Combination of a retinoid and an extract of <i>Silybum marianum </i>(L.) gaertn

Abstract

The present invention relates to the combination of a retinoid and an extract of Silybum marianum (L.) Gaertn., the dermatological and dermocosmetic compositions containing such a combination, as well as their uses as a medicinal product, in particular in the treatment of acne, seborrhea, seborrhoeic dermatitis and/or rosacea.

Claims

1. An active ingredient combination consisting of a retinoid and an extract of Silybum marianum (L.) Gaertn., wherein the retinoid is retinaldehyde, and wherein the extract of Silybum marianum (L.) Gaertn. is an extract of achenes of Silybum marianum (L.) Gaertn. comprising less than 0.2% by weight of silymarin based on the weight of the dry extract.

2. The combination according to claim 1, wherein the extract of Silybum marianum (L.) Gaertn. is an achene extract of Silybum marianum (L.) Gaertn. comprising less than 0.1% by weight of silymarin based on the weight of the dry extract.

3. The combination according to claim 1, wherein the extract of Silybum marianum (L.) Gaertn. contains between 0.5% and 2.5% by weight of beta-sitosterol based on the weight of the dry extract.

4. The combination according to claim 1, wherein the extract of Silybum marianum (L.) Gaertn. contains between 1.0% and 2.0% by weight of beta-sitosterol based on the weight of the dry extract.

5. The combination according to claim 1, wherein the extract of Silybum marianum (L.) Gaertn. contains between 2 and 7% by weight of sterols based on the weight of the dry extract.

6. The combination according to claim 1, wherein the extract of Silybum marianum (L.) Gaertn. contains between 3 and 5% by weight of sterols based on the weight of the dry extract.

7. The combination according to claim 1, wherein the extract of Silybum marianum (L.) Gaertn. contains between 3% and 15% by weight of free linoleic acid based on the weight of the dry extract.

8. The combination according to claim 1, wherein the extract of Silybum marianum (L.) Gaertn. contains between 4% and 10% by weight of free linoleic acid based on the weight of the dry extract.

9. The combination according to claim 1, wherein the extract of Silybum marianum (L.) Gaertn. contains between 10% and 50% by weight of free fatty acids based on the weight of the dry extract.

10. The combination according to claim 1, wherein the extract of Silybum marianum (L.) Gaertn. contains between 10% and 30% by weight of free fatty acids based on the weight of the dry extract.

11. The combination according to claim 1, wherein the extract of Silybum marianum (L.) Gaertn. comprises between 0.01% and 0.5% by weight of tocopherols based on the weight of the dry extract.

12. A method for treating acne, seborrhea, rosacea or seborrheic dermatitis comprising administering to a person in need thereof an effective amount of a combination according to claim 1.

13. A dermatological or dermocosmetic composition comprising the active ingredient combination according to claim 1 and at least one dermatologically or dermocosmetically acceptable excipient, wherein the sole active ingredient of the composition consists of the active ingredient combination.

14. The dermatological or dermocosmetic composition according to claim 13, comprising 0.01 to 10% by weight of the extract of Silybum marianum (L.) Gaertn. by weight of dry extract based on the total weight of the composition.

15. The dermatological or dermocosmetic composition according to claim 13, comprising 0.001 to 5% by weight of the retinoid based on the total weight of the composition.

16. The dermatological or dermocosmetic composition according to claim 13, being intended for topical application.

17. A method for treating acne, seborrhea, rosacea or seborrheic dermatitis comprising administering to a person in need thereof an effective amount of a dermatological or dermocosmetic composition according to claim 13.

Description

FIGURES

(1) FIG. 1 shows the UPLC chromatogram of extract I of Silybum marianum obtained according to Protocol 1.

(2) FIGS. 2A, 2B and 2C represent respectively a UPLC chromatogram of extract M, extract E and extract I of Silybum marianum obtained according to Protocol 2.

(3) FIGS. 3A, 3B and 3C represent respectively the 10-22 min zone (corresponding to the fatty acid zone) of a GC/MS chromatogram of extract E, extract M and extract I of Silybum marianum obtained according to Protocol 3.

