# Patent

## Title
Treatment of superficial and cutaneous mycoses with a pure form of 1,7-bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione

## Abstract

Disclosed are novel and enhanced anti-fungal properties of greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione in comparison with the corresponding 95% pure form, said greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione being devoid of 5-Hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-3-heptanone and 3,5-Dihydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-heptane. The inventors disclose the uses of greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione as anti-fungal agents capable of inhibiting superficial and cutaneous mycoses in humans and treatment methods thereof. Also disclosed is the enhanced potential of greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione as compared to the corresponding 95% pure form against bacterial and fungal phytopathogens and applications thereof in agriculture.

## Background
BACKGROUND OF THE INVENTION
This application is a non-provisional filing of provisional application No. 60/945,254, filed on Jun. 20, 2007 the contents of which are incorporated herein by reference.


FIELD OF THE INVENTION
The present invention in general relates to 1,7-Bis (4-hydroxy-3-methoxyphenyl)-3,5-heptanedione and its applications. More specifically, the present invention relates to novel and enhanced anti-fungal properties of greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione in comparison with the corresponding 95% pure form, said greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione being devoid of 5-Hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-3-heptanone and 3,5-Dihydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-heptane. The inventors disclose the uses of greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione as anti-fungal agents capable of inhibiting superficial and cutaneous mycoses in humans and treatment methods thereof. Also disclosed is the enhanced potential of greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione as compared to the corresponding 95% pure form against bacterial and fungal phytopathogens and applications thereof in agriculture. Fungal infections are responsible for several disease conditions in human and plant kingdom. Some pathogenic fungi such as Malassezia furfur give rise to an unhigenic scaling of the skull. Other systemic infections produce debilitating health conditions such as fever and pain. Some fungi are specifically plant pathogens and stunt the growth of plants either sharing their nutrients or interfering with their metabolic pathways. 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione is novel inhibitor of fungi growth. It is an effective inhibitor of both human and plant pathogenic fungi. A remarkable purity dependence of the activity was uncovered in this invention. It was found that 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione with >99% purity had manifold activity than when it is >95%. Such a large dependence of activity for a small change in purity is a totally unexpected outcome.
DESCRIPTION OF PRIOR ART
Human mycoses may be broadly classified based on the location of the infection within the body as (i) Superficial, cutaneous and subcutaneous; and (ii) Systemic.
Superficial mycoses include cosmetic fungal infections of the skin or hair shaft with no involvement of living tissues or cellular (immunological or pathological) responses from the host. Cutaneous mycoses are superficial fungal infections of the skin, hair or nails. No living tissue is invaded, however a variety of pathological changes occur in the host because of the presence of the infectious agent and its metabolic products.
An overview of the common pathogens associated with Superficial and cutaneous mycoses is presented herein below as Table A.












Disease
Causative organisms
Incidence















I. SUPERFICIAL MYCOSES









Pityriasis versicolor
Malassezia furfur
Common


Seborrhoeic dermatitis
(a lipophilic yeast)


including Dandruff


and Follicular


pityriasis


Tinea nigra
Exophiala werneckii
Rare


White piedra
Trichosporon beigelii
Common


Black piedra
Piedraia hortae
Rare







II. CUTANEOUS MYCOSES









Dermatophytosis
Dermatophytes (Microsporum,
Common


Ringworm of the
Trichophyton, Epidermophyton)


scalp, glabrous


skin and nails.


Candidiasis of skin,
Candida albicans and related species
Common


mucous membranes


and nails.


