Compound Solubility and Dissolution Profiles.

Saturation solubility studies were performed in simulated lung fluid, a phosphate-buffered saline containing lecithin and bovine serum albumin at naturally occurring lung lining fluid pH. The amount of three different salt forms of GSK2292767 (version A = free base, version C = sodium salt, and version K = cinnamate salt) and nemiralisib added into the media was excessive, wetted by sonication for 5 minutes, and all suspensions were kept shaking at 100 rpm on an orbital shaker (37°C) for 24 hours. The suspensions were centrifuged at 14,000 rpm for 10 minutes and filtered through a 0.22 µm polyvinylidene difluoride filter.

For dissolution testing, an abbreviated Next Generation Impactor (NGI) was used to collect dose. The fine particle fraction of micronized GSK2292767 salt forms was collected above next-generation impactor stage 3 (Riley et al., 2012) onto a filtrete for aerosol dissolution. Micronized active pharmaceutical ingredients were filled into gelatin capsules and dispersed with a HandiHaler (Boehringer Ingelheim) into the next-generation impactor at a 60 l/min airflow rate with 4-second exposure. A previously reported USP intravenous apparatus was used to perform aerosol dissolution work (Davies and Feddah, 2003) with simulated lung fluid as the dissolution media. After dose collection, the drug-loaded filtrete was transferred to the dissolution cell, placed in the dissolution oven, and allowed to equilibrate. Dissolution testing was performed at 37°C with a simulated lung fluid flow rate of 1 ml/min. At the end of the experimental procedure all parts of the dissolution cell were washed with a defined amount of solvent [acetonitrile:water (50:50)] to determine total recovery.

All saturation solubility and dissolution samples were quantified via high-performance liquid chromatography. High-performance liquid chromatography analysis was performed using an Agilent 1100 system with UV-visible detection.

Cell Permeability.

GSK2292767A and nemiralisib (GSK2269557B, HCl salt form) were applied to the apical side of a confluent monolayer of Madin-Darby canine kidney cells transfected with the efflux transporter MDR1 (MDR1/Madin-Darby canine kidney II cells), in duplicate at a concentration of 3 µM Dulbecco’s Modified Eagle’s Medium (DMEM) buffer at pH 7.4 as both donor and receiver solutions. Incubations were carried out in an atmosphere of 5% CO₂ with relative humidity of 95% at 37°C for 90 minutes. The permeability was measured from the apical to the basolateral side of the cell membrane in the presence of the MDR1 inhibitor GF120918A, (N-(4-(2-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)phenyl)-5-methoxy-9-oxo-9,10-dihydroacridine-4-carboxamide). Quantification of parent compound in transport media was determined following analysis by liquid chromatography–tandem mass spectrometry (LC-MS/MS).

Lung Tissue Binding.

All animal studies were ethically reviewed and carried out in accordance with the Animals (Scientific Procedures) Act 1986 and the GlaxoSmithKline policy on the care, welfare, and treatment of animals.

The binding of GSK2292767A and nemiralisib (GSK2269557B) was measured in male CD rat lungs and in female BALB/c mouse lungs and blood in vitro, using rat and mouse lung homogenate and mouse whole blood, by rapid equilibrium dialysis at the nominal concentration of 1 µg/ml. The test compound was spiked into lung homogenate diluted 1:9, with dialysis buffer (PBS −100 mM sodium phosphate + 150 mM sodium chloride, pH 6.9–7.2) or whole blood (diluted 1:1 with dialysis buffer) and incubated for 4 hours at 37°C in five replicates using rapid equilibrium dialysis plates (Thermo Scientific, Rockford, IL). The matrix was on one side on the dialysis membrane with the dialysis buffer on the other. All buffer, lung homogenate, and blood samples (50 µl) were matrix matched by diluting 2-fold with control lung homogenate, blood, or buffer, and the resultant samples were extracted by protein precipitation. The lung homogenate and blood binding of GSK2292767A was determined by peak area ratio following LC-MS/MS analysis. The lung tissue binding was then determined using an in-house validated method previously described for brain homogenates (Kalvass and Maurer, 2002).

Lung and Blood PK Profiles Following Intranasal Delivery to the Mouse.

