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Title:
ANTIMYCOTIC
Document Type and Number:
WIPO Patent Application WO/2021/013931
Kind Code:
A1
Abstract:
The present invention relates to methods and means for inhibiting or preventing the growth of non-filamentous biofilm forming fungal cells with at least one flavone of formula (I) wherein R1, R2, R3, R4, R5, R6 and R7 are independently from each other H or OH.

Inventors:
KAINZ KATHARINA (AT)
ZIMMERMANN ANDREAS (AT)
CARMONA-GUTIERREZ DIDAC (AT)
MADEO FRANK (AT)
BAUER MARIA (AT)
Application Number:
PCT/EP2020/070808
Publication Date:
January 28, 2021
Filing Date:
July 23, 2020
Export Citation:
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Assignee:
KARL FRANZENS UNIV GRAZ (AT)
International Classes:
A61P31/10; C07D311/30
Foreign References:
CN105669625A2016-06-15
CN105669625A2016-06-15
Other References:
RICARDO SALAZAR-ARANDA ET AL: "Activity of Polyphenolic Compounds against Candida glabrata", MOLECULES, vol. 20, no. 10, 29 September 2015 (2015-09-29), pages 17903 - 17912, XP055462836, DOI: 10.3390/molecules201017903
WEIDENBORNER M ET AL: "Antifungal activity of flavonoids against storage fungi of the genus Aspergillus", PHYTOCHEMISTRY, PERGAMON PRESS, GB, vol. 29, no. 4, 1 January 1990 (1990-01-01), pages 1103 - 1105, XP026605263, ISSN: 0031-9422, [retrieved on 19900101], DOI: 10.1016/0031-9422(90)85412-9
WANG X G ET AL: "Antifungal Flavonoids from Ficus sarmentosa var. henryi (King) Corner", AGRICULTURAL SCIENCES IN CHINA,, vol. 9, no. 5, 1 May 2010 (2010-05-01), pages 690 - 694, XP027059802, ISSN: 1671-2927, [retrieved on 20100501]
SALAZAR-ARANDA ET AL., MOLECULES, vol. 20, 2015, pages 17903 - 17912
WEIDENBORNER ET AL., PHYTOCHEM, vol. 29, 1990, pages 1103 - 1105
WANG ET AL., AGR SCI CHINA, vol. 9, 2010, pages 690 - 694
DELATTIN N ET AL., ANTIMICROB CHEMOTHER., vol. 69, 2014, pages 1035 - 1044
WAGNERFARKAS: "The Flavonoids", 1975, SPRINGER, article "Synthesis of Flavonoids"
ALTEMIMI ET AL., PLANTS, vol. 6, no. 4, 2017, pages 42
REX JH: "Clinical and Laboratory Standards Institute", 2008, NATIONAL COMMITTEE FOR CLINICAL LABORATORY STANDARDS, article "Reference method for broth dilution antifungal susceptibility testing of yeasts: approved standard"
DELATTIN N ET AL., J ANTIMICROB CHEMOTHER, vol. 69, 2014, pages 1035 - 1044
"Maintenance of C. elegans", WORMBOOK, 2006
PORTA-DE-LA-RIVA M ET AL., JOVE, vol. 64, 2012, pages e4019 - e4019
CLSI PROTOCOL M27-A3 (M27-A3, vol. 28, no. 14, 2008, ISBN: 1-56238-666-2
DELATTIN N ET AL., J ANTIMICROB CHEMOTHER., vol. 69, 2014, pages 1035 - 1044
BREGER J ET AL., PLOS PATHOG., vol. 3, no. 2, 2007, pages e18
Attorney, Agent or Firm:
SCHWARZ & PARTNER PATENTANWÄLTE OG et al. (AT)
Download PDF:
Claims:
CLAIMS :

1. Use of a flavone of formula (I)

for inhibiting or preventing the growth of a non-filamentous biofilm forming fungal cell, wherein Ri, R2, R3, R4, R5, R6 and R7 are independently from each other H or OH.

2. Use according to claim 1, wherein the flavone is selected from the group consisting of 3, 6-dihydroxyflavone, 3,3'-dihy- droxyflavone, 6, 7-dihydroxyflavone, 2 ' , 3-dihydroxyflavone,

3, 7-dihydroxyflavone, 3' , 4' -dihydroxyflavone, 3-hydroxyfla vone, 6-hydroxyflavone, 7-hydroxyflavone, 3' -hydroxyflavone,

3' , 4' , 5, 7-tetrahydroxyflavone, 3 ' , 4 ' , 7-trihydroxyflavone and 3 ' , 4 ' -dihydroxyflavone .

3. Use according to claim 1 or 2, wherein the non-filamentous fungal cell is selected from the group consisting of Candida

4. Use according to any one of claims 1 to 3, wherein the fla- vone of formula (I) is used in combination with at least one further antimycotic compound.

5. Use according to claim 4, wherein the antimycotic compound is selected from the group consisting of azoles, echinocandins and polyenes.

6. Use according to any one of claims 1 to 5, wherein the fla- vone is applied to a plant or parts thereof, in particular fruits or leaves.

7. Composition comprising at least one flavone of formula (I)

for the use in the treatment of a fungal infection in a human or animal, preferably mammalian, subject wherein Ri, R2, R3, R4, R5, Re and R7 are independently from each other H or OH, and wherein said fungal infection is caused by a non-filamentous biofilm forming cell.

8. Composition for use according to claim 7, wherein the fla vone is selected from the group consisting of 3, 6-Dihy- droxyflavone, 3, 3' -Dihydroxyflavone, 6, 7-Dihydroxyflavone, 2 ' , 3-Dihydroxyflavone, 3, 7-Dihydroxyflavone, 3',4'-Dihy- droxyflavone, 3-Hydroxyflavone, 6-Hhydroxyflavone, 7-Hy- droxyflavone, 3' -hydroxyflavone, 3' , 4' , 5, 7-tetrahydroxyfla vone, 3 , 4 , 7-trihydroxyflavone and 3 , 4 -dihydroxyflavone .

9. Composition for use according to claim 7 or 8, wherein the composition comprises at least one further antimycotic com pound .

