The Molecular Neurobiology and Human Genetics of Specific Addictive Diseases: Implications for Treatments Mary Jeanne Kreek, M.D

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, 2019 New York, NY

Funded primarily by NIH-NIDA (P60-05130), The Adelson Medical Research Foundation, Robertson Therapeutic Development Fund, Tri-Institutional Therapeutics Discovery Institute, NCATS-NIH-CTSA UL1-TR000043 (RUH – Dr B. Coller) The Molecular Neurobiology and Human Genetics of Specific Addictive Diseases: Implications for Treatments

I. “The Opioid Crisis” (2001 increasing through 2018); early treatment research; current status and needs II. Molecular neurobiology of addictive diseases: current work on oxycodone effects in adult and adolescent mice III. 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 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 self- administration, 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), 79:122-136, 1966. (including discussion)

Dole, V.P., Nyswander, M.E. and Kreek, M.J.: Narcotic blockade. Arch. Intern. Med., 118:304-309, 1966. Dole, Nyswander and Kreek, 1964, 1966, 2018 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

Apparent Plasma Terminal

Drug or Half-life and Duration of "High"

Medication Desired Effects

HEROIN 3 min for prodrug "Straight"

30 min for active (Heroin)

"Sick" compound, mono-acetyl

Functional Functional State morphine (fast on-set and AM PM AM PM AM off-set) Days

6 hours for active

metabolites (morphine "High" and others)

"Straight" METHADONE 24h for racemic (rs) medication (slow on-set

and off-set – steady-state (Methadone)

Functional Functional State "Sick" achieved)

AM PM AM PM AM 48h for active (r) H Days enantiomer

Dole, Nyswander and Kreek, 1966; Kreek et al., 1973; 1976; 1977; 1979; 1982; Inturrisi et al, 1973; 1984; 2018

• Drug overdoses, primarily opioids, killed more than 72,300 Americans in 2017, a record and a rise of approximately 10% over 2016 • Drug overdose deaths in 2017 were higher than the peak yearly deaths from HIV, car accidents, or gun Overdose Deaths in Thousands in Preceding 12 months deaths 30 thousand Synthetic opioids • Overdose deaths have begun to fall in Massachusetts, Vermont, and Rhode 20 Heroin Island following major Other public health opioids campaigns, including Cocaine increased access to 10 treatment, in response Other Psychostimulants to the early arrival of fentanyl in those states Methadone 0 Sanger-Katz, NY Times, 2015 2016 2017 Aug 15, 2018 Prevalence of Specific Drug Abuse and Vulnerability to Develop Addictions – 2019 National Household Survey and Related Surveys – 2007 – 2016 Heroin Use – ever ~ 5.2 million Heroin Addiction ~ 626,000 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 49,068 (in 2017)*

Cocaine Use – ever ~ 40.5 million Cocaine Addiction ~ 966,000

Alcohol Use – ever ~ 216 million Alcoholism ~ 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 (20% 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, 2017; Others, 2007-18; *Nat’l Center for Health Statistics (CDC), 2019

Research and Clinical Evidence Contributes to Specific Actions for “Prevention”, “Reversal”, and “Reduction” of Illicit Opiate Use and Overdose Deaths (53,332 in 2016) “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 (60-90m 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 (60-80% 12-month voluntary retention with reduction or elimination of opiate use in over 80% of patients; half life 24-36 hours; oral dose 80-150mg per day)

– Buprenorphine-naloxone maintenance pharmacotherapy: 30 years of clinical research and practice evidence of effectiveness (40-60% 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, 2000, 2002, 2017, 2019

Identification of HIV-1 Infection in Intravenous Drug Users New York City: 1983 – 1984 Study Protective Effect of Methadone Maintenance Treatment

100

Percent of IV Drug Users Infected with HIV-1 75

% 50

0 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1992 50% – 60% Untreated, street heroin addicts: positive for HIV-1 antibody

9% Methadone maintained since <1978 (beginning of AIDS epidemic): less than 10% positive for HIV-1 antibody

