Effect of an NHE3 inhibitor in combination with an NPT2b inhibitor on gastrointestinal phosphate absorption in Rodent models.

Many of the pathological consequences of chronic kidney disease can be attributed to an elevation in serum phosphate levels. Current therapies focused on decreasing intestinal phosphate absorption to treat hyperphosphatemia are inadequate. The most effective therapeutic strategy may be to target multiple absorptive pathways. In this study, the ability of a novel inhibitor of the intestinal sodium hydrogen exchanger 3 (NHE3), LY3304000, which inhibits paracellular, diffusional uptake of phosphate, to work in combination with an inhibitor of the active transporter, sodium dependent phosphate cotransporter 2b (NPT2b), LY3358966, was explored. LY3304000 modestly inhibited the acute uptake of phosphate into plasma of rats, while surprisingly, it doubled the rate of phosphate uptake in mice, an animal model dominated by NPT2b mediated acute phosphate uptake. In rats, LY3004000 and LY3358966 work in concert to inhibit acute phosphate uptake. On top of LY3358966, LY3304000 further decreased the acute uptake of phosphate into plasma. Studies measuring the recovery of radiolabeled phosphate in the intestine demonstrated LY3304000 and LY3358966 synergistically inhibited the absorption of phosphate in rats. We hypothesize the synergism is because the NHE3 inhibitor, LY3304000, has two opposing effects on intestinal phosphate absorption in rats, first it decreases diffusion mediated paracellular phosphate absorption, while second, it simultaneously increases phosphate absorption through the NPT2b pathway. NHE3 inhibition decreases proton export from enterocytes and raises the cell surface pH. In vitro, NPT2b mediated phosphate transport is increased at higher pHs. The increased NPT2b mediated transport induced by NHE3 inhibition is masked in rats which have relatively low levels of NPT2b mediated phosphate transport, by the more robust inhibition of diffusion mediated phosphate absorption. Thus, the inhibition of NPT2b mediated phosphate transport in rats in the presence of NHE3 inhibition has an effect that exceeds its effect in the absence of NHE3 inhibition, leading to the observed synergism on phosphate absorption between NPT2b and NHE3 inhibition.

Effects of pharmacological inhibition of the sodium-dependent phosphate cotransporter 2b (NPT2b) on intestinal phosphate absorption in mouse and rat models.

An excess phosphate burden in renal disease has pathological consequences for bone, kidney, and heart. Therapies to decrease intestinal phosphate absorption have been used to address the problem, but with limited success. Here, we describe the in vivo effects of a novel potent inhibitor of the intestinal sodium-dependent phosphate cotransporter NPT2b, LY3358966. Following treatment with LY3358966, phosphate uptake into plasma 15 min following an oral dose of radiolabeled phosphate was decreased 74% and 22% in mice and rats, respectively, indicating NPT2b plays a much more dominant role in mice than rats. Following the treatment with LY3358966 and radiolabeled phosphate, mouse feces were collected for 48 h to determine the ability of LY3358966 to inhibit phosphate absorption. Compared to vehicle-treated animals, there was a significant increase in radiolabeled phosphate recovered in feces (8.6% of the dose, p < .0001). Similar studies performed in rats also increased phosphate recovered in feces (5.3% of the dose, p < .05). When used in combination with the phosphate binder sevelamer in rats, there was a further small, but not significant, increase in fecal phosphate. In conclusion, LY3358966 revealed a more prominent role for NPT2b on acute intestinal phosphate uptake into plasma in mice than rats. However, the modest effects on total intestinal phosphate absorption observed in mice and rats with LY3359866 when used alone or in combination with sevelamer highlights the challenge to identify new more effective therapeutic targets and/or drug combinations to treat the phosphate burden in patients with renal disease.

Identification of 4-Aminopyrazolopyrimidine Metabolite That May Contribute to the Hypolipidemic Effects of LY2584702 in Long Evans Diet-Induced Obese Rats.

LY2584702 is an inhibitor of p70 S6 kinase-1 previously developed for the treatment of cancer. In two phase 1 trials in oncology patients, significant reductions of total cholesterol, low-density lipoprotein cholesterol (LDL-C), and triglyceride were observed. In the current study, we sought to understand the potential mechanism of action of this compound in regulating lipid metabolism. In Long Evans diet-induced obese (DIO) rats, oral administration of LY2584702 for 3-4 weeks led to robust reduction of LDL-C up to 60%. An unexpected finding of liver triglyceride (TG) increase implicated a metabolite of LY2584702, 4-aminopyrazolo[3,4-day]pyrimidine (4-APP), in modulation of lipid metabolism in these rats. We showed that low-dose 4-APP, when administered orally for 3-4 weeks to Long Evans DIO rats, produced lipoprotein profile changes that were strikingly similar to LY2584702. Kinetic studies suggested that both LY2584702 and 4-APP had no effect on chylomicron-TG secretion and only exerted a modest effect on hepatic very low-density lipoprotein (VLDL)-TG secretion. In human hepatoma HepG2 cells, 4-APP, but not LY2584702, increased LDL uptake. We hypothesize that generation of the 4-APP metabolite may contribute to the efficacy of LY2584702 in lowering LDL-C in rats and potentially in humans as well. This mechanism of LDL-C lowering may include inhibition of VLDL production and increase in LDL clearance.

