Lack of authentic atrial fibrillation in commonly used murine atrial fibrillation models.

The mouse is a useful preclinical species for evaluating disease etiology due to the availability of a wide variety of genetically modified strains and the ability to perform disease-modifying manipulations. In order to establish an atrial filtration (AF) model in our laboratory, we profiled several commonly used murine AF models. We initially evaluated a pharmacological model of acute carbachol (CCh) treatment plus atrial burst pacing in C57BL/6 mice. In an effort to observe micro-reentrant circuits indicative of authentic AF, we employed optical mapping imaging in isolated mouse hearts. While CCh reduced atrial refractoriness and increased atrial tachyarrhythmia vulnerability, the left atrial (LA) excitation patterns were rather regular without reentrant circuits or wavelets. Therefore, the atrial tachyarrhythmia resembled high frequency atrial flutter, not typical AF per se. We next examined both a chronic angiotensin II (Ang II) infusion model and the surgical model of transverse aortic constriction (TAC), which have both been reported to induce atrial and ventricular structural changes that serve as a substrates for micro-reentrant AF. Although we observed some extent of atrial remodeling such as fibrosis or enlarged LA diameter, burst pacing-induced atrial tachyarrhythmia vulnerability did not differ from control mice in either model. This again suggested that an AF-like pathophysiology is difficult to demonstrate in the mouse. To continue searching for a valid murine AF model, we studied mice with a cardiac-specific deficiency (KO) in liver kinase B1 (Cardiac-LKB1), which has been reported to exhibit spontaneous AF. Indeed, the electrocardiograms (ECG) of conscious Cardiac-LKB1 KO mice exhibited no P waves and had irregular RR intervals, which are characteristics of AF. Histological evaluation of Cardiac-LKB1 KO mice revealed dilated and fibrotic atria, again consistent with AF. However, atrial electrograms and optical mapping revealed that electrical activity was limited to the sino-atrial node area with no electrical conduction into the atrial myocardium beyond. Thus, Cardiac-LKB1 KO mice have severe atrial myopathy or atrial standstill, but not AF. In summary, the atrial tachyarrhythmias we observed in the four murine models were distinct from typical human AF, which often exhibits micro- or macro-reentrant atrial circuits. Our results suggest that the four murine AF models we examined may not reflect human AF well, and raise a cautionary note for use of those murine models to study AF.

Discovery of Novel Oral Protein Synthesis Inhibitors of Mycobacterium tuberculosis That Target Leucyl-tRNA Synthetase.

The recent development and spread of extensively drug-resistant and totally drug-resistant resistant (TDR) strains of Mycobacterium tuberculosis highlight the need for new antitubercular drugs. Protein synthesis inhibitors have played an important role in the treatment of tuberculosis (TB) starting with the inclusion of streptomycin in the first combination therapies. Although parenteral aminoglycosides are a key component of therapy for multidrug-resistant TB, the oxazolidinone linezolid is the only orally available protein synthesis inhibitor that is effective against TB. Here, we show that small-molecule inhibitors of aminoacyl-tRNA synthetases (AARSs), which are known to be excellent antibacterial protein synthesis targets, are orally bioavailable and effective against M. tuberculosis in TB mouse infection models. We applied the oxaborole tRNA-trapping (OBORT) mechanism, which was first developed to target fungal cytoplasmic leucyl-tRNA synthetase (LeuRS), to M. tuberculosis LeuRS. X-ray crystallography was used to guide the design of LeuRS inhibitors that have good biochemical potency and excellent whole-cell activity against M. tuberculosis Importantly, their good oral bioavailability translates into in vivo efficacy in both the acute and chronic mouse models of TB with potency comparable to that of the frontline drug isoniazid.

Nicotinic acid and DP1 blockade: studies in mouse models of atherosclerosis.

