Drug-induced Phospholipidosis Research Articles

Inhibition of lysosomal phospholipase A2 predicts drug-induced phospholipidosis

Phospholipidosis, the excessive accumulation of phospholipids within lysosomes, is a pathological response observed following exposure to many drugs across multiple therapeutic groups. A clear mechanistic understanding of the causes and implications of this form of drug toxicity has remained elusive. We previously reported the discovery and characterization of a lysosome-specific phospholipase A2 (PLA2G15) and later reported that amiodarone, a known cause of drug-induced phospholipidosis, inhibits this enzyme. Here, we assayed a library of 163 drugs for inhibition of PLA2G15 to determine whether this phospholipase was the cellular target for therapeutics other than amiodarone that cause phospholipidosis. We observed that 144 compounds inhibited PLA2G15 activity. Thirty-six compounds not previously reported to cause phospholipidosis inhibited PLA2G15 with IC50 values less than 1 mM and were confirmed to cause phospholipidosis in an in vitro assay. Within this group, fosinopril was the most potent inhibitor (IC50 0.18 μM). Additional characterization of the inhibition of PLA2G15 by fosinopril was consistent with interference of PLA2G15 binding to liposomes. PLA2G15 inhibition was more accurate in predicting phospholipidosis compared with in silico models based on pKa and ClogP, measures of protonation, and transport-independent distribution in the lysosome, respectively. In summary, PLA2G15 is a primary target for cationic amphiphilic drugs that cause phospholipidosis, and PLA2G15 inhibition by cationic amphiphilic compounds provides a potentially robust screening platform for potential toxicity during drug development.

Analysis of Live Single Cells by Confocal Microscopy and High-Resolution Mass Spectrometry to Study Drug Uptake, Metabolism, and Drug-Induced Phospholipidosis.

The analysis of large numbers of cells from a population results in information that does not reflect differences in cell phenotypes. Individual variations in cellular drug uptake, metabolism, and response to drug treatment may have profound effects on cellular survival and lead to the development of certain disease states, drug persistence, and resistance. Herein, we present a method that combines live cell confocal microscopy imaging with high-resolution mass spectrometry to achieve absolute cell quantification of the drug amiodarone (AMIO) and its major metabolite, N-desethylamiodarone (NDEA), in single liver cells (HepG2 and HepaRG cells). The method uses a prototype system that integrates a confocal microscope with an XYZ stage robot to image and automatically sample selected cells from a sample compartment, which is kept under growth conditions, with nanospray tips. Besides obtaining the distributions of AMIO and NDEA cell concentrations across a population of individual cells, as well as variabilities in drug metabolism, the effect of these on phospholipidosis and cell morphology was studied. The method was suited to identify subpopulations of cells that metabolized less drug and to correlate cell drug concentrations with cell phospholipid content, cell volume, sphericity, and other cell phenotypic features. Using principal component analysis (PCA), the treated cells could be clearly distinguished from vehicle control cells (0 μM AMIO) and HepaRG cells from HepG2 cells. The potential of using multidimensional and multimodal information collected from single cells to build predictive models for cell classification is demonstrated.

Incidental finding of cornea verticillata or lamellar inclusions in kidney biopsy: measurement of lyso-Gb3 in plasma defines between Fabry disease and drug-induced phospholipidosis

IntroductionTherapy with cationic amphiphilic drugs (Amiodarone or hydroxychloroquine) may result in biochemically and ultrastructurally similar lipid inclusions in many cells also affected by Fabry disease (FD). In addition, it often results in similar clinical manifestations such as cornea verticillata. This may lead to a FD misdiagnosis, especially when a complete medical history is not available to the ophthalmologist confronted with cornea verticillata or to the pathologist examining a kidney biopsy. When enzymatic/genetic test or pathological studies are not conclusive, a specific biomarker may help clarify this dilemma. The plasma globotriaosylsphingosine (lyso-Gb3) assay has high sensitivity and specificity and is elevated above normal levels in FD. Materials and methodsWe measured plasma lyso-Gb3 levels in male patients receiving Amiodarone or hydroxychloroquine and compared it with male patients with classic and late onset variant of FD. ResultsIn all Fabry patients (classic and late onset variant) α-GalA activity was deficient in dried blood spot and plasma lyso-Gb3 was above normal levels. Patients on treatment with Amiodarone or hydroxychloroquine had normal values for α-GalA activity and lyso-Gb3 in plasma. ConclusionsEven when Amiodarone or hydroxychloroquine may decrease α-GalA activity in vitro or in cell culture, our results showed that in all patients lyso-Gb3 plasma levels remain normal with no evidence of reduction in α-GalA activity, confirming the specificity of this biomarker for the diagnosis of FD.

