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Electrophysiologic in-vitro effects of cocaine and its metabolites
International Journal of Cardiology 46 (1994) 235-242
Electrophysiologic in-vitro effects of cocaine and its metabolites Jun Wang’,
Robert
G. Carpentier*
Department of Physiology and Biophysics, College of Medicine. Howard University, 520 W Street N. W., Washington, DC 2OOS9, USA
Received 27 December 1993; accepted 14 June 1994
Abstract We studied the in-vitro electrophysiologic effects of equimolar concentrations of cocaine and its metabolites on rat cardiac tissues. The effects on the sinus node rate were studied in spontaneously active sinoatrial preparations. The order of magnitude of the effects was: ethylcocaine > cocaine > benzoylecgonine and ecgonine methyl ester > ecgonine. The effects of cocaine and ethylcocaine were not additive. The actions of cocaine and ethylcocaine on membrane potentials were studied in papillary muscles driven at 5 Hz. Both compounds depressed to similar degrees the resting potential and the amplitude of the action potential, and increased the duration of the action potential. Simultaneous exposure to the two drugs did not result in effects greater than those of ethylcocaine or cocaine alone. It is concluded that (a) cocaine and its metabolites depressed the sinus node rate. Only cocaine and ethylcocaine exerted actions that may be of clinical significance. (b) Ethylcocaine had an effect greater than that of cocaine on the sinus node rate, and similar to that of the parent compound on ventricular membrane potentials. Thus, ethylcocaine may play a significant role in the cardiac electrophysiologic actions of cocaine, when the latter is used in combination with ethanol. (c)
The effects of cocaine and ethylcocaine were not additive. Keywords: Cocaine; Ethylcocaine; Cocaethylene;
Sinoatrial node; Myocardium;
1. Introduction The acute toxic effects of cocaine on the cardiovascular system are well known and have been associated with sudden cardiac death [l]. Almost one third of the deaths related to cocaine overdose ’ Visiting Scholar from the Xuzhou Medical College, Xuzhou, Jiangsu, People’s Republic of China.
* Corresponding author.
Membrane
potentials
in humans occur 2-5 h after the exposure to the drug [2]. Cocaine is rapidly metabolized, which makes it difficult to rule out a causative role by one or more of its metabolites in the cardiac effects of cocaine intoxication [3]. Even though cocaine metabolites may accumulate shortly after cocaine administration, there is little information on the effects of these metabolites on cardiac electrical activity. Crumb and Clarkson reported that norcoCaine, one of the metabolites, is also a potent
Ol67-5273/94/$07.00 0 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0167-5273(94)02123-Z
236
J. Wang. R.G. Carpmtier.
/ht.
sodium channel blocker in single cardiac myocytes, while benzoylecgonine and ecgonine are ineffective blockers at clinically relevant concentrations 141. Concurrent ingestion of ethanol is common in cocaine abusers [5] and a recent epidemiological study reported that it results in a signi~cant increase in the risk of sudden death related to cocaine use [6,7]. Ethylcocaine (Cocaethylene, benzoylecgonine ethyl ester), an ethylated metabolite of cocaine, is formed in individuals after combined administration of ethanol and cocaine, but not in individuals given cocaine or ethanol alone [8-IO]. The plasma concentrations of ethylcocaine may be as great and even greater than those of cocaine [7,11,12]. It has been reported that ethylcocaine may be of greater potency than cocaine in producing lethality [8] and in its negative inotropic action [13], but there is no information on the cardiac electrophysiologic actions of this metabolite. The purpose of the present study was to study the effects of cocaine and its metabolites, including ethylcocaine, on the electrical activity of the heart. 2. Materials and methods 2.1. Animals and equipment Adult (300-400 g) male Sprague-Dawley rats from Charles River Breeding Labs Co. were used in this study. The animals were euthanized under barbiturate anesthesia (sodium pentobarbital, Sigma Chemical Co., 60 mg/kg i.p.). The heart was quickly excised through open chest and immersed in oxygenated and buffered Tyrode’s solution (see below for composition) kept at 31°C. Preparations beating spontaneously (sinoatrial strips) and preparations to be stimulated electrically at a constant rate (papillary muscles) were isolated and mounted horizontally in a Lucite tissue bath holding 2 ml of physiological solution flowing at 5 mlimin at a constant temperature (37’C). The preparations were superfused with oxygenated and buffered (95% O2 - 5% COZ; pH 7.4). Tyrode’s solution, contained the following (mM): NaCl 137, KC1 5.4, CaCl, 2.7, MgCl* 0.5. NaHCOs 11.9, NaH2P04 0.45 and glucose 5.55. All preparations were equilibrated with the Tyrode’s solution for 2 60
J Curdiol. 46 (IY94)
-735-242
min before any experimental procedure was carried out. 2.2. Studies with s~noafria~preparations spontaneously active A small strip of the right atrium containing the sinus node and a small portion of the crista terminalis was mounted, endocardial side up, in the tissue bath. Membrane potentials were recorded with standard intracellular glass microelectrodes filled with 3 M KC1 (tip resistance 20-30 MQ), using multiple impalements in different areas of the preparation. It was possible to impale sinus node subsidiary pacemaker fibers located in the periphery of the sinus node as well as atria1 contractile fibers located in the crista terminalis. However, it was difficult to impale true pacemaker fibers, which are located mainly intramurally in the rat preparation. The procedure allowed us to establish the control sinus node rate and to determine if there were irregularities or areas of conduction block. Only preparations with a regular and steady control sinus node rate and without areas of block were used in this study. After the control sinus node rate was recorded, the superfusate was changed to Tyrode’s containing one or more drugs (see below). The sinus node rate was recorded again during and after (recovery) a 20-min exposure to the drug(s). 2.3. Studies with papillary muscles driven at a constant rate
A left ventricle papillary muscle was mounted in the tissue bath. The preparation was driven electrically at a constant frequency of 5 Hz, approximately 20% above the rate of the preparations beating spontaneously, by a Grass stimulator (S88) with 2 SIUS isolation units. Square pulses of voltage 50°/ above threshold of l-2 ms duration were delivered through a pair of silver electrodes in close contact with one end of the preparation. Membrane potentials were recorded from one cell; only fibers for which the impalement was maintained for the entire protocol were used for subsequent analysis. After the control membrane potentials were recorded, the superfusate was
