Abstract:
The invention relates to the use of spermidine or a pharmaceutically acceptable derivative thereof as the active principle in a pharmaceutical, cosmetic or dietetic composition. The composition is used for promoting pigmentation of the hair, particularly the shaft of the hair. The invention also relates to the composition which promotes this pigmentation effect, the composition containing spermidine or a derivative thereof (such as a salt) as an active principle and is intended for topical or oral administration.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to hair pigmentation in man. 
       BACKGROUND ART 
       [0002]    The human hair follicle is a complex organ wherein there are interactions of epithelial (for example, different lines of keratinocytes, endothelium), mesenchymal (for example dermal papilla fibroblasts, connective tissue sheath fibroblasts), neuroectodermal (nerves, melanocytes) cellular populations and transitory migrating cells (immune cells, mastocytes). 
         [0003]    The growth and pigmentation of hair fibers are affected by several intrinsic factors comprising changes depending on hair cycle, body distribution, age and genre differences, variable hormone sensitivity, genetic defects and age-related changes. The study of hair growth is also complicated by the effects of extrinsic variables that comprise climate and seasons, polluting substances, toxins and exposure to chemicals. The differences found between the pigmentation regulation in the epidermis and in hair follicles reflect the division into compartments of the mammal skin pigmentation system. 
         [0004]    The melanocytes of epidermis, of the hair follicle bulb and of the sheath of the hair follicle outer root are very different from each other, despite the fact that the skin pigmentation in mammals must be understood as an open system. The major differences are those of the nature of their respective melanocyte-keratinocyte functional units. The melamine unit of the hair bulb is found in the proximal anagen bulb, which is an immunologically distinct region of skin and overall, is formed by one melanocyte every 5 keratinocytes in the hair bulb and of one melanocyte for each keratinocyte in the basal layer of the hair bulb matrix. On the contrary, each epidermal melanocyte is associated to 36 vital keratinocytes in the immunocompetent epidermal melamine unit. 
         [0005]    However, the most obvious difference between these two melanocyte skin populations and with considerable implications for the regulation of the hair pigmentation, is the observation that the hair bulb melanocyte activity is subject to a cyclic control and that melanogenesis is strictly associated to the hair growth cycle. On the other hand, the skin melanogenesis appears to be continuous. 
       SUMMARY OF THE INVENTION 
       [0006]    It has now surprisingly been found, and this is the object of the present invention, that 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 melanogenesis activity towards hair and can therefore be effectively used for promoting their pigmentation, in particular the shaft pigmentation. 
         [0007]    Such activity allows configuring the use of the active compound in man as a natural pigmentation agent free from negative side effects, for example typical of hair dies. 
         [0008]    The object of the invention is also a pharmaceutical or cosmetic or dietetic composition suitable to promote such pigmentation effect and therefore containing spermidine as active principle, as such or in the form of a pharmaceutically acceptable derivative such as a salt, for either topical or oral administration. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0009]    A preferred salt according to the invention is spermidine trichlorohydrate, namely N-(3-aminopropyl)butan-1,4-diamine.3HCl. A composition of the invention preferably comprises spermidine trichlorohydrate in a solution formulated for topical use. Suitable forms for topical use are, for example, a lotion, a conditioner, a shampoo, a mask. 
         [0010]    A different composition of the invention preferably comprises spermidine trichlorohydrate in administration unit formulated for oral use. Suitable forms for oral use for example are a tablet or a capsule, either coated or not, or a granulate to disperse in water or other liquid. 
         [0011]    Spermidine, as such or in the form of a pharmaceutically acceptable derivative, as a salt, is contained in a composition of the invention according to an amount preferably comprised within the following ranges:
       10 −7  to 1 g/100 ml, corresponding to 0.004 to 4·10 4  μM   10 −5  to 1 g/100 ml, corresponding to 0.4 to 4·10 4  μM   10 −4  to 2.4·10 −2  g/100 ml, corresponding to 4 to 9·10 2  μM.       
 
