'a healthsciences. Corn bination antifungal therapy against Candida species: the new frontier - are we there yet? Review

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1 Medical Mycology October 2003, 4 1, ' Taylor&Francis 'a healthsciences Review Corn bination antifungal therapy against Candida species: the new frontier - are we there yet? J, A. VAZQUEZ Division of Infectious Diseases, Wayne State University School of Medicine, Detroit, Michigan, USA In the past decade, we have seen a significant increase in the incidence of invasive fungal infections. In addition, opportunistic fungal infections resistant to antifungal agents have become increasingly common and their frequency will more than likely continue to increase. The antifungal armamentarium for the treatment of serious fungal infections remains limited. A possible approach to overcoming antifungal drug resistance and high mortality rates seen in severe fungal infections is to combine two or three classes of antifungals, especially if the drugs have different mechanisms of action. The unique properties of newer antifungals now provide us with the opportunity to investigate antifungal combinations that may become the standard of care for serious fungal infections. Combinations of new agents along with more traditional antifungals have now been shown to possess some synergistic or at least additive activity against Candida in clinical trials. On the other hand, caution is still needed since other antifungal combinations have demonstrated antagonistic activity in uitro. Well-controlled clinical trials are needed to define the most efficacious antifungal regimen. Furthermore, these trials should also evaluate the side effect potential of combination regimens and the pharmacoeconomic impact these regimens may have. Thus, while much optimism exists for combination therapy, there is much yet to be done. Introduction The increase in fungal infections and the change in fungal epidemiology is due to the extensive use of antifungal agents to treat fungal infections that are being diagnosed in severely immunocompromised host. The leading causes of these invasive fungal infections include Candida species, Aspergillus spp., and Fusariun~ spp. [I]. Candida are the most frequently encountered cause of medically significant fungal infections. Of the Candida species, lion-albicaizs species of Caizdida, especially C. glabrata and C. krusei, have recently increased in isolation frequency [2]. These Caizdida Accepted 21 August 2003 Correspondence: Jose A. Vazquez, M.D., Harper University Hospital, Division of Infectious Diseases, 3990 John R, Suite 4810, Detroit, Michigan , USA. Tel: ; Fax: ; jvazquez@intmed.wayne.edu species are unique because they are either intrinsically resistant to or less susceptible to azole antifungals [2]. In addition, C. lusitaniae is known to possess intrinsic resistance to amphotericin B [3]. Until recently, our antifungal armamentarium for the treatment of serious fungal infections has been limited to amphotericin B, fluconazole, and itraconazole, all of which target the ergosterol in the fungal cell membrane. Recently, new antifungal agents with a broader spectrum of activity have been developed. Some of these newer antifungals have novel mechanisms of action, and are highly active against isolates that are resistant to current polyenes and azoles. The unique properties of these newer antifungals provide us with the opportunity to investigate antifungal combinations that may become the standard of care for serious fungal infections. DOI: 10. lo8oil I6528

2 A possible approach to overcoming antifungal drug resistance is to combine two or three classes of drugs, especially if the drugs have different mechanisms of action. There are several possible reasons for using two or more antifungals simultaneously instead of a single agent. One common reason is to achieve fungicidal activity with two agents that may not be accomplished with one agent. Thus, the fungicidal activity may be more likely to eradicate an infection than either drug used alone. Secondly, simultaneous use of combinations may enable us to diminish drug dosages, and thus diminish side effects and toxicity while maintaining efficacy. Another possible reason may be to delay the emergence of resistant mutants that may become resistant to a single drug. Additionally, it may also be used to provide broad coverage in seriously ill patients suspected of having either mixed infections or resistant infections. In other words, it can provide coverage for the two or three most probable pathogens simultaneously. On the other hand, there are several disadvantages to using antifungals combinations. First of all, the cost of therapy generally increases. Secondly, the more drugs that are administered, the greater the chance of drug reactions and side effects. On occasion, one drug may antagonize the effect of another drug. Additionally, many drug combinations accomplish no more than one effective drug. Finally, the use of drug combinations may set up a sense of false security, since the physician may feel that all possible pathogens are being covered. Current antifungal agents Available antifungal agents include the azoles (fluconazole, itraconazole, voriconazole) and the polyenes (amphotericin B, nystatin) all of which act either by inhibition of lanosterol-ergosterol synthesis by inhibiting 14-a lanosterol demethylase or by blocking ergosterol on the cellular membrane [4]. Flucytosine, on the other hand, acts intracellularly by inhibiting the protein synthesis of the fungal cell. A new addition to the antifungal regimens is caspofungin acetate, which has been FDA approved for the treatment of refractory aspergillosis, invasive candidiasis and candidemia. Echinocandins function by inhibiting 1-3 P glucan synthase in the fungal cell wall [5]. The advent of new antifungals, especially the echinocandin family of compounds, which possess a different mechanism