Pet studies of enzyme activity monoamine oxidase and aromatase
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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

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Outline
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


MAO

RCH2NH2 + O2 + H2O RCHO + NH3 + H2O2

Monoamine Oxidase

  • Oxidizes neurotransmitters

  • Produces hydrogen peroxide

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

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


Cellular Locations of MAO A and MAO B

MAO A

MAO A

DA

DA

MAO B

DA

MAO B

DA

DA

DA

signal



Uptake Images (multiple time points)

Amount of tracer injected

Blood Flow

# receptor, enzyme binding sites

Modeling (also requires input function)

# receptor, enzyme binding sites


Quantifying MAO in the brain: A useful model

K1

k’3

k’3~all processes involvedin forming E-Sinact

Sp

E+ Sb

E-Sinact

k2

PET region of interest

K1plasma to tissue

k2 tissue to plasma

Sb brain tracer concentration

Sp plasma tracer concentration

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

k’3E = f(SE , kM , kM’ ,kinact)

SE endogenous substrate (assume constant)

kM Michaelis-Menten constant substrate kM’ Michaelis-Menten constant tracer

Kinact rate constant for inactivation


Differential equations for the 2T irreversible model

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

d E-Sinact/dt = k3Sb

Use k3 or the composite term lk3 as an index for MAO.

(l = K1/k2 which does not depend upon blood flow)

Because estimates of k2and k3 are highly correlated

we have found lk3 to be a more reliable estimate of MAO activity.

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

Cp(t) is the arterial concentration of radiotracer at time t

ROI(t) is the radioactivity measured in a region of interest at t including both reversible and irreversibly bound tracer

Kiis an index of tracer uptake

Solution: reduce k3


Deuterium Isotope Effect

[

bond broken by MAO

[11C]L-Deprenyl-H2

bond broken by MAO

[11C]L-Deprenyl-D2

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.


Deuterium Isotope Effects and MAO Mapping

[11C]L-deprenyl

[11C]L-deprenyl-D2


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

[11C]L-Deprenyl

KiD2= .12 Ki H2= .29

% Dose/cc

[11C]L-Deprenyl-D2

K1D2 = .42 K1H2 = .41

Time


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

K1

MAO B

Using λk3from the 2 tissue irreversible model

Fowler et al., 1997


Human brain MAO B increases with age

Tobacco smoke inhibits human brain MAO B

male 86 yrs

non-smoker (female 48 yrs)

male 43 yrs

smoker (female 51 yrs)

MAO B level

L-deprenyl treatment (male, 43 yrs)

male 27 yrs

Fowler et al, Nature, 1995


[11C]clorgyline Uptake in Thalamus

% Dose/cc

Time min

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


Distribution of MAO A and MAO B

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

MAO A

MAO B

non-smoker smoker

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

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


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

brain

thyroid

lungs

heart

kidneys

[11C]CLG

[11C]DEP

CDH007WB1

325x09232DH

MAO A MAO B


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

0.03

0.025

H

D

0.02

0.015

0.01

0.005

CDH211

0

0

10

20

30

40

50

60

heart-CLG

lungs-CLG

kidneys-CLG

% dose/cc

time

heart-DEP

lungs-DEP

kidneys-DEP


K imaging in peripheral organs 1

k3

F

T

k2

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

capillary

model of lung Model for peripheral organs

K1plasma to tissue

k2tissue to plasma

k3(MAO) α k'E

or combination parameter λk3


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

λk3

thyroid

heart

lungs

spleen

kidneys


MAO in Smokers vs Nonsmokers Isotope effect

MAO in Smokers vs Nonsmokers

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


Nonsmoker H DEP Isotope effect

Nonsmoker H DEP

Smoker H DEP

Smoker H DEP

0.020

0.020

0.012

Nonsmoker D CLG

0.015

Smoker D CLG

0.015

0.008

0.020

0.010

0.010

Nonsmoker D DEP

0.015

Smoker D DEP

Time (min)

0.004

0.005

0.005

0.010

0.000

0.000

0.000

0

20

40

60

0

0

20

20

40

40

60

60

Time (min)

0.005

0.000

0.012

Nonsmoker H CLG

0

10

20

30

40

50

60

Smoker H CLG

0.008

0.004

0.000

0

20

40

60

Lung uptake averaged over subjects

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

% Dose/cc

% Dose/cc

Time (min)

% Dose/cc

% Dose/cc

Time (min)

Time (min)


Smoker Isotope effect

16

Nonsmoker

0.12

0.16

20

12

0.10

0.12

15

0.08

8

0.08

10

0.06

4

3

3

k

k

0.04

5

0.04

0

0

0.00

0.02

0.00

[11C]clorgyline MAO A

[11C]Ldeprenyl (MAO B)

λ

λ

DEP

D-DEP

CLG

D-CLG

λ: S > NS = tracer is retained in tissue of smokers longer than nonsmokers. k3: NS>S (Logan, Fowler 2005)

>



MAO Isotope effect

RCH2NH2 + O2 + H2O RCHO + NH3 + H2O2

MAO A Deletion and Aggression

x

  • 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)

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


High and Low MAOA Genotypes in Humans Isotope effect

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/Low = 60/40 in males

From 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)


1 Isotope effect

0.75

0.5

0.25

0

-0.25

-0.5

Low MAOA

Activity , n=163

High MAOA

Activity , n=279

Composite Index of

Antisocial Behavior (z scores)

None Probable Severe

Childhood Maltreatment

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

Antisocial behavior requires the low MAO A genotype AND childhood maltreatment

This is a gene-environment interaction!


High and Low MAO A Genotypes and Aggression Isotope effect

What is the mechanism?

  • 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)?