10. Composition for use according to claim 9, wherein the at least one further antimycotic compound is selected from the group consisting of azoles, echinocandins and polyenes.

11. Composition for use according to any one of claims 7 to

10, wherein the composition comprises at least one pharmaceu tically acceptable excipient.

12. Composition for use according to any one of claims 7 to

11, wherein the at least one flavone is administered to the human or animal, preferably mammalian, subject together or subsequently with at least one further antimycotic compound as defined in claim 5.

13. Composition for use according to any one of claims 7 to

12, wherein the composition or the flavone and/or the at least one further antimycotic compound are administered orally, top ically or intravenously.

14. Method for inhibiting or preventing the growth of a non- filamentous biofilm fungal cell comprising the step of con tacting fungal cells with at least one flavone as defined in claim 1 or 2 and/or a composition as defined in any one of claims 7 to 11.

Description:
ANTIMYCOTIC

TECHNICAL FIELD

[0001] The present invention relates to the field of antimy- cotics .

BACKGROUND ART

[0002] Fungal infections are rapidly developing into an in creasing medical and socioeconomic problem. Both the num ber of infections as well as resulting deaths are in creasing rapidly, so pandemic proportions may arise in the near future. The eukaryotic nature of the fungal cell and the resulting close relationship to human cells makes the search for new antifungal agents even more difficult.

[0003] Currently, there are only a very limited number of antimycotics on the market, which, however, sometimes cause serious side effects or are not as efficient as re quired. The effectiveness is increasingly limited as the incidence of resistance to it increases rapidly. There fore, the search for new antifungal active substances is of major importance.

[0004] Salazar-Aranda et al . (Molecules 20 (2015) : 17903- 17912) describe an antifungal activity of certain flavo- noids, such as baicalein and myricetin, against flucona zole-resistant C. glabrata.

[0005] Weidenborner et al . (Phytochem 29 (1990) : 1103-1105) found that certain flavones and flavanones display fungi cidal activity against the filamentous fungus Aspergil lus. Similarly, Wang et al. (Agr Sci China 9(2010) : 690- 694) describe an inhibitory activity of certain flavo- noids on the filamentous fungi F. graminearum and S.

zeicola .

[0006] Besides the search of novel antimycotics, the search for so-called "potentiators", i.e. substances that sig nificantly increase the antifungal effect of antimycot ics, has become increasingly important in recent years. Such substances may be able not only to increase the ef fect of antimycotics on fungal cells but also to broaden the effect of antimycotics on fungal cells whose viabil ity cannot be effectively reduced using such antimycot ics .

[0007] CN 105 669 625 A discloses a synergistic antifugal effect of certain flavonoids and fluconazole on Candida albicans .

[0008] It is an object of the present invention to provide methods, compounds and combination of compounds to effec tively inhibit the growth of fungal cells ex vivo as well as in vivo and to affect their viability. Another object of the present invention is to provide compounds that are able to enhance the efficacy of antimycotics.

SUMMARY OF THE INVENTION

[0009] Thus, the present invention relates to the use of a flavone of formula (I)

for inhibiting or preventing the growth of a non-filamen- tous biofilm forming fungal cell, wherein Ri, R 2 , R 3 , R 4 , R 5 , Re and R 7 are independently from each other H or OH.

[0010] Flavonoids are phytochemicals that are ubiquitous in plants and therefore also present in human food. Fla- vones, a class of flavonoids, have a 2-phenylchromen-4- one (2-phenyl-l-benzopyran-4-one) backbone and are com monly present in the food supply, mainly from spices, and red-purple fruits and vegetables. It turned surprisingly out that flavones having or consisting of formula (I) are able to inhibit the growth of non-filamentous fungal cells so that the viability and the number of fungal cells is significantly decreased. These antimycotic prop erties may not only be used for decreasing the number of viable fungal cells but also to prevent that fungal cells grow or reproduce.

[0011] The flavones of the present invention induce cell

death in fungal cells. This was exemplarily confirmed in the pathogenic yeast Candida albicans and Candida gla- brata. Surprisingly, the flavones of the present inven tion can be used in the treatment and in the control of fungal cells being part of a biofilm. The flavones of the present invention are also suitable for the treatment and control of planktonic fungal cells. Treatment of both planktonic cells and biofilms of C. albicans and/or C. glabrata with the flavones of the present invention re sulted in a decreased proliferation of the pathogens as well as inhibition of biofilm formation. The antifungal potential of the flavones of the present invention could be demonstrated in vivo using a Caenorhabditis elegans infection model. In particular di-substituted flavones, like 3, 6-dihydroxyflavone (DHF) , turned out to show good antimycotic properties.

[0012] Another aspect of the present invention relates to a composition comprising at least one flavone of formula (I), for the use in the treatment of a fungal infection in a human or animal, preferably mammalian, subject, wherein Ri, R 2 , R 3 , R 4 , R5, R 6 and R 7 are independently from each other H or OH, and wherein said fungal infection is caused by a non-filamentous biofilm forming cell.

[0013] Besides the antimycotic properties of the flavones of the present invention it turned surprisingly out that these flavones enhance the antifungal activity of other antimycotics . [0014] A further aspect of the present invention relates to a method for inhibiting or preventing the growth of non- filamentous biofilm forming fungal cells comprising the step of contacting fungal cells with at least one flavone and/or a composition of the present invention, wherein this method is preferably an in vitro method.

DESCRIPTION OF THE FIGURES

[0015] Fig. 1 shows that DHF interferes with planktonic and biofilm cells of Candida spp . in vitro. (A) 3, 6-DHF inhibits growth of planktonic C. albicans (MIC50 = 9.5 ± 0.1 mM) and (B) C. albicans biofilm formation (BIC50 =

55.2 ± 8.1 mM) . (C) DHF is active against planktonic C. glabrata (MIC50 = 7.9 ± 1.0 mM) and (D) C. glabrata biofilm cells (BIC50 = 23.4 ± 9.7 mM) . Untreated controls were set to 100%. For dose-response data, sigmoidal curves were generated using non-linear regression and the IC50 values were derived from the whole dose-response curves. Data represent means ± s.e.m of at least 3 independent experiments. Data analyzed compared to the untreated control using one-way ANOVA and corrected for multiple comparison using a Bonferroni post-hoc test.

