Intravenous lipid emulsion prolongs survival in rats intoxicated with digoxin

    Intravenous lipid emulsion prolongs survival in rats intoxicated with digoxin ¨ B¨ulent Serhan Yurtlu M.D., S¸ule Ozbilgin M.D., Derya Arslan Yurtlu M.D., Nilay Boztas¸ M.D., Gonca Kamacı D.V.M., Mahmut Akaltun, Volkan Hancı M.D., Osman Yılmaz PhD PII: DOI: Reference:

S0735-6757(16)00242-4 doi: 10.1016/j.ajem.2016.03.038 YAJEM 55687

To appear in:

American Journal of Emergency Medicine

Received date: Accepted date:

17 February 2016 14 March 2016

¨ Please cite this article as: Yurtlu B¨ ulent Serhan, Ozbilgin S¸ule, Yurtlu Derya Arslan, Bozta¸s Nilay, Kamacı Gonca, Akaltun Mahmut, Hancı Volkan, Yılmaz Osman, Intravenous lipid emulsion prolongs survival in rats intoxicated with digoxin, American Journal of Emergency Medicine (2016), doi: 10.1016/j.ajem.2016.03.038

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ACCEPTED MANUSCRIPT TITLE: Intravenous lipid emulsion prolongs survival in rats intoxicated with digoxin

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SHORT TITLE: Digoxin Toxicity and Lipid

AUTHORS: Bülent Serhan Yurtlu*1 M.D., Şule Özbilgin1 M.D., Derya Arslan Yurtlu2 M.D.,

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Nilay Boztaş1M.D., Gonca Kamacı3 D.V.M., Mahmut Akaltun1, Volkan Hancı1 M.D., Osman

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Yılmaz3, PhD.

AFFILIATIONS 1

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: Dokuz Eylül University, School of Medicine, Department of Anesthesiology and Reanimation 2

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: Katip Celebi University, School of Medicine, Department of Anesthesiology and Reanimation

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: Dokuz Eylül University, Institute of Health Science, Department of Laboratory Animals Science

* CORRESPONDING AUTHOR: Bülent Serhan Yurtlu MD Dokuz Eylül Universitesi, Tıp Fakültesi, Dokuz Eylül Universitesi Hastanesi, Ameliyathane, Kat: 1, Narlıdere, İzmir,Turkey Tel:

+90.535.887.17.23, +90.232.412.28.01

e-mail: FINANCIAL SUPPORT: None CONFLICT OF INTEREST: None

[email protected], [email protected]

ACCEPTED MANUSCRIPT TITLE: Intravenous lipid emulsion prolongs survival in rats intoxicated with digoxin ABSTRACT

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Background

Intravenous lipid emulsion eliminates the toxicity related symptoms for several drugs. We

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hypothesized that intravenous lipid emulsion prolongs the survival time in digoxin-intoxicated

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rats. Methods

Electrocardiograms of 14 anesthesized Wistar rats were monitored. All of the rats received digoxin infusion at a rate of 12 ml/h (0.25 mg/ml). Five minutes after the start of digoxin

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infusion, animals were treated either with 12.4 ml/kg intravenous lipid emulsion (Group L) or

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saline (Group C). The primary outcome variable was time elapsed until asystole development.

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Cumulative dose of digoxin required to induce asystole was also recorded. Results

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Mean time until asystole development in Groups C and L were 21.28 ± 8.61 min and 32.00 ± 5.41 min, respectively (p<0.05). The mean lethal doses of digoxin in the Groups C and L were 3.97±1.54 mg/kg, and 6.09±0.96 mg/kg, respectively (p<0.05). Conclusion Intravenous lipid emulsion prolonged the time until asystole development and cumulative lethal dose in rats intoxicated with digoxin. Keywords: Lipid rescue, Digoxin, Toxicity

ACCEPTED MANUSCRIPT INTRODUCTION

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Treatment of local anesthetic toxicity with the aid of intravenous lipid emulsion (ILE) became a standart of care within the recent years (1). At the first years of the discovery, it was thought

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that ILE forms a “lipid sink” in the intravenous compartment and this lipid sink incorporates highly lipid soluble bupivacaine inside itself so that bupivacaine is removed from the tissues

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where it exerts its toxic effects (2). This hypothesis has led to an idea that ILE therapy could have similar therapeutic effect at the toxic conditions caused by highly lipid soluble drugs. Thereafter, successful treatment reports of cardiovarcularly collapsed or central nervous system dysoriented patients due to drug intoxications have appeared in the literature (3-7).

