Cholesteryl ester transfer protein (CETP) inhibitors based on cyclic urea, bicyclic urea and bicyclic sulfamide cores.

Cholesteryl ester transfer protein (CETP) inhibitors reduce the transfer of cholesteryl esters from the high-density lipoprotein (HDL-C) to apolipoprotein such as VLDL/LDL, with exchange of triglycerides. Thus, this inhibition increases the HDL-C levels, which is believed to lower the risk for heart disease and stroke. We report here a series of CETP inhibitors based on the cyclic, bicyclic urea and sulfamide cores. These CETP inhibitors exemplified by 15, 31, and 45 demonstrated in vitro potency in inhibiting the CETP transfer activity, and 15, 31 showing in vivo efficacy to increase HDL-C levels in cynomolgus-CETP transgenic mice. The synthesis and biological evaluations of these CETP inhibitors are described.

Accelerating the discovery of DGAT1 inhibitors through the application of parallel medicinal chemistry (PMC).

The parallel medicinal chemistry (PMC) was effectively applied to accelerate the optimization of diacylglycerol O-acyltransferase I (DGAT-1) inhibitors. Through a highly collaborative and iterative library design, synthesis and testing, a benzimidazole lead was rapidly and systematically advanced to a highly potent, selective and bioavailable DGAT1 inhibitor with the potential for further development.

Benzimidazole-based DGAT1 inhibitors with a [3.1.0] bicyclohexane carboxylic acid moiety.

Previously disclosed benzimidazole-based DGAT1 inhibitors containing a cyclohexane carboxylic acid moiety suffer from isomerization at the alpha position of the carboxylic acid group, generating active metabolites which exhibit DGAT1 inhibition comparable to the corresponding parent compounds. In this report, we describe the design, synthesis and profiling of benzimidazole-based DGAT1 inhibitors with a [3.1.0] bicyclohexane carboxylic acid moiety. Our results show that single isomer 3A maintains in vitro and in vivo inhibition against DGAT1. In contrast to previous lead compounds, 3A does not undergo isomerization during in vitro hepatocyte incubation study or in vivo mouse study.

Effects of small interfering RNA-mediated hepatic glucagon receptor inhibition on lipid metabolism in db/db mice.

Hepatic glucose overproduction is a major characteristic of type 2 diabetes. Because glucagon is a key regulator for glucose homeostasis, antagonizing the glucagon receptor (GCGR) is a possible therapeutic strategy for the treatment of diabetes mellitus. To study the effect of hepatic GCGR inhibition on the regulation of lipid metabolism, we generated siRNA-mediated GCGR knockdown (si-GCGR) in the db/db mouse. The hepatic knockdown of GCGR markedly reduced plasma glucose levels; however, total plasma cholesterol was increased. The detailed lipid analysis showed an increase in the LDL fraction, and no change in VLDL HDL fractions. Further studies showed that the increase in LDL was the result of over-expression of hepatic lipogenic genes and elevated de novo lipid synthesis. Inhibition of hepatic glucagon signaling via siRNA-mediated GCGR knockdown had an effect on both glucose and lipid metabolism in db/db mice.

Use of [13C18] oleic acid and mass isotopomer distribution analysis to study synthesis of plasma triglycerides in vivo: analytical and experimental considerations.

We have previously reported on a liquid chromatography-mass spectrometry method to determine the disposition of [(13)C18]-oleic acid following intravenous and oral administration in vivo. This approach has enabled us to study a variety of aspects of lipid metabolism including a quantitative assessment of triglyceride synthesis. Here we present a more rigorous evaluation of the constraints imposed upon the analytical method in order to generate accurate data using this stable-isotope tracer approach along with more detail on relevant analytical figures of merit including limits of quantitation, precision, and accuracy. The use of mass isotopomer distribution analysis (MIDA) to quantify plasma triglyceride synthesis is specifically highlighted, and a re-evaluation of the underlying mathematics has enabled us to present a simplified series of equations. The derivation of this MIDA model and the significance of all underlying assumptions are explored in detail, and examples are given of how it can successfully be applied to detect differences in plasma triglyceride synthesis in lean and high-fat diet fed mouse models. More work is necessary to evaluate the applicability of this approach to triglyceride stores with slower rates of turnover such as in adipose or muscle tissue; however, the present report provides investigators with the tools necessary to conduct such studies.

Command hallucinations and clinical characteristics of suicidality in patients with psychotic spectrum disorders.

