Lamellar Inclusion Bodies Research Articles

Zebra Bodies in the Kidney: Is it a Pathognomonic Finding of Fabry Disease?

Zebra bodies are intralysosomal lamellar inclusion bodies. It is accepted as a specific feature of Fabry disease. However, it has been reported in many hereditary and acquired conditions. We are reporting Zebra bodies in the kidneys of cases with Rheumatoid Arthritis and hydroxychloroquine-induced phospholipidosis. Case 1: A 55-year-old male presented with hypertension and renal dysfunction. Serum ANA and anti-CCP antibodies were positive. A kidney biopsy revealed chronic tubulointerstitial nephritis with Zebra Bodies in the podocytes. Genetic analysis was negative for Fabry disease. Case 2: A 34-year-old female with Systemic Lupus Erythematosus on Hydroxychloroquine for a year presented with subnephrotic proteinuria. Serum ANA and anti-dsDNA antibodies were positive. Electron microscopy showed lamellated osmiophilic inclusion bodies in the tubular and visceral epithelial cells. Thus, Zebra bodies are not pathognomonic for Fabry disease and Rheumatoid Arthritis should also be considered in the differential diagnosis, particularly if family or drug history is negative.

An Unusual Case of Kidney Infiltration by Heavy Chain Disease with Lamellar Inclusion Bodies

An Unusual Case of Kidney Infiltration by Heavy Chain Disease with Lamellar Inclusion Bodies

Fabry disease exacerbates renal interstitial fibrosis after unilateral ureteral obstruction via impaired autophagy and enhanced apoptosis.

BackgroundFabry disease is a rare X-linked genetic lysosomal disorder caused by mutations in the GLA gene encoding alpha-galactosidase A. Despite some data showing that profibrotic and proinflammatory cytokines and oxidative stress could be involved in Fabry disease-related renal injury, the pathogenic link between metabolic derangement within cells and renal injury remains unclear.MethodsRenal fibrosis was triggered by unilateral ureteral obstruction (UUO) in mice with Fabry disease to investigate the pathogenic mechanism leading to fibrosis in diseased kidneys.ResultsCompared to kidneys of wild-type mice, lamellar inclusion bodies were recognized in proximal tubules of mice with Fabry disease. Sirius red and trichrome staining revealed significantly increased fibrosis in all UUO kidneys, though it was more prominent in obstructed Fabry kidneys. Renal messenger RNA levels of inflammatory cytokines and profibrotic factors were increased in all UUO kidneys compared to sham-operated kidneys but were not significantly different between UUO control and UUO Fabry mice. Protein levels of Nox2, Nox4, NQO1, catalase, SOD1, SOD2, and Nrf2 were not significantly different between UUO control and UUO Fabry kidneys, while the protein contents of LC3-II and LC3-I and expression of Beclin1 were significantly decreased in UUO kidneys of Fabry disease mouse models compared with wild-type mice. Notably, TUNEL-positive cells were elevated in obstructed kidneys of Fabry disease mice compared to wild-type control and UUO mice.ConclusionThese findings suggest that impaired autophagy and enhanced apoptosis are probable mechanisms involved in enhanced renal fibrosis under the stimulus of UUO in Fabry disease.

Exploiting ensemble learning to improve prediction of phospholipidosis inducing potential

Phospholipidosis is characterized by the presence of excessive accumulation of phospholipids in different tissue types (lungs, liver, eyes, kidneys etc.) caused by cationic amphiphilic drugs. Electron microscopy analysis has revealed the presence of lamellar inclusion bodies as the hallmark of phospholipidosis. Some phospholipidosis causing compounds can cause tissue specific inflammatory/retrogressive changes. Reliable and accurate in silico methods could facilitate early screening of phospholipidosis inducing compounds which can subsequently speed up the pharmaceutical drug discovery pipelines. In the present work, stacking ensembles are implemented for combining a number of different base learners to develop predictive models (a total of 256 trained machine learning models were tested) for phospholipidosis inducing compounds using a wide range of molecular descriptors (ChemMine, JOELib, Open babel and RDK descriptors) and structural alerts as input features. The best model consisting of stacked ensemble of machine learning algorithms with random forest as the second level learner outperformed other base and ensemble learners. JOELib descriptors along with structural alerts performed better than the other types of descriptor sets. The best ensemble model achieved an overall accuracy of 88.23%, sensitivity of 86.27%, specificity of 90.20%, mcc of 0.765, auc of 0.896 with 88.21 g-means. To assess the robustness and stability of the best ensemble model, it is further evaluated using stratified 10×10 fold cross validation and holdout testing sets (repeated 10 times) achieving 84.83% mean accuracy with 0.708 mean mcc and 88.46% mean accuracy with 0.771 mean mcc respectively. A comparison of different meta classifiers (Generalized linear regression, Gradient boosting machines, Random forest and Deep learning neural networks) in stacking ensemble revealed that random forest is the better choice for combining multiple classification models.

