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The use of cannabidiol (CBD) for treating brain disorders has gained increasing interest. While the mechanism of action of CBD in these conditions is still under investigation, CBD has been shown to affect numerous different drug targets in the brain that are involved in brain disorders. Here we review the preclinical and clinical evidence on the potential therapeutic use of CBD in treating various brain disorders. Moreover, we also examine various drug delivery approaches that have been applied to CBD. Due to the slow absorption and low bioavailability with the current oral CBD therapy, more efficient routes of administration to bypass hepatic metabolism, particularly pulmonary delivery, should be considered. Comparison of pharmacokinetic studies of different delivery routes highlight the advantages of intranasal and inhalation drug delivery over other routes of administration (oral, injection, sublingual, buccal, and transdermal) for treating brain disorders. These two routes of delivery, being non-invasive and able to achieve fast absorption and increase bioavailability, are attracting increasing interest for CBD applications, with more research and development expected in the near future., (© 2023. The Author(s).)

Purpose: The probability of agglomerate-to-wall collision was quantified using a unique image processing technique applied to high-speed microscopic images. The study aimed to investigate the effects of flow rate and particle size on the percentage of colliding agglomerates detected within an in-house powder dispersion device., Method: The device consists of a swirl chamber and two tangential inlets in various configurations, designed to emulate the geometric features of commercial devices such as the Aerolizer® and Osmohaler®. The test cases were conducted with constant flow rates of 30 SLPM and 60 SLPM. Four powder samples were tested, including carrier Respitose® SV010 (median volume diameter 104 µm, span 1.7) and mannitol of three constituent primary particle sizes (3 µm, 5 µm and 7 µm; span 1.6 - 1.9)., Results: At the lower flow rate of 30 SLPM, collision frequencies were significantly different between powders of different constituent particle sizes, but the effects of powder properties diminished on increasing the flow rate to 60 SLPM. At the higher flow rate, all powders experienced a significant increase in the proportion of colliding particles., Conclusion: Analysis of collision events showed that the probability of collision for each agglomerate increased with agglomerate diameter and velocity. Experimental data of agglomerate-to-wall collision were utilised to develop a logistic regression model that can accurately predict collisions with various powders and flow rates., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Dry powder inhalation formulations have become increasingly popular for local and systemic delivery of small molecules and biotherapeutics. Powder formulations provide distinct advantages over liquid formulations such as elimination of cold chain due to room temperature stability, improved portability, and the potential for increasing patient adherence. To become a viable product, it is essential to develop formulations that are stable (physically, chemically and/or biologically) and inhalable over the shelf-life. Physical particulate properties such as particle size, morphology and density, as well as chemical properties can significantly impact aerosol performance of the powder. This review will cover these critical attributes that can be engineered to enhance the dispersibility of inhalation powder formulations. Challenges in particle engineering for biotherapeutics will be assessed, followed by formulation strategies for overcoming the hurdles. Finally, the review will discuss recent examples of successful dry powder biotherapeutic formulations for inhalation delivery that have been clinically assessed., (© 2022. The Author(s).)

Purpose: This study aims to understand the impact of spray drying nozzles on particle surface composition and aerosol stability., Methods: The combination formulations of colistin and azithromycin were formulated by 2-fluid nozzle (2 N) or 3-fluid (3 N) spray drying in a molar ratio of 1:1. A 3-factor, 2-level (2 3 ) factorial design was selected to investigate effects of flow rate, inlet temperature and feed concentration on yield of spray drying and the performance of the spray dried formulations for the 3 N., Results: FPF values for the 2 N formulation (72.9 ± 1.9% for azithromycin & 73.4 ± 0.8% for colistin) were higher than those for the 3 N formulation (56.5 ± 3.8% for azithromycin & 55.1 ± 1.6% for colistin) when stored at 20% RH for 1 day, which could be attributed to smaller physical size for the 2 N. There was no change in FPF for both drugs in the 2 N formulation after storage at 75% RH for 90 days; however, there was a slight increase in FPF for colistin in the 3 N formulation at the same storage conditions. Surface enrichment of hydrophobic azithromycin was measured by X-ray photoelectron spectroscopy for both 2 N and 3 N formulations and interactions were studied using FTIR., Conclusions: The 3-fluid nozzle provides flexibility in choosing different solvents and has the capability to spray dry at higher feed solid concentrations. This study highlights the impact of hydrophobic azithromycin enrichment on particle surface irrespective of the nozzle type, on the prevention of moisture-induced deterioration of FPF for hygroscopic colistin.

Purpose: Dissolution behavior of dry powder inhaler (DPI) antibiotic formulations in the airways may affect their efficacy especially for poorly-soluble antibiotics such as azithromycin. The main objective of this study was to understand the effects of surface composition on the dissolution of spray dried azithromycin powders by itself and in combination with colistin., Methods: Composite formulations of azithromycin (a poorly water-soluble molecule) and colistin (a water-soluble molecule) were produced by spray drying. The resultant formulations were characterized for particle size, morphology, surface composition, solid-state properties, solubility and dissolution., Results: The results demonstrate that surfaces composition has critical impacts on the dissolution of composite formulations. Colistin was shown to increase the solubility of azithromycin. For composite formulations with no surface colistin, azithromycin released at a similar dissolution rate as the spray-dried azithromycin alone. An increase in surface colistin concentration was shown to accelerate the dissolution of azithromycin. The dissolution of colistin from the composite formulations was significantly slower than the spray-dried pure colistin. In addition, FTIR spectrum showed intermolecular interactions between azithromycin and colistin in the composite formulations, which could contribute to the enhanced solubility and dissolution of azithromycin., Conclusions: Our study provides fundamental understanding of the effects of surface concentration of colistin on azithromycin dissolution of co-spray-dried composite powder formulations.

Purpose: Nose-to-brain drug administration along the olfactory and trigeminal nerve pathways offers an alternative route for the treatment of central nervous system (CNS) disorders. The characterization of particle deposition remains difficult to achieve in experiments. Alternative numerical approach is applied to identify suitable aerosol particle size with maximized inhaled doses., Methods: This study numerically compared the drug delivery efficiency in a realistic human nasal cavity between two aerosol drug administration systems targeting the olfactory region: the aerosol mask system and the breath-powered bi-directional system. Steady inhalation and exhalation flow rates were applied to both delivery systems. The discrete phase particle tracking method was employed to capture the aerosol drug transport and deposition behaviours in the nasal cavity. Both overall and regional deposition characteristics were analysed in detail., Results: The results demonstrated the breath-powered drug delivery approach can produce superior olfactory deposition with peaking olfactory deposition fractions for diffusive 1 nm particles and inertial 10 μm. While for particles in the range of 10 nm to 2 μm, no significant olfactory deposition can be found, indicating the therapeutic agents should avoid this size range when targeting the olfactory deposition., Conclusions: The breath-powered bi-directional aerosol delivery approach shows better drug delivery performance globally and locally, and improved drug administration doses can be achieved in targeted olfactory region.

Purpose: To compare titer reduction and delivery rate of active anti-tuberculosis bacteriophage (phage) D29 with three inhalation devices., Methods: Phage D29 lysate was amplified to a titer of 11.8 ± 0.3 log 10 (pfu/mL) and diluted 1:100 in isotonic saline. Filters captured the aerosolized saline D29 preparation emitted from three types of inhalation devices: 1) vibrating mesh nebulizer; 2) jet nebulizer; 3) soft mist inhaler. Full-plate plaque assays, performed in triplicate at multiple dilution levels with the surrogate host Mycobacterium smegmatis, were used to quantify phage titer., Results: Respective titer reductions for the vibrating mesh nebulizer, jet nebulizer, and soft mist inhaler were 0.4 ± 0.1, 3.7 ± 0.1, and 0.6 ± 0.3 log 10 (pfu/mL). Active phage delivery rate was significantly greater (p < 0.01) for the vibrating mesh nebulizer (3.3x10 8  ± 0.8x10 8 pfu/min) than for the jet nebulizer (5.4x10 4  ± 1.3x10 4 pfu/min). The soft mist inhaler delivered 4.6x10 6  ± 2.0x10 6 pfu per 11.6 ± 1.6 μL ex-actuator dose., Conclusions: Delivering active phage requires a prudent choice of inhalation device. The jet nebulizer was not a good choice for aerosolizing phage D29 under the tested conditions, due to substantial titer reduction likely occurring during droplet production. The vibrating mesh nebulizer is recommended for animal inhalation studies requiring large amounts of D29 aerosol, whereas the soft mist inhaler may be useful for self-administration of D29 aerosol.

