Induction Of Phospholipidosis Research Articles

Side effects of oxysterols: cytotoxicity, oxidation, inflammation, and phospholipidosis

Oxysterols are 27-carbon atom molecules resulting from autoxidation or enzymatic oxidation of cholesterol. They are present in numerous foodstuffs and have been demonstrated to be present at increased levels in the plasma of patients with cardiovascular diseases and in atherosclerotic lesions. Thus, their role in lipid disorders is widely suspected, and they might also be involved in important degenerative diseases such as Alzheimer's disease, osteoporosis, and age-related macular degeneration. Since atherosclerosis is associated with the presence of apoptotic cells and with oxidative and inflammatory processes, the ability of some oxysterols, especially 7-ketocholesterol and 7beta-hydroxycholesterol, to trigger cell death, activate inflammation, and modulate lipid homeostasis is being extensively studied, especially in vitro. Thus, since there are a number of essential considerations regarding the physiological/pathophysiological functions and activities of the different oxysterols, it is important to determine their biological activities and identify their signaling pathways, when they are used either alone or as mixtures. Oxysterols may have cytotoxic, oxidative, and/or inflammatory effects, or none whatsoever. Moreover, a substantial accumulation of polar lipids in cytoplasmic multilamellar structures has been observed with cytotoxic oxysterols, suggesting that cytotoxic oxysterols are potent inducers of phospholipidosis. This basic knowledge about oxysterols contributes to a better understanding of the associated pathologies and may lead to new treatments and new drugs. Since oxysterols have a number of biological activities, and as oxysterol-induced cell death is assumed to take part in degenerative pathologies, the present review will focus on the cytotoxic activities of these compounds, the corresponding cell death signaling pathways, and associated events (oxidation, inflammation, and phospholipidosis).

NMR-based urine analysis in rats: Prediction of proximal tubule kidney toxicity and phospholipidosis

Introduction The aim of safety pharmacology is early detection of compound-induced side-effects. NMR-based urine analysis followed by multivariate data analysis (metabonomics) identifies efficiently differences between toxic and non-toxic compounds; but in most cases multiple administrations of the test compound are necessary. We tested the feasibility of detecting proximal tubule kidney toxicity and phospholipidosis with metabonomics techniques after single compound administration as an early safety pharmacology approach. Methods Rats were treated orally, intravenously, inhalatively or intraperitoneally with different test compounds. Urine was collected at 0–8 h and 8–24 h after compound administration, and 1H NMR-patterns were recorded from the samples. Variation of post-processing and feature extraction methods led to different views on the data. Support Vector Machines were trained on these different data sets and then aggregated as experts in an Ensemble. Finally, validity was monitored with a cross-validation study using a training, validation, and test data set. Results Proximal tubule kidney toxicity could be predicted with reasonable total classification accuracy (85%), specificity (88%) and sensitivity (78%). In comparison to alternative histological studies, results were obtained quicker, compound need was reduced, and very importantly fewer animals were needed. In contrast, the induction of phospholipidosis by the test compounds could not be predicted using NMR-based urine analysis or the previously published biomarker PAG. Discussion NMR-based urine analysis was shown to effectively predict proximal tubule kidney toxicity after single compound administration in rats. Thus, this experimental design allows early detection of toxicity risks with relatively low amounts of compound in a reasonably short period of time.

Proteomic analysis of phospholipidosis in citalopram treated U937 cells – Support for the cholesterol biosynthesis hypothesis

Excessive accumulation of phospholipids leads to phospholipidosis (PL), which disrupts cellular functions, in extreme cases leading to acute or chronic disease. Citalopram and many other cationic amphiphilic drugs (CADs) have been shown to cause PL both in vitro and in vivo. Recent toxicogenomic studies suggest four hypothetical mechanisms for PL (lysosomal enzyme transport decrease, lysosomal phospholipase activity decrease, phospholipids biosynthesis increase or cholesterol biosynthesis increase). However, the post-genomic steps remain largely unknown and proteomic analyses hold significant promise for defining mechanisms of PL induction. In this study U937 monoblastoid cells were exposed to citalopram hydrobromide for 24 h (0, 20, 100 or 200 microM as citalopram free base) and then harvested for whole cell proteomic analysis using 2-D gel electrophoresis, or transmission electron microscopy (TEM). Protein spots that were significantly altered versus controls were analysed by MALDI-TOF mass spectrometry. Up-regulated proteins were Glyoxalase-I (Glo 1) and 3-Hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (HMGCS1) in cells with PL shown by TEM (favouring the cholesterol biosynthesis increase hypothesis for citalopram induced PL). Other altered proteins were catalase (up-regulated), beta-actin (up-regulated) and 14-3-3 protein (down-regulated). The function of several of the successfully identified proteins indicates a potential perturbed lipid metabolism.

