ProbeTime

Molecular Imaging Concept

Biochemical Target

Administer probe Image probe

Imaging Device

Sensitivity - Ability to either detect the probe signal

at the target (direct response) or change in a signal that is dependent on the quantity of probe at the target (indirect response)

Specificity - Ability to distinguish the target fromnontarget processes or tissues

Diagnostic Utility

Molecular Imaging Modalities• Gamma ray emission

•Positron emission – annihilation photons (PET)•Single photon emission (SPECT)•Direct signal from tissue in response to probe concentration

• Gamma ray absorption•Used in CT scans• Contrast with high atomic number nuclei that absorb gamma rays (Iodine)

• Magnetic resonance• Protons provide signal in clinical MRI scans• Perturb proton relaxation with Gd contrast agents• Use paramagnetic nuclei (e.g., 13C, 19F) labeled probes

•Optical•Fluorescent molecules (luciferase/luciferin; GFP)•Visible wavelengths have limited depth of detectability•Infrared extends depth a bit

•Ultrasound•Acoustic absorption/modification (microbubbles)•Thermoacoustic stimulation (absorption probes)

CT Contrast Agents

• High atomic number – x-ray absorption– Ba, I, Gd, Au

• Intraintestinal or intravascular (extracellular)

Examples:

Barium sulfate (oral, rectal admin.)

iopromide (iodinated IV contrast agent)

Stomach

Kidneys

Ovarian

liver

Radioapague Contrast Agent Enhance the Organ Delineation

Without contrast administration With contrast administration

-100

0

100

200

300

400

500

600

700

0 50 100 150 200 250

Time in seconds

En

ha

nc

em

en

t in

HU

kidney

input (heart)

muscle

The representative CT images of a dynamic sequence with iodinated contrast enhancement. From top to the bottom, one can see the flowing path of the contrast medium within the blood stream: from tail vein to vena cava, to heart, and then to kidney. These three images are associated with the first 8 seconds of the sequence.

spinekidney

heartvena cava

vena cava

Catheter

Opportunities in microCT:Anatomy and physiology

• Positive contrast agents (appearing bright on MRI) Small molecular weight compounds containing as their active element Gadolinium (Gd), Manganese or Iron

Unpaired electron spins in their outer shells and long relaxivities, which make them good T1 relaxation agents.

Examples:

GD-DTPA, Gadopentetate dimeglumine, gadoteridol, and gadoterate meglumine are utilized for the central nervous system and whole body

Mangafodipir trisodium for lesions of the liverGadodiamide for the central nervous system.

Proton MR Contrast Agents

• Negative contrast agents (appearing predominantly dark on MRI) are small particulate aggregates often termed superparamagnetic iron oxide (SPIO). These agents produce predominantly spin-spin relaxation effects, but very small particles smaller than 300 nm also produce substantial T1 relaxation.

• A special group of negative contrast agents (appearing dark on MRI) are perfluorocarbons because their presence excludes the hydrogen atoms responsible for the signal in MR imaging.

Proton MR Contrast Agents

• Some MR contrast agents require biocompatible carriers/capsules– Reduce toxicity – hide the bad guy inside– Target specific cells/proteins/processes

Examples:Ferumoxide – SPIO core particles (~150nm), dextran T-10 coveringNanomag – SPIO particles (50nm) with cross-linked dextran and amino acid sequences to form bonds to organic compoundsP7228 – SPIO, anionic dextran layer – can be encapsulated by positively charged liposomes

MR Molecular Probes

Biologically Important NMR NucleiBiologically Important NMR Nuclei1H - Wall thickness, ejection fraction, wall motion,

perfusion, coronary artery angiography. (large signal from ~50M concentration in tissues)

31P – ATP, PCr, Pi, PDE, PME, pHi, [Mg2+], kinetics of creatine kinase and ATP hydrolysis.

23Na – Transmembrane Na+ gradient, tissue and cartilage structure.

13C – Glycogen, metabolic rates, substrate preference, drug metabolism, etc.

19F – Drug metabolism, pH, Ca2+ and other metal ion concentration, pO2, temperature, etc.

2H – Perfusion, drug metabolism, tissue and cartilage structure.

In vivo detection sensitivity limits use of C-13 and F-19 molecular probes (C-13 requires >0.1mM, F-19 >5 mM)

Advantages of PET

• PET has high sensitivity (~pmol of probe can be detected)

• PET images biochemistry. Small radionuclides (C-11,F-18) label small biological molecules with retention of biological specificity.