40% – 90% Heroin addicts in treatment positive for Hepatitis C infection (But less than 10% 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, 261:1008, 1989. Methadone Maintenance Treatment for Opiate (Heroin) Addiction – 2019

Number of patients currently in treatment: ~ 1.4 million worldwide

 USA: ~ 360,000  Europe: ~ 600,000  Rest of world: ~ 400,000 Efficacy in “good” methadone treatment programs using adequate doses (80 to 150mg/d): Voluntary retention in treatment (1 year or more) 60 – 80% Continuing use of illicit heroin 5 – 20% Actions of methadone treatment: • Prevents withdrawal symptoms and “drug hunger” • Blocks euphoric effects of short-acting narcotics • Allows normalization of disrupted physiology 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; 2019 Status of Methadone, Buprenorphine, and Extended Release Naltrexone Treatments for Opioid Addiction in the United States: Decrease, then Increase, of Numbers in Treatment 2015-2017 (2015, 2016, and 2017 data, SAMHSA, 2018)* US Patients in Treatment

Treatment 2015 2016 2017 Methadone 356,843 345,443 382,867 Maintenance (-11,400; -3.2%) (+37,424; +10.8%)

Buprenorphine 75,723 61,486 112,223 Maintenance (-14,237; -18.8%) (+50,737; +82.5%)

Extended 7,035 10,128 23,065 Release (+3,093; +44.0%) (+12,937; +128.7%) Naltrexone

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), 2018; Kreek 2019 Limited Targeted Pharmacotherapies Available for Specific Addictive Diseases I. Opiate Addiction (Heroin and Illicit Use of Prescription Opiates) a. METHADONE (60-80%)** b. BUPRENORPHINE (+ NALOXONE) (40-50%)** [c. NALTREXONE / SUSTAINED RELEASE NALTREXONE (<15%)*]

II. Alcohol Addiction and Excessive Alcohol Use a. NALTREXONE (30-40%)* b. NALMEFENE (approved in Europe only, 2012) c. ACAMPROSATE (low in USA)

III. 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 $600 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, 2019 Targets of Currently Approved Treatments for Addictive Disorders

Kreek et al, Journal of Clinical Investigation, 12: 3387, 2012 Natural History of Drug and Alcohol Abuse and Addictions relapse to addiction without pharmacotherapy 90% - opiate; Primary Possible Utility of Vaccines Medications Useful 60% - cocaine, alcohol Prevention and Selected Medications and Needed

Initial Use of Sporadic Regular Addiction Early Protracted Drug of Abuse Intermittent Use Withdrawal Abstinence Use (abstinence)

Progression sustain abstinence with no specific medications ADDICTION: Compulsive drug seeking behavior 10% - opiate; and drug self-administration, without regard to 40% - cocaine, alcohol negative consequences to self or others (adapted from WHO). Adapted from Kreek et al., Nature Reviews Drug Discovery, 1:710, 2002; 2019 Factors Contributing to Vulnerability to Develop a Specific Addiction

use of the drug of abuse essential (100%)

Genetic Environmental (25-80%) (very high) • DNA • prenatal • SNPs • postnatal • repeats • epigenetics • other • cues polymorphisms • peer pressure • comorbidity • stress-responsivity

• mRNA levels • neurochemistry • peptides • synaptogenesis • proteomics Drug-Induced Effects • behaviors (very high) Kreek et al., 2000; 2005; 2019 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 1992-2015, 2019

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 10h 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 Kreek et al., 1987; agonist in rodent to long-acting human pharmacokinetic profile) 1992; 2001; 2005; 2019

Frontal Striatum cortex Substantia DOPAMINE nigra 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, 2016 Maisonneuve, 1994 Development of an Addiction: Stress and Atypical Responsivity to Stressors – HPA Axis

Atypical responsivity to stress and stressors hypothalamus may contribute to the – Arginine persistence of, and CRF Vasopressin relapse to, self- + administration of drugs Anterior of abuse and thus to pituitary Endogenous – + addictive diseases. Opioids POMC (mu – inhibition) (kappa – ? activation) Such atypical stress b-End responsivity in some Cortisol ACTH individuals may exist prior to use of addictive adrenal + drugs on a genetic or acquired basis, and increase the vulnerability to develop

