Delvin R. Knight

Torcetrapib Induces Aldosterone and Cortisol Production by an Intracellular Calcium-Mediated Mechanism Independently of Cholesteryl Ester Transfer Protein Inhibition

ILLUMINATE (Investigation of Lipid Level Management to Understand its Impact in Atherosclerotic Events), the phase 3 morbidity and mortality trial of torcetrapib, a cholesteryl ester transfer protein (CETP) inhibitor, identified previously undescribed changes in plasma levels of potassium, sodium, bicarbonate, and aldosterone. A key question after this trial is whether the failure of torcetrapib was a result of CETP inhibition or of some other pharmacology of the molecule. The direct effects of torcetrapib and related molecules on adrenal steroid production were assessed in cell culture using the H295R as well as the newly developed HAC15 human adrenal carcinoma cell lines. Torcetrapib induced the synthesis of both aldosterone and cortisol in these two in vitro cell systems. Analysis of steroidogenic gene expression indicated that torcetrapib significantly induced the expression of CYP11B2 and CYP11B1, two enzymes in the last step of aldosterone and cortisol biosynthesis pathway, respectively. Transcription profiling indicated that torcetrapib and angiotensin II share overlapping pathways in regulating adrenal steroid biosynthesis. Hormone-induced steroid production is mainly mediated by two messengers, calcium and cAMP. An increase of intracellular calcium was observed after torcetrapib treatment, whereas cAMP was unchanged. Consistent with intracellular calcium being the key mediator of torcetrapibs effect in adrenal cells, calcium channel blockers completely blocked torcetrapib-induced corticoid release and calcium increase. A series of compounds structurally related to torcetrapib as well as structurally distinct compounds were profiled. The results indicate that the pressor and adrenal effects observed with torcetrapib and related molecules are independent of CETP inhibition.

Selective adenosine A3 receptor stimulation reduces ischemic myocardial injury in the rabbit heart

OBJECTIVE The aim of this study was to determine whether selective activation of the adenosine A3 receptor reduces infarct size in a Langendorff model of myocardial ischemia-reperfusion injury. METHODS Buffer-perfused rabbit hearts were exposed to 30 min regional ischemia and 120 min of reperfusion. Infarct size was measured by tetrazolium staining and normalized for area-at-risk (IA/AAR). RESULTS Preconditioning by 5 min global ischemia and 10 min reperfusion reduced infarct size (IA/AAR) to 19 +/- 4% (controls: 67 +/- 5%). Replacing global ischemia with 5 min perfusion of the rabbit A3-selective agonist, IB-MECA (A3 Ki: 2 nM; A1 Ki: 30 nM) elicited a concentration-dependent reduction in infarct size; 50 nM IB-MECA reduced IA/AAR to 24 +/- 4%. The A1-selective agonist, R-PIA (25 nM) reduced IA/AAR to a similar extent (21 +/- 6%). However, while the cardioprotective effect of R-PIA was significantly inhibited (54 +/- 7% IA/AAR) by the rabbit A1-selective antagonist, BWA1433 (50 nM), the IB-MECA-dependent cardioprotection was unaffected (28 +/- 6% IA/AAR). A non-selective (A1 vs. A3) concentration of BWA1433 (5 microM) significantly attenuated the IB-MECA-dependent cardioprotection (61 +/- 7% IA/AAR). CONCLUSIONS These data clearly demonstrate that selective A3 receptor activation provides cardioprotection from ischemia-reperfusion injury in the rabbit heart. Furthermore, the degree of A3-dependent cardioprotection is similar to that provided by A1 receptor stimulation or ischemic preconditioning.

Measurement of organ blood flow with coloured microspheres in the rat

OBJECTIVE The aim was to establish a method for measuring organ blood flow in rats using commercially available, coloured, dye extraction microspheres. METHODS A mixture of radiolabelled and dye extraction microspheres was infused into rats at rest (basal) and during intravenous administration of either angiotensin II (0.5 microgram.kg-1.min-1) or isoprenaline [12.5 ng.(g0.74)-1.min-1]. Tissues were removed and placed in test tubes, counted for radioactivity, then digested with 2N sodium hydroxide. Within the same tube, microspheres were isolated using centrifugation and the dye was extracted with dimethylformamide. The dye was quantified by spectrophotometry. RESULTS Recovery of microspheres averaged greater than 95% for all tissues studied; larger reagent volumes were required to achieve this level of recovery from white adipose tissue. Statistical analyses showed excellent correlations between blood flow values obtained by the dye extraction and radiolabelled microsphere techniques. Blood flow values obtained with the radioactive technique tended to be slightly higher. There were no differences in the results obtained with the two techniques when they were simultaneously used to measure changes in organ blood flow induced by angiotensin II or isoprenaline. CONCLUSIONS The coloured, dye extraction microsphere technique accurately measured organ blood flow in rats. This technique is potentially useful for estimating blood flow in any animal, even if tissue sample size is limited.

