In Vitro Assessment of Uptake and Lysosomal Sequestration of Respiratory Drugs in Alveolar Macrophage Cell Line NR8383

Ufuk, Ayşe; Somers, Graham; Houston, J. Brian; Galetin, Aleksandra

doi:10.1007/s11095-015-1753-8

Cited by 18 publications

(40 citation statements)

References 54 publications

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“…In Silico Prediction of the Extent of Lysosomal Trapping. For an accurate in silico prediction of lysosomal trapping, comprehensive information on the acidity and the fractional volume of the endo-/ lysosomal system is needed due to tremendous variations between cell types ranging from 0.23% up to 7.8% of the cellular volume (de Duve et al, 1974;Blouin et al, 1977;MacIntyre and Cutler, 1988a;Ufuk et al, 2015) with pH values reported in the range of 4.65-5.18 (Regec et al, 1989;Tietz et al, 1990;Kharbanda et al, 1997). We have, for the first time, determined a full profile of the pH values and corresponding fractional volumes of the endo-/lysosomal system in rat hepatocytes (Fig.…”

Section: Downloaded Frommentioning

confidence: 99%

“…For simplification, all acidic cellular compartments are hereinafter referred to as lysosomes. Despite the typical minor volume of ,2% of the cellular volume (with exceptions in certain cell types, e.g., macrophages of up to 8%) (Ufuk et al, 2015), the contribution to cellular drug uptake can be significant since trapping can theoretically lead to as much as 160,000-fold higher drug concentrations within the lysosome relative to the cytosol (MacIntyre and Cutler, 1988a,b). Besides contributing to the distribution into lysosome-rich tissues, lysosomal sequestration also affects the intracellular localization of drugs.…”

Section: Introductionmentioning

confidence: 99%

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Quantitation of Lysosomal Trapping of Basic Lipophilic Compounds Using In Vitro Assays and In Silico Predictions Based on the Determination of the Full pH Profile of the Endo-/Lysosomal System in Rat Hepatocytes

et al. 2018

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Lysosomal sequestration may affect the pharmacokinetics, efficacy, and safety of new basic lipophilic drug candidates potentially impacting their intracellular concentrations and tissue distribution. It may also be involved in drug-drug interactions, drug resistance, and phospholipidosis. However, currently there are no assays to evaluate the lysosomotropic behavior of compounds in a setting fully meeting the needs of drug discovery. We have, therefore, integrated a set of methods to reliably rank order, quantify, and calculate the extent of lysosomal sequestration in rat hepatocytes. An indirect fluorescence-based assay monitors the displacement of the fluorescence probe LysoTracker Red by test compounds. Using a lysosomal-specific evaluation algorithm allows one to generate IC 50 values at lower than previously reported concentrations. The concentration range directly agrees with the concentration dependency of the lysosomal drug content itself directly quantified by liquid chromatography-tandem mass spectrometry and thus permits a quantitative link between the indirect and the direct trapping assay. Furthermore, we have determined the full pH profile and corresponding volume fractions of the endo-/lysosomal system in plated rat hepatocytes, enabling a more accurate in silico prediction of the extent of lysosomal trapping based only on pK a values as input, allowing early predictions even prior to chemical synthesis. The concentration dependency-i.e., the saturability of the trappingcan then be determined by the IC 50 values generated in vitro. Thereby, a more quantitative assessment of the susceptibility of basic lipophilic compounds for lysosomal trapping is possible. This work was financed by Bayer AG, Germany. https://doi.org/10.1124/dmd.118.084541. s This article has supplemental material available at dmd.aspetjournals.org. ABBREVIATIONS: DAPI -49,6-diamidino-2-phenylindole; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LTR, LysoTracker Red DND-99; PBS, phosphate-buffered saline. 49

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Section: Downloaded Frommentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Quantitation of Lysosomal Trapping of Basic Lipophilic Compounds Using In Vitro Assays and In Silico Predictions Based on the Determination of the Full pH Profile of the Endo-/Lysosomal System in Rat Hepatocytes

et al. 2018

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“…29 Interestingly, clarithromycin, a macrolide antibiotic, is not amphiphilic, but behaves like a cationic amphiphilic drug in that it causes phospholipidosis because it is sequestered in lysosomes. 77 Lysosomal sequestration occurs when a hydrophobic weak base enters the lysosome, is protonated in the acid environment, and is unable to cross the membrane. 38 Results of a large-scale gene expression analysis performed in human hepatoma Hep G2 cells with 12 compounds known to cause phospholipidosis suggest that several processes might be involved.…”

