The Molecular Neurobiology and Human Genetics of Specific ... · Actions of methadone treatment: • Prevents withdrawal symptoms and “drug hunger” • Blocks euphoric effects

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)

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

The Molecular Neurobiology and Human Genetics of Specific Addictive Diseases:

Implications for Treatments

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.

Initial Research on the Biology of Addictive Diseases at the Rockefeller University (then Institute), 1964: Development of a

Pharmacotherapy for Heroin Addiction

Dole, Nyswander and Kreek, 1964, 1966, 2018

First research paper describing methadone maintenance treatment research

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: 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.

Vincent P. Dole, Jr., MD; Marie Nyswander, MD; and Mary Jeanne Kreek, MD

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

Drug or

Medication

Apparent Plasma Terminal

Half-life and Duration of

Desired Effects

HEROIN 3 min for prodrug

30 min for active

compound, mono-acetyl

morphine (fast on-set and

off-set)

6 hours for active

metabolites (morphine

and others)

Fu

ncti

on

al

Sta

te

(Hero

in)

"High"

"Straight"

"Sick"

Days

AM PM AM PM AM

Fu

ncti

on

al

Sta

te

(Meth

ad

on

e) "High"

"Straight"

"Sick"

Days

AM PM AM PM AM

H

METHADONE 24h for racemic (rs)

medication (slow on-set

and off-set – steady-state

achieved)

48h for active (r)

enantiomer

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

Overdose Deaths in Thousands in Preceding 12 months

• 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

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, 2018

Synthetic

opioids

Other

opioids

Other Psychostimulants

Methadone

Heroin

Cocaine

30 thousand

20

10

0

2015 2016 2017

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

Prevalence of Specific Drug Abuse and Vulnerability to Develop Addictions – 2019

SAMHSA Nat’l Survey on Drug Use and Health, 2017; Others, 2007-18; *Nat’l Center for Health Statistics (CDC), 2019

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%)

Development of Addiction After Self-Exposure to Specific Drugs

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

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.

100

75

50

25

0

%

1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1992

Percent of IV Drug Users Infected with HIV-1

Number of patients currently in treatment:

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 Maintenance Treatment for Opiate (Heroin) Addiction – 2019

Kreek, 1972; 1973; 2019

~ 1.4 million worldwide

• 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

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)*

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

US Patients in Treatment

Treatment 2015 2016 2017

Methadone

Maintenance

356,843 345,443

(-11,400; -3.2%)

382,867

(+37,424; +10.8%)

Buprenorphine

Maintenance

75,723 61,486

(-14,237; -18.8%)

112,223

(+50,737; +82.5%)

Extended

Release

Naltrexone

7,035 10,128

(+3,093; +44.0%)

23,065

(+12,937; +128.7%)

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

Limited Targeted Pharmacotherapies Available for Specific Addictive Diseases

Kreek, 2019

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.

(%) 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.

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

Initial Use of

Drug of Abuse

Sporadic

Intermittent

Use

Regular

Use Addiction Early

Withdrawal

(abstinence)

Protracted

Abstinence

Primary

Prevention

Possible Utility of Vaccines

and Selected Medications

Medications Useful

and Needed

Adapted from Kreek et al., Nature Reviews Drug Discovery, 1:710, 2002; 2019

Progression

relapse to addiction without

pharmacotherapy

90% - opiate;

60% - cocaine, alcohol

sustain abstinence with no

specific medications

10% - opiate;

40% - cocaine, alcohol

ADDICTION: Compulsive drug seeking behavior

and drug self-administration, without regard to

negative consequences to self or others

(adapted from WHO).

Factors Contributing to Vulnerability to Develop a Specific Addiction

use of the drug of abuse essential (100%)

Genetic

(25-80%) • DNA

• SNPs

• repeats

• other

polymorphisms

Drug-Induced Effects

(very high)

Environmental

(very high) • prenatal

• postnatal

• epigenetics

• cues

• peer pressure

• comorbidity

• stress-responsivity

Kreek et al., 2000; 2005; 2019

• mRNA levels

• peptides

• proteomics

• neurochemistry

• synaptogenesis

• behaviors

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

• “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

agonist in rodent to long-acting human pharmacokinetic profile)

Bidirectional-Translational Research: Novel and Conventional Rodent Models

to Mimic Human Patterns of Abuse

Kreek et al., 1987;

