U.S. patent application number 10/799550 was filed with the patent office on 2005-01-06 for magnetic resonance imaging agents for the detection of physiological agents.
Invention is credited to Fraser, Scott, Meade, Thomas J..
Application Number | 20050002866 10/799550 |
Document ID | / |
Family ID | 27556895 |
Filed Date | 2005-01-06 |
United States Patent
Application |
20050002866 |
Kind Code |
A1 |
Meade, Thomas J. ; et
al. |
January 6, 2005 |
Magnetic resonance imaging agents for the detection of
physiological agents
Abstract
The invention relates to novel magnetic resonance imaging
contrast agents and methods of detecting physiological signals or
substances.
Inventors: |
Meade, Thomas J.; (Altadena,
CA) ; Fraser, Scott; (La Canada, CA) |
Correspondence
Address: |
DORSEY & WHITNEY LLP
INTELLECTUAL PROPERTY DEPARTMENT
4 EMBARCADERO CENTER
SUITE 3400
SAN FRANCISCO
CA
94111
US
|
Family ID: |
27556895 |
Appl. No.: |
10/799550 |
Filed: |
March 11, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10799550 |
Mar 11, 2004 |
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09866512 |
May 24, 2001 |
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6770261 |
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09866512 |
May 24, 2001 |
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09405046 |
Sep 27, 1999 |
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09405046 |
Sep 27, 1999 |
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09134072 |
Aug 13, 1998 |
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5980862 |
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09134072 |
Aug 13, 1998 |
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08971855 |
Nov 17, 1997 |
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09134072 |
Aug 13, 1998 |
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08486968 |
Jun 7, 1995 |
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5707605 |
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08486968 |
Jun 7, 1995 |
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08460511 |
Jun 2, 1995 |
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60063328 |
Oct 27, 1997 |
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Current U.S.
Class: |
424/9.322 ;
530/400 |
Current CPC
Class: |
A61K 49/122 20130101;
A61K 49/103 20130101; A61K 49/128 20130101; A61K 49/10 20130101;
A61K 49/146 20130101; A61K 49/14 20130101; A61K 49/085 20130101;
A61K 49/106 20130101 |
Class at
Publication: |
424/009.322 ;
530/400 |
International
Class: |
A61K 049/00 |
Claims
We claim:
1. A composition comprising at least one MRI agent having the
formula: 21wherein is a polymer; M is a chelator comprising a
paramagnetic metal ion, or a salt thereof; n is an integer of at
least one; R.sub.26 a first linker; R.sub.27 is an optional second
linker; and wherein upon interaction of said peptide blocking
moiety with a target protease, the T.sub.1 of said MRI agent is
decreased.
2. A composition according to claim 1, having the formula: 22
3. A composition according to claim 1 wherein R.sub.26 is a
cleavable linker.
4. A composition according to claim 3 is a wherein said cleavable
linker is a protease labile linker.
5. A composition according to claim 3 wherein said cleavable linker
is an esterase labile linkage.
6. A composition according to claim 1 or 2 wherein said polymer
further comprises a targeting moiety.
7. A composition according to claim 1 wherein said chelator is
DOTA.
8. A composition according to claim 1 wherein said target protease
is selected from the group consisting of serine proteases, cysteine
proteases, aspartyl proteases and matrix metalloproteases
(MMPs).
9. A composition according to claim 8 wherein said cysteine
proteases are selected from the group consisting of cathepsins,
calpains, caspases, and interleukin-converting enzyme (ICE).
10. A composition according to claim 8 wherein said serine
proteases are selected from the group consisting of trypsin,
chymotrypsin, and tissue plasminogen activator and (tPA).
11. A composition according to claim 8 wherein said
metalloproteases are selected from the group consisting of MMP-1,
MMP-2, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-9, and
MMP-10.
12. A composition according to claim 1 wherein said paramagnetic
metal ion is selected from the group consisting of gadolinium
(Gd3+), iron III (Fe+3), manganese II (Mn+2), yttrium III (Y+3),
dysprosium (Dy+3), and chromium (Cr(III)).
13. A method comprising administering a composition according to
claim 1 to a tissue, cell or patient, under conditions whereby said
peptide blocking moiety interacts with a target protease in said
tissue, cell or patient such that the T.sub.1 of said MRI agent is
decreased, and, acquiring a magnetic resonance image of said cell,
tissue or patient.
14. A method according to claim 13 wherein R.sub.26 is a cleavable
linker.
15. A method according to claim 14 wherein said cleavable linker is
a protease labile linker.
16. A method according to claim 14 wherein said cleavable linker is
an esterase labile linkage.
17. A method according to claim 14 wherein said polymer further
comprises a targeting moiety.
18. A method according to claim 14, wherein said chelator is
DOTA.
19. A method according to claim 13, wherein said target protease is
selected from the group consisting of serine proteases, cysteine
proteases, aspartyl proteases and metalloproteases (MMPs).
20. A method according to claim 19, wherein said cysteine proteases
are selected from the group consisting of cathepsins, calpains,
caspases, and interleukin-converting enzyme (ICE).
21. A method according to claim 19, wherein said serine proteases
are selected from the group consisting of trypsin, chymotrypsin,
and tissue plasminogen activator and (tPA).
22. A method according to claim 19, wherein said MMPs are selected
from the group consisting of metalloprotease-1 (MMP), MMP-2, MMP-3,
MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-9, and MMP-10.
Description
[0001] This application is a continuation of Ser. No. 09/866,512,
filed May 24, 2001; which is a continuation-in-part application of
Ser. No. 09/405,046, filed Sep. 27, 1999; which is a
continuation-in-part of application of Ser. No. 09/134,072, filed
Aug. 13, 1998, now U.S. Pat. No. 5,980,862; which is a
continuation-in-part of Ser. No. 08/971,855, filed Nov. 17, 1997,
now abandoned; which claims the benefit of the filing date under 35
U.S.C. .sctn. 119(e) of provisional Ser. No. 60/063,328, filed Oct.
27, 1997 and PCT/US96/08548, filed Jun. 3, 1996; and is a
continuation-in-part of Ser. No. 08/486,968, filed Jun. 7, 1995,
now U.S. Pat. No. 5,707,605; which is a continuation-in-part of
Ser. No. 08/460,511, filed Jun. 2, 1995, now abandoned.
FIELD OF THE INVENTION
[0002] The invention relates to novel magnetic resonance imaging
contrast agents and methods of detecting physiological signals or
substances.
BACKGROUND OF THE INVENTION
[0003] Magnetic resonance imaging (MRI) is a diagnostic and
research procedure that uses high magnetic fields and
radio-frequency signals to produce images. The most abundant
molecular species in biological tissues is water. It is the quantum
mechanical "spin" of the water proton nuclei that ultimately gives
rise to the signal in all imaging experiments. In MRI the sample to
be imaged is placed in a strong static magnetic field (1-12 Tesla)
and the spins are excited with a pulse of radio frequency (RF)
radiation to produce a net magnetization in the sample. Various
magnetic field gradients and other RF pulses then act on the spins
to code spatial information into the recorded signals. MRI is able
to generate structural information in three dimensions in
relatively short time spans.
[0004] The Image.
[0005] MR images are typically displayed on a gray scale with black
the lowest and white the highest measured intensity (I). This
measured intensity I=C*M, where C is the concentration of spins (in
this case, water concentration) and M is a measure of the
magnetization present at time of the measurement. Although
variations in water concentration (C) can give rise to contrast in
MR images, it is the strong dependence of the rate of change of M
on local environment that is the source of image intensity
variation in MRI. Two characteristic relaxation times, T.sub.1
& T.sub.2, govern the rate at which the magnetization can be
accurately measured. T.sub.1 is the exponential time constant for
the spins to decay back to equilibrium after being perturbed by the
RF pulse. In order to increase the signal-to-noise ratio (SNR) a
typical MR imaging scan (RF & gradient pulse sequence and data
acquisition) is repeated at a constant rate for a predetermined
number of times and the data averaged. The signal amplitude
recorded for any given scan is proportional to the number of spins
that have decayed back to equilibrium since the previous scan.
Thus, regions with rapidly decaying spins (i.e. short T.sub.1
values) will recover all of their signal amplitude between
successive scans.
[0006] The measured intensities in the final image will accurately
reflect the spin density (i.e. water content). Regions with long
T.sub.1 values compared to the time between scans will
progressively lose signal until a steady state condition is reached
and will appear as darker regions in the final image. Changes in
T.sub.2 (spin-spin relaxation time) result in changes in the signal
linewidth (shorter T.sub.2 values) yielding larger linewidths. In
extreme situations the linewidth can be so large that the signal is
indistinguishable from background noise. In clinical imaging, water
relaxation characteristics vary from tissue to tissue, providing
the contrast which allows the discrimination of tissue types.
Moreover, the MRI experiment can be setup so that regions of the
sample with short T.sub.1 values and/or long T.sub.2 values are
preferentially enhanced so called T.sub.1 -weighted and
T.sub.2-weighted imaging protocol.
[0007] MRI Contrast Agents.
[0008] There is a rapidly growing body of literature demonstrating
the clinical effectiveness of paramagnetic contrast agents
(currently 8 are in clinical trials or in use). The capacity to
differentiate regions/tissues that may be magnetically similar but
histologically distinct is a major impetus for the preparation of
these agents [1, 2]. In the design of MRI agents, strict attention
must be given to a variety of properties that will ultimately
effect the physiological outcome apart from the ability to provide
contrast enhancement [3]. Two fundamental properties that must be
considered are biocompatability and proton relaxation enhancement.
Biocompatability is influenced by several factors including
toxicity, stability (thermodynamic and kinetic), pharmacokinetics
and biodistribution. Proton relaxation enhancement (or relaxivity)
is chiefly governed by the choice of metal and rotational
correlation times.
[0009] The first feature to be considered during the design stage
is the selection of the metal atom, which will dominate the
measured relaxivity of the complex. Paramagnetic metal ions, as a
result of their unpaired electrons, act as potent relaxation
enhancement agents. They decrease the T.sub.1 and T.sub.2
relaxation times of nearby (r.sup.6 dependence) spins. Some
paramagnetic ions decrease the T.sub.1 without causing substantial
linebroadening (e.g. gadolinium (III), (Gd.sup.3+)), while others
induce drastic linebroadening (e.g. superparamagnetic iron oxide).
The mechanism of T.sub.1 relaxation is generally a through space
dipole-dipole interaction between the unpaired electrons of the
paramagnet (the metal atom with an unpaired electron) and bulk
water molecules (water molecules that are not "bound" to the metal
atom) that are in fast exchange with water molecules in the metal's
inner coordination sphere (are bound to the metal atom).
[0010] For example, regions associated with a Gd.sup.3+ ion
(near-by water molecules) appear bright in an MR image where the
normal aqueous solution appears as dark background if the time
between successive scans in the experiment is short (i.e. T.sub.1
weighted image). Localized T.sub.2 shortening caused by
superparamagnetic particles is believed to be due to the local
magnetic field inhomogeneities associated with the large magnetic
moments of these particles. Regions associated with a
superparamagnetic iron oxide particle appear dark in an MR image
where the normal aqueous solution appears as high intensity
background if the echo time (TE) in the spin-echo pulse sequence
experiment is long (i.e. T.sub.2-weighted image). The lanthanide
atom Gd.sup.3+ is by the far the most frequently chosen metal atom
for MRI contrast agents because it has a very high magnetic moment
(u.sup.2=63BM.sup.2), and a symmetric electronic ground state,
(S.sup.8). Transition metals such as high spin Mn(II) and Fe(III)
are also candidates due to their high magnetic moments.
[0011] Once the appropriate metal has been selected, a suitable
ligand or chelate must be found to render the complex nontoxic. The
term chelator is derived from the Greek word chele which means a
"crabs claw", an appropriate description for a material that uses
its many "arms" to grab and hold on to a metal atom (see DTPA
below). Several factors influence the stability of chelate
complexes include enthalpy and entropy effects (e.g. number, charge
and basicity of coordinating groups, ligand field and
conformational effects). Various molecular design features of the
ligand can be directly correlated with physiological results. For
example, the presence of a single methyl group on a given ligand
structure can have a pronounced effect on clearance rate. While the
addition of a bromine group can force a given complex from a purely
extracellular role to an effective agent that collects in
hepatocytes.
[0012] Diethylenetriaminepentaacetic (DTPA) chelates and thus acts
to detoxify lanthanide ions. The stability constant (K) for
Gd(DTPA).sup.2- is very high (logK=22.4) and is more commonly known
as the formation constant (the higher the logK, the more stable the
complex). This thermodynamic parameter indicates the fraction of
Gd.sup.3+ ions that are in the unbound state will be quite small
and should not be confused with the rate (kinetic stability) at
which the loss of metal occurs (k.sub.f/k.sub.d). The water soluble
Gd(DTPA).sup.2- chelate is stable, nontoxic, and one of the most
widely used contrast enhancement agents in experimental and
clinical imaging research. It was approved for clinical use in
adult patients in June of 1988. It is an extracellular agent that
accumulates in tissue by perfusion dominated processes.
[0013] To date, a number of chelators have been used, including
diethylenetriaminepentaacetic (DTPA),
1,4,7,10-tetraazacyclododecane'-N,N- 'N",N'"-tetracetic acid
(DOTA), and derivatives thereof. See U.S. Pat. Nos. 5,155,215,
5,087,440, 5,219,553, 5,188,816, 4,885,363, 5,358,704, 5,262,532,
and Meyer et al., Invest. Radiol. 25: S53 (1990).
[0014] Image enhancement improvements using Gd(DTPA) are well
documented in a number of applications (Runge et al., Magn, Reson.
Imag. 3:85 (1991); Russell et al., AJR 152:813 (1989); Meyer et
al., Invest. Radiol. 25:S53 (1990)) including visualizing
blood-brain barrier disruptions caused by space occupying lesions
and detection of abnormal vascularity. It has recently been applied
to the functional mapping of the human visual cortex by defining
regional cerebral hemodynamics (Belliveau et al., (1991)
254:719).
[0015] Another chelator used in Gd contrast agents is the
macrocyclic ligand
1,4,7,10-tetraazacyclododecane-N,N',N"N'"-tetracetic acid (DOTA).
The Gd-DOTA complex has been thoroughly studied in laboratory tests
involving animals and humans. The complex is conformationally
rigid, has an extremely high formation constant (logK=28.5), and at
physiological pH possess very slow dissociation kinetics. Recently,
the GdDOTA complex was approved as an MRI contrast agent for use in
adults and infants in France and has been administered to over 4500
patients.
[0016] As noted above, these MRI contrast agents have a variety of
uses. However, there are no MRI contrast agents that report on
physiologic or metabolic processes within a biological or other
type of sample. Accordingly, it is an object of the present
invention to provide MRI contrast or enhancement agents which allow
the visualization and detection of physiological agents within an
animal, tissue or cells.
SUMMARY OF THE INVENTION
[0017] In accordance with the above objects, the invention provides
MRI agents comprising a paramagnetic metal ion bound to a complex.
The complex comprises a chelator and a blocking moiety in at least
a first coordination sites of said metal ion. The blocking moiety
is covalently attached to the chelator, and capable of interacting
with a target substance such that the exchange of water in at least
said first coordination site in the metal ion complex is
altered.
[0018] In one aspect, the invention provides MRI agents comprising
a) a Gd(III) ion bound to a chelator such that the Gd(III) ion has
coordination atoms in at least 5 coordination sites, and b) a
blocking moiety covalently attached to the chelator which hinders
the rapid exchange of water in the remaining coordination sites.
The blocking moiety is capable of interacting with a target
substance such that the exchange of water in the remaining
coordination sites is increased.
[0019] In an additional aspect, the invention provides MRI agents
having the formula: 1
[0020] wherein M is a paramagnetic metal ion selected from the
group consisting of Gd(III), Fe(III), Mn(II), Yt(III), Cr(III) and
Dy(III); A, B, C and D are either single bonds or double bonds;
X.sub.1, X.sub.2, X.sub.3 and X.sub.4 are --OH, --COO--,
--CH.sub.2OH --CH.sub.2COO--, or a blocking moiety;
R.sub.1-R.sub.12 are hydrogen, alkyl, aryl, phosphorus moiety, or a
blocking moiety; wherein at least one of X.sub.1-X.sub.4 and
R.sub.1-R.sub.12 is a blocking moiety.
[0021] In a further aspect, the invention provides MRI contrast
agents comprising a first paramagnetic metal ion bound to a first
complex, and at least a second paramagnetic metal ion bound to a
second complex. The first and second complexes each comprise a
chelator with a covalently attached blocking moiety. The complexes
can be attached via a linker, for example a polymer.
[0022] In an additional aspect, the MRI agent comprise a) a first
chelator comprising a first paramagnetic metal ion; b) a second
chelator comprising a second paramagnetic metal ion; and c) a
blocking moiety covalently attached to at least one of the first or
second chelators. The blocking moiety provides at least a first
coordination atom of each of the first and second metal ions, or
serves as a coordination site barrier. As above, the blocking
moiety is capable of interacting with a target substance such that
the exchange of water in at least a first coordination site of at
least one of the metal ions is increased.
