Thesis (Ph. D.)--Virginia Commonwealth University, 2007. / Prepared for: Dept. of Microbiology and Immunology. Bibliography: leaves 92-108. Also available online via the Internet.

Thesis (Ph. D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Microbiology and Immunology . Bibliography: leaves 118-133 . Also available online via the Internet.

Thesis (Ph. D.)--Virginia Commonwealth University, 2006. / Prepared for: Dept. of Neurology. Bibliography: leaves 180-205. Also available online.

Cisplatin is a widely used chemotherapeutic drug for the treatment of solid tumors. However, the drug accumulates in the cochlea, and damages inner ear structures, resulting in bilateral andpermanent hearing loss. Previous data from our laboratory indicate that activation of the transient receptor potential vanilloid 1 (TRPV1) receptor (by capsaicin) increases the NOX3 isoform of NADPH oxidase, leading to the generation of reactive oxygen species (ROS) in the cochlea, transient cochlear inflammation and transient hearing loss. We also demonstrated that the transient inflammation was produced by ROS-mediated activation of signal transducer and activator of transcription 1 (STAT1). Surprisingly, over time, this response desensitizes and capsaicin was subsequently able to protect against cisplatin ototoxicity. The goal of this study was to determine the mechanism of otoprotection against cisplatin ototoxicity following the administration of capsaicin. For this study we utilize both an immortalized organ of Corti outer hair cells and rat cochlea. Capsaicin (2.5 µM) increased both Ser727 p-STAT1 and Tyr705 p-STAT3 implicating its role in inflammation. Expression of cannabinoid receptors were observed in UB/OC-1 cells as well as rat outer hair cells (OHCs). However, inhibition of CB2 receptors (by AM630) reduced capsaicin-mediated Tyr705 p-STAT3, but had little effect on Ser727 STAT1. Capsaicin protected UB/OC-1 cells against cisplatin-induced apoptosis. This protection was reversed by CB2 antagonist but potentiated by TRPV1 inhibition. Significant cell death was observed following treatment of UB/OC-1 cells with AM630 alone, underscoring the importance of CB2 receptors in survival of these cells. CB2 agonist, JWH, significantly increased the protective signal, STAT3. Furthermore, capsaicin-mediated protection was reversed by the inhibition of STAT3, implicating STAT3 in otoprotection. In animal studies, oral administration of capsaicin (0.5% solution) induced transient inflammation but led to a long term recovery. Animals pre-treated with oral capsaicin were protected against cisplatin-induced hearing loss as compared to vehicle-treated animals, suggesting protection against hearing loss. Capsaicin increased the expression of both CB1 and CB2 receptors in the organ of Corti, which might confer the long term protective actions of this agent against hearing loss. In rats pretreated with AM630, the protective action of capsaicin was abolished. We conclude that otoprotection mediated by capsaicin is produced by activation of CB receptors in the cochlea which are coupled to both STAT1 and STAT3 activation. However, our data support the conclusion that activation of STAT3 confers the otoprotective action of capsaicin. In contrast, activation of STAT1 by capsaicin could contribute to the transient inflammatory response previously observed in vivo. The net protective action of capsaicin could result from an increase in the STAT3/STAT1 ratio of cells in the cochlea, which antagonizes the ability of cisplatin lower this ratio and promote cell death.