Purpose: This work investigated the effect of relative humidity (RH) on bipolar electrostatic charge profiles of dry powder inhaler aerosols using the Bipolar Charge Analyzer (BOLAR)., Methods: Two commercial products, Pulmicort® (400 μg, budesonide) and Bricanyl® (500 μg, terbutaline sulfate) Turbuhaler®, were used as model dry powder inhalers (DPIs) in this study. Three individual doses from each Turbuhaler® were sampled at 15, 40, 65 and 90% RH. Subsequently, charge and mass profiles were determined for each dispersion., Results: The aerosols from these two Turbuhaler® DPI were bipolarly charged, with larger particles carrying negative charge and smaller particles positive charge. Particles changed polarity around 2.60-4.17 μm and 0.95-2.60 μm for Pulmicort® and Bricanyl®, respectively. The effect of RH on particles differed between DPIs even though the mass output was not significantly affected. The net charge profiles of Pulmicort® were relatively independent of RH, whereas those of Bricanyl® showed a reduction in the charge magnitude with increasing RH. Both positive and negative charge profiles followed a similar trend with the change in RH and individually they had higher magnitudes than the measured net charge., Conclusions: This study showed drug-specific bipolar charging of the Turbuhaler® DPI aerosols at varied RHs. Bricanyl® was more susceptible to RH and showed decreased bipolar and net charge levels with increasing RH, in comparison to Pulmicort®.

Purpose: In order to attenuate the drug release rate, a single freeze-thaw step was previously shown to convert encapsulated drug into a single nanocrystal within each liposome vesicle. The goal of this study was to alter the nanocrystalline character, and thus the drug encapsulation state and release profile, by addition of surfactant prior to freeze-thaw., Methods: A liposomal ciprofloxacin (CFI) formulation was modified by the addition of surfactant and frozen. After thawing, these formulations were characterized in terms of drug encapsulation by centrifugation-filtration, liposome structure by cryo-TEM imaging, vesicle size by dynamic light scattering, and in vitro release (IVR) performance., Results: The addition of increasing levels of polysorbate 20 (0.05 to 0.4%) or Brij 30 (0.05 to 0.3%) to the CFI preparations followed by subsequent freeze-thaw, resulted in a greater proportion of vesicles without drug nanocrystals and reduced the extent of growth of the nanocrystals thus leading to modified release rates including an increase in the ratio of non-encapsulated to sustained release of drug., Conclusions: This study provides another lever to achieve the desired release rate profile from a liposomal formulation by addition of surfactant and subsequent freeze-thaw, and thus may provide a personalized approach to treating patients.

Purpose: This study was undertaken to evaluate the biopharmaceutical properties of cyclosporine A (CsA)-loaded nano-matrix particles for inhalation., Methods: Nano-matrix particles of CsA with mannitol (nCsAm) were prepared by a flash nano-precipitation technique employing a multi-inlet vortex mixer and evaluated in terms of physicochemical properties, anti-inflammatory effect in the rat model of airway inflammation, pharmacokinetic behavior, and distributions of CsA to side-effect-related organs after intratracheal administration., Results: In nCsAm, spherical nano-particles of CsA were covered with mannitol and the mean particle size was 1.3 μm. The in vitro Next Generation Impactor analysis demonstrated fine inhalation performance with a fine particle fraction value of 65.8%. Intratracheal nCsAm (100 μg-CsA/rat) significantly attenuated the recruitment of inflammatory cells into the airway in the rat model of airway inflammation, followed by suppression of the inflammatory biomarkers. After intratracheal nCsAm at a pharmacologically effective dose (100 μg-CsA/rat), there was a 42-47-fold decrease in the distribution of CsA to side-effect-related organs such as the kidney and liver compared with oral CsA at a toxic dose (10 mg-CsA/kg), potentially leading to avoidance of systemic side-effects of CsA., Conclusion: Upon these findings, nCsAm prepared with the flash nano-precipitation technique could be a novel dosage form of CsA for inhalation therapy of airway inflammation with a better safety margin.

Purpose: Previously, a respirable powder (RP) formulation of pirfenidone (PFD) was developed for reducing phototoxic risk; however, PFD-RP demonstrated unacceptable in vitro inhalation performance. The present study aimed to develop a new RP system of PFD with favorable inhalation properties by spray-drying method., Methods: Spray-dried PFD (SD/PFD) was prepared by spray-drying with L-leucine, and the physicochemical properties and efficacy in an antigen-sensitized airway inflammation model were assessed. A pharmacokinetic study was also conducted after intratracheal and oral administration of PFD formulations., Results: Regarding powder characterization, SD/PFD had dimpled surface with the mean diameter of 1.793 μm. In next generation impactor analysis, SD/PFD demonstrated high in vitro inhalation performance without the need of carrier particles, and the fine particle fraction of SD/PFD was calculated to be 62.4%. Insufflated SD/PFD (0.3 mg-PFD/rat) attenuated antigen-evoked inflammatory events in the lung, including infiltration of inflammatory cells and myeloperoxidase activity. Systemic exposure level of PFD after insufflation of SD/PFD at the pharmacologically effective dose was 600-fold lower than that after oral administration of PFD at the phototoxic dose., Conclusion: SD/PFD would be suitable for inhalation, and the utilization of an RP system with SD/PFD would provide a safer medication compared with oral administration of PFD.

Purpose: The potential of aerosol phage therapy for treating lung infections has been demonstrated in animal models and clinical studies. This work compared the performance of two dry powder formation techniques, spray freeze drying (SFD) and spray drying (SD), in producing inhalable phage powders., Method: A Pseudomonas podoviridae phage, PEV2, was incorporated into multi-component formulation systems consisting of trehalose, mannitol and L-leucine (F1 = 60:20:20 and F2 = 40:40:20). The phage titer loss after the SFD and SD processes and in vitro aerosol performance of the produced powders were assessed., Results: A significant titer loss (~2 log) was noted for droplet generation using an ultrasonic nozzle employed in the SFD method, but the conventional two-fluid nozzle used in the SD method was less destructive for the phage (~0.75 log loss). The phage were more vulnerable during the evaporative drying process (~0.75 log further loss) compared with the freeze drying step, which caused negligible phage loss. In vitro aerosol performance showed that the SFD powders (~80% phage recovery) provided better phage protection than the SD powders (~20% phage recovery) during the aerosolization process. Despite this, higher total lung doses were obtained for the SD formulations (SD-F1 = 13.1 ± 1.7 × 10(4) pfu and SD-F2 = 11.0 ± 1.4 × 10(4) pfu) than from their counterpart SFD formulations (SFD-F1 = 8.3 ± 1.8 × 10(4) pfu and SFD-F2 = 2.1 ± 0.3 × 10(4) pfu)., Conclusion: Overall, the SD method caused less phage reduction during the powder formation process and the resulted powders achieved better aerosol performance for PEV2.

