Sensitization to epinephrine-induced ventricular fibrillation produced by probucol in dogs

TOXICOLOGY

AND APPLIED

Sensitization

PHARMACOLOGY

24, 594-602

(1973)

to Epinephrine-Induced Produced by Probucol F.N.

MARSHALL

Ventricular in Dogs

Fibrillation

AND J. E. LEWIS

Department of Pharmacology, Human Health Research and Development Laboratories, The Dow ChemicaI Company, ZionsvilIe, Indiana 46077 Received May 25: 1972

Sensitization to Epinephrine-Induced Ventricular Fibrillation Produced by Probucol in Dogs. MARSHALL, F. N. AND LEWIS, J. E. (1973). ToxicoI. Appl. Pharmacol. 24, 594-602. Probucol administered iv in the form of an emulsified oil preparation was found to “sensitize” the canine myocardium but not the simian myocardium to epinephrine-induced ventricular fibrillation. Probucol differs from other known ‘sensitizing” agents in several respects.Dogs treated with probucol have been observed to respond to an iv epinephrine challenge with ventricular fibrillation which reverted spontaneously to a sinus rhythm. “Sensitization” to sustained ventricular fibrillation is slow in onset, requiring approximately 2 weeks of treatment. “Sensitivity” once established, is slow to dissipate, disappearing between 16 and 23 days. Not all dogs were capable of being “sensitized” to produce sustained ventricular fibrillation in response to epinephrine challenge even after 3 weeks of probucol treatment. In these dogs, sustained ventricular fibrillation could be induced with epinephrine after administration of nhexane. Premonitory changes in the electrocardiogram signaling the presence of the “sensitized” state were not seen in probucol-treated dogs. “Sensitivity” to epinephrine-induced ventricular fibrillation was not seenin rhesus monkeys even after 4 weeks of treatment with probucol. In toxicity studies and in other long-term experiments, a significant number of deaths have occurred in dogs which received the new blood cholesterol-lowering agent probucol[4,4’-(isopropylidenedithio)bis(2,6-di-Ze~~-butylphenol)]. Extensive clinical chemistries and comprehensive postmortem examination including complete histologic investigation of animals which died, showed no cause for death (Mole110 et al., 1973). Death produced by probucol in dogs has not been correlated with the magnitude of the dose administered nor with the length of treatment. Only three of the dogs have thus far been observed in the process of dying. The first two were observed before this investigation was started, and the last, after its completion. The dogs, which were not unduly restrained, suddenly collapsed, became cyanotic and made a few gasps. Subsequently, no vital signs could be found. In the case of the last dog to be observed dying, the chest was opened at the time of apparent death and ventricular fibrillation was observed. At the time this investigation was started, the observation of ventricular fibrillation had not been made and it was necessary to form a working hypothesis as a first step in the Copyright Q 1973 by Academic Press, Inc. 594 All rights of reproduction in zmy form reserved.

