HBK-10, A Compound with α1-Adrenolytic Properties, Showed Antiarrhythmic and Hypotensive Effects in Rats

Arrhythmia, an irregular heartbeat, might be a life-threatening condition but also a risk factor for stroke or worsen the prognosis after myocardial infarction. The limited efficacy and proarrhythmic potential of the available drugs require searching for new, more effective, and safer pharmacotherapies. Studies indicate that the blockade of α1-adrenoceptors could be effective in treating heart rhythm abnormalities. In this study, we aimed to assess the antiarrhythmic and hypotensive potential of HBK-10, a novel 2-methoxyphenylpiperazine derivative, as well as its binding to the selected adrenergic receptors. Radioligand binding studies demonstrated that HBK-10 showed a high affinity for α1 but not for α2 or β1 receptors. Next, we evaluated the ability of HBK-10 to protect against an adrenaline-induced arrhythmia in rats. The compound showed potent prophylactic antiarrhythmic properties in this arrhythmia model. Notably, the compound did not show proarrhythmic potential in normotensive rats since it did not influence the ECG parameters at antiarrhythmic doses. Finally, the compound showed hypotensive properties in rats, which were not observed after coadministration with adrenaline, noradrenaline, or methoxamine, which suggests α1-adrenolytic properties of HBK-10. Our results confirm that compounds with a 2-methoxyphenylpiperazine group show a high affinity for α1-adrenoceptors and a significant antiarrhythmic effect. Given the promising results of our study, further evaluation of HBK-10 is necessary to unravel the mechanisms behind its pharmacological effects and evaluate the safety profile.


Introduction
According to the World Health Organization, cardiovascular diseases are the leading cause of death globally [1]. Approximately 85% of deaths are due to myocardial infarction or stroke [1]. Cardiac-related risk factors, such as heart rhythm disturbances, are often involved in the mechanisms of these two conditions [2]. For example, atrial fibrillation increases fivefold the risk for acute ischemic stroke [3,4]. On the other hand, 90% of patients with acute myocardial infarction have some cardiac rhythm abnormalities [5]. Thus, restoring normal rhythm in these patients is of utmost importance. Antiarrhythmic drugs either block β 1 receptors or influence sodium, potassium, or calcium channels [6]. Unfortunately, the above pharmacotherapy is often ineffective in treating heart rhythm abnormalities [7,8]. Furthermore, antiarrhythmic drugs have proarrhythmic potential, which may increase the risk of a cardiac event [9][10][11]. Thus, searching for novel compounds with different mechanisms of action devoid of proarrhythmic potential is necessary.
Several studies have proved that α 1 -adrenoceptors might play a vital role in arrhythmias [12]. Experiments have shown that during both early ischemia and reperfusion, there is an enhanced responsivity to α-adrenergic stimulation [13,14]. The increased α-adrenergic responsivity may be due to an increase in α 1 receptors in the ischemic myocardium originating from a site distinct from the intracellular site for trafficking of β-adrenergic receptors, possibly within or near the sarcolemma [12]. Interestingly, an α 1 receptor blockade induced a potent antiarrhythmic effect in several species [13][14][15][16][17][18][19][20][21]. Overall, these findings indicate that targeting α 1 -adrenoceptors could be an effective strategy to reduce the incidence of sudden cardiac death due to heart rhythm abnormalities.

HBK-10 Showed a High Affinity for α 1 -Adrenoceptors
The studied compound possessed a high affinity for α 1 -adrenoceptors but not for α 2 or β 1 receptors (Table 1).

HBK-10 Showed a Prophylactic Antiarrhythmic Activity in the Adrenaline-Induced Arrhythmia
The administration of HBK-10 before the arrhythmogen decreased the number of extrasystoles caused by adrenaline injection (ED 50 = 0.125 (0.061-0.256), Table 2, Figure 1). Rats were anesthetized intraperitoneally (ip) with thiopental (75 mg/kg). The tested compound was administered ip 45 min before the experiment. The control group received no treatment except the administration of arrhythmogen. The observation was performed for 15 min after the intravenous (iv) injection of adrenaline (20 µg/kg), i.e., during the first 2 min, and in the 5, 10, and 15th min. Results are presented as a percentage of the occurrence of extrasystoles, n = 6 animals.

