U.S. patent application number 10/837504 was filed with the patent office on 2004-12-02 for imaging of enzymatic activity.
This patent application is currently assigned to The General Hospital Corporation, a Massachusetts corporation. Invention is credited to Bogdanov, Alexei, Weissleder, Ralph.
Application Number | 20040241096 10/837504 |
Document ID | / |
Family ID | 26934397 |
Filed Date | 2004-12-02 |
United States Patent
Application |
20040241096 |
Kind Code |
A1 |
Bogdanov, Alexei ; et
al. |
December 2, 2004 |
Imaging of enzymatic activity
Abstract
The invention features methods of detecting enzymatic activity
(e.g., in a magnetic resonance image). In general, the methods
include: (1) providing a monomeric substrate (e.g., a substrate
that is polymerizable in the presence of an enzyme or as a result
of an enzyme-catalyzed reaction), having the generic structure
X-Y-Z, where X includes a chelator moiety having a chelated
paramagnetic or superparamagnetic metal atom or ion, Y includes a
linker moiety (e.g., to provide a covalent or non-covalent chemical
bond or bonds between X and Z), and Z includes a polymerizing
moiety; (2) contacting the substrate with a target tissue, wherein
the substrate undergoes polymerization to form a paramagnetic or
superparamagnetic polymer, the polymerization being catalyzed by an
enzyme in an extracellular matrix or bound to the surfaces of cells
of the target tissue; and (3) detecting an increase in relaxivity
for the polymer relative to an equivalent amount of unpolymerized
substrate. The invention also features substrate compositions.
Inventors: |
Bogdanov, Alexei;
(Arlington, MA) ; Weissleder, Ralph; (Charlestown,
MA) |
Correspondence
Address: |
FISH & RICHARDSON PC
225 FRANKLIN ST
BOSTON
MA
02110
US
|
Assignee: |
The General Hospital Corporation, a
Massachusetts corporation
|
Family ID: |
26934397 |
Appl. No.: |
10/837504 |
Filed: |
April 30, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10837504 |
Apr 30, 2004 |
|
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|
09999665 |
Oct 19, 2001 |
|
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|
6737247 |
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60241566 |
Oct 19, 2000 |
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60310335 |
Aug 6, 2001 |
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Current U.S.
Class: |
424/9.34 ;
435/6.16; 530/400 |
Current CPC
Class: |
G01R 33/48 20130101;
G01R 33/465 20130101 |
Class at
Publication: |
424/009.34 ;
530/400; 435/006 |
International
Class: |
A61K 049/00; C12Q
001/68 |
Claims
1-27. (canceled).
28. A composition comprising a compound of formula X-Y-Z, wherein X
comprises a chelator moiety, Y comprises a linker moiety, and Z
comprises a polymerizing moiety.
29. The composition of claim 28, wherein the compound further
comprises a paramagnetic or superparamagnetic metal atom or
ion.
30. The composition of claim 29, wherein the paramagnetic or
superparamagnetic metal atom or ion is a transition metal atom or
ion.
31. The composition of claim 29, wherein the paramagnetic or
superparamagnetic metal atom or ion is a lanthanide atom or
ion.
32. The composition of claim 29, wherein the metal ion is selected
from the group consisting of an iron ion, a dysprosium ion, a
europium ion and a manganese ion.
33. The composition of claim 29, wherein the metal ion is a
gadolinium ion.
34. The composition of claim 28, wherein the polymerizing moiety
comprises any chemical group that can be chemically modified as a
result of the catalytic activity of an enzyme to form a covalent
chemical bond between either (1) Z and another compound of formula
X-Y-Z or (2) Z and any polymer or macromolecule.
35. The composition of claim 28, wherein Z is a moiety that can be
accommodated by the catalytic center of an enzyme.
36. The composition of claim 28, wherein X comprises a structure
selected from the group consisting of:
1,4,7,10-tetraazacyclodo-decane-N,N',N",N'"- -tetraacetic acid;
1,4,7,10-tetraaza-cyclododecane-N,N',N"-triacetic acid;
1,4,7-tris(carboxymethyl)-10-(2'-hydroxypropyl)-1,4,7,10-tetraazocyclodec-
ane; 1,4,7-triazacyclonane-N,N',N"-triacetic acid;
1,4,8,11-tetraazacyclot- etra-decane-N,N',N",N'"-tetraacetic acid;
diethylenetriamine-pentaacetic acid (DTPA); ethylenedicysteine;
bis(aminoethanethiol)carboxylic acid;
triethylenetetraamine-hexaacetic acid; ethylenediamine-tetraacetic
acid (EDTA); 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid;
N-(hydroxy-ethyl)ethylenediaminetriacetic acid; nitrilotriacetic
acid; and ethylene-bis(oxyethylene-nitrilo)tetraacetic acid.
37. The composition of claim 28, wherein Z comprises the following
structure: 4wherein: R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5
are selected independently from the group consisting of H; R.sup.6,
wherein R.sup.6 is C.sub.1-C.sub.6 unsubstituted alkyl;
NHC(O)R.sup.6; OH; or NR.sup.7R.sup.8, wherein R.sup.7 and R.sup.8
are H or R.sup.6; provided that at least one of R.sup.1, R.sup.2,
R.sup.3, R.sup.4 and R.sup.5 is OH.
38. The composition of claim 37, wherein R.sup.1, R.sup.2, R.sup.3,
R.sup.4 or R.sup.5 is at an ortho position relative to the OH
substituent, and is selected from the group consisting of OH and
OCH.sub.3.
39. The composition of claim 37, wherein R.sup.1, R.sup.2, R.sup.3,
R.sup.4 or R.sup.5 is at a meta position relative to the OH
substituent, and is selected from the group consisting of
NHC(O)R.sup.6 and NR.sup.7R.sup.8.
40. The composition of claim 28, wherein Y comprises a structure
selected from the group consisting of: an amino acid, an
oligopeptide comprising 2-6 amino acid residues, a nucleotide, an
oligonucleotide comprising 2-6 nucleotide residues, a
C.sub.3-C.sub.12 alkyl group, a polyethyleneimine, a saccharide, an
oligosaccharide, a medium chain fatty acid, a polyamidoamine, a
polyacrylic acid, and a polyalcohol.
41. The composition of claim 28, wherein Y comprises an amino acid
or oligopeptide containing 2-6 amino acid residues.
42. The composition of claim 41, wherein the oligopeptide comprises
a glycine residue.
43. The composition of claim 29, wherein the compound comprises the
formula: 5where R1 is selected from the group consisting of H, OH,
and OCH.sub.3.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/241,566, filed Oct. 19, 2000, and U.S.
