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Cannabis plant has been used from ancient times with therapeutic purposes for treating human pathologies, but the identification of the cellular and molecular mechanisms underlying the therapeutic properties of the phytocannabinoids, the active compounds in this plant, occurred in the last years of the past century. In the late 1980s and early 1990s, seminal studies demonstrated the existence of cannabinoid receptors and other elements of the so-called endocannabinoid system. These G protein-coupled receptors (GPCRs) are a key element in the functions assigned to endocannabinoids and appear to serve as promising pharmacological targets. They include CB 1 , CB 2 , and GPR55, but also non-GPCRs can be activated by endocannabinoids, like ionotropic receptor TRPV 1 and even nuclear receptors of the PPAR family. Their activation, inhibition, or simply modulation have been associated with numerous physiological effects at both central and peripheral levels, which may have therapeutic value in different human pathologies, then providing a solid experimental explanation for both the ancient medicinal uses of Cannabis plant and the recent advances in the development of cannabinoid-based specific therapies. This chapter will review the scientific knowledge generated in the last years around the research on the different endocannabinoid-binding receptors and their signaling mechanisms. Our intention is that this knowledge may help readers to understand the relevance of these receptors in health and disease conditions, as well as it may serve as the theoretical basis for the different experimental protocols to investigate these receptors and their signaling mechanisms that will be described in the following chapters., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

GPR55 is an orphan G-protein coupled receptor involved in various pathophysiological conditions. However, there are only a few noncannabinoid GPR55 ligands reported so far. The lack of potent and selective GPR55 ligands precludes a deep exploration of this receptor. The studies presented here focused on a thienopyrimidine scaffold based on the GPR55 antagonist ML192, previously discovered by high-throughput screening. The GPR55 activities of the new synthesized compounds were assessed using β-arrestin recruitment assays in Chinese hamster ovary cells overexpressing human GPR55. Some derivatives were identified as GPR55 antagonists with functional efficacy and selectivity versus CB1 and CB2 cannabinoid receptors., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Cannabinoids act as pleiotropic compounds exerting, among others, a broad-spectrum of neuroprotective effects. These effects have been investigated in the last years in different preclinical models of neurodegeneration, with the cannabinoid type-1 (CB 1 ) and type-2 (CB 2 ) receptors concentrating an important part of this research. However, the issue has also been extended to additional targets that are also active for cannabinoids, such as the orphan G-protein receptor 55 (GPR55). In the present study, we investigated the neuroprotective potential of VCE-006.1, a chromenopyrazole derivative with biased orthosteric and positive allosteric modulator activity at GPR55, in murine models of two neurodegenerative diseases. First, we proved that VCE-006.1 alone could induce ERK1/2 activation and calcium mobilization, as well as increase cAMP response but only in the presence of lysophosphatidyl inositol. Next, we investigated this compound administered chronically in two neurotoxin-based models of Parkinson's disease (PD), as well as in some cell-based models. VCE-006.1 was active in reversing the motor defects caused by 6-hydroxydopamine (6-OHDA) in the pole and the cylinder rearing tests, as well as the losses in tyrosine hydroxylase-containing neurons and the elevated glial reactivity detected in the substantia nigra. Similar cytoprotective effects were found in vitro in SH-SY5Y cells exposed to 6-OHDA. We also investigated VCE-006.1 in LPS-lesioned mice with similar beneficial effects, except against glial reactivity and associated inflammatory events, which remained unaltered, a fact confirmed in BV2 cells treated with LPS and VCE-006.1. We also analyzed GPR55 in these in vivo models with no changes in its gene expression, although GPR55 was down-regulated in BV2 cells treated with LPS, which may explain the lack of efficacy of VCE-006.1 in such an assay. Furthermore, we investigated VCE-006.1 in two genetic models of amyotrophic lateral sclerosis (ALS), mutant SOD1, or TDP-43 transgenic mice. Neither the neurological decline nor the deteriorated rotarod performance were prevented with this compound, and the same happened with the elevated microglial and astroglial reactivities, albeit modest spinal motor neuron preservation was achieved in both models. We also analyzed GPR55 in these in vivo models and found no changes in both TDP-43 transgenic and mSOD1 mice. Therefore, our findings support the view that targeting the GPR55 may afford neuroprotection in experimental PD, but not in ALS, thus stressing the specificities for the development of cannabinoid-based therapies in the different neurodegenerative disorders.

