Trabajo presentado en el ASPET Annual Meeting at Experimental Biology 2022, celebrado en Philadelphia, PA (Estados Unidos), del 2 al 5 de abril de 2022This research by the Grant PID2020-118127RB-I00 funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe” to S.V. Financial support from Fundació Bosch i Gimpera, Universitat de Barcelona (F2I grant), to S.V., and from the Xunta de Galicia (ED431G 2019/02 and ED431C 2018/21) to M.I.L. are acknowledged. Partial support was provided by NIH-NIEHS River Award R35 ES03443, NIH-NIEHS Superfund Program P42 ES004699, NINDS R01 DK107767, and NIDDK R01 DK103616 to B.D.H. S.C. acknowledges a PhD fellowship from the Universitat de Barcelona (APIF grant)

Bartra Cabré, Clara

Brea, José

Cobos, Enrique J.

Codony, Sandra

Entrena, José M.

González-Cano, Rafael

Hammock, Bruce D.

Jora, Beatrice

Loza, María Isabel

Morisseau, Christophe

Sanfeliu, Coral

Vázquez, Santiago

English

Digital.CSIC

Soluble Epoxide Hydrolase Inhibitors: Design, Synthesis, in vitro Profiling and in vivo Evaluation in Murine Models of PainSantiago Vázquez1, Sandra Codony1, José Entrena2, Beatrice Jora1, Rafael González-Cano2, M. Isabel Loza3, José M. Brea3, Christophe Morisseau4, Clara Bartra5, Coral Sanfeliu5, Bruce D. Hammock4, and Enrique J. Cobos21Laboratori de Química Farmacèutica, Faculty of Pharmacy and Food Sciences, Universitat de Barcelona and IBUB, Barcelona, Spain; 2Department of Pharmacology, School of Medicine, University of Granada, Granada, Spain; 3Drug Screening Platform/Biofarma Research Group, CIMUS Research Center. University of Santiago de Compostela (USC), Santiago de Compostela, Spain; 4Department of Entomology and Nematology, UC Davis Comprehensive Cancer Center, University of California, Davis, CA, USA. 5Institut d’Investigacions Biomèdiques de Barcelona (IIBB), CSIC and IDIBAPS, Barcelona, Spain.INTRODUCTIONThe inhibition of soluble epoxide hydrolase (sEH) hasbeen suggested as a novel pharmacological approachfor the treatment of pain-related disorders and variousinflammatory diseases.1OBJECTIVERecently, we discovered that a selected member of anew family of benzohomoadamantane-based sEHinhibitors (sEHI), 1, showed in vivo efficacy in a murinemodel of acute pancreatitis.2 Taking into account thestructure of the clinical candidate sEHI for neuropathicpain EC5026,3 herein we report further structure-activity relationships within the series ofbenzohomoadamantane-derived sEHI with the aim toconduct a screening cascade and to perform an in vivoproof of concept in murine models of pain with theselected candidate.SYNTHESISFurther in vitro profiling (human and murine sEH inhibition, human and mice microsomal stability,solubility, cytotoxicity, cytochromes inhibition, Caco-2 permeability, selectivity and hERG inhibition)allowed us to select compounds 8, 14 and 15 for in vivo studies.CpdsEH IC50a(nM)Microsomalstabilityb (%)Cytochromeinhibition(% at 10 µM)Solubilityc(µM)PAMPA-BBBCytotoxicity LD50(µM)PId MTTeHuman Murine Human Mouse CYP 2C198 0.4 1.0 47 64 38 ± 4 57 CNS + >100 >10014 0.4 0.5 66 84 32 ± 4 95 CNS +/- >100 >10015 0.4 0.4 58 60 26 ± 5 92 CNS + >100 >100SCREENING CASCADEPHARMACOKINETIC STUDIES IN MICE (5 mg/Kg, S.C.)0 1 2 3 4 5 60123Time (h)Concentration (ug/ml).Figure 2. Plasma concentration vs time for compounds 8 and 14, respectively. 