[0023] The invention also provides methods of magnetic resonance
imaging of a cell, tissue, experimental animal or patient
comprising administering an MRI agent of the invention to a cell,
tissue, experimental animal or patient and rendering a magnetic
resonance image of said cell, tissue, experimental animal or
patient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 depicts a representative complex of the invention,
where the blocking moiety is tethered at one end only. The blocking
moiety comprises a enzyme substrate and a coordination site
barrier. The R group is the coordination site barrier.
[0025] FIG. 2 depicts a representative complex of the invention,
wherein the blocking moiety is tethered at two ends. The R group is
the coordination site barrier.
[0026] FIG. 3 depicts a representative synthesis of
Do3a-hydroxyethyl-.beta.-galactose, which has a single galactose
moiety attached to the DOTA ring.
[0027] FIG. 4 depicts a representative synthesis of a
.beta.-galactose-DOTA derivative that has two galactose moieties
attached to the DOTA ring.
[0028] FIG. 5 depicts the synthesis of a BAPTA-DOTA derivative.
[0029] FIG. 6 depicts the syntheis of a FURA-DOTA derivative.
[0030] FIG. 7 depicts a synthetic scheme for the synthesis of
BAPTA-DTPA.
[0031] FIG. 8 depicts an alternative synthesis of a BAPTA-DTPA
derivative.
[0032] FIG. 9 depicts the change in T.sub.1 observed upon the
.beta.-galactosidase catalyzed cleavage of the galactopyranose
residue (n=3). The first Column of each pair (1,3,5) represents the
T.sub.1 of the galactose-DOTA complex and .beta.-galactosidase
mixture immediately after addition. The second column represents
the T.sub.1 of the solution after a period of time in the presence
of .beta.-galactosidase. Each column is reported as a ratio to a
control containing only the complex. Column 1 and 2: 2.0 mM Gd
complex plus 1.7 uM .beta.-galactosidase phosphate buffer (25 mM)
pH 7.3. Column 3 and 4: 2.0 mM Gd plus 5.1 uM .beta.-galactosidase
phosphate buffer (25 mM) pH 7.3. Column 5 and 6: 2 mM Gd complex
plus 5.1 uM heat inactivated .beta.-galactosidase (10 minutes at 80
degrees) phosphate buffer (25 mM) pH 7.3. The complexes were
incubated with the enzyme for 7 days and HPLC traces indicated
greater than 95% cleavage. A minimal concentration of enzyme was
used in these experiments to reduce potential effects of any
contrast agent-enzyme interactions. T.sub.1 were carried out using
a Bruker AMX 500 spectrometer at 26 degrees using a standard
inversion-recovery sequence. The solution was placed in a 40 ul
round bottomed NMR tube insert (Wilmad glass) and inserted into a
tube containing d.sub.3-chloroform. A two dimensional data file was
collected containing 16 different inversion delays with 8 scans
each. The raw nmr data was processed (Felix, BIOSYM/Molecular
Simulations, San Diego, Calif.) and the peak heights were fitted to
an exponential rise to a max to obtain T.sub.1. The R value was
always greater than 0.999.
[0033] FIGS. 10A, 10B, 10C, 10D, 10E, 10F, and 10G depict several
of the possible conformations of the dimer embodiments. Boxes
represent chelators, with M being the paramagnetic metal ions.
FIGS. 10A and 10B represent two possible duplex conformations. In
FIG. 10A, R.sub.27 can be a linker, such as described herein as
R.sub.26, a cleavable moiety such as an enzyme substrate such as a
peptide, or a blocking moiety that will preferentially interact
with the target molecule. R.sub.28, which may or may not be present
depending on R.sub.27, is a coordination site barrier similar to
R.sub.23 or a blocking moiety. FIG. 10B has R.sub.28 blocking
moieties or coordination site barriers attached via an R.sub.27
group to two chelators. FIG. 10C is similar to FIG. 10A, but at
least one of the R.sub.27 groups must be a cleavable moiety. FIG.
10D depicts the case where two blocking moieties or coordination
site barriers are present; if R.sub.27 is a blocking moiety,
R.sub.28 need not be present. FIG. 10E is similar to 10B but the
chelators need not be covalently attached. FIGS. 10F (single MRI
agents) and and 10G (duplex agents) are multimers of MRI contrast
agents, wherein n can be from 1 to 1000, with from 1 to about 20
being preferred, and from about 1 to 10 being especially preferred.
FIGS. 10H and 10I depict polymer 10 as defined herein being
attached to either single MRI agents (10H) or duplex MRI agents
(10I).
[0034] FIGS. 11A, 11B and 11C depicts precursors for making MRI
duplexes for Ca.sup.+2 detection using BAPTA derivatives as the
blocking moiety, each with a different R.sub.26 linkers. FIG. 11A
depicts AEPA, which when Gd is present exhibits a q of 0.7 (q is
the number of water molecules associated with the complex, which is
an indicator of the ability of the blocking moiety to block the
exchange of water; the lower the q the better). The q values were
determined using fluorescence lifetime measurements using Terbium
(Tb.sup.3+) as the metal ion in D.sub.2O and H.sub.2O (data not
shown). FIG. 11B depicts APPA, which has a q of 0.3. FIG. 11C
depicts ABPA, which has a q of 0.7.
[0035] FIG. 12 depicts the synthesis of AEPA. As will be
appreciated by those in the art, the full duplex can be made by
functionalizing the other ortho position on the nitrobenzyl
ring.
[0036] FIG. 13 depicts the synthesis of APPA and ABPA. As will be
appreciated by those in the art, the full duplexes can be made by
functionalizing the other ortho position on the nitrobenzyl
ring.
[0037] FIG. 14 depicts the synthesis of Gd3+-BAPTA-DO3A.sub.2
("CalGad").
[0038] FIG. 15 schematically depicts the structural changes in
CalGad that occur upon binding of calcium.
[0039] FIG. 16 depicts the relaxivity of the CalGad complex as a
function of calcium ion concentration.
[0040] FIG. 17 depicts MRI detection of .beta.-galactosidase mRNA
expression in living Xenopus laevis embryos. Background corrected
MR images of two embryos injected with EGadMe at the 2-cell stage.
The embryo on the right was also injected with .beta.-gal mRNA,
resulting in the higher intensity regions. The observed contrast
enhancement makes it possible to recognize the eye, head, brachial
arches, and somites of both embryos, but the signal strength is
45-65% greater in the embryo on the right containing .beta.-gal
(contrast-to-noise ratio ranges from 3.5 to 6). The cement gland
has intrinsically short T.sub.1, thus is visible as a bright
structure on both embryos. d: dorsal; v: ventral; r: rostral; e:
eye; c: cement gland; s: somite; b: brachial arches. Scale Bar=1
mm.
[0041] FIG. 18 depicts MRI detection of regions positive for
.beta.-galactosidase within a single living Xenopus laevis embryo.
Embryos were injected at the 2-cell stage. Both cells received
EGadMe; one cell also received mRNA for .beta.-galactosidase. The 2
cells at this stage represent the future left and right sides of
the animal. The head of embryo is to the left in all three panels.
FIG. 18A depicts GFP fluorescence image of a living embryo. In this
dorsal view, fluorescence is clearly localized to the right side of
the embryo (top), although absent from the head, indicating the
position of the descendants of the mRNA injected blastomere. FIG.
18B depicts MR image of the same living embryo depicted in A, with
signal from water made transparent. The high intensity region on
the right side indicates cleavage of EGadMe. Blocks of somites are
visible on the right side. Labeling is present in the skin on both
sides, with fainter label in the skin on the left side. MRI also
visualizes internal regions of the animal rather than the
surface-only views offered by light microscopy (A, C), and detects
expression deep in the head that is not visible from the surface
view offered by light microscopy. FIG. C depicts the same embryo
stained for .beta.-galactosidase after completion of the MR
imaging. Whole-mount cytochemistry reveals that
.beta.-galactosidase is located primarily on the right side of the
embryo, with less in the head, with some skin label toward the
ventral half of the left side. The embryo curled slightly during
fixation. Scale Bar=1 mm
[0042] FIG. 19 demonstrates use of EGadMe for MR detection of lacZ
gene expression. FIG. 19A depicts MR image of living embryo
injected with plasmids carrying the lacZ gene. Plasmid carrying the
lacZ gene was injected to one cell at the two-cell stage and
subsequent enzyme expression was on the left side of the embryo as
shown. Regions of high signal intensity are found in the bright
stripe of endoderm (e), regions of the head (h) and ventrally,
including two distinct spots (red arrows) found just ventral to the
cement gland (c). FIG. 19B depicts bright field image of same
embryo fixed and stained for .beta.-galactosidase. Whole mount
cytochemistry shows that regions demonstrating enzyme expression
correlate with the regions of high intensity in the MR image.
[0043] FIGS. 20A, 20B, 20C, 20D and 20E depict several different
linkers that are cleavable by esterases. FIG. 20A depicts schematic
esterase enzyme mechanisms. FIGS. 20B, 20C, 20D and 20E depict
several different linkers, using a blocking moiety, although as
will be appreciated, other blocking moieties can be used as
well.
[0044] FIG. 21 depicts a synthetic scheme for the synthesis of the
compositions of the invention that have peptides as a component of
the system (either as a blocking moiety, linker or targeting
moiety, for example).
[0045] FIG. 22 depicts a representative structure utilizing a
blocking moiety comprising a peptide and a HIV-TAT peptide as a
targeting moiety, attached with an aryl amine linker, although as
outlined herein, any number of different linkers may be used.
[0046] FIG. 23 depicts a variety of configurations for adding
targeting moieties to the compositions of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0047] The present invention provides magnetic resonance imaging
contrast agents which can detect physiological agents or target
substances. The MRI agents of the invention are relatively
inactive, or have weak relaxivity, as contrast enhancement agents
in the absence of the physiological target substance, and are
activated, thus altering the MR image, in the presence of the
physiological target substance.
[0048] Viewed simplistically, this "trigger" mechanism, whereby the
contrast agent is "turned on" (i.e. increases the relaxivity) by
the presence of the target substance, is based on a dynamic
equilibrium that affects the rate of exchange of water molecules in
one or more coordination sites of a paramagnetic metal ion
contained in the MRI contrast agents of the present invention. In
turn, the rate of exchange of the water molecule is determined by
the presence or absence of the target substance in the surrounding
environment. Thus, in the absence of the target substance, the
metal ion complexes of the invention which chelate the paramagnetic
ion have reduced coordination sites available which can rapidly
exchange with the water molecules of the local environment. In such
a situation, the water coordination sites are substantially
occupied or blocked by the coordination atoms of the chelator and
at least one blocking moiety. Thus, the paramagnetic ion has
essentially no water molecules in its "inner-coordination sphere",
i.e. actually bound to the metal when the target substance is
absent. It is the interaction of the paramagnetic metal ion with
the protons on the inner coordination sphere water molecules and
the rapid exchange of such water molecules that cause the high
observed relaxivity, and thus the imaging effect, of the
paramagnetic metal ion. Accordingly, if all the coordination sites
of the metal ion in the metal ion complex are occupied with
moieties other than water molecules, as is the case when the target
substance is absent, there is little if any net enhancement of the
imaging signal by the metal ion complexes of the invention.
However, when present, the target substance interacts with the
blocking moiety or moities of the metal ion complex, effectively
freeing at least one of the inner-sphere coordination sites on the
metal ion complex. The water molecules of the local environment are
then available to occupy the inner-sphere coordination site or
sites, which will cause an increase in the rate of exchange of
water and relaxivity of the metal ion complex toward water thereby
producing image enhancement which is a measure of the presence of
the target substance.
[0049] Generally, a 2 to 5% change in the MRI signal used to
generate the image is sufficient to be detectable. Thus, it is
preferred that the agents of the invention in the presence of a
target substance increase the MRI signal by at least 2 to 5% as
compared to the signal gain the absence of the target substance.
Signal enhancement of 2 to 90% is preferred, and 10 to 50% is more
preferred for each coordination site made available by the target
substance interaction with the blocking moiety. That is, when the
blocking moiety occupies two or more coordination sites, the
release of the blocking moiety can result in double the increase in
signal or more as compared to a single coordination site.
[0050] It should be understood that even in the absence of the
target substance, at any particular coordination site, there will
be a dynamic equilibrium for one or more coordination sites as
between a coordination atom of the blocking moiety and water
molecules. That is, even when a coordination atom is tightly bound
to the metal, there will be some exchange of water molecules at the
site. However, in most instances, this exchange of water molecules
is neither rapid nor significant, and does not result in
significant image enhancement. However, upon exposure to the target
substance, the blocking moiety dislodges from the coordination site
and the exchange of water is increased, i.e. rapid exchange and
therefore an increase in relaxivity may occur, with significant
image enhancement.
[0051] The complexes of the invention comprise a chelator and a
blocking moiety. The metal ion complexes of the invention comprise
a paramagnetic metal ion bound to a complex comprising a chelator
and a blocking moiety. By "paramagnetic metal ion", "paramagnetic
ion" or "metal ion" herein is meant a metal ion which is magnetized
parallel or antiparallel to a magnetic field to an extent
proportional to the field. Generally, these are metal ions which
have unpaired electrons; this is a term understood in the art.
Examples of suitable paramagnetic metal ions, include, but are not
limited to, gadolinium III (Gd+3 or Gd(III)), iron III (Fe+3 or
Fe(III)), manganese II (Mn+2 or Mn(II)), yttrium III (Y+3 or
Y(III)), dysprosium (Dy+3 or Dy(III)), and chromium (Cr(III) or
Cr+3). In a preferred embodiment the paramagnetic ion is the
lanthanide atom Gd(III), due to its high magnetic moment
(u.sup.2=63BM2), a symmetric electronic ground state (S8), and its
current approval for diagnostic use in humans.
[0052] In addition to the metal ion, the metal ion complexes of the
invention comprise a chelator and a blocking moiety which may be
covalently attached to the chelator. Due to the relatively high
toxicity of many of the paramagnetic ions, the ions are rendered
nontoxic in physiological systems by binding to a suitable
chelator. Thus, the substitution of blocking moieties in
coordination sites of the chelator, which in the presence of the
target substance are capable of vacating the coordination sites in
favor of water molecules, may render the metal ion complex more
toxic by decreasing the half-life of dissociation for the metal ion
complex. Thus, in a preferred embodiment, only a single
coordination site is occupied or blocked by a blocking moeity.
However, for some applications, e.g. analysis of tissue and the
like, the toxicity of the metal ion complexes may not be of
paramount importance. Similarly, some metal ion complexes are so
stable that even the replacement of one or more additional
coordination atoms with a blocking moiety does not significantly
effect the half-life of dissociation. For example, DOTA, described
below, when complexed with Gd(III) is extremely stable.
Accordingly, when DOTA serves as the chelator, several of the
coordination atoms of the chelator may be replaced with blocking
moieties without a significant increase in toxicity. Additionally
such an agent would potentially produce a larger signal since it
has two or more coordination sites which are rapidly exchanging
water with the bulk solvent.
[0053] There are a variety of factors which influence the choice
and stability of the chelate metal ion complex, including enthalpy
and entropy effects (e.g. number, charge and basicity of
coordinating groups, ligand field and conformational effects).
[0054] In general, the chelator has a number of coordination sites
containing coordination atoms which bind the metal ion. The number
of coordination sites, and thus the structure of the chelator,
depends on the metal ion. The chelators used in the metal ion
complexes of the present invention preferably have at least one
less coordination atom (n-1) than the metal ion is capable of
binding (n), since at least one coordination site of the metal ion
complex is occupied or blocked by a blocking moeity, as described
below, to confer functionality on the metal ion complex. Thus, for
example, Gd(III) may have 8 strongly associated coordination atoms
or ligands and is capable of weakly binding a ninth ligand.
Accordingly, suitable chelators for Gd(III) will have less than 9
coordination atoms. In a preferred embodiment, a Gd(III) chelator
will have 8 coordination atoms, with a blocking moiety either
occupying or blocking the remaining site in the metal ion complex.
In an alternative embodiment, the chelators used in the metal ion
complexes of the invention have two less coordination atoms (n-2)
than the metal ion is capable of binding (n), with these
coordination sites occupied by one or more blocking moieties. Thus,
alternative embodiments utilize Gd(III) chelators with at least 5
coordination atoms, with at least 6 coordination atoms being
preferred, at least 7 being particularly preferred, and at least 8
being especially preferred, with the blocking moiety either
occupying or blocking the remaining sites. It should be appreciated
that the exact structure of the chelator and blocking moiety may be
difficult to determine, and thus the exact number of coordination
atoms may be unclear. For example, it is possible that the chelator
provide a fractional or non-integer number of coordination atoms;
i.e. the chelator may provide 7.5 coordination atoms, i.e. the 8th
coordination atom is on average not fully bound to the metal ion.
However, the metal ion complex may still be functional, if the 8th
coordination atom is sufficiently bound to prevent the rapid
exchange of water at the site, and/or the blocking moiety impedes
the rapid exchange of water at the site.
[0055] There are a large number of known macrocyclic chelators or
ligands which are used to chelate lanthanide and paramagnetic ions.
