- Email: [email protected]
Istaroxime: A New Luso-Inotropic Agent for Heart Failure
Istaroxime: A New Luso-Inotropic Agent for Heart Failure Giovan Giuseppe Mattera, MD,a,* Pietro Lo Giudice, BSc,a Francesca M. P. Loi, BSc,a Emilio Vanoli, MD,b,c Jean-Pierre Gagnol, MD,d Franco Borsini, PhD,a and Paolo Carminati, MDa Istaroxime is a new luso-inotropic compound selected for the treatment of acute heart failure syndromes, which reduces sodium-potassium adenosine triphosphatase (ATPase) activity and stimulates the sarcoplasmic calcium ATPase isoform 2 reuptake function. The aim of this study was to evaluate the safety profile of istaroxime. For this purpose, istaroxime was administered during a 24-hour infusion to conscious dogs with chronic heart failure and to genetically cardiomyopathic BIO TO.2 hamsters for 34 weeks orally. The parameters recorded were arrhythmic events and hemodynamic effects in dogs and mortality in hamsters. In dogs, istaroxime at 1, 3, and 4 g/kg per min did not trigger arrhythmic events or magnify preexisting events. It increased left ventricular (LV) dP/dtmax (about 50% at 3 g/kg per min) and LV ⴚ dP/dtmax (about 20% at 3 g/kg per min) without changing heart rate, blood pressure, or double product. At 4 g/kg per min, istaroxime increased dP/dtmax >100% but induced intense emesis in all animals. In cardiomyopathic hamsters, the dose of 30 mg/kg prolonged the survival rate to 32%. In conclusion, istaroxime seems to be a promising and safe new drug for improving cardiac performance in the failing heart. © 2007 Elsevier Inc. All rights reserved. (Am J Cardiol 2007;99[suppl]:33A– 40A)
Acute heart failure syndromes (AHFS) represent a significant public health burden, resulting in 1 million hospitalizations annually in the United States.1 Patients hospitalized for treatment of AHFS have a readmission rate as high as 30%– 60% within 3– 6 months after initial discharge,2 and the mortality rate in the same period remains very high (25%–55%). Moreover, clinical trials show that dobutamine and phosphodiesterase inhibitors, despite their efficacy in acute symptomatic relief, lead to increased mortality rates.3,4 In fact, such agents are associated with increased ventricular ectopy, tachyarrhythmias, ischemia, and hypotension, especially at high doses. Digitalis has long been the main instrument for improving contractility of the failing heart by increasing intracellular calcium, as a consequence of its inhibition of sodium-potassium adenosine triphosphatase (ATPase). However, its use is progressively decreasing because of its proarrhythmic potential, specifically in the ischemic heart. Indeed, although increased intracellular calcium concentrations improve contractility, they also enhance calcium-dependent arrhythmia. Overall, cardiac contractility support therapy remains among the most critical aspects of acute cardiac decompensation management. Istaroxime, previously called PST2744, is a compound a Research and Development Division, sigma-tau Industrie Farmaceutiche Riunite SpA, Pomezia, Italy; bDepartment of Cardiology, University of Pavia, Pavia, Italy; cDepartment of Cardiology, Policlinico di Monza, Monza, Italy; and dUniversity Hospital CNRS, Montpellier, France. *Address for reprints: Giovan Giuseppe Mattera, MD, Research and Development, sigma-tau Industrie Farmaceutiche Riunite SpA, Via Pontina Km 30,500, 00040 Pomezia, Italy. E-mail address: [email protected].
0002-9149/07/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.amjcard.2006.09.004
with a novel mechanism of action, exerting its effects by inhibiting sodium-potassium ATPase activity5–7 and, in parallel, by stimulating sarcoplasmic reticulum calcium ATPase (SERCA) isoform 2a (SERCA2a). Whereas inhibition of sodium-potassium ATPase activity produces cytosolic calcium accumulation and an inotropic response, SERCA2a stimulation results in rapid clearance of cytosolic calcium, thus preventing calcium accumulation (calcium intoxication) and facilitating myocardial relaxation (lusitropism).8 These 2 properties invest istaroxime with the unique characteristic of acting as a calcium cycling modulator. Short-time istaroxime infusion improves left ventricular (LV) function and is safe in animals with normal heart or myocardial damage.5,7,9,10 Therefore, it is necessary to test this principle in experimental conditions (prolonged duration of administration) that better mimic clinical reality. Accordingly, the aims of the present experiments were (1) to assess the safety and efficacy of istaroxime in conscious dogs with chronic heart failure (HF) during 24-hour infusion, and (2) to evaluate the effects of long-term treatment with istaroxime on mortality in genetically cardiomyopathic Syrian BIO TO.2 hamsters.11
Materials and Methods All experiments were conducted according to international guidelines and approved by the Italian Ministry of Health. Animals were routinely monitored by a veterinarian. www.AJConline.org
34A
The American Journal of Cardiology (www.AJConline.org) Vol 99 (2A) January 22, 2007
Figure 1. Type of electrocardiographic changes (tachyarrhythmias and bradyarrhythmias) in animals with different doses of istaroxime. AV ⫽ atrioventricular; n ⫽ number of dogs in each group; PVC ⫽ premature ventricular contractions; SA ⫽ sinoatrial; VT ⫽ ventricular tachycardia.
