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Repeated amiodarone exposure in the rat: toxicity and effects on hepatic and extrahepatic monooxygenase activities JONATHANM. DANIELS, RANDALLG. LEBDBR,AND JAMBSE BUBN Department of Phannacokogy and T o x ~ c Q ~Queen's o ~ ~ , University, Kingston, Ont., Canada K7L 3N6 AND

'THOMAS E. MASSEY Department of P h a m c o l o g y and Toxicology and Department of Medicine, Queen's University, Kingston, On&., Canada K7L 3 N 6 Received December 2 1, 1989 DANIELS,J. M., EEEDER,R. G., BNEN,J, E , and MASSEY, T. E. 1990. Repeated amidarone exposure in the rat: toxicity and effects on hepatic and extrahepatic monosxygenase activities. Can. 3. Physiol. Phamacol. 68: 1261 - 1268. Amidarone is a potent and efficadsus antiarrhythmic agent, yet associated with its use are life-threatening pulmonary fibrosis and hepatotoxicity. We Rave investigated the susceptibility of the male Sprague -Dawley rat to pulmonary and hepatic toxicity after repeated exposure to amiodarone and the effects sf such exposure on hepatic and extrahepatic drug metabolizing enzymes. Animals received amiodarone (288 mg kg-' day-' i.p., 5 days/week) for 1 week followed by 158 mg = kg-" day-' (5 dayslweek) for 3 additional weeks. No signs of pulmonary fibrosis or hepatotoxicity were observed, based on histological examination, lung hydrsxyproline content9 and plasm alanine aminotransferase activity. Analysis of tissues revealed extensive accumulation of amiodarone and desethylamidarone 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-' . day-' i.p. (5 dayslweek) for 1 or 4 weeks. No differences in tissue concentrations of amidarone and desethylamiodarone were detected between animals treated for 1 or 4 weeks. This regimen did not affect hepatic or extrahegatic monooxygenase activities. These results indicate that, in the m l e Sgrague-Dawley rat, there is no observable pulmonary or hepatic toxicity folowing short-term amiodarone exposure, and there is enhanced elimination of amiodaroane and desethyamidarone when the daily dose of amiodarsne is decreased after 1 week from 200 to 150 rng/kg. Key words: amiodarone, desethylamiodarorme, pulmonary fibrosis, rat, lung.

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DANIELS, J. M., EEEDER,R. G., BRHEN, J. E , et MASSEY, T. E. 1990. Repeated amiodarone exposure in the rat: toxicity and effects on hepatic and extrahepatic monooxygenase activities. Can. J. Physiol. Phamacol. 68 : 1261 - 1268. L'misdarone est un antiaqthrnique d'une grande efficacitk, dont l'usage a CtC associk h la fibrose pulmonaire et B I9hCpatotoxicit&.Nous avons examink la susceptibditk du rat Sgrague-Dawley d l e h la toxicite hkpatique et pulmonaire aprks une exposition rCp6tCe h 19amidarone,et les effets d9unetelle exposition sur les enzymes extrahtpdiques et htpatiques participant au metabolisrne de la drogue. Les animaux ont reGu de l'amiodarone (2W mg . kg-' jour-' i.p., 5 jourslsemaine) pendant une semaine, puis 150 mg . kg-' - jour-I (5 jourslsemaine) pendant 3 autres semaines. Aucun signe de fibrose pulmonaire ou d'ht5patotoxicid n'a CtC observC, d 9 a p r bun examen histologique, k teneur en hydroxyproline pulmonaire et 19activitC d9alanine aminotransfCrase plasmtique. L'analyse des tissus a rCvClC une forte accumulation d'anniodarsne et de BCsCthylamidarone dans le poumon et le foie, les concentrations &antsignificativement plus faibles chez les anirnaux trait& pendant 4 semaines que chez ceux traitts pendant 1 semaine. Dans une experience distincte, les rats ont reGu de 19arniodarone, 150 mg kg-l .joursE i.p. (5 jours/semaine), pendant 1 semaine ou 4 semaines. Aucune diE6rence dans les concentrations tissulaires d'amidarsne et de dCsCthylamic4darone n'a CtC dCtectCe entre les animaux traitis pendant 1 ou 4 semaines. Ce dosage n9a p a affect6 les activit6s de monooxygCnase extrah6patiques ou hkpatiques. Ces rksultats indiquent que, chez le rat Sprague -Dawley mile, une expsition de courte durCe B l'arniodarone we produit pas de toxicite hkpatique ou pulmonaire, et que l'6limination d'amidaroane et de dCsCthylamiodarone augmente lorsque la dose quotidienne d'amiodarone est rCduite, apr&s 1 semaine, de 200 2i 150 mglkg. Mots cl6s : amidarone, dCsCthylan-iodarone, fibrose pulmonaire, rat, poumon. [Tradbait par la revue]

