piperidines and disobutamide

In the investigation of cellular changes associated with intracellular drug storage, we incubated cultured rabbit aorta muscle cells with various amphiphilic agents. Disobutamide, chloroquine, and desipramine each increased cellular content of rhamnose, arabinose, mannose, glucose, and total saccharides; these agents also elevated total and individual phospholipid of all classes. Amiodarone did not alter total saccharide content, but increased total phospholipid. Tilorone, in contrast, decreased total saccharides, but phospholipid content was unchanged. All test agents decreased xylose content. By light microscopy, disobutamide, chloroquine, and tilorone induced clear cytoplasmic vacuoles; desipramine induced dense cytoplasmic granules; and amiodarone induced both cytoplasmic changes. By electron microscopy, the content of the cellular alterations induced by disobutamide was primarily electron lucent; that of the alterations induced by desipramine was primarily concentric lamellar bodies/flocculent electron-dense structures; and that of the alterations induced by amiodarone was a mixture of both. There was no correlation, therefore, between the induced cellular chemical contents and morphologic changes. Despite the physicochemical similarity of the amphiphilic drugs (all have cationic and lipophilic moieties), the chemical responses they induced were different. The results suggest that amphiphilic drugs alter processes involving saccharides as well as those of phospholipid metabolism. The origin of the saccharide moieties associated with the induced changes in monosaccharide contents is not known. Increased content of phosphatidylinositol, mannose, and glycosyl residues is consistent with the suggestion that amphiphilic drugs may cause an increase in membrane anchor synthesis. The inhibition of lysosomal enzyme activities responsible for the degradation of phospholipid and other anchors may also account for the observed increase in monosaccharides and phosphatidylinositol content.

Topics: Amines; Amiodarone; Animals; Anti-Arrhythmia Agents; Aorta; Cells, Cultured; Chloroquine; Desipramine; Ethylenediamines; Kinetics; Microscopy, Electron; Monosaccharides; Muscle, Smooth, Vascular; Phospholipids; Piperidines; Rabbits; Tilorone

Disobutamide, a bis tertiary amine (pKa1 = 8.6; pKa2 = 10.2) cationic amphiphilic compound, and a putative cardiac antiarrhythmic drug induced clear cytoplasmic vacuoles in dogs and rats. Ultrastructurally, the vacuoles were membrane-bound vesicles containing primarily electron-lucent material. Some concentric lamellar bodies indicative of phospholipidosis were also present. Although numerous vacuoles were seen in one-year toxicity studies in dogs and rats, there was no apparent evidence of necrosis, inflammation, atrophy, hypoplasia, hyperplasia, or metaplasia. Clinical signs or laboratory findings indicative of functional impairment were also not apparent. The picture of the vacuolation in vivo was one of storage. In cultured cells vacuoles were shown to be storage sites for disobutamide and specifically in distended vesicles of the cytoplasmic acidic compartments, such as lysosomes, endocytic, and probably transport vesicles. Storage of the drug in acidic vesicles is compatible with the dibasic nature of the cationic moiety of disobutamide. The intrinsic cell chemicals which accumulate in the vacuoles along with disobutamide remain unknown. Disobutamide may be a useful agent for defining experimentally the borderline between physiologic limits (normal function) and toxicity (functional impairment) in the condition of intracellular drug storage abnormalities and for advancing knowledge of storage mechanisms.

Topics: Animals; Anti-Arrhythmia Agents; Cells, Cultured; Coronary Vessels; Culture Media; Cytoplasm; Dogs; Drug Evaluation, Preclinical; Gastric Mucosa; Intestinal Mucosa; Kidney; Microscopy, Electron; Muscle, Smooth; Piperidines; Rats; Staining and Labeling; Structure-Activity Relationship; Uvea; Vacuoles

In the investigation of the dynamic nature of intracellular drug-induced storage disorder associated with clear cytoplasmic vacuoles (CCV), rat urinary bladder carcinoma cells (RBT CC-8) were cultured with [14C]disobutamide. Cell monolayers were then harvested and analytical cell fractionation techniques were employed to examine the association of disobutamide with the various subcellular fractions. Disobutamide distributed into two modes: as a free, organelle-independent fraction and as a light membrane-associated fraction that overlapped with markers for the plasma membrane, endoplasmic reticulum, and lysosomes. The similarity in buoyant densities of these organelles derived from RBT CC-8 cells precluded resolution of these structures by isopycnic centrifugation. In additional experiments, disobutamide was incubated in vitro with a suspension of isolated rabbit renal proximal tubules. In these cells, analytic fractionation showed that the drug localized predominantly to lysosomes and to a separate light membrane fraction that was clearly resolved from the markers for the endoplasmic reticulum, brush border, mitochondria, and peroxisomes; this fraction overlapped with the most buoyant aspects of the Golgi apparatus and basolateral plasma membrane. The buoyant density of this disobutamide-associated nonlysosomal fraction was 1.11 g/ml. Electron microscopy of the disobutamide-exposed tubules showed a substantial formation of apical vesicles, especially small, smooth-surfaced vesicles, typical of the endocytic apparatus. From these findings and based on the physicochemical properties of the cationic moiety of disobutamide, we conclude that the drug localizes in lysosomal and nonlysosomal acidic vesicles.

