Medical Mycology, 2014, 52, 320–325 doi: 10.1093/mmy/myt027 Advance Access Publication Date: 24 March 2014 Short Communication
Short Communication
ˆ Raimunda Samia Nogueira Brilhante1,2,∗ , ˆ ´ Angela Donato Maia Malaquias2 , Erica Pacheco Caetano1 , ´ Debora de Souza Collares Maia Castelo-Branco1 , Rita Amanda Chaves de Lima1 , Francisca Jakelyne de Farias Marques1 , Natalya Fechine Silva2 , Lucas Pereira de Alencar1,3 , Andre´ Jalles Monteiro4 , Zoilo Pires de Camargo5 , Tereza de Jesus Pinheiro Gomes Bandeira1,6 , Anderson Messias Rodrigues5 , Rossana de Aguiar Cordeiro1,2 , ´ Costa Sidrim1,2 and Jose´ Luciano Bezerra Moreira1 , Jose´ Julio ´ Marcos Fabio Gadelha Rocha1,3 1
´ 2 Postgraduate Program in Medical Specialized Medical Mycology Center, Federal University of Ceara, 3 ´ Postgraduate Program in Veterinary Sciences, State University of Sciences, Federal University of Ceara, ´ Fortaleza, Ceara, ´ ´ 4 Department of Statistics and Applied Mathematics, Federal University of Ceara, Ceara, 5 ˜ Paulo Federal University, Brazil, Department of Microbiology, Immunology and Parasitology, Sao ´ Brazil ˜ Paulo, Brazil and 6 School of Medicine, Christus College, Fortaleza, Ceara, Sao
˜ de Caninde, ´ 210; Montese, CEP: 60.425-540, *To whom correspondence should be addressed. R.S.N. Brilhante, Rua Barao Fortaleza, CE, Brazil. Tel: 55 (85) 3366-8319; E-mail: [email protected]. Received 26 April 2013; Revised 7 October 2013; Accepted 23 November 2013
Abstract Miltefosine (MIL), originally developed for use in cancer chemotherapy, has been shown to have important antifungal activity against several pathogenic fungi. Our aim in this study was to determine the in vitro activity of MIL against the dimorphic fungi Histoplasma capsulatum and Sporothrix spp. This was done using the broth microdilution method. MIL had an in vitro inhibitory effect against all strains of H. capsulatum var. capsulatum and Sporothrix spp. analyzed. The minimal inhibitory concentrations (MIC) varied from 0.25 µg/ml to 2 µg/ml for H. capsulatum var. capsulatum in the filamentous phase and from 0.125 µg/ml to 1 µg/ml in the yeast phase. The MIC interval for Sporothrix spp. in the filamentous phase was 0.25–2 µg/ml. The minimal fungicidal concentrations (MFCs) were ≤4 µg/ml for isolates of both analyzed species. This study demonstrates that MIL has an antifungal effect in vitro against two potentially pathogenic fungi and that more studies should be performed in order to evaluate its applicability in vivo. Key words: Histoplasma capsulatum, Sporothrix spp., miltefosine, inhibitory effect.
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C The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal
Mycology. All rights reserved. For permissions, please e-mail: [email protected]
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In vitro inhibitory effect of miltefosine against strains of Histoplasma capsulatum var. capsulatum and Sporothrix spp.
Brilhante et al.
Introduction
Materials and methods Microorganisms Fifty-two filamentous strains of H. capsulatum var. capsulatum (50 recovered from humans and 2 from animals) and 15 yeast strains (13 from humans and 2 from animal) were obtained from the collection of the Specialized Medical Mycology Center of the Federal University of Ceara. ´ Sixty-two filamentous-phase species of the Sporothrix schenckii complex used in this investigations consisted of 48 S. brasiliensis, 6 S. schenckii, 5 S. mexicana, and 3 S. globosa. Of this total, 25 were recovered from humans, 34 from animals, and 3 from the environment and all were obtained from
the Molecular Medical Mycology Laboratory of Federal University, Sao ˜ Paulo. The strains Candida parapsilosis, American Type Culture Collection (ATCC) 22019, and C. krusei, ATCC 6258, were included in the study as control strains.
