Mechanisms of Obesity-Induced Changes in Pharmacokinetics of IgG in Rats.

PURPOSE: To evaluate how obesity affects the pharmacokinetics of human IgG following subcutaneous (SC) and intravenous (IV) administration to rats and the homeostasis of endogenous rat IgG. METHODS: Differences in body weight and size, body composition, and serum concentration of endogenous rat IgG in male Zucker obese (ZUC-FA/FA) and control (ZUC-LEAN) rats were measured from the age of 5 weeks up to 30 weeks. At the age of 23-24 weeks animals received a single IV or SC dose of human IgG (1 g/kg of total body weight), and serum pharmacokinetics was followed for 7 weeks. A mechanistic model linking obesity-related changes in pharmacokinetics with animal growth and changes in body composition was developed. RESULTS: Significant differences were observed in both endogenous and exogenous IgG pharmacokinetics between obese and control groups. The AUC for human IgG was lower in obese groups (57.6% of control after IV and 48.1% after SC dosing), and clearance was 1.75-fold higher in obese animals. The mechanistic population model successfully captured the data and included several major components: endogenous rat IgG homeostasis with age-dependent synthesis rate; competition of human IgG and endogenous rat IgG for FcRn binding and its effect on endogenous rat IgG concentrations following injection of a high dose of human IgG; and the effect of body size and composition (changing over time and dependent on the obesity status) on pharmacokinetic parameters. CONCLUSIONS: We identified important obesity-induced changes in the pharmacokinetics of IgG. Results can potentially facilitate optimization of the dosing of IgG-based therapeutics in the obese population.

Superior weight loss with once-weekly semaglutide versus other glucagon-like peptide-1 receptor agonists is independent of gastrointestinal adverse events.

INTRODUCTION: Gastrointestinal (GI) adverse events (AEs) are the most common AEs with glucagon-like peptide-1 receptor agonists (GLP-1RAs). Weight loss (WL) is slightly greater in people who experience GI AEs than those who do not. A previous mediation analysis of the SUSTAIN 1-5 trials indicated minor contribution of nausea/vomiting to the greater WL with once-weekly semaglutide versus comparators. Semaglutide demonstrated superior glycated hemoglobin and body weight (BW) reductions versus other GLP-1RAs in SUSTAIN 3 (versus exenatide extended release 2.0 mg), SUSTAIN 7 (versus dulaglutide) and SUSTAIN 10 (liraglutide 1.2 mg). The objective of this analysis was to assess if significantly greater WL with semaglutide versus other GLP-1RAs is mediated by nausea/vomiting and other GI AEs (diarrhea, constipation, dyspepsia) during dose escalation (baseline to week 12, when GI AEs are generally most prevalent) and from baseline to end of treatment (EOT: week 56 (SUSTAIN 3), 40 (SUSTAIN 7) or 30 (SUSTAIN 10)). RESEARCH DESIGN AND METHODS: Subjects within trials were subdivided into those who reported (yes/no) nausea/vomiting or any other GI AE. Change from baseline in BW was assessed within each trial and subgroup. A mediation analysis separated WL into direct or indirect (mediated by GI AEs) effects. RESULTS: From baseline to week 12 or EOT, the nausea/vomiting-mediated difference in WL was, respectively: 0.05 or 0.09 kg of 3.78 kg at EOT (SUSTAIN 3); 0.06 or 0.03 kg of 2.26 kg at EOT (low-dose comparison) and 0.08 or 0.04 kg of 3.55 kg at EOT (high-dose comparison) (SUSTAIN 7) and 0.05 or 0.09 kg of 3.82 kg at EOT (SUSTAIN 10). CONCLUSIONS: In SUSTAIN 3, 7 and 10, nausea/vomiting by week 12 (end of dose escalation) or throughout treatment contributed minimally (<0.1 kg) to the superior WL with semaglutide versus GLP-1RA comparators at EOT.

Lymphatic Distribution of Etanercept Following Intravenous and Subcutaneous Delivery to Rats.