PROBUCOL-EPINEPHRINE

INTERACTION

IN

DOGS

595

investigation of sudden death occurring in dogs treated with probucol. It was hypothesized that probucol produced a “sensitization” of the canine myocardium to epinephrine such that epinephrine would evoke ventricular fibrillation. No drug-related deaths have been observed in long-term studies in which rats and monkeys were treated with probucol. For this reason, it was of interest to extend the investigation of the myocardial “sensitizing” properties of probucol to monkeys. METHODS In these studies, probucol was administered iv in an emulsion vehicle because of the low solubility of the compound in water. Probucol was dissolved in a purified coconut oil fraction (Neobee M-5, Drew Chem. Corp.) at its maximum solubility and an oil/ water emulsion prepared using the Lipomul IV formula (Thompson et ui., 1963) employing ultrasonic energy. This resulted in a final concentration of probucol of 22 rngj ml of emulsion. A similar emulsion was prepared without probucol for use as a control. Five mongrel dogs of either sex were used to determine the acute effects of the iv infusion of the emulsion and of the probucol-containing emulsion. Dogs were anesthetized with pentobarbital sodium (32 mg/kg, iv) and were prepared for the recording of ventilation rate, arterial blood pressure and lead II electrocardiogram. Emulsions were infused at the rate of 2 ml/kg/hr iv for a total of 3 hr. The response of the dog to bilateral carotid occlusion (30 set), anoxia (tracheal occlusion, 60 set) and epinephrine bitartrate (Nutritional Biochemicals. 10 and 5 pg/kg, iv) were recorded twice before infusion of the emulsion and at 30-min intervals after the infusion was begun. Three experiments were performed, one using the control emulsion and two with the emulsion containing probucol. Six distemper-immune, mongrel dogs of either sex were used to determine the effect of prolonged administration of probucol-containing emulsion. Dogs were distributed into 2 groups of 3 each and placed in individual cages with food and water ad libitum. On day 0 each dog was anesthetized with pentobarbital sodium (24 mg/kg, iv). Using sterile technique, a plastic catheter (Venocath-18, Abbott Laboratories) was placed in a cephalic vein. Similarly, a siliconized, 20-gage hypodermic needle was used to puncture a femoral artery. The needle was connected via plastic tubing to a pressure transducer (P23A, Statham Instruments, Inc.) filled with heparinized (lOpg/ml) saline. Ventilation rate, blood pressure and lead II electrocardiogram were recorded on a Grass polygraph. A freshly prepared solution of epinephrine bitartrate (0.2 mg/ml) was used to administer 10 pg/kg of the drug iv as rapidly as possible. The response of the dog to this challenge was recorded and when blood pressure returned to pre-epinephrine values, a challenge of 5 pg/kg of epinephrine bitartrate was administered. After this procedure the dogs recovered uneventfully. Each dog then received 0.5 ml/kg of an emulsion each day. One group (dogs 1, 2, and 3) initially received emulsion containing no probucol while the other group (dogs 4,5 and 6) received emulsion containing probucol. After 3 weeks of treatment, dogs 1,2 and 3 began receiving probucol-containing emulsion. The dogs received a dose of 11 mg/kg/day of probucol. The dogs did not receive emulsions on the days on which they received the epinephrine challenges. Challenges were administered at weekly intervals throughout the experiment. After the 0 time challenges and the challenges after 1 week of dosing with the emulsions, it was decided to administer 3

596

MARSHALL

AND

LEWIS

additional doses of epinephrine bitartrate (10 pg/kg). Electrical defibrillation, when used, was performed with an Electrodyne defibrillator. Utilizing an experimental design similar to that described above for the prolonged administration of probucol to dogs, 5 male rhesus monkeys were randomly assigned to 2 groups. Three monkeys received emulsion containing probucol(O.5 ml/kg, I I mg/kg, iv) daily and 2 monkeys received emulsion containing no probucol. The experimental protocol differed from that described in the dog experiment in that the dose of epinephrine bitartrate administered was 30 pg/kg rather than 10 and 5 pg/kg. This dose was administered 5 times at each determination and the animals were challenged at the end of the second and fourth week of dosing rather than at weekly intervals. RESULTS

No consistent changes in the blood pressure, heart rate or ventilatory rate responses to the injection of epinephrine, bilateral carotid occlusion or anoxia were produced by the infusion of either emulsion or emulsion containing probucol. In no case was ventricular fibrillation observed during the course of any of the experiments. In addition, no marked changes in the electrocardiogram occurred as a result of the infusion of probucol-containing emulsion.

FIG. 1. Spontaneouslyreverting ventricular fibrillation in responseto an injection of epinephrine bitartrate (10 pg/kg) in a probucol-treated dog (dog 3, 1 week of probucol treatment).

