A myelin-specific contrast agent for magnetic resonance imaging of myelination

Article abstract of DOI:10.1021/ja1040896

Myelination is one of the most fundamental biological processes in the development of vertebrate nervous systems. Abnormal or disrupted myelination occurs in many acquired or inherited neurodegenerative diseases, including multiple sclerosis (MS) and vari

Full text of DOI:10.1021/ja1040896

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A Myelin-Specific Contrast Agent for Magnetic Resonance
Imaging of Myelination
Luca Frullano,† Changning Wang,† Robert H. Miller,‡ and Yanming Wang*,†
†Division of Radiopharmaceutical Science, Case Center for Imaging Research, Department of Radiology, and
‡Department of Neuroscience, Case Western Reserve University, Cleveland, Ohio 44106, United States
S Supporting Information
b
a dual-echo T2-weighted sequence reflects a change in tissue water
content, which is a nonspecific measure of the overall changes in
macroscopic tissue injury ranging from edema to inflammation,
demyelination, and axonal loss.8 As a result, the use of MRI as a
primary measure of disease activity still has not been accepted by
the FDA, as it was shown to be dissociated from the clinical attack
rate in disease-modifying therapies. This dissociation was evi-
denced by a clinical study of interferon-βmeasured by conventional
MRI parameters.9 Although additional MRI techniques, such as
magnetization transfer (MT)10 and diffusion tensor imaging
(DTI),11 have been developed, use of these techniques still lacks
specificity for more destructive myelin pathology.
Thus, a long-standing goal has been to develop a direct measure
of myelination that will effectively correlate clinical outcomes with
myelin changes. This requires the development of contrast agents
for MR imaging of myelin. Recently, an attempt has been made to
use Luxol Fast Blue (LFB), a histological stain for myelin with a
paramagnetic copper core, as a MR probe.12 However, LFB shows
low relaxivity (r1 = 0.09 s-1 mM-1, room temperature, 4.7 and
14 T), modest solubility, and limited tissue permeability and
requires post-incubation tissue processing to differentiate mye-
linated regions.
ABSTRACT: Myelination is one of the most fundamental
biological processes in the development of vertebrate
nervous systems. Abnormal or disrupted myelination oc-
curs in many acquired or inherited neurodegenerative
diseases, including multiple sclerosis (MS) and various
leukodystrophies. To date, magnetic resonance imaging
(MRI) has been the primary tool for diagnosing and
monitoring the progression of MS and related diseases;
however, any change in signal intensity of conventional
MRI reflects a change only in tissue water content, which is a
nonspecific measure of the overall changes in macroscopic
tissue injury. Thus, the use of MRI as a primary measure of
disease activity was shown to be disassociated from the clinical
outcome due to the lack of specificity for myelination. In
order to increase the MRI specificity for myelin pathologies,
we designed and synthesized the first Gd-based T1 MR
contrast agent (MIC) that binds to myelin with high
specificity. In this Communication, we demonstrate that
MIC localizes in brain regions in proportion to the extent of
myelination. In addition, MIC possesses promising MR
contrast properties, which allow for direct detection of
myelin distribution through T1 mapping in the mouse brain.
For this reason, we set out to develop a small-molecule MR
contrast agent that is myelin specific with suitable MR-imaging
properties that could potentially be applied in vivo. In this Com-
munication, we report, for the first time, the design and synthesis of
a Gd-based T1 MR contrast agent that binds specifically to myelin
membranes and demonstrate its efficacy to discriminate myelinated
regions in mouse brains by MRI.
n this Communication, we report a myelin-specific Gd-based
magnetic resonance contrast agent for imaging of myelination.
I
Myelination is one of the most fundamental biological processes
in nervous system development that defines the vertebrate
species.1 Myelin sheaths provide a unique structure in the
nervous system that fosters rapid and efficient conduction of
impulses along axons.2 Abnormalities or destruction of myelin
occur in many acquired or inherited neurodegenerative diseases,
including multiple sclerosis (MS) and various leukodystrophies.3
MS, which affects an estimated 350 000 people in the United
States and 2 million people worldwide, is the most commonly
acquired myelin disease in humans.4 The leukodystrophies, on
the other hand, are the result of inherited enzyme deficiencies
that cause abnormal formation, destruction, and/or abnormal
turnover of myelin sheaths within the central nervous system
(CNS) white matter.5 One major challenge has been assessing
and quantifying changes in myelin content in vivo.