Discovery of zoniporide: A potent and selective sodium–hydrogen exchanger type 1 (NHE-1) inhibitor with high aqueous solubility

Zoniporide (CP-597,396) is a potent and selective inhibitor of NHE-1, which exhibits high aqueous solubility and acceptable pharmacokinetics for intravenous administration. The discovery, synthesis, activities, and rat and dog pharmacokinetics of this compound are presented. The potency and selectivity of zoniporide may be due to the conformation that the molecule adopts due to the presence of a cyclopropyl and a 5-quinolinyl substituent on the central pyrazole ring of the molecule.

One hour of myocardial ischemia in conscious dogs increases β-adrenergic receptors, but decreases adenylate cyclase activity

An increased myocardial beta-adrenergic receptor density has been reported following myocardial ischemia. However, it is not clear whether these receptors are effectively coupled to adenylate cyclase which would be necessary for enhanced physiological responsiveness. We, therefore, examined the effects of myocardial ischemia in six conscious dogs (4 intact and 2 with posterior wall denervation) in which the left circumflex coronary artery was occluded. Ischemia was verified by measurement of regional blood flow by radioactive microspheres. After 1 h of coronary artery occlusion, the dogs were anesthetized with pentobarbital and the left ventricle was divided into normal, intermediate and ischemic regions. A crude membrane fraction was prepared from each region. beta-Adrenergic receptors were quantitated with 125I-cyanopindolol binding and adenylate cyclase activity was measured. In all six animals studied, beta-adrenergic receptor density increased progressively and adenylate cyclase activity decreased progressively, when the ischemic myocardium was compared to the intermediate and the non-ischemic myocardium. Since adenylate cyclase activity declined, these results do not support the concept that the increased beta-receptor density induced by myocardial ischemia is causally related to enhanced beta-adrenergic sensitivity.

Zoniporide: a potent and highly selective inhibitor of human Na(+)/H(+) exchanger-1.

We evaluated the in vitro pharmacological profile of a novel, potent and highly selective Na(+)/H(+) exchanger-1 (NHE-1) inhibitor, [1-(Quinolin-5-yl)-5-cyclopropyl-1H-pyrazole-4-carbonyl]guanidine hydrochloride monohydrate (zoniporide or CP-597,396). The potency and selectivity of zoniporide were determined via inhibition of 22Na(+) uptake by PS-120 fibroblast cell lines overexpressing human NHE-1, -2 or rat NHE-3. Additionally, potency for endogenous NHE-1 was confirmed via ex vivo human platelet swelling assay (PSA), in which platelet swelling was induced by exposure to sodium propionate. The pharmacological profile of zoniporide was compared with that of eniporide and cariporide. Zoniporide inhibited 22Na(+) uptake in fibroblasts expressing human NHE-1 in a concentration-dependent manner (IC(50) = 14 nM) and was highly selective (157-fold and 15,700-fold vs. human NHE-2 and rat NHE-3, respectively). Zoniporide was 1.64- to 2.6-fold more potent at human NHE-1 than either eniporide or cariporide (IC(50) = 23 and 36 nM, respectively). Zoniporide was also more selective at inhibiting human NHE-1 vs. human NHE-2 than either eniporide or cariporide (157-fold selective compared with 27- and 49-fold, respectively). All three compounds inhibited human platelet swelling with IC(50) values in low nanomolar range. From these results, we conclude that zoniporide represents a novel, potent and highly selective NHE-1 inhibitor.

