María Gómez-Cañas

Prospects for cannabinoid therapies in basal ganglia disorders

Cannabinoids are promising medicines to slow down disease progression in neurodegenerative disorders including Parkinsons disease (PD) and Huntingtons 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 Δ9‐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 CB1 and CB2 receptors and involving the control of endogenous antioxidant defences. On the other hand, the activation of CB2 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 CB2 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 CB2 receptor up‐regulation has been found in many neurodegenerative disorders including HD and PD, which supports the beneficial effects found for CB2 receptor agonists in both disorders. In conclusion, the evidence reported so far supports that those cannabinoids having antioxidant properties and/or capability to activate CB2 receptors may represent promising therapeutic agents in HD and PD, thus deserving a prompt clinical evaluation.

A Cannabigerol Quinone Alleviates Neuroinflammation in a Chronic Model of Multiple Sclerosis

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 CB1, CB2 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.

Targeting CB2-GPR55 Receptor Heteromers Modulates Cancer Cell Signaling

Background: Cannabinoid receptor CB2 (CB2R) and GPR55 are overexpressed in cancer cells and control cell fate. Results: In cancer cells, CB2R and GPR55 form heteromers that impact the signaling of each protomer. Conclusion: CB2R-GPR55 heteromers drive biphasic signaling responses as opposed to the individual receptors via cross-antagonism. Significance: These heteromers may explain some of the biphasic effects of cannabinoids and, therefore, constitute potential new targets in oncology. 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.

Prenatal exposure to cannabinoids evokes long-lasting functional alterations by targeting CB1 receptors on developing cortical neurons

Significance Marijuana is the most commonly used illicit drug, and its consumption constitutes a serious health concern. The psychoactivity of the plant is exerted by its cannabinoid constituents, especially Δ9-tetrahydrocannabinol (THC), which acts by engaging CB1 cannabinoid receptors. Despite the large knowledge accumulated on how THC affects the adult brain, its molecular and functional impact on neuronal development remains obscure. This study demonstrates that remarkable detrimental consequences of embryonic THC exposure on adult-brain function, which are evident long after THC withdrawal, are solely due to the impact of THC on CB1 receptors located on developing cortical neurons. Our findings thus delineate the risk of cannabis consumption during pregnancy and contribute to identify precise neuronal lineages targeted by prenatal THC exposure. The CB1 cannabinoid receptor, the main target of Δ9-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.

Selective, nontoxic CB2 cannabinoid o-quinone with in vivo activity against triple-negative breast cancer

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.

Chromenopyrazoles: Non‐psychoactive and Selective CB1 Cannabinoid Agonists with Peripheral Antinociceptive Properties

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.

Synthetic cannabinoid quinones: preparation, in vitro antiproliferative effects and in vivo prostate antitumor activity.

Chromenopyrazolediones have been designed and synthesized as anticancer agents using the multi-biological target concept that involves quinone cytotoxicity and cannabinoid antitumor properties. In cell cytotoxicity assays, these chromenopyrazolediones have antiproliferative activity against human prostate cancer and hepatocellular carcinoma. It has been shown that the most potent, derivative 4 (PM49), inhibits prostate LNCaP cell viability (IC₅₀ = 15 μM) through a mechanism involving oxidative stress, PPARγ receptor and partially CB₁ receptor. It acts on prostate cell growth by causing G₀/G₁ phase arrest and triggering apoptosis as assessed by flow cytometry measurements. In the in vivo treatment, compound 4 at 2 mg/kg, blocks the growth of LNCaP tumors and reduces the growth of PC-3 tumors generated in mice. These studies suggest that 4 is a good potential anticancer agent against hormone-sensitive prostate cancer.

Novel antiobesity agents: Synthesis and pharmacological evaluation of analogues of Rimonabant and of LH21

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.

Tricyclic pyrazoles. Part 8. Synthesis, biological evaluation and modelling of tricyclic pyrazole carboxamides as potential CB2 receptor ligands with antagonist/inverse agonist properties

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.

The cannabinoid quinol VCE-004.8 alleviates bleomycin-induced scleroderma and exerts potent antifibrotic effects through peroxisome proliferator-activated receptor-γ and CB2 pathways

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.