Safety and nonclinical and clinical pharmacokinetics of PC945, a novel inhaled triazole antifungal agent

Abstract PC945 is a novel antifungal triazole formulated for nebulized delivery to treat lung Aspergillus infections. Pharmacokinetic and safety profiles from nonclinical studies and clinical trials in healthy subjects, and subjects with mild asthma were characterized. Toxicokinetics were assessed following daily 2‐hour inhalation for 14 days. Potential for drug‐drug interactions was evaluated using pooled human liver microsomes. Clinical safety and pharmacokinetics were assessed following (a) single inhaled doses (0.5‐10 mg), (b) 7‐day repeat doses (5 mg daily) in healthy subjects; (c) a single dose (5 mg) in subjects with mild asthma. C max occurred 4 hours (rats) or immediately (dogs) after a single dose. PC945 lung concentrations were substantially higher (>2000‐fold) than those in plasma. PC945 only inhibited CYP3A4/5 substrate metabolism (IC50: 1.33 µM [testosterone] and 0.085 µM [midazolam]). Geometric mean C max was 322 pg/mL (healthy subjects) and 335 pg/mL (subjects with mild asthma) 4‐5 hours (median tmax) after a single inhalation (5 mg). Following repeat, once daily inhalation (5 mg), Day 7 C max was 951 pg/mL (0.0016 µM) 45 minutes after dosing. Increases in C max and AUC0–24h were approximately dose‐proportional (0.5‐10 mg). PC945 administration was well tolerated in both healthy subjects and subjects with mild asthma. Treatment‐emergent adverse events were mild/moderate and resolved before the study ended. No clinically significant lung function changes were observed. PC945 pharmacokinetics translated from nonclinical species to humans showed slow absorption from lungs and low systemic exposure, thereby limiting the potential for adverse side effects and drug interactions commonly seen with systemically delivered azoles.


| INTRODUC TI ON
Incidence of fungal infections has increased substantially over the past two decades. 1 Immunocompromised or immunosuppressed patients are particularly susceptible to such infections, and invasive forms remain a leading cause of morbidity and mortality for these patients. 2 Pulmonary aspergillosis, caused by Aspergillus, is particularly problematic. 3 Aspergillus fumigatus is one of the primary causative agents of lung infections in humans. 4 Chronic Aspergillus infections can leave patients with permanent lung damage, requiring life-long management using oral azole treatment. 5 Existing treatments for fungal infections are administered orally (azoles) or intravenously (azoles, amphotericin B or the echinocandins). 6 Azole antifungals are potent inhibitors of cytochrome P450 (CYP450) enzymes, especially the CYP3A4 isoenzyme, which is responsible for the metabolism of a broad range of drugs. 7 This inhibition presents a significant risk of interactions with other comedicated drugs. To allow orally or intravenously administered antifungal agents to achieve high local concentrations sufficient for pathogen clearance, systemic exposure must be high, resulting in poor safety profiles. 8,9 Nebulized delivery of antifungal agents results in higher local exposure in the epithelial lining fluid compared with intravenous administration. 10 PC945 is the first antifungal triazole specifically designed to treat pulmonary infection via inhaled administration. 11 In common with other triazole agents, PC945 inhibits the enzyme lanosterol 14α-demethylase (CYP51A1) in fungus, which prevents conversion of lanosterol to ergosterol. 12,13 Reduction of ergosterol causes disruption to the structure and function of fungal membranes, hence inhibiting fungal growth and spread. Using a method from the European Committee on Antimicrobial Susceptibility Testing (EUCAST), against 96 clinically isolated A. fumigatus strains obtained in France and the United Kingdom, geometric mean minimum inhibitory concentration (MIC) of PC945 was 0.17 μg/mL, and MIC 50 and MIC 90 values were 0.125 and 1.0 μg/mL, respectively; the potency of PC945 was superior to that of voriconazole and comparable to that of posaconazole. 13 PC945 was also found to inhibit A. fumigatus infection in an in vitro human alveolus bilayer model. 14 In animal models, PC945 delivered a sustained and persistent antifungal effect in the lung when administered intranasally. 15 In addition, PC945 was more effective than alternative antifungal agents because of a higher local exposure at the infected site, which was attributed to the intranasal administration delivering the compound directly to the lung. 13,15 In this article, we report the pharmacokinetic profile of PC945 in nonclinical studies after single and repeat inhaled doses in rats and dogs. We also present the results from a Phase 1 study, which evaluated the safety, tolerability and pharmacokinetics of single (escalating), and repeat inhaled doses of PC945 in healthy subjects, as well as the safety and tolerability of a single dose of inhaled PC945 in subjects with mild asthma (ClinicalTrials.gov Identifier: NCT02715570). It was important to demonstrate a low systemic exposure (ie, in pg/mL level) following inhaled delivery to ensure a favorable safety profile for PC945 compared with the current systemic (oral and intravenous) treatments.

