D'ENFERT, Christophe (8 rue des Francs Bourgeois, Paris, Paris, F-75003, FR)
DUMONTET, Vincent (169 rue du Temple, Paris, Paris, F-75003, FR)
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS) (3 rue Michel Ange, Paris, Paris, F-75016, FR)
VEDIYAPPAN, Govindsamy (6103, 18th StreetLubbock, TX, 79416, US)
D'ENFERT, Christophe (8 rue des Francs Bourgeois, Paris, Paris, F-75003, FR)
DUMONTET, Vincent (169 rue du Temple, Paris, Paris, F-75003, FR)
| CLAIMS: 1. A method of modulating morphogenesis of a fungus, comprising contacting said fungus with a compound produced by the plant Gymnema sylvestre. 2. The method of claim 1 , wherein said modulation comprises one or more of the following: (a) inhibition of yeast-to-hypha transition; (b) inhibition of filamentation; (c) prevention of hyphal extension; (d) triggering production of yeast cells from hyphae; (e) downregulating the expression of the HWP1 gene; and (f) downregulating the expression of the TEC1 gene. 3. The method of claim 1 or 2, wherein said compound consists of one or more gymnemic acids. 4. The method of claim 3, wherein said one or more gymnemic acid is represented by Formula I: I wherein Ri-R5 are independently selected from hydrogen, acyl groups and sugar moieties. 5. The method of claim 4, wherein RrR5 are independently selected as follow: Ri is (S)-2-Methylbutyryl or tigloyl; R2 is hydroxyl or O-N-methylanthraniloxy; R3 is hydrogen; R4 is hydrogen or beta-D-glucopyranosyl; and R5 is beta-D-glucuronide. 6. The method of claim 4, wherein Ri is O-methylbutyroyl; R2 is OH; R3 is H; R4 is H; and R5 is Beta-D-glucopyranosyl. 7. The method of claim 4, wherein R1 is O-tiglyol; R2 is OH; R3 is H; R4 is H; and R5 is Beta-D-glucopyranosyl. 8. The method of any one of claims 4 to 7, wherein the gymnemic acid of Formula I is extracted or purified from the plant Gymnema sylvestre. 9. The method of any one of claims 3 to 8, wherein the one or more gymnemic acids is at a concentration of about 0.001 % w/w to about 0.01 % w/w. 10. The method any one of claims 1 to 9, wherein the fungus is a yeast, preferably a Candida, more preferably Candida albicans. 11. Use of a C. θlegans infection model for inhibiting hyphal growth of C. albicans. 12. A method for interfering with the hyphal growth of a fungus, comprising exposing the fungus to one or more gymnemic acids, or a pharmaceutically acceptable salt thereof. 13. A method for preventing and/or treating a mycosis infection in a subject, comprising administering to said subject an effective amount of one or more gymnemic acids, or a pharmaceutically acceptable salt thereof. 14. The method of claim 12 or 13, wherein the one or more gymnemic acid is represented by Formula I: I wherein Ri-R5 are independently selected as follow: R1 is (S)-2-Methylbutyryl or tigloyl; R2 is hydroxyl or O-Λ/-methylanthraniloxy; R3 is hydrogen; R4 is hydrogen or beta-D-glucopyranosyl; and R5 is beta-D-glucuronide. 15. The method of any one of claims 13 or 14, wherein the mycosis infection is a Candida albicans infection. 16. The method of any one of claims 13 to 15, further comprising administering said one or more gymnemic acids in combination with one or more additional antifungal agent. 17. The method of claim 16, wherein said one or more additional antifungal agent is selected from the group consisting of azoles, polyenes, echinocandins, flucytosine and allylamines. 18. Use of one or more gymnemic acids, or a pharmaceutically acceptable salt thereof, in the prevention and/or treatment of a mycosis infection. 19. Use of one or more gymnemic acids, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating a patient infected with Candida, preferably Candida albicans. 20. An antifungal composition comprising an effective amount of one or more gymnemic acids, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. 21. The antifungal composition of claim 20, wherein said composition further comprises one or more additional antifungal agent selected from the group consisting of azoles, polyenes, echinocandins, flucytosine and allylamines. 22. The antifungal composition of claim 20 or 21 , wherein said composition is formulated for an intravenous, oral or topical administration. 23. A method of reducing virulence of fungi in a subject, comprising administering to a susceptible individual an effective amount of an antifungal composition according to any one of claims 19 to 22 for achieving a prophylactic and/or therapeutic antifungal treatment. 24. Use of one or more gymnemic acids, or a pharmaceutically acceptable salt thereof, for reducing and/or preventing fungus-related biofilm formation. |
FIELD OF THE INVENTION
The invention relates to fungal pathogens. More particularly, it relates to antifungals and to methods of inhibiting yeast-to-hypha transition.