Antimicrobial agents For the susceptibility assays, a commercial formulation of MIL (Sigma Chemical Corporation, St. Louis, MO, USA) was diluted with sterile distilled water, as recommended by the manufacturer, and tested at a concentration range from 0.0312 µg/mL to 16 µg/ml. AMB (Sigma Chemical Corporation, St. Louis, MO, USA) and ITC (Janssen Pharmaceutica, Beerse, Belgium) were included as control drugs. These drugs were diluted with 100% Dulbecco’s modified Eagle’s medium as recommended by the Clinical and Laboratory Standards Institute (CLSI, Wayne, PA, USA) [19,20] and tested at a concentration range from 0.03125 µg/mL to 16 µg/mL [19,20]. Serial dilutions of each antimicrobial agent were prepared in RPMI 1640 medium (Sigma Chemical Corporation, St. Louis, MO, USA), with L-glutamine and without sodium bicarbonate, buffered to pH 7.0 with 0.165 M 4-morpholinepropanesulfonic acid (Sigma Chemical Corporation, St. Louis, MO, U.S.A.) [19,20].
Preparation of the inocula Inocula of the strains of H. capsulatum var. capsulatum and Sporothrix spp. in the filamentous phase were prepared from brain heart infusion (BHI) agar (Himedia, India) cultures incubated for 6–7 d at 25◦ C–28◦ C [12,21]. H. capsulatum var. capsulatum yeast phase strains were grown on BHI agar supplemented with 10% sheep blood and incubated for 7 d at 35◦ C [12]. A sterile saline solution was added to each fungal culture and scrapings were taken from the mycelial surface. Then, each fungal suspension was adjusted to 0.5 on the McFarland scale for H. capsulatum var. capsulatum, in both phases [12], and adjusted to 2 on the McFarland scale for Sporothrix spp. [21]. The suspensions were diluted in the proportion of 1:10 with RPMI medium to obtain inocula with final concentrations of 0.5 × 103 to 2.5 × 104 CFU/ml for H. capsulatum var. capsulatum [12] and 1–5 × 105 CFU/ml for Sporothrix spp. [21].
Susceptibility assay Susceptibility was analyzed using the broth microdilution assay, based on reference methods issued by the CLSI [19,20], with adaptations for H. capsulatum var. capsulatum and Sporothrix spp. All strains were tested in
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Histoplasma capsulatum var. capsulatum and Sporothrix spp. are pathogenic dimorphic fungi that cause histoplasmosis and sporotrichosis, respectively, in humans and other animals [1,2]. Classic histoplasmosis occurs throughout the Americas, with reports mainly from the United States and Brazil [3,4]. In turn, sporotrichosis is a cosmopolitan disease that predominantly occurs in tropical and subtropical areas and is endemic to the Americas, with high prevalence in Brazil [5,6]. The treatment of histoplasmosis includes use of itraconazole (ITC) in cases with slight to moderate symptoms and amphotericin B (AMB) in more severe cases [3]. The drugs typically used to treat sporotrichosis are potassium iodide for localized sporotrichosis, ITC for lymphocutaneous infections, and AMB for serious infections [1,7]. Although the antifungal therapy that is commonly used is efficient in treating these mycoses, there are some drawbacks, such as the need for prolonged treatment and the high nephrotoxicity of AMB [8,9]. Also, despite the application of these antifungals, persistent and recurrent cases of these diseases have been described [9–11]. This situation has prompted the search for new agents with potential antifungal properties. Some studies have demonstrated that certain drugs currently used to treat nonfungal diseases can also have relevant antifungal activity [12,13]. Miltefosine (MIL) was originally developed for cancer chemotherapy. Subsequent studies have found that it has relevant inhibitory activity against Leishmania spp. and Trypanosoma cruzi [14–16] as well as against some fungal pathogens [17,18]. Although the antifungal properties of this drug have previously been demonstrated, no study has been conducted to show its potential against these dimorphic fungi. Based on this, our objective was to evaluate the in vitro inhibitory activity of MIL against H. capsulatum var. capsulatum and Sporothrix spp.
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through analysis of variance. Results with P < 0.05 were considered statistically significant.
