Time-dependent effects of hydrophobic amine-containing drugs on lysosome structure and biogenesis in cultured human fibroblasts.

Many weakly basic amine-containing drugs are known to be extensively sequestered in acidic lysosomes by an ion trapping-type mechanism. The entrapment of drugs in lysosomes has been shown to influence drug activity, cancer cell selectivity, and pharmacokinetics and can cause the hyperaccumulation of various lipids associated with lysosomes. In this work, we have investigated the prolonged time-dependent effects of drugs on lysosomal properties.

Amine-containing molecules and the induction of an expanded lysosomal volume phenotype: a structure-activity relationship study.

Many weakly basic amine-containing compounds have a strong propensity to become highly concentrated in lysosomes by virtue of an ion-trapping-type mechanism; the substrates for this are referred to as lysosomotropic. We have previously shown that many lysosomotropic drugs can produce a significant expansion in the apparent volume of lysosomes, which can ultimately result in an intracellular distribution-based drug-drug interaction. In this study, we have systematically evaluated the physicochemical and structural features of weakly basic molecules that correlate with their ability to induce an expanded lysosomal volume phenotype (ELVP) in cultured human fibroblasts.

Evaluating the roles of autophagy and lysosomal trafficking defects in intracellular distribution-based drug-drug interactions involving lysosomes.

Many currently approved drugs possess weakly basic properties that make them substrates for extensive sequestration in acidic intracellular compartments such as lysosomes through an ion trapping-type mechanism. Lysosomotropic drugs often have unique pharmacokinetic properties that stem from the extensive entrapment in lysosomes, including an extremely large volume of distribution and a long half-life. Accordingly, pharmacokinetic drug-drug interactions can occur when one drug modifies lysosomal volume such that the degree of lysosomal sequestration of secondarily administered drugs is significantly altered.

Lysosomotropic properties of weakly basic anticancer agents promote cancer cell selectivity in vitro.

Drug distribution in cells is a fundamentally important, yet often overlooked, variable in drug efficacy. Many weakly basic anticancer agents accumulate extensively in the acidic lysosomes of normal cells through ion trapping. Lysosomal trapping reduces the activity of anticancer drugs, since anticancer drug targets are often localized in the cell cytosol or nucleus.

Drug-drug interactions involving lysosomes: mechanisms and potential clinical implications.

Introduction: Many commercially available, weakly basic drugs have been shown to be lysosomotropic, meaning they are subject to extensive sequestration in lysosomes through an ion trapping-type mechanism. The extent of lysosomal trapping of a drug is an important therapeutic consideration because it can influence both activity and pharmacokinetic disposition. The administration of certain drugs can alter lysosomes such that their accumulation capacity for co-administered and/or secondarily administered drugs is altered.

Cationic amphiphilic drugs cause a marked expansion of apparent lysosomal volume: implications for an intracellular distribution-based drug interaction.

How a drug distributes within highly compartmentalized mammalian cells can affect both the activity and pharmacokinetic behavior. Many commercially available drugs are considered to be lysosomotropic, meaning they are extensively sequestered in lysosomes by an ion trapping-type mechanism. Lysosomotropic drugs typically have a very large apparent volume of distribution and a prolonged half-life in vivo, despite minimal association with adipose tissue.

Intracellular Distribution-based Anticancer Drug Targeting: Exploiting a Lysosomal Acidification Defect Associated with Cancer Cells.

The therapeutic usefulness of anticancer agents relies on their ability to exert maximal toxicity to cancer cells and minimal toxicity to normal cells. The difference between these two parameters defines the therapeutic index of the agent. Towards this end, much research has focused on the design of anticancer agents that have optimized potency against a variety of cancer cell types; however, much less effort is spent on the design of drugs that are minimally toxic to normal cells.

Mechanisms of amine accumulation in, and egress from, lysosomes.

The human body is continuously exposed to small organic molecules containing one or more basic nitrogen atoms. Many of these are endogenous (i.e.

Uptake, distribution and diffusivity of reactive fluorophores in cells: implications toward target identification.

There is much recent interest in the application of copper-free click chemistry to study a wide range of biological events in vivo and in vitro. Specifically, azide-conjugated fluorescent probes can be used to identify targets which have been modified with bioorthogonal reactive groups. For intracellular applications of this chemistry, the structural and physicochemical properties of the fluorescent azide become increasingly important.

The role of lysosomes in limiting drug toxicity in mice.

