50 Years of INRC: 1969 to 2019 – 55 Years of Our Rockefeller University Research and 50 to 60 Years of Opioid Research Mary Jeanne Kreek, M.D

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50 Years of INRC: 1969 to 2019 – 55 Years of our Rockefeller University Research and 50 to 60 Years of Opioid Research 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 International Narcotics Research Conference July 8, 2019 New York, NY

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 International Narcotics Research Conference Founders Lecturers (First Awardee 1999)

1999 Eric J. Simon 2009 R. Alan North 2000 Brian M. Cox 2010 Masamichi Satoh 2001 Philip S. Portoghese 2011 Charles Chavkin 2002 Lars Terenius 2012 F. Ivy Carroll 2003 Bernard Roques 2013 Graeme Henderson 2004 Ji-Sheng Han 2014 Christopher Evans 2005 Mary Jeanne Kreek Brigitte Kieffer 2006 Huda Akil 2015 Gavril Pasternak (d. 2019) Stan Watson 2016 Lakshmi Devi 2007 Volker Hoellt 2017 John Traynor Horace Loh 2018 Macdonald Christie 2008 Jan van Ree 2019 Sol Snyder

Kreek 2019 International Narcotics Research Conference Founders Lecturers at this 50th Anniversary Meeting

Kreek 2019 Ji-Sheng Han cannot attend this 50th Anniversary meeting because of a family illness and sends his regards, along with this photo of him with his family. Ivy Carroll and Bernard Roques are unable to attend this year because of illness. Brigitte Kieffer and Huda Akil could not attend because of university business.

All send their regards! Kreek 2019 Early Opioid Research of Martin (US) and Collier (UK) – 1961 Hypothesis and experimental evidence of the development of tolerance to and dependence on opiates with chronic exposure (heroin, morphine, methadone, other)

Martin, W.R., Frasier, H.F. A comparative study of physiological and subjective effects of heroin and morphine administered intravenously in postaddicts. JPET, 133:388-99. (1961)

Collier, H.O., Warner, B.T., Skerry, R. Multiple toe-pinch method for testing analgesic drugs. Br J Pharmacol Chemother. 17:28-40. (1961) (in rodents)

Frasier, H.F., Van Horn, G.D., Martin, W.R., Wolbach, A.B., Isbell, H. Methods for evaluating addiction liability. (A) “Attitude” of opiate addicts toward opiate-like drugs. (B) a short-term “direct” addiction test. JPET, 133:371-87. (1961)

Martin, W.R., Eades, C.G. Demonstration of tolerance and physical dependence in the dog following short-term infusion of morphine. JPET, 133:262-70. (1961)

Kreek 2019 55th Anniversary of the Beginning of Our Rockefeller Institute (now University) Research: 1963-1964 Treatment of Heroin Addiction: Identification of Need, Formulation of Hypothesis, Basic Clinical and Related Laboratory Research, Translational Research, and Evaluations Autumn Vincent P. Dole, Jr., MD recruitment of two new staff 1963 members to his Laboratory of Physiology and Metabolism at The Rockefeller Institute for Medical Research: 1) Marie Nyswander, MD – psychiatrist, years of work with heroin addicts in New York City and Lexington, KY. Author of book The Addict as a Patient. 2) Mary Jeanne Kreek, MD – second year Resident in (Internal) Medicine (“PGY-2”) at Cornell-New York Hospital with experience in clinical and laboratory based research at NIH and Columbia P&S. January – New team formed and first patients admitted to the July 1964 Rockefeller Hospital; initial research on potential novel pharmacological treatment of heroin addiction, accomplished by July 1964. Kreek 2019 Development of Methadone Maintenance Treatment – 1964-1966 1964 HYPOTHESIS –NOW EVIDENCE-BASED FACT: 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. First publications describing methadone maintenance treatment research 1) 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: Dole, V.P., Nyswander, M.E. and Kreek, M.J.: Narcotic blockade. Arch. Intern. Med., 118:304-309, 1966. (also recorded in the Association of American Physicians meeting transcription of discussion) 2) 1965: Translational applied clinical research performed at Manhattan General Hospital: Dole, V.P. and Nyswander, M.E.: A medical treatment for diacetylmorphine (heroin) addiction. JAMA, 193:646-650, 1965. Kreek 2019 Impact of Short-Acting Heroin versus Long-Acting Methadone Administered on a Chronic Basis in Humans (1964 through 1973 Studies): Opioid Agonist Pharmacokinetics – Heroin Versus Methadone “On-Off” Apparent Plasma Terminal

"High" Drug or Half-life and Duration of Medication Desired Effects HEROIN 3 min for prodrug "Straight"

30 min for active (Heroin) "Sick" compound, mono-acetyl

morphine (fast on-set and Functional Functional State AM PM AM PM AM off-set) Days “Steady State” 6 hours for active metabolites (morphine "High" and others) METHADONE 24h for racemic (rs) "Straight" medication (slow on-set and off-set – steady-state (Methadone) achieved)