BACKGROUND OF THE INVENTION
Candida albicans is one of the most prevalent fungal pathogens in humans, affecting humans at all ages, particularly immunocompromised individuals and patients undergoing organ transplants. Candida albicans virulence can be attributed to its ability to survive and thrive in multiple organs, the mucosa, and the bloodstream of the host and to virulence factors that aid in adherence to and invasion of epithelial and endothelial cell types. C albicans cells exist in different morphological states (yeasts, pseudohypha, hypha) and a well established virulence trait is its ability to switch from yeast or pseudohyphal growth to hyphal growth Moreover, biofilm-mediated resistance to various antifungal agents is well known in C albicans and many hyphal growth related genes are involved in biofilm formation.
Despite the availability of conventional antifungals such as azoles, polyenes, and echinocandins, the mortality associated to disseminated candidiasis remains high (30- 50%). Thus, new therapeutic approaches are needed for the treatment of C. albicans infections Furthermore, the molecular mechanisms that underlie the morphogenetic pathways in C albicans and other yeasts and their potential cross talk are not fully understood and remain to be exploited for the discovery of novel antifungal compounds.
The plant Gymnema sylvestre (family: Asclepiadaceae) is extensively used in traditional systems of medicine. In India, it has been used in Ayurveda for several centuries for the management of diabetes and related disorders and it is well-known for its anti-sweet properties The active principles was isolated in 1887 and named Gymnemic acids (GA) Gymnemic acids have multiple pharmacological activities such as anti-sweet, anti- hyperglycemic, glucose uptake inhibitory, anti obesity, antiviral, gut glycosidase inhibitory activities and antimicrobial activities (E. Porchezhian, R. M. Dobnyal, Pharmazie 58, 5 (2003); P. Kanetkar, R. Singhal, M Kamat, J CIm Biochem Nutr Aλ , 11 (2007); I. Kimura, Yakugaku Zasshi 126, 133 (2006), and M. J. Leach, J Altern Complement Med 13, 977 (2007)). One additional example of extracts from Gymnema sylvestre for use in antimicrobial compositions is US patent publication 2008/0241281 which describes gymnemic acids and other associated saponins to modulate bacterial biofilm virulence, especially biofilms associated with Staphylococcus aureus.
Notwithstanding the discoveries about the medicinal properties of the plant Gymnema sylvestre or its extracts, it was unknown prior to the present invention that gymnemic acids may be therapeutically effective agents in the prevention and/or treatment of fungal infections. It was also unknown that gymnemic acids may be effective in inhibiting yeast- to-hypha transition of C albicans or other fungi.
The present invention addresses the needs for new methods, compounds, and pharmaceutical compositions for use in the prevention and/or treatment of fungal infections. The present invention also addresses the need for antifungal agents capable of reducing virulence of yeasts and hyphal growth related genes involved in biofilm formation.
Additional features of the invention will be apparent from review of the disclosure, figures and description of the invention below. BRIEF SUMMARY OF THE INVENTION
The present invention is derived from the discovery that extracts from the plant Gymnema sylvestre, and more particularly gymnemic acids, are potent inhibitors of the yeast-to- hypha transition in C albicans.
Therefore, one aspect of the invention relates to methods for treating, improving, or alleviating a fungal infection in a subject. In one embodiment, the method comprises administering to the subject (e.g. a human having a mycosis infection) a therapeutically effective amount of one or more of gymnemic acids in a patient.
Another aspect of the invention relates to a method of modulating morphogenesis of a fungus, comprising contacting said fungus with a compound extracted or purified from the plant Gymnema sylvestre. Preferably, the compound consists of one or more gymnemic acids, more preferably of gymnemic acιd(s) represented by Formula I as defined hereinafter The gymnemic acιd(s) of Formula I may also be synthesized chemically.
Examples of modulation of the fungus morphogenesis according to the invention include, but is not limited to, (a) inhibition of yeast-to-hypha transition; (b) inhibition of filamentation; (c) prevention of hyphal extension; (d) triggering production of yeast cells from hyphae, (e) inhibition of HWP1 gene expression; (g) inhibition of TEC1 gene expression; (h) inhibition of hyphal formation in a non-mammalian host model such as Caenorhabditis elegans and (i) survival of C. a/ib/cans-i nfected C. elegans.
