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.

Predicting the Risk of Phospholipidosis with in Silico Models and an Image-Based in Vitro Screen.

The drug-induced accumulation of phospholipids in lysosomes of various tissues is predominantly observed in regular repeat dose studies, often after prolonged exposure, and further investigated in mechanistic studies prior to candidate nomination. The finding can cause delays in the discovery process inflicting high costs to the affected projects. This article presents an in vitro imaging-based method for early detection of phospholipidosis liability and a hybrid approach for early detection and risk mitigation of phospolipidosis utilizing the in vitro readout with in silico model prediction. A set of reference compounds with phospolipidosis annotation was used as an external validation set yielding accuracies between 77.6% and 85.3% for various in vitro and in silico models, respectively. By means of a small set of chemically diverse known drugs with in vivo phospholipidosis annotation, the advantages of combining different prediction methods to reach an overall improved phospholipidosis prediction will be discussed.

Hydrophilic interaction chromatography with a focus on the drug-phosphate interaction in drug screening to determine the phospholipidosis induction risk.

Cationic amphiphilic drugs (CADs) can induce the hyperaccumulation of phospholipids in cells and tissues. This side effect, which is known as drug-induced phospholipidosis, is sometimes problematic in the development and clinical use of CADs. It is known that CADs generally interact with phospholipids via both hydrophobic and acid-base interactions, and CADs with the larger affinity to phospholipid exhibit the larger induction risk. To develop a chromatographic assay system to predict the phospholipidosis-inducing potential with considering the acid-base interaction between CAD and phosphate group of phospholipid, hydrophilic interaction chromatographic (HILIC) methods were tested in this study. First, a PC HILIC column with phosphocholine groups on a packed material was used. The acid-base or other hydrophilic interactions to the stationary phase differed among basic drugs, and retention to the PC HILIC column did not accurately reflect the induction potential of phospholipidosis. As an alternative HILIC approach, the elution of CADs with the phosphate buffer from an amide column was tested. The elution effect, which is expressed as ratio of retention factors between different phosphate content in the mobile phase, closely correlated with the induction potential. Using the elution effect and retention factor to a reversed-phase HPLC column, the phospholipidosis-inducing drugs were clearly discriminated from the non-inducers. These results suggest that the proposed chromatographic approach can screen phospholipidosis-inducing drugs.

Arachidonic acid-containing phosphatidylcholine characterized by consolidated plasma and liver lipidomics as an early onset marker for tamoxifen-induced hepatic phospholipidosis.

Lipid profiling has emerged as an effective approach to not only screen disease and drug toxicity biomarkers but also understand their underlying mechanisms of action. Tamoxifen, a widely used antiestrogenic agent for adjuvant therapy against estrogen-positive breast cancer, possesses side effects such as hepatic steatosis and phospholipidosis (PLD). In the present study, we administered tamoxifen to Sprague-Dawley rats and used lipidomics to reveal tamoxifen-induced alteration of the hepatic lipid profile and its association with the plasma lipid profile. Treatment with tamoxifen for 28 days caused hepatic PLD in rats. We compared the plasma and liver lipid profiles in treated vs. untreated rats using a multivariate analysis to determine differences between the two groups. In total, 25 plasma and 45 liver lipids were identified and altered in the tamoxifen-treated group. Of these lipids, arachidonic acid (AA)-containing phosphatidylcholines (PCs), such as PC (17:0/20:4) and PC (18:1/20:4), were commonly reduced in both plasma and liver. Conversely, tamoxifen increased other phosphoglycerolipids in the liver, such as phosphatidylethanolamine (18:1/18:1) and phosphatidylinositol (18:0/18:2). We also examined alteration of AA-containing PCs and some phosphoglycerolipids in the pre-PLD stage and found that these lipid alterations were initiated before pathological alteration in the liver. In addition, changes in plasma and liver levels of AA-containing PCs were linearly associated. Moreover, levels of free AA and mRNA levels of AA-synthesizing enzymes, such as fatty acid desaturase 1 and 2, were decreased by tamoxifen treatment. Therefore, our study demonstrated that AA-containing PCs might have potential utility as novel and predictive biomarkers for tamoxifen-induced PLD. Copyright © 2017 John Wiley & Sons, Ltd.