See for example, Alexander, Chem. Rev. 95:273-342 (1995) and
Jackels, Pharm. Med. Imag, Section III, Chap. 20, p645 (1990),
expressly incorporated herein by reference, which describes a large
number of macrocyclic chelators and their synthesis. Similarly,
there are a number of patents which describe suitable chelators for
use in the invention, including U.S. Pat. Nos. 5,155,215,
5,087,440, 5,219,553, 5,188,816, 4,885,363, 5,358,704, 5,262,532,
and Meyer et al., Invest. Radiol. 25: S53 (1990), all of which are
also expressly incorportated by reference. Thus, as will be
understood by those in the art, any of the known paramagnetic metal
ion chelators or lanthanide chelators can be easily modified using
the teachings herein to further comprise at least one blocking
moiety.
[0056] When the metal ion is Gd(III), a preferred chelator is
1,4,7,10-tetraazacyclododecane-N,N',N", N'"-tetracetic acid (DOTA)
or substituted DOTA. DOTA has the structure shown below: 2
[0057] By "substituted DOTA" herein is meant that the DOTA may be
substituted at any of the following positions, as shown below:
3
[0058] Suitable R substitution groups include a wide variety of
groups, as will be understood by those in the art. For example,
suitable substitution groups include substitution groups disclosed
for DOTA and DOTA-type compounds in U.S. Pat. Nos. 5,262,532,
4,885,363, and 5,358,704. These groups include hydrogen, alkyl
groups including substituted alkyl groups and heteroalkyl groups,
aryl groups including substituted aryl and heteroaryl groups,
phosphorus moieties, and blocking moieties. As will be appreciated
by those skilled in the art, each position designated above may
have two R groups attached (R' and R"), although in a preferred
embodiment only a single non-hydrogen R group is attached at any
particular position; that is, preferably at least one of the R
groups at each position is hydrogen. Thus, if R is an alkyl or aryl
group, there is generally an additional hydrogen attached to the
carbon, although not depicted herein. In a preferred embodiment,
one R group is a blocking moiety and the other R groups are
hydrogen.
[0059] By "alkyl group" or grammatical equivalents herein is meant
a straight or branched chain alkyl group, with straight chain alkyl
groups being preferred. If branched, it may be branched at one or
more positions, and unless specified, at any position. Also
included within the definition of alkyl are heteroalkyl groups,
wherein the heteroatom is selected from nitrogen, oxygen,
phosphorus, sulfur and silicon. Also included within the definition
of an alkyl group are cycloalkyl groups such as C5 and C6 rings,
and heterocycloalkyl.
[0060] Additional suitable heterocyclic substituted rings are
depicted in U.S. Pat. No. 5,087,440, expressly incorporated by
reference. In some embodiments, two adjacent R groups may be bonded
together to form ring structures together with the carbon atoms of
the chelator, such as is described in U.S. Pat. No. 5,358,704,
expressly incorporated by reference. These ring structures may be
similarly substituted.
[0061] The alkyl group may range from about 1 to 20 carbon atoms
(C1-C20), with a preferred embodiment utilizing from about 1 to
about 10 carbon atoms (C1-C10), with about C1 through about C5
being preferred. However, in some embodiments, the alkyl group may
be larger, for example when the alkyl group is the coordination
site barrier.
[0062] By "alkyl amine" or grammatical equivalents herein is meant
an alkyl group as defined above, substituted with an amine group at
any position. In addition, the alkyl amine may have other
substitution groups, as outlined above for alkyl group. The amine
may be primary (--NH.sub.2R), secondary (--NHR.sub.2), or tertiary
(--NR.sub.3). When the amine is a secondary or tertiary amine,
suitable R groups are alkyl groups as defined above. A preferred
alkyl amine is p-aminobenzyl. When the alkyl amine serves as the
coordination site barrier, as described below, preferred
embodiments utilize the nitrogen atom of the amine as a
coordination atom, for example when the alkyl amine includes a
pyridine or pyrrole ring.
[0063] By "aryl group" or grammatical equivalents herein is meant
aromatic aryl rings such as phenyl, heterocyclic aromatic rings
such as pyridine, furan, thiophene, pyrrole, indole and purine, and
heterocyclic rings with nitrogen, oxygen, sulfur or phosphorus.
[0064] Included within the definition of "alkyl" and "aryl" are
substituted alkyl and aryl groups. That is, the alkyl and aryl
groups may be substituted, with one or more substitution groups.
For example, a phenyl group may be a substituted phenyl group.
Suitable substitution groups include, but are not limited to,
halogens such as chlorine, bromine and fluorine, amines, hydroxy
groups, carboxylic acids, nitro groups, carbonyl and other alkyl
and aryl groups as defined herein. Thus, arylalkyl and hydroxyalkyl
groups are also suitable for use in the invention. Preferred
substitution groups include alkyl amines and alkyl hydroxy.
[0065] By "phosphorous moieties" herein is meant moieties
containing the --PO(OH)(R.sub.25).sub.2 group. The phosphorus may
be an alkyl phosphorus; for example, DOTEP utilizes ethylphosphorus
as a substitution group on DOTA. R.sub.25 may be alkyl, substituted
alkyl, hydroxy. A preferred embodiment has a --PO(OH).sub.2R.sub.25
group.
[0066] The substitution group may also be hydrogen or a blocking
moiety, as is described below. In an alternative embodiment, when
the metal ion is Gd(III), a preferred chelator is
diethylenetriaminepentaacetic acid (DTPA) or substituted DTPA. DPTA
has the structure shown below: 4
[0067] By "substituted DPTA" herein is meant that the DPTA may be
substituted at any of the following positions, as shown below:
5
[0068] See for example U.S. Pat. No. 5,087,440.
[0069] Suitable R substitution groups include those outlined above
for DOTA. Again, those skilled in the art will appreciate that
there may be two R groups (R' and R") at each position designated
above, although as described herein, at least one of the groups at
each position is hydrogen, which is generally not depicted
herein.
[0070] In an alternative embodiment, when the metal ion is Gd(III),
a preferred chelator is
1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraeth- ylphosphorus
(DOTEP) or substituted DOTEP (see U.S. Pat. No. 5,188,816). DOTEP
has the structure shown below: 6
[0071] DOTEP may have similar R substitution groups as outlined
above.
[0072] Other suitable Gd(III) chelators are described in Alexander,
supra, Jackels, supra, U.S. Pat. Nos. 5,155,215, 5,087,440,
5,219,553, 5,188,816, 4,885,363, 5,358,704, 5,262,532, and Meyer et
al., Invest. Radiol. 25: S53 (1990), among others.
[0073] When the paramagnetic ion is Fe(III), appropriate chelators
will have less than 6 coordination atoms, since Fe(III) is capable
of binding 6 coordination atoms. Suitable chelators for Fe(III)
ions are well known in the art, see for example Lauffer et al., J.
Am. Chem. Soc. 109:1622 (1987); Lauffer, Chem. Rev. 87:901-927
(1987); and U.S. Pat. Nos. 4,885,363, 5,358,704, and 5,262,532, all
which describe chelators suitable for Fe(III).
[0074] When the paramagnetic ion is Mn(II) (Mn+2), appropriate
chelators will have less than 5 or 6 coordination atoms, since
Mn(II) is capable of binding 6 or 7 coordination atoms. Suitable
chelators for Mn(II) ions are well known in the art; see for
example Lauffer, Chem. Rev. 87:901-927 (1987) and U.S. Pat. Nos.
4,885,363, 5,358,704, and 5,262,532.
[0075] When the paramagnetic ion is Y(III), appropriate chelators
will have less than 7 or 8 coordination atoms, since Y(III) is
capable of binding 8 or 9 coordination atoms. Suitable chelators
for Y(III) ions include, but are not limited to, DOTA and DPTA and
derivatives thereof (see Moi et al., J. Am. Chem. Soc.
110:6266-6267 (1988)) and those chelators described in U.S. Pat.
No. 4,885,363 and others, as outlined above.
[0076] When the paramagnetic ion is Dy+3 (Dy(III)), appropriate
chelators will have less than 7 or 8 coordination atoms, since
DyIII is capable of binding 8 or 9 coordination atoms. Suitable
chelators are known in the art, as above.
[0077] In a preferred embodiment, the chelator and the blocking
moiety are covalently linked; that is, the blocking moiety is a
substitution group on the chelator. In this embodiment, the
substituted chelator, with the bound metal ion, comprises the metal
ion complex which in the absence of the target substance has all
possible coordination sites occupied or blocked; i.e. it is
coordinatively saturated.
[0078] In an alternative embodiment, the chelator and the blocking
moiety are not covalently attached. In this embodiment, the
blocking moiety has sufficient affinity for the metal ion to
prevent the rapid exchange of water molecules in the absence of the
target substance. However, in this embodiment the blocking moiety
has a higher affinity for the target substance than for the metal
ion. Accordingly, in the presence of the target substance, the
blocking moiety will have a tendency to be dislodged from the metal
ion to interact with the target substance, thus freeing up a
coordination site in the metal ion complex and allowing the rapid
exchange of water and an increase in relaxivity.
[0079] What is important is that the metal ion complex, comprising
the metal ion, the chelator and the blocking moiety, is not readily
able to rapidly exchange water molecules when the blocking moeities
are in the inner coordination sphere of the metal ion, such that in
the absence of the target substance, there is less or little
substantial image enhancement.
[0080] By "blocking moiety" or grammatical equivalents herein is
meant a functional group associated with the chelator metal ion
complexes of the invention which is capable of interacting with a
target substance and which is capable, under certain circumstances,
of substantially blocking the exchange of water in at least one
inner coordination site of the metal ion of the metal ion complex.
For example, when bound to or associated with the metal ion
complexes of the invention, the blocking moiety occupies or blocks
at least one coordination site of the metal ion in the absence of
the target substance. Thus, the metal ion is coordinately saturated
with the chelator and the blocking moiety or moieties in the
absence of the target substance.
[0081] A blocking moiety may comprise several components. The
blocking moiety has a functional moiety which is capable of
interacting with a target substance, as outlined below. This
functional moiety may or may not provide the coordination atom(s)
of the blocking moiety. In addition, blocking moieties may comprise
one or more linker groups to allow for correct spacing and
attachment of the components of the blocking moiety. Furthermore,
in the embodiment where the functional group of the blocking moiety
does not contribute a coordination atom, the blocking moiety may
comprise a coordination site barrier, which serves to either
provide a coordination site atom or sterically prevent the rapid
exchange of water at the coordination site; i.e. the coordination
site barrier may either occupy or block the coordination site.
[0082] By "capable of interacting with a target substance" herein
is meant that the blocking moiety has an affinity for the target
substance, such that the blocking moiety will stop blocking or
occupying at least one coordination site of the metal ion complex
when the target substance is present. Thus, as outlined above, the
blocking moiety is blocking or occupying at least one coordination
site of the metal ion in the absence of the target substance.
However, in the presence of the target substance, the blocking
moiety associates or interacts with the target substance and is
released from its association with the metal ion, thus freeing at
least one coordination site of the metal ion such that the rapid
exchange of water can occur at this site, resulting in image
enhancement.
[0083] The nature of the interaction between the blocking moiety
and the target substance will depend on the target substance to be
detected or visualized via MRI. For example, suitable target
substances include, but are not limited to, enzymes; proteins;
peptides; nucleic acids; ions such as Ca+2, Mg+2, Zn+2, K+, Cl-,
and Na+; cAMP; receptors such as cell-surface receptors and
ligands; hormones; antigens; antibodies; ATP; NADH; NADPH;
FADH.sub.2; FNNH.sub.2; coenzyme A (acyl CoA and acetyl CoA); and
biotin, among others.
[0084] In some embodiments, the nature of the interaction is
irreversible, such that the blocking moiety does not reassociate to
block or occupy the coordination site; for example, when the
blocking moiety comprises an enzyme substrate which is cleaved upon
exposure to the target enzyme. Alternatively, the nature of the
interaction is reversible, such that the blocking moiety will
reassociate with the complex to hinder the exchange of water; for
example, when the blocking moiety comprises an ion ligand, or a
receptor ligand, as outlined below.
[0085] The corresponding blocking moieties will be enzyme
substrates or inhibitors, receptor ligands, antibodies, antigens,
ion binding compounds, substantially complementary nucleic acids,
nucleic acid binding proteins, etc.
[0086] In a preferred embodiment, the target substance is an
enzyme, and the blocking moiety is an enzyme substrate. In this
embodiment, the blocking moiety is cleaved from the metal ion
complex of the invention, allowing the exchange of water in at
least one coordination site of the metal ion complex. This
embodiment allows the amplification of the image enhancement since
a single molecule of the target substance is able to generate many
activated metal ion complexes, i.e. metal ion complexes in which
the blocking moiety is no longer occupying or blocking a
coordination site of the metal ion.
[0087] As will be appreciated by those skilled in the art, the
possible enzyme target substances are quite broad. The target
substance enzyme may be chosen on the basis of a correlation to a
disease condition, for example, for diagnositic purposes.
Alternatively, the metal ion complexes of the present invention may
be used to establish such correlations.
[0088] Suitable classes of enzymes include, but are not limited to,
hydrolases such as proteases, carbohydrases, lipases and nucleases;
isomerases such as racemases, epimerases, tautomerases, or mutases;
transferases, kinases and phophatases.
[0089] As will be appreciated by those skilled in the art, the
potential list of suitable enzyme targets is quite large. Enzymes
associated with the generation or maintenance of arterioschlerotic
plaques and lesions within the circulatory system, inflammation,
wounds, immune response, tumors, may all be detected using the
present invention. Enzymes such as lactase, maltase, sucrase or
invertase, cellulase, .alpha.-amylase, aldolases, glycogen
phosphorylase, kinases such as hexokinase, proteases such as
serine, cysteine, aspartyl and metalloproteases may also be
detected, including, but not limited to, trypsin, chymotrypsin, and
other therapeutically relevant serine proteases such as tPA and the
other proteases of the thrombolytic cascade; cysteine proteases
including: the cathepsins, including cathepsin B, L, S, H, J, N and
O; and calpain;; metalloproteinases including MMP-1 through MMP-10,
particularly MMP-1, MMP-2, MMP-7 and MMP-9; and caspases, such as
caspase-3, -5, -8 and other caspases of the apoptotic pathway, and
interleukin-converting enzyme (ICE). Similarly, bacterial and viral
infections may be detected via characteristic bacterial and viral
enzymes. As will be appreciated in the art, this list is not meant
to be limiting.
[0090] Once the target enzyme is identified or chosen, enzyme
substrate blocking moieties can be designed using well known
parameters of enzyme substrate specificities.
[0091] For example, when the enzyme target substance is a protease,
the blocking moieity may be a peptide or polypeptide which is
capable of being cleaved by the target protease. By "peptide" or
"polypeptide" herein is meant a compound of about 2 to about 15
amino acid residues covalently linked by peptide bonds. Preferred
embodiments utilize polypeptides from about 2 to about 8 amino
acids, with about 2 to about 4 being the most preferred.
Preferably, the amino acids are naturally occurring amino acids,
although amino acid analogs and peptidomimitic structures are also
useful. Under certain circumstances, the peptide may be only a
single amino acid residue.
[0092] Preferred target substance/peptide blocking moiety pairs
include, but are not limited to, cat B and GGGF; cat B and
GFQGVQFAGF; cat B and GFGSVGFAGF; cat B and GLVGGAGAGF; cat B and
GGFLGLGAGF; cat D and GFGSTFFAGF; caspase-3 and DEVD; MMP-7 and
PELR; MMP-7 and PLGLAR; MMP-7 and PGLWA-(D-arg); MMP-7 and PMALWMR;
and MMP-7 and PMGLRA.
[0093] Similarly, when the enzyme target substance is a
carbohydrase, the blocking moiety will be a carbohydrate group
which is capable of being cleaved by the target carbohydrase. For
example, when the enzyme target is lactase or .beta.-galactosidase,
the enzyme substrate blocking moiety is lactose or galactose.
Similar enzyme/blocking moiety pairs include sucrase/sucrose,
maltase/maltose, and .alpha.-amylase/amylose. In addition, the
addition of carbohydrate moieties such as galactose, outlined
herein, can alter the biodistribution of the agents; for example,
the galactose blocking moieties outlined herein cause concentration
in liver, kidneys and spleen.
[0094] In another embodiment, the blocking moiety may be an enzyme
inhibitor, such that in the presence of the enzyme, the inhibitor
blocking moiety disassociates from the metal ion complex to
interact or bind to the enzyme, thus freeing an inner coordination
sphere site of the metal ion for interaction with water. As above,
the enzyme inhibitors are chosen on the basis of the enzyme target
substance and the corresponding known characteristics of the
enzyme.
[0095] In a preferred embodiment, the blocking moiety is a
phosphorus moiety, as defined above, such as
--(OPO(OR.sub.2)).sub.n, wherein n is an integer from 1 to about
10, with from 1 to 5 being preferred and 1 to 3 being particularly
preferred. Each R is independently hydrogen or a substitution group
as defined herein, with hydrogen being preferred. This embodiment
is particularly useful when the target molecule is alkaline
phosphatase or a phosphodiesterase, or other enzymes known to
cleave phosphorus containing moieties such as these.
[0096] In one embodiment, the blocking moiety is a nucleic acid.
The nucleic acid may be single-stranded or double stranded, and
includes nucleic acid analogs such as peptide nucleic acids and
other well-known modifications of the ribose-phosphate backbone,
such as phosphorthioates, phosphoramidates, morpholino structures,
etc. The target molecule can be a substantially complementary
nucleic acid or a nulceic acid binding moiety, such as a
protein.
