Magnetic Resonance Imaging to Assess Tissue

Oxygenation and Redox Status

.
Hyperpolarized (by EPR) MRI
Electron Paramagnetic Resonance (EPR) Imaging
Redox Sensitive Paramagnetic Contrast Agents in MRI

Murali C Krishna
Radiation Biology Branch
Center for Cancer Research
National Cancer Institute
NIH, Bethesda, MD
 Electron Paramagnetic Resonance
EPR spectroscopy is similar to NMR
spectroscopy.

NMR spectroscopy detects nuclei with magnetic

moments.
ex: 1H, 13 C, 19 F, 31 P etc.
EPR spectroscopy detects species with unpaired
electrons.
ex: free radicals, transition metal complexes

At a given magnetic field, EPR is more sensitive

than NMR
NMR spectroscopy detects nuclei with magnetic moments.
ex: 1H, 13 C, 19 F, 31 P etc.

EPR spectroscopy is similar to NMR spectroscopy.

EPR spectroscopy detects species with unpaired electrons.

ex: free radicals, transition metal complexes

At a given magnetic field, EPR is more sensitive than NMR

In MRI, proton NMR spectra are used for anatomic imaging.

Reason: simple NMR spectrum.

For EPR Imaging we need species with simple EPR spectrum.

candidates: free radicals
Can we image free radicals in biological systems?

Spatially-resolved (anatomical) information can be

obtained using EPR imaging, similar to MRI
MRI EPR imaging

Spin Probes Tissue protons Free radicals

(endogenous) (>50 M) (<< nM)

Probe Stability Ideal < nanoseconds

No suitable endogenous spin probes exist for EPR imaging.

Non-toxic exogenous paramagnetic probes needed for in vivo EPR imaging.

 Molecular Oxygen Provides Contrast to Paramagnetic Probes in
EPRI

• Molecular oxygen is paramagnetic and provides contrast to paramagnetic probes.

This causes spectral broadening (increase in line width)

Line width changes from oxygen contrast > 200 % in EPR

In NMR such changes may be ~10%

EPR Line Width vs pO2

600
EPR Line-width (mG)

400

200

0
0 25 50 75 100
Oxygen (pO2)

• It is possible to image spatial distribution of paramagnetic spin

probe by EPR and obtain pO2 information
 EPR Imaging with infusible probes may provide a more
global assessment of pO2

Desirable Features

Chemical and spectroscopic:

Water soluble
Kinetically and metabolically stable
Single and narrow line resonance
Linewidth ∝ pO2
Pharmacologic:
In vivo life time  imaging time

Toxicology:
Non-toxic at concentrations required for imaging
 Physical Basis for Hyperpolarization of Nuclei
Dynamic Nuclear Polarization with Paramagnetic Agents
Overhauser Effect

Overhauser, A; Phys. Rev. (1953)

Dynamic nuclear polarization

Φ C
.
“…applicable to conduction electrons in alkali metals”
Φ

Φ
Hyperpolarized MRI using EPR and Paramagnetic Contrast Agents
Lurie DJ, Bussell DM, Bell LH, Mallard JR ,
Proton- Electron Double Magnetic-Resonance Imaging of Free-Radical Solutions
J. Magnetic Resonance 76, 366-370 (1988)
Contrast Agents for Hyperpolarization
of 1H, 13 C
Trityl Radicals

Gomberg, M; JACS. (1900)

Triphenyl methyl: A case of trivalent carbon
…. “I reserve the field for myself.”

Golman, K et. al.

Overhauser-enhanced MR imaging (OMRI)
Acta Radiologica 39, 10-17(1998)
 Contrast Agent - Trityl Radical

OX063 • Mouse MTD = 2.5 - 5 mmol/kg

.
Φ C Φ
• T1/2 in mouse blood and
kidney: 9-10 min
Φ
• Linewidth: 100 - 300 mG
Oxygen tension: 0 - 21 %
HOCH2 S S CH2OH
Φ =
HOCH2 S S CH2OH Nycomed Imaging
Nycomed Amersham
COO- Na+ Amersham
GE
 Overhauser MRI
Combination of EPRI + MRI
MRI for anatomy and EPR for Spectral information

MR Imaging of Hyperpolarized Water Protons by EPR

Low magnetic field (~10 mT)
Uses Free Radical Paramagnetic contrast agents
Coil tuned to both NMR and EPR frequencies
Scanner
• Field strengths and Pulse Frequencie

EPR NMR
Magnet (mT): 8 15
Resonators: 226 MHz 640 kHz

• Magnet and resonator dimensions

Resonator Magnet
Diameter (cm): 2.5 80
Length (cm): 8 125

• Frequency Encoding
Gradient: 1.5 G/cm
• Phase Encoding
Gradient 64 steps
MRI – Contrast Agent MRI + Contrast Agent
3.0