The distribution behavior of a drug within a cell is an important, yet often overlooked, variable in both activity and differential selectivity. In normal cells, drugs with weakly basic properties are known to be extensively compartmentalized in acidic organelles such as lysosomes via ion trapping. Several cancer cell lines have been shown to have defective acidification of endocytic organelles and therefore have a diminished capacity to sequester such lysosomotropic agents.

Niemann-Pick C1 functions independently of Niemann-Pick C2 in the initial stage of retrograde transport of membrane-impermeable lysosomal cargo.

The rare neurodegenerative disease Niemann-Pick Type C (NPC) results from mutations in either NPC1 or NPC2, which are membrane-bound and soluble lysosomal proteins, respectively. Previous studies have shown that mutations in either protein result in biochemically indistinguishable phenotypes, most notably the hyper-accumulation of cholesterol and other cargo in lysosomes. We comparatively evaluated the kinetics of [(3)H]dextran release from lysosomes of wild type, NPC1, NPC2, and NPC1/NPC2 pseudo-double mutant cells and found significant differences between all cell types examined.

cIBR effectively targets nanoparticles to LFA-1 on acute lymphoblastic T cells.

Leukocyte function associated antigen-1 (LFA-1) is a primary cell adhesion molecule of leukocytes required for mediating cellular transmigration into sites of inflammation via the vascular endothelium. A cyclic peptide, cIBR, possesses high affinity for LFA-1, and conjugation to the surface of poly(DL-lactic-co-glycolic acid) nanoparticles can specifically target and deliver the encapsulated agents to T cells expressing LFA-1. The kinetics of targeted nanoparticle uptake by acute lymphoblastic leukemia T cells was investigated by flow cytometry and microscopy and compared to untargeted nanoparticles.

Effect of modification of the physicochemical properties of ICAM-1-derived peptides on internalization and intracellular distribution in the human leukemic cell line HL-60.

The objective of this work is to test the hypothesis that increasing the hydrophilicity of DOX-peptide conjugates may modify their entry mechanisms into HL-60 cells from passive diffusion to receptor-mediated uptake. To test this hypothesis, the entry mechanisms and the intracellular disposition of DOX-cIBR7, DOX-PEGcIBR7, FITC-cIBR, and FITC-cIBR7 were evaluated in HL-60 cells. To increase the hydrophilicity of the peptide, the cIBR peptide (cyclo(1,12)Pen-PRGGSVLVTGC) was modified to cIBR7 peptide (cyclo(1,8)CPRGGSVC) by removing the hydrophobic residues at the C-terminus.

Niemann-Pick C1 functions in regulating lysosomal amine content.

Mutations in the late endosomal/lysosomal membrane protein Niemann-Pick C1 (NPC1) are known to cause a generalized block in retrograde vesicle-mediated transport, resulting in the hyper-accumulation of multiple lysosomal cargos. An important, yet often overlooked, category of lysosomal cargo includes the vast array of small molecular weight amine-containing molecules that are substrates for ion trapping in the highly acidic organelle lumen. We show here that the introduction of amine-containing molecules in lysosomes can significantly stimulate NPC1-mediated late endosome/lysosome fusion, and subsequently the secretion of lysosomal cargo.

Assessment of golgi apparatus versus plasma membrane-localized multi-drug resistance-associated protein 1.

Traditionally, proteins belonging to the ATP-binding cassette superfamily have been thought to function exclusively at the plasma membrane (PM) of cells. We have previously shown multidrug resistance-associated protein 1 (MRP1) to reside on the Golgi apparatus of the multidrug resistant (MDR) human leukemic cell line HL-60 (HL-60/ADR); however, neither the prevalence of this abnormal localization nor the functionality of the transporter at the Golgi has been thoroughly addressed. To assess the functionality of MRP1, with respect to its localization in the cell, we transfected MRP1-deficient HeLa cells with an MRP1-enhanced green fluorescent protein (MRP1-EGFP) plasmid.

Mechanism of internalization of an ICAM-1-derived peptide by human leukemic cell line HL-60: influence of physicochemical properties on targeted drug delivery.

