Dietary omega-3 fatty acids modulate the production of platelet-derived microvesicles in an in vivo inflammatory arthritis model.

PURPOSE: The aim of this study was to investigate the effects of different ω-3 polyunsaturated fatty acid (PUFA) enriched diets, including a novel renewable plant source of ω-3 fatty acids (Buglossoides arvensis), on the development and progression of rheumatoid arthritis (RA). METHODS: RA was induced in mice consuming experimental diets using the K/BxN model. The experimental diets consisted of either a western control diet (control), diets containing B. arvensis oil or fish oil. The effects of the diets on platelets, platelet microvesicles (PMVs), and inflammatory markers such as clinical index, ankle thickness and cytokine/chemokine release were measured. RESULTS: While ω-3 PUFA-enriched diets did not prevent the development of arthritis in the K/BxN model, a significant decrease in ankle swelling was observed compared to the control group. Platelets isolated from mice consuming either low content of B. arvensis oil or fish oil diets exhibited significantly decreased PMVs production compared to mice consuming the control diet. CONCLUSION: Our study provides insight into the contribution of ω-3 PUFA supplementation in modulating the pro-inflammatory phenotype of platelets in RA pathology. Furthermore, our study suggests that low concentrations of dietary B. arvensis oil may have similar anti-inflammatory potential seen with dietary fish oil supplementation.

A silent agonist of α7 nicotinic acetylcholine receptors modulates inflammation ex vivo and attenuates EAE.

Nicotinic acetylcholine receptors (nAChRs) are best known to function as ligand-gated ion channels in the nervous system. However, recent evidence suggests that nicotine modulates inflammation by desensitizing non-neuronal nAChRs, rather than by inducing channel opening. Silent agonists are molecules that selectively induce the desensitized state of nAChRs while producing little or no channel opening. A silent agonist of α7 nAChRs has recently been shown to reduce inflammation in an animal model of inflammatory pain. The objective of this study was to determine whether a silent agonist of α7 nAChRs can also effectively modulate inflammation and disease manifestation in an animal model of multiple sclerosis. We first evaluated the effects of various nAChR ligands and of an α7 nAChR-selective silent agonist, 1-ethyl-4-(3-(bromo)phenyl)piperazine (m-bromo PEP), on the modulation of mouse bone marrow-derived monocyte/macrophage (BMDM) numbers, phenotype and cytokine production. The non-competitive antagonist mecamylamine and the silent agonist m-bromo PEP reduced pro-inflammatory BMDM numbers by affecting their viability and proliferation. Both molecules also significantly reduced cytokine production by mouse BMDMs and significantly ameliorated disease in experimental autoimmune encephalomyelitis. Finally, m-bromo PEP also reduced chronic inflammatory pain in mice. Taken together, our results further support the hypothesis that nAChRs may modulate inflammation via receptor desensitization rather than channel opening. α7 nAChR-selective silent agonists may thus be a novel source of anti-inflammatory compounds that could be used for the treatment of inflammatory disorders.

Infiltration of CCR2+Ly6Chigh Proinflammatory Monocytes and Neutrophils into the Central Nervous System Is Modulated by Nicotinic Acetylcholine Receptors in a Model of Multiple Sclerosis.

Myeloid cells, including proinflammatory monocytes and neutrophils, have important roles in the pathology of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). These cells infiltrate the CNS in the early stages of disease development and contribute to the inflammatory response that is associated with symptom severity. It is thus crucial to identify and understand new mechanisms that can regulate the CNS infiltration of proinflammatory myeloid cells. Nicotinic acetylcholine receptors (nAChRs) have been increasingly studied for their immune-regulatory properties. In this study, we assessed the ability of nicotine, an nAChR ligand, to modulate proinflammatory myeloid cell numbers within the bone marrow, spleen, blood, and CNS of EAE mice. We found that nicotine significantly inhibits the infiltration of proinflammatory monocytes and neutrophils into the CNS at time points where these cells are known to play critical roles in disease pathology. In contrast, nicotine does not affect the expansion of other monocytes. We also show that nicotine exerts these effects by acting on α7 and α9 nAChR subtypes. Finally, mRNA transcript levels for CCL2 and CXCL2, chemokines involved in the chemotaxis of proinflammatory monocytes and neutrophils, respectively, are reduced in the brain of nicotine-treated EAE mice before the massive infiltration of these cells. Taken together, our data provide evidence that nAChRs can regulate proinflammatory cell infiltration into the CNS, which could be of significant value for the treatment of neuroinflammatory disorders.

