Suppression of superoxide/hydrogen peroxide production at mitochondrial site IQ decreases fat accumulation, improves glucose tolerance and normalizes fasting insulin concentration in mice fed a high-fat diet.

We developed S1QEL1.719, a novel bioavailable S1QEL (suppressor of site IQ electron leak). S1QEL1.719 prevented superoxide/hydrogen peroxide production at site IQ of mitochondrial complex I in vitro. The free concentration giving half-maximal suppression (IC50) was 52 nM. Even at 50-fold higher concentrations S1QEL1.719 did not inhibit superoxide/hydrogen peroxide production from other sites. The IC50 for inhibition of complex I electron flow was 500-fold higher than the IC50 for suppression of superoxide/hydrogen peroxide production from site IQ. S1QEL1.719 was used to test the metabolic effects of suppressing superoxide/hydrogen peroxide production from site IQin vivo. C57BL/6J male mice fed a high-fat chow for one, two or eight weeks had increased body fat, decreased glucose tolerance, and increased fasting insulin concentrations, classic symptoms of metabolic syndrome. Daily prophylactic or therapeutic oral treatment of high-fat-fed animals with S1QEL1.719 decreased fat accumulation, strongly protected against decreased glucose tolerance and prevented or reversed the increase in fasting insulin level. Free exposures in plasma and liver at Cmax were 1-4 fold the IC50 for suppression of superoxide/hydrogen peroxide production at site IQ and substantially below levels that inhibit electron flow through complex I. These results show that the production of superoxide/hydrogen peroxide from mitochondrial site IQin vivo is necessary for the induction and maintenance of glucose intolerance caused by a high-fat diet in mice. They raise the possibility that oral administration of S1QELs may be beneficial in metabolic syndrome.

Increased stress associated with head-out plethysmography testing can exacerbate respiratory effects and lead to mortality in rats.

INTRODUCTION: DSM421, a dihydroorotate dehydrogenase inhibitor, was in preclinical development as a potential treatment option for malaria. When tested in a core battery of safety pharmacology assays, DSM421 did not produce any effects at oral doses up to 750 mg/kg in an Irwin test in rats, but a respiratory study in rats using head-out plethysmography resulted in substantial changes in respiratory function as well as moribundity and mortality at that and lower doses. An investigation was performed to determine the source of this discrepancy. METHODS: Potential testing errors, differences in types of plethysmography testing chambers, effects on stress indicators, and off-target activity were investigated. RESULTS: Respiratory changes and toxicity (resulting in euthanasia in extremis) were confirmed in a repeat, head-out plethysmography test, but the effects of DSM421 were much less severe overall when the rats were tested in whole-body chambers. Additionally, at the end of the 5-h post-dosing respiratory monitoring periods, levels of stress-related hormones (particularly corticosterone) were higher overall in the head-out, than in the whole-body, tested rats. Furthermore, DSM421 was found to produce changes in cardiovascular function in unrestrained rats, and it was shown to have off-target binding affinity at the adenosine A3 receptor (which is associated with bronchoconstriction). DISCUSSION: The generalized stress inherent to head-out plethysmography testing exacerbated the respiratory effects of DSM421 and was possibly compounded by DSM421's cardiovascular effects, thus artifactually resulting in moribundity and mortality in rats. Care should be taken when choosing whether to use head-out versus whole-body plethysmography chambers during respiratory function testing in animals.

Synthesis and Pharmacology of (Pyridin-2-yl)methanol Derivatives as Novel and Selective Transient Receptor Potential Vanilloid 3 Antagonists.

Transient receptor potential vanilloid 3 (TRPV3) is a Ca(2+)- and Na(+)-permeable channel with a unique expression pattern. TRPV3 is found in both neuronal and non-neuronal tissues, including dorsal root ganglia, spinal cord, and keratinocytes. Recent studies suggest that TRPV3 may play a role in inflammation, pain sensation, and skin disorders. TRPV3 studies have been challenging, in part due to a lack of research tools such as selective antagonists. Herein, we provide the first detailed report on the development of potent and selective TRPV3 antagonists featuring a pyridinyl methanol moiety. Systematic optimization of pharmacological, physicochemical, and ADME properties of original lead 5a resulted in identification of a novel and selective TRPV3 antagonist 74a, which demonstrated a favorable preclinical profile in two different models of neuropathic pain as well as in a reserpine model of central pain.

