Evaluation of Enzyme Substrate Radiotracers as PET/MRS Hybrid Imaging Agents

Article abstract of DOI:10.1021/acsmedchemlett.8b00402

The development of a positron emission tomography (PET)/magnetic resonance spectroscopy (MRS) hybrid imaging agent allows for functional imaging by both methods with a single imaging agent. Enzyme substrates that are cleaved to form two metabolites present an interesting opportunity, as the unique metabolites generated might each be detected by a different modality. To be successful, such enzyme substrates would require administration of doses that (a) reach the in vivo target tissue at concentrations necessary for MRS imaging, (b) do not show substrate inhibition of tissue uptake or enzymatic activity, and (c) provide PET images that still reflect the action of the enzyme. We report in vitro and in vivo proof-of-concept studies of a carbon-11 small molecule substrate for brain monoamine oxidases that, upon enzyme-mediated cleavage, produces two metabolites, one detectable by PET and the other by MRS.

Full text of DOI:10.1021/acsmedchemlett.8b00402

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Letter
Evaluation of Enzyme Substrate Radiotracers
as PET/MRS Hybrid Imaging Agents.
Allen F Brooks, Lindsey R Drake, Xia Shao, Austin Zhao, Peter J. H. Scott, and Michael R. Kilbourn
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00402 • Publication Date (Web): 03 Oct 2018
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Evaluation of Enzyme Substrate Radiotracers as PET/MRS Hybrid
Imaging Agents.
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Allen F. Brooks,a,† Lindsey R. Drake,b,† Xia Shao,a Austin Zhao,a Peter J. H. Scott,a,b,* Michael R. Kil-
bourn.a,*
a Department of Radiology, University of Michigan Medical School, 1301 Catherine Street, Ann Arbor, Michigan 48109,
United States.
b Department of Medicinal Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, Michigan
48109, United States.
KEYWORDS. Hybrid Imaging • Carbon-11 • Positron Emission Tomography • Magnetic Resonance Spectroscopy • Neu-
roimaging.
ABSTRACT: The development of a PET/MRS hybrid imaging agent allows for functional imaging by both methods with a
single imaging agent. Enzyme substrates that are cleaved to form two metabolites present an interesting opportunity, as the
unique metabolitesgenerated might each be detected bya different modality. To be successful, such enzyme substrates would
require administration of doses which (a) reach the in vivo target tissue at concentrations necessary for MRS imaging, (b) do
not show substrate inhibition of tissue uptake or enzyme activity, and (c) provide PET images that still reflect the action of
the enzyme. We report in vitro and in vivo proof-of-concept studies of a carbon-11 small molecule substrate for brain mono-
amine oxidases that upon enzyme-mediated cleavage produces two metabolites, one detectable by PET and the other byMRS.
Positron emission tomography (PET) imaging is a non-in-
vasive molecular imaging method that has been utilized for
decades in studies of physiology and pharmacology in living
animals and human subjects.1,2 Most applications of PET
target high affinity but low capacity sites on proteins (e.g.
receptors, enzymes and transporters) and typically require
high molar activities (radioactivity per mole), with clear de-
creases of specific binding as the mass of the injected radio-
tracer is increased.3 The molar activities commonly accepta-
ble for PET imaging (>37 GBq/µmol; >1,000 Ci/mmol) are
thus usually considered incompatible with the higher con-
centrations (mmol) of imaging agents required for alternate
functional imaging methods like magnetic resonance spec-
troscopy (MRS).4
The recent development of PET/MR imaging instrumen-
tation has made it possible to simultaneously acquire imag-
ing data for both modalities.5-7 However, combining PET im-
aging with functional magnetic resonance spectroscopy
(MRS) imaging has proven challenging. This would require
a PET imaging agent that can function successfully at the
high massconcentrations requiredfor MRS imaging, despite
the resulting low molar activityknowntobe problematic for
PET radioligands. We hypothesized that the technique of
metabolic trapping, whereby the product of an enzymatic
reaction is trapped and retained in tissues at the site of the
enzyme, might provide a suitable approach to combining
PET and MRS: if the enzyme does not exhibit substrate inhi-
bition in vivo, then a high mass/low molar activity PET radi-
otracer should still function well. To test this hypothesis, we
have examined potential metabolically-trapped substrates
for monoamine oxidases (MAO-A and –B), enzymes of con-
siderable interest in studies of brain biochemistry using
PET, and report an example of a small molecule hybrid
PET/MRS imaging agent.
