The in vitro effects on the aerosol performance of poor lip sealing around the mouthpiece of pressurised metered dose inhalers

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WOOLCOCK INSTITUTE OF MEDICAL RESEARCH

The in vitro effects on the aerosol

performance of poor lip sealing around

the mouthpiece of pressurised metered

dose inhalers

Final Report 1.0

Jasper Lamers

/08/2014

1

Figure 2 A sectional of the human respiratory system with indications of the main parts

Figure 3 Mass deposition profiles of emitted particles from Ventolin®_{suspension formulation with different actuators and}

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The in vitro effects on the aerosol performance of poor lip sealing around the mouthpiece of pressurised metered dose inhalers

Version 1.0

Date 04/08/2014

Home institution Avans University of Applied Science Lovensdijkstraat 61/63

4818 AJ, Breda The Netherlands

Internship Institution Woolcock Institute of Medical Research,

Department Respiratory Technology 431 Glebe Point Road 2037, Glebe

Australia

Student Jasper Lamers j.lamers@studentavans.nl

Supervisors a/Prof Paul M. Young Paul.young@sydney.edu.au a/Prof Daniela Traini

daniela.traini@sydney.edu.au Dr. Bing Zhu

bzhu9570@uni.sydney.edu.au

Coordinator Dr. Robert Sijbrandi r.sijbrandi@avans.nl

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Acknowledgments

At first I want to thank Paul Young and Daniela Traini to give me the opportunity to join their group and for teaching me so much about this research area and the academic world. Secondly I want to thank Bing Zhu for all his time, support and wise advices during this internship. He really helped me with making decisions about my future and taught me to look critically at several aspects that I will certainly encounter in later phases in my life. Furthermore I appreciate the support by “Steunfonds Technisch Hoger Onderwijs (STHO)”. I could never enjoy this experience without their financial support. I also want to thank Robert Sijbrandi and Ans Arets to supervise and help me from a distance.

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Table of commonly used abbreviations

ACI Andersen Cascade Impactor

API Active Pharmaceutical Ingredient

BDP Beclomethasone Dipropionate

DUSA Dose Unit Sampling Dose

ED Emitted Dose

FDA U.S. Food and Drug Administration

FPD Fine Particle Dose

GSD Geometric Standard Deviation

HPLC High performance Liquid Chromatography

MMAD Median Mass Aerodynamic Diameter

LOD Limit of Detection

LOQ Limit of Quantitation

pMDI pressurised Metered Dose Inhaler

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Abstract

A problem associated with the use of pressurised metered dose inhalers (pMDI) is the inappropriate seal between a patient’s lips and the inhaler mouthpiece. Such incorrect use of the inhaler may affect its aerosol performance due to unpredicted changes of the flow dynamics in the inhaler. The current study investigates the in vitro aerosol performance of a commercial Ventolin® suspension pMDI and a beclomethasone dipropionate solution pMDI using cascade impaction method, at different experimental setup to simulate conditions where the correct/incorrect use of inhalers. In addition, effects of modified Ventolin® actuators, with high and low flow resistances, on the pMDI aerosol performances with the above configurations were also evaluated. This study demonstrated that different seal conditions with either a suspension or solution pMDIs did not significantly affect the aerosol performance of the formulation (p<0.05). No significant change (p<0.05) in fine particle dose was observed under different combinations of actuators and mouthpiece adaptors. The aerodynamic diameter and distribution of emitted particles from all experimental conditions did not show any significant change (p>0.05), probably due to the similar size of pre-engineered particles in the suspension formulation. It is hypothesized that the additional airflow induced by improper seals between patients’ lips and actuator mouthpiece may not influence the aerodynamic performance of suspension pMDIs. Resistance of air flowing through the actuator influenced particle deposition in the actuator mouthpiece and USP induction port, possibly due to different degree of flow turbulence at the exit of the actuator mouthpiece. However the particle deposition profiles in the cascade impactor did not exhibit any significant changes.