Experiments in dogs: ANIMALS. A total of 7 male beagles (Harlan, Udine, Italy) weighing 14 –18 kg were used; 1 of 7 of these dogs was kept as a reserve. ANESTHESIA. Overnight-fasted dogs were preanesthetized with an intramuscular injection of Zoletil 100 (tiletamine-zolazepam; Virbac Laboratories, Carros, France). After endotracheal intubation, the surgical anesthesia level was obtained and maintained by ventilation with isoflurane (1%–2%) in an oxygen–nitrous oxide mixture. Anesthesia was used to implant telemetric transmitters and for microembolization. TELEMETRIC TRANSMITTER IMPLANT. Body temperature was maintained at 38°C ⫾ 1°C. The apex of the heart was reached through a transdiaphragmatic approach by laparotomy. A small incision was made in the muscular part of the diaphragm, exposing the apex of the heart; then, a telemetric transducer catheter was inserted into the left ventricle. The second pressure catheter was introduced into the mesenteric artery to monitor arterial blood pressure. Then, 2 electrocardiographic wires were positioned subcutaneously in the upper left and in the lower right thoracic area. Finally, the telemetric transducer was fastened into the abdomen and secured. The animals were allowed to recover for ⱖ4 weeks
before the microembolization procedure. After all surgical procedures, the antibiotic Spectrum 1 g/day (ceftazidime; sigma-tau, SpA, Pomezia, Italy) and the analgesic Contramal (tramadol; Formenti s.r.l., Milan, Italy) 50 mg twice daily were administered for 1 week. MICROEMBOLIZATION PROCEDURE. Chronic LV dysfunction was produced by multiple sequential intracoronary embolizations with polystyrene latex microspheres (Polysciences, Warrington, PA) according to Sabbah et al.12 Briefly, under general anesthesia, angiography of the left coronary artery was performed. After adequate visualization of the coronary bed, the embolization procedure was initiated. A solution (1.5 mL) containing microspheres 45–90 m in diameter was embolized into the coronary artery through the preformatted femoral catheter. Embolizations were performed 1–3 weeks apart. In all, 2–3 embolizations were necessary to reach an LV ejection fraction ⬎0.35, which was controlled by echocardiography. ISTAROXIME ADMINISTRATION. The experiment was performed by using a 3 ⫻ 3 Latin square design. Istaroxime (at 1 or 3 g/kg per min) or vehicle was administered via the jugular vein. The intravenous infusion lasted 24 hours, with
Mattera et al/Istaroxime: A New Luso-Inotropic Agent for Heart Failure
35A
Figure 2. Effects of istaroxime on left ventricular ⫹dP/dtmax. Points are mean ⫾ SEM of 6 dogs.
a ⱖ1-week washout period between infusions. Administration volume was 50 mL. Because 1 and 3 g/kg per min istaroxime did not cause proarrhythmic effects, a third dose of 4 g/kg per min was infused for 24 hours in 4 dogs from the original group (ancillary study). OBSERVATIONS AND TIMING. The recorded parameters were as follows: electrocardiographic DII, systolic blood pressure (SBP) and diastolic blood pressure (DBP), LV pressure and derivatives (LV ⫾ dP/dtmax), heart rate, and double product (SBP ⫻ heart rate). All telemetric signals were collected and processed by a validated telemetric computerized system (Dataquest ART Gold V 3.01; Data Sciences International, St. Paul, MN). Parameters were recorded for 1 minute every 15 minutes, starting 2 hours before administration, then during the following 24-hour period, and for 4 hours after termination of infusion. DATA EVALUATION. Data were visually checked to eliminate possible artifacts, and the values were averaged per hour. Electrocardiograms were inspected and arrhythmias classified as shown in Figure 1. STATISTICS. The experimental 3 ⫻ 3 Latin square design was repeated twice, and analysis of variance was applied by pooling results from the 2 experiments. Experiments in hamsters: ANIMALS. Cardiomyopathic BIO TO.2 and BIO F1B, 8- to 10-week-old Syrian hamsters (BIO Breeders, Watertown, MA) were used.
SURVIVAL. BIO TO.2 hamsters were randomized to enter daily treatment with 30 mg/kg of oral istaroxime (n ⫽ 50) or with the oral vehicle (5 mL/kg, n ⫽ 50) from the age of 12– 46 weeks. BIO F1B hamsters (n ⫽ 20) served as healthy controls. The number of dead hamsters was recorded weekly until the 54th week of age. LEVELS OF LV TUMOR NECROSIS FACTOR–␣. BIO TO.2 hamsters were randomized to daily treatment with oral 30 mg/kg istaroxime (n ⫽ 10) or oral vehicle (n ⫽ 10) from 12 weeks of age for about 4 months. BIO F1B hamsters (n ⫽ 10) were used as healthy controls. On the day after the last istaroxime administration (day 28 ⫾ 1 week), animals were anesthetized, hearts rapidly excised, and left ventricles dissected and homogenated. The supernatant was divided into aliquots, frozen, and stored at a temperature of ⫺80°C. Tumor necrosis factor–␣ (TNF-␣) analysis was performed on the supernatant with an enzyme-linked immunosorbent assay kit (Endogen Inc., Rockford, IL) according to the method suggested by the provider. Results are expressed as picograms per milligram of ventricular proteins. STATISTICS. Survival was analyzed by Kaplan-Meier analysis with a log-rank test. Because TNF-␣ values did not meet the homogeneity of variances, they were expressed as median (and interquartile range) and were compared using the Kruskal-Wallis test.
36A
The American Journal of Cardiology (www.AJConline.org) Vol 99 (2A) January 22, 2007
Figure 3. Effects of istaroxime on left ventricular – dP/dtmax. Points are mean ⫾ SEM of 6 dogs.
