Preclinical Pharmacokinetics and Pharmacodynamic Target of SCY-078, a First-in-Class Orally Active Antifungal Glucan Synthesis Inhibitor, in Murine Models of Disseminated Candidiasis

ABSTRACT SCY-078 (MK-3118) is a novel, semisynthetic derivative of enfumafungin and represents the first compound of the triterpene class of antifungals. SCY-078 exhibits potent inhibition of β-(1,3)-d-glucan synthesis, an essential cell wall component of many pathogenic fungi, including Candida spp. and Aspergillus spp. SCY-078 is currently in phase 2 clinical development for the treatment of invasive fungal diseases. In vitro disposition studies to assess solubility, intestinal permeability, and metabolic stability were predictive of good oral bioavailability. Preclinical pharmacokinetic studies were consistent with once-daily administration to humans. After intravenous delivery, plasma clearance in rodents and dogs was low, representing <15% and <25% of hepatic blood flow, respectively. The terminal elimination-phase half-life was 5.5 to 8.7 h in rodents, and it was ∼9.3 h in dogs. The volume of distribution at steady-state was high (4.7 to 5.3 liters/kg), a finding suggestive of extensive tissue distribution. Exposure of SCY-078 in kidney tissue, a target organ for invasive fungal disease such as candidiasis, exceeded plasma by 20- to 25-fold for the area under the concentration-time curve from 0 h to infinity (AUC0–∞) and Cmax. SCY-078 achieved efficacy endpoints following oral delivery across multiple murine models of disseminated candidiasis. The pharmacokinetic/pharmacodynamic indices Cmax/MIC and AUC/MIC correlated with outcome. Target therapeutic exposure, expressed as the plasma AUC0–24, was comparable across models, with an upper value of 11.2 μg·h/ml (15.4 μM·h); the corresponding mean value for free drug AUC/MIC was ∼0.75. Overall, these results demonstrate that SCY-078 has the oral and intravenous (i.v.) pharmacokinetic properties and potency in murine infection models of disseminated candidiasis to support further investigation as a novel i.v. and oral treatment for invasive fungal diseases.

classes of antifungals (echinocandins [ECHs], azoles, and polyenes) are available, a significant unmet need remains for patients with invasive fungal infections due to the increasing frequency of resistance to these therapies, in particular the emergence of multidrug-resistant strains and the lack of therapeutic options that allow for oral (p.o.) administration. Sadly, the overall mortality rate of invasive candidiasis remains high despite therapy.
SCY-078 (MK-3118) is an investigational antifungal agent currently in clinical development for the treatment of invasive infections caused by Candida and Aspergillus spp. SCY-078 is a novel, first-in-class, semisynthetic derivative of the naturally occurring hemiacetal triterpene glycoside enfumafungin that incorporates a pyridine triazole at position 15 of the core phenanthropyran carboxylic acid ring system and a 2-amino-2,3,3-trimethyl-butyl ether at position 14 ( Fig. 1) to enhance its antifungal potency and pharmacokinetic (PK) properties. In vitro, SCY-078 has demonstrated potent, broadspectrum activity against multiple clinical isolates of Candida spp. (2) and isolates of Aspergillus spp. (3), including those with resistance to azoles or ECHs, the latter containing mutations in the fks gene(s) (4).
Like the ECHs, SCY-078 disrupts fungal cell wall formation by inhibiting ␤-1,3-glucan synthesis. The clinical success of this mechanism of action has established ECHs as first-line treatment for invasive candidiasis, although the poor oral bioavailability of ECHs has restricted their clinical utility to parenteral administration. Despite SCY-078 targeting the same fungal site as other ECHs, it is structurally distinct, and in light of its activity against ECH-resistant strains, thought to interact differently at the enzyme target. Thus, SCY-078 distinguishes itself from ECHs by being a first-in-class glucan synthesis inhibitor (GSI) based on enfumafungin, having activity against ECH-and azole-resistant isolates, and being orally bioavailable. It thereby holds promise as an antifungal agent with broad spectrum of activity against Candida and Aspergillus spp. that can be administered both orally and intravenously (i.v.). To date, single-and multiple-dose phase 1 and 2 clinical studies showed that SCY-078 was well tolerated with oral exposure, PK properties, and a safety profile consistent with achieving clinical efficacy (5).