Provisional Application No. 60/310,335, filed Aug. 6, 2001, both of
which are incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates to biochemistry and magnetic
resonance imaging.
BACKGROUND OF THE INVENTION
[0003] Non-invasive imaging of molecular expression in vivo with
high resolution and high sensitivity would be a useful tool in
clinical diagnostics and in biomedical research. A detectable
label, e.g., a radioactive atom, can be linked to a targeting
moiety, e.g., an antibody, which binds specifically a molecular
target (molecule of interest). Such targeting can be used for
imaging cells or tissues that display the molecular target.
Magnetic resonance imaging (MRI) offers certain well-known
advantages as a non-invasive imaging technology. For example, MRI
can potentially provide exceptionally high anatomic resolution
approaching single-cell levels (voxel of 20-40 .mu.m.sup.3).
Moreover, recent innovations in instrument design and contrast
agent development indicate that above level of resolution can be
achieved non-invasively in vivo. One of the major future directions
of in vivo MRI research includes mapping of specific molecules
(e.g. receptors) and detecting patterns of their expression.
[0004] However, the inherently low sensitivity of MRI to the
presence of magnetic labels, and consequently low
signal-to-background ratio, has limited the usefulness of MRI for
detection and imaging of low-abundance, molecular targets such as
cell surface receptor molecules. MRI of receptor-specific contrast
agents has been challenging because of relatively low sensitivity
to the presence of paramagnetic metal labels. For example, the
detectability limit for paramagnetic gadolinium complexes is
estimated to be approximately 100 .mu.mol Gd per gram of tissue.
Therefore, a way of amplifying an MRI signal from a targeted,
magnetic label is needed.
[0005] A number of different amplification schemes have been
pursued to increase specific MR signal. Most commonly,
amplification is achieved by covalent attachment of several
signal-generating paramagnetic cations or a superparamagnetic
particle to a targeting molecule (e.g., a receptor ligand).
However, affinity molecules that are not bound to the target
(circulating in the bloodstream or retained non-specifically) can
generate high background signal due to indiscriminate shortening of
water proton relaxation times. Nonspecific signal can obscure the
target due to the low target/background ratio. This is especially
relevant in the case of vascular targeting.
SUMMARY OF THE INVENTION
[0006] The invention is based on the discovery that enzyme activity
can be used to amplify the decrease in local proton relaxation
rates produced by chelated gadolinium (Gd) or other metals. This
amplification has been demonstrated to result from enzyme-dependent
polymerization of a monomeric substrate in which the metal atom or
ion is chelated.
[0007] Based on this development, the invention features methods of
detecting enzymatic activity (e.g., in a magnetic resonance image).
In general, the methods include: (1) providing a monomeric
substrate (e.g., a substrate that is polymerizable in the presence
of an enzyme or as a result of an enzyme-catalyzed reaction),
having the generic structure X-Y-Z, where X includes a chelator
moiety having a chelated paramagnetic or superparamagnetic metal
atom or ion, Y includes a linker moiety (e.g., to provide a
covalent or non-covalent chemical bond or bonds between X and Z),
and Z includes a polymerizing moiety; (2) contacting the substrate
with a target tissue, wherein the substrate undergoes
polymerization to form a paramagnetic or superparamagnetic polymer,
the polymerization being catalyzed by an enzyme in an extracellular
matrix or bound to the surfaces of cells of the target tissue; and
(3) detecting an increase in relaxivity for the polymer relative to
an equivalent amount of unpolymerized substrate.
[0008] As used herein, "an equivalent amount of unpolymerized
substrate" means the number of monomeric substrate molecules
represented by a polymer having a particular molecular size or
mass.
[0009] Examples of chelating moieties that can be incorporated into
a monomeric substrate for use in the invention include the
following: 1,4,7,10-etraazacyclododecane-N,N',N",N'"-tetraacetic
acid (DOTA); 1,4,7,10-tetraaza-cyclododecane-N,N',N"-triacetic
acid;
1,4,7-tris(carboxymethyl)-10-(2'-hydroxypropyl)-1,4,7,10-tetraazacyclodec-
ane, 1,4,7-triazacyclonane-N,N',N"-triacetic acid; and
1,4,8,11-tetraazacyclotetra-decane-N,N',N",N'"-tetraacetic acid;
diethylenetridiamine-pentaacetic acid (DTPA);
triethylenetetraamine-hexaa- cetic acid;
ethylenediamine-tetraacetic acid (EDTA); EGTA;
1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid;
N-(hydroxyethyl)ethylenediaminetriacetic acid; nitrilotriacetic
acid; and ethylene-bis(oxyethylene-nitrilo)tetraacetic acid.
[0010] The paramagnetic or superparamagnetic metal atom or ion can
be, for example, a transition metal or lanthanide atom or ion
having paramagnetic properties (e.g., Fe.sup.3+, Gd.sup.3+,
Dy.sup.3+, Eu.sup.3+, Mn.sup.2+).
[0011] Examples of suitable linker moieties include: amino acids,
oligopeptides (e.g., oligopeptides having 2-6 amino acid residues),
nucleotides, an oligonucleotides (e.g., oligonucleotides having 2-6
nucleotide residues), C.sub.3-C.sub.12 alkyl groups,
polyethyleneimines, saccharides, oligosaccharides, medium chain
fatty acids, polyamidoamines, polyacrylic acids, and polyalcohols.
In some embodiments of the invention, the linker moiety can contain
an amino acid or oligopeptide containing 2-6 amino acid residues.
Thus, in certain embodiment of the invention, the monomeric
substrate can have the structure: 1
[0012] where R.sup.1 is H, OH, or OCH.sub.3.
[0013] As used herein, a "polymerizing moiety" can be any chemical
group (e.g., a phenolic moiety or a modified nucleotide) that can
be chemically modified in the presence of and as a result of the
catalytic activity of an enzyme to form a covalent chemical bond
between (1) the modified polymerizing moiety and another substrate
of the invention or (2) the modified polymerizing moiety and any
other macromolecule present during the reaction, including (but not
limited to) the enzyme itself. As used herein, "chemically
modified" means subjected to any rearrangement of electron density,
including addition or withdrawal of electrons.
[0014] Examples of polymerizing moieties that can be incorporated
into a monomeric substrate for use in the invention include
phenolic moieties and other moieties that can be accommodated by
the catalytic center of the enzyme (e.g., a chemical structure
having a suitable size, shape, and functional groups such as
hydrogen bond donors and/or acceptors, hydrophobic and/or
hydrophilic groups, aromatic rings and/or other functional groups
as appropriate for creating hydrogen bonding, van der Waals
interactions, ionic bonding, and/or pi stacking or other
interactions between the substrate and the enzyme; such parameters
can be identified using known or future methods including, but not
limited to, computer-based molecular modeling and computational
methods).
