Drug-induced Phospholipidosis Research Articles

Dogs with diet-associated dilated cardiomyopathy have higher urine di-docosahexaenoyl (22:6)-bis(monoacylglycerol)phosphate, a biomarker of phospholipidosis.

In dogs with diet-associated dilated cardiomyopathy (DCM), we have identified electron microscopic changes suggestive of abnormal lysosomal accumulation of phospholipids and consistent with the appearance of drug-induced phospholipidosis in people and other animals. The objective of this study was to compare concentrations of urine di-docosahexaenoyl (22:6)-bis(monoacylglycerol)phosphate (BMP), a biomarker of drug-induced phospholipidosis, in dogs with DCM eating high-pulse (HP) diets, dogs with DCM eating low-pulse (LP) diets, and healthy controls (control-HP and control-LP). In this cross-sectional study, voided urine was collected from client-owned dogs with DCM from September 2018 through March 2020. Urine di-22:6-BMP was measured by LC-MS-MS and normalized to urine creatinine. Normalized di-22:6-BMP concentrations were compared among groups using mixed-effects-model analysis. 53 dogs were included: DCM-HP (n = 25), DCM-LP (n = 4), control-HP (n = 10), and control-LP (n = 14). Mixed-effects models adjusted for age and sex showed that HP diet was significantly associated with higher normalized urine di-22:6-BMP concentrations. A 1-way ANOVA identified a significant difference among the 4 groups, with Tukey post hoc analysis showing that the DCM-HP group had significantly higher normalized urine di-22:6-BMP concentrations compared to the control-LP group. Normalized di-22:6-BMP concentrations were significantly positively correlated with diet pulse scores (r = 0.52). High-pulse diets were significantly associated with higher normalized urine di-22:6-BMP concentrations. These results support the possible presence of primary or secondary phospholipidosis in dogs with diet-associated DCM and provide a plausible mechanism for further investigation.

The Bis(monoacylglycero)-phosphate Hypothesis: From Lysosomal Function to Therapeutic Avenues.

Lysosomes catabolize and recycle lipids and other biological molecules to maintain cellular homeostasis in diverse nutrient environments. Lysosomal lipid catabolism relies on the stimulatory activity of bis(monoacylglycero)phosphate (BMP), an enigmatic lipid whose levels are altered across myriad lysosome-associated diseases. Here, we review the discovery of BMP over half a century ago and its structural properties that facilitate the activation of lipid hydrolases and recruitment of their coactivators. We further discuss the current, yet incomplete, understanding of BMP catabolism and anabolism. To conclude, we discuss its role in lysosome-associated diseases and the potential for modulating its levels by pharmacologically activating and inhibiting the BMP synthase to therapeutically target lysosomal storage disorders, drug-induced phospholipidosis, Alzheimer's disease, Parkinson's disease, frontotemporal dementia, cancer, and viral infection.

Label-Free Screening of Drug-Induced Liver Injury Using Stimulated Raman Scattering Microscopy and Spectral Phasor Analysis.

Hepatic toxicity is a leading cause of the termination of clinical trials and the withdrawal of therapeutics following regulatory approval. The detection of drug-induced liver injury (DILI) is therefore of importance to ensure patient safety and the effectiveness of novel small molecules and drugs. DILI encompasses drug-induced steatosis (DIS) and drug-induced phospholipidosis (DIPL) which involve the accumulation of excess intracellular lipids. Here, we develop hyperspectral stimulated Raman scattering (SRS) microscopy as a label-free methodology for discriminating DIS and DIPL in mammalian cell culture. We demonstrate that hyperspectral SRS imaging in tandem with spectral phasor analysis is capable of discriminating DIS and DIPL based on the nature and distribution of intracellular lipids resulting from each process. To demonstrate the practical application of this methodology, we develop a panel of alkyne-tagged propranolol analogues that display varying DILI effects. Using hyperspectral SRS imaging together with spectral phasor analysis, our label-free methodology corroborated the standard fluorescence-based assay for DILI. As a label-free screening method, it offers a convenient and expedient methodology for visualizing hepatotoxicity in cell cultures which could be integrated into the early stages of the drug development process for screening new chemical entities for DILI.