         [0015]    Further preferred concentration ranges are as follows:
       10 −6  to 10 −1  g/100 ml   10 −5  to 10 −2  g/100 ml   10 −4  to 10 −3  g/100 ml   10 −7  to 10 −6  g/100 ml   10 −6  to 10 −5  g/100 ml   10 −5  to 10 −4  g/100 ml   10 −4  to 10 −3  g/100 ml   10 −3  to 10 −2  g/100 ml   10 −2  to 10 −1  g/100 ml   10 −1  to 1 g/100 ml       
 
         [0026]    The following formulation examples illustrate the invention, but are not intended to be limiting in any manner. The component amounts are expressed in grams or milligrams and in the case of examples 1 to 4, by concentration ranges. 
       Example 1 
       [0027]      
         [0000]    
       
         
               
             
               
             
               
               
               
             
               
               
             
           
               
                   
               
               
                 Shampoo 
               
             
          
           
               
                 Composition for 100 ml solution 
               
               
                 Component (INCI nomenclature) 
               
               
                   
               
             
          
           
               
                 Magnesium laureth sulfate 
                 2-10 
                 g 
               
               
                 Sodium lauroyl sarcosinate 
                 2-10 
                 g 
               
               
                 Disodium laureth sulfosuccinate 
                 0.5-5 
                 g 
               
               
                 PEG-200 hydrogenated glyceryl palmate 
                 0.5-5 
                 g 
               
               
                 Cocamide MIPA 
                 0.5-5 
                 g 
               
               
                 Glycol distearate 
                 0.5-5 
                 g 
               
               
                 Glycerin 
                 0.5-5 
                 g 
               
               
                 Laureth-7 
                 0.1-3 
                 g 
               
               
                 PEG-7 glyceryl cocoate 
                 0.1-3 
                 g 
               
               
                 Lauryl methyl gluceth-10 hydroxypropyldimonium 
                 0.1-3 
                 g 
               
               
                 chloride 
               
               
                 Polyquaternium-10 
                 0.1-3 
                 g 
               
               
                 Potassium undecilenoyl wheat protein 
                 0.1-3 
                 g 
               
               
                 Panthenol 
                 0.1-3 
                 g 
               
               
                 Tetrasodium EDTA 
                 0.1-3 
                 g 
               
               
                 Spermidine trihydrochloride 
                 10 −7 -1 
                 g 
               
               
                 Preservative 
                 q.s. 
               
               
                 pH corrector (to a final pH of 5.0-5.5) 
                 q.s. 
               
               
                 Parfum 
                 q.s. 
               
             
          
           
               
                 Aqua 
                 q.s. to 100 ml 
               
               
                   
               
             
          
         
       
     
       Example 2 
       [0028]      
         [0000]    
       
         
               
             
               
             
               
               
               
             
               
               
             
           
               
                   
               
               
                 Hair mask 
               
             
          
           
               
                 Composition for 100 ml solution 
               
               
                 Component (INCI) 
               
               
                   
               
             
          
           
               
                 Glycerin 
                 1-10 
                 g 
               
               
                 Ammonium acrylolyl-dimethyltaurate/vp copolymer 
                 1-10 
                 g 
               
               
                 Cyclopentasiloxane 
                 1-10 
                 g 
               
               
                 Silicone quaternium-15 
                 0.1-3 
                 g 
               
               
                 Tocopheryl acetate 
                 0.1-3 
                 g 
               
               
                 Dimethicone 
                 0.1-3 
                 g 
               
               
                 Sericin 
                 0.1-3 
                 g 
               
               
                 Methylparaben 
                 0.05-0.1 
                 g 
               
               
                 C11-15 pareth-5 
                 0.05-0.1 
                 g 
               
               
                 C11-15 pareth-9 
                 0.05-0.1 
                 g 
               
               
                 Trideceth-12 
                 0.05-0.1 
                 g 
               
               
                 Decyl glucoside 
                 0.01-0.5 
                 g 
               
               
                 Panthenyl ethyl ether 
                 0.01-0.5 
                 g 
               
               
                 Disodium EDTA 
                 0.01-0.5 
                 g 
               
               
                 Ethyl hexyl methoxycinnamate 
                 0.01-0.5 
                 g 
               
               
                 Lactic acid 
                 q.s. 
               
