Paula Morales

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.

Cannabinoid receptor 2 (CB2) agonists and antagonists: a patent update.

ABSTRACT Introduction: Modulation of the CB2 receptor is an interesting approach for pain and inflammation, arthritis, addictions, neuroprotection, and cancer, among other possible therapeutic applications, and is devoid of central side effects. Areas covered: This review highlights the novel scaffolds for CB2 ligands and the diverse therapeutic applications for CB2 modulators disclosed in patents published since 2012. Expert opinion: Structural diversity of CB2 modulator scaffolds characterized the patent literature. Several CB2 agonists reached clinical Phase II for pain management and inflammation. Other therapeutic applications need to be explored such as neuroprotection and/or neurodegeneration.

Description of a Bivalent Cannabinoid Ligand with Hypophagic Properties

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.

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.

Combining rimonabant and fentanyl in a single entity: preparation and pharmacological results

Based on numerous pharmacological studies that have revealed an interaction between cannabinoid and opioid systems at the molecular, neurochemical, and behavioral levels, a new series of hybrid molecules has been prepared by coupling the molecular features of two wellknown drugs, ie, rimonabant and fentanyl. The new compounds have been tested for their affinity and functionality regarding CB1 and CB2 cannabinoid and μ opioid receptors. In [35S]-GTPγS (guanosine 5′-O-[gamma-thio]triphosphate) binding assays from the post-mortem human frontal cortex, they proved to be CB1 cannabinoid antagonists and μ opioid antagonists. Interestingly, in vivo, the new compounds exhibited a significant dual antagonist action on the endocannabinoid and opioid systems.

Chromenopyrazole, a Versatile Cannabinoid Scaffold with in Vivo Activity in a Model of Multiple Sclerosis.

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 Theilers 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.

Identification of Novel GPR55 Modulators Using Cell-Impedance-Based Label-Free Technology

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.

Cannabidiol skews biased agonism at cannabinoid CB1 and CB2 receptors with smaller effect in CB1-CB2 heteroreceptor complexes

Graphical abstract Figure. No Caption available. ABSTRACT Currently, biased agonism is at the center stage of drug development approaches. We analyzed effects of a battery of cannabinoids plus/minus cannabidiol (CBD) in four functional parameters (cAMP levels, phosphorylation of extracellular signal–regulated kinases (ERK1/2), &bgr;‐arrestin recruitment and label‐free/DMR) in HEK‐293T cells expressing cannabinoid receptors, CB1 or CB2, or CB1‐CB2 heteroreceptor complexes. In all cases two natural agonists plus two selective synthetic agonists were used. Furthermore, the effect of cannabidiol, at a dose (100 nM) that does not allow significant binding to the orthosteric center of either receptor, was measured. From the huge amount of generated data, we would like to highlight that the two psychotropic molecules (&Dgr;9‐tetrahydrocannabinol/THC and CP‐55940) showed similar bias in CB1R and that the bias of THC was particularly relevant toward MAPK pathway. Furthermore, THC did not activate the Gi protein coupled to CB2R. Interestingly, the biased agonism was reduced when assays were performed in cells expressing the two receptors, thus suggesting that the heteromer allows less functional selectivity. In terms of cannabidiol action, the phytocannabinoid altered the functional responses, likely by allosteric means, and modified potency, agonist IC50/EC50 values and biased agonism in qualitative and/or quantitative different ways depending on the agonist. The effect of cannabidiol on anandamide actions on both cannabinoid receptors was particularly noteworthy as was significantly different from that of other compounds. Results are a compendium of data on biased agonism on cannabinoid receptors in the absence and presence of cannabidiol. In addition, for the first time, GPCR biased agonism is characterized in an heteromeric context.