Lance R. McMahon, Ph.D. - US grants

DESCRIPTION: (Applicant's Abstract) The acute behavioral effects of cocaine are mediated, in part, by monoamine transmitters located in mesolimbic circuits. The presence of high concentrations of the serotonin (5HT) receptor subtypes, 5HT4 and 5HT6, in the nucleus accumbens and striatum suggests that 5HT systems may modulate the locomotor and discriminative stimulus properties of cocaine. The intent of the present studies is to assess this hypothesis by measuring the locomotor and discriminative stimulus effects of cocaine following either activation or blockade of the brain 5HT4 receptor with systemic administration of 5HT4 receptor ligands as well as following depletion of the brain 5HT6 receptor with intracranial infusion of specific antisense oligonucleotides.

DESCRIPTION (provided by applicant): The general goal of this R21 application is to develop and characterize a novel drug discrimination procedure to study dependence and withdrawal resulting from A9-tetrahydrocannabinol (delta9-THC), a cannabinoid agonist that underlies the reinforcing, subjective, analgesic, and antiemetic effects of the marijuana plant. Marijuana is used by a large segment of the population and recent clinical studies have reported dependence on and withdrawal from delta9-THC and related cannabinoid agonists. Marijuana dependence could promote long-term marijuana use and polydrug abuse and could be a prominent factor underlying high comorbidity between marijuana use and certain mental disorders including panic, anxiety, depression, and psychosis. Very little is known about mechanisms underlying delta9-THC dependence and withdrawal because there are few pre-clinical assays that can be used to define these mechanisms. The procedures developed under this proposal are intended to satisfy this need for new pre-clinical assays that can be used to study delta9-THC dependence. Drug discrimination is uniquely sensitive to drug withdrawal and can measure aspects of withdrawal in non-human animals that cannot be directly observed. One strategy that has been applied successfully to the study of drug dependence and withdrawal is to train drug dependent animals to discriminate a competitive antagonist. This proposal will use standard two-lever operant procedures to train SR 141716A as a discriminative stimulus in a group of delta9-THC treated rhesus monkeys and in another group of untreated monkeys. Experiments will be designed to determine if the discriminative stimulus effects of SR 141716A in delta9-THC treated monkeys are related to delta9-THC withdrawal. Several criteria must be satisfied for this to be the case. First, the SR 141716A discriminative stimulus in A9-THC treated monkeys should be pharmacologically distinct from the SR 141716A discriminative stimulus in untreated monkeys, or SR 141716A should be demonstrated to maintain stimulus control in A9-THCtreated monkeys but not in untreated monkeys. Second, suspension of delta9-THC treatment should substitute for SR141716A in treated monkeys. Third, supplemental doses of delta9-THC should attenuate the SR 141716A discriminative stimulus or re-administration of delta9-THC during delta9-THC deprivation should reverse SR 141716A-appropriateresponding. The outcome of these experiments will determine whether this discrimination assay is related to delta9-THCwithdrawal and whether this assay can be used to define pharmacological mechanisms underlying delta9-THC dependence.