Natural cannabidiol ((-)-CBD) and its derivatives have increased interest for medicinal applications due to their broad biological activity spectrum, including targeting of the cannabinoid receptors type 1 (CB 1 R) and type 2 (CB 2 R). Herein, we synthesized the (+)-enantiomer of CBD and its derivative (+)-CBD hydroxypentylester ((+)-CBD-HPE) that showed enhanced CB 1 R and CB 2 R binding and functional activities compared to their respective (-) enantiomers. (+)-CBD-HPE Ki values for CB 1 R and CB 2 R were 3.1 ± 1.1 and 0.8 ± 0.1 nM respectively acting as CB 1 R antagonist and CB 2 R agonist. We further tested the capacity of (+)-CBD-HPE to prevent hyperglycemia and its complications in a mouse model. (+)-CBD-HPE significantly reduced streptozotocin (STZ)-induced hyperglycemia and glucose intolerance by preserving pancreatic beta cell mass. (+)-CBD-HPE significantly reduced activation of NF-κB by phosphorylation by 15% compared to STZ-vehicle mice, and CD3 + T cell infiltration into the islets was avoided. Consequently, (+)-CBD-HPE prevented STZ-induced apoptosis in islets. STZ induced inflammation and kidney damage, visualized by a significant increase in plasma proinflammatory cytokines, creatinine, and BUN. Treatment with (+)-CBD-HPE significantly reduced 2.5-fold plasma IFN-γ and increased 3-fold IL-5 levels compared to STZ-treated mice, without altering IL-18. (+)-CBD-HPE also significantly reduced creatinine and BUN levels to those comparable to healthy controls. At the macroscopy level, (+)-CBD-HPE prevented STZ-induced lesions in the kidney and voided renal fibrosis and CD3 + T cell infiltration. Thus, (+)-enantiomers of CBD, particularly (+)-CBD-HPE, have a promising potential due to their pharmacological profile and synthesis, potentially to be used for metabolic and immune-related disorders., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The 3-hydroxyquinone derivative of the non-psychotrophic phytocannabinoid cannabigerol, so-called VCE-003.2, and some other derivatives have been recently investigated for neuroprotective properties in experimental models of Parkinson's disease (PD) in mice. The pharmacological effects in those models were related to the activity on the peroxisome proliferator-activated receptor-γ (PPAR-γ) and possibly other pathways. In the present study, we investigated VCE-004.8 (formulated as EHP-101 for oral administration), the 3-hydroxyquinone derivative of cannabidiol (CBD), with agonist activity at the cannabinoid receptor type-2 (CB 2 ) receptor in addition to its activity at the PPAR-γ receptor. Studies were conducted in both in vivo (lesioned-mice) and in vitro (SH-SY5Y cells) models using the classic parkinsonian neurotoxin 6-hydroxydopamine (6-OHDA). Our data confirmed that the treatment with VCE-004.8 partially reduced the loss of tyrosine hydroxylase (TH)-positive neurons measured in the substantia nigra of 6-OHDA-lesioned mice, in parallel with an almost complete reversal of the astroglial (GFAP) and microglial (CD68) reactivity occurring in this structure. Such neuroprotective effects attenuated the motor deficiencies shown by 6-OHDA-lesioned mice in the cylinder rearing test, but not in the pole test. Next, we explored the mechanism involved in the beneficial effect of VCE-004.8 in vivo, by analyzing cell survival in cultured SH-SY5Y cells exposed to 6-OHDA. We found an important cytoprotective effect of VCE-004.8 at a concentration of 10 µM, which was completely reversed by the addition of antagonists, T0070907 and SR144528, aimed at blocking PPAR-γ and CB 2 receptors, respectively. The treatment with T0070907 alone only caused a partial reversal, whereas SR144528 alone had no effect, indicating a major contribution of PPAR-γ receptors in the cytoprotective effect of VCE-004.8 at 10 µM. In summary, our data confirmed the neuroprotective potential of VCE-004.8 in 6-OHDA-lesioned mice, and in vitro studies confirmed a greater relevance for PPAR-γ receptors rather than CB 2 receptors in these effects.