Compounds: (EZ)-3-(2-aminoethoxyimino)androstane-6, 17-dione hydrochloride (istaroxime), synthesized in sigmatau’s chemistry department, was dissolved in saline solution (dog experiment) or water (hamster experiment). Pentobarbital sodium (Sigma Chemical Co., St. Louis, MO) was dissolved in saline solution. Results Conscious dogs with chronic LV dysfunction: CLINICAL SIGNS AND BEHAVIOR. Dogs did not show signs of intolerance or discomfort during administration of vehicle or 1 g/kg per min istaroxime for 24 hours and presented regurgitation of food within 4 –5 hours only at an infusion rate of 3 g/kg per min. EFFECTS ON HEMODYNAMICS. Istaroxime-induced hemodynamic effects were independent of treatment sequence (F(2,17) ⫽ 1.34, p ⫽ 0.38). Istaroxime dose dependently increased (F(2,17) ⫽ 11.80, p ⬍0.004) cardiac inotropism (LV ⫹dP/dtmax), which reached a peak after 8 –10 hours and remained constant throughout the experiment (Figure 2). Lusitropism (LV ⫺dP/dtmax) (Figure 3) showed a nonstatistically significant trend to increase during infusions of 1 and 3 g/kg per min (p ⫽ 0.213). Heart rate (p ⫽ 0.703) was not affected by istaroxime (Figure 4). Likewise, SBP (p ⫽ 0.974) and DBP (p ⫽ 0.957) were not changed, even if SBP values were slightly higher in dogs infused with 3 g/kg per min
istaroxime in the first hours (Figure 5). Double product values, which represent an indirect evaluation of oxygen consumption, showed nonstatistically significant (p ⫽ 0.468) variations during istaroxime infusion (Figure 6). In the group receiving istaroxime at 4 g/kg per min, dP/dtmax was increased by ⬎100%, and vomiting was intense. ELECTROCARDIOGRAPHIC LEAD II. As seen in Figure 1, 4 of 7 microembolized dogs presented with spontaneous, low-degree arrhythmias (premature ventricular contractions, couplet, bigeminism, or sinoatrial block). Administration of istaroxime at 1 and 3 g/kg per min for 24 hours did not worsen the basal picture in these 4 dogs, nor was it associated with proarrhythmia in dogs without baseline arrhythmias. The evidence collected at 1 and 3 g/kg per min made it reasonable to test the higher dose of 4 g/kg per min in 4 dogs to further challenge the tolerability, safety, and efficacy of this compound; 2 of 4 microembolized dogs used in this ancillary study presented with basal spontaneous, low-degree arrhythmias (premature ventricular contractions, couplet, bigeminism, or sinoatrial block). In these dogs, administration of istaroxime at 4 g/kg per min for 24 hours did not induce a worsening of the basal situation. Similar findings were obtained in 1 of the 2 dogs without spontaneous arrhythmias at baseline; the other presented a short period of bigeminism after 13 hours of istaroxime infusion.
Mattera et al/Istaroxime: A New Luso-Inotropic Agent for Heart Failure
37A
Figure 4. Effects of istaroxime on heart rate. Points are mean ⫾ SEM of 6 dogs.
Hamsters: SURVIVAL. None of the healthy BIO F1B hamsters died during the observation period. Figure 7 shows that all vehicle-treated BIO TO.2 hamsters died at 26 –52 weeks of age. On the contrary, 16 (32%) istaroxime-treated BIO TO.2 hamsters survived until the age of 54 weeks. Kaplan-Meier survival analysis shows that istaroxime treatment significantly prolonged the survival of cardiomyopathic hamsters (p ⬍0.05). LV TNF-␣. TNF-␣ levels were markedly augmented in cardiomyopathic BIO TO.2 hamsters (median [interquartile range]: 124.8 pg/g protein [119.8 –142.2 pg/g protein]) when compared with healthy BIO F1B animals (median [interquartile range]: 61.0 pg/g protein [53.0 –70.8 pg/g protein]). Istaroxime prevented an increase in TNF-␣ levels in cardiomyopathic BIO TO.2 hamsters (median [interquartile range]: 47.2 pg/g protein [40.4 – 69.0 pg/g protein]); Kruskal-Wallis test: 22 ⫽ 15.77, p ⬍0.001).
Discussion This study, which was conducted in conscious animals with LV dysfunction, shows that both short-term (24hour infusion) and long-term (oral) istaroxime adminis-
tration have powerful beneficial effects on cardiac performance and survival, without inducing relevant side effects. The combined mechanisms of action by sodiumpotassium ATPase activity inhibition and SERCA2a stimulation in the same molecule may explain these findings. Compared with digoxin, istaroxime induced positive inotropic effects with a wider therapeutic ratio in anesthetized guinea pigs and healthy dogs,5 as well as in conscious dogs with a small anterior myocardial infarction during treadmill exercise.7 Similar findings were observed in short-term (1-hour) infusion in anesthetized dogs with acute LV dysfunction10 and in conscious dogs with chronic LV dysfunction.9 The present study goes beyond the previous studies by exploring the effects of istaroxime in experimental settings (prolonged duration of intravenous administration) that better mimic clinical reality. In conscious dogs with HF, istaroxime exerted a sustained positive action on LV performance (increase in LV ⫹dP/dtmax) that peaked at the 10th hour of infusion and remained constant throughout the 24-hour infusion period. This positive hemodynamic activity occurred without relevant side effects. The primary risk in inotropic
38A
The American Journal of Cardiology (www.AJConline.org) Vol 99 (2A) January 22, 2007
Figure 5. Effects of istaroxime on (A) systolic blood pressure (SBP) and (B) diastolic blood pressure (DBP). Points are mean ⫾ SEM of 6 dogs.
support is proarrhythmia. This is true for all currently used agents, from digoxin to dobutamine to phosphodiesterase inhibitors. Istaroxime did not trigger “de novo” ventricular arrhythmia events or magnify any preexisting events. In fact, life-threatening arrhythmias were never seen even with the high, powerful dose of 4 g/kg per min. The only side effect at this high dose was vomiting. The discomfort associated with vomiting may partially explain the slight alteration in blood pressure evident only with the higher dose. Thus, it appears that during the 24 hours of infusion, the maximum tolerated infusion rate of istaroxime is 3 g/kg per min. At this dose, istaroxime induced lusitropic and inotropic effects in the absence of relevant changes in systemic blood pressure, heart rate,
and oxygen consumption as derived from the double product index. The BIO TO.2 hamster is a genetic model of dilated cardiomyopathy that develops up to the end-stage level through well-defined steps. In fact, the prenecrotic phase is observable at 4 – 6 weeks of age; the necrotic phase, from 10 –16 weeks; the healing stage, from 18 –22 weeks; and cardiac hypertrophy, from 25–33 weeks; then, progressive dilatation and terminal HF develop.13 A significant imbalance of autonomic nervous system activity related to the evolution of cardiomyopathy was also described in this hamster strain.14 The longer-duration survival rate of istaroxime-treated hamsters (32%) compared with vehicle-treated animals suggests that the compound
Mattera et al/Istaroxime: A New Luso-Inotropic Agent for Heart Failure
39A
Figure 6. Effects of istaroxime on double product. Points are mean ⫾ SEM of 6 dogs.