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Experimental animal models of cardiac arrest due to drug toxicity with verapamil,

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clomipramine and amiptriptylin have also yielded supporting evidence for ILE therapy (8-10). Thus, ILE has become a promising therapy of future for drug toxicities (11,12).

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Digoxin is one of the oldest drugs that is used for the treatment of heart failure. Although digoxin is prescribed frequently, its therapeutic margin of safety is narrow. It is lipid soluble

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cardiac glycoside that is readly absorbed from the gastrointestinal tract (13). Since its therapeutic index is narrow, dose adjustment is a major concern in the elderly (14). Patients with digoxin overdose can be seen at ICU services. Mortality rate during the hospital course can be as high as 7% in patients intoxicated with digoxin (14). Since digoxin is lipid soluble, we have hypothesized that ILE therapy may delay cardiac arrest due to digoxin intoxication. In order to test this hypothesis digoxin intoxicated rats were treated either with ILE or saline. The primary outcome variable was determined as time until asystole.

ACCEPTED MANUSCRIPT METHODS

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We have used a model that was an established way of studying drug toxicity (8, 15). The

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study protocol was approved by the Animal Ethics Committee of School of Medicine, Dokuz Eylul University (date 04/03/2014, number: 03/05). The study was carried out at the Dokuz

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Eylul University Faculty of Medicine, Department of Labaratory Animals. All of the rats included in the study were obtained from institution’s Labaratory Animals Department, all fed

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with standart rat pellets and housed in temperature and humidity controlled (22–24°C and 60% relative humidity) rooms that were lit on a daily schedule (12:12 h light/dark) until the day of experiment. During the experimental period, the care of the laboratory animals was in accord with international guidelines. Fourteen Wistar rats weighing between 245 to 291 gr

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were anesthesized with intraperitoneal injection of 60 mg/kg ketamin. Electrocardiogram was

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applied to monitor the heart rate and and two intravenous cannula were placed at the lateral tail veins. A venous blood sample from the tail vein was drawn to control blood pH and gas

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tensions. One lt/min oxygen was supplied to all of the rats. Digoxin (0,25 mg/ml), at a rate of

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12 ml/h, was started to be infused to all of the rats at time zero (T0). Digoxin infusion continued until rat’s death (defined as asystole lasting 1 min together with absence of respiratory efforts). This infusion rate of digoxin was taken from a previous experiment to enable death of animals within a reasonable time frame (15). At the 5th min of infusion (T5), rats were administered either 12.4 ml/kg 20% ILE (Clinoleic %20 Lipid Emülsiyonu, Eczacıbaşı-Baxter, İstanbul) or saline at the equal volume over 5 min through the second venous route (8). Duration of the time elapsed between start of digoxin infusion and death of the rat was defined as time until asystole and recorded in minutes. Times of first appearence of dysrhtymia (atrioventricular conduction delay) and widening of QRS complex were

ACCEPTED MANUSCRIPT recorded in minutes. The total dose of digoxin infused until asystole development was recorded.

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Statistical Analysis

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Data was analysed by a statistician who is blinded to treatment groups. SPSS 15.0 programme was used for the analysis of data. Data were expressed as mean ± SD. Mann-Whitney U test

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was used for the analysis of baseline and toxicologic characteristic properties of the groups. A

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p value less than 0.05 was considered as statistically significant.

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RESULTS

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The mean age and weight of the rats in all the groups included in the study and their mean heart and respiratory rates prior to drug infusion were similar (Table 1). In control blood gases

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analyses, none of the animals had developed respiratory acidosis, hypoxia or hypercarbia. In all animals, the terminal event was respiratory arrest, most often preceded by primary apnea

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and then gasping respirations. The T5 heart rates of Groups C and L prior to lipid infusion were determined as 372.42 ± 45.69, and 361.00 ± 50.57 respectively and there were no statistical difference between them (p>0.05).

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Time until asystole at Groups C and L were determined as 21,28 ± 8,61 min and 32.00 ± 5.41 min respectively. Time until asystole in Group L was significantly longer than that in Group

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C (p<0.05) (Table 2, Figure 1, 2). While 4 rats in the Group L survived until the 30th minute,

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only one rat survived longer than 30 minutes in Group C (Figure 1, 2, 3). The mean lethal doses of digoxin in control and lipid groups were 3,97±1,54 mg/kg, and

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6,09±0,96 mg/kg respectively. The mean lethal dose of digoxin was significantly higher in Group L than in Group C (p <0.05). Heart rate tended to decrease in both groups during the digoxin infusion; however, the rate of decrease was faster in Group C in comparison with Group L (Figure 4). ECG analysis showed the dominant rhythm was sinus bradycardia together with atrioventricular conduction block and widening of the QRS complex followed that later on. Some of the rats had junctional, idioventricular rhythms or varying degrees of heart block prior to asystole.