Kidney
2.4

1.8 Bladder

1.2 Tumor

0.6

Int

Image Intensity enhanced by:

1) Contrast Agent concentration
2) Hypoxic/Ischemic regions
 Oxygen Mapping with OMRI
Rat, respiratory model
1.5 mmol/kg
100

mm Hg

Time 0:45 6:20 12:00 17:30 23:20

(min.)
% Oxygen in 21% 9.5% 21% 9.5% 21%
breathing air

Oxygen Maps from OMRI Correspond with Changes in Tissue Oxygenation

Dynamic changes in pO2 can be monitored

Air 140

(Oxygen, mm Hg)
120
100
80
60
40
20
0
Carbogen
140

(Oxygen, mm Hg)
120
100
80
60
40
20
0
Overhauser MRI/Summary

Currently implemented in mice, rats.

For human applications:

Contrast agents/Safety
SAR/Localized coils
 Hyperpolarized 13 C MRI
Implications for Molecular Imaging

Challenges in Imaging of 13 C labeled molecules with MRI.

1) Lower concentration compared to protons

2) Lower magnetic moment than protons (25%)
3) Lower Polarization than protons (25%)
 Sensitivity Considerations in MRI of 1H and 13 C at 3 Tesla
Magnetic Field

1
H 13
C 13
C Hyperpolarized

C, M 80 0.1 0.1

γ (MHz/T) 42.5 10.7 10.7

Polariz., P 1.10-5 2.10-6 0.5

cγ P 0.034 2.14.10-6 0.535

Molecular Imaging With Endogenous Substances.

Golman et al: PNAS Vol 100, 10158-10163, 2003.
Golman et al: PNAS Vol 100, 10435-10439, 2003.
Strategy for Hyperpolarization of 13 C labeled Molecules by EPR
for in vivo imaging

• Mix the 13 C labeled compound with trityl radical

• Freeze to 1. 4 K and put it in 2.7 T magnet
• Irradiate with 95 GHz radiation (EPR)
• Thaw it to room temperature, inject and image in< 2min!

Golman et al PNAS 2003

Ardenkjaer et al PNAS 2003
The DNP C polariser 13
Solid material doped with
13
C unpaired electrons
13
C
13
13
C C
13
C 13
C
13
C
13
C
13
C 13
C
13
C e-
13
C 13
C
13
C
13
C
13
C 13
C
13
C
13
C
13
C 13
C
13
C
13
C
13
C 13
C
13
C
13
C
13
C 13
C
13
C e-
e- 13
C
13
C
13
C
13
C 13
C
13
C
13
C
13
C 13
C
13
C
13
C 13
C 13
C
13
C
13
C
13
C 13
C
Pe= 94% and PC=130.086%
C

3.35 T and
Pellet 1.2K
of 13C
3. 35 T
molecule doped Microwaves transfer
95 GHz
with the polarisation
1.2 K
paramagnetic
from electrons to nuclei
substance
 NMR Spectrum of 13 C Urea (natural Abundance)
9.4 Tesla (400 MHz)
C Urea at normal polarization
13

A) Acquisition time: 65 Hours

Polarization: 7.5 ppm

Single Shot NMR Spectrum

Acquisition time: 1s
Polarization of 13 C Urea 20%
B)
Signal Loss of hyperpolarized 13 C Urea in a mouse after iv bolus
120

100

80

60

40

20

0
0 10 20 30 40 50 60 70

Time (s)

There is ~ minute for image data

acquisition before polarization is lost.
 Metabolic fates of
pyruvate
Pyruvate
Transamination
Reduction

Lac
nine tate
Al a

Carboxylation Oxidative
decarboxylation

Oxaloacetate Acetyl-CoA

Withis suitable
Pyruvate hyperpolarized
converted into lactate, alanine, molecules,
oxaloacetate or Acetyl-CoA depending on the
it is possible to distinguish breakdown
needs of the tissues.
products based on their chemical shifts
by 13 C MRI.
In vivo metabolic mapping
using 13 C-pyruvate

3D surface rendering
Imaging pO2 using EPR Imaging and
paramagnetic molecules
Direct detection of contrast agent by EPRI
Image collection using static magnetic field gradients
Images of spin probe distribution
pO2 maps obtained by T2* weighted imaging
 TIME DOMAIN EPR EXPERIMENTS

dead time
0.3 - 1.0 µs
pulse width
10 - 100 ns
FID
FT

1.0 - 4.0 µs frequency

The EPR Signals last ~ microseconds after RF pulse

 EPR/Single Point Imaging
After the dead time, the phase of one point after a fixed time delay is monitored at
different gradient magnitudes per direction.
The resultant envelope is equivalent of a Gradient Recalled Echo.
FT of this envelop gives the spatial projection
RF pulse

τ
time
Dead

r
time

Gradient ramping and pseudo-echo

Gx
FT
Gy
Gz
Resolution independent of line width.
Fourier reconstruction possible with static gradients

Frequency ( 1D spatial profile)

We implemented Fourier Imaging Capabilities in EPRI.

