Charge is an important determinant of hemodynamic and adverse cardiovascular effects of cationic drugs

Pharmacological Research 102 (2015) 46–52

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Charge is an important determinant of hemodynamic and adverse cardiovascular effects of cationic drugs Michael K Pugsley a,∗ , Simon Authier a , Michael J Curtis b a b

CiToxLAB Research Inc., 445 Armand Frappier, Laval, QC H7V 4B3 Canada Cardiovascular Division, King’s College London, Rayne Institute, St Thomas’ Hospital, London SE17EH, UK

a r t i c l e

i n f o

Article history: Received 15 July 2015 Received in revised form 9 August 2015 Accepted 9 September 2015 Available online 11 September 2015 Keywords: Cationic Hypotension Cardiodepressant QT Hemoconcentration Protamine Hexadimethrine Tetraethylammonium Poly-l-lysine

a b s t r a c t Cationic compounds are diverse and atypical therapeutic substances. In the present study we examined whether a prototypical class effect of cationic drugs in the cardiovascular system exists and whether this might be predictable on the basis of chemistry. The dose-dependent effects of cationic compounds of varying molecular weights and charge were examined on the blood pressure (BP), heart rate (HR) and the ECG in anesthetized rats. The compounds examined were protamine, hexadimethrine, tetraethylammonium (TEA), low molecular weight poly-L-lysine (LMW-PLL) and high molecular weight PLL (HMW-PLL). All of the compounds examined except TEA produced a dose-dependent reduction in BP. No changes occurred in HR even when high doses were administered. The ECG effects of these cationic compounds included a dose-dependent prolongation of the QT interval, especially at higher doses. All compounds transiently decreased the size of the P-wave after i.v. bolus administration whereas only protamine and hexadimethrine prolonged the PR and QRS intervals and only at the highest dose (32 mg/kg) administered. All cationic compounds, except TEA and saline, evoked ventricular premature beats (VPB), and protamine and HMW-PLL also evoked brief episodes of ventricular tachycardia (VT). The incidence and frequency of arrhythmias was not dose-dependent and no animals experienced protracted episodes of arrhythmia incidence. These dose dependent effects of the polycationic compounds tested suggest a collective mechanism of action that relates the effect of charge of the compound to the onset and persistence of observed cardiovascular toxicity, and adverse cardiovascular effect risk appears to be predictable on this basis. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction In the present study we evaluated the cardiovascular actions of a range of cationic compounds in order to explore the possible existence of class effects (potentially beneficial or adverse). Although many cationic compounds have been developed they have a disparate spectrum of use within medicine and no ‘class effect’ whether advantageous or adverse has yet been characterized. The most widely-used cationic compound is protamine, a highly basic, polycationic protein that neutralizes the acidic charge of heparin and thus reverses its anticoagulant effects [1,2,3]. Protamine also exhibits adverse effects on the cardiovascular system including hypotension, vascular leak, bradycardia [4] and reduced contractility [5,6,7]. Furthermore, in vivo studies show that

∗ Corresponding author. E-mail address: [email protected] (M.K. Pugsley). http://dx.doi.org/10.1016/j.phrs.2015.09.008 1043-6618/© 2015 Elsevier Ltd. All rights reserved.

protamine dose-dependently elicits hypotension, alters the ECG and may cause arrhythmias [8,9,10]. A number of cationic compounds have been developed as pharmacological tools or as marketed products. Tetraethylammonium (TEA) is a quaternary ammonium compound that was originally used as a ganglion blocker [11] and then as a pharmacological research tool to characterize the neuromuscular junction as well as function of sympathetic nerve terminals in autonomic pharmacology. Studies have shown that TEA blocks voltage-gated neuronal potassium channels [12,13] such as the delayed rectifier family of currents (Kv1.x) with a range of potencies [14]. Hexadimethrine (Polybrene) is a cationic polymer used by researchers to improve the efficiency of retroviral cellular infection rates in cell culture [15,16] and can also be used to transfect mammalian cells with DNA [17]. PLL is a highly basic, cationic homo-polypeptide where the charge depends upon the molecular weight (or number of repeating units of L-lysine). Like hexadimethrine, it can be used as an attachment factor for histochemical procedures. PLL can be used to augment the electrostatic interaction that develops between

M.K. Pugsley et al. / Pharmacological Research 102 (2015) 46–52 Table 1 Some physicochemical properties and molecular charge of cationic compounds. MW (g/mol) TEA 130 Hexadimethrine 374 Protamine 5,500–13,000a LMW-PLL 5,500–15,000a HMW-PLL >30,000a

Charge

Dose Range (mg/kg) Reference(s)

+1 +58 ∼ =+21–65 ∼ =+25–70 ≥+144

1–128 1–32 1–32 1–32 1–8

[13] [73,74] [1,10,75] [76,77] [18,77]

a Molecular weight is based on viscosity determinations by the manufacturer (Sigma–Aldrich, LLC) according to Yaron & Berger [78]. All polyamino acid molecular weights are an average molecular weight and not absolute. PLL charge is determined from literature sources and is also based upon the number of L-lysine repeating units as per Alamanda Polymers (http://www.alamanda-polymers.com/ ). TEA = tetraethylammonium; LMW-PLL = Low Molecular Weight Poly-L-Lysine; HMW-PLL = High Molecular Weight Poly-L-Lysine.

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administered as bolus doses through the jugular vein cannula. Test article dosing began 5 min after blood samples were obtained for determination of the pre-dose (i.e., time 0) packed red cell volume. Cumulative dose-response curves were constructed for each compound until a maximum tolerated dose was achieved (Table 1). The maximum tolerated dose achieved was defined as that resulting in a mean BP reduction to 25 mmHg or marked conduction difficulties in the heart resulting in arrhythmias. If neither effect occurred the dosing was terminated once the required dose volume of a single dose of injected test article exceeded 1.5 mL. ECG changes, BP and HR were monitored for 10 min after each bolus dose, whereupon the next dose was administered. At the end of dosing, a second approximate 0.5 mL sample of whole blood was collected for determination of packed red cell volume.

the negatively-charged cell membrane protein components and positively-charged cell culture surface [18]. Like most cationic proteins, PLL is polydisperse meaning that in aqueous solution it is a composition of multiple molecules in solution. There is some indication that cytotoxicity of cationic compounds is correlated with molecular size and charge [19,20] a phenomenon that is typically observed with common cytotoxic drugs [21]. However a class profile has yet to be elaborated. In the present study we characterized the dose-dependent effects of several cationic compounds of varying MW and molecular charge on BP, HR and the ECG of anesthetized rats. The compounds examined were protamine, hexadimethrine, TEA, LMW-PLL (5.5–15 kDa) and HMW-PLL (>30 kDa).