PURPOSE: The purpose of this work was to investigate the role of the lymphatic system in the pharmacokinetics of etanercept, a fusion protein. METHODS: Etanercept 1 mg/kg was administered intravenously (IV) and subcutaneously (SC) to thoracic lymph duct-cannulated and sham-operated control rats. Blood and lymph samples were obtained for up to 6 days. RESULTS: Model-based SC bioavailability of etanercept was 65.2% in the control group. In lymph-cannulated rats, etanercept concentration in the lymph was consistently lower than in serum following IV dosing; and the concentration in the lymph was significantly higher than in serum after SC injection. The absorption occurred predominantly through the lymphatic pathway (82.7%), and only 17.3% by direct uptake into the central compartment (blood pathway). Lymphatic cannulation reduced the area under the serum concentration-time curve by 28% in IV group and by 91% in SC group. A mechanistic pharmacokinetic model that combined dual absorption pathways with redistribution of the systemically available protein drug into lymph was developed. The model successfully captured serum and lymph data in all groups simultaneously, and all parameters were estimated with sufficient precision. CONCLUSIONS: Lymphatic system was shown to play an essential role in systemic disposition and SC absorption of etanercept.

Impact of drug distribution into adipose on tissue function: The cholesteryl ester transfer protein (CETP) inhibitor anacetrapib as a test case.

Anacetrapib is an inhibitor of cholesteryl ester transfer protein (CETP) previously under development as a lipid-modifying agent that reduces LDL-cholesterol and increases HDL-cholesterol in hypercholesterolemic patients. Anacetrapib demonstrates a long terminal half-life and accumulates in adipose tissue, which contributes to a long residence time of anacetrapib. Given our previous report that anacetrapib distributes into the lipid droplet of adipose tissue, we sought to understand whether anacetrapib affected adipose function, using a diet-induced obese (DIO) mouse model. Following 20 weeks of treatment with anacetrapib (100 mg/kg/day), levels of the drug increased to approximately 0.6 mmol/L in white adipose tissue. This level of anacetrapib was not associated with any impairment in adipose functionality as evidenced by a lack of any reduction in biomarkers of adipose functionality (plasma adiponectin, leptin, insulin; adipose adiponectin, leptin mRNA). In DIO wild-type (WT) mice treated with anacetrapib for 2 weeks and then subjected to 30% food restriction during washout to induce weight loss (18%) and fat mass loss (7%), levels of anacetrapib in adipose and plasma were not different between food restricted and ad lib-fed mice. These data indicate that despite deposition and long-term residence of ~0.6 mmol/L levels of anacetrapib in adipose tissue, adipose tissue function appears to be unaffected in mice. In addition, these data also indicate that even with severe caloric restriction and acute loss of fat mass, anacetrapib does not appear to be mobilized from the fat depot, thereby solidifying the role of adipose as a long-term storage site of anacetrapib.

Effect of Insulin Degludec Versus Insulin Glargine U100 on Hypoglycemia in Hispanic Patients With Type 2 Diabetes: Results From the SWITCH 2 Trial.

IN BRIEF Hispanic patients with type 2 diabetes have poorer glycemic control and are at higher risk of severe diabetes complications and mortality than non-Hispanic white patients. This post hoc analysis investigated the safety and efficacy of insulin degludec versus insulin glargine 100 units/mL (glargine U100) in the Hispanic patient subpopulation from the SWITCH 2 trial. In Hispanic patients, hypoglycemia was consistently lower and nocturnal hypoglycemia was significantly lower with degludec versus glargine U100 at similar levels of glycemic control. Overall, results in Hispanic patients in SWITCH 2 were consistent with those in non-Hispanic patients.

Semaglutide Added to Basal Insulin in Type 2 Diabetes (SUSTAIN 5): A Randomized, Controlled Trial.