Vaginocervical stimulation attenuates the sensitization of appetitive sexual behaviors by estradiol benzoate in the ovariectomized rat.

The acute administration of estradiol benzoate (EB) to the ovariectomized (OVX) rat induces low levels of lordosis while sexually appetitive behaviors (e.g., hops, darts, solicitations) are absent, yet the repeated administration of EB results in a behavioral sensitization in which lordosis is potentiated and sexually appetitive behaviors are induced. We have shown that repeated copulation attenuates the sensitization of appetitive sexual behaviors. Here, we assessed which component of male stimulation during copulation is involved in the attenuation. On 8 occasions, sexually experienced OVX Long-Evans rats were treated with 10µgEB and 48h later assigned to one of six groups that differed in their experience on intermediates tests (2-7). One was given repeated access to a male (EB/Male), and another was placed in the copulation chamber alone (EB/Alone) on intermediate tests. Three groups were given one of three somatosensory stimuli by the experimenter: manual flank stimulation (FLS), clitoral stimulation (CLS), or vaginocervical stimulation (VCS). Finally, the control group was left undisturbed in the animal care facility (ACF). Sexual behaviors were measured on Tests 1 and 8. VCS received from the experimenter (VCS) or from the male during copulation (EB/Male) attenuated the magnitude of the sensitization of appetitive sexual behaviors compared with those that were not brought to the testing rooms (ACF), and the effect was most pronounced on sexual solicitations. These results suggest that VCS received during penile intromission inhibits the sensitization of sexually appetitive behaviors by repeated administration of EB. As such, repeated administration of EB may oppose those mechanisms that induce estrous termination, perhaps by sensitizing inhibitory processes within the ventromedial hypothalamus that typically prevent the display of sexual behaviors (i.e., by facilitating disinhibition).

The discovery and synthesis of potent zwitterionic inhibitors of renin.

The incorporation of a carboxylic acid within in a series of 3-amido-4-aryl substituted piperidines (represented by general structure 32) led to the discovery of potent, zwitterionic, renin inhibitors with improved off-target profiles (CYP3A4 time-dependent inhibition and hERG affinity) relative to analogous non-zwitterionic inhibitors of the past (i.e., 3). Strategies to address the oral absorption of these zwitterions are also discussed within.

Trisubstituted ureas as potent and selective mPGES-1 inhibitors.

A novel series of trisubstituted ureas has been identified as potent and selective mPGES-1 inhibitors. These compounds are selective over other prostanoid enzymes such as PGF synthase and TX synthase. This series of inhibitors was developed by lead optimization of a hit from an internal HTS campaign. Lead compound 42 is potent in A549 cell assay (IC(50) of 0.34 µM) and in human whole blood assay (IC(50) of 2.1 µM). An efficient and versatile one-pot strategy for the formation of ureas, involving a reductive amination, was developed to generate these inhibitors.

Evaluating the mitochondrial activity and inflammatory state of dimethyl sulfoxide differentiated PLB-985 cells.

