Role of phospholipase A inhibition in amiodarone pulmonary toxicity in rats

Amiodarone is effective in the treatment of ventricular and supraventricular arrhythmias. In man its clinical use is associated with the accumulation of phospholipid-rich multilamellar inclusions in various tissues including lung, liver and others. This report presents evidence showing that amiodarone is a potent inhibitor of lysosomal phospholipase A from rat alveolar macrophages, J-744 macrophages and rat liver. When compared with other cationic amphiphilic agents which are known to produce phospholipidosis, amiodarone is one of the most potent inhibitors yet discovered. The subcellular localization of amiodarone has been determined in lung and its distribution was consistent with a lysosomal localization. It is hypothesized that amiodarone causes cellular phospholipidosis by concentrating in lysosomes and inhibiting phospholipid catabolism.     

In vitro inhibition of lysosomal phospholipase A1 of rat lung by amiodarone and desethylamiodarone

Amiodarone causes phospholipid storage in the lysosomes of various types of lung cell in animals and man. It has been proposed that this is due to its ability to inhibit lysosomal phospholipase A. To investigate this further, a crude lysosomal fraction from rat lung was prepared and phospholipase A was isolated and its positional specificity was determined. Analysis of the products formed after incubation with 2-[1-14C]oleoylphosphatidylcholine showed that only phospholipase A1 activity is present. This soluble preparation of lung lysosomal phospholipase A1 was used to study inhibition by amiodarone and desethylamiodarone, in vitro. Both were extremely potent inhibitors of the lung acid phospholipase A1. To evaluate the levels of amiodarone in lung lysosomes, rats were treated with the agent for 3 days and the combined mitochondrial/lysosomal fraction of lung tissue was prepared by differential centrifugation. This fraction had been shown previously to be highly enriched in amiodarone. Purified mitochondria and lysosomes were isolated from the combined mitochondrial/lysosomal fraction with Percoll gradients and analyzed for their drug content by HPLC. Amiodarone and desethylamiodarone were present in roughly equal amounts, relative to protein, in mitochondria and lysosomes, respectively. Amiodarone appears to differ from other cationic amphiphilic drugs which cause lipidosis because the latter are more highly lysosomotropic. Although amiodarone does not appear to be highly lysosomotropic in lung, it causes lysosomal phospholipid storage because of its ability to concentrate in lung and because it inhibits lysosomal phospholipase A to a much greater extent than other cationic amphiphiles such as diethylaminoethoxyhexestrol, chloroquine and chlorphentermine.     

Repeated amiodarone exposure in the rat: toxicity and effects on hepatic and extrahepatic monooxygenase activities

Amiodarone is a potent and efficacious antiarrhythmic agent, yet associated with its use are life-threatening pulmonary fibrosis and hepatotoxicity. We have investigated the susceptibility of the male Sprague-Dawley rat to pulmonary and hepatic toxicity after repeated exposure to amiodarone and the effects of such exposure on hepatic and extrahepatic drug metabolizing enzymes. Animals received amiodarone (200 mg.kg-1.day-1 i.p., 5 days/week) for 1 week followed by 150 mg.kg-1.day-1 (5 days/week) for 3 additional weeks. No signs of pulmonary fibrosis or hepatotoxicity were observed, based on histological examination, lung hydroxyproline content, and plasma alanine aminotransferase activity. Analysis of tissues revealed extensive accumulation of amiodarone and desethylamiodarone in lung and liver, but concentrations were significantly lower in animals treated for 4 weeks than for 1 week. In a separate experiment, rats received amiodarone 150 mg.kg-1.day-1 i.p. (5 days/week) for 1 or 4 weeks. No differences in tissue concentrations of amiodarone and desethylamiodarone were detected between animals treated for 1 or 4 weeks. This regimen did not affect hepatic or extrahepatic monooxygenase activities. These results indicate that, in the male Sprague-Dawley rat, there is no observable pulmonary or hepatic toxicity following short-term amiodarone exposure, and there is enhanced elimination of amiodarone and desethylamiodarone when the daily dose of amiodarone is decreased after 1 week from 200 to 150 mg/kg.     

