Article pubs.acs.org/molecularpharmaceutics
Deposition, Diffusion and Transport Mechanism of Dry Powder Microparticulate Salbutamol, at the Respiratory Epithelia Mehra Haghi,† Daniela Traini,† Mary Bebawy,‡ and Paul M. Young*,† †
Advanced Drug Delivery Group, Faculty of Pharmacy, The University of Sydney, Sydney, NSW 2006, Australia School of Pharmacy, Graduate School of Health, The University of Technology, Sydney, PO Box 123, Broadway, NSW 2007, Australia
‡
ABSTRACT: The deposition, dissolution and transport of salbutamol base (SB) and salbutamol sulfate (SS) inhalation powders were investigated using the Calu-3 air interface cell culture model and Franz diffusion cell. Drug uptake by cells was studied with respect to deposited dose, drug solubility and hydrophobicity. Furthermore, the role of active transport via organic cationic transporters (OCTs) was studied. SB and SS were processed to have similar diameters (3.09 ± 0.06 μm and 3.07 ± 0.03 μm, respectively) and were crystalline in nature. Analysis of drug wetting, dissolution and diffusion using a conventional in vitro Franz cell (incorporating a cell culture support Transwell polyester membrane) showed diffusion of SB to be slower than that of SS (98.57 ± 4.23 μg after 4 h for SB compared to 98.57 ± 4.01 μg after 15 min for SS). Such observations suggest dissolution to be the rate-limiting step. In comparison, the percentage transfer rate using the air interface Calu-3 cell model suggested SB transport to be significantly faster than SS transport (92.02 ± 4.47 μg of SB compared to 63.76 ± 8.84 μg of SS transported over 4 h), indicating that passive diffusion through the cell plays a role in transport. Furthermore, analysis of SB and SS transport, over a range of deposited doses, suggested the transport rate in the Franz diffusion cell to be limited by wetting of the particle and dissolution into the medium. However, for the cell monolayer, the cell membrane properties regulate the diffusion and transport rate. Analysis of the drug transport in the presence of triethylamine (TEA), a known inhibitor of OCTs, resulted in a significant decrease in drug transport, suggesting an active transport mechanism. The presence of OCTs in this cell line was further validated by Western blot analysis. Finally, the transport of SS from a commercial product (Ventolin Rotacaps) was studied and showed good agreement with the model SS system studied here. KEYWORDS: salbutamol, Calu-3 epithelial cell line, air interface model, organic cation transporters (OCT), dry powder inhaler
1. INTRODUCTION The delivery of dry powder inhaler formulations (DPI) to the respiratory tract, for the treatment of local disease states, is an established therapeutic avenue.1 The human bronchial airways are composed of a range of pseudostratified, ciliated and mucus producing cells, covered with a ca. 8 μm mucus layer, with a volume of approximately ∼1 μL per cm2.2−4 Following deposition in the lung, the aerosol particle interacts with the lining fluid and must dissolve for further absorption and/or transport to achieve a therapeutic outcome. However, the fate of particles after deposition in the lung is not fully understood since there are many complex mechanisms and factors with respect to the local histology of the epithelial surface and physicochemical properties of the particle.5 © 2012 American Chemical Society
To study the fate of particles after deposition in the bronchial regions of the lung, there are two widely accepted in vitro approaches; nonbiological pharmacopeial apparatuses and biological “cell” based approaches commonly used for prediction of in vivo oral drug release and absorption6,7 and cell based in vitro models are used to investigate drug absorption, transport or efflux.8 1.1. The Use of Conventional Noncellular Based in Vitro Apparatuses To Study Drug Dissolution. While Received: Revised: Accepted: Published: 1717
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Figure 1. Chemical structure of salbutamol base and sulfate.
