Anti-dandruff agents

Abstract

A novel anti-dandruff agent being a fumagillin derivative according to formula (I), and use of said agent as an anti-dandruff actives in anti-dandruff compositions, particularly shampoos and conditioners. The active is particularly effective against Malassezia yeasts and Malassezia furfur which may cause dandruff. A method of obtaining the fumagillin derivative from culturing of Peyronellaea sp. strain RKDO347 is also described. ##STR00001##

Claims

1. An anti-dandruff composition comprising an effective amount of an anti-dandruff active comprising a fumagillin derivative according to formula (I): ##STR00007##

2. The anti-dandruff composition according to claim 1, wherein the MIC cytotoxicity of the fumagillin derivative against Malassezia furfur cells is less than 80 g per ml.

3. The anti-dandruff composition according to claim 1, wherein the IC.sub.50 cytotoxicity of the fumagillin derivative against M. furfur cells is less than 40 g per ml.

4. The anti-dandruff composition according to claim 1, wherein said composition comprises in the range from 0.001 wt. % to 20 wt of the fumagillin derivative, based on the total weight of the composition.

5. The anti-dandruff composition according to claim 1, wherein the fumagillin derivative is the only anti-dandruff active present in the anti-dandruff composition.

6. The anti-dandruff composition according to claim 1, wherein the fumagillin derivative is used in combination with at least one other anti-dandruff active material.

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

8. A method of forming an anti-dandruff composition which comprises mixing together: (i) an anti-dandruff composition according to claim 1; (ii) at least one surfactant; and (iii) water.

9. An anti-dandruff shampoo or conditioner comprising: an anti-dandruff composition according to claim 1; at least one surfactant; and optionally one or more of a betaine, a non-ionic surfactant, an amphoteric surfactant, and a cationic surfactant.

10. A method of providing anti-dandruff efficacy which comprises the steps of: (i) wetting the hair with water; (ii) applying an effective amount of an anti-dandruff composition comprising an anti-dandruff composition according to claim 1 to the hair; (iii) rinsing the anti-dandruff composition from the hair using water; and (iv) optionally repeating steps (ii) and (iii).

11. The method according to claim 10, wherein the anti-dandruff composition further comprises Pseurotin A.

12. A method for killing or retarding the growth of Malassezia spp., the method comprising the step of contacting the Malassezia spp. with an amount of the composition according to claim 1 effective to kill or retard the growth of Malassezia spp.

13. A method of obtaining an anti-dandruff active comprising the steps of: culturing Peyronellaea sp. in a medium under conditions which promote the metabolic synthesis of an anti-dandruff composition according to claim 1 from the Peyronellaea sp.; and purifying the synthesised anti-dandruff composition from the cultured medium.

Description

EXAMPLE 1FORMING & EXTRACTION OF ANTI-DANDRUFF ACTIVE

(1) Bioassay-guided fractionation of culture extracts lead to the isolation of a biologically active metabolite produced by the fungus Peyronellaea sp. The isolate RKDO347 was plated out on YM (Yeast extract Malt extract) 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 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.

(2) 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 plates, incubated for 3 days at 22 C. and inspected to ensure inoculum purity. An additional 500 L of seed inoculum from strain RKDO347 was dispensed into a capped 250 mL Erlenmyer flask containing autoclave sterilised Mannitol Murashuge & Skoog Salts broth medium and incubated at 22 C. for 14 days. After the incubation period, colony biomass was removed from the growth medium and the remaining broth was filtered through Whatman #3 filter paper using a glass vacuum chamber with a Buchner funnel and then partitioned into an equivalent volume of EtOAc using a separatory funnel. The solvent extract was dried down under a stream of air prior to further chemical purification.

(3) The EtOAc extract was re-suspended in a mixture Water:MeOH (2:8) and partitioned with hexane. The polar layer was then subjected to an automated low pressure chromatography (Combiflash) on a Diol Redisep column (30 g). The eluent system at a 20 mL/min flow rate included a 5 minute isocratic step with 5% aqueous MeOH followed by a continuous gradient to 100% MeOH over 15 minutes and an final 100% MeOH isocratic step during 5 minutes. The twelve resulting fractions were analysed by LC-HRMS and their antimicrobial and cytotoxic activities were evaluated. Fraction 10 yielded to a pure compound demonstrating anti-Malassezia activity, designated as the fumagillin derivative.

