International Journal of Impotence Research (2007) 19, 55–61 & 2007 Nature Publishing Group All rights reserved 0955-9930/07 $30.00 www.nature.com/ijir

ORIGINAL ARTICLE The phosphodiesterase-5 inhibitor reduces myocardial infarct size

C Sesti1,4, V Florio2, EG Johnson3 and RA Kloner1

1Department of Medicine, The Heart Institute, Keck School of Medicine, University of Southern California, Good Samaritan Hospital, Los Angeles, CA, USA; 2ICOS Corporation, Bothell, WA, USA and 3Lilly Research Laboratories, , , IN, USA

The aim of this study was to determine, in an animal model, the effects of tadalafil on myocardial infarct size (IS), hemodynamics and regional myocardial blood flow after myocardial ischemia and reperfusion. Patients with (ED) often have risk factors for coronary artery disease. Tadalafil, a long-acting inhibitor of the phosphodiesterase-5 (PDE5), is used for the treatment of ED; there are no previous data regarding tadalafil in the setting of coronary artery occlusion (CAO). Sprague–Dawley male rats were treated with tadalafil or vehicle (10 mg/kg, by gastric gavage), 2 h before a 30 min CAO. Heart rate was comparable between tadalafil and control groups. Tadalafil reduced mean arterial pressure (P ¼ 0.009), systolic (P ¼ 0.035) and diastolic (P ¼ 0.009) blood pressures during ischemia/reperfusion. Tadalafil significantly reduced IS (4272%) versus controls (5473%) (P ¼ 0.006). For the first time, we showed that the PDE5 inhibitor, tadalafil, was well tolerated and cardioprotective in the setting of an experimental , by substantially reducing ischemic cell death. International Journal of Impotence Research (2007) 19, 55–61. doi:10.1038/sj.ijir.3901497; published online 20 July 2006

Keywords: acute myocardial infarction; coronary occlusion; myocardial infarct size; phosphodies- terase-5 inhibitor; erectile dysfunction; tadalafil

Introduction an early manifestation of .3,4 Indeed, patients with ED have many of the risk Tadalafil (Cialis) is an inhibitor of the enzyme factors associated with coronary artery disease phosphodiesterase-5 (PDE5), a class of mild vaso- (CAD), such as smoking, hypertension, diabetes active drugs developed for the treatment of erectile and hyperlipidemia.1,5 The association between ED dysfunction (ED); the other available PDE5 inhibi- and CAD has raised concern regarding the cardio- tors are citrate (Viagra) and HCl vascular safety of PDE5 inhibitors. PDE5 catalyzes (Levitra). ED is a common disorder that affects the breakdown of the potent smooth muscle-relaxing almost 200 million men worldwide.1,2 Studies have agent cyclic guanosine monophosphate (cGMP), shown that vascular endothelial dysfunction is a a second messenger of nitric oxide. Inhibition of main cause of ED, thus suggesting that ED might be PDE5 increases cGMP levels, reduces intracellular calcium and induces vasodilation.6 So far, clinical studies have shown that PDE5 inhibitors are safe and do not increase cardiovascular risk in patients Correspondence: Dr RA Kloner, Department of Medicine, with CAD.7–9 Recent studies have shown that these The Heart Institute, Keck School of Medicine, University medicines improve endothelial function and may of Southern California, Good Samaritan Hospital, 1225 also have vascular and myocardial protective Wilshire Boulevard, 9th Floor Research, Los Angeles, CA effects.10–13 Tadalafil differs from sildenafil and 90017-2395, USA. vardenafil by its pharmacokinetic profile, 17.5 h E-mail: [email protected] 4 half-life, maximum plasma concentration at 2 h and Current address: Department of Medicine, Division 14 of Cardiology/Cardiovascular Research, University of efficacy for up to 36 h after dosing. , , WA, USA. As there is no previous information on tadalafil in Received 26 April 2006; revised 31 May 2006; accepted 8 the setting of myocardial infarction, we studied the June 2006; published online 20 July 2006 effects of tadalafil on infarct size (IS), hemody- Tadalafil reduces myocardial infarct size C Sesti et al 56 namics and regional myocardial blood flow (RMBF) for 3 h. At the end of the reperfusion phase, the after myocardial ischemia and reperfusion.15,16 As artery was reoccluded and 0.8 ml of 50% Unisperse studies on sildenafil have implicated the mitochon- blue pigment suspension (Ciba–Geigy, Hawthorne, drial ATP-sensitive K (mKATP) channels in the car- NY, USA) was injected via the jugular vein, to dioprotective effect of the drug, we also investigated identify the ischemic risk area (not perfused by the whether tadalafil’s protective effects on the heart blue dye). While the rats were deeply anesthetized, 10,13 occur through an mKATP channel mechanism. 2 ml of potassium chloride (2 mEq/ml) was injected into the same vein to stop the heart in diastole and the heart was excised. Materials and methods

