Pitambar Somani

Evaluation of amiodarone free radical toxicity in rat hepatocytes

The possible roles of free radicals and lipid peroxidation in the mechanism of toxicity of amiodarone (AD) [2-butyl-3-(3,5-diiodo-4 alpha-diethylaminoethoxybenzoyl)benzofuran] and its principle metabolite, desethylamiodarone (DE), were examined in primary cultured Sprague-Dawley male rat hepatocytes. AD (20 and 40 micrograms/ml) and DE (10 and 25 micrograms/ml) killed hepatocytes in concentration- and time-dependent fashions. Several antioxidants [Cu,Zn-superoxide dismutase (200 U/ml), catalase (200 U/ml), N,N-diphenylphenylenediamine (DPPD; 25 microM), butylated hydroxytoluene (0.1 mM), and N-acetylcysteine (5 mM)] were incapable of preventing AD and DE hepatocyte toxicity. Only vitamin E (VE, d,l-alpha-tocopherol acetate; 20-200 microM) prevented AD and DE toxicity. No correlation between the onset of hepatocyte death by AD and DE and hepatocyte lipid peroxidation was seen. Both drugs inhibited NADPH-dependent rat liver microsomal superoxide production. These results, excluding the preventive effects of VE, do not support a free radical/lipid peroxidation mechanism of hepatocyte toxicity by AD and DE. VE may have prevented hepatocyte toxicity through non-antioxidant effects.

Unidirectional absorption of gentamicin from the peritoneum during continuous ambulatory peritoneal dialysis

Gentamicin kinetics were determined after intravenous or intraperitoneal injection in five patients undergoing continuous ambulatory peritoneal dialysis (CAPD). Our objective was to determine rate of absorption of gentamicin from the peritoneum into the systemic circulation and vice versa. After intraperitoneal instillation of 1 mg/kg in the CAPD fluid during a 6‐hr dwell time, the antibiotic appeared in the serum within 15 min in four of five patients. Peak serum concentrations ranged between 1.6 and 7.2 mg/l (x̄ ± SD = 3.52 ± 2.22) in all five patients and the time to reach peak concentration was 3.8 ± 1.5 hr. Peritoneal gentamicin clearance was 13 ml/min. Percent extraction of gentamicin from the PD fluid within the 6 hr of intraperitoneal exposure ranged from 65% to 100% (x̄ ± SD = 86.8 ± 13.2). The fraction of the intraperitoneal dose absorbed into systemic circulation was found to be 0.84 independently by calculating the ratio of AUCip and AUCiu. When the same dose of gentamicin was injected intravenously (1 mg/kg), no gentamicin could be detected in the peritoneal fluid in three of five patients and only a very small amount of the drug was present for a brief period of time in the remaining two. The kinetic parameters of intravenous gentamicin were: volume of distribution, 0.3 l/kg; elimination rate constant, 0.028 hr−1; plasma clearance 0.009 l/kg · min−1; and half‐life 27.4 hr. In two patients with acute peritonitis treated with intraperitoneal gentamicin, peak serum concentrations were found to range between 3.5 and 4.5 mg/l. These data suggest that gentamicin is rapidly absorbed from the peritoneal fluid into the blood compartment, but that occurrence of the reverse exchange is negligible. Thus, CAPD would not be expected to alter the elimination characteristics of intravenous gentamicin. Instillation of gentamicin in CAPD fluid may allow rapid absorption to reach therapeutic serum concentrations.

Amiodarone and desethylamiodarone toxicity in isolated hepatocytes in culture.