[0097] In a preferred embodiment, the target substance is a
physiological agent. As for the enzyme/substrate embodiment, the
physiological agent interacts with the blocking moiety of the metal
ion complex, such that in the presence of the physiological agent,
there is rapid exchange of water in at least one inner sphere
coordination site of the metal ion complex. Thus, the target
substance may be a physiologically active ion, and the blocking
moiety is an ion binding ligand. For example, as shown in the
Examples, the target substance may be the Ca+2 ion, and the
blocking moiety may be a calcium binding ligand such as is known in
the art (see Grynkiewicz et al., J. Biol. Chem. 260(6):3440-3450
(1985); Haugland, R. P., Molecular Probes Handbook of Fluorescent
Probes and Research Chemicals (1989-1991)). Other suitable target
ions include Mn+2, Mg+2, Zn+2, Na+, and Cl-.
[0098] When Ca+2 is the target substance, preferred blocking
moieties include, but are not limited to, the acetic acid groups of
bis(o-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA),
ethylene glycol bis(.beta.-aminoethyl ether)-N,N,N',N'-tetraacetic
acid (EGTA); ethylenediaminetetracetic acid (EDTA); and derivatives
thereof, such as disclosed in Tsien, Biochem. 19:2396-2404 (1980).
Other known chelators of Ca+2 and other divalent ions, such as
quin2 (2-[[2-[bis(carboxymethyl)-
amino]-5-methylphenoxy]methyl-6-methoxy-8-[bis(carboxymethyl)amino]quinoli-
ne; fura-1, fura-2, fura-3, stil-1, stil-2 and indo-1 (see
Grynkiewicz et al., supra).
[0099] As for the enzyme/substrate embodiments, the metabolite may
be associated with a particular disease or condition within an
animal. For example, as outlined below, BAPTA-DOTA derivatives may
be used to diagnose Alzeheimer's disease and other neurological
disorders.
[0100] In a preferred embodiment, the blocking moiety is a ligand
for a cell-surface receptor or is a ligand which has affinity for a
extracellular component. In this embodiment, as for the
physiological agent embodiment, the ligand has sufficient affinity
for the metal ion to prevent the rapid exchange of water molecules
in the absence of the target substance. Alternatively, there may be
R groups "locking" the ligand into place, as described herein,
resulting in either the contribution of a coordination atom or that
the ligand serves as a coordination site barrier. In this
embodiment the ligand blocking moiety has a higher affinity for the
target substance than for the metal ion. Accordingly, in the
presence of the target substance, the ligand blocking moiety will
interact with the target substance, thus freeing up at least one
coordination site in the metal ion complex and allowing the rapid
exchange of water and an increase in relaxivity. Additionally, in
this embodiment, this may result in the accumulation of the MRI
agent at the location of the target, for example at the cell
surface. This may be similar to the situation where the blocking
moiety is an enzyme inhibitor, as well.
[0101] In a preferred embodiment, the blocking moiety is a
photocleavable moiety. That is, upon exposure to a certain
wavelength of light, the blocking moiety is cleaved, allowing an
increase in the exchange rate of water in at least one coordination
site of the complex. This embodiment has particular use in
developmental biology fields (cell lineage, neuronal development,
etc.), where the ability to follow the fates of particular cells is
desirable. Suitable photocleavable moieties are similar to "caged"
reagents which are cleaved upon exposure to light. A particularly
preferred class of photocleavable moieties are the O-nitrobenzylic
compounds, which can be synthetically incorporated into a blocking
moiety via an ether, thioether, ester (including phosphate esters),
amine or similar linkage to a heteroatom (particularly oxygen,
nitrogen or sulfur). Also of use are benzoin-based photocleavable
moieties. A wide variety of suitable photocleavable moieties is
outlined in the Molecular Probes Catalog, supra.
[0102] In a preferred embodiment, the compounds have a structure
depicted below in Structure 18, which depicts a nitrobenzyl
photocleavable group, although as will be appreciated by those in
the art, a wide variety of other moieties may be used: 7
[0103] Structure 18 depicts a DOTA-type chelator, although as will
be appreciated by those in the art, other chelators may be used as
well. R.sub.26 is a linker as defined below. Similarly, the X.sub.2
group may be as defined above, although additional structures may
be used, for example a coordination site barrier as outlined
herein. Similarly, there may be substitutent groups on the aromatic
ring, as is known in the art.
[0104] The blocking moiety itself may block or occupy at least one
coordination site of the metal ion. That is, one or more atoms of
the blocking moiety (i.e. the enzyme substrate, ligand, moiety
which interacts with a physiological agent, photocleavable moiety,
etc.) itself serves as a coordination atom, or otherwise blocks
access to the metal ion by steric hinderance. For example, it
appears that one or more of the atoms of the galactose blocking
moiety outlined in the Examples may be direct coordination atoms
for the Gd(III) metal ion. Similarly, peptide based blocking
moieties for protease targets may contribute coordination
atoms.
[0105] In an alternative embodiment, the blocking moiety further
comprises a "coordination site barrier" which is covalently
tethered to the complex in such a manner as to allow disassociation
upon interaction with a target substance. For example, it may be
tethered by one or more enzyme substrate blocking moieties. In this
embodiment, the coordination site barrier blocks or occupies at
least one of the coordination sites of the metal ion in the absence
of the target enzyme substance. Coordination site barriers are used
when coordination atoms are not provided by the functional portion
of the blocking moiety, i.e. the component of the blocking moiety
which interacts with the target substance. The blocking moiety or
moieties such as an enzyme substrate serves as the tether,
covalently linking the coordination site barrier to the metal ion
complex. In the presence of the enzyme target, the enzyme cleaves
one or more of the enzyme substrates, either within the substrate
or at the point of attachment to the metal ion complex, thus
freeing the coordination site barrier. The coordination site or
sites are no longer blocked and the bulk water is free to rapidly
exchange at the coordination site of the metal ion, thus enhancing
the image. As will be appreciated by those in the art, a similar
result can be accomplished with other types of blocking
moieties.
[0106] In one embodiment, the coordination site barrier is attached
to the metal ion complex at one end, as is depicted in FIG. 1. When
the enzyme target cleaves the substrate blocking moiety, the
coordination site barrier is released. In another embodiment, the
coordination site barrier is attached to the metal ion complex with
more than one substrate blocking moiety, as is depicted in FIG. 2
for two attachments. The enzyme target may cleave only one side,
thus removing the coordination site barrier and allowing the
exchange of water at the coordination site, but leaving the
coordination site barrier attached to the metal ion complex.
Alternatively, the enzyme may cleave the coordination site barrier
completely from the metal ion complex.
[0107] In a preferred embodiment, the coordination site barrier
occupies at least one of the coordination sites of the metal ion.
That is, the coordination site barrier contains at least one atom
which serves as at least one coordination atom for the metal ion.
In this embodiment, the coordination site barrier may be a
heteroalkyl group, such as an alkyl amine group, as defined above,
including alkyl pyridine, alkyl pyrroline, alkyl pyrrolidine, and
alkyl pyrole, or a carboxylic or carbonyl group. The portion of the
coordination site barrier which does not contribute the
coordination atom may also be consider a linker group. Preferred
coordination site barriers are depicted in FIG. 2.
[0108] In an alternative embodiment, the coordination site barrier
does not directly occupy a coordination site, but instead blocks
the site sterically. In this embodiment, the coordination site
barrier may be an alkyl or substituted group, as defined above, or
other groups such as peptides, proteins, nucleic acids, etc.
[0109] In this embodiment, the coordination site barrier is
preferrably linked via two enzyme substrates to opposite sides of
the metal ion complex, effectively "stretching" the coordination
site barrier over the coordination site or sites of the metal ion
complex, as is depicted in FIG. 2.
[0110] In some embodiments, the coordination site barrier may be
"stretched" via an enzyme substrate on one side, covalently
attached to the metal ion complex, and a linker moeity, as defined
below, on the other. In an alternative embodiment, the coordination
site barrier is linked via a single enzyme substrate on one side;
that is, the affinity of the coordination site barrier for the
metal ion is higher than that of water, and thus the blocking
moiety, comprising the coordination site barrier and the enzyme
substrate, will block or occupy the available coordination sites in
the absence of the target enzyme.
[0111] In some embodiments, the metal ion complexes of the
invention have a single associated or bound blocking moiety. In
such embodiments, the single blocking moiety impedes the exchange
of water molecules in at least one coordination site.
Alternatively, as is outlined below, a single blocking moiety may
hinder the exchange of water molecules in more than one
coordination site, or coordination sites on different
chelators.
[0112] In alternative embodiments, two or more blocking moieties
are associated with a single metal ion complex, to implede the
exchange of water in at least one or more coordination sites.
[0113] It should be appreciated that the blocking moieties of the
present invention may further comprise a linker group as well as a
functional blocking moiety. That is, blocking moieties may comprise
functional blocking moieties in combination with a linker group
and/or a coordination site barrier.
[0114] Linker groups (sometimes depicted herein as R.sub.26) will
be used to optimize the steric considerations of the metal ion
complex. That is, in order to optimize the interaction of the
blocking moiety with the metal ion, linkers may be introduced to
allow the functional blocking moiety to block or occupy the
coordination site. In general, the linker group is chosen to allow
a degree of structural flexibility. For example, when a blocking
moiety interacts with a physiological agent which does not result
in the blocking moiety being cleaved from the complex, the linker
must allow some movement of the blocking moiety away from the
complex, such that the exchange of water at at least one
coordination site is increased.
[0115] Generally, suitable linker groups include, but are not
limited to, alkyl and aryl groups, including substituted alkyl and
aryl groups and heteroalkyl (particularly oxo groups) and
heteroaryl groups, including alkyl amine groups, as defined above.
Preferred linker groups include p-aminobenzyl, substituted
p-aminobenzyl, diphenyl and substituted diphenyl, alkyl furan such
as benzylfuran, carboxy, and straight chain alkyl groups of 1 to 10
carbons in length. Particularly preferred linkers include
p-aminobenzyl, methyl, ethyl, propyl, butyl, pentyl, hexyl, acetic
acid, propionic acid, aminobutyl, p-alkyl phenols,
4-alkylimidazole. The selection of the linker group is generally
done using well known molecular modeling techniques, to optimize
the obstruction of the coordination site or sites of the metal ion.
In addition, as outlined in the Examples, the length of this linker
may be very important in order to achieve optimal results. As shown
in FIG. 11, the length of the linker, i.e the spacer between the
chelator and the coordination atom(s) of the blocking moiety,
contributes to the steric conformation and association of the
coordination atoms with the metal ion, thus allowing excellent
blocking of the metal ion by the blocking moiety.
[0116] In a preferred embodiment, a coordination site barrier can
be attached by a cleavable linker as outlined herein. A preferred
embodiment utilizes esterase linkages such as are generally
depicted in FIG. 20. Esterase linkages are particularly preferred
when the blocking moiety is attached via an "arm" of the chelate,
as the product of an esterase reaction is a carboxylic acid, which
thus allows the regeneration of a stable chelate (and, in the case
of DOTA and DPTA, chelates that are approved for human use).
Alternatively, cleavable peptide linkers can also be used.
[0117] The blocking moiety is attached to the metal ion complex in
a variety of ways. In a preferred embodiment, as noted above, the
blocking moiety is attached to the metal ion complex via a linker
group. Alternatively, the blocking moiety is attached directly to
the metal ion complex; for example, as outlined below, the blocking
moiety may be a substituent group on the chelator.
[0118] In a preferred embodiment at least one of the R groups
attached to the "arms" of the chelator, for example R.sub.9,
R.sub.10, R.sub.11 or R.sub.12 of the DOTA structures, or R.sub.13,
R.sub.14, R.sub.17, R.sub.20 or R.sub.21 of the DTPA structures,
comprises an alkyl (including substituted and heteroalkyl groups),
or aryl (including substituted and heteroaryl groups), i.e. is a
group sterically bulkier than hydrogen. This is particular useful
to drive the equilibrium towards "locking" the coordination atom of
the arm into place to prevent water exchange, as is known for
standard MRI contrast agents. Preferred groups include the C1
through C6 alkyl groups with methyl being particularly
preferred.
[0119] This is particularly preferred when the blocking moiety is
attached via one of the "arms", for example when a blocking moiety
is at position X.sub.1 to X.sub.4 (Structure 6), position S, T, U
or V (Structure 8) or position H, I, J or K of Structure 16.
[0120] However the inclusion of too many groups may drive the
equilibrium in the other direction effectively locking the
coordination atom out of position, as is shown in Example 3.
Therefore in a preferred embodiment only 1 or 2 of these positions
is a non-hydrogen group, unless other methods are used to drive the
equilibrium towards binding.
[0121] The blocking moieties are chosen and designed using a
variety of parameters. In the embodiment which uses a coordination
site barrier, i.e. when the functional group of the blocking moiety
does not provide a coordination atom, and the coordination site
barrier is fastened or secured on two sides, the affinity of the
coordination site barrier of the blocking moiety for the metal ion
complex need not be great, since it is tethered in place. That is,
in this embodiment, the complex is "off" in the absence of the
target substance. However, in the embodiment where the blocking
moiety is linked to the complex in such a manner as to allow some
rotation or flexibility of the blocking moiety, for example, it is
linked on one side only, such as the galactose embodiment of the
examples, the blocking moiety should be designed such that it
occupies the coordination site a majority of the time. Thus, for
example, the galactose-DOTA structure of Example 1 gives roughly a
20% increase in the signal in the presence of galactosidase, thus
indicating that the galactose blocking moiety is in equilibrium
between blocking or occupying the coordination site and rotating
free in solution. However, as described herein and shown in Example
3, these agents may be "locked" off using R groups on the
carboxylic acid "arms" of a chelator, to reduce the rotational
freedom of the group and thus effectively drive the equilibrium to
the "off" position, and thus result in a larger percentage increase
in the signal in the presence of the target.
[0122] When the blocking moiety is not covalently tethered on two
sides, as is depicted in FIG. 1, it should be understood that
blocking moieties and coordination site barriers are chosen to
maximize three basic interactions that allow the blocking moiety to
be sufficiently associated with the complex to hinder the rapid
exchange of water in at least one coordination site of the complex.
First, there may be electrostatic interactions between the blocking
moiety and the metal ion, to allow the blocking moiety to associate
with the complex. Secondly, there may be Van der Waals and
dipole-dipole interactions. Thirdly, there may be ligand
interactions, that is, one or more functionalities of the blocking
moiety may serve as coordination atoms for the metal. In addition,
linker groups may be chosen to force or favor certain
conformations, to drive the equilibrium towards an associated
blocking moiety. Similarly, removing degrees of fredom in the
molecule may force a particular conformation to prevail. Thus, for
example, the addition of alkyl groups, and particularly methyl
groups, at positions equivalent to the R.sub.9 to R.sub.12
positions of Structure 7 when the blocking moiety is attached at W,
X, Y or Z, can lead the blocking moiety to favor the blocking
position. Similar restrictions can be made in the other
embodiments, as will be appreciated by those in the art.
[0123] Furthermore, effective "tethering" of the blocking moiety
down over the metal ion may also be done by engineering in other
non-covalent interactions that will serve to increase the affinity
of the blocking moiety to the chelator complex, as is depicted
below.
[0124] Potential blocking moieties may be easily tested to see if
they are functional; that is, if they sufficiently occupy or block
the appropriate coordination site or sites of the complex to
prevent rapid exchange of water. Thus, for example, complexes are
made with potential blocking moieties and then compared with the
chelator without the blocking moiety in imaging experiments. Once
it is shown that the blocking moiety is a sufficient "blocker", the
target substance is added and the experiments repeated, to show
that interaction with the target substance increases the exchange
of water and thus enhances the image.
[0125] Thus, as outlined above, the metal ion complexes of the
present invention comprise a paramagnetic metal ion bound to a
chelator and at least one blocking moiety. In a preferrred
embodiment, the metal ion complexes have the formula shown in
Structure 6: 8
[0126] In Structure 6, M is a paramagnetic metal ion selected from
the group consisting of Gd(III), Fe(III), Mn(II), Yt(III), and
Dy(III). A, B, C and D are each either single or double bonds. The
R.sub.1 through R.sub.12 groups are alkyl or aryl groups, as
defined above, including substituted alkyl and aryl groups,
phosphorus groups, or a blocking moiety, as described above.
X.sub.1 through X.sub.4 are --OH, --COO--, --(CH.sub.2).sub.nOH
(with --CH.sub.2OH being preferred), --(CH.sub.2).sub.nCOO-- (with
CH.sub.2COO-- being preferred) or a blocking moiety. n is from 1 to
10, with from 1 to 5 being preferred. At least one of R.sub.1 to
R.sub.12 and X.sub.1 to X.sub.4 is a blocking moiety.
[0127] Structure 6 includes Structures 7 and 8, shown below: 9
[0128] In this embodiment, W, X, Y and Z are as defined above for
X, and at least one of the R.sub.1 to R.sub.12 groups is a blocking
moiety.
[0129] As applied to DOTA, the four nitrogens of the DOTA ring, and
the W, X, Y and Z groups provide 8 of the coordination atoms for
the paramagnetic metal ion. The ninth coordination atom is provided
by a blocking moiety which is substituted at one of the R.sub.1 to
R.sub.12 positions. In a preferred embodiment, the other R groups
are either hydrogen or methyl; in a particularly preferred
embodiment the chelator is Gd-MCTA, which has a single methyl group
on the DOTA ring (see Meyer et al., Invest. Radiol. 25:S53
(1990)).