Animals Hemodynamics Sprague–Dawley male rats (250–350 g) were main- Heart rate (HR), mean arterial pressure (MAP), tained in accordance with the policies and guide- systolic blood pressure (SBP) and diastolic blood lines of the Position of the American Heart pressure (DBP) were measured through a pressure Association on research animal use and Committee transducer connected to the catheter inserted into on Care and Use of Laboratory Animals of the the carotid artery (ADInstruments, Colorado Institute of Laboratory Animal Resources, National Springs, CO, USA) and recorded at different time Research Council (Department of Health, Education 17 points in the course of the experiment. Episodes of and Welfare Publication No. 85-230). Animal arrhythmias, if manifested (based on HR and blood procedures were approved by the Institutional Care pressure tracings), were also recorded. and Use Committee at Good Samaritan Hospital.

Treatment RMBF 6 Rats were randomized to receive tadalafil or vehicle RMBF was assessed by using 1  10 radioactive 141 103 (control group) at the dose of 10 mg/kg, which was microspheres labeled with Ce or Ru (Perkin- indicated by Lilly ICOS LLC (Bothell, WA, USA). Elmer Life Sciences, Boston, MA, USA). RMBF was The treatment was given by gastric gavage 2 h before measured twice, at 25 min of CAO and at the end of coronary artery occlusion (CAO). Another group of reperfusion, by injecting 0.5 ml of sucrose solution animals treated with tadalafil or vehicle also containing the microspheres into the left ventricle received 5 mg/kg of the selective blocker of mKATP (LV) and by simultaneously withdrawing a reference channel, 5-hydroxydecanoate (5-HD), 10 min before blood sample from the carotid artery at a rate of CAO. The study was conducted in a blinded 0.37 ml/min. manner, with solutions provided in bottles labeled with a secret code by Lilly ICOS LLC. Both tadalafil and vehicle (hydroxypropyl methylcellulose phtha- late) were formulated in 1% hydroxypropyl methyl- Post-mortem evaluation cellulose/1% Tween 80. The excised hearts were cleared of the right ventricle and sliced into five transverse sections from apex to base, which were weighed and photographed to identify the non-perfused region Acute myocardial infarction or area at risk (AR). The slices were then incubated The rats were anesthetized with intraperitoneal in a 1% solution of triphenyltetrazolium chloride at injection of 75 mg/kg ketamine and 5 mg/kg xyla- 371C for 15 min, kept in formalin overnight and zine, endotracheally intubated and ventilated with a rephotographed the following morning to demon- rodent respirator. A temperature probe was inserted strate the necrotic area. Computerized planimetry into the rectum to monitor body/rectal temperature, of the photographs was used to determine the maintained at 361C with a heating pad. A cut-down AR and of necrosis (AN), expressed as % of LV, was made in the neck to expose the carotid artery which were used to calculate the IS, expressed as and the jugular vein and fluid-filled catheters were % of AR (IS ¼ necrosis/risk  100). inserted into the vessels. A thoracotomy was performed in the fourth left intercostal space and the pericardium removed. A suture was placed under the proximal portion of the Measurement of RMBF left coronary artery, the ends of the suture were Samples from infarcted and non-infarcted areas threaded through a piece of plastic tubing, forming were dissected from the heart slices and weighed. a snare that when tightened and clamped occluded The radioactivity of these tissue samples and the the artery. The coronary artery was occluded for reference blood sample were counted in a gamma 30 min, and then reperfused (by releasing the clamp) counter and RMBF was calculated.18