Figure 7. Survival curves of Bio TO.2 hamsters treated with vehicle or istaroxime. Treatment started when hamsters were 12 weeks old. (p ⬍0.001 Kaplan-Meier test.)
slows down some critical mechanisms involved in the progression of HF. This is supported by the fact that the increase in cardiac TNF-␣ levels found in Bio TO2 hamsters compared with BIO F1B hamsters was decreased by istaroxime when the animals were 28 weeks old. Conclusion Istaroxime is a powerful luso-inotropic agent that, in animal models of HF, does not enhance the risk of ar-
rhythmia. The lusitropic effect of the compound could be of interest in patients with diastolic dysfunction. The experiment in cardiomyopathic hamsters suggests that long-term oral treatment with istaroxime may interfere with critical mechanisms associated with disease progression and may ultimately result in improved survival. Very recent investigations documented that istaroxime does not increase myocardial oxygen consumption in dogs with advanced HF15 and stimulates SERCA2a activity in myocytes obtained from patients with congestive
40A
The American Journal of Cardiology (www.AJConline.org) Vol 99 (2A) January 22, 2007
HF.16 The sum of this evidence, together with the fact that, in contrast to dobutamine, istaroxime maintains its activity in infarcted dogs treated with -blockers,17 makes istaroxime a promising and safe new drug for improving cardiac performance in acute HF. 1. American Heart Association. Heart Disease and Stroke Statistics– 2004 Update. Dallas, TX: American Heart Association, 2004:49. 2. Krumholz HM, Parent EM, Tu N, Vaccarino V, Wang Y, Radford MJ, Hennen J. Readmission after hospitalization for congestive heart failure among Medicare beneficiaries. Arch Intern Med 1997;157:99 –104. 3. Siostrzonek P, Koreny M, Delle-Karth G, Haumer M, Koller-Strametz J, Heinz G. Milrinone therapy in catecholamine-dependent critically ill patients with heart failure. Acta Anaesthesiol Scand 2000;44:403– 409. 4. Silver MA, Horton DP, Ghali JK, Elkayam U. Effect of neseritide versus dobutamine on short-term outcomes in the treatment of patients with acutely decompensated heart failure. J Am Coll Cardiol 2002;39:798–803. 5. Micheletti R, Mattera GG, Rocchetti M, Schiavone A, Loi MF, Zaza A, Gagnol JP, De Munari S, Melloni P, Carminati P, Bianchi G, Ferrari P. Pharmacological profile of the novel inotropic agent (E,Z)-3-((2aminoethoxy)imino)androstane-6,17-dione hydrochloride (PST2744). J Pharmacol Exp Ther 2002;303:592– 600. 6. De Munari S, Cerri A, Gobbini M, Almirante N, Banfi L, Carzana G, Ferraro P, Marazzi G, Micheletti R, Schiavone A, et al. Structurebased design and synthesis of novel potent Na⫹/K⫹ ATPase inhibitors derived from a 5␣,14␣-androstane scaffold as positive inotropic compounds. J Med Chem 2003;56:3644 –3654. 7. Rocchetti M, Besana A, Mostacciuolo G, Ferrari P, Micheletti R, Zaza A. Diverse toxicity associated with cardiac Na⫹/K⫹ pump inhibition: evaluation of electrophysiological mechanisms. J Pharmacol Exp Ther 2003;305:765–771. 8. Rocchetti M, Besana A, Mostacciuolo G, Micheletti R, Ferrari P, Sarkozi S, Szegedi C, Jona I, Zaza A. Modulation of sarcoplasmic reticulum function by Na⫹/K⫹ pump inhibitors with different toxicity: digoxin and PST2744 [(E,Z)-3-((2-aminoethoxy)imino)androstane6,17-dione hydrochloride]. J Pharmacol Exp Ther 2005;313:207–215.
9. Adamson PB, Vanoli E, Mattera GG, Germany R, Gagnol JP, Carminati P, Schwartz PJ. Hemodynamic effects of a new inotropic compound, PST-2744, in dogs with chronic ischemic heart failure. J Cardiovasc Pharmacol 2003;42:169 –173. 10. Mattera GG, Loi F, Adamson P, Gagnol JP, Vanoli E, Borsini F, Carminati P. Istaroxime, a novel luso-inotropic agent, improves cardiac function in dogs with acute LV dysfunction: a pilot study [abstract]. J Card Fail 2005;11:S160. 11. Ikeda Y, Martone M, Gu Y, Hoshijima M, Thor A, Oh SS, Peterson KL, Ross J Jr. Altered membrane proteins and permeability correlate with cardiac dysfunction in cardiomyopathic hamsters. Am J Physiol Heart Circ Physiol 2000;278:H1362–H1370. 12. Sabbah HN, Stein PD, Kono T, Gheorghiade TB, Jafri S, Hawkins ET, Goldstein S. A canine model of chronic heart failure produced by multiple sequential coronary microembolizations. Am J Physiol 1991; 260:H1379 –H1384. 13. Lambert C, Massillon Y, Meloche S. Upregulation of cardiac angiotensin II AT1 receptors in congenital cardiomyopathic hamsters. Circ Res 1995;77:1001–1007. 14. Lo Giudice P, Gagnol JP, Bellucci A, Buffone G, Careddu A, Magni G, Quagliata T, Pacifici L, Carminati P. Autonomic nervous system activity imbalance in cardiomyopathic hamster. J Cardiovasc Pharmacol 2000;36:369 –375. 15. Imai M, Gheorghiade M, Sabbah HN. Intravenous istaroxime improves left ventricular lusitropic properties in dogs with advanced heart failure without increasing oxygen consumption. Presented at the 2005 Annual Scientific Meeting of the Heart Failure Society of America; September 18 –21, 2005; Boca Raton, Florida. 16. Barassi P, Ferandi M, Bianchi G, Ferrari F. Istaroxime stimulates SERCA2a activity in animal and human failing heart preparations [abstract]. J Card Fail 2005;11:S154. 17. Mattera GG, Loi FM, Alivernini ML, Botarelli M, Gagnol JP, Vanoli E, Carminati P. Inotropes and beta blockers: PST2744, a new inotropic agent, maintains its effects in infracted bisprolol treated dogs. Presented at the 64th National Meeting of the Italian Society of Cardiology; December 6 –10, 2003; Rome, Italy.
