IA 2013 | Posters

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In vitro and in vivo evaluation of ciprofloxacin liposome for pulmonary administration
Aiping Zheng

The term pulmonary drug delivery system (PDDS) refers to drug release to the lung producing local or systemic therapeutic effects. As an administration of treatment of localized disease of the respiratory system, it has been widely used in clinical, such as cystic fibrosis, bronchiectasis, chronic obstructive pulmonary disease, pneumonia, emphysema. Ciprofloxacin is a broad-spectrum antibiotic of the quinolone family, and has good antibacterial activity against most gram-negative bacteria and gram-positive cocci. It has been approved by the FDA as an anti-infective agent and is widely used for the treatment of a variety of bacterial infections. Currently, ciprofloxacin is delivered through oral or intravenous administration and reaches the lungs, which are important infection sites for inspiratory bacteria. However, ciprofloxacin does not preferentially accumulate in the lungs and may therefore not reach high, sustained therapeutic levels at the site. Delivering this potent antibiotic directly to the lung may improve its safety and efficacy in the treatment of pulmonary infections. Liposomes have been shown to be a promising delivery system for a number of antimicrobial drugs including antibiotics. Liposomes as drug carriers for pulmonary delivery are composed of phospholipids bimolecular layer formation, the same as the lung of endogenous substances both of which have good biocompatibility.

The successful treatment of pulmonary infection using ciprofloxacin depends on high, sustained drug concentrations in tissues where the bacteria reside and multiply. Furthermore, liposome encapsulated ciprofloxacin, which directly targets the lung could prolong the residence time of the drug in the lung, thereby reducing the pulmonary irritation and decreasing the systemic side effects. The aim of this study was to investigate the ciprofloxacin liposome of high encapsulation efficiency with optimal physical properties for pulmonary administration and to test its in vivo potential in rats. The optimized ciprofloxacin liposome, which had a high encapsulation efficiency of 93.96%, and an average particle size of 349.65 nm with a span of 0.42, showed sustained in vitro release. The optimized ciprofloxacin liposome was further examined in the in vivo study in rats. The concentration of ciprofloxacin in lung and blood was simultaneously determined in each rat. The ratio of the AUClung value between ciprofloxacin liposome and ciprofloxacin solution was 288.333, whereas the relative bioavailability was 72.42%, and the drug targeting efficiency of ciprofloxacin liposome and ciprofloxacin solution by intratracheal administration were 799.707 and 2.009, respectively. The results showed that ciprofloxacin liposome for pulmonary administration could be delivered to a specific target site and achieve a high local concentration.

In conclusion, ciprofloxacin liposome for pulmonary administration offered an attractive alternative which was able to deliver high concentrations of antibiotic directly to the chosen target site while minimizing local irritation and systemic bioavailability.


Comparison of in-vitro equivalence approaches for inhaler-based products
Anuj Srivastava

It is well known that the EMA and the FDA follow contrasting regulatory approaches for the assessment of bioequivalence for orally inhaled nasal drug products (OINDPs). The EMA currently recommends a step-wise approach for the determination of equivalence of product performance. Comparative in-vitro tests are to be performed first, followed, if needed, by pharmacokinetic (PK) studies for safety and efficacy, followed, if needed, by pharmacodynamic (PD) or clinical studies. If equivalence is demonstrated at any of these stages, the subsequent steps are not required. Thus the EMA follows an equivalence approach which can be termed as quite liberal. In contrast, the FDA prescribes a conservative approach in this regard. The FDA currently recommends a "Weight-of-Evidence" approach which incorporates qualitative (Q1) and quantitative (Q2) formulation sameness, in vitro testing, PK studies for safety evaluation and PD/clinical studies for efficacy evaluation.

The important in-vitro tests to be performed are fairly consistent in both the EMA and FDA approaches. For example, both dose uniformity and Aerodynamic Particle Size Distribution (APSD) characterization are required. Considerable differences do, however, emerge in the recommended specific statistical approaches, particularly in regard to comparison of Cascade Impactor profiles for APSD characterization. This is particularly important since APSD characterization is generally recognized to the most important in-vitro test for OINDPs and which has therefore garnered the lion's share of scientific debate in this area.

This presentation will provide an overview of the EMA and FDA recommended statistical approaches for in-vitro equivalence based on Cascade Impaction studies. The presentation will also highlight the key challenges related to the practical implementation of these approaches.


Bioequivalence study of two inhaled insulin formulations in healthy volunteers
Ashish T. Shedage

Aim: The present study was performed to compare the bioavailability of two inhaled insulin 3mg formulations (test formulation by Cipla Ltd., India and reference formulation by Pfizer Ltd., UK). Secondary objective of this study was to monitor the safety and tolerability of a single dose of inhaled insulin 3mg.

Methods: This was a randomized, open label, balanced, two-treatment, two-cohort, two-period, crossover, single dose study which included 38 healthy human adult male subjects (aged between 18 to 45 years) under fasting condition with a washout period of 7 days. Healthy subjects with clinically acceptable profile, chest X ray, ECG and pulmonary function tests were enrolled into the study. As per randomization, subjects were administered inhaled insulin 3mg formulation through the mouth using a single unit dose dry powder Inhaler device. The powder for inhalation is aerosolized from the blister cavity of the inhalation device to deliver efficacious dose into the deep lungs. Blood samples were collected up to 6 hours post dose for pharmacokinetic evaluations. Plasma concentrations of the total insulin were determined by the MEIA [micro particle enzyme immunoassay] methodology. Mean of the three pre-dose blood glucose levels was maintained at the target clamp level until 6 hours post dose using glucose clamp method in each subject.

Results: The ratios and 90% confidence intervals for the difference between least-square means of the log transformed parameters Cmax, AUC0-t and AUC0-inf were 95.10% (81.86 to 110.49%) for Cmax, 93.71% (83.41 to 105.28%) for AUC0-t, 98.60% (84.93 - 114.46%) for AUC0-inf.

The 90% confidence intervals calculated for Cmax, AUC0-t and AUC0-inf of total insulin were within the standard bioequivalence range (80-125%). No serious adverse event was reported during the course of study.
Conclusions: It was concluded that the two inhaled insulin 3mg formulations (test formulation by Cipla Ltd. India and reference formulation by Pfizer Ltd. UK) were bioequivalent in terms of the rate and extent of absorption. Based on the observations and evaluation of adverse events, clinical laboratory evaluation and vital signs, both the test and reference products were well tolerated and were found to be safe.


Challenges and opportunities in phasing out CFC inhalers in China
Bill Liang

The inhalation drug delivery industry in China has undergone dramatic development over the past decade. In compliance to the Montreal Environmental Pact, China's SFDA has carried out a package plan to facilitate the phase out of CFC-MDIs. This poster summarizes the challenges and opportunities in developing HFA-based metered dose inhalers in China.

Industrial Dilemma:

(1) China's non-CFC MDI industry face patent protection from worldwide leading companies in the aspects of formulation, process and devices.
(2) The key packaging materials, manufacturing, analytical equipment are all imported from abroad.

(3) China is short of experienced professional and technical personnel in inhalation industry.

(4) The supply cost of HFAs-MDI materials is significantly higher that of CFCs-MDI. The reimbursement by the national Medicare for HFA-MDI is uncertain.

Along with these challenges opportunities exist in China for non-CFCs MDI products.
For example, SFDA classified the API of CFCs-MDI products into 7 classes and implemented a parallel phase-out program for CFC MDI products by 2015-2016. The domestic manufacturers have to discontinue the sale of their CFC inhalers if the HFA formulated inhalers are approved. Such mandate opens the room for both innovators and foreign inhalation companies with HFA-MDIs to grow their market share. In addition, several blockbuster non-CFCs MDIs from multinational companies will face patent expiry in the coming years, which creates the opportunity to develop generic MDIs.

CF PharmTech. Inc is an emerging leader in developing and manufacturing inhalation drugs in China. Its research programs have been led by the management team with over 50 years of combined experience in American pharmaceutical industry. The company has been actively engaged in developing HFA metered inhalers including short-acting beta-agonist bronchodilators (SABA), inhaled corticosteroid (ICS), long-acting beta-agonist bronchodilators (LABA) and ICS+LABA. The company employs state-of-the-art technology to develop various HFA inhalers that can achieve in vitro equivalence to the branded drugs, and is capable of manufacturing HFA MDIs at both pilot and pivotal batch scales at its facility. There will be numerous submissions for its MDI inhalers in the next few years, enabling the company to exert positive impact in the phase out of CFC metered inhalers in China.


Beta-2 receptor agonists are selective inhibitors of organic cation transporter 1 in respiratory epithelial cells
Carsten Ehrhardt

In this work, the uptake mechanism of β2-agonistic drugs (i.e., salmeterol, formoterol and salbutamol) at the respiratory epithelial barrier was studied in vitro. All compounds are present as cations at physiological pH, allowing the hypothesis that organic cation transporters (OCT) are involved in their mucosal absorption.

The inhibitory potential of β2-agonists uptake of the bona fide OCT substrate, [14C]-TEA, was investigated in alveolar (A549) and bronchial (Calu-3) epithelial cells. In HEK-293 cells over-expressing hOCT1, hOCT2 or hOCT3, the uptake of [3H]-1-methyl-4-phenylpyridinium (MPP+) was studied in the presence of β2-agonists. Furthermore, uptake of [3H]-salbutamol into A549 and Calu-3 cells was assessed in the presence of OCT modulators. [3H]-salbutamol transport studies were carried out across Calu-3 cell monolayers.

[14C]-TEA uptake into A549 cells was reduced to 17.7% (100 μM salmeterol), 15.4% (500 μM formoterol) and 54.5% (500 μM salbutamol), respectively. [14C]-TEA uptake into Calu-3 cells was 52.9% (100 μM salmeterol), 52.4% (500 μM formoterol) and 78.1% (500 μM salbutamol) of control, respectively

[3H]-MPP+ uptake into OCT-transfected HEK293 cells was inhibited by β2-receptor agonists in the order salmeterol>formoterol>>salbutamol. The most pronounced inhibition was observed in hOCT1 expression systems, followed by hOCT3, whereas hOCT2 was only marginally affected. [3H]-salbutamol uptake was time and concentration dependent, and sensitive to extracellular pH. Intriguingly, [3H]-salbutamol uptake was only weakly inhibited by typical OCT modulators in A549 and Calu-3 cell lines. [3H]-salbutamol transport across Calu-3 cell monolayers revealed net-secretive Papp values of 6.8±0.9·10-7 cm/s (apical-to-basolateral) and 6.2±1.4·10-6 cm/s (basolateral-to-apical), suggesting the involvement of an efflux system. However, addition of the P-glycoprotein inhibitor, verapamil did not change the drug's permeability.

In summary, β2-agonists were identified as specific inhibitors of hOCT1 function. This suggests that OCT-mediated uptake of endogenous and exogenous OCT substrates could be attenuated by β2-agonists in respiratory epithelial cells, potentially resulting in unwanted side effects.


Computational fluid dynamic studies in the Andersen cascade impactor
Janewit Dechraksa

Cascade impactors are commonly used to determine the size characteristics of pharmaceutical aerosols. However, airflow pattern and its effect on wall shear stress, pressure gradient and velocity contour are still unclear. Airflow and those affected parameters can be observed by computational modelling.
The objectives of this study were to develop a validated computational fluid dynamics simulation (CFD) model of the Mark II Andersen Cascade Impactor (ACI) and investigate the effects of various parameters (nozzle velocity, airflow pattern, and wall shear).

The flow field was simulated using a commercial CFD software (ANSYS) to produce an incompressible laminar flow. Velocity streamlines were used to describe airflow profiles and examine velocity magnitude at each specific flow rate. The inlet of the pre-separator or the cone of the induction port was used as the streamline starting point. Wall shear stress on the collection plate (the collection plate of Stage 0) with velocity magnitude as a contour plot was observed. Prediction error of the nozzle velocity for the eight ACI stages was found to be within 3.56% of the existing data from the manufacturer. The pre-separator accelerated airflow velocity at the induction port was 3.71±0.09, 8.68±0.16 m/s at 30 and 60 L/min of air flow rate, respectively. This accelerated velocity promoted large particle deposition on the pre-separator. Moreover, pre-separator nozzle wall shear stress ranged from 0.08-0.34 Pa at 30 L/min on the collection plate, while the cone of metal inlet spread wall shear from the plate's center in the range of 0.11-0.37 Pa at 30 L/min. The wall shear stress generated an ellipse like shape. The velocity at the nozzle increased steadily from Stage 0 to Stage 7, which agreed with the theoretical principles of an impactor. Additionally, the nozzle velocities increased with distance from the middle of the collection plate to the periphery. Some recirculation zone was observed on each stage.

In conclusion, the CFD simulation data showed that the ACI pre-separator accelerates the airflow to induce deposition of large particles on the pre-separator and generates smooth wall shear stress at the collection plates to minimize undesirable small particle trapping. The induction port generates stronger airflow (i.e. higher airflow velocity) and more condensed than the pre-separator that resulted in higher wall shear stress on the collection plates of the lower stages.


