Establishment of an in vitro test strategy for orally inhaled drug products

This thesis describes and evaluates three animal experiment free approaches for the safety and efficacy assessment of pulmonary drug formulation in the pre-clinical phase. In the first approach the complex cell culture system MucilAir™ was exposed to pollen through the Vitrocell® Powder Chamber to simulate in vitro an allergic reaction and predict drug safety. However, cytokine response was not sufficient, and the setup has to be optimized for safety studies. Two in vitro setups were further developed for estimating drug safety and inflammatory response. For safety studies, an in vitro model should predict human data by using approved FDA excipient concentrations. From these excipients the in vitro IC50 was determined with pulmonary cell lines and ranked in the thus established SAFE classification. Consequently, SAFE allows for predicting human safety data by in vitro testing. For estimating potential inflammatory reactions located in the respiratory area, the alveolar cell line hAELVi was exposed to the pro-inflammatory cytokines TNF-α and IFN-γ. Similar to an inflammatory in vivo reaction, a loss of epithelial barrier function was observed. Furthermore, the anti-inflammatory response to hydrocortisone of the stimulated model underlines its po-tential for in vitro drug testing. The described in vitro models are a promising tool for replacing animal experiments, whereby their standardization and validation are future challenges according to recognized guidelinesDiese Arbeit beschreibt und bewertet drei tierversuchsfreie Ansätze zur Sicherheits- und Wirk-samkeitsbewertung von Inhalativa in der präklinischen Phase. Im ersten Ansatz wurde das Zellsystem MucilAir™ durch die Vitrocell® Powder Chamber Pol-len ausgesetzt, um in vitro eine allergische Reaktion zu simulieren. Die Zytokinreaktion war jedoch nicht ausreichend und der Aufbau muss für Sicherheitsstudien optimiert werden. Folg-lich wurden zwei in vitro Modelle entwickelt, um die Arzneimittelsicherheit und Entzündungs-reaktion abzuschätzen. Für eine Sicherheitsbewertung sollte ein in vitro Modell Humandaten unter Verwendung von FDA-Hilfsstoffkonzentrationen vorhersagen. Aus diesen Hilfsstoffen wurde der in vitro IC50 Wert für Lungenzellen bestimmt und in die etablierte SAFE-Klassifi-kation eingestuft. SAFE ermöglicht so die Vorhersage von Humandaten durch in vitro Tests. Zur Bewertung von Entzündungen wurden hAELVi Zellen den Zytokinen TNF-α und IFN-γ ausgesetzt. Ähnlich wie bei einer entzündlichen in vivo Reaktion wurde ein Verlust der epithe-lialen Barrierefunktion beobachtet. Darüber hinaus unterstreicht die entzündungshemmende Reaktion des Modells auf Hydrocortison sein Potenzial für eine in vitro Sicherheitsbewertungen von potenziellen Wirkstoffen. Die etablierten in vitro Modelle bieten einen vielversprechenden Ansatz, um Tierversuche zu ersetzen, wobei ihre Standardisierung und Validierung künftige Herausforderungen zur Auf-nahme in anerkannten Richtlinien darstellen

Pathway-based predictive approaches for non-animal assessment of acute inhalation toxicity

New approaches are needed to assess the effects of inhaled substances on human health. These approaches will be based on mechanisms of toxicity, an understanding of dosimetry, and the use of in silico modeling and in vitro test methods. In order to accelerate wider implementation of such approaches, development of adverse outcome pathways (AOPs) can help identify and address gaps in our understanding of relevant parameters for model input and mechanisms, and optimize non-animal approaches that can be used to investigate key events of toxicity. This paper describes the AOPs and the toolbox of in vitro and in silico models that can be used to assess the key events leading to toxicity following inhalation exposure. Because the optimal testing strategy will vary depending on the substance of interest, here we present a decision tree approach to identify an appropriate non-animal integrated testing strategy that incorporates consideration of a substance's physicochemical properties, relevant mechanisms of toxicity, and available in silico models and in vitro test methods. This decision tree can facilitate standardization of the testing approaches. Case study examples are presented to provide a basis for proof-of-concept testing to illustrate the utility of non-animal approaches to inform hazard identification and risk assessment of humans exposed to inhaled substances

