Synergisms of cardiovascular effects between iptakalim and amlodipine, hydrochlorothiazide or propranolol in anesthetized rats

Hong-min ZHOU, Ming-li ZHONG, Ru-huan WANG, Chao-liang LONG, Yan-fang ZHANG, Wen-yu CUI, Hai WANG

1. Cardiovascular Drug Research Center, Institute of Health and Environmental Medicine, Academy of Military Medical Sciences, Beijing 100850, China; 2. Thadweik Academy of Medicine, Beijing 100039, China

Synergisms of cardiovascular effects between iptakalim and amlodipine, hydrochlorothiazide or propranolol in anesthetized rats

Hong-min ZHOU1+, Ming-li ZHONG1+, Ru-huan WANG2+, Chao-liang LONG1, Yan-fang ZHANG1, Wen-yu CUI2, Hai WANG1

1. Cardiovascular Drug Research Center, Institute of Health and Environmental Medicine, Academy of Military Medical Sciences, Beijing 100850, China; 2. Thadweik Academy of Medicine, Beijing 100039, China

doi 10.13459/j.cnki.cjap.2015.06.008

The primary object of this fundamental research was to survey the synergistic cardiovascular effects of iptakalim, a novel ATP-sensitive potassium channel (KATP) opener, and clinical first-line antihypertensive drugs, such as calcium antagonists, thiazide diuretics and β receptor blockers by a 2×2 factorial-design experiment. It would provide a theoretical basis for the development of new combined anhypertensive therapy program aer iptakalim is applied to the clinic. Amlodipine besylate, hydrochlorothiazide and propranolol were chosen as clinical first-line antihypertensive drugs. Blood pressure, heart rate (HR) and cardiac functions were observed in anesthetized normal rats by an eightchannel physiological recorder. The results showed that iptakalim monotherapy in a low dose could produce significant antihypertensive effect. There was no interaction between iptakalim and amlodipine on the maximal changes of systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial blood pressure (MABP), the leventricular systolic pressure (LVSP), and the leventricular end-diastolic pressure (LVEDP) (P＞0.05). However, the effects of combination iptakalim/amlodipine on the maximal changes of SBP, DBP, MABP, LVSP and LVEDP were more obvious than those of iptakalim or amlodipine monotherapy. And there was strong posive interacon between iptakalim and amlodipine on the maximal changes of HR (P＞0.05). According to the maximal changes of DBP, MABP, LVSP and LVEDP (P＜0.05) of combinaon iptakalim with hydrochlorothiazide, there was strong posive interacon between them. But there was no interacon between iptakalim and hydrochlorothiazide on the maximal drop of SBP and HR (P＞0.05). According to the maximal drops of DBP, MABP of combinaon iptakalim with propranolol, there was strong positive interaction between them (P＜0.05). But there was no interaction between iptakalim and propranolol on the maximal changes of SBP, LVSP，LVEDP and HR (P＞0.05). In conclusion, it was the first time to study the effects of amlodipine, hydrochlorothiazide or propranolol, which had different mechanisms of action from iptakalim, on cardiovascular effects of iptakalim in anesthetized normal rats. This study proved that the combinaon of iptakalim with hydrochlorothiazide or propranolol respecvely had signif i cant synergism on lowering blood pressure, while the combinaon of iptakalim/amlodipine had addive acon on lowering blood pressure. Meanwhile the anhypertensive ef f ect was explicit, stable and long-lasng. Iptakalim thus appears suitable for the clinical treatment of hypertensive people who need two or more kinds of anhypertensive agents.

