The renal outer medullary potassium (ROMK) channel mediates potassium recycling and facilitates sodium reabsorption through the Na + /K + /2Cl − cotransporter in the loop of Henle and potassium secretion at the cortical collecting duct. Evidence from the phenotype of humans and rodents with functional ROMK deficiency supports the contention that selective ROMK inhibitors (ROMKi) will represent a novel diuretic with potential of therapeutic benefit for hypertension. ROMKi have recently been synthesized by Merck & Co, Inc. The present studies were designed to examine the effects of ROMKi B on systemic hemodynamics, renal function and structure, and vascular function in Dahl salt-sensitive rats. Four experimental groups—control, high-salt diet alone; ROMKi B 3 mg·kg − 1 ·d − 1 ; ROMKi B 10 mg·kg − 1 ·d − 1 ; and hydrochlorothiazide 25 mg·kg − 1 ·d − 1 —were included in prophylactic (from week 1 to week 9 on high-salt diet) and therapeutic studies (from week 5 to week 9 on high-salt diet), respectively. ROMKi B produced sustained blood pressure reduction and improved renal and vascular function and histological alterations induced by a high-salt diet. ROMKi B was superior to hydrochlorothiazide at reducing blood pressure. Furthermore, ROMKi B provided beneficial effects on both the plasma lipid profile and bone mineral density. Chronic ROMK inhibition not only prevented but also reversed the development of hypertension and end-organ damage in Dahl salt-sensitive rats. Our findings suggest a potential utility of ROMKi B as a novel antihypertensive agent, particularly for the treatment of the salt-sensitive hypertension patient population.

We report the discovery of a benzimidazole series of CYP11B2 inhibitors. Hit-to-lead and lead optimization studies identified compounds such as 32, which displays potent CYP11B2 inhibition, high selectivity versus related CYP targets, and good pharmacokinetic properties in rat and rhesus. In a rhesus pharmacodynamic model, 32 produces dose-dependent aldosterone lowering efficacy, with no apparent effect on cortisol levels.

Introduction: In vivo profiles of aldosterone synthase inhibitors (ASIs) have been investigated utilizing various rodent models. Due to lack of CYP17 activity, rodents produce corticosterone rather than cortisol as that of humans, which raised concern to their effectiveness in translational pharmacological characterization of ASI. Methods: A rhesus monkey model that combines a low sodium diet with adrenocorticotropin (ACTH) treatment was developed. Plasma concentrations of steroid metabolites associated with reactions catalyzed by CYP11B2 and CYP11B1 were measured concurrently by a UPLC/MS method. Results: Plasma concentration of aldosterone in regular diet fed rhesus monkeys was low at 109 pg/mL. Aldosterone concentrations were increased to 252 pg/mL when animals were maintained on a low sodium diet for 3 weeks, and to 300 pg/mL with ACTH treatment at 0.3 mg/kg. The combination of low sodium diet with ACTH treatment further increased plasma concentration of aldosterone to 730 pg/mL and other steroid metabolites at various levels. Intravenous administration of ASI, fadrozole (0.001-1 mg/kg) or LCI699 (0.003-3 mg/kg), led to dose-dependent reductions in aldosterone and 18-hydroxycorticosterone, increases in 11-deoxycorticosterone and 11-deoxycortisol, and bell-shaped changes in cortisol and corticosterone. In vivo selectivity of CYP11B2/CYP11B1 for fadrazole was 26-fold and LCI-699 was 27-fold, which was consistent with relative selectivity using in vitro values from recombinant cells transfected with rhesus monkey CYP11B2 and CYP11B1. Discussion: This model enables concurrent characterization of pharmacokinetics, pharmacodynamics and selectivity of CYP11B2 over CYP11B1 inhibition in the same animal. It may be used as a translational model for pharmacological characterization of ASI. (C) 2014 Elsevier Inc. All rights reserved.

In an effort to understand the origin of blood-pressure lowering effects observed in recent clinical trials with 11β-HSD1 inhibitors, we examined a set of 11β-HSD1 inhibitors in a series of relevant in vitro and in vivo assays. Select 11β-HSD1 inhibitors reduced blood pressure in our preclinical models but most or all of the blood pressure lowering may be mediated by a 11β-HSD1 independent pathway.

