Dr. Erik rstad, KLB room 2.11 (e.arstad@ucl.ac.

uk)

Chem 2601/2012

Molecular Imaging
Lecture 5 and 6: Radiotracers and applications for diagnostic imaging

Overview (lecture 5 and 6): 1) 2) 3) 4) 5) 6) Radiotracers key principles Properties of radiotracers Interactions of tracers with biological targets Nuclear imaging of cancer Nuclear imaging of brain diseases Nuclear imaging in research and drug discovery

Radiotracers key principles: 1) 2) 3) Principles of imaging with radiotracers Properties of radiotracers Interactions of radiotracers with biological targets

George De Hevesy (1911): My boy, if you are worth your salt, you try to separate Radium-D from all that lead. After two years on non-successful work: . . . .I failed completely. In order to make the best of this depressing situation, I decided to use Radium-D as an indicator of lead. . . Later it was found that Radium-D is 210Pb.

A radiotracer (also known as a tracer or radiopharmaceutical) is a chemical compound that allows investigation of a biochemical process without influencing the process.
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Basic principles of radiotracers: To image a biological target or process, the following criteria must be fulfilled: - The radiotracer must be able to reach its intended target in vivo - The radiotracer must interact with its intended target so that its distribution changes in a concentration dependent manner - The radiotracer must not influence the process it measures (= high specific binding)
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Properties of radiotracers: 1) 2) 3) Affinity and binding potential Lipophilicity Pharmacokinetics and time-activity curves

Tracer interaction with target depends on binding affinity: [Ligand][Receptor] Affinity (Kd) = [Ligand-Receptor complex]

and target concentration: Binding potential (BP) = [Receptor] Kd Affinity ideally between 0.5-5 nM Binding Potential > 2
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The ability of a tracer to reach its target depends on: Physiochemical properties (e.g. lipophilicity) Metabolic stability Ability to cross biological barriers (membranes etc.)

Lipophilicity and Log P Lipophilicity is a measure of the polarity of a compound (water vs. fat solubility) Lipophilicity is measured as the logarithm of the partition coefficient between water and octanol [organic phase] [aqueous phase]

Log P = log

Brain uptake as function of Log P:

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Question: A compound is shaken with equal amounts of water and octanol. You find that the aqueous phase contains 0.1% of the compound and the organic phase contains 99.9%. What is the Log P? What would be the measured Log P if the compound would contain 1% of a water soluble impurity?

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Pharmacokinetics is the distribution of a tracer over time (referred to as time-activity curves) Increased biological half-life (retention) in target tissue is the basis of the signal

time-activity curve
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Interactions of radiotracers with biological targets 1) 2) 3) 4) Imaging of receptor binding Imaging of transporters Imaging of enzymes Imaging metabolic pathways

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Imaging of receptors
Radiotracer Receptor Receptor-radiotracer complex

= washout of tracer

= retention of tracer

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O
123

I NH H

D2 Receptor imaging with [123I]IBF SPECT:

[123I]IBF

Uptake of [123I]IBF over 2.5 h in humans

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Time-activity curves:
in blood in target (striatum) and non-target (frontal cortex)

Basis of the signal

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Imaging transporter function Tracer substrate Transporter transporter function

And transporter expression Tracer inhibitor Transporter Transporter-radiotracer complex

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Imaging of transporter function in brain tumour: [18F]FET

[18F]FET
18

ONH2

F
O

Time-activity curve
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Imaging enzyme function Tracer substrate Multiple tracer molecules trapped

Enzyme Imaging enzyme expression Tracer inhibitor 1 Tracer is trapped per enzyme

Enzyme

Imaging Metabolic Processes Tracer substrate Multiple tracer molecules trapped

Enzyme

= transporter function + enzyme function

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Applications in biomedical research oncology

1) Imaging glucose metabolism with [18F]FDG 2) Imaging membrane synthesis with Choline 3) Imaging protein synthesis with amino acids 4) Imaging of cell proliferation with [18F]FLT
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Imaging of glucose metabolism with FDG

OH O HO HO OH
18

Phosphate group prevents leakage from the cell

[18F]FDG:

PO32O

Hexokinase

Glucose Transporters

HO HO
18

OH F

Cancer cells have increased rate of proliferation, and tend to be in an anaerobic environment = increased glucose metabolism

Fluoride blocks further metabolism.

