Evaluation of Amebicidal and Cysticidal Activities of Antifungal Drug Isavuconazonium Sulfate against Acanthamoeba T4 Strains

Acanthamoeba species of amebae are often associated with Acanthamoeba keratitis, a severe corneal infection. Isavuconazonium sulfate is an FDA-approved drug for the treatment of invasive aspergillosis and mucormycosis. This prodrug is metabolized into the active isavuconazole moiety. Isavuconazole was previously identified to have amebicidal and cysticidal activity against Acanthamoeba T4 strains, but the activity of its prodrug, isavuconazonium sulfate, against trophozoites and cysts remains unknown. Since it is not known if isavuconazonium can be metabolized into isavuconazole in the human eye, we evaluated the activities of isavuconazonium sulfate against trophozoites and cysts of three T4 genotype strains of Acanthamoeba. Isavuconazonium displayed amebicidal activity at nanomolar concentrations as low as 1.4 nM and prevented excystation of cysts at concentrations as low as 136 μM. We also investigated the cysticidal activity of isavuconazonium sulfate in combination with a currently used amebicidal drug polyhexamethylene biguanide (PHMB). Although combination of isavuconazonium with PHMB did not elicit an obvious synergistic cysticidal activity, the combination did not cause an antagonistic effect on the cysts of Acanthamoeba T4 strains. Collectively, these findings suggest isavuconazonium retains potency against Acanthamoeba T4 strains and could be adapted for Acanthamoeba keratitis treatment.


Introduction
Acanthamoeba castellanii is a causative agent of Acanthamoeba keratitis (AK). It is a serious infection of the eye that causes inflammation in the cornea and can result in permanent visual impairment or blindness. Acanthamoeba is common in nature and can be found in soil, air and water, including insufficiently chlorinated pools, hot tubs, tap and shower water. In unfavorable environments, the ameboid form of the organism called a 'trophozoite' transforms into a drug-resistant double-walled cyst. Cyst resistance to therapeutic agents, and recurrence of infection due to Acanthamoeba excystment, remain challenges for disease prevention and cures. Infection recurrence occurs in approximately 10% of cases [1], due possibly to excystment. No single drug has yet been shown effective at therapeutic concentrations against both the trophozoite and cyst stages of Acanthamoeba. Current treatment of AK involves an aggressive disinfectant chlorhexidine, in combination with diamidines, polyhexamethylene biguanide (PHMB) and neomycin. Combination therapies have proven more successful than monotherapies [2][3][4]. The most aggressive and severe cases of AK require corneal grafts or surgical removal of the eye [5]. Despite advances in combination therapies and surgery, the resistance of cysts to therapeutic agents poses challenges that are yet to be addressed [6]. Therefore, discovering and identifying therapeutics that are effective against both stages of the parasite would be critical to reducing AK recurrence and improving existing therapies.
Earlier, we identified isavuconazole as amebicidal and cysticidal [7], but clinically the prodrug isavuconazonium sulfate is administered orally or intravenously for the treatment of fungal infections. Isavuconazonium sulfate is metabolized by plasma esterase enzymes, specifically butyrylcholinesterase, into isavuconazole [8]. However, it is unknown if isavuconazonium can be metabolized into isavuconazole in the human eye. As such, we evaluated the activity of isavuconazonium sulfate activity against the trophozoites and cysts of three separate T4 genotype strains of Acanthamoeba.

Determination of Amebicidal Activity
The amebicidal activity of isavuconazonium sulfate was tested against three T4 genotype strains of Acanthamoeba (strains Ma, CDC:V240, and MEEI 0184). The trophozoites were exposed to serial dilutions of isavuconazonium sulfate with final concentrations ranging from 50 µM to 0.006 nM. All three strains of Acanthamoeba trophozoite appeared to be highly susceptible to isavuconazonium sulfate. Isavuconazonium displayed an EC 50 of 0.001 µM against Acanthamoeba strain Ma ( Figure 1A), which was about 1700-to 5000-fold more potent than the current standards of care chlorhexidine and PHMB. The EC 50 of isavuconazonium against Acanthamoeba strain CDC:V240 was 0.037 µM ( Figure 1B), which was about 30-to 300-fold more potent than chlorhexidine and PHMB, respectively. Isavuconazonium exhibited an EC 50 of 0.024 µM against clinical strain MEEI 0184 ( Figure 1C). This EC 50 was about 1.5-fold better than the EC 50 demonstrated against the CDC:V240 strain (Table 1). Overall, these nanomolar potencies demonstrate that isavuconazonium retains its potency against trophozoites of Acanthamoeba T4 strains.

