2014
Mary Goud
An investigation into delivery of
theophylline for inhalation
An investigation into delivery of theophylline for inhalation
Graduation internship at the Woolcock Institute of Medical Research
Date: 06-01-2014 to 08-08-2014
Final report Version: 1.0 08-08-2014
Student details:
Mary Goud (+61) 449 068341 ma.goud@avans.nl
marygoud1993@hotmail.com
Supervisor details:
Avans University:
Nicole van den Braak (+31) 6 13693853 npwcj.vandenbraak@avans.nl (+31) 765 238007
Woolcock Institute:
A/Prof Daniela Traini (+61) 2 911 40352 daniela.traini@sydney.edu.au Dr Mehra Haghi (+61) 2 911 40366 mehra.haghi@sydney.edu.au
Company details:
Avans University of Applied Science s The University of Sydney Medical Diagnostics Woolcock Institute of Medical Research
Lovensdijkstraat 61-63 Respiratory Technology 4800 RA Breda 431 Glebe Point Road, Glebe,NSW,2037
The Netherlands Sydney, Australia +31 7 65250500 +61 2 91140352
Foreword
After eight fantastic months, my time at the Woolcock has come to an end. It was a big step for me to travel so far away on my own while I never even went on a holiday by myself or lived alone. It was very exciting and I ran into a lot of common problems and even had to move to a different home in the first week, but I quickly adapted to the new situation and actually thought it was fun. When I first arrived at the Woolcock, I had already met Jasper but only knew a few others by name only. The people were incredibly welcoming and before I knew it, I knew everyone and had a lot of fun inside and outside the laboratory.
I had an unforgettable time and I want to thank Daniela and Paul for the opportunity to do my internship here, for the help throughout the project and for the many new possibilities that they opened for my future. I also want to thank Eric and Mehra for teaching me all the techniques and for the fantastic guidance and fun during the project. I also want to thank the Steunfonds Technisch Hoger Onderwijs (STHO) for the money I received to support me during this great experience.
I want to thank Jasper, Wilco, Nadine, Sumit, Michele, Matteo, Valentina, Angelo, Emelie, Yang, Judy, Nessa, Wing, Giulia and Khanh for the great times we had together and for always being there for me.
Lastly, I want to thank my supervisor in the Netherlands, Nicole van den Braak, for the support during my internship.
The time I spent in Australia is the best time I ever had and I will always carry it in my heart. Thank you all for the fantastic time!
Abstract
Theophylline (TP) is a drug used to treat Chronic Obstructive Pulmonary Disease (COPD) and has been associated with multiple side effects, lessening its present use. This study aims to improve COPD treatment by creating a low-dose pressurized Metered Dose Inhaler (pMDI) and a Dry Powder Inhaler (DPI) formulation with theophylline (TP). Aerosol performance was assessed using Andersen Cascade Impaction (ACI) and Multi-stage liquid impinge (MSLI) for the DPI formulation. Morphology of the particles was analyzed with a Scanning Electron Microscope (SEM) and Calu-3 cell viability assay and cell transport were conducted to study the impact of the formulation on lung epithelial cells. The mass deposition profile of the formulation showed an emitted dose of 250.04 ± 14.48 µg per 5 actuations for the pMDI, achieving the designed nominal dose (50µg/dose). The aerosol performance of the DPI formulation showed an emitted dose of 3.2 ± 0,058 mg. SEM showed that the emitted particles of the pMDI were crystalline, spherical and hollow and the DPI particles were also spherical and crystalline but not hollow and the particles were bigger with a few clustered particles. Suitable solution pMDI and DPI formulations of TP were developed that could be used for the treatment of COPD.