Molecular biomarkers of phospholipidosis in rat blood and heart after amiodarone treatment.

Phospholipidosis (PLD) is characterized by an intracellular accumulation of phospholipids in lysosomes and concurrent development of concentric lamellar bodies. It is induced in humans and in animals by drugs with a cationic amphiphilic structure. The purpose of the present study was to identify a set of molecular biomarkers of PLD in rat blood and heart, hypotheticallya pplicable in preclinical screens within the drug development process. A toxicological study was set up in rats orally treated up to 11 days with 300 mg kg(­1) per day(­1) amiodarone (AMD). Light and transmission electron microscopy investigations were performed to confirm the presence of lamellar bodies indicative of phospholipid accumulation. The effects of AMD upon the transcriptome of these tissues were estimated using DNA microarray technology. Microarray data analysis showed that a total of 545 and 8218 genes were modulated by AMD treatment in heart and blood, respectively. Some genes implicated in the phospholipid accumulation in cells, such as phospholipase A2, showed similar alterations of gene expression. After transcriptome criteria of analysis and target selection, including also the involvement in the onset of PLD, 7 genes (Pla2g2a, Pla2g7, Gal, Il1b, Cebpb, Fcgr2b, Acer 2) were selected as candidate biomarkers of PLD in heart and blood tissues, and their potential usefulness as a sensitive screening test was screened and confirmed by quantitative Real-Time PCR analysis. Collectively, these data underscore the importance of transcriptional profiling in drug discovery and development, and suggest blood as a surrogate tissue for possible phospholipid accumulation in cardiomyocytes.

Long-term chronic toxicity testing using human pluripotent stem cell-derived hepatocytes.

Human pluripotent stem cells (hPSC) have the potential to become important tools for the establishment of new models for in vitro drug testing of, for example, toxicity and pharmacological effects. Late-stage attrition in the pharmaceutical industry is to a large extent caused by selection of drug candidates using nonpredictive preclinical models that are not clinically relevant. The current hepatic in vivo and in vitro models show clear limitations, especially for studies of chronic hepatotoxicity. For these reasons, we evaluated the potential of using hPSC-derived hepatocytes for long-term exposure to toxic drugs. The differentiated hepatocytes were incubated with hepatotoxic compounds for up to 14 days, using a repeated-dose approach. The hPSC-derived hepatocytes became more sensitive to the toxic compounds after extended exposures and, in addition to conventional cytotoxicity, evidence of phospholipidosis and steatosis was also observed in the cells. This is, to the best of our knowledge, the first report of a long-term toxicity study using hPSC-derived hepatocytes, and the observations support further development and validation of hPSC-based toxicity models for evaluating novel drugs, chemicals, and cosmetics.

Evaluation and validation of multiple cell lines and primary mouse macrophages to predict phospholipidosis potential.

Phospholipidosis (PLD) in preclinical species can lead to regulatory delays thereby creating incentives to screen for PLD during drug discovery. The objective of this work was to compare, optimize, and validate in vitro PLD assays in primary mouse macrophages and hepatocyte- (HepG2, HuH7) or macrophage-derived cells lines (I.13.35, RAW264.7) and to evaluate whether primary cells were better at predicting PLD. Assay precision, determined by a measure of signal to noise window (Z'), within assay variability, and day-to-day variability, using amiodarone, was generally acceptable for all cell types; however, precision limits for HepG2 and HuH7 were slightly below assay acceptance criteria. Up to 66 known PLD inducers and non-inducers were subsequently tested to validate the assays. The concordance for predicting PLD in primary macrophages, I-13.35, RAW264.7, HuH7, and HepG2 cells was 91%, 74%, 73%, 62%, and 62% respectively using a decision limit of EC50≤125 µM as a positive finding. Increasing the number of negative controls tested in RAW264.7 cells and changing the decision limit to ≥4-fold increase in PLD, improved the specificity and overall concordance to 88%. RAW264.7 cells were selected as the primary screen for predicting PLD, and together with the primary macrophages, were integrated into an overall testing paradigm proposed for use in PLD risk identification.