Results MIL inhibited the growth of all tested strains, with MICs ≤ 2 µg/ml and MFCs ≤ 4 µg/ml (Table 1). More specifically, the MICs of H. capsulatum var. capsulatum in its hyphal form varied from 0.25 to 2 µg/ml, whereas the yeast phase MIC was from 0.125 to 1 µg/ml. The MFCs for the mycelial stage of H. capsulatum var. capsulatum were 0.5 to 4 µg/ml and 0.25 to 4 µg/ml in the yeast phase. For MIL, the MICs and MFCs against filamentous form were statistically higher than those found against the yeast-like form (MIC, P = 0.0002; MFC, P = 0.0015; Table 1). The MIC interval for MIL against Sporothrix spp. was 0.25−2 µg/ml and the MFC interval was 1−4 µg/ml. Statistically significant differences were observed between the MICs found against different Sporothrix species (P < 0.01), but not between the MFCs. The MICs of MIL were higher against S. globosa, when compared with
Table 1. In vitro antifungal activity of miltefosine against filamentous and yeast-like Histoplasma capsulatum and Sporothrix spp. Drugs MIL Inhibitory Concentration (µg/ml) Species
n
50% Inhibition
80% Inhibition (MIC)
100% Inhibition
MFC
Histoplasma capsulatum (M)
52
0.125 (8)∗ 0.25 (9) 0.5 (32) 1 (3)
0.25 (8) 0.5 (9) 1 (32) 2 (3)
0.5 (8) 1 (9) 2 (32) 4 (3)
0.5 (6) 1 (5) 2(32) 4 (9)
H. capsulatum (Y)
15
Sporothrix brasiliensis
48
0.125 (7) 0.25 (21) 0.5 (20)
0.25 (7) 0.5 (21) 1 (20)
0.5 (7) 1 (21) 2 (20)
1 (19) 2 (21) 4 (8)
S. schenckii
6
0.25 (2) 0.5 (4)
0.5 (2) 1 (4)
1 (2) 2 (4)
1 (1) 2 (4) 4 (1)
S. mexicana
5
0.25 (2) 0.5 (2) 1 (1)
0.5 (2) 1 (2) 2 (1)
1 (2) 2 (2) 4 (1)
1 (1) 2 (3) 4 (1)
S. globosa
3
0.5 (1) 1 (2)
1 (1) 2 (2)
2 (1) 4 (2)
2 (1) 4 (2)
0.0625 (1) 0.125 (8) 0.25 (4) 0.5 (2)
0.125 (1) 0.25 (8) 0.5 (4) 1 (2)
0.25 (1) 0.5 (8) 1 (4) 2 (2)
0.25 (1) 0.5 (3) 1 (7) 2 (4)
M, mycelial phase; MFC, minimum fungicidal concentration; MIC, minimum inhibitory concentration; MIL, miltefosine; Y, yeast-like phase. ∗ Number of tested strains. Control strains: Candida krusei, American Type Culture Collection (ATCC) 6258 (amphotericin B [AMB], 1 µg/ml; itraconazole [ITC], 0.25 µg/ml; MIL, 1 µg/ml); C. parapsilosis, ATCC 22019 (AMB, 1 µg/ml; ITC, 0.25 µg/ml; MIL, 1 µg/ml).
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duplicate, and the results were read visually after 4 and 7 d of incubation at 35◦ C for H. capsulatum var. capsulatum in the yeast phase and filamentous phase, respectively, and after 3 d of incubation at 35◦ C for Sporothrix spp. [21]. The minimum inhibitory concentration (MIC) for MIL was defined as the lowest drug concentration capable of inhibiting 80% of fungal growth [17] when compared with the growth control; however, the minimum concentrations that caused 50% and 100% inhibition of fungal growth were also observed. The MIC for AMB was defined as the lowest concentration capable of inhibiting 100% of fungal growth, and the MIC for ITC was defined as the lowest drug concentration capable of inhibiting 50% of fungal growth when compared with the respective growth control [12]. After the MIC reading, 100-µL aliquots from the wells that did not show visible fungal growth were transferred to tubes containing potato agar and incubated for 7 d at 35◦ C. The minimum fungicidal concentration (MFC) was defined as the lowest concentration able to prevent growth of fungal colonies [22]. The MICs (80% inhibition) and MFCs obtained for MIL against H. capsulatum in filamentous and yeast-like forms and the Sporothrix species were compared
Brilhante et al.
S. brasiliensis (P < 0.0001), S. schenckii (P = 0.0009), and S. mexicana (P = 0.0085), and the MICs against S. mexicana were higher than those of S. brasiliensis (P = 0.0417). In addition, for MIL, the MICs and MFCs against Sporothrix spp. were similar to those of the filamentous stage of H. capsulatum but higher than those of yeastlike H. capsulatum (MIC, P = 0.0021; MFC, P = 0.0017). The control strains C. parapsilosis ATCC 22019, and C. krusei ATCC 6258 presented MICs for MIL, AMB, and ITC of 1, 1, and 0.25 µg/ml, respectively, which is consistent with the data described in the literature (Table 1).