Application of risk-based evaluation strategies to ensure materials quality in OINDP
Lee M. Nagao

Throughout the lifecycle of a pharmaceutical product decisions are made to select, qualify and control the materials that are in direct contact with the dosage form. Orally inhaled and nasal drug products (OINDP) are considered high risk dosage forms due to the route of administration and likelihood of component-dosage form interaction. The dosage form may be a liquid or a solid delivered via metered dose inhalers (MDIs), dry powder inhalers (DPIs), nebulizers or other devices. OINDP packaging and delivery systems consist of a variety of polymeric, elastomeric and metal components, some of which are in direct contact with the drug formulation during storage or when using the drug product, most of which are integral and necessary to the product's proper functioning. OINDP manufacturers and global regulatory authorities therefore hold the materials and components used to a high standard of quality. A risk-based evaluation strategy can be taken to support decisions regarding these materials and components.

To aid in developing risk-based approaches the IPAC-RS Materials working group has developed evaluation tools. These include a compilation of requirements from global regulations and a testing paradigm that takes into account the phase of product development and supply chain complexity. Examples from a variety of OINDP will be used to illustrate the use of these tools and to discuss risk-mitigation strategies for purposes of material selection, control and change management that can assist companies in meeting global regulatory expectations.


The role of DOTAP in siRNA containing cationic PLGA lipopolymeric nanoparticles intended for inhalation
Dongmei Cun

The primary obstacle for RNA-interference based therapeutics is the efficient delivery to target cells. Nanoparticles fabricated from polymer poly (DL-lactide-co-glycolide acid) (PLGA) are gaining increasing attention for their potential to be a clinically suitable, safe and effective siRNA delivery vehicle due to favourable safety profiles, sustained release properties and improved colloidal stability. However, the major drawback of PLGA nanoparticles is the low siRNA delivery efficiency.

The incorporation of cationic lipid dioleoyltrimethylammoniumpropane (DOTAP) could effectively enhance the gene silencing efficiency of PLGA nanoparticles, and this formulation could be spray-dried with mannitol into nanocomposite microparticles of an aerodynamic size appropriate for lung deposition. However nanoparticles containing more than 5% (w/w) DOTAP reduced the cell viability.

The purpose of this study was to explore the mechanism of DOTAP enhancing the transfection efficiency to provide support information for further rational formulation optimization. For this purpose, various amounts of siRNA were loaded into the combinated matrix of PLGA and DOTAP by using the double emulsion solvent evaporation method to prepare lipid and polymer hybrid nanoparticles with different n/p ratio.

The effects of n/p ratio on the physical-chemical properties including the size, zeta-potential, encapsulation efficiency and content of siRNA, the distribution of siRNA and release behaviour of siRNA was investigated systematically. A mixed solution of heparin and detergent octyl-β-D-glucopyranoside was used to identify the existence of lipoplex in both of the nanoparticles matrix and release samples. The in vitro transfection efficiency and toxicity of nanoparticles with different n/p ratio were also investigated. The results suggested that at high n/p ratio (>5), DOTAP could not only decorate the surface of nanoparticle to enable the net positive surface charge but also form lipoplex with siRNA. As a result, the efficiency of delivery siRNA delivery was enhanced significantly by the inclusion of DOTAP in the way of n/p ratio and siRNA-dose dependence. In addition, it was shown that increasing the loading of siRNA within an appropriate range is a reasonable approach to decrease the cytotoxicity of this system. This study demonstrates that the developed hybrid nanoparticles merge the benefit of both lipoplex and polymeric nanoparticles and incorporating cationic DOTAP into biodegradable PLGA nanoparticles to form a sort of lipid and polymer hybrid nanoparticles, is a promising approach for effective delivery siRNA to target cell and generate gene silencing effects.


Optimising the performance of dry powder inhaler formulations through improved powder characterisation techniques
Eike Cordts

Delivering an active ingredient to lungs requires the precise design of the formulation ingredients and their processing route. Thus considerable efforts are made to optimise the device and the appropriate formulation with respect to its local lung deposition. However, the complexity of interactions that govern powder dispersion and therefore its inhalable fraction, challenge research groups around the world. Recent studies have indicated that partially saturating the high energy sites of carrier particles with (carrier) fines will enhance the detachment of the fine API molecules and improve deep lung deposition.

This presentation will examine the characteristics of a DPI formulation commonly used in the treatment of asthma and specifically how the fine API particle fraction is released. Binary lactose blends (LH300) and adhesive ternary powder mixtures containing additional budesonide fines were produced and analysed using conventional dispersion testers (Helos and NGI) and by means of permeability and aeration measurements conducted using an FT4 Powder Rheometer® (Freeman Technology).

By comparing the results of the bulk property and dispersion tests, it was expected to gain a better understanding of the effect of adding excipient fines to an adhesive powder mixture. The graph below shows there is an interesting change in the fine particle fraction (FPF) release at fines contents above 7.5%. Below this level there is a linear relationship between FPF and Aerated Energy at air velocities in the range 5 to 7mm/s and the correlation, at 6mm/s is shown below. Above this level of fines there is a distinct change in behaviour; a reduction in available FPF, which is also observed as a change in the aeration behaviour.

From this data it can be deduced that small amounts of excipient fines are able to displace API fines toward sites on the surface of the lactose carrier which have lower binding energy. This leads to an increase in FPF up to an excipient fines fraction of ~7.5%. Above this level, the correlation does not hold as there is likely a change in mechanism of the particle interactions.

This change in mechanism can also be observed in the powder rheometer aeration results and suggests the formation of stable, and therefore less dispersible, fines agglomerates with the further, successive increase in excipient fines within a ternary adhesive mixture. This agglomerate theory takes account of the decrease in drug FPF seen in the dispersion test results and the aeration performance.

This work clearly shows the need for a carefully composed powder mixture in order to maximise the resulting lung deposition upon inhalation. Powder rheology measurements can help to gain a better understanding about the impact of complex interactions between powder particles within a mixture and enable better prediction of DPI performance.


Capsule piercing and aerodynamic performance in DPIs
Francesca Buttini

The fluidization, deaggregation, and aerodynamic size of drug particles for inhalation are controlled by the relationship between the physicochemical properties of the powder and the design of the dry powder inhaler (DPI). Many DPIs based on capsule reservoir, together with their own intrinsic resistance show different operation methods for drug administration.

In this work three different capsule-based DPIs were selected in order to investigate how the capsule piercing position and capsule movement inside the device influenced the powder emission and the aerodynamic performance of a formoterol fumarate powder blended with lactose. We performed a "design game" in which the pierced capsules were used in different devices. Specifically, the capsule was pierced with a selected device and was then transferred to another in order to aerosolize its content. All the possible combinations of device-pierced capsule were tested.

A lactose blend containing formoterol fumarate (Foradil®, Novartis) was used as a model formulation. Three devices were selected to aerosolize the powder: RS01® (Plastiape Spa, Italy, Figure 1), Turbospin® (PH&T Pharma; Italy) and HandiHaler® (Boehringer Ingelheim, Germany). The piercing mechanism of all three selected inhalers consisted of two pins but the piercing position on a size 3 capsule, is different.

Initially, reference data were collected by piercing and aerosolizing the capsule with the same device. Afterwards, a capsule pierced with one device was aerosolized with another one. This procedure was repeat- ed for all possible combinations. Aerodynamic performance of the formoterol fumarate blend was investigated using a Next Generation Impactor (Copley Scientific UK) following USP 34 at a specific flow rate in conformity with the device resistance.

The three devices used according to their original instructions gave the following performances: RS01® showed the highest emitted dose and fine particle dose (FPD) of 10.71μg and 4.15μg, respectively. The HandiHaler® emitted dose and FPD were 9.92μg and 3.13μg whereas the Turbospin® gave 9.00μg and 3.35μg, respectively.

Furthermore, when inhalers were combined with capsules pierced with other devices, RS01® again showed the best performance with a fine particle fraction (FPF) value higher than 37%, not significantly different between the various combinations. However, it must be observed that with this device the delivered dose decreased when the capsule was pierced with the other two. Turbospin® and HandiHaler® loaded with capsules pierced in other devices showed lower FPF values ranging between 22% and 35%. In addition, FPD was significantly higher in the case of RS01® compared to other devices.

The capsule piercing position and the movement of the capsule are critical for the efficacy of emitted dose and aerosolization performance. In particular the holes made at opposite ends of the capsule and the rotating movement around the major axis of the capsule gave the highest emitted dose value.


Understanding best practice approaches to materials quality for inhalation delivery systems
Lee Nagao

The International Pharmaceutical Aerosol Consortium on Regulation and Science (IPAC-RS) has a long history and experience with the management of materials quality and extractables and leachables, issues all of which affect inhalation products. Since 2000, IPAC-RS has: worked with a number of international regulators, suppliers and pharmaceutical organizations to conduct discussion forums and workshops on materials quality and extractables/leachables; initiated and led the Product Quality Research Institute (PQRI) effort to develop safety thresholds and best practices for extractables and leachables for inhalation products; worked with global suppliers to develop a GMP guideline for component suppliers that was recently incorporated into PS9000:2011 (a globally available GMP guideline for suppliers of packaging and container closure systems); submitted comments on world-wide regulatory guidelines and standards focused on extractables and leachables issues; published a number of scientific papers addressing challenges and best practices in this area; and interacted with all parts of the supply chain globally to develop and produce a documentdetailing baseline requirements for materials quality for developers and manufacturers of inhalation products.

The common and guiding thread among all of these activities is the concept of a holistic, rationalized approach to managing the quality of materials used in inhalation product packaging, container closure systems, devices, etc., which incorporates ICH Q8, Q9 and Q10 concepts. This approach considers the supply chain sources of the materials (which are often complex) and encourages the maximum possible transfer of knowledge and information about the material between supplier and drug product manufacturer. This in turn, leads to early and risk-based safety assessments of potential leachables and thorough knowledge-based and scientifically rigorous controlled extraction studies. Such studies inform, help define next steps, and, where needed, strongly link to development and performance of routine extractables testing and, if necessary, testing and specifications for leachables. IPAC-RS recognized very early on that, especially for inhalation products, designing quality and safety into the container closure systems and delivery devices made scientific sense and would benefit industry and most importantly, the patient.

This poster will describe this holistic, rationalized approach, highlighting tools to assist companies that are considering how they might implement these approaches. We will also describe the global regulatory context within which these tools and concepts have been developed. The relevance and use of key guidelines such as those from EMA/Health Canada, FDA, ICH and the recently issued Chinese SFDA guideline on plastic packaging will be discussed.


Evaluation of systemic exposure between two formulations of beclomethasone dipropionate HFA pMDI in healthy subjects
J S Yewale

To investigate the bioequivalence of Beclomethasone dipropionate (BDP) HFA pMDI 250 mcg/actuation (test product of Cipla Ltd) with the reference product Clenil Modulite (containing beclometasone dipropionate 250 mcg/actuation), supplied by Chiesi Pharmaceuticals Ltd, UK, 24 healthy adult male subjects received a single dose of 8 puffs of both the treatments on two treatment days in a randomized, open label, crossover study. Blood samples were collected for the estimation of parent drug (BDP), its active metabolite i.e. beclomethasone-17-monopropionate (B-17-MP) and plasma cortisol after administration of the treatments. Blood samples were also collected for baseline plasma cortisol estimation (24 hour profile) prior to administration of the treatment. The plasma samples of 18 subjects (i.e. excluding data of 3 subjects who were non-compliant to dosing and 3 subjects who were withdrawn from the study due to adverse events) were analysed and considered for the final pharmacokinetic and statistical evaluations. The 90% confidence interval (CI) for the ratios (%) of the least square mean (LSM) of the test and reference product of the log transformed data for Cmax, AUC0-6 and AUC0-∞ of BDP was not within the bioequivalence limits. Bioequivalence for the parent drug i.e. BDP was difficult to demonstrate due to high variability in the absorption and due to its rapid clearance from the systemic circulation. Further, the systemic bioavailability of BDP is only 2%, whereas the active metabolite B-17-MP which has a systemic bioavailability of 62%. The 90% CI for the ratio (%) of the LSM of the test and reference product of log transformed data for Cmax (82.16-105.70), AUC0-24 (93.95-108.71) and AUC0-∞ (94.44-108.89) of B-17-MP was within the bioequivalence limits. B-17-MP plasma concentration-time profile following administration of the test product was similar in shape to that of the reference product. The 95% CI for the difference between treatments for change from baseline in plasma cortisol AUC0-24 (85.99-110.81) and Cmin (91.03-102.44) was also within the bioequivalence limits. A good correlation was observed between B-17-MP levels and cortisol suppression. Both the products were well tolerated after administration of a single dose of 2000mcg. Thus, it was concluded that the test product has a systemic exposure equivalent to the reference product.


