PET Studies of Enzyme Activity: Monoamine oxidase and Aromatase

Outline

Monoamine oxidase (MAO) human studies modeling (difficulties) age smoking status in peripheral organs genotype and personality depression and MAO inhibitor drugs epigenetics

Aromatase (converts androgens to estrogens) human studies distribution in brain modeling (difficulties) in peripheral organs

Joanna Fowler

Anat Biegon

Monoamine Oxidase

RCH2NH2 + O2 + H2O RCHO + NH3 + H2O2

MAO

MAO inhibitor drugs are used to treat depression and Parkinson's disease.

Oxidizes neurotransmitters

Produces hydrogen peroxide

Two subtypes which are different gene products: MAO A (NE, 5HT, DA) and MAO B (PEA, DA)

DA

DA

DADA

DA

DA

signal

MAO B

MAO AMAO A

MAO B

Cellular Locations of MAO A and MAO B

Radiotracers for MAO A and B

Uptake Images (multiple time points)

Amount of tracer injected

Blood Flow

# receptor, enzyme binding sites

# receptor, enzyme binding sites

Modeling (also requires input function)

SpK1

k2

E + Sb E-Sinact

PET region of interest

k’3

Quantifying MAO in the brain: A useful model

2 Tissue irreversible model K1, k2 k3=k’3E

k’3~all processes involvedin forming E-Sinact

k’3E = f(SE , kM , kM’ ,kinact)

SE endogenous substrate (assume constant) kM Michaelis-Menten constant substrate kM’ Michaelis-Menten constant tracerKinact rate constant for inactivation

Sb brain tracer concentration Sp plasma tracer concentration

K1 plasma to tissuek2 tissue to plasma

dSb/dt = K1Sp(t) - k2Sb - k3Sb

d E-Sinact/dt = k3Sb

Use k3 or the composite term k3 as an index for MAO. = K1/k2 which does not depend upon blood flow)

Because estimates of k2 and k3 are highly correlatedwe have found k3 to be a more reliable estimate of MAO activity.

Differential equations for the 2T irreversible model

Equations are solved by numerical integration . Nonlinear least squares optimization to determine model parameter values.

The problem with irreversible ligands

If k3 is too large (k3 >> k2), tracer uptake depends only upon K1 (called flow limited)

Patlak et al

2

1

31

31

32

31

k

Kλ

λkK

λkK

kk

kKKi

Ki is an index of tracer uptake

Cp(t) is the arterial concentration of radiotracer at time tROI(t) is the radioactivity measured in a region of interest at t including both reversible and irreversibly bound tracer

)()(

')'(

)(

)( 0pe

t

VVtCp

dttCp

KitCp

tROI

Solution: reduce k3

Deuterium Isotope Effect[

A C-D bond is harder to break than a C-H bond: Comparing the H and D tracers allows to determine whether MAO is involved in the formation of the PET image.

[11C]L-Deprenyl-D2

bond broken by MAO

bond broken by MAO

[11C]L-Deprenyl-H2

Deuterium Isotope Effects and MAO Mapping

[11C]L-deprenyl

[11C]L-deprenyl-D2

[11C]L-Deprenyl-D2

[11C]L-Deprenyl

KiD2 = .12 Ki H2= .29

K1 D2 = .42 K1 H2 = .41 Time

% D

ose/

cc

Sensitivity of [11C]L-deprenyl-D2 is greater than that of [11C]L-deprenyl-H2 the rate of trapping (k3) is reduced

improving quantification

MAO B K1

[11C]L-deprenyl-D2 reveals brain MAO B increases and blood flow decreases in normal aging (n=21)

Fowler et al., 1997

Using λk3 from the 2 tissue irreversible model

Tobacco smoke inhibits human brain MAO B

non-smoker (female 48 yrs)

smoker (female 51 yrs)

L-deprenyl treatment (male, 43 yrs)M

AO B

leve

l

Fowler et al, Nature, 1995

male 86 yrs

male 43 yrs

male 27 yrs

Human brain MAO B increases with age

[11C]clorgyline Uptake in Thalamus

[11C]clorgyline was found to be superior to [11C]clorgyline-D2

% D

ose/

cc

Time min

non-smoker smoker

[11C]clorgyline (MAO A) and [11C]L-deprenyl-D2 (MAOB) images of smokers and non-smokers at the level of the thalamus

Both MAO –B and A are inhibited by tobacco smoke (B>A). Former smokers have normal MAO levels; nicotine does not inhibit MAO.