MIL is an alkyl-phospholipid drug, an analogue of phosphocholine, that was initially developed to treat cancer [14–16]. Even though several cytotoxic antineoplastic drugs have been shown to present antiparasitic and antifungal activity, MIL was chosen for this work because it has been approved for the treatment of leishmaniasis and has been demonstrated to be an effective inhibitor of other protozoans and emerging fungal pathogens, including antifungalresistant strains [17,23]. Several mechanisms have been proposed to explain the activity of MIL in recent years, but no specific method has been definitively identified. The large number of potential mechanisms and contradictory experimental results suggest that MIL has more than one site of molecular action [24]. Similar molecular targets have been identified for Leishmania parasites and human cancer cells. These observations suggest that the activity of MIL is essentially associated with apoptosis and disturbance of lipid-dependent cell-signaling pathways [24]. It has been proposed that MIL uses lipid rafts to enter cancerous cells, where it inhibits the synthesis of phosphocholine and protein kinase B, which is supposedly sufficient to induce apoptosis [23,24]. MIL’s potential rupturing of the cell membrane is consistent with the broad activity spectrum of MIL against pathogenic fungi and protozoa as well as some types of tumor cells. According to Moreira et al. [23], MIL penetrates the plasmatic membrane through its lipid component and interacts with the hydrophobic side chains at the lipid–protein interface. Through detergent action, MIL can form structures that are similar to micelles with proteins that favor more expanded and dynamic conformations. Proteins with greater hydrophobicity can cause the hydrophilic groups of MIL to penetrate the membrane and cause its rupture. Considering the structural and molecular similarities between fungal and mammalian cells [25], it is plausible that similar mechanisms are involved in the inhibitory action of MIL against fungi. Additionally, Zuo et al. [26], in a study involving Saccharomyces cerevisiae,
demonstrated that MIL is quickly absorbed and penetrates the internal mitochondrial membrane, where it interrupts the membrane potential and causes dose-dependent inhibition of cytochrome c oxidase, leading to apoptosis. The results presented here demonstrate that MIL could inhibit the development of all test isolates of the dimorphic fungi H. capsulatum var. capsulatum and Sporothrix spp. Lower MICs were found against H. capsulatum var. capsulatum in the yeast phase when compared with the fungus’ filamentous phase and Sporothrix species. Additionally, it was observed that S. globosa was less susceptible to MIL when compared with the other Sporothrix species included in this investigation. It was observed that the MIC interval found for MIL (0.125 ≤ MIC ≤ 2 µg/ml) against these dimorphic fungi was similar to or lower than those reported for other fungal species [17]. For example, Tong et al. [18] found an MIC interval for MIL of 0.25−2 µg/ml against different genera of dermatophytes (Trichophyton, Epidermophyton, and Microsporum). Higher MICs were presented by Widmer et al. [17] against Cryptococcus spp. (MICs: 0.25−4 µg/ml), Candida spp. (MICs: 1−8 µg/ml), as well as several Aspergillus species (MICs: 2−16 µg/ml). These reports indicate the importance of our results regarding the inhibitory activity of MIL against the pathogens H. capsulatum var. capsulatum and Sporothrix spp. Even though experiments conducted by Widmer et al. [17] confirmed the inhibitory activity of MIL against Cryptococcus neoformans in a murine model of disseminated cryptococcosis, more recently, Shubitz et al. [27] and Wiederhold et al. [28] demonstrated the limited efficacy of MIL in murine models of coccidioidomycosis and cryptococcosis (meningoencephalitis and disseminated infection), respectively. The authors stated that these discrepant findings were most likely associated with the drug’s pharmacokinetic aspects and the animal host’s immune status [27,28]. It is important to note that the MICs and MFCs obtained for MIL in this research (0.125–4 µg/ml) are within the range of attainable in vivo concentrations, which presents an average concentration of 30.8 µg/mL in humans [29], and that MIL presents extensive tissue distribution [28]. In addition, this drug is successfully used to treat cutaneous, mucocutaneous, and visceral leishmaniasis [28,30] and, at therapeutic concentrations, it is less toxic than AMB [30]. Finally, considering the clinical similarities between leishmaniasis, histoplasmosis, and sporotrichosis, that is, intracellular parasitism and development of cutaneous and subcutaneous lesions [31–33], and the similar in vitro inhibitory concentrations of MIL against Leishmania spp. [34], we believe that MIL could potentially be used to treat sporotrichosis and histoplasmosis. Thus, more studies should be performed in order to evaluate the in vivo
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Discussion
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applicability of MIL and validate its potential use as a therapeutic alternative. 13.
Acknowledgments This study was supported by National Council for Scientific and Technological Development (CNPq), Brazil (PROTAX 562296/2010-7, 552161/2011-0, 504189/2012–3) and Coordination for the Improvement of Higher Education Personnel (CAPES); Brazil (PNPD 2103/2009, AE1-0052-000630100/11).
14. 15. 16.
