Phase 1 Dose Escalation Study of Aerosolized Pirfenidone in Normal Healthy Volunteers, Smokers, and IPF Patients Ian Glaspole MBBS, PhD FRACP(1) , Jun Keng Khoo MBBS (1), Kelly Otto MS (2), Bruce Montgomery MD (2) (1) Alfred Hospital and Monash University, Melbourne AU, (2) Avalyn Pharma, Seattle WA, USA ABSTRACT RESULTS DISCUSSION BACKGROUND: We evaluated the pharmacokinetics and safety/tolerability of a pirfenidone solution optimized for inhalation using a vibrating plate nebulizer in normal healthy volunteers (NHV), smokers (higher risk for intolerance), and patients with IPF. METHODS: 44 adults in 6 cohorts (n=2 placebo; n=6 pirfenidone; no placebo in BAL and IPF cohorts) were consented to receive single doses of a 12.5 mg/mL solution of pirfenidone administered with an eFlow® Nebulizer; (PARI Pharma GMBH, Starnberg, Germany).  Cohorts 1, 2, and 3 (NHV) received a single ascending dose of 25, 50, and 100 mg pirfenidone, respectively.    Cohort 4 (NHV) received 100 mg of pirfenidone and underwent BAL to measure epithelial lining fluid (ELF) pirfenidone concentrations.   Cohort 5 (smokers), and Cohort 6 (IPF), received 100 mg of pirfenidone. RESULTS: Administration time ranged from 1.4 to 1.8 min/mL.  No clinically relevant adverse effects on respiratory rate, spirometry, or oxygenation were observed.  Drug related adverse events were predominantly cough, n=8/44 (1 in IPF cohort), all mild, transient and not dose limiting.  Mean plasma pirfenidone Cmax levels in the 25, 50, 100 mg NHV, 100 mg smoker, and IPF cohorts were 202, 292, 802, 1370,1016 and 1026 ng/mL respectively. In the BAL cohort, the ELF Cmax was estimated to be 135.9±54.5 µg/mL. In the BAL and IPF cohorts, 24-hour urine excretion of pirfenidone and metabolites data suggests similar amounts of alveolar deposition. . CONCLUSIONS: Aerosol pirfenidone was well tolerated in normal volunteers, smokers and IPF patients. High epithelial lining fluid concentrations were achieved in normal healthy volunteers with a 100 mg nebulizer dose. The 100 mg nebulizer dose had on average a 15-fold lower systemic pirfenidone exposure than reported with oral administration of the licensed 801 mg oral dose. Demographics and doses of each cohort are reported on Table 1; All treatments were well tolerated with no clinically meaningful changes in oximetry, spirometry, vital signs’ biochemistry or hematology; All drug related adverse effects were transient and mild, except for one moderate dizziness occurring 45 minutes after drug administration (Table 1); Delivery time ranged 1.4 to 1.8 minutes per mL of the pirfenidone solution or placebo. 100mg nebulized pirfenidone equates to 1/15 the systemic exposure of the usual oral dose. Following 100 mg nebulized pirfenidone, approximately 45% systemic absorption occurs, as determined by 24 hour urine collection of pirfenidone and metabolites, equating to 45 mg; Following an 801 mg oral dose, systemic absorption is approximately 85%, equating to 680 mg exposure, with a peak plasma mean concentration is 7.9 ug/ml (Rubino et al, 2009). For 100 mg inhaled dose, the ELF Cmax would be on average 35 times that for the usual oral dose. Using 100 mg nebulized pirfenidone, in the BAL cohort, the predicted Cmax ±SD in ELF is 135.9±108 ug/ml; As 50% of plasma pirfenidone is protein bound, the estimated epithelial lining fluid (ELF) Cmax after 801 mg oral dose is 3.9 ug/mL. IPF patients are likely to have higher ELF Cmax than the normal human volunteer cohort. The 24-hour urine carboxypirfenidone (the predominate form of urinary excretion) (mean±SD) was 40.2±20.5 and 42.2±14 in the BAL and IPF cohorts respectively. At peak plasma