Our prior radiotracer development efforts yielded several
carbon-11 labeled N-methyl-4-aryloxy-1,2,3,6-tetrahydro-
pyridines,8 based on the work of Castagnoli and cowork-
ers,9,10 which are in vitro and in vivo substrates for MAO-A or
MAO-B. These function in vivo as metabolic trapping PET
agents, as the enzymatic action releases a carbon-11 labeled
N-methylpyridinone that is retained in brain tissues. Inclu-
sion of a coumarin group as the phenoxy substituent pro-
duced a radiotracer that released both radiolabeled and flu-
orescent metabolic products (Figure 1a), similar to a previ-
ous report of a MAO-B selective fluorogenic probe (but
which was not radiolabeled).11
To produce a combined PET/MRS radiotracer, we desired
a 4-aryloxy substituent which would provide a metabolite
with a unique signal visible by MRS following MAO oxida-
tion. For this, we turned to the work of Yamaguchi et. al. who
reported a MAO substrate described as a chemical shift-
switching agent (Figure 1b), as the 19F NMR signal of the
substrate (a 2-fluoro-4-nitrophenyl ether) could be distin-
guished from that of the metabolite (2-fluoro-4-nitrophe-
nol).12 We thus hypothesized that combining 2-fluoro-4-ni-
trophenol with the N-[11C]methyl-1,2,3,6-tetrahydro-pyri-
dine moiety should provide a molecule suitable for testing
our hypothesisthat a combined PET/MRS radiotracer isfea-
sible using the metabolic trapping concept (Figure 1c).
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To next demonstrate that the compound would be useful
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a) PET/Fluorescence (Our Previous Work8):
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as a chemical shift-switching agent for MRS, enzymatic re-
actions with hMAO-A and 1 were run for 90 min and
quenched by addition of an excess of the irreversible inhib-
itor clorgyline. 19F NMR analyses showed that over the time
period the signal for substrate 1 at -130 ppm was replaced
by that of the phenol metabolite 4 at -136 ppm, as well as a
secondary metabolite at -129 ppm (Figure 3).
O
MAO, H2O
O
O
O
+
N
HO
O
O
11CH3
N
PET Trapped Fluorescent Metabolite
Metabolite
11CH3
b) Chemical Shift-Switching (Yamaguchi12):
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NO2
F
NO2
MAO, H2O
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H2N
O
HO
19F MRI Metabolite
c) A PET/MR Hybrid Probe (This Work):
F
NO2
O
F
NO2
O
MAO, H2O
+
N
11CH3
HO
N
11CH3
PET Trapped
Metabolite (3)
19F MRI Metabolite (4)
[
11C]1
Figure 1. Design of a PET/MRS hybrid probe for monoamine
oxidases, building on published examples of PET/fluorescence
and chemical shift-switching in vitro MAO substrates.
Figure 3. 19F NMR experiments (376 MHz) demonstrat-
ing time-dependent formation of 2-fluoro-4-nitrophenol
metabolite (-136 ppm) & secondary metabolite (-129 ppm)
via oxidation of substrate 1 (-130 ppm) by MAO-A.
The authentic standard 4-(2-fluoro-4-nitrophenoxy)-1-
methyl-1,2,3,6-tetrahydropyridine (1) and corresponding
N-desmethyl compound (2) were prepared using methods
previously reported for syntheses of 4-aryloxy substituted
tetrahydropyridines.8,13 To first demonstrate that the pro-
posed radiotracer was a substrate for monoamine oxidases,
kinetic studies were done with solutions of human MAO
(hMAO)-A or –B and using the change in absorbance from
substrate to metabolite in a spectrophotometric assay. As
shown in Figure 2, 1 is a substrate for both hMAO-A and –B,
with similar catalytic efficiencies (kcat/KM) for both iso-
zymes.
With 1 confirmed in vitro as a MAO chemical shift-switch-
ing agent, the last step wasto radiolabel with carbon-11 and
evaluate whether [11C]1 could be utilized as a PET imaging
agent at low molar activity. The procedure to radiolabel
[11C]1 was similar to the method previously described.8,13
Briefly, [11C]carbon dioxide was converted by standard pro-
cedure into [11C]CH3OTf and sparged through a solution of
the pyridine precursor (1 mg) in ethanol (0.2mL). The
[11C]methyl pyridinium intermediate was transferred to a
conical vial containing NaBH4 (2 mg) in ethanol (0.3mL) and
stirred for 5 min. [11C]1 was then purified by HPLC and re-
formulated into a 10% ethanol in saline dose (392±207
MBq; non-decay corrected 1.2% radiochemical yield based
on starting [11C]CH3OTf; 30 min from end of bombardment)
for use in animal imaging studies. Final product was ob-
tained in high molar activities(>259 GBq/µmol), and to pre-
pare doses of low molar activity [11C]1, unlabeled standard
was added to the saline used for reformulation. Final molar
activities were confirmed using HPLC analysis of the UV-
254nm absorbances.
kcat
(min-1)
KM
kcat/KM
(min-1µM-1)
0.0022
MAO is known to be a highly conserved enzyme between
species with, for example, high sequence homology be-
tween hMAO and rat MAO (rMAO).14 Therefore kinetic PET
imaging studieswere conducted inSprague Dawleyrats. Ex-
periments conducted with high (259 GBq/µmol) and low
(0.0185 GBq/µmol) molar activity [11C]1 gave very similar
kinetic curves for whole brain radioactivity (Figure 4).