Purpose: To investigate the applicability of Bipolar Charge Analyzer (BOLAR), a new commercial instrument developed by Dekati Ltd., in simultaneously characterizing the bipolar electrostatic charge profile and measuring the size distribution of commercial metered dose inhalers (MDIs)., Methods: Intal Forte(®) (sodium cromoglycate), Tilade(®) (nedocromil sodium), Ventolin(®) (salbutamol sulphate), and QVAR(®) (beclomethasone dipropionate) were used as model MDIs in this study. Three individual actuations of each MDI were introduced into the BOLAR at an air flow rate of 60 l/min. Charge and mass profiles for each actuation were determined., Results: The BOLAR was found to have better performance in collecting valid charge data (≥80%) than valid mass data (≥50%). In all tested products, both positively and negatively charged particles were found in five defined size fractions between zero and 11.6 μm, with the charge magnitude decreased with increasing particle size. The net charge profiles obtained from the BOLAR qualitatively agreed with the results reported previously. In all suspension type MDIs, negligible masses were detected in the smallest size fraction (<0.95 μm), for which the charge was most likely caused by the propellant and excipients. QVAR was the only solution MDI tested and the charge and mass profiles were significantly different from the suspension-type MDIs. Its mass profile was found to follow closely with the charge profile., Conclusions: Positively and negatively charged MDI particles of different size fractions and their corresponding charge-to-mass profiles were successfully characterized by the BOLAR.

Purpose: This study investigated the effect of different active pharmaceutical ingredients (API) on aerosol electrostatic charges and aerosol performances for pressurized metered dose inhalers (pMDIs), using both insulating and conducting actuators., Methods: Five solution-based pMDIs containing different API ingredients including: beclomethasone dipropionate (BDP), budesonide (BUD), flunisolide (FS), salbutamol base (SB) and ipratropium bromide (IPBr) were prepared using pressure filling technique. Actuator blocks made from nylon, polytetrafluoroethylene (PTFE) and aluminium were manufactured with 0.3 mm nominal orifice diameter and cone nozzle shape. Aerosol electrostatics for each pMDI formulation and actuator were evaluated using the electrical low-pressure impactor (ELPI) and drug depositions were analysed using high performance liquid chromatography (HPLC)., Results: All three actuator materials showed the same net charge trend across the five active drug ingredients, with BDP, BUD and FS showing positive net charges for both nylon and PTFE actuators, respectively. While SB and IPBr had significantly negative net charges across the three different actuators, which correlates to the ionic functional groups present on the drug molecule structures., Conclusions: The API present in a pMDI has a dominant effect on the electrostatic properties of the formulation, overcoming the charge effect arising from the actuator materials. Results have shown that the electrostatic charges for a solution-based pMDI could be related to the interactions of the chemical ingredients and change in the work function for the overall formulation.

Purpose: Carrier-based dry powder inhalers (DPIs) are widely used for rapid and convenient delivery of drug to the site of action. This work aimed to predict powder aerosolisation in DPIs through numerical modelling., Methods: A multi-scale modelling technique based on the combined computational fluid dynamics (CFD) and discrete element method (DEM) approach was developed., Results: The simulation results of the detachments of the drug particles from single carrier under different impact velocities and angles were comparable with those measured in the experiments in terms of fine particle fraction FPF loaded . Empirical equations were developed to link the detachment performance with impact velocity and impact angle. Then the dynamics of the carrier particles in Aerolizer® was simulated. The results indicated that the carrier-wall impaction was the dominant mechanism for drug aerosolisation performance. By linking the empirical equations with the carrier-wall impact energy, the predictions showed that for a given formulation mass with a fixed carrier/drug ratio, the inhaler performance decreased with carrier size and increased with air flow rate. Device empty efficiency, however, was independent with carrier size and flow rate., Conclusions: The multi-scale model was able to provide quantitative information to better understand the aerosolisation mechanisms of carrier-based formulation.

Purpose: To investigate the influence of different actuator nozzle designs on aerosol electrostatic charges and aerosol performances for pressurised metered dose inhalers (pMDIs)., Methods: Four actuator nozzle designs (flat, curved flat, cone and curved cone) were manufactured using insulating thermoplastics (PET and PTFE) and conducting metal (aluminium) materials. Aerosol electrostatic profiles of solution pMDI formulations containing propellant HFA 134a with different ethanol concentration and/or model drug beclomethasone dipropionate (BDP) were studied using a modified electrical low-pressure impactor (ELPI) for all actuator designs and materials. The mass of the deposited drug was analysed using high performance liquid chromatography (HPLC)., Results: Both curved nozzle designs for insulating PET and PTFE actuators significantly influenced aerosol electrostatics and aerosol performance compared with conducting aluminium actuator, where reversed charge polarity and higher throat deposition were observed with pMDI formulation containing BDP. Results are likely due to the changes in plume geometry caused by the curved edge nozzle designs and the bipolar charging nature of insulating materials., Conclusions: This study demonstrated that actuator nozzle designs could significantly influence the electrostatic charges profiles and aerosol drug deposition pattern of pMDI aerosols, especially when using insulating thermoplastic materials where bipolar charging is more dominant.

Purpose: Use isothermal microcalorimetry to directly evaluate the effects of excipients and water content to produce a stable amorphous glycopyrrolate pressurized metered dose inhaler (pMDI) formulation., Methods: Amorphous glycopyrrolate particles with and without excipients (Distearoyl-sn-glycero-3-phosphatidylcholine (DSPC) or β-cyclodextrin (βCD)) were spray dried and cold filled along with HFA 134a into customized thermal activity monitor (TAM) measurement ampoules. When applicable, a known amount of water was also pipetted into the ampoule. Sample ampoules were hermetically sealed, equilibrated to 25°C and measured isothermally for at least 24 h using the TAM III (TA Instruments, Sollentuna, Sweden)., Results: Amorphous glycopyrrolate particles were highly unstable and crystallized rapidly when suspended in HFA 134a. Co-spray drying the glycopyrrolate with DSPC failed to mitigate this instability, but co-spray drying with βCD protected the amorphous glycopyrrolate from crystallization, resulting in a stable formulation at low water contents (≤ 100 ppm)., Conclusions: This study shows that isothermal microcalorimetry can easily differentiate between physically stable and unstable pMDI formulations of glycopyrrolate within a few hours. Furthermore, it allows rapid screening of various formulation factors (drug form, excipients, water ingress), which can greatly reduce the time required to develop marketable products with acceptable shelf life.

Purpose: To demonstrate the novel application of nano X-ray computed tomography (NanoXCT) for visualizing and quantifying the internal structures of pharmaceutical particles., Methods: An Xradia NanoXCT-100, which produces ultra high-resolution and non-destructive imaging that can be reconstructed in three-dimensions (3D), was used to characterize several pharmaceutical particles. Depending on the particle size of the sample, NanoXCT was operated in Zernike Phase Contrast (ZPC) mode using either: 1) large field of view (LFOV), which has a two-dimensional (2D) spatial resolution of 172 nm; or 2) high resolution (HRES) that has a resolution of 43.7 nm. Various pharmaceutical particles with different physicochemical properties were investigated, including raw (2-hydroxypropyl)-beta-cyclodextrin (HβCD), poly (lactic-co-glycolic) acid (PLGA) microparticles, and spray-dried particles that included smooth and nanomatrix bovine serum albumin (BSA), lipid-based carriers, and mannitol., Results: Both raw HβCD and PLGA microparticles had a network of voids, whereas spray-dried smooth BSA and mannitol generally had a single void. Lipid-based carriers and nanomatrix BSA particles resulted in low quality images due to high noise-to-signal ratio. The quantitative capabilities of NanoXCT were also demonstrated where spray-dried mannitol was found to have an average void volume of 0.117 ± 0.247 μm(3) and average void-to-material percentage of 3.5%. The single PLGA particle had values of 1993 μm(3) and 59.3%, respectively., Conclusions: This study reports the first series of non-destructive 3D visualizations of inhalable pharmaceutical particles. Overall, NanoXCT presents a powerful tool to dissect and observe the interior of pharmaceutical particles, including those of a respirable size.

