Novel Imaging Modalities EPR/ERP http://iminds.creativemediadays.be/session/novel-imaging-modalities

1. LDRI
RESEARCH
INSTITUTE
LOUVAIN
DRUG
In vivo EPR and EPR Imaging
Bernard Gallez
Biomedical Magnetic Resonance Research Group
Louvain Drug Research Institute
Université catholique de Louvain
Brussels
November 2012

2. 1 cm 1 cm
cm
1 cm
a b

3. 1 cm 1 cm
EPR / ESR
Magnetic Resonance Technique
cm
1 cm
a that detects electron spins
b
in impaired electron compounds
(paramagnetic species)

4. Aims of this talk
To give you a flavour
about the challenges and opportunities
linked to the development
of in vivo EPR and EPR imaging

5. Comparison between NMR and EPR
 Frequency / magnetic field ratio
 Paramagnetic materials
 Short relaxation times

6. EPR vs NMR
Frequency / Magnetic Field Ratio
Gyromagnetic ratio of unpaired electron
659 times larger than that of a proton
Frequency / Magnetic Field ratio
Electron: 28 GHz/T
Proton: 42.5 MHz/T

7. EPR vs NMR
Frequency / Magnetic Field Ratio
Standard EPR spectrometers operate
at much higher frequencies and lower fields
than conventional NMR spectrometers
9.5 GHz (X-Band) / ~ 34 mT
Non resonant absorption of the electromagnetic radiation
by the liquid water of the biological samples
Need for reducing the operating frequency:
Increase the penetration depth
200 MHz to 1.5 GHz (L-Band)

8. EPR vs NMR
Paramagnetic materials
 In vivo, lack of sufficient amounts of naturally
occurring paramagnetic materials
 Short half life of most free radicals
 Stable paramagnetic materials should be
introduced in the system
 Beneficial aspects:
 absence of background signals (except melanin)
 Sensitivity of EPR: around 700 times greater than NMR on
a molar basis

9. EPR vs NMR
Short relaxation times
 Time scale of relaxation:
 Electron: nanoseconds
 Proton: milliseconds-seconds
Most EPR spectra obtained through continuous wave
experiments
 Linewidth:
 EPR: kHz-MHz
 NMR: Hz
EPR Imaging: gradients orders of magnitude larger than
those used in MRI

10. EPRI is capable of measuring the distribution
of paramagnetic and free radical species in
samples
EPR Spectroscopy Spatial Imaging Spectral-spatial Imaging
Spatially-unresolved Spatially-resolved Spatially-resolved
Spin density Spectral shape
0 + 1 dimensional 3 + 0 dimensional 3+1
dimensional
From P. Kuppusamy

11. What can we learn
from an EPR spectrum and image ?

12. What can we learn
from an EPR spectrum and image ?
 Is there a signal? In which condition?
 Intensity of the signal: amount of paramagnetic compounds
 Position of the signal (g-value): characterization of the chemical
entity
 Shape of the EPR signal
 Dependent on the physical environment (mobility, viscosity,…)
 Spin-spin coupling
 Coupling electron – nucleus
 Coupling electron – electron
 Relaxation time
 Oxygen-broadening of the EPR linewidth

13. Biomedical applications of EPR/EPRI
 Characterization / Mapping of stable
paramagnetic free radicals
 Characterization / mapping of reactive free
radicals by spin trapping
 Spin labeling
use of paramagnetic reporters sensitive to
their environment

14. Characterization/Mapping of stable
paramagnetic free radicals

15. O
HO
N
H
EPRI of melanin in melanoma
E. Vanea et al, NMR Biomed. 2008, 21, 296-300

16. First in vivo EPR Image of an endogenous radical
E. Vanea et al, NMR Biomed. 2008, 21, 296-300
Characterization of primary tumors
and distant metastases
Q. Godechal et al, CMMI 2011, 6, 282-288
Applications to human melanoma samples
Q. Godechal et al, Exp. Dermatol 2012, 21, 341-346
Q. Godechal et al., Mol. Imaging. 2012 in press