1992; 2001; 2005; 2019

DOPAMINE SYNAPSE

Kreek, 2016

Maisonneuve, 1994

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

National Institute on Drug Abuse

Frontal

cortex

Nucleus

accumbens

VTA

Striatum

Substantia

nigra

Atypical responsivity to

stress and stressors

may contribute to the

persistence of, and

relapse to, self-

administration 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. Kreek, 1972; 1981; 1982; 1984 … 2019

Development of an Addiction: Stress and Atypical Responsivity to Stressors –

HPA Axis

–

–

b-End

adrenal

POMC

hypothalamus

ACTH

Anterior pituitary +

Cortisol

+

Endogenous

Opioids

(mu – inhibition)

(kappa – ? activation)

Arginine

Vasopressin

+

CRF

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

Zhang et al, Psychopharmacology, 231, 1277, 2013

4-hr Sessions n=11

1-hr Sessions n=8

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

0

5

1 0

1 5

2 0

2 5

3 0

0

2

4

6

8

S e s s io n s

No

se

Po

ke

s /

1 h

ou

r s

es

sio

n

Mg

/kg

/infu

sio

n

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

0

5

1 0

1 5

2 0

2 5

3 0

3 5

0

2

4

6

8

A c tiv e H o le

In a c tiv e H o le

S e s s io n s

No

se

Po

ke

s /

4 h

ou

r s

es

sio

ns

mg

/kg

/infu

sio

n

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)

Mayer-Blackwell…Zhang, Neuroscience, 258:280, 2013

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

0

5

1 0

1 5

2 0

2 5

1

2

3

4

5

6A d u lt O x y (n = 1 1 )

A d o le s c e n t O x y (n = 1 2 )

S e s s io n s

Nu

mb

er o

f N

os

e P

ok

es

at

Ac

tiv

e H

ole

(F

R1

)

Ox

yc

od

on

e m

g/k

g

0

2 0 0

4 0 0

6 0 0

Y o k e d S a l

O x y c o d o n e

O x y c o d o n e D o s e (m g /k g )

Inc

re

as

ed

Tim

e (

se

c)

on

Ox

yc

od

on

e S

ide

0 1 3

*

3 .3 5 m g /k g 5 m g /k g 7 .5 m g /k g

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

5 0 0 0

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 )

AU

C %

MP

E (

10

-6

0 m

in)

p < 0 .0 5

Yoked Saline

Oxycodone Self-Administration

Adolescent Oxycodone SA in Male Mice Leads Both to

Tolerance in Oxycodone-Induced Anti-Nociception and

Increases in Oxycodone-Induced CPP in Adulthood

Zhang et al., Neuropharmacology, 111:314, 2016

Hot Plate Analgesia Test Condition Place Preference (liking)

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

ADULT MOUSE HUMAN SNPs

Gene Symbol Protein Gene

Expression Change

SNP Location Opiate

Addiction 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

HTR3A 5-hydroxytryptamine (serotonin) receptor 3A

↑ rs897687 Intron x

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

ADOLESCENT MOUSE HUMAN SNPs

Gene Symbol Protein Gene

Expression Change

SNP Location Opiate

Addiction 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

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

Control

1

Control

3

Cocaine

2

Cocaine

4

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

7

6

5

4

1

0

pg

pp

Dy

n m

RN

A /

µg

to

tal R

NA

Caudate Putamen Nucleus

Accumbens

Saline

14 day cocaine

(15mg/kg x 3)

COUNTERMODULATION – KAPPA OPIOID RECEPTOR-DYNORPHIN SYSTEM: Cocaine Increases Kappa Opioid

Receptor Density in Rat, But Kappa Opioid Receptor Directed “Dynorphins” Also Increase Persistently

Dynorphin Acting at the Kappa Opioid Receptor Lowers

Dopamine Levels and Prevents Surge After Cocaine

COUNTERMODULATION OF REWARD: Natural Dynorphin A1-17 (Kappa Opioid Receptor Agonist) Infusion into Mouse Striatum Lowers Basal and Cocaine

Induced Dopamine Levels

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

20-min Sample

10

8

6

4

2

0 60 120 180

Infusion

Do

pam

ine

in

Dia

lysa

te (

nM

)

Dynorphin Dose (nmol) 0 1.0

2.0 4.4

4.4+nBNI

(antagonist) Dynorphin (4.4nmol)

