A Soluble Guanylate Cyclase Activator Inhibits the Progression of Diabetic Nephropathy in the ZSF1 Rat.

Therapies that restore renal cGMP levels are hypothesized to slow the progression of diabetic nephropathy. We investigated the effect of BI 703704, a soluble guanylate cyclase (sGC) activator, on disease progression in obese ZSF1 rats. BI 703704 was administered at doses of 0.3, 1, 3, and 10 mg/kg/d to male ZSF1 rats for 15 weeks, during which mean arterial pressure (MAP), heart rate (HR), and urinary protein excretion (UPE) were determined. Histologic assessment of glomerular and interstitial lesions was also performed. Renal cGMP levels were quantified as an indicator of target modulation. BI 703704 resulted in sGC activation, as evidenced by dose-dependent increases in renal cGMP levels. After 15 weeks of treatment, sGC activation resulted in dose-dependent decreases in UPE (from 463 ± 58 mg/d in vehicle controls to 328 ± 55, 348 ± 23, 283 ± 45, and 108 ± 23 mg/d in BI 703704-treated rats at 0.3, 1, 3, and 10 mg/kg, respectively). These effects were accompanied by a significant reduction in the incidence of glomerulosclerosis and interstitial lesions. Decreases in MAP and increases in HR were only observed at the high dose of BI 703704. These results are the first demonstration of renal protection with sGC activation in a nephropathy model induced by type 2 diabetes. Importantly, beneficial effects were observed at doses that did not significantly alter MAP and HR.

(1aR,5aR)1a,3,5,5a-Tetrahydro-1H-2,3-diaza-cyclopropa[a]pentalene-4-carboxylic acid (MK-1903): a potent GPR109a agonist that lowers free fatty acids in humans.

G-protein coupled receptor (GPCR) GPR109a is a molecular target for nicotinic acid and is expressed in adipocytes, spleen, and immune cells. Nicotinic acid has long been used for the treatment of dyslipidemia due to its capacity to positively affect serum lipids to a greater extent than other currently marketed drugs. We report a series of tricyclic pyrazole carboxylic acids that are potent and selective agonists of GPR109a. Compound R,R-19a (MK-1903) was advanced through preclinical studies, was well tolerated, and presented no apparent safety concerns. Compound R,R-19a was advanced into a phase 1 clinical trial and produced a robust decrease in plasma free fatty acids. On the basis of these results, R,R-19a was evaluated in a phase 2 study in humans. Because R,R-19a produced only a weak effect on serum lipids as compared with niacin, we conclude that the beneficial effects of niacin are most likely the result of an undefined GPR109a independent pathway.

Potent tricyclic pyrazole tetrazole agonists of the nicotinic acid receptor (GPR109a).

Tricyclic pyrazole tetrazoles which are potent partial agonists of the high affinity niacin receptor, GPR109a, have been discovered and optimized. One of these compounds has proven to be effective at lowering free fatty acids in vitro and in vivo.

Solubilized phenyl-pyrazole ureas as potent, selective 5-HT(2A) inverse-agonists and their application as antiplatelet agents.

Potent 5-HT(2A) inverse-agonists containing phenyl-pyrazole ureas with an amino side chain were identified. Optimization of this series resulted in selective compounds that proved effective in modulating 5HT-induced amplification of ADP-stimulated human platelet aggregation.

5-N,N-Disubstituted 5-aminopyrazole-3-carboxylic acids are highly potent agonists of GPR109b.

A series of 5-N,N-disubstituted-5-aminopyrazole-3-carboxylic acids were prepared and found to act as highly potent and selective agonists of the G-Protein Coupled Receptor (GPCR) GPR109b, a low affinity receptor for niacin and some aromatic d-amino acids. Little activity was observed at the highly homologous higher affinity niacin receptor, GPR109a.

3-(1H-tetrazol-5-yl)-1,4,5,6-tetrahydro-cyclopentapyrazole (MK-0354): a partial agonist of the nicotinic acid receptor, G-protein coupled receptor 109a, with antilipolytic but no vasodilatory activity in mice.

The discovery and profiling of 3-(1H-tetrazol-5-yl)-1,4,5,6-tetrahydro-cyclopentapyrazole (5a, MK-0354), a partial agonist of GPR109a, is described. Compound 5a retained the plasma free fatty acid lowering effects in mice associated with GPR109a agonism, but did not induce vasodilation at the maximum feasible dose. Moreover, preadministration of 5a blocked the flushing effect induced by nicotinic acid but not that induced by PGD2. This profile made 5a a suitable candidate for further study for the treatment of dyslipidemia.

Anti-thrombotic and vascular effects of AR246686, a novel 5-HT2A receptor antagonist.