(4) FIGS. 4A, 4B and 4C represent respectively the 22-28 min zone (corresponding to the sterol zone) of a GC/MS chromatogram of extract E, extract M and extract I of Silybum marianum obtained according to Protocol 3.

(5) FIG. 5 represents the amount produced by skin cells of total retinoids as a function of the treatment received, i.e. vehicle (EtOH) alone, 2 μM retinaldehyde (2 μM RAL), 50 μg/mL Silybum marianum extract (50 μg/mL SYL) or the combination of 2 μM retinaldehyde and 50 μg/mL Silybum marianum extract (RAL+SYL).

(6) FIG. 6 represents the amount produced by skin cells of total retinoids as a function of the treatment received, i.e. vehicle (EtOH) alone, 2 μM retinol (2 μM ROL), 50 μg/mL Silybum marianum extract (50 μg/mL SYL) or the combination of 2 μM retinol and 50 μg/mL Silybum marianum extract (ROL+SYL).

EXAMPLES

(7) I—Examples of Preparations of Extracts According to the Invention

(8) Isopropanolic extract 90 (extract I) obtained by the following process: cold pressing of Silybum marianum achenes to obtain an oil from Silybum marianum achenes, extraction of the oil from Silybum marianum achenes by an isopropanol/water mixture (90/10 v/v) with 1 gram of the isopropanol/water mixture per gram of oil for 2 hours at 20° C., recovery of the isopropanolic phase, and evaporation of the solvent.

(9) Methanolic (extract M) and ethanolic 90 (extract E) extracts were obtained in a similar manner by replacing the isopropanol/water mixture (90/10 v/v) with methanol and an ethanol/water mixture (90/10 v/v) respectively. The extraction of the oil from Silybum marianum achenes is carried out with 3 volumes of methanol and 3 volumes of the ethanol/water mixture (90/10 v/v) for 1 volume of oil for 2 hours at 20° C., respectively.

(10) These different extracts were characterized by ultra high-performance liquid chromatography (UPLC) or gas chromatography coupled with mass spectrometry (GC-MS) according to the protocols detailed below.

(11) Protocols for Evaluating the Extracts Obtained: 1. Protocol 1: Evaluation of silymarin content by UPLC Preparation of sample and control: Silymarin control: Prepare a 5 mg solution of silymarin in 10 mL of methanol/water mixture (60:40) (v/v). Sample: Extract A: Prepare a solution of 100 mg of extract to be analyzed in 10 mL of a methanol/dichloromethane mixture (70:30) (v/v) Extracts M, E, I: Heat the dry extract to be analyzed to 35° C. with stirring until a homogeneous and clear solution is obtained. Weigh exactly 200 mg (pe) of the extract, solubilize it in 10 mL of a methanol/dichloromethane mixture allowing total solubilization of the extract and homogenize the solution. This mixture ranges from a methanol/dichloromethane ratio (1:1) (v/v) to pure methanol. Analytical conditions: Column: Acquity BEH Shield C18 150 mm×2.1 mm-1.7 μm (Waters) Mobile phase: A: Water+0.1% formic acid B: Acetonitrile+0.1% formic acid Gradient:

(12) TABLE-US-00001 T (min) A (%) B (%) 0 90 10 15 60 40 20 0 100 39.5 0 100 40 90 10 45 90 10 Column temperature: 40° C. Flow rate: 0.4 mL/min Detection: 287 nm Injection volume: 1 μL 2. Protocol 2: Evaluation of linoleic acid content by UPLC Preparation of sample and control Linoleic acid control: Prepare a solution of 10 mg linoleic acid in 10 mL of a 1:1 (v/v) methanol/dichloromethane mixture. Sample: Extracts M, E, I: Heat the dry extract to be analyzed to 35° C. with stirring until a homogeneous and clear solution is obtained. Weigh exactly 50 mg (pe) of the extract, solubilize it in 1 mL of a methanol/dichloromethane mixture allowing total solubilization of the extract and homogenize the solution. This mixture ranges from a methanol/dichloromethane ratio (1:1) (v/v) to pure methanol. Analytical conditions Column: Acquity BEH Shield C18 150 mm×2.1 mm-1.7 μm (Waters) Mobile phase: A: Water+0.1% formic acid B: Acetonitrile+0.1% formic acid Gradient:

