Real-Time Interrogation of Aspirin Reactivity, Biochemistry, and Biodistribution by Hyperpolarized Magnetic Resonance Spectroscopy

Article abstract of DOI:10.1002/anie.201812759

Hyperpolarized magnetic resonance spectroscopy enables quantitative, non-radioactive, real-time measurement of imaging probe biodistribution and metabolism in vivo. Here, we investigate and report on the development and characterization of hyperpolarized acetylsalicylic acid (aspirin) and its use as a nuclear magnetic resonance (NMR) probe. Aspirin derivatives were synthesized with single- and double-¹³C labels and hyperpolarized by dynamic nuclear polarization with 4.7 % and 3 % polarization, respectively. The longitudinal relaxation constants (T₁) for the labeled acetyl and carboxyl carbonyls were approximately 30 seconds, supporting in vivo imaging and spectroscopy applications. In vitro hydrolysis, transacetylation, and albumin binding of hyperpolarized aspirin were readily monitored in real time by ¹³C-NMR spectroscopy. Hyperpolarized, double-labeled aspirin was well tolerated in mice and could be observed by both ¹³C-MR imaging and ¹³C-NMR spectroscopy in vivo.

Full text of DOI:10.1002/anie.201812759

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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International Edition
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Accepted Article
Title: Real-Time Interrogation of Aspirin Reactivity, Biochemistry,
and Biodistribution by Hyperpolarized Magnetic Resonance
Spectroscopy
Authors: Niki M Zacharias, Argentina Ornelas, Jaehyuk Lee,
Jingzhe Hu, Jennifer Davis, Md. Nasir Uddin, Shivanand
Pudakalakatti, David Menter, Jose A Karam, Christopher
G. Wood, Ernest Hawk, Scott Kopetz, Eduardo Vilar, Pratip
Bhattacharya, and Steven Millward
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201812759
Angew. Chem. 10.1002/ange.201812759
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Real-Time Interrogation of Aspirin Reactivity, Biochemistry, and
Biodistribution
Spectroscopy
by
Hyperpolarized
Magnetic
Resonance
Niki M. Zachariasa,b‡, Argentina Ornelasa‡, Jaehyuk Leea, Jingzhe Hua, Jennifer Davisc, Md. Nasir Uddina,
Shivanand Pudakalakattia, David Menterd, Jose A. Karamb, Christopher G. Woodb, Ernest T. Hawke,
Scott Kopetzd, Eduardo Vilard,e, Pratip K. Bhattacharyaa, Steven W. Millwarda*
Abstract: Hyperpolarized magnetic resonance spectroscopy (HP-
MR) enables quantitative, non-radioactive, real-time measurement of
imaging probe biodistribution and metabolism in vivo. Here, we
investigate and report on the development and characterization of
hyperpolarized acetylsalicylic acid (aspirin) and its use as a nuclear
magnetic resonance (NMR) probe. Aspirin derivatives were
synthesized with single- and double-13C labels and hyperpolarized by
dynamic nuclear polarization with 4.7% and 3% polarization,
respectively. The longitudinal relaxation constants (T1) for the labeled
acetyl and carboxyl carbonyls were approximately 30 seconds,
supporting in vivo imaging and spectroscopy applications. In vitro
hydrolysis, transacetylation, and albumin binding of hyperpolarized
aspirin were readily monitored in real time by 13C-NMR spectroscopy.
Hyperpolarized, double-labeled aspirin was well tolerated in mice and
could be observed by both 13C-MR imaging and 13C-NMR
spectroscopy in vivo.
Given this metabolic and toxicologic variability, it is desirable
to develop method to monitor aspirin metabolism and
a
biodistribution in real time in patients. While radiotracer-based
methods have been used to interrogate aspirin biodistribution and
pharmacokinetics,[7] these approaches cannot distinguish
between the intact compound and its metabolic byproducts and
are poorly adaptable to routine patient imaging.
The advent of hyperpolarized nuclear magnetic resonance
(NMR) spectroscopy enables the real-time interrogation of
imaging probe metabolism in living systems.[8] In 13C
hyperpolarized NMR experiments, a 13C-labeled compound (e.g.,
[1-13C]-pyruvate) is polarized by one of a variety of methods (e.g.,
dynamic nuclear polarization [DNP],[9] parahydrogen and
synthesis allow dramatically enhanced nuclear alignment
[PASADENA][10], or brute force[11]), rapidly injected into the animal
or phantom, and its metabolism observed by 13C spectroscopy or
spectroscopic imaging.[12]
Recent epidemiologic evidence strongly suggests that long-
term low-dose acetylsalicylic acid (aspirin) use significantly
decreases the incidence of many cancers.[1] While this effect may
be mediated by cyclooxygenase-2 (COX-2) inhibition in the tumor
and surrounding stroma,[2] aspirin’s highly promiscuous
transacetylation activity[3] suggests alternative mechanisms of
action.