activity in respiratory cell models,26 only a limited number of studies have investigated expression and function of OCTs in cell models of the airway epithelium. OCTs may be important since many common respiratory medicines adopt a positive charge at physiological pH; these include the majority of β2-adrenergic agonists (formoterol and salbutamol)24 and also anticholinergic bronchodilators (ipratropium).27 Five main subtypes of OCTs (OCT1, OCT2, OCT3, OCTN1 and OCTN2) have been detected in healthy human lungs.28 Most studies using the Calu-3 cells have confirmed the presence of these transporters using RT-PCR, with the assumption of protein translation. However, only in few studies has the specific subtype involved in transport across the Calu-3 cell line been identified.24−26 Furthermore, it is important to note that these previous mechanistic studies utilized the liquid covered Calu-3 model, the properties of which differ significantly from those of the air interface Calu-3 model.29 1.4. Drug Delivery Using Air Interface Models. Air interface cell models are more appropriate to study the fate of particles after deposition in the lung since the histology and structure is more consistent with that observed in vivo. Furthermore, these models allow for deposition of aerosol particles directly onto the epithelial surface and measurement of drug dissolution and paracellular or trancellular processes across the pulmonary epithelia.4,30,31 The air interface model has been used in combination with modified impactor technology to capture aerosolized inhalation formulations and evaluate drug diffusional processes. The twin stage impinger (TSI), multistage liquid impinger (MSLI) and Anderson cascade impactor (ACI) have been used in different investigations to deposit various microparticles and formulations and study their transport across Calu-3 epithelial cells.4,30,32−35 Interestingly, none of these previous air-interface studies investigated the potential effect of transporters on drug uptake after deposition. 1.5. Study Outline. As part of an ongoing study, the authors evaluate the in vitro Calu-3 air interface model and
there are established pharmacopeial in vitro noncellular techniques within the field of gastrointestinal drug delivery, there are no pharmacopeial methods approved for the testing of drug release in the lung. Furthermore, the main focus in pharmacopeial in vitro methodology development has focused on evaluating the aerodynamic size of the inhaled formulation to predict total and regional lung deposition.7 Recently, however, there has been interest in the fate of particles after deposition in the lung.5 Many researchers have studied the potential dissolution rates of small molecules and their salts,9 larger molecular weight biomolecules10−12 and a wide range of excipients.6,13 Of note, among the noncellular in vitro methods available for evaluating drug dissolution/diffusion is the Franz cell.14,15 The Franz cell was primarily designed to evaluate transdermal formulations. Interestingly, in recent studies the Franz cell has been used to differentiate drug release profiles of inhalation dry powder formulations containing small molecular weight drugs combined with controlled release agents.14,16,17 The ability to predict the fate of drug particles using noncellular in vitro apparatuses, such as the Franz cell, have limitations that must be considered.18 Specifically, it is important to note that such techniques generally utilize artificial membranes and/or dissolution media compared to the more complex lung epithelia. 1.2. The Use of Cell Based in Vitro Apparatuses To Study Drug Dissolution. Among human airway epithelial cell lines, Calu-3 (derived from bronchial adenocarcinoma of the airway) has been extensively studied.14,19−23 Importantly, permeability characteristics of a liquid covered culture of Calu-3 can be correlated with in vivo models.22 Furthermore, studies have shown organic cationic transporters (OCTs) to be present and active in this cell line;24,25 however, we have shown protein expression in Calu-3 cell line to be passage and time dependent.23 1.3. Membrane Bound Protein Transport: Importance in Respiratory Drug Delivery. Although investigations can be found in literature with respect to transporter expression and 1718
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using a D5000 XRD (Siemens, Munich, Germany). Settings were as follows: temperature 25 °C, Cu Kα radiation source at 30 mA and 40 kV, angular increment of 0.04°/s and count time of 2 s. 2.2.4. Differential Scanning Calorimetery. The thermal response of SS and SB was studied using differential scanning calorimetery (DSC) (DSC823e; Mettler-Toledo, Schwerzenbach, Switzerland). Approximately 5 mg of powder was placed into a DSC sample pan and crimp-sealed. The lid was pierced (to ensure constant pressure), and the thermal properties were studied using a 10 °C/min temperature ramp from 40 to 400 °C. STARe V9.0x software (Mettler-Tolledo GmbH) was used to determine specific endothermic or exothermic events, and data was normalized for initial mass. 2.2.5. Solubility