Gene expression profiling in rat liver treated with compounds inducing phospholipidosis

We have constructed a large-scale transcriptome database of rat liver treated with various drugs. In an effort to identify a biomarker for diagnosis of hepatic phospholipidosis, we extracted 78 probe sets of rat hepatic genes from data of 5 drugs, amiodarone, amitriptyline, clomipramine, imipramine, and ketoconazole, which actually induced this phenotype. Principal component analysis (PCA) using these probes clearly separated dose- and time-dependent clusters of treated groups from their controls. Moreover, 6 drugs (chloramphenicol, chlorpromazine, gentamicin, perhexiline, promethazine, and tamoxifen), which were reported to cause phospholipidosis but judged as negative by histopathological examination, were designated as positive by PCA using these probe sets. Eight drugs (carbon tetrachloride, coumarin, tetracycline, metformin, hydroxyzine, diltiazem, 2-bromoethylamine, and ethionamide), which showed phospholipidosis-like vacuolar formation in the histopathology, could be distinguished from the typical drugs causing phospholipidosis. Moreover, the possible induction of phospholipidosis was predictable by the expression of these genes 24 h after single administration in some of the drugs. We conclude that these identified 78 probe sets could be useful for diagnosis of phospholipidosis, and that toxicogenomics would be a promising approach for prediction of this type of toxicity.

P.1.c.010 Gender-dependent influence of bupropion pretreatment on locomotor activity and the activity of hepatic CYP2D2 in rats

One of the most important enzymatic systems participating in drug metabolism is cytochrome P450 (CYP450). CYP450 consists of many isoforms which activity of is influenced by numerous endogenous (e.g. gender, pathophysiological status, age) and exogenous factors (e.g. xenobiotical inhibitors and inducers). Although bupropion is not metabolized by CYP 2D6 (human ortologue of rat CYP2D2), there is certain potential for interaction when bupropion is co-administered with other drugs metabolized by this isoenzyme, too. The aim of the present study was to determine the influence of gender and administration of bupropion on the CYP450 isoform 2D2 (CYP2D2) activity. As selective markers of the metabolic activity of CYP2D2 dextromethorphan (DEM) and its metabolite dextrorphane (DOR) were used. In animals treated with bupropion compared to control animals administered with saline the inhibitory effect of bupropion was expressed as a decrease of DOR in the 30th, 60th and 120th minutes of perfusion. The influence of gender on locomotor activity has shown that activity in intact females were similar as in the intact males. In the bupropion treated animals females were significantly more active in subsequent open field tests then in males.

Adaptation of an In Vitro Phospholipidosis Assay to an Automated Image Analysis System

ABSTRACTPhospholipidosis is the excessive intralysosomal accumulation of phospholipids and is induced in humans and animals by the chronic administration of cationic amphiphilic drugs. To identify compounds that may induce phospholipidosis early in the discovery process, we have developed a predictive fluorescent cell-based assay amenable to automated high content screening using the 2-(4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (ß-BODIPY C12-HPC) dye and primary rat hepatocytes. ß-BODIPY C12-HPC localized to lysosomes that accumulate phospholipids and not to lipid droplets, indicating the selectivity for phospholipid-containing granules. Accumulation of ß-BODIPY C12-HPC was monitored in primary rat hepatocytes plated onto 96-well plates and 24 h after exposure to increasing concentrations of 13 drugs known to induce phospholipidosis and four negative compounds. Fluorescent images were captured and analyzed using the Discovery-1 automated cellular imaging system. Eleven out of the 12 selected positive compounds and all negative compounds were properly assigned as positive and negative inducers of phospholipidosis, respectively, indicating the high degree of sensitivity and specificity of this assay. The ability of ß-BODIPY C12-HPC to detect and quantify phospholipidosis is similar to that of the well-established probe, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dipalmitoylphosphatidylethanolamine (NBD-PE).