The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper.
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Declaration of interest
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Short Communication
ˆ Raimunda Samia Nogueira Brilhante1,2,∗ , ˆ ´ Angela Donato Maia Malaquias2 , Erica Pacheco Caetano1 , ´ Debora de Souza Collares Maia Castelo-Branco1 , Rita Amanda Chaves de Lima1 , Francisca Jakelyne de Farias Marques1 , Natalya Fechine Silva2 , Lucas Pereira de Alencar1,3 , Andre´ Jalles Monteiro4 , Zoilo Pires de Camargo5 , Tereza de Jesus Pinheiro Gomes Bandeira1,6 , Anderson Messias Rodrigues5 , Rossana de Aguiar Cordeiro1,2 , ´ Costa Sidrim1,2 and Jose´ Luciano Bezerra Moreira1 , Jose´ Julio ´ Marcos Fabio Gadelha Rocha1,3 1
´ 2 Postgraduate Program in Medical Specialized Medical Mycology Center, Federal University of Ceara, 3 ´ Postgraduate Program in Veterinary Sciences, State University of Sciences, Federal University of Ceara, ´ Fortaleza, Ceara, ´ ´ 4 Department of Statistics and Applied Mathematics, Federal University of Ceara, Ceara, 5 ˜ Paulo Federal University, Brazil, Department of Microbiology, Immunology and Parasitology, Sao ´ Brazil ˜ Paulo, Brazil and 6 School of Medicine, Christus College, Fortaleza, Ceara, Sao
˜ de Caninde, ´ 210; Montese, CEP: 60.425-540, *To whom correspondence should be addressed. R.S.N. Brilhante, Rua Barao Fortaleza, CE, Brazil. Tel: 55 (85) 3366-8319; E-mail: [email protected]. Received 26 April 2013; Revised 7 October 2013; Accepted 23 November 2013
Abstract Miltefosine (MIL), originally developed for use in cancer chemotherapy, has been shown to have important antifungal activity against several pathogenic fungi. Our aim in this study was to determine the in vitro activity of MIL against the dimorphic fungi Histoplasma capsulatum and Sporothrix spp. This was done using the broth microdilution method. MIL had an in vitro inhibitory effect against all strains of H. capsulatum var. capsulatum and Sporothrix spp. analyzed. The minimal inhibitory concentrations (MIC) varied from 0.25 µg/ml to 2 µg/ml for H. capsulatum var. capsulatum in the filamentous phase and from 0.125 µg/ml to 1 µg/ml in the yeast phase. The MIC interval for Sporothrix spp. in the filamentous phase was 0.25–2 µg/ml. The minimal fungicidal concentrations (MFCs) were ≤4 µg/ml for isolates of both analyzed species. This study demonstrates that MIL has an antifungal effect in vitro against two potentially pathogenic fungi and that more studies should be performed in order to evaluate its applicability in vivo. Key words: Histoplasma capsulatum, Sporothrix spp., miltefosine, inhibitory effect.
320
C The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal
Mycology. All rights reserved. For permissions, please e-mail: [email protected]
Downloaded from http://mmy.oxfordjournals.org/ at University of Wisconsin-Madison Libraries on September 30, 2014
In vitro inhibitory effect of miltefosine against strains of Histoplasma capsulatum var. capsulatum and Sporothrix spp.
Brilhante et al.
Introduction
Materials and methods Microorganisms Fifty-two filamentous strains of H. capsulatum var. capsulatum (50 recovered from humans and 2 from animals) and 15 yeast strains (13 from humans and 2 from animal) were obtained from the collection of the Specialized Medical Mycology Center of the Federal University of Ceara. ´ Sixty-two filamentous-phase species of the Sporothrix schenckii complex used in this investigations consisted of 48 S. brasiliensis, 6 S. schenckii, 5 S. mexicana, and 3 S. globosa. Of this total, 25 were recovered from humans, 34 from animals, and 3 from the environment and all were obtained from
the Molecular Medical Mycology Laboratory of Federal University, Sao ˜ Paulo. The strains Candida parapsilosis, American Type Culture Collection (ATCC) 22019, and C. krusei, ATCC 6258, were included in the study as control strains.
