MK-8719, a Novel and Selective O-GlcNAcase Inhibitor That Reduces the Formation of Pathological Tau and Ameliorates Neurodegeneration in a Mouse Model of Tauopathy.

Deposition of hyperphosphorylated and aggregated tau protein in the central nervous system is characteristic of Alzheimer disease and other tauopathies. Tau is subject to O-linked N-acetylglucosamine (O-GlcNAc) modification, and O-GlcNAcylation of tau has been shown to influence tau phosphorylation and aggregation. Inhibition of O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc moieties, is a novel strategy to attenuate the formation of pathologic tau. Here we described the in vitro and in vivo pharmacological properties of a novel and selective OGA inhibitor, MK-8719. In vitro, this compound is a potent inhibitor of the human OGA enzyme with comparable activity against the corresponding enzymes from mouse, rat, and dog. In vivo, oral administration of MK-8719 elevates brain and peripheral blood mononuclear cell O-GlcNAc levels in a dose-dependent manner. In addition, positron emission tomography imaging studies demonstrate robust target engagement of MK-8719 in the brains of rats and rTg4510 mice. In the rTg4510 mouse model of human tauopathy, MK-8719 significantly increases brain O-GlcNAc levels and reduces pathologic tau. The reduction in tau pathology in rTg4510 mice is accompanied by attenuation of brain atrophy, including reduction of forebrain volume loss as revealed by volumetric magnetic resonance imaging analysis. These findings suggest that OGA inhibition may reduce tau pathology in tauopathies. However, since hundreds of O-GlcNAcylated proteins may be influenced by OGA inhibition, it will be critical to understand the physiologic and toxicological consequences of chronic O-GlcNAc elevation in vivo. SIGNIFICANCE STATEMENT: MK-8719 is a novel, selective, and potent O-linked N-acetylglucosamine (O-GlcNAc)-ase (OGA) inhibitor that inhibits OGA enzyme activity across multiple species with comparable in vitro potency. In vivo, MK-8719 elevates brain O-GlcNAc levels, reduces pathological tau, and ameliorates brain atrophy in the rTg4510 mouse model of tauopathy. These findings indicate that OGA inhibition may be a promising therapeutic strategy for the treatment of Alzheimer disease and other tauopathies.

A selective and potent CXCR3 antagonist SCH 546738 attenuates the development of autoimmune diseases and delays graft rejection.

BACKGROUND: The CXCR3 receptor and its three interferon-inducible ligands (CXCL9, CXCL10 and CXCL11) have been implicated as playing a central role in directing a Th1 inflammatory response. Recent studies strongly support that the CXCR3 receptor is a very attractive therapeutic target for treating autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis and psoriasis, and to prevent transplant rejection. We describe here the in vitro and in vivo pharmacological characterizations of a novel and potent small molecule CXCR3 antagonist, SCH 546738. RESULTS: In this study, we evaluated in vitro pharmacological properties of SCH 546738 by radioligand receptor binding and human activated T cell chemotaxis assays. In vivo efficacy of SCH 546738 was determined by mouse collagen-induced arthritis, rat and mouse experimental autoimmune encephalomyelitis, and rat cardiac transplantation models. We show that SCH 546738 binds to human CXCR3 with a high affinity of 0.4 nM. In addition, SCH 546738 displaces radiolabeled CXCL10 and CXCL11 from human CXCR3 with IC50 ranging from 0.8 to 2.2 nM in a non-competitive manner. SCH 546738 potently and specifically inhibits CXCR3-mediated chemotaxis in human activated T cells with IC90 about 10 nM. SCH 546738 attenuates the disease development in mouse collagen-induced arthritis model. SCH 546738 also significantly reduces disease severity in rat and mouse experimental autoimmune encephalomyelitis models. Furthermore, SCH 546738 alone achieves dose-dependent prolongation of rat cardiac allograft survival. Most significantly, SCH 546738 in combination with CsA supports permanent engraftment. CONCLUSIONS: SCH 546738 is a novel, potent and non-competitive small molecule CXCR3 antagonist. It is efficacious in multiple preclinical disease models. These results demonstrate that therapy with CXCR3 antagonists may serve as a new strategy for treatment of autoimmune diseases, including rheumatoid arthritis and multiple sclerosis, and to prevent transplant rejection.

Inflammatory Signals shift from adipose to liver during high fat feeding and influence the development of steatohepatitis in mice.