Dermatomycosis
Non-dermatophyte moulds
Rare



Hendersonula toruloidea



Scytalidium hyalium



Scopulariopsis brevicaulis









Therapeutic management of superficial and cutaneous fungal diseases has posed a tremendous challenge owing to the fact that fungal cells, like other living organisms, may become resistant to toxic compounds. Antifungal resistance may be defined as a stable, inheritable adjustment by a fungal cell to an antifungal agent, resulting in a less than normal sensitivity to that antifungal. Variable results on the anti-fungal resistance (both clinical and in-vitro resistance patterns) among dermatophytes has been indicated in many prior art references. Some important ones are included herein below.
I. Mycoses. 2007 July; 50(4):286-9
In vitro activities of four antifungal drugs against Trichophyton rubrum isolates exhibiting resistance to fluconazole. Santos D A, Hamdan J S. Department of Microbiology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
Summary:
Variable MIC ranges for drugs Terbinafine, itakonazole, ketokonazole and griseofulvin against T. rubrum, in vitro. Efficacy in the order of Terbinafine>itaconazole>ketoconazole>griseofulvin. Much work is still needed to correlate the MICs of these drugs with clinical outcomes to develop interpretative breakpoints for T. rubrum and other dermatophytes.
II. Dermatology. 2003; 207(4):375-80.
Evaluation of in vitro resistance in patients with onychomycosis who fail antifungal therapy.
Gupta A K, Kohli Y. Division of Dermatology, Department of Medicine, Sunnybrook and Women's College Health Science Center, Sunnybrook site and the University of Toronto, Toronto, Ontario, Canada.
Summary:
With the more common use of anti-fungals to treat various fungal infections, development of increased resistance in the causative organisms remains a possibility. However, factors other than fungal resistance may also be implicated in treatment failure.
III. Rev Iberoam Micol. 2007 Dec. 1; 24(4):320-2.
Resistance to azolic compounds in clinical Trichophyton spp. Strains. Laboratorio de Investigación Médica en Dermatología y Micología “Dr. Ernesto Macotela”, Hospital de Especialidades “Dr. Bernardo Sepúlveda”, UMAE Centro Médico Nacional Siglo XXI, IMSS, México D. F., México.
Summary:
Among 36 clinical isolates tested, the resistance to one or more antifungal drugs was demonstrated in seven isolates (19.4%) as follows: three Trichophyton rubrum, three T. mentagrophytes and one T. tonsurans. A T. rubrum isolate was resistant to the three azolic drugs; the other six only to fluconazole. It is important to establish the antifungal susceptibility as part of the study procedures in patients with dermatophytosis and a poor antifungal response.
Agricultural mycosis involves the complex relationship between pathogenic fungi and plant hosts in terms of infestation and disease. Agricultural mycosis has also serious implications in terms of the deleterious effects of “mycotoxins” found in agricultural products during the post harvest period. Important mycotoxins include

    
    
        A. Aflatoxins are produced by Aspergillus species, and are largely associated with commodities such as groundnuts, other edible nuts, figs, spices and maize. Aflatoxin B1 is a potent carcinogen associated with liver cancer.
        B. Ochratoxin A produced by Penicillium verrucosum and Aspergillus ochraceus, and a contaminant of a wide range of commodities including cereals and their products, fruit and a wide range of beverages and spices.
        C. Fusarium toxins (fumonisins, trichothecenes and zearalenone) produced by members of the genus Fusarium which infect the grain of developing cereals such as wheat and maize.
    
    


In an attempt to study significant differences in the biological properties of greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione in comparison with the corresponding 95% pure form, wherein the greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione does not occur along with 5-Hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-3-heptanone and 3,5-Dihydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-heptane, the present inventors have surprisingly proved that the anti-fungal properties of the greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione is significantly enhanced in comparison with the corresponding 95% pure form. Hence the present inventors disclose the use of greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione in the manufacture of medicaments for the treatment of superficial and cutaneous mycoses in humans and also treatment methods thereof. Further, the inventors also disclose the use of greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione to control phytopathogenic infestation (bacteria and fungi) in agricultural crops.
Accordingly, it is the principle object of the present invention to disclose the enhanced anti-fungal properties of greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione in comparison to the corresponding 95% pure form, wherein the greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione does not occur along with 5-Hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-3-heptanone and 3,5-Dihydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-heptane.
It is another object of the present invention to disclose the use of greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione not occurring along with 5-Hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-3-heptanone and 3,5-Dihydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-heptane in the manufacture of medicaments for the treatment of superficial/cutaneous mycoses and treatment methods thereof with no residual toxicity and minimal clinical resistance.
It is yet another object of the present invention to disclose methods of controlling phytopathogenic infestations with no residual toxicity by bringing into contact affected parts of plant with compositions comprising greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione, wherein the greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione does not occur along with 5-Hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-3-heptanone and 3,5-Dihydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-heptane
The present invention fulfills the aforesaid objectives and provides further related advantages.
SUMMARY OF THE INVENTION
The present invention relates to novel and enhanced anti-fungal properties of greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione in comparison with the corresponding 95% pure form, said greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione being devoid of 5-Hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-3-heptanone and 3,5-Dihydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-heptane. The inventors disclose the uses of greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione as anti-fungal agents capable of inhibiting superficial and cutaneous mycoses in humans and treatment methods thereof. Also disclosed is the enhanced potential of greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione as compared to the corresponding 95% pure form against bacterial and fungal phytopathogens and applications thereof in agriculture. Fungal infections are responsible for several disease conditions in human and plant kingdom. Some pathogenic fungi such as Malassezia furfur give rise to an unhigenic scaling of the skull. Other systemic infections produce debilitating health conditions such as fever and pain. Some fungi are specifically plant pathogens and stunt the growth of plants either sharing their nutrients or interfering with their metabolic pathways. 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione is novel inhibitor of fungi growth. It is an effective inhibitor of both human and plant pathogenic fungi. A remarkable purity dependence of the activity was uncovered in this invention. It was found that 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione with >99% purity had manifold activity than when it is >95%. Such a large dependence of activity for a small change in purity is a totally unexpected outcome.
The present invention provides the following advantages.