Alveolus-like organoid from isolated tip epithelium of embryonic mouse lung

Embryonic lungs were obtained from embryonic day 13.5 ICR mice. The lung-tip epithelium isolated using dispase treatment was embedded in low-growth factor Matrigel, cultured in DMEM/F12 medium containing 0.1% bovine serum albumin, supplemented with insulin, transferrin, and selenium (ITS), with or without fibroblast growth factor 7 (FGF7), and were observed for 14 days. With the addition of FGF7, the tip epithelium grew to form a cyst by culture day 7. Then, tubular tufts-like alveolus appeared around the cyst surface. Reverse transcription-polymerase chain reaction revealed that, with the addition of FGF7, the cultured lung explants expressed alveolar-type 1 cell markers, such as HopX and Aquaporin5, and type 2 cell markers, such as Lamp3 and Surfactant apoproteins (Sftp) C and D. Paraffin-embedded sections were stained with hematoxylin and eosin, and alveolar structures at culture day 14 were composed of squamous and cuboidal epithelial cells. Immunohistochemical studies showed that the squamous epithelial cells were positive for HopX, and the cuboidal epithelial cells were positive for pro-SftpC. Furthermore, transmission electron microscopic observation confirmed that the squamous epithelial cells were alveolar-type 1 cells and the cuboidal cells were type 2 cells, because they had many lamellar inclusion bodies. Embryonic lung-tip epithelium forms an alveolus-like organoid through the self organization with the aid of Matrigel, ITS, and FGF7. This method to make alveolus-like organoid in vitro is easy, reproducible, and economical. This method could have potential to solve many issues in alveolar epithelial cells in normal and pathological conditions.

Lamellar inclusion bodies in the retinal pigment epithelium of diurnal rodents

The ultrastructure of the retinal pigment epithelium of a diurnal rodent (Brandt's vole) was described taking into account 1) the functions of the pigment epithelium as a participant in the renewal of photoreceptor outer segment and. 2) digestion of outer segment membranes into phagosomes of the retinal pigment epithelium. The myeloid bodies were observed after exposure of the pigment epithelium to light (200 lux, 4 hours) and darkness (0,1 lux, 1,5-hour). In the cytoplasm of the pigment epithelium of the vole no myeloid bodies were observed. Instead of it small lamellar bodies, which have the spiral form and size (from - 200 to 400 nm) were found. The structure of these lamellar bodies was described. Furthermore, the structures, which were presumably responsible for the transport of the digested material, were revealed. The evidence of it is the presence of 1) dense precipitate in the apical domain of the pigment epithelium and 2) microtubules which participate in transport of this precipitate.

Role of N-glycosylation of human lysosomal phospholipase A2 for the formation of catalytically active enzyme

To understand the role of N-glycosylation of lysosomal phospholipase A2 (LPLA2), four potential N-glycosylation sites in human LPLA2 (hLPLA2) were individually modified replacing asparagine (Asn) with alanine by site-direct mutagenesis. COS-7 cells transiently transfected with wild-type (WT) hLPLA2 gene produced catalytically active LPLA2. A single mutation at 273-, 289-, or 398-Asn partially reduced production of active LPLA2. A single mutation at 99-Asn and quadruple mutations at all four Asn sites resulted in a marked reduction of active LPLA2 and loss of active LPLA2, respectively. Western blot analysis using anti-hLPLA2 antibody showed that the LPLA2 expression level was similar between all transfectants. N-glycosidase F digestion revealed that multiple forms of LPLA2 found in individual transfectants are due to different N-glycans linked to the core protein. The LPLA2 activity in individual transfectants was mostly recovered in the soluble fraction and correlated to the quantity of LPLA2 detected in the soluble fraction. LPLA2 mutated at 99-Asn was mostly retained in the membrane fraction. The WT transfectants treated with tunicamycin markedly lost LPLA2 activity. These data indicate that the 99-Asn is the most critical N-glycosylation site for formation of native hLPLA2 in vivo and that the N-glycosylation of LPLA2 is crucial for biosynthesis of catalytically active hLPLA2.