In the experiments in which probucol was administered chronically, ventricular fibrillation was sometimes observed in response to the iv injection of epinephrine bitartrate. In this regard, it was observed that ventricular fibrillation occasionally reverted spontaneously to a sinus rhythm. This unusual phenomenon is illustrated in Fig. 1. This figure shows a 6-set period of ventricular fibrillation precipitated by the iv injection of epinephrine bitartrate (10 pg/kg), which spontaneously reverted to a sinus rhythm. At the time of the epinephrine challenge, this dog had been treated with probucol-containing emulsion (0.5 ml/kg/day, 11 mg/kg/day of probucol) for 6 days. This phenomenon was also observed in 2 other probucol-treated dogs. In those cases in which spontaneous reversion was observed, the electrocardiogram was indistinguishable from those in which ventricular fibrillation was the terminal event. Furthermore, during the period of ventricular fibrillation, blood pressure was seen to approach 0 with no evidence of ventricular contraction. In these respects, the tracings in Fig. 1 are typical.

PROBUCOL-EPINEPHRINE

INTERACTION

IN

DOGS

597

The results of the experiment in which emulsion and probucol-containing emulsion were administered chronically to dogs are summarized in Table I. The figures in the table indicate the total numbers of “abnormal” beats produced in response to each iv challenge of epinephrine bitartrate. “Abnormal” beats were found to be of the ectopic type originating in the ventricle or atrioventricular node. Six dogs were used in the experiments. Three were initially assigned to the control group (which received emulsion containing no probucol), and 3 were assigned to the group which received probucolcontaining emulsion. Response to the iv challenge of IO and 5 pg/kg of epinephrine bitartrate was determined before treatment with emulsion (0 time). Treatment was then initiated as indicated under Methods. The challenges of 10 and 5 pg/kg of epinephrine bitartrate were used at time 0 and after I week of treatment, but after the second week of treatment and subsequently thereafter, 3 additional challenges of 10 pg/kg were administered. The additional challenges were deemed desirable in order to increase the probability of detecting the ‘Ssensitized” state. Administration of probucol-containing emulsion was initiated with dogs I. 2 and 3 after the challenges administered at the end of 3 weeks of treatment. These dogs had previously been receiving emulsion containing no probucol. As can be seen, the numbers of abnormal beats in response to the epinephrine challenges was highly variable, quite independent of treatment, both within the same dog and among dogs. In dogs which proved to be “sensitized,” ventricular fibrillation usually did not occur on the first challenge with epinephrine. In those dogs receiving emulsion containing no probucol (dogs I, 2 and 3) ventricular fibrillation was never observed in response to any of the epinephrine challenges through 3 weeks of treatment. After I week of probucol treatment, dog 5 responded to the first epinephrine challenge with ventricular fibrillation. The fibrillation. however, lasted 40 set with spontaneous reversion to a sinus rhythm. After 2 weeks of treatment, this same dog responded to the first epinephrine challenge with sustained ventricular tibrillation resulting in death. At this time, dog 6, which also had received probucol for 2 weeks, responded to the third epinephrine challenge with persistent ventricular fibrillation which also resulted in death. Dog 4 accepted all 5 epinephrine challenges without ventricular fibrillation. This dog. after 3 weeks of treatment with probucol, was again tested for sensitivity to epinephrine-induced ventricular fibrillation and no ventricular fibrillation was observed in response to the 5 challenges. When challenged with n-hexane (0.04 mljkg, intratracheally) followed by epinephrine bitartrate 15 set later (10 pg/kg. iv), the dog responded with sustained ventricular fibrillation and death. Dog 3. after the fourth week of treatment (after I week of treatment with probucolcontaining emulsion), responded to the fourth epinephrine challenge with 20 set of ventricular fibrillation which reverted spontaneously to a sinus rhythm. Similarly, the fifth epinephrine challenge resulted in the production of 6 set of ventricular fibrillation, which also reverted spontaneously (this is illustrated in Fig. I). Dogs 1 and 2, at this time. did not show ventricular fibrillation in response to any of the challenges. After an additional week of treatment with probucol-containing emulsion, dog 1 responded with persistent ventricular fibrillation and death to the third epinephrine challenge. Dog 2. when tested did not respond with ventricular fibrillation to any ofthe epinephrine ChaTlenges. At this time, dog 3 responded with persistent ventricular fibrillation to the second challenge with epinephrine bitartrate (5 pg/kg). The dog was successfully defibrillated