The preclinical PK properties of SCY-078 have been described to support early PK/pharmacodynamic (PD) studies in murine models of disseminated candidiasis (6,7); however, these studies used a limited number of animals per treatment group and may have only measured exposure after a single oral dose. Here, we present further characterization of SCY-078, including in vitro studies to demonstrate properties consistent with oral bioavailability and single-and multiple-dose PK across preclinical species and disposition studies to assess the impact of protein binding on the volume of distribution (V d ss ) for SCY-078 as a predictor of tissue penetration. Single-dose PK studies were performed in mice, rats, and dogs after oral and i.v. doses to determine PK parameters and bioavailability. In addition, multiple oral-dose studies were conducted in mice to compare the dose-dependent steady-state exposure in plasma and kidney tissue after twice per day (i.e., every 12 h [Q12h]) treatment for 7 days, which reflects dosing regimens used for murine infection models. Lastly, we present preliminary exposure efficacy targets based on free drug the area under the concentration-time curve (AUC)/MIC ratios determined across murine models of disseminated candidiasis.

RESULTS
Antifungal activity in a murine model of disseminated candidiasis. The in vivo activity of SCY-078 was evaluated across three murine models of disseminated candidiasis that either evaluated efficacy after 7 days of twice-daily (BID) oral treatment initiated immediately after infection or identified the PK/PD measures associated with a stasis endpoint after either a single dose or the same dose fractionated as half or quarter doses administered 16 h after infection.
Immediate treatment model. The in vivo efficacy of SCY-078 was evaluated based on a murine model of disseminated candidiasis utilized previously for the characterization of caspofungin (8). SCY-078 demonstrated potent and reproducible activity across four independent studies following BID oral treatment initiated on the day of C. albicans infection with MY1055 (MIC, 0.03 g/ml). Initial studies demonstrated 100% clearance in kidney fungal burden with SCY-078 doses of Ն12.5 mg/kg. In contrast, treatment with FLU up to 5 mg/kg administered p.o. did not result in clearance of fungal infection in any of the animals tested. Caspofungin was effective in clearing kidney fungal burden at doses of Ն0.125 mg/kg; however, it was administered intraperitoneally because it is not orally bioavailable (data not shown).
Pharmacokinetic analysis was performed in three studies, and the efficacious exposure was defined as that affording complete clearance of measurable infection in Ͼ50% of treated animals and a Ͼ4-log reduction in CFU in kidney tissue compared to the sham-treated animals ( Table 1). The plasma exposure of SCY-078 associated with efficacy expressed as the interstudy mean AUC from 0 to 24 h (AUC 0 -24 ) Ϯ the standard deviation (SD) was 15.4 Ϯ 2.21 M·h (11.2 Ϯ 1.61 g/ml·h).
Single delayed-dose treatment model. Single-dose SCY-078 treatment was assessed in a more delayed treatment infection model of disseminated candidiasis where infection with three C. albicans strains (MY1055 at 2.72 ϫ 10 4 CFU/mouse, CLY724 at 1.71 ϫ 10 4 CFU/mouse, or CLY18600 at 4.62 ϫ 10 4 CFU/mouse) was allowed to Caspofungin and fluconazole were not evaluated in this model. Fractionated delayed-dose treatment model. The fractionated delayed treatment model was used to compare the efficacy of SCY-078 when administered as divided doses of either two half doses or four quarter doses, relative to the outcome of a single dose. The total doses were 12.5, 25, and 50 mg/kg delivered by bolus intraperitoneal injection. Divided doses were administered at either 0 and 48 h or at 0, 24, 48, and 72 h relative to the single dose. Over the range of doses studied the change in CFU/g kidney tissue correlated to C max /MIC and AUC 0 -96 /MIC, with R 2 values of 0.91 and 0.91, respectively (Fig. 2). For all groups, the plasma concentrations were greater than the MIC throughout the treatment period. In all total dose levels the single dose was slightly more efficacious than the divided doses, suggesting that the C max is associated with outcome, although the differences did not achieve statistical significance except for the quarter doses of either 6.25 or 3.125 mg/kg relative to the single doses of 25  Overall, SCY-078 demonstrated potent and reproducible activity in each of the murine models of disseminated candidiasis with C. albicans. Therapeutic exposure expressed as plasma AUC 0 -24 , was comparable across the tested murine models, i.e., 15.4 Ϯ 2.2, 14.1, and 13.41 M·h in the immediate-treatment, delayed-single-dose, and delayed-fractionated-dose models, respectively. The mean therapeutic exposure across all models was 14.3 M·h.
