Fosmanogepix (APX001) Is Effective in the Treatment of Pulmonary Murine Mucormycosis Due to Rhizopus arrhizus

Mucormycosis is a life-threatening infection with high mortality that occurs predominantly in immunocompromised patients. Manogepix (MGX) is a novel antifungal that targets Gwt1, a protein involved in an early step in the conserved glycosylphosphotidyl inositol (GPI) posttranslational modification pathway of surface proteins in eukaryotic cells. Inhibition of fungal inositol acylation by MGX results in pleiotropic effects, including inhibition of maturation of GPI-anchored proteins necessary for growth and virulence.

Glycosylphosphatidylinositol (GPI)-anchored proteins are known to have several functions, including mediating adhesion to and invasion of host tissues by microorganisms (7,8). Therefore, they play a pivotal role in pathogenesis of infectious diseases. Fosmanogepix (formerly APX001 and E1210) is the prodrug of manogepix (MGX, formerly APX001A and E1210), a broad-spectrum investigational antifungal agent that inhibits inositol acyltransferase, thereby preventing GPI-anchored protein maturation (9). This inhibition results in pleiotropic effects on fungal growth and virulence.
Previous studies have shown that MGX has broad in vitro activity against Candida spp., Aspergillus spp., and some rare molds (9)(10)(11). The MGX MEC values against limited numbers of Mucorales strains were more moderate and variable, with reported ranges of 0.12 to 16 g/ml against Rhizopus arrhizus; 2 to 16 g/ml against R. microsporus; 1 to 16 g/ml against Rhizomucor pusillus; and 0.5 to 8 g/ml against Mucor circinelloides using CLSI methodology (9,12). These MEC values are similar to the ranges of MIC values seen for isavuconazole (ISA) in a study of worldwide isolates from 2015 to 2016 conducted by two reference laboratories (13). In that study, a wide range in MIC values was observed as follows: 0.25 to 4 g/ml against R. arrhizus; 0.5 to 32 g/ml against R. microsporus; 0.5 to 8 g/ml against Rhizomucor pusillus; and 2 to 32 g/ml against M. circinelloides (13).
The correlation between MIC or MEC values and outcome in clinical studies has not been well established, especially for values Ͼ16 g/ml (14). However, correlations in MIC (Candida) or MEC (Aspergillus) values have generally been observed in pharmacokinetic/pharmacodynamic (PK/PD) mouse studies that evaluated isolates with MGX MIC or MEC values below 0.125 g/ml (15). Thus, we sought to assess the activity of fosmanogepix in neutropenic murine invasive pulmonary mucormycosis (16) models using two strains of Rhizopus, the most common cause of mucormycosis, in which the MEC values of MGX were low (0.25 g/ml for R. arrhizus var. delemar) and high (4.0 g/ml for R. arrhizus var. arrhizus).
We utilized fosmanogepix treatment regimens in mice that resulted in MGX exposures (the area under the plasma drug concentration-time curve [AUC]) similar to what was observed in phase 1 clinical studies (15,17,18). This was accomplished by preadministration of 50 mg/kg 1-aminobenzotriazole (ABT), a nonselective suicide inhibitor of cytochrome P450 (CYP) enzymes (19). We have previously shown that ABT administered 2 h prior to fosmanogepix enhanced MGX exposures 16-to 18-fold and enhanced serum half-life from ϳ1 to 9 h, more closely mimicking human pharmacokinetic values (2 to 2.5 days) observed in phase 1 clinical studies in healthy volunteers (15,17,18). As a comparator in the efficacy studies, we orally administered the prodrug isavuconazonium sulfate (ISA) using a dosing regimen that resulted in isavuconazole exposures in mice similar to what has been observed in human clinical studies (20,21). ISA was utilized as the comparator since it was approved by the FDA in 2015 for the treatment of invasive aspergillosis and invasive mucormycosis (CRESEMBA [isavuconazonium sulfate]). In addition, ISA was approved by the European Medicines Agency (EMA) for treating patients with mucormycosis who cannot be treated with amphotericin B. The efficacy endpoints in the mouse models were survival and reduction in fungal burden in target organs.