Acute heart failure syndromes (AHFS) represent a significant public health burden, resulting in 1 million hospitalizations annually in the United States.1 Patients hospitalized for treatment of AHFS have a readmission rate as high as 30%– 60% within 3– 6 months after initial discharge,2 and the mortality rate in the same period remains very high (25%–55%). Moreover, clinical trials show that dobutamine and phosphodiesterase inhibitors, despite their efficacy in acute symptomatic relief, lead to increased mortality rates.3,4 In fact, such agents are associated with increased ventricular ectopy, tachyarrhythmias, ischemia, and hypotension, especially at high doses. Digitalis has long been the main instrument for improving contractility of the failing heart by increasing intracellular calcium, as a consequence of its inhibition of sodium-potassium adenosine triphosphatase (ATPase). However, its use is progressively decreasing because of its proarrhythmic potential, specifically in the ischemic heart. Indeed, although increased intracellular calcium concentrations improve contractility, they also enhance calcium-dependent arrhythmia. Overall, cardiac contractility support therapy remains among the most critical aspects of acute cardiac decompensation management. Istaroxime, previously called PST2744, is a compound a Research and Development Division, sigma-tau Industrie Farmaceutiche Riunite SpA, Pomezia, Italy; bDepartment of Cardiology, University of Pavia, Pavia, Italy; cDepartment of Cardiology, Policlinico di Monza, Monza, Italy; and dUniversity Hospital CNRS, Montpellier, France. *Address for reprints: Giovan Giuseppe Mattera, MD, Research and Development, sigma-tau Industrie Farmaceutiche Riunite SpA, Via Pontina Km 30,500, 00040 Pomezia, Italy. E-mail address: [email protected].
0002-9149/07/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.amjcard.2006.09.004
with a novel mechanism of action, exerting its effects by inhibiting sodium-potassium ATPase activity5–7 and, in parallel, by stimulating sarcoplasmic reticulum calcium ATPase (SERCA) isoform 2a (SERCA2a). Whereas inhibition of sodium-potassium ATPase activity produces cytosolic calcium accumulation and an inotropic response, SERCA2a stimulation results in rapid clearance of cytosolic calcium, thus preventing calcium accumulation (calcium intoxication) and facilitating myocardial relaxation (lusitropism).8 These 2 properties invest istaroxime with the unique characteristic of acting as a calcium cycling modulator. Short-time istaroxime infusion improves left ventricular (LV) function and is safe in animals with normal heart or myocardial damage.5,7,9,10 Therefore, it is necessary to test this principle in experimental conditions (prolonged duration of administration) that better mimic clinical reality. Accordingly, the aims of the present experiments were (1) to assess the safety and efficacy of istaroxime in conscious dogs with chronic heart failure (HF) during 24-hour infusion, and (2) to evaluate the effects of long-term treatment with istaroxime on mortality in genetically cardiomyopathic Syrian BIO TO.2 hamsters.11
Materials and Methods All experiments were conducted according to international guidelines and approved by the Italian Ministry of Health. Animals were routinely monitored by a veterinarian. www.AJConline.org
34A
The American Journal of Cardiology (www.AJConline.org) Vol 99 (2A) January 22, 2007
Figure 1. Type of electrocardiographic changes (tachyarrhythmias and bradyarrhythmias) in animals with different doses of istaroxime. AV ⫽ atrioventricular; n ⫽ number of dogs in each group; PVC ⫽ premature ventricular contractions; SA ⫽ sinoatrial; VT ⫽ ventricular tachycardia.
Experiments in dogs: ANIMALS. A total of 7 male beagles (Harlan, Udine, Italy) weighing 14 –18 kg were used; 1 of 7 of these dogs was kept as a reserve. ANESTHESIA. Overnight-fasted dogs were preanesthetized with an intramuscular injection of Zoletil 100 (tiletamine-zolazepam; Virbac Laboratories, Carros, France). After endotracheal intubation, the surgical anesthesia level was obtained and maintained by ventilation with isoflurane (1%–2%) in an oxygen–nitrous oxide mixture. Anesthesia was used to implant telemetric transmitters and for microembolization. TELEMETRIC TRANSMITTER IMPLANT. Body temperature was maintained at 38°C ⫾ 1°C. The apex of the heart was reached through a transdiaphragmatic approach by laparotomy. A small incision was made in the muscular part of the diaphragm, exposing the apex of the heart; then, a telemetric transducer catheter was inserted into the left ventricle. The second pressure catheter was introduced into the mesenteric artery to monitor arterial blood pressure. Then, 2 electrocardiographic wires were positioned subcutaneously in the upper left and in the lower right thoracic area. Finally, the telemetric transducer was fastened into the abdomen and secured. The animals were allowed to recover for ⱖ4 weeks
before the microembolization procedure. After all surgical procedures, the antibiotic Spectrum 1 g/day (ceftazidime; sigma-tau, SpA, Pomezia, Italy) and the analgesic Contramal (tramadol; Formenti s.r.l., Milan, Italy) 50 mg twice daily were administered for 1 week. MICROEMBOLIZATION PROCEDURE. Chronic LV dysfunction was produced by multiple sequential intracoronary embolizations with polystyrene latex microspheres (Polysciences, Warrington, PA) according to Sabbah et al.12 Briefly, under general anesthesia, angiography of the left coronary artery was performed. After adequate visualization of the coronary bed, the embolization procedure was initiated. A solution (1.5 mL) containing microspheres 45–90 m in diameter was embolized into the coronary artery through the preformatted femoral catheter. Embolizations were performed 1–3 weeks apart. In all, 2–3 embolizations were necessary to reach an LV ejection fraction ⬎0.35, which was controlled by echocardiography. ISTAROXIME ADMINISTRATION. The experiment was performed by using a 3 ⫻ 3 Latin square design. Istaroxime (at 1 or 3 g/kg per min) or vehicle was administered via the jugular vein. The intravenous infusion lasted 24 hours, with
Mattera et al/Istaroxime: A New Luso-Inotropic Agent for Heart Failure
35A
Figure 2. Effects of istaroxime on left ventricular ⫹dP/dtmax. Points are mean ⫾ SEM of 6 dogs.
