Advanced therapeutic inhalation aerosols of a Nrf2 activator and RhoA/Rho kinase (ROCK) inhibitor for targeted pulmonary drug delivery in pulmonary hypertension: design,characterization,aerosolization,in vitro 2D/3D human lung cell cultures,and in vivo efficacy

Preparation of respirable powders by organic solution advanced spray drying (no water) in closed mode

Using conditions similar to those previously reported,^47,48 advanced organic solution spray drying processing in the absence of water was performed using a Büchi B-290 Mini Spray Dryer with a high performance cyclone in closed-mode using UHP dry nitrogen gas as the atomizing and drying gas and connected to a B-295 Inert Loop (Büchi Labortechnik AG, Flawil, Switzerland). Sim was spray dried under: (a) feed concentration (FC) (0.1% w/v and 0.5% w/v in methanol); and (b) pump rate (PR) (25%, 50%, 75% and 100%). The feed solutions were prepared by dissolving the components in methanol using a Branson 7500 ultrasonicator to assist the dissolution. Table 1 lists the spray drying parameters and Table 2 lists the outlet temperatures. The SD particles were separated from the nitrogen drying gas in the high-performance cyclone and collected in a small sample collector. All SD powders were carefully stored in sealed scintillation glass vials and stored in sealed desiccators over indicating Drierite/Drierite desiccant at −20°C.

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Table 1.

Spray drying parameters for particle engineering design.

Spray drying parameter	Value
Inlet temperature	150°C
Aspirator rate	100% (40 m3/hour)
Pump rate	25% (7.5 mL/min) 50% (15 mL/min) 75% (22.5 mL/min) 100% (30 mL/min)
Gas flow	670 L/hour (55 mmHg)
Feed solution concentration	0.1% w/v 0.5% w/v
Solvent	Methanol
Atomizer and drying gas	UHP nitrogen
Nozzle type and diameter	Stainless steel (0.7 mm)

UHP; ultra-high purity.

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Table 2.

Spraying drying pump rates and feed solution concentrations with corresponding outlet temperatures.

System composition	Outlet T (°C)
Raw Sim	N/A
0.1% w/v SD Sim (25% PR)	80
0.1% w/v SD Sim (50% PR)	67
0.5% w/v SD Sim (25% PR)	86–89
0.5% w/v SD Sim (50% PR)	71–72

PR, pump rate; SD, spray dried; Sim, simvastatin.

Scanning electron microscopy

Using conditions similar to previously reported^47,49 visual imaging and analysis of particle size, particle morphology (shape), and surface structure were achieved by scanning electron microscopy (SEM) using a FEI Inspect S microscope (FEI, Brno, Czech Republic). Samples were placed on double-sided adhesive carbon tabs (TedPella, Inc., Redding, CA, USA), which were adhered to aluminum stubs (TedPella, Inc.) and were coated with a gold thin film using a Hummer 6.2 sputtering system from Anatech (Union City, CA, USA). The coating process was operated at 15 AC milliAmperes with about 7 kV of voltage for 90 s. The electron beam with an accelerating voltage of 30 kV was used at a working distance of 9–12.5 mm. Several magnification levels were used.

Particle sizing and size distribution image analysis using SEM micrographs

The mean size, standard deviation, and size range were determined using SigmaScan Pro 5.0.0 (SYSTAT Software, Inc., San Jose, CA, USA) based on their scanning electron micrographs using a similar procedure that we have previously reported.^49,50 Representative micrographs for each SD powder at 3000× magnification were analyzed by measuring the diameter of at least 100 particles per sample which is the same number of particles as previously reported.^48–51

X-Ray powder diffraction

Using conditions similar to those previously reported,^47,49 the degree of long-range molecular order (crystallinity) of all powders was measured by X-ray powder diffraction (XRPD). XRPD patterns of samples were collected at room temperature with a PANalytical X’pert diffractometer (PANalytical Inc., Westborough, MA, USA) equipped with a programmable incident beam slit and an X’Celerator Detector. The X-ray radiation used was Ni-filtered Cu Kα (45 kV, 40 Ma, and λ = 1.5418 Å). Measurements were taken between 5.0° and 60.0° (2θ) with a scan rate of 2°/min. The powder samples were loaded on zero background silicon sample holder.