Eighteen female BALB/c mice were placed under anesthesia using isoflurane to facilitate intranasal administration of GSK2292767A or nemiralisib (GSK2269557B) at 1 mg base equivalent/kg, as a 50 μl volume of a 0.4 mg/ml suspension formulated in 0.2% Tween 80, which was in saline adjusted to pH 4.5 with 1 M HCl (∼7 μl). Terminal lung and blood samples were collected at 10 and 30 minutes, and 1, 3, 5, and 8 hours after dosing (n = 3 per time point). Concentration-time profiles were used to determine the PK parameters in both blood and lung via noncompartmental analysis. Unbound concentrations were derived by correcting for in vitro binding data generated in mouse whole blood and mouse lung homogenate.

Mouse lungs along with 1 ml water were homogenized with ceramic beads using a Precellys 24 homogenizer (Bertin Technologies) for two 20 second cycles at 6500rpm separated by a 10 second pause, with a further 1 ml water then added. Lung homogenates and blood samples underwent protein precipitation with 100% acetonitrile containing internal standard followed by centrifugation. Aliquots of the resultant supernatants were dried under heated nitrogen (37°C), and then reconstituted in 200 µl of acetonitrile:water (10:90 v/v) before being analyzed by LC-MS/MS.

Lung Retention Profiles Following Inhaled Delivery to the Rat.

The PK in lung was measured for the free base, sodium salt, and cinnamate salt forms of GSK2292767 after a single administration of each version as a micronized dry powder, blended to 15% active pharmaceutical ingredients with lactose, to the male Wistar Han rat at a nominal dose of 0.6 mg/kg. For each version, the drug was delivered via an aerosolized dust generation inhalation tower connected to a small cup Wrights Dust Feeder II aerosol generator, with a target inhalation period of 20 minutes. Twenty-one rats were exposed for each study, with termination (n = 3 rats/time point) scheduled at the following time points measured from the end of the aerosol delivery: −10 (half-way through drug administration), and 0 minutes immediately after dosing and 2, 4, 8, 12, and 24 hours. Aqueous tissue homogenates were obtained from all lung samples, using a Precellys 24 device (two 20 second cycles at 6500 rpm separated by a 10 second pause).

The PK in lung was measured for nemiralisib (GSK2269557H, hemisuccinate salt), after a single intratracheal administration by Penn-Century insufflation as a micronized dry powder, blended to 15% active pharmaceutical ingredients with lactose, to the male Brown Norway rat at a nominal dose of 0.67 mg/kg. Nine rats were exposed in total, with termination and collection of lungs (n = 3 rats/time point) scheduled at the following times: 0 minutes immediately after dosing 7 and 24 hours. Aqueous tissue homogenates were obtained from all lung samples, using a Precellys device as described previously. Levels of GSK2292767A and nemiralisib were measured in lung homogenate samples prepared by protein precipitation followed by LC-MS/MS analysis.

House Dust Mite Rechallenge Model of Pulmonary Inflammation.

Female BALB/c mice were sensitized to house dust mite [(HDM), Dermatophagoides pteronyssinus; Greer Laboratories, Lenoir, NC] by intranasal dosing, once a day for 5 days a week over a 21-day period with 25 µg HDM extract (Greer Laboratories). Pulmonary inflammation was then allowed to resolve until day 33. Inflammation was then reinitiated by an intranasal rechallenge of HDM using 100 µg of HDM. GSK2292767A and nemiralisib (GSK2269557H) were administered as repeat daily doses of inhaled dry powder lactose blends for 7 days prior to rechallenge and their effect on inflammatory eosinophils was ascertained by flow cytometry on bronchoalveolar lavage (BAL) samples.

First Time in Human Subject Population.

This first time in human study (GSK Protocol 202062, NCT03045887) was conducted at the Clinical Unit Cambridge (Cambridge, UK), from February 2017 to August 2017. Male and female (nonchildbearing potential) subjects between the ages of 18 and 50 were eligible to be recruited, if they were deemed to be healthy by an experienced physician, had a body mass index within the range of 18–31 kg/m², and had blood chemistry, electrocardiogram, and spirometry results within normal range. Subjects were required to be current daily cigarette smokers and had to be able to produce >100 mg of sputum on saline induction at screening. Subjects were excluded if they had significant ongoing medical conditions, a recent history of asthma, or currently had asthma. Written informed consent was obtained prior to study participation. The study was conducted in accordance with the Declaration of Helsinki, and relevant ethics committee/institutional review boards and regulatory authorities reviewed and approved the study protocols.

