Michael J. Forrest

Impaired neuropathic pain responses in mice lacking the chemokine receptor CCR2

Mice lacking the chemokine receptor chemotactic cytokine receptor 2 (CCR2) have a marked attenuation of monocyte recruitment in response to various inflammatory stimuli and a reduction of inflammatory lesions in models of demyelinating disease. In the present study, we compared nociceptive responses in inflammatory and neuropathic models of pain in CCR2 knockout and wild-type mice. In acute pain tests, responses were equivalent in CCR2 knockout and wild-type mice. In models of inflammatory pain, CCR2 knockout mice showed a 70% reduction in phase 2 of the intraplantar formalin-evoked pain response but only a modest (20–30%) and nonsignificant reduction of mechanical allodynia after intraplantar Freunds adjuvant (CFA). In a model of neuropathic pain, the development of mechanical allodynia was totally abrogated in CCR2 knockout mice. CFA administration induced marked up-regulation of CCR2 mRNA in the skin and a moderate increase in the sciatic nerve and dorsal root ganglia (DRG). In response to nerve ligation, persistent and marked up-regulation of CCR2 mRNA was evident in the nerve and DRG. Disruption of Schwann cells in response to nerve lesion resulted in infiltration of CCR2-positive monocytes/macrophages not only to the neuroma but also to the DRG. Chronic pain also resulted in the appearance of activated CCR2-positive microglia in the spinal cord. Collectively, these data suggest that the recruitment and activation of macrophages and microglia peripherally and in neural tissue may contribute to both inflammatory and neuropathic pain states. Accordingly, blockade of the CCR2 receptor may provide a novel therapeutic modality for the treatment of chronic pain.

The discovery of rofecoxib, [MK 966, VIOXX®, 4-(4′-methylsulfonylphenyl)-3-phenyl-2(5H)-furanone], an orally active cyclooxygenase-2 inhibitor

The development of a COX-2 inhibitor rofecoxib (MK 966, Vioxx) is described. It is essentially equipotent to indomethacin both in vitro and in vivo but without the ulcerogenic side effect due to COX-1 inhibition.

Peroxisome Proliferator-Activated Receptors γ and α Mediate in Vivo Regulation of Uncoupling Protein (UCP-1, UCP-2, UCP-3) Gene Expression

A role for peroxisome proliferator-activated receptors, PPAR gamma and PPAR alpha, as regulators of energy homeostasis and lipid metabolism, has been suggested. Recently, three distinct uncoupling protein isoforms, UCP-1, UCP-2, and UCP-3, have also been identified and implicated as mediators of thermogenesis. Here, we examined whether in vivo PPAR gamma or PPAR alpha activation regulates the expression of all three UCP isoforms. Rats or lean and db/db mice were treated with PPAR gamma [thiazolidinedione (TZD)] or PPAR alpha (WY-14643) agonists, followed by measurement of messenger RNAs (mRNAs) for UCP-1, UCP-2, and UCP-3 in selected tissues where they are expressed. TZD treatment (AD 5075 at 5 mg/kg x day) of rats (14 days) increased brown adipose tissue (BAT) depot size and induced the expression of each UCP mRNA (3x control levels for UCP-1 and UCP-2, 2.5x control for UCP-3). In contrast, UCP-2 and UCP-3 mRNA levels were not affected in white adipose tissue or skeletal muscle. Chronic (30 days) low-dose (0.3 mg/kg x day) TZD treatment induced UCP-1 mRNA and protein in BAT (2.5x control). In contrast, chronic TZD treatment (30 mg/kg x day) suppressed UCP-1 mRNA (>80%) and protein (50%) expression in BAT. This was associated with further induction of UCP-2 expression (>10-fold) and an increase in the size of lipid vacuoles, a decrease in the number of lipid vacuoles in each adipocyte, and an increase in the size of the adipocytes. TZD treatment of db/db mice (BRL 49653 at 10 mg/kg x day for 10 days) also induced UCP-1 and UCP-3 (but not UCP-2) expression in BAT. PPAR alpha is present in BAT, as well as liver. Treatment of rats or db/db mice with WY-14643 did not affect expression of UCP-1, -2, or -3 in BAT. Hepatic UCP-2 mRNA was increased (4x control level) in db/db and lean mice, although this effect was not observed in rats. Thus, in vivo PPAR gamma activation can induce expression of UCP-1, -2, and -3 in BAT; whereas chronic-intense PPAR gamma activation may cause BAT to assume white adipose tissue-like phenotype with increased UCP-2 levels. PPAR alpha activation in mice is sufficient to induce liver UCP-2 expression.