The use of nicotinic acid to treat dyslipidemia is limited by induction of a "flushing" response, mediated in part by the interaction of prostaglandin D(2) (PGD(2)) with its G-protein coupled receptor, DP1 (Ptgdr). The impact of DP1 blockade (genetic or pharmacologic) was assessed in experimental murine models of atherosclerosis. In Ptgdr(-/-)ApoE(-/-) mice versus ApoE(-/-) mice, both fed a high-fat diet, aortic cholesterol content was modestly higher (1.3- to 1.5-fold, P < 0.05) in Ptgdr(-/-)ApoE(-/-) mice at 16 and 24 weeks of age, but not at 32 weeks. In multiple ApoE(-/-) mouse studies, a DP1-specific antagonist, L-655, generally had a neutral to beneficial effect on aortic lipids in the presence or absence of nicotinic acid treatment. In a separate study, a modest increase in some atherosclerotic measures was observed with L-655 treatment in Ldlr(-/-) mice fed a high-fat diet for 8 weeks; however, this effect was not sustained for 16 or 24 weeks. In the same study, treatment with nicotinic acid alone generally decreased plasma and/or aortic lipids, and addition of L-655 did not negate those beneficial effects. These studies demonstrate that inhibition of DP1, with or without nicotinic acid treatment, does not lead to consistent or sustained effects on plaque burden in mouse atherosclerotic models.

Niacin lipid efficacy is independent of both the niacin receptor GPR109A and free fatty acid suppression.

Nicotinic acid (niacin) induces beneficial changes in serum lipoproteins and has been associated with beneficial cardiovascular effects. Niacin reduces low-density lipoprotein, increases high-density lipoprotein, and decreases triglycerides. It is well established that activation of the seven-transmembrane G(i)-coupled receptor GPR109A on Langerhans cells results in release of prostaglandin D2, which mediates the well-known flushing side effect of niacin. Niacin activation of GPR109A on adipocytes also mediates the transient reduction of plasma free fatty acid (FFA) levels characteristic of niacin, which has been long hypothesized to be the mechanism underlying the changes in the serum lipid profile. We tested this "FFA hypothesis" and the hypothesis that niacin lipid efficacy is mediated via GPR109A by dosing mice lacking GPR109A with niacin and testing two novel, full GPR109A agonists, MK-1903 and SCH900271, in three human clinical trials. In mice, the absence of GPR109A had no effect on niacin's lipid efficacy despite complete abrogation of the anti-lipolytic effect. Both MK-1903 and SCH900271 lowered FFAs acutely in humans; however, neither had the expected effects on serum lipids. Chronic FFA suppression was not sustainable via GPR109A agonism with niacin, MK-1903, or SCH900271. We conclude that the GPR109A receptor does not mediate niacin's lipid efficacy, challenging the long-standing FFA hypothesis.

(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.

Pharmacological activation and genetic manipulation of cystathionine beta-synthase alter circulating levels of homocysteine and hydrogen sulfide in mice.

Hydrogen sulfide (H(2)S) is a recently discovered gasotransmitter found in mammalian tissues and blood. Treatment with H(2)S donor molecules has shown promising results in preclinical models of inflammatory and cardiovascular diseases. Augmentation of H(2)S levels thus holds promise as a novel therapeutic approach for treatment of disease in man. Cystathionine ß-synthase (CBS) has been shown to catalyze H(2)S production in vitro. CBS enzyme activity is allosterically regulated by the endogenous activator S-adenosyl methionine. This mode of regulation suggests the possibility for designing a small molecule activator of CBS to enhance H(2)S production. This hypothesis, however, has not been directly tested in vivo. We show here that CBS contributes significantly to endogenous H(2)S production in mice: adenovirus mediated over expression of CBS in the liver significantly increased circulating levels of H(2)S, whereas CBS deficiency resulted in reduced levels. We demonstrate that CBS enzyme from endogenous sources can be activated by S-adenosyl methionine to a greater extent compared to recombinant enzyme, suggesting greater potential for activation than previously anticipated. Importantly, we show that circulating H(2)S levels are increased by pharmacological activation of CBS in vivo; i.e. in the presence of the endogenous activator. Together, our data demonstrate that CBS activity partially regulates endogenous H(2)S in mice, and suggest that pharmacological activation of CBS is a promising approach for enhancing endogenous production of H(2)S for the treatment of cardiovascular and other diseases.