J. Wang. R.G. Carpentier. / I~I. J. Cur&l.
system
The recording system consisted of a Winston 1090 amplifier and a Nicolet Explorer II digital oscilloscope. The tracings were stored on floppy disks. In the experiments in which spontaneously active sinoatrial preparations were used, the cycle length was read directly from the oscilloscope and was used to calculate the sinus node rate. In the experiments with papillary muscles driven at a constant rate, the parameters measured during the single impalement were: resting membrane potential, overshoot of the action potential, total action potential amplitude and action potential duration measured at -50 mV of repolarization.
2.6. Statistical
(cocaine
hydrochloride)
and its metab-
r
COCAINE
10
20
MINUTES
10
2c
MINUTES
analysis
Only one concentration of a single drug was tested in each preparation. A single preparation was dissected from each animal, so that in all series of experiments the number of preparations indicates also the number of rats used. The mean value and the standard error of the mean (S.E.M.) were calculated for each parameter measured in each sin-
2.5. Drugs Cocaine
237
235-242
olites ecgonine (ecgonine hydrochloride), ecgonine methyl ester (ecgonine methyl ester hydrochloride), benzoylecgonine (benzoylecgonine) and cocaethylene (ethylcocaine, ecgonine ethyl ester benzoate), all purchased from Sigma Chemical Co., MO, were tested. Stock solutions of the drugs were freshly prepared. All stock and final solutions were kept in the dark and protected from direct light during the superfusion. All compounds, with the only exception of ethylcocaine, are water soluble, and Tyrode’s solution was used as the vehicle to reconstitute the drugs. On the other hand, a small volume of ethanol was used initially to reconstitute ethylcocaine.
changed to Tyrode’s containing one or more drugs (see below). The membrane potentials were recorded again during and after (recovery) a 3-min exposure to the drug(s). 2.4. Recording
46 /I9941
_BENZOYLECGONINE
ECGONINE METHYL ESTER
1
ETHYLCOCAINE
I
I
I
/
I
I
10
20
10
20
10
20
( ‘1’ ITES
MINUTES
MINUTES
Fig. 1. Percent change in sinus node rate (SNR) induced by cocaine and its metabolites (58 FM). Each bar is the mean and S.E.M. of values obtained from six preparations. There was no difference (P > 0.05) between the control values (action potentialsimin) for the five compounds, as well as between the control and recovery values for each compound, which were, respectively: cocaine. 262.2 f 5.4 and 265.7 * 6.7; ecgonine, 264.3 f 7.1 and 271.2 f 10.9; benzoylecgonine, 245.5 f 6.8 and 249.8 f 8.9; ecgonine methyl ester, 256.0 f 14.9 and 260.3 f 13.4; ethylcocaine. 252 f 10.0 and 255.0 f 9.7. *Significant change, F(2,lO) = 19.9, P < 0.0005. F( 1.10) = 13.6. P < 0.005; ecgonine methyl ester, +Signiticantly smaller than change induced by cocaine: benzoylecgonine. F(l,lO) = 16.8, P < 0.003. **Not significantly different from change induced by cocaine, F(l,lO) = 0.36. P > 0.56.
238
J. Wang. R.G. Carpmtier.
/ Int. J
ECGONINE
Cm&l.
46 i 19941 235-242
BENZOYLECGONlNE
ECGONINE METHYL ESTER
ET~COC~NE
** I
I
10
20
MINUTES
I
10 MINUTES
I
/
r
20
10
20
I
10
MINUTES
MtNUTES
-10
20
MINUTES
Fig. 2. Percent change in sinus node rate (SNR) induced by cocaine and its metabolites (87 PM). Each bar is the mean and S.E.M. of values obtained from six preparations. There was no difference (P > 0.05) between the control values (action potentialsimin) for the five compounds, as well as between the control and recovery values for each compound, which were, respectively: cocaine, 259.7 f 5.7 and 256.3 f 3.3; ecgonine, 274.8 f 14.4 and 275.0 f 14.3; benzoylecgonine. 253.7 f 9.2 and 253.3 f 9.1; ecgonine methyl ester, 269.0 + 13.4 and 265.3 f 13.6; ethylcocaine, 263.4 f 7.2 and 260.5 f 5.8. *Significant change. F(2.10) = 16.5. P < 0.001. +Signiftcantly smaller than change induced by cocaine: ecgonine, F( I.lO) = II.3, P < 0.0009; benzoylecgonine. F( IJO) = 8.6. P < 0.02; ecgonine methyl ester, F( 1.10) = 10.0, P =c0.001. **Not significantly different from change induced by cocaine. F(l.lO) = 0.34, P > 0.58.