Study Design.

This study was a single-center, double-blind, placebo-controlled two-part study to evaluate the safety, tolerability, PK, and PD of GSK2292767A in healthy participants who smoke cigarettes. Part A (n = 16 per cohort) used a two interlocking cohort, three treatment period crossover design to evaluate ascending single doses (50, 100, 200, 500, 1000, and 2000 μg) of GSK2292767A or placebo, with a 4-week washout period between doses. Part B used a parallel group design (n = 12; 3:1 ratio; note: 11 subjects from part A participated in part B) to evaluate the highest tolerated dose from part A (2000 μg) or placebo for 14 days of repeat dosing. In both parts, GSK2292767A was administered as a dry powder blended with lactose, and 1% magnesium stearate using an ELLIPTA dry powder inhaler; placebo (lactose only) was administered using a matching device.

In part A, hypertonic saline–induced sputum samples were collected 3 and 24 hours after administration of inhaled treatment and blood samples for PK analysis were collected at predose, 5, 30, and 45 minutes, and 1, 2, 3, 4, 6, 8, 12, and 24 hours postdose. In part B, hypertonic saline–induced sputum samples were collected predose, 3 hours postdose on day 1, and 3 and 24 hours postdose on day 12 of treatment. Blood samples for PK analysis were collected predose and 5 minutes postdose on days 2–13 inclusive, and predose, 5, 30, and 45 minutes, and 1, 2, 3, 4, 6, 8, and 12 hours postdose on days 1 and 14. Bronchoscopy for the collection of BAL samples was performed on day 15 at 24 hours (trough) after the final (day 14) dose. Saline washes (3 × 50 ml) were used for the lavage procedure via a bronchoscope that was positioned at the second bifurcation of the right middle lobe, and each return was recorded and kept separately on ice prior to processing.

Blood Sample for PK.

For the PK analysis of GSK2292767A, whole blood samples of approximately 3 ml were collected for measurement of plasma concentrations of GSK2292767A. Blood samples were collected via venipuncture or a cannula into uniquely labeled 3 ml K2EDTA collection tubes (Greiner) and placed in ice water, centrifuged, and refrigerated at approximately 1500g for 10 minutes at 4°C. Resultant plasma was transferred into Nunc tubes (ThermoFisher Scientific) and stored at −20°C before shipment to the bioanalysis site.

BAL Sample for PK.

BAL supernatant and cells were prepared by centrifuging the BAL at 750g for 10 minutes. The supernatant was then removed (BAL supernatant), and the remaining pellet was reconstituted in deionized water as BAL cells. The volume of deionized water for reconstitution was equivalent to 20% of the recovered lavage volume following bronchoscopy. Prior to use, the reconstituted pellet was frozen at −80°C to ensure complete lysing of the cells and stored at −80°C. The reconstituted BAL cell sample was defrosted thoroughly and mixed prior to analysis for total cell fraction concentration and intracellular drug level detection.

Drug Concentration Measurements in Plasma and BAL Cells and Fluid.

Human plasma, BAL supernatant, and BAL cell pellet samples were analyzed for GSK2292767A using a validated analytical method based on protein precipitation, followed by high-performance liquid chromatography (LC-MS/MS) analysis. The lower limit of quantification was 20 pg/ml and the higher limit of quantification was 10,000 pg/ml when using a 50 µl aliquot of human plasma, lung BAL supernatant, and BAL cell pellet.

Human plasma and lung BAL supernatant samples were analyzed for urea by a standard validated analytical method based on the Roch-Ramel enzymatic reaction, using the urease and glutamate dehydrogenase methodology on the Siemens Advia 1800 chemistry analyzer. In plasma, the lower limit of quantification was 0.2 mmol/l with a higher limit of quantification of 239.5 mmol/l, and in the BAL supernatant the lower limit of quantification was 0.01 mmol/l with a higher limit of quantification of 2.8 mmol/l.

PK Analysis of Concentration Data.

