Anti-dandruff composition comprising pycnidione and epolone

Abstract

Use of pycnidione, epolone A, and epolone B as anti-dandruff actives either alone or in combination in anti-dandruff compositions, particularly shampoos and conditioners. The actives are particularly effective against Malassezia yeasts and Malassezia furfur. A method of obtaining pycnidione, epolone A, and epolone B from culturing of Neosetophoma samarorum is also described.

Claims

1. An anti-dandruff composition comprising 0.001 wt. % to 20 wt. % of at least one of pycnidione, epolone A, or epolone B, or any combination thereof, based on the total weight of the composition.

2. The anti-dandruff composition according to claim 1, which has a MIC cytotoxicity against M. furfur cells of less than 130 g per ml.

3. The anti-dandruff composition according to claim 1, which has an IC50 cytotoxicity against M. furfur cells of less than 100 g per ml.

4. The anti-dandruff composition according to claim 1, wherein the pycnidione, epolone A, and/or epolone B are the only anti-dandruff actives present in said composition.

5. The anti-dandruff composition according to claim 1, wherein said composition comprises at least one other anti-dandruff active material.

6. The anti-dandruff composition according to claim 5, wherein said other anti-dandruff active material is selected from the group consisting of ketoconazole, zinc pyrithione (ZPT), piroctone olamine, octopirox, salicylic acid, selenium sulphide, coal tar, azelaic acid, climbazole, undecylenic acid, and mixtures thereof.

7. The anti-dandruff composition according to claim 5, which comprises 0.01 wt. % to 15 wt. % of said other anti-dandruff active material.

8. The anti-dandruff composition according to claim 5, wherein said other anti-dandruff active material is zinc pyrithione (ZPT).

9. The anti-dandruff composition according to claim 1, which comprises 0.01 wt. % to 10 wt. % of least one of pycnidione, epolone A, or epolone B, or any combination thereof, based on the total weight of the composition.

10. The anti-dandruff composition according to claim 1, which comprises 0.001 wt. % to 10 wt. % of least one of pycnidione, epolone A, or epolone B, or any combination thereof, based on the total weight of the composition.

11. The anti-dandruff composition according to claim 1, which comprises 0.001 wt. % to 5 wt. % of least one of pycnidione, epolone A, or epolone B, or any combination thereof, based on the total weight of the composition.

12. The anti-dandruff composition according to claim 1, which contains pycnidione.

13. The anti-dandruff composition according to claim 1, which contains pycnidione and does not contain epolone A or epolone B.

14. The anti-dandruff composition according to claim 1, which is in the form of a member selected from the group consisting of shampoos, conditioners, hair styling products, soaps, lotions, ointments, medicated wipes, anti-fungal sprays, elixirs, suspensions, emulsions, solutions, syrups, aerosols, aqueous solutions, aqueous or oil suspensions, emulsions, hair coloring products, leave-on hair tonics, hair sunscreen products, creams, pastes and gels.

15. The anti-dandruff composition according to claim 1, which further comprises at least one surfactant.

16. The anti-dandruff composition according to claim 15, wherein said surfactant is a non-ionic surfactant, an amphoteric surfactant, or a cationic surfactant.

17. The anti-dandruff composition according to claim 15, which comprises 0.1 wt. % to 50 wt. % of said surfactant.

18. The anti-dandruff composition according to claim 15, which further comprises at least one betaine.

19. The anti-dandruff composition according to claim 1, wherein the pycnidione, epolone A, or epolone B are obtained from Neosetophoma samarorum.

20. A method of treating dandruff in hair, comprising applying an effective amount of the composition according to claim 1 to the hair.

21. A method of preparing the composition according to claim 16, comprising combining at least one of pycnidione, epolone A, or epolone B, or any combination thereof, and said surfactant.

Description

EXAMPLE 1FORMING & EXTRACTION OF ANTI-DANDRUFF ACTIVES

(1) Bioassay-guided fractionation of culture extracts led to the isolation of three biologically active metabolites produced by the fungus N. samarorum. The isolate RKDO834 along with reference strains of N. samarorum including the type strain were obtained from the Centraalbureau voor Schimmelcultures (Utrecht, Netherlands), plated out on YM (Yeast extract Malt extract) and OA (oatmeal) agar, and incubated for 14 days at 22 C. Colony morphology was observed and eight explants (approximately 10 mm.sup.3) were aseptically removed into glass scintillation vials, to which 15 mL of EtOAc was added and the vials were shaken for 1 hr at 200 rpm. The resulting EtOAc extract was then removed from the vial and dried down under a stream of air and retained for chemical analysis.