a ⱖ1-week washout period between infusions. Administration volume was 50 mL. Because 1 and 3 g/kg per min istaroxime did not cause proarrhythmic effects, a third dose of 4 g/kg per min was infused for 24 hours in 4 dogs from the original group (ancillary study). OBSERVATIONS AND TIMING. The recorded parameters were as follows: electrocardiographic DII, systolic blood pressure (SBP) and diastolic blood pressure (DBP), LV pressure and derivatives (LV ⫾ dP/dtmax), heart rate, and double product (SBP ⫻ heart rate). All telemetric signals were collected and processed by a validated telemetric computerized system (Dataquest ART Gold V 3.01; Data Sciences International, St. Paul, MN). Parameters were recorded for 1 minute every 15 minutes, starting 2 hours before administration, then during the following 24-hour period, and for 4 hours after termination of infusion. DATA EVALUATION. Data were visually checked to eliminate possible artifacts, and the values were averaged per hour. Electrocardiograms were inspected and arrhythmias classified as shown in Figure 1. STATISTICS. The experimental 3 ⫻ 3 Latin square design was repeated twice, and analysis of variance was applied by pooling results from the 2 experiments. Experiments in hamsters: ANIMALS. Cardiomyopathic BIO TO.2 and BIO F1B, 8- to 10-week-old Syrian hamsters (BIO Breeders, Watertown, MA) were used.
SURVIVAL. BIO TO.2 hamsters were randomized to enter daily treatment with 30 mg/kg of oral istaroxime (n ⫽ 50) or with the oral vehicle (5 mL/kg, n ⫽ 50) from the age of 12– 46 weeks. BIO F1B hamsters (n ⫽ 20) served as healthy controls. The number of dead hamsters was recorded weekly until the 54th week of age. LEVELS OF LV TUMOR NECROSIS FACTOR–␣. BIO TO.2 hamsters were randomized to daily treatment with oral 30 mg/kg istaroxime (n ⫽ 10) or oral vehicle (n ⫽ 10) from 12 weeks of age for about 4 months. BIO F1B hamsters (n ⫽ 10) were used as healthy controls. On the day after the last istaroxime administration (day 28 ⫾ 1 week), animals were anesthetized, hearts rapidly excised, and left ventricles dissected and homogenated. The supernatant was divided into aliquots, frozen, and stored at a temperature of ⫺80°C. Tumor necrosis factor–␣ (TNF-␣) analysis was performed on the supernatant with an enzyme-linked immunosorbent assay kit (Endogen Inc., Rockford, IL) according to the method suggested by the provider. Results are expressed as picograms per milligram of ventricular proteins. STATISTICS. Survival was analyzed by Kaplan-Meier analysis with a log-rank test. Because TNF-␣ values did not meet the homogeneity of variances, they were expressed as median (and interquartile range) and were compared using the Kruskal-Wallis test.
36A
The American Journal of Cardiology (www.AJConline.org) Vol 99 (2A) January 22, 2007
Figure 3. Effects of istaroxime on left ventricular – dP/dtmax. Points are mean ⫾ SEM of 6 dogs.
Compounds: (EZ)-3-(2-aminoethoxyimino)androstane-6, 17-dione hydrochloride (istaroxime), synthesized in sigmatau’s chemistry department, was dissolved in saline solution (dog experiment) or water (hamster experiment). Pentobarbital sodium (Sigma Chemical Co., St. Louis, MO) was dissolved in saline solution. Results Conscious dogs with chronic LV dysfunction: CLINICAL SIGNS AND BEHAVIOR. Dogs did not show signs of intolerance or discomfort during administration of vehicle or 1 g/kg per min istaroxime for 24 hours and presented regurgitation of food within 4 –5 hours only at an infusion rate of 3 g/kg per min. EFFECTS ON HEMODYNAMICS. Istaroxime-induced hemodynamic effects were independent of treatment sequence (F(2,17) ⫽ 1.34, p ⫽ 0.38). Istaroxime dose dependently increased (F(2,17) ⫽ 11.80, p ⬍0.004) cardiac inotropism (LV ⫹dP/dtmax), which reached a peak after 8 –10 hours and remained constant throughout the experiment (Figure 2). Lusitropism (LV ⫺dP/dtmax) (Figure 3) showed a nonstatistically significant trend to increase during infusions of 1 and 3 g/kg per min (p ⫽ 0.213). Heart rate (p ⫽ 0.703) was not affected by istaroxime (Figure 4). Likewise, SBP (p ⫽ 0.974) and DBP (p ⫽ 0.957) were not changed, even if SBP values were slightly higher in dogs infused with 3 g/kg per min
istaroxime in the first hours (Figure 5). Double product values, which represent an indirect evaluation of oxygen consumption, showed nonstatistically significant (p ⫽ 0.468) variations during istaroxime infusion (Figure 6). In the group receiving istaroxime at 4 g/kg per min, dP/dtmax was increased by ⬎100%, and vomiting was intense. ELECTROCARDIOGRAPHIC LEAD II. As seen in Figure 1, 4 of 7 microembolized dogs presented with spontaneous, low-degree arrhythmias (premature ventricular contractions, couplet, bigeminism, or sinoatrial block). Administration of istaroxime at 1 and 3 g/kg per min for 24 hours did not worsen the basal picture in these 4 dogs, nor was it associated with proarrhythmia in dogs without baseline arrhythmias. The evidence collected at 1 and 3 g/kg per min made it reasonable to test the higher dose of 4 g/kg per min in 4 dogs to further challenge the tolerability, safety, and efficacy of this compound; 2 of 4 microembolized dogs used in this ancillary study presented with basal spontaneous, low-degree arrhythmias (premature ventricular contractions, couplet, bigeminism, or sinoatrial block). In these dogs, administration of istaroxime at 4 g/kg per min for 24 hours did not induce a worsening of the basal situation. Similar findings were obtained in 1 of the 2 dogs without spontaneous arrhythmias at baseline; the other presented a short period of bigeminism after 13 hours of istaroxime infusion.