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Amiodarone is a potent and efficacious antiarrhythmic agent used for the treatment of ventricular and supraventricular arrhythmias refractory to other therapy (Mason 1987). Hswever, associated with its uses are a Barge number of adverse effects (Mason 1987; Martin and Rosenow 1988), with pulmonary and hepatic toxicity being of greatest concern because of their potentid for causing morbidity and death (Greene et a&.1983; Mason 1987). The rat is the experimental animal that has received the most attention with respect to amiodarone phamacokinetics (Plsmp et a!. 1985a, 1985b; Weir and Ueda 1987) and phsspholipidosis (Chatelain and Brotelle 1985; Reasor et a&.1988). From these studies, it has emerged that the rat parallels the human in many important aspects: ( i ) desethylamiodarone, the major Printed in Canada / Imprim&au Canada

organic amiodarone metabolite, is found at high concentrations in tissues (Plornp et a&.1985a, 1985Eo; Weir and Ueda 1986, 1987). The formation of desethylamiodarone has been shown to be cytochrome PA50 mediated (Young and Mehend d e 1986, 1987; Rafeiro et &el. 1990); ( i i ) tissue concentrations, particularly in lung and lives, of both amiodasone and desethylamiodarone, are many times those of semm (Plomp et al. 1985a, 19858; Weir and Ueda 1986); (ikk) the elimination half-life of amiodarone is long (Weir m d Ueda 1986, 1987, 1988); (iv) there is no significant renal elimination of amiodarone or desethylarniodarone w e i r and U d a 1986, 1987); and (Q) amiodarone treatment causes pulmonary phospholipidosis (Chatelain and Brotelle 1985; Reasor et al. 1988).

The etiologies of miodarone-induced pulmonary and hepatic

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toxicity are unknown. As the rat is similar to the human with respect to its responses to misdarone, it was suspected that the rat would manifest the pulmonary fibrotic and hepatotoxic effects of miodarone following repeated administration. The purposes of the present investigations were as follows: (i) to examine tRe susceptibility, as assessed by biwhemicd and morphologicd criteria, of the mde Sprague-Dawley rat to pulmonary and hepatic toxicity following repeated administration of miodairone and to determine whether a correlation exists between toxicity and tissue concentrations of amiodmone and (or) desethylamiodarone; and (ii) to determine the effects of repeated amiodarone exposure on hepatic and extrahepatic dmg-metabolizing enzyme activities. Our initial experiment indicated the possibility of induction of biotransformation activities following amiodarone treatment. In light of this observation and previous evidence indicating the formation of a complex between amiohrone and cytochrome P-450 (Larrey et al. 1986), it was of interest to determine the effects of repeated miodairone administration on monooxygenase activities.

Materials and methods Chemicals Chemicals were obtained from the following suppliers: chloramine T (N-chloro-p-toluenesulfonamidesodium salt), Ehrlich9s reagent (p-dimethylaminobemaldehyde), sodium thiosulfate, alanine, heparin (sodium salt), 10% phosphate-buffered formalin (pH 7.41, reduced micotinaide adenine dinucleotide phosphate (NABPH), and alanine aminotransferase activity Brit (ALT 10) from Sigma Chemical Co., St. Louis, MO; tmns-4-hydroxy-L-proline and 4-dimethylaminwntipyrine (aminopyrine) from Aldrich Chemical Co., Milwaukee, WB; Harris9 hematoxylin, 176 ((wv) alcoholic eosin, analytical grade boric acid, sodium hydroxide, potassium chloride, high-performance liquid chromatography (HPLC) grade acetonitrile, and HPLC grade methanol from BDH Chemicals, Toronto, Ont.; amiodarone hydrochloride (HCl), desethylamiodarone HCl, and didesethylamidarone HCl from Ayerst Laboratories, Montreal, Que.; ethoxyresomfin and rescsmfin from Molecular Probes, Inc., Junction City, OR. All other chemicals were of reagent grade and were obtained from common commercial suppliers. The amiodarone dosing solution was prepared by dissolving amidarone HCl in heated distilled water (60°C) to a concentration sf 50 mg/mL and then cooling it to room temperature. Experiment A: Susceptibility of the m t to pulmonary and hepatic tmicidies fillowing repeated amioBarone exposure Anirm&freatment Male Sprague-Dawley rats weighing 225 -250 g (Charles River Canada, Hnc., St. Constant, Que.) were maintained in group wire cages on a 12-h light - 12-h dark cycle and were given food and water ad Bibiturn. Animals were acclimatized for 4 days prior to my manipulation. Twenty rats initially received 200 mg . kg-' day-' aqueous amidarone HCl i.p. (5 day sfweek) for 1 week followed by 150 mg kg-' . day-' (5 daysfweek) for 3 weeks. Injections were made on alternating sides of the abdomen. Twenty control animals received an equivalent volume of distilled water. Rats were terminated by cervical dislocation at 1 or 4 weeks of treatment 72 h after receiving their final doses. Tissue preparation Upon termination, each animal's heart and Bungs were exposed via thoracotomy, and blood was removed by cardiac puncture with a 3 - d syringe containing 10 pL of sdium heparin solution ( 8 W U f d in distilled water). Plasma was prepared by centrifugation at 1000 X g for 5 min at 4°C. Approximately 0.5 m% of plasma was frozen in liquid nitrogen and stored at -20°C until determination of amio-

