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INTRODUCTION
Candida species are commensal fungi that live on the skin and the oral, vaginal and intestinal mucous membranes of the human body. The genus Candida is related to a wide range of clinical manifestations, mainly when the immune defense mechanisms of the individual are compromised by several risk factors, including the use of corticosteroids, systemic antibiotics, internal medical devices, total parenteral nutrition, surgeries and others [1-3]. Although the most prevalent species of this genus involved in invasive fungal infections is C. albicans, infections caused by non-albicans species have increased significantly, further raising a worrying scenario because such infections are often more severe, rapidly progressive, treatment-refractory and associated the highest mortality and morbidity [1, 4]. Antifungals available to treat infections caused by Candida spp. include topical or systemic drugs, showing 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 [8-12].
Ibuprofen is a non-steroidal anti-inflammatory inhibitor of cyclooxygenase (COX-1 and COX-2) isoenzymes, which specifically blocks mammalian prostaglandin biosynthesis [13]. This anti-inflammatory 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.
MATERIALS AND METHODS
Strains
For this study, 14 Candida strains were used, including clinical isolates (LM) and standard strains (American Type Culture Collection - ATCC). Amongst them, C. albicans (LM-13, LM-410, LM-178, LM-703, ATCC 76485, ATCC 40042); C. tropicalis (LM-10, ATCC 13803); C. guilliermondii (LM-703, LM-103); C. krusei (LM-120, LM-13); C.parapsilosis (ATCC 22019, ATCC 20019). All strains were provided by the Mycology Laboratory of the Federal University of Paraíba, João Pessoa-PB, Brazil.
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. Thus, the final inoculum concentration obtained was 1-5 x 106 CFU/mL [17, 18].
Minimum inhibitory concentration (MIC)
MIC determination of ibuprofen and antifungals was performed by the 96-well plate microdilution 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 [17-20]. 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 [17-20].
Drug association
The association assay between ibuprofen and antifungals was conducted using the checkerboard method [22]. Initially, 100 μL of double concentrated RPMI-1640 was added to the wells of the plate. Then 50 μL of ibuprofen at different concentrations (MICx8, MICx4, MICx2, MIC, MIC/2, MIC/4 and MIC/8) were added horizontally and 50 μL amphotericin B or ketoconazole, also at different concentrations (MICx8, MICx4, MICx2, MIC, MIC/2, MIC/4 and MIC/8) were added vertically to the plate. Thus, different ibuprofen concentrations were tested in the presence of various antifungal concentrations individually. Subsequently, 20 μL of the corresponding inoculum, previously adjusted to 0.5 McFarland scale, were added. The plates were incubated at 37 °C and read after 24-48 h to observe the presence or absence of visible fungal growth [22], as indicated after 12 h of the addition of 20 μL of 1% TTC [21]. All assays were performed in triplicate and the negative (RPMI-1640 only) and positive (RPMI-1640 and microorganism) controls tested.
The effect produced between the combination of anti-inflammatory and amphotericin B or ketoconazole was determined by the fractional inhibitory concentration index (Fid). This index was calculated by the sum of fractional inhibitory concentrations (FIC), where FICA= (MIC of substance A in combination)/(MIC of substance A alone) and FICB= (MIC of substance B in combination)/(MIC of substance B alone), thus FICI= FICA + FICB. The association was defined as synergistic for FICI < 0.5, as additive for 0.5 <FICI<1, as indifferent for 1 ≤ FICI < 4, and as antagonistic for FICI ≥ 4 [23, 24].
RESULTS AND DISCUSSION
The MIC values of ibuprofen against Candida species are shown in table 1.
Table 1 MIC (μg/mL) of ibuprofen against Candida spp.
| Strains | Ibuprofen |
| C. albicans | |
| LM-13 | 32 |
| LM-410 | 128 |
| LM-178 | 128 |
| LM-703 | 256 |
| ATCC 76485 | 32 |
| ATCC 40042 | >2.048 |
| C. tropicalis | |
| LM-10 | 128 |
| ATCC 13803 | >2.048 |
| C. guilliermondii | |
| LM-703 | 512 |
| LM-103 | >2.048 |
| C. krusei | |
| LM-120 | >2.048 |
| LM-13 | >2.048 |
| C. parapsilosis | |
| ATCC 22019 | 128 |
| ATCC 20019 | 512 |
>: MIC higher than the concentrations tested.