Qualified Method for the Estimation of di-18:1 Bis(Monoacylglycero)Phosphate in Urine, a Noninvasive Biomarker to Monitor Drug-Induced Phospholipidosis

Aim: Our objective was to develop and qualify a bioanalytical method for the estimation of di-18:1-bis(monoacylglycero)phosphate (di-18:1BMP) as a urinary biomarker for the assessment of drug-induced phospholipidosis and demonstrate its application in a preclinical study. Methodology/results: di-18:1 BMP was extracted by liquid-liquid extraction using n-butanol and analyzed by LC-MS/MS. The qualified method was selective, precise, robustand accurate across the linearity range (0.2-250ng/ml). Qualified method was then used to assess chloroquine-induced phospholipidosis in rats dosed at 120mg/kg for 5 days. A fivefold increase in di-18:1 BMP was observed onDay5 compared with predose. Conclusion: Di-18:1 BMP can be used as a noninvasive biomarker to assess/screen compounds that could cause drug-induced phospholipidosis in rats.

Bis(monoacylglycero)phosphate, a new lipid signature of endosome-derived extracellular vesicles

Bis(monoacylglycero)phosphate (BMP), also known as lysobisphosphatidic acid (LBPA), is a phospholipid specifically enriched in the late endosome-lysosome compartment playing a crucial role for the fate of endocytosed components. Due to its presence in extracellular fluids during diseases associated with endolysosomal dysfunction, it is considered as a possible biomarker of disorders such as genetic lysosomal storage diseases and cationic amphiphilic drug-induced phospholipidosis. However, there is no true validation of this biomarker in human studies, nor a clear identification of the carrier of this endolysosome-specific lipid in biofluids. The present study demonstrates that in absence of any sign of renal failure, BMP, especially all docosahexaenoyl containing species, are significantly increased in the urine of patients treated with the antiarrhythmic drug amiodarone. Such urinary BMP increase could reflect a generalized drug-induced perturbation of the endolysosome compartment as observed in vitro with amiodarone-treated human macrophages. Noteworthy, BMP was associated with extracellular vesicles (EVs) isolated from human urines and extracellular medium of human embryonic kidney HEK293 cells and co-localizing with classical EV protein markers CD63 and ALIX. In the context of drug-induced endolysosomal dysfunction, increased BMP-rich EV release could be useful to remove excess of undigested material. This first human pilot study not only reveals BMP as a urinary biomarker of amiodarone-induced endolysosomal dysfunction, but also highlights its utility to prove the endosomal origin of EVs, also named as exosomes. This peculiar lipid already known as a canonical late endosome-lysosome marker, may be thus considered as a new lipid marker of urinary exosomes.

Investigating the role of endogenous opioid system in chloroquine-induced phospholipidosis in rat liver by morphological, biochemical and molecular modelling studies.

