Inhaled drug delivery: Mechanisms by which pressurised metered dose inhaler excipients affect respiratory membrane biophysics.
Lead Research Organisation:
King's College London
Department Name: Pharmaceutical Sciences
Abstract
Strategi Research Priority: Bioscience for Health
Background: Glycerol was added to some products as a non-volatile component to modify aerosol particle size when pressurised metered dose inhalers were reformulated with hydrofluoroalkanes in the 1990's to comply with the Montreal treaty on greenhouse gases. It was recently discovered that the addition of glycerol to inhaler formulations to increase aerosol particle size may affect drug bioavailability even when pharmaceutical aerosols are aerodynamically equivalent. Previous studies comparing glycerol free and glycerol containing formulations have observed differences in the pharmaceutical properties and performances of these formulations. It is hypothesized that the effects of glycerol on the disposition of deposited drug goes beyond changes in particle size. Not only may glycerol have effects on the release of drug from the aerosol particle, it might also play a role in transiently modifying the permeability of the lung cell membranes. The aim of this study is to gain more insight into the effect glycerol present in inhaled drug particles would have on the permeability of the airway epithelium. These effects will be studied in vitro using the Calu-3 cell continuous cell line. Specific objectives include evaluation of Calu-3 cells tolerability to glycerol. The outcome of the tolerability studies will be used to establish a concentration range for further investigations into the effect of glycerol on the permeability of cell layers using transport markers in vitro.
Method: The MTT assay was used to evaluate the toxicity of glycerol to the cells and to determine an LC50 value. Glycerol concentrations ranged between 1% to 35% v/v. The effect of glycerol on the cell layer integrity was investigated via trans-epithelial electrical resistance (TEER) measurements. Cell layer permeability of two transport markers; 3H-digoxin and 14C-Mannitol (transcellular and paracellular transport markers respectively) was also assessed. The glycerol concentrations chosen for the permeability studies were such that gave cell viability between 60% and 100% as reported by the MTT studies.
Results: LC50 value was determined by to be 14% v/v. TEER values showed that the tight junctions were perturbed with increasing glycerol concentration and upon prolonged glycerol exposure. Permeability across cell layers for both markers was also enhanced by glycerol (24-fold for mannitol and 6-fold for digoxin). This was expected for mannitol especially because the tight junctions which constitute the paracellular route had been compromised.
Conclusion: The LC50 of glycerol with Calu-3 cells was determined and the range of concentrations used in the toxicity assay showed the extent of tolerability of the cell line to glycerol. Glycerol also appeared to enhance the permeability of cell monolayers to transport markers. Further studies are required to establish optimum conditions for the investigation of BDP transport and whether this observed glycerol effect on membrane permeability translates to effects on drug transport in lung tissue.
Background: Glycerol was added to some products as a non-volatile component to modify aerosol particle size when pressurised metered dose inhalers were reformulated with hydrofluoroalkanes in the 1990's to comply with the Montreal treaty on greenhouse gases. It was recently discovered that the addition of glycerol to inhaler formulations to increase aerosol particle size may affect drug bioavailability even when pharmaceutical aerosols are aerodynamically equivalent. Previous studies comparing glycerol free and glycerol containing formulations have observed differences in the pharmaceutical properties and performances of these formulations. It is hypothesized that the effects of glycerol on the disposition of deposited drug goes beyond changes in particle size. Not only may glycerol have effects on the release of drug from the aerosol particle, it might also play a role in transiently modifying the permeability of the lung cell membranes. The aim of this study is to gain more insight into the effect glycerol present in inhaled drug particles would have on the permeability of the airway epithelium. These effects will be studied in vitro using the Calu-3 cell continuous cell line. Specific objectives include evaluation of Calu-3 cells tolerability to glycerol. The outcome of the tolerability studies will be used to establish a concentration range for further investigations into the effect of glycerol on the permeability of cell layers using transport markers in vitro.
Method: The MTT assay was used to evaluate the toxicity of glycerol to the cells and to determine an LC50 value. Glycerol concentrations ranged between 1% to 35% v/v. The effect of glycerol on the cell layer integrity was investigated via trans-epithelial electrical resistance (TEER) measurements. Cell layer permeability of two transport markers; 3H-digoxin and 14C-Mannitol (transcellular and paracellular transport markers respectively) was also assessed. The glycerol concentrations chosen for the permeability studies were such that gave cell viability between 60% and 100% as reported by the MTT studies.
Results: LC50 value was determined by to be 14% v/v. TEER values showed that the tight junctions were perturbed with increasing glycerol concentration and upon prolonged glycerol exposure. Permeability across cell layers for both markers was also enhanced by glycerol (24-fold for mannitol and 6-fold for digoxin). This was expected for mannitol especially because the tight junctions which constitute the paracellular route had been compromised.
Conclusion: The LC50 of glycerol with Calu-3 cells was determined and the range of concentrations used in the toxicity assay showed the extent of tolerability of the cell line to glycerol. Glycerol also appeared to enhance the permeability of cell monolayers to transport markers. Further studies are required to establish optimum conditions for the investigation of BDP transport and whether this observed glycerol effect on membrane permeability translates to effects on drug transport in lung tissue.
People |
ORCID iD |
B Forbes (Primary Supervisor) | |
Precious Akhuemokhan (Student) |
Studentship Projects
Project Reference | Relationship | Related To | Start | End | Student Name |
---|---|---|---|---|---|
BB/M009513/1 | 01/10/2015 | 31/03/2024 | |||
1764834 | Studentship | BB/M009513/1 | 01/10/2016 | 30/12/2020 | Precious Akhuemokhan |
Description | Using in vitro techniques, we have demonstrated that non volatile excipient present in inhaled formulations may affect inhaled drug disposition |
Exploitation Route | More in vivo studies on different inhaled formulations may need to be carried out to fully ascertain excipient effects on product performance. |
Sectors | Pharmaceuticals and Medical Biotechnology |
Description | Faculty of Life Sciences and Medicine Travel Bursary |
Amount | £500 (GBP) |
Organisation | King's College London |
Sector | Academic/University |
Country | United Kingdom |
Start | 10/2019 |
End | 12/2019 |