darone and desethylamiodarone concentrations by HPLC within 3 weeks. The remainder of the plasma (approximately 1.5 mL) was stored at 4°C until assayed for alanine aminotransferase activity within 24 h. The trachea was exposed and cannulated. The right bronchus was ligated and the right lungs were removed, weighed. frozen in liquid nitrogen, and stored at -2Q°C until analyzed for amiodarone and desethylamiodarme concentrations, and for hydroxyproline content within 4 weeks. Through the trachea, the left lung was inflated in situ to a pressure of 20 cmH,O (1 cmH,8 = 98.1 Pa) with 10% buffered fomalin for 1 h. The trachea was then ligated and the lung placed in 10% buffered fomalin for 3 additional days. The liver was removed and weighed. A portion sf each lobe was removed and placed in 10% buffered fomalin for 3 days. The remainder of the liver was frozen in liquid nitrogen and stored at -20°C until analyzed for amiodarone and desethylamiodarone concentrations within 3 weeks.

His topafk01og-y Following fixation, lung and liver samples were dehydrated, embedded in paraffin, and 5-pm sections cut and stained with hematoxylin and eosin by standard techniques. To evaluate morphological changes in lung tissue in a quantitative manner, a disease index was computed for each animal based on a previously published protocol (Snider et a1. 1978; Daniels et a&. 1989). Each lung section was scanned without prior knowledge of experimental treatment, at a magnification of 125% with an eyepiece grid consisting of 180 equalsized squares. The animal's disease index vdue (indicative of the percentage of grid squares containing cellular infiltration of alveolar spaces, interstitial thickening, cellular infiltration of interstitium, or fibrosis) was determined by taking the mean of values from equal numbers of sections from apical and posterior lung. The mean number of squares examined per section was 675 f 212 (mean f SB) and the disease index for each animal was based on examination of 102%f 742 squares. Morphologic evaluation of amiodarone-induced hepatic injury was assessed by light microscopic examination of liver sections without knowledge of treatment. Liver sections were examined for infiltration by inflammatory cells, fibrosis, and necrosis. Hydr0;&9)prcbline contend The right lung was pulverized in liquid nitrogen and the hydroxyproline content was determined by the spectrophotometric method of Lindenschmidt and Witscki (1985). Tissue amiodarone and &sethy1amio&rone concenfrations Lung, liver, and plasma concentrations of amiodarone and desethylamiodarone were determined by the HPLC procedures of Brien et a&.(1983, 1987). Authentic amiodarone HCl, desethylamiodarone HCl, and didesethylamiaiarone HC1 were dissolved in methanol and used to construct aqueous standard curves. For HPLC analysis of plasma, the mobile phase was a modification of that previously published (Brien et ak. 1983) and was found to greatly prolong the life of the HPLC column. It consisted sf methanol - distilled water - ammonium hydroxide, 94.$:5.1:0.1 (vfvfv) and was pumped at a flow rate of 1 -5 mEfmin. Plasm alanine aminotrarlsferase activity Plasma alanine aminotransferase (ALT) activity was determined at room temperature by a kinetic spectrophotometric method using a commercially available ALT kit (Sigma ALT- 10). Statistical analysis Lung disease indices were compared statistically by a randomized design two-way analysis of variance (ANOVA)after confirming homogeneity sf variance by Bartlett9stest (Gad and Weil1982). Lung hydroxyproline content data from control and amiodarone-treated rats were compared by Student's unpaired t-test, as were alanine aaninotransferase activities and mean body weights. Amiodarone and desethylamiodarone concentrations of lung, liver, and plasm from 1- and 4-week treatment groups were compared using a Cochran t-test for heterogeneous data (Gad and Weil 198%)after determining

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DANIELS BT A&.

heterogeneity of variance by Bartlett's test. Lung, liver, and plasma desea%ayla~&rone/a~odaroneratios, as well as lunglplasma and liverlplasm arPliodarone and desethylamiodarone ratios of animals treated for 4 weeks, were compared with those of animls treated for 1 week using Student's unpaired t-test after first confirming homogeneity of variance by Bartlett's test. En all cases, p < 0.05 was considered statistically significant.