International Journal of Impotence Research Tadalafil reduces myocardial infarct size C Sesti et al 57 Statistics RMBF. Either during occlusion or during reperfu- Data were presented as mean7s.e.m. and analyzed sion, RMBF in the ischemic area was lower than that using SAS (version 6.04, Cary, NC, USA). Student’s in the non-ischemic area (Table 1). However, there t-tests were performed to compare discrete values were no significant differences in RMBF values between tadalafil and control groups. The relation- between the two groups in each area (Table 1). ship between AR and AN was analyzed using analysis of covariance (ANCOVA). Hemodynamic Infarct size. Planimetric analysis showed that the values recorded at different time points were ischemic AR (% of LV) was not significantly analyzed using repeated-measures analysis of var- different between the two groups (Table 1). IS (% iance (ANOVA) to compare variables by group and of AR) was decreased significantly in the tadalafil over the time; followed by CONTRAST post hoc group (4272%) compared to controls (5473%) test for comparisons between corresponding time (P ¼ 0.006). Correlation between AR and AN in the points in the two groups. Po0.05 was considered tadalafil versus control group showed that for any statistically significant. AR, rats treated with tadalafil had smaller percen- tage of necrosis (P ¼ 0.004; Figure 4).

Results Did tadalafil act through an mKATP channel Effects of tadalafil after myocardial ischemia/ mechanism? Hemodynamics and RMBF. The two groups, reperfusion tadalafil þ 5-HD (n ¼ 10) and control þ 5-HD (n ¼ 6), Hemodynamics. There were no significant differ- showed no significant differences in HR, MAP, SBP, ences in HR between tadalafil (n ¼ 15) and control DBP, frequency of arrhythmias and RMBF (data not (n ¼ 15) groups either at baseline or during occlu- shown). sion or reperfusion (Figure 1). Tadalafil reduced MAP, SBP and DBP throughout the experiment (P ¼ 0.009, P ¼ 0.035 and P ¼ 0.009, respectively) Infarct size. The two groups had similar AR: (Figures 2 and 3). In both groups, MAP, SBP and tadalafil þ 5-HD, 3571%; control þ 5-HD, 3573%; DBP were significantly lower during the occlusion AN was 1871% in the tadalafil þ 5-HD group; and and reperfusion phases than at baseline (Po0.001, 2272% in the control þ 5-HD group. IS (% of AR) P ¼ 0.026 and Po0.001, respectively). There were no was significantly smaller in the tadalafil þ 5-HD differences in the frequency of arrhythmias between group (5273%) compared to control þ 5-HD the two groups. (6271%) (P ¼ 0.003).

300 HR-Control 280 HR-Tadalafil

260

240

220

200 beats per minute (bpm) beats per minute

0 baseline 15min O 30min O 30min R 60min R 90min R 120min R 175min R Figure 1 HR in the control (open symbol) and tadalafil group (black symbol) measured at different time points during ischemia and reperfusion. Statistical analysis by repeated-measures ANOVA between groups (NS) and over the time (NS). O ¼ occlusion; R ¼ reperfusion. n ¼ 15 for both groups.

International Journal of Impotence Research Tadalafil reduces myocardial infarct size C Sesti et al 58 100 MAP - Control MAP - Tadalafil 90 ** p = 0.009

80 *

70 * mmHg *

60

50

40 baseline 15min O 30min O 30min R 60min R 90min R 120min R 175min R Figure 2 MAP in the control (open symbol) and tadalafil group (black symbol) measured at different time points during ischemia and reperfusion. Statistical analysis by repeated-measures ANOVA, followed by CONTRAST for post hoc comparisons between corresponding time points in the two groups. *Po0.05 and **Po0.01 versus control. O ¼ occlusion; R ¼ reperfusion. n ¼ 15 for both groups.

120 SBP - Control * SBP - p = 0.035 SBP - Tadalafil DBP - Control 100 DBP - Tadalafil ** ** * * 80 * ** ** mmHg 60 *

40 DBP - p = 0.009

20 baseline 15min O 30min O 30min R 60min R 90min R 120min R 175min R Figure 3 SBP and DBP in the control (open symbol) and tadalafil group (black symbol) at different time points during ischemia and reperfusion. Statistical analysis by repeated-measures ANOVA, followed by CONTRAST as post hoc test. *Po0.05 and **Po0.01 versus control. O ¼ occlusion; R ¼ reperfusion. n ¼ 15 for both groups.