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1. Introduction

Pressurised metered dose inhalers (pMDIs) are the most prescribed inhalation therapy to deliver pulmonary medication for pulmonary diseases1_{, like asthma and chronic obstructive} pulmonary disease (COPD). This device is widely used due to its low cost, portability and independence from patient’s inspiratory flow. The short dispersion duration and rapid uptake of active pharmaceutical ingredients (APIs) in the lungs make the pMDI an efficient device. However the poor patient handling remains a problem that reduces the efficacy of the device2–4_{. Research regarding patient handling shows that the correct sealing of the lips} around the mouthpiece of the device is performed poorly among test subjects, but to date no research has been done to the effects of this poor sealing on aerosol performances.

The main aim of the current research was to investigate the in vitro aerosols performances of two formulations (Ventolin®_{and a beclomethasone dipropionate (BDP) formulation) with} different actuator setups using aerosol cascade impaction method. Standard Ventolin® actuators with modified airflow resistances were tested in sealed (“non-leaky”) and unsealed (“leaky”) conditions. The “non-leaky” condition was simulated by using a 3D-printed adapter, supplied by Clement-Clark International (UK). BDP formulations were investigated under “non-leaky” and “leaky” conditions and with/without the addition of glycerol in the formulation.

This project proposal consists of six chapters. Error: Reference source not found contains the theoretical background of this project. The materials and methods are described in the third chapter, followed by the results and discussion in chapter four. Chapter five and six contain the conclusion and the recommendations respectively.

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2. Theoretical Background

2.1 Respiratory System

In humans inhalation is put in motion by the respiratory muscles which increase or decrease the volume of the chest cavity5_{. By increasing the volume, the air pressure} decreases and air flows, according to Boyle’s law, into the lungs. While exhaling a counteraction occurs. At resting conditions humans breath approximately 12-15 times a minute, moving half a litre of air per breath6_{. This results in an uptake of 0.25 L oxygen and} give off of 0.20 L carbon dioxide. The respiratory system is divided into two parts; the upper- and lower respiratory tract6_{. The upper tract consists of the nose, nasal cavity,} sinuses and pharynx. The main function of the upper respiratory tract within the respiratory system is to be an entrance, filter, heater and humidifier of the air before it enters the lower respiratory tract. After reaching the pharynx the air flows to the larynx which is part of the lower respiratory tract. The trachea, bronchi, bronchioles and the alveoli are located below the larynx (Figure 2). The alveoli are the final stage of the respiratory system where the particles and molecules from the air diffuse through the air-blood membrane, a small membrane that prevents formation of air bubbles and damage to tissue while breathing5_. Alveoli have an average surface area of 1 m2_{per kilogram of bodyweight}7_{. This relatively} large surface area results, in combination with the fact that the lungs are directly connected to the blood vessels, in a rapid offset of molecules to the body. These factors make this organ an ideal route to administer active pharmaceutical ingredients (APIs) to the body. Furthermore pulmonary drug administration provides the opportunity to deposit a relatively high amount of APIs locally and it provides a method to administer APIs that are problematic to deliver through other administration routes. These properties make

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pulmonary drug administration efficient against respiratory diseases like asthma and COPD8_.

2.2 Pulmonary Drug Delivery

Nowadays three types of devices are used to administer APIs through pulmonary route: nebulizers, dry powder inhalers (DPIs) and pMDIs1_{. Each device has unique properties that} enable an optimized drug administration in different patient populations (Nowadays three types of devices are used to administer APIs through pulmonary route: nebulizers, dry powder inhalers (DPIs) and pMDIs 1 . Each device has unique properties that enable an optimized drug administration in different patient populations (Nowadays three types of devices are used to administer APIs through pulmonary route: nebulizers, dry powder inhalers (DPIs) and pMDIs 1 . Each device has unique properties that enable an optimized drug administration in different patient populations (Table 1). Nebulizers have good drug delivery properties, but the size of the device, the need of an electrical source and a long dispersion duration time are disadvantages. The main advantage of the pMDIs and the DPIs is their portability; their main disadvantage are the patient coordination for pMDI and relatively high inspiratory flow for DPIs, respectively, which can be problematic for patients