Mattera et al/Istaroxime: A New Luso-Inotropic Agent for Heart Failure
37A
Figure 4. Effects of istaroxime on heart rate. Points are mean ⫾ SEM of 6 dogs.
Hamsters: SURVIVAL. None of the healthy BIO F1B hamsters died during the observation period. Figure 7 shows that all vehicle-treated BIO TO.2 hamsters died at 26 –52 weeks of age. On the contrary, 16 (32%) istaroxime-treated BIO TO.2 hamsters survived until the age of 54 weeks. Kaplan-Meier survival analysis shows that istaroxime treatment significantly prolonged the survival of cardiomyopathic hamsters (p ⬍0.05). LV TNF-␣. TNF-␣ levels were markedly augmented in cardiomyopathic BIO TO.2 hamsters (median [interquartile range]: 124.8 pg/g protein [119.8 –142.2 pg/g protein]) when compared with healthy BIO F1B animals (median [interquartile range]: 61.0 pg/g protein [53.0 –70.8 pg/g protein]). Istaroxime prevented an increase in TNF-␣ levels in cardiomyopathic BIO TO.2 hamsters (median [interquartile range]: 47.2 pg/g protein [40.4 – 69.0 pg/g protein]); Kruskal-Wallis test: 22 ⫽ 15.77, p ⬍0.001).
Discussion This study, which was conducted in conscious animals with LV dysfunction, shows that both short-term (24hour infusion) and long-term (oral) istaroxime adminis-
tration have powerful beneficial effects on cardiac performance and survival, without inducing relevant side effects. The combined mechanisms of action by sodiumpotassium ATPase activity inhibition and SERCA2a stimulation in the same molecule may explain these findings. Compared with digoxin, istaroxime induced positive inotropic effects with a wider therapeutic ratio in anesthetized guinea pigs and healthy dogs,5 as well as in conscious dogs with a small anterior myocardial infarction during treadmill exercise.7 Similar findings were observed in short-term (1-hour) infusion in anesthetized dogs with acute LV dysfunction10 and in conscious dogs with chronic LV dysfunction.9 The present study goes beyond the previous studies by exploring the effects of istaroxime in experimental settings (prolonged duration of intravenous administration) that better mimic clinical reality. In conscious dogs with HF, istaroxime exerted a sustained positive action on LV performance (increase in LV ⫹dP/dtmax) that peaked at the 10th hour of infusion and remained constant throughout the 24-hour infusion period. This positive hemodynamic activity occurred without relevant side effects. The primary risk in inotropic
38A
The American Journal of Cardiology (www.AJConline.org) Vol 99 (2A) January 22, 2007
Figure 5. Effects of istaroxime on (A) systolic blood pressure (SBP) and (B) diastolic blood pressure (DBP). Points are mean ⫾ SEM of 6 dogs.
support is proarrhythmia. This is true for all currently used agents, from digoxin to dobutamine to phosphodiesterase inhibitors. Istaroxime did not trigger “de novo” ventricular arrhythmia events or magnify any preexisting events. In fact, life-threatening arrhythmias were never seen even with the high, powerful dose of 4 g/kg per min. The only side effect at this high dose was vomiting. The discomfort associated with vomiting may partially explain the slight alteration in blood pressure evident only with the higher dose. Thus, it appears that during the 24 hours of infusion, the maximum tolerated infusion rate of istaroxime is 3 g/kg per min. At this dose, istaroxime induced lusitropic and inotropic effects in the absence of relevant changes in systemic blood pressure, heart rate,
and oxygen consumption as derived from the double product index. The BIO TO.2 hamster is a genetic model of dilated cardiomyopathy that develops up to the end-stage level through well-defined steps. In fact, the prenecrotic phase is observable at 4 – 6 weeks of age; the necrotic phase, from 10 –16 weeks; the healing stage, from 18 –22 weeks; and cardiac hypertrophy, from 25–33 weeks; then, progressive dilatation and terminal HF develop.13 A significant imbalance of autonomic nervous system activity related to the evolution of cardiomyopathy was also described in this hamster strain.14 The longer-duration survival rate of istaroxime-treated hamsters (32%) compared with vehicle-treated animals suggests that the compound
Mattera et al/Istaroxime: A New Luso-Inotropic Agent for Heart Failure
39A
Figure 6. Effects of istaroxime on double product. Points are mean ⫾ SEM of 6 dogs.
