cyp2d activity in the brain acts independently from …cyp2d activity in the brain acts...

CYP2D activity in the brain acts independently from

CYP2D in the liver to alter CNS drug levels and response

Rachel Tyndale and Sharon Miksys, CAMH and University of Toronto

Drug metabolism in the Liver

Main organ for pharmacokinetics:

alters drug levels and treatment response

Drug metabolism in the Brain:

CNS drug response and neurotoxicity

Disclosure

Rachel Tyndale has consulted for Apotex on material unrelated to the content of this presentation

Sharon Miksys has no disclosures

Tyndale LaboratorySharon MiksysEwa HoffmannQian ZhouBin ZhoaMaria NovalenFariba BaghaiAman MannJibran KhokharKristine GarciaMeghan Chenoweth Kaidi ZhouAndy ZhuDoug McMillianJulie-Anne TannerTaraneh TaghaviEdgor Cole TolledoAnnie PengMarlaina StuccoAhmed ElborieNicole Arguelles

Funding FromCentre for Addiction and Mental HealthCIHR: MOP-97751; TMH-109787, SCN 9/4256, TMD 132557, MOP-136937 Chair in Addictions, Canada Research Chair in PharmacogenomicsNIH PA CA148110; NIH DA 031815, NIDA DA029160NIH PNAT PGRN Pharmacogenetics Network NIDA: DA 020830Consulted for a Pharmaceutical company during the past 3 years

Acknowledgements for CNS work

CNS CollaboratorsDeborah Mash,

Roberta Palmour,

AD Le,

Megan Shram,

Derek van der Kooy,

Imad Damaj,

FJ Gonzalez,

M Quik,

C Omiecinski,

A Dagher

CYPs may metabolize drugs and neurotoxins

locally in the brain

Brain CYPs are active in vivo

In some cell types, levels are as high as in heptocytes

Response to CNS-acting drugs does not always correlate

with drug plasma levels

Miksys & Tyndale, 2009; Miksys et al., 2000; Michels & Marzuk, 1993

Local metabolism by

brain CYPsDrug response

Local drug and

metabolite levels

CYP enzymes in the Brain: Understanding variation in drug and toxin response

Toxin InactivationMPP+ and CYP2D

Parkinson’s Disease

Haloperidol and CYP2D

Tardiff dyskinesia

Drug InactivationPropofol and CYP2B

Sedation

Nicotine and CYP2B

Self-Administration

Toxin ActivationChlorpyrifos and CYP2B

Pesticide Neurotoxicity

Drug ActivationCodeine and CYP2D

Analgesia and Abuse Liability

Substrates of Polymorphic CYP2D6Including clinical and non-clinical drugs,

toxins and endogenous compounds

Opioids (e.g.) Codeine Hydrocodone

Oxycodone

Ethylmorphine

Thebaine

Dextromethorphan*

SSRIs (e.g.)

Fluoxetine

Antidepressants (e.g.)

Fluoxetine

Desipramine

Amitriptyline

Nortriptyline

Imipramine

Clomipramine

Misc.

Methoxyphenamine

Methylphenydate

Phencyclidine

Toxins MPTP (demethylation, detoxification)

TIQ (hydroxylation, detoxification,

less TIQ with chronic nicotine/smoke

Carbolines / 5-Methoxyindolethylamine

(demethylation, detoxification)

Harmaline

Harmine

Pinoline

5-methoxy-N,N-dimethyltryptamine

Pesticides

parathion, diazinon, chlorpyrifos

Endogenous / Neurotransmitters

Tyramine to Dopamine

Octamine to Norepinephrine

Synephrine to Epineprine

5-Methoxytryptamine to serotonin

21 OH’n of Progesterone

21 OH’n of Allopregnanalone

Neuroleptics (e.g.)

Perhenazine

Thioridazine

Zuclopenthixol

Antiarrhythmics (e.g.)

Propafenone

Sparteine*

Flecainide

n-Propylajmaline

Beta Blockers (e.g.)

Metoprolol*

Timolol

Amphetamines (e.g.)

p-Methoxy-Amphetamine

Methamphetamine

Amphetamine

Methylene-dioxy-

methamphetamine

(MDMA, ecstasy)

Mouse brain CYP2DConnie Chung , Frank Gonzalez, Sharon Miksys, Rachel Tyndale

I

IV

V

II

III

HippocampusFrontal Cortex

200 m20 m

CYP2D6 in Human Brain(Immunohistochemical Studies)

No 1o Antibody

50 µm

Hippocampus

P

Frontal Cortex

Miksys et al., J Neurochem 2002

CYP2D6*4 splice site variant results in

human brains without CYP2D6 protein

0

1

2

3

4

5

FC TC CG OC HC EC CD PT NA GP SN CV CH

Miksys et al., J Neurochem 2002

13 Human Brain Regions

Cerebellum

*4/*4 *1/*4 *1/*1

2D6 Liver

*1/*4 *1/*1 *4/*4

De

nsity o

f C

YP

2D

6 (

S.D

.)