Simultaneous measurement of arterial and left ventricular pressure in conscious freely moving rats by telemetry.

Comprehensive cardiovascular assessment in conscious rodents by utilizing telemetry has been limited by the restriction of current devices to one pressure channel. The purpose of this study was to test and validate a dual pressure transmitter that allows the simultaneous measurement of arterial pressure (AP) and left ventricular pressure (LVP) in conscious freely moving rats. Six rats were surgically implanted with dual pressure transmitters. Baseline hemodynamics and circadian rhythm were observed to return within 7days. AP, heart rate (HR), LVP and indices of left ventricular contractility were stable and demonstrated a prominent circadian rhythm over a two-week period of uninterrupted recordings. Administration of the vasodilator nifedipine produced the anticipated dose-dependent decrease in AP which was accompanied by a baroreflex mediated increase in HR and cardiac contractility. The negative inotrope verapamil produced the expected dose-dependent decreases in AP and cardiac contractility. Finally, a terminal validation of the dual pressure transmitter was performed under anesthesia by measuring AP and LVP simultaneously via telemetry and from a fluid filled arterial catheter and an intraventricular Millar catheter, respectively. A range of pressures and cardiac contractility were studied by administering sequential intravenous infusions of the positive inotrope dobutamine followed by verapamil. Linear regression analysis revealed a high level of agreement between pressures measured by the dual pressure transmitter and the exteriorized catheters. Histopathologic analysis of the heart revealed mild peri-catheter fibrosis. In conclusion, the simultaneous measurement of AP and LVP offers the potential for more detailed cardiovascular assessment in conscious rats.

TRPV1 ligands with hyperthermic, hypothermic and no temperature effects in rats.

Transient receptor potential vanilloid 1 (TRPV1) is a multifunctional ion channel playing important roles in a numerous biological processes including the regulation of body temperature. Within distinct and tight chemical space of chromanyl ureas TRPV1 ligands were identified that exhibit distinctive pharmacology and a spectrum of thermoregulatory effects ranging from hypothermia to hyperthermia. The ability to manipulate these effects by subtle structural modifications of chromanyl ureas may serve as a productive approach in TRPV1 drug discovery programs addressing either side effect or desired target profiles of the compounds. Because chromanyl ureas in the TRPV1 context are generally antagonists, we verified observed partial agonist effects of a subset of compounds within that chemotype by comparing the in vitro profile of Compound 3 with known partial agonist 5'-I-RTX.

Discovery of (R)-1-(7-chloro-2,2-bis(fluoromethyl)chroman-4-yl)-3-(3-methylisoquinolin-5-yl)urea (A-1165442): a temperature-neutral transient receptor potential vanilloid-1 (TRPV1) antagonist with analgesic efficacy.

The synthesis and characterization of a series of selective, orally bioavailable 1-(chroman-4-yl)urea TRPV1 antagonists is described. Whereas first-generation antagonists that inhibit all modes of TRPV1 activation can elicit hyperthermia, the compounds disclosed herein do not elevate core body temperature in preclinical models and only partially block acid activation of TRPV1. Advancing the SAR of this series led to the eventual identification of (R)-1-(7-chloro-2,2-bis(fluoromethyl)chroman-4-yl)-3-(3-methylisoquinolin-5-yl)urea (A-1165442, 52), an analogue that possesses excellent pharmacological selectivity, has a favorable pharmacokinetic profile, and demonstrates good efficacy against osteoarthritis pain in rodents.

Pharmacology of modality-specific transient receptor potential vanilloid-1 antagonists that do not alter body temperature.

