Classical Cannabinoids Research Articles

Safety and efficacy of a novel cannabinoid chemotherapeutic, KM-233, for the treatment of high-grade glioma

To test in vitro and in vivo the safety and efficacy of a novel chemotherapeutic agent, KM-233, for the treatment of glioma. In vitro cell cytotoxicity assays were used to measure and compare the cytotoxic effects of KM-233, Delta(8)-tetrahydrocannabinol (THC), and bis-chloroethyl-nitrosurea (BCNU) against human U87 glioma cells. An organotypic brain slice culture model was used for safety and toxicity studies. A human glioma-SCID mouse side-pocket tumor model was used to test in vivo the safety and efficacy of KM-233 with intratumoral and intra-peritoneal administration. KM-233 is a classical cannabinoid with good blood brain barrier penetration that possesses a selective affinity for the CB2 receptors relative to THC. KM-233 was as efficacious in its cytotoxicity against human U87 glioma as Delta(8)-tetrahydrocannabinol, and superior to the commonly used anti-glioma chemotherapeutic agent, BCNU. The cytotoxic effects of KM-233 against human glioma cells in vitro occur as early as two hours after administration, and dosing of KM-233 can be cycled without compromising cytotoxic efficacy and while improving safety. Cyclical dosing of KM-233 to treat U87 glioma in a SCID mouse xenograft side pocket model was effective at reducing the tumor burden with both systemic and intratumoral administration. These studies provide both in vitro and in vivo evidence that KM-233 shows promising efficacy against human glioma cell lines in both in vitro and in vivo studies, minimal toxicity to healthy cultured brain tissue, and should be considered for definitive preclinical development in animal models of glioma.

Therapeutic Potential of the Endocannabinoid System in the Brain

Cannabinoids have been predominantly considered as the substances responsible of the psychoactive properties of marijuana and other derivatives of Cannabis sativa. However, these compounds are now being also considered for their therapeutic potential, since the term "cannabinoid" includes much more compounds than those present in Cannabis sativa derivatives. Among them, there are numerous synthetic cannabinoids obtained by modifications from plant-derived cannabinoids, but also from the compounds that behave as endogenous ligands for the different cannabinoid receptor subtypes. Within the family of "cannabinoid-related compounds", one should also include some prototypes of selective antagonists for these receptors, and also the recently developed inhibitors of the mechanism of finalization of the biological action of endocannabinoids (transporter + FAAH). All this boom of the cannabinoid pharmacology has, therefore, an explanation in the recent discovery and characterization of the endocannabinoid signaling system, which plays a modulatory role mainly in the brain but also in the periphery. The objective of the present article will be to review, from pharmacological and biochemical points of view, the more recent advances in the study of the endocannabinoid system and their functions in the brain, as well as their alterations in a variety of pathologies and the proposed therapeutic benefits of novel cannabinoid-related compounds that improve the pharmacokinetic and pharmacodynamic properties of classic cannabinoids.

Adamantyl Cannabinoids: A Novel Class of Cannabinergic Ligands

Structure-activity relationship studies have established that the aliphatic side chain plays a pivotal role in determining the cannabinergic potency of tricyclic classical cannabinoids. We have now synthesized a series of analogues in which a variety of adamantyl substituents were introduced at the C3 position of Delta(8)-THC. Our lead compound, (-)-3-(1-adamantyl)-Delta(8)-tetrahydrocannabinol (1a, AM411), was found to have robust affinity and selectivity for the CB1 receptor as well as high in vivo potency. The X-ray crystal structure of 1a was determined. Exploration of the side chain conformational space using molecular modeling approaches has allowed us to develop cannabinoid side chain pharmacophore models for the CB1 and CB2 receptors. Our results suggest that although a bulky group at the C3 position of classical cannabinoids could be tolerated by both CB1 and CB2 binding sites, the relative orientation of that group with respect to the tricyclic component can lead to receptor subtype selectivity.

Cannabinoids for the Treatment of Pain: An Update on Recent Clinical Trials

Over the past five years, there has been a considerable increase in clinical research on cannabinoid use for a range of pain syndromes. Cannabinoid products are becoming available for research and clinical use, and pharmaceutical industry interest in the potential for cannabinoids in therapeutics is also gaining momentum. The present article summarizes recent clinical trial data in the field of pain management and suggests that the potential for cannabinoid therapy for chronic pain states is encouraging. Clinicians working in pain management should be aware of the options becoming available from the cannabinoid class of medications.

