Cynthia Bosquillon

Influence of formulation excipients and physical characteristics of inhalation dry powders on their aerosolization performance

The objective of this study was to determine the effects of formulation excipients and physical characteristics of inhalation particles on their in vitro aerosolization performance, and thereby to maximize their respirable fraction. Dry powders were produced by spray-drying using excipients that are FDA-approved for inhalation as lactose, materials that are endogenous to the lungs as albumin and dipalmitoylphosphatidylcholine (DPPC); and/or protein stabilizers as trehalose or mannitol. Dry powders suitable for deep lung deposition, i.e. with an aerodynamic diameter of individual particles <3 microm, were prepared. They presented 0.04--0.25 g/cm(3) bulk tap densities, 3--5 microm geometric particle sizes, up to 90% emitted doses and 50% respirable fractions in the Andersen cascade impactor using a Spinhaler inhaler device. The incorporation of lactose, albumin and DPPC in the formulation all improved the aerosolization properties, in contrast to trehalose and the mannitol which decreased powder flowability. The relative proportion of the excipients affected aerosol performance as well. The lower the bulk powder tap density, the higher the respirable fraction. Optimization of in vitro aerosolization properties of inhalation dry powders can be achieved by appropriately selecting composition and physical characteristics of the particles.

Drug transporters in the lung--do they play a role in the biopharmaceutics of inhaled drugs?

The role of transporters in drug absorption, distribution and elimination processes as well as in drug-drug interactions is increasingly being recognised. Although the lungs express high levels of both efflux and uptake drug transporters, little is known of the implications for the biopharmaceutics of inhaled drugs. The current knowledge of the expression, localisation and functionality of drug transporters in the pulmonary tissue and the few studies that have looked at their impact on pulmonary drug absorption is extensively reviewed. The emphasis is on transporters most likely to affect the disposition of inhaled drugs: (1) the ATP-binding cassette (ABC) superfamily which includes the efflux pumps P-glycoprotein (P-gp), multidrug resistance associated proteins (MRPs), breast cancer resistance protein (BCRP) and (2) the solute-linked carrier (SLC and SLCO) superfamily to which belong the organic cation transporter (OCT) family, the peptide transporter (PEPT) family, the organic anion transporter (OAT) family and the organic anion transporting polypeptide (OATP) family. Whenever available, expression and localisation in the intact human tissue are compared with those in animal lungs and respiratory epithelial cell models in vitro. The influence of lung diseases or exogenous agents on transporter expression is also mentioned.

Evaluation of Differentiated Human Bronchial Epithelial Cell Culture Systems for Asthma Research

The aim of the current study was to evaluate primary (human bronchial epithelial cells, HBEC) and non-primary (Calu-3, BEAS-2B, BEAS-2B R1) bronchial epithelial cell culture systems as air-liquid interface- (ALI-) differentiated models for asthma research. Ability to differentiate into goblet (MUC5AC+) and ciliated (β-Tubulin IV+) cells was evaluated by confocal imaging and qPCR. Expression of tight junction/adhesion proteins (ZO-1, E-Cadherin) and development of transepithelial electrical resistance (TEER) were assessed. Primary cells showed localised MUC5AC, β-Tubulin IV, ZO-1, and E-Cadherin and developed TEER with, however, a large degree of inter- and intradonor variation. Calu-3 cells developed a more reproducible TEER and a phenotype similar to primary cells although with diffuse β-Tubulin IV staining. BEAS-2B cells did not differentiate or develop tight junctions. These data highlight the challenges in working with primary cell models and the need for careful characterisation and selection of systems to answer specific research questions.

Sparing methylation of β-cyclodextrin mitigates cytotoxicity and permeability induction in respiratory epithelial cell layers in vitro