Measurement of Salbutamol Base and Sulfate. Saturated solutions of SB and SS were prepared by adding excess powder to purified water, HBSS and simulated lung fluid (SLF) containing 0.02% DPPC followed by storage at 37 °C in a water bath for 24 h before analysis. The saturated solutions were filtered using a 0.45 μm membrane filter (MF Membrane Filters: Millipore, Bedford, MA, USA), and the filtrate was analyzed via HPLC for calculation of solubility. Solubility measurements for SB and SS at 37 °C were repeated 6 times. The total solubility of SS and SB in SLF with 0.02% DPPC is calculated from the sum of drug dissolved in SLF (aqueous phase) and DPPC (lipid phase). Therefore, to calculate the amount of dissolved SS and SB in SLF modified with 0.02% DPPC, samples were first diluted with ethanol (1:4) and measured by HPLC at 276 nm. 2.2.6. High Performance Liquid Chromatography. Chemical analysis of SB or SS was determined via high performance liquid chromatography (HPLC). Briefly, analysis of SS and SB was conducted using a Shimadzu Prominence UFLC system equipped with an SPD-20A UV−vis detector (operating at 276 nm), LC-20AT solvent delivery unit, SIL-20A HT autosampler (Shimadzu Corporation, Japan) and NovaPak C18 column (5 μm, 150 × 3.9 mm). The mobile phase was a mixture of methanol:0.1% w/v aqueous sodium dodecyl sulfate solution (60:40%, v/v). The flow rate was 1 mL·min−1, and injection volume was 100 μL. Standard linearity was obtained between 0.1 and 25 μg·mL−1 (R2 = 0.9999) with a retention time of approximately 6 min. 2.3. In Vitro Drug Diffusion Studies Using the Franz Cell. The rate of drug dissolution and diffusion of the micronized SB and SS was evaluated using a modified Franz diffusion cell (VB6; PermeGear Inc., Hellertown, PA, USA) (Figure 2). Briefly, 50 mL of degassed dissolution medium (HBSS or SLF with 0.02% DPPC), at 37 ± 0.5 °C in a water bath, was pumped at 5 mL·min−1 via a peristaltic pump (Minipuls 3, Gilson, Middleton, WI, USA) into the inlet port of the receptacle compartment of the Franz cell. A second pump, operating at the same flow rate, was connected to the outlet port of the receptacle compartment to return the medium to the sink, forming a closed loop. This setup ensured the volume in the Franz cell remained constant and the membrane remained constantly and uniformly wetted. The Transwell polyester insert (Corning Costar, USA) with 0.4 μm pore size and 0.33 cm2 area available for diffusion was used as model membrane. Twin stage impinger (TSI) (Radleys, Essex, U.K.) was used to deposit each powder on the Transwell polyester membranes. The TSI was modified, as described previously by Grainger et al.,4 to allow the attachment of a
Franz diffusion cell to study drug dissolution and diffusion after deposition in the lung. Specifically, the study focused on the effect of particle solubility and lipophilicity as well as the role of uptake transporters on influencing transport kinetics through the epithelia. Salbutamol (albuterol in the USA) was chosen as a model drug since it is cationic, it has been shown to be actively transported in liquid cell culture,25 and is available as a freely soluble salt and sparingly soluble base (Figure 1). Furthermore, salbutamol is one of the most commonly used inhalation medicines for the treatment of asthma, whose therapeutic activity requires diffusion through the bronchial epithelia to the smooth muscle cells beneath. Salbutamol can be found in commercial products as the sulfate form (e.g., Ventolin, GSK) and base (Ventide, Allen & Hanburys).
2. MATERIALS AND METHODS 2.1. Materials. Salbutamol sulfate was purchased from S & D Chemicals (Sydney, Australia) and salbutamol base from Inter-Chemical Ltd. (Shenzhen, China). Both salbutamol base and sulfate were micronized using an air jet mill (Trost Air Impact Pulveriser, Trost Equipment Corporation, USA) at a feed pressure of 280 kPa and grinding pressure of 680 kPa. The Calu-3 cell line (HTB-55) was purchased from the American Type Cell Culture Collection (ATCC, Rockville, USA). Dulbecco’s modified Eagle’s medium (DMEM, without phenol red and L-glutamine, including sodium bicarbonate and 15 mM HEPES), tetraethylammonium (TEA), nonessential amino acids solution, Trypan blue solution (0.4% w/v), CelLytic M Cell Lysis (50 mM Tris−HCl, pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS), mammalian protease inhibitor cocktail, fluorescein-sodium (flu-Na) and dipalmitoylphosphatidylcholine (DPPC) were obtained from SigmaAldrich (Sydney, Australia). Fetal bovine serum (FBS), trypsin−EDTA solution (2.5 g/L trypsin, 0.5 g/L EDTA), phosphate buffered saline (PBS), L-glutamine solution (200 mM), and Hanks balanced salt solution (HBSS) were from Invitrogen (Australia). Transwell cell culture supports (0.33 cm2 polyester, 0.4 μm pore size) were purchased from Corning Costar (Lowell, MA, USA), and all other sterile culture plastic wares were obtained from Sarstedt (Adelaide, Australia). Water was purified by reverse Osmosis (Milli-Q, Molsheim, France). All solvents were analytical grade and were supplied by Sigma (Sydney, NSW, Australia). Unless otherwise stated in the text, salbutamol sulfate is referred to as SS, and salbutamol base as SB. 2.2. Physical and Chemical Characterization. 