Context: Combination therapy with insulin and glucagon-like peptide-1 receptor agonists (GLP-1RAs) is important for treating type 2 diabetes (T2D). This trial assesses the efficacy and safety of semaglutide, a GLP-1RA, as an add-on to basal insulin. Objective: To demonstrate the superiority of semaglutide vs placebo on glycemic control as an add-on to basal insulin in patients with T2D. Design: Phase 3a, double-blind, placebo-controlled, 30-week trial. Setting: This study included 90 sites in five countries. Patients: We studied 397 patients with uncontrolled T2D receiving stable therapy with basal insulin with or without metformin. Interventions: Subcutaneous semaglutide 0.5 or 1.0 mg once weekly or volume-matched placebo. Main Outcome Measures: Primary endpoint was change in glycated Hb (HbA1c) from baseline to week 30. Confirmatory secondary endpoint was change in body weight from baseline to week 30. Results: At week 30, mean HbA1c reductions [mean baseline value, 8.4% (67.9 mmol/mol)] with semaglutide 0.5 and 1.0 mg were 1.4% (15.8 mmol/mol) and 1.8% (20.2 mmol/mol) vs 0.1% (1.0 mmol/mol) with placebo [estimated treatment difference (ETD) vs placebo, -1.35 (14.8 mmol/mol); 95% CI, -1.61 to -1.10 and ETD, -1.75% (19.2 mmol/mol); 95% CI, -2.01 to -1.50; both P < 0.0001]. Severe or blood glucose-confirmed hypoglycemic episodes were reported in 11 patients (17 events) and 14 patients (25 events) with semaglutide 0.5 and 1.0 mg, respectively, vs seven patients (13 events) with placebo (estimated rate ratio vs placebo, 2.08; 95% CI, 0.67 to 6.51 and estimated rate ratio vs placebo, 2.41; 95% CI, 0.84 to 6.96 for 0.5 and 1.0 mg; both P = nonsignificant). Mean body weight decreased with semaglutide 0.5 and 1.0 mg vs placebo from baseline to end of treatment: 3.7, 6.4, and 1.4 kg (ETD, -2.31; 95% CI, -3.33 to -1.29 and ETD, -5.06; 95% CI, -6.08 to -4.04 kg; both P < 0.0001). Premature treatment discontinuation due to adverse events was higher for semaglutide 0.5 and 1.0 mg vs placebo (4.5%, 6.1%, and 0.8%), mainly due to gastrointestinal disorders. Conclusions: Semaglutide, added to basal insulin, significantly reduced HbA1c and body weight in patients with uncontrolled T2D vs placebo.

Effect of Insulin Degludec vs Insulin Glargine U100 on Hypoglycemia in Patients With Type 2 Diabetes: The SWITCH 2 Randomized Clinical Trial.

