Inhaled drug delivery: Mechanisms by which pressurised metered dose inhaler excipients affect respiratory membrane biophysics.

背景：20世纪90年代，为遵守关于温室气体的《蒙特利尔条约》，用氢氟烷烃重新配制加压计量吸入器时，在一些产品中添加了甘油作为非挥发性成分，以改变气溶胶颗粒大小。最近发现，在吸入器配方中添加甘油以增加气溶胶颗粒大小可能会影响药物的生物利用度，即使药物气溶胶在空气动力学上是等效的。以前的研究比较了不含甘油和含甘油的配方，观察到这些配方在药物特性和性能上的差异。据推测，甘油对沉积药物处置的影响超出了颗粒大小的变化。甘油不仅对气溶胶颗粒的药物释放有影响，它还可能在短暂地改变肺细胞膜的通透性方面起作用。本研究的目的是为了更深入地了解吸入药物颗粒中甘油对气道上皮通透性的影响。这些影响将在体外使用Calu-3细胞连续细胞系进行研究。具体目的包括评价Calu-3细胞对甘油的耐受性。耐受性研究的结果将用于建立一个浓度范围，以便在体外使用运输标记物进一步研究甘油对细胞层通透性的影响。方法：采用MTT法测定甘油对细胞的毒性，并测定LC50值。甘油浓度在1%至35% v/v之间。通过跨上皮电阻（TEER）测量来研究甘油对细胞层完整性的影响。两种转运标志物的细胞层通透性；还评估了3h -地高辛和14c -甘露醇（分别为跨细胞和细胞旁运输标志物）。正如MTT研究报告的那样，为渗透性研究选择的甘油浓度使细胞存活率在60%到100%之间。结果：测定LC50值为14% v/v。TEER值显示，随着甘油浓度的增加和甘油暴露时间的延长，紧密连接受到干扰。甘油也增强了这两种标记物在细胞层间的通透性（甘露醇为24倍，地高辛为6倍）。这在甘露醇中是预料之中的，特别是因为构成细胞旁通路的紧密连接已经受损。结论：测定了Calu-3细胞对甘油的LC50，用浓度范围测定了Calu-3细胞对甘油的耐受程度。甘油似乎还能增强细胞单层对运输标记物的渗透性。研究BDP转运的最佳条件，以及观察到的甘油对膜通透性的影响是否转化为对肺组织中药物转运的影响，还需要进一步的研究。

Strategi Research Priority: Bioscience for HealthBackground: 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.