gle series of experiments. Significance of differences between pair of means was calculated by using Student’s r-test for paired or unpaired data, as appropriate. A one-way analysis of variance was used to analyze the effect of one concentration of a single drug over a period of time, which involved comparison of several means. A two-way analysis of variance was used to compare the effects observed in two different groups of experiments. The level of significance is given each time a comparison is made. 3. Results
The concentration of cocaine used initially in this study (58 PM) was selected from results obtained in a previous investigation using the same preparation [ 141.Concentrations of cocaine I 2.9 pM did not affect the sinus node rate. Cocaine L 87 PM
resulted in reduction in sinus node rate associated with development of sinoatrial block. On the other hand, cocaine 58 PM reduced the sinus node rate without producing sinoatrial block. The changes in sinus node rate produced by this concentration of cocaine and its metabolites (58 PM) are presented in Fig. 1. Ecgonine did not have any effect. The depressant effects of benzoylecgonine and ecgonine methyl ester were signi~cantly smaller than that of the parent compound. On the other hand the effect of ethylcocaine was not significantly different from that of cocaine. The data presented in the legend for the figure also show that the effects of ail compounds were completely reversed within 30 min. The effects of a higher concentration of cocaine and its metabolites (87 PM) are presented in Fig. 2. At this concentration, all compounds depressed the sinus node rate. Ecgonine, benzoylecgonine and ecgonine methyl ester exerted a si~i~~antly smaller effect than their parent compound, whereas ethylco-
J. Wang, R.G. Carpentier. /ht.
J. Cardiol. 46 (1994) 235-242
m
I
29 COCAINE + ETHYLCOCAINE
ETHYLCOCAINE
1 +
239
+
I
I
I
I
I
I
10
20
10
20
10
20
MINUTES
MINUTES
MINUTES
Fig. 3. Percent change in sinus node rate induced by cocaine and ethylcocaine (2.9 and 29 pM), alone and in combination. There was no difference (P > 0.05) between the control values for the different experimental groups, as well as between the control and recovery values for each group, which were, respectively, for the 2.9 PM concentrations: cocaine, 273.0 + 8.2 and 273.8 + 7.7; ethylcocaine, 267.3 f 7.3 and 270 & 6.2; cocaine + ethylcocaine, 271.3 f 7.3 and 272.5 f 7.0. For the 29 PM concentrations the control and recovery values were, respectively, cocaine, 273.3 f 4.9 and 273.8 * 4.3; ethylcocaine, 279.8 f 6.0 and 274.3 * 5.7; cocaine + ethylcocaine, 275.5 f 4.4 and 277.8 f 5.9. Each bar is the mean and S.E.M. of values obtained from six preparations. *Significant change: F(2.6) = 36.5, P < 0.0008. +Significant change, and significantly different from change induced by cocaine, F( 1.6) = 24.4, P < 0.001 and F( 1.6) = 13.2. P C 0.02. **Significant change, but not significantly different from change induced by ethylcocaine alone, F(l.6) = 2.5. P > 0.16 and F(1,6)=0.11, P > 0.74.
Caine had an action similar to that of cocaine. All the compounds tested acted in a fully reversible manner, as shown by the data presented in the legend for the figure. The results obtained with two lower concentrations of cocaine and ethylcocaine, administered alone and in combination, are presented in Fig. 3. Cocaine 2.9 PM did not depress the sinus node rate, whereas ethylcocaine produced a significant effect. Cocaine 29 PM depressed the sinus node rate, but again ethylcocaine had a significantly greater effect. It is also clear that the effects of the two compounds were not additive.
3.2. Studies with papillary constant rate
muscles driven at a
The effects of cocaine and ethylcocaine,
alone and
in combination, on the membrane potentials of papillary muscles driven at 5 Hz, are presented in Fig. 4. Both cocaine and ethylcocaine depressed the resting potential, the overshoot and the total amplitude of the action potential, without signilicant differences between the magnitude of their effects. The figure also shows that the effects of the two compounds were not additive. The parameters measured recovered completely after withdrawal of the drug(s) from the superfusate, as shown by the recovery values. Cocaine and ethylcocaine prolonged also the action potential duration, by 12.4 f 3.1 and 17.0 f 4.1 ms, respectively. Superfusion of the preparations with the two compounds in combination prolonged the action potential by 19.6 f 2.0 ms. There was no significant difference between the effects of cocaine and ethylcocaine, or between the effects of the two drugs in combination and either one alone.
240
J. Wang,
mV JIllSTINGMEMB~~E _ -65 c
POTENTIAL
R.G.
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J.
Curcliol.
A5 11994) 235-242
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Fig. 4. Effects of cocaine and ethylcocaine (58 pM) on membrane potentials of papillary muscles driven at 5 Hz. CON. control values: DRUG, values recorded at the end of a 3-min exposure to the respective drug; REC. recovery values. Each data point is the mean and S.E.M. of values obtained from six preparations. *Significantly different from the respective control value (P < 0.01).