Differential scanning calorimetry

Using conditions similar to those previously reported,^47,49 thermal analysis and phase transition measurements were performed on a TA Q1000 differential scanning calorimeter (DSC) (TA Instruments, New Castle, DE, USA) equipped with T-Zero technology, a RSC90 automated cooling system, and an auto sampler. The instrument was previously calibrated with indium. Approximately 1–5 mg of powder was placed into an anodized aluminum hermetic DSC pan. The T-Zero DSC pans were hermetically sealed with the T-Zero hermetic press (TA Instruments). For all the experiments, an empty hermetically sealed aluminum pan was used as reference. UHP nitrogen was used as the purging gas at a rate of 40 mL/min. The samples were heated from at least 0.00°C to 200.00°C at a scanning rate of 5.00°C/min. All measurements were carried out in triplicate (n = 3).

Hot-stage microscopy under cross-polarizers

Hot-stage microscopy (HSM) was performed using a Leica DMLP cross-polarized microscope (Wetzlar, Germany) equipped with a Mettler FP 80 central processor heating unit and Mettler FP82 hot stage (Columbus, OH, USA). Using similar conditions as previously reported,^47,49 samples were fixed on a glass slide and heated from 25.0°C to 200.0°C at a heating rate of 5.00°C/min. The images were digitally captured using a Nikon Coolpix 8800 digital camera (Nikon, Tokyo, Japan) under 10× optical objective and 10× digital zoom.

Karl Fischer titration

Using conditions similar to those previously reported,^47,49 the residual water content of all powders was quantified analytically by coulometric Karl Fischer titration (KFT) using a TitroLine 7500 trace titrator (SI Analytics, Weilheim, Germany) following similar conditions reported in other studies. Approximately 1–5 mg of powder was added to the titration cell containing Hydranal Coulomat AD reagent.

Raman spectroscopy

Using similar conditions to those previously reported, ⁴⁹ Raman ⁵² spectra were obtained at 514 nm laser excitation using a Renishaw InVia Reflex (Gloucestershire, UK) at the surface using a 20× magnification objective on a Leica DM2700 optical microscope (Wetzlar, Germany). This Renishaw system has a 2400 l/mm grating, with a slit width of 65 μm and a thermoelectrically cooled Master Renishaw CCD detector. The laser power was adjusted to achieve 5000 counts per second for the 520 cm⁻¹ line of the internal Si Reference. Raman spectra were achieved using 1% of laser power, and 10 seconds of exposure in all samples.

Attenuated total reflectance-Fourier-transform infrared spectroscopy

A Nicolet Avatar 360 Fourier-transform infrared (FTIR) spectrometer (Varian Inc., CA, USA) equipped with a DTGS detector and a Harrick MNP-Pro (Pleasantville, NY, USA) attenuated total reflectance (ATR) accessory was used for this kind of spectroscopy. Each spectrum was collected for 32 scans at a spectral resolution of 2 cm⁻¹ over the wavenumber range of 4000–400 cm⁻¹. A background spectrum was carried out under the same experimental conditions. Spectral data were acquired with EZ-OMNIC software. These conditions are similar to those in previous reports.^47,49

In vitro aerosol dispersion performance

According to USP Chapter <601> specifications on aerosols, ⁵³ the aerosol dispersion performance of SD Sim formulations were tested using the next generation impactor (NGI) (MSP Corporation, Shoreview, MN, USA) with a stainless steel induction port (USP throat) attachment (NGI model 170; MSP Corporation) equipped with specialized stainless steel NGI gravimetric insert cups (MSP Corporation). Three different FDA-approved human DPI unit-dose capsule-based devices an airflow rate (Q) of 60 L/min were used which were the HandiHaler (Boehringer Ingelheim, Ingelheim, Germany), the NeoHaler (Novartis AG, Stein, Switzerland), and the Aerolizer (Novartis Pharma AG, Basel, Switzerland) which varied in shear stress from highest to medium to lowest, respectively. Using similar conditions described previously,^47,49 Q was adjusted and measured before each experiment using a Copley DFM 2000 digital flow meter (Copley Scientific, Nottingham, UK). The NGI was connected to a Copley HCP5 high capacity vacuum pump (Copley Scientific, Nottingham, UK) with a Copley TPK 2000 critical flow controller (Copley Scientific, Nottingham, UK).