Effects of amiodarone on elastin biosynthesis in primary hamster lung cell cultures

Amiodarone is a Class III antiarrhythmic agent that has been implicated as a cause of human pulmonary fibrosis. Pulmonary fibrosis is associated with increased levels of connective tissue proteins such as collagen and elastin. The purpose of this investigation was to determine whether elastin synthesis would be altered by in vitro amiodarone administration. Primary hamster lung cell cultures were utilized. Cultures were treated with 2, 10, and 20 micrograms/ml amiodarone. Following treatment, elastin synthesis was monitored by a biochemical tracer assay based on the presence of the cross-linking amino acids: desmosine/isodesmosine. These cross-links are found only in elastin. Addition of [14C] lysine to cultures results in uptake of the radiolabel into the cross-links. Cross-links were isolated and identified using chromatography and electrophoresis. At all doses of amiodarone, elastin synthesis was seen to increase above control levels. Light and electron microscopy confirmed the presence of an extracellular matrix. The morphologic studies also revealed the presence of cytoplasmic inclusion bodies and vacuoles that are often associated with cationic, amphiphilic drugs such as amiodarone.     

Effects of chronic amiodarone treatment on cat myocardial phospholipid content and on in vitro phospholipid catabolism

Amiodarone is used extensively for the chronic treatment of life-threatening arrhythmias caused by ischemic heart disease. However, chronic therapy with this agent results in phospholipidosis in various tissues and it has been suggested that the inhibition of lysosomal phospholipase A by this drug contributes to this abnormality. Exogenous amiodarone has been shown to inhibit purified rat liver lysosomal phospholipase A₁, as well as acid phospholipase activities of alveolar macrophage homogenates and those of snake venom phospholipase A₂ and bacterial phospholipase C. The effects of drug treatment on heart have not been explored. The results described here demonstrate that amiodarone also significantly increases (37%, p < 0.001) phospholipid content in cat hearts. This increase is proportionately distributed to all major phospholipid classes, with the exception of sphingomyelin which appears to increase more than the others. In addition, the data also show that following amiodarone treatment, the endogenous drug levels in the heart were sufficient to reduce in vitro losses of membrane phospholipid at 37°C by inhibiting a variety of endogenous phospholipases at physiological (7.4), ischemic (6.2) and acidic (5.0) pH values. This protection is more pronounced at acidic pH values than at physiological pH. Endogenous amiodarone also affects myocardial phospholipase activities towards exogenous phosphatidylcholine and again the extent of inhibition is more at acidic pH. These results suggest that amiodarone induces phospholipidosis in the heart by inhibiting phospholipid catabolism and that its antiarrhythmic properties may reside in its ability to modulate alkaline, neutral and acid phospholipase activities in ischemia. To what extent amiodarone metabolites (desethylamiodarone and bis-desethylamiodarone) are involved in these actions remains to be determined.     

Distribution of amiodarone and its metabolite, desethylamiodarone, in human tissues

The distribution of the antiarrhythmic drug amiodarone and its principal lipophilic metabolite, desethylamiodarone, was determined in postmortem tissues of six patients who received amiodarone therapy (treatment period, 6-189 days; total dose, 4.8-127.0 g). Amiodarone concentration was highest in liver, lung, adipose tissue, and pancreas, followed by kidney, heart (left ventricle), and thyroid gland, and lowest in antemortem plasma. There was no measurable amiodarone in brain (less than 1.0 microgram/g). Desethylamiodarone concentration was highest in liver and lung, followed by pancreas, adipose tissue, kidney, heart, thyroid gland, and brain, and lowest in plasma. For most patients, the desethylamiodarone concentration was higher than the amiodarone concentration in liver, lung, kidney, heart, thyroid gland, and brain, whereas the parent drug concentration was higher than the metabolite concentration in adipose tissue, pancreas, and plasma. Tissue amiodarone and desethylamiodarone concentrations appeared to be related more closely to the total dose of amiodarone than to their respective plasma concentrations. One patient died of apparent amiodarone-induced pulmonary toxicity after an 18-day period of pharmacotherapy. Clinical evidence of pulmonary dysfunction appeared at 15 days after the initiation of amiodarone therapy, and the patient died at 23 days. Histologic assessment of a lung necropsy specimen revealed acute alveolar interstitial damage. This case represents the earliest reported incident of amiodarone-induced pulmonary toxicity.     