Figure 7. Survival curves of Bio TO.2 hamsters treated with vehicle or istaroxime. Treatment started when hamsters were 12 weeks old. (p ⬍0.001 Kaplan-Meier test.)
slows down some critical mechanisms involved in the progression of HF. This is supported by the fact that the increase in cardiac TNF-␣ levels found in Bio TO2 hamsters compared with BIO F1B hamsters was decreased by istaroxime when the animals were 28 weeks old. Conclusion Istaroxime is a powerful luso-inotropic agent that, in animal models of HF, does not enhance the risk of ar-
rhythmia. The lusitropic effect of the compound could be of interest in patients with diastolic dysfunction. The experiment in cardiomyopathic hamsters suggests that long-term oral treatment with istaroxime may interfere with critical mechanisms associated with disease progression and may ultimately result in improved survival. Very recent investigations documented that istaroxime does not increase myocardial oxygen consumption in dogs with advanced HF15 and stimulates SERCA2a activity in myocytes obtained from patients with congestive
40A
The American Journal of Cardiology (www.AJConline.org) Vol 99 (2A) January 22, 2007
HF.16 The sum of this evidence, together with the fact that, in contrast to dobutamine, istaroxime maintains its activity in infarcted dogs treated with -blockers,17 makes istaroxime a promising and safe new drug for improving cardiac performance in acute HF. 1. American Heart Association. Heart Disease and Stroke Statistics– 2004 Update. Dallas, TX: American Heart Association, 2004:49. 2. Krumholz HM, Parent EM, Tu N, Vaccarino V, Wang Y, Radford MJ, Hennen J. Readmission after hospitalization for congestive heart failure among Medicare beneficiaries. Arch Intern Med 1997;157:99 –104. 3. Siostrzonek P, Koreny M, Delle-Karth G, Haumer M, Koller-Strametz J, Heinz G. Milrinone therapy in catecholamine-dependent critically ill patients with heart failure. Acta Anaesthesiol Scand 2000;44:403– 409. 4. Silver MA, Horton DP, Ghali JK, Elkayam U. Effect of neseritide versus dobutamine on short-term outcomes in the treatment of patients with acutely decompensated heart failure. J Am Coll Cardiol 2002;39:798–803. 5. Micheletti R, Mattera GG, Rocchetti M, Schiavone A, Loi MF, Zaza A, Gagnol JP, De Munari S, Melloni P, Carminati P, Bianchi G, Ferrari P. Pharmacological profile of the novel inotropic agent (E,Z)-3-((2aminoethoxy)imino)androstane-6,17-dione hydrochloride (PST2744). J Pharmacol Exp Ther 2002;303:592– 600. 6. De Munari S, Cerri A, Gobbini M, Almirante N, Banfi L, Carzana G, Ferraro P, Marazzi G, Micheletti R, Schiavone A, et al. Structurebased design and synthesis of novel potent Na⫹/K⫹ ATPase inhibitors derived from a 5␣,14␣-androstane scaffold as positive inotropic compounds. J Med Chem 2003;56:3644 –3654. 7. Rocchetti M, Besana A, Mostacciuolo G, Ferrari P, Micheletti R, Zaza A. Diverse toxicity associated with cardiac Na⫹/K⫹ pump inhibition: evaluation of electrophysiological mechanisms. J Pharmacol Exp Ther 2003;305:765–771. 8. Rocchetti M, Besana A, Mostacciuolo G, Micheletti R, Ferrari P, Sarkozi S, Szegedi C, Jona I, Zaza A. Modulation of sarcoplasmic reticulum function by Na⫹/K⫹ pump inhibitors with different toxicity: digoxin and PST2744 [(E,Z)-3-((2-aminoethoxy)imino)androstane6,17-dione hydrochloride]. J Pharmacol Exp Ther 2005;313:207–215.
9. Adamson PB, Vanoli E, Mattera GG, Germany R, Gagnol JP, Carminati P, Schwartz PJ. Hemodynamic effects of a new inotropic compound, PST-2744, in dogs with chronic ischemic heart failure. J Cardiovasc Pharmacol 2003;42:169 –173. 10. Mattera GG, Loi F, Adamson P, Gagnol JP, Vanoli E, Borsini F, Carminati P. Istaroxime, a novel luso-inotropic agent, improves cardiac function in dogs with acute LV dysfunction: a pilot study [abstract]. J Card Fail 2005;11:S160. 11. Ikeda Y, Martone M, Gu Y, Hoshijima M, Thor A, Oh SS, Peterson KL, Ross J Jr. Altered membrane proteins and permeability correlate with cardiac dysfunction in cardiomyopathic hamsters. Am J Physiol Heart Circ Physiol 2000;278:H1362–H1370. 12. Sabbah HN, Stein PD, Kono T, Gheorghiade TB, Jafri S, Hawkins ET, Goldstein S. A canine model of chronic heart failure produced by multiple sequential coronary microembolizations. Am J Physiol 1991; 260:H1379 –H1384. 13. Lambert C, Massillon Y, Meloche S. Upregulation of cardiac angiotensin II AT1 receptors in congenital cardiomyopathic hamsters. Circ Res 1995;77:1001–1007. 14. Lo Giudice P, Gagnol JP, Bellucci A, Buffone G, Careddu A, Magni G, Quagliata T, Pacifici L, Carminati P. Autonomic nervous system activity imbalance in cardiomyopathic hamster. J Cardiovasc Pharmacol 2000;36:369 –375. 15. Imai M, Gheorghiade M, Sabbah HN. Intravenous istaroxime improves left ventricular lusitropic properties in dogs with advanced heart failure without increasing oxygen consumption. Presented at the 2005 Annual Scientific Meeting of the Heart Failure Society of America; September 18 –21, 2005; Boca Raton, Florida. 16. Barassi P, Ferandi M, Bianchi G, Ferrari F. Istaroxime stimulates SERCA2a activity in animal and human failing heart preparations [abstract]. J Card Fail 2005;11:S154. 17. Mattera GG, Loi FM, Alivernini ML, Botarelli M, Gagnol JP, Vanoli E, Carminati P. Inotropes and beta blockers: PST2744, a new inotropic agent, maintains its effects in infracted bisprolol treated dogs. Presented at the 64th National Meeting of the Italian Society of Cardiology; December 6 –10, 2003; Rome, Italy.