Efficacy of APX2039 in a Rabbit Model of Cryptococcal Meningitis

Abstract

Cryptococcal Meningitis (CM) is uniformly fatal if not treated, and treatment options are limited. We previously reported on the activity of APX2096, the prodrug of the novel Gwt1 inhibitor APX2039, in a mouse model of CM. Here, we investigated the efficacy of APX2039 in mouse and rabbit models of CM. In the mouse model, the controls had a mean lung fungal burden of 5.95 log₁₀ CFU/g, whereas those in the fluconazole-, amphotericin B-, and APX2039-treated mice were 3.56, 4.59, and 1.50 log₁₀ CFU/g, respectively. In the brain, the control mean fungal burden was 7.97 log₁₀ CFU/g, while the burdens were 4.64, 7.16, and 1.44 log₁₀ CFU/g for treatment with fluconazole, amphotericin B, and APX2039, respectively. In the rabbit model of CM, the oral administration of APX2039 at 50 mg/kg of body weight twice a day (BID) resulted in a rapid decrease in the cerebrospinal fluid (CSF) fungal burden, and the burden was below the limit of detection by day 10 postinfection. The effective fungicidal activity (EFA) was -0.66 log₁₀ CFU/mL/day, decreasing from an average of 4.75 log₁₀ CFU/mL to 0 CFU/mL, over 8 days of therapy, comparing favorably with good clinical outcomes in humans associated with reductions of the CSF fungal burden of -0.4 log₁₀ CFU/mL/day, and, remarkably, 2-fold the EFA of amphotericin B deoxycholate in this model (-0.33 log₁₀ CFU/mL/day). A total drug exposure of the area under the concentration-time curve from 0 to 24 h (AUC_0-24) of 25 to 50 mg · h/L of APX2039 resulted in near-maximal antifungal activity. These data support the further preclinical and clinical evaluation of APX2039 as a new oral fungicidal monotherapy for the treatment of CM. IMPORTANCE Cryptococcal meningitis (CM) is a fungal disease with significant global morbidity and mortality. The gepix Gwt1 inhibitors are a new class of antifungal drugs. Here, we demonstrated the efficacy of APX2039, the second member of the gepix class, in rabbit and mouse models of cryptococcal meningitis. We also analyzed the drug levels in the blood and cerebrospinal fluid in the highly predictive rabbit model and built a mathematical model to describe the behavior of the drug with respect to the elimination of the fungal pathogen. We demonstrated that the oral administration of APX2039 resulted in a rapid decrease in the CSF fungal burden, with an effective fungicidal activity of -0.66 log₁₀ CFU/mL/day, comparing favorably with good clinical outcomes in humans associated with reductions of -0.4 log₁₀ CFU/mL/day. The drug APX2039 had good penetration of the central nervous system and is an excellent candidate for future clinical testing in humans for the treatment of CM.

Keywords: APX001; APX2039; Cryptococcus; Gwt1; antifungal; fosmanogepix; gepix; manogepix; meningitis.

FIG 1

Mouse delayed-treatment model (log₁₀ CFU per gram of mouse brain and lung tissue). Each point represents data from a single mouse. Data were analyzed with a Kruskal-Wallis test followed by a Wilcoxon rank sum test with Holm’s correction. Dosing groups were as follows: control (vehicle [p.o.]) (n = 14), ABT (50 mg/kg ABT [p.o., QD]) (n = 11), FLU (150 mg/kg fluconazole [p.o., QD]) (n = 9), AMB (3 mg/kg amphotericin B deoxycholate [i.p., QD]) (n = 10), and 60 mg/kg APX2039 (p.o., QD) plus 50 mg/kg ABT administered 2 h prior to dosing with APX2039 (n = 7). (A) Data from brain tissue. (B) Data from lung tissue.

FIG 2

Average log₁₀ CFU per milliliter of rabbit CSF over time. (A) CFU per milliliter of CSF data were averaged and then log₁₀ transformed. Error bars are SEM. Data are pooled from three independent experiments (control, n = 9 for day 2, n = 11 for day 7, n = 9 for day 10, and n = 5 for day 14; fluconazole, n = 5 for all time points; amphotericin B, n = 5 for all time points; fosmanogepix, n = 6 for all time points; APX2039 [25 mg/kg, QD], n = 5 for days 2, 7, and 10 and n = 4 for day 14; APX2039 [50 mg/kg, QD], n = 4 for all time points; APX2039 [75 mg/kg, QD] n = 5 for day 2, day 7, and day 10 and n = 2 for day 14; APX2039 [50 mg/kg, BID], n = 8 for day 2, day 7, and day 10 and n = 7 for day 14). (B) All data from each rabbit plotted individually, with each line corresponding to a single rabbit.

FIG 3

Log₁₀ CFU per milliliter of rabbit CSF over time: plot of slopes and intercepts from a linear calculation, a linear model, and a linear mixed-effects model. Fungal clearance was calculated with four separate methods. Dots are log₁₀ CFU per milliliter of CSF data collected during the study and are the same for each panel. Line labels are slopes of selected lines: control, FLU, AMB, and APX2039 (50 mg/kg, BID). (A) Slopes were manually calculated for each rabbit. The mean values of the slopes and intercepts were used to create the lines using the formula log₁₀ CFU/mL = mean change in log₁₀ CFU/mL/day · day postinfection + intercept mean. (B) Estimated marginal means from a linear model that includes an interaction between day postinfection and treatment (log₁₀ CFU/mL ~ day postinfection · treatment). (C) Estimated marginal means from a linear mixed-effects model with day postinfection and treatment group as the fixed effects and rabbit with a random slope as the random effects [log₁₀ CFU/mL ~ day postinfection · treatment + (0 + day postinfection|rabbit ID)]. (D) Means of coefficients extracted from a linear mixed model with only day postinfection as the fixed effect and random slopes for each rabbit [log₁₀ CFU/mL ~ day postinfection + (0 + day postinfection|rabbit ID)].

FIG 4

Brain tissue fungal burden in the rabbit model of cryptococcal meningitis. Each point represents data from a single rabbit. Group effects were tested using a Kruskal-Wallis test. Pairwise comparisons were made using a Wilcoxon rank sum test with Holm’s adjustment (control, n = 10; FLU, n = 5; AMB, n = 5; fosmanogepix, n = 6; APX2039 at 25 mg/kg QD, n = 5; APX2039 at 50 mg/kg QD, n = 5; APX2039 at 75 mg/kg QD, n = 5; APX2039 at 50 mg/kg BID, n = 8).

FIG 5

Plasma levels of APX2039. Each line shows data from a single rabbit. Plasma was collected from each rabbit for the following treatments: APX2039 at 25 mg/kg QD (n = 5) (top left), APX2039 at 50 mg/kg QD (n = 5) (top right), APX2039 at 75 mg/kg QD (n = 5) (bottom left), and APX2039 at 50 mg/kg BID (n = 8) (bottom right).

FIG 6

Levels of APX2039 in rabbit CSF. Concentrations of APX2039 in micrograms per milliliter of CSF were quantified by LC-MS. Each line shows data from a separate rabbit. All CSF samples were collected 1 to 2 h after dosing with APX2039. The black dotted line is the log₁₀-transformed value of the MIC of APX2039 (0.008 μg/mL).

FIG 7

Average total drug AUC_0–24 and decline in the log₁₀ CFU per milliliter as estimated by the area under the fungal density (log₁₀ CFU per milliliter)-versus-time curve for each rabbit.