New Treatment Methods in Verapamil Poisoning: Experimental Studies

NEW TREATMENT METHODS IN VERAPAMIL POISONING: EXPERIMENTAL STUDIES

Jan Magdalan Chair and Department of Pharmacology, Wroc³aw Medical University, J. Mikulicza-Radeckiego 2, PL 50-345 Wroc³aw, Poland

New treatment methods in verapamil poisoning: experimental studies. J. MAGDALAN. Pol. J. Pharmacol., 2003, 55, 425–432.

The aim of this study was to evaluate the effectiveness of the treatment with 4-aminopyridine (4-AP, potassium channel inhibitor) and Bay K 8644 (calcium channel activator) in experimentally evoked verapamil poisoning in rats and to compare the results of this treatment with the effectiveness of widely accepted methods (adrenaline, calcium compounds). The experiment was carried out on male and female Wistar rats which were divided into 4 experimental (A, B, C, D) and a control (K) groups. Rats were anesthetized and the abdominal aorta was cannulated for mean arterial pressure and heart rate measurements while caudal vein was cannulated for drug administration. All animals were infused with verapamil (150 mg/kg/h) until 50% reduction of mean arterial pressure and/or heart rate was observed. After verapamil, control animals were given 0.9% NaCl solution and the other groups received 687.5 mg/kg/h of calcium glucolactobionicum (group A), 0.3 mg/kg/h of adrenaline (group B), 2 mg/kg/h of 4-AP (group C) or 2 mg/kg/h of Bay K 8644 (group D). The mean blood pressure and heart rate was checked and ECG was recorded every 10 min. A statistically significant decrease in mortality compared with the control group was observed in animals treated with adrenaline (p £ 0.05), Bay K 8644 (p £ 0.01) and 4-AP (p £ 0.005). The treatment of experimentally evoked poisoning in rats using 4-AP or Bay K 8644 resulted in fast receding of poisoning symptoms: increase in blood pressure and heart rate, receding of bradyarrhythmia and return of sinus rhythm. The results of the study suggest the usefulness of 4-AP and Bay K 8644 in the treatment of verapamil poisoning.