In vitro characterization of SCY-078 for oral administration. (i) In vitro permeability and metabolic stability of SCY-078. Caco-2 cell monolayers were used to assess the in vitro absorptive permeability of SCY-078 as a predictor of absorption across the gut following oral delivery. The mean apparent permeability for 5 M SCY-078 in the apical-to-basolateral direction was 8.9 Ϯ 0.78 ϫ10 Ϫ6 cm/s (89 Ϯ 7.8 nm/s), indicating good oral absorption since in this assay permeability values of Ͼ10 Ϫ6 cm/s are considered predictive of good absorption after oral administration (9).
The metabolic stability of SCY-078 was assessed after incubation with mouse, rat, dog, and human liver microsomes ( Table 2) (10). The values for in vitro intrinsic clearance (CL int ) in rodents (mouse and rat), dog, and human liver microsomes were Յ11, Յ48, and 34 l/min/mg, respectively, indicating that SCY-078 has low clearance with rodent microsomes and low to moderate clearance in dogs and humans. The corresponding values for in vitro intrinsic clearance scaled to in vivo intrinsic clearance (CL= int ) in rodent, dog, and human liver microsomes were Ͻ40, Յ69, and 38 l/min/kg, respectively. The half-lives in rodent, dog, and human liver microsomes were Ն125, Ն29, and Ն42 min, respectively. Overall, these data indicate that SCY-078 is most stable in rodent microsomes and most extensively metabolized by dog microsomes. The CL int and half-life values for the control compounds 7-ethoxycoumarin, propranolol, and verapamil were consistent with previous findings in our laboratory and demonstrated the metabolic competency of the microsomes. Changes in the log CFU/g kidney tissue were determined for five animals/group and are expressed relative to animals receiving sham treatment. The lines fitted through the data represent the best fit over the concentrations and doses studied. R 2 is the coefficient of determination. Control (untreated) and sham-treated animals achieved either a mean 4.18 or 5.95 log 10 increase in CFU/g of kidney tissue during the study. (ii) In vitro solubility. The solubility of SCY-078 was inversely related to pH consistent with pKa values of 9.0 (primary amine), 5.5 (carboxylic acid), and 2.4 (pyridine). At 24 h, amorphous SCY-078 free-base achieved good solubility in simulated gastric fluid (SGF; Ͼ5.2 mg/ml) and fed-state intestinal fluid (FeSSIF; Ͼ3.0 mg/ml) but was sparingly soluble in fasted-state simulated intestinal fluid (FaSSIF). Solubility improved with the citrate salt form used for i.v. and oral PK studies. SCY-078 salt achieved Ͼ20 mg/ml solubility in SGF and FeSSIF at 24 h and Ͼ4.2 mg/ml solubility at 24 h in FaSSIF.
In vitro plasma protein binding and blood distribution. In vitro reversible protein binding and blood/plasma partition ratio were determined for SCY-078 in mouse, rat, dog, and human plasma and whole blood ( Table 3). The extent of protein binding was high, as anticipated for a lipophilic compound (logD ϳ 6.4 at pH 7.3 and 4.90 at PI). The unbound fraction of SCY-078 in plasma determined by means of equilibrium dialysis was 0.2 to 0.5% (99.8 to 99.5% bound) and appeared to be independent of concentration over the range studied (0.1 to 10 M). Within the limitations of the assay method, binding perhaps appeared modestly higher in rodent plasma than in dog or human plasma; however, the study was not powered to identify differences between species. The mean blood/plasma partition ratio across species was 0.64 Ϯ 0.079 over the same concentration range, indicating that SCY-078 associates primarily with the plasma compartment of whole blood.
Pharmacokinetics after intravenous and oral administration to rodents and dogs. Summary pharmacokinetic parameters determined in plasma after administration of SCY-078 to rodents and dogs by i.v. and oral routes are presented in Tables 4  and 5 Table 4). Plasma concentrations declined in a linear manner in all three species (Fig. 3).