Neutrophils play a key role in the innate immunity with their ability to generate and release inflammatory mediators that promote the inflammatory response and consequently restore the hemostasis. As active participants in several steps of the normal inflammatory response, neutrophils are also involved in chronic inflammatory diseases such as asthma, atherosclerosis, and arthritis. Given their dual role in the modulation of inflammation, regulating the inflammatory response of neutrophils has been suggested as an important therapeutic approach by numerous researchers. The neutrophils have a relatively short lifespan, which can be problematic for some in vitro experiments. To address this issue, researchers have used the human monomyelocyte cell line PLB-985 as an in vitro model for exploratory experiments addressing neutrophil-related physiological functions. PLB-985 cells can be differentiated into a neutrophil-like phenotype upon exposure to several agonists, including dimethyl sulfoxide (DMSO). Whether this differentiation of PLB-985 affects important features related to the neutrophil's normal functions (i.e., mitochondrial activity, eicosanoid production) remains elusive, and characterizing these changes will be the focal point of this study. Our results indicate that the differentiation affected the proliferation of PLB-985 cells, without inducing apoptosis. A significant decrease in mitochondrial respiration was observed in differentiated PLB-985 cells. However, the overall mitochondria content was not affected. Immunoblotting with mitochondrial antibodies revealed a strong modulation of the succinate dehydrogenase A, superoxide dismutase 2, ubiquinol-cytochrome c reductase core protein 2 and ATP synthase subunit α in differentiated PLB-985 cells. Finally, eicosanoids (leukotriene B4, 12-hydroxyheptadecatrienoic and 15-hydroxyeicosatetraenoic acids) production was significantly increased in differentiated cells. In summary, our data demonstrate that the differentiation process of PLB-985 cells does not impact their viability despite a reduced respiratory state of the cells. This process is also accompanied by modulation of the inflammatory state of the cell. Of importance, our data suggest that PLB-985 cells could be suitable in vitro candidates to study mitochondrial-related dysfunctions in inflammatory diseases.

Identification of a new biaryl scaffold generating potent renin inhibitors.

The discovery and SAR of a series of potent renin inhibitors possessing a novel biaryl scaffold are described herein. Molecular modeling revealed that the cyclopropylamide spacer present in 1 can be replaced by a simple, substituted aromatic ring such as a toluene in 2. The resulting compounds exhibit subnanomolar renin IC(50) and good oral bioavailability in rats.

Pentastarch 10% (250 kDa/0.45) is an independent risk factor of acute kidney injury following cardiac surgery.

UNLABELLED: OBJECTIVE, DESIGN AND PATIENTS: The risk of acute kidney injury (AKI) associated with hydroxyethyl starch may be limited to higher molecular weight agents. We retrospectively evaluated the risk of AKI using pentastarch 10% (250 kDa, 0.45) in a random cohort of 563 patients operated for a cardiac surgery at a university hospital. MEASURES: We assessed previously identified preoperative, perioperative, and postoperative risk factors, and the volume of pentastarch given until the end of the first postoperative day. We defined AKI by a 50% rise in serum creatinine within 4 days after surgery. Different propensity adjustment methods were used to further assess the selection bias. RESULTS: Fifty-four (10%) patients developed AKI. Risk factors of AKI were age, female gender, preoperative creatinine clearance, hypertension, diuretic use, left ventricular ejection fraction, valvular surgery, duration of extracorporeal circulation, duration and dose of postoperative vasopressor support, and the number of red blood cells and fresh frozen plasma transfusions. Patients with AKI received 16 +/- 9 mL/kg of pentastarch as opposed to 10 +/- 7 mL/kg in controls (p < 0.001). Pentastarch remained independently predictive of AKI, with an adjusted odds ratio per mL/kg of 1.08 (95% confidence interval 1.04-1.12, p = 0.001). This risk was dose-dependent, and the optimal cutoff volume predicting AKI was 14 mL/kg. Different propensity adjustment methods were tested, and pentastarch as a risk factor of AKI was identified. CONCLUSIONS: This study identified a dose-dependent risk of AKI with pentastarch following cardiac surgery, given until the end of the first postoperative day.

Discovery of potent and selective DP1 receptor antagonists in the azaindole series.

Azaindole based structures were evaluated as DP1 receptor antagonists. This work has lead to the discovery of potent, selective and distinct DP1 receptor antagonists.

Intramitochondrial Src kinase links mitochondrial dysfunctions and aggressiveness of breast cancer cells.