598

MARSHALL

AND

TABLE NUMBERS

LEWIS 1

OF ABNORMAL BEATS FOLLOWING INTRAVENOUS ADMINISTRATION EPINEPHRINE TO Does TREATED CHRONICALLY WITH EMLJLSION AND EMULSION CONTAINING PROBUCOL“

OF

~Weeks of EB doses* dosing bdd 0

1

2

1

2

3

4

124 0 -

43 1 -

78 73 -

Emulsion (0.5 ml/kg/day)*

72 125 75 43 5 0 Emulsion (0.5 ml/kg/day) with probucol(l1 mg/kg)

10 5 -

151 2 -

5 0

84 50 -

VF*-40 set 4 -

5 11 -

10 5 IO 10

42 0 3 1 0 29 1 3 2

6 7 1 0 1 2 1 1 0 2 7 3 1 0 I 1 3 1 1 0 0 Emulsion (0.5 ml/kg/day) with probucol (I 1 mg/kg)

27 26 25 56 15 80 65 99 99 144

VF

31 5 VF

10 5 -

10

3

10 5 10 10 10

4

10 5 10 10 10 10 5 10 10 IO

5

6

Dog number

11 18 5 19 11 15 6 VF

10

5 IO IO 10 a Ectopic beats beats. * EB, epinephrine

originating bitartrate.

in the VF,

10 1 -

-

5

6

1 20 1 4 2 9 1 VF-20 set 0 VF-6 set 15 4 17 VF 10 4 4 85 Probucol 28 adminis8 tration 21 stopped VF-4 sec. ventricle ventricular

and

atrioventricular

fibrillation,

node

were

considered

abnormal

PROBUCOL-EPINEPHRINE

INTERACTION

599

IN DOGS

electrically and the administration of probucol-containing emulsion was stopped. This dog was retested 6 days later (see below), Dog 2 was treated for an additional week with probucol-containing emulsion, and it responded to the last epinephrine challenge with only 4 set of ventricular fibrillation which reverted spontaneously to a sinus rhythm. This dog, when treated with n-hexane and epinephrine as with dog 4, responded with a persistent and fatal ventricular fibrillation. b-i the experiments summarized in Table 1, there were no significant changes in mean arterial blood pressure, heart rate, or ventilation rate as a result of daily treatment with emulsion or probucol-containing emulsion. Also, no disturbances in cardiac rhythm could be noted. Conspicuous changes in the electrocardiogram configuration were also not apparent, including changes in QRS width or changes in QT interval. Furthermore. alterations in mean arterial blood pressure or heart rate in response to epinephrine chailenge did not appear to be different before or after administration of either the emulsion or probucol-containing emulsion. In addition, previous studies with probucol indicate that the compound has little or no effect on the cardiovascular response to iv administered histamine, acetylcholine, norepinephrine or epinephrine. The results of challenging dog 3 with epinephrine at various times after the cessation of probucol treatment are summarized in Table 2. Six days after the last dose of probucoL this dog responded with persistent ventricular fibrillation following the first epinephrine challenge. The dog was defibrillated electrically and challenged again 4 days later (IO days after the last dose of probucol). At this time, persistent ventricular fibrillation was observed with the second challenge with epinephrine bitartrate (5 pg/kg). As before. the dog was defibrillated electrically. Sixteen days after cessation of probucol treatment, the dog responded with persistent ventricular fibrillation to the third epinephrine challenge. The dog was defibrillated electrically once more and challenged again on day 23 and day 31 after the last dose of probucol. At neither ofthese times did the dog respond with ventricular fibrillation to any of the 5 epinephrine challenges. TABLE DURATION OF SENSITIZATION FIBRILLATION PRODUCED

EB doses’ hdk)

2

TO EPINEPHRINE-INDUCED VENTRICULAR BY PROBUCOL IN DOG NUMBER 3

Number of days elapsed since last dose of probucol -~

10

-

-

10

-

-

16 23 ~~. - -~.--.~-~ I2 11 7 3 VF 15 9

10

-

-

11

10

5

’ EB,

epinephrine

6

10

VFa -

4 VF

bitartrate.