 EPR Experiment in Time-Domain.

Dead-time Line width, ν = 1/(π T2)

FT

Time (ns) Frequency (MHz)

We have Developed EPR Instrumentation with

nanosecond time resolution
EPR can be used for pO2 imaging by post-processing for T2* effects.

τ
RF pulse

time Int.

cm
Gx

Using several times points in the echo for image reconstruction

it is possible to estimate oxygen dependent line width of the
contrast agent
 Oxygen Provides Excellent T2 Contrast to
Paramagnetic Contrast Agents
Phantom Schematic
-20 0.16

5% 2.5 %
-15 Intensity Image 0.14

-10 0.12

-5
0.1

mm
0
0% 1% 0.08
5
0.06
10
0.04
15
0.02
LineWidthMapping
20
250
0
-20 -20 -15 -10 -5 0 5 10 15 20
240
-15 pO2 dependent T2 Map 230
mm

-10
220 250
-5 240
210
230
Lw in mG
mm

220
0 200

5 190 210
180
200
190
10

15
170
180
160 170
20

150
160
-20 -15 -10 -5 0 5 10 15 20
150
0 1 2 3 4 5
mm

Oxygen concentration %
Intensity Image
Sagital View ( 1mm Slices)
 Line Width Image
Sagital View ( 1mm Slices)
Nitroxides can provide redox status dependent contrast in MRI

R R

(n) (n)
Reduction

Oxidation

N. N
O OH

n = 1, piperidine nitroxides eg. Tempol, Tempo, Tempone

n = 0, pyrrolidine nitroxides. Eg. Carbamoyl proxyl, carboxy proxyl

Nitroxide radical Hydroxylamine

Paramagnetic Diamagnetic
Provides enhancement in T1 based MRI Does not provide T1 contrast in MRI
 Time Course of Tempol-Induced
Tempol-Induced T1 contrast in Image Intensity in Tumor Bearing
Tumor Bearing Mouse Mouse

GEFI: TR75, TE3, FA45

B C D

Tumor Normal

Evo = 1/60 Evo = 8/60 Evo = 15/60

E F G
Evo = 1/60

Evo = 1/60 A
Evo = 22/60 Evo = 29/60 Evo = 60/60
%Difference in Intensity Green: +; Red: -

Tempol-Induced T1 contrast in Tumor Bearing Mouse changes with time after administration.

Time-intensity profiles in muscle and tumor are different

 Change in Tempol-induced MR Intensity Enhancement as a Function of Time

3 y = -0.8592x + 5.2778

ln(% change)
R2 = 0.9954

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

y = -0.2735x + 3.126
3
ln(% change)

R2 = 0.7954

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

Intensity change with time is faster in tumor than normal tissue.

Nitroxides are reduced faster in tumors than in normal tissue
 Time Course of 3-CP-Induced
3CP-Induced T1 contrast in Image Intensity in Tumor Bearing
Tumor Bearing Mouse Mouse

MSME: TR4000, TE450 GEFI: TR75, TE3, FA45

GEFI: TR75, TE3, FA45

Tumor Normal
Evo = 1/60 Evo = 8/60 Evo = 15/60

Evo = 1/60

Evo = 1/60 Evo = 22/60 Evo = 29/60 Evo = 60/60

3CP-Induced T1 contrast in Tumor Bearing Mouse changes with time after administration.

Time-intensity profiles in muscle and tumor are different

%Difference
 4

ln(% change)
2
y = -0.1074x + 4.074
1 R2 = 0.9978

0
0 5 10 15 20
Time (min)
Tumor

3
ln(% change)

2
y = -0.0698x + 3.9395
R2 = 0.9933
1

0
0 5 10 15 20
Time (min)
Normal
 ACKNOWLEDGEMENTS
John Cook, Ph. D
Deva Devasahayam, MS (EE)
Fuminori Hyodo, Ph. D
Janusz Koscielniak, Ph. D
Atsuko Matsumoto M.S
Ken-Ichiro Matsumoto, Ph. D
Sankaran Subramanian, Ph. D
James B. Mitchell, Ph. D
David Wink, Ph. D
Angelo Russo, Ph. D, M. D
Amram Samuni, Ph. D
Klaes Golman, Amersham, Sweden
Jan-Henrik Ardenkjaer, Amersham,
Sweden