The packed red blood cell volume was determined before test article administration and upon completion of the dosing interval [10]. Approximately 50 ␮L of whole blood was collected using non-heparinized glass microcapillary (hematocrit) tubes. The tubes were placed in Seal-Ease® , a microhematocrit tube sealer and holder (Becton Dickinson, Franklin Lakes, NJ) at room temperature for 15 min before being microcentrifuged using an IEC MB microcentrifuge (Damon/IEC, Needham Heights, MA). The packed red blood cell volume of the sample was determined from the 50 ␮L sample of whole blood collected using a CRITOCAPSTM microhematocrit capillary tube reader (Oxford Labs, St. Louis, MO).

2. Materials

6. Arrhythmia analysis

Male Sprague-Dawley rats (Charles River Labs, Hollister, CA) weighing 200–300 g were used for studies. All studies conducted were performed according to the guidelines established by the Institutional Animal Care & Use Committee (IACUC), the American Association of Laboratory Animal Sciences (AALAS) [22] and the EU Directive 2010/63/EU. The study design and animal ethics conform with ARRIVE [23] and more recent guidance on experimental design and analysis [24]

Arrhythmias were categorized according to guidelines established by the Lambeth conventions [26,27]. VPBs were defined as single QRS complexes which occurred before any identifiable P wave. Doublets (bigeminal) or salvo (runs of 2 or 3 VPBs) variations in the single complex were not classed as distinct arrhythmias but rather were summed for each group [26]. VPB occurrence (number of VPBs per animal) was log10 transformed to a normal distribution [28]. VT was defined as 4 or more consecutive VPBs and not sub-classified according to rate. VT incidence was classified by characteristic changes in ECG morphology, typically accompanied by step function elevation in HR and fall in mean BP. Ventricular fibrillation (VF) was defined as a sequence of at least 4 consecutive ventricular complexes without intervening diastolic pauses, in which the intrinsic shape, peak–peak interval and height vary, and the variation between each is non-progressive [26].

3. Animal preparation In vivo dose-response curves were constructed (Table 1). Male Sprague-Dawley rats were randomly selected weighed and administered an intraperitoneal injection of anesthetic (65.0 mg/kg sodium pentobarbital at dose volume of 1.0 mL/kg). The trachea was cannulated for artificial ventilation (at a stroke volume of 12 mL/kg and rate of 60 strokes/min) and polyethylene catheters were implanted into the right jugular vein and left carotid artery of each animal. Cannulation of the right jugular vein allowed for administration of test articles while cannulation of the left carotid artery allowed for recording of BP and whole blood collection. The ECG was recorded in a Lead II configuration [25]. Silver wire needle electrodes were placed along the anatomical axis (right atrium to apex) of the heart as determined by palpation. All animals were monitored post-surgically prior to administration of test articles, for approximately 10 min. Animal body temperature was maintained between 35 and 38 ◦ C using a heating pad and rectal thermistor (Digital Long Probe Thermometer, Nasco, Fort Atkinson, WI) 4. Dose administration Animals randomly received either an intravenous (i.v.) injection of 0.9% phosphate buffered saline (PBS) or cationic test article

5. Packed red cell volume (Hematocrit)

7. Drugs Tetraethylammonium (PubChem CID: 5413), Protamine (CAS Number: 9007-31-2), hexadimethrine (1,3-dibromopropane;N,N,N ,N -tetramethylhexane-1,6-diamine; PubChem CID: 24769), LMW-PLL (Poly[imino](2S)-2-amino-1oxo-1,6-hexanediyl; PubChem CID: 53628747) and HMW-PLL (Poly[imino](2S)-2-amino-1-oxo-1,6-hexanediyl; CAS Number 26124-78-7) were purchased from Sigma–Aldrich Co. LLC (St. Louis, MO). Note that the PLL molecules used in these studies are positively charged amino acid polymers with approximately one HBr or HCl per lysine residue. The HBr or HCl allows the PLL to be in a crystalline form soluble in phosphate buffered saline (PBS). All drugs were dissolved in PBS immediately prior to administration. Table 1 provides details on some physicochemical and charge properties of the cationic compounds selected for evaluation. These cationic compounds have only minimal pharmacology literature available (Table 1) but were easily purchased for use.

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marked dose-dependent reductions in BP. Maximal reductions in BP were greater with more potivively-charged compounds at maximal doses administered (Table 2). At a maximal dose of 32 mg/kg, HMW-PLL reduced BP by 71% (from 101 ± 7.8 mmHg pre-drug to 29 ± 4.2 mmHg). Both protamine and hexadimethrine showed comparable dose-dependent profiles of reduction in BP (−55 ± 10 and −65 ± 18 mmHg, respectively). LMW-PLL slightly elevated BP (∼11–17%) while high doses of TEA reduced blood pressure by ∼27%. Table 2 shows the maximal change in BP (MaxBP) observed after dosing each cationic compound. When polycationic compounds caused marked reductions in blood pressure this was not necessarily accompanied by compensatory changes in HR. HR was actually reduced at the maximal doses given (Table 1) for HMW-PLL (from 405 ± 21 pre-drug to 331 ± 29 beats/min), protamine (from 395 ± 28 pre-drug to 360 ± 16 beats/min) and hexadimethrine (from 391 ± 25 pre-drug to 314 ± 8 beats/min). LMW-PLL did not alter HR (from 383 ± 11 pre-drug to 391 ± 12 beats/min) while TEA increased HR only at the maximal dose given (from 398 ± 11 pre-drug to 430 ± 18 beats/min).