[0016] Fig. 2 shows that DHF confers prolonged protection against C. albicans infection in vivo. Survival curves of non-infected (ui Ctrl) and infected (i Ctrl) nematodes treated or not with DHF are shown in (A) , and the

survival of living nematodes on day 5 is shown in (B) . Synchronized C. elegans Aglp-4 lsek-1 larvae were

infected and treated as described in Breger J et al . ) . At least 3 wells for each condition were prepared. Worms were treated with 50 mM DHF or the corresponding volume of solvent (i Ctrl) immediately after infection (single application) . Worm survival was monitored daily over the period of 5 days. Additionally, the survival of non- infected worms (ui Ctrl) was monitored as a control. Data show means ± SEM of 4 independent experiments. In (B) , worm survival is expressed as the percentage of viability at day 5 compared to day 0, with data analyzed compared to i Ctrl using one-way ANOVA and corrected for multiple comparison using a Bonferroni post-hoc test. * p<0.05,

**** p<0.0001.

[0017] Fig. 3 shows that 3, 6-DHF enhances the activity of azoles against C. albicans in vitro and in vivo. (A, D) Checkerboard assays were performed and a representative combination (0.4 mM miconazole, 3.1 mM 3, 6-DHF in (A);

0.5 mM fluconazole, 3.1 mM 3, 6-DH F in (D) ) resulting in growth inhibition is shown. (B, C, E, F) C. elegans infection experiments were performed as described

previously. Worms were treated with either 25 mM 3, 6-DHF or 0.125 mM miconazole (B, C) / 0.5 mM fluconazole (E, F) alone or in combination or with the corresponding volume of solvent (i Ctrl) immediately after infection (single application) . Worm survival was monitored daily over the period of five days. The survival of non-infected worms (ui Ctrl) was monitored as a control. Survival curves for the combination of miconazole with 3, 6-DHF are shown in (B) , and the survival of living nematodes on day 5 is shown in (C) . Survival curves for the combination of fluconazole with 3, 6-DHF are shown in (E) , and the survival of living nematodes on day 5 is shown in (F) . In (C, F) , worm survival is expressed as the percentage of viability at day 5 compared to day 0 and asterisk above each bar indicate the significance level compared to i Ctrl. Data show means ± s.e.m. of at least 3 independent experiments. Data were analyzed using one-way ANOVA and corrected for multiple comparison using a Bonferroni post-hoc test, ns: not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Micon, miconazole; Flu, fluconazole .

[0018] Fig. 4 shows that 3, 6-DHF potentiates the antifungal activity of AMB against C. albicans in vitro and in vivo. (A) 3, 6-DHF acts synergistically with AMB on C. albicans biofilms in vitro. Checkerboard assays were performed and biofilm inhibition of substances alone and combinations were measured with CTB staining. A representative

combination (0.16 mM AMB, 25mM 3, 6-DHF) resulting in synergism is shown in (A) . (B, C) 3, 6-DHF enhances the antifungal activity of AMB against C. albicans in vivo. Survival curves of non-infected (ui Ctrl) and infected (i Ctrl) nematodes treated or not with 3, 6-DHF or AMB alone or in combination are shown in (B) , and the survival of living nematodes on day 5 is shown in (D) . Synchronized C. elegans Aglp-4 Asek-1 larvae were infected as

described in Delattin N, et al . (Antimicrob Chemother. 69(2014) : 1035-1044) . Worms were treated with either 25 mM 3, 6-DHF or 0.5 mM AMB alone or in combination, or with the corresponding volume of solvent (i Ctrl) immediately after infection (single application) . Worm survival was monitored daily over the period of five days.

Additionally, the survival of non-infected worms (ui Ctrl) was monitored as a control. In (C) , worm survival is expressed as the percentage of viability at day 5 compared to day 0 and asterisk above each bar indicates the significance level compared to i Ctrl. Data show means ± s.e.m. of at least 3 independent experiments.

Data were analyzed using one-way ANOVA and corrected for multiple comparison using a Bonferroni post-hoc test, ns: not significant, * p<0.05, *** p<0.001, **** p<0.0001.

[0019] Fig. 5 shows that 3, 6-DHF enhances the activity of Caspofungin against C. albicans . (A) A representative combination (4.9 nM Caspo, 5 mM 3, 6-DHF resulting in growth inhibition of planktonic cells is shown.

Preliminary data. (B) Checkerboard assays were performed and biofilm inhibition of substances alone and

combinations were measured with CTB staining. A

representative combination (39 nM Caspo, 3. ImM 3, 6-DHF) resulting in reduced biofilm formation is shown. Data show means ± s.e.m. of at least 3 independent

experiments. Data were analyzed using one-way ANOVA and corrected for multiple comparison using a Bonferroni post-hoc test. ** p<0.01, *** p<0.001.

[0020] Fig. 6 shows that polyhydroxylated flavones enhance the antifungal activity of azole antifungals. In vitro antifungal acitivity of luteolin (Lut; 3' , 4' , 5, 7-tetrahy- droxyflavone ) or 3 ' , 4 ' , 7-trihydroxyflavone ( 3 ' , 4 ' , 7-THF) in combination with miconazole (M) or fluconazole (F) was analyzed by monitoring growth of yeast cells via OD490 measurement, and the untreated control was set to 100%.

(A, C) In vitro antifungal activity of Lut or 3', 4', 7-THF in combination with M. C. albicans cells were either treated with 0.1 mM M or 12.5 mM (A)/ 6.25 mM (B) flavone alone or in combination. (B, D) In vitro antifungal ac tivity of Lut or 3', 4', 7-THF in combination with F. C. albicans cells were either treated with 1 mM F or 12.5 mM (A)/25 mM (B) flavone alone or in combination. Data rep resent means ± SEM of at least 3 independent experiments. (A) shows preliminary data of two independent experi ments. ns: not significant; * p<0.05; ** p<0.01; *** p<0.001.

