APX001 Pharmacokinetic/Pharmacodynamic Target Determination against Aspergillus fumigatus in an In Vivo Model of Invasive Pulmonary Aspergillosis

In vitro susceptibility testing and in vivo fitness.

The minimum effective concentration (MEC) values of APX001A, genetic mutations, and relative fitness in the in vivo murine model of each isolate are shown in Table 1. APX001A exhibited similar potency against wild-type and Cyp51 and Fks1 mutant strains with similar MEC values (0.03 to 0.06 mg/liter). The organisms also demonstrated similar in vivo fitness in the animal models, as defined by relatively equal increases in the lung organism burden in untreated mice over the study period.

Pharmacokinetics.

Data from our prior PK study of APX001 were used for the current study analysis (27). The area under the concentration-time curve (AUC) and peak concentrations (C_max) over the dose range were linear (coefficient of determination [R²] = 0.96), including when accounting for drug accumulation due to the frequent administration. Thus, for dose levels that were not directly measured, the AUC and C_max were estimated using linear extrapolation or interpolation. A plasma protein binding value for mice of 98.3%, based upon prior unpublished data from the sponsor, was used in the current study to calculate free drug concentrations for analysis.

PK/PD index determination.

At the start of therapy, mice had 4.85 ± 0.12 log₁₀ conidial equivalents (CE)/ml of lung homogenate, and the organism burden increased to 5.61 ± 0.26 log₁₀ CE/ml of lung homogenate after 96 h in untreated control mice. Escalating doses of APX001 resulted in dose-dependent killing (Fig. 1). The highest doses studied reduced the organism burden by approximately 0.5 log₁₀ CE/ml of lung homogenate compared to that at the start of therapy. Visually, the similarity of the dose-response curves for each of the three dosing fractionations in Fig. 1 suggests that AUC/MEC is likely the most predictive PK/PD parameter of efficacy. To determine this statistically, the dose-response data were modeled using each of the three PK/PD indices in relation to the microbiological effect and applying nonlinear regression analysis using the Hill equation. This is shown in Fig. 2. The strongest relationship was noted when efficacy results were correlated with the AUC/MEC ratio, with an R² value of 0.79. Regression with C_max/MEC and the percentage of the time that the plasma free drug concentrations exceed the MEC (%T>MEC) resulted in less strong relationships, with R² values of 0.52 and 0.23, respectively.

Treatment efficacy and pharmacodynamic magnitude determination.

Dose-response results for all strains are shown in Fig. 3. There was congruence among the dose-response curves, which would be expected, given the narrow MEC range observed in vitro. In general, at the highest APX001 dose studied, a 1- to 2-log kill was observed. The dose-response data were modeled to characterize the relationship between the 24-h free-drug AUC/MEC (fAUC/MEC) and treatment effect (Fig. 4). A sigmoid exposure-response relationship was observed for each isolate, with a strong fit for fAUC/MEC and treatment effect (R² = 0.80), similar to the dose fractionation results. The dose and fAUC/MEC needed to produce growth suppression compared to the growth at the start of therapy (static dose) and in the regimens associated with a 1-log reduction in organism burden (1-log₁₀ kill) for each isolate are reported in Table 2. The median static dose fAUC/MEC ratio was 47.6. The 1-log₁₀-kill fAUC/MEC ratio was roughly 2-fold higher than the stasis PD target, with a median value of 89.4.

Organisms, media, and antibiotic.

Six A. fumigatus isolates were chosen, including three isolates with Cyp51 mutations and one laboratory isolate with an Fks1 mutation. The organisms were grown and subcultured on potato dextrose agar (PDA; Difco Laboratories, Detroit, MI). The organisms were chosen based on similar fitness, as determined by growth in the lungs of untreated animals over 96 h (Table 1). APX001A for in vitro studies and APX001 for in vivo studies were supplied by the study sponsor (Amplyx Pharmaceuticals, Inc., San Diego, CA). APX001A was prepared on the day of use by dissolving with 700 mM HCl and subsequent dilution in RPMI to the required concentrations. APX001 was prepared in 0.21 M NaOH at a concentration of 100 mg/ml and then diluted with sterile 5% glucose solution to the required concentrations.