• PET images are quantitative

General Aspects of PET Tracers

• Understanding of targeted biochemical process

• Practical synthesis: sufficient yield and purity, automated

• Tissue uptake and kinetics are specific to targeted process

• Fate of radiolabel understood for metabolized tracers

• Tracer distribution is sensitive to answer clinical questions relevant to diagnosis, prognosis or monitoring of therapy

• Tissue kinetics amenable to mathematical modeling to give quantitative indices

nuclide half-lifeC-11 20.3 minN-13 10 minO-15 124 secF-18 110 minI-124 4.2 d (+ high Energy photon)

e.g., 18F 18O + e+ +

Positron Decay

ZAXN Z 1

AYN1 e+

Biochemical/Physicological Targets for PET Imaging

• Substrate metabolism carbohydrates, fatty acids, amino acids, oxygen, nucleosides,

oligonucleotides• Receptor binding

adrenergics, cholinergics, neurotransmitters, hormone receptors, growth factor receptors

• Ionic transportNa, K, Ca, F, Cl, I

• Perfusionwater, ammonia, butanol

• pH• Blood volume (11CO, C15O)• Hypoxia (misonidazoles)• Redox potentials• Protein-protein interactions

monoclonal antibodies• Gene expression

reporter genes and probes

Challenge #1:Radiochemical limitations

>90% of PET probes are synthesize by simple 1 or 2 step labelingfollowed by purification and formulation

•Short radionuclide half-life (<2 hr)•Limited radionuclide availability•Radiation exposure to chemist

Synthesis times typically under 45 min for C-11 (t 1/2 = 20 min)and under 2 hr for F-18 (t 1/2 = 110 min)

TARGET

Challenge #2:Biochemical Complexity

Challenge #3:The radioimaging signal is chemically nonspecific

*TARGET*Probe *(Intermediates)

*systemic metabolites (i.e. hepatic)

*Nonspecific binding to proteins or membranes

*alternative binding or metabolic products

Specificity! Specificity! Specificity!

Challenge #4:Physiological Barriers To Delivery of Probe to the Target

TARGET

Arterial Blood

(Subcellular compartmentation may also limit delivery)

Transport of probe to target should not be rate-limitingLimits utility of technique in poorly perfused tissues

Challenge #4B:Subcellular Barriers of Delivery

TARGET

Again, transport of probe to target should not be rate-limiting,And probe must be able to leave cell if not acted upon.

• 11C-acetylene (C2H2) may be useful as a radio-labeling intermediate for organic molecules in physiology studies

• [11C]C2H2, by itself, can be used in perfusion studies (i.e. brain)

11C-Acetylene as PET Probe and Labeling Intermediate

Comparison of PET Tracers for Measuring Tissue Perfusion

TracerPhysical half-life

Octanol/Water partition coeff. (log P)

Solubility in water (g/ 100 ml)

Cost per synthesis*

Ease of Synthesis

[15O]water[13N]ammonia[11C]CH3F

[18F]CH3F

[11C]C2H2

[11C]butanol[15O]butanol

2.019.9720.4109.820.420.42.01

-1.38-1.380.510.510.370.880.88

340.230.230.1067.87.8

HighLowModerateModerateLowModerateModerate

SimpleSimpleDifficultModerateSimpleModerateDifficult

* Using proton accelerator and most common nuclear reaction for production

1) Trap on Ba2) 900 C / H211CO2 + 12CO2 Ba*CC *CCH2

Bu-LiH*CCLi

ON

O

O

O

*

*

+

NaOH

*

(Madsen et al., 1981)

+*

[3,4-11C]-2-oxo-butynoic acid (COBA)

O

O

O

O

O

O

O

OH

O

O

OH

O

Sieve Trap

Trap inlet

ThermocoupleWires Trap Outlet

Helium

Quartz RxnVessel

Dose Calibrator

ad

c

V3

C-11 CO2

Waste

RecirculationPump

C10 0

1C

C

0 1

Dose Calibrator

Sampling Bag

Draw Samplefor Analysis

Hydrogen

V4

V5

V1a

V1b

V2

Waste

Soda LimeTrap

Furnace

b

0 1

0

1C

Computer-controlled Apparatus for synthesis of C-11 Acetylene

0 1 2 3 4 5 6 7 8 9Time (min)

Num

ber

of I

ons

Col

lect

ed (

Mil

lion

s)

0

1

2

3

4

Gas Chromatography/ Mass Spec of C-11 Acetylene Product

StandardProduct

0 1 2 3 4 5 6 7 8 9 10 11 12Time (min)

0

1

2

3

4

5R

adio

activ

ity (A

rbitr

ary

Uni

ts)

Gas Chromatography / Rad. Detection of C-11 Acetylene Product

Target: Myocardial Fatty Acid Oxidation

• Long-chain fatty acids are the predominant substrates for production of ATP in heart.