Kreek, 1972; 1981; 1982; 1984 … 2019 an addictive disease. Oxycodone – Mu Opioid Receptor Mediated Reward: Studies in Rodent Models REWARD– Self-Administration of Oxycodone (0.25 mg/kg/infusion) in C57BL6 Adult Male Mice: Long Access vs Short Access

4-hr Sessions 1-hr Sessions

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Zhang et al, Psychopharmacology, 231, 1277, 2013 REWARD – Self-Administration of Oxycodone (0.25 mg/kg/infusion) in C57BL6 Adolescent Male (35d-49d) Compared with Adult Male (11-13 weeks) Mice (FR1: 2 hours/day Over 14 Days)

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Mayer-Blackwell…Zhang, Neuroscience, 258:280, 2013 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 Condition Place Preference (liking)

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Yoked Saline Oxycodone Self-Administration

Zhang et al., Neuropharmacology, 111:314, 2016 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 ADOLESCENT MOUSE HUMAN SNPs Gene Opiate Gene Symbol Protein Expression SNP Location Addiction Change EA AA NPY1R Neuropeptide Y receptor Y1 ↑ rs4518200 5' near gene x NPY5R Neuropeptide Y receptor Y5 ↑ rs6536721 intergenic x

MC2R Melanocortin 2 receptor ↑ rs1893219 5' near gene x ADULT MOUSE HUMAN SNPs Gene Opiate Gene Symbol Protein Expression SNP Location Addiction Change EA AA NPY1R Neuropeptide Y receptor Y1 ↑ rs4518200 5' near gene x NPY5R Neuropeptide Y receptor Y5 ↑ rs6536721 intergenic x CHRM5 Cholinergic receptor, muscarinic 5 ↑ rs4041435 5'-UTR x rs4779656 5'-UTR x rs2684941 5'-UTR x 5-hydroxytryptamine (serotonin) ↑ rs897687 Intron HTR3A x receptor 3A Levran et al., Gene Brain & Behav., 8:531, 2009; Levran et al., Psychoneuroendocrinol., 45:67, 2014 Mayer-Blackwell et al., Neurosci., 258:280, 2014; Levran et al., Ann Hum Genet., 78:290, 2014; Zhang et al., Psychopharmacol., 231:1277, 2014; Levran et al., CNS Neurosci Ther., 11:898, 2015; Levran et al., Prog in Neuro-Psychopharmacol & Biol Psych, 11:898, 2016 Chronic (14d) Oxycodone Self-Administration (4h/d; 0.25mg/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, 2019 Countermodulation of Reward – Reversal or Modulation of Drug or Task-induced Stress:

Kappa Opioid Receptor-Dynorphin Neurobiology COUNTERMODULATION – KAPPA OPIOID RECEPTOR- DYNORPHIN SYSTEM: Cocaine Increases Kappa Opioid Receptor Density in Rat, But Kappa Opioid Receptor Directed “Dynorphins” Also Increase Persistently

7 1 2 Saline 14 day cocaine (15mg/kg x 3) 6

Control Cocaine 5

3 4

pg ppDyn mRNA / µg total RNA total µg / mRNA ppDyn pg Control Cocaine 0 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., 19:323-327, 1993; Unterwald, Rubenfeld, and Kreek , NeuroReport, 5:1613, 1994; Spangler, Ho, Zhou, Maggos, Yuferov, and Kreek , Mol. Brain Res., 38:71, 1996 COUNTERMODULATION OF REWARD: Natural Dynorphin A1-17 (Kappa Opioid Receptor Agonist) Infusion into Mouse Striatum Lowers Basal and Cocaine Induced Dopamine Levels

10 10

8 8

6 6

4 4

Dopamine in Dopamine

Dopamine Dopamine in Dialysate (nM) 2 Dialysate (nM) 2

0 0 60 120 180 60 120 180 Infusion Infusion Injection 20-min Sample 20-min Sample Dynorphin Dose (nmol) Infusion and Injection 0 2.0 Control 1.0 4.4 Cocaine (15mg/kg) 4.4+nBNI Dynorphin (4.4nmol) (antagonist) + Cocaine (15mg/kg)

