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In vitro cytotoxicity of two novel oral formulations of Amphotericin B (iCo-009 and iCo-010) against… Leon, Carlos G; Lee, Jinkyung; Bartlett, Karen; Gershkovich, Pavel; Wasan, Ellen K; Zhao, Jinying; Clement, John G; Wasan, Kishor M Aug 20, 2011

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RESEARCH Open AccessIn vitro cytotoxicity of two novel oralformulations of Amphotericin B (iCo-009 and iCo-010) against Candida albicans, human monocyticand kidney cell linesCarlos G Leon1†, Jinkyung Lee1†, Karen Bartlett2, Pavel Gershkovich1, Ellen K Wasan3, Jinying Zhao1,John G Clement4 and Kishor M Wasan1*AbstractBackground: Invasive fungal infections such as candidiasis constitute an increasingly important medical problem.Drugs currently used for the treatment of candidiasis include polyenes (such as Amphotericin B) and azoles.Amphotericin B (AmpB) presents several limitations such as its nephrotoxicity and limited solubility. We havedeveloped two novel lipid-based AmpB formulations which in vivo show less nephrotoxicity and enhancedsolubility compared to Fungizone™ a commercial AmpB formulation.The purpose of this study was to determine the cytotoxicity of Fungizone™, Ambisome™ and two novel AmpBformulations (iCo-009 and iCo-010) against Candida albicans, human kidney (293T) cells and monocytic (THP1) cells.Methods: Cell cytotoxicity to the AmpB formulations was evaluated by MTS and LDH assays. In vitro anti-Candidaalbicans activity was assessed after a 48 h drug incubation.Results: None of the AmpB formulations tested showed cytotoxicity against 293T cells. In the case of THP1 cellsonly Fungizone™ and Ambisome™ showed cytotoxicity at 500 μg/L (n = 4-10, p < 0.05).The calculated EC50 to Candida albicans for the different formulations was as follows: 26.8 ± 2.9 for iCo-010, 74.6 ±8.9 for iCo-009, 109 ± 31 for Ambisome™ and 87.1 ± 22 for Fungizone™ (μg of AmpB/L, n = 6-12, p < 0.05).Conclusions: The AmpB formulations analyzed were not cytotoxic to 293T cells. Cytotoxicity in THP1 cells wasobserved for Fungizone™ and Ambisome™, but not with the novel AmpB formulations. iCo-010 had higherefficacy compared to other three AmpB formulations in the Candida albicans model.The absence of cytotoxicity as well as its higher efficacy for the Candida model compared to Fungizone™ andAmbisome™ suggest that iCo-010 has potential in treating candidiasis.Keywords: Amphotericin B, Candida, cytotoxicity, monocytes.1. BackgroundInvasive fungal infections constitute an increasinglyimportant medical problem due to the growth of immu-nodeficient populations, the development of antifungalresistance and limitations in the efficacy and toxicity ofcurrent antifungals [1]. Candida species are the mostcommon cause of nosocomial invasive mycosis and arethe leading cause of related mortality [2]. An increasingrate of candidaemia [3] as well as the emergence ofdrug resistant strains [4] support the efforts in discover-ing novel therapeutic approaches [1].Drugs currently used for the treatment of candidiasisinclude polyenes (such as Amphotericin B AmpB),azoles (fluconazole), echinocandins and flucytosine. Lim-ited therapeutic efficiency and drug resistance have led* Correspondence: kwasan@mail.ubc.ca† Contributed equally1Division of Pharmaceutics and Biopharmaceutics, Faculty of PharmaceuticalSciences, The University of British Columbia, Vancouver British Columbia, V6T1Z3, CanadaFull list of author information is available at the end of the articleLeon et al. Lipids in Health and Disease 2011, 10:144http://www.lipidworld.com/content/10/1/144© 2011 Leon et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.to consider new therapeutic approaches, in particularthe use of new analogs of existing drugs [1].Amphotericin B (AmpB) is a treatment of choice forsystemic fungal infections [5]. The mechanism of actionof AmpB involves its binding to ergosterol in the fungalcell membrane, producing pores in the membranewhich leads to ion loss and cell death [6]. Recently ithas been described that AmpB