FOREWORD...2
ABSTRACT...3
TABLE OF CONTENTS...4
1. INTRODUCTION...5
2. THEORETICAL BACKGROUND...6
2.1 THELUNGSANDPARTICLEDISTRIBUTION...6
2.2 PRESSURIZED METERED-DOSE INHALERS...6
2.3 DRY POWDER INHALERS...8
2.4 THEOPHYLLINE... 9
2.4.1 The drug...9
2.4.2 Mechanisms of action...9
2.5 TECHNIQUES... 10
2.5.1 Dosage unit sampling apparatus...10
2.5.2 Reversed-phase high performance liquid chromatography...10
2.5.3 Cascade Impaction...11
2.5.4 Scanning Electron Microscope...13
2.5.5 Particle micronization...14
2.5.7 Thermogravimetric analysis...15
3. PURPOSE AND HYPOTHESIS...16
3.1 PURPOSE... 16
3.2 HYPOTHESIS... 16
4. MATERIALS AND METHODS...17
4.1 PMDI... 17
4.1.1 Formulation...17
4.1.2 Morphology study...17
4.2.3 Calu-3 cell viability assay...17
4.1.4 Transport of theophylline across Calu-3 epithelium...17
4.2 DPI... 18 4.2.1 Jet mill... 18 4.2.2 Spray dry... 18 4.2.3 Shot weight...18 4.2.4 Aerosol performance...18 4.2.5 Transport studies...18 4.2.6 Thermogravimetric analysis...18
5. RESULTS AND DISCUSSION...19
5.1 PMDI... 19
5.2 DPI... 21
CONCLUSION...24
1. Introduction
Theophylline (TP) is a drug used to treat respiratory diseases like asthma and COPD. It is cheap and soluble in water. TP is available in syrup and tablet form for oral
administration, but can also be administered intravenously. When taken as a syrup, TP has to be metabolized before it is absorbed into the systemic circulation. This process takes time and this is also known to cause side effects like headaches, insomnia and irritability.
In order to reduce the side effects of the drug, this study aimed to make inhalable formulations of theophylline to provide local treatment and therefore lower the dose and reduce the side effects. A High Performance Liquid Chromatography (HPLC) method was developed to detect and analyse the amount of TP. Furthermore, the solubility of
theophylline in the pMDI formulation was determined and used to calculate the quantities of TP, hidrofluoro alcane propellant (HFA) and ethanol (EtOH) required in the formulation to develop an inhalable formulation with optimal aerodynamic particle size. The
morphology was investigated using Scanning Electron Microscopy (SEM). The aerodynamic particle size distribution was determined using the Andersen Cascade Impactor (ACI) for the pMDI and the Multi-stage liquid impinger (MSLI) was used for the DPI. Calu-3 sub-bronchial epithelial cells, grown in the air liquid interface model, were placed at bronchial level of the ACI and deposition and subsequent transport of the microparticles across the Calu-3 cells was studied.
Chapter 2 describes the Theoretical Background of the inhalation systems, followed by information about TP and the techniques that were used in this study. Chapter 3 includes the purpose and hypothesis of this study and Chapter 4 describes the materials and methods used. The conclusions are listed in chapter 5, followed by the references.
2. Theoretical background
2.1 The lungs and particle distribution
The airways are designed in such a way that most particles larger than 10 µm are unable to enter the lungs, as in figure 1.
Figure 1 Particle distribution in the respiratory tract by particle aerodynamic size.
In order to deliver a drug to the lungs effectively, the particles have to be in the size range of 1~5 µm [1] and have the ability to transport across the lung epithelium to reach its site of action. Figure 2 shows the cell structure of the lungs and its major cell types.
2.2 Pressurized Metered-Dose Inhalers
Pressurized Metered Dose Inhalers, pMDIs, originally formulated with chlorofluorcarbon (CFC) as the system propellant. However, since the year 1989, due to ozone-damaging effects caused by CFCs, these molecules were replaced with hydrofluoro alkane (HFA) propellants. [3] The advantages of pMDIs are their portability, their ease of operation and their familiarity. The disadvantages of the pMDI are the low concentration of the
delivered drug, a training requirement, the use of an propellant and the requires patient cooperation. Unlike DPIs, pMDIs are active devices, and therefore coordination between activation and inhalation is required; this is one of the major issues related to patients. If the patient inhales before or after the actuation, the majority of the medicine will impact on the throat and be transported to the digestive system.
To overcome this issue, patients receive training and spacers are used in attachment to the pMDI and breath-actuated pMDIs are developed. Spacers are add-ons that allow the inhalation of a dose over multiple breaths. Because spacers are bulky additions to the pMDIs, they might cause portability limitations for the patients. Furthermore, spacers have specific cleaning instructions. If these instructions are not carried out as described, for example if the spacer is cloth-dried, the performance may be affected by static-charge build-up on the walls of the device, attracting some aerosol particles. Figure 3 shows the schematic components of a pMDI.