Modeling Phospholipidosis Induction: Reliability and Warnings

Drug-induced phospholipidosis (PLD) is characterized by accumulation of phospholipids, the inducing drugs and lamellar inclusion bodies in the lysosomes of affected tissues. These side effects must be considered as early as possible during drug discovery, and, in fact, numerous in silico models designed to predict PLD have been published. However, the quality of any in silico model cannot be better than the quality of the experimental data set used to build it. The present paper reports an overview of the difficulties and errors encountered in the generation of databases used for the published PLD models. A new database of 466 compounds was constructed from seven literature sources, containing only publicly available compounds. A comparison of the PLD assignations in selected databases proved useful in revealing some inconsistencies and raised doubts about the previously assigned PLD+ and PLD- classifications for some chemicals. Finally, a Partial Least Squares Discriminant Analysis (PLS-DA) approach was also applied, revealing further anomalies and clearly showing that metabolism as well as data quality must be taken into account when generating accurate methods for predicting the likelihood that a compound will induce PLD. A new curated database of 331 compounds is proposed.

Increased levels of urinary phenylacetylglycine associated with mitochondrial toxicity in a model of drug-induced phospholipidosis.

Phospholipidosis (PLD) is a lysosomal storage disorder induced by a class of cationic amphiphilic drugs. However, drug-induced PLD is reversible. Evidence of PLD from animal studies with some compounds has led to discontinuation of development. Regulatory authorities are likely to request additional studies when PLD is linked to toxicity. We conducted a trial to investigate urinary phenylacetylglycine (uPAG) as a biomarker for PLD. Five groups of 12 male Wistar rats were dosed once with vehicle, 300 mg/kg or 1500 mg/kg of compound A (known to induce PLD), or 300 mg/kg or 1000 mg/kg of compound B (similar structure, but does not induce PLD) to achieve similar plasma exposures. Following dosing, urine and blood samples underwent nuclear magnetic resonance (NMR), proteomic, and biochemical analyses. Necropsies were performed at 48 and 168 h, organ histopathology evaluated, and gene expression in liver analyzed by microarray. Electron microscopic examination of peripheral lymphocytes was performed. For compound A, uPAG increased with dose, correlating with lamellar inclusion bodies formation in peripheral lymphocytes. NMR analysis showed decreased tricarboxylic acid cycle intermediates, inferring mitochondrial toxicity. Mitochondrial dysfunction was suggested by uPAG increase, resulting from a switch to anaerobic metabolism or disruption of the urea cycle. uPAG shows utility as a noninvasive biomarker for mitochondrial toxicity associated with drug-induced PLD, providing a mechanistic hypothesis for toxicity associated with PLD likely resulting from combined direct and indirect mitochondrial toxicity via impairment of the proton motor force and alteration of fatty acid catabolism.

Cardiomyopathy Caused by Longterm Treatment with Chloroquine: A Rare Disease, or a Rare Diagnosis?

