The Molecular Neurobiology and Human Genetics of Specific Addictive Diseases: Implications for Treatments

Transcription

1 The Molecular Neurobiology and Human Genetics of Specific Addictive Diseases: Implications for Treatments Mary Jeanne Kreek, M.D. Patrick E. and Beatrice M. Haggerty Professor Head of Laboratory The Laboratory of the Biology of Addictive Diseases The Rockefeller University Senior Physician, The Rockefeller University Hospital NYSAM February 2, 219 New York, NY Funded primarily by NIH-NIDA (P6-513), The Adelson Medical Research Foundation, Robertson Therapeutic Development Fund, Tri-Institutional Therapeutics Discovery Institute, NCATS-NIH-CTSA UL1-TR43 (RUH Dr B. Coller)

2 The Molecular Neurobiology and Human Genetics of Specific Addictive Diseases: Implications for Treatments I. The Opioid Crisis (21 increasing through 218); early treatment research; current status and needs II. III. Molecular neurobiology of addictive diseases: current work on oxycodone effects in adult and adolescent mice Translational work to attempt to develop a potential treatment for cocaine addiction and alcoholism: focus on kappa opioid system IV. Human molecular genetic studies related primarily to opiate addiction, but also to cocaine addiction and alcoholism

3 Initial Research on the Biology of Addictive Diseases at the Rockefeller University (then Institute), 1964: Development of a Pharmacotherapy for Heroin Addiction HYPOTHESIS: Heroin (opiate) addiction is a disease a metabolic disease of the brain with resultant behaviors of drug hunger and drug selfadministration, despite negative consequences to self and others. Heroin addiction is not simply a criminal behavior or due alone to antisocial personality or some other personality disorder. Vincent P. Dole, Jr., MD; Marie Nyswander, MD; and Mary Jeanne Kreek, MD 1964: Initial clinical research on development of treatment using methadone maintenance pharmacotherapy and on elucidating mechanisms of efficacy performed at The Rockefeller Hospital of The Rockefeller Institute for Medical Research: First research paper describing methadone maintenance treatment research Dole, V.P., Nyswander, M.E. and Kreek, M.J.: Narcotic blockade: a medical technique for stopping heroin use by addicts. Transactions of the Association of American Physicians (May 1966), 79: , (including discussion) Dole, V.P., Nyswander, M.E. and Kreek, M.J.: Narcotic blockade. Arch. Intern. Med., 118:34-39, Dole, Nyswander and Kreek, 1964, 1966, 218

4 Functional State (Heroin) Functional State (Methadone) Impact of Short-Acting Heroin versus Long-Acting Methadone Administered on a Chronic Basis in Humans (1964 through 1978 Studies): Opioid Agonist Pharmacokinetics Heroin Versus Methadone "High" Drug or Medication Apparent Plasma Terminal Half-life and Duration of Desired Effects "Straight" "Sick" "High" AM PM AM PM AM Days HEROIN 3 min for prodrug 3 min for active compound, mono-acetyl morphine (fast on-set and off-set) 6 hours for active metabolites (morphine and others) "Straight" "Sick" METHADONE 24h for racemic (rs) medication (slow on-set and off-set steady-state achieved) AM PM AM PM AM Days H 48h for active (r) enantiomer Dole, Nyswander and Kreek, 1966; Kreek et al., 1973; 1976; 1977; 1979; 1982; Inturrisi et al, 1973; 1984; 218

5 Drug overdoses, primarily opioids, killed more than 72,3 Americans in 217, a record and a rise of approximately 1% over 216 Overdose Deaths in Thousands in Preceding 12 months Synthetic opioids 3 thousand 2 1 Cocaine Heroin Other opioids Other Psychostimulants Methadone Drug overdose deaths in 217 were higher than the peak yearly deaths from HIV, car accidents, or gun deaths Overdose deaths have begun to fall in Massachusetts, Vermont, and Rhode Island following major public health campaigns, including increased access to treatment, in response to the early arrival of fentanyl in those states Sanger-Katz, NY Times, Aug 15, 218

6 Prevalence of Specific Drug Abuse and Vulnerability to Develop Addictions 219 National Household Survey and Related Surveys Heroin Use ever ~ 5.2 million Heroin Addiction ~ 626, Illicit Use of Opiate Medication ever ~ 37.1 million (i.e., 14.2% of the population 12 and over) Dependence on Opiate Medication Use ~ 2.1 million Opiate (heroin, fentanyl, other) Overdose Deaths 49,68 (in 217)* Cocaine Use ever ~ 4.5 million Cocaine Addiction ~ 966, Alcohol Use ever Alcoholism ~ 216 million ~ 14.5 million Marijuana Use ever ~ 123 million Marijuana Daily Use ~ 4 million Development of Addiction After Self-Exposure to Specific Drugs Opiate Addiction ~ 1 in 5 to 1 in 15 (2% to 6.5%) Alcoholism, Marijuana, and Cocaine Dependency ~ 1 in 8 to 1 in 15 (12.5% to 6.5%) SAMHSA Nat l Survey on Drug Use and Health, 217; Others, 27-18; *Nat l Center for Health Statistics (CDC), 219