    
    
        1. Disclosure of the enhanced biological (anti-fungal) properties of greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione in comparison to the corresponding 95% pure form.
        2. Disclosure of a new therapeutic use for greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione with minimal toxicity in the management of superficial and cutaneous mycoses in the light of increasing anti-fungal resistance to Terbinafine, itakonazole, ketokonazole and griseofulvin and concerns over residual toxicity.
        3. Disclosure of novel applications for greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione with minimal toxicity in controlling phytopathogens (bacteria and fungi).
    
    


Other features and advantages of the present invention will become apparent from the following more detailed description, taken in conjunction with the accompanying images, which illustrate, by way of example, the principle of the invention.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 (Photograph 1) shows the anti-dermatophytic activity of greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione at concentrations of 0.15%, 0.31%, 0.625%, 1.25%, 2.50% and 5.0%, completely inhibiting the growth of Trichophyton rubrum, Microsporum gypseum and Epidermophyton floccosum both at low and higher concentrations.
FIG. 2 (Photograph 2) shows the ability of dermatophytes Trichophyton rubrum, Microsporum gypseum and Epidermophyton floccosum to grow in the presence of lower concentrations (0.15%) of 95% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione. Epidermophyton floccosum is also shown to be tolerant to a slightly higher concentration (0.31%) of 95% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione.
FIG. 3 (Photograph 3) shows no effect of Dimethylsulfoxide (DMSO) used to dissolve the test compounds namely 95% and greater than 99% pure forms of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione on the growth of dermatophytes Trichophyton rubrum, Microsporum gypseum and Epidermophyton floccosum. 



DETAILED DESCRIPTION OF THE MOST PREFERRED EMBODIMENT
In the most preferred embodiment, the present invention relates to the use of greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione represented by STR#1 in the manufacture of a medicament for the treatment of cutaneous mycoses in humans, said greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione showing enhanced anti-fungal activity than the corresponding 95% pure form, on account of being devoid of 5-Hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-3-heptanone represented by STR#II or 3,5-Dihydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-heptane represented by STR#III.