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with respiratory diseases. The pMDI will be the focus of this research and further chapters will include specific information about his device.). Nebulizers have good drug delivery properties, but the size of the device, the need of an electrical source and a long dispersion duration time are disadvantages. The main advantage of the pMDIs and the DPIs is their portability; their main disadvantage are the patient coordination for pMDI and relatively high inspiratory flow for DPIs, respectively, which can be problematic for patients with respiratory diseases. The pMDI will be the focus of this research and further chapters will include specific information about his device.). Nebulizers have good drug delivery properties, but the size of the device, the need of an electrical source and a long dispersion duration time are disadvantages. The main advantage of the pMDIs and the DPIs is their portability; their main disadvantage are the patient coordination for pMDI and relatively high inspiratory flow for DPIs, respectively, which can be problematic for patients with respiratory diseases. The pMDI will be the focus of this research and further chapters will include specific information about his device.

Table 1 Guidelines for the use of different inhalation devices based on different patient age1 Category Age (years) Recommended device

Infants and children <3 pMDI with a facemask or a nebulizer 3-5 pMDI with a spacer or a nebulizer 5-18 Nebulizer, pMDI or DPI Adolescents and adults >18 Nebulizer, pMDI or DPI

2.3 Pressured Metered Dose Inhalers

A pMDI consists of four major components9_{(Figure 2) these are; (1) the canister that} contains the formulation, (2) the actuator that holds the can in position, (3) the nozzle which atomizes the content when released and (4) the mouthpiece which allows a good transfer of the released content to the patient10_.

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Since pMDIs are used to administer APIs, the delivery of a constant dose is of great importance. This is regulated by the metering valve, a chamber inside the canister of a fixed volume, which is in contact with the formulation while the actuator is in rest position. When actuation occurs, the valve closes to the rest of the canister, while he valve stem opens. The propellant, hydrofluoroalkanes (HFA), evaporates immediately by the decreased air pressure and the content is driven through the nozzle. The content will accelerate quickly to 14 m/s11_{and causes mainly deposition via impaction of particles >6 µm in the upper} respiratory tract12_{. This is the major disadvantage of a pMDI and research has been} performed to lower its throat deposition13_{. Significant improvements can be achieved by} improving patient handling, i.e. either changing the design of the actuator to improve the aerosol delivery performance14_{or the ease of handling}13_.

Multiple investigations into patient handling have shown that a significant number of patients are unable to use the pMDI correctly2,4,15–18_{. An area requiring attention is the effect} of incorrect sealing of the patient lips around the pMDI mouthpiece, a factor that is performed incorrectly by 3.818_{- 31.8}4_{of the patients. This incorrect sealing results possibly} in unpredicted airflow dynamics in the actuator and the upper respiratory tract19_{, which} could results in a different amount of particle deposition within the lung10_.

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2.3.1 Types of pMDI

There are two types of pMDIs: solution and suspension pMDIs9,10_{. Solution pMDIs can} contain up to 10% ethanol to solubilise APIs, excipients and surfactants. Suspension pMDIs consist of pre-engineered particles that are insoluble in the propellant. It is expected that the improper sealing of the lips around the mouthpiece of a pMDI will have different outcomes in aerosol performance with the use of different pMDIs.

Since solution pMDIs consist of a homogeneous solution, the size of the particles is related to the size of the droplets and the formulation composition12,20–22_{. The use of} chlorofluorocarbons (CFCs), which were commonly used before the Montreal-protocol (1987), as propellant resulted in larger droplets than the later HFA formulations12,20–22_{. In the} later developed HFA formulations, excipients (i.e. glycerol) are added to increase the particle size of solution pMDIs to achieve equal aerosol performances as with the CFC formulations.

The droplets of suspension pMDIs are grouped in three different scenarios23_{, these are; (1)} droplets without particles, (2) droplets with one particle and (3) droplets with multiple particles. The particle distribution in the droplets has a significant effect on the deposition pattern, since the multiple particles in the third situation act as one big particle and will be deposited at higher areas within the respiratory tract23_.

2.4 Particle Deposition

The mass deposition profile in the airways is very important for the efficacy of a product. This profile is influenced by the anatomy of the respiratory system and the physical properties of aerosols, like particle size, surface geometry and composition of the particles24,25_{. The main mechanisms that causes particle deposition are impaction,} sedimentation and Brownian Diffusion6,25,26_{. These mechanisms influences mainly particles} bigger than 10 µm, between 1-8 µm and <1 µm respectively. Smaller particles can reach

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lower parts of the lungs and be retained there longer due to low air velocity in the lower part of the respiratory system27_.