For the NGI, Q = 60 L/min, the D_a50 aerodynamic cut-off diameter for each NGI stage was calibrated by the manufacturer and stated as: stage 1 (8.06 µm); stage 2 (4.46 µm); stage 3 (2.82 µm); stage 4 (1.66 µm); stage 5 (0.94 µm); stage 6 (0.55 µm); and stage 7 (0.34 µm). For each NGI stage, powder deposition was quantified gravimetrically with A/E 55 mm diameter glass fiber filters (PALL Corporation, Port Washington, NY, USA) and 75 mm diameter glass fiber filter (Advantec, Japan) for stage 1. Inhalation grade capsules were used and were clear hydroxypropylmethylcellulose (HPMC) size 3 (Qual V, Qualicaps, NC, USA). Each inhalation capsule contained ~10 mg of powder. Three inhalation capsules were used in each experiment. Under ambient conditions, in vitro aerosolization was evaluated in triplicate (n = 3).

Several aerosol dispersion parameters were determined. The emitted dose (ED) was determined as the difference between the initial mass of powder loaded in the capsules and the remaining mass of powder in the capsules following the aerosolization. The ED (%) in Equation 1 was used to express the percentage of ED based on the total dose (TD) used. The fine particle dose (FPD) was defined as the dose deposited on NGI stages 2–7. The fine particle fraction (FPF %) in Equation 2 was expressed as the percentage of FPD to ED. The respirable fraction (RF %) in Equation 3 was used as the percentage of FPD to total deposited dose (DD) on all NGI stages.

E m i t t e d d o s e f r a c t i o n ( E D % ) = E D T D × 100 %

(1)

F i n e p a r t i c l e f r a c t i o n ( F P F % ) = F P D E D × 100 %

(2)

R e s p i r a b l e f r a c t i o n ( R F % ) = F P D D D × 100 %

(3)

The mass median aerodynamic diameter (MMAD) of aerosol particles and geometric standard deviation (GSD) were calculated using a Mathematica (Wolfram Research, Inc., Champaign, IL, USA) program written by Dr. Warren Finlay.

In vitro 2D human cell culture

The drug dose effect on cell viability was analyzed at different drug concentrations using similar conditions previously reported.^50,54 The A549 pulmonary cell line is a human alveolar epithelial lung cell line and is also used as a model of the alveolar type I pneumocyte.^55–57 The H358 pulmonary cell line is a human bronchioalveolar epithelial cell line similar to alveolar type II cells and expresses lung surfactant associated protein A (SP-A).^55,56 These cell lines were grown in a growth medium including DMEM, advanced 1×, 10% (v/v) FBS, Pen-Strep (100 U/mL penicillin, 100 µg/mL), Fungizone (0.5 µg/mL amphotericin B, 0.41 µg/mL sodium deoxycholate), and 2 mM l-glutamine in a humidified incubator at 37°C and 5% carbon dioxide (CO₂).

After confluence, A549 and H358, cells were seeded in 96 black well plates at a concentration of 5000 cells/well and 100 μL/well. They were incubated for 48 h to allow attachment to the surface of the plates. Cells were then exposed to different concentrations of the Raw and SD formulations. The drug solutions were prepared by dissolving the powders in 10% ethanol and 90% of non-supplemented DMEM media. A volume of 100 µL of the different drug solution concentrations or control solution (10% ethanol and 90% advanced DMEM) were added to each well. Seventy-two hours after exposure under incubation at 37°C and 5% CO₂, 20 μL of 20 µM resazurin sodium salt was added to each well and incubated for 4 h. At this point, the fluorescence intensity of the resorufin (metabolite) produced by only viable cells was detected at 544 nm (excitation) and 590 nm (emission) as previously reported, using the Synergy H1 Multi-Mode Reader (BioTek Instruments, Inc., Winooski, VT, USA). The relative viability of the cell lines was calculated as follow by Equation (4):

R e l a t i v e v i a b i l i t y ( % ) = S a m p l e f l u o r e s c e n c e i n t e n s i t y C o n t r o l f l u o r e s c e n c e i n t e n s i t y × 100 %

(4)

The statistical method used to compare the relative viability between the treated versus the non-treated cells was analysis of variance (ANOVA) using SigmaPlot 13 (SYSTAT Software, Inc,, San Jose, CA, USA).