Aspirin is initially metabolized in vivo by hydrolysis of the O-
acetyl group (position 8, Figure 1) to form acetate and salicylic
acid. Following hydrolysis, salicylic acid is conjugated to glycine
or glucuronic acid at the exocyclic carboxyl group (position 7).
Aspirin metabolism is heterogeneous and can vary as much as
20-fold among individuals[4], potentially modulating its
chemopreventive effect within patient populations[5]. Long-term
aspirin use can also result in gastrointestinal bleeding and
intracranial hemorrhages in some patients,[6] further complicating
its routine use as a cancer chemopreventive.
[a]
Dr. A. Ornelas, Dr. J. Lee, Dr. J. Hu, Dr. M. Uddin, Dr. S.
Pudakalakatti, Prof. P. K. Bhattacharya, and Prof. S.W. Millward.
Department of Cancer Systems Imaging
E-mail: smillward@mdanderson.org
[b]
[c]
[d]
[e]
Prof. N. Zacharias, Prof. J. Karam, Prof C. Wood.
Department of Urology
Dr. J. Davis
Figure 1: 13C-labeled acetylsalicylic acid (aspirin) derivatives synthesized
in this study. 13C-labeled carbons are represented by a red dot. The carbon
numbering scheme used throughout this manuscript is shown. Percent
polarization values (Php) and median longitudinal relaxation constants (T1) for
single-labeled (n=2) and double-labeled (n=2) aspirin at 7T are provided ±
standard error of the mean.
Department of Epidemiology
Prof. D. Menter, Prof. S. Kopetz, Prof. E. Vilar
Department of Gastrointestinal Medical Oncology
Prof. E. T. Hawk, Prof. E. Vilar
Department of Clinical Cancer Prevention
The University of Texas MD Anderson Cancer Center
1515 Holcombe Blvd., Houston, TX 77030
Here, we report the successful hyperpolarization of 13C-
[‡]
These authors contributed equally to this work.
labeled aspirin; real-time monitoring of its in vitro hydrolysis,
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transacetylation, and albumin-binding reactions; and visualization
of its early biodistribution in mice. This work represents one of the
first demonstrations of 13C-NMR spectroscopy as a tool to track
the biodistribution of a clinical drug in vivo as well as the first use
of aspirin as a hyperpolarized MR probe.
mM.
Approximately 168 µmoles labeled aspirin were
hyperpolarized in each experiment with gadolinium III complex
(Prohance, 1.8 mM, Bracco Diagnostics) added to each sample
to enhance solid-state polarization. Samples were polarized for 1
to 1.5 hours and then dissolved in 4 mL of either phosphate-
buffered saline (PBS) in deuterium oxide (pH = 7.2) or PBS in
10% deuterium oxide to a final concentration of ~42 mM
hyperpolarized aspirin. Following dissolution, the hyperpolarized
material was rapidly injected in either a 7T horizontal-bore MR
scanner or a vertical-bore 7T NMR spectrometer (See Figure S7
for a schematic of the experimental setup)
In the case of hyperpolarized (1), a single major peak at 173.3
ppm was observed by 13C-NMR spectroscopy (Figure 2a). The
chemical shift of the major peak was consistent with the O-13C
acetyl carbon of intact aspirin (Figure S2). There was no indication
of hydrolysis of the hyperpolarized compound after dissolution or
during spectroscopy. The percent polarization (Php) was
approximately 4.7% at the time of detection by comparison to the
signal from the same, thermally polarized sample.
This
represents a >3000-fold signal enhancement relative to the
unpolarized material at 7T field strength[14]. Double-labeled aspirin
(2) showed enhanced polarization buildup in the solid state
relative to the single-labeled compound, likely resulting from the
addition of the second 13C nucleus. 13C-NMR spectroscopy of
hyperpolarized (2) showed two partially resolved resonances at
173.3 and 173.6 ppm (Figure 2d) corresponding to the O-13C
acetyl carbonyl (position 8) and carboxylic acid carbonyl carbons
(position 7), respectively (Figure S4). The combined polarization
for both carbonyls in (2) was approximately 3%[14]
.