Given that neuronal degeneration in Alzheimer's disease (AD) is caused by the combination of multiple neurotoxic insults, current directions in the research of novel therapies to treat this disease attempts to design multitarget strategies that could be more effective than the simply use of acetylcholinesterase inhibitors; currently, the most used therapy for AD. One option, explored recently, is the synthesis of new analogues of cannabinoids that could competitively inhibit the acetylcholinesterase (AChE) enzyme and showing the classic neuroprotective profile of cannabinoid compounds. In this work, molecular docking has been used to design some cannabinoid analogues with such multitarget properties, based on the similarities of donepezil and Δ 9 -tetrahydrocannabinol. The analogues synthesized, compounds 1 and 2, demonstrated to have two interesting characteristics in different in vitro assays: competitive inhibition of AChE and competitive antagonism at the CB 1 /CB 2 receptors. They are highly lipophilic, highlighting that they could easily reach the CNS, and apparently presented a low toxicity. These results open the door to the synthesis of new compounds for a more effective treatment of AD., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The quinone derivative of the non-psychotropic cannabinoid cannabigerol (CBG), so-called VCE-003.2, has been recently investigated for its neuroprotective properties in inflammatory models of Parkinson's disease (PD) in mice. Such potential derives from its activity at the peroxisome proliferator-activated receptor-γ (PPAR-γ). In the present study, we investigated the neuroprotective properties of VCE-003.2 against the parkinsonian neurotoxin 6-hydroxydopamine (6-OHDA), in comparison with two new CBG-related derivatives, the cannabigerolic acid quinone (CBGA-Q) and its sodium salt CBGA-Q-Salt, which, similarly to VCE-003.2, were found to be active at the PPAR-γ receptor, but not at the cannabinoid CB 1 and CB 2 receptors. First, we investigated their cytoprotective properties in vitro by analyzing cell survival in cultured SH-SY5Y cells exposed to 6-OHDA. We found an important cytoprotective effect of VCE-003.2 at a concentration of 20 μM, which was not reversed by the blockade of PPAR-γ receptors with GW9662, supporting its activity at an alternative site (non-sensitive to classic antagonists) in this receptor. We also found CBGA-Q and CBGA-Q-Salt being cytoprotective in this cell assay, but their effects were completely eliminated by GW9662, thus indicating that they are active at the canonical site in the PPAR-γ receptor. Then, we moved to in vivo testing using mice unilaterally lesioned with 6-OHDA. Our data confirmed that VCE-003.2 administered orally (20 mg/kg) preserved tyrosine hydroxylase (TH)-positive nigral neurons against 6-OHDA-induced damage, whereas it completely attenuated the astroglial (GFAP) and microglial (CD68) reactivity found in the substantia nigra of lesioned mice. Such neuroprotective effects caused an important recovery in the motor deficiencies displayed by 6-OHDA-lesioned mice in the pole test and the cylinder rearing test. We also investigated CBGA-Q, given orally (20 mg/kg) or intraperitoneally (10 mg/kg, i.p.), having similar benefits compared to VCE-003.2 against the loss of TH-positive nigral neurons, glial reactivity and motor defects caused by 6-OHDA. Lastly, the sodium salt of CBGA-Q, given orally (40 mg/kg) to 6-OHDA-lesioned mice, also showed benefits at behavioral and histopathological levels, but to a lower extent compared to the other two compounds. In contrast, when given i.p., CBGA-Q-Salt (10 mg/kg) was poorly active. We also analyzed the concentrations of dopamine and its metabolite DOPAC in the striatum of 6-OHDA-lesioned mice after the treatment with the different compounds, but recovery in the contents of both dopamine and DOPAC was only found after the treatment with VCE-003.2. In summary, our data confirmed the neuroprotective potential of VCE-003.2 in 6-OHDA-lesioned mice, which adds to its previous activity found in an inflammatory model of PD (LPS-lesioned mice). Additional phytocannabinoid derivatives, CBGA-Q and CBGA-Q-Salt, also afforded neuroprotection in 6-OHDA-lesioned mice, but their effects were lower compared to VCE-003.2, in particular in the case of CBGA-Q-Salt. In vitro studies confirmed the relevance of PPAR-γ receptors for these effects., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background and Purpose: Δ 9 -Tetrahydrocannabinolic acid (Δ 9 -THCA-A), the precursor of Δ 9 -THC, is a non-psychotropic phytocannabinoid that shows PPARγ agonist activity. Here, we investigated the ability of Δ 9 -THCA-A to modulate the classic cannabinoid CB 1 and CB 2 receptors and evaluated its anti-arthritis activity in vitro and in vivo., Experimental Approach: Cannabinoid receptors binding and intrinsic activity, as well as their downstream signalling, were analysed in vitro and in silico. The anti-arthritis properties of Δ 9 -THCA-A were studied in human chondrocytes and in the murine model of collagen-induced arthritis (CIA). Plasma disease biomarkers were identified by LC-MS/MS based on proteomic and elisa assays., Key Results: Functional and docking analyses showed that Δ 9 -THCA-A can act as an orthosteric CB 1 receptor agonist and also as a positive allosteric modulator in the presence of CP-55,940. Also, Δ 9 -THCA-A seemed to be an inverse agonist for CB 2 receptors. In vivo, Δ 9 -THCA-A reduced arthritis in CIA mice, preventing the infiltration of inflammatory cells, synovium hyperplasia, and cartilage damage. Furthermore, Δ 9 -THCA-A inhibited expression of inflammatory and catabolic genes on knee joints. The anti-arthritic effect of Δ 9 -THCA-A was blocked by either SR141716 or T0070907. Analysis of plasma biomarkers, and determination of cytokines and anti-collagen antibodies confirmed that Δ 9 -THCA-A mediated its activity mainly through PPARγ and CB 1 receptor pathways., Conclusion and Implications: Δ 9 -THCA-A modulates CB 1 receptors through the orthosteric and allosteric binding sites. In addition, Δ 9 -THCA-A exerts anti-arthritis activity through CB 1 receptors and PPARγ pathways, highlighting its potential for the treatment of chronic inflammatory diseases such as rheumatoid arthritis., (© 2020 The British Pharmacological Society.)