Evaluation of pulmonary deposition between two formulations of beclomethasone dipropionate HFA pMDI in healthy subjects
J S Yewale

Beclometasone-17-monopropionate (B-17-MP) is an active metabolite of Beclometasone dipropionate (BDP). This pharmacokinetic study was conducted to evaluate the pulmonary deposition for B-17-MP between the test product (BDP HFA pMDI 250 mcg/actuation of Cipla Ltd) with the reference product (Clenil Modulite, containing BDP 250 mcg/actuation, Chiesi Pharmaceuticals Limited, UK) using the charcoal blockade method. A total of 24 healthy adult male human subjects received a single dose of 4 puffs of both the treatments on two treatment days in a randomized, open label, crossover study using charcoal blockade method. Activated charcoal was administered at pre defined intervals to prevent the gastrointestinal absorption following dosing to evaluate the pulmonary deposition of B-17-MP. The plasma samples of all 24 subjects were analysed and considered for the final pharmacokinetic and statistical evaluations. The 90% confidence interval for ratios (%) of the least square mean of the test and reference product of log transformed data for Cmax and AUC0-t of B-17-MP was 79.50 to 99.23 and 79.77 to 93.63 respectively. B-17-MP plasma concentration-time profile following administration of the test product was similar in shape to that of the reference product. For both the formulations, B-17-MP exhibited a median Tmax of around 30 minutes. Hence the two formulations can be concluded to have a similar bioavailability. Both the products were well tolerated after administration of a single dose of 1000mcg.

In summary, the test product has similar pulmonary exposure to that of the reference product.


The tissue distribution of polymeric micelles after intratracheal administration to rats
Y.H. Liao

Introduction: Little is known about their extra-pulmonary distribution of polymeric micelles after delivery to the lungs. Understanding their pulmonary fate and translocation to other tissues is important for the rational design of targeting delivery. Therefore, we encapsulated curcumin acetate (CA) into polymeric micelles and studied the pulmonary retention and tissue distribution after intravenous (IV) and intratracheal (IT) administration.

Materials and Methods: CA was loaded into mPEG2000-PLGA5000 micelles in the presence of leucine (1% of the polymer) by solvent evaporation. The rats were administered with formulations at a CA dose of 2.0 mg/kg by IV injection or IT spraying. Three rats were sacrificed at each predetermined time point. Whole blood and various tissues were collected for analysis. The content of CA and curcumin in the blood and tissues were determined by a validated high performance liquid chromatography method.

Results and Discussion: The encapsulation of CA inside the micellar cores was measured by differential scanning calorimetry and nuclear magnetic resonance to be > 97%. The drug-loaded micelles exhibited core-shell morphology with a hydrodynamic size of about 24 nm and achieved sustained drug release in vitro.
The bioavailability of drug (area under the curve; AUC) from the IT micelles was broadly comparable to that from IV for most tissues, except in the lung, where the AUC for IT was more than 100-fold higher than that for IV. This may be due to the local retention of the IT micelles. The IV free CA also resulted in higher lung distribution than IV micelles. Interestingly, the AUC increased by 5.32-fold and 8.48-fold in the brain, and 4.91-fold and 4.69-fold in the lymph nodes for IV and IT micelles, respectively, compared to those for IV free CA.

Conclusion: The results demonstrate that CA-loaded micelles facilitated the uptake of CA by the brain and lymph nodes. The bioavailability of the drug from the micelles to the brain was comparable to that from IV administration. This suggests that micelles delivered by IT spraying could be absorbed from the lungs into the blood and then cross the blood-brain barrier. Thus inhaled nanoparticles may achieve non-invasive targeted delivery to the brain.

Acknowledgements: This work was supported by grants from the NSFC and MOST of China (No 81172997 and 2011DFA32730).


Formulation and evaluation of curcumin dry powder inhaler
Namdeo R. Jadhav

Curcumin, a naturally occurring highly lipophilic molecule, has a wide range of pharmacological actions. However, its limited aqueous solubility and degradation at alkaline pH restricts its bioavailability. To enable it's delivery in the form of DPI, and to overcome aforesaid limitations, solid dispersion of curcumin with PVP K30 were prepared by spray drying and evaluated for solubility, flow properties, particle size and distribution (Malvern mastersizer), SEM, FTIR, DSC, XRPD twin impinger analysis, anti-angiogenesis and toxicity in cell lines. Carr's index, Hausner's ratio and angle of repose indicated good flowability of spray dried microparticles. The particle size and size distribution data of optimised batch (CP3) showed 90% of microparticles measuring less than 5μ, which can be deposited maximally in the lung. Twin Impinger studies showed that 29% of fine particle fraction was generated, which can directly be delivered to the lungs. The CP3 exhibited absence of chemical interactions and decreased crystallinity. Angiolytic activity and MTT assay has demonstrated enhancement in the anti-tumour potential of spray dried microparticles.

Consequently, from the above result it can be concluded that spray dried microparticles of Curcumin-PVP K30 can be used to improve the bioavailability and be used as a DPI for pulmonary delivery application.


Production and characterisation of HSA particles for pulmonary delivery
Keishi Yamasaki

Background: Human serum albumin (HSA) is a major protein component of blood plasma and plays an important role in the regulation of colloidal osmotic pressure, the antioxidant capacity of human plasma, and the transport of numerous endogenous compounds such as fatty acids, hormones, toxic metabolites (e.g. bilirubin), bile acids, amino acids, and metals. Recently, HSA has emerged as a versatile carrier for drug targeting and for improving the pharmacokinetic profile of chemical-, peptide- or protein-drugs. The biodegradability, and lack of toxicity and immunogenicity make HSA an ideal candidate for drug delivery.

We have applied S-nitrosylated HSA, HSA-insulin conjugate and albumin-thioredoxin fusion protein for treatment of cancer, diabetes and pulmonary fibrosis with animal model, respectively. These are administered as injection and in future inhalation may be an alternative method both for the topical and systemic treatments. In this present study, as a first step to develop the pulmonary delivery system of HSA based drugs, spray-dried particles of HSA were produced in several conditions, and their structure and aerosol performance of the powder were characterised. Furthermore, stability of HSA during particle production was also investigated.

Methods: Aqueous solutions of HSA (1~10 w/v%) in the presence and absence of stabilizers (sodium caprylate and sodium N-acetyl tryptophanate) were spray-dried at 50~110 °C (inlet temperature). Spray-dried particle of pegylated HSA was also produced. The size and morphology of obtained particles were evaluated by scanning electron microscopy (SEM). Solid-state characterization was carried out using X-ray diffraction (XRD).
Aerosol performance was measured using a Next Generation Impactor. Structural change of HSA during spray-drying, which reflects the stability, was monitored using circular dichroism (CD) and electrophoresis.

Results and Discussion: Based on SEM micrographs, particle obtained by spray-drying at lower HSA concentration dis- played corrugated surface, which is different from the smooth surface particles obtained by spray-drying at high- er concentration. Addition of stabilizers or pegylation of HSA made particle surface smoother. The particle sur- face became more corrugated by spray-drying at higher inlet temperature. Sizes of obtained particles were comparable (diameter; 2~4 μm), and XRD data suggested that each spray-dried powder was amorphous. The powder constructed from corrugated particles had better aerosol performance than that from smooth particles, which might be due to differences in surface roughness of the particles. Under the spray-drying condition of higher inlet temperature, the decrease in α-helical structure of HSA and the formation of aggregates, which may imply denaturation, were suggested from CD and electrophoresis data. The changes in HSA structure were controlled by addition of stabilizers, primarily sodium caprylate which binds strongly to HSA (association constant: 5.5 x 105 M-1). These results show that HSA concentrations, the additives (or bindings to HSA), and modification of HSA, and temperature for spray-drying should be considered as important factors to control the aerosol performance and stability of HSA based-drug particles for pulmonary delivery.

Conclusion: These results are useful information to manufacture suitable particles for pulmonary delivery of HSA based drugs. However further studies are required to optimise condition for manufacturing.


Insulin microparticles for nasal and lung deposition
Paolo Colombo

Recent technologies employed to produce highly respirable powders focus on the ability to create microparticles with low density and irregular shape that can be easily carried in the air streams during patient inhalation. A one-step spray drying process has been employed to construct micronized insulin powders. These powders have been obtained by drying solutions at pH lower than the isoelectric point of the hormone. Using this technique insulin crystals are transformed in protein microparticles.

These microparticles are characterized by wrinkled morphology and low density, which reduce cohesion and allow for easy loading and dispersion out of the inhalation device. Once aerosolized these powders show respirable fractions up to 80-90% of the emitted dose. Stability studies over a one year period have showed that insulin microparticles remain active and in vitro promptly dissolvable.

Since the formulation is virtually made of active agent, this allows a minimal quantity of the dosage to be administered, and potential side effects associated with excipients are eliminated. The insulin powder obtained can be used for other administration routes such as nasal or oral. Whereas for oral use requires the employment of an appropriate container, for nasal use it requires the transient transformation of the micronized powder in a powder useful for nasal deposition. This was obtained by mixing with a coarse carrier substance. The insulin powder manufacturing process here described highlights several key advantages, including excipient free formulation, excellent aerodynamic behaviour for pulmonary deposition and improved stability.


Fluid flow study in dry powder inhalers device
Tan Suwandecha

One of the widely used pulmonary drug delivery formulation is dry powder inhalers (DPIs). Differences in the mouthpiece, air inlet channel and dispersion chamber may affect drug delivery performance. Computational fluid dynamic (CFD) allows researchers to understand air-flow pattern and particles dispersion process in the inhaler device. Cyclohaler® and salbutamol sulfate-lactose blend were used as the model inhaler and formulation in this study, respectively. The Cyclohaler® consists of a three-part mouthpiece tube with grid, cyclone chamber and capsule holder. Dispersion was carried out at 30, 60, and 90 L/min. Cyclohalers® with or without the grid was used with or without a capsule. Fluid dynamic parameters and particle tracking were observed. Velocity and frequency of particles impaction to device wall were extracted from particle tracking and calculated to obtain the probability of deagglomeration.

Fine particle fractions (FPF) varied from 7 - 30% and increased with air flow rate. CFD simulation indicated that the turbulence kinetic energy (TKE) was directly related to the flow-rate. Grid structure was assisted to create a turbulence kinetic energy and fluid shearing while introducing some additional flow resistance. TKE elevated the level of particle impaction because of the rapid swirling of the airflow in the cyclone part of the device that introduced a greater probability of impaction of the particles on the wall. The impaction counts per 200 injected particles varied with the flow-rate. There were 10299, 13651 and 18588 impactions at 30, 60 and 90 L/min, respectively. Increasing the flow rate led to higher impaction counts per injected particle. Probability of deagglomeration was high in the capsule chamber and grid part. Carrier size had minimal effect on the probability of deagglomeration. In short, maximizing the turbulence, kinetic energy, and particle impaction rate by adding a cyclone-like design and grid were key factors for high particle deagglomeration.


The relationship between spray pattern and deposition of pressurized metered dose inhalers
Qi Shao

Background: Spray pattern is an important parameter which can influence the Fine Particle Fraction result obtained from pressurized Metered-Dose Inhalers. This parameter depends on different formulations and actuators but the relationship between spray pattern and deposition are rarely mentioned.

Methods: We studied 3 different kinds of aerosol products with 4 types of actuators.
The spray pattern was recorded and analyzed using the Envision Pharma Laser Measurement System, Oxford Lasers UK. The Fine particle fraction was obtained using an Anderson Cascade Impactor, Copley Scientific UK and a Next Generation Impactor, Copley Scientific UK. Drug collected from both impactors were analyzed by using a HPLC system.

Results: The critical attributes determined by the Envision Pharma measurement system were the cone angle and the processed spray pattern. The cone angle was affected by the diameter and the length of the hole inside the actuator. The larger the hole diameter, the larger the cone angle was. When test products with large hole actuators were tested with the ACI and NGI, the drug deposition in the induction port was higher. Since each dose were all the same according to the labelled content, the fine particle fraction would be less than we expected.

Conclusion: Different products have different kinds of spray patterns which currently depend on whether the formulation is solution or suspension and the actuator may have a great effect on the spray pattern.

The diameter and the length of the hole may not only affect the cone angle but also change the fine particle fraction.


Chitosan and its derivatives as the carriers for intranasal drug delivery
Shirui Mao

In the past two decades, intranasal drug delivery has drawn great attention and been increasingly investigated. In recent years, chitosan has been widely used in the field of intranasal drug delivery not just owing to its biocompatibility, biodegradation, non-immunogenicity, the most important point is that chitosan serves as both mucoadhesive and absorption enhancers with great safety. The purpose of this paper is to study the potential of a series of chitosan derivatives as novel permeation enhancer for the intranasal absorption of isosorbide dinitrate (ISDN). A series of N-succinyl chitosan (NSCS) with different degrees of succinylation (DS) and molecular weights were synthesized. An in situ nasal perfusion technique in rats was utilized to investigate the effect of NSCS substitution degree, NSCS molecular weight and concentration on the intranasal absorption of ISDN. The absorption enhancing effect of NSCS was compared with that of chitosan. It was found that all the NSCS investigated improved the intranasal absorption of ISDN remarkably. Better promoting effect was observed for 0.1% NSCS 50 (63) compared with 0.5% chitosan 50. In nasal ciliotoxicity test, both NSCS and chitosan investigated showed good safety profiles. Thereafter, in vivo studies of the selected formulations were carried out in rats and the pharmacokinetic parameters were calculated and compared with that of intravenous injection. Both in situ and in vivo studies demonstrated that NSCS was more effective than chitosan in promoting intranasal absorption of ISDN. Taking both absorption enhancing and safety reason into account, we concluded that NSCS is a promising intranasal absorption enhancer.