HBK-10 Did Not Influence Negatively the Normal Electrocardiogram in Rats
HBK-10 at the dose 10 mg/kg had no effect on PR [F (9,

Discussion
In this study, we found that HBK-10, a novel 2-methoxyphenylpiperazine derivative, possessed a high affinity for α 1 -adrenoceptors. Furthermore, the tested compound elicited antiarrhythmic and hypotensive activity in normotensive rats, and its cardiovascular effects were most likely related to α 1 -adrenolytic properties. Notably, HBK-10 did not affect normal ECG in rodents.
Numerous studies on 2-methoxyphenylpiperazine derivatives indicate their various biological properties, including cardiovascular effects, such as antiarrhythmic or antihypertensive [22][23][24]. Moreover, the compounds containing the 2-methoxyphenylpiperazine group often show a high affinity for the α 1 receptors [28]. The α 1 -adrenoceptor, in turn, might play a vital role not only in the regulation of blood pressure, but also in antiarrhythmic effect [29,30]. Thus, we selected a novel 2-methoxyphenylpiperazine derivative-HBK-10-to determine its binding with the α 1 receptor and the potential antiarrhythmic effect, as well as the influence on blood pressure. The compound in our previous studies demonstrated antagonistic properties at 5-HT 1A and D 2 receptors and the antidepressant-like effect in mice [25].
First, we investigated the compound's affinity towards adrenergic receptors, i.e., α 1 -, α 2 -, and β 1 -adrenoceptors, using radioligand binding assays. Our studies demonstrated that HBK-10 showed a high affinity for α 1 -adrenergic receptors, more robust than that of the reference compound, phentolamine. Compared with the α 1 receptor, its affinities towards α 2 -, and β 1 -adrenoceptors were over 600-or 1000-fold lower, respectively. The radioligand binding studies suggest that among the adrenergic receptors, HBK-10 shows selectivity for α 1 -adrenoceptor. Furthermore, our studies confirmed that compounds containing the 2-metoxyphenylpiperazine group show a high affinity for α 1 receptors. Our results align with our previous experiments [31] as well as other studies on 2-methoxyphenylpiperazine derivatives [28].
Since studies demonstrated a possible role of α 1 -adrenoceptors in the treatment of arrhythmia [23], in the following step, we investigated the potential antiarrhythmic activity of HBK-10 in the model of adrenaline-induced arrhythmia. In this model, adrenaline was used as an arrhythmogen, leading to arrhythmias manifested by extrasystoles, conduction blocks, bradycardia, and animal mortality. The tested compound showed prophylactic antiarrhyth-mic activity, reducing the number of post-adrenaline extrasystoles. It is worth highlighting that the calculated ED 50 value for HBK-10 administered intraperitoneally was almost 3-fold lower than for carvedilol administered intravenously (ED 50 = 0.36 mg/kg) [24]. Our results are even more promising given that compared with iv injection, after ip administration, a drug is subject to a hepatic first-pass metabolism, which decreases its systemic exposure. Moreover, as carvedilol is a potent β 1 -and α 1 -adrenoceptors blocker [32] and HBK-10 targets only α 1 -type adrenergic receptors, our results suggest that the α 1 -adrenoceptors might play a more significant role in the adrenaline-induced arrhythmia model than the β 1 -adrenoceptors. Since no fully effective antiarrhythmic drug is on the market, our results encourage further studies on HBK-10 as a model structure for synthesizing more effective drugs to treat heart rhythm disturbances.
Unfortunately, the available antiarrhythmic drugs show proarrhythmic potential that can manifest by the occurrence of torsades de pointes, an increased number of premature heartbeats, or reentry [33][34][35]. In particular, elderly patients with impaired heart muscle contractility and patients with electrolyte disturbances are susceptible to arrhythmogenic effects of antiarrhythmic drugs [36,37]. Therefore, next, we evaluated the influence of HBK-10 on normal ECG in rats. Our experiments revealed that HBK-10 influenced normal ECG at the dose 160-fold higher than the median effective dose in the adrenaline-induced arrhythmia model. The tested compound shortened the calculated QTc interval and decreased heart rate. The QT interval reflects depolarization and repolarization phases in the myocardium and its shortening indicates that the studied derivative may, to some extent, disrupt the heart rhythm at higher doses. The negative chronotropic effect of HBK-10 may be associated with the blockade of α 1 -receptors. However, considering that the dose necessary to disturb normal ECG is 160-fold higher than the antiarrhythmic dose, we can conclude that the compound did not show proarrhythmic potential at pharmacologically active doses.
Since α 1 -adrenergic receptors regulate blood pressure and α 1 -adrenolytics are being used in the treatment of hypertension [38], we next evaluated the effect of HBK-10 on systolic and diastolic blood pressure after a single intraperitoneal administration in normotensive rats. Our experiment demonstrated that HBK-10 reduced blood pressure at the dose 10-fold higher than the median effective dose in the adrenaline-induced model. The results suggest that the antiarrhythmic effect of HBK-10 requires lower doses than the hypotensive effect. The fact that these effects can be separated is beneficial. Compounds with such properties could be used in a larger group of patients, i.e., in patients with heart rhythm irregularities with or without hypertension.
Radioligand binding studies indicated the strength of a compound's affinity for a given receptor without specifying the type of interaction. To prove the agonistic or antagonistic effect toward α 1 -adrenoceptors, we conducted the experiment with catecholamines. The inhibition of the pressor response to epinephrine, norepinephrine, or methoxamine by the pretreatment with the studied compound at the lowest hypotensive dose indicates its α 1 -adrenolytic properties. HBK-10 decreased the effect induced by epinephrine, norepinephrine, and methoxamine, suggesting that its hypotensive effect is due to the blockade of α 1 -adrenoceptors. However, we need to emphasize that HBK-10 targets serotonin 5-HT 1A and dopamine D 2 receptors, which also regulate blood pressure. Therefore, the observed overall effect on blood pressure might result from the interaction with several receptors.
Considering the results of our experiments, the hypotensive effect of HBK-10 depended on the blockade of α 1 -adrenoceptors. Several studies showed significant antiarrhythmic effects of α 1 -adrenolytics [23,39,40]. Since HBK-10 showed a high affinity for the α 1 -adrenoceptors and negligible affinity for the β 1 -adrenoceptors, we may suspect that the observed antiarrhythmic effect was due to the interaction with α 1 -adrenoceptors. Nevertheless, to prove our hypothesis, we need further studies, as HBK-10 is a multimodal compound, and the observed antiarrhythmic effect might also be associated with the interaction with other biological targets.
Limitations to our study include assessing the antiarrhythmic and hypotensive activity of HBK-10 only after an acute administration. The repeated administration would verify the tested compound's long-lasting effects on preventing attacks of arrhythmia, as well as increases in blood pressure in animal models. Moreover, to unravel the full antiarrhythmic potential of HBK-10, its activity in other arrhythmia models needs further investigation. Similarly, to understand the mechanism of action of HBK-10, electrophysiological studies should be performed and more potential molecular targets (e.g., ion channels) should be investigated in future studies.

