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Pharmacological profile of the new inotropic agent AT-11
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European Journal of Pharmacology 580 (2008) 224 – 230 www.elsevier.com/locate/ejphar
Pharmacological profile of the new inotropic agent AT-11 An-Sheng Lee a , Tian-Shung Wu b,c , Ming-Jai Su a,⁎ a
Institute of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC b Department of Chemistry, National Cheng Kung University, Tainan, Taiwan, ROC c National Research Institute of Chinese Medicine, Taipei, Taiwan, ROC Received 21 June 2007; received in revised form 2 October 2007; accepted 22 October 2007 Available online 30 October 2007
Abstract Although there are many classes of drugs, including cardiac glycosides, sympathomimetic inotropes, β-adrenergic antagonists, angiotensinconverting enzyme inhibitors (ACE inhibitors) and spironolactone etc. used for the treatment of heart failure, the morbidity and mortality rates of patients after these treatments are not ameliorated. Chronic administration of Sympathomimetic inotropes also increased the arrhythmogenic effects. Consequently, improvement of treatment for heart failure remains a major medical challenge for the coming years. In this present experiment, the novel Na+–K+ ATPase inhibitor AT-11 was characterized for its inotropic and toxic properties. Comparing AT-11 with ouabain, we found that AT-11 concentration-dependently increased contractility in guinea pig heart preparations, and the safety index of AT-11 was better than ouabain in vitro. In the in vivo study, AT-11 was also safer than ouabain at the equieffective dose. Moreover, AT-11 slowed heart rate more than ouabain did. This may be due to a larger AT-11-induced increase in vagal reflex than with ouabain and an indirect decrease in sympathetic tone to prevent Ca2+ overload. © 2007 Elsevier B.V. All rights reserved. Keywords: Digitalis; Cardiac glycoside; Heart failure; Sodium pump; Na/K ATPase
1. Introduction Heart failure is clearly a major clinical and public health problem. It is estimated that nearly 23 million people worldwide suffer from heart failure (Cleland et al., 2001). Despite recent innovations and improvements used for treating heart failure, it remains highly lethal once established (Stewart et al., 2001). Consequently, improvement of heart failure treatment remains a major medical challenge in the coming years. In chronic heart failure, the aims are to relieve symptoms, improve hemodynamic status, prevent hospitalization, and, above all, to reduce mortality. There are many clinical drugs used in combination for treatment of heart failure, including cardiac glycosides, sympathomimetic inotropes, β-adrenergic antagonists, angiotensin-converting enzyme inhibitors (ACE inhibitors), and ⁎ Corresponding author. Institute of Pharmacology, College of Medicine, National Taiwan University, No.1, Sec.1, Jen-Ai Rd. Taipei 100, Taiwan, ROC, Tel.: +886 2 23123456x8317; fax +886 2 23971403. E-mail address: [email protected] (M.-J. Su). 0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2007.10.056
now spironolactone (1999). Among these agents, digitalis is the only one with both positive inotropic effect and inhibition of neurohumoral responses. Differing from other inotropes which tend to cause tachycardia, digitalis has bradycardic action by increasing vagal tone. Digitalis has played a prominent role in the therapy of congestive heart failure since William Withering codified its use in his classic thesis on the efficacy of the common foxglove plant (Digitalis purpurea) in 1785. In the 1990s, digoxin was the most commonly prescribed drug due to the availability of techniques for measuring its serum levels, flexible routes of its administration, and its intermediate duration of action (Valente et al., 2003). Digitalis binds to and inhibits the sarcolemma Na+ pump (Na+–K+-ATPase) (Akera and Ng, 1991). This leads to a transient increase in intracellular Na+ concentration, which in turn increases intracellular Ca2+ concentration by the sodium–calcium exchange mechanism. Thus, the result is an enhanced myocardial contractility. However, when cardiac muscle is exposed to toxic concentrations of digitalis, Na+ pump inhibition and intracellular
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Ca2+ become excessive. In digitalis toxicity, the excessive Ca2+ concentration may cause afterdepolarization, and even more lifethreatening ventricular tachycardia and ventricular fibrillation. Because the therapeutic positive inotropic effect of these drugs is also caused by the enhanced Ca2+ loading, the therapeutic and toxic effects are inseparable. The narrow margin of safety index (therapeutic index) is the restriction of this class of positive inotropic drugs. That is the reason even with improvement of the whole heart function, digitalis could not reduce the rate of mortality (The Digitalis Investigation Group, 1997). In the present study the effects of the novel agent AT-11 (Fig. 1), a derivative of digitalis, are compared with the effects of ouabain in in vitro and whole animal experiments.
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2. Materials and methods
deeply anesthetized, tracheotomy was performed for artificial respiration with a stroke volume of approximately 15 ml/kg body weight and at a rate of 60 strokes/min by a respirator (model 683, Harvard Rodent ventilator). Polyethylene catheter (PE50) was inserted into the carotid vein for administration of ouabain and AT-11, and a Millar catheter with a high-fidelity pressure sensor (model SPC 320, size 2F, Millar Instruments, Houston, TX, U.S. A.) was inserted through the carotid artery into left ventricle to measure the pulsatile left ventricle pressure. The electrocardiogram (ECG) of lead II was also recorded. Left ventricular pressure, maximum velocity of pressure rise (+dP / dtmax) and fall (−dP / dtmax), heart rate, and electrocardiogram changes were calculated and recorded on a personal computer with a wave form data analysis software (PowerLab data acquisition system; AD Instruments Pty. Ltd).
2.1. In vitro assay
2.3. Electrophysiological recording
The investigation was performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Cardiac contractions were measured according to the procedure described previously (Chen et al., 2007). Adult male guinea pigs (300–500 g) were intraperitoneally injected with sodium pentobarbital (25 mg/kg) plus heparin (16 mg/kg). After the guinea pigs were deeply anesthetized, hearts were excised, and retrograde coronary perfusion with normal Tyrodes solution containing (in mM): 137 NaCl, 11.9 NaHCO3, 11 glucose, 2 CaCl2, 1.1 MgCl2, 0.33 NaH2PO4, and 5.4 KCl. Left atria strips and left papillary muscles were separated from the heart. One end of the muscle was attached to a transducer (Type BG; Gould, Cleveland, Ohio, USA) and the other end was fixed to a rigid support by a silk thread in a 10 ml organ bath. Each tissue was placed under 1 g resting tension and stimulated at 2 Hz with rectangular voltage pulses of 2 ms duration and amplitude twice threshold via an isolated Grass SD9 stimulator (Grass Instruments Co., Quiney, Mass., USA). The twitch tension was recorded on a Gould 2200s recorder.