[0015] In certain embodiments, for example, the polymerizing moiety
can be a phenolic moiety such as the following: 2
[0016] where R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5,
independently, can be H; R.sup.6, wherein R.sup.6 is
C.sub.1-C.sub.6 unsubstituted alkyl; NHC(O)R.sup.6; OH; or
NR.sup.7R.sup.8, wherein R.sup.7 and R.sup.8 are H or R.sup.6;
provided that at least one of R.sup.1, R.sup.2, R.sup.3, R.sup.4
and R.sup.5 is OH.
[0017] In some embodiments of the invention, R.sup.1, R.sup.2,
R.sup.3, R.sup.4 or R.sup.5 is at an ortho position relative to the
OH substituent, and is either OH or OCH.sub.3. In other
embodiments, R.sup.1, R.sup.2, R.sup.3, R.sup.4 or R.sup.5 is at a
meta position relative to the OH substituent, and is either
NHC(O)R.sup.6 or NR.sup.7R.sup.8.
[0018] The enzyme employed to catalyze polymerization of the
monomeric substrate can be, in some cases, covalently linked to a
targeting moiety, and the targeting moiety can in turn bind
noncovalently to a target molecule in an intercellular matrix or on
the surface of a cell of the target tissue. In some embodiments,
the enzyme is an oxidoreductase, e.g., a peroxidase such as
lactoperoxidase and horseradish peroxidase, or a laccase. In
alternative embodiments, the enzyme is a monophenol oxidase,
monophenol monooxygenase, or catechol oxidase. An exemplary
monophenol oxidase is tyrosinase.
[0019] Examples of useful targeting moieties are a primary
antibody, a secondary antibody, a cell adhesion molecule, a
cytokine, a cell surface receptor molecule, or a fragment thereof
that recognizes a preselected binding partner. A primary antibody
and a secondary antibody are preferred targeting moieties.
[0020] Compositions that include the compounds X-Y-Z described
above, with or without a chelated metal atom or ion, are also
considered to be an aspect of the invention.
[0021] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. In case
of conflict, the present application, including definitions, will
control. All publications, patents and other references mentioned
herein are incorporated by reference.
[0022] Although methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the present invention, the preferred methods and materials are
described below. The materials, methods and examples are
illustrative only and not intended to be limiting. Other features
and advantages of the invention will be apparent from the detailed
description and from the claims.
BRIEF DESCRIPTION OF DRAWINGS
[0023] FIG. 1 is a synthetic pathway used for synthesis of an
enzyme-responsive, paramagnetic monomeric substrate.
[0024] FIG. 2 is a chromatogram summarizing results of size
exclusion analysis of polymerized reaction products.
[0025] FIG. 3 is a graph summarizing data from MRI of
tyramine-[1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic
acid, gadolinium salt] (tyramine-DOTA(Gd)) at various Gd
concentrations before peroxidase treatment (open circles), and
after peroxidase treatment (closed circles). These data show the
dependence of signal intensity on gadolinium concentration and
peroxidase-dependent polymerization.
[0026] FIG. 4 is a graph showing magnetic resonance signal
intensity enhancement as a function of peroxidase amount.
Circles--Dopamine-DOTA(Gd- ); squares--Tyramine-DOTA(Gd). Imaging
was performed at 1.5 T (Signa GE), 400 .mu.M 1% fetal calf serum,
0.005% H.sub.2O.sub.2 detected using a spin-echo (SE) sequence.
[0027] FIG. 5 is a graph showing magnetic resonance-ELISA signal
intensity as a function of DIG-labeled antibody amount.
Circles--Dopamine-DOTA(Gd); squares--Tyramine-DOTA(Gd). Imaging was
performed at 1.5 T (Signa GE), 400 .mu.M 1% fetal calf serum,
0.005% H.sub.2O.sub.2 detected using a SE sequence.
[0028] FIG. 6 is a graph showing confidence intervals of the
corresponding pixel signal intensity distribution (SI) within
region-of-interest (ROI) from photographs illustrating MRI of human
umbilical vein endothelial cells (HUVEC) at 1.5 T. Image 1 shows a
positive control solution of 50 .mu.M Gd. Image 2 shows HUVEC cells
treated with IL-1.beta., anti-E-selectin-DIG antibody and anti-DIG
peroxidase conjugate followed by 400 .mu.M Tyr-DOTA(Gd) (1 hour,
room temperature). Image 3 shows control HUVEC cells with no
IL-1.beta. stimulation. Image 4 shows control HUVEC cells receiving
IL-1.beta. stimulation with no anti-E-selectin-DIG antibody.
DETAILED DESCRIPTION OF THE INVENTION
[0029] The invention features an MRI method that can be employed
for non-invasive detection and imaging of a selected "marker"
enzyme activity in tissues of an experimental animal or a human
patient. The basis of the method is an enhancement of the effect on
local proton relaxation rates (decrease in T1 and T2 relaxation
times) exerted by a chelated (super)paramagnetic metal or metal
oxide. This enhancement occurs when a monomeric substrate
containing the chelated (super)paramagnetic metal or metal oxide
undergoes polymerization catalyzed by the marker enzyme. The
decreased relaxation times (increased relaxivity) associated with
the polymerized product, relative to an equivalent amount of
unpolymerized substrate, translates into an amplified MRI signal at
the site of enzymatic activity.
[0030] While not intending to be bound by theory of the invention's
mechanism, the inventors believe the increased relaxivity occurs
because the polymerized product has an increased rotational
correlation time (.tau..sub.r), relative to that of the monomeric
substrate.
[0031] Monomeric substrates used in method of the invention include
four basic components: three structural moieties: (1) a chelating
moiety, (2) a linker moiety, and (3) a polymerizing moiety. The
fourth component is a bound paramagnetic or superparamagnetic metal
atom or metal oxide. Each of the three structural moieties performs
a separate function. The chelating moiety binds or chelates the
paramagnetic or superparamagnetic metal atom or metal oxide. The
phenolic moiety serves as an electron donor that participates in a
free radical polymerization reaction catalyzed by the marker
enzyme. The linker moiety provides a chemical bond between the
chelating moiety and the polymerizing moiety, so that, when the
polymerizing moiety undergoes polymerization, the chelating moiety,
with its bound paramagnetic or superparamagnetic label, is
polymerized concomitantly.