Key words: verapamil, poisoning, 4-aminopyridine, rats J. Magdalan

INTRODUCTION 32]. We still have not got a specific antidote. New treatment methods for CCB poisonings that would The common use of calcium channel blockers be more specific and effective than those used so (CCB) in the treatment of cardiovascular diseases far are still being sought. Over the recent years, has resulted in an increasing number of poisonings there have been some sparse information on at- by these drugs over recent years [2, 5, 25]. tempts at treatment of verapamil poisonings with Poisoning by CCB, derivatives of dihydropyri- 4-aminopyridine (4-AP) [15, 26, 36, 38] and the dine (DHP) (e.g. nifedipine, nitrendipine, amlodip- compound Bay K 8644 [38]. Preliminary reports ine, felodipine) is characterized by hypotension have been encouraging, however, there have been (shock) whereas the contractive heart function re- too few experimental studies and clinical trials to mains relatively undisturbed, because the drugs of state whether those substances are useful or not. this group show a high vessel selectivity [15, 21]. The aim of this study was to evaluate the effec- The most serious CCB poisonings are those result- tiveness of the treatment with 4-AP (potassium ing from verapamil (an arylalkylamine derivative) channel blocker) and Bay K 8644 (calcium channel overdosing due to its very strong cardiodepressive activator) in experimentally evoked verapamil poi- effect [15]. The influence of this drug on the heart soning in rats and to compare the results of this function is similar to that of b-blockers. The sinus treatment with the effectiveness of widely accepted bradycardia, sinus arrest, asystole, atrioventricular methods (adrenaline, calcium compounds). blocks, including a complete block are not rare symptoms in a poisoning incident [2, 14, 15, 25, 37]. The cardiodepressive action combined with MATERIALS and METHODS the dilatation of blood vessels leads to shock and Animals cardiogenic pulmonary oedema [15]. From toxicological point of view, verapamil The experiments were carried out on male and poisonings are much more difficult to treat than female white Wistar rats with mean body weight of those caused by CCB of DHP derivatives group. 330.1 ± 16.1 g. Until the experiment started the ani- Patients suffering from poisoning by DHP deriva- mals had been kept in a special compartment at tives can be successfully treated with calcium pre- temperature 18–22°C, with food and water accessi- parations and catecholamines [15, 25]. Patients, es- ble ad libitum. pecially those who taken sustained-release verapa- Drugs and procedure mil, should be admitted to an intensive care unit. Calcium is a drug of choice. The optimal calcium Rats were anesthetized using 10% solution of dosage required for the treatment of verapamil tox- ethyl urethane administered intraperitoneally at icity has not been determined. Hypercalcemia may the dose of 1.2 ml/100 g. The urethane anesthesia be significant if more than 45 mEq of calcium is was chosen due to its insignificant influence on the administered acutely. Atropine is commonly used electrocardiogram (ECG) in rat [12]. Besides, for the treatment of bradycardia and atrioventricu- a strong anesthetic was required because 4-AP used lar nodal blockade. Glucagon exhibits chronotropic in the experiment has a very strong analeptic activ- and inotropic effects in verapamil overdose. Various ity resulting in a considerable lessening or even re- catecholemines and sympathomimetic agents, in- ceding of the general anesthesia [22, 39]. cluding isoproterenol, dopamine, dobutamine, epi- The animals were divided into 4 experimental nephrine, and norepinephrine, have been used to (A, B, C, D) and a control (K) groups, with 10 ani- counter the hypotensive effects of calcium channel mals in each. After urethane anesthesia, the ani- overdose [2, 4, 7, 15, 20]. mals were placed on a table keeping constant tem- Unfortunately, routine administration of cate- perature (36–38°C). The caudal vein of the animals cholamines and calcium compounds in verapamil was catheterized (0.8 × 25 mm) for administration poisoning is usually insufficient. In most serious of verapamil and other tested drugs. The peritoneal cases, even endocavitary electrode stimulation and cavity was opened with central cut and the abdomi- intra-aortic contrapulsation or extracorporal meth- nal aorta was cannulated (1.1 × 25 mm) with the ods of drug elimination from the blood (hemodialy- cannula linked to MCK-4011-meter, to monitor the sis, hemoperfusion) prove equally ineffective [18, heart rate and the mean blood pressure expressed