Antimicrobial agents For the susceptibility assays, a commercial formulation of MIL (Sigma Chemical Corporation, St. Louis, MO, USA) was diluted with sterile distilled water, as recommended by the manufacturer, and tested at a concentration range from 0.0312 µg/mL to 16 µg/ml. AMB (Sigma Chemical Corporation, St. Louis, MO, USA) and ITC (Janssen Pharmaceutica, Beerse, Belgium) were included as control drugs. These drugs were diluted with 100% Dulbecco’s modified Eagle’s medium as recommended by the Clinical and Laboratory Standards Institute (CLSI, Wayne, PA, USA) [19,20] and tested at a concentration range from 0.03125 µg/mL to 16 µg/mL [19,20]. Serial dilutions of each antimicrobial agent were prepared in RPMI 1640 medium (Sigma Chemical Corporation, St. Louis, MO, USA), with L-glutamine and without sodium bicarbonate, buffered to pH 7.0 with 0.165 M 4-morpholinepropanesulfonic acid (Sigma Chemical Corporation, St. Louis, MO, U.S.A.) [19,20].
Preparation of the inocula Inocula of the strains of H. capsulatum var. capsulatum and Sporothrix spp. in the filamentous phase were prepared from brain heart infusion (BHI) agar (Himedia, India) cultures incubated for 6–7 d at 25◦ C–28◦ C [12,21]. H. capsulatum var. capsulatum yeast phase strains were grown on BHI agar supplemented with 10% sheep blood and incubated for 7 d at 35◦ C [12]. A sterile saline solution was added to each fungal culture and scrapings were taken from the mycelial surface. Then, each fungal suspension was adjusted to 0.5 on the McFarland scale for H. capsulatum var. capsulatum, in both phases [12], and adjusted to 2 on the McFarland scale for Sporothrix spp. [21]. The suspensions were diluted in the proportion of 1:10 with RPMI medium to obtain inocula with final concentrations of 0.5 × 103 to 2.5 × 104 CFU/ml for H. capsulatum var. capsulatum [12] and 1–5 × 105 CFU/ml for Sporothrix spp. [21].
Susceptibility assay Susceptibility was analyzed using the broth microdilution assay, based on reference methods issued by the CLSI [19,20], with adaptations for H. capsulatum var. capsulatum and Sporothrix spp. All strains were tested in
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Histoplasma capsulatum var. capsulatum and Sporothrix spp. are pathogenic dimorphic fungi that cause histoplasmosis and sporotrichosis, respectively, in humans and other animals [1,2]. Classic histoplasmosis occurs throughout the Americas, with reports mainly from the United States and Brazil [3,4]. In turn, sporotrichosis is a cosmopolitan disease that predominantly occurs in tropical and subtropical areas and is endemic to the Americas, with high prevalence in Brazil [5,6]. The treatment of histoplasmosis includes use of itraconazole (ITC) in cases with slight to moderate symptoms and amphotericin B (AMB) in more severe cases [3]. The drugs typically used to treat sporotrichosis are potassium iodide for localized sporotrichosis, ITC for lymphocutaneous infections, and AMB for serious infections [1,7]. Although the antifungal therapy that is commonly used is efficient in treating these mycoses, there are some drawbacks, such as the need for prolonged treatment and the high nephrotoxicity of AMB [8,9]. Also, despite the application of these antifungals, persistent and recurrent cases of these diseases have been described [9–11]. This situation has prompted the search for new agents with potential antifungal properties. Some studies have demonstrated that certain drugs currently used to treat nonfungal diseases can also have relevant antifungal activity [12,13]. Miltefosine (MIL) was originally developed for cancer chemotherapy. Subsequent studies have found that it has relevant inhibitory activity against Leishmania spp. and Trypanosoma cruzi [14–16] as well as against some fungal pathogens [17,18]. Although the antifungal properties of this drug have previously been demonstrated, no study has been conducted to show its potential against these dimorphic fungi. Based on this, our objective was to evaluate the in vitro inhibitory activity of MIL against H. capsulatum var. capsulatum and Sporothrix spp.
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through analysis of variance. Results with P < 0.05 were considered statistically significant.