Drug-induced phospholipidosis (DIPL) is characterized by phospholipid storage in the lysosomes of affected tissues. Many severe effects and toxicities have been linked to DIPL. The aim of this study was to determine whether the endogenous opioid system is involved in chloroquine-induced phospholipidosis. The effect of naltrexone as an antagonist of opioid receptors in chloroquine-induced phospholipidosis in rat liver was investigated by morphological, biochemical, and molecular modelling studies. Transmission electron microscopy (TEM) showed that morphological characteristic changes of rat liver, including the number of lamellar bodies, grade of vacuolization and cell steatosis, were markedly attenuated in rats treated with naltrexone alone or in combination with chloroquine, in comparison with chloroquine-treated rats. The results of liquid chromatography mass spectrometry (LC/MS) showed that the concentrations of phenylacetylglycine (PAG) and hippuric acid (HA) were significantly decreased and increased, respectively, in target groups. Besides, the concentration ratio of PAG/HA was significantly decreased. Spectrophotometry resulted in a notable decrease in alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activities in target groups. The results from the molecular docking and molecular dynamic simulation studies demonstrated clear chloroquine interaction with the active site cavity of the µopioid receptor. These data suggest that administration of naltrexone alone, or in combination with chloroquine, notably attenuates the side effects of chloroquine-induced phospholipidosis, as well as demonstrating an increased probability of the endogenous opioid system involvement in chloroquine-induced phospholipidosis in rat liver.

Synchrotron Photothermal Infrared Nanospectroscopy of Drug-Induced Phospholipidosis in Macrophages.

Synchrotron resonance-enhanced infrared atomic force microscopy (RE-AFM-IR) is a near-field photothermal vibrational nanoprobe developed at Diamond Light Source (DLS), capable of measuring mid-infrared absorption spectra with spatial resolution around 100 nm. The present study reports a first application of synchrotron RE-AFM-IR to interrogate biological soft matter at the subcellular level, in this case, on a cellular model of drug-induced phospholipidosis (DIPL). J774A-1 macrophages were exposed to amiodarone (10 μM) or medium for 24 h and chemically fixed. AFM topography maps revealed amiodarone-treated cells with enlarged cytoplasm and very thin regions corresponding to collapsed vesicles. IR maps of the whole cell were analyzed by exploiting the RE-AFM-IR overall signal, i.e., the integrated RE-AFM-IR signal amplitude versus AFM-derived cell thickness, also on lateral resolution around 100 nm. Results show that vibrational band assignment was possible, and all characteristic peaks for lipids, proteins, and DNA/RNA were identified. Both peak ratio and unsupervised chemometric analysis of RE-AFM-IR nanospectra generated from the nuclear and perinuclear regions of untreated and amiodarone-treated cells showed that the perinuclear region (i.e., cytoplasm) of amiodarone-treated cells had significantly elevated band intensities in the regions corresponding to phosphate and carbonyl groups, indicating detection of phospholipid-rich inclusion bodies typical for cells with DIPL. The results of this study are of importance to demonstrate not only the applicability of Synchrotron RE-AFM-IR to soft biological matters with subcellular spatial resolution but also that the spectral information gathered from an individual submicron sample volume enables chemometric identification of treatment and biochemical differences between mammalian cells.

Use of 3D Human Liver Organoids to Predict Drug-Induced Phospholipidosis

Drug-induced phospholipidosis (PL) is a storage disorder caused by the formation of phospholipid-drug complexes in lysosomes. Because of the diversity of PL between species, human cell-based assays have been used to predict drug-induced PL in humans. We established three-dimensional (3D) human liver organoids as described previously and investigated their liver characteristics through multiple analyses. Drug-induced PL was initiated in these organoids and in monolayer HepG2 cultures, and cellular changes were systemically examined. Organoids that underwent differentiation showed characteristics of hepatocytes rather than HepG2 cells. The organoids also survived under PL-inducing drug conditions for 48 h and maintained a more stable albumin secretion level than the HepG2 cells. More cytoplasmic vacuoles were observed in organoids and HepG2 cells treated with more potent PL-induced drugs, but to a greater extent in organoids than in HepG2 cells. Lysosome-associated membrane protein 2, a marker of lysosome membranes, showed a stronger immunohistochemical signal in the organoids. PL-distinctive lamellar bodies were observed only in amiodarone-treated organoids by transmission electron microscopy. Human liver organoids are thus more sensitive to drug-induced PL and less affected by cytotoxicity than HepG2 cells. Since PL is a chronic condition, these results indicate that organoids better reflect metabolite-mediated hepatotoxicity in vivo and could be a valuable system for evaluating the phospholipidogenic effects of different compounds during drug development.

Emerging mechanisms of drug-induced phospholipidosis.

