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.

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 the drug-phospholipid interaction: correlation to phospholipidosis.

Phospholipid bilayers represent a complex, anisotropic environment fundamentally different from bulk oil or octanol, for instance. Even "simple" drug association to phospholipid bilayers can only be fully understood if the slab-of-hydrocarbon approach is abandoned and the complex, anisotropic properties of lipid bilayers reflecting the chemical structures and organization of the constituent phospholipids are considered. The interactions of drugs with phospholipids are important in various processes, such as drug absorption, tissue distribution, and subcellular distribution. In addition, drug-lipid interactions may lead to changes in lipid-dependent protein activities, and further, to functional and morphological changes in cells, a prominent example being the phospholipidosis (PLD) induced by cationic amphiphilic drugs. Herein we briefly review drug-lipid interactions in general and the significance of these interactions in PLD in particular. We also focus on a potential causal connection between drug-induced PLD and steatohepatitis, which is induced by some cationic amphiphilic drugs.

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.

Phospholipid membrane abnormalities and reduced niacin skin flush response in schizophrenia.

OBJECTIVES: Reduced n-3 and n-6 polyunsaturated fatty acids (PUFAs) content in red blood cell (RBC) membranes and abnormal membrane phospholipid metabolism were repeatedly implicated in the etiology of schizophrenia. FINDINGS: Prenatal and perinatal depletion of PUFAs interferes with normal brain development and function. The lack of docosahexaenoic acid - DHA in the brain is reflected in lower membrane DHA/AA (AA - arachidonic acid) ratio, increased activity of AA-metabolizing enzymes, and disturbance of downstream metabolic pathways involved in signaling, growth modulation, brain glucose uptake, immune functions, neurotransmission, synaptogenesis and neurogenesis. Preliminary high-throughput metabolomic studies revealed abnormal biochemical profile in patients with schizophrenia or brief psychotic disorder when compared to healthy controls. The results of both metabolomic and proteomic studies pointed to energy metabolism and lipid biosynthesis being impaired in schizophrenia. The usefulness of antipsychotic medication and supplementation with PUFAs in reverting to the normal metabolic state has been suggested in early treatment of the first psychotic episode. Abnormalities of phospholipid metabolism can be also detected as attenuated niacin skin flush response in the variety of neuropsychiatric disorders. CONCLUSIONS: Disturbances of lipid homeostasis could represent biochemical markers in the preclinical phase of neuropsychiatric illnesses and could serve as triggers in genetically vulnerable individuals. The assessment of patients' lipid status may also help in monitoring the course of the disease and treatment response. In this regard, simple, cheap and fast niacin skin flush test might be valuable. It might help in diagnosis of adolescents and young adults with psychotic behaviour, or in defining the necessity for long-term antipsychotic therapy. Along with antipsychotic medication schizophrenic patients need specific medical nutrition therapies.

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.

Protective effect of Chinese prescription Kangen-karyu and its crude drug Tanjin against age-related lipidosis in rats.

We have investigated the effect of the Chinese prescription Kangen-karyu and its crude drug Tanjin against age-related lipidosis in-vivo in a rat model. The serum and hepatic triglyceride levels were remarkably elevated in 12-month-old compared with two-month-old rats. However, the administration of Kangen-karyu and Tanjin extracts significantly decreased these levels. This suggested a protective role against related pathological conditions as well as hyperlipidaemia. On the other hand, the reduction of the levels of adiponectin in serum with ageing did not show significant changes in rats given diets supplemented with Kangen-karyu and Tanjin extracts. Furthermore, the expression of transcription factors in nuclear hepatic tissue related to lipid metabolism was investigated. The decline in the expression of nuclear peroxisome proliferator-activated receptor alpha protein in hepatic tissue with age was ameliorated by the administration of Kangen-karyu and Tanjin supplements. On the other hand, the overexpression of sterol regulatory element-binding proteins (SREBP)-1 and SREBP-2 in old rats compared with young rats showed a tendency to decrease with Kangen-karyu and Tanjin administration. The decline of hepatic function with ageing was attenuated by Kangen-karyu and Tanjin, suggesting the beneficial role of Kangen-karyu and Tanjin on lipid metabolism through the improvement of hepatic function. This study has demonstrated that Kangen-karyu and Tanjin inhibited the accumulation of triglyceride with regulation of related protein expressions and they improved hepatic function. Evidence has been provided for the anti-ageing activity of Kangen-karyu and its crude drug Tanjin against age-related lipidosis.

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.

Drug-induced phospholipidosis: issues and future directions.

Numerous drugs containing a cationic amphiphilic structure are capable of inducing phospholipidosis in cells under conditions of in vivo administration or ex vivo incubation. The principal characteristics of this condition include the reversible accumulation of polar phospholipids in association with the development of unicentric or multicentric lamellated bodies within cells. There is an abundance of data providing an understanding of potential mechanisms for the induction of phospholipidosis; however, the process is likely to be complex and may differ from one drug to another. The functional consequences of the presence of this condition on cellular or tissue function are not well understood. The general consensus is that the condition is an adaptive response rather than a toxicological manifestation; however, additional studies to examine this question are needed. Until this issue is resolved, concerns about phospholipidosis will continue to exist at regulatory agencies. Procedures for the screening of potential phospholipogenic candidate compounds are available. In contrast, a clear need exists for the identification of valid biomarkers to assess the development of phospholipidosis in preclinical and clinical studies.

