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In vitro assessment of drug sequestration into lysosomes and in silico prediction of implications for drug disposition and therapeutic efficacy


Project Description

Intracellular concentration of a drug is an important determinant of its efficacy, toxicity, and potential drug-drug interactions [1]. Cationic amphiphilic drugs can accumulate in the lysosome organelle due to its acidic pH which facilitates a high degree of ionization, trapping drugs because of limited diffusion of the ionized form back into the cytosol [2,3]. This can result in significantly higher lysosomal drug concentration in comparison to the cytosol with potentially important therapeutic consequence for many clinically used basic drugs; lysosomal accumulation may be either a prerequisite for therapeutic efficacy (e.g. antimalarial drugs) or may lead to phospholipidosis [4]. Over the years, our laboratory has investigated lysosomal sequestration in hepatocytes and alveolar macrophages [3, 5]; current projects involve isolation of lysosomes for direct assessment of this phenomenon [3,5].

The aim of the project is to provide novel quantitative in vitro and in silico assessment of lysosomal sequestration and its impact on unbound drug concentration in the liver and lung, including detailed characterisation and quantification of sequestration mechanisms. Immortalised human hepatocytes (e.g., Fa2N4), alveolar macrophages and isolated lysosomes from these (using in-house methodology) will be used as experimental cell/subcellular systems. Lysosomal sequestration of a range of drugs with different physicochemical properties and with reported evidence of phospholipidosis will be considered for the analysis.

The project will also have a substantial mechanistic modelling component to complement the existing in house data and data yet to be generated experimentally. The project will apply and further refine existing in-house mechanistic in silico cell model (set up in MATLAB software), which enables prediction of drug subcellular distribution and accumulation using specific cell parameters (e.g., lysosomal volume) and relevant drug physicochemical properties (logP, pKa); lipid partitioning and electrostatic interactions are also accounted for. The current experimental approach combined with modelling aims to develop a novel strategy in predicting cellular and lysosomal drug concentrations and implications on therapeutic efficacy and target engagement.

The successful candidate will be trained to perform cell isolation and cell culture techniques developed in-house to provide cells for in vitro studies. In addition, isolation of lysosomes using a novel in-house technique will be performed. Experimental work will also include design and planning of in vitro drug kinetic studies, preparation of relevant reagents together with quantification of drug in experimental samples by LC-MS/MS.

In silico work will involve kinetic analysis of data computational software packages such as MATLAB to further refine existing in silico cell models.

Throughout the PhD project, the student will be provided with opportunities to (and be expected to) develop a high level of presentation of their work, both orally and in writing, for both internal and external audiences. Publications in leading peer reviewed journals are expected.

We are looking for enthusiastic and motivated students that hold (or expect to obtain) a minimum 2.1 degree (or equivalent) in pharmacology, biochemistry, or a related chemistry/biological science area. A strong mathematical background is essential. Previous experience of in vitro systems (e.g., hepatocytes) or cell culturing and mathematical modelling would be an advantage.

Funding Notes

This project has a Band 3 fee. Details of our different fee bands can be found on our website (View Website). For information on how to apply for this project, please visit the Faculty of Biology, Medicine and Health Doctoral Academy website (View Website). Informal enquiries may be made directly to the primary supervisor.

References

1. Chu X, Korzekwa KR, Elsby R, Fenner KS, Galetin A, Lai Y, et al. Intracellular Drug Concentrations and Transporters: Measurement, Modeling and Implications in the Liver. Clin Pharmacol Ther. 94, 126-141 (2013).
2. Kazmi F, Hensley T, Pope C, Funk RS, Loewen GJ, Buckley DB, et al. Lysosomal Sequestration (Trapping) of Lipophilic Amine (Cationic Amphiphilic) Drugs in Immortalized Human Hepatocytes (Fa2N-4 cells). Drug Metab Dispos. 41(4), 897-905 (2013).
3. Hallifax D and Houston JB. Saturable uptake of lipophilic amine drugs into isolated hepatocytes: mechanisms and consequences for quantitative clearance prediction. Drug Metab Dispos. 35, 1325-1332 (2007).
4. Logan R, Funk RS, Axcell E and Krise JP. Drug-drug interactions involving lysosomes: mechanisms and potential clinical implications. Expert Opin Drug Metab Toxicol. 8, 943-958 (2012).
5. Ufuk A, Somers G, Houston JB and Galetin A. In Vitro Assessment of Uptake and Lysosomal Sequestration of Respiratory Drugs in Alveolar Macrophage Cell Line NR8383. Pharm Res 32(12):3937-51 (2015).

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