Background: As a library of cannabinoid (CB) derivatives with (-)- trans -cannabidiol (CBD) or (-)- trans -cannabidivarin (CBDV) scaffold, we synthesized nine novel cannabinoids: 2-hydroxyethyl cannabidiolate (2-HEC), 2-hydroxypentyl cannabidiolate (2-HPC), 2,3-dihydroxypropyl cannabidiolate (GCBD), cyclohexyl cannabidiolate (CHC), n -hexyl-cannabidiolate (HC), 2-(methylsulfonamido)ethyl cannabidiolate (NMSC), 2-hydroxyethyl cannabidivarinolate (2-HECBDV), cyclohexyl cannabidivarinolate (CHCBDV), and n -hexyl cannabidivarinolate (HCBDV). Their binding and intrinsic effects at the CB1- and CB2-receptors and the effects on inflammatory signaling cascades were investigated in in vitro and ex vivo cell models. Materials and Methods: Binding affinity was studied in membranes isolated from CB-receptor-transfected HEK293EBNA cells, intrinsic functional activity in Chinese hamster ovary (CHO) cells, and activation of nuclear factor κB (NF-κB) and nuclear factor of activated T-cells (NFAT) in phorbol 12-myristate 13-acetate (PMA)/ionomycin (IO)-treated Jurkat T-cells. Inhibition of interleukin (IL)-17-induced pro-inflammatory cytokines and chemokines [IL-6, IL-1β, CC-chemokine ligand 2 (CCL2), and tumor necrosis factor (TNF)-α] was studied in RAW264.7 macrophages at the RNA level. Pro-inflammatory cytokine (IL-1β, IL-6, IL-8, and TNF-α) expression and prostaglandin E2 (PGE 2 ) expression were investigated at the protein level in lipopolysaccharide (LPS)-treated primary human monocytes. Results: Derivatives with long aliphatic side chains at the ester position at R 1 [HC ( 5 )] as well as the ones with polar side chains [2-HECBDV ( 7 ), NMSC ( 6 ), and 2-HEC ( 1 )] can be selective for CB2-receptors. The CBDV-derivatives HCBDV and CHCBDV demonstrated specific binding at CB1- and CB2-receptors at nanomolar concentrations. 2-HEC, 2-HPC, GCBD, and NMSC were agonists at CB2-receptor and antagonists at CB1-receptor. CHC bound both receptors at submicromolar ranges and was an agonist for these receptors. 2-HECBDV was an agonist at CB2-receptor and an antagonist at the CB1-receptor despite its modest affinity at this receptor (micromolar range). NMSC inhibited NF-κB and NFAT activity, and 2-HEC, 2-HPC, and GCBD dose-dependently inhibited PMA/IO-stimulated NFAT activation. CHC and HC dose-dependently reduced IL-1β and CCL2 messenger RNA (mRNA) expression. NMSC inhibited IL-1β, CCL2, and TNF-α at lower doses. At higher doses, it induced a pronounced increase in IL-6 mRNA. 2-HEC, 2-HPC, and GCBD dose-dependently inhibited LPS-induced IL-1β, TNF-α, and IL-6 synthesis. NMSC further increased LPS-stimulated IL-1β release but inhibited IL-8, TNF-α, and PGE 2 . Conclusion: The CBD- and CBDV-derivatives studied are suitable for targeting CB-receptors. Some may be used as selective CB2 agonists. The length of the aliphatic rest at R 2 of CBD (pentyl) and CBDV (propyl) did not correlate with the binding affinity. Higher polarity at R 1 appeared to favor the agonistic activity at CB2-receptors., (Copyright © 2019 Götz, Collado, Fernández-Ruiz, Fiebich, García-Toscano, Gómez-Cañas, Koch, Leha, Muñoz, Navarrete, Pazos and Holzgrabe.)

In a recent study, we described the neuroprotective properties of VCE-003.2-an aminoquinone derivative of the non-psychotropic phytocannabinoid cannabigerol (CBG)-administered intraperitoneally (i.p.) in an inflammatory model of Parkinson's disease (PD). We also demonstrated that these properties derive from its activity on the peroxisome proliferator-activated receptor-γ, in particular at a regulatory site within this receptor type. In the present study, we wanted to further confirm this neuroprotective potential using an oral lipid formulation of VCE-003.2, developed to facilitate the clinical development of this phytocannabinoid derivative. To this end, we evaluated VCE-003.2, administered orally at two doses (10 and 20 mg/kg), to mice subjected to unilateral intrastriatal injections of lipopolysaccharide (LPS), a classic model of inflammation-driven neuronal deterioration that recapitulates characteristics of PD. The administration of VCE-003.2 to these mice showed, as expected, poor activity in the different motor tests (rotarod, computer-aided actimeter) used in experimental parkinsonism, in general due to the lack of evident changes in these behaviors by LPS lesion. However, VCE-003.2, at 20 mg/kg, was highly active in improving the changes detected in LPS-lesioned mice in the cylinder rearing test. In addition, the histopathological analysis of the basal ganglia revealed a trend towards recovery at 20 mg/kg VCE-003.2 in the loss of tyrosine hydroxylase-containing nigrostriatal neurons, as well as a complete reduction in the elevated LAMP-1 immunolabeling (reflecting autophagy impairment) caused by LPS lesion. These effects were not seen at 10 mg/kg. This was associated with a partial reduction in the intense glial reactivity provoked by LPS in the substantia nigra, in particular the astroglial reactivity labeled with glial fibrillary acidic protein. The analysis using qPCR in the striatum of proinflammatory mediators, such as tumor necrosis factor-α, interleukin-1β, inducible nitric oxide synthase, and cyclooxygenase-2, showed that the marked elevations provoked by the LPS lesion tended to be, in general, attenuated by VCE-003.2 treatment, with the greatest effects normally found with the highest dose of 20 mg/kg. In summary, our data confirm the neuroprotective potential of an oral formulation of VCE-003.2 against neuronal injury in an in vivo model of PD based on neuroinflammation, and this study opens the possibility to further the development of oral VCE-003.2 in the clinic.

Background and Purpose: The endocannabinoid system and PPARγ are important targets for the development of novel compounds against fibrotic diseases such as systemic sclerosis (SSc), also called scleroderma. The aim of this study was to characterize VCE-004.3, a novel cannabidiol derivative, and study its anti-inflammatory and anti-fibrotic activities., Experimental Approach: The binding of VCE-004.3 to CB 1 and CB 2 receptors and PPARγ and its effect on their functional activities were studied in vitro and in silico. Anti-fibrotic effects of VCE-004.3 were investigated in NIH-3T3 fibroblasts and human dermal fibroblasts. To assess its anti-inflammatory and anti-fibrotic efficacy in vivo, we used two complementary models of bleomycin-induced fibrosis. Its effect on ERK1/2 phosphorylation induced by IgG from SSc patients and PDGF was also investigated., Key Results: VCE-004.3 bound to and activated PPARγ and CB 2 receptors and antagonized CB 1 receptors. VCE-004.3 bound to an alternative site at the PPARγ ligand binding pocket. VCE-004.3 inhibited collagen gene transcription and synthesis and prevented TGFβ-induced fibroblast migration and differentiation to myofibroblasts. It prevented skin fibrosis, myofibroblast differentiation and ERK1/2 phosphorylation in bleomycin-induced skin fibrosis. Furthermore, it reduced mast cell degranulation, macrophage activation, T-lymphocyte infiltration, and the expression of inflammatory and profibrotic factors. Topical application of VCE-004.3 also alleviated skin fibrosis. Finally, VCE-004.3 inhibited PDGF-BB- and SSc IgG-induced ERK1/2 activation in fibroblasts., Conclusions and Implications: VCE-004.3 is a novel semisynthetic cannabidiol derivative that behaves as a dual PPARγ/CB 2 agonist and CB 1 receptor modulator that could be considered for the development of novel therapies against different forms of scleroderma., (© 2018 The British Pharmacological Society.)