4. Discussion Previous studies in our laboratory have demonstrated the in vitro cardiac electrophysiologic and inotropic effects of cocaine [14- 161. The present study compared the effects of cocaine and metabolites, including its ethanol derived metabolite cocaine ethyl ester (ethylcocaine), on the sinus node rate and membrane potentials of ventricular fibers. This is the first study comparing the cardiac electrophysiologic effects of cocaine and its metabolites. The results suggest that most metabohtes of cocaine are of little, if any, clinical significance. However, they also suggest that ethylcocaine may play an important role in the cardiac toxic effects of the parent compound when ethanol and cocaine are administered concurrently.
Cocaine depressed the sinus node rate in sinoatrial preparations beating spontaneously. It also decreased the resting potential, the overshoot and the total amplitude of the action potential, and increased the action potential duration, of papillary muscle fibers driven at a constant rate, con~rming our previous findings [14]. The mechanism of the depressant action on the automaticity of the sinus node is still not clear. There is no info~ation on possible actions of cocaine on the time-dependent currents IK and If, responsible for most of the slow diastolic depolarization in the sinus node. On the other hand, there is some evidence that cocaine depresses the slowly activated current I, [ 17, If?], which contributes to the latter portion of phase 4. The changes in the action potential amplitude are easily explained by the anesthetic action of cocaine
J. tVaq,
R.G. Curpenrier.
on the fast sodium system [19] and the fall in the resting potential [20]. The prolongation of the action potential is abolished by phentolamine and by 4-aminopyridine, suggesting that it is an cu-adrenergic mediated action resulting in the inhibition of the transient outward current I,, [14]. The results presented here indicate that the metabolites ecgonine, benzoylecgonine and ecgonine methyl ester had a signi~~antly smaller effect than cocaine on the sinus node rate. Ecgonine had the smallest action, with the other two metabolites showing similar effects, but still well below the effect of the parent compound. These results suggest that these three metabolites most probably play very little role, if any, in the sinus bradycardia observed in cocaine intoxication. Crumn and Clarkson also found that both benzoylecgonine and ecgonine methyl ester, in clinically relevant concentrations (i.e., < 100 PM), are ineffective blockers of the fast sodium channels in single cardiac myocytes [4]. The results obtained with ethylcocaine are of great interest. The ethanol derived metabolite of cocaine depressed the sinus node rate at a concentration (2.9 PM) at which cocaine had no effect. Also, 29 PM ethylcocaine had a significantly greater depressant effect than cocaine. It has been recently reported that ethylcocaine produces more potent inhibition than cocaine of the intracellular calcium transient and the myocardial contraction in single cardiac myocytes [ 131. Our results provide additional support to the hypothesis that ethylcocaine may exert greater effects than its parent compound on the heart. Although it is virtually impossible to precisely compare the superfusate levels of cocaine and ethylcocaine with those present in vivo, the concentrations used in this study were comparable to those found in blood in acute cocaine intoxication [3,7,11,12,21]. Cocaine is rapidly metabolized [3], but almost one third of the deaths related to cocaine overdose occur 2-5 h after the exposure to the drug [2]. Moreover, ethylcocaine formed after combined use of ethanol and cocaine may reach concentrations in plasma as great and even greater than those of cocaine [7,11,12]. Thus, it seems reasonable to propose that ethylcocaine may play a significant role in the cardiac effects of cocaine when the latter and ethanol are used concurrently.
/Iat.
J. Cur&f.
46 (19941 23%.?42
24!
Acknowledgement This research was supported by Grant S06GM08016 from the National Institute of General Medical Sciences.
References ill
Billman GE. Mechanisms responsible for the cardiotoxic effects of cocaine. FASEB J 1990; 4: 2469-2475. 121 Finkle BS, McCloskey KL. The forensic toxicology of cocaine. J Forensic Sci 1977; 23: 173-189. 131 Wilkinson P, Van Dyke C, Jatlow P, Barash P, Byck R. Intranasal and oral cocaine kinetics. Clin Pharmacol Ther 1980; 27: 386-394. of the sodi141 Crumb WJ, Clarkson CW. Characterization um channel blocking properties of the major metabolites of cocaine in single cardiac myocytes. J Pharmacol Exp Ther 1992; 261: 910-917. ML, Michael JL. 151 Weiss RD, Mirin SM, Griffin Psychopathology in cocaine abusers: changing trends. J Nerv Ment Dis 1988; 176: 719-725. 161 Rose S, Hearn WL, Hime GW, Wetli CV, Ruttenber AJ, Mash D. Cocaine and ~ocaethyIene concentrations in human post mortem cerebral cortex. Neurosci Abstr 1990; 16: 11.6. I71 Hearn WJ, Rose S, Wagner J, Ciarleglio A. Mash DC. Cocaethylene is more potent than cocaine in mediating lethality. Pharmacol Biochem Behav 1991; 39: 531-533. behavioral 181 Katz JL, Terry P, Witkin JM. Comparative pharmacology and toxicology of cocaine and its ethanol derived metabolite, cocaine ethyl-ester (cocaethylene). Life Sci 1992; 50: 1351-1361. 191 Dean RA. Harper ET, Dumaual N. Stoeckel DA, Bosron WF. Effects of ethanol on cocaine metabolism: formation of cocaethylene and norcocaethylene. Toxic01 Appl Pharmacol 1992; 117: l-8. EF, Price LH, McDougle CJ, Kosten TR, I101 McCance-Katz Black JE. Jatlow PI. Concurrent cocaine-ethanol ingestion in humans: pharmacology, physiology, behavior, and
1111
1121
1131
1141
the role of cocaethylene. Psychopharmacology 1993; I 1 I: 39-46. De La Torre R, Farre M, Ortuno J, Cami J. Segura J. The relevance of urinary cocaethylene following the simultaneous administration of alcohol and cocaine. J Anal Toxicol 1991; 15: 223-228. Jatlow P, Elsworth JD, Bradberry CW et al. Cocaethylene: a neuropharmacologically active metabolite associated with concurrent cocaine-ethanol ingestion. Life Sci 1991: 48: 1787-1794. Qiu 2, Morgan JP. Differential effects of cocaine and cocaethylene on intracellular CaZ+ and myocardial contraction in cardiac myocytes. Br J Pharmacol 1993: 109: 293-298. Sherief Z-IT, Carpentier RG. Electrophysiological mechanisms of cocaine-induced cardiac arrest: a possible