Preparation and characterization of salmon calcitonin nanoparticle formulation for nasal delivery
Sung-Joo Hwang

The overall aim of this study was to prepare nasal nanoparticle formulations of salmon calcitonin (sCT) using supercritical fluid assisted spray-drying (SASD), with the objective of improving the stability and bioavailability of salmon calcitonin. In this work, nanoparticle formulations for nasal delivery of sCT have been studied using various absorption enhancer and stabilizer. Nanoparticles were prepared by twodifferent methods; conventional spray-drying (SD) and supercritical fluid assisted spray-drying to compare the particle formation. The stability of prepared sCT nanoparticles were determined by HPLC. The crystallinity was characterized by powder X-ray diffractometry (PXRD). The SD and SASD processed sCT nanoparticles morphology was determined by scanning electron microscopy (SEM). The particle size distribution was evaluated by HELOS laser diffraction analyzer and dynamic light scattering (DLS). In vivo absorption tests were carried out on S.D rat. Quantitative analysis indicated that sCT was chemically stable upon both SD and SASD process. Results of PXRD, SEM did not indicate a strong interaction with excipients or defragmentation of sCT through the process. The SASD processed particles have narrow particle distribution and the smaller particle size than SD processed particles. In vivo absorption test, SASD processed sCT exhibited higher nasal absorption when compared with SD processed sCT in all formulations due to reduction of particle size. The results from this study would indicate that the preparation of nasal nanoparticle formulations using SASD process could be a promising approach to improve nasal absorption of sCT.


Engineering of inhaled antibiotic powder formulation of colistin for the treatment of respiratory infections
David A. V. Morton

Objectives: Directly delivering antibiotics to the respiratory infection sites holds great potential in maximising bacterial-killing activity and minimising drug resistance and adverse effects. This study aimed to develop and evaluate inhalable dry powder formulations of colistin with high aerosol efficiency for respiratory infections caused by multidrug-resistant Gram-negative pathogens. Methods: The inhalable dry powder formulations of colistin (sulfate) were produced via particle engineering by spray drying. The susceptibility of Acinetobacter baumannii to the developed formulations was determined using broth microdilution. The physico-chemical properties were characterised by particle sizing, scanning electron microscopy, dynamic vapor sorption and powder X-ray diffraction. The aerosolisation performance in the Aerolizer® at 100 L/min was evaluated by a pharmacopeial multi-stage liquid impinger method. Results: The MICs of the spray-dried formulations against A. baumannii was comparable to that of the supplied colistin. The produced colistin formulations had particle sizes within the inhalable range (< 5 μm). All supplied and developed formulations were amorphous and absorbed significant amount of water up to 30% (w/w) at the high RH > 70% environment. There were no changes in amorphous form or water sorption behavior after spray drying process. The spray-dried formulations were physically stable after two-month storage at RH 60% and 25°C. The engineered formulations achieved high aerosol efficiency with > 85% drug released from the inhaler and > 80% drug may reach lower respiratory tract based on the in vitro aerosolisation results.

Conclusions: The spray-dried colistin powder formulations have high aerosolisation efficiency and maintain antibacterial activity. The dry powder inhalation formulations are very promising for the respiratory delivery of colistin.


Use of computational modeling to understand the effect of device design on the aerosolisation of a carrier-based dry powder inhaler
Hak-Kim Chan

Objective: To observe the changes of antiasthmatic drugs for the outpatients in our hospital. Method: Surveying the prescribed quantity of oral β2-receptor agonists, oral theophylline, oral leukotriene receptor antagonist, inhaled short-acting β2-receptor agonists, inhaled long-acting β2-agonist, inhaled corticosteroid and the complex of inhaled long-acting β2-agonist and inhaled corticosteroid in our hospital from 2001 to 2011. Based on the drugs limit the daily dose (DDD value) and the equivalent dose for inhaled steroid, DDDs of drugs and the number of equivalent dose for inhaled corticosteroid were calculated. Making the data of 2001 as the base "1", the ratios of the quantities of these drugs each year to those in 2001 were determined.

Result: Comparing the increase in the number of respiratory outpatient cases to the total outpatient cases in the our hospital, there was an increase from 2001 to 2011. The changes of antiasthmatic drugs followed 6 trends. (a) Among oral antiasthmatic drugs, only oral leukotriene receptor antagonist had a higher increase in usage rate than the increase rate of respiratory outpatient and total outpatient cases. The usage for 2011 was 542 times higher as that of 2001. (b) Among inhaled drugs, only ICS had a higher increase usage rate than the increase rate of respiratory outpatient and total outpatient cases. The usage of 2011 was 18.33 times as high as that of 2001 while the use of inhaled bronchodilators (including inhaled SABA, LABA and complex preparation of anticholinergic agents and SABA) didn't increase. (c) Among inhaled glucocorticoids, the complex preparation of long-acting _2 receptor agonists and glucocorticoids (ICS/LABA) had a higher increase usage rate than the increase rate of respiratory outpatient and total outpatient cases. The usage of 2011 was 56.29 times as high as that of 2001. While ICS unilateral prescription had a lower increase usage rate than the increase rate of respiratory outpatient and total outpatient cases. (d) Among inhalants, DPI had a higher increase usage rate than the increase rate of respiratory outpatient and total outpatient cases. The usage of 2011 was 41.05 times as high as that of 2001. While MDI had a lower increase usage rate than the increase rate of respiratory outpatient and total outpatient cases. However, the absolute value of the total amount of MDI exceeded that of DPI. (e) The usage of ICS devices was changed gradually to DPI and the share of MDI decreased from 55.3 % (2001) to 5.4 % (2008). In particular, there was no MDI of ICS to prescribe after 2009. (f) Among the inhaled bronchodilators, all inhaled short-acting bronchodilators were MDI, with just the inhaled long-acting bronchodilators as DPI.

Conclusion: The tendency of the changes of antiasthmatic drugs for the outpatients in our hospital is consistent with the modification of the guidelines of asthma and COPD prevention and treatment.


Best practice in cascade impactor measurements
Lee Nagao

For orally inhaled products (OIPs) such as pressurized metered dose inhalers, dry powder inhalers and nebulizing systems, one of the key attributes needed to assure quality, safety and efficacy of the drug product to the patient is aerodynamic particle size distribution (APSD), which ensures that the OIP delivers a consistent inhalable mass fraction of active pharmaceutical ingredient (API) throughout its entire lifetime. APSD measurements are usually conducted by using cascade impactors (CI) that are described in the respective Pharmacopeias. The results of these measurements could be reported as a fine particle dose (as required by the Ph.Eur.) or as stage groupings (as required by the US FDA). One of the advantages of the CI method is that it provides chemical identification of the API in specified size ranges based on aerodynamic diameter, and therefore linked with likely deposition in the respiratory tract. On the other hand, CI measurements have been long recognized to be prone to bias, high method variability (relative to the expected product variability) and error associated with their set-up and operation. These methodological errors and measurement uncertainties could be minimized or avoided through the use of Good Cascade Impaction Practices augmented under certain circumstances by the careful use of more rapid and simpler-to-execute non-CI particle sizing methods. This presentation describes both the background problems and suggested solutions. An overview of factors influencing the accuracy and precision of APSD-based measurements by the widely used Andersen Cascade Impactor (ACI) and the Next Generation Impactor (NGI) has been undertaken by the IPAC-RS CI-WG. Significant sources of error and variability arise from the following sources: CI operator/analyst: incorrect CI assembly, inconsistent impactor handling and sample introduction; CI system: in particular, mensuration procedures; set-up and measurement of the flow rate or flow profile; preparation and application of the stage coating material; API recovery and analysis procedure: inadequate recovery or robustness of the chosen method; Drug product: variability arising both from the formulation and/or device and/or their interaction. In response to this complex-to-resolve situation, the CI-WG has developed the Abbreviated Impactor Measurement (AIM) concept that greatly reduces the number of operations involved with full resolution CI techniques, simplifying the method to provide only those metrics that are needed to assure product quality, This development has been augmented by research into potential non-CI-based alternative methods for particle sizing that have the advantage of being rapid and involving fewer steps in their implementation.


The survey on the trend of antiasthmatic drugs in the department of outpatients of our hospital from 2001 to 2011
Ye Xiaofen

Objective: To observe the changes of antiasthmatic drugs for the outpatients in our hospital. Method: Surveying the prescribed quantity of oral β2-receptor agonists, oral theophylline, oral leukotriene receptor antagonist, inhaled short-acting β2-receptor agonists, inhaled long-acting β2-agonist, inhaled corticosteroid and the complex of inhaled long-acting β2-agonist and inhaled corticosteroid in our hospital from 2001 to 2011. Based on the drugs limit the daily dose (DDD value) and the equivalent dose for inhaled steroid, DDDs of drugs and the number of equivalent dose for inhaled corticosteroid were calculated. Making the data of 2001 as the base "1", the ratios of the quantities of these drugs each year to those in 2001 were determined.

Result: Comparing the increase in the number of respiratory outpatient cases to the total outpatient cases in the our hospital, there was an increase from 2001 to 2011. The changes of antiasthmatic drugs followed 6 trends. (a) Among oral antiasthmatic drugs, only oral leukotriene receptor antagonist had a higher increase in usage rate than the increase rate of respiratory outpatient and total outpatient cases. The usage for 2011 was 542 times higher as that of 2001. (b) Among inhaled drugs, only ICS had a higher increase usage rate than the increase rate of respiratory outpatient and total outpatient cases. The usage of 2011 was 18.33 times as high as that of 2001 while the use of inhaled bronchodilators (including inhaled SABA, LABA and complex preparation of anticholinergic agents and SABA) didn't increase. (c) Among inhaled glucocorticoids, the complex preparation of long-acting _2 receptor agonists and glucocorticoids (ICS/LABA) had a higher increase usage rate than the increase rate of respiratory outpatient and total outpatient cases. The usage of 2011 was 56.29 times as high as that of 2001. While ICS unilateral prescription had a lower increase usage rate than the increase rate of respiratory outpatient and total outpatient cases. (d) Among inhalants, DPI had a higher increase usage rate than the increase rate of respiratory outpatient and total outpatient cases. The usage of 2011 was 41.05 times as high as that of 2001. While MDI had a lower increase usage rate than the increase rate of respiratory outpatient and total outpatient cases. However, the absolute value of the total amount of MDI exceeded that of DPI. (e) The usage of ICS devices was changed gradually to DPI and the share of MDI decreased from 55.3 % (2001) to 5.4 % (2008). In particular, there was no MDI of ICS to prescribe after 2009. (f) Among the inhaled bronchodilators, all inhaled short-acting bronchodilators were MDI, with just the inhaled long-acting bronchodilators as DPI.

Conclusion: The tendency of the changes of antiasthmatic drugs for the outpatients in our hospital is consistent with the modification of the guidelines of asthma and COPD prevention and treatment.


Quantitative and In Vitro deposition of zanamivir DPI by HPLC
Yang Yang

Dry powder formulations for inhalation are often composed of micron-sized drug particles and inert carrier particles. Currently, nearly all DPI products (Relenza®, Seretide®, Spiriva®, Symbicort®, Beclophar®, Flixotide®) already on the market rely on lactose as a carrier material. However, the use of lactose has some disadvantages, such as its sugar-associated reducing function that may interact with functional groups of drugs such as budesonide or peptides and proteins. Mannitol is widely used in pharmaceutical formulations and food products. It may be an attractive alternative carrier to lactose because it does not have the reducing effect and is less hygroscopic. Zanamivir is a new anti-flu drug, which is being trumpeted by Glaxo Wellcome. Although the zanamivir dry powder (Relenza®) presented good aerosolization properties, drawbacks are linked to lactose. In our previous study, a number of dry powder formulations were produced by jet mill zanamivir with mannitol as the carrier. In this study, a high performance liquid chromatographic method was developed for the estimation of the content of zanamivir dry powder inhalation (DPI) and its in vitro deposition. The separation was achieved by Luna SCX column (250x4.6 mm, 5 μm, make: Phenomenex), a mixture of acetonitrile / buffer (60/40 and pH=3.7) as mobile phase, at flow rate of 1 ml/min. Detection was carried out at 233 nm. The calibrated linear plot of zanamivir was within 0.05- 0.15 mg/ml with the mean recovery within 99.60%- 102.81% and within-day and among-days RSD of less than 2%. The relative standard deviation of the repeatability test was 0.12%. Developed method was found to be accurate, precise, selective and rapid for estimation of the content of zanamivir DPI and its in vitro deposition. The in vitro deposition was also evaluated after the aerosolization of powders at 60 l/min via Aerolizer® inhaler into a Twin-Stage Impinger (TSI) and a Next Generation Impactor (NGI) (analysed using high performance liquid chromatography). It was found that the trends in the FPF values from the NGI data broadly mirror those of the TSI study. The FPF values for tanamivir DPI were lower by NGI than TSI, an effect most probably due to the lower aerodynamic cut-off for the NGI.