More specifically, the cutaneous mycoses condition is one selected from the group consisting of Tinea pedis, Tines corporis, Tinea incognito, Tinea unguium (dermatophyte onychomycosis), Tinea capitis, Tinea barbae and candidosis of skin, hair or nails. Further, the cutaneous mycosis condition is dermatophytosis caused by the members of the genus Trichophyton, Microsporum and Epidermophyton. Still further, the cutaneous mycoses condition is candidosis of hair, skin and nails causes by members of the genus Candida. Preferably, said medicament for the treatment of cutaneous mycoses comprises greater than 99% pure form of 1,7-bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione at a concentration range of about 0.15% to about 5%. More preferably, the said medicament for the treatment of cutaneous mycoses comprises greater than 99% pure form of 1,7-bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione at a concentration of about 0.15%. Further, the said medicament additionally comprises 0.01-0.1% tetrahydropiperine as a bioavailability enhancer.
In another preferred embodiment, the present invention relates to the method of treating cutaneous mycoses in humans, said method involving the step of topically applying to the affected area a formulation comprising 0.1-10% w/w of greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione represented by STR#1, said greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione showing enhanced anti-fungal activity than the corresponding 95% pure form, on account of being devoid of 5-Hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-3-heptanone represented by STR#II and 3,5-Dihydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-heptane represented by STR#III. More preferably, the said formulation comprises from about 0.15% to about 5.0% of greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione represented by STR#1. Most preferably, the said formulation comprises 0.15% of greater than 99% pure form of 1,7-Bis (4-hydroxy-3-methoxyphenyl)-3,5-heptanedione represented by STR#1. Further, the said medicament additionally comprises 0.01-0.1% tetrahydropiperine as a bioavailability enhancer.
In yet another preferred embodiment, the present invention relates to the use of greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione represented by STR#1 in the manufacture of a medicament for the treatment of superficial mycoses in humans, said greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione showing enhanced anti-fungal activity than the corresponding 95% pure form, on account of being devoid of 5-Hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-3-heptanone represented by STR#II and 3,5-Dihydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-heptane represented by STR#III.




In specific, the superficial mycoses condition is one selected from the group consisting of Pityriasis versicolor, Seborrhoeic dermatitis including Dandruff and Follicular pityriasis, Tinea nigra, White piedra and Black piedra. More specifically, the superficial mycoses condition is caused by Malassezia furfur. Still more specifically, the superficial mycoses condition is caused by Exophiala werneckii. Still more specifically, the superficial mycoses condition is caused by Trichosporon beigelii. Still more specifically, the superficial mycoses condition is caused by Piedraia hortae. Preferably, said medicament for the treatment of superficial mycoses comprises greater than 99% pure form of 1,7-bis (4-hydroxy-3-methoxyphenyl)-3,5-heptanedione at a concentration range of about 0.15% to about 5%. More preferably, the said medicament for the treatment of superficial mycoses comprises greater than 99% pure form of 1,7-bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione in a concentration range of about 0.15%. Further, the said medicament additionally comprises 0.01-0.1% tetrahydropiperine.
In another preferred embodiment, the present invention relates to the method of treating superficial mycoses in humans, said method comprising the step of topically applying to the affected area a formulation comprising greater than 99% pure form of 1,7-bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione represented by STR#1 in a concentration range of about 0.15% to about 5%, wherein said greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione shows enhanced anti-fungal activity than the corresponding 95% pure form, on account of being devoid of 5-Hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-3-heptanone represented by STR#II and 3,5-Dihydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-heptane represented by STR#III.




In an alternate embodiment, the present invention also relates to a method of controlling pathogenic infestation of agricultural crops, said method comprising the step of bringing into contact the infected parts of the plants with 100 ppm (parts per million) to 2000 ppm (parts per million) of a composition comprising greater than 99% pure 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione, wherein the said greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione shows enhanced anti-phytopathogenic properties in comparison to the corresponding 95% pure form, on account of being devoid of 5-Hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-3-heptanone and 3,5-Dihydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-heptane. Specifically, the phytopathogens are soil borne pathogens or foliar pathogens. More specifically, the phytopathogens are bacteria, fungi and oomycetes. Still more specifically, the phytopathogenic fungi are members of the Phylum Zygomycota, Phylum Ascomycota, Phylum Basidiomycota and Deuteromycetes [Fungi imperfecti]. Further, the phytopathogenic fungi also produce mycotoxins.
The enhanced anti-fungal properties of 0.15% to about 5.0% of 99.5% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione represented by STR#1 in comparison to the corresponding 95% pure form, on account of being devoid of 5-Hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-3-heptanone represented by STR#II or 3,5-Dihydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-heptane represented by STR#III has been highlighted in specific examples included herein below.
Example 1
Anti-Dermatophytic Activity
The anti-dermatophyte efficacy testing for greater than 99% pure form of 1,7-Bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione (coded TP) in comparison to the corresponding 95% pure form (coded SW) was done by the agar dilution method. The medium used was Sabouraud's dextrose agar (SDA). Doubling concentration ranges from about 0.15% to about 5% of TP and TW was prepared in 5 ml Dimethylsulfoxide and added to 95 ml of SDA to form a total volume of 100 ml of the medium. The test organisms Trichophyton rubrum, Microsporum gypseum and Epidermophyton floccosum encoded as TR, MG and EF were inoculated in biosafety level 2 standards into the media comprising test compounds in the specified concentration ranges. Triplicate experiments were set up for each of the three fungi for each concentration of the test compounds to ensure reproducibility of results. DMSO and SDA controls were also included in triplicates for the test.
The test results are represented as Table I (FIG. 1, FIG. 2 and FIG. 3). The table may be read as follows. For example, TPTR1 represents growth of Trichophyton rubrum in greater than 99% pure form of 1,7-Bis (4-hydroxy-3-methoxyphenyl)-3,5-heptanedione in the first test. Similarly duplicate and triplicate testing are represented as TPTR2 and TPTR3.