Functional Functional State "Sick" 48h for active (r) AM PM AM PM AM enantiomer Days H Dole, Nyswander and Kreek, 1966; Kreek et al., 1973; 2019 1973

Kreek, New York State Journal of Medicine, Vol. 73, No. 23, December 1, 1973 “On-Off” versus “Steady-State”: Relationship Between Blood (and Brain) Levels of Drugs of Abuse and Their Effects

Disruption versus Normalization • levels of gene expression • receptor mediated events • physiology • behaviors

Rates of rise of blood (and presumable brain) levels of drugs of abuse are related positively to their reinforcing effects Rates of fall of blood (and presumably brain) levels of drugs of abuse are related positively to the onset of withdrawal symptoms and/or acute “craving”

Kreek, 1973 on, 2019 1965-1968: Early Expansion of Methadone Maintenance Treatment

1964-1967 – Initially all patients admitted as active or recently abstinent heroin addicts to the Rockefeller Hospital, unlocked research ward for inpatient induction into methadone maintenance treatment (minimum 6- 8 weeks). Follow-up in Rockefeller Hospital Outpatient Department.

1965-1966 – Replication of methadone maintenance treatment research model in an inpatient unit of the Manhattan General Hospital, a fee-for- service proprietary hospital (later to become the Bernstein Institute of Beth Israel Hospital), which had been engaged in 10-14 day detoxification “treatment” for opiates (“revolving door”).

1967-1968 – Introduction of outpatient induction into methadone maintenance treatment (at the Rockefeller Hospital clinic, Dr. MJ Kreek).

Kreek 2019 Prospective studies of first 214 patients admitted to methadone maintenance research and treatment (January 1964 – July 1966, and followed-up for three years or more) and Retrospective Study of 1435 methadone maintenance patients.

1973 1967-1973: Further Expansion of Methadone Maintenance Treatment and FDA Approval 1967-1972 – Development of multiple satellite outpatient methadone maintenance treatment programs at Harlem, Beth Israel, St. Luke’s, Roosevelt, and Einstein Hospitals in New York City (Dole, Nyswander, Kreek, Lowinson, et al.).

1967-1972 – First replication of methadone maintenance program outside of New York City – Department of Psychiatry, University of Uppsala, Uppsala, Sweden (Dr. Lars Gunne and later Drs. Leif Grönbladh and Olof Blix). 1969-1973 – Opening of the “Adolescent Development Program” (self- administration for > three years in age 15-21) (Elizabeth Khuri and Robert Millman) and “Adult Program” (Aaron Wells) at New York Hospital- Cornell Medical Center.

1973 – Approval by US-FDA of long-term moderate to high dose methadone maintenance treatment for opiate addiction. Kreek 2019 Synthesis and Early Studies of Mu Opioid Receptor Antagonists Naloxone, and Subsequently Naltrexone and Nalmefene: Use in Reversal of Opiate Overdose and Later Treatment of Addictive Diseases (1967 onward) In 1967, Dr. Jack Fishman, a chemist and endocrinologist working with several institutions in New York City, visited with one of his former medical students (MJK) for lunch at The Rockefeller University. At that lunch, he shared that he had synthesized a new compound hoping that it would be a “non-addicting pain medication,” but in fact it did not relieve pain, instead it reversed the effect of short-acting opiate agonists such as heroin and morphine.

“Would there be a clinical use for such a compound?” (JF) That compound was naloxone (Narcan™), which is now used world over for the reversal of opiate overdose, as well as planned narcotic effects during surgery and related procedures.

Dr. Fishman also synthesized and assisted in the development of nalmefene and naltrexone. Naltrexone was studied and subsequently FDA approved for treatment of opiate addiction and, later, alcoholism. Nalmefene was studied and subsequently FDA approved for perioperative effects of opiates and, in Europe, for the treatment of alcoholism. He later became a Professor at The Rockefeller University before pursuing drug development in the pharmaceutical industry. He died in 2013. Kreek 2019 International Narcotics Research Conference Meeting Sites, Presidents, and Local Chairs – 1969-1979 Local and Program Dates Venue Chairperson(s) INRC President 1969 Basel, Switzerland H. Collier, H. Collier (d 1983), H. Kosterlitz H.Kosterlitz (d 1996) 1970 NO MEETING 1971 Aberdeen, Scotland H. Kosterlitz H. Kosterlitz 1972 San Francisco, CA, USA A. Goldstein A. Goldstein (d 2012) 1973 Chapel Hill, NC, USA A. Goldstein A. Goldstein 1974 Cocoyoc, Mexico A. Goldstein A. Goldstein 1975 Airlie House, VA, USA A. Goldstein A. Goldstein 1976 Aberdeen, Scotland (#1 mjk) H. Kosterlitz S. Archer (d 1996) 1977 Brewster Academy, NH, USA E. Simon S. Archer (Gordon Conference) 1978 Noordwijkerhout, J. Van Ree, S. Archer Netherlands L. Terenius (mjk missed; birth of EAKS) 1979 N. Falmouth, MA, USA E.L. Way (d 2017) S. Archer Kreek 2019 Harry Collier Hanz Kosterlitz