Fig. 1. The dose-related changes in blood pressure produced by i.v. administration of the cationic compounds protamine (䊐), hexadimethrine (O), TEA (), low molecular weight poly-L-lysine () and high molecular weight poly-L-lysine () in anesthetized rats. Values are mean ± SEM for n = 7 animals/group. *Indicates a statistically significant difference from pre-dose values (p < 0.05). Vehicle administration had no effect on BP.

8. Statistical analysis Values are shown as the mean ± s.d. for group sizes (n = 7). Statistical analyses were performed using StatView® (SAS Institute Inc., Cary, NC) at an ␣-level of 0.05. A repeated measures analysis of variance (ANOVA) and (if F reached significance and there was no variance in homogeneity) subsequent Dunnett’s multiple comparison test were used for in vivo studies. For arrhythmia quantification, the VPB number was log10 transformed prior to statistical analysis. Mainland’s contingency tables were used to determine significance between the incidences of events in different arrhythmia groups [29].

9.3. Effect of cationic compounds on ECG parameters High doses of the cationic compounds produced similar effects on the ECG. None of the compounds examined, except hexadimethrine and protamine, produced significant changes in either the PR interval (Fig. 2A) or QRS width (data not shown). Hexadimethrine significantly prolonged the PR interval from 54 ± 3 ms pre-drug to 64 ± 2 ms and the QRS width from 31 ± 2 ms pre-drug to 39 ± 2 ms at a dose of 32 mg/kg. Protamine also significantly prolonged the PR interval from 57 ± 5 ms pre-drug to 74 ± 6 ms and the QRS width from 33 ± 2 ms pre-drug to 40 ± 2 ms at the same dose. There was a dose-dependent prolongation of the QT interval (Fig. 2B), indicative of a delay in ventricular repolarization by all the polycationic compounds, especially at higher doses. Fig. 2B shows that the cationic compounds prolonged the QT interval between 12% (HMW-PLL) and 45% (TEA) at the highest doses administered. No changes in the R-wave voltage were observed with the cationic compounds tested at any doses examined. Vehicle administration had no effect on ECG variables.

9. Results 9.1. Effect of cationic compounds on packed red blood cell volume Previous studies have shown that the percentage of packed red cell volume in rats administered cationic compounds may be elevated due to reduced vascular resistance between endothelial cells resulting in vascular leak [30,31]. All cationic compounds, except TEA, increased the packed cell volumes from pre-dose levels by ∼20% (Table 2) suggesting a reduced plasma volume in all dose groups. The vehicle control (saline) did not affect packed red cell volume.

9.4. Administration of cationic compounds produces arrhythmias High doses of the cationic compounds produced a transient reduction of the P-wave size (Fig. 3A–C). Arrhythmias occurred with greater frequency after administration of higher doses of cationic compounds (Table 3). Arrhythmias were ectopic in nature (Fig. 3B) and manifest as either single, bigeminal or runs of 2 or 3 consecutive VPBs (defined as a salvo, Fig. 3D) [24]. Protamine and HMW-PLL produced brief episodes of VT (Table 3). While no VF was observed it appears from the data in Table 2 that the more severe the arrhythmia (i.e., greater number of VPBs and occurrence of VT) the greater the charge of the cationic compound.

9.2. Effect of cationic compounds on BP and HR The vehicle control did not affect BP or HR for the duration of the dosing intervals (data not shown) permitting drug effects to be determined as versus pre-drug values. All of the cationic compounds tested produced a reduction in mean arterial BP as has been shown with synthetic protamine-like peptides of varying cationic charge (+8 to +21) [3]. Fig. 1 shows that bolus doses of protamine, hexadimethrine and HMW-PLL all produce Table 2 The effects of cationic compounds on packed red cell volume and blood pressure. Group Saline TEA Hexadimethrine Protamine LMW-PLL HMW-PLL

Max Dose (mg/kg) 0 128 32 32 32 8.0

RCV Pre-Dose (%) 43 40 42 41 43 42

± ± ± ± ± ±

2.3 1.5 2.8 1.2 1.8 1.9

RCV Post-Dose (%) 46 45 52 49 49 51

± ± ± ± ± ±

1.6 3.4 3.2a 1.4a 1.0a 2.9a

Max BP (mmHg) 0 −27 ± 7.0 −55 ± 10 −65 ± 18 −10 ± 14 −71 ± 15

a Indicates a statistically significant difference from pre-dose values (p < 0.05). Vehicle administration (saline) had no effect on red cell volume. TEA = tetraethylammonium; MW = molecular weight; LMW-PLL = Low Molecular Weight Poly-L-Lysine; HMW-PLL = High Molecular Weight Poly-L-Lysine; RCV = red cell volume; BP = blood pressure.

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Fig. 2. The dose-related effect of the cationic compounds protamine (䊐), hexadimethrine (O), TEA (), low molecular weight poly-L-lysine () and high molecular weight poly-L-lysine () on the (A) the PR and (B) the QT interval in anesthetized rats. Values are mean ± SEM for n = 7 animals/group. *Indicates a statistically significant difference from pre-dose values (p < 0.05). Vehicle administration had no effect on the QT interval.

Pre-dose

Hexadimethrine (8mg/kg)

Protamine (2mg/kg)

B

A

P-wave VPB

C

D

LMW-PLL (8mg/kg)

HMW-PLL (1mg/kg)

VPB (Salvo)

Fig. 3. Lead II ECG traces recorded before (pre-dose) and 10 s after the administration of hexadimethrine (8 mg/kg, i.v.) (Panel A). In the control ECG there are clear P, Q, R, S, and T waves present. After hexadimethrine there is a transient suppression of P-wave size. Panel B shows the predominant type of arrhythmia observed after a single i.v. dose of protamine (2 mg/kg). Ventricular premature beats (VPB) occur in the presence of reduced atrial P-wave wave size but are not dose-dependent. Panel C shows an example ECG trace after low molecular weight poly-L-lysine (LMW-PLL, 8 mg/kg i.v.) administration and Panel D shows a similar reduction in P-wave size and a run of serial VPB’s (salvo) after high molecular weight poly-L-lysine (HMW-PLL, 1 mg/kg, i.v.). The calibration bars in Panel A indicate 50 ms duration and 1 mV voltage.