Zhang, Butelman, Schlussman, Ho, and Kreek, Psychopharmacology, 172:422, 2004 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

60 nor-BNI Dose

50 0 mg/kg (n=6) * 5 mg/kg (n=6)

40 10 mg/kg (n=6)

Swim 15 Minutes Day 1; 30 1 hour later pre-treatment Score nor-BNI Swim 5 Minutes Day 2 20 (* - p<0.05)

Immobility

Reed et al., Neuroscience, 220:109-118, 2012 COUNTERMODULATION – KAPPA OPIOID RECEPTOR – DYNORPHIN SYSTEM: “BIOMARKER” Dynorphin A Lowers Tuberoinfundibular Dopaminergic Tone, which Tonically Inhibits Prolactin Release

Dose-Response Effects of Dynorphin A1-13 on

Prolactin Levels (BIOMARKER) in Normal Volunteers

500 µg/kg Dynorphin A1-13 35 120 µg/kg – Hypothalamus 30 Placebo (n=10) + TIDA 25

10 –

anterior pituitary 5 lactotropes (ng/ml) Levels Prolactin 0

-10 0 10 20 30 40 50 60 75 90 120 150 180 240

Time after injection (min)

Kreek et al., 1994; 1999; 2016 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

Naloxone Naltrexone Nalmefene Buprenorphine MOP-r affinity 0.66 0.11 0.24 0.21 Ki (nM) KOP-r affinity 1.2 0.19 0.083 0.62 Ki (nM) DOP-r affinity 120 60 16 2.1 Ki (nM) Kreek, Reed, Butelman 2014 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 side- effects (? 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 • 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, 2019 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: U50,488 and U69,593) activate G-protein β-arrestin both G-protein vs. β-arrestin pathways ? ? Analgesia Sedation Motor incoordination • Biased agonists: differentially activate intracellular pathways ? (G-protein vs. β-arrestin) Anhedonia Reed, AD Dunn, Butelman, Kreek, CPDD 2017 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 2017, 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: ~200 novel compounds synthesized (Kreek Lab/WuXi); to date, ~60 identified with Ki <100 nM, and ~10 of these also meeting our second criteria of G protein efficacy in range of 20-80%. Continuing iterative syntheses. Reed, Dunn, et al, in progress, 2019

REVERSAL OF DYNORPHIN-KAPPA INDUCED STRESS – Short-Acting Selective Kappa Opioid Receptor Antagonists

LY2444296 – “Tool” compound LY2456302 – “OpraKappa” (Lilly – Laboratory) (Lilly – Clinical)

CERC-501 (now owned by Jansen of Johnson & Johnson and under study for treatment of depression) Kreek, 2019

Pretreatment with “tool compound” (LY2444296) reduces anxiety- and depression-like behaviors in rats 30h in withdrawal from extended-access (18h/d, 14d) cocaine self-administration

FORCED SWIM TEST ELEVATED PLUS MAZE 150 Vehicle 50 VehicleLY2444296 3 mg/kg vehicle 120 LY2444296 3 mg/kg 40 LY2444296 3 mg/kg 90 30 * 60 20 * Time 30 10

Immobility (s) Immobility

0 arms (s) open in 0 300300 300

225225 225

150 150 ** 150 75 75 75

Latency to Latency

immobility (s) immobility ***

immobility (s) immobility 0 0 enter to Latency arms (s) open in 0 Valenza et al., Psychopharmacology, 234: 2219, 2017 “OpraK” Clinical Study (LY2456302): 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, LY2456302 (which we call “OpraK”) in normal volunteers (n=40) and in volunteers diagnosed with cocaine dependence (DSM IV) (n=30). Four days of drug administration caused no adverse effects. Reed et al, Neuropsychopharmacology, 43:739, 2017 “OpraK”: Prolactin Levels – Male Normal Volunteers vs. Early Abstinence Cocaine Dependent Volunteers – No Evidence of Kappa Partial Agonism 25 Baseline, Day 1 20 1st OpraK (10mg), Day 2 th 15 4 OpraK (10mg), Day 5

