Effects of age on noninvasive assessments of vascular function in nonhuman primates: implications for translational drug discovery.

BACKGROUND: Endothelium-dependent flow mediated dilation (FMD) and pulse-wave velocity (PWV), are used as measures of vascular health and predictors of cardiovascular risk in clinical studies, and both are age-dependent. Numbers of circulating endothelial microparticles (EMPs) and endothelial progenitor cells (EPCs) are also associated with cardiovascular risk, but independent of age in humans. The use of these measurements for pre-clinical assessment of drug cardiovascular safety and efficacy in non-human primates (NHPs) may promote the translation of drug-induced effects on vascular function to clinic outcomes. However, in NHPs, the age effects on the non-invasive measurements of FMD and PWV and the relationship of EMPs/EPCs with FMD are unknown. METHODS: A non-invasive, clinically-relevant approach to assess FMD and PWV was used to examine their relationship with age and with EMPs/EPCs in NHPs. The effects on FMD of nicotine and rosiglitazone were evaluated in senescent primates in an effort to validate our FMD method for pre-clinical assessment of vascular function. RESULTS: FMD and PWV methods were established in a colony (n = 25) of metabolically healthy, cynomolgus monkeys ranging in age from 6 to 26 years. FMD, defined as the percent change, at 1 min of cuff release, from baseline vascular diameter (0.15 ± 0.03 cm), had a strong, negative correlation with age (r = -0.892, p < 0.0001), ranging from 6% to 33%. PWV positively correlated with age (r = 0.622, p < 0.002) in the same healthy monkeys. Nicotine and rosiglitazone, were evaluated in subsets of senescent primates (mean age 16.3 ± 1.5[SEM] years). Rosiglitazone significantly improved FMD (21.0 ± 1.6% vs. vehicle 16.3 ± 1.6%, p < 0.01) without changing baseline diameters, and coincided with a significant increase in circulating numbers of endothelial progenitor cells (CD45-CD31 + CD34 + VEGFR2+ 7.1 ± 1.3 vs. 4.8 ± 1.1 counts/µl) and a decrease in endothelial microparticles (CD45-CD42a-CD54+ 26.7 ± 11.1 vs. 62.2 ± 9.8 counts/µl)(p < 0.05). Conversely, FMD was significantly reduced with nicotine (8.7 ± 1.4% vs. vehicle 20.1 ± 2.2%, p < 0.05). CONCLUSIONS: Adult NHPs demonstrate the characteristic linear relationship between age and vascular function using the non-invasive clinically-related measurements of FMD and PWV. However, numbers of circulating EMPs and EPCs did not correlate with age. Endothelial function assessed with FMD, together with EMPs/EPCs assessment, may serve as a novel approach for translational research and therapeutic discovery. Age should be considered in the study design or data analyses when FMD or PWV is used in NHPs.

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.

Novel variants in human and monkey CETP.

Variation in CETP has been shown to play an important role in HDL-C levels and cardiovascular disease. To better characterize this variation, the promoter and exonic DNA for CETP was resequenced in 189 individuals with extreme HDL-C or age. Two novel amino acid variants were found in humans (V-12D and Y361C) and an additional variant (R137W) not previously studied in vitro were expressed. D-12 was not secreted and had no detectable activity in cells. C361 and W137 retained near normal amounts of cholesteryl ester transfer activity when purified but were less well secreted than wild type. Torcetrapib, a CETP inhibitor in clinical development with atorvastatin, was found to have a uniform effect on inhibition of wild type CETP versus W137 or C361. In addition, the level of variation in other species was assessed by resequencing DNA from nine cynomolgus monkeys. Numerous intronic and silent SNPs were found as well as two variable amino acids. The amino acid altering SNPs were genotyped in 29 monkeys and not found to be significantly associated with HDL-C levels. Three SNPs found in monkeys were identical to three found in humans with these SNPs all occurring at CpG sites.

Comparisons between electromagnetic and X-beam transit-time flow measurements for evaluating drug actions on cardiac output in the conscious dog.

INTRODUCTION: Cardiac output remains an important preclinical measurement for evaluating the cardiovascular effects of drugs. We evaluated the performance of the Triton Active Redirection Transit-Time, ART(2), which represents a new class of X-beam flow systems and compared it in vivo and in vitro to an electromagnetic flow (EMF) system for measuring large vessel flow. METHODS: In vivo, simultaneous aortic flow measurements were obtained during alpha- and beta-adrenergic receptor stimulation in 5 conscious dogs instrumented with both ART(2) and EMF probes on their ascending aortas. In vitro, simultaneous measurements of volume flow using the ART(2), EMF, and timed-volume collection were made using a novel benchtop flow apparatus that ensured probe alignment and precise timed-volume flow measurements. Accuracy and sensitivity of both systems were assessed by recording flow measurements while varying rates, temperature and hematocrit. RESULTS: In vivo aortic flow measurements between ART(2) and EMF were closely correlated (linear regression r(2) values ranged from 0.84 to 0.99), with the ART(2) system recording lower flow values than the EMF. In vitro ART(2) flow rates were in excellent agreement with timed-volume flow, while EMF flow rates were lower (p<0.05) and exhibited more variation and dependency upon temperature or hematocrit than the ART(2). Saline flows measured by ART(2) and EMF averaged 97+/-2% and 91+/-5% accuracy, respectively, over the temperature range 32 degrees C to 42 degrees C. For blood hematocrit values between 35% and 45%, ART(2) accuracy averaged 98+/-2%, compared to 89+/-5% accuracy with the EMF. DISCUSSION: The ART(2) flow measurements in conscious dogs correlated closely to concurrent measurements obtained with the EMF over a wide range of flow rates, even though the absolute aortic flow values differed. Since it accurately measured flow in vitro, the ART(2) system is an appropriate alternative for evaluating cardiovascular effects of disease progression or drug administration in experimental animals.