The role of halogen substitution in classical cannabinoids: A CB1 pharmacophore model

The presence of halogens within the classical cannabinoid structure leads to large variations in the compounds' potencies and affinities for the CB1 receptors. To explore the structure activity relationships within this class of analogs we have used a series of halogen-substituted (-)-Delta8-tetrahydrocannabinol analogs and compared their affinities for the CB1 cannabinoid receptor. Our results indicate that halogen substitution at the end-carbon of the side chain leads to an enhancement in affinity with the bulkier halogens (Br, I) producing the largest effects. Conversely, 2-iodo substitution on the phenolic ring leads to a 2-fold reduction in affinity while iodo-substitution in the C1'-position of the side chain lowers the compound's affinity for CB1 by more than 8-fold. The pharmacophoric requirements resulting from halogen-substitution are explored using computer modeling methods.

Cannabinoids as neuroprotective agents in traumatic brain injury.

The name Cannabinoid applies to a large and diverse family of compounds including plant derived, synthetic and endogenously produced chemicals, some but not all of which are psychotropic. Cannabinoids of all classes have the ability to protect neurons from a variety of insults that are believed to underlie delayed neuronal death after traumatic brain injury (TBI), including excitotoxicity, calcium influx, free radical formation and neuroinflammation. The pathways and experimental models supporting a neuroprotective role for the various classes of cannabinoids are critically reviewed vis a vis their potential to support the development of a clinically viable neuroprotective agent for human TBI.

Theoretical analysis of the NMR and electronic structure of novel Δ 8-THC derivatives

Several novel classical cannabinoids have been studied using a combination of density functional theory and semi-empirical calculations. Potential energy surfaces for each of these novel compounds were calculated using the PM3 semi-empirical Hamiltonian and show a wide range of conformational freedom for all the compounds in the study. The electronic structure for each of these derivatives has been studied using B3LYP/6-31G( p,d) calculations. Effects of the electronic structure on NMR experiments have been analyzed and clearly demonstrate that an understanding of these molecules requires a detailed description of the electronic structure. Additionally, we show that there is significant potential for these compounds to exhibit fluorescence, making them possible candidates for probing receptor sites in vivo.

Synthesis and Testing of Novel Phenyl Substituted Side‐Chain Analogues of Classical Cannabinoids.

Abstract A series of novel phenyl substituted side-chain analogues of classical cannabinoids were synthesized and their CB1 and CB2 binding affinities were evaluated relative to Δ 8 -THC and compound 2 . CB1 and CB2 binding assays indicate that the dimethyl and ketone analogues ( 3 ) and ( 6 ) display selectivity for the CB2 receptor in comparison to Δ 8 -THC and compound 2 . This study provides newer insights into the geometrical and functional group requirements of the ligand binding pockets of the CB1 and the CB2 receptors.

Synthesis and testing of novel phenyl substituted side-chain analogues of classical cannabinoids.

A series of novel phenyl substituted side-chain analogues of classical cannabinoids were synthesized and their CB1 and CB2 binding affinities were evaluated relative to Delta(8)-THC and compound 2. CB1 and CB2 binding assays indicate that the dimethyl and ketone analogues (3) and (6) display selectivity for the CB2 receptor in comparison to delta(8)-THC and compound 2. This study provides newer insights into the geometrical and functional group requirements of the ligand binding pockets of the CB1 and the CB2 receptors.

Pharmacophoric requirements for the cannabinoid side chain. Probing the cannabinoid receptor subsite at C1'.

Earlier work from our laboratories has provided evidence for the existence of a subsite within the CB1 and CB2 cannabinoid receptor binding domain corresponding to substituents at the benzylic side chain position of classical cannabinoids. The existence and stereochemical features of this subsite have now been probed through the synthesis of a novel series of (-)-Delta(8)-tetrahydrocannabinol analogues bearing C1'-ring substituents. Of the compounds described here, those with C1'-dithiolane (1c), C1'-dioxolane (2d), and cyclopentyl (2a) substituents exhibited the highest affinities for CB1 and CB2. We used molecular modeling approaches to better define the stereochemical limits of the putative subsite. In vitro pharmacological testing found 1c to be a potent CB1 agonist.

Synthesis and testing of novel classical cannabinoids: exploring the side chain ligand binding pocket of the CB1 and CB2 receptors

A series of C3 cyclic side-chain analogues of classical cannabinoids were synthesized to probe the ligand binding pocket of the CB1 and CB2 receptors. The analogues were evaluated for CB1 and CB2 receptor binding affinities relative to Δ 8-THC. The C3 side-chain geometries of the analogues were studied using high field NMR spectroscopy and quantum mechanical calculations. The results of these studies provide insights into the geometry of the ligand binding pocket of the CB1 and CB2 receptors.