[0032] Various chelating moieties are known, and can be
incorporated into a monomeric substrate useful in the invention,
without undue experimentation. In addition, novel chelating
moieties may be discovered in the future, and can be used in the
invention. Preferably, the chelating moiety does not form a
covalent bond with the paramagnetic or superparamagnetic metal or
metal oxide. In preferred embodiments, the chelating moiety forms a
thermodynamically and kinetically stable, non-covalent coordination
complex or ionic complex with Fe.sup.3+, Gd.sup.3+, Dy.sup.3+,
Eu.sup.3+, Mn.sup.2+, or other useful metal or metal oxide.
[0033] Numerous chelating moieties suitable for incorporation into
a monomeric substrate useful in the invention are known in the art.
Examples of chelating moieties useful in the invention include:
[0034] 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid
(DOTA);
[0035] 1,4,7,10-tetraaza-cyclododecane-N,N',N"-triacetic acid;
[0036]
1,4,7-tris(carboxymethyl)-10-(2'-hydroxypropyl)-1,4,7,10-tetraazocy-
clodecane;
[0037] 1,4,7-triazacyclonane-N,N',N"-triacetic acid;
[0038] 1,4,8,11-tetraazacyclotetra-decane-N,N',N",N'"-tetraacetic
acid;
[0039] diethylenetriamine-pentaacetic acid (DTPA);
[0040] triethylenetetraamine-hexaacetic acid;
[0041] ethylenediamine-tetraacetic acid (EDTA);
[0042] 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid;
[0043] N-(hydroxyethyl)ethylenediaminetriacetic acid;
[0044] nitrilotriacetic acid; and
[0045] ethylene-bis(oxyethylene-nitrilo)tetraacetic acid;
[0046] The polymerizing moiety can be any biocompatible moiety that
undergoes enzyme-dependent polymerization. Exemplary polymerizing
moieties are a phenolic moiety, a modified nucleotide moiety, and a
saccharide moiety. The marker enzyme and polymerizing moiety are
selected for functional compatibility, i.e., the polymerizing
moiety is recognized as a substrate by the marker enzyme.
[0047] As used herein, "phenolic moiety" means a moiety containing
a phenolic ring. As used herein, a "phenolic ring" is a phenyl ring
wherein at least one ring position is substituted with a hydroxyl
(OH) group, and other ring positions are optionally substituted,
provided that at least one ring position is unsubstituted. A
phenolic ring can participate in a free radical polymerization
reaction, under certain conditions.
[0048] A preferred phenolic moiety has the structure: 3
[0049] where R.sup.1 is H, OH, or OCH.sub.3. Numerous structural
variations are permissible in the phenolic moiety. For example, in
addition to the foregoing substitutions at one of the para
positions, the other para position can be substituted as well,
e.g., with H, OH, or OCH.sub.3. When both para positions are
substituted, the substituents can be the same or different. In
another variation, an amino group or an amido group is substituted
at a meta position on the phenolic ring. The effect(s) of the
various substitutions possible on the phenolic ring can be
predicted by one of skill in the art according to known principles
of organic chemistry, based on the identities of the substituents
and their relative positions on the ring. See, e.g., L. G. Wade,
Jr., 1988, Organic Chemistry, Prentice-Hall, Inc., Englewood
Cliffs, N.J. at 666-669. For example, an amino group at the meta
position (relative to the hydroxyl group) is strongly activating,
i.e., it makes the ring a better electron donor, and thus more
reactive.
[0050] Based on well-known chemistry, it is predicted that in the
present invention phenolic polymerization occurs when a phenolic
free radical is generated by loss of an electron from a phenolic
moiety. This occurs, for example, when each of two phenolic
moieties donates one electron apiece in the reaction
H.sub.2O.sub.2.fwdarw.2H.sub.2O catalyzed by peroxidase. Two
phenolic free radicals then react with each other to form a
covalent linkage. The phenolic free radicals include several
resonance forms in which the unpaired electron is present at
different positions on the aromatic ring, as well as on the oxygen.
This results in covalent coupling of the free radicals in various
linkages, giving rise to a mixture of different polymerized
products. Information concerning phenolic polymerization reactions
and mechanisms of enzymes such as peroxidases, laccases, and
tyrosinases is known in the art. See, e.g., Akkara et al., 1994,
Biomimetics 2:331-339; Saunders et al., 1963, Peroxidase,
Butterworth, Washington, D.C.; Akkara et al., 1991, J. Polymer.
Sci. 29:1561-1574; Crestini et al., 2000, Bioorg. Med. Chem.
8:433-438; Guerra et al., 2000, Enzyme Microb. Technol.
26:315-323.
[0051] In practicing the present invention, knowledge of the exact
structure of the polymerized product is not necessary. Without
wishing to be bound by theory, it is believed that operation of the
invention relates to the difference in atomic relaxivity between
the monomeric substrate and the polymerized product, and does not
depend on any particular structural arrangement of the subunit
residues in the polymer. It is predicted that the polymerized
product is a mixture of numerous, differently branching
polymers.
[0052] Although the structural arrangement of the substrate
residues in the polymerized product usually is not known, the range
in the number of residues per polymer molecule can be determined in
in vitro reactions, e.g., by size exclusion (gel filtration)
chromatography. Such in vitro tests utilizing a particular
substrate/enzyme combination can be employed to make useful
predictions concerning the size of the polymers that will be formed
in vivo. While the exact number of residues (or range of number of
residues) per polymer is not critical, preferably the product
mixture contains polymers whose length ranges up to 6, 7, 8, 10,
12, or 14 residues. In general, longer polymers are preferred. In
preferred embodiments of the invention, the monomeric substrate is
chosen so that: (1) neither the monomer nor the resulting polymers
display significant toxicity in the amounts used for imaging, and
(2) both the monomer and the resulting polymer are excreted or
biologically degraded within hours to days after the monomer is
administered to a patient.
[0053] Because its function is simply to connect the chelating
moiety to the polymerizing moiety, there are no strict structural
requirements for the linker moiety. Once incorporated in the
monomeric substrate, the linker moiety need not participate in any
chemical reaction or any particular binding interaction. Thus, the
linker moiety can be chosen or designed primarily on factors such
as convenience of synthesis, lack of steric hindrance, and
biodegradation properties. A linker moiety containing one or more,
e.g., 2-6, L-amino acids is preferred, because their carboxyl
groups and amino groups are convenient for employment in synthesis
of the monomeric substrate, the peptide bonds are biodegradable,
and the products of polypeptide degradation are non-toxic. Amino
acids such as glycine and alanine are preferred amino acids,
because they do not have bulky or reactive side chains.
[0054] Although the invention is described here in terms of three
distinct structural moieties in the monomeric substrate, those of
skill in the art will recognize that there may not be a clearly
defined dividing line between the chelating moiety and the linker
moiety, and/or between the linker moiety and the polymerizing
moiety. For example, in the monomeric substrate shown in FIG. 1,
there are two methylene groups between the glycine residue in the
linker moiety and the phenol ring in the polymerizing moiety.