Determination of Cysticidal Activity
Acanthamoeba T4 cysts are also clinically relevant, as they are often more difficult to treat than trophozoites and require higher concentrations of antimicrobial compounds for efficacy. In order to determine the activity of isavuconazonium sulfate against cysts of Acanthamoeba T4 strains, cysts from all three strains were tested against higher concentrations of isavuconazonium sulfate than for evaluating trophozoites. Final concentrations of isavuconazonium ranged from 200 µM to 100 µM in increments of 10 µM, and a treatment was considered to be cysticidal if there was no evidence of trophozoite proliferation or excystation by day 7, which is a commonly used end point for cysticidal assays [9][10][11][12][13].  Different concentrations of isavuconazonium were tested in triplicate for activity against trophozoites of Acanthamoeba T4 strains. The data points represent mean percentage growth inhibition of (A) Ma strain, (B) CDC:V240, and (C) MEEI 0184 of different concentrations of isavuconazonium. EC50 curves were generated from mean values of percentage growth inhibition of isavuconazonium against Acanthamoeba.

Determination of Cysticidal Activity
Acanthamoeba T4 cysts are also clinically relevant, as they are often more difficul treat than trophozoites and require higher concentrations of antimicrobial compounds efficacy. In order to determine the activity of isavuconazonium sulfate against cysts Acanthamoeba T4 strains, cysts from all three strains were tested against higher concen tions of isavuconazonium sulfate than for evaluating trophozoites. Final concentrati of isavuconazonium ranged from 200 μM to 100 μM in increments of 10 μM, and a tre ment was considered to be cysticidal if there was no evidence of trophozoite proliferat or excystation by day 7, which is a commonly used end point for cysticidal assays [9-1 Acanthamoeba T4 cysts were treated with isavuconazonium and allowed to reco in PYG growth media for 7 days and evaluated for cysticidal activity. Isavuconazoni displayed cysticidal activity against all three tested strains, as no excystation was served at day 7 (Figure 2A  Acanthamoeba T4 cysts were treated with isavuconazonium and allowed to recover in PYG growth media for 7 days and evaluated for cysticidal activity. Isavuconazonium displayed cysticidal activity against all three tested strains, as no excystation was observed at day 7 (Figure 2A,D,G). Isavuconazonium exhibited an average minimum cysticidal concentration (MCC) of 167.1 ± 23.6 µM against Acanthamoeba Ma strain. Acanthamoeba strain CDC:V240 had an average MCC of 136.0 ± 11.4 µM. Acanthamoeba strain MEEI 0184 displayed an average MCC of 187.5 ± 5.0 µM.

Effect of Combination of Isavuconazonium and PHMB on Cysts
While isavuconazonium has low nanomolar potency against trophozoites, it appears to display cysticidal activity only at high micromolar concentrations. Since cysts require higher isavuconazonium concentrations to prevent excystation, we wanted to evaluate if isavuconazonium in combination with other currently used drugs can display synergy to reduce the isavuconazonium concentration required to treat cysts.
Isavuconazonium was combined with PHMB and qualitatively assessed for excystation after 7 days of incubation in growth media (Figure 3). Isavuconazonium displayed an MCC of 167.1 µM. Combined with 40.41 µM PHMB, isavuconazonium was able to be lowered to 20.52 µM and still have minimal excystation ( Figure 4A). Monotherapy of 20.52 µM isavuconazonium was confluent with trophozoites by day 7 ( Figure 4B). Monotherapy of 40.41 µM PHMB had minimal to no excystation ( Figure 4C). Since the combination of 20.52 µM of isavuconazonium with 40.41 µM of PHMB elicited a similar effect to what was caused by 40.41 µM of PHMB alone, it is apparent that the combination of two compounds did not have a synergistic effect able to suppress excystation or kill cysts. It was clear that the combination of these two compounds at this concentration did not cause an antagonistic effect on the cysts of Ma strain of Acanthamoeba.