RESULTS
Antifungal susceptibility. The microbiological activities of MGX, posaconazole (POSA, another antifungal drug used to treat mucormycosis), and ISA were evaluated against 17 and 19 clinical isolates of R. arrhizus var. arrhizus and R. arrhizus var. delemar, respectively. MGX MEC interpretive criteria for molds were as described for the echinocandins and MIC values were determined for the comparators ISA and POSA (22). R. arrhizus var. delemar strains were more susceptible to MGX than R. arrhizus var. arrhizus, with MEC geometric means (GM) of 0.75 g/ml and 3.84 g/ml, respectively (Table 1). In contrast, R. arrhizus var. arrhizus isolates were more sensitive to ISA (GM ϭ 0.85 g/ ml) than R. arrhizus var. delemar strains (GM ϭ 2.5 g/ml). Both sets of clinical isolates demonstrated similar susceptibility to POSA with GMs of 0. 15  Fosmanogepix demonstrates efficacy in a highly immunosuppressed mouse model of pulmonary mucormycosis caused by R. arrhizus var. delemar. In order to assess fosmanogepix in our established model of neutropenic murine mucormycosis, ICR mice were immunosuppressed with cyclophosphamide (200 mg/kg) and cortisone acetate (500 mg/kg) on days Ϫ2, ϩ3, and ϩ8, relative to intratracheal infection with 2.5 ϫ 10 5 cells of R. arrhizus var. delemar 99-880 on day 0 (17). Treatment with placebo (diluent control), fosmanogepix (52, 78, or 104 mg/kg, once daily orally [p.o.]), or ISA (110 mg/kg three times a day [TID], p.o.) began 16 h postinfection and continued for 7 days. To extend the half-life of MGX in mice, 50 mg/kg of the cytochrome P450 inhibitor 1-aminobenzotriazole (ABT) was administered 2 h prior to fosmanogepix administration, as previously described (23). Control mice did not receive ABT because ABT has no in vitro activity against Rhizopus strains and we previously demonstrated no difference in survival of mice infected with pulmonary invasive aspergillosis and treated with or without ABT (24). ISA was dosed at 110 mg/kg p.o. TID to achieve an exposure in mice that is equivalent to clinical exposures achieved in humans (20,21). Similarly, ABT plus fosmanogepix (78 mg/kg and 104 mg/kg once daily) achieved exposures in mice that approximate exposures achieved in clinical doses in phase 1 trials (17,18), whereas the 52 mg/kg dose achieved a lower exposure.
(i) Survival. Mice survival was assessed over 21 days (n ϭ 10 mice/cohort). Doses of fosmanogepix in mice that achieved clinically relevant exposures (78 mg/kg and 104 mg/kg) demonstrated significantly improved survival compared to the placebo control (P Ͻ 0.01 by log rank test), whereas the low dose of 52 mg/kg fosmanogepix, although numerically better than placebo control, did not achieve significance (P ϭ 0.07) (Fig. 1A). As we previously reported (20), a clinically relevant exposure of ISA in mice also improved survival compared to placebo (P ϭ 0.001), but was not significantly different from any of the three fosmanogepix dosing groups (P Ͼ 0.25) (Fig. 1A). The two highest dosing groups of fosmanogepix resulted in 40% and 50% survival at day 21 postinoculation, respectively, similar to ISA (40% survival at this time point) (Fig.  1A). Furthermore, fosmanogepix demonstrated a dose-dependent prolongation in median survival time of mice (9, 13, and 21 days) versus placebo (6 days). The median survival time for ISA was 13 days (Fig. 1B). (ii) Tissue fungal burden. Mice were immunosuppressed and infected as described above; however, mice (n ϭ 10/group) were sacrificed at day ϩ4 after infection to determine conidial equivalents (CE)/g of lung and brain tissue by quantitative PCR (qPCR) using 18S primers. In the placebo group, fungal burdens (CE) at day ϩ4 were log 10 4.74 Ϯ 0.96 (lung) and log 10 3.48 Ϯ 0.67 (brain) (Fig. 2). For the 78 mg/kg and 104 mg/kg fosmanogepix dosing groups, a 1.3 and 1.97 log 10 reduction in CE/g of lung tissue was observed, respectively, which was similar to what was observed for ISA (1.79 log 10 reduction in CE). Reductions in log 10 CE/g of brain tissue were 0.93, 1.78, and 1.65 for 78 mg/kg fosmanogepix, 104 mg/kg fosmanogepix, and 110 mg/kg TID ISA, respectively. In all three treatment groups, the CE counts observed for tissue fungal burden in lung and brains were significantly lower than the placebo control group (Fig. 2). However, the 104 mg/kg fosmanogepix dosing regimen reduced tissue fungal burden to a greater degree than the 78 mg/kg fosmanogepix treatment (P ϭ 0.001), and was equivalent to tissue burden reductions observed for the ISA treatment (P Ͼ 0.13).