Over the past years, we have developed a wide array of small-
molecule probes for myelin imaging. One of these imaging agents,
termed CMC, readily enters the brain and selectively binds to
myelinated fibers.13 In addition, our ongoing studies have shown
that the structure of CMC is amendable and methylation or
alkylation of the amino group has no negative impact on its
myelin-binding property. We thus hypothesized that the structure
of CMC could be modified by conjugation with a Gd complex for
MR imaging while preserving its ability to bind specifically to
myelin. To test this hypothesis, we designed and synthesized MIC
as shown in Scheme 1.
The coumarin derivative with a nucleophilic hydroxyl group (4)
was prepared in two steps via a Perkin condensation of 1 and 2,
followed by hydrolysis. A three-carbon spacer was then introduced
To date, magnetic resonance imaging (MRI) has been the
primary tool for diagnosing and monitoring the progression of
myelin diseases.6,7 Unfortunately, any change in signal intensity on
Received: May 12, 2010
Revised:
December 7, 2010
Published: January 25, 2011
r
2011 American Chemical Society
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Scheme 1. Synthesis of MIC, a MR Contrast Agent
for Myelin
Figure 1. (A) MIC staining of the whole mouse brain tissue section in
vitro and (B) subsequent immunohistochemical staining for MBP of the
same section. (C) MIC staining of the cerebellum and (D) subsequent
immunohistochemical staining for MBP of the same section. For clarity,
the MIC staining and the subsequent MBP staining of the myelin-deficient
subcortical gray matter region (E,H) and the myelin-rich corpus callosum
(F,I) and striatumregions (G,J) are highlighted separately. Scale bar: A,B =
500 μm, C,D,F,I = 300 μm, E,H = 100 μm, G,J = 50 μm.
by reacting 4 with Boc-protected 3-bromopropylamine. After
deprotection, 6 was coupled with DOTA-tris-tert-butyl ester (7).
Treatment with neat trifluoroacetic acid yielded the deprotected
ligand, which gave the myelin-targeting probe MIC upon com-
plexation with GdCl3 in water.
MIC's longitudinal relaxivity (r1 = 5.2 s-1 mM-1, 21 °C, 9.4 T)
is more than 50 times greater than that of LFB. At clinically relevant
magnetic fields such as 1.41 and 0.47 T, MIC's longitudinal
relaxivity (r1) at 40 °C is 5.1 and 5.8 s-1 mM-1, respectively,
which is ca. 1.5 times greater than those of commercial MR contrast
agents such as Magnevist and Dotarem at the same magnetic
fields.14 The transmetalation stability of MIC toward Zn proved
to be excellent and in line with other DOTA-monoamide
complexes.15
Figure 2. Representative MR T1 maps of wild-type (A) and shiverer
(B) mouse brain tissues. (C) Average water proton relaxation rate in
MIC-treated wild-type (black) and shiverer (dashed) mouse brains and
untreated wild-type (white) and shiverer (gray) mouse brains (n = 3). (D)
Average fluorescent intensity of wild-type mouse brain sections treated with
MIC (n = 5). The error bars represent the standard error of the mean.