Effects of metabolites and analogs of amiodarone on alveolar macrophages: structure-activity relationship

Amiodarone, an antiarrhythmic drug toxic toward the lung, is metabolized through sequential modifications of the diethylaminoethoxy group to mono-N-desethylamiodarone (MDEA), di-N-desethylamiodarone (DDEA), and amiodarone-EtOH (B2-O-EtOH), whose effects on lung cells are unclear. To clarify this, we exposed rabbit alveolar macrophages to analogs with different modifications of the diethylaminoethoxy group and then searched for biochemical signs of cell damage, formation of vacuoles and inclusion bodies, and interference with the degradation of surfactant protein A, used as a tracer of the endocytic pathway. The substances studied included MDEA, DDEA, and B2-O-EtOH, analogs with different modifications of the diethylaminoethoxy group, fragments of the amiodarone molecule, and the antiarrhythmic agents dronedarone (SR-33589) and KB-130015. We found the following: 1). MDEA, DDEA, and B2-O-EtOH rank in order of decreasing toxicity toward alveolar macrophages, indicating that dealkylation and deamination of the diethylaminoethoxy group represent important mechanisms of detoxification; 2). dronedarone has greater, and KB-130015 has smaller, toxicity than amiodarone toward alveolar macrophages; and 3). the benzofuran moiety, which is toxic to liver cells, is not directly toxic toward alveolar macrophages.     

Influence of amiodarone on urinary excretion of 6beta-hydroxycortisol in humans

The present study was undertaken to evaluate the use of cortisol 6beta-hydroxylation in defining the effect of amiodarone on cytochrome CYP3A activity. To accomplish this goal, the in vivo activity of CYP3A was estimated by measuring the 24-hour urinary excretion of 6beta-hydroxycortisol (6beta-OHC) and by calculating 24-hour ratio of 6beta-hydroxycortisol to urinary free cortisol (6beta-OHC/UFC ratio). Nine cardiac patients scheduled for amiodarone treatment were recruited to participate in this study. Urine was collected over a 24-hour period from each subject before the first amiodarone administration and during the third day of oral administration of amiodarone (200 mg four times daily as a loading dose). Three days of amiodarone treatment caused a significant decrease (p<0.05) in both the 6beta-OHC/UFC ratio and the 24-hour urinary excretion of 6beta3-OHC. These results suggest that amiodarone is an inhibitor of CYP3A activity.     

Combined treatment with <Emphasis Type="SmallCaps">l</Emphasis>-carnitine and a pan-caspase inhibitor effectively reverses amiodarone-induced injury in cultured human lung epithelial cells

Amiodarone is an effective class III antiarrhythmic drug, however, the pulmonary toxicity is one of the most life-threatening complications of its use. The present study was designed to determine the mechanisms underlying pulmonary toxicity of amiodarone. In cultured human lung epithelial cells A549, amiodarone caused cell injury characterized by mitochondrial membrane depolarization, ATP depletion, enhanced propidium iodide (PI) uptake and increase in the number of Annexin-V positive cells, although the population of PI-stained cells appeared earlier and was not identical to that of Annexin-V stained cells, suggesting that the apoptosis and necrosis appeared in different cells. The apoptosis was accompanied with the activation of caspase-2, -3 and -8 but not caspase-9, and reversed by these caspase inhibitors. However, the caspase inhibitors had no influence on mitochondrial membrane potential or PI uptake after exposure of A549 cells to amiodarone. In contrast, mitochondrial cofactors such as L-carnitine and acetyl-l-carnitine attenuated mitochondrial membrane depolarization, abrogated cellular ATP depletion and reversed PI uptake without affecting Annexin-V positive cells. These finding suggest that different intracellular events operate to cause apoptosis and necrosis after exposure of pulmonary epithelial cells to amiodarone.     