               
                 Preservative 
                 q.s. 
               
               
                 Spermidine trihydrochloride 
                 10 −7 -1 
                 g 
               
               
                 Parfum 
                 q.s. 
                 g 
               
             
          
           
               
                 Aqua 
                 q.s. to 100 ml 
               
               
                   
               
             
          
         
       
     
       Example 3 
       [0029]      
         [0000]    
       
         
               
             
               
             
               
               
               
             
               
               
             
           
               
                   
               
               
                 Hair conditioner 
               
             
          
           
               
                 Composition for 100 ml solution 
               
               
                 Component (INCI) 
               
               
                   
               
             
          
           
               
                 Cetearyl alcohol 
                 1-10 
                 g 
               
               
                 Glyceryl stearate 
                 1-10 
                 g 
               
               
                 Dimethicone 
                 1-10 
                 g 
               
               
                 C12-13 alkyl lactate 
                 0.5-5 
                 g 
               
               
                 Cetrimonium chloride 
                 0.5-5 
                 g 
               
               
                 PEG-100 stearate 
                 0.5-5 
                 g 
               
               
                 Cyclopentasiloxane 
                 0.5-5 
                 g 
               
               
                 Hydroxyethylcellulose 
                 0.1-3 
                 g 
               
               
                 Dimethiconol 
                 0.1-3 
                 g 
               
               
                 Panthenol 
                 0.1-3 
                 g 
               
               
                 Bis-isobutyl Peg/Ppg-20/35/amodimethicone copolymer 
                 0.05-2 
                 g 
               
               
                 Phytantriol 
                 0.05-2 
                 g 
               
               
                 Cetyl ethylhexanoate 
                 0.05-2 
                 g 
               
               
                 Butylene glycol 
                 0.05-2 
                 g 
               
               
                 Disodium edta 
                 0.05-2 
                 g 
               
               
                 Polysorbate 80 
                 0.05-2 
                 g 
               
               
                 Sericin 
                 0.05-2 
                 g 
               
               
                 Spermidine trihydrochloride 
                 10 −7 -1 
                 g 
               
               
                 Preservative 
                 q.s. 
                 g 
               
               
                 Parfum 
                 q.s. 
                 g 
               
             
          
           
               
                 Aqua 
                 q.s. to 100 ml 
               
               
                   
               
             
          
         
       
     
       Example 4 
       [0030]      
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
             
               
               
               
             
           
               
                   
               
               
                 Hair lotion 
               
             
          
           
               
                   
                 Composition for 100 ml solution 
               
               
                   
                 Component (INCI) 
               
               
                   
                   
               
             
          
           
               
                   
                 Alcohol 
                 10-20 
                 g 
               
               
                   
                 PEG-40 Hydrogenated Castor Oil 
                 0.2-2 
                 g 
               
               
                   
                 Disodium EDTA 
                 0.01-0.5 
                 g 
               
               
                   
                 Parfum 
                 q.s. 
               
               
                   
                 Spermidine trihydrochloride 
                 10 −7 -1 
                 g 
               
             
          
           
               
                   
                 Aqua 
                 q.s. to 100 ml 
               
               
                   
                   
               
             
          
         
       
     
       Example 5 
       [0031]      
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
             
           
               
                   
               
               
                 Hard gelatine capsules 
               
             
          
           
               
                   
                 Composition for a single capsule 
               
               
                   
                 Component 
               
               
                   
                   
               
             
          
           
               
                   
                 Lactose Monohydrate 
                 85 
                 mg 
               
               
                   
                 Corn starch 
                 25 
                 mg 
               
               
                   
                 Talc 
                 5 
                 mg 
               
               
                   
                 Spermidine trihydrochloride 
                 1.25 
                 mg 
               
               
                   
                 Magnesium stearate 
                 1.5 
                 mg 
               
               
                   
                 Hard gelatine capsule shell N. 4 
                 1 
                 n. 
               
