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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 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 RCH 2 NH 2 + O 2 + H 2 O RCHO + NH 3 + H 2 O 2 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 signal MAO B MAO 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 Modeling (also requires input function)
Sp K1K1 k2k2 E + SbE-S inact PET region of interest k’ 3 Quantifying MAO in the brain: A useful model 2 Tissue irreversible model K 1, k 2 k 3 =k’ 3 E k’ 3 ~all processes involved in forming E-S inact k’ 3 E = f(S E, k M, k M’,k inact ) S E endogenous substrate (assume constant) k M Michaelis-Menten constant substrate k M’ Michaelis-Menten constant tracer K inact rate constant for inactivation Sb brain tracer concentration Sp plasma tracer concentration K 1 plasma to tissue k 2 tissue to plasma
dSb/dt = K 1 Sp(t) - k 2 Sb - k 3 Sb d E-S inact /dt = k 3 Sb Use k 3 or the composite term k 3 as an index for MAO. = K 1 /k 2 which does not depend upon blood flow) Because estimates of k 2 and k 3 are highly correlated we have found k 3 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 k 3 is too large ( k 3 >> k 2 ), tracer uptake depends only upon K 1 (called flow limited) Patlak et al Ki is an index of tracer uptake 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 Solution: reduce k 3
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. [ 11 C]L-Deprenyl-D2 bond broken by MAO [ 11 C]L-Deprenyl-H2
Deuterium Isotope Effects and MAO Mapping [ 11 C]L-deprenyl [ 11 C]L-deprenyl-D2
[ 11 C]L-Deprenyl-D2 [ 11 C]L-Deprenyl Ki D2 =.12 Ki H2 =.29 K 1 D2 =.42 K 1 H2 =.41 Time % Dose/cc Sensitivity of [ 11 C]L-deprenyl-D2 is greater than that of [ 11 C]L-deprenyl-H2 the rate of trapping ( k 3 ) is reduced improving quantification
MAO B K1K1 [ 11 C]L-deprenyl-D2 reveals brain MAO B increases and blood flow decreases in normal aging (n=21) Fowler et al., 1997 Using λk 3 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) MAO B level Fowler et al, Nature, 1995 male 86 yrs male 43 yrs male 27 yrs Human brain MAO B increases with age
[ 11 C]clorgyline Uptake in Thalamus [ 11 C]clorgyline was found to be superior to [ 11 C]clorgyline-D2 % Dose/cc Time min
non-smoker smoker [ 11 C]clorgyline (MAO A) and [ 11 C]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
brain thyroid lungs heart kidneys [ 11 C]CLG[ 11 C]DEP CDH007WB1325x09232DH 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 0102030405060 CDH211 H D % dose/cc time Comparing H and D was also used to validate MAO A and B imaging in peripheral organs
capillary K1K1 k2k2 k3k3 T F tissue (T and F)+ blood vol + metabolites? + (air in lungs) model of lung Model for peripheral organs k 3 (MAO) α k'E or combination parameter λk 3 K 1 plasma to tissue k 2 tissue to plasma
λk3λk3 thyroid heartlungsspleenkidneys 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
Lung uptake averaged over subjects 0204060 0.000 0.005 0.010 0.015 0.020 Nonsmoker H DEP Smoker H DEP Time (min) 0204060 0.000 0.005 0.010 0.015 0.020 Nonsmoker H DEP Smoker H DEP Time (min) 0204060 0.000 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 0204060 0102030405060 0.000 0.005 0.010 0.015 0.020 Nonsmoker D DEP Smoker D DEP Time (min) % Dose/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 λ λ [ 11 C]clorgyline MAO A[ 11 C]Ldeprenyl (MAO B) 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.00 0.04 0.08 0.12 0.16 Nonsmoker k 3 Smoker k 3 λ: S > NS = tracer is retained in tissue of smokers longer than nonsmokers. k 3 : NS>S (Logan, Fowler 2005) > CLGD-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 RCH 2 NH 2 + O 2 + H 2 O RCHO + NH 3 + H 2 O 2 x 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 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)
Antisocial behavior requires the low MAO A genotype AND childhood maltreatment Composite Index of Antisocial Behavior (z scores) Childhood Maltreatment None Probable Severe 1 0.75 0.5 0.25 0 -0.25 -0.5 Low MAOA Activity, n=163 High MAOA Activity, 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 [ 11 C]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. High (n=26) Average k 3 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?
O Cl Cl N 11 C H 3 HH 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 k 3 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 k 3 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 Epigenetics??? cytosine 5-Methylcytosine Hypothesis: epigenetic mechanisms will influence gene expression and therefore MAO A activity in the brain.
Methylation state vs k 3 Brain MAO A levels MAOA promoter methylation (%) 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 [ 11 C]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 [ 11 C]harmine (Meyer et al., 2006) depressed Healthy
Patients with MDD have elevation MAO A 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.
RCH 2 NH 2 + O 2 + H 2 O RCHO + NH 3 + H 2 O 2 MAO X MAO 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: [ 11 C]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)
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 CH 3 CH 3 CH 3 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 (K i = 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: [ 11 C]Vorozole Sunny Kim
Distribution of [ 11 C]vorozole in the human brain Summed images from 60 to 90 min overlaid on structural MRI B. Anterior Hypothalamus/preoptic area C.Amygdala D.Dorsomedial thalamus E.Thalamus F.Medulla
Metabolic Stability in human plasma Uptake TACs in human brain
C2C2 C1C1 K1K1 k2k2 k3k3 k4k4 CACA 2 Tissue Compartment model (with small k 4 ) V T =3.16 ( k 4 =.01 min -1 ) V T =5.35 ( k 4 =.005 min -1 ) Problem: V T is very sensitive to variations in k 4 for regions of high uptake. % Dose/cc Time min
Methods [ 11 C] 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 V T NLL 2TC 4 parameter model. ( k 4 estimated from 4 highest regions combined and this value was used for these individual “high” regions). For the “low” regions k 4 was allowed to vary. Graphical analysis (GA) MA1 (Ichise) Tissue to plasma tracer ratio (TR)
Comparison of methods for estimating V T 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 VTVT
Baseline/blocked (2.5 mg letrozole (AI) oral 2 hours prior) Thalamus base 4.00 Thalamus blk 1.56 Cerebellum base 1.91 Cerebellum blk 1.67 VTVT % Dose/cc Time min
Blocked Baseline Parametric images of NLL estimates of V T for [ 11 C]VOR Images were generated using a clustering method for initial kinetic parameters. Non enzyme parameters were fixed at the cluster value so that only k 3 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
[ 11 C]Vorozole uptake in liver Baseline Blocked (letrozole) % Dose/cc Time min
The high liver uptake raises the possibility that [ 11 C]vorozole might be a good tracer for imaging the liver and its blood supply.
Cluster analysis of [ 11 C]vorozole binding in the torso Because of its distinctive binding the liver is easily separated from other organs by cluster analysis.
Liver Blood Supply 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
Avg peak time 0.52 min This subject has a much greater arterial contribution Liver Blood Signal Time min
Subject VTVT K1K1 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) V T and K 1 for 12 clusters of liver uptake in 5 subjects V T liver > V T brain
Parametric image of [ 11 C]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 theoretical Engineering Genetics Medicine Nursing Pharmacology Psychology Physics Plant biology Program Support from NIH and DOE-OBER (infrastructure), SBU-BNL Partnership, pharmaceutical industry
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