Category: Formulation and Quality
Purpose: Glycerol, a non-volatile component of the beclomethasone dipropionate (BDP) Clenil Moduliteâ pressurized metered dose inhaler (pMDI) formulation plays the role of modulating the aerosol particle size produced from the hydrofluoroalkane propellant formulated product (1). Studies comparing BDP formulations that contained glycerol and glycerol-free formulations have shown differences in the absorptive profiles of drug after deposition on in vitro respiratory epithelial cell models (2).
To explore this phenomenon further, several techniques have been utilized, including the parallel artificial membrane permeability Assay (PAMPA) which offers an alternative platform to cellular models to investigate the effect of glycerol on drug transport. In this assay, drug flux is measured across a membrane consisting of lipids and phospholipids separating a donor and an acceptor compartment allowing drug transport to be determined under controlled conditions (3).
This study aimed to compare a PAMPA (Parallel Artificial Membrane Permeability Assay) to an established Calu-3 technique for evaluating the effect of glycerol on drug transport.
Methods: Transport studies - For both models, two radioactively labelled transport probes were used: mannitol, a small hydrophilic molecule and propranolol which is a marker for transcellular transport.
Calu-3 model: Cell layers were grown and matured over a 14-day period. Mannitol and propranolol were diluted in HBSS and applied to the apical compartment and sampling was carried out at specific intervals from the basolateral compartment. Samples were analyzed by scintillation counting.
PAMPA: The artificial membrane was generated by coating a transwell semi permeable insert with a solution of POPC (palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline) dissolved with cholesterol in dodecane in a 2:1 ratio. An aliquot of the mixture, 10 μL, was applied to the insert for 30 min at ambient laboratory temperature before permeability experiments were carried out as described for the Calu-3 cell layers.
Microscopy and electrical measurements - Cell layers were also treated with glycerol concentrations ranging from 1% v/v to 10% v/v before observing staining for zona occludens-1 (ZO-1) by microscopy and measuring trans-epithelial electrical resistance (TEER).
Results: TEER measurements indicated a progressive disruption of tight junctions between the cells in the Calu-3 model with increasing glycerol concentration. This was evidenced by the decline in electrical resistance between cell layers exposed to 0% v/v glycerol and 10% v/v glycerol after 1 hour of exposure; 196.9 Ω.cm2 and 50.6 Ω.cm2 respectively. Microscopy showed a concomitant loss in defined boundaries formed by ZO-1 between cells suggesting a depletion in tight junction proteins.
The effect of glycerol on the transport probes (mannitol and propranolol) in both the Calu-3 model and the PAMPA models were comparable with similar trends apparent in both models. Glycerol retarded propranolol transport in the Calu-3 model (Control Papp = 15.2 x 10-6 cm/s, 2% v/v glycerol Papp = 11.5 x 10-6 cm/s) and in PAMPA (Control Papp = 4.7 x 10-6 cm/s, 2% v/v glycerol Papp = 3.4 x 10-6 cm/s). This reduction in transcellular transport can be attributed a stiffening effect of glycerol on phospholipid membranes as suggested by published biophysical and in silico data (4). Mannitol transport was enhanced in the presence of glycerol in the cellular models (Control Papp = 0.17 cm/s, 2% v/v glycerol Papp = 0.31 cm/s) corresponding to glycerol’s disruptive effect on tight junctions which would leave the paracellular route more permeable to the movement of small hydrophilic molecules.
Conclusion: This study has shown that glycerol’s stiffening effect on biological membranes can impact drug transport using in both cell models (Calu-3) and artificial membranes used for PAMPA. BDP, being mostly transcellularly transported, is expected to show similar trends to propranolol in the presence of glycerol. Finally, these studies are a step towards establishing PAMPA as a complementary and alternative method for the investigation of the effect of glycerol on the transport of BDP and, more generally, the effect of other excipients that alter membrane biophysics on lung permeability.
Ben Forbes– Professor of Pharmaceutics, King's College London, London, England, United Kingdom
Josie Williams– Waterloo, England, United Kingdom
Magda Swedrowska– London, England, United Kingdom
Richard Harvey– Waterloo, England, United Kingdom