Marsha L. Roach

Impaired inflammatory and pain responses in mice lacking an inducible prostaglandin E synthase

Prostaglandin (PG)E2 is a potent mediator of pain and inflammation, and high levels of this lipid mediator are observed in numerous disease states. The inhibition of PGE2 production to control pain and to treat diseases such as rheumatoid arthritis to date has depended on nonsteroidal antiinflammatory agents such as aspirin. However, these agents inhibit the synthesis of all prostanoids. To produce biologically active PGE2, PGE synthases catalyze the isomerization of PGH2 into PGE2. Recently, several PGE synthases have been identified and cloned, but their role in inflammation is not clear. To study the physiological role of the individual PGE synthases, we have generated by targeted homologous recombination a mouse line deficient in microsomal PGE synthase 1 (mPGES1) on the inbred DBA/1lacJ background. mPGES1-deficient (mPGES1-/-) mice are viable and fertile and develop normally compared with wild-type controls. However, mPGES1-/- mice displayed a marked reduction in inflammatory responses compared with mPGES1+/+ mice in multiple assays. Here, we identify mPGES1 as the PGE synthase that contributes to the pathogenesis of collagen-induced arthritis, a disease model of human rheumatoid arthritis. We also show that mPGES1 is responsible for the production of PGE2 that mediates acute pain during an inflammatory response. These findings suggest that mPGES1 provides a target for the treatment of inflammatory diseases and pain associated with inflammatory states.

The prostaglandin E2 EP1 receptor mediates pain perception and regulates blood pressure

The lipid mediator prostaglandin E2 (PGE2) has diverse biological activity in a variety of tissues. Four different receptor subtypes (EP1-4) mediate these wide-ranging effects. The EP-receptor subtypes differ in tissue distribution, ligand-binding affinity, and coupling to intracellular signaling pathways. To identify the physiological roles for one of these receptors, the EP1 receptor, we generated EP1-deficient (EP1-/-) mice using homologous recombination in embryonic stem cells derived from the DBA/1lacJ strain of mice. The EP1-/- mice are healthy and fertile, without any overt physical defects. However, their pain-sensitivity responses, tested in two acute prostaglandin-dependent models, were reduced by approximately 50%. This reduction in the perception of pain was virtually identical to that achieved through pharmacological inhibition of prostaglandin synthesis in wild-type mice using a cyclooxygenase inhibitor. In addition, systolic blood pressure is significantly reduced in EP1 receptor-deficient mice and accompanied by increased renin-angiotensin activity, especially in males, suggesting a role for this receptor in cardiovascular homeostasis. Thus, the EP1 receptor for PGE2 plays a direct role in mediating algesia and in regulation of blood pressure.

High-throughput Screening in Embryonic Stem Cell-derived Neurons Identifies Potentiators of α-Amino-3-hydroxyl-5-methyl-4-isoxazolepropionate-type Glutamate Receptors

Stem cell biology offers advantages to investigators seeking to identify new therapeutic molecules. Specifically, stem cells are genetically stable, scalable for molecular screening, and function in cellular assays for drug efficacy and safety. A key hurdle for drug discoverers of central nervous system disease is a lack of high quality neuronal cells. In the central nervous system, α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) subtype glutamate receptors mediate the vast majority of excitatory neurotransmissions. Embryonic stem (ES) cell protocols were developed to differentiate into neuronal subtypes that express AMPA receptors and were pharmacologically responsive to standard compounds for AMPA potentiation. Therefore, we hypothesized that stem cell-derived neurons should be predictive in high-throughput screens (HTSs). Here, we describe a murine ES cell-based HTS of a 2.4 × 106 compound library, the identification of novel chemical “hits” for AMPA potentiation, structure function relationship of compounds and receptors, and validation of chemical leads in secondary assays using human ES cell-derived neurons. This reporting of murine ES cell derivatives being formatted to deliver HTS of greater than 106 compounds for a specific drug target conclusively demonstrates a new application for stem cells in drug discovery. In the future new molecular entities may be screened directly in human ES or induced pluripotent stem cell derivatives.

The Human Specific CCR1 Antagonist CP-481,715 Inhibits Cell Infiltration and Inflammatory Responses in Human CCR1 Transgenic Mice

We previously described the in vitro characteristics of the potent and selective CCR1 antagonist, CP-481,715. In addition to being selective for CCR1 vs other chemokine receptors, CP-481,715 is also specific for human CCR1 (hCCR1), preventing its evaluation in classical animal models. To address this, we generated mice whereby murine CCR1 was replaced by hCCR1 (knockin) and used these animals to assess the anti-inflammatory properties of CP-481,715. Cells isolated from hCCR1 knockin mice were shown to express hCCR1 and migrate in response to both murine CCR1 and hCCR1 ligands. Furthermore, this migration is inhibited by CP-481,715 at dose levels comparable to those obtained with human cells. In animal models of cell infiltration, CP-481,715 inhibited CCL3-induced neutrophil infiltration into skin or into an air pouch with an ED50 of 0.2 mg/kg. CP-481,715 did not inhibit cell infiltration in wild-type animals expressing murine CCR1. In a more generalized model of inflammation, delayed-type hypersensitivity, CP-481,715 significantly inhibited footpad swelling and decreased the amount of IFN-γ and IL-2 produced by isolated spleen cells from sensitized animals. It did not, however, induce tolerance to a subsequent challenge. These studies illustrate the utility of hCCR1 knockin animals to assess the activity of human specific CCR1 antagonists; demonstrate the ability of the CCR1 antagonist CP-481,715 to inhibit cell infiltration, inflammation, and Th1 cytokine responses in these animals; and suggest that CP-481,715 may be useful to modulate inflammatory responses in human disease.