Whether those methylene groups are regarded as part of the linker
moiety or part of the polymerizing moiety is essentially arbitrary.
Moreover, those of skill in the art will recognize that the linker
moiety does not necessarily represent a separate synthetic reagent.
For example, in the monomeric substrate shown in FIG. 1, one
glycine residue of the linker moiety derives from a portion of the
glycylmethyl-DOTA tri-tBu ester reagent, and the other glycine
residue derives from the tyramine or dopamine.
[0055] In the practice of this invention, in general, chelating
moieties are interchangeable, phenolic moieties are
interchangeable, and linkers are interchangeable. Thus, numerous
different combinations of a chelating moiety, a phenolic moiety,
and a linker are within the scope of the invention.
[0056] Each of the three structural moieties can be obtained
commercially or synthesized according to conventional, organic
chemical synthesis methods. Suitable covalent linkage of the three
moieties can be carried out by one of skill in the art, employing
conventional methods, without undue experimentation.
[0057] The marker enzyme can be any enzyme capable of catalyzing
polymerization of a monomeric substrate containing a chelated
(super)paramagnetic metal or metal oxide. This means that the
marker enzyme is chosen for compatibility with a given monomeric
substrate, or that the monomeric substrate is designed for
compatibility with a given type of marker enzyme. For example, the
marker enzyme can be a template-independent RNA or DNA polymerase,
and the monomeric substrate can be a polymerizable nucleotide
derivative. Alternatively, the marker enzyme can be an
oxidoreductase, and the monomeric substrate can be an electron
donor that undergoes polymerization upon oxidation by the
oxidoreductase. Useful oxidoreductases include peroxidases such as
hydrogen peroxide-oxidoreductase (E.C. 1.11.1.7), lactoperoxidase,
and horseradish peroxidase.
[0058] When a peroxidase is used, methods of the invention include
providing a suitable amount of hydrogen peroxide in the tissue to
be imaged. The hydrogen peroxide can be supplied directly.
Alternatively, it can be generated in situ, e.g., using glucose
oxidase. If the hydrogen peroxide is enzymatically generated in
situ, the generating enzyme can be administered directly (as a
pre-formed enzyme) or can be expressed in the tissue from a
suitable nucleic acid vector introduced into the tissue.
[0059] In principle, the marker enzyme can be an endogenous enzyme
that occurs naturally in the tissue to be imaged. Typically,
however, the marker enzyme is an exogenous enzyme linked to a
targeting moiety. The targeting moiety causes selective
accumulation of the marker enzyme in the tissue to be imaged. In
general, the targeting moiety binds selectively to a molecule
exposed in an extracellular matrix or on the surface of one or more
cell types found in the tissue to be imaged. An example of a useful
targeting moiety is an antibody directed against a cell surface
protein or carbohydrate. Alternatively, the targeting moiety can
be, for example, a cell adhesion molecule, a cytokine, a cell
surface receptor molecule, or a fragment thereof that recognizes
the intended binding partner. In some embodiments, the targeting
moiety and marker enzyme are covalently linked to form a single
molecule. For example, a peroxidase enzyme can be covalently
coupled to a primary targeting antibody, using a conventional
coupling reaction. In other embodiments, the marker enzyme is
coupled to a secondary targeting moiety, e.g., a secondary
antibody, which recognizes a primary targeting moiety, e.g., a
primary antibody. This approach represents an adaptation of
conventional "sandwich ELISA" techniques.
[0060] Enzyme-catalyzed reactions that result in polymeric products
are not limited to oxidation-reduction reactions. Many enzymes
(polymerases) catalyze formation of chemical bond between
individual monomers.
[0061] As demonstrated by the Examples below, we have observed and
characterized a paramagnetic relaxation phenomenon that can be
utilized for magnetic resonance imaging signal amplification
(MRAMP). The effect was observed using paramagnetic gadolinium held
in a chelating moiety covalently bound to phenols that serve as
electron donors during peroxidase-catalyzed hydrogen peroxide
reduction. Instead of hydrogen peroxide itself, a hydrogen-peroxide
system was also used including, for example, a mixture of glucose
oxidase and glucose that produces hydrogen peroxide as a result of
glucose oxidase-mediated oxidation of glucose. The monomers
underwent rapid condensation into paramagnetic oligomers containing
approximately eight residues of the monomeric substrate.
Condensation resulted in a 2.5-fold to 3-fold increase of atomic
relaxivity (R1/Gd). The observed relaxation effect could be
explained by the increase of rotational correlation time
.tau..sub.r of magnetic moieties comprising the product resulting
in higher gadolinium atomic relaxivity (r1 or r2). Condensation of
substrate monomers facilitated the detection of enzymatic activity
by magnetic resonance imaging both spatially (qualitatively) and
quantitatively. The feasibility of MRAMP in detecting nanomolar
amounts of peroxidase was demonstrated in enzyme-linked
immunoadsorbent assay format. MRAMP was further utilized in
detecting E-selectin expression on the surface of IL-1.beta.
treated endothelial cells.
[0062] We assumed that the oxidoreductase (e.g., peroxidase
(donor:hydrogen peroxide-oxidoreductase E.C. 1.11.1.7), or
lactoperoxidase) would catalyze reduction of peroxide using a
paramagnetic substrate (AH) as a donor of electrons (reaction 1).
We also predicted that oxidized donors would then polymerize
(oligomerize) into the larger paramagnetic polymers (reaction
2).
2AH+[EH.sub.2O.sub.2].fwdarw.2[*A]+2H.sub.2O+E (reaction 1)
n[*A].fwdarw.[A]n (reaction 2)
[0063] We demonstrated that oxidoreductases oxidize tyramine- and
dopamine-linked chelated gadolinium leading to formation of
polymers. We observed a resultant 2.7-fold to 3.5-fold increase of
atomic relaxivity and demonstrated that this relaxation phenomenon
can be utilized to visual marker enzyme activity using MRI.
EXAMPLES
[0064] The invention is further illustrated by the following
Examples. The Examples are provided for illustrative purposes only,
and are not to be construed as limiting the scope or content of the
invention in any way.
Example 1
[0065] Substrate Synthesis
[0066] Using glycylmethyl-DOTA, tri tBu ester, we linked a carboxyl
group of glycine to the amino group of tyramine or dopamine
(hydroxytyramine) by reacting equimolar amounts (0.25 mmol) in the
presence of 1.1-fold molar excess of dicyclohexylcarbodiimide (FIG.