The role of lysosomal phospholipase A2 in the catabolism of bis(monoacylglycerol)phosphate and association with phospholipidosis

Bis(monoacylglycerol)phosphate (BMP) is an acidic glycerophospholipid localized to late endosomes and lysosomes. However, the metabolism of BMP is poorly understood. Because many drugs that cause phospholipidosis inhibit lysosomal phospholipase A2 (LPLA2, PLA2G15, LYPLA3) activity, we investigated whether this enzyme has a role in BMPcatabolism. The incubation of recombinant human LPLA2 (hLPLA2) and liposomes containing the naturally occurring BMP (sn-(2-oleoyl-3-hydroxy)-glycerol-1-phospho-sn-1’-(2′-oleoyl-3′-hydroxy)-glycerol (S,S-(2,2′,C18:1)-BMP) resulted in the deacylation of this BMP isomer. The deacylation rate was 70 times lower than that of dioleoyl phosphatidylglycerol (DOPG), an isomer and precursor of BMP. The release rates of oleic acid from DOPG and four BMP stereoisomers by LPLA2 differed. The rank order of the rates of hydrolysis were DOPG>S,S-(3,3′,C18:1)-BMP>R,S-(3,1′,C18:1)-BMP>R,R-(1,1′,C18:1)>S,S-(2,2′)-BMP. The cationic amphiphilic drug amiodarone (AMD) inhibited the deacylation of DOPG and BMP isomers by hLPLA2 in a concentration-dependent manner. Under these experimental conditions, the IC50s of amiodarone-induced inhibition of the four BMP isomers and DOPG were less than 20 μM and approximately 30 μM, respectively. BMP accumulation was observed in AMD-treated RAW 264.7 cells. The accumulated BMP was significantly reduced by exogenous treatment of cells with active recombinant hLPLA2 but not with diisopropylfluorophosphate-inactivated recombinant hLPLA2. Finally, a series of cationic amphiphilic drugs known to cause phospholipidosis were screened for inhibition of LPLA2 activity as measured by either the transacylation or fatty acid hydrolysis of BMP or phosphatidylcholine as substrates. Fifteen compounds demonstrated significant inhibition with IC50s ranging from 6.8 to 63.3 μM. These results indicate that LPLA2 degrades BMP isomers with different substrate specificities under acidic conditions and may be the key enzyme associated with BMP accumulation in drug-induced phospholipidosis.

Livogrit prevents Amiodarone-induced toxicity in experimental model of human liver (HepG2) cells and Caenorhabditis elegans by regulating redox homeostasis

Treatment with cationic amphiphilic drugs like Amiodarone leads to development of phospholipidosis, a type of lysosomal storage disorder characterized by excessive deposition of phospholipids. Such disorder in liver enhances accumulation of drugs and its metabolites, and dysregulates lipid profiles, which subsequently leads to hepatotoxicity. In the present study, we assessed pharmacological effects of herbal medicine, Livogrit, against hepatic phospholipidosis-induced toxicity. Human liver (HepG2) cells and in vivo model of Caenorhabditis elegans (N2 and CF1553 strains) were used to study effect of Livogrit on Amiodarone-induced phospholipidosis. In HepG2 cells, Livogrit treatment displayed enhanced uptake of acidic pH-based stains and reduced phospholipid accumulation, oxidative stress, AST, ALT, cholesterol levels, and gene expression of SCD-1 and LSS. Protein levels of LPLA2 were also normalized. Livogrit treatment restored Pgp functionality which led to decreased cellular accumulation of Amiodarone as observed by UHPLC analysis. In C. elegans, Livogrit prevented ROS generation, fat-6/7 gene overexpression, and lysosomal trapping of Amiodarone in N2 strain. SOD-3::GFP expression in CF1553 strain normalized by Livogrit treatment. Livogrit regulates phospholipidosis by regulation of redox homeostasis, phospholipid anabolism, and Pgp functionality hindered by lysosomal trapping of Amiodarone. Livogrit could be a potential therapeutic intervention for amelioration of drug-induced phospholipidosis and prevent hepatotoxicity.

Constructing a graph neural network-based artificial intelligence model to predict drug-induced phospholipidosis potential

Constructing a graph neural network-based artificial intelligence model to predict drug-induced phospholipidosis potential

AMALPHI: A Machine Learning Platform for Predicting Drug-Induced PhospholIpidosis.