Figure 5 shows the AR/AN correlation for all the trol þ 5-HD rats the biggest percentage of necrosis. It four groups: tadalafil, control, tadalafil þ 5-HD and also shows how the lines of regression of control and control þ 5-HD. It is evident that for any AR, tadalafil þ 5-HD groups are very close; in fact, the IS tadalafil-treated rats have the smallest and con- are very similar (54 and 52%, respectively).

International Journal of Impotence Research Tadalafil reduces myocardial infarct size C Sesti et al 59 Discussion Tadalafil is the most recently approved PDE5 inhibitor used for the treatment of ED. A significant In this study, we showed that the PDE5 inhibitor percentage of men with ED also suffer from tadalafil reduced myocardial IS in a rat model of cardiovascular disease and men with increased myocardial ischemia without having unsafe hemo- cardiovascular risk have a higher incidence of dynamic effects that might increase oxygen demand. ED.1,5 Because of this correlation between ED and Tadalafil induced an expected small reduction in cardiovascular disease, and because tadalafil has arterial pressure, did not cause a coronary artery a half-life of 17.5 h, it would be important to know steal phenomenon and was not associated with what effect tadalafil might have in men who develop arrhythmias during coronary occlusion. myocardial ischemia or infarction while this med- icine is ‘on board’. By inhibiting PDE5, tadalafil induces accumulation of cGMP, resulting in dilata- tion of blood vessels and decrease in blood pressure. Table 1 RMBF and planimetric variables during ischemia/ The theoretical concern is that the drop in blood reperfusion pressure could exacerbate ischemia and infarction Control Tadalafil P-values in men with severely obstructed vessels, as in this (n ¼ 15) (n ¼ 15) case myocardial blood flow is dependent on perfu- sion pressure. A second theoretical concern with RMBF (ml/min/g) any vasodilator is that there will be a coronary artery Occlusion steal phenomenon, whereby coronary flow will be 7 7 Infarct tissue 0.06 0.02 0.05 0.03 NS diverted through collaterals from the ischemic Normal tissue 3.2470.82 4.5570.99 NS Reperfusion coronary bed to the non-ischemic bed – another Infarct tissue 0.8470.20 0.7970.14 NS potential mechanism for worsening ischemia. Normal tissue 3.5570.55 2.4270.42 NS To date, no one has investigated the effects of tadalafil on IS and hemodynamics in a myocardial IS AR (% LV) 38724373NSischemia model. In our study, we determined what AN (% LV) 21721872NShappens during myocardial infarction when this IS (% AR) 54734272 0.006 drug is present by examining the effects of tadalafil on myocardial IS, hemodynamics and RMBF in a rat Abbreviations: AN, necrotic area; AR, risk area; IS, infarct size; model of coronary ischemia/reperfusion. Our data LV, left ventricle; NS, nonsignificant; RMBF, regional myocardial showed that the size of the myocardial infarct was blood flow. Values are mean7s.e.m. Tadalafil versus control by Student’s significantly reduced after tadalafil treatment, and t-test. that for any AR, rats treated with tadalafil had a Po0.05 statistically significant. smaller percentage of necrosis. We cannot draw the

40

Tadalafil Control

30

20 Area of Necrosis (% LV) 10

p = 0.004

0 010203040506070 Area at Risk (% LV) Figure 4 Correlation between AR and AN. For any AR, AN was significantly lower in the tadalafil (black symbol) than in the control group (open symbol). Statistical analysis of covariance by ANCOVA.

International Journal of Impotence Research Tadalafil reduces myocardial infarct size C Sesti et al 60 40 Tadalafil Control Tadalafil + 5-HD Control + 5-HD 30

20 Area of Necrosis (% LV) 10

0 010203040506070 Area at Risk (% LV) Figure 5 Correlation between risk and necrotic areas for all the four groups: tadalafil, control, tadalafil þ 5-HD and control þ 5-HD.