Figure 3 Schematic components of a pMDI [4].
Typically, the canister is a single layer of formed aluminium with a metering valve, crimped to the canister. The metering valve keeps the pressurized formulation separated from the ambient environment and releases a known quantity of propellant (usually 25-100µL) upon activation. The active ingredient is suspended or solubilized in the
propellant and the concentration in the canister determines the therapeutic dose. In suspension-based pMDIs, the stability of the suspension is of great importance. To maintain the bulk formulation, the pMDI has to be metered while exposing a small amount of pressurized liquid to standard pressure. An aerosol is generated upon exposure, when this small volume expands rapidly. To sustain the bulk formulation, the formulation should either be able to remain homogenous during storage, or it should be readily resuspended prior to actuation. Unfortunately, HFAs have a low dielectric constant and only exert a weak double-layer force on suspended particles. To overcome this, formulations must alter the drug properties and media or modify the contact dynamics between particulate systems.
Solution-based pMDIs do not encounter the same issues as the suspension-based formulations. They are dispersions in which individual drug molecules can interact with the HFA at molecular level and thus have a higher potential chemical degradation than their suspension counterparts. The molecules of drugs used in pMDIs are not readily soluble in HFA and therefore require a co-solvent. When solubilized, these formulations require a good physical stability and may not precipitate when exposed to different temperatures. Ethanol can be used as a co-solvent as it is mixable with HFA and most drugs are also soluble in it. pMDIs utilizing volatile co-solvents result in higher fine-particle fractions because of the small fine-particle size of the dried aerosols.
2.3 Dry Powder Inhalers
Dry powder inhalers (DPIs) contain and deliver the active drug as a dry powder with suitable aerodynamic size for respiratory therapy. These dry powder particles can be produced by micronization, like jet milling, or spray drying. These particles have high surface area to mass ratios and consequently, are highly cohesive and need to be formulated in a specific way to avoid this factor during inhalation. The size of the particles is generally less than 6 microns and the efficiencies of many commercial products are relatively low by pharmaceutical standards. Often only 20% of the drug is being delivered to the lung.
Even though the efficiency is relatively low, Dry Powder Inhalers have become popular because of the relatively chemical stability and portability. Furthermore, they do not require coordination between system actuation and inhalation [5].
2.4 Theophylline
2.4.1 The active pharmaceutical ingredient
The drug of interest in this investigation, TP, is a drug of the xanthine family used to treat the symptoms of respiratory diseases like asthma, bronchitis, emphysema, COPD and other lung diseases. Recently, it has been shown to have anti-inflammatory and bronchodilator effects [6]. Its chemical structure and pharmacological effects are very similar to caffeine, as shown in figure 4.
Figure 4 The chemical structure of TP (left) and caffeine (right).
TP is usually given orally as slow-release preparations for chronic treatment and intravenously for acute exacerbations of asthma. The efficiency of TP is related to the concentration in the blood which is mainly determined by hepatic metabolism and may be variable in patients [6]. TP is still being used as an add-on therapy because it is inexpensive and widely available. It’s side effects include headache, insomnia, irritability, dizziness and light-headedness. In severe cases it can cause seizure [7], which is
considered a neurological emergency.
2.4.2 Mechanisms of action
TP is a competitive nonselective phosphodiesterase inhibitor (PDE). It increases Cyclisch adenosinemonofosfaat (cAMP) levels in the cells, activates Protein Kinase A (PKA), inhibits Tumor Necrose Factor-alpha (TNF-alpha) and leukotriene synthesis and reduces inflammation and innate immunity. TP is also a nonselective adenosine receptor [8] antagonist, antagonizing A1, A2, and A3 receptors [9] almost equally resulting in its cardiac side effects. Theophylline inhibits Transforming growth factor beta (TGF-beta) -mediated conversion of pulmonary fibroblasts into myofibroblasts in COPD and asthma via cAMP-PKA pathway and suppresses COL1 mRNA, which codes for the protein collagen [10].