A dose normalized AUC 0 -∞ to a 1-mg/kg dose was 1.4 g/ml·h in mice and ϳ2-fold higher in rats and dogs (ϳ2.4 g/ml·h). SCY-078 was well absorbed into plasma after oral administration (Table 5). After single oral doses associated with efficacy in murine models of invasive candidiasis, absolute bioavailability values in mice, rats, and dogs were approximately Ͼ51, 45, and 35%, respectively; the corresponding half-life values were approximately 8.3, 9.1, and 15.2 h, respectively. Maximum plasma concentrations were generally reached between 4 and 6 h after dosing across all species, likely reflecting an extended absorption phase from the formulation used in these studies. The bioavailability for mice represents an underestimate because the AUC could only be measured to 12 h rather than to infinity because the animals received a second dose immediately after collection of the 12-h sample. The dose normalized AUC 0 -12 across species was between 0.5 and 0.8 g/ ml·h/mg/kg dose (exposure normalized to a 1-mg/kg dose), although variability was observed among dogs.
Comparison of exposures after a single oral dose and 7 days of twice-per-day (Q12h) oral treatment to mice revealed an ϳ2-fold increase in C max and AUC. The mean values for C max and AUC 0 -12 increased from 0.39 Ϯ 0.13 to 0.64 Ϯ 0.12 g/ml and from 3.11  Biological distribution to kidney tissue after oral delivery to mice. The biological distribution of SCY-078 between plasma and kidney tissue was examined in mice at steady state after oral dose 13 of either 6.25 or 12 mg/kg on day 7 of BID treatment ( Table 6). Within each treatment group, kidney exposures, based on the AUC 0 -∞ and C max values, were 20-to 25-fold greater than the corresponding parameters for plasma, indicating marked distribution into kidney tissue. The elimination half-lives determined from plasma and kidney tissue were similar (8.3 to 8.9 h, respectively), although the enhanced distribution into kidney tissues would have allowed concentrations to remain measurable to approximately 100 and 115 h for the 6.25-and 12-mg/kg doses, respectively, compared to approximately 50 and 60 h postdose, respectively, in plasma (Fig. 4). The kidney tissue distribution at doses of Ͼ12.5 mg/kg BID suggested at least equivalent portioning; however, samples obtained beyond 60 h postdose would be required to accurately determine the AUC in renal tissue. The kidney concentrations were measured as g/g of tissue homogenate; the density of the kidney homogenate was assumed to be 1 g/ml. C 12 , C at 12 h. b The PK parameters were calculated from a single composite mean profile for each dose group. The AUC 0 -24 was measured after dose 13 on day 7 of BID oral treatment.

DISCUSSION
SCY-078 (formerly MK-3118, Fig. 1) is a derivative of enfumafungin and represents the first compound of the triterpene class of ␤-1,3-glucan synthesis inhibitors to reach clinical development for the treatment of invasive candidiasis. The experiments reported here demonstrate good oral bioavailability of SCY-078 across multiple species and preclinical pharmacokinetic properties consistent with once-per-day oral dosing in humans, together with a high volume of distribution suggestive of excellent tissue distribution. In murine models of disseminated candidiasis, SCY-078 demonstrated consistent anti-Candida activity following oral administration, and the preclinical exposure targets and PK/PD measures associated with achieving efficacy endpoints have been defined.
The inhibition of ␤-(1,3)-D-glucan synthesis is a proven antifungal target in the treatment of pathogenic fungi, including Aspergillus and Candida spp., and is the mechanism of action for the ECH class of antifungal agents (caspofungin, micafungin, and anidulafungin) now used as first-line treatments for invasive candidiasis (1).
Unlike SCY-078, echinocandins are high-molecular-mass cyclic lipopeptides (ϳ1,200 kDa) and not orally bioavailable. Thus, they are restricted to parenteral delivery, thereby limiting their use in clinical settings where oral treatment would be desirable or as a potential oral step-down agent after initial i.v. therapy. Although the ECHs continue to be generally very effective in vitro against Candida and Aspergillus spp., resistance has been on the rise for Candida infections, particularly in C. glabrata (9). SCY-078 retains in vitro activity against many ECH-resistant isolates of Candida that contain mutations in the fks gene(s) (4); this suggests that SCY-078 shows promise as an oral and i.v. antifungal agent with broad-spectrum anti-Candida activity.