We have evaluated the anti-platelet and vascular pharmacology of AR246686, a novel 5-hydroxytryptamine2A (5-HT2A) receptor antagonist. AR246686 displayed high affinity binding to membranes of HEK cells stably expressing recombinant human and rat 5-HT2A receptors (Ki=0.2 nM and 0.4 nM, respectively). Functional antagonism (IC50=1.9 nM) with AR246686 was determined by inhibition of ligand-independent inositol phosphate accumulation in the 5-HT2A stable cell line. We observed 8.7-fold and 1360-fold higher affinity of AR246686 for the 5-HT2A receptor vs. 5-HT2C and 5-HT2B receptors, respectively. AR246686 inhibited 5-HT-induced amplification of ADP-stimulated human platelet aggregation (IC50=21 nM). Similar potency was observed for inhibition of 5-HT stimulated DNA synthesis in rat aortic smooth muscle cells (IC(50)=10 nM) and 5-HT-mediated contraction in rat aortic rings. Effects of AR246686 on arterial thrombosis and bleeding time were studied in a rat model of femoral artery occlusion. Oral dosing of AR246686 to rats resulted in prolongation of time to occlusion at 1 mg/kg, whereas increased bleeding time was observed at a dose of 20 mg/kg. In contrast, both bleeding time and time to occlusion were increased at the same dose (10 mg/kg) of clopidogrel. These results demonstrate that AR246686 is a high affinity 5-HT2A receptor antagonist with potent activity on platelets and vascular smooth muscle. Further, oral administration results in anti-thrombotic effects at doses that are free of significant effects on traumatic bleeding time.

3-Nitro-4-amino benzoic acids and 6-amino nicotinic acids are highly selective agonists of GPR109b.

A series of 3-nitro-4-substituted-aminobenzoic acids were prepared and found to act as potent and highly selective agonists of the orphan human GPCR GPR109b, a low affinity receptor for niacin. No activity was observed at the closely homologous high affinity niacin receptor, GPR109a. A second series, comprising 6-amino-substituted nicotinic acids was, also prepared and several analogues showed comparable activity to the nitroaryl series.

Fluorinated pyrazole acids are agonists of the high affinity niacin receptor GPR109a.

A series of 5-alkyl pyrazole-3-carboxylic acids were prepared and found to act as potent and selective agonists of the human GPCR, GPR109a, the high affinity nicotinic acid receptor. No activity was observed at the highly homologous low affinity niacin receptor, GPR109b. A further series of 4-fluoro-5-alkyl pyrazole-3-carboxylic acids were shown to display similar potency. One example from the series was shown to have improved properties in vivo compared to niacin.

Recent progress in the discovery of niacin receptor agonists.

Niacin (nicotinic acid) favorably affects multiple serum lipid parameters that are believed to be cardiovascular risk factors, but is also associated with a cutaneous flushing adverse effect. The recent discovery of a G-protein-coupled receptor target for niacin (termed GPR109alpha) has stimulated interest in the discovery of new compounds with niacin-like effects on lipids, but with fewer adverse effects. This review discusses the progress made toward the discovery of such molecules and highlights the pharmacological models used to enable their identification. The advances made in both these areas may help to determine whether GPR109alpha is the molecular target responsible for the beneficial anti-atherogenic effects of niacin.

Comparison of rat and dog models of vasodilatation and lipolysis for the calculation of a therapeutic index for GPR109A agonists.

INTRODUCTION: GPR109A is the receptor mediating both the antilipolytic and vasodilatory effects of nicotinic acid. In order to develop agonists for GPR109A with improved therapeutic indices we have sought to optimize animal models that evaluate both nicotinic acid-mediated inhibition of lipolysis and stimulation of vasodilatation. The rat and the dog have previously been used to study the antilipolytic effects of nicotinic acid, but no optimal vasodilatation model exits in either species. METHODS: We have developed a vasodilatation model in the rat that measures changes in ear perfusion using laser Doppler flowmetry. In the dog, we have developed a model of vasodilatation measuring changes in red color values in the ear, using a spectrocolorimeter. Effects of GPR109A agonists on lipolysis were measured in both species after oral dosing of compounds, and measuring plasma levels of free fatty acids. RESULTS: In both rat and dog, GPR109A agonists induce dose- and time-dependent vasodilatation, similar to that observed in humans. Vasodilatation is inhibited in both species with cyclooxygenase inhibitors or a specific DP1 receptor antagonist, indicating that, as in man, nicotinic acid-induced vasodilatation in rats and dogs is mainly mediated by the release of PGD(2). DISCUSSION: Our results show that both rat and dog are useful models for the characterization of GPR109A agonists. A therapeutic index for GPR109A agonists can be calculated in either species.

Agonist lead identification for the high affinity niacin receptor GPR109a.

A strategy for lead identification of new agonists of GPR109a, starting from known compounds shown to activate the receptor, is described. Early compound triage led to the formulation of a binding hypothesis and eventually to our focus on a series of pyrazole acid derivatives. Further elaboration of these compounds provided a series of 5,5-fused pyrazoles to be used as lead compounds for further optimization.

Nicotinic acid receptor agonists differentially activate downstream effectors.

