Anti-dandruff compositions comprising a spirofuranone lactam tetramic acid derivative

Abstract

Anti-Dandruff Agents A novel anti-dandruff composition comprising spirofuranone-lactam tetramic acid or derivatives thereof, and optionally at least one biologically derivable meroterpene. The spirofuranone-lactam tetramic acid is preferably a bio-active heterospirocyclic secondary metabolite of Aspergillus, such as pseurotin A. The biologically derivable meroterpene may be selected from fumagillin, fumagillin derivative, chlovalicin, or ovalicin. Use of said agents as anti-dandruff actives in anti-dandruff compositions, particularly shampoos and conditioners is also provided. The active is particularly effective against Malassezia yeasts and Malassezia furfur which may cause dandruff. A method of obtaining the anti-dandruff actives from culturing of Peyronellaea sp. strain RKDO347 is also described.

Claims

1. An anti-dandruff composition comprising an effective amount of: at least one compound selected from the group consisting of pseurotin A, pseurotin A.sub.1, pseurotin A.sub.2, pseurotin B, pseurotin C, pseurotin E, pseurotin D, pseurotin F.sub.1, pseurotin F.sub.2, asaspirenen, and synerazol; and at least one meroterpene represented by formula (I): ##STR00002## wherein: R.sup.1 represents hydrogen and R.sup.2 represents chlorine, or R.sup.1 and R.sup.2 together represent a single bond to form an epoxide ring; R.sup.3 represents hydrogen or hydroxyl; R.sup.4 represents a hydroxyl, oxygen, or OC(O)[CHCH].sub.nR.sup.5 group wherein R.sup.5 represents a C.sub.2-C.sub.4 alcohol or diol, a C.sub.2-C.sub.4 optionally substituted epoxide, or a carboxyl group; and n represents an integer in the range from 1 to 3.

2. The anti-dandruff composition according to claim 1, wherein the meroterpene comprises at least one of chlovalicin and ovalicin.

3. The anti-dandruff composition according to claim 1, wherein R.sup.4 represents the OC(O)[CHCH].sub.nR.sup.5 group.

4. The anti-dandruff composition according to claim 3, wherein n is 1.

5. The anti-dandruff composition according to claim 3, wherein n is 2.

6. The anti-dandruff composition according to claim 3, wherein n is 3.

7. The anti-dandruff composition according to claim 1, wherein R.sup.1 represents hydrogen and R.sup.2 represents chlorine.

8. The anti-dandruff composition according to claim 1, wherein R.sup.1 and R.sup.2 together represent a single bond to form an epoxide ring.

9. The anti-dandruff composition according to claim 1, wherein R.sup.4 represents hydroxyl or oxygen.

10. The anti-dandruff composition according to claim 1, wherein the compound is at least one of pseurotin A, pseurotin A.sub.1, pseurotin A.sub.2 or synerazol.

11. The anti-dandruff composition according to claim 1, wherein the compound is pseurotin A.

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

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

14. The anti-dandruff composition according to claim 1, further comprising at least one surfactant.

15. The anti-dandruff composition according to claim 1, further comprising at least one or more of a betaine, a non-ionic surfactant, an amphoteric surfactant, and a cationic surfactant.

16. The anti-dandruff composition according to claim 1, which is a shampoo.

17. The anti-dandruff composition according to claim 1, which is obtained from a culture of Peyronellaea.

Description

EXAMPLE 1FORMING & EXTRACTION OF ANTI-DANDRUFF ACTIVES

(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 d at 22 C.

(2) 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. A 500 L aliquot of seed inoculum was removed into a sterile 2 mL Eppendorf tube, centrifuged at 10000 rpm for 5 min 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.

(3) An additional 500 L of seed inoculum from strain RKDO347 was dispensed into a capped 250 mL Erlenmyer flask containing autoclave sterilized 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 seperatory funnel. The solvent extract was dried down under a stream of air prior to further chemical purification.

(4) 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 min isocratic step with 5% aqueous MeOH followed by a continuous gradient to 100% MeOH over 15 min and a final 100% MeOH isocratic step during 5 min. The twelve resulting fractions were analyzed by LC-HRMS and NMR and their antimicrobial and cytotoxic activities were evaluated.

(5) Fraction 6 yielded pure pseurotin A and 10 yielded pure fumagillin derivative as identified by characteristic mass spectrometry signals (m/z corresponding to [M+H]+ and [M+Na]+ ions) and proton chemical shifts and correlations with neighbouring carbon atoms. Both compounds demonstrated anti-Malassezia activity.

(6) 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.

(7) Anti-Fungal Activity Examples

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

(9) Minimum Inhibitory Concentration (MIC)

(10) 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.

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

(12) 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 & PSEUROTIN A ANTI-FUNGAL ACTIVITY

(13) Malassezia furfur (ATCC #38593) was cultured on Media C agar for 7 d 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.

(14) Extract fractions and pure compounds were tested in triplicate against each organism. Extract fractions and pure compounds were resuspended 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.

(15) Each plate contained eight uninoculated 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 5d after which growth within the wells were visualized 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.

(16) 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.

(17) 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.

(18) 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.

(19) Experimentation was carried out to assess the anti-fungal effect of the produced fumagillin derivative and the pseurotin A each on their own, and in combination against M. furfur in a shampoo type formulation.

(20) Initially the M. furfur efficacy assay was tested with both J&J Baby Shampoo as well as Head & Shoulders to establish a baseline and assess the compatibility of the assay using a shampoo. 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 (concentration combinations are listed in Table 1).

(21) The results of the anti-dandruff actives of the invention are shown in Table 1.

(22) TABLE-US-00002 TABLE 1 Anti-dandruff active comparison (all values in g/mL) MIC (g/ml) IC.sub.50 Fumagillin derivative 2 0.75 Pseurotin A (sample 1) 128 55 Pseurotin A (sample 2) 75 Fumagillin derivative & Pseurotin A 0.31 & 37.5

(23) From this data, it can be seen that Pseurotin A alone produces an MIC response and so does the fumagillin derivative alone. A combination of 0.31 g/ml of the fumagillin derivative and 37.5 g/ml of pseurotin A (sub-inhibitory concentrations) produced an MIC response which suggests a synergistic effect. Each of Pseurotin A and the fumagillin derivative alone are sufficient for anti-dandruff effects, a combination of the fumagillin derivative and pseurotin A allowed for the low effective dose of the fumagillin derivative and pseurotin A to 0.31 g/ml. These MIC values when using the actives together show a synergistic effect when compared to the MIC values of each component on their own.

(24) This data demonstrates that when used in combination 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 than a shampoo formulation containing either of the two active ingredients alone.

(25) 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.