The invention also relates to a method for interfering with the hyphal growth of a fungus, comprising exposing the fungus to one or more gymnemic acids, or a pharmaceutically acceptable salt thereof.
Another aspect of the invention concerns the use of one or more gymnemic acids, or a pharmaceutically acceptable salt thereof, in the prevention and/or treatment of a mycosis infection. A related aspect concerns the use of such compound(s) for the manufacture of a medicament for treating a patient infected with Candida, preferably Candida albicans. Yet, an additional related aspect concerns a method for preventing and/or treating a mycosis infection in a subject, comprising administering to said subject an effective amount of one or more gymnemic acids, or a pharmaceutically acceptable salt thereof. The mycosis infection may be superficial or systemic. In some embodiments the mycosis infection is a Candida infection, and more particularly an infection by Candida albicans. The invention also concerns an antifungal composition comprising an effective amount of one or more gymnemic acids, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. Preferably, the antifungal composition is formulated for an oral, topical or intravenous administration.
An additional aspect of the invention concerns the use of one or more gymnemic acids, or a pharmaceutically acceptable salt thereof, for reducing virulence of fungi during a fungal infection in a subject.
Yet, another aspect of the invention concerns the use of one or more gymnemic acids, or a pharmaceutically acceptable salt thereof, for reducing and/or preventing fungus-related biofilm formation. The methods, extracts, gymnemic acid(s) and antifungal compositions according to the invention may be used in combination with additional conventional antifungal agent(s).
Another aspect of the invention concerns the use of a C. elegans infection model for inhibiting hyphal growth of C. albicans and methods for inhibiting hyphal growth of C. albicans using a C. elegans infection model. Additional aspects, advantages and features of the present invention will become more fully understood from the detailed description given herein and from the accompanying drawings, which are exemplary and should not be interpreted as limiting the scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS
FIGURES 1A, 1 B and 1C are pictures showing that Gymnnemic acids (GA) inhibit hyphal formation and extension by C. abicans. Figure 1A shows the effect of GA addition on the yeast-to-hypha transition induced by RPMI or 10% serum at 37°C in liquid. Arrowheads point to vesicular bodies that are represented in yeast cells exposed to GA. Figure 1 B shows the effect of GA on filamentation of C. abicans grown in solid RPMI. Figure 1C shows the effect of GA on hyphae.
FIGURE 2 are pictures showing that Gymnnemic acids (GA) trigger the accumulation of vesicular bodies in S cerevisiae.
FIGURE 3 are pictures showing that gymnemic acids (GA) inhibit hyphal formation and virulence of C albicans upon interaction with C elegans Figure 3A shows that in the absence of GA 1 worms feeded by C albicans are invaded and killed by hyphal cells In contrast, Figure 3B shows that in the presence of GA worms remain viable.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The invention relates to fungal pathogens and more particularly to antifungals and to methods of inhibiting yeast-to-hypha transition Indeed, the present inventors have unexpectedly found that compounds from the plant Gymnema sylvestre are potent inhibitors of the yeast-to-hypha transition in C albicans.
Compounds according to the invention include those which have been produced by the plant Gymnema sylvestre As used herein, the term 'produced" encompasses those compounds which are synthesized by the plant Gymnema sylvestre. This comprises not only compounds which can be purified or isolated form G sylvestre, but also compounds which exist in G sylvestre and which are chemically synthesized.
Gymnemic acids
Some aspects of the invention relates to an antifungal agent comprising a compound extracted or purified from the plant Gymnema sylvestre. As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a compound" includes one or more of such compounds and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein. In some embodiment, the compound consists of substantially pure gymnemic acid(s). The term "substantially pure" refers to a preparation comprising at least 60-75% by weight of a given material (e.g., one or more gymnemic acids). More preferably, the preparation comprises at least 85% by weight, and most preferably 95-99% by weight of gymnemic acid(s). Purity is measured by methods appropriate for the given compound (e.g. chromatographic methods, HPLC analysis, and the like). The present invention encompasses substantially pure gymnemic acid(s) and compositions comprising the same.
The basic molecular structure of GAs has been reported in the literature (E. Porchezhian, R. M. Dobriyal, Pharmazie 58, 5 (Jan, 2003); H. M. Liu, F. Kiuchi, Y. Tsuda, Chem Pharm Bull (Tokyo) 40, 1366 (Jun, 1992)). Isomers of GA (MW of about 800 dalton) are known to have similar genin with variable side groups.
