Anti-Aging Compositions

Abstract

The present invention relates to a composition comprising at least one first compound of the formula (I), or a polymer of the first compound and at least one second compound of formula (I), of the following formula (II) or of the following formula (III), the second compound of formula (I) being different from the first compound of formula (I).

Claims

1. A composition comprising at least one first compound of the following formula (I), or a polymer of the first compound: ##STR00048## wherein: R.sub.1 and R.sub.4, identical or different, represent H, OH, or OCH.sub.3, R.sub.2 and R.sub.3, identical or different, represent H, OH, OSO.sub.3H, O-hexoside, or OR.sub.31, OCOR.sub.31 or CH.sub.2OCOR.sub.31, wherein R.sub.31 represents a saturated or unsaturated, linear or branched, aliphatic group comprising from 1 to 30 carbon atoms, optionally substituted by at least one O, OH or COOH group, or R.sub.2 and R.sub.3 represent together a group of the following formula (Ia) or (Ib): ##STR00049## wherein: R.sub.7 represents H, OH, OCH.sub.3 or an aryl group comprising from 5 to 8 carbon atoms optionally substituted by at least one-OH group or oxyalkyl group comprising from 1 to 5 carbon atoms, R.sub.8 represents H, OH or OCH.sub.3, ##STR00050## wherein R.sub.9, R.sub.10, R.sub.11 et R.sub.12, identical or different, represent H, OH or OCH.sub.3, one of R.sub.5 and R.sub.6 represents H, OH or OCH.sub.3 and the other represents a saturated or unsaturated, linear or branched, aliphatic group comprising from 1 to 10 carbon atoms, optionally substituted by at least one O, OH or COOH group, or a group of the following formula (Ic): ##STR00051## wherein n=0 or 1, k=0 or 1, when k=1 the above cycle is aromatic (Ar) and when k=0 the above cycle is not aromatic, m=1 when k=0 and m=0 when k=1, X represents O, CH or CH.sub.2, R.sub.13 represents O, a group R.sub.23 or a group OR.sub.23 wherein R.sub.23 represents a saturated or unsaturated, linear or branched, aliphatic group comprising from 1 to 5 carbon atoms, optionally substituted by at least one O group, R.sub.14 represents H, OH, OCH.sub.3 or a group of the following formula (Id): ##STR00052## wherein R.sub.19, R.sub.20, R.sub.21 and R.sub.22, identical or different, represent OH, CH.sub.2OH, an alkyl group comprising from 1 to 5 carbon atoms or an oxyalkyl group comprising from 1 to 5 carbon atoms, R.sub.15, R.sub.16, R.sub.17 and R.sub.18, identical or different, represent H, OH, OSO.sub.3H, CH.sub.2OH, COOH, O-glucuronide, OR.sub.30, OCOR.sub.30 or CH.sub.2OCOR.sub.30, wherein R.sub.30 represent a saturated or unsaturated, linear or branched, aliphatic group comprising from 1 to 30 carbon atoms, optionally substituted by at least one O, OH or COOH group, and at least one second compound of formula (I), of the following formula (II) or of the following formula (III), the second compound of formula (I) being different from the first compound of formula (I): ##STR00053## wherein: a represents a single bond or a double bond, Y represents O or CO, W represents CH.sub.2 or CO, R.sub.24 represents an aryl group comprising from 5 to 8 carbon atoms, optionally substituted by at least one-OH, OCH.sub.3, O-hexoside or O-glucuronide group, R.sub.25 represents H, OH, or an oxycarbonylaryl group comprising from 6 to 9 carbon atoms, optionally substituted by at least one-OH, OCH.sub.3, O-hexoside or O-glucuronide group, or R.sub.24 and R.sub.25 represent together, with the carbon atoms to which they are attached and a representing a double bond, an aryl group comprising 6 carbon atoms, optionally substituted by at least one-OH or CH.sub.2OH group, R.sub.26, R.sub.27 and R.sub.28, identical or different, represent H, OH, OCH.sub.3, O-hexoside or O-glucuronide, R.sub.29 represents H, OH or a group of the following formula (IIa): ##STR00054## wherein: j=0 or 1, when j=1 the above cycle is aromatic (Ar) and when j=0 the above cycle is not aromatic, m=1 when j=0 and m=0 when j=1, when j=1, R.sub.32 represents an alkyl group having from 1 to 5 carbon atoms and when j=0, R.sub.32 together with R.sub.33 represent a group of the following formula (IIIa): ##STR00055## wherein: n=0 or 1, R.sub.45 represents H or CH.sub.3, R.sub.46 represents CH.sub.2OH or a group of the following formula (IIIb) or (IIIc): ##STR00056## R.sub.47 and R.sub.48, identical or different, represent H or OH R.sub.34, R.sub.35, R.sub.38, and R.sub.43, identical or different, represent H or CH.sub.3, R.sub.36 represents H, R.sub.42 represents CH.sub.3, or together R.sub.36 and R.sub.42 represent a group of formula OCO, R.sub.37 represents H or O-hexoside, b represents a single bond or a double bond, R.sub.39 represents CH.sub.3, R.sub.40 represents H, or together R.sub.39 and R.sub.40 represent a group of the following formula (IIId): ##STR00057## R.sub.41 represents H, OH or a group of the following formula (IIIe) or (IIIf): ##STR00058## R.sub.44 represents H or OH, or cosmetically or nutritionally acceptable salts or esters thereof.

