Patent Application
20160067161
The present invention relates to a cosmetic or pharmaceutical composition for resisting the effects of skin ageing.
The human skin is constantly exposed to air, to solar radiation, to environmental pollutants, physical and chemical agents that are able to induce the production of free radicals in the body.
Oxidative damage made by free radicals is a main cause of physical ageing in general, and of the skin in particular. The ageing process consists in a deterioration of metabolic balance, or homeostasis, accompanied by an alteration of the physiological systems, including the immune system.
One of the targets of oxidative damage within the cell is mitochondrion, which generates most of the energy for cellular requirements and which consumes more than 90% of the oxygen. The process of mitochondrial oxidation is therefore directly associated with ageing.
The changes in the immune function related to advancing age originate from oxidative and inflammatory stress. Numerous studies show that chronic inflammation is the primary responsible for skin ageing.
The two major pro-inflammatory stimuli are the alteration or destruction of the skin barrier and ultraviolet radiation. In general, the environmental insults inducers of free radicals lead to a destruction of the skin barrier that activates the release of TNF-alpha and interleukins IL-1 and IL-8, as well as other pro-inflammatory molecules that induce an acute inflammatory reaction. Acute inflammation does not seem to be an inducer of direct damage to the skin, but it aggravates skin ageing in progress.
On the other hand, the repeated alteration of the skin barrier, even if slight, activates chronic inflammation. Considering this evidence, it seemed possible to slow or delay the skin ageing process by acting on chronic inflammation with treatments based on antioxidants and anti-inflammatory products.
Although at the skin level there are endogenous systems of protection from free radicals, such as melanin and some enzyme and non antioxidant systems, it must be considered that skin antioxidants defense mechanisms during the ageing process decreases physiologically.
This physiological decline adds up to a further important depletion of the pool of endogenous antioxidants due to UV exposure. Faced with this reduction, many studies point out that topical treatment with antioxidants is useful in restoring the defenses against free radicals, in some cases even reversing the ageing process.
WO2005013932 by the same Applicant describes a composition for pharmaceutical, dieting or cosmetic use for human use adapted to slow down the ageing of the skin and skin adnexa, including spermine, spermidine or salts thereof as active principle. In particular, this document describes experimental studies related to the effects of such polyamines on the elasticity, hydration and the cell renewal action of the skin. However, no effects or anti-inflammatory or antioxidants are described or suggested therein by the active compounds spermine, spermidine or salts thereof.
It should also be noted that in the case of topical formulations, spermidine, as well as other polyamines, is subject to oxidation because when applied topically on the skin, before being absorbed by the skin to perform its action, it remains for some time in extended contact with air. A possible oxidation of spermidine during that time before the absorption would lead to oxidation products that are no longer active.
According to the present invention, it has now been surprisingly found that the effects of skin ageing may be resisted through an anti-inflammatory action using a compound of general formula (I): R—N1-spermidine as defined below, as such or in the form of a pharmaceutically acceptable derivative such as a salt.
The object of the invention is the use of compounds of formula (I) R—N1-spermidine, i.e. 1.4-butanediamine,N-(3-aminopropyl)-N1—R,
H2N—(CH2)3—N1(R)—(CH2)4—NH2 (I)
wherein R is a substituent bound to the secondary amine function of spermidine, selected from:
saturated or unsaturated, linear or branched alkyl groups consisting of 1 to 6 carbon atoms, wherein optionally one or more carbon atoms are substituted by fluorine, such as methyl, ethyl, trifluoromethyl, trifluoroethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, ethylene, vinyl, propylene, butylene;
aryl or arylalkyl groups such as naphthyl, phenyl, benzyl, tolyl, wherein optionally one or more carbon atoms are substituted by fluorine, and wherein said arylalkyl groups include saturated or unsaturated, linear or branched alkyl groups consisting of 1 to 6 carbon atoms, wherein optionally one or more carbon atoms are substituted by fluorine, such as methyl, ethyl, trifluoromethyl, trifluoroethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, ethylene, vinyl, propylene, butylene;
saturated or unsaturated cycloalkyl groups consisting of 3 to 8 carbon atoms, optionally substituted with saturated or unsaturated, linear or branched alkyl groups consisting of 1 to 6 carbon atoms, wherein optionally one or more carbon atoms are substituted by fluorine, such as methyl, ethyl, trifluoromethyl, trifluoroethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, ethylene, vinyl, propylene, butylene; or a corresponding pharmaceutically acceptable salt,
such use being directed at resisting the effects of skin ageing through an anti-inflammatory action.
As shown in the experimental part below in the present description, such anti-inflammatory activity is also accompanied by an antioxidant activity against oxygen free radicals (ROS), comparable with that of α-tocopherol.
Moreover, the compounds of general formula (I) are both active according to the purposes of the present invention, and stable to air so as to allow an efficient application for topical use on the skin without being transformed into a different, not active substance due to the effect of oxidation.
