COMPOSITION FOR COSMETIC USE SUITABLE TO PRODUCE A PIGMENTATION EFFECT ON HAIR

Abstract

The invention concerns the use of compounds with formula (I) RN1-spermidine, or 1,4-butandiamin,N-(3-amino propyl)-N1-R, (I) H.sub.2N(CH.sub.2).sub.3N.sup.1(R)(CH.sub.2).sub.4NH.sub.2 either as such or in the form of a pharmaceutically acceptable derivative, such as an active ingredient in a cosmetic composition designed to promote hair pigmentation, particularly the pigmentation of the shaft, and a composition suitable to achieve said pigmentation effect and containing said active ingredient, either as such or in the form of a pharmaceutically acceptable derivative such as a salt, for topical administration.

Claims

1-18. (canceled)

19. Method for producing a melanogenetic action in the hair and to promote its pigmentation and pigmentation of the stem, comprising the step of administering to a subject in need thereof an effective dose of a compound of formula (I) RN.sup.1-spermidine, or 1,4-butandiamin,N-(3-amino propyl)-N.sup.1R,
H.sub.2N(CH.sub.2).sub.3N.sup.1(R)(CH.sub.2).sub.4NH.sub.2 (I) wherein R is a substituent that is bound to the secondary amine group of spermidine, chosen from saturated or unsaturated, linear or branched alkyl groups formed from 1 to 6 atoms of carbon, wherein one or more carbon atoms are optionally replaced by fluorine, namely methyl, ethyl, trifluoromethyl, trifluoroethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, ethylene, vinyl, propylene, butylene; aryl or aryl-alkyl groups, such as phenyl, naphthyl, benzyl, tolyl, wherein one or more carbon atoms are optionally replaced by fluorine, and wherein said aryl-alkyl groups include saturated or unsaturated, linear or branched alkyl groups formed by 1 to 6 atoms of carbon, wherein one or more carbon atoms are optionally replaced by fluorine, namely methyl, ethyl, trifluoromethyl, trifluoroethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, ethylene, vinyl, propylene, butylene; saturated or unsaturated cycloalkyl groups formed by 3 to 8 atoms of carbon that are optionally replaced by saturated or unsaturated, linear or branched alkyl groups formed by 1 to 6 atoms of carbon, wherein one or more carbon atoms are optionally replaced by fluorine, namely methyl, ethyl, trifluoromethyl, trifluoroethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, ethylene, vinyl, propylene, butylene; or their pharmaceutically acceptable salt.

20. Method according to claim 19, wherein said formula (I) is applied topically to the scalp.

21. Method according to claim 19, wherein said compound of formula (I) is N.sup.1-methyl-spermidine of formula:
H.sub.2N(CH.sub.2).sub.3N.sup.1(CH.sub.3)(CH.sub.2).sub.4NH.sub.2 (II)

22. Method according to claim 19, wherein said compound of formula (I) is N.sup.1-cyclohexyl-spermidine with formula:
H.sub.2N(CH.sub.2).sub.3N.sup.1(C.sub.6H.sub.11)(CH.sub.2).sub.4NH.sub.2 (III)

23. Method according to claim 19, wherein said compound of formula (I) is N.sup.1-ethyl-spermidine of formula:
H.sub.2N(CH.sub.2).sub.3N.sup.1(C.sub.2H.sub.5)(CH.sub.2).sub.4NH.sub.2 (IV)

24. Method according to claim 19, wherein said compound of formula (I) is N.sup.1-propyl-spermidine of formula:
H.sub.2N(CH.sub.2).sub.3N.sup.1(C.sub.3H.sub.7)(CH.sub.2).sub.4NH.sub.2 (V)

25. Method according to claim 19, wherein said pharmaceutically acceptable salt of said compound with formula (I) is a salt with maleic acid.

26. Method according to claim 19, wherein said pharmaceutically acceptable salt of said compound with formula (I) is a salt with hydrochloric acid.

Description

DESCRIPTION OF THE DRAWINGS

[0053] FIGS. 1 and 2 of the enclosed drawings show the results of an experimental study. FIGS. 3 and 4 of the enclosed drawings show the results of another experimental study.

RESULTS

[0054] FIGS. 1 and 2 of the enclosed drawings show the results of the above-described experimental study.

[0055] N.sup.1-methyl-spermidine is identified herein with the initials MS. The initials Spd identify spermidine used as a control compound.