MIC is a fluorescent compound with maximal excitation and
emission at 460 and 570 nm, respectively. We thus examined the
myelin-binding property of MIC by fluorescent staining of mouse
brain tissue sections. For comparison, the same sections were then
subjected to immunohistochemical staining for myelin basic protein
(MBP), a distinctive protein that is characteristic of myelin mem-
branes. Axial sections of the whole brain were used to examine
myelin-binding properties of MIC in different brain regions, either
rich or poor in myelin. As shown in Figure 1A,B, MIC staining (blue)
is very similar to immunohistochemical staining (red). At a 100 μM
concentration, MIC selectively labeled myelinated fibers in myelin-
rich white matter regions such as the cerebellum (Figure 1C), corpus
callosum (Figure 1F), and striatum (Figure 1G). Subsequent
immunohistochemical staining for MBP in the same regions
(Figure 1B,D,I,J, respectively) allowed us to determine if MIC
binds to myelin, oligodendrocytes, or both. From the data
presented, we concluded that MIC selectively binds to myelin
but not oligodendrocytes, which accounts for the small difference
between MIC and immunohistochemical staining for MBP in the
images shown below. For example, the myelin-deficient subcortical
gray matter region showed little MIC staining (Figure 1E) but more
MBP staining (Figure 1H). These studies demonstrate that MIC
selectively binds to myelin present in the white matter regions, while
MBP staining also labels oligodendrocyte cell soma and processes.
Based on the high binding specificity of MIC for myelin, the MR
properties of MIC were then comprehensively evaluated in the
brains of wild-type mice and C3Fe.SWV-Mbpshi/J shiverer mice,
an autosomal recessive mutant with myelin deficiency in the CNS.
After 24 h of incubation in 1.0 mM water solution of MIC (0.4 mL)
followed by extensive washing with saline, the brain tissues were
imaged by MR for both proton density weighted images and T1
mapping.
For MR imaging, T1 was measured at 9.4 T and room tempera-
ture using a multiple TR spin-echo acquisition (TE 7.0 ms, TR
100-2500 ms, 0.098 Â 0.098 cm/pixel, matrix size 256 Â 256), and
T1 maps were generated using the QuickVol II plug-in in ImageJ
(Figure 2A,B).16 Prior to incubation with MIC, T1 maps were
acquired in both wild-type and shiverer mouse brains. In MIC
untreated brains, different regions could not be conspicuously
distinguished in T1 maps. After incubation with MIC, contrast
was observed in T1 maps of wild-type mouse brains in proportion
to the myelin content. In the MIC-treated wild-type mouse
brains, the longitudinal relaxation rates and fluorescent intensi-
ties in the myelinated white matter regions (i.e., corpus callosum
and the external capsule) are significantly higher than those in the
subcortical region (p < 0.05) (Figure 2C,D). In the MIC-treated
myelin-deficient shiverer mouse brains, T1 is more uniform
throughout the brain. The T1 maps showed no statistical
difference between the gray and white matter (p > 0.6) within
the same shiverer mouse brains. Compared to the wild-type mice,
T1 of the shiverer mice is significantly longer in the white matter
regions (p < 0.05). Thus, the MR data suggest that retention of
MIC is proportional to the level of myelination in the brain.
Following the MR imaging studies, the stability of MIC in the
incubated tissues was verified by HPLC analysis of extracts of the
pulverized brain tissues.
In conclusion, we developed the first Gd-based MRI contrast
agent that selectively localizes in myelinated brain regions and allows
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COMMUNICATION
for myelin mapping in MR images. The exact binding mechanism of
MIC remains to be delineated. Such a MR contrast agent is expected
to improve the specificity of MR imaging of myelination in both
peripheral and central nervous systems. Further studies are under way
to investigate the in vivo MR properties, biodistribution, and pharma-
cokinetics of MIC in animal models of myelin-related diseases.
’ ASSOCIATED CONTENT
S
Supporting Information. Detailed experimental proce-
b
dures, including synthetic methods, protocols for animal preparation,
chemical and immunohistochemical staining protocols, and MR
imaging procedures; statistical analysis of MIC distribution; analysis
of MIC stability in incubated tissues; fluorescent imaging; MIC
transmetalation stability; and relaxivity measurements. This material
is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
yanming.wang@case.edu
’ ACKNOWLEDGMENT
We gratefully acknowledge financial support from the Depart-
ment of Defense (MS090082), National Multiple Sclerosis
Society, and NIH/NINDS (R01 NS061837). We thank Dr.
Christopher Flask for his advice and expertise on MR imaging
protocols.
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