Linda P. Dwoskin - US grants

DESCRIPTION: (Applicant's Abstract) Cigarette smoking is the number one health problem accounting for more illnesses and deaths than any other single factor. The abuse liability of the major tobacco alkaloid, nicotine (NIC), stems from its intrinsic reinforcing properties suggested to be the result of activation of dopamine (DA) pathways in brain. Elucidation of NIC's mechanism of action and determination of the effects of chronic administration are important for understanding the initiation/maintenance of smoking behavior, and for overcoming problems that smokers experience in extinguishing this behavior. Considering the plethora of information on the peripheral metabolism of NIC, little is known about NIC metabolism in brain and about the potential pharmacologic activity of NIC metabolites. The overall objective of this proposal is to test the hypothesis that CNS effects resulting from NIC exposure during tobacco usage are not solely due to NIC, but result at least in part from actions of NIC metabolites. We have recently demonstrated that NIC metabolites (cotinine, nornicotine (norNIC), norcotinine, and an as yet unidentified fourth metabolite) are present in brain 4 hours after acute peripheral [2'-14C]NIC administration. Also, we have found that norNIC increases DA release from superfused rat striatal slices in a concentration-dependent, Ca++ -dependent, mecamylamine-sensitive, dihydro-beta-erythroidine-sensitive, and stereo-selective manner, indicating nicotinic receptor mediation. Also similar to NIC, repeated S(-) or R(+)norNIC administration were capable of activating the neural mechanism(s) responsible for behavioral sensitization, even though R(+)norNIC produced no overt behavioral effects after its repeated administration. The proposed project will determine the kinetics of accumulation of NIC metabolites in brain, the pharmacological activity and mechanism of action of NIC metabolites, and the time-dependent behavioral and neurochemical changes in response to repeated norNIC administration. The results of the proposed studies will increase our knowledge of the molecular mechanisms that are involved in the neurobehavioral effects resulting from tobacco use and NIC exposure.

This K02 application proposes to provide me with release from teaching and administrative responsibilities and afford me a period of intensive research focus as a means of enhancing my research career. My career goal is to become a leading researcher in the field of drug abuse. In this application, I plan to develop a new class of subtype-selective nicotinic antagonists by determining the structure activity relationship (SAR) of pyridine-N substituted nicotine (NIC) analogues. Preliminary results demonstrate that N-octylnicotinium iodide (NONI) is a potent, selective, and competitive antagonist of NIC-evoked [3H]dopamine ([3H]DA) release. N-Decylnicotinium iodide (NDNI) competitively, and with high affinity, binds to the [3H]NIC binding site, but does not inhibit NIC-evoked [3H]DA release. These preliminary data demonstrate that pyridine-N substitution of the NIC molecule confers with nicotinic-receptor antagonist activity, and moreover, suggests that NONI and NDNI are selective for different nicotinic receptor subtypes. The proposed research will test the hypothesis that SAR directed at the pyridine-N substituent will optimize the potency, efficacy and nicotinic-receptor subtype selectivity of this novel class of nicotinic receptor antagonist. Structural modifications to be studied include varying pyridine-N substituent steric bulk and lipophilicity, alteration of C-2' chirality, enlargement of the pyrrolidine ring to a piperidine ring and rotameric preference about the C(3)-C(2') bond. The major goal of the research is to determine structural features of the novel NIC analogues that convert the NIC molecule from an agonist to an antagonist at specific nicotinic receptor subtypes. These subtype-selective nicotinic receptor antagonists would be invaluable neuropharmacologic agents for basic and clinical research. As evidence of Institutional support of this endeavor, I will be relieved of 75 percent of my teaching and committee assignments, and I will be provided with a postdoctoral research fellow and the work time and travel funds necessary to learn new techniques, including in vivo and in vitro electrochemistry from collaborative investigators at the University of Kentucky and Indiana University. Electrochemistry is a state-of-the-art technique which will provide outstanding spacial (within a brain region) and temporal resolution (msec) to measure neurotransmitter release both in the brain slice preparation and in the anesthetized, as well as awake and behaving animal. Thus, redistribution of my effort will allow me to concentrate on my research, including learning additional methodologies which will significantly extend my research capabilities and enhance my scientific career in drug abuse research.

