U.S. patent application number 14/620847 was filed with the patent office on 2015-09-17 for magnetic resonance imaging and/or spectroscopy contrast agents and methods of use thereof.
The applicant listed for this patent is Duke University. Invention is credited to Warren S. Warren.
Application Number | 20150258220 14/620847 |
Document ID | / |
Family ID | 41610972 |
Filed Date | 2015-09-17 |
United States Patent
Application |
20150258220 |
Kind Code |
A1 |
Warren; Warren S. |
September 17, 2015 |
MAGNETIC RESONANCE IMAGING AND/OR SPECTROSCOPY CONTRAST AGENTS AND
METHODS OF USE THEREOF
Abstract
The presently disclosed subject matter demonstrates that a spin
state which has zero magnetic resonance signal, but an extremely
long lifetime, can be used to store magnetization, which can then
be recovered into an observable transition. Coupled with
hyperpolarization techniques, this permits the preparation of a
wide range of contrast agent molecules for use in magnetic
resonance imaging (MRI) techniques that have long effective
relaxation time.
Inventors: |
Warren; Warren S.; (Durham,
NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Duke University |
Durham |
NC |
US |
|
|
Family ID: |
41610972 |
Appl. No.: |
14/620847 |
Filed: |
February 12, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13056795 |
Apr 25, 2011 |
8980225 |
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PCT/US09/52393 |
Jul 31, 2009 |
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14620847 |
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61085178 |
Jul 31, 2008 |
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Current U.S.
Class: |
424/9.3 ;
204/157.6; 204/157.71; 204/157.72; 204/157.81; 204/157.87;
204/157.89; 204/157.9; 204/157.93; 568/412 |
Current CPC
Class: |
A61K 49/18 20130101;
C07C 49/12 20130101; G01R 33/5605 20130101; G01R 33/5601 20130101;
A61K 49/08 20130101; A61K 49/1812 20130101; G01R 33/282
20130101 |
International
Class: |
A61K 49/08 20060101
A61K049/08; A61K 49/18 20060101 A61K049/18; C07C 49/12 20060101
C07C049/12 |
Goverment Interests
GOVERNMENT INTEREST
[0002] This presently disclosed subject matter was made with U.S.
Government support under Grant No. EB02122 awarded by National
Institutes of Health. Thus, the U.S. Government has certain rights
in the presently disclosed subject matter.
Claims
1-42. (canceled)
43. A contrast agent comprising a contrast agent molecule prepared
by (a) providing a precursor molecule comprising two J-coupled,
non-zero-spin, non-equivalent nuclei, wherein said precursor
molecule is chemically convertible to a contrast agent molecule
wherein the two J-coupled, non-zero-spin, non-equivalent nuclei are
converted to equivalent nuclei; (b) hyperpolarizing the precursor
molecule to provide a hyperpolarized precursor molecule; (c)
applying one or more radiofrequency pulse(s) to the hyperpolarized
precursor molecule to create one or both of a non-equilibrium
.alpha..beta. nuclear spin state population and a non-equilibrium
.beta..alpha. nuclear spin state population; and (d) chemically
converting the hyperpolarized precursor molecule into a contrast
agent molecule by converting the two J-coupled, non-zero-spin,
non-equivalent nuclei to two J-coupled, non-zero-spin, equivalent
nuclei, wherein the contrast agent molecule comprises a
non-equilibrium nuclear singlet spin state population and is
chemically convertible to a detection molecule wherein said
equivalent nuclei are converted to non-equivalent nuclei; wherein
said contrast agent can be used to enhance a signal in one of
magnetic resonance imaging or magnetic resonance spectroscopy.
44. The contrast agent of claim 43, wherein the contrast agent
molecule is encapsulated in a biodegradable drug delivery format
that prevents water contact with the contrast agent molecule.
45. The contrast agent of claim 43, wherein the contrast agent
molecule is selected from the group consisting of diacetyl, oxolin,
alendronate, amitryptyline, nortriptyline, succinate, fumarate,
maleimide, catechol, naphthalene, naphthoquinone, phenylbutazone,
pyridazine, phthalazine, dopamine, L-dihydroxyphenylalanine
(L-DOPA) and derivatives thereof.
46. The contrast agent of claim 43, wherein the contrast agent
molecule is encapsulated in a liposome or in a delivery agent that
swells in water.
47. The contrast agent of claim 43, wherein the contrast agent
molecule is encapsulated in a delivery agent that comprises
targeting groups for directing the contrast agent to a tissue,
organ or cell.
48. The contrast agent of claim 43, wherein the contrast agent
molecule is incorporated in a pharmaceutically acceptable
carrier.
49. The contrast agent of claim 43, wherein the contrast agent
molecule is convertible to the detection molecule under
physiological conditions.
50. The contrast agent of claim 43, wherein the contrast agent
molecule is convertible to the detection molecule via contact with
water or via an enzymatic reaction.
51. The contrast agent of claim 43, wherein the contrast agent
molecule is in chemical equilibrium with the precursor molecule or
the detection molecule.
52. The contrast agent of claim 43, wherein the precursor molecule
and the detection molecule have the same molecular structure.
53. The contrast agent of claim 43, wherein the two J-coupled,
non-zero-spin non-equivalent nuclei of the precursor molecule are
selected from the group consisting of .sup.1H, .sup.13C, .sup.15N,
and .sup.31P.
54. The contrast agent of claim 43, wherein the hyperpolarizing is
performed by dynamic nuclear polarization (DNP).
55. The contrast agent of claim 43, wherein chemically converting
the hyperpolarized precursor molecule into the contrast agent
molecule comprises dehydrating the hyperpolarized precursor
molecule.
56. The contrast agent of claim 43, wherein one or both of the
precursor molecule and the detection molecule is the monohydrate of
diacetyl.
57. The contrast agent of claim 43, wherein the two J-coupled,
non-zero-spin, equivalent nuclei of the contrast agent molecule are
free of directly bonded hydrogen atoms.
58. The contrast agent of claim 43, wherein the two J-coupled,
non-zero-spin, equivalent nuclei of the contrast agent molecule are
directly bonded to hydrogen atoms and are J-coupled to two other,
additional equivalent nuclei, wherein the two other, additional
equivalent nuclei are free of directly bonded hydrogen atoms, and
whereby application of one or more radiofrequency pulse(s) can
transfer a spin state population between the two J-coupled,
non-zero-spin, equivalent nuclei and the two other, additional
equivalent nuclei.
59. The contrast agent of claim 43, wherein the non-equilibrium
nuclear singlet spin state population can persist for a time that
is substantially greater than T.sub.1.
60. The contrast agent of claim 59, wherein the non-equilibrium
nuclear singlet spin state population can persist for a time that
is greater than 3 times T.sub.1.
61. The contrast agent of claim 60, wherein the non-equilibrium
nuclear singlet spin state population can persist for a time that
is greater than 10 times T.sub.1.
62. The contrast agent of claim 59, wherein the non-equilibrium
nuclear singlet spin state population can persist for a time that
is between about 3 times T.sub.1 and about 10 times T.sub.1.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/056,795, filed on Apr. 25, 2011, which is a
national stage of PCT International Application No.
PCT/US2009/052393, filed on Jul. 31, 2009, the disclosure of each
of which is incorporated herein by reference in its entirety, which
is based on and claims priority to U.S. Provisional Application
Ser. No. 61/085,178, filed Jul. 31, 2008, herein incorporated by
reference in its entirety.
TECHNICAL FIELD
[0003] The presently disclosed subject matter relates to magnetic
resonance imaging (MRI) and/or nuclear magnetic resonance (NMR)
spectroscopy contrast agents having long effective relaxation
times. Also provided are methods of preparing and using the
contrast agents.
Abbreviations
[0004] .sup.13C=carbon-13
[0005] DNP=dynamic nuclear polarization
[0006] FID=free induction decay
[0007] HVA=homovanillic acid
[0008] Hz=hertz
[0009] L-DOPA=L-dihydroxyphenylalanine
[0010] MRI=magnetic resonance imaging
[0011] ms=millisecond
[0012] PET=positron emission tomagraphy
[0013] ppm=parts-per-million
[0014] rf=radiofrequency
[0015] s=second
[0016] t=time
[0017] T=Tesla
[0018] T.sub.1=longitudinal relaxation time constant
[0019] T.sub.2=spin-spin relaxation time constant
[0020] TCA=tricarboxylic acid cycle
BACKGROUND
[0021] Nuclear magnetic resonance (NMR), or magnetic resonance
spectroscopy, is a powerful, well-established tool for studying
chemical samples and sample interactions. In NMR, the spin and
magnetism of atomic nuclei are exploited to provide information
about the chemical composition, spatial distribution, or molecular
motion of molecules or atoms. The imaging analog of NMR, magnetic
resonance imaging (MRI), is a powerful technique in biomedical
sample imaging.