IMPORTANCE: Hypoglycemia, a serious risk for insulin-treated patients with type 2 diabetes, negatively affects glycemic control. OBJECTIVE: To test whether treatment with basal insulin degludec is associated with a lower rate of hypoglycemia compared with insulin glargine U100 in patients with type 2 diabetes. DESIGN, SETTING, AND PARTICIPANTS: Randomized, double-blind, treat-to-target crossover trial including two 32-week treatment periods, each with a 16-week titration period and a 16-week maintenance period. The trial was conducted at 152 US centers between January 2014 and December 2015 in 721 adults with type 2 diabetes and at least 1 hypoglycemia risk factor who were previously treated with basal insulin with or without oral antidiabetic drugs. INTERVENTIONS: Patients were randomized 1:1 to receive once-daily insulin degludec followed by insulin glargine U100 (n = 361) or to receive insulin glargine U100 followed by insulin degludec (n = 360) and randomized 1:1 to morning or evening dosing within each treatment sequence. MAIN OUTCOMES AND MEASURES: The primary end point was the rate of overall symptomatic hypoglycemic episodes (severe or blood glucose confirmed [<56 mg/dL]) during the maintenance period. Secondary end points were the rate of nocturnal symptomatic hypoglycemic episodes (severe or blood glucose confirmed, occurring between 12:01 am and 5:59 am) and the proportion of patients with severe hypoglycemia during the maintenance period. RESULTS: Of the 721 patients randomized (mean [SD] age, 61.4 [10.5] years; 53.1% male), 580 (80.4%) completed the trial. During the maintenance period, the rates of overall symptomatic hypoglycemia for insulin degludec vs insulin glargine U100 were 185.6 vs 265.4 episodes per 100 patient-years of exposure (PYE) (rate ratio = 0.70 [95% CI, 0.61-0.80]; P < .001; difference, -23.66 episodes/100 PYE [95% CI, -33.98 to -13.33]), and the proportions of patients with hypoglycemic episodes were 22.5% vs 31.6% (difference, -9.1% [95% CI, -13.1% to -5.0%]). The rates of nocturnal symptomatic hypoglycemia with insulin degludec vs insulin glargine U100 were 55.2 vs 93.6 episodes/100 PYE (rate ratio = 0.58 [95% CI, 0.46-0.74]; P < .001; difference, -7.41 episodes/100 PYE [95% CI, -11.98 to -2.85]), and the proportions of patients with hypoglycemic episodes were 9.7% vs 14.7% (difference, -5.1% [95% CI, -8.1% to -2.0%]). The proportions of patients experiencing severe hypoglycemia during the maintenance period were 1.6% (95% CI, 0.6%-2.7%) for insulin degludec vs 2.4% (95% CI, 1.1%-3.7%) for insulin glargine U100 (McNemar P = .35; risk difference, -0.8% [95% CI, -2.2% to 0.5%]). Statistically significant reductions in overall and nocturnal symptomatic hypoglycemia for insulin degludec vs insulin glargine U100 were also seen for the full treatment period. CONCLUSIONS AND RELEVANCE: Among patients with type 2 diabetes treated with insulin and with at least 1 hypoglycemia risk factor, 32 weeks' treatment with insulin degludec vs insulin glargine U100 resulted in a reduced rate of overall symptomatic hypoglycemia. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT02030600.

Effects of carvedilol on structural and functional outcomes and plasma biomarkers in the mouse transverse aortic constriction heart failure model.

INTRODUCTION: Despite the widespread use of the mouse transverse aortic constriction heart failure model, there are no reports on the characterization of the standard-of-care agent carvedilol in this model. METHODS: Left ventricular pressure overload was produced in mice by transverse aortic constriction between the innominate and left common carotid arteries. Carvedilol was administered at multiple dose levels (3, 10 and 30 mg/kg/day per os; yielding end-study mean plasma concentrations of 0.002, 0.015 and 0.044 µM, respectively) in a therapeutic design protocol with treatment initiated after the manifestation of left ventricular remodeling at 3 weeks post transverse aortic constriction and continued for 10 weeks. RESULTS: Carvedilol treatment in transverse aortic constriction mice significantly decreased heart rate and left ventricular dP/dt (max) at all dose levels consistent with ß-adrenoceptor blockade. The middle dose of carvedilol significantly decreased left ventricular weight, whereas the higher dose decreased total heart, left and right ventricular weight and wet lung weight compared to untreated transverse aortic constriction mice. The higher dose of carvedilol significantly increased cardiac performance as measured by ejection fraction and fractional shortening and decreased left ventricular end systolic volume consistent with the beneficial effect on cardiac function. End-study plasma sST-2 and Gal-3 levels did not differ among sham, transverse aortic constriction control and transverse aortic constriction carvedilol groups. Plasma brain natriuretic peptide concentrations were elevated significantly in transverse aortic constriction control animals (~150%) compared to shams in association with changes in ejection fraction and heart weight and tended to decrease (~30%, p = 0.10-0.12) with the mid- and high-dose carvedilol treatment. CONCLUSION: A comparison of carvedilol hemodynamic and structural effects in the mouse transverse aortic constriction model versus clinical use indicates a strong agreement in effect profiles preclinical versus clinical, providing important translational validation for this widely used animal model. The present plasma brain natriuretic peptide biomarker findings support the measurement of plasma natriuretic peptides in the mouse transverse aortic constriction model to extend the translational utility of the model.

Early echocardiographic predictors of outcomes in the mouse transverse aortic constriction heart failure model.