TABLE I





(FIG. 1, FIG. 2 and FIG. 3)

















ANTIFUNGAL EFFICACY - AGAR DILUTION METHOD (Sabourauds Dextrose Agar)











COMPOUND SW




DMSO SOLVENT



COMPOUND TP
WITH FINAL


TEST
DMSO SOLVENT WITH FINAL CONCENTRATION
CONCENTRATION















ORGANISM
TRIAL
5%
2.5%
1.25%
0.625%
0.31%
0.15%
5%





Trichophyton
TR1
TPTR1-5
TPTR1-2.5
TPTR1-1.25
TPTR1-0.625
TPTR1-0.31
TPTR1-0.15
SWTR1-5


rubrum

Negative
Negative
Negative
Negative
Negative
Negative
Negative



TR2
TPTR2-5
TPTR2-2.5
TPTR2-1.25
TPTR2-0.625
TPTR2-0.31
TPTR2-0.15
SWTR2-5




Negative
Negative
Negative
Negative
Negative
Negative
Negative



TR3
TPTR3-5
TPTR3-2.5
TPTR3-1.25
TPTR3-0.625
TPTR3-0.31
TPTR3-0.15
SWTR3-5




Negative
Negative
Negative
Negative
Negative
Negative
Negative


Microsporum
MG1
TPMG1-5
TPMG1-2.5
TPMG1-1.25
TPMG1-
TPMG1-0.31
TPMG1-0.15
SWMG1-5


gypseum

Negative
Negative
Negative
0.625
Negative
Negative
Negative







Negative



MG2
TPMG2-5
TPMG2-2.5
TPMG2-1.25
TPMG2-0.625
TPMG2-0.31
TPMG2-0.15
SWMG2-5




Negative
Negative
Negative
Negative
Negative
Negative
Negative



MG3
TPMG3-5
TPMG3-2.5
TPMG3-1.25
TPMG3-0.625
TPMG3-0.31
TPMG3-0.15
SWMG3-5




Negative
Negative
Negative
Negative
Negative
Negative
Negative


Epidermophyton
EF1
TPEF1-5
TPEF1-2.5
TPEF1-1.25
TPEF1-
TPEF1-0.31
TPEF1-0.15
SWEF1-5


flocossun

Negative
Negative
Negative
0.625
Negative
Negative
Negative







Negative



EF2
TPEF2-5
TPEF2-2.5
TPEF2-1.25
TPEF2-0.625
TPEF2-0.31
TPEF2-0.15
SWEF2-5




Negative
Negative
Negative
Negative
Negative
Negative
Negative



EF3
TPEF3-5
TPEF3-2.5
TPEF3-1.25
TPEF3-0.625
TPEF3-0.31
TPEF3-0.15
SWEF3-5




Negative
Negative
Negative
Negative
Negative
Negative
Negative













ANTIFUNGAL EFFICACY - AGAR DILUTION METHOD




(Sabourauds Dextrose Agar)