The international pharmaceutical aerosol consortium on regulation and science: advancing the science of inhalation products through collaboration and knowledge sharing
Ying Li

This poster will describe the International Pharmaceutical Aerosol Consortium on Regulation and Science (IPAC-RS) and its global efforts to bring together industry experts and regulators to develop and share good science and best practices to advance the quality, safety and efficacy of inhalation products for the patient. IPAC-RS is an international consortium of innovator and generic companies that develop, manufacture, and market orally inhaled and nasal drug products for the treatment of diseases such as asthma, chronic obstructive pulmonary disease, and diabetes. IPAC-RS is committed to advancing consensus-based, scientifically driven standards and regulations for inhalation products, with the purpose of facilitating the availability of high-quality, safe, and efficacious drug products to patients.

IPAC-RS has formed working groups and collaborations to initiate and advance efforts on materials quality and extractables and leachables issues; patient adherence; supply chain GMP/quality; particle size distribution; delivered dose uniformity; and many others. IPAC-RS has worked collaboratively with industry and regulators across the globe, including the United States Food and Drug Administration (FDA), the European Medicines Agency, and Health Canada -- for example, working with FDA on development of the Product Quality Research Institute (PQRI) recommendations on extractables and leachables, and collaborating with the UK-based Pharmaceutical Quality Group to develop the PS 9000:2011, a Good Manufacturing Practices standard for pharmaceutical packaging materials.

IPAC-RS regularly holds educational forums, conferences, and workshops addressing key scientific and technical topics, led by IPAC-RS members and bringing together industry, regulators, academia and patients. IPAC-RS regularly comments on new draft guidelines from international regulatory agencies. Recently IPAC-RS submitted comments to the State Food and Drug Administration (SFDA) Draft Guidance "Technical Guideline for Compatibility Studies between Chemical Drug Injections and Plastic Packaging Materials," and has commented on the SFDA "Technical Guideline for Quality Control of Inhalation Preparation." IPAC-RS is initiating outreach to countries in the Asia Pacific regions to establish contacts and collaborations through knowledge exchange; communication with regulatory agencies, standard setting bodies, and industry organizations; and engaging more IPAC-RS members based in Asia. The consortium has formed working groups to specifically advance this outreach.


Development and evaluation of a dry powder formulation of liposome-encapsulated oseltamivir phosphate for inhalation
Yue Tang

Oseltamivir phosphate (OP) is recommended by the World Health Organization for both treatment and prophylaxis of influenza, which can be extensively converted by hepatic carboxylesterases to the active metabolite oseltamivir carboxylate (OSCA), a potent and selective inhibitor of influenza virus neuraminidase. The study aims to develop a dry powder formulation of liposome-encapsulated OP for inhalation by spray-drying based on the optimized conditions of single-factor experimental analysis and orthogonal experimental analysis. Firstly, the OP liposomes were prepared by an active loading technique that relied on formation of an ammonium sulfate gradient and the optimized preparation conditions were as following: drug/lipid, 1:10; (NH4)2SO4 concentration, 0.3 mol·l-1; time of incubation and dialysis, 25 min, 12 h; incubation temperature, 40oC. Secondly, the identified conditions of oseltamivir phosphate and liposome ratio (1:10), the inlet temperature (110°C), aspiration (100%) and airflow rate (5%) resulted in optimal physical properties of OP liposomal dry powders, including particle size and distribution, morphology, yield of the powder, angle of repose and powder density. Finally, the aerosol performance was studied by dispersing the powders using the Aerolizer® at 60 L/min into a twin-stage liquid impinger. Furthermore, the pharmacokinetics of OP and OSCA in rats plasma of different groups have been determined by liquid chromatographic-tandem mass spectrometric method (LC-MS/MS)and performed via pharmacokinetic software DAS 2.0. The OP liposomes were characterized by particle size 105.9 ± 3.4 nm, polydispersity index, 0.191 ± 0.012 and the average entrapment efficiency, 88.75% ± 3.09% (RSD=0.03) determined by the Sephadex G-50 gel microcolumn centrifugation method. The OP liposomal spray-dried powders displayed an average particle size around 3.5 μm with fine particle fraction (FPF=35.40%) and the powder yield was 55.37%, which could be stored for 3 years at room temperature. In vitro evaluation also demonsrated a sustained release pattern with 20% drug release in 2 h compared to that of OP solution up to 90% drug release in 2 h. And the cumulative release percentage was 50% in 20 h, which suggests its better efficacy in treatment of influenza and less side effects on other tissues. In vivo pharmacokinetic atrioventricular fitting results indicated that the data of plasma OSCA from all groups were best fitted with a two-compartment model. There was significant difference in the MRT and Tmax(P<0.01) between the two groups of OP liposomal dry powders and OP dry powders with T-test, which indicated that the drug released slowly from OP liposomal dry powders in the lungs. To sum up, dry powder formulation of liposome-encapsulated oseltamivir phosphate for inhalation was suitable for pulmonary administration for depositing predominately in the central and peripheral regions of the lung with minimal oropharyngeal deposition, which offers the opportunity to reduce dosing frequency.


Novel nano spray-dried hollow particles for pulmonary drug delivery
Jing Guo

The pulmonary delivery has attracted increasing attention for the treatment of local and systemic diseases such as cancers, diabetes and myocardial infarction. The dry powder inhaler formulations offer advantages over other pulmonary delivery devices including enhanced stability, ease of administration and relatively simple formulation. The deposition site for inhaled particles in the respiratory airway is closely related to the particle properties. Although nano- and submicro- particles have the size in the inhalation size range, these particles generally possess high surface energy and are easily aggregated to form large agglomerates, which may reduce the powder flowability. Particle engineering techniques may be used to optimize the particle properties and overcome the problems of cohesiveness and poor flowability often associated with nano- and submicro- particles. Hollow sphere particles have low densities, and potentially can reduce interparticulate attractive forces and improved flowability characteristics. These porous particles may exhibit smaller aerodynamic diameters than their geometric diameters, facilitating improved deposition in the lower pulmonary region. In this study, we reported a novel approach to prepare porous nano spray-dried particles with the ideal properties for inhalation. The innovative nano spray drying technology (Nano Spray Dryer B-90, Buchi Labortechnik AG, Switzerland) was utilized to prepare hollow particles containing the salbutamol sulfate as the model drug, β-cyclodextrin as the thermal protectant, L-leucine as the dispersibility enhancer and ammonium bicarbonate (NH4HCO3) as porogen. The increase of NH4HCO3 concentration in the spray-drying solution will generate much hollower nano spray-dried particles with the mean size increased from 1.01 ± 0.01 μm to 3.05 ± 0.16 μm in diameter, and the tap density had a sharp decline from 0.40 ± 0.05 g/cm3 to 0.02 ± 0.003 g/cm3, indicating an improved flowability and a greater capability to be delivered to the alveolar regions in the airflow. The in vitro aerosolisation performance of hollow particles was investigated in use of next generation impactor (NGI, Copley Scientific, UK) under the standard operation parameters. The optimized hollow nano spray-dried powders showed a noticeably increased deposition in the lower respiratory tract, and the fine particle fraction (FPF, effective cut off diameter < 4.46 μm) can be achieved as high as 82.7 %, strongly suggesting an ideal dry powder inhaler formulation for efficient delivery to the respiratory target regions. The hollow nano spray-dried powders have been successfully prepared by the addition of NH4HCO3 in the spray-drying solution. The generated porous particles showed a very low tap density, excellent flowability and dispersibility, and subsequently offered an extremely high deposition level in the lower respiratory tract. The porous nano spray-dried dry powders may provide an ideal platform for efficient pulmonary drug delivery for the treatment of local and systemic diseases.


The effect of feedstock co-solvent on the morphology and delivered dose performance of spray-dried insulin powder formulations for inhalation
Keith T. Ung

Spray-drying is becoming more widely used in the manufacturing process of dry powder formulations for inhalation, as it provides a way to engineer low-density particles that are easily dispersed and efficiently delivered to the lung. This study evaluated a co-solvent spray-drying approach to modulate particle morphology and dose delivery performance of spray-dried insulin powders for inhalation. The binary co-solvent system studied included water as the primary solvent mixed with an organic co-solvent, e.g. ethanol. Factors such as the relative rate of evaporation of each component of a binary co-solvent mixture, and insulin solubility in each component were considered in selecting feedstock compositions for the experiment. A water-ethanol co-solvent mixture with a composition range considered suitable for modulating particle shell formation during drying was selected for experimental investigation. Six spray-dried insulin powders were manufactured to target a range of bulk power densities and primary particle sizes, and their dose delivery performance assessed using a blister-based dry powder inhaler. Bulk powder compacted densities varied from 0.15 - 0.30 g/cm3, and the median primary particle size varied from 1.36 - 2.58 μm. The measured fine particle dose <4.0 μm cut-sizes varied from 38 - 77% of nominal dose, and performance trended with increasing particle size and decreasing density. The results demonstrate that the co-solvent spray-drying approach may be used to modulate particle morphology and dose delivery characteristics of engineered powder formulations of insulin.


In vitro assessment of dose delivery performance of engineered dry powders for inhalation
Keith T. Ung

The dose delivery performance of inhalable porous particles as a function of powder properties was assessed using the "Alberta" throat. Engineered placebo powders were prepared using the PulmoSphereTM technology, which is based on spray-drying an emulsion feedstock to produce porous particles with well-controlled size and density. These placebo powders are useful surrogates for a class of potent active formulations. As part of study design, particles with range of particle size and density were manufactured, representing a particle design space relevant to dry powder inhalers. The Alberta throat provides an in-vitro estimate of "lung dose," and was used to study the effect of flow rate, particle size and density on dose delivery performance using the Simoon blister-based inhaler. Commercially available inhalers with lactose-blend formulations were also evaluated to provide a comparative benchmark. The in-vitro lung doses measured with the Alberta throat were compared to predictions from semi-empirical numerical models. Data from the Alberta throat and numerical models were used to rank order dose delivery performance over a pressure drop range of 1 - 6 kPa. Powder fluidization and aerosol emission for the PulmoSphere powders was favored by low particle density and large geometric diameter, while improved lung dose and flow rate dependence were favored by powders with median particle diameter ≥2.5 microns. In comparison, traditional lactose blend formulations showed significantly lower lung dose and increased airflow dependence. The rank-ordered in-vitro results for the different formulation types are consistent with published in-vivo results from clinical trials with similar formulations.


Surface energy distributions - a promising technique for understanding materials and processes used for inhalation
Shyamal C. Das

Inverse gas chromatography (IGC) has been used to determine surface energy characterization of materials and pharmaceutical processes used for inhalation. While the surface energy determined using IGC at infinite dilution is often criticized to have little practical meaning and relevance, surface energy distributions determined using finite dilution experiment have shown promising applications. Surface energy distributions determined at finite dilution reveal information about different surface energies present on material surface rather than a single value. Thus, surface energy distributions can be used for understanding surface changes during different pharmaceutical processes such as micronization, relaxation, mixing, dry coating, hydration, dispersion, dissolution and storage at different relative humidity.

Alkane probes such as decane, nonane, octane, heptane and hexane were used for nonpolar surface energy while a monopolar acidic probe, dichloromethane and a monopolar basic probe, ethyl acetate were used for polar surface energy. All these probes were run at concentrations of 0.03, 0.10, 0.25, 0.35, 0.55, 0.70, 0.80, 0.90 and 0.95 p/p0 to construct adsorption isotherms. Indomethacin was micronized by air jet mill, and milled by Pulverisette 5 Fritsch Planetary Ball mill. Lactose was mixed with magnesium stearate in Turbula mixer and dry coated with Hosakawa mechanofusion apparatus. The X-ray powder diffraction patterns were obtained using a powder diffractometer. Morphology was determined by scanning electron microscope.

The non-polar surface energy significantly increased after micronization of indomethacin. The nonpolar, polar and total surface energy changes were supported by molecular modeling. The micronized indomethacin relaxed and reached in stable form within three days as shown by nonpolar surface energy distributions and supported by differential scanning calorimetric study. The decrease in surface energy and work of cohesion of lactose was more pronounced after dry coating with magnesium stearate compared to mixing in Turbula which was consistent with the flow properties. The surface energies of dihydrate form of magnesium stearate were higher than anhydrate form, and the surface energies of different commercial magnesium stearates were different. The nonpolar surface energies of micronized lactose decreased after storage at high RH for three months. The surface energy distributions were used to calculate agglomerate strength distributions which explained the dispersibility behaviour of micronized lactose and dissolution of indomethacin from interactive mixtures.