BACKGROUND: Obesity and inflammation are highly integrated processes in the pathogenesis of insulin resistance, diabetes, dyslipidemia, and non-alcoholic fatty liver disease. Molecular mechanisms underlying inflammatory events during high fat diet-induced obesity are poorly defined in mouse models of obesity. This work investigated gene activation signals integral to the temporal development of obesity. METHODS: Gene expression analysis in multiple organs from obese mice was done with Taqman Low Density Array (TLDA) using a panel of 92 genes representing cell markers, cytokines, chemokines, metabolic, and activation genes. Mice were monitored for systemic changes characteristic of the disease, including hyperinsulinemia, body weight, and liver enzymes. Liver steatosis and fibrosis as well as cellular infiltrates in liver and adipose tissues were analyzed by histology and immunohistochemistry. RESULTS: Obese C57BL/6 mice were fed with high fat and cholesterol diet (HFC) for 6, 16 and 26 weeks. Here we report that the mRNA levels of macrophage and inflammation associated genes were strongly upregulated at different time points in adipose tissues (6-16 weeks) and liver (16-26 weeks), after the start of HFC feeding. CD11b+ and CD11c+ macrophages highly infiltrated HFC liver at 16 and 26 weeks. We found clear evidence that signals for IL-1ß, IL1RN, TNF-α and TGFß-1 are present in both adipose and liver tissues and that these are linked to the development of inflammation and insulin resistance in the HFC-fed mice. CONCLUSIONS: Macrophage infiltration accompanied by severe inflammation and metabolic changes occurred in both adipose and liver tissues with a temporal shift in these signals depending upon the duration of HFC feeding. The evidences of gene expression profile, elevated serum alanine aminotransferase, and histological data support a progression towards nonalcoholic fatty liver disease and steatohepatitis in these HFC-fed mice within the time frame of 26 weeks.

Kinetic assessment and therapeutic modulation of metabolic and inflammatory profiles in mice on a high-fat and cholesterol diet.

The kinetics of metabolic and inflammatory parameters associated with obesity were evaluated in a murine diet-induced obesity (DIO) model using a diet high in fat and cholesterol. Cellular infiltration and mediator production were assessed and shown to be therapeutically modulated by the PPARgamma agonist rosiglitazone. C57BL/6 mice were maintained on a 45% fat/ 0.12% cholesterol (HF/CH) or Chow diet for 3, 6, 16, or 27 weeks. Flow cytometry was employed to monitor peripheral blood monocytes and adipose tissue macrophages (ATM). Gene expression and protein analysis methods were used to evaluate mediator production from total epididymal fat (EF), stromal vascular fraction (SVF), and sorted SVF cells. To investigate therapeutic intervention, mice were fed a HF/CH diet for 12 weeks and then a diet formulated with rosiglitazone (5 mg/kg) for an additional 6 weeks. A HF/CH diet correlated with obesity and a dramatic proinflammatory state. Therapeutic intervention with rosiglitazone attenuated the HF/CH induced inflammation. In addition, a novel population was found that expressed the highest levels of the pro-inflammatory mediators CCL2 and IL-6.

Expression of chemokine receptor CXCR3 by lymphocytes and plasmacytoid dendritic cells in human psoriatic lesions.

In psoriasis, leukocytes that infiltrate skin lesions have been shown to be involved in the pathogenesis of this disease. Previous investigations reporting the presence of CXCR3(+) T lymphocytes in psoriatic lesional skin have suggested a role of this receptor in the recruitment of T cells into the lesion. The purpose of this study was to quantify the mRNA levels of CXCR3 and to perform a systematic analysis of the cell populations that express CXCR3 in human lesional and non-lesional psoriatic biopsies. We showed by real-time reverse transcriptase-polymerase chain reaction that the mRNA levels of CXCR3 and its ligands, CXCL9-11, were significantly elevated in psoriatic lesions, as compared to non-lesional samples. Serial cryostat sections of psoriasis skin biopsies were evaluated by immunohistochemistry. The number of CXCR3(+) cells was low in non-lesional tissues. Quantitative image analysis demonstrated significant increases in the number of both epidermal and dermal CXCR3(+) cells in lesional compared with non-lesional biopsies. The majority of CXCR3(+) cells were located in the dermis of the lesional skin and 74% were demonstrated to be CD3(+) T lymphocytes. A small number of CXCR3(+) cells were CD68(+) myeloid cells. In addition, we found that nearly all BDCA-2(+) plasmacytoid dendritic cells in the psoriatic biopsies were CXCR3(+). These findings support and extend prior reports suggesting the potential role for CXCR3 in the pathophysiology of plaque psoriasis, by mediating the recruitment of plasmacytoid dendritic cells and T cells into the developing lesions.