426 Pol. J. Pharmacol., 2003, 55, 425432 NEW TREATMENT IN VERAPAMIL POISONING as a mean of the systolic and diastolic pressure In all groups, the mean blood pressure and heart systolic pressure + diastolic pressure ()2 . Then needle electro- rate were checked and ECG was recorded every des were implanted into four limbs of the tested 10 min starting from T0. The animals were ob- animals to monitor the ECG using Simplicard E10 served for 120 min or till dead, whichever came apparatus, adjusted to experiments on rats, made by first. The parameters checked every 10 min were Roentgen Apparatus and Medical Instruments Plant compared with the initial values (Ti). in Warszawa (Poland). Upon completion of the testing, the mortality Ten minutes after the meter and electrodes were rate, average survival time and the ECG in groups connected (time needed for the circulation system A, B, C, D were compared with the results in the to stabilize), the mean blood pressure and heart rate control group K. were measured and the ECG was recorded. These The ECG was measured using standard ex- measurements were the initial parameters (mean tremital electrodes to determine the type of rhythm blood pressure 10–12 kPa; sinus rhythm with heart and conduction disturbances as well as the pace of rate 300–400/min). sinus rhythm recovery in rats treated with different Next, all animals were infused with verapamil preparations. hydrochloride (Isoptin, 5 mg/2 ml, ampoules, made Statistical analysis by Knoll, serial no. 8581) via the caudal vein dosed at 2.5 mg/kg/min until a 50% reduction of mean The results are presented as means ± SEM. The blood pressure and/or heart rate (bradycardia) as significance of the differences between the para- compared to initial parameters (Ti) was observed metric data was calculated by a one-way analysis for at least 60 s. Then the aortal pressure, heart rate of variance (ANOVA), followed, when appropriate, and ECG were recorded. That was the poisoning by individual comparison with the control using c starting point (To), verapamil infusion was stopped Student’s t-test. For nonparametric data square and the administration of antidotes to animals be- was used. gan. Rats of the control group (K) were administered only 0.9% NaCl solution 10 ml/kg/h. RESULTS The doses of the antidotes were chosen based on preliminary studies and on studies of Tuncok et All the rats poisoned with verapamil and given al. [38]. Antidotes were dosed intravenously (iv)as no treatment (group K) died within the first hour of follows: experiment. Continuous reduction in the blood — group A: calcium (1.375 g/10 ml, ampoules, ma- pressure and heart rate was observed from the very de by Polfa, Kraków, serial no. 40296) 5 ml/kg/h first minutes of poisoning (Fig. 1, 2). The average (687.5 mg calcium glucolactobionicum/kg/h survival time was very short and amounted to 34 min 2+ which is an equivalent of 45.36 mg Ca /kg /h); (Tab. 1). Major conduction disturbances, mainly II- — group B: adrenaline (1.8 mg adrenalinum hydro- and III-degree atrioventricular blocks, appeared tartaricum/1 ml, ampoules made by Polfa Warsza- throughout the experiment with no animal showing wa, serial no. 31296) 0.3 mg/kg/h (5 mg/kg/min); spontaneous recovery of the sinus rhythm. — group C: 4-AP (Sigma, Japan, cat. no. A-0152) The mortality rate in the group A (rats treated – 1 mg/kg bolus, and then 2 mg/kg/h; to avoid with calcium glucolactobionicum) was high with the convulsions, the infusion of 4-AP was only 20% of the animals surviving the experiment stopped immediately after conduction distur- (Tab. 2), the average survival time was longer than bances receded and sinus rhythm returned, then that of the control group and amounted to 75 min the animals were given 0.9% NaCl solution iv. (Tab. 1). Initially, the treatment resulted in an im- — group D: BAY K 8644 (1,4-dihydro-2,6-dime- provement of the animals condition and increase in thyl-5-nitro-4-[2-(trifluoromethyl)-phenyl]-3-py- blood pressure and heart rate, however, from the ridine-carboxylic acid methyl ester, ICN Phar- 30th minute of the experiment, gradual decrease in maceuticals, USA, cat. no. 153628) – 1 mg/kg the mean blood pressure and heart rate was noted bolus, then 2 mg/kg/h. (Fig. 1, 2) in spite of continued administration of All the substances were diluted in 0.9% NaCl the calcium preparation. The II-degree atrioventri- solution so that the volumes of liquids administered cular block was the major conduction disturbance were the same for all groups (10 ml/kg/h). observed during the experiments. A recovery of

ISSN 1230-6002 427 J. Magdalan

14 K

A 12 B

C 10 D

Mean blood pressure (kPa) 4

0 Ti T0 10 20 30 40 50 60 70 80 90 100 110 120 Time (min) Empty fields – non-significant in comparison with Ti (initial values)

Fig. 1. Mean blood pressure in K, A, B, C, D groups

400 K

A 350 B

300 C D

250

200

150 Heart rate (n/min)

100

0 Ti T0 10 20 30 40 50 60 70 80 90 100 110 120

Time (min) Empty fields – non-significant in comparison with Ti (initial values)