+ Cocaine (15mg/kg)

Infusion and Injection

Control

Cocaine (15mg/kg)

8

6

4

2

0 60 120 180

10

Infusion Injection D

op

am

ine i

n

Dia

lysa

te (

nM

) 20-min Sample

Reed et al., Neuroscience, 220:109-118, 2012

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

Swim 15 Minutes Day 1;

1 hour later pre-treatment

nor-BNI

Swim 5 Minutes Day 2

0 mg/kg (n=6)

5 mg/kg (n=6)

10 mg/kg (n=6)

nor-BNI Dose

0

10

20

30

40

50

60

Immobility

(* - p<0.05)

*

Sc

ore

Kreek et al., 1994; 1999; 2016

+

Dynorphin A1-13

anterior pituitary

lactotropes

Hypothalamus

TIDA

–

–

COUNTERMODULATION – KAPPA OPIOID RECEPTOR – DYNORPHIN SYSTEM: “BIOMARKER” Dynorphin A Lowers Tuberoinfundibular Dopaminergic Tone,

which Tonically Inhibits Prolactin Release

Time after injection (min)

35

30

25

20

15

10

5

0

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

500 µ g/kg

120 µ g/kg

Placebo (n=10) P

rola

cti

n L

evels

(n

g/m

l)

Dose-Response Effects of Dynorphin A1-13 on

Prolactin Levels (BIOMARKER) in Normal Volunteers

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

Kreek, Reed, Butelman 2014

MOP-r

affinity

Ki (nM)

0.66 0.11 0.24 0.21

KOP-r

affinity

Ki (nM)

1.2 0.19 0.083 0.62

DOP-r

affinity

Ki (nM)

120 60 16 2.1

Naloxone Naltrexone Nalmefene Buprenorphine

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

both G-protein vs. β-arrestin

pathways

• Biased agonists: differentially

activate intracellular pathways

(G-protein vs. β-arrestin)

G-protein β-arrestin

Analgesia Sedation

Motor incoordination

? ?

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

LY2444296 – “Tool” compound

(Lilly – Laboratory)

LY2456302 – “OpraKappa”

(Lilly – Clinical)

REVERSAL OF DYNORPHIN-KAPPA

INDUCED STRESS – Short-Acting Selective

Kappa Opioid Receptor Antagonists

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

La

ten

cy

to

im

mo

bil

ity

(s

)

0

75

150

225

300

Vehicle

LY2444296 3 mg/kg

**

Valenza et al., Psychopharmacology, 234: 2219, 2017

La

ten

cy

to

en

ter

in o

pe

n a

rms

(s

)

0

75

150

225

300

***

ELEVATED PLUS MAZE

Tim

e

in o

pe

n a

rms

(s

)

0

10

20

30

40

50vehicle

LY2444296 3 mg/kg

*

La

ten

cy

to

im

mo

bil

ity

(s

)

0

75

150

225

300

Vehicle

LY2444296 3 mg/kg

Imm

ob

ilit

y (

s)

0

30

60

90

120

150FORCED SWIM TEST

*

“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

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

0

5

10

15

20

25

seru

m p

ro

lacti

n (

ng

/mL

)

Time (min)

Normal Volunteers

(n=24)

Early Abstinence

Cocaine Dependent

Volunteers

(n=19)

“OpraK”: Prolactin Levels – Male Normal Volunteers vs. Early

Abstinence Cocaine Dependent Volunteers – No Evidence of Kappa Partial Agonism

-30 0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 450 4800

5

10

15

20

25

seru

m p

rola

cti

n (

ng

/mL

)

Time (min)

Baseline, Day 1

1st OpraK (10mg), Day 2

4th OpraK (10mg), Day 5

Time (min)

Baseline, Day 1

1st OpraK (10mg), Day 2

4th OpraK (10mg), Day 5

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

Baseline-Day 1 1st OpraKappa-Day 2 4th OpraKappa-Day 5

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

p<0.05

Are

a U

nd

er

the

Cu

rve

, 0

-48

0 m

inu

tes

, A

CT

H

Day

p<0.005

HV (n=39)

EACD (n=23)

Baseline-Day 1 1st OpraKappa-Day 24th OpraKappa-Day 5

0

1000

2000

3000

4000

5000

6000 p<0.0005

Are

a U

nd

er

the

Cu

rve

, 0

-48

0 m

inu

tes

, c

ort

iso

l

Day

p<0.0005

HV (n=16)

