Wade Berrettini, MD, PhD

(1)

Genetics and Pharmacogenetics in

Addictions

Wade Berrettini, MD, PhD

Karl E. Rickels Professor of Psychiatry

Director, Center for Neurobiology and

Behavior, Department of Psychiatry,

School of Medicine

University of Pennsylvania

(2)

Definitions: Addiction & Dependence

Dependence:

is defined by withdrawal and tolerance

Tolerance:

higher dose needed to achieve same effect; 2

types:

Pharmacokinetic tolerance:

Change in drug metabolism

Pharmacodynamic tolerance:

Change in cellular response

to the drug.

Withdrawal:

characteristic syndrome which appears

shortly after abrupt cessation of daily drug intake.

Addiction:

chronic, compulsive, maladaptive pattern of

drug use, despite adverse consequences due to drug use:

loss of job, family, development of medical problems,

(3)

Two Topics to be Covered



Mu opioid receptor variant predicts therapeutic

response to naltrexone in alcohol addiction

(

or what I learned at my visit to the FDA

)



Alpha3/5 nicotinic receptor subunit genes in

(4)

Naltexone Treatment of Alcohol Addiction

Naltrexone (NTX), a mu opioid receptor antagonist, is

efficacious (at 50-100 mg daily) in the treatment of ethanol addiction.

It reduces risk for relapse to heavy drinking (> 4 drinks daily for a woman, > 5 drinks daily for a man), but does not

influence abstinence.

It reduces the euphoria from ethanol, and thereby reduces the drive to drink excessively.

Because alcohol addiction is so common in our country, widespread use of naltrexone could improve public health considerably.

(5)

Studies supporting efficacy Studies not supporting efficacy

Study # Ss Notes Study # Ss Notes

Volpicelli et al 1992 70 None Oslin et al 1997 44 Older O’Malley et al 1992 97 None Kranzler et al 2000 183 None Volpicelli et al 1997 97 None Krystal et al 2001 627 VA only Kranzler et al 1998 20 Depot Lee et al 2001 (Singapore) 53 None Anton et al 1999 131 None Gastpar et al 2002 (Germ.) 171 None Chick et al 2000 (UK) 169 Adherence Kranzler et al 2004 315 Depot Monterosso et al 2001 183 None Killeen et al 2004 145 None Morris et al 2001 (Australia) 111 None Oslin et al in press 240 None Heinala et al 2001 (Finland) 121 Nonabst.

Latt et al 2002 (Australia) 107 None Ahmadi and Ahmadi 2002 (Iran) 116 None Guardia et al 2002 (Spain) 202 None Balldin 2003 118 None Kiefer et al 2003 (Germany) 160 None Kranzler et al 2003 153 None Kranzler et al 2004 315 For drinking not

relapse Anton et al 2004 270 None

Randomized Placebo Controlled NTX Trials

(6)

Mu Opioid Receptor Amino Acid Sequence

cytoplasm

Extracellular Space N40D (A118G) SNP; N is a glycosylation site

(7)

A MAJOR CNS REWARD SYSTEM

Dopamine neurons, with cell bodies in the VTA project to the

n. Acc. & the medial

prefrontal cortex.

Activation of these neurons is a key brain signal of reward.

(n. Acc.)

(ventral

(8)

(9)

OPRM1 A118G EFFECT

ON TRANSCRIPTION

(10)

OPRM1 A118G EFFECT

ON TRANSLATION

(11)

G Allele Effects Miotic Response to a mu

Opioid Receptor Agonist in vivo

0 5 10 15 20 25 30 35 40 45 AA AG GG OPRM1 GENOTYPE miosis 45 +/- 8 33 +/- 6* 24 +/- 7* •p<0.001 AA vs AG/GG Lotsch et al, 2006 (8) (3) (40) (n)

Are G allele carriers hyporesponsive to mu receptor agonists?

(12)

G Allele Carriers are Hyporesponsive to

mu Opioid Receptor Agonists

• Romberg et al. Polymorphism of mu-opioid receptor gene

(OPRM1:c.118AG) does not protect against opioid-induced

respiratory depression

despite reduced analgesic response

.

Anesthesiology 2005;102:522-30.