Table 3 The administration of cationic compounds produces ventricular arrhythmias. Compound

VPB #

VPB (N)

Log10 VPB

VT # (duration, sec)

VT (N)

Saline TEA Hexadimethrine Protamine LMW-PLL HMW-PLL

0 0 28 110 69 249

0 0 3 5 4 6

1.4 2.0 1.8 2.4

– – – 4 (2.0) – 16 (11)

0 0 0 2 0 2

Note that VPB incidence is the total number of VPB’s that occurred during the entire dosing period for each compound up to the maximum tolerated dose (n = 7). The arrhythmias in all groups are those that resulted before the mean arterial blood pressure was reduced to a level (less than 25 mmHg) which resulted in A–V block. VT is the number and duration of all episodes of ventricular tachycardia observed during the dosing period. VPB (N) and VT (N) are the number of animals within the dose groups experiencing arrhythmias. Vehicle administration (saline) did not produce arrhythmias.

10. Discussion The purpose of this study was to characterize the actions of several chemically diverse cationic compounds on blood pressure, heart rate and the ECG in anesthetized rats. This study shows

that cationic compounds of low and high molecular weight and charge all produce a dose-dependent reduction in BP with limited effects on HR until high doses were administered. The cationic compounds examined did not affect either the PR interval or QRS width; however, they dose-dependently prolonged the QT interval. The cationic compounds, except TEA, also produced irregular, aberrant VPBs.

10.1. Effect of cationic compounds on BP, HR, Packed Red Cell Volume and the ECG Maximum reductions in systemic mean arterial BP were greater with more positively charged compounds (+8 to +21) when a series of protamine-like compounds were synthesized and tested [3]. Needham et al. [20] also showed that the vascular endothelial cell effects observed with polycations were not related to the charge nature of the polycation. The present study confirmed a dose-dependent hypotensive action of positively charged compounds when administered to animals. Our data, when hypotension is examined, shows that HMW-PLL (charge > +144) produced a greater reduction in blood pressure than TEA (charge

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+1) with the remaining cationic compounds producing hypotension similarly according to total charge. This hypotensive effect has primarily been revealed in many different rodent and nonrodent animal species (with highly variable sensitivity) conducted for protamine alone [1,32]. Dogs appear to be the most sensitive non-rodent species that develop protamine-related hypotension while rats, mice, guinea pigs and rabbits require much higher doses (many multiples above the clinical therapeutic dose range) [1]. Hypotension is not consistently produced in humans at the much lower heparin-neutralizing therapeutic doses of protamine (usually ∼1.5 mg per 100 USP units of heparin which should not exceed 1.0–2.0 mg/kg) [1,10,33,34]. Of the cationic compounds examined, only protamine is used clinically, to reverse the effects of heparin in post-surgical cardiac patients [35]. Hexadimethrine was developed as an alternative, novel anti-heparin agent for use in patients with a propensity to develop allergic reactions when given protamine [36,37] but is not readily used in clinical practice today. When these cationic compounds were administered to animals they each elicited a dose-dependent hypotension that was greatest in magnitude for hexadimethrine, HMW-PLL and protamine. Egerton & Robinson [38] found that hexadimethrine produced a more protracted reduction in blood pressure than protamine in dogs. Both TEA and LMW-PLL reduced blood pressure only at high doses (≥30 mg/kg). The hypotension, when it developed in the present study, persisted for the duration of the dosing interval prior to administration of the next higher dose and was a common effect with all the cationic compounds. This effect accords with the findings of Sakharov et al. [39] who show showed that protamine and PLL were ubiquitously bound throughout the luminal layer of the vascular wall and were retained by the tissue for long periods. The manner by which these charged compounds mediate this hypotensive action may reflect a variety of direct and indirect mechanisms but the responses observed are consistent with the literature [1,10,40,41]. The mechanism may be vascular in nature and has been suggested to result from a direct receptor-mediated vasodilatory action on endothelial cells due to activation of the nitric oxide synthase (NOS) enzyme system [7,42,43]. Other studies suggest that the hypotension results from either the release of histamine from basophils and/or mast cells [38,44,45] or from activation of the complement system or synthesis of eicosanoids from the cyclooxygenase pathway [46,47,48]. This mechanism is particularly well characterized in dogs which are highly sensitive to drug-induced histamine release [49]. Doses of cationic compounds that produced hypotension did not stimulate compensatory increases in HR which may be a consequence of a direct action on cardiac contracility [50]. While these may be plausible mechanisms to account for the observed effects on BP, these effects could be simply due to the positive charge nature of the molecules directly imparting an effect on endothelial and myocardial cells where the greater the charge on the cationic compound the greater the effects on the heart and cardiovascular system. A charge-related mechanism could account for the observed hypotension. Direct myocardial depression may be the outcome since Pugsley et al. [10] showed that protamine produced a dosedependent reduction in cardiac output and stroke volume in anesthetized rats but also a direct decrease in systolic pressure and the maximal rate of left ventricular pressure development (+dP/dtmax ) and relaxation (−dP/dtmax ) in Langendorff hearts. These effects in isolated hearts are independent of autonomic nervous system effects (since the Langendorff preparation is an isolated heart) as well as blood and its cellular constituents. These findings corroborate those of many researchers using different in vitro cardiac preparations [8,51,52,53,54]. While the majority of the literature on cationic drugs is focused on protamine, Mackenzie et al. [55] found that intravenous hexadimethrine