2.4.1 Deposition in the upper airways

As mentioned before, the upper airways first filter the particles from incoming air via impaction, before the air enters the lower respiratory tract30_{. The efficacy of this filtering is} related to (1) morphology, (2) the inhalation velocity and (3) particle size31_.

(1) The morphology of the oropharyngeal cavity influences to the fluid dynamics and inspiration flow4,32 _{within this area. A higher oropharyngeal deposition is observed in cases} with a relatively small volume of the oral cavity33_{or a relatively short distance between the} lips and the pharynx11,34_{. (2) A lower deposition in the oropharyngeal area is observed with a} lower velocity of the inspiration flow11,34_{, since the deposition via impaction is decreased}35_. (3) The contribution of this physical effect is also related to the size of the particles31_{. The} complex coherence of multiple factors in the upper respiratory tract will result in a deposition of 3.311_{– 40%}36_{of the total delivered dose in this area.}

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2.4.2 Drug Deposition in the lower airways

The lower airways are the preferred deposition area for inhalable APIs, since the absorption of APIs is optimal in this area. Especially particles with a size of <5 µm penetrate deep in the lower airways and will remain there longer due to the low air velocity37_{. Particles are} likely to deposit via impaction around the branches of the lungs38_{. The deposition in this} area is, like the upper respiratory tract, related to the morphology, particle size and the air velocity38,39_.

In the lungs the particles will be subject to the clearing mechanism to remove foreign particles from the lung26,27_{. The first barrier of this mechanism is mucus, produced by the} goblet cells in the epithelium. Mucus is produced to enclose foreign particles; thereafter mucus is transported by the ciliated epithelial layer finally swallowed or coughed out26_. Smaller particles (<6 µm) penetrate and dissolve in the mucus and diffuse ending up in the epithelium, which is beyond the coverage of the mucociliary clearance27_{. The second barrier} consist of alveolar macrophages, which phagocytise particles and transport them to the mucus, lymphatic system or digest the particles inside them by the lysosomes. The hypothesis is that slowly dissolving particles with a size between 1.5µm- 3µm26_{in the alveoli} are removed by this process.

When deposited on the lung surface, APIs enter the blood circulation via passive and active transport26,27_._{Passive transport occurs when particles are deposited at the lung surface and} proceed to the blood circulation due to the concentration gradient. The rate of this transport is mainly dependent of the composition of the particle, the local thickness of the air-blood barrier and the blood perfusion at that location26_{. It is assumed that lipophilic molecules} diffuse through transcellular diffusion and hydrophilic compounds by paracelluar diffusion5_.

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2.5 In vitro aerosol performance: Andersen Cascade Impactor

The American and European Pharmacopeia prescribe a Cascade Impactor28,29_{to analyse} the aerodynamic particle size and predict the in vivo mass deposition profile. An Andersen Cascade Impactor (ACI) is an apparatus used to determine the particle size and is based on airborne particle inertia28,29,40_. _{In vitro measurement of aerodynamic particle size is an} indication of in vivo particle deposition region. This system consists of an induction port, stages with jets of certain aerodynamic diameters and collection plates. A sectional of this apparatus is shown in Figure 3. A vacuum pump is used to generate a constant airflow. The airflow through the ACI accelerates at every stage due to the decreasing diameter of the air-inlets. The increasing flow separates particles on the basis of their aerodynamic diameter, since particles of a certain diameter will deposit due to inertia at a certain air velocity, while smaller particles remain entrapped in the airflow, as the force is too small to deposit them. This process proceeds till the last stage, where the particles with an aerodynamic diameter of <0.4 µm are deposited in the filter. The ACI can separate particles within a range of 0.4-12µm28_.

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Error: Reference source not found

_{3. Materials and Methods}

3.1 Ventolin

®

3.1.1 Determination of the air resistance

The air resistance of the investigated conditions (Table 2) was measured with a TSI flowmeter (Model 4040, TSI instruments, USA). The flow was generated with a flow simulator (1120, Hans and Rudolph Inc., USA) at different air velocities (30, 60, 90, 120 and 150 L/min). The airflow resistance for each condition (figure 4) was calculated as square root of the pressure drop in kPa divided by the flowrate.