Purpose: The techniques available to study formulation stability in pressurized metered dose inhalers (pMDIs) are limited, due to the challenging conditions of working with high pressure propellants. Isothermal microcalorimetry is a valuable tool used to screen and aid in formulation development of solid and solution drug formulations; however there are currently no available methods to evaluate pMDIs. In this paper, we have developed a method that allows measurement of such pressurized systems., Methods: Samples were prepared by cold filling ampoules with propellant (HFA 134a) and drugs of interest. Ampoule caps were fitted with a specific O-ring, coated with paraffin and pre-conditioned prior to measurement. Samples were equilibrated at 25°C, placed in a Thermal Activity Monitor III (TAM III) system and measured isothermally at 25°C for a period of at least 24 h., Results: Using well-defined procedures and ampoule preparation techniques we were able to safely contain the volatile propellant and acquire a stable measurement baseline. We were able to rapidly determine, within 6 h, the physical stability of amorphous and crystalline drug forms of beclomethasone dipropionate and formoterol fumarate dihydrate when formulated with HFA 134a., Conclusions: Isothermal microcalorimetry in pressurized HFA propellant systems was shown to be a rapid screening tool to evaluate pMDI formulation physical stability. This method can potentially be applied to study pMDI formulation factors to expedite product development.

Purpose: This study aimed to characterize inherent charge generated by micron-sized drug-only formulations of amorphous and crystalline salbutamol sulfate (SS)., Methods: Amorphous SS was produced by spray-drying whilst crystalline SS was produced by conditioning spray-dried SS with supercritical CO2 and menthol. Electrostatic charge of the powders was characterized in two ways. Firstly, the charge profile of the aerosols dispersed from an Aerolizer® was measured using a modified Electrostatic Low Pressure Impactor (ELPI™). Secondly, the net charge of the bulk powders generated from tumbling in containers composed of different materials (polyethylene, polyvinyl chloride, Teflon, nylon and stainless steel) was measured by a Faraday pail., Results: Following aerosolization, crystalline SS appeared to show more consistent charging and mass deposition than amorphous SS. In the tumbling experiment crystalline SS had a significant correlation between net charge and work function, which was absent in amorphous SS. This may be due to the long-range crystal packing which was reflected as more predictable charging. In addition, the polarity of charging was attributed to the arrangement of SS molecules in the crystal lattice., Conclusions: The effect of crystallinity on the electrostatic charge behavior of inhalable micron-sized spherical drug particles with well-defined particle size distribution was investigated for the first time. The knowledge gained may assist in the development of optimized inhaled pharmaceutical products.

Purpose: The potential for rifapentine-containing oral therapeutic regimens to significantly shorten the current six-month anti-tubercular treatment regimen is confounded by high plasma protein binding of rifapentine. Inhaled aerosol delivery of rifapentine, a more potent anti-tubercular antibiotic drug, in combination with other first-line antibiotics may overcome this limitation to deliver a high drug dose at the pulmonary site of infection., Methods: A formulation consisting of rifapentine, moxifloxacin and pyrazinamide, with and without leucine, was prepared by spray-drying. This formulation was assessed for its physico-chemical properties, in vitro aerosol performance and antimicrobial activity., Results: The antibiotic powders, with and without leucine, had similar median aerodynamic diameters of 2.58 ± 0.08 μm and 2.51 ± 0.06 μm, with a relatively high fine particle fraction of 55.5 ± 1.9% and 63.6 ± 2.0%, respectively. Although the powders were mostly amorphous, some crystalline peaks associated with the δ polymorph for the spray-dried crystalline pyrazinamide were identified., Conclusions: Stabilisation of the powder with 10% w/w leucine and protection from moisture ingress was found to be necessary to prevent overt crystallisation of pyrazinamide after long-term storage. In vitro biological assays indicated antimicrobial activity was retained after spray-drying. Murine pharmacokinetic studies are currently underway.

Purpose: To investigate the influence of different actuator materials and nozzle designs on the electrostatic charge properties of a series of solution metered dose inhaler (pMDI) aerosols., Methods: Actuators were manufactured with flat and cone nozzle designs using five different materials from the triboelectric series (Nylon, Polyethylene terephthalate, Polyethylene-High density, Polypropylene copolymer and Polytetrafluoroethylene). The electrostatic charge profiles of pMDI containing beclomethasone dipropionate (BDP) as model drug in HFA-134a propellant, with different concentrations of ethanol were studied. Electrostatic measurements were taken using a modified electrical low-pressure impactor (ELPI) and the deposited drug mass assayed chemically using HPLC., Results: The charge profiles of HFA 134a alone have shown strong electronegativity with all actuator materials and nozzle designs, at an average of -1531.34 pC ± 377.34. The presence of co-solvent ethanol significantly reduced the negative charge magnitude. BDP reduced the suppressing effect of ethanol on the negative charging of the propellant. For all tested formulations, the flat nozzle design showed no significant differences in net charge between different actuator materials, whereas the charge profiles of cone designs followed the triboelectric series., Conclusion: The electrostatic charging profiles from a solution pMDI containing BDP and ethanol can be significantly influenced by the actuator material, nozzle design and formulation components. Ethanol concentration appears to have the most significant impact. Furthermore, BDP interactions with ethanol and HFA have an influence on the electrostatic charge of aerosols. By choosing different combinations of actuator materials and orifice design, the fine particle fractions of formulations can be altered.

Purpose: While most examples of nanoparticle therapeutics have involved parenteral or IV administration, pulmonary delivery is an attractive alternative, especially to target and treat local infections and diseases of the lungs. We describe a successful dry powder formulation which is capable of delivering nanoparticles to the lungs with good aerosolization properties, high loadings of nanoparticles, and limited irreversible aggregation., Methods: Aerosolizable mannitol carrier particles that encapsulate nanoparticles with dense PEG coatings were prepared by a combination of ultrasonic atomization and spray freeze drying. This process was contrasted to particle formation by conventional spray drying., Results: Spray freeze drying a solution of nanoparticles and mannitol (2 wt% solids) resulted in particles with an average diameter of 21 ± 1.7 μm, regardless of the fraction of nanoparticles loaded (0-50% of total solids). Spray freeze dried (SFD) powders with a 50% nanoparticle loading had a fine particle fraction (FPF) of 60%. After formulation in a mannitol matrix, nanoparticles redispersed in water to < 1 μm with hand agitation and to < 250 nm with the aid of sonication. Powder production by spray drying was less successful, with low powder yields and extensive, irreversible aggregation of nanoparticles evident upon rehydration., Conclusions: This study reveals the unique advantages of processing by ultrasonic spray freeze drying to produce aerosol dry powders with controlled properties for the delivery of therapeutic nanoparticles to the lungs.

Purpose: We hypothesize that the USP induction port may de-agglomerate carrier-free powder emitting from dry powder inhalers (DPIs)., Methods: Aerosols emitting from a range of DPIs (Spinhaler®, Turbuhaler® and Osmohaler™) and induction ports (USP throat, straight tube, Alberta idealized mouth-throat geometry (AG)) were sized by laser diffraction. Total drug recovery was obtained by HPLC and fine particle fraction computed. Air flow patterns were simulated using Computational Fluid Dynamics (CFD)., Results: The straight tube did not de-agglomerate emitted powder. However, the USP throat and AG further de-agglomerated powders from the Spinhaler, but not the Turbuhaler and Osmohaler. While budesonide powder deposited similarly in all induction ports, deposition was significantly higher in the AG for both DSCG and mannitol. CFD revealed agglomerates impacting on the USP throat with higher localized velocity compared with the straight tube. CFD further showed a more complex flow pattern with high-velocity air jets in the AG, which explains the higher FPF for DSCG and the lower FPF for mannitol using the AG., Conclusion: The USP throat further de-agglomerated the emitted powder from the DPI when it did not sufficiently disperse the powder. Other tools such as laser diffraction may be used for cross-examining to avoid artifacts in the results.