17. Free radicals in dental resins
 Photopolymerisation
 Long-lived free radicals in the polymer matrix
P. Leveque et al, J. Magn. Reson. 2012 ;220:45-53

18. Spectral-spatial imaging in dental resins
High power Low power High power Low power
cm
Commercial Experimental
Resin Resin Commercial Experimental
Resin Resin
P. Leveque et al,
J. Magn. Reson. 2012 ;220:45-53

19. EPRI and dosimetry
 Radiations (γ or X rays) induce free radicals
formation
 In some samples, CO3- radical can be detected
 Bones
 Teeth
 Nails
 EPR spectroscopy is internationally recognised
as a standard method for dosimetry
 EPRI is now investigated for application in dosimetry
(brachytherapy)

20. EPRI and dosimetry
EPR signal is due to CO2- radicals induced by
radiations in hydroxyapatite Ca10(PO4)6(OH)2
2.003 (g )
(teeth, bones) ⊥
1.997 (g//)
3300 3320 3340 3360 3380 3400 3420 3440
Field (Gauss)

21. Dose gradient in irradiated bones
95
Ir-192
I-125
75
Rel. Int. (%)
55
35
15
-5
-4 -3 -2 -1 0 1 2 3 4 5 6 7 8
P. Leveque et al, Med. Phys 2009, 36, 4223-4229
dist (mm)

22. EPRI dosimetry in brachytherapy
• Lithium formate and ammonium formate are radiosensitive materials
giving a suitable EPR signal
• Tablets were pressed, and holes drilled inside
• 125I brachytherapy sources were positionned in holes
• After irradiation, tablet were measured by EPRI
Dose gradient can be measured
and compared to Monte Carlo
simulation
E. Vanea et al, Magn Reson Med. 2009 : 61, 1225-31
N. Kolbun et al, Med. Phys. 2010;37:5448-55.

23. Sunflower seed
Licorice flavored sweets
Coffee bean
Frog’s leg
Peppercorn
P. Leveque et al, Isr. J. Chem 2008, 48, 19-26

24. Characterization/mapping of reactive
free radicals by spin trapping

25. Indirect free radical detection by « spin trapping »
 To be applied on reactive free radicals
 Trapping by a nitrone to form a stable spin adduct
 Detection – identification - Quantification

26. EPR Imaging of Nitric Oxide
Spin trapping in vivo
S. Fujii et al, Am J Physiol 274: G857-G862, 1998

27. Spin labeling
use of paramagnetic reporters
sensitive to their environment

28. Molecular dynamics / microviscosity
Application in drug delivery systems
 I0 I0   I0 
τ c = 6.5 x10 −10
* ∆H 0 *  + − 2 τ c = 6.5 x10 −10
* ∆H 0 *  − 1
 I +1 I −1   I +1 
mmePEG750-p(CL-co-TMC) 50/50 mmePEG750-p(CL-co-TMC) 50/50
50mM
2A max 50mM
2A min
5mM
5mM
I+1 I0 I-1
PEG400/MOG/SA (45/5/50) PEG400/MOG/SA (45/5/50)
50mM
η= 3kT τ c / 4 πr 3 2A max
50mM
5mM 2A min
5mM
NaOH
NaOH
3280 3300 3320 3340
3280 3300 3320 3340
H (Gauss)
H (Gauss)
N. Beghein et al, J. Control. Release 2007, 117, 196-203

29. pH measurements
Change in hyperfine splitting
Recent development
Trityl probe for extracellular pH
B. Driesschaert et al,
Chem. Commun., 2012,48, 4049-4051
First in vivo application
Effect of anti-acids on pH of stomach
B. Gallez et al,
Magn. Reson.Med 1996, 36, 694-697

30. EPR Oximetry
B. Gallez, NMR Biomed. 2004,17, 240
 O2 dependent broadening of
nitrogen
the EPR linewidth of a
paramagnetic O2 sensor air
implanted in the tumor
 A particular material can be
calibrated in terms of the 3168 3318 3468
Magnetic Field (G)
effect of oxygen on the LW
40
 When introduced in vivo, the 30
LW (G)
measurement of LW can be 20
interpreted in terms of 10
oxygenation in the vicinity of 0
0 7 14 21
the probe % O2