[0022] One of the limitations of NMR and MRI is low intrinsic
signal strength. Some attempts to overcome this limitation have
involved the use of hyperpolarized contrast agents, which have very
large nuclear polarizations and, therefore, sensitivities that are
orders of magnitude higher than ordinary molecules. For a few
molecules, polarization can persist for 100 or more seconds before
the polarized nuclei return to thermal equilibrium. However, for
the majority of molecules, including those that could be used as in
vivo contrast agents for MRI, polarization lasts more in the range
of seconds or tens of seconds.
[0023] While such lifetimes can be sufficient for some imaging
and/or spectroscopy studies, contrast agents with longer lifetimes
are highly desirable to study additional processes of interest, for
example processes related to diffusion, flow, slow molecular
motion, chemical reactions, metabolism, and drug targeting and
distribution, among others. The relaxation of nuclear spins back to
thermal equilibrium is characterized by a time constant, T.sub.1,
known as the longitudinal relaxation time constant or as the spin
lattice relaxation time constant. The development of contrast
agents having polarization that persists for times longer than
T.sub.1 would be beneficial for both NMR and MRI.
SUMMARY
[0024] The presently disclosed subject matter pertains to a method
of providing a contrast agent for magnetic resonance imaging or
magnetic resonance spectroscopy, the method comprising:
[0025] providing a precursor molecule comprising two J-coupled,
non-zero-spin, non-equivalent nuclei, wherein said precursor
molecule is chemically convertible to a contrast agent molecule
wherein the two J-coupled, non-zero-spin, non-equivalent nuclei are
converted to equivalent nuclei;
[0026] hyperpolarizing the precursor molecule to provide a
hyperpolarized precursor molecule;
[0027] applying one or more radiofrequency pulse(s) to the
hyperpolarized precursor molecule to create one or both of a
non-equilibrium .alpha..beta. nuclear spin state population and a
non-equilibrium .beta..alpha. nuclear spin state population;
and
[0028] chemically converting the hyperpolarized precursor molecule
into a contrast agent molecule by converting the two J-coupled,
non-zero-spin, non-equivalent nuclei to two J-coupled,
non-zero-spin, equivalent nuclei, wherein the contrast agent
molecule comprises a non-equilibrium nuclear singlet spin state
population and is chemically convertible to a detection molecule
wherein said equivalent nuclei are converted to non-equivalent
nuclei.
[0029] In some embodiments, the contrast agent molecule is
chemically convertible to the detection molecule under
physiological conditions. In some embodiments, the contrast agent
molecule is converted to the detection molecule via contact with
water. In some embodiments, the contrast agent molecule is
converted to the detection molecule via an enzymatic reaction.
[0030] In some embodiments, the contrast agent molecule is in
chemical equilibrium with the hyperpolarized precursor molecule or
the detection molecule. In some embodiments, the chemical
equilibrium is perturbed to interconvert the contrast agent
molecule and the hyperpolarized precursor molecule, the contrast
agent molecule and the detection molecule, or both. In some
embodiments, the precursor molecule and the detection molecule have
the same molecular structure.
[0031] In some embodiments, the two J-coupled, non-zero-spin,
non-equivalent nuclei of the precursor molecule are selected from
the group including, but not limited to, .sup.1H, .sup.13C,
.sup.15N and .sup.31P.
[0032] In some embodiments, the hyperpolarizing is performed by
dynamic nuclear polarization (DNP).
[0033] In some embodiments, chemically converting the
hyperpolarized precursor molecule into the contrast agent molecule
comprises dehydrating the hyperpolarized precursor molecule. In
some embodiments, the dehydrating is accelerated or slowed by
changing the pH of an aqueous solvent in which the hyperpolarized
precursor molecule is dissolved.
[0034] In some embodiments, one or both of the precursor molecule
and the detection molecule is the monohydrate of diacetyl. In some
embodiments, the contrast agent molecule is selected from the group
including, but not limited to, diacetyl, oxolin, alendronate,
amitryptyline, nortriptyline, succinate, fumarate, maleimide,
catechol, naphthalene, naphthoquinone, phenylbutazone, pyridazine,
phthalazine, dopamine, L-dihydroxyphenylalanine (L-DOPA) and
derivatives thereof.
[0035] In some embodiments, the two J-coupled, non-zero-spin,
equivalent nuclei of the contrast agent molecule are free of
directly bonded hydrogen atoms. In some embodiments, the two
J-coupled, non-zero-spin, equivalent nuclei of the contrast agent
molecule are directly bonded to hydrogen atoms and are J-coupled to
two other, additional equivalent nuclei, wherein the two other,
additional equivalent nuclei are free of directly bonded hydrogen
atoms, and whereby application of one or more radiofrequency
pulse(s) can transfer a spin state population between the two
J-coupled, non-zero-spin, equivalent nuclei and the two other,
additional equivalent nuclei.
[0036] In some embodiments, the method further comprises
incorporating the contrast agent molecule into a pharmaceutically
acceptable carrier to provide a pharmaceutically acceptable
formulation suitable for administration to a subject. In some
embodiments, the subject is a mammal. In some embodiments, the
method further comprises encapsulating the contrast agent molecule
into a biodegradable delivery format that prevents water contact
with the contrast agent molecule prior to complete or partial
biodegradation of said delivery format.
[0037] In some embodiments, the non-equilibrium nuclear singlet
spin state population can persist for a time that is substantially
greater than T.sub.1. In some embodiments, the non-equilibrium
nuclear singlet spin state population can persist for a time that
is greater than 3 times T.sub.1 or greater than 10 times T.sub.1.
In some embodiments, the non-equilibrium nuclear singlet spin state
population can persist for a time that is between about 3 times
T.sub.1 and about 10 times T.sub.1.
[0038] In some embodiments, the presently disclosed subject matter
provides a method of imaging a target, the method comprising:
[0039] providing a contrast agent molecule having a non-equilibrium
singlet state nuclear spin population, wherein providing the
contrast agent molecule comprises providing a precursor molecule
comprising two J-coupled, non-zero-spin, non-equivalent nuclei,
wherein said precursor molecule is chemically convertible to a
contrast agent molecule wherein the two J-coupled, non-zero-spin,
non-equivalent nuclei are converted to equivalent nuclei;
hyperpolarizing the precursor molecule to provide a hyperpolarized
precursor molecule; applying one or more radiofrequency pulse(s) to
the hyperpolarized precursor molecule to create one or both of a
non-equilibrium .alpha..beta. nuclear spin state population and a
non-equilibrium .beta..alpha. nuclear spin state population; and
chemically converting the hyperpolarized precursor molecule into
the contrast agent molecule by converting the two J-coupled,
non-zero-spin, non-equivalent nuclei to two J-coupled,
non-zero-spin, equivalent nuclei, wherein the contrast agent
molecule comprises a non-equilibrium nuclear singlet spin state
population and is chemically convertible to a detection molecule
wherein the two J-coupled, non-zero-spin equivalent nuclei are
converted to non-equivalent nuclei;
[0040] contacting the contrast agent molecule with the target;
[0041] allowing the contrast agent molecule to be chemically
converted into the detection molecule;
[0042] generating a nuclear magnetic resonance signal; and
[0043] detecting the nuclear magnetic resonance signal, thereby
imaging the target.
[0044] In some embodiments, the target is one of a cell, a tissue,
an organ, and a subject. In some embodiments, the contacting
comprises administering a pharmaceutical formulation comprising the
contrast agent to a subject. In some embodiments, the subject is a
mammal.
[0045] In some embodiments, the presently disclosed subject matter
provides a contrast agent comprising a contrast agent molecule
prepared by the method comprising:
[0046] providing a precursor molecule comprising two J-coupled,
non-zero-spin, non-equivalent nuclei, wherein said precursor
molecule is chemically convertible to a contrast agent molecule
wherein the two J-coupled, non-zero-spin, non-equivalent nuclei are
converted to equivalent nuclei; hyperpolarizing the precursor
molecule to provide a hyperpolarized precursor molecule; applying
one or more radiofrequency pulse(s) to the hyperpolarized precursor
molecule to create one or both of a non-equilibrium .alpha..beta.
nuclear spin state population and a non-equilibrium .beta..alpha.
nuclear spin state population; and chemically converting the
hyperpolarized precursor molecule into a contrast agent molecule by
converting the two J-coupled, non-zero-spin, non-equivalent nuclei
to two J-coupled, non-zero-spin, equivalent nuclei, wherein the
contrast agent molecule comprises a non-equilibrium nuclear singlet
spin state population and is chemically convertible to a detection
molecule wherein said equivalent nuclei are converted to
non-equivalent nuclei; wherein said contrast agent can be used to
enhance a signal in one of magnetic resonance imaging or magnetic
resonance spectroscopy.
[0047] In some embodiments, the contrast agent molecule is
encapsulated in a biodegradable drug delivery format that prevents
water contact with the contrast agent molecule. In some
embodiments, the contrast agent molecule is selected from the group
including, but not limited to, diacetyl, oxolin, alendronate,
amitryptyline, nortriptyline, succinate, fumarate, maleimide,
catechol, naphthalene, naphthoquinone, phenylbutazone, pyridazine,
phthalazine, dopamine, L-dihydroxyphenylalanine (L-DOPA) and
derivatives thereof.