In vitro susceptibility testing.

The MECs of APX001A for the various isolates were determined using a broth microdilution method, in accordance with the guidelines presented in Clinical and Laboratory Standards Institute (CLSI) document M38-A2 (40). All MEC assays were performed in duplicate on three separate occasions. The MEC values of APX001A were defined as the lowest concentration that led to the growth of small, rounded, compact hyphal forms compared to the hyphal growth seen in the growth control well. All MEC results were determined at 48 h, as recommended by CLSI document M38-A2 (40). The median MEC for replicate assays was reported and utilized in the PK/PD analysis.

Infection model.

Six-week-old, specific-pathogen-free, female ICR/Swiss mice weighing 23 to 27 g were used for all studies (Harlan Sprague-Dawley, Indianapolis, IN). Animals were maintained in accordance with the criteria of the Association for Assessment and Accreditation of Laboratory Animal Care. All animal studies were approved by the Animal Research Committee of the William S. Middleton Memorial VA Hospital.

A neutropenic, corticosteroid-immunosuppressed murine IPA model was utilized as previously described (34–36). Briefly, mice were rendered neutropenic (polymorphonuclear cell counts, <100/mm³) by the subcutaneous (s.c.) injection of 150 mg/kg of body weight cyclophosphamide on days −4, −1, and +3 to ensure the maintenance of neutropenia until the end of the study (96 h). Additionally, cortisone acetate was administered at 250 mg/kg s.c. on day −1. Throughout the 4-day experiment, mice were also given ceftazidime at 50 mg/kg/day s.c. to prevent opportunistic bacterial infection.

Organisms were prepared by subculturing on PDA 5 days prior to infection and incubated at 37°C. On the day of infection, the inoculum was prepared by flooding the culture plate with 5 ml of normal saline containing 0.05% Tween 20. Gentle agitation was used to release the conidia. The conidial suspension was collected and quantified by use of a hemocytometer (Bright-Line; Hausser Scientific, Horsham, PA). The suspension was diluted in saline to a final concentration of 1 × 10⁷ conidia/ml. Viability was confirmed by plating the suspension and performing CFU counts.

Infection was produced in animals using an aspiration pneumonia model that has been successfully utilized in previous studies (34–36). Briefly, mice were anesthetized with a combination of ketamine and xylazine. Fifty microliters of the conidial suspension was pipetted into the anterior nares with the mice, held upright to allow aspiration into the lungs. Drug treatment commenced 2 h after the initiation of infection.

Tissue fungal burden.

Pulmonary fungal burden was determined by real-time quantitative PCR (qPCR) using previously reported methods (34–36, 41, 42). Briefly, animals were euthanized if showing signs of distress or at the end of the 96-h experiment. Both lungs were immediately removed by aseptic technique and placed into sterile Whirl-Pak bags (Nasco, Fort Atkinson, WI) containing 2 ml of sterile 0.9% normal saline. Samples were then homogenized or frozen until homogenization could occur. Homogenization of lung tissue occurred in two steps. First, the lungs were manually homogenized using direct pressure to yield a primary homogenate (43). One milliliter of the primary homogenate was then transferred to a sterile 2-ml screw-cap microcentrifuge tube (Sarstedt, Newton, NC) with 700 μl of 425- to 600-μm acid-washed glass beads (Sigma-Aldrich, St. Louis, MO). The primary homogenate was mechanically disrupted by vigorous agitation in a BioSpec Mini-BeadBeater (Bartlesville, OK) for 90 s at 4,200 rpm to yield a secondary homogenate. This secondary homogenate was stored at −20°C until DNA extraction.