Background: Neuroprotection with cannabinoids in Parkinson's disease (PD) has been afforded predominantly with antioxidant or anti-inflammatory cannabinoids. In the present study, we investigated the anti-inflammatory and neuroprotective properties of VCE-003.2, a quinone derivative of the non-psychotrophic phytocannabinoid cannabigerol (CBG), which may derive its activity at the peroxisome proliferator-activated receptor-γ (PPARγ). The compound is also an antioxidant., Methods: We evaluated VCE-003.2 in an in vivo [mice subjected to unilateral intrastriatal injections of lipopolysaccharide (LPS)] model of PD, as well as in in vitro (LPS-exposed BV2 cells and M-213 cells treated with conditioned media generated from LPS-exposed BV2 cells) cellular models. The type of interaction of VCE-003.2 at the PPARγ receptor was furtherly investigated in bone marrow-derived human mesenchymal stem cells (MSCs) and sustained with transcriptional assays and in silico docking studies., Results: VCE-003.2 has no activity at the cannabinoid receptors, a fact that we confirmed in this study using competition studies. The administration of VCE-003.2 to LPS-lesioned mice attenuated the loss of tyrosine hydroxylase (TH)-containing nigrostriatal neurons and, in particular, the intense microgliosis provoked by LPS in the substantia nigra, measured by Iba-1/Cd68 immunostaining. The analysis by qPCR of proinflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and inducible nitric oxide synthase (iNOS) in the striatum showed they were markedly elevated by the LPS lesion and strongly reduced by the treatment with VCE-003.2. The effects of VCE-003.2 in LPS-lesioned mice implied the activation of PPARγ receptors, as they were attenuated when VCE-003.2 was co-administered with the PPARγ inhibitor T0070907. We then moved to some in vitro approaches, first to confirm the anti-inflammatory profile of VCE-003.2 in cultured BV2 cells exposed to LPS. VCE-003.2 was able to attenuate the synthesis and release of TNF-α and IL-1β, as well as the induction of iNOS and cyclooxygenase-2 (COX-2) elicited by LPS in these cells. However, we found such effects were not reversed by GW9662, another classic PPARγ antagonist. Next, we investigated the neuroprotective effects of VCE-003.2 in cultured M-213 neuronal cells exposed to conditioned media generated from LPS-exposed cultured BV2 cells. VCE-003.2 reduced M-213 cell death, but again, such effects were not reversed by T0070907. Using docking analysis, we detected that VCE-003.2 binds both the canonical and the alternative binding sites in the PPARγ ligand-binding pocket (LBP). Functional assays further showed that T0070907 almost abolished PPARγ transcriptional activity induced by rosiglitazone (RGZ), but it did not affect the activity of VCE-003.2 in a Gal4-Luc system. However, T0070907 inhibited the effects of RGZ and VCE-003.2 on the expression of PPARγ-dependent genes upregulated in MSCs., Conclusions: We have demonstrated that VCE-003.2 is neuroprotective against inflammation-driven neuronal damage in an in vivo model of PD and in in vitro cellular models of neuroinflammation. Such effects might involve PPARγ receptors, although in silico and in vitro experiments strongly suggest that VCE-003.2 targets PPARγ by acting through two binding sites at the LBP, one that is sensitive to T0070907 (canonical binding site) and other that is not affected by this PPARγ antagonist (alternative binding site).

A series of sulfenamide and sulfonamide derivatives was synthesized and evaluated for the affinity at CB 1 and CB 2 receptors. The N-bornyl-S-(5,6-di-p-tolylpyridazin-3-yl)-sulfenamide, compound 11, displayed good affinity and high selectivity for CB 1 receptors (K i values of 44.6 nM for CB 1 receptors and >40 μM for CB 2 receptors, respectively). The N-isopinocampheyl-sulfenamide 12 and its sulfonamide analogue 22 showed similar selectivity for CB 1 receptors with K i values of 75.5 and 73.2 nM, respectively. These novel compounds behave as antagonists/inverse agonists at CB 1 receptor in the [ 35 S]-GTPγS binding assays, and none showed adequate predictive blood-brain barrier permeation, exhibiting low estimated LD 50 . However, testing compound 12 in a supraspinal analgesic test (hot-plate) revealed that it was as effective as the classic CB 1 receptor antagonist rimonabant, in reversing the analgesic effect of a cannabinoid agonist., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In the last few years, cannabinoid type-2 receptor (CB 2 R) selective ligands have shown a great potential as novel therapeutic drugs in several diseases. With the aim of discovering new selective cannabinoid ligands, a series of pyridazinone-4-carboxamides was designed and synthesized, and the new derivatives tested for their affinity toward the hCB 1 R and hCB 2 R. The 6-(4-chloro-3-methylphenyl)-2-(4-fluorobenzyl)-N-(cis-4-methylcyclohexyl)-3-oxo-2,3-dihydropyridazine-4-carboxamide (9) displayed high CB 2 -affinity (K i CB 2  = 2.0 ± 0.81 nM) and a notable selectivity (K i CB 1 /K i CB 2  > 2000). In addition, 9 and other active new synthesized entities have demonstrated to behave as CB 2 R inverse agonists in [ 35 S]-GTPγS binding assay. ADME predictions of the newly synthesized CB 2 R ligands suggest a favourable pharmacokinetic profile. Docking studies disclosed the specific pattern of interactions of these derivatives. Our results support that pyridazinone-4-carboxamides represent a new promising scaffold for the development of potent and selective CB 2 R ligands., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