| Drug
For nonclinical studies, PC945 was synthesized by Sygnature Discovery Ltd (Nottingham, UK) and micronized at JetPharma (Balerna, Switzerland). PC945 powder was directly suspended in sodium phosphate-buffered saline containing wetting agents to 10 mg/mL and further diluted with physiological saline after sonication to obtain the desired dose concentration.
For the clinical study, PC945 was manufactured by Onyx Scientific Ltd (Sunderland, UK), micronized at JetPharma and supplied as a powder at a single strength of 14 mg/vial (Juniper Pharma, Nottingham, UK) for reconstitution using placebo solution. Placebo was supplied in a similar vial (Nova Laboratories, Leicester, UK). PC945 and placebo were administered by oral inhalation using a PARI LC SPRINT ® nebuliser and PARI TurboBoy SX ® compressor (PARI Medical Ltd., Surrey, UK). The particle size distribution of the drug substance in the product has been designed to be typical of inhaled medicines that are used to treat lung disease and is expected to be able to penetrate small airways, ie, a d50 of <2 µm and a d90 of <4.5 µm when measured by laser diffraction.

| Nonclinical toxicokinetic studies
All animal studies were designed to meet the requirements of

| In vitro plasma protein binding
Plasma protein binding of PC945 was determined by ultrafiltration in pooled plasma samples from human, dog, rat, and mouse. PC945 was tested at 0.1, 1, 5, 25, and 50 µmol/L in plasma for each species.
PC945-containing plasma samples were centrifuged at 238 859 g for 20 hours at 37°C. Supernatant was removed and diluted. The concentration of unbound PC945 was determined using LC-MS/ MS, from which the extent of protein binding was calculated.

| Trial design
This PC945 first-in-human clinical trial (ClinicalTrials.gov Identifier: NCT02715570) was a two-part randomized, placebo-controlled study to assess the safety and tolerability of PC945 in both healthy subjects and subjects with mild asthma, conducted at Parexel Early Phase Clinical Unit (Harrow, UK). The study was considered singleblind as the appearance of active and placebo doses were different, however, it was conducted in a double-blind manner (employing separate dosing and assessment teams).
Part one consisted of a single dose escalation study (Cohort 1) and a repeat dose study (Cohort 2) of inhaled PC945 in healthy subjects. Part two comprised a single dose study of inhaled PC945 in subjects with mild asthma (Cohort 3). The doses selected were predicted to achieve target lung concentrations above the IC 90 /MIC 90 for A. fumigatus (based on data from a range of in vitro and in vivo systems) throughout a 24-hour period and with a suitable safety margin based on animal toxicology data.
The study was carried out in accordance with the Declaration of Helsinki, the International Council for Harmonisation consolidated guideline for Good Clinical Practice, and local regulations and were approved by an Independent Ethics Committee. All subjects signed a written informed consent form before enrollment.
In Cohort 1, 11 healthy subjects (three males and eight females; mean age: 48.5 years, mean weight: 65.32 kg, mean body mass index [BMI]: 23.62 kg/m 2 ), attended the single ascending dose study. Eight healthy subjects started the study in which two subjects were randomized to each of the four ascending dose treatment sequences (Table 1); two subjects withdrew due to adverse events unrelated to study medication and both were replaced. One of the replacement subjects subsequently withdrew from the study for personal reasons and was replaced by another subject (Figure 1). Overall, six subjects each received three of four doses of PC945 (0.5, 2, 5, and 10 mg) and one dose of placebo.
These doses were administered with a minimum of 13 days interval to allow for washout.
Nine healthy subjects (four males and five females; mean age: 33.2 years, mean weight: 66.5 kg, mean BMI: 23.53 kg/m 2 ) whose forced expiratory volume over 1 second (FEV 1 ) and forced vital capacity (FVC) were ≥80% of predicted values with an FEV 1 /FVC ratio >0.7, received once daily doses of PC945 5 mg or placebo for 7 days (Cohort 2, PC945:placebo = 2:1). to a fall in FEV 1 of ≥20% of personal best) ≤8 mg/mL at screening and an FEV 1 >60% of predicted normal value at least 6 hours after the last use of a short acting β-agonist.

Subjects returned to the study unit for a final follow-up visit
10 days after the last administration of study medication.

| Sample collection and handling
Serial blood samples were collected to determine plasma PC945 con-

| Safety assessment
Safety was evaluated based on assessments of adverse events, physical examination, vital signs, 12-lead electrocardiogram, spirometry and clinical laboratory tests. The verbatim terms used to identify adverse events were coded using the Medical Dictionary for Regulatory Activities (version 20.1). All adverse events were mapped to system organ class and preferred term. (Fordham, UK) after the drug was extracted from plasma samples.
Lower limit of quantification of the assay was 10 pg/mL for clinical samples and 100 pg/mL for rat and dog plasma samples.

| Pharmacokinetic analysis
Plasma pharmacokinetic parameters of PC945 were estimated

Attainment of steady state was assessed by visual inspection
of the predose plots during the 7-day treatment period in human subjects.

| Analysis of lung samples
Portions of rat lung tissue were homogenized in methanol:water (50:50; 15 mL for 1 g tissue). PC945 concentrations in tissue homogenates were analyzed using LC-MS/MS. A control rat lung sample (B&K Universal Ltd, Hull, UK) was used as standards and quality control.

| Nomenclature of Targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guide topha rmaco logy. Systemic exposure to PC945 was consistently higher in female than in male rats ( Table 2). C max occurred 6 hours after the start of dosing and PC945 was detectable throughout the 24-hour sampling period after dosing.