To the Editor: Chloroquine (CQ) has been used for the treatment and prophylaxis of malaria since World War II. Ten years later, the administration of CQ and hydroxychloroquine (HCQ) was introduced for the longterm therapy of rheumatic diseases. Although no reliable statistical data are available, HCQ, which differs from CQ only in a hydroxyl group at the end of the side chain, but not in pharmacokinetics or metabolism, is described in the literature as less toxic and has been staging a comeback, particularly in the treatment of systemic lupus erythematosus (SLE)1. CQ and HCQ induce a dysfunction of the lysosomal enzymes, leading to the impairment of intracellular degradation processes in conjunction with the accumulation of pathological metabolic products (glycogen and phospholipids). These appear histologically as granulovacuolar cell mutations and ultrastructurally as lamellar inclusion bodies (“myeloid bodies”) and as “curvilinear bodies” in cytoplasm (remnants of poorly digested membranes)2,3. Their pharmacokinetics are characterized by a long half-life and a high volume of distribution; they follow a multicompartment model with very slow distribution between plasma and tissue, leading to sustained organ sequestration and sometimes irreversible organ damage4. Severe toxicity in the form of irreversible retinopathy5 is well known under longterm treatment with these substances. In contrast, neuromyopathy6 and especially cardiac damage7 receive scant mention in the literature. Cardiac complications comprise conduction disturbances [bundle-branch block, atrioventricular (AV) block] and cardiomyopathy — often with hypertrophy, restrictive physiology, and congestive heart failure (literature reviews are found in Nord, et al 8 and Costedoat-Chalumeau, et al 9). Because the clinical features of cardiotoxicity are unspecific, the followup of potentially affected patients is of utmost importance. We report a case of a patient with rheumatoid arthritis (RA) and heart disease who was treated with CQ. A … Address correspondence to Dr. E. Tonnesmann, Kaiser-Karl-Klinik, Graurheindorfer Str. 137, D-53117 Bonn, Germany. E-mail: e.toennesmann{at}t-online.de

Predicting Phospholipidosis Using Machine Learning

Phospholipidosis is an adverse effect caused by numerous cationic amphiphilic drugs and can affect many cell types. It is characterized by the excess accumulation of phospholipids and is most reliably identified by electron microscopy of cells revealing the presence of lamellar inclusion bodies. The development of phospholipidosis can cause a delay in the drug development process, and the importance of computational approaches to the problem has been well documented. Previous work on predictive methods for phospholipidosis showed that state of the art machine learning methods produced the best results. Here we extend this work by looking at a larger data set mined from the literature. We find that circular fingerprints lead to better models than either E-Dragon descriptors or a combination of the two. We also observe very similar performance in general between Random Forest and Support Vector Machine models.

What’s Your Diagnosis?

A 14-month-old girl presented for further evaluation for glaucoma with a history of esotropia and concern for corneal clouding first seen by her mother at 9 months of age without associated epiphora or photophobia. At 11 months of age, an examination under anesthesia recorded the presence of suspected epithelial edema with bilateral borderline elevated intraocular pressures. After removal of the temporal corneal epithelium, strikingly clear visualization of the filtration angle was present. Bilateral goniotomies were then performed. One month postoperatively, the examination under anesthesia was repeated and moderately improved intraocular pressures were measured and persistent epithelial opacification was present. She had just begun standing and her pediatrician had noted mild global developmental delay. Her parents are both of Ashkenazim ancestry. On examination, she possessed mild coarse facial features and generalized hypotonia without hepatosplenomegaly or skin abnormalities. She fixed and followed well without nystagmus. Her corneas were diffusely cloudy and thickened bilaterally (Figure). Corneal diameter measurements, intraocular pressures, gonioscopic findings, and lenses were normal. The fundi were normal, without evidence of retinal vessel attenuation or optic atrophy. Conjunctival and corneal epithelial biopsies were performed. Electron microscopic analysis revealed lysosomal membrane-bound intracytoplasmic lamellar inclusion bodies that confirmed the suspected diagnosis.

Cardiovascular Manifestations in Fabry Disease: A Clinical and Echocardiographic Study

This study reviews the clinical and echocardiographic findings in a cohort of Fabry patients (n=12) and compares echocardiographic findings to normal controls. Almost all patients had extracardiac manifestations. Five out of 12 patients had cardiovascular symptoms. Nine out of 12 patients had left ventricular hypertrophy (LVH) on the electrocardiogram (ECG) and one patient had short PR interval. Three patients had epicardial coronary disease. Four patients had 'rat-tail' appearance on left ventriculogram. Six patients who had myocardial biopsy showed extensive vacuolation of the myocytes on light microscopy and concentric, myelinoid lamellar cytoplasmic inclusion bodies on electron microscopy. On echocardiography, LV mass was significantly increased in the Fabry group compared to normal controls. Traditional parameters of diastolic function including peak E velocity, peak A velocity and deceleration time were no different to normal controls. The IVRT was significantly prolonged in the Fabry subjects. The PV atrial reversal duration exceeded that of mitral A wave duration in the Fabry group. The septal E' velocity with Doppler tissue imaging (DTI) was significantly lower in the Fabry group than the normal controls. Fabry disease should be considered in the differential diagnosis in patients with unexplained LVH and late onset hypertrophic cardiomyopathy. Extracardiac manifestations are common.

