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Electropharmacology of amiodarone: Absence of relationship to serum, myocardial, and cardiac sarcolemmal membrane drug concentrations
November1966 Lababidi
and Weinhaus
American
3. Lababidi ZA, Wu JR, Walls JT. Percutaneous balloon aortic valvuloplasty: results in 23 patients. Am J Cardiol 1983; 53:194. 4. Rupprath G, Neuhaus K. Percutaneous balloon valvuloplasty for aortic valve stenosis in infancy. Am J Cardiol 1985; 55:1655. 5. Keane JF, Bernhard WF, Nadas AS. Aortic stenosis surgery in infancy. Circulation 1975;52:1138. 6. Edmunds LH Jr, Wagner HR, Heymann MA. Aortic valvulotomy in neonates. Circulation 1980;61:421. 7. Kugler JD, Campbell E, Vargo TA, McNamara DG, Hallman GL. Results of aortic valvulotomy in infants with isolated aortic valvular stenosis. J Thorac Cardiovasc Surg 1979; 78:553.
Heart
Journal
8. Trinkle JK, Grover FL, Arom KV. Closed aortic valvulotomy in infants. J Thorac Cardiovasc Surg 1978;76:198. 9. Brown JW, Robinson RJ, Waller BF. Transventricular balloon catheter aortic valvulotomy in neonates. Ann Thorac Surg 1985;39:376. 10. Waller BF, Girod DA, Dillon JC. Transverse aortic wall tears in infants after balloon angioplasty for aortic valve stenosis. J Am Co11 Cardiol 1984;4:1235. 11. Sanchez GR, Mehta AV, Ewing LL, Brickley SE, Anderson TM, Black IF. Successful percutaneous balloon valvuloplasty of the aortic valve in an infant. Pediatr Cardiol 1985;6:103.
Electropharmacology of amiodarone: Absence of relationship to serum, myocardial, and cardiac sarcolemmal membrane drug concentrations Plasma concentrations are often of major consideration in the evaluation of therapeutic efficacy of cardiovascular drugs. This approach is based on the assumptions that the concentration of the drug in the cardiac muscle is in equilibrium with the plasma drug level and that pharmacologic efficacy is proportional to the myocardial drug concentration. The more pronounced pharmacologic efficacy of amiodarone following chronic administration, despite low plasma drug concentrations, and the lesser effects of the drug after acute intravenous administration, when drug levels are maximum, has not been explained on the basis of the pharmacokinetic behavior of the drug. Data obtained from the transmembrane action potential recordings from rabbit ventricular myocardium were therefore correlated with drug concentrations in the serum, myocardium, and myocardial sarcolemma following acute intravenous administration and after 4 weeks of oral administration of 20 mg/kg/day of amiodarone. Following 15 minutes of acute drug administration, when amiodarone concentrations were maximal in the serum (4.72 + 1.23 As/ml), cardiac muscle (34.5 k 7.6 pg/gm), and sarcolemma (1.94 mg/gm protein), the electrophysiologic changes were insignificant. However, following chronic treatment, when levels of amiodarone were low in the serum (0.05 + 0.01 Ag/ml amiodarone, 0.25 t 0.08 pg/ml desethylamiodarone), cardiac muscle (1.91 + 0.9 Ag/gm amiodarone, 1.35 ? 1.33 pg/gm desethylamiodarone), and myocardial membranes (0.043 mg/gm protein [amiodarone], 0.097 mg/gm protein [desethylamiodarone], there was a 54.3% increase in action potential duration at 90% repolariration (p < 0.01) and 65% increase in the effective refractory period (p < 0.01) of rabbit ventricular myocardium. Therefore, the magnitude of electrophysiologic effects induced by amiodarone are not explained by the pharmacokinetics of the drug but are probably related to the drug-induced changes in cellular metabolism. (AM HEART J 1986;112:916.)
Nagammal Venkatesh, M.D., Pitambar Somani, M.D., Ph.D., Malcolm Bersohn, M.D., Ph.D., Richard Phair, Rinya Kato, M.D., and Bramah N. Singh, M.D., Ph.D. Los Angeles, Calif,, and Toledo, Ohio From the Departments Medical Centers and Clinical Pharmacology, Supported Veterans Angeles
916
of Cardiology, the UCLA School Medical College
VA Wadsworth of Medicine. and of Ohio.
in part by grants from the Medical Administration and the American Heart Affiliate.
Research Association
and Sepulveda the Division of Received Service of the Greater Los
for publication
April
Reprint requests: Nagammal lllE, VA Wadsworth Hospital, CA 90073.
1, 1986; Venkatesh, Wilshire
accepted
May
5. 1986.
M.D., Cardiology and Sawtelle Blvds.,
Section 6911 Los Angeles.
With modern methods such as gas-liquid chromatography, high-pressure liquid chromatography (HPLC), and radioimmunoassay, plasma levels of drugs can be measured easily and specifically. However, this information is only useful where there is a reasonably linear relationship between the plasma levels and the various pharmacologic effects of a drug. Drugs such as amiodarone, which exhibit multicompartmental kinetics, cannot be monitored by single serum level determinations. Data from single-dose studies show that amiodarone has a very large volume of distribution and a relatively low total clearance rate.’ The drug is cleared more rapidly from the well-perfused tissues of the central compartment in which its receptor is likely to be located but is eliminated much more slowly from a poorly perfused tissue compartment.2 This might partially explain the long elimination half-life of amiodarone.2 It is also known that there is a poor correlation between plasma levels and the pharmacologic effects of the drug.” It has been shown that a finite period of drug treatment is necessary before the manifestation of maximum pharmacologic action of amiodarone.4.” This latency of onset of action has been attributed to: (1) time to reach maximal concentrations at the site of action, the myocardium, (2) slow accumulation of the pharmacologically active metabolite, desethylamiodarone, (3) drug-induced T, deficiency resulting in secondary changes in cardiac membrane, and (4) alterations in cellular lipid metabolism. Experiments in animals have not shown a direct relationship between serum or myocardial levels of amiodarone and its pharmacologic effects.6 The question thus arose whether the observed electrophysiologic differences between the acute and chronic effects of amiodarone might be the result of a time-dependent drug uptake by the myocardial sarcolemmal membranes. We therefore determined the levels of amiodarone and its metabolite desethylamiodarone in the ventricular sarcolemma of the rabbit and explored the relationship between the concentrations of amiodarone and desethylamiodarone in the serum, cardiac muscle, and myocardial sarcolemma and the magnitude of electrophysiologic responses following acute imtravenous and chronic oral administration of the drug. Data obtained from transmembrane action potential recordings of the ventricular myocardium were correlated with drug concentrations in the serum, myocardium, and myocardial sarcolemmal preparations. METHODS New Zealand white male rabbits, weighing 2.0 to 2.5 kg, were used for studies involving the measurement of
ventricular transmembrane action potentials and for the measurements of drug levels in the plasma. ventricular myocardium, and the cardiac sarcolemmal preparation. Since the entire right and the left ventricular myocardium was used for the sarcolemmal preparation, a different set of animals was used for electrophysiologic experiments. Measurements of ventricular transmembrane action potentials. Four groups of rabbits were studied. Two
groups (l[n = 51 and 2[n = 91) were used ah control subjects for the acute and chronic experiments. Groups lA(n = 5) and ZA(n = 8) were used for acute and chronic experiments following amiodarone treatment. Rabbits used in the acute experiments were giv~tn 20 mg/kg amiodarone (5”; aqueoussolution) or an equivalent VOIume of deionized distilled water intravenously. Rabbits used in the chronic experiments were treated with 20 mg/kg/day amiodaroneor an equivalent volume of deionized distilled water orally for 4 weeks. Fifteen minutes following acute intravenous administration and 24 hours following the last oral dosein the chronic experiments, the rabbits were given an overdoseof intravenous pentobarbital sodium,and the hearts were quickly excisedand placed in cool oxygenated (95°C O2 5’( CO,) Tyrode’s solut,ion. The right ventricular outflow tract, measuring approximately 1 cm X 0.5 cm, was carefully dissected and was pinned, with the endocardium facing up, 10 i be paraffin base of a 10 ml Lucite chamber. Preparations were stimulated by meansof Teflon-coated biptllar silver-wire electrodes connected via a stimulus isolation unit to a Grass S-88 simulator. Impulses of ‘Lmsecduration and twice diastolic threshold current were used Ventricles were stimulated at 1.0 Hz frequency. The pH of Tyrade’s solution was7.4 i 0.02 and the composition (mM) was as follows: NaCl 130: KC1 4.0; CaCI. 1.~ MgSO, 0.5; NaH,PO, 1.8; glucose5.5; and NaHCO, I &!I. The flow rate was 10 to 15 ml/min. The temperat,ure wab maintained at 35.5 ~tr0.5” C for the acute experiments attd at 37 + 0.5” C for the chronic experiments. Transmembrane action potentials were recorded by meansof 3M K(:‘l-filled glass capillary microelectrodeshaving tip resistancesof 10to 30 MO. The microelectrodes were coupled tr+ a Ag-AgCl junction and an intracellular probe system to an amplifier with a high-input impedance and variable input capacity neutralization (Mentor N-950). An electrrjde differentiating circuit (time constant: 2Oyset) was usedto determine the maximum rate of rise of phase zero of the action potential (V,,J. The system was calibrated by meansof a 10 mV internal square-wavesignal. The a&m potentials and V,,, were displayed on a storage oscilloscope(Tektronix R564B) and photographed on poiaroid film. The action potentials were recorded from ii site near the stimulating electrode. Effective refract.ory periods were determined by introducing, after every 8 (o IO basicbeats, extra stimuli with progressively shorter coupkingintervals. The effective refractory period wasdefined asthe shortest premature interval producing a responsewhich depolar-ized to values more positive than 0 ml’. En four acute preparations ventricular myocardial samples were obtained prior to and following superfuslon to determine if there were differences in the mvocerdia! drug concentra-
918
Venkatesh
et al.