iptakalim; KATPopener; amlodipine; hydrochlorothiazide; propranolol; synergisms

Introduction

At the start of the 21st century, over a quarter adult population (972 million) in the world suf f ered from hypertension, and it is predicted the number will increase by about 60% to 1.56 billion in 2025[1]. In China，the prevalence of hypertension is higher than 18.8% and the number of patients has been reached up to 200 million, whereas the control rate ofhypertension is only 6.1%, which have posed a serious threat to the health of population2. Because of the high prevalence of hypertension and the serious cardiovascular consequences of untreated, inadequately treated, and uncontrolled hypertension, hypertension control continues to be an important public health concern.us, the selection of drugs for initial and continuing long-term therapy of hypertension has large-scale public health and economic implications. At present, blood pressure targets are achieved in only a small number of hypertensive patients using monotherapy, while the majority requires two or more kinds of antihypertensive agents[3, 4]. Iptakalim is a new KATPopener which has a unique chemical structure that differs from other KATPopeners. Animal studies and Phase III clinical trials have shown that iptakalim had explicit, stable and long-lasting antihypertensive effect; besides, it had good safety without treatment-related serious adverse events[5-7]. Amlodipine besylate --- dihydropyridine calcium antagonists, hydrochlorothiazide --- thiazide diuretics or propranolol --- β blockers are the most commonly used antihypertensive drugs in clinical currently[8, 9]. As a new antihypertensive agent, the interactions between iptakalim and clinical fi rst-line antihypertensive agents have not been documented. In this research, effects of combined treatment of iptakalim with amlodipine, hydrochlorothiazide or propranolol on cardiovascular in normal anesthetized rats were observed.erefore we could provide a theoretical basis for the clinical development of new combined antihypertensive therapy program.

Material and Methods

Chemicals

Iptakalim was synthesized by Nhwa Thad Pharmaceutical Co Ltd (Beijing, China). All other chemicals and materials were obtained from local commercial sources.

Experimental groups

This is a 2×2 factorial-design intervention experiment, with iptakalim as the fi rst factor and amlodipine, hydrochlorothiazide or propranolol as the second.

All procedures were performed in accordance with the protocol outlined in the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH publication no. 85-23, revised 1996) and approved by the local animal care and use committee. Experiments were performed on healthy adult male Wistar rats (weight range 300±30 g). All animals were divided into 11 groups (n=8): (1)Normal saline (NS) group, (2)Cre group: 3 % acetone-5 % Cremophor group (for Hyd’s dissolving), (3) iptakalim 0.125 mg/kg group, (4)iptakalim 0.5 mg/kg group, (5) iptakalim 2.0 mg/kg group, (6) amlodipine 1.0 mg/kg group, (7) iptakalim 0.5 + amlodipine 1.0 mg/kg group, (8)hydrochlorothiazide 2.5 mg/ kg group, (9)iptakalim0.5 + hydrochlorothiazide 2.5 mg/kg group, (10)propranolol 2.0 mg/kg group, (11) iptakalim 0.5 + propranolol 2.0 mg/kg group.

Cardiac functions and peripheral blood pressure measurement

All animals were anaesthetized with pentobarbital sodium (45 mg/kg) by intraperitoneal injection, and given endotracheal intubation, femoral venous, right carotid and lefemoral artery catheterizations.e right carotid and lefemoral arteries were cannulated with polyethylene catheters connected to a Statham transducer to measure cardiac functions and the blood pressures including systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial blood pressure (MABP), as the femoral venous catheterization was used for administration.en the catheter was inserted along the right carotid artery into the leventricle, and the signals were recorded on an eight-channel direct-writing oscillograph (RM-6000, Nihon Kohden Kogyo Co., Ltd, Japan) and digitally sampled (1 kHz) on an personal computer equipped with an analogue to digital interface (SMUP-PC bioanalysis system, Japan). The left ventricular systolic pressure (LVSP), leventricular end-diastolic pressure (LVEDP), the maximal rate of rise/decrease of leventricular pressure (±dp/dtmax) and heart rate (HR) were recorded at dif f erent time (0 min, 3 min, 5 min, 10 min, 15 min, 20 min, 30 min and 45 min).

Statistical analysis

The experimental results were expressed as means ± standard deviation, and SPSS13.0 software were used. Variance analysis was carried out on factorial experimental data to analyze the interaction between iptakalim and amlodipine, hydrochlorothiazide or propranolol.e one-way ANOVA was used for each experimental group. Statistical significance was accepted atP＜0.05.