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FDG for diagnostic imaging of cancer

Healthy control

B-cell lymphoma

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Recap: imaging of Glut5 transport with fluorescently labelled fructose

Cells lacking Glut5, cells with Glut5, after addition of fructose

NBD derivative

Cy5.5. derivative

Question: Why is imaging of glucose metabolism so much more successful with nuclear imaging than with fluorescence?

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FDG PET: Limitations for diagnostic applications High background in the brain and bladder Low uptake in slow growing tumours (e.g. prostate) FDG relates to metabolic activity only - limited biochemical information

Healthy control

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Imaging metabolism vs. receptor expression

18F-FDG

PET

68Ga-DOTANOC

PET

Metabolic activity

SSR receptor expression

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Imaging of Choline metabolism

N

+
OH

Phosphate group prevents leakage from the cell

Choline

Choline kinase

Choline Transporters

2
N

+
O

PO32-

Cancer cells have increased rate of proliferation = increased membrane lipid synthesis = increased uptake of Choline

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Imaging prostate cancer with choline tracers:

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Imaging cell proliferation with [18F]FLT Allows assessment of tumour aggressiveness Can predict response to chemotherapy Can distinguish cancer from infection
O

However, much lower sensitivity than FDG

NH N O
18

HO

Salskaov et al., Semin Nucl Med. 2007, 37(6):429-439

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Imaging response to chemotherapy with [18F]FLT:

Before chemotherapy

7 days after chemotherapy

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Imaging of transporter function/protein synthesis in brain tumours: [11C]methionine

H311CS OH H2N C O
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Nuclear imaging of brain diseases

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Nuclear imaging of brain diseases 1) Imaging of Amyloid plaque in Alzheimers disease 2) Imaging of movement disorders (Parkinson disease)

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Alzheimers disease is a form of dementia Disease frequency increases rapidly with age 15% of population aged >80 years have AD No known treatments but several therapies in clinical trials Need for imaging to enable diagnosis and to evaluate treatments

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Alzheimers disease is associated with Amyloid plaques:

HO S
11CH 3

NH N

[11C] PIB

Amyloid plaque

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[11C]PIB vs. [18F]FDG

Healthy (67) [11C]PIB AD patient (79)

[18F]FDG

Klunk, Engler, Nordberg, et al Annal Neurol 2004, 55, 306-319

Review: A Nordberg; The Lancet Neurology 36 Vol 3, September 2004, 519-527

PIB PETScans scans in elderly volunteers (n=32) [11 C]PIB inhealthy healthy elderly volunteers

78%
-ve scan

8%

9%
+ve scans

5%

In memory subjects amyloid plaque scored lower on Healthytests controls with with +ve scans had memory Z-scores average than than subjects without amyloid plaque 0.5 lower controls with ve scans 37

Parkinsons disease (PD) A class of movement disorders Caused by neurodegeneration (death of nerve cells) Selective loss of dopamine producing cells = classical PD Wider loss of nerve cells, including dopamine producing cells = atypical PD

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Imaging Parkinsons disease with [123I]FP-CIT SPECT

Dopamine transporter Healthy subject

F N

O OMe I

[123I]FP-CIT

Parkinsons disease

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Imaging PD D2 receptor expression with [123I]IBZM

Dopamine D2 receptor

Classical PD

I OH

H N N OMe O

[123I]IBZM

Atypical PD

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Nuclear imaging in research and drug discovery 1) 2) Use of PET to study receptor occupancy Use of PET to study drug behaviour

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Imaging of drug receptor occupancy (and release of endogenous neurotransmitters) Drug Tracer Tracer

Receptors at synapse

Receptors at synapse = Drug competes with tracer for binding 42

Imaging of drug receptor occupancy: time-activity curves

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Effect of Smoking on nicotinic acetylcholine receptors

Conclusion: Smoking results in complete block of nAChRs

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Profiling drugs in man: What is the optimal dose?