Extensive metabolizers N=13

Poor metabolizers (*4/*4)

CYP2D6 genetic fast metabolizers

differ from slow metabolizers

23

24

25

26

27

52

53

54

55

56

57

58 Other distinguishing features:

Portion of Type A vs B

Psychasthenia

Inhibition of Aggression

Novelty seeking

Harm avoidance

Fear of Uncertainty

Shyness

Fast FastSlow Slow

Psychic Anxiety Socialization

Bertilsson et al., 1989; Llerena et al., 1989; Llerana et al., 1993; Roberts et al., 2004; Gan et al., 2004

Healthy individuals (N=188): CBP may reflect an ongoing biological process regulating the reactivity of

the individual to emotional stimuli and the detection of signals evoking fear.

Kirchheinner et al. Molecular Psychiatry (2011) 16, 333–341

CYP2D6 genotype effects on

resting cerebral brain perfusion

Cere

bra

l B

loo

d flo

w





Tardiff dyskinesia


Sedation

Nicotine and CYP2B

Self-Administration





Codeine is activated to Morphine by

CYP2D6 Morphine (MOR) has greater affinity for μ-opioid receptors

COD analgesia comes from conversion to MOR

CYP2D6 poor metabolizers: no analgesia

Pharmacological Inhibitors: decrease analgesia

Adler et al., 1955; Pert et al., 1973; Chen et al., 1991; Sindrup et al., 1991; Sindrup et al., 1992

COD MORCYP2D6

O-demethylation

COD MOR

CYP2D6

Codeine enters brain

faster than morphine

Oldendorf et al., 1972; Bouw et al., 2000

Codeine is metabolized mainly by the liver, morphine then crosses into the brain

Codeine Morphine Morphine less lipophilic than codeine, less permeable across BBB

Brain uptake of morphine slower than codeine

Morphine actively transported out of brain

CYP activity in the brain:Mechanism-Based Inhibitors (suicide inhibitors)

CYP

Substrate (Mechanism based inhibitor)

Metabolite, covalently bound to the enzyme

Inhibition of brain CYP2D

Propranolol (PRL): mechanism-based inhibitor of CYP2D

− Reactive metabolite binds covalently to enzyme

− Irreversible loss of enzyme function, requiring synthesis of new enzyme

before activity is restored

Masubuchi et al., 1994

ACSF

COD MORCYP2D

analgesia

Zhou et al, 2013

Propranolol

COD MOR

PRL

CYP2D

PRL

analgesia

Tail-flick test:

rat model of nociception

Tail-flick latency, TFL: time from start of heat exposure to withdrawal of tail

Lengthening of TFL is interpreted as analgesia

Le Bars et al., 2001

Radiant heat source

Timer

Morphine

(10-15%)

Morphine

P-glycoprotein

µ-OR

CYP2D6 and Codeine

BBBCYP2D6 activates Codeine

Morphine has greater affinity for µ-OR

CYP2D6 poor metabolizers: no analgesia

CYP2D6 Inhibitors: less analgesia

Hepatic

CYP2D

Codeine

Adler et al. 1955; Pert et al. 1973; Chen et al. 1991; Sindrup et al. 1990,

1992; Oldendorf et al. 1972; Bouw et al. 2000

Doug McMillian

Morphine

(10-15%)

P-glycoprotein

CYP2D6 and Codeine

BBB

Hepatic

CYP2D

Codeine Morphine

Brain

CYP2D

Codeine

Adler et al. 1955; Pert et al. 1973; Chen et al. 1991; Sindrup et al. 1990,


µ-OR

Morphine

(10-15%)

P-glycoproteinAdler et al. 1955; Pert et al. 1973; Chen et al. 1991; Sindrup et al. 1990,


CYP2D6 and Codeine

BBB

Hepatic

CYP2D

Codeine Morphine

Brain

CYP2D

µ-OR

CodeinePrimary Objective

Examine the role of rat brain CYP2D in the

metabolic activation and resulting analgesia

of peripherally administered codeine

Study Design

BaselineOpioid Drug

AdministrationAnalgesia

TestingSacrifice

Analgesia- Baseline Tail-Flick Latency

PretreatmentCodeine

(20 mg/kg in saline)