Drug-induced phospholipidosis is a lysosomal storage disorder characterized by excessive accumulation of phospholipids. Its cellular mechanism is still not well understood, but it is known that cationic amphiphilic drugs can induce it. These drugs have a hydrophilic amine head group that can be protonated in the endolysosomal compartment. As cationic amphiphiles, they are trapped in lysosomes, where they interfere with negatively charged intralysosomal vesicles, the major platforms of cellular sphingolipid degradation. Metabolic principles observed in sphingolipid and phospholipid catabolism and inherited sphingolipidoses are of great importance for lysosomal function and physiological lipid turnover at large. Therefore, we also propose intralysosomal vesicles as major platforms for degradation of lipids and phospholipids reaching them by intracellular pathways like autophagy and endocytosis. Phospholipids are catabolized as components of vesicle surfaces by protonated, positively charged phospholipases, electrostatically attracted to the negatively charged vesicles. Model experiments suggest that progressively accumulating cationic amphiphilic drugs inserting into the vesicle membrane with their hydrophobic molecular moieties disturb and attenuate the main mechanism of lipid degradation as discussed here. By compensating the negative surface charge, cationic enzymes are released from the surface of vesicles and proteolytically degraded, triggering a progressive lipid storage and the formation of inactive lamellar bodies.

Comparison of Oral, Intranasal and Aerosol Administration of Amiodarone in Rats as a Model of Pulmonary Phospholipidosis.

‘Foamy’ alveolar macrophages (FAM) observed in nonclinical toxicology studies during inhaled drug development may indicate drug-induced phospholipidosis, but can also derive from adaptive non-adverse mechanisms. Orally administered amiodarone is currently used as a model of pulmonary phospholipidosis and it was hypothesized that aerosol administration would produce phospholipidosis-induced FAM that could be characterized and used in comparative inhalation toxicology. Han-Wistar rats were given amiodarone via (1) intranasal administration (6.25 mg/kg) on two days, (2) aerosol administration (3 mg/kg) on two days, (3) aerosol administration (10 mg/kg) followed by three days of 30 mg/kg or (4) oral administration (100 mg/kg) for 7 days. Alveolar macrophages in bronchoalveolar lavage were evaluated by differential cell counting and high content fluorescence imaging. Histopathology and mass-spectrometry imaging (MSI) were performed on lung slices. The higher dose aerosolised amiodarone caused transient pulmonary inflammation (p < 0.05), but only oral amiodarone resulted in FAM (p < 0.001). MSI of the lungs of orally treated rats revealed a homogenous distribution of amiodarone and a putative phospholipidosis marker, di-22:6 bis-monoacylglycerol, throughout lung tissue whereas aerosol administration resulted in localization of both compounds around the airway lumen. Thus, unlike oral administration, aerosolised amiodarone failed to produce the expected FAM responses.

The Limited Contribution of the Analyte Partition to the Water-Rich Layer in Immobilized Artificial Membrane Chromatography with an Acetonitrile-Rich Binary Mobile Phase

Drug-induced phospholipidosis, a side effect of drugs, is associated with drug–membrane binding. Hydrophobic interactions of the drug with the membrane and polar interactions of the drug with the membrane are involved. For risk predictions, membrane binding studies often give false results for strongly hydrophobic compounds and basic hydrophilic compounds because the effects of the hydrophobic interactions overshadow the effects of the polar interactions. Therefore, separate assessments of hydrophobic partitioning and polar interactions are needed. However, a screening method focusing on the polar interactions between drugs and membranes has not yet been reported. In the present study, the retention behavior of an immobilized artificial membrane (IAM) column using a binary mobile phase with a high acetonitrile content was studied to clarify the contribution of the analyte partition to the water-rich layer on the surface of the packed material, which may interfere with the assessment of the polar interactions with the membrane. For acetonitrile contents greater than 50%, the retention factor of propranolol increased as a result of the hydrophilic interactions. The Van’t Hoff plot suggested that the adsorptive retention of the analyte became dominant with high acetonitrile content in the mobile phase, in contrast to the hydrophobic partitioning that occurred for low acetonitrile content. Hydrophilic neutral compounds (e.g., thiourea and mesityl oxide) were not retained. The addition of salts to the mobile phase decreased the propranolol retention. Even with the addition of a kosmotropic salt, the decreased retention indicated that hydrophilic partitioning to the water-enriched layer was unlikely to be the major mechanism. In addition to ionic interactions, other polar interactions were presumed to be involved. The relationships between the retention factor and the log P or pKa values also supported this conclusion. The results of this study suggest that there is potential for IAM chromatography to be used as a tool to estimate the contribution of the polar interactions in overall drug–membrane interactions, which can aid in predicting the risk of phospholipidosis.