American
November 1986 Heart Journal
1. Electrophysiologic effects of acute and chronic treatment with amiodaronein isolated rabbit ventricles: Comparisonwith a control group* Table
A i’A fmV)
Treatment
RMP CmVI
r;;,,,” (v/see)
APD,, (msec)
ERP (mwc)
AP& (msec)
(Group 1) 1 ml DD H,O intravenouslylfj min (n = 5) (Group 1A) Amiodarone20 mg/kg intravenouslyfor 15 min (n = 5) (Group2) Control 1 ml DD H,O/day orally for 4 weeks (n =9) (Group2A) Amiodarone20 mg/kg/day orally for 4 weeks (n = 8)
Control
90 i: 90
164
+ 52
851
120& 4.0
89+
4
153
k 69
80
1002
4
78k
4
150
i
16
7
78k
5
155
r
56
123
IL 12
99+
Ahbrreviations: APA = action potential amplitude; RMP = resting membrane repolarization times; ERP API>,,, = action potential durations at 50rC and 90’; *There were no significant changes in any of the parameters measured following treatment, there were significant increases in action potential durations at 50’,
8
115
+
8.0
+ 24
121
zt 27
66
+
18
94 I
103
I
26t
145
17
-t
36t
119
t
7.1
l??<. + 27
100
i
17
165
-t 31t
rate of rise of phase 0: Al’DS,, and potential; vnax = maximum = effective refractory period: DD H,O = deionized distilled water. 15 minutes of treatment with amiodarone. However, following 4 weeks 01 and 90’, repolarization times and effective refractory period.
tp < 0.01.
tions resulting from washout of amiodarone from the tissue during superfusion. Preparation of myocardial sarcolemma. Two groupsof animalstreated in an identical manner to those in groups IA and 2A wereused.Following an overdoseof intravenous sodium pentobarbital, the hearts were quickly removed through a thoracotomy incision and washed in ice-cold Tris-maleate buffer. Following dissection and removal of the atria, great vessels, fat, and connective tissue, the ventricles were blotted dry and weighed;the myocardium was minced in ice-cold Tris-maleate buffer and homogenized in a Polytron homogenizer(Brinkmann Instruments, Inc., Westbury, N.Y.). Then KC1 and pyrophosphate (final concentrations 300 mM and 25 mM, respectively) were added and the homogenate was rehomogenized. The homogenatewascentrifuged at 4’ C and 167,000 X g for 40 minutes. The pellet was then treated with DNase (2000 units/gm heart weight). This mixture was incubated in a shakerbath at 40” C for 45 minutes. Following a short burst of homogenization, the homogenatewaslayered over 27% sucrosesolution and centrifuged at 4’ C and 122,000X g for 20 minutes. The supernate was removed and, following Teflon homogenization, centrifugation was carried out at 4OC and 167,000X g for 45 minutes. Then the pellet was suspendedin 2 ml of the Tris-maleate buffer. In separate preparations not used for drug level determinations, the sarcolemmawaspurified eight- and threefold compared to crude homogenate, as determined by the sarcolemmal marker K+-dependent p-nitrophenylphosphatase. Serum levels of amiodarone
and desethylamiodarone.
To 500~1aliquots of serum, 100 ~1of the internal standard L8040 hydrochloride (0.01pg/ml in methanol) and 25~~1 of 1.5M potassium dihydrogen phosphate (pH 4.5) were added and then mixed thoroughly. Ten microliters of
isoamyl alcohol and 5 ml of n-pentane were added to the preceding mixture and, after thorough mixing, centrifugation at 1500X g for 5 minutes was carried out. The pentane layer was saved, and 100 11 of 5M sodium hydroxide was added and then vortexed for 90 seconds. The mixture wasthen centrifuged at 600 X g for 1 minute; the aqueouslayer was frozen in dry ice-methanol, and the organic layer was carefully decanted into a 10 ml Lauertipped glass syringe fitted with a Millipore filtration system loaded with a 0.2 pm pore-size nylon filter. The filtrate was collected and evaporated to dryness at 85’ C. Methanol-perchloric acid (100 ~1of the mobile phase)was added to the tube and vortexed. An aliquot (50 ~1) was injected into the HPLC and peak heights of amiodarone, desethylamiodarone,and the internal standard were measured. At a flow rate of 1.5 ml/min, the retention times for desethylamiodarone, L8040, and amiodarone were 6.3, 7.86, and 10.06minutes, respectively. Standard curves in plasma and tissue samples were prepared and the unknown concentrations were calculated from the peakheight ratios. Determination of levels of amiodarone and desethylamiodarone in cardiac muscle and sarcolemma. A small
piece of ventricular myocardium, 0.5 cm x 0.5 cm, was removed, washed, and blotted dry. A 0.3 gm/ml tissue homogenate was prepared with a Polytron homogenizer and L8040 was added to the homogenate as internal standard. Standard calibration curves for the myocardial tissue and sarcolemmawere prepared by adding 2.5 to 40 pg/gm of amiodaroneand desethylamiodaroneand L8040 (0.85 pg/gm) to the cardiac muscle and sarcolemma obtained from a control animal. Extraction wascarried out on 2 ml of homogenateand 500 ~1of sarcolemmalsuspension buffered with 70~1of phosphatebuffer; the procedure
Volume
112
Number
5
of nnrmdaronc
Electropharr;,acology
CONTROL
AM (20mg/kg,
I.V. 15min)
AM (20mg/kg/day,
919
p.o. 4wks)
L-----._I 100
msec
Fig. 1. Action potentials from ventricles of control and amiodarone-treated rabbits (acute and chronic) are shown. The lengthening of action potential duration following 15 minutes of intravenous treatment was insignificant. A significant prolongation of action potential duration was observed with 4 weeks of treatment.