The present invention is not limited to any particular gymnemic acid and any known and unknown gymnemic acid having a desirable antifungal activity is encompassed by the present invention. Examples of methods for assessing antifungal activity are known in the art and provided in the Exemplification section, and include for instance filamentation assay. In some embodiments, the gymnemic acids are selected from the gymnemic acids described in E. Porchezhian, R. M. Dobriyal, Pharmazie 58, 5 (2003); P. Kanetkar, R. Singhal, M. Kamat, J CHn Biochem Nutr Λϊ , 77 (2007); I. Kimura, Yakugaku Zasshi 126, 133 (2006); and/or M. J. Leach, J Altern Complement Med 13, 977 (2007).
In preferred embodiments, the gymnemic acid(s) of the present invention is represented by Formula I:
I
wherein RrR 5 are independently selected hydrogen, acyl groups and sugar moieties.
In one particular embodiment, the gymnemic acid is represented by Formula I, wherein R1-R5 are independently selected as follow:
R 1 is (S)-2-Methylbutyryl or tigloyl;
R 2 is hydroxyl or O-Λ/-methylanthraniloxy;
R 3 is hydrogen;
R 4 is hydrogen or beta-D-glucopyranosyl; and
R 5 is beta-D-glucuronide.
In one subset of gymnemic acids of Formula I, R 1 is (S)-2-Methylbutyryl.
In one subset of gymnemic acids of Formula I, R 1 is tigloyl. In one subset of gymnemic acids of Formula I, R2 is hydroxyl. In one subset of gymnemic acids of Formula I, R 2 Is O-Λ/-methylanthraniloxy. In one subset of gymnemic acids of Formula I, R 3 is hydrogen. In one subset of gymnemic acids of Formula I, R 4 is hydrogen.
In one subset of gymnemic acids of Formula I, R 4 is beta-D-glucopyranosyl. In one subset of gymnemic acids of Formula I, R 5 is beta-D-glucuronide.
In one particular embodiment, R 1 is O-methylbutyroyl; R2 is OH; R 3 is H; R 4 is H; and R 5 is Beta-D-glucopyranosyl. In another particular embodiment, Ri is O-tiglyol; R 2 is OH; R 3 is H; R 4 is H; and R 5 is Beta- D-glucopyranosyl.
The one or more gymnemic acid(s) of the present invention, or their pharmaceutically acceptable salts may contain one or more asymmetric centers, chiral axes and chiral planes and may thus give rise to enantiomers, diastereomers, and other stereoisomer^ forms and may be defined in terms of absolute stereochemistry, such as (R)- or (S)- or, as (D)- or (L)- for amino acids. The present invention is intended to include all such possible isomers, as well as, their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as reverse phase HPLC. The racemic mixtures may be prepared and thereafter separated into individual optical isomers or these optical isomers may be prepared by chiral synthesis. The enantiomers may be resolved by methods known to those skilled in the art, for example by formation of diastereoisomeric salts which may then be separated by crystallization, gas-liquid or liquid chromatography, selective reaction of one enantiomer with an enantiomer specific reagent. It will also be appreciated by those skilled in the art that where the desired enantiomer is converted into another chemical entity by a separation technique, an additional step is then required to form the desired enantiomeric form. Alternatively specific enantiomers may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts, or solvents or by converting one enantiomer to another by asymmetric transformation.
Certain gymnemic acids may exist in Zwitterionic form and the present invention includes Zwitterionic forms of these compounds and mixtures thereof.
The present invention encompasses all acids, salts and other ionic and non-ionic forms of the gymnemic acids of Formula I. As used herein, the term "pharmaceutically acceptable salt" refers more particularly to base addition salt which include those salts which retain the biological effectiveness and properties of the free acids, and which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Example of pharmaceutically acceptable salts are also described, for example, in Berge er a/., "Pharmaceutical Salts", J. Pharm. Sci. 66, 1-19 (1977).
In general, gymnemic acids of Formula I may be purified from the plant Gymnema sylvestre using methods and techniques known in the art [see for instance H. M. Liu, F. Kiuchi, Y. Tsuda, Chem Pharm Bull (Tokyo) 40, 1366 (1992)] or as described hereinafter in the Exemplification section.
It is also conceivable that Gymnemic acids of Formula I may be chemically synthesized. Accordingly, the present invention also encompasses chemically synthesized GAs.