of action, suggests that antifungal combinations may be possible, and may actually have some synergistic 01- additive activity without any antagonistic activity. In addition, there are also several investigational antifungal agents that are currently in phase I1 or 111, which may also be considered when designing antifungal drug combinations. These include the triazoles, posaconazole and ravuconazole, which possess a broad spectrum of activity, specifically against fluconazoleresistant Candida species such as C. krusei and C. glabrata, and some of the emerging moulds such as Fusarium, Alternaria, and Scedosporium [4]. In addition, there are also two echinocandins in phase I11 trials, micafungin and anidulafungin, which possess similar activity to caspofungin. Thus, in the near future there may be up to five new antifungals added to the current antifungal armamentarium [5]. Definitions In vitro susceptibility methods A great deal of effort has gone into developing a standardized, reproducible, and clinically relevant susceptibility testing method for fungi. This has resulted in the development of the NCCLS M27-A2 methodology for determining the susceptibility of yeast [6]. Data driven interpretive breakpoints using this method are available for testing the susceptibility of Carzdida species to fluconazole, itraconazole, and flucytosine. Reliable interpretive breakpoints are not yet available for amphotericin B. The breakpoints place a strong emphasis on interpretation in the context of the delivered dose of the antifungal agent. The category S-DD (susceptible-dose dependent) indicates the maximization of dosage and bioavailability that are critical to successful therapy. Resistance In vitro antifungal resistance is frequently divided into two categories, primary and secondary resistance. Primary or innate resistance identifies organisms that are intrinsically resistant to an antifungal without any exposure to prior antifungals. Intrinsic amphotericin B resistance is seen in some isolates of C. guilliermondii, C. lusitarziae, Pseudallesclzeria boydii and some Fusarium species. Secondary resistance indicates that resistance developed during or after antifungal drug exposure. This form of resistance has been frequently observed in HIV-positive patients with low CD4 cell counts who have suffered recurrent bouts of mucosal candidiasis and have received multiple courses of azole antifungal 2003 ISHAM, Medical Mycology, 4 1,

3 Combination antifungal therapy against Candida species Drug interactions and definitions The definitions of what combinations may be clinically effective and which may be ineffective have certain in vitro definitions that are generally followed. These terms include synergy, additive, antagonism, and indifference [7]. refers to the situation when the combined action of two drugs is significantly greater than the sum of both drugs. A drug combination is additive when the combined action is equivalent to the sum of the actions of each drug when used alone. implies that the combined effect is less than that of the more effective agent when used alone. Indifference signifies that the combined effect is no greater than that of the more effective agent when used alone (Table I). Frequently, the nature of the interaction between the two drugs can be determined quantitatively by using the fractional inhibitory concentration index (FICI) [8]. The formula used is as follows: FICI = [(MIC A in combination)/mic A] + [(MIC B in combination)/mic B]. The FIC Index interpretation defines synergy as an FIC I 0.5; additive as an FICI of ; indifference as an FICI of > 1 but I 2; and antagonism as an FICI of Although this method has been used for more than 20 years to assess in vitro drug interactions, we must remember that there is still great variability in the FIC Index, as there is with standard MICs. For all practical purposes, synergy using this calculation is equal to a reduction of at least two dilution steps in the MIC of each drug when they are combined [8] (Table I). Antifungal combinations In vitro antifungal combination studies Since Candida species are the most common cause of fungal infections, it stands to reason that has been the Candida species most commonly evaluated in the majority of in vitro antifungal combination studies. Some of the non-albicans Candida species, such as C. glabrata, C. parapsilosis, C. tropicalis and C. krusei have also been evaluated. Until recently, the most commonly evaluated combinations were amphotericin B and flucytosine and, on occasion, some of the older azoles such as miconazole, clotrimazole and ketoconazole. There are more than 30 published reports describing various in vitro antifungal combination studies against Candida species to date. Clinical trial data evaluating combination therapy is limited; hence, combination therapy is frequently considered only as salvage therapy. Unfortunately, in vitro combination stddies have provided mixed results (a summary is provided in Table 2). In addition, because of the numerous drug combinations currently available, the most effective strategy has not been elucidated. One of the most common combinations described in the literature is the polyeneazole combination. As with all other combination studies published to date, this combination has also reported a variety of results from synergistic activity to antagonism, indifference and additive effect. One of the earliest in vitro studies was published in 1978 by Cosgrove et al., who evaluated the combination of amphotericin B and either miconazole or clotrimazole against, C. glabrata and C. kefyr [9]. Both combinations demonstrated antagonistic activity against all three Candida species. Similarly, over the next few years, several other investigators also evaluated the polyene-azole combinations, and all have essentially demonstrated the same antagonistic effect. These combinations included amphotericin B with either miconazole or ketoconazole [lo]; amphotericin B with either miconazole, ketoconazole, itraconazole or fluconazole [11], and amphotericin B and fluconazole [12,13]. In addition, Sud and Feingold also showed that C. albicans developed in vitro resistance to amphotericin B after an overnight pre-incubation of Candida strains in ketoconazole [14]. Furthermore, they also showed that the organisms became depleted of ergosterol in parallel with the generation of amphotericin B resistance. In 1995, Scheven et al. also evaluated the effect of azole pre-incubation on the in vitro activity of amphotericin B [15]. They evaluated the effect of pre-incuba- Table 1 Drug interaction definitions Drug interactions Definition uantjtative definition PI C* index The combined action of two drugs is greater than the sum of both effects independently Additive The combined action is equivalent to the sum of the actions of each drug when used alone. > The combined effect is less than that of the more effective agent when used alone. 24 Indifference The combined effect is no greater than that of the more effective agent when used alone. > "Fractional inhibitory 2003 ISHAM, Medical Mycology 4 1,

4 Table 2 In vitro susceptibility assays against various Candida species Organism In vitro assays Reference Drug regimen Drug 1 Drug 2 Result, C. glabrata, C. kefyr, C. tropicalis, C. guilliermondii, C. krusei, C. tropicalis, C. tropicalis, C. krusei, C. tropicalis, C. krusei, C. glabrata, C. tropicalis, C parapsilosis C. albicarzs, C. lusitaniae C. parapsilosis, C. tropicalis C. albicarzs, C. glabrata, C. tropicalis - resistant to fluconazole and voriconazole C. glabrata - biofilm Ketoconazole (pre-incubation) Nikkomycin Z (pre-exposure) Fluvastatin Terbinafine (pre-exposure) Terbinafine Lactoferrin Terbinafine (prexposure - 18 h) (prexposure - 18 h) Terbinafine Caspofungin Caspofungin Amlodipne Miconazole Clotrimazole Rifampin Miconazole Ketoconazole Miconazole Ketoconazole Ketoconazole Tioconazole 5-FC Rifampin Ketoconazole Miconazole (pre-incubation) Ketoconazole Miconazole (co-incubation) Quinolone Quinolone 5-FC Additive - synergism Antagonistic Indifference lantagonism Cyclosporine [381 Voriconazole [ /amphotericin B Indifference Voriconazole Posaconazole - 17% [32] - 21% - 33% - 12% Additive [331 Indifference 2003 ISHAM, Medical Mycology 4 1,

5 Combination antifungal therapy against Candida species Table 2 (Continued) Organism In uitro assays Reference Drug regimen Drug 1 Drug 2 Result Terbinafine, C. tropicalis, C. parapsilosis Milbemycins 1411 C. glabrata, C. krusei, C. lusitaniae, (ABC) efflux inhibitor C. krusei Trichostatin A, itraconazole, 441 miconazole Histone deacetylase Terbinafine - Inhibitors, 5-FC, Indifference caspofungin, C. krusei, C. glabrata FK-506 Terbinafine [391 tion with ketoconazole, fluconazole, itraconazole and miconazole. As expected, they found antagonism when was pre-incubated with either ketoconazole, itraconazole or miconazole. However, they did not detect any antagonism when the strains were pre-incubated with fluconazole. In addition, they also observed antagonism when the other azoles tested were simultaneously co-incubated with amphotericin B. Thus, they concluded that the consecutive or serial exposure of Candida to fluconazole followed by the exposure to amphotericin B does not necessarily result in absolute antagonism or the development of resistance as opposed to other azoles. In a follow-up study, Scheven and Scheven describe additional results supporting their earlier hypothesis. The authors state that because fluconazole is a hydrophilic azole and thus behaves differently than the lipophilic azoles (ketoconazole, clotrimazole and itraconazole), the simultaneous or serial use with amphotericin B does not always result in antagonism. Their results seem to indicate that the cytosolic components of the yeast are able to absorb amounts of lipophilic azoles, but are unable to absorb similar amounts of fluconazole [16]. Contrary to Scheven et al., Vazquez et al. were able to demonstrate that pre-incubation of several strains of C. albicans in fluconazole resulted in some degree of resistance to amphotericin B [15,17]. Furthermore, the same investigators were also able to show that preincubation of either C. lusitaniae, C. parapsilosis or C. tropicalis also generated the same degree of ampliotericin B resistance [18]. In addition, they also showed that itraconazole was more effective than fluconazole in generating cells that were resistant to amphotericin B and that these cells were able to tolerate an otherwise lethal exposure to amphotericin B. Louie et al. published a similar study, demonstrating that fluconazole pre-exposure of Candida cells generated amphotericin B resistance [19]. However, if after the fluconazole pre-incubation, the fluconazole was continued along with the addition of amphotericin B, there was no evidence of antagonism or resistance. Another antifungal combination evaluated is the simultaneous use of amphotericin B and flucytosine. As early as 1974, investigators have reported on the in vitro synergistic activity of both of these agents against a variety of Candida species [20,21]. Scalarone and colleagues demonstrated that besides the synergistic activity of these two antifungal agents in combination, they also demonstrated a significant postantifungal effect lasting up to 14.7 h [22]. Because of synergistic interaction demonstrated in numerous in vitro studies, treatment of experimental candidiasis in various animal models has also evaluated using this antifungal combination. Because of the toxicity and lack of efficacy of monotherapy with either amphotericin B or flucytosine other agents have also been evaluated in uitro. In 1974, Beggs et al. demonstrated in vitro synergistic activity with the simultaneous combination of rifampin and amphotericin B against [23]. Several years later Edwards et al. demonstrated in vitro additive to synergistic activity with the combination of rifampin and amphotericin B in approximately 50% of the Candida isolates evaluated. In this study, they used a modified checkerboard dilution microtitration assay against several species of Candida that included C. albicans, C. parapsilosis, C. tropicalis, C. stellatoidea, C. guilliermondii and C. krusei [24]. The investigators showed an increase in the fungicidal activity of amphotericin B of about 50% when used in combination. Several other investigators have also 2003 ISHAM, Medical Mycology, 4 1,