wassimilar to that of plasmaexcept no isoamyl alcoholwas added. The resultswere expressedasmicrogramsper gram wet weight of cardiac muscle and milligrams of drug per gram of protein concentration in the sarcolemmalpreparation. The method of Lowry wasusedfor determination of protein in the sarcolemmalpreparation. Statistical analysis. Data were expressedas means-t SD. Linear correlations were determined by correlation coefficient using the Biomedical Data Package (BMDP) programs on the PDP-11 computer system. The significance of differences observed in the electrophysiologic data in the drug levels in the plasma,cardiac muscle,and myocardial sarcolemma were determined by unpaired Student’s t test. RESULTS Effects of acute and chronic amiodarone treatments on rabbit ventricular action potentials. Following acute
intravenous administration of 20 mg/kg of amiodarone, there was a 5.2% (NS) increase in the action
potential duration at 90% repolarization time and a 12.6% increase (NS) in the effective refractory period (Table I and Fig. 1). There were no significant changes in the 6,,, and resting membrane potential. However, following 4 weeks of oral administration of 20 mg/kg/day of amiodarone, there was a 65% increase (p < 0.01) in the effective refractory period and a 54.3% increase (p < 0.01) in the action potential duration at 90% repolarization. Once again, no changes were observed in other electrophysiologic parameters such as the $“,,= and resting membrane potential. Concentrations of amiodarone and desethylamiodarone in rabbit serum, cardiac muscle, and myocardial sarcolemmai membranes. The concentrations of the
drugs in the serum ,cardiac muscle, and myocardial sarcolemma following acute and chronic treatment with amiodarone are presented in Fig. 2. In the blood samples obtained at 15 minutes following intravenous administration of amiodarone, the
serum levels were 4.72 -t 1.23 pg/ml of amiodarone and 0.14 it- 0.08 pg/ml of desethylamiodarone. The corresponding concentrations of amiodarone and desethylamiodarone in the cardiac muscle were 34.5 +- 7.6 pg/gm and 2.13 2 1.37 ,ug/gm, respectively. In the left ventricular sarcolemmal preparation the concentration of amiodarone was 1.94 +- 0.445 mg/gm of protein and that of desethylamiodarone was 0.034 it 0.011 mg/gm of protein. Following 4 weeks of oral administration of the same dose of amiodarone, the serum levels were 0.05 + 0.01 pg/ml of amiodarone and 0.25 -t 0.08 &ml of desethylamiodarone. Myocardial concentrations were 1.91 + 0.9 pg/gm of amiodarone and 1.35 + 1.33 &gm of desethylamiodarone. Measurements in the sarcolemma1 preparations showed 0.043 + 0,023 mg of amiodarone /gm of protein and 0.097 f 0.047 mg of desethylamiodarone /gm of protein. DISCUSSION
Our study confirms the previous findings that at a constant dose of amiodarone, the electrophysiologic effects of amiodarone, dominated by increases in the action potential duration and refractoriness in the rabbit ventricular myocardium, develop as a function of time.i Recently, Morady et al.B have shown the lack of correlation between serum levels of the drug and changes in ventricular refractoriness, conduction, and ventricular tachycardia induction. In experimental animals the latency in the onset of pharmacologic action could not be explained by the drug concentrations in the plasma or the myocardium.6 In the present study, measurements of drug levels in the ventricular myocardial sarcolemmal preparation demonstrated a similar pattern in that, following acute administration of amiodarone, the levels were significantly higher than those following long-term administration. However, drug levels in the intracellular components were not measured.
November
920
Venkatesh
et al.
American
0
-
L,
AMIODARONE
1
AC Ch
AC Ch
m
Heart
1986 Journal
DESETHYLAMlODARO,,E
0AC Ch AC Ch
2. Concentrations of amiodaroneand desethylamiodaronein the serum, myocardium, and ventricular sarcolemmal preparation are shown. Note the significant reduction in the concentrations of amiodarone following chronic (Ch) treatment in all of the tissuesanalyzed, and the levels of desethylamiodarone in the serum and sarcolemmafollowing chronic treatment; p < 0.0001.AC = acute. Fig.
The fact that following acute administration the levels of amiodarone were high in the plasma, myocardium, and myocardial membrane when the electrophysiologic effects were minimal or absent and the converse occurred following chronic oral administration of the drug makes it difficult to explain the chronic efficacy of amiodarone based on its pharmacokinetics. We and other investigators have shown that the acute effects may, in part, result from the nonspecific adrenolytic properties of the drug,6pg-11whereas the long-term action is related to the prolongation of action potential duration and refractoriness in addition to the enhancement of adrenergic inhibition.7 Gloor et a1.12 have presented data to indicate that the acute effects of amiodarone on the sinoatrial and atrioventricular nodes in dogs might also be the result of inhibition of the slow calcium channel. Further work is clearly necessary to elucidate the mechanisms involved in mediating the acute electrophysiologic changes induced by amiodarone. In contrast, the electrophysiologic effects of long-term amiodarone therapy are reasonably uniform and consistent.4s5 Myocardial uptake of amiodarone. Connolly et al.13 showed that amiodarone concentrations in the plasma and the myocardium were different during the first 30 minutes following intravenous administration of the drug. While the plasma level fell significantly, the myocardial concentration of amiodarone increased during this period. Maximal myocardial concentrations were observed between 20 and 30
minutes. They also showed that myocardial concentrations of amiodarone more closely reflected the electrophysiologic effects of the drug in the dog than do plasma concentrations. However, the electrophysiologic parameters that were measured were sinus cycle length, atrioventricular node, Wenkebath cycle length, and functional refractory period, all of which are under adrenergic influence and therefore may be attributed to the drug’s ability to exert a direct nonspecific adrenergic antagonism. Latini et alI4 have shown that myocardial uptake of amiodarone was rapid and maximum levels were found at 15 to 30 minutes following acute intravenous administration. This was also shown by Somani et a1.15 who, in addition, found a close correlation between the time course of myocardial uptake and uptake in white blood cells. It was suggested that measurement of drug concentration in white blood cells might predict myocardial tissue concentration. Somani’s study also showed a significant decrease in premature ventricular contractions and rate of ventricular tachycardia following experimental myocardial infarction in dogs at 6 hours following intravenous administration of amiodarone. Again, this may have been secondary to the beta-adrenergic-blocking action of amiodarone since, in studies of sudden death in a canine model, Patterson et a1.16showed that the chronically administered drug was more effective in preventing inducible ventricular fibrillation than the acutely administered drug, a difference that could not be explained on the basis of myocardial drug levels.
