Enhancing antisense efficacy with multimers and multi-targeting oligonucleotides (MTOs) using cleavable linkers.

The in vivo potency of antisense oligonucleotides (ASO) has been significantly increased by reducing their length to 8-15 nucleotides and by the incorporation of high affinity RNA binders such as 2', 4'-bridged nucleic acids (also known as locked nucleic acid or LNA, and 2',4'-constrained ethyl [cET]). We now report the development of a novel ASO design in which such short ASO monomers to one or more targets are co-synthesized as homo- or heterodimers or multimers via phosphodiester linkers that are stable in plasma, but cleaved inside cells, releasing the active ASO monomers. Compared to current ASOs, these multimers and multi-targeting oligonucleotides (MTOs) provide increased plasma protein binding and biodistribution to liver, and increased in vivo efficacy against single or multiple targets with a single construct. In vivo, MTOs synthesized in both RNase H-activating and steric-blocking oligonucleotide designs provide ≈4-5-fold increased potency and ≈2-fold increased efficacy, suggesting broad therapeutic applications.

Preclinical PK/PD modeling and human efficacious dose projection for a glucokinase activator in the treatment of diabetes.

Human Hexokinase IV, or glucokinase (GK), is a regulator of glucose concentrations in the body. It plays a key role in pancreatic insulin secretion as well as glucose biotransformation in the liver, making it a potentially viable target for treatment of Type 2 diabetes. Allosteric activators of GK have been shown to decrease blood glucose concentrations in both animals and humans. Here, the development of a mathematical model is presented that describes glucose modulation in an ob/ob mouse model via administration of a potent GK activator, with the goal of projecting a human efficacious dose and plasma exposure. The model accounts for the allosteric interaction between GK, the activator, and glucose using a modified Hill function. Based on model simulations using data from the ob/ob mouse and in vitro studies, human projections of glucose response to the GK activator are presented, along with dose and regimen predictions to maintain clinically significant decreases in blood glucose in a Type 2 diabetic patient. This effort serves as a basis to build a detailed mechanistic understanding of GK and its role as a therapeutic target for Type 2 diabetes, and it highlights the benefits of using such an approach in a drug discovery setting.

Pharmacological characterization of a small molecule inhibitor of c-Jun kinase.

c-Jun NH(2)-terminal kinase (JNK) plays an important role in insulin resistance; however, identification of pharmacologically potent and selective small molecule JNK inhibitors has been limited. Compound A has a cell IC(50) of 102 nM and is at least 100-fold selective against related kinases and 27-fold selective against glycogen synthase kinase-3beta and cyclin-dependent kinase-2. In C57BL/6 mice, compound A reduced LPS-mediated increases in both plasma cytokine levels and phosphorylated c-Jun in adipose tissue. Treatment of mice fed a high-fat diet with compound A for 3 wk resulted in a 13.1 +/- 1% decrease in body weight and a 9.3 +/- 1.5% decrease in body fat, compared with a 6.6 +/- 2.1% increase in body weight and a 6.7 +/- 2.1% increase in body fat in vehicle-treated mice. Mice pair fed to those that received compound A exhibited a body weight decrease of 7 +/- 1% and a decrease in body fat of 1.6 +/- 1.3%, suggesting that reductions in food intake could not account solely for the reductions in adiposity observed. Compound A dosed at 30 mg/kg for 13 days in high-fat fed mice resulted in a significant decrease in phosphorylated c-Jun in adipose tissue accompanied by a decrease in weight and reductions in glucose and triglycerides and increases in insulin sensitivity to levels comparable with those in lean control mice. The ability of compound A to reduce the insulin-stimulated phosphorylation of insulin receptor substrate-1 (IRS-1) von Ser307 and partially reverse the free fatty acid inhibition of glucose uptake in 3T3L1 adipocytes, suggests that enhancement of insulin signaling in addition to weight loss may contribute to the effects of compound A on insulin sensitization in vivo. Pharmacological inhibition of JNK using compound A may therefore offer an effective therapy for type 2 diabetes mediated at least in part via weight reduction.

Antisense inhibition of 11betahydroxysteroid dehydrogenase type 1 improves diabetes in a novel cortisone-induced diabetic KK mouse model.