Peptide-mediated targeted delivery offers an attractive strategy for selective delivery of cytotoxic drugs to cancer cells. In this work, we have investigated the mechanism of internalization of cIBR peptide [cyclo(1,12)PenPRGGSVLVTGC] that is conjugated with fluorescein isothiocyanate (FITC) and doxorubicin (DOX) to give FITC-cIBR and DOX-cIBR conjugates, respectively. Internalization mechanisms of FITC-cIBR and DOX-cIBR were studied in LFA-1-expressing cells (HL-60) and LFA-1-deficient cells (HUVEC) under the following conditions: (a) at two different temperatures (4 and 37 degrees C), (b) in the presence of ATP-depleting agents (sodium azide and 2-deoxy- d-glucose), and (c) in the presence of a microtubule-disrupting agent (nocodazole).

Lysosomes contribute to anomalous pharmacokinetic behavior of melanocortin-4 receptor agonists.

Purpose: A series of melanocortin-4 receptor (MC4R) agonists, developed for use as anti-obesity agents, were found to have unusual pharmacokinetic behavior arising from excessive retention in the liver, with nearly undetectable levels in plasma following oral administration in mice. This work investigates the molecular basis of the prolonged liver retention that provided a rational basis for the design of an analog with improved behavior.

Materials And Methods: The livers of mice were harvested and techniques were utilized to fractionate them into pools differentially enriched in organelles.

Exposure of cells to hydrogen peroxide can increase the intracellular accumulation of drugs.

One of the fastest growing areas of scientific research involves aspects of oxidative stress, either causes of or results from. Despite the enormous quantity of literature on the topic, surprisingly, the effects of oxidative stress on the pharmacokinetics of drugs have not been previously investigated. This is an extremely important concern, considering that the degree of oxidative stress that the human body experiences is known to be widely variable.

A new approach for enhancing differential selectivity of drugs to cancer cells.

The degree to which anticancer agents selectively target cancer cells is a key determinant in successful therapeutic outcomes. Inhibitors of the Hsp90 molecular chaperone represent an important new class of anticancer agents. We propose here a novel mechanism by which physiochemical properties of Hsp90 inhibitors can be optimized to increase selectivity towards cancer cells.

Lysosomal sequestration of amine-containing drugs: analysis and therapeutic implications.

Amine-containing drugs represent a very important class of therapeutic agents, with the majority of all drugs containing at least one basic nitrogen. For many decades, it has been known that weakly basic compounds can be sequestered into acidic organelles such as lysosomes. Some amines can achieve very high concentrations and induce a dramatic expansion (vacuolization) of the compartment.

A novel assay reveals that weakly basic model compounds concentrate in lysosomes to an extent greater than pH-partitioning theory would predict.

Many weakly basic drugs incubated with cells have been shown to specifically accumulate in lysosomes. The mechanistic basis and substrate specificity for this sequestration have not been rigorously evaluated; however, conditions are favorable for a pH-partitioning type accumulation. In some circumstances, this compartmentalization can be very extensive, which can impact the therapeutic efficacy of a drug.

A chemical strategy to manipulate the intracellular localization of drugs in resistant cancer cells.

A number of multidrug-resistant (MDR) cancer cells have been shown to have acquired an increased capacity to sequester weakly basic anticancer drugs in their lysosomes relative to drug-sensitive counterparts. In this report we have comparatively evaluated the concentrations of the anticancer agent daunorubicin (DNR) in intracellular compartments of drug-sensitive and MDR HL-60 cell lines, both of which do not express common efflux transporters such as P-glycoprotein at the plasma membrane. Our results suggest that lysosomal sequestration plays a significant role in the emergence of MDR since it effectively limits the drug's ability to interact with target molecules located in the nucleus.

Niemann-Pick C1 protein facilitates the efflux of the anticancer drug daunorubicin from cells according to a novel vesicle-mediated pathway.

Niemann-Pick C1 (NPC1) is a late endosomal/lysosomal membrane protein originally reported on for its role in cholesterol trafficking in mammalian cells. NPC1 has been shown recently to share significant structural homology with a family of prokaryotic permeases and was proposed to play a role in intracellular drug transport; however, the mechanism for this has not been fully understood. We provide evidence here that is consistent with NPC1's involvement in a vesicle-mediated clearance of the anticancer agent daunorubicin from cells.

Intracellular drug sequestration events associated with the emergence of multidrug resistance: a mechanistic review.

The acquisition of multi-drug resistance (MDR) in cancer cells subjected to anticancer agents remains a formidable obstacle to successful therapeutic outcomes in cancer patients. As the name implies, the resistance phenotype (MDR) is not typically limited to the drug initially used to eradicate cancer but is often transferred to structurally unrelated chemotherapeutic agents. The mechanisms underlying the development of MDR have been extensively studied and are considered multifactorial.