Volume Number
112 5
Electropharmacology
Significance of sarcolemmal amiodarone tions. Following chronic administration
concentra-
of the drug we found the sarcolemmal concentration of amiodarone to be 40 times less than that following acute administration, a phenomenon identical to that seen in the cardiac muscle. It is known that most drugs exhibit a reasonably simple and linear concentraconcentration is tion-response curve. l7 A minimum usually required for a threshold response, with a stepwise increase in effect as the drug concentration is elevated. Our data show that this does not hold for complex pharmacologic molecules such as amiodarone, which is thought to act by influencing various metabolic parameters of the cell as a function of time. Long-term amiodarone treatment has been shown to inhibit the peripheral conversion of T, I8 to T.3, resulting in a decrease in T3, an increase in rT,, and a minimal increase in T, in the plasma.lg This alteration in the thyroid hormone metabolism, as well as a direct inhibition of T, nuclear binding by amiodarone and/or its metabolite desethylamiodarone,*“. *I is thought to result in a hypothyroid state at a cellular level. l8 Since the electrophysiologic effects of hypothyroidism22-25 are almost identical to those observed following long-term amoidarone treatment, this phenomenon is thought to exhibit some cardiospecificity.7. ~326 Gross and Somani2? have shown that amiodarone alters the lipid metabolism of the cardiac cell, as evidenced by the development of lysosomal and myelinoid inclusion bodies. Whether the membrane lipids are altered by the presence of the drug and its metabolite is, however, not known at present. Neither is it clear whether such changes might result from prolonged hypothyroidism. Pharmacologic
significance
921
been found that the therapeutic efficacy of amiodarone may be better monitored by rT,, levels’” in the plasma, and the incidence of toxicity correlates more closely with the plasma levels of desethylamiodaTone4 than with those of amiodarone. In summary, our study confirms the previous findings that measurement of drug levels may not constitute a reliable index for the evaluation of therapeutic efficacy of amiodarone, but our data do not exclude it as a basis of drug toxicity during protracted treatment. We have shown that. there is a poor correlation between the myocardial drug levels as well as the membrane levels and the magnitude of pharmacologic action of the drug. Therefore, mechanisms other than tissue drug concentrations are likely to be responsible for the magnitude and temporally related enhancement of the electrophysiologic and antiarrhythmic actions of arniodarone. We are indebted to Lawrence preparation of this manuscript.
Kimble
i’or ~~‘cu’x~,I!
help in t hc
REFERENCES
1. Heger
JJ, Prystowsky EN, Miles W-M, Zipes L)t’. Clinlcai use pharmacology of amiodarone. Meci ( ‘I~II North Am 1984;68:1339-67. Halt DW, Tucker GT, Jackson PR, St,orey (;;.:A .4miodarone pharmacokinetics. AM HEART d 1983;106:840-846. Mostow ND, Rakita L. Vrobel TR, Noon DI,. Blumer ,I. Amiodarone: Correlation of serum concemration with suppression of complex ventricular ectropic as?ivitv. Am J Cardiol 1984;54:569-74. Heger JJ, Prystowsky EN, *Jackman WM. er al. Amiodarone. Clinical efficacy and electrophysiology durin:: lijng-term therapy for recurrent ventricular tachycardia or \-ell.tricular tihrillation. N Engl J Med 1981;305:539-545. Nademanee K. Singh BN, Hendrickson JA, t-7 al. Amiodartrnt in refractory life-threatening ventricular tirrhvthmias. Ann Intern Med 1983;98:577-84. Venkatesh N. Padbury JF. Singh BN. Effec,i.s ($I amiodarimr. and desethylamiodarone on rabbit myocarrhai beta-adreno ceptors and serum thyroid hormones. Absence (II relationship to serum and myocardial drug concrntrar i# in+. .I (‘ardiovasl, Pharmacol. (In press.) Singh BN. Amiodarone: Historical developmen; and pharmacologic profile. AM HEART J 1983;106:788-9;. Morady F, Dicarlo LA, Krol RB, Berman ,JXl, Huitleir M. Acute and chronic effects of amiodaronc, !UI vent,ricular refractoriness. intraventricular ronducticm and ventricular tachycardia induction. J Am Co11 Cardiol lifH6;7:148-57. Polster P, Broekhuysen .J. The adrenerpcc antagonism of amiodarone. Biochem Pharmacol 1976::!S: I :l 1%. Nokin P, Clinet M, Shoenfeld P. Cardial f<-adrem,ceptol modulation b> amiodarone. Biocbc~n, F’harmaccsl 1983;32:2473-‘457. Gagnol ,JP, Devos (.‘. (‘linrt M, NcBk!n 1’ Ami~lclaronr: 13iochemical aspects and hemodvnami 2 tfects. I)ruph 1985;29(suppl 3):1-l& (iloor HO. LTrthaler F. James ‘I’ti. Acute i,tftec‘ts uf amioda rone upon the canine sinus node and arrilrve~ltric:ular jum tional region. J Clin Invest 1983;71:1457-I 17’:. Connolly SJ, Latini R, Kates RE. Ph:lrm;ir:odynamIcs ~)i intravenous amiodarone in the dog. -I (‘arriiovalsc I’harmacg ,i 1984;6:531-535. Latini R. Connnllv S,J, Katcs RE. Mv{bcar.:i;ti dispcrsltion 111 and
2. 3.
4.
5.
6.
of desethylamiodarone.
The metabolism of amiodarone results in the formation of its prinicipal metabolite desethylamiodarone, which we have shown to be pharmacologically active.“~“” In our study, following chronic treatment with amiodarone, the desethylamiodarone levels were higher than those of amiodarone in the myocardial sarcolemmal membrane and equivalent in the cardiac muscle and serum. Thus, the question arises whether the manifestation of the pharmacologic actions of amiodarone is dependent upon the attainment of a critical level of the metabolite in the myocardial membrane. However, we have shown that a latency in the onset of action, almost similar to that observed with amiodarone treatment, is also seen with desethylamiodarone.6 We have also shown that, with regard to adrenergic antagonism and electrophysiologic changes, the metabolite was at least as potent as the parent compound.6’2g It has
of nmrorlurlmc
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American
Heart
1986 Journal
23. Johnson PN, Freedberg AS, Marshall JM. Action of thyroid hormone on the transmembrane potentials from sino atria1 nodal cells and atria1 muscle cells in isolated atria of rabbits. Cardiology 1973;58:273-289. 24. Gavrilescu S, Luca C, Streian C, Lungu G, Deutsch G. Monophasic action potential of right atrium and electrophysiologic properties of AV conducting system in patients with hypothyroidism. Br Heart J 1976;38:1350-1354. 25. Sharp NA, Neel DS, Parsons RL. Influence of thyroid hormone levels on the electrical and mechanical properties of rabit uapillarv muscle. J Mol Cell Cardiol 1985:17:119-132. 26. Singh’BN, Vaughan Williams EM. The effect ofamiodarone, a new antianginal drug, on cardiac muscle. Br J Pharmacol 1970;39:657-667. 27. Gross SA, Somani P. Amiodarone-induced ultrastructural changes in the canine myocardial fibers. AM HEART J (in press 1986). 28. Venkatesh N, Al-Sarraf L, Singh BN. Digoxin-desethylamiodarone interaction in rats. Comparison with that of amiodarone. J Cardiovasc Pharmacol. (In press.) 29. Kato R, Venkatesh N, Yabek S, Takikawa R, Kannan R, Singh BN. Electrophysiologic effects of desethylamiodarone, an active metabolite of amiodarone: Comparison with amiodarone during chronic administration in rabbits. (Submitted to J Am Co11 Cardioll 30. Venkatesh N, Al-sarraf L, Hershman JM, Singh BN. Effects of desethylamiodarone on thyroid hormone metabolism in rats: Comparison with the effects of amiodarone. Proc SOC Exp Biol Med 1986;181:233-236.
and Weinhaus
American
3. Lababidi ZA, Wu JR, Walls JT. Percutaneous balloon aortic valvuloplasty: results in 23 patients. Am J Cardiol 1983; 53:194. 4. Rupprath G, Neuhaus K. Percutaneous balloon valvuloplasty for aortic valve stenosis in infancy. Am J Cardiol 1985; 55:1655. 5. Keane JF, Bernhard WF, Nadas AS. Aortic stenosis surgery in infancy. Circulation 1975;52:1138. 6. Edmunds LH Jr, Wagner HR, Heymann MA. Aortic valvulotomy in neonates. Circulation 1980;61:421. 7. Kugler JD, Campbell E, Vargo TA, McNamara DG, Hallman GL. Results of aortic valvulotomy in infants with isolated aortic valvular stenosis. J Thorac Cardiovasc Surg 1979; 78:553.
