title: In Vitro and in Silico Tools To Assess Extent of Cellular Uptake and Lysosomal Sequestration of Respiratory Drugs in Human Alveolar Macrophages.
abstract: Accumulation of respiratory drugs in human alveolar macrophages (AMs) has not been extensively studied in vitro and in silico despite its potential impact on therapeutic efficacy and/or occurrence of phospholipidosis. The current study aims to characterize the accumulation and subcellular distribution of drugs with respiratory indication in human AMs and to develop an in silico mechanistic AM model to predict lysosomal accumulation of investigated drugs. The data set included 9 drugs previously investigated in rat AM cell line NR8383. Cell-to-unbound medium concentration ratio (Kp,cell) of all drugs (5 μM) was determined to assess the magnitude of intracellular accumulation. The extent of lysosomal sequestration in freshly isolated human AMs from multiple donors (n = 5) was investigated for clarithromycin and imipramine (positive control) using an indirect in vitro method (±20 mM ammonium chloride, NH4Cl). The AM cell parameters and drug physicochemical data were collated to develop an in silico mechanistic AM model. Three in silico models differing in their description of drug membrane partitioning were evaluated; model (1) relied on octanol-water partitioning of drugs, model (2) used in vitro data to account for this process, and model (3) predicted membrane partitioning by incorporating AM phospholipid fractions. In vitro Kp,cellranged >200-fold for respiratory drugs, with the highest accumulation seen for clarithromycin. A good agreement in Kp,cellwas observed between human AMs and NR8383 (2.45-fold bias), highlighting NR8383 as a potentially useful in vitro surrogate tool to characterize drug accumulation in AMs. The mean Kp,cellof clarithromycin (81, CV = 51%) and imipramine (963, CV = 54%) were reduced in the presence of NH4Cl by up to 67% and 81%, respectively, suggesting substantial contribution of lysosomal sequestration and intracellular binding in the accumulation of these drugs in human AMs. The in vitro data showed variability in drug accumulation between individual human AM donors due to possible differences in lysosomal abundance, volume, and phospholipid content, which may have important clinical implications. Consideration of drug-acidic phospholipid interactions significantly improved the performance of the in silico models; use of in vitro Kp,cellobtained in the presence of NH4Cl as a surrogate for membrane partitioning (model (2)) captured the variability in clarithromycin and imipramine Kp,cellobserved in vitro and showed the best ability to predict correctly positive and negative lysosomotropic properties. The developed mechanistic AM model represents a useful in silico tool to predict lysosomal and cellular drug concentrations based on drug physicochemical data and system specific properties, with potential application to other cell types.
authors.affiliation: Centre for Applied Pharmacokinetic Research, School of Health Sciences, University of Manchester , Manchester, U.K.
authors.forename: Ayşe
authors.initials: A
authors.lastname: Ufuk
authors.affiliation: Centre for Applied Pharmacokinetic Research, School of Health Sciences, University of Manchester , Manchester, U.K.
authors.forename: Frauke
authors.initials: F
authors.lastname: Assmus
authors.affiliation: Centre for Applied Pharmacokinetic Research, School of Health Sciences, University of Manchester , Manchester, U.K.
authors.forename: Laura
authors.initials: L
authors.lastname: Francis
authors.affiliation: Respiratory and Allergy Clinical Research Facility, University Hospital of South Manchester , Manchester, U.K.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Plumb
authors.affiliation: Computational Modeling Sciences, DDS, GlaxoSmithKline , Upper Merion, Pennsylvania 19406, United States.
authors.forename: Valeriu
authors.initials: V
authors.lastname: Damian
authors.affiliation: Centre for Applied Pharmacokinetic Research, School of Health Sciences, University of Manchester , Manchester, U.K.|Pharmaceutical Sciences, pRED, Roche Innovation Center , Basel, Switzerland.
authors.forename: Michael
authors.initials: M
authors.lastname: Gertz
authors.affiliation: Centre for Applied Pharmacokinetic Research, School of Health Sciences, University of Manchester , Manchester, U.K.
authors.forename: J Brian
authors.initials: JB
authors.lastname: Houston
authors.affiliation: Centre for Applied Pharmacokinetic Research, School of Health Sciences, University of Manchester , Manchester, U.K.
authors.forename: Aleksandra
authors.identifier: 0000-0002-3933-5217
authors.initials: A
authors.lastname: Galetin
mesh_terms: D000368:Aged; D000818:Animals; D002460:Cell Line; D017291:Clarithromycin / Q000008:administration & dosage; D003198:Computer Simulation; D005260:Female; D006801:Humans; D007099:Imipramine / Q000008:administration & dosage; D008247:Lysosomes / Q000378:metabolism*; D016676:Macrophages, Alveolar / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D004364:Pharmaceutical Preparations / Q000008:administration & dosage*; D010743:Phospholipids / Q000378:metabolism; D051381:Rats; D014018:Tissue Distribution
chemical_list: D004364:Pharmaceutical Preparations; D010743:Phospholipids; D017291:Clarithromycin; D007099:Imipramine
keywords: NR8383; drug accumulation; human alveolar macrophages; in silico cell model; lysosomal sequestration