6 similar degrees of synergistic activity using this same combination [25]. Another antimicrobial combination that has been evaluated in vitro is the use of nikkomycin Z with azole antifungals. Although never FDA approved for use as an antifungal compound, numerous combination studies have been published. Nikkomycin Z, is a peptidyl nucleoside antifungal and a strong inhibitor of chitin synthase, a major component of fungal cell walls that is not present in mammalian cells, and thus makes it attractive for use in humans [26]. Although irz vitro and in vivo studies have demonstrated poor activity against many clinically relevant fungi when used alone [27], when used in combination with compounds that inhibit lanosterol demethylase, such as azoles and polyenes, nikomycin Z has been shown to have potent synergistic activity in vitro. Milewski and co-workers were able to show synergistic activity using nikomycin Z and either fluconazole, ketoconazole, or tioconazole against C. albicans [28]. Terbinafine is an approved oral antifungal used to treat dermatophytes and onychomycosis, and has also been evaluated in combination with other agents. Terbinafine has a broad spectrum with excellent activity against many moulds and yeast, including Carzdida species. Terbinafine acts at the cellular membrane level by inhibiting squalene oxidase, a precursor to lanosterol. Early studies using a checkerboard microdilution method evaluated the combination of terbinafine with either fluconazole, itraconazole or amphotericin B against. Investigators were able to demonstrate excellent synergistic activity in vitro [29,30]. More recently, terbinafine has also been evaluated in combination with the new broad-spectrum triazoles such as voriconazole and posaconazole. In a recent study by Weig et al., the investigators evaluated the activity of terbinafine and voriconazole against 39 clinical isolates recovered from HIV-positive patients that suffered form refractory mucosal candidiasis and demonstrated in vitro resistance to fluconazole and voriconazole [3 11. Synergistic activity was shown in 100% of the isolates tested. Perea and co-workers, evaluated the combination of terbinafine and either fluconazole, itraconazole, voriconazole or posaconazole against C. glabrata isolates [32]. The investigators demonstrated synergistic activity in 12% of the isolates when combined with posaconazole, 17% of the isolates with fluconazole, 21% when combined with itraconazole and 33% of the isolates when combined with voriconazole. Recently, caspofungin acetate (cancidasb) was approved for the treatment of esophageal candidiasis, candidemia and invasive candidiasis. Caspofungin is unique because it offers a completely novel mechanism of action, exerting its effect at the cell-wall level by inhibiting glucan synthesis, required for the formation of the fungal cell wall. A recently presented study evaluated the use of caspofungin and either amphotericin B or fluconazole against in biofilms [33]. In this study, investigators were able to demonstrate an additive effect when caspofungin and amphotericin B were used in combination. However, they found that fluconazole inhibited the effects,f caspofungin activity against C. albicaizs biofilms, an effect that is not generally seen under planktonic conditions. In comparison, the investigators noted indifference when the combination of amphotericin B and fluconazole were studied under similar conditions. In addition to antifungal combinations, several investigators have evaluated other agents in combination with an antifungal with the hope of increasing the potency of monotherapy. Some of these agents include quinolones, cliolesterol lowering agents, lactoferrin, cyclosporine, amlodipine, milbemycins, trichostatin A, histone deacetylase inhibitors and, more recently, calcineurin inhibitors (a summary is provided in Table 3). Chin and co-workers evaluated fluvastatin, a cholesterol lowering agent, in combination with either fluconazole or itraconazole against several strain? of C. albicarzs, C. tropicalis and C. parapsilosis [34]. Fluvastatin per se has poor intrinsic antifungal activity. The combination of fluvastatin with either fluconazole or itraconazole demonstrated excellent synergistic activity against the different species of Candida. An investigational quinolone, DU-6859a, with potent antibacterial properties was also evaluated in combination with fluconazole or amphotericin B against several species of Caizdida including C. albicaizs, C. tropicalis, C. krusei and C. glabrata [35]. The possible rationale is the inhibition of fungal topoisomerase I1 by some of the newer quinolones. In this study, the combination of the quinolone with either fluconazole or amphotericin B demonstrated synergistic activity. Lactoferrin is an iron-binding glycoprotein present in high concentrations in many exocrine fluids including saliva. The protein has a broad spectrum of activity against many bacteria, viruses and yeast [36]. It is considered to be an important host defence mechanism on mucosal surfaces. Kuippers et al. evaluated the in vitro antifungal effects of lactoferrin in combination with either fluconazole, amphotericin B or flucytosine against clinical isolates of, C. glabrata and C. tropicalis [37]. The combination of lactoferrin and fluconazole appeared to have the most 2003 ISHAM, Medical Mycology 4 1,