BACKGROUND: Suicide is a leading cause of death among patients with psychotic illnesses. Several researchers have suggested that specific illness symptoms may better predict suicide risk. An ability to identify high-risk patients would aid clinicians in instituting risk-reduction measures to decrease suicidal behavior in this population. METHODS: We examined the association between psychotic symptoms and suicidal behavior among 148 inpatients with psychosis using the Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV), the Scale for the Assessment of Positive Symptoms, and the Positive and Negative Syndrome Scale. Measures of suicidality were obtained from risk assessment clinical data routinely collected during intake. RESULTS: For individuals with a DSM-IV diagnosed psychotic spectrum disorder, 40% (n=57) endorsed suicidal ideation on admission and 23% (n=33) endorsed a recent suicide attempt. The presence of command auditory hallucinations was significantly associated with active suicidal ideation across diagnostic categories. Similarly, a greater percentage of patients endorsed a recent suicide attempt in the presence of command hallucinations. These correlations with CAH are noteworthy, as we found no significant difference in the prevalence of SI among those with and without general auditory hallucinations (42.5% and 37.7%). CONCLUSIONS: The presence of command auditory hallucinations, in particular, but not auditory hallucinations, in general, was associated with suicidal behavior. These results indicate that command auditory hallucinations may identify or even place psychotic individuals at greater risk for acute, suicidal behavior--these symptoms should be the target of immediate and aggressive characterization and treatment.

Omega-3 fatty acid treatment, with or without cytidine, fails to show therapeutic properties in bipolar disorder: a double-blind, randomized add-on clinical trial.

OBJECTIVE: This study aimed to test the effects of omega-3 fatty acids (O3FA), given as fish oil capsules, with and without oral cytidine (CYT), a pyrimidine with reported preclinical and clinical antidepressant-like effects, in patients with bipolar disorder (BD). METHODS: A total of 45 outpatients with diagnosed BD (type I) according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition - Text Revision, were recruited for this 4-month, randomized, double-blind, placebo-controlled, add-on study. Treatment groups were (1) oral CYT + O3FA, (2) placebo + O3FA, and (3) placebo + placebo control. O3FA was given 2 g twice a day and CYT was administered as 1 g twice a day. RESULTS: There was no statistically significant difference among the groups in the primary outcome: study retention. Clinical measures improved in all treatment groups, and there were no significant differences between groups, including change in probability of symptoms of depression or mania, change in positive ratings of depression or mania, or change in Global Assessment of Functioning scores. Neither CYT + O3FA nor placebo + O3FA treatment was superior to placebo treatment. Rather, there was a statistically nonsignificant trend for both groups treated with O3FA to do worse than the placebo group. CONCLUSIONS: Despite preclinical studies suggesting that the effect of O3FA might be augmented with pyrimidines, add-on CYT did not substantially improve mood symptoms in BD. In addition, although a power analysis indicated that the sample size would be adequate to see beneficial effects similar to those previously reported, O3FA treatment by itself was not superior to placebo for BD.

A rapid method for cross-species quantitation of apolipoproteins A1, B48 and B100 in plasma by ultra-performance liquid chromatography/tandem mass spectrometry.

Apolipoprotein B100 (apoB100) and apolipoprotein A1 (apoA1) are the primary protein components of low density lipoprotein (LDL) and high density lipoprotein (HDL) particles, respectively, and plasma levels of these proteins are associated with risks of cardiovascular disease. Existing apoB100 quantitation methods for animal models have been limited to affinity capture techniques such as enzyme-linked immunosorbent assay (ELISA) and Western blot which require specialized reagents for each species and in many cases are not readily available. Here we demonstrate a single translatable ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) assay that is fast and robust and can be used to measure apolipoprotein concentrations in plasma for six species. When possible, peptide sequences that are conserved across species were identified for this assay. The sample preparation is limited and can be carried out in 96-well microtiter plates and thus allows for multiplexed preparation of samples for analysis of large numbers of samples in a short time frame when combined with UPLC/MS/MS. Separation and quantitation of the tryptic peptides is carried out at 700 µL/min using a 1.7 µm core shell C18 column (2.1 × 50 mm). The chromatography is designed for the analysis of over 100 samples per day, and the UPLC run is less than 10 min. This assay is capable of supporting cardiovascular research by providing a single assay to measure critical biomarkers across multiple species without the need for antibodies, and does so in a high-throughput manner.

Discovery of benzodihydroisofurans as novel, potent, bioavailable and brain-penetrant prolylcarboxypeptidase inhibitors.

A series of benzodihydroisofurans were discovered as novel, potent, bioavailable and brain-penetrant prolylcarboxypeptidase (PrCP) inhibitors. The structure-activity relationship (SAR) is focused on improving PrCP activity and metabolic stability, and reducing plasma protein binding. In the established diet-induced obese (eDIO) mouse model, compound ent-3a displayed target engagement both in plasma and in brain. However, this compound failed to induce significant body weight loss in eDIO mice in a five-day study.