Cardiomyocytes were isolated by using the enzymatic method as previously described (Chen et al., 2007). Adult male guinea pigs (200–250 g) were intraperitoneally injected with sodium pentobarbital (25 mg/kg) plus heparin (16 mg/kg). After the guinea pig was deeply anesthetized, heart was excised and the coronary artery was antegradely perfused with oxygenated Ca2+-free HEPES solution containing (in mM): 137 NaCl, 22 glucose, 6 HEPES, 1.2 MgSO4, 1.2 KH2PO4, and 5.4 KCl; pH was adjusted to 7.4 using NaOH. The heart was then perfused with the same solution containing 0.4 mg/ml collagenase (type II, Sigma Chemical Co., St. Louis, Mo, USA), 0.06 mg/ml protease (type XIV, Sigma) and bovine serum albumin 1 mg/ml. After 4–5 mins of digestion, enzymes were washed out in Kruftbruhe solution containing (in mM): 10 taurine, 10 oxylate, 70 glutamate, 25 KCl, 10 KH2PO4, 11 glucose, 0.5 EGTA; pH was adjusted to 7.4 using KOH (Isenberg and Klockner, 1982). The ventricles were then chopped and resuspended under gentle mechanical agitation and stored in Kruftbruhe solution at room temperature. The whole-cell patch clamp technique was used to record ionic currents in voltage clamp mode with a Dagan 8900 voltage clamp amplifier (Dagan Co., Minneapolis, Minn., U.S.A.). A droplet of the cell suspension was placed in a chamber mounted on the stage of an inverted microscope (Nikon, Diaphot, Japan). After settling down, cells were finally exposed to the bath solution containing (in mM):137 NaCl, 5.4 KCl, 2.9 MgCl2, 6 HEPES, 22 glucose, 0.33 NaH2PO4, 2 BaCl2, and 0.2 CdCl; pH was adjusted to 7.4 using NaOH. All experiments were performed at 30 ± 0.5 °C. For the measurement of Ipump, a pipette was filled with the internal solution containing (mM): 80 CsOH, 50 NaOH, 3 MgCl2, 20 TEA-Cl, 100 aspartic acid, 10 HEPES, 10 ATP-Mg, 0.2 GTP-Na3, 5.5 glucose, 5 Na-creatine phosphate, and 5 pyruvic acid; pH was adjusted to 7.2 using CsOH as previous described (Gadsby et al., 1985). Heatpolished glass electrodes (tip resistances about 1 MΩ when filled with pipette internal solution) were used. Junction potentials were zeroed before the formation of the membrane– pipette seal in bath solution. The series resistance was electronically compensated by about 80% to minimize the duration
2.2. In vivo assay Adult male guinea pigs (400–550 g) were intraperitoneally injected with urethane (600 mg/kg). After the guinea pigs were
Fig. 1. Chemical structure of AT-11.
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Fig. 2. Comparison of twitch amplitude between AT-11 and ouabain. Concentration-dependent increases in twitch amplitude caused by both AT-11 (filled circles, n = 5) and ouabain (open circles, n = 5) in isolated left atria (A), and papillary muscles (B). (C) Twitch amplitude trace of papillary muscle in the presence of AT-11. Shortly after addition, the concentration of 5 μM caused arrhythmias.
of the capacitive surge on the current recorded and the voltage drop across the pipette. Currents were elicited and acquired using Digidata 1200 data acquisition system controlled using pClamp software (Axon Instruments). Recordings were lowpass filtered at 10 kHz and stored on the hard disk of a computer. 2.4. Statistical analysis The results were presented as means ± standard error of the mean (S.E.M.). Significant differences between treatment groups were determined by one-way ANOVA with post hoc analysis using the Bonferroni t-test. 3. Results 3.1. Inotropic effects of AT-11 and ouabain in isolated cardiac muscles In this experiment, both of AT-11 and ouabain concentrationdependently increased the twitch amplitudes relative to control in left atria and papillary muscles (Fig. 2A and B, respectively). Maximal twitch amplitudes of left atria and papillary muscles are expressed as percentages of basal values (0.7± 0.1 g and 0.8 ± 0.1 g of left atria, 0.2 ± 0.1 g and 0.2± 0.1 g of papillary muscles in AT-
11 and ouabain groups, respectively) and plotted as functions of concentration of AT-11 and ouabain. At higher concentrations, further enhancements of inotropic effects were often accompanied with irregular twitches (Fig. 2C). In detail, the minimal effective concentration to increase myocardial contraction, the arrhythmogenic concentration to induce arrhythmia, and the onset of AT-11 and ouabain were measured as shown in Table 1. The maximal positive inotropic effect was determined at a concentration level immediately before the occurrence of cardiac arrhythmia and the safety index was calculated from the ratio of the arrhythmogenic concentration to the minimal effective positively inotropic concentration. The onsets of AT-11 in the left atria and papillary muscles are both shorter than those of ouabain (51.7 ± 4.0 versus 76.7 ± 3.3s and 44.0 ± 2.5 versus 83.3 ± 14.5s, respectively). The time to steady state of inotropic effect at therapeutic concentration, or the time to cause arrhythmia at arrhythmogenic concentration, was not significantly different between these two agents (data not shown). In the left atria, both the minimum effective concentration and arrhythmogenic concentration of AT-11 were 5fold higher than those of ouabain (0.25 versus 0.05 μM and 5 versus 1 μM, n = 5). The maximum inotropic effects were roughly similar of these two agents (660 ± 88 versus 775 ± 128%, n = 5, N.S.). However, in papillary muscles, the maximum
Table 1 Comparison of AT-11 and ouabain on onset, minimal effective concentration, arrhythmogenic concentration, maximal positive inotropic effect, and safety index in guinea pig isolated left atria and papillary muscles
Left atria Papillary muscle
AT-11 Ouabain AT-11 Ouabain
Onset (sec)
Minimal effective concentration (μM)
Arrhythmogenic concentration (μM)
Maximal positive inotropic effect (%)
Safety index
51.7 ± 4.0a 76.7 ± 3.3 44.0 ± 2.5a 83.3 ± 14.5
0.25 0.05 0.25 0.05
5 1 6 1
660 ± 88 775 ± 128 464 ± 89a 249 ± 26
20 20 24 20
Values of onset and maximal positive inotropic effect are shown as mean ± S.E.M. aP b 0.05 between AT-11 and ouabain. (ANOVA).The safety index was calculated from the ratio of the arrhythmogenic concentration to the minimal effective positively inotropic concentration.