Abstract Amiodarone, a class III antiarrhythmic drug, has been found to be effective in the management of patients with life-threatening ventricular arrhythmias. Recent reports describe the presence of myelinoid inclusion bodies following amiodarone therapy in liver, myocardium, white blood cells, lung, cornea, skin, and lymph nodes; their relationship to toxicity is unclear. The exact role of desethylamiodarone, the major metabolite, of amiodarone in systemic toxicity of the parent drug is not known. Concentration-response relationships for amiodarone and desethylamiodarone were investigated by adding 1–50 μg/ml of the compounds of dimethyl sulfoxide (controls) to hepatocytes isolated from Sprague-Dawley rats and cultured in Lelbovitz L-15 medium. Using lactate dehydrogenase release into the medium to quantitate cell death, both drugs were found to cause cell death in a concentration-dependent manner within 24 hr of incubation; this data showed desethylamiodarone to be significantly more toxic than amiodarone. In experiments with 50-μg/ml concentrations of amiodarone or desethylamiodarone, we found desethylamiodarone to produce a significantly greater release of lactate dehydrogenase as compared with amiodarone within 2–4 hr. Electron microscopic studies indicated the presence of myelinoid inclusion bodies at early culture stages followed by progressive swelling of mitochondria and rough endoplasmic reticula, disruption of membranes, aggregation of subcellular structures, and ultimately cell death. Ultrastructural changes occurred sooner in the hepatocytes treated with desethylamiodarone than with amiodarone. These data demonstrate that (i) desethylamiodarone is more toxic than amiodarone; (ii) acute toxicity of desethylamiodarone and amiodarone can be quantitated by lactate dehydrogenase release; (iii) both desethylamiodarone and amiodarone can induce myelinoid inclusion bodies in cultured hepatocytes; and (iv) toxicity is characterized by progressive subcellular changes leading to cell death.

Enhancement of phenytoin elimination by multiple-dose activated charcoal.

The effect of multiple-dose activated charcoal on the elimination of intravenously administered phenytoin was studied. Seven normal volunteers received phenytoin sodium 15 mg/kg IV with and without activated charcoal. During the charcoal phase, a total dose of 300 g was administered in repeated doses over 48 hours with sufficient sorbitol to produce one to two bowel movements per day. Serum phenytoin concentrations were determined from one to 72 hours after the infusions and were fitted to a one-compartment linear elimination model. The administration of multiple-dose activated charcoal reduced the phenytoin half-life from 44.5 to 22.3 hours. In addition, phenytoin area under the curve was decreased and the elimination rate was increased. Multiple-dose activated charcoal is effective in enhancing the elimination of phenytoin in normal volunteers. Although future studies are needed to determine its role in treating patients with phenytoin toxicity, multiple-dose activated charcoal may provide a readily available, inexpensive therapeutic intervention.

Clinical and electrophysiologic effects of chronic lorcainide therapy in refractory ventricular tachycardia

The clinical, electrophysiologic and pharmacologic effects of chronic lorcainide therapy in patients with refractory ventricular tachycardia were evaluated using programmed electrical stimulation. Twelve patients with recurrent refractory ventricular tachycardia and organic heart disease, 10 men and 2 women aged 41 to 76 years, with no evidence of prior high degree atrioventricular or bifascicular block were studied. Programmed electrical stimulation was performed in the control, drug-free state and after chronic administration of lorcainide (dose range 200 to 600 mg/day, duration 48 to 240 hours, mean 106 hours) in 11 patients. One patient developed intolerable drug side effects and treatment was discontinued after 36 hours. In the 11 other patients, there was a significant increase in PR interval (187 ± 55 to 219 ± 56 ms; p 0.2). The ventricular effective refractory period increased from 234 ± 21 to 266 ± 15 ms (p Long-term lorcainide therapy was continued in the four responders. During a 2 to 12 month follow-up period, two patients have remained arrhythmia-free, one patient developed intolerable side effects requiring discontinuation of the drug and one patient with advanced renal failure died. It is concluded that chronic lorcainide therapy has significant electrocardiographic and electrophysiologic effects, but has a limited role in the long-term treatment of patients with refractory sustained ventricular tachycardia.