2. The composition according to claim 1, wherein the first compound is selected from the group consisting of the compounds of the following formulae: ##STR00059## wherein A.sub.1, A.sub.2 and A.sub.3, identical or different, represent OH, OSO.sub.3H, O-glucuronide, or OCO-A.sub.4, wherein A.sub.4 represents an aliphatic group comprising from 1 to 30 carbon atoms, saturated or unsaturated, linear or branched, optionally substituted by at least one O, OH or COOH group, ##STR00060## ##STR00061##

3. The composition according to claim 1, wherein the second compound is selected from the group consisting of the compounds of the following formulae: ##STR00062## wherein: B.sub.1 represents OH, OCH.sub.3 or O-glucuronide, B.sub.2 represents OH or OCH.sub.3, B.sub.3 represents H or OH, B.sub.4 represents OH, O-glucuronide or a group of the following formula: ##STR00063## wherein B.sub.6 represents OH, OCH.sub.3 or O-glucuronide, B.sub.5 represents OH, O-hexoside or O-glucuronide, ##STR00064## ##STR00065##

4. The composition according to claim 1, further comprising at least one third compound of formula (I), (II) or (III), provided the third compound is different from the first and second compounds.

5. The composition according to claim 1, comprising at least two different compounds, or at least three different compounds, selected respectively from: a compound of formula (I.1), (I.2) or (I.3), a compound of formula (II.1), and a compound of formula (II.3) or (II.4).

6. The composition according to claim 1, wherein the compounds of formula (I), (II) and (III) are comprised in at least one vegetal extract.

7. The composition according to claim 1, further comprising at least one cosmetically or nutritionally acceptable carrier or excipient.

8. A method of preventing, attenuating, alleviating, or treating skin aging, comprising administering a cosmetic agent to the skin or a human or animal food supplement to the diet, wherein the cosmetic agent or human or animal food supplement comprises the composition of claim 1.

9. The method of claim 8, wherein at least one sign of skin aging is alleviated.

10. The method of claim 8, wherein the cosmetic agent or a human or animal food supplement composition comprises at least one first compound of formula (I), or of at least one polymer of the first compound, in combination with at least one second compound of formula (I), of formula (II), or of formula (III), the second compound of formula (I) being different from the first compound of formula (I).

11. The method of claim 8, wherein the cosmetic agent or a human or animal food supplement composition comprises at least one first compound of formula (I), or of at least one polymer of the first compound, in combination with at least one second compound of formula (I), of formula (II), or of formula (III), the second compound of formula (I) being different from the first compound of formula (I), to alleviate at least one sign of skin aging.

