Jason A. Segreti

Selective blockade of TRPA1 channel attenuates pathological pain without altering noxious cold sensation or body temperature regulation

&NA; Despite the increasing interest in TRPA1 channel as a pain target, its role in cold sensation and body temperature regulation is not clear; the efficacy and particularly side effects resulting from channel blockade remain poorly understood. Here we use a potent, selective, and bioavailable antagonist to address these issues. A‐967079 potently blocks human (IC50: 51 nmol/L, electrophysiology, 67 nmol/L, Ca2+ assay) and rat TRPA1 (IC50: 101 nmol/L, electrophysiology, 289 nmol/L, Ca2+ assay). It is >1000‐fold selective over other TRP channels, and is >150‐fold selective over 75 other ion channels, enzymes, and G‐protein‐coupled receptors. Oral dosing of A‐967079 produces robust drug exposure in rodents, and exhibits analgesic efficacy in allyl isothiocyanate‐induced nocifensive response and osteoarthritic pain in rats (ED50: 23.2 mg/kg, p.o.). A‐967079 attenuates cold allodynia produced by nerve injury but does not alter noxious cold sensation in naive animals, suggesting distinct roles of TRPA1 in physiological and pathological states. Unlike TRPV1 antagonists, A‐967079 does not alter body temperature. It also does not produce locomotor or cardiovascular side effects. Collectively, these data provide novel insights into TRPA1 function and suggest that the selective TRPA1 blockade may present a viable strategy for alleviating pain without untoward side effects. Selective blockade of TRPA1 channel attenuates pathological pain without altering noxious cold sensation or body temperature regulation.

Effect of bradykinin metabolism inhibitors on evoked hypotension in rats: rank efficacy of enzymes associated with bradykinin-mediated angioedema

Inhibition of bradykinin metabolizing enzymes (BMEs) can cause acute angioedema, as demonstrated in a recent clinical trial in patients administered the antihypertensive, omapatrilat. However, the relative contribution of specific BMEs to this effect is unclear and confounded by the lack of a predictive pre‐clinical model of angioedema.

Vitamin D Receptor Activation Mitigates the Impact of Uremia on Endothelial Function in the 5/6 Nephrectomized Rats

Endothelial dysfunction increases cardiovascular disease risk in chronic kidney disease (CKD). This study investigates whether VDR activation affects endothelial function in CKD. The 5/6 nephrectomized (NX) rats with experimental chronic renal insufficiency were treated with or without paricalcitol, a VDR activator. Thoracic aortic rings were precontracted with phenylephrine and then treated with acetylcholine or sodium nitroprusside. Uremia significantly affected aortic relaxation (−50.0 ± 7.4% in NX rats versus −96.2 ± 5.3% in SHAM at 30 μM acetylcholine). The endothelial-dependent relaxation was improved to –58.2 ± 6.0%, –77.5 ± 7.3%, and –90.5 ± 4.0% in NX rats treated with paricalcitol at 0.021, 0.042, and 0.083 μg/kg for two weeks, respectively, while paricalcitol at 0.042 μg/kg did not affect blood pressure and heart rate. Parathyroid hormone (PTH) suppression alone did not improve endothelial function since cinacalcet suppressed PTH without affecting endothelial-dependent vasorelaxation. N-omega-nitro-L-arginine methyl ester completely abolished the effect of paricalcitol on improving endothelial function. These results demonstrate that VDR activation improves endothelial function in CKD.

Evaluation of tissue perfusion in a rat model of hind-limb muscle ischemia using dynamic contrast-enhanced magnetic resonance imaging

To evaluate the feasibility of using dynamic contrast‐enhanced magnetic resonance imaging (MRI) for assessment of muscle perfusion in a rat model of hind‐limb ischemia.

ETA receptor blockade with atrasentan prevents hypertension with the multitargeted tyrosine kinase inhibitor ABT-869 in telemetry-instrumented rats.