Basic and Clinical Pharmacology of Amiodarone: Relationship of Antiarrhythmic Effects, Dose and Drug Concentrations to Intracellular Inclusion Bodies

Amiodarone is a unique class III antiarrhythmic drug with several unusual pharmacokinetic, pharmacodynamic, and toxicological actions which are quite distinct from those of the standard antiarrhythmic drugs. Extensive animal and clinical studies have demonstrated that amiodarone and its major metabolite, desethylamiodarone, both produce a marked increase in the duration of transmembrane action potential which may be related to their antiarrhythmic as well as clinical electrophysiological activity. Unlike most other cardiovascular drugs, it has been recognized for more than 20 years that optimal antiarrhythmic effects may take several days to weeks after onset of oral therapy. Amiodarone is highly lipid soluble and exhibits at least three separate compartments of drug distribution, with a long elimination half‐life of 14–120 days after chronic therapy. The pharmacokinetic profile of desethylamiodarone is qualitatively similar to that of amiodarone, but its elimination half‐life is even longer and its tissue distribution may be slightly different. Although there may not be any correlation between serum drug levels and clinical toxicity of amiodarone during long‐term therapy, recent animal as well as clinical data suggest that multilamellar intracellular inclusions can be dissociated from cell death or clinical toxicity. Thus, it is possible that amiodarone toxicity can be minimized with low doses or low serum drug concentrations. The metabolite(s) of amiodarone may play a major role in its pharmacological and toxicological actions.

Ceftizoxime elimination kinetics in continuous ambulatory peritoneal dialysis

We investigated the kinetics of ceftizoxime, a β‐lactamase stable cephalosporin, in eight subjects undergoing continuous ambulatory peritoneal dialysis (CAPD). A single 500‐mg or 1‐gm dose was injected IV, or a 500‐mg dose was given intraperitoneally in the CAPD fluid during a 6‐hr dwell time. The ceftizoxime (500 mg) serum kinetic parameters were as follows: peak concentrations, 21 to 46 mg/l; volume of distribution, 0.27 l/kg; elimination rate constant, 0.0784 hr−1; plasma clearance, 1.66 l/kg hr−1; and t½, 10.2 hr. The t½ after 1 gm was 12 hr. Dialysate ceftizoxime concentrations rose rapidly between 0.25 and 2 hr and slowly over the next 4 hr, but only 4.04 ± 7.5 and 7.4 ± 2.9 mg ceftizoxime/hr was eliminated by the peritoneal route over a 6‐hr dwell time after 500 mg or 1 gm IV. This represents only 4% to 5% of the dose. After intraperitoneal instillation, the antibiotic appeared in the serum within 15 min in all four subjects, and the peak serum concentrations ranged from 12 to 19.8 mg/l (mean ± SD = 16.4 ± 3.3) between 5 and 6 hr. Approximately 78% of ceftizoxime was absorbed from the peritoneal dialysis fluid during a single 6‐hr dwell time. Rate constant for absorption, ka, was 0.3959 hr−1 and absorption t½ was 1.75 hr (as calculated by the residual equation). These data suggest that ceftizoxime has bidirectional exchange characteristics through the peritoneal membrane. Instillation of ceftizoxime in CAPD fluid alone may permit rapid absorption to reach therapeutic serum concentrations.