Fosmanogepix demonstrates efficacy in a highly immunosuppressed mouse model of pulmonary mucormycosis caused by R. arrhizus var. arrhizus. The efficacy of fosmanogepix was assessed in the immunosuppressed murine pulmonary mucormycosis model using a strain of R. arrhizus var. arrhizus that had a 16-folder higher MEC value than the R. arrhizus var. delemar 99-880 strain. ICR mice were immunosuppressed and infected intratracheally as above with 2.5 ϫ 10 5 spores of R. arrhizus var. arrhizus 99-892/mouse on day 0 (with confirmed lung-delivered inoculum of 1.1 ϫ 10 4 spores). Due to the better performance of 104 mg/kg fosmanogepix versus the 78 mg/kg treatment group in the R. arrhizus var. delemar mucormycosis model, only the higher dosing regimen was evaluated versus ISA in this efficacy model.  efficacy in this mucormycosis model with 30% overall survival compared to 0% survival for placebo-treated mice (P Ͻ 0.05 versus placebo control [ Fig. 3]). Despite the difference in MEC/MIC values against this strain, fosmanogepix (4 g/ml) and ISA (2 g/ml) survival curves were not significantly different from each other (P ϭ 0.80) (Fig. 3).
(ii) Tissue fungal burden. Mice were infected and treated as above but sacrificed on day ϩ4 postinfection. Lung and brain tissue were harvested to determine conidial equivalents (CE)/g of lung and brain tissue by qPCR using 18S primers. Fungal burdens at day ϩ4 for the placebo group were log 10 4.21 Ϯ 1.0 (lung) and 3.18 Ϯ 0.4 log 10 (brain) (Fig. 4). Compared to placebo, both fosmanogepix and ISA treatment groups demonstrated significant reductions in lung (P Յ 0.001) and brain (P Ͻ 0.0001) CE. Treatment with 104 mg/kg fosmanogepix reduced lung and brain CE by log 10 1.15 (lung) and log 10 1.14 (brain), whereas ISA reduced CE by log 10 1.69 (lung) and log 10 1.14 (brain). ISA and fosmanogepix CE reductions were not significantly different (P Ͼ 0.28) for either brain or lung.

DISCUSSION
Although relatively rare, mucormycosis is difficult to treat and is frequently lifethreatening, with all-cause mortality estimated to be 54% (25). These infections are most often seen in solid organ and stem cell transplant recipients, diabetics, neutropenic patients, or patients treated with corticosteroids. Guidelines for the treatment of mucormycoses recommend high dose liposomal amphotericin B, posaconazole, or isavuconazole as first line therapy, and these infections often require surgical debridement and immune recovery to improve the chances of a successful outcome (26). Isavuconazole (administered as the prodrug isavuconazonium sulfate, CRESEMBA) is approved for the treatment of adults with invasive mucormycosis. In a phase 3, open-label, noncomparative trial (VITAL) for isavuconazole, the most common organisms identified were Rhizopus arrhizus (oryzae) and Mucormycetes (27). Of   Intergroup P values are shown as a dark line. All dosing groups resulted in a statistically significant reduction in brain and lung fungal burden versus placebo control using the Wilcoxon rank sum test.