[0021] Fig. 7 shows that 3 ' , 4 ' -dihydroxyflavone enhances the antifungal activity of antimycotics . In vitro antifungal acitivity of 3 ' , 4 ' -dihydroxyflavone (3',4'-DHF) in combi nation with miconazole (M) , fluconazole (F), ketoconazole (K) and clotrimazole (Clot) against C. albicans was ana lyzed by monitoring growth of yeast cells via OD490 meas urement, and the untreated control was set to 100%. (A)

Cells were either treated with 0.4 mM M or 25 mM 3', 4'- DHF alone or in combination. (B) Cells were either treated with 0.5 mM F or 25 mM 3',4'-DHF alone or in com bination. (C) Cells were either treated with 1 mM K or 12.5 mM 3',4'-DHF alone or in combination. (D) Cells were either treated with 0.2 mM Clot or 25 mM 3',4'-DHF alone or in combination. (A-D) Data represent means ± SEM of at least 3 independent experiments, ns: not significant, * p < 0.05, ** p < 0.01, **** p < 0.0001.

DESCRIPTION OF EMBODIMENTS

[0022] According to the present invention substituents Ri,

R2, R3, R4, R5, Re and R7 of the flavones of formula (I) may be independently from each other H or OH. In a par ticular preferred embodiment of the present invention one or more of substituents Ri, R 2 , R 3 , R 4 , R5, R 6 and R 7 may be OH. Hence, it is preferred that at least one, preferably at least two, more preferably at least three, more pref erably at least five, of substituents Ri, R 2 , R 3 , R 4 , R5,

R 6 and R 7 are OH. In a particularly preferred embodiment of the present invention one, two, three, four, five or six substituents are OH. If substituents Ri, R 2 , R 3 , R 4 ,

R 5 , Re and R 7 are not OH the substituents are by default H.

[0023] Methods for producing and/or isolating the flavones disclosed herein with the aforementioned substituents are well known in the art (Wagner and Farkas (1975) Synthesis of Flavonoids. In: Harborne, Mabry, Mabry (eds.) The Fla- vonoids, Springer, Boston (MA, USA); Altemimi et al .

(2017) , Plants 6(4) : 42) .

[0024] The flavones of the present invention influence the physiology and viability of fungal cells, so that these flavones may be used for inhibiting or preventing the growth of fungal cells. The flavones of the present in vention may be used to control the growth of fungal cells wherever the presence of fungal cells is not desired. For instance, the flavones may be used in liquids, in suspen sions, on surfaces or in any other composition. The fla vones can also be used to inhibit the growth of fungal cells in food or feed. Also any kind of surface can be treated with the flavones of the present invention. On the other side the flavones of the present invention can also be used therapeutically in humans and animals, pref erably mammals, in order to influence the viability and growth of fungal cells. Hence, the flavones of the pre sent invention can be used in preventing and treating diseases caused by or associated with fungal cells, pref erably non-filamentous fungal cells, more preferably non- filamentous biofilm-forming fungal cells.

[0025] The present invention relates to the use of a flavone of formula (I) for inhibiting or preventing the growth of a non-filamentous biofilm forming cell. [0026] Also disclosed herein is the use of a flavone of for mula (I) for inhibiting or preventing the growth of a non-filamentous fungal cell.

[0027] According to a preferred embodiment of the present invention Ri and R2; R2 and R5; Ri and R3; R2 and R4; R2 and R3; R5 and Re; Ri; R2; R3 or R5 are OH. Thereby it is pre ferred that the other substituents are H.

[0028] According to a further preferred embodiment of the present invention the flavone is selected from the group consisting of 3, 6-dihydroxyflavone, 3 , 3 ' -dihydroxyfla- vone, 6, 7-dihydroxyflavone, 2 ' , 3-dihydroxyflavone, 3,7- dihydroxyflavone, 3' , 4' -dihydroxyflavone, 3-hydroxyfla vone, 6-hydroxyflavone, 7-hydroxyflavone, 3 ' -hydroxyfla vone, 3' , 4' , 5, 7-tetrahydroxyflavone, 3 ' , 4 ' , 7-trihy- droxyflavone and 3' , 4' -dihydroxyflavone, whereby 3, 6-di hydroxyflavone is particularly preferred.

[0029] According to a preferred embodiment of the present invention the non-filamentous biofilm forming fungal cell is of the class Saccharomycetes , preferably of the family Saccharomycetaceae, more preferably of the genus Candida.

[0030] The flavones of the present invention can modulate the growth of non-pathogenic as well as of pathogenic fungal cells. Pathogenic fungal cells are of major im portance so that it is particularly preferred that the fungal cell is a pathogenic fungal cell.

[0031] The fungal cells to be contacted with the flavones of the present invention are non-filamentous biofilm forming fungal cells. In other aspects disclosed herein, the fun gal cells may be single cells, cell colonies or plank tonic cells.

[0032] According to a preferred embodiment of the present invention the non-filamentous biofilm forming fungal cell is selected from the group consisting of Candida albi mata, Candida glabrata, Candida krusei r Candida lusita- niae, Candida auris, Cryptococcus neoformans, and Crypto coccus gattii.

[0033] It turned surprisingly out that the flavones of the present invention are not only able to influence or pre vent the growth of non-filamentous biofilm forming fungal cells but show significant synergistic effects on fungal cells, filamentous as well as non-filamentous fungal cells, when combined with other antimycotic compounds. Hence, it is particularly preferred to use the flavone having formula (I) in combination with at least one fur ther antimycotic compound.

[0034] According to a preferred embodiment disclosed herein the filamentous fungal cell is selected from the group consisting of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus terreus .

[0035] According to a preferred embodiment of the present invention the at least one further antimycotic compound is selected from the group of azoles, echinocandins and polyenes .

[0036] It turned out that the flavones of the present inven tion show particular synergistic effects in regard to the growth and viability of fungal cells when used in combi nation with azoles, echinocandins and polyenes.

[0037] According to a preferred embodiment of the present invention the azole is an imidazole, a triazole or a thi- azole .

[0038] According to a further preferred embodiment of the present invention the imidazole is selected from the group consisting of bifonazole, butoconazole, clotrima zole, econazole, fenticonazole, isoconazole, ketocona- zole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole and tioconazole.