31. Spectral spatial imaging:
Each voxel yields a spectrum
whose line width increases linearly
with local oxygen concentration
Oxygen map
EPR line broadening for current
narrow line spin probes: approximately
0.5 mG/torr O2
From H. Halpern

32. Tumor-hypoxia guided combination of treatments
Combination of oxygen modulator with Radiation Therapy
C. Diepart et al, Cancer Res 2012, 72, 482
60
As2O3 12
50
CTRL
pO2 (mmHg)
10
pO2 (mmHg)
40
8
30 6
20
4
2
10
0
0
0 15 30 45 60 75 90 105 120 135 CTRL As2O3
Time (min)
Effect of As2O3 and radiation
18
18
18
on TLT tumor regrowth
16
16
16
Tumor size (mm)
14
14
14
12
12
12
10
10
10
8
88
6
6
0
00 5
55 10 15 20 25 30 35 40 45
10 15 20 25 30 35 40 45
10 15 20 25 30 35 40 45
Time (days)

33. EPR oximetry
applied in biomedical sciences
Collaborations of our group: 2007-2012
Tumors Tumors Brain
KULeuven Duke University KULeuven
P. Carmeliet-M. Mazzone M. Dewhirst P. Carmeliet
Cell 2009, 136, 839-851 PNAS 2010, 107, 20477-20482 J. Neurosci 2010, 30, 15052-15066
Nature Genetics 2008, 40, 170-180
Tumors Muscles Liver
UCL KULeuven KULeuven
O. Feron – P. Sonveaux Mazzone P. Carmeliet
Mol. Cancer Res. 2009, 7, 1056-1063 Nature 2011, 479,122-126 Gastroenterology 2010, 138, 1143-1154
FEBS 2009, 276, 509-518
J. Clin. Invest. 2008, 118, 3930-3942
Clin. Cancer Res. 2008, 14, 2768-2774
Pancreas islets grafts Ovarian grafts
UCL UCL
Am. J. Pathol. 2007, 171, 1619-1628
D. Dufrane J. Donnez
IJROBP 2007, 67, 1155-1162
Biomaterials 2011, 32, 5945-5956 Fertil. Steril. 2009, 92, 374-381
Tumors Tissue Eng A 2010, 16, 1503-1513
VUB
M. Deridder
Submitted/In preparation
IJROBP 2010, 76, 1520-1527 Pancreas, Endometrium

34. Perspectives of EPR
Clinical Applications in EPR oximetry
 Biocompatibility of
the oxygen sensors
 Instrumental
developments

35. Biocompatibility of the oxygen sensors
Clearance of oxygen sensors for use in human subjects
 Nitroxides and Trityl radicals:
usual procedures via FDA, EMEA, …
 India ink:
grandfathered for human use
H.M. Swartz
MRM 1994, 31, 229
 Other particulate materials:
encapsulate with approved permeable biocompatible material / remove
short term after use
B. Gallez et al, MRM 1999, 42, 193 N. Charlier et al,
B. Gallez et al, Free Rad. Biol. Med. 2000, 29, 1078 NMR Biomed 2004, 17, 303
J. He et al, MRM 2001, 46, 610
J. He et al, Phys Med. Biol. 2001, 46, 3323 Biocompatible « ink »
M. Dinguizli et al, Biosens. Bioelectr. 2006, 21, 1015 used in first human EPR studies
M. Dinguizli et al, Physiol. Meas. 2008, 29, 1247

36. EPR Oximetry : Clinical Hardware
By courtesy of B. Williams and H.M. Swartz
Dartmouth Medical School
Air ing
th
brea
n
ge
r bo ing
Ca ath
bre
N. Khan, Antiox. Redox. Signal. 2007, 9, 1169

37. Clinical EPR…
Does it make sense?...

38. MRI evolution
J.Hutchinson
Bloch, Purcell Lauterbur, Nature
and J. Mallard
46 1973 1978-1980 1990-2012

39. Evolution of in vivo EPR and EPRI
Zavoisky Berliner, Science H.M. Swartz
?
1945 1985 2004 ?

40. Clinical EPR in Europa