CCR2 receptor blockade alters blood monocyte subpopulations but does not affect atherosclerotic lesions in apoE-/- mice

OBJECTIVE The CCR2 receptor plays a crucial role in monocyte recruitment and has been implicated as a contributing factor to atherosclerosis. CCR2 receptor deletion leads to significant inhibition of lesion development. Our objective was to determine if CCR2 receptor blockade with a small molecule would have a beneficial effect of decreasing established lesions. METHODS AND RESULTS We demonstrated that CCR2 blockade had no significant effect on advanced lesions or the progression of fatty streaks. CCR2 blockade in mice resulted in elevations in plasma CCL2 levels and a significant reduction in the plasma Ly-6C(hi) subpopulations of monocytes expressing the CCR2 receptor. Neither CCL2 elevation nor margination of the Ly-6C(hi) population was observed in CCR2(-/-) mice. CONCLUSIONS CCR2 receptor blockade with a small molecule antagonist at dose levels showing efficacy in several inflammatory models did not show a beneficial effect in murine models of atherosclerosis. Elevations in CCL2 and margination of Ly-6C(hi) cells demonstrate that the role of CCR2 in controlling monocyte levels goes beyond the control of monocyte emigration.

Impact of Delayed Reperfusion of Myocardial Hibernation on Myocardial Ultrastructure and Function and Their Recoveries After Reperfusion in a Pig Model of Myocardial Hibernation

UNLABELLED This study examined the effect of delayed reperfusion of myocardial hibernation from 24 hours to 7 days on myocardial ultrastructural and functional changes and their recoveries after reperfusion. BACKGROUND We have previously shown in pigs that after reperfusion the functional and structural alterations in short-term myocardial hibernation which was reperfused in 24 hours can recover in 7 days. The effect of delayed reperfusion of hibernating myocardium on the extent and severity of cellular and extracellular structural changes of hibernating myocardium, and their recoveries after reperfusion is not known. METHODS AND RESULTS A severe LAD stenosis was created in 27 pigs, reducing resting flow by 30-40% immediately after placement of the stenosis and producing acute ischemia as evidenced by regional lactate production, a decrease in regional coronary venous pH, reduced regional wall thickening (from 38.5 +/- 5.1% to 10.4 +/- 8.0%) and a 33% reduction of regional oxygen consumption. The stenosis was maintained either for 24 hours in 9 pigs (group 1) with LAD flow of 0.65 +/- 0.13 ml/min/g (38% reduction), or for 7 days in 17 pigs (group 2) with LAD flow of 0.67 +/- 0.14 ml/min/g (36% reduction). There were no differences (p = NS) in the reduction of wall thickening, rate-pressure product, lactate production, or regional oxygen consumption between group 1 and group 2. Quantitative morphometric evaluation of the ultrastructure on electromicrographs revealed a greater decrease in sarcomere volume and a higher incidence of myocytes with reduced sarcomere volume in 7-day than in 24-hour hibernating regions (53 +/- 19% versus 33 +/- 14%, p < 0.05). Patchy myocardial necrosis with replacement fibrosis was common, but 6 of the 18 pigs had no myocardial necrosis or replacement fibrosis in the 7-day hibernating group, and 4 of 9 pigs had no patchy myocyte necrosis in the 24 hour hibernating group. In 6 pigs in group 1 in which the stenosis was then released and hibernating myocardium reperfused in 24 hours, regional wall thickening recovered to 30 +/- 6% (p = NS compared to baseline) after one week of reperfusion. In 12 pigs in group 2 in which the stenosis was released and hibernating myocardium reperfused in 7 days, regional wall thickening recovered slowly, from 10.1 +/- 7.2% to 18.1 +/- 8.3% at one week (n = 5) and to 28.0 +/- 3.6% at 3-4 weeks of reperfusion (n = 7, p < 0.05 compared to baseline). Similarly, the sarcomere volume or myofilament recovered significantly (p < 0.01) and was not different compared to the normal region (p = NS) in the 24-hour hibernating region of group 1, but the recovery was much slower and was incomplete at 4 weeks (p < 0.01) compared to baseline in the 7-day hibernating region of group 2. Recovery of regional wall thickening correlated with ultrstructural recovery (p < 0.01). By multivariate stepwise regression analysis, the degree of LAD flow reduction, the extent of fibrosis, and myofilament loss were independent predictors of the extent of functional recovery. CONCLUSIONS In a porcine model of myocardial hibernation with myocardial hypoperfusion, systolic dysfunction, and metabolic adaptations, a longer period of myocardial hibernation with delayed reperfusion was associated with more severe abnormalities of myocytes. an increasing interstitial fibrosis, and more protracted myofibrillar and functional recoveries after reperfusion. The extent of functional recovery is related to the degree of coronary flow reduction, the severity of the ultrastructural changes, and the extent of interstitial fibrosis.