also depressed the myocardium. No literature is available that characterizes the effects of LMW-PLL and HMW-PLL on the cardiovascular system. However, our study shows that both reduce blood pressure dose-dependently and are likely to produce effects through charge-related mechanisms, albeit these effects are observed at different potencies. Since large interspecies variability (10–100-fold) has been reported for direct cardiac toxicity associated with polycationic protamine derivatives [56] we selected the anesthetized rat, a well characterized animal model with which to assess the effects of these cationic compounds on the cardiovascular system and limit the potential variability to druginduced hypotension reported when other animal species are used. The cationic compounds tested span a range of charges (+1 to >+144) when compared to standard small molecular therapeutic agents. These compounds electrostatically repel mono- and divalent cations such as sodium, potassium and calcium. This effect is termed ‘charge shielding’ and biophysical studies suggest that multivalent compounds in close proximity to the cell surface microenvironment may alter cellular structure and ion channels involved in cell–cell coupling and maintenance of resting membrane potentials [30,57]. The highly cationic nature of these compounds has been shown to reduce the electrostatic barrier that maintains ionic gradients and prevents the transmural movement of protein across the endothelium, and thus affect multiple physiological systems [30,31]. In this and previous studies with cationic compounds such as protamine, hexadimethrine and polylysine it has been observed that the packed red cell volume is significantly increased after administration [10,31] and protein levels in the lumen of vessels are reduced [30,31,58,59,60]. These findings suggest that cationic compounds may enhance endothelial cell leak by neutralizing outer cellular membrane charge which would result in altered membrane potential while simultaneously affecting cell–cell coupling. A ‘leaky’ cell membrane would cause an increase in cell membrane conductance which likely would affect critical ionic pump exchangers as well as ion channels. There is evidence for this, for example, Yoshida et al. [61] showed that protamine inhibits the Na/K-ATPase membrane exchange pump while polylysine can induce a rapid calcium release from cardiac sarcoplasmic reticulum [62]. Similarly, protamine is a potent, reversible inhibitor of the calcium-activated calcium release channel (ryanodine receptor) and has also been shown to augment the stretch induced calcium transient in human umbilical vein endothelial cells [63,64]. Furthermore, electrophysiology studies show that protamine can directly block cardiac calcium currents by affecting the voltage-dependence of channel activation [65]. 10.2. Effect of cationic compounds on the ECG The i.v. bolus dose administration of these cationic compounds produced effects on several intervals of the ECG. Primarily, the compounds with greater cationic charge (e.g., HMW-PLL and hexadimethrine) produced a transient suppression of the P-wave size after each dose administered. However, all were devoid of effect on the PR interval or QRS width of the ECG, an effect similar to that observed by Goldman et al. [50] for protamine and Kimura et al. [37] for hexadimethrine. The effect is only transient in nature which likely relates to the tight binding of the cationic compound to vascular endothelial glycosaminoglycans, rapid accumulation within the luminal layer of the vessel wall and rapid hepatic clearance mechanisms [10,39]. Repolarization of cardiac muscle occurs as a result of the heterogeneous opening and closing of multiple voltage-gated potassium channels. The transient outward potassium channel (Ito ) is the

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major repolarizing current found in the rat ventricle that has, in the literature, been shown to be sensitive to blockade by TEA [13]. Blockade of this potassium channel by this drug was observed only at high doses (>64 mg/kg); however, similar effects on QT were found for those cationic compounds tested with greater positive charge at lower administered doses (>8 mg/kg) in the present study. Thus, block of Ito may account for the QT prolongation observed in the rat and may be a class effect of cationic compounds. Further work beyond the scope of the present study would be required to test this electrophysiologically. These effects are consistent with previous findings for protamine [10,53]. We note that if one is assessing a drug for a torsades de pointes (TdP) liability, the rat is not an appropriate species owing to lack of functional IKr [66]. However, drugs that target other potassium currents found in the heart such as the inward rectifier (IK1 ), Ito and ATP-dependent (IKATP ) potassium currents all prolong QT in rat hearts [67]. Thus, the rat heart QT interval provides data of interest that can be used to help direct subsequent mechanism studies conducted in other species. Thus, QT prolongation in rabbit, for example, but not rat would imply IKr or IKs is the target responsible for the effect [68]. QT interval in rats is measured uncorrected for HR since in this species the QT/HR relationship is flat over the physiological HR range [68,69]. Thus, the present results suggest that all of the cationic compounds examined may interact with one or more potassium channels expressed in the rat heart.

10.3. Administration of cationic compounds produces ventricular arrhythmias The bolus dose administration of cationic compounds resulted in a transient (at low doses) and longer duration (as associated with the HMW-PLL at higher doses) reduction of the size of the P-wave. While only transient in nature, these effects suggest that the positive charge associated with these compounds based on rank order of effect vs. charge as shown in all the Tables from low charge (+1 for TEA) to high charge (>+144 for HMW-PL), when given at a rapid i.v. bolus dose, modulates conduction of atrial electrical activity but does not suppress either sinoatrial or atrioventricular nodal activity. All of the animals administered the cationic compounds experienced spontaneous, single extra beat arrhythmias (VPBs), except for those given TEA. TEA has been used to pharmacologically characterize K channels based upon their sensitivity to block. It has been specifically shown to have an extracellular binding site and does not produce arrhythmias at high doses [12,70,71]. Protamine and HMW-PLL produced some episodes of VT. While this effect has been reported in the literature, the arrhythmogenic mechanism is not understood. Caplan and Su [8] observed that exposure of isolated cardiac tissue to protamine consistently produced extrasystoles at a concentration one fifth of that producing a reduction of peak developed tension. They found that the atria were more sensitive to protamine effects than the ventricles. Goldman et al. [50] also observed occasional arrhythmias in dogs given protamine. Bruins et al. [48] also showed that in cardiopulmonary bypass (CPB) patients given protamine post-surgically, atrial arrhythmia episodes were more frequently observed. The present study is the first to show that a broad range of cationic compounds can suppress atrial electrical activity and also produce ventricular arrhythmias when given either at low or high bolus doses. Suppression of the atrial P-wave likely results from anomalous A–V excitation due to aberrant complexation of ventricular activation [10]; however, this is only a transient phenomenon as the dose of administered drug becomes rapidly diluted by the circulating blood and is rapidly sequestered by myocardial and vascular tissue [39,72].