TP has been shown to reverse steroid insensitivity in patients experiencing oxidative stress [10]. It can restore reduced histone deacetylase activity which restores steroid responsiveness toward normal levels. It also activates Histone deacetylase 2 (HDAC2) which blocks the inflammatory response [11].
2.5 Techniques
2.5.1 Dosage unit sampling apparatus
A dosage unit sampling apparatus (DUSA) can be used with a flow rate up to 100 L/min for both DPI and pMDI [12] to determine the dosage of a drug released from a
formulation. The device consists of an actuator, adapter, DUSA and filter. The actuator for the DPI or pMDI formulation is placed into the adapter and actuated into the tube and onto the filter. The drug contends from DUSA parts can be re-constituted in suitable solvents and analysed with HPLC. Figure 5 shows the setup for the DUSA and its individual parts.
Figure 5 DUSA setup with its separate parts.
2.5.2 Reversed-phase high performance liquid chromatography
High performance liquid chromatography (HPLC) is an analytic method to analyse the components in a liquid mixture. The system consists of pumps that lead a pressurized liquid and sample mixture through a column with a solid absorbent material that is typically made of silica or polymers. Each component reacts differently to the material in the column resulting in separation of the components. The mobile phase usually
consisted of a mixture of water, methanol or acetonitrile. The composition and
temperature of the mobile phase and the column that is used are very important in the separation process.
An HPLC system includes a sampler, pumps and a detector. The sampler transports the sample to the tubing containing pressurized mobile phase where the pumps bring it to the column and the detector analyses the signal of the sample component as it is eluted from the column.
Reversed phase HPLC (RP-HPLC) uses a non-polar column and an aqueous, moderately polar mobile phase. With this setup, the retention time of les polar molecules is longer while polar molecules are eluted from the column early in the analysis. Since TP is only very slightly polar, TP is eluted from the column later than its solvent, water. To increase the retention times, more water can be added to the mobile phase. This increases the affinity of the hydrophobic sample for the hydrophobic column relative to the now more hydrophilic mobile phase [13].
2.5.3 Cascade Impaction
Cascade Impactors are used to simulate the impaction of inhaled particles in different areas of the respiratory tract. Each stage, after the throat piece, comprises a series of nozzles or jets through which the airborne sample is drawn and directed towards the surface of the collection plate for each stage. The aerodynamic size determines whether a particle will impact on a certain stage, or remain in the air stream to continue to the next stage where this is repeated. The stages are assembled in a stack or a row and each stage has a smaller particle size that will impact on the surface due to increasing air velocity and smaller passages. At the end of the experiment, the stages are all washed with a suitable solvent to recover the particles from each stage. To determine the amount of drug present, the solvent is analysed using HPLC.
2.5.3.1 Andersen Cascade Impactor
The Andersen Cascade Impactor (ACI) is most commonly used in the pharmaceutical industry for the testing of inhaled medicaments [14]. This system consists of 8 stages and can be used to test the aerodynamic particle size distribution (APSD) of both Dry Powder Inhalers (DPI) and pressurized Metered Dose Inhalers (pMDI). Figure 6 displays the ACI system with the MDI and aerodynamic cut-off size per stage.
Figure 6 Schematic diagram of the cascade impactor with aerosol aerodynamic size
range at each impactor stage 0-7 and at the final filter (F). MDI = metered-dose inhaler; VHC = valved holding chamber; USP = United States Pharmacopeia. [15]
2.5.3.2 Next generation impactor
The next generation impactor (NGI) was introduced in 2000 and was accepted in 2005 in the Pharmacopoeia as a system to analyse aerodynamical size distribution. This system contains 5 stages which have a range of 0.5 to 5 µm [16][17] and a final filter to collect the particles that are too small. The airflow follows a sawtooth pattern through the nozzles with decreasing jet diameters. The samples are collected in collection cups that can be removed and washed. Figure 7 displays the NGI and its collection plates.
Figure 7 NGI with collection plates
2.5.3.3 Multi-stage liquid impinger
The multi-stage liquid impinger (MSLI) is used to determine aerodynamical size distribution for the DPI, MDI and nebulizer. The system consists of 5 stages that each contains 20 mL of liquid that is used to capture the particles and reduces the
re-entrainment that is sometimes a problem in the NGI and ACI systems. The design allows a flow rate of 30-100 L/min [18]. Figure 8 displays the MSLI system with its cut-off diameters at a flow of 60 L/min.