ABT-869 is a novel multitargeted inhibitor of vascular endothelial growth factor and platelet-derived growth factor receptor tyrosine kinases (RTKs) with potent antiangiogenic properties that slow tumor progression. Vascular endothelial growth factor receptor blockade has been shown to produce hypertension. Atrasentan is a potent and selective endothelin (ETA) receptor antagonist that lowers blood pressure and affects tumor growth. To assess the utility of ETA receptor blockade in controlling hypertension with RTK inhibition, we evaluated the ability of atrasentan to block hypertension with ABT-869 in conscious, telemetry-instrumented rats. Changes in mean arterial pressure (MAP) and heart rate (HR) were evaluated using mean values and the area under the curve (AUC). Atrasentan (0.5, 1.5, and 5.0 mg kg−1 d−1 for 5 days) elicited dose-dependent decreases in MAP-AUC (−16.7 ± 1.3, −20.94 ± 3.68, and −30.12 ± 3.57 mm Hg·day, respectively) compared with vehicle. ABT-869 (1, 3, 10, 30 mg kg−1 d−1 for 5 days) increased MAP compared with vehicle (MAP-AUC values of −5.52 ± 3.75, 12.7 ± 8.4, 37.5 ± 4.4, and 63.8 ± 3.3 mm Hg·day, respectively). Pretreatment with atrasentan (5 mg/kg for 5 days) prevented and abolished the hypertensive effects of ABT-869. Thus, ETA receptor blockade effectively alleviated hypertension with RTK inhibition and may serve a dual therapeutic role by preventing hypertension and slowing tumor progression.

Diacylglycerol Acyltransferase 1 Inhibition Lowers Serum Triglycerides in the Zucker Fatty Rat and the Hyperlipidemic Hamster

Acyl CoA/diacylglycerol acyltransferase (DGAT) 1 is one of two known DGAT enzymes that catalyze the final and only committed step in triglyceride biosynthesis. The purpose of this study was to test the hypothesis that chronic inhibition of DGAT-1 with a small-molecule inhibitor will reduce serum triglyceride concentrations in both genetic and diet-induced models of hypertriglyceridemia. Zucker fatty rats and diet-induced dyslipidemic hamsters were dosed orally with A-922500 (0.03, 0.3, and 3-mg/kg), a potent and selective DGAT-1 inhibitor, for 14 days. Serum triglycerides were significantly reduced by the 3 mg/kg dose of the DGAT-1 inhibitor in both the Zucker fatty rat (39%) and hyperlipidemic hamster (53%). These serum triglyceride changes were accompanied by significant reductions in free fatty acid levels by 32% in the Zucker fatty rat and 55% in the hyperlipidemic hamster. In addition, high-density lipoprotein-cholesterol was significantly increased (25%) in the Zucker fatty rat by A-922500 administered at 3 mg/kg. This study provides the first report that inhibition of DGAT-1, the final and only committed step of triglyceride synthesis, with a selective small-molecule inhibitor, significantly reduces serum triglyceride levels in both genetic and diet-induced animal models of hypertriglyceridemia. The results of this study support further investigation of DGAT-1 inhibition as a novel therapeutic approach to the treatment of hypertriglyceridemia in humans, and they suggest that inhibition of triglyceride synthesis may have more diverse beneficial effects on serum lipid profiles beyond triglyceride lowering.

Evoked Changes in Cardiovascular Function in Rats by Infusion of Levosimendan, OR-1896 [(R)-N-(4-(4-Methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)acetamide], OR-1855 [(R)-6-(4-Aminophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one], Dobutamine, and Milrinone: Comparative Effects on Peripheral Resistance, Cardiac Output, dP/dt, Pulse Rate, and Blood Pressure

Levosimendan enhances cardiac contractility primarily via Ca2+ sensitization, and it induces vasodilation through the activation of ATP-sensitive potassium channels and large conductance Ca2+-activated K+ channels. However, the concentration-dependent hemodynamic effects of levosimendan and its metabolites (R)-N-(4-(4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)acetamide (OR-1896) and (R)-6-(4-aminophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (OR-1855) have not been well defined. Thus, levosimendan (0.03, 0.10, 0.30, and 1.0 μmol/kg/30 min; n = 6) was infused as four escalating 30-min i.v. doses targeting therapeutic to supratherapeutic concentrations of levosimendan (Cmax, ∼62.6 ng/ml); metabolites were infused at one-half log-unit lower doses and responses compared to dobutamine (β1-agonist) and milrinone (phosphodiesterase 3 inhibitor). Peak concentrations of levosimendan, OR-1896, and OR-1855 at the end of the high dose were 323 ± 14, 83 ± 2, and 6 ± 2 ng/ml, respectively (OR-1855 rapidly metabolized to OR-1896; peak = 82 ± 3 ng/ml). Levosimendan and OR-1896 produced dose-dependent reductions in blood pressure and peripheral resistance with a rank potency, based on ED15 values, of OR-1896 (0.03 μmol/kg) > OR-1855 > levosimendan > milrinone (0.24 μmol/kg); an ED15 for dobutamine could not be defined. Only dobutamine produced increases in pulse pressure (30 ± 5%) and rate-pressure product (34 ± 4%). All of the compounds, with the exception of OR-1855, elicited dose-dependent increases in dP/dt with a rank potency, based on ED50 values, of dobutamine (0.03 μmol/kg) > levosimendan > OR-1896 > milrinone (0.09 μmol/kg), although only levosimendan produced sustained increases in cardiac output (9 ± 4%). Thus, levosimendan and OR-1896 are hemodynamically active at sub- to supratherapeutic concentrations (whereas the effects of OR-1855 in the rat are thought to be predominantly mediated by conversion to OR-1896) and produce direct inotropic effects and also direct relaxation of the peripheral vasculature, which clearly differentiates them from dobutamine, which does not elicit K+ channel activation, suggesting a more balanced effect on the cardiac-contractile state and K+ channel-mediated changes in vascular resistance.