10 Normal Volunteers

5 (n=24)

-30 0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 serum prolactin (ng/mL) prolactin serum 25 Time (min) Baseline, Day 1 20 1st OpraK (10mg), Day 2 4th OpraK (10mg), Day 5 15

10 Early Abstinence Cocaine Dependent 5 Volunteers

0 (n=19)

-30 0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 serum prolactin (ng/mL) serum TimeTime (min)(min) Reed et al, Neuropsychopharmacology, 43:739, 2017 “OpraK”: Serum ACTH and Cortisol Levels AUC – Normal Volunteers vs. Early Abstinence Cocaine Dependent Volunteers: Evidence of Partial Mu Opioid Receptor Antagonism

ACTH p<0.05 Cortisol p<0.0005 18000 p<0.005 6000 p<0.0005

16000 5000 14000

12000 4000

10000 3000 8000

6000 2000

4000 1000 2000

0 cortisol 0-480Curve,minutes, Area the Under 0 Area Under the Curve, 0-480 minutes, ACTH 0-480Curve,minutes, Area the Under Baseline-DayBaseline 1 1st OpraKappa-Day1st OpraK 2 4th OpraKappa-Day4th OpraK 5 Baseline-DayBaseline 1 1st OpraKappa-Day1st OpraK 24th OpraKappa-Day4th OpraK 5 Day 1 (10mg)Day (10mg) Day 1 (10mg)Day (10mg) Day 2 Day 5 Day 2 Day 5 HV (n=16) HV (n=39) EACD (n=13) EACD (n=23) Reed et al, Neuropsychopharmacology, 43:739, 2017 Human Molecular Genetics (1998-2019):

Three Functional Polymorphisms from Mu Opioid Receptor and Dynorphin Opioid Peptide Genes Genetic Variants of the Human Mu Opioid Receptor: Single Nucleotide Polymorphisms in the Coding Region Including the Functional A118G (N40D) Variant

HYPOTHESIS (C17T)

Gene variants: (A118G) • Alter physiology “PHYSIOGENETICS”

• Alter response to medications “PHARMACOGENETICS”

• Are associated with specific addictions

Bond, LaForge… Kreek, Yu, PNAS , 95:9608, 1998; Kreek, Yuferov and LaForge, 2000

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

100

A118G A118G 1.0 Prototype Prototype 80

0.5 40

Percent Bound Percent 20

Current Response Current Fraction Maximum Fraction 0 0

-11 -10 -9 -8 -7 -9 -8 -7 -6 Log [b Endorphin (M)] Log [b Endorphin (M)]

Bond, LaForge… Kreek, Yu, PNAS , 95:9608, 1998; Kreek, Yuferov and LaForge, 2000

FUNCTIONAL MOP-r (A118G) VARIANT – “Physiogenetics” Related to A118G Variant of Human Mu Opioid Receptor Gene – Alters Stress Responsivity in Healthy Control Volunteers

P = Placebo

N = Naloxone

2500 Cortisol 24 A/A (n=29)

22 A/G (n=7) N N

2000 P < 0.05 AUC

20 - 1500 18 16 N 1000 14

10:30am 10:30am 90min) + P - 500 40 19 12 I N

Serum Cortisol (ug/dl) Cortisol Serum 10 (no (no food for 9 hours)

Cortisol Cortisol Levels 0 8 (9:30am Prototype A118G 50 0 50 100 150 200

Time (min)

Bart et al. Neuropsychopharmacology, Wand et al., Neuropsychopharmacol, 26:106, 2002 31:2313-2317, 2006 Chong…Wand, Neuropsychopharmacology, 31:204, 2006 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) Control (n=170)

118G Allele Frequency 0.155 0.074 (15.5%) (7.4%) Odds Ratio=2.86 p=0.00025 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, 9:547-549, 2004