Alternative approaches for acute inhalation toxicity testing to address global regulatory and non-regulatory data requirements: an international workshop report

Inhalation toxicity testing, which provides the basis for hazard labeling and risk management of chemicals with potential exposure to the respiratory tract, has traditionally been conducted using animals. Significant research efforts have been directed at the development of mechanistically based, non-animal testing approaches that hold promise to provide human-relevant data and an enhanced understanding of toxicity mechanisms. A September 2016 workshop, “Alternative Approaches for Acute Inhalation Toxicity Testing to Address Global Regulatory and Non-Regulatory Data Requirements”, explored current testing requirements and ongoing efforts to achieve global regulatory acceptance for non-animal testing approaches. The importance of using integrated approaches that combine existing data with in vitro and/or computational approaches to generate new data was discussed. Approaches were also proposed to develop a strategy for identifying and overcoming obstacles to replacing animal tests. Attendees noted the importance of dosimetry considerations and of understanding mechanisms of acute toxicity, which could be facilitated by the development of adverse outcome pathways. Recommendations were made to (1) develop a database of existing acute inhalation toxicity data; (2) prepare a state-of-the-science review of dosimetry determinants, mechanisms of toxicity, and existing approaches to assess acute inhalation toxicity; (3) identify and optimize in silico models; and (4) develop a decision tree/testing strategy, considering physicochemical properties and dosimetry, and conduct proof-of-concept testing. Working groups have been established to implement these recommendations

Development of a Dry Powder Inhaler and Nebulised Nanoparticle-Based Formulations of Curcuminoids for the Potential Treatment of Lung Cancer. Development of Drug Delivery Formulations of Curcuminoids to the Lungs using Air Jet Milling and Sonocrystallisation Techniques for Dry Powder Inhaler Preparations; and Nanoemulsion and Microsuspension for Nebuliser Formulations

Curcuminoids have strong anticancer activities but have low bioavailability. The highest rate of cancer deaths comes from lung tumours; therefore, inhaled curcuminoids could treat lung cancer locally. To date, there are no nebulised formulations of curcuminoids, and there are no inhalable curcuminoids particles without excipients using air jet mill and sonocrystallisation methods for DPI formulations. It is the first time; the aerodynamic parameters of curcumin, demethoxycurcumin and bisdemethoxycurcumin were measured individually using NGI. The size, shape, free surface energy, and the crystal polymorphism of the produced inhalable curcuminoid particles were characterised using laser diffraction, SEM, IGC, DSC and XRPD, respectively. Several DPI formulations with a variable particle size of curcuminoids were prepared in two drug-carrier ratios (1:9 and 1:67.5). The best performance of the DPI formulations of the sonocrystallised particles, which exist in crystal structure form1, were obtained from ethanol- heptane, as illustrated FPF 43.4%, 43.6% and 43.4% with MMAD of 3.6µm, 3.5µm and 3.4µm, whereas the best DPI formulation of the air jet milled particles was presented FPF 38.0%, 38.9%, and 39.5% with MMAD of 3.6µm, 3.4µm and 3.2µm for curcumin, demethoxycurcumin and bisdemethoxycurcumin, respectively. Nebulised curcuminoids using nanoemulsion and microsuspension formulations were prepared. The physical properties, such as osmolality, pH and the viscosity of the aerosolised nanoemulsion and the microsuspension formulations were determined. The FPF% and MMAD of nebulised nanoemulsion ranged from 44% to 50% and from 4.5µm to 5.5µm respectively. In contrast, the FPF% of microsuspension ranged from 26% to 40% and the MMAD from 5.8µm to 7.05µm. A HPLC method was developed and validated in order to be used in the determination of curcuminoids from an aqueous solution