• Klepstad et al. The 118 A G polymorphism in the human

mu-opioid receptor gene

may increase morphine

requirements in patients with pain

caused by malignant

(13)

Wand et al, Neuropsychopharm 26:106–114, 2002

(14)

0 5 10 15 20 25 30 35 40 45 50 0.02 0.04 0.06 AA allele AG allele Se lf -r e p o rt e d S ti mu la ti o n ( S H A S )

Breath Alcohol Concentration

Alcohol effects by genotype

(15)

NTX Blunts Alcohol-Induced High in OPRM1 118G Carriers

…But not in OPRM1 AA Homozygotes (Ray & Hutchinson, 2004)

(16)

Ethnicity & A118G Allele Frequency



 Based on multiple _{Based on multiple}

studies, allele studies, allele frequencies differ frequencies differ markedly across markedly across

ethnicities for the

A118G SNP in the

mu opioid receptor

gene.

gene. It arose after It arose after the out

the out--ofof--Africa Africa migration.

migration.



 Crowley et al, 2003_{Crowley et al, 2003}



 Gelernter et al, Gelernter et al, 1999

1999



 Tan et al, 2003_{Tan et al, 2003}



 Bart et al, 2004_{Bart et al, 2004}

African

1%

Koreans

31%

African

-

-American

American

3%

Chinese

35%

Swedish

17%

Malaysian

45%

European

-

-origin US

origin US

15%

Indian

47%

ETHNICITY f(G) ETHNICITY f(G)

(17)

Adapted from Cavalli-Sforza et al, 2003

Human

migration

(18)

SUMMARY



The A118G mis

_{The A118G mis}

-

sense SNP changes asparagine to

aspartate, with resultant loss of an N

-

glycosylation

site, and with

functional consequences.



The G allele results in a marked decrease in OPRM1

_{The G allele results in a marked decrease in OPRM1}

transcription and translation

ex vivo

and

in vitro.



G allele carriers are

_{G allele carriers are}

hypo

-

responsive to mu opioid

receptor agonists and

hyper

-

responsive to antagonists

in vivo.



The A118G SNP shows marked ethnicity

_{The A118G SNP shows marked ethnicity}

-

variations in allele frequency.

(19)

Does A118G genotype influence

treatment response to naltrexone

among alcohol addicted patients?

(20)

Naltrexone (NTX) in Clinical

Trials for Alcoholism

Patients were selected from 3 NTX multiple double-blind, placebo-controlled trials. These trials were 12 weeks, and include

self-report measures of drinking along with biochemical verification (liver enzymes and carbohydrate deficient transferrin). Primary endpoint was relapse to heavy drinking.

Medication was given in blister packs and compliance was determined by pill counts. Participants had twice weekly abstinence counseling sessions.

Only those persons completing the trial, with outcome data,

(21)

OPRM1 VARIANTS IN NALTREXONE

Rx FOR ALCOHOL DEPENDENCE

Naltrexone / Asp40 Allele (A/G, G/G), N = 23*

Naltrexone Asn40 Allele (A/A), N = 48

Placebo / Asp40 Allele (A/G, G/G), N = 41 Placebo / Asn40 Allele (A/A), N = 18 Cumulative Survival (time to relapse) Days Oslin et al 2003 *p = 0.04 vs NTX Asn40 odds ratio = 3.4

(22)

Fig 6: 118G Allele Predicts Good NTX Response

A/A G/A, G/G % G O O D O U T C O M E NTX PLA P = 0.005, genotype x medication interaction odds ratio = 5.8, A/A vs. G/* The COMBINE study was a multi-site trial of NTX, acamprosate or both drugs, +/- addiction counseling, for alcohol addiction. The NTX & placebo groups were

compared by

A118G genotype for outcome after 14

weeks of treatment.

NTX

(23)

Questions for the FDA

 What types of trials (retrospective and prospective, double-blind,

placebo-controlled, etc.) would be required for the FDA to modify the current naltrexone indication to a genotype-specific indication for

alcoholism?

 ANS: 2 prospective placebo-controlled trials in which persons with the ‘response’ allele, treated with naltrexone, have outcomes statistically

superior to persons without the ‘response’ allele, treated with naltrexone. Further, in both trials, placebo-treated groups do not differentiate from the naltrexone-treated group without the ‘response’ allele. After one such trial, given the two retrospective studies, labeling could be changed to indicate the advantage of naltrexone pharmacotherapy in persons with the ‘response’ allele.

 Would such an indication lead to the genetic test itself being FDA approved as a diagnostic test?

 ANS: Yes

 Are there separate requirements for approval of different formulations of the medication - depot vs oral naltrexone?

