Study of the antifungal activity of ibuprofen and its association with amphotericin B or ketoconazole against Candida spp. Short title: Antifungal activity of ibuprofen and its association with other drugs against Candida spp

1 Academic Unit of Health, Education and Health Center, Federal University of Campina Grande, 58175-000, Cuité, Paraíba, Brazil. E-mail: paullamaryanne@hotmail.com 2 Academic Unit of Health, Education and Health Center, Federal University of Campina Grande, 58175-000, Cuité, Paraíba, Brazil. E-mail: jeffersonrodriguesn@hotmail.com 3 PhD student of Programa de Pós-Graduação em Produtos Naturais e Sintéticos Bioativos at Universidade Federal da Paraíba, 58033-455, João Pessoa, Paraíba, Brazil. E-mail: laisavilar@gmail.com 4 Master in natural and synthetic bioactive products at Universidade Federal da Paraíba, 58033-455, João Pessoa, Paraíba, Brazil. E-mail: juniorfarmacia.ufcg@outlook.com 5 Professor PhD at Universidade Federal de Campina Grande, 58429-900, Cuité, Paraíba, Brazil. Email: wylly@ufcg.edu.br

fungistatic or fungicidal action [5]. However, the low number of antifungal drugs available, the high rates of resistant microorganisms, as well as the inherent toxicity of these drugs have underlined the importance for researching new strategies that lead to effective treatments for the control of fungal infections [6,7]. With this propose, recent studies have focused on the association between conventional non-antifungal pharmacological agents and conventional antifungal agents [8][9][10][11][12].
Ibuprofen is a non-steroidal anti-inflammatory inhibitor of cyclooxygenase (COX-1 and COX-2) isoenzymes, which specifically blocks mammalian prostaglandin biosynthesis [13]. This antiinflammatory is classically used due to its antipyretic, analgesic, and anti-inflammatory effects [14]. The antimicrobial potency of ibuprofen has been demonstrated in its ability to reverse resistance related to efflux pump activity in C. albicans [15]. Recently, ibuprofen showed in vitro antifungal activity against Cryptococcus [16]. Based on this, the present study aimed to evaluate the antifungal activity of ibuprofen alone and when associated with amphotericin B or ketoconazole against Candida species.

Strains
For this study, 14 Candida strains were used, including clinical isolates (LM) and standard strains

Substances
The substances to which antifungal activity was performed were ibuprofen, amphotericin B and ketoconazole. In addition, sabouraud dextrose agar (SDA) and RPMI-1640 broth were purchased from Difco laboratories and Inlab, respectively. All substance solutions were prepared only at the time of testing by dissolving them in sterile distilled water with the addition of 50 μL dimethylsulfoxide (DMSO). DMSO controls were tested at the same concentrations.

Inoculum
Suspensions were prepared from fresh Candida fungal cultures, kept in SDA, and incubated at 37 °C for 24-48h. After this period, colonies of these cultures were suspended in 4 mL of sterile saline (0.85%).
Finally, these suspensions were homogenized, and the turbidity was adjusted to 0.5 McFarland scale.

Minimum inhibitory concentration (MIC)
technique. Initially, 100 μL of double concentrated RPMI-1640 was added to the wells of the plate. Then 100 μL of the substance was distributed in the first-row wells of the plate. Through a serial dilution in the ratio of 2, concentrations ranged from 2048 to 8 μg/mL for ibuprofen and from 512 to 0.0625 μg/mL for antifungals. Then 10 μL of the inoculum was added to each well. Finally, the plates were incubated at 37 °C and read after 24-48 h, observing the presence or absence of visible fungal growth [17][18][19][20].
Then 20 μL of 1% 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma-Aldrich ® ) was added to each well of the plate to prove fungal growth and the plate incubated for a further 12 h [21]. The MIC of the tested drugs was defined as the lowest concentration capable of producing visible inhibition of fungal growth, as indicated by TTC. The following controls were tested: negative controls (RPMI-1640 only) and positive controls (RPMI-1640 and microorganism) to evaluate medium sterility and inoculum viability, respectively. All assays were performed in triplicate [17][18][19][20].

Results and discussion
The MIC values of ibuprofen against Candida species are shown in table 1.     Table 4 shows the effects of the combination of ibuprofen and ketoconazole against Candida spp.  [26], and antifungal activity against Trichosporon asahii (MIC 500 to 2000 µg/mL) [9], besides inhibiting the growth of 10 clinical isolates of Candida, among them C. albicans, C. krusei, C. tropicalis and C. guilliermondii, with MIC value similar to that found in this study [27].
The effect of the association with ibuprofen has been investigated in several studies, including synergism in 43.5% of the combinations of ibuprofen and amphotericin B against Fusarium spp. strains [28], this same association showed indifferent results against Aspergillus spp. [29]. Recently, this association showed synergistic effects in 86.67% and indifferent effects in 13.33% of the associations against clinical isolates of Trichosporon asahii [9]. Amphotericin B leads to rapid death of fungal cells by causing plasma membrane damage when interacting with ergosterol, resulting in pore formation, surface adsorption and ergosterol extraction from the fungal membrane [30]. Ketoconazole interferes with ergosterol synthesis, which prevents the conversion of lanosterol to ergosterol by inhibiting 14α-demethylase enzyme of cytochrome P450 [31]. In addition to a major problem in the eradication of nosocomial infections, resistance to these drugs is multifactorial and causes several complications in therapy [32]. For this reason, it could be useful to increase the effectiveness of these drugs through combinations with non-antifungal medicines.

Conclusions
This study showed that ibuprofen exerted antifungal activity against most Candida species tested, and this information provides more enlightened expectations for future studies that detail the mechanisms of action and resistance involved to ensure its clinical applicability in the treatment of fungal infections caused by Candida spp. The combinations of ibuprofen and antifungals promoted synergistic effects.
However, antagonistic results were evidenced too, which would hinder its clinical applicability in this case. Therefore, studies of this combined activity should be investigated, as the use of these combinations would bring positive points in antifungal therapy.