Efficacy of Fosmanogepix against Mucormycosis
Antimicrobial Agents and Chemotherapy reference laboratories (13). Although no correlations were observed between trough ISA plasma concentrations, MIC values, and key outcomes, the authors indicate that this is possibly due to the relatively small number of patients in the study. Similarly, Andes et al. evaluated the efficacy of ISA and voriconazole against Aspergillus spp. and found that although the drugs were clearly ineffective against strains where MICs were  Ն16 g/ml, there was no clear relationship with clinical outcomes in cases where the MIC was Ͻ16 g/ml for either drug (14). Fosmanogepix is an intravenously (i.v.) and orally (p.o.) available antifungal prodrug that is currently in clinical development for the treatment of life-threatening invasive fungal infections. The broad-spectrum activity of this first-in-class agent has been demonstrated in vivo using multiple mouse models of invasive pulmonary and disseminated fungal infections, including those of Candida spp., Coccidioides spp., Cryptococcus spp., Aspergillus spp., Fusarium spp., and Scedosporium spp. (15,24,(28)(29)(30)(31). These studies demonstrated increased survival, reduced fungal burden in lung, kidney, and brain tissues of infected mice, as well as histological improvement (15,23,30,32,33). Here, we extend these findings to Rhizopus, where we evaluated the efficacy against two different strains with MGX MEC values of 0.25 g/ml and 4.0 g/ml.
We previously evaluated the efficacy of ISA in murine models of pulmonary mucormycosis due to R. arrhizus var. delemar 99-880 or M. circinelloides and demonstrated improved mouse survival and clearance of fungal burden versus placebo control (20,34). In this study, as a comparator in both experiments, we also utilized 110 mg/kg ISA (TID), a dose which gives rise to exposures in mice that are similar to exposures achieved clinically by ISA. MIC values of ISA against R. arrhizus var. delemar 99-880 and R. arrhizus var. arrhizus 99-892 were similar (1.0 and 2.0 g/ml, respectively). Similar to our historical data (20,34), in this study ISA significantly improved survival and reduced fungal burden in brain and lung by 1 to 2 log reductions versus the placebo control. Importantly, in the current study, similar results were seen for the 104 mg/kg fosmanogepix cohorts in both infection models. Total exposures for the 78 mg/kg and 104 mg/kg doses in mice (14) are ϳ200 and ϳ280 g · h/ml, respectively (K. J. Shaw, unpublished data) and were previously shown to be associated with A. fumigatus stasis and 1 log kill in PK/PD studies (28). These exposures in mice are consistent with exposures achieved in fosmanogepix phase 1 single and multiple ascending-dose studies (17,18) and anticipated in future fosmanogepix clinical trials. Thus, although the MGX MEC values of the two strains differed by 16-fold (0.25 versus 4.0 g/ml), the efficacy of fosmanogepix at clinically relevant total exposures was equivalent to ISA, which is approved for the treatment of mucormycosis. Fosmanogepix has now been shown to be efficacious against both varieties of Rhizopus arrhizus, which together are the main cause of the disease and responsible for ϳ50% of cases of lethal mucormycosis (25).
In this study, we used a highly immunocompromised mouse model to show that fosmanogepix has equivalent efficacy to ISA, a current standard of care antifungal drug, in treating pulmonary mucormycosis. Findings from both oral and i.v. fosmanogepix phase 1 clinical studies have shown favorable PK, allowing once-daily dosing, with high bioavailability (ϳ90%), and no food effect (17,18). These data support further investigations into the development of this first-in-class agent for a broad range of difficultto-treat invasive fungal infections.

MATERIALS AND METHODS
Antifungal agents. For in vitro studies, the active moiety manogepix (Amplyx Pharmaceuticals), ISA active compound, and POSA (both from Sigma-Aldrich Corp. St. Louis, MO, USA) were used. For efficacy studies, the water-soluble N-phosphonooxymethyl prodrug fosmanogepix (Amplyx Pharmaceuticals) was dissolved in a final prodrug solution of 5% dextrose. The water soluble isavuconazonium sulfate (Astellas Pharam US) was purchased from Bellavida Pharmacy, Torrance, CA. POSA (Merck & Co., Inc., Rahway, NJ) was purchased as an oral suspension (200 mg/5 ml) and kept at room temperature. All drugs were prepared fresh and orally (p.o.) dosed per gram mouse body weight on a daily basis. A 5 mg/ml solution of ABT (Fisher Scientific, Hampton, NH) in water was administered orally 2 h prior to administration of fosmanogepix at 10 l per gram mouse body weight resulting in a dose of 50 mg/kg.
