Cardiovascular pharmacology

Cardioprotection of the enkephalin analog Eribis peptide 94 in a ratmodel of ischemia and reperfusion is highly dependent on dosingregimen and timing of administration

Elias Spanos a,n, Lars O. Karlsson a, Björn Redfors a, Yangzhen Shao a, Elmir Omerovic a,Irina Bobrova c, Lars Grip a, Niklas Bergh b

a Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, SE-413 45 Gothenburg, Swedenb Department of Acute and Cardiovascular Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, SE-423 45 Gothenburg, Swedenc Eribis Pharmaceuticals AB, SE-754 50 Uppsala, Sweden

a r t i c l e i n f o

Article history:Received 23 October 2014Received in revised form10 November 2014Accepted 13 November 2014Available online 29 November 2014

Keywords:Myocardial ischemiaReperfusion injuryEnkephalinOpioid receptor

a b s t r a c t

Eribis Peptide 94 (EP94) is an enkephalin analog with cardioprotective properties in ischemia andreperfusion. The aim of the present study was to define the optimal timing and dosing of theadministration of EP94 during ischemia and reperfusion in a rat model. 172 anesthetized andmechanically ventilated male Sprague–Dawley rats were randomly assigned to different administrationprotocols of EP94 and subjected to 30 or 40 min of coronary artery occlusion followed by 2 h ofreperfusion. EP94 was administered intravenously at different doses and time intervals. Area at risk(AAR) and infarct size (IS) were determined by staining with Evans Blue (EB) and Triphenyl tetrazoliumchloride (TTC), respectively. EP94 reduced IS/AAR when administered as a double bolus (0.5 mg/kg perdose), whereas single (1 μg/kg) or triple boluses (0.5 μg/kg per dose) did not confer any protection.Reduction of IS/AAR was of highest magnitude if EP94 was administered 5 and 0 min before the 30 minischemic period (47% reduction, Po0.05), with declining cardioprotective effect with later administra-tion during ischemia. When EP94 was administered after 15 and 20 min of a 40-min ischemic period,reduction of IS/AAR was of the same magnitude as when given after 5 and 10 min of a 30-min ischemicperiod. It is concluded that EP94 confers cardioprotection after double bolus administration. The effectsare highly dependent on the timing of administration in relation to ischemia and reperfusion. Time ofreperfusion from drug administration seems to be more critical than the total duration of ischemia.

& 2014 Elsevier B.V. All rights reserved.

1. Introduction

Myocardial ischemia induces tissue injury in a time-dependentmanner (DeBoer et al., 1983; Reimer and Jennings, 1979). In theclinical setting, an acute myocardial infarction is caused by anocclusive thrombotic lesion in a coronary artery. Although reperfu-sion therapy reduces the size of the infarction and subsequentmortality, it may induce cardiomyocyte death. This phenomenon,often referred to as myocardial reperfusion injury, can paradoxicallyreduce the beneficial effect of myocardial reperfusion (The GUSTOinvestigators, 1993; Braunwald and Kloner, 1985). Several studies ofmyocardial ischemia and reperfusion in animal models have demon-strated cardioprotection by different pharmacological compounds(Gross et al., 2004; Louttit et al., 1999; Park et al., 2006). However,

these findings have not been unequivocally reproduced in the clinicalsetting (Yellon and Hausenloy, 2007). In a majority of the studieswith pharmacological intervention the drug has been administeredintravenously or intracoronary only minutes before, at, or immedi-ately after reperfusion (Atar et al., 2009; Bates et al., 2008; Piot et al.,2008). However, the optimal regimen in terms of timing of pharma-cological intervention has not yet been defined.

Opioids have been demonstrated to exert cardioprotection inanimal models. Schultz et al. (1996) demonstrated in a rat model ofmyocardial ischemia and reperfusion that morphine reduced the sizeof the infarction. Furthermore, Gross et al. (2012) have demonstratedsimilar cardioprotective effects of the enkephalin analog EP94, anendogenous opioid peptide. We have previously shown a similarcardioprotective effect of EP94, on top of morphine, in a porcinemodel (Karlsson et al., 2012). In these studies, EP94 was administratedintracoronary just before and during the early phase of reperfusion, orintravenously with repetitive boluses during the early or late phases ofthe ischemic period. The positive effects of EP94 in terms of reduced

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European Journal of Pharmacology

http://dx.doi.org/10.1016/j.ejphar.2014.11.0180014-2999/& 2014 Elsevier B.V. All rights reserved.

n Correspondence to: Department of Cardiology, Sahlgrenska University Hospital,SE-413 45 Gothenburg, Sweden. Tel.: þ46 31 342 75 36; fax: þ46 31 820062.