In one embodiment, crude GAs extract are obtained from a crude extract of G. sylvestre by acidic extraction and solvent partitions. GAs are purified on 18C preparative chromatography columns using MeOH-H 2 O to yield pure GA. GAs may be characterized using 1 D and 2D 1 H NMR and 13 C NMR and compared with literature data. Sugar moiety assignments can be confirmed by sodium borohydride reduction of GAs in methanol and methanolysis of the reduction mixture. GAs purification can be guided by C.albicans filamentation assay. Biological and pharmaceutical applications
As indicated hereinbefore and exemplified hereinafter, the gymnemic acids of Formula I have interesting biological properties against fungus and these compounds may have useful pharmaceutical applications, especially in the prevention and/or treatment of various fungal infections in a subject. Biological applications contemplated by the inventors include, but are not limited to, modulation of morphogenesis of fungi, and interfering with the hyphal growth of fungi More particular uses include a) inhibition of yeast-to-hypha transition; (b) inhibition of filamentation; (c) prevention of hyphal extension; (d) triggering production of yeast cells from hyphae; (e) inhibition of HWP1 gene expression; and (f) inhibition of TECI gene expression. Medical and pharmaceutical applications contemplated by the inventors include, but are not limited to, the use of gymnemic acids in antifungal compositions and for the manufacture of such compositions, and more particularly for preventing and/or treating a mycosis infection in a subject. As used herein, the term 'fungus" or 'fungi" refers to heterotrophic organisms possessing a chitinous cell wall, and more particularly to those fungal species growing as multicellular filaments called hyphae. As used herein, the term "fungus" or "fungi" encompass yeast, mould, and mildews. In some embodiments, the fungus is a single-celled yeast of the genera Saccharomyces (e.g. S. cerevisiae) or Candida (e.g. C. albicans). In preferred embodiments, the yeast is from the Candida species, and more preferably Candida albicans.
As used herein, the term "fungal infection" or "mycosis" broadly is defined as an infection that is caused by a fungus In some embodiments the fungal infection is by a fungus from the following genus: Candida, Aspergillus, Penicillium, Cryptococcus, Mucor, Scedosporium, Magnaporthe, Ustilago, Fusarium, Botrytis, Mycosphaerella. In some embodiments the fungal infection is by a yeast of the following genus: Candida Cryptococcus, Ustilago. In some embodiments the fungal infection is by a mould of the following genus: Aspergillus, Penicillium, Mucor, Scedosporium. In some embodiments the fungal infection is by a mildew of the following families Magnaporthe, Fusarium, Botrytis, Mycosphaerella As used herein, the term 'fungal infections" includes but is not limited to athlete's foot (Tinea pedis), jock itch, ringworm (Tinea corporis), skin infections, nail infections, scalp infections, candidemia and candidiasis. Candidiasis refers to those infections involving Candida albicans and includes, but is not limited to, oral intertrigo, vulvovaginitis, diaper rashes, paronychia and chronic mucocutaneous candidiasis. Candidemia refers to the presence of fungi or yeast in the blood.
The term ' subject' includes living organisms in which a fungus infection can occur, or which are susceptible to such conditions. The term "subject" includes animals (e.g , mammals, e.g , cats dogs, horses, pigs, cows, goats, sheep, rodents (e.g , mice or rats, rabbits, squirrels), bears, primates (e.g., chimpanzees, monkeys, gorillas, and humans)), as well as wild and domestic bird species (e g. chickens, ducks, Peking ducks, geese), and transgenic species thereof Preferably, the subject is a mammal More preferably, the subject is a human. Even more preferably, the subject is a human patient in need of treatment, e.g. a human having a mycosis infection. As used herein, "preventing" or "prevention" is intended to refer to at least the reduction of likelihood of the risk of (or susceptibility to) acquiring or suffering from a fungus infection.
The terms "treatment" or "treating" of a subject includes the application or administration of one or more gymnemic acids of Formula I to a subject (or application or administration of such gymnemic acid(s) to a cell or tissue from a subject) with the purpose of stabilizing, curing, healing, alleviating, relieving, altering, remedying, less worsening, ameliorating, improving, or affecting the disease or condition, the symptom of the disease or condition, or the risk of (or susceptibility to) the disease or condition. The term "treating" refers to any indication of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; lessening of the rate of worsening; lessening severity of the disease, stabilization, diminishing of symptoms or making the injury, pathology or condition more tolerable to the subject; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a subject s physical or mental well-being In some embodiments, the term "treating" can include increasing a subject's life expectancy and/or delay before additional treatments are required (e.g. administration of a second antifungal agent).