3'-Aminoadenosine-5'-uronamides: discovery of the first highly selective agonist at the human adenosine A3 receptor.

Selective adenosine A(3) agonists have potential utility for the prevention of perioperative myocardial ischemic injury. Herein, we report on the discovery and synthesis of compound 7. This amino nucleoside agonist possesses unprecedented levels of selectivity for the human adenosine A(3) receptor.

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.

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 torcetrapib's 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.

The synthesis of highly potent, selective, and water-soluble agonists at the human adenosine A3 receptor.

Using a combination of parallel and directed synthesis, the discovery of a highly potent and selective series of adenosine A3 agonists was achieved. High aqueous solubility, required for the intended parenteral route of administration, was achieved by the presence of one or two basic amine functional groups.

The sulfonylurea glipizide does not inhibit ischemic preconditioning in anesthetized rabbits.

The K(ATP) channel blocker glibenclamide inhibits cardioprotection afforded by ischemic preconditioning (IPC), raising concern about sulfonylurea use by patients with cardiovascular disease. We examined the effects of the widely prescribed sulfonylurea glipizide (Glucotrol XL(R) ) on IPC in anesthetized rabbits. Initially, in parallel studies in pentobarbital-anesthetized rabbits, we identified doses of glipizide (GLIP, 0.17 mg/kg + 0.12 mg/kg/h, IV) and glibenclamide (GLIB, 0.05 mg/kg + 0.03 mg/kg/h, IV) that produced steady-state, clinically relevant plasma levels of both drugs; these doses also significantly increased plasma insulin by 51 +/- 17% (GLIP) and by 57 +/- 17% (GLIB, both p < 0.05 vs. their respective baseline levels). Subsequent parallel studies in ketamine-xylazine-anesthetized rabbits examined the effects of these doses of GLIP and GLIB on IPC. Myocardial injury (30 min coronary occlusion/120 min reperfusion), either with or without IPC (5 min occlusion/10 min reperfusion) was induced midway during a 2 h infusion of vehicle (VEH), GLIP or GLIB (n = 10-11 each). Infarct area (IA) normalized to area-at-risk (%IA/AAR) was 62 +/- 3% in the VEH group, and was significantly reduced to 39 +/- 5% by IPC (p < 0.05 vs. VEH). Neither GLIP nor GLIB treatment had any effect on %IA/AAR in the absence of IPC (p > 0.05). IPC-induced cardioprotection was preserved in the GLIP + IPC treatment group (45 +/- 4%) when compared to VEH alone (p < 0.05), but was attenuated in the presence of GLIB (GLIB+IPC: 53 +/- 4% IA/AAR, p > 0.05 vs. VEH). Thus, at a clinically relevant plasma concentration, glipizide did not limit the cardioprotective effects of IPC, and is unlikely to increase the severity of cardiac ischemic injury.

Zoniporide: a potent and selective inhibitor of the human sodium-hydrogen exchanger isoform 1 (NHE-1).

The sodium-hydrogen exchanger isoform-1 (NHE-1) plays an important role in the myocardial response to ischemia-reperfusion; inhibition of this exchanger protects against ischemic injury, including reduction in infarct size. Herein we describe a novel, potent, and highly selective NHE-1 inhibitor, zoniporide (CP-597,396; [1-(quinolin-5-yl)-5-cyclopropyl-1H-pyrazole-4-carbonyl] guanidine). Zoniporide inhibits human NHE-1 with an IC(50) of 14 nM, has >150-fold selectivity vs. other NHE isoforms, and potently inhibits ex vivo NHE-1-dependent swelling of human platelets. This compound is well tolerated in preclinical animal models, exhibits moderate plasma protein binding, has a t(1/2) of 1.5 h in monkeys, and has one major active metabolite. In both in vitro and in vivo rabbit models of myocardial ischemia-reperfusion injury, zoniporide markedly reduced infarct size without adversely affecting hemodynamics or cardiac function. In the isolated heart (Langendorff), zoniporide elicited a concentration-dependent reduction in infarct size (EC(50) = 0.25 nM). At 50 nM it reduced infarct size by 83%. This compound was 2.5-20-fold more potent than either eniporide or cariporide (EC(50)s of 0.69 and 5.11 nM, respectively), and reduced infarct size to a greater extent than eniporide. In open chest, anesthetized rabbits, zoniporide also elicited a dose-dependent reduction in infarct size (ED(50) = 0.45 mg/kg/h) and inhibited NHE-1-mediated platelet swelling (93% inhibition at 4 mg/kg/h). Furthermore, zoniporide attenuated postischemic cardiac contractile dysfunction in conscious primates, and reduced both the incidence and duration of ischemia-reperfusion-induced ventricular fibrillation in rats. Zoniporide represents a novel class of potent and selective human NHE-1 inhibitors with potential utility for providing cardioprotection in a clinical setting.