Results

Ef f ects of iptakalim in dif f erent doses on blood pressure, HR, and hemodynamic indexes in anesthetized rats

Iptakalim lowered blood pressure in a dose-dependent manner: iptakalim (0.125 mg/kg) had no ef f ect on blood pressure, and iptakalim (0.5 mg/kg) signif i cantly lowered blood pressure for long duration. When the dose of iptakalim increased to 2.0 mg/kg, the blood pressure was decreased more remarkable. Iptakalim had the same ef f ects on HR and hemodynamics indexes as blood pressure (Data not shown). Iptakalim (0.5 mg/kg) had a few effect on HR with a slight decrease only in 5 minutes aer administration and then gradually returned to normal, and the maximal decrease of HR was only 7.3 %. Iptakalim at the dose of 0.5 mg/kg had a few ef f ect on LVSP with a slight decrease and the maximal decrease of LVSP was only 7.0 %, whereas LVEDP was signif i cantly increased and the maximal increase of LVEDP was 43.7 % (Data not shown).erefore iptakalim (0.5 mg/kg) was chosen as the combined dose with amlodipine, hydrochlorothiazide or propranolol.

Ef f ects of iptakalim combined with amlodipine on blood pressure, HR, and hemodynamic indexes in anesthetized normal rats

LVSP, +dp/dtmax, LVEDP and -dp/dtmaxwere ventricular function indexes. The two former represented left ventricular systolic function, and the two later represented left ventricular diastolic function. Because ±dp/dtmaxwas more sensitive than LVSP and LVEDP, the changes of ±dp/dtmaxwere more signif i cant than those of LVSP and LVEDP.

Amlodipine (1.0 mg/kg) by intravenous injection decreased blood pressure significantly in normal anesthetized rats with the long duration and the values of maximal decreasing of SBP, DBP, MABP were 38.8 mmHg (25.3 %), 37.1 mmHg (37.9 %), 38.5 mmHg (33.1 %) respectively (P＜0.01vsbaseline values, Fig.1, 2; Tab.1). Amlodipine had no ef f ect on HR (P＞0.05vsbaseline value, Tab.2). Amlodipine had few effect on LVSP with a slight decrease, whereas +dp/dtmaxwas significantly declined, and the maximal change was 35.7 %. LVEDP was significantly increased and the maximal rise was 22.4 %; -dp/dtmaxwas signif i cantly decreased, and the maximal change was 40.4 % (Tab.3,4).

Blood pressure was reduced signif i cantly aer the rats were injected intravenously with iptakalim (0.5 mg/kg) and amlodipine (1.0 mg/kg), and the values of the maximal decreasing of SBP, DBP, and MABP were 69.0 mmHg (44.9 %), 51.7 mmHg (53.2 %), 59.1 mmHg (50.9 %) respectively (P＜0.01 compared with the baseline values) (Fig.1, Tab.1). Iptakalim and amlodipine of simultaneous administration had a few effect on HR with a slight decrease only in 5 minutes after administration and then gradually returned to normal, and the maximal decreasing of HR was only 9.2% (P＜0.01 compared with baseline values, Fig.1, Tab.1). Coadministration of iptakalim and amlodipine had few effect on LVSP with a slight decrease, whereas +dp/dtmaxwas significantly declined and the maximal change was 43.8 %. LVEDP was signif i cantly increased and -dp/dtmaxwas significantly decreased, and the maximal changes were 41.5 % and 43.7 % respectively (Tab.3, 4).

According to the maximal decreasing of HR (P＜0.05) of combination iptakalim with amlodipine, there was strong positive interaction between them. But there was no interaction between iptakalim and amlodipine on the maximal changes of SBP, DBP, MABP, LVSP and LVEDP (P＞0.05).