7 Research and Clinical Evidence Contributes to Specific Actions for Prevention, Reversal, and Reduction of Illicit Opiate Use and Overdose Deaths (53,332 in 216) Prevention of Overdose FDA recommended by outside experts to approve opioid prescriptions for acute pain for only seven days (1-3 days adequate for most patients; now prescriptions usually are for 21 days) and possibly to allow chronic opioid prescriptions for cancer pain only. Reversal of Overdose Naloxone (IM, IV, or pernasal) is the primary way to reverse overdose (6-9m duration of action); two other mu opioid receptor antagonists, naltrexone and nalmefene, if available in an IV form, would also work. Reduction by Long-Term, Effective Treatment Methadone maintenance pharmacotherapy: 55 years of clinical research and practice evidence of effectiveness (6-8% 12-month voluntary retention with reduction or elimination of opiate use in over 8% of patients; half life hours; oral dose 8-15mg per day) Buprenorphine-naloxone maintenance pharmacotherapy: 3 years of clinical research and practice evidence of effectiveness (4-6% 6-month voluntary retention; receptor occupancy of 24 hours; 24-32mg per day sublingual or film; naloxone added to prevent intravenous abuse) Kreek, Adelson, and other colleagues 1966, 1972, 1973, 1975, 1978, 2, 22, 217, 219

8 Identification of HIV-1 Infection in Intravenous Drug Users New York City: Study Protective Effect of Methadone Maintenance Treatment 1 75 Percent of IV Drug Users Infected with HIV-1 % % 6% Untreated, street heroin addicts: positive for HIV-1 antibody 9% Methadone maintained since <1978 (beginning of AIDS epidemic): less than 1% positive for HIV-1 antibody 4% 9% Heroin addicts in treatment positive for Hepatitis C infection (But less than 1% in treatment for Hepatitis C) Des Jarlais, Kreek, et al., MMWR: Morbid. Mortal. Wkly Rep., 33:377, 1984; Novick, Khan, Kreek, United Nations Bulletin on Narcotics, 38:15, 1986; Des Jarlais, Kreek et al., JAMA, 261:18, 1989.

9 Methadone Maintenance Treatment for Opiate (Heroin) Addiction 219 Number of patients currently in treatment: USA: ~ 36, Europe: ~ 6, Rest of world: ~ 4, Efficacy in good methadone treatment programs using adequate doses (8 to 15mg/d): Voluntary retention in treatment (1 year or more) 6 8% Continuing use of illicit heroin 5 2% Actions of methadone treatment: Prevents withdrawal symptoms and drug hunger Blocks euphoric effects of short-acting narcotics Allows normalization of disrupted physiology ~ 1.4 million worldwide Mechanism of action: Long-acting medication (24h half-life for racemate in humans) provides steady levels of opioid at specific receptor sites. methadone found to be a full mu opioid receptor agonist which internalizes like endorphins (beta-endorphin and enkephalins) methadone also has modest NMDA receptor complex antagonism Kreek, 1972; 1973; 219

10 Status of Methadone, Buprenorphine, and Extended Release Naltrexone Treatments for Opioid Addiction in the United States: Decrease, then Increase, of Numbers in Treatment (215, 216, and 217 data, SAMHSA, 218)* Source: Center for Behavioral Health Statistics and Quality, Substance Abuse and Mental Health Services Administration (SAMHSA), National Survey of Substance Abuse Treatment Services (N-SSATS), 218; Kreek 219 US Patients in Treatment Treatment Methadone Maintenance 356, ,443 (-11,4; -3.2%) 382,867 (+37,424; +1.8%) Buprenorphine Maintenance Extended Release Naltrexone 75,723 61,486 (-14,237; -18.8%) 7,35 1,128 (+3,93; +44.%) 112,223 (+5,737; +82.5%) 23,65 (+12,937; %)

11 Limited Targeted Pharmacotherapies Available for Specific Addictive Diseases I. Opiate Addiction (Heroin and Illicit Use of Prescription Opiates) a. METHADONE (6-8%)** b. BUPRENORPHINE (+ NALOXONE) (4-5%)** [c. NALTREXONE / SUSTAINED RELEASE NALTREXONE (<15%)*] II. III. Alcohol Addiction and Excessive Alcohol Use a. NALTREXONE (3-4%)* b. NALMEFENE (approved in Europe only, 212) c. ACAMPROSATE (low in USA) Cocaine, Amphetamines, Methamphetamines and Other Stimulants NONE (%) is % of unselected persons with specific addictions who can be retained voluntarily in treatment for 3 months (*) or 12 months (**), with success in eliminating specific drug use. According to the National Institute on Drug Abuse, every year drug and alcohol misuse costs the United States $64 billion in healthcare and a total of $6 billion in healthcare, crime and criminal justice, and loss of productivity costs. By comparison, cancer costs $172 billion annually. Effective treatment saves around $12 for every $1 spent. Kreek, 219

12 Targets of Currently Approved Treatments for Addictive Disorders Kreek et al, Journal of Clinical Investigation, 12: 3387, 212