(2) Additionally eight colony explants (approximately 3 mm.sup.3) were aseptically removed into 15 mL of YM broth in a sterile, capped 50 ml test tube containing 2 sterile glass coverslips and shaken at 200 rpm, 22 C. for 5 days to create a seed inoculum. A 500 L aliquot of seed inoculum was removed into a sterile 2 mL Eppendorf tube, centrifuged at 10000 rpm for 5 minutes to pellet the mycelia and allow for the removal of the broth by pipetting, and stored frozen at 20 C. prior to DNA extraction. The seed inoculum was also streak plated onto YM and LB (Lysogeny Broth) agar (25 g LB Broth MILLER, 18 g agar in 1 L diH2O) plates, incubated for 3 days at 22 C. and inspected to ensure inoculum purity.

(3) An additional 500 L of seed inoculum from strain RKDO834 was dispensed into a capped 250 mL Erlenmyer flask containing an autoclave sterilised rice-base growth medium (10 g brown rice and 25 mL of YNB broth (6.7 g YNB powder, 5 g sucrose, 18 g instant ocean, 1 L diH2O)) and incubated at 22 C. for 21 days. After the incubation period, colony growth upon the rice-based medium was disturbed using a spatula and 40 mL of a 1:1 (v:v) EtOAc:MeOH solution was added and the capped flask was shaken for 60 minutes at 175 rpm. The contents of the flask were then filtered through Whatman #3 filter paper using a glass vacuum chamber with a Buchner funnel and the filtered solvent extract was dried down under a stream of air prior to further chemical purification.

(4) The extract obtained from the rice-based culture of RKDO834 was fractionated into 4 fractions on a Thermo HyperSep C18 column (500 mg C-18, 6 ml column volume) using a vacuum manifold by eluting with 14 mL of 4 different solvent combinations: 8:2 diH.sub.2O:MeOH (fraction 1), 1:1 diH.sub.2O:MeOH (fraction 2), EtOH (fraction 3), and 1:1 MeOH:DCM (fraction 4). The eluent representing fractions 2-4 were retained and dried down under air, weighed and submitted for antimicrobial testing against a pathogen panel. Fraction 3 was further fractionated on a Thermo HyperSep Diol column (500 mg Diol, 6 ml column volume) using a vacuum manifold by eluting with 14 mL of three different solvent combinations: 9:1 hexane:tBME (diol fraction 1), 9:1 tBME:MeOH (diol fraction 2), MeOH (diol fraction 3). Each fraction was retained, dried down under air and submitted for bioassay.

(5) Preparative HPLC fractionation of diol fraction 2 was performed on a reverse phase HPLC column (Gemini 5, C18 column, 10250 mm) using a Thermo electron HPLC coupled with UV and evaporative light scattering detector (ELSD). Initial fractionation of diol fraction 2 was carried out with isocratic elution of 85% aqueous MeCN with a flow rate 2.5 mL/min yielding eight sub fractions. Bioactivity was determined for each of the sub-fractions and followed up by an additional fractionation step. Fractionation of sub-fraction 2 with isocratic elution of 85% aqueous MeCN (2.5 mL/min) yielded pure epolone B. Both sub-fraction 3 and sub-fraction 4 were further fractionated using isocratic conditions of 70% aqueous MeCN (2.5 mL/min) to yield pure pycnidione and epolone A respectively.

(6) NMR spectra were recorded on a Bruker Avance III 600 MHz NMR spectrometer operating at 600 and 150 MHz for .sup.1H and .sup.13C, respectively. Spectra were referenced to residual undeuterated solvent peaks. Optical rotation was measured on a Rudolp Autopol III polarimeter. Analytical mass spectrometry of all samples was carried out on a Thermo Scientific Accela UHPLC coupled with a Thermo Exactive electrospray mass spectrometer (ESI-MS), with a SEDEX 80LT ELSD and a Thermo photodiode array (PDA) detector. Chromatography was carried out on a Kinetex 1.7 C18, 2.150 mm column using a gradient of 95:5% diH.sub.2O:MeCN with 0.1% formic acid-100% MeCN with 0.1% formic acid in 4 min, held at 100% MeCN with 0.1% formic acid for 5 min and returned to 95:5% diH.sub.2O:MeCN with 0.1% formic acid-100% MeCN with 0.1% formic acid and held at these conditions for 1 min.