Fig. 2. Heart rate in K, A, B, C, D groups

428 Pol. J. Pharmacol., 2003, 55, 425432 NEW TREATMENT IN VERAPAMIL POISONING

Table 1. Average survival time of verapamil-poisoned rats turned in 7 animals. The return of the sinus rhythm Group Average survival SEM p was in the group C the fastest – on average at 29.4 time (min) min of observation. In two cases, convulsions appeared several minutes after 4-AP infusion were K (control) 34.0 4.58 stopped and all experimental animals demonstrated A (calcium) 75.0 11.25 £ 0.005 weakening of urethane anesthesia (fasciculation, B (adrenaline) 92.5 8.24 £ 0.001 tremor of whiskers, periodical head twitches). C (4-AP) 100.5 11.56 £ 0.001 Bay K 8644 (group D) enabled to save 50% of D (BAY K 8644) 98.5 7.78 £ 0.001 poisoned rats (Tab. 2). The average survival time was long and reached 98.5 min (Tab. 1). The most p comparison to K; number of animals within the group 10 frequent rhythm disturbance was a sinus bradycardia occurring in 70% of experimental animals and Table 2. Effectiveness of verapamil poisoningtreatment II-degree atrioventricular block occurring in as much as 90% of animals. As a result of the treatment the Group W c2 p sinus rhythm was recovered in 5 animals. The sinus K (control) 0 rhythm returned on average at the 33rd minute. A (calcium) 2 2.222 NS In all groups no differences in mortality rate and efficacy of treatment between male and female B (adrenaline) 4 5.0 £ 0.05 rats were observed. C (4-AP) 7 10.769 £ 0.005 D (BAY K 8644) 5 6.6667 £ 0.01 DISCUSSION p comparison to K; number of animals within the group 10; W number of animals within the group surviving 120 min of Verapamil does not change the serum calcium observation concentration [15]. Administration of calcium preparations in poisonings caused by this drug aims to increase the calcium ions gradient via cell mem- the sinus rhythm was observed only in 2 rats, at the brane and increase the calcium ion influx and intra- 53 min of experiment on average. cellular calcium concentration. One thing to re- The survival rate of the animals treated with member is that even under normal conditions the adrenaline (group B) was 40% (Tab. 2). The aver- calcium concentration in the extracellular fluid is age survival time was 92.5 min (Tab. 1). Similarly 5000–10000 times greater than in the cytoplasm, to group A, administration of adrenaline resulted in therefore, the gradient is very high. However, a an improvement of the blood pressure and heart considerable increase in intracellular calcium con- rate especially at the beginning of the treatment, centration occurs only after stimulation when the with its values gradually decreasing in the second voltage-dependent calcium channels open as a con- hour of experiment (Fig. 1, 2). The II-degree atrio- sequence of depolarization [24]. In verapamil poi- ventricular block was the major conduction distur- soning conditions, those channels are blocked and bance, transiently occurring in all animals. The si- increasing the extracellular calcium concentration nus rhythm returned in 4 animals, at the 56 min of does not add significantly to the increase in cal- experiment on average. cium ion concentration in the cytoplasm. This The treatment with 4-AP (group C) enabled to seems to be the reason for minor effectiveness of save as much as 70% of the animals (Tab. 2). Unlike calcium preparations used as drugs in the treatment adrenaline or calcium preparation, the effectiveness of severe verapamil poisoning. of 4-AP manifested itself first of all in the second In the heart, adrenaline is bound with b1-adreno- hour of treatment; then a considerable improve- ceptor and stimulates via G protein the adenyl cy- ment in the blood pressure values and heart rate clase activity leading to activation of appropiate ki- was noted (Fig. 1, 2). The average survival time in nases by cyclic AMP which phosphorylate the cal- treated animals was long and reached on average cium channel a1 subunit. Phosphorylation of a1 100.5 min (Tab. 1). The most common conduction subunit opens the channel and lets calcium ions in- disturbance was II-degree atrioventricular block side the cell, thus, increasing their concentration in occurring in 80% of animals. The sinus rhythm re- cytoplasm which not only improves the myocar-