Individual plasma GSK2292767A concentration-time data using actual times were analyzed using Phoenix WinNonlin (Certara Inc.) to derive PK parameters, including maximum observed plasma concentration (C_max), time to C_max (t_max), trough concentration (C_min), and area under the plasma concentration-time curve (AUC_(0–t)). Assessment of dose proportionality was carried out for the plasma PK data using a power model. An estimate of the mean slope of log_e (dose) was reported for C_max and AUC_(0–t), along with corresponding 90% confidence intervals (CIs). Further exploratory analysis was performed using the ANOVA method after normalizing the parameters by a chosen nominal dose of 100 µg. Adjusted geometric means and 90% CIs were calculated for each dose along with estimated treatment ratios and corresponding 90% CIs.

Assessment of accumulation for the plasma PK data was carried out using data from the single (day 1) and repeat dose phases in part B. Accumulation ratios were calculated for both peak (geometric mean C_max) and trough (geometric mean C_min) values using the day 14 and 1 values. Peak:trough ratios were also calculated for days 1 and 14.

No formal assessment of steady-state was performed. Visual analysis of the concentration-time profiles taken daily during the studies was used to assess the achievement of steady-state concentration in the plasma of dosed subjects.

BAL samples (cells and supernatant) were analyzed for GSK2292767A concentrations for derivation of lung epithelial lining fluid (ELF) and cell pellet concentrations (part B only). Urea levels were measured in the BAL supernatant samples together with the corresponding time-matched plasma sample for calculation of the ELF dilution factor (using urea as a dilution marker). The dilution factor was used to multiply the BAL GSK2292767A concentration to derive the volume of ELF within the sample, and subsequently concentrations within ELF. The derived ELF concentrations of GSK2292767A for each BAL wash were calculated separately. A pooled analysis was calculated per subject by calculating the total mass of GSK2292767A in all three washes and dividing by the total volume of ELF within all three washes. The cell pellet concentration (part B only) was derived using the resulting ELF concentration and the ratio of the raw (not urea corrected) sample results between supernatant and cell pellet (reconstituted and lysed).

Analysis of Intracellular Drug Detection.

The intracellular drug assessment was based on the LiveCell mass spectrometry protocol described previously (Masujima, 2009). BAL samples for intracellular mass spectrometry analysis were stored at 4°C and assayed within 24 hours of collection. Briefly, 500 µl was mixed with 1.5 ml of PBS and plated on 35-mm-diameter, 0.17-mm base thickness cell culture dishes and maintained at 37°C for 12 hours. The latter was for the cells to settle at the bottom of the dish, since sampling is dependent on cell adherence to the dish. Intracellular contents were captured using N-150 CT-1 Humanix tips and an Olympus X81 microscope coupled to a Scientifica Motorized Micromanipulator. Sampled tips were frozen at −80°C and kept in dry ice until the mass spectrometry analysis was performed. Just before the analysis, 8 µl of internal standard solution (20 nM fumaric acid and 20 nM taurocholic acid in 50:50 methanol) was added to the back of the Humanix tip and the intracellular contents were analyzed using a Fusion Lumos Orbitrap mass spectrometer using a direct infusion setup. The analysis was performed in full scan–only mode, and the mass resolution of the Orbitrap was set to 250,000. Data were analyzed using Compound discoverer 2.1 using both the KEGG and Chemspider processing nodes.

Phosphatidylinositol-Biphosphate and Phosphatidylinositol-Trisphosphate Detection in Induced Sputum Samples.

Phosphatidylinositol-trisphosphate (PIP3) peak area proportion, i.e., PIP3 peak area/[PIP3 peak area + phosphatidylinositol-biphosphate (PIP2) peak area], was calculated from the mass spectrometer peak areas for PIP3 and PIP2, derived using established methodology (Clark et al., 2011). The PIP3 proportion, using PIP2, was calculated to correct for changes in cell numbers between different sputum samples, assuming PIP2 was correlated with the cell number (Clark, 2011).

Statistical Analysis of PIP3 Proportion.

For study samples, Log_e (PIP3 peak area proportion) was analyzed using mixed effects, repeated measures analyses. Treatment ratios (and 90% CI) of adjusted geometric means for GSK2292767A versus placebo are presented. Statistical models were used to determine Bayesian posterior probabilities (assuming a noninformative prior); a Student’s t test cumulative distribution function was used to obtain the probabilistic statements.