Heart
Journal
8. Trinkle JK, Grover FL, Arom KV. Closed aortic valvulotomy in infants. J Thorac Cardiovasc Surg 1978;76:198. 9. Brown JW, Robinson RJ, Waller BF. Transventricular balloon catheter aortic valvulotomy in neonates. Ann Thorac Surg 1985;39:376. 10. Waller BF, Girod DA, Dillon JC. Transverse aortic wall tears in infants after balloon angioplasty for aortic valve stenosis. J Am Co11 Cardiol 1984;4:1235. 11. Sanchez GR, Mehta AV, Ewing LL, Brickley SE, Anderson TM, Black IF. Successful percutaneous balloon valvuloplasty of the aortic valve in an infant. Pediatr Cardiol 1985;6:103.
Electropharmacology of amiodarone: Absence of relationship to serum, myocardial, and cardiac sarcolemmal membrane drug concentrations Plasma concentrations are often of major consideration in the evaluation of therapeutic efficacy of cardiovascular drugs. This approach is based on the assumptions that the concentration of the drug in the cardiac muscle is in equilibrium with the plasma drug level and that pharmacologic efficacy is proportional to the myocardial drug concentration. The more pronounced pharmacologic efficacy of amiodarone following chronic administration, despite low plasma drug concentrations, and the lesser effects of the drug after acute intravenous administration, when drug levels are maximum, has not been explained on the basis of the pharmacokinetic behavior of the drug. Data obtained from the transmembrane action potential recordings from rabbit ventricular myocardium were therefore correlated with drug concentrations in the serum, myocardium, and myocardial sarcolemma following acute intravenous administration and after 4 weeks of oral administration of 20 mg/kg/day of amiodarone. Following 15 minutes of acute drug administration, when amiodarone concentrations were maximal in the serum (4.72 + 1.23 As/ml), cardiac muscle (34.5 k 7.6 pg/gm), and sarcolemma (1.94 mg/gm protein), the electrophysiologic changes were insignificant. However, following chronic treatment, when levels of amiodarone were low in the serum (0.05 + 0.01 Ag/ml amiodarone, 0.25 t 0.08 pg/ml desethylamiodarone), cardiac muscle (1.91 + 0.9 Ag/gm amiodarone, 1.35 ? 1.33 pg/gm desethylamiodarone), and myocardial membranes (0.043 mg/gm protein [amiodarone], 0.097 mg/gm protein [desethylamiodarone], there was a 54.3% increase in action potential duration at 90% repolariration (p < 0.01) and 65% increase in the effective refractory period (p < 0.01) of rabbit ventricular myocardium. Therefore, the magnitude of electrophysiologic effects induced by amiodarone are not explained by the pharmacokinetics of the drug but are probably related to the drug-induced changes in cellular metabolism. (AM HEART J 1986;112:916.)
Nagammal Venkatesh, M.D., Pitambar Somani, M.D., Ph.D., Malcolm Bersohn, M.D., Ph.D., Richard Phair, Rinya Kato, M.D., and Bramah N. Singh, M.D., Ph.D. Los Angeles, Calif,, and Toledo, Ohio From the Departments Medical Centers and Clinical Pharmacology, Supported Veterans Angeles
916
of Cardiology, the UCLA School Medical College
VA Wadsworth of Medicine. and of Ohio.
in part by grants from the Medical Administration and the American Heart Affiliate.
Research Association
and Sepulveda the Division of Received Service of the Greater Los
for publication
April
Reprint requests: Nagammal lllE, VA Wadsworth Hospital, CA 90073.
1, 1986; Venkatesh, Wilshire
accepted
May
5. 1986.
M.D., Cardiology and Sawtelle Blvds.,
Section 6911 Los Angeles.
With modern methods such as gas-liquid chromatography, high-pressure liquid chromatography (HPLC), and radioimmunoassay, plasma levels of drugs can be measured easily and specifically. However, this information is only useful where there is a reasonably linear relationship between the plasma levels and the various pharmacologic effects of a drug. Drugs such as amiodarone, which exhibit multicompartmental kinetics, cannot be monitored by single serum level determinations. Data from single-dose studies show that amiodarone has a very large volume of distribution and a relatively low total clearance rate.’ The drug is cleared more rapidly from the well-perfused tissues of the central compartment in which its receptor is likely to be located but is eliminated much more slowly from a poorly perfused tissue compartment.2 This might partially explain the long elimination half-life of amiodarone.2 It is also known that there is a poor correlation between plasma levels and the pharmacologic effects of the drug.” It has been shown that a finite period of drug treatment is necessary before the manifestation of maximum pharmacologic action of amiodarone.4.” This latency of onset of action has been attributed to: (1) time to reach maximal concentrations at the site of action, the myocardium, (2) slow accumulation of the pharmacologically active metabolite, desethylamiodarone, (3) drug-induced T, deficiency resulting in secondary changes in cardiac membrane, and (4) alterations in cellular lipid metabolism. Experiments in animals have not shown a direct relationship between serum or myocardial levels of amiodarone and its pharmacologic effects.6 The question thus arose whether the observed electrophysiologic differences between the acute and chronic effects of amiodarone might be the result of a time-dependent drug uptake by the myocardial sarcolemmal membranes. We therefore determined the levels of amiodarone and its metabolite desethylamiodarone in the ventricular sarcolemma of the rabbit and explored the relationship between the concentrations of amiodarone and desethylamiodarone in the serum, cardiac muscle, and myocardial sarcolemma and the magnitude of electrophysiologic responses following acute imtravenous and chronic oral administration of the drug. Data obtained from transmembrane action potential recordings of the ventricular myocardium were correlated with drug concentrations in the serum, myocardium, and myocardial sarcolemmal preparations. METHODS New Zealand white male rabbits, weighing 2.0 to 2.5 kg, were used for studies involving the measurement of
ventricular transmembrane action potentials and for the measurements of drug levels in the plasma. ventricular myocardium, and the cardiac sarcolemmal preparation. Since the entire right and the left ventricular myocardium was used for the sarcolemmal preparation, a different set of animals was used for electrophysiologic experiments. Measurements of ventricular transmembrane action potentials. Four groups of rabbits were studied. Two
groups (l[n = 51 and 2[n = 91) were used ah control subjects for the acute and chronic experiments. Groups lA(n = 5) and ZA(n = 8) were used for acute and chronic experiments following amiodarone treatment. Rabbits used in the acute experiments were giv~tn 20 mg/kg amiodarone (5”; aqueoussolution) or an equivalent VOIume of deionized distilled water intravenously. Rabbits used in the chronic experiments were treated with 20 mg/kg/day amiodaroneor an equivalent volume of deionized distilled water orally for 4 weeks. Fifteen minutes following acute intravenous administration and 24 hours following the last oral dosein the chronic experiments, the rabbits were given an overdoseof intravenous pentobarbital sodium,and the hearts were quickly excisedand placed in cool oxygenated (95°C O2 5’( CO,) Tyrode’s solut,ion. The right ventricular outflow tract, measuring approximately 1 cm X 0.5 cm, was carefully dissected and was pinned, with the endocardium facing up, 10 i be paraffin base of a 10 ml Lucite chamber. Preparations were stimulated by meansof Teflon-coated biptllar silver-wire electrodes connected via a stimulus isolation unit to a Grass S-88 simulator. Impulses of ‘Lmsecduration and twice diastolic threshold current were used Ventricles were stimulated at 1.0 Hz frequency. The pH of Tyrade’s solution was7.4 i 0.02 and the composition (mM) was as follows: NaCl 130: KC1 4.0; CaCI. 1.~ MgSO, 0.5; NaH,PO, 1.8; glucose5.5; and NaHCO, I &!I. The flow rate was 10 to 15 ml/min. The temperat,ure wab maintained at 35.5 ~tr0.5” C for the acute experiments attd at 37 + 0.5” C for the chronic experiments. Transmembrane action potentials were recorded by meansof 3M K(:‘l-filled glass capillary microelectrodeshaving tip resistancesof 10to 30 MO. The microelectrodes were coupled tr+ a Ag-AgCl junction and an intracellular probe system to an amplifier with a high-input impedance and variable input capacity neutralization (Mentor N-950). An electrrjde differentiating circuit (time constant: 2Oyset) was usedto determine the maximum rate of rise of phase zero of the action potential (V,,J. The system was calibrated by meansof a 10 mV internal square-wavesignal. The a&m potentials and V,,, were displayed on a storage oscilloscope(Tektronix R564B) and photographed on poiaroid film. The action potentials were recorded from ii site near the stimulating electrode. Effective refract.ory periods were determined by introducing, after every 8 (o IO basicbeats, extra stimuli with progressively shorter coupkingintervals. The effective refractory period wasdefined asthe shortest premature interval producing a responsewhich depolar-ized to values more positive than 0 ml’. En four acute preparations ventricular myocardial samples were obtained prior to and following superfuslon to determine if there were differences in the mvocerdia! drug concentra-
918
Venkatesh
et al.