Effect of Combination of Isavuconazonium and PHMB on Cysts
While isavuconazonium has low nanomolar potency against trophozoites, it appears to display cysticidal activity only at high micromolar concentrations. Since cysts require higher isavuconazonium concentrations to prevent excystation, we wanted to evaluate if isavuconazonium in combination with other currently used drugs can display synergy to reduce the isavuconazonium concentration required to treat cysts.
Isavuconazonium was combined with PHMB and qualitatively assessed for excystation after 7 days of incubation in growth media (Figure 3). Isavuconazonium combination of two compounds did not have a synergistic effect able to suppress excystation or kill cysts. It was clear that the combination of these two compounds at this concentration did not cause an antagonistic effect on the cysts of Ma strain of Acanthamoeba.

Discussion
Isavuconazole has previously been evaluated and demonstrated potent amebicidal and cysticidal activity [7]. Since isavuconazole is typically administered as the prodrug isavuconazonium sulfate, there is a possibility that it may not be metabolized in the human eye and that Acanthamoeba T4 strains would not be susceptible to isavuconazole.

Discussion
Isavuconazole has previously been evaluated and demonstrated potent amebicidal and cysticidal activity [7]. Since isavuconazole is typically administered as the prodrug isavuconazonium sulfate, there is a possibility that it may not be metabolized in the human eye and that Acanthamoeba T4 strains would not be susceptible to isavuconazole. Previously, isavuconazonium was identified as effective against Acanthamoeba T4 trophozoites [14], but its effect was not investigated against multiple strains and, more importantly, on the cysts of Acanthamoeba T4 strains. In this work, we evaluated the amebicidal and cysticidal activity of isavuconazonium against multiple strains of Acanthamoeba T4, and also explored the possibility of combining isavuconazonium with PHMB against the cysts of Acanthamoeba.
Isavuconazonium had mean EC 50 values against trophozoites ranging from 0.001 µM (strain Ma) to 0.037 µM (strain CDC:V240) ( Table 1). Our reported values are lower than those reported by Rice et al. against Acanthamoeba strain Ma (EC 50 of 0.09 ± 0.02 µM [14]. This could be due to the differences in experimental conditions. We also previously evaluated the active drug isavuconazole against trophozoites and reported an EC 50 of <0.001 µM (strain CDC:V240), 0.005 µM (strain Ma), and 0.026 µM (strain MEEI 0184) [7]. The EC 50 values of isavuconazonium for strains Ma and MEEI 0184 are comparable to those previously reported values for isavuconazole. Interestingly, the EC 50 of isavuconazonium against CDC:V240 was approximately 40× higher than that of the previously reported isavuconazole EC 50 value. Taken together, this suggests Acanthamoeba T4 trophozoites are still susceptible to the prodrug isavuconazonium.
To our knowledge, this is the first reported evaluation of the cysticidal activity of isavuconazonium sulfate against Acanthamoeba T4 strains. We previously reported that isavuconazole, the active form of isavuconazonium, displayed cysticidal activity against Acanthamoeba Ma at 70 µM [7]. In this work, we determined the MCC of isavuconazonium against various Acanthamoeba T4 strains. The MCC of isavuconazonium varies from 1.9× (CDC:V240) to 2.6× (MEEI 0184) higher than previously reported about isavuconazole [7]. Since isavuconazonium is a prodrug that must be metabolized to active isavuconazole, it is possible that higher concentrations of the prodrug are required as not all of the isavuconazonium is metabolized into isavuconazole.
AK therapies frequently rely on chlorhexidine or PHMB as monotherapy, or in combination with propamidine isethionate and hexamidine. Commonly used combinations include PHMB with propamidine, chlorhexidine with propamidine, chlorhexidine with PHMB, and PHMB with propamidine and neomycin [15]. Since combination therapies were found to be more successful than monotherapies, we investigated the effect of the combination of isavuconazonium and PHMB on cysts of an Acanthamoeba T4 strain. In spite of the challenges associated with the excystation-based cysticidal assay that depends on a "cysticidal-or-not" readout rather than percentage inhibition [16], we identified that the combination of isavuconazonium and PHMB did not cause antagonistic or synergistic cysticidal effects on the cysts of the Ma strain of Acanthamoeba. Future studies will require confirmation of the effect of the combination of isavuconazonium and PHMB on trophozoites and cysts of different strains. Whether isavuconazonium can be combined with other commonly used drugs will require further investigation.
Isavuconazonium sulfate is FDA-approved for the treatment of invasive aspergillosis and mucormycosis [17]. In terms of fungal infections, isavuconazonium sulfate inhibits lanosterol 14α-demethylase [8], which prevents the biosynthesis of ergosterol and results in its eventual depletion. It is available in both oral and intravenous formulations and, following administration, it is rapidly cleaved to the active isavuconazole. The tissue distribution of isavuconazole was evaluated in animals after oral and intravenous administrations of isavuconazonium sulfate, and a low concentration of isavuconazole was detected in the eye lens [17,18]. This low concentration of isavuconazole may not be sufficient to kill Acanthamoeba T4 cysts if the drug is administered orally or intravenously. The preferable route of administration of drugs for the treatment of AK is topical, but the distribution of isavuconazole in the eye has not been evaluated when administered topically. Therefore, it was important to determine the effect of the prodrug isavuconazonium sulfate in case topical administration of prodrug isavuconazonium sulfate does not lead to the formation of active isavuconazole in human eyes. The potent amebicidal and cysticidal activities of the prodrug isavuconazonium sulfate against multiple T4 strains of Acanthamoeba provide confidence that the FDA-approved isavuconazonium sulfate is a promising lead for the treatment of AK.