Drug-induced phospholipidosis (PLD) involves the accumulation of phospholipids in cells of multiple tissues, particularly within lysosomes, and it is associated with prolonged exposure to druglike compounds, predominantly cationic amphiphilic drugs (CADs). PLD affects a significant portion of drugs currently in development and has recently been proven to be responsible for confounding antiviral data during drug repurposing for SARS-CoV-2. In these scenarios, it has become crucial to identify potential safe drug candidates in advance and distinguish them from those that may lead to false in vitro antiviral activity. In this work, we developed a series of machine learning classifiers with the aim of predicting the PLD-inducing potential of drug candidates. The models were built on a high-quality chemical collection comprising 545 curated small molecules extracted from ChEMBL v30. The most effective model, obtained using the balanced random forest algorithm, achieved high performance, including an AUC value computed in validation as high as 0.90. The model was made freely available through a user-friendly web platform named AMALPHI (https://www.ba.ic.cnr.it/softwareic/amalphiportal/), which can represent a valuable tool for medicinal chemists interested in conducting an early evaluation of PLD inducer potential.

A machine learning and live-cell imaging tool kit uncovers small molecules induced phospholipidosis

Drug-induced phospholipidosis (DIPL), characterized by excessive accumulation of phospholipids in lysosomes, can lead to clinical adverse effects. It may also alter phenotypic responses in functional studies using chemical probes. Therefore, robust methods are needed to predict and quantify phospholipidosis (PL) early in drug discovery and in chemical probe characterization. Here, we present a versatile high-content live-cell imaging approach, which was used to evaluate a chemogenomic and a lysosomal modulation library. We trained and evaluated several machine learning models using the most comprehensive set of publicly available compounds and interpreted the best model using SHapley Additive exPlanations (SHAP). Analysis of high-quality chemical probes extracted from the Chemical Probes Portal using our algorithm revealed that closely related molecules, such as chemical probes and their matched negative controls can differ in their ability to induce PL, highlighting the importance of identifying PL for robust target validation in chemical biology.

Drug-induced phospholipidosis is not correlated with the inhibition of SARS-CoV-2 - inhibition of SARS-CoV-2 is cell line-specific.

Recently, Tummino etal. reported that 34 compounds, including Chloroquine and Fluoxetine, inhibit SARS-CoV-2 replication by inducing phospholipidosis, although Chloroquine failed to suppress viral replication in Calu-3 cells and patients. In contrast, Fluoxetine represses viral replication in human precision-cut lung slices (PCLS) and Calu-3 cells. Thus, it is unlikely that these compounds have similar mechanisms of action. Here, we analysed a subset of these compounds in the viral replication and phospholipidosis assays using the Calu-3 cells and PCLS as the patient-near system. Trimipramine and Chloroquine induced phospholipidosis but failed to inhibit SARS-CoV-2 replication in Calu-3 cells, which contradicts the reported findings and the proposed mechanism. Fluoxetine, only slightly induced phospholipidosis in Calu-3 cells but reduced viral replication by 2.7 orders of magnitude. Tilorone suppressed viral replication by 1.9 orders of magnitude in Calu-3 cells without causing phospholipidosis. Thus, induction of phospholipidosis is not correlated with the inhibition of SARS-CoV-2, and the compounds act via other mechanisms. However, we show that compounds, such as Amiodarone, Tamoxifen and Tilorone, with antiviral activity on Calu-3 cells, also inhibited viral replication in human PCLS. Our results indicate that antiviral assays against SARS-CoV-2 are cell-line specific. Data from Vero E6 can lead to non-transferable results, underlining the importance of an appropriate cell system for analysing antiviral compounds against SARS-CoV-2. We observed a correlation between the active compounds in Calu-3 cells and PCLS.

Fluorescent macrocycle-dye-anchor conjugates for sensing phospholipids in biomembranes

Negatively charged phospholipids can serve as biomarkers for a number of biological processes allowing detection of apoptosis in animal cells, drug-induced phospholipidosis as well as detection of bacteria. Selective detection of phospholipids thus represents an important but challenging goal. The development of fluorescent probes capable of selective recognition of specific phospholipids can be a promising solution to the problem. Along this line, we have developed the macrocycle-dye-anchor conjugates, in which the macrocycle recognizes the phosphate residue, hydrocarbon anchors ensure hydrophobic interaction with the membrane, and environment-sensitive fluorescent dye functions as a reporter unit. To the best of our knowledge, this is the first report of synthetic fluorescent probes for specific phospholipids using the macrocycle-dye-anchor concept. We have studied the effect of the structure of the anchor and the macrocycle on the resulting fluorescent response in the presence of two types of lipid vesicles: phosphatidylcholine as a model of healthy cells and phosphatidylcholine and phosphatidylserine (1:1), mimicking apoptotic cells. The properties of the obtained compounds were analyzed in various model media and in HeLa cells. The response of fluorescent probes has been found to be strongly dependent on the macrocycle structure, length of the anchor, nature of the buffer, and pH. The probes showed significant fluorescence enhancement in apoptotic cells, thus providing the proof of concept for the macrocycle-dye-anchor design.