Biomarkers to monitor drug-induced phospholipidosis

Di-docosahexaenoyl (C22:6)-bis(monoacylglycerol) phosphate (BMP) was identified as a promising phospholipidosis (PL) biomarker in rats treated with either amiodarone, gentamicin, or azithromycin. Sprague-Dawley rats received either amiodarone (150 mg/kg), gentamicin (100 mg/kg) or azithromycin (30 mg/kg) once daily for ten consecutive days. Histopathological examination of tissues by transmission electron microscopy (TEM) indicated different degrees of accumulation of phospholipidosis in liver, lung, mesenteric lymph node, and kidney of drug-treated rats but not controls. Liquid chromatography coupled to mass spectrometry (LC/MS) was used to identify levels of endogenous biochemical profiles in rat urine. Urinary levels of di-docosahexaenoyl (C22:6)-bis(monoacylglycerol) phosphate (BMP) correlated with induction of phospholipidosis for amiodarone, gentamicin and azithromycin. Rats treated with gentamicin also had increased urinary levels of several phosphatidylinositol (PI), phosphatidylcholine (PC), and phosphatidylethanolamine (PE) species.

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.

Improved toxicogenomic screening for drug-induced phospholipidosis using a multiplexed quantitative gene expression ArrayPlate assay

We previously showed that a toxicogenomics analysis of drug-induced phospholipidosis enabled the identification of 12 specific gene markers and the establishment of an in vitro real-time PCR screening assay for the assessment of the phospholipidosis-inducing potential of compounds. The purpose of this study was to transfer our PCR-based assay into a 96-well microplate-based multiple mRNAs measuring assay (ArrayPlate™ assay) in order to increase throughput. Specifically, we determined the expression of the 12 marker genes using real-time PCR and ArrayPlate in human hepatoma HepG2 cells that were treated for 24 h with each of amiodarone and 80 proprietary compounds. The following three performance criteria were satisfied in the ArrayPlate analysis: 1. Sensitivity—the expression of mRNA for all target genes was detected at quantifiable levels. 2. Repeatability—signal intensities and fold change values of each marker gene were highly repeatable. 3. Correlation—fold change values and their average values, which were used as indices of phospholipidosis induction potential, showed apparent correlation between the ArrayPlate and real-time PCR assays. Thus, the in vitro screening assay for compound-induced phospholipidosis should be transferable from a PCR-based assay to the higher-throughput ArrayPlate-based method.

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.

A toxicogenomic approach to drug-induced phospholipidosis: analysis of its induction mechanism and establishment of a novel in vitro screening system.

Phospholipidosis is a lipid storage disorder in which excess phospholipids accumulate within cells. Some cationic amphiphilic compounds are known to have the potential to induce phospholipidosis. This study was undertaken to examine the molecular mechanisms that contribute to the development of phospholipidosis and to identify specific markers that might form the basis of an in vitro screening test. Specifically, we performed a large-scale gene expression analysis using DNA microarrays on human hepatoma HepG2 cells after they were treated with each of 12 compounds known to induce phospholipidosis. In electron microscopy, HepG2 cells developed lamellar myelin-like bodies in their lysosomes, the characteristic change of phospholipidosis, after treatment with these compounds for 72 h. DNA microarray analysis performed 6 and 24 h after treatment showed alterations in gene expression reflecting the inhibition of lysosomal phospholipase activity and lysosomal enzyme transport, and the induction of phospholipid and cholesterol biosynthesis. Seventeen genes that showed a similar expression profile following treatment were selected as candidate markers. Real-time PCR analysis confirmed that 12 gene markers showed significant concordance with lamellar myelin-like body formation. Furthermore, the average fold change values of these markers correlated well with the magnitude of this pathological change. In conclusion, microarray analysis revealed that factors such as alterations in lysosomal function and cholesterol metabolism were involved in the induction of phospholipidosis. Furthermore, comprehensive gene expression analysis enabled us to identify biomarkers of this condition that we then used to develop a rapid and sensitive in vitro screening test for drug-induced phospholipidosis.

Use of physicochemical calculation of pKa and CLogP to predict phospholipidosis-inducing potential: A case study with structurally related piperazines