2.2.1. Scanning Electron Microscopy. The morphology of each of the micronized powders was studied using a fieldemission scanning electron microscope (SEM) at 5 keV (Zeiss Ultra plus, Carl Zeiss Pty Ltd., Sydney, Australia). Prior to imaging, samples were dispersed onto carbon sticky tabs and sputter-coated (sputter coater S150B, Edwards High Vacuum, Sussex, U.K.) with gold to a thickness of approximately 20 nm. 2.2.2. Particle Sizing via Laser Diffraction. The particle size of both micronized drug powders was studied using laser diffraction (Malvern Mastersizer 2000, Malvern Instruments Ltd., U.K.). Samples were dispersed using a dry powder dispersion unit (Scirocco, Malvern, U.K.) at a feed pressure of 400 kPa and feed rate of 50%. Samples were analyzed in triplicate with an obscuration value between 0.3% and 10% and a reference refractive index of 1.553. 2.2.3. X-ray Powder Diffraction. The X-ray powder diffraction pattern for each micronized powder was analyzed 1719
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the Transwell membrane with methanol:water (60:40) at the end of the experiment. 2.4. In Vitro Drug Diffusion/Transport Studies Using the Calu-3 Cell Culture. 2.4.1. Cell Culture. The Calu-3 cell line (passage 35−40) was grown in 75 cm2 flasks in Dulbecco’s modified Eagle’s medium: F-12 containing 10% (v/v) fetal calf serum, 1% (v/v) nonessential amino acid solution and 1% (v/ v) L-glutamine solution. Cells were maintained in a humidified 95% air 5% CO2 atmosphere at 37 °C and were propagated and subcultured according to ATCC recommendations. Cells were seeded onto 24 well Transwell polyester inserts at a density of 5 × 105 cells·cm−2 in 100 μL of apical and 500 μL of basolateral medium. For the air-interfaced model, the apical medium was removed after 24 h and the culture was fed every alternate day with fresh basolateral medium only. The monolayers were allowed to differentiate under air-interface conditions for 12−14 days. This time period was previously determined sufficient to ensure a suitable tight junction, optimal efflux transporter expression and activity and homogeneous mucus coverage.23 After 12 days, the transepithelial electrical resistance (TEER) of the cell line was evaluated using a voltohmmeter (EVOM with STX-2 chopstick electrodes, World Precision Instruments, Sarasota, FL, USA). Briefly, prewarmed medium was added to the apical and basolateral sides of the Calu-3 monolayer, which was then equilibrated for 30 min prior to resistance measurements. Three wells were tested and TEER was calculated by subtracting the resistance of a blank insert and corrected for the Transwell surface area as previously described.23 TEER was measured 10 times for each well. In addition, these wells were further tested to confirm cell viability. After drug deposition, the cells were harvested and a Trypan blue staining method was used to enumerate viable cells.36 The percentage of the viable cells after drug deposition compared to the control cells following sham deposition (air flow) was calculated. No significant difference in cell viability was observed for all experiments 2.4.2. Drug Deposition, Dissolution and Diffusion/Transport. In order to investigate the mechanism of drug deposition, dissolution and diffusion/transport in the lung, an in vitro aerosol testing apparatus was used to deposit each powder on the Calu-3 cell line. A twin stage impinger (TSI) (Radleys, Essex, U.K.) was modified, as described above, to allow the attachment of a Transwell containing the Calu-3 epithelial cell line (Figure 3). Between 1 and 6 mg (±0.05 mg) of either SB or SS was weighed (for studying the effect of different amounts of drug deposited on cells) into a size 3 HPMC capsule. After deposition of the powders, particles adhering to the outer surface of the Transwell were washed away and the Transwell was dried before transferring to a 24 well plate containing 600 μL of fresh prewarmed HBSS. At different time points the Transwell was moved to a well containing fresh HBSS to study the diffusion/transport of the drug particles over time. After 4 h the surface of the monolayer was washed with HBSS buffer and collected for analysis of residual apical drug via HPLC. This allowed for the calculation of total drug deposited via sum of all samples. Afterward, the cells were harvested, washed 3 times with ice cold PBS and lysed using cell lysis buffer containing 1% protease inhibitor so that intracellular drug could be evaluated. The cells were sheared using a 21-gauge needle and left on ice for 1 h following a centrifugation at 10 000g for 10 min. The supernatant was collected for HPLC analysis. The initial
Figure 2. Schematic of the modified Franz cell set up to incorporate the Transwell.
Transwell (Figure 3). The TSI consists of two stages and, when operated at 60 L·min−1, separates an aerosol cloud based on
Figure 3. Schematic of the modified TSI set up to incorporate the Transwell insert at the stage 2 jet assembly. Particles