Effect of amiodarone on Na+-, K+-ATPase and Mg2+-ATPase activities in rat brain synaptosomes

Amiodarone hydrochloride is a diiodinated antiarrhythmic agent widely used in the treatment of cardiac disorders. With the increasing use of amiodarone, several untoward effects have been recognized and neuropathy following amiodarone therapy has recently been reported. The present studies were carried out to study the effect of amiodarone on rat brain synaptosomal ATPases in an effort to understand its mechanism of action. Na+, K+-ATPase and oligomycin sensitive Mg2+ ATPase activities were inhibited by amiodarone in a concentration dependent manner with IC50 values of 50 microM and 10 microM respectively. [3H]ouabain binding was also decreased in a concentration dependent manner with an IC50 value of 12 microM, and 50 microM amiodarone totally inhibited [3H]ouabain binding. Kinetics of [3H]ouabain binding studies revealed that amiodarone inhibition of [3H]ouabain binding is competitive. K+-activated p-nitrophenyl phosphatase activity showed a maximum inhibition of 32 per cent at 200 microM amiodarone. Synaptosomal ATPase activities did not show any change in rats treated with amiodarone (20 mg kg-1 day-1) for 6 weeks, when compared to controls. The treatment period may be short, since the reported neurological abnormalities in patients were observed during 3-5 years of treatment. The present results suggest that amiodarone induced neuropathy may be due to its interference with sodium dependent phosphorylation of Na+, K+-ATPase reaction, thereby affecting active ion transport phenomenon and oxidative phosphorylation resulting in low turnover of ATP in the nervous system.     

Ultrastructure of yeast cell <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> after amiodarone treatment

The ultrastrcutre of Saccharomyces cerevisiae cells (wild-type and ysp2 mutant cells) was studied after amiodarone treatment. Amiodarone is used as a pharmaceutical substance for treating a number of diseases; however, it is known that amiodarone causes structural and functional disturbances in patient tissues. Here, the peculiarities of the amiodarone effect are studied in Saccharomyces cerevisiae yeast, in which amiodarone has been shown to cause apoptosis. Electron-microscopic study of yeast cells after amiodarone treatment reveals a significant increase in the number of lipid particles, which can lead to the formation of a structural complex by interacting with cell membranous organelles. Amiodarone causes the appearance of small and slightly swollen mitochondria. Chromatin displacement to the periphery of the nucleus, nuclear sectioning, and nuclear envelope disturbances are observed in the cells under these conditions. The detected changes int eh ultrastructure of the cell in Saccharomyces cerevisiae are considered to be a specific response to phospholipidosis and apoptosis caused by amiodarone.     

Genotoxic studies in hypertensive and normotensive rats treated with amiodarone

Amiodarone, a benzofuran derivative, is a very effective antiarrhythmic medication, but has potential to cause side effects. Although its cytotoxicity potential is very well-known, there are few reports about its genotoxicity effects. Since amiodarone has not been investigated in genotoxicity studies, and the spontaneously hypertensive rat (SHR) is a well-characterized model for hypertension, the aim of the present study was to perform cytogenetic analysis on chromosome aberrations in bone marrow cells of SHRs and normotensive Wistar-Kyoto rats (WKYs) that received oral amiodarone treatment for 4 weeks. Amiodarone activity was also monitored using electrocardiograms. The presence of bradycardia in amiodarone-treated rats confirmed that this drug was really active. Metaphase analysis on bone marrow cells showed that there were significant differences in total chromosomal damage and percentage abnormal metaphase between WKY and SHR negative controls. In the SHR negative control, the frequencies of basal chromosomal aberrations and abnormal metaphases were significantly higher (p < 0.05). There were high numbers of chromosomal aberrations in all amiodarone-treated groups, compared with negative controls. In amiodarone-treated groups, the most frequent chromosomal aberration was chromatid breaks. More chromosomal aberrations were found in WKYs that received amiodarone, with a statistically significant difference in comparison with negative controls (p < 0.05). However, in SHR rats there was no significant difference between the amiodarone and negative groups regarding chromosomal damage induction. These results showed that treatment with amiodarone was genotoxic in WKYs, but not in SHRs. Further studies are needed to confirm whether amiodarone is genotoxic or efficient and harmless, among humans undergoing therapy.     