12. The method of claim 8, wherein the cosmetic agent or a human or animal food supplement composition comprises a first compound selected from the group consisting of the compounds of the following formulae: ##STR00066## wherein A.sub.1, A.sub.2 and A.sub.3, identical or different, represent OH, OSO.sub.3H, O-glucuronide, or OCO-A.sub.4, wherein A.sub.4 represents an aliphatic group comprising from 1 to 30 carbon atoms, saturated or unsaturated, linear or branched, optionally substituted by at least one O, OH or COOH group, ##STR00067## ##STR00068##

13. The method of claim 8, wherein the cosmetic agent or a human or animal food supplement composition comprises a second compound selected from the group consisting of the compounds of the following formulae: ##STR00069## wherein: B.sub.1 represents OH, OCH.sub.3 or O-glucuronide, B.sub.2 represents OH or OCH.sub.3, B.sub.3 represents H or OH, B.sub.4 represents OH, O-glucuronide or a group of the following formula: ##STR00070## wherein B.sub.6 represents OH, OCH.sub.3 or O-glucuronide, B.sub.5 represents OH, O-hexoside or O-glucuronide, ##STR00071## ##STR00072##

14. The method of claim 8, wherein the cosmetic agent or a human or animal food supplement composition comprises at least one compound of formula (I), (II) or (III) in combination with the first and second compounds, provided the third compound is different from the first and second compounds.

15. The method of claim 8, wherein the cosmetic agent or a human or animal food supplement composition comprises at least two different compounds, or at least three different compounds, respectively selected from: a compound of formula (I.1), (I.2) or (I.3), a compound of formula (II.1), and a compound of formula (II.3) or (II.4).

16. The method of claim 9, wherein the at least one sign of skin aging that is alleviated is selected from the group consisting of at least one of forehead lines, frown lines, crow's feet wrinkles, bunny lines, purse-string wrinkles, nasolabial folds, tear troughs, marionette lines, mental crease and neck lines.

Description

DESCRIPTION OF THE FIGURES

[0174] FIG. 1 represents a photograph of a western blot of proteins revealed by anti-lamin A/C, anti-prelamin A and anti-HDJ2 antibodies after having been extracted from human skin fibroblasts cultured in the presence of the following compounds: [0175] 1: DMSO (negative control) [0176] 2: Alendronate/Pravastatin (positive control) [0177] 3: Alendronate [0178] 4: Alendronate/Compound I.1a [0179] 5: Alendronate/Compound I.7a [0180] 6: Alendronate/Compound I.6a [0181] 7: Alendronate/Compound I.4a [0182] 8: Alendronate/Compound I.8a [0183] 9: DMSO [0184] 10: Alendronate/Pravastatin [0185] 11: Pravastatin [0186] 12: Pravastatin/Compound II.1 [0187] 13. Pravastatin/Compound II.3a [0188] 14. Pravastatin/Compound II.2 [0189] 15. Pravastatin/Compound I.12 [0190] 16. Pravastatin/Compound II.5

[0191] FIG. 2 represents the LC3II/I protein ratio (vertical axis, in percent change from negative control) in human skin fibroblasts exposed to: [0192] 1: Compound I.1a alone [0193] 2: Compound II.1 alone [0194] 3: Compound II.3a alone [0195] 4: Combination compound I.1a+compound II.1+compound II.3a [0196] Lane 5 represents the theoretical expected effect observed in case of additive effect of the compounds (addition of the respective effects of compound I.1a alone, compound II.1 alone and compound II.3a alone).

[0197] FIG. 3 represents the amount of Lamin B.sub.1 (vertical axis, in percent change from negative control) in human skin fibroblasts exposed to: [0198] 1: Compound I.1a alone [0199] 2: Compound II.1 alone [0200] 3: Compound II.3a alone [0201] 4: Combination compound I.1a+compound II.1+compound II.3a [0202] Lane 5 represents the theoretical expected effect observed in case of additive effect of the compounds (addition of the respective effects of compound I.1a alone, compound II.1 alone and compound II.3a alone).