In vitro TEER at the ALI on particle exposure to lung epithelial cells

Calu-3, a human lung epithelial cell line derived from bronchial submucosal airway region, was used as a model for monolayer integrity in the upper airways and is known to form tight junctions. Using previously reported similar conditions,^50,54 cells were grown in a growth medium including EMEM, 10% (v/v) FBS, Pen-Strep (100 U/mL penicillin, 100 µg/mL), Fungizone (0.5 µg/mL amphotericin B, 0.41 µg/mL sodium deoxycholate) in a humidified incubator at 37°C and 5% CO₂. After confluence, the cells were seeded at a concentration of 500,000 cells/mL in Costar (Costar 3460, Corning, NY, USA) Trans-well inserts (0.4 μm polyester membrane, 12 mm for a 12-well plate) from Fisher Scientific (Hampton, NH, USA) with 0.5 mL of media on the apical side and 1.5 mL of media on the basolateral side. Media was changed every other day from the basolateral side. After approximately 1 week of growth, when the cells looked packed and a complete monolayer was visible under the microscope, transepithelial electrical resistance (TEER) values were measured using an EndOhm 12 mm Culture Cup (World Precision Instruments, Sarasota, FL, USA). TEER values of 1000 Ω.cm², were an indicator of a confluent monolayer at liquid-covered culture (LCC). At this point, the media was removed from the apical side in order to facilitate air-liquid interface (ALI) conditions. The TEER responses of the cells were also measured with an EndOhm 12 mm Culture Cup (World Precision Instruments, Sarasota, FL, USA). For TEER measurements at ALI, 0.5 mL of media was added to the apical side of the each Transwell insert 30 min before the measurement and then immediately removed to return the cells to ALI conditions. After the TEER values reached 500 Ω.cm² (indicating a confluent monolayer at ALI conditions), the cells were exposed to 100 µM of representative SD formulations dissolved in 90:10 media:ethanol to facilitate dissolution. The liquid aerosol formulations were delivered to the Calu-3 cells at ALI using a Penn-Century MicroSprayer Aerosolizer model IA-1B (Penn-Century, Inc., Wyndmoor, PA, USA).^50,55 TEER values were then recorded after 3 h of exposure and then recorded every 24 h up to 7 days after drug exposure using an EndOhm 12 mm Culture Cup (World Precision Instruments, Sarasota, FL, USA), as previously reported.^50,54

The statistical method used to compare the TEER values between the treated versus the non-treated cells was ANOVA using SigmaPlot 13 (SYSTAT Software, Inc., San Jose, CA, USA).

In vitro cell dose response assay in 3D cell culture at the ALI

The 3D small airway epithelia (SmallAir, Epithelix, Geneva, Switzerland) reconstituted in vitro using primary small airways human cells were fully differentiated and functional. The cells were received in 24-well Transwell inserts in a gel matrix. Once the cells were received, they were transferred into a new 24-well plate with 700 µL of the SmallAir media in the basal surface. Media was changed every other day.

After 3 days of incubation at 37°C and 5% CO₂, experiments were performed. For the in vitro cell dose response, the cells were exposed to different concentrations of the drug formulation dissolved in 90:10 media:ethanol to facilitate dissolution. After 72 h of incubation, the inserts were rinsed with a 6 µM Resazurin solution in order to eliminate the remaining red phenol from the cell growth media. The inserts were transferred to a new 24-well plate filled will 500 µL/well of Resazurin solution and then 200 µL/well was added in the apical surface. After 1 h of incubation, 100 µL from the apical side was transferred to a 96 black well plate. At this point, the fluorescence intensity of the resorufin (fluorescent metabolite) produced by viable cells was detected at 544 nm (excitation) and 590 nm (emission) using the Synergy H1 Multi-Mode Reader (BioTek Instruments, Inc., Winooski, VT, USA). The relative viability of the cell line was calculated with Equation 4. This protocol was provided by the vendor. ⁵⁸

In vitro TEER analysis upon particle exposure to 3D human small airway epithelia at the ALI

As described above, after receiving the cells, they were transferred into a new 24-well plate pre-filled with 700 µL of SmallAir media on the basal side. After 3 days of incubation the experiments were performed. Cells were exposed to a different concentration of drug solution (1000 µM, 100 µM and 50 µM). TEER values were measured using EVOMX (Epithelial VoltOhmMeter) and electrode (STX2) (World Precision Instruments, Sarasota, FL, USA). To measure TEER, 200 µL of the cell media was added to the apical surface of the inserts. The long part of the electrode was inserted through the gap of the insert and leaned on the bottom of the well, and the short stem was above in the apical surface, inside the culture media. TEER values were obtained before exposure to the drug solution and after exposure to them. The response was measured after 3 h of exposure and then every 24 h for 5 days. Every time the TEER measurement was finished, the media was removed from the apical surface in order to leave the cells in ALI conditions. This was followed by the protocol given by the vendor. ⁵⁸