A combination of molecular modeling and structure-activity relationship studies has been used to fine-tune CB2 selectivity in the chromenopyrazole ring, a versatile CB1/CB2 cannabinoid scaffold. Thus, a series of 36 new derivatives covering a wide range of structural diversity has been synthesized, and docking studies have been performed for some of them. Biological evaluation of the new compounds includes, among others, cannabinoid binding assays, functional studies, and surface plasmon resonance measurements. The most promising compound [43 (PM226)], a selective and potent CB2 agonist isoxazole derivative, was tested in the acute phase of Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD), a well-established animal model of primary progressive multiple sclerosis. Compound 43 dampened neuroinflammation by reducing microglial activation in the TMEV.

Previous studies have investigated the relevance and structure-activity relationships (SARs) of pyrazole derivatives in relation with cannabinoid receptors, and the series of tricyclic 1,4-dihydroindeno[1,2-c]pyrazoles emerged as potent CB2 receptor ligands. In the present study, novel 1,4-dihydroindeno[1,2-c]pyrazole and 1H-benzo[g]indazole carboxamides containing a cyclopropyl or a cyclohexyl substituent were designed and synthesized to evaluate the influence of these structural modifications towards CB1 and CB2 receptor affinities. Among these derivatives, compound 15 (6-cyclopropyl-1-(2,4-dichlorophenyl)-N-(adamantan-1-yl)-1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamide) showed the highest CB2 receptor affinity (Ki = 4 nM) and remarkable selectivity (KiCB1/KiCB2 = 2232), whereas a similar affinity, within the nM range, was seen for the fenchyl derivative (compound 10: Ki = 6 nM), for the bornyl analogue (compound 14: Ki = 38 nM) and, to a lesser extent, for the aminopiperidine derivative (compound 6: Ki = 69 nM). Compounds 10 and 14 were also highly selective for the CB2 receptor (KiCB1/KiCB2 > 1000), whereas compound 6 was relatively selective (KiCB1/KiCB2 = 27). The four compounds were also subjected to GTPγS binding analysis showing antagonist/inverse agonist properties (IC50 for compound 14 = 27 nM, for 15 = 51 nM, for 10 = 80 nM and for 6 = 294 nM), and this activity was confirmed for the three more active compounds in a CB2 receptor-specific in vitro bioassay consisting in the quantification of prostaglandin E2 release by LPS-stimulated BV2 cells, in the presence and absence of WIN55,212-2 and/or the investigated compounds. Modelling studies were also conducted with the four compounds, which conformed with the structural requirements stated for the binding of antagonist compounds to the human CB2 receptor., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

The orphan G protein-coupled receptor GPR55 has been proposed as a novel receptor of the endocannabinoid system. However, the validity of this categorization is still under debate mainly because of the lack of potent and selective agonists and antagonists of GPR55. Binding assays are not yet available for GPR55 screening, and discrepancies in GPR55 mediated signaling pathways have been reported. In this context, we have designed and synthesized novel GPR55 ligands based on a chromenopyrazole scaffold. Appraisal of GPR55 activity was accomplished using a label-free cell-impedance-based assay in hGPR55-HEK293 cells. The real-time impedance responses provided an integrative assessment of the cellular consequence to GPR55 stimulation taking into account the different possible signaling pathways. Potent GPR55 partial agonists (14b, 18b, 19b, 20b, and 21-24) have been identified; one of them (14b) being selective versus classical cannabinoid receptors. Upon antagonist treatment, chromenopyrazoles 21-24 inhibited lysophosphatidylinositol (LPI) effect. One of these GPR55 antagonists (21) is fully selective versus classic cannabinoid receptors. Compared to LPI, the predicted physicochemical parameters of the new compounds suggest a clear pharmacokinetic improvement.

Scleroderma is a group of rare diseases associated with early and transient inflammation and vascular injury, followed by fibrosis affecting the skin and multiple internal organs. Fibroblast activation is the hallmark of scleroderma, and disrupting the intracellular TGFβ signaling may provide a novel approach to controlling fibrosis. Because of its potential role in modulating inflammatory and fibrotic responses, both PPARγ and CB2 receptors represent attractive targets for the development of cannabinoid-based therapies. We have developed a non-thiophilic and chemically stable derivative of the CBD quinol (VCE-004.8) that behaves as a dual agonist of PPARγ and CB2 receptors, VCE-004.8 inhibited TGFβ-induced Col1A2 gene transcription and collagen synthesis. Moreover, VCE-004.8 inhibited TGFβ-mediated myofibroblast differentiation and impaired wound-healing activity. The anti-fibrotic efficacy in vivo was investigated in a murine model of dermal fibrosis induced by bleomycin. VCE-004.8 reduced dermal thickness, blood vessels collagen accumulation and prevented mast cell degranulation and macrophage infiltration in the skin. These effects were impaired by the PPARγ antagonist T0070907 and the CB2 antagonist AM630. In addition, VCE-004.8 downregulated the expression of several key genes associated with fibrosis, qualifying this semi-synthetic cannabinoid as a novel compound for the management of scleroderma and, potentially, other fibrotic diseases.