The discovery of high affinity agonists of GPR109a with reduced serum shift and improved ADME properties.

Amino-anthranilic acid derivatives have been identified as a new class of low serum shifted, high affinity full agonists of the human orphan G-protein-coupled receptor GPR109a with improved ADME properties.

GPR109a agonists. Part 2: pyrazole-acids as agonists of the human orphan G-protein coupled receptor GPR109a.

5-Alkyl and aryl-pyrazole-acids have been identified as a new class of selective, small-molecule, agonists of the human orphan G-protein-coupled receptor GPR109a, a high affinity receptor for the HDL-raising drug nicotinic acid.

Anthranilic acid replacements in a niacin receptor agonist.

Niacin is an effective drug for raising HDL cholesterol. However, niacin must be taken in large doses and significant side effects are often observed, including facial flushing, loss of glucose tolerance, and liver toxicity. An anthranilic acid was identified as an agonist of the niacin receptor. In order to improve efficacy and provide structural diversity, replacements for the anthranilic acid were investigated and several compounds with improved properties were identified.

Discovery of pyrazolyl propionyl cyclohexenamide derivatives as full agonists for the high affinity niacin receptor GPR109A.

A series of pyrazolyl propionyl cyclohexenamides were discovered as full agonists for the high affinity niacin receptor GPR109A. The structure-activity relationship (SAR) studies were aimed to improve activity on GPR109A, reduce Cytochrome P450 2C8 (CYP2C8) and Cytochrome P450 2C9 (CYP2C9) inhibition, reduce serum shift and improve pharmacokinetic (PK) profiles.

Discovery of a biaryl cyclohexene carboxylic acid (MK-6892): a potent and selective high affinity niacin receptor full agonist with reduced flushing profiles in animals as a preclinical candidate.

Biaryl cyclohexene carboxylic acids were discovered as full and potent niacin receptor (GPR109A) agonists. Compound 1e (MK-6892) displayed excellent receptor activity, good PK across species, remarkably clean off-target profiles, good ancillary pharmacology, and superior therapeutic window over niacin regarding the FFA reduction versus vasodilation in rats and dogs.

Discovery of novel tricyclic full agonists for the G-protein-coupled niacin receptor 109A with minimized flushing in rats.

Tricyclic analogues were rationally designed as the high affinity niacin receptor G-protein-coupled receptor 109A (GPR109A) agonists by overlapping three lead structures. Various tricyclic anthranilide and cycloalkene carboxylic acid full agonists were discovered with excellent in vitro activity. Compound 2g displayed a good therapeutic index regarding free fatty acids (FFA) reduction and vasodilation effects in rats, with very weak cytochrome P450 2C8 (CYP2C8) and cytochrome P450 2C9 (CYP2C9) inhibition, and a good mouse pharmacokinetics (PK) profile.

Extended-release niacin/laropiprant: reducing niacin-induced flushing to better realize the benefit of niacin in improving cardiovascular risk factors.

Treatment with niacin effectively improves multiple lipid parameters and cardiovascular outcomes. Widespread use of niacin, however, is limited by flushing, which is mediated primarily by prostaglandin D2 (PGD2). Laropiprant is a selective PGD2 receptor 1 (DP1) antagonist that reduces objective measures of niacin-induced flushing symptoms upon initiation of therapy and with more chronic use. Results from early dosing and formulation studies have culminated in the development of a combination extended-release (ER) niacin/laropiprant tablet aimed at providing the beneficial lipid-modifying effects of niacin, while reducing niacin-induced flushing. The improvement in the tolerability of niacin with ER niacin/laropiprant allows niacin dosing to initiate directly at 1 g and rapidly advance to a 2-g target dose. ER niacin/laropiprant generally is tolerated well and represents a new treatment option for dyslipidemia that offers the potential for more patients to receive the lipid-modifying and cardiovascular benefits of niacin.

Role of GPR81 in lactate-mediated reduction of adipose lipolysis.