The results were quite variable, showing a higher antifungal activity against C. albicans strains, different from that observed for C. krusei where ibuprofen showed lower activity. The concentration of 512 [μg/mL inhibited approximately 65% of the tested strains, whereas against 35% of the strains the MIC values were above 2048 μg/mL.
Table 2 presents the MIC for antifungals against the various strains tested.
Table 2 MIC (μg/mL) antifungals against Candida spp.
| Strains | Amphotericin B | Ketoconazole |
|---|---|---|
| C. albicans | ||
| LM-13 | 0.5 | 64 |
| C. tropicalis | ||
| ATCC 13803 | 2 | 0.5 |
| C. guilliermondii | ||
| LM-703 | 0.5 | 0.125 |
| C. krusei | ||
| LM-120 | 2 | 64 |
| C. parapsilosis | ||
| ATCC 20019 | 2 | 0.5 |
Amphotericin B showed the best activity, where the concentration of 2 μg/mL was able to inhibit 100% of the strains. Ketoconazole presented MIC ranging from 0.125 to 64 μg/mL. From the individual antifungal MIC, it was possible to make the associations with ibuprofen. The results of the combination of ibuprofen and amphotericin B against Candida strains are shown in table 3.
Table 3 Minimum inhibitory concentration (MIC) in the combination; fractional inhibitory concentration (FIC); and fractional inhibitory concentration index (FICI) of the association between ibuprofen and amphotericin B against Candida spp.
| Strains | MIC (μg/mL) in combination | FIC of drugs | FICI | Result | ||
|---|---|---|---|---|---|---|
| Ibuprofen | Amphotericin B | Ibuprofen | Amphotericin B | |||
| C. albicans LM-13 | 256 | 0.25 | 8 | 0.5 | 8.5 | Antagonism |
| C. tropicalis ATCC 13803 | 512 | 0.25 | 0.25 | 0.125 | 0.375 | Synergism |
| C. guilliermondii LM-703 | 4096 | 4 | 8 | 8 | 16 | Antagonism |
| C. krusei LM-120 | 512 | 1 | 0.25 | 0.5 | 0.75 | Additivity |
| C. parapsilosis ATCC 20019 | 256 | 0.25 | 0.5 | 0.125 | 0.625 | Additivity |
The association of ibuprofen with amphotericin B against C. tropicalis ATCC 13803 showed a synergistic effect. Additivity was observed in the combinations against C. krusei LM-20 and C. parapsilosis ATCC 20019. Antagonism was evidenced in 40% of the combinations.
Table 4 shows the effects of the combination of ibuprofen and ketoconazole against Candida spp.
Table 4 Minimum inhibitory concentration (MIC) in the combination; fractional inhibitory concentration (FIC); and fractional inhibitory concentration index (FICI) of the association between ibuprofen and ketoconazole against Candida spp.
| Strains | MIC (μg/mL) in combination | FIC of drugs | FICI | Result | ||
|---|---|---|---|---|---|---|
| Ibuprofen | Amphotericin B | Ibuprofen | Amphotericin B | |||
| C. albicans LM-13 | 128 | 8 | 4 | 0.125 | 4.125 | Antagonism |
| C. tropicalis ATCC 13803 | 256 | 4 | 0.125 | 8 | 8.125 | Antagonism |
| C. guilliermondii LM-703 | 64 | 0.125 | 0.125 | 1 | 1.125 | indifference |
| C. krusei LM-120 | 256 | 8 | 0.125 | 0.125 | 0.25 | Synergism |
| C. parapsilosis ATCC 20019 | 256 | 0.25 | 0.5 | 0.5 | 1 | indifference |
Different forms of interactions between anti-inflammatory and ketoconazole were observed, among them: synergism in 20% of the associations, indifference in 40% and, finally, 40% of the combinations had an antagonistic effect. Non-steroidal anti-inflammatory drugs (ibuprofen, indomethacin, diclofenac sodium and acetylsalicylic acid) are therapeutic options for Candida-related infections by inhibiting COX-1 and/ or COX-2 that are involved in prostaglandin E2 biosynthesis, which is a virulence factor in promoting fungal colonization and chronic infections [25].
Studies conducted to evaluate the antimicrobial activity of ibuprofen have shown antibacterial action against methicillin-resistant Staphylococcus aureus (MIC 2500 μg/mL), Salmonella choleraesuis, Pseudomonas aeroginosa, Klebsiella pneumoniae, Escherichia coli (MIC> 5000 μg/mL) [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 14a-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.