Avram Goldstein Sydney Archer Eddie Way Major Scientific Discoveries or Advances Concerning the Endogenous Opioid System or the Treatment of Opioid Addiction – 1969-1979: Unsuccessful Attempts to Delineate Opiate Receptors

1970-1971 Hypotheses and attempts to delineate selective endogenous opioid *** receptors using the technique of stereo-selective binding

Ingoglia, N. A., & Dole, V. P. Localization of d- and l-methadone after intreventricular injection into rat brains. JPET, 175(1), 84-87. (1970)

Goldstein, A., Lowney, L. I., & Pal, B. K. Stereospecific and nonspecific interactions of the morphine congener levorphanol in sebcellelar fractions of mouse brain. PNAS, 68(8), 1742-1747. (1971)

Kreek 2019 Major Scientific Discoveries or Advances Concerning the Endogenous Opioid System or the Treatment of Opioid Addiction – 1969-1979: Delineation of Specific Opiate Receptors 1973 Clear delineation of the existence of endogenous opioid *** receptors, successfully using stereo-selective binding of an opioid agonist or opioid antagonist

Pert, C. B., & Snyder, S. H. Opiate receptor: demonstration in nervous tissue. Science, 179(4077), 1011-1014. (1973)

Terenius, L. Stereospecific interaction between narcotic analgesics and a synaptic plasma membrane fraction of rat cerebral cortex. Acta pharmacol toxicol, 32, 317-320. (1973)

Simon, E. J., Hiller, J. M., & Edelman, I. Stereospecific binding of the potent narcotic analgesic [3H] etorphine to rat-brain homogenate. PNAS, 70(7), 1947-1949. (1973) Development and Discontinuation of a Second Effective Pharmacotherapy for Opiate Addiction – LAAM (1970-2003)

1970-1971 Initial development of l-alpha-acetylmethadol (LAAM) for the treatment of opiate addiction

Jaffe, J. H., Schuster, C. R., Smith, B. B., & Blachley, P. H. Comparison of acetylmethadol and methadone in the treatment of long-term heroin users: A pilot study. Jama, 211(11), 1834-1836. (1970)

Jaffe, J. H., & Senay, E. C. Methadone and l-methadyl acetate: Use in management of narcotics addicts. Jama, 216(8), 1303-1305. (1971)

1993 LAAM approved by FDA for treatment of opiate addiction

2002 Borg, L., Ho, A., Wells, A., Joseph, H., Appel, P., Moody, D., and Kreek, M.J. The use of levo-alpha-acetylmethadol (LAAM) in methadone patients who have not achieved heroin abstinence. J. Addict. Dis. 21:13-22. (2002)

2003 Availability of LAAM discontinued in USA

Kreek 2019 Major Scientific Discoveries or Advances Concerning the Endogenous Opioid System or the Treatment of Opioid Addiction – 1969-1979

1975 First elucidation of endogenous opioid peptides: *** • delta-opioid receptor-directed enkephalins Hughes, J., Smith, T. W., Kosterlitz, H. W., Fothergill, L. A., Morgan, B. A., & Morris, H. R. Identification of two related pentapeptides from the brain with potent opiate agonist activity. Nature, 258(5536), 577. (1975)

• mu opioid receptor directed endorphin; POMC, containing beta-endorphin,as well as ACTH and other important neuropeptides Terenius, L., & Wahlström, A. Search for an endogenous ligand for the opiate receptor. Acta Physiologica Scandinavica, 94(1), 74-81. (1975)

1979 • kappa opioid receptor directed dynorphins *** Goldstein, A., Tachibana, S., Lowney, L. I., Hunkapiller, M., & Hood, L. Dynorphin-(1-13), an extraordinarily potent opioid peptide. PNAS, 76(12), 6666-6670. (1979) Development of Novel Techniques and Studies of the Pharmacokinetics and Pharmacodynamics of Methadone – 1976-1981 1977-1979 Techniques developed to measure stereo-selective pharmacokinetics of dl(sr) methadone using gas liquid chromatography in conjunction with mass spectrometry utilizing stable isotope technology Hachey, D. L., Kreek, M. J., & Mattson, D. H. Quantitative analysis of methadone in biological fluids using deuterium-labeled methadone and GLC- chemicalionization mass spectrometry. J Pharm Sci, 66(11), 1579-1582. (1977) Kreek, M. J., Hachey, D. L., & Klein, P. D. Stereoselective disposition of methadone in man. Life Sci, 24(10), 925-932. (1979)

