TRPV1 Channel Activated by the PGE2/EP4 Pathway Mediates Spinal Hypersensitivity in a Mouse Model of Vertebral Endplate Degeneration

Low back pain (LBP) is the primary cause of disability globally. There is a close relationship between Modic changes or endplate defects and LBP. Endplates undergo ossification and become highly porous during intervertebral disc (IVD) degeneration. In our study, we used a mouse model of vertebral endplate degeneration by lumbar spine instability (LSI) surgery. Safranin O and fast green staining and μCT scan showed that LSI surgery led to endplate ossification and porosity, but the endplates in the sham group were cartilaginous and homogenous. Immunofluorescent staining demonstrated the innervation of calcitonin gene-related peptide- (CGRP-) positive nerve fibers in the porous endplate of LSI mice. Behavior test experiments showed an increased spinal hypersensitivity in LSI mice. Moreover, we found an increased cyclooxygenase 2 (COX2) expression and an elevated prostaglandin E2 (PGE2) concentration in the porous endplate of LSI mice. Immunofluorescent staining showed the colocalization of E-prostanoid 4 (EP4)/transient receptor potential vanilloid 1 (TRPV1) and CGRP in the nerve endings in the endplate and in the dorsal root ganglion (DRG) neurons, and western blotting analysis demonstrated that EP4 and TRPV1 expression significantly increased in the LSI group. Our patch clamp study further showed that LSI surgery significantly enhanced the current density of the TRPV1 channel in small-size DRG neurons. A selective EP4 receptor antagonist, L161982, reduced the spinal hypersensitivity of LSI mice by blocking the PGE2/EP4 pathway. In addition, TRPV1 current and neuronal excitability in DRG neurons were also significantly decreased by L161982 treatment. In summary, the PGE2/EP4 pathway in the porous endplate could activate the TRPV1 channel in DRG neurons to cause spinal hypersensitivity in LSI mice. L161982, a selective EP4 receptor antagonist, could turn down the TRPV1 current and decrease the neuronal excitability of DRG neurons to reduce spinal pain.

817 DT095895, a selective EP4 receptor antagonist with monotherapy efficacy in syngeneic mouse model(s) and best-in-class properties

BackgroundElevated levels of Prostaglandin E2 (PGE2), an eicosanoid notably synthesized by the cyclooxygenase-2 (COX-2), exert strong immunosuppressive effects in the tumor microenvironment. COX-2-positive solid tumors have the ability to use this pathway as a resistance mechanism, especially to escape from the host immune system, thus limiting the anti-tumor effects of immune checkpoint inhibitors (ICI). These immunosuppressive effects are largely mediated by the EP4 receptor, expressed on multiple immune cells.MethodsA novel series of EP4 receptor antagonists has been developed, with improved pharmacokinetic properties when compared to the EP4 receptor antagonists currently being evaluated in clinical trials. An intensive lead optimization program led to the identification of DT095895, a small molecule development candidate with a ‘best-in class’ potential. DT095895 was assessed in multiple syngeneic mouse tumor models selected for their COX-2 expression profile.ResultsDT095895 preclinical package will be presented in the poster. Efficacy was seen both in a monotherapy setting, as well as in combination with an ICI. Additionally, a specific biomarker program was implemented and validated in order to show target engagement. A phospho-flow murine whole blood assay was set-up to assess the ability of DT095895 to inhibit CREB phosphorylation induced by a selective EP4 receptor agonist in CD3+ cells. This biomarker was further developed for human whole blood to support Phase 1 and clinical trials studies.ConclusionsDT095895 is a selective EP4 receptor antagonist and demonstrates strong anti-tumor effects in multiple syngeneic mouse tumor models, both as a monotherapy and in combination with ICI, through the inhibition of the PGE2-induced immunosuppression. DT095895 progresses in regulatory development.