2.5.4 Scanning Electron Microscope
A Scanning Electron Microscope (SEM) produces images of a sample by scanning it with a high-voltage beam of electrons that is controlled by lenses and powerful magnets for high resolution and accuracy, which travel in vacuum. When the beam strikes the sample, the backscattered electrons, secondary electrons and X-rays are refracted from the sample and detected by the detectors of the Scanning Electron Microscope. These detectors convert the signal into an image that is displayed on a screen. Figure 9 displays a schematic overview of the parts of the SEM that were described before.
Figure 9 Schematic view of the SEM [19]
Prior to scanning a sample, the sample has to be properly prepared for the conditions in the SEM. All non-metals need to be made conductive by coating them with a very thin layer of conductive material. Sputter coater uses an electrical field and argon gas to coat the samples. The sample is placed in a vacuum chamber. An electric field removes the electrons from argon gas, making the atoms positively charged. The argon ions are then attracted to the negatively charged gold foil where it knocks gold atoms from the surface of the foil. These atoms then fall and settle onto the surface of the sample, producing a thin gold coating, thus making it conductive. [20]
2.5.5 Particle engineering
In order to produce a dry powder with suitable aerodynamical characteristics for lung deposition, several techniques can be used.
2.5.5.1 Jet milling
Jet milling is a technique used to micronize particles by grinding the particles at nearly sonic velocity. In order to achieve this, the particles are put into a toroidal chamber where several air or steam jets create a high velocity in the chamber, grinding the particles due to high-velocity collision between the particles [21]. The milled particles migrate to the central collection vial. No solvent is required for this technique, which increases the purity of the produced material [22].
2.5.6 Closed-loop Spray drying
When a flammable organic solvent is used to dissolve a drug for spray drying,
oxygen/atmospheric air cannot be applied as a drying medium. For this reason, a closed loop is used. Closed loop spray drying applies an inert gas like nitrogen which also allows re-use of the gas and it avoids pollution [23]. The solution is pumped through a nozzle where it is atomized into the drying chamber with a hot gas that is usually nitrogen. The generated droplets size can range from 20 to 180 µm depending on the nozzle. In the drying chamber, the droplets evaporate in the warm current and form dry particles that are hollow or solid. The dried particles enter the cyclone where the circular airflow separates them based on size. The bigger particles are collected in the collecting chamber and the particles the are too small are sucked into the waste [24]. Figure 10 shows the spray dry setup and its parts.
Figure 10 Spray dry setup [25].
2.5.7 Thermogravimetric analysis
Thermogravimetric analysis (TGA) is a thermal analysis method that can be used to measure the changes in physical and chemical properties of materials a function of
increasing temperature and/or time. The system can measure mass and temperature change through a very precise balance. A sample is loaded into a pan which is hanged into a furnace which heats the sample, increasing at a constant rate, while measuring the weight [26]. From the results, the difference in mass can be used to determine
vaporisation, degradation, decomposition and other mass changing processes. Figure 11 displays the schematic of a TGA.
3. Aim and hypothesis
3.1 Aim
TP has been used for the treatment of respiratory diseases such as asthma and COPD over 80 years. It is available in oral dosage form. Therefore it requires a higher dose and longer time to achieve therapeutic level that increases the risk of negative side effects. This study aims to improve the treatment of COPD by formulating inhalable formulations of TP in order to reduce the required dose via local treatment and therefore reducing side effects.
3.2 Hypothesis
TP is slightly polar and dissolves in water with a solubility of 8,3 mg/mL. Theoretically, it should be possible to develop a TP pMDI formulation with optimal aerodynamic size distribution if TP dissolves in HFA 134a or in HFA 134a with Ethanol as a co-solvent. The toxicity for the airway epithelial cells of this formulation depends on the composition of the formula. If the ethanol concentration is too high, it could be toxic to the airway epithelial cells and cause tissue damage when inhaled.
Since TP is a non-specific inhibitor of the enzymes that break down cAMP, it may enhance bronchodilator action.
TP exerts anti-inflammatory actions and can inhibit inflammation by up-regulating the same anti-inflammatory proteins considered important for the repressive effects of corticosteroids.