DESCRIPTION: (Applicant's Abstract) The abuse of methamphetamine (METH) has escalated in recent years and effective treatments to curtail its use are not yet available. Our preliminary results demonstrate that a-lobeline (LOB), a weakly basic lipophilic alkaloid, binds to the high affinity nicotinic receptor binding site (a4P2 subtype) and acts as an antagonist at the nicotinic receptor subtype which evokes dopamine (DA) release (purportedly, the a3P2 subtype). LOB also inhibits the dopamine transporter (DAT) and the vesicular monoamine transporter (VMAT2), but does not inhibit monoamine oxidase (MAO). Moreover, LOB inhibits amphetamine (AMPT)-evoked DA release and METH self-administration in rats. Our preliminary results suggest that VMAT2 may be an important new therapeutic target for the treatment of METH abuse. With this in mind, we have begun synthesizing a series of LOB analogs to target VMAT2. The long-term goal of this proposal is to develop our lead LOB analog, 2R,6S-N-methyl-2,6-

DESCRIPTION (provided by applicant): Tobacco smoking is the number one health problem accounting for more illnesses and deaths in the US than any other factor. Despite efficacy of some current pharmacotherapies (i.e., nicotine replacement and bupropion), relapse rates continue to be high, indicating that novel medications are needed. Research in the current application proposes to develop a new class of subtype-selective nicotinic receptor (nAChR) antagonists as therapeutic agents with efficacy for tobacco use cessation and for treatment of nicotine dependence. As much as tobacco use behavior and nicotine addiction have links to depression, these novel drug candidates may also prove to be new treatments for depression. Based on the observations that the bupropion acts as a nAChR antagonist and that the nonselective nAChR antagonist, mecamylamine, has some efficacy as a tobacco use cessation agent, but is limited by its peripherally-mediated side-effect of constipation, we predict that the subtype-selective nAChR antagonists, which we propose to develop, may have therapeutic advantages and efficacy as tobacco use cessation agents in the treatment of nicotine addiction. We hypothesize that quaternizing the pyridine-N atom of the nicotine molecule with a lipophillic N-substituent to afford N-nicotinium analogs and/or by connecting these quaternary ammonium moieties with a lipophillic linker to afford N,N'-bis-analogs will result in subtype-selective nAChR antagonists, which will inhibit either nicotine-evoked dopamine, norepinephrine or serotonin release, and thus, inhibit nicotine-induced behaviors, indicating their potential as nicotine addiction treatments. Brain bioavailability, pharmacokinetics and metabolism of the lead candidates will also be evaluated. Comparison of results using native and recombinant nAChRs will provide new insights into the subunit composition of nAChRs mediating these functions. Drug candidates will be assessed for their ability to decrease nicotine self-administration, to decrease nicotine-induced reinstatement of nicotine seeking behavior, and to precipitate withdrawal in nicotine-dependent animals. Thus, an integrative approach (i.e., medicinal chemistry, pharmacokinetics, metabolism, pharmacology, psychology and neuroscience) will be used to increase our understanding of the underlying mechanisms of tobacco use and nicotine addiction, with focus on pharmacotherapeutic candidate development.

