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This item is the archived peer-reviewed author-version of: Combination of miconazole and domiphen bromide is fungicidal against biofilms of resistant Candida spp. Reference: Tits Jana, Cools Freya, De Cremer Kaat, De Brucker Katrijn, Berman Judith, Verbruggen Kristof, Gevaert Bert, Cos Paul, Cammue Bruno P.A., Thevissen Karin.- Combination of miconazole and domiphen bromide is fungicidal against biofilms of resistant Candida spp. Antimicrobial agents and chemotherapy - ISSN 0066-4804 - 64:10(2020), e01296-20 Full text (Publisher's DOI): https://doi.org/10.1128/AAC.01296-20 To cite this reference: https://hdl.handle.net/10067/1705020151162165141 Institutional repository IRUA 1 Combination of miconazole and domiphen bromide is fungicidal against biofilms of resistant 2 Candida spp. 3 Jana Titsa, Freya Coolsb, Kaat De Cremera, Katrijn De Bruckera, Judith Bermanc, Kristof 4 Verbruggend, Bert Gevaertd, Paul Cosb, Bruno P.A. Cammuea, Karin Thevissena# 5 6 aCentre of Microbial and Plant Genetics, KU Leuven, Leuven, Belgium 7 bLaboratory for Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Antwerp, 8 Belgium 9 cDepartment of Molecular Microbiology & Biotechnology, School of Molecular Cell Biology and 10 Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel. 11 dPurna pharmaceuticals, Puurs, Belgium 12 13 Running Head: Fungicidal combination against resistant Candida spp. 14 15 #Address correspondence to Karin Thevissen, [email protected] 1 16 Abstract 17 The occurrence and recurrence of mucosal biofilm-related Candida infections, such as oral and 18 vulvovaginal candidiasis, is a serious clinical issue. Vaginal infections caused by Candida spp., for 19 example, affect 70‐75% of women at least once during their lives. Miconazole (MCZ) is the 20 preferred topical treatment against these fungal infections, yet it has only moderate antibiofilm 21 activity. Through screening of a drug repurposing library we identified the quaternary ammonium 22 compound domiphen bromide (DB) as a MCZ potentiator against Candida biofilms. DB displayed 23 synergistic C. albicans antibiofilm activity with MCZ, reducing viable biofilm cells 1000-fold. In 24 addition, the MCZ-DB combination also resulted in significant killing of biofilm cells of azole- 25 resistant C. albicans, C. glabrata, and C. auris isolates. In vivo, the MCZ-DB combination had 26 significantly improved activity in a vulvovaginal candidiasis rat model as compared to single 27 compound treatments. Data from an artificial evolution experiment indicated that resistance 28 development against the combination was not occurring, highlighting the potential of MCZ-DB 29 combination therapy to treat Candida biofilm-related infections. 30 31 Introduction 32 Various fungal species have the capacity to form biofilms, which are structured microbial 33 communities, surrounded by a self-produced extracellular polymer matrix and attached to biotic 34 or abiotic surfaces (1–3). Fungal biofilms are characterized by an increased tolerance to 35 commonly used antimycotics and are therefore difficult to eradicate (1, 3–6). The number of 36 people suffering from biofilm-related fungal infections is increasing, mainly due to rising numbers 2 37 of immunocompromised individuals and increased use of indwelling medical devices, like 38 implants and catheters, on which biofilms can form (7–11). 39 Biofilm-related fungal infections can occur in the oral cavity, the respiratory -and urinary tract, on 40 reproductive organs, in wounds or on medical devices, and are dominated by the genus Candida, 41 of which Candida albicans is the most prevalent (12–17). However, the number of fungal 42 infections caused by non-albicans Candida species is rising with Candida glabrata infections being 43 first in frequency (18–24). C. glabrata is characterized by an innate resistance to azoles, which is 44 the preferred topical treatment against mucosal biofilm-related fungal infections. Hence, C. 45 glabrata mucosal biofilm-related infections are difficult to treat (24–26). Moreover, other 46 emerging Candida spp. present new challenges for antifungal drug development. Candida auris, 47 for instance, is known for its high levels of resistance to multiple antifungal drug classes and its 48 very efficient human-to-human transmissibility (27–31) . 49 Despite the increasing occurrence of fungal biofilm-related infections and their huge impact on 50 the healthcare system, only few novel antifungals with potent antibiofilm activity have been 51 developed during the last decades (12, 32–38). In addition to searches for novel types of 52 antimycotics a promising strategy to develop effective antibiofilm compounds is to increase the 53 activity of conventional antimycotics against fungal biofilms by combining them with a so-called 54 potentiator. This strategy may result in a fungicidal action of the combination against biofilms 55 (39–41). Potentiators can for example induce increased uptake of the antimycotic in the biofilm 56 cells or inhibit biofilm-specific tolerance pathways (42–47). 