Invention of MK-8262, a Cholesteryl Ester Transfer Protein (CETP) Inhibitor Backup to Anacetrapib with Best-in-Class Properties.

Cholesteryl ester transfer protein (CETP) represents one of the key regulators of the homeostasis of lipid particles, including high-density lipoprotein (HDL) and low-density lipoprotein (LDL) particles. Epidemiological evidence correlates increased HDL and decreased LDL to coronary heart disease (CHD) risk reduction. This relationship is consistent with a clinical outcomes trial of a CETP inhibitor (anacetrapib) combined with standard of care (statin), which led to a 9% additional risk reduction compared to standard of care alone. We discuss here the discovery of MK-8262, a CETP inhibitor with the potential for being the best-in-class molecule. Novel in vitro and in vivo paradigms were integrated to drug discovery to guide optimization informed by a critical understanding of key clinical adverse effect profiles. We present preclinical and clinical evidence of MK-8262 safety and efficacy by means of HDL increase and LDL reduction as biomarkers for reduced CHD risk.

Pyrido[2,3-b]pyrazines, discovery of TRPV1 antagonists with reduced potential for the formation of reactive metabolites.

The transient receptor potential cation channel, subfamily V, member 1 (TRPV1) is a non-selective cation channel that can be activated by a wide range of noxious stimuli, including capsaicin, acid, and heat. Blockade of TRPV1 activation by selective antagonists is under investigation in an attempt to identify novel agents for pain treatment. During pre-clinical development, the 1,8-naphthyridine 2 demonstrated unacceptably high levels of irreversible covalent binding. Replacement of the 1,8-naphthyridine core by a pyrido[2,3-b]pyrazine led to the discovery of compound 26 which was shown to have significantly lower potential for the formation of reactive metabolites. Compound 26 was characterized as an orally bioavailable TRPV1 antagonist with moderate brain penetration. In vivo, 26 significantly attenuated carrageenan-induced thermal hyperalgesia (CITH) and dose-dependently reduced complete Freund's adjuvant (CFA)-induced chronic inflammatory pain after oral administration.

Series of Novel and Highly Potent Cyclic Peptide PCSK9 Inhibitors Derived from an mRNA Display Screen and Optimized via Structure-Based Design.

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) is a key regulator of plasma LDL-cholesterol (LDL-C) and a clinically validated target for the treatment of hypercholesterolemia and coronary artery disease. In this paper, we describe a series of novel cyclic peptides derived from an mRNA display screen which inhibit the protein-protein interaction between PCSK9 and LDLR. Using a structure-based drug design approach, we were able to modify our original screening lead 2 to optimize the potency and metabolic stability and minimize the molecular weight to provide novel bicyclic next-generation PCSK9 inhibitor peptides such as 78. These next-generation peptides serve as a critical foundation for continued exploration of potential oral, once-a-day PCSK9 therapeutics for the treatment of cardiovascular disease.

AMP-activated protein kinase and nitric oxide regulate the glucose sensitivity of ventromedial hypothalamic glucose-inhibited neurons.

The mechanisms by which glucose regulates the activity of glucose-inhibited (GI) neurons in the ventromedial hypothalamus (VMH) are largely unknown. We have previously shown that AMP-activated protein kinase (AMPK) increases nitric oxide (NO) production in VMH GI neurons. We hypothesized that AMPK-mediated NO signaling is required for depolarization of VMH GI neurons in response to decreased glucose. In support of our hypothesis, inhibition of neuronal nitric oxide synthase (nNOS) or the NO receptor soluble guanylyl cyclase (sGC) blocked depolarization of GI neurons to decreased glucose from 2.5 to 0.7 mM or to AMPK activation. Conversely, activation of sGC or the cell-permeable analog of cGMP, 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), enhanced the response of GI neurons to decreased glucose, suggesting that stimulation of NO-sGC-cGMP signaling by AMPK is required for glucose sensing in GI neurons. Interestingly, the AMPK inhibitor compound C completely blocked the effect of sGC activation or 8-Br-cGMP, and 8-Br-cGMP increased VMH AMPKalpha2 phosphorylation. These data suggest that NO, in turn, amplifies AMPK activation in GI neurons. Finally, inhibition of the cystic fibrosis transmembrane regulator (CFTR) Cl(-) conductance blocked depolarization of GI neurons to decreased glucose or AMPK activation, whereas decreased glucose, AMPK activation, and 8-Br-cGMP increased VMH CFTR phosphorylation. We conclude that decreased glucose triggers the following sequence of events leading to depolarization in VMH GI neurons: AMPK activation, nNOS phosphorylation, NO production, and stimulation of sGC-cGMP signaling, which amplifies AMPK activation and leads to closure of the CFTR.