Discovery of 6-(4-{[5-Cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl]methoxy}piperidin-1-yl)-1-methyl-1H-indole-3-carboxylic Acid: A Novel FXR Agonist for the Treatment of Dyslipidemia.

The farnesoid X receptor (FXR) is a member of the "metabolic" subfamily of nuclear receptors. Several FXR agonists have been reported in the literature to have profound effects on plasma lipids in animal models. To discover novel and effective therapies for dyslipidemia and atherosclerosis, we have developed a series of potent FXR agonists that robustly lower plasma LDL and vLDL in LDLr-/- mice. To this end the novel piperidinylisoxazole system LY2562175 was discovered. This molecule is a potent and selective FXR agonist in vitro and has robust lipid modulating properties, lowering LDL and triglycerides while raising HDL in preclinical species. The preclinical ADME properties of LY2562175 were consistent with enabling once daily dosing in humans, and it was ultimately advanced to the clinic for evaluation in humans. The synthesis and biological profile of this molecule is discussed.

Design and synthesis of a novel series of [1-(4-hydroxy-benzyl)-1H-indol-5-yloxy]-acetic acid compounds as potent, selective, thyroid hormone receptor ß agonists.

The design, synthesis, and structure activity relationships for a novel series of indoles as potent, selective, thyroid hormone receptor ß (TRß) agonists is described. Compounds with >50× binding selectivity for TRß over TRα were generated and evaluation of compound 1c from this series in a model of dyslipidemia demonstrated positive effects on plasma lipid endpoints in vivo.

Varespladib (A-002), a secretory phospholipase A2 inhibitor, reduces atherosclerosis and aneurysm formation in ApoE-/- mice.

The family of secretory phospholipase A2 (sPLA2) enzymes has been associated with inflammatory diseases and tissue injury including atherosclerosis. A-001 is a novel inhibitor of sPLA2 enzymes discovered by structure-based drug design, and A-002 is the orally bioavailable prodrug currently in clinical development. A-001 inhibited human and mouse sPLA2 group IIA, V, and X enzymes with IC50 values in the low nM range. A-002 (1 mg/kg) led to high serum levels of A-001 and inhibited PLA2 activity in transgenic mice overexpressing human sPLA2 group IIA in C57BL/6J background. In addition, the effects of A-002 on atherosclerosis in 2 ApoE mouse models were evaluated using en face analysis. (1) In a high-fat diet model, A-002 (30 and 90 mg/kg twice a day for 16 weeks) reduced aortic atherosclerosis by 50% (P < 0.05). Plasma total cholesterol was decreased (P < 0.05) by 1 month and remained lowered throughout the study. (2) In an accelerated atherosclerosis model, with angiotensin II-induced aortic lesions and aneurysms, A-002 (30 mg/kg twice a day) reduced aortic atherosclerosis by approximately 40% (P < 0.05) and attenuated aneurysm formation (P = 0.0096). Thus, A-002 was effective at significantly decreasing total cholesterol, atherogenesis, and aneurysm formation in these 2 ApoE mouse models.

Genetic ablation of IRAK4 kinase activity inhibits vascular lesion formation.

Inflammation is critically involved in atherogenesis. Signaling from innate immunity receptors TLR2 and 4, IL-1 and IL-18 is mediated by MyD88 and further by interleukin-1 receptor activated kinases (IRAK) 4 and 1. We hypothesized that IRAK4 kinase activity is critical for development of atherosclerosis. IRAK4 kinase-inactive knock-in mouse was crossed with the ApoE-/- mouse. Lesion development was stimulated by carotid ligation. IRAK4 functional deficiency was associated with down-regulation of several pro-inflammatory genes, inhibition of macrophage infiltration, smooth muscle cell and lipid accumulation in vascular lesions. Reduction of plaque size and inhibition of outward remodeling were also observed. Similar effects were observed when ApoE-/- mice subjected to carotid ligation were treated with recombinant IL-1 receptor antagonist thereby validating the model in the relevant pathway context. Thus, IRAK4 functional deficiency inhibits vascular lesion formation in ApoE-/- mice, which further unravels mechanisms of vascular inflammation and identifies IRAK4 as a potential therapeutic target.