Jeffrey P. Krise

Prodrugs of phosphates, phosphonates, and phosphinates

Abstract The objective of this paper is to review the literature on the use of prodrugs to overcome the drug delivery obstacles associated with phosphate, phosphonate and phosphinate functional group-containing drugs. This is an important area of research because, although we have been successful at identifying numerous phosphate and phosphonate functional group-containing drugs as antiviral and anticancer agents, as well as for other uses, our ability to orally deliver these drugs and to target them to desired sites has led to limited success. Various acyloxymethyl- and aryl-ester prodrugs have shown promise. Alternative and imaginative approaches may be necessary before complete success is realized. It is our hope that this review will stimulate further innovative prodrug research into overcoming the barriers to the delivery of these important drugs.

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 [3H]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. Specifically, NPC1 or NPC2 mutant fibroblasts treated with NPC1 or NPC2 siRNA (to create NPC1/NPC2 pseudo-double mutants) secreted dextran less efficiently than did either NPC1 or NPC2 single mutant cell lines, suggesting that the two proteins may work independently of one another in the egress of membrane-impermeable lysosomal cargo. To investigate the basis for these differences, we examined the role of NPC1 and NPC2 in the retrograde fusion of lysosomes with late endosomes to create so-called hybrid organelles, which is believed to be the initial step in the egress of cargo from lysosomes. We show here that cells with mutated NPC1 have significantly reduced rates of late endosome/lysosome fusion relative to wild type cells, whereas cells with mutations in NPC2 have rates that are similar to those observed in wild type cells. Instead of being involved in hybrid organelle formation, we show that NPC2 is required for efficient membrane fission events from nascent hybrid organelles, which is thought to be required for the reformation of lysosomes and the release of lysosomal cargo-containing membrane vesicles. Collectively, these results suggest that NPC1 and NPC2 can function independently of one another in the egress of certain membrane-impermeable lysosomal cargo.

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., neurotransmitters, polyamines and biogenic amines), while others are exogenously supplied in the form of drugs, foods and pollutants. It is well-known that many amines have a strong propensity to specifically and substantially accumulate in highly acidic intracellular compartments, such as lysosomes, through a mechanism referred to as ion trapping. It is also known that cells have acquired the unique ability to sense and respond to amine accumulation in lysosomes in an effort to prevent potential negative consequences associated with hyperaccumulation. We describe here methods that are used to evaluate the dynamics of amine accumulation in, and egress from, lysosomes. Moreover, we highlight specific proteins that are thought to play important roles in these pathways. A theoretical model describing lysosomal amine dynamics is described and shown to adequately fit experimental kinetic data. The implications of this research in understanding and treating disease are discussed.

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. In this report we tested the prediction that the accumulation of one drug (perpetrator) in lysosomes could influence the accumulation of a secondarily administered one (victim), resulting in an intracellular distribution-based drug interaction. To test this hypothesis cells were exposed to nine different hydrophobic amine-containing drugs, which included imipramine, chlorpromazine and amiodarone, at a 10 μM concentration for 24 to 48 h. After exposure to the perpetrators the cellular accumulation of LysoTracker Red (LTR), a model lysosomotropic probe, was evaluated both quantitatively and microscopically. We found that all of the tested perpetrators caused a significant increase in the cellular accumulation of LTR. Exposure of cells to imipramine caused an increase in the cellular accumulation of other lysosomotropic probes and drugs including LyosTracker Green, daunorubicin, propranolol and methylamine; however, imipramine did not alter the cellular accumulation of non-lysosomotropic amine-containing molecules including MitoTracker Red and sulforhodamine 101. In studies using ionophores to abolish intracellular pH gradients we were able to resolve ion trapping-based cellular accumulation from residual pH-gradient independent accumulation. Results from these evaluations in conjunction with lysosomal pH measurements enabled us to estimate the relative aqueous volume of lysosomes of cells before and after imipramine treatment. Our results suggest that imipramine exposure caused a 4-fold expansion in the lysosomal volume, which provides the basis for the observed drug interaction. The imipramine-induced lysosomal volume expansion was shown to be both time- and temperature-dependent and reversed by exposing cells to hydroxypropyl-β-cyclodextrin, which reduced lysosomal cholesterol burden. This suggests that the expansion of lysosomal volume occurs secondary to perpetrator-induced elevations in lysosomal cholesterol content. In support of this claim, the cellular accumulation of LTR was shown to be higher in cells isolated from patients with Niemann-Pick type C disease, which are known to hyperaccumulate cholesterol in lysosomes.

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. Ideal fluorophores should extensively accumulate within cells, have even intracellular distribution, and be free (unbound), allowing them to efficiently participate in bimolecular reactions. We report here on the synthesis and evaluation of a set of structurally diverse fluorescent probes to examine their potential usefulness in intracellular click reactions. Total cellular uptake and intracellular distribution profiles were comparatively assessed using both quantitative and qualitative approaches. The intracellular diffusion coefficients were measured using a fluorescence recovery after photobleaching (FRAP)-based method. Many reactive fluorophores exhibited suboptimal properties for intracellular reactions. BODIPY- and TAMRA-based azides had superior cellular accumulation, whereas TAMRA-based probes had the most uniform intracellular distribution and best cytosolic diffusivity. Collectively, these results provide an unbiased comparative evaluation regarding the suitability of azide-linked fluorophores for intracellular click reactions.

