Post on 16-Jan-2016
description
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