(7) Antifungal activity of the initial HyperSep C-18 fractions of the crude culture extract generated from the fermentation of RKDO834 on rice medium was observed from fraction 3. Fraction 3 was selected for further bioassay-guided fractionation by HPLC to ultimately afford three separate metabolites demonstrating anti-Malassezia activity. Purification of the first compound yielded 0.44 mg and mass spectrometrical analysis confirmed a protonated molecular ion ([M+H].sup.+) of 385.2375 m/z ([M+MeCN+H].sup.+425.2498 m/z also observed). NMR data and an optical rotation of [].sub.D ([].sub.D.sup.27.8+81.1 (0.05, CH.sub.2Cl.sub.2) matched with literature data confirming the metabolite as epolone B (Cal et al., 1998).

(8) Purification of the second bioactive metabolite yielded 3.47 mg of a compound having a [M+H].sup.+ ion of 549.2853 m/z ([M+MeCN+H].sup.+589.2973 m/z also observed) and NMR signals matched reported literature values confirming the identity of the compound as pycnidione. An optical rotation [].sub.D ([].sub.D.sup.27.8+260.7 (0.3, CH.sub.2Cl.sub.2) confirmed the stereochemistry of the molecule. Purification of the third metabolite yielded 0.64 mg of a compound having a [M+H].sup.+ of 521.2898 m/z ([M+MeCN+H].sup.+561.3018 m/z). NMR and specific optical rotation data Gab ([].sub.D.sup.27.8+210.2 (0.07, CH.sub.2Cl.sub.2)) matched with literature values for epolone A.

(9) Ethyl acetate culture extracts generated for RKDO834 and each of the representative N. samarorum strains were analysed and compared for secondary metabolite production. All strains produced the compounds epolones A and B and pycnidione as confirmed by mass spectral and UV data. All four strains differed in the relative quantities of epolone A and B produced compared to pycnidione, as inferred from ELSD data. In all of the culture extracts examined, pycnidione was one of the more predominant metabolites observed after 14 days growth on solid YM media.

(10) Anti-Fungal Activity Examples

(11) The terms Minimum Inhibitory Concentration and Half Maximal Inhibitory Concentration will be understood to have the following meanings. Minimum Inhibitory Concentration (MIC)
The minimum inhibitory concentration (MIC) will be understood to represent the lowest concentration of an anti-microbial that will inhibit the visible growth of a microorganism after overnight incubation. Half Maximal Inhibitory Concentration (IC.sub.50)
The half maximal inhibitory concentration (IC.sub.50) is a measure of the effectiveness of a substance in inhibiting a spec biological or biochemical function. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological process (or component of a process, i.e. an enzyme, cell, cell receptor or microorganism) by half. IC.sub.50 represents the concentration of an active that is required for 50% inhibition in-vitro.

EXAMPLE 2 PYCNIDIONE ANTI-FUNGAL ACTIVITY

(12) Malassezia furfur (ATCC #38593) was cultured on Media C agar for 7 days at 37 C. Yeast colonies were then harvested into 0.9% saline sterile diH2O and diluted to approximately 1.5106 CFU/mL using a 0.5 MacFarland standard (Fisher #R20410) to create an assay inoculum. Assay inoculum was added to sterile Media C broth to a final concentration of 4.5104 CFU/mL. Assays were carried out in 96 well plates with a final well volume of 100 L.

(13) Extract fractions and pure compounds were tested in triplicate against each organism. Extract fractions and pure compounds were re-suspended in sterile 20% DMSO. Extract fractions were assayed at two concentrations (50 and 250 g/mL) with a final well volume concentration of 2% DMSO, while pure compounds were serially diluted to generate a range of eight concentrations (128 g/mL to 1 g/mL) in a final well volume concentration of 2% DMSO.

(14) Each plate contained eight un-inoculated positive controls (media+20% DMSO), eight untreated negative controls (Media+20% DMSO+organism), and one column containing a concentration range of a ketoconazole as a control antibiotic. The assay plate was incubated at 37 C. for 5 days after which growth within the wells were visualised and photographed with a UVP Biospectrum 500 imaging system. Alamar blue was then added to each well at 10% of the culture volume (11 L in 100 L). Fluorescence was monitored using a BioTek Synergy HT plate reader at 530/25 excitation, 590/35 emission and 35 sensitivity at both time zero and 4 hours after Alamar blue was added. After subtracting the time zero emission 590 nm measurement from the final reading the inferred percentage of microorganism survival relative to vehicle control wells were calculated and the IC50 was determined.