DESCRIPTION (provided by applicant): Methamphetamine (METH) abuse continues to escalate and effective treatments are not currently available. METH interacts with the vesicular monoamine transporter-2 (VMAT2), promoting both dopamine (DA) release into the cytosol and reversal of the DA transporter to increase extracellular DA concentrations, which is thought to be associated with its abuse liability. The overall objective of this project is to provide a clinical candidate for the treatment of METH abuse. Recently, we identified a novel small molecule (GZ-793A), which potently and selectively inhibits DA uptake by VMAT2, inhibits METH-evoked DA release from synaptic striatal vesicles and slices, and exhibits selectivity does not interact with nicotinic receptors or DA transporters. Off-target evaluation at a cadre of neurotransmitter-related, steroid and ion channel sites, second messenger, prostaglandin, growth factor and hormones, brain/gut peptides and enzymes revealed outstanding selectivity. Translation to whole animals models revealed that GZ-793A specifically decreases METH self-administration without altering responding for sucrose, does not produce tolerance to the decrease in METH self- administration upon repeated administration, and decreases METH seeking in the cue-induced reinstatement. Acute GZ793A pretreatment also protects against the neurotoxic effects of METH as measured by striatal DA depletion. Although the physicochemical properties of GZ-793A are favorable in terms of druggability, GZ-793A has only 2-3% oral bioavailability, likely due to the presence of the hydroxyl functionalities in the molecule which are likely sites for high first-pass metabolism. Thus, new optimized analogs of GZ-793A are the focus of the current application with the goal of identifying analogs with the required pharmacological properties, druggability, increased oral bioavailability, and acceptable pharmacokinetic profile. Compounds with these characteristics would be high value preclinical candidates for advancement toward clinical trials as treatments for METH abuse. Thus, we propose to expand our existing molecular library of analogs by performing the final steps of optimization of GZ-793A to identify a high value preclinical candidate as a potential therapeutics for METH abuse. In future work, the best candidate will undergo comprehensive toxicological evaluation for subsequent preparation of an Investigational New Drug application to the FDA as a proposed treatment for methamphetamine abuse.

DESCRIPTION (provided by applicant): Tobacco use is the number one preventable cause of death in the US. The reinforcing efficacy of nicotine (NIC), the most abundant alkaloid in tobacco, plays a major role in the maintenance of tobacco smoking. Furthermore, tobacco use and depressive disorders are highly comorbid, and NIC maybe be used in part to alleviate depression in this clinical population. Research on the neurobiology of reward and drug addiction has focused on mesocorticolimbic and nigrostriatal dopamine (DA) pathways. NIC activates nicotinic receptors which increases extracellular DA concentrations at both terminal and cell body regions of these pathways. Extracellular DA concentration is the net result of neurotransmitter release from the presynaptic terminal and neurotransmitter clearance from the extracellular space. DA clearance is mediated primarily by the plasma membrane dopamine transporter (DAT). Although there is a wealth of information on NIC stimulated DA release, the ability of NIC to modulate DAT function and the underlying mechanisms responsible for this effect have not been studied in detail. Preliminary data show that in prefrontal cortex, acute NIC increases DA uptake into synaptosomes, DA clearance in in vivo voltammetry studies and DAT trafficking to the cell surface. Thus, NIC augmentation of DAT function appears to sharpen the kinetics of the NIC-induced increase in extracelluar DA concentration in prefrontal cortex. The proposed study will begin to elucidate the underlying mechansims responsible for the NIC-induced enhancement of DAT function. The hypothesis to be tested in the current application is that NIC, via nicotinic receptor activation, mediates the trafficking of DAT. This hypothesis will be tested using the rat as the animal model, and will determine (1) the dose-related effects of acute in vivo administration of NIC on DAT trafficking in striatum, nucleus accumbens, prefrontal cortex and amygdala; (2) if nicotinic receptors mediate the effect of NIC on DAT trafficking; and (3) the effect of intermittent and continuous NIC administration on DAT trafficking. Results of these experiments will begin to elucidate the cellular mechanisms by which NIC and nicotinic receptors modulate DAT function, and thereby, contribute to the regulation of extracellular DA concentration. Thus, insight will be provided with respect to the effect of NIC on DA neurotransmission in brain regions associated with drug abuse.