4. Materials and methods
Anhydrous TP was used as supplied (MP Biomedicals, France). Methanol (HPLC grade) and Ethanol (100%) were obtained from Biolab (Clayton, Victoria, Australia). The propellant —1,1,1,2 tetrafluoralkane (HFA 134a) was obtained from Solvay Chemicals (Bruxelles, Belgium). The water was purified through reverse osmosis (MiliQ, Millipore, France).
4.1 pMDI
4.1.1 Formulation
The Solubility apparatus was used with a DUSA (Copley, Nottingham, UK) to determine the solubility of TP in HFA 134a with Ethanol concentrations of 10 and 15%. TP pMDI formulation was formulated to contain 15% (w/w) ethanol, due to the solubility limit of TP in the mixture of HFA 134a, to deliver 50 µg of active medication per actuation. The aerosol performance of the pMDI formulation (actuator orifice size 0.3 mm) was
evaluated using an Andersen cascade impactor at 28.3 L/min, firing 5 dose into the ACI in order to overcome the limit of detection of chemical quantification. After cascade
impaction, the ACI was disassembled and each part was thoroughly rinsed with deionized water into proper volumetric flasks followed by sample collection for chemical
quantification using High Performance Liquid Chromatography (HPLC), using a validated method. The samples were quantified using a reverse phase C18 column (4.6 x 150 mm and 5 µm, XBridge™ Shield, Waters, USA) with methanol aqueous solution (55%, v/v), at a flow rate of 1 mL/min, detection wavelength 275 nm and injection volume 100 µL. The standard solutions were prepared daily and the linearity of standard solutions in the concentration of 0.01~100 µg mL was confirmed with a R2 value > 0.999.
4.1.2 Morphology study
The morphology of deposited TP was studied using scanning electron microscopy (SEM) (Zeiss Ultra Plus, Carl Zeiss NTS GmbH, Germany). The particles on stage 5 of the ACI were collected on adhesive carbon tape and sputter coated with 15 nm gold (Sputter coater S150B, Edwards High Vacuum, Sussex, UK) according to a previously reported method [27][28].
4.2.3 Calu-3 cell viability assay
Human airway Calu-3 cells is an immortalized cell line used as a respiratory model of human respiratory function, structure and inflammatory response. The toxicity of TP was evaluated by exposing Calu-3 cells to increasing concentrations of TP phosphate buffered saline (PBS) solution. The cell viability was measured in a liquid covered culture (LCC) following a 72-hour incubation, according to the previously published method [29]. Briefly, Calu-3 cells were seeded in a volume of 100 µL into a 96 well plate and incubated
overnight at 37°C in 5% CO2 atmosphere. To assess the viability, 20 µL of the CellTiter
96® Aqueous assay (MTS reagent) (Promega, Madison, USA) was added to each well. The
plates were incubated for 3 hours at 37°C in humidified 5% CO2 atmosphere. The
absorbance was measured at 490 nm using a Wallac 1420 VICTOR 2 Multilabel Counter (Wallac, Waltham, USA). The drug concentration that resulted in a decrease of 50% in cell viability compared to the untreated control was deemed as the half maximal inhibitory concentration (IC50).
4.1.4 Transport of theophylline across Calu-3 epithelium
The modified ACI setup described by Haghi et al. [30], as seen in figure 12, was used to deposit the TP pMDI formulation on Calu-3 epithelial cells. 17 days post seeding, the Snapwells (0.4 μm pore size polyester membrane, 1.12 cm2 Surface area) (Corning Costar, Lowell, MA, USA) with the Calu-3 cells grown at air-liquid interface, were placed on the ACI stage 5. Two to four actuations of the TP pMDI were actuated into the modified ACI to confirm the dose consistency. Following TP deposition, the Snapwells® were
transferred to a 6-well plate containing Hank’s balanced salt solution (HBSS) (Gibco-Invitrogen, Sydney, Australia) with incubation of 2 hours. At 10, 30, 45 and 60-minute time points, 200 µl samples were taken from the basal compartment and was replaced with same volume of fresh HBSS. Prior to analysis with HPLC, the epithelium was washed with 125 µL HBSS and then collected for chemical quantification. Finally, the cells were lysed using cell lysis reagents (CelLytic®, Sigma-Aldrich, Sydney, Australia) and sheared
using a 21-gauge needle as described previously by Haghi et al. [31]. The supernatant was collected after ultracentrifugation for analysis of the intracellular drug component.
