Five-year results of the INSPIRATION study for the INCRAFT low-profile endovascular aortic stent graft system.

OBJECTIVE: We present the 5-year results of a prospective regulatory study of the INCRAFT device, a low-profile endovascular stent graft system for repair of abdominal aortic aneurysms. METHODS: This was an open-label prospective nonrandomized single-arm study enrolling in centers in the United States and Japan. The primary effectiveness outcome was successful aneurysm treatment and the primary safety outcome was the incidence of major adverse events at 30 days after the procedure. Major long-term outcomes were mortality, reintervention, adverse limb outcomes, and suprarenal stent fracture. RESULTS: One hundred and ninety patients (mean age, 73.8 ± 7.6 years; 90% male; 69% white and 30% Asian) were enrolled from 32 centers throughout the United States and Japan. Minimal access vessel size was less than 7 mm on both sides in 43.9% of the study cohort. Thirty-day major adverse events occurred in 3.2% of patients (6/190). Periprocedural technical success was 94.1% (176/187). Successful aneurysm treatment was 100% at 30 days and 87.9% at 1 year. Two patients required open conversion for thromboembolic complications, 3 developed new type I or III endoleaks, and 7 experienced graft or limb occlusion. Freedom from graft occlusion was 96 ± 2% at 1 year and 94 ± 2% at 5 years. Freedom from stent fracture was 97 ± 1% at 1 year and 87 ± 3% at 5 years. Freedom from aneurysm-related mortality was 99 ± 1% at 1 and 5 years. CONCLUSIONS: This study demonstrates good efficacy and safety and a very low rate of aneurysm related deaths with the INCRAFT device in a population with a high proportion of challenging anatomy.

Validation of diacyl glycerolacyltransferase I as a novel target for the treatment of obesity and dyslipidemia using a potent and selective small molecule inhibitor.

A highly potent and selective DGAT-1 inhibitor was identified and used in rodent models of obesity and postprandial chylomicron excursion to validate DGAT-1 inhibition as a novel approach for the treatment of metabolic diseases. Specifically, compound 4a conferred weight loss and a reduction in liver triglycerides when dosed chronically in DIO mice and depleted serum triglycerides following a lipid challenge in a dose-dependent manner, thus, reproducing major phenotypical characteristics of DGAT-1(-/-) mice.

Characterization of ghrelin receptor activity in a rat pituitary cell line RC-4B/C.

Ghrelin, a 28 amino acid, octanoylated peptide, is an endogenous ligand for the growth hormone secretagogue receptor (GHS-R). In addition to various endocrine functions, including stimulation of GH release, ghrelin has been characterized as an important regulator of energy homeostasis. Ghrelin administration has been shown to increase adiposity in rodents and stimulate food intake in humans. Studies suggest that these orexigenic effects are mediated primarily through GHS-R expression in hypothalamic and pituitary neuronal pathways. In this context, GHS-R has been recognized as a potential target for the treatment of GH deficiency and body weight disorders. Cell lines provide convenient in vitro systems to identify and characterize potential pharmacophores and to analyze GHS-R functional activity. While recombinant cell lines that overexpress GHS-R have served as effective research tools for these studies, such cell lines may differ in signaling response to ghrelin compared with hypothalamic or pituitary cells expressing GHS-R. We show here that a cell line derived from a rat anterior pituitary adenoma, RC-4B/C, expresses endogenous GHS-R as judged by reverse transcriptase-PCR. In a Ca(2+)mobilization assay, RC-4B/C cells demonstrate a dose-dependent increase in intracellular [Ca(2+)] on stimulation with rat ghrelin and a related peptide agonist, hexarelin (EC(50), 1.0 nM and 1.7 nM respectively), but are unresponsive to treatment with inactive des-octanoyl rat ghrelin. A subclone, RC-4B/C.40, with a more robust and stable ghrelin response, was isolated from the parental population of cells to allow further analysis of GHS-R signal transduction. Using pertussis toxin and the phospholipase C inhibitor U-73122, we show that ghrelin signals through the Gq pathway in the RC-4B/C.40 cells. We also demonstrate that the ghrelin-induced rise of intracellular [Ca(2+)] in RC-4B/C.40 cells involves initial Ca(2+)release from intracellular stores followed by a sustained elevation that occurs via influx of extracellular Ca(2+) through ion channels. In addition, unlike observations reported in recombinant cell systems, the RC-4B/C.40 cells do not exhibit a high level of GHS-R constitutive activity as determined in a phosphatidylinositol hydrolysis assay. Overall, the data presented here suggest that the RC-4B/C parental and RC-4B/C.40 cells provide novel in vitro systems for the characterization of GHS-R pharmacophores and ghrelin signaling.