20,21 conclusion that the mKATP channels are involved in omy. The incidence of cardiovascular adverse the tadalafil effect on myocardial IS in our study, events was not found higher in patients receiving because the vehicle þ 5-HD group had an IS bigger tadalafil and cardiac mortality in tadalafil-treated than the group treated with vehicle alone; thus patients was similar to that expected in the general suggesting that 5-HD by itself might have a negative male population.7–9,15 effect on IS in this model. Whereas the involvement Sildenafil has been shown to be beneficial in of the mKATP channels has been demonstrated for and cardiac hyper- the cardioprotective effect of sildenafil, the possibi- trophy.22,23,12 Tadalafil and sildenafil have also lity of the same mechanism of action being involved been shown to improve endothelial dysfunction in for the tadalafil effect, at least in view of our results, patients with high cardiovascular risk, chronic heart will require further investigation.10,13 failure, diabetes and in smokers.11,24–26 It has been suggested that elevated levels of cGMP, There is evidence that PDE5 inhibitors may have caused by PDE5 inhibitors, could inhibit PDE3, cardioprotective effects. Kukreja and associates have an enzyme responsible for the hydrolysis of cyclic shown that sildenafil induced cardioprotection adenosine monophosphate (cAMP) and increase by reducing IS in an ischemia/reperfusion rabbit cAMP levels, resulting in elevated HR and contrac- model.13 Our research group has also investigated tility.19 Our results showed that tadalafil did not the cardiovascular effects of sildenafil, and observed increase HR nor induce arrhythmias. other positive effects, such as reduction of LV As expected from a PDE5 inhibitor, tadalafil diastolic pressure during occlusion, improvement induced a moderate reduction in MAP, SBP and of myocardial blood flow in the reperfused myocar- DBP during ischemia/reperfusion. Other studies dium and no increase in arrhythmias.27 have reported that sildenafil and vardenafil also In conclusion, the present study, which was the cause a small drop in SBP and DBP.7 The reason is first to investigate the effect of tadalafil in an that PDE5 is found not only in smooth muscle cells experimental model of myocardial infarction, of the vasculature of the corpus cavernosum but showed that the moderate vasodilation induced by also in smooth muscle cells of arteries and veins tadalafil could be potentially beneficial in the systemically. There were also no differences in setting of ischemic heart disease. Our findings RMBF between tadalafil and control rats, suggesting demonstrated that the PDE5 inhibitor, tadalafil, coronary steal did not occur. was well tolerated in an experimental model of Tadalafil has been shown to improve erectile coronary occlusion, as indicated by no evidence for function in a broad population of men with ED increase in HR, arrhythmias or coronary steal. In and in men with ED and comorbid conditions addition, tadalafil was cardioprotective, as revealed (cardiovascular disease, hypertension, diabetes, by a significant reduction of ischemic cell death in depression, hyperlipidemia) or after prostatect- the setting of acute CAO.