13 Natural History of Drug and Alcohol Abuse and Addictions Primary Prevention Possible Utility of Vaccines and Selected Medications Medications Useful and Needed relapse to addiction without pharmacotherapy 9% - opiate; 6% - cocaine, alcohol Initial Use of Drug of Abuse Sporadic Intermittent Use Regular Use Addiction Early Withdrawal (abstinence) Protracted Abstinence Progression ADDICTION: Compulsive drug seeking behavior and drug self-administration, without regard to negative consequences to self or others (adapted from WHO). sustain abstinence with no specific medications 1% - opiate; 4% - cocaine, alcohol Adapted from Kreek et al., Nature Reviews Drug Discovery, 1:71, 22; 219

14 Factors Contributing to Vulnerability to Develop a Specific Addiction use of the drug of abuse essential (1%) Genetic (25-8%) DNA SNPs repeats other polymorphisms Environmental (very high) prenatal postnatal epigenetics cues peer pressure comorbidity stress-responsivity mrna levels peptides proteomics Drug-Induced Effects (very high) neurochemistry synaptogenesis behaviors Kreek et al., 2; 25; 219

15 Development of an Addiction: Neurobiology Drugs (and alcohol) which may lead to chemical addictions alter normal brain networks and neurochemicals, primarily the dopaminergic and endogenous opioid systems, in the substantia nigra compacta (SNc), ventral tegmental area (VTA), dorsal (caudate putamen) and ventral (nucleus accumbens) striatum, and also amygdala, anterior cingulate, and prefrontal cortex Rewarding, pleasurable, or reinforcing effects of drugs involve: Dopamine release (opioids), or blockaded reuptake (cocaine), with progressively lower levels of synaptic dopamine during chronic administration or self-administration of opiate or cocaine (Mu opioid receptor agonists inhibit GABAergic neurons which inhibit dopamine release) Beta Endorphin and the Enkephalins ( Endorphins ) acting at Mu Opioid Receptors, with progressive beta-endorphin deficiency with chronic administration or self-administration of opiate or cocaine Countermodulatory response to reward involves: Dynorphins, acting at Kappa Opioid Receptors, lower dopamine levels (heroin or other short-acting opiates and cocaine increase dynorphin mrna levels and peptides in a persistent manner and increase kappa opioid receptors) Kreek , 219

16 Bidirectional-Translational Research: Novel and Conventional Rodent Models to Mimic Human Patterns of Abuse Binge Pattern Cocaine Parenteral Administration (Rat or Mouse): Constant or Ascending Dose (mimics most common pattern of human use in addiction) Intermittent Heroin (Morphine) Parenteral Administration (Rat or Mouse): Constant or Ascending Dose (mimics most common pattern of human use in addiction) Binge Pattern Oral Alcohol Self-Administration (Mouse): (mimics common pattern of human excessive use) Chronic Escalation (24h Access every other day) Alcohol Self-Administration (Rat or Mouse): (mimics common pattern of human excessive use) Intravenous Self-Administration (Rat) Extended Access 1h or 18h with Individual Selection of Dose Escalation (Heroin, Oxycodone, or Cocaine) Intravenous Self-Administration (Mouse) Extended Access 4h for adult mice; 2h maximum for adolescent mice (Heroin, Oxycodone, or Cocaine) Intravenous Pump Methadone Administration (Rat): (converts short-acting pharmacokinetic properties of opioid agonist in rodent to long-acting human pharmacokinetic profile) Kreek et al., 1987; 1992; 21; 25; 219

17 Frontal cortex Striatum Substantia nigra DOPAMINE SYNAPSE Nucleus accumbens VTA National Institute on Drug Abuse Heroin (or other opiates) increases dopamine release by acting at mu opioid receptors to inhibit GABAergic inhibition in the SNc and VTA Cocaine blocks dopamine re-uptake at synapse Kreek, 216 Maisonneuve, 1994

18 Development of an Addiction: Stress and Atypical Responsivity to Stressors HPA Axis Endogenous Opioids (mu inhibition) (kappa? activation) Kreek, 1972; 1981; 1982; hypothalamus Anterior + pituitary + POMC Cortisol adrenal CRF + ACTH Arginine Vasopressin b-end Atypical responsivity to stress and stressors may contribute to the persistence of, and relapse to, selfadministration of drugs of abuse and thus to addictive diseases. Such atypical stress responsivity in some individuals may exist prior to use of addictive drugs on a genetic or acquired basis, and increase the vulnerability to develop an addictive disease.