American
November 1986 Heart Journal
1. Electrophysiologic effects of acute and chronic treatment with amiodaronein isolated rabbit ventricles: Comparisonwith a control group* Table
A i’A fmV)
Treatment
RMP CmVI
r;;,,,” (v/see)
APD,, (msec)
ERP (mwc)
AP& (msec)
(Group 1) 1 ml DD H,O intravenouslylfj min (n = 5) (Group 1A) Amiodarone20 mg/kg intravenouslyfor 15 min (n = 5) (Group2) Control 1 ml DD H,O/day orally for 4 weeks (n =9) (Group2A) Amiodarone20 mg/kg/day orally for 4 weeks (n = 8)
Control
90 i: 90
164
+ 52
851
120& 4.0
89+
4
153
k 69
80
1002
4
78k
4
150
i
16
7
78k
5
155
r
56
123
IL 12
99+
Ahbrreviations: APA = action potential amplitude; RMP = resting membrane repolarization times; ERP API>,,, = action potential durations at 50rC and 90’; *There were no significant changes in any of the parameters measured following treatment, there were significant increases in action potential durations at 50’,
8
115
+
8.0
+ 24
121
zt 27
66
+
18
94 I
103
I
26t
145
17
-t
36t
119
t
7.1
l??<. + 27
100
i
17
165
-t 31t
rate of rise of phase 0: Al’DS,, and potential; vnax = maximum = effective refractory period: DD H,O = deionized distilled water. 15 minutes of treatment with amiodarone. However, following 4 weeks 01 and 90’, repolarization times and effective refractory period.
tp < 0.01.
tions resulting from washout of amiodarone from the tissue during superfusion. Preparation of myocardial sarcolemma. Two groupsof animalstreated in an identical manner to those in groups IA and 2A wereused.Following an overdoseof intravenous sodium pentobarbital, the hearts were quickly removed through a thoracotomy incision and washed in ice-cold Tris-maleate buffer. Following dissection and removal of the atria, great vessels, fat, and connective tissue, the ventricles were blotted dry and weighed;the myocardium was minced in ice-cold Tris-maleate buffer and homogenized in a Polytron homogenizer(Brinkmann Instruments, Inc., Westbury, N.Y.). Then KC1 and pyrophosphate (final concentrations 300 mM and 25 mM, respectively) were added and the homogenate was rehomogenized. The homogenatewascentrifuged at 4’ C and 167,000 X g for 40 minutes. The pellet was then treated with DNase (2000 units/gm heart weight). This mixture was incubated in a shakerbath at 40” C for 45 minutes. Following a short burst of homogenization, the homogenatewaslayered over 27% sucrosesolution and centrifuged at 4’ C and 122,000X g for 20 minutes. The supernate was removed and, following Teflon homogenization, centrifugation was carried out at 4OC and 167,000X g for 45 minutes. Then the pellet was suspendedin 2 ml of the Tris-maleate buffer. In separate preparations not used for drug level determinations, the sarcolemmawaspurified eight- and threefold compared to crude homogenate, as determined by the sarcolemmal marker K+-dependent p-nitrophenylphosphatase. Serum levels of amiodarone
and desethylamiodarone.
To 500~1aliquots of serum, 100 ~1of the internal standard L8040 hydrochloride (0.01pg/ml in methanol) and 25~~1 of 1.5M potassium dihydrogen phosphate (pH 4.5) were added and then mixed thoroughly. Ten microliters of
isoamyl alcohol and 5 ml of n-pentane were added to the preceding mixture and, after thorough mixing, centrifugation at 1500X g for 5 minutes was carried out. The pentane layer was saved, and 100 11 of 5M sodium hydroxide was added and then vortexed for 90 seconds. The mixture wasthen centrifuged at 600 X g for 1 minute; the aqueouslayer was frozen in dry ice-methanol, and the organic layer was carefully decanted into a 10 ml Lauertipped glass syringe fitted with a Millipore filtration system loaded with a 0.2 pm pore-size nylon filter. The filtrate was collected and evaporated to dryness at 85’ C. Methanol-perchloric acid (100 ~1of the mobile phase)was added to the tube and vortexed. An aliquot (50 ~1) was injected into the HPLC and peak heights of amiodarone, desethylamiodarone,and the internal standard were measured. At a flow rate of 1.5 ml/min, the retention times for desethylamiodarone, L8040, and amiodarone were 6.3, 7.86, and 10.06minutes, respectively. Standard curves in plasma and tissue samples were prepared and the unknown concentrations were calculated from the peakheight ratios. Determination of levels of amiodarone and desethylamiodarone in cardiac muscle and sarcolemma. A small
piece of ventricular myocardium, 0.5 cm x 0.5 cm, was removed, washed, and blotted dry. A 0.3 gm/ml tissue homogenate was prepared with a Polytron homogenizer and L8040 was added to the homogenate as internal standard. Standard calibration curves for the myocardial tissue and sarcolemmawere prepared by adding 2.5 to 40 pg/gm of amiodaroneand desethylamiodaroneand L8040 (0.85 pg/gm) to the cardiac muscle and sarcolemma obtained from a control animal. Extraction wascarried out on 2 ml of homogenateand 500 ~1of sarcolemmalsuspension buffered with 70~1of phosphatebuffer; the procedure
Volume
112
Number
5
of nnrmdaronc
Electropharr;,acology
CONTROL
AM (20mg/kg,
I.V. 15min)
AM (20mg/kg/day,
919
p.o. 4wks)
L-----._I 100
msec
Fig. 1. Action potentials from ventricles of control and amiodarone-treated rabbits (acute and chronic) are shown. The lengthening of action potential duration following 15 minutes of intravenous treatment was insignificant. A significant prolongation of action potential duration was observed with 4 weeks of treatment.