[0039] According to another preferred embodiment of the pre sent invention the triazole is selected from the group consisting of albaconazole, efinaconazole, epoxiconazole, fluconazole, isavuconazole, itraconazole, posaconazole, propiconazole, ravuconazole, terconazole and voricona zole.

[0040] According to a preferred embodiment of the present invention the thiazole is abafungin.

[0041] According to another preferred embodiment of the pre sent invention the echinocandin is selected from the group consisting of anidulafungin, caspofungin and mica- fungin .

[0042] According to a further preferred embodiment of the present invention the polyene is selected from the group consisting of amphotericin B, candicidin, filipin, hamy- cin, natamycin, nystatin and rimocidin.

[0043] According to a particularly preferred embodiment of the present invention the antimycotic compound is se lected from the group consisting of amphotericin B, miconazole, ketoconazole, fluconazole, clotrimazole and caspofungin .

[0044] The at least one flavone of the present invention and optionally the at least one additional antimycotic com pound can be used to prevent or inhibit the growth of fungal cells, preferably non-filamentous fungal cells, even more preferably non-filamentous biofilm forming fun gal cells, for various purposes. Hence, the aforemen tioned compounds can be applied in various ways depending where and how the growth and viability of fungal cells shall be inhibited or prevented. The at least one flavone may be applied on surfaces, added to suspensions and liq uids or even incorporated into polymers, for instance.

[0045] The effective concentration of the flavones of the present invention is preferably from 1 to 500 mM, more preferably from 2 to 400 mM, more preferably from 5 to 300 mM. In particular, at theses concentrations the fla vones of the present invention show to be effective against fungal cells. Hence, it is particularly preferred to apply or administer the flavones of the present inven tion at these concentrations.

[0046] In combination with an antimycotic compound, in par ticular with one or more azoles, the flavones and the further antimycotic compound (s) are preferably applied or administered at a concentration from 0.1 to 100 mM, pref erably from 0.2 to 80 mM, more preferably from 0.5 to 50mM .

[0047] According to a preferred embodiment of the present invention the flavones of the present invention are com bined with antimycotic compounds, preferably azole, in a molar ratio of 2:1 to 100:1 ( flavone : antimycotic com pound) .

[0048] Plants and parts thereof are often affected by fungal cells. Hence, the flavone of the present invention is preferably applied alone or in combination with a further antimycotic compound as defined herein to a plant or parts thereof, in particular fruits or leaves.

[0049] According to a preferred embodiment of the present invention the plant is selected from the group consisting of wheat, barley, millet, oat, corn and rice.

[0050] Another aspect of the present invention relates to a composition comprising at least one flavone of formula (I) for the use in the treatment of a fungal infection in a human or animal, preferably mammalian, subject, wherein Rl, R2, R3, R4, R5, R6 and R7 are independently from each other H or OH, and wherein said fungal infection is caused by a non-filamentous biofilm forming cell.

[0051] Also disclosed herein is a composition comprising at least one flavone of formula (I) and optionally at least one further antimycotic compound, wherein Ri, R 2 , R 3 , R 4 ,

R 5 , Re and R 7 are independently from each other H or OH.

[0052] According to a preferred embodiment of the present invention at least one, preferably at least two, more preferably one or two, of Ri, R 2 , R 3 , R 4 , R5, R 6 and R 7 are OH.

[0053] In a particularly preferred embodiment of the present invention the substituents Ri and R 2 ; R 2 and R5; Ri and R 3 ; R 2 and R 4 ; R 2 and R 3 ; Rs and R 6 ,- Ri; R 2 ; R 3 or R5 are OH.

[0054] According to a further preferred embodiment of the present invention the flavone is selected from the group consisting of 3, 6-Dihydroxyflavone, 3 , 3 ' -Dihydroxyfla- vone, 6, 7-Dihydroxyflavone, 2 ' , 3-Dihydroxyflavone, 3,7- Dihydroxyflavone, 3' , 4' -Dihydroxyflavone, 3-Hydroxyfla vone, 6-Hhydroxyflavone, 7-Hydroxyflavone, 3 ' -hydroxyfla vone, 3' , 4' , 5, 7-tetrahydroxyflavone, 3 ' , 4 ' , 7-trihy- droxyflavone and 3' , 4' -dihydroxyflavone, whereby 3, 6-Di- hydroxyflavone is particularly preferred.

[0055] It was surprisingly found that the flavones of the present invention show antimycotic effects and exhibit synergistic effects in combination with other antimycotic compounds on fungal cells, preferably on non-filamentous fungal cells, more preferably on non-filamentous biofilm forming fungal cells. Thus, the flavones of the present invention exhibit synergistic effects in combination with other antimycotic compounds on non-filamentous biofilm forming fungal cells. In addition thereto, it was found that antimycotic compounds which do not show an effect against certain fungal cells exhibit an antimycotic ef fect when combined with the flavones of the present in vention .

[0056] A "synergistic effect", as used herein, is defined as the response of two variables which is greater than the sum of both parts alone.

[0057] According to a preferred embodiment, the composition for use according to the present invention comprises at least one further antimycotic compound.

[0058] According to a preferred embodiment of the present invention the at least one further antimycotic compound is selected from the group of azoles, echinocandins and polyenes .

[0059] According to a preferred embodiment of the present invention the azole is an imidazole, a triazole or a thi- azole .

[0060] According to a further preferred embodiment of the present invention the imidazole is selected from the group consisting of bifonazole, butoconazole, clotrima- zole, econazole, fenticonazole, isoconazole, ketocona- zole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole and tioconazole.

[0061] According to another preferred embodiment of the pre sent invention the triazole is selected from the group consisting of albaconazole, efinaconazole, epoxiconazole, fluconazole, isavuconazole, itraconazole, posaconazole, propiconazole, ravuconazole, terconazole and voricona zole.

[0062] According to a preferred embodiment of the present invention the thiazole is abafungin.

[0063] According to a further preferred embodiment of the present invention the echinocandin is selected from the group consisting of anidulafungin, caspofungin and mica- fungin .

