Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
  • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
  • A61K8/41—Amines

Definitions

• the present invention relates to the use of a skin bleaching composition.
• melanin characterize a large number of skin diseases, such as acquired hyperpigmentation, for example melasma, post-inflammatory melanodermia, sun freckles.
• Skin and dermal hyperpigmentation may depend on a larger number of melanocytes or on the activity of melanogenic enzymes.
• An ideal depigmenting compound must have a bleaching and powerful, quick and selective effect on hyperactivated melanocytes and no short- or long-term side effects and lead to a removal of the undesired pigment acting in one or more steps of the pigmenting process.
• the spermidine compound that is N-(3-aminopropyl)butan-1 ,4-diamine, as such or in the form of a pharmaceutically acceptable derivative such as a salt, is provided with a pigmentation inhibition activity in the skin basal layer, and can therefore be effectively used to promote a skin bleaching effect.
• Such activity allows configuring the use of the active compound in man as a natural skin depigmenting agent free from negative side effects.
• the object of the invention is also a pharmaceutical or cosmetic or dietetic composition suitable to promote such skin bleaching effect and therefore containing spermidine as active principle, as such or in the form of a pharmaceutically acceptable derivative such as a salt.
• a preferred salt according to the invention is spermidine trichlorohydrate, namely N-(3-aminopropyl)butan-1 ,4-diamine ⁇ 3HCI.
• a composition of the invention preferably comprises spermidine trichlorohydrate formulated for topical use on the skin.
• Suitable forms for topical use are, for example, a cream or a mask or an emulsion or a solution.
• Spermidine as such or in the form of a pharmaceutically acceptable derivative, as a salt, is contained in a composition of the invention formulated for topical use according to a concentration preferably comprised between 10 '5 and 1 g/100 ml, corresponding to 0.4 to 4-10 4 ⁇ M.
• composition of the invention comprises spermidine trichlorohydrate formulated for oral use.
• Spermidine as such or in the form of a pharmaceutically acceptable derivative, as a salt, is contained in a composition of the invention formulated for oral use according to an amount comprised between 0.125 and 25 mg spermidine as such, or base, per single administration unit.
• the component amounts are expressed in grams or milligrams and in the case of examples 1 to 3, by concentration ranges.
• the tissues subject to biopsy with 3-4 mm cylindrical scalpel were cultured at 37°C for 6 days in Williams E medium (Biochrom, Cambridge, U.K.), integrated with 100
• Spermidine HCI or the vehicle as a reference substance, was then administered at a concentration of 0.1 ⁇ M; 0.5 ⁇ M; 1 ⁇ M once at each medium change (i.e., every 48 hours).
• the Masson-Fontana staining was carried out for the histochemical display of melanin on frozen sections. Melanin was stained in the form of brown granules and the pigmentation level was determined through the quantitative Masson- Fontana technique (Ito N., lto T., Kromminga A., Bettermann A., Takigawa M., Kees F., Straub R. H., and Paus R. (2005): Human hair follicles display a functional equivalent of the hypothalamic-pituitary-adrenal axis and synthesize Cortisol. FASEB J 19, 1332-4).
• This method is a particularly sensitive and reliable indicator of melanin synthesis variations, as proven by enzymatic activity assays and standard tyrosinase expression (Kauser S., Slominski A., Wei E. T., and Tobin D. J. (2006): Modulation of the human hair follicle pigmentary unit by corticotropin-releasing hormone and urocortin peptides. FASEB J 20, 882-95).
• the staining intensity was analyzed in a defined reference region of the skin pigmentation unit using the ImageJ software (National Institute of Health).
• the TUNEL (terminal dUTP nick-end labeling) dual staining method with Ki-67 was used.
• the cryostat sections were fixed in paraformaldehyde and ethanol- acetic acid (2:1 ) and marked with a digoxigenin-deoxy-UTP (kit for the identification of apoptosis in situ with ApopTag fluorescein; Intergen, Purchase, NY) in the presence of terminal deoxynucleotidyl transferase, followed by incubation with a murine anti-Ki-67 antiserum (1 :20 in PBS overnight at 4°C; Dako, Glostrup, Denmark).
• TUNEL-positive cells were displayed by a conjugate isothiocyanate fluorescein anti digoxigenin antibody (kit ApopTag), whereas Ki-67 was detected by a goat anti-mouse antibody marked with rhodamine (Jackson ImmunoResearch, West Grove, PA). Negative controls were carried out omitting the terminal deoxynucleotidyl transferase and the Ki-67 antibody. The counter- staining was carried out with 4',6-diamidino-2-phenylindole (DAPI) (Roche Molecular Biochemicals GmbH, Mannheim, Germany). The quantitative histomorphometric assessment was carried out; Ki-67, TUNEL or DAPI-positive cells were counted in a reference region defined beforehand of the skin matrix and the percentage of positive Ki-67/TUNEL cells was determined.
• DAPI 4',6-diamidino-2-phenylindole
• tyramide signal amplification method described before was used for examining the expression of keratin K15 (Kloepper et al., 2008).
• cryosections fixed with acetone were washed three times for 5 minutes using the TNT (tris-HCL NaCI Tween) buffer (0.1 mol/l Tris-HCI, pH 7.5; containing 0.15 mol/l NaCI and 0.05% Tween 20). Radish peroxidase was then blocked through wash with 3% H 2 O 2 in an isotonic phosphate buffer (PBS) for 15 minutes.
• PBS isotonic phosphate buffer
• Preincubation was carried out with the incubation of avidin and biotin for 15 minutes and with 5% normal goat serum in TNT for 30 minutes with intermediate washing steps.
• Murine anti-human K15 (clone LHK15, Chemicon, Billerica, USA) were diluted in TNT and incubated overnight at 4°C, followed by a secondary goat anti-mouse biotinylate antibody (1 :200 in TNT) for 45 minutes at room temperature. Radish streptavidin-peroxidase was then administered (kit TSA; Perkin-Elmer, Boston, MA, USA) (1 :100 in TNT) for 30 minutes at room temperature. The reaction was amplified with a FITC-tyramide amplification agent at room temperature for 5 minutes (1 :50 in an amplification diluent supplied with the kit). The intensity of this immuno-staining was quantified by the ImageJ (National Institutes of Health) software. The staining intensity of reference regions in the skin basal layer was measured and compared between the control groups treated with vehicle only and the groups treated with spermidine.
• the statistical analysis was carried out using a bilateral Student t-test for unpaired samples.
• Fig. 1 shows the relevant images taken from the hystochemical display of melanin through Masson-Fontana staining, compared between the control groups treated with vehicle only and the groups treated with spermidine HCI at the concentrations of 0.1 ⁇ M; 0.5 ⁇ M; 1 ⁇ M.
• Fig. 2 shows a corresponding diagram relating to the pigmentation intensity found in the skin basal layer.
• a pigmentation inhibition effect is clear from both figures in the case of treatment with spermidine HCI, depending on the concentration and therefore in progressive increase from the concentration of 0.1 ⁇ M to 1 ⁇ M.

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• 2009
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