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Drug development: The microdosing concept

Early clinical evaluation using radiolabelled drug in tracer amounts

Drug concentration too low to cause pharmacological or toxic effects

Early ADME data at relatively low cost

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Microdosing concept: brain uptake and drug metabolism

Does the drug penetrate the blood-brain barrier?

No

hmmm

Yes!

What is the metabolic profile??

What is the biological half-life?

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Lappin and Garner (2003). Nature reviews, Drug Discovery. Vol 2; 233-240.

Synopsis: Radiotracers
A radiotracer is a chemical compound that allow investigation of a biochemical process without influencing the process. Tracers can interact with different biological targets, e.g. receptors, transporters, enzymes and metabolic processes. The information obtained depends on the mechanism for tracer accumulation. Important properties of tracers are: affinity, lipophilicity, selectivity, and pharmacokinetics. Also important with choice of radionuclide, labelling chemistry and specific activity.

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Synopsis: Applications of nuclear imaging

FDG PET is limited by high background in brain and bladder, low uptake in slow growing tumours and because it does not provide specific biochemical information. For diagnostic imaging of cancer, imaging of receptor expression can provide increased sensitivity, FLT can enable imaging of proliferation, choline can allow diagnosis of prostate cancer, FET can enable imaging of brain tumours. For brain imaging, PIB enables detection of amyloid plaque as a marker of Alzheimers disease, and a combination of imaging of dopamine transporters with FP CIT and imaging of D2 receptors with IBZM allows accurate diagnosis of Parkinsons disease. For drug development, microdosing studies allows early evaluation of ADME properties, and drug occupancy studies allows determination of the effective dose.
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PET in drug development:

Microdosing: Radiolabelled drug in tracer amount Good for: ADME data Not good for: Dose optimisation Mechanism of action Pharmacological effect Toxicity Receptor occupancy studies: Radiotracer + drug Good for: Optimising dose Mechanistic studies

Microdosing + receptor occupancy: Very few subjects needed + big cost savings! NB: Large scale trials still needed but much lower risk
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Learning outcomes - you should understand: The limitations of FDG, and how the use of other tracers, including FLT, Choline, FET and RGD peptides can provide additional information. The principle of imaging amyloid plaque with PIB and how this relates to Alzheimers disease. The principle of imaging Dopamine transporters and D2 receptors and how this enables diagnosis of Parkinsons disease. What microdosing studies are and how they can be used in drug development The principles of drug occupancy studies and how these can be used for drug development

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Assessment you should be able to apply your knowledge of radioactivity and nuclear imaging to explain underlying principles, solve practical problems and provide rationale explanations related to: Imaging with FDG and its limitations , and how the use of other tracers, including FLT, Choline, and amino acids can provide additional information. The principle of imaging amyloid plaque with PIB and how this relates to Alzheimers disease. The principle of imaging Dopamine transporters and D2 receptors and how this enables diagnosis of Parkinsons disease. What microdosing studies are and how they can be used in drug development The principles of drug occupancy studies and how these can be used for drug development
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Further reading:
PET in Oncology: Ashley M Groves AM, Win T, Haim SB, Ell PJ (2007). Lancet Oncol., 8: 82230 Buerkle A, Weber WA (2208), Cancer Metastasis Rev (2008) 27:545554 Salskaov et al., Semin Nucl Med. 2007, 37(6):429-439 PET in microdosing and drug development: Lappin G, Garner RC (2003). Nat Rev Drug Discov. 2(3):233-40. Brooks DJ (2005). NeuroRx., 2; 226236. Mamo D et al. (2007). Am J Psychiatry, 164: 14111417. Brain imaging: Thobois S et al. (2001). Neurophysiol Clin., 31:321-40 Tasch K, Ell PJ (2006). Clin Med., 6:25962 Henriksen G, Willoch F (2007). Brain., 131(Pt 5):1171-96.

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