The Parkinson-associated human P5B-ATPase ATP13A2 modifies lipid homeostasis

Mutations in the ATP13A2 gene (PARK9, CLN12, OMIM 610513) were initially associated with a form of Parkinson's Disease (PD) known as Kufor Rakeb Syndrome (KRS). However, the genetic spectrum of ATP13A2-associated disorders was expanded in the last years, because it has been found to underlay variants of neuronal ceroid-lipofuscinoses (NCLs) and hereditary spastic paraplegia. As ATP13A2 seems to be a key component of the endo-lysosome pathway, the fact that these pathologies are commonly characterized by endo-lysosomal dysfunction is not surprising.Here we report that increasing the level of functional ATP13A2 in a stable SH-SY5Y cell line disrupts lipid homeostasis. ATP13A2 overexpression increases the fluorescence intensity of the fluorescent analog phosphatidylethanolamine (NBD-PE) and the formation of multilamellar bodies, resembling the so-called “drug-induced phospholipidosis”. We also found that expression of ATP13A2 reduces the ceramide-fluorescence intensity and the content of bis(monoacylglyceryl)phosphate (BMP). BMP is required for lipid degradation and exosome biogenesis inside acidic compartments, so this result suggests that ATP13A2 may be modifying the lipid digestion capacity and/or the redistribution of lipids in these subcellular organelles. In addition, ATP13A2-overexpression decreased the total content of triglycerides (TGs), cholesterol and lipid droplets. As TGs are necessary for the synthesis of new membranes, this observation suggests that increasing the function of ATP13A2 switches the endo-lysosomal system towards vesicle secretion.

Synthesis of di‐docosahexaenoyl (C22:6)‐bis(monoacylglycerol) phosphate in unlabelled and C‐13 labelled forms for use as a biomarker of drug induced phospholipidosis

Di-docosahexaenoyl (C22:6)-bis(monoacylglycerol) phosphate (BMP) has been identified as a promising biomarker for drug-induced phospholipidosis (DIPL). Both unlabelled and stable isotope labelled versions of BMP were desired for use as internal standards. Isopropylideneglycerol was converted to 4-methoxyphenyldiphenylmethyl-3-PMB-glycerol in three steps. Initially, the 2-postion of the glycerol was protected as a t-butyldiphenylsilyl ether, which proved to be a mistake; deprotection of the ether resulted in the decomposition of the compound. A switch to a t-butyldimethylsilyl ether protecting group resulted in an intermediate that could be deprotected to the alcohol to give the target compound after salt exchange. The same procedure was used to prepare [13 C6 ]BMP from [13 C3 ]glycerol.

A multiplexed UPLC-MS/MS assay for the simultaneous measurement of urinary safety biomarkers of drug-induced kidney injury and phospholipidosis