Cyclodextrins (CDs) are promising solubility enhancers for inhaled drug delivery. However, they have dose-dependent effects on the respiratory epithelium, which may have advantages for permeability enhancement but also gives rise to safety concerns. In this study, the methyl thiazol tetrazolium (MTT) assay was used to compare a new sparingly methylated beta-CD, Kleptose Crysmebeta (Crysmeb) with the more established CD derivatives hydroxypropyl-gamma-cyclodextrin (HPgammaCD), randomly methylated beta-cyclodextrin (Rameb) and hydroxypropyl-beta-cyclodextrin (HPbetaCD). The betaCD derivatives affected cell metabolism in A549 cells in a concentration dependent manner with LD(50) of 56, 31 and 11 mM obtained for HPbetaCD, Crysmeb and Rameb, respectively. Calu-3 cells were less susceptible to betaCD with an LD(50) of 25 mM being obtained for Rameb only. Permeability increases in Calu-3 cell layers were observed with betaCD derivatives and a concentration dependency shown. The mechanism of permeability enhancement and its reversibility was investigated. Rameb produced an irreversible loss of cell layer barrier function at > or = 25 mM, but perturbations of epithelial integrity were moderate and reversible in the case of HPbetaCD and Crysmeb (25-50 mM). Given its high solubilisation capacity, the low toxicity and transient absorption promoting properties, this study identifies Crysmeb as a promising adjuvant in formulations for inhalation.

Confocal imaging of rat lungs following intratracheal delivery of dry powders or solutions of fluorescent probes

The overall pulmonary disposition of various fluorescent probes was viewed by confocal imaging following intratracheal delivery in the rat in vivo. The green fluorescent dyes, coumarin-6, a 350 Da lipophilic molecule; calcein, a 623 Da hydrophilic molecule; or FITC-albumin, a 65000 Da hydrophilic molecule; were insufflated as a dry powder or instilled as a solution in the lungs of rat in vivo. Immediately, 2 or 24 h following delivery, the lungs were colored with sulforhodamine and fixed by vascular perfusion. The lungs were then removed, grossly sliced and examined by confocal laser scanning fluorescence microscopy. Coumarin-6 diffused within minutes across the trachea, airways and alveolar tissue but was also retained for hours in type II alveolar epithelial cells. The diffusion of calcein across the tissue was fast as well, with no particular affinity for specific cells. FITC-albumin slowly permeated the tissue. It remained in the airspaces for hours and was intensively captured by alveolar macrophages. Compared to the powder, the solution bypassed dissolution and therefore shortened the lag time for diffusion and cellular capture. The technique allowed to obtain an overview of the fate of fluorescent probes locally in each region of the lungs and highlighted the strong dependence of the localization behavior on physico-chemical properties of molecules as well as a capture by particular cells of the pulmonary tissue.

In-vitro respiratory drug absorption models possess nominal functional P-glycoprotein activity

OBJECTIVES The P-glycoprotein (P-gp) efflux pump is known to be present within several major physiological barriers including the brain, kidney, intestine and placenta. However, the function of P-gp in the airways of the lung is unclear. The purpose of this study was to use the highly specific P-gp inhibitor GF120918A to investigate the activity of the P-gp transporter in the airways to determine whether P-gp could influence inhaled drug disposition. METHODS P-gp activity was measured as a change in digoxin transport in the presence of GF120918A in normal human bronchial epithelial (NHBE) cells, Calu-3 cell layers and the ex-vivo rat lung. KEY FINDINGS The efflux ratios (ERs) in NHBE and Calu-3 cells were between 0.5 and 2, in contrast to 10.7 in the Caco-2 cell control. These low levels of GF120918A-sensitive polarised digoxin transport were measured in the absorptive direction in NHBE cells (ER = 0.5) and in the secretory direction in Calu-3 cells (ER = 2), but only after 21 days in culture for both cell systems and only in Calu-3 cells at passage > 50. The airspace to perfusate transfer kinetics of digoxin in the ex-vivo rat lung were unchanged in the presence of GF120918A. CONCLUSIONS These results demonstrated that although low levels of highly culture-dependent P-gp activity could be measured in cell-lines, these should not be interpreted to mean that P-gp is a major determinant of drug disposition in the airways of the lung.

Evaluation of air-interfaced Calu-3 cell layers for investigation of inhaled drug interactions with organic cation transporters in vitro

A physiologically pertinent in vitro model is urgently needed for probing interactions between inhaled drugs and the organic cation transporters (OCT) in the bronchial epithelium. This study evaluated OCT expression, functionality, inhibition by common inhaled drugs and impact on formoterol transepithelial transport in layers of human bronchial epithelial Calu-3 cells grown at an air-liquid interface. 21 day old Calu-3 layers expressed OCT1, OCT3, OCTN1 and OCTN2 whereas OCT2 could not be detected. Quantification of the cellular uptake of the OCT substrate ASP(+) in presence of inhibitors suggested several OCT were functional at the apical side of the cell layers. ASP(+) uptake was reduced by the bronchodilators formoterol, salbutamol (albuterol), ipratropium and the glucocorticoid budesonide. However, the OCT inhibitory properties of the two β(2)-mimetics were suppressed at therapeutically relevant concentrations. The absorptive permeability of formoterol across the cell layers was enhanced at a high drug concentration shown to decrease ASP(+) uptake by ∼50% as well as in presence of the OCT inhibitor tetraethylammonium (TEA). Secretory transport was unaffected by the drug concentration but was reduced by TEA. Our data indicate air-interfaced Calu-3 layers offer a low-cost in vitro model suitable for assessing inhaled drug-OCT interactions in the bronchial epithelium.

