The main psychoactive and therapeutic effects of Δ9-tetrahydrocannabinol (THC) are mediated through cannabinoid type 1 receptors (CB1Rs). The therapeutic uses of THC are mitigated by the development of tolerance to these therapeutic effects, whereas tolerance does not readily develop to some of the side-effects of THC, like motor impairment and reward. The development of tolerance occurs through adaptations at CB1Rs, which include desensitization (G-protein uncoupling) and downregulation (receptor degradation). Brain region-dependent differences in THC-mediated adaptations are proposed to explain the differences in tolerance to various THC-mediated effects. These studies focused on whether ΔFosB, a stable transcription factor, could regulate CB1R adaptations since regions resistant to CB1R adaptations, like the basal ganglia, exhibit THC-mediated ΔFosB induction. The studies in this dissertation tested the hypothesis that THC-mediated induction of ΔFosB is regulated through interactions between cannabinoid and dopamine systems and that brain region-dependent differences in ΔFosB transcriptional regulation could explain some aspects of long-term CB1R signaling and CB1R adaptations. Results determined that THC induced ΔFosB primarily in forebrain areas, like striatum, that are innervated by midbrain dopamine neurons. An inverse, brain region-dependent correlation was found between CB1R desensitization and ΔFosB induction. Studies utilizing bitransgenic mice with overexpression of ΔFosB, or its dominant negative ∆cJun, determined that ΔFosB regulates CB1R signaling and reduces CB1R desensitization. Based on this regional profile, studies determined the role of dopamine signaling in THC-mediated ∆FosB induction. Results showed that THC-mediated induction of ΔFosB required dopamine type 1 receptors, but not the dopamine-and cAMP-dependent phosphoprotein of Mr 32kDA. Finally, the functional consequences of THC-mediated ΔFosB induction were assessed by measuring expression of known targets of ΔFosB following both acute and repeated THC administration. Results found that, in prefrontal cortex, known targets of ΔFosB exhibited functionally different signaling expression patterns when comparing acute THC with THC-challenge in THC-experienced mice, which enhanced ΔFosB induction. These studies establish a role for ΔFosB in regulating long-term CB1R signaling/adaptation following repeated THC administration and could have implications for changes in the effects of THC during repeated administration, including the development of differential tolerance to motor-impairing and rewarding effects of THC versus other pharmacological effects.

In the past ten years, synthetic cannabinoids (SC) have emerged as drugs of abuse. Unlike D9-tetrahydrocannabinol (THC), many SCs are associated with serious health complications and death. One way in which THC and SCs differ lies with their enhanced potency and efficacy at the CB1 receptor. No current methods exist to measure efficacy at the CB1 receptor in vivo, and the abuse-related properties of SC cannabinoids are not well explored. Here, we utilized CB1 wild type (WT), heterozygous (HET), and knockout (KO) mice. By employing CB1 ligands which differ in efficacy we have developed a method to explore the relationship between efficacy and the ability to produce cannabimimetic (catalepsy, hypothermia, and antinociception) effects when CB1 expression was reduced by half. Additionally, the intracranial self-stimulation procedure (ICSS) was utilized to investigate the effects of enhanced efficacy at CB1 upon reward processes using representative SC CP55,940. As predicted, the potency shift between WT and HET mice inversely correlated with the efficacy of the test drug for both hypothermia and antinociception, but not catalepsy. This efficacy stratification was correlated with the agonist-stimulated [35S]GTPgS binding assay, demonstrating this model as an effective tool to ascertain in vivo efficacy differences at CB1. In ICSS, CP55,940 elicited only rate-decreasing effects acutely, although tolerance developed following repeated dosing, with no evidence for spontaneous or rimonabant-precipitated withdrawal. Together, these data indicate that highly efficacious cannabinoid ligands require few receptors to produce cannabimimetic effects, and that the model provides an effective means to quickly ascertain differences in efficacy.