ACCEPTED MANUSCRIPT DISCUSSION

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The results of the current study demonstrated that administration of ILE prior to a catastrophic

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cardiac event incresases the dosage of digoxin required to produce asystole. Additionally, ILE therapy increases the time until asystole development. To the best of our knowledge, these

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results are the first findings in the literature giving clues about the potential role of ILE therapy for digoxin intoxication.

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Digoxin has a long story in the treatment of chronic heart failure. Although new theurapeutic options have emerged for the treatment of heart failure during the last decades, digoxin remains as an important tool and intoxication reports with this drug still are still frequently encountered in the literature. Since theurapeutic index of digoxin is small and overdose is a

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common problem, digoxin binding antibodies were developed as an antidote. Thus most of

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the previous studies focus on the effects of digoxin binding antibodies. The other examples focus on the effect of glucose-insulin infusion, anticalin administration or nano-magnet based,

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electrochemical immun-sensor technology to remove digoxin molecules from the plasma (16-

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19). Unfortunately it is not possible to make direct comparisons between the results of this study and those previous ones due to methodologic heterogenicities. The improvement in survival with ILE therapy in a drug toxicity other than a local anesthetic was first demonstrated by Krieglstein (20) et al. They had shown that rabbits which have been intoxicated with chlorpromazine had improved survival if they were pre-treated with ILE (20). Two decades later, the therapeutic efficiacy of ILE for local anesthetic systemic toxicity was established both with the experimental studies and case reports. First experimental evidence of ILE therapy came up with the lipophilic local anesthetic bupivacaine and clinical reports have confirmed this finding later on (21-23). Since drug’s lipophilicity is suggested as a key point for the efficiacy of ILE, it is thought that the same therapy could have beneficial

ACCEPTED MANUSCRIPT effects on toxidromes with other lipophilic agents. However accumulated experimental evidences about drugs other than local anesthetics are limited at the moment. Most of the

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other drugs investigated for ILE therapy’s effect are belonging to cardiovascular group of

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drugs such as beta blockers, calcium channel blockers. One of these studies was conducted with verapamil. Tebbutt et al (8) has administered intravenous 37.5 mg/kg/h verapamil

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infusion to the rats and then at the 5th min of infusion rats were either treated with ILE or saline. They have found that the survival time in ILE treated rats were almost twice times

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longer than the saline treated rats (44 ± 21 vs 24 ± 9 min). They had also found out that there was also a similar difference in the mean lethal dose (27.4 vs 14.7 mg) of verapamil for both groups. The results of the current study demonstrates a similar increase in both survival time (32.00 vs 21.28 min) and the mean lethal doses of digoxin (3.97 vs 6.09 mg) for saline or ILE

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treated rats respectively. Tebbutt’s (8) findings were later replicated by an another study in

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which dogs were used and resuscitated (24). In this second study, resuscitation has survived 100% of animals treated with ILE, whereas only 14% survived who received saline (24). The

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results of our study gave the initial clues about the role of ILE for digoxin intoxication. In

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case of life threating toxidromes, it doesn’t seem ethical to conduct a randomized controlled study on volunteers, so our findings needs a similar confirmation and advancement with a resuscitation model.

The effect of ILE therapy for lipophilic β blockers are not similar to the results obtained with verapamil. For example, propranolol and metoprolol are lipophilic β blockers and if “lipid sink” is the sole mechanism of ILE therapy, theoretically their toxicity would have benefit from ILE. However in the several animal experiments, supportive evidence for ILE therapy in lipophilic β blocker toxicities could not be identified (25-28). On the other hand, ILE therapy resulted in better recovery scores when compared with standart bicarbonate therapy in lipophilic clomipramine toxicity model (29). In order to clarify underlying mechanism of this

ACCEPTED MANUSCRIPT extra-ordinary success of ILE in clomipramine toxicity, the same authors have conducted a new study in which they administered ILE therapy either alone or in combination with plasma

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exchange (30). They have found that there was no difference in survival time between sole

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ILE and ILE plus plasma exchange treatment groups. According to these results authors concluded that it cannot be just lipid sink explaining the action of ILE therapy (30). These

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findings together with demonstrated inefficiacy of ILE for lipophilic β blockers, suggests that an another mechanism other than “lipid sink” exists. Possible mechanisms of effect are still a

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subject of investigation without a conclusive end (20,21,30,31) . Controversial issues about the underlying mechanisms of ILE therapy were summarized in a recent paper (31). Authors state that lipid sink theory is not the sole mechanism and it is not enough to explain success/failure dilemma for several lipophilic drugs (31). Lipid sink theory or one of the other

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proposed mechanisms could be responsible for the delay in asystole duration observed in this

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current experiment. Present study does not proposes any underlying mechanism of the observed effect but rather establishes a connection in between digoxin overdose and lipid

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emulsions.