MAO A

MAO B

BNL Group, Nature, 1996; BNL group, PNAS, 1996)

Distribution of MAO A and MAO B

brainthyroidlungsheart

kidneys

[11C]CLG [11C]DEPCDH007WB1 325x09232DH

MAO A and B can be quantified in brain and in most peripheral organs

MAO A MAO B

heart-DEP

kidneys-CLGlungs-CLGheart-CLG

lungs-DEP

kidneys-DEP

0

0.005

0.01

0.015

0.02

0.025

0.03

0 10 20 30 40 50 60

CDH211

HD

% d

ose/

cc

time

Comparing H and D was also used to validate MAO A and B imaging in peripheral organs

capillary

K1

k2

k3TF

tissue (T and F)+ blood vol + metabolites? + (air in lungs)

model of lung Model for peripheral organs

k3 (MAO) α k'E

or combination parameter λk3

K1 plasma to tissuek2 tissue to plasma

0.0

0.2

0.4

0.6

0.8

1.0

1.2HD

λk3

thyroid heart lungs spleen kidneys

MAO A activity in organs as demonstrated by the Deuterium Isotope effect

Smokers have

reduced MAO A and B in brain

reduced MAO B but not A in heart, kidney

MAO A and MAO B are present in lungs (H/D effect)

but lung uptake is similar for both smokers and nonsmokers

MAO in Smokers vs Nonsmokers

MAO in Smokers vs Nonsmokers

Lung uptake averaged over subjects

0 20 40 600.000

0.005

0.010

0.015

0.020

Nonsmoker H DEP Smoker H DEP

Time (min)

0 20 40 600.000

0.005

0.010

0.015

0.020Nonsmoker H DEP Smoker H DEP

Time (min)

0 20 40 600.000

0.004

0.008

0.012 Nonsmoker D CLG Smoker D CLG

Nonsmoker H CLG Smoker H CLG

0.000

0.004

0.008

0.012

0 20 40 60

0 10 20 30 40 50 600.000

0.005

0.010

0.015

0.020

Nonsmoker D DEP Smoker D DEP

Time (min)Time (min)

Time (min)

% D

ose/

cc%

Dos

e/cc

% D

ose/

cc%

Dos

e/cc

Lung uptake for smokers and non-smokers comparing clorgyline H,D and deprenyl H,D

0

5

10

15

20

0

4

8

12

16

λλ

[11C]clorgyline MAO A [11C]Ldeprenyl (MAO B)

0.000.020.040.060.080.100.12

0.00

0.04

0.08

0.12

0.16

Nonsmokerk

3Smoker

k3

λ: S > NS = tracer is retained in tissue of smokers longer than

nonsmokers. k3: NS>S (Logan, Fowler 2005)>

CLG D-CLG D-DEPDEP

MAO A Genotype and Brain MAO A

MAO A Deletion and Aggression

• A single Dutch family with MAO A deletion is prone to

violence (Brunner et al., 1993)

• MAO A knockout mice are aggressive to an intruder in the

home cage (Cases et al., 1995)

• MAO inhibition during pregnancy in rodents

produces an aggressive phenotype (Whitaker et al.,

1994; Mejia et al., 2002)

MAO

RCH2NH2 + O2 + H2O RCHO + NH3 + H2O2x

Mechanism: Low MAO A would impair monoamine regulation in development and also in adulthood (in response to maltreatment)