Human β3-adrenergic receptor agonists containing 1,2,3-triazole-substituted benzenesulfonamides

Compounds containing a 1,2,3-triazole-substituted benzenesulfonamide were prepared and found to be potent and selective human beta3-adrenergic receptor agonists. The most interesting compound, trifluoromethylbenzyl analogue 12e (beta3 EC50 = 3.1 nM with >1500-fold selectivity over binding to both beta1- and beta2 receptors), stimulates lipolysis in the rhesus monkey (ED50 = 0.36 mg/kg) and is 25% orally bioavailable in the dog.

Sphingosine-1-Phosphate Receptor Agonism Impairs the Efficiency of the Local Immune Response by Altering Trafficking of Naive and Antigen-Activated CD4+ T Cells

FTY720 (2-amino-[2-(4-octylphenyl) ethyl]-1,3-propanediol hydrochloride) is an immunosuppressive agent that inhibits allograft rejection. We recently demonstrated that FTY-phosphate, the active metabolite of FTY720, acts as a full agonist for sphingosine-1-phosphate (S1P) receptors. Furthermore, activation of S1P receptors with their natural ligand, S1P, as well as pharmacological ligands leads to lymphopenia, probably due to sequestration of lymphocytes in secondary lymphoid organs. In the present study we used a local Ag-challenged mouse model to examine the effects of FTY720 on T cell activation in the draining lymph node (DLN) and on the release of activated T cells to the peripheral blood compartment. We showed that the number of Ag-activated CD4+ T cells in the DLN after injection of Ag and CFA into a footpad was dramatically reduced after FTY720 treatment. However, T cell proliferation, both in vitro and in vivo, was not impaired by FTY720. Our results suggest that the reduced efficiency of T cell responses in the DLN in response to a local Ag is probably due to a defective recirculation of naive T cells caused by FTY720 treatment. Furthermore, we found that the numbers of naive and Ag-activated CD4+ T cells in the peripheral blood of Ag-challenged mice were equally reduced with FTY720 treatment, suggesting that both T cell subsets are sequestered in the DLNs. Thus, FTY720 induces immunosuppression through inhibition of both the recirculation of naive T cells and the release of Ag-activated T cells from the DLN to lymph and to the blood compartment.

Sphingosine-1-Phosphate Agonists Increase Macrophage Homing, Lymphocyte Contacts, and Endothelial Junctional Complex Formation in Murine Lymph Nodes

The sphingosine-1-phosphate (S1P) receptor agonist, phosphorylated FTY720 (FTY-P), causes lymphopenia, lymphocyte sequestration in mesenteric lymph nodes (MLNs), and immunosuppression. Using multiple techniques to analyze MLN cells harvested from mice treated with S1P receptor agonists, we saw a redistribution of lymphocytes out of nodal sinuses and an expansion of follicles. Although changes in circulating monocytes were not observed with overnight exposure to FTY720, we saw a significant increase in S1P receptor 1 (S1P1)-expressing CD68+ macrophages in subcapsular sinuses of FTY-P-treated MLNs. This was confirmed by quantitative analysis of F4/80+ cells in MLN suspensions. The sinus volume and number of S1P1-positive cells within sinuses were also increased by FTY-P. High endothelial venules and lymphatic endothelium expressed high levels of S1P1, and treatment with FTY-P resulted in intense staining and colocalization of CD31, β-catenin, and zona occludens 1 in junctions between sinus cells. Transmission electron microscopy showed that FTY-P greatly reduced lymphocyte microvilli and increased cell-cell contacts in the parenchyma. Immunoelectron microscopy revealed that intranodal lymphocytes lacked surface expression of S1P1, whereas S1P1 was evident on the surface and within the cytoplasm of macrophages, endothelial cells, and stromal cells. This subcellular pattern of intranodal receptor distribution was unchanged by treatment with FTY-P. We conclude that S1P1 agonists have profound effects on macrophages and endothelial cells, in addition to inducing lymphopenia.