VF,

ventricular

--.-~-~-

31

-3 I 6

10

IO

fibrillation

The results of the experiment in which emulsion and probucol-containing emulsion were administered chronically to 5 rhesus monkeys are summarized in Table 3. The figures in the table indicate the total number of abnormal beats produced in response to each 30 pg/kg iv challenge of 5 consecutive injections of epinephrine bitartrate. Tests

600

MARSHALL AND LEWIS

were conducted before administration of the emulsions and after 2 and 4 weeks of treatment. At no testing period was ventricular fibrillation observed in any of the animals in response to epinephrine challenge. TABLE 3 NUMBERS OF ABNORMALBEATS FOLLOWING INTRAVENOUS ADMINISTRATION OF EPINEPHIUNE BITARTRATE(~~~~/~~)TO MONKEYS TREATED WITH EMULSION AND EMULSION CONTAINING PROBUCOL~ Monkey Control Weeks of treatment Pre-emulsion

control

2

4

’ Ectopic beats originating

number Probucol(l1

1

2

0 0 0 1 0 0 0 0 0 0 0 0 0 0 0

0 0 99 164 159 0 2 0 4 0 69 125 113 104 78

in the ventricle and atrioventricular

mg/kg/day)

3

4

5

0 43 111 132 120 72 99 92 131 121 70 87 65 63 80

0 0 0 0 0 0

8 31 23 200 288 42 51 55 40 57 152 116 88 71 75

1

0 0 24 38 14 0 0 0

node were considered abnormal beats.

DISCUSSION

The studies presented here illustrate an effort to investigate the possibility that probucol produces a “sensitization” of the myocardium to epinephrine-induced ventricular fibrillation. The first experiments were performed to determine whether the acute administration of probucol would produce this phenomenon as has been described for several other agents. Besides the well known hydrocarbon-epinephrine interaction, the compounds U-0882 (Moore and Swain, 1959a) and amarine (Moore and Swain, 1959b) have been reported as “sensitizers.” Probucol was not found to cause myocardial “sensitization” to epinephrine during acute infusion over a 3-hr period. Thus, probucol differs from the other known “sensitizing” agents in that it does not act acutely. The experiments in which probucol was administered to dogs chronically demonstrate that “sensitization” can occur as a result of long-term administration. This is unlike that seen with most hydrocarbons, which require 15 set or less when administered intratracheally (Garb and Chenoweth, 1948), U-0882, which requires only 30 set after iv administration (Moore and Swain, 1959a) and amarine, which requires approximately