(4) NMR spectra were recorded on a Bruker Avance III 600 MHz NMR spectrometer and chemical shifts () were referenced to the CD.sub.3OD residual peaks at .sub.H 3.31 ppm and .sub.C 49.0 ppm. Analytical mass spectrometry of all samples was carried out on a Thermo Scientific Accela UHPLC coupled with a Thermo Exactive mass spectrometer (ESI-MS) with an ESI ion source, a SEDEX 80LT ELSD, and a Thermo photodiode array (PDA) detector.

(5) HRMS analysis of the fumagillin derivative supported the molecular formula C.sub.22H.sub.32O.sub.6 ([M+H].sup.+ m/z 393.22717, [M+Na].sup.+ m/z 415.20911). The chemical structure of the fumagillin derivative was determined based on 1D and 2D NMR data and the key HMBC and COSY correlations as indicated in Figure (II). The observed coupling constant (1.9 Hz) for both protons (.sub.H 3.26 and 3.00) suggest a trans configuration for epoxide present on the fatty acid side chain.

(6) ##STR00006##

Anti-Fungal Activity Examples

(7) The terms Minimum Inhibitory Concentration and Half Maximal Inhibitory Concentration will be understood to have the following meanings.

(8) Minimum Inhibitory Concentration (MIC)

(9) 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.

(10) Half Maximal Inhibitory Concentration (IC.sub.50)

(11) The half maximal inhibitory concentration (IC.sub.50) is a measure of the effectiveness of a substance in inhibiting a specific 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 2FUMAGILLIN DERIVATIVE 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 diH.sub.2O and diluted to approximately 1.510.sup.6 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 4.510.sup.4 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 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.

(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 cm.sup.2 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 cm.sup.2 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 hrs 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 uninoculated positive controls (media+20% DMSO), eight untreated negative controls (Media +20% DMSO+cells), and one column containing a concentration range of zinc pyrithione. 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 active of the invention are shown in Table 1. Keratinocyte and fibroblast are both skin cells representing various layers of the epidermis, so clearly any cytotoxicity against these cells should be within acceptable limits.

(21) TABLE-US-00002 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 Fumagillin 2 0.7 >64 >64 >64 >64 derivative

(22) Solvent culture extracts were found to have an antifungal effect when screened against the dandruff causing Malassezia yeasts; where antifungal activity was associated both with the fungal mycelia as well as the fermentation broth.

(23) Purification efforts demonstrated that the observed potent anti-Malassezia activity was associated with the novel fumagillin derivative. The fumagillin derivative has selective antifungal activity against Malassezia spp., demonstrating an approximate minimal inhibitory concentration (MIC) of 2 g/mL and a half maximal inhibitory concentration (IC.sub.50) of 0.7 g/mL; antifungal activities that are comparable to existing anti-fungal actives. However, unlike existing anti-fungal actives, this good anti-fungal activity is combined with little cytotoxicity to either keratinocyte or fibroblast cells at the highest concentration tested (>64 g/mL).

EXAMPLE 3FUMAGILLIN DERIVATIVE IN COMBINATION WITH PSEUROTIN A

(24) Experimentation was carried out to assess the anti-fungal effect of the fumagillin derivative alone and in combination with pseurotin A against M. furfur in a shampoo type formulation.

(25) Compositions of the fumagillin derivative with and without pseurotin A were created and suspended into the baby shampoo. The shampoo mixture was then diluted out into the assay in a similar manner to Head & Shoulders, so that various dilution points in the assay represented multiple sub-inhibitory concentrations of the fumagillin derivative and pseurotin A.

(26) The results of the anti-dandruff activity are shown in Table 2.

(27) TABLE-US-00003 TABLE 2 fumagillin derivative in combination with Pseurotin A MIC (g/ml) Fumagillin derivative 1.25 Fumagillin derivative & Pseurotin A 0.31 & 37.5

(28) An antifunimgal effect against M. furfur was observed for the pseurotin A on its own. However, when the fumagillin derivative and Pseurotin A compounds were assayed together as a mixture, at concentrations of and below the expected MIC for both compounds, a synergistic increase in efficacy was observed at sub-MIC concentrations in comparison to results for each compound tested individually. When the same experiment was run against the keratinocyte cell line, no cytotoxicity was observed for any of the concentration combinations tested.

(29) From this data, the combination of 0.31 g/ml of fumagillin derivative and 37.5 g/ml of pseurotin A (sub-inhibitory concentrations) produced an MIC response and allows for a lowering of the effective dose of fumagillin derivative from 1.25 g/ml to 0.31 g/ml whilst maintaining the anti-fungal properties.

(30) This data shows that less fumagillin derivative and pseurotin A are needed in a shampoo formulation to achieve the desired effect of M. furfur inhibition similar to Head & Shoulders when compared to shampoo formulation containing either of the two active ingredients alone.

(31) 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.