Such compounds of general formula (I), being therefore provided with an anti-inflammatory activity and slowing the skin ageing processes, can be effectively used for topical application on the skin in the form of suitable compositions for cosmetic or pharmaceutical use for the achievement of such effects.
Therefore, an object of the invention is also a composition for, pharmaceutical or cosmetic use adapted to carry out this anti-inflammatory effect and slowing the skin ageing processes and containing at least one compound of formula (I) as active principle as such or in the form of a pharmaceutically acceptable derivative such as a salt, for topical administration on the skin.
Suitable forms for topical use are, for example, a cream, a serum, a balm, a mask, a gel for the face or the body.
If said compound of formula (I) is in the form of a pharmaceutically acceptable derivative such as a salt, it is preferably a maleic acid salt, such as trimaleate, or a hydrochloric acid salt, such as trichydrochloride.
Any other organic or inorganic acid salt pharmaceutically acceptable for a formulation for topical application is suitable.
A preferred compound of formula (I) according to the present invention is N1-methylspermidine, or N-(3-aminopropyl)-N1-methyl-1,4-butanediamine (CAS Registry Number 51460-23-2), of formula:
H2N—(CH2)3—N1(CH3)—(CH2)4—NH2 (II)
used in a composition according to the invention as such or as a pharmaceutically acceptable salt, such as trimaleate (3C4H4O4) or trihydrochloride (3HCl).
A further preferred compound of formula (I) according to the present invention is N1-cyclohexylspermidine, or N-(3-aminopropyl)-N1-cyclohexyl-1,4-butanediamine (CAS Registry Number 183070-28-2), of formula:
H2N—(CH2)3—N1(C6H11)—(CH2)4—NH2 (III)
used in a composition according to the invention as such or as a pharmaceutically acceptable salt, such as trimaleate (3C4H4O4) or trihydrochloride (3HCl).
Further preferred compounds of formula (I) according to the present invention are: N1-ethylspermidine, of formula
H2N—(CH2)3—N1(C2H5)—(CH2)4—NH2 (IV)
N1-propylspermidine, of formula
H2N —(CH2)3—N1(C3H7)—(CH2)4—NH2 (V)
N1-isobutylspermidine, of formula
H2N—(CH2)3—N1(C4H9)—(CH2)4—NH2 (VI)
A compound of formula (I), as such or in the form of pharmaceutically acceptable derivative such as a salt, is contained in a composition of the invention in an amount preferably within the following ranges in percentage by weight, w/w (%): 0.0001 to 0.30; preferably from 0.010 to 0.30 and from 0.0001 to 0.15.
Compositions according to the invention formulated for topical use on the skin of the body or face are described hereinafter, by way of non-limiting examples.
The amounts of the components, indicated by the INCl nomenclature, are expressed as a percentage by weight variable within the ranges indicated therein.
Limnanthes alba seed oil
Vitis vinifera seed extract
Helianthus annuus seed oil
Olea europaea leaf extract
Citrus Aurantium Amara flowfer water
Limnanthes alba seed oil
Butyrospermum parkii butter
Butyrospermum parkii
Limnanthes alba seed oil
Limnanthes alba seed oil
Experimental Tests
The following tests were carried out in order to study the activity of the compounds of formula (I) for the proposed use according to the present invention.
Description of the Drawings
The results of these tests are shown in the graphs in
Anti-Inflammatory Test
With reference to
Culture and Propagation of the Cell Line
An immortalized line of human keratinocytes NCTC2544 is used (Perry, V. P., Sanford, K. K., Evans, V. J., Hyatt, G. W., Earle, W. R., 1957: Establishment of clones of epithelial cells from human skin. J Natl Cancer Inst. 18 (5): 709-717) cultured in sterile flasks (25 cm3), incubated at 37° C. in a humid atmosphere at 5% CO2 in MEM (Minimum Essential Medium) added with bovine fetal serum (FBS), 2 mm L-glutamine, 1% non-essential amino acids, in the presence of 1% penicillin and streptomycin.
A 1:3 split is done every 2 days upon achieving the monolayer by washing with 1× PBS (phosphate buffer without Ca2+ and Mg2+) and detachment of cells with a trypsin-EDTA solution at 37° C. for 2 minutes.
Control
Negative control: human keratinocytes NCTC2544 cultured in EMEM (EBSS) at 2.5% FBS, supplemented with 1% L-glutamine 2 mM, 1% solution of amino acids and 1% mixture of penicillin (10,000 U/ml)/streptomycin (10,000 Ug/ml) at 37° C., 5% CO2. Positive control: human keratinocytes NCTC2544 treated with lipopolysaccharide (LPS, 5 μg/ml) in EMEM (EBSS) at 2.5% FBS, supplemented with 1% L-glutamine 2 mM, 1% solution of amino acids and 1% mixture of penicillin (10,000 U/ml)/streptomycin (10,000 Ug/ml) at 37° C., 5% CO2.
Methods
The gene expression of TNF-α on NCTC2544 cells treated with the samples was evaluated by qRT-PCR.