The inhibition of 11betahydroxysteroid dehydrogenase 1 (11betaHSD1), an enzyme that catalyzes the conversion of inactive cortisone to active cortisol, is an attractive target to treat diabetes by suppressing hepatic gluconeogenesis. To test this hypothesis, we developed a novel glucocorticoid-induced diabetic KK mouse model and used 11betaHSD1 antisense oligonucleotide (ASO) as an inhibitory tool. KK mice were treated with 25 or 50mg/kg/day of 11betaHSD1 ASO for 28 days. On day 25, cortisone pellets were surgically implanted to induce diabetes. In the ASO-treated mice, plasma blood glucose levels were significantly reduced by up to 54%. In parallel, cortisol and other diabetes endpoints were also significantly reduced. Hepatic 11betaHSD1 mRNA was suppressed by up to 84% with a concomitant respective decrease of up to 49% in the expression of PEPCK. The results suggest that inhibition of 11betaHSD1 activity reduces the availability of cortisol to activate the glucocorticoid receptor, down regulates gluconeogenesis and thus reduces plasma glucose levels in cortisone-induced diabetic KK mice.

RAR and RXR modulation in cancer and metabolic disease.

Retinoic acid receptors (RARs) are ligand-controlled transcription factors that function as heterodimers with retinoid X receptors (RXRs) to regulate cell growth and survival. The success of RAR modulation in the treatment of acute promyelocytic leukaemia (APL) has stimulated considerable interest in the development of RAR and RXR modulators. This has been aided by recent advances in the understanding of the biological role of RARs and RXRs and in the design of selective receptor modulators that might overcome the limitations of current drugs. Here, we discuss the challenges and opportunities for therapeutic strategies based on RXR and RAR modulators, with a focus on cancer and metabolic diseases such as diabetes and obesity.

Pyridine-2-propanoic acids: Discovery of dual PPARalpha/gamma agonists as antidiabetic agents.

A series of novel pyridine-2-propanoic acids was synthesized. A structure-activity relationship study of these compounds led to the identification of potent dual PPARalpha/gamma agonists with varied isoform selectivity. Based on the results of efficacy studies in diabetic (db/db) mice, and the desired pharmacokinetic parameters, compound (S)-13 was selected for further profiling.

Pyridine-3-propanoic acids: Discovery of dual PPARalpha/gamma agonists as antidiabetic agents.

A series of novel pyridine-3-propanoic acids was synthesized. A structure-activity relationship study of these compounds led to the identification of potent dual PPARalpha/gamma agonists with varied isoform selectivity. Based on the results of efficacy studies in diabetic (db/db) mice, and the desired pharmacokinetic parameters, compounds (S)-14 and (S)-19 were selected for further profiling.

Biological characterization of a heterodimer-selective retinoid X receptor modulator: potential benefits for the treatment of type 2 diabetes.

Specific retinoid X receptor (RXR) agonists, such as LG100268 (LG268), and the thiazolidinedione (TZD) PPARgamma agonists, such as rosiglitazone, produce insulin sensitization in rodent models of insulin resistance and type 2 diabetes. In sharp contrast to the TZDs that produce significant increases in body weight gain, RXR agonists reduce body weight gain and food consumption. Unfortunately, RXR agonists also suppress the thyroid hormone axis and generally produce hypertriglyceridemia. Heterodimer-selective RXR modulators have been identified that, in rodents, retain the metabolic benefits of RXR agonists with reduced side effects. These modulators bind specifically to RXR with high affinity and are RXR homodimer partial agonists. Although RXR agonists activate many heterodimer partners, these modulators selectively activate RXR:PPARalpha and RXR:PPARgamma, but not RXR:RARalpha, RXR:LXRalpha, RXR:LXRbeta, or RXR:FXRalpha. We report the in vivo characterization of one RXR modulator, LG101506 (LG1506). In Zucker fatty (fa/fa) rats, LG1506 is a potent insulin sensitizer that also enhances the insulin-sensitizing activities of rosiglitazone. Administration of LG1506 reduces both body weight gain and food consumption and blocks the TZD-induced weight gain when coadministered with rosiglitazone. LG1506 does not significantly suppress the thyroid hormone axis in rats, nor does it elevate triglycerides in Sprague Dawley rats. However, LG1506 produces a unique pattern of triglycerides elevation in Zucker rats. LG1506 elevates high-density lipoprotein cholesterol in humanized apolipoprotein A-1-transgenic mice. Therefore, selective RXR modulators are a promising approach for developing improved therapies for type 2 diabetes, although additional studies are needed to understand the strain-specific effects on triglycerides.

Conversion of human-selective PPARalpha agonists to human/mouse dual agonists: a molecular modeling analysis.

To understand the species selectivity in a series of alpha-methyl-alpha-phenoxy carboxylic acid PPARalpha/gamma dual agonists (1-11), structure-based molecular modeling was carried out in the ligand binding pockets of both human and mouse PPARalpha. This study suggested that interaction of both 4-phenoxy and phenyloxazole substituents of these ligands with F272 and M279 in mouse PPARalpha leads to the species-specific divergence in ligand binding. Insights obtained in the molecular modeling studies of these key interactions resulted in the ability to convert a human-selective PPARalpha agonist to a human and mouse dual agonist within the same platform.