7 Combination antifungal therapy against Candida species Table 3 Summary of unconventional antifungal drug combinations and their activity against candida species Drug regimen Study type Activity Reference Additive Indifference Quinolones +AmB Quinolones +AmB Quinolones +FLZ Quinolones + FLZ Lactoferrin + FLZ Lactoferrin + 5-FC Lactoferrin + AmB FLZ +cyclosporin FLZ + cyclosporiii Ainlodipine + FLZ Amlodipine + ITZ Amlodipine +Tbf Milbemycins + FLZ Trichostatin + FLZ Trichostatin + ITZ Trichostatin +Tbf Trichostatin + AmB Trichostatin + 5-FC Trichostatin +CSP FK-506 +Tbf Immunocompetent mice 111 vitro Immunocompetent mice IIZ citro In uitro IH citro Endocarditis in rats IIZ citro hz ritro 111 citro In vitro activity, although all three combinations demonstrated some synergy against all Candida species. Non-antimicrobial inhibitors of mammalian multidrug efflux transporters have a wide range of usage in medicine today. Cyclosporine, a potent immunosuppressive agent used to prevent graft versus host reactions in transplant recipients, is a well-known immunosuppressive agent. In addition, it also posses the properties of an efflux pump inhibitor. Marchetti and co-workers evaluated the combination of fluconazole and cyclosporine in vitro against several strains of [38]. Using an agar disk diffusion assay, they were able to demonstrate that cyclosporine in concentrations that are achievable in vitro had significant synergistic activity when given in combination with fluconazole. The hypothesis behind this combination is unclear, but cyclosporine does inhibit cyclophilins and calcineurin, which may affect different steps in fungal cell metabolism. Cyclosporine also has the ability to alter the cellular membrane of fungal cells, and thus its functional properties. In another recently published study, Onyewu and co-workers demonstrated that the calcineurin inhibitor, tacrolimus (FK-506), when combined with ergosterol biosynthesis inhibitors such as terbinafine and fenpropimorph, were able to enhance the activity of the antifungals [39]. In fact, antifungals that are normally fungistatic against Candida species become fungicidal when combined with tacrolimus. The drug combination was also fungicidal against C. glabrata and C. krusei. Amlodipine is a calcium pump inhibitor used for the management of hypertension. Amlodipine has nc measurable antifungal activity. However, with either fluconazole, itraconazole or terbinafine, the combinations demonstrated in vitro synergistic activity against many different strains of. Specifically, in vitro experiments detailed by Vitale and co-workers studied the combination of fluconazole and amlodipine, which appears to have excellent synergistic activity against strains of Candida that were either resistant to or dose-dependent susceptible to either itraconazole or fluconazole [40]. Interestingly enough, in the Candida strains that were susceptible to both drugs, the combinations were indifferent. As with the other agents that inhibit various pumps, the mechanism of action is still unknown. Recent research in the area of antifungal resistance mechanisms has led to the development of several new avenues which may be useful in either overcoming resistance in fungi or augmenting the effect of commonly used antifungals. Milbemycins are newly described CDR1/2 efflux pump inhibitors. Andrade and co-workers demonstrated irz vitro synergistic activity using these newly described compounds when used in combination with fluconazole [41] against several different strains of Candida including, C. tropicalis, C. parapsilosis, C. dublirziensis, C. glabrata, C. krusei and C. lusitaniae. Recently, liistone acetylation and deacetylation have been described to play a significant role in the 2003 ISHAM, Medical Mycology 4 1,

8 tion of eukaryotic genes [42,43]. Histone deacetylase inhibitors have been used in molecular biology to study histone deacetylases. These compounds include trichostatin A, apicidin and trapoxin, and have been studied to evaluate their effects on mammalian cells. Specifically, their ability to reverse phenotypes has been studied extensively in human tumour cells. Recently, Smith and Edlind evaluated the activity of several histone deacetylase inhibitors and their effects on fungal cells [44]. Their initial studies evaluated the effects of these compounds on the in vitro growth of C. albicalzs. Although the compounds alone had no effect on growth, when trichostatin A was used simultaneously in combination with either fluconazole, itraconazole, miconazole or terbinafine, the investigators noted that there was increased antifungal activity of all of the antifungals. There was no effect seen when trichostatin A was used with either caspofungin, amphotericin B or flucytosine, as was seen with the other antifungals. Theoretically, this enhanced antifungal effect may be due to the fact that these four antifungals target the ergosterol biosynthetic pathway, while amphotericin B, caspofungin and flucytosine have different mechanisms of action. To further evaluate the effect of the histone deacetylase inhibitors, the researchers evaluated the expression of ERG genes that encode several enzymes along the sterol pathway (ERG1 and ERGll) and CDRIMDRI genes, which encode multi-drug transporters. cells exposed to either fluconazole or terbinafine with or without trichostatin A were evaluated for gene expression. As expected, both antifungals induced the expression of ERGl, ERGl 1, CDRl and CDR2. The addition of trichostatin A to either fluconazole or terbinafine decreased the upregulation by %. Thus, the authors hypothesize that the upregulation of these genes is a plausible explanation of the trailing phenomenon seen with azoles. This appears to be due to the upregulation of these target genes and the multi-drug transporters. Experimental animal studies After demonstrating in vitro efficacy of an antifungal combination, the next logical step is to attempt to validate the same results in an animal model to confirm the efficacy