EVALUATION OF THE REGIONAL DRUG DEPOSITION OF NASAL DELIVERY DEVICES USING IN VITRO REALISTIC NASAL MODELS

The overall objectives of this research project were i) to develop and evaluate methods of characterizing nasal spray products using realistic nasal airway models as more clinically relevant in vitro tools and ii) to develop and evaluate a novel high-efficiency antibiotic nanoparticle dry powder formulation and delivery device. Two physically realistic nasal airway models were used to assess the effects of patient-use experimental conditions, nasal airway geometry and formulation / device properties on the delivery efficiency of nasal spray products. There was a large variability in drug delivery to the middle passages ranging from 17 – 57 % and 47 – 77 % with respect to patient use conditions for the two nasal airway geometries. The patient use variables of nasal spray position, head angle and nasal inhalation timing with respect to spray actuation were found to be significant in determining nasal valve penetration and middle passage deposition of Nasonex®. The developed test methods were able to reproducibly generate similar nasal deposition profiles for nasal spray products with similar plume and droplet characteristics. Differences in spray plume geometry (smaller plume diameter resulted in higher middle passage drug delivery) were observed to have more influence on regional nasal drug deposition than changes to droplet size for mometasone furoate formulations in the realistic airway models. Ciprofloxacin nanoparticles with a mean (SD) volume diameter of 120 (10) nm suitable for penetration through mucus and biofilm layers were prepared using sonocrystallization technique. These ciprofloxacin nanoparticles were then spray dried in a PVP K30 matrix to form nanocomposite particles with a mean (SD) volume diameter of 5.6 (0.1) µm. High efficiency targeted delivery of the nanocomposite nasal powder formulation was achieved using a modified low flow VCU DPI in combination with a novel breathing maneuver; delivering 73 % of the delivered dose to the middle passages. A modified version of the nasal airway model accommodating Transwell® inserts and a Calu-3 monolayer was developed to allow realistic deposition and evaluation of the nasal powder. The nanocomposite formulation was observed to demonstrate improved dissolution and transepithelial transport (flux = 725 ng/h/cm2) compared to unprocessed ciprofloxacin powder (flux = 321 ng/h/cm2)

Risk Exposure to Particles – including Legionella pneumophila – emitted during Showering with Water-Saving Showers

The increase in legionellosis incidence in the general population in recent years calls for a better characterization of the sources of infection, such as showering. Water-efficient shower systems that use water atomization technology may emit slightly more inhalable bacteria-sized particles than traditional systems, which may increase the risk of users inhaling contaminants associated with these water droplets. To evaluate the risk, the number and mass of inhalable water droplets emitted by twelve showerheads—eight using water-atomization technology and four using continuous-flow technology— were monitored in a shower stall. The water-atomizing showers tested not only had lower flow rates, but also larger spray angles, less nozzles, and larger nozzle diameters than those of the continuous-flow showerheads. A difference in the behavior of inhalable water droplets between the two technologies was observed, both unobstructed or in the presence of a mannequin. The evaporation of inhalable water droplets emitted by the water-atomization showers favored a homogenous distribution in the shower stall. In the presence of the mannequin, the number and mass of inhalable droplets increased for the continuous-flow showerheads and decreased for the water-atomization showerheads. The water-atomization showerheads emitted less inhalable water mass than the continuous-flow showerheads did per unit of time; however, they generally emitted a slightly higher number of inhalable droplets—only one model performed as well as the continuous-flow showerheads in this regard. To specifically assess the aerosolisation rate of bacteria, in particular of the opportunistic water pathogen Legionella pneumophila, during showering controlled experiments were run with one atomization showerhead and one continuous-flow, first inside a glove box, second inside a shower stall. The bioaerosols were sampled with a Coriolis® air sampler and the total number of viable (cultivable and noncultivable) bacteria was determined by flow cytometry and culture. We found that the rate of viable and cultivable Legionella aerosolized from the water jet was similar between the two showerheads: the viable fraction represents 0.02% of the overall bacteria present in water, while the cultivable fraction corresponds to only 0.0005%. The two showerhead models emitted a similar ratio of airborne Legionella viable and cultivable per volume of water used. Similar results were obtained with naturally contaminated hoses tested in shower stall. Therefore, the risk of exposure to Legionella is not expected to increase significantly with the new generation of water-efficient showerheads