EXAMPLES

Example 1

[0203] The topical administration of an alendronate-pravastatin composition on the face of human volunteers has shown an anti-skin aging effect in a double blind, randomized and placebo controlled comparative study, by reducing crow's feet wrinkles and restoring cheek volume. This effect is believed to be carried, at least in part, by the inhibition of the synthesis of farnesylpyrophosphate, the precursor of the farnesyl group which anchors the pro-aging factor progerin (Scaffidi & Misteli (2006) Science 312:1059-1063) to the nuclear envelop in cells from aged subjects.

[0204] Inhibition of progerin farnesylation has been shown to reduce progerin toxicity and its pro-aging consequences (Glynn & Glover (2005) Human Molecular Genetics 14:2959-2969; Varela et al. (2008) Nature Medicine 14:767-772). Farnesylated progerin sequesters and mislocalizes NRF2 within nuclear lamina, resulting in impaired NRF2 transcriptional activity which consequently increases chronic oxidative stress (Kubben et al. (2016). Cell 165, 1361-1374).

[0205] In the following, the inventors assessed the capacity of various tested compounds to substitute for either alendronate or pravastatin in the inhibition of the farnesylation of prelamin A and of HDJ2, which is indicative of anti-aging and antioxidant effects.

Material and Methods

Tested Compounds

[0206] The following compounds were tested:

TABLE-US-00008 Structure Name CAS Number [00038] I.1a 501-36-0 [00039] I.4a 327-97-9 [00040] I.6a 4773-96-0 [00041] I.7a 13241-33-3 [00042] I.8a 10236-47-2 [00043] I.12a 10338-51-9 [00044] II.1 520-36-5 [00045] II.2 481-72-1 [00046] II.3a 154-23-4 [00047] II.5 117-39-5

[0207] The tested compounds were obtained from Euromedex, Souffelweyersheim, France for I.1a and Sigma, Saint-Quentin Fallavier, France for I.4a, I.6a, I.7a, I.8a, I.12a, II.1, II.2, II.3a and II.5. The tested compounds were diluted in DMSO.

[0208] Alendronate and pravastatin were obtained from AbMole/Euromedex, Souffelweyersheim, France and Alsachim, Illkirch-Graffenstaden, France respectively. Alendronate and pravastatin were diluted in H.sub.2O.

Cell Culture

[0209] Control Human fibroblast from skin (AG13334) were obtained from Coriell Institute. Cells were maintained in culture according to supplier information at 37 C. in a humidified atmosphere containing 5% CO.sub.2.

[0210] Fibroblasts were cultured 72 h without culture-media renewal.

[0211] To assess cell viability, cells were treated 72 h with the tested compounds, with concentrations ranging from 1 pM to 1000 M. Each tested compound was combined with Alendronate (10 M) or Pravastatin (10 M).

[0212] To assess efficacy, cells were treated by a combination of Alendronate or Pravastatin (10 M) with the tested compounds at their highest non-toxic doses or half of this concentration (Compound I.1a, 50 M; Compound I.7a, 500 M; Compound I.6a, 500 M; Compound I.4a, 500 M; Compound I.8a, 1000 M; Compound II.1, 60 M; Compound II.3a, 500 M; Compound II.2, 30 M; Compound I.12a 500 M; Compound II.5, 250 M).

[0213] The following combinations were tested: [0214] 1: DMSO (negative control) [0215] 2: Alendronate/Pravastatin (positive control) [0216] 3: Alendronate [0217] 4: Alendronate/Compound I.1a [0218] 5: Alendronate/Compound I.7a [0219] 6: Alendronate/Compound I.6a [0220] 7: Alendronate/Compound I.4a [0221] 8: Alendronate/Compound I.8a [0222] 9: DMSO [0223] 10: Alendronate/Pravastatin [0224] 11: Pravastatin [0225] 12: Pravastatin/Compound II.1 [0226] 13. Pravastatin/Compound II.3a [0227] 14. Pravastatin/Compound II.2 [0228] 15. Pravastatin/Compound I.12 [0229] 16. Pravastatin/Compound II.5

[0230] Ritonavir (4 M) and Lopinavir (20 M) were added in all tested conditions to improve the lower limit of detection of prenylated prelamin A in western blots.

