The Combination Treatment of Fosmanogepix and Liposomal Amphotericin B Is Superior to Monotherapy in Treating Experimental Invasive Mold Infections

Antifungal susceptibility.

The MIC values for amphotericin B (AMB) and minimum effective concentration (MEC) values for MGX for the 3 strains used in the infection models are shown in Table 1. A. fumigatus strain AF293 and F. solani strain 95-2478 each had a low MGX MEC of 0.03 μg/mL, whereas R. arrhizus var. delemar strain 99-880 had an MGX MEC of 0.25 μg/mL. For AMB, A. fumigatus AF293 and R. arrhizus var. delemar 99-880 each had a MIC of 0.25 μg/mL, while F. solani 95-2478 was slightly more resistant, with the MIC registering at 4.0 μg/mL (Table 1). Previous synergy measurement by checkerboard analysis of 18 strains of A. fumigatus and 4 strains of A. flavus has shown that the combination of MGX and AMB is indifferent (26).

Combination therapy of FMGX and L-AMB is superior to monotherapy in treating murine IPA.

The efficacies of monotherapies of FMGX (78 mg/kg) and L-AMB (5 mg/kg) and of the combination of FMGX and L-AMB were evaluated versus the outcomes for the vehicle control (placebo) in a severe delayed-treatment IPA model, where dosing was initiated 48 h postinfection. A dose of 50 mg/kg ABT plus 78 mg/kg FMGX was chosen since previous studies demonstrated that the resulting exposures in mice mimicked those obtained in humans in phase 1 clinical studies of healthy volunteers (11, 21, 22). Similarly, a dose of 5 mg/kg of L-AMB in mice gives rise to a peak plasma concentration close to that resulting from a 4-mg/kg dose in humans (23, 24).

In a separate experiment, the effect of the combination of drugs on the exposure (AUC from dosing to the time of the last measured concentration [AUC_last]) was assessed in mice. A dose of 10 mg/kg L-AMB was administered alone, with 50 mg/kg ABT, with 26 mg/kg FMGX, and with FMGX+ABT. A dose of 26 mg/kg FMGX was administered with and without 50 mg/kg ABT. There was no difference in the AMB AUC_last values without or with ABT (1.37 and 1.38 mg · h/mL, respectively), whereas as anticipated, a large (19-fold) increase in AUC_last was observed for MGX when ABT was present. The AMB AUC_last value in the presence of both ABT and MGX was 14% higher than the AMB value when dosed with ABT but without MGX (1.60 versus 1.38 mg · h/mL). The MGX AUC_last value in the presence of both ABT and L-AMB was 17% lower than the value when MGX+ABT was administered without L-AMB (60.1 versus 72.2 μg · h/mL). These results suggest that there are limited changes in the drug exposures observed when given in combination.

In the severe delayed-treatment IPA model, after treatment for 7 days with FMGX and 4 days with L-AMB, 60% survival was observed at day 21 for the combination therapy cohort, whereas 25%, 25%, and 5% survival were observed for the FMGX, L-AMB, and placebo cohorts, respectively (Fig. 1A). Furthermore, the median survival times were determined to be 7 days for the placebo cohort, 10 days for the FMGX cohort, 8.5 days for the L-AMB cohort, and >21 days for the FMGX+L-AMB cohort (Fig. 1B). While all treatments resulted in significant prolongations of median survival times and increased overall survival compared to the outcomes for placebo-treated mice, combination therapy significantly enhanced the survival outcomes of mice even compared to those of L-AMB or FMGX monotherapy (P < 0.02) (Fig. 1).