The surface energy distributions determined at finite dilution provided useful information on surface energy changes in materials undergoing different pharmaceutical processes such as micronization, relaxation, mixing, dry coating, storage at high RH; distinguishing different hydrates forms of magnesium stearates and for understanding dispersion and dissolution of drugs.


Intranasal delivery of antipsychotics: CNS pharmacokinetics and pharmacodynamics
Yin Cheong Wong

Purpose: Antipsychotics are versatile drugs. Intranasal route could provide efficient delivery for certain therapeutic agents; however, studies on intranasal antipsychotics are limited. Moreover, the systemic and central nervous system (CNS) dispositions of active metabolites after intranasal drug administration are seldom investigated. The current project aims to 1) identify the antipsychotics that are more suitable to be developed into intranasal medications; and 2) characterize the CNS pharmacokinetic (PK) and pharmacodynamic (PD) profiles of the selected antipsychotic delivered by intranasal route, with a special attention to the role of drug metabolism in PK and PD outcomes.

Methods: To select an antipsychotic with greater potential for intranasal delivery, a systematic approach was adopted to screen antipsychotic candidates with in silico evaluations and then in vitro permeability assays. The systemic and CNS PK and PD profiles of the selected antipsychotic would be investigated in different intranasal delivery models and compared to that after oral and intravenous (IV) administrations.

Results: Twenty two antipsychotics were included in the primary in silico screening. Chlorpromazine, fluphenazine, prochlorperazine, and loxapine, which possess more favorable physicochemical and clinical properties required for intranasal delivery, were selected. Secondary screening in the Calu-3 cell monolayer model demonstrated that the apparent permeability coefficients (Papp) correlated inversely to the antipsychoitc's lipophilicity and total recovery. Loxapine, which demonstrated the highest permeability, was selected for further in vivo investigations.

A novel LC-MS/MS assay method was first developed for quantification of loxapine and its metabolites including 7-hydroxy-loxapine (7-OH-loxapine) in rat brain and plasma. The systemic PKs of loxapine in conscious and anesthetized rat models of intranasal delivery were then studied and compared. While intranasal loxapine achieved high absolute bioavailabilities in both conscious (~50%) and anesthetized (~100%) models, anesthesia and nasal surgery were found to exert profound effects on the systemic disposition of loxapine and its metabolites, and such effects were dependent on the administration route.

The CNS PK and PD outcomes after intranasal and oral loxapine administrations were characterized. Intranasally administered loxapine was efficiently absorbed into systemic circulation followed by entering brain, with a tmax less than 15 min in all the studied brain regions. In contrast, oral route delivered minimal amounts of loxapine to plasma and brain. Intranasal and oral loxapine achieved similar brain levels of 7-OH-loxapine, the major metabolite, and these two routes induced similar changes in the striatal levels of dopamine, serotonin, and their metabolites. The extrapyramidal symptoms (EPS), a motor side effect frequently associated with antipsychotics, were evaluated by the catalepsy models. The severity and incidence of catalepsy were consistent higher after oral than after intranasal loxapine administration. Individual IV injections of loxapine and 7-OH-loxapine to rats revealed that 7-OH-loxapine was even more cataleptogenic than the loxapine, while co-injection of loxapine tended to lower the catalepsy induced by 7-OH-loxapine.

Conclusion: Loxapine seems to be a promising antipsychotic for further development into intranasal medication. 7-OH-loxpaine, rather than the parent loxapine, could be the culprit in loxapine-induced EPS. Our results demonstrated that the active metabolites could have considerable contribution to the clinical effects of antipsychotics.


CNS target delivery of acetylcholinesterase inhibitors via intranasal administration
Shuai Qian

Background: Acetylcholinesterase (AChE) inhibitors remain the mainstay of treatment for Alzheimer's disease (AD). Tacrine (THA), as the first approved drug for AD, was limited in clinical application by its extensive liver first-pass metabolism which leads to low oral bioavailability and metabolite-induced hepatotoxicity. To improve the efficacy and reduce the side effect, application of new drug delivery system such as intranasal route for brain-targeted delivery was proposed.

Purpose: The project aims to develop an effective intranasal formulation of THA to achieve CNS targeting effect as well as avoid liver first-pass metabolism.

Methods: Pharmacokinetics profiles and brain distributions of intranasally delivered THA were compared with that from oral and intravenous routes, and an optimized THA intranasal formulation was developed.

Results: Comparing with oral THA, intranasal THA significantly enhanced Cmax and AUC0-6h of THA in both plasma and brain (by 1.8 to 3.0-fold) and reduced the plasma concentrations of bioactive THA metabolites. In addition, intranasal THA dramatically enhanced the central cholinergic responses (hypothermia and tremor) but only marginally enhanced the unwanted peripheral cholinergic response (lacrimation) in comparison to that of oral THA. Moreover, AChE inhibition in cortex was six times higher after intranasal than after oral THA at 1 h post dosing. Further studies demonstrated that the developed thermosensitive in situ gel could prolong the retention time of THA in nasal cavity which enhanced its intranasal bioavailability and brain uptake.

Conclusion: Improved bioavailability and enhanced CNS pharmacodynamic effects of the intranasally delivered THA suggested that intranasal administration could be an alternative and effective way for delivering THA for AD treatment.


Sildenafil citrate - cyclodextrin complexes for use in pMDIs as a vasodilator
Somchai Sawatdee

Sildenafil citrate is a selective phosphodiesterase-5 inhibitor used for the treatment of erectile dysfunction and pulmonary hypertension. The delivery of sildenafil to the lung directly could have several advantages over conventional treatments for pulmonary hypertension because of onsite delivery, a more rapid onset of response, and reduced side effects. The major problem of sildenafil citrate is its limited solubility in water. Sildenafil citrate was complexed with hydroxypropyl-β-cyclodextrin (HP-β-CD) to enhance water solubility before development as inhaled preparation. Sildenafil citrate-HP-β-CDs complexed pressurized metered dose inhaler (pMDI) formulations were prepared by a bottom-up process using dried ethanol as a solvent and HFA-134a propellant as antisolvent to produce a nanosuspension. All formulations consist of sildenafil citrate equivalent to sildenafil 20 μg/puff. A 3 x 3 factorial design was applied for the contents of dried ethanol and HFA-134a propellant in order to produce suitable aerosol properties. PEG400 and sorbitan monooleate were used as stabilizing agents. All formulations were evaluated according to standard guideline of inhalation products. Vasodilation testing of the selected formulations was determined in arterial blood vessel of chicken egg embryo compared with normal saline and sildenafil solutions. The arterial blood vessels were determined the effects of sildenafil pMDIs to blood pressure changes.

The complexes of sildenafil citrate with HP-β-CDs were characterized by phase solubility diagrams. The phase solubility profiles of sildenafil and CDs were described as AL type with a mole ratio of 1:1. The particle diameters of the sildenafil citrate-HP-β-CDs complexed suspensions in pMDIs were within a nanosuspension size range (300-600 nm). An assay of sildenafil content was close to 100%. The emitted doses (EDs) varied within 92.5 ± 11.4%, the fine particle fractions (FPFs) were 64.4 ± 10.8%, and the mass median aerodynamic diameters (MMADs) were 1.88 ± 0.36 μm. Dried ethanol was used as a solvent and medium for the sildenafil-HP-β-CD complex. The delivered dose was used as a parameter to determine the effects of various factors. A medium to high concentration of dried ethanol (10% and 15%) resulted in releasing of a highly accurate content with a low variation. The high content of ethanol enhanced the solubility of sildenafil in the formulations resulting in effective delivery of the pMDI. The dried ethanol content is necessary to facilitate the complex formation. A low content of HFA-134a propellant (85%) resulted in insufficient energy to release the contents from the container. However, medium and high contents of HFA-134a propellant (90% and 95%) would likely to result in sufficient energy to release the contents from the container (100% sildenafil content). The formulation containing 9.2% of dried ethanol and 87.6% of HFA-134a propellant (calculated from total weight of the formulation) is the best sildenafil pMDI in this study. Vasodilation activity of that formulation reduced arterial blood pressure by 68.4% in comparison with sildenafil solutions (55.1%) (p < 0.05).


Investigation of therapeutic efficacy and mechanisms of polysaccharide of dendrobium officinale in alleviating cigarette-induced pulmonary inflammation
Yanbo Zhang

Dendrobium officinale is a medicinal plant from the Orchidaceae family. It has been listed in the latest edition of the Chinese Pharmacopoeia as medicinal material Dendrobii Officinalis Caulis (Tiepishihu) because of its treatment efficacy. According to the Pharmacopoeia it benefits the stomach, promotes the production of body fluid, nourishes yin and eliminates evil-heat. Consequently it commands high prices and is frequently adulterated. Our recent study revealed that a compound, the polysaccharides, from Dendrobium officinale can upregulate the expression of AQP5 in molecular level from smoking cells and the lungs of C57 mouse which have been damaged by exposure to cigarette smoke.

In this study, we hypothesize that patients who have been smoking for years exhibit pulmonary inflammation and the precipitation of cigarette dust particles within the tracheal wall and the lungs and thus a downregulated expression of aquaporin 5 (AQP5) and an increased gene expression of MUC5AC (a protein that stimulates phlegm formation), resulting in excessive phlegm secretion in the lungs. Dendrobium officinale polysaccharides could promote AQP5 expression in the lungs of smokers and thereby inhibit expression of MUC5AC. Finally, the cigarette smoke-induced tracheal and pulmonary inflammation could be alleviated.

Our research group has 10 years of experience in studying the medicinal plants of genus Dendrobium. Since 2006, we have conducted investigations on the effect of Dendrobium species in promoting the expression of AQP5 gene. Based on the results of the investigations, four papers have been published in international journals.

Eventually, this study will elucidate the mechanisms and pathway with which Dendrobium officinale polysaccharides alleviates cigarette smoke-induced pulmonary inflammation. The results of this study could facilitate the establishment of a platform for the screening of more drugs from Chinese medicine and the provision of early intervention and therapy to patients with cigarette smoke-induced pulmonary inflammation.


Formulation and in vitro evaluation of reverse aqueous microemulsions containing salmon calcitonin in hydrofluoroalkane propellants
Ziyun Shan

Inhalation has been a traditional delivering route for drugs that exert therapeutic effects locally in the lung. The lungs also represent an outstanding route for systemic drug delivery because of its large alveolar surface area, thin epithelial barrier, extensive vascularization, and relatively low proteolytic activity. Pressurized metered-dose inhalers (pMDIs) are the most economical and efficient vehicles for delivery of drugs to the respiratory tract. For development of pMDIs, formulating the drug within microemulsion cores can avoid drug loss caused by the interactions between the drug and the canister walls. In addition, this approach may provide an opportunity to deliver drug combinations by saving extensive reformulation. Therefore, the objective of this study was to develop and investigate reverse aqueous microemulsions as a potential strategy for salmon calcitonin in 1,1,1,2-tetrafluoroethane (HFA134a). Salmon calcitonin, water, surfactant, and ethanol were added to pressure-proof glass vials. Subsequently, a 50 μL metering valve was crimped onto the vial using a semi-automatic bottle crimper and HFA134a (10 g) was filled into the vial using a propellant filler. The performance of different kinds of non-ionic ethoxylated copolymers in the HFA propellants was studied and Pluronic P85 was chosen as the most appropriate candidate surfactant for the formation of microemulsions of salmon calcitonin in the presence of ethanol. Central composite design-response surface methodology was used to optimize the formulation. Microemulsions containing 12.95 wt% of Pluronic P85/ethanol (with a Pluronic P85:ethanol ratio of 1:2), 0.58 wt% of water, and 1.3 mg of salmon calcitonin showed optimal physical stability and aerosolisation properties. The microemulsions were clear and capable of scattering laser light a few weeks after preparation. The aerosolisation properties of the pMDIs were assessed by the Next Generation Impinger (NGI) at 30 L/min and the Twin-Stage Impinger (TSI) at 60 L/min. High Performance Liquid Chromatography (HPLC) was used to quantify the fractions on each stage. Fine particle fractions (FPF) of 24.0 ± 0.4% and 28.0 ± 0.3% were achieved on the NGI and TSI, respectively. Cytotoxicity studies were performed on A549 cells, a Type II alveolar epithelial cell line. The results indicated that within the concentration range of interest (Pluronic P85 up to 0.5 mg/mL), the cell viability was only slightly decreased. This study suggests that reverse aqueous microemulsions are potential candidates for the delivery of therapeutic proteins and peptides as pMDI formulations.


A novel rotating fluidized powder dispensing process for the preparation of carrier-Free DPIs
Mingxi Qiao

For drugs to be delivered to the lung their sizes are normally controlled to less than 5 μm. Due to the extremely small particle size, the forces exerted by the neighboring particles causes the difficulty in dispensing powders. One widely accepted solution is to use excipient such as lactose with larger particle size to facilitate the dispensing of the extra-fine powders. However, the presence of lactose in the dry powder inhalation formulation generates the problems such as low delivery efficiency, complexity in formulation preparation and uncertainty of lung deposition.