(R)-methanandamide and Delta 9-THC as discriminative stimuli in rats: tests with the cannabinoid antagonist SR-141716 and the endogenous ligand anandamide.

(R)-methanandamide (AM-356), a metabolically more stable chiral analog of the endocannabinoid ligand anandamide, was used as a representative of fatty acid ethanolamide CB1 receptor ligands to characterize the discriminative stimulus functions of anandamides. Rats discriminated between 10 mg/kg (R)-methanandamide and vehicle administered IP 15 min prior to session onset. Another group of rats was initially trained to discriminate between 3 mg/kg Delta9-THC and vehicle given IP 30 min prior to session onset; for anandamide testing, the animals were retrained with 1.8 and 5.6 mg/kg Delta9-THC. A two lever operant methodology (FR10) was used. Delta9-THC was more potent than (R)-methanandamide at both 15 and 30 min post-injection, irrespective of the training drug used. Additional tests with 10 and 18 mg/kg (R)-methanandamide suggested that the effects were declining by 1 h. The cannabinoid antagonist SR 141716 (0.3 and 1 mg/kg) produced rightward shifts in the Delta9-THC dose-response curve for Delta9-THC-appropriate responding and for (R)-methanandamide-appropriate responding (surmountable antagonism). SR-141716 (0.3 and 1 mg/kg) antagonized the ability of (R)-methanandamide to occasion either Delta9-THC-appropriate responding or (R)-methanandamide-appropriate responding. This antagonism was surmountable only at a dose of 0.3 mg/kg SR-1421716 in the (R)-methanandamide-trained rats. SR-141716 did not antagonize the rate-decreasing effects of (R)-methanandamide in either the Delta9-THC or the (R)-methanandamide trained rats. Response suppression precluded testing doses higher than 30 mg/kg (R)-methanandamide. Tests with SR-141716 (1 and 10 mg/kg) alone resulted in <3% Delta9-THC-appropriate responding. With 10 mg/kg SR-141716, response rate was significantly lower as compared to the rate observed during a vehicle test. Tests with anandamide (10 and 18 mg/kg) resulted in 41% and 85% (R)-methanandamide-appropriate responding at a 3-min pre-treatment time, but in a maximum of 15% (R)-methanandamide-appropriate responding at a longer (15 min) pre-treatment time. In the Delta9-THC (1.8 and 5.6 mg/kg) trained rats, anandamide never produced more than about 20% Delta9-THC-appropriate responding. The results add to a growing body of evidence indicating that there are both similarities and dissimilarities between classical cannabinoids such as THC and endogenous fatty acid ethanolamides.

High-resolution NMR and computer modeling studies of the cannabimimetic aminoalkylindole prototype WIN-55212-2

Aminoalkylindoles (AAIs), although structurally dissimilar from the classical cannabinoids (CCs), are known to be capable of binding to cannabinoid receptors and of evoking cannabimimetic responses. However, their mode of binding remains unknown. In this communication, we have carried out further studies on the AAI prototype (R)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone (WIN-55212-2, 1) by the combined use of high-resolution 2D NMR and computer modeling. Our results suggest that the minimum energy conformations of the molecule 1 have distinct pharmacophoric features: (i) The naphthyl ring is oriented off the plane of the benzoxazine ring by approximately 59° with the carbonyl CO group pointing toward the C2−CH3 group. (ii) At the C10-position the axial morpholinomethyl conformation is preferred over the equatorial in order to relieve a steric interaction with the C2-methyl group. The preferred conformer as defined by the three key pharm...

High-resolution NMR and computer modeling studies of the cannabimimetic aminoalkylindole prototype WIN-55212-2.

Aminoalkylindoles (AAIs), although structurally dissimilar from the classical cannabinoids (CCs), are known to be capable of binding to cannabinoid receptors and of evoking cannabimimetic responses. However, their mode of binding remains unknown. In this communication, we have carried out further studies on the AAI prototype (R)-[2, 3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1, 4-benzoxazin-6-yl](1-naphthalenyl)methanone (WIN-55212-2, 1) by the combined use of high-resolution 2D NMR and computer modeling. Our results suggest that the minimum energy conformations of the molecule 1 have distinct pharmacophoric features: (i) The naphthyl ring is oriented off the plane of the benzoxazine ring by approximately 59 degrees with the carbonyl C=O group pointing toward the C2-CH(3) group. (ii) At the C10-position the axial morpholinomethyl conformation is preferred over the equatorial in order to relieve a steric interaction with the C2-methyl group. The preferred conformer as defined by the three key pharmacophores, naphthyl, morpholino, and 3-keto groups, shows that the morpholinyl ring of the molecule 1 deviates from the plane of the benzoxazine ring by about 32 degrees and orients in the left molecular quadrant. This model supports the hypothesis that a certain deviation of the morpholino group from the plane of the indole ring in compound 1 is essential for cannabimimetic activity. We postulate that such an alignment by the respective pharmacophores allows them to interact optimally with the receptor. The results should help us to better understand the pharmacophoric requirements of the AAIs and serve as a basis for future SAR studies and drug design.