Characterization of a murine keyhole limpet hemocyanin (KLH)-delayed-type hypersensitivity (DTH) model: role for p38 kinase.

Molecular and cellular assessment of dermal delayed-type hypersensitivity (DTH) responses is a useful approach for evaluating the mechanism of action (MOA) of immunomodulatory agents. In the present report, we characterized the delayed-type hypersensitivity response induced by keyhole limpet hemocyanin (KLH), and validated its utility by evaluating an immunomodulator, BIRB-796. Intradermal KLH challenge of the ear pinna following subcutaneous antigen sensitization resulted in a pronounced skin inflammation that peaked at 24-48h. At the molecular level, there was an activation of 3 mitogen-activated protein kinases (MAPKs: p38, JNK and ERK), an induction of the chemokines CCL2/JE, CXCL2/Mip-2, CXCL1/KC, CCL3/Mip-1alpha CCL4/Mip-1beta and CXCL10/IP-10, and expression of the cytokines IL-1beta and IL-10 in the ear parenchyma. Modulation of TNFalpha protein level was only detected in ex-vivo ear whole organ cultures (EWOC). Consistent with this inflammatory profile there was an infiltration of neutrophils and mononuclear cells into the ear parenchyma. BIRB-796, a potent allosteric p38 MAPK inhibitor attenuated the ear swelling response, which correlated with a reduced inflammatory profile. BIRB-796 inhibited p38 but not JNK or ERK kinase activation, decreased multiple chemokines which correlated with a decrease in the infiltration of neutrophils and macrophages; CD4 T cells were modesty reduced. Similarly, there was a decrease of levels of cytokines including IL-1beta, IL-10 and TNFalpha. These data support the utility of this model for evaluating immunomodulators on skin inflammation and suggest that modulation of p38 kinase may be of therapeutic value for the treatment of inflammatory skin conditions.

Characterization of peripheral human cannabinoid receptor (hCB2) expression and pharmacology using a novel radioligand, [35S]Sch225336.

Studies to characterize the endogenous expression and pharmacology of peripheral human cannabinoid receptor (hCB2) have been hampered by the dearth of authentic anti-hCB2 antibodies and the lack of radioligands with CB2 selectivity. We recently described a novel CB2 inverse agonist, N-[1(S)-[4-[[4-methoxy-2-[(4methoxyphenyl)sulfonyl] phenyl]sulfonyl] phenyl]ethyl]methane-sulfonamide (Sch225336), that binds hCB2 with high affinity and excellent selectivity versus hCB1. The precursor primary amine of Sch225336 was prepared and reacted directly with [(35)S]mesyl chloride (synthesized from commercially obtained [(35)S]methane sulfonic acid) to generate [(35)S]Sch225336. [(35)S]Sch225336 has high specific activity (>1,400 Ci/mmol) and affinity for hCB2 (65 pm). Using [(35)S]Sch225336, we assayed hemopoietic cells and cell lines to quantitate the expression and pharmacology of hCB2. Lastly, we used [(35)S]Sch225336 for detailed autoradiographic analysis of CB2 in lymphoid tissues. Based on these data, we conclude that [(35)S]Sch225336 represents a unique radioligand for the study of CB2 endogenously expressed in blood cells and tissues.

Ectopic expression of the murine chemokines CCL21a and CCL21b induces the formation of lymph node-like structures in pancreas, but not skin, of transgenic mice.

The CC chemokine CCL21 is a potent chemoattractant for lymphocytes and dendritic cells in vitro. In the murine genome there are multiple copies of CCL21 encoding two CCL21 proteins that differ from each other by one amino acid at position 65 (either a serine or leucine residue). In this report, we examine the expression pattern and biological activities of both forms of CCL21. We found that although both serine and leucine forms are expressed in most tissues examined, the former was the predominant form in lymphoid organs while the latter was predominantly expressed in nonlymphoid organs. When expressed in transgenic pancreas, both forms of CCL21 were capable of inducing the formation of lymph node-like structures composed primarily of T and B cells and a few dendritic cells. Induction of lymph node-like structures by these CCL21 proteins, however, could not be reproduced in every tissue. For instance, no lymphocyte recruitment or accumulation was observed when CCL21 was overexpressed in the skin. We conclude that both forms of CCL21 protein are biologically equivalent in promoting lymphocyte recruitment to the pancreas, and that their ability to induce the formation of lymph node-like structures is dependent on the tissues in which they are expressed.