Purpose: To produce and examine the aerosol performance of protein nano-matrix particles with different surface roughness., Methods: Aqueous lysozyme solutions were poured into isopropanol during high-shear mixing to produce nanoparticles by precipitation. The size of the nanoparticles was varied by adjusting the precipitation conditions. The resultant suspensions were spray-dried to obtain micron-sized aggregates (nano-matrices). Smooth particles were made by spray-drying a lysozyme solution. The aggregate size distribution, surface roughness, and cohesion were evaluated. The aerosol performance was assessed by dispersing 10 mg of powder from a Rotahaler(®) at 60 L/min or an Aerolizer® at 100 L/min into a Next Generation Impactor, followed by chemical assay (n=3)., Results: The median volume diameter and span of the nano-matrix particles were 1.0-1.2 μm and 1.5-1.6, respectively, which were comparable to those of the smooth particles. Surface roughness increased with the size of the primary nanoparticles. The nano-matrix particles were significantly less cohesive than the smooth particles. The fine particle fraction increased linearly with increasing surface roughness and decreasing cohesion., Conclusions: Nano-matrix particles with controlled surface architecture were successfully produced by spray-drying nanosuspensions. Aerosol performance was enhanced with increasing surface roughness due to the reduction in cohesion forces.

Purpose: To measure aerosol performance of a lactose carrier/salbutamol sulphate powder blend and identify contributions of non-formulation and formulation components on the resulting aerosol charge., Methods: A 67.5:1 (%w/w) blend of 63-90 microm lactose with salbutamol sulphate, and lactose alone (with and without the blending process), was dispersed from a Cyclohaler into the electrical Next Generation Impactor at 30, 60 and 90 L/min. Mass and charge profiles were measured from each dispersion, as a function of impactor stage. The charge profile from an empty capsule in the Cyclohaler was also studied., Results: Lactose deposition from the blend was significantly greater, and net charge/mass ratios were smaller, in the pre-separator compared to formulations without drug. Fine particle fraction of salbutamol sulphate increased with flow rate (9.2 +/- 2.5% to 14.7 +/- 2.7%), but there was no change in net charge/mass ratio. The empty capsule produced a cycle of alternating net positive and negative discharges ( approximately 200 pC to 4 nC)., Conclusions: Capsule charge can ionize surrounding air and influence net charge measurements. Detachment of fine drug during aerosolisation may reduce net specific charge and lead to increased lactose deposition in the pre-separator. Increase in FPF may be due to increased force of detachment rather than electrostatic forces.

Purpose: This study utilized a combination of computational fluid dynamics (CFD) and standardized entrainment tubes to investigate the influence of turbulence on the break-up and aerosol performance of a model inhalation formulation., Methods: Agglomerates (642.8 mum mean diameter) containing 3.91 mum median diameter primary spherical mannitol particles were prepared by spheronisation. A series of entrainment tubes with different Venturi sections were constructed in silico, and the flow pattern and turbulence/impaction parameters were predicted using CFD. The entrainment models were constructed from the in silico model using three-dimensional printing. The aerosol performance of the mannitol was assessed by entraining the agglomerates into the experimental tubes at a series of flow rates and assessing the size distribution downstream of the venturi via in-line laser diffraction., Results: A series of parameters (including Reynolds number (Re), turbulence kinetic energy, turbulence eddy frequency, turbulence length-scale, velocity and pressure drop) were calculated from the CFD simulation. The venturi diameter and volumetric flow rate were varied systematically. The particle size data of the agglomerated powders were then correlated with the CFD measurements. No correlation between turbulence and aerosol performance could be made (i.e. at a Reynolds number of 8,570, the d(0.1) was 52.5 mum +/- 19.7 mum, yet at a Reynolds number of 12,000, the d(0.1) was 429.1 mum +/- 14.8 mum). Lagrangian particle tracking indicated an increase in the number of impactions and the normal velocity component at the wall, with increased volumetric airflow and reduced venturi diameter. Chemical analysis of the mannitol deposited on the walls showed a linear relationship with respect to the theoretical number of impactions (R(2) = 0.9620). Analysis of the relationship between the CFD results and the experimental size data indicated a critical impact velocity was required to initiate agglomerate break-up ( approximately 0.4 m.s(-1))., Conclusion: While this study focussed on the effect of turbulence on agglomerate break-up, the small amount of impaction, which inevitably occurs in the venturi assembly, appeared to dominate agglomerate break-up in this dry powder system.

Purpose: The capability of the electrostatic next generation impactor (eNGI) has been investigated as a tool capable of measuring the electrostatic charge of single (Flixotide; containing fluticasone propionate (FP)) and combination (Seretide; FP and salmeterol xinafoate (SX)) pressurised metered dose inhalers (pMDIs) at different flow rates., Methods: Aerosol mass distributions were investigated at 30, 60 and 90 l.min(-1) and simultaneous charge measurements recorded., Results: Analysis of the mass distribution data indicated a flow dependent relationship, where the aerosol performance (aerodynamic diameter <5 mum) of FP significantly increased between 30 l.min(-1) and 60 l.min(-1) for both formulations. No significant increase in SX was observed for Seretide with increased flow rate. Analysis of the charge distribution indicated both formulations to primarily charge negatively with a concurrent increase in charge with increased flow rate. Interestingly, the charge-tomass ratio remained relatively constant between 30 l.min(-1) and 60 l.min(-1) and increased at 90 l.min(-1), indicating that charging was majorly influenced at the highest flow rate., Conclusions: This study has shown how the eNGI could be used as a simple Pharmacopeia based methodology for the evaluation of mass and charge profiles of single and combination pMDIs at a series of flow rates.

Purpose: Lactose dry powder inhaler (DPI) carriers, constructed of smaller sub units (composite carriers), were evaluated to assess their potential for minimising drug-carrier adhesion, variability in drug-carrier forces and influence on drug aerosol performance from carrier-drug blends., Methods: Lactose carrier particles were prepared by fusing sub units of lactose (either 2, 6 or 10 microm) in saturated lactose slurry. The resultant composite particles, as well as supplied lactose, were sieve fractioned to obtain a 63-90 microm carriers. The carriers were evaluated in terms of size (laser diffraction) morphology (electron microscopy and atomic force microscopy), crystallinity and drug adhesion (colloid probe microscopy). In addition, blends containing drug and carrier were prepared and evaluated in terms of drug aerosol performance., Results: The surface morphology and physico-chemical properties of the composite carriers were significantly different. Depending on the initial primary lactose size, the composite particles could be prepared with different surface roughness. Variation in composite roughness could be related to the change in drug adhesion (via modification in contact geometry) and thus drug aerosol performance from drug-lactose blends., Conclusion: Composite based carriers are a potential route to control drug-carrier adhesion forces and variability thus allowing more precise control of formulation performance.

Purpose: To introduce the design of the electrical next generation impactor (eNGI), and validate its proposed function as a method of electrostatic characterization for pressurized metered dose inhaler (pMDI) formulations., Methods: Flixotide (fluticasone propionate), ventolin (salbutamol sulphate), and qvar (beclomethasone dipropionate) were used as model pMDIs in this study. At an airflow rate of 30 l/min, five individual actuations of each pMDI were introduced into the electrical low-pressure impactor (ELPI), Next Generation Impactor (NGI), and the eNGI. Charge profiles for each actuation were measured by the ELPI and eNGI, while mass profiles were recorded by the all three impactors., Results: The difference in estimated mass median aerodynamic diameters and geometric standard deviations for all pMDIs using the NGI and eNGI were not found to be statistically significant (p<0.05). The mean charge profiles from the ELPI and eNGI overlap well between 0.54 and 6.61 microm (flixotide and ventolin), and between 0.615 and 11.72 microm (qvar), where the majority of the impacted doses were collected., Conclusion: For the analysis of pMDIs, the eNGI is comparable to the NGI in measuring particle size distribution, while still being comparable to the ELPI in measuring charge distribution.