Cannabis (Marijuana) has multiple effects on the human body, such as analgesia, euphoria and memory impairment. Delta-9 tetrahydrocannabinol (D9-THC), the active ingredient in cannabis, binds to cannabinoid receptors, seven-transmembrane G protein-coupled receptors (GPCRs) that mediate a variety of physiological functions. GPCRs were believed to function only in homomeric forms, however, recent findings show that different GPCRs can also form heteromeric complexes that may expand their signaling properties. In this study, we focused on Cannabinoid CB1 receptor (CB1R) heteromers with the mu-opioid receptor (MOR) and the Dopamine type 2 receptor (D2R), respectively. We utilized a variety of techniques, such as the calcium mobilization assay, a luciferase complementation assay and an electrophysiology assay to study the pharmacology of the CB1R-MOR and CB1R-D2R heteromers. Our data demonstrate that co-expression of CB1R enhances the Gi signaling through MOR and inhibits the beta-arrestin recruitment to MOR. We also show that co-application of CB1R ligands can further accentuate the MOR signaling modulation. Co-expression of a CB1R transmembrane domain 5 (TM5), but not a TM1, mini-gene abrogated the signaling change suggesting that it is likely due to heteromerization of MOR and CB1R. Utilizing this herteromeric signaling could provide a novel therapeutic approach that may yield potent analgesic effects with reduced side effects. We have also found that CB1R switched its signaling specificity from Gi to Gs upon its heteromerizaiton with D2R. In conclusion, our data show that CB1R expands its signaling repertory and modulates the partner receptor signaling upon heteromerization.

Glucose and oxygen supply to the brain is critical for its proper function and when it is restricted as during a stroke, neurons and glial cells quickly become necrotic leading to structural damage as well as functional impairment and even death. To date there are few effective therapies that inhibit the neurodegenerative process and improves the outcome for the affected individual. One possible target is the cannabinoid system. Cannabinoid receptor agonists reduce ischemic volume, endogenous cannabinoid levels are elevated during neurodegenerative insults and mice devoid of the central cannabinoid receptor are more seriously affected by experimental stroke than wild type mice. The cannabinoids are also ascribed anti-inflammatory properties and post ischemic inflammation has been proposed to contribute to the evolution of the ischemic damage. In this thesis mechanisms that can contribute to cannabinoid neuroprotection have been studied. In papers I and II the chick was used as a model species, since preparation of embryonic primary neuronal cultures from chick is relatively simple and time efficient compared to rodent primary cultures. Both adult and embryonic chick brain membranes contain functional CB1 receptors and in the cultures they are coupled to inhibition of cAMP production. In embryonic primary cultures, neurons were not protected from glutamate toxicity by preincubation with CB receptor agonists suggesting that postsynaptic cannabinoid mediated neuroprotection is not effective in this system. The effect of cannabinoid agonists on neutrophil chemotaxis and transmigration was investigated in paper III. The CB1/CB2 agonist WIN 55,212-2 inhibited TNF-α-induced transmigration across ECV304 cell monolayers. The effect of WIN 55,212-2 on this process which was mediated by a reduction of IL-8 release from the ECV304 cells rather than a direct effect upon the migratory response to IL-8 was not possible to abolish with CB1 or CB2 agonists suggesting a mechanism distinct from the cannabinoid receptors is operative. In paper IV the photothrombotic ring stroke model was evaluated to determine if it is suitable in intervention studies targeting the cannabinoid system. Three major endpoints were of interest, ischemic volume, neutrophil infiltration and CB1 receptor function. Consistent with previous studies the ischemic volume peaked at 48 hours after irradiation. Neutrophil infiltration was quantified using a myeloperoxidase activity assay. The assay revealed an increase in myeloperoxidase activity 48 hours after irradiation, albeit at a modest level. The function of the CB1 receptor was assessed by radioligand binding and there was no change in either total binding or functional G-protein coupling following photothrombosis. Taken together these results indicate that it is feasible to undertake cannabinoid intervention studies in this model.