The MAOA gene is located on the x chromosome. There are two common alleles in the MAOA promoter: 4-

repeat allele (High MAOA) has a five-fold higher transcriptional induction in non-neural cells than the 3-repeat allele (Low MAOA) in vitro (Sabol et al., 1998)

High and Low MAOA Genotypes in Humans

High/Low = 60/40 in malesFrom Huang et al., 2004

Low MAOA genotype is associated with an antisocial behavior whereas the high MAOA genotype appears to be protective in individuals maltreated as children (Caspi et al, 2002); replicated (Foley et al., 2004)

Antisocial behavior requires the low MAO A genotype AND childhood maltreatment

Com

pos

ite

Ind

ex o

f A

nti

soci

al B

ehav

ior

(z s

core

s)

Childhood Maltreatment None Probable Severe

1

0.75

0.5

0.25

0

-0.25

-0.5

Low MAOAActivity , n=163

High MAOAActivity , n=279

Source: Caspi, A. et al., Science 297, 2 August 2002.

This is a gene-environment interaction!

High and Low MAO A Genotypes and Aggression

• Do high and low MAO A genotypes have different levels of brain MAO A Activity?

• Is there a relationship between MAO A level and personality traits (aggression, anger etc)?

What is the mechanism?

•We measured brain MAO A and negative personality traits in 38 normal healthy volunteers (26 high and 12 low MAOA genotype) with [11C]clorgyline and PET.

•We excluded smokers due to brain MAO A inhibition by cigarette smoke.

Study Design

Could this be a developmental effect? Note the large intersubject variability in brain MAO A.

Could this be a developmental effect? Note the large intersubject variability in brain MAO A.

High(n=26)

Average k3 images

Low(n=12)

Brain MAO A level does not differ with genotype (Fowler et al., 2007)

Are brain MAO A levels related to trait aggression?

OCl

Cl

N

11CH3

H H

The protein product, MAO A, not the genotype predicts trait aggression (Alia-Klein et al., 2008)

Non-aggression-prone subjects

Aggression-prone subjects

Gene-Brain-Behavior Relationships

Nelly Alia-Klein

Variability in brain MAO A levels in healthy males.

Parametric k3 images

Cluster analysis was applied to group voxels with similar kinetics. Model parameters for the cluster to which a voxel belongs are used as the starting point. Assuming that the non-enzyme parameters are similar for the cluster only k3 needs to be determined for each voxel.

What accounts for the variable brain MAO A levels in humans?

Measure DNA methylation (regulation of gene expression) pattern in subjects in whom we also have measured brain MAO A enzyme levels with PET. Elena Shumay

N

N

NH2

N

N

NH2

CH3DNA methyltransferase

Epigenetics???

cytosine 5-Methylcytosine

Hypothesis: epigenetic mechanisms will influence gene expression and therefore MAO A activity in the brain.

Methylation state vs k3

Brain MAO A levels

MAO

A pr

omot

er m

ethy

latio

n (%

) Shumay, Logan, Volkow, Fowler (2012)

Strategy: Use DNA methylation patterns on white blood cells (WBC) as a proxy for brain DNA methylation to relate to PET measures of MAO A levels. Assume that environmental exposure will have a global impact on the epigenome.

MAO A and Depression

For many years it was thought that depression was linked to low levels of the monoamines serotonin, dopamine and norepinephrine but the mechanism for the loss was unclear.

Figure 1. Time activity curves for [11C]harmine demonstrating reversible kinetics. Time activity curves for a representative: depressed individual (closed circles) and a healthy individual (open circles) are shown.

Brain MAO A in major depressive disorder: a study with [11C]harmine (Meyer et al., 2006)

depressedHealthy

Patients with MDD have elevation MAO A

depressedhealthy

This PET study was a major milestone in characterizing the neurobiology of depression and in explaining why monoamine elevating drugs alleviate symptoms (Meyer et al., 2006); replicated in PPD.