Ef f ects of iptakalim combined with hydrochlorothiazide on blood pressure, HR, and hemodynamic indexes in anesthetized normal rats

3% acetone and 5% Cremophor CO40 Ethoxylated hydrogenated castor oil (Cre) were added to dissolve hydrochlorothiazide because of its poor dissolubility. Cre by intravenous injection showed no effects on HR, blood pressure and hemodynamic indexes in normal anesthetized rats (Fig.1, Tab.1, 2, 3, 4). Hydrochlorothiazide (2.5 mg/kg) by intravenous injection did not affect HR, LVSP and ±dp/dtmaxin normal anesthetized rats.ere was no decrease on blood pressure until 30 minutes after intravenous injection with hydrochlorothiazide, and the values of maximal decrease of SBP, DBP, and MABP were only 20.4 mmHg (12.6%), 10.1 mmHg (9.9%) and 13.5 mmHg (11.3%) respectively (P＜0.01 compared with the baseline values) (Fig.1, 2 Tab.1). LVEDP was increased aer 45 minutes and the maximal rise was 28.1% (P＜0.05 compared with the baseline values, Tab.3).

Blood pressure was reduced signif i cantly aer the rats were injected intravenously with iptakalim (0.5 mg/kg) and hydrochlorothiazide (2.5 mg/kg) in 3 minutes and the values of maximal decrease of SBP, DBP, and MABP were 56.5 mmHg (34.6%), 41.2 mmHg (37%) and 46.3 mmHg (36%) respectively (P＜0.01 compared with the baseline values, Fig.1, 2,Tab,1).ere were less ef f ect on HR and the maximal decrease was only 8.1 % (P＜0.01 compared with the baseline value, Tab.2). Iptakalim and hydrochlorothiazide of simultaneous administration had a few ef f ect on LVSP with a slight decrease, whereas +dp/dtmaxwas signif i cantly declined, and the maximal changes of them were 11.3% and 33.1% respectively. LVEDP was significantly increased and -dp/dtmaxwas signif i cantly decreased, and the maximal changes were 38.4% and 36.4% respectively (Tab.3, 4).

According to the maximal changes of DBP, MABP, LVSP and LVEDP of combination iptakalim with hydrochlorothiazide, there was strong positive interaction between them. But there was no interaction between iptakalim and hydrochlorothiazide on the maximal decrease of SBP and HR.

Effects of iptakalim combined with propranolol on blood pressure, HR, and hemodynamic indexes in anesthetized normal rats

Propranolol (2.0 mg/kg) by intravenous injection had a few ef f ects on blood pressure, and the values of the maximal decrease of SBP, DBP, and MABP were only 16.3 mmHg (11.1%), 7.1 mmHg (7.1%) and 10 mmHg (8.4%) respectively (P＜0.05,P＞0.05,P＞0.05 compared with the baseline values, Fig.1, Tab.1). Propranolol had a few ef f ects on leventricular systolic and diastolic function, especially diastolic function, and the maximal changes of LVSP, +dp/dtmax, -dp/ dtmaxand LVEDP were 8.6 %, 17.8 %, 19.9 % and 2.5 % respectively (P＞0.05,P＜0.01,P＜0.01P＞0.05 compared with the baseline values, Tab.3, 4). HR was reduced significantly by propranolol in 3 minutes with 98 beats per minute (22.8 %), and it was lowered slowly and sustained (P＜0.01 compared with the baseline values, Tab.2).

Blood pressure was reduced signif i cantly aer the rats were injected intravenously with iptakalim (0.5 mg/kg) and propranolol (2.0 mg/kg) in 3 minutes and the values of maximal decrease of SBP, DBP, and MABP were 55.3 mmHg (34.4%), 44.3 mmHg (41%) and 49.1 mmHg (38.8%) respectively (P＜0.01 compared with the baseline values) (Fig.1, 2, Tab.1). Iptakalim and propranolol of simultaneous administration had a few ef f ect on LVSP with a slight decrease, whereas +dp/dtmaxwas signif i cantly declined and the maximal change was 35.9%. LVEDP was signif i cantly increased and -dp/dtmaxwas significantly decreased, and the maximal changes were 47.1% and 39.6% respectively (Tab.3, 4). Iptakalim and propranolol of simultaneous administration signif i cantly decreased HR in 3 minutes with 98 beats per minute (28%,P＜0.01 compared with the baseline value, Tab.2).

According to the maximal decrease of DBP and MABP aer treated with combination iptakalim and propranolol, there was strong positive interaction between them. But there was no interaction between iptakalim and propranolol on the maximal changes of SBP, LVSP, LVEDP and HR.