can also elicit cell deaththrough the induction of a strong oxidative burst [7].Acquired resistance to Amphotericin B in Candida spe-cies, though rare, has been reported previously [8,9].This AmpB resistance has been correlated withdecreased levels of ergosterol in the plasma membraneand has been accompanied with azole resistance [10,11].One of the major limitations associated with AmpB isits nephrotoxicity [12]. Another issue related to AmpBis its poor solubility that limits its route of administra-tion [13]. The development of an effective, safe, andinexpensive oral formulation of amphotericin B wouldhave many applications for the treatment of fungal dis-eases. The new oral amphotericin B formulations devel-oped by our group, iCo-009 and iCo-010, show efficacyin vivo against leishmaniasis [14,15], aspergillosis andcandidiasis [16].The purpose of the present study was to determine iffour different formulations of AmpB (iCo-009, iCo-010,Fungizone™ and Ambisome™) were cytotoxic inhuman monocytic, human kidney cells and Candidaalbicans.2. Results and DiscussionThe first objective of our study was to determine theAmpB-induced cytotoxicity in two human cell lines,293T kidney and THP1 monocytic cells. No evidence ofcytotoxicity was found in any of the three AmpB formu-lations used (Fungizone™, iCo-009 and iCo-010) at thedoses tested (Figure 1, n = 4) using a respiration assay(MTS) in the kidney 293T cells. The vehicle controls forboth iCo-009 and iCo-010 did not show a toxic effect(data not shown). At the highest AmpB dose analyzed(10,000 μg/L), there was a reduction of 20% in therespiration rate of the cells treated with Fungizone™,however, it didn’t reach statistical significance. In termsof cytotoxicity in THP1 monocytic cells of the fourAmpB formulations at the doses analyzed (0 to 500 μg/L), differences were shown between the MTS (Figure 2a,n = 4-10) and LDH (Figure 2b, n = 6) results. Nochanges in viability were found with the LDH assay atany of the AmpB concentrations analyzed. The LDHresults were corrected by the amount of protein in eachtreatment group. There was also no difference in proteincontent in the different treatment groups. However, theNormalized MTS results exhibited a reduced respirationrate at 500 μg/L for both Fungizone™ (0.71 ± 0.04) andAmbisome™ (0.72 ± 0.17) compared to controls (1.00).Previously, others [17] have described a difference inthe degree of cellular activation by AmpB in the 293and THP1 cell lines. This disparity was accounted bythe differential expression of the TLR2, highly expressedin THP1 cells and expressed at very low levels in 293cells. The new oral formulations, iCo-009 and iCo-010,did not show any evidence of toxicity at the highestconcentration analyzed in 293T cells (10,000 μg/L, Fig-ure 1). The limited TLR2 expression in 293T cells mayexplain this lack of cytotoxicity that was observed evenat large doses of AmpB compared with a cytotoxic effectof AmpB in THP1 cells at a lower concentration (500μg/L).The difference in cytotoxicity found in THP1 cellsbetween the LDH and MTS assays could be accountedby the fact that the MTS assay relies on the accessibilityof the substrate to the respiratory machinery of the cell(i.e. expression and activity of transporters). In the caseof LDH there is no active transport factor(s) as it is anindex of the cell breakdown and the release of LDHenzyme to the medium. The drug concentration that eli-cits toxicity in the THP1 monocytes with Fungizone™and Ambisome™ can be compared with the serum druglevels obtained in patients. After a standard dose ofAmpB (0.3 mg/kg on days 1 and 2 followed by increas-ing doses to 0.5 mg/kg) for patient therapy, the expectedserum AmpB levels can reach 200 μg/L [18]. Few stu-dies have assessed the AmpB-induced cytotoxicity inmonocytes and macrophages. Sesana et al., [19] exam-ined the in vitro activity of AmpB cochleates againstLeishmania chagasi and the cytotoxic effect on mouseperitoneal macrophages using AmpB deoxycholate (Fun-gizone™) as a control. They found that the AmpB deox-ycholate (Fungizone™) was