In Vitro Pharmacological Characterization and In Vivo Validation of LSN3172176 a Novel M1 Selective Muscarinic Receptor Agonist Tracer Molecule for Positron Emission Tomography.

In the search for improved symptomatic treatment options for neurodegenerative and neuropsychiatric diseases, muscarinic acetylcholine M1 receptors (M1 mAChRs) have received significant attention. Drug development efforts have identified a number of novel ligands, some of which have advanced to the clinic. However, a significant issue for progressing these therapeutics is the lack of robust, translatable, and validated biomarkers.

The Discovery, Preclinical, and Early Clinical Development of Potent and Selective GPR40 Agonists for the Treatment of Type 2 Diabetes Mellitus (LY2881835, LY2922083, and LY2922470).

The G protein-coupled receptor 40 (GPR40) also known as free fatty acid receptor 1 (FFAR1) is highly expressed in pancreatic, islet β-cells and responds to endogenous fatty acids, resulting in amplification of insulin secretion only in the presence of elevated glucose levels. Hypothesis driven structural modifications to endogenous FFAs, focused on breaking planarity and reducing lipophilicity, led to the identification of spiropiperidine and tetrahydroquinoline acid derivatives as GPR40 agonists with unique pharmacology, selectivity, and pharmacokinetic properties. Compounds 1 (LY2881835), 2 (LY2922083), and 3 (LY2922470) demonstrated potent, efficacious, and durable dose-dependent reductions in glucose levels along with significant increases in insulin and GLP-1 secretion during preclinical testing.

Design, synthesis, and evaluation of hydroxamic acid-based molecular probes for in vivo imaging of histone deacetylase (HDAC) in brain.

Hydroxamic acid-based histone deacetylase inhibitors (HDACis) are a class of molecules with therapeutic potential currently reflected in the use of suberoylanilide hydroxamic acid (SAHA; Vorinostat) to treat cutaneous T-cell lymphomas (CTCL). HDACis may have utility beyond cancer therapy, as preclinical studies have ascribed HDAC inhibition as beneficial in areas such as heart disease, diabetes, depression, neurodegeneration, and other disorders of the central nervous system (CNS). However, little is known about the pharmacokinetics (PK) of hydroxamates, particularly with respect to CNS-penetration, distribution, and retention.

Akt activation, but not extracellular signal-regulated kinase activation, is required for SDF-1alpha/CXCR4-mediated migration of epitheloid carcinoma cells.

Emerging evidence shows that the stromal cell-derived factor 1 (SDF-1)/CXCR4 interaction regulates multiple cell signaling pathways and a variety of cellular functions such as cell migration, proliferation, and survival. There is little information linking the cellular functions and individual signaling pathways mediated by SDF-1 and CXCR4 in human cancer cells. In this study, we have shown that human epitheloid carcinoma HeLa cells express functional CXCR4 by reverse transcription-PCR, immunofluorescent staining, and 125I-SDF-1alpha ligand binding analyses.

Substituted 3-imidazo[1,2-a]pyridin-3-yl- 4-(1,2,3,4-tetrahydro-[1,4]diazepino-[6,7,1-hi]indol-7-yl)pyrrole-2,5-diones as highly selective and potent inhibitors of glycogen synthase kinase-3.

Glycogen synthase kinase-3 (GSK3) is involved in signaling from the insulin receptor. Inhibitors of GSK3 are expected to effect lowering of plasma glucose similar to insulin, making GSK3 an attractive target for the treatment of type 2 diabetes. Herein we report the discovery of a series of potent and selective GSK3 inhibitors.

Localization of the ROMK protein on apical membranes of rat kidney nephron segments.

The ATP-sensitive, inwardly rectifying K+ channel, ROMK, has been suggested to be the low-conductance ATP-sensitive K+ channel identified in apical membranes of mammalian renal thick ascending limb (TAL) and cortical collecting duct (CCD). Mutations in the human ROMK gene (KIR 1.2) have been identified in kindreds with neonatal Bartter's syndrome.

Endogenous IL-1 receptor antagonist protein (IRAP) regulates schistosome egg granuloma formation and the regional lymphoid response.

This study examined the role of IL-1 receptor antagonist protein (IRAP) in the regulation of the immune/inflammatory response to schistosome eggs. Initial screening for IRAP-specific mRNA transcripts by reverse transcriptase and primer-directed polymerase chain reactions suggested significant endogenous IRAP synthesis in lungs with Schistosoma mansoni egg-induced hypersensitivity granulomas but not in normal lungs or lungs with non-immune bead granulomas. Direct detection using mIRAP-specific antibodies corroborated the RNA studies.

Cloning, heterologous expression and characterization of murine interleukin 1 receptor antagonist protein.

A cDNA coding for the human interleukin 1 receptor antagonist protein (IL 1Ra) was used to clone the corresponding murine cDNA. The nucleotide sequence of the open reading frame coding for the processed form of mIL 1Ra predicted a 152-residue protein that was 77% identical to human IL 1Ra. The cellular and tissue distribution of murine IL 1Ra (mIL 1Ra) transcripts showed high levels in macrophages and skin while lower levels were detected in tissues that contain significant numbers of resident macrophages.