Table 2 Summary of the investigated Ventolin® combinations (figure 4)

Figure 5 Schematic overview of the used combinations

Condition Actuator Mouthpiece

A Commercial Non-leaky

B Commercial Leaky

C High resistance (custom made) Non-leaky D High resistance (custom made) Leaky E Low resistance (custom made) Non-leaky F Low resistance (custom made) Leaky

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3.1.2 HPLC Method Validation

An high performance liquid chromatography (HPLC) method was validated for the detection of salbutamol sulphate, the API present in commercail Ventolin®_{, according to the FDA} guidelines 41,42_{(Appendix I). The target concentration was 5 µg/mL.}

The linearity was determined in the range of 0.1 – 10 µg/mL. These concentrations were prepared in triplicate by serial dilutions starting from three different stock solutions of 100 µg/mL salbutamol base (Alibaba, Co. Ltd., China). 100 mL volumetric flasks were used to prepare these dilutions. The relative standard deviation (RSD) of the linearity, deviation of the slope, Limit of Detection (LOD) and Limit of Quantification (LOQ) were calculated. The accuracy was calculated by analysing samples with a concentration of 4,5 and 6 µg/mL in triplicate. The RSD and recovery were calculated to determine the accuracy. The precision was determined by measuring the target level in hexaplicate and calculating the RSD of these samples. The used HPLC method is described in chapter 3.1.3.

3.1.3 High performance liquid chromatography

A Shimadzu Prominence UPLC system consisting of a LC 20AD solvent delivery Unit, SIL 20A HT Autosampler and a SPD m20A photodiode array detector (Shimadzu Corporation, Japan) was used to analyse the samples with a C-18 chromatographic column (00F-4251-E0, Phenomenex®_{, USA). The used mobile phase was 92:8 (}v_/

v) 0.05 M K2HPO4:C2H3N (Clyde Industries Limited, Australia : Honeywell Burdick & Jackson®_{, USA) with a pH of 4.5.} The needlewash existed of 92:8 (v_/

v) water:C2H3N, this was also used as washing liquid for the dose unit sampling apparatus (DUSA) and the ACI. The used injection volume was 100 µL, the flowrate 1.0 mL/min and the detection wavelength 270 nm. The HPLC system was used at room temperature (22,5 ± 1,5 ºC).

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3.1.4 Dose Unit Sampling Apparatus (DUSA)

A DUSA (Copley Scientific, UK) was used to validate the dose of salbutamol sulphate delivered by a pMDI. The airflow was set at 28.3 L_/

min that was generated with a vacuum pump (Wp-0330, Westech Scientific Instruments, UK) and measured with a flowmeter (1120, Hans and Rudolph Inc., USA). The canister was primed according to the European Pharmacopoeia43_{. 5 shots, with 5 seconds shaking in between, were actuated in the DUSA.} The DUSA and the actuator were washed separately with 5 mL washing liquid. This experiment was performed in triplicate. The samples were analysed with the HPLC, as described in chapter 3.1.3.

3.1.5 Andersen Cascade Impaction

An ACI (Copley Scientific, UK) was used to determine the aerosol performances of the different conditions, as shown in Table 2. First the flow through the ACI was set at 28.3 L_/

min. As shown in Figure 5.

Figure 6 Schematic overview of the setup while setting the flow. The arrow indicates the flow direction.

After adjusting the flow, the pMDI canister was primed and actuated 10 times, with shaking in between, into the ACI. After the actuations, the ACI was dissembled and the collection plates were washed with the stages above. The actuator, the adapter and the throat were also washed with 100 mL washing liquid except stage 6 and 7, which were washed with 50 mL and 5 mL, respectively. The filter was sonicated in 50 mL washing liquid, which was centrifuged at 13.3 x103_{g for 10 minutes. The chemical quantification was performed with a} HPLC using the method described in chapter 3.1.2. Statistical analysis of the results was

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performed with the One-way Anova followed by the Tukey-test using SPSS (IBM, USA), with a p-level of <0.05 considered significant.