19 Oxycodone Mu Opioid Receptor Mediated Reward: Studies in Rodent Models

20 N o s e P o k e s / 4 h o u r s e s s io n s N o s e P o k e s / 1 h o u r s e s s io n REWARD Self-Administration of Oxycodone (.25 mg/kg/infusion) in C57BL6 Adult Male Mice: Long Access vs Short Access 4-hr Sessions n=11 1-hr Sessions n= A c tiv e H o le In a c tiv e H o le m g /k g /in fu s io n M g /k g /in fu s io n S e s s io n s S e s s io n s Zhang et al, Psychopharmacology, 231, 1277, 213

21 N u m b e r o f N o s e P o k e s a t A c tiv e H o le (F R 1 ) REWARD Self-Administration of Oxycodone (.25 mg/kg/infusion) in C57BL6 Adolescent Male (35d-49d) Compared with Adult Male (11-13 weeks) Mice (FR1: 2 hours/day Over 14 Days) 2 5 A d u lt O x y ( n = 1 1 ) A d o l e s c e n t O x y ( n = 1 2 ) O x y c o d o n e m g /k g S e s s io n s Mayer-Blackwell Zhang, Neuroscience, 258:28, 213

22 A U C % M P E (1-6 m in ) In c re a s e d T im e (s e c ) o n O x y c o d o n e S id e Adolescent Oxycodone SA in Male Mice Leads Both to Tolerance in Oxycodone-Induced Anti-Nociception and Increases in Oxycodone-Induced CPP in Adulthood Hot Plate Analgesia Test p < Condition Place Preference (liking) * m g /k g 5 m g /k g 7.5 m g /k g O x y c o d o n e d o s e (a n tin o c ic e p tio n p ro b e ) Y o k e d S a l O x y c o d o n e 1 3 O x y c o d o n e D o s e (m g /k g ) Yoked Saline Oxycodone Self-Administration Zhang et al., Neuropharmacology, 111:314, 216

23 REWARD BIDIRECTIONAL TRANSLATIONAL RESEARCH mrna Levels of Several Genes Increased by Oxycodone (SA) in the Dorsal Striatum in Adolescent and Adult Mice are SNPs Found to be Associated with Opiate Addiction in Humans Gene Symbol Protein ADULT MOUSE HUMAN SNPs Gene Symbol Protein Gene Expression SNP Location Opiate Addiction Change EA AA NPY1R Neuropeptide Y receptor Y1 rs ' near gene x NPY5R Neuropeptide Y receptor Y5 rs intergenic x CHRM5 Cholinergic receptor, muscarinic 5 rs '-UTR x HTR3A ADOLESCENT MOUSE 5-hydroxytryptamine (serotonin) receptor 3A Gene Expression Change rs '-UTR x rs '-UTR x rs Intron HUMAN SNPs x Levran et al., Gene Brain & Behav., 8:531, 29; Levran et al., Psychoneuroendocrinol., 45:67, 214 Mayer-Blackwell et al., Neurosci., 258:28, 214; Levran et al., Ann Hum Genet., 78:29, 214; Zhang et al., Psychopharmacol., 231:1277, 214; Levran et al., CNS Neurosci Ther., 11:898, 215; Levran et al., Prog in Neuro-Psychopharmacol & Biol Psych, 11:898, 216 SNP Location Opiate Addiction EA AA NPY1R Neuropeptide Y receptor Y1 rs ' near gene x NPY5R Neuropeptide Y receptor Y5 rs intergenic x MC2R Melanocortin 2 receptor rs ' near gene x

24 Chronic (14d) Oxycodone Self-Administration (4h/d;.25mg/kg per adm.; FR1) Alters Expression of Reward- and Stress-Related Genes in Ventral and Dorsal Striatum in C57BL/6J Male Mice: RNAseq Analysis Transcription-wide Sequencing (RNAseq) Focus on: Selected genes (based on our human genetic and rodent studies) were analyzed : 123 genes, mainly from opioid, stress-responsive, and neurotransmitter systems Genes possibly involved in development of opioid addiction Significant changes in mrna levels (difference between oxycodone SA and yoked saline-controlled mice, 5 mice/group): Ventral Striatum: Changes in mrna levels of 32 genes 15 increased; 17 decreased Dorsal Striatum: Changes in mrna levels of 7 genes 5 increased; 2 decreased Expression (mrna levels) of five genes in the ventral striatum showed experiment-wise changes: Increased mrna levels: Proopiomelanocortin (Pomc) and Serotonin 5-HT-2A receptor (Htr2a) Decreased mrna levels: Serotonin receptor 7 (Htr7), Galanin receptor1 (Galr1) and Glycine receptor 1 (Glra1) Confirmation of gene-expression changes of 2 of the 5 experiment-wise findings (Pomc and Htr7) using qpcr; other 3 not analyzed Y Zhang, Y Liang, C Zhao, et al, Neuroscience, in press, 219

25 Countermodulation of Reward Reversal or Modulation of Drug or Task-induced Stress: Kappa Opioid Receptor-Dynorphin Neurobiology

26 pg ppdyn mrna / µg total RNA COUNTERMODULATION KAPPA OPIOID RECEPTOR- DYNORPHIN SYSTEM: Cocaine Increases Kappa Opioid Receptor Density in Rat, But Kappa Opioid Receptor Directed Dynorphins Also Increase Persistently 7 Saline day cocaine (15mg/kg x 3) 5 Control Cocaine Control Cocaine Caudate Putamen Nucleus Accumbens Dynorphin Acting at the Kappa Opioid Receptor Lowers Dopamine Levels and Prevents Surge After Cocaine Spangler and Kreek, Brain Res. Mol. Brain Res., 19: , 1993; Unterwald, Rubenfeld, and Kreek, NeuroReport, 5:1613, 1994; Spangler, Ho, Zhou, Maggos, Yuferov, and Kreek, Mol. Brain Res., 38:71, 1996