Polyethylene glycol-drug ester conjugates for prolonged retention of small inhaled drugs in the lung.

Typically, inhaled drugs are rapidly absorbed into the bloodstream, which results in systemic side effects and a brief residence time in the lungs. PEGylation was evaluated as a novel strategy for prolonging the retention of small inhaled molecules in the pulmonary tissue. Hydrolysable ester conjugates of PEG₁₀₀₀, PEG₂₀₀₀, ₂₀₀₀, PEG₃₄₀₀ and prednisolone, a model drug cleared from the lungs within a few minutes, were synthesised and thoroughly characterised. The conjugates were stable in buffers with hydrolysis half-lives ranging from 1h to 70 h, depending on the pH and level of substitution. With the exception of PEG₃₄₀₀-prednisolone, conjugates did not induce a significant lactate dehydrogenase (LDH) release from Calu-3 cells after a 20 h exposure. Following nebulisation to isolated perfused rat lungs (IPRL), the PEG₂₀₀₀ and mPEG₂₀₀₀ conjugates reduced the maximum prednisolone concentration in the perfusate (Cmax) by 3.0 and 2.2 fold, respectively. Moreover, while prednisolone was undetectable in the perfusion solution beyond 20 min when the free drug was administered, prednisolone concentrations were still quantifiable after 40 min following delivery of the conjugates. This study is the first to demonstrate hydrolysable PEG drug ester conjugates are a promising approach for optimising the pharmacokinetic profile of small drugs delivered by inhalation.

Relationship between the affinity of PEO-PPO-PEO block copolymers for biological membranes and their cellular effects

ABSTRACTPurposeThe interactions of poly(ethylene oxide)-co-poly(propylene oxide) tri-block copolymers (PEO-PPO-PEO block copolymers, Pluronics®, Synperonics®, Poloxamers) of differing chemical composition with cell membranes were systematically investigated in order to clarify the mechanisms behind their previously reported various cellular responses.MethodsRelationships between the structural components of a defined series of PEO-PPO-PEO block copolymers and i) their interactions with biological membranes; ii) their cytotoxic potential were probed using a combination of haemolysis studies and cytotoxicity assays in the Caco-2 and HMEC-1 cell lines.ResultsThe length of the PPO block as well as the PEO/PPO ratio were determinants of their membrane binding constant and cytotoxicity endpoints measured in the MTS and LDH assays. Similar 2D parabolic relationships were found between polymer composition and their affinity for membranes or their cytotoxicity potential. Cytotoxicity was related to the ability of the copolymers to form ion transversable pores within the cell membrane.ConclusionsThe data suggest a link between the affinity of certain Pluronics for biological membranes and their cellular adverse effects. This first cell-based investigation of the interactions of Pluronics with biological membranes is an important step towards unravelling the complex mechanisms which govern the biological effects of widely used amphiphilic materials.

The Isolated Perfused Lung for Drug Absorption Studies

Over the past 20 years, the isolated perfused lung (IPL) technique has been developed for the evaluation of pulmonary drug absorption and disposition. The procedure for establishing the model requires a skilled operator, a validated technique for intra-tracheal drug delivery and a system for maintenance and monitoring of the preparation. Most absorption studies to date have utilised the ex vivo rat lung maintained by ventilation and perfusion in an artificial thorax. Techniques for delivery of drugs into the airspaces of the IPL have been developed and the drug transfer (air-to-perfusate) profiles of a variety of actively and passively transported compounds have been measured. Recent developments include the reporting of in vitro-in vivo correlation for air-to-perfusate transfer in the IPL with pulmonary absorption in the rat in vivo, the use of the IPL to model active transport mechanisms and the use of a human lung reperfusion model. The value of the IPL is in discerning lung-specific drug absorption and disposition kinetics that may be difficult to interpolate from in vivo data and cannot be modelled with physiological relevance using reductive in vitro techniques such as cell culture.