Targeted PEG-poly(glutamic acid) complexes for inhalation protein delivery to the lung.

Pulmonary delivery is increasingly seen as an attractive, non-invasive route for the delivery of forthcoming protein therapeutics. In this context, here we describe protein complexes with a new 'complexing excipient' - vitamin B12-targeted poly(ethylene glycol)-block-poly(glutamic acid) copolymers. These form complexes in sub-200nm size with a model protein, suitable for cellular targeting and intracellular delivery. Initially we confirmed expression of vitamin B12-internalization receptor (CD320) by Calu-3 cells of the in vitro lung epithelial model used, and demonstrated enhanced B12 receptor-mediated cellular internalization of B12-targeted complexes, relative to non-targeted counterparts or protein alone. To develop an inhalation formulation, the protein complexes were spray dried adopting a standard protocol into powders with aerodynamic diameter within the suitable range for lower airway deposition. The cellular internalization of targeted complexes from dry powders applied directly to Calu-3 model was found to be 2-3 fold higher compared to non-targeted complexes. The copolymer complexes show no complement activation, and in vivo lung tolerance studies demonstrated that repeated administration of formulated dry powders over a 3 week period in healthy BALB/c mice induced no significant toxicity or indications of lung inflammation, as assessed by cell population count and quantification of IL-1ß, IL-6, and TNF-α pro-inflammatory markers. Importantly, the in vivo data appear to suggest that B12-targeted polymer complexes administered as dry powder enhance lung retention of their protein payload, relative to protein alone and non-targeted counterparts. Taken together, our data illustrate the potential developability of novel B12-targeted poly(ethylene glycol)-poly(glutamic acid) copolymers as excipients suitable to be formulated into a dry powder product for the inhalation delivery of proteins, with no significant lung toxicity, and with enhanced protein retention at their in vivo target tissue.

Enhanced expression of Organic Cation Transporters in bronchial epithelial cell layers following insults associated with asthma - Impact on salbutamol transport.

Increasing evidence suggests Organic Cation Transporters (OCT) might facilitate the absorption of inhaled bronchodilators, including salbutamol, across the lung epithelium. This is essentially scarred and inflamed in asthma. Accordingly, the impact of epithelial insults relevant to asthma on OCT expression and salbutamol transport was evaluated in air-liquid interfaced layers of the human broncho-epithelial cell line Calu-3. These were physically injured and allowed to recover for 48h or exposed to the pro-inflammatory stimulant lipopolysaccharide (LPS) for 48h and the aeroallergen house dust mite (HDM) for 8h twice over 48h. Increases in transporter expression were measured following each treatment, with the protein levels of the OCTN2 subtype consistently raised by at least 50%. Interestingly, OCT upregulation upon LPS and HDM challenges were dependent on an inflammatory event occurring in the cell layers. Salbutamol permeability was higher in LPS exposed layers than in their untreated counterparts and in both cases, was sensitive to the OCT inhibitor tetraethylammonium. This study is the first to show epithelial injury, inflammation and allergen abuse upregulate OCT in bronchial epithelial cells, which might have an impact on the absorption of their substrates in diseased lungs.

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.

In-cell Western™ detection of organic cation transporters in bronchial epithelial cell layers cultured at an air-liquid interface on Transwell(®) inserts.

INTRODUCTION: Organic cation transporters (OCT) have been shown to mediate the transport of inhaled drugs in bronchial epithelial cells and might have important physiological functions in the airway epithelium. However, a quantitative method to evaluate OCT protein expression in physiologically relevant airway epithelial cell culture models is currently lacking. In-cell Western™ (ICW) techniques might fill that gap but to date, have only been performed on cells grown on 96 or 384-well microplates. METHODS: An ICW assay was designed for measuring levels of the different OCT subtypes in intact layers of the human bronchial epithelial Calu-3 cell line cultured at an air-liquid interface on Transwell(®) inserts. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal standard for normalisation of cell number between the layers. The protocol was subsequently validated by exposing cell layers to compounds known to cause variations in OCT expression. RESULTS: Antibody signals above the background fluorescence were detected for OCT1, OCT3, OCTN1 and OCTN2 but not for OCT2 in 21day old Calu-3 layers, in agreement with previous studies which had reported OCT2 was absent in the Calu-3 cell line. Furthermore, increases in the fluorescence signal associated with OCT1, OCTN1 and OCTN2 were obtained following treatment of the layers with, respectively, the nitric oxide inducer sodium nitroprusside, the peroxisome proliferator activated receptor α (PPARα) agonist fenofibrate or the PPARγ agonist rosiglitazone, confirming the reliability of the ICW method developed. However, a suitable positive control for OCT3 could not be identified. DISCUSSION: This novel ICW assay can be exploited to quantify basal OCT protein expression as well as changes in transporter levels following external stimuli in various in vitro models. It can also be easily adapted to probe any protein in epithelial layers maintained on permeable filters.

Evaluation of layers of the rat airway epithelial cell line RL-65 for permeability screening of inhaled drug candidates.

A rat respiratory epithelial cell culture system for in vitro prediction of drug pulmonary absorption is currently lacking. Such a model may however enhance the understanding of interspecies differences in inhaled drug pharmacokinetics by filling the gap between human in vitro and rat in/ex vivo drug permeability screens. The rat airway epithelial cell line RL-65 was cultured on Transwell inserts for up to 21 days at an air-liquid (AL) interface and cell layers were evaluated for their suitability as a drug permeability measurement tool. These layers were found to be morphologically representative of the bronchial/bronchiolar epithelium when cultured for 8 days in a defined serum-free medium. In addition, RL-65 layers developed epithelial barrier properties with a transepithelial electrical resistance (TEER) >300 Ω cm(2) and apparent (14)C-mannitol permeability (P(app)) values between 0.5-3.0 × 10(-6)cm/s; i.e., in the same range as established in vitro human bronchial epithelial absorption models. Expression of P-glycoprotein was confirmed by gene analysis and immunohistochemistry. Nevertheless, no vectorial transport of the established substrates (3)H-digoxin and Rhodamine123 was observed across the layers. Although preliminary, this study shows RL-65 cell layers have the potential to become a useful in vitro screening tool in the pre-clinical development of inhaled drug candidates.

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 ß(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.

Sparing methylation of beta-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.

Comparison of particle sizing techniques in the case of inhalation dry powders.

The objectives of this work were (i) to validate electrical zone sensing and laser diffraction for the analysis of primary particle size in the case of inhalation dry powders and (ii) to study the influence of the aggregation state of the powder on the sizing techniques. Free-flowing dry powders were prepared by spray-drying with a combination of albumin, lactose, and dipalmitoylphosphatidylcholine. The replacement of lactose by mannitol, the removal of albumin, and the atomization at high relative humidity all increased powder cohesion. Automated measurements were compared with primary particle sizes collected by light and electron microscopy. The mass mode obtained by electrical zone sensing and the mass median diameter measured by laser diffraction following dispersion with compressed air at a pressure of 3 bar or following suspension in water and ultrasonic dispersion at a power of 60 W for 30 s each provided primary particle sizes close to microscopy measurements. However, these conditions only applied in the case of slightly to moderately aggregated powders. For strongly agglomerated powders, an exact measurement of the size was only collected by laser diffraction in the wet state combined with ultrasonic dispersion. Our study underlies how measurement of primary particle size highly depends on both powder material and proper particle dispersion.

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.