Morphine (3.5 mg/kg in dH2O)

Analgesia- Tail-Flick Reflex

Drug Levels- Plasma

- Saphenous Vein

- Whole Brain

- Sacrifices

Washout (One Week)

IP

Wistar

ICV

SC

Propranolol (Inhibitor)(20 µg in 4 µl cyclodextrin)

Nicotine (Inducer)(7 x 1 mg/kg in saline)

0 30 6015

Time (Minutes)

Morphine


BBB

Hepatic

CYP2D

Codeine Morphine

Brain

CYP2D

Codeine

4-OH-PLPropranolol

ICV

Zhou et al. 2013

mu-OR

(Codeine tested 24 hr after propranolol)

Morphine


BBB

Hepatic

CYP2D

Codeine

Brain

CYP2D

Codeine

Zhou et al. 2013

mu-OR


Morphine

Morphine


BBB

Hepatic

CYP2D

Codeine

Brain

CYP2D

Codeine

Zhou et al. 2013

mu-OR


Morphine

0

2

4

6

8

10

Pre Post (24 h)

Ta

il F

lic

k L

ate

nc

y (

s, m

ea

n

+ S

EM

)

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

15 min

Pla

sm

a M

orp

hin

e (

fold

ch

an

ge v

s.

ve

hic

le)

Propranolol inhibition of brain CYP2D

decreases codeine-induced analgesia

**

0

10

20

30

40

50

60

70

80

90

100

0 15 30 45 60

An

alg

es

ia (

%M

PE

, m

ea

n +

SE

M)

Time after codeine injection (min)

*

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

0-30 minA

UC

(fo

ld c

ha

ng

e v

s.

ve

hic

le)

Analgesia

Within animal (n=12)

Plasma

Morphine

0

500

1000

1500

2000

2500

0 1000 2000 3000

Pla

sm

a c

od

ein

e (

ng

/ml)

Brain codeine (ng/g)

0

10

20

30

40

50

60

70

0 50 100 150P

lasm

a m

orp

hin

e (

ng

/ml)

Brain morphine (ng/g)

Codeine, but not morphine, levels at 15 min after a codeine injection

correlate between peripheral (plasma) and central (brain) compartments

r=0.60p<0.001

r=0.14p=0.26

Analgesia correlated with brain, not plasma,

morphine levels at 15 min after codeine-injection

0

20

40

60

80

100

0 20 40 60 80C

od

ein

e A

nalg

esia

(%

MP

E)

Morphine (ng/g)

r=0.41p<0.02

Brain

0

20

40

60

80

100

0 50 100 150

Co

dein

e A

nalg

esia

(%

MP

E)

Morphine (ng/ml)

r=0.01p=0.48

Plasma

0

10

20

30

40

50

60

70

80

90

100

0 10 20 30 40 50 60

An

alg

esia

(%

MP

E,

mean

+ S

EM

)

Time after Morphine injection (min)

Vehicle

Propranolol

Propranolol pretreatment did not effect

morphine-induced analgesia

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

0-60 min

AU

C (

fold

ch

an

ge v

s.

veh

icle

)

Between animal (n=6/group)

CYP2D6 levels are higher in

some brain regions of smokers

0123456789

10

FC TC CG OC HC EC CD PT NA GP SN Cv Ch

Bra

in C

YP

2D

6(S

mo

kers

/ N

on

-Sm

okers

)

*

** **

***

Smokers (n=10); Non-smokers (n=3)

Mann et al. 2008

No difference in hepatic CYP2D levels between

smokers and non-smokers

Nicotine induces CYP2D6 in Monkeys (Nicotine 0.3 mg/kg s.c. bid, 21 days)

CY

P2D

6 in b

rain

regio

ns

Frontal Cortex Western Blot

Saline (N=6) Nicotine (N=6)

Substantia

nigra

Putamen Cerebellum

1

X 2.7p=0.04 X 2.4

p=0.01

0Frontal

cortex

2

3

X 1.7p=0.001 X 1.6

p=0.006

Male African Green Monkey

(Cercopithecus aethiops)

Morphine

Induction of brain CYP2D

BBB

Hepatic

CYP2D

Codeine Codeine

SCYue et al. 2008

mu-OR

(Codeine tested 8 hr after chronic nicotine)