DESCRIPTION (provided by applicant): Drug abuse continues to cause great societal burden. Despite intensive efforts, effective pharmacotherapeutic treatments for drug dependence are still lacking, indicating new strategies and targets are needed. A growing body of evidence supports our hypothesis that selective antagonism at the M5 muscarinic acetylcholine receptor (mAChR) represents a novel target for the treatment of drug dependence. The rewarding effects of most drugs of abuse are believed to be mediated by the mesolimbic dopaminergic pathway projecting from the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) to the nucleus accumbens (NAc) and striatum, respectively. The M5 receptor is the only subtype localized on dopaminergic neurons of the VTA and SNc. Microinfusion of the non-selective mAChR antagonist scopolamine into the VTA or SNc substantially reduces DA release in NAc or striatum, respectively. Although there are no selective M5 receptor antagonists currently available, behavioral studies using mice lacking functional M5 receptors have shown a reduction in reward and withdrawal responses following morphine and cocaine administration, and also a reduction in the rate of cocaine self-administration. These results show a role of the M5 receptor in modulating cocaine and opiate addiction. Furthermore, co-administration of scopolamine with cocaine reduces self-administration of cocaine by rhesus monkeys, and scopolamine has been shown to be effective for detoxification of heroin addicts in clinical studies. We reasoned that the effectiveness of scopolamine is due to antagonism of the M5 receptor;however, the involvement of other subtypes can not be ruled out. To date, the physiological and pharmacological roles of the M5 mAChR are still obscure and research has been hampered by the lack of subtype-selective M5 ligands. The objective of this application is to discover potent and selective M5 receptor antagonists, which, in our long-term goal, can be developed into medical treatments for drug dependence. We expect that compounds developed in this research project will also serve as pharmacological tools useful for studying physiological functions of the M5 receptor. New compounds will be designed and synthesized based on the structural scaffold of literature reports on low selectivity and low potency M5 antagonists. Compounds synthesized will be characterized for receptor binding affinities in recombinant Chinese hamster ovarian (CHO- K1) cells expressing the individual human muscarinic receptor subtypes, ability to inhibit carbachol-induced phosphatidylinositol (PI) hydrolysis in CHO cells expressing hM5 receptors and by inhibition of oxotremorine- evoked dopamine release from superfused rat striatal slices (functional assay). PUBLIC HEALTH RELEVANCE: These studies will pioneer the development of selective M5 receptor antagonists which have potential as efficacious treatments for drugs of abuse.

DESCRIPTION (provided by applicant): This application requests a second renewal of five years of support for a successful interdisciplinary and translational training program in broad-based contemporary aspects of drug abuse research. Training the next generation of drug abuse researchers is critical to current and future public health challenges associated with drug addiction. The goal is to prepare trainees for productive and successful careers in drug abuse research. This program proposes to support 4 predoctoral and 2 postdoctoral trainees. We will vigorously recruit individuals from underrepresented minorities, disadvantaged backgrounds, and individuals with disabilities to increase diversity. This program provides a highly collaborative environment of interdisciplinary and translational training in drug abuse research. The 17 training faculty represent 7 academic units, have excellent training records, and will provide a rich interdisciplinary training environment. The program fosters the development of essential experimental and critical thinking skills, and provides the opportunity to gain an in depth understanding of and expertise in the interrelationships of the molecular/cellular aspects of receptors and signaling mechanisms involved in the neural and behavioral response to drugs of abuse and to become immersed in drug discovery and development in the pursuit of novel treatments for drug abuse. The overarching theme of the program is that drug addiction alters fundamental cellular and macromolecular processes resulting in long term changes in neural plasticity and behavior, which can be treated using pharmacotherapeutic intervention. The curriculum provides knowledge from physicochemical properties of molecules to structural biology, neurophysiology, neurochemistry, to animal and human behavior, with numerous opportunities for in depth study of focused areas of drug abuse research. The breadth of drug abuse research opportunities is enhanced by strong links to the University of Kentucky's (UK's) Center on Drug and Alcohol Research, the Center for Drug and Alcohol Research Translation, the Center for Clinical and Translational Science, the Center for Pharmaceutical Research and Innovation, the Laboratory on Human Behavioral Pharmacology and the Residential Research Facility. This program provides value added by serving as a linchpin to networking, interactions and collaborations with other trainees and training faculty focused on human behavioral and clinical aspects of drug abuse research and supported by a second T32 program directed by Dr. Craig Rush at UK. All positions in both T32 programs have been filled completely during the last funding period, indicative of the large number of promising trainees in drug abuse research at UK. UK provides solid infrastructure and institutional support, optimizing the training environment. The majority of program graduates continue to actively pursue drug abuse research and advance towards independent investigator status. In the upcoming funding period, we propose to continue our record of success and develop responsible and ethical drug abuse researchers, who will move the field of drug abuse forward.