RCH2NH2 + O2 + H2O RCHO + NH3 + H2O2

MAO

XMAO A inhibitor drugs elevate serotonin, norepinephrine, and dopamine

Serotonin: mood

Norepinephrine: arousal

Dopamine: reward

MAO A Inhibitor Drugs for Depression

The first generation of non-selective, irreversible MAO inhibitors require dietary restrictions – foods high in tyramine (metabolized by MAO-A) caused hypertensive events. Replaced by reuptake inhibitors.

New Antidepressant Drugs (Reversible

Inhibitors of MAO A RIMAs)

• What dose of CX-157 (Tyrima) is needed to inhibit >60% of brain MAO A?

• How often does it need to be given?

Planning clinical trials for the New Reversible MAO A inhibitor, Tyrima (CX157, CeNeRx Biopharma)

Fowler et al., Neuropsychopharmacology, 2010

Subjects: 15 healthy males (33.4 9.0 yrs)

Dosing with CX157:

20-80 mg (single dose) (n=12)

40 mg BID for 1 week (n=3)

PET scans at baseline and 2, 5, 8, 12 and 24 hours after dosing

Radiotracer: [11C]clorgyline

PK Samples for [CX157]: at time of PET scan

Study Protocol

Baseline

60 mg Tyrima - 2 hrs

60 mg Tyrima – 12 hrs

MAO A Activity in Human Brain and After Tyrima (CeNeRx)

Tyrima shows robust and reversible MAO A blockade

Plasma levels of CX-157 predict brain MAO A Inhibition

These PET studies have formed the basis for dosing for the Phase II studies of CX157 for efficacy in depression treatment (www.clinicaltrials.gov)

xEC

x EmaxI

50

The MAOA gene predicts happiness in women

Chen et al, (2013) Prog in Neuro-Psychopharm & Biol Psy

Association between happiness and MAOA-L in women but not in men.

Outline

Monoamine oxidase (MAO) human studies age smoking status in peripheral organs genotype and personality depression and MAO inhibitor drugs epigenetics

Aromatase (converts androgens to estrogens) human studies distribution in brain modeling difficulties in peripheral organs

Joanna Fowler

Anat Biegon

Aromatase (Estrogen synthase, CYP19A1)

HO

CH3

CH3

CH3

O

aromatase

OH OH

testosterone 17-estradiol

Anat Biegon

• Mediates sexual differentiation of the brain during development (Wu et al., Cell 139, 139: 61, 2009)

• Is elevated in brain injury (neuroprotective effects of estrogen)

• Aromatase inhibitor (AI) drugs are used to treat breast cancer

• AI’s are used by body builders to avoid the feminizing effects of testosterone

• Since they cross the BBB AI’s are useful tools for investigating brain aromatase with PET

Crystal structure: Ghosh et al., Nature 457: 219, 2009

• (S)-Vorozole is a specific and potent (Ki= 0.1nM) non-steroidal aromatase inhibitor originally developed as an antineoplastic agent.

• First labeled with carbon-11 by Lidstrom et al.(1998).• Synthesis and purification optimized by Kim et al. (2009)

Aromatase PET tracer: [11C]Vorozole

Sunny Kim

Distribution of [11C]vorozole in the human brain

Summed images from 60 to 90 min overlaid on structural MRI

B. Anterior Hypothalamus/preoptic areaC.AmygdalaD.Dorsomedial thalamusE.ThalamusF.Medulla

Metabolic Stability in human plasma

Uptake TACs in human brain

0 20 40 60 80 100

0.000

0.001

0.002

0.003

0.004

C2C1

K1

k2

k3

k4

CA

2 Tissue Compartment model (with small k4)

VT=3.16 (k4=.01 min-1) VT=5.35 (k4=.005 min-1)

Problem: VT is very sensitive to variations in k4 for regions of high uptake.