The T1 of the hyperpolarized O-acetyl 13C-carbonyl in
°
hyperpolarized (1) was measured in 100% D2O and at 37 C by
NMR using a 12° flip angle every 6 seconds and found to be 27 ±
2 seconds in longitudinal relaxation experiments, sufficient for
approximately 1 minute of imaging in vivo. These experiments
showed nearly identical values for the O-acetyl 13C-carbonyl
carbon in hyperpolarized (2) (30 ± 3 seconds) and a T1 of 33 ± 2
seconds for the 13C-labeled carboxylic acid carbon (position 7,
Figure 1). Previous work has shown that incorporation of
deuterium nuclei within 1-2 bonds of 13C nuclei eliminates
relaxation pathways and enhances the T1 of the 13C label.[15] With
this in mind, derivative (3), which is deuterated at the O-acetyl
methyl carbon, was synthesized and hyperpolarized by DNP.
Unfortunately, no significant effect on the T1 of the O-acetyl
carbonyl carbon was observed which may indicate that the methyl
protons of aspirin do not play a significant role in the spin-lattice
relaxation of this nucleus (Figure S8).
Figure 2. In vitro reactions with hyperpolarized (1) and (2). a)
Hyperpolarized (1) in phosphate-buffered saline (100% D2O, 7T). b)
Hyperpolarized (1) in 1 M KOH hydrolyzes rapidly to form hyperpolarized
acetate (4), seen at 181.5 ppm. c) Hyperpolarized (1) in 1 M KOH and 250 mM
Nα-acetyl lysine undergoes rapid transacetylation to form Nα, 13C-Nε-diacetyl
lysine (6), seen at 174.1 ppm. d) Hyperpolarized (2) in phosphate-buffered
saline (100% D2O). Both 13C-labeled carbons were observed as a partially
resolved doublet at 173.3-173.6 ppm (7T). e) Hydrolysis of hyperpolarized (2)
in excess KOH resulted in the formation of 13C-acetate (4) and 13C-salicylic acid
(7) (178.7 ppm). f) Reaction with hyperpolarized (2) and Nα-acetyl lysine
showed the expected Nα, 13C-Ne-diacetyl lysine (6) as well as 13C-acetate and
13C-salicylic acid.
We reasoned that labeling the O-acetyl (position 8) and
carboxyl carbonyl (position 7) carbons of aspirin with 13C would
allow us to monitor both aspirin transacetylation and metabolism
in vivo by changes in the chemical shift of these nuclei. Single-
(1) and double-labeled (2) aspirin (Figure 1) were readily obtained
by the reaction of salicylic acid or labeled [7-13C]-salicylic acid with
[1,1-13C2]-acetic anhydride or [1-13C]-acetyl chloride followed by
silica gel chromatography and recrystallization from ethanol[13]
(Figures S1-S6). Both aspirin derivatives were polarized using
We next sought to determine whether hydrolysis and
transacetylation reactions with hyperpolarized (1) could be
observed by hyperpolarized 13C-NMR. Initial NMR and mass
spectrometry studies of non-hyperpolarized, 13C-labeled aspirin
showed rapid transacetylation of glycine and Nα-acetyl lysine
(Figures S9 and S10), as evidenced by the appearance of peaks
at 173.9 ppm and 173.8 ppm corresponding to acetylation of the
α-amino group of glycine and the Nε-amino group of Nα-acetyl
lysine, respectively. The chemical shifts of the acetylated
products are well-resolved from the intact aspirin O-acetyl
carbonyl resonance at 173.3 ppm and are consistent with those
DNP (HyperSense, Oxford Instruments). In
a
typical
hyperpolarization experiment, 13C-labeled aspirin was dissolved
in a 63% (v/v) dimethyl sulfoxide-d6 and water solution to a final
concentration of 1.5 M. Oxo63 free radical (Oxford Instruments)
was added directly to the mixture to a final concentration of 15.6
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10.1002/anie.201812759
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observed in previous transacetylation studies with non-
hyperpolarized 13C-labeled aspirin.[16] Reaction of hyperpolarized
(1) with excess KOH (pH = 10-11) showed rapid hydrolysis to
acetate (Figure 2b). In contrast, no hydrolysis was observed at
biological pH (pH = 7.2). Addition of Nα-acetyl lysine (Figure 2c)
in the presence of hyperpolarized (1) and excess KOH showed
rapid and complete conversion to the expected N, N-diacetyl
product within approximately 20 seconds. A parallel set of
experiments with hyperpolarized (2) showed rapid conversion to
acetate and salicylic acid (181.3 and 178.7 ppm, respectively) in
the presence of KOH (Figure 2e) and conversion to acetate,
salicylic acid, and Nα, 13C-Nε-diacetyl lysine in the presence of
KOH and Nα-acetyl lysine (Figure 2f). The resonances for
acetate and salicylic acid were well resolved from the parent
resonance (∆δ = 8.0 and 5.4 ppm respectively), suggesting that
these products can be readily detected in vivo by MR
spectroscopy. The T1 values for aspirin derivatives and reaction
products (Table S1) were similar to those of the carbons at
positions 7 and 8 of aspirin, while the T1 of the acetate carbonyl
carbon was significantly higher, as expected on the basis of
previous studies.[17] These experiments demonstrated that
transacetylation and hydrolysis reactions of both hyperpolarized
aspirin derivatives could be monitored in real time by 13C-NMR on
the time scale of the hyperpolarized signal.