0 1 2 3 4 5 60102030Time (h)Concentration (ug/ml)CpdHL(h)Tmax(h)Cmax(µg/mL)AUClast(µg*h/mL)AUCINF (µg*h/mL)8 3.42 0.75 1.2 2.4 2.514 0.70 0.25 19.1 13.5 13.6Table 2. Pharmacokinetic parameters for compounds 8 and 14.Table 1. In vitro profiling of selected sEHIs. aReported IC50 values are the average of three replicates.bPercentage of remaining compound after 60 min of incubation with pooled human and mouse microsomes in thepresence of NADPH at 37 °C. cSolubility measured in a 1% DMSO: 99% PBS buffer solution. dCytotoxicity tested bypropidium iodide (PI) staining after 24h incubation in SH-SY5Y cells. eCytotoxicity tested by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay after 24h incubation in SH-SY5Y cells.IN VIVO EFFICACY STUDIESA first in vivo efficacy study was performed in thecapsaicin-induced secondary mechanical hypersensitivity(allodynia) model in mice.Scheme 1. Synthesis of the new sEHIs. aReagents and conditions: a) triphosgene, NaHCO3, DCM, 30 min; b)DCM, overnight; c) n-BuLi, anh. THF, anh. DCM, overnight.Figure 1. Objective of this work.Figure 2. Reduction of capsaicin-induced secondarymechanical hypersensitivity in mice by the systemicadministration of AS2586114, and compounds 8 and 14,is due to sEH inhibition. The data shown represent the effectof the subcutaneous (s.c.) administration of AS2586114, 8 and 14administered alone or associated to the CYP450 epoxidaseinhibitor MS-PPOH (s.c.) on paw withdrawal latency in micetreated intraplantarly (i.pl.) with capsaicin. **p < 0.01 betweennonsensitized mice (open bar) and the other experimentalgroups; #p < 0.05, ##p < 0.01 between capsaicin-treated miceinjected with the sEHI or their solvent (black bar); ⴕⴕp < 0.01 sEHI-treated mice associated or not with MS-PPOH.CONCLUSIONS• We have further explored medicinal chemistry aroundnew benzohomoadamantane-based piperidinederivatives, analogs of the clinical candidate EC5026.• An in vitro screening cascade and pharmacokineticstudies allowed us to select two candidates for in vivoefficacy studies.• The administration of compounds 8 and 14 reducedpain in the capsaicin-induced murine model ofallodynia in a dose-dependent manner andoutperformed AS2586114, a standard sEHI.• Hence, this study opens a whole range of applicationsof the benzohomoadamantane-based sEHIs in the painfield.ACKNOWLEDGEMENTSThis research by the Grant PID2020-118127RB-I00 funded byMCIN/AEI/10.13039/501100011033 and by “ERDF A way of makingEurope” to S.V. Financial support from Fundació Bosch i Gimpera,Universitat de Barcelona (F2I grant), to S.V., and from the Xunta deGalicia (ED431G 2019/02 and ED431C 2018/21) to M.I.L. areacknowledged. Partial support was provided by NIH-NIEHS River AwardR35 ES03443, NIH-NIEHS Superfund Program P42 ES004699, NINDS R01DK107767, and NIDDK R01 DK103616 to B.D.H. S.C. acknowledges a PhDfellowship from the Universitat de Barcelona (APIF grant).REFERENCES1Morisseau, C.; Hammock, B.D. Annu. Rev. Pharmacol. Toxicol. 2013,53, 37–58.2Codony S.; Calvó-Tusell C.; Valverde E.; Osuna S.; Moriseau C.; LozaM.I.; Brea J.; Pérez C.; Rodríguez-Franco M.I.; Pizarro-Delgado J.;Corpas R.; Griñán-Ferré C.; Pallàs M.; Sanfeliu C.; Vázquez-Carrera M.;Hammock B. D.; Feixas F.; Vázquez S. J. Med. Chem., 2021, 64, 5429–5446.3Hammock, B. D.; McReynolds, C. B.; Wagner, K.; Buckpitt, A.; Cortes-Puch, I.; Croston, G.; Lee, K. S. S.; Yang, J.; Schmidt, W. K.; Hwang, S. H.J. Med. Chem. 2021, 64, 1856-1872...ArachidonicacidCYP2C19CYP1A2CYP2J2Epoxyeicosatrienoicacids (EETs)Soluble Epoxide Hydrolase (sEH)Dihydroxyeicosatrienoicacids (DHETs)sEH inhibitorsAnti-inflammatoryFigure 1. Metabolism pathway of arachidonic acid.8 14MS-PPOH(20 mg/kg, s.c.)sEHI(mg/kg, s.c.)