or usefulness of the various combination regimens. There have been numerous animal models used to establish the efficacy of antifungals against various forms of candidiasis (Table 4). Some models establish localized mucosal infections, while others generate immunosuppression followed by the induction of invasive candidiasis. It is not uncommon to demonstrate in vitro activity and be unable to demonstrate any form of improved activity in an animal model of experimental candidiasis. Early animal trials used non-immunocompromised mice in which invasive candidiasis was generated by injecting Candida microorganisms into the animals tail vein, followed by subsequent parenteral administration of the various antifungals. In 1982, Polak et al. published one of the first in vivo antifungal combination trials in mice [45]. In their study, the investigators were able to demonstrate some synergistic activity against, using various concentrations of amphotericin B and flucytosine. This was followed by a similar study by Polak et al. of invasive candidiasis in mice, in which the investigators used several different antifungal combinations [46]. The combinations studied included flucytosine and itraconazole, flucytosine and fluconazole, and amphotericin B and itraconazole. All of the combinations were compared to the traditional flucytosine and amphotericin B combination that had been previously evaluated. In their study, the combination of flucytosine and itraconazole was found to be synergistic to additive, with the most enhanced synergistic activity detected in isolates that were resistant to flucytosine. This degree of synergy was similar to that seen with the amphotericin B1 flucytosine combination. In addition, the combination of fluconazolelflucytosine was also synergistic, while the combination of amphotericin Blitraconazole was found to be indifferent to antagonistic. Although the degree of antagonism was not as profound as with the amphotericin Blketoconazole combination that had been previously described. In addition, Polak also investigated localized candidiasis infections in a mouse model immunosuppressed by cyclophosphamide [47]. The authors have attempted to create an animal model mimicking oral candidiasis infections in HIV-positive patients. In this model, which had previously demonstrated the efficacy of various antifungals, the investigator used the combinations of amphotericin Blflucytosine, as well as flucytosine with either ketoconazole, fluconazole or itraconazole. After treatment, the Candida CFUs of infected cysts were counted and compared with those of untreated animals. The amphotericin Blflucytosine combination was shown to be highly effective and demonstrated synergistic activity. In contrast, the azolel flucytosine was not as effective and the additive or synergistic effect was not detected. In 1991, Sugar published the results of a new combination that included amphotericin B and SCH 39304, a new investigational triazole [48]. In this study a strain of was injected into mice via the lateral tail vein without any form of 2003 ISHAM, Medical Mycology; 4 1,

9 Combination antifungal therapy against Candida species Table 4 Animal trials evaluating combination antifungal therapy against Cundida species Animal model Organism Drug regimen Outcome Reference Immunosuppressed mice Localized candidiasis mouse Immunosuppressed mice with disseminated candidiasis Immunocompetent mice Neutropenic mice with disseminated candidiasis Neutropenic mice with Caizdidu endocarditis Immunosuppressed mice Aortic valve endocarditis in rats Immunocompetent mice with systemic candidiasis Immunocompetent mice with systemic candidiasis Rabbit endocarditis or pyelonephritis C. albicuiis C. alhicans C. albicaiis C. ulhicaizs C. ulbicuns C. ulhicans C. ulhicam C. ulbicuns AmB +ITZ AmB + 5-FC or 5-FC +FLZ 5-FC+ITZ or 5-FC +KTZ AmB + FLZ AmB or FLZ + quinolone AmB + FLZ AmB + ITZ FLZ + cyclosporine AmB +PCZ Indifference - [461 antagonisms Additive - synergy [47] Indifference Additive [481 [351 Indifference to additive [49] Indifference to additive Indifference [~OI 1541 Indifference [531 caspofungin + FLZ Indifference 1521 AmB (preincubation ) followed by AmB+FLZ Indifference [511 FLZ (preincubation - 24 h) followed by AmB FLZ (preincubation ) followed by AmB +FLZ Indifference AmB - amphotericin B; ITZ - itraconazole; 5-FC - flucytosine; PCZ - posaconazole; FLZ - fluconazole; KTZ - ketoconazole. sion. In contrast to other studies that demonstrated antagonism with the azolelpolyene combination, Sugar found no evidence of antagonism but found some evidence of an additive to synergistic effect. Thus he concluded that the antagonism described previously with the polyenelazole combination is not necessarily true with all azoles. In addition, some combinations might have an additive or synergistic effect. In a second study the same investigators using immunosuppressed mice with invasive candidiasis demonstrated that the combination of amphotericin Blfluconazole was also additive to synergistic [48]. In two additional trials that evaluated invasive candidiasis in a rabbit endocarditis model and in a neutropenic-mouse model, investigators using the amphotericin Blfluconazole combination were unable to detect any antagonism using this antifungal combination in either infection [49]; however, they were unable to document any additive or synergistic effect. Thus, it appears that in animal models evaluating candidiasis, the antagonistic effect seen with some of the combinations such as itraconazole/amphotericin B appears to be azole-specific and not complete across all azoles. In addition, it appears that the amphotericin B/fluconazole combination appears to be the safest combination, since it has not been found to be antagonistic in any animal models. To complicate matters, Sugar and Liu evaluated the activity of amphotericin B and itraconazole as mono- therapy, and compared it to simultaneous combination