[0048] Accordingly, it is an object of the presently disclosed
subject matter to provide contrast agents for use in NMR
spectroscopy and MRI.
[0049] Certain objects of the presently disclosed subject matter
having been stated hereinabove, which are addressed in whole or in
part by the presently disclosed subject matter, other objects and
aspects will become evident as the description proceeds when taken
in connection with the accompanying Examples as best described
herein below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] FIG. 1 is a single-shot hyperpolarized .sup.13C nuclear
magnetic resonance (NMR) spectrum of 2,3-.sup.13C diacetyl in
water. Hyperpolarized 2,3-.sup.13C diacetyl was produced by a
commercially available hyperpolarizer (Oxford HYPERSENSE.TM.,
Oxford Instruments Molecular Biotools Ltd., Tubney Woods, Abingdon,
Oxfordshire, United Kingdom). The sample has about 20% nuclear
polarization. The thermally polarized spectrum (not shown) is
similar after many averages.
[0051] FIG. 2 is a series of free induction decay (FID) signals of
the thermally polarized monohydrate of diacetyl after inverting one
line, thereby locking the population into a long-lived singlet
state. The FIDs should vanish at time=1/2 J (dotted line).
[0052] FIG. 3 is a series of free induction decay (FID) signals of
the monohydrate of diacetyl in a hyperpolarized
diacetyl-water-acetone mixture after inversion of one line.
[0053] FIG. 4A is a portion of the .sup.13C nuclear magnetic
resonance (NMR) spectrum of 2-.sup.13C diacetyl showing the
carbonyl region. The coupling constant J.sub.C-H=6.4 Hz, -1.1
Hz.
[0054] FIG. 4B is a portion of the .sup.13C nuclear magnetic
resonance (NMR) spectrum of acetone showing the carbonyl region.
The coupling constant J.sub.C-H=5.8 Hz.
[0055] FIG. 4C is a portion of the .sup.13C nuclear magnetic
resonance (NMR) spectrum of 2,3-.sup.13C diacetyl showing the
carbonyl region. For reference the bar in the upper right hand
corner represents 10 Hz.
[0056] FIG. 4D is a portion of a simulated .sup.13C nuclear
magnetic resonance (NMR) spectrum of 1,2-.sup.13C diacetyl showing
the carbonyl region when the coupling constant J.sub.C-C between
the two carbons is zero.
[0057] FIG. 4E is a portion of a simulated .sup.13C nuclear
magnetic resonance (NMR) spectrum of 1,2-.sup.13C diacetyl showing
the carbonyl region when the coupling constant J.sub.C-C is larger
than all of the other couplings.
[0058] FIG. 4F is a portion of a simulated .sup.13C nuclear
magnetic resonance (NMR) spectrum of 1,2-.sup.13C diacetyl showing
the carbonyl region when the coupling constant J.sub.C-C is 50
Hz.
DETAILED DESCRIPTION
[0059] The presently disclosed subject matter will now be described
more fully hereinafter with reference to the accompanying Examples,
in which representative embodiments are shown. The presently
disclosed subject matter can, however, be embodied in different
forms and should not be construed as limited to the embodiments set
forth herein. Rather, these embodiments are provided so that this
disclosure will be thorough and complete, and will fully convey the
scope of the embodiments to those skilled in the art.
[0060] All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety.
[0061] Throughout the specification and claims, a given chemical
formula or name shall encompass all optical and stereoisomers, as
well as racemic mixtures where such isomers and mixtures exist,
unless otherwise noted.
I. Definitions
[0062] While the following terms are believed to be well understood
by one of ordinary skill in the art, the following definitions are
set forth to facilitate explanation of the presently disclosed
subject matter.
[0063] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which the presently disclosed subject
matter belongs. Although any methods, devices, and materials
similar or equivalent to those described herein can be used in the
practice or testing of the presently disclosed subject matter,
representative methods, devices, and materials are now
described.
[0064] The term "contrast agent" refers to a contrast agent
molecule or a composition comprising a contrast agent molecule that
increases the contrast of a tissue, organ, cell or other biological
structure being examined, for example using nuclear magnetic
resonance imaging (MRI). The contrast agent molecules of the
presently disclosed subject matter can also be used to determine
the progress of chemical reactions or non-biological phenomena
(e.g., chemical diffusion) via nuclear magnetic resonance (NMR)
spectroscopy.
[0065] The term "J-coupling" can also be referred to as spin-spin
coupling or scalar coupling. As used herein, J-coupling can refer
to a J coupling which is larger than the reciprocal of the
spin-spin relaxation time, T.sub.2.
[0066] As used herein the term "equivalent nuclei" can refer to
nuclei having exactly the same chemical shift. If the chemical
shifts are identical because the two nuclei are related by some
symmetry element, such as a mirror plane, such nuclei can be
referred to as "chemically equivalent." Alternatively, the
equivalent nuclei can be "effectively equivalent," wherein, by
coincidence or choice of field strength, the resonance frequencies
of the nuclei will differ by less than the J coupling between them.
For example, in some embodiments, the effectively equivalent nuclei
can have a chemical shift difference that is at least three times
(or at least five times) smaller than the J coupling between
them.
[0067] "Non-equivalent nuclei" are generally nuclei wherein one
nucleus is bonded to at least one chemical group that is different
in structure than the chemical group or groups bonded to the other
non-equivalent nuclei. Thus, non-equivalent nuclei have a resonance
frequency difference which exceeds the J coupling between them.
[0068] The term "derivatives" refers to compounds that differ from
a named parent compound by the addition or subtraction of one or
more atoms or chemical groups. Thus, the term "derivatives"
includes, but is not limited to, compounds wherein one or more
hydrogen atom of the parent compound has been replaced by one or
more alkyl, aralkyl, aryl, acyl, halo, nitro, cyano, hydroxyl,
alkoxyl, aryloxyl, or amino groups. For example, the derivative can
be the ester or amide of a parent molecule that includes a carbonyl
(i.e., a--C(.dbd.O)--), carboxylic acid (i.e., a --C(.dbd.O)OH
group), amine (i.e., a --NH.sub.2 or --NHR group, where R is an
alkyl or aryl moiety) or hydroxyl (--OH) group. Other derivatives
include ethers of hydroxyl-containing parent molecules and
N-alkylated amines of amino-containing parent molecules. In
addition, derivatives of the presently disclosed contrast agent
molecules are derivatives wherein the derivatization does not
change the equivalency of the equivalent nuclei therein. Thus, any
derivative of a named contrast agent molecule includes two
J-coupled, non-zero-spin, equivalent nuclei.
[0069] The terms "chemically converting" and "chemical
transformation" refer to chemical and biochemical reactions wherein
one or more bonds are formed or broken. In some embodiments, the
term chemically converting refers to a non-photo catalyzed
reaction.
[0070] The term "physiological conditions" refers to biologically
relevant pH, temperature, and salt conditions, such as might be
present in vivo (i.e., in a living organism) or in vitro (e.g., in
a cell, tissue, organ or mixture of biological molecules outside a
living organism). Physiological conditions can refer to the
presence of an aqueous solvent (e.g., water or saline), which can
be buffered or unbuffered by the presence of a buffering agent
(e.g., sodium bicarbonate, sodium carbonate, monopotassium
phosphate, dipotassium phosphate, sodium citrate, citric acid,
sodium acetate, acetic acid, bicine, cacodylate,
tris(hydroxymethyl)methylamine (Tris),
N-tris(hydroxymethyl)methylglycine (Tricine),
4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES),
piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES),
2-(N-morpholino)ethanesulfonic acid (MES),
3-(N-morpholino)propanesulfonic acid (MOPS), etc). One or more
biological molecules (e.g., proteins, peptides, enzymes, nucleic
acids, lipids, carbohydrates, or the like) can be present under
physiological conditions.
II. General Considerations
[0071] Nuclear magnetic resonance (NMR) and the imaging analog,
magnetic resonance imaging (MRI), have become extraordinarily
important techniques. MRI has become a very powerful clinical
imaging modality, for two fundamental reasons. First, the hardware
is mature: modern MRI machines routinely give complex sequences of
arbitrarily shaped radiofrequency pulses to create precise
excitation, and give magnetic field gradient pulses to suppress
magnetization or obtain spatial resolution. More importantly,
however, the theoretical framework is mature. No other modern
spectroscopy has such a strong theoretical basis, which of course
is used to understand the structures of molecules as complicated as
proteins in solution. This maturity is even more important in MRI:
complications associated with imaging in vivo can often be reduced
or eliminated by clever pulse sequence design.
[0072] However, the maturity of that theoretical framework also
implies that the known limitations of MRI are rather fundamental.
The Boltzmann distribution implies that the net fractional
magnetization is small at room temperature, so in most MRI studies,
the signal arises mostly from water. Contrast then arises primarily
from parameters that can be traced back to the spin physics
explorations of the 1940s and 1950s (the local bulk magnetization
M.sub.o, the relaxation parameters T.sub.1, T.sub.2 and T.sub.2*,
and local values of diffusion, sometimes in different directions),
which often only have very indirect clinical relevance or
correlation with metabolism and cell biochemistry. MRI contrast
agents generally have limited specificity, and usually need to be
present in high concentration to affect the signal.