(15) Human foreskin BJ fibroblast cells (ATCC CRL-2522) were grown and maintained in 15 mL of Eagle's minimal essential medium (Sigma M5650) supplemented with 10% fetal bovine serum (VWR #CA95043-976) and 100 U penicillin and 0.1 mg/mL streptomycin (VWR #CA12001-692) in T75 cm2 cell culture flasks (VWR #CABD353136) at 37 C. in a humidified atmosphere of 5% CO.sub.2. Culture medium was refreshed every two to three days and cells were not allowed to exceed 80% confluency.

(16) Adult human epidermal keratinocytes (Heka) isolated from skin (Invitrogen #C-005-5C) were grown and maintained in 15 mL of EPilife medium (Invitrogen #M-EPI-500) supplemented with HKGS growth supplements (Invitrogen #S-001-5) (0.2% v/v bovine pituitary extract (BPE), 5 g/mL bovine insulin, 0.18 g/mL hydrocortisone, 5 g/mL bovine transferrin, 0.2 ng/mL human epidermal growth factor) and 50 g/mL gentamicin (Sigma #G1397-10ML) in T75 cm2 cell culture flasks (VWR #CABD353136) and incubated at 37 C. in a humidified atmosphere of 5% CO.sub.2. Growth medium was refreshed every 2 days until the cells reached 50% confluency and then the medium was refreshed every 24 hours until 80% confluency was obtained.

(17) At 80% confluency, the cells were counted, diluted and plated into 96 well treated cell culture plates (VWR #29442-054) at a cell density of 10000 cells per well in 90 L of respective growth medium. All media used for the assay were the same without the addition of antibiotics. The plates were incubated at 37 C. in a humidified atmosphere of 5% CO.sub.2 to allow cells to adhere to the plates for 24 hrs before treatment. DMSO was used as the vehicle at a final concentration of 1% in the wells.

(18) All compounds to be tested were resolublised in sterile DMSO (Sigma #D2438) and a dilution series was prepared for each cell line using the respective cell culture growth medium of which 10 L were added to the respective assay plate well yielding eight final concentrations ranging from 128 g/mL to 1 g/mL per well (final well volume of 100 L) and incubated at 37 C. in a humidified atmosphere of 5% CO.sub.2 for 24 hrs.

(19) All samples were tested in triplicate. Each plate contained eight un-inoculated positive controls (media+20% DMSO), eight untreated negative controls (Media+20% DMSO+cells). Alamar blue (Invitrogen #Dal1100) was added, 24 hrs after treatment, to each well at 10% of the culture volume (11 L in 100 L). Fluorescence was monitored using a BioTek Synergy HT plate reader at 530/25 excitation, 590/35 emission and 35 sensitivity at both time zero and 4 hrs after Alamar blue was added. After subtracting the time zero emission 590 nm measurement from the final reading the inferred percentage of microorganism survival relative to vehicle control wells were calculated and the IC.sub.50 was determined.

(20) The results of the anti-dandruff actives of the invention are shown in Table 1. Keratinocyte and fibroblast are both skin cells representing various layers of the epidermis, so clearly cytotoxicity against these cells should be within acceptable limits.

(21) TABLE-US-00001 TABLE 1 Anti-dandruff active comparison (all values in g/mL) M. furfur Keratinocyte Fibroblast Active Used MIC IC.sub.50 MIC IC.sub.50 MIC IC.sub.50 Pycnidione 8 6 64 15 64 11 Epolone A 64 48 32 13 32 14 Epolone B 32 24 >128 50 >128 >128

(22) Antifungal activity against the dandruff causing fungus M. furfur as observed for each of the compounds tested, most notably pycnidione which showed an MIC of 8 g/ml (1050 of 6 g/ml).

(23) The compounds pycnidione, epolone A, and epolone B were found to have varying degrees of cytotoxicity against both the keratinocyte and the fibroblast cell lines. Regarding pycnidione, no cell line cytotoxicity was observed at lower concentrations where inhibition of Malassezia yeasts is still retained (ie. 8 g/ml). Therefore a therapeutic window exists for the safe use of pycnidione in the treatment of Malassezia yeasts.

(24) Pycnidione (MIC 64 g/ml; IC.sub.50 15 m/ml) is has very low toxicity to keratinocyte cells compared to existing active zinc pyrithione (MIC 1 g/ml; IC50 0.75 g/ml), the most commonly used active ingredient found in anti-dandruff shampoo formulations.

(25) When formulating pycnidione in an off-the-shelf shampoo composition (J&J baby shampoo) it was found that the composition remained stable after a two month period.

(26) It is to be understood that the invention is not to be limited to the details of the above embodiments, which are described by way of example only. Many variations are possible.