Drug-induced kidney injury (DIKI) is a major concern in drug risk assessment given its clinical importance and the absence of a sensitive/specific method of diagnosis. Pharmaceutical regulatory agencies have qualified and issued letters of support for new biomarkers to better evaluate DIKI in nonclinical toxicity and clinical studies. Additional efforts have focused on drug-induced phospholipidosis (DIPL) and its potential link with collateral renal damage. The combined use of urinary biomarkers is an efficient way to evaluate renal safety in nonclinical and clinical studies. Eight FDA/EMA/PMDA qualified (or supported) urinary biomarkers, including kidney injury molecule-1 (KIM-1), β2-microglobulin (B2M), clusterin (CLU), cystatin C (CysC), trefoil factor 3 (TFF3), neutrophil gelatinase-associated lipocalin (NGAL), osteopontin (OPN), and alpha-glutathione S-transferase (α-GST), were quantified by multiplex UPLC-MS/MS in a repeat dose study of gentamicin in rats. Rats administered gentamicin at 100 mg/kg/day for 2 weeks developed renal lesions detected by histopathology. Biomarkers of tubular damage (CLU, KIM-1, OPN) increased 9.8, 34.7, and 35.6-fold (relative to concurrent controls), respectively, after 2 weeks of dosing. Biomarkers of glomerular damage and/or impairment of tubular reabsorption (CysC, B2M) increased 11.7 and 22.6-fold. NGAL and α-GST increased <3-fold after 2 weeks of dosing. TFF3 was comparable to concurrent controls. The elevated biomarker concentrations met PSTC threshold criteria and were consistent with mechanisms of gentamicin nephrotoxicity. Increased urinary di-22:6-BMP indicated concomitant DIPL as confirmed by TEM. This work provides evidence supporting the combined use of the DIKI biomarker panel and di-22:6-BMP as a biomarker of DIPL in drug risk assessment.

Inkjet-printed micro-calibration standards for ultraquantitative Raman spectral cytometry.

Herein we report the development of a cytometric analysis platform for measuring the contents of individual cells in absolute (picogram) scales; this study represents the first report of Raman-based quantitation of the absolute mass - or the total amount - of multiple endogenous biomolecules within single-cells. To enable ultraquantitative calibration, we engineered single-cell-sized micro-calibration standards of known composition by inkjet-printer deposition of biomolecular components in microarrays across the surface of silicon chips. We demonstrate clinical feasibility by characterizing the compositional phenotype of human skin fibroblast and porcine alveolar macrophage cell populations in the respective contexts of Niemann-Pick disease and drug-induced phospholipidosis: two types of lipid storage disorders. We envision this microanalytical platform as the foundation for many future biomedical applications, ranging from diagnostic assays to pathological analysis to advanced pharmaco/toxicokinetic research studies.

Imaging Mass Microscopy of Kidneys from Azithromycin-Treated Rats with Phospholipidosis

Drug-induced phospholipidosis is a lysosomal storage disorder characterized by the excess accumulation of tissue phospholipids. Although azithromycin can be used to induce phospholipidosis, no experimental studies evaluating the relationship between drug accumulation and phospholipid localization have been performed. In this study, azithromycin was orally administered to rats for 7 days, and the relationship between drug and phospholipid accumulation was performed using imaging mass microscopy. The administration of azithromycin induced tubular epithelial vacuolation in the inner stripe of the outer medulla of the kidney, consistent with the lamellar bodies that are typical manifestations of drug-induced phospholipidosis. Azithromycin and phospholipid tissue levels were extensively elevated in the kidneys of azithromycin-treated rats. Imaging mass microscopy revealed that both azithromycin and its metabolites were found in the kidneys of azithromycin-treated rats but not in control animals. The vacuolated areas of the kidneys were primarily found in the inner stripe of the outer medulla, consistent with the areas of high azithromycin concentration. Azithromycin was colocalized with several phospholipids-phosphatidylinositol (18:0/20:4), phosphatidylethanolamine (18:0/20:4 and 16:0/20:4), and possibly didocosahexaenoyl (C22:6)-bis(monoacylglycerol) phosphate, a putative biomarker of drug-induced phospholipidosis. In summary, we found correlations between regions of kidney damage and the accumulation of azithromycin, its metabolites, and phospholipids using imaging mass microscopy. Such analyses may help reveal the mechanism and identify putative biomarkers of drug-induced phospholipidosis.