[0073] These limitations have recently been partially surmounted by
the ready commercial availability of hyperpolarized reagents, which
have very large nuclear polarizations and thus orders of magnitude
higher sensitivity than ordinary molecules. Among the recent
methods are techniques to create spin polarized .sup.3He (see
McFall et al., Radiology, 200, 553-558 (1996); and Salerno et al.,
Eur. J. Radiol., 40, 33-44 (2001)), para-H.sub.2 addition across
double bonds (see Bowers and Weitekamp, Phys. Rev. Lett., 57,
2645-2648 (1986); Bowers and Weitekamp, J. Am. Chem. Soc., 109,
5541-5542 (1987); Natterer and Bargon, Progr. Nucl., Magn. Res.
Spectrosc., 31, 293-315 (1997); Duckett and Sleigh, Progr. Nucl.,
Magn. Res. Spectrosc., 34, 71-92 (1999); and Golman et al.,
Magnetic Resonance in Medicine, 46, 1-5 (2001)), and dynamic
nuclear polarization (DNP). See Abragam and Goldman, Rep. Prog.,
Phys., 41, 395-467 (1978); de Boer et al., Journal of Low
Temperature Physics, 15, 249-267 (1974); de Boer and Niinikoski,
Nuclear Instruments and Methods, 114, 495-498 (1974); Hall et al.,
Science, 276, 930-931 (1997); Bajaj et al., Journal of Magnetic
Resonance, 160, 85-90 (2003); Johansson et al., Magn. Reson. Med.,
51, 464-472 (2004); and Ardenkjaer-Larsen et al., Proc. Natl. Acad.
Sci., USA, 100, 10158-10163 (2003). All of these methods have
demonstrated large nuclear magnetization (>10%, as compared to
typical thermal magnetization of 10.sup.-5), with the polarization
persisting as long as 100 s in some molecules. Many different
research groups have been examining potential uses of such
hyperpolarized molecules both in vivo and in vitro. See Kurhanewicz
et al., J. Nucl. Med. 49(3), 341-344 (2008); Golman et al., Cancer
Research, 66, 10855-10860 (2006); Merritt et al., Proc. Natl. Acad.
Sci., USA, 104, 19773-19777 (2007); Day et al., Mag. Res. Chem.,
45(12), 1018-1021 (2007); Gabellieri et al., J. Am. Chem. Soc.,
130(14), 4598 (2008).
[0074] DNP methodology in particular is very versatile, and
hundreds of different molecules have been polarized. However, most
DNP studies have focused on .sup.13C in pyruvate, largely because
the T.sub.1 relaxation time for the C1 position is relatively long
(40 s at 14.1 T), so the polarized nuclei can potentially undergo
many reactions before the NMR signal returns to thermal equilibrium
and becomes undetectable. Generically, carbon-13 T.sub.1 values are
expected to be tens of seconds for carbons without attached
protons, and much shorter with attached protons. While this
lifetime permits some important metabolic processes to be studied,
it is vastly shorter than the lifetimes associated with other
molecular imaging modalities (e.g., .sup.18F PET) and provides a
fundamental limitation to the ultimate generality of the
technique.
[0075] Previous studies have demonstrated the use of singlet states
comprised of non-symmetry related spins to lengthen T.sub.1. See
Ahuja, et al., J. Chemical Physics, 127, 134112 (2007); Carravetta
et al., Physical Review Letters, 92, 153003 (2004); Carravetta and
Levitt, J. Am. Chem. Soc., 126, 6228-6229 (2004); and Carravetta
and Levitt, J. Chem. Physics, 122, 214505 (2005). In the studies,
molecules with broken symmetry (for example, a single carbon-13)
are used. The non-symmetry related spins are manipulated to appear
to be equivalent by removing frequency differences with multiple
spin echoes (which requires excessive radiofrequency power
dissipation in vivo) or by lowering the magnetic field so much that
the resonance frequencies are essentially the same (which requires
removing the sample from the magnet). The signal is then observed
by permitting free evolution in a high field. Both approaches give
interesting demonstrations of lifetime increases, but neither is
practical for MRI. In addition, at the microscopic level, both of
these approaches have certain limitations. For example, relaxation
is dominated by the local components of the magnetic field
fluctuating near the Larmor frequency, and, if two sites are
physically inequivalent, these fluctuations are expected to be
poorly correlated, even if the resonance frequencies are nearly the
same.
[0076] PCT International Patent Application Publication No. WO
2005/015253 relates to an approach that involves lowering the field
to create a pseudo-singlet state and to reacting an unsaturated
symmetric molecule with parahydrogen to provide a quasi-equilibrium
nuclear spin ensemble estate. See also, Carravetta et al., Physical
Review Letters, 92, 153003 (2004); Carravetta and Levitt, J. Am.
Chem. Soc., 126, 6228-6229 (2004); and Carravetta and Levitt, J.
Chem. Physics, 122, 214505 (2005).
III. Contrast Agents and Methods of Preparing Contrast Agents
[0077] The presently disclosed subject matter provides novel
methods to make certain molecules have vastly longer effective
relaxation times, thus facilitating their practical use in clinical
and preclinical magnetic resonance imaging. A central concept is
that of storing population in a "singlet state", the only
antisymmetric spin state created when two magnetically equivalent
nuclear spins are present in the same molecule. In such a system,
quantum mechanics predicts that the singlet energy level
2.sup.1/2(.alpha..beta.-.beta..alpha.) is disconnected from the
other three (triplet) energy levels. This has various consequences.
For example, the water molecule has two nuclear spins, but a very
simple NMR spectrum (i.e., one line) because one of the four
possible energy levels is disconnected from all of the others. The
singlet state also should have no significant interactions with
external magnetic fields, and, thus an extremely long relaxation
time, as long as the field does not break the symmetry between the
spins. Unfortunately, the signal is also unobservable for the same
reasons. The methods of the presently disclosed subject matter take
advantage of chemistry to break the symmetry in a controlled way so
that signal can be detected. Thus, under certain circumstances,
combinations of chemical action and radiofrequency pulses can make
these spin states accessible, so that they can be used as a "safe
storage" for hyperpolarization.
[0078] Prior to the presently disclosed subject matter, most
general methods for preparing hyperpolarized reagents do not
directly prepare substantial population in singlet states. Further,
parahydrogen addition has been demonstrated to work in a very small
number of molecules, only one of which at this time has any
relevance to biochemistry. The dynamic nuclear polarization method,
on the other hand, has been used to hyperpolarize hundreds of
different small molecules, for example, via the commercially
available Oxford HYPERSENSE.TM. (Oxford Instruments Molecular
Biotools Ltd., Tubney Woods, Abingdon, Oxfordshire, United Kingdom)
system. Other methods to hyperpolarize a sample with equivalent
carbons, nitrogens, or phosphorus (for example, transfer of
polarization from a highly spin polarized gas or reduction of
temperature to achieve increased polarization) also tend not to
produce singlet population. The presently disclosed methods can be
used for preparing substantial excess population, which requires
creation of a precursor that is chemically inequivalent;
perturbation of a line in the spectrum (or other methods using rf
pulses on allowed transitions); and then chemical transformation
into the singlet. The presently disclosed subject matter further
encompasses many examples of classes of chemical reactions that
would be capable in vivo of transforming population between singlet
and observable signal and is applicable, in some embodiments, to
carbon, nitrogen, or phosphorus atoms without attached hydrogens,
which are known to have an appreciably longer relaxation time.
[0079] In some embodiments, the presently disclosed subject matter
provides a hyperpolarized contrast agent molecule. Generically,
hyperpolarized contrast agent molecules that can be used in methods
of the presently disclosed subject matter satisfy one or more of
the following three conditions. First, the contrast agent molecule
can have two equivalent nuclei. For example, the molecule can have
two nearby equivalent H, C (e.g., .sup.13C), N (e.g., .sup.15N), or
P (e.g., .sup.31P) nuclei. Proximity is important because the
nuclei need to have a coupling. In solution phase, this coupling
would normally be the so-called scalar or J coupling in NMR. The
equivalent nuclei will have a resonance frequency difference much
less than the scalar coupling (or zero) and much weaker coupling to
other spins.
[0080] Secondly, there can be a method for preparing excess (or
depleted) population in the singlet state. This is possible if the
contrast agent molecule has a precursor where the two nuclei are
non-equivalent, and if the precursor can be hyperpolarized by a
method such as, but not limited to, dynamic nuclear polarization
(DNP). Then combinations of radiofrequency pulses can be used to
perturb the population in the .alpha..beta. and .beta..alpha.
energy levels in the hyperpolarized precursor to increase or
decrease the singlet population. Chemical transformation of the
hyperpolarized precursor to a species with equivalent nuclei can
then lock population in the singlet. Ideally, this chemical
transformation is achievable in a time shorter than the normal
T.sub.1.