[0130] In an alternative embodiment, the metal ion complexes have
the formula depicted in Structure 8: 10
[0131] In this embodiment, S, T, U, and V are --OH, --COO--,
--(CH2).sub.nOH (with --CH.sub.2OH being preferred),
--(CH2).sub.nCOO-- (with CH.sub.2COO-- being preferred) or a
blocking moiety. In this embodiment, the four nitrogens of the DOTA
ring, and three of the S, T, U or V groups provide 7 of the
coordination atoms for the paramagnetic metal ion. The remaining
coordination atoms are provided by a blocking moiety which is
substituted at one of the S, T, U or V positions. Alternatively,
the coordination sites are either filled by coordination atoms
provided by the S, T, U or V groups, or blocked by the S, T, U or V
structure, or both. In addition, Structure 8 does not depict the A,
B, C and D bonds, but as for the other embodiments, these bonds may
be either single or double bonds.
[0132] As applied to DOTA, the four nitrogens of the DOTA ring, and
the (generally) three S, T and U groups provide 7 of the
coordination atoms for the Gd(III) paramagnetic metal ion. The
eigth and ninth coordination atoms are provided by a blocking
moiety which is substituted at one of the S, T, U and V positions.
As above, the other R groups are preferably either hydrogen or
methyl, with Gd-MCTA being especially preferred.
[0133] In the Structures depicted herein, any or all of A, B, C or
D may be a single bond or a double bond. It is to be understood
that when one or more of these bonds are double bonds, there may be
only a single substitutent group attached to the carbons of the
double bond. For example, when A is a double bond, there may be
only a single R.sub.1 and a single R.sub.2 group attached to the
respective carbons; in a preferred embodiment, as described below,
the R.sub.1 and R.sub.2 groups are hydrogen. In a preferred
embodiment, A is a single bond, and it is possible to have two
R.sub.1 groups and two R.sub.2 groups on the respective carbons. In
a preferred embodiment, these groups are all hydrogen with the
exception of a single blocking moiety, but alternate embodiments
utilize two R groups which may be the same or different. That is,
there may be a hydrogen and a blocking group attached in the
R.sub.1 position, and two hydrogens, two alkyl groups, or a
hydrogen and an alkyl group in the R.sub.2 positions.
[0134] It is to be understood that the exact composition of the
X.sub.1-X.sub.4 (Structure 6) S, T, U, V (Structure 8) or W, X, Y
and Z (Structure 7) groups will depend on the presence of the metal
ion. That is, in the absence of the metal ion, the groups may be
--OH, --COOH, --(CH.sub.2).sub.nOH, or (CH.sub.2).sub.nCOOH;
however, when the metal is present, the groups may be --OH,
--COO--, --(CH.sub.2).sub.nO--, or (CH.sub.2).sub.nCOO--.
[0135] In a preferred embodiment, the compositions have the formula
shown in Structure 9: 11
[0136] In this embodiment, there is a single blocking moiety
attached to the metal ion complex. That is, all but one of the R
groups are hydrogen. It should be appreciated that the blocking
moiety may be at any of the R positions.
[0137] In a preferred embodiment, the magnetic resonance imaging
agents are used to detect Ca+2 ions, and have the structure
depicted in Structure 10: 12
[0138] In this embodiment, the blocking moiety comprises a linker
and the BAPTA molecule, although any of the fura-type Ca.sup.+2
ligands may be substituted. Without being bound by theory, it
appears that one of the carboxy groups of the BAPTA moiety serves
to provide a coordination atom in the absence of Ca+2. However, in
the presence of Ca+2, the carboxy group chelates Ca+2, and thus is
unavailable as a coordination group, thus allowing the rapid
exchange of water. Preferably, the metal ion is Gd(III), the R
groups are all hydrogen, and the W, X, Y and Z groups are
carboxy.
[0139] In one embodiment the carboxylic acid groups of the BAPTA
molecule may be protected with acetate protecting groups, resulting
a neutral molecule that may then cross membranes. Once inside a
cell, intracellular esterases can cleave off the acetate protecting
groups, allowing the detection of Ca.sup.+2. See Li et al.,
Tetrahedron 53(35):12017-12040 (1997).
[0140] In a preferred embodiment, the compositions have the formula
shown in Structure 11: 13
[0141] In this embodiment, there is a single blocking moiety
attached to the metal ion complex. It should be appreciated that
the blocking moiety may be at any of the S, T, U or V positions.
Similarly, a single blocking moiety may be attached to DTPA.
[0142] In a preferred embodiment, the magnetic resonance imaging
contrast agents have the structure shown in Structure 12: 14
[0143] In this embodiment, the blocking moiety comprises a linker
and a carbohydrate, attached to the complex via a .beta.(1, 4)
linkage such as is recognized by lactose or .beta.-galactosidase.
Without being bound by theory, it is apparent that the galactose
moiety provides a coordination atom, such that in the absence of
.beta.-galactosidase there is reduced exchange of water in the
complex. Upon exposure to .beta.-galactosidase, the carbohydrate
blocking moiety is cleaved off, removing the coordination atom and
allowing the rapid exchange of water. Preferably, the R groups are
hydrogen, and the W, X, Y and Z groups are carboxy.
[0144] In another embodiment, the metal ion complexes have the
formula depicted in Structure 13: 15
[0145] In this embodiment, R.sub.22, R.sub.23 and R.sub.24 comprise
a blocking moiety, with R.sub.23 being a coordination site barrier
which also serves to contribute a coordination atom. It is to be
understood that the R.sub.22 and R.sub.24 groups may be attached at
any of the R.sub.1 to R.sub.12 positions. Preferred R.sub.23 groups
include, but are not limited to, compounds listed above that
provide a coordination atom, blocking moieties, and those shown in
FIG. 2. R.sub.22 and R.sub.24 may also comprise a linker, as
defined above and as shown in Structure 14, below. Preferred
R.sub.22 and R.sub.24 groups include enzyme substrates which are
cleaved upon exposure to the enzyme, such as carbohydrates and
peptides. Accordingly, when the target substance is a carbohydrase
such as .beta.-galactosidase, the compositions have the formula
shown in Structure 14: 16
[0146] In this embodiment, the blocking moiety comprises two
linkers, two carbohydrates, and a coordination site barrier. The
carbohydrates are attached to the complex via a linkage which will
be recognized by a carbohydrase such as a .beta.(1, 4) linkage such
as is recognized by lactose or .beta.-galactosidase. The R.sub.22
group provides a coordination atom in the absence of the
carbohydrase such there is no rapid exchange of water in the
complex. Upon exposure to the carbohydrase, such as
.beta.-galactosidase, one or both of the carbohydrate blocking
moieties are cleaved off, removing the coordination atom and
allowing the rapid exchange of water. Preferably, the R groups are
hydrogen, and the W, X, Y and Z groups are carboxy. Alternatively,
the blocking moiety could comprise peptides for a protease target
substance.
[0147] In place of the carbohydrates in Structure 14, an
alternative embodiment utilizes peptides. That is, a peptide
comprising 2 to 5 amino acids or analogs may be "stretched" from
one side of the complex to the other, and linker groups may or may
not be used. Similarly, nucleic acids may be used.
[0148] Alternatively, there may not be covalent attachment at both
ends. As discussed above, effective "tethering" of the blocking
moiety down over the metal ion may also be done by engineering in
other non-covalent interactions that will serve to increase the
affinity of the blocking moiety to the chelator complex. Thus, for
example, electrostatic interactions may be used, as is generally
depicted below for a DOTA derivative in Structure 15: 17
[0149] In Structure 15, the blocking moeity/coordination site
barrier occupies the X.sub.3 position, although any position may be
utilized. E.sub.1 and E.sub.2 and electrostatic moieties bearing
opposite charges. In Structure 15, the E.sub.2 group is shown a
position R.sub.8, although any position may be used.
[0150] A further embodiment utilizes metal ion complexes having the
formula shown in Structure 16: 18
[0151] It is to be understood that, as above, the exact composition
of the H, I, J, K and L groups will depend on the presence of the
metal ion. That is, in the absence of the metal ion, H, I, J, K and
L are --OH, --COOH, --(CH.sub.2).sub.nOH, or (CH.sub.2).sub.nCOOH;
however, when the metal is present, the groups are --OH, --COO--,
--(CH.sub.2).sub.nOH, or (CH.sub.2).sub.nCOO--.
[0152] In this embodiment, R.sub.13 through R.sub.21 are alkyl or
aryl, including substituted and hetero derivatives, a phosphorus
moiety or a blocking moiety, all as defined above. In a preferred
embodiment, R.sub.12 to R.sub.21 are hydrogen. At least one of
R.sub.13-R.sub.21, H, I, J, K or L is a blocking moiety, as defined
above.
[0153] In a preferred embodiment, the MRI contrast agents of the
invention comprise more than one metal ion, such that the signal is
increased. As is outlined below, this may be done in a number of
ways, some of which are shown in FIG. 10.
[0154] In a preferred embodiment, the MRI agents of the invention
comprise at least two paramagnetic metal ions, each with a chelator
and blocking moiety; that is, multimeric MRI agents are made. In a
preferred embodiment, the chelators are linked together, either
directly or through the use of a linker such as a coupling moiety
or polymer. For example, using substitution groups that serve as
functional groups for chemical attachment on the chelator,
attachment to other chelators may be accomplished. As will be
appreciated by those in the art, attachment of more than one MRI
agent may also be done via the blocking moieties (or coordination
site barriers, etc.), although these are generally not
preferred.
[0155] In a preferred embodiment, the chelators of the invention
include one or more substitution groups that serve as functional
groups for chemical attachment. Suitable functional groups include,
but are not limited to, amines (preferably primary amines), carboxy
groups, and thiols (including SPDP, alkyl and aryl halides,
maleimides, .alpha.-haloacetyls, and pyridyl disulfides) are useful
as functional groups that can allow attachment.
[0156] In one embodiment, the chelators are linked together
directly, using at least one functional group on each chelator.
This may be accomplished using any number of stable bifunctional
groups well known in the art, including homobifunctional and
heterobifunctional linkers (see Pierce Catalog and Handbook, 1994,
pages T155-T200, hereby expressly incorporated by reference). This
may result in direct linkage, for example when one chelator
comprises a primary amine as a functional group and the second
comprises a carboxy group as the functional group, and carbodiimide
is used as an agent to activate the carboxy for attach by the
nucleophilic amine (see Torchilin et al., Critical Rev. Therapeutic
Drug Carrier Systems, 7(4):275-308 (1991). Alternatively, as will
be appreciated by those in the art, the use of some bifunctional
linkers results in a short coupling moiety being present in the
structure. A "coupling moiety" is capable of covalently linking two
or more entities. In this embodiment, one end or part of the
coupling moiety is attached to the first MRI contrast agent, and
the other is attached to the second MRI agent. The functional
group(s) of the coupling moiety are generally attached to
additional atoms, such as alkyl or aryl groups (including hetero
alkyl and aryl, and substituted derivatives), to form the coupling
moiety. Oxo linkers are also preferred. As will be appreciated by
those in the art, a wide range of coupling moieties are possible,
and are generally only limited by the ability to synthesize the
molecule and the reactivity of the functional group. Generally, the
coupling moiety comprises at least one carbon atom, due to
synthetic requirements; however, in some embodiments, the coupling
moiety may comprise just the functional group.
[0157] In a preferred embodiment, the coupling moiety comprises
additional atoms as a spacer. As will be appreciated by those in
the art, a wide variety of groups may be used. For example, a
coupling moiety may comprise an alkyl or aryl group substituted
with one or more functional groups. Thus, in one embodiment, a
coupling moiety containing a multiplicity of functional groups for
attachment of multiple MRI contrast agents may be used, similar to
the polymer embodiment described below. For example, branched alkyl
groups containing multiple functional groups may be desirable in
some embodiments.
[0158] In an additional embodiment, the linker is a polymer. In
this embodiment, a polymer comprising at least one MRI contrast
agent of the invention is used. As will be appreciated by those in
the art, these MRI contrast agents may be monomeric (i.e. one metal
ion, one chelator, one blocking moiety) or a duplex, as is
generally described below (i.e. two metal ions, two chelators, one
blocking moiety). Preferred embodiments utilize a plurality of MRI
agents per polymer. The number of MRI agents per polymer will
depend on the density of MRI agents per unit length and the length
of the polymer.
[0159] The character of the polymer will vary, but what is
important is that the polymer either contain or can be modified to
contain functional groups for the the attachment of the MRI
contrast agents of the invention. Suitable polymers include, but
are not limited to, functionalized dextrans, styrene polymers,
polyethylene and derivatives, polyanions including, but not limited
to, polymers of heparin, polygalacturonic acid, mucin, nucleic
acids and their analogs including those with modified
ribose-phosphate backbones, the polypeptides polyglutamate and
polyaspartate, as well as carboxylic acid, phosphoric acid, and
sulfonic acid derivatives of synthetic polymers; and polycations,
including but not limited to, synthetic polycations based on
acrylamide and 2-acrylamido-2-methylpropanetrimethylamine,
poly(N-ethyl-4-vinylpyridine) or similar quarternized polypyridine,
diethylaminoethyl polymers and dextran conjugates, polymyxin B
sulfate, lipopolyamines, poly(allylamines) such as the strong
polycation poly(dimethyldiallylammonium chloride),
polyethyleneimine, polybrene, spermine, spermidine and polypeptides
such as protamine, the histone polypeptides, polylysine,
polyarginine and polyornithine; and mixtures and derivatives of
these. Particularly preferred polycations are polylysine and
spermidine, with the former being especially preferred. Both
optical isomers of polylysine can be used. The D isomer has the
advantage of having long-term resistance to cellular proteases. The
L isomer has the advantage of being more rapidly cleared from the
subject. As will be appreciated by those in the art, linear and
branched polymers may be used.
[0160] A preferred polymer is polylysine, as the --NH.sub.2 groups
of the lysine side chains at high pH serve as strong nucleophiles
for multiple attachment of activated chelating agents. At high pH
the lysine monomers are coupled to the MRI agents under conditions
that yield on average 5-20% monomer substitution.
[0161] In some embodiments, particularly when charged polymers are
used, there may be a second polymer of opposite charge to the first
that is electrostatically associated with the first polymer, to
reduce the overall charge of polymer-MRI agent complex. This second
polymer may or may not contain MRI agents.
[0162] The size of the polymer may vary substantially. For example,
it is known that some nucleic acid vectors can deliver genes up to
100 kilobases in length, and artificial chromosomes (megabases)
have been delivered to yeast. Therefore, there is no general size
limit to the polymer. However, a preferred size for the polymer is
from about 10 to about 50,000 monomer units, with from about 2000
to about 5000 being particularly preferred, and from about 3 to
about 25 being especially preferred. It should be understood that
the multimeric MRI agents of the invention may be made in a variety
of ways, including those listed above. What is important is that
manner of attachment does not significantly alter the functionality
of the agents; that is, the agents must still be "off" in the
absence of the target substance and "on" in its presence.
[0163] In a preferred embodiment, the MRI contrast agents of the
invention are "duplexes". In this embodiment, the MRI duplex
comprises two chelators, each with a paramagnetic metal ion, and at
least one blocking moiety that restricts the exchange of water in
at least one coordination site of each chelator. In this way, a
sort of signal amplification occurs, with two metal ions increasing
the signal with a single target molecule. While "duplex" implies
two chelators, it is intended to refer to complexes comprising a
single blocking moiety donating coordination atoms to more than 1
metal ion/chelator complex. As will be appreciated by those in the
art, the MRI agents of this embodiment may have a number of
different conformations, as is generally shown in FIG. 10. As will
be appreciated by those in the art, the R.sub.26, R.sub.27 and
R.sub.28 groups of the figure can be attached to any of the
positions described herein, to any R groups or X.sub.1-X.sub.4, S,
T, U, V, W, X, Y, or Z groups.
[0164] As outlined above, the MRI duplex moieties may also be
combined into higher multimers, either by direct linkage or via
attachment to a polymer.
[0165] In a preferred embodiment, the blocking moiety is BAPTA, as
is generally depicted below in Structure 17, with propyl linking
groups between the chelators and the BAPTA derivative: 19
[0166] As will be appreciated by those in the art, the structure
depicted in Structure 17 may be altered, for example, replacing the
phenyl groups of the BAPTA derivative with cycloalkyl groups, or
removing them entirely, as is generally depicted in Structure 19:
20
[0167] As noted above, the carboxylic acids of the BAPTA molecule
may also be protected using acetate protecting groups, to render a
neutral molecule for entry into cells, that then can be reactivated
via cleavage by intracellular esterases.
[0168] In addition, although Structures 17 and 19 have ethylene
groups between the oxygens of the bridge of BAPTA, methylene and
propylene may also be used, as well as substituted derivatives of
these.
[0169] In a preferred embodiment, A, B, C and D are single bonds,
R.sub.1-R.sub.12 are hydrogen, and each R.sub.26 is --CH.sub.2O--,
with the CH.sub.2 group being attached to the macrocycle. In
addition, the complexes and metal ion complexes of the invention
may further comprise one or more targeting moieties. That is, a
targeting moiety may be attached at any of the R positions (or to a
linker, including a polymer, or to a blocking moiety, etc.),
although in a preferred embodiment the targeting moiety does not
replace a coordination atom. By "targeting moiety" herein is meant
a functional group which serves to target or direct the complex to
a particular location, cell type, diseased tissue, or association.