High content screening analysis of phospholipidosis: validation of a 96-well assay with CHO-K1 and HepG2 cells for the prediction of in vivo based phospholipidosis.

Drug-induced phospholipidosis is marked by an excessive accumulation of phospholipids in lysosomes which can occur after exposure to cationic amphiphilic drugs. Phospholipidosis is considered as an adverse side effect and may delay or negatively affect registration of drug candidates. Currently, the gold standard method of phospholipidosis detection is electron microscopy on tissue samples. This technique is time consuming and only performed relatively late in drug development. Therefore, in vitro screening methods for phospholipidosis are essential in early drug development. In this study, an in vitro phospholipidosis detection assay is developed with CHO-K1 and HepG2 cells by using the fluorescent marker NBD-PE and high content screening analysis. Lysosomal localization of NBD-PE was demonstrated by colocalization with Lysotracker and lamellar body formation by electron microscopy. Upon drug exposure, lysosomal NBD-PE accumulation can be visualized and quantified. Validation with 56 reference compounds, divided in 25 phospholipidosis inducers and 31 negative compounds, showed that this new in vitro assay has a high sensitivity (CHO-K1=92.0% and HepG2=88.0%) and specificity (CHO-K1=87.1% and HepG2=80.6%) for predicting phospholipidosis in vivo. Thus a selective screening tool has been developed for early selection of drug candidates with low probability for phospholipidosis.

Screening for phospholipidosis induced by central nervous drugs: comparing the predictivity of an in vitro assay to high throughput in silico assays.

Drug-induced phospholipidosis is a side effect for which drug candidates can be screened in the drug discovery phase. The numerous in silico models that have been developed as a first line of screening are based on the characteristic physicochemical properties of phospholipidosis-inducing drugs, e.g. high logP and pK(b) values. However, applying these models on a predominantly high lipophilic, basic CNS chemistry results in a high false positive rate and consequently in a wrong classification of a large number of valuable drug candidates. Here, we tested 33 CNS-compounds (24 in vivo negative and 9 in vivo positive phospholipidosis-inducers) in our in house developed in vitro phospholipidosis screening assay (Mesens et al., 2009) and compared its predictivity with the outcome of three different, well established in silico prediction models. Our in vitro assay demonstrates an increased specificity of 79% over the in silico models (29%). Moreover, by considering the proposed plasma concentration at the efficacious dose we can show a clear correlation between the in vitro and in vivo occurrence of phospholipidosis, improving the specificity of prediction to 96%. Through its high predictive value, the in vitro low throughput assay is thus preferred above high throughput in silico assays, characterized by a high false positive rate.

A strategy for risk management of drug-induced phospholipidosis.

Drug-induced phospholipidosis (PL) is an excessive accumulation of phospholipids and drug in lysosomes. Phospholipidosis signals a change in cell membrane integrity and accumulation of intracellular drug or metabolite in tissues. The sensitivity and susceptibility of preclinical models to detect PL vary with therapeutic agents, and PL is expected to be reversible after discontinuation of drug treatment. The prevailing scientific opinion is that PL by itself is not adverse; however, some regulatory authorities consider PL to be adverse because a small number of chemicals are able to cause PL and concurrent organ toxicity. Until a greater understanding of PL emerges, a well-thought-out risk management strategy for PL will increase confidence in safety and improve selection and development of new drugs. This paper provides a tiered approach to risk management of drug-induced PL. It begins with use of in silico and in vitro tools to design and select compounds with reduced potential to produce PL. Early in vivo studies in two species are used to better characterize potential for toxicity and PL. Finally, routine risk management tools (i.e., translational biomarkers, assessment of reversibility) are used to support confidence in safety of compounds that induce PL in animals.

Phospholipidosis in neurons caused by posaconazole, without evidence for functional neurologic effects.