Discussion

It is probable that a great many interrelated factors contribute to the raised blood pressure in hypertensive patients, and their relative roles may differ between individuals[10]. Recent estimates indicated that only 34% of those taking antihypertensive medication achieved adequate blood pressure control.e Seventh Report of the Joint National Committee on the Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC 7) guidelines suggested consideration of initial antihypertensive therapy with two or more antihypertensive drugs with different and complementary mechanisms of action in hypertensive patients[11]. As expected, the blood pressure-lowering ef ficacy of the dif f erent antihypertensive drug classes is also accompanied by reductions in the risk of developing coronary heart disease. And a reduction in adverse events is frequently observed with specific combination strategies. In addition, compared with initiating treatment with monotherapy, combination therapy has been associated with a lower rate of discontinuation[12].

It had been demonstrated that KATPplayed important roles in the cardiovascular system and various antihypertensive ef f ects of its openers.e KATPconsists of inward rectifier potassium channel subunits (Kir6s) and regulative sulfonylurea receptors (SURs). The subunit constitution of KATPvaries in different tissues. In vascular smooth muscle cells and endothelial cells, they consist of SUR2B and Kir6.1 subunits, which modulate vascular smooth muscle tone[13, 14]. Iptakalim, a new KATPopener, selectively opens the SUR2B/Kir6.1 channel subtype. It had been shown that iptakalim had explicit and stable antihypertensive effect in animal experiments[5, 15, 16]. For now, all three phases of clinical trials had been accomplished in 888 subjects and the results suggested that iptakalim had no ef f ect on blood pressure in healthy volunteers; however, it could significantly lower the blood pressure in hypertensive people which possessed the merits of stable and long-lasting anti-hypertensive effect. In addition, good security was frequently observed without treatment-related serious adverse events and changes of heart rate[6].This experiment proved that iptakalim (0.5 mg/kg) by intravenous injection significantly reduced the blood pressure in normal anesthetized rats, slightly declined heart rate only within 5 minutes and then gradually returned to normal, mildly suppressed the cardiac systolic and diastolic function. Our previous studies showed that iptakalim had no effect on HR and cardiac systolic and diastolic functions in normal conscious rats and the isolated working hearts of normal awake rats[17, 18]; iptakalim inhibited the cardiac contraction functions and pumping function of the spontaneously hypertensive rats and had no effect on HR[16, 17].erefore, it was believed that the ef f ects of iptakalim on HR and systolic and diastolic function were correlated with the state (awake or anesthesia) and the level of blood pressure of animals.

Fig. 1 The anti-hypertensive effect of the combination of iptakalim (Ipt) and amlodipine besylate(Aml) (A), hydrochlorothiazide (Hyd) (B) or propranolol (Pro) (C) on MABP at different time in normal anesthesia rats . Data are expressed as Means+SD,n=8. *P＜0.05, **P＜0.01;#P＜0.05,##P＜0.01;&P＜0.05,&&P＜0.01vsbaseline values.

Fig. 2 The anti-hypertensive effect of combination of iptakalim (Ipt) and amlodipine besylate(Aml) (A), hydrochlorothiazide (Hyd) (B) or propranolol (Pro) (C) on the maximal decrease of MABP in normal anesthesia rats. Data are expressed as Means+SD,n=8. *P＜0.05, **P＜0.01vsNS group or Cre group;#P＜0.05,##P＜0.01vsIpt 0.5mg/kg group;&P＜0.05,&&P＜0.01vsAml 1.0 mg/kg, Hyd 2.5 mg/kg or pro 2.0 mg/kg group.