In general, the targeting moiety is directed against a target
molecule. As will be appreciated by those in the art, the MRI
contrast agents of the invention are generally injected
intraveneously; thus preferred targeting moieties are those that
allow concentration of the agents in a particular localization.
Thus, for example, antibodies, cell surface receptor ligands and
hormones, lipids, sugars and dextrans, alcohols, bile acids, fatty
acids, amino acids, peptides and nucleic acids may all be attached
to localize or target the contrast agent to a particular site.
[0170] In a preferred embodiment, the targeting moiety allows
targeting of the MRI agents of the invention to a particular tissue
or the surface of a cell. That is, in a preferred embodiment the
MRI agents of the invention need not be taken up into the cytoplasm
of a cell to be activated.
[0171] As will be appreciated by those in the art, the targeting
moieties can be attached in a large number of different ways, and
in a variety of configurations, as are schematically depicted in
FIG. 23.
[0172] In a preferred embodiment, the targeting moiety is a
peptide. For example, chemotactic peptides have been used to image
tissue injury and inflammation, particularly by bacterial
infection; see WO 97/14443, hereby expressly incorporated by
reference in its entirety.
[0173] In a preferred embodiment, the targeting moiety is an
antibody. The term "antibody" includes antibody fragments, as are
known in the art, including Fab Fab.sub.2, single chain antibodies
(Fv for example), chimeric antibodies, etc., either produced by the
modification of whole antibodies or those synthesized de novo using
recombinant DNA technologies.
[0174] In a preferred embodiment, the antibody targeting moieties
of the invention are humanized antibodies or human antibodies.
Humanized forms of non-human (e.g., murine) antibodies are chimeric
immunoglobulins, immunoglobulin chains or fragments thereof (such
as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of
antibodies) which contain minimal sequence derived from non-human
immunoglobulin. Humanized antibodies include human immunoglobulins
(recipient antibody) in which residues from a complementary
determining region (CDR) of the recipient are replaced by residues
from a CDR of a non-human species (donor antibody) such as mouse,
rat or rabbit having the desired specificity, affinity and
capacity. In some instances, Fv framework residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Humanized antibodies may also comprise residues which are found
neither in the recipient antibody nor in the imported CDR or
framework sequences. In general, the humanized antibody will
comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the FR regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin [Jones et
al., Nature 321:522-525 (1986); Riechmann et al., Nature
332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596
(1992)].
[0175] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers [Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988);
Verhoeyen et al., Science 239:1534-1536 (1988)], by substituting
rodent CDRs or CDR sequences for the corresponding sequences of a
human antibody. Accordingly, such "humanized" antibodies are
chimeric antibodies (U.S. Pat. No. 4,816,567), wherein
substantially less than an intact human variable domain has been
substituted by the corresponding sequence from a non-human species.
In practice, humanized antibodies are typically human antibodies in
which some CDR residues and possibly some FR residues are
substituted by residues from analogous sites in rodent
antibodies.
[0176] Human antibodies can also be produced using various
techniques known in the art, including phage display libraries
[Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al.,
J. Mol. Biol. 222:581 (1991)]. The techniques of Cole et al. and
Boerner et al. are also available for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J.
Immunol. 147(1):86-95 (1991)]. Similarly, human antibodies can be
made by introducing of human immunoglobulin loci into transgenic
animals, e.g., mice in which the endogenous immunoglobulin genes
have been partially or completely inactivated. Upon challenge,
human antibody production is observed, which closely resembles that
seen in humans in all respects, including gene rearrangement,
assembly, and antibody repertoire. This approach is described, for
example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,661,016, and in the following scientific
publications: Marks et al., Bio/Technology 10:779-783 (1992);
Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature
368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51
(1996); Neuberger, Nature Biotechnology, 14:826 (1996); Lonberg and
Huszar, Intern. Rev. Immunol. 13:65-93 (1995).
[0177] Bispecific antibodies are monoclonal, preferably human or
humanized, antibodies that have binding specificities for at least
two different antigens. In the present case, one of the binding
specificities is for a first target molecule and the other one is
for a second target molecule.
[0178] Methods for making bispecific antibodies are known in the
art. Traditionally, the recombinant production of bispecific
antibodies is based on the co-expression of two immunoglobulin
heavy-chain/light-chain pairs, where the two heavy chains have
different specificities [Milstein and Cuello, Nature 305:537-539
(1983)]. Because of the random assortment of immunoglobulin heavy
and light chains, these hybridomas (quadromas) produce a potential
mixture of ten different antibody molecules, of which only one has
the correct bispecific structure. The purification of the correct
molecule is usually accomplished by affinity chromatography steps.
Similar procedures are disclosed in WO 93/08829, published May 13,
1993, and in Traunecker et al., EMBO J. 10:3655-3659 (1991).
[0179] Antibody variable domains with the desired binding
specificities (antibody-antigen combining sites) can be fused to
immunoglobulin constant domain sequences. The fusion preferably is
with an immunoglobulin heavy-chain constant domain, comprising at
least part of the hinge, CH2, and CH3 regions. It is preferred to
have the first heavy-chain constant region (CH1) containing the
site necessary for light-chain binding present in at least one of
the fusions. DNAs encoding the immunoglobulin heavy-chain fusions
and, if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transfected into a suitable
host organism. For further details of generating bispecific
antibodies see, for example, Suresh et al., Methods in Enzymology
121:210 (1986).
[0180] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target immune system cells to unwanted cells [U.S.
Pat. No. 4,676,980], and for treatment of HIV infection [WO
91/00360; WO 92/200373; EP 03089]. It is contemplated that the
antibodies may be prepared in vitro using known methods in
synthetic protein chemistry, including those involving crosslinking
agents. For example, immunotoxins may be constructed using a
disulfide exchange reaction or by forming a thioether bond.
Examples of suitable reagents for this purpose include
iminothiolate and methyl-4-mercaptobutyrimidate and those
disclosed, for example, in U.S. Pat. No. 4,676,980.
[0181] In a preferred embodiment, the antibody is directed against
a cell-surface marker on a cancer cell; that is, the target
molecule is a cell surface molecule. As is known in the art, there
are a wide variety of antibodies known to be differentially
expressed on tumor cells, including, but not limited to, HER2,
VEGF, etc.
[0182] In addition, antibodies against physiologically relevant
carbohydrates may be used, including, but not limited to,
antibodies against markers for breast cancer (CA15-3, CA 549, CA
27.29), mucin-like carcinoma associated antigen (MCA), ovarian
cancer (CA125), pancreatic cancer (DE-PAN-2), and colorectal and
pancreatic cancer (CA 19, CA 50, CA242).
[0183] In a preferred embodiment, the targeting moiety is all or a
portion (e.g. a binding portion) of a ligand for a cell surface
receptor. Suitable ligands include, but are not limited to, all or
a functional portion of the ligands that bind to a cell surface
receptor selected from the group consisting of insulin receptor
(insulin), insulin-like growth factor receptor (including both
IGF-1 and IGF-2), growth hormone receptor, glucose transporters
(particularly GLUT 4 receptor), transferrin receptor (transferrin),
epidermal growth factor receptor (EGF), low density lipoprotein
receptor, high density lipoprotein receptor, leptin receptor,
estrogen receptor (estrogen); interleukin receptors including IL-1,
IL-2, IL-3, IL4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13,
IL-15, and IL-17 receptors, human growth hormone receptor, VEGF
receptor (VEGF), PDGF receptor (PDGF), transforming growth factor
receptor (including TGF-.alpha. and TGF-.beta.), EPO receptor
(EPO), TPO receptor (TPO), ciliary neurotrophic factor receptor,
prolactin receptor, and T-cell receptors. In particular, hormone
ligands are preferred. Hormones include both steroid hormones and
proteinaceous hormones, including, but not limited to, epinephrine,
thyroxine, oxytocin, insulin, thyroid-stimulating hormone,
calcitonin, chorionic gonadotropin, cortictropin,
follicle-stimulating hormone, glucagon, leuteinizing hormone,
lipotropin, melanocyte-stimutating hormone, norepinephrine,
parathryroid hormone, thyroid-stimulating hormone (TSH),
vasopressin, enkephalins, seratonin, estradiol, progesterone,
testosterone, cortisone, and glucocorticoids and the hormones
listed above. Receptor ligands include ligands that bind to
receptors such as cell surface receptors, which include hormones,
lipids, proteins, glycoproteins, signal transducers, growth
factors, cytokines, and others.
[0184] In a preferred embodiment, the targeting moiety is a
carbohydrate. By "carbohydrate" herein is meant a compound with the
general formula Cx(H2O)y. Monosaccharides, disaccharides, and
oligo- or polysaccharides are all included within the definition
and comprise polymers of various sugar molecules linked via
glycosidic linkages. Particularly preferred carbohydrates are those
that comprise all or part of the carbohydrate component of
glycosylated proteins, including monomers and oligomers of
galactose, mannose, fucose, galactosamine, (particularly
N-acetylglucosamine), glucosamine, glucose and sialic acid, and in
particular the glycosylation component that allows binding to
certain receptors such as cell surface receptors. Other
carbohydrates comprise monomers and polymers of glucose, ribose,
lactose, raffinose, fructose, and other biologically significant
carbohydrates. In particular, polysaccharides (including, but not
limited to, arabinogalactan, gum arabic, mannan, etc.) have been
used to deliver MRI agents into cells; see U.S. Pat. No. 5,554,386,
hereby incorporated by reference in its entirety.
[0185] In a preferred embodiment, the targeting moiety is a lipid.
"Lipid" as used herein includes fats, fatty oils, waxes,
phospholipids, glycolipids, terpenes, fatty acids, and glycerides,
particularly the triglycerides. Also included within the definition
of lipids are the eicosanoids, steroids and sterols, some of which
are also hormones, such as prostaglandins, opiates, and
cholesterol.
[0186] In addition, as will be appreciated by those in the art, any
moiety which may be utilized as a blocking moiety can be used as a
targeting moiety. Particularly preferred in this regard are enzyme
inhibitors, as they will not be cleaved off and will serve to
localize the MRI agent in the location of the enzyme.
[0187] In a preferred embodiment, the targeting moiety may be used
to either allow the internalization of the MRI agent to the cell
cytoplasm or localize it to a particular cellular compartment, such
as the nucleus.
[0188] In a preferred embodiment, the targeting moiety is all or a
portion of the HIV-1 Tat protein, and analogs and related proteins,
which allows very high uptake into target cells. See for example,
Fawell et al., PNAS USA 91:664 (1994); Frankel et al., Cell 55:1189
(1988); Savion et al., J. Biol. Chem. 256:1149 (1981); Derossi et
al., J. Biol. Chem. 269:10444 (1994); and Baldin et al., EMBO J.
9:1511 (1990), all of which are incorporated by reference.
[0189] In a preferred embodiment, the targeting moiety is a nuclear
localization signal (NLS). NLSs are generally short, positively
charged (basic) domains that serve to direct the moiety to which
they are attached to the cell's nucleus. Numerous NLS amino acid
sequences have been reported including single basic NLS's such as
that of the SV40 (monkey virus) large T Antigen (Pro Lys Lys Lys
Arg Lys Val), Kalderon (1984), et al., Cell, 39:499-509; the human
retinoic acid receptor-.beta. nuclear localization signal (ARRRRP);
NF.kappa.B p50 (EEVQRKRQKL; Ghosh et al., Cell 62:1019 (1990);
NF.kappa.B p65 (EEKRKRTYE; Nolan et al., Cell 64:961 (1991); and
others (see for example Boulikas, J. Cell. Biochem. 55(1):32-58
(1994), hereby incorporated by reference) and double basic NLS's
exemplified by that of the Xenopus (African clawed toad) protein,
nucleoplasmin (Ala Val Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln
Ala Lys Lys Lys Lys Leu Asp), Dingwall, et al., Cell, 30:449-458,
1982 and Dingwall, et al., J. Cell Biol., 107:641-849; 1988).
Numerous localization studies have demonstrated that NLSs
incorporated in synthetic peptides or grafted onto reporter
proteins not normally targeted to the cell nucleus cause these
peptides and reporter proteins to be concentrated in the nucleus.
See, for example, Dingwall, and Laskey, Ann, Rev. Cell Biol.,
2:367-390, 1986; Bonnerot, et al., Proc. Natl. Acad. Sci. USA,
84:6795-6799, 1987; Galileo, et al., Proc. Natl. Acad. Sci. USA,
87:458-462, 1990.
[0190] In a preferred embodiment, targeting moieties for the
hepatobiliary system are used; see U.S. Pat. Nos. 5,573,752 and
5,582,814, both of which are hereby incorporated by reference in
their entirety.
[0191] In a preferred embodiment, the metal ion complexes of the
present invention are water soluble or soluble in aqueous solution.
By "soluble in aqueous solution" herein is meant that the MRI agent
has appreciable solubility in aqueous solution and other
physiological buffers and solutions. Solubility may be measured in
a variety of ways. In one embodiment, solubility is measured using
the United States Pharmacopeia solubility classifications, with the
metal ion complex being either very soluble (requiring less than
one part of solvent for 1 part of solute), freely soluble
(requiring one to ten parts solvent per 1 part solute), soluble
(requiring ten to thirty parts solvent per 1 part solute),
sparingly soluble (requiring 30 to 100 parts solvent per 1 part
solute), or slightly soluble (requiring 100-1000 parts solvent per
1 part solute).
[0192] Testing whether a particular metal ion complex is soluble in
aqueous solution is routine, as will be appreciated by those in the
art. For example, the parts of solvent required to solubilize a
single part of MRI agent may be measured, or solubility in gm/ml
may be determined.
[0193] The complexes of the invention are generally synthesized
using well known techniques. See, for example, Moi et al., supra;
Tsien et al., supra; Borch et al., J. Am. Chem. Soc., p2987 (1971);
Alexander, (1995), supra; Jackels (1990), supra, U.S. Pat. Nos.
5,155,215, 5,087,440, 5,219,553, 5,188,816, 4,885,363, 5,358,704,
5,262,532; Meyer et al., (1990), supra, Moi et al., (1988), and
McMurray et al., Bioconjugate Chem. 3(2):108-117 (1992)).
[0194] For DOTA derivatives, the synthesis depends on whether
nitrogen substitution or carbon substitution of the cyclen ring
backbone is desired. For nitrogen substitution, such as is
exemplified by the galactose-DOTA structures of the examples, the
synthesis begins with cyclen or cyclen derivatives, as is well
known in the art; see for example U.S. Pat. Nos. 4,885,363 and
5,358,704. FIGS. 3 and 4 depict the nitrogen substitution as
exemplified by galactose-DOTA derivatives.
[0195] For carbon substitution, such as is exemplified by the
BAPTA-DOTA structures of the examples, well known techniques are
used. See for example Moi et al., supra, and Gansow, supra. FIGS. 5
and 6 depict the carbon substitution as exemplified by the
BAPTA-DOTA type derivatives.
[0196] The contrast agents of the invention are complexed with the
appropriate metal ion as is known in the art. While the structures
depicted herein all comprise a metal ion, it is to be understood
that the contrast agents of the invention need not have a metal ion
present initially. Metal ions can be added to water in the form of
an oxide or in the form of a halide and treated with an equimolar
amount of a contrast agent composition. The contrast agent may be
added as an aqueous solution or suspension. Dilute acid or base can
be added if need to maintain a neutral pH. Heating at temperatures
as high as 100.degree. C. may be required.
[0197] The complexes of the invention can be isolated and purified,
for example using HPLC systems.
[0198] Pharmaceutical compositions comprising pharmaceutically
acceptable salts of the contrast agents can also be prepared by
using a base to neutralize the complexes while they are still in
solution. Some of the complexes are formally uncharged and do not
need counterions.
[0199] Once synthesized, the metal ion complexes of the invention
have use as magnetic resonance imaging contrast or enhancement
agents. Specifically, the functional MRI agents of the invention
have several important uses. First, they may be used to diagnose
disease states of the brain, as is outlined below. Second, they may
be used in real-time detection and differentiation of myocardial
infraction versus ischemia. Third, they may be used in in vivo,
i.e. whole organism, investigation of antigens and
immunocytochemistry for the location of tumors. Fourth, they may be
used in the identification and localization of toxin and drug
binding sites. In addition, they may be used to perform rapid
screens of the physiological response to drug therapy.
[0200] The metal ion complexes of the invention may be used in a
similar manner to the known gadolinium MRI agents. See for example,
Meyer et al., supra; U.S. Pat. No. 5,155,215; U.S. Pat. No.
5,087,440; Margerstadt et al., Magn. Reson. Med. 3:808 (1986);
Runge et al., Radiology 166:835 (1988); and Bousquet et al.,
Radiology 166:693 (1988). The metal ion complexes are administered
to a cell, tissue or patient as is known in the art. A "patient"
for the purposes of the present invention includes both humans and
other animals and organisms, such as experimental animals. Thus the
methods are applicable to both human therapy and veterinary
applications. In addition, the metal ion complexes of the invention
may be used to image tissues or cells; for example, see Aguayo et
al., Nature 322:190 (1986).