Section: Phospholipidosismentioning

confidence: 99%

Drug-induced corneal epithelial changes

Raizman

Hamrah

Holland

et al. 2017

Survey of Ophthalmology

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Drugs across many pharmacologic classes induce corneal epithelial changes. Many of these drugs have cationic amphiphilic structures, with a hydrophobic ring and hydrophilic cationic amine side chain that allow them to cross cell membranes. These drugs lead to intracellular phospholipid accumulation, often manifested in the cornea by vortex keratopathy, with no effect on visual acuity and few ocular symptoms. Other drugs, notably antineoplastic agents, produce a fine diffuse corneal haze, sometimes accompanied by decreased vision that can be dose limiting. Still other medications cause crystalline epithelial precipitation that might require debridement for resolution. An understanding of the variety of drugs involved, the multiple mechanisms responsible, and the systemic diseases that produce similar changes can lead to improved management strategies for patients with corneal epithelial deposits. In most cases, drug therapy need not be modified or discontinued, but if visual acuity is affected, close collaboration with the prescribing physician can result in determining an optimized dose that treats systemic disease and minimizes these deposits. Additionally, close monitoring might be required if the drug is also associated with other ocular findings, such as optic neuropathy or retinopathy.

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“…29 Also, half of the terbutaline uptake into human alveolar macrophages is mediated by active transport. 49 However, apical uptake transporter activity in the intestine has not been reported. The experimental profile in Figure 6E is consistent with the report that absorption occurs primarily in the jejunum 50 and not consistent with the low passive permeability of terbutaline ( P app = 0.47 × 10 −6 cm s −1 ).…”

Section: Discussionmentioning

confidence: 99%

Continuous Intestinal Absorption Model Based on the Convection–Diffusion Equation

Nagar

Korzekwa

2017

Mol. Pharmaceutics

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Prediction of the rate and extent of drug absorption upon oral dosing needs models that capture the complexities of both the drug molecule and intestinal physiology. We report here the development of a continuous intestinal absorption model based on the convection–diffusion equation. The model includes explicit enterocyte apical membrane and intracellular lipid radial compartments along the length of the intestine. Physiologic functions along length x are built into the model and include velocity, diffusion, surface areas, and pH of the intestine. Also included are expression levels of the intestinal active uptake transporter OATP2B1 and efflux transporter P-gp. Oral dosing of solution as well as solid (with a dissolution function) was modeled for several drugs. The fraction absorbed (FA) and concentration–time (C–t) profiles were predicted and compared with clinical data. Overall, FA was well predicted upon oral (n = 21) or colonic dosing (n = 11), with four outliers. The overall accuracy (prediction of the correct bin) was 81% with outliers and 90% without outliers. Of the nine solution dosing data sets, six drugs were very well predicted with an exposure overlap coefficient (EOC) > 0.9 and predicted Cmax and Tmax values similar to those observed. Of the six solid dose formulations evaluated, the EOC values were > 0.9 for all drugs except budesonide. The observed precipitation of nifedipine at high doses was predicted by the model. Most of the poor predictions were for drugs that are known to be transporter substrates. As proof of concept, incorporating OATP2B1 and P-gp markedly improved the EOC and predicted Cmax and Tmax for fexofenadine. Finally, the continuous intestinal model accurately recapitulated the known relationships between drug absorption and permeability, solubility, and particle size. Together, these results indicate that this preliminary intestinal absorption model offers a simple and straightforward framework to build in complexities such as drug permeability, lipid partitioning, solubility, metabolism, and transport for improved prediction of the rate and extent of drug absorption.

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In Vitro Assessment of Uptake and Lysosomal Sequestration of Respiratory Drugs in Alveolar Macrophage Cell Line NR8383

Cited by 18 publications

References 54 publications

Quantitation of Lysosomal Trapping of Basic Lipophilic Compounds Using In Vitro Assays and In Silico Predictions Based on the Determination of the Full pH Profile of the Endo-/Lysosomal System in Rat Hepatocytes

Quantitation of Lysosomal Trapping of Basic Lipophilic Compounds Using In Vitro Assays and In Silico Predictions Based on the Determination of the Full pH Profile of the Endo-/Lysosomal System in Rat Hepatocytes

Drug-induced corneal epithelial changes

Continuous Intestinal Absorption Model Based on the Convection–Diffusion Equation

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