Another aspect of the invention relates to a method of reducing virulence of fungi prior to or during a fungal infection. Examples of effective virulence reduction may comprises (ι) reducing the ability of the fungus to survive and thrive in multiple organs, the mucosa, and the bloodstream of the host; (ii) decreasing the presence of virulence factors that aid in adherence to and invasion of epithelial and endothelial cell types, (ιιι) preventing or inhibiting the fungus ability to switch from yeast or pseudohyphal growth to hyphal growth (ιv) inhibiting the expression of hyphal specific genes (eg. HWP1, TEC1 etc.), (v) preventing the formation of biofilms; and (vi) synergizing the activity of conventional or other antimicrobials In one embodiment the method comprises administering to a susceptible individual an extract, compound or composition as described herein for achieving a prophylactic and/or therapeutic antifungal treatment.
Biofilm-mediated resistance to various antifungal agents is well known in C albicans and many hyphal growth related genes are involved in biofilm formation Preliminary results indicate that GA prevents biofilm formation, possibly because of its effect on the yeast-to- hypha transition Accordingly, contemplated in the scope of the present invention is the use of gymnemic acid(s) in preventing biofilm formation and or for the manufacture of compositions for preventing and/or reducing biofilm formation The gymnemic acid(s) and compositions comprising the same may be useful at any of the following stages of biofilm development, i.e. initial attachment; irreversible attachment maturation I; maturation II; and dispersion. The invention is particularly concerned with biofilms which contain fungi and with the use of gymnemic acid(s) where biofilm formation is involved in fungal infections such as oropharyngeal candidiasis, vulvovaginalcandidiasis, disseminated candidiasis. Antifungal compositions
A related aspect of the invention concerns antifungal compositions comprising a therapeutically effective amount of a compound extracted or purified from the plant Gymnema sylvestre. In preferred embodiments, the term "antifungal composition" refers to the presence of gymnemic acid(s) in the composition, and more preferably gymnemic acids of Formula I.
As used herein, the term "therapeutically effective amount" means the amount of compound that, when administered to a subject for treating or preventing a particular disorder, disease or condition, is sufficient to effect such treatment or prevention of that disorder, disease or condition. Dosages and therapeutically effective amounts may vary for example, depending upon a variety of factors including the activity of the specific agent employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and any drug combination, if applicable, the effect which the practitioner desires the compound to have upon the subject and the properties of the compounds (e.g. bioavailability, stability, potency, toxicity, etc), and the particular disorder(s) the subject is suffering from. In addition, the therapeutically effective amount may depend on the subject's blood parameters (e.g. lipid profile, insulin levels, glycemia), the severity of the disease state, organ function, or underlying disease or complications. Such appropriate doses may be determined using any available assays. When one or more of the compounds of the invention is to be administered to humans, a physician may for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
The antifungal compositions of the invention may comprise one or more gymnemic acids in a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the gymnemic acιd(s). The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, and intraperitoneal routes. In some embodiments, the antifungal compositions of the invention are formulated to be administered topically to a subject, e.g., by the direct laying on or spreading of the composition on the epidermal or epithelial tissue of the subject, or transdermal^ via a "patch". Such compositions include, for example, lotions, creams, solutions, gels and solids. These topical compositions may comprise an effective amount, usually at least about 0 001 % (ou 0 1 mg/10ml), or about 0.01 % (ou 1 mg/10ml), or about 0 1 % (ou 10 mg/10ml), or about 1 % (ou 100 mg/10ml), or more of a compound of the invention. Suitable carriers for topical administration typically remain in place on the skin as a continuous film, and resist being removed by perspiration or immersion in water. Generally, the carrier is organic in nature and capable of having dispersed or dissolved therein the therapeutic agent. The carrier may include pharmaceutically acceptable emollients, emulsifiers, thickening agents, solvents and the like.