Differentiated Human Alveolar Epithelial Cells and Reversibility of their Phenotype In Vitro

Cultures of differentiating fetal human type II cells have been available for many years. However, studies with differentiated adult human type II cells are limited. We used a published method for type II cell isolation and developed primary culture systems for maintenance of differentiated adult human alveolar epithelial cells for in vitro studies. Human type II cells cultured on Matrigel (basolateral access) or a mixture of Matrigel and rat tail collagen (apical access) in the presence of keratinocyte growth factor, isobutylmethylxanthine, 8-bromo-cyclicAMP, and dexamethasone (KIAD) expressed the differentiated type II cell phenotype as measured by the expression of surfactant protein (SP)-A, SP-B, SP-C, and fatty acid synthase and their morphologic appearance. These cells contain lamellar inclusion bodies and have apical microvilli. In both systems the cells appear well differentiated. In the apical access system, type II cell differentiation markers initially decreased and then recovered over 6 d in culture. Lipid synthesis was also increased by the addition of KIAD. In contrast, type II cells cultured on rat tail collagen (or tissue culture plastic) slowly lose their lamellar inclusions and expression of the surfactant proteins and increase the expression of type I cell markers. The expression of the phenotypes is regulated by the culture conditions and is, in part, reversible in vitro.

Positional specificity of lysosomal phospholipase A2

Lysosomal phospholipase A(2) (Lpla2) is highly expressed in alveolar macrophages and may mediate the phospholipid metabolism of surfactant. Studies on the properties of this phospholipase are consistent with the presence of both phospholipase A(1) and phospholipase A(2) activities. These activities were studied through the production of O-acyl compounds, produced by the transacylase activity of Lpla2. Liposomes containing POPC and N-acetylsphingosine (NAS) were incubated with the soluble fraction obtained from MDCK cells stably transfected with the mouse Lpla2 gene. Two 1-O-acyl-NASs, 1-O-palmitoyl-NAS and 1-O-oleoyl-NAS, were produced by Lpla2. The formation rate of 1-O-oleoyl-NAS was 2.5-fold that of 1-O-palmitoyl-NAS. When 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC) was used, the formation rate of 1-O-oleoyl-NAS was 5-fold higher than that of 1-O-palmitoyl-NAS. Thus, Lpla2 can act on acyl groups at both sn-1 and sn-2 positions of POPC and OPPC. When 1-palmitoyl-2-unsaturated acyl-sn-glycero-3-phosphocholines were used as acyl donors, the transacylation of the acyl group from the sn-2 position to NAS was preferred to that of the palmitoyl group from the sn-1 position. An exception was observed for 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), for which the formation rate of 1-O-palmitoyl-NAS from PAPC was 4-fold greater than that of 1-O-arachidonoyl-NAS. Thus, Lpla2 has broad positional specificity for the sn-1 and sn-2 acyl groups in phosphatidylcholine and phosphatidylethanolamine.

Lysosomal Phospholipase A2 and Phospholipidosis

A lysosomal phospholipase A2, LPLA2, was recently characterized and shown to have substrate specificity for phosphatidylcholine and phosphatidylethanolamine. LPLA2 is ubiquitously expressed but is most highly expressed in alveolar macrophages. Double conditional gene targeting was employed to elucidate the function of LPLA2. LPLA2-deficient mice (Lpla2-/-) were generated by the systemic deletion of exon 5 of the Lpla2 gene, which encodes the lipase motif essential for the phospholipase A2 activity. The survival of the Lpla2-/- mice was normal. Lpla2-/- mouse mating pairs yielded normal litter sizes, indicating that the gene deficiency did not impair fertility or fecundity. Alveolar macrophages from wild-type but not Lpla2-/- mice readily degraded radiolabeled phosphatidylcholine. A marked accumulation of phospholipids, in particular phosphatidylethanolamine and phosphatidylcholine, was found in the alveolar macrophages, the peritoneal macrophages, and the spleens of Lpla2-/- mice. By 1 year of age, Lpla2-/- mice demonstrated marked splenomegaly and increased lung surfactant phospholipid levels. Ultrastructural examination of Lpla2-/- mouse alveolar and peritoneal macrophages revealed the appearance of foam cells with lamellar inclusion bodies, a hallmark of cellular phospholipidosis. Thus, a deficiency of lysosomal phospholipase A2 results in foam cell formation, surfactant lipid accumulation, splenomegaly, and phospholipidosis in mice.