3 57 The preferred antimycotics for topical treatment against mucosal biofilm-related fungal 58 infections are azoles. They include imidazoles (e.g. miconazole, MCZ) and triazoles (e.g. 59 fluconazole), and interfere with the biosynthesis of ergosterol by inhibiting the enzyme lanosterol 60 14‐alpha‐demethylase. Since ergosterol is a major constituent of the fungal membrane, its 61 depletion results in growth inhibition (48, 49). Additionally, MCZ causes an accumulation of 62 reactive oxygen species in planktonic fungal cultures and distinguishes itself from most other 63 fungistatic azoles by its fungicidal action (50–54). MCZ is a preferred topical treatment of mucosal 64 Candida infections, as intravenously administered MCZ is linked with liver toxicity and phlebitis 65 (55). MCZ at its normal therapeutic level is characterized by fungicidal activity against planktonic 66 Candida cultures, but only at very high doses (5 mM), it is fungicidal against Candida biofilm cells 67 (51, 56). Such high doses can only be achieved in antifungal lock therapy but not therapeutically 68 (57, 58). Hence, increasing miconazole’s fungicidal antibiofilm activity by combining it with a 69 potentiator is highly relevant. This study aimed at identifying compounds that increase the 70 fungicidal activity of MCZ against Candida biofilm cells. 71 We therefore screened 1,311 compounds of a drug repurposing library in combination with a sub- 72 inhibitory concentration of MCZ against mature C. albicans biofilms. The quaternary ammonium 73 compound domiphen bromide (DB) was selected as most promising MCZ potentiator and the 74 MCZ-DB combination was further characterized with regard to in vitro and in vivo activity. 75 Furthermore, as resistance to azoles is occurring (25, 59–62), resistance development against 76 MCZ-DB was studied by experimental evolution of C. glabrata cultures. 77 4 78 Results 79 Screening for MCZ potentiators against C. albicans biofilms. Analogous to our previous study 80 (39), we screened another 1,311 off-patent drugs and bioactive agents (Pharmakon repositioning 81 library) to identify compounds that can enhance the antibiofilm activity of miconazole (MCZ) 82 against mature C. albicans biofilms, after which we assessed potential fungicidal activity of 83 synergistic antibiofilm MCZ-based combinations. 84 In this way, we identified five compounds that resulted in reduced viability of MCZ-treated C. 85 albicans biofilm cells and had primary applications other than as antimycotics (Table 1). 86 Checkerboard analyses and FICI determination confirmed that, out of these five potential hits, 87 only staurosporine, domiphen bromide (DB) and NSC-317926 act synergistically with MCZ to 88 reduce metabolic activity of C. albicans biofilms. These synergistic combinations were 89 characterized by FICI values of 0.49, 0.42 and 0.16, respectively. To determine whether these 90 synergistic MCZ-based combination treatments are fungicidal against C. albicans biofilm cells, 91 survival of biofilm cells after 24 h of drug exposure was assessed by determining CFUs. Maximal 92 concentrations of MCZ and the potentiators that did not result in significant killing of biofilm cells 93 upon single compound treatment were used in combination. These concentrations were 94 determined based on dose-response curves for fungicidal activity of the single compounds (as 95 assessed by CFU determination) and may vary slightly depending on the experimental setup, the 96 tested compounds and the organism in question. When potentiator concentrations are used that 97 are too low, potentiation will not occur, whereas the use of high, active potentiator 98 concentrations leaves little room for improvement, thereby complicating the analyses. DB was 99 most effective in potentiating MCZ toward fungicidal activity against biofilms, resulting in up to 5 100 1000-fold reduction in viable biofilm cells (Fig. 1 and Fig. S1) relative to MCZ alone, and was 101 selected for further analysis. 102 Combination treatment of imidazoles and DB is fungicidal against C. albicans biofilms. To 103 investigate the compound specificity of this combination, the fungicidal activity of combinations 104 consisting of other azoles or other quaternary ammonium compounds against Candida biofilms 105 was investigated. Maximal concentrations of azoles and quaternary ammonium compounds that 106 did not result in significant killing of biofilm cells upon

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