DESCRIPTION (provided by applicant): Marijuana use is a public health concern. Withdrawal that occurs in over one-half of daily marijuana users is responsible, in part, for marijuana smoking. However, cannabinoids in marijuana produce a variety of therapeutic effects (analgesic and anti-emetic effects). While progress has been made toward establishing receptor mechanisms underlying the behavioral effects of cannabinoids, it is not clear whether the clinically useful actions and abuse liability of cannabinoids vary as a function of pharmacologic efficacy at cannabinoid receptors. Moreover, it is not clear whether pharmacologic modulation (e.g. decreased metabolism or cellular uptake) of endogenous cannabinoid agonists (e.g. anandamide) produces therapeutic effects and less of the non-preferred effects associated with direct cannabinoid agonism. This competing continuation of an R01 examines cannabinoid and non-cannabinoid approaches for treating marijuana withdrawal. This application further examines relationships between behavioral effects, pharmacologic (agonist) efficacy, and pharmacologic manipulation of endocannabinoid levels in assays predictive of marijuana-like effects in humans. Aim 1 uses a drug discrimination assay of rimonabant-induced cannabinoid withdrawal in rhesus monkeys to characterize the neuropharmacology of withdrawal that emerges upon discontinuation of treatment. Aim 2 explores relationships between pharmacologic (agonist) efficacy at cannabinoid receptors and behavioral effects. Tolerance and cross-tolerance among cannabinoids that vary in efficacy will be examined in rhesus monkeys discriminating 9-tetrahydrocannabinol ( 9-THC). This aim also establishes a discrimination assay with a high efficacy cannabinoid agonist and examines dependence to a high efficacy cannabinoid agonist, indexed by discriminative stimulus effects and overt signs of withdrawal. The 9-THC discrimination assay in rhesus monkeys was highly sensitive to exogenously administered anandamide, and this assay is used in Aim 3 to examine pharmacologic manipulation of endogenous cannabinoids and interactions between endocannabinoids and 9-THC. Aim 3 also examines modification of cannabinoid withdrawal by anandamide and inhibitors of its metabolism (URB 597) and uptake (AM 404). This competing continuation addresses a need for understanding the neuropharmacology of cannabinoids in behavioral assays predictive of marijuana-like intoxication and dependence. Collectively, studies in this competing continuation provide a framework for developing novel pharmacotherapies of marijuana withdrawal and cannabinoid-based therapeutics that could produce fewer adverse effects (i.e. abuse and dependence liability) than marijuana. PUBLIC HEALTH RELEVANCE: Marijuana use continues to be a public health concern. However, cannabinoids in marijuana produce a variety of therapeutic effects (analgesic and anti-emetic effects). This competing continuation addresses a need to understand mechanisms at cannabinoid receptors that mediate the dependence liability of marijuana and the potential therapeutic utility of the cannabinoids.

DESCRIPTION (provided by applicant): Cigarette smoking is a leading cause of cancer as well as cardiovascular and respiratory disease and is the leading preventable cause of death in the United States. Many factors contribute to cigarette smoking, including nicotine, other chemicals in tobacco smoke, and conditioned reinforcers. This competing continuation of an R01 proposal focuses on nicotine and nicotinic acetylcholine receptors (nAChR) as critical determinants of smoking behavior and smoking cessation pharmacotherapy. Drug discrimination assays in rhesus monkeys will be used to examine the impact of drug history (nicotine treatment) on the effects of low and high efficacy nAChR agonists and allosteric nAChR modulators. In the previous funding period, whereas varenicline substituted for the discriminative stimulus effects of nicotine administered acutely, varenicline was no longer able to substitute for nicotine under conditions relevant to the clinical setting (i.e., chronic nicotin treatment). Aim 1 examines nAChR efficacy (intrinsic activity) as the critical determinant of the ability of nAChR agonists to mimic the discriminative stimulus effects of nicotine. Novel nAChR agonists with higher efficacy than varenicline will be examined for their ability to mimic the effects of nicotine during chronic treatment, whereas novel nAChR agonists with lower efficacy than varenicline will be examined for their ability to antagonize the effects of nicotine during chronic nicotine treatment. In a second experiment conducted during the previous funding period, the dual positive allosteric nAChR modulator and competitive, reversible AChE inhibitor galantamine fully substituted for the discriminative stimulus effects of nicotine in rhesus monkeys. Aim 2 compares the effects of orthosteric and allosteric ligands at nAChR and, in particular, examines the potential of allosteric nAChR modulators to modify the effects of nicotine. Both positive and negative allosteric nAChR modulators with selectivity for subtypes of nAChR will be tested under acute and chronic nicotine treatment conditions. Although currently available pharmacotherapies for smoking cessation are effective in some, there is considerable margin for improvement. These pre-clinical studies will help identify pharmacologic dimensions and novel directions upon which to develop novel medications that could further reduce the devastating consequences of cigarette smoking.