pirfenidone levels in the BAL and IPF cohorts were 1.4±0.7 and 1.0±0.1 ng/mL respectively. This study supports the potential use of either 25, 50 or 100 mg aerosol doses in future studies. If the target tissue is the alveolar epithelium, the Cmax in the ELF with the aerosol doses would be 8, 17, 35 fold higher than what is achieved with an 801 mg oral dose. In the IPF cohort, due to the decreased lung surface area from disease and slower absorption had higher ELF Cmax than the normal human volunteer cohort. Table 1: Demographic Characteristics and Drug Related Treatment Emergent Adverse Events   All Placebo Subjects (N=8) Cohort 1: Active 25 mg (N=6) Cohort 2: 50 mg Cohort 3: 100 mg Cohort 4: Cohort 5: (N=6): Cohort 6: Age (years) n 8 6 Mean (SD) 31.1 (12.3) 25.8 (6.2) 24.3 (2.4) 27.8 (4.1) 31.8 (5.9) 44.8 (5.8) 69.5 (4.3) Gender M/F 2/6 2/4 1/5 3/3 4/2 5/1 FVC (L) 3.99 (1.03) 4.17 (1.08) 4.14 (0.86) 4.96 (1.42) 3.90 (1.35) 2.93 (0.83) Subjects with at least one Drug- Related [1] TEAE 3 2 1 Respiratory, thoracic and mediastinal disorders Cough Increased upper airway secretion Dysphonia Nervous system disorders  Dizziness Headache BACKGROUND FVC was not performed in Cohort 4 due to BAL procedure [1] All adverse events were transient. Severity was mild in all events with the exception of dizziness in IPF cohort that was moderate. This event occurred 45 minutes after drug administered. TEAE: Treatment emergent adverse event Dysphonia: classification for hoarseness Aerosol administration of multiple classes of drugs, including bronchodilators, corticosteroids, and antibiotics have been proven to improve both efficacy and safety by increasing delivery to lung tissue and decreasing systemic exposure (Clarke et al 1984). We investigated the safety and pharmacokinetics of a single administration aqueous formulation of pirfenidone delivered by a high efficiency vibrating plate nebulizer to assess whether aerosol delivery of pirfenidone would be suitable for a longer term clinical trial that would assess safety and efficacy. Pirfenidone Plasma Pharmacokinetics The pirfenidone plasma pharmacokinetics are presented in Table2. Nebulized pirfenidone was rapidly absorbed and cleared: Cmax occurring usually at the 10 minute time point and pirfenidone levels were below the level of quantification at 24 hours. The mean Cmax increased with dose. The volunteers with smoking history and IPF patients had similar Cmax to normal human volunteers. The T1/2 in the IPF patients was longer, likely reflecting slower absorption from the lung rather than slower clearance. Pirfenidone Urine Pharmacokinetics The carboxypirfenidone recovered in urine is shown in Table 2. Carboxypirfenidone represented at least 98% of total pirfenidone and metabolites in every subject urine (Data not shown). This indicates on average about 45% systemic absorption of the nebulizer dose with the eFlow nebulizer system. The predominant amount of pirfenidone was collected in the initial two 6 hour aliquots. CONCLUSIONS MATERIALS AND METHODS Aerosol Pirfenidone is well tolerated by human volunteers, including those with a smoking history and IPF patients; PARI eFlow nebulizer is 40-45% efficient in delivering drug from nebulizer to lung; Systemic exposure from a 100 mg nebulizer dose is 1/15 of that reported for a 801 mg oral dose; High ELF levels are achieved after aerosol administration; Similar systemic absorption is seen in normal volunteers and IPF patients. A phase 1 randomized, double-blind, placebo-controlled, dose-escalation study in volunteers and patients with idiopathic pulmonary fibrosis; 12.5 milligrams/milliliter (mg/mL) pirfenidone, 5 