Purification and characterization of interleukin 1 receptor level antagonist proteins from THP-1 cells.

Culture medium conditioned by phorbol 12-myristate 13-acetate-differentiated THP-1 cells contained interleukin 1 (IL-1) antagonist activity as measured by inhibition of both IL-1 beta binding to receptors on YT cells and inhibition of IL-1/phytohemagglutinin-stimulated IL-2 synthesis by LBRM-33-1A5 T cells. Based on their ability to compete for 125I-IL-1 beta binding to receptors on YT cells, four distinct antagonist proteins were purified from THP-1 cell conditioned medium using a combination of ion-exchange, hydrophobic interaction, and size exclusion chromatographies. The four proteins had different isoelectric points with molecular masses in the range 22-26 kDa and had similar specific activities for inhibition of IL-1 beta binding to cell surface receptors (Ki values 0.

Binding and biological effects of tumor necrosis factor alpha on cultured human neonatal foreskin keratinocytes.

Tumor necrosis factor alpha (TNF alpha) localizes to the epidermis when injected in vivo, but its role in the skin has heretofore not been evaluated. As a first approach to assessing the role of TNF alpha in the skin, we evaluated the binding and biological effects of TNF alpha on human neonatal foreskin keratinocytes maintained in culture. We found that TNF alpha at 0.

Purification and characterization of early pregnancy factor from human pregnancy sera.

Early pregnancy factor (EPF) is a pregnancy-associated protein detected in the maternal serum by using the rosette inhibition assay and by evaluating the suppression of adoptive transfer of contact sensitivity. Because of its inhibitory effect on the functional reactivity of immunocompetent cells, EPF is thought to be involved in immunoregulation of the maternal immune system during early pregnancy. EPF was purified six million-fold from the serum of pregnant women between 5 and 12 weeks of gestation.

Amplified expression of the HER2/ERBB2 oncogene induces resistance to tumor necrosis factor alpha in NIH 3T3 cells.

Functional characterization of oncogene products that induce cellular transformation has progressed rapidly in recent years. However, less is known about the mechanism(s) by which the transformed cells may escape destruction by host immune defenses and form tumors. A recently described oncogene that has an important association with aggressive human breast carcinoma is "HER2," for human epidermal growth factor receptor 2.

The effects of human recombinant tumor necrosis factor on glioma-derived cell lines: cellular proliferation, cytotoxicity, morphological and radioreceptor studies.

To determine whether tumor necrosis factor is of potential value for the treatment of human malignant gliomas, we studied the effects of human recombinant tumor necrosis factor (rTNF-alpha) on the morphology, incorporation of tritiated thymidine, and proliferation of 5 established cell lines derived from human malignant gliomas and 3 normal human brain cell cultures. A radioreceptor analysis for rTNF-alpha was performed on all cell lines and cultures. Two of the 5 human glioma cell lines (SF-188 and U 343 MG-A) demonstrated a marked decrease (60% or less of untreated controls) in the uptake of tritiated thymidine when treated with rTNF-alpha at a concentration of 40 U/ml; rTNF-alpha at 100 U/ml had antiproliferative and cytotoxic effects on both cell lines.

Characterization of the in vitro and in vivo species preference of human and murine tumor necrosis factor-alpha.

The species preference of human and murine tumor necrosis factor-alpha (TNF-alpha) was evaluated in human and murine systems for cytotoxic/cytostatic effects and receptor binding in vitro and murine systems for toxicity and antitumor activity in vivo. The in vitro cytotoxic/cytostatic effects of both species TNF-alpha on human and murine cell lines as well as the receptor binding studies using 125I-labeled recombinant human TNF-alpha demonstrated homologous species preferences. Species preference of TNF-alpha was also apparent in toxicity studies with BALB/c nu/nu and CB6F1 mice, and antitumor responses of CB6F1 mice to s.

Effect of phorbol esters on down-regulation and redistribution of cell surface receptors for tumor necrosis factor-alpha.

The effects of various phorbol esters on the interaction of human cells with recombinant human tumor necrosis factor-alpha (rTNF-alpha) was investigated. Preexposure of several different types of cells with only biologically active tumor promoter, i.e.

Modulation of the growth of transformed cells by human tumor necrosis factor-alpha and interferon-gamma.

Recombinant human tumor necrosis factor-alpha (rHuTNF-alpha) inhibited growth of the cervical carcinoma cell line, ME-180neo, at doses greater than 50 units/ml, but stimulated the growth of these cells at low doses (0.1-10 units/ml). ME-180neo variants selected for resistance to the cytotoxic effects of rHuTNF-alpha retained the ability to be growth stimulated at all concentrations tested.

Tumor necrosis factors alpha and beta differ in their capacities to generate interleukin 1 release from human endothelial cells.