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inotropic effect of AT-11 was significantly higher than that of ouabain (464 ± 89 versus 249 ± 26%, n = 5). The safety index of AT-11 is also higher than that of ouabain (24 versus 20, n = 5). 3.2. Inotropic Effects of AT-11 and Ouabain in Anesthetized Guinea Pigs Not only in-vitro experiments but also the in vivo study revealed that the safety of AT-11 was better than that of ouabain. Fig. 3 shows comparison of dose responses of maximal left ventricle pressure between AT-11 and ouabain. Left ventricular pressure is expressed as percentage of basal value (54.6 ± 1.2 mmHg and 51.9± 1.9 mmHg in AT-11 and ouabain groups, respectively) and plotted as a function of various AT-11 (0.016, 0.053, 0.16, 0.53, 1.6, and 5.3 mg/kg) and ouabain (0.0029, 0.0058, 0.029, and 0.058 mg/kg) doses. At the dose of 0.0029 mg/kg, ouabain began to enhance pressure to 106.17± 1.84% compared to the control. However, when the dose was increased to 0.029 mg/kg, ouabain reached the maximal inotropic effect (142.0± 6.5%), and even at higher dose (0.058 mg/kg) which resulted in 50% animal death (5 of 10 rats died due to arrhythmia) could not increase more ventricular pressure (140.2 ± 5.6%). On the other hand, AT-11 started to increase pressure to 106.18 ± 2.49% at the dose (0.016 mg/kg) higher than ouabain and reached the maximum inotropic effect (177.5 ±14.7%) at the dose of 5.3 mg/kg with 43% animal death (3 of 7 rats died due to arrhythmia). The effects of the maximal velocity of pressure rise (+dP /dtmax) and fall (−dP / dtmax) were also calculated. +dP /dtmax and −dP /dtmax are expressed as percentage of basal value (3043.9± 176.4 mmHg/s and 2901.4 ± 188.2 mmHg/s of +dP / dtmax and −2771.7 ± 133.5 mmHg/s and −2547.0 ± 125.1 mmHg/s of −dP /dtmax in AT-11 and ouabain groups, respectively) and plotted as functions of various AT-11 (0.016, 0.053, 0.16, 0.53, 1.6, and 5.3 mg/kg) and ouabain (0.0029, 0.0058, 0.029, and 0.058 mg/kg) doses. The effects of AT-11 and ouabain on −dP /dtmax were similar to those on left ventricular pressure (Fig. 4B). However, the effect of AT-11 on +dP /dtmax was different from that on ventricular pressure and −dP / dtmax. AT-11 induced increase of +dP / dtmax reached the
Fig. 3. Comparison of dose-dependent increase of maximal left ventricular pressure between AT-11 (circles, n = 10, 10, 10, 10, 10, 4) and ouabain (triangles, n = 10, 10, 10, 5) in anesthetized guinea pigs. Left ventricular pressure is expressed as percentage of basal value and plotted as a function of various doses of AT-11 (0.016, 0.053, 0.16, 0.53, 1.6, 5.3 mg/kg) and ouabain (0.0029, 0.0058, 0.029, 0.058 mg/kg).
Fig. 4. Comparison of dose-dependent increases of maximal velocity of pressure rise (+dP / dtmax, A) and fall (− dP / dtmax, B) between AT-11 (circles, n = 10, 10, 10, 10, 10, 4) and ouabain (triangles, n = 10, 10, 10, 5) in anesthetized guinea pigs. +dP / dtmax and − dP / dtmax are expressed as percentages of basal values and plotted as functions of various doses of AT-11 (0.016, 0.053, 0.16, 0.53, 1.6, 5.3 mg/kg) and ouabain (0.0029, 0.0058, 0.029, 0.058 mg/kg).
peak at the dose of 1.6 mg/kg and did not increase as the dose increased (Fig. 4A). Although the dose needed to reach the equieffective inotropic effect of AT-11 was 27-fold higher than that of ouabain, AT-11 was safer than ouabain. Lethal arrhythmias occurred in 50% (5 of 10) of animals treated with ouabain at a dose of 0.058 mg/kg. In contrast, no arrhythmias occurred in the animals treated with
Fig. 5. Effect of equieffective concentrations of AT-11 and ouabain on maximal left ventricular pressure in anesthetized guinea pigs. Left ventricular pressure is expressed as percentage of basal value and plotted as a function of time at the dose of AT-11 (1.6 mg/kg, filled circles, n = 10), ouabain (0.029 mg/kg, open circles, n = 10), and vehicle (triangles, n = 10). ⁎, P b 0.05 between AT-11 and vehicle; #, P b 0.05 between ouabain and vehicle. (ANOVA)
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AT-11 at the equieffective dose (1.6 mg/kg). AT-11 began to cause arrhythmia (43%) at the dose of 5.3 mg/kg, and the safety index was calculated as 331.25 which was much higher than that of ouabain (20). Fig. 5 shows time courses of AT-11 and ouabain inotropic effects at equieffective doses (1.6 and 0.029 mg/kg, respectively). AT-11 induced an increase in left ventricular pressure that reached the peak of 144.7 ± 7.8% relative to basal at 2 min after intravenous administration of the agent. After the peak, a decrease toward control value occurred throughout the period. A comparable increase (142.0 ± 6.5%) was afforded by administration of ouabain. AT-11 at this dose also caused increases in + dP / dtmax and −dP / dtmax related to control status with peaks of 198.2 ± 17.8 and 156.87 ± 18.34%, respectively whereas ouabain increased + dP / dtmax and − dP / dtmax with peaks of 193.7 ± 17.1 and 141.0 ± 6.9%, respectively (Fig. 6). Ouabaininduced increase of + dP / dtmax did not fall to control value even at 20 min after the administration of the agent, but AT-11induced increase did. Differing from other inotropes which tend to cause tachycardia, digitalis has bradycardic action by increasing vagal tone. The effects of AT-11 and ouabain on heart rate were also measured (Fig. 7). Heart rate is expressed as percentage of a basal value (273.1 ± 7.3 bpm and 263.2 ± 5.6 bpm in AT-11 and ouabain groups, respectively) and plotted as a function of time at
Fig. 7. Effect of equieffective concentrations of AT-11 and ouabain on heart rate in anesthetized guinea pigs. Heart rate is expressed as percentage of basal value and plotted as a function of time at the dose of AT-11 (1.6 mg/kg, filled circles, n = 10), ouabain (0.029 mg/kg, open circles, n = 10), and vehicle (triangles, n = 10). ⁎, P b 0.05 between AT-11 and vehicle. (ANOVA)
the dose of AT-11 (1.6 mg/kg, filled circles), ouabain (0.029 mg/ kg, open circles) and vehicle (triangles). Three minutes after administration of ouabain, the heart rate started to be decreased 3% related to vehicle (98.7 ± 1.2 versus 101.8 ± 1.3%, N.S.) but without statistical significance until the end of this experiment. In contrast, AT-11 decreased 7% heart rate (94.6 ± 2.3%) through this period. 3.3. Na+/K+ pump current (Ipump) recordings To confirm that the inotropic effect of AT-11 was through the inhibition of Na+/K+ ATPase like other digitalis, sodium pump current (Ipump) was measured by whole-cell patch clamp technique (Fig. 8). After rupture of the patch, the holding potential was set at − 40 mV to inactivate Na+ channels and the cell interior was allowed to equilibrate 5 min with the pipette solution. Then membrane currents were elicited by voltage ramps from + 60 mV to − 120 mV (see inset of Fig. 8). Ipump was defined as the difference of current between after and before AT11 or ouabain treatment. Fig. 8 shows the effects on Ipump current–voltage relationships of ouabain at 10 μM (filled squares, n = 6), AT-11 at 10 μM (open circles, n = 4), AT-11 at 30 μM (open squares, n = 4), and vehicle (filled circles, n = 4) sensitive currents. Both ouabain and AT-11 sensitive currents
Fig. 6. Effect of equieffective concentrations of AT-11 and ouabain on maximal velocity of pressure rise (+ dP / dtmax, A) and fall (− dP / dtmax, B) in anesthetized guinea pigs. +dP / dtmax and − dP / dtmax are expressed as percentages of basal values and plotted as functions of time at the dose of AT11 (1.6 mg/kg, filled circles, n = 10), ouabain (0.029 mg/kg, open circles, n = 10), and vehicle (triangles, n = 10). ⁎, P b 0.05 between AT-11 and vehicle; #, P b 0.05 between ouabain and vehicle. (ANOVA)
Fig. 8. Drug effects on Ipump. Current–voltage relationships of ouabain at 10 μM (filled squares, n = 6), AT-11 at 10μM (open circles, n = 4), AT-11 at 30 μM (open squares, n = 4), and vehicle (filled circles, n = 4) sensitive currents were obtained by applying slow hyperpolarizing ramps as inset showed.