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Experiment B: Eflect of treatment with 158 mg . kg-' day-' amioh o n e for 4 weeh on tissue amiodarane and desethylamiohrone concentrations an$ on hepatic and extrahepatic monooxygenme activities in the rat Animal treatment Eighteen male Sprague-Dawley rats (225 -250 g) received aqueous amiodarone HC1, 850 mg * kg-' . day-' i.p., 5 days/week. Eighteen control animds received an equal volume of distilled water. A n h d s were terminated at 1 or 4 weeks of treatment by cervical dislocation 72 h after their find doses. Plasma was obtained and stored as described for experiment A. Lungs, livers, kidneys, and epididyma1 adipose tissue were removed, weighed, frozen in liquid nitrogen, and stored at -20°C. Plasma and tissue amidarone and desethylamidarone concentrations were determined within 3 weeks as described for experiment A. Preparation of tissue microsmes and measurement of mnosxygemse activities Microsomes from liver, kidney, and lung were prepared as follows. Tissues were perfused in situ with ice-cold 1.15% KC1. Minced tissues were homogenized in four volumes of 1.15% (w/v) KC1 0.1 M potassium phosphate buffer (pH 7.4) using a Potter -Elvehjem glass -tenon tissue homogenizer. Each homogenate was centrifuged at 80 OOQ x g for 20 min at 4°C. The supernatant was centrifuged at 100 OOO x g for g0 f i n at 4°C. The rPnicrosoma1 pellet was resuspended in 0.25 M sucrose - 0.1 M potassium phosphate buffer (pH 7.4). Microsomes were frozen in liquid nitrogen and stored at -70°C. Protein concentration was determined in duplicate using the method of Lowry et al. (1958). Microsom1 7-ethoxyresorufin 0-Beethylase activity (approximately 1 mg microsoml protein1mL of reaction mixture) was determined in duplicate by a kinetic procedure previously described (Burke and Mayer 1974). Aminopyrine N-demethylase activity (approximately 2 mg microsoma1 protein1mL reaction mixture) was determined in duplicate using the method of Maze1 (1971). Cytochrome P-450 content of microsomes from the various organs was determined by the spectrophotometric method of Omura and Sats (1964). Statistical analysis Microsomal 7-ethoxyresorufin 0-deethylase and amninopyrine N-demethylase activities of control and amiodarone-treated animals were compared by Student's unpaired t-test. Kidney aminopyrine N-demethylase activities of control and treated animals were compared using Cochran's t-test for heterogeneous data, after determining heterogeneity of variance by Bartlett's test. Lung, liver, kidney, plasma, and adipose tissue amiodarone and desethylamidarone concentrations, and weights of animals treated with amiodarone for 4 weeks, were compared with those of animals that received 1 week of drug treatment, using the Student's unpaired t-test. Liver 7-ethoxyresomfin 0-deethylase and aminopyrine N-demethykse activities of microsomes from 1- and 4-week control and treated animals were compared using a two-way ANOVA of transformed (natural logarithm) dab (Zivin and Bartko l976), after first determining heterogeneity of variance by Bartlett's test. In all cases, p < 0.05 was considered statistically significant.

Results Experiment A

Mean M y weights of rats that received adodarone or vehicle alone are presented in Fig. 1. Treatment of animals with 200 rng kg-' . day -hmiodarone i.p. (5 daysiweek)

1

;

;

0

2

4

:

:

6

:

8

:

:

:

:

:

:

:

:

;

10 12 94 16 18 20 22 2 4 26 28

T r e a t m e n t Day Fig. 1. Mean b d y weights (fSD) of rats that received i.p. administration of aqueous amiodarone HC1, 200 mg . kg-I day-' (5 dayslweek), for 1 week followed by 150 mg kg-' . daysa (5 dayslweek) for 3 weeks (a), or vehicle alone (0). The number of animals at each time point is indicated. With the exception of day 0, the body weights of amidafone-treated animals were significantly lower than those of control animals ( p < 0,BB5, Student's unpaired t-test).

resulted in the death of 11 of 28 animals by day 7 of treatment, with profound weight loss and diarrhea occurring in surviving animals. No lethality was observed in animals receiving d s tilled water. After the 1st week, the arraiodarone dose was decreased to I50 mg . kg-' - dayo1 (5 daysiweek) to attenuate the observed lethality. In four of the cases of animal mortality, necropsies were performed (gross examination of the abdominal cavity and light microscopic examination of the lung and liver). In all cases, no changes in normal lung or liver histology were observed. Peritonitis was present in one of these animals, but with this exception, the cause of death was not discernable. Upon adjusting the dose, animals showed increases in body weight over the remainder of the study. There was no indication of pulmonary fibrosis in my animal, as assessed by light microscopic exmination. Furthermore, lung hydroxyproline contents (mgiright lung) of rats treated with amiodarone were not significantly different from those of vehicle control animals in the 1-week treatment group (2.25 f 8.54 (n = 4) vs. 1.73 f 0.32 (pa = 5)) or the 4-week treatment group (2.23 f 0.26 (PF= 3) VS. 1.79 f 0.17 (n = 5)). The results of quantitative histopathologicd andysis of lung tissue are presented in Fig. 2. Animals treated with amiodarone for 1 week showed a small increase in the number of macrophages in alvmlu spaces. However, statistical comparison of the group means revealed no significant differences ( p > 0.05). Histologicd examination of livers from amiodarone-treated a n i d s did not reveal any signs of toxicity at either 1 or 4 weeks. This apparent lack of a m i h r o n e hepatotoxicity was further supported by plasma alanine aminotransferase activities (UiE), in which no significant differences were observed between miodarone-treated animals and vehicle control animals in the 1-week treatment group (25.7 f 6.1 (n = 4) vs. 30.0 f 5.0 (n = 5)) or the 4-week treatment group (28.3 f 15.0 (n = 3) vs. 27.2 f 5.7 (a8 = 5)). The amiodarone and desethylamiodarone concentrations in lung, liver, and plasma of rats treated with amiodarone for 1 or 4 weeks are presented in Fig. 3. No didesethylamiodarone,