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.

Dietary L-carnitine supplementation in obese cats alters carnitine metabolism and decreases ketosis during fasting and induced hepatic lipidosis.

This study was designed to determine whether dietary carnitine supplement could protect cats from ketosis and improve carnitine and lipid metabolism in experimental feline hepatic lipidosis (FHL). Lean spayed queens received a diet containing 40 (CL group, n = 7) or 1000 (CH group, n = 4) mg/kg of L-carnitine during obesity development. Plasma fatty acid, beta-hydroxybutyrate and carnitine, and liver and muscle carnitine concentrations were measured during experimental induction of FHL and after treatment. In control cats (CL group), fasting and FHL increased the plasma concentrations of fatty acids two- to threefold (P < 0.0001) and beta-hydroxybutyrate > 10-fold (from a basal 0.22 +/- 0.03 to 1.70 +/- 0.73 after 3 wk fasting and 3.13 +/- 0.49 mmol/L during FHL). In carnitine-supplemented cats, these variables increased significantly (P < 0.0001) only during FHL (beta-hydroxybutyrate, 1.42 +/- 0.17 mmol/L). L-Carnitine supplementation significantly increased plasma, muscle and liver carnitine concentrations. Liver carnitine concentration increased dramatically from the obese state to FHL in nonsupplemented cats, but not in supplemented cats, which suggests de novo synthesis of carnitine from endogenous amino acids in control cats and reversible storage in supplemented cats. These results demonstrate the protective effect of a dietary L-carnitine supplement against fasting ketosis during obesity induction. Increasing the L-carnitine level of diets in cats with low energy requirements, such as after neutering, and a high risk of obesity could therefore be recommended.

Testing a short-term feeding trial to assess compositional and histopathological changes in hearts of rats fed vegetable oils.

Male, female and castrated rats, three wk of age, were fed a low-fat diet for 14 wk followed by high-fat diets (20% by weight) for one wk containing graded levels of erucic acid from 1 to 50%, to evaluate the effect of short-term feeding and interaction of male sex hormones on formation of heart lesions. Some rats within each group were returned to the low-fat diet for one wk after the test period. For comparison, one group of three-wk-old male rats was fed the high fat 50% erucic acid diet for 15 wk. Erucic acid depressed growth rate and food consumption and increased cardiac lipidosis and triglycerides proportional to the erucic acid content of the diet. There were no sex differences, and the effects disappeared once rats were returned to the low-fat diet for one week. There was a significance (P less than 0.05) in the incidence of myocardial necrosis among male rats fed increased levels of erucic acid for one week, but the response was not linear to the increase in dietary erucic acid. Furthermore, the response was much less than in males fed the 50% erucic acid diet continually for 15 weeks. These results suggest that the short-term model is not a suitable substitute for the long-term feeding trial to test the cardiopathogenicity of a vegetable oil. The significantly lower incidence in myocardial lesions in female and castrated male rats compared with male rats suggests involvement of sex hormones.(ABSTRACT TRUNCATED AT 250 WORDS)

Selenium content of tissues in Finnish infants and adults with various diseases, and studies on the effects of selenium supplementation in neuronal ceroid lipofuscinosis patients.

A low blood selenium level has previously been observed in healthy inhabitants of Finland (WESTERMARCK et al. 1977). In this study even lower blood selenium values were observed in patients with acrodermatitis enteropathica, dystrophia musculorum progressiva (Duchenne), infantile and juvenile type of neuronal ceroid lipofuscinosis (NCL), severe mental retardation caused by various factors, and myocardial infarction. The selenium content of the brain, heart, kidney and liver in patients of different ages was also determined. The highest selenium level was found in the kidney. The mean liver selenium concentrations in stillborn, premature and full-term neonates were 1.11 +/- 0.23 (8), 1.21 +/- 0.17 (12) and 0.93 +/- 0.16 microgram/g dry weight (12) respectively (the number of subjects in parentheses). The selenium values are considerably higher than those in infants of from one to nine months of age and adults, whose liver selenium values were 0.58 +/- 0.21 (8) and 0.67 +/- 0.08 microgram/g dry weight (8) respectively. The vitamin E levels of serum in patients with NCL, as well as in subjects with severe mental retardation (controls), were low compared with values in healthy normal subjects. Sodium selenite supplementation in patients with NCL produced at least a transitory improvement without causing any toxic effects during one year of administration.

Carnitine effect on heart steatosis induced in rats by rapeseed oil.

Myocardial triglyceride levels in rats fed a high erucic acid rapeseed oil diet for three days were five times higher than in controls. The incorporation of erucic acid and, to a lower extent, some unsaturated fatty acids was increased, as well as total cholesterol content, compared to controls. The presence of 5% carnitine in the diet partially prevented these effects. It is assumed that carnitine may be a rate limiting factor in the myocardial catabolism of unsaturated fatty acids and particularly erucic acid, when these substance are ingested in supraoptimal amount.