The CB1 cannabinoid receptor, the main target of Δ(9)-tetrahydrocannabinol (THC), the most prominent psychoactive compound of marijuana, plays a crucial regulatory role in brain development as evidenced by the neurodevelopmental consequences of its manipulation in animal models. Likewise, recreational cannabis use during pregnancy affects brain structure and function of the progeny. However, the precise neurobiological substrates underlying the consequences of prenatal THC exposure remain unknown. As CB1 signaling is known to modulate long-range corticofugal connectivity, we analyzed the impact of THC exposure on cortical projection neuron development. THC administration to pregnant mice in a restricted time window interfered with subcerebral projection neuron generation, thereby altering corticospinal connectivity, and produced long-lasting alterations in the fine motor performance of the adult offspring. Consequences of THC exposure were reminiscent of those elicited by CB1 receptor genetic ablation, and CB1-null mice were resistant to THC-induced alterations. The identity of embryonic THC neuronal targets was determined by a Cre-mediated, lineage-specific, CB1 expression-rescue strategy in a CB1-null background. Early and selective CB1 reexpression in dorsal telencephalic glutamatergic neurons but not forebrain GABAergic neurons rescued the deficits in corticospinal motor neuron development of CB1-null mice and restored susceptibility to THC-induced motor alterations. In addition, THC administration induced an increase in seizure susceptibility that was mediated by its interference with CB1-dependent regulation of both glutamatergic and GABAergic neuron development. These findings demonstrate that prenatal exposure to THC has long-lasting deleterious consequences in the adult offspring solely mediated by its ability to disrupt the neurodevelopmental role of CB1 signaling.

During the last years, there has been a continuous interest in the development of cannabinoid receptor ligands that may serve as therapeutic agents and/or as experimental tools. This prompted us to design and synthesize analogues of the CB2 receptor antagonist N-fenchyl-5-(4-chloro-3-methyl-phenyl)-1-(4-methyl-benzyl)-1H-pyrazole-3-carboxamide (SR144528). The structural modifications involved the bioisosteric replacement of the pyrazole ring by a pyrrole ring and variations on the amine carbamoyl substituents. Two of these compounds, the fenchyl pyrrole analogue 6 and the myrtanyl derivative 10, showed high affinity (Ki in the low nM range) and selectivity for the CB2 receptor and both resulted to be antagonists/inverse agonists in [(35)S]-GTPγS binding analysis and in an in vitro CB2 receptor bioassay. Cannabinoid receptor binding data of the series allowed identifying steric constraints within the CB2 binding pocket using a study of Van der Waals' volume maps. Glide docking studies revealed that all docked compounds bind in the same region of the CB2 receptor inactive state model., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Triple-negative breast cancer (TNBC) represents a subtype of breast cancer characterized by high aggressiveness. There is no current targeted therapy for these patients whose prognosis, as a group, is very poor. Here, we report the synthesis and evaluation of a potent antitumor agent in vivo for this type of breast cancer designed as a combination of quinone/cannabinoid pharmacophores. This new compound (10) has been selected from a series of chromenopyrazolediones with full selectivity for the nonpsychotropic CB2 cannabinoid receptor and with efficacy in inducing death of human TNBC cell lines. The dual concept quinone/cannabinoid was supported by the fact that compound 10 exerts antitumor effect by inducing cell apoptosis through activation of CB2 receptors and through oxidative stress. Notably, it did not show either cytotoxicity on noncancerous human mammary epithelial cells nor toxic effects in vivo, suggesting that it may be a new therapeutic tool for the management of TNBC.