The transient receptor potential vanilloid-1 (TRPV1) channel is involved in the development and maintenance of pain and participates in the regulation of temperature. The channel is activated by diverse agents, including capsaicin, noxious heat (≥ 43°C), acidic pH (< 6), and endogenous lipids including N-arachidonoyl dopamine (NADA). Antagonists that block all modes of TRPV1 activation elicit hyperthermia. To identify efficacious TRPV1 antagonists that do not affect temperature antagonists representing multiple TRPV1 pharmacophores were evaluated at recombinant rat and human TRPV1 channels with Ca(2+) flux assays, and two classes of antagonists were identified based on their differential ability to inhibit acid activation. Although both classes of antagonists completely blocked capsaicin- and NADA-induced activation of TRPV1, select compounds only partially inhibited activation of the channel by protons. Electrophysiology and calcitonin gene-related peptide release studies confirmed the differential pharmacology of these antagonists at native TRPV1 channels in the rat. Comparison of the in vitro pharmacological properties of these TRPV1 antagonists with their in vivo effects on core body temperature confirms and expands earlier observations that acid-sparing TRPV1 antagonists do not significantly increase core body temperature. Although both classes of compounds elicit equivalent analgesia in a rat model of knee joint pain, the acid-sparing antagonist tested is not effective in a mouse model of bone cancer pain.

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.

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

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 (IC(50): 51 nmol/L, electrophysiology, 67 nmol/L, Ca(2+) assay) and rat TRPA1 (IC(50): 101 nmol/L, electrophysiology, 289 nmol/L, Ca(2+) 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 (ED(50): 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.

In vivo efficacy of acyl CoA: diacylglycerol acyltransferase (DGAT) 1 inhibition in rodent models of postprandial hyperlipidemia.

Postprandial serum triglyceride concentrations have recently been identified as a major, independent risk factor for future cardiovascular events. As a result, postprandial hyperlipidemia has emerged as a potential therapeutic target. The purpose of this study was two-fold. Firstly, to describe and characterize a standardized model of postprandial hyperlipidemia in multiple rodent species; and secondly, apply these rodent models to the evaluation of a novel class of pharmacologic agent; acyl CoA:diacylglycerol acyltransferase (DGAT) 1 inhibitors. Serum triglycerides were measured before and for 4h after oral administration of a standardized volume of corn oil, to fasted C57BL/6, ob/ob, apoE(-/-) and CD-1 mice; Sprague-Dawley and JCR/LA-cp rats; and normolipidemic and hyperlipidemic hamsters. Intragastric administration of corn oil increased serum triglycerides in all animals evaluated, however the magnitude and time-course of the postprandial triglyceride excursion varied. The potent and selective DGAT-1 inhibitor A-922500 (0.03, 0.3 and 3 mg/kg, p.o.), dose-dependently attenuated the maximal postprandial rise in serum triglyceride concentrations in all species tested. At the highest dose of DGAT-1 inhibitor, the postprandial triglyceride response was abolished. This study provides a comprehensive characterization of the time-course of postprandial hyperlipidemia in rodents. In addition, the ability of DGAT-1 inhibitors to attenuate postprandial hyperlipidemia in multiple rodent models, including those that feature insulin resistance, is documented. Exaggerated postprandial hyperlipidemia is inherent to insulin-resistant states in humans and contributes to the substantially elevated cardiovascular risk observed in these patients. Therefore, by attenuating postprandial hyperlipidemia, DGAT-1 inhibition may represent a novel therapeutic approach to reduce cardiovascular risk.

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 muM 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 mug/kg for two weeks, respectively, while paricalcitol at 0.042 mug/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.

Tumour-selective antivascular effects of the novel anti-mitotic compound A-318315: An in vivo rat regional haemodynamic study.