1) in 2 ml dimethylformamide (DMF) for 24 hours. The reaction
mixture was filtered through glass fiber filter, dissolved in 100
ml chloroform and washed with water. The product was recovered by
vacuum evaporation and treated with 50% trifluoroacetic acid (TFA)
for 1 hour. Deprotected acid was washed with diethyl ether and
dried by vacuum evaporation. Crude tyraminyl- or
hydroxytyraminyl-glycylmethylDOTA was dissolved in a solution of
equimolar amount of Gd citrate (pH 3.5), heated at 75.degree. C.
for 1 hour under argon and purified by using a Vydac C-18 HPLC
column eluted by a gradient of acetonitrile in 0.1% TFA. The major
peak at 280 nm was collected and dried. Analysis of the free acid
by matrix-assisted laser desorption ionization/time-of-flight mass
spectrometry (MALDI-TOF MS) gave a mass (m/z) of 594 (593
calculated). Analysis of purified gadolinium salt gave m/z 748
corresponding to the formation of monogadolinium salt.
Example 2
[0067] Cell Culture
[0068] Human umbilical vein endothelial cells (Endothelial Biology,
Brigham and Women's Hospital, Boston, Mass.) were isolated using
conventional techniques (see, e.g., Saba et al., Series
Haematologica 6:456). Cells were plated on gelatin-covered plastic
and cultured in 10% fetal bovine serum (FBS) in endothelial basal
medium (EDM) (Clonetics) with endothelial growth supplements.
Treatment of cells with human recombinant IL-1.beta. (10 pg/ml) was
performed at 37.degree. C. for 4 hours. E-selectin expression on
the surface of IL-1.beta. treated cells has been proven by
fluorescent microscopy using monoclonal anti-human E selectin
antibody H18/7 (Vascular Research Dept. of Pathology, Brigham and
Women's Hospital, Boston, Mass.), followed by anti-mouse-rhodamine
conjugate (Pierce Chem. Co.).
Example 3
[0069] Peroxidase Catalysis and Imaging
[0070] Substrates I and II at concentrations of 10-50 .mu.M were
treated by peroxidase (0.1-100 nM) and an excess of hydrogen
peroxide (3.5 mM) in 10 mM phosphate-buffered saline (PBS) or 0.05
M sodium phosphate pH 6.8. In some experiments, a
peroxide-generating system (5 mM glucose, glucose oxidase and
lactoperoxidase) was used. In inhibition studies, 2 mM Gd-free
substrate, tyramine, dopamine, or methyldopamine were added as
competitive inhibitors. The reaction was monitored by
spectrophotometry at 400 nm and by NMR spectrometry (Minispec 120
Bruker). Magnetic resonance imaging was performed using a 1.5 T
Signa GE system and surface or knee coils. Inversion-recovery pulse
sequences (TE 11 ms/TR 1000 ms/TI 50-600 ms) were used for T1
measurements. Spin-echo sequence (TE 13 ms/TR 400 ms/2 NEX, Matrix
256.times.160) was optimal for magnetic resonance imaging of signal
amplification. Magnetic resonance signal intensity was measured
using region-of-interest approach and 16-bit TIFF images. Mean
pixel values were compared using a Student t-test.
Example 4
[0071] MRI of Peroxidase Conjugate-Mediated Catalysis
[0072] Fab.sub.2' fragment of H18/7 monoclonal anti-human E
selectin antibody was prepared using pepsin digestion, and then
purified. Labeling of Fab.sub.2'-fragment with digoxigenin (DIG)
hydroxysuccinimide ester (HSE) (Roche Molecular Diagnostics) was
carried out according to the vendor's instructions. One ng to 1000
ng of DIG-labeled antibody was serially diluted with 0.01 M sodium
carbonate (pH 9) in a 96-well plate (Nunc) and adsorbed at
37.degree. C. overnight. Wells were washed with PBS containing 0.1%
Tween 20 (PBST) blocked with BSA solution, and anti-DIG
antibody-peroxidase conjugate (Roche, diluted 1:1000) was incubated
in wells in PBS-B for 1hour. Washed wells were filled with 200
.mu.l of 0.4 mM substrate I or II and hydrogen peroxide (3.5 mM)
and incubated for 30 min before imaging. Cells (2 million/sample)
were treated sequentially with IL-1.beta., anti-E selectin
DIG-labeled Fab.sub.2'-fragment, and anti-DIG antibody-peroxidase.
Cell suspensions were prepared in PBS and substrates were used as
above. Cells were pelleted in Eppendorf tubes (0.5 ml), and then
imaged as described above. Control samples were prepared by using
no IL-1.beta. treatment or in the absence of the first antibody in
treated cells. Magnetic resonance signal intensity was quantified
as described above and compared to that of aqueous Gd
solutions.
Example 5
[0073] Enzyme-Mediated Oxidation and Relaxation Phenomena
[0074] Kinetics of oxidation of tyraminyl-DOTA(Gd), I, or
hydroxytyraminyl-DOTA(Gd), II, in the presence of the excess of
hydrogen peroxide was studied using spectrophotometry. The increase
in absorbance at 400 nm in the case of both gadolinium-labeled
substrates was rapid, indicating efficient oxidation of both
substrates, and gave similar pseudo-first order kinetic constants:
k.sub.1app=0.0125 s.sup.-1 (I) and 0.013 s.sup.-1 (II).
[0075] The measurements of relaxation time changes (T1 and T2)
performed in parallel to spectrophotometry by using H1 NMR
relaxometry at 20 MHz (0.47 T) and 60 MHz (1.5 T) showed a
concomitant rapid decrease in relaxation times after the addition
of the enzyme. By plotting relaxation data against the
concentration of gadolinium, a raise of 1/T1 and 1/T2 of 2 fold (at
0.47 T) and 2.7 fold (1.5 T) in the case of substrate I, and 3.5
(at 0.47 T) in the case of substrate II has been measured (Table
1). Incubation of substrates in the presence of peroxide only did
not result in any measurable change of gadolinium relaxivity. To
find out whether the increase in relaxivity is a result of
dissociation of gadolinium cation from a DOTA(Gd) complex, we
treated Tyr-DOTA(Gd) or Dopamine-DOTA(Gd) with Chelex-100 resin and
compared it to the control with no peroxidase added. No difference
in T1 relaxation time of substrate solutions before and after the
treatment was observed.