(24)

Planned Study Design Features for an FDA

Pharmacogenetic Registration Trial of

Naltrexone in Alcohol Addiction



150 G allele subjects (190 A/A)



Placebo controlled



Randomization stratified by gender and genotype

(compare A/A vs A/G + G/G)



Measures of stimulation / high and craving



12 weeks, relapse to heavy drinking is the primary

endpoint



Medication Management as psychosocial platform

(25)

SUMMARY



For a genotype-specific indication, the FDA may

require two independent studies, both of which are

placebo-controlled, double-blind, with randomization

stratified by genotype.



Both studies must show:



1) that outcome for persons with the favorable allele,

randomized to the drug, differentiates from persons

with the favorable allele randomized to placebo; and



2) outcomes for persons without the favorable allele,

randomized to the drug, do not differentiate from

persons without the favorable allele randomized to

placebo.

(26)

Two Topics to be Covered



Mu opioid receptor variant predicts therapeutic

response to naltrexone in alcohol addiction



Alpha3/5 nicotinic receptor subunit genes in

(27)

Public Health Perspectives



If current trends continue, the annual

number of deaths (world-wide) from

tobacco-related diseases will double

from 5 million in the year 2000 to 10

million in 2020 (

WHO, http://www.who.int/whosis

).



In the US, ~ 13% of adults are ND.



_{Smoking is the largest preventable}

cause of morbidity and mortality in

the US, with obesity being the second

largest.

(28)

Patch Efficacy

Effectiveness of Patch therapy (vs. Placebo)

No. of studies No. of participants Odds Ratio ( 95% CI)

37 16228 1.84 [1.65, 2.06]

(29)

Bupropion

300mg used for 9 weeks Mechanism may involve blockade of DA/NE reuptake Effective for only a

subset of smokers

Effectiveness of Bupropion (vs. Placebo)

No. of studies No. of participants Odds Ratio (95% CI)

21 7171 1.99 [1.73, 2.30]

(30)

Varenicline

 nicotinic receptor partial agonist

 Eliminates reward from smoking and craving and withdrawal  AEs are comparable to placebo (nausea is biggest concern)

17.1 33 28.6 37.3 48 0 10 20 30 40 50 60 Placebo Bupropion 0.3mg Once/day 1mg Once/day 1mg Twice/day Onckene et al., 2005 4-week Continuous Abstinence %

(31)

**Drug Dependence* is**

>

3 of These

 1. Tolerance (e.g., the need to take more drug to achieve the desired effect)

 2. Withdrawal: Daily use for at least several weeks; for nicotine, following abrupt cessation, four or more of the following signs:

• (A) dysphoric or depressed mood • (B) insomnia

• (C) irritability, difficulty managing anger • (D) anxiety

• (E) difficulty in concentration • (F) restlessness

• (G) decreased heart rate

• (H) increased appetite or weight gain

 3. Repeated unsuccessful attempts to quit or reduce using

 4. Continued drug use despite medical or psychological harm

 5. Drug use is often greater than intended or longer than intended.

 6. Reduction or elimination of social, recreational or occupational activities because of drug use

 7. Much time is spent obtaining and using the drug, & recovering from use.

For nicotine dependence (ND) #6 & #7 are not very relevant. Thus, DSM criteria for drug dependence are not ideal for ND.

(32)

(33)

(34)

NachRs Influence Transmitter Release

NachRs modulate

neurotransmitter release via pre-synaptic receptors.

Nicotine’s ability to modulate release of

dopamine may be key to its addictive quality.

Post-synaptic NachRs

modulate the response of the post-synaptic neuron.

NachR

dopamine molecules

dopamine receptors

(35)

Electrophysiology of Nicotine Reward

• Nicotine increases release of dopamine in the n.

Accumbens and medial prefrontal cortex.

• Dopamine is released at low levels tonically in

the n. Accumbens, but at high levels during

phasic (burst) firing

.

• The difference (delta) of dopamine released

between tonic and

phasic

modes is thought to be

a signature of reward.

• Electrophysiologic studies show that nicotine

increases dopamine release during

phasic

, as

opposed to tonic, firing.

• This may underlie the rewarding effect of

nicotine.