We also evaluated the efficacy of combination therapy in reducing the tissue fungal distribution in the lungs of mice infected with A. fumigatus. Immunosuppressed mice were infected through inhalation, and treatment with either FMGX or L-AMB monotherapy or FMGX+L-AMB combination therapy was initiated 48 h postinfection and lasted for 4 days. Lung fungal burdens (determined as the Log₁₀ conidial equivalent using qPCR) (27) were determined in lungs harvested from mice euthanized on day 4 postinfection (i.e., the third day of treatment, ~6 h after the last treatment). As can be seen by the results in Fig. 2A, only combination therapy resulted in a significant, ~5-Log₁₀ reduction in lung fungal burden compared to the lung fungal burden in placebo-treated mice. In addition, combination treatment of FMGX+L-AMB resulted in 3- to 4-Log₁₀ reductions in fungal burdens compared to either monotherapy (Fig. 2A). These results were corroborated by the histopathological examination of lungs harvested on the same day as tissue processed for quantification of the fungal burden. Specifically, while lungs harvested from placebo-treated mice showed extensive fungal hyphae surrounded by phagocytes, with signs of fungal pneumonia and a substantial degree of tissue edema, lungs from mice treated with FMGX or combination therapy showed normal tissue architecture with no apparent fungal hyphae (Fig. 2B). Lungs harvested from mice treated with L-AMB showed some fungal abscesses with lesser signs of pneumonia and tissue edema. Collectively, these data demonstrate the benefit of combination therapy of FMGX+L-AMB in treating murine IPA compared to the outcomes for placebo- and monotherapy-treated mice.

Combination therapy of FMGX and L-AMB is superior to monotherapy in treating murine IM.

The efficacies of monotherapies of FMGX (78 mg/kg) and L-AMB (10 mg/kg) and of the combination of FMGX and L-AMB were evaluated versus the outcomes for the placebo vehicle control in a severe model of invasive pulmonary mucormycosis (infection with R. arrhizus var. delemar via intratracheal instillation) (28), where dosing was initiated 48 h postinfection. A higher dose of L-AMB was used in this severe IM model than in the IPA model. After treatment for 7 days with FMGX and 4 days with L-AMB, 70% survival was observed at day 21 for the combination therapy cohort, whereas 30%, 35%, and 5% survival were observed for FMGX-, L-AMB-, and placebo-treated mice, respectively (Fig. 3A). Moreover, the median survival times were determined to be 6.5 days for the placebo cohort, 11. 5 days for the FMGX cohort, 13 days for the L-AMB cohort, and >21 days for the FMGX+L-AMB cohort. Although all treatments resulted in significant prolongations of median survival times and increases in overall survival compared to the outcomes for placebo-treated mice, the combination therapy significantly enhanced the survival of mice even compared to either monotherapy (P < 0.03) (Fig. 3).

Because combination therapy of FMGX+L-AMB enhanced survival over the survival in the placebo and monotherapy cohorts, we investigated the effects of this treatment regimen on the tissue fungal burdens of target organs in a separate experiment. Immunosuppressed mice were infected intratracheally with R. arrhizus var. delemar and treated as described above. On day 4 postinfection (i.e., ~6 h after the third treatment), mice were euthanized, lungs (primary target organ) and brains (secondary target organ) were harvested, and the fungal burdens determined as the Log₁₀ spore equivalent per gram of tissue using qPCR (29). While neither of the monotherapy treatments resulted in significant reductions in tissue fungal burdens, the combination therapy of FMGX+L-AMB reduced the lung and brain fungal burdens by ~2 log compared to the burdens in placebo-treated mice. Furthermore, the combination therapy significantly reduced the fungal burdens of both organs, by ~1.0 to 1.5 log, compared to the burdens after monotherapy of either FMGX or L-AMB (Fig. 4A and B). Finally, histopathological examination of lung tissues taken from mice euthanized at the same time point as for the tissue fungal burden assessment showed that only the combination treatment resulted in resolution of fungal abscesses and of signs of pneumonia and tissue edema (Fig. 4C). Collectively, these data clearly show a benefit of the combination therapy of FMGX+L-AMB over monotherapy in treating murine IM.

Combination therapy of FMGX and L-AMB is superior to monotherapy in treating hematogenously disseminated murine fusariosis.

The efficacies of monotherapies of FMGX (78 mg/kg) and L-AMB (10 mg/kg) and of the combination of FMGX+L-AMB were evaluated versus the outcomes for the placebo vehicle control in a severe model of hematogenously disseminated fusariosis where treatment was initiated ~20 h postinfection. Similar to the IM model, a higher dose of L-AMB was used than for the IPA model. After treatment for 7 days with FMGX and 4 days with L-AMB, 50% survival was observed at day 21 for the combination therapy cohort, whereas 20%, 25%, and 0% survival were observed for FMGX-, L-AMB-, and placebo-treated mice, respectively (Fig. 5A). The median survival times were determined to be 7 days for the placebo cohort, 8.5 days for the FMGX cohort, 7.5 days for the L-AMB cohort, and 20 days for the FMGX+L-AMB cohort (Fig. 5B). Both FMGX and L-AMB monotherapies enhanced survival versus the survival in the placebo control group in this severe model (P = 0.003 and P = 0.007, respectively). Similar to what we found in murine IPA and IM, the combination of FMGX+L-AMB resulted in a significant survival enhancement versus the survival after treatment with either monotherapy (P ≤ 0.04) or the vehicle control (P < 0.0001) (Fig. 5).