The compound(s) of the invention can be orally administered. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Formulations of the invention suitable for oral administration may be in the form of capsules (e g. hard or soft shell gelatin capsule), cachets, pills, tablets, lozenges, powders, granules, pellets, dragees, e.g., coated (e.g., enteric coated) or uncoated, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oιl-ιn-water or water-ιn-oιl liquid emulsion, or as an elixir or syrup, or as pastilles or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. A compound of the present invention may also be administered as a bolus, electuary or paste, or incorporated directly into the subject's diet. Moreover, in certain embodiments these pellets can be formulated to (a) provide for instant or rapid drug release {i e , have no coating on them); (b) be coated, e.g., to provide for sustained drug release over time; or (c) be coated with an enteric coating for better gastrointestinal tolerability. Coating may be achieved by conventional methods, typically with pH or time-dependent coatings, such that the compound(s) of the invention is released in the vicinity of the desired location, or at various times to extend the desired action. Such dosage forms typically include, but are not limited to, one or more of cellulose acetate phthalate, polyvinylacetate phthalate, hydroxypropyl methyl cellulose phthalate, ethyl cellulose, waxes, and shellac.
The compound(s) of the invention may also be administered intravenously, parenterally, intraperitoneal^, intraspinally, or intracerebrally using acceptable methods and formulations known in the art.
Those skilled in the art will be able to determine the best formulation and the best route of administration which will vary depending upon a variety of factors. For instance, for a systemic infection, an IV administration is generally preferred On the other hand, a topical administration may be suitable for localized infections (e.g Oropharyngeal Candidiasis (OPC), or vulvovaginal candidiasis (WC)).
The methods and uses of the present invention may also include co-administration of an extract of Gymnema sylvestre, of a compound and/or of gymnemic acιd(s) according to the invention, or of a pharmaceutically acceptable salt thereof, together with the administration of additional antifungal agent(s) Therefore, an additional aspect of the invention relates to methods of concomitant therapeutic treatment of a subject, comprising administering to a subject in need thereof an effective amount of a first agent and a second agent, wherein the first agent is an extract and/or gymnemic acid(s), or a pharmaceutically acceptable salt thereof, as defined herein, and the second agent is a compound having antifungal activities. As used herein, the term "concomitant" or "concomitantly" as in the phrases "concomitant therapeutic treatment" or concomitantly with ' includes administering the first agent in the presence of a second agent. A concomitant therapeutic treatment method includes methods in which the first agent is co-administered with second, third or additional agents. A concomitant therapeutic treatment method also includes methods in which the first agent is administered in the presence of a second or additional agents, wherein the second or additional agents, for example, may have been previously administered. A concomitant therapeutic treatment method may be executed step-wise by different actors. For example, one actor may administer a first agent to a subject and a second actor may administer a second agent to the subject and the administering steps may be executed at the same time, or nearly the same time, or at distant times, so long as the first agent is in the presence of the second agent (and/or additional agents) after their respective administration. The actor and the subject may be the same entity (e.g. a human). The additional antifungal agent(s) may be selected from known compounds which are currently being used or in development for preventing or treating fungal infections Examples of such known compounds include but are not limited to azoles (e g fluconazole, voriconazole, posoconazole), polyenes (e g amphotericin B, Nystatin), echinocandins (e.g caspofungin, micafungin, aminocandin), flucytosine (e.g. 5- fluorocytosin), allylamines and others.
The invention further relates to a screening method for identifying antifungals In one embodiment, the screening method comprises contacting C. albicans yeast cells with a compound to be tested; and assessing activity of said compound on HWP1 and/or TEC1 activity and/or mRNA levels. Headings are included herein for reference and to aid in locating certain sections These headings are not intended to limit the scope of the concepts described therein under, and these concepts may have applicability in other sections throughout the entire specification Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, concentrations, properties, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about " At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present specification and attached claims are approximations that may vary depending upon the properties sought to be obtained. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the embodiments are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contain certain errors resulting from variations in experiments, testing measurements, statistical analyses and such. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of this invention and covered by the claims appended hereto. The invention is further illustrated by the following examples, which should not be construed as further limiting EXAMPLES
EXAMPLE 1. Characterization of the active molecule(s) in Gymnema sylvestre extracts
The inventors have selected a collection of medicinal plant derived extracts for screening of inhibitors of hyphal growth. By using a multiwell plate cell based assay the inventors screened for compounds that inhibit C albicans yeast-to-hypha transition and their hyphal growth. Primary screening results were confirmed by secondary screening and/or with different hypha-inducing assay conditions. One promising plant source (Gymnema sylvestre) was selected for large-scale purification and characterization. Ethanol extract (50Og) of G. sylvestre (GS) leaves was sourced from LaksBiotech (India). Dried crude extract was dissolved in 70% methanol and mixed with charcoal powder at 8O 0 C for 1 h. The de-pigmented extract was acid precipitated and extracted sequentially with n-butanol-water (1 :1 ), petroleum ether and methanol. After solvent evaporation, the GA mixture was purified on preparative reverse phase (RP) & HPLC chromatography using MeOH-water gradient. At least 4 different fractions with hyphal inhibitory activity were recovered from preparative HPLC. Fractions 6 and 7 contained Gymnemic acid (GA) GA III and IV, respectively, as determined by LC/DAD/DEDL and other spectroscopic methods (For GA III, Formula I wherein R 1 is O-methylbutyroyl; R 2 is OH; R 3 is H; R 4 is H; and R 5 is Beta-D-glucopyranosyl; for GA IV, Formula I wherein R 1 is O-tiglyol; R 2 is OH; R 3 is H; R 4 is H; and R 5 is Beta-D-glucopyranosyl). These latter two compounds may be related to GA I and GA II, respectively. All these compounds are 90-95% pure as determined by analytical HPLC.