In vivo healthy rat study

Fifteen male Sprague–Dawley rats with a body weight of 220–270 g and 3 weeks of age were purchased from Charles River Laboratories International, Inc. (Wilmington, MA, USA), for this study. Rats were housed in the University of Arizona animal care (UAC) facility for at least 1 week before being used in the experiments. Animals were kept in a 12-hour light/dark cycle at an ambient temperature of 22°C and received standard rodent food and water ad libitum. All experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Arizona (IACUC). The local delivery of the aerosols was done non-invasively without any incision into the animal using the Penn-Century Dry Powder Insufflator model DP-4R (Penn-Century Inc., Wyndmoor, PA, USA), which is customized to the respiratory tract of the rat. Three groups with five rats per group were defined for the study which were the following: (a) control (air); (b) SD Sim 25% PR; and (c) SD Sim 50% PR. A single dose of 10 mg/kg of SD Sim 25% PR and SD Sim 50% PR formulations was administered under local short-acting anesthesia using 1–5% isoflurane diluted in air or oxygen. The rodent insufflator atomizer was placed on the tongue of the animal and slowly fed down into the larynx/trachea region located at the front of the esophagus/neck area to just above the first bifurcation of the bronchi. A small-animal rodent laryngoscope (Penn-Century Inc., Wyndmoor, PA, USA) was used directly to visualize the trachea of the rats. The particles were gently puffed into the lungs of the animals using the small-animal air pump (Penn-Century Inc., Wyndmoor, PA, USA) and the atomizer portion of the insufflator was gently removed from the throat of the animal. Total aerosol treatment took approximately 5–7 min per rat. Rats were closely monitored after recovery to be sure they have no difficulty with respiration, even though this was not expected.

After 3 days, animals were euthanized by an anesthetic cocktail overdose of 100 mg/kg ketamine/10 mg/kg xylazine by intraperitoneal injection which was followed by thoracic incision to remove the lungs, collect terminal blood, and bronchoalveolar lavage fluid (BALF) by three times flushing with isotonic saline. The lungs and respiratory tree were removed and fixed for histopathology.

Enzyme-linked immunosorbent assay

The frozen BALF was thawed on ice and centrifuged at 2000 g for 60 min at 4°C. The concentrations of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in the BALF supernatants were measured by sandwich enzyme-linked immunosorbent assay (ELISA) using pair-matched antibodies according to the manufacturer’s instructions. The ELISA kits were obtained from eBioscience, Inc. (San Diego, CA, USA) and R&D (Minneapolis, MN, USA). ⁴⁵

The statistical method used to compare the concentrations of IL-6 and TNF-α in the BALF supernatants of the treated versus the non-treated rats was ANOVA using SigmaPlot 13 software (SYSTAT Software, Inc., San Jose, CA, USA). Three repeats (i.e. triplicates) of the ELISA for each rat were done.

Tissue processing and histological analysis

Whole lungs were taken from rats under terminal anesthesia, as described in the previous section. The whole lungs were inflated with 10% of formalin and immersed in the same solution. The intact whole lungs in formalin were sent in 15 mL centrifuge tubes sealed with parafilm at room temperatureto HistoWiz, Inc. (Brooklyn, NY, USA), an expert professional histology service vendor, where the intact whole lungs were paraffin-embedded, sectioned horizontally into 5 µm slices, and hematoxylin and eosin (H&E) stained using their standard procedure.

In vivo efficacy in the shunt lamb model of PH

A lamb model of PH associated with increased pulmonary blood flow and pressure (PBF) was created, as described.^59,60 At 4 weeks of age, a shunt lamb was anesthetized and dosed with SD Sim (25% PR) as aerosol in a dose of 6.5 mg/kg. Endothelial function was demonstrated by measuring the pulmonary vascular resistance (PVR) in response to acetylcholine (Ach). The Penn-Century Microsprayer Aerosolizer (Penn Century, Inc., Wyndmoor, PA, USA) was used to deliver simvastatin. The lamb study was performed at UCSF under an approved animal safety protocol.

Statistical analysis

Design of experiments was conducted using Design-Expert 8.0.7.1 software (Stat-Ease Corporation, Minneapolis, MN, USA). A multi-factorial design for SD Sim was utilized for in vitro aerosol testing. Interaction of process parameters and the different devices were evaluated using the ANOVA test performed using Design-Expert software. The different interactions on the performance of the formulations were evaluated using the 3D surface plots generated from Design Expert. All experiments were performed at least in triplicate (n = 3). Results are expressed as mean ± standard deviation.