Activation of the retinoid X receptor suppresses appetite in the rat.

The retinoid X receptor (RXR), a ubiquitously expressed intracellular receptor, regulates pathways controlling glucose, triglycerides, cholesterol, and bile acid metabolism. In addition to its role in those metabolic pathways, we reported that RXR activation with a pan agonist [e.g. LG100268 (LG268)] decreases both body weight gain (BWG) and food consumption (FC) in obese, insulin-resistant rodents. In parallel with those changes in energy balance, we show here that activation of RXR pathways results in adipose tissue remodeling, particularly within sc fat where the rate of apoptosis is increased 5-fold. This change may underlie the selective decrease in fat mass observed in Zucker fatty rats treated with LG268 for 6 wk. Because FC is strongly correlated with BWG in treated animals, we hypothesized that regulation of FC might be the primary mechanism underlying reduced BWG during RXR agonist administration. Importantly, decreased FC is due to decreased meal size, suggestive of induced satiety rather than malaise and/or aversion to food. Furthermore, administration of LG268 directly into the brain via intracerebroventricular injection also reduces FC, BWG, and insulin, whereas the elevation in triglycerides observed after oral administration is absent. The latter observation suggests that RXR actions on energy balance and lipid homeostasis are separable. Therefore, ligand-mediated activation of either an RXR homodimer or an unidentified heterodimeric complex regulates pathways controlling energy balance at least in part via a central nervous system-mediated mechanism.

Mechanism of selective retinoid X receptor agonist-induced hypothyroidism in the rat.

The retinoid X receptor (RXR) agonist bexarotene can cause clinically significant hypothyroidism in cutaneous T cell lymphoma patients. The mechanism by which the RXR agonist produces this effect is unclear. We have studied the impact of a selective RXR agonist (rexinoid), LG100268, on rat thyroid axis hormones and show that the acute phase of hypothyroidism is associated with reduced pituitary TSH secretion. A single oral administration of LG100268 to naive Sprague Dawley rats causes a rapid and statistically significant decline in TSH levels (apparent in 0.5-1 h). Total T(4) and T(3) levels decline more gradually, reaching statistical significance 24 h after treatment. Increasing doses of LG100268 produce greater suppression of thyroid axis hormones. To investigate the mechanism(s) mediating this suppression, we determined pituitary TSHbeta mRNA, TSH protein levels, and TRH-stimulated TSH secretion. Two hours after treatment, neither TSHbeta mRNA nor TSH protein levels were altered by LG100268. However, LG100268 treatment reduced the area under the curve for TRH-stimulated TSH secretion by 54%. We have identified an unexpected mechanism by which rexinoids induce hypothyroidism by acutely reducing TSH secretion from the anterior pituitary. This mechanism is independent of the rexinoid's previously demonstrated inhibition of TSHbeta gene transcription.

A tailored therapy for the metabolic syndrome: the dual peroxisome proliferator-activated receptor-alpha/gamma agonist LY465608 ameliorates insulin resistance and diabetic hyperglycemia while improving cardiovascular risk factors in preclinical models.

A novel nonthiazolidinedione dual peroxisome proliferator- activated receptor (PPAR)-alpha/gamma agonist, LY465608, was designed to address the major metabolic disturbances of type 2 diabetes. LY465608 altered PPAR-responsive genes in liver and fat of db/db mice and dose-dependently lowered plasma glucose in hyperglycemic male Zucker diabetic fatty (ZDF) rats, with an ED(50) for glucose normalization of 3.8 mg small middle dot kg(-1) small middle dot day(-1). Metabolic improvements were associated with enhanced insulin sensitivity, as demonstrated in female obese Zucker (fa/fa) rats using both oral glucose tolerance tests and hyperinsulinemic-euglycemic clamps. Further characterization of LY465608 revealed metabolic changes distinct from a selective PPAR-gamma agonist, which were presumably due to the concomitant PPAR-alpha agonism, lower respiratory quotient, and less fat accumulation, despite a similar impact on glycemia in male ZDF rats. In addition to these alterations in diabetic and insulin-resistant animals, LY465608 dose-dependently elevated HDL cholesterol and lowered plasma triglycerides in human apolipoprotein A-I transgenic mice, demonstrating that this compound significantly improves primary cardiovascular risk factors. Overall, these studies demonstrate that LY465608 beneficially impacts multiple facets of type 2 diabetes and associated cardiovascular risk, including those facets involved in the development of micro- and macrovascular complications, which are the major sources for morbidity and mortality in these patients.