[0081] Thirdly, for the contrast agent molecule to give desired
contrast, there can be a mechanism (e.g., a biological or chemical
pathway) which makes the equivalent nuclei non-equivalent again,
thus permitting detection of the hyperpolarization. For example,
there might be a biological pathway that involves an enzymatic
reaction that transforms the contrast agent molecule into a
detection molecule in which the nuclei are non-equivalent. As
another example, the contrast agent molecule might be encapsulated
in a delivery system which excludes water. At an appropriate time,
opening or degrading the capsules causes hydration of the contrast
agent molecule and, thus, causes the equivalent nuclei to become
non-equivalent (e.g., by breaking the symmetry of the contrast
agent molecule). Such a mechanism can be employed, for example,
when diacetyl is used as a contrast agent molecule.
[0082] Some contrast agent molecules, such as, but not limited to
diacetyl, comprise no hydrogens attached directly to the equivalent
nuclei. Partitioning between hydrophobic and hydrophilic phases can
control the rate of conversion of such contrast agent molecules to
detectable molecules. For instance, partitioning in vivo modulates
the rate of conversion between diacetyl and its first metabolite,
acetoin (i.e., CH.sub.3C(.dbd.O)CH(OH)CH.sub.3), which has
inequivalent carbons.
[0083] In addition to diacetyl, many other molecular systems can be
used as contrast agent molecules. Simple examples include oxolin
(an antiviral compound with two equivalent carbons and no attached
hydrogens) and FOSAMAX.TM. (i.e., alendronate, Merck & Co.,
Inc., Whitehouse Station, N.J., United States of America), which
has two equivalent P atoms separated by a single carbon.
Derivatives of pyridazine or phthalazine, which have recently been
shown to have VEGFR-2 inhibitory activity (see Kisselyov et al.,
Chem. Biol. Drug Des., 68, 308-313 (2006)), and which can have
equivalent nitrogen atoms, can also be used. Also, in some
embodiments, deuteration or very weak irradiation can essentially
eliminate the coupling to outside nuclei.
[0084] As directly bonded H atoms are not needed in the DNP method
to create hyperpolarization, isotopic substitution of hydrogen with
deuterium is possible and thus additional, more complex, molecular
systems can also be used as contrast agent molecules according to
the presently disclosed subject matter. These molecules include,
but are not limited to, the antidepressant amitryptyline and its
primary metabolite nortriptyline, whose seven-member ring gets
hydroxylated in the liver (see Oleson and Linnet, Drug Metabolism
and Disposition, 25, 740-744 (1997)), as well as succinate and
fumarate, which become asymmetric as they get converted to malate
and oxaloacetate in the tricarboxylic acid (TCA) cycle. Succinate
can be prepared directly as the (hydrogen) singlet via parahydrogen
addition (see Bhattacharya et al., J. Mag. Resonance, 186(1),
150-155 (2007)), but, in the past, has generally been singly
carbon-13 labeled, intentionally to break the symmetry. In the 2,3-
or 1,2,3,4-labeled compound, the singlet could be transferred by
radiofrequency (rf) pulses between H and the 2,3-carbons, or to the
1,4 carbons (which are long lived), then back to the 2,3 carbons to
have a detectable coupling. Thus, hyperpolarized succinate is
accessible by para-hydrogen addition, as well as the DNP
hyperpolarization method.
[0085] At moderate fields, such as at about 1 Tesla or at between
about 0.5 to about 4.0 Tesla (i.e., the field range of commercial
MRI machines), even molecules with not quite chemically equivalent
nuclei, such as the 3,4-.sup.13C versions of L-DOPA or dopamine can
be used as contrast agent molecules. The degradation pathways of
L-DOPA and dopamine lead to compounds such as homovanillic acid
(HVA), which has significant asymmetry.
[0086] Thus, the presently disclosed subject matter provides, in
some embodiments, a contrast agent comprising a contrast agent
molecule that allows for retention or "storage" of
hyperpolarization for an extended period of time, for example, so
that the contrast agent can be used to detect a number of
biologically or chemically relevant events. In some embodiments,
the presently disclosed subject matter relates to a method of
providing a contrast agent for magnetic resonance imaging or
magnetic resonance spectroscopy, the method comprising:
[0087] providing a precursor molecule comprising two J-coupled,
non-zero-spin, non-equivalent nuclei, wherein said precursor
molecule is chemically convertible to a contrast agent molecule
wherein the two J-coupled, non-zero-spin, non-equivalent nuclei are
converted to equivalent nuclei;
[0088] hyperpolarizing the precursor molecule to provide a
hyperpolarized precursor molecule (which also comprises the two
J-coupled, non-zero-spin, non-equivalent nuclei);
[0089] applying one or more radiofrequency pulse(s) to the
hyperpolarized precursor molecule to create one or both of a
non-equilibrium .alpha..beta. nuclear spin state population and a
non-equilibrium pa nuclear spin state population; and
[0090] chemically converting the hyperpolarized precursor molecule
into a contrast agent molecule by converting the two J-coupled,
non-zero-spin, non-equivalent nuclei to two J-coupled,
non-zero-spin, equivalent nuclei, wherein the contrast agent
molecule comprises a non-equilibrium nuclear singlet spin state
population and is chemically convertible to a detection molecule
wherein said equivalent nuclei are converted into non-equivalent
nuclei. In some embodiments, the presently disclosed subject matter
relates to the contrast agent molecule prepared according to the
above-described method.
[0091] In some embodiments, the contrast agent molecule is
chemically convertible to the detection molecule under
physiological conditions. Thus, in some embodiments, the contrast
agent molecule is converted to the detection molecule via contact
with water. In some embodiments, the contrast agent molecule is
converted to the detection molecule via an enzymatic reaction. In
some cases, conversion between equivalent and inequivalent states
can be as simple as a change in pH (e.g., in perdeuterated urea or
arginine) or removal of water (as in diacetyl), and such systems
can be used as reporters in incapsulated delivery systems.
[0092] In some embodiments, the contrast agent molecule is in
chemical equilibrium with the hyperpolarized precursor molecule or
the detection molecule. In some embodiments, the chemical
equilibrium can be perturbed (e.g., changed or manipulated) to
interconvert the contrast agent molecule and the hyperpolarized
precursor molecule, the contrast agent and the detection molecule,
or both. For example, the hyperpolarized precursor molecule and/or
the detection molecule can be dehydrated to form the contrast agent
molecule. In such an embodiment, the pH of an aqueous solvent in
which the hyperpolarized precursor molecule or detection molecule
is dissolved can be adjusted to accelerate or slow chemical
conversion to the contrast agent molecule.
[0093] In some embodiments, the contrast agent molecule is selected
from the group including, but not limited to, diacetyl, oxolin,
alendronate, amitryptyline, nortriptyline, succinate, fumarate,
maleimide, catechol, naphthalene, naphthoquinone, phenylbutazone,
pyridazine, phthalazine, dopamine, L-dihydroxyphenylalanine
(L-DOPA) and derivatives thereof. In some embodiments, the
precursor molecule and the detection molecule can have the same
molecular structure. In some embodiments, the contrast agent
molecule is diacetyl (i.e., CH.sub.3C(.dbd.O)C(=O)CH.sub.3) and one
or both of the precursor molecule and the detection molecule is the
monohydrate of diacetyl (ie., CH.sub.3CH(OH)C(.dbd.O)CH.sub.3). In
some embodiments, both the precursor molecule and the detection
molecule are the monohydrate of diacetyl.
[0094] In some embodiments, the two J-coupled, non-zero-spin,
non-equivalent nuclei of the precursor molecule are selected from
the group consisting of .sup.1H, .sup.13C, .sup.15N and .sup.31P.
In some embodiments, the nuclei are selected from .sup.13C and
.sup.31P. In some embodiments, providing the precursor molecule
comprises synthetically doping or labeling a molecule with
.sup.13C, .sup.1H, .sup.15N or .sup.31P.
[0095] In some embodiments, the hyperpolarizing is performed by
DNP. DNP refers to transferring spin polarization from electrons to
nuclei. DNP can be performed by doping a material with a free
radical. The unpaired electrons in the free radical can be
polarized, for example, by exposure to a high magnetic field and
low temperature. Irradiation at the electron paramagnetic resonance
frequency can then serve to transfer polarization to the nuclei. By
"hyperpolarization," it is meant that the sample is polarized to a
level over that found at room temperature and 1 Tesla, preferably
polarized to a polarization degree in excess of 0.1%, more
preferably 1%, even more preferably 10%.
[0096] In some embodiments, the two J-coupled, non-zero-spin,
equivalent nuclei of the contrast agent molecule are free of
directly bonded hydrogen atoms. In some embodiments, the two
J-coupled, non-zero-spin, equivalent nuclei of the contrast agent
molecule are directly bonded to hydrogen atoms and are J-coupled to
two other, additional equivalent nuclei within the molecule,
wherein the two other, additional equivalent nuclei are free of
directly bonded hydrogen atoms, and whereby application of one or
more radiofrequency pulse(s) can transfer a spin state population
between the two J-coupled, non-zero-spin, equivalent nuclei and the
two other, additional equivalent nuclei.