Nicotine

Brain

CYP2D

Morphine

Morphine


BBB

Hepatic

CYP2D

Codeine Morphine

Brain

CYP2D

Codeine

SCYue et al. 2008

mu-OR


Nicotine

Morphine


BBB

Hepatic

CYP2D

Codeine Morphine

Brain

CYP2D

Codeine

SCYue et al. 2008

mu-OR


Nicotine

0

2

4

6

8

10

Pre Post (8 h)

Tail

Fli

ck L

ate

ncy (

s,

me

an

+ S

EM

)Day 1

Day 7

Morphine


BBB

Hepatic

CYP2D

Codeine Morphine

Brain

CYP2D

Codeine

SCYue et al. 2008

mu-OR


Nicotine

0

50

100

150

200

250

0 2 4 6 8

Pla

sm

a level

(ng

/ml,

me

an

+ S

EM

)

Time after nicotine injection (h)

0

2

4

6

8

10

Pre Post (8 h)

Tail F

lick

Late

ncy

(s,

mean

+ S

EM

)Day 1

Day 7

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

15 min

Pla

sm

a M

orp

hin

e (

fold

ch

an

ge v

s.

veh

icle

)

Nicotine induction of brain CYP2D

increases codeine-induced analgesia

**

0

10

20

30

40

50

60

70

80

90

100

0 15 30 45 60

An

alg

es

ia (

%M

PE

, m

ea

n +

SE

M)


*

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

0-30 minA

UC

(fo

ld c

ha

ng

e v

s.

ve

hic

le)


Plasma

Morphine

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

15 min

Pla

sm

a M

orp

hin

e (

fold

ch

an

ge v

s.

veh

icle

)



**

**

*

0

10

20

30

40

50

60

70

80

90

100

0 15 30 45 60

An

alg

es

ia (

%M

PE

, m

ea

n +

SE

M)


***

*

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

0-30 minA

UC

(fo

ld c

ha

ng

e v

s.

ve

hic

le)


Plasma

Morphine

Nicotine pretreatment did not effect

morphine-induced analgesia

0

10

20

30

40

50

60

70

80

90

100

0 10 20 30 40 50 60

An

alg

esia

(%

MP

E,

mean

+ S

EM

)

Time after Morphine injection (min)

Vehicle

Nicotine


0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

0-60 min

AU

C (

fold

ch

an

ge v

s.

veh

icle

)

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

15 min

Pla

sm

a M

orp

hin

e (

fold

ch

an

ge v

s.

veh

icle

)



**

**

*

0

10

20

30

40

50

60

70

80

90

100

0 15 30 45 60

An

alg

es

ia (

%M

PE

, m

ea

n +

SE

M)


***

*

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

0-30 minA

UC

(fo

ld c

ha

ng

e v

s.

ve

hic

le)


Plasma

Morphine

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

15 min

Pla

sm

a M

orp

hin

e (

fold

ch

an

ge v

s.

veh

icle

)

Effect of nicotine (brain CYP2D inducer) on

codeine-induced analgesia was reversible

**####

##

**

* #

0

10

20

30

40

50

60

70

80

90

100

0 15 30 45 60

An

alg

es

ia (

%M

PE

, m

ea

n +

SE

M)


***

##

#

*

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

0-30 minA

UC

(fo

ld c

ha

ng

e v

s.

ve

hic

le)


Plasma

Morphine

Brain CYP2D contributes to codeine-induced analgesia

at early time points

Brain CYP2D plays a significant role in the metabolism and initialanalgesia from peripheral codeine

Despite first-pass metabolism (intraperitoneal administration)

Inhibiting and/or inducing brain CYP2D did not effect:

Baseline tail-flick latencies (nociception)

Plasma morphine levels after codeine-injection

Morphine-induced analgesia (antinociception)

Brain CYP2D activity

Brain metabolism

of codeine to morphine

Brain morphine

levels

Codeine analgesia





Tardiff dyskinesia


Sedation

Nicotine and CYP2B

Self-Administration





Qian Zhou Sharon Miksys Bin Zhao Fariba Baghai Ewa Hoffmann

Andy Zhu, Meghan Chenoweth, Evelyn Craig, Taraneh Taghavi,

Ning Chen Francesca Toselli, Douglas McMillan, Julie-Anne Tanner

Catherine Wassenaar, Kristine Garcia

Maria NovalenKaidi Zhou

Jibran Khokhar

Charmaine Ferguson

Rebecca Miller

Amandeep Mann

cyp2d activity in the brain acts independently from …cyp2d activity in the brain acts...

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