27 Dopamine in Dialysate (nm) COUNTERMODULATION OF REWARD: Natural Dynorphin A 1-17 (Kappa Opioid Receptor Agonist) Infusion into Mouse Striatum Lowers Basal and Cocaine Induced Dopamine Levels Dopamine in Dialysate (nm) Infusion min Sample Infusion Injection 2-min Sample Dynorphin Dose (nmol) nBNI (antagonist) Infusion and Injection Control Cocaine (15mg/kg) Dynorphin (4.4nmol) + Cocaine (15mg/kg) Zhang, Butelman, Schlussman, Ho, and Kreek, Psychopharmacology, 172:422, 24

28 Score Stress Manifested by Immobility During Forced Swim in Rats is Due To Increased Dynorphin Levels: Effect Reversed by Kappa Opioid Receptor Antagonist (nor-bni) in Dose-Dependent Manner * nor-bni Dose mg/kg (n=6) 5 mg/kg (n=6) 1 mg/kg (n=6) 3 2 Swim 15 Minutes Day 1; 1 hour later pre-treatment nor-bni Swim 5 Minutes Day 2 (* - p<.5) 1 Immobility Reed et al., Neuroscience, 22:19-118, 212

29 COUNTERMODULATION KAPPA OPIOID RECEPTOR DYNORPHIN SYSTEM: BIOMARKER Dynorphin A Lowers Tuberoinfundibular Dopaminergic Tone, which Tonically Inhibits Prolactin Release Prolactin Levels (ng/ml) Dose-Response Effects of Dynorphin A 1-13 on Prolactin Levels (BIOMARKER) in Normal Volunteers Dynorphin A Hypothalamus TIDA µ g/kg 12 µ g/kg Placebo (n=1) 2 15 anterior pituitary lactotropes Time after injection (min) Kreek et al., 1994; 1999; 216

30 Compounds Approved for Use in Human Therapeutics with KOPr Partial Agonism in Addition to Mu-Opioid Receptor Antagonism or Partial Agonism and Binding Affinity in Cloned Human Receptors MOP-r affinity Ki (nm) KOP-r affinity Ki (nm) DOP-r affinity Ki (nm) Naloxone Naltrexone Nalmefene Buprenorphine Kreek, Reed, Butelman 214

31 Kappa Opioid Receptor Partial Agonist (antagonist and agonist) reduces dopaminergic surge and modulates dopaminergic tone? thus reduction of reward after cocaine, alcohol, or opiate use and? reduction of depressive symptoms Kreek, AD Dunn, AM Dunn, Butelman, Reed, in preparation, 219 CURRENT TRANSLATIONAL RESEARCH: POTENTIAL TARGET FOR NOVEL PHARMACOTHERAPIES FOR COCAINE ADDICTION OR ALCOHOLISM KAPPA OPIOID RECEPTOR / DYNORPHIN SYSTEM Novel target for possible treatment of cocaine addiction, alcoholism, and stimulant or alcohol co-dependence with opioid addiction. (Our laboratory and others have shown that heroin, morphine, cocaine, and alcohol and also stress (e.g., forced swim test) increase dynorphin gene expression in rodents.) Kappa Opioid Receptor Full Agonist decreases dopaminergic surge and lowers dopaminergic tone? thus reduction of reward after cocaine, alcohol, or opiate use? but possibly with persistent dysphoric sideeffects (? avoid by use of biased kappa agonist) Kappa Opioid Receptor Antagonist? reduction of depressive symptoms, stress-related, spontaneous, or drug use-induced,? decreased relapse to drug use; possible increases in dopamine surge after drug or alcohol self-administration

32 Kappa Opioid Receptor Agonists or Partial Agonists: Unbiased or Biased Agonism Kappa Opioid Receptor member of 7-transmembrane GPCR family Unbiased kappa opioid receptor agonists (prototypes: U5,488 and U69,593) activate both G-protein vs. β-arrestin pathways Biased agonists: differentially activate intracellular pathways (G-protein vs. β-arrestin) G-protein?? Analgesia? β-arrestin Anhedonia Sedation Motor incoordination Reed, AD Dunn, Butelman, Kreek, CPDD 217

33 Development of Novel Selective Kappa Opioid Receptor Partial Agonist (Unbiased or Biased) Goal: Synthesize, screen, and thus discover compound(s) with kappa partial agonist activity, without activity at other receptors. Test in animal models of addiction. [Funded by Robertson Therapeutic Development Fund with introduction to a contract synthesis and analysis company and, as of September 217, also funded by the Tri-Institutional Therapy Discovery Institute] Approach: Synthesize analogs of different candidate backbone structures Initial studies in vitro binding and signaling Determine Kappa Opioid Receptor Binding Elucidate G-protein and b-arrestin Signaling Efficacy (? unbiased or biased) Determine binding to the mu and delta opioid receptors Current Project Status: ~2 novel compounds synthesized (Kreek Lab/WuXi); to date, ~6 identified with K i <1 nm, and ~1 of these also meeting our second criteria of G protein efficacy in range of 2-8%. Continuing iterative syntheses. Reed, Dunn, et al, in progress, 219

