• The Development of levo-Tetrahydropalmatine as a Treatment for Nicotine Addiction

      Faison, Shamia; Wang, Jia Bei; 0000-0002-4355-8624 (2015)
      The negative consequences of nicotine use are well known and documented, however, abstaining from nicotine use and achieving abstinence poses a major challenge for the majority of nicotine users trying to quit. l-Tetrahydropalmatine (l-THP), a compound extracted from the Chinese herb Corydalis, has been used clinically in China for over 40 years as a sedative and analgesic. l-THP has also displayed utility in the treatment of drug addiction, particularly cocaine and heroin addiction. The objective of this dissertation was to investigate if the utility of l-THP could be extended to include treatment of nicotine addiction. As such, the effect of l-THP treatment on abuse-related effects of nicotine was assessed in various behavioral models of nicotine addiction. Once the utility of l-THP was determined, the efficacy of l-THP was tested against varenicline and bupropion, FDA approved treatments for nicotine addiction. To better understand neurochemical changes that occur with l-THP administration, microdialysis experiments were performed where it was found that l-THP interacts with the mesolimbic dopamine system, a prominent pathway of drug addiction. The efficacy of l-THP in the treatment of various models of nicotine addiction and its interaction with the mesolimbic dopamine system make l-THP an attractive compound to study concerning nicotine addiction. Given the long standing safe use of l-THP in China and this new preclinical data, l-THP warrants further study and development for the treatment of nicotine addiction.
    • The in vitro and in vivo studies of the structure and functional regulation of the mu-opioid receptor

      Deng, Hong Bing; Wang, Jia Bei (1999)
      A prominent characteristic of opioid drugs is their ability to induce tolerance and dependence in human. The mu receptor phosphorylation and desensitization are important cellular mechanisms that may contribute to these neuroadaptive processes. The overall goal of the present research is to understand the molecular mechanisms of mu opioid receptor phosphorylation and desensitization as well as study the molecular structure of mu opioid receptor. We employed molecular biology and cell biology techniques combined with pharmacological methods to accomplish these research goals in three aspects. In the first part, we characterized agonist-induced mu opioid receptor phosphorylation events in brain tissues. We found that in the thalamus of morphine-tolerant rats, both the basal and agonist stimulated receptor phosphorylation levels were enhanced compared with those observed in normal rats. The enhanced phosphorylation level correlated well with the desensitization of their cellular response. These findings demonstrate that agonist induced mu opioid receptor phosphorylation Plays an important role in receptor desensitization, which may contribute to the mechanisms underlying tolerance and dependence. In the second part, we determined the role of the C-terminus in mu opioid receptor phosphorylation and desensitization by C-terminal deletion and point mutation of the receptor. Our data suggest that the C-terminus is an important domain involved in the 4 opioid receptor phosphorylation and desensitization. We further found that threonine 394 is a crucial residue that is required for agonist-induced mu opioid receptor phosphorylation and desensitization. In the last part, we investigated the effects of sulfhydryl reagents on mu receptor binding and identified that the cysteine residues in TMIII (C161), TMIV (C192), TMV (C237) and TMVII (C332) of the receptor that are critical cysteines responsible for the sensitivity of mu opioid receptor binding to sulfhydryl specific reagents. These four cysteines may be located in or near the receptor binding site. These findings provide important information for understanding the molecular mechanisms of interaction between the mu opioid receptor and its ligands.
    • levo-tetrahydropalmatine (l-THP) and low dose naltrexone (LDN) a novel combination treatment for the prevention of cocaine relapse