High levels and activity of Src kinase are common among breast cancer subtypes, and several inhibitors of the kinase are currently tested in clinical trials. Alterations in mitochondrial activity is also observed among the different types of breast cancer. Src kinase is localized in several subcellular compartments, including mitochondria where it targets several proteins to modulate the activity of the organelle. Although the subcellular localization of other oncogenes modulates the potency of known treatments, nothing is known about the specific role of intra-mitochondrial Src (mtSrc) in breast cancer. The aim of this work was to determine whether mtSrc kinase has specific impact on breast cancer cells. We first observed that activity of mtSrc is higher in breast cancer cells of the triple negative subtype. Over-expression of Src specifically targeted to mitochondria reduced mtDNA levels, mitochondrial membrane potential and cellular respiration. These alterations of mitochondrial functions led to lower cellular viability, shorter cell cycle and increased invasive capacity. Proteomic analyses revealed that mtSrc targets the mitochondrial single-stranded DNA-binding protein, a regulator of mtDNA replication. Our findings suggest that mtSrc promotes aggressiveness of breast cancer cells via phosphorylation of mitochondrial single-stranded DNA-binding protein leading to reduced mtDNA levels and mitochondrial activity. This study highlights the importance of considering the subcellular localization of Src kinase in the development of potent therapy for breast cancer.

Identification of prostaglandin D2 receptor antagonists based on a tetrahydropyridoindole scaffold.

A new series of indole-based antagonists of the PGD(2) receptor subtype 1 (DP1 receptor) was identified and the progress of the structure-activity relationship study to the identification of potent and selective antagonists is presented. Selective DP1 antagonists with high potency and selectivity were prepared. Of particular interest is the DP1 antagonist 26 with a K(i) value of 1 nM for the DP1 receptor and an IC(50) value of 4.6 nM in a DP1 functional assay for the inhibition of the PGD(2) induced cAMP production in platelet rich plasma (PRP).

Prescription patterns of pharmacological agents for left ventricular systolic dysfunction among hemodialysis patients.

BACKGROUND: Recommendations for the management of left ventricular (LV) systolic dysfunction in the general population and patients with end-stage renal disease (ESRD) include the use of angiotensin-converting enzyme (ACE) inhibitors and beta-blockers. Limited data from the literature suggest that these pharmacological agents may be underused in patients with ESRD. The goal of this study is to describe the use of these medications and investigate barriers to their use in dialysis patients. METHODS: We prospectively studied 420 hemodialysis patients. Diagnosis of systolic dysfunction was based on echocardiogram results. Use of cardiac medication was recorded for all patients with systolic dysfunction, and a questionnaire was administered to nephrologists to determine the basis for decisions concerning ACE-inhibitors and beta-blockers use/nonuse. RESULTS: Forty-seven patients (11%) were found to have an LV ejection fraction of 40% or less. Of those, 72% were administered a beta-blocker and 36% were administered an ACE inhibitor. Only 12 patients (25.5%) were administered a combination of beta-blocker and ACE inhibitor. Reasons indicated by nephrologists for not prescribing these medications were "concern about adverse reactions (eg, hypotension, hyperkalemia)" in 88% of cases, "adequate control of symptoms with ultrafiltration" in 38%, "unproven benefit" in 25%, and "unfavorable risk-benefit ratio" in 17%. Medication costs and concern about patient compliance were not identified as significant concerns by physicians. CONCLUSION: Only 25.5% of patients with ESRD with LV systolic dysfunction receive appropriate treatment. Concern regarding adverse reactions was the most frequent reason indicated by nephrologists for not prescribing ACE inhibitors and beta-blockers.

Nicotinic Acetylcholine Receptors Modulate Bone Marrow-Derived Pro-Inflammatory Monocyte Production and Survival.