The new Training and Pilot Core for the Center for Drug Abuse Research Translation (CDART) has the specific goal of encouraging trainees to develop, implement and evaluate evidence-based preventive interventions that rely on multi-disciplinary perspectives and research. The central theme of CDART is that different facets of impulsivity (urgency, disinhibition and sensation seeking) are associated most strongly with different phases of drug use/abuse (problem use, escalation and initiation, respectively). As a key component of CDART, this core will require trainees to build bridges that connect different areas of research and translate basic research into prevention practice. Pilot investigators will be encouraged to design and implement preventive interventions that fit within the CDART theme. Thus, we will expand and formalize the roles of training and pilot project development through the following Specific Aims: 1) Provide the structure to train graduate students and postdoctoral scholars in translational research relevant to drug abuse prevention; 2) Promote and manage our pilot project program that introduces faculty and research associates to translational research relevant to drug abuse prevention and CDART's research mission; 3) Identify promising minority candidates for recruitment into the training and pilot project programs relevant to drug abuse prevention. As part of the approach, pre- and post-doctoral trainees will be introduced to the field of drug abuse prevention, with a special emphasis on translational work by attending and presenting in monthly trainee data presentations, attending seminars by researchers in drug abuse, and retreats designed to brainstorm with CDART faculty about future research plans, taking relevant topic-specific courses and ethics in research training, and attending informal seminars. The pilot program will prioritize eariy career investigators and minority scholars for funding, as well as more advanced faculty new to the drug abuse field. Proposals will be required to demonstrate the relevance of the project to the science and practice of drug abuse prevention. As a result, CDART trainees and pilot project principal investigators will be prepared to take lead roles in bridging the gap between basic and applied researchers. RELEVANCE (See instructions): Prevention strategies are important for reducing the incidence of drug abuse. The Training and Pilot Core will will require CDART trainees to build bridges connecting and translating basic reearch into prevention science. As such, the core will be instrumental in preparing trainees and fostering pilot project investigators to take leading roles in bridging the gap between basic and applied research in prevention science.

Methamphetamine (METH) use disorder is linked to dire health and societal consequences. Effective treat- ments for METH addiction are not available. METH interacts with the vesicular monoamine transporter-2 (VMAT2), promoting dopamine (DA) release into the cytosol and reversal of the DA transporter to increase ex- tracellular DA, resulting in abuse liability. This project focuses on VMAT2 as a novel therapeutic target, with the overall goal of obtaining a treatment for METH use disorder. Phase 1b studies with lobeline, the initial lead, were completed; however, bitter taste and multiple daily doses were expected to reduce compliance. Lobeline was modified to obtain GZ-793A, which had the desired pharmacology, but also had hERG toxicity. We next identified novel small molecules, GZ-11608 and JPC-077, with greatly reduced hERG interaction, and with po- tent and selective inhibition of VMAT2 function and METH-evoked DA release. These new leads specifically decrease METH self-administration and METH-induced reinstatement in rats at doses that do not alter food reinforcement, suggesting efficacy against relapse. Tolerance does not develop upon repeated dosing. These analogs protect against or do not exacerbate METH-induced striatal DA neurotoxicity. Despite this favorable pharmacologic profile, these analogs have low oral bioavailability. Thus, this proposal focuses on the discov- ery/development of optimized GZ-11608 and JPC-077 analogs with increased oral bioavailability. First, we will substitute deuterium for hydrogen at sites of metabolic liability. If deuterium substitution is not successful, we will use rational design to synthesize a focused library based on each lead. Chemoinformatics, incorporating refined models based on our library of ~520 VMAT2-targeted compounds, was used to predict optimized ana- logs with decreased metabolic liability and desirable drug-like properties. Each analog, from deuterium substi- tution and medchem approaches, will be synthesized and evaluated for selective inhibition of VMAT2, as well as metabolism. We will then assess the pharmacokinetics (PK) to determine oral bioavailability, plasma and brain concentrations and estimate PK parameters following IV, PO and SC dosing. Metabolites exceeding 10% will be synthesized and evaluated for toxicity and potential use as active pharmaceutical ingredient. We will then determine the dose-related inhibition of METH self-administration and reinstatement. Development of tol- erance following repeated administration will be determined. We will conduct abbreviated toxicology studies and assess the potential for off-target interactions and CYP450 inhibition/induction. We will relate the pharma- cokinetics of optimized analogs to pharmacodynamic effects, and determine the maximum tolerated dose, ther- apeutic index and behavioral specificity. Completion of this optimization program will allow for immediate tran- sition of our leads into IND-enabling studies. Successful completion of the project should have a tremendously beneficial health and socioeconomic impact on society, since currently there are no available treatments for METH use disorder.