The azole antifungal drug posaconazole caused phospholipidosis in neurons of the central nervous system, dorsal root ganglia of the spinal cord, and myenteric plexus in chronic toxicity studies in dogs. The time of onset, light and electron microscopic features, neurologic and electrophysiologic effects on the central and peripheral nervous systems, and potential for regression were investigated in a series of studies with a duration of up to one year. Nuclei of the medulla oblongata were the prominently affected areas of the brain. Neurons contained cytoplasmic vacuoles with concentrically whorled plasma membrane-like material (i.e., multilamellar bodies) morphologically identical to that commonly caused in other tissues by cationic amphiphilic drugs. Some axons in the brain and spinal cord were swollen and contained granular eosinophilic, electron-dense lysosomes. There were no features suggesting degeneration or necrosis of neurons or any associated elements of nervous tissue. The earliest and most consistent onset was in neurons of dorsal root ganglia. The observed neural phospholipidosis did not result in any alteration in the amplitude or latency of the auditory, visual, or somatosensory evoked potentials. The histopathologic changes did not progress or regress within the three-month postdose period. The results indicate that phospholipidosis can be induced in central and peripheral neurons of dogs by administration of posaconazole, but this change is not associated with functional effects in the systems evaluated.

A 96-well flow cytometric screening assay for detecting in vitro phospholipidosis-induction in the drug discovery phase.

Drug-induced phospholipidosis is caused by lysosomal accumulation of the drug, resulting in the disturbance of phospholipid degradation and a consequent excessive phospholipid accumulation. Depending on the type and number of tissues affected, phospholipidosis occurrence in test animals can raise safety issues, which may be critical for the risk assessment. Safety profiling of potential phospholipidosis-inducing drugs in the drug discovery phase can predict these late obstructions of drug development. For this purpose, a flow cytometric assay based on the difference in fluorescent phospholipid accumulation in a human monocyte cell line was initially established. Modifying the assay studying degradation of the fluorescent phospholipids instead of accumulation drastically improved sensitivity. By testing various phospholipidosis-inducers and negative compounds, it was found that the assay could detect the occurrence of phospholipidosis by a 2-fold difference in fluorescence compared to control cells, demonstrating the superior sensitivity of the novel approach. Implementation of a higher throughput 96-well flow cytometric set up did not affect the sensitivity of detection or the reproducibility of the assay. Based on an extended test set of reference compounds a profiling approach was introduced, by which we show we can rank our drug candidates according to their phospholipidosis-inducing potential.

Monitoring the accumulation of fluorescently labeled phospholipids in cell cultures provides an accurate screen for drugs that induce phospholipidosis.

A large number of cationic amphiphilic drugs (CADs) are known to cause phospholipidosis (PLD) in vivo. In the present study, we have built upon our previous findings to further qualify the use of a fluorescently labeled phospholipid-based cell-culture assay to detect PLD-inducing drugs. In this paper, we demonstrate that 12 PLD-negative compounds and 11 drugs known to cause PLD in vivo are all correctly identified by using this assay. Interestingly, we found that in cells treated with certain CADs, the fluorescent phospholipid was sequestered in a very specific punctate pattern, which overlapped strongly with the staining pattern seen with a lysosomal marker protein. Our data also show that false positives can be generated with the fluorescence assay when compounds are used at concentrations that cause a >30% decrease in cell number in this assay. Confocal microscopy demonstrated that the staining pattern of fluorescent phospholipids in these cases may be differentiated from those of true positives by the fact that diffuse, rather than punctuate, fluorescence is observed. These studies confirm and expand our previous results showing that the fluorescent phospholipid assay is a highly sensitive, specific tool for detecting PLD-inducing drugs, if care is taken to rule out cytotoxicity-related artifact.

Urinary metabolic fingerprinting for amiodarone-induced phospholipidosis in rats using FT-ICR MS.

In the research and development for new therapeutic compounds, there has been a focus on detecting the changes of metabolites induced by drug administration and finding surrogate markers to assess its toxicity. We examined the suitability of urinary metabolic fingerprinting using Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) for toxicological assessment in the amiodarone (AMD)-induced phospholipidosis (PLD) rat model. There were more than 400 different ion peaks detected in the negative ion mode analysis with FT-ICR MS. About 20% of the detected ions were altered more than 1.5 fold by AMD-treatment. On the scores plot of principal component analysis (PCA), the ion profiles of the treated were separated time-dependently. The loading plot revealed that the metabolites causing PCA results were m/z 178.05101, 191.01979, 192.06676, 212.00239, 258.9944 and 283.0820. The ion at m/z 178.05101 is considered to be hippurate (HA), 192.06676 is phenylacetylglycine (PAG) and 212.00239 is indican (IDN). These results indicate that PAG, IDN and HA are biomarkers for AMD-induced PLD in urinary metabolic fingerprinting using FT-ICR MS. These markers may be useful for evaluation of chemicals, which have the potential to induce PLD.

