In vivo EPR and EPR Imaging

Bernard Gallez

Biomedical Magnetic Resonance Research GroupLouvain Drug Research InstituteUniversité catholique de Louvain

Brussels

November 2012

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EPR / ESREPR / ESR

Magnetic Resonance TechniqueMagnetic Resonance Technique

that detects electron spins that detects electron spins

in impaired electron compoundsin impaired electron compounds

(paramagnetic species)(paramagnetic species)

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

Comparison between NMR and EPR

Frequency / magnetic field ratioFrequency / magnetic field ratio

Paramagnetic materialsParamagnetic materials

Short relaxation timesShort relaxation times

EPR vs NMRFrequency / 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

EPR vs NMRFrequency / 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)

EPR vs NMRParamagnetic 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

EPR vs NMRShort 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

EPRI is capable of measuring the distribution of paramagnetic and free radical species in samples

EPR SpectroscopySpatially-unresolved

0 + 1 dimensional

Spatial ImagingSpatially-resolved

Spin density3 + 0 dimensional

Spectral-spatial ImagingSpatially-resolved

Spectral shape

3 + 1 dimensional

From P. Kuppusamy

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

What can we learn

from an EPR spectrum and image ?

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

Characterization/Mapping of stable

paramagnetic free radicals

EPRI of melanin in melanoma

E. Vanea et al, NMR Biomed. 2008, 21, 296-300

NH

O

HO

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 metastasesQ. Godechal et al, CMMI 2011, 6, 282-288

Applications to human melanoma samplesQ. Godechal et al, Exp. Dermatol 2012, 21, 341-346Q. Godechal et al., Mol. Imaging. 2012 in press

PhotopolymerisationPhotopolymerisation Long-lived free radicals in the polymer matrixLong-lived free radicals in the polymer matrix

Free radicals in dental resins

P. Leveque et al, J. Magn. Reson. 2012 ;220:45-53

Spectral-spatial imaging in dental resins

P. Leveque et al, J. Magn. Reson. 2012 ;220:45-53

High power Low power

Commercial ExperimentalResin Resin

cm

High power Low power

Commercial ExperimentalResin Resin

EPRI and dosimetry

Radiations ( 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)

EPR signal is due to COEPR signal is due to CO22-- radicals induced by radicals induced by

radiations in hydroxyapatite radiations in hydroxyapatite CaCa1010(PO(PO44))66(OH)(OH)22 (teeth, bones)(teeth, bones)

3300 3320 3340 3360 3380 3400 3420 3440

Field (Gauss)

1.997 (g//)

2.003 (g)

EPRI and dosimetryEPRI and dosimetry

Dose gradient in irradiated bones

-5

15

35

55

75

95

-4 -3 -2 -1 0 1 2 3 4 5 6 7 8

dist (mm)

Re

l. In

t. (

%)

Ir-192

I-125

P. Leveque et al, Med. Phys 2009, 36, 4223-4229

• 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 measuredand compared to Monte Carlo simulation

EPRI dosimetry in brachytherapy

E. Vanea et al, Magn Reson Med. 2009 : 61, 1225-31N. Kolbun et al, Med. Phys. 2010;37:5448-55.

Coffee bean

Peppercorn

Frog’s leg

Sunflower seed

Licorice flavored sweets

P. Leveque et al, Isr. J. Chem 2008, 48, 19-26

Characterization/mapping of reactive

free radicals by spin trapping

Indirect free radical detection by « spin trapping »

To be applied on reactive free radicalsTo be applied on reactive free radicals Trapping by a nitrone to form a stable spin adductTrapping by a nitrone to form a stable spin adduct Detection – identification - QuantificationDetection – identification - Quantification

EPR Imaging of Nitric Oxide

Spin trapping in vivo

S. Fujii et al, Am J Physiol 274: G857-G862, 1998

Spin labeling

use of paramagnetic reporters use of paramagnetic reporters

sensitive to their environmentsensitive to their environment

Molecular dynamics / microviscosityApplication in drug delivery systems

2**105.61

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10

I

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IHxc

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00

10

I

IHxc

= 3kT c / 4 r3

3280 3300 3320 3340

H (Gauss)

NaOH

PEG400/MOG/SA (45/5/50)

mmePEG750-p(CL-co-TMC) 50/50

5mM

50mM

5mM

50mM

I0I+1 I-1

3280 3300 3320 3340

H (Gauss)

NaOH

PEG400/MOG/SA (45/5/50)

mmePEG750-p(CL-co-TMC) 50/50

5mM

50mM

5mM

50mM2Amax

2Amax

2Amin

2Amin

N. Beghein et al, J. Control. Release 2007, 117, 196-203

B. Gallez et al, Magn. Reson.Med 1996, 36, 694-697

pH measurementsChange in hyperfine splitting

First in vivo applicationEffect of anti-acids on pH of stomach

Recent developmentTrityl probe for extracellular pH

B. Driesschaert et al,Chem. Commun., 2012,48, 4049-4051

EPR Oximetry

OO22 dependent broadening of dependent broadening of

the the EPREPR linewidth of a linewidth of a

paramagnetic Oparamagnetic O22 sensor sensor

implanted in the tumorimplanted in the tumor

A particular material can be A particular material can be

calibrated in terms of the calibrated in terms of the

effect of oxygen on the LWeffect of oxygen on the LW

When introduced in vivo, the When introduced in vivo, the

measurement of LW can be measurement of LW can be

interpreted in terms of interpreted in terms of

oxygenation in the vicinity of oxygenation in the vicinity of

the probethe probe

3168 3318 3468

Magnetic Field (G)