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11. Conclusion This study provides the initial basis to predict adverse effects of cationic compounds on the cardiovascular system. All the polycationic compounds examined produced dose-dependent changes in BP and the ECG. Total cationic charge, as we have seen when TEA (+1) effects are compared to HMW-PLL (>+144), may be an important determinant for hemodynamic and arrhythmic toxicities. The hypotensive and QT prolongation observed are common but dose dependent effects of all polycationic compounds tested and suggest a collective mechanism of action that relates the effect of charge of the compound to observed cardiovascular toxicity. Conflict of interest The authors declare that there are no conflicts of interest. References [1] J.C. Horrow, Protamine a review of its toxicity, Anesth. Analg. 64 (1985) 348–361. [2] A. Bouraghda, P. Gillois, P. Albaladejo, Alternatives to heparin and protamine anticoagulation for cardiopulmonary bypass in cardiac surgery, Can. J. Anaesth. 62 (5) (2015) 518–528. [3] A. DeLucia, T.W. Wakefield, P.C. Andrews, B.J. Nichol, A.M. Kadell, et al., Efficacy and toxicity of differently charged polycationic protamine-like peptides for heparin anticoagulation reversal, J. Vasc. Surg. 18 (1993) 49–60. [4] K. Chilukuri, C.A. Henrikson, D. Dalal, D. Scherr, E.C. MacPherson, A. Cheng, D. Spragg, et al., Incidence and outcomes of protamine reactions in patients undergoing catheter ablation of atrial fibrillation, J. Interv. Card. Electrophysiol. 25 (3) (2009) 175–181. [5] N.D. Kien, D.D. Quam, J.A. Reitan, D.A. White, Mechanism of hypotension following rapid infusion of protamine sulfate in anesthetized dogs, J. Cardiothorac. Vasc. Anesth. 6 (2) (1992) 143–147. [6] D. Pevni, I. Frolkis, A. Iaina, Y. Wollman, T. Chernichovski, et al., Protamine cardiotoxicity and nitric oxide, Eur. J. Cardiothorac. Surg. 20 (1) (2001) 147–152. [7] P.J. Pearson, P.R.B. Evora, K. Ayrancioglu, H.V. Schaff, Protamine releases endothelium-derived relaxing factor from systemic arteries. A possible mechanism of hypotension during heparin neutralization, Circulation 86 (1) (1992) 289–294. [8] R.A. Caplan, J.Y. Su, Differences in threshold for protamine toxicity in isolated atrial and ventricular tissue, Anesth. Analg. 63 (12) (1984) 1111–1115. [9] M.K. Pugsley, V. Kalra, S. Froebel-Wilson, K. Aardalen, Cardiovascular and antiarrhythmic properties of protamine in rats, Proc. West. Pharmacol. Soc. 44 (2001) 33–36. [10] M.K. Pugsley, V. Kalra, SF, roebel-W, ilson, Protamine is a low molecular weight polycationic amine that produces actions on cardiac muscle, Life Sci. 72 (3) (2002) 293–305. [11] G.K. Moe, W.A. Freyburger, Ganglionic blocking agents, Pharmacol. Rev. 2 (1950) 61–95. [12] A. Al-Sabi, S.K. Kaza, J.O. Dolly, J. Wang, Pharmacological characteristics of Kv1. 1- and Kv1. 2-containing channels are influenced by the stoichiometry and positioning of their ␣ subunits, Biochem. J. 454 (1) (2013) 101–108. [13] C.M. Armstrong, Interaction of tetraethylammonium ion derivatives with the potassium channels of giant axons, J. Gen. Physiol. 58 (1971) 413–437. [14] G.A. Gutman, K.G. Chandy, S. Grissmer, M. Lazdunski, D. McKinnon, L.A. Pardo, G.A. Robertson, B. Rudy, M. Sanguinetti, W. Stuehmer, X. Wang, International Union of Pharmacology. LIII. Nomenclature and molecular relationships of voltage-gated potassium channels, Pharmacol. Rev. 57 (2005) 473–508. [15] S.A. Homburger, D.M. Fekete, High efficiency gene transfer into the embryonic chicken CNS using B-subgroup retroviruses, Dev. Dyn. 206 (1) (1996) 112–120. [16] S. Kawai, M. Nishizawa, New procedure for DNA transfection with polycation and dimethyl sulfoxide, Mol. Cell. Biol. 4 (6) (1984) 1172–1174. [17] W.C. Shen, H.J. Ryser, Conjugation of poly-L-lysine to albumin and horseradish peroxidase: a novel method of enhancing the cellular uptake of proteins, Proc. Natl. Acad. Sci. 75 (4) (1978) 1872–1876. [18] B.S. Jacobson, D. Branton, Plasma membrane: rapid isolation and exposure of the cytoplasmic surface by use of positively charged beads, Science 195 (4275) (1977) 302–304. [19] L.J. Arnold, A. Dagan, J. Gutheil, N.O. Kaplan, Antineoplastic activity of poly(L-lysine) with some ascites tumor cells, Proc. Natl. Acad. Sci. U. S. A. 76 (7) (1979) 3246–3250. [20] L. Needham, P.G. Hellewell, T.J. Williams, J.L. Gordon, Endothelial functional responses and increased vascular permeability induced by polycations, Lab. Invest. 59 (4) (1988) 538–548. [21] Y. Miyamoto, T. Oda, H. Maeda, Comparison of the cytotoxic effects of the high- and low-molecular-weight anticancer agents on multidrug-resistant Chinese Hamster Ovary cells in vitro, Cancer Res. 50 (1990) 1571–1575.