Cell-based fluorescence assay for evaluation of new-drugs potential for phospholipidosis in an early stage of drug development.

To evaluate new-drugs potential for phospholipidosis (PL), we developed a cell-based fluorescence assay using a fluorescent-labeled phospholipid analogue (NBD-PE). CHL/IU cells derived from newborn hamster lung were exposed to positive reference compounds (amiodarone, imipramine, chloroquine, propranolol, chlorpromazine and amantadine) in the presence of NBD-PE, and the level of PL, as indicated by accumulation of fluorescent inclusions in the cytoplasm, was evaluated using fluorescence microscopy and fluorometry. All positive reference compounds induced accumulation of fluorescent inclusions in a concentration-dependent manner with an increase in fluorescence intensity. Fluorescence microscopically, the positive dose of test compound was determined as the concentration with a grade equivalent to or above that of 3.13 microM of amiodarone. Based on this criterion, 8 of 20 test compounds including PL-positive or -negative compounds were judged positive that were concurrent with the pathological results from rat toxicity studies. Furthermore, a positive criterion for fluorometry was decided as equivalent to or above 25% of maximum intensity induced by 1.56-25.0 microM amiodarone. In comparison of fluorometry methods with fluorescence microscopy method, 19 of 20 compounds were judged same. From these findings, we concluded that the assay developed in this study is a rapid and reliable method to predict new-drugs potential for PL at an early stage of drug development.

In vitro assays and biomarkers for drug-induced phospholipidosis.

Drug-induced phospholipidosis is the cytoplasmic accumulation of phospholipids as a result of xenobiotic exposure. This accumulation results in a unique histological effect in cells noted as electron-dense lamellar inclusions or whorls in the cytoplasm when observed with transmission electron microscopy. Electron microscopy has been the widely accepted standard for classification of the phospholipidosis effect. Molecules that have been prone to induce such an effect are made up of a lipophilic region and a positively charged region. Phospholipidosis has most commonly been associated with drugs that are cationic, amphiphilic drugs and can occur in a variety of tissues. Although phospholipidosis is not considered adverse in isolation, depending on the tissue affected or the occasional circumstance of concurrent toxicity, phospholipidosis can be perplexing if identified in early drug development. In most circumstances, characterisation of the effect with in vivo studies allows for determination of exposure and the magnitude of the effect. On occasion in drug development, there may be an interest to screen early stage compounds to minimise phospholipidosis. In such circumstances, in silico and in vitro assays can be employed in a strategy with in vivo assessments. In addition, there may be an interest to monitor for the potential development of phospholipidosis in longer-term animal studies. In such cases, biomarker approaches could be used. The challenge in the overall assessment of phospholipidosis remains the question of the possible relevance to any toxicity, and, therefore, any screening approach, while assessing the potential to induce phospholipidosis, must be considered in relation to prediction of findings in vivo. The status of current assays and biomarkers is presented with strategies for screening.

Validation of an in vitro screen for phospholipidosis using a high-content biology platform.

Several cationic amphiphilic drugs cause local or systemic phospholipidosis (PLD) after chronic exposure in preclinical species. PLD is characterized by the accumulation of drug, phospholipid, and concentric lamellar bodies in cellular lysosomes. We have developed a fluorescence-based in vitro screen that is predictive of PLD using the Cellomics ArrayScan high-content screening platform, which captures and analyzes images from 96-well cell culture microtiter plates using multichannel fluorescence microscopy. I-13.35 adherent mouse spleen macrophage cells were cultured with drug and a fluorescently tagged phospholipid, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE). Drug concentrations were used in a range from 1 to 100 micro mol/L. After 24 h incubations, the cells were fixed with formalin. NBD-PE uptake was quantified in controls and treated cells. Nuclei were identified by Hoechst 33258 staining and dead cells were identified using ethidium homodimer-2 incorporation. Thus, confounding accumulation of NBD-PE due to cytotoxicity that produces false-positive results at high concentrations was eliminated from quantitation by ethidium staining and employing cell gating (dead cell rejection). The assay was found to be both sensitive and selective in that 26 of 28 positive, phospholipidogenic controls and 8 of 8 negative, non-phospholipidogenic controls were correctly called.