• Abnormalities of fatty acid oxidation by the myocardium are associated with ischemic heart disease, congestive heart failure, cardiomyopathies, and deficiencies of carriers, enzymes or co-factors required for fatty acid transport or oxidation.

• The lack of a specific radiolabeled probe of fatty acid oxidation has impeded the development of a non-invasive technique for assessment of fatty acid oxidation.

Myocardial Metabolism of Fatty Acids

LCFA

LCFA

Myocyte

ACS

LCFA-CoA

Lipids

lipase

LCFA-carn

CPT-I

CAT

CPT-II

LCFA-CoA MTP

VLAD

MCFA-CoA

Acetyl-CoA

-ox.

-ox.

Mitochondrion

FATr hyd.LCFA-carn

18FS OH

O

S OH

O18F

18F S OH

O

6-Thia Analogs 4-Thia Analog

14-[18F]fluoro-6-thia-heptadecanoicacid (14F6THA)

17-[18F]fluoro-6-thia-heptadecanoicacid (17F6THA)

16-[18F]fluoro-4-thia-hexadecanoicacid (FTP)

Plasma

Myocyte

(LC-AcylCoA synthetase) (LC-AcylCoA hydrolase)

Mitochondrion

(CPT-I)

(VLC-acylCoA dehydrogenase)

slow(Mit. Trifunctional Protein)

Protein Binding

(spontaneous)

Complex Lipids(acyl transferase)

(Translocase, CPT-II)

Outer Membrane

Inner Membrane

S O-

O18F

S O-

O18F

S S

O18F CoA

S O

O18F Carn

S S

O18F CoA

S S

O18F CoA

S S

O18F CoA

OH

SH18F

ORGAN

0.0

0.5

1.0

1.5

Upt

ake

(% d

ose

kg/ g

)ControlEtomoxir-treated

Biodistribution in Fasted Rats at 30 min p.i.

* * **

*

Effects of CPT-I Inhibition on 18F-FTP

* p<0.05 versus control

0 2 4 6 8 10 12 14 16 18 20

Time (min)

0

10

20

30

40A

DV

(m

l/g d

ry)

Normoxic

Hypoxic

Kinetics of [18F]FTP in Isolated Rat Heart

k2k1

k3

k4

C1

C2

dC1(t)/dt = k1 Cp(t) - (k2 + k3) C1(t) (1) dC2(t)/dt = k3 C1(t) - k4 C2 (t) (2)Ctot(t) = (1-BV) (C1+ C2) + BV Cp (3)

Reversible

Trapped

Cp

2-Compartment Model Fit to FTP Kinetics in Isolated Rat Heart

0 2 4 6 8 10 12 14 16 18 20

Time (min)

0

10

20

30

40

AD

V (

ml/g

dry

)

DataModel

BV=0.98 ml/g dryk1= 5.01 (ml/min/g dry)

k2=0.56/min

k3=0.36/min

k4=0.0050/min

*

**

*p<0.01

18F S OH

O

S OH

O18F

F-18 FTP in Normal Human Subject Short-axis Images of Heart at 50-55 min p.i.

Duke University Medical Center

0 10 20 30 40 50 60 70 80

Time (min)

0.1

1.0

68

2

34568

2

3456

LiverRenal CortexHeart (septum)Blood PoolFTP Input FunctionBrainlung

F-18 FTP Kinetics in Normal Human Subject

% d

ose

/ 100

ml

0 5 10 15 20 25 30

Time (min)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Frac

tion

F-18 FTP Nonmetabolized Fraction

Normal Human Subject (11-18-1998)

y = 1 - (Ax / (B + x + C/x))

A = 0.653, B = -1.737, C = 112.1

Nonmetabolized fraction of F-18 FTP in Plasma

0 10 20 30 40

Time (min)

0.0

200.0

400.0

600.0

800.0

1000.0

1200.0

1400.0

1600.0

1800.0

2000.0DataModel

2-Compartment Model Fit to FTP Kinetics in Normal Human Myocardium

nci /

cc

TBV = 0.26; k1 = 0.164; k2=0.069;k3 = 0.0704; k4 =0.005

FRFTP = 0.0827 (ml blood/min/ml tissue)

F-18 FTP (Fatty Acid Oxidation) SPECT Tc-99m Myoview Perfusion Scan

Diabetic Cardiomyopathy Patient

0 10 20 30 40

Time (min)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7 DataModel

nci /

cc

TBV = 0.323; k1 = 0.128; k2=0.811;k3 = 0.206; k4 =0.000 (fit)

FRFTP = 0.0280 (ml blood/min/ml tissue)

F-18 FTP Kinetics - 2-Compartment Model Fit

Diabetic, Ischemic Cardiomyopathy (11-8-1999)