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The results of this study should be interpreted carefully in that these results do not present an evidence about the return of spontaneous circulation after digoxin intoxication. The rats in the study were not resuscitated to enable return of spontaneous circulation since we had no piece of evidence signaling that such a resuscitation could be helpful. In addition, there are opposite findings about epinephrine inclusion in resuscitation protocols of ILE treated cardiovascular collapse models, which could be important for the success of resuscitation after digoxin toxicity (32,33). Digoxin intoxications are continuous pictures of emergency services and intensive care units. According to the results of a recent retrospective analysis, there was a 7% mortality rate during the hospital course of definitely “digoxin intoxication” diagnosed patients (14). On the

ACCEPTED MANUSCRIPT other hand, results of a hospital based questionarre conducted at the developed world, Ontario/Canada, has revealed that only 9% of the acute care hospitals had an adequate supply

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of life saving digoxin immune FAB antibody fragments in their stores (34). The availability of

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lipid emulsions elsewhere makes ILE therapy a valuable alternative where the other theurapeutic choices are unavailable and the time is limited to increase survival.

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Limitations

First, researchers conducted this study by themselves so the study was single blinded,

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however, analysis of the data was performed by an another researcher (VH), who did not directly involved in experiments and he was unaware of the group assignments. Secondly, rats had spontaneous ventilation in the model and this can be a confounding factor, as acid-base status and thus survival duration of the rats could be effected by their ventilation patterns.

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However, there was no statistical difference at the initial blood gas measurements of the

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groups before the digoxin infusion, reflecting a similar baseline acid-base status. On the other hand, this situation mimics the condition in the real life where patients have spontaneous

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breathing during the toxin intake.

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In conclusion, intravenous lipid emulsion increases time required to asystole development and cumulative lethal dose in digoxin intoxicated rats. These results exhibite the potential of ILE therapy in cardiovascularly collapsed digoxin overdose patients when specific antibodies are unavailable. Supporting further evidence is necessary to make conclusive statements.

ACCEPTED MANUSCRIPT ACKNOWLEDGEMENTS

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The authors would like to thank to Dokuz Eylul University, Department of Laboratory

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Animals Science for their supply of the animals in order to conduct this study.

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FIGURES:

Figure 1. Box-and-whisker plot of asystole duration by lipid and control group status. Median indicated by black line, and upper and lower quartiles indicated by box edges.

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Figure 2. Dot plot of lethal dose by digoxin in lipid and control groups status.

ACCEPTED MANUSCRIPT Lipid

Control

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2 1 0 0

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Number of live rats

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Digoxin Dose (mg/kg)

Figure 3. Dose-response curves for lipid and control groups.

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*: p<0.05 compared with Control Group, chi-squared test.

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Figure 4. Heart rate versus time for control and lipid groups

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*: p<0.05 compared with Group Control, Mann - Whitney U-test

ACCEPTED MANUSCRIPT TABLES: Table 1: Baseline characteristics of groups (mean ± SD) Control (n=7)

Lipid (n=7)

p

Age (days)

126,00±9,89

127,00±10,24 0,902

Weight (g)

266,28±15,46

262,57±16,02 0,710

Heart rate (beat/min)

388,28±35,59

Respiratory rate (breath/min)

86,14±5,08

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387,14±62,37 0,649 85,00±4,32

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Mann - Whitney U-test

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Group

0,700

ACCEPTED MANUSCRIPT Table 2: Toxicological characteristics of groups (mean ± SD) Control (n=7)

Lipid (n=7)

p

Dysrhytmia duration (min)

14,28±6,99

22,28±8,69

0,165

QRS change duration (min)

16,71±8,63

21,57±7,95

0,259

Time until asystole (min)

21,28±8,61

32,00±5,41

0,026*

Lethal dose of digoxin (mg/kg)

3,97±1,54

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Group

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6,09±0,96

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*: p<0.05 compared with Control Group, Mann - Whitney U-test

0,017*