Phenylacetylglycine,a putative biomarker of phospholipidosis: Its origins and relevance to phospholipid accumulation using amiodarone treated rats as a model

Amiodarone was given to male Sprague–Dawley rats at a dose of 150 mg kg^?1 day^?1 for 7 consecutive days to induce phospholipidosis in the lungs of treated rats. Amiodarone was given alone or concurrently with phenobarbitone. Animals given amiodarone had raised total phospholipid in serum, lung and lymphocytes, and elevated lyso(bis)phosphatidic acid (LBPA) in all tissues. Urinary and plasma phenylacetylglycine (PAG) and hepatic portal:aortal phenylacetate (PA) ratio were increased, whereas hepatic phenylalanine hydroxylase (PAH) activity and plasma phenylalanine:tyrosine ratio were not affected. Phenobarbitone treatment increased hepatic total P450 content and induced 7-pentoxyres-orufin O-dealkylatian (PROD) activity, as expected, but had no effect on any other biochemical parameter. Plasma amiodarone concentration was reduced in rats co-administered both drugs and phospholipid accumulation in target tissues was attenuated compared with rats treated with amiodarone alone. However, phenobarbitone co-administration failed to alter the magnitude of response with regards to urinary PAG excretion and plasma concentration of its precursors after amiodarone treatment. Increased intestinal absorption of PAG precursors probably resulted in the raised urinary PAG after amiodarone treatment. Urinary PAG correlated weakly with serum, lymphocyte and lung phospholipids. However, urinary PAG excretion was similar in rats dosed solely with amiodarone or in combination with phenobarbitone, despite the fact that the degree of phospholipid accumulation was far less in rats given the combined treatment. Nevertheless, urinary PAG was raised only in animals exhibiting abnormal phospholipid accumulation in target tissues and may thus be useful as a surrogate biomarker for phospholipidosis.     

Immunofluorescence Identification of Thermopolyspora polyspora,the Causative Agent of Farmer's Lung

Farmer''s lung is a serious disabling pulmonary disease found in agricultural workers. The disease is believed to be a hypersensitivity to the thermophilic actinomycetes, principally Thermopolyspora polyspora. This organism is difficult to stain with the usual bacteriological stains and thus far has not been demonstrated in the lung tissue by microscopic methods. In this paper, it is demonstrated that the fluorescent-antibody technique is a simple method for the positive identification of T. polyspora. The technique can also be used as a rapid screening test for the detection of antibodies to T. polyspora in the patient''s serum. In addition, it opens up the possibility of the identification of T. polyspora in the lung tissue of patients with farmer''s lung and makes available a means for the study of the immunological reaction in the lung parenchyma. No false positive or cross-reactions with Thermoactinomyces vulgaris or Streptomyces griseus could be demonstrated.     

Neuropathy and fatal hepatitis in a patient receiving amiodarone.