A novel rotating fluidized bed powder dispensing device was developed to dispense extra fine drug particles into a dry powder inhaler (DPI). The dispensing device has a rotating porous cylindrical chamber with alternating distributor and ceiling, and a flexible nozzle for additional agitation. It is designed to prevent the agglomeration of extra-fine particles in the absence of any large excipient particles. With such an arrangement, the extra-fine particles can be fluidized inside the chamber to produce a uniform gas-particle suspension which can be withdrawn from the fluidized chamber as a gas-particle plume. Tests were carried out with various 1-5 micron powders such as salbutamol sulphate, insulin, lactose, and mannitol. The dispensing device is designed to permit filling of up to 60 doses per minute, with weights ranging from 20 micrograms - 2000 micrograms metered into a multi-dose blister pack, integrated into the DPI. Testing results revealed that the rotating fluidized bed powder dispensing device can provide a dilute gas-solid suspension with more than 90% of the particles present in discrete (non-aggregated) suspended form. TSI sizing clearly indicates that the starting powder can be broken into single particles effectively. The dose uniformity of each powder filled by the dispensing device was tested using disks with different dosage sizes (20 μg - 2000 μg). The relative standard deviation (within the disk) of the filled weights into the different size dosage disk was less than 5%. In vitro tests have shown that the newly designed DPI exhibits a high fine particle fraction (<4.7 μm) of approximately 60%-80%, using both Andersen cascade impactor and Next Generation Impactor. The study suggests that the novel rotating fluidized bed powder dispensing process is capable of efficiently dispensing and delivery of extremely small quantities (as low as 20 μg) of extra-fine drug particles with the RSD below 8%. The novel dispensing process may provide a solution for lactose free dry powder inhalation.


Long term treatment of S-Bambuterol, an inactive enantiomer, causes bronchial hyper-responsiveness and drug tolerance
Qing Liu

Aim: It is known that chronic use of beta-agonists leads to drug tolerance and adverse effects such as bronchial hyper-responsiveness. Bambuterol, the third-generation long acting beta-agonist (LABA), is commercially available as a racemic mixture of two enantiomers, R- and S-bambuterol. In this study, we examined the bronchial hyper-responsiveness and the drug tolerance after long-term treatments of R- or S-bambuterol in Guinea pigs, and confirm the greater safety and less drug tolerance of R-bambuterol than S-bambuterol.

Method: Guinea pigs were divided into four groups: saline-treated group, S-bambuterol treated group S-bambuterol treated group, and racemic bambuterol treated group. All the drugs (10mg/kg) were intragastrically administered once a day for ten days. On the eleventh day, bronchospasm was induced by histamine (i.v.), and tidal volume, intrathoracic pressure, oral cavity flow velocity and breathing frequency were measured. Lung resistance (Rl ) and dynamic lung compliance ( Cdyn ) were calculated based on those parameters. Following treatment of aerosol R-terbutaline, the active metabolite of R-bambuterol, bronchoconstriction was induced by histamine again, and the changes in lung resistance and dynamic lung compliance were determined. Alveolar lavage was collected at the end of the experiments. Total and differential white blood cells were counted and the lavage was further analyzed by mass spectrometry.

Result: The survival rate of S-bambuterol treated group is significantly decreased, comparing to the R-bambuterol group. The response to histamine challenge is always stronger in S-Bambuterol treated group, comparing to other groups. The total amount of white blood cells is increased and inflammatory factors are found in the alveolar lavage by mass spec analysis, which indicates that chronic use of S-bambuterol can lead to bronchial hyperresponsiveness and inflammation.

Furthermore, long-term treatment of S-bambuterol reduces bronchodilating effects of R-Terbutaline, suggesting drug tolerance to the active metabolite of R-bambuterol.

Conclusion: S-bambuterol is the major ingredient that is involved in the bronchial hyperresponsiveness and drug tolerance following long-term treatment of racemic bambuterol, and the administration of R-bambuterol significantly improves drug safety and reduces adverse effects.


Engineering of excipient-free composite particles containing both hydrophilic and hydrophobic drugs for inhalation by co-spray drying
Huamei Wang

The aim of this study was to engineer excipient-free composite powders containing different ratios of hydrophilic (ginsenoside; Rb1) and hydrophobic (tanshinone; IIA) drugs for inhalation. Powders were produced by spray drying different ratios of Rb1 and TA from ethanol/acetone co-solvent solutions at similar spray-drying conditions. Each sample was characterized using laser diffraction, scanning electron microscopy, laser scanning confocal microscopy, dynamic vapour sorption, differential scanning calorimetry, and their aerodynamic behavior were evaluated by a Next Generation Impactor. The results show that all particles have hollow internal structures. The morphology varied from spherical to polygonal, and the span of particle size also slightly increased from 1.18 to 1.38 with the increase of TA content. The increase in the proportion of the hydrophobic drug improved the stability and emptying dose of the particles but not the fine particle fraction. The combination sample formulated at the ratio of 3 Rb1 to 1 TA had the best aerosol performance. In conclusion, composite particles containing both hydrophilic and hydrophobic drugs can be prepared for inhalation by using their complementary physical and chemical properties at appropriate ratios. This research provides a new way for the preparation of dry powder particles with multiple active ingredients at high doses.


Effect of crystallinity on electrostatic charging in DPI formulations
Philip Chi Lip Kwok

Introduction: Many physicochemical, mechanical and environmental factors can influence charging of dry powder inhaler (DPI) formulations - crystallinity is one of these factors. Due to differences in crystal packing and surface energies, there may be differences in charge transfer behaviours between amorphous and crystalline materials. Although the effect of crystallinity on electrostatic charging in DPI formulations has been investigated previously by other researchers, the reported data were inconclusive since the samples not only differed in crystallinity, but also in particle morphology and size distributions. Therefore, these variables are controlled in the present study to determine the role of crystallinity in electrostatic charging of DPI formulations. The objective of this study was to characterize inherent charges generated by amorphous and crystalline micron-sized salbutamol sulfate (SS) powders.

Materials and Methods: Amorphous SS was spray dried, whereas crystalline SS was produced by conditioning spray dried SS in the presence of supercritical CO2 and menthol. Solid-state characterization was carried out using laser diffraction, scanning electron microscopy (SEM), X-ray diffraction (XRD), dynamic vapour sorption (DVS), atomic force microscopy (AFM) and nitrogen adsorption for multipoint BET measurement. In vitro aerosol performance and electrostatic charge were measured simultaneously by a modified Electrostatic Low Pressure Impactor (ELPITM). Triplicate samples (approximately 5 ± 0.2 mg) were dispersed by an Aerolizer®. Charge was defined as the area under the curve in the current-versus time plot. Net charge was defined as the sum of charges on a particular impactor stage. Specific charge was defined as the quotient of the net charge and deposited drug mass.

Results and Discussion: Amorphous and crystalline SS had comparable particle size distributions, where SEM images confirmed formulations showed comparable particle size and shape. The rougher surface of crystalline SS indicated that crystallisation had occurred.

Subsequent XRD and DVS data confirmed the crystalline nature of supercritically conditioned SS. On the other hand, amorphous SS showed the typical halo pattern and crystalline SS had peaks that generally overlapped with those of raw SS. AFM and BET measurements quantified the increased roughness and specific surface area, respectively, of crystalline SS. Both powders exhibited similar bipolar charging pattern, with a reversal in polarity was observed around the 1.60 μm cut off diameter. Crystalline SS charged to higher magnitudes with more consistent charge and mass deposition profiles, and an improved aerosol performance. There were significant differences in specific charges, in particular for particles with cut off diameters ≤ 0.615 μm.

Conclusion: A difference in charging behavior was observed between amorphous and crystalline SS. In vitro deposition studies found crystalline SS charged to higher magnitudes with more consistent charge and mass deposition. These results suggest that crystalline SS could lead to more predictable charging with improved aerosol performance.

Acknowledgements: The authors thank Ms Ellen Braybon and Mr Matthew Foley, from The University of Sydney.


Aerosol generation and deposition in the human lung
Chih Chieh Chen

The human lung is not only an aerosol collector but also an aerosol generator. Previous lung deposition studies have shown that aerosol deposition efficiency was strongly dependent on particle size, breathing pattern, aerosol charge distribution, and lung morphometric parameters. On the other hand, the generation rate of exhaled breath aerosols (EBA) was mainly controlled by the tidal volume. The main objective of this work was to study the relationship between exhaled breath aerosols and lung deposition efficiency.

The experimental system consisted of a test chamber, a mouthpiece, a pneumotachograph flow meter, and a particle counter. Both tests shared the same experimental apparatus except the aerosol generating system. For regional lung deposition measurement, a stable aerosol output was essential, while aerosol-free air was supplied to the chamber when conducting the EBA measurements. The volunteers were asked to follow the sinusoidal breathing patterns generated by a piston-cylinder breathing simulator. The subjects were instructed to perform various percentages of forced vital capacity (20, 40, 60% FVC) and fixed tidal volumes (500, 750 and 100 mL) with different breathing frequencies (10, 12, 14, 15 breath/min).

The results showed that the EBA counts increased with the tidal volume but was not majorly affected by the breathing frequency. Regional deposition data showed that local deposition efficiency increased with penetration volume. Correlation analysis showed that the total lung deposition efficiency increased with increasing EBA counts. This indicates that subjects who collect aerosols more efficiently generate more aerosol particles through tidal breathing, which is a puzzling finding.


Development of amphotericin B incorporated in lipid derivatives as a reconstituted powder for nebulisation
Kajiram R. Adhikari

Amphotericin B (AmB) is the drug of choice for the treatment of lung fungal infections caused by Cryptococcosis, Candidiasis, Histoplamosis, and invasive Aspergillosis. It is a broad spectrum, low resistance, and natural polyene macrolide antibiotic. It is insoluble in water and soluble in very few organic solvents. AmB can cause nephrotoxicity and other adverse effects (fever, chills, vomiting, haemolysis) when it is given intravenously. To reduce these effects, nebulisation is an attractive option since high local drug concentrations are achieved with minimal systemic exposure. Jet nebulisers are inexpensive and do not require trained personnel.

Our aim of this study was to prepare a highly water soluble and stable form of AmB for nebulization.

This study employed lipid derivatives to formulate a AmB-lipids system that was subsequently transformed to a dry powder by lyophilization. The three lipid derivatives were sodium deoxycholate (SDC), potassium deoxycholate (KDC), and potassium cholate (KC). The AmB:lipid molar ratio was 1:1.9. The physicochemical properties of AmB dry powders were analyzed by dynamic light scattering, UV spectroscopy, differential scanning calorimetry (DSC), and Fourier transform infrared spectroscopy (FTIR). The pH of the reconstituted liquids was measured with a pH meter. In vitro drug deposition from a jet nebuliser was determined using an Andersen Cascade Impactor (ACI).

Lyophilized AmB dry powders were solid cake, porous, light and free flowing. All these formulations were readily dissolved into distilled water within 45 s and formed clear yellowish liquids. The pH was between 7.4 and 7.7. The particle size was in the range of 17 to 51 nm after reconstitution and was stable within one week. Zeta potentials were -32 to -41 mV. UV spectra recorded between 300 to 450 nm confirmed that the spectrum did not shift markedly within the seven days after reconstitution. DSC thermograms indicated that KC and KDC carriers were amorphous. SDC showed an exothermic peak at 198.5oC. AmB formulations were amorphous powders. FTIR indicated that there was AmB formed complexes with the lipid carriers. The mass median aerodynamic diameter of the nebulised particles was 1.36-1.86 μm. The fine particle fraction < 5 μm of all the formulations was 74-80%.


Controlled antisolvent precipitation and characterization of itraconazole nanoparticles
Ka Yee Wan

Purpose: This study aimed to develop a consistent and stable nanoparticle formulation of itraconazole (ITZ), a triazole antifungal agent.

Methods: ITZ nanosuspensions were prepared using a four-stream multi-inlet vortex mixer (MIVM), inside which an organic solvent (dimethylformamide, DMF) stream containing ITZ and stabilizers/costabilizers was efficiently mixed with three other independent aqueous antisolvent streams (in a defined organic solvent to water ratio of 1:19 v/v) to create a supersaturation level high enough for triggering extremely rapid nanoprecipitation. Polyethylene glycol - polylactic acid (PEG-PLA) block copolymers and d-alpha tocopheryl polyethylene glycol 1000 succinate (TPGS) were employed as primary stabilizers and cholesterol (CLT) as costabilizer. The resulting nanosuspensions were subjected to ultrafiltration to remove residual organic solvent, followed by addition of a protectant [sucrose, mannitol, lactose or hydroxypropyl-β-cyclodextrin (HP-β-CD)] prior to spray drying. Surface composition and morphology of the nanoparticles were analyzed by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM), respectively. Particle size of the nanoparticles was determined by dynamic light scattering (DLS) technique before drying and after reconstitution of the dried powder with water.