(13) TABLE-US-00002 T (min) A (%) B (%) 0 50 50 1 50 50 10 0 100 15 0 100 15.5 50 50 20 50 50 Column temperature: 40° C. Flow rate: 0.4 mL/min Detection: 215 nm Injection volume: 1 μL 3. Protocol 3: Evaluation of fatty acid and sterol content by GC-MS Sample preparation Heat the dry extract to be analyzed to 35° C. while stirring until a clear homogeneous liquid is obtained. Solubilize 20 mg of the extract in 800 μL of methanol/dichloromethane (1:1) (v/v) mixture Add 200 μL of the derivatization reagent N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA)+Trimethylchlorosilane (TMCS) (99:1) (Supelco—Sigma Aldrich) Vortex for 1 minute Gas chromatography (GC) conditions Column: DB-5 ms (Agilent technologies); 30 m×0.25 mm; 0.25 μm Injection: T=300° C.; Mode=Split; Split ratio=100:1 Oven: Temperature gradient (° C.): Initial temperature=150° C. Gradient=7° C./min up to final temperature=340° C. Maintain at 340° C. for 10 minutes Carrier gas flow rate: 1 mL/min Detection: MS-EI; T=300° C.; Scan Time=0.2 sec; Full Scan Start Mass=40; Full Scan End Mass=600 Injection volume: 1 μL

(14) Results:

(15) Extract I contain mostly substances with a retention time between 13 and 30 minutes per UPLC (see FIG. 1). It has the following characteristics:

(16) TABLE-US-00003 Components % by weight Silymarin 0.06 Free fatty acids (mainly palmitic, 24.6 oleic and linoleic acids) of which linoleic acid 5.1 Sterols 3.6 of which beta-sisterol 1.5

(17) The UPLC and GC/MS chromatograms of the 3 extracts (extracts I, M and E) of Silybum marianum are similar (see FIGS. 2 and 3).

(18) II—Examples of Compositions Comprising a Retinoid and/or an Extract of Silybum marianum According to the Invention

(19) Composition 1 (Cream) Based on Retinaldehyde=RAL1

(20) TABLE-US-00004 INCI designation Percentage by weight Glycylglycine oleamide  0.1% Tocopheryl glucoside  0.1% Retinaldehyde 0.05% Gelling carbomer 0.35% Squalane   15% Mineral oil Caprylic capric triglycerides Water q.s. 100%

(21) Composition 2 (Cream) Based on Retinaldehyde=RAL2

(22) TABLE-US-00005 INCI designation Percentage by weight Water 100% Q.S. Potassium sorbate 0.3 Ethanol 3 Glycolic acid 6 Ceteareth 33 & cetearyl alcohol 3 Polysorbate 60 6 Cetyl alcohol 11 Butylhydroxytoluene 0.01 Cyclopentasiloxane 10 Sodium hydroxide q.s. Retinaldehyde 0.1 Undecyl rhamnoside 0.2

(23) Composition 3 (Cream) Based on Retinaldehyde=RAL3

(24) TABLE-US-00006 INCI designation Percentage by weight Water 100% Q.S. Glycerin 6 Disodium EDTA 0.1 Pentylene glycol 3 Glyceryl Stearate & PEG-100 Stearate 3 Isododecane 7 Butylhydroxytoluene 0.02 Carbomer papain crosspolymer 1 Retinaldehyde 0.1 Caprylic/capric triglycerides 7 2-Hydroxy-octyl 1α-linolenate 0.6

(25) Composition 4 (Cream) Based on Retinaldehyde=RAL4

(26) Retinaldehyde was formulated at 0.1% (w/w) in a mixture comprising:

(27) 5% glycerin

(28) 1.5% gelling agent

(29) 1% silica

(30) q.s. water.

(31) Composition 5 (Cream) Based on Silybum marianium Extract=SYL1

(32) Extract I prepared in Example 1 was formulated at 7% (w/w) in a mixture of isopropanol/PEG 300 (1:1) (w/w).

