Rix et. al.Pharmacokinetics, Pharmacodynamics, Safety and Tolerability of Nebulized Sodium Nitrite (AIR001) Following Repeat-Dose Inhalation in Healthy Subjects
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SUPPLEMENTARY METHODS
Subject Inclusion/Exclusion Criteria: Healthy male and female subjects between 18 and 55 years of age with clinical laboratory assessments within normal limits were enrolled. Otherwise healthy subjects were excluded from studies of AIR001 if they had any of the following: clinically significant illness (including lower respiratory tract infection) in prior 4 weeks, current smoker or a past history of smoking >10 pack years, history of bronchial asthma or sleep apnea, evidence of restrictive or obstructive lung disease, family history of primary pulmonary hypertension, history of pulmonary embolism, evidence of supine hypertension, hypotension, orthostatic hypotension or significant postural symptoms (dizziness, lightheadedness, vertigo) when going from the supine to the standing position; personal or family history of congenital or acquired methemoglobinemia, RBC CYPB5-reductase deficiency or any hemoglobinopathy; glucose-6-phosphate dehydrogenase (G6PD) deficiency or any contraindication to receiving methylene blue.
Definitions of Adverse Events and Dose Limiting Toxicities (DLT): Adverse events were prospectively documented by the investigators using Common Terminology Criteria for Adverse Events (ctcae) v. 4.0, and classified as mild, moderate, or severe, and as definitely, probably, or possibly related to study drug. The MTD was defined by the occurrence of DLTs of a moderate nature in three or more subjects in a cohort, or a severe DLT seen in one of 3 subjects in a cohort. Severe DLTs were defined as follows: forced expiratory volume (FEV1) or forced vital capacity (FVC) <65% of pre-dose baseline; symptomatic bronchospasm requiring therapy; symptomatic supine systolic or diastolic blood pressure drop of ≥ 30 or 20mmHg, respectively from pre-dose baseline; symptomatic orthostatic systolic or diastolic blood pressure drop of ≥ 30 or 20mmHg, respectively from supine value; incapacitating dyspnea or coughing, hypoxemia (SaO2 ≤ 90%); venous MetHb ≥ 7%; Or any grade 3 or 4 toxicity.
Efficiency of Solo-Idehaler Device: The efficiency of the Solo-Idehaler, defined as the ratio of the emitted dose (delivered at the mouthpiece) to the dose of AIR001 Inhalation Solution loaded into the nebulizer medication chamber, was determined at simulated dosage levels of 4 and 200 mg via in vitro breathing simulation experiments utilizing a large animal ventilator (Harvard Apparatus; Holliston, Massachusetts) with the following parameters: respiratory rate of 15 breaths per minute, a tidal volume of 500 mL, and an inhalation/exhalation ratio of 1:1. Emitted doses were captured on RespirGard-II 303 Bacterial Filters (Vital Signs, Inc.; Totowa, NJ), extracted with ethanol and distilled water and analyzed via HPLC.
Primary LC-Fluorometric Assay for Nitrite in Plasma: In all clinical studies of AIR001, a primary LC-fluorometric assay was utilized for determination of nitrite in heparinized plasma. Plasma samples (50µL) were dispensed into a 0.8mL 96-well plate. Saturated ammonium sulfate solution (100µL) was added to precipitate proteins, and the plate was vortexed and centrifuged. A 50µL aliquot of each sample was transferred to a microtiter plate and 10µL of the derivatizing reagent 1,2-diaminonapthalene (Cayman Chemical, Ann Arbor MI) was added. After incubation for 20 minutes at 37°C, 20µL 2.8mol/L sodium hydroxide was added, followed by 25µL of the internal standard solution (500ng/mL fluorescein). Samples were injected onto a Varian Pursuit C18 column (4.6 x 100mm) using an isocratic methanol/sodium phosphate buffer mobile phase at a flow rate of 0.9 – 1.0mL/min. The excitation and emission wavelength (λ) were set to 494nm and 518nm, respectively, for detection of fluorescein and 365nm and 430nm, respectively, for detection of the nitrite derivative. Due to the endogenous levels of nitrite in “blank” plasma, the assay was based on a proxy calibration relative to ultrapure water. A correction was then made for the combined nitrite contributions of the proxy matrix and the assay. The lower limit of quantitation (LLOQ) of the assay ranged from 0.4 – 0.6 µmol/L, depending on the amount of background nitrite present in the proxy matrix.