EACD (n=13)

ACTH Cortisol

Reed et al, Neuropsychopharmacology, 43:739, 2017

Baseline

Day 1

1st OpraK

(10mg)

Day 2

4th OpraK

(10mg)

Day 5

Baseline

Day 1

1st OpraK

(10mg)

Day 2

4th OpraK

(10mg)

Day 5

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

Gene variants:

• Alter physiology

“PHYSIOGENETICS”

• Alter response to

medications

“PHARMACOGENETICS”

• Are associated with

specific addictions

(A118G)

(C17T)

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

Log [b Endorphin (M)]

1.0

0.5

0

-9 -8 -7 -6

A118G

Prototype

Fra

cti

on

Maxim

um

Cu

rren

t R

esp

on

se

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

Log [b Endorphin (M)]

100

80

60

40

20

0

-11 -10 -9 -8 -7

Pe

rce

nt

Bo

un

d A118G

Prototype

FUNCTIONAL MOP-r (A118G) VARIANT – “Physiogenetics” Related to A118G Variant of Human

Mu Opioid Receptor Gene – Alters Stress Responsivity in Healthy Control Volunteers

Se

rum

Co

rtis

ol (u

g/d

l)

24

22

20

18

16

14

12

10 8

50 0 50 100 150 200

Time (min)

P

I

A/A (n=29) A/G (n=7) N N

N

N

N = Naloxone P = Placebo

Wand et al., Neuropsychopharmacol, 26:106, 2002

Chong…Wand, Neuropsychopharmacology, 31:204, 2006

2500

2000

1000

500

1500

0

Cortisol

P < 0.05

Prototype A118G

40 19

Co

rtis

ol

Levels

- A

UC

(9:3

0am

-10:3

0am

+ 9

0m

in)

(no

fo

od

fo

r 9 h

ou

rs)

Bart et al. Neuropsychopharmacology,

31:2313-2317, 2006

Association Between a Functional Polymorphism (SNP) in the Mu Opioid Receptor Gene (A118G) and Opiate Addiction and Also Alcoholism in Central Sweden

Alcohol Dependent (n=389) Control (n=170)

118G Allele Frequency * 0.125

(12.5%)

0.074

(7.4%)

Odds Ratio=1.92 p=0.0074

In the entire study group in this central Swedish population:

Attributable Risk due to genotypes with a G allele: 11.1%

* Overall 118G Allele Frequency = 0.109 (10.9%)

Bart et al., Neuropsychopharmacology, 30:417, 2005

Opiate Dependent (n=139) Control (n=170)

118G Allele Frequency 0.155

(15.5%)

0.074

(7.4%)

Odds Ratio=2.86 p=0.00025

Bart et al., Molecular Psychiatry, 9:547-549, 2004

In the entire study group in this central Swedish population:

Attributable Risk due to genotypes with a G allele: 18%

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

14

12

10

8

6

4

2

0

10mg/kg

Heroin

20mg/kg

Heroin

GG (6)

AA (6)

Do

pa

min

e (

nM

)

Females 14

12

10

8

6

4

2

0

10mg/kg

Heroin

20mg/kg

Heroin

GG (6)

AA (7)

Do

pa

min

e (

nM

)

Males

Zhang, Blendy…Kreek et al., Neuropsychopharmacology, 40:1091, 2015

Zhang, Blendy…Kreek et al., Neuropsychopharm, 40:1091, 2015; unpublished data presented at CPDD 2018

1 2 3 4 5 6 7 8 9 1 0

0

1 0

2 0

3 0

4 0

0

2

4

6

8

1 0

Da

ily

He

ro

in S

A (

mg

/kg

)

A A (1 5 )

G G (1 2 )

No

se

po

ke

s (

FR

=1

, 0

.25

mg

/kg

)

1 2 3 4 5 6 7 8 9 1 0

0

1 0

2 0

3 0

4 0

0

2

4

6

8

1 0

Da

ily

He

ro

in S

A (

mg

/kg

)

A A (12)

G G (1 2 )

No

se

po

ke

s (

FR

=1

, 0

.25

mg

/kg

)

1 2 3 4 5 6 7 8 9 1 0

0

2 0

4 0

6 0

0

5

1 0

1 5

No

se

po

ke

s (

FR

=1

,0

.25

mg

/kg

)