Enfumafungin was identified as having antifungal activity during a natural product screening campaign (11), and SCY-078 was selected for development following a lead optimization program to improve potency, breadth of activity, and drug-like properties including oral bioavailability (12)(13)(14). It is well established that oral bioavailability reflects contributions from multiple processes, including solubility at physiologically relevant pHs in the stomach and intestinal lumen, absorptive permeability, and firstpass metabolism.
SCY-078 exhibited pH-dependent solubility, achieving the highest concentrations in acidic media consistent with simulated gastric fluid (solubility in SGF Ͼ 20 mg/ml) and intestinal fluids (solubility in FeSSIF, Ͼ20 mg/ml; solubility in FaSSIF, Ͼ4.2 mg/ml). In vitro SCY-078 is readily permeable through Caco-2 monolayers with an apparent permeability coefficient (P app ) of approximately 8.9 ϫ 10 Ϫ6 Ϯ 0.78 cm/s (89 Ϯ 7.8 nm/s), where values of Ͼ10 Ϫ6 cm/s are consistent with drugs that are well absorbed across the intestinal epithelium (9). Incubations performed with liver microsomes demonstrated that SCY-078 undergoes only moderate turnover with ca. 85% remaining (t 0.5 , Ն135 min) after 30 min with rodent microsomes and approximately 50% remaining (t 0.5 , Ն29 min) with dog or human microsomes. The corresponding values for intrinsic clearance are Յ10 and Ͻ48 l/min/mg for rodents and larger species, respectively. Scaling intrinsic clearance to hepatic clearance based on liver blood flow indicates low clearance in rodents and moderate clearance in dogs and humans (15). These solubility, permeability, and metabolic stability data are consistent with SCY-078 being well absorbed from the intestine and able to achieve clinically relevant exposures following oral delivery.
As predicted by in vitro data, i.v. delivery of SCY-078 showed a low level of clearance in rodents representing approximately 10 to 15% of hepatic blood flow (16). In dogs, clearance was modestly higher; although still low, representing approximately 25% of the hepatic blood flow (16). Estimates for the terminal elimination-phase half-life ranged from 5.5 to 8.7 h in rodents to ϳ9.3 h in dogs. These values for low clearance and long half-life in preclinical species are consistent with once daily dosing to humans. Interestingly, values for V d ss (4.7 to 5.3 liters/kg) were ϳ10-fold body water in all species, an observation suggestive of extensive distribution into compartments other than plasma. This was somewhat unanticipated because the binding of SCY-078 to plasma proteins measured by means of equilibrium dialysis was 99.5 to 99.8%, and extensive, high-affinity binding to plasma proteins is usually associated with low values for V d ss since the drug is restrictively retained within the vasculature. Consequently, these data indicate that although SCY-078 is bound extensively to plasma proteins, the binding affinity may be weak, thereby allowing distribution to tissues. In vitro blood distribution studies indicated that SCY-078 is largely associated with the plasma compartment of whole blood in all species studied (blood/plasma ratio, 0.5 to 0.7), which is desirable because erythrocytes are not a pharmacologically relevant compartment for invasive candidiasis. Moreover, limited association with erythrocytes suggests that the high values for V d ss are predictive of excellent distribution into tissues, which is desirable for treating invasive disease.
SCY-078 was well absorbed, demonstrating good oral bioavailable in mice (Ͼ51%), rats (45%), and dogs (35%). Estimates for the terminal elimination-phase half-lives ranged from 7.8 to 9.1 h in rodents to 15 h in dogs and were consistent with values following i.v. administration. At the doses studied, the values for C max were ϳ1 g/ml after a single dose and generally observed at 4 to 6 h postdose. In mice, the accumulation factors for C max and AUC 0 -∞ after 7 days of twice-daily dosing on a Q12h regimen were ϳ2-fold. The group mean SCY-078 concentrations were similar in plasma samples collected predose and 4 h postdose following dose 7 on day 4 relative to dose 13 on day 7, indicating that steady-state exposure had been achieved by day 4 of treatment. Moreover, comparable exposures on days 4 and 7 suggests that SCY-078 does not induce drug-metabolizing enzymes responsible for its metabolism.