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were near linear, and at negative potentials they declined with voltage towards an extrapolated zero-current potential near about − 140 mV. The current densities at 0 mVof ouabain, AT-11 10 μM, AT-11 30 μM, and vehicle were 0.53 ± 0.09, 0.63 ± 0.08, 0.71 ± 0.10, and 0.03 ± 0.03 pA/pF, respectively (with cell capacitance = 83.9 ± 6.4 pF). 4. Discussion There are many classes of drugs which are used for the treatment of heart failure (1999). Among them, β-adrenergic antagonists are known to have the potential to worsen both ventricular function and symptoms in patients with heart failure. Although sympathomimetic inotropes cause a marked improvement in cardiac systolic function and may alleviate symptom and improve exercise capacity, long term treatment with these agents has been shown to increase mortality. Therefore, cardiac glycosides such as digoxin and ouabain remain as important inotropes used in patients with heart failure. Digitalis derivatives are found in several plants, including foxglove, lily and oleander. In spite of several advances in the treatment of cardiovascular disorders, digitalis has been widely used for centuries as a therapeutic agent for the treatment of congestive heart failure and has remained one of the most commonly prescribed drugs in many countries (The Digitalis Investigation Group, 1997). However, as the safety index of digitalis is narrow, arrhythmias are common problems in clinical practice (Khatter et al., 1986, 1989). Therefore, improvements of digitalis and other heart failure treatments remain a major medical challenge for the coming years. In recent years, some novel agents (Micheletti et al., 2002; Rocchetti et al., 2003; Wahed et al., 2004) and mechanisms (Chierchia and Deferrari, 2004; Mengi and Dhalla, 2004; von Harsdorf et al., 2004) for treatment of heart failure have been reported. This present study demonstrated a novel inotropic agent AT-11, which has been extracted from Antiaris toxicaria, and investigated its pharmacological properties. Results from both in vitro and in vivo studies showed that AT11 is a potent inotropic agent. More importantly, all experiments showed that AT-11 had greater safety than ouabain did. In isolated papillary muscles, no arrhythmia occurred with AT-11 at the concentration able to increase muscle contraction up to 464%, whereas with ouabain, arrhythmia ensued already at a 249% increase in the force. Moreover, in the anesthetized guinea pigs, AT-11 also had a larger safety index. Although the onset between AT-11 and ouabain were different in vitro, most properties of these two agents on inotropic effects in vivo were similar, and the time courses of drugs' effects were almost the same. However, there is a difference between these two agents because the ouabain-induced increase of + dP / dtmax did not fall to control value even at 20 min after the administration of the agent, but AT-11-induced increase fell to basal value. Although the safety index of digitalis is narrow and arrhythmias are common problems in clinical practice, the underlying cellular mechanisms are complicated. The reason why AT-11 has a wider safety index than ouabain and the difference between these two agents also still needs to be determined. As with
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the positive inotropic effect of digitalis, the major effect of these agents on baroreflex and cardiac rhythm are also believed to result from the inhibition of the sodium pump. However, cells in different regions of the heart and the nerve have different subtypes of sodium pumps and sensitivities to digitalis, and both neurally mediated effects and direct effects on the myocytes are known to occur. At a therapeutic dose of digitalis, these agents decrease heart rate and increase maximum diastolic filling of the heart, whereas toxic doses decrease maximum diastolic filling and increase automaticity. These effects are also due to both neurally mediated increases in autonomic activity and direct effects on the myocardium (Hauptman and Kelly, 1999). Common supraventricular arrhythmias associated with digitalis toxicity originate from enhanced atrial automaticity, and several central pathways and receptors including opioid, β1 adrenergic, α2 adrenergic, dopamine, GABAA and I1-imidazoline receptors are involved in these neurally mediated arrhythmia (Demiryurek and Demiryurek, 2005). At the aspect of direct effects on the myocardium, both systolic and diastolic intracellular Ca2+ increase dramatically during digitalis-induced arrhythmias. This effect leads to Ca2+ overloading and then results in afterdepolarizations and aftercontractions via Ca2+activated transient inward current. Besides, Demiryurek and Demiryurek (2005) summarized some peripheral receptors, channels or other cellular components involving the digitalisinduced arrhythmia. Histamine, lipoxygenase, leukotrienes, cyclooxygenase, nitric oxide, β adrenoceptor, polyunsaturated fatty acids, platelet-activating factors, endothelin, angiotensin, and reactive oxygen species all play roles in arrhythmia. Furthermore, some researches indicated that the open probability of ryanodine-receptor isoform 2 (RyR2) increased and played an important role in digitalis-induced arrhythmia (McGarry and Williams, 1993; Sagawa et al., 2002). However, it has recently been shown that ouabain toxicity is not directly related to its classical action as a sodium pump inhibitor, but seems to be associated with signal transduction via formation of superoxide anion and sustained increase in tyrosine phosphorylation and Ras expression (Valente et al., 2003). Above all, there are many possibilities to explain why there are some differences between ouabain and AT-11. Whether AT11 and ouabain bind to different subtype of Na+–K+ ATPase or they bind to other receptors still needs to be further determined. As previously reported, besides the inotropic effect, digitalis had a unique property. It slowed the ventricular rate, especially in atrial fibrillation, which allows better ventricular filling. Sinus slowing and AV nodal inhibition may be due to the decrease of sympathetic tone or the increase of the vagal tone induced by digitalis (Gheorghiade and Pitt, 1997; The Digitalis Investigation Group, 1997). However, excess vagal stimulation predisposed to sinus bradycardia and AV block and caused arrhythmia. In conclusion, as a novel inotropic agent, AT-11 has a wider safety index than ouabain at comparable positive inotropic effects. On the other hand, AT-11 also enhanced more vagal reflex which may result in its greater safety. Finally, whether the difference in safety index resulted from the difference in numbers of –OH group on the steroid ring or the difference in glycoside residue remains to be determined.