Characterization of a neuronal cell line expressing native human melanin-concentrating hormone receptor 1 (MCHR1).

Melanin-concentrating hormone (MCH), an orexigenic neuropeptide in mammals, activates a G-protein coupled receptor, MCHR1. It is expected that antagonists of MCHR1 function will prove therapeutically useful as anti-obesity agents. Intracellular signaling by MCHR1 has been investigated primarily using non-neural cell lines expressing the recombinant receptor, in which MCHR1 has been shown to couple to G alpha(i/o) and G alpha(q) G-proteins. While these cell lines have been widely utilized to discover and optimize small molecule antagonists, it is unknown whether the intracellular signaling pathways in these cells accurately reflect those in neurons. Thus, we sought to develop a neurally derived cell line endogenously expressing MCHR1. IMR32, a human neuroblastoma cell line, has been shown to express MCHR1 mRNA; however, we were unable to detect either MCH-binding or MCH-stimulated Ca++-mobilization in these cells. Following transfection of IMR32 cells with a plasmid encoding human G alpha(16) G-protein, we isolated a cell line, I3.4.2, which responded to MCH in Ca++-mobilization assays. We found that the expression level of MCHR1 mRNA in I3.4.2 cells was 2000-fold higher than in the parent cell line. Using [125I]MCH saturation-binding to I3.4.2 cell membranes, we estimated the Bmax as 0.72 pmol/mg protein and the Kd as 0.35 nM. We report that Ca++-mobilization in I3.4.2 cells was insensitive to pertussis toxin (Ptx) treatment, indicating that signaling was via G alpha(q) G-proteins. Furthermore, negative results in cAMP accumulation assays confirmed the lack of signaling via the G alpha(i/o) G-proteins. Our results suggest that the I3.4.2 cell line may be useful for characterization of MCHR1 activity in a neural-derived cell line.

Discovery and characterization of aminopiperidinecoumarin melanin concentrating hormone receptor 1 antagonists.

4-(1-Benzo[1,3]dioxol-5-ylmethylpiperidine-4-ylmethyl)-6-chlorochromen-2-one (7) is a potent, orally bioavailable melanin concentrating hormone receptor 1 (MCHr1) antagonist that causes dose-dependent weight loss in diet-induced obese mice. Further evaluation of 7 in an anesthetized dog model of cardiovascular safety revealed adverse hemodynamic effects at a plasma concentration comparable to the minimally effective therapeutic concentration. These results highlight the need for scrutiny of the cardiovascular safety profile of MCHr1 antagonists.

Synthesis and evaluation of 2-amino-8-alkoxy quinolines as MCHr1 antagonists. Part 1.

A high-throughput screen was performed in order to identify chemotypes that are bound by the melanin concentrating hormone receptor-1 (MCHr1). A novel 2-amino-8-alkoxyquinoline compound (1) was identified and subsequently optimized using a parallel and automated procedure for the rapid production of multiple analogs. The structure-activity relationships that emerged from this effort are described, along with selected pharmacokinetic parameters of compound (d)-61 when dosed orally in diet-induced obese mice.

Synthesis and evaluation of 2-amino-8-alkoxy quinolines as MCHr1 antagonists. Part 2.

The continued SAR investigation of 2-amino-8-alkoxy quinolines as melanin concentrating hormone receptor-1 (MCHr1) antagonists is reported. Prior hit-to-lead efforts resulted in the identification of 1 as a robust MCHr1 antagonist. Further delineation of the structural parameters essential for MCHr1-binding affinity of this class of nontraditional GPCR ligands resulted in the identification of compounds such as 33, 34 and 37, which demonstrate single digit nanomolar antagonism of MCHr1-mediated Ca(2+) release. The synthesis and biological evaluation of these compounds are reported.

Synthesis and evaluation of 2-amino-8-alkoxy quinolines as MCHr1 antagonists. Part 3.

Prior SAR studies on 2-amino-8-alkoxyquinoline MCHr1 antagonists demonstrated that compounds with acyclic amide-containing sidechains displayed exceptional binding and functional potency, but negligible CNS penetration. Related analogs with acyclic benzylamine-containing sidechains showed greatly improved CNS exposure, but suffered in functional potency. In this report, we demonstrate that cyclization of these benzylic amine sidechains affords compounds that combine the best elements of potency and CNS penetration among this class of antagonists. This is exemplified by compound 21, which has sub-nanomolar MCHr1 binding affinity, good functional potency, and excellent CNS exposure over 24h.