3.2 Beclomethasone Dipropionate

3.2.1 Formulation production

Two different formulations were developed in triplicate; both formulations consist of a 10 mL BDP solution in 7% (w/w) ethanol, one containing an additional 0.18% (w/w) glycerol (Table 3). Both formulations contain HFA-134a (Solvay Fluor GmbH, Germany) as propellant. The amount of BDP per shot was determined for each canister using a DUSA, as described in chapter 3.1.4. The used washing liquid was 80:20 (v_/

v) methanol:water.

Table 3 Summary of the investigated BDP conditions

Condition Mouthpiece BDP Ethanol Glycerol Metering valve Actuator Orofice size

G Non-leaky 20 mg 7% (w/w) - 50 µL BESPAK (UK) 0.3 mm

H Leaky 20 mg 7% (w/w) - 50 µL BESPAK (UK) 0.3 mm

I Non-leaky 20 mg 7% (w/w) 0.18% (w/w) 50 µL BESPAK (UK) 0.3 mm J Leaky 20 mg 7% (w/w) 0.18% (w/w) 50 µL BESPAK (UK) 0.3 mm

3.2.2 High performance liquid chromatography

The method validation was performed as described in chapter 3.1.2. The chemical quantification of BDP was performed with the same HPLC setup as described in chapter 3.1.3. 80:20 (v_/

v) methanol:water was used as mobile phase and needlewash. The injection volume was 100 µL, the flowrate was 1.0 mL/min and the detection wavelength was 240 nm. The column was used at room temperature (22,5 ± 1,5 ºC).

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The ACI was performed as described in chapter 3.1.5 . 80:20 (v_/

v) methanol:water was used as washing liquid.

The ACI was all washed with 100 ml washing liquid, except for stage 1,2,7 and the filter. These parts were washed with 50 ml. Stage 2, 6 7 and the filter of the ACI used with the glycerol containing canister were washed with 50 ml washing liquid, all other parts with 100 ml. The filters were sonicated and centrifuged (10 min at 13,3 x 103_{rpm) before analysis.} The used method for chemical quantification is described in chapter 3.2.2. The statistical analysis of the results was performed with the One-way Anova followed by the Tukey-test using SPSS, with p-level <0.05 considered significant.

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4. Results and discussion

4.1 Ventolin

®

4.1.1 Determination of the air resistance

The airflow resistances for different actuator and mouthpiece combinations are shown in Table 4.

Table 4 Calculated air resistances of the different conditions (n=5)

4.1.2 Method Validation

Linearity was measured within a range of 0.1 – 10 µg/mL salbutamol sulphate (Table 5). The calculated theoretical limit of qualification and detection were 0.32 and 0.10 µg/mL, respectively. The tailing factor was 1.25 and the theoretical plate number was 2497. All these results were within the recommendations of the FDA-guidelines.

Condition Resistance (mmH2O0.5 * L/min-1)

A 4.94 B 4.51 C 10.3 D 6.50 E 4.71 F 4.78

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Table 5 The data of the linearity, accuracy and precision of the used HPLC method for salbutamol sulphate.

Concentration (µg/mL) Average Area (SD) RSD(%) Recovery

Li n e ar it y 0.1 2558 (±51) 2.00% 0.5 13358 (±245) 1.84 % 1 26688 (±124) 0.47 % 5 135336 (±1441) 1.07 % 10 274664 (±3569) 1.30 % A cc u ra cy 4 108230 (±303) 0.28% 98.18% 5 137235 (±76) 0.06% 99.54% 6 163029 (±461) 0.28% 98.51% P re ci si o n 5 132324 (±455) 0.34%

4.1.3 Dose Unit Sampling Apparatus

The average shot weight of salbutamol (as sulphate) (n=3) that was determined by HPLC was 110 ± 7.4 µg. This was 10% more than the theoretical input. This value was within the maximum acceptable deviation for suspension pMDIs of ±25%44_.