242
J. Wang, R.G. Carpenrier.
/ht.
cause of sudden cardiac death. J Electrocardiol 1991: 24: 247-255. [ 151 Carpentier RG, Coleman BR, Pate1 DJ. Cocaine enhances postrest and paired-stimulation potentiation in rat papillary muscle. J Pharmacol Exp Ther 1993; 266: 41-46. [ 161 Carpentier RG, Coleman BR, Pate1 DJ. Mechanism of the negative inotropic action of cocaine in rat papillary muscle. Toxicol Appl Pharmacoi 1993; 123: 293-298. [17] Josephson I, Sperelakis N. Local anesthetic blockade of Ca*+ mediated action potentials in cardiac muscle. Eur J Pharmacol 1976; 40: 201-208. [IS] Tracy CM, Bachenheimer L, Solomon A et al. Evidence
J. Cur&l.
46 (1994)
235-242
that cocaine slows cardiac conduction by an action on both AV nodal and His-Purkinje tissue in the dog. J Electrocardiol 1991; 24: 257-262. [I91 Crumb WJ, Clarkson CW. Characterization of cocaineinduced block of cardiac sodium channels. Byophys J 1990; 57: 589-599. [20] Weidmann S. The effect of the cardiac membrane potential on the rapid availability of the sodium carrying system. J Physiol 1955; 127: 213-224. [21] Virmani R, Robinowitz M, Smialek JE, Smyth DF. Cardiovascular effects of cocaine: an autopsy study of 40 patients. Am Heart J 1988; 115: 1068-1076.
Electrophysiologic in-vitro effects of cocaine and its metabolites Jun Wang’,
Robert
G. Carpentier*
Department of Physiology and Biophysics, College of Medicine. Howard University, 520 W Street N. W., Washington, DC 2OOS9, USA
Received 27 December 1993; accepted 14 June 1994
Abstract We studied the in-vitro electrophysiologic effects of equimolar concentrations of cocaine and its metabolites on rat cardiac tissues. The effects on the sinus node rate were studied in spontaneously active sinoatrial preparations. The order of magnitude of the effects was: ethylcocaine > cocaine > benzoylecgonine and ecgonine methyl ester > ecgonine. The effects of cocaine and ethylcocaine were not additive. The actions of cocaine and ethylcocaine on membrane potentials were studied in papillary muscles driven at 5 Hz. Both compounds depressed to similar degrees the resting potential and the amplitude of the action potential, and increased the duration of the action potential. Simultaneous exposure to the two drugs did not result in effects greater than those of ethylcocaine or cocaine alone. It is concluded that (a) cocaine and its metabolites depressed the sinus node rate. Only cocaine and ethylcocaine exerted actions that may be of clinical significance. (b) Ethylcocaine had an effect greater than that of cocaine on the sinus node rate, and similar to that of the parent compound on ventricular membrane potentials. Thus, ethylcocaine may play a significant role in the cardiac electrophysiologic actions of cocaine, when the latter is used in combination with ethanol. (c)
The effects of cocaine and ethylcocaine were not additive. Keywords: Cocaine; Ethylcocaine; Cocaethylene;
Sinoatrial node; Myocardium;
1. Introduction The acute toxic effects of cocaine on the cardiovascular system are well known and have been associated with sudden cardiac death [l]. Almost one third of the deaths related to cocaine overdose ’ Visiting Scholar from the Xuzhou Medical College, Xuzhou, Jiangsu, People’s Republic of China.
* Corresponding author.