1TABLE 1 Relaxivity enhancement 0.47 T (20 MHz) 1.5 T (63 MHz) r1
[mM.sup.-1s.sup.-1] r2 [mM.sup.-1s.sup.-1] r1 [mM.sup.-1s.sup.-1]
r2 [mM.sup.-1s.sup.-1] Substrate control peroxidase control
peroxidase control peroxidase control peroxidase Tyraminyl- 3.30
7.10 3.60 8.00 4.5 10.1 ND ND DOTA(Gd) Hydroxytyraminyl- 3.75 11.50
4.10 12.46 5.2 14.1 ND ND DOTA(Gd)
[0076] To investigate whether the observed changes in atomic
relaxivity were associated with the production of high molecular
weight products, we incubated reaction mixture for different times
ranging from 10 minutes to 1 hour, and analyzed reaction products
using size-exclusion HPLC. We then compared elution profiles to
that of a control substrate in the absence of peroxidase (FIG. 2).
The comparison of elution profiles before and after
peroxidase-mediated catalysis clearly pointed to the formation of a
higher-molecular weight product with a hydrodynamic radius
corresponding to a 6-7 kDa molecule (median=6.8 kDa). The measured
mass suggests that the product was formed as a result of the
condensation of eight oxidized substrate monomers. This was
confirmed by MALDI-TOF analysis of reaction products.
[0077] To determine if the molecular mass of the condensation
product depended on the initial concentration of the substrate, we
varied the substrate concentration (10-60 .mu.M) but observed no
change in elution times of final condensation product. Finally, the
addition of equimolar amount of non-labeled substrates as well as
tyramine, dopamine, methyl-dopamine or tyrosine did not influence
gadolinium relaxivity observed initially.
Example 6
[0078] Magnetic Resonance Imaging
[0079] The first MRI experiment was designed to test the
feasibility of visualization of enzyme-mediated conversion of
paramagnetic substrates. An array of tubes containing different
dilutions of the substrates in the presence or in the absence of
peroxidase and the substrate was used in this experiment. The
enhancement of magnetic resonance signal in samples containing
peroxidase and peroxide was clearly visible after applying
spin-echo T1 weighted sequences. A median 1.6-fold enhancement of
magnetic resonance signal was measured in a gadolinium
concentration range of 0.05-0.4 mM after peroxidase treatment (FIG.
4). The signal intensity of reaction mixtures was brighter than
aqueous gadolinium solution standards due to a higher atomic
relaxivity.
[0080] To determine the sensitivity of the amplification method to
the presence of peroxidase we varied the concentration of the
enzyme in reaction mixtures containing 0.1-0.2 mM substrate I or II
(FIG. 4). In both cases, amounts above 1 ng (e.g., 10 ng of
peroxidase in the volume of 200 .mu.l) produced clearly visible
relaxation effects.
[0081] In the next series of experiments, we determined whether
MRAMP could be utilized to detect a model ligand in an ELISA-like
assay. Different amounts of the model protein (Fab2' fragment of
monoclonal antibody) covalently labeled with digoxigenin were
adsorbed on the surface of a 96-well plate and incubated in the
presence of anti-digoxigenin-peroxidase conjugate. We found that
the sensitivity of the MRAMP assay was optimized by using substrate
II (1 ng antibody fragment detected at threshold) (FIG. 5). The
sensitivity of standard ELISA assay was similar, also giving 1 ng
as the threshold amount.
[0082] The latter experiment suggested feasibility of further MRI
involving the detection of specific antigen expression on the
surface of cells. We utilized a model system involving the highly
specific expression of E selectin on the surface of human
endothelial cells (HUVEC) as a response to interleukin-1.beta.
treatment. First, we demonstrated that E-selectin was indeed
specifically expressed on the cell surface. The binding of
anti-E-selectin Fab'2 was highly specific and detectable only in
the case of IL-1.beta.-treated cells as demonstrated by microscopy
using fluorescent-labeled secondary antibody. The enzyme-mediated
magnetic resonance signal enhancement was detected only in
precipitates of cells that were treated with IL-1.beta. followed by
digoxigenin-labeled antibody and anti-dig-peroxidase. In controls,
non-treated cells or IL-1.beta. treated cells that were not
incubated with anti-E-selectin antibody no enhancement over the
background signal was seen. Specific magnetic resonance enhancement
induced by the enzyme bound to the cell surface was typically
2-fold and was equivalent to the signal intensity of a 50 .mu.M
gadolinium phantom.
Example 7
[0083] Detection of Tyrosinase by Incorporation of
Dopamine-DOTA(Gd) in Melanins
[0084] Murine melanoma cells (B16 amelanotic melanoma, B16-F10,
PC1, and PC1A) were plated in 10 cm dishes at 0.5 million
cells/plate in 10% FCS, Dubecco's modified eagle medium (DMEM). At
subconfluency (80%), medium was supplemented with 1 mM
dopamine-DOTA, Gd salt, in the presence of 25 .mu.M sodium
ascorbate. At various time points, cells were harvested by
trypsinization, washed by passing through a step of 40%
Hypaque-1077 in Hank's solution, sedimented in 0.2 ml tubes, and
changes in T1 and T2 relaxation times in cell precipitates were
measured using 1.5 T Signa clinical imaging MR system using a 3 in
surface coil.
Example 8
[0085] Synthesis of dUTP or UDP Substituted in 5-ring Position with
Allylamino-DOTA(Gd)
[0086] Equimolar amounts (25 .mu.mole) of GlyMeDOTA, tri-t-Bu ester
and N-hydroxysuccinimide were treated with 1.1-molar excess of
dicyclohexylcarbodiimide in 1 ml DMF for 4 hours under argon.
Precipitate of dicyclohexylurea was removed by filtration and DMF
removed in vacuum. 5-allylamino-2'-deoxyuridine-5'-triphosphate or
5-allylamino-2'-uridine-5- '-diphosphate (25 .mu.mol) was dissolved
in a mixture of dioxane:water (1:1) and Gly-MeDOTA, tri-tBu HSE
ester was added. The mixture was incubated for 18 hours and treated
with 70% TFA (by volume). After 3 hours at room temperature, the
mixture was dried, in vacuo, dissolved in water, and extracted with
chloroform. The aqueous phase was collected and mixed with 50
.mu.mol Gd citrate in water. The reaction mixture was kept at
60.degree. C. for two hours and purified using Nucleosil-4000 PE17
HPLC column eluted with a 0.02-1 M gradient of ammonium acetate in
water, pH 6. Fractions containing triphosphate (third major 260 nm
positive peak) or diphosphate (second peak) were collected and
lyophilized to constant weight.
Example 9
[0087] Nick-translation (NT) Labeling Using DNA Polymerase
I--Preparation of Paramagnetic DNA
[0088] Series of individual NT reactions were set in PCR tubes (0.2
ml) using the following reagents: 5 .mu.g pCMV-Luc double-stranded
plasmid DNA for labeling (concentration c>1 .mu.g/.mu.l)
DOTA(Gd)-dUTP 1 nmol/.mu.l; dNTPs (regular nucleotides): dATP,
dCTP, dGTP, 0.5 mM each, dTTP 0.1 mM NT reaction buffer 10.times.