52

M.K. Pugsley et al. / Pharmacological Research 102 (2015) 46–52

[22] National research council, guide for the care and use of laboratory animals: Eighth edition. Washington, DC: The National Academies Press (2011). [23] C. Kilkenny, W.J. Browne, I.C. Cuthill, M. Emerson, D.G. Altman, Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research, PLoS Biol 8 (2010) e1000412. [24] M.J. Curtis, R.A. Bond, D. Spina, A. Ahluwalia, S.P.A. Alexander, M.A. Giembycz, A. Gilchrist, D. Hoyer, et al., Experimental design and analysis and their reporting: new guidance for publication in BJP, Br. J. Pharmacol. 172 (2015) 2671–2674. [25] W.P. Penz, M.K. Pugsley, M. Hsieh, M.J.A. Walker, A new ECG measure (RSh) for detecting possible sodium channel blockade in vivo in rats, J. Pharmacol. Toxicol. Meth. 27 (1992) 51–58. [26] M.J. Curtis, J.C. Hancox, A. Farkas, C.L. Wainwright, C.L. Stables, D.A. Saint, et al., The Lambeth Conventions (II): guidelines for the study of animal and human ventricular and supraventricular arrhythmias, Pharmacol. Ther. 139 (2013) 213–248. [27] M.J.A. Walker, M.J. Curtis, D.J. Hearse, R.W.F. Campbell, M.J. Janse, D.M. Yellon, S.M. Cobbe, et al., The Lambeth Conventions: guidelines for the study of arrhythmias in ischaemia, infarction, and reperfusion, Cardiovasc. Res. 22 (1988) 447–455. [28] M.J. Curtis, M.J.A. Walker, Quantification of arrhythmias using scoring systems: an examination of seven scores in an in vivo model of regional myocardial ischaemia, Cardiovasc. Res. 22 (9) (1988) 656–665. [29] D. Mainland, L. Herrera, M.I. Sutcliffe, Statistical tables for use with binomial samples—Contingency tests, in: Confidence Limits and Sample Size Estimates, Department of Medical Statistical Publishers, New York, 1956. [30] K.P. Sunnergren, R.P. Fairman, G.G. deBlois, F.L. Glauser, Effects of protamine, heparinase, and hyaluronidase on endothelial permeability and surface charge, J. Appl. Physiol. 63 (5) (1987) 1987–1992. [31] V.M. Vehaskari, C.T. Chang, J.K. Stevens, A.M. Robson, The effects of polycations on vascular permeability in the rat. A proposed role for charge sites, J. Clin. Invest. 73 (4) (1984) 1053–1061. [32] I.A. Michaels, P.G. Baresh, Hemodynamic changes during protamine administration, Anesth. Analg. 62 (9) (1983) 831–851. [33] C.E. Green, C.B. Higgins, M.J. Kelley, W.S. Schmidt, F.H. Haigler, J.D. Newell, Cardiovascular effects of protamine sulfate, Invest. Radiol. 16 (4) (1981) 324–329. ˜ Fernandez, A. Hernandez Fernandez, J.M. Borrego Dominguez, P. [34] A. Ordonez Garcia Tejero, J. Perez Bernal, R. Hinojosa, J. Lopez Hidalgo, The systemic vasodilatory action of protamine: is it inhibited or mediated by heparin, Res. Exp. Med. (Berl) 197 (6) (1998) 337–547. [35] M. Codispoti, P.S. Mankad, Management of anticoagulation and its reversal during paediatric cardiopulmonary bypass: a review of current UK practice, Perfusion 15 (3) (2000) 191–201. [36] A. Cooney, T.J. Mann, Recent experiences with hexadimethrine for neutralizing heparin after cardiopulmonary bypass, Anaesth. Intensive Care 27 (3) (1999) 298–300. [37] E.T. Kimura, P.R. Young, D.M. Ebert, Further studies on hexadimethrine bromide (polybrene)—an antiheparin agent, Tox. Appl. Pharmacol. 1 (1959) 560–575. [38] W.S. Egerton, C.L. Robinson, The anti-heparin, anticoagulant and hypotensive properties of hexadimethrine and protamine, Lancet 2 (1961) 635–637. [39] D.V. Sakharov, A.F.H. Jie, M.E.A. Bekkers, J.J. Emeis, D.C. Rijken, Polylysine as a vehicle for extracellular matrix-targeted local drug delivery, providing high accumulation and long-term retention within the vascular wall, Arterioscler. Thromb. Vasc. Biol. 21 (6) (2001) 943–948. [40] R.W. Frater, Y. Oka, Y. Hong, T. Tsubo, P.G. Loubser, R. Masone, Protamine-induced circulatory changes, J. Thorac. Cardiovasc. Surg. 87 (5) (1984) 687–692. [41] E. Antunes, M. Mariano, G. Cirino, S. Levi, G. de Nucci, Pharmacological characterization of polycation-induced rat hind-paw oedema, Br. J. Pharmacol. 101 (4) (1990) 986–990. [42] L.J. Ignarro, M.E. Gold, G.M. Buga, R.E. Byrns, K.S. Wood, G. Chaudhuri, G. Frank, Basic polyamino acids rich in arginine, lysine, or ornithine cause both enhancement of and refractoriness to formation of endothelium-derived nitric oxide in pulmonary artery and vein, Circ. Res. 64 (1989) 315–329. [43] K. Takakura, M. Mizogami, S. Fukuda, Protamine sulfate causes endothelium-independent vasorelaxation via inducible nitric oxide synthase pathway, Can. J. Anaesth. 53 (2) (2006) 162–167. [44] D. Lagunoff, T.W. Martin, G. Read, Agents that release histamine from mast cells, Ann. Rev. Pharmacol. Toxicol. 23 (1983) 331–351. [45] R.S. Parsons, K. Mohandas, The effect of histamine-receptor blockade on the hemodynamic responses to protamine, J. Cardiothorac. Anesth. 3 (1) (1989) 37–43. [46] R. Rent, N. Ertel, R. Eisenstein, H. Gewurz, Complement activation by interaction of polyanions and polycations, I. Heparin-protamine induced consumption of complement, J. Immunol. 114 (1975) 120–124. [47] J. Hobbhahn, H. Habazettl, P.F. Conzen, K. Peter, Complications caused by protamine. 1: pharmacology and pathophysiology, Anaesthetist 40 (1991) 365–374. [48] P. Bruins, H. te Velthuis, A.P. Yazdanbakhsh, P.G. Jansen, F.W. van Hardevelt, et al., Activation of the complement system during and after cardiopulmonary bypass surgery: postsurgery activation involves C-reactive protein and is associated with postoperative arrhythmia, Circulation 96 (10) (1997) 3542–3548.