therapy and sequential antifungals therapy [50]. The interactions were studied in mice infected by an intravenous injection of. Concomitant administration of amphotericin B and itraconazole resulted in a 100% mortality rate. In contrast, 20% and 40% of mice treated with either amphotericin B or itraconazole as monotherapy survived. Overall, all combinations of amphotericin B and itraconazole resulted in a decrease in efficacy when compared to monotherapy. In tissue cultures of kidneys removed on day 30, there was also a decrease in efficacy when the mice were treated in combination or sequential therapy when compared to monotherapy. A similar study evaluating sequential antifungal therapy in experimental candidiasis was also published by Louie and coworkers [19]. In this study, a rabbit model of endocarditis and pyelonephritis was used to evaluate the impact that the order of exposure of either fluconazole or amphotericin B has on candidal infections when used as sequential or combination therapy. Overall, the investigators found that amphotericin B monotherapy and sequential therapy using amphotericin B alone for the first 24 h, followed by simultaneous therapy with amphotericin Blfluconazole was essentially the same and produced a rapid sterilization of kidney and cardiac vegetations but no antagonism was detected. On the other hand, preincubation with fluconazole monotherapy, followed by the simultaneous use 2003 ISHAM, Medical Mycology 4 1,

10 amphotericin B/fluconazole, was fungistatic and not as effective as amphotericin B alone or the regimen using amphotericin B prexposure, followed by simultaneous amphotericin B/fluconazole. In addition, preincubation with fluconazole, followed by amphotericin B montherapy produced a slower killing effect that was due to the generation of a transient amphotericin B resistance, which in vitro, can last from 8-40 h, depending on the length of preincubation. This is theoretically due to the ergosterol depletion in the cell membrane and the amount of time it takes the cell to re-accumulate ergosterol. Thus, it appears that the simultaneous exposure of cells to amphotericin B1 fluconazole in these models do not have any benefit to monotherapy with amphotericin B. Whereas prexposure to fluconazole may actually delay the fungicidal activity of amphotericin B by hrs. With the recent introduction of echinocandins and their unique mechanism of action, it stands to reason that they would also be evaluated in combination with other antifungals. One recently published animal trial evaluated the simultaneous use of caspofungin acetate and fluconazole in a murine model of invasive candidiasis. Bocanegra and co-workers were unable to show any significant additive or synergistic activity with this combination [51]. On the other hand, they did not observe any antagonism. In a similar experiment, Cacciapuoti and co-workers evaluated the triazole, posaconazole, in combination with amphotericin B in a murine model of systemic candidiasis [52]. They did not observe any antagonistic effects with this combination. In fact, they observed greater efficacy in some of the treatment groups that received the combination. Recent in vitro studies have demonstrated that cyclosporine in combination with antifungals was fungicidal against [53]. As previously stated, the effect may be due an increased intracellular fluconazole concentration produced by the inhibition of intrinsic efflux pumps that are found in Candida. Using a rat endocarditis model, the same investigators attempted to demonstrate similar synergistic interactions with the combination of fluconazole/cyclosporine in animals. In their experiments, they evaluated the CFUs from vegetations and kidneys of animals 72 h after the last dose of the antifungal. In this study, as in the in vitro experiments, they were able to demonstrate that the combination of fluconazole/cyclosporine was superior to monotherapy with either fluconazole or amphotericin B in sterilizing the organs. Table 5 provides a summary of results of all the antifungal combination trials, both in vitro and in animals. Clinical trials Clinical trials are generally the final step in evaluating and establishing efficacy, and comparing antifungal monotherapy versus combination therapy against clinical infections due to Candida. Unfortunately, there have been few clinical trials that have evaluated combination antifungal therapy in humans. Mucosal candidiasis A small clinical trial was conducted in 1991 by Brockwleyer et al. in 20 patients with Candida esophagitis [54]. In their study, 10 patients were randomized to receive amphotericin B 0.4 mglkglday IV and flucytosine 150 mglkglday orally for 8 days, while the second cohort of 10 patients received fluconazole 400 mglday for 8 days. Both groups showed a complete clinical and microbiologic response at 10 days post end of therapy. The relapse rate 7-24 months after therapy was essentially the same for both groups while both groups had a similar number of side effects. Overall, there was no advantage to using combination therapy versus monotherapy in this study. In a small open-labelled trial, the combination of fluconazole and terbinafine was evaluated against highdose terbinafine in fluconazole-refractory oropharyngeal candidiasis [55]. In this study, 48 HIV-positive patients who had failed at least 200 mglday of fluconazole were enrolled into one of three cohorts. Cohort 1 consisted of therapy with terbinafine alone at 1.5 glday, cohort 2 received terbinafine alone at 2.0 g/ day, and cohort 3 received terbinafine 1.0 glday plus fluconazole 200 mglday. The overall clinical cure rate was 66% and 62% for cohorts 1 and 2, respectively, and 62% for cohort 3. In addition, there was also marked improvement in 17%, 15%, and 23% of patients in cohorts 1, 2 and 3, respectively. The drawback of the study, however, was the small cohort sizes, which included 18 patients in cohort 1, 14 in cohort 2 and 16 in cohort 3. On the other hand, the trial did demonstrate that the combination of terbinafinelfluconazole produced either an improvement 01- cure in > 80% of patients with this refractory infection. Candidemia and invasive candidiasis In an open label, prospective, randomized clinical trial, Kujath et al. compared the efficacy of fluconazole 300 mglday against amphotericin B 0.5 mglkglday IV and flucytosine 7.5 glday orally in forty surgical patients with deep-seated candidiasis [56]. In their study, 20 patients were randomized to each cohort. At the end of study, Candida was eradicated in 12 of 20 patients in 2003 ISHAM, Medical Mycology, 4 1,