EXAMPLE 2: Activity of Gymnemic acid on Candida albicans and Saccharomyces cerevisiae
Gymnemic acids (GA) identified using the purification scheme described in Example 1 was used in a variety of biological assays in order to study its activity on C. albicans cells.
The results of these experiments are shown in Figures 1 and 2 and hereinafter:
(1 ) GA (at 10 μM) have no effect on the growth and viability of C. albicans yeast cells;
(2) GA (at least at 10 μM) inhibit C. albicans yeast-to-hypha transition under several hypha inducing conditions (liquid and solid RPMI, 37 0 C; 10% serum, 37°C; Figs. 1A and B). As previously reported, this effect is associated to a defect in the yeast-to- hypha dependent up regulation of the HWP1 hypha-specific gene (Staab et al. (1999), Science 283, 1535) as well as the TEC1 gene encoding a transcription factor involved in the yeast-to-hypha transition (Brown and Gow, (1999), Trends Microbiol 7, 333).
(3) GA (at 10 μM) prevents hyphal extension and triggers the production of yeast cells from hyphae (Fig. 1C);
(4) Exposure of C. albicans to GA triggers the appearance of uncharacterized intracellular vesicular bodies (Fig. 1A). Preliminary results suggest that these bodies might be related to vacuoles.
The effect of GA on Saccharomyces cerevisiae was also tested. Data presented in Figure 2 show that exposure of S. cerevisiae to GA did not impair growth of that yeast but resulted in the appearance of numerous vesicular bodies. This phenotype is reminiscent of that seen in S. cerevisiae mutants that are defective in vacuolar biogenesis suggesting that GA might target this process, a process which is known to be involved in the yeast-to- hypha transition in C. albicans. The hyphal inhibitory activity of GA was examined in a non-mammalian animal (Caenorhabditis elegans) model as decribed by J. Breger et al. PLoS Pathog 3, e18 (2007) and E. Tampakakis et al. Nat . Protocol. 3, 1925 (2008) (Figure 3). In the absence of GA (Figure 3A), C. albicans produced hyphae from the gut of worms and killed the worms by piercing through their cuticle. However, in the presence of GA (Figure 3B), not only the C. albicans hyphal formation was inhibited but also the worms survived indicating that GA was not toxic to the host and prevented hyphal formation in the host.
Taken together, these results indicate that GA is a potent inhibitor of the yeast-to-hypha transition in C. albicans in vitro and in a non-mammalian animal model. Moreover, GA triggers the appearance of vesicular bodies in yeast cells suggesting a possible relationship with vacuole biogenesis. In this regard, C. albicans mutants that have defects in vacuolar biogenesis show defects in the yeast-to-hypha transition and have reduced virulence in an animal model of systemic candidiasis (M. Cornet et al., Infect lmmun 73, 7977 (2005); S. M. Bernardo et al., Fungal Genet Biol 45, 861 (2008); C. J. Barelle, et al., Eukaryot Cell 5, 359 (2006); A. Kitanovic et al., FEMS Yeast Res 5, 431 (2005)). Moreover, these mutants show hypersensitivity to conventional antifungals such as azoles and echinocandins (M. Cornet et al., Infect lmmun 73, 7977 (2005); S. M. Bernardo et al., Fungal Genet Biol 45, 861 (2008)). Thus, GA might represent a promising lead compound for an antivirulence approach to be used alone or in combination with conventional antifungals. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of this invention and covered by the claims appended hereto. The invention is further illustrated by the following examples, which should not be construed as further limiting.