Membrane
potentials
in humans occur 2-5 h after the exposure to the drug [2]. Cocaine is rapidly metabolized, which makes it difficult to rule out a causative role by one or more of its metabolites in the cardiac effects of cocaine intoxication [3]. Even though cocaine metabolites may accumulate shortly after cocaine administration, there is little information on the effects of these metabolites on cardiac electrical activity. Crumb and Clarkson reported that norcoCaine, one of the metabolites, is also a potent
Ol67-5273/94/$07.00 0 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0167-5273(94)02123-Z
236
J. Wang. R.G. Carpmtier.
/ht.
sodium channel blocker in single cardiac myocytes, while benzoylecgonine and ecgonine are ineffective blockers at clinically relevant concentrations 141. Concurrent ingestion of ethanol is common in cocaine abusers [5] and a recent epidemiological study reported that it results in a signi~cant increase in the risk of sudden death related to cocaine use [6,7]. Ethylcocaine (Cocaethylene, benzoylecgonine ethyl ester), an ethylated metabolite of cocaine, is formed in individuals after combined administration of ethanol and cocaine, but not in individuals given cocaine or ethanol alone [8-IO]. The plasma concentrations of ethylcocaine may be as great and even greater than those of cocaine [7,11,12]. It has been reported that ethylcocaine may be of greater potency than cocaine in producing lethality [8] and in its negative inotropic action [13], but there is no information on the cardiac electrophysiologic actions of this metabolite. The purpose of the present study was to study the effects of cocaine and its metabolites, including ethylcocaine, on the electrical activity of the heart. 2. Materials and methods 2.1. Animals and equipment Adult (300-400 g) male Sprague-Dawley rats from Charles River Breeding Labs Co. were used in this study. The animals were euthanized under barbiturate anesthesia (sodium pentobarbital, Sigma Chemical Co., 60 mg/kg i.p.). The heart was quickly excised through open chest and immersed in oxygenated and buffered Tyrode’s solution (see below for composition) kept at 31°C. Preparations beating spontaneously (sinoatrial strips) and preparations to be stimulated electrically at a constant rate (papillary muscles) were isolated and mounted horizontally in a Lucite tissue bath holding 2 ml of physiological solution flowing at 5 mlimin at a constant temperature (37’C). The preparations were superfused with oxygenated and buffered (95% O2 - 5% COZ; pH 7.4). Tyrode’s solution, contained the following (mM): NaCl 137, KC1 5.4, CaCl, 2.7, MgCl* 0.5. NaHCOs 11.9, NaH2P04 0.45 and glucose 5.55. All preparations were equilibrated with the Tyrode’s solution for 2 60
J Curdiol. 46 (IY94)
-735-242
min before any experimental procedure was carried out. 2.2. Studies with s~noafria~preparations spontaneously active A small strip of the right atrium containing the sinus node and a small portion of the crista terminalis was mounted, endocardial side up, in the tissue bath. Membrane potentials were recorded with standard intracellular glass microelectrodes filled with 3 M KC1 (tip resistance 20-30 MQ), using multiple impalements in different areas of the preparation. It was possible to impale sinus node subsidiary pacemaker fibers located in the periphery of the sinus node as well as atria1 contractile fibers located in the crista terminalis. However, it was difficult to impale true pacemaker fibers, which are located mainly intramurally in the rat preparation. The procedure allowed us to establish the control sinus node rate and to determine if there were irregularities or areas of conduction block. Only preparations with a regular and steady control sinus node rate and without areas of block were used in this study. After the control sinus node rate was recorded, the superfusate was changed to Tyrode’s containing one or more drugs (see below). The sinus node rate was recorded again during and after (recovery) a 20-min exposure to the drug(s). 2.3. Studies with papillary muscles driven at a constant rate
A left ventricle papillary muscle was mounted in the tissue bath. The preparation was driven electrically at a constant frequency of 5 Hz, approximately 20% above the rate of the preparations beating spontaneously, by a Grass stimulator (S88) with 2 SIUS isolation units. Square pulses of voltage 50°/ above threshold of l-2 ms duration were delivered through a pair of silver electrodes in close contact with one end of the preparation. Membrane potentials were recorded from one cell; only fibers for which the impalement was maintained for the entire protocol were used for subsequent analysis. After the control membrane potentials were recorded, the superfusate was
J. Wang. R.G. Carpentier. / I~I. J. Cur&l.
system
The recording system consisted of a Winston 1090 amplifier and a Nicolet Explorer II digital oscilloscope. The tracings were stored on floppy disks. In the experiments in which spontaneously active sinoatrial preparations were used, the cycle length was read directly from the oscilloscope and was used to calculate the sinus node rate. In the experiments with papillary muscles driven at a constant rate, the parameters measured during the single impalement were: resting membrane potential, overshoot of the action potential, total action potential amplitude and action potential duration measured at -50 mV of repolarization.
2.6. Statistical
(cocaine
hydrochloride)
and its metab-
r
COCAINE
10
20
MINUTES
10
2c
MINUTES
analysis
Only one concentration of a single drug was tested in each preparation. A single preparation was dissected from each animal, so that in all series of experiments the number of preparations indicates also the number of rats used. The mean value and the standard error of the mean (S.E.M.) were calculated for each parameter measured in each sin-
2.5. Drugs Cocaine
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olites ecgonine (ecgonine hydrochloride), ecgonine methyl ester (ecgonine methyl ester hydrochloride), benzoylecgonine (benzoylecgonine) and cocaethylene (ethylcocaine, ecgonine ethyl ester benzoate), all purchased from Sigma Chemical Co., MO, were tested. Stock solutions of the drugs were freshly prepared. All stock and final solutions were kept in the dark and protected from direct light during the superfusion. All compounds, with the only exception of ethylcocaine, are water soluble, and Tyrode’s solution was used as the vehicle to reconstitute the drugs. On the other hand, a small volume of ethanol was used initially to reconstitute ethylcocaine.
