Synergistic effect of ibuprofen with itraconazole and fluconazole against Cryptococcus neoformans

The present study investigated the association of the non-steroidal anti-inflammatory drug ibuprofen with itraconazole, fluconazole and amphotericin B against Cryptococcus neoformans isolates. The minimal inhibitory concentration (MIC) was found according to M27-A3 protocol and in vitro interactions were evaluated using checkerboard microdilution method. Synergism was demonstrated between azoles and ibuprofen for most isolates. However, no synergistic effects were seen when amphotericin B was combined with ibuprofen. Therefore, our results suggest that ibuprofen presents clinical potential when combined with azole drugs in the treatment of cryptococcosis.


INTRODUCTION
Opportunistic pathogenic fungi such as Candida, Aspergillus and Cryptococcus species are responsible for systemic infections affecting mainly immunodeficient patients such as neonates, transplanted and patients with acquired immunodeficiency syndrome (AIDS) (Grimaldi et al., 2010). Cryptococcosis is an infection mainly caused by encapsulated yeast fungus such as Cryptococcus neoformans. This microorganism is found in bird droppings and contaminated soil with higher prevalence in tropical and subtropical regions (Ramose-Silva et al., 2012). It has the airway as portal of entry causing pulmonary infection and can be disseminated to the brain resulting in severe meningoencephalitis (Prates et al., 2013;Chen et al., 2015). It is estimated that cryptococcal meningitis result in 120.000 to 240.000 deaths per year worldwide (Rajasingham et al., 2017).
The classical treatment of cryptococcosis is based on amphotericin B alone or combined with 5-fluorocytosine or azoles (Reichert-Lima et al. 2016;Rossato et al., 2016). However, amphotericin B formulations have restricted use due to nephrotoxicity problems and must be administered by intravenous infusion (Kagan et al., 2012;Xie et al., 2014;Lai et al., 2016); and 5-flucytosine is expensive and is not present in therapeutic protocols in several countries, making this combination difficult to administer, particularly in resource-poor settings (Smith et al., 2015;Lai et al., 2016). Fluconazole is cheap, safe, and easy to administer and is the drug of choice in maintenance therapy, typically after cerebrospinal fluid cultures are negative (Lai et al., 2016). Nonetheless, fluconazole monotherapy is not recommended because it has shown ineffectiveness due development of resistant strains (Gullo et al., 2013). Ttriazoles voriconazole and posaconazole are highly active in vitro but have unpredictable bioavailability and experience with their use for cryptococcosis is still lacking. Furthermore, echinocandins, has no useful activity against Cryptococcus (Chen et al., 2012). Thus, mortality remains high and there are still considerable rates of permanent neurological sequels including seizures, headache, memory loss, blindness, and personality disorders (Chen et al., 2012;Lai et al., 2016).
Currently few antifungals are commercially available and the development of new drugs does not accompany the high incidence of the development of resistant strains and the market needs (Liu et al., 2014). Combination therapy with two or more antifungals has the potential to reduce antifungal resistance and decrease toxicity of each drug, but its side effects should be evaluated with caution (Hatipoglu, Hatipoglu, 2013). Thus, in vitro association studies with non-antifungal agents and antifungal drugs have been performed and are still required to delineate in vivo assays and consequent clinical trials (Venturini et al., 2011;Hatipoglu, Hatipoglu, 2013). Ibuprofen is a non-steroidal anti-inflammatory drug commonly used for its antipyretic, analgesic, and anti-inflammatory effects (Arai, Sugita, Nishikawa, 2005). Ibuprofen inhibits inflammation by suppressing cyclooxygenase 1 and 2 (COX-1 and COX-2) activity with subsequent inhibition of prostaglandin (PG) synthesis (Matos, Jordan, 2015). Ibuprofen is easily accessible because it is inexpensive and has shown synergistic effect when combined with fluconazole in Candida strains (Hatipoglu, Hatipoglu, 2013;Liu et al., 2014). So, the present study aims to test the association of ibuprofen with itraconazole, fluconazole and amphotericin B against C. neoformans isolates.

Fungal strains
A total of twenty five clinical isolates of C. neoformans isolated from cerebrospinal fluid were included in this study. All isolates were previously confirmed by PCR (Polymerase Chain Reaction) using primers CNa-70S (5'-ATTGCGTCCACCAAGGAGCTC-3') and CNa-70A (5'-ATTGCGTCCATGTTACGTGGC-3'). The isolates were provided by the Clinical Analysis Department of the Federal University of Rio Grande do Sul, Porto Alegre, RS. All isolates were grown on Sabouraud dextrose agar at 35 ° C for 48 h prior to the experiments.

Drugs
The drugs were prepared according to Clinical and Laboratory Standards Institute (CLSI) recommendations. Fluconazole (FLC) stock solution (Metrochem Api Private Limited, India) was prepared in distilled water. Ibuprofen (IBP; Sigma-Aldrich, USA), itraconazole (ITC; MetrochemApi Private Limited), and amphotericin B (AMB; MetrochemApi Private Limited) stock solution were prepared in dimethylsulfoxide (DMSO; Nuclear, Brazil). For the experiments, the compounds were diluted in Roswell Park Memorial Institute 1640 medium (RPMI 1640; Sigma-Aldrich) to obtain a maximum concentration of 2% DMSO.

