1 / 66

PET Studies of Enzyme Activity: Monoamine oxidase and Aromatase

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

fern
Download Presentation

PET Studies of Enzyme Activity: Monoamine oxidase and Aromatase

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. PET Studies of Enzyme Activity: Monoamine oxidase and Aromatase

  2. 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

  3. 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.

  4. Cellular Locations of MAO A and MAO B MAO A MAO A DA DA MAO B DA MAO B DA DA DA signal

  5. Radiotracers for MAO A and B

  6. Uptake Images (multiple time points) Amount of tracer injected Blood Flow # receptor, enzyme binding sites Modeling (also requires input function) # receptor, enzyme binding sites

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. Deuterium Isotope Effects and MAO Mapping [11C]L-deprenyl [11C]L-deprenyl-D2

  12. 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

  13. [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

  14. 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

  15. [11C]clorgyline Uptake in Thalamus % Dose/cc Time min [11C]clorgyline was found to be superior to [11C]clorgyline-D2

  16. 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)

  17. 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

  18. 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

  19. K1 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

  20. MAO A activity in organs as demonstrated by the Deuterium Isotope effect λk3 thyroid heart lungs spleen kidneys

  21. MAO in Smokers vs Nonsmokers 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

  22. Nonsmoker H DEP 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)

  23. Smoker 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) >

  24. MAO A Genotype and Brain MAO A

  25. MAO 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)

  26. High and Low MAOA Genotypes in Humans 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)

  27. 1 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!

  28. High and Low MAO A Genotypes and Aggression 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)?

  29. Study Design • 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.

  30. Average 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.

  31. Are brain MAO A levels related to trait aggression?

  32. 1 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)

  33. 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.

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

  35. Methylation state vs 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

  36. 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.

  37. Brain MAO A in major depressive disorder: a study with [11C]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.

  38. 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.

  39. MAO 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.

  40. New Antidepressant Drugs (Reversible Inhibitors of MAO A RIMAs) 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

  41. Study Protocol 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

  42. MAO A Activity in Human Brain and After Tyrima (CeNeRx) Baseline 60 mg Tyrima - 2 hrs 60 mg Tyrima – 12 hrs Tyrima shows robust and reversible MAO A blockade

  43. 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)

  44. 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.

  45. 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

  46. O 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

  47. 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

  48. Aromatase PET tracer: [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)

  49. Distribution of [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

  50. Metabolic Stability in human plasma Uptake TACs in human brain

More Related