Heavy exercise or oxygen deficit often links with higher levels of arterial lactate and lower levels of plasma free fatty acids (FFA). Treatment with lactate reduces circulating levels of FFA in vivo and lipolysis in adipose tissues in vitro. However, the underlying mechanism has remained unclear. Here we employ pharmacological and genetic approaches to show that GPR81, an orphan G-protein-coupled receptor with relatively restricted expression in the adipose tissues, functions as a receptor for lactate and can mediate an anti-lipolytic effect of lactate. GPR81 may thus function as a sensor of lactate that can modulate the FFA pool under exercise or conditions of oxygen deficit.

Molecular modeling aided design of nicotinic acid receptor GPR109A agonists.

A homology model of the nicotinic acid receptor GPR109A was constructed based on the X-ray crystal structure of bovine rhodopsin. An HTS hit was docked into the homology model. Characterization of the binding pocket by a grid-based surface calculation of the docking model suggested that a larger hydrophobic body plus a polar tail would improve interaction between the ligand and the receptor. The designed compounds were synthesized, and showed significantly improved binding affinity and activation of GPR109A.

Discovery of pyrazolopyrimidines as the first class of allosteric agonists for the high affinity nicotinic acid receptor GPR109A.

Pyrazolopyrimidines were discovered as the first class of allosteric agonists for the high affinity nicotinic acid receptor GPR109A. In addition to its intrinsic activity, compound 9n significantly enhances nicotinic acid binding to the receptor, thereby potentiating the functional efficacy of nicotinic acid.

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.

Tetrahydro anthranilic acid as a surrogate for anthranilic acid: application to the discovery of potent niacin receptor agonists.

The design, synthesis, and biological activity of a series of cycloalkene acid-based niacin receptor agonists are described. This led to the discovery that tetrahydro anthranilic acid is an excellent surrogate for anthranilic acid. Several compounds were identified that were potent against the niacin receptor, had enhanced cytochrome P450 selectivity against subtypes CYP2C8 and CYP2C9, and improved oral exposure in mice.

Effects of a niacin receptor partial agonist, MK-0354, on plasma free fatty acids, lipids, and cutaneous flushing in humans.

BACKGROUND: Development of niacin-like agents that favorably affect lipids with an improved flushing profile would be beneficial. OBJECTIVE: To evaluate a niacin receptor partial agonist, MK-0354, in Phase I and II studies. METHODS: The pharmacokinetic/pharmacodynamic effects of single and multiple doses (7 days) of MK-0354 (300-4000 mg) were evaluated in two Phase I studies conducted in healthy men. A Phase II study assessed the effects of MK-0354 2.5 g once daily on lipids during 4 weeks in 66 dyslipidemic patients. RESULTS: MK-0354 single doses up to 4000 mg and multiple doses (7 days) up to 3600 mg produced robust dose-related reductions in free fatty acid (FFA) over 5 hours. Single doses of MK-0354 300 mg and extended release-niacin (Niaspan) 1 g produced comparable reductions in FFA. Suppression of FFA following 7 daily doses of MK-0354 was similar to that after a single dose. In the Phase II study, MK-0354 2.5 g produced little flushing but no clinically meaningful effects on lipids (placebo-adjusted percent change: high-density lipoprotein cholesterol, 0.4%, 95% confidence interval -5.2 to 6.0; low-density lipoprotein cholesterol, -9.8%, 95% confidence interval -16.8 to -2.7; triglyceride, -5.8%, 95% confidence interval -22.6 to 11.9). CONCLUSION: Treatment with MK-0354 for 7 days resulted in plasma FFA suppression with minimal cutaneous flushing. However, 4 weeks of treatment with MK-0354 failed to produce changes in high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, or triglycerides.

Discovery of biaryl anthranilides as full agonists for the high affinity niacin receptor.

Biaryl anthranilides are reported as potent and selective full agonists for the high affinity niacin receptor GPR109A. The SAR presented outlines approaches to reduce serum shift and both CYPCYP2C8 and CYP2C9 liabilities, while improving PK and maintaining excellent receptor activity. Compound 2i exhibited good in vivo antilipolytic efficacy while providing a significantly improved therapeutic index over vasodilation (flushing) with respect to niacin in the mouse model.