Antifungal susceptibility testing
Minimum inhibitory concentrations (MICs) of IBP and antifungal agents were determined in duplicate by the broth microdilution method according to M27-A3 protocol (CLSI, 2008). Serial two-fold dilutions were made in RPMI 1640 medium (Sigma-Aldrich) buffered with morpholinepropansulfonic acid (MOPS; Sigma-Aldrich) and concentrations' ranges tested were: 0.0312 -16 µg/mL of ITC, 0.125 -64 µg/mL of FLC, 0.0312 -16 µg/mL of AMB and 1 -512 µg/mL of IBP. The experiments were carried out in duplicate. MICs values were defined as the lowest concentration of compounds at which the microorganisms tested did not show visible growth (AMB) or reduced 50% of visible growth (FLC, IBP and ITC) in 72 h.

Checkerboard assay
The interaction between IBP and each antifungal was evaluated for eight randomly selected C. neoformans isolates using the checkerboard method (Johnson et al., 2004) where MICA and MICB are the MICs of ibuprofen and antifungal agent, respectively (Mukherjee et al., 2005). Synersgim was defined when FICI ≤ 0.5, indifference when 0.5 < FICI ≤ 4 and antagonism when FICI > 4 (Odds, 2003).

RESULTS AND DISCUSSION
MIC values of each antifungal agents against twenty-five C. neoformans isolates were determined. MIC range, Geometric means (GM), MIC50 (MIC value which inhibits 50% of the isolates) and MIC90 (MIC value that inhibits 90% of the isolates) for itraconazole (ITC), fluconazole (FLC), amphotericin B (AMB) and ibuprofen (IBP) are presented in Table  I. Standardizations of susceptibility tests for C. neoformans are less developed than for Candida spp.; so that, there are no breakpoints established. The isolates showed variable susceptibility to antifungal agents and isolates with low sensitivity were found. The isolates showed low MICs range for ITC (0.03125 -1 µg/mL) compared to FLC (0.25 -8 µg/mL) and AMB (0.5 -16 µg/mL). The geometric mean of MIC was also lower for ITC (0.66 µg/mL) than for FLC (1.74 µg/mL) and AMB (6.17 µg/mL). Since IBP is not an antifungal and there is no standardization in relation to the evaluation of its inhibitory effect, we consider as MIC the concentration that reduces 50% fungal growth. Based on the high MICs, the non-antifungal agent showed weak antifungal activity against C. neoformans. Table II presents the effects of antifungal agent combination, which demonstrated synergism or indifference. The combination of azoles (ITC and FLC) with IBP resulted predominantly in synergism, which was detected in 75% of isolates for combination with FLC and in 62% of isolates for combination with ITC. On the other hand, AMB associated with IBP resulted in 100% of indifference against C. neoformans. Antagonism was not detected against both groups. MIC for AMB combined with IBP was chosen when fungal growth was reduced in 100%.
The limited efficacy and the difficulty to introduce new antifungal drugs into the market make the drugs association an important therapeutic strategy to treat potentially life-threatening invasive fungal infections (Fuentefria et al., 2018). Previous studies have detected synergism between IBP and azole against C. albicans (Ricardo et al., 2009;Costa-de-Oliveira et al., 2015;Sharma et al., 2015) and C. neoformans (Ogundeji, Pohl, Sebolai, 2016) increasing the susceptibility of the isolates to these antifungal agents, and corroborating with research. Other non-steroidal anti-inflammatory drugs, such as tenoxicam, diclofenac sodium and sodium salicylate have also shown synergistic effect when combined with azoles (Yücesoy, Oktem, Güllay, 2000). However, studies are commonly performed with Candida species.
Several mechanisms may be involved in the selection of azole resistant strains, such as mutations causing structural changes in enzyme affinity, overproduction of enzymes and overexpression of efflux pumps (Gullo et al., 2013). Efflux pumps are transporter proteins present in the plasma membrane and are involved in the removal of azoles from the cytoplasm. When efflux pumps are overexpressed there is expulsion of the drug out of the cell reducing the drug concentration at the action site. These mechanisms are responsible for Cryptococcus resistance against most azoles (Basso Jr et al., 2015).  Understanding the resistance mechanisms of azoles and the action of IBP helps to explain our findings of in vitro synergy. IBP is an efflux pump blocker and can prevent the output of azole from the fungal cell. Thus, the high susceptibility of cells to IBP + azoles association may be attributed to the increase in intracellular concentration of the antifungal (Pina-Vaz et al., 2005). On the other hand, AMB does not require internalization into fungal cells for exerting their antifungal activity and so they escape from efflux systems (Vandeputte, Ferrari, Coste, 2012). This may justify the indifferent effect of IBP + AMB association found in the present study.
Besides, previous studies showed that IBP causes fungal membrane damage and can be considered, depending on the dose, fungicide or fungistatic (Argenta et al., 2012;Arai, Sugita, Nishikawa, 2005). Our results corroborate these studies, since IBP alone was able to inhibit cell growth of C. neoformans isolates. The antiinflammatory effect of IBP can also be relevant in the treatment of fungal infections, since prostaglandins may be involved in fungal colonization's and its anti-inflammatory mechanism works mostly by inhibiting cyclooxygenase isoenzymes (Rusu et al., 2014).
In addition to the advantageous effects mentioned above, IBP has a good record of efficacy and safety, and thus it is the most commonly used nonsteroidal anti-inflammatory drug. It can be used even for the most vulnerable patient populations. Furthermore, pharmacokinetic studies of ibuprofen showed that it penetrates into the cerebrospinal fluid, which may be advantageous in treatments of cryptococcal meningitis (Bannwarth et al., 1995;Kokki et al., 2007). Thus, IBP may improve the action of azoles and may still present clinical benefits due to anti-inflammatory action and its favorable pharmacokinetics.

CONCLUSION
The results of this present study suggest that the combination of IBP and azole drugs may be suitable for cryptococcosis therapy since synergism was demonstrated. Further in vivo studies in clinical Page 5/6 situations are still required to prove the effects of the combination of ibuprofen and azoles antifungals.