Biological characterization of PM226, a chromenoisoxazole, as a selective CB2 receptor agonist with neuroprotective profile.

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.

Central versus peripheral antagonism of cannabinoid CB1 receptor in obesity: effects of LH-21, a peripherally acting neutral cannabinoid receptor antagonist, in Zucker rats.

The endogenous cannabinoid system plays an important modulatory role in feeding behaviour and metabolism, acting at both central and peripheral levels. Chronic administration of cannabinoid CB(1) receptor antagonists has been found to be effective in experimental obesity. However, clinically available cannabinoid receptor antagonists are inverse agonists that can target CB(1) receptors located in both central circuits regulating appetite and motivation and in peripheral organs regulating metabolism and energy expenditure. This profile complicates understanding of cannabinoid CB(1) receptor blockade as a therapeutic strategy in obesity and metabolic disorders. This review aims to explore the relevance of both inverse agonism and peripheral cannabinoid receptor blockade on the beneficial actions of chronic cannabinoid receptor blockade, by comparing the actions of the reference antagonist/inverse agonist rimonabant and the newly designed drug LH-21. LH-21 is a triazol derivative and a neutral cannabinoid receptor antagonist; it has a poor penetration rate into the central nervous system. When given acutely it decreases food intake and enhances the anorectic actions of oleoylethanolamide, a feeding suppressant lipid that acts on peripheral sensory terminals in a similar way as rimonabant. Unlike rimonabant, chronic administration of LH-21 (3 mg/kg) reduces feeding but does not improve hypertriglyceridaemia or hypercholesterolaemia; nor does it reduce liver fat deposits in Zucker rats. These results suggest that the inverse agonism and/or the antagonism of central cannabinoid CB(1) receptors are necessary for the metabolic benefits of cannabinoid CB(1) receptor blockade, but not for the appetite reduction.

Cannabinoids and neuropathic pain.

After a brief overview of the endocannabinoid system (CB receptors, and endocannabinoids) and of the cannabinergic ligands, some general issues related to cannabinoids and pain are commented. Finally, the most important findings regarding cannabinoids and neuropathic pain are discussed in detail.

A solid-state NMR, X-ray diffraction, and ab initio computational study of hydrogen-bond structure and dynamics of pyrazole-4-carboxylic acid chains.

Using high-resolution solid-state (15)N CMAS NMR, X-ray crystallography, and ab initio calculations, we have studied the structure of solid pyrazole-4-carboxylic acid (1). The crystal structure was determined at 295 and 150 K. Molecules of 1 are located on a two-fold axis, implying proton disorder of the NH and OH groups; no phase transition was observed between these two temperatures. The compound forms quasi-linear ribbons in which the molecules are linked by cyclic hydrogen bonds between pyrazole and carboxylic acid groups with disordered hydrogen-bonded protons. Crystallography is unable to decide whether the disorder is dynamic or static. NMR shows that this disorder is dynamic, that is, consisting of very fast degenerate double proton transfers between two rapidly interconverting O-H.N and O.H-N hydrogen bridges. However, at low temperature, NMR shows a proton disorder-order transition where the protons are preferentially localized on given nitrogen and oxygen atoms. An amorphous phase exhibiting proton order is observed when the compound is precipitated rapidly. In this case, the defects are annealed by moderate heating. Ab initio calculations performed on oligomers of 1 show that the O-H.N hydrogen bridge is about 0.064 A shorter and less bent ( approximately 171 degrees ) than the O.H-N hydrogen bridge ( approximately 150 degrees ). For an isolated ribbon, this result leads to structures with localized protons, either to a cycle with about 200 molecules, or to a quasi-linear ribbon involving an undulated structure, or to a combination of both motifs. Only the undulated structure is compatible with the linear ribbon observed by X-ray crystallography, where the fast proton transfer in the high-temperature phase is assisted by the motions of the undulated chain. A disordered structure is assigned to the amorphous phase, which exhibits the combination of the curved and the undulated motifs.

Photosensitization with derivatives of chlorin p6.

Biophysical and photobiological properties of three derivatives of chlorin p6 were examined. These agents can be considered as lysyl analogs of the aspartyl chlorin NPe6. Lysyl chlorin p6 diester (LCP) and the triester analog (LCP2) were readily accumulated by murine leukemia L1210 cells, localized in lysosomes, and were relatively inefficient photosensitizing agents in vitro. In contrast, lysyl chlorin e6 imide (LCI) was poorly accumulated, concentrated in mitochondrial and plasma membranes, but was more efficacious. LCI was the most effective agent with regard to photosensitization of a murine tumor in vivo, but all three agents caused substantially more toxicity than was observed with NPe6.