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McGarry, S.J., Williams, A.J., 1993. Digoxin activates sarcoplasmic reticulum Ca(2+)-release channels: a possible role in cardiac inotropy. Br. J. Pharmacol. 108, 1043–1050. Mengi, S.A., Dhalla, N.S., 2004. Carnitine palmitoyltransferase-I, a new target for the treatment of heart failure: perspectives on a shift in myocardial metabolism as a therapeutic intervention. Am. J. Cardiovasc. Drugs 4, 201–209. Micheletti, R., Mattera, G.G., Rocchetti, M., Schiavone, A., Loi, M.F., Zaza, A., Gagnol, R.J., De Munari, S., Melloni, P., Carminati, P., Bianchi, G., Ferrari, P., 2002. Pharmacological profile of the novel inotropic agent (E,Z)-3-((2aminoethoxy)imino) androstane-6,17-dione hydrochloride (PST2744). J. Pharmacol. Exp. Ther. 303, 592–600. Rocchetti, M., Besana, A., Mostacciuolo, G., Ferrari, P., Micheletti, R., Zaza, A., 2003. Diverse toxicity associated with cardiac Na+/K+ pump inhibition: evaluation of electrophysiological mechanisms. J. Pharmacol. Exp. Ther. 305, 765–771. Sagawa, T., Sagawa, K., Kelly, J.E., Tsushima, R.G., Wasserstrom, J.A., 2002. Activation of cardiac ryanodine receptors by cardiac glycosides. Am. J. Physiol. 282, H1118–H1126. Stewart, S., MacIntyre, K., Hole, D.J., Capewell, S., McMurray, J.J., 2001. More 'malignant' than cancer? Five-year survival following a first admission for heart failure. Eur. J. Heart Fail. 3, 315–322. The Digitalis Investigation Group, 1997p. The effect of digoxin on mortality and morbidity in patients with heart failure. N. Engl. J. Med. 336, 525–533. Valente, R.C., Capella, L.S., Monteiro, R.Q., Rumjanek, V.M., Lopes, A.G., Capella, M.A., 2003. Mechanisms of ouabain toxicity. FASEB J. 17, 1700–1702. von Harsdorf, R., Poole-Wilson, P.A., Dietz, R., 2004. Regenerative capacity of the myocardium: implications for treatment of heart failure. Lancet 363, 1306–1313. Wahed, M.I., Watanabe, K., Ma, M., Nakazawa, M., Takahashi, T., Hasegawa, G., Naito, M., Yamamoto, T., Kodama, M., Aizawa, Y., 2004. Effects of pranidipine, a novel calcium channel antagonist, on the progression of left ventricular dysfunction and remodeling in rats with heart failure. Pharmacology 72, 26–32.
European Journal of Pharmacology 580 (2008) 224 – 230 www.elsevier.com/locate/ejphar
Pharmacological profile of the new inotropic agent AT-11 An-Sheng Lee a , Tian-Shung Wu b,c , Ming-Jai Su a,⁎ a
Institute of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC b Department of Chemistry, National Cheng Kung University, Tainan, Taiwan, ROC c National Research Institute of Chinese Medicine, Taipei, Taiwan, ROC Received 21 June 2007; received in revised form 2 October 2007; accepted 22 October 2007 Available online 30 October 2007
Abstract Although there are many classes of drugs, including cardiac glycosides, sympathomimetic inotropes, β-adrenergic antagonists, angiotensinconverting enzyme inhibitors (ACE inhibitors) and spironolactone etc. used for the treatment of heart failure, the morbidity and mortality rates of patients after these treatments are not ameliorated. Chronic administration of Sympathomimetic inotropes also increased the arrhythmogenic effects. Consequently, improvement of treatment for heart failure remains a major medical challenge for the coming years. In this present experiment, the novel Na+–K+ ATPase inhibitor AT-11 was characterized for its inotropic and toxic properties. Comparing AT-11 with ouabain, we found that AT-11 concentration-dependently increased contractility in guinea pig heart preparations, and the safety index of AT-11 was better than ouabain in vitro. In the in vivo study, AT-11 was also safer than ouabain at the equieffective dose. Moreover, AT-11 slowed heart rate more than ouabain did. This may be due to a larger AT-11-induced increase in vagal reflex than with ouabain and an indirect decrease in sympathetic tone to prevent Ca2+ overload. © 2007 Elsevier B.V. All rights reserved. Keywords: Digitalis; Cardiac glycoside; Heart failure; Sodium pump; Na/K ATPase
1. Introduction Heart failure is clearly a major clinical and public health problem. It is estimated that nearly 23 million people worldwide suffer from heart failure (Cleland et al., 2001). Despite recent innovations and improvements used for treating heart failure, it remains highly lethal once established (Stewart et al., 2001). Consequently, improvement of heart failure treatment remains a major medical challenge in the coming years. In chronic heart failure, the aims are to relieve symptoms, improve hemodynamic status, prevent hospitalization, and, above all, to reduce mortality. There are many clinical drugs used in combination for treatment of heart failure, including cardiac glycosides, sympathomimetic inotropes, β-adrenergic antagonists, angiotensin-converting enzyme inhibitors (ACE inhibitors), and ⁎ Corresponding author. Institute of Pharmacology, College of Medicine, National Taiwan University, No.1, Sec.1, Jen-Ai Rd. Taipei 100, Taiwan, ROC, Tel.: +886 2 23123456x8317; fax +886 2 23971403. E-mail address: [email protected] (M.-J. Su). 0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2007.10.056
now spironolactone (1999). Among these agents, digitalis is the only one with both positive inotropic effect and inhibition of neurohumoral responses. Differing from other inotropes which tend to cause tachycardia, digitalis has bradycardic action by increasing vagal tone. Digitalis has played a prominent role in the therapy of congestive heart failure since William Withering codified its use in his classic thesis on the efficacy of the common foxglove plant (Digitalis purpurea) in 1785. In the 1990s, digoxin was the most commonly prescribed drug due to the availability of techniques for measuring its serum levels, flexible routes of its administration, and its intermediate duration of action (Valente et al., 2003). Digitalis binds to and inhibits the sarcolemma Na+ pump (Na+–K+-ATPase) (Akera and Ng, 1991). This leads to a transient increase in intracellular Na+ concentration, which in turn increases intracellular Ca2+ concentration by the sodium–calcium exchange mechanism. Thus, the result is an enhanced myocardial contractility. However, when cardiac muscle is exposed to toxic concentrations of digitalis, Na+ pump inhibition and intracellular
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Ca2+ become excessive. In digitalis toxicity, the excessive Ca2+ concentration may cause afterdepolarization, and even more lifethreatening ventricular tachycardia and ventricular fibrillation. Because the therapeutic positive inotropic effect of these drugs is also caused by the enhanced Ca2+ loading, the therapeutic and toxic effects are inseparable. The narrow margin of safety index (therapeutic index) is the restriction of this class of positive inotropic drugs. That is the reason even with improvement of the whole heart function, digitalis could not reduce the rate of mortality (The Digitalis Investigation Group, 1997). In the present study the effects of the novel agent AT-11 (Fig. 1), a derivative of digitalis, are compared with the effects of ouabain in in vitro and whole animal experiments.
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2. Materials and methods
deeply anesthetized, tracheotomy was performed for artificial respiration with a stroke volume of approximately 15 ml/kg body weight and at a rate of 60 strokes/min by a respirator (model 683, Harvard Rodent ventilator). Polyethylene catheter (PE50) was inserted into the carotid vein for administration of ouabain and AT-11, and a Millar catheter with a high-fidelity pressure sensor (model SPC 320, size 2F, Millar Instruments, Houston, TX, U.S. A.) was inserted through the carotid artery into left ventricle to measure the pulsatile left ventricle pressure. The electrocardiogram (ECG) of lead II was also recorded. Left ventricular pressure, maximum velocity of pressure rise (+dP / dtmax) and fall (−dP / dtmax), heart rate, and electrocardiogram changes were calculated and recorded on a personal computer with a wave form data analysis software (PowerLab data acquisition system; AD Instruments Pty. Ltd).