Structure and Receptor Activity for Classical Cannabinoids

In this decade, classical cannabinoid SAR has increasingly been interpreted in terms of ligand-receptor interaction. Membrane receptor binding affinity, inhibition of receptor coupled processes, functional in vitro assays that distinguish between agonists and antagonists, transfected receptor systems, and distinction among receptor subtypes have been applied to cannabinoid pharmacology studies. These studies have revealed information about the pivotal receptor interaction that mediates the neurochemical system characterized by the receptor. The positive correlation between binding affinity and behavioral effects validates ligand-receptor interaction as a focus for developing new cannabinoids with the potential for selective pharmacological effects. Added to these direct receptor studies are the computational methods that compare active and inactive ligands for steric, electronic, and conformational similarities that reveal an SAR for activity with a sophistication and a perspective not possible before computer methodology. Merging these studies with knowledge of the receptor sequence, consequences of mutations of the receptor, and a calculated structure of the receptor are evolving a physical picture of the interaction of cannabinoids with its receptor(s). This picture of a ligand-receptor interaction guides medicinal chemists in their interpretation of classical cannabinoid SAR and the design of new analogs for selective pharmacological activity and as probes of the cannabinoid receptor. It is from a receptor perspective that this review of the SAR of classical cannabinoids is presented.

The Development of Cannabinoid Antagonists

The discovery of two distinct cannabinoid receptors (CB1 and CB2) in the early 1990's has revived the research on cannabinoid antagonists. While the search for antagonists based on the structure of agonists (classical cannabinoids or aminoalkylindoles) appeared rather disappointing, the first potent cannabinoid antagonists were developed in a new chemical series: the diarylpyrazoles. Since its discovery in 1994, the selective CB1 antagonist SR 141716 has became a major pharmacological tool to elucidate the physiological role of the CB1 cannabinoid receptor and its endogenous ligand. The selective CB2 antagonist SR 144528 is expected to play the same role for the CB2 receptors, while the recent development of cannabinoid antagonists belonging to other chemical series illustrates the interest of these compounds which are now considered as interesting therapeutic targets by many pharmaceutical companies.

Evaluation of cannabimimetic effects of structural analogs of anandamide in rats

Arachidonylethanolamide (anandamide), an endogenous ligand for the cannabinoid receptor, binds competitively to brain cannabinoid receptors and shares many, but not all, of the in vivo effects of Δ 9-tetrahydrocannabinol. In this study, the cannabinoid effects of anandamide analogs in which the anandamide molecule was altered were assessed in a drug discrimination model. Structural manipulations of the anandamide molecule included saturation of the arachidonyl moiety with fluorination (O-586), substitution for either the ethanolamide moiety (O-612 and O-595) or C2′ hydroxyl (O-585), and addition of a methyl group at various positions (O-610, O-680, and O-689). Despite the low binding affinities of the non-methylated compounds ( K i values >2000 nM), all of the analogs had previously shown cannabinoid activity in mice. In the present study, these analogs were tested in a more pharmacologically specific Δ 9-tetrahydrocannabinol discrimination procedure in rats. This animal model is predictive of the subjective effects of marijuana intoxication in humans. Whereas Δ 9-tetrahydrocannabinol and an aminoakylindole fully substituted for the training dose of 3 mg/kg Δ 9-tetrahydrocannabinol, anandamide and its non-methylated analogs were not cannabimimetic in this procedure. Methylation appeared to increase binding affinity ( K i values <150 nM) and efficacy; however, the greatest substitution produced by the methylated analogs occurred only at doses that decreased overall rates of responding, suggesting that these analogs are not fully Δ 9-tetrahydrocannabinol-like. The rapid metabolism of anandamide and some of its analogs undoubtedly contribute to the differences between the pharmacological profiles of the anandamides and classical cannabinoids. These results support the prediction that the subjective effects of anandamide analogs that have been developed thus far would not be cannabimimetic except at high doses.

Importance of the C-1 substituent in classical cannabinoids to CB2 receptor selectivity: synthesis and characterization of a series of O,2-propano-delta 8-tetrahydrocannabinol analogs.