Repeat dose study in healthy subjects
Single dose study in subjects with mild asthma (N=9) • Two subjects withdrew and were replaced • One replacement subject withdrew and was replaced by another subjects The magnitude of exposure obtained in dogs was lower than that in rats at similar dose/kg levels. There was a slightly less than   Table 8. In total, seven adverse events required intervention; only one of these was considered drug-related (headache, following a single 10 mg dose of PC945).
No clinically significant changes in lung function were observed in healthy subjects. Importantly, no evidence of acute bronchospasm or significant change in lung function (defined as >15% change from baseline) was observed in any subject with mild asthma who received PC945. One subject had a transient reduction in FEV 1 values >15% compared with baseline 10 minutes after receiving a dose of placebo.
There were no notable differences in mean laboratory values, vital signs, electrocardiogram results or spirometry values (Figure 2) between the placebo and PC945 groups. No clinically significant abnormal laboratory results considered to be related to study drug were reported in any cohort.

| Pharmacokinetics
Single dose in healthy subjects (Cohort 1) Following single inhaled administration of PC945 at 0.5, 2, 5 and 10 mg in healthy subjects, geometric mean C max values of 54.7, 128, 322, and 619 pg/mL in plasma were achieved at 1, 4, 5, and 2 hours (median t max ) postend of inhalation, respectively (Table 9 and Figure 3). Geometric mean t ½ ranged from 27.9 to 110 hours over the entire dose range. However, the period over which estimated t ½ was calculated was less than twofold the t ½ itself and the area extrapolated was greater than 20% in most cases; estimated t ½ was therefore considered to be unreliable at each dose level. Between-subject variability in the extent of systemic exposure to PC945 at 0.5, 2, 5 and 10 mg was moderate to high, as revealed by geometric coefficient of variations for C max and AUC 0-24h ranging from 39.1% to 107%. Systemic exposure to PC945 in female subjects was not appreciably different to that in male subjects.

Single dose in subjects with mild asthma (Cohort 3)
Following single inhaled administration of PC945 at 5 mg, a geometric mean C max of 335 pg/mL in plasma was achieved 4 hours (median t max ) postdose ( Figure 4) Abbreviations: AUC 0-24h , area under the plasma concentration curve from time zero to 24 hours postdose; C max , maximum observed concentration; R o , accumulation ratio. a Standard Deviation values were omitted for reasons of clarity in presentation of mean data.

Dose proportionality
Dose proportionality was only assessed for single doses using C max and

| DISCUSS ION
PC945 is a novel antifungal triazole, designed specifically for inhaled administration with physico-chemical properties for sustained lung retention and persistent antifungal activity. 13 Drug delivery directly to the lung to treat pulmonary disease is well es- Systemic exposure to PC945 in humans (C max and AUC 0-24h ) increased with escalating doses in a dose-proportional manner and following 7-day, once daily dosing with 5 mg PC945 and accumulation was observed. Visual inspection of predose plasma concentrations indicated that steady state had not been reached by Day 7, and was predicted to be attained approximately 5 weeks after once daily dosing. The observed prolonged plasma t ½ was consistent with slow absorption from the lung, demonstrating that a typical lung-dominant process was controlling systemic kinetic behavior. The t max data across the cohorts suggested rapid exposure to the respiratory epithelium following the inhaled delivery.
TA B L E 6 Summary of mean plasma C max and mean lung tissue concentrations of PC945 in male and female rats following 14 days of inhaled dosing of 2.0, 7. In rat lung samples on Day 15 after 14-day inhaled dosing, mean concentrations of PC945 were approximately proportional to daily doses and >2000-fold (range 2340-5020) higher than the Preferential uptake of antifungal agents into alveolar cells (which are principally macrophages) could be clinically relevant, as macrophages are a host defence mechanism against alveolar infection by Aspergillus and scavenge to remove particulate matter. 7,29 It is well known that all antifungal azoles are potent inhibitors of CYP450 enzymes, leading to a significant risk of interactions with TA B L E 8 Summary of subjects with treatment-emergent adverse events considered by investigators to be related to study drug (First-in-Human Study)   have a much lower drug-drug interaction risk than that of orally administered triazole antifungals.
In the first-in-human study, reported herein, PC945 was well tolerated in healthy subjects at either single doses up to 10 mg or at repeat doses of 5 mg once daily for 7 days in addition to subjects with mild asthma at a single dose of 5 mg. No evidence of irritancy was observed in subjects with mild asthma.
In summary, PC945 was well tolerated at all doses tested and for a duration up to 7 days; no safety signals were identified. The   The data that support the findings of this study are available from the corresponding author upon reasonable request.