CAN. J. PHYSIOL. PHARMACOL. VOL. 48, 1990

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TABLE1. Lung, liver, and plasm desethyla~ohronelamiodarone ratios of rats treated with amiodarone for 1 or 4 weeks" Weeks of treatment

No. of animals

1 4

4

3

Tissue DAIA ratiob Lung

Liver

Plasma

0.64k0.35 0 . 2 Q f0.81

0.42k0.49 8.23 k 0 . M

0 . 5 2 f 0.24 0.23C

"Rats received 200 mg . kg-' . day-' (5 dayslweek) aqueous amidarone HCI i.p. for 1 week followed by 150 mg - kg-' . day-' (5 dayslweek) for 3 weeks.

Control

Amiodarone

Control

Amiodarsne

4 Weeks D u r a t i o n sf T r e a t m e n t 1 Week

FIG. 2. Disease indices of lungs from rats following i.p. administration of aqueous awaiobrone HCl, 200 wag . kg-' . days1 (5 dayslwek), for 1 week followed by 150 mg kg-' . days1 (5 dayslweek) for 3 weeks ( 0 , A), or vehicle alone for 1 or 4 weeks

-

(0, A).

w

-?.200

a;? 100

m .-

I--

'

Lung

Liver

Plasma

Lung

1 Week

Liver

Plasma

4 Weeks

D u r a t i o n of T r e a t m e n t FIG. 3 . Tissue and plasm a~niodaroneand desetkylawaiodarome concentrations of rats following i.p. administration of aqueous amiodarone WGl, 200 mg * k g B- days1 (5 dayslweek), for 1 week followed by 150 mg . kg-' . day-' for an additional 3 weeks (5 dayslweek). Bars represent means _+ SD for PB = 4 and m = 3 animals for I and 4 weeks, respectively. * Significantly different from the I-week value ( p < 0.05, Gochran t-test). Detected but below level of quantitative sensitivity of the HPLC assay.

+

another amiodarone metabolite found in certain species (Latini et sl. 1984; Daniels et al. 1989), was detected in any tissues analyzed. Mean amiodarone concentrations were significantly lower in lungs, liver, and plasma of the 4-week treatment group as compared with those from the 1-week group (by 89-9, 87.8, and 89.2% , respectively). Mean desethylamiodarone concentrations were also significantly decreased in lung and liver of the 4-week group (by 97.1 and 94.0%, respectively). Plasma desethylmiodarone concentrations of the 4-week group were below the limit of quantitative sensitivity (0.10 pg1mL) and could not be compared statistically with the plasma desethylmiodarone concentrations of rats in the 1-week treatment group (2.2 f 1.6 pg/mL, pa = 4). Lung and liver dese~ylmidaronelamiodaroneratios were not significantly different between animds treated with miodarone for 1 or 4 weeks (Table 1). As plasma desethylamiodarone con-

bRatios were cdculatd as micrograms Qesethylamiodarone per gram wet tissue weight (or mL piasma) divided by micrograms amidarone per gram web tissue weight (or mL plasma) for the individual animds. The data are presented as group means f SB. A, amiobrone; DA, desethykrniodarone. 'Plasma desethylamiodarone after 4 weeks o f treatment was below the limit sf quantitative sensitivitg: (0.10pglmL), yet was dekechble (above 0.02 pglmk). The ratio presented is the maximum possible desethyla~da~one/ami&rone ratio with an assigned desethyiamihrsne concentration o f 0.10 pg1mL.

centrations of rats treated for 4 weeks were below the limit of quantitative sensitivity, only an estimate of the desethylamio&rone/miodarone ratios could be calculated assuming a desethylamiodarone concentration of 0.10 pg/mL, the limit of sensitivity of the assay. Lunglplasma and liverlplasma amiodaone and desethylmiodarone ratios of animals treated for 1 or 4 weeks are presented in Table 2. Lung and liver for both treatment periods showed extensive accumulation of m i o darone and desethylmiodarone, based on tissuelplasma ratios, with lung and liver amiodarone accumulation not k i n g significantly different between animds treated for 1 or 4 weeks. Although the 4-week plasma desethylmiodarone concentration was below the limit of quantitative sensitivity of the HPLC assay, minimum tissuelplasma ratios have been reported in Table 2 based on an assigned value of 0.10 pg1mE for plasma desethylmiodarone concentration. These ratios demonstrated extensive tissue accumulation, but they could not be compared statistically with 1-week vdues.