L-770,644 : A potent and selective human β3 adrenergic receptor agonist with improved oral bioavailability

L-770,644 (9c) is a potent and selective agonist of the human beta3 adrenergic receptor (EC50 = 13 nM). It shows good oral bioavailability in both dogs and rats (%F = 27), and is a full agonist for glycerolemia in the rhesus monkey (ED50 = 0.21 mg/kg). Based on its desirable in vitro and in vivo properties, L-770,644 was chosen for further preclinical evaluation.

Human β3 andrenergic receptor agonists containing imidazolidinone and imidazolone benzenesulfonamides

The cyclopentylpropylimidazolidinone L-766,892 is a potent beta3 AR agonist (EC50 5.7 nM, 64% activation) with 420- and 130-fold selectivity over binding to the beta1 and beta2 ARs, respectively. In anesthetized rhesus monkeys, L-766,892 elicited dose-dependent hyperglycerolemia (ED50 0.1 mg/kg) with minimal effects on heart rate.

3-pyridylethanolamines: Potent and selective human β3 adrenergic receptor agonists

The 3-pyridylethanolamine L-757,793 is a potent beta 3 AR agonist (EC50 6.3 nM, 70% activation) with 1,300- and 500-fold selectivity over binding to the beta 1 and beta 2 ARs, respectively. L-757,793 stimulated lipolysis in rhesus monkeys (ED50 0.2 mg/kg) with a maximum response equivalent to that elicited by isoproterenol.

On- and off-target pharmacology of torcetrapib: current understanding and implications for the structure activity relationships (SAR), discovery and development of cholesteryl ester-transfer protein (CETP) inhibitors.

Lowering of serum low-density lipoprotein cholesterol (LDL-C) levels remains the primary aim of lipid management. Much progress has been made in reducing rates of cardiovascular disease morbidity and mortality, largely through increased awareness of lipid-lowering therapies and particularly through the use of high-efficacy LDL-C-lowering HMG-CoA reductase inhibitors (statins). While statins have been effective in reducing cardiovascular disease risk, many patients do not adequately achieve guideline-recommended LDL-C goals and may benefit from additional cholesterol management therapies. Low serum levels of high-density lipoprotein cholesterol (HDL-C) are considered another important determinant of cardiovascular disease risk, and increased serum HDL-C levels have been shown to be associated with reductions in the incidence of cardiovascular disease. One approach toward raising serum HDL-C levels is the inhibition of cholesteryl ester-transfer protein (CETP), a plasma protein that promotes the transfer of cholesteryl ester from HDL particles and other lipoprotein fractions to pro-atherogenic apolipoprotein B-containing lipoproteins. The inhibition of this protein raises HDL-C levels and also reduces LDL-C levels. The concept of raising HDL-C levels through pharmacological intervention of this target was validated in preclinical and clinical studies with torcetrapib, the first CETP inhibitor to be assessed inlate-stage clinical trials. The large clinical outcomes trial, ILLUMINATE, was prematurely terminated due to other unexpected pharmacological effects of torcetrapib that led to an increased risk of cardiovascular events and deaths. Thus, the ultimate effect of CETP inhibition on cardiovascular disease outcomes remains to be determined. Other CETP inhibitors currently in development do not have the adverse effects of increased blood pressure and circulating levels of aldosterone shown to be structurally related to torcetrapib. Preclinical and pharmacology studies have shown that these CETP inhibitors are distinct compared with torcetrapib and lack the features related to its off-target pharmacology. These findings indicate that the off-target activities of torcetrapib are not necessarily class effects of CETP inhibitors. Recent clinical trials have shown that dalcetrapib, anacetrapib and evacetrapib, the most advanced of these compounds in development, effectively raise HDL-C levels and lower LDL-C in the absence of off-target activities. The results of these trials are encouraging within the limits of study size and duration and provide a rationale for conducting further studies, including large clinical outcomes trials to assess whether CETP inhibition can lead to cardioprotective effects. This review summarizes the data supporting the development of CETP inhibitors as HDL-C-raising therapy, including structureactivity relationships and preclinical and clinical pharmacology studies ofknown CETP inhibitors.