title: Biological characterization of PM226, a chromenoisoxazole, as a selective CB2 receptor agonist with neuroprotective profile.
abstract: Cannabinoids have emerged as promising neuroprotective agents due to their capability to activate specific targets, which are involved in the control of neuronal homeostasis and survival. Specifically, those ligands that selectively target and activate the CB2 receptor may be useful for their anti-inflammatory and neuroprotective properties in various neurological disorders, with the advantage of being devoid of psychotropic effects associated with the activation of CB1 receptors. The aim of this work has been to investigate the neuroprotective properties of 7-(1,1-dimethylheptyl)-4,4-dimethyl-9-methoxychromeno[3,4-d]isoxazole (PM226), a compound derived from a series of chromeno-isoxazoles and -pyrazoles, which seems to have a promising profile related to the CB2 receptor. The compound binds selectively to this receptor with an affinity in the nanomolar range (Ki=12.8±2.4nM). It has negligible affinity for the CB1 receptor (Ki>40000nM) and no activity at the GPR55. PM226 was also evaluated in GTPγS binding assays specific to the CB2 receptor showing agonist activity (EC50=38.67±6.70nM). In silico analysis of PM226 indicated that it has a good pharmacokinetic profile and a predicted ability to cross the blood-brain barrier. Next, PM226 was investigated in an in vitro model to explore its anti-inflammatory/neuroprotective properties. Conditioned media were collected from LPS-stimulated cultures of BV2 microglial cell line in the absence or presence of different doses of PM226, and then media were added to cultured M213-2O neuronal cells to record their influence on cell viability evaluated using MTT assays. As expected, cell viability was significantly reduced by the exposure to these conditioned media, while the addition of PM226 attenuated this reduction in a dose-dependent manner. This effect was reversed by co-incubating with the CB2 antagonist SR144528, thus confirming the involvement of CB2 receptors, whereas the addition of PM226 to neuronal cultures instead cultured BV2 cells was not effective. PM226 has also been studied in an in vivo model of mitochondrial damage generated by intrastriatal application of malonate in rats. MRI analysis showed that PM226 administration decreased the volume of the striatal lesion caused by malonate, effect that was confirmed after the histopathological evaluation (Nissl staining, Iba-1 immunostaining and TUNEL assay) of striatal sections derived from malonate-lesioned rats in the absence or presence of PM226. Again, the beneficial effects of PM226 were dependent on the activation of CB2 receptors as they were reversed by blocking these receptors with AM630. Overall, PM226 has shown to have a promising neuroprotective profile derived from its ability to selectively activate CB2 receptor, so that it could be a useful disease-modifying agent in those neurodegenerative pathologies in which the activation of these receptors may have therapeutic value.
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad Complutense, Madrid, Spain; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain; Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Gómez-Cañas
authors.affiliation: Instituto de Química Médica, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors.forename: P
authors.initials: P
authors.lastname: Morales
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad Complutense, Madrid, Spain; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain; Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain.
authors.forename: L
authors.initials: L
authors.lastname: García-Toscano
authors.affiliation: Vivacell Biotechnology Spain, Córdoba, Spain.
authors.forename: C
authors.initials: C
authors.lastname: Navarrete
authors.affiliation: Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC)/Hospital Universitario Reina Sofía, Universidad de Córdoba, Córdoba, Spain.
authors.forename: E
authors.initials: E
authors.lastname: Muñoz
authors.affiliation: Instituto de Química Médica, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors.forename: N
authors.initials: N
authors.lastname: Jagerovic
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad Complutense, Madrid, Spain; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain; Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain. Electronic address: jjfr@med.ucm.es.
authors.forename: J
authors.initials: J
authors.lastname: Fernández-Ruiz
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad Complutense, Madrid, Spain; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain; Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain. Electronic address: moisesgar@med.ucm.es.
authors.forename: M
authors.initials: M
authors.lastname: García-Arencibia
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad Complutense, Madrid, Spain; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain; Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain. Electronic address: ruthpazos@med.ucm.es.
authors.forename: M R
authors.initials: MR
authors.lastname: Pazos
mesh_terms: D000818:Animals; D001665:Binding Sites; D001812:Blood-Brain Barrier / Q000378:metabolism; D001921:Brain / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D001930:Brain Injuries / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D063386:Cannabinoid Receptor Agonists / Q000138:chemical synthesis / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D002460:Cell Line; D004195:Disease Models, Animal; D006801:Humans; D007555:Isoxazoles / Q000138:chemical synthesis / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D008297:Male; D008314:Malonates; D051379:Mice; D008954:Models, Biological; D009410:Nerve Degeneration; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D018696:Neuroprotective Agents / Q000138:chemical synthesis / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D010539:Permeability; D011485:Protein Binding; D017208:Rats, Wistar; D043885:Receptor, Cannabinoid, CB2 / Q000819:agonists* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: C588309:CNR2 protein, human; D063386:Cannabinoid Receptor Agonists; C571431:Cnr2 protein, mouse; C469526:Cnr2 protein, rat; D007555:Isoxazoles; D008314:Malonates; D018696:Neuroprotective Agents; C000625621:PM226; D043885:Receptor, Cannabinoid, CB2; C030290:malonic acid
keywords: Anti-inflammatory effects; Cannabinoids; Chromenoisoxazole; Glial-mediated effects; Neuronal death; Neuroprotection; PM226