wassimilar to that of plasmaexcept no isoamyl alcoholwas added. The resultswere expressedasmicrogramsper gram wet weight of cardiac muscle and milligrams of drug per gram of protein concentration in the sarcolemmalpreparation. The method of Lowry wasusedfor determination of protein in the sarcolemmalpreparation. Statistical analysis. Data were expressedas means-t SD. Linear correlations were determined by correlation coefficient using the Biomedical Data Package (BMDP) programs on the PDP-11 computer system. The significance of differences observed in the electrophysiologic data in the drug levels in the plasma,cardiac muscle,and myocardial sarcolemma were determined by unpaired Student’s t test. RESULTS Effects of acute and chronic amiodarone treatments on rabbit ventricular action potentials. Following acute
intravenous administration of 20 mg/kg of amiodarone, there was a 5.2% (NS) increase in the action
potential duration at 90% repolarization time and a 12.6% increase (NS) in the effective refractory period (Table I and Fig. 1). There were no significant changes in the 6,,, and resting membrane potential. However, following 4 weeks of oral administration of 20 mg/kg/day of amiodarone, there was a 65% increase (p < 0.01) in the effective refractory period and a 54.3% increase (p < 0.01) in the action potential duration at 90% repolarization. Once again, no changes were observed in other electrophysiologic parameters such as the $“,,= and resting membrane potential. Concentrations of amiodarone and desethylamiodarone in rabbit serum, cardiac muscle, and myocardial sarcolemmai membranes. The concentrations of the
drugs in the serum ,cardiac muscle, and myocardial sarcolemma following acute and chronic treatment with amiodarone are presented in Fig. 2. In the blood samples obtained at 15 minutes following intravenous administration of amiodarone, the
serum levels were 4.72 -t 1.23 pg/ml of amiodarone and 0.14 it- 0.08 pg/ml of desethylamiodarone. The corresponding concentrations of amiodarone and desethylamiodarone in the cardiac muscle were 34.5 +- 7.6 pg/gm and 2.13 2 1.37 ,ug/gm, respectively. In the left ventricular sarcolemmal preparation the concentration of amiodarone was 1.94 +- 0.445 mg/gm of protein and that of desethylamiodarone was 0.034 it 0.011 mg/gm of protein. Following 4 weeks of oral administration of the same dose of amiodarone, the serum levels were 0.05 + 0.01 pg/ml of amiodarone and 0.25 -t 0.08 &ml of desethylamiodarone. Myocardial concentrations were 1.91 + 0.9 pg/gm of amiodarone and 1.35 + 1.33 &gm of desethylamiodarone. Measurements in the sarcolemma1 preparations showed 0.043 + 0,023 mg of amiodarone /gm of protein and 0.097 f 0.047 mg of desethylamiodarone /gm of protein. DISCUSSION
Our study confirms the previous findings that at a constant dose of amiodarone, the electrophysiologic effects of amiodarone, dominated by increases in the action potential duration and refractoriness in the rabbit ventricular myocardium, develop as a function of time.i Recently, Morady et al.B have shown the lack of correlation between serum levels of the drug and changes in ventricular refractoriness, conduction, and ventricular tachycardia induction. In experimental animals the latency in the onset of pharmacologic action could not be explained by the drug concentrations in the plasma or the myocardium.6 In the present study, measurements of drug levels in the ventricular myocardial sarcolemmal preparation demonstrated a similar pattern in that, following acute administration of amiodarone, the levels were significantly higher than those following long-term administration. However, drug levels in the intracellular components were not measured.
November
920
Venkatesh
et al.
American
0
-
L,
AMIODARONE
1
AC Ch
AC Ch
m
Heart
1986 Journal
DESETHYLAMlODARO,,E
0AC Ch AC Ch
2. Concentrations of amiodaroneand desethylamiodaronein the serum, myocardium, and ventricular sarcolemmal preparation are shown. Note the significant reduction in the concentrations of amiodarone following chronic (Ch) treatment in all of the tissuesanalyzed, and the levels of desethylamiodarone in the serum and sarcolemmafollowing chronic treatment; p < 0.0001.AC = acute. Fig.
The fact that following acute administration the levels of amiodarone were high in the plasma, myocardium, and myocardial membrane when the electrophysiologic effects were minimal or absent and the converse occurred following chronic oral administration of the drug makes it difficult to explain the chronic efficacy of amiodarone based on its pharmacokinetics. We and other investigators have shown that the acute effects may, in part, result from the nonspecific adrenolytic properties of the drug,6pg-11whereas the long-term action is related to the prolongation of action potential duration and refractoriness in addition to the enhancement of adrenergic inhibition.7 Gloor et a1.12 have presented data to indicate that the acute effects of amiodarone on the sinoatrial and atrioventricular nodes in dogs might also be the result of inhibition of the slow calcium channel. Further work is clearly necessary to elucidate the mechanisms involved in mediating the acute electrophysiologic changes induced by amiodarone. In contrast, the electrophysiologic effects of long-term amiodarone therapy are reasonably uniform and consistent.4s5 Myocardial uptake of amiodarone. Connolly et al.13 showed that amiodarone concentrations in the plasma and the myocardium were different during the first 30 minutes following intravenous administration of the drug. While the plasma level fell significantly, the myocardial concentration of amiodarone increased during this period. Maximal myocardial concentrations were observed between 20 and 30
minutes. They also showed that myocardial concentrations of amiodarone more closely reflected the electrophysiologic effects of the drug in the dog than do plasma concentrations. However, the electrophysiologic parameters that were measured were sinus cycle length, atrioventricular node, Wenkebath cycle length, and functional refractory period, all of which are under adrenergic influence and therefore may be attributed to the drug’s ability to exert a direct nonspecific adrenergic antagonism. Latini et alI4 have shown that myocardial uptake of amiodarone was rapid and maximum levels were found at 15 to 30 minutes following acute intravenous administration. This was also shown by Somani et a1.15 who, in addition, found a close correlation between the time course of myocardial uptake and uptake in white blood cells. It was suggested that measurement of drug concentration in white blood cells might predict myocardial tissue concentration. Somani’s study also showed a significant decrease in premature ventricular contractions and rate of ventricular tachycardia following experimental myocardial infarction in dogs at 6 hours following intravenous administration of amiodarone. Again, this may have been secondary to the beta-adrenergic-blocking action of amiodarone since, in studies of sudden death in a canine model, Patterson et a1.16showed that the chronically administered drug was more effective in preventing inducible ventricular fibrillation than the acutely administered drug, a difference that could not be explained on the basis of myocardial drug levels.
Volume Number
112 5
Electropharmacology
Significance of sarcolemmal amiodarone tions. Following chronic administration
concentra-
of the drug we found the sarcolemmal concentration of amiodarone to be 40 times less than that following acute administration, a phenomenon identical to that seen in the cardiac muscle. It is known that most drugs exhibit a reasonably simple and linear concentraconcentration is tion-response curve. l7 A minimum usually required for a threshold response, with a stepwise increase in effect as the drug concentration is elevated. Our data show that this does not hold for complex pharmacologic molecules such as amiodarone, which is thought to act by influencing various metabolic parameters of the cell as a function of time. Long-term amiodarone treatment has been shown to inhibit the peripheral conversion of T, I8 to T.3, resulting in a decrease in T3, an increase in rT,, and a minimal increase in T, in the plasma.lg This alteration in the thyroid hormone metabolism, as well as a direct inhibition of T, nuclear binding by amiodarone and/or its metabolite desethylamiodarone,*“. *I is thought to result in a hypothyroid state at a cellular level. l8 Since the electrophysiologic effects of hypothyroidism22-25 are almost identical to those observed following long-term amoidarone treatment, this phenomenon is thought to exhibit some cardiospecificity.7. ~326 Gross and Somani2? have shown that amiodarone alters the lipid metabolism of the cardiac cell, as evidenced by the development of lysosomal and myelinoid inclusion bodies. Whether the membrane lipids are altered by the presence of the drug and its metabolite is, however, not known at present. Neither is it clear whether such changes might result from prolonged hypothyroidism. Pharmacologic
significance
921
been found that the therapeutic efficacy of amiodarone may be better monitored by rT,, levels’” in the plasma, and the incidence of toxicity correlates more closely with the plasma levels of desethylamiodaTone4 than with those of amiodarone. In summary, our study confirms the previous findings that measurement of drug levels may not constitute a reliable index for the evaluation of therapeutic efficacy of amiodarone, but our data do not exclude it as a basis of drug toxicity during protracted treatment. We have shown that. there is a poor correlation between the myocardial drug levels as well as the membrane levels and the magnitude of pharmacologic action of the drug. Therefore, mechanisms other than tissue drug concentrations are likely to be responsible for the magnitude and temporally related enhancement of the electrophysiologic and antiarrhythmic actions of arniodarone. We are indebted to Lawrence preparation of this manuscript.