(0.5 M Tris pH 8, 50 mM MgCl.sub.2, 0.5 mg/ml BSA) DTT 0.1 M DNase
(stock solution 3 mg/ml) diluted 1:2000 diluted in water.
DNA-polymerase 5 U/.mu.l (e.g., Boehringer Mannheim); EDTA (0.5 M,
pH 8.0) A 50 .mu.l reaction mixture was prepared on ice, using for
one NT reaction 5 .mu.l of DNA is used; 5 .mu.l NT (10.times.); 5
.mu.l DTT; 5 .mu.l dNTP; 2 .mu.l DOTA(Gd)-dUTP; 1 .mu.l DNAse I; 1
.mu.l DNA polymerase; water to 50 .mu.l. The mixture was incubated
0.25-3 hours at 15.degree. C., and then stopped with 2.5 .mu.l EDTA
(0.5 M, pH 8.0). A control mixture was prepared containing EDTA in
the buffer. T1 changes were monitored using a 1.5 T Signa MR
imaging system.
Example 10
[0089] Random-primer Paramagnetic Labeling of DNA Driven by Klenow
Fragment of DNA Polymerase I
[0090] Series of individual random-primer labeling reactions were
set in PCR tubes (0.2 ml) using the following reagents: 10 .mu.l
DNA template (100 ng-1 .mu.g); 10 .mu.l random primer in 5.times.
reaction buffer deionized water was added to increase the volume to
40 .mu.l . The contents of these tubes were mixed and spun down.
Tubes were heated in a boiling water bath for 5-10 minutes and
cooled on ice. The following components were then added: 3 .mu.l
Non-radioactive Labeling Mix (0.5 mM); 2 .mu.l 1 mM DOTA(Gd)-dUTP;
and 1 .mu.l Klenow fragment, exo-(5U/reaction).
[0091] Tubes were incubated for 0-20 hours at 37.degree. C.
Reactions were terminated using 1 .mu.l 0.5M EDTA, pH 8.0.
Time-dependent T1 changes were monitored using a 1.5 T Signa MR
imaging system.
Example 11
[0092] PCR Labeling Driven by Thermostable DNA
Polymerase--Synthesis of Paramagnetic DNA Fragments
[0093] A mixture was prepared using the following reagents: 1-2 ul
DNA template (0.1-100 ng pCMV-GFP plasmid DNA); 2.5 .mu.l of
10.times. PCR buffer (Roche); 1 .mu.l primer(s) (20-50 .mu.M
forward and reversed GFP amplification primes from the stock); 0.25
.mu.l d(ACG)TP (33.3 mM each); 0.7 .mu.l 5 mM dTTP; 0.3-1.6 .mu.l 1
mM DOTA(Gd)-dUTP; 0.2-0.4 .mu.l Taq polymerase (5U/.mu.l stock);
and water to 25 .mu.l. PCR was run using the following scheme: 1-2
cycles: 45 sec/94.degree. C.-45 sec/15.degree. C.-12 min/37.degree.
C.; 5 cycles: 40 sec/94.degree. C.-45 sec/37.degree. C.-4
min/66.degree. C.; 24 cycles 40 sec/94.degree. C.-45 sec/54.degree.
C.-4 min/66.degree. C. Control reaction mixture was not subjected
to PCR. The reaction was stopped at different phases of PCR and T1
changes were monitored using a 1.5 T Signa MR imaging system at
room temperature.
Example 12
[0094] Reversed Transcription (Reaction is Driven by RNA-dependent
DNA Polymerase). Synthesis of Paramagnetic DNA Using mRNA as a
Template
[0095] Total RNA was extracted from 9L-GFP cells using the RNA
STAT-60 according standard protocol. The extracted RNA was
re-precipitated in the presence of 0.2 M sodium chloride and 2
volumes of absolute ethanol before finally being dissolved in 20
.mu.l of RNase-free sterile water. The following reagents were
combined on ice: 8.0 .mu.l 5.times. First Strand Buffer
(Superscript II, Life Technologies); 1.5 .mu.l anchored mRNA primer
(5'-T20 100 pmol/.mu.l); 3.0 .mu.l 20 mM dNTP-dTTP (6.7 mM each of
dATP, dCTP, dGTP); 3.0 .mu.l 2 mM d TTP; 3.0 .mu.l 2 mM
DOTA(Gd)-dUTP; 4.0 .mu.l 0.1 M DTT; 10 .mu.g total RNA and water to
40 .mu.l. Labeling reaction was incubated at 65.degree. C. for 5
minutes, and then at 42.degree. C. for 5 minutes. 200 U of reverse
transcriptase (Superscript II, Life Technologies) was added and the
mixture was incubated at 42.degree. C. for 2 hours. T1 changes were
monitored using a 1.5 T Signa MR imaging system at room
temperature.
Example 13
[0096] Terminal Nucleotide transferase (TdT)-mediated Synthesis of
Paramagnetic DNA
[0097] To a 50 .mu.l reaction mixture containing 10.mu. of 5.times.
reaction buffer was added: (1.times. reaction buffer: 20 mM Tris
Acetate pH 7.9; 50 mM potassium acetate, 1 mM CoCl.sub.2, 0.1 mM
DTT, 0.01% Triton X-100, 10 .mu.M oligo(dT)10) added dTTP (or dATP)
to 0.2 mM and 3 .mu.l 2 mM DOTA(Gd)-dUTP. Forty units of terminal
deoxynucleotidyl transferase were added, and the reaction mixture
was incubated for 30 minutes at 37.degree. C. The reaction was
stopped by heating to 70.degree. C. with subsequent cooling to
40.degree. C., and relaxivity was determined. Control reaction
included heat-treated enzyme.
Example 14
[0098] Synthesis of Paramagnetic Polyribonucleotides Using
Polymerization Catalyzed by Polynucleotide Phosphorylase
(Polyribonucleotide Nucleotidyltransferase)
[0099] 0.5 mM 5-(DOTA(Gd)allylamido-substituted
uridine-5'-diphosphate in 50 mM Tris acetate; 50 mM NaCl; 6.7 mM
UDP, 6.7 mM MgCl.sub.2; and 0.1 mM MnCl.sub.2 at pH 8.5 were
reacted in the presence of polynucleotide phosphorylase from E.
coli (40 PK units) at 37.degree. C. for 30 min. The reaction was
continuously monitored by measuring T1 changes every 5 minutes.
Other Embodiments
[0100] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. For example, two or more chelating moieties
can be incorporated into a single monomeric substrate molecule.
Accordingly, other embodiments are within the scope of the
following claims.
* * * * *