cytotoxic to mouseperitoneal macrophages at a concentration of 1250 μg/L,while the AmpB cochleates were not toxic at that AmpBconcentration.The second objective of our study was to determinethe efficacy of the novel AmpB formulations in an invitro Candida albicans model. Unfortunately, frequentlythere is limited correlation between in vitro susceptibil-ity determination and patient response [20]. However,use of RPMI medium supplemented with dextroseshowed improvement in assessing Candida albicansantibiotic susceptibility [21]. Thus this methodology wasused in the present study. The results (Figure 3 andTable 1) showed that iCo-010 had a lower EC50 thanFungizone™, Ambisome™ and iCo-009 for this ATCCCandida albicans reference strain (n = 6-12 experi-ments). The EC50 calculated for AmpB (as Fungi-zone™) for the Candida albicans reference strain 18804Leon et al. Lipids in Health and Disease 2011, 10:144http://www.lipidworld.com/content/10/1/144Page 2 of 7that was assessed using this system is similar to thatpreviously obtained for this strain in an Etest [22].iCo-009 was previously compared to Abelcet® in an invivo study in Candida albicans-infected male rats [16].In that report, iCo-009 exhibited significant reduction inviable colony forming units of Candida from kidneyhomogenates compared to Abelcet®.3. ConclusionsWe have tested the cytotoxicity and efficacy of fourAmpB formulations in two human cell lines and a Can-dida albicans model, respectively. Fungizone™ andAmbisome™, but not iCo-009 and iCo-010, show cyto-toxicity in human monocytes at a concentration of 500μg/L. iCo-010 shows a higher efficacy compared to iCo-009, Ambisome™ and Fungizone™ in the Candidaalbicans model. The lack of toxicity and efficacy of iCo-010, a novel oral AmpB formulation, warrant future invivo Candida albicans studies to determine its therapeu-tic index.4. Materials and methods4.1. Novel lipid oral Amphotericin B formulationsThe description of the composition and development ofthe two novel lipid oral Amphotericin B formulationscan be found elsewhere iCo-009 [16] and iCo-010 [15].AmpB concentration was analyzed by HPLC as pre-viously described [16]. For the cytotoxicity and efficacystudies we used as negative controls medium alone andmedium containg the vehicle controls for both iCo-009and iCo-010. No cytotoxicity was found when any ofthese controls were used (data not shown).4.2. Cytotoxicity assay on 293T cellsHuman embryonic kidney cells (293T ATCC CRL11268) were kindly provided by Dr JS Hill (St Paul’sFigure 1 Cytotoxicity in 293T cells: Dose-response effects of Fungizone™, iCo-009 and iCo-010 on cytotoxicity on human embryonic (293T)kidney cells. The cells were exposed for 48 h with different drug concentrations (0, 250, 500, 1000, 5000 and 10,000 μg AmpB/L). MTS valueswere corrected for protein determination and normalized to the control cells. Data are reported as means ± standard deviation of fourexperiments (eight replicates per experiment).Leon et al. Lipids in Health and Disease 2011, 10:144http://www.lipidworld.com/content/10/1/144Page 3 of 7Figure 2 Dose-response effects of Fungizone™, Ambisome™, iCo-009 and iCo-010 on cytotoxicity in human monocytic (THP1) cellsas assessed using an MTS assay (Figure 2a). The cells were exposed for 48 h with different drug concentrations (31.25, 62.5, 125, 250 and500 μg AmpB/L). The cells were washed and incubated with the MTS reagent for 90 min. Data are reported as means ± standard deviation offour to ten experiments, * p < 0.05, eight replicates per experiment). Dose-response effects of Fungizone™, Ambisome™, iCo-009 and iCo-010on cytotoxicity in human monocytic (THP1) cells as assessed using an LDH assay (Figure 2b). The cells were exposed for 48 h with differentdrug concentrations (31.25, 62.5, 125, 250 and 500 μg AmpB/L). An aliquot of the medium was incubated with the LDH reagent. Data arereported as means ± standard deviation of six experiments (four replicates per experiment).Leon et al. Lipids in Health and Disease 2011, 10:144http://www.lipidworld.com/content/10/1/144Page 