[0201] Generally, sterile aqueous solutions of the contrast agent
complexes of the invention are administered to a patient in a
variety of ways, including orally, intrathecally and especially
intraveneously in concentrations of 0.003 to 1.0 molar, with
dosages from 0.03, 0.05, 0.1, 0.2, and 0.3 millimoles per kilogram
of body weight being preferred. Dosages may depend on the
structures to be imaged. Suitable dosage levels for similar
complexes are outlined in U.S. Pat. Nos. 4,885,363 and
5,358,704.
[0202] In addition, the contrast agents of the invention may be
delivered via specialized delivery systems, for example, within
liposomes (see Navon, Magn. Reson. Med. 3:876-880 (1986)) or
microspheres, which may be selectively taken up by different organs
(see U.S. Pat. No. 5,155,215).
[0203] In some embodiments, it may be desirable to increase the
blood clearance times (or half-life) of the MRI agents of the
invention. This has been done, for example, by adding carbohydrate
polymers to the chelator (see U.S. Pat. No. 5,155,215). Thus, one
embodiment utilizes polysaccharides as substitution R groups on the
compositions of the invention.
[0204] A preferred embodiment utilizes complexes which cross the
blood-brain barrier. Thus, as is known in the art, a DOTA
derivative which has one of the carboxylic acids replaced by an
alcohol to form a neutral DOTA derivative has been shown to cross
the blood-brain barrier. Thus, for example, neutral complexes are
designed that cross the blood-brain barrier with blocking moieties
which detect Ca+2 ions. These compounds are used in MRI of a
variety of neurological disorders, including Alzeheimer's disease.
Currently it is difficult to correctly diagnosis Alzeheimer's
disease, and it would be useful to be able to have a physiological
basis to distinguish Alzeheimer's disease from depression, or other
treatable clinical symptoms for example.
[0205] The following examples serve to more fully describe the
manner of using the above-described invention, as well as to set
forth the best modes contemplated for carrying out various aspects
of the invention. It is understood that these examples in no way
serve to limit the true scope of this invention, but rather are
presented for illustrative purposes. The references cited herein
are expressly incorporated by reference.
EXAMPLES
Example 1
Synthesis and Characterization of Galactose-DOTA Derivative
[0206] Synthesis of Do3a-hydroxyethyl-beta-galactose Gadolinium
complex (FIG. 4). Acetyl protected bromo-galactose (Aldrich) was
reacted with bromoethanol. Difference ratios of the alpha- and
beta-bromoethyl ether of the acetylgalactose were obtained in good
yield. The isomers were separated using silica gel chromatography
and their assigments were made by hydrolyzing the acetyl protecting
groups and comparing the proton NMR coupling constants to known
compounds. Recently an x-ray structure was done confirming these
assignments (data not shown).
[0207] The beta-isomer was reacted with cyclen at reflux in
chloroform with monitoring of the reaction by TLC. Hydrolysis of
the acetates was acheived with TEA/MCOH/H.sub.2O overnight, and the
solvent was removed under low vacuum. The resulting product was
reacted directly with bromoacetic acid and then maintained at pH
10-10.5 until the pH remained constant. The possible products all
would have different charges in ammonia acetate buffer and thus
were separated by anion exchange chromatography. An ammonium
acetate buffer gradient was used during FPLC anion exchange to
elute the desired compound, with detection at 218 nm. Gadolinium
oxide in water at 80.degree. C. was used to insert the metal into
the complex. The reaction was followed using fluorescence
spectroscopy. The product was purified by HPLC reverse phase
chromatography using fluorescence spectroscopy for detection and
the structure was confirmed using high resolution mass
spectrometry. The overall yield for this essentially one pot
synthesis was greater than 25%.
[0208] Synthesis of aceto-1-ethylbromo-.beta.-galactose (FIG. 3):
1-Bromoethane-2-ol was reacted with
2,3,4,6-aceto-1-a-bromo-galactose to produce a mixture of .alpha.
and .beta. anomers (10/90) of aceto-1-ethylbromo-.beta.-galactose
in 68% yield (8.3 g). The purified .beta. anomer could be obtained
using flash chromatography. Stereochemical assignments were made
via a X-ray crystal structure of the .beta. anomer.
[0209] Aceto-1-ethylbromo-.beta.-galactose was reacted with cyclen
(Aldrich Chemical Co.) to produce the monosubsituted product. The
acetate protecting groups were cleaved and the 3 carboxylic acid
substituents were added using bromoacetic acid at pH 10.5. The
product was isolated by anion exchange fast performance liquid
chromatography (FPLC) observed by fluoroscence spectrascopy in 37%
yield. Gd.sup.3+ or Tb.sup.3+ was inserted into the complexes and
were purified using repeated collections on a reverse phase HPLC
analytic C.sub.18 column with a water/acetonitrile gradient (0-10%)
as the elute and fluorescence for detection (274 nm-ex and 315
nm-em) in 70% yield. High resolution mass spectrum analysis of the
solid provided a parent molecular ion for the (M+Na).sup.+ which
exhibited the correct exact mass and the predicted isotope
ratios.
[0210] Alternate synthetic route: Do3a methyl ester was synthesized
by literature methods. Do3a methyl ester was reacted with
beta-bromoethyl ether of the acetylgalactose obtained as described
in D.sub.2O/d4 methanol while maintaining the reaction at basic pH.
The reaction was followed by NMR. First the acetate methyl ester
cleaved and the sugar became water soluble as judged by allowing
the methanol to evaporate. Next the methyl ester was absorbed to
cleave and finally at around pH 10 a shift consistant with the
formation of the sugar Do3a was observed.
[0211] Summary of the synthesis of
Do2a-hydroxyethyl-di-beta-galactose: The reaction of cyclen with
beta-bromoethyl ether of the acetylgalactose in chloroform was
done. The reaction mixture was purified using silica gel
chromatography. While the alpha isomer gave monosubstitution only
di-substituted products were obtained for the beta isomer as shown
in FIG. 5. The acetic acid derived arm was added as described for
the monosubstituted compound above and purified by FPLC cation
exchange using an acid water gradient. Individual fractions were
detected by TLC spotting.
[0212] Characterization: The ability of .beta.-galactosidase to
remove the galactopyranosal blocking group from GadGal was examined
by HPLC. The cleavage reaction was monitored using the distinct
retention times of the complex and the complex without the
galactopyranose residue. Upon incubation with native
.beta.-galactosidase, a peak with an elution time of 15 min
appeared that corresponds to the complex without galactopyranose.
In a control experiment, using heat-inactivated
.beta.-galactosidase, the retention time of the peak remained
constant. Thus, the HPLC experiments confirm the enzymatic
processing of the complex by native but not heat-treated
enzyme.
[0213] The effect the presence of the galactopyranosal residue on
the water exchange rate of the complex was tested by measuring the
fluorescence spectra of the terbium (Tb.sup.3+) derivative ( 545
nm) in water/deuterium oxide mixtures. Terbium was substituted for
Gd because of the more intense fluorescence signal and long
lifetime when chelated. The fluorescence of the terbium complex is
quenched by H.sub.2O but not by D.sub.2O. This effect occurs
because the excited state of the terbium is coupled to the OH
oscillator but not the OD oscillator. Therefore, the lifetime of
the fluorescence signal is longer in D.sub.2O than in H.sub.2O. A
plot of 1/lifetime versus the percentage of H.sub.2O allows the
calculation of the number of water molecules, q, that are fast
exchange with the complex (Kumar et al., Pure and Appl. Chem.
65:515-520 (1993); Lie et al., J. Am. Chem. Soc. 117:8123-8138
(1995); Zhang et al., Inorg. Chem. 31:5597-5600 (1992)). The q
values for the terbium complexes in the presence and absence of the
galactopyranose were 0.7 and 1.2, respectively. Therefore,
spectrofluorimetry confirms that the galactopyranose blocking group
hinders the fast exchange of water.
[0214] The effect of the enzymatic cleavage of the galactopyranose
on the T.sub.1 of the complex was assessed using NMR spectroscopy.
The molar quantity related to these T.sub.1 values is the
relaxivity, R. R values at 500 MHz were determined for the complex
plus galactose (1800 mM s.sup.-1) and minus galactose (2400 mM
s.sup.-1) and compared to that of the related species Prohance
(2700 mM s.sup.-1). The difference in observed relaxivity parallels
the results obtained from the T.sub.1 measurements for complexes.
The increase in water exchange, demonstrated in the
spectrofluorimetry experiments, suggested that the T.sub.1 of a
solution of the agent should decrease upon enzymatic processing. A
20% difference between the measured T.sub.1 values in the presence
and absence of .beta.-galactosidase confirmed this prediction. The
complex exposed to .beta.-galactosidase at two different
concentrations showed identical and significant decreases in the
solution T.sub.1. A 20% change in observed T.sub.1 accompanies
cleavage of the galactopyranose from the complex, consistent with
the change in measured hydration number, q, obtained from
fluorescence measurements. Control solutions of the complex
together with heat inactivated enzyme show no decrease; in fact,
the T.sub.1 appeared to increase slightly. MRI microscopy was used
to examine if the observed difference in T.sub.1 between the
complex in the presence and absence of the galactose would be
sufficient to serve as a MRI contrast agent. Images obtained using
a standard inversion recovery sequence revealed that the T.sub.1
change generated by enzymatic processing could be visualized in a
MR image (FIG. 9). The complex was placed in 1.5-1.8 mm capillary
tubes, either with or without .beta.-galactosidase. The images
displayed in FIG. 9 show that the T.sub.1 mediated contrast was
altered by the action of .beta.-galactosidase, yielding the
expected increase in the image contrast.
Example 2
Synthesis of BAPTA-DTPA and BAPTA-DOTA Derivatives
[0215] Two representative synthetic schemes are shown for the
synthesis of a BAPTA-DTPA derivative in FIGS. 7 and 8. In FIG. 7
(the preferred method), structure I was prepared by modification of
published procedures (Tsien et al., supra) and coupled to
hexamehtylenediaamine using NaCNBrH.sub.3 in dry methanol. The
ratio of reactants used was 6:1:0.6 (diamine:BAPTA
aldehyde:NaCNBrH.sub.3). The reaction was quenched with the
addition of concentrated HCl and the product purified by HPLC (II).
This material was reacted with the mono (or bis) anhydride of DTPA
with the protecting groups left on the BAPTA until after the
Gd(III)Cl.sub.3 or Gd.sub.2O.sub.3 was added (elevated pH, heat).
The final product was purified by ion-exchange HPLC.
[0216] In FIG. 8, the monoanhydride of DTPA was prepared and
reacted with a bisalkylamine (e.g.
NH.sub.2(CH.sub.2).sub.6(NH.sub.2). This material was purified by
ion-exchange HPLC and placed in a round bottom flask equipped with
argon inlet and pressure equalizing funnel. The BAPTA aldehyde in
dry methanol was added dropwise to a solution of alkylamine-DTPA in
dry methanol and 6 equivalents of HCl:MeOH was added. The reaction
mixture was purified by HPLC, Gd(III) inserted as above, and the
protecting groups removed by literature procedures.
Example 3
The Use of R Groups to Increase Signal
[0217] The Example 1 compound exhibits an enhancement of roughly
20% upon exposure to the target analyte, in this case
.beta.-galactosidase. In order to increase the MR contrast
enhancement, our intention was to further decrease the access of
bulk water to the Gd(III) site by stabilizing the position of the
galactopyranose unit on top of the macrocyclic framework. Several
studies dealing with intramolecular dynamic processes in
tetraazacarboxyclic macrocycles were recently reported (see Kang et
al., Inorg. Chem. 36:2912 (1993); Aime et al., Inorg. Chem. 36:2095
(1997); Pittet et al., J. Am. Chem. Soc. Dalton Trans. 1997,
895-900; Spirlet et al., J. Am. Chem. Soc. Dalton Trans. 1997,
497-500, all of which are incorporated by reference. This work
demonstrated that introducing .alpha.-methyl groups to the
ethylenic groups of carboxyclic arms increases the rigidity of the
amino-carboxylate macrocyclic framework. We therefore added
sterically bulky .alpha.-methyl groups to two distinct sites of the
molecule to make two new compounds. The first, "EGADMe", is the
GADGAL of Example 1 with a single methyl group on the DOTA arm
containing the galactosyl blocking moiety. The second, "CarboxyMe",
is the GADGAL of Example 1 with three methyl groups on the other
three DOTA arms, leaving the arm containing the galoctosyl blocking
moiety alone. The final products EGadMe and CarboxyMe as well as
the intermediates were characterized by NMR- and mass
spectrometry.
[0218] The successful and complete enzymatic cleavage of the
galactopyranose blocking group from EGadMe and CarboxyMe,
respectively, was followed by TLC chromatography (C18 reverse phase
plates in 20 mM tris-acetate, 10 mM EDTA buffer pH 7.0, 8%
acetonitrile), to produce EGADMecl and CarboxyMecl. While 90% of
the galactopyranose units were enzymatically cleaved from EGadMe
within 3 days in an aequous solution containing 0.5 mM EgadMe and 5
.mu.M .beta.-galactosidase at 37C, the same effect was observed for
CarboxyMe within a period of 24 hrs under the same conditions. This
result implied that the galactopyranose unit of CarboxyMe might be
more exposed and accessible for the enzyme, therefore leading to a
higher cleavage rate.
[0219] The effect of the enzymatic cleavage of the galactopyranose
unit from EGadMe and CarboxyMe on relaxation time T1 was determined
by NMR spectroscopy at 500 MHz and 24 C. Various aqueous solutions
of 0.5 mM EGadMe and CarboxyMe, respectively, were prepared,
containing either: (a) no enzyme; (b) heat inactivated 5 .mu.M
.beta.-galactosidase that was treated at 80C for 10 min; (c) 5
.mu.M .beta.-galactosidase where T1 was measured immediately after
mixing; or (d) 5 .mu.M .beta.-galactosidase that was reacted with
the complex for 3.5 days at 37C. A remarkably difference between
the T1 of solutions containing EGadMe and those containing EGadMecl
is clearly obvious. In the presence of EGadMecl the T1 of water
protons is enhanced by 55% with respect to solutions containing
EGadMe. These results indicate that EGadMe is a highly effective,
fuctional or "smart" MRI contrast agent. A large difference in T1
between uncleaved and cleaved states represents the crucial factor
for successful in vivo applications. Preliminary in vivo studies
indicate that the compound fulfils these high expectations.
[0220] Interestingly, for solutions (a)-(d) containing CarboxyMe no
significant variations in T1 were detected. However, for all
CarboxyMe solutions the determined T1 values compare well to those
obtained for solutions containing EGadMecl. Since the relaxation
time of water protons is in the same order of magnitude for
CarboxyMe and CarboxyMecl it must be assumed that the
galactopyranose unit does not block the Gd(III) site. It is
therefore not effective in limiting the access of bulk water to the
metal site.
[0221] Molecular modeling studies support this hypothesis. The
calculated configurations of EGadMe and CarboxyMe were evaluated.
In EGadMe the galactopyranose unit is placed on top of the
macrocyclic framework, thereby shielding the metal center. When the
galactopyranose unit is cleaved off, the metal site becomes readily
exposed and accessible for bulk water molecules to complete the
Gd(III) coordination sphere. However, with CarboxyMe the
galactopyranose unit is facing away from the macrocyclic unit
instead of being located on top of it. The steric influence of the
.alpha.-methyl groups on the carboxyclic arms seems to prevent the
galactopyranose unit from taking a position on top of the Gd(III)
site. Therefore the metal site is easily accessible for bulk water
molecules in the uncleaved state as much as in the cleaved state,
leading to comparable T1 data for both structures. Furthermore, a
mass spectrum obtained for CarboxyMe reveals that two chloride
anions are coordinated to the molecule that complete the two vacant
Gd(III) coordination sites. In EGadMe these coordination sites are
filled by the galactopyranose unit.
[0222] To determine the efficiency of the galactopyranose unit in
blocking the access of water molecules to Gd(III), thereby
monitoring the water exchange rate of EGadMe, the lifetime of the
fluorescence signal of the corresponding terbium derivative (ETbMe)
was investigated. The fluorescence of the terbium complex is
quenched by H2O, since terbium is strongly coupled to the OH
oscillator. This effect is not observed for the OD-oscillator. As a
consequence, the lifetime of the fluorescence signal is longer in
D.sub.2O than in H.sub.2O. Measuring ETbMe (lex=460 nm, lem=545 nm)
in various H.sub.2O/D.sub.2O mixtures and plotting the resulting
lifetimes vs. the D.sub.2O concentration leads to the number of
water molecules q, that are in fast exchange with the complex (see
Kumar et al., Pure Appl. Chem 65:515-520 (1993); Li et al, J. Am.
Chem. Soc. 117:8132 (1995); Horrocks et al., J. Am. Chem. Soc.
101:334 (1979); Zhang et al., Iorg. Chem. 31:5597 (1992), all of
which are incorporated by reference. For ETbMe a value of q=0.6 was
determined, which was compared to a value of q=1.2 observed for the
tetraaceticacid macrocycle Gd-DOTA (see Lauffer, Chem. Rev. 901-927
(1997). The change in the number of coordinating water molecules is
clearly obvious. For the corresponding cleaved complex, ETbMecl,
the exponential decay of the fluorescence signal was much faster,
indicating a number of q<1. However, attempts to detemine q for
ETbMecl correctly were limited by the time-resolution of the
fluorimeter.