To investigate whether the enhanced survival seen with combination therapy over monotherapy would be corroborated by the effect on tissue fungal burdens, in an independent experiment, we infected immunosuppressed mice intravenously with F. solani and treated them as described above. On day 4 postinfection (i.e., ~6 h after the third treatment), we euthanized the mice and harvested their kidneys and brains (target organs) to determine the tissue fungal burdens (using qPCR to determine the Log₁₀ spore equivalent per gram of tissue [18]) and for histological examination. Both monotherapies were equally effective in lowering the kidney and brain fungal burdens, by ~1 to 2 log compared to the burdens in placebo-treated mice. Combination therapy resulted in augmented reductions in both kidney and brain fungal burdens compared to the burdens in mice treated with placebo or either monotherapy (~3-log reductions versus the burdens in mice treated with the placebo and ~1-log reductions versus mice treated with either monotherapy) (P ≤ 0.01) (Fig. 6A and B). Histological examination of kidneys harvested from placebo-treated mice showed significant fungal abscesses, where none of the treated mice (monotherapy or combination therapy) showed any signs of fungal infection (Fig. 6C). Collectively, these results show that combination therapy of FMGX+L-AMB is effective in treating hematogenously disseminated murine fusariosis due to F. solani and is superior to either monotherapy.

Antifungal agents.

For in vitro studies, MGX (APX001A; Amplyx Pharmaceuticals) was used, while for efficacy studies, the water soluble N-phosphonooxymethyl prodrug FMGX (APX001; Amplyx Pharmaceuticals) was used. The final prodrug solution was in 5% dextrose and dosed orally (per os [PO]) on a per gram of mouse daily body weight basis. A 5-mg/mL solution of ABT (Fisher Scientific, Hampton, NH) in water was administered orally 2 h prior to treatment as 10 μL per gram of mouse body weight, resulting in a dose of 50 mg/kg. L-AMB was obtained from Bella Vida Pharmacy (Torrance, CA) and manufactured by Gilead Sciences (Foster City, CA) and was dosed at 5 mg/kg for IPA models or 10 mg/kg for IM and IF models.

Microorganisms.

In this study, we used A. fumigatus strain AF293 (a generous gift of P. Magee), R. arrhizus var. delemar strain 99-880 (a brain isolate obtained from the Fungus Testing Laboratory at the University of Texas Health Sciences Center at San Antonio [UTHSCSA]), and F. solani strain 95-2478 (a blood isolate provided by P. Ferrieri, University of Minnesota). Routine culturing of the fungal isolates was conducted on Sabouraud dextrose agar plates at 37°C for 5 days for R. arrhizus var. delemar 99-880 and F. solani and for 10 days for A. fumigatus until cells were confluent. Conidia were collected by flooding the plates with sterile phosphate-buffered saline containing either 0.2% (vol/vol) Tween 80 for A. fumigatus or 0.01% (vol/vol) Tween 80 for R. arrhizus var. delemar 99-880 or F. solani. The conidia were concentrated by centrifugation, washed in the same buffer, diluted, and counted using a hemocytometer.

In vitro testing.

The in vitro susceptibilities of agents of aspergillosis, mucormycosis, and fusariosis to MGX were evaluated using the Clinical Laboratory and Standards Institute (CLSI) M38-A2 method (41), using minimum effective concentration (MEC) endpoints, as for the echinocandins. For AMB, MIC endpoints were utilized.

Efficacy models.