               
                   
                   
               
             
          
         
       
     
       Example 6 
       [0032]      
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
             
           
               
                   
               
               
                 Soft gelatine capsules 
               
             
          
           
               
                   
                 Composition for a single capsule 
               
               
                   
                 Component 
               
               
                   
                   
               
             
          
           
               
                   
                 Soy oil 
                 263.07 
                 mg 
               
               
                   
                 Gelatine 
                 129.7 
                 mg 
               
               
                   
                 Glycerine 
                 36.5 
                 mg 
               
               
                   
                 Sorbitol 70% 
                 24.2 
                 mg 
               
               
                   
                 Water 
                 23.274 
                 mg 
               
               
                   
                 Yellow wax 
                 22 
                 mg 
               
               
                   
                 Fatty acids mono-diglycerides 
                 20 
                 mg 
               
               
                   
                 Soy lecithin 
                 10 
                 mg 
               
               
                   
                 Titanium Dioxide 
                 0.686 
                 mg 
               
               
                   
                 Spermidine trihydrochloride 
                 0.25 
                 mg 
               
               
                   
                   
               
             
          
         
       
     
       Example 7 
       [0033]      
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
             
           
               
                   
               
               
                 Tablet 
               
             
          
           
               
                   
                 Composition for a single tablet 
               
               
                   
                 Component 
               
               
                   
                   
               
             
          
           
               
                   
                 Microcrystalline cellulose 
                 168.97 
                 mg 
               
               
                   
                 Lactose 
                 150 
                 mg 
               
               
                   
                 Methylcellulose 
                 45 
                 mg 
               
               
                   
                 Mono- and diglycerides of fatty acids 
                 9 
                 mg 
               
               
                   
                 Colloidal silicon dioxide 
                 8 
                 mg 
               
               
                   
                 Magnesium stearate 
                 2 
                 mg 
               
               
                   
                 Spermidine trihydrochloride 
                 1.0 
                 mg 
               
               
                   
                   
               
             
          
         
       
     
       Example 8 
       [0034]      
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
             
           
               
                   
               
               
                 Slow-release coated tablet 
               
             
          
           
               
                   
                 Composition for a single tablet 
               
               
                   
                 Component 
               
               
                   
                   
               
             
          
           
               
                   
                 Microcrystalline cellulose 
                 105 
                 mg 
               
               
                   
                 Calcium Phosphate bibasic dihydrate 
                 85 
                 mg 
               
               
                   
                 Hydroxypropylmethylcellulose K100 
                 45 
                 mg 
               
               
                   
                 Sepifilm TM LP 770 white 
                 15 
                 mg 
               
               
                   
                 Magnesium stearate 
                 8 
                 mg 
               
               
                   
                 Hydroxypropylmethylcellulose 
                 6 
                 mg 
               
               
                   
                 Colloidal silicon dioxide 
                 3.5 
                 mg 
               
               
                   
                 Spermidine trihydrochloride 
                 0.55 
                 mg 
               
               
                   
                   
               
             
          
         
       
     
       Example 9 
       [0035]      
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
             
           
               
                   
               
               
                 Effervescent granulate in sachet for making an improptu solution 
               
             
          
           
               
                   
                 Composition for a single sachet 
               
               
                   
                 Component 
               
               
                   
                   
               
             
          
           
               
                   
                 Mannitol 
                 960 
                 mg 
               
               
                   
                 Tartaric acid 
                 530 
                 mg 
               
               
                   
                 Anhydrous sodium bicarbonate 
                 280 
                 mg 
               
               
                   
                 Flavor 
                 130 
                 mg 
               
               
                   
                 Polyvinyl pyrrolidone 
                 45 
                 mg 
               
               
                   
                 Trometamol 
                 32 
                 mg 
               
               
                   
                 Aspartame 
                 20 
                 mg 
               
               
                   
                 Spermidine trihydrochloride 
                 1.25 
                 mg 
               
               
                   