1976-1981 Identification of interactions of other drugs with methadone as used in chronic maintenance treatment of addiction in humans Kreek, M.J., Garfield, J.W., Gutjahr, C.L. and Giusti, L.M.: Rifampin-induced methadone withdrawal. N. Engl. J. Med., 294:1104-1106. (1976) Tong, T.G., Benowitz, N.L. and Kreek, M.J.: Methadone-disulfiram interaction during methadone maintenance. J. Clin. Pharmacol., 20:506-513 (1980) Tong, T.G., Pond, S.M., Kreek, M.J., Jaffery, N.F. and Benowitz, N.L.: Pheny- toin-induced methadone withdrawal. Ann. Intern. Med., 94:349-351 (1981) Opiate (Opioid) Analgesic Agents (1975 onward) • Numerous – all mu opioid receptor-directed • Diverse formulations – many long-acting (but also some still friable) • Most available in oral, sublingual, or patch preparations Examples – mostly short-acting Sustained Release/Long-Acting(*) Morphine Codeine Oxycodone (e.g., OxyContin®) Hydromorphone (e.g., HydromorphoneContin®)** Oxymorphone Hydrocodone and Acetaminophen (e.g., Vicodin®) Meperidine (e.g., Demerol®) Fentanyl (e.g., Duragesic®) Methadone* (long-acting) (e.g., Dolophine®) – full mu agonist Buprenorphine* (long-acting) (e.g., Subutex® sl) – partial mu agonist Buprenorphine-Naloxone* (long-acting) (e.g., Suboxone® sl) – partial mu agonist ** Removed from market by FDA shortly after approval Kreek, 1975 on, 2019 Use of Methadone as an Analgesic: Doses Usually Range from 0.5mg tid to 10mg tid (rarely over 60 mg/d) • Very few indications for use of methadone in management of acute pain – because of long-acting pharmacokinetic properties, accumulation of medication must be anticipated over the first four days or longer. Accidental overdose deaths frequently occur when methadone is so prescribed.

• Methadone is a superb (and often superior) opioid analgesic for chronic pain, including neoplastic and non-neoplastic pain – because tolerance develops more slowly than to other opioid analgesics, steady doses may be used over long periods of time and escalation of dose may be gradual; no drowsiness, no dulled responses. Kreek, 1975 on; 2019 International Narcotics Research Conference Meeting Sites, Presidents, and Local Chairs – 1980-1989 Local and Program Dates Venue Chairperson(s) INRC President 1980 Plymouth State College, E. Simon E. Simon NH, USA (Gordon Conference) 1981 Kyoto, Japan H. Takagi E. Simon 1982 N. Falmouth, MA, USA B. Cox, A. Takemori E. Simon 1983 Garmisch, Germany A. Herz E. Simon 1984 Cambridge, England J. Hughes E.L. Way (d 2017) 1985 N. Falmouth, MA, USA A. North E.L. Way 1986 San Francisco, CA, USA H. Loh E.L. Way 1987 Adelaide, Australia S. Johnson E.L. Way 1988 Albi, France J. Cros, J-C. Meunier A. Herz (d 2018) 1989 St. Adele, Canada R. Quirion, K. Jhamandas, A. Herz C. Gianoulakis Major Scientific Discoveries or Advances Concerning the Endogenous Opioid System or the Treatment of Opioid Addiction – 1982: Cloning of the Genes Encoding Endogenous Opioid Peptides 1982 Cloning of the genes encoding the three classes of endogenous opioid peptide

Noda M, Yasuji F, Takahashi H, Toyosato M, Hirose T, Inayama S, Shigetada N, Numa S. Cloning and sequence analysis of cDNA for bovine adrenal preproenkephalin. Nature. 295:202-06. (1982)

Kakidani H, Furutani Y, Takahashi H, Noda M, Morimoto Y, Hirose T, Asai M, Inayama S, Nakanishi S, Numa S. Cloning and sequence analysis of cDNA for porcine beta-neo-endorphin/dynorphin precursor. Nature. 298:245-9. (1982)

Civelli O, Birnberg N, Herbert E. Detection and Quantitation of Pro-Opiomelanocortin mRNA in Pituitary and Brain Tissues from Different Species. J Biol Chem. 257:6783-87. (1982)

Kreek 2019 Mechanisms for Development of an Addiction

• Drugs alter normal brain networks and chemicals

• “Rewarding” or “pleasurable” effects of drugs (the so-called “reinforcing effects”) involve: – Dopamine – Endorphins (acting at Mu Opioid Receptors)

• “Countermodulatory” response to reward involves: – Dynorphins (acting at Kappa Opioid Receptors)

• “Atypical stress responsivity” Kreek, 1970s; 1984; 2019 STRESS RESPONSIVITY: Hypothesis – Atypical Responsivity to Stressors, A Possible Etiology of Addictions – HPA Axis