11 Combination antifungal therapy against Candida species Table 5 Summary of antifungal drug combinations and their activity against Candida species Drug regimen Activity Zrz vitro trials (number of studies) Animal trials (number of studies) Additive Indifference Additive Indifference AmB +MCZ AmB + CLZ AmB + KTZ AmB + FLZ AmB + ITZ AmB + 5-FC AmB + rifampin AmB +Tbf AmB +CSP AmB +PCZ 5-FC + KTZ 5-FC + ITZ Nik Z + FLZ Nik Z + KTZ Tbf + FLZ Tbf + ITZ Tbf + VCZ Tbf + PCZ CSP + FLZ AmB - amphotericin B, MCZ - miconazole, CLZ - clotrimazole, KTZ - ketoconazole, FLZ - fluconazole, ITZ - itraconazole, VCZ - voriconazle, PCZ - posaconazole, 5-FC - flucytosine, Tbf - terbinafine, Nik Z - nikkomycin Z, CSP - caspofungin. fluconazole group with a median time to eradication of 8.5 days, while the organisms were eradicated in 14 of the 20patients in the combination arm, with a median time to elimination of 5.5 days. Two patients in the combination arm were switched to the fluconazole arm because of adverse events. Overall, the mortality rates and cure rates were similar in both groups. The only difference was in the combination arm, where there was earlier eradication of the organism from the infected sites. A second prospective, randomized clinical trial, yublished in 1996 by Abele-Horn et al., compared fluconazole 200 mglday versus combination therapy with amphotericin B mglkg every other day and flucytosine 7.5 glday in intensive care unit patients with the diagnosis of systemic candidiasis [57]. Thirtysix patients were randomized to each arm and treated for 14 days. Overall, there was no difference in clinical outcome between the fluconazole and combination arms when treating pneumonia and sepsis, with cure rates of 64% and 63%, respectively. However, for the treatment of Candida peritonitis the combination arm proved more effective than fluconazole, 55% vs. 25%, respectively. In addition, microbiological cure rates were superior in the combination arm (86% vs. 50%). On the other hand, the fluconazole arm was associated with lower toxicity than the combination arm. The most important clinical trial evaluating the combination of fluconazole and amphotericin B, was published recently by Rex and the Mycosis Study Group [58]. This randomized, blinded, multi-centre clinical trial compared fluconazole 800 mglday versus fluconazole 800 mglday plus amphotericin B given for the first 5-6 days as therapy for candidemia in adults. A total of 219 patients eventually met the criteria for analysis. As a whole, both groups were similar with the exception that the group that received fluconazole plus placebo had a slightly higher APACHE score when compared to the combination therapy arm. The overall success rates were found to be 56% for those who received fluconazole and 69% for those who received the combination therapy. Moreover, although not statistically significant, the combination arm had a slight trend toward improved outcome and a morerapid clearing of the candidemia than those patients who received amphotericin B alone. In addition, the combination arm did not show any antagonism. Overall, although only a few clinical trials have been conducted evaluating combination antifungal therapy, it appears that combination therapy does not appear to add much to our antifungal armamentarium. In fact, in studies where amphotericin B was used, it usually added to the side effect profile of that particular 2003 ISHAM, Medical Mycology 4 1,

12 Conclusion Future studies will no doubt take advantage of the novel mechanisms of action and broad-spectrum activity of the new antifungals agents in an attempt to reduce the high mortality rate associated with invasive fungal infections. Combinations of the new agents and older antifungals have been shown to have some synergistic or at least additive activity against Candida. On the other hand, we must also use caution in this approach, since several antifungal combinations have demonstrated antagonistic activity in vitro [59]. In addition, we now know from several of the in vitro combination trials that sequential antifungal therapy may also have to be considered when choosing an antifungal agent. Paying attention to which antifungal the patient has been on after the patient has either failed one type of antifungal or has broken through an antifungal regimen may also have an impact on the outcome of the patient. The current IDSA guidelines do not recommend the use of combination therapy except for occasional patients with Candida meningitis or endocarditis [60]. However, because of the advent of new antifungals such as caspofungin and voriconazole, combination strategy has become commonplace in seriously ill patients with documented candidemia and disseminated candidiasis. Several combinations deserve a more careful evaluation for the treatment of candidiasis since they appear to have some synergistic activity in vitro and in animal studies. Certainly, echinocandins have demonstrated some synergy with amphotericin B. Nikkomycin Z and terbinafine have also demonstrated excellent synergistic activity with most azoles. Data from well-controlled randomized clinical trials using the new antifungal in combination is still needed to define the most efficacious antifungal regimen. 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