Kimble
i’or ~~‘cu’x~,I!
help in t hc
REFERENCES
1. Heger
JJ, Prystowsky EN, Miles W-M, Zipes L)t’. Clinlcai use pharmacology of amiodarone. Meci ( ‘I~II North Am 1984;68:1339-67. Halt DW, Tucker GT, Jackson PR, St,orey (;;.:A .4miodarone pharmacokinetics. AM HEART d 1983;106:840-846. Mostow ND, Rakita L. Vrobel TR, Noon DI,. Blumer ,I. Amiodarone: Correlation of serum concemration with suppression of complex ventricular ectropic as?ivitv. Am J Cardiol 1984;54:569-74. Heger JJ, Prystowsky EN, *Jackman WM. er al. Amiodarone. Clinical efficacy and electrophysiology durin:: lijng-term therapy for recurrent ventricular tachycardia or \-ell.tricular tihrillation. N Engl J Med 1981;305:539-545. Nademanee K. Singh BN, Hendrickson JA, t-7 al. Amiodartrnt in refractory life-threatening ventricular tirrhvthmias. Ann Intern Med 1983;98:577-84. Venkatesh N. Padbury JF. Singh BN. Effec,i.s ($I amiodarimr. and desethylamiodarone on rabbit myocarrhai beta-adreno ceptors and serum thyroid hormones. Absence (II relationship to serum and myocardial drug concrntrar i# in+. .I (‘ardiovasl, Pharmacol. (In press.) Singh BN. Amiodarone: Historical developmen; and pharmacologic profile. AM HEART J 1983;106:788-9;. Morady F, Dicarlo LA, Krol RB, Berman ,JXl, Huitleir M. Acute and chronic effects of amiodaronc, !UI vent,ricular refractoriness. intraventricular ronducticm and ventricular tachycardia induction. J Am Co11 Cardiol lifH6;7:148-57. Polster P, Broekhuysen .J. The adrenerpcc antagonism of amiodarone. Biochem Pharmacol 1976::!S: I :l 1%. Nokin P, Clinet M, Shoenfeld P. Cardial f<-adrem,ceptol modulation b> amiodarone. Biocbc~n, F’harmaccsl 1983;32:2473-‘457. Gagnol ,JP, Devos (.‘. (‘linrt M, NcBk!n 1’ Ami~lclaronr: 13iochemical aspects and hemodvnami 2 tfects. I)ruph 1985;29(suppl 3):1-l& (iloor HO. LTrthaler F. James ‘I’ti. Acute i,tftec‘ts uf amioda rone upon the canine sinus node and arrilrve~ltric:ular jum tional region. J Clin Invest 1983;71:1457-I 17’:. Connolly SJ, Latini R, Kates RE. Ph:lrm;ir:odynamIcs ~)i intravenous amiodarone in the dog. -I (‘arriiovalsc I’harmacg ,i 1984;6:531-535. Latini R. Connnllv S,J, Katcs RE. Mv{bcar.:i;ti dispcrsltion 111 and
2. 3.
4.
5.
6.
of desethylamiodarone.
The metabolism of amiodarone results in the formation of its prinicipal metabolite desethylamiodarone, which we have shown to be pharmacologically active.“~“” In our study, following chronic treatment with amiodarone, the desethylamiodarone levels were higher than those of amiodarone in the myocardial sarcolemmal membrane and equivalent in the cardiac muscle and serum. Thus, the question arises whether the manifestation of the pharmacologic actions of amiodarone is dependent upon the attainment of a critical level of the metabolite in the myocardial membrane. However, we have shown that a latency in the onset of action, almost similar to that observed with amiodarone treatment, is also seen with desethylamiodarone.6 We have also shown that, with regard to adrenergic antagonism and electrophysiologic changes, the metabolite was at least as potent as the parent compound.6’2g It has
of nmrorlurlmc
7. 8.
9. 10.
11.
12.
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14.
November
922
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17. 18.
19. 20.
21.
22.
Venkatesh
et al.
amiodarone in the dog. J Pharmacol Exp Ther 1983;224:603608. Somani P, Simon VA, McManus K. Pharmacokinetics antiarrhythmic effects and tissue concentrations of amiodarone and desethylamiodarone in dogs with acute coronary artery occlusion. Can J Cardiol 1985;1:56-65. Patterson E, Eller BT, Abrams GD, Vasilades J, Lucchesi BR. Ventricular fibillation in conscious canine preparation of sudden coronary death. Prevention by short and long-term amiodarone administration. Circulation 1983;68:85-92. Evans WE, Schentag JJ, Jusko EJ, eds. Applied pharmacokinetics: Principles of therapeutic drug monitoring. San Francisco: Applied Therapeutics, Inc, 1980. Sogol PB, Hershman JM, Reed AW. The effects of amiodarone a serum thyroid hormones and hepatic thyroxine 5’ monodeiodination in rats. Endocrinology 1983;113:14641469. Singh BN, Nademanee K. Amiodarone and thyroid function: Clinical implications during antiarrhythmic therapy. AM HEART J 1983;106:857-869. Wiersinga WM, Broenik MM. In vitro inhibition of nuclear thyroid hormone binding by amiodarone and desethylamiodarone in rat liver and cardiac muscle [Abstract]. In: Fiftyninth American Thyroid Association, New Orleans, Ott 5-8, 1983. Latham KR, Sellittie DF, Goldstein RE. Interaction of amiodarone and desethylamiodarone with nuclear thyroid hormone receptors [Abstract]. J Am Co11 Cardiol 1985;5:466. Freedberg AS, Papp GJ, Vaughan Williams EM: The effects of altered thyroid state on atria1 intracellular potentials. J Physiol 1970;207:357-69.
American
Heart
1986 Journal
23. Johnson PN, Freedberg AS, Marshall JM. Action of thyroid hormone on the transmembrane potentials from sino atria1 nodal cells and atria1 muscle cells in isolated atria of rabbits. Cardiology 1973;58:273-289. 24. Gavrilescu S, Luca C, Streian C, Lungu G, Deutsch G. Monophasic action potential of right atrium and electrophysiologic properties of AV conducting system in patients with hypothyroidism. Br Heart J 1976;38:1350-1354. 25. Sharp NA, Neel DS, Parsons RL. Influence of thyroid hormone levels on the electrical and mechanical properties of rabit uapillarv muscle. J Mol Cell Cardiol 1985:17:119-132. 26. Singh’BN, Vaughan Williams EM. The effect ofamiodarone, a new antianginal drug, on cardiac muscle. Br J Pharmacol 1970;39:657-667. 27. Gross SA, Somani P. Amiodarone-induced ultrastructural changes in the canine myocardial fibers. AM HEART J (in press 1986). 28. Venkatesh N, Al-Sarraf L, Singh BN. Digoxin-desethylamiodarone interaction in rats. Comparison with that of amiodarone. J Cardiovasc Pharmacol. (In press.) 29. Kato R, Venkatesh N, Yabek S, Takikawa R, Kannan R, Singh BN. Electrophysiologic effects of desethylamiodarone, an active metabolite of amiodarone: Comparison with amiodarone during chronic administration in rabbits. (Submitted to J Am Co11 Cardioll 30. Venkatesh N, Al-sarraf L, Hershman JM, Singh BN. Effects of desethylamiodarone on thyroid hormone metabolism in rats: Comparison with the effects of amiodarone. Proc SOC Exp Biol Med 1986;181:233-236.