[49] S. Qiu, Z. Liu, L. Hou, Y. Li, J. Wang, H. Wang, W. Du, W. Wang, Y. Qin, Z. Liu, Complement activation associated with polysorbate 80 in beagle dogs, Int. Immunopharmacol. 15 (1) (2013) 144–149. [50] B.S. Goldman, J. Joison, W.G. Austen, Cardiovascular effects of protamine sulfate, Ann. Thorac. Surg 7 (1969) 459–471. [51] R.B. Hird, T.W. Wakefield, R. Mukherjee, B.U. Jones, F.A. Crawford, P.C. Andrews, J.C. Stanley, F.G. Spinale, Direct effects of protamine sulfate on myocyte contractile processes cellular and molecular mechanisms, Circulation 92 (9) (1995) 433–446. [52] N. Iwatsuki, S. Matsukawa, K. Iwatsuki, A weak negative inotropic effect of protamine sulfate upon the isolated canine heart muscle, Anesth. Analg. 59 (2) (1980) 100–102. [53] R. Kertesz, M.J.A. Walker, The cardiovascular effects of protamine in rats and isolated rat heart, Proc. West. Pharmacol. Soc. 36 (1993) 53–57. [54] T.W. Wakefield, S.K. Wrobleski, B.J. Nichol, A.M. Kadell, J.C. Stanley, Heparin-mediated reductions of the toxic effects of protamine sulfate on rabbit myocardium, J. Vasc. Surg. 16 (1) (1992) 47–53. [55] G.J. Mackenzie, J.D. Wade, S.H. Davies, S. Zellos, The circulatory effects of hexadimethrine bromide (Polybrene) in dogs, Am. Heart J. 62 (1961) 511–518. [56] L.B. Jacques, A study of the toxicity of the protamine, salmine, Br. J. Pharmacol. 4 (1949) 135–144. [57] W.N. Green, O.S. Andersen, Surface charges and ion channel function, Annu. Rev. Physiol. 53 (1991) 341–359. [58] D.N. Granger, P.R. Kvietys, M.A. Perry, A.E. Taylor, Charge selectivity of rat intestinal capillaries. Influence of polycations, Gastroenterology 91 (6) (1986) 1443–1446. [59] S.W. Chang, J.Y. Westcott, J.E. Henson, N.F. Voelkel, Pulmonary vascular injury by polycations in perfused rat lungs, J. Appl. Physiol. 62 (5) (1987) 1932–1943. [60] C.J. Tzan, J. Berg, S.A. Lewis, Effect of protamine sulfate on the permeability properties of the mammalian urinary bladder, J. Membr. Biol. 133 (3) (1993) 227–242. [61] H. Yoshida, H. Fujisawa, Y. Ohi, Influences of protamine on the Na+ , K+ -dependent ATPase and on the active transport processes of potassium and of L-DOPA into brain slices, Can. J. Biochem. 43 (7) (1965) 841–849. [62] M. Yano, T. Yamamoto, M. Kohno, T. Hisaoka, K. Ono, T. Tanigawa, T. Ueyama, T. Ohkusa, M. Matsuzaki, Polylysine-induced rapid Ca2+ release from cardiac sarcoplasmic reticulum, J. Cardiovasc. Pharmacol. 32 (1) (1998) 96–100. [63] P. Koulen, B.E. Ehrlich, Reversible block of the calcium release channel/ryanodine receptor by protamine, a heparin antidote, Mol. Biol. Cell 11 (7) (2000) 2213–2219. [64] K. Murase, K. Naruse, A. Kimura, K. Okumura, T. Hayakawa, M. Sokabe, Protamine augments stretch induced calcium increase in vascular endothelium, Br. J. Pharmacol. 134 (7) (2001) 1403–1410. [65] W.K. Park, J.J. Pancrazio, C. Lynch, Mechanical and electrophysiological effects of protamine on isolated ventricular myocardium: evidence for calcium overload, Cardiovasc. Res. 28 (4) (1994) 505–514. [66] M.J. Curtis, Characterisation, utilisation and clinical relevance of isolated perfused heart models of ischaemia-induced ventricular fibrillation, Cardiovasc. Res. 39 (1998) 194–215. [67] S.A. Rees, M.J. Curtis, Which cardiac potassium channel subtype is the preferable target for suppression of ventricular arrhythmias? Pharmacol. Therap. 69 (1996) 199–217. [68] S.A. Rees, M.J. Curtis, Selective IK blockade as an antiarrhythmic mechanism in ischaemic heart disease: effects of UK66,914 in rat and rabbit, Br. J. Pharmacol. 108 (1993) 139–145. [69] S.A. Rees, K. Tsuchihashi, D.J. Hearse, M.J. Curtis, Combined administration of an IK(ATP) activator and an Ito blocker increases coronary flow independently of effects on heart rate, QT interval and ischaemia-induced ventricular fibrillation, J. Cardiovasc. Pharmacol. 22 (1993) 343–349. [70] L. Heginbotham, R. MacKinnon, The aromatic binding site for tetraethylammonium ion on potassium channels, Neuron 8 (1992) 483–491. [71] S.A. Doggrell, B.E. Bishop, Effects of potassium channel blockers on the action potentials and contractility of the rat right ventricle, Gen. Pharmacol. 27 (2) (1996) 379–385. [72] J. Butterworth, Y.A. Lin, R. Prielipp, J. Bennett, R. James, The pharmacokinetics and cardiovascular effects of a single intravenous dose of protamine in normal volunteers, Anesth. Analg. 94 (3) (2002) 514–522. [73] R. Hohf, F.W. Preston, O. Trippel, The neutralization of heparin with polybrene, Q. Bull. Northwest Univ. Med. Sch. 30 (2) (1956) 138–143. [74] P. Lalezari, A new method for detection of red blood cell antibodies, Transfusion (Paris) 8 (6) (1968) 372–380. [75] H. Goto, T. Kushihashi, K.T. Benson, H. Kato, D.K. Fox, K. Arakawa, Heparin, protamine, and ionized calcium in vitro and in vivo, Anesth. Analg. 64 (11) (1985) 1081–1084. [76] J. Hiraki, T. Ichikawa, S. Ninomiya, H. Seki, K. Uohama, H. Seki, S. Kimura, Y. Yanagimoto, J.W. Barnett, Use of ADME studies to confirm the safety of epsilon-polylysine as a preservative in food, Regul. Toxicol. Pharmacol. 37 (2) (2003). [77] P.J. Symonds, C. Murray, A.C. Hunter, G. Debska, A. Szewczyk, S. Moein Moghimi, Low and high molecular weight poly(L-lysine) s/poly(L-lysine)–DNA complexes initiate mitochondrial-mediated apoptosis differently, FEBS Lett. 579 (27) (2005) 6191–6198. [78] A. Yaron, A. Berger, The effect of urea and guanidine on the helix content of poly-NS-(3-hydroxyproply)-glutamine in aqueous solvent systems, Biochim. Biophys. Acta 69 (1963) 430–432.