Determination of Amebicidal Activity
Stock 10 mM isavuconazonium sulfate (Cayman Chemical, Ann Arbor, MI, USA) and 10 mM chlorhexidine were prepared in DMSO. Isavuconazonium sulfate was serially diluted two-fold to generate solutions ranging in concentration from 10 mM to 1.2 nM. Next, 0.5 µL of each of these isavuconazonium sulfate dilutions was added to 96-well white, flat bottom microplates (Greiner Bio-One, Kremsmünster, Austria). This was followed by the addition of 5 × 10 3 trophozoites in 99.5 µL of PYG media to each well, giving final isavuconazonium sulfate concentrations ranging from 50 µM to 5.96 pM. Additionally, 0.5 µL of DMSO was added as a negative control (0.5% (v/v) DMSO), while 0.5 µL of chlorhexidine (50 µM) was added as a positive control. The plates were incubated for 48 h at 28 • C and 5% CO 2 . At 48 h, viability measurements were taken using the CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI, USA) [7]. For the measurements, 25 µL of CellTiter-Glo was added to each well and shaken on an orbital shaker at 360 RPM for 10 min prior to luminescence readings on an EnVision 2104 Multilabel Reader (PerkinElmer, Waltham, MA, USA). Data from a minimum of three independent experiments (biological replicates) conducted in triplicate were analyzed on GraphPad Prism 6 to determine EC 50 values.

Cyst Generation
Encystment of Acanthamoeba T4 strains was induced by culturing trophozoites in an encystation media (95 mM NaCl; 5 mM KCl; 8 mM MgSO 4 ; 0.4 mM CaCl 2 ; 1 mM NaHCO 3 ; 20 mM Tris-HCl, pH 9.0) [19]. The trophozoite harvesting and encystation protocols were conducted as previously described by Shing et al. [7]. Briefly, trophozoites of Acanthamoeba T4 strains were centrifuged at 200× g for 5 min and washed in phosphate-buffered saline (PBS) three times prior to resuspension in encystation media. Then, 5 × 10 3 cells in 99.5 µL were added to each well of a 96 well clear-bottom plate (Corning, Corning, NY, USA). The cells were incubated in encystation media to facilitate the encystation of trophozoites into cysts for 48 h prior to any cysticidal or combination experiments.

Determination of Cysticidal Activity
Cyst plate generation was conducted as previously described in Section 4.3 [7]. After 48 h, 0.5 µL of isavuconazonium sulfate solution was added to a final concentration of 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, or 100 µM. To serve as negative and positive controls, 0.5% (v/v) DMSO and 461.85 µM PHMB were used, respectively. The cysts were incubated for 48 h. Afterwards, the wells were washed four times with 100 µL of PBS before the addition of 100 µL of PYG medium. The cysts were then incubated for one week and imaged by an ImageXpress Micro XLS (Molecular Devices, San Jose, CA, USA) at 200× magnification. The PYG growth media were exchanged for fresh media on day 3 and day 5. The images were manually reviewed for excysted trophozoites, and cysticidal activity was defined as having no trophozoites by day 7. Image brightness and contrast were adjusted