Experiment B In this experiment, in which rats received i.p. administration of arnidarone, 150 rng kg-" day-' (5 daysiweek) for up to 4 weeks, lethality occurred in 4 of 18 animals by 7 days of treatment. No lethality occurred in animals receiving distilled water. Mean body weights of animals treated with miodarone or distilled water over the course of this experiment are presented in Fig. 4, with the differences between the two groups not being as large or as long-lasting as seen in experiment A (i.e., significantly different only at days 7 and 14 in experiment B). Lung, liver, kidney, adipose, and plasma amiohrone and desethyl~odaroneconcentration data are presented in Fig. 5. No significant differences were found between tissue and plasm drug and metabolite concentrations of animals &at received amiodarone for 4 weeks when compared with those treated for 1 week. This dosage regimen also had no apparent effect on liver, kidney, or lung microsomd 7-ethoxyresomfin 0-deethylase activity (a selective measure of the cytochrome P45OIA subfmily of enzymes), aminopyrine N-demethylase activity (a nonselective isozyme assay used as an index of total cytochrome P-450 dependent activity), and tot& cytocbome P 4 5 0 content, when compared with the data of the vehicle control animds (Table 3).

Discussion %nthe present study, mde Sprague-Dawley rats received 200 rng kg-' day-' aqueous amiodarone i.p. (5 dayslweek)

DANIELS ET AL.

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TABLE2. Lunglplasm and liverlplasma arniodarone (A) and desethylarnidarone (DA) ratios of rats treated with amiohrone for 1 or 4 weeksa Weeksof treatment

No.of animals

Lunglplasma ratio

kiverlplasma ratio

A

A

DA

DA

'Rats received 208 mg . kg-' . days' (5 dayslweek) aqueous amisdarone HCl i.p. for 1 week folbwed by 150 mg . kg-' . day-' (5 byslweek) for 3 weeks. Ratios (468 misdarone or desethylmisdaronelg wet tissue weight divided by pp asnidarone or desethyl~sdaronelmLplasma) were calculated for individual animals. The data are ex r e s d as means f SD. 'Minimum pwiMe tis.sue/plasma desethylamiodaroneratios, based on an assigned deaethylamiodarone concentration of 8.18 pglmL. The actual plasm concentrations were below the limit of quantitative sensitivity (0.10 ypimL), but they were detectable (above 0.02 pg1mL).

k

0

-

2

4

~

6

8

;

~

;

10 12 14 16 18 20 22 24 26 28

T r e a t m e n t Day

~

~

~

~ 4 Weeks D u r a t i o n sf T r e a t m e n t

I

~

Week

;

:

~

FIG. 4. Mean body weights (fSD) of rats that received i.p. administration of 150 mg kg-' . day-' (5 dayslweek) aqueous amidarone HCl for 4 weeks ( 0 ) or vehicle done (a).The number of animals at each time point is indicated. The body weights of amiochrone-treated animals were significantly Bower than those of control mianials at days 7 and 14 ( p < 64.645, Student's unpaired t-test).

FIG. 5. Tissue and plasma amiodarone and desethylamiodarone concentrations of rats following 1.p. administration of aqueous amiodarcane WCl, 150 mg . kg-I . day-l (5 dayslweek), for 1 or 4 weeks. Bars represent means f SD for la = 4 animals. No significant differences were detected between tissue concentrations of a n i d s treated for 4 weeks and 1 week ( p > 0.05, Student's unpaired t-test). AD, adipose tissue; LU, lung; %I, liver; KI, kidney; PL, plasma.

for 1 week, followed by 150 mg . kg-' - day-' (5 dayslweek) for 3 additional weeks (experiment A). Under these conditions, no signs of pulmonary fibrosis or hepatotoxicity were observed, based on histological and biochemical criteria. The rat has received much attention as a model for both m i o darone pharmacokinetics and miodarone-induced phospholipidosis, with the duration of these studies seldom exceeding 3 weeks or employing dosing regimens above 100 mg . kg-' - days1. Furthermore, the majority of investigators have administered arniodarone orally, which has been shown to result in slow and incomplete absorption in the rat (Blomp et ak. 1985a). As a result, animals may not have received sustained exposure to high levels of amiodarone. Thus, the dose and route of administration employed initially in our study were selected to subject the animals to high levels of amiodarone for a prolonged period. Furthermore, as tissue concentrations of parent drug or desethylmiodarone of animals used in the investigation of miodarone-induced toxicity have not been previously measured concomitantly with other parameters, we have attempted to determine if there was a correlation between tissue odarone and desethylamiodarone concentrations and amiodarone-induced pulmonary and hepatic toxicity.

The high lethality (11 of 20 animals by 7 days) and pronounced weight loss encountered, suggested that i.p. administration of 208 mg kg-l - day-' amiodarone exceeded the maximum tolerable for this strain of rat by this route. Reduction of the dose to 150 mg . kg-I day-' (5 dayslweek) for 3 additional weeks still resulted in the deaths of two of the five remaining animals. However, 150 mg kg-" day-' (5 days1 week) for 4 weeks (experiment B) caused lethality in 4 of 18 animals by 7 days, with 2 subsequent deaths of 8 animals receiving amiodarone for 3 additional weeks. The cause of death is unknown (Costa-Jussa et al. 1984; Heath et ak. 1985; Riva et al. 1987), yet may be the result of adverse cardiovascular effects of amiodarone and (or) desethylamiodarone. Histological examination of the lungs sf rats treated with amiohone (experiment A) failed to show any signs of pulmonary toxicity, including pulmonary fibrosis or alveolar infiltration of lymphocytes, which are often seen in humans with amio&rone-induced pulmonary toxicity. This lack of pulmonary lesions in the rat is consistent with previously published studies in which mimals received various doses of h o d a r o n e orally and were followed for up to 1 month (Costa-Jwsa et al. 1984; Heath et al. 1985; Riva et a&.1987). The results of our study suggest that either the rat is not suse