Positive precepting: preparation can reduce the stress.

Preceptors are critical participants in the education of student nurses. They establish role expectations and evaluate performance based on faculty input and student responses. When guidance is provided to establish realistic expectations, positive clinical experiences that proactively deal with potentially significant issues can result.

Conceptualization, Assessment, and Treatment of Traumatic Stress in First Responders: A Review of Critical Issues.

First responders are regularly confronted with exposure to traumatic events, including potentially life-threatening situations as well as the grave injuries and deaths of colleagues and civilians. Evidence indicates that the prevalence of posttraumatic stress disorder (PTSD) is substantially higher among first responders than the general population. This article provides information about the outpatient trauma services at McLean Hospital's LEADER (Law Enforcement, Active Duty, Emergency Responder) program to assist clinicians who encounter these first responders in their practices or who are specifically interested in working with this patient population. We begin by synthesizing the literature on the prevalence of PTSD in first responders following work-related exposure to traumatic stress, and by addressing the occupation-specific risk factors and the third-variable risk factors that may contribute to potentiated risk. We then discuss assessment strategies and treatment options used in our program, which is tailored for individuals who are dealing with mental health issues stemming from occupation-specific traumatic-stress exposure. We also address the unique challenges of treating traumatized first responders with more complex issues such as traumatic stress exposure across the lifespan and safety issues, including acute suicidality. We conclude by discussing notable gaps in the literature, including the need to investigate why and how women present with different PTSD symptoms than men and how these differences need to be taken into account in determining appropriate treatment for women.

Discovery and Pharmacology of a Novel Somatostatin Subtype 5 (SSTR5) Antagonist: Synergy with DPP-4 Inhibition.

We report new SSTR5 antagonists with enhanced potency, subtype selectivity, and minimal off-target activities as compared to previously reported compounds. Starting from the reported SSTR5 antagonist 1, we systematically surveyed changes in the central core and head piece while maintaining the diphenyl tail group constant. From this study the azaspirodecanone 10 emerged as a new highly potent and selective SSTR5 antagonist. Compound 10 lowered glucose excursion by 94% in an oral glucose tolerance test (OGTT) in mice following a 3 mg/kg oral dose. The compound increased both total and active circulating incretin hormone GLP-1 levels in mice at a dose of 10 mg/kg. A synergistic effect was also demonstrated when compound 10 was coadministered with a DPP-4 inhibitor, substantially increasing circulating active GLP-1[7-36] amide and insulin in response to a glucose challenge.

Optimization of Preclinical Metabolism for Somatostatin Receptor Subtype 5-Selective Antagonists.

A series of structurally diverse azaspirodecanone and spirooxazolidinone analogues were designed and synthesized as potent and selective somatostatin receptor subtype 5 (SSTR5) antagonists. Four optimized compounds each representing a subseries showed improvement in their metabolic stability and pharmacokinetic profiles compared to those of the original lead compound 1 while maintaining pharmacodynamic efficacy. The optimized cyclopropyl analogue 13 demonstrated efficacy in a mouse oral glucose tolerance test and an improved metabolic profile and pharmacokinetic properties in rhesus monkey studies. In this Communication, we discuss the relationship among structure, in vitro and in vivo activity, metabolic stability, and ultimately the potential of these compounds as therapeutic agents for the treatment of type 2 diabetes. Furthermore, we show how the use of focused libraries significantly expanded the structural class and provided new directions for structure-activity relationship optimization.

DGAT2 Inhibition Alters Aspects of Triglyceride Metabolism in Rodents but Not in Non-human Primates.

Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step in triglyceride (TG) synthesis and has been shown to play a role in regulating hepatic very-low-density lipoprotein (VLDL) production in rodents. To explore the potential of DGAT2 as a therapeutic target for the treatment of dyslipidemia, we tested the effects of small-molecule inhibitors and gene silencing both in vitro and in vivo. Consistent with prior reports, chronic inhibition of DGAT2 in a murine model of obesity led to correction of multiple lipid parameters. In contrast, experiments in primary human, rhesus, and cynomolgus hepatocytes demonstrated that selective inhibition of DGAT2 has only a modest effect. Acute and chronic inhibition of DGAT2 in rhesus primates recapitulated the in vitro data yielding no significant effects on production of plasma TG or VLDL apolipoprotein B. These results call into question whether selective inhibition of DGAT2 is sufficient for remediation of dyslipidemia.