[0097] In some embodiments, such as when the contrast is for use in
an in vivo MRI study, the method can further comprise incorporating
the contrast agent molecule into a pharmaceutically acceptable
carrier to provide a pharmaceutically acceptable formulation
suitable for administration to a subject. Administration can be by
any suitable means, such as, but not limited to, oral,
intraperitoneal or intravenous administration. In some embodiments,
the contrast agent can be delivered directly to a target organ or
tissue of interest in the subject directly via injection or topical
application (e.g., to a wound or surgical incision).
[0098] In some embodiments, the subject is a mammal. In some
embodiments, the mammal is a human. However, the presently
disclosed subject matter also relates to the administration of
contrast agents to any vertebrate animal (e.g., dogs, cats, horses,
cows, goats, sheep, and the like, including both animals kept on
farms and in zoos). Thus, the presently disclosed MRI contrast
agents can be used in both medical and veterinary practice. In some
embodiments, the term "pharmaceutically acceptable carrier" refers
to a carrier that is pharmaceutically acceptable in humans,
including water, saline, and aqueous solutions that can comprise
other diluents in addition to water, including, but not limited to,
ethanol, propylene glycol, glycerin, and the like.
[0099] In some embodiments, the contrast agent molecule can be
encapsulated into a delivery format or vehicle to protect the
contrast agent molecule from one or more conditions in an in vivo
environment (e.g., for at least a period of time) or to aid in
delivery of the contrast agent molecule to a specific location in a
subject (e.g., to a particular organ or to tumor). Thus, the
delivery format can include one or more targeting groups (e.g., an
antibody, antigen, receptor ligand, enzyme substrate, or the like)
that directs the contrast agent to a particular tissue, organ or
type of cell (e.g., a cancer or other type of diseased cell) due to
the presence of receptors or enzymes present on or nearby the
tissue, organ or cell.
[0100] In some embodiments, the method comprises encapsulating the
contrast agent molecule into a biodegradable delivery format that
prevents water contact with the contrast agent molecule prior to
complete or partial biodegradation of said delivery format.
Therefore, in some embodiments, the rate of biodegradation of the
delivery format can be chosen or manipulated to control the rate of
conversion of contrast agent molecule to detection molecule.
Suitable encapsulation agents include, but are not limited to,
various biocompatible, biodegradable polymers such as polyglycolic
acid (PGA) and copolymers thereof, polylactic acid (PLA) and
copolymers thereof, other polyesters and polyamide esters,
polyvinyl esters, and polyanhydrides. In some embodiments, the
encapsulation agent is not biodegradable, but can swell in the
presence of water to allow conversion of the contrast agent
molecule to the detection molecule and/or diffusion of the contrast
agent molecule or detection molecule out of the encapsulation
agent. In some embodiments, the delivery agent is a liposome. For
example, the liposome can have a lipophillic interior to protect
the contrast agent molecule from physiological conditions until
delivery to a site of interest.
[0101] In some embodiments, the non-equilibrium nuclear singlet
spin state population can persist for a time that is substantially
greater than T.sub.1. For example, in some embodiments, the
non-equilibrium nuclear singlet spin state population can persist
for a time that is greater that about 3 times T.sub.1. In some
embodiments, the non-equilibrium nuclear singlet spin state
population can persist for a time that is greater that about 10
times T.sub.1. In some embodiments, the non-equilibrium nuclear
singlet spin state population can persist for a time that is
between about 3 times T.sub.1 and about 10 times T.sub.1 (e.g.,
about 3 times T.sub.1, about 4 times T.sub.1, about 5 times
T.sub.1, about 6 times T.sub.1, about 7 times T.sub.1, about 8
times T.sub.1, about 9 times T.sub.1 or about 10 times
T.sub.1).
IV. Imaging Methods
[0102] In some embodiments, the presently disclosed subject matter
provides a method of imaging a target, the method comprising:
[0103] providing a contrast agent molecule having a non-equilibrium
singlet state nuclear spin population;
[0104] contacting the contrast agent molecule with the target;
[0105] allowing the contrast agent molecule to be chemically
converted into the detection molecule;
[0106] generating a nuclear magnetic resonance signal; and
[0107] detecting the nuclear magnetic resonance signal, thereby
imaging the target; wherein providing the contrast agent molecule
comprises: providing a precursor molecule comprising two J-coupled,
non-zero-spin, non-equivalent nuclei, wherein said precursor
molecule is chemically convertible to a contrast agent molecule
wherein the two J-coupled, non-zero-spin, non-equivalent nuclei are
converted to equivalent nuclei; hyperpolarizing the precursor
molecule to provide a hyperpolarized precursor molecule; applying
one or more radiofrequency pulse(s) to the hyperpolarized precursor
molecule to create one or both of a non-equilibrium .alpha..beta.
nuclear spin state population and a non-equilibrium .beta..alpha.
nuclear spin state population; and chemically converting the
hyperpolarized precursor molecule into the contrast agent molecule
by converting the two J-coupled, non-zero-spin, non-equivalent
nuclei to two J-coupled, non-zero-spin, equivalent nuclei, wherein
the contrast agent molecule comprises a non-equilibrium nuclear
singlet spin state population and is chemically convertible to a
detection molecule wherein the two J-coupled, non-zero-spin
equivalent nuclei are converted to non-equivalent nuclei.
[0108] In some embodiments, the target is one of a cell, a tissue,
an organ, and a subject. In some embodiments, the contrast agent
can be used in an NMR study and the target can be a chemical
composition (e.g., a non-biochemical reaction mixture or an
environmental sample, such as a water sample from a lake, stream,
river, ocean, residential water supply, or industrial site).
[0109] In some embodiments, the contacting comprises administering
a pharmaceutical formulation comprising the contrast agent to a
subject. The subject of the presently disclosed imaging methods can
be, in many embodiments, a human subject, although it is to be
understood the methods described herein are effective with respect
to all vertebrate species, which are intended to be included in the
term "subject." The methods described herein are particularly
useful in the imaging of warm-blooded vertebrates. Thus, the
methods can be used as medical or veterinary diagnostic methods in
mammals and birds.
[0110] More particularly, provided herein is the nuclear magnetic
imaging of mammals, such as humans, as well as those mammals of
importance due to being endangered (such as Siberian tigers), of
economical importance (animals raised on farms for consumption by
humans) and/or social importance (animals kept as pets or in zoos)
to humans, for instance, carnivores other than humans (such as cats
and dogs), swine (pigs, hogs, and wild boars), ruminants (such as
cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), and
horses. Also provided herein is the imaging of birds, including the
treatment of those kinds of birds that are endangered, kept in zoos
or as pets, as well as fowl, and more particularly domesticated
fowl, i.e., poultry, such as turkeys, chickens, ducks, geese,
guinea fowl, and the like, as they also are of economical
importance to humans. Thus, embodiments of the methods described
herein include the imaging of livestock, including, but not limited
to, domesticated swine (pigs and hogs), ruminants, horses, poultry,
and the like.
[0111] In some embodiments, providing the contrast agent molecule
further comprises incorporating the contrast agent molecule into a
pharmaceutically acceptable carrier. In some embodiments, the term
"pharmaceutically acceptable carrier" refers to a carrier that is
pharmaceutically acceptable in humans, including water, saline, and
aqueous solutions that can comprise other diluents in addition to
water, including, but not limited to, ethanol, propylene glycol,
glycerin, and the like. In some embodiments, the contrast agent
molecule is formulated for oral, intravenous, or interperitoneal
administration to the subject. In some embodiments, the contrast
agent is formulated for administration directly to a site of
interest (e.g., a wound or a tumor site accessible via a surgical
incision).
[0112] In some embodiments, the contrast agent molecule can be
incorporated into a delivery format that effects delivery of the
agent to a specific site in vivo (e.g., to a particular type of
tissue, organ or cell). In some embodiments, providing the contrast
agent molecule further comprises encapsulating the contrast agent
molecule into a biodegradable delivery format that prevents water
contact with the contrast agent molecule prior to complete or
partial biodegradation of said delivery format. Thus, in some
embodiments, upon complete or partial degradation of the delivery
format, the contrast agent molecule will be converted into the
detection molecule.
[0113] Suitable encapsulation agents for use in preparing the
delivery format include, but are not limited to, various
biocompatible, biodegradable polymers such as polyglycolic acid
(PGA) and copolymers thereof, polylactic acid (PLA) and copolymers
thereof, other polyesters and polyamide esters, polyvinyl esters,
and polyanhydrides. In some embodiments, the encapsulation agent is
not biodegradable, but can swell in the presence of water to allow
conversion of the contrast agent molecule to the detection molecule
and/or diffusion of the contrast agent molecule or detection
molecule out of the encapsulation agent. In some embodiments, the
delivery agent is a liposome. For example, the liposome can have a
lipophillic interior to protect the contrast agent molecule from
physiological conditions until delivery to a site of interest.