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TABLE3. Effect of i.p. administration of amidamne, 150 mg kg-' day-' (5 days/week), for 1 or 4 weeks on rnicrosomal monooxygenase activities in the liver, kidney, and lung of the rat" Tissue

Treatment

AHgljinopyrim Ndemehy laseb

7-Ethoxyressmfin 0-dmtBrylaseh

Cytceehrsme P450b

1 week Liver Kidney Lung

Control Amiadarone Control Afiodarone Control Amisdarone

3.84k1.70 (5) 4.46f2.50 (4) 0.02 &0.02 (5)

0.59k0.60 (4) NB ND

4 weeks Liver Kidney Lung

Control Amidarsne Control Ananiodarrane Control Amidarone

'The data are presented as group means f SD. with the number of animals examined in parentheses. 'The 7-ehoxyresomfin 0-dmthylase and aminopyrine N-demethylase activities are reported as picomoles of resomfin formed per minute per milligram protein and nanomoles of fomddehyde fomed per minute per milligram protein, respectively, and the cytochrome P450 content reported as taanomoles P458imilligram progein. ND, not determined.

ceptible to the fibrogenic effects of miodarone or that such a response requires a much longer duration of dmg exposure. In humans, pneumonitis rarely develops before 4 months during amiodarone therapy (Martin and Rosenow 1988). Despite the fact that the animals treated in our experiments received between 13 and 50 times the daily human therapeutic amiodarone dose on a milligram per kilogram basis, the lack of detectable pulmonary toxicity may be the result of the short Wilson duration of treatment. In a recently published report-$, et al. (1989) treated rats ordally with 175 mg - kg-' day-I amiodarone for 1%weeks. No changes in lung hydroxyproline content were detected in the lungs of miodarone-treated animals during their investigation, yet at 6 weeks of treatment md beyond, thickening of alveolar segtae and infiltration of alveolar air spaces with f o m y macrophages were observed. These results are consistent with our findings, indicating the extreme resistance of the Sprague -Dawley rat to amiodaroneinduced pulmonary fibrosis. The small increase in the number of macrophages we observed in the alveolar spaces of rats treated with miodarone for E week (experiment A) probably -induced phospholipidosis, as the dveolar macrophage plays a role in the turnover of phospholipidrich surfactant (Reasor et a&.1988). Phospholipidosis has been observed in the rat afer even acute exposure to low-dose arniochrone (Heath et aI. 1985; Hostetler et a!. 1986; Reasor et a!. 1988). In OUT study, the lack of macrophages in the lungs of rats treated with amiodarone for 4 weeks may be due to the low lung amiodarone and desethylarniodarone concentrations seen in these a n h d s . The lack of pulmonary fibrosis was observed biochemically, as well as histologically. As lung hydroxyproline is found dmost exclusively in the connective tissue protein collagen (Fuller a d Mann 1981; Lindenschmidt and Witschi 1985), and pulmonary fibrosis is considered to be the abnormal deposition of collagen in the lung (Reiser and Last 1979; Fuller and Mann 1981; Weiser and Last 19861, the hydroxyproline content of lungs was measured as a biochemical index

of pulmonary fibrosis. Lung hydroxyproline content sf rats treated with amiodarone was not elevated compared with vehicle control animals. No signs of hepatotoxicity, as assessed by light microscopic examination and measurement of plasma alanine aminotransferase activity, were detected under the conditions of our study. Although severe hepatic injury, including profound necrosis and fibrosis, is a rare occurrence in amiodaronetreated patients, elevated semm transminase activities occur relatively frequently in up to 55% of patients (Heger et al. 1983; Harris et al. 1983; Adams et al. 1986). Gross e&a&. (1989) have recently demonstrated the in viko cytotoxicity (lactate dehydrogenase release) of both arniodarone and desethylmiodarone in cultured Sprague --Dawley rat hepatocytes. These in vitro cytotoxic effects were observed at media concentrations of amiodarone (50 pg/mL), which were lower than hose found in the livers of animals after receiving amiodarone for 1 week (experiment A). We are unable to explain the lack of arniodarone-induced hepatotoxicity in our study, but it could conceivably be related to the sequestration of drug in nontarget organelles, with target organelles not being exposed to prolonged high amiodarone concentrations in vivo. Despite the lack of detectable pulmonary or hepatic toxicity, we observed a marked accumulation of both arniodarone and its major organic metabolite, desethylamiodarone, in lung and liver tissue. This observation is of interest, as it has been demonstrated that desethylmiodarone is more toxic than amiodarone following intratrached administration to hamsters (Daniels et al. 1989) or incubation with cultured rat hepatocytes (Gross et al. 1989). AccumuHation of amiohrone and desethylmidarone in lung and liver may be due ody partly to extensive blood flow to these organs, as other well-perfused tissues such as the heart characteristically have much lower levels (Greene et al. 1983; Plomp et aE. 1985a9 1985b). In experiment B, in which rats received amiodarone, 150 mg kg-' day-' (5 days/wmHc)l for 4 weeks, adipose tissue had we concentration with ody limited desethyl-

