Torcetrapib impairs endothelial function in hypertension

BASIC SCIENCE European Heart Journal (2012) 33, 1615–1624 doi:10.1093/eurheartj/ehr348 Torcetrapib impairs endothelial function in hypertension Branko Simic1†, Matthias Hermann1,2†, Sidney G. Shaw3, Laurent Bigler4, Urs Stalder4, Carola Dörries1, Christian Besler1, Thomas F. Lüscher1,2, and Frank Ruschitzka1,2* Received 14 April 2011; revised 27 July 2011; accepted 15 August 2011; online publish-ahead-of-print 14 September 2011 This paper was guest edited by Prof. Dr Thomas Münzel, Johannes Gutenberg Universität, Mainz, Germany See page 1548 for the editorial comment on this article (doi:10.1093/eurheartj/ehr408) Aims A marked increase in HDL notwithstanding, the cholesterol ester transfer protein (CETP) inhibitor torcetrapib was associated with an increase in all-cause mortality in the ILLUMINATE trial. As underlying mechanisms remain elusive, the present study was designed to delineate potential off-target effects of torcetrapib. ..................................................................................................................................................................................... Methods Spontaneously hypertensive rats (SHRs) and Wistar–Kyoto (WKY) rats were treated with torcetrapib (100 mg/kg/ day; SHR-T and WKY-T) or placebo (SHR-P and WKY-P) for 3 weeks. Blood pressure transiently increased during and results the first 3 days of torcetrapib administration in SHRs and returned to baseline thereafter despite continued drug administration. Acetylcholine-induced endothelium-dependent relaxations of aortic rings were markedly impaired, and endothelial nitric oxide synthase (eNOS) mRNA and protein were down-regulated after 3 weeks of torcetrapib treatment in SHR (P , 0.0001, ,0.01, and ,0.05, resp. vs. SHR-P). Torcetrapib reduced NO release in cultured aortic endothelial cells (P , 0.01 vs. vehicle-treated cells) and increased generation of reactive oxygen species in aortas of SHR-T (P , 0.05, vs. SHR-P). Vascular reactivity to endothelin-1 (ET-1) and aortic ET-1 tissue content were increased in SHR-T (P , 0.05 vs. SHR-P). Importantly, the ET-1 receptor A/B (ETA/B) antagonist bosentan normalized endothelial function in SHR-T (P , 0.05). ..................................................................................................................................................................................... Conclusion Torcetrapib induces a sustained impairment of endothelial function, decreases eNOS mRNA, protein as well as NO release, stimulates vascular ROS and ET production, an effect that is prevented by chronic ETA/B-receptor blockade. These unexpected off-target effects of torcetrapib need to be ruled out in the clinical development of novel CETP inhibitors, particularly before a large patient population at increased cardiovascular risk is exposed to these compounds. ----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords HDL † CETP inhibition † Endothelin † Endothelial dysfunction † Nitric oxide Introduction The plasma levels of high-density lipoprotein (HDL) cholesterol are inversely related to cardiovascular risk.1 – 3 Currently available HDL-raising therapies are associated with undesirable side effects, limited efficacy, or have not yet been shown to improve morbidity and mortality on top of statins in clinical outcome trials.4 – 6 A novel pharmacological target for raising circulating HDL-C levels is the cholesterol ester transfer protein (CETP), an enzyme involved in † the physiological process of reverse cholesterol transport in humans, by which excess cholesterol is removed from peripheral tissues, and then returned to the liver for secretion into the bile. Conceptually, inhibition of CETP thus provides an attractive therapeutic target.7 Indeed, three pharmacological small-molecule inhibitors of CETP, i.e. dalcetrapib (JTT-705; Roche), anacetrapib (Merck), and torcetrapib (Pfizer), have been developed. The molecules effectively raise HDL-C by 60–100% in humans either when used as a monotherapy or in combination with statins. B.S. and M.H. contributed equally to this work. *Corresponding author. Tel: +41 44 255 39 57, Fax: +41 255 87 01, Email: Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2011. For permissions please email: 1 Department of Cardiovascular Research, Institute of Physiology, University of Zurich, Zurich, Switzerland; 2Department of Cardiology, Cardiovascular Center, University Hospital Zurich, CH-8091 Zurich, Switzerland; 3Department of Clinical Research, Medical Faculty of the University of Bern, Bern, Switzerland; and 4Department of Organic Chemistry, University of Zurich, Zurich, Switzerland 1616 Methods Animals Male spontaneously hypertensive rats (SHRs) and Wistar– Kyoto (WKY) rats, 16– 20 weeks old, mean weight 320 g, were purchased from Charles River Laboratories (Research Models and Services, Germany GmbH). Animals were fed a normal chow diet and had ad libitum access to food and water, maintained at 248C under a 12 h light/dark cycle. Torcetrapib (CP-529414 Spray-dried dispersion, LOT ID FP-04-069, kindly provided by Pfizer Inc., Groton, CT, USA) or placebo was given as a suspension in 0.5% methyl cellulose in a final concentration 100 mg/kg/day p.o. (oral gavage), for a total duration of 3 weeks. Dosage of torcetrapib was adjusted for body weight and calculated on a daily basis. In addition, animals were co-treated with the endothelin (ET)-receptor antagonist bosentan 100 mg/kg/day (Actelion Pharmaceuticals Ltd, Switzerland) together with torcetrapib. Systolic BP and heart rate were measured by tail-cuff method (model LE 5002, Storage Pressure Meter, Letica, Spain) after intensive training of the animals. Study design and experimental protocols fully complied with the guidelines for research animal use by the American Heart Association and were approved by the institutional animal care committee (Licence Nr. 17/2008, Kommission für Tierversuche des Kantons Zürich, Switzerland). Tissue harvesting and organ chamber experiments At the end of the 21-day treatment, animals were first anaesthetized using pentobarbital (50 mg/kg i.p.) and sacrificed by blood exsanguinations. Blood was drawn into Li-Heparin-coated vacutainers (Vacutainerw Heparin Tubes, Additive Lithium Heparin (68 USP), BD) and plasma was isolated. Aorta was removed and placed in cold (48C) modified Krebs – Ringer bicarbonate solution. The aorta was cleaned from connective tissue and cut into rings of 3 mm length. The remaining aortic tissue was snap frozen in liquid nitrogen for further analysis. Aortic rings were placed in an organ bath for isometric tension recording as described elsewhere.18 Shortly after 60 min of an equilibration, the rings were progressively stretched to their optimal passive tension (3 g). Rings were preconstricted with norepinephr (...truncated)