Figure 12 Modified ACI setup for Transport studies.
4.2 DPI
4.2.1 Jet milling
The raw TP was milled with an air jet mill (Food Pharma Systems, Como, Italia) once and twice at 6 bar and once at 8 bar. Due to the high amount of unsuitable particles, >10 µm or <1 µm, and the low yield, <30%, it was decided to use spray drying instead.
4.2.2 Spray drying
A closed loop setup with nitrogen gas was used to micronize the TP particles. Briefly, theophylline was dissolved in 50% (v/v) aqueous Ethanol solution at a concentration of 20 mg/mL and a pump rate of 40 L/min-1 was used. The aspiration rate was 100% and the
inlet temperature was 100 ºC with an outlet temperature of 48 ºC. A 140 mm nozzle was used with a standard drying chamber. The collected powder was analysed for its size and morphology with SEM.
4.2.3 Shot weight
5 mg of spray dried TP was weighted into a gelatine capsule size 3 (Capsugel®,
Greenwood, US) and loaded into a RS01 high resistance device (Plastitape®,Osnago,
Italy) for actuation. The actuator and the capsule with the drug were weighted and the NGI (Westech Scientific Instruments, Bedfordshire, UK) was actuated into the NGI because this was the dive initially used for aerosol performance. A flow rate of 60 L/min for 4 seconds was used. After the actuation, the device was weighted again to calculated the delivered dose.
4.2.4 Aerosol performance
MSLI (Copley, Nottingham, UK) was used to assess the aerosol performance of the spray dried TP. Each stage of the MSLI contained 20 mL deionized water. 5 mg spray-dried TP was weighted into a capsule. The content in the capsule was actuated into the MSLI with a flow rate of 60 L/min for 4 seconds. Chemical quantification was conducted using HPLC, with a validated method.
4.2.5 Transport studies
The same ACI setup as the pMDI transport study was used, but for this formulation, 5 mg spray-dried TP was weighted into a gelatine capsule and actuated at a flow rate of 60 L/minfor 4 seconds [32] with the modified plate at stage 4 instead of stage 5.
4.2.6 Thermogravimetric analysis
TGA was used to analyse the amount of remaining moisture that may be present in the spray-dried powder, since this will affect the chemical stability and aerolisation of the powder and could potentially explain the high drug retention whithin the capsule post aerosolization. 9.898 mg of the spray-dried TP was weighted into a pen and analysed from 25-250 ºC over 23 minutes.
5. Results and discussion
5.1 pMDI
The solubility test for the pMDI solution showed that TP is able to dissolve at a rate of 427 µg TP per gram of HFA, with 10% Ethanol. However, 2673,12 µg TP per gram of HFA with 15% Ethanol can be dissolved which is required for a suitable dosage of 50 µg TP per actuation.
The cascade impaction study showed TP aerosols emitted from the pMDI formulation had suitable aerodynamic characteristics, with an MMAD (mass median aerodynamic diameter) of 1.3 ± 0.01 µm, GSD (geometric standard deviation) of 2.1 ± 0.2 and fine particle fraction (FPF, defined as the fraction less than 10 micron) of 38.0 ± 1.1%. The total emitted dose from 5 actuations was 250.0 ± 14.5 µg, achieving the targeted dose of 50 µg/actuation, in line with pharmacopeia dose requirement (i.e. ±25% of nominal dose) [33]. Figure 13 shows the mass deposition profile of the TP pMDI formulation.
with results from previous studies, due to the ballistic nature of this type of inhalers [34]. In addition, results showed that up to 50% of the produced aerosols were captured in the USP induction port. However, particles in the size range < 3.3 µm (i.e. from stage 4 to filter) showed a good deposition profile, accounting for the high FPF value observed.
Figure 13 Mass deposition profile of TP pMDI formulation from 5 actuations (n=3
± SD)
Figure 14 Representative electron micrographs of (A) raw TP material and (B) TP
particles collected after pMDI deposition on ACI stage 5
Figure 14, shows the morphology of the aerosolised pMDI TP particles collected on the ACI stage 5. These particles have a spherical morphology with a hollow core.