Endocannabinoid (eCB) system is composed of endogenous CB ligands including anandamide (AEA) and 2-Arachidonyl glycerol (2-AG), enzymes involved in their biosynthesis and degradation such as diacylglycerol lipase-α (DAGL- α), and CB receptors. Primarily, there are three types of CB receptors - CB receptor 1(CB1), CB receptor 2 (CB2) and non CB1 non CB2 types of CB receptor (e.g. GPR, TRPV1) and they belong to G-protein (Gi/o) coupled receptors (GPCR) family.CB1 receptors are abundant in the brain where they modulate neuronal activities. On the other hand, CB2 receptors are predominantly expressed in the immune cells and regulate the growth and proliferation of different immune cells and modulate the activities of cytokines network and anti-oxidant machinery in stress conditions. Inflammation plays a central role in hearing loss (HL) caused by different ototoxic insults including anti-neoplastic agents such as cisplatin, aminoglycosides and acoustic trauma. These insults can trigger chronic production of reactive oxygen species (ROS) in regions of cochlea such as organ of Corti, stria vascularis (SVA), spiral ligament (SL) and spiral ganglion neurons (SG). This leads to increased synthesis of pro-inflammatory cytokines, disruption of mitochondrial membrane integrity, activation of DNA damage/repair pathways and activation of pro-apoptotic enzymes. Jeong et al. (2007) have shown that CB2 receptor specific agonist (JWH-015) protects the HEI-OC1 hair cell cultures against cisplatin-induced cytotoxicity in-vitro. The goal of the current study was to examine the distribution and function of CB receptors (mainly CB2) in the cochlea and determine whether activation of these receptors could protect the cochlea by altering the expression of ROS generating proteins, along with pro-inflammatory and pro-apoptotic proteins. This study also investigated whether inhibition of eCB synthesis can causes HL. Aim 1 of the current study investigated the expression of CB receptors in the cochlea using different in-vivo models such as male Wistar rat and knock-in mice with GFP-tagged CB2 receptors, in-vitro models such as organotypic culture of neonatal mouse (C57BL/6) cochlea and University of Bristol organ of Corti (UB/OC1) cells. We show that both CB1 and CB2 receptors are expressed in the outer and the inner hair cells (OHCs and IHCs), SV, SG and supporting cells (SCs) included outer and inner pillar cells. The distribution of DAGL- α was also examined in the male Wistar rats and we found the similar distribution pattern of this enzymes as CB2. DAGL- α catalyzes the hydrolysis of DAG to synthesize 2-AG, which acts as a chief endogenous CB2 ligand. Our initial studies suggested a role of CB2 and not CB1 in protection, leading us to focus on CB2 receptors for subsequent studies. Aim 2 examined the otoprotective role of trans-tympanic application of CB2 specific agonist (JWH-015) against cisplatin-induced hearing loss in male Wistar rats. Activation of CB2 receptors restored cisplatin-induced elevations in ABR thresholds which was significantly reversed by CB2 antagonist AM-630. Pre-treatment with JWH-015 protected against cisplatin-induced loss of hair cell and synaptic ribbons. In-vitro studies in UB/OC-1 cells demonstrated that pre-treatment of JWH-015 modulates the activities of signal transducer and activator of transcription 1 and 3 (STAT1 and STAT3), increases the expression of anti-apoptotic protein Bcl-xL, indicating its role in regulating the apoptosis Activation of CB2 also abrogated cisplatin-induced decrease in Na+/K+ATPase- α in the SV and SL fibrocytes and ameliorated the expression of different pro-inflammatory genes including TRPV1, COX2, NOX3, KIM1, iNOS and TNF- α. We also found that blocking of CB2 by AM630 itself resulted in hearing loss and loss in CB2 receptors, indicating eCB system is tonically active and could be important for physiological function of the cochlea. Indeed, we observed that inhibition of DAGL- α by RHC80267 results in HL. Aim 3 of this current study investigated whether pre-treatment of CB2 agonist will interfere with anti-cancer efficacy of cisplatin against various cancer cell lines head and neck cancer cells (UMSCC10B), and colon cancer cells (HCT116). Our data indicate that JWH-015 did not interfere with cisplatin-induced apoptosis in these cells. Overall, this study provides novel insights into the essential role eCBs plays in protection the cochlea under non-stressed conditions and following exposure to ototoxic agents. It also demonstrates that application of exogenous CB2 agonist (JWH-015) could serve as an effective protective agent against cisplatin ototoxicity These data suggest that localized delivery of CB2 agonists should be studied in human for protection against hearing loss.

The co-abuse of alcohol and marijuana is widespread, although the mechanisms underlying this behavior are unclear. There is some evidence of a relationship between the neural processes that mediate the effects of ethanol and marijuana. For example, research has shown that exposure to marijuana increases responding for, and intake of, ethanol. The alcohol deprivation effect is an anima l model of alcoholism that suggests that the reinforcing efficacy of ethanol, as measured by intake, increases following a period of deprivation. Recent research indicates that rats chronically exposed to marijuana during periods of alcohol deprivation consume ethanol above and beyond deprivation alone. It is unclear, however, whether the marijuana exposure or the repeated deprivations increased motivation to consume ethanol. In the present experiment, rats were trained to self-administer ethanol on a progressive ratio schedule and subjected to two separate periods of deprivation during which either drug or saline was chronically administered for 7 days. Breakpoint (i.e., last ratio completed) was recorded as a measure of the reinforcing efficacy of ethanol. Following deprivations, breakpoint was initially lower than baseline, regardless of whether the drug or saline was administered. Breakpoint recovered to, but did not exceed, baseline levels following both deprivations, indicating a lack of increased reinforcing efficacy of ethanol after repeated deprivation or chronic exposure to marijuana. The lack of an expression of an alcohol deprivation effect following deprivation may have been due to the length and number of deprivations employed. Furthermore, lowered breakpoint recorded following chronic drug administration during deprivation may have been due to the dose administered or stress generated by chronic injections . Further investigation is necessary to separate and clarify the variables responsible for the present results.