34 REVERSAL OF DYNORPHIN-KAPPA INDUCED STRESS Short-Acting Selective Kappa Opioid Receptor Antagonists LY Tool compound (Lilly Laboratory) LY OpraKappa (Lilly Clinical) CERC-51 (now owned by Jansen of Johnson & Johnson and under study for treatment of depression) Kreek, 219

35 Pretreatment with tool compound (LY ) reduces anxiety- and depression-like behaviors in rats 3h in withdrawal from extended-access (18h/d, 14d) cocaine self-administration Immobility (s) Latency to immobility (s) Latency to immobility (s) FORCED SWIM TEST Vehicle Vehicle LY mg/kg LY mg/kg * ** Time in open arms (s) Latency to enter in open arms (s) ELEVATED PLUS MAZE vehicle LY mg/kg *** Valenza et al., Psychopharmacology, 234: 2219, 217 *

36 OpraK Clinical Study (LY245632): kappa opioid receptor antagonist We have hypothesized that a selective kappa opioid receptor (KOPr) antagonist might be helpful in managing the dysphoric and depressive symptoms of early and protracted abstinence from cocaine or alcohol. However, we also have hypothesized that a KOPr antagonist might increase the reward of cocaine or alcohol by increasing baseline dopaminergic tone and drug-induced dopamine surges. There is only limited data available on the impact of selective KOP-r antagonism in humans. We have conducted an in-patient basic research study that examined neuroendocrine and behavioral effects of a novel short-acting selective KOP-r antagonist, LY (which we call OpraK ) in normal volunteers (n=4) and in volunteers diagnosed with cocaine dependence (DSM IV) (n=3). Four days of drug administration caused no adverse effects. Reed et al, Neuropsychopharmacology, 43:739, 217

37 OpraK : Prolactin Levels Male Normal Volunteers vs. Early Abstinence Cocaine Dependent Volunteers No Evidence of Kappa Partial Agonism serum prolactin (ng/ml) serum prolactin (ng/ml) Baseline, Day 1 1 st OpraK (1mg), Day 2 4 th OpraK (1mg), Day 5 Normal Volunteers (n=24) Time (min) Time (min) Baseline, Day 1 1 st OpraK (1mg), Day 2 4 th OpraK (1mg), Day 5 Early Abstinence Cocaine Dependent Volunteers (n=19) Reed et al, Neuropsychopharmacology, 43:739, 217

38 Area Under the Curve, -48 minutes, ACTH Area Under the Curve, -48 minutes, cortisol OpraK : Serum ACTH and Cortisol Levels AUC Normal Volunteers vs. Early Abstinence Cocaine Dependent Volunteers: Evidence of Partial Mu Opioid Receptor Antagonism ACTH Cortisol p<.5 18 p<.5 p<.5 6 p< Baseline-Day 1 1st OpraKappa-Day 2 4th OpraKappa-Day 5 Baseline Day 1 1 st OpraK (1mg) Day Day 2 HV (n=16) EACD (n=13) 4 th OpraK (1mg) Day 5 Baseline-Day 1 1st OpraKappa-Day 24th OpraKappa-Day 5 Baseline Day 1 1 st OpraK (1mg) Day Day 2 HV (n=39) EACD (n=23) 4 th OpraK (1mg) Day 5 Reed et al, Neuropsychopharmacology, 43:739, 217

39 Human Molecular Genetics ( ): Three Functional Polymorphisms from Mu Opioid Receptor and Dynorphin Opioid Peptide Genes

40 Genetic Variants of the Human Mu Opioid Receptor: Single Nucleotide Polymorphisms in the Coding Region Including the Functional A118G (N4D) Variant HYPOTHESIS Gene variants: Alter physiology PHYSIOGENETICS (C17T) (A118G) Alter response to medications PHARMACOGENETICS Are associated with specific addictions Bond, LaForge Kreek, Yu, PNAS, 95:968, 1998; Kreek, Yuferov and LaForge, 2

41 Percent Bound FUNCTIONAL MOP-r (A118G) VARIANT Enhanced Binding and Coupling to G Protein-Activated, Inwardly Rectifying K + (GIRK) Channels by Beta-Endorphin Acting at A118G Variant Compared with Prototype A118A Fraction Maximum Current Response A118G Prototype 1. A118G Prototype Log [b Endorphin (M)] Log [b Endorphin (M)] Bond, LaForge Kreek, Yu, PNAS, 95:968, 1998; Kreek, Yuferov and LaForge, 2