ISSN 1230-6002 429 J. Magdalan diac contraction, but largely enhances conduction Verapamil poisoning accompanied by shock may in the atrioventricular system as well. The calcium lead to acute respiratory distress syndrome. The channel activation within this zone is believed to major role in the pathogenesis of this syndrome is largely depend on catecholamines [24]. Adrenaline attributed to diminished production of surfactant by proves to be a more effective drug in verapamil lung epithelial type II cells resulting in liquid exu- poisoning treatment than calcium compounds pro- dating to alveoli and generating vitreous mem- bably due to its indirect action, i.e. via intracellular branes with focal atelectasis. Bay K 8644 used in transmitter system, as calcium channel “opener”, treatment of verapamil poisoning complicated with leading to influx of calcium ions into the cell. There shock could probably prevent acute respiratory in- are opinions that the effectiveness of the treatment sufficiency as the substance stimulates surfactant could be further improved by administration of the production by increasing intracellular calcium con- calcium preparation prior to adrenaline [19]. centration and activating the protein kinase C bound Bay K 8644 opens N- and L-type channels ex- with cellular membrane [35]. In many cases vera- erting direct action on their a1 subunit at the spot pamil poisoning leads to hyperglycemia and inhibi- where specific inhibitors are bound (e.g. nifedipine, tion of insulin release from pancreas. Treatment nitrendipine). Activation of the Ca2+ ion influx with adrenaline, and especially glucagon, can en- combined with release of calcium from endoplas- hance those disturbances [37]. The likelihood of mic reticulum [27–29] evoked by Bay K 8644 in- hyperglycemia in the treatment of verapamil poi- creases cellular concentrations of those ions lead- soning with Bay K 8644 seems to be smaller as the ing to improvement of contraction of the vascular substance enhances glucose-stimulated insulin production probably by opening L-type calcium chan- smooth muscle [17, 33] and myocardium [3, 30], b and electrical conduction in the heart [31]. In in vi- nels in pancreas cells [23]. The influence of 4-AP on calcium channels is tro tests, the substance showed to enhance the heart indirect. It blocks potassium K channels on cyto- electrical conduction and eliminate the unidirec- 1 plasm side [6] which makes potassium stay inside tional nifedipine-related block liquidate the nega- the cell leading to depolarization and opening of tive chronotropic effect caused by nifedipine, dilti- voltage-dependent calcium channels [8, 9]. azem or verapamil poisoning [31]. The substance is believed to be an effective an- In the experiment carried out by Tuncok et al., tidote in treatment of tetradotoxine and saxitoxine only transient and short in duration improvement of poisonings, because toxicity of saxitoxine and tet- mean blood pressure without significant influence radotoxine is a consequence of sodium channel on the heart rate was observed in verapamil-poiso- blocking which makes depolarization of cell im- ned rats treated with Bay K 8644 dosed at 0.3–0.6 possible, whereas 4-AP can evoke depolarization mg/kg/h [38]. In this experiment, Bay K 8644 used of poisoned cells by inhibiting outflow of potas- at much higher doses proved to be much more ef- sium ions from cytoplasm [10, 11]. Also 4-AP im- fective in verapamil poisoning treatment leading to proves conduction in demyelinated axons, there- increase in the mean blood pressure values and re- fore, it tends to be used in symptomatic treatment treat of the conduction disturbances and recovery of sclerosis multiplex, Alzheimer’s disease [1] and of sinus rhythm in 50% of animals. in the therapy of spinal cord injuries complicated No convulsions were observed during admini- with plegia [16]. In some case reports, 4-AP re- stration of BAY K 8644. However, Wielosz et al. in stored blood pressure and cardiac rhythm in their experiment proved that one-off iv administra- verapamil-poisoned patients unresponsive to atro- tion of Bay K 8644 dosed at 2 mg/kg did evoke pine, calcium and catecholamines [26, 36]. convulsions in rats. Prior administration of atropine The drawback of 4-AP is its strong and dose- inhibited this effect, with pilocarpine enhancing it dependent epileptogenic action resulting from sti- and nifedipine having no influence on the convul- mulation of glutamic acid release in the central sions evoked by Bay K 8644. Hence, its epilepto- nervous system. Next, the glutamic acid stimulates genic action seems to depend mainly on the influ- NMDA receptors enhancing calcium ion influx to ence it exerts on the cholinergic system with its in- neurons [13]. Antiepileptic drugs, e.g. phenytoin, fluence on the calcium channels being of lesser phenobarbital and diazepam, prove to be effective significance [40]. in suppressing the 4-AP-evoked convulsions [34].

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