INTRODUCTION: Mouse transverse aortic constriction (TAC) is a widely-used model of pressure overload-induced heart failure. An intrinsic limitation of the model is variability in the response to pressure overload even when employing a standard severity of stenosis. Few literature studies have explicitly reported the use of entry criteria or early predictors to mitigate variability and enrich outcomes in this model. METHODS: Eleven-week-old male C57BL/6J mice underwent TAC or sham surgery. Left ventricular (LV) function and dimensions were assessed by M-mode echocardiography at baseline (pre) and 3, 9 and 12weeks post-procedure (end-study). At 24h post-procedure, transverse aortic flow velocities were obtained for estimating trans-TAC pressure gradients. Invasive LV hemodynamic assessments were performed and terminal heart and lung weights obtained at end-study. RESULTS: TAC mice displayed early development of LV hypertrophy and wall thickening followed by the later development of LV chamber dilation, and progressive development of LV systolic and diastolic dysfunction. The use of a pre-defined trans-TAC pressure gradient criterion of 45-60mmHg did not affect end-study organ weight, echocardiographic and invasive hemodynamic outcomes. A post-hoc receiver operator characteristic (ROC) analysis identified early 3week echocardiographic measures of LVmass(echo) and ejection fraction, with threshold changes of ~+30% and -10% normalized to baseline respectively, as good predictors for multiple end-study organ weight, echocardiographic and invasive hemodynamic outcomes. DISCUSSION: This ROC analysis has identified early predictive threshold changes which may serve, alone or in combination, as entry criteria to enrich outcome in this model.

Tracking fatty acid kinetics in distinct lipoprotein fractions in vivo: a novel high-throughput approach for studying dyslipidemia in rodent models.

Isotopic tracers have been used to examine lipid trafficking for many years, and data from those studies have typically yielded novel insight regarding the pathophysiology of dyslipidemia. Previous experimental designs were suitable for studies in humans because relatively large volumes of plasma could be regularly sampled. We have expanded on the earlier logic by applying high-throughput analytical methods that require reduced sample volumes. Specifically, we have examined the possibility of coupling gel-based separations of lipoproteins (e.g., lipoprint) with LC-MS/MS analyses of complex lipid mixtures as a way to routinely measure the labeling profiles of distinct lipids in discrete lipoprotein subfractions. We demonstrate the ability to measure the incorporation of [U-(13)C]oleate into triglycerides (TG), PLs (PL), and cholesterol esters (CE) in VLDL, LDL, and HDL particles in mice. Although rodent models of dyslipidemia are inherently different from humans because of alterations in enzyme activities and underlying metabolism, rodent models can be used to screen novel compounds for efficacy in altering a given biochemical pathway and therein enable studies of target engagement in vivo. We expect that it is possible to translate our approach for application in other systems, including studies in humans.

Quantifying apoprotein synthesis in rodents: coupling LC-MS/MS analyses with the administration of labeled water.

Stable isotope tracer studies of apoprotein flux in rodent models present difficulties as they require working with small volumes of plasma. We demonstrate the ability to measure apoprotein flux by administering either (2)H- or (18)O-labeled water to mice and then subjecting samples to LC-MS/MS analyses; we were able to simultaneously determine the labeling of several proteolytic peptides representing multiple apoproteins. Consistent with relative differences reported in the literature regarding apoprotein flux in humans, we found that the fractional synthetic rate of apoB is greater than apoA1 in mice. In addition, the method is suitable for quantifying acute changes in protein flux: we observed a stimulation of apoB production in mice following an intravenous injection of Intralipid and a decrease in apoB production in mice treated with an inhibitor of microsomal triglyceride transfer protein. In summary, we demonstrate a high-throughput method for studying apoprotein kinetics in rodent models. Although notable differences exist between lipoprotein profiles that are observed in rodents and humans, we expect that the method reported here has merit in studies of dyslipidemia as i) rodent models can be used to probe target engagement in cases where one aims to modulate apoprotein production and ii) the approach should be adaptable to studies in humans.

2-(4-Carbonylphenyl)benzoxazole inhibitors of CETP: attenuation of hERG binding and improved HDLc-raising efficacy.