COMPOUND SW
CONTROL










TEST
DMSO SOLVENT WITH FINAL CONCENTRATION
DMSO
SDA















ORGANISM
TRIAL
2.5%
1.25%
0.625%
0.31%
0.15%
Control
Control





Trichophyton
TR1
SWTR1-2.5
SWTR1-1.25
SWTR1-0.625
SWTR10.31
SWTR1-0.15
DMSOTR1
SDATR1


rubrum

Negative
Negative
Negative
Negative
Growth
Growth
Growth



TR2
SWTR2-2.5
SWTR2-1.25
SWTR2-0.625
SWTR2-0.31
SWTR2-0.15
DMSOTR2
SDATR2




Negative
Negative
Negative
Negative
Growth
Growth
Growth



TR3
SWTR3-2.5
SWTR3-1.25
SWTR3-0.625
SWTR3-0.31
SWTR3-0.15
DMSOTR3
SDATR3




Negative
Negative
Negative
Negative
Growth
Growth
Growth


Microsporum
MG1
SWMG1-2.5
SWMG1-1.25
SWMG1-0.625
SWMG1-0.31
SWMG1-0.15
DMSOMG1
SDAMG1


gypseum

Negative
Negative
Negative
Negative
Growth
Growth
Growth



MG2
SWMG2-2.5
SWMG2-1.25
SWMG2-0.625
SWMG2-0.31
SWMG2-0.15
DMSOMG2
SDAMG2




Negative
Negative
Negative
Negative
Growth
Growth
Growth



MG3
SWMG3-2.5
SWMG3-1.25
SWMG3-0.625
SWMG3-0.31
SWMG3-0.15
DMSOMG3
SDAMG3




Negative
Negative
Negative

Growth
Growth
Growth


Epidermophyton
EF1
SWEF1-2.5
SWEF1-1.25
SWEF1-
SWEF1-0.31
SWEF1-0.15
DMSOEF1
SDAEF1


flocossun

Negative
Negative
0.625
Growth
Growth
Growth
Growth






Negative



EF2
SWEF2-2.5
SWEF2-1.25
SWEF2-0.625
SWEF2-0.31
SWEF2-0.15
DMSOEF2
SDAEF2




Negative
Negative
Negative
Growth
Growth
Growth
Growth



EF3
SWEF3-2.5
SWEF3-1.25
SWEF3-0.625
SWEF3-0.31
SWEF3-0.15
DMSOEF3
SDAEF3




Negative
Negative
Negative
Growth
Growth
Growth
Growth









It was clearly evident that greater than 99% pure form of 1,7-bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione (coded TP) inhibits the growth of dermatophytes Trichophyton rubrum, Microsporum gypseum and Epidermophyton flocossum at all tested concentrations in contrast to 95% pure form of 1,7-bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione (coded SW) which was tolerated by the fungi at lower concentrations (0.15%). Epidermophyton flocossum was able to grow at both 0.15% and 0.31% of SW.
Example II
Activity Against Malassezia Furfur (Table II)









TABLE II









ANTIFUNGAL EFFICACY - AGAR DILUTION METHOD



(Sabourauds Dextrose Agar with an overlay of coconut oil)



COMPOUND TP



DMSO SOLVENT WITH FINAL CONCENTRATION














TEST ORGANISM
TRIAL
5%
2.5%
1.25%
0.625%
0.31%
0.15%





Malassezia furfur
MF1
TPMF1 - 5
TPMF1 -
TPMF1 -
TPMF1 -
TPMF1 -
TPMF1 -


(MF)

Negative
2.5
1.25
0.625
0.31
0.15





Negative
Negative
Negative
Negative
Negative



MF2
TPMF2 - 5
TPMF2 -
TPMF2 -
TPMF2 -
TPMF2 -
TPMF2 -




Negative
2.5
1.25
0.625
0.31
0.15





Negative
Negative
Negative
Negative
Negative



MF3
TPMF3 - 5
TPMF3 -
TPMF3 -
TPMF3 -
TPMF3 -
TPMF3 -




Negative
2.5
1.25
0.625
0.31
0.15





Negative
Negative
Negative
Negative
Negative












ANTIFUNGAL EFFICACY - AGAR DILUTION METHOD



(Sabourauds Dextrose Agar with an overlay of coconut oil)



COMPOUND SW



DMSO SOLVENT WITH FINAL CONCENTRATION














TEST ORGANISM
TRIAL
5%
2.5%
1.25%
0.625%
0.31%
0.15%





Malassezia furfur
MF1
SWMF1 -
SWMF1 -
SWMF1 -
SWMF1 -
SWMF10.31
SWMF1


(MF)