• Rice & Cragg, Nature Neurosci 7:583, 2004

(36)

Nicotine Increases Dopamine Release

During Phasic Firing in n. Accumbens

Rice & Cragg, Nature Neurosci 7:583, 2004 D O P A M I N E

At low frequency stimulation of VTA dopaminergic neurons (eg, tonic

activity), nicotine decreases the amount of dopamine released. At high

frequency stimulation (eg, phasic activity), nicotine increases the amount

(37)

Nicotine Increases Dopamine Release

During Phasic Firing in n. Accumbens

Rice & Cragg, Nature Neurosci 7:583, 2004

Nicotine increases the phasic

(burst firing) dopamine release

from accumbal slices and inhibits

the tonic firing dopamine release,

thereby creating a larger

difference (delta) between basal

synaptic dopamine levels and

phasic firing synaptic dopamine

levels. The magnitude of this delta

may be an electrophysiologic

measure of the rewarding valence

of a drug.

(38)

Single Nucleotide Polymorphisms (SNPs)

The most common variation in DNA is the

SNP. An example of a SNP is shown below.

At a hypothetical point in the human genome

(the fifth base pair in the sequence), humans

can have a

T:A

base pair or a

C:G

base pair .

ALLELE 1

ALLELE 2

CGAT

T

GCACC CGAT

C

GCACC

GCTA

A

CGTGG GCTA

G

CGTGG

SNPs are the variants which increase risk for common

diseases. A common SNP occurs

every 1000 base pairs

(39)

The Human Genome Project

Among the ~3 million common SNPs, which are functional?

(40)

ADVANCES in SNP TECHNOLOGY

These 1,000,000 SNP CHIPs provide on average 1 SNP every ~

4000 base pairs across the genome, allowing study of most

chromosomal regions, with assessment of

copy number variations (

insertions, deletions, duplications) and

(41)

Genome-Wide Association Study

(GWAS) for Cigarettes per Day

• GWAS of common diseases (eg, asthma, hypertension)

have been done (or are in process) at GSK to find new

targets for medication development.

• Some of these studies collected crude smoking data,

including the answer to the question: If you ever smoked

daily, what was the greatest number of cigarettes per day

(CPD) you smoked for at least a month?

• This CPD variable is related to a DSM-IV concept of

nicotine dependence (ND) in a complex manner, but

virtually everyone who ever smoked > 25 CPD would

satisfy criteria for ND.

(42)

NESARC: National Epidemiologic Survey on Alcohol and Related Conditions

 Survey methods

 Interviews conducted 2001-02

 Conducted by National Institute on Alcohol Abuse and Alcoholism (NIAAA)

 Data were collected in personal interviews conducted in respondents’ homes

 One sample adult 18 or over was selected randomly for interview in each household

 Population

 Represents the civilian, non-institutionalized adult population of US

– Including DC, Alaska and Hawaii

 African Americans, Hispanics and adults aged 18-24 years over-sampled

 Overall response rate 81% (n=43,093)

 Smoking measures

 Nicotine Dependence using DSM-IV criteria

 Number of Cigarettes smoked – usual quantity currently smoked or when you did smoke

(43)

Sensitivity and Specificity for Usual Quantity Smoked by Lifetime History of Nicotine Dependence among Smokers (n=18,930)

Cutoff Sensitivity Specificity

1 0.97 0.0809 5 0.8855 0.308 10 0.7877 0.4307 15 0.603 0.3936 20 0.5373 0.6499 25 0.1813 0.8933 30 0.1676 0.899 35 0.1013 0.9367 40 0.0984 0.9378 45 0.0282 0.98 50 0.0276 0.9804 55 0.0197 0.9846 60 0.0194 0.9847 70 0.0046 0.9963

~ 65% specificity for persons

habitually smoking >20 CPD for criteria for DSM-IV ND.

This suggests that the concept of DSM-IV ND might require

revision, so that a greater fraction of people who habitually smoke > 20 CPD would meet criteria. When the CPD value is > 25, specificity improves to 90% for DSM-IV criteria for ND.