E-mail address: elias.spanos@vgregion.se (E. Spanos).

European Journal of Pharmacology 747 (2015) 1–6

infarct size have been consistent in different protocols but the magni-tude of the effects have varied, raising the question of optimal timingand dosing regimen of EP94, an issue that has not been properlyresolved.

The aim of the present study was to define the optimal timingof the administration and dosage regimen of EP94 in a rat model ofmyocardial ischemia and reperfusion.

2. Materials and methods

The study was carried out at Experimental Bio Medicine at theUniversity of Gothenburg, Sweden. The local ethical committee on

animal research approved the studies and animal experiments wereperformed in accordance with the European Convention for theProtection of Vertebrate Animals used for Experimental and otherScientific Purposes (Council of Europe No. 123, Strasbourg 1985).

2.1. Animal preparation

Male Sprague–Dawley (SD) rats, weighing between 250–350 g(Taconic, Denmark) were used in the study protocols. All the ratswere housed in cages, four in each, for at least one week before theexperiments. During the acclimatization period the rats wereprovided food and water ad libitum and maintained on a 12:12 h

Fig. 1. (A) Protocol I. EP94 0.5 μg/kg given in two doses 5 and 10 min into the ischemia period versus control. (B) Protocol II. EP94 given at different dose and time intervalsinto the ischemia period versus control. (C) Protocol III. EP94 0.5 μg/kg given in two doses at different time intervals before, during and after 30 min of ischemia period versuscontrol. (D) Protocol IV. EP94 0.5 μg/kg given 15 and 20 min into a prolonged 40-min ischemia period versus control.

E. Spanos et al. / European Journal of Pharmacology 747 (2015) 1–62

light–dark cycle. The room temperature was maintained between21–24 1C with the help of a central ventilation system. At commence-ment of the experiments the animals were sedated by intraperitonealadministration of sodium pentobarbital (60 mg/kg) and placed on aheating pad, intubated and mechanically ventilated with room air(positive pressure of 14 mm H2O and 70 breaths/min) using a rodentventilator (SAR-830/AP). Deep anesthesia was maintained throughoutthe ischemic as well as reperfusion period by intraperitoneal admin-istrations of sodium pentobarbital 30mg/kg every 45 min. Stable andgood anesthetic control was maintained throughout the whole experi-ment evaluated by regularly pain provocation as well as continuousblood and pulse monitoring. The carotid artery was cannulated and acatheter was introduced through which a pressure guide wire wasplaced for continuous monitoring of the arterial pressure by aRadiAnalyzer system (St. Jude Medical, Uppsala, Sweden). A drop inmean arterial pressure (MAP) below 65mmHg was treated withvolume substitution (crystalloid fluid). The right jugular vein was usedfor injection of EP94, drugs or vehicle. Heart rate was monitored by anelectrocardiogram (ECG). The temperature was continuously moni-tored rectally and regulated between 37.170.5 1C.

2.2. Intervention procedure

The heart was accessed through a median sternotomy. After astabilization period of 15 min, the left anterior descending artery(LAD) was encircled by a 6.0 prolene suture one to two millimetersbelow the tip of the left atrial appendage and the ends of thesuture were threaded through a polyethylene tubing (PE-50) toform a snare for reversible coronary artery occlusion. A pale areabelow the suture and ST-T elevation on the ECG confirmed cardiacischemia. At the end of the ischemia the snare was released andreperfusion was characterized by rapid disappearance of cyanosisand vascular blush. Thereafter, the rats were hemodynamicallystabilized for another two hours before kill.

2.3. Determination of area at risk and infarct size

After two hours of reperfusion the LAD was permanentlyligated at the same position as during the ischemia inductionand 1 ml of 2% Evans blue dye was injected through the jugularvein. The heart was then removed and sliced transversely in aplane perpendicular to the apical–basal axis into five sections. Theboarders of the total transverse area and the area at risk, identifiedas the areas not stained by Evans blue were photographed on eachside. All heart slices were then weighed and incubated with 0.8%2,3,5-triphenyltetrazolium chloride (TTC) (Sigma) for 15 min at37 1C. The slices were then once again photographed and infarctedmyocardium was identified as myocardium not stained by TTC(Fig. 3). By planimetry (Image J software version 1.45s, WayneRasband, National Institutes of Health, USA), the total transverseareas, the area at risk, and the infarct size were calculated from allthe slices. By cognition of the areas on both aspects of each slice,mean areas for each slice were calculated. The relative masses ofinfarct size and area at risk were then calculated. An independentperson blinded to the experiments performed the planimetry andcalculations.