PROBUCOL-EPINEPHRINE

INTERACTlON

IN

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120 set after iv administration (Moore and Swain, 1959b). Also, spontaneous reversion to a sinus rhythm was observed in some cases after ventricular fibrillation had been established. This is not a usual finding with other “sensitizing” agents. Probucol does not appear to be as potent a “sensitizing” agent as any of the above, considering that 2 of 6 dogs responded with episodes of spontaneously reverting ventricular fibrillation following epinephrine challenge after 1 week of probucol treatment. Four of 6 dogs responded with persistent ventricular fibrillation and death to epinephrine challenge after 2 weeks of probucol treatment. Furthermore, those dogs that would not respond to epinephrine with persistent ventricular fibrillation even after 3 weeks of probucol treatment could be readily “sensitized” with n-hexane to produce persistent ventricular fibrillation and death in response to epinephrine challenge. With regard to cardiovascular effects, probucol did not produce a hypotensive response and stimulation of ventilation as does amarine and U-0882. The latter is also reported to produce slight bradycardia (Moore and Swain, 1959a) which was not seen with probucol. Probucol was not found to produce arrhythmias by itself during longterm administration. U-0882 has also been reported to produce no disturbance of cardiac rhythm (Moore and Swain, 1959a). Nodal and idioventricular beats have been reported to occur after amarine administration to dogs (Moore and Swain, l959b). In marked contrast to the above agents, probucol produced no conspicuous lengthening of the QT interval or T wave configurational changes in dogs. These experiments also demonstrate that the “sensitization” produced by probucol is reversible upon withdrawal of the drug. Just as sensitization to sustained ventricular fibrillation is slow in onset, taking approximately 2 weeks to develop, so “sensitivity’. seems to gradually dissipate, disappearing between day 16 and day 23. If “sensitivity” to epinephrine-induced ventricular fibrillation were to serve as an acceptable explanation for the sudden deaths observed in dogs, then this phenomenon should not occur in monkeys, a species in which drug-related deaths have not been observed. in preliminary experiments, it was found that a IO pg/kg challenge of epinephrine bitartrate did not produce a pressor effect of comparable magnitude to that observed in dogs given the same dose. Exploration of various doses showed that 30 pg/kg constituted an appropriate challenge. In general, it can be seen that the differences between monkeys which showed large numbers of abnormal beats in response to epinephrine challenge and those which showed few or none were striking and occurred without respect to treatment. At no time was either sustained or spontaneously reverting ventricular fibrillation observed in response to epinephrine challenge in any monkey. In all monkeys, an increase in QT interval from their pretreatment controls was observed at both the 2- and 4-week time periods. It was also observed that heart rate decreased from pretreatment controls at the same times. The correlation between the QT interval and heart rate was found to be highly significant (Y= -0.92; p = =:O.OOl). That an inverse relationship exists between QT interval and heart rate is well known (Ruth and Fuhon, 1960). Thus, the significance of the prolongation of the QT interval is in doubt. There was no consistent pattern of change in blood pressure at the times that testing for ‘*sensitivity” was carried out. It was not apparent from the pressor responses to

602

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epinephrine at any time period that there was either an antagonism or an enhancement of this effect of epinephrine. The reason for the species difference with regard to the “sensitizing” activity of probucol is not known. No differences have thus far been found in the mode of biotransformation of probucol between dogs and monkeys. REFERENCES S. AND CHENOWETH, M. B. (1948). Studies on hydrocarbon-epinephrine induced ventricular fibrillation. J. Pharmacol. Exp. Ther. 94, 12-18. MOLELLO, J. A., GE~IG, C. G. AND ROBINSON, V. B. (1973). Toxicity of [4,4’-(isopropylidenedithio)bis(2,6-di-f-butylphenol)], probucol, in mice, rats, dogs and monkeys: Demonstration of a species-specificphenomenon. Toxicol. Appl. Pharmacol, 24, 590-593. MOORE, J. I. AND SWAIN, H. H. (1959a). Sensitization to ventricular fibrillation. I. Sensitization by a substituted propiophenone, U-0882. J. Pharmacol. Exp. Ther. 128, 243-252. MOORE, J. I. AND SWAIN, H. H. (1959b). Sensitization to ventricular fibrillation. II. Sensitization by amarine and congeners of U-0882. J. Pharmacol. Exp. Ther. 128,253-258. RUCH, T. C. AND FULTON, J. F, (1960). Medical PhysioZogy and Biophysics, 18th Ed., p. 617 Saunders, Philadelphia, Pennsylvania. THOMPSON, S. W., II, HARTWIG,Q. L., ATIK, M., Fox, M. A. AND COHN,I., JR. (1963). Some long-term effects following daily infusion of intravenous fat emulsions into dogs. ToxicoZ. GARB,

Appl. Pharmacol.

5, 306-318.