Novel N6-substituted adenosine 5'-N-methyluronamides with high selectivity for human adenosine A3 receptors reduce ischemic myocardial injury.

We recently reported the identification of a novel human adenosine A3 receptor-selective agonist, (2S,3S,4R,5R)-3-amino-5-[6-[5-chloro-2-(3-methylisoxazol-5-ylmethoxy)benzylamino]purin-9-yl]-4-hydroxytetrahydrofuran-2-carboxylic acid methylamide (CP-608,039), with 1,260-fold selectivity for the human A3 versus human A1 receptor (DeNinno et al., J Med Chem 46: 353-355, 2003). However, because the modest (20-fold) rabbit A3 receptor selectivity of CP-608,039 precludes demonstration of A3-mediated cardioprotection in rabbit models, we identified another member of this class, (2S,3S,4R,5R)-3-amino-5-[6-(2,5-dichlorobenzylamino)purin-9-yl]-4-hydroxytetrahydrofuran-2-carboxylic acid methylamide (CP-532,903), which both retained human A3 receptor selectivity (210-fold; human A3/human A1 Ki: 23/4,800 nM) and had improved rabbit A3 receptor selectivity (90-fold; rabbit A3/rabbit A1 Ki: 23/2,000 nM). Infarct size was measured in Langendorff hearts or in vivo after 30 min of regional ischemia and 120 min of reperfusion. Five-minute perfusion with CP-532,903 before ischemia-reperfusion elicited a concentration-dependent reduction in infarct size in isolated hearts (EC50: 0.97 nM; maximum reduction in infarct size: 77%, P < 0.05 vs. control). Furthermore, administration of CP-532,903 (150 nM) at reperfusion also significantly reduced infarct size by 64% (P < 0.05 vs. control), which was not different (P > or = 0.05) from the cardioprotection provided by the same concentration of drug given before ischemia. The selective rabbit A1 receptor antagonist BWA1433 did not affect CP-532,903-dependent cardioprotection. In vivo, CP-532,903 (1 mg/kg) reduced infarct size by 50% in the absence of significant hemodynamic effects (mean arterial pressure, heart rate, rate-pressure product). CP-532,903 and CP-608,039 represent a novel class of human A3 receptor-selective agonists that may prove suitable for investigation of the clinical cardioprotective efficacy of A3 receptor activation.

Cardioprotective effects of ingliforib, a novel glycogen phosphorylase inhibitor.

Interventions such as glycogen depletion, which limit myocardial anaerobic glycolysis and the associated proton production, can reduce myocardial ischemic injury; thus it follows that inhibition of glycogenolysis should also be cardioprotective. Therefore, we examined whether the novel glycogen phosphorylase inhibitor 5-Chloro-N-[(1S,2R)-3-[(3R,4S)-3,4-dihydroxy-1-pyrrolidinyl)]-2-hydroxy-3-oxo-1-(phenylmethyl)propyl]-1H-indole-2-carboxamide (ingliforib; CP-368,296) could reduce infarct size in both in vitro and in vivo rabbit models of ischemia-reperfusion injury (30 min of regional ischemia, followed by 120 min of reperfusion). In Langendorff-perfused hearts, constant perfusion of ingliforib started 30 min before regional ischemia and elicited a concentration-dependent reduction in infarct size; infarct size was reduced by 69% with 10 microM ingliforib. No significant drug-induced changes were observed in either cardiac function (heart rate, left ventricular developed pressure) or coronary flow. In open-chest anesthetized rabbits, a dose of ingliforib (15 mg/kg loading dose; 23 mg.kg(-1).h(-1) infusion) selected to achieve a free plasma concentration equivalent to an estimated EC(50) in the isolated hearts (1.2 microM, 0.55 microg/ml) significantly reduced infarct size by 52%, and reduced plasma glucose and lactate concentrations. Furthermore, myocardial glycogen phosphorylase a and total glycogen phosphorylase activity were reduced by 65% and 40%, respectively, and glycogen stores were preserved in ingliforib-treated hearts. No significant change was observed in mean arterial pressure or rate-pressure product in the ingliforib group, although heart rate was modestly decreased postischemia. In conclusion, glycogen phosphorylase inhibition with ingliforib markedly reduces myocardial ischemic injury in vitro and in vivo; this may represent a viable approach for both achieving clinical cardioprotection and treating diabetic patients at increased risk of cardiovascular disease.