Carr_2025_Biochem.Biophys.Rep_42_102009

The treatment of orofacial pain disorders is poor. Both opioids and serotonin agonists are commonly used; however, they produce dangerous and unpleasant side effects. Therefore, there is an urgent need to identify new pharmacological treatments that can resolve orofacial pain. Moreover, a treatment that engages multiple mechanisms using one compound may be advantageous. Fatty acid amide hydrolase (FAAH) and soluble epoxide hydrolase (sEH) are two enzymes that can regulate both pain and inflammation via independent pathways. Small molecules that inhibit both enzymes simultaneously were previously synthesized and produced antinociception in vivo. Quinolinyl-based dual inhibitors of FAAH and sEH can inhibit acute inflammatory pain in rats. Here, following on these findings, we generated 7 new isoquinolinyl- and 7 pyridinyl-based analogs and tested their inhibition at both enzymes. Structure-activity relationship study coupled with docking experiments, revealed that the isoquinoline moiety is well-tolerated in the binding pockets of both enzymes, yielding several analogs with nanomolar activity in enzymatic assays. All newly synthesized analogs were assessed in the solubility assay at pH 7.4, and we determined that isoquinolinyl- and substituted pyridinyl-analogs exhibit limited solubility under the experimental conditions. The most potent inhibitor, 4f, with IC(50) values in the low nanomolar range for both enzymes, was evaluated in a plasma stability assay in human and rat plasma where it showed a moderate stability. Primary binding assays revealed that 4f does not engage any opioid or serotonin receptors. A high dose (3 mg/kg) of 4f reversed orofacial hyperalgesia following pretreatment with nitroglycerin and orofacial injection of formalin; however, this same dose did not inhibit acute orofacial inflammatory pain or restore pain-depressed wheel running. These findings indicate that simultaneous inhibition of FAAH and sEH using isoquinolinyl-based dual inhibitors may only reverse certain components of orofacial hyperalgesia.