Soluble epoxide hydrolase inhibitors: design, synthesis, in vitro profiling and in vivo evaluation in murine models of pain

Epoxyeicosatrienoic acids (EETs) are endogenous chemical mediators that show anti-inflammatory, antihypertensive, and analgesic effects. Soluble epoxide hydrolase (sEH) converts EETs to their corresponding dihydroxyeicosatrienoic acids, whereby the biological effects of EETs are modified. Therefore, inhibition of sEH has been suggested as a novel pharmacological approach for the treatment of inflammatory and pain-related disorders.1 Recently, we have discovered a new family of sEH inhibitors (sEHI) featuring a unique benzohomoadamantane scaffold. In a murine model of cerulein-induced acute pancreatitis, the administration of a selected candidate significantly reduced pancreatic damage and improved the health status of the animals.2 Herein we report further structure-activity relationships within this series of benzohomoadamantane-derived sEHI. Most of the novel derivatives were endowed with low nanomolar or even subnanomolar IC50 values at the human, murine and rat sEH. Further in vitro profiling (solubility, cytotoxicity, metabolic stability, selectivity, permeability, etc.) and pharmacokinetic studies permitted us to select a candidate for in vivo efficacy studies. This candidate reduced pain in the capsaicin-induced model of allodynia in a dose-dependent manner and outperformed other sEHI tested. In summary, these novel results and the previously reported studies using other families of sEHI, strongly suggest that sEH may be a target of clinical interest for managing pain.3 References: 1-Sun, C.-P.; Zhang, X.-Y.; Morisseau, C.; Hwang, S. H.; Zhang, Z.-J.; Hammock, B. D.; Ma, X.-C. J. Med. Chem. 2021, 64, 184-215. 2-Codony, S.; Calvó-Tusell, C.; Valverde, E.; Osuna, S.; Morisseau, C.; Loza, M.I.; Brea, J.; Pérez, C.; Rodríguez-Franco, M.I.; Pizarro-Delgado, J.; Corpas, R.; Griñán-Ferré, C.; Pallàs, M.; Sanfeliu, C.; Vázquez-Carrera, M.; Hammock, B. D.; Feixas, F.; Vázquez, S. J. Med. Chem., 2021, 64, 5429-5446. 3-McReynolds, C.; Morisseau, C.; Wagner, K.; Hammock, B. D. Adv. Exp. Med. Biol. 2020, 1274, 71-99.This research was funded by Grant PID2020-118127RB-I00 funded by MCIN/AEI/ 10.13039/501100011033 and by “ERDF A way of making Europe” to S.V. Financial support from Fundació Bosch i Gimpera (F2I grant), to S.V., and from the Xunta de Galicia (ED431G 2019/02 and ED431C 2018/21) to M.I.L. are acknowledged. Partial support was provided by NIH-NIEHS River Award R35 ES03443, NIH-NIEHS Superfund Program P42 ES004699, NINDS R01 DK107767, and NIDDK R01 DK103616 to B.D.H

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Soluble Epoxide Hydrolase Inhibitors: Design, Synthesis, in vitro Profiling and in vivo Evaluation in Murine Models of Pain

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Soluble epoxide hydrolase inhibitors: design, synthesis, in vitro profiling and in vivo evaluation in murine models of pain

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