NO modulates myocardial O2consumption in the nonhuman primate: an additional mechanism of action of amlodipine

Recent evidence from our laboratory and others suggests that nitric oxide (NO) is a modulator of in vivo and in vitro oxygen consumption in the murine and canine heart. Therefore, the goal of our study was twofold: to determine whether NO modulates myocardial oxygen consumption in the nonhuman primate heart in vitro and to evaluate whether the seemingly cardioprotective actions of amlodipine may involve an NO-mediated mechanism. Using a Clark-type O2 electrode, we measured oxygen consumption in cynomologous monkey heart at baseline and after increasing doses of S-nitroso-N-acetylpenicillamine (SNAP; 10(-7)-10(-4) M), bradykinin (10(-7)-10(-4) M), ramiprilat (10(-7)-10(-4) M), and amlodipine (10(-7)-10(-5) M). SNAP (-38 +/- 5.8%), bradykinin (-19 +/- 3.9%), ramiprilat (-28 +/- 2.3%), and amlodipine (-23 +/- 4.5%) each caused significant (P < 0.05) reductions in myocardial oxygen consumption at their highest dose. Preincubation of tissue with nitro-L-arginine methyl ester (10(-4) M) blunted the effects of bradykinin (-5.4 +/- 3.2%), ramiprilat (-4.8 +/- 5.0%), and amlodipine (-5.3 +/- 5.0%) but had no effect on the tissue response to SNAP (-38 +/- 5.8%). Our results indicate that NO can reduce oxygen consumption in the primate myocardium in vitro, and they support a role for the calcium-channel blocker amlodipine as a modulator of myocardial oxygen consumption via a kinin-NO mediated mechanism.Recent evidence from our laboratory and others suggests that nitric oxide (NO) is a modulator of in vivo and in vitro oxygen consumption in the murine and canine heart. Therefore, the goal of our study was twofold: to determine whether NO modulates myocardial oxygen consumption in the nonhuman primate heart in vitro and to evaluate whether the seemingly cardioprotective actions of amlodipine may involve an NO-mediated mechanism. Using a Clark-type O2 electrode, we measured oxygen consumption in cynomologous monkey heart at baseline and after increasing doses of S-nitroso- N-acetylpenicillamine (SNAP; 10-7-10-4M), bradykinin (10-7-10-4M), ramiprilat (10-7-10-4M), and amlodipine (10-7-10-5M). SNAP (-38 ± 5.8%), bradykinin (-19 ± 3.9%), ramiprilat (-28 ± 2.3%), and amlodipine (-23 ± 4.5%) each caused significant ( P < 0.05) reductions in myocardial oxygen consumption at their highest dose. Preincubation of tissue with nitro-l-arginine methyl ester (10-4 M) blunted the effects of bradykinin (-5.4 ± 3.2%), ramiprilat (-4.8 ± 5.0%), and amlodipine (-5.3 ± 5.0%) but had no effect on the tissue response to SNAP (-38 ± 5.8%). Our results indicate that NO can reduce oxygen consumption in the primate myocardium in vitro, and they support a role for the calcium-channel blocker amlodipine as a modulator of myocardial oxygen consumption via a kinin-NO mediated mechanism.

Effects of CP-532,623 and torcetrapib, cholesteryl ester transfer protein inhibitors, on arterial blood pressure.

ILLUMINATE, the phase 3 morbidity and mortality trial of the cholesteryl ester transfer protein (CETP) inhibitor, torcetrapib, plus atorvastatin terminated in 2006. The underlying morbidity and mortality cause remains undetermined. In addition to lipoprotein changes, off-target increases in blood pressure (BP), sodium, bicarbonate, and aldosterone and potassium decreases were described. We report nonclinical and clinical studies using torcetrapib and a closely related CETP inhibitor, CP-532,623, to further characterize this pharmacology. Pressor effects of torcetrapib and CP-532,623 were observed in monkeys and human subjects. CETP inhibition and high-density lipoprotein cholesterol elevation were demonstrated. In humans, high- versus low-dose CP-532,623 produced significantly greater pressor effects despite similar maximal CETP inhibition. Inhibition of CETP was seen 48 hours post dose, whereas BP elevation dissipated by 24 hours, temporally dissociating CETP inhibition from BP changes. These data, and structural similarities between the compounds, support the conclusion that the BP effects are related to chemotype. We also observed an acute aldosterone increase without changes in renin in monkeys. Continuous BP measurements showed persistent elevations, whereas aldosterone changes were transient, suggesting that increases in BP were not directly the result of renin-angiotensin-aldosterone system activation and may, in part, be due to direct effects on blood vessels or other nongenomic effects.