Cell Viability and Assessment of Cell-Toxicity

[0231] Assays were carried out in 96-well microplates. After 72 hours of treatment, the cells were washed once with 100 L DPBS (no calcium, no magnesium). Then 100 l of PrestoBlue solution (Life Technologies) diluted at 10% in DPBS was added to each well. Plates were incubated at 37 C. for 30 minutes. The fluorescence intensity was measured by multiwell plate reader (Glomax microplate reader, Promega, Charbonnires les Bains, France) using green filter (Excitation 525 nm/emission 580-640 nm). Fluorescence intensities values were analysed in Prism Software (GraphPad, San Diego, CA).

Protein Extraction

[0232] Total fibroblast proteins were extracted after 72 hours treatment in 100 l of NP40 (Invitrogen) with 1 protease and phosphatase inhibitor cocktail (Life Technologies). Lysates were incubated on ice for 30 minutes, with vortexing at 10-minute intervals. Finally, they were sonicated four times (20 sec each) and then centrifuged at 13000 rpm for 10 minutes at 4 C.

[0233] Protein concentrations were determined with the BCA Protein Assay (Life Technologies).

Antibodies

[0234] The following antibodies were used in this study: rabbit monoclonal anti-lamin A/C (ab108922, 1/1000, Abcam, Amsterdam, Netherlands), mouse monoclonal anti-prelamin A (MABT858, Millipore, Molsheim, France), mouse monoclonal anti-HDJ2 (MA5-12748, Thermoscientific, France). Secondary antibodies conjugated with IR-Dye 800CW or 680 were used according to the manufacturer's instructions (926-32213 and 926-68072, 1/5000, Li-COR Biosciences).

Western Blotting

[0235] Protein lysates were separated on NuPAGE Novex 8% Bis-Tris Midi Protein Gels (Life Technologies) and transferred to Immobilon-FL PVDF membranes (Millipore, Molsheim, France). Membranes were blocked for one hour in 1:1 diluted blocking buffer for near fluorescent western blotting (Rockland, Le Perray, France). Blocked membranes were incubated with primary antibody anti-prelamin A overnight at 4 C., following which they were washed and incubated with conjugated secondary antibody for one hour at room temperature. Then, this step was repeated with primary antibodies for HDJ2 and LMNA/C, and conjugated secondary antibodies.

[0236] Bound antibodies were detected and analyzed on an Odyssey imaging system (Li-COR Biosciences, Bad Homburg, Germany) according to the manufacturer's instructions. Revert Protein Stain (Li-COR Biosciences) was used as a total protein loading control.

Results

[0237] The results are shown in FIG. 1.

[0238] Prelamin A and unfarnesylated HDJ2 are absent in lanes 1 and 9, negative control(s) (DMSO) and essentially absent in lanes 3 for alendronate and 11 for pravastatin alone.

[0239] In contrast, a strong band can be seen for prelamin A for the alendronate/pravastatin combination (positive control, lanes 2 and 10). Indeed, prelamin A accumulates due to the inhibition of farnesylation which prevents its maturation to lamin A. A clear band can also be seen for unfarnesylated HDJ2.

[0240] Clear bands are also visible for prelamin A and for unfarnesylated HDJ2 for the combinations of the tested compounds I.1a, I.4a, I.6a, I.7a and I.8a (lanes 4, 7, 6, 5 and 8 respectively) with alendronate and of compounds I.12, II.1, II.2, II.3a and II.5 (lanes 15, 12, 14, 13 and 16 respectively) with pravastatin, which indicates an inhibition of farnesylation and therefore an anti-aging effect.

[0241] This also indicates that combining compounds I.1a, I.4a, I.6a, I.7a and I.8a with compounds I.12, II.1, II.2, II.3a and II.5 should also yield an inhibition of farnesylation and anti-aging and an antioxidant effects.