The capacity of the tumor necrosis factors, TNF-alpha and TNF-beta, products of activated macrophages and lymphocytes, respectively, to stimulate interleukin 1 (IL-1) release from endothelial cells derived from human umbilical veins was examined in vitro. Recombinant TNF-alpha caused IL-1 release by 4 hr with maximal levels of 17 U/ml by 24 hr; half-maximal stimulation occurred at approximately 80 pM. In contrast, recombinant TNF-beta was a relatively poor stimulus for IL-1 release.

Induction of receptors for tumor necrosis factor-alpha by interferons is not a major mechanism for their synergistic cytotoxic response.

We have investigated the effects of various interferons on the receptors for recombinant tumor necrosis factor-alpha (rTNF-alpha) and also their effects on rTNF-alpha-mediated cytotoxicity on human cervical carcinoma cell line ME-180. Preincubation of cells with interferon (IFN)-gamma causes a concentration- and time-dependent increase in rTNF-alpha receptor number without any change in the affinity constant of the receptors. The increase in receptor number is caused only by IFN-gamma and not by IFN-alpha or IFN-beta.

Receptor binding and activation of polymorphonuclear neutrophils by tumor necrosis factor-alpha.

The interaction of highly purified recombinant human tumor necrosis factor-alpha (rTNF-alpha) with human polymorphonuclear neutrophils (PMNs) was investigated. Binding of 125I-rTNF-alpha to PMN reached maximum levels in 30 min at 37 degrees C and in 2 h at 4 degrees C. Scatchard analysis of competitive binding data indicated approximately 6000 receptor sites per cell and a Kd of 1.

Effects of growth factors on the antiproliferative activity of tumor necrosis factors.

Tumor necrosis factors (TNFs) are a class of cytokines secreted by activated effector cells involved in host defense against tumor progression. Epidermal growth factor (EGF) and recombinant human transforming growth factor-alpha (rHuTGF-alpha) were shown to interfere with the in vitro antiproliferative effects of recombinant human tumor necrosis factor-alpha (rHuTNF-alpha) and -beta on a human cervical carcinoma cell line, ME-180. The inhibitory effect could be observed at EGF or rHuTGF-alpha concentrations of 100 pg/ml, and was maximal between 1 and 10 ng/ml.

Interferons and tumor necrosis factors have similar catabolic effects on 3T3 L1 cells.

The effect of a variety of cytokines on lipid metabolism in 3T3 L1 mouse fibroblasts and adipocytes was studied. Uptake of [3H]acetate by adipocytes and heparin-releasable lipoprotein lipase activity was inhibited after treatments of the cells with picomolar concentrations of recombinant human tumor necrosis factor alpha (rHuTNF-alpha), human tumor necrosis factor beta (rHuTNF-beta, also called lymphotoxin), murine interferon-gamma (rMuIFN-gamma), and a human hybrid interferon-alpha [rHuIFN-alpha 2/alpha 1 (Bgl II)]. Recombinant human interferon-gamma (rHuIFN-gamma), natural human colony-stimulating factor (HuCSF), and human interleukin 2 (HuIL-2) had no effect.

Modulation of receptors and cytotoxic response of tumor necrosis factor-alpha by various lectins.

The effects of various lectins on the interaction of the human cervical carcinoma cell line ME-180 with recombinant human tumor necrosis factor-alpha (rTNF-alpha) was investigated. rTNF-alpha is known to have cytotoxic effects on this tumor cell line and has been reported to interact with these cells through a single class of specific high affinity receptors (Kd = 0.45 nM; approximately 1790 binding sites/cell).

Hormone dependency of transformation of rat liver glucocorticoid receptor in vitro: effects of heat, salt, and nucleotides.

A majority of the untransformed glucocorticoid-receptor complexes (GRc) from rat liver cytosol sedimented in the 9S region in 5-20% sucrose gradients containing 0.15 M KCl and 20 mM Na2MoO4. Incubation of the cytosol at 23 degrees C, or at 0 degree C with 10 mM ATP or 0.

Characterization of receptors for human tumour necrosis factor and their regulation by gamma-interferon.

Tumour necrosis factors, TNF-alpha and TNF-beta (previously called lymphotoxin), are the products of activated monocytes and lymphocytes, respectively, and both have recently been purified, sequenced and cloned by recombinant DNA methods, revealing 35% identity and 50% homology in the amino-acid sequence. Both proteins have been found to be specifically toxic to many tumour cells. Furthermore, it has been reported that various interferons are synergistic with TNF for anti-tumour effects in vitro, while activities attributed to the two proteins have also been shown to necrotize various tumours in vivo.

Interaction of chick oviduct progesterone receptor with immobilized aurintricarboxylic acid.

Aurintricarboxylic acid (ATA) was immobilized on Sepharose 4B via a carbodiimide coupling mechanism. A majority of the chick oviduct progesterone receptor was retained on the affinity resin and could be recovered upon washing the column with buffer containing free ligand or 3 M guanidine-HCl. The [3H]progesterone-receptor complex retained its integrity following the chromatography on ATA-Sepharose as judged by sedimentation analysis.