Nicotinic acid remains the most effective therapeutic agent for the treatment and prevention of atherosclerosis resulting from low high density lipoprotein cholesterol. The therapeutic actions of nicotinic acid are mediated by GPR109A, a Gi protein-coupled receptor, expressed primarily on adipocytes, Langerhans cells, and macrophage. Unfortunately, a severe, cutaneous flushing side effect limits its use and patient compliance. The mechanism of high density lipoprotein elevation is not clearly established but assumed to be influenced by an inhibition of lipolysis in the adipose. The flushing side effect appears to be mediated by the release of prostaglandin D2 from Langerhans cells in the skin. We hypothesized that the signal transduction pathways mediating the anti-lipolytic and prostaglandin D2/flushing pathways are distinct and that agonists may be identified that are capable of selectively eliciting the therapeutic, anti-lipolytic pathway while avoiding the activation of the parallel flush-inducing pathway. We have identified a number of GPR109A pyrazole agonists that are capable of fully inhibiting lipolysis in vitro and in vivo and not only fail to elicit a flushing response but can antagonize the ability of nicotinic acid to elicit a flush response in vivo. In contrast to flushing agonists, exposure of cells expressing GPR109A to the non-flushing agonists fails to induce internalization of the receptor or to activate ERK 1/2 mitogen-activated protein kinase phosphorylation.

Analogues of acifran: agonists of the high and low affinity niacin receptors, GPR109a and GPR109b.

Recently identified GPCRs, GPR109a and GPR109b, the high and low affinity receptors for niacin, may represent good targets for the development of HDL elevating drugs for the treatment of atherosclerosis. Acifran, an agonist of both receptors, has been tested in human subjects, yet until recently very few analogs had been reported. We describe a series of acifran analogs prepared using newly developed synthetic pathways and evaluated as agonists for GPR109a and GPR109b, resulting in identification of compounds with improved activity at these receptors.

Langerhans cells release prostaglandin D2 in response to nicotinic acid.

Nicotinic acid, used for atherosclerosis treatment, has an adverse effect of skin flushing. The flushing mechanism, thought to be caused by the release of prostaglandin D(2) (PGD(2)), is not well understood. We aimed to identify which cells mediate the flushing effect. Nicotinic acid receptor (GPR109A) gene expression was assessed in various tissues and cell lines. Cells expressing GPR109A mRNA were further assayed for PGD(2) release in response to nicotinic acid. Of all samples, only skin was able to release PGD(2) upon stimulation with nicotinic acid. The responsive cells were localized to the epidermis, and immunocytochemical studies revealed the presence of GPR109A on epidermal Langerhans cells. CD34+ cells isolated from human blood and differentiated into Langerhans cells (hLC-L) also showed GPR109A expression. IFNgamma treatment increased both mRNA and plasma membrane expression of GPR109A. IFNgamma-stimulated hLC-Ls released PGD(2) in response to nicotinic acid in a dose-dependant manner (effector concentration for half-maximum response=1.2 mM+/-0.7). Acifran, a structurally distinct GPR109A ligand, also increased PGD(2) release, whereas isonicotinic acid, a nicotinic acid analog with low affinity for GPR109A, had no effect. These results suggest that nicotinic acid mediates its flushing side effect by interacting with GPR109A on skin Langerhans cells, resulting in release of PGD(2).

1-Alkyl-benzotriazole-5-carboxylic acids are highly selective agonists of the human orphan G-protein-coupled receptor GPR109b.

1-Substituted benzotriazole carboxylic acids have been identified as the first reported examples of selective small-molecule agonists of the human orphan G-protein-coupled receptor GPR109b (HM74), a low-affinity receptor for the HDL-raising drug niacin. No activity was observed at the highly homologous high-affinity niacin receptor GPR109a (HM74A). The high degree of selectivity was attributed to a difference in the amino acid sequence adjacent to a key arginine-ligand interaction allowing somewhat larger ligands to be tolerated by GPR109b.

Alpha-2 adrenergic receptors stimulate actin organization in developing fetal rat cardiac myocytes.

Expression of alpha(2)-adrenergic receptors (alpha(2)-AR) is very high in fetal rat heart although numbers decline with increasing gestational age. The current experiments were designed to identify the subtypes of alpha(2)-AR expressed and the function of these receptors in fetal cardiac myocytes. Expression of alpha(2)A and alpha(2)C, but not alpha(2)B, was confirmed in the myocyte population by indirect immunofluorescence microscopy with subtype-specific antibodies and by Western blot. Both dexmedetomidine, an alpha(2)-selective agonist, and norepinephrine, increased actin cytoskeleton organization and this increase was blocked by the alpha(2)-selective antagonist, atipamezole. Furthermore, dexmedetomidine inhibited isoproterenol-stimulated cAMP accumulation in isolated fetal rat heart and this was blocked by rauwolscine. Therefore, functional alpha(2)A and alpha(2)B subtypes are present in the fetal rat heart where they may have a role in cardiac development.