ORIGINAL ARTICLE Antiatherosclerotic Properties of EP2302, a Novel Squalene Synthase Inhibitor, in the Cholesterol-fed Rabbit Anna Tavridou, PhD,* Loukas Kaklamanis, MD, PhD,† Apostolos Papalois, PhD,‡ Angeliki P. Kourounakis, PhD,§ Eleni A. Rekka, PhD,¶ Panos N. Kourounakis, PhD,¶ Avgui Charalambous, PhD,‡ and Vangelis G. Manolopoulos, PhD* Abstract: EP2302 is a novel nitric oxide donor compound that inhibits squalene synthase. We hypothesized that EP2302 can reduce atherosclerosis in the cholesterol-fed rabbit atherosclerosis model. Animals were fed a high-cholesterol (HC) diet for 4 weeks. Animals subsequently received drug or placebo for 4 (n = 15) or 12 weeks (n = 15) while on HC diet. A third group (n = 16) received drug or placebo for 4 weeks while on regular diet (regression group). No significant differences were observed in circulating lipids among any of the treatment groups at each time point during HC intake. The perimeter and area of the ascending aorta covered by lesions were significantly decreased in animals treated with 2 mg/kg EP2302 for 4 weeks (44% and 42% reduction, respectively). Moreover, a significant decrease in the perimeter of the ascending and descending aorta covered by lesions was observed in animals treated with 2 mg/kg EP2302 for 12 weeks (73% and 44% reduction, respectively) as well as in the regression group (61% and 65% reduction, respectively). Treatment with EP2302 did not cause any toxicity in animal vital organs. We have shown that EP2302 inhibits atherosclerosis in the cholesterol-fed rabbit and therefore may serve as a candidate drug to be tested in humans for atherosclerosis-related disorders. Key Words: EP2302, squalene synthase inhibitor, nitric oxide donor, atherosclerosis, lipids, cholesterol-fed rabbit (J Cardiovasc Pharmacol TM 2008;51:573–580) INTRODUCTION Atherosclerosis is unquestionably the main underlying pathology of cardiovascular disease, the leading cause of morbidity and mortality worldwide. 1 Among major risk factors for the development of atherosclerosis are increased levels of low-density lipoprotein (LDL) cholesterol, oxidative modification of LDL, and dysregulation of endogenous nitric oxide (NO) activity. 2,3 Lipid-lowering with 3-hydroxy- 3-methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors, hereafter referred to as statins, is the main phar- macological intervention for dyslipidemia and reduces mor- bidity and mortality from coronary heart disease. Moreover, statins have direct antiatherosclerotic effects on the vessel wall. 4 While statins significantly reduce the risk of future acute coronary syndromes by 30%, 5 the addition of drugs that act with different mechanisms of action may improve risk reduc- tion and achieve cardiovascular protection more safely. Apart from inhibiting HMG-CoA reductase, statins also suppress the production of mevalonate, a precursor of non- sterol products that are vital for diverse cellular functions. Both major known side effects of statins (hepatotoxicity and myo- toxicity) have been associated with inhibition of the synthesis of the non-sterol products. 6,7 The first committed step in the de novo cholesterol biosynthesis is catalyzed by squalene synthase. Squalene synthase inhibitors decrease circulating LDL-cholesterol by an increased expression of hepatic LDL receptors in a manner similar to statins. 8 However, they leave other non-sterol products of mevalonate metabolism unaffected and, therefore, have at least one theoretical advantage compared with statins as hypocholesterolemic and antiatherosclerotic drugs because of reduced side effects. Several squalene synthase inhibitors have been reported in the literature. 8 Among them, TAK-475 has been shown to be an effective lipid-lowering agent in a variety of animal models 9–12 and has become the first squalene synthase inhibitor to enter phase III clinical trials to assess its ability to reduce LDL-cholesterol alone or in com- bination with statins or ezetimibe. 13 However, to the best of our knowledge, there is a lack of data on possible antiatherosclerotic effects of squalene synthase inhibitors in animals or humans. A series of novel 2-biphenylmorpholine derivatives has been synthesized, and preliminary results for their hypolipi- demic and antioxidant properties have been reported. 14 The most potent of these compounds, 2-(4-biphenyl)-4-methyl- octahydro-1,4-benzoxazin-2-ol, hydrobromide, was named EP2306. It was postulated that if these morpholine derivatives could be manipulated into becoming NO donors without loosing their other properties, their activity on atherosclerosis might be even more pronounced. Thus a series of NO-releasing morpholine derivatives was synthesized. 15 2-(4-biphenyl)-2-(3- nitrooxypropoxy)-4-methylmorpholine, hydrobromide (named Received for publication February 13, 2008; accepted April 1, 2008. From the *Laboratory of Pharmacology, Medical School, Democritus University of Thrace, Alexandroupolis, Greece; †Onassis Cardiac Surgery Center, Department of Pathology, Athens, Greece; ‡ELPEN Pharmaceu- tical Co Inc., Pikermi, Greece; §Department of Pharmaceutical Chemistry, School of Pharmacy, University of Athens, Athens, Greece; {Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki, Greece. The authors have no conflicts of interest in connection with this article. This work was financially supported by ELPEN Pharm. Co. Inc which holds exclusive rights on EP2302. Reprints: Vangelis G. Manolopoulos, PhD, Laboratory of Pharmacology, Medical School, Democritus University of Thrace, Dragana Campus, 68100 Alexandroupolis, Greece (e-mail: emanolop@med.duth.gr). Copyright Ó 2008 by Lippincott Williams & Wilkins J Cardiovasc Pharmacol ä  Volume 51, Number 6, June 2008 573