[0114] In some embodiments, the contrast agent molecule is selected
from the group including, but not limited to, diacetyl, oxolin,
alendronate, amitryptyline, nortriptyline, succinate, fumarate,
maleimide, catechol, naphthalene, naphthoquinone, phenylbutazone,
pyridazine, phthalazine, dopamine, L-dihydroxyphenylalanine
(L-DOPA) and derivatives thereof.
EXAMPLES
[0115] The following Examples have been included to provide
guidance to one of ordinary skill in the art for practicing
representative embodiments of the presently disclosed subject
matter. In light of the present disclosure and the general level of
skill in the art, those of skill can appreciate that the following
Examples are intended to be exemplary only and that numerous
changes, modifications, and alterations can be employed without
departing from the scope of the presently disclosed subject
matter.
Example 1
.sup.13C-Labelled Diacetyl
[0116] .sup.13C-labelled diacetyl was prepared from .sup.13C.sub.2
oxalate, which was reacted with N, N'-dimethylethylenediamine to
afford 2,3-.sup.13C.sub.2-1,4-dimethylpiperazine-2,3-dione in 97%
yield. Two equivalents of methyl magnesium bromide were added to
the piperazine dione to produce the dimethyl piperazine N,O-acetal.
Crude acetal was hydrolyzed with 10% aqueous HCl to yield
2,3-.sup.13C.sub.2-diacetyl, which was purified by fractional
distillation. The carbonyl carbons in diacetyl are magnetically
equivalent by symmetry, so at modest resolution (such as that
commonly achieved in an imaging system), the spectrum is expected
to have only a single line. Neat 2,3-.sup.13C diacetyl does have a
single line carbon-13 NMR spectrum, however the carbon spectrum in
water has five lines. See FIG. 1. In water, the monohydrate (i.e.,
CH.sub.3--(.sup.13C.dbd.O)(.sup.13C(OH).sub.2)CH.sub.3) with
classic AX doublets (splitting J.sub.C-C=50 Hz) is the majority
species. See Bell, Adv. Phys. Org. Chem., 4, 1 (1966); Bell and
McDougall, Trans. Faraday Soc., 56, 1281-1285 (1960); and Greenzaid
et al., J. Am. Chem. Soc., 89, 749 (1967). The dihydrate is
undetectable. Equilibrium can be shifted back to diacetyl by
changing solvent, and the rate of interconversion is pH dependent.
At pH 7, inversion of the diacetyl alone causes recovery in 8 s,
which gives the rate of dehydration. Inversion of all lines causes
diacetyl to recover in 22 s.
Example 2
Long-Lived Singlet State of Diacetyl
[0117] Preparation of the singlet state of diacetyl requires
perterbation of the .alpha..beta. and .beta..alpha. populations
from their equilibrium 25%. Hyperpolarization does not do this
efficiently by itself. For example, 20% nuclear polarization (60%
.alpha., 40% .beta.) would imply an .alpha..beta. population of
24%, only 1% from equilibrium, wasting most of the potential
signal. However, since all the energy levels in the hydrate are
accessable, suitable pulse sequences can manipulate the
.alpha..beta. and .beta..alpha. populations. The simplest is
inversion of one line in one of the doublets (e.g.
.alpha..alpha..fwdarw..alpha..beta.), which in this example would
interchange the 36% .alpha..alpha. and 24% .alpha..beta.
populations. Dehydration converts the sum of the .alpha..beta. and
.beta..alpha. populations (in this case, 60%) evenly among the
singlet .alpha..beta.-.beta..alpha. and triplet
.alpha..beta.+.beta..alpha. of diacetyl. The singlet population in
this case is 30%, six times farther from equilibrium than is
produced by DNP alone. After this dehydration, the population
should be locked for a very long time, unless it exchanges back to
the hydrate.
[0118] FIGS. 2 and 3 demonstrate this singlet lifetime extension,
both with and without hyperpolarization. FIG. 2 shows the results
of inverting only one line in thermally polarized monohydrate of
diacetyl, then checking populations later with a small flip angle
pulse. Deviations of the .alpha..beta. and .beta..alpha. population
from equilibrium cause a characteristic alternation of the peak
intensities (or, equivalently, FID signal at t=1/(2J)=10 ms, which
is absent in the signal from the unperturbed multiplets). FIG. 2
shows that population flows rapidly into diacetyl as expected, but
when it returns to the monohydrate (after about 30 s), it has
excess .alpha..beta. and .beta..alpha. population consistent only
with a long-lived state (the singlet).
[0119] With hyperpolarization, this population could be readily
converted back to observable signal in the hydrate (excess
population in .alpha..beta. and .beta..alpha. implies dipolar
order, which can be optimally converted to observable signal with a
45.degree. pulse and delay), dramatically extending the effective
hyperpolarized lifetime. This differs from most hyperpolarization
studies previously reported, where small pulse flip angles are used
to conserve the signal for multiple shots. Singlet diacetyl is
unaffected by rf pulses, so large flip angles do not deplete the
stored population. Equilibrium is readily shifted away from the
hydrate by the addition of acetone, thus permitting the singlet
state to last longer as a spin reservoir. For the data shown in
FIG. 3, diacetyl was hyperpolarized in water, then followed
inversion of one line of the monohydrate with immediate 3:1
dilution with acetone. See Warren et al., Science, 323, 1711-1714
(2009). The individual spectra were acquired by 45.degree. pulses
with 10 s time separation. The dynamics are complex (for example,
as the equilibrium shifts). Thus, one way to follow what is going
on is to look only at the FID from hydrate peaks. See FIG. 3. The
first FIDs have excess signal at t=1/2 J (dotted lines), expected
from the selective inversion. At intermediate times (sufficient for
dehydration and mixing of the acetone), the FIDs look similar to
the thermal ones (averaged over 360 shots). The later FIDs have
signal higher than thermal, and the structure is very complex. In
the case of FID 14 in FIG. 3, the preceding 45.degree. pulses
should have depleted all but 1% of the hyperpolarized signal, the
relaxation should have depleted all but 0.1%, so less than
10.sup.-5 should remain in the absence of singlet effects.
[0120] Shifting the equilibrium with acetone in vivo is not
feasible. However, as diacetyl does not have a dipole moment, it
can migrate to the nonpolar phase. For example, it is mostly found
in the fatty phase in butter. See Hoecker and Hammer, J. Dairy
Sci., 25, 175-185 (1942). Accordingly one application of diacetyl
is as a "reporter molecule" in a delivery system, including, but
not limited to, functionalized or temperature sensitive liposomes
or functionalized ultrasound contrast agents, including those based
on encapsulated perfluorocarbons. See Jakobsen, et al., Eur.
Radiol, 15, 941-945 (2005).
Example 3
Effectively Equivalent Nuclei
[0121] In a high resolution spectrometer, the spectra of diacetyl
is complicated. See Warren et al., Science, 323, 1711-1714 (2009).
The carbonyl carbon spins are not strictly fully equivalent. Each
carbonyl carbon is coupled differently to the two methyl groups
(the C--H couplings are 6.4 Hz and -1.1 Hz). However, the C--C
coupling constant (approximately 50 Hz) significantly exceeds these
couplings. The effect of this can be seen in FIGS. 4A-4F.
[0122] FIG. 4A shows the carbonyl region .sup.13C NMR spectrum of
2-.sup.13C diacetyl, where the carbon has two different couplings
to three hydrogens each and, thus, becomes a quartet of quartets.
FIG. 4B shows the carbonyl region .sup.13C NMR spectrum of acetone,
which is a septet due to the equivalence of the six hydrogens. FIG.
4C shows the complicated carbonyl region of the .sup.13C NMR
spectrum of 2,3-.sup.13C diacetyl, which suggests that the carbon
singlet state is nearly disconnected.
[0123] FIGS. 4D-4F show simulations of .sup.13C NMR spectra of
1,2-.sup.13C diacetyl which further illustrate the near
disconnectedness of the carbon singlet state by varying the
coupling J.sub.C--C between the two carbons. The simulations were
prepared using the WindNMR-Pro program, freeware available from the
website of Professor Hans Reich at the University of
Wisconsin-Madison. When J.sub.C-C=0, as shown in FIG. 4D, the
doubly labelled and singly labeled spectra (FIG. 4A) are
comparable, and inspection of the energy levels shows that the
carbon singlet is not an eigenstate. The two different scalar
couplings readily connect this state to others with the same
overall symmetry, but with (.alpha..beta.+.beta..alpha.) as the
carbon component. However, if J.sub.C-C is much larger than all
other couplings (see FIG. 4E), the spectrum changes dramatically.
It collapses back into a septet, similar to the acetone spectrum,
excepting that the splitting is not a real coupling. It is the 2.65
Hz average of the couplings between the near and far methyl groups.
This result can be explored by exact calculations, which indicate
that the spectrum comes from transitions involving
(.alpha..beta.+.beta..alpha.) as a carbon state, which is
delocalized over the two carbons and is coupled equally to each
hydrogen. The two carbon spins achieve effective magnetic full
equivalence and the (.alpha..beta.+.beta..alpha.) state is
completely disconnected and, thus, long-lived.