In this study, the effects of antagonists to the cannabinoid receptors in MCL cell lines were studied. Results presented in this study show that signalling through cannabinoid receptor with antagonists such as SR141716, SR144528 decreases cell viability but hemopressin when analyzing with XTT. The decrease in cell viability by SR141716 is caused by apoptosis triggered after 5 hours of treatment. The CB1 expression was confirmed in all MCL cell lines tested via western blotting but the expression of CB2 and GPR55 – another receptor to which SR141716 has affinity - was not confirmed due to lack of reliable antibodies. Specific agonist to GPR55 – LPI (l-α-lysophosphatidylinositol) showed different response compared to SR141716 which suggests that the effect seen by SR141716 was not induced through GPR55. The effect induced by CB1/CB2 agonist AEA is shown to be neither through CB1 or CB2 alone but possibly on another receptor yet to be described.

Thesis (Ph.D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Pharmacology and Toxicology. Title from title-page of electronic thesis. Bibliography: leaves 130-143.

The central hypothesis of the dissertation is that “the design and synthesis of a codrug of an opiate and a cannabinoid can be achieved which is stable in the gastrointestinal tract and shows a superior pharmacological and pharmacokinetic profile when compared to a physical mixture of the two parent drugs.” To prove the hypothesis, a series of novel codrugs were prepared by conjugation of the opiate drug codeine with Δ9-tetrahydrocannabinol (Δ9-THC), cannabidiol, abn-cannabidiol and an opiate prodrug 3-O-acetylmorphine with Δ9-THC. Codeine-cannabinoid codrugs were evaluated for analgesic activity in the rat after oral administration. The Cod-THC codrug showed greater effectiveness as well as prolonged pain management properties as compared to the parent drugs. The stability of Cod-THC codrug in aqueous solutions from pH 1-9, in simulated gastrointestinal fluids, in brain homogenate and the hydrolysis of the carbonate ester linkage in rat plasma suggested that after oral administration, the codrug would be absorbed intact from the GI tract and then hydrolyze in the plasma to generate both parent drugs. The enzymes present in rat brain homogenate were incapable of cleaving the codrug into the parent drugs. The pharmacokinetic profiles of the Cod-THC codrug and an equimolar physical mixture of the parent drugs were evaluated in rats. The plasma concentrations of codeine and Δ9-THC were much higher after codrug administration compared to the plasma concentrations of these drugs after oral administration of an equimolar physical mixture. The parent drugs were also present in the plasma for longer period of time compared to the physical mixture, probably due to the sustained release of the parent drugs from codrug in the plasma. The concentrations of codeine and Δ9-THC were much higher in rat brain after oral administration of the Cod-THC codrug as compared to brain concentrations of these drugs after oral administration of the physical mixture. Thus, the design and synthesis of an opiate and a cannabinoid codrug was achieved which was stable in the gastrointestinal tract, showed enhanced analgesic effects as compared to the parent drugs, and also showed a superior pharmacokinetic profile when compared to a physical mixture or the two parent drugs.