[0064] According to another preferred embodiment of the pre sent invention the polyene is selected from the group consisting of amphotericin B, candicidin, filipin, hamy- cin, natamycin, nystatin and rimocidin.

[0065] According to a particularly preferred embodiment of the present invention the antimycotic compound is se lected from the group consisting of amphotericin B, miconazole, ketoconazole, fluconazole, clotrimazole and caspofungin .

[0066] The composition of the present invention may be used for controlling the growth and viability of fungal cells, preferably of non-filamentous fungal cells, even more preferably of non-filamentous biofilm forming fungal cells, in animals, preferably mammals, and humans.

[0067] In a preferred embodiment, the composition for use according to the present invention may comprise at least one pharmaceutically acceptable excipient which is mainly dependent from the route of administration and well known to a person skilled in the art and commonly used for an timycotic compositions.

[0068] It was surprisingly found that the flavone as well as the composition of the present invention can be used as a medicament for treating or preventing fungal infections in humans and animals, preferably mammals.

[0069] The at least one flavone of the present invention is administered preferably to the human or animal, prefera bly mammalian, subject alone or together or subsequently with at least one further antimycotic compound as defined above .

[0070] Thus, according to a preferred embodiment, the compo sition for use according to the present invention is ad ministered to the human or animal, preferably mammalian, subject together or subsequently with at least one fur ther antimycotic compound as defined above.

[0071] According to a particularly preferred embodiment of the present invention the composition or the flavone and/or the at least one further antimycotic compound are administered orally, topically or intravenously.

[0072] In a preferred embodiment, the composition for use according to the present invention is administered orally, topically or intravenously.

[0073] The composition as well as the flavone of the present invention can be formulated for any kind of mode of de livery to the patient including oral, topical, by inhala tion, intravenous or parenteral administration. Thus, de pending upon chosen mode of administration, the composi tion and the flavone of the present invention can be for mulated with common excipients, diluents or carriers, and formed into tablets, capsules, solutions, suspensions, powders, aerosols and the like. Examples of excipients, diluents, and carriers that are suitable for such formu lations include buffers, as well as fillers and extenders such as starch, cellulose, sugars, mannitol and silicic derivatives. Binding agents can also be included such as carboxymethyl cellulose, hydroxymethylcellulose, hydroxy- propyl methylcellulose and other cellulose derivatives, alginates, gelatin, and polyvinyl-pyrrolidone .

[0074] Moisturizing agents can be included such as glycerol, disintegrating agents such as calcium carbonate and so dium bicarbonate. Agents for retarding dissolution can also be included such as paraffin. Resorption accelera tors such as quaternary ammonium compounds can also be included. Surface active agents such as cetyl alcohol and glycerol monostearate can be included. Adsorptive carri ers such as kaolin and bentonite can be added. Lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols can also be included. Preservatives may also be added. The compositions of the invention can also contain thickening agents such as cellulose and/or cellu lose derivatives. They may also contain gums such as xan- than, guar or carbo gum or gum arabic, or alternatively polyethylene glycols, bentones and montmorillonites , and the like.

[0075] For oral administration, the flavone and optionally the further antimycotic compound may be present as a pow der, a granular formulation, a solution, a suspension or an emulsion or may be presented as a bolus, electuary or paste .

[0076] Tablets containing the compounds of the present in vention can include buffering agents such as calcium car bonate, magnesium oxide and magnesium carbonate. Tablets can also include inactive ingredients such as cellulose, pre-gelatinized starch, silicon dioxide, hydroxy propyl methyl cellulose, magnesium stearate, microcrystalline cellulose, starch, talc, titanium dioxide, benzoic acid, citric acid, corn starch, mineral oil, polypropylene gly col, sodium phosphate, zinc stearate, and the like. Hard or soft gelatin capsules containing the compounds of the present invention can contain inactive ingredients such as gelatin, microcrystalline cellulose, sodium lauryl sulfate, starch, talc, and titanium dioxide and the like, as well as liquid vehicles such as polyethylene glycols (PEGs) and vegetable oil. Moreover, enteric-coated tab lets or capsules are also provided and designed to resist disintegration in the stomach and dissolve in the more neutral to alkaline environment of the duodenum. Orally administered therapeutic compounds of the present inven tion can also be formulated for sustained release. [0077] A sustained-release formulation can be designed to release the compounds of the present invention, for exam ple, in a particular part of the intestinal or respira tory tract, possibly over a period of time. Coatings, en velopes, and protective matrices may be made, for exam ple, from polymeric substances, such as polylactide-gly- colates, liposomes, microemulsions, microparticles, nano particles, or waxes.

[0078] The flavones and optionally the at least one further antimycotic compounds of the present invention can also be formulated as elixirs or solutions for convenient oral administration or as solutions appropriate for parenteral administration, for instance by intramuscular, subcutane ous, intraperitoneal or intravenous routes.

[0079] For parenteral administration, e.g. by injection, for example, bolus injection or continuous infusion, the com pounds of the present invention may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion containers or in multi-dose containers. Preserv atives can be added to help maintain the shelve life of the dosage form. The compounds of the present invention and other ingredients may form suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

[0080] It is particularly preferred to administer the fla vones and compositions of the present invention topically or to mucosal surfaces such as the vagina, the rectum, eyes, nose and the mouth. For topical administration, the therapeutic agents may be formulated as is known in the art for direct application to a target area. For topical application creams, milks, gels, dispersion or microemul sions, lotions thickened to a greater or lesser extent, impregnated pads, ointments or sticks, aerosol formula tions (e.g., sprays or foams), soaps, detergents, lotions or cakes of soap are particularly preferred. Other con ventional forms for this purpose include wound dressings, coated bandages or other polymer coverings, ointments, creams, lotions, pastes, jellies, sprays, and aerosols.

[0081] Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suita ble thickening and/or gelling agents. Lotions may be for mulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents. Topical applications may also comprise an adjuvant. Compositions for topical applica tion to skin may include an adjuvant, solvent, or co-sol- vent to assist the flavone and optionally the additional antimycotic compounds with penetrating the outer dermal layers. An exemplary adjuvant, solvent, or co-solvent is dimethyl sulfoxide (DMSO) .