Blockade of aldosterone action on the kidney with mineralocorticoid receptor antagonist spironolactone (SPL) or eplerenone (EPL) leads to increased natriuresis and decreased kaliuresis and has proven clinically effective for the treatment of hypertension and heart failure. However, a direct correlation between blood pressure (BP) lowering and the increased natriuretic effect in response to chronic administration of MR antagonist has not been well characterized. In the present study, our goal was to understand whether MR antagonist induces increased natriuresis overtime and therefore correlates to BP lowering in a hypertensive rat model with renal mass reduction (remnant kidney). Animals with the remnant kidney developed sustained systemic hypertension (140–180 mmHg), and their BP and urine chemistry was continuously monitored before and during the drug treatment with vehicle or SPL (30–100mg/kg) administered orally for 4 weeks. Following the 4 week chronic administration of SPL, systolic BP was dose‐dependently reduced by up to 15 mmHg, which was also accompanied with a marked attenuation in proteinuria. However, SPL induced initial increases in natriuresis and decreases in kaliuresis were gradually diminished over the course of the study. These findings clearly demonstrate that the BP lowering effect of SPL is not correlated with its chronic natriuretic effect in the hypertensive rat with remnant kidney.

The use of stable isotopically labeled substrates and analysis by mass spectrometry have provided substantial insight into rates of synthesis, disposition, and utilization of lipids in vivo. The information to be gained from such studies is of particular benefit to therapeutic research where the underlying causes of disease may be related to the production and utilization of lipids. When studying biology through the use of isotope tracers, care must be exercised in interpreting the data to ensure that any response observed can truly be interpreted as biological and not as an artifact of the experimental design or a dilutional effect on the isotope. We studied the effects of dosing route and tracer concentration on the mass isotopomer distribution profile as well as the action of selective inhibitors of microsomal tri-glyceride transfer protein (MTP) in mice and diacylglycerol acyltransferase 1 (DGAT1) in nonhuman primates, using a stable-isotopically labeled approach. Subjects were treated with inhibitor and subsequently given a dose of uniformly ¹³C-labeled oleic acid. Samples were analyzed using a rapid LC-MS technique, allowing the effects of the intervention on the assembly and disposition of triglycerides, cholesteryl esters, and phospholipids to be determined in a single 3 min run from just 10 μl of plasma.

Aldosterone (Aldo) is a steroid hormone that promotes Na reabsorption by binding to the mineralocorticoid receptor (MR). MR antagonists, spironolactone (SPL) and eplerenone (EPL) are used for the treatment of hypertension and heart failure. One measure of anti‐MR activity is urinary Na/K ratio derived from adrenalectomized rats infused with Aldo. Our goal was to develop an improved approach to measure anti‐MR activity in rats with intact adrenal glands. Plasma Aldo levels and anti‐MR activity of SPL were determined at different times of the day in WKY rats. Circulating Aldo levels and urinary Na, K excretion showed diurnal dependence. During the 24 hr cycle (light period 6AM: 6PM), plasma Aldo was significantly higher in the active phase than in the inactive phase. Na excretion was significantly lower during the period of 4PM:10PM than from 9AM:3PM. In contrast, kaliuresis was inversely correlated with natriuresis. The anti‐MR activity of SPL within 6hrs post dose was much greater when dosed prior to the active phase than when dosed during the early inactive phase. Results from 4PM dosing showed that natriuresis continuously increased up to 14hrs but declined during 15–17hrs post dose. These findings demonstrate that the anti‐MR activity of SPL varies with its time of administration. In summary, the use of a chronopharmacological approach proved effective in defining an improved natriuresis model of MR antagonism.

Synthesis and structure-activity relationships of cannabinoid-1 receptor (CB1R) inverse agonists based on dihydro-pyrano[2,3-b] pyridine and tetrahydro-1,8-naphtyridine scaffolds are presented. Rat food intake and pharmacokinetic evaluation of 13g, 13i, 13k and 17a revealed these compounds to be highly efficacious orally active modulators of CB1R.

The synthesis, SAR and binding affinities of cannabinoid-1 receptor (CB1R) inverse agonists based on furo[2,3-b]pyridine scaffolds are described. Food intake, mechanism specific efficacy, pharmacokinetic, and metabolic evaluation of several of these compounds indicate that they are effective orally active modulators of CB1R.

Bombesin receptor subtype 3 (BRS-3) is a G protein coupled receptor whose natural ligand is unknown. We developed potent, selective agonist (Bag-1, Bag-2) and antagonist (Bantag-1) ligands to explore BRS-3 function. BRS-3-binding sites were identified in the hypothalamus, caudal brainstem, and several midbrain nuclei that harbor monoaminergic cell bodies. Antagonist administration increased food intake and body weight, whereas agonists increased metabolic rate and reduced food intake and body weight. Prolonged high levels of receptor occupancy increased weight loss, suggesting a lack of tachyphylaxis. BRS-3 agonist effectiveness was absent in Brs3(-/Y) (BRS-3 null) mice but was maintained in Npy(-/-)Agrp(-/-), Mc4r(-/-), Cnr1(-/-), and Lepr(db/db) mice. In addition, Brs3(-/Y) mice lost weight upon treatment with either a MC4R agonist or a CB1R inverse agonist. These results demonstrate that BRS-3 has a role in energy homeostasis that complements several well-known pathways and that BRS-3 agonists represent a potential approach to the treatment of obesity.