2.4. Experimental protocols

The study was performed in four different protocols comprisinga total of 172 rats assigned to 16 different groups. In each protocol,the rats were randomly assigned a treatment group. The rats weresubjected to 30 (protocols I–III) or 40 min (protocol IV) of ischemia,followed by two hours of reperfusion before kill.

2.4.1. Protocol ISixteen rats were randomised into two groups. The rats were

administered intravenous boluses of vehicle (0.9% NaCl) or EP94(0.5 μg/kg) for 5 and 10 min into the ischemic period, respectively(Fig. 1A).

2.4.2. Protocol IIFifty-six rats were randomized into four groups. The rats were

administered intravenous boluses of vehicle (0.9% NaCl), after5 and 10 min of ischemia, 0.5 μg/kg EP94 after 5 and 10 min ofischemia, 1 μg/kg EP94 after 5 min of ischemia or 0.5 μg/kg EP94after 5, 10 and 15 min of ischemia (Fig. 1B).

2.4.3. Protocol IIIEighty rats were randomized into eight groups. Rats in the

control group received neither vehicle nor EP94. In the other groups,EP94 was administered as two intravenous boluses of 0.5 μg/kgeach 5 min apart. In the different groups, the first dose of EP94 wasadministered 5 min before, at the start of, or 10, 15, 20, 25 or 30 mininto the ischemic period or 5 min into the reperfusion period,respectively (Fig. 1C).

2.4.4. Protocol IVTwenty rats were randomized into two groups. The rats were

administered intravenous boluses of vehicle (0.9% NaCl) or EP94(0.5 μg/kg) for 15 and 20 min into a 40 min ischemic period (Fig. 1D).

2.5. Statistical analysis

Data are expressed as medians and percentiles. All data wereanalyzed using SPSS v22. Student's t-test was used for comparisonof hemodynamics between the different groups. A probabilityvalue of Po0.05 was considered statistically significant.

After consulting a statistician Mann–Whitney U-test was usedfor comparisons regarding infarct size as proportion of area at riskbetween intervention groups and control groups. For evaluation ofexperiments with more than one intervention group the result ofthis test was adjusted with Bonferroni's correction.

3. Results

A total of 172 rats were entered into the different protocols. In thedifferent protocols the number of rats in each group was 8 (protocol I),14 (protocol II) and 10 (protocol III and IV). In protocol I no animalswere excluded. In protocol II 8 animals were excluded due topremature death owing to ventricular fibrillation (VF) (n¼2), asystole(n¼4) or failed coronary occlusion (n¼2). In protocol III 13 animalswere excluded due to premature death owing to ventricular fibrilla-tion (VF) (n¼8), asystole (n¼3) or failed coronary occlusion (n¼2). Inprotocol IV 2 animals were excluded due to premature death owing toventricular fibrillation (VF) (n¼1) or failed coronary occlusion (n¼1).These animals were excluded from the analysis. Failed coronaryocclusion presented as reduced paleness during ischemia and verysmall or no infarction at TTC staining. A person blinded to groupallocation decided which animals to exclude for this reason. Thus, 149rats were included in the data analysis.

3.1. Hemodynamics

There were no significant differences in heart rate, mean arterialblood pressure or temperature between any of the groups at baseline,15 and 30 min of ischemia, 15 min of reperfusion, or at the end of theexperiment (Table 1). Blood gases were randomly analyzed and werewithin the normal range throughout the experiments Table 2.

E. Spanos et al. / European Journal of Pharmacology 747 (2015) 1–6 3

3.1.1. Protocol IEP94, 5 and 10 min into the ischemic period significantly

reduced infarct size as a proportion of area at risk compared withsaline (Fig. 2A).

3.1.2. Protocol IIA reduction of infarct size as a proportion of area at risk could

be demonstrated by EP94 compared with saline if EP94 wasadministered as two doses 5 and 10 min after ischemia inductionbut not when given as a single dose. When EP94 was given asthree separate doses 5, 10 and 15 min into the ischemic period thiseven resulted in significant larger infarct sizes as compared to inthe control group (Fig. 2B).