Systemic Activation of the Calcium Sensing Receptor Produces Acute Effects on Vascular Tone and Circulatory Function in Uremic and Normal Rats: Focus on Central versus Peripheral Control of Vascular Tone and Blood Pressure by Cinacalcet

Calcium-sensing receptor (CaR) activation decreases serum parathyroid hormone (PTH) and Ca2+ and, despite long-term reductions in mean arterial blood pressure (MAP), may produce acute hypertension in rats, an effect we hypothesized was mediated by constriction of multiple vascular beds. Rats were subjected to 5/6 nephrectomy (NX) or no surgery (Normal); at 7 to 8 weeks, uremia animals were anesthetized and instrumented to record MAP and regional blood flow (carotid, mesenteric, and hindlimb). Cinacalcet [N-(1-naphthalen-1-ylethyl)-3-[3-(trifluoromethyl)phenyl]-propan-1-amine; 1, 3, and 10 mg/kg; 30 min/dose] was infused over 90 min. In NX rats, cinacalcet dose-dependently decreased ionized calcium (iCa2+), elicited a 90% reduction in PTH, and produced dose-dependent self-limiting increases in MAP (from 119 ± 6to129 ± 5, 142 ± 4, and 145 ± 3 mm Hg at the end of each infusion). At 1 mg/kg, carotid vascular resistance (CVR) and mesenteric vascular resistance (MVR) increased to 16 ± 6 and 18 ± 6% above baseline, respectively. Hindlimb vascular resistance (HVR) also trended upward (13 ± 8%). At 3 mg/kg, increases in CVR (38 ± 10%), MVR (40 ± 8%), and HVR (39 ± 14%) were exacerbated; at 10 mg/kg, values remained at or near these levels. The effects of cinacalcet in Normal rats were similar to NX and were attenuated by ganglionic blockade with hexamethonium at low doses but remained significantly elevated at higher doses. Thus, CaR activation acutely increases MAP in uremic and nonuremic rats, responses that occur in parallel to vasoconstriction in multiple vascular beds through both a central and peripheral mechanism of action. Moreover, subsequent mechanistic studies suggest that increases in MAP produced by cinacalcet may be mediated by reduced tonic NO synthase-dependent NO production subsequent to reductions in blood iCa2+.

Effects of selective dopamine receptor subtype agonists on cardiac contractility and regional haemodynamics in rats

1. Activation of dopamine (DA) receptors produces cardiovascular responses such as vasodilation and hypotension. However, knowledge of the role of specific dopamine receptor subtypes (especially D3 and D4) in the cardiovascular system is limited. The objective of the present study was to characterize the haemodynamic and cardiac responses to agonists with selectivity for D1, D2, D3 and D4 receptor subtypes.

Identification and preliminary characterization of a potent, safe, and orally efficacious inhibitor of acyl-CoA:diacylglycerol acyltransferase 1.

A high-throughput screen against human DGAT-1 led to the identification of a core structure that was subsequently optimized to afford the potent, selective, and orally bioavailable compound 14. Oral administration at doses ≥0.03 mg/kg significantly reduced postprandial triglycerides in mice following an oral lipid challenge. Further assessment in both acute and chronic safety pharmacology and toxicology studies demonstrated a clean profile up to high plasma levels, thus culminating in the nomination of 14 as clinical candidate ABT-046.