PROJECT SUMMARY T32 Training Grant supplement entitled ?Training in Drug Abuse Related Research?, requests 1 year of support for a highly collaborative translational program in broad-based contemporary aspects of substance use disorder (SUD) research. Training the next generation of SUD researchers is critical to current and future public health challenges associated with SUD. The overarching goal is to prepare trainees for successful careers in SUD research. The overarching theme is that abused substances alter fundamental cellular and macromolecular proteins and signaling processes in brain, resulting in long term changes in neural plasticity and behavior, and that these macromolecular proteins serve as targets for novel therapeutic discovery/development that ultimately will be effective in mitigating harm to the individual and the population. The program will: 1) foster the development of essential experimental and critical thinking skills; 2) provide opportunities to gain an in depth understanding of and expertise in molecular/cellular aspects of receptors/brain signaling mechanisms mediating the behavioral response to abused drugs; 3) become immersed in the pathway of discovery/development/translation/implementation of medications to treat SUD; and 4) understand population-level pharmaceutical outcomes of these interventions. We propose to continue to support 2 predoctoral trainees for 1 year. In the upcoming period, we propose to continue our record of success in developing ethical SUD researchers, who will move the SUD field forward.

ABSTRACT Knowledge generated by academic research is a major source of health-related innovations. Transforming innovative discoveries into commercialized products that improve human health is a difficult process. However, all stakeholders (universities, funding bodies, pharmaceutical and medical device industries, federal and state governments and the public) have a vested interest in identifying scalable approaches that accelerate the translation of academic innovations into biomedical products. The goal of the Kentucky Network for Innovation and Commercialization (KYNETIC) is to build a Research Evaluation and Commercialization Hub (REACH) network of commercialization resources accessible across the entire Commonwealth of Kentucky to accelerate translation of academic innovations into biomedical products. To reach this goal, the following aims will be conducted: 1) establish KYNETIC?s organizational and collaborative structure; 2) Find, fund and manage product definition/development projects; 3) Provide mentoring, skills development, experiential education and networking opportunities with industry experts, entrepreneurs and investors; and 4) Devise and implement a plan to transition to a self-sustaining entity. KYNETIC will build on lessons learned from developing a regional IDeA technology transfer accelerator (at UK) and a current REACH hub (at UL). KYNETIC will be led by the University of Kentucky (UK), in partnership with the University of Louisville (UL) and the Commonwealth Commercialization Center (C3), a private 501(c)3 non-profit commercialization organization. Also, KYNETIC will include all of Kentucky?s public regional universities and community and technical college system (KCTCS). KYNETIC?s mission is to nurture innovations and academic innovators by providing funding, mentoring, education and a network of relevant expertise. Required matching funds for KYNETIC have been committed in full by Kentucky Commonwealth Economic Development (CED) and the state R1 universities. KYNETIC will be directed by a leadership team consisting of 2 academic innovators, 2 technology transfer professionals (from UK and UL) and 2 representatives from partnering entities (C3 and CED). A steering committee including the leadership team and representatives from each participating institution, an External Review Board and the NIH will review and evaluate KYNETIC projects and fund up to 13 projects/yr using an innovative ?quick kill? management strategy. Our services will be available to as many individuals as possible. Diverse role models including innovators and entrepreneurs from underrepresented groups will be sought. The proposed Kentucky-wide REACH program will leverage our collective wealth of resources, ecosystems and extensive experience to apply and expand best practices for innovator education and mentoring. The overall impact of KYNETIC will be unprecedented opportunities for collaboration and synergy, well-prepared innovators, a statewide pro-entrepreneurial culture and delivery of de-risked technologies to benefit health and overcome the state?s health disparities.