2.1. In vitro assay
2.3. Electrophysiological recording
The investigation was performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Cardiac contractions were measured according to the procedure described previously (Chen et al., 2007). Adult male guinea pigs (300–500 g) were intraperitoneally injected with sodium pentobarbital (25 mg/kg) plus heparin (16 mg/kg). After the guinea pigs were deeply anesthetized, hearts were excised, and retrograde coronary perfusion with normal Tyrodes solution containing (in mM): 137 NaCl, 11.9 NaHCO3, 11 glucose, 2 CaCl2, 1.1 MgCl2, 0.33 NaH2PO4, and 5.4 KCl. Left atria strips and left papillary muscles were separated from the heart. One end of the muscle was attached to a transducer (Type BG; Gould, Cleveland, Ohio, USA) and the other end was fixed to a rigid support by a silk thread in a 10 ml organ bath. Each tissue was placed under 1 g resting tension and stimulated at 2 Hz with rectangular voltage pulses of 2 ms duration and amplitude twice threshold via an isolated Grass SD9 stimulator (Grass Instruments Co., Quiney, Mass., USA). The twitch tension was recorded on a Gould 2200s recorder.
Cardiomyocytes were isolated by using the enzymatic method as previously described (Chen et al., 2007). Adult male guinea pigs (200–250 g) were intraperitoneally injected with sodium pentobarbital (25 mg/kg) plus heparin (16 mg/kg). After the guinea pig was deeply anesthetized, heart was excised and the coronary artery was antegradely perfused with oxygenated Ca2+-free HEPES solution containing (in mM): 137 NaCl, 22 glucose, 6 HEPES, 1.2 MgSO4, 1.2 KH2PO4, and 5.4 KCl; pH was adjusted to 7.4 using NaOH. The heart was then perfused with the same solution containing 0.4 mg/ml collagenase (type II, Sigma Chemical Co., St. Louis, Mo, USA), 0.06 mg/ml protease (type XIV, Sigma) and bovine serum albumin 1 mg/ml. After 4–5 mins of digestion, enzymes were washed out in Kruftbruhe solution containing (in mM): 10 taurine, 10 oxylate, 70 glutamate, 25 KCl, 10 KH2PO4, 11 glucose, 0.5 EGTA; pH was adjusted to 7.4 using KOH (Isenberg and Klockner, 1982). The ventricles were then chopped and resuspended under gentle mechanical agitation and stored in Kruftbruhe solution at room temperature. The whole-cell patch clamp technique was used to record ionic currents in voltage clamp mode with a Dagan 8900 voltage clamp amplifier (Dagan Co., Minneapolis, Minn., U.S.A.). A droplet of the cell suspension was placed in a chamber mounted on the stage of an inverted microscope (Nikon, Diaphot, Japan). After settling down, cells were finally exposed to the bath solution containing (in mM):137 NaCl, 5.4 KCl, 2.9 MgCl2, 6 HEPES, 22 glucose, 0.33 NaH2PO4, 2 BaCl2, and 0.2 CdCl; pH was adjusted to 7.4 using NaOH. All experiments were performed at 30 ± 0.5 °C. For the measurement of Ipump, a pipette was filled with the internal solution containing (mM): 80 CsOH, 50 NaOH, 3 MgCl2, 20 TEA-Cl, 100 aspartic acid, 10 HEPES, 10 ATP-Mg, 0.2 GTP-Na3, 5.5 glucose, 5 Na-creatine phosphate, and 5 pyruvic acid; pH was adjusted to 7.2 using CsOH as previous described (Gadsby et al., 1985). Heatpolished glass electrodes (tip resistances about 1 MΩ when filled with pipette internal solution) were used. Junction potentials were zeroed before the formation of the membrane– pipette seal in bath solution. The series resistance was electronically compensated by about 80% to minimize the duration
2.2. In vivo assay Adult male guinea pigs (400–550 g) were intraperitoneally injected with urethane (600 mg/kg). After the guinea pigs were
Fig. 1. Chemical structure of AT-11.
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Fig. 2. Comparison of twitch amplitude between AT-11 and ouabain. Concentration-dependent increases in twitch amplitude caused by both AT-11 (filled circles, n = 5) and ouabain (open circles, n = 5) in isolated left atria (A), and papillary muscles (B). (C) Twitch amplitude trace of papillary muscle in the presence of AT-11. Shortly after addition, the concentration of 5 μM caused arrhythmias.
of the capacitive surge on the current recorded and the voltage drop across the pipette. Currents were elicited and acquired using Digidata 1200 data acquisition system controlled using pClamp software (Axon Instruments). Recordings were lowpass filtered at 10 kHz and stored on the hard disk of a computer. 2.4. Statistical analysis The results were presented as means ± standard error of the mean (S.E.M.). Significant differences between treatment groups were determined by one-way ANOVA with post hoc analysis using the Bonferroni t-test. 3. Results 3.1. Inotropic effects of AT-11 and ouabain in isolated cardiac muscles In this experiment, both of AT-11 and ouabain concentrationdependently increased the twitch amplitudes relative to control in left atria and papillary muscles (Fig. 2A and B, respectively). Maximal twitch amplitudes of left atria and papillary muscles are expressed as percentages of basal values (0.7± 0.1 g and 0.8 ± 0.1 g of left atria, 0.2 ± 0.1 g and 0.2± 0.1 g of papillary muscles in AT-
11 and ouabain groups, respectively) and plotted as functions of concentration of AT-11 and ouabain. At higher concentrations, further enhancements of inotropic effects were often accompanied with irregular twitches (Fig. 2C). In detail, the minimal effective concentration to increase myocardial contraction, the arrhythmogenic concentration to induce arrhythmia, and the onset of AT-11 and ouabain were measured as shown in Table 1. The maximal positive inotropic effect was determined at a concentration level immediately before the occurrence of cardiac arrhythmia and the safety index was calculated from the ratio of the arrhythmogenic concentration to the minimal effective positively inotropic concentration. The onsets of AT-11 in the left atria and papillary muscles are both shorter than those of ouabain (51.7 ± 4.0 versus 76.7 ± 3.3s and 44.0 ± 2.5 versus 83.3 ± 14.5s, respectively). The time to steady state of inotropic effect at therapeutic concentration, or the time to cause arrhythmia at arrhythmogenic concentration, was not significantly different between these two agents (data not shown). In the left atria, both the minimum effective concentration and arrhythmogenic concentration of AT-11 were 5fold higher than those of ouabain (0.25 versus 0.05 μM and 5 versus 1 μM, n = 5). The maximum inotropic effects were roughly similar of these two agents (660 ± 88 versus 775 ± 128%, n = 5, N.S.). However, in papillary muscles, the maximum
Table 1 Comparison of AT-11 and ouabain on onset, minimal effective concentration, arrhythmogenic concentration, maximal positive inotropic effect, and safety index in guinea pig isolated left atria and papillary muscles
Left atria Papillary muscle
AT-11 Ouabain AT-11 Ouabain
Onset (sec)
Minimal effective concentration (μM)
Arrhythmogenic concentration (μM)
Maximal positive inotropic effect (%)
Safety index
51.7 ± 4.0a 76.7 ± 3.3 44.0 ± 2.5a 83.3 ± 14.5
0.25 0.05 0.25 0.05
5 1 6 1
660 ± 88 775 ± 128 464 ± 89a 249 ± 26
20 20 24 20
Values of onset and maximal positive inotropic effect are shown as mean ± S.E.M. aP b 0.05 between AT-11 and ouabain. (ANOVA).The safety index was calculated from the ratio of the arrhythmogenic concentration to the minimal effective positively inotropic concentration.