[0223] Assuming a number of 1<q<2 for EGadMecl is in total
agreement with the T1 data observed for EGadMe and EGadMecl and
structural arrangements, where the hydroxy group in EGadMecl is not
tightly coordinating the metal site. Theory predicts that an
increase in q is related to an increase in T1. It can therefore be
assumed that the large difference in T1 of 55% is a synergetic
effect, i.e. effective blocking the access of bulk water to the
metal site by the galactopyranose unit in EGadMe and assuming a
number of water molecules that are in fast exchange with the
complex 1<q<2 in EGadMecl.
Example 4
Synthesis of Gd.sup.3+-BAPTA-DO3A.sub.2 ("CalGad")
[0224] The synthetic scheme for Calgad is shown in FIG. 14.
[0225] Compound 1: 2-Nitroresorcinol (2 g, 12.9 mmol) was dissolved
in 95% ethanol (15 mL), 1 equivalent of NaOH was added slowly.
After the addition the solvent was removed under vacuum and the
resulting solid was redissolved in 4 mL DMF with 1 equivalent of
3-bromopropanol. After heating the solution at 100.degree. C. for 7
hours, the reaction was quenched with a few drops of acetic acid.
After removing the solvent under vacuum, the residue was suspended
in methylene chloride and filtered. Flash chromotography
(CH.sub.2Cl.sub.2/MeOH, 20:1) afforded 1.08 g (42%) of 1.
.sup.1H-NMR (300 MHz, CDCl.sub.3): 2.14(m, 2H,
CH.sub.2CH.sub.2CH.sub.2), 3.94(t, 2H, CH.sub.2OH), 4.26(t,
OCH.sub.2), 6.6(d, 1H, aromatic H), 6.72(d, IH, aromatic H), 7.41
(t, 1H, aromatic H).
[0226] Compound 2: Compound 1 (0.8 g, 3.76 mmol) was dissolved in 8
mL DMF. 1,2Dibromoethane (0.16 mL, 1.88 mmol) and K.sub.2CO3 (0.28
g) was then added and the mixture was heated at 120.degree. C. for
10 h. The reaction was quenched with a few drops of acetic acid and
the solvent was evaporated under vacuum. The residue was purified
by flash chromatography (CH.sub.2Cl.sub.2/MeOH, 20:1) and 0.46 g of
product (55%) was obtained. .sup.1H-NMR (CDCl.sub.3): 2.0(m, 4H,
CH.sub.2CH.sub.2CH.sub.2), 3.85(m, 4H, CH.sub.2OH), 4.25(t, 4H,
CH.sub.2O), 4.46(s, 4H, OCH.sub.2CH.sub.2O), 6.66(m, 4H, aromatic
H), 7.4(t, 2H, aromatic H).
[0227] Compound 3: Compound 2 (0.15 g) was suspended in a mixture
of ethyl acetate (10 mL) and 95% ethanol (10 mL). After adding
Palladium catalyst (Pa/carbon, 10%, 50 mg), the solution was
hydrogenated at 1 atm overnight. The catalyst was filtered off and
the filtrate was concentrated under vacuum. The residue was used
directly for the next step.
[0228] Compound 4: The above residue was mixed with acetonitrile (2
mL), DIEA (0.25 mL, 1.37 mmol) and bromoethylacetate (0.15 mL, 1.37
mmol). The solution was refluxed under argon for 24 h and then
cooled down to RT. Toluene (20 mL) was added to precipitate the
DIEA salt. After filtering off the precipitation, the filtrate was
purified on flash chromotography (CH.sub.2Cl.sub.2/MeOH, 20:1) and
0.15 g of product (61% for 2 steps) was obtained. MS (Electrospray)
m/z (M+H).sup.+, calcd 737 (C.sub.36H.sub.53O.sub.14N.sub.2), obsd
737.6, 759.4 (M+Na).sup.+. .sup.1H-NMR (CDC1.sub.3): 1.25(t, 12H,
CH.sub.3), 2.08(m, 4H, CH.sub.2CH.sub.2CH.sub.2), 3.9(m, 4H,
CH.sub.2OH), 4.05-4.4(m, 24H), 6.62(m, 4H, aromatic H), 7.0(m, 2H,
aromatic H).
[0229] Compound 5: Compound 4 (245 ma, 0.33 mmol),
triphenylphosphine (262 ma, 1 mmol) and carbon tetrabromide (332
ma, Immol) were dissolved in diethyl ether (3 mL). After stirring
at RT for 40 min, flash chromatography (CH.sub.2C1.sub.2 to
CH.sub.2Cl.sub.2/MeOH, 20:1) purification gave 0.19 g of product
(67%). .sup.1H-NMR (CDC1.sub.3): 1.21(t, 12H, CH.sub.3), 2.34(m,
4H, CH.sub.2CH.sub.2CH.sub.2), 3.67(t, 4H, CH.sub.2Br),
4.05-4.34(m, 24H), 6.62(m,4H, aromatic H), 7.0(m, 2H, aromatic
H).
[0230] Compound 6: Compound 5 (42 ma, 49 .mu.mol) was reacted with
cyclen (43 ma, 0.25 mmol) in CHCl.sub.3 (0.5 mL) for 30 hours.
Flash chromatography (CHCl.sub.3/MeOH/NH.sub.3 H.sub.2O 12:4:1)
afforded the product as a clear glass (41 ma, 80%). MS
(Electrospray) m/z (M+H).sup.+, calcd 1046
(C5.sub.2H.sub.89O,.sub.2N,o), obsd 1046.0(M+H).sup.+,
1067.8(M+Na).sup.+, 1089.8(M+2Na--H).sup.+, 523.4(M+2H).sup.2+,
534.4(M+H+Na).sup.2+, .sup.1H-NMR (CDC1.sub.3): 1.2(t, 12H,
CH.sub.3), 2.0(br, 4H, CH.sub.2CH.sub.2CH.sub.2), 2.6-2.85(br.
36H), 4.0-4.4(br, 24H), 6.64(br, 4H. aromatic H), 6.95(br, 2H,
aromatic H).
[0231] Compound 7: Compound 6 (38 ma, 38 ,umol) was mixed with
bromoacetic acid (37 ma, 266 .mu.mol) in H.sub.2O (0.2 mL). Sodium
hydroxide (SN) was slowly added to keep the pH of the solution
above 10. When the pH of the solution reached stable, the reaction
was quenched with small amount of acetic acid. The product was
purified by reverse phase chromatography (LiChroprep RP-18,
CH.sub.3CN/H.sub.2O, 5% -50%) and 38 mg (82%) of white powder was
obtained after lyophilization. MS (Electrospray) m/z (M+H).sup.+,
calcd 1280 (C.sub.56H.sub.83N.sub.10O.sub.24), obsd
1279.4(M-H).sup.-, 639.3(M-2H).sup.2-. .sup.1H-NMR (D.sub.2O):
2.32(br, 4H, CH.sub.2CH.sub.2CH.sub.2), 3.05-3.83(br, 48H), 4.05(s,
8H), 4.27(br, 4H), 4.7(s, 4H), 6.8(d, 2H, aromatic H), 6.95(d, 2H,
aromatic H), 7.4(t, 2H, aromatic H)
[0232] Gd3.sup.+-complex of compound 7: The above ligand (compound
6, 16.5 ma, 12.9 .mu.mol) was dissolved in H.sub.2O (0.5 mL)
containing GdCl3 (10.6 ma, 28.4 .mu.mol). NaOH (1N) was slowly
added to keep the pH around 5-6. The pH of the solution reached
stable within 2 h indicating the completion of the reaction. The
mixture was passed through a column packed with the Chelex resin
(Biorad, Chelex 100, Na.sup.+ form) and the fractions containing
the product were further purified by reverse phase chromatography
(LiChroprep RP-18, CH.sub.3CN/H.sub.2O, 5%-50%). The final product
was obtained as a white powder (17 ma, 81%). MS (Electrospray) m/z
(M+H).sup.+: calcd 1583-1597
(C.sub.56H.sub.78N.sub.10O.sub.24Gd.sub.- 2, 1590 highest
abundance). obsd (the peak of the highest abundance) 1611.4
(M-2H+Na).sup.-, 1633.2(M3H+2Na).sup.-, 804.8(M-3H+Na).sup.2-,
793.6(M-2H).sup.2-.
[0233] The Effect of Ca2.sup.+ on the Relaxivity of the Complex
[0234] In the presence of Ca.sup.2+, R=5.53 mM.sup.-1
sec.sup.-1
[0235] In the absence of Ca.sup.2+, R=3.03 mM.sup.-1 sec.sup.-1
[0236] The Effect of pH on the Relaxivity of the Complex
[0237] The T1 of the Gd3.sup.+-complex (0.4 mM in the buffer
containing 100 mM KC1, 10 mM MOPS, 2 mM K.sub.2H.sub.2EGTA or 2mM
K.sub.2CaEGTA) was measured under different pH. Changing pH from
6.80 to 7.40 in 0.2 pH unit steps had minimum effects on the
relaxivity of the complex, either in the presence or in the absence
of Ca.sup.2+.
1 pH 6.80 7.00 7.20 7.40 T1 (msec, K.sub.2H.sub.2EGTA) 600 605 604
608 T1 (msec, K.sub.2CaEGTA) 390 393 394 397
[0238] The Effect of Mg.sup.2+ on the Relaxivity of the Complex
[0239] The T1 of the Gd.sup.3+-complex (0.4 mM in the buffer
containing 132 mM KC1, 10 mM MOPS, 1 mM K.sub.2H.sub.2EGTA, pH
7.20) was measured. Changing Mg.sup.2+ concentration from 0 to 20
mM had minimum effects on the relaxivity of the complex.
2 Mg.sup.2+ (mM) 0 1 2 5 10 20 T1 (msec) 607 602 601 609 607
610
Example 5
MR Imaging of Xenopus laevis Embryos
[0240] In vivo experiments using EGadMe were carried out in Xenopus
laevis embryos. To permit an independent tracking of which cells
were descendants of the .beta.-gal injected cell, mRNA for
nuclearly localized GFP (nGFP) was co-injected. Since the first
mitosis approximately defines the plane dividing the future left
and right sides of the embryo, these injections introduce EGadMe to
both sides of the embryo; while .beta.-galactosidase and nGFP will
only be expressed in a subset of the cells located primarily on one
side of the animal. Therefore, each embryo will contain cells in
which EGadMe is expected to be enzymatically processed and other
cells with unprocessed contrast agent.
[0241] Synthetic mRNA was transcribed using the SP6 mMessage
Machine Kit (Ambion, Austin, Tex.) according to the manufacturer's
protocol. Embryos at the 2-cell stage in 1.times. Marc's Modified
Ringer's Solution (MMR) were injected with 4 nL of 400 mM EGadMe to
both cells. One cell was injected with either 4 nL 0.5 .mu.g/.mu.l
.beta.-galactosidase mRNA, 0.025 .mu.g/.mu.l nGFP mRNA, or water.
This yields approximately 13,000 copies of the 3,000 bp sequence
per cell in the 100,000 cells stage animal, this is in the range of
a high copy endogenous gene. The bolus volume was controlled using
a pulsed constant-pressure picoinjector system (PLI-100, Medical
Systems, Greenvale, N.Y.). Additional embryos were injected with
water alone as a control for viability of the given batch of
embryos. Embryos were incubated at 16.degree. C. following
injections. Twenty minutes to one hour after injection, embryos
were moved to 0.1.times.MMR. After gastrulation the embryos were
moved to room temperature for 24 hours prior to imaging.
[0242] Images were acquired using a Bruker AMX500 (Bruker
Instruments, Billerica, Mass.) microimaging system with a wide-bore
(89 mm) 11.7 T magnet and a laboratory-built solenoid imaging
probe. Multiple embryos were imaged simultaneously in a 2.5 mm
diameter quartz tube containing 0.1.times.MMR solution. In order to
minimize embryo movement during imaging, MS-222 anaesthestic was
added to a final concentration of 0.01% (Finquel, Argent, Redmond,
Wash.) and the embryos were maintained at 15.degree. C. A 3D
spin-echo sequence was used to acquire images with
T.sub.R/T.sub.E=400/21 ms, 512.times.128.times.128 image points,
and a field of view of 1.4.times.0.4.times.0.4 cm (acquisition
time=3 hours 45 minutes). The data was zero-filled to
512.times.256.times.256 points before Fourier Transformation
yielding a final voxel size of 27.times.16.times.16 .mu.m.
[0243] Images were processed using VoxelView (Vital Images. Inc.,
Minneapolis, Minn.) on a Silicon Graphics O2 workstation. To make
water transparent, the opacity was adjusted to zero for those
voxels with the signal intensity expected of Ringer's solution
(region surrounding the embryos).
[0244] .beta.-galactosidase images were obtained on embryos fixed
for 15 minutes in 250 mM Na cacodylate, 1% glutaraldehyde, and
washed for 5 minutes with CMAP [7.2 mM Na.sub.2HPO.sub.4, 2.8 mM
NaH.sub.2PO.sub.4, 150 mM NaCl, 1 mM MgCl.sub.2, 3 mM
K.sub.3Fe(CN)6, 3 mM K.sub.4Fe(CN).sub.6 3H.sub.2O], followed by
staining for 60 minutes at 37.degree. C. in 0.027% X-gal and 0.1%
Triton X-100. Embryos were photographed using a Zeiss Stemi SV 11
equipped with a Prog/Res/3012 camera (Kontron Elektronik, Eching,
Germany) controlled by a Roche Image Analysis System and processed
with Photoshop 4.0 (Adobe, San Jose, Calif.).
[0245] Fluorescence images were obtained with a Zeiss Axioplan
epifluorescence microscope (5.times. magnification) equipped with a
Hammamatsu C2400 SIT camera, and an image processor (Imaging
Technology 151, Bedford, Mass.) controlled by the VidIm software
package (Belford, Stollberg, and Fraser, unpublished data) using
boxcar averaging of 16 frames. Acquired images were rendered using
Photoshop 4.0 (Adobe, San Jose, Calif.).
[0246] Images of the embryos (collected using a three-dimensional,
spin-echo, T.sub.1-weighted pulse sequence) revealed the expected
increase in the signal intensity for those regions of the embryo
containing the mRNA for .beta.-galactosidase (FIG. 17). This
increase depends on the presence of EGadMe; embryos injected with
mRNA alone (no contrast agent) resembled those injected with water
alone. Comparison of the two embryos in FIG. 17 illustrates the
significant signal enhancement achieved in those cells with both
EgadMe and .beta.-galactosidase.
[0247] The difference between the cleaved and uncleaved states of
EGadMe is clearly demonstrated by comparing regions within a single
embryo. FIG. 18 compares the dorsal views of a living embryo by
fluorescence (nGFP; FIG. 18A) and MRI (FIG. 18B). The embryo was
subsequently fixed and stained for .beta.-gal activity (FIG. 18C).
More intense signal is evident on the right side of the embryo by
all techniques and there is a good correlation between the regions
showing nGFP-fluoresence, X-gal staining for .beta.-galactosidase,
and regions of intense signal in the MR image. The MRI shows both
the region of heavy labeling on the right side of the embryo and a
region of sparse labeling on the left side that are confirmed in
the .beta.-galactosidase staining of the fixed embryo (FIG. 18C).
Autofluorescence in the embryo and the limited depth of penetration
by light microscopy make it difficult to perform a point to point
comparision between the nGFP-fluoresence, the .beta.-galactosidase
labeling, and the 3D MR images. There is a 57% enhancement of MRI
signal intensity in the somites expressing .beta.-galactosidase
compared to the equivalent somites not expressing
.beta.-galactosidase (signal intensity is approximately 10-15 times
the noise level in the image).
[0248] These results document the ability of EGadMe to render
three-dimensional images of marker expression. The present results
emphasize that EgadMe does not serve merely as a lineage tracer but
is sensitive to beta-galactose activity.
Example 6
MRI Detection of Gene Expression in Xenopus laevis Embryos
[0249] To test the ability of this approach to detect in vivo gene
expression (i.e. transcription and translation), both cells of
Xenopus embryos at the two cell stage were injected with EgadMe and
then one of the blastomeres was injected with a DNA construct
carrying the lacZ gene. Comparison of the MR image for a live
embryo (FIG. 19A) with the image of the same embryo after fixation
and staining for .beta.-galactosidase activity (FIG. 19B) shows
good correlation between areas of intense MRI signal and those
staining positive for the enzyme after fixation. Both images show
strong labeling in the stripe of endoderm running the length the
embryo and in two dinstinct spots located ventral to the cement
gland (arrows). Both techniques also detect .beta.-galactosidase
expression in the eye, regions of the head, and ventral
regions.
[0250] Expression of injected DNA is known to be mosaic; injected
DNA is not expressed in every cell in which it is introduced.
Therefore, these results emphasize that EgadMe does not serve
merely as an inert lineage tracer but is sensitive to gene
expression. Furthermore, these images demonstrate the advantages of
MRI for visualizing deep tissue. These data demonstrate that MR
imaging with EGadMe offers both the contrast and spatial resolution
to clearly detect positive domains at cellular resolution in living
animals.
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