(33) Composition 6 (Cream) Based on Silybum marianium Extract=SYL2 (Percentage w/w)

(34) 25% SYL1

(35) 5% glycerin

(36) 1.5% gelling agent

(37) 1% silica

(38) q.s. water.

(39) Composition 7 (Cream) Comprising the Combination of an Extract of Silybum marianum and Retinaldehyde=ASSO1 (Percentage w/w)

(40) 25% SYL1

(41) 0.1% retinaldehyde

(42) 5% glycerin

(43) 1.5% gelling agent

(44) 1% silica

(45) q.s. water.

(46) Composition 8 (Cream) Comprising the Combination of an Extract of Silybum marianum and Retinaldehyde=ASSO2 (Percentage w/w)

(47) 12.5% SYL1

(48) 0.1% retinaldehyde

(49) 5% glycerin

(50) 1.5% gelling agent

(51) 1% silica

(52) q.s. water.

(53) III—Clinical Studies

(54) The different creams tested were applied to the face (cleansed skin), 2 applications/day morning and evening by gentle massage except for compositions containing RAL: in the evening only.

(55) The analysis of the therapeutic effects obtained was carried out according to the Investigator Global Assessment (IGA) method accepted by the Food and Drug Administration (FDA)—for acne and suitable for rosacea and seborrheic dermatitis—and applied by a clinical dermatologist (Guidance for Industry Acne Vulgaris: Developing Drugs for Treatment).

(56) With such a method, the following IGA Grades are assigned according to the severity of the acne observed:

(57) TABLE-US-00007 Grade IGA Patient's condition 0 Clear skin without inflammatory or non-inflammatory lesions 1 Nearly clear skin with rare non-inflammatory lesions and not more than one small inflammatory lesion 2 Mild acne of severity greater than grade 1: some non-inflammatory lesions with no more than a few inflammatory lesions (papules and pustules, no nodular lesions) 3 Moderate acne of severity greater than grade 2: up to many non-inflammatory lesions and potentially some inflammatory lesions but no more than one small nodular lesion. 4 Severe acne of severity greater than grade 3: up to numerous non- inflammatory and inflammatory lesions but no more than a few nodular lesions

(58) The results of the various analyses are presented below.

(59) Analysis 1: Before Treatment Versus Silybum marianum Extract without RAL Versus Combination RAL+Silybum marianum Extract

(60) TABLE-US-00008 Protocol (number of successive IGA Grade weeks of the same treatment and After treatment After treatment second treatment applied on the day Silybum RAL + Silybum Patients following the end of the 1.sup.st treatment) Before treatment marianum extract marianum extract Man with rosacea 35 weeks SYL1 2 1 0 and seborrheic then 15 weeks SYL1 + RAL1 dermatitis Woman with rosacea 9 weeks SYL1 4 2 0 and seborrheic then 50 weeks SYL1 + RAL1 dermatitis Man with rosacea 3 weeks SYL1 3 1 0 and seborrheic then 18 weeks SYL1 + RAL1 dermatitis Woman with late 5 weeks SYL1 3 2 1 acne then 8 weeks SYL1 + RAL2 Teenager (male) with 5 weeks SYL1 3 1 0 acne then 5 weeks SYL1 + RAL3 Teenager (female) 4 weeks SYL1 2 1 0 with acne then 28 weeks SYL1 + RAL3 Teenager (male) with 52 weeks SYL1 3 1 0 acne then 16 weeks SYL1 + RAL3 Woman with late 10 weeks SYL1 3 2 1 acne then 20 weeks SYL1 + RAL3 Woman with late 8 weeks SYL1 3 2 2 acne then 8 weeks SYL1 + RAL3 Woman with late 24 weeks SYL2 3 2 1 acne then 4 weeks ASSO1 Teenager (female) 4 weeks SYL2 2 1 0 with acne then 6 weeks ASSO1 Teenager (male) with 16 weeks SYL2 3 2 1 acne then 3 weeks ASSO1 Woman with late 6 weeks SYL2 3 2 1 acne then 3 weeks ASSO1 Woman with late 8 weeks SYL2 3 2 1 acne then 8 weeks ASSO2 Teenager (female) 12 weeks SYL1 4 2 1 with acne then 10 weeks ASSO2