This paper describes the discovery of N-[(4R)-6-(4-chlorophenyl)-7-(2,4-dichlorophenyl)-2,2-dimethyl-3,4-dihydro-2H-pyrano[2,3-b]pyridin-4-yl]-5-methyl-1H-pyrazole-3-carboxamide (MK-5596, 12c) as a novel cannabinoid-1 receptor (CB1R) inverse agonist for the treatment of obesity. Structure activity relationship (SAR) studies of lead compound 3, which had off-target hERG (human ether-a-go-go related gene) inhibition activity, led to the identification of several compounds that not only had attenuated hERG inhibition activity but also were subject to glucuronidation in vitro providing the potential for multiple metabolic clearance pathways. Among them, pyrazole 12c was found to be a highly selective CB1R inverse agonist that reduced body weight and food intake in a DIO (diet-induced obese) rat model through a CB1R-mediated mechanism. Although 12c was a substrate of P-glycoprotein (P-gp) transporter, its high in vivo efficacy in rodents, good pharmacokinetic properties in preclinical species, good safety margins, and its potential for a balanced metabolism profile in man allowed for the further evaluation of this compound in the clinic.

A novel series of 1-sulfonyl-4-acylpiperazines as selective cannabinoid-1 receptor (CB1R) inverse agonists was discovered through high throughput screening (HTS) and medicinal chemistry lead optimization. Potency and in vivo properties were systematically optimized to afford orally bioavailable, highly efficacious, and selective CB1R inverse agonists that caused food intake suppression and body weight reduction in diet-induced obese rats and dogs. It was found that the receptor binding assay predicted in vivo efficacy better than functional antagonist/inverse agonist activities. This observation expedited the structure-activity relationship (SAR) analysis and may have implications beyond the series of compounds presented herein.

To develop pharmaceutical treatments for obesity and diabetes, researchers carry out food intake studies and more in-depth assessments of the feeding behavior in rodents. To facilitate such studies, the authors designed and developed a rodent behavior monitoring system that simultaneously measures food intake, water consumption and motor activity. When tested, their Automated Water Food Activity System (AWFAS) substantially increased throughput for routine rat food intake studies and also improved user ergonomics and safety. The authors describe their system and suggest that others could design similar systems or adapt certain features of the AWFAS to fit existing rodent caging systems.

The synthesis, SAR and binding affinities are described for cannabinoid-1 receptor (CB1R) specific inverse agonists based on pyridopyrimidine and heterotricyclic scaffolds. Food intake and pharmacokinetic evaluation of several of these compounds indicate that they are effective orally active modulators of CB1R.

Selective inhibitors of biogenic amine (e.g., serotonin, norepinephrine, and dopamine) uptake exhibit varying degrees of safety and efficacy as antiobesity agents. Moreover, preclinical findings suggest that the combined inhibition of monoamine neurotransmitter transporters synergistically enhances antiobesity activity. (1R,5S)-(+)-1-(3,4-Dichlorophenyl)-3-azabicyclo-[3.1.0] hexane hydrochloride (DOV 21947) inhibits norepinephrine, 5-hydroxytryptamine, and dopamine uptake, and it reduces body weight in rodent models of diet-induced obesity (DIO). DIO rats treated orally with DOV 21947 for 1 to 24 days showed significantly lower body weights than vehicle-treated DIO rats. The decrease in body weight resulted specifically from a loss of retroperitoneal and mesenteric depots of white adipose tissue. DOV 21947 also reduced daily food intake in DIO rats, but consumption returned to control levels after 11 days of treatment. With the exception of a decrease in triglyceride levels, blood chemistry was unaltered after 24 days of DOV 21947 treatments. DOV 21947 had no effect on motor activity. Although DOV 21947 increased respiratory rate and decreased the tidal volume of normal rats, it did not alter the minute volume. In addition, DOV 21947 did not significantly affect blood pressure, heart rate, electrocardiographic indices or body temperature in telemeterized dogs. However, it caused a sustained, but reversible reduction in the rate of body weight gain for as long as 6 months in normal rats, and for up to 1 year in normal dogs. In summary, DOV 21947 is effective in causing a sustained and selective reduction in fat content and triglyceride levels in animal models of obesity without significantly altering vital organ function.

Sulfonamide analogues of the potent CB1R inverse agonist taranabant were prepared and optimized for potency and selectivity for CB1R. They were variably more potent than the corresponding amide analogues. The most potent representative 22 had good pharmacokinetic and brain levels, but was modestly active in blocking CB1R agonist-mediated hypothermia.