Establishment of an in vitro high-throughput screening assay for detecting phospholipidosis-inducing potential.

Excessive accumulation of phospholipids results in phospholipidosis (PL), which may interfere with cellular functions, leading to acute or chronic disease or even death. Electron-microscopic detection of cytoplasmic lamellar bodies is often used as a diagnostic criterion of PL, but a faster, more convenient procedure is required for high-throughput assay of the PL-inducing potential of candidate drugs. We have developed a 96-well microplate cell-culture method for detecting PL, using a phosphatidylcholine-conjugated dye (NBD-PC) and a fluoro-microplate reader. The fluorescence intensity due to NBD-PC was normalized to that of Hoechst33342, used as an indicator of cell number, to obtain the amount of NBD-PC taken up per living cell. To select a suitable cell type, we examined the PL-detection sensitivity of five cell lines, as well as human and rat primary hepatocyte cultures, with five cationic amphiphilic drugs (CAD) as PL inducers and a negative control compound. The cell lines CHO-K1 and CHL/IU gave the best results. The NBD-PC uptake per CHO-K1 cell showed a high correlation with the pathological score of PL for 24 compounds, including PL-positive and negative compounds. This high-throughput screening assay for PL-inducing potential (HTS-PL assay) offers high sensitivity and accuracy, and it allows simultaneous determination of cytotoxicity.

Myopathy related to administration of a cationic amphiphilic drug and the use of multidose drug distribution analysis to predict its occurrence.

Many cationic amphiphilic (phospholipidosis-inducing) drugs (CADs) accumulate in tissues following repeated dosing in preclinical models, and this is sometimes associated with dose-limiting toxicities. Plasma drug levels cannot be used to estimate tissue accumulation of CADs since it occurs in tissues despite stabilization of plasma levels. Severe myopathy was found in skeletal muscles of rats during the initial safety evaluation of a dopamine D3 receptor antagonist, PNU-177864, and was associated with phospholipidosis in numerous tissues. The myopathy was observed only when plasma levels of PNU-177864 remained essentially constant throughout the 24-hour dosing period. A repeat dose drug distribution study using whole body autoradiography demonstrated that drug-related material did not accumulate in skeletal muscle or other tissues following repeated doses at levels considered within the therapeutic range and showing toxicokinetic profiles acceptable for further development. These observations provided support for the continued development of and longer-term toxicity studies with this candidate compound.

Epididymal and systemic phospholipidosis in rats and dogs treated with the dopamine D3 selective antagonist PNU-177864.

Repeat dose oral toxicity studies were conducted in rat and dog to assess the safety for human clinical testing of the potent dopamine D3 receptor antagonist, PNU-177864. Systemic phospholipidosis was the principal treatment-related change with epididymal epithelial cell phospholipidosis being the most prominent finding in rats and dogs. Epididymal epithelial cells had no histologic evidence of degeneration; sperm density and morphology were normal histologically in both species; and sperm concentration, morphology, and motility in the dog were comparable to dogs given vehicle. Other sites with phospholipidosis included lymphoid tissues (lymph nodes, Peyer's patches, and/or spleen), pulmonary alveolar macrophages, and peripheral blood lymphocytes in rats and dogs and adrenal cortex, liver, pituitary, hair follicles, bone marrow lymphocytes and plasma cells, and skeletal muscle in rats only. The phospholipidosis was resolved after a 6-week recovery period in all tissues but epididymis. There was no evidence of cell injury in tissues that had phospholipid accumulations except in rat skeletal muscle that had multifocal myofiber degeneration and necrosis. For clinical trials, serum AST and CK and peripheral blood lymphocyte vacuolation were considered potential safety biomarkers for skeletal muscle degeneration and phospholipidosis, respectively.