Topics: Animals; Cations; Cell Fractionation; Kidney Tubules, Proximal; Lysosomes; Piperidines; Rabbits; Rats; Subcellular Fractions; Tumor Cells, Cultured; Vacuoles

Analogues of the dibasic antiarrhythmic agent disobutamide (2) were prepared and evaluated for antiarrhythmic efficacy, myocardial depression, and anticholinergic activity. The replacement of an isopropyl group in disobutamide by an acetyl group led to the monobasic analogue SC-40230, 7a, which demonstrated good antiarrhythmic activity accompanied by less myocardial depressant and anticholinergic activities. In addition, it did not induce clear cytoplasmic vacuoles as did the parent compound. SC-40230 was chosen from among other analogues as a candidate for clinical evaluation. Other compounds prepared and evaluated included indolizidinones and a secondary amine isomer of disobutamide.

Topics: Animals; Anti-Arrhythmia Agents; Binding Sites; Dogs; Drug Evaluation; Hydrogen-Ion Concentration; Isomerism; Piperidines; Rats; Receptors, Muscarinic; Sodium Channels; Structure-Activity Relationship

Topics: Animals; Cells; Dogs; Organoids; Pharmacokinetics; Piperidines; Structure-Activity Relationship; Vacuoles

Disobutamide (D), an antiarrhythmic cationic amphiphilic amine (CAA), was withdrawn from clinical testing when clear cytoplasmic vacuoles (CCV) were found in the rat and dog during toxicity studies. To delineate the structural determinants of amines that induce CCV, we exposed cultured rat urinary bladder carcinoma and rabbit aorta muscle cells to numerous cationic drugs and chemicals and examined cells by phase light microscopy. The cationic moiety of these CAA was responsible for the induction of CCV. The very potent inducers were compounds that had two strongly basic amine (cationic) centers. The bis tertiary amines were particularly potent inducers. Aliphatic diamines of minimal lipophilicity-induced CCV, thus showing that an "amphiphilic" structural feature, though present in many CAA drugs, is not necessary for CCV induction. The distance between the two cationic centers was irrelevant to the induction of CCV. These results support the concept that CCV are a manifestation of intracellular (e.g., intralysosomal) drug storage. These structural delineations will be useful in future drug design and for further understanding of drug-cell interactions. Based on these findings, we were able to synthesize an antiarrhythmic CAA which did not induce CCV.

Topics: Amines; Animals; Aorta; Cell Line; Cells, Cultured; Diamines; Muscle, Smooth, Vascular; Organoids; Piperidines; Rabbits; Rats; Structure-Activity Relationship; Surface-Active Agents; Urinary Bladder Neoplasms; Vacuoles

A subchronic oral toxicity study of disobutamide, a piperidine ring compound with antiarrhythmic activity, was conducted at doses of 30, 100, and 250 mg/kg in rats, 45 mg/kg in dogs, and 90 mg/kg in monkeys. Numerous vacuoles were observed in various organs such as the liver, kidneys, heart, lungs, spleen, thymus, stomach, and choroid plexus in these animals. The epithelium of the choroid plexus (CP), however, showed severe vacuolation in rats and monkeys but not in dogs. The vacuoles corresponded to enlarged and myelin-figured lysosomes observed by electron microscopy, revealing morphological characteristics which have been reported as drug-induced phospholipidosis. In a further study, the drug penetration to cerebrospinal fluid (CSF) and the drug concentration in CP were examined in these animals. Daily po doses of 250, 45, and 90 mg/kg were, respectively, administered to rats, dogs, and monkeys to maintain approximate equivalency in peak blood concentrations across species, over a course of 35 days. The concentration of the drug in the CP was higher in rats and monkeys than in dogs, and the CSF/serum ratio of the drug concentration was extremely high in rats. The uptake of the drug by the CP in vitro was high in rats, monkeys, and dogs, in this order. In dogs, both direct contact of the drug with the CP during incubation and intraventricular administration induced vacuolation in the epithelium. From these results it was concluded that differences of the drug's penetration into the CSF and its uptake by the choroid plexus epithelium are responsible for the species differences of CP vacuolation in the animals.