The separation of the mood-altering effects of cannabinoids from their therapeutic effects has been long sought. Results reported here for a series of C-9 analogs of the cyclic ether O,2-propano-delta 8-tetrahydrocannabinol (O,2-propano-delta 8-THC) point to the C-1 position in classical cannabinoids as a position for which CB2 subtype selectivity occurs within the cannabinoid receptors. O,2-Propano-11-delta 8-THC, O,2-propano delta 9,11-THC, O,2-propano-9-oxo-11-nor-hexahydrocannabinol (O,2-propano-9-oxo-11-nor-HHC), and O,2-propano-9 alpha- and O,2-propano-9 beta-OH-11-nor-HHC were synthesized and evaluated in radioligand displacement assays for affinity at the CB1 and CB2 receptors and in the mouse vas deferens in vitro assay and the mouse tetrad in vivo assay for cannabinoid activity. Evaluation of binding affinity at the CB1 and CB2 receptors revealed that each compound possesses a modest increased affinity for the CB2 receptor. Analogs which contained an oxygen attached to C-9 (i.e., oxo and hydroxy derivatives) showed the highest affinity and selectivity for CB2 (for O,2-propano-9-oxo-11-nor-HHC, Ki(CB1) = 90 nM, Ki(CB2) = 23 nM, selectivity ratio 3.9; for O,2-propano-9 beta-OH-11-nor-HHC, Ki(CB1) = 26 nM, Ki(CB2) = 5.8 nM, selectivity ratio 4.5). Each compound was found to produce a dose-dependent inhibition of electrically-evoked contractions of the mouse isolated vas deferens when administered at submicromolar concentrations. This inhibition could readily be prevented by the selective CB1 cannabinoid receptor antagonist SR-141716A. The analogs exhibited unique in vivo profiles with O,2-propano-delta 9,11-THC exhibiting antinociception with reduced activity in three other in vivo measures and O,2-propano-9 beta-OH-HHC exhibiting lack of dose responsiveness in all measures. The CB2 selectivities in the O,2-propano analogs may be due to differences in solvation/desolvation that occur when the ligands enter the CB1 vs CB2 binding site. Alternatively, the CB2 selectivities may be a results of an amino acid change from a hydrogen bond-accepting residue in CB1 to a hydrogen bond-donating residue in CB2.

Studies on the thermotropic effects of cannabinoids on phosphatidylcholine bilayers using differential scanning calorimetry and small angle X-ray diffraction

We have studied the thermotropic properties of a wive variety of cannabinoids in DPPC bilayers. The molecules unher study were divived into four classes: (a) classical cannabinoids possessing a phenolic hydroxyl group; (b) Δ 9-THC metabolites with an additional hydroxyl group on the C ring; (c) non-classical cannabinoids, and (d) cannabinoids with a protected phenolic hydroxyl group. The results showed that the first three groups have similar effects on the thermotropic properties of DPPC bilayers up to x = 0.05 (molar ratio) and that these effects do not parallel their biological activity. For concentrations less than x = 0.01, cannabinoids affect mainly the pretransition temperature in a progressive manner until its final abolishment. At x = 0.05, they further affect the main phase transition by lowering its phase transition temperature and broavening its half width. At high concentrations the thermograms have multiple components, indicating that membranes are no longer homogeneous but rather consist of different domains. At these concentrations cannabinoids with more hydroxyl groups give simpler thermograms. Low concentrations of cannabinoids in group d affect significantly the pretransition temperature, while high concentrations affect only marginally the main phase transition by slightly lowering its temperature and broavening its half width. These results point out the importance of the phenolic hydroxyl group in inducing membrane perturbations. The d-spacing data from our small angle X-ray diffraction experiments show that Δ 8-THC produces significant structural changes in the lipid bilayer, including the gel-phase tilting angle, the intermolecular cooperativity and the gauche:trans conformer ratio. Conversely, the inactive analog Me- Δ 8-THC does not cause drastic changes to the bilayer structure.

Pharmacophoric requirements for cannabinoid side chains. Naphthoyl and naphthylmethyl substituted Δ8‐tetrahydrocannabinol analogs

AbstractWe have synthesized hybrids of classical cannabinoids and aminoalkylindoles by combining the dibenzopyran structure of Δ8‐tetrahydrocannabinol with the ketonaphthyl or 1‐naphthylmethyl moieties found in aminoalkylindoles in order to investigate the role of the ketonaphthyl and 1‐naphthylmethyl pharmacophores in cannabimimetic activity.