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DANIELS ET AL.

miodarone present. It has been proposed that adipose tissue acts as a depot with a large storage capacity from which amiodarone is redistributed to plasma and other tissues in the human (Greene et wl. 1983; Heger et al. 1983; Blomp et al. 1985a, 1985b) and in the rat (Plomp et al. 1985a, 1985b). This pattern of distribution of the two compounds into adipose tissue indicates that the pronounced accumulation of desethylamiodarone in the lungs and liver may not be dependent solely on lipid solubility. In experiment A, use of the amiodarone dosage regimen of 200 mg . kg-I . day-' (5 hyslweek) for 1 week followed by 150 mg . kg-' - day-I (5 dayslweek) for 3 weeks resulted in much lower amiodarone and desethylamiodarone lung, liver, and plasma concentrations at 4 weeks when compared with 1 week. These results are consistent with enhanced elimination, yet previous studies using the rat have either failed to show any differences between disposition characteristics of amiodarone after 5 -6 weeks of continuous drug administration compared with single dose regimens (Weir and Ueda 1986, 1987) or have shown small decreases in elimination aker repeated exposure to amiodarone (Weir and Ueda 1986). While there are reports of a depression of hepatic oxidative metabolism in rats after amiodarone exposure (Grech-Belanger 1984; Barry et al. 1986, 1987), increased activity in the kidney has been observed, showing the potentid for extrahepatic biotransfomation to compensate for this depression (Barry et al. 1987). The observation of decreased amiodarone and desethylamioh r o n e concentrations after 4 weeks of treatment prompted our second experiment, in which animals received amiodarone, 150 mg kg-' - day-' (5 dayslweek) for 4 weeks, and effects on hepatic and extrahepatic microsomd biotransformation activities were measured. Use of this dosing regimen did not result in differences between 1- and 4-week arniodarone or desethylamiodarone concentrations in adipose tissue, lung, liver, or plasma. These data suggest that the additional 50 mg - kg-I - day-I used for the 1st week in experiment A in some way led to decreased tissue drug levels at 4 weeks. While the mechanism by which this occurred is unknown, it may have involved an increase in drug biotransformation capacity, rend or fecal drug elimination, or a combination of these. Treatment with 150 mg kg-' - day-' (5 dayslweek) for 4 weeks did not result in detectable changes in hepatic and extrahepatic microsomal monooxygenase activities or in total cytochrome P-450 content. Another factor that could have played a part in decreased tissue amiodarone and desethylamiodarone concentrations is diminished absorption of amiodarone after injection into the peritoneal cavity. However, there was no evidence of precipitated amidarone in the abdominal cavity upon termination of the animals. A further possibility is that administering 200 mg . kg-' - dayy-Ifor the first 5 days altered the distribution or excretion of amiodarone. Furthermore, profound weight loss may have reduced adipose amiodarone stores, hence perturbing tissue distribution. Despite the lower tissue miodarone and desethylmiodarone concentrations at 4 weeks, no significant differences were seen between lung, liver, or plasma desethylamiodaronelmiodarone ratios of animals treated for 4 weeks as compared with those treated for 1 week. As the in vivo formation of desethylarniodarone was likely to be first order with our treatment protocol (Weir and Ueda B986), this implies that the capacity of mechanisms for the elimination of miodarone and desethylamiodarone are either affected in an identical manner

or are not affected at d l . This is consistent with induction of a pathway or pathways for amiodarone and desethylamiodarone elimination. There is evidence to suggest that alternate pathways of amiohrone metabolism are present, resulting in formation of metabolites (as yet unidentified), which are more water soluble than miodarone, desethylamiodarone, and didesethylamiodarone (Staubli et a!. 1985; Weir and Ueda 1987). The HBLC assays employed in our laboratory and in other laboratories are capable of separating and quantitating the latter three compounds. The studies indicating the existence of other metabolites have utilized radiolabelled amiodarone, with the observation that the radiolabel is present in forms not associated with miodarone or its two known organic metabolites. The complete elucidation of the disposition of miodarone, including the identification of all its metabolites, remains to be determined. In the present investigation, male Sprague -Dawley rats have been treated with amiodarone for 4 weeks without observable pulmonary or hepatic toxicity resulting, based upon biochemical or histological criteria (experiment A). In this experiment, tissue levels of amiodarone and its metabolite, desethylamiodarone were lower after 4 weeks of treatment than aker 1 week, suggesting amiodarone-induced alterations in monooxygenase activities. However, no differences were observed in hepatic or extrahepatic monooxygenase activities in a subsequent experiment after repeated amiodarone exposure (experiment B). Our resdts, in conjunction with those of Wilson et a!. (1989), indicate that systemic administration of amiodarone to rats is unlikely to produce a model useful for exmining amiodarone-induced pulmonary fibrosis or hepatotoxicity. -

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