0

10

20

30

40

0 7 14 21

% O2

LW

(G

)

air

nitrogen

B. Gallez, NMR Biomed. 2004,17, 240

Spectral spatial imaging:

Each voxel yields a spectrum whose line width increases linearly with local oxygen concentration

EPR line broadening for current narrow line spin probes: approximately 0.5 mG/torr O2

Oxygen map

From H. Halpern

Tumor-hypoxia guided combination of treatmentsCombination of oxygen modulator with Radiation

Therapy

0 15 30 45 60 75 90 105 120 1350

10

20

30

40

50

60As2O3

CTRL

Time (min)

pO

2 (

mm

Hg

)

CTRL As2O3

pO2 (

mm

Hg)

121086420

Effect of As2O3 and radiation on TLT tumor regrowth

0 5 10 15 20 25 30 35 40 456

8

10

12

14

16

18

Time (days)

Tu

mo

r si

ze (

mm

)

0 5 10 15 20 25 30 35 40 456

8

10

12

14

16

18

0 5 10 15 20 25 30 35 40 456

8

10

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0 5 10 15 20 25 30 35 40 456

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C. Diepart et al, Cancer Res 2012, 72, 482

EPR oximetryapplied in biomedical sciences

Collaborations of our group: 2007-2012

TumorsKULeuven

P. Carmeliet-M. Mazzone

Cell 2009, 136, 839-851Nature Genetics 2008, 40, 170-180

MusclesKULeuvenMazzone

Nature 2011, 479,122-126

Pancreas islets graftsUCL

D. Dufrane

Biomaterials 2011, 32, 5945-5956Tissue Eng A 2010, 16, 1503-1513

BrainKULeuven

P. Carmeliet

J. Neurosci 2010, 30, 15052-15066

TumorsDuke University

M. Dewhirst

PNAS 2010, 107, 20477-20482

TumorsVUB

M. Deridder

IJROBP 2010, 76, 1520-1527

LiverKULeuven

P. Carmeliet

Gastroenterology 2010, 138, 1143-1154

Ovarian graftsUCL

J. Donnez

Fertil. Steril. 2009, 92, 374-381

TumorsUCL

O. Feron – P. Sonveaux

Mol. Cancer Res. 2009, 7, 1056-1063FEBS 2009, 276, 509-518

J. Clin. Invest. 2008, 118, 3930-3942Clin. Cancer Res. 2008, 14, 2768-2774Am. J. Pathol. 2007, 171, 1619-1628

IJROBP 2007, 67, 1155-1162

Submitted/In preparationPancreas, Endometrium

Perspectives of EPRClinical Applications in EPR oximetry

Biocompatibility of the oxygen sensors

Instrumental developments

Biocompatibility of the oxygen sensors

Clearance of oxygen sensors for use in human subjectsClearance of oxygen sensors for use in human subjects

Nitroxides and Trityl radicals:Nitroxides and Trityl radicals:

usual procedures via FDA, EMEA, …usual procedures via FDA, EMEA, … India ink:India ink:

grandfathered for human usegrandfathered for human use

Other particulate materials:Other particulate materials:

encapsulate with approved permeable biocompatible material / remove encapsulate with approved permeable biocompatible material / remove short term after useshort term after use

B. Gallez et al, MRM 1999, 42, 193B. Gallez et al, MRM 1999, 42, 193

B. Gallez et al, Free Rad. Biol. Med. 2000, 29, 1078B. Gallez et al, Free Rad. Biol. Med. 2000, 29, 1078

J. He et al, MRM 2001, 46, 610J. He et al, MRM 2001, 46, 610

J. He et al, Phys Med. Biol. 2001, 46, 3323J. He et al, Phys Med. Biol. 2001, 46, 3323

M. Dinguizli et al, Biosens. Bioelectr. 2006, 21, 1015M. Dinguizli et al, Biosens. Bioelectr. 2006, 21, 1015

M. Dinguizli et al, Physiol. Meas. 2008, 29, 1247M. Dinguizli et al, Physiol. Meas. 2008, 29, 1247

H.M. SwartzMRM 1994, 31, 229

N. Charlier et al, NMR Biomed 2004, 17, 303

Biocompatible « ink » used in first human EPR studies

EPR Oximetry : Clinical HardwareBy courtesy of B. Williams and H.M. Swartz

Dartmouth Medical School

Carbogen

breathing

N. Khan, Antiox. Redox. Signal. 2007, 9, 1169

Air

breathing

Clinical EPR…Does it make sense?...

MRI evolution

1946 1973 1978-1980 1990-2012

Lauterbur, NatureJ.Hutchinson and J. Mallard

Bloch, Purcell

Evolution of in vivo EPR and EPRI

1945 1985 2004 ?

Zavoisky Berliner, Science H.M. Swartz

?

Clinical EPR in Europa