3.1.3. Protocol IIITwo doses of 0.5 mg/kg of EP94 administered intravenously

5 min apart significantly reduced infarct size as a proportion of

Fig. 2. Results of different doses and times of administration of EP94 compared to control treatment with vehicle. The results of the experiments performed according toprotocol I are presented in (A), protocol II in (B), protocol III in (C) and protocol IV in (D). Asterisk (n) denotes significance at the Po0.05 level.

Fig. 3. TTC stain of the heart. The pale area represents the infarcted myocardium.

E. Spanos et al. / European Journal of Pharmacology 747 (2015) 1–64

area at risk. There was a time-dependant reduction, with the bestcardioprotective effect observed with earlier administration of thedrug (Fig. 2C). The largest reduction was seen with the first dosegiven 5 min before ischemia. There was no statistically significantbenefit from EP94 compared with saline if EP94 was given 10 minor later into the ischemic period.

3.1.4. Protocol IVWhen EP94 was given as two intravenous boluses 15 and

20 min into a 40-min ischemic period there was a borderlinesignificance in the reduction of infarct size as a proportion of areaat risk compared with vehicle. The differences between groupswere, however, of the same magnitude as in protocol I. (Fig. 2D).

4. Discussion

The results of the present study confirm that EP94 reducesinfarct size in an in vivo rat model of myocardial ischemia andreperfusion. The maximum reduction of infarct size was observedafter an intravenous administration of two boluses of 0.5 μg/kg ofEP94 given 5 min prior to, and at the onset of, a 30-min period ofischemia. The effect was gradually lost the later into the ischemicperiod EP94 was administered. To our knowledge, this highly timeand dose regimen dependency has not previously been shown.

In order to validate our model we initially repeated theprotocol by Gross et al. (2012). We could reproduce their findingswith a beneficial cardioprotective effect of EP94 given in a two-dose regimenwith 0.5 μg/kg of EP94 in each dose. In the next step,we could demonstrate a lack of effect when EP94 was given as asingle dose of 1 μg/kg or as a triple dose regimen with 0.5 μg/kg ineach dose. The latter regimen compared with vehicle even seemedto increase infarct size. It cannot be ruled out that the latterregimen was too high since Gross et al. (2012) also demonstratedthat the dose–response relationship may follow a U-formed curve,meaning that there may be an optimal dose and that both lowerand higher doses attenuate the benefits.

Previous animal studies with numerous substances targetingdifferent possible mechanisms for reperfusion injury have shownpromising results and a significant reduction in infarct size whengiven before, during or after the ischemic period. Yao and Gross,1994 showed that intracoronary administration of adenosine beforethe onset of cardiac ischemia in dogs significantly reduced infarctsize and these results were similar to those obtained after ischemicpreconditioning . On the other hand, Jonassen et al. have shown thatglucose–insulin–potassium administrated at reperfusion reducesinfarct size in the in vivo rat heart but that there is no reductionin infarct size when glucose–insulin–potassium is administeredbefore and under the ischemic period (Jonassen et al., 2001). In1991, Higginson showed in a canine model of ischemia/reperfusionthat intracoronary diltiazem given during occlusion or just beforereperfusion increases the salvage of myocardium compared with thesalvage achieved by reperfusion alone (Brieger et al., 2000; Gross etal., 2004; Higginson et al., 1991).

These positive effects have not, however, been observed whenthese substances were tested in clinical trials in patients withacute myocardial infarction (AMI), but were observed in patientspre-treated with these drugs before coronary artery bypass graft-ing surgery (CABG) (Boyce et al., 2003; Mentzer et al., 2008). Apossible explanation may be that these substances are not effec-tive against ischemia and reperfusion injury in the case of AMI inhumans. Another explanation for the inconsistent results may bethat drugs may not reach the area at risk in sufficiently highconcentrations prior to reperfusion when given either intrave-nously or intracoronary shortly before reperfusion.

Furthermore, this concept is supported by pre-clinical studieswith NHE-inhibitors. Rohmann et al. found that the selective NHE-1isoform inhibitor HOE 694 significantly reduced infarct size in pigswhen administered 15 min before occlusion and 15 min before theonset of reperfusion, although the cardioprotective effect was greaterin the pre-treated group (Rohmann et al., 1995). Other studies withNHE inhibitors have shown similar results (Guminaet al., 1998). In recent years, studies on FX06 and cyclosporine Ahave shown some, but rather marginal, effects and the δ-proteinkinase C inhibitor KAI9803 conferred no protection in clinical studiesin patients with acute myocardial infarction. In these studies, thepharmacological agent was administered intravenously or intracor-onary immediately before or after reperfusion (Bates et al., 2008).