Detection of nanocarrier potentiation on drug induced phospholipidosis in cultured cells and primary hepatocyte spheroids by high content imaging and analysis

Considerable effort has been made to develop nanocarriers for controlled drug delivery over the last decade, while it remains unclear how the strength of adverse drug effect will be altered when a drug is loaded on the nanocarrier. Drug-induced phospholipidosis (DIP) is characterized with excessive accumulation of phospholipids in cells and is common for cationic amphiphilic drugs (CAD). Previously, we have reported that PEGylated graphene oxide (PEG-GO) loaded with several CAD can potentiate DIP. In current study, we extended our study on newly identified phospholipidosis (PLD) inducers that had been identified from the Library of Pharmacologically Active Compounds (LOPAC), to investigate if PEO-GO loaded with these CAD can alter DIP. Twenty-two CAD were respectively loaded on PEG-GO and incubated with RAW264.7, a macrophage cell line. The results showed that when a CAD was loaded on PEG-GO, its strength of PLD induction can be enhanced, unchanged or attenuated. PEG-GO loaded with Ifenprodil exhibited the highest PEG-GO potentiation effect compared to Ifenprodil treatment alone in RAW264.7 cells, and this effect was confirmed in human hepatocellular carcinoma HepG2, another cell line model for PLD induction. Primary hepatocyte culture and spheroids mimicking in vivo conditions were used to further validate nanocarrier potentiation on DIP by Ifenprodil. Stronger phospholipid accumulation was found in PEG-GO/Ifenprodil treated hepatocytes or spheroids than Ifenprodil treatment alone. Therefore, evidences were provided by us that nanocarriers may increase the adverse drug effects and guidance by regulatory agencies need to be drafted for the safe use of nanotechnology in drug delivery.

Hepatic cells derived from human skin progenitors show a typical phospholipidotic response upon exposure to amiodarone

Phospholipidosis is a metabolic disorder characterized by intracellular accumulation of phospholipids. It can be caused by short-term or chronic exposure to cationic amphiphilic drugs (CADs). These compounds bind to phospholipids, leading to inhibition of their degradation and consequently to their accumulation in lysosomes. Drug-induced phospholipidosis (DIPL) is frequently at the basis of discontinuation of drug development and post-market drug withdrawal. Therefore, reliable human-relevant in vitro models must be developed to speed up the identification of compounds that are potential inducers of phospholipidosis. Here, hepatic cells derived from human skin (hSKP-HPC) were evaluated as an in vitro model for DIPL. These cells were exposed over time to amiodarone, a CAD known to induce phospholipidosis in humans. Transmission electron microscopy revealed the formation of the typical lamellar inclusions in the cell cytoplasm. Increase of phospholipids was already detected after 24 h exposure to amiodarone, whereas a significant increase of neutral lipid vesicles could be observed after 72 h. At the transcriptional level, the modulation of genes involved in DIPL was detected. These results provide a valuable indication of the applicability of hSKP-HPC for the quick assessment of drug-induced phospholipidosis in vitro, early in the drug development process.

Oxymorphone Hydrochloride Extended-Release (OPANA®) Associated With Acute Kidney Injury in a Chronic Pain Patient

Oxymorphone hydrochloride extended-release (OPANA®) is an opioid prescribed for the treatment of moderate-to-severe chronic pain. Kidney injury related to its use has not previously been reported. We present a case of a chronic pain patient with underlying chronic renal insufficiency who developed superimposed acute kidney injury when his opioid analgesic was changed from morphine sulfate extended-release to OPANA. Electron microscopy of his renal tissue revealed lamellated podocytes typically seen with drug-induced phospholipidosis.

Tools to Measure Cell Health and Cytotoxicity Using High Content Imaging and Analysis.

High content screening (HCS)-based multiparametric measurements are very useful in early toxicity testing and safety assessment during drug development, and useful in evaluating the impact from new food supplements and environmental toxicants. Mitochondrial membrane potential, plasma membrane permeability, oxidative stress, phosphoplipidosis, and steatosis are a few of the important markers routinely studied for the assessment of drug-induced liver injury and toxicity. Mitochondrial dysfunction leads to oxidative stress and cell death. Liver injury from drug-induced phospholipidosis and steatosis is routinely studied in hepatotoxicity investigations to determine the risk factors and fate of drugs or chemical compounds as some drugs can lead to defects in lipid metabolism and accumulation of lipids in lysosomes. In this chapter, we describe fluorescent reagents and the protocols for the measurement of various parameters such as mitochondrial membrane potential, plasma membrane permeability, oxidative stress, phospholipidosis, and steatosis using high content imaging-based methodologies and instrumentation.