Results MIL inhibited the growth of all tested strains, with MICs ≤ 2 µg/ml and MFCs ≤ 4 µg/ml (Table 1). More specifically, the MICs of H. capsulatum var. capsulatum in its hyphal form varied from 0.25 to 2 µg/ml, whereas the yeast phase MIC was from 0.125 to 1 µg/ml. The MFCs for the mycelial stage of H. capsulatum var. capsulatum were 0.5 to 4 µg/ml and 0.25 to 4 µg/ml in the yeast phase. For MIL, the MICs and MFCs against filamentous form were statistically higher than those found against the yeast-like form (MIC, P = 0.0002; MFC, P = 0.0015; Table 1). The MIC interval for MIL against Sporothrix spp. was 0.25−2 µg/ml and the MFC interval was 1−4 µg/ml. Statistically significant differences were observed between the MICs found against different Sporothrix species (P < 0.01), but not between the MFCs. The MICs of MIL were higher against S. globosa, when compared with
Table 1. In vitro antifungal activity of miltefosine against filamentous and yeast-like Histoplasma capsulatum and Sporothrix spp. Drugs MIL Inhibitory Concentration (µg/ml) Species
n
50% Inhibition
80% Inhibition (MIC)
100% Inhibition
MFC
Histoplasma capsulatum (M)
52
0.125 (8)∗ 0.25 (9) 0.5 (32) 1 (3)
0.25 (8) 0.5 (9) 1 (32) 2 (3)
0.5 (8) 1 (9) 2 (32) 4 (3)
0.5 (6) 1 (5) 2(32) 4 (9)
H. capsulatum (Y)
15
Sporothrix brasiliensis
48
0.125 (7) 0.25 (21) 0.5 (20)
0.25 (7) 0.5 (21) 1 (20)
0.5 (7) 1 (21) 2 (20)
1 (19) 2 (21) 4 (8)
S. schenckii
6
0.25 (2) 0.5 (4)
0.5 (2) 1 (4)
1 (2) 2 (4)
1 (1) 2 (4) 4 (1)
S. mexicana
5
0.25 (2) 0.5 (2) 1 (1)
0.5 (2) 1 (2) 2 (1)
1 (2) 2 (2) 4 (1)
1 (1) 2 (3) 4 (1)
S. globosa
3
0.5 (1) 1 (2)
1 (1) 2 (2)
2 (1) 4 (2)
2 (1) 4 (2)
0.0625 (1) 0.125 (8) 0.25 (4) 0.5 (2)
0.125 (1) 0.25 (8) 0.5 (4) 1 (2)
0.25 (1) 0.5 (8) 1 (4) 2 (2)
0.25 (1) 0.5 (3) 1 (7) 2 (4)
M, mycelial phase; MFC, minimum fungicidal concentration; MIC, minimum inhibitory concentration; MIL, miltefosine; Y, yeast-like phase. ∗ Number of tested strains. Control strains: Candida krusei, American Type Culture Collection (ATCC) 6258 (amphotericin B [AMB], 1 µg/ml; itraconazole [ITC], 0.25 µg/ml; MIL, 1 µg/ml); C. parapsilosis, ATCC 22019 (AMB, 1 µg/ml; ITC, 0.25 µg/ml; MIL, 1 µg/ml).
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duplicate, and the results were read visually after 4 and 7 d of incubation at 35◦ C for H. capsulatum var. capsulatum in the yeast phase and filamentous phase, respectively, and after 3 d of incubation at 35◦ C for Sporothrix spp. [21]. The minimum inhibitory concentration (MIC) for MIL was defined as the lowest drug concentration capable of inhibiting 80% of fungal growth [17] when compared with the growth control; however, the minimum concentrations that caused 50% and 100% inhibition of fungal growth were also observed. The MIC for AMB was defined as the lowest concentration capable of inhibiting 100% of fungal growth, and the MIC for ITC was defined as the lowest drug concentration capable of inhibiting 50% of fungal growth when compared with the respective growth control [12]. After the MIC reading, 100-µL aliquots from the wells that did not show visible fungal growth were transferred to tubes containing potato agar and incubated for 7 d at 35◦ C. The minimum fungicidal concentration (MFC) was defined as the lowest concentration able to prevent growth of fungal colonies [22]. The MICs (80% inhibition) and MFCs obtained for MIL against H. capsulatum in filamentous and yeast-like forms and the Sporothrix species were compared
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S. brasiliensis (P < 0.0001), S. schenckii (P = 0.0009), and S. mexicana (P = 0.0085), and the MICs against S. mexicana were higher than those of S. brasiliensis (P = 0.0417). In addition, for MIL, the MICs and MFCs against Sporothrix spp. were similar to those of the filamentous stage of H. capsulatum but higher than those of yeastlike H. capsulatum (MIC, P = 0.0021; MFC, P = 0.0017). The control strains C. parapsilosis ATCC 22019, and C. krusei ATCC 6258 presented MICs for MIL, AMB, and ITC of 1, 1, and 0.25 µg/ml, respectively, which is consistent with the data described in the literature (Table 1).