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inotropic effect of AT-11 was significantly higher than that of ouabain (464 ± 89 versus 249 ± 26%, n = 5). The safety index of AT-11 is also higher than that of ouabain (24 versus 20, n = 5). 3.2. Inotropic Effects of AT-11 and Ouabain in Anesthetized Guinea Pigs Not only in-vitro experiments but also the in vivo study revealed that the safety of AT-11 was better than that of ouabain. Fig. 3 shows comparison of dose responses of maximal left ventricle pressure between AT-11 and ouabain. Left ventricular pressure is expressed as percentage of basal value (54.6 ± 1.2 mmHg and 51.9± 1.9 mmHg in AT-11 and ouabain groups, respectively) and plotted as a function of various AT-11 (0.016, 0.053, 0.16, 0.53, 1.6, and 5.3 mg/kg) and ouabain (0.0029, 0.0058, 0.029, and 0.058 mg/kg) doses. At the dose of 0.0029 mg/kg, ouabain began to enhance pressure to 106.17± 1.84% compared to the control. However, when the dose was increased to 0.029 mg/kg, ouabain reached the maximal inotropic effect (142.0± 6.5%), and even at higher dose (0.058 mg/kg) which resulted in 50% animal death (5 of 10 rats died due to arrhythmia) could not increase more ventricular pressure (140.2 ± 5.6%). On the other hand, AT-11 started to increase pressure to 106.18 ± 2.49% at the dose (0.016 mg/kg) higher than ouabain and reached the maximum inotropic effect (177.5 ±14.7%) at the dose of 5.3 mg/kg with 43% animal death (3 of 7 rats died due to arrhythmia). The effects of the maximal velocity of pressure rise (+dP /dtmax) and fall (−dP / dtmax) were also calculated. +dP /dtmax and −dP /dtmax are expressed as percentage of basal value (3043.9± 176.4 mmHg/s and 2901.4 ± 188.2 mmHg/s of +dP / dtmax and −2771.7 ± 133.5 mmHg/s and −2547.0 ± 125.1 mmHg/s of −dP /dtmax in AT-11 and ouabain groups, respectively) and plotted as functions of various AT-11 (0.016, 0.053, 0.16, 0.53, 1.6, and 5.3 mg/kg) and ouabain (0.0029, 0.0058, 0.029, and 0.058 mg/kg) doses. The effects of AT-11 and ouabain on −dP /dtmax were similar to those on left ventricular pressure (Fig. 4B). However, the effect of AT-11 on +dP /dtmax was different from that on ventricular pressure and −dP / dtmax. AT-11 induced increase of +dP / dtmax reached the
Fig. 3. Comparison of dose-dependent increase of maximal left ventricular pressure between AT-11 (circles, n = 10, 10, 10, 10, 10, 4) and ouabain (triangles, n = 10, 10, 10, 5) in anesthetized guinea pigs. Left ventricular pressure is expressed as percentage of basal value and plotted as a function of various doses of AT-11 (0.016, 0.053, 0.16, 0.53, 1.6, 5.3 mg/kg) and ouabain (0.0029, 0.0058, 0.029, 0.058 mg/kg).
Fig. 4. Comparison of dose-dependent increases of maximal velocity of pressure rise (+dP / dtmax, A) and fall (− dP / dtmax, B) between AT-11 (circles, n = 10, 10, 10, 10, 10, 4) and ouabain (triangles, n = 10, 10, 10, 5) in anesthetized guinea pigs. +dP / dtmax and − dP / dtmax are expressed as percentages of basal values and plotted as functions of various doses of AT-11 (0.016, 0.053, 0.16, 0.53, 1.6, 5.3 mg/kg) and ouabain (0.0029, 0.0058, 0.029, 0.058 mg/kg).
peak at the dose of 1.6 mg/kg and did not increase as the dose increased (Fig. 4A). Although the dose needed to reach the equieffective inotropic effect of AT-11 was 27-fold higher than that of ouabain, AT-11 was safer than ouabain. Lethal arrhythmias occurred in 50% (5 of 10) of animals treated with ouabain at a dose of 0.058 mg/kg. In contrast, no arrhythmias occurred in the animals treated with
Fig. 5. Effect of equieffective concentrations of AT-11 and ouabain on maximal left ventricular pressure in anesthetized guinea pigs. Left ventricular pressure is expressed as percentage of basal value and plotted as a function of time at the dose of AT-11 (1.6 mg/kg, filled circles, n = 10), ouabain (0.029 mg/kg, open circles, n = 10), and vehicle (triangles, n = 10). ⁎, P b 0.05 between AT-11 and vehicle; #, P b 0.05 between ouabain and vehicle. (ANOVA)
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AT-11 at the equieffective dose (1.6 mg/kg). AT-11 began to cause arrhythmia (43%) at the dose of 5.3 mg/kg, and the safety index was calculated as 331.25 which was much higher than that of ouabain (20). Fig. 5 shows time courses of AT-11 and ouabain inotropic effects at equieffective doses (1.6 and 0.029 mg/kg, respectively). AT-11 induced an increase in left ventricular pressure that reached the peak of 144.7 ± 7.8% relative to basal at 2 min after intravenous administration of the agent. After the peak, a decrease toward control value occurred throughout the period. A comparable increase (142.0 ± 6.5%) was afforded by administration of ouabain. AT-11 at this dose also caused increases in + dP / dtmax and −dP / dtmax related to control status with peaks of 198.2 ± 17.8 and 156.87 ± 18.34%, respectively whereas ouabain increased + dP / dtmax and − dP / dtmax with peaks of 193.7 ± 17.1 and 141.0 ± 6.9%, respectively (Fig. 6). Ouabaininduced increase of + dP / dtmax did not fall to control value even at 20 min after the administration of the agent, but AT-11induced increase did. Differing from other inotropes which tend to cause tachycardia, digitalis has bradycardic action by increasing vagal tone. The effects of AT-11 and ouabain on heart rate were also measured (Fig. 7). Heart rate is expressed as percentage of a basal value (273.1 ± 7.3 bpm and 263.2 ± 5.6 bpm in AT-11 and ouabain groups, respectively) and plotted as a function of time at
Fig. 7. Effect of equieffective concentrations of AT-11 and ouabain on heart rate in anesthetized guinea pigs. Heart rate is expressed as percentage of basal value and plotted as a function of time at the dose of AT-11 (1.6 mg/kg, filled circles, n = 10), ouabain (0.029 mg/kg, open circles, n = 10), and vehicle (triangles, n = 10). ⁎, P b 0.05 between AT-11 and vehicle. (ANOVA)
the dose of AT-11 (1.6 mg/kg, filled circles), ouabain (0.029 mg/ kg, open circles) and vehicle (triangles). Three minutes after administration of ouabain, the heart rate started to be decreased 3% related to vehicle (98.7 ± 1.2 versus 101.8 ± 1.3%, N.S.) but without statistical significance until the end of this experiment. In contrast, AT-11 decreased 7% heart rate (94.6 ± 2.3%) through this period. 3.3. Na+/K+ pump current (Ipump) recordings To confirm that the inotropic effect of AT-11 was through the inhibition of Na+/K+ ATPase like other digitalis, sodium pump current (Ipump) was measured by whole-cell patch clamp technique (Fig. 8). After rupture of the patch, the holding potential was set at − 40 mV to inactivate Na+ channels and the cell interior was allowed to equilibrate 5 min with the pipette solution. Then membrane currents were elicited by voltage ramps from + 60 mV to − 120 mV (see inset of Fig. 8). Ipump was defined as the difference of current between after and before AT11 or ouabain treatment. Fig. 8 shows the effects on Ipump current–voltage relationships of ouabain at 10 μM (filled squares, n = 6), AT-11 at 10 μM (open circles, n = 4), AT-11 at 30 μM (open squares, n = 4), and vehicle (filled circles, n = 4) sensitive currents. Both ouabain and AT-11 sensitive currents
Fig. 6. Effect of equieffective concentrations of AT-11 and ouabain on maximal velocity of pressure rise (+ dP / dtmax, A) and fall (− dP / dtmax, B) in anesthetized guinea pigs. +dP / dtmax and − dP / dtmax are expressed as percentages of basal values and plotted as functions of time at the dose of AT11 (1.6 mg/kg, filled circles, n = 10), ouabain (0.029 mg/kg, open circles, n = 10), and vehicle (triangles, n = 10). ⁎, P b 0.05 between AT-11 and vehicle; #, P b 0.05 between ouabain and vehicle. (ANOVA)
Fig. 8. Drug effects on Ipump. Current–voltage relationships of ouabain at 10 μM (filled squares, n = 6), AT-11 at 10μM (open circles, n = 4), AT-11 at 30 μM (open squares, n = 4), and vehicle (filled circles, n = 4) sensitive currents were obtained by applying slow hyperpolarizing ramps as inset showed.