It is increasingly clear that nicotinic acetylcholine receptors (nAChRs) are involved in immune regulation, and that their activation can protect against inflammatory diseases. Previous data have shown that nicotine diminishes the numbers of peripheral monocytes and macrophages, especially those of the pro-inflammatory phenotype. The goal of the present study was to determine if nicotine modulates the production of bone marrow -derived monocytes/macrophages. In this study, we first found that murine bone marrow cells express multiple nAChR subunits, and that the α7 and α9 nAChRs most predominant subtypes found in immune cells and their precursors. Using primary cultures of murine bone marrow cells, we then determined the effect of nicotine on monocyte colony-stimulating factor and interferon gamma (IFNγ)-induced monocyte production. We found that nicotine lowered the overall number of monocytes, and more specifically, inhibited the IFNγ-induced increase in pro-inflammatory monocytes by reducing cell proliferation and viability. These data suggested that nicotine diminishes the ratio of pro-inflammatory versus anti-inflammatory monocyte produced in the bone marrow. We thus confirmed this hypothesis by measuring cytokine expression, where we found that nicotine inhibited the production of the pro-inflammatory cytokines TNFα, IL-1ß and IL-12, while stimulating the secretion of IL-10, an anti-inflammatory cytokine. Finally, nicotine also reduced the number of pro-inflammatory monocytes in the bone marrow of LPS-challenged mice. Overall, our data demonstrate that both α7 and α9 nAChRs are involved in the regulation of pro-inflammatory M1 monocyte numbers.

The structure of PTP-1B in complex with a peptide inhibitor reveals an alternative binding mode for bisphosphonates.

Inhibitors of PTP-1B could be therapeutically beneficial in the treatment of type 2 diabetes. Owing to the large number of phosphatases in the cell, inhibitors against PTP-1B must not only be potent but selective as well. N-Benzoyl-L-glutamyl-[4-phosphono(difluoromethyl)]-L-phenylalanine-[4-phosphono(difluoro-methyl)]-L-phenylalanineamide (BzN-EJJ-amide) is a low nanomolar inhibitor of PTP-1B that shows selectivity over several protein tyrosine phosphatases. To gain an insight into the basis of its potency and selectivity, we evaluated several analogues of the inhibitor and introduced amino acid substitutions into PTP-1B by site-directed mutagenesis. We also determined the crystal structure of PTP-1B in complex with BzN-EJJ-amide at 2.5 A resolution. Our results indicate that the high inhibitory potency is due to interactions of several of its chemical groups with specific protein residues. An interaction between BzN-EJJ-amide and Asp48 is of particular significance, as substitution of Asp48 to alanine resulted in a 100-fold loss in potency. The crystal structure also revealed an unexpected binding orientation for a bisphosphonate inhibitor on PTP-1B, where the second difluorophosphonomethyl phenylalanine (F(2)PMP) moiety is bound close to Arg47 rather than in the previously identified second aryl phosphate site demarked by Arg24 and Arg254. Our results suggest that potent and selective PTP-1B inhibitors may be designed by targeting the region containing Arg47 and Asp48.

Discovery of a potent and selective COX-2 inhibitor in the alkoxy lactone series with optimized metabolic profile.

The COX-2 inhibitor DFP [5,5-dimethyl-3-(2-propoxy)-4-methanesulfonylphenyl)-2(5H)-furanone] was found to have a long half-life in humans. Analogues have been characterized in order to optimize pharmacokinetics. This has lead to the discovery of 5(S)-(5-ethyl-5-methyl-3-(2-propoxy)-4-methanesulfonylphenyl)-2(5H)-furanone analogue 11 a potent and selective COX-2 inhibitor which is metabolized to a greater extent than DFP upon incubation with rat and human hepatocytes, suggesting a shorter half-life in humans.

The development of potent non-peptidic PTP-1B inhibitors.

The SAR from our peptide libraries was exploited to design a series of potent deoxybenzoin PTP-1B inhibitors. The introduction of an ortho bromo substituent next to the difluoromethylphosphonate warhead gave up to 20-fold increase in potency compared to the desbromo analogues. In addition, these compounds were orally bioavailable and active in the animal models of non-insulin dependent diabetes mellitus (NIDDM).