Topics: Animals; Behavior, Animal; Blood; Choroid Plexus; Dogs; Epithelium; Female; In Vitro Techniques; Kidney; Lipids; Liver; Macaca fascicularis; Male; Microscopy, Electron; Organ Size; Organoids; Piperidines; Rats; Rats, Inbred Strains; Sex Factors; Species Specificity; Time Factors; Tissue Distribution; Vacuoles

Cellular uptake of disobutamide (D), and clear cytoplasmic vacuoles (CCV) induction by D in cultured rat urinary bladder carcinoma cells were dependent on the culture medium pH. At pH 6.0-6.7, drug uptake was slow and no CCV formed in 24 hr. At pH 7.0-8.0, the rate of D uptake and early appearance of CCV were directly proportional to increased basicity. This was explained by the increasing fraction of un-ionized D molecules at increasing basicity of the culture medium. It is only these electrically neutral D molecules which can penetrate the lipoidal cell membrane to induce formation of CCV. Intracellular presence of D was demonstrated by mass spectrometry methods. The results indicate that D is incorporated intracellularly, that D and not its metabolite(s) is in cells, and suggest that CCV are a result of drug sequesteration.

Topics: Animals; Cell Line; Culture Media; Cytoplasm; Hydrogen-Ion Concentration; Kinetics; Magnetic Resonance Spectroscopy; Mass Spectrometry; Organoids; Piperidines; Rats; Urinary Bladder Neoplasms; Vacuoles

Smooth muscle cells released by protease from the coronary artery of dogs were cultured in M199 supplemented with 10% fetal calf serum. Cells were grown on glass coverslips to semiconfluence and exposed to disobutamide at 0, 1, 2, 3, 6, 8, and 10 X 10(-4) M for 24, 48, and 72 h, examined in situ by light microscopy, then fixed in 100% methanol, stained with May-Grünwald Giemsa and examined by light microscopy. Cells were also exposed to drug for 24 h, pelleted, fixed and prepared routinely for electron microscopy. Control cells and cells exposed to 6 X 10(-4) M disobutamide were examined. Vacuole formation was dose and time dependent between 2 X 10(-4) M and 10(-3) M. By light microscopy, morphologic alterations induced by the drug were clear cytoplasmic vacuoles in live cells, vacuoles in dead cells and dead cells without vacuoles. Small round vacuoles were an early change. Dark granules were dispersed among the vacuoles. The vacuoles increased in size at higher doses or longer times. Eventually all the cytoplasm was occupied by vacuoles and the cells were enlarged. By electron microscopy, the vacuoles were round, primarily membrane-bound, contained mostly electron-lucent material and occasionally small flocculent bodies. There was vacuolar coalescence. The dose response of vacuole induction in confluent and semiconfluent cells was similar. Cytoplasmic vacuoles without cell death can be induced by disobutamide at 2 or 4 X 10(-4) M during a 3-day exposure. Cell culture is a suitable biological system for studying cytoplasmic vacuoles of the type induced by disobutamide.

Topics: Animals; Anti-Arrhythmia Agents; Cells, Cultured; Coronary Vessels; Cytoplasm; Dogs; Muscle, Smooth, Vascular; Organoids; Piperidines; Time Factors; Vacuoles

The effects of disopyramide (DP) and a new antiarrhythmic agent, disobutamide (DB) on cardiac conduction were studied using His bundle recording from modified rabbit Langendorff preparations electrically driven at 3 and 4 Hz. Both disopyramide (4-16 microgram/mL) and disobutamide (1-30 microgram/ml) showed conduction throughout the atrioventricular conduction system, i.e., SA, AH, and HV intervals were increased in a dose-related manner. Conversion of the conduction time changes to percent changes indicates that disobutamide had a relatively equal effect on each part of the system whereas disopyramide exhibited significantly less effect on AV nodal conduction. Slowing of conduction in the AV node by DP was clearly related to rate. Changes in SA and HV intervals were rate related to a lesser degree. No such rate-related effect was evident with disobutamide. Block of arterial conduction occurred in two out of six hearts when the rate was increased 8 microgram/mL of DP and in three additional hearts at 16 microgram/mL. This was interpreted to indicate a change in atrial excitability such that 2 X threshold currents no longer excited the tissues. This was not observed at any concentration of DB.

Topics: Animals; Anti-Arrhythmia Agents; Disopyramide; Dose-Response Relationship, Drug; Heart Conduction System; Heart Rate; Piperidines; Pyridines; Rabbits