1. It has been shown that tubulin-binding agents can destabilize cellular microtubules and suppress tumour growth; but it has also become apparent that some compounds can exert anti-vascular effects within the neovasculature of a solid tumour. To date, the difficulty with these targets has been the ability to selectivity induce vascular damage to the tumour while leaving normal vasculature unaffected. The data presented here characterizes the in vivo, tumour selective, anti-vascular effects of the novel tubulin-binding agent A-318315. 2. To that purpose, we have used an anaesthetized in vivo rat model designed to quantify acute changes in regional vascular resistance (VR) in both tumour and non-tumour vascular beds, simultaneously. Tissue-isolated tumours (approximately 1.25 gm) with blood flow supplied by a single epigastric artery were grown in the hindlimb of adult male rats. Blood flow to the tumour, mesenteric, renal and normal (non-tumour epigastric) arteries was measured pre-dose and post-dose under anaesthesia. 3. A-318315 was tested at 3, 10 and 30 mg/kg, i.v. These doses produced modest, transient increases in mean arterial pressure with little to no effect on heart rate. At peak effect, tumour VR increased to 175 +/- 47, 337 +/- 77 and 751 +/- 151% above the baseline, for the 3, 10 and 30 mg/kg doses, respectively, whereas VR was only modestly and transiently increased in normal epigastric (88 +/- 19%), mesenteric (33 +/- 3.3%) and renal arteries (17 +/- 8.6%). 4. These data demonstrate that A-318315 produces marked reductions in tumour blood flow in the rat at doses that exert minor effects on normal vascular function.

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.

Effect of the multitargeted receptor tyrosine kinase inhibitor, ABT-869 [N-(4-(3-amino-1H-indazol-4-yl)phenyl)-N'-(2-fluoro-5-methylphenyl)urea], on blood pressure in conscious rats and mice: reversal with antihypertensive agents and effect on tumor growth inhibition.

ABT-869 [N-(4-(3-amino-1H-indazol-4-yl)phenyl)-N'-(2-fluoro-5-methylphenyl)urea] is a novel multitargeted inhibitor of the vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptor tyrosine kinase family members. ABT-869 demonstrates tumor growth inhibition in multiple preclinical animal models and in early clinical trials. VEGF receptor inhibition is also associated with reversible hypertension that may limit its benefit clinically. To evaluate optimal therapeutic approaches to prevent hypertension with VEGF receptor inhibition, we characterized the dose-dependent effects of seven antihypertensive agents from three mechanistic classes [angiotensin-converting enzyme inhibitors (ACEis), angiotensin receptor blockers (ARBs), calcium channel blockers (CCBs)] on hypertension induced by ABT-869 in conscious telemetry rats. We report that ABT-869-induced hypertension can be prevented and reversed with subtherapeutic or therapeutic doses of antihypertensive drugs with a general rank order of ACEi > ARB > CCB. In SCID mice, the ACE inhibitor, enalapril (C(20)H(28)N(2)O(5) x C(4)H(4)O(4)) at 30 mg/kg, prevented hypertension, with no attenuation of the antitumor efficacy of ABT-869. These studies demonstrate that the adverse cardiovascular effects of the VEGF/PDGF receptor tyrosine kinase inhibitor, ABT-869, are readily controlled by conventional antihypertensive therapy without affecting antitumor efficacy.

Antagonism of TRPV1 receptors indirectly modulates activity of thermoregulatory neurons in the medial preoptic area of rats.

In order to enhance understanding of TRPV1 contributions to thermoregulation, we measured the effects of a TRPV1 receptor antagonist, A-889425, on thermoregulatory neurons in the medial preoptic area of the hypothalamus (mPOA) of rats while simultaneously monitoring rectal temperature (T(r)). Administration of A-889425 (4 micromol/kg, i.v.) significantly increased T(r) by 0.42+/-0.02 degrees C in anesthetized rats. Warm-sensitive (WS) neurons in the mPOA increase firing in response to body warming, and when active stimulate heat loss and inhibit heat production. WS neurons were initially inhibited by A-889425. Subsequently, WS neuronal activity diverged, differentiating WS neurons into two subgroups. One group of WS neurons continued to be inhibited during the recording period while another group of "biphasic" WS neurons increased firing as T(r) increased. Cold-sensitive (CS) neurons fire at a higher rate during cooling of the body, and when active, may contribute to heat production. Injection of A-889425 affected CS neurons in a manner opposite to the biphasic WS neurons; activity was initially increased followed by a later decrease. Direct administration of A-889425 into the mPOA (10 and 30 nmol) or spinal cord (30 nmol) did not affect T(r). Disruption of abdominal TRPV1 receptor function by injection of the TRPV1 receptor agonist, resiniferatoxin (20 microg/kg, i.p.), 9-15 days prior to experiments, blocked the effects of systemically injected A-889425 on T(r) and mPOA neuronal activity. These data demonstrate that antagonist block of abdominal TRPV1 receptors indirectly modulates activity of thermoregulatory neurons in the mPOA in a manner that is consistent with producing an acute rise in body temperature.