[0082] The pharmaceutical formulations of the present inven tion may include, as optional ingredients, pharmaceuti cally acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are avail able in the art. Examples of such substances include nor mal saline solutions such as physiologically buffered sa line solutions and water. Specific non-limiting examples of the carriers and/or diluents that are useful in the pharmaceutical formulations of the present invention in clude water and physiologically acceptable buffered sa line solutions such as phosphate buffered saline solu tions pH 7.0-8.0.

[0083] Another aspect of the present invention relates to a method for inhibiting or preventing the growth of a non- filamentous biofilm forming fungal cell comprising the step of contacting fungal cells with at least one flavone and/or a composition as defined above.

[0084] Another aspect of the present invention relates to a method for inhibiting or preventing the growth of a fun gal cell comprising the step of contacting fungal cells with at least one flavone and/or a composition as defined above . [0085] According to a preferred embodiment of the present invention the at least one flavone is contacted together or subsequently with at least one further antimycotic compound as defined above.

EXAMPLES

[0086] Material & Methods

[0087] Yeast strains, fungal strains and growth conditions

[0088] Candida strains were maintained in YPD media (10 g/L Yeast extract, 20 g/L Peptone, 40 g/L Pextrose/glucose ) . Experiments were performed in YPD media or in PRMI media (Sigma Aldrich, Germany) buffered with MOPS (TCI, Germany or ABCR, Germany) Agar (20 g/L; BD, Germany) was added to prepare solid media. For long time storage yeast strains in YPD were mixed 1:1 with 50% glycerol as antifreeze and stored at -80°C.

[0089] In vitro antifungal susceptibility testing

[0090] Minimal inhibitory concentration

[0091] The minimal inhibitory concentration (MIC) was deter mined following the standard Clinical and Laboratory Standards Institute (CLSI) protocol M27 (Rex JH, and Clinical and Laboratory Standards Institute (2008) . Ref erence method for broth dilution antifungal susceptibil ity testing of yeasts: approved standard, 3rd ed. Na tional Committee for Clinical Laboratory Standards,

Wayne, PA) for yeast strains or the standard CLSI proto col M38 (Clinical and Laboratory Standards Institute (2008) . Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. Clinical and Laboratory Standards Institute, Wayne, PA) for filamen tous fungi .

[0092] For yeast strains media was inoculated with an over night culture to an Oϋ d oo of 0.001 (l 04 cells/mL) . Serial dilutions of substances were added and cells were incu bated at 37°C without shaking for 48 ± 1 h.

[0093] For filamentous fungi, ½ PDB media was inoculated with respective spores to a concentration of 4xl0 4 spores/mL. Serial dilutions of substances were added and spores were incubated at 22 °C without shaking for three to four days .

[0094] MIC tests were performed in flat-bottom 96 well

plates (Greiner Bio One, Austria) using 100 pL suspension per well sealed with gas-permeable foils. After incuba tion, OD490 values were measured using 96-well plate reader and growth capacity was analyzed by setting the corresponding control to 100%.

[0095] Biofilm inhibition concentration

[0096] The biofilm inhibition concentration (BIC) was deter mined as described in Delattin N et al . (J Antimicrob Chemother 69(2014) : 1035-1044) . In short, RPMI medium was inoculated with an overnight culture to an OD (optical density) of 0.1, transferred to a U-bottom 96-well plate (MLS, Belgium) and treated with serial dilutions of sub stances. After the adherence phase (1 h, 37°C), superna tant was removed, adherent cells were washed with PBS and fresh media (including substances) was added. Biofilms were then allowed to grow for 24 h at 37°C.

[0097] Formed biofilms were then analyzed for their meta bolic activity. Therefore, C. albicans biofilm cells were treated with CTB (cell titer blue®; Promega, Germany) , diluted 1:100 in PBS. After incubation for 1 h at 37°C in the dark fluorescence intensity was measured using a 96- well plate reader (Ex: 535, Em: 590, sens 65) . For C. glabrata, XTT ( 2H-Tetrazolium, 2 , 3-bis ( 2-methoxy-4-nitro- 5-sulfophenyl ) -5- [ (phenylamino ) carbonyl] -hydroxide;

Sigma, Germany or ABCR, Germany) was used instead.

Thereby, biofilm cells were treated with an XTT solution (0.25 mg/mL XTT + 0.1 mM menadione in PBS) at 37°C in the dark and OD490 was measured.

[0098] Additionally, biofilm inhibition was determined by plating experiments. Therefore, grown biofilms were washed, biofilm cells were resuspended in PBS + 1% Triton and serial dilutions were plated on YPD agar plates.

Plates were incubated at 37 °C for one day and CFUs were counted . [0099] As for MIC, biofilm inhibition was analyzed by set ting the corresponding control to 100%.

[0100] Biofilm eradication concentration

[0101] To determine the biofilm eradication concentration (BEC) , biofilms were grown as described above in the ab sence of substances. Pre-grown biofilms (24 h, 37°C) were washed with PBS before adding fresh media and serial di lutions of substances. After incubation for 24 h at 37°C, metabolic activity of remaining biofilm cells was ana lyzed as described above.

[0102] Checkerboard assay (Synergy Testing)

[0103] For synergy testing of different combinations, check erboard assays were performed. Therefore, serial dilu tions of two different compounds were combined for MIC,

BIC or BEC testing as described above. Antifungal activ ity of substances alone or in combination was determined by setting the corresponding control to 100%.

[0104] In vivo antifungal susceptibility testing using Cae- norhabditis elegans

[0105] C. elegans maintenance and long-time storage

[0106] The C. elegans Aglp-4 Asek-l strain (generously pro vided by Valerie Defraine, Centre of Microbial and Plant Genetics, KU Leuven, Belgium) was used for in vivo test ing of antifungal activity. Worms were maintained on NGM plates (nematode growth medium; 2.5 g/L Bacto Peptone, 3 g/L NaCl, 17 g/L agar, supplemented with 5 mg/L choles terol, 1 mM CaCl 2 , 1 mM MgS0 4 and 25 mM KPO4 buffer pH 6.0), seeded with a thin layer of E. coli OP50, at 16°C and worms were chunked onto fresh plates every three to four days .