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. To illustrate the physiological importance of this NPC1-mediated transport pathway, we show that NPC1-deficient cells are more susceptible to the toxic effects of a lysosomotropic polyamine metabolite 3-aminopropanal. Moreover, NPC fibroblasts are shown to have higher levels of polyamine oxidase, an enzyme involved in the formation of 3-aminopropanal. Collectively, these findings provide strong support for a novel functional role for NPC1 and may also provide clues toward understanding NPC disease progression.

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. In this study, we tested the hypothesis that the low lysosomal pH of normal cells plays an important role in protecting normal tissues from the toxic effects of lysosomotropic anticancer drugs. The influence of lysosomal pH status on the toxicity of inhibitors of the molecular chaperone Hsp90 that did or did not possess lysosomotropic properties was evaluated in mice. Toxicity of Hsp90 inhibitors was evaluated in normal mice and in mice treated with chloroquine to elevate lysosomal pH by assessing morbidity and utilizing biochemical assays to diagnose hepatic and renal toxicity. Toxicity of the lysosomotropic inhibitor 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG) was significantly enhanced in mice with elevated lysosomal pH relative to mice with normal lysosomal pH. In contrast, elevation of lysosomal pH had no significant impact on toxicity of the nonlysosomotropic inhibitor geldanamycin. These results support the notion that the low lysosomal pH of normal cells plays an important role in protecting these cells from the toxic effects of anticancer agents with lysosomotropic properties and has implications for the design/selection of anticancer drugs with improved safety and differential selectivity.

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. Some cancer cells have defective acidification of lysosomes, which causes a redistribution of trapped drugs from the lysosomes to the cytosol. We have previously established that such differences in drug localization between normal and cancer cells can contribute to the apparent selectivity of weakly basic drugs to cancer cells in vitro. In this work, we tested whether this intracellular distribution-based drug selectivity could be optimized based on the acid dissociation constant (pKa) of the drug, which is one of the determinants of lysosomal sequestration capacity. We synthesized seven weakly basic structural analogs of the Hsp90 inhibitor geldanamycin (GDA) with pKa values ranging from 5 to 12. The selectivity of each analog was expressed by taking ratios of anti-proliferative IC50 values of the inhibitors in normal fibroblasts to the IC50 values in human leukemic HL-60 cells. Similar selectivity assessments were performed in a pair of cancer cell lines that differed in lysosomal pH as a result of siRNA-mediated alteration of vacuolar proton ATPase subunit expression. Optimal selectivity was observed for analogs with pKa values near 8. Similar trends were observed with commercial anticancer agents with varying weakly basic pKa values. These evaluations advance our understanding of how weakly basic properties can be optimized to achieve maximum anticancer drug selectivity towards cancer cells with defective lysosomal acidification in vitro. Additional in vivo studies are needed to examine the utility of this approach for enhancing selectivity.

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. Areas covered: In this review the authors explore what is known regarding the mechanistic basis for drug-drug interactions involving lysosomes. Specifically, the authors address the influence of drugs on lysosomal pH, volume and lipid processing. Expert opinion: Many drugs are known to extensively accumulate in lysosomes and significantly alter their structure and function; however, the therapeutic and toxicological implications of this remain controversial. The authors propose that drug-drug interactions involving lysosomes represent an important potential source of variability in drug activity and pharmacokinetics. Most evaluations of drug-drug interactions involving lysosomes have been performed in cultured cells and isolated tissues. More comprehensive in vivo evaluations are needed to fully explore the impact of this drug-drug interaction pathway on therapeutic outcomes.

Lysosomes Contribute to Anomalous Pharmacokinetic Behavior of Melanocortin-4 Receptor Agonists

PurposeA 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 MethodsThe livers of mice were harvested and techniques were utilized to fractionate them into pools differentially enriched in organelles. The distribution of organelles in the fractions was determined using organelle-specific enzymatic assays. Livers from mice dosed with drug were fractionated and comparisons with organelle distributions assisted in determining the subcellular localization of the drug. Further analysis in cell culture systems was used to confirm results from liver fractionation studies and also allowed for more extensive evaluations to examine the mechanism for organelle compartmentalizationResultsFractionation of livers following oral administration of the agonist showed sequestration in lysosomes. Subsequent evaluations in a cell culture system confirmed this finding. Agents used to disrupt acidification of lysosomes led to decreased lysosomal accumulation of the drug, which implicated a pH-partitioning type sequestration mechanism. These findings led to the rational synthesis of an analog of the parent compound with properties that reduced lysosomal sequestration. When this compound was examined in mice, the liver retention was found to be greatly reduced and plasma levels were significantly elevated relative to the parent compound.ConclusionsWeakly basic drugs with optimal physicochemical properties can be extensively sequestered into lysosomes according to a pH-partitioning type mechanism. When administered orally in animals, this particular sequestration event can manifest itself in long term retention in the liver and negligible levels in blood. This work revealed the mechanism for liver retention and provided a rational platform for the design of a new analog with decreased liver accumulation and better opportunity for pharmacokinetic analysis and therapeutic activity.