      Sushchyk, Sarah Ashley; Wang, Jia Bei; 0000-0001-5523-5876 (2015)
      To date, FDA is yet to approve a medication for the treatment of cocaine dependence or for the prevention of cocaine relapse. One promising potential treatment is l-THP, primarily a modest dopamine antagonist. However, l-THP possesses unwanted sedative side effects, which could be difficult to overcome clinically. The present study aims to develop an improved cocaine relapse treatment, creating an l-THP based combination medication. Our preliminary experiments determined l-THP when co-administered with LDN, decreased the sedative effect and increased the efficacy of l-THP. As a result, the focus of this dissertation was placed on the development of l-THP & LDN as a combination medication for the prevention of cocaine relapse. Specific aims used to accomplish the objective were: 1) determine the efficacy of l-THP & LDN combination for attenuation of cocaine seeking behavior as well as minimization of sedative effect of l-THP and 2) investigate the mechanism of l-THP & LDN through β-endorphin release and POMC expression. The combination of l-THP & LDN attenuated reinstatement of conditioned place preference as well as drug-seeking behavior in the reinstatement of cocaine self-administration. Additionally, the l-THP sedative effect observed at the 3mg/kg and 5mg/kg l-THP doses was ameliorated through co-administration of 0.1mg/kg LDN. Taken together, results of the behavioral studies indicate 3mg/kg l-THP & 0.1mg/kg LDN has the greatest potential as a cocaine relapse prevention treatment. This dosage was used to examine the effect of l-THP & LDN on endogenous β-endorphin release and POMC expression. In animals treated with 3mg/kg l-THP & 0.1mg/kg LDN, we correlated the reduction of drug seeking with an increase of plasma β-endorphin and hypothalamic POMC mRNA expression. This to our knowledge is the first study investigating the underlying mechanism of LDN. The research presented in this dissertation establishes l-THP & LDN as novel treatment for the prevention of cocaine relapse and dependence with great potential for future clinical translation.
    • Molecular Mechanisms Underlying the Regulation of Mu Opioid Receptor Function by Protein Kinase C and Histidine Triad Nucleotide Binding Protein 1

      Feng, Bo; Wang, Jia Bei (2010)
      Mu opioid receptor (MOPr) belongs to G protein-coupled receptor superfamily and is the primary target through which opioid drugs exert their biological activities. Multiple lines of evidence support that agonist-induced adaptive changes of MOPr are important molecular mechanisms underlying the development of opioid tolerance and dependence. It has also been shown that phosphorylation of MOPr, especially phosphorylation of C-terminus, plays a critical role in the regulation of receptor adaptive changes. In addition to G protein-coupled receptor kinase (GRK), second messenger protein kinases, including protein kinase C (PKC), are also involved in the phosphorylation of MOPr. Although previous research has shown that PKC is capable of regulating MOPr adaptive changes and the development of opioid tolerance and dependence, functional impacts of PKC-mediated receptor phosphorylation on MOPr signaling remain unclear. To test the hypothesis that PKC-mediated regulation of MOPr signaling is achieved through phosphorylation of the receptor C-terminus. In vitro phosphorylation results demonstrated that PKC is capable of phosphorylating MOPr C-terminus and Ser363 is the primary PKC phosphorylation site. This result was further confirmed in CHO cells stably expressing full-length MOPr. Mutating the PKC-phosphorylation site to Ala did not affect the receptor-ligand binding and receptor-G protein coupling. However, this mutation inhibited PMA-induced, but not DAMGO-induced, decrease of receptor-G protein coupling. In all, these results indicated that PKC phosphorylates MOPr C-terminus, and induces the receptor desensitization at G protein coupling level. Previous research of our lab has identified an intracellular protein, histidine triad nucleotide binding protein 1 (HINT1), interacts with MOPr and modulates receptor functions. Although HINT1 inhibits PMA-induced MOPr phosphorylation, which suggests that HINT1 may regulate MOPr function through a PKC-related manner, the molecular mechanism underlying the regulation of MOPr by HINT1 is still unknown. It is suspected that HINT1-nucleotide interaction is crucial for HINT1 biological functions, and interrupting the HINT1-nucleotide interaction will provide an opportunity to reveal the mechanism for HINT1 regulation of MOPr. Nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography studies were performed to reveal the structure requirement of HINT1-nucleotide interaction. Fluorescence titration and isothermal titration calorimetry (ITC) assays further confirmed and quantified the HINT1-nucleotide interaction. Moreover, the interaction of HINT1 with AICAR, a novel non-phosphate nucleotide analogue, was also investigated. Structure information and methodologies achieved in this research provide a basis for future research aimed at revealing the mechanism of HINT1 function.