Purpose: Many existing and new drugs fail to be fully utilized because of their limited bioavailability due to poor solubility in aqueous media. Given the emerging importance of using nanoparticles as a promising way to enhance the dissolution rate of these drugs, a method must be developed to adequately reflect the rate-change due to size reduction. At present, there is little published work examining the suitability of different dissolution apparatus for nanoparticles., Methods: Four commonly-used methods (the paddle, rotating basket and flow-through cell from the US Pharmacopia, and a dialysis method) were employed to measure the dissolution rates of cefuroxime axetil as a model for nanodrug particles., Results: Experimental rate ratios between the nanoparticles and their unprocessed form were 6.95, 1.57 and 1.00 for the flow-through, basket and paddle apparatus respectively. In comparison, the model-predicted value was 7.97. Dissolution via dialysis was rate-limited by the membrane., Conclusions: The data showed the flow-through cell to be unequivocally the most robust dissolution method for the nanoparticulate system. Furthermore, the dissolution profiles conform closely to the classic Noyes-Whitney model, indicating that the increase in dissolution rate as particles become smaller results from the increase in surface area and solubility of the nanoparticles.

Purpose: At present, there is no published data examining the effect of relative humidity on the electrostatic charges of dry powder inhaler aerosols. The charging behaviour of two commercial products, Pulmicort and Bricanyl Turbuhalers, were investigated using an electrical low pressure impactor (ELPI)., Methods: ELPI was successfully modified to disperse the aerosols at 60 l/min. Four doses from each new inhaler were sampled at 15, 40, 65, and 90% RH. Particles deposited on the impactor stages according to their aerodynamic diameters and their charges were measured simultaneously by the electrometers. The drug in each size fraction was quantified using HPLC., Results: Both products generated bipolar charges. The charging behaviour of the two types of inhaler showed different humidity dependence although the mass output was not significantly affected. The absolute specific charge of budesonide fine particles from Pulmicort was the lowest at 40% RH but increased at lower and higher RHs. In contrast, the terbutaline sulfate fine particles from Bricanyl followed the expected trend of charge reduction with increasing RH., Conclusions: The distinct trends of charging of aerosols from Pulmicort and Bricanyl Turbuhalers was explained by differences in hygroscopicity and other physicochemical factors between the two drugs.

Purpose: The current study aimed to examine the pharmaceutical applications of the focused-ion-beam (FIB) in the inhalation aerosol field, particularly to particle porosity determination (i.e. percentage of particles having a porous interior)., Materials and Methods: The interior of various spray dried particles (bovine serum albumin (BSA) with different degrees of surface corrugation, mannitol, disodium cromoglycate and sodium chloride) was investigated via FIB milling at customized conditions, followed by viewing under a high resolution field-emission scanning electron microscope. Two sets of ten particles for each sample were examined., Results: For the spray-dried BSA particles, a decrease in particle porosity (from 50 to 0%) was observed with increasing particle surface corrugation. Spray-dried mannitol, disodium cromoglycate and sodium chloride particles were determined to be 90-100%, 0-10% and 0% porous, respectively. The porosity in the BSA and mannitol particles thus should be considered for the aerodynamic behaviour of these particles., Conclusions: The FIB technology represents a novel approach useful for probing the interior of particles linking to the aerosol properties of the powder. Suitable milling protocols have been developed which can be adapted to study other similar particles.

Purpose: To investigate the influence of mouthpiece geometry on the amount of throat deposition and device retention produced using a dry powder inhaler (Aerolizer), along with the subsequent effect on the overall inhaler performance., Materials and Methods: Computational Fluid Dynamics analysis of the flowfield generated in the Aerolizer with various modified mouthpiece geometries (including cylindrical, conical and oval designs) was used in conjunction with experimental dispersions of mannitol powder using a multi-stage liquid impinger to determine how the overall inhaler performance varied as the mouthpiece geometry was modified., Results: Geometry of the inhaler mouthpiece had no effect on device retention or the inhaler dispersion performance. In contrast, the mouthpiece geometry strongly affected the amount of throat deposition by controlling the axial component of the exit air flow velocity. The radial motion of the emitted aerosol jet was found to have little effect on throat deposition in representative mouth-throat models. Despite the reduced throat deposition, there was no difference in the overall inhaler performance., Conclusions: For cases where low throat deposition is a key design parameter, this study demonstrates that the amount of throat deposition can be reduced by making minor modifications to the inhaler mouthpiece design.

To investigate the influence of storage relative humidity (RH) on the aerosolisation efficiency and tribo-electrification of carrier based dry powder inhaler (DPI) formulations using the next generation impactor (NGI) in vitro methodology and the electrostatic low pressure impactor (ELPI). Micronised salbutamol (d (0.5) 1.48 +/- 0.03 microm) was blended with 63-90 microm sieve fractioned alpha-lactose monohydrate carrier and stored at a range of humidities (0-84% RH). The aerosolisation efficiency after storage for 24 h periods was investigated using the NGI. The same experiment was conducted using the ELPI, with corona charger switched off, to measure the net charge vs. mass deposition profile. Significant variations in the aerosolisation efficiency of the formulation were observed with respect to storage RH. In general, the fine particle fraction aerosol performance measured by NGI and ELPI (fraction with mass median aerodynamic diameter <4.46 and 4.04 microm, respectively) followed a positive parabola with aerosol performance increasing over the range 0-60% RH before decreasing >60% RH. Analysis of the ELPI charge data suggested that the micronised salbutamol sulphate had an electronegative charge when aerosolised from lactose based carriers, which was most electronegative at low RH. Increased storage RH resulted in a reduction in net charge to mass ratio with the greatest reduction at RH >60%. The aerosol performance of this binary system is dependent on both electrostatic and capillary forces. The use of the ELPI allows a degree of insight into how these forces affect formulation performances after storage at different RH.

Purpose: The aims of the study are to analyze the influence of air flow on the overall performance of a dry powder inhaler (Aerolizer and to provide an initial quantification of the flow turbulence levels and particle impaction velocities that maximized the inhaler dispersion performance., Methods: Computational fluid dynamics (CFD) analysis of the flow field in the Aerolizer, in conjunction with experimental dispersions of mannitol powder using a multistage liquid impinger, was used to determine how the inhaler dispersion performance varied as the device flow rate was increased., Results: Both the powder dispersion and throat deposition were increased with air flow. The capsule retention was decreased with flow, whereas the device retention first increased then decreased with flow. The optimal inhaler performance was found at 65 l min(-1) showing a high fine particle fraction (FPF) of 63 wt.% with low throat deposition (9.0 wt.%) and capsule retention (4.3 wt.%). Computational fluid dynamics analysis showed that at the critical flow rate of 65 l min(-1), the volume-averaged integral scale strain rate (ISSR) was 5,400 s(-1), and the average particle impaction velocities were 12.7 and 19.0 m s(-1) at the inhaler base and grid, respectively. Correlations between the device flow rate and (a) the amount of throat deposition and (b) the capsule emptying times were also developed., Conclusions: The use of CFD has provided further insight into the effect of air flow on the performance of the Aerolizer. The approach of using CFD coupled with powder dispersion is readily applicable to other dry powder inhalers (DPIs) to help better understand their performance optimization.