[0124] Similar spin systems with the same properties, such as but
not limited to, the A.sub.2X.sub.2 case (e.g., 1,2-dichlorobenzene)
have been previously studied. See Pople et al., Can. J. Chem., 35,
1060 (1957); and McConnell et al., Chem. Phys., 23, 1152 (1955). In
general, it is known that strong couplings can produce deceptively
simple spectra. See Abraham and Bernstein, Can. J. Chem., 39, 216
(1961); Anet, Can. J. Chem., 39, 2262 (1961); Musher and Corey,
Tetrahedron, 18, 791 (1962); and Becker, High Resolution NMR:
Theory and Chemical Applications (Academic, San Diego, 2000), page
171-175. In the A.sub.2X.sub.2 case, 12 lines associated with the A
transitions would be expected, not one. As described previously
(see Pople et al., Can. J. Chem., 35, 1060 (1957); and McConnell et
al., Chem. Phys., 23, 1152 (1955)), one important parameter is the
ratio (J.+-.J')/(J.sub.A-A-J.sub.X-X), where J and J' are the two
different A-X couplings (the minus sign gives the larger value in
cases where the couplings have opposite signs). When this ratio is
small, simple perturbation theory analysis shows the worst overlap
of a carbon singlet state with a true spin eigenstate to be
{1-0.25((.sub.J-J')/J.sub.A-A-J.sub.X-X)).sup.2}, and T.sub.1
lengthening is expected to be on the order of
((JA-A-JX-X)/(J-J')).sup.2.
[0125] Perturbation theory analysis can be extended to the case of
diacetyl. Even though the spectra in FIGS. 4C and 4F are quite
complex, assuming the couplings have the same value as in the
hydrate shows that the average overlap of the singlet state with an
eigenstate is better than 0.97. This can be verified by precise
numerical analysis of this eight-spin system, predicting more than
an order of magnitude lengthening of the spin lifetime. In effect,
the strong coupling between the two carbons quenches communication
with other spins. Thus, without being bound to any one theory, it
is believed that virtually all the spectral complexity comes from
the other three carbon states, and singlet to triplet
interconversion is slow. Perdeuteration, which can be readily
achieved in this system via keto-enol tautomerization, can
dramatically reduce even this limited singlet-triplet mixing and
further increase the lifetime.
REFERENCES
[0126] All references listed below, as well as all references cited
in the instant disclosure, including but not limited to all
patents, patent applications and publications thereof, and
scientific journal articles, are incorporated herein by reference
in their entireties to the extent that they supplement, explain,
provide a background for, or teach methodology, techniques, and/or
compositions employed herein.
[0127] Abragam, A. and Goldman, M., Rep. Prog. Phys. 41, 395-467
(1978).
[0128] Abraham, R. J., and Bernstein, H. J., Can. J. Chem., 39, 216
(1961).
[0129] Ahuja, P., Sarkar, R., Vasos, P. R., Bodenhausen, G., J.
Chem. Physics, 127, 134112 (2007).
[0130] Anet, F. A. L., Can. J. Chem., 39, 2262 (1961).
[0131] Ardenkjaer-Larsen, J. H., Fridlund, B., Gram, A., Hansson,
G., Hansson, L., Lerche, M. H., Servin, R., Thaning, M., Golman,
K., Proc. Natl. Acad. Sci. U.S.A., 100, 10158-10163 (2003).
[0132] Bajaj, V. S., Farrar, C. T., Hornstein, M. K., Mastovsky,
I., Vieregg, J., Bryant, J., Elena, B., Kreischer, K. E., Temkin,
R. J., Griffin, R. G., Journal of Magnetic Resonance, 160, 85-90
(2003).
[0133] Becker, E. D., High Resolution NMR: Theory and Chemical
Applications, (Academic, San Diego, 2000), 171-175.
[0134] Bell. R. P., Adv. Phys. Org. Chem., 4, 1 (1966).
[0135] Bell, R. P., and McDougall, A. O., Trans. Faraday Soc. 56,
1281-1285 (1960).
[0136] Bhattacharya, P., et al., Journal of Magnetic Resonance,
186(1), 150-155 (2007).
[0137] Bowers, C. R., and Weitekamp, D. P., Phys. Rev. Lett., 57,
2645-2648 (1986).
[0138] Bowers, C. R., and Weitekamp, D. P., J. Am. Chem. Soc.,109,
5541-5542 (1987).
[0139] Carravetta, M., Johannessen, O. G., Levitt, M. H., Physical
Review Letters, 92, 153003 (2004). Carravetta, M. and Levitt, M.
H., J. Am. Chem. Soc., 126(20), 6228-6229 (2004).
[0140] Carravetta, M. and Levitt, M. H., J. Chem. Physics, 122,
214505 (2005).
[0141] Day, L. J.; Mitchell, J. C.; Snowden, M. J.; Davis, A. L.,
Magnetic Resonance in Chemistry 45(12), 1018-1021 (2007).
[0142] de Boer, W., Borghini, M., Morimoto, K., Niinikoski, T. O.,
Udo, T., Journal of Low Temperature Physics 15, 249-267 (1974).
[0143] de Boer, W., and Niinikoski, T. O., Nuclear Instruments and
Methods 114, 495-498 (1974).
[0144] Duckett, S. B., and Sleigh, C. J., Progr. Nucl. Magn. Reson.
Spectrosc., 34, 71-92 (1999).
[0145] Gabellieri, C., Reynolds, S., Lavie, A., Payne, G. S.,
Leach, M. O., Eykyn, T. R., J. Am. Chem. Soc., 130(14), 4598
(2008).
[0146] Golman, K., Ardenkjaer-Larsen, J. H., Petersson, J. S.,
Mansson, S., Leunbach, I., Proc. Natl. Acad. Sci. U.S.A., 100,
10435-10439 (2003).
[0147] Golman, K., Axelsson, O., Johannesson, H., M{dot over
(a)}nsson, Olofsson, C., Petersson, J. S., Magnetic Resonance in
Medicine, 46, 1-5 (2001).
[0148] Golman, K., in't Zandt, R., Lerche, M., Pehrson, R.,
Ardenkjaer-Larsen, J. H., Cancer Research, 66, 10855-10860,
(2006).
[0149] Greenzaid, P., Luz, Z., Samuel, D., J. Am. Chem. Soc., 89,
749 (1967).
[0150] Hall, D. A., Maus, D. C., Gerfen, G. J., Inati, S. J.,
Becerra, L. R., Dahlquist, F. W., Griffin, R. G., Science, 276,
930-931 (1997).
[0151] Hoecker, W. H., and Hammer, B. W., J. Dairy Sci., 25,
175-185 (1942).
[0152] Jakobsen, J. A., Oyen, R., Thomsen, H. S., Morcos, S. K.,
Eur. Radiol., 15, 941-945 (2005).
[0153] Johansson E., Mansson, S., Wirestam, R., Svensson, J.,
Petersson, J. S., Golman, K., Stahlberg, F., Magn. Reson. Med., 51,
464-472 (2004).
[0154] Kiselyov, A. S., Semenov, V. V., Milligan, D., Chem. Biol.,
Drug Des., 68, 308-313 (2006).
[0155] Kurhanewicz, J., Bok, R., Nelson, S. J., Vigneron, D. B., J.
Nucl. Med., 49(3), 341-344 (2008).
[0156] MacFall, J. R., Charles, H. C., Black, R. D., Middleton, H.,
Swartz, J. C., Saam, B., Driehuys, B., Erickson, C., Happer, W.,
Cates, G. D., Johnson, G. A., Ravin, C. E., Radiology, 200, 553-558
(1996).
[0157] McConnell, H. M., McLean, A. D., Reilly, C. A., J. Chem.
Phys., 23, 1152 (1955).
[0158] Merritt, M. E., Harrison, C., Storey, C., Jeffrey, F. M.,
Sherry, A. D., Malloy, C. R., Proc. Natl. Acad. Sci. U.S.A., 104,
19773-19777 (2007).
[0159] Musher, J. I., and Corey, E. J., Tetrahedron, 18, 791
(1962).
[0160] Natterer, J., and Bargon. J., Progr. Nucl. Magn. Reson.
Spectrosc., 31, 293-315, (1997).
[0161] Oleson, O. V., and Linnet, K., Drug Metabolism and
Disposition, 25, 740-744 (1997).
[0162] Pople, J. A., Schneider, W. G., Bernstein, H. J., Can. J.
Chem., 35, 1060 (1957).
[0163] PCT International Patent Publication Number WO
2005/015253.
[0164] Salerno, M., Altes, T. A., Mugler 3.sup.rd, J. P., Nakatsu,
M., Hatabu, H., de Lange, E. E., Eur. J Radiol., 40, 33-44
(2001).
[0165] Warren, W. S., Jenista, E. R., and Branca, R. T., Science,
323, 1711-1714 (2009).
[0166] It will be understood that various details of the presently
disclosed subject matter may be changed without departing from the
scope of the presently disclosed subject matter. Furthermore, the
foregoing description is for the purpose of illustration only, and
not for the purpose of limitation.
* * * * *