Muscle weakness, neuropathy, and transient rises in hepatic enzyme activity have been reported with the use of the antiarrhythmic agent amiodarone. A 68 year old teetotaller with normal liver function was given amiodarone for resistant supraventricular arrhythmias. He presented 19 months later with vomiting, muscle weakness and wasting, sensory neuropathy, and hepatomegaly. Liver biopsy showed fibrosis and the presence of hyaline. The amiodarone was withdrawn. Three months later he developed ascites. Oesophageal varices were found and he later died. The liver showed micronodular cirrhosis. The large volume of distribution and long half life of amiodarone may explain the persistence of toxicity, which may have been aggravated by simultaneously administered doxepin in this case. Amiodarone should be withdrawn if abnormal liver function or neuropathy develops.     

Ponatinib: An update on its drug targets,therapeutic potential and safety

Leukemia is a malignancy of the hematopoietic system, and as its pathogenesis has become better understood, three generations of tyrosine kinase inhibitors (TKIs) have been developed. Ponatinib is the third-generation breakpoint cluster region (BCR) and Abelson (ABL) TKI, which has been influential in the leukemia therapy for a decade. Moreover, ponatinib is a potent multi-target kinase inhibitor that acts on various kinases, such as KIT, RET, and Src, making it a promising treatment option for triple-negative breast cancer (TNBC), lung cancer, myeloproliferative syndrome, and other diseases. The drug's significant cardiovascular toxicity poses a significant challenge to its clinical use, requiring the development of strategies to minimize its toxicity and side effects. In this article, the pharmacokinetics, targets, therapeutic potential, toxicity and production mechanism of ponatinib will be reviewed. Furthermore, we will discuss methods to reduce the drug's toxicity, providing new avenues for research to improve its safety in clinical use.     

Amiodarone inhibits multiple drug resistance in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Amiodarone is a widely used antiarrhythmic drug. There is also evidence that amiodarone decreases multidrug resistance in human cell lines. In this paper, we have shown that amiodarone has similar effect on yeast, Saccharomyces cerevisiae, decreasing multiple drug resistance. Amiodarone stimulates the accumulation of ethidium bromide by inhibiting its efflux from the cells. The effect of amiodarone is much stronger on wild-type cells compared to the mutant with inactivated ABC-transporters. Interestingly, the action of amiodarone is additive with the one of chloroquine, a known inhibitor of ABC-transporters. We speculate that these findings could help in the development of antifungal drug mixes.     

High-performance liquid chromatographic isolation and fast atom bombardment mass spectrometric identification of Di-N-desethylamiodarone, a new metabolite of amiodarone in the dog

Amiodarone is an antiarrhythmic drug which has received considerable attention in recent years. It has been suggested that the unusual pharmacodynamic characteristics of this drug may be due in part to the influence of active metabolites. Using fast atom bombardment (FAB) mass spectrometry we have identified a new metabolite of amiodarone, the di-N-desethyl analog (DDEA). This metabolite was present in the blood of dogs treated with the parent drug, and showed a greater affinity for myocardium than did the parent drug. The unique features of FAB mass spectrometry over electron impact mass spectrometry was an essential element in facilitating the identification of this new metabolite. Whether or not this metabolite has pharmacologic activity or is responsible for some of the side effects occurring during amiodarone administration is not known.     

Comparison of the effects of amiodarone and ipodate on the rat heart

Chronic treatment of rats with amiodarone has been shown to produce hypothyroid-like effects such as a reduction in body and heart weight and increased synthesis of the low ATPase V3-cardiac isomyosin (Bagchi, Brown, Schneider and Banerjee 1987). In this report, we have tested the hypothesis that amiodarone causes these effects through the inhibition of intracellular production of triiodothyronine (T3) from thyroxine (T4) by comparing the effects of amiodarone with those of ipodate, a potent inhibitor of T4 to T3 conversion. Separate groups of rats were given dietary ipodate and amiodarone respectively for six weeks. Both agents increased serum T4 and T4/T3 ratios, a finding consistent with the inhibition of peripheral T4 to T3 conversion. However, ipodate failed to produce hypothyroid-like effects on body weight, heart weight and isomyosin transitions similar to those found in the amiodarone group. These data indicate that the hypothyroid-like effects of amiodarone on the rat heart are not due to the inhibition of intracellular generation of T3 from T4.