Fosfolipidoza indukowana lekami – przyczyny, skutki, identyfikacja

Niepożądane działania leków stanowią istotny problem w farmakoterapii. Zaobserwowane działania uboczne mogą mieć wiele przyczyn. Mogą one zależeć od dawki i rodzaju zastosowanego leku. Mogą wystąpić w trakcie terapii lub pojawić się z opóźnieniem, po jej zakończeniu. Mogą zniknąć po przerwaniu leczenia lub utrzymywać się. W tym przeglądzie podsumowujemy obszerną literaturę dotyczącą fosfolipidozy, która jest główną konsekwencją stosowania kationowych leków amfifilowych (CAD). Ich specyficzna budowa chemiczna powoduje gromadzenie się fosfolipidów wewnątrz lizosomów, co wpływa na ich prawidłową funkcję, jednak mechanizm powstawania fosfolipidozy na poziomie molekularnym nie jest do końca poznany. Istnieje kilka hipotez mechanizmu powstawania fosfolipidozy - dotyczą one głównie: tworzenia kompleksów pomiędzy fosfolipidami a lekami CAD, kompetycyjnej inhibicji lizosomalnych fosfolipaz przez leki CAD oraz zwiększonej biosyntezy fosfolipidów i cholesterolu pod wpływem działania leku. W wyniku kumulacji fosfolipidów w lizosomach w obrazie mikroskopowym widoczne są tzw. ciałka blaszkowate, które mogą pojawić się w tkankach w kilka dni lub tygodni po podaniu CAD in vivo i jest to proces dawko-zależny. Uważa się, że fosfolipidoza jest procesem odwracalnym i przyjmuje się, że stanowi adaptacyjną - a nie toksyczną - reakcję na leki. Jeśli stężenie CAD lub toksycznej substancji skumulowanej w lizosomach przekroczy wartość krytyczną, może dojść do aktywacji procesu apoptozy i autofagii. Fosfolipidozę mogą wywołać także inne leki niż CAD, oksysterole oraz niektóre nanocząstki. Fosfolipidoza stanowi jeden z najsłabiej dotychczas poznanych powikłań farmakoterapii - nadal poszukiwane są zarówno sposoby jej wykrywania na początkowym etapie, jak również pełne spektrum zaburzeń czynnościowych organizmu. Identyfikacja procesu fosfolipidozy w komórkach jest możliwa poprzez badania w mikroskopii elektronowej stwierdzającej obecność ciałek blaszkowatych w tkankach pochodzących z biopsji lub za pomocą technik real-time PCR badającej ekspresję genów skorelowanych z występowaniem fosfolipidozy. Potencjalnym biomarkerem wykrywającym ten proces w krwi i moczu pacjentów jest bis(monoacyloglicero)fosforan (BMP). Fosfolipidoza wywołana działaniem substancji leczniczej może zostać wykryta na etapie badań przedklinicznych. Jednak akumulacja fosfolipidów i powstanie ciałek blaszkowatych stwierdzone w badaniach in vitro, czy w badaniach na zwierzętach, nie musi oznaczać uszkodzenia narządów w organizmie człowieka. W ostatnich latach obserwuje się wzrost zainteresowania mechanizmem procesu fosfolipidozy oraz badaniem nowych substancji leczniczych pod kątem wywoływania tego niepożądanego działania. Niniejsza praca przeglądowa przedstawia omówienie najistotniejszych dotychczasowych badań związanych z mechanizmem powstawania fosfolipidozy, metodami jej identyfikacji oraz skutkami na poziomie komórkowym, a także całego organizmu.