                 Anhydrous colloidal silicon 
                 2 
                 mg 
               
               
                   
                   
               
             
          
         
       
     
         [0036]    Below is the description of an experimental study relating to the activity in the use of spermidine according to the present invention. 
       Activity Study 
     Tissue Samples 
       [0037]    The skin of normal human scalp was taken from a woman who had undergone a routine face lifting surgery after receiving the informed consent. All the experiments were carried out according to the Helsinki principles, with the ethical committee&#39;s approval. 
         [0000]    Skin Organ Culture with Complete Thickness 
         [0038]    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 IU ml −1  penicillin, 10 μg ml −1  streptomycin (Gibco, Karlsruhe, Germany), 10 μg ml −1  insulin (Sigma, Taukfirchen, Germany), 10 ng ml −1  hydrocortisone (Sigma) and 2 mmol L −1  (L-glutamine (Invitrogen, Paisley, U.K.). 
         [0039]    Spermidine trichlorohydrate, or the vehicle as a reference substance, was then administered at a concentration of 0.1 μM, once at each medium change (i.e., every 48 hours). 
       Micro-Dissection of Hair Follicles and Organ Culture 
       [0040]    The hair follicles (HF) in anagen VI phase with normal pigmentation (gray/white hair follicles were excluded from the study) were micro-dissected from normal human scalp skin and subject to organ culture based on the Philpott model. Spermidine or the vehicle were administered once at each medium change (i.e., every 48 hours). 
       LDH Measurement 
       [0041]    The LDH activity in the supernatant served as a cytotoxicity indicator and was measured every day according to the manufacturer&#39;s instructions (Cytotoxicity Detection Kit; Roche, Mannheim, Germany). The sample absorbance was measured at 490 nm using an ELISA plate reader. 
       Hair Shaft Elongation 
       [0042]    The measurements of the hair follicle shaft length were taken every second day on the single hair follicles using a Zeiss inverted binocular microscope with an ocular measurement reticle. 
       Determination of the Hair Follicle Cycle Stage 
       [0043]    The determination of the hair follicle cycle stage was carried out based on the morphological criteria defined before, and the percentage of hair follicles in anagen phase and in early, intermediate or late catagen phase was determined. 
       Hair Pigmentation 
       [0044]    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., Ito 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). 
         [0045]    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). 
         [0046]    The staining intensity was analyzed in a defined reference region of the hair follicle pigmentation unit using the ImageJ software (National Institute of Health). 
       Proliferation and Apoptosis Measurement 
       [0047]    In order to evaluate the apoptotic cells in co-location with a proliferation marker Ki-67, 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, N.Y.) 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 hair follicle and skin matrix and the percentage of positive Ki-67/TUNEL cells was determined. 
       Quantitative Immunohistochemistry of K15 
       [0048]    The tyramide signal amplification method described before was used for examining the expression of keratin K15 (Kloepper et al., 2008). In brief, cryosections fixed with acetone were washed three times for 5 minutes using the TNT (tris-HCL NaCl Tween) buffer (0.1 mol/l Tris-HCl, pH 7.5; containing 0.15 mol/l NaCl 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. 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, Mass., 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 defined in hair follicles was measured and compared between the control groups treated with vehicle only and the groups treated with spermidine. 
       Statistical Analysis 
       [0049]    The statistical analysis was carried out using a bilateral Student t-test for unpaired samples. 
       Results 
       [0050]    The figures of the annexed drawings show the results of the experimental study described. 
       BRIEF DESCRIPTION OF THE FIGURES 
       [0051]      FIG. 1  shows a diagram relating to the pigmentation intensity in hair follicles as measured and compared between the control group treated with vehicle only and the group treated with spermidine 3HCl in a concentration of 0.1 μM. 
         [0052]      FIG. 2  shows the corresponding images taken from the hystochemical display of melanin through Masson-Fontana staining. 
         [0053]    The increase of melanin is clear from both figures in the case of treatment with spermidine, therefore a significant melanogenesis activity towards hair treated with such compound compared to the reference vehicle.