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

Kreek, 1982, 2019 STRESS RESPONSIVITY – Heroin, Cocaine, and Alcohol Profoundly Alter Stress Responsive Hypothalamic-Pituitary-Adrenal (HPA) Axis: Normalization During Methadone Treatment • Acute effects of opiates Suppression of HPA Axis • Chronic effects of short-acting (decrease levels of HPA opiates (e.g., heroin addiction) hormones)

• Opiate withdrawal effects * Activation of HPA Axis • Opioid antagonist effects (increase levels of HPA • Cocaine effects * Hormones) • Alcohol effects

• Chronic effects of long-acting opiate (e.g. methadone in Normalization of HPA Axis maintenance treatment)

* Our challenge studies have shown that a relative and functional “endorphin deficiency” develops.

Kreek, 1984, 2019 Identification of HIV-1 Infection in Drug Users in New York City and Protective Value of Methadone Maintenance Treatment: (1969 –) 1978 – 1992; 1983 – 1984 Study and Beyond 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

Kreek with Des Jarlais and others, Des Jarlais, MMWR: Morbid. Mortal. Wkly Rep., 33:377-379, 1984; 2019 Development of a Third and Fourth Pharmacotherapy for Opiate Addiction – Naltrexone (not effective) and Buprenorphine-Naloxone (effective) (1984-1988) 1984 FDA approval of the mu opioid antagonist naltrexone for opiate addiction (effective in <15% of unselected opiate addicted patients) under the trade name Trexan™. Clinical studies for opioid dependence began in 1973. Found to cause dose-dependent hepatotoxicity. 1984-1988 Development of buprenorphine (sublingual) and buprenorphine- naloxone (sublingual; naloxone added to prevent IV misuse) for treatment of opiate addiction (effective in 40%-50% of unselected opiate addicted patients); buprenorphine originally developed as an IV or IM or SC pain medication (Lewis, Cowan of Reckitt Colman) Jasinski, D.R., Pevnick, J.S., Griffith, J.D. Human pharmacology and abuse potential of the analgesic buprenorphine: a potential agent for treating narcotic addiction. Arch Gen Psychiatry. 35:501-16. (1978) [subcutaneous] Preston K.L., Bigelow G.E., Liebson I.A. Buprenorphine and naloxone alone and in combination in opioid-dependent humans. Psychopharmacology (Berl). 94:484-90. (1988) 2002 FDA approval of sublingual buprenorphine and buprenorphine- naloxone for treatment of opiate addiction Kreek 1984, 2019 International Narcotics Research Conference Meeting Sites, Presidents, and Local Chairs – 1990-1999 Local and Program Venue Chairperson(s) INRC President 1990 Noordwijkerhout, Netherlands J. Van Ree A. Herz 1991 Copper Mountain, CO, USA T. Burks, B. Cox, H. Akil (with FASEB) J. Holaday 1992 Keystone, CO, USA H. Akil H. Akil (with CPDD) 1993 Skövde, Sweden F. Nyberg H. Akil 1994 N. Falmouth, MA, USA E. Simon H. Akil 1995 St. Andrews, Scotland J. Traynor B. Cox 1996 Long Beach, CA, USA S. Watson B. Cox 1997 Hong Kong, China T. Wong, H. Loh B. Cox 1998 Garmisch-Partenkirchen, V. Höllt B. Cox Germany* 1999 Saratoga Springs, NY, USA S. Childers, J. C. Chavkin Bidlack * NY Times call to MJK re: Mayor Giuliani Major Scientific Discoveries or Advances Concerning the Endogenous Opioid System or the Treatment of Opioid Addiction – 1990-1999: Cloning of the Delta Opioid Receptor 1992 Cloning of delta opioid receptor *** Evans CJ, et al. Cloning of a delta opioid receptor by functional expression. Science. 258:1952-5. (1992)

Kieffer BL et al. The delta opioid receptor: Isolation of a cDNA by expression cloning and pharmacological characterization. Proc Natl Acad Sci USA. 89:12048-52. (1992) Major Scientific Discoveries or Advances Concerning the Endogenous Opioid System or the Treatment of Opioid Addiction – 1990-1999: Cloning of the Mu and Kappa Opioid Receptors

1993 Cloning of the mu opioid receptor. *** Chen Y… Yu L. Molecular Cloning and Functional Expression of a mu Opioid Receptor from Rat Brain. Mol Pharmacol. 44:8-12. (1993)

Wang JB… Uhl GR. Mu opiate receptor: cDNA cloning and expression. Proc Natl Acad Sci USA. 90:10230-34. (1993)

1993 Cloning of the kappa opioid receptor. *** Yasuda K… Bell GI. Cloning and functional comparison of kappa and delta opioid receptors from mouse brain. Proc Natl Acad Sci USA. 90:6736-40. (1993)