Animals
The experiments were performed on male normotensive Wistar rats, weighing 200-250 g. Animals were kept in standard plastic cages (42.7 cm × 26.7 cm) in groups of 3, at constant room temperature (22 ± 2 • C), on a 12 h light/dark cycle with ad libitum access to food and water. Each experimental and control group consisted of six animals. All injections were administered in a volume of 1 mL/kg. Rats were used only once in each test and immediately after each experiment, animals were euthanized.

Prophylactic Antiarrhythmic Activity in Adrenaline-Induced Arrhythmia
The experiment was carried out according to the method described by Szekeres and Papp [41]. Normotensive rats were anesthetized with thiopental (75 mg/kg ip). Adrenaline was administered at the dose of 20 µg/kg to induce heart rhythm disturbances. The studied compound was injected intraperitoneally 45 min before the arrhythmogen. The electrocardiogram (ECG) was recorded during the first 2 min and in the 5th, 10th, and 15th min after the adrenaline injection. The criterion of prophylactic antiarrhythmic activity was the lack of extrasystoles and inhibition of cardiac arrhythmia in comparison with the control group. The ED 50 (a dose producing a 50% inhibition of ventricular contractions) was determined by computer log-probit analysis according to Litchfield and Wilcoxon [42].

Effect on a Normal Electrocardiogram in Rats
To verify the influence of the tested compound on the normal electrocardiogram (ECG), the experiment was performed. Aspel ASCARD apparatus (standard II lead, with tape speed 50 mm/s and voltage calibration 1 mV = 1 cm) was used for ECG measurements. Normotensive rats were anesthetized with thiopental (75 mg/kg ip). The ECG was recorded prior and 5, 10, 15, 20, 30, 40, 50, 60, and 80 min after intraperitoneal administration of the tested compound. The influence on PQ, QT c interval, QRS complex, and heart rate was evaluated. Bazzett's formula, QT c = QT/ √ RR, was used to calculate QT c [27].

Influence on Blood Pressure in Normotensive Rats
Normotensive rats were anesthetized with thiopental (75 mg/kg ip). The right carotid artery was cannulated with a polyethylene tube filled with heparin solution to allow pressure measurements, using a Datamax apparatus (Columbus Instruments, Columbus, OH, USA) [41]. The tested compound was administered intraperitoneally after 15 min of stabilization period and its effect on blood pressure was measured.

Influence on Blood Vasopressor Response in Rats
To establish agonistic or antagonistic toward α 1 -adrenergic receptors, we studied the influence of the studied compound on the pressor response to adrenaline (2 µg/kg), noradrenaline (2 µg/kg), and methoxamine (150 µg/kg). Normotensive rats were anesthetized with thiopental (75 mg/kg ip). The right carotid artery was cannulated with a polyethylene tube filled with heparin solution to allow pressure measurements, using a Datamax apparatus (Columbus Instruments, Columbus, OH, USA) [41]. The experiment was carried out after 15 min of the stabilization period. The pressor response to adrenaline, noradrenaline, and methoxamine was measured before (control) and 5 min after the administration of the tested compound.

Statistical Analysis
The number of animals in groups was based on our previous studies [24]. Results are presented as either means ± SD or as a percentage of occurrence of specific events (extrasystoles). Where it was necessary, the normality of data sets and homogeneity of variance were determined using Shapiro-Wilk and Brown-Forsythe test, respectively. In our analysis, we used one-way repeated measures ANOVA, followed by Dunnet's or Bonferroni post hoc. The reported p values of all ANOVAs used the Geisser-Greenhouse correction when the sphericity assumption was not met.