Iptakalim primarily activates SUR2B/Kir6.1 subtype of vascular endothelial cells so as to promote the secretion of NO. NO activates adenylate cyclase, which could promote the generation of cGMP. As a result, the reduction of blood pressure is observed. In addition, it also directly activates SUR2B/Kir6.1 subtype of vascular smooth muscle cells, which causes hyperpolarization of membrane.is ef f ect lowers the opening probability of L-type Ca2+channels, decreases restraint in [Ca2+]iintracellular. As a result, there is vasodilatation and the drop of blood pressure[7, 16]. Calcium antagonists, thiazide diuretics and β-receptor blockers are the recommended fi rstline therapy agents for most hypertensive patients in clinical currently, and they all have different mechanisms of antihypertensive action from KATPopener[9, 11]. So the anti-hypertensive ef f ects of iptakalim combination with amlodipine, hydrochlorothiazide or propranolol were really to be expected.

Amlodipine, the third-generation dihydropyridine calcium antagonists, could selectively block voltage-dependent Ca2+channels and inhibit extracellular Ca2+influx to relax the vascular smooth muscles, therefore peripheral vascular resistance wasreduced, and blood pressure was lowered[19]. The combination of iptakalim/amlodipine could produce more signif i cant antihypertensive ef f ect than iptakalim or amlodipine monotherapy. However there was no synergistic effect but additive action on blood pressure, which probably was relevant to their target sites: there was partial overlap in the smooth muscle between iptakalim and amlodipine. Hydrochlorothiazid, a classic thiazide diuretic, could reduce the blood volume thought natriuresis, therefore cardiac output was declined, and blood pressure was lowered [20]. When treated with hydrochlorothiazid, the blood pressure of rats was decreased so slowly that it couldn’t reach up to the maximal antihypertensive effect until 30 minutes passed, and more important, the antihypertensive ef f ect was dissatisfactory. But when it was used in combination with iptakalim, the blood pressure was decreased quickly and signif i cantly and it reached up to the maximal antihypertensive ef f ect in 3 minutes. Propranolol, the representative drug of non-selective β receptor blockers, could inhibit cardiac contractility and slowed the HR thought blocking the β1receptor of heart, so cardiac output was declined and blood pressure was lowered[21]. The antihypertensive ef f ect of propranolol was weak, and it signif i cantly reduced HR to 98 beats per minute in 3 minutes and then the HR gradually fell.e combined application of iptakalim/propranolol had noticeable synergistic antihypertensive ef f ect which was quickly and durably, but it didn’t increase the drop of propranolol on HR signif i cantly.

Tab. 1 Effects of iptakalim, amlodipine besylate, hydrochlorothiazide and propranolol on blood pressure of normal anesthetized rats (Mean±SD,n=8).

Tab. 2 Effects of iptakalim, amlodipine besylate, hydrochlorothiazide and propranolol on HR of normal anesthetized rats (Mean±SD,n=8).

Tab. 3 Effects of iptakalim, amlodipine besylate, hydrochlorothiazide and propranolol on left ventricular systolic function of normal anesthetized rats (Mean±SD,n=8).

In conclusion, it was the first time to investigate the synergistic cardiovascular ef f ects of iptakalim and amlodipine, hydrochlorothiazide or propranolol in anesthetized normal rats. This research proved that the combination of iptakalim with hydrochlorothia-zide or propranolol respectively had signif i cant synergism on lowering blood pressure, while the combination of iptakalim/amlodipine had additive action on lowering blood pressure. Meanwhile the antihypertensive ef f ect was explicit, stable and long-lasting. Iptakalim thus appears suitable for the clinical treatment of hypertensive people who need two or more kinds of antihypertensive agents.

Tab. 4 Effects of iptakalim, amlodipine besylate, hydrochlorothiazide and propranolol on left ventricular diastolic function of normal anesthetized rats (Mean±SD,n=8).

Acknowledgements

This study was supported by grants from the State“973” Research Project (2012CB518200) and State Key Project (AWS11J003).

Disclosures

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Hai WANG, Chao-liang LONG, MD, PhD, Professor, Cardiovascular Drug Research Center, Institute of Health and Environmental Medicine, Academy of Military Medical Sciences, Beijing 100850, China. Tel: 86-10-66932651, Fax: 86-10-66931557; E-mail: wh9588@sina.com (Hai WANG); longchaoliang@sohu.com (Chao-liang LONG)

+ese three authors contributed equally to this work

Received 2015-11-12; accepted 2015-11-20