Alcohol Dependent (n=389) Control (n=170) 118G Allele Frequency * 0.125 0.074 (12.5%) (7.4%) Odds Ratio=1.92 p=0.0074 * Overall 118G Allele Frequency = 0.109 (10.9%) 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, 30:417, 2005 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

Males Females

14 14

GG (6) 12 GG (6) 12 10 10 8 8 6 6 4 4 AA (6) AA (7)

2 2

Dopamine (nM) Dopamine Dopamine (nM) Dopamine 0 0

10mg/kg 20mg/kg 10mg/kg 20mg/kg Heroin Heroin Heroin Heroin

Zhang, Blendy…Kreek et al., Neuropsychopharmacology, 40:1091, 2015 Genetically Modified A112G Mice (Asparagine to Aspartic Acid), A Model of the Human A118G Mu Opioid Receptor Functional Variant: Heroin versus Oxycodone Self-Administration (10d, 4h/d) by

Wild-Type AA ( ) versus Genetically Modified GG ( ) versus Mice )

4 0 1 0 ) 4 0 1 0

) g

Males – Heroin Females – Heroin )

g m

8 8 g 5

3 0 5 3 0

(

A ,

6 6 A

= n

R 2 0

2 0 n

(

r 4

1 0 o 1 0 o A A (12)

A A (1 5 ) y

y p

2 l p

2 l

i e

e G G (1 2 )

a a

s G G (1 2 )

D N 0 0 N 0 0

1 2 3 4 5 6 7 8 9 1 0 1 2 3 4 5 6 7 8 9 1 0

)

) )

g Day (4h/d) Day (4h/d)

g g 6 0 1 5 g

k 6 0 1 5

/ g

Males – Oxycodone g Females – Oxycodone

(

0 0

4 0 1 0 S 4 0 1 0

(

c s

2 0 5 2 0 5 c

O O

O x y -A A (7 ) O x y -A A (8 )

i s

O x y -G G (6 ) s O x y -G G (8 )

a o

0 0 o 0 0

D N 1 2 3 4 5 6 7 8 9 1 0 N 1 2 3 4 5 6 7 8 9 1 0 Day (4h/d) Day (4h/d) Zhang, Blendy…Kreek et al., Neuropsychopharm, 40:1091, 2015; unpublished data presented at CPDD 2018 Human prodynorphin gene: exon / intron organization, repeats, and single nucleotide polymorphisms

Promoter 3′-UTR 68-bp tandem I II III IV repeats

ATG

rs6045819 rs35286281 rs10485703 rs34535593 rs6035222 rs910080_T/C rs1997794 rs910079_T/C rs2235749_C/T

- 68 base pair tandem repeat – 1 to 5 copies per allele +/- 1 SNP

- Three 3′UTR SNPs (rs910080, rs910079, and rs2235749) are in complete linkage disequilibrium (LD), and comprise two haplotype blocks: T-T-C or C-C-T

Yuferov et al, Neuropsychopharmacology, 34:1185, 2009; Rouault et al., Addict. Biol. 16: 334, 2011; Yufererov et al, Neuropsychiatric Disease and Treatment, 14:1025, 2018 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, 2007)

Transcription start TGACTTA

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 Short Short/Long Long Short Short/Long Long Genotype (SS) (SL)* (LL) (SS) (SL) (LL) African n 19 16 14 10 26* 25** American % 39% 33% 29% 16% 43% 42% (61 cases/ 49 controls) *SS versus SL – Fisher 1-sided mid-p = 0.013 (= 0.01, rounded) **SS versus LL – Fisher 1-sided mid-p = 0.009 ***SS versus SL + LL is significant in cocaine-alcohol dependent group; Chi-square p=0.0081) Williams, Ott, et al, Addict. Biol. 12:496, 2007; further statistical analysis, Ott and Butelman 2018 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

1 2 5 114 1 2 5

s t

t  2 = 6 .0 8 : p < 0 .0 5 n

n  2 : N S a

a 1 0 0 1 0 0

t r

r 7 5 7 5

a p

p 55 55 54

f 54 o

o 5 0 5 0

r e

e 28 26 b

b 26

2 5 2 5

m u

u 5 9 7

N N 0 0 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 0 -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 ) " H ig h " E x p o s u re (d e p e n d e n c e ; K M S K 1 0 -1 4 ) SS - 1/1, 1/2 or 2/2 copies African American volunteer subjects with SL - 1/3 or 2/3 copies cannabis KMSK scores of 0–4, 5-9, and 10–14, LL - 3/3 or 3/4 copies of the 68 bp repeat respectively (KMSK 10 is cut-off for dependence for cannabis).