These data were also used in conjunction with specific ac-
tivity to generate curves estimating mass of tracer in the
brain (inset, Figure 4). For the low specific activity PET
studies, 1.29 ± 0.86 µmol of 1 was administered, similar to
(µM)
MAO-A 0.0416 ± 0.0016
19 ± 2.3
67 ± 4.5
MAO-B
0.240 ± 0.048
0.0036
Figure 2. Michaelis-Menten kinetic analysis data for
MAO-A and –B by absorbance assay.
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ACS Medicinal Chemistry Letters
the mass administered for prior rodent 19F-MRI studies,15
the tissues will reflect the transporter function, as previ-
ously described for the glutathione-conjugated metabolites
of radiolabeled purines.18 Future work will address the in-
teresting possibilities of designing bifunctional PET-MRS
brain imaging tracers based on MAO, and the extension to
other enzymes that can be studied using the metabolic trap-
ping principle.
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and peak uptake of 1 in the rodent brain was ~0.02 mM.
Given that the total concentration of MAO (A + B) in the ro-
dent brain is ~11 pmol/mg protein,16 and that rat brain is
~10% protein,17 this corresponds to an estimated total
rMAO concentration of ~1.1 pmol/mg tissue (0.0011 mM).
Thus [1]/[rMAO] ~18 and, given the high catalytic effi-
ciency of MAO (Figure 2), we would not expect to over-
whelm MAO at this concentration of 1 in the brain. Reflect-
ing this, there was indeed no evidence that the MAO en-
zymes in the brain tissues were subject to in vivo substrate
inhibition at this concentration.
ASSOCIATED CONTENT
Supporting Information
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The Supporting Information is available free of charge on the
ACS Publications website at DOI: XXX:
Experimental procedures for chemistry and radiochemistry
as well as spectra / chromatograms for all novel compounds
synthesized. Procedures for in vitro assays and in vivo rodent
PET imaging.
AUTHOR INFORMATION
Corresponding Author
* Email: pjhscott@umich.edu
* Email: mkilbour@umich.edu
Author Contributions
The manuscript was written through contributions of all au-
thors, and all authors have given approval to the final version
of the manuscript. † These authors contributed equally.
Funding Sources
We thank NIH (R21NS075553 (MRK); T32GM007767 (PJHS
and LRD)) and the University of Michigan (Energy Insti-
tute/Michigan MemorialPhoenixProject (AFB), Michigan Insti-
tute for Clinical and Health Research/TREC (LRD), College of
Pharmacy (LRD and PJHS), Dept of Radiology (PJHS) and Rack-
ham Graduate School (LRD)) for financial support.
Figure 4. Main panel: averaged (n = 2) rodent whole brain
time-radioactivity curves (0 – 60 min post-iv-injection of
Notes
The authors declare no competing financial interest.
£
high (259 GBq/µmol, ) and low (0.0185 GBq/µmol, n)
molar activity doses of [11C]1). Inset: whole brain concen-
ACKNOWLEDGMENTS
We thank Phillip Sherman, Janna Arteaga and Jenelle Stauff for
conducting pre-clinical PET imaging.
Å
tration curve (0 – 60 min post-iv-injection of low ( ) molar
activity doses of [11C]1).
In summary, enzyme substrates that form trapped me-
tabolites appear as a promising way forward to design
PET/MRS hybrid imaging agents. In the system evaluated
here, the enzyme reaction produces two metabolites that
are separatelyvisible byPET or NMR. Whenconsidering po-
tential in vivo applications of such a hybrid PET-MRS imag-
ing agent inthe future, ifboth metabolitesare trappedin the
tissue they will yield a measure of the same parameter –
MAO oxidation. Of more interest would be if the two meth-
ods gave different biochemical measures. How might that
be achieved? Two possibilities come to mind. First, if the
MRS-visible metabolite is freely diffusible across the BBB
but is designed to have high affinity but reversible binding
to a specific CNS site (e.g., receptor), then the retention and
delayed clearance from the brain tissue might reflect the
amount of specific binding (the pharmacokinetics of deliv-
ery are identical to the radiotracer trapping curve). As an
alternative, if the metabolite is designed not to be BBB per-
meable but instead is a substrate for an efflux transporter
(e.g., P-glycoprotein - Pgp), then the rate of clearance from
ABBREVIATIONS
BBB, Blood-brain barrier; Bq, Becquerel; CNS, Central nervous
system; MAO, Monoamine oxidase; PET, Positron emission to-
mography; Pgp, P-glycoprotein.
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NO2
O
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NO2
O
MAO, H2O
+
N
11CH3
HO
N
11CH3
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PET Trapped
Metabolite
19F MRI Metabolite
MAO Substrate
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