Example 2

[0242] Autophagy is an evolutionarily conserved, lysosome-dependent catabolic process whereby cytoplasmic components, including damaged organelles, protein aggregates and lipid droplets, are degraded and their components recycled. Autophagy has an essential role in maintaining cellular homeostasis in response to intracellular stress; however, the efficiency of autophagy declines with age (Kitada et al. (2021) Nat Rev Endocrinol. 17:647-661). Conversely, autophagy activation promotes longevity (Hansen et al. (2018) Nat Rev Mol Cell Biol. 9:579-593).

[0243] Autophagy activation can be monitored through the conversion of microtubule-associated protein light chain 3 I (LC3I) to LC3II, in particular by measuring the LC3II/LC3-I ratio (also named LC3II/I ratio) by immunoblot analysis, whereby an increased ratio indicates an increased autophagy (Klionsky et al. (2021) Autophagy 17:1-382).

Material and Methods

Cell Culture

[0244] Control Human fibroblast from skin (AG13334) were obtained from Coriell Institute. Cells were maintained in culture according to supplier information at 37 C. in a humidified atmosphere containing 5% CO.sub.2.

[0245] Fibroblasts were cultured 72 h without culture-media renewal.

[0246] To assess efficacy, cells were treated 72 h with the tested molecules alone or in combination (Compound I.1a, 50 M; Compound II.1, 60 M; Compound II.3a, 500 M). As for the detection of prenylated prelamin A, Ritonavir (4 M) and Lopinavir (20 M) were added in all tested conditions and negative control.

[0247] The following conditions were tested: [0248] 1: Compound I.1a alone [0249] 2: Compound II.1 alone [0250] 3: Compound II.3a alone [0251] 4: Combination Compound I.1a+Compound II.1+Compound II.3a [0252] 5: Theoretical additive compounds effect (Compound I.1a+Compound II.1+Compound II.3)

Protein Extraction

[0253] Total fibroblast proteins were extracted after 72 hours treatment in 100 l of NP40 (Invitrogen) with 1 protease and phosphatase inhibitor cocktail (Life Technologies). Lysates were incubated on ice for 30 minutes, with vortexing at 10-minute intervals. Finally, they were sonicated four times (20 sec each) and then centrifuged at 10000 g for 10 minutes at 4 C.

[0254] Protein concentrations were determined with the Pierce BCA Protein Assay (Life Technologies).

Antibodies

[0255] The following antibodies were used in this study: mouse monoclonal anti-LC3B (sc271625, 1/200, Santa Cruz, Heidelberg, Germany). Secondary antibodies conjugated with IR-Dye 800CW were used according to the manufacturer's instructions (926-32212, 1/5000, Li-COR Biosciences).

Western Blotting

[0256] Protein lysates were separated on NuPAGE Novex 8% Bis-Tris Midi Protein Gels (Life Technologies) and transferred to Immobilon-FL PVDF membranes (Millipore, Molsheim, France). Membranes were blocked for one hour in 1:1 diluted blocking buffer for near fluorescent western blotting (Rockland, Le Perray, France). Blocked membranes were incubated with primary antibody anti-LC3B overnight at 4 C., following which they were washed and incubated with conjugated secondary antibody for one hour at room temperature.

[0257] Bound antibodies were detected and analyzed on an Odyssey imaging system (Li-COR Biosciences, Bad Homburg, Germany) according to the manufacturer's instructions. LC3II and LC3I were detected.

[0258] Results were expressed as percent change from negative control of LC3II/I ratio.

Results

[0259] The results are shown in FIG. 2.

[0260] In can be seen that the tested combination of compounds I.1a, II.1, and II.3a yields an increase in the LC3II/I ratio with respect to the negative control (DMSO) (lane 5) which is greater than the increase expected from the addition of the respective increases in the LC3II/I ratio yielded by each compound alone (lane 6).

[0261] Accordingly, the effect of the tested combination of compounds I.1a, II.1, and II.3a on the activation of autophagy in human skin fibroblasts is supra-additive, i.e. it is synergistic.

[0262] The tested combination is therefore expected to exert anti-aging effects on the skin.