[0056] FIG. 1 shows a graph of the (absolute) intensity of pigmentation in hair follicles measured and compared between the control group treated only with the carrier, the groups treated with N.sup.1-methyl-spermidine (MS) in a concentration of 0.5 and 1.5 M, respectively, and, lastly, the reference group treated with spermidine (Spd) in a concentration of 0.5 M.

[0057] FIG. 2 shows the corresponding images based on the histochemical study of melanin with the Masson-Fontana stain for the control group treated only with the carrier, and for the groups treated with N.sup.1-methyl-spermidine (MS) in a concentration of 0.5 and 1.5 M, respectively.

[0058] The image of the carrier distinguishes the proximal and distal areas of the pigmented region, as indicated.

[0059] Both FIGS. 1 and 2 highlight the increase in melanin produced in case of treatment with N.sup.1-methyl-spermidine at both concentrations tested in a dose-dependent manner; hence, the considerable melanogenetic activity in hair treated with this compound, compared to the reference carrier.

[0060] The graph in FIG. 1 also shows the higher increase in absolute pigmentation intensity in hair follicles when N.sup.1-methyl-spermidine (MS) is used in a concentration of 0.5 M, compared to the reference group treated with spermidine (Spd) in the same concentration, 0.5 M.

[0061] Enzymatic Assay of Tyrosinase

[0062] FIGS. 3 and 4 of the enclosed drawings show the results of the additional experimental study described below.

[0063] The following compounds with formulae (II), (III), (IV) and (V) were tested as defined above, all in a concentration of 5 mM:

TABLE-US-00007 Methyl-spermidine (base) Formula (II) Ethyl-spermidine Formula (IV) Propyl-spermidine Formula (V) Ciclohexyl-spermidine Formula (III)

[0064] Principle of the Assay

[0065] Tyrosinase is a membrane glycoprotein that is expressed in melanosomes, specialised organelles in the endocytic pathway through which the synthesis and assembly of melanin takes place. The synthesis of melanin from tyrosine takes place in pigment-producing cells, namely melanocytes. Melanin produced in melanosomes is transferred to keratinocytes through said processes. Melanin is a polymer that absorbs light within a broad spectrum of wavelengths, and protects keratinocytes from the harmful effects of UV radiations. A peroxidase is involved in the first phase of the reaction, which converts the amino acid into 2,3-dihydroxyphenyl alanine or DOPA. The enzyme tyrosinase is involved in the subsequent phase of the biosynthesis pathway that produces dopaquinone. Melanin is synthesised starting from precursors of tyrosine and DOPA, through the enzyme tyrosinase, which presents both hydrolase and oxidase functions. Tyrosine is converted into L-DOPA (dihydroxyphenilalanine) and, then, into dopaquinone, which is red, until the brown melanin polymer is obtained. In a first branch of the pathway dopaquinone is converted into black polymeric melanins, while the reaction with cysteine forms a series of polymers that are similar among them and are called red polymeric melanins.

[0066] Materials

[0067] Monobasic potassium phosphate (Sigma P5379-100G)

[0068] L-tyrosine (Sigma T3754-50G)

[0069] Tyrosinase (Sigma T3824-25KU)

[0070] Potassium hydroxide, standard solution (Sigma 35113-1L)

[0071] Water (BioChemika, for molecular biology, DEPC-treated and sterile filtered)

[0072] (Sigma, 95284)

[0073] Instruments

[0074] Cuvettes 100-QS (Helima, Z600105)

[0075] Sterile nozzle tips with filter

[0076] Spectrophotometer (Jenway UVNIS MOD:6715, BS-671560)

[0077] pH-meter 827 pH Lab (Metrhom)

[0078] Procedure

[0079] REAGENT A (potassium phosphate buffer, 50 mM, pH 6.5, 25 C.):

[0080] Correct the pH (approx. 4.5) to pH 6.5 with KOH 1M;

[0081] Store at 4 C. until use.

[0082] REAGENT B (L-tyrosine solution 1 mM):

[0083] Store at 4 C. until use.

[0084] REAGENT C (tyrosinase solution):

[0085] Just before use:

[0086] The enzyme has a volume of xxxx units/mg solid (indicated on the bottle and, therefore, it differs every time a different bottle is used).

[0087] Calculate the corresponding liquid=xxxx units/mL liquid.

[0088] =xxxx units/mL liquid/xxxx units/mg solid=x mg/mL

[0089] Prepare portions of about 1 mL and preserve in aluminium paper at 20 C. until use.

[0090] Prepare a solution A+B as described below:

[0091] 9 mL of deionised water

[0092] 10 mL of Reagent A (potassium phosphate buffer 50 mM)

[0093] 10 mL of Reagent B (L-tyrosine 1 mM)

[0094] Vortex the solution and, if necessary, restore pH 6.5, at 25 C. with HCl 1M or NaOH.