changed to Tyrode’s containing one or more drugs (see below). The membrane potentials were recorded again during and after (recovery) a 3-min exposure to the drug(s). 2.4. Recording
46 /I9941
_BENZOYLECGONINE
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Fig. 1. Percent change in sinus node rate (SNR) induced by cocaine and its metabolites (58 FM). Each bar is the mean and S.E.M. of values obtained from six preparations. There was no difference (P > 0.05) between the control values (action potentialsimin) for the five compounds, as well as between the control and recovery values for each compound, which were, respectively: cocaine. 262.2 f 5.4 and 265.7 * 6.7; ecgonine, 264.3 f 7.1 and 271.2 f 10.9; benzoylecgonine, 245.5 f 6.8 and 249.8 f 8.9; ecgonine methyl ester, 256.0 f 14.9 and 260.3 f 13.4; ethylcocaine. 252 f 10.0 and 255.0 f 9.7. *Significant change, F(2,lO) = 19.9, P < 0.0005. F( 1.10) = 13.6. P < 0.005; ecgonine methyl ester, +Signiticantly smaller than change induced by cocaine: benzoylecgonine. F(l,lO) = 16.8, P < 0.003. **Not significantly different from change induced by cocaine, F(l,lO) = 0.36. P > 0.56.
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J. Wang. R.G. Carpmtier.
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46 i 19941 235-242
BENZOYLECGONlNE
ECGONINE METHYL ESTER
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Fig. 2. Percent change in sinus node rate (SNR) induced by cocaine and its metabolites (87 PM). Each bar is the mean and S.E.M. of values obtained from six preparations. There was no difference (P > 0.05) between the control values (action potentialsimin) for the five compounds, as well as between the control and recovery values for each compound, which were, respectively: cocaine, 259.7 f 5.7 and 256.3 f 3.3; ecgonine, 274.8 f 14.4 and 275.0 f 14.3; benzoylecgonine. 253.7 f 9.2 and 253.3 f 9.1; ecgonine methyl ester, 269.0 + 13.4 and 265.3 f 13.6; ethylcocaine, 263.4 f 7.2 and 260.5 f 5.8. *Significant change. F(2.10) = 16.5. P < 0.001. +Signiftcantly smaller than change induced by cocaine: ecgonine, F( I.lO) = II.3, P < 0.0009; benzoylecgonine. F( IJO) = 8.6. P < 0.02; ecgonine methyl ester, F( 1.10) = 10.0, P =c0.001. **Not significantly different from change induced by cocaine. F(l.lO) = 0.34, P > 0.58.
gle series of experiments. Significance of differences between pair of means was calculated by using Student’s r-test for paired or unpaired data, as appropriate. A one-way analysis of variance was used to analyze the effect of one concentration of a single drug over a period of time, which involved comparison of several means. A two-way analysis of variance was used to compare the effects observed in two different groups of experiments. The level of significance is given each time a comparison is made. 3. Results
The concentration of cocaine used initially in this study (58 PM) was selected from results obtained in a previous investigation using the same preparation [ 141.Concentrations of cocaine I 2.9 pM did not affect the sinus node rate. Cocaine L 87 PM
resulted in reduction in sinus node rate associated with development of sinoatrial block. On the other hand, cocaine 58 PM reduced the sinus node rate without producing sinoatrial block. The changes in sinus node rate produced by this concentration of cocaine and its metabolites (58 PM) are presented in Fig. 1. Ecgonine did not have any effect. The depressant effects of benzoylecgonine and ecgonine methyl ester were signi~cantly smaller than that of the parent compound. On the other hand the effect of ethylcocaine was not significantly different from that of cocaine. The data presented in the legend for the figure also show that the effects of ail compounds were completely reversed within 30 min. The effects of a higher concentration of cocaine and its metabolites (87 PM) are presented in Fig. 2. At this concentration, all compounds depressed the sinus node rate. Ecgonine, benzoylecgonine and ecgonine methyl ester exerted a si~i~~antly smaller effect than their parent compound, whereas ethylco-
J. Wang, R.G. Carpentier. /ht.
J. Cardiol. 46 (1994) 235-242
m
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ETHYLCOCAINE
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239
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Fig. 3. Percent change in sinus node rate induced by cocaine and ethylcocaine (2.9 and 29 pM), alone and in combination. There was no difference (P > 0.05) between the control values for the different experimental groups, as well as between the control and recovery values for each group, which were, respectively, for the 2.9 PM concentrations: cocaine, 273.0 + 8.2 and 273.8 + 7.7; ethylcocaine, 267.3 f 7.3 and 270 & 6.2; cocaine + ethylcocaine, 271.3 f 7.3 and 272.5 f 7.0. For the 29 PM concentrations the control and recovery values were, respectively, cocaine, 273.3 f 4.9 and 273.8 * 4.3; ethylcocaine, 279.8 f 6.0 and 274.3 * 5.7; cocaine + ethylcocaine, 275.5 f 4.4 and 277.8 f 5.9. Each bar is the mean and S.E.M. of values obtained from six preparations. *Significant change: F(2.6) = 36.5, P < 0.0008. +Significant change, and significantly different from change induced by cocaine, F( 1.6) = 24.4, P < 0.001 and F( 1.6) = 13.2. P C 0.02. **Significant change, but not significantly different from change induced by ethylcocaine alone, F(l.6) = 2.5. P > 0.16 and F(1,6)=0.11, P > 0.74.