Several cationic amphiphilic compounds are known to induce phospholipidosis, a condition primarily characterized by excessive accumulation of phospholipids in different cell types, giving the affected cells a finely foamy appearance. Excessive storage of lamellar membranous intralysosomal inclusion bodies is the hallmark for phospholipidosis on the electron microscopic level. In case of alveolar phospholipidosis, foamy macrophages accumulate within the alveolar spaces of the lung. Based on such findings in a one-year toxicity study with gepirone in rats, we studied the molecular properties of this compound and compounds suspected of being phospholipidosis inducers by means of physicochemical calculations. Physicochemical molecular calculations of molecular weight, ClogP (partition coefficient octanol/water), logD at pH 7.4, and pKa were performed, for the cationic amphiphilic compounds chlorpromazine, amiodarone, imipramine, propranolol and fluoxetine, and for the structurally related compounds 1-phenylpiperazine (1-PHP), gepirone (and its major metabolites, 3-OH-gepirone and 1-pyrimidinylpiperazine [1-PP]), and buspirone. ClogP and calculated pKa cluster differently for the amphiphilic drugs compared to the chemical series of piperazines. In line with this analysis, lamellar inclusion bodies were found in an in vitro validation experiment in the human monoblastoid cell line U-937, incubated for 96 h at 10 microg/mL with cationic amphiphilic drugs (amiodarone, imipramine, or propranolol). No such lamellar inclusion bodies were seen for any of the compounds from the chemical series of piperazines including gepirone and its metabolites. The data presented support the use of simple physicochemical calculations of ClogP and pKa to discriminate rapidly between compounds suspected of being phospholipidosis inducers. Finally, the discriminative power of these physicochemical ClogP and pKa calculations to predict phospholipidosis-inducing potential was further validated by extension of the set of compounds.

Use of physicochemical calculation of pKa and CLogP to predict phospholipidosis-inducing potentialA case study with structurally related piperazines

Several cationic amphiphilic compounds are known to induce phospholipidosis, a condition primarily characterized by excessive accumulation of phospholipids in different cell types, giving the affected cells a finely foamy appearance. Excessive storage of lamellar membranous intralysosomal inclusion bodies is the hallmark for phospholipidosis on the electron microscopic level. In case of alveolar phospholipidosis, foamy macrophages accumulate within the alveolar spaces of the lung. Based on such findings in a one-year toxicity study with gepirone in rats, we studied the molecular properties of this compound and compounds suspected of being phospholipidosis inducers by means of physicochemical calculations. Physicochemical molecular calculations of molecular weight, ClogP (partition coefficient octanol/water), logD at pH 7.4, and pKa were performed, for the cationic amphiphilic compounds chlorpromazine, amiodarone, imipramine, propranolol and fluoxetine, and for the structurally related compounds 1-phenylpiperazine (1-PHP), gepirone (and its major metabolites, 3-OH-gepirone and 1-pyrimidinylpiperazine [1-PP]), and buspirone. ClogP and calculated pKa cluster differently for the amphiphilic drugs compared to the chemical series of piperazines. In line with this analysis, lamellar inclusion bodies were found in an in vitro validation experiment in the human monoblastoid cell line U- 937, incubated for 96 h at 10 μg/mL with cationic amphiphilic drugs (amiodarone, imipramine, or propranolol). No such lamellar inclusion bodies were seen for any of the compounds from the chemical series of piperazines including gepirone and its metabolites. The data presented support the use of simple physicochemical calculations of ClogP and pKa to discriminate rapidly between compounds suspected of being phospholipidosis inducers. Finally, the discriminative power of these physicochemical ClogP and pKa calculations to predict phospholipidosis-inducing potential was further validated by extension of the set of compounds.

A Quantitative Image-Based in vitro Assay for Induction of Phospholipidosis in Hepatocytes

A Quantitative Image-Based in vitro Assay for Induction of Phospholipidosis in Hepatocytes

Analysis of two matrix metalloproteinase inhibitors and their metabolites for induction of phospholipidosis in rat and human hepatocytes

ABT-770 [( S)- N-[1-[[4′-trifluoromethoxy-[1,1′-biphenyl]-4-yl]oxy]methyl-2-(4,4-dimethyl-2,5-dioxo-1-imidazolidinyl)ethyl]- N-hydroxyformamide], a matrix metalloproteinase inhibitor (MMPI), produced generalized phospholipidosis in rats. Phospholipid accumulation was accompanied by retention of drug-related material and was associated with increased mortality. Generation of a successful drug candidate depended upon understanding the cause of the phospholipidosis and redesigning the chemical structure accordingly. ABT-770 and other MMPIs, plus several metabolites of each, were assayed for their ability to induce phospholipidosis in primary cultured rat and human hepatocytes. Phospholipid accumulation was detected by following the incorporation of a fluorescent phospholipid analogue into intracytoplasmic inclusion bodies characteristic of phospholipid storage disorders. At 24 and 48 hr, none of the parent compounds induced phospholipidosis in vitro in rat or human hepatocytes. Phospholipidosis was associated primarily with an amine metabolite of ABT-770. The amine metabolite of another MMPI, ABT-518 ([ S-( R∗, R∗)]- N-[1-(2,2-dimethyl-1,3-dioxol-4-yl)-2-[[4-[4-(trifluoromethoxy)-phenoxy]phenyl]sulfonyl]ethyl]- N-hydroxyformamide), produced little phospholipidosis in rat and human hepatocytes even at concentrations up to 100 μM. The presence or absence of phospholipidosis in the in vitro assay correlated well with ultrastructural findings and drug accumulation in rat tissues. ABT-770, which produced phospholipidosis associated with its amine metabolite in vitro and in vivo, also generated a higher tissue to plasma distribution of metabolites particularly in tissues where phospholipidosis was observed. ABT-518 and its amine metabolite, however, produced low tissue to plasma ratios and induced little to no phospholipidosis in vitro or in vivo. These results demonstrate that the phospholipidosis observed for ABT-770 could be attributed to a cationic metabolite, and that altering the properties of such a metabolite, by modification of the parent compound, alleviated the disorder.