DNA was extracted from secondary homogenates with a DNeasy Blood and Tissue kit (Qiagen, Valencia, CA) following the manufacturer’s protocol and stored at −20°C until the day of qPCR analysis. Extracted DNA was subjected to quantitative, real-time PCR. qPCR plates were prepared on the day of the assay. Standard amounts of conidia were prepared by the use of hemocytometer counts and were spiked into blank uninfected lungs, used for generating standard curves. Samples were assayed in duplicate using a Bio-Rad CFX96 real-time system (Bio-Rad, Hercules, CA). A single-copy gene, Fks1, was chosen for quantitation (41). Primer sequences included 5′-GCCTGGTAGTGAAGCTGAGCGT-3′ for the forward primer and 5′-CGGTGAATGTAGGCATGTTGTCC-3′ for the reverse primer, and the probe sequence was 5′–6-carboxyfluorescein–AGCCAGCGGCCCGCAAATG-MGB-3′ (Integrated DNA Technologies, Coralville, IA). The Fks1 mutation in strain EMFR S678P was not located in the primer-probe area of the genome and has previously been shown to not affect the quantitation reaction for that isolate (35). The primer-probe set was validated for all isolates by determining the kinetics and quantitative results for known quantities of conidia over the dynamic range (10² to 10⁸) (34–36). The cycling conditions were as follows: activation, 50°C for 2 min; heat inactivation, 95°C for 10 min for 1 cycle; denaturation, 95°C for 15 s; annealing and extension, 65°C for 1 min for 40 cycles. Quantitation standards were run in conjunction with each set of samples. The threshold cycle (C_T) of each sample was interpolated from a five-point standard curve of C_T values prepared by spiking uninfected mouse lungs with known amounts of conidia (10³ to 10⁷) from each isolate being tested. Results were reported as the number of conidial equivalents (CE) per milliliter of lung homogenate.

Pharmacokinetics.

Murine APX001 pharmacokinetic data, including the AUC and C_max, were derived from our previous study (27).

PK/PD index determination.

A dose fractionation study design was undertaken to determine the PK/PD index (AUC/MEC, C_max/MIC, or time above the MEC) that was predictive of efficacy for APX001 against A. fumigatus. Four doses (80, 192, 512, and 768 mg/kg) of APX001 were fractionated into dosing regimens in which the drug was administered every 3 h (q3h), every 6 h (q6h), and every 8 h (q8h). Mice were infected with isolate AF293 as described above and administered APX001 by oral gavage. After 96 h the mice were euthanized, and the lungs were collected for DNA quantification. To determine which PK/PD index was most closely linked with efficacy, the number of CE per milliliter of lung homogenate at the end of 96 h of therapy was correlated with (i) the 24-h free drug AUC/MEC, (ii) the free drug C_max/MEC ratio, and (iii) the percentage of time over 24 h during which the plasma free drug levels exceeded the MEC (%T>MEC) for each of the dosage regimens studied. The correlation between efficacy and each of the three PK/PD indices was determined by nonlinear regression based on the Hill equation. The coefficient of determination (R²) was used to estimate the variance that might be due to regression with each of the PK/PD indices.

PK/PD index magnitude studies.

Dose-response experiments were performed for the five remaining A. fumigatus isolates using the invasive pulmonary aspergillosis model, as described above. Six dose levels (consisting of 5, 10, 24, 64, 96, and 192 mg/kg/3 h) were administered by the oral route. The treatment duration was 96 h. The dose-response relationships were quantified, and the relationship between the PK/PD index and treatment efficacy was determined using the sigmoid maximal effect (E_max) model. These PK/PD relationships were examined utilizing the plasma free drug concentrations from pharmacokinetic studies. The coefficient of determination (R²) from this model was used to assess the strength of this relationship. The doses required to produce a net static effect (static dose) and a 1-log-kill effect compared to the number of CE per milliliter of lung homogenate at the start of therapy for multiple pathogens in the invasive pulmonary aspergillosis model were determined utilizing the plasma free drug concentrations and the following equation: log₁₀D = log₁₀[E/(E_max − E)]/N + log₁₀ ED₅₀, where D is the drug dose, E is the growth (measured by qPCR and represented as the number of CE per milliliter of lung homogenate) in untreated control mice, E_max is the maximal effect, N is the slope of the dose-response relationship, and ED₅₀ is the dose needed to achieve 50% of the maximal effect.