[0095] Prepare the solutions with the various concentrations of the compounds to be tested, and preserve at 4 C. until use.

[0096] Defrost the tyrosinase solution and keep it on ice in the dark until use.

[0097] Mix, at ambient temperature, the solution of potassium phosphate buffer (50 mM, pH 6.5), the above solution A+B and the solutions that include the compounds of the formula (I) to be tested.

[0098] Start up the spectrophotometer for calibration.

[0099] Use the following parameters:

[0100] Reading: 280 nm;

[0101] Measuring mode: absorbance;

[0102] Execution settings: 700 seconds.

[0103] The rest is as per the default procedure.

[0104] First read the enzymatic activity-of-tyrosinase when there is only L-tyrosine to verify the actual enzyme/mL units and have a basic reference to later assess the effects of the compound examined on enzyme activity:

[0105] 1 replicates for the blank test (one blank test for each concentration to be tested);

[0106] 2 replicates for the test (one test for each concentration to be tested).

[0107] Perform a reading of the blank test of the instrument (no cuvette).fwdarw.Cal.

[0108] Prepare the mixtures in separate cuvettes, as described below:

TABLE-US-00008 Order TEST TUBE Blank Test Test 1 Solution A + B 2.90 mL 2.90 mL 2 Reagent A (buffer) 0.1 mL / 2 Reagent C / 0.1 mL (Tyrosinase solution) kept on ice

[0109] Test the examined compounds with formulae (II), (III), (IV) and (V), as specified below:

[0110] 1 replicates for the blank test (one blank test for each concentration to be tested);

[0111] 2 replicates for the test (one test for each concentration to be tested).

[0112] Prepare the mixtures in separate cuvettes, as described below:

TABLE-US-00009 Order TEST TUBE Blank Test Test 1 Solution A + B 2.90 mL 2.80 mL Reagent A (buffer) / / 3 Reagent C / 0.1 mL (Tyrosinase solution) kept on ice 2 Compound with formula (I) 0.1 mL 0.1 mL to be tested

[0113] Calculating the Inhibition or Induction of Enzymatic Activity

[00001]$\mathrm{Units}.Math.\text{/}.Math.\mathrm{mL}.Math..Math.\mathrm{of}.Math..Math.\mathrm{enzyme}=\frac{\left(.Math..Math.A_{280.Math..Math.n.Math..Math.m}/\min .Math..Math.\mathrm{Test}-.Math..Math.A_{280.Math..Math.n.Math..Math.m}/\min .Math..Math.\mathrm{Blank}.Math..Math.\mathrm{Test}\right).Math..Math.\left(\mathrm{df}\right)}{\left(0.001\right).Math..Math.\left(0.1\right).Math.}$

[0114] df=dilution factor (30 because 0.1 mL/3 mL tot)

[0115] 0.001=change of A.sub.280 nm/min per unit of L-tyroxine at pH 6.5, 25 C. in a 3 mL reaction mixture;

[0116] 0.1=volume (mL) of enzyme used

[0117] Unit/mg solid=unit/mL of enzyme: mg solid/mL of enzyme

[0118] Unit/mg protein=unit/mL of enzyme: mg protein/mL of enzyme

[0119] Definition of unit=increment of A.sub.280 nm/min equal to 0.001, at 25 C., pH 6.5 in a 3 mL reaction volume containing L-tyrosine.

[0120] The percentage of enzymatic activity was calculated for each tested compound, compared to the activity of the enzyme (100% enzymatic activity) failing the presence of active compounds.

[0121] The increment percentage of enzymatic activity was later calculated (% enzymatic activity 100).

TABLE-US-00010 Increment in Enzymatic enzymatic activity activity Results (%) (%) Tyrosinase base 100.00 Methyl-spermidine 5 mM 105.16 5.16 Ethyl-spermidine 5 mM 121.50 21.50 Propyl-spermidine 5 mM 131.29 31.29 Cyclohexyl-spermidine 5 mM 135.31 35.31

[0122] Data reported in the above tables are summarised in the graphs in FIGS. 3 and 4.

[0123] FIG. 3 shows the absolute data of enzymatic activity (%).

[0124] FIG. 4 shows the respective increments in enzymatic activity (%) thus measured, compared to the control.

[0125] All tested compounds of formula (I) show an evident increase in the enzymatic activity of tyrosinase and, therefore, in the melanogenetic activity concerned by the invention.