Imaging of enzymatic activity

Details Imaging of enzymatic activity Imaging of enzymatic activity

2001-10-19

2004-05-18

Weber, Jon P. (Department: 1651)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving oxidoreductase

C435S015000, C435S018000, C435S019000, C435S026000, C435S027000, C435S028000

Reexamination Certificate

active

06737247

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to biochemistry and magnetic resonance imaging.
BACKGROUND OF THE INVENTION
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 &mgr;m
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.
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 &mgr;mol Gd per gram of tissue. Therefore, a way of amplifying an MRI signal from a targeted, magnetic label is needed.
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
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.
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.
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.
Examples of chelating moieties that can be incorporated into a monomeric substrate for use in the invention include the following: 1,4,7,10-tetraazacyclododecane-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-tetraazocyclodecane, 1,4,7-triazacyclonane-N,N′,N″-triacetic acid; and 1,4,8,11-tetraazacyclotetra-decane-N,N′,N″,N′″-tetraacetic acid; diethylenetriamine-pentaacetic acid (DTPA); triethylenetetraamine-hexaacetic 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.
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
3+
, Gd
3+
, Dy
3+
, Eu
3+
, Mn
2+
).
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
3
-C
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:
where R
1
is H, OH, or OCH
3
.
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.
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).
In certain embodiments, for example, the polymerizing moiety can be a phenolic moiety such as the following:
where R
1
, R
2
, R
3
, R
4
and R
5
, independently, can be H; R
6
, wherein R
6
is C
1
-C
6
unsubstituted alkyl; NHC(O)R
6
; OH; or NR
7
R
8
, wherein R
7
and R
8
are H or R
6
; provided that at least one of R
1
, R
2
, R
3
, R
4
and R
5
is OH.
In some embodiments of the invention, R
1
, R
2
, R
3
, R
4
or R
5
is at an ortho position relative to the OH substituent, and is either OH or OCH
3
. In other embodiments, R
1
, R
2
, R
3
, R
4
or R
5
is at a meta position relative to the OH substituent, and is either NHC(O)R
6
or NR
7
R
8
.
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 lactoperoxi

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