Da

ily

Ox

yc

od

on

e S

A (

mg

/kg

)

O x y -A A (7 )

O x y -G G (6 )

1 2 3 4 5 6 7 8 9 1 0

0

2 0

4 0

6 0

0

5

1 0

1 5

No

se

po

ke

s (

FR

=1

,0

.25

mg

/kg

)

Da

ily

Ox

yc

od

on

e S

A (

mg

/kg

)

O x y -A A (8 )

O x y -G G (8 )

Males – Oxycodone Females – Oxycodone

Males – Heroin Females – Heroin

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

Day (4h/d)

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

Day (4h/d)

I II III IV

Promoter 68-bp tandem

repeats

rs1997794

3′-UTR

rs6045819

Human prodynorphin gene:

exon / intron organization, repeats, and

single nucleotide polymorphisms

rs34535593

rs35286281

rs10485703

rs910080_T/C

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

ATG

rs6035222

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)

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

*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

TGACTTA

68 bp repeat

Transcription start

CAAT TATA

box

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%

10

16%

26*

43%

25**

42%

S S + S L L L

0

2 5

5 0

7 5

1 0 0

1 2 5

F e m a le s

G e n o ty p e

Nu

mb

er o

f p

arti

cip

an

ts

2 : N S

S S + S L L L

0

2 5

5 0

7 5

1 0 0

1 2 5

M a le s

G e n o ty p e

Nu

mb

er o

f p

arti

cip

an

ts 2 = 6 .0 8 : p < 0 .0 5

" 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 )

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

African American volunteer subjects with

cannabis KMSK scores of 0–4, 5-9, and 10–14,

respectively (KMSK 10 is cut-off for dependence

for cannabis).

Yuferov, Butelman, Kreek, Neuropsychiatr Dis Treat., 14:1025, 2018

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

40

114

26 28

5

55 55

9

54 54

7

26

Shared SNPs, e.g.:

DRD2: rs1076563

rs2587546 Kreek 2018 after Levran 2015

African

Descent

European

Descent

African cluster

European cluster

European

sample

(24 genes)

African

sample

(35 genes)

12 genes

Stress Dopamine

Serotonin

GABA Opioid

Signal Transduction

Adrenergic

Cholinergic

Circadian

Rhythms

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

OPRM1 (mu opioid receptor)

OPRD1

(delta opioid receptor)

OPRK1

(kappa opioid receptor)

PDYN (dynorphin peptide)

AVPR1A (arginine vasopressin receptor 1A)

FKBP5

(FK506-binding protein 51/ corticosterone chaperone)

GAL

(galanin)

CSNK1E

(casein kinase 1, epsilon)

CRHBP

(Corticotropin Releasing Hormone Binding Protein)

OPIOID SYSTEM (selected genes) STRESS SYSTEM (selected genes)

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

Opioid Stress

Neuro-

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

ASSOCIATION WITH OPIATE ADDICTION IN

CAUCASIANS, AFRICAN AMERICANS, AND BOTH

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

Opioid Stress

Neuro-

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

ASSOCIATION WITH OPIATE ADDICTION IN

CAUCASIANS, AFRICAN AMERICANS, AND BOTH

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)

The Laboratory of the Biology of Addictive Diseases – 2019

Research Nurse

Practitioner

Kate Brown

Rachel Conybeare

Administrative Team

Kitt Lavoie

Abigail Sintim

Assistants for Research

Ariel Ben-Ezra

Jose Erazo

Michelle Morochnik

Carina Chen

Bryan Elroy

Laboratory Manager

Matthew Randesi

Laboratory Scientists

Eduardo Butelman

Yan Zhou

Orna Levran

Yong Zhang

Vadim Yuferov

Brian Reed

Postdoctoral Fellows

Kyle Windisch

Devon Collins

Guest Investigators

Miriam Adelson

Gavin Bart

Lawrence Brown

Don Des Jarlais

David Novick

Einat Peles

Ellen Unterwald

Graduate Students

Amy Dunn

Statistics & Informatics

Collaborators

Jurg Ott

Yupu Liang

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

Other Rockefeller

University Collaborators

Brian Chait

Jeff Friedman

Paul Greengard

Bruce McEwen

Don Pfaff

Sid Strickland

Tom Tuschl