mMol citrate buffer, 150 mMol NaCl, and 0.75 mMol sodium saccharine and water, pH 6.0 or placebo containing 5 mMol citrate buffer, 150 mMol NaCl, 0.225 mMol sodium saccharine at pH 6.0; Delivered via an eFlow® Nebulizer (PARI Pharma GMBH, Starnberg, Germany); 44 adults in 6 cohorts (n=2 placebo; n=6 pirfenidone; no placebo in BAL and IPF cohorts); Cohorts 1, 2, and 3 (NHV) received a single ascending dose of 25, 50, and 100 mg pirfenidone, respectively;   Cohorts 4 (BAL), 5 (smokers) and 6 (IPF) received 100mg pirfenidone; Alfred Hospital Human Research Ethics Committee approved and registered with the Australian New Zealand Clinical Trials registry (ACTRN 12617001501336). Inclusion criteria: Males or females 18 ≤ age ≤ 55 years; Cohort 5 was current or past smokers with > 20 pack year history of smoking.; Cohort 6: Diagnosis of IPF by ATS/ERS/JRS/ALAT criteria, age < 80 years, and for at least two subjects a greater than 20 pack-year smoking history. Exclusion criteria: history of previous allergy or sensitivity to pirfenidone, use of oral pirfenidone in the 3 days prior; history of obstructive airways disease. Measurements: Pirfenidone plasma and urine concentrations: pre-dose, 10 minutes, 30 minutes 1, 2, 4, 6 and 24 (± 2) hours post dose; 24 hour urines, in four six hour collections for pirfenidone concentrations; Bronchoalveolar lavage (cohort 4): as soon as practical after dosing to measure epithelial lining fluid levels. The urea method was used to correct for BAL fluid dilution. Pirfenidone concentrations were determined by HPLC MS/MS method. (personal communication, To estimate the Cmax concentrations in ELF of the BAL cohort immediately post dose,a ELF standard curve of clearance derived from serial BAL’s in a sheep model following inhalation of a pirfenidone formulation was used. (personal communication Lisa Kaminskas ) Table 2: Pirfenidone Cmax, T ½ and Urine Excretion Pirfenidone   Cohort 1: Active 25 mg (N=6) Cohort 2: 50 mg (N=5) Cohort 3: 100 mg Cohort 4: Cohort 5: Cohort 6: n 6 5 Cmax (ng/mL) Mean (SD) 202 (100.0) 292 (228) 803 (605) 1370 (770) 1016 (179) 1026 (118) t ½ (hr) 2.48 (0.346) 3.21 (0.751) 2.34 (0.537) 2.53 (0.701) 2.01 (0.433) 3.87 (1.15) Ae0-241 (mg) 10.4 (5.5) 11.1 (8.4) 49.0 (22.6) 40.2 (21.5) 46.7 (5.3) 42.2 (14.7) REFERENCES Clarke SW, Newman SP. Therapeutic aerosols 2--Drugs available by the inhaled route. Thorax. 1984;39:1-7. Rubino CM, Bhavnani SM, Ambrose PG, et al. Effect of food and antacids on the pharmacokinetics of pirfenidone in older healthy adults Pulmonary Pharmacology & Therapeutics 2009;22: 279–285. Noble PW, Albera C, Bradford WZ,, et al.; CAPACITY Study Group. Pirfenidone in patients with idiopathic pulmonary fibrosis (CAPACITY): two randomised trials. Lancet 2011: 377:1760-1769 Surber MW, Poulin D, McInally K, et al. , Ask K. Inhaled pirfenidone improves animal efficacy through superior pulmonary and vascular pharmacokinetics. Am Thoracic Soc. 2014. A6599. 1Ae0-24 is Carboxy Pirfenidone, the predominate excreted metabolite. Pirfenidone Epithelial Lining Fluid Levels The pirfenidone epithelial lining fluid levels are presented in Table 3. ELF concentrations from the pooled aliquot after correction for dilution by the urea method, and then extrapolated back to immediate post dose assuming after assuming the T ½ of pirfenidone in the ELF is 7.9 minutes. FUNDING Supported by Avalyn Pharma, Seattle WA Table 3: ELF Pirfenidone from Pooled BAL Aliquots Subject Number Ratio Serum Urea BAL Urea Collection Time Post Dose ELF Cmax1 (µg/mL) 1 8.9 46 115.5 2 17.6 33 13.6 3 59.1 336.0 4 173 42 120.3 5 37.4 39 97.7 6 72.7 54 129.4 Mean (SD) µg/mL   135.4 (106.9)