The development of 2-phenylbenzoxazoles as inhibitors of cholesteryl ester transfer protein (CETP) is described. Efforts focused on finding suitable replacements for the central piperidine with the aim of reducing hERG binding: a main liability of our benchmark benzoxazole (1a). Replacement of the piperidine with a cyclohexyl group successfully attenuated hERG binding, but was accompanied by reduced in vivo efficacy. The approach of substituting a piperidine moiety with an oxazolidinone also attenuated hERG binding. Further refinement of this latter scaffold via SAR at the pyridine terminus and methyl branching on the oxazolidinone led to compounds 7e and 7f, which raised HDLc by 33 and 27mg/dl, respectively, in our transgenic mouse PD model and without the hERG liability of previous series.

siRNA-induced liver ApoB knockdown lowers serum LDL-cholesterol in a mouse model with human-like serum lipids.

Increased serum apolipoprotein (apo)B and associated LDL levels are well-correlated with an increased risk of coronary disease. ApoE⁻/⁻ and low density lipoprotein receptor (LDLr)⁻/⁻ mice have been extensively used for studies of coronary atherosclerosis. These animals show atherosclerotic lesions similar to those in humans, but their serum lipids are low in apoB-containing LDL particles. We describe the development of a new mouse model with a human-like lipid profile. Ldlr CETP⁺/⁻ hemizygous mice carry a single copy of the human CETP transgene and a single copy of a LDL receptor mutation. To evaluate the apoB pathways in this mouse model, we used novel short-interfering RNAs (siRNA) formulated in lipid nanoparticles (LNP). ApoB siRNAs induced up to 95% reduction of liver ApoB mRNA and serum apoB protein, and a significant lowering of serum LDL in Ldlr CETP⁺/⁻ mice. ApoB targeting is specific and dose-dependent, and it shows lipid-lowering effects for over three weeks. Although specific triglycerides (TG) were affected by ApoB mRNA knockdown (KD) and the total plasma lipid levels were decreased by 70%, the overall lipid distribution did not change. Results presented here demonstrate a new mouse model for investigating additional targets within the ApoB pathways using the siRNA modality.

The use of stable-isotopically labeled oleic acid to interrogate lipid assembly in vivo: assessing pharmacological effects in preclinical species.

The use of stable isotopically labeled substrates and analysis by mass spectrometry have provided substantial insight into rates of synthesis, disposition, and utilization of lipids in vivo. The information to be gained from such studies is of particular benefit to therapeutic research where the underlying causes of disease may be related to the production and utilization of lipids. When studying biology through the use of isotope tracers, care must be exercised in interpreting the data to ensure that any response observed can truly be interpreted as biological and not as an artifact of the experimental design or a dilutional effect on the isotope. We studied the effects of dosing route and tracer concentration on the mass isotopomer distribution profile as well as the action of selective inhibitors of microsomal tri-glyceride transfer protein (MTP) in mice and diacylglycerol acyltransferase 1 (DGAT1) in nonhuman primates, using a stable-isotopically labeled approach. Subjects were treated with inhibitor and subsequently given a dose of uniformly ¹³C-labeled oleic acid. Samples were analyzed using a rapid LC-MS technique, allowing the effects of the intervention on the assembly and disposition of triglycerides, cholesteryl esters, and phospholipids to be determined in a single 3 min run from just 10 µl of plasma.

2-(4-carbonylphenyl)benzoxazole inhibitors of CETP: scaffold design and advancement in HDLc-raising efficacy.

The development of 2-phenylbenzoxazoles as inhibitors of cholesteryl ester transfer protein (CETP) is described. Initial efforts aimed at engineering replacements for the aniline substructures in the benchmark molecule. Reversing the connectivity of the central aniline lead to a new class of 2-(4-carbonylphenyl)benzoxazoles. Structure-activity studies at the C-7 and terminal pyridine ring allowed for the optimization of potency and HDLc-raising efficacy in this new class of inhibitors. These efforts lead to the discovery of benzoxazole 11v, which raised HDLc by 24 mg/dl in our transgenic mouse PD model.