The cell viability assay indicated that Calu-3 cells were viable across all TP tested
concentrations (2.5 mM – 19 nM), confirming that TP is non-toxic to Calu-3 cells as shown in figure 15.
Figure 15 Cell viability of Calu-3 cells after incubation with TP (n= 3 ± SD).
Transport studies showed that 98.2% of the deposited drug was transported across Calu-3 cells over 180 minutes. This indicates that TP was able to be transported across the epithelial cell barrier and reach the A3 Adenosine receptors (TP site of action) located on the smooth muscle cells after 3 hours. Figure 16 displays the transported percentage of TP over time.
Figure 16 Transport of TP across Calu-3 cells grown in the air-liquid interface (n= 3 ±
5.2 DPI
As displayed in figure 17, particles produced by jet milling were not in the suitable size range for inhalation purpose. In addition, the yield after the raw material was processed was low with only 40%, which made spray drying a better option.
Figure 17 TP particles when milled by jet mill.
TP was spray-dried under the conditions listed in table 1. Initially, deionized water was used as a solvent for TP. This solvent resulted in a low yield and unsuitable particles which are fused together as shown in Figure 18 A. Increasing the concentration with a 50% (v/v) ethanol solution as solvent, resulted in a yield of 73,1% and also produced suitable particles in the desired aerodynamical size range. The particles were globes of 1~3 µm with a crystalline structure, as shown in figure 18 B, which implies good powder stability. To increase the particle size, a bigger nozzle of 150 mm was used. This resulted in large fused particles not suitable for inhalation as shown in figure 18 C. Increasing the inlet temperature instead reduced the particles size to less than 1 µm, making them too small for the DPI formulation. The particles for the high temperature are shown in figure 18 D.
1 2 3 4
Nozzle 140 mm 140 mm 150 mm 140 mm
Pump rate 40 mL/min 40 L/min 40 L/min 40 L/min
Aspiration rate 100% 100% 100% 100%
Temperature 100ºC 100ºC 100ºC 150ºC
Concentration 5 mg/mL 20 mg/mL 20 g/mL 20 g/mL
Drying chamber Standard Standard Standard Standard
Solvent 100% EtOH 50% EtOH 50% H2O 50% EtOH 50% H2O 50% EtOH 50%
H2O
Yield 28% 73,1% 60% 59,4%
Table 1 Spray drying conditions of TP.
The cascade impaction study for the DPI showed suitable characteristics for the dry powder particles. The particles had an MMAD of 2.9 ± 0.02 µm, GSD of 2.6 ± 0.02 and fine particle fraction of 50.61 ± 0.16%. The total emitted dose was 3.2 ± 0,058 mg, which is a consistent 64% of the amount in the capsule. However, particles in the size range < 3.0 µm (i.e. from stage 3 to filter) showed a good deposition profile, accounting for the high FPF value observed. Figure 19 shows the mass deposition profile of the TP DPI formulation.
Figure 19 Mass deposition profile of TP DPI formulation (n=3 ± SD)
Transport studies showed that 22.3% of the deposited TP was transported across Calu-3 cells over 3 hours. However, 60% remains in the cells which indicates that TP is only partially able to transport across the epithelial cell barrier over 3 hours as is shown in figure 20.
Figure 20 Transport of DPI TP over 3 hours.
Thermogravimetric analysis has shown that the DPI formulation of TP contains only 0.14% moisture, which is a negligible amount of moisture, which implies that the charge of the dry powder is the reason for the high capsule retention.
Conclusions
In this investigation two inhalable formulations of TP were successfully developed. Both pMDI and DPI formulations showed suitable physiochemical and aerodynamic
characteristics for lung delivery, as confirmed by the morphology studies and aerosol performance investigations. Cell studies confirmed TP delivered on Calu-3 cell to be safe at the concentrations investigated. Drug delivered as solution pMDI was able to be transported across Calu-3 epithelial cells to reach its primary site of action on the smooth muscles. The DPI formulation was instead only able to transport partially across the cell. The reason for these differences are not well understood yet and are currently being investigated further.
Further studies will be investigating TP anti-inflammatory properties and its effective dose in vivo.
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