This R01, submitted in response to RFA-DA-09-001 (Medications Development for Cannabis-Related Disorders), has been modified to accommodate a 2-year budget. The grant proposes using pre-clinical measures of cannabinoid withdrawal in non-human primates to identify potential pharmacotherapies of marijuana dependence. Over one-half of daily marijuana users experience withdrawal upon discontinuation of use, which is reported to drive marijuana smoking. Pharmacotherapy of marijuana withdrawal is associated with decreased relapse to marijuana use in the clinical laboratory and, therefore, is a viable strategy for promoting abstinence. This application addresses a compelling need for pre-clinical assays that can provide rapid and efficient testing of drugs for their capacity to attenuate cannabinoid withdrawal. The cannabinoid antagonist rimonabant will provide an index of withdrawal in rhesus monkeys receiving chronic treatment with [unreadable]9-tetrahydrocannabinol, the cannabinoid primarily responsible for the abuse and dependence liability of marijuana. Discriminative stimulus effects will provide a measure of the private experience of withdrawal. Medications will be examined for their ability to modify not only discriminative stimulus effects but also other signs of withdrawal including head shaking and increased night activity in the home cage (i.e. sleep disruption). Aim 1 will examine modification of cannabinoid withdrawal by [unreadable]9-tetrahydrocannabionol alone and in combination with [unreadable]2-adrenergic agonists (clonidine and lofexidine). The combination has been reported to attenuate marijuana withdrawal more effectively than either drug alone. Quantitative pharmacologic (i.e. isobolographic) analysis will be used to examine whether attenuation of cannabinoid withdrawal by the drug combination is additive, less than additive, or greater than additive (synergistic). Synergistic drug combinations could be especially effective therapeutics. Aim 2 evaluates pharmacotherapy of sleep disruption as a strategy for attenuating cannabinoid withdrawal indexed by discriminative stimulus effects and head shaking during the day. Drugs to be studied for their capacity to attenuate sleep disruption as well as next-day expression of cannabinoid withdrawal signs will include a benzodiazepine (zolpidem or Ambien(), the 5HT2A antagonist M100907, and the melatonin agonist ramelteon (Rozerem(). Collectively, these specific aims provide a framework for developing novel pharmacotherapies of marijuana withdrawal that could markedly decrease marijuana use and dependence.

Mitragyna speciosa (kratom) has exploded onto the US market, with an estimated 55,000 kg entering the US between 2014 and 2016, corresponding to 12 million kratom doses and one million users. The FDA recently used computational modeling to predict that 22 kratom constituents bind to opioid receptors; however, these predictions await translation into whole animals. There is an urgent need for systematic, pharmacological testing of kratom alkaloids to help inform abuse risk. We feel it is important to assess the behavioral effects of not only individual alkaloids, but also alkaloid mixtures representative of plant material in commercially used products. We will extract, isolate, and purify alkaloids from two kratom sources: dried leaf material of Mitragyna speciosa trees and a US commercial product (i.e., OPMS Gold capsules). We will quantify up to ten kratom alkaloids simultaneously using our bioanalytical methods. In a pharmacokinetic-based Aim 1, we will quantify kratom alkaloids in multiple biological matrices (urine, plasma, brain, liver) after p.o. and i.v. administration in rats to obtain a comprehensive ADME profile. Liver microsomes, recombinant CYP450 enzymes, and specific chemical CYP450 inhibitors will be used to identify pathways of metabolism and biologically active metabolites. In our pharmacodynamic-based Aim 2, we will use drug discrimination to identify receptor mechanism(s) underlying abuse-related effects. Four separate groups of rats will be trained to discriminate one of the following: 1) alkaloid mixture in proportion to kratom dried leaf material, 2) alkaloid mixture in proportion to commercially available kratom product (both containing 32 mg/kg mitragynine, 3) 32 mg/kg mitragynine alone, or 4) 3.2 mg/kg 7- hydroxymitragynine alone. Moreover, we will use i.v. drug self-administration to assess abuse risk, and to assess how kratom alkaloids modify the reinforcing effects of abused opioids (i.e., heroin). Male and female rats will be used throughout to assess sex as a biological variable. The overarching hypothesis is that Mitragyna speciosa has a complex pharmacology resulting from multiple alkaloids differentially interacting with both opioid and non- opioid receptors. The following specific hypotheses will be tested: 1) alkaloids interact (i.e., exert synergistic and antagonist effects) with each other and with other abused opioids in drug discrimination and self-administration assays; 2) some of the interactions are due to PK; 3) behavioral effects of the parent alkaloid are due in part to both the parent compound and its behaviorally active metabolites; 4) no single alkaloid accounts for the discriminative stimulus and reinforcing effects of the mixtures; and 5) both opioid and adrenergic receptors mediate the effects of alkaloid mixtures representative of natural plant material. Subtraction and addition of individual alkaloids will help identify the alkaloids most responsible for kratom's integrated pharmacology. After 5 years, we expect to demonstrate that kratom alkaloids exert differential effects through multiple receptor systems, and we further expect to identify key pharmacological mechanisms responsible for the widespread use of kratom.