Efficacy in murine candidiasis models has been associated with the distribution and persistence of antifungals in the key tissues associated with invasive mycoses (kidney, lung, liver, and spleen) (17)(18)(19). Prolonged exposure in kidney beyond when measurable levels of drug have been cleared from plasma has also been attributed to the postantifungal effect, where extended protection is observed against infectious challenge posttreatment (19)(20)(21). At steady state, after 7 days of BID oral treatment, the exposure of SCY-078 in kidney tissue exceeded plasma by approximately 20-to 25-fold for AUC 0 -∞ and C max ( Table 6). In comparison, after i.v. delivery to rodents, the values for the kidney/plasma ratios for the ECHs were approximately 2.9 for caspofungin (22) or 2.8 based on the total radioactivity over 24 h (23), 10.7 for anidulafungin (17), and 3.4 for micafungin (24). Comparison of available values for V d ss between SCY-078 and the ECHs indicates a correlation with drug distribution into kidney tissue. For SCY-078 the V d ss values ranged from 4.7 to 5.3 liters/kg, while for the ECHs the V d ss values ranged from 0.5 to 2.16 liters/kg for anidulafungin, 0.14 to 0.67 liters/kg for caspofungin, and 0.21 to 0.56 liters/kg for micafungin (Table 7). Distribution into erythrocytes likely contributes to the higher V d ss value for anidulafungin compared to the other ECHs (17).
The PK/PD parameters C max /MIC and AUC/MIC were found to best correlate with outcome following a comparison of single and fractionated doses in the current efficacy studies and in those performed by Andes et al. (34) and Lepak et al. (6) for GSIs in a murine neutropenic model of disseminated candidiasis. This is consistent with a Source references for various data are indicated within parentheses. Preclinical data were also summarized by Gumbo et al. (33). NA, not applicable. b Calculated using previously published data (17).
general observations for ECH GSIs where, although C max /MIC does correlate with efficacy, the AUC/MIC is considered a more predictive PK/PD measure, perhaps because it correlates more closely to the more consistent exposure achieved in key tissues at steady state (19,35). Four independent studies have been performed in the immediate-treatment model, and SCY-078 met or exceeded efficacy targets in each of the following 7 days of BID treatment with oral doses of either 6.25 or 12.5 mg/kg of SCY-078 in the "fit-for-purpose" formulation. The mean plasma SCY-078 steady-state AUC 0 -24 required for efficacy Ϯ the SD, across the three studies where PK was measured, was 15.4 Ϯ 2.21 M·h (11.2 Ϯ 1.61 g/ml·h), and the corresponding total drug AUC/MIC ratio was 373 Ϯ 54. PD studies with the ECH GSIs (34) have indicated that the free-drug AUC/MIC (fAUC/MIC) may be a more reliable predictor of efficacy when comparing therapeutic targets for a given isolate across compounds or when making predictions between species (36)(37)(38). The total AUC/MIC ratio of SCY-078 with MY1055 (in vitro MIC, 0.03 g/ml) is equivalent to a free-drug (f) AUC 0 -24 of 0.0224 Ϯ 0.0032 g/ml·h (f b [fraction bound], 99.8% in mouse plasma) and corresponds to a target fAUC 0 -24 /MIC ratio in plasma of ϳ0.75 Ϯ 0.107. Recently Lepak et al. (6) evaluated the efficacy of SCY-078 in a murine neutropenic invasive candidiasis model following infections with four C. albicans isolates, four C. glabrata isolates, and three C. parapsilosis isolates, covering an in vitro MIC range of 0.03 to 0.25 g/ml. SCY-078 afforded potent activity in vivo against each isolate, and the mean fAUC/MIC ratio for stasis was 0.70 Ϯ 0.54 (median, 0.60; range, 0.10 to 1.71), which is generally consistent with the ratio determined in the present study for MY1055 (0.75 Ϯ 0.107). Interestingly, a C. glabrata isolate (CG 5592) and a C. parapsilosis isolate (CP 20519.069) with the highest MIC values (0.25 g/ml) required the lowest fAUC/MIC ratios (0.11 and 0.10, respectively) to meet the stasis endpoints, indicating some degree of variability in the fAUC/MIC target between isolates. The total drug AUC 0 -24 target for these two isolates was ϳ18.8 M·h. In a comparison to ECHs that target the same fungal enzyme as SCY-078, the therapeutic efficacy, based on a stasis endpoint, was typically observed when the fAUC/MIC values were 11 to 28 for anidulafungin, 3 to 22 for caspofungin, or 4 to 13 for micafungin (34). When tested by Lepak et al. (6), the range in fAUC/MIC values for SCY-078 against a representative subset of the same panel of isolates (n ϭ 11 of 30 isolates) was 0.1 to 1.7 (6). Although further work is required to elucidate the mechanistic basis for the lower fAUC/MIC value with SCY-078, the current data suggest that the fAUC/MIC measured in plasma is lower because of enhanced distribution into tissues, as indicated by the markedly higher V d ss and the higher kidney/plasma exposures determined for SCY-078 relative to the echinocandins.