(61) Analysis 2: Before Treatment Versus RAL without Silybum marianum Extract Versus Combination RAL+Silybum marianum Extract

(62) TABLE-US-00009 Protocol (number of successive weeks of the same treatment and IGA Grade second treatment applied on the day After treatment following the end of the first After treatment RAL + Silybum Patients treatment) Before treatment RAL marianum extract Man with seborrheic 35 weeks RAL1 3 2 1 dermatitis + bald then 43 weeks SYL1 + RAL1 scalp Woman with late 6 weeks RAL 3 4 3 1 acne then 6 weeks SYL1 + RAL3 Woman with late 8 weeks RAL2 3 2 1 acne Then 8 weeks SYL1 + RAL2 Teenager (female) 60 weeks RAL2 3 2 1 with acne Then 16 weeks SYL1 + RAL2 Teenager (male) with 6 weeks RAL3 3 2 1 acne then 20 weeks SYL1 + RAL3 Woman with late 8 weeks RAL3 4 3 2 acne Then 4 weeks ASSO1

(63) Conclusion

(64) The results obtained with the different treatments based respectively on a combination of retinaldehyde and Silybum marianum extract, retinaldehyde alone or Silybum marianum extract alone, in the clinical studies described above, show that retinaldehyde in combination with Silybum marianum extract, allows a better clinical result to be obtained.

(65) IV—In Vitro Studies: Evaluation of the Correction of Vitamin A Deficiency by the Combination Retinaldehyde (RAL) or Retinol (ROL)+Silybum marianum Extract (SYL)

(66) Materials and Methods:

(67) Culture: 3 days prior to the experiment, culture A431 epidermoid carcinoma cells in 2 6-well plates at a density of 40,000 cells per well (2 mL per well).

(68) Cell treatment (3 wells per condition, 2 mL per well; final concentrations): Ethanol (solvent): 1% ethanol: ROL: 10 μM ROL in 1% ethanol: RAL: 2 μM RAL in 1% ethanol: SYL: 50 μg/mL Extract I of Silybum marianum in 1% ethanol: ROL+SYL: 10 μM ROL+50 μg/mL Silybum marianum extract in 1% ethanol; RAL+SYL: 2 μM RAL+50 μg/mL Silybum marianum extract in 1% ethanol.

(69) Incubation: 1 h

(70) Cell Harvest: Remove the medium, wash twice with PBS (phosphate saline buffer); Add 500 μL per well of EDTA-NaOH (0.02% ethylene diamine tetraacetic acid (EDTA)+200 μM NaOH), allow to stand for 10-15 minutes, then detach the cells with a rubber scraper and transfer to extraction glass tubes; Add 10 μL of 20 mM HCl, sonicate briefly, then transfer 10 μL of suspension to 1.5 mL Eppendorf tubes for protein determination; To the glass tubes add 500 μL of 200 μM butylated hydroxytoluene (BHT)/ethanol, 20 μL of 20 μM retinyl acetate (internal standard), then 4 mL of hexane; Vortex vigorously for 30 sec, then centrifuge the tubes (900 g, 5 min); Transfer the supernatant into uncapped glass tubes, evaporate dry under nitrogen; Reconstitute in 200 μL of methanol, then transfer to high-performance liquid chromatography (HPLC) vials.

(71) Retinoid Analysis by HPLC: Agilent 1100 HPLC chain with quaternary pump and DAD detector Macherey-Nagel column Nucleodur C18 pyramid 3 μm mobile phase: 0-6 min: 100% methanol 6-8 min: linear gradient to methanol/THF (4:1) 8-18 min: methanol/THF (4:1) 18-20 min: linear gradient to 100% methanol 20-30 min: 100% methanol detection: 325 nm (retinol, retinyl esters); 383 nm (retinal).

(72) Results:

(73) The results obtained are presented in FIGS. 5 and 6.

(74) Conclusion:

(75) Silybum marianum extract alone does not induce significant retinoid production.

(76) On the other hand, skin cells produce significantly more retinoids in the presence of the combination of an extract of Silybum marianum with retinaldehyde or retinol than in the presence of retinaldehyde or retinol alone, demonstrating the synergistic effect of the combination according to the invention.