MIL is an alkyl-phospholipid drug, an analogue of phosphocholine, that was initially developed to treat cancer [14–16]. Even though several cytotoxic antineoplastic drugs have been shown to present antiparasitic and antifungal activity, MIL was chosen for this work because it has been approved for the treatment of leishmaniasis and has been demonstrated to be an effective inhibitor of other protozoans and emerging fungal pathogens, including antifungalresistant strains [17,23]. Several mechanisms have been proposed to explain the activity of MIL in recent years, but no specific method has been definitively identified. The large number of potential mechanisms and contradictory experimental results suggest that MIL has more than one site of molecular action [24]. Similar molecular targets have been identified for Leishmania parasites and human cancer cells. These observations suggest that the activity of MIL is essentially associated with apoptosis and disturbance of lipid-dependent cell-signaling pathways [24]. It has been proposed that MIL uses lipid rafts to enter cancerous cells, where it inhibits the synthesis of phosphocholine and protein kinase B, which is supposedly sufficient to induce apoptosis [23,24]. MIL’s potential rupturing of the cell membrane is consistent with the broad activity spectrum of MIL against pathogenic fungi and protozoa as well as some types of tumor cells. According to Moreira et al. [23], MIL penetrates the plasmatic membrane through its lipid component and interacts with the hydrophobic side chains at the lipid–protein interface. Through detergent action, MIL can form structures that are similar to micelles with proteins that favor more expanded and dynamic conformations. Proteins with greater hydrophobicity can cause the hydrophilic groups of MIL to penetrate the membrane and cause its rupture. Considering the structural and molecular similarities between fungal and mammalian cells [25], it is plausible that similar mechanisms are involved in the inhibitory action of MIL against fungi. Additionally, Zuo et al. [26], in a study involving Saccharomyces cerevisiae,
demonstrated that MIL is quickly absorbed and penetrates the internal mitochondrial membrane, where it interrupts the membrane potential and causes dose-dependent inhibition of cytochrome c oxidase, leading to apoptosis. The results presented here demonstrate that MIL could inhibit the development of all test isolates of the dimorphic fungi H. capsulatum var. capsulatum and Sporothrix spp. Lower MICs were found against H. capsulatum var. capsulatum in the yeast phase when compared with the fungus’ filamentous phase and Sporothrix species. Additionally, it was observed that S. globosa was less susceptible to MIL when compared with the other Sporothrix species included in this investigation. It was observed that the MIC interval found for MIL (0.125 ≤ MIC ≤ 2 µg/ml) against these dimorphic fungi was similar to or lower than those reported for other fungal species [17]. For example, Tong et al. [18] found an MIC interval for MIL of 0.25−2 µg/ml against different genera of dermatophytes (Trichophyton, Epidermophyton, and Microsporum). Higher MICs were presented by Widmer et al. [17] against Cryptococcus spp. (MICs: 0.25−4 µg/ml), Candida spp. (MICs: 1−8 µg/ml), as well as several Aspergillus species (MICs: 2−16 µg/ml). These reports indicate the importance of our results regarding the inhibitory activity of MIL against the pathogens H. capsulatum var. capsulatum and Sporothrix spp. Even though experiments conducted by Widmer et al. [17] confirmed the inhibitory activity of MIL against Cryptococcus neoformans in a murine model of disseminated cryptococcosis, more recently, Shubitz et al. [27] and Wiederhold et al. [28] demonstrated the limited efficacy of MIL in murine models of coccidioidomycosis and cryptococcosis (meningoencephalitis and disseminated infection), respectively. The authors stated that these discrepant findings were most likely associated with the drug’s pharmacokinetic aspects and the animal host’s immune status [27,28]. It is important to note that the MICs and MFCs obtained for MIL in this research (0.125–4 µg/ml) are within the range of attainable in vivo concentrations, which presents an average concentration of 30.8 µg/mL in humans [29], and that MIL presents extensive tissue distribution [28]. In addition, this drug is successfully used to treat cutaneous, mucocutaneous, and visceral leishmaniasis [28,30] and, at therapeutic concentrations, it is less toxic than AMB [30]. Finally, considering the clinical similarities between leishmaniasis, histoplasmosis, and sporotrichosis, that is, intracellular parasitism and development of cutaneous and subcutaneous lesions [31–33], and the similar in vitro inhibitory concentrations of MIL against Leishmania spp. [34], we believe that MIL could potentially be used to treat sporotrichosis and histoplasmosis. Thus, more studies should be performed in order to evaluate the in vivo
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Discussion
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applicability of MIL and validate its potential use as a therapeutic alternative. 13.
Acknowledgments This study was supported by National Council for Scientific and Technological Development (CNPq), Brazil (PROTAX 562296/2010-7, 552161/2011-0, 504189/2012–3) and Coordination for the Improvement of Higher Education Personnel (CAPES); Brazil (PNPD 2103/2009, AE1-0052-000630100/11).
14. 15. 16.
The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper.
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