Yuferov, Butelman, Kreek, Neuropsychiatr Dis Treat., 14:1025, 2018 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 Circadian Adrenergic Rhythms Stress Dopamine Cholinergic Serotonin

Signal Transduction GABA Opioid

African European 12 African African cluster sample genes sample Descent (24 genes) (35 genes)

European Descent European cluster Shared SNPs, e.g.: DRD2: rs1076563 rs2587546 Kreek 2018 after Levran 2015 ASSOCIATION WITH OPIATE ADDICTION IN CAUCASIANS, AFRICAN AMERICANS, AND BOTH Neuro- Opioid Stress transmitters Total Genes 5/0 13/11 42/34 60/45 SNPs in Genes 11/0 22/12 48/54 81/66 Genes Replicated 3/0 6/3 13/17 22/20 SNPs Replicated 6/0 7/1 2/30 15/31 OPIOID SYSTEM (selected genes) STRESS SYSTEM (selected genes) OPRM1 AVPR1A (mu opioid receptor) (arginine vasopressin receptor 1A)

OPRD1 FKBP5 (delta opioid receptor) (FK506-binding protein 51/ corticosterone chaperone) GAL OPRK1 (galanin) (kappa opioid receptor) CSNK1E PDYN (casein kinase 1, epsilon) (dynorphin peptide) CRHBP (Corticotropin Releasing Hormone Binding Protein)

updated after Reed et al., Current Psychiatry Reports, 16(11): 504, 2014 Kreek Lab: Bond, Yu, LaForge, Nielsen, Levran, Randesi, Yuferov, and others; Kreek 2019 ASSOCIATION WITH OPIATE ADDICTION IN CAUCASIANS, AFRICAN AMERICANS, AND BOTH Neuro- Opioid Stress transmitters Total Genes 5/0 13/11 42/34 60/45 SNPs in Genes 11/0 22/12 48/54 81/66 Genes Replicated 3/0 6/3 13/17 22/20 SNPs Replicated 6/0 7/1 2/30 15/31 NEUROTRANSMITTER SYSTEMS (selected genes)

COMT GABRG1 (catechol-o-methyltransferase) (gamma-aminobutyric acid (GABA) A receptor) HTR1B (serotonin receptor 1B) GRIN2A (glutamate receptor, ionotropic, N- BDNF methyl D-aspartate 2A) (brain-derived neurotrophic factor) GAD1 (glutamate decarboxylase 1)

updated after Reed et al., Current Psychiatry Reports, 16(11): 504, 2014 Kreek Lab: Bond, Yu, LaForge, Nielsen, Levran, Randesi, Yuferov, and others; Kreek 2019 The Laboratory of the Biology of Addictive Diseases – 2019 Laboratory Scientists Postdoctoral Fellows Guest Investigators Eduardo Butelman Kyle Windisch Miriam Adelson Yan Zhou Devon Collins Gavin Bart Orna Levran Lawrence Brown Yong Zhang Graduate Students Don Des Jarlais Vadim Yuferov Amy Dunn David Novick Brian Reed Einat Peles Assistants for Research Ellen Unterwald Laboratory Manager Ariel Ben-Ezra Matthew Randesi Jose Erazo Other Rockefeller University Collaborators Research Nurse Michelle Morochnik Brian Chait Practitioner Carina Chen Jeff Friedman Kate Brown Bryan Elroy Paul Greengard Rachel Conybeare Statistics & Informatics Bruce McEwen Administrative Team Collaborators Don Pfaff Kitt Lavoie Jurg Ott Sid Strickland Abigail Sintim Yupu Liang 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