Meng F… Goldstein A, Watson SJ, Akil K. Cloning and pharmacological characterization of a rat kappa opioid receptor. Proc Natl Acad Sci USA. 90:9954-58. (1993) Major Scientific Discoveries or Advances Concerning the Endogenous Opioid System or the Treatment of Opioid Addiction – 1994-1998: Cloning and Studies of the Orphan Opioid Receptor 1994 Cloning of the nociceptin/orphanin FQ (NOP receptor) *** Mollereau C… Meunier JC. ORL1, a novel member of the opioid receptor family. Cloning, functional expression and localization. FEBS Lett. 341:33-8. (1994) Bunzow JR, Saez C, Mortrud M, Bouvier C, Williams JT, Low M, Grandy DK. Molecular cloning and tissue distribution of a putative member of the rat opioid receptor gene family that is not a mu, delta or kappa opioid receptor type. FEBS Lett. Jun 27;347:284-8. (1994)

1995-1998 Isolation, structure, and distribution of nociceptin/orphanin in human central nervous system

Meunier JC…Toll L, et al. Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptor. Nature. 377:532-5. (1995)

Peluso, J., LaForge, K.S., Matthes, H.W., Kreek, M.J., Kieffer, B.L., and Gaveriaux-Ruff, C.: Distribution of nociceptin/orphanin FQ receptor transcript in human central nervous system and immune cells. J. Neuroimmunol., 81:184-192. (1998) New York Times, June 3, 1997 New York Times, July 21, 1998 INRC, July 20-25, Garmisch, Germany New York Times, July 21, 1998 INRC, July 20-25, Garmisch, Germany This mayoral challenge initiated an interesting sequence of brief intransient distractions from our research of molecular neurobiology, human molecular genetics, and epigenetics of opiate addiction and other specific addictions. On the third Saturday in January of 1999, Mayor Giuliani reversed himself! New York Times, January 16, 1999 Genetic Vulnerability to (or Protection from) Development of an Addiction (Kreek Lab)

Genetic vulnerability to develop an addiction once self-exposed probably due to: • Multiple variants (SNPs and other types) of • Multiple genes (as with any complex disorder, e.g., hypertension, diabetes) • Probably both shared and also unique variants for each specific addiction • Genetic contributions of comorbid conditions and personality types may play a role No gene variant or group of variants causes an addictive disease alone; self-administration of a

drug is essential. Kreek, 1998, 2019 Single Nucleotide Polymorphisms of the Mu Opioid Receptor in the Coding Region, Including the Functional and Heroin Addiction Associated A118G (N40D) Variant

HYPOTHESIS

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

• Alter response to medications “PHARMACOGENETICS”

• Are associated with specific addictions

Bond, LaForge… Kreek, Yu, PNAS, 95:9608, 1998 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 Prototype 1.0 Prototype 80

40 0.5

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 Levels 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 Genetically Modified A112G Mice (Asparagine to Aspartic Acid), A Model of the Human A118G Mu Opioid Receptor Functional Variant: Heroin Self-Administration (10d, 4h/d) by

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Zhang, Blendy…Kreek et al., Neuropsychopharm, 40:1091, 2015; Kreek 1998, 2015; 2018 Major Scientific Discoveries or Advances Concerning the Endogenous Opioid System or the Treatment of Opioid Addiction – 2000-2010

Local and Program Dates Venue Chairperson(s) INRC President 2000 Seattle, WA, USA C. Chavkin C. Chavkin 2001 Helsinki, Finland E. Kalso, L. Ahtee C. Chavkin 2002 Monterey, CA, USA C. Evans, L. Toll C. Chavkin (with ICRS) 2003 Perpignan, France B. Kieffer, P. Jauzac M.J. Kreek 2004 Kyoto, Japan H. Ueda, M. Satoh M.J. Kreek 2005 Annapolis, MD USA G. Uhl, J.B. Wang M.J. Kreek 2006 St. Paul, MN, USA P.L. Law , G. Wilcox M.J. Kreek 2007 Berlin, Germany C. Stein L. Devi 2008 Charleston, SC, USA J. McGinty L. Devi 2009 Portland, OR, USA J. Williams, S. Ingram L. Devi Scientific Discoveries Concerning the Endogenous Opioids (Kreek Lab) – 2000-2009

2000 Distribution of the mu opioid receptor in human brains using PET (Positron Emission Tomography) with 18F cyclofoxy in healthy subjects and methadone maintained former heroin addicts (Kling, Carson, Kreek)

2000 Mu, delta, and kappa opioid receptor populations found to be differentially altered in distinct areas in post-mortem brains of Alzheimer’s disease patients (Mathieu-Kia, Kreek, Simon, Hiller)

2000 Opioid receptor and peptide gene polymorphisms, potential implications for development of addictive diseases (LaForge, Yuferov, Kreek)