Secondary Reductive Chemiluminescence Assays: In Part A of study AIR001-CS04 only, a reductive chemiluminescence assay system was utilized for determination of nitrate metabolite in plasma, as well as determination of R-SNO species (plasma and blood) and Hb(Fe)-NO (blood only) as previously reported [35,36]. Blood samples were collected in heparin flushed syringes and immediately treated with heparin (10units/mL) to prevent clotting. For collection of plasma, blood was immediately centrifuged (750×g for 2 min; 4°C) and plasma removed and flash frozen. For determinations in whole blood, blood was immediately added to a nitrite stabilization cocktail (4 parts blood: 1 part cocktail) consisting of 25mmol/L potassium ferricyanide (K3Fe(CN)6), Nonidet-40 substitute (NP40) detergent (1%v/v), 10mmol/L N-ethylmaleimide (NEM) and 0.1mmol/L diethylenetriaminepentaacetic acid (DTPA - copper chelator). Plasma samples were divided into three 50µl aliquots. The first aliquot was treated with PBS (5µL), the second was treated with acidified sulfanilamide (5%w/v in 1mol/L HCl), and the third underwent addition of mercuric chloride (5mmol/L) followed by acidified sulfanilamide. Each aliquot was then injected into tri-iodide solution purged with helium gas in a vessel connected in line to a Seivers nitric oxide chemiluminescence detector (Agilent Technologies, Santa Clara, CA). Whole blood samples were de-proteinated with ice cold methanol (1:1 v:v) and centrifuged (750×g for 2 min; 4°C). Samples were then subjected to the same treatment as outlined for plasma above. Area under the curve was calculated and the concentration of NO generated was determined by comparison to a standard curve generated with authentic nitrite. The untreated aliquot represents the combined concentration of nitrite, R-SNO and Hb(Fe)-NO species, while the second and third aliquots represent the concentrations of R-NSO + Hb(Fe)-NO and Hb(Fe)-NO, respectively. For nitrate measurements, plasma samples were injected into a vanadium chloride-HCL solution (90°C) which was connected in line to the chemiluminescence detector. Since this method measures the total concentration of nitrite and nitrate, the nitrite concentration measured in tri-iodide solution (above) was subtracted to calculate nitrate concentration in the sample. The LLOQ of the assays were as follows: plasma nitrite and nitrate – 0.002 µmol/L; plasma R-SNO – 0.003 µmol/L; blood nitrite – 0.004 µmol/L; blood R-SNO – 0.004 µmol/L and Hb(Fe)-NO – 0.004 µmol/L.
Bioanalytical Methods for Sildenafil and N-desmethyl Sildenafil: Sildenafil and N-desmethyl sildenafil were quantified in heparinized human plasma using a validated LC-MS/MS assay (PPD, Richmond, VA). Briefly, a 250 μL aliquot of heparinized plasma was fortified with 50 μL of internal standard working solution (containing N-CD3 sildenafil as internal standard for both analytes). Analytes were isolated through solid phase extraction using Oasis HLB SPE cartridges. The eluate was evaporated and the residue reconstituted with 250 μL of mobile phase. The final extract was analyzed via HPLC with tandem MS/MS detection. Samples were stored at -80°C prior to analysis, and freezer stability was independently confirmed to cover the storage period. All acceptance criteria for the runs passed. The LLOQ of the assays was 1.0 ng/mL for both sildenafil and N-desmethyl sildenafil in heparinized human plasma. The validation also included an assessment of assay interference from either nitrite or nitrate at low and high QC concentrations of sildenafil and N-desmethyl sildenafil.
Pharmacokinetic Analysis: Pharmacokinetic analyses were performed on individual subject plasma nitrite concentration versus time data using the non-compartmental analysis function of WinNonlin (ver 5.2, Certara, L.P., St Louis, MO). In studies AIR001-CS01 and AIR001-CS02, where a majority of subjects had quantifiable pre-dose plasma nitrite concentrations, a baseline correction method was applied to the plasma versus time data prior to pharmacokinetic analysis. If the pre-dose value was greater than the assay LLOQ, this value was subtracted from all subsequent data points for that subject. However, if the pre-dose value was less than LLOQ, the LLOQ value was subtracted from all subsequent data points for that subject. Following baseline correction, any negative values were treated as zero for the purpose of calculations. In the case of study AIR001-CS04, baseline nitrite was below the LLOQ for all study subjects, and baseline subtraction was therefore not employed. A similar baseline correction process was employed for nitrate metabolite concentration data. Nominal sampling times were used for calculations, and all time-points were relative to the start of the 10 minute inhalation period. Area under the plasma concentration time curve from pre-dose to the time of the last quantifiable concentration (AUC0-t) was calculated using the linear trapezoidal rule. AUCinf was calculated as the sum of AUC0-t + Clast/λz, where Clast is the last observed quantifiable concentration and λz is the terminal elimination rate constant based on regression of a minimum of three post-tmax concentrations. Terminal half-life (t1/2) was determined from ln2/λz. For multiple dose data from study AIR001-CS04, AUC was reported as AUCtau (ie. AUC0-8h), which was used in calculation of other steady-state parameters. Apparent total clearance (CL/F) was given by [Dose/AUCinf] following a single dose, or [Dose/AUCtau] for repeat-dose data, using the molar (salt corrected) mass of nitrite in the nebulizer medication chamber further corrected for the device efficiency of 76%. The apparent volume of distribution at steady-state (Vss/F) was given by [CL/F·MRT], where MRT (mean residence time) was given by [(AUMC/AUC) – Inhalation time/2] (ie. subtraction of 0.083 hours for the standard 10 minute inhalation). Both CL/F and Vss/F were normalized by the pre-dose body weights of individual subjects in kg. Other processing of descriptive statistics, such as means, standard deviations and significance testing, were performed with Microsoft Excel ver. 2007 (Redmond, WA). Graphics and figures were prepared using Prism ver 6.0 (GraphPad Software, La Jolla, CA).
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