Example 3

[0263] Depletion of Lamin B.sub.1 from the nuclear envelope is an important event during senescence. Lamin B.sub.1 is crucial to maintain the structural integrity of the nucleus. However, this integrity is lost in senescent cells, which allows the release of cytoplasmic chromatin fragments outside the nucleus, which is believed to fuel the SASP through the cGAS/STING pathway (Gonzalez-Gualda et al. (2021) The FEBS Journal 288:56-80). The loss of Lamin B.sub.1 is thus a hallmark of senescent cells (Wang et al. (2017) Sci Rep. 7:15678).

[0264] The loss of Lamin B1 can be detected immunoassayed and detected by imaging or immunoblotting.

Material and Methods

Cell Culture

[0265] Control Human fibroblast from skin (AG13334) were obtained from Coriell Institute. Cells were maintained in culture according to supplier information at 37 C. in a humidified atmosphere containing 5% CO.sub.2.

[0266] Fibroblasts were cultured 72 h without culture-media renewal.

[0267] To assess efficacy, cells were treated 72 h with the tested molecules alone or in combination (Compound I.1a, 50 M; Compound II.1, 60 M; Compound II.3a, 500 M). As for the detection of prenylated prelamin A, Ritonavir (4 M) and Lopinavir (20 M) were added in all tested conditions and negative control.

[0268] The following conditions were tested: [0269] 1: Compound I.1a alone [0270] 2: Compound II.1 alone [0271] 3: Compound II.3a alone [0272] 4: Combination Compound I.1a+Compound II.1+Compound II.3a [0273] 5: Theoretical additive compounds effect (Compound I.1a+Compound II.1+Compound II.3a)

Protein Extraction

[0274] Total fibroblast proteins were extracted after 72 hours treatment in 100 L of NP40 (Invitrogen) with 1 protease and phosphatase inhibitor cocktail (Life Technologies). Lysates were incubated on ice for 30 minutes, with vortexing at 10-minute intervals. Finally, they were sonicated four times (20 sec each) and then centrifuged at 10000 g for 10 minutes at 4 C.

[0275] Protein concentrations were determined with the Pierce BCA Protein Assay (Life Technologies).

Antibodies

[0276] The following antibodies were used in this study: rabbit polyclonal anti-laminB1 (ab16048, 1/1000, Abcam, Amsterdam, Netherlands). Secondary antibodies conjugated with IR-Dye 680RD were used according to the manufacturer's instructions (926-68073, 1/5000, Li-COR Biosciences).

Western Blotting

[0277] Protein lysates were separated on NuPAGE Novex 8% Bis-Tris Midi Protein Gels (Life Technologies) and transferred to Immobilon-FL PVDF membranes (Millipore, Molsheim, France). Membranes were blocked for one hour in 1:1 diluted blocking buffer for near fluorescent western blotting (Rockland, Le Perray, France). Blocked membranes were incubated with primary antibody anti-lamin B.sub.1 overnight at 4 C., following which they were washed and incubated with conjugated secondary antibody for one hour at room temperature.

[0278] Bound antibodies were detected and analyzed on an Odyssey imaging system (Li-COR Biosciences, Bad Homburg, Germany) according to the manufacturer's instructions. All bands detected with anti LaminB1 were considered as specific (KO validated antibody reference). Revert Protein Stain (Li-COR Biosciences) was used as a total protein loading control.

[0279] Results were expressed as percent change from negative control of Lamin B1.

Results

[0280] The results are shown in FIG. 3.

[0281] In can be seen that the tested combination of compounds I.1a, II.1, and II.3a yields an increase in the amount of Lamin B.sub.1 with respect to the negative control (DMSO) (lane 5) which is greater than the increase expected from the addition of the respective increases in the amount of LaminB1 yielded by each compound alone (lane 6).

[0282] Accordingly, the effect of the tested combination of compounds I.1a, II.1, and II.3a on the reversal of senescence signs in human skin fibroblasts is supra-additive, i.e. it is synergistic.

[0283] The tested combination is therefore expected to exert anti-aging effects on the skin.