(44)

GEMS and Lausanne Genes Correlated with CPD SNP ChromoStartPos Gene GEMS p Laus p pooled p RS6495308 15 76694711 CHRNA3 0.008723 0.000601 6.90E-05 RS7804771 7 1.37E+08 DGKI 0.007254 0.001059 9.81E-05 RS2645339 5 1.78E+08 GRM6 0.000916 0.02548 0.000272 RS5522 4 1.5E+08 NR3C2 0.000589 0.001751 1.52E-05 RS5525 4 1.5E+08 NR3C2 0.01019 0.000269 3.78E-05 RS10869409 9 76313209 RORB 0.01129 0.000365 5.53E-05 RS7846903 9 76304931 RORB 0.009396 0.001039 0.000122 RS13293006 9 76326716 RORB 0.02351 0.002327 0.000592 RS7873840 9 76340109 RORB 0.01027 0.01714 0.001698 RS4932598 15 90338849 SLCO3A1 0.0249 0.000639 0.000192 RS4932597 15 90338621 SLCO3A1 0.03262 0.000637 0.000245 RS12439738 15 90336555 SLCO3A1 0.00379 0.01282 0.000531 RS12439765 15 90336606 SLCO3A1 0.004227 0.01374 0.000625

GEMS (2000 people) and Lausanne (6000 people) are two studies of

cardiovascular disease risk, in which CPD was treated as a quantitative variable. CHRNA3 is the alpha 3 subunit of the nicotinic receptor,

(45)

Comparison of GEMS/Lausanne (Using CPD as a Quantitative Trait) and HITDIP Analysis in a Case (> 25 CPD) Control (< 5 CPD) Mode

GENE GEMS/Lausanne*

combined p Value

eHITDIP^ p Value

CHRNA3 (alpha 3 subunit of nicotinic receptor) 0.000069 (rs6495308) 0.0000026 (rs1317286) GSK3B (glycogen synthase

kinase 3, beta subunit):

P=0.008 p = 0.004

ADAMTS1: ADAM metallopeptidase with thrombospondin type 1 motif, 1

3 SNPs 0.0005<p<0.005

3 SNPs** 0.006<p<0.034 ABCC11: ATP-binding cassette, sub-

family C (CFTR/MRP), member 11

p = 0.012 P = 0.008

*GEMS: ~1000 dyslipidemia cases + ~ 1000 controls; Lausanne: ~5600 persons ^HITDIP: ~1740 cases and ~6200 controls

**No overlapping SNPs

CHRNA3 significant in a candidate gene study (Saccone et al, HMG 16:34, 2007) of ~1000 ND cases and ~ 1000 controls (p = 0.0003, same SNP not tested)

(46)

CHRNA3 Gene Structure & SNPs in LD with CPD Rs578776: 76,675,454 Risk allele = C F(G) = 0.72 Saccone et al, 2007 P = 0.0003 Rs1051730: 76,681,392 Risk allele = T F(A) = 0.32 Saccone et al, 2007 P = 0.001 RS1317286: 76683184 eHITDIP Risk allele = G F(C) = 0.33 P = 0.0000026 RS6495308: 76694711 GEMS/Lausanne Risk allele = T F(T) = 0.77 P = 0.000069 Berrettini et al, 2008

(47)

Saccone, et al, 2007, P=0.0003 Thorgeirsson et al, 2008, P=10-20

Berrettini et al, 2008 (P=0.000003

Berrettini et al, 2008 (p = 0.00007)

Risk alleles are boxed and all are found on a common

haplotype (frequency = 38%)

Bierut et al, 2008, P = 0.007; Chen et al, in press

P=0.003 and 0.007; Weiss et al, 2008 p = 0.0009 Saccone et al, 2007 P=0.003 CHRNA5 CHRNB4 CHRNA3

(48)

Prominent Neuronal NachR Subtypes

Alpha5 subunits cannot

form functioning receptors alone or in combination with only one other subtype. Alpha 5 subunits

do not participate in forming the

acetylcholine binding site.

(49)

5 mRNA in Human Brain (GeneLogic)

(50)

3 mRNA in Human Brain (GeneLogic)

(51)

CHRNA5 GENE STRUCTURE: Mis-sense SNP in Exon 5

A/G (N/D) SNP with A as the minor allele (~35% frequency in individuals of European origin). This SNP occurs in a region of high conservation, and may be functional. The ‘A’ allele increases risk for ND.

(52)

Biol Psychiatry, 2008 Mol Psychiatry, 2008

(53)

CHRNA5 SNP rs588765 Affects mRNA Transcription

Wang et al, Mol Psychiatry, 2008

(54)

CHRNA3/5 Alleles Increase Lung CA Risk (Hung et al, Nature, 2008)

This was found even though cases and controls were closely matched for smoking history!

(55)

CHRNA3/5 Alleles Increase Risk for Lung CA # 2

Amos et al,

Nat Genetics, 2008

CHRNA5 CHRNA3

This was found even though cases and controls were closely matched for smoking history!