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 x 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 x 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.

Repeated dosing of ABT-102, a potent and selective TRPV1 antagonist, enhances TRPV1-mediated analgesic activity in rodents, but attenuates antagonist-induced hyperthermia.

Transient receptor potential vanilloid type 1 (TRPV1) is a ligand-gated ion channel that functions as an integrator of multiple pain stimuli including heat, acid, capsaicin and a variety of putative endogenous lipid ligands. TRPV1 antagonists have been shown to decrease inflammatory pain in animal models and to produce limited hyperthermia at analgesic doses. Here, we report that ABT-102, which is a potent and selective TRPV1 antagonist, is effective in blocking nociception in rodent models of inflammatory, post-operative, osteoarthritic, and bone cancer pain. ABT-102 decreased both spontaneous pain behaviors and those evoked by thermal and mechanical stimuli in these models. Moreover, we have found that repeated administration of ABT-102 for 5-12 days increased its analgesic activity in models of post-operative, osteoarthritic, and bone cancer pain without an associated accumulation of ABT-102 concentration in plasma or brain. Similar effects were also observed with a structurally distinct TRPV1 antagonist, A-993610. Although a single dose of ABT-102 produced a self-limiting increase in core body temperature that remained in the normal range, the hyperthermic effects of ABT-102 effectively tolerated following twice-daily dosing for 2 days. Therefore, the present data demonstrate that, following repeated administration, the analgesic activity of TRPV1 receptor antagonists is enhanced, while the associated hyperthermic effects are attenuated. The analgesic efficacy of ABT-102 supports its advancement into clinical studies.

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 Ca(2+) sensitization, and it induces vasodilation through the activation of ATP-sensitive potassium channels and large conductance Ca(2+)-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 mumol/kg/30 min; n = 6) was infused as four escalating 30-min i.v. doses targeting therapeutic to supratherapeutic concentrations of levosimendan (C(max), approximately 62.6 ng/ml); metabolites were infused at one-half log-unit lower doses and responses compared to dobutamine (beta(1)-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 ED(15) values, of OR-1896 (0.03 mumol/kg) > OR-1855 > levosimendan > milrinone (0.24 mumol/kg); an ED(15) 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 ED(50) values, of dobutamine (0.03 mumol/kg) > levosimendan > OR-1896 > milrinone (0.09 mumol/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 +/- 6 to 129 +/- 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+.

Structure-activity studies of a novel series of 5,6-fused heteroaromatic ureas as TRPV1 antagonists.

Novel 5,6-fused heteroaromatic ureas were synthesized and evaluated for their activity as TRPV1 antagonists. It was found that 4-aminoindoles and indazoles are the preferential cores for the attachment of ureas. Bulky electron-withdrawing groups in the para-position of the aromatic ring of the urea substituents imparted the best in vitro potency at TRPV1. The most potent derivatives were assessed in in vivo inflammatory and neuropathic pain models. Compound 46, containing the indazole core and a 3,4-dichlorophenyl group appended to it via a urea linker, demonstrated in vivo analgesic activity upon oral administration. This derivative also showed selectivity versus other receptors in the CEREP screen and exhibited acceptable cardiovascular safety at levels exceeding the therapeutic dose.