Results: ITZ nanosuspensions with mean particle sizes less than 200 nm and polydispersity indices less than 0.3 were readily and reproducibly fabricated by the MIVM. TPGS and CLT in combination afforded the most stable ITZ nanosuspension with a mean particle size of ~90 nm, a drug loading of ~45% and an encapsulation efficiency >99%. XPS analysis revealed an increase in the ratio of hydrophilic to hydrophobic segments of the TPGS on the nanoparticle surface after two hours of storage, indicative of the molecular rearrangement of TPGS with their hydrophilic groups orientating towards the particle surface. AFM revealed that the ITZ nanoparticles were roughly spherical in shape and the observed particle size was consistent with the DLS data. Among all the protectants tested, only HP-β-CD was found to be effective for preserving the size (<50% increase) of the nanoparticles when subjected to spray drying. As shown by SEM, the spray-dried product appeared as micron-sized particles with smooth surfaces, reflecting a homogeneous dispersion of the nanoparticles within the HP-β-CD carrier matrix. A higher concentration of nanoparticles or HP-β−CD used in spray drying tended to afford better particle size preservability for the dried nanoparticles.

Conclusion: The present work clearly demonstrated that the MIVM is an effective tool for generating consistent ITZ nanoparticles with particle size below 200 nm, and judicious selection of stabilizers and protectants is critical to ensuring the stability of nanoparticles following precipitation from solutions and during the subsequent spray drying process.


Synergistic Combination Dry Powders for Inhaled Antimicrobial Therapy: Formulation, Characterization and In Vitro Evaluation
Desmond Heng

Combination products play an important role in medicine as they offer improved clinical effectiveness, enhanced patient adherence, and reduced administrative costs. In combination antimicrobial therapy, the desired outcome is to broaden the antimicrobial spectrum and to achieve a possible synergistic effect. However, adverse antagonistic species may also emerge from such combinations, leading to treatment failure with serious consequences. It is therefore imperative to screen the drug candidates for compatibility and possible antagonistic interactions. The aim of this work was to develop a novel synergistic dry powder inhaler (DPI) formulation for antimicrobial combination therapy via the pulmonary route. Binary (ciprofloxacin hydrochloride and gatifloxacin hydrochloride) and ternary (ciprofloxacin hydrochloride, gatifloxacin hydrochloride and lysozyme) combinations were prepared via spray drying on a BUCHI® Nano Spray Dryer B-90. The powder particle morphologies were spherical with a slightly corrugated surface and all were within the respirable size range. The powers yielded fine particle fractions (of the loaded dose) of over 40% when dispersed using an Aerolizer® at 60 L/min. Time-kill studies carried out against the respiratory tract pathogenic bacteria Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumonia and Acinetobacter baumannii at 1x the minimum inhibitory concentration (MIC) over 24 hours revealed no antagonistic behavior for both the binary and ternary combinations. While the interactions were generally found to be indifferent, a favorable synergistic effect was detected in the binary combination when it was tested against Pseudomonas aeruginosa bacteria. The inclusion of lysozyme in the ternary formulation is potentially beneficial for its dual mucolytic and anti-inflammatory properties.


The development of dimple shape dry powder carrier for ethambutol dihydrochloride
Md Iftekhar Ahmad

The objective of this study was to prepare dimple shape dry powder carrier of ethambutol dihydrochloride to administer by inhalation to the infected site with high concentration in lung while simultaneously minimizing both peak serum levels and exposure to other body tissues.

Ethambutol is primarily a bacteriostatic and anti-tuberculosis agent. The oral dose of ethambutol dihydrochloride is 20 mg/kg with MIC of 2 μg/ml. Due to this high dose; it causes visual impairment and lead to complete blindness. However, if it is administered in aerosol form as a dry powder inhaler (DPI), the dose of ethambutol dihydrochloride is expected to be reduced drastically. Chitosan is a de-N-acetylated form of chitin; consist mainly of β(1-4)-2 amino-2-deoxy-D-glucose units. It is a biodegradable, biocompatible, non-toxic and mucoadhesive in nature. Spray drying was carried out to prepare dimple shape micro-size (1.2 μm) carrier powder. Carrier particles with rough surface particles are likely to reduce Van der Waals force of attraction and increase aerosolization properties of powder. The dimple shapes have grooves all over its body surface which provides good space for drug to bind over its surface and its dispersion in an oral cavity. The ethambutol dihydrochloride solution (1% w/v) was nanosprayed by nano spray dryer B-90 at 110°C and 85% spray rate. The chitosan solution (1% w/v) was spray dried through a 1.5 mm nozzle using a mini-spray dryer B-290 under different inlet temperatures (80-150°C) with a feeding rate of 3 ml/min. Five carriers of different ratios were prepared by physical mixing. The physico-chemical properties of different formulations were analyzed by SEM, DSC, and FT-IR. The depositions of drug in different regions of lungs were determined by the Anderson Cascade Impactor (ACI). The separation of drug with the carrier inside the lung was determined by using ultracentrifugation method. The drug exhibited smooth, free flowing and spherical surface having size of 222 nm. The carrier showed the dimple surface when solution was sprayed at high inlet temperature (150°C) with low feed rate. The SEM image showed the adhesion of drug with the carrier on its groove as well as on its surface. The binding of drug with carrier depends on the amount of carrier available in the formulation. DSC thermograms. All formulations had shown amorphous halo because at high temperature carrier got melt and acted as a solvent for the drug to dissolve in it. FT-IR indicated the complex formation between drug and carrier. All the formulations showed content uniformity in acceptable range (99-107%). The emitted doses (ED) of different formulations were between 80-85% and have the mass median aerodynamic diameter of 1.9 μm. The separation of drug with carrier was depended on the drug: carrier ratios in formulation; formulation with low ratio of carrier got separated at low RCF while higher ratio separated at higher RCF.


Design of dry powder formulations of pH responsive peptide/plasmid DNA complexes for pulmonary delivery
Wanling Liang

Respiratory diseases are substantial public health problems around the world. Recently, nucleic acid was developed as a potential therapeutic strategy to tackle a series of lung diseases. Delivery still poses one of the major challenges for their clinical application. pH responsive peptides containing either histidine or derivatives of 2,3-diaminopropionic acid (Dap) can mediate effective DNA transfection in lung epithelial cells with the latter remaining effective even in the presence of lung surfactant containing bronchoalveolar fluid (BALF), which make them promising vectors for delivering therapeutic nucleic acid to the airways.

To investigate the suitability of pH responsive peptide system for pulmonary delivery of nucleic acids, inhalable dry powder formulations of peptides/plasmid DNA complexes, with mannitol as carrier, were prepared by spray drying (SD) and spray freeze drying (SFD). The physicochemical properties and biological activity of dry powder formulations were evaluated by scanning electron microscopy (SEM), next generation impaction (NGI), gel retardation and in vitro transfection via a twin-stage impinger (TSI) following aerosolisation by a dry powder inhaler (Osmohaler™). The result showed that SD powders are spherical particles with wrinkled surface, while SFD powders have porous structure with larger geometric diameters. SFD particles possess low aerodynamic diameters because of their low densities and similar FPFs of the two powders were observed. The integrity of plasmid DNA was maintained in both SD and SFD formulations. Most importantly, the in vitro DNA transfection efficiency was well preserved after spray drying and spray drying process with SD have superior transfection efficacy to SFD.

In summary, this study demonstrated that inhalable, pH responsive, peptide-based, plasmid DNA dry powder formulations can be successfully produced by spray drying and spray freeze drying using mannitol as carrier. Both formulations showed promising aerodynamic properties and biological activities but spray dried powders were physically more robust with better transfection efficiency and higher industrial potential.


Spray dried oleanolic acid powder for pulmonary delivery
Shuangning Chen

Introduction: Oleanolic acid (OA), well known for its hepatoprotective effect, has been shown in vitro to be cytotoxic in A549 human non-small-cell lung cancer cell line. Thus it may be potentially useful for lung cancer treatment. Being a BCS Class IV drug, it has low oral bioavailability. Therefore, inhalation is the preferred route of administration for local delivery. The aim of this study is to develop an inhalable oleanolic acid dry powder formulation.

Methods: OA was spray dried from an acetone solution using a Büchi B-290 Mini Spray Dryer. The spray dried powder was characterised and compared with raw OA. Particle morphology was observed by scanning electron microscopy (SEM), whereas aerodynamic performance was measured by dispersion from an Osmohaler™ into a Next Generation Impactor (NGI) according to the British Pharmacopoeial method.

Results and Discussion: Raw OA particles were needle-like, while the spray dried ones were relatively spherical of 0.5‒3 µm in diameter. The spray dried formulation exhibits a significantly higher fine particle fraction (FPF) (63.4 ± 2.1%) than that of the raw material (16.1 ± 6.3%), indicating an enhanced dispersion efficiency. The apparently large mass median aerodynamic diameter (MMAD) of 13.51 ± 4.46 µm of raw OA was due to the large agglomerates. In contrast, spray dried OA could be dispersed into primary particles, resulting in a reduced apparent MMAD of 1.71 ± 0.11 µm. Spray dried OA showed higher retention in the capsule and inhaler, indicating that it is more adhesive than raw OA and may be amorphous.

Conclusion: An OA dry powder formulation was successfully prepared by spray drying. It showed excellent aerosol performance (63% FPF) and may be useful for pulmonary delivery.


Antibacterial activity of D-antimicrobial peptides against Mycobacterium tuberculosis: In vitro and ex vivo study
Y Lan

Tuberculosis is a severe infectious disease caused by Mycobacterium tuberculosis (Mtb), an intracellular pathogen which primarily infects human pulmonary macrophages. Despite the control strategy implemented by the World Health Organization (WHO), tuberculosis still leads to millions of death each year, with cases of multiple drug resistant (MDR) and extensive-drug-resistant-tuberculosis (XDR) increasing at an alarming rate.

Antimicrobial peptides (AMPs) are originally produced by all the living organisms, serving an essential role in host innate immune system. Various studies have demonstrated that both natural occurring AMPs and their synthetic analogs have a very wide spectrum of pathogenic organisms. A novel class of AMPs, the D-formed cationic amphipathic α-helical LAK peptides, is being investigated as potential anti-tuberculosis agent. These peptides contain 24 to 25 D-amino acid residues and have the ability to penetrate and destabilize the bacterial membrane. The antimicrobial potency and selectivity between host membrane and pathogenic membrane are determined by their structure characteristics, including charge angle (angle subtended by the positively charged amino acids), hydrophobicity, and the incorporation of proline residues. To investigate the structure-activity relationships, in vitro anti-tuberculosis assays of a series of AMPs were carried out on eight different strains of Mtb, including the clinical strains, in order to identify the most potent peptide candidates. To investigate whether these peptides remain active against the intracellular mycobacteria, ex vivo studies were also carried out. The cytotoxicity of the AMPs was evaluated by MTT assay.

It was found that D-AMPs could greatly inhibit the extracellular growth of Mtb, including the MDR and XDR strains. Peptides with a charge angle of 120° had the most potent antibacterial activity but they were also more cytotoxic to the host human macrophage-like cells (THP-1). Increasing hydrophobicity decreased the antimicrobial potency, and due to the reduction of self-association of the peptides in solution, a significant decrease of cytotoxicity was also observed. The ex vivo studies showed that D-AMPs were effective in inhibiting the intracellular growth of Mtb.
Our findings suggested that the hydrophilic D-AMPs with charge angle 120° conferred the highest potency against extracellular Mtb, and remained active against intracellular Mtb. The incorporation of proline residue may have a role in the improved potency against intracellular bacteria, but further studies are required to confirm. Overall, this study showed the potential of using antimicrobial peptides as a novel therapeutic agent for the treatment of tuberculosis.


Learning from Nature: Novel Alternatives to Reduce Powder Retention in the Dry Powder Inhaler during Aerosolization
Desmond Heng

In the natural world, turbulent skin friction drag reduction by 'additives' is evident in the movement of Avians and Nektons (i.e. fliers and swimmers) in fluids. For example, drag-reducing surfactants (e.g. lipids, phospholipids, lipoproteins) are known to be a constituent of fish slime. Can we learn from nature in order to develop more efficient inhalers? Currently, there has been a lot of 'downstream' work focused on improving dispersion and deaggregation of drug powders via carriers and/or fines with little attention paid to the 'upstream' mechanics. This study seeks to examine the biomimetic approach to improving drug retention at the inhaler device and capsule region. Capsule-based DPIs are presently limited by powder retention in the capsule and/or device, which leads to a reduction in the emitted dose. In an in-vivo setting, inhaler retention of around 15-60% of the administered drug had been reported. Via innovative coating (e.g. painting) of the drug capsule and/or delivery device with a suitable excipient, adhesion between the drug particles and the internal surfaces of the DPI could be reduced. Drug wastage was also minimized as there was efficient channelling of the residual drug into the useful inhaled therapeutic fraction. Remarkably, the fine particle dose could be increased by as much as 300%. Future uptake/refinement of this technology by industry (e.g. device and capsule manufacturers) could be via direct incorporation of the surface modifier into the device and/or capsule manufacturing process.