hyperpolarized material (42 mM, 3% polarized) via a tail-vein
catheter. An 8-M 13C-urea phantom was used for chemical shift
referencing. 13C imaging was performed using an echo-planar
imaging (EPI) sequence, and hyperpolarized signal was observed
in the inferior vena cava (<5 seconds post-injection) and the heart
(>5 seconds post-injection) (Figures 4a and b).
In preparation for animal studies, we investigated the
interaction of hyperpolarized aspirin with serum albumin using
hyperpolarized NMR. Human albumin contains multiple aspirin-
binding sites[18] and is covalently acetylated by aspirin at lysine
Figure 3. Interaction between hyperpolarized (1) and bovine serum
albumin (BSA). a) Stacked 13C-NMR spectra of hyperpolarized (1) in
phosphate buffered-saline (PBS, 10% D2O) and 50 mg/mL BSA in PBS (10%
D2O). Median T1 values for the major resonances in each spectrum are shown
± standard deviation (n = 2). b) Average linewidths of the major peaks (W1/2 in
Hz) in each hyperpolarized spectrum are shown along with the standard
deviation (n = 2). c) Stacked non-hyperpolarized 13C-NMR spectra of (1) in PBS,
PBS+BSA, and PBS+BSA with 3-fold excess 12C-aspirin (1000 scans per
spectrum). d) Median linewidths of the major aspirin resonance in each
spectrum along with the standard deviation (n=4). Two-way analysis of variance
(ANOVA, multiple comparisons) was used to assess statistical significance (****,
P < 0.0001).
residues in
a
pH-dependent manner.[19] Incubation of
hyperpolarized (1) with a 5% solution of bovine serum albumin
(BSA) in PBS resulted in the appearance of a broad resonance
centered at 172.9 ppm that was partially resolved from a sharp
minor resonance at 173.1 ppm (Figure 3a). The linewidth of the
broad resonance was 15 Hz, and the T1 was approximately 15
seconds. In contrast, the linewidth of the sharp minor resonance
was 5 Hz, consistent with hyperpolarized (1) in PBS (Figure 3b).
Analogous 13C-NMR experiments with non-hyperpolarized (1)
showed a similar line-broadening effect in the presence of BSA
which was significantly reduced by competition with excess 12C-
aspirin (Figures 3c, d).
Images with an acceptable signal-to-noise ratio could be acquired
for up to 10 seconds following injection of the hyperpolarized
material. A series of slice-selective 13C spectra were collected 1-
10 seconds after injection of hyperpolarized material. A single
slice was placed over the majority of the animal, and single free
induction decay (FID) signals were acquired every 2 seconds. The
in vivo spectroscopy showed the expected double peak of (2), but
no hydrolysis or transacetylation products were observed
(Figures 4c and d). The full time courses of both imaging
experiments are shown in Figures S11 and S12 and enlarged
versions of 4a and 4b are shown in Figures S13 and S14.
These experiments suggest that the increased linewidth and
relaxation rate of (1) in the presence of BSA arises from exchange
between the free and bound form. This finding is consistent with
previous studies showing that the aromatic protons of aspirin
undergo increased line-broadening and T1 reduction upon
albumin binding,[20] likely owing to a decreased tumbling rate in
the bound form. Further support for exchange-mediated line
broadening is seen in the decreased linewidth of (1) in 12C-aspirin
competition experiments where binding site occupancy by the
unlabeled aspirin inhibits exchange of (1) with the protein.
Although these observations support exchange-mediated line-
broadening, we cannot rule out contributions from dipolar
coupling between bound (1) and BSA.