The analysis of gene expression involves four steps:
1. Treatment of cells with the samples for 16 and 24 hours;
2. RNA extraction;
3. Retrotranscription in cDNA;
4. Quantitative RT-PCR.
Treatment of NCTC2544 Cells
In the experimental conditions, in relation with the results obtained in a previous MTT test, samples of spermidine and N1-methyl-spermidine were tested at the following concentrations: 1 μM, 500 nM and 1 mM (final concentration in the medium). Said positive and negative controls were also tested.
Anti-Inflammatory Test on NCTC2544
Day 1: Cell Seeding
When the human keratinocyte cells NCTC 2544 have reached about 80% confluence, they are detached with trypsin/EDTA and seeded at a density of 1×106 cells/ml in 12-well plates and incubated at 37° C., 5% CO2 (24 h).
Day 2: Exposure to the Samples for 16 and 24 h
The above samples of spermidine and N1-methyl-spermidine were dissolved in EMEM, supplemented with 2.5% FCS, 1% 2 mM L-glutamine, 1% NEAA solution and 1% penicillin (10,000 U/ml)/streptomycin (10,000 pg/ml).
The controls, containing only culture medium (negative control) and culture medium plus LPS (5 pg/ml) (positive control), were included in each plate. The cells were exposed to the action of the samples of spermidine and N1-methyl-spermidine at the above concentrations of 1 μM, 500 nM and 1 mM: a total of six samples.
LPS at a concentration of 5 μg/ml was added to each well (except in the negative control). Each sample was tested in replicate.
Results
The results are shown in the graphs of
In general, the results obtained show an anti-inflammatory effect of N1-methyl-spemidine at all concentrations examined.
In
In particular, the improved anti-inflammatory effect is remarkable at the concentration of 500 nM.
In
ROS Antioxidant Test
Samples of a compound of the invention, N1-methylspermidine trihydrochloride salt, i.e. N-(3-aminopropyl)-N1-methyl-1,4-butanediamine 3HCl, were subjected to the following antioxidant test to reactive oxygen species (ROS).
The production of ROS was monitored spectrofluorometrically using 2′,7′-dichlorofluorescein diacetate (DCFH-DA), as described by Tobi et al. (Tobi S E, Paul N, T McMillan J. J Photochem Photobiol, B 2000: 57: 102-112).
The NCTC 2544 cells (about 80% confluence) were detached with trypsin/EDTA and seeded at a density of 5×104 cells per well in 96-well plates. Subsequently, the cells were treated with samples of N1-methylspermidine, at the following concentrations: 500 nM, 1 μm and 500 μm, 1 mM (final concentration in the culture medium).
α-tocopherol (250 μg/ml, 580 μm) was used as a comparison of the antioxidant activity.
The plates were incubated at 37° C. in 5% CO2 for 1.5 hours. Cells cultured on basal medium with 2.5% FBS were used as a control.
Results
The results are shown in the graph of
Compared with the control cells treated with H2O2 both cells treated with α-tocopherol and those treated with the compound of the invention at the various concentrations indicated therein showed a decrease substantially comparable to the intracellular concentration of ROS produced. In particular, it may be seen that at the concentration of 500 μm, N1-methylspermidine trichydrochloride salt a shows a higher antioxidant activity than that of α-tocopherol (580 μM).
From the complex of the experimental evidence summarized above, it is noted that the compounds of formula (I) are suitable for a pharmaceutical or cosmetic use aimed at resisting the effects of skin ageing mainly through an anti-inflammatory action.
Such anti-inflammatory activity is also accompanied by an antioxidant activity against oxygen free radicals (ROS) comparable with that of α-tocopherol.
Moreover, the compounds of general formula (I) are both active according to the purposes of the present invention, and stable to air so as to allow an efficient application for topical use on the skin without being transformed into a different, not active substance due to the effect of oxidation.
Anti-Inflammatory Test
With reference to the graphs in
Tested Compounds
N1-ethylspermidine, of formula
H2N—(CH2)3—N1(C2H5)—(CH2)4—NH2 (IV)
N1-propylspermidine, of formula
H2N—(CH2)3—N1(C3H7)—(CH2)4—NH2 (V)
N1-isobutylspermidine, of formula
H2N—(CH2)3—N1(C4H9)—(CH2)4—NH2 (VI)
Controls
The controls containing only culture medium (negative control) and culture medium plus LPS (5 μg/ml) (positive control), were included in each plate.
Tests and Results
The cells were exposed to the action of samples of the compounds N1-ethylspermidine, N1-propylspermidine, N1-isobutylspermidine at the concentrations of 1 nM and 1 μM.
The results are shown in the graphs in
The gene expression data were obtained by RT-PCR. The data are interpreted as relative decrease compared to the treatment with LPS alone and the data expressed as RQ (relative quantification) or as a percentage are directly comparable.
In general, the results obtained show a remarkable anti-inflammatory effect of the compounds of the invention at all the examined concentrations.
| Number | Date | Country | Kind |
|---|---|---|---|
| MI2013A000555 | Apr 2013 | IT | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2014/060555 | 4/9/2014 | WO | 00 |