Purpose: To study the fundamental effects of the spinning capsule on the overall performance of a dry powder inhaler (Aerolizer)., Methods: The capsule motion was visualized using high-speed photography. Computational fluid dynamics (CFD) analysis was performed to determine the flowfield generated in the device with and without the presence of different sized capsules at 60 l min(-1). The inhaler dispersion performance was measured with mannitol powder using a multistage liquid impinger at the same flowrate., Results: The capsule size (3, 4, and 5) was found to make no significant difference to the device flowfield, the particle-device impaction frequency, or the dispersion performance of the inhaler. Reducing the capsule size reduced only the capsule retention by 4%. In contrast, without the presence of the spinning capsule, turbulence levels were increased by 65%, FPF(Em) (wt% particles < or =6.8 microm in the aerosol referenced against the amount of powder emitted from the device) increased from 59% to 65%, while particle-mouthpiece impaction decreased by 2.5 times. When the powder was dispersed from within compared to from outside the spinning capsule containing four 0.6 mm holes at each end, the FPF(Em) was increased significantly from 59% to 76%, and the throat retention was dropped from 14% to 6%., Conclusions: The presence, but not the size, of a capsule has significant effects on the inhaler performance. The results suggested that impaction between the particles and the spinning capsule does not play a major role in powder dispersion. However, the capsule can provide additional strong mechanisms of deagglomeration dependent on the size of the capsule hole.

Purpose: The current study aimed to quantify the different degree of particle surface corrugation and correlate it to the aerosol performance of powders., Methods: Powders of different degree of surface corrugation were prepared by spray drying under varying conditions. The solid-state properties of the powders including particle size, morphology, crystal form, true density, and moisture content were characterized. The degree of surface corrugation was quantified by the surface fractal dimension (Ds) obtained by light scattering. The aerosol performance was studied by dispersing the powders using the Rotahaler at 60 L/min into a multi-stage liquid impinger. Fine particle fraction (FPF) was expressed as the wt% of BSA particles of size < or =5 microm in the aerosol., Results: Four powders of increasing degree of particle surface corrugation were prepared, with Ds ranging from 2.06 for the least corrugated to 2.41 for the most corrugated. The powders had a similar size distribution (VMD 3 microm, span 1.4-1.5) and solid-state properties. Increasing the surface corrugation, Ds, slightly from 2.06 to 2.18 enhanced the FPF significantly from 27% to 41%. This was explained by the reduced area of contacts and increased separation distance between the particles. Further increase of corrugation (Ds > or = 2.18) did not improve FPF., Conclusions: Powders with varying degrees of corrugation were successfully obtained by spray drying with their surface roughness quantified by fractal analysis. It was shown that only a relatively small degree of surface corrugation was sufficient to accomplish a considerable improvement in the aerosol performance of the powder.

Purpose: To investigate the feasibility of using the Aerosol Solvent Extraction System (ASES) to produce fine powders of recombinant human deoxyribonuclease (rhDNase), lysozyme-lactose and rhDNase-lactose powders from aqueous based solutions., Methods: The ASES technique using high pressure carbon dioxide modified with ethanol or ethanol and triethylamine was used for the generation of rhDNase powders and protein-lactose powders from aqueous based solutions. Particle size, morphology, size distributions, crystallinity, and powder aerosol performance were measured. The biochemical integrity of the processed rhDNase was assessed by testing the monomer content and the degree of deamidation., Results: RhDNase precipitated as spherical particles in the size range between 50 and 500 nm. The primary nano-sized particles were agglomerated to micron-sized clumps of particles during the precipitation process. The median particle size and the fine particle fraction were functions of the operating temperature and the nozzle system used. RhDNase was substantially denatured in the ASES process using carbon dioxide modified with ethanol as anti-solvent. However almost complete recovery of the monomer was achieved using carbon dioxide modified with ethanol-triethylamine as an anti-solvent. Lysozyme-lactose and rhDNase-lactose powders were also precipitated as agglomerated spheres using the ASES process. The powders were amorphous except for those with lactose content higher than 45%., Conclusions: Micron-sized particles of rhDNase suitable for inhalation delivery were generated from aqueous based solutions using the modified ASES technique. The biochemical integrity of the rhDNase powder is a function of the antisolvent and the operating temperature.

Purpose: To study the dispersion performance of non-porous corrugated particles, with a focus on the effect of particle surface morphology on aerosolization of bovine serum albumin (BSA) powders., Methods: The solid-state characteristics of the spray-dried BSA powders, one consisting of smooth spherical particles and another corrugated particles, were characterized by laser diffraction, X-ray powder diffraction, scanning electron microscopy, confocal microscopy, thermogravimetric analysis, surface area analyzer, and buoyancy method. The powders were dispersed using the Rotahaler and the Dinkihaler coupled to a four-stage liquid impinger operating at 30 to 120 L/min. Fine particle fraction (FPF) was expressed as the wt. % of BSA particles of size < or =5 microm collected from the liquid impinger., Results: Apart from the morphology and morphology-related properties (specific surface area, envelope density), the corrugated particles and spherical particles of BSA had very similar solid-state characteristics (particle size distribution, water content, true density, amorphous nature). Using the Dinkihaler, the FPFs of the corrugated particles were 10-20 wt. % higher than those of the smooth particles. Similar FPF differences were found for the powders dispersed by the Rotahaler, but the relative changes were larger. In addition, the differences were inversely proportional to the air flows (17.3% at 30 L/min, 25.2% at 60 L/min, 13.8% at 90, 8.5% at 120 L/min). Depending on the inhaler, capsule and device retention and impaction loss at the impinger throat were lower for the corrugated particles., Conclusions: Enhanced aerosol performance of powders can be obtained by surface modification of the particles. The surface asperities of the corrugated particles could lower the true area of contact between the particles, and thus reduce the powder cohesiveness. A distinct advantage of using corrugated particles is that the inhaler choice and air flow become less critical for these particles.

Purpose: To investigate the feasibility of using the Aerosol Solvent Extraction System (ASES) to generate microparticles of proteins suitable for aerosol delivery from aqueous-based solutions., Methods: The ASES technique using high-pressure carbon dioxide modified with ethanol was utilised for the generation of microparticles of proteins (lysozyme, albumin, insulin and recombinant human deoxyribonuclease (rhDNase)) from aqueous solutions. Particle size, morphology, size distributions and powder aerosol performance were examined. The biochemical integrity of the processed proteins was assessed by testing the level of molecular aggregation using size exclusion chromatography and by bioassay technique for lysozyme., Results: Proteins were precipitated as spherical particles ranging in size from 100 to 500 nm. The primary nano-sized particles agglomerated to form micron-sized particles during the precipitation process. The median size of the particles was a function of the operating conditions. In-vitro aerosol performance tests showed that the percent fine particle mass (< 5 microm) was approximately 65%, 40% and 20% for lysozyme, albumin and insulin, respectively. Negligible loss in the monomer content or biological activity was observed for lysozyme. Insulin exhibited slight aggregation and 93% of the monomer was retained after processing. Albumin was affected by processing and only 50-75% of the monomer was retained compared with 86% in the original material. However, rhDNase was substantially denatured during processing as shown by the significantly reduced monomer content., Conclusions: Micron-sized particles of lysozyme, albumin and insulin with satisfactory inhalation performance were successfully generated from aqueous solutions using the modified ASES technique. The biochemical integrity of the processed proteins was a function of the operating conditions and the nature of the individual protein.

Purpose: To study the effect of particle size, air flow and inhaler type on the dispersion of spray dried mannitol powders into aerosols., Methods: Mannitol powders were prepared by spray drying. The solid state properties of the powders were determined by laser diffraction, X-ray powder diffraction, scanning electron microscopy, freeze fracture, Karl Fischer titration and gas pycnometry. The powders were dispersed using Rotahaler and Dinkihalerg, connected to a multistage liquid impinger at different air flows., Results: Three crystalline mannitol powders with primary particle size (MMD) 2.7, 5.0, 7.3 microm and a similar polydispersity were obtained. The particles were spherical with a density of 1.5 g/cm3 and a moisture content of 0.4 wt.%. At an air flow of 30 L/min all the powders were poorly dispersed by both inhalers. With the Rotahaler increasing the flow (60-120 L/min) increased the fine particle fraction (FPF) in the aerosols for the 2.7 microm powder, and decreased the FPF for the 7.3 microm powder; whereas the FPF for 5.0 microm powder was unaffected. With the Dinkihaler, all the powders were near complete dispersion at > or = 60 L/min., Conclusions: The FPF in the mannitol powder aerosols was determined by an interplay of the particle size, air flow and inhaler design.

