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

战略研究重点：生物科学促进健康背景：甘油作为一种非挥发性成分被添加到一些产品中，以改变气溶胶颗粒尺寸，当时加压计量吸入器在20世纪90年代被重新配制为氢氟烷烃，以遵守关于温室气体的蒙特利尔条约。最近发现，在吸入器配方中添加甘油以增加气雾剂颗粒尺寸可能会影响药物的生物利用度，即使药物气雾剂在空气动力学上是等效的。以前比较不含甘油和含甘油的制剂的研究已经观察到这些制剂的药学性质和性能的差异。据推测，甘油对药物沉积的影响不仅仅是颗粒大小的变化。甘油不仅可能影响药物从气雾剂颗粒中的释放，它还可能在瞬时改变肺细胞膜的通透性方面发挥作用。这项研究的目的是更深入地了解吸入药物颗粒中的甘油对呼吸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。具体目标包括评估CALU-3细胞对甘油的耐受性。耐受性研究的结果将被用来建立一个浓度范围，为进一步研究甘油对细胞层通透性的影响提供体外转运标志。方法：采用四甲基偶氮唑盐比色法评价甘油对细胞的毒性，并测定LC_(50)值。甘油浓度范围为1%~35%v/v。通过跨上皮电阻(TEER)测量，研究甘油对细胞层完整性的影响。测定了两种转运标志物--~3H-地高辛和~(14)C-甘露醇的细胞层通透性(分别为跨细胞转运和细胞旁转运)。根据四甲基偶氮唑盐的研究，选择的甘油浓度使细胞存活率在60%到100%之间。结果：确定的LC_(50)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志物的细胞层渗透性(甘露醇是24倍，地高辛是6倍)。对于甘露醇来说，这是意料之中的，特别是因为构成细胞旁路的紧密连接已经被破坏。结论：测定了甘油与CALU-3细胞的LC_(50)，毒性试验中使用的浓度范围反映了细胞对甘油的耐受程度。甘油似乎也增强了细胞单层对运输标志物的渗透性。需要进一步的研究来确定BDP转运的最佳条件，以及这种观察到的甘油对膜通透性的影响是否会转化为对肺组织中药物转运的影响。

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.