There are several lines of evidence supporting the existence of a pivotal role of the cannabinoid system in mediating the reinforcing effects of nicotine. Characterization of the crosstalk between nicotine addiction and the cannabinoid system may have significant implications for our understanding of the neurobiological mechanisms underlying nicotine dependence. Objectives: The current series of experiments, we investigated the effects of activating CB1 receptors, modulating CB2 receptors as well as elevating levels of the endogenous cannabinoid ligand anandamide on nicotine taking and reinstatement of nicotine seeking behaviour. METHODS: In the first series of experiments, we investigated the effects of pretreatment with the CB receptor agonist WIN 55, 212-2 (0.1-1mg/kg), on nicotine self-administration and on the reinstatement of nicotine seeking behaviour. In the next series of experiments, we used a selective CB1 inverse agonist rimonabant (0.3mg/kg) and CB2 antagonist AM630 (5mg/kg) to delineate wether the effects obsereved with WIN 55, 212-2 are CB1 or CB2 meidated. Moreover, we investigated the effect of selective CB2 receptor activation (AM1241 1-10 mg/kg) and inhibition (AM630 1.25-5 mg/kg) on nicotine self-administration under fixed ratio (FR) and progressive (PR) schedules of reinforcement and on reinstatement of nicotine seeking induced by nicotine associated cues and nicotine priming. Finally, the effects of activation of CB receptors through administration of anandamide reuptake inhibitor VDM11 (1-10 mg/kg) on nicotine self-administration and on reinstatement of nicotine seeking were investigated. RESULTS: WIN 55,212-2 enhanced the break points for nicotine self-administration under a PR schedule of reinforcement, reinstated nicotine seeking behaviour and enhanced cue induced reinstatement of nicotine seeking. Neither activation nor blockade of CB2 receptors affected the responding of the animals for nicotine self-administration under FR or PR schedules of reinforcement or for reinstatement of nicotine seeking induced by nicotine associated cues and priming. Pretreatment with VDM11 dose dependently attenuated the reinstatement of nicotine seeking behaviour induced by nicotine associated cues and priming without affecting stable nicotine self administration. CONCLUSION: CB1 but not CB2 receptors appear to play a pivotal role in modulating the reinforcing effects of nicotine. Inhibition of anandamide reuptake could be a potentially useful tool in modulating relapse to smoking

There are several lines of evidence supporting the existence of a pivotal role of the cannabinoid system in mediating the reinforcing effects of nicotine. Characterization of the crosstalk between nicotine addiction and the cannabinoid system may have significant implications for our understanding of the neurobiological mechanisms underlying nicotine dependence. Objectives: The current series of experiments, we investigated the effects of activating CB1 receptors, modulating CB2 receptors as well as elevating levels of the endogenous cannabinoid ligand anandamide on nicotine taking and reinstatement of nicotine seeking behaviour. METHODS: In the first series of experiments, we investigated the effects of pretreatment with the CB receptor agonist WIN 55, 212-2 (0.1-1mg/kg), on nicotine self-administration and on the reinstatement of nicotine seeking behaviour. In the next series of experiments, we used a selective CB1 inverse agonist rimonabant (0.3mg/kg) and CB2 antagonist AM630 (5mg/kg) to delineate wether the effects obsereved with WIN 55, 212-2 are CB1 or CB2 meidated. Moreover, we investigated the effect of selective CB2 receptor activation (AM1241 1-10 mg/kg) and inhibition (AM630 1.25-5 mg/kg) on nicotine self-administration under fixed ratio (FR) and progressive (PR) schedules of reinforcement and on reinstatement of nicotine seeking induced by nicotine associated cues and nicotine priming. Finally, the effects of activation of CB receptors through administration of anandamide reuptake inhibitor VDM11 (1-10 mg/kg) on nicotine self-administration and on reinstatement of nicotine seeking were investigated. RESULTS: WIN 55,212-2 enhanced the break points for nicotine self-administration under a PR schedule of reinforcement, reinstated nicotine seeking behaviour and enhanced cue induced reinstatement of nicotine seeking. Neither activation nor blockade of CB2 receptors affected the responding of the animals for nicotine self-administration under FR or PR schedules of reinforcement or for reinstatement of nicotine seeking induced by nicotine associated cues and priming. Pretreatment with VDM11 dose dependently attenuated the reinstatement of nicotine seeking behaviour induced by nicotine associated cues and priming without affecting stable nicotine self administration. CONCLUSION: CB1 but not CB2 receptors appear to play a pivotal role in modulating the reinforcing effects of nicotine. Inhibition of anandamide reuptake could be a potentially useful tool in modulating relapse to smoking

Diss. (sammanfattning) Umeå : Univ., 2005. / Härtill 5 uppsatser.