Study Design Isotope effect

  • We measured brain MAO Aand 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.


Average Isotope effectk3 images

High

(n=26)

Low

(n=12)

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

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



1 Isotope effect

1

O

N

C

C

l

H

3

H

H

C

l

Gene-Brain-Behavior Relationships

Nelly Alia-Klein

Non-aggression-prone subjects

Aggression-prone subjects

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


Variability in brain MAO A levels in healthy males. Isotope effect

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? Isotope effect

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

Epigenetics???

5-Methylcytosine

cytosine

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


Methylation state vs k 3
Methylation state vs Isotope effectk3

Shumay, Logan, Volkow, Fowler (2012)

MAOA promoter methylation (%)

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.

Brain MAO A levels


MAO A and Depression Isotope effect

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.


Brain MAO A in major depressive disorder: a study with [ Isotope effect11C]harmine (Meyer et al., 2006)

depressed

Healthy

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.


Patients with MDD have elevation MAO A Isotope effect

depressed

healthy

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.


MAO Isotope effect

RCH2NH2 + O2 + H2O RCHO + NH3 + H2O2

X

MAO A Inhibitor Drugs for Depression

MAO A inhibitor drugs elevate serotonin, norepinephrine, and dopamine

Serotonin: mood

Norepinephrine: arousal

Dopamine: reward

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
New Antidepressant Drugs (Reversible Inhibitors of MAO A Isotope effectRIMAs)

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

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

  • How often does it need to be given?

Fowler et al., Neuropsychopharmacology, 2010


Study Protocol Isotope effect

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


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

Baseline

60 mg Tyrima - 2 hrs

60 mg Tyrima – 12 hrs

Tyrima shows robust and reversible MAO A blockade


Plasma levels of CX-157 predict brain MAO A Inhibition Isotope effect

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


The MAOA gene predicts happiness in women Isotope effect

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

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


Outline1
Outline Isotope effect

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


O Isotope effect

H

O

H

C

H

C

H

3

3

a

r

o

m

a

t

a

s

e

C

H

3

H

O

t

e

s

t

o

s

t

e

r

o

n

e

1

7

-

e

s

t

r

a

d

i

o

l

b

Aromatase (Estrogen synthase, CYP19A1)

O

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


Aromatase PET tracer: [ development (Wu et al., Cell 139, 139: 61, 2009)11C]Vorozole

Sunny Kim

  • (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)


Distribution of [ development (Wu et al., Cell 139, 139: 61, 2009)11C]vorozole in the human brain

  • B. Anterior Hypothalamus/preoptic area

  • Amygdala

  • Dorsomedial thalamus

  • Thalamus

  • Medulla

Summed images from 60 to 90 min overlaid on structural MRI


Metabolic Stability in human plasma development (Wu et al., Cell 139, 139: 61, 2009)

Uptake TACs in human brain


K development (Wu et al., Cell 139, 139: 61, 2009)1

k3

C2

C1

CA

k2

k4

2 Tissue Compartment model (with small k4)

% Dose/cc

Time min

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.


Methods [ development (Wu et al., Cell 139, 139: 61, 2009)11C] Vorozole PET Studies

  • Experimental

  • 27 normal volunteer subjects (baseline)

  • 5 subjects (baseline/block 2.5mg letrozole)

  • 90 min uptake

  • Arterial plasma radioactivity corrected for metabolites

  • Modeling (region of interest) total tissue distribution volume VT

  • NLL 2TC 4 parameter model. (k4estimated 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)


Comparison of methods for estimating V development (Wu et al., Cell 139, 139: 61, 2009)T

VT

Hyp Thl Amy Inf Put Cb

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


Baseline/blocked (2.5 mg letrozole (AI) oral 2 hours prior) development (Wu et al., Cell 139, 139: 61, 2009)

VT

% Dose/cc

Thalamus base 4.00

Thalamus blk 1.56

Cerebellum base 1.91

Cerebellum blk 1.67

Time min


Parametric images of NLL estimates of V development (Wu et al., Cell 139, 139: 61, 2009)T for [11C]VOR

Blocked

Baseline

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.


Aromatase Distribution in the Female Body development (Wu et al., Cell 139, 139: 61, 2009)

Postmenopausal

Woman, 54

Woman at

Midcycle, 38


Aromatase Distribution in the Male Body development (Wu et al., Cell 139, 139: 61, 2009)

Baseline

Blocked


[ development (Wu et al., Cell 139, 139: 61, 2009)11C]Vorozole uptake in liver

% Dose/cc

Baseline

Blocked (letrozole)

Time min


The high liver uptake raises the possibility that [ development (Wu et al., Cell 139, 139: 61, 2009)11C]vorozole might be a good tracer for imaging the liver and its blood supply.


Cluster analysis of [ development (Wu et al., Cell 139, 139: 61, 2009)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 development (Wu et al., Cell 139, 139: 61, 2009)

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

Avg peak time 0.79 min

Time min

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).


Liver Blood Signal development (Wu et al., Cell 139, 139: 61, 2009)

This subject has a much greater arterial contribution

Avg peak time 0.52 min

Time min


V development (Wu et al., Cell 139, 139: 61, 2009)T and K1 for 12 clusters of liver uptake in 5 subjects

VT liver > VT brain


Parametric image of [ development (Wu et al., Cell 139, 139: 61, 2009)11C]vorozole in liver


Brookhaven imaging group 2011
Brookhaven development (Wu et al., Cell 139, 139: 61, 2009)Imaging Group 2011

  • Chemistry

  • organic

  • inorganic

  • nuclear

  • theoretical

  • Engineering

  • Genetics

  • Medicine

  • Nursing

  • Pharmacology

  • Psychology

  • Physics

  • Plant biology

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.

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


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