The G protein-coupled receptors CB2 (CB2R) and GPR55 are overexpressed in cancer cells and human tumors. Because a modulation of GPR55 activity by cannabinoids has been suggested, we analyzed whether this receptor participates in cannabinoid effects on cancer cells. Here we show that CB2R and GPR55 form heteromers in cancer cells, that these structures possess unique signaling properties, and that modulation of these heteromers can modify the antitumoral activity of cannabinoids in vivo. These findings unveil the existence of previously unknown signaling platforms that help explain the complex behavior of cannabinoids and may constitute new targets for therapeutic intervention in oncology., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Designing drugs with a specific multi-target profile is a promising approach against multifactorial illnesses as Alzheimer's disease. In this work, new indazole ethers that possess dual activity as both cannabinoid agonists CB2 and inhibitors of BuChE have been designed by computational methods. On the basis of this knowledge, the synthesis, pharmacological evaluation and docking studies of a new class of indazoles has been performed. Pharmacological evaluation includes radioligand binding assays with [(3)H]-CP55940 for CB1R and CB2R and functional activity for cannabinoid receptors on isolated tissue. Additionally, in vitro inhibitory assays of AChE/BuChE and the corresponding competition studies have been carried out. The results of pharmacological tests have revealed that three of these derivatives behave as CB2 cannabinoid agonists and simultaneously show BuChE inhibition. In particular, compounds 3 and 24 have emerged as promising candidates as novel cannabinoids that inhibit BuChE by a non-competitive or mixed mechanism, respectively. On the other hand, both molecules show antioxidant properties., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Searching for novel antiobesity agents, a series of cannabinoid LH21 and of Rimonabant-fatty acid amide analogues have been prepared. Synthesis of pyrazoles 2a-2c was achieved by a two steps simple methodology via α,β-unsaturated ketones. Carboxamides 8a-8h were obtained in good yields from esters 7a-7c by a one-pot procedure which takes place under mild conditions. New compounds have been evaluated in vivo as anorectic agents. Some of them showed interesting properties reducing food intake in rats by a mechanism which does not involve the endocannabinoid system., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A series of bivalent cannabinoid ligands is proposed. The synthesis of double amides based on the rimonabant structure separated by an alkyl chain and the evaluation of their affinities for cannabinoid receptors are reported. The data of 4d confirmed that a bivalent structure is a suitable scaffold for CB1 cannabinoid receptor binding. The compound 4d was selected for in vitro and in vivo pharmacological evaluations. Moreover, intraperitoneal administration of 4d to food-deprived rats resulted in a dose-dependent inhibition of feeding that was maintained up to 240 min., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Phytocannabinoids like ∆(9)-tetrahydrocannabinol (THC) and cannabidiol (CBD) show a beneficial effect on neuroinflammatory and neurodegenerative processes through cell membrane cannabinoid receptor (CBr)-dependent and -independent mechanisms. Natural and synthetic cannabinoids also target the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARγ), an attractive molecular target for the treatment of neuroinflammation. As part of a study on the SAR of phytocannabinoids, we have investigated the effect of the oxidation modification in the resorcinol moiety of cannabigerol (CBG) on CB(1), CB(2) and PPARγ binding affinities, identifying cannabigerol quinone (VCE-003) as a potent anti-inflammatory agent. VCE-003 protected neuronal cells from excitotoxicity, activated PPARγ transcriptional activity and inhibited the release of pro-inflammatory mediators in LPS-stimulated microglial cells. Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis (MS) was used to investigate the anti-inflammatory activity of this compound in vivo. Motor function performance was evaluated and the neuroinflammatory response and gene expression pattern in brain and spinal cord were studied by immunostaining and qRT-PCR. We found that VCE-003 ameliorated the symptoms associated to TMEV infection, decreased microglia reactivity and modulated the expression of genes involved in MS pathophysiology. These data lead us to consider VCE-003 to have high potential for drug development against MS and perhaps other neuroinflammatory diseases.

The unwanted psychoactive effects of cannabinoid receptor agonists have limited their development as medicines. These CB₁-mediated side effects are due to the fact that CB₁ receptors are largely expressed in the central nervous system (CNS). As it is known that CB₁ receptors are also located peripherally, there is growing interest in targeting cannabinoid receptors located outside the brain. A library of chromenopyrazoles designed analogously to the classical cannabinoid cannabinol were synthesized, characterized, and tested for cannabinoid activity. Radioligand binding assays were used to determine their affinities at CB₁ and CB₂ receptors. Structural features required for CB₁/CB₂ affinity and selectivity were explored by molecular modeling. Some compounds in the chromenopyrazole series were observed to be selective CB₁ ligands. These modeling studies suggest that full CB₁ selectivity over CB₂ can be explained by the presence of a pyrazole ring in the structure. The functional activities of selected chromenopyrazoles were evaluated in isolated tissues. In vivo behavioral tests were then carried out on the most effective CB₁ cannabinoid agonist, 13 a. Chromenopyrazole 13 a did not induce modifications in any of the tested parameters on the mouse cannabinoid tetrad, thus discounting CNS-mediated effects. This lack of agonistic activity in the CNS suggests that this compound does not readily cross the blood-brain barrier. Moreover, 13 a can induce antinociception in a rat peripheral model of orofacial pain. Taking into account the negative results obtained with the hot-plate test, the antinociception induced by 13 a in the orofacial test could be mediated through peripheral mechanisms., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cannabinoids are promising medicines to slow down disease progression in neurodegenerative disorders including Parkinson's disease (PD) and Huntington's disease (HD), two of the most important disorders affecting the basal ganglia. Two pharmacological profiles have been proposed for cannabinoids being effective in these disorders. On the one hand, cannabinoids like Δ(9) -tetrahydrocannabinol or cannabidiol protect nigral or striatal neurons in experimental models of both disorders, in which oxidative injury is a prominent cytotoxic mechanism. This effect could be exerted, at least in part, through mechanisms independent of CB(1) and CB(2) receptors and involving the control of endogenous antioxidant defences. On the other hand, the activation of CB(2) receptors leads to a slower progression of neurodegeneration in both disorders. This effect would be exerted by limiting the toxicity of microglial cells for neurons and, in particular, by reducing the generation of proinflammatory factors. It is important to mention that CB(2) receptors have been identified in the healthy brain, mainly in glial elements and, to a lesser extent, in certain subpopulations of neurons, and that they are dramatically up-regulated in response to damaging stimuli, which supports the idea that the cannabinoid system behaves as an endogenous neuroprotective system. This CB(2) receptor up-regulation has been found in many neurodegenerative disorders including HD and PD, which supports the beneficial effects found for CB(2) receptor agonists in both disorders. In conclusion, the evidence reported so far supports that those cannabinoids having antioxidant properties and/or capability to activate CB(2) receptors may represent promising therapeutic agents in HD and PD, thus deserving a prompt clinical evaluation., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)