Technetium-99m DTPA inhalation scintigraphy in patients treated with fluoxetine and maprotiline: preliminary results.

Drug-metabolising enzymatic activities have been detected in tracheobronchiolar, bronchiolar and alveolar regions in the lungs. Induction of phospholipidosis by amine drugs such as clorphentermine has also been shown. This study aimed to investigate the effect of fluoxetine and maprotiline, which contain amine groups in their structure, on pulmonary epithelial membrane permeability. Twenty-seven patients (mean age 36+/-12 years) with various psychiatric problems, of whom 17 were treated with fluoxetine and 10 with maprotiline, were included in this study. Technetium-99m diethylene triamine penta-acetic acid (DTPA) aerosol inhalation scintigraphy was performed before and after 4-6 weeks of therapy. Following the inhalation of 1480 MBq 99mTc-DTPA for 3 min, lung images in a 64x64 matrix were obtained every minute for 30 min. Regions of interest were drawn around the periphery of the lungs and on the major airways. Clearance half-times (T 1/2) were calculated by placing a mono-exponential fit on the curves. Penetration index (PI) was calculated on the first-minute image. There was no difference between the clearance rates of 99mTc-DTPA before and after therapy for either the fluoxetine or the maprotiline group. After therapy, a significant decrease in PI was found in patients treated with fluoxetine (PI values before and after therapy: 0.53+/-0.03 and 0.49+/-0.05 respectively, P< or =0.05). This finding might have been due to the induction of increased synaptic serotonin (5-HT) by fluoxetine, which acts by inhibiting the re-uptake of 5-HT on presynaptic membranes. Bronchoconstriction of small and medium airways may be caused by direct and indirect effects of 5-HT on smooth muscle contraction.

Cationic amphiphilic drug-induced phospholipidosis.

Phospholipidosis, a phospholipid storage disorder, defines an excessive accumulation of intracellular phospholipids. Phospholipids are structural components of mammalian cytoskeleton and cell membranes. The metabolism of this essential cell component is regulated by the individual cell and may be altered by drugs that interact with phospholipids or the enzymes that affect their metabolism. Xenobiotics or their metabolites that induce phospholipidosis include a wide variety of pharmacologic agents, including antibacterials, antipsychotics, antidepressants, antiarrhythmics, antianginals, antimalarials, anorexic agents, cholesterol-lowering agents, and others. Each of these drugs shares several common physiochemical properties: hydrophobic ring structure on the molecule and a hydrophilic side chain with a charged cationic amine group, hence the class term cationic amphiphilic drugs (CADs). This paper reviews the phospholipid metabolism, physiochemical characteristics of CADs, specificity of phospholipidosis in animals and humans, functional effects of phospholipidosis, interaction of CADs with biologic membranes and lysosome metabolism, influence of CADs on phospholipases and phospholipid synthesis, and a proposed mechanism for induction of phospholipidosis in the lung. In human risk assessment, investigators should consider the many factors in evaluating a drug that induces phospholipidosis in animals. These include: the therapeutic class of drug, presence of active metabolites, tissue or organ selectivity in animals and humans, influence of concurrently administered drugs, reversibility of effect, and other factors that increase or decrease the induction of phospholipidosis. Generalities regarding the etiology, incidence, and effect of the drug on a specific host may not be made. Each drug must be evaluated separately to identify the risk when administered for therapeutic effect in humans.