Overall, these studies have identified key attributes that may result in a clinical benefit when SCY-078 is used in the treatment of Candida infection, including low to moderate clearance, oral bioavailability, and tolerability across preclinical species. It demonstrates a high volume of distribution, indicating extensive tissue penetration, as shown by the kidney exposure, and met efficacy endpoints across multiple murine models of invasive candidiasis at concentrations that have been safely achieved after oral administration in humans. Collectively, these data support the further clinical development of SCY-078 for the treatment of infections by Candida spp.

MATERIALS AND METHODS
Chemicals. SCY-078 as a soluble monocitrate salt was prepared by SCYNEXIS (Jersey City, NJ) and was characterized by 1 H nuclear magnetic resonance and liquid chromatography-mass spectrometry (LC-MS) to demonstrate Ͼ99% purity. SCY-078 used for "fit for purpose" formulations were produced by Merck to purity Ͼ97%. Control plasma and tissues for bioanalysis were obtained either from satellite (untreated) animals during the murine efficacy studies or purchased as control tissues from Bioreclamation, Inc. (Westbury, NY).
Murine models of disseminated candidiasis. The in vivo activity of SCY-078 was evaluated using two murine models of disseminated candidiasis to establish the pharmacokinetic exposure target and PK/PD measures associated with efficacy.
In the first model, the target therapeutic exposure was established across four independent experiments, each employing 7 days of twice-daily (BID) oral treatment initiated shortly after infection. To establish the PK/PD parameters associated with the outcome, the efficacy of SCY-078 was compared after single or fractionated doses starting 16 h after the infectious challenge.
A disseminated Candida infection was induced in C=5-deficient DBA/2N mice (Taconic Farms, Germantown, NY), weighing on average 20 g, by i.v. inoculation with C. albicans MY1055 (Merck Culture Collection). C. albicans MY1055 was cultured on Sabouraud dextrose agar (SDA; BBL, Cockeysville, MD) plates at 35°C for 24 h. Yeast cells were washed from the surface of agar plates into sterile saline, and the cell concentrations were quantitated by using a hemocytometer. Viable cell counts were confirmed by a serial 10-fold dilution of the cell suspension and plating on SDA plates. Plates were incubated for 24 to 48 h at 35°C, whereupon the numbers of CFU were determined. The in vitro activity of SCY-078 against the MY1055 isolate was evaluated using broth microdilution assays as described in CLSI M27-A3 (39). The MIC endpoints were based on 50% inhibition of fungal growth at 24 h.
For infection, 0.2 ml of a blastospore suspension containing between 2.44 ϫ 10 4 and 3.56 ϫ 10 4 CFU of C. albicans MY1055 was inoculated into the lateral tail vein. Mice were housed in groups of up to 10 animals in sterile microisolator cages with sterile bedding. Water and food were provided ad lib. The infected and nonmedicated (sham-treated control) animals (n ϭ 20) received vehicle only. Treatment groups comprised five animals each, with an additional three animals included for PK analysis for SCY-078. Blood samples were collected from infected satellite PK mice at typically 0, 0.25, 0.5, 1, 2, 4, 6, and 24 h after dose 13 on day 7. Kidneys from five mice were aseptically removed from each treatment group at day 7 after infectious challenge, unless otherwise indicated.