2001 Kappa opioid receptor agonist induced prolactin levels in non-human primates blocked by dopamine 2-like receptor agonists (Butelman) STRESS RESPONSIVITY – Normalization of Heroin Disrupted Physiology During Methadone Maintenance Treatment: PET Studies of mu Opioid Receptors in Human Brain Regions Using the Antagonist [18F] Cyclofoxy

Normal volunteers n=14 16 MMTP volunteers n=14 (~20-30 percent reduction in 14 mu-opioid receptor binding due to methadone occupancy) 12

2 Specific Binding (ml plasma/ml tissue) plasma/ml (ml Binding Specific 0 Thl Amy Caud Ins ACg Put MT MFr Par Crb IT Hip WMt Area related to pain response Region of Interest Kling et al., . J. Area with dopamine terminals involved in “reward”, mood, and decision-making Pharmacol. Exp. Area with dopamine terminals involved in memory, learning, and movement Ther., 295, 2000 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 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 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

Short = 1,1; 1,2; 2,2 Long/Short = 1,3; 1,4; 2,3; 2,4 Long = 3,3; 3,4; 4,4

Controls Cocaine/Alcohol Dependent Short Short Long Long (SS) Short/Long (LL) (SS) Short/Long (LL) Genotype (>dynorphin) (SL)* (dynorphin) (SL) (Montreal, Canada L. Gendron J. Traynor 2015 Phoenix, Arizona, USA T. Vanderah F. Nyberg (with CPDD) 2016 Bath, England, UK C. Bailey F. Nyberg 2017 Hotel, Chicago, IL, USA A. Pradhan F. Nyberg 2018 San Diego, CA, USA L. Toll F. Nyberg (with CPDD) (mjk missed; RU graduation of Devon Collins, PhD) 2019 New York, NY, USA M.J. Kreek, L. Toll L. Devi 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 ~ 2.1 million Opiate (heroin, fentanyl, other) Overdose Deaths ~ 72,300 (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 • 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 Research and Clinical Evidence Contributes to Specific Actions for “Prevention”, “Reversal”, and “Reduction” of Illicit Opiate Use and Overdose Deaths (72,300 in 2017) “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 Limited Targeted Pharmacotherapies Available for Specific Addictive Diseases I. Opiate Addiction (Heroin and Illicit Use of 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 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, 2018 Methadone synthetic mu opioid receptor full agonist NMDA partial antagonist

Buprenorphine thebaine-derived mu opioid receptor partial agonist kappa opioid receptor partial agonist

Naltrexone thebaine-derived mu opioid receptor full antagonist kappa opioid receptor partial agonist

Kreek 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 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 Targets of Currently Approved Treatments for Addictive Disorders

Kreek et al, Journal of Clinical Investigation, 12: 3387, 2012 DYNORPHIN-KAPPA OPIOID RECEPTOR SYSTEM: COUNTERMODULATION OF MU OPIOID RECEPTOR-DOPAMINE MEDIATED REWARD Cocaine, Heroin, and Alcohol Increase Dynorphin Gene Expression Levels in an Acute and Chronic Persistent Manner

7 Saline * 14 day cocaine (15mg/kg x 3) 6 e.g., 14 cocaine administration persistently 5 increases dynorphin gene expression in 4 the caudate putamen of rat

1 pg ppDyn mRNA / µg total RNA total µg / mRNA ppDyn pg

0 Caudate Putamen Nucleus Accumbens Dynorphin Acting at the Kappa Opioid Receptor Lowers Dopamine Levels and Prevents Surge After Cocaine Spangler, Ho, Zhou, Maggos, Yuferov, and Kreek , Mol. Brain Res., 38:71, 1996 ; 2019 DYNORPHIN-KAPPA OPIOID RECEPTOR SYSTEM: COUNTERMODULATION OF MU OPIOID RECEPTOR-DOPAMINE MEDIATED 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 Dopamine in

Dopamine Dopamine in Dialysate 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-Induced Immobility During Forced Swim Test in Rats Caused by Increases in Dynorphin Gene Expression: Kappa Opioid Receptor Antagonist (nor-BNI) Reduces Immobility in a 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; 2019 KAPPA OPIOID RECEPTOR – DYNORPHIN SYSTEM ACTIVATION: PROLACTIN AS A “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 2030 4050 60 75 90 120 150 180 240 Time after injection (min)