2-Arylbenzoxazoles as CETP inhibitors: raising HDL-C in cynoCETP transgenic mice.

We describe structure-activity studies leading to the discovery of 2-arylbenzoxazole 3, the first in a series to raise serum high-density lipoprotein cholesterol levels in transgenic mice. Replacement of the 4-piperidinyloxy moiety with piperazinyl provided a more synthetically tractable lead, which upon optimization resulted in compound 4, an excellent inhibitor of cholesteryl ester transfer protein function with good pharmacokinetic properties and in vivo efficacy.

Interleukin-2 gene variation impairs regulatory T cell function and causes autoimmunity.

Autoimmune diseases are thought to result from imbalances in normal immune physiology and regulation. Here, we show that autoimmune disease susceptibility and resistance alleles on mouse chromosome 3 (Idd3) correlate with differential expression of the key immunoregulatory cytokine interleukin-2 (IL-2). In order to test directly that an approximately twofold reduction in IL-2 underpins the Idd3-linked destabilization of immune homeostasis, we show that engineered haplodeficiency of Il2 gene expression not only reduces T cell IL-2 production by twofold but also mimics the autoimmune dysregulatory effects of the naturally occurring susceptibility alleles of Il2. Reduced IL-2 production achieved by either genetic mechanism correlates with reduced function of CD4(+) CD25(+) regulatory T cells, which are critical for maintaining immune homeostasis.

SAR studies: designing potent and selective LXR agonists.

Counterscreening compounds from a Merck PPAR program discovered lead 1, as a nanomolar LXR/PPAR dual agonist. SAR optimization developed a series of heterocyclic LXR agonists having excellent selectivity over all PPAR isoforms and possessing high LXR affinity and strong in vivo potency.

Different roles of liver X receptor alpha and beta in lipid metabolism: effects of an alpha-selective and a dual agonist in mice deficient in each subtype.

Liver X receptor (LXR) alpha and LXRbeta are closely related nuclear receptors that respond to elevated levels of intracellular cholesterol by enhancing transcription of genes that control cholesterol efflux and fatty acid biosynthesis. The consequences of inactivation of either LXR isoform have been thoroughly studied, as have the effects of simultaneous activation of both LXRalpha and LXRbeta by synthetic compounds. We here describe the effects of selective activation of LXRalpha or LXRbeta on lipid metabolism. This was accomplished by treating mice genetically deficient in either LXRalpha or LXRbeta with an agonist with equal potency for both isoforms (Compound B) or a synthetic agonist selective for LXRalpha (Compound A). We also determined the effect of these agonists on gene expression and cholesterol efflux in peritoneal macrophages derived from wild-type and knockout mice. Both compounds raised HDL-cholesterol and increased liver triglycerides in wild-type mice; in contrast, in mice deficient in LXRalpha, Compound B increased HDL-cholesterol but did not cause hepatic steatosis. Compound B induced ATP-binding cassette transporter (ABC) A1 expression and stimulated cholesterol efflux in macrophages from both LXRalpha and LXRbeta-deficient mice. Our data lend further experimental support to the hypothesis that LXRbeta-selective agonists may raise HDL-cholesterol and stimulate macrophage cholesterol efflux without causing liver triglyceride accumulation.

Type 1 diabetes genes and pathways shared by humans and NOD mice.

The identification of causative genes for the autoimmune disease type 1 diabetes (T1D) in humans and candidate genes in the NOD mouse has made significant progress in recent years. In addition to sharing structural aspects of the MHC class II molecules that confer susceptibility or resistance to T1D, genes and pathways contributing to autoimmune pathogenesis are held in common by the two species. There are data demonstrating a similar need to establish central tolerance to insulin. Gene variants for the interacting molecules IL2 and CD25, members of a pathway that is essential for immune homeostasis, are present in mice and humans, respectively. Variation of two molecules that negatively regulate T cells, CTLA-4 and the tyrosine phosphatase LYP/PEP, are associated with susceptibility to human and NOD T1D. These observations underscore the value of the NOD mouse model for mechanistic studies on human T1D-associated molecular and cellular pathways.