4 of 7Hospital, UBC, Vancouver, BC). Cells were grown incomplete DMEM without phenol red at 37°C (5% CO2)in T75 flasks. At near confluency, the cells were trypsi-nized and transferred to either additional flasks or to96-well plates (previously coated with Poly L-lysine andseeded at a density of 10,000 cells/well). Twenty fourhours post seeding, the adherent cells were washed andincubated with different concentrations of AmpB formu-lations (250 to 10,000 μg/L of AmpB) for 48 h. PercentAmpB-mediated cytotoxicity was determined using theMTS conversion assay (Promega Corp, Madison, WI), aspreviously described [23].4.3. Cytotoxicity assay on THP1 cellsHuman monocytic THP1 cells (ATCC TIB-202) werekindly provided by Dr JS Hill (St Paul’s Hospital, UBC,Vancouver, BC). Cells were grown in complete RPMIwithout phenol red at 37°C (5% CO2) in T75 flasks.Before the cells reached a concentration of one millionper mL, the cells were transferred to either additionalflasks or to 96-well plates and seeded at a density of20,000 cells/well with the addition of PMA (10 ng/mLfinal) to allow the cells to differentiate overnight.Twenty four hours post seeding, the differentiated cellswere washed and the different concentrations of AmpBformulations were added (31 to 500 μg/L of AmpB) for48 h. After a 90 min incubation with the MTS reagent,percent AmpB-mediated cytotoxicity was determinedFigure 3 In vitro Candida efficacy studies. Candida was exposed for 48 h to different doses of Fungizone™, iCo-009 and iCo-010 (0, 15.6,31.25, 62.5, 125 and 250 μg AmpB/L). A vehicle control with the same lipid composition as iCo-010 was also used at comparable volumes.Growth was normalized by the corresponding absorbance of the untreated Candida. The vehicle control showed no effect on Candida growth.(* p < 0.05 n = 6-12 independent experiments, each experiment includes eight replicates).Table 1 EC50 for AmpB formulations (mean ± SD,expressed in μg/L of AmpB, n = 6-12, * p < 0.05) testedin the Candida modelMean ± SDFungizone ™ 87.1 ± 22Ambisome™ 109 ± 31iCo-009 74.6 ± 8.9iCo-010 26.8 ± 2.9 *Leon et al. Lipids in Health and Disease 2011, 10:144http://www.lipidworld.com/content/10/1/144Page 5 of 7using the MTS conversion assay (Promega Corp, Madi-son, WI), as previously described [23] or the LDH assay(Promega Corp, Madison, WI) as outlined by the manu-facturer specifications. The MTS and LDH results werecorrected by the amount of protein present in therespective wells. Purified LDH from the manufacturerkit was used as a positive control (data not shown).4.4. Minimum inhibitory concentration determination ofthe AmpB formulations on Candida growth in liquidmediumRPMI without phenol red and supplemented with 10%Fetal bovine serum, 2% dextrose plus penicillin andstreptomycin was used to prepare dilutions of AmpB(31.25, 62.5, 125, 250 and 500 μg/L- final drug concen-tration) from Fungizone™, Ambisome™, iCo-009 andiCo-010 (AmpB concentration in the formulations wasdetermined by HPLC). A vehicle control of iCo-009 andiCo-010 was used as a negative control. RPMI is thestandard medium used in microplate drug susceptibilityassays and is commonly used for testing of drug sensi-tivity for both Candida albicans and Cryptococcus neo-formans [24]. A limitation of the RPMI medium is thatthere are frequent overlaps between the EC50 ranges forisolates that are putatively resistant or susceptible toAmphotericin B. The supplementation of RPMI mediumwith 2% glucose has lead to higher fungal growth ratesand better separation of EC50s for isolates that are puta-tively resistant to fluconazole [21]. This is the methodol-ogy that we have used for our experiments.The Candida albicans (ATCC reference strain 18804)inoculum was prepared as follows. The fungal cell con-centration was determined using a hematocytometer.One hundred microlitres of a 5 × 105/mL preparation ofCandida in RPMI was placed in the 96 well plates for afinal concentration of 2.5 × 105 cells/mL. Each concen-tration was analyzed in eight replicates per experiment(n = 6-12 independent experiments).The plates were incubated for 48 h at 37°C protectedfrom the light before analyzing the growth pattern usinga spectrophotometer at 650 nm. A new Candida agarplate was streaked each week to ensure the viability ofthe culture.AcknowledgementsThis work was supported by a grant-in-aid from iCo Therapeutics Inc.,Vancouver, BC Canada and a Canadian Institutes of Health Research (CIHR)grant awarded to KMW.Author details1Division of Pharmaceutics and Biopharmaceutics, Faculty of PharmaceuticalSciences, The University of British Columbia, Vancouver British Columbia, V6T1Z3, Canada. 