Phospholipid storage in the myocardium of a unique Japanese case of idiopathic cardiomyopathy

Background A unique adult male patient who developed cardiomyopathy was first suspected to have cardiac Fabry disease based on the pathological findings in heart tissues obtained on biopsy, but the α-galactosidase activity in his leukocytes was normal and no mutation was detected in the coding region of the α-galactosidase gene. We identified accumulated materials in the myocardium of this patient. Methods Pathological and biochemical analyses were performed using the autopsied heart tissues as samples. Results Although numerous lamellar and concentric inclusion bodies were ultrastructurally found in the autopsied myocardium, the α-galactosidase activity in the heart tissues was not decreased. Lipid analysis revealed the accumulation of phospholipids including phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol, but not globotriaosylcereamide or gangliosides. Conclusions We found that a large amount of phospholipids accumulated in the myocardium of a patient with idiopathic cardiomyopathy, and electron microscopic findings of lamellar and concentric inclusion bodies in cardiomyocytes. A cardiac phospholipid storage disorder should be considered as an important candidate disease on differential diagnosis of myocardiac disorders including cardiac Fabry disease.

Structural anomalies in the peripheral lung of Mecp2 knockout mice

Mice that have had the methyl-CpG-binding transcriptional repressor gene Mecp2 knocked out are an established model of Rett syndrome, a neurodevelopmental disorder in humans. Previous studies have indicated that Mecp2 is highly expressed not only in brain tissues, but also in the lung. This expression pattern may play a role in the findings that Rett patients suffer respiratory impairment, and respiratory failure is a common cause of death. The present study was undertaken to examine the morphology of the peripheral lung, in the Mecp21lox mouse model (Chen et al. 2001, Nat. Genet. 27:327). . Hemizygous-null and wildtype males were anesthetized: the lungs were removed rapidly, chopped and fixed by immersion, and processed for epoxide embedding. They were sectioned, and examined by light and electron microscopy. Light microscopic observation revealed that the overall architecture of the lung appeared normal. However, the type II alveolar epithelial cells were often enlarged, and appeared to contain many, large, lamellar inclusion bodies. Electron microscopic observation confirmed that these cells often contained very large lamellar bodies, some of which appeared abnormally lamellated. In addition, the mitochondria of the type II cells often appeared unusually large and sometime misshapen. Some type II cells contained glycogen deposits, and there were lipid droplets within some interstitial cells. The results suggest that type II cell development and/or function may be affected by the Mecp2-null genotype. Glycogen within type II cells, and interstitial lipid droplets suggest that the post-natal development of the peripheral lung may also be effected. This study supported by IRSA and the Brachman Hoffman Fellowship of Wellesley College.

Comparison of the effects of agalsidase alfa and agalsidase beta on cultured human Fabry fibroblasts and Fabry mice

We compared two recombinant alpha-galactosidases developed for enzyme replacement therapy for Fabry disease, agalsidase alfa and agalsidase beta, as to specific alpha-galactosidase activity, stability in plasma, mannose 6-phosphate (M6P) residue content, and effects on cultured human Fabry fibroblasts and Fabry mice. The specific enzyme activities of agalsidase alfa and agalsidase beta were 1.70 and 3.24 mmol h(-1) mg protein(-1), respectively, and there was no difference in stability in plasma between them. The M6P content of agalsidase beta (3.6 mol/mol protein) was higher than that of agalsidase alfa (1.3 mol/mol protein). The administration of both enzymes resulted in marked increases in alpha-galactosidase activity in cultured human Fabry fibroblasts, and Fabry mouse kidneys, heart, spleen and liver. However, the increase in enzyme activity in cultured fibroblasts, kidneys, heart and spleen was higher when agalsidase beta was used. An immunocytochemical analysis revealed that the incorporated recombinant enzyme degraded the globotriaosyl ceramide accumulated in cultured Fabry fibroblasts in a dose-dependent manner, with the effect being maintained for at least 7 days. Repeated administration of agalsidase beta apparently decreased the number of accumulated lamellar inclusion bodies in renal tubular cells of Fabry mice.

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.