[0107] For longtime storage, starved worms were harvested from plates, diluted 1:1 with 50% glycerol as an anti freeze and stored at -80°C.

[0108] Egg collecting and synchronization of C. elegans

[0109] Egg collecting was performed by bleaching according to Stiernagle T ("Maintenance of C. elegans". WormBook. doi: 10.1895/wormbook .1.101 (2006)) and Porta-de-la-Riva

M et al . JoVE 64(2012) : e4019-e4019) . In short, adult worms were collected from plates and treated with a bleaching solution (10 mL household bleach + 5 mL 5M NaOH) , thereby worms disintegrate and eggs are released. After vigorous shaking for 5 min, M9 medium (3 g/L KH2PO4,

6 g/L Na2HPC>4, 5 g/L NaCl, supplemented with 1.25 mM

MgS04) was added and worms were immediately put on ice. After three washing steps, residual eggs were incubated in M9 medium over night at 16°C on a tube roller. The next day, eggs were collected, transferred onto fresh NGM/OP50 plates and incubated at 25°C for 3-4 days until nematodes have reached the L3/L4 stage prior to infec tion .

[0110] Infection with C. albicans and efficacy testing

[0111] Synchronized (L3/L4 stage) worms were collected,

washed three times with M9 media and spotted onto pre pared YPD plates with a thin layer of C. albicans SC5314. Nematodes were fed on YPD/C. albicans plates for of 2 h at 25°C. To remove residual yeast on the cuticula of the worms, nematodes were collected and washed several times with MilliQ water using a sterilized membrane (pore size ~ 20 mM) . Collected nematodes were then resuspended in growth medium (M9 medium supplemented with 10 pg/mL cho lesterol, 100 pg/mL kanamycin and 75 pg/mL ampicillin) and diluted to a concentration of ~ 40 worms/750 pL .

[0112] For efficacy testing of compounds, 750 pL of the worm suspension were transferred into each well of a 24-well plate and substances were added at indicated concentra tions (1% DMSO) . Nematodes were immediately counted (tO) and survival was monitored over five days post infection by counting living worms.

[0113] Data analysis

[0114] Presented data represent mean ± s.e.m of at least three independent experiments. For dose-response experi ments, sigmoidal curves were generated using non-linear regression (formula: Y=Bottom + (Top-Bot

tom) /( 1+10 L ( (LogIC50-X) *HillSlope) ) ) and IC50 values were derived from the whole dose-response curves. Data were analyzed by one-way ANOVA and corrected for multiple com parison using a Bonferroni post-hoc test and a confidence level of 0.05. All data were analyzed using GraphPad Prism 6.

[0115] Example 1: Antimycotic effect of flavones

[0116] Different flavones were randomly chosen and tested for their effects on planktonic cells and biofilms of C. albicans and C. glabrata to determine corresponding IC50 values (the concentration that is required to reduce growth (minimal inhibitory concentration = MIC) or bio film formation (biofilm inhibitory concentration = BIC) to 50% compared to the untreated control) .

[0117] The antifungal activities of the tested compounds differed greatly depending on the type and respective po sition of the substitution ( s ) (see Table 1) .

[0118] Table 1. Summary of antifungal susceptibility testing of selected compounds in C. albicans and C. glabrata.

n.d. - not determined

Activities of flavones on planktonic and biofilm cells of C.

albicans and C. glabrata were determined as described in the CLSI protocol M27-A3 (M27-A3; ISBN 1-56238-666-2; 2008; Vol . 28 No. 14) and in Delattin N, et al . (J Antimicrob Chemother. 69(2014) : 1035-1044) .

[0119] These results show that not all flavones exhibit an timycotic properties since some substances were not able to reduce growth or biofilm formation of either of both Candida spp . beneath 50% (compared to the untreated con trol) in the used setup (see e.g. 4 ' , 5-dihydroxyflavone and 5, 7-dihydroxyflavone) . The flavones of the present invention showed antimycotic effects in specific set tings, e.g. against biofilms and fungal cells in suspen sion. However, some flavones seemed to show slightly bet ter effects on fungal cells within biofilms compared to fungal cells in suspension (planktonic cells) . In partic ular 3, 6-dihydroxyflavone (DHF) turned out to exhibit outstanding antimycotic effects on fungal cells within a biofilm as well as in suspension (see Table 1 and Fig.

1) .

[0120] To verify the antifungal potential of DHF in vivo, the well-established C. elegans infection model (Breger J, et al . (2007) PLOS Pathog. 3(2) : el8.) was used.

Therefore, synchronized L3/L4 larvae were infected by feeding with C. albicans SC5314 and treatment was started right after infection. Survival of infected as well as non-infected nematodes was monitored daily over the time period of 5 days (Fig. 2A) . DHF was able to counteract the infection of C. albicans in vivo, reflected in the improved survival of treated worms compared to the untreated infection control. For instance, at day 5 post infection, 45.7% of the worms treated with 50 mM DHF were still alive compared to only 20.5% in the infected control (Fig. 2B) .

[0121] Example 2: Flavones potentiate the antimycotic effect of anitmycotics

[0122] In example 1 the antimycotic effect of flavones ac cording to the present invention, like 3, 6-DHF, is de scribed. In this example it was examined whether the fla vones of the present invention exhibit a potentiating ef fect in combination with antimycotics of other substance classes like azoles in vitro and in vivo (see Fig. 3) .

[0123] Furthermore, the results show that 3, 6-DHF exhibits a potentiating effect also in combination with other com mercially available antimycotics, for example Amphoteri cin B (AMB) , as a representative of the polyene class, or caspofungin (Caspo) , as a representative of the echi- nocandin class (Figs. 4 and 5) .

[0124] As shown in Figs. 6 and 7 also the flavones

3' , 4' , 5, 7-tetrahydroxyflavone, 3' , 4' , 7-trihydroxyflavone and 3 ' , 4 ' -dihydroxyflavone show a synergistic antimycotic effect in combination with other known antimycotics.




 
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