Amiodarone-induced ultrastructural changes in canine myocardial fibers

Chronic clinical toxicity with amiodarone, a unique antiarrhythmic drug, is associated with intracellular myelinoid inclusion bodies in skin, cornea, lung, liver and lymph nodes. The present study was designed to develop a canine animal model to study amiodarone concentration and onset of formation of myelinoid inclusion bodies in the cardiac tissue. Amiodarone was given intravenously in a single dose (40 mg/kg body weight) or multiple doses (40 mg/kg IV + 10 mg/kg IV X 7 days). Amiodarone concentration in the heart was high after a single dose but electron microscopic examination showed a normal ultrastructure. Numerous myelinoid inclusion bodies were found in the myofibrils of the left atria and right and left ventricles after only 7 days of amiodarone treatment. Myelinoid inclusion bodies were identified in several subcellular locations including intercalated disc but most were in close proximity of the mitochondria, sometimes touching and indenting the mitochondrial membrane. The antiarrhythmic effect of the schedule of intravenous amiodarone for 7 days used in this study was minimal, and this correlated with unexpectedly low myocardial levels of the drug and its metabolite. The results are consistent with our previous data of an antiarrhythmic effect with a single intravenous dose of 40 mg/kg body weight associated with high myocardial levels of amiodarone. We conclude that a single large dose of amiodarone with high tissue level may not cause myelinoid inclusion bodies, but they can be readily identified in all heart chambers after only 1 week of amiodarone treatment. This model would be useful to study amiodarone-induced ultrastructural changes in the heart.

Amiodarone-associated changes in human neutrophils☆

Amiodarone and its major metabolite, desethylamiodarone, were measured in the plasma, white blood cells (WBCs) and red blood cells (RBCs) of 14 patients receiving chronic amiodarone therapy. The mean plasma concentrations (+/- standard error of the mean) of amiodarone and desethylamiodarone were 2.4 +/- 0.6 and 1.6 +/- 0.4 microgram/ml, respectively. The drug level in the WBCs was 62 +/- 12 micrograms/g protein during the early loading phase and 106 +/- 33 micrograms/g protein during maintenance phase of amiodarone therapy. Desethylamiodarone concentration in the WBCs was 42 +/- 18 and 190 +/- 33 micrograms/g protein during the loading and maintenance phases, respectively. Although a trend in WBC to plasma concentration was seen, there was no linear correlation between these levels. In 1 patient with severe neuropathy, biopsy of the nerve and muscle showed high concentrations of both amiodarone and desethylamiodarone. Although there was a decrease in tissue drug levels, proportionately high tissue:plasma drug levels were detected at the time of necropsy approximately 6.5 months after amiodarone was discontinued in this patient. Neutrophils from all patients receiving chronic amiodarone therapy showed multiple myelin-like polymorphic inclusion bodies (onionoid bodies) upon electron microscopic examination. Our observations suggest that WBC drug concentrations and electron microscopic changes may provide a means of correlating tissue concentrations and of following patients receiving chronic amiodarone therapy.

[3H]Yohimbine binding to human platelet membranes: evidence for high and low affinity binding sites with negative cooperativity.

Evidence is presented that [3H]yohimbine binding to human platelet membranes does not follow the simple mass action kinetics. Although [3H]yohimbine binding was saturable and stereospecific, Scatchard analysis of the equilibrium binding data produced a curvilinear plot. Competitive displacement of [3H]yohimbine from the binding sites by unlabeled yohimbine and other alpha 2-antagonists produced shallow inhibition curves. Further, the apparent Hill coefficients of equilibrium binding and competitive displacement data were found to be less than unity. Factors such as binding to nonreceptor sites or to alpha1-adrenoceptors, dopamine receptors, or 5-HT receptors that may explain the curvilinear curve were excluded. The rank order for inhibiting [3H]yohimbine was rauwolscine greater than yohimbine greater than phentolamine greater than clonidine much greater than prazosin, suggesting that the binding sites had the characteristics of alpha2-adrenoreceptors. The affinity of the alpha2-antagonist for the receptor was enhanced by Na+ but not by guanine nucleotide, suggesting that the binding of the antagonist is modulated only by Na+. Graphic analysis of the specific binding data resulted in two components: one with high affinity and low capacity sites, and second with low affinity and high capacity sites. The experiments on dissociation kinetics, however, suggest that the observed deviation of [3H]yohimbine binding from the simple mass action kinetics is most likely due to negative cooperative interactions among alpha2-adrenoceptor sites.