% D

ose/

cc

Time min

Methods [11C] Vorozole PET Studies

Experimental27 normal volunteer subjects (baseline) 5 subjects (baseline/block 2.5mg letrozole)90 min uptakeArterial plasma radioactivity corrected for metabolites

Modeling (region of interest) total tissue distribution volume VT

• NLL 2TC 4 parameter model. (k4 estimated from 4 highest regions combined and this value was used for these individual “high” regions). For the “low” regions k4 was allowed to vary.• Graphical analysis (GA)• MA1 (Ichise)• Tissue to plasma tracer ratio (TR)

0

2

4

6

8

10

NLLMA1GARatio

Comparison of methods for estimating VT

NLL – 2T model, MA1 and GA are graphical estimations and Ratio is the ratio of radioactivity in tissue to plasma for times from 60 to 90 min

Hyp Thl Amy Inf Put Cb

V T

Baseline/blocked (2.5 mg letrozole (AI) oral 2 hours prior)

0 20 40 60 80 100-0.001

0.000

0.001

0.002

0.003

0.004

0.005

0.006

0.007

Thalamus base 4.00Thalamus blk 1.56Cerebellum base 1.91Cerebellum blk 1.67

VT

% D

ose/

cc

Time min

Blocked

Baseline

Parametric images of NLL estimates of VT

for [11C]VOR

Images were generated using a clustering method for initial kinetic parameters. Non enzyme parameters were fixed at the cluster value so that only k3 was varied at each voxel.

Postmenopausal Woman, 54

Woman at Midcycle, 38

Aromatase Distribution in the Female Body

Baseline

Blocked

Aromatase Distribution in the Male Body

0 20 40 60 80 100

0.000

0.005

0.010

0.015

0.020

0.025

0.030

[11C]Vorozole uptake in liver

Baseline Blocked (letrozole)

% D

ose/

cc

Time min

The high liver uptake raises the possibility that [11C]vorozole might be a good tracer for imaging the liver and its blood supply.

Cluster analysis of [11C]vorozole binding in the torso

Because of its distinctive binding the liver is easily separated from other organs by cluster analysis.

Liver Blood Supply

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.40.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

Avg peak time 0.79 min

Histogram plot of times of peak radioactivity from blood voxels in the liver

The voxels contributing to the blood supply within the liver were extracted by clustering the early time points (5 sec scans for the first minute).

Time min

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.40.00

0.05

0.10

0.15

0.20

0.25

Avg peak time 0.52 min

This subject has a much greater arterial contribution

Liver Blood Signal

Time min

Subject VT K1

4757 6.3(7.0-5.0) 0.61(0.69-0.48)

4809 9.8(11.2-8.6) 0.69(0.78-0.52)

4870 13.2(14.6-11.1) 0.74(0.50-0.83)

5046 17.2(20.6-16.5) 0.66(0.75-0.52)

5094 15.6(16.5-14.1) 0.55(0.61-0.53)

VT and K1 for 12 clusters of liver uptake in 5 subjects

VT liver > VT brain

Parametric image of [11C]vorozole in liver

Brookhaven Imaging Group 2011

David Alexoff, Karen Apelskog, Helene Benveniste, Anat Biegon, E. Caparelli, Pauline Carter, Congwu Du, Richard Ferrieri, Joanna Fowler, Andrew Gifford, Rita Goldstein, Jacob Hooker, Bud Jayne, Dohyun Kim, Sunny Kim, Payton King, Nelly Klein, So Jeong Lee, Jean Logan, Lisa Muench, Alicia Reid, Colleen Shea, David Schlyer, Mike Schueller, Young Jun Seo,Elena Shumay, Peter Thanos, Dardo Tomasi, Frank Telang, Paul Vaska, Nora Volkow, Gene-Jack Wang, Donald Warner, Chris Wong, Youwen Xu, Wei Zhu.

Chemistry•organic•inorganic•nuclear•theoreticalEngineeringGeneticsMedicineNursingPharmacologyPsychologyPhysicsPlant biology

Program Support from NIH and DOE-OBER (infrastructure), SBU-BNL Partnership, pharmaceutical industry