2Faculty of Environmental and Occupational Health, TheUniversity of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada.3School of Health Sciences, British Columbia Institute of Technology, 3700Willingdon Avenue, Burnaby, British Columbia, V5G 3H2, Canada. 4iCoTherapeutics Inc., 760-777 Hornby, Vancouver, British Columbia, V6Z 1S4,Canada.Authors’ contributionsCGL carried out the 293T cytotoxicity assays and drafted the manuscript. JLcarried out the THP1 cytotoxicity assays and the Candida efficacyexperiments. KB participated in the design of the study. PG prepared theAmpB formulations and edited the manuscript. EKW designed and preparedthe AmpB formulations. JZ prepared and characterized the novel AmpBformulations. JGC edited the manuscript. KMW participated in the design ofthe study and helped to draft the manuscript. All authors read andapproved the final manuscript.Competing interestsJ. G. C. is an employee/co-founder/shareholder and director of iCoTherapeutics Inc. All other authors: none to declare.Received: 15 July 2011 Accepted: 20 August 2011Published: 20 August 2011References1. Calugi C, Trabocchi A, Guarna A: Novel small molecules for the treatmentof infections caused by Candida albicans: a patent review 2. Expert OpinTher Pat 2011, 21(3):381-97.2. Marr KA: Fungal infections in oncology patients: update onepidemiology, prevention, and treatment. Curr Opin Oncol 2010,22(2):138-42.3. 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Sesana AM, Monti-Rocha R, Vinhas SA, Morais CG, Dietze R, Lemos EM: Invitro activity of amphotericin B cochleates against Leishmania chagasi.Mem Inst Oswaldo Cruz 2011, 106(2):251-3.20. Rex JH, Pfaller MA, Galgiani JN, Bartlett MS, Espinel-Ingroff A,Ghannoum MA, Lancaster M, Odds FC, Rinaldi MG, Walsh TJ, Barry AL:Development of interpretive breakpoints for antifungal susceptibilitytesting: conceptual framework and analysis of in vitro-in vivo correlationdata for fluconazole, itraconazole, and candida infections. Subcommitteeon Antifungal Susceptibility Testing of the National Committee forClinical Laboratory Standards. Clin Infect Dis 1997, 24(2):235-47.21. Rodriguez-Tudela JL, Martinez-Suarez JV: Improved medium forfluconazole susceptibility testing of Candida albicans. Antimicrob AgentsChemother 1994, 38(1):45-8.22. Chang HC, Chang JJ, Chan SH, Huang AH, Wu TL, Lin MC, Chang TC:Evaluation of Etest for direct antifungal susceptibility testing of yeasts inpositive blood cultures. J Clin Microbiol 2001, 39(4):1328-33.23. Zager RA: Polyene antibiotics: relative degrees of in vitro cytotoxicityand potential effects on tubule phospholipid and ceramide content. AmJ Kidney Dis 2000, 36(2):238-49.24. Anaissie EJ, Paetznick VL, Ensign LG, Espinel-Ingroff A, Galgiani JN,Hitchcock CA, LaRocco M, Patterson T, Pfaller MA, Rex JH, Rinaldi MG:Microdilution antifungal susceptibility testing of Candida albicans andCryptococcus neoformans with and without agitation: an eight-centercollaborative study. Antimicrob Agents Chemother 1996, 40(10):2387-91.doi:10.1186/1476-511X-10-144Cite this article as: Leon et al.: In vitro cytotoxicity of two novel oralformulations of Amphotericin B (iCo-009 and iCo-010) against Candidaalbicans, human monocytic and kidney cell lines. Lipids in Health andDisease 2011 10:144.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitLeon et al. Lipids in Health and Disease 2011, 10:144http://www.lipidworld.com/content/10/1/144Page 7 of 7


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