Caine had an action similar to that of cocaine. All the compounds tested acted in a fully reversible manner, as shown by the data presented in the legend for the figure. The results obtained with two lower concentrations of cocaine and ethylcocaine, administered alone and in combination, are presented in Fig. 3. Cocaine 2.9 PM did not depress the sinus node rate, whereas ethylcocaine produced a significant effect. Cocaine 29 PM depressed the sinus node rate, but again ethylcocaine had a significantly greater effect. It is also clear that the effects of the two compounds were not additive.
3.2. Studies with papillary constant rate
muscles driven at a
The effects of cocaine and ethylcocaine,
alone and
in combination, on the membrane potentials of papillary muscles driven at 5 Hz, are presented in Fig. 4. Both cocaine and ethylcocaine depressed the resting potential, the overshoot and the total amplitude of the action potential, without signilicant differences between the magnitude of their effects. The figure also shows that the effects of the two compounds were not additive. The parameters measured recovered completely after withdrawal of the drug(s) from the superfusate, as shown by the recovery values. Cocaine and ethylcocaine prolonged also the action potential duration, by 12.4 f 3.1 and 17.0 f 4.1 ms, respectively. Superfusion of the preparations with the two compounds in combination prolonged the action potential by 19.6 f 2.0 ms. There was no significant difference between the effects of cocaine and ethylcocaine, or between the effects of the two drugs in combination and either one alone.
240
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Fig. 4. Effects of cocaine and ethylcocaine (58 pM) on membrane potentials of papillary muscles driven at 5 Hz. CON. control values: DRUG, values recorded at the end of a 3-min exposure to the respective drug; REC. recovery values. Each data point is the mean and S.E.M. of values obtained from six preparations. *Significantly different from the respective control value (P < 0.01).
4. Discussion Previous studies in our laboratory have demonstrated the in vitro cardiac electrophysiologic and inotropic effects of cocaine [14- 161. The present study compared the effects of cocaine and metabolites, including its ethanol derived metabolite cocaine ethyl ester (ethylcocaine), on the sinus node rate and membrane potentials of ventricular fibers. This is the first study comparing the cardiac electrophysiologic effects of cocaine and its metabolites. The results suggest that most metabohtes of cocaine are of little, if any, clinical significance. However, they also suggest that ethylcocaine may play an important role in the cardiac toxic effects of the parent compound when ethanol and cocaine are administered concurrently.
Cocaine depressed the sinus node rate in sinoatrial preparations beating spontaneously. It also decreased the resting potential, the overshoot and the total amplitude of the action potential, and increased the action potential duration, of papillary muscle fibers driven at a constant rate, con~rming our previous findings [14]. The mechanism of the depressant action on the automaticity of the sinus node is still not clear. There is no info~ation on possible actions of cocaine on the time-dependent currents IK and If, responsible for most of the slow diastolic depolarization in the sinus node. On the other hand, there is some evidence that cocaine depresses the slowly activated current I, [ 17, If?], which contributes to the latter portion of phase 4. The changes in the action potential amplitude are easily explained by the anesthetic action of cocaine
J. tVaq,
R.G. Curpenrier.
on the fast sodium system [19] and the fall in the resting potential [20]. The prolongation of the action potential is abolished by phentolamine and by 4-aminopyridine, suggesting that it is an cu-adrenergic mediated action resulting in the inhibition of the transient outward current I,, [14]. The results presented here indicate that the metabolites ecgonine, benzoylecgonine and ecgonine methyl ester had a signi~~antly smaller effect than cocaine on the sinus node rate. Ecgonine had the smallest action, with the other two metabolites showing similar effects, but still well below the effect of the parent compound. These results suggest that these three metabolites most probably play very little role, if any, in the sinus bradycardia observed in cocaine intoxication. Crumn and Clarkson also found that both benzoylecgonine and ecgonine methyl ester, in clinically relevant concentrations (i.e., < 100 PM), are ineffective blockers of the fast sodium channels in single cardiac myocytes [4]. The results obtained with ethylcocaine are of great interest. The ethanol derived metabolite of cocaine depressed the sinus node rate at a concentration (2.9 PM) at which cocaine had no effect. Also, 29 PM ethylcocaine had a significantly greater depressant effect than cocaine. It has been recently reported that ethylcocaine produces more potent inhibition than cocaine of the intracellular calcium transient and the myocardial contraction in single cardiac myocytes [ 131. Our results provide additional support to the hypothesis that ethylcocaine may exert greater effects than its parent compound on the heart. Although it is virtually impossible to precisely compare the superfusate levels of cocaine and ethylcocaine with those present in vivo, the concentrations used in this study were comparable to those found in blood in acute cocaine intoxication [3,7,11,12,21]. Cocaine is rapidly metabolized [3], but almost one third of the deaths related to cocaine overdose occur 2-5 h after the exposure to the drug [2]. Moreover, ethylcocaine formed after combined use of ethanol and cocaine may reach concentrations in plasma as great and even greater than those of cocaine [7,11,12]. Thus, it seems reasonable to propose that ethylcocaine may play a significant role in the cardiac effects of cocaine when the latter and ethanol are used concurrently.
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46 (19941 23%.?42
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Acknowledgement This research was supported by Grant S06GM08016 from the National Institute of General Medical Sciences.
References ill
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