Purpose: Development of a mathematical model to provide information about the amount of water associated with a protein and an excipient in a lyophilized product., Methods: The moisture content of the product and the mass fraction of each component were used to derive a model for the calculation of the mass of water associating with each component. The model was applied to lyophilized formulations of rhDNase containing various amounts of mannitol or lactose. The total water content was investigated by thermogravimetry, crystalline properties by X-ray powder diffraction and water uptake behaviour using a moisture microbalance system., Results: Calculations based on the model suggest that in a lyophilized rhDNase-mannitol formulation where the sugar is crystalline, most of the water is taken up by the protein. However, in the lyophilized rhDNase-lactose formulation where the sugar is amorphous, water is taken up by both the sugar and protein to a comparative extent. At high relative humidities when the amorphous sugar undergoes crystallization, the model can accommodate such a change by allowing for the formation of an additional crystalline phase., Conclusions: The rhDNase-sugar formulations show excellent conformity to the model which provides quantitative information about the distribution of water in the lyophilized binary protein-excipient products.

Purpose: The mobility of protein in powders at different hydration levels was studied in relation to aggregation and activity., Methods: Magic angle spinning 13C, 15N, 1H, 2H, and 17O NMR techniques were used to determine changes in the mobility of surface residues in proteins as a function of hydration and related to changes in activity. NMR relaxation measurements of high frequency (omega0, T1) and low frequency (omega1,T1p) motions have been carried out on lyophilized DNase, insulin and lysozyme stored at different relative humidities. Moisture-induced aggregation and enzymatic activity of the lyophilized proteins was determined by high performance size exclusion chromatography and bioassays., Results: There was little change in T1p observed with increasing humidity. The results show, however, that there is a decrease in T1 for DNase, insulin and lysozyme at relative humidities ranging from 0-98%, and we propose that the reduction in T1 is related to the aggregation susceptibility of proteins during storage at different humidities. The water mobility was determined directly using 17O NMR experiments. We found that as the amount of weakly-bound water increases, the protein surface mobility decreases and is coupled with increased aggregation. Aggregation measurements at different humidities were correlated with bioassays for lysozyme and found to be consistent with the hydration data., Conclusions: Mobility of protein molecules was determined by solid-state NMR over a wide range of % RH and it was found that water content leads to a change in mobility of protein molecules. The aggregation and activity of proteins were strongly correlated to change in molecular mobility.

Purpose: We have used rhDNase to investigate the feasibility of developing a dry protein powder aerosol for inhalation delivery., Methods: Powders of rhDNase alone and with sodium chloride were prepared by spray drying. Powder blends were obtained by mixing (tumbling and sieving) pure rhDNase powder with 'carrier' materials (lactose, mannitol or sodium chloride). The weight percent of drug in the blends was between 5 and 70%. The particle size distributions and crystallinity of the spray dried powders were obtained by laser diffraction and X-ray powder diffraction, respectively. Particle morphology was examined by scanning electron microscopy. The ability of the powders and powder blends to be dispersed into respirable aerosols was measured using a Rotahaler connected to a multistage liquid impinger operating at 60 L/min., Results: Pure rhDNase powder was quite cohesive with a fine particle fraction (FPF or "respirable fraction': % wt. of particles < 7 microns in the aerosol cloud) of about 20%. When particles also contained NaCl, the powders were dispersed better to form aerosols. A linear relationship was observed between the NaCl content and FPF for a similar primary size (approximately 3 microns volume median diameter) of particles. The particle morphology of these powders varied systematically with the salt content. For the blends, SEM revealed a monolayer-like adhesion of the fine drug particles to the carriers at drug contents > or = 50% wt. An overall 2-fold increase in FPF of rhDNase in the aerosol cloud was obtained for all the blends compared to the pure drug aerosols., Conclusions: The aerosol properties of spray dried rhDNase powders can be controlled by incorporation of a suitable excipient, such as NaCl, and its relative proportion. Coarse carriers can also enhance the performance of rhDNase dry powder aerosols.

Purpose: To study the thermal stability of recombinant human deoxyribonuclease I (rhDNase) in aqueous solutions., Methods: Differential scanning calorimetry (DSC) was used to measure the denaturation or melting temperature (T(m)) and enthalpy (H(m)) of rhDNase. The effects of denaturants (guanidine HCl and urea) and additives (mainly divalent cations and disaccharides) were investigated at pH 6-7., Results: The T(m) and H(m) of rhDNase in pure water were measured as 67.4 degrees C and 18.0 J/g respectively, values typical of globular proteins. The melting peak disappeared on re-running the sample after cooling to room temperature, indicating that the thermal denaturation was irreversible. The latter was due to the occurrence of aggregation accompanying the unfolding process of rhDNase. Size exclusion chromatography indicated that during heat denaturation, rhDNase formed soluble high molecular weight aggregates with a molecular size >300kD estimated by the void volume. Of particular interest are the divalent cations: Ca(2+) stabilizes rhDNase against thermal denaturation and elevates T(m) and H(m) while Mg(2+), Mn(2+) and Zn(2+) destabilize it. Sugars also stabilize rhDNase. As expected, denaturants destabilize the protein and lower the T(m) and H(m). All destabilization of rhDNase can be prevented by adding Ca(2+) to the solutions., Conclusions: CaCl(2) and sugars were found to stabilize rhDNase against thermal denaturation while divalent cations, urea and guanidine HCl destabilize the protein. The effects could be explained by a mixture of mechanisms. For Ca(2+) the protective effect is believed to be due to an ordering of the rhDNase structure in its native state, and by prevention of breaking of a disulfide bridge, thus making it less susceptible to unfold under thermal stress.

Purpose: To determine if a protein changes when it is compressed into a KBr pellet for FTIR spectroscopy measurement in the solid state, using recombinant human deoxyribonuclease I (rhDNase) as an example., Methods: Lyophilized rhDNase with KBr compressed at different pressures were analyzed by FTIR spectroscopy, size exclusion HPLC and enzymatic activity assay. Different protein/KBr weight ratios and residual water contents were studied for their possible effects on aggregation., Results: Depending on the pressure, a loss of enzymatic activity accompanied by an increase in soluble high molecular weight aggregates of the protein (up to approximately 15%) was demonstrated. Aggregation was reduced to less than 5% by a suitable dilution of the protein in KBr (1 in 1000). In contrast, water content variability (1-11 wt. %) did not affect aggregation., Conclusions: The findings emphasize the importance to examine for protein integrity when using the KBr method for FTIR sample preparation. Protein aggregation may be minimized by optimizing the sample preparation condition such as changing the protein/KBr weight ratio.

The habit (external morphology) of a crystal is controlled by both the external (environmental) conditions of crystallization and the internal (structural) factors of the crystal. In order to separate the effects of the crystal structure and of the solvent and other external factors on the experimentally observed growth habit, the theoretical habit can be derived from the crystal structure using the periodic bond chain (PBC) theory and attachment energy considerations. According to the PBC theory the crystal habit is governed by a set of uninterrupted chains of strong bonds formed in the crystal lattice. In addition, the attachment energy (E(att)) is defined as the energy released per mole when a new layer is deposited on a crystal face. Since the habit of a crystal is determined by the relative growth rate (R) of the various faces, by taking R proportional to E(att), the theoretical habit can thus be derived from E(att). Using this approach, we obtained the theoretical crystal habit of an antitumor drug, hexamethylmelamine (HMM). The possible effect of solvents on the habit modification of HMM is discussed. This technique, based purely on the knowledge of the crystal structure, is directly applicable to other pharmaceuticals in deriving their theoretical crystal habit.