PROJECT SUMMARY This project is submitted under Funding Opportunity Announcement (FOA) Number: RFA-DA-19-002. Opioids have been significantly over-prescribed and are associated with numerous deaths, resulting in the Nation?s current opioid crisis. The FDA recently approved the ?2 adrenergic agonist lofexidine as a non-addictive, non-opioid treatment for opioid use disorder. This preclinical drug development effort stems from the psychoactive, natural product, Mitragyna speciosa (kratom), a Thai medicinal herb used as a self-treatment for opioid use disorder. Mitragynine, the plant?s most abundant alkaloid, is a low efficacy µ receptor agonist with G- protein signaling bias. Our preliminary studies suggest that mitragynine has limited abuse liability, and interacts with non-opioid CNS targets including ?2 adrenergic receptors, which have not been exploited in its unique mechanism. A single drug (mitragynine) that interacts with both opioid and ?2 adrenergic receptors would offer a highly innovative approach for treating opioid use disorder. The work planned here, involving a collaborative, interdisciplinary team, will examine the pharmacophoric elements of mitragynine through synthetic derivatives in an approach that led to the understanding of the essential pharmacophore of morphine. We will use a combination of chemical and prodrug synthesis, in vitro metabolic stability, affinity and efficacy analysis, behavioral assays predictive of receptor mechanism (drug discrimination), abuse (self-administration), and untoward effects (respiratory depression, tolerance, and dependence), and in vivo ADME assays. Mitragynine analogs are expected to yield innovative compounds with a pharmacological mechanism that includes opioid and adrenergic activity. Our efforts to identify the pharmacophoric requirements of mitragynine will lead to templates for the design of novel opioid receptor ligands; this will greatly improve the knowledge of interactions of these structurally novel compounds with opioid receptors and facilitate the development of these ligands as treatments for opioid use disorders. The specific aims of the 2-year UG3 phase are as follows. AIM 1: Identify opioid pharmacophoric requirements of mitragynine analogs through deletion design and analog stability; identify mitragynine prodrugs. AIM 2: Investigate mitragynine analogs in drug discrimination, self-administration, and respiration assays. Analogs exhibiting desired metabolic stability, bioavailability, blood-brain-barrier penetration, binding characteristics, and behavioral activity will be further studied in the UH3 phase as follows. AIM 3: Establish comprehensive in vivo ADME of mitragynine analogs and prodrugs. AIM 4: Assess mitragynine analogs and prodrugs in tolerance, dependence, and withdrawal assays. The results of this project will provide a more comprehensive understanding of the chemical requirements of the putative recognition elements of mitragynine-related ligands at opioid and ?2 adrenergic receptors. Ultimately, the potential use of mitragynine and its analogs as templates for the development of a new treatment for opioid use disorders will be realized that may have the potential to yield a safe, effective FDA-approved pharmacotherapy.