Identification of novel antiviral drug candidates using an optimized SARS-CoV-2 phenotypic screening platform

Reliable, easy-to-handle phenotypic screening platforms are needed for the identification of anti-SARS-CoV-2 compounds. Here, we present caspase 3/7 activity as a readout for monitoring the replication of SARS-CoV-2 isolates from different variants, including a remdesivir-resistant strain, and of other coronaviruses in numerous cell culture models, independently of cytopathogenic effect formation. Compared to other models, the Caco-2 subline Caco-2-F03 displayed superior performance. It possesses a stable SARS-CoV-2 susceptibility phenotype and does not produce false-positive hits due to drug-induced phospholipidosis. Aproof-of-concept screen of 1,796 kinase inhibitors identified known and novel antiviral drug candidates including inhibitors of phosphoglycerate dehydrogenase (PHGDH), CDC like kinase 1 (CLK-1), and colony stimulating factor 1 receptor (CSF1R). The activity of the PHGDH inhibitor NCT-503 was further increased in combination with the hexokinase II (HK2) inhibitor 2-deoxy-D-glucose, which is in clinical development for COVID-19. In conclusion, caspase 3/7 activity detection in SARS-CoV-2-infected Caco-2-F03 cells provides a simple phenotypic high-throughput screening platform for SARS-CoV-2 drug candidates that reduces false-positive hits.

Cell-Based Imaging Assay for Detection of Phospholipidosis.

Accumulation of lysosomal phospholipids in cells exposed to cationic amphiphilic drugs is characteristic of drug-induced phospholipidosis. The morphological hallmark of phospholipidosis is the appearance of unicentric or multicentric-lamellar bodies when viewed under an electron microscope (EM). The EM method, the gold standard of detecting cellular phospholipidosis, has downsides, namely, low-throughput, high-costs, and unsuitability for screening a large chemical library. This chapter describes a cell-based high-content phospholipidosis assay using the LipidTOX reagent in a high-throughput screening (HTS) platform. This assay has been optimized and validated in HepG2 and HepRG cells, and miniaturized into a 1536-well plate, thus can be used for high-throughput screening (HTS) to identify chemical compounds that induce phospholipidosis.

Raman and fluorescence imaging of phospholipidosis induced by cationic amphiphilic drugs in endothelial cells

Cationic amphiphilic drugs (CADs) are known from lysosomotropism, drug-induced phospholipidosis (DIPL), activation of autophagy, and decreased cell viability, but the relationship between these events is not clear and little is known about DIPL in the endothelium. In this work, the effects of fluoxetine, amiodarone, clozapine, and risperidone on human microvascular endothelial cells (HMEC-1) were studied using a combined methodology of label-free Raman imaging and fluorescence staining. Raman spectroscopy was applied to characterize biochemical changes in lipid profile and their distribution in the cellular compartments, while fluorescence staining (LysoTracker, LipidTOX, LC3B, and JC-1) was used to analyze lysosome volume expansion, activation of autophagy, lipid accumulation, and mitochondrial membrane depolarization. We demonstrated that fluoxetine, amiodarone, and clozapine, but not risperidone, at non-toxic concentrations induced lipid accumulations in the perinuclear and cytoplasmic regions of endothelial cells. Spectroscopic markers of DIPL included a robust increase in the ratio (lipid/(protein + lipid)), an increase in choline-containing lipid, fatty acids, and the presence of cholesterol esters, while starvation-induced activated autophagy revealed a spectroscopic signature associated with subtle changes in the lipid profile only. Interestingly, lysosomal volume expansion, occurrence of DIPL, and activation of autophagy induced by selected CADs all depended on drug-accumulation in acidic pH of lysosome cellular compartments whereas reduced endothelial viability did not, and was attributed to mitochondrial mechanisms as evidenced by a decreased mitochondrial transmembrane potential. In conclusion, drug-induced phospholipidosis in the endothelium did not reduce endothelial viability per se and can be efficiently assayed by Raman imaging.

Drug-Induced Lysosomal Impairment Is Associated with the Release of Extracellular Vesicles Carrying Autophagy Markers.

Amiodarone is a cationic amphiphilic drug used as an antiarrhythmic agent. It induces phospholipidosis, i.e., the accumulation of phospholipids within organelles of the endosomal–lysosomal system. Extracellular vesicles (EVs) are membrane-enclosed structures released by any type of cell and retrieved in every fluid of the body. EVs have been initially identified as a system to dispose cell waste, but they are also considered to be an additional manner to transmit intercellular signals. To understand the role of EVs in drug-induced phospholipidosis, we investigated EVs release in amiodarone-treated HEK-293 cells engineered to produce fluorescently labelled EVs. We observed that amiodarone induces the release of a higher number of EVs, mostly of a large/medium size. EVs released upon amiodarone treatment do not display significant morphological changes or altered size distribution, but they show a dose-dependent increase in autophagy associated markers, indicating a higher release of EVs with an autophagosome-like phenotype. Large/medium EVs also show a higher content of phospholipids. Drugs inducing lysosomal impairment such as chloroquine and bafilomycin A1 similarly prompt a higher release of EVs enriched in autophagy markers. This result suggests a mechanism associated with amiodarone-induced lysosomal impairment more than a connection with the accumulation of specific undigested substrates. Moreover, the implementation of the lysosomal function by overexpressing TFEB, a master gene regulator of lysosomal biogenesis, prevents the amiodarone-induced release of EVs, suggesting that this could be a feasible target to attenuate drug-induced abnormalities.