Microorganisms. R. arrhizus var. delemar 99-880 and R. arrhizus var. arrhizus 99-892 are brain and lung isolates obtained from the Fungus Testing Laboratory at the University of Texas Health Sciences Center at San Antonio (UTHSCSA). Other strains from R. arrhizus var. delemar and R. arrhizus var. arrhizus were also obtained from the culture collection of the Fungus Testing Laboratory, and species identification was confirmed as previously described (13). Strains were routinely grown on potato dextrose agar plates for 4 days until confluent at 37°C. Spores were collected by flooding the plates with sterile Efficacy of Fosmanogepix against Mucormycosis Antimicrobial Agents and Chemotherapy phosphate-buffered saline (PBS) containing 0.01% (vol/vol) Tween 80. The spores were concentrated by centrifugation, washed in the same buffer, diluted, and counted using a hemocytometer.
In vitro testing. The in vitro susceptibility to manogepix, ISA, and POSA by fungal agents of mucormycosis was evaluated using the Clinical Laboratory and Standards Institute (CLSI) M38 method using minimum effective concentration (MEC) endpoints, as per the echinocandins, and MIC endpoints for POSA and ISA (22).
Efficacy models. The pulmonary mucormycosis model has been previously described (16). Briefly, ICR mice (Envigo) were immunosuppressed with cyclophosphamide (200 mg/kg) and cortisone acetate (500 mg/kg) on days Ϫ2, ϩ3, and ϩ8 relative to infection. This regimen was shown to result in ϳ16 days of leucopenia (16). To prevent bacterial infection, 50 g/ml enrofloxacin (Baytril; Bayer, Leverkusen, Germany) was added to the drinking water from day Ϫ3 to day 0. Ceftazidine (5 g/dose/0.2 ml) replaced enrofloxacin treatment on day 0 and was administered daily by subcutaneous injection from day 0 until day ϩ4 (tissue fungal burden) or day ϩ13 (survival). Mice were challenged with R. arrhizus var. delemar or R. arrhizus var. arrhizus (2.5 ϫ 10 5 /mouse) through intratracheal instillation of 25 l after sedation with isoflurane gas (16). Immediately after infection, a subset of mice was sacrificed and lungs were removed to determine conidial equivalent (CE) by qPCR. All drug treatments (by oral gavage) were initiated at 16 h postinfection and continued for 7 consecutive days (survival) or 4 days (tissue burden assessment). To extend the half-life of MGX after fosmanogepix administration, 50 mg/kg of ABT was administered 2 h prior to each daily fosmanogepix dose. The prodrug fosmanogepix was dosed at 78 mg/kg and 104 mg/kg once daily by oral gavage. Using a conversion factor of 1.3 to account for the methyl phosphate group in the prodrug, the doses were equivalent to MGX at 60 mg/kg and 80 mg/kg, respectively. ISA was dosed at 110 mg/kg TID, the dose that gives rise to exposures equivalent to the human clinical dose (21). For the survival experiments, mice were monitored for 21 days. To assess tissue fungal burden, mice were sacrificed on day ϩ4 and organs processed for conidial equivalent (CE) by real-time qPCR using 18S primers (sense amplification primer, 5=-GCGGATCGCATGGCC-3=; antisense amplification primer, 5=-CCATGATAGGGCAGAAAATCG-3=.) Ethics statement. All animal-related study procedures were compliant with the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Office of Laboratory Animal Welfare and were conducted under an IACUC-approved protocol by The Lundquist Institute for Biomedical Innovations at Harbor-UCLA Medical Center.
Statistical analyses. The nonparametric log rank test was used to determine differences in survival times. Differences in lung and brain CE were compared by the nonparametric Wilcoxon rank sum test. All analyses were corrected for multiple comparisons with the Bonferroni correction. A P value of Ͻ0.05 was considered significant.