42 Cortisol Levels - AUC (9:3am-1:3am + 9min) (no food for 9 hours) FUNCTIONAL MOP-r (A118G) VARIANT Physiogenetics Related to A118G Variant of Human Mu Opioid Receptor Gene Alters Stress Responsivity in Healthy Control Volunteers Serum Cortisol (ug/dl) Cortisol P < Prototype A118G P = Placebo N = Naloxone A/A (n=29) A/G (n=7) N P I N N Time (min) N Bart et al. Neuropsychopharmacology, 31: , 26 Wand et al., Neuropsychopharmacol, 26:16, 22 Chong Wand, Neuropsychopharmacology, 31:24, 26

43 Association Between a Functional Polymorphism (SNP) in the Mu Opioid Receptor Gene (A118G) and Opiate Addiction and Also Alcoholism in Central Sweden Opiate Dependent (n=139) 118G Allele Frequency.155 (15.5%) Control (n=17).74 (7.4%) Odds Ratio=2.86 p=.25 In the entire study group in this central Swedish population: Attributable Risk due to genotypes with a G allele: 18% Bart et al., Molecular Psychiatry, 9: , 24 Alcohol Dependent (n=389) Control (n=17) 118G Allele Frequency *.125 (12.5%).74 (7.4%) Odds Ratio=1.92 p=.74 * Overall 118G Allele Frequency =.19 (1.9%) In the entire study group in this central Swedish population: Attributable Risk due to genotypes with a G allele: 11.1% Bart et al., Neuropsychopharmacology, 3:417, 25

44 Genetically Modified A112G Mice, A Model of the Human A118G Mu Opioid Receptor Functional Variant: Microdialysis in Striatum of Wild-Type AA ( ) versus Genetically Modified GG ( ) Mice: Absolute Dopamine Levels (Three Baseline Samples) and Levels of Dopamine after Heroin Injections Dopamine (nm) Dopamine (nm) Males GG (6) AA (7) Females GG (6) AA (6) 1mg/kg Heroin 2mg/kg Heroin 1mg/kg Heroin 2mg/kg Heroin Zhang, Blendy Kreek et al., Neuropsychopharmacology, 4:191, 215

45 N o s e p o k e s (F R = 1,.2 5 m g /k g ) D a ily O x y c o d o n e S A (m g /k g ) N o s e p o k e s (F R = 1,.2 5 m g /k g ) D a ily O x y c o d o n e S A (m g /k g ) N o s e p o k e s ( F R = 1,.2 5 m g /k g ) D a ily H e ro in S A (m g /k g ) N o s e p o k e s ( F R = 1,.2 5 m g /k g ) D a ily H e ro in S A (m g /k g ) Genetically Modified A112G Mice (Asparagine to Aspartic Acid), A Model of the Human A118G Mu Opioid Receptor Functional Variant: Heroin versus Oxycodone Self-Administration (1d, 4h/d) by Wild-Type AA ( ) versus Genetically Modified GG ( ) versus Mice 4 Males Heroin 1 4 Females Heroin A A ( 1 5 ) G G ( 1 2 ) 2 1 A A (12) G G (1 2 ) Day (4h/d) Males Oxycodone Day (4h/d) Females Oxycodone O x y - A A (7 ) O x y -A A (8 ) O x y - G G ( 6 ) O x y -G G (8 ) Day (4h/d) Day (4h/d) Zhang, Blendy Kreek et al., Neuropsychopharm, 4:191, 215; unpublished data presented at CPDD 218

46 Yuferov et al, Neuropsychopharmacology, 34:1185, 29; Rouault et al., Addict. Biol. 16: 334, 211; Yufererov et al, Neuropsychiatric Disease and Treatment, 14:125, 218 Human prodynorphin gene: exon / intron organization, repeats, and single nucleotide polymorphisms Promoter 68-bp tandem repeats I II III IV 3 -UTR ATG rs rs rs rs rs rs rs918_t/c rs9179_t/c rs _c/t - 68 base pair tandem repeat 1 to 5 copies per allele +/- 1 SNP - Three 3 UTR SNPs (rs918, rs9179, and rs ) are in complete linkage disequilibrium (LD), and comprise two haplotype blocks: T-T-C or C-C-T

47 In African Americans, the 68 Base Pair Repeat Polymorphism of Dynorphin Gene Long (LL) and also Short/Long (SL) (probably yielding relatively lower amounts of dynorphin peptide) Associated with Cocaine/Alcohol Dependence (Early Study, 27) TGACTTA Transcription start 68 bp repeat CAAT TATA box Long = 3,3; 3,4; 4,4 Long/Short = 1,3; 1,4; 2,3; 2,4 Short = 1,1; 1,2; 2,2 Controls Cocaine/Alcohol Dependent Genotype Short (SS) Short/Long (SL)* Long (LL) Short (SS) Short/Long (SL) Long (LL) African American (61 cases/ 49 controls) n % 19 39% 16 33% 14 29% 1 16% 26* 43% 25** 42% *SS versus SL Fisher 1-sided mid-p =.13 (=.1, rounded) **SS versus LL Fisher 1-sided mid-p =.9 ***SS versus SL + LL is significant in cocaine-alcohol dependent group; Chi-square p=.81) Williams, Ott, et al, Addict. Biol. 12:496, 27; further statistical analysis, Ott and Butelman 218