Defending Antiviral Cationic Amphiphilic Drugs That May Cause Drug-Induced Phospholipidosis.

A recent publication in Science has proposed that cationic amphiphilic drugs repurposed for COVID-19 typically use phosholipidosis as their antiviral mechanism of action in cells but will have no in vivo efficacy. On the contrary, our viewpoint, supported by additional experimental data for similar cationic amphiphilic drugs, indicates that many of these molecules have both in vitro and in vivo efficacy with no reported phospholipidosis, and therefore, this class of compounds should not be avoided but further explored, as we continue the search for broad spectrum antivirals.

Screening for drugs that don't work

Coronavirus In the battle against COVID-19, drugs discovered in repurposing screens are of particular interest because these could be rapidly implemented as treatments. However, Tummino et al. deliver a cautionary tale, finding that many leads from such screens have an antiviral effect in cells through phospholipidosis, a phospholipid storage disorder that can be induced by cationic amphiphilic drugs (see the Perspective by Edwards and Hartung). There is a strong correlation between drug-induced phospholipidosis and inhibition of severe acute respiratory syndrome coronavirus 2 replication in cells. Unfortunately, drugs that have an antiviral effect in cells through phospholipidosis are unlikely to be effective in vivo. Screening out such drugs may allow a focus on drugs with better clinical potential. Science , abi4708, this issue p. [541][1]; see also abj9488, p. [488][2] [1]: /lookup/doi/10.1126/science.abi4708 [2]: /lookup/doi/10.1126/science.abj9488

Drug-induced phospholipidosis confounds drug repurposing for SARS-CoV-2.

Repurposing drugs as treatments for COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has drawn much attention. Beginning with sigma receptor ligands and expanding to other drugs from screening in the field, we became concerned that phospholipidosis was a shared mechanism underlying the antiviral activity of many repurposed drugs. For all of the 23 cationic amphiphilic drugs we tested, including hydroxychloroquine, azithromycin, amiodarone, and four others already in clinical trials, phospholipidosis was monotonically correlated with antiviral efficacy. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the physicochemical properties of drugs and does not reflect specific target-based activities—rather, it may be considered a toxic confound in early drug discovery. Early detection of phospholipidosis could eliminate these artifacts, enabling a focus on molecules with therapeutic potential.

Development of acidic phospholipid containing immobilized artificial membrane column to predict drug-induced phospholipidosis potency

Drug-induced phospholipidosis (DIPLD) represents a big concern for both regulatory authorities and pharmaceutical companies in drug discovery. Many researches pointed out that the negatively charged intralysosomal lipids play an important role in the formation of DIPLD. To better mimic this negatively charged lipid surface, a novel immobilized artificial membrane (IAM) column was prepared via in situ copolymerization of 12-methacryloyl n-dodecylphosphocholine (MDPC) and 12-methacryloyl n-dodecylphosphoric acid (MDPA). By introducing MDPA, the surface of the resulting monolithic column can be maintained negatively charged over a broad pH range. Scanning electron microscopy, elemental analysis and nano-HPLC experiments were carried out to characterize the physicochemical properties and chromatographic performance of the obtained monolithic IAM column. The results of ζ-potential and retention mechanism studies indicate that both hydrophobic and electrostatic interactions contribute greatly to the retention of cation analytes owing to the existence of the negatively charged MDPA under acidic conditions. To better assess the DIPLD potency of drug, the molar ratio between MDPC and MDPA in the monolithic column was carefully optimized. The results show that the poly(MDPC70PA30-co-EDMA) column has the best predictability with only two false-positives (donepezil, flecainide) in qualitative analysis of 61 drugs.

Zebra Body Formation Due to Drug-Induced Phospholipidosis Observed on Electron Micrographs.

Zebra Body Formation Due to Drug-Induced Phospholipidosis Observed on Electron Micrographs.