Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.799235
Title: Drug accumulation in alveolar macrophages : in vitro and in silico assessment of the contribution of lysosomal trapping
Author: Ufuk, Ayse
ISNI:       0000 0004 8510 1555
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2015
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Abstract:
Alveolar macrophages (AMs) are involved in phagocytosis and clearance of inhaled particles including drugs from the lungs which may result in lack of efficacy of new drug candidates. In addition, drug accumulation in AMs is associated with safety concerns due to formation of foamy AMs which can occur as a consequence of "trapping" of cationic amphiphilic drugs in the lysosomes (e.g., in phospholipidosis). Currently minimal in vitro data exist on accumulation and lysosomal trapping of respiratory drugs in AMs. Ten drugs were selected for the present project, namely clarithromycin, rifampicin, ciprofloxacin, formoterol, fenoterol, terbutaline, budesonide, ipratropium and tiotropium bromide and imipramine (positive control for lysosomal trapping). Rationale for inclusion was that these were respiratory drugs with a wide range of physicochemical properties and therapeutic classes, have a potential for lysosomal accumulation and limited in vitro accumulation data reported. The aims of this project were to characterise the accumulation of selected drugs in rat AM cell line NR8383 and primary human AMs and investigate the contribution of lysosomal trapping to their cellular accumulation. Total uptake clearance (CLuptake) of drugs was assessed up to 10 minutes at 5 µM drug concentration at 37 and 4°C to delineate active uptake (CLactive) and passive diffusion (CLdiff) clearances. Cell accumulation was determined from cell-to-medium concentration ratios (Kp) at 37°C. Lysosomal trapping of drugs was evaluated by the reduction in CLuptake and Kp of drugs in the presence of NH4Cl, monensin and nigericin which abolish intracellular pH gradients. Wide range of CLuptake in both NR8383 (0.04 - 16.7 µL/min/106 cells) and human AMs (0.02 - 66.2 µL/min/106 cells) was evident; the lowest and highest values were observed for terbutaline and imipramine, respectively. Uptake of most drugs was driven by an active process as the CLactive/CLdiff ratio ranged between 2 and >150-fold for imipramine and clarithromycin, respectively in NR8383. Similar trends were seen in human AMs where this ratio ranged between 2 and >500-fold for tiotropium bromide and clarithromycin, respectively. Comparison of CLuptake and Kp data revealed a 3-fold bias between NR8383 and human AMs; in general values were higher in NR8383. Among all drugs, imipramine and clarithromycin (LogP >3, pKa 9-9.5) were shown to significantly accumulate in the lysosomes of both cells; overall >60% reduction in CLuptake and Kp was observed by the chemical agents. Overall, NR8383 was shown to be a useful in vitro model to study accumulation and lysosomal trapping of drugs. Lysosomal trapping of investigated drugs was further assessed using a published in silico mechanistic cell model which was modified for AMs. Drug physicochemical data (logP, pKa) and AM specific parameters where available (e.g., lysosomal volume, pH) were used to predict the extent of lysosomal trapping based on the pH differences between external medium, cytosol and lysosome. Cell and lysosomal drug concentrations were predicted, allowing the estimation of Kp for these compartments. The predictive performance of the model was evaluated by comparing the predicted Kp,cell with experimental Kp,NR8383. An overall 7-fold bias was observed; Kp,NR8383 was under-predicted by 2.4 to approximately 14-fold for half of the drugs investigated. However, the model could correctly predict lysosomal trapping of basic clarithromycin and imipramine, and lysosomal non-targeting of the majority of basic, zwitterionic, neutral and positively charged drugs. The strengths and the limitations of the in silico model are highlighted, the areas for improvements identified and recommendations made for future applications of this approach to predict subcellular accumulation.
Supervisor: Houston, James ; Galetin, Aleksandra Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.799235  DOI: Not available
Keywords: alveolar macrophages ; NR8383 ; lysosomal trapping ; drug accumulation
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