The development and characterization of theophylline and budesonide co-encapsulated poly (lactic acid) (pla) nanoparticles

Inhaled drug delivery is ideal for treatment of asthma and chronic obstructive pulmonary disease (COPD) as it allows a local action of the medication at the disease site. Biodegradable polymeric nanoparticles which might allow extended/sustained release of inhaled drugs are synthesized using various methods however; these do not permit high encapsulation efficiency for hydrophilic drugs. The aim of the project was to test the hypothesis that it was possible to develop an efficient method for the co-encapsulation of a hydrophilic and lipophilic drug (theophylline and budesonide respectively) into nanoparticles. In order to improve the loading efficiency of both hydrophilic and hydrophobic drugs, a modified double emulsification solvent diffusion (DESD) method was developed and both co-encapsulated and mono-encapsulated nanoparticles (containing either drug) were synthesized. Improved loading efficiency, studied using high performance liquid chromatography (HPLC), for both drugs was obtained. Dynamic light scattering (DLS) and scanning electron microscopy (SEM) showed that particles were in the sub-micron range (150-400 nm). Measurement of zeta potential showed that the particles had a negative surface charge and additionally Fourier-transform infra-red (FT-IR) spectroscopy confirmed that this was due to the polymer and no drug was adsorbed on the external surface of the nanoparticles. Resemblance of nanoparticles thermograms, obtained using differential scanning calorimetry (DSC), to those of the polymer alone suggested successful encapsulation of the drugs. Stability studies of the drug encapsulated nanoparticles conducted at different temperatures indicated that storage conditions of 2-8°C over a period of 6 months showed minimal changes in the particle size, zeta potential and morphological characteristics of the nanoparticles. Storage (of the nanoparticles) at 40°C over the course of 6 months resulted in larger variations on the particle size and zeta potential but also loss of morphological features of the nanoparticles, suggestive of changes in the polymer state at this temperature. Franz diffusion cells were used to study the release of drugs from the nanoparticles over 24 hours at room temperature and at 37oC. The results showed that release of theophylline and budesonide from nanoparticles was biphasic and sustained compared to release of drug from solutions containing an equivalent concentration of drug. The effect of the nanoparticles on the viability of airway epithelial cells was studied using a human bronchial epithelial cell line (16HBE14o-) using a 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. The nanoparticles had no significant effect ii on cell viability except at the highest concentration of the suspension studied (5 mg/mL) (P <0.05). The permeability of 16HBE14o- cells, cultured at an air-liquid interface, to theophylline and budesonide applied in solution and as mono-encapsulated and co-encapsulated nanoparticles was studied. The nanoparticles and drug solutions did not affect the tight junctions of the cells and similar to the results obtained in the Franz diffusion cells, both drugs crossed the cells more slowly when applied as nanoparticles in comparison to the solutions. To study deposition of the nanoparticles; nebulized suspensions of the nanoparticles in de-ionized water and dry powder formulations using different grades of lactose were compared. The prepared formulations were studied using a multi-stage liquid impinger (MSLI). The results indicated that drug deposition was greatest in stages 1 and 2 of the MSLI where particle size was greater than 6.8μm from the dry powder formulations in contrast to deposition throughout the five stages of the MSLI from the nebulized suspension. Morphological assessment of the dry powder formulations using SEM showed nanoparticles adhered to the lactose but also included nanoparticles in the absence of lactose and vice versa. In conclusion, theophylline and budesonide nanoparticles were successfully formulated using PLA by application of the DESD method. Nanoparticles possessed desired physicochemical properties including submicron size range and negatively charged surface; however a higher loading efficiency of the hydrophobic drug was obtained despite modifications to the DESD method. Low toxicity of the nanoparticles to human bronchial epithelial cells and sustained release over a period of 24 hours was achieved. Nanoparticles were delivered successfully in the target site at a desired particle size range when formulated as nebulized suspensions