There may be some different mechanisms behind a timedependent effect of EP94 administration. First, even in case of acoronary occlusion that reduces circulation and causes ischemia

Table 1Mean arterial pressure (MAP)a during experiment in all study groups. Data aremean7S.D.

Protocol I Preischemia Ischemia150

Ischemia300

Reperfusion150

End

Control 9178 8774 8076EP94 9476 9275 8179

Protocol IIControl 10979 111717 108713 10677 10476EP94 0.5 μg/kg 10879 10477 10379 10579 10578EP94 1 μg/kg 10575 10477 10278 10376 10176EP94 0.3#3 μg/kg 11076 11076 10876 10977 10575

Protocol IIIControl 10876 10778 10774 10974 10472EP94-50þ00 11078 10479 10776 10876 10474EP94 00þ50 11378 105711 10677 10876 10476EP94 100þ150 10877 10275 10675 10976 10274EP94 150þ200 10876 10678 10876 10777 10274EP94 200þ250 10977 10476 10478 10774 10373EP94 250þ300 10978 10578 11074 11075 10574EP94 300þþ50 10978 10777 10478 10975 10475

Protocol IVControl 10778 11078 11177 10976 10275EP94 10677 10976 10976 10774 10175

a The mean arterial pressure is in mmHg.

Table 2Heart ratea during experiment in all study groups. Data are mean7S.D.

Protocol I Preischemia Ischemia150

Ischemia300

Reperfusion150

End

Control 29778 297715 297723EP94 297711 29877 29575

Protocol IIControl 294710 297716 293717 294712 292719EP94 0.5 μg/kg 292713 294712 293711 291710 293713EP94 1 μg/kg 29177 29277 29377 29575 29679EP94 0.3#3 μg/kg 298713 298711 29879 29379 294710

Protocol IIIControl 286711 28679 290710 28878 28879EP94 -50þ00 293711 29379 29477 29577 29476EP94 00þ50 28779 28877 29277 29078 29174EP94 100þ150 28976 29078 29076 29174 29275EP94 150þ200 287710 29079 28975 29077 29377EP94 200þ250 28878 29079 29079 29078 29077EP94 250þ300 287711 29179 293711 294711 29276EP94 300þþ50 290714 291712 294712 29379 29377

Protocol IVControl 29279 29379 29479 29378 28875EP94 29177 29377 29476 29277 28977

a Heart rate is referred as beats per minute (BPM).

E. Spanos et al. / European Journal of Pharmacology 747 (2015) 1–6 5

and necrosis some circulation may be maintained through smallcollaterals or by a retrograde route (Rivas et al., 1976). This mayallow for some penetration of a cardioprotective compound in atime dependent manner. Second the mechanisms of EP94 are notclearly defined. Our previous findings in a porcine model of acardioprotective effect of local intracoronary administration at thetime of reperfusion may speak in favor of a direct effect inmyocardial tissue. The molecular targets may well be the opioidreceptors that have been demonstrated in the myocardium by ourgroup and others (Karlsson et al., 2012). Findings by Gross et al.(2012) may however, also indicate that a central nervous mechan-ism may be involved which may also be compatible with a timedependent effect of administration. Further studies are needed tosort out this issue.

Our results show that it is crucial at what time point EP94 isadministered. A borderline statistical significant reduction afterlater administration of EP94 into a prolonged ischemic period mayspeak in favor of the notion that time to reperfusion is critical.

This is, however, our study is based on only one experimentand has to be verified in larger studies exploring different admin-istration times in relation to different times of ischemia.

5. Conclusion

In the present study, an intravenous two-bolus regimen of theenkephalin analog EP94 exerted cardioprotective effects in anischemia reperfusion rat model that was not seen after single ortriple dose regimens. Furthermore this cardioprotective effect washighly time-dependent with respect to timing of EP94 adminis-tration. The results indicate that time to reperfusion as well astime on ischemia might be important. However, this issue war-rants further studies.

Acknowledgments

The study was financed by grants from the Swedish state underthe agreement between the Swedish government and the countycouncils concerning economic support for research and the educa-tion of doctors (ALF-agreement) and the Swedish Heart-LungFoundation (ALFGBG-150351). EP94 was provided by Eribispharmaceuticals.

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