Therapy with SCY-078, caspofungin or fluconazole was initiated within 15 to 30 min after challenge. Mice were treated with SCY-078 with BID p.o. doses of 6.25, 12.5, or 25 mg/kg administered in a "fit-for-purpose" formulation. Caspofungin was administered twice daily via the intraperitoneal route at doses of 0.0078, 0.03, 0.125, and 0.5 mg/kg. Fluconazole was administered p.o. BID at doses of 0.078, 0.31, 1.25, and 5.0 mg/kg (39). At day 7 after challenge, the mice (n ϭ 5/group) were euthanized, and both kidneys were aseptically removed, placed in sterile Whirl-Pak bags (Fisher Scientific, Fairlawn, NJ), weighed, and homogenized in 5 ml of sterile physiological saline. Kidney homogenates were serially 10-fold diluted in sterile saline and plated on SDA. Plates were incubated at 35°C and counted after 30 to 48 h of incubation. The CFU/g of kidney were determined, and counts from treatment groups were compared to counts from sham-treated controls using a paired two-tailed t test (Microsoft Excel). The percent clearance was determined as the number of mice with no detectable yeast, with a limit of detection of 50 yeast cells per pair of kidneys because of the dilution scheme. For data from individual mice where no detectable yeast were recovered from paired kidneys, 9.8 was entered into a Microsoft Excel spreadsheet formula [log 10 (5 ϫ raw count)/paired kidney weight)] so that the counts would be one less than the limit of detection or 49 cells per pair of kidneys.
Pharmacokinetic analysis was performed on samples collected from satellite-infected mice (n ϭ 3/group) after SCY-078 dose 13 on treatment day 7. Tail bleeds were obtained (20 l collected into 60 l of 0.1 M sodium citrate) at time points from 15 min to 6 h, concluding with a terminal bleed at 24 h. Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after protein precipitation. Plasma exposure for SCY-078 was calculated from the in vitro plasma/whole-blood distribution ratio (see below).
Efficacy was determined in a delayed treatment model based on the protocol described above using typically 5 mice per treatment group. For the single delayed-dose treatment model, a single dose of SCY-078 was administered 16 h after infection. The target endpoint for efficacy in this model was a static effect on the tissue burden measured at 96 h posttreatment. Caspofungin and fluconazole were not evaluated in this model. In the fractionated delayed-dose treatment model, SCY-078 was administered as divided doses of either two half doses or four quarter doses relative to the single dose. The total doses were 12.5, 25, and 50 mg/kg administered as a suspension in a "fit-for-purpose" formulation. Divided doses were administered at either 0 or 48 h or at 0, 24, 48, and 72 h relative to the single dose for the half and quarter doses, respectively. Caspofungin and fluconazole were not evaluated in this model.
In vitro permeability through Caco2 cell monolayers. Caco2 cells (ATCC CRL-2102) were cultured in Dulbecco modified Eagle medium with the dipeptide form of L-glutamine (GlutaMAX), 10% (vol/vol) fetal bovine serum, and 1% (vol/vol) penicillin-streptomycin at 10,000 U/ml in a 75-ml flask at 37°C in a humidified atmosphere of 5% CO 2 . Near-confluent Caco-2 cell cultures were harvested by trypsinization with 0.25% trypsin at 37°C for 5 min and resuspended in culture medium. The cells were seeded onto semipermeable filter inserts (catalog no. 3401; Corning, Corning, NY) at a density of approximately 200,000 cells/cm 2 . The cell culture medium was changed every 2 to 3 days over a total of 21 days of culture. On the day of the assay, the cell monolayers were rinsed with transport medium (Hanks balanced salt solution with 25 mM glucose and 25 mM HEPES [MediaTech Corning, Tewksbury, MA]), and the absorptive permeation of SCY-078 was evaluated by measuring the flux from the apical to the basolateral compartments. Cell monolayers were incubated with SCY-078 (5 M) in triplicate for 2 h at 37°C. Samples were removed from the apical and basolateral compartments after incubation and assayed for test compound concentrations by LC-MS/MS. The apparent permeability coefficient (P app ; cm/s) was calculated as follows: P app ϭ 1/A·C 0 (dQ/dt), where dQ/dt is the rate of drug appearance in the basolateral compartment (mol/s), C 0 is the initial drug concentration in the donor compartment (M), and A is the surface area of the monolayer (cm 2 ). The results were expressed as the mean Ϯ the SD from triplicate samples (n ϭ 3).
Solubility. The solubility of SCY-078 was measured in simulated gastric fluid (SGF), fasted-state simulated intestinal fluid (FaSSIF), and fed-state intestinal fluid (FeSSIF) by Crystal Pharmatech (North Brunswick, NJ). In general, approximately 15 mg of solid was weighed into a 4-ml vial, 3.0 ml of medium was added, and the suspensions were stirred on a rolling incubator (25 rpm) at an ambient room temperature for 24 h. After incubation, 0.5 ml of suspension was centrifuged and filtered (0.45-m pore