The pulmonary aspergillosis, pulmonary mucormycosis, and disseminated F. solani models have been previously described (16–18). For all models, CD-1 male mice weighing 20 to 23 g (Envigo) were used. Mice were immunosuppressed with cyclophosphamide (200 mg/kg) and cortisone acetate (500 mg/kg) on days −2 and +3 relative to infection for the IPA and IF models and on days −2, +3, and +8 for the IM model. To prevent bacterial infection, Baytril (50 μg/mL) was added to the drinking water from day −3 to day zero. Ceftazidime (5 μg/dose/0.2 mL) replaced Baytril treatment on day zero and was administered daily by subcutaneous injection from day zero until day +8 in the IPA and IF models and from day zero until day +13 for the IM model. To extend the half-life of MGX, mice were administered 50 mg/kg of the cytochrome P450 inhibitor 1-aminobenzotriazole (ABT) 2 h prior to FMGX administration. ABT was also added to the L-AMB treatment to control for any introduced variables. The prodrug FMGX was dosed at 78 mg/kg once daily by oral gavage to yield an actual dose of MGX of 60 mg/kg (a conversion of 1.3 to account for the methyl phosphate group in the prodrug). This dose results in exposures in mice that mimic exposures in humans achieved in phase I clinical trials using healthy volunteers (21, 22). The L-AMB doses administered via tail vein injections and used in these models included 5 mg/kg/day for IPA and 10 mg/kg/day for IM and IF.

Infection and treatment. (i) IPA.

Immunosuppressed mice were infected with Aspergillus fumigatus in an inhalation chamber by aerosolizing 12 mL of a 1 × 10⁹ mL suspension of conidia with a small particle nebulizer driven by compressed air (38). A standard exposure time of 1 h was used for all experiments. Immediately after infection, a subset of the mice were euthanized, and the lungs were removed for quantitative culture. Treatment with FMGX (78 mg/kg/day, by oral gavage), L-AMB (5 mg/kg, administered in the tail vein), or the combination of both drugs started 48 h postinfection and continued for 7 days for FMGX and 4 days for L-AMB for survival studies. For tissue fungal burden studies, treatment started 48 h postinfection and continued for 3 days. On the third day of treatment, mice were treated in the morning and euthanized 6 h later, and target tissues were processed for fungal burden determination (see below).

(ii) IM.

Immunosuppressed mice were challenged with Rhizopus arrhizus var. delemar (2.5 × 10⁵/mouse) through intratracheal instillation of 25 μL after sedation with isoflurane gas (16). Immediately after infection, a subset of mice were euthanized, lungs were removed and harvested to determine the infectious inoculum by plating on potato dextrose agar (PDA) plates containing 0.1% Triton X-100, and colonies were counted after a 24-h period at 37°C. Treatment with FMGX (78 mg/kg/day, by oral gavage), L-AMB (10 mg/kg, administered in the tail vein), or the combination of both drugs started 48 h postinfection and continued for 7 days for FMGX and 4 days for L-AMB for survival studies. For tissue fungal burden studies, treatment started 48 h postinfection and continued for 3 days. On the third day of treatment, mice were treated in the morning and euthanized 6 h later, and target tissues were processed for fungal burden determination (see below).

(iii) IF.

Immunosuppressed mice were infected with a targeted inoculum of 8.0 × 10² cells of F. solani by tail vein injection (18). Treatment with FMGX (78 mg/kg/day, by oral gavage), L-AMB (10 mg/kg, administered in the tail vein), or the combination of both drugs started ~20 h postinfection and continued for 7 days for FMGX and 4 days for L-AMB for survival studies. For tissue fungal burden studies, treatment started 48 h postinfection and continued for 3 days. On the third day of treatment, mice were treated in the morning and euthanized 6 h later, and target tissues were processed for fungal burden determination (see below).

Tissue fungal burden studies and histopathological examination.

In all three models, we used qPCR to determine the effects of the treatments on target tissue fungal burdens, expressed as conidial equivalents, using 18S rRNA primers for IPA or IM and 28S rRNA primers for IF (17, 18, 27). Mouse DNA was detected with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers (Table 2). Tissues harvested from mice to assess fungal burdens were also processed for histopathological examination. Briefly, tissues were fixed in 10% zinc-buffered formalin, embedded in paraffin, sectioned, and stained with Grocott’s methenamine silver (GMS) stain for microscopic examination.

Statistical analysis.

The nonparametric log rank test was used to determine differences in survival times. Differences in tissue fungal burdens were compared by the nonparametric Wilcoxon rank sum test. A P value of <0.05 was considered significant.

All animal-related study procedures were compliant with the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals (42), and the Office of Laboratory Animal Welfare, NIH, and were conducted under an IACUC-approved protocol by The Lundquist Institute for Biomedical Innovation at Harbor-UCLA Medical Center.