50

Hard-capusle of gelatine of 0-side as primary package.