International Journal of Impotence Research Tadalafil reduces myocardial infarct size C Sesti et al 61 References 14 Rosen RC, Kostis JB. Overview of phosphodiesterase 5 inhibition in erectile dysfunction. Am J Cardiol 2003; 1 Feldman HA, Goldstein I, Hatzichristou DG, Krane RJ, 92(Suppl): 9M–18M. McKinlay JB. Impotence and its medical and psychosocial 15 Kloner RA, Mitchell M, Emmick JT. Cardiovascular effects of correlates: results of the Massachusetts Male Aging Study. tadalafil. Am J Cardiol 2003; 92(Suppl): 37M–46M. J Urol 1994; 151: 54–61. 16 Kloner RA, Mitchell M, Emmick JT. Cardiovascular effects of 2 Laumann EO, Paik A, Rosen RC. in United tadalafil in patients on common antihypertensive therapies. States: prevalence and predictors. JAMA 1999; 281: 537–544. Am J Cardiol 2003; 92(Suppl): 47M–57M. 3 Blumentals WA, Gomez-Caminero A, Joo S, Vannappaggari V. 17 American Heart Association. Position of the American Heart Should erectile dysfunction be considered as a marker for Association on research animal use. Circulation 1985; 71: acute myocardial infarction? Results from a retrospective 849A–850A. cohort study. Int J Impot Res 2004; 16: 350–353. 18 Heymann MA, Payne BD, Hoffman JE, Rudolph AM. Blood 4 Kaiser DR, Billups K, Mason C, Wetterling R, Lundberg JL, flow measurement with radionuclide-labeled particles. Prog Bank AJ. Impaired brachial artery endothelium-dependent Cardiovasc Dis 1977; 20: 55–79. and -independent vasodilation in men with erectile dysfunc- 19 Bischoff E. Potency, selectivity, and consequences of non- tion and no other clinical cardiovascular disease. J Am Coll selectivity of PDE inhibition. Int J Impot Res 2004; 16(Supp 1): Cardiol 2004; 43: 179–184. S11–S14. 5 Kloner RA, Jarow JP. Erectile dysfunction and sildenafil citrate 20 Carson CC, Rajfer J, Eardley I, Carrier S, Dennes JS, Walker DJ and cardiologists. Am J Cardiol 1999; 83: 576–582. et al. The efficacy and safety of tadalafil: an update. BJU Int 6 Aversa A, Pili M, Fabbri A, Spera E, Spera G. Erectile 2004; 39: 1276–1281. dysfunction: expectations beyond phosphodiesterase type-5 21 Montorsi F, Padma Nathan H, McCullough A, Brock GB, inhibition. J Endocrinol Invest 2004; 27: 192–206. Broderick G, Ahuja S et al. Tadalafil in the treatment of 7 Kloner RA. Cardiovascular effects of the 3 phosphodiesterase- erectile dysfunction following bilateral nerve-sparing radical 5 inhibitors approved for the treatment of erectile dysfunction. retropubic prostatectomy: a randomized, double-blind, place- Circulation 2004; 110: 3149–3155. bo-controlled trial. J Urol 2004; 172: 1036–1041. 8 Jackson G, Kloner RA, Costigan TM, Warner MR, Emmick JT. 22 Sastry BK, Narasimhan C, Reddy NK, Raju BS. Clinical Update on clinical trials of tadalafil demonstrates no increased efficacy of sildenafil in primary pulmonary hypertension: a risk of cardiovascular adverse effects. J Sex Med 2004; 1: randomized, placebo-controlled, double-blind, crossover 161–167. study. J Am Coll Cardiol 2004; 43: 1149–1153. 9 Kloner RA, Padma-Nathan H. Erectile dysfunction in patients 23 Tantini B, Manes A, Fiumana E, Pignatti C, Guarnieri C, with coronary artery disease. Int J Impot Res 2005; 17: Zannoli R et al. Antiproliferative effect of sildenafil on human 209–215. pulmonary artery smooth muscle cells. Basic Res Cardiol 10 Kukrejia RC, Ockaili R, Salloum F, Yin C, Hawkins J, Das A 2005; 100: 131–138. et al. Cardioprotection with phosphodiesterase-5 inhibition – 24 Katz SD, Balidemaj K, Homma S, Wu H, Wang J, Maybaum S. a novel preconditioning strategy. J Mol Cell Cardiol 2003; 36: Acute type 5 phosphodiesterase inhibition with sildenafil 165–173. enhances flow-mediated vasodilation in patients with chronic 11 Rosano GMC, Aversa A, Vitale C, Fabbri A, Fini M, Spera G. . J Am Coll Cardiol 2000; 36: 845–851. Chronic treatment with tadalafil improves endothelial 25 Desouza C, Parulkar A, Lumpkin D, Akers D, Fonseca VA. function in men with increased cardiovascular risk. Eur Urol Acute and prolonged effects of sildenafil on brachial artery 2005; 47: 214–222. flow-mediated dilatation in type-2 diabetes. Diabetes Care 12 Takimoto E, Champion HC, Li M, Belardi D, Ren S, Rodriguez 2002; 25: 1336–1339. ER et al. Chronic inhibition of cyclic GMP phosphodiesterase 26 Kimura M, Higashi Y, Hara K, Noma K, Sasaki S, Nakagawa K 5A prevents and reverses cardiac hypertrophy. Nat Med 2005; et al. PDE5 inhibitor sildenafil citrate augments endothelium- 11: 214–222. dependent vasodilation in smokers. Hypertension 2003; 41: 13 Ockaili R, Salloum F, Hawkins J, Kukreja RC. Sildenafil 1106–1110. (Viagra) induces powerful cardioprotective effect via opening 27 Reffelmann T, Kloner RA. Effects of sildenafil on myocardial of mitochondrial KATP channels in rabbits. Am J Physiol infarct size, microvascular function, and acute ischemic left Heart Circ Physiol 2002; 283: H1263–H1269. ventricular dilation. Cardiovasc Res 2003; 59: 441–449.

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