5
2.5
1.25
0.625
Growth
0.15




Negative
Negative
Negative
Growth

Growth



MF2
SWMF2 -
SWMF2 -
SWMF2 -
SWMF2 -
SWMF2 -
SWMF2




5
2.5
1.25
0.625
0.31
0.15




Negative
Negative
Growth
Growth
Growth
Growth



MF3
SWMF3 -
SWMF3 -
SWMF3 -
SWMF3 -
SWMF3 -
SWMF3




5
2.5
1.25
0.625
0.31
0.15




Negative
Negative
Negative
Growth
Growth
Growth









It was clearly evident that greater than 99% of 1,7-bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione in contrast to the corresponding 95% pure form showed enhanced activity against Malassezia furfur at all tested concentration (Table II). It was evident that the 95% pure form could inhibit the growth of the fungus only at higher concentrations (1.25% and above).
Example III
Activity Against Candida Species (Table III)







TABLE III





Organism: Candida albicans NCIM 3471 (yeast)


















Concentration of organism




(cfu/ml)
Percent Reduction (%)















1,7-bis



1,7-bis



1,7-
(4-


1,7-bis
(4-



bis(4-
hydroxy-


(4-
hydroxy-



hydroxy-
3-


hydroxy-
3-



3-
methoxyphenyl)-


3-
methoxyphenyl)-



methoxyphenyl)-
3,5-


methoxyphenyl)-
3,5-



3,5-
heptanedione


3,5-
heptanedione


Test
heptanedione
(about


heptanedione
(about


interval
(>99%
95%
Nutrient

(>99%
95%


(days)
purity)
purity)
Base
Control
purity)
purity)





 0
14.6 × 105
  25 × 105
16.3 × 105
21.7 × 106
—
—


 7
  72 × 105
19.7 × 105
83.5 × 105
30.8 × 104
50.6
21.2


14
  20 × 108
  16 × 104
73.5 × 104
12.7 × 104
99.8
99.3


21
  75 × 102
  53 × 105
76.0 × 105
10.5 × 104
99.94
78.6


28
<130
31.5 × 105
82.0 × 106
6.25 × 108
99.99
87.4












Log Reduction
















1,7-






1,7-
bis(4-





bis(4-
hydroxy-





hydroxy-
3-





3-
methoxyphenyl)-





methoxyphenyl)-
3,5-




Percent
3,5-
heptanedione



Test
Reduction (%)
heptanedione
(about















interval
Nutrient

(>99%
95%
Nutrient




(days)
Base
Control
purity)
purity)
Base
Control







 0
—
—



 7
48.7
98.5
0.5
0.2
0.5
1.8



14
95.4
99.4
2.8
2.3
1.5
2.4



21
53.3
99.5
3.4
0.7
0.53
2.5



28
45.6
99.9
5.3
0.8
0.5
3.7










The enhanced efficacy of greater than 99% pure form of 1,7-bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione in reducing the colony counts of Candida albicans NCIM3471 (Yeast) to less than 100 CFU/ml with an overall percentage reduction of 99.99% over a test interval time of 28 days is indicated in Table III.
Example 4
Activity Against Fusarium oxysporum (Table 4)







TABLE 4







1,7-bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione (99%) Test


Compound













Inoculation
Day 0
Day 7
Day 14
Day 28



Level
count
count
count
Count


ORGANISMS
(CFU/g)
(CFU/g)
(CFU/g)
(CFU/g)
(CFU/g)





Fusarium
1 × 107
1 × 107
<10
<10
<10


oxysporum


(mycotoxin


producing


fungus)









While the invention has been described with reference to a preferred embodiment, it is to be clearly understood by those skilled in the art that the invention is not limited thereto. Rather, the scope of the invention is to be interpreted only in conjunction with the appended claims.

## Claims

1. A method of treating superficial and cutaneous mycoses in a human, said method comprising topically applying to the affected area of said human a formulation comprising greater than 99% pure form of 1,7-bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione.

2. The method according to claim 1, wherein the superficial and cutaneous mycoses condition is selected from a group consisting of Pityriasis versicolor, Seborrhoeic dermatitis, Dandruff, Follicular pityriasis, Tinea nigra, White piedra, Black piedra and onychomycosis.

3. The method according to claim 1, wherein the superficial and cutaneous mycoses are caused by Malassezia furfur, Trichophyton species, Epidermophyton species, Microsporum species, Candida or Aspergillus.

4. The method according to claim 1, wherein the formulation topically applied additionally contains 0.01-0.1% w/w of tetrahydropiperine.