Both 13C-labeled aspirin derivatives synthesized in this work
were efficiently polarized by DNP within 90 minutes and retained
this polarization after dissolution. The longitudinal relaxation times
for both 13C-labeled carbonyls were approximately 30 seconds,
which was sufficient for in vivo imaging and spectroscopy. In vitro
base-catalyzed hydrolysis and transacetylation reactions of both
hyperpolarized derivatives could be followed in real time by NMR
spectroscopy, suggesting that this technique could be used to
observe analogous reactions in vivo. Binding of hyperpolarized
(1) to serum albumin was readily observed by changes in
Finally, we sought to monitor the in vivo biodistribution and
metabolism of hyperpolarized (2), which offered the possibility of
monitoring metabolism at both the O-acetyl and carboxylic acid
groups. Normal male nude mice were injected with 200 µL of
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10.1002/anie.201812759
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linewidth, relaxation time, and chemical shift, suggesting that Experimental Section
hyperpolarized NMR spectroscopy can be used for label-free real-
Detailed synthetic protocols and experimental methods along
with Figures S1-S16 and Table S1 are available in the Supporting
Information document.
time analysis of aspirin-protein binding interactions. This finding
is consistent with previous work showing the utility of
hyperpolarized 19F-NMR for the study of ligand-protein binding
interactions in the slow- and fast-exchange regimes.[21]
Acknowledgements
This work was supported by MD Anderson start-up funds (S.W.M.,
P.B.), R21CA181994 (S.W.M.), 2R44CA206771 (S.W.M.),
R21CA185536 (P.B.), CPRIT RP180164 (P.B.), CDMRP
PC110065 (N.Z., J.L., P.B.), Colleen’s Dream Foundation (NZ),
an MD Anderson Institutional Research Grant (N.Z., P.B.), and a
grant from the Gulf Coast Consortium (J.L., P.B.). Additional
funding was provided by a G.E. In-kind Multi-investigator Imaging
(MI2) Research Award (S.W.M, P.B.), the Boone Pickens
Distinguished Chair for Early Prevention of Cancer, The Duncan
Family Institute, the Colorectal Moon Shot, 1R01CA187238,
5R01CA1722670, and 1R01CA184843. A.O. was supported by a
cancer prevention educational award (R25T CA057730, Dr. Shine
Chang, PI). S.P. was supported by Gulf Coast Consortia/Keck
Center postdoctoral fellowship CPRIT RP170593. The MD
Anderson Nuclear Magnetic Resonance Facility and Small Animal
Imaging Facility (SAIF) are supported by the MD Anderson
Cancer Center Support Grant 5P30CA016672 (Dr. Peter Pisters).
We also thank the staff at the SAIF and the NMR facility for their
assistance with the magnetic resonance imaging experiments
and the Department of Scientific Publications at MD Anderson
Cancer Center for reading and editing this manuscript.
Figure 4. In vivo imaging and spectroscopy of hyperpolarized double-labeled
aspirin (2). a) Overlaid 1H and 13C (false color) coronal image immediately after
injection of hyperpolarized (2). The posterior of the mouse was placed inside
the volume coil. b) Same as a) except the anterior of the mouse occupies the
volume coil and imaging was initiated 5 seconds after injection. Single 13C
transient arrays following injection of hyperpolarized (2) with posterior c) or
anterior d) of the mouse placed in the volume coil. The resonance for (2) is
readily observed at 174.5 ppm for approximately 30 seconds when
spectroscopy is initiated immediately after injection (c).
Keywords: aspirin • hyperpolarization • magnetic resonance
imaging • magnetic resonance spectroscopy • chemopreventive
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COMMUNICATION
A new trick for an old drug:
Zacharias, Ornelas, and co-
workers demonstrate the
successful hyperpolarization
of 13C-labeled aspirin by
DNP. NMR spectroscopy
was used to monitor in vitro
hydrolysis, transacetylation,
and albumin-binding
reactions of hyperpolarized
aspirin in real-time and MRI
was employed to visualize its
early biodistribution in mice.
Niki M. Zacharias,
Argentina Ornelas, Jaehyuk
Lee, Jingzhe Hu, Jennifer
Davis, Md. Nasir Uddin,
Shivanand Pudakalakatti,
David Menter, Jose A.
Karam, Christopher G.
Wood, Ernest T. Hawk,
Scott Kopetz, Eduardo
Vilar, Pratip K.
Bhattacharya, Steven W.
Millward*
Page No. – Page No.
Real-Time Interrogation
of Aspirin Reactivity,
Biochemistry, and
Biodistribution by
Hyperpolarized Magnetic
Resonance Spectroscopy
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