Ketoconazole-induced hepatic phospholipidosis in the mouse and its association with de- N-acetyl ketoconazole

Ketoconazole (KC), an orally effective systemic antifungal agent, has been associated with symptomatic hepatotoxicity with an incidence as low as 1 in 2000. Studies from this laboratory have shown that in the mouse ketoconazole elicit a biphasic effect on drug metabolism and induced phospholipidosis. The pathogenesis of the latter, however, has never been established. Studies in mice demonstrated that ketoconazole administration induced phospholipid accumulation in the liver in a dose and time dependent fashion; and de- N-acetyl ketoconazole (DAKC), a major hepatic metabolite of KC was associated with this biochemical change. A comparative biochemical study following equimolar (0.47 nmol/kg p.o.× 7 days) administration of these two compounds indicated that hepatic phospholipids were elevated to a greater extent by DAKC treatment that by KC. Hepatic profiles of KC, DAKC, and other metabolites at 2, 7.5 and 24 h following single and multiple dosing regimens with either KC or DAKC indicated that KC was readily metabolized to DAKC whereas, DAKC appeared to be recalcitrant to metabolism and accumulated in the liver. In contrast to the biphasic effects of KC on hepatic enzyme activity observed previously following the administration of KC (enzyme inhibition as well as induction), the biological effects of DAKC were consistent with only an enzyme inhibitory effect: liver microsomal protein was not elevated; cytochrome P-450 was depressed; and ethylmorphine N-demethylase and benzphetamine N-demethylase were inhibited. Consequently the induction of phospholipidosis and the inhibition of drug metabolism associated with ketoconazole treatment were attributed to DAKC, whereas the inductive properties of KC were ascribed to the unchanged drug. The dramatic difference in the biological effects of these two compounds was attributed to differences in the orientation of these agents in lipid membranes. These results offer an explanation for the previously observed apparent inhibitory effects of KC on enzyme activities (Whitehouse et al. (1990b) Hepatic effects of ketoconazole in the male Swiss Webster mouse: temporal changes in drug metabolic parameters. Can. J. Physiol. Pharmacol., 68, 1136–1142) and suggest that DAKC may be the chemical entity responsible for the induction of phospholipidosis following ketoconazole administration.

In vivo toxicity and pulmonary effects of promazine and chlorpromazine in rats

Cationic amphiphilic drugs induce a phospholipid storage disorder known as phospholipidosis. Halogenated analogs of the drugs are more potent inducers of phospholipidosis when compared to nonhalogenated analogs. Two such antipsychotic drugs, promazine and chlorpromazine, are effectively taken up by the lungs and induce lamellar inclusions in vitro. We compared the in vivo toxicity and efficacy of promazine and chlorpromazine to induce phospholipidosis in the lung and in pulmonary alveolar macrophages. Male Sprague-Dawley rats were given promazine or chlorpromazine (25 mg/kg/day, P.O., in water) for 5 weeks. Food intake was decreased in promazine- and chlorpromazine-treated rats, chlorpromazine rats being affected more than promazine rats. To minimize experimental error due to starvation, control rats were pair-fed. The body weight gain was decreased in chlorpromazine rats in comparison to pair-fed controls. Chlorpromazine-treated rats, but not promazine-treated rats, showed increased mortality over the 5-week treatment period. Histopathologic examination of lung revealed loss of alveolar macrophages with no other gross abnormalities in chlorpromazine-treated rats. Quantitative analysis of lung lavage also showed significant reduction in the number of macrophages. This finding is in contrast to other cationic amphiphilic drugs, which induce phospholipidosis as well as accumulation of alveolar macrophages. Phospholipid level increased in alveolar macrophages but not in lavaged lung following chlorpromazine treatment. Acid phosphatase activity in lavaged lung homogenate and macrophages of promazine- and chlorpromazine-treated rats, taken as an index of toxicity to cells, did not differ significantly from control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological profile of tazasubrate, A new cholesterol-lowering agent

The properties of tazasubrate (2-phenyl-2-[(6-ethoxy-2-benzothiazolyl)thiolpropionic acid), a potent hypocholesterolemic agent, were studied in rats. Tazasubrate was found to be a reliable and highly effective hypocholesterolemic agent. There was a marked and reproducible reduction of serum cholesterol in various rat models differing in age, sex and diet, an improvement of the pathological lipoprotein pattern, slight but variable effects on serum triglycerides and phospholipids, no accumulation of intermediates of cholesterol biosynthesis, no inhibition of phospholipid metabolism (i.e. no induction of phospholipidosis), no interaction with the thyroid gland, and in contrast to fibrates, only minimal induction of peroxisomes in hepatocytes.