There is significant sequence homology and anatomical co-distribution between cannabinoid (CB1) and sphingosine-1-phosphate (S1P) receptors in the CNS, but potential functional relationships between these lysolipid receptors have not been examined. Therefore, to investigate possible relationships between these two systems at the level of G-protein activation, agonist-stimulated [35S]GTPγS binding and autoradiography were conducted. Autoradiographic studies were first performed to localize receptor-mediated G-protein activation in mouse brain. Coronal brain slices were processed for stimulation of [35S]GTPγS binding using the synthetic cannabinoid agonist WIN 55,212-2 (WIN) or SIP. High levels of WIN- and S1P-stimulated [35S]GTPγS binding were observed in the caudate putamen, hippocampus, substantia nigra, and cerebellum. To further characterize the relationship between S1P-and CB1-mediated G-protein activation, spinal cords from adult male CB1 receptor knockout mice, CNS-deleted S1Pl receptor knockout mice and wild type C57 mice were collected, and assessed using agonist-stimulated [35S]GTPγS binding. Results from this experiment revealed that the S1Pl receptor is predominant in mouse spinal cord. To further investigate potential CBl and SIP receptor interactions spinal cords were collected from adult male ICR mice. Additivity studies were preformed using agonist-stimulated [35S]GTPγs binding. Results showed significantly less than additive stimulation when spinal cord tissue was treated with both WIN and SIP. These results suggest an interaction between the CB1 and S1P receptors in the mouse spinal cord. The effect of cannabinoid antagonists, SR141716A (CB1) and SR144528 (CB2) on S1P-and WIN-stimulated [35S]GTPγS binding were also examined in mouse spinal cord homogenates. These results showed that there was no significant difference between S1P-stimulated [35S]GTPγS binding in the presence of SR141716A or SR144528 compared to vehicle control. This shows that S1P produced stimulation independent of the CBl or CB2receptor. In addition WIN-stimulated [35S]GTPγS binding was not affected by SR144528, but was inhibited by SR141716A, confirming that this action is due to the CB1 receptor. The combined results of this project demonstrate an interaction between CB1 and S1P receptors in certain CNS regions where they are co-distributed, such as the caudate putamen, hippocampus, substantia nigra, cerebellum and spinal cord. These results may be due to convergence on a common pool of G-proteins via dimerization or co-localization in lipid rafts, or a possible direct ligand-receptor interaction.

Stroke is the most common cause of acquired epilepsy in persons 35 and older. The massive increase in extracellular glutamate during stroke causes a cascade of intracellular events that can lead to cell death or the molecular changes that initiate the development of epilepsy. In addition, many studies point to a modulatory role of the endocannabinoid system in controlling seizures. Animal models of stroke induced acquired epilepsy have been difficult to develop. Therefore, this dissertation was initiated to develop an organotypic hippocampal slice culture model of acquired epilepsy and examine the changes in distribution and function of the endogenous CB1 receptor system. We utilized 4-aminopyridine and glutamate to induce separate excitotoxic injuries to slice cultures. Both injuries produced significant cell death acutely following the injury. After a latency period, we observed a significant increase in the number of slice cultures that displayed electrographic seizures in both injury models. Western blot analysis demonstrated that the cannabinoid CB1 receptor protein was significantly upregulated following injury with glutamate. Immnohistochemical studies demonstrated that this receptor upregulation was likely specific to the glutamatergic terminals. Electrophysiological experiments were performed to study endocannabinoid modulation of inhibitory and excitatory signaling in the CA3 pyramidal cells. We demonstrated that depolarization induced suppression of excitation (DSE) was enhanced in slice cultures that had undergone glutamate injury. This indicated that the upregulation of CB1 receptors following glutamate injury was physiologically functional, as it enhanced cannabinoid control of the excitatory signaling. These studies support the hypothesis that there is a functional alteration of CB1 receptors in the epileptic state that acts to suppress seizures. The development of an organotypic hippocampal slice culture model of stroke acquired epilepsy provides a unique tool to study the neuronal plasticity changes associated with epileptogenesis. It also provides a practical model to study pharmacological agents that may be useful in preventing or treating epilepsy.

