AZ-3451

It has been reported the presence of components from the coagulation thrombin‐generating pathway and prostaglandins (PGs) in human ovarian follicular fluid (FF) but with unclear functions. Moreover, thrombin can induce COX‐2 expression linking to PG synthesis in several cell types. Therefore, this study sought to explore the thromboxane (TX) generation in FF and the correlation between FF TX levels and oocyte maturation. The FF TXB 2 (a stable metabolite of TXA 2 ) levels exhibited a negative correlation between large preovulatory leading and small mid‐antral follicles from in vitro fertilization (IVF) patients, indicating a requirement of TX for a small follicle/oocyte to grow/mature. Further receiver operating characteristic curve analysis identified that intrafollicular TXB 2 level could predict oocyte maturity. Thrombin was found expressed in a similar pattern/trend to TXB 2 in intrafolliclar FF, where it could induce TXB 2 production/secretion in human ovarian follicular GCs via proteinase‐activated receptor‐2 (PAR‐2) and PAR‐3 cooperation/transactivation and Erk/p38 MAPK/JNK signaling to cause COX‐2/TXB 2 induction. Accordingly, PAR‐2 and ‐3 were present in human ovarian follicular GCs and thrombin, PAR‐2 agonist, and TXA 2 analog caused a substantial enhancement in follicle development, which could be blocked by the PAR‐2, COX‐2, and TXA 2 prostanoid (TP) receptor inhibitors in an ex vivo cultured murine ovary model. Collectively, we first demonstrated that FF thrombin regulates PAR‐2/‐3 cooperation and MAPK signaling pathways to induce COX‐2 expression and TX production in follicular GCs, possibly supporting intrafollicular TX levels and triggering TP‐related pathways to enhance follicle development. The FF TX levels also can be a predictor for oocyte maturation during IVF.

Oral cancer is notoriously painful. Activation of protease-activated receptor 2 (PAR₂, encoded by F2RL1) by proteases in the cancer microenvironment is implicated in oral cancer pain. PAR₂ is a G protein-coupled receptor (GPCR) expressed on neurons and cells in the cancer microenvironment. Sustained signaling of PAR₂ from endosomes of neurons mediates sensitization and nociception. We focused on the differential contribution of PAR₂ on oral cancer cells and neurons to oral cancer pain and whether encapsulation of a PAR₂ inhibitor, AZ3451 in nanoparticles (NP) more effectively reverses PAR₂ activation. We report that F2RL1 was overexpressed in human oral cancers and cancer cell lines. Deletion of F2RL1 on cancer cells reduced cancer-associated mechanical allodynia. A third-generation polyamidoamine dendrimer, functionalized with cholesterol was self-assembled into NPs encapsulating AZ3451. NP encapsulated AZ3451 (PAMAM-Chol-AZ NPs) more effectively reversed activation of PAR₂ at the plasma membrane and early endosomes than free drug. The PAMAM-Chol-AZ NPs showed greater efficacy in reversing nociception than free drug, with respect to both level and duration, in three preclinical mouse models of oral cancer pain. The antinociceptive efficacy was confirmed with an operant orofacial assay. Genetic deletion of F2RL1 on cancer cells or F2rl1 on neurons each partially reversed mechanical cancer allodynia. The remaining nociception could be effectively reversed by PAMAM-Chol-AZ NPs. These findings suggest that PAR₂ on oral cancer cells and neurons contribute to oral cancer nociception and NPs loaded with a PAR₂ antagonist provide increased antinociception and improved oral function compared to free drug.