Kreek et al., 1994; 1999; 2016 TRANSLATIONAL RESEARCH: IDENTIFICATION OF POTENTIAL TARGET FOR NOVEL PHARMACOTHERAPIES FOR COCAINE ADDICTION OR ALCOHOLISM – KAPPA OPIOID RECEPTOR / DYNORPHIN SYSTEM Cocaine, alcohol, heroin, and morphine, and also stress (e.g., forced swim test), increase dynorphin gene expression in rodents. • Kappa Opioid Receptor Full Agonist – lowers basal dopaminergic tone and decreases drug-induced dopaminergic surge – ? reduction of reward after drug or alcohol use – ? possibly persistent dysphoric side-effects (? avoid by use of biased or partial kappa agonist) • Kappa Opioid Receptor Antagonist – reduction of depressive symptoms due to increase in dynorphin levels due to stress, opiate, cocaine, alcohol, or spontaneous (shown in rodents), ? decreased relapse to drug use but possible increases in dopamine surge after drug or alcohol self-administration • Kappa Opioid Receptor Partial Agonist (antagonist and agonist activity) – ? reduces dopaminergic surge and modulates dopaminergic tone – ? thus reduction of reward after drug use, and ? reduction of depressive symptoms – ? reduction of drug self-administration (>200 novel compounds synthesized for our studies) 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

• Prototype unbiased kappa opioid receptor agonists G-protein β-arrestin U50,488 and U69,593 ? ? • Biased agonism: differentially Analgesia Sedation activate intracellular Motor incoordination pathways ? (e.g., G-protein vs. β-arrestin) Anhedonia Reed, AD Dunn, Butelman, Kreek, CPDD 2017, in preparation, 2019 Compounds Approved for Use in Human Therapeutics with KOPr Partial Agonism, but also with Mu-Opioid Receptor Antagonism* or Partial Agonism**; 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)

Bidlack Laboratory (U Rochester); Bart… Bidlack et al, Neuropsychopharmacol, 30:2254, 2005 REVERSAL OF DYNORPHIN-KAPPA INDUCED STRESS – Short-Acting Selective Kappa Opioid Receptor Antagonists

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

sold to Cerecor (CERC-501), then sold to Janssen-Johnson & Johnson (JNJ-67953964)

Kreek, 2019 Pretreatment with kappa opioid receptor selective antagonist (LY2444296 – “tool compund”) reduces anxiety- and depression-like behaviors in rats in withdrawal (30h) following extended-access cocaine self-administration (0.5mg/kg/infusion, 18h/d, 14d)

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 * p< 0.05 225225 225 ** p< 0.01 *** p< 0.001 150 150 ** 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; 2019 “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 abstinence from cocaine or alcohol, but that such use might increase cocaine or alcohol-induced reward by increasing baseline dopaminergic tone and drug-induced dopamine surges. (Data of impact of selective KOP-r antagonist limited.)

We have conducted a five-day, in-patient study that examined neuroendocrine and behavioral effects of a novel short-acting selective KOP-r antagonist, LY2456302 (“OpraK”) in normal volunteers (n=40) and in volunteers with cocaine dependence (n=30).

Reed et al, Neuropsychopharmacology, 43:739, 2017 “OpraK” Study: Prolactin Levels – Male Normal Volunteers vs. Early Abstinence Cocaine Dependent Volunteers – No Evidence of Kappa Partial Agonism

25 OpraK 10mg/d PO for 4 days Baseline, Day 1 20 1st OpraK, Day 2 th 15 4 OpraK, 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, Day 2 th 15 4 OpraK, Day 5 Early Abstinence 10 Cocaine Dependent 5 Volunteers (n=19)

0 (minimal symptom -30 0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480

serum prolactin (ng/mL) serum changes) TimeTime (min)(min) Reed et al, Neuropsychopharmacology, 43:739, 2017; 2019 GENE VARIANTS ASSOCIATED WITH OPIATE ADDICTION IN CAUCASIANS, AFRICAN AMERICANS, OR 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 Systems (Selected Genes) Opioid Stress-Responsive Neurotransmitter OPRM1 AVPR1A COMT (mu opioid receptor) (arginine vasopressin receptor 1A) (catechol-o-methyltransferase) OPRD1 FKBP5 HTR1B (delta opioid receptor) (FK506-binding protein 51/ (serotonin receptor 1B) corticosterone chaperone) OPRK1 GAL GABRG1 (kappa opioid receptor) (galanin) (gamma-aminobutyric acid (GABA) A receptor) PDYN CRHBP GRIN2A (dynorphin peptide) (corticotropin releasing hormone (glutamate receptor, ionotropic, binding protein) NMDA 2A)

updated after Reed et al., Current Psychiatry Reports, 16(11): 504, 2014 Kreek Lab: Levran, LaForge, Nielsen, Randesi, Yuferov, and others; Kreek 2019 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 2019 after Levran 2015 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 Amelia Dunn David Novick Brian Reed Einat Peles Assistants for Research Ellen Unterwald Laboratory Manager Ariel Ben-Ezra Matthew Randesi Michelle Morochnik Other Rockefeller Phil Pikus University Collaborators Research Nurse Carina Chen Brian Chait Practitioner Bryan McElroy Jeff Friedman Kate Brown Paul Greengard (d. 2019) 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 Kreek Laboratory 2019