48 N u m b e r o f p a rtic ip a n ts N u m b e r o f p a rtic ip a n ts In African American Males, but not Females, the 68 Base Pair Repeat Polymorphism of Dynorphin Gene Short Short (SS) or Short Long (SL) (probably yielding higher amounts of dynorphin peptide) is Associated with Greater Lifetime Self-Exposure to Cannabis M a le s F e m a le s = 6. 8 : p < : N S S S + S L L L S S + S L L L G e n o ty p e G e n o ty p e " L o w " c a n n a b is e x p o s u re (K M S K -4 ) " M e d iu m " c a n n a b is e x p o s u re (K M S K 5-9 ) SS - 1/1, 1/2 or 2/2 copies SL - 1/3 or 2/3 copies LL - 3/3 or 3/4 copies of the 68 bp repeat " H ig h " E x p o s u re (d e p e n d e n c e ; K M S K ) African American volunteer subjects with cannabis KMSK scores of 4, 5-9, and 1 14, respectively (KMSK 1 is cut-off for dependence for cannabis). Yuferov, Butelman, Kreek, Neuropsychiatr Dis Treat., 14:125, 218

49 Recent Published Results from Our Laboratory on Association of Specific Gene Variants with Opiate and/or Cocaine Addiction (over 145) Using AIMS Markers to Define Ethnicity Stress Cholinergic Adrenergic Circadian Rhythms Dopamine Serotonin Signal Transduction Opioid GABA African Descent African cluster European sample (24 genes) 12 genes African sample (35 genes) European Descent European cluster Shared SNPs, e.g.: DRD2: rs rs Kreek 218 after Levran 215

50 ASSOCIATION WITH OPIATE ADDICTION IN CAUCASIANS, AFRICAN AMERICANS, AND BOTH Opioid Stress Neurotransmitters Total Genes 5/ 13/11 42/34 6/45 SNPs in Genes 11/ 22/12 48/54 81/66 Genes Replicated 3/ 6/3 13/17 22/2 SNPs Replicated 6/ 7/1 2/3 15/31 OPIOID SYSTEM (selected genes) STRESS SYSTEM (selected genes) OPRM1 (mu opioid receptor) AVPR1A (arginine vasopressin receptor 1A) OPRD1 (delta opioid receptor) OPRK1 (kappa opioid receptor) PDYN (dynorphin peptide) FKBP5 (FK56-binding protein 51/ corticosterone chaperone) GAL (galanin) CSNK1E (casein kinase 1, epsilon) CRHBP (Corticotropin Releasing Hormone Binding Protein) updated after Reed et al., Current Psychiatry Reports, 16(11): 54, 214 Kreek Lab: Bond, Yu, LaForge, Nielsen, Levran, Randesi, Yuferov, and others; Kreek 219

51 ASSOCIATION WITH OPIATE ADDICTION IN CAUCASIANS, AFRICAN AMERICANS, AND BOTH Opioid Stress Neurotransmitters Total Genes 5/ 13/11 42/34 6/45 SNPs in Genes 11/ 22/12 48/54 81/66 Genes Replicated 3/ 6/3 13/17 22/2 SNPs Replicated 6/ 7/1 2/3 15/31 NEUROTRANSMITTER SYSTEMS (selected genes) COMT (catechol-o-methyltransferase) HTR1B (serotonin receptor 1B) BDNF (brain-derived neurotrophic factor) GABRG1 (gamma-aminobutyric acid (GABA) A receptor) GRIN2A (glutamate receptor, ionotropic, N- methyl D-aspartate 2A) GAD1 (glutamate decarboxylase 1) updated after Reed et al., Current Psychiatry Reports, 16(11): 54, 214 Kreek Lab: Bond, Yu, LaForge, Nielsen, Levran, Randesi, Yuferov, and others; Kreek 219

52 The Laboratory of the Biology of Addictive Diseases 219 Laboratory Scientists Eduardo Butelman Yan Zhou Orna Levran Yong Zhang Vadim Yuferov Brian Reed Laboratory Manager Matthew Randesi Research Nurse Practitioner Kate Brown Rachel Conybeare Administrative Team Kitt Lavoie Abigail Sintim Postdoctoral Fellows Kyle Windisch Devon Collins Graduate Students Amy Dunn Assistants for Research Ariel Ben-Ezra Jose Erazo Michelle Morochnik Carina Chen Bryan Elroy Statistics & Informatics Collaborators Jurg Ott Yupu Liang Guest Investigators Miriam Adelson Gavin Bart Lawrence Brown Don Des Jarlais David Novick Einat Peles Ellen Unterwald Other Rockefeller University Collaborators Brian Chait Jeff Friedman Paul Greengard Bruce McEwen Don Pfaff Sid Strickland Tom Tuschl Funded primarily by Dr. Miriam and Sheldon Adelson Medical Research Foundation, NIH-NIDA, NIH-NIAAA, NIH-CRR, Tri-Institutional Therapeutics Discovery Institute, Robertson Therapeutic Development Fund, and others

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