The mass deposition profiles and the aerosol performances of the Ventolin®_suspension formulation are displayed in Figure 6 and Table 6, respectively. Aerosol performance for suspension pMDIs is mainly determined by the initial size of pre-engineered particles suspended in the liquefied propellant13_{. Therefore, the mass median aerodynamic diameter} (MMAD; the median size of the particles that are deposited on the stage) and geometric standard deviation (GSD; indicates the spread of the particle size in the formulation. <1.6 is considered as a monodisperse formulation) under different experimental configurations did not exhibit any significant change (p<0.05). This was consistent to previously published results45_{. However the flow dynamics, and mainly the intensity of the turbulence, in these}

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regions influences the particle deposition in the actuator and the throat heavily via impaction46,47_.

Table 6 Emitted aerosol results with different actuators and mouthpiece adaptors conditions (n=3 ± Standard deviation)

Condition MMAD (µm) GSD Total dose (mg) Emitted Dose (mg) Fine Particle Dose (mg) A 3.11 (±0.24) 1.76 (±0.11) 1.23 (±0.08) 1.08 (±0.07) 0.40 (±0.03) B 3.24 (±0.09) 1.80 (±0.17) 1.27 (±0.02) 1.13 (±0.09) 0.40 (±0.03) C 2.94 (±0.11) 1.69 (±0.02) 1.12 (±0.08) 0.85 (±0.02)* 0.33 (±0.02) D 3.09 (±0.12) 1.69 (±0.03) 1.07 (±0.06) 0.81 (±0.03)* 0.36 (±0.04) E 3.38 (±0.33) 1.63 (±0.10) 1.27 (±0.07) 1.09 (±0.05) 0.37 (±0.03) F 3.10 (±0.07) 1.67 (±0.02) 1.17 (±0.08) 1.02 (±0.01) 0.45 (±0.04)

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4.1.4.1 Airflow Resistances

In comparing the different actuators, analysis showed that there was no significant difference (p<0.05) in aerosol performance (Table 6) between the conventional actuator and the low resistance actuator. This was expected, since the airflow resistances of standard and low-resistance actuators (A and B, E, Table 3) were similar, which resulted probably in similar flow conditions at the mouthpiece exit.

However a significant increase in the flow resistance and pressure drop for the high-resistance actuator (C and D, Table 2) was noticed, possibly resulting in different flow patterns in the mouthpiece. This was observed since the use of either condition C or D results in a significantly lower emitted dose (ED; the dose that reach the patient) than the other actuators (A, B, E and F), caused by a high actuator deposition due to a change in the ex-orifice plume geometry, presumably due to a higher degree of airflow turbulence46_{. This} is demonstrated since the intensity of the turbulence is positively related to the deposition of, especially large, particles48_{and the deposition on stages 0-3 was significant lower with} either conditions C or D than to conditions A,B, E and F, while the fine particle dose (FPD; particles <4.7 µm) was significantly equal.

4.1.4.2 Sealing condition

No significant differences (p<0.05) in aerosol performances or the mass deposition profile were observed between the sealing conditions with the use of a conventional Ventolin® actuator. However the FPD of the “leaky” conditions (D and F, table 2) of the modified actuators was slightly higher than the “non-leaky” conditions (C and D, table 2) of the modified actuators. This effect was possibly caused by additional flows introduced by the leaky mouthpiece adaptor that may serve as ‘protective’ flow sheets to contain the ex-valve plume geometry in the USP induction port, resulting in a lower throat deposition profile.

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Furthermore no significant differences are observed between condition C and D and E and F, respectively.

However, modified actuators (B,C and F, table 3) introduced significant variations (~32%) between the triplicate experiments performed for the mass deposition profile of the actuator and up to ~11% in the cumulative deposition in the throat and actuator. This may be caused by the introduced non-predictable airflows in these areas.