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were near linear, and at negative potentials they declined with voltage towards an extrapolated zero-current potential near about − 140 mV. The current densities at 0 mVof ouabain, AT-11 10 μM, AT-11 30 μM, and vehicle were 0.53 ± 0.09, 0.63 ± 0.08, 0.71 ± 0.10, and 0.03 ± 0.03 pA/pF, respectively (with cell capacitance = 83.9 ± 6.4 pF). 4. Discussion There are many classes of drugs which are used for the treatment of heart failure (1999). Among them, β-adrenergic antagonists are known to have the potential to worsen both ventricular function and symptoms in patients with heart failure. Although sympathomimetic inotropes cause a marked improvement in cardiac systolic function and may alleviate symptom and improve exercise capacity, long term treatment with these agents has been shown to increase mortality. Therefore, cardiac glycosides such as digoxin and ouabain remain as important inotropes used in patients with heart failure. Digitalis derivatives are found in several plants, including foxglove, lily and oleander. In spite of several advances in the treatment of cardiovascular disorders, digitalis has been widely used for centuries as a therapeutic agent for the treatment of congestive heart failure and has remained one of the most commonly prescribed drugs in many countries (The Digitalis Investigation Group, 1997). However, as the safety index of digitalis is narrow, arrhythmias are common problems in clinical practice (Khatter et al., 1986, 1989). Therefore, improvements of digitalis and other heart failure treatments remain a major medical challenge for the coming years. In recent years, some novel agents (Micheletti et al., 2002; Rocchetti et al., 2003; Wahed et al., 2004) and mechanisms (Chierchia and Deferrari, 2004; Mengi and Dhalla, 2004; von Harsdorf et al., 2004) for treatment of heart failure have been reported. This present study demonstrated a novel inotropic agent AT-11, which has been extracted from Antiaris toxicaria, and investigated its pharmacological properties. Results from both in vitro and in vivo studies showed that AT11 is a potent inotropic agent. More importantly, all experiments showed that AT-11 had greater safety than ouabain did. In isolated papillary muscles, no arrhythmia occurred with AT-11 at the concentration able to increase muscle contraction up to 464%, whereas with ouabain, arrhythmia ensued already at a 249% increase in the force. Moreover, in the anesthetized guinea pigs, AT-11 also had a larger safety index. Although the onset between AT-11 and ouabain were different in vitro, most properties of these two agents on inotropic effects in vivo were similar, and the time courses of drugs' effects were almost the same. However, there is a difference between these two agents because the ouabain-induced increase of + dP / dtmax did not fall to control value even at 20 min after the administration of the agent, but AT-11-induced increase fell to basal value. Although the safety index of digitalis is narrow and arrhythmias are common problems in clinical practice, the underlying cellular mechanisms are complicated. The reason why AT-11 has a wider safety index than ouabain and the difference between these two agents also still needs to be determined. As with
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the positive inotropic effect of digitalis, the major effect of these agents on baroreflex and cardiac rhythm are also believed to result from the inhibition of the sodium pump. However, cells in different regions of the heart and the nerve have different subtypes of sodium pumps and sensitivities to digitalis, and both neurally mediated effects and direct effects on the myocytes are known to occur. At a therapeutic dose of digitalis, these agents decrease heart rate and increase maximum diastolic filling of the heart, whereas toxic doses decrease maximum diastolic filling and increase automaticity. These effects are also due to both neurally mediated increases in autonomic activity and direct effects on the myocardium (Hauptman and Kelly, 1999). Common supraventricular arrhythmias associated with digitalis toxicity originate from enhanced atrial automaticity, and several central pathways and receptors including opioid, β1 adrenergic, α2 adrenergic, dopamine, GABAA and I1-imidazoline receptors are involved in these neurally mediated arrhythmia (Demiryurek and Demiryurek, 2005). At the aspect of direct effects on the myocardium, both systolic and diastolic intracellular Ca2+ increase dramatically during digitalis-induced arrhythmias. This effect leads to Ca2+ overloading and then results in afterdepolarizations and aftercontractions via Ca2+activated transient inward current. Besides, Demiryurek and Demiryurek (2005) summarized some peripheral receptors, channels or other cellular components involving the digitalisinduced arrhythmia. Histamine, lipoxygenase, leukotrienes, cyclooxygenase, nitric oxide, β adrenoceptor, polyunsaturated fatty acids, platelet-activating factors, endothelin, angiotensin, and reactive oxygen species all play roles in arrhythmia. Furthermore, some researches indicated that the open probability of ryanodine-receptor isoform 2 (RyR2) increased and played an important role in digitalis-induced arrhythmia (McGarry and Williams, 1993; Sagawa et al., 2002). However, it has recently been shown that ouabain toxicity is not directly related to its classical action as a sodium pump inhibitor, but seems to be associated with signal transduction via formation of superoxide anion and sustained increase in tyrosine phosphorylation and Ras expression (Valente et al., 2003). Above all, there are many possibilities to explain why there are some differences between ouabain and AT-11. Whether AT11 and ouabain bind to different subtype of Na+–K+ ATPase or they bind to other receptors still needs to be further determined. As previously reported, besides the inotropic effect, digitalis had a unique property. It slowed the ventricular rate, especially in atrial fibrillation, which allows better ventricular filling. Sinus slowing and AV nodal inhibition may be due to the decrease of sympathetic tone or the increase of the vagal tone induced by digitalis (Gheorghiade and Pitt, 1997; The Digitalis Investigation Group, 1997). However, excess vagal stimulation predisposed to sinus bradycardia and AV block and caused arrhythmia. In conclusion, as a novel inotropic agent, AT-11 has a wider safety index than ouabain at comparable positive inotropic effects. On the other hand, AT-11 also enhanced more vagal reflex which may result in its greater safety. Finally, whether the difference in safety index resulted from the difference in numbers of –OH group on the steroid ring or the difference in glycoside residue remains to be determined.
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