Inflammation is a multifactorial response towards noxious stimuli, however appropriate regulation and resolution of inflammation is crucial for the prevention of chronic inflammatory diseases such as atherosclerosis. The endocannabinoid (eCB) system is an endogenous immunomodulatory system which consists of a series of lipophilic ligands that signal via two G-protein-coupled receptors. Cannabinoid receptor 1 (CB1R) is mainly expressed in the central nervous system and its activation has psychoactive effects. Cannabinoid receptor 2 (CB2R) is mainly expressed on leukocytes and receptor activation has anti-inflammatory actions in mouse models of atherosclerosis and chronic inflammatory pain. It is considered that CB2R activation is involved in modulation of the recruitment of inflammatory cells, especially monocytes/macrophages; however the exact mechanism of action has not been fully elucidated. We hypothesised that activation of CB2R modulates monocyte/ macrophage recruitment and signalling, thus providing a homeostatic mechanism to limit macrophage activation in inflammatory responses. The high lipophilicity of cannabinoid ligands and their lack of selectivity for CB2R over CB1R limits CB2R drug development. In collaboration with Dr Angela Russell, we used virtual screening and a CB2R cAMP assay that we validated to discover a novel CB2R agonist, 3-((2’-Cyanobenzyl)thio)-5H-[1,2,4]triazino[5,6-b]indole, (DIAS2). In collaboration with Dr Russell’s group who did chemical synthesis, we extended this novel scaffold to include over 80 compounds. Using the same hCB2R cAMP screening assay we demonstrated that 16 compounds with the same scaffold are at active at CB2R in the nanomolar range. At least 3 compounds, including DIAS2, were found to be ≥ 300-fold selective for CB2R over CB1R in cAMP assays and radioligand binding studies. In the inflammatory model of zymosan-induced peritonitis, DIAS2 dose-dependently inhibited inflammatory monocyte recruitment by 50% at highest dose of 5 mg/kg with no effect on neutrophils. In further zymosan-induced peritonitis experiments 5 mg/kg of DIAS2 and a structurally-similar CB2R agonist from the same family of triazino-indoles inhibited monocyte recruitment while a different CB2R agonist (JWH-133) at 5 mg/kg did not inhibit monocyte recruitment. Analysis of peritoneal exudates showed that the inhibition of monocyte recruitment was not associated with changes in the levels of JE, MIP-1α and nitric oxide but was associated with increased levels of the chemokine KC. Using in vitro cell biology approaches, we demonstrated that 10μΜ dose of both DIAS2 and JWH-133 reduced forskolin-induced cAMP production in primary murine macrophages. Also 2.5 to 10 μΜ οf JWH-133 and HU-308 dose-dependently induced primary murine macrophage chemotaxis which could be blocked a CB2R antagonist (SR 144528, 1 μΜ) while DIAS2 at doses up to 10 μΜ was not a chemoattractant. Accordingly HU-308 and JWH-133 were at least 3-fold more efficacious than DIAS2 at recruiting β-arrestin to the murine CB2R. Moreover in studies with primary murine macrophages 10 μΜ dose of JWH-133 and HU-308 induced ERK1/2 and Akt phosphorylation within 30 minutes, while 2-AG (an endogenous eCB ligand) and DIAS2 at 10 μΜ had no such effect. In summary, we have discovered a novel family CB2R agonists and demonstrated that some devoid of chemotactic active CB2R agonists can reduce monocyte recruitment in vivo while other chemoattractant CB2R agonists have no in vivo anti-inflammatory effect. We propose that non-chemotactic CB2R agonists represent a new class of anti-inflammatory drugs with a novel mode of action.

Amphibian alkaloids are attractive targets for synthesis due to their biological activity. An important class of amphibian alkaloids is the 2,5-disubstituted pyrrolidine-based family of compounds. There are many synthetic approaches for the preparation of the trans-2,5- disubstituted pyrrolidines, but methods for the construction of the cis-2,5-pyrrolidines are limited. Therefore, it was desired to develop an enantioselective approach for the preparation of cis-2,5-disubsituted pyrrolidines. (+)-Tropin-2-one derived from cocaine was used as starting material to exploit the inherent stereochemistry for construction of the cis-pyrrolidine ring. This permitted the unequivocal assignment of the absolute configuration of the target pyrrolidine. The structurally simple pyrrolidine alkaloid, 225H, was selected as a target to develop a general synthetic approach. The enantioselective synthesis of 225H was achieved in nine steps and good overall yield. The search for potent cannabinoid receptor partial agonist ligands as potential marijuana addiction therapeutic agents has led to an investigation of the synthesis of diaryl ether hybrid analogues of BAY 59-3074. A series of 2-(3-alkyl-5-hydroxyphenoxy)-6- (trifluoromethyl)benzonitriles, 3-(2-cyano-3-(trifluoromethyl)phenoxy)phenylalkanoates, and (3- (benzyloxy)phenoxy)-6-(trifluoromethyl)benzonitriles were synthesized and evaluated in vitro for CB1 affinity. The olivetol diaryl ether analogue was the most potent ligand of the alkyl series, but the diaryl ester analogues exhibited modest affinity for CB1 receptors. The most potent compound of the series was the 2-(3-(benzyloxy)phenoxy)-6- (trifluoromethyl)benzonitrile.