4.2 Beclomethasone Dipropionate

4.2.1 Manufacture of BDP pMDI solution formulations The content of the canisters is displayed in Table 7.

Table 7 Content of the canisters and the results from the DUSA

Canister BDP (g) Ethanol (ml) Glycerol (ml) HFA 134a (mL) BDP per shot (µg) (n=1)

G and H 1 0.02 1.05 0 9.45 95.05 2 0.02 1.05 0 9.19 99.40 3 0.02 1.05 0 9.76 99.69 I and J 1 0.02 1.05 0.023 9.22 91.58 2 0.02 1.05 0.023 9.36 92.36 3 0.02 1.05 0.023 9.08 95.91 4.2.2 Method Validation

The linearity for the BDP samples was confirmed within a range of 0.1 – 10 µg/mL (Table 8). The RSD of these readings were within the limits of the FDA-guidelines. The calculated LOQ and LOD were respectively 0.24 and 0.08 µg/mL. The tailing factor was 1.39 and the theoretical plate number 1011. All results, except for the theoretical plate number, were within the recommendations of the FDA-guidelines. In order to obtain a perfect theoretical plate number the retention time of the analyte should be advanced by 14 seconds, but this was not considered as a problem since all other parameters were within the recommendations and there is no interference with other peaks.

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Table 8 Data of the method validation for BDP

Concentration (µg/mL) Average Area (SD) RSD(%) Recovery

Li n e ar it y 0.1 16369 (±296) 1.81% 0.5 77190 (±813) 1.05% 1 163535 (±913) 0.56% 5 815234 (±8362) 1.03% 10 1615405 (±9305) 0.58% A cc u ra cy 4 650617 (±608) 0.09% 100.49% 5 823846 (±1679) 0.20% 101.38% 6 978966 (±917) 0.09% 100.81% P re ci si o n 5 819540 (±7166) 0.87%

The mass deposition profiles and the aerosol performances of the BDP solution are displayed in Figure 7 and Table 9 respectively. Non-volatile excipients increase the particle size of solution pMDIs since the particle size is related to the formulation composition and the size of the droplets. Therefore conditions I and J (with glycerol, table 3) had a significant larger MMAD and GSD (p<0.05) than condition G and H (Without glycerol, table 3). However glycerol increase the particle size, no significant change in FPD was observed for all investigated conditions, since the addition of glycerol only slightly increase the amount of particles larger than an aerodynamic diameter of 4.7 µm. This is consistent to previously reported results49_{. However the addition of glycerol had the predicted effects on the aerosol} performance, there are no significant differences between the “leaky” and “non-leaky” conditions.

Table 9 Emitted aerosol results with different formulations and mouthpiece adaptors conditions (n=3 ± Standard deviation)

Condition MMAD (µm) GSD Total dose (mg) Emitted Dose (mg) FPD (mg) G 1.42 (±0.05) 1.87 (±0.03) 0.91 (±0.01) 0.83 (±0.01) 0.45 (±0.02) H 1.50 (±0.10) 1.80 (±0.02) 0.86 (±0.02) 0.79 (±0.01) 0.43 (±0.05) I 1.97 (±0.03) 1.99 (±0.05) 0.96 (±0.03) 0.86 (±0.03) 0.40 (±0.01) J 2.08 (±0.13) 1.98 (±0.02) 0.95 (±0.06) 0.87 (±0.00) 0.42 (±0.02)

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Figure 7 Mass deposition profiles of emitted particles from BDP solution formulations with different formulations and mouthpiece adaptors combinations (n=3, ± standard deviation)

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5. Conclusions

The increased airflow resistance within the actuator of a suspension pMDI resulted in a higher actuator deposition and a lower throat deposition. The FPD, MMAD and GSD with using this actuator remained the same compared to the conventional actuator, a possible benefit against throat irritation, a common problem associated with the use of a pMDI50_.

The addition of 0.18% glycerol in a solution pMDI increased the MMAD with ~0.55 µm and the GSD with ~0.1. A formulation with glycerol will target other areas in the lung, since larger particles cannot penetrate as deep in the lower respiratory tract as smaller particles can.

The aerosol performance of two different types of pMDIs has been investigated in vitro. In both cases the ED, FPD, MMAD and GSD did not change according different mouth sealing conditions. This in vitro research gives an indication that the sealing of the lips around the mouthpiece of a pMDI does not have an significant impact on pMDIs aerosol performances.

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6. Future work

In the future, computational fluid dynamics (CFD) would be recommended to investigate the airflows within all the investigated different actuator-mouthpiece conditions and the USP throat. A CFD study could provide insight on the hypothesized “protective” flows suggested in this current investigation.

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The in vitro effects on the aerosol performance of poor lip sealing around the mouthpiece of pressurised metered dose inhalers