Note: Descriptions are shown in the official language in which they were submitted.
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MAGNETIC RESONANCE IMAGING AGENTS
FOR THE DELIVERY OF THERAPEUTIC AGENTS
This application is a continuing application of U.S.S.N.s 60/063,328 filed
October 27, 1997;
09/179,927, filed October 27, 1998; and~601203,224, filed June 6, 2000.
FIELD OF THE INVENTION
The invention relates to novel magnetic resonance imaging contrast agents and
methods of delivering
therapeutically active substances.
BACKGROUND OF THE INVENTION
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.
The Image.
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~ & T2,
govern the rate at which the
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magnetization can be accurately measured. T1 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~ values)
will recover all of their signal amplitude between successive scans.
The measured intensities in the final image will accurately reflect the spin
density (i.e. water content).
Regions with long T~ 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 TZ
(spin-spin relaxation time) result in changes in the signal linewidth (shorter
TZ 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, values and/or long T2
values are preferentially
enhanced so called T~ -weighted and TZ weighted imaging protocol.
MRI Contrast Agents.
There is a rapidly growing body of literature demonstrating the clinical
effectiveness of paramagnetic
contrast agents (currently S 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.
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, and TZ
relaxation times of nearby (r6 dependence) spins. Some paramagnetic ions
decrease the T~ without
causing substantial linebroadening (e.g. gadolinium (III), (Gd3+)), while
others induce drastic
linebroadening (e.g. superparamagnetic iron oxide). The mechanism of T~
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).
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For example, regions associated with a Gd3+ 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 if the experiment is short (i.e. T, weighted image).
Localized TZ 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. T2-weighted image). The lanthanide atom Gd3+ is by
the far the most
frequently chosen metal atom for MRI contrast agents because it has a very
high magnetic moment
(u2 = 63BM2), and a symmetric electronic ground state, (S8). Transition metals
such as high spin
Mn(II) and Fe(III) are also candidates due to their high magnetic moments.
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.
Diethylenetriaminepentaacetic (DTPA) chelates and thus acts to detoxify
lanthanide ions. The stability
constant (K) for Gd(DTPA) ~~ is very high (IogK = 22.4) and is more commonly
known as the formation
constant (the higher the IogK, the more stable the complex). This
thermodynamic parameter indicates
the fraction of Gd3+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 (kf/kd).
The water soluble Gd(DTPA)Z-
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.
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. Patent 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).
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
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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).
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 (IogK = 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.
Previous work has resulted in MRI contrast agents that report on physiologic
or metabolic processes
within a biological or other type of sample. As described in U.S. Patent No.
5,707,605, PCT
US96/08549, and U.S.S.N. 09/134,072, MRI contrast agents have been constructed
that allow an
increase in contrast as a result of the interaction of a blocking moiety
present on the agent with a
target substance. That is, in the presence of the target substance, the
exchange of water in one or
more inner sphere coordination sites of the contrast agent is increased,
leading to a brighter signal; in
the absence of the target substance, the exchange of water is hindered and the
image remains dark.
Thus, the previous work enables imaging of physiological events rather than
just structure.
However, it would be a further improvement to be able to deliver therapeutic
agents and follow their
delivery via MRI. Accordingly, it is an object of the present invention to
provide MRI contrast or
enhancement agents which allow the visualization and detection of the delivery
of therapeutic agents
within an animal, tissue or cells.
SUMMARY OF THE INVENTION
In accordance with the above objects, the invention provides MRl agents
comprising a chelator and a
paramagnetic metal ion that is coordinatively saturated by the chelator and a
therapeutic blocking
moiety, wherein said therapeutic blocking moiety is covalently attached to
said chelator such that the
rapid exchange of water in at least one coordination site is hindered, wherein
said therapeutic blocking
moiety comprises at least a therapeutically active agent, wherein the exchange
of water in at least one
coordination site is increased upon delivery of said therapeutically active
agent to its physiological
target resulting in a therapeutic effect.
In an additional aspect, the invention provides MRI agents having the formula:
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Rt t
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~, X2, X3 and X4 are -OH, -COO-, -CH20H -CHZCOO-, a substitution group, a
therapeutic blocking
moiety or a targeting moiety;
R, - R,Z are hydrogen, a substitution group, a therapeutic blocking moiety or
a targeting moiety;
wherein at least one of X~-X4 and R~ - R~2 is a therapeutic blocking moiety.
In a further aspect, the MRI agents have the formula:
M
R13 Rzo
Rie R~s
K
N N
N
Rts ~Rt~ l9 R2t
J
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);
H, I, J, K and L are -OH, -COO-, -CH20H -CH2C00-, a substitution group, a
therapeutic blocking
moiety or a targeting moiety;
R,3 - Rz, are hydrogen, a substitution group, a therapeutic blocking moiety or
a targeting moiety;
wherein at least one of H, I, J, K and L nd R,3 - R2, is a therapeutic
blocking moiety.
In an additional aspect, the invention provides methods of treating a disorder
associated with a
therapeutically active agent comprising administering an MRI agent of the
invention to produce a
magnetic resonance image of said cell, tissue or patient.
5
X1
Rt R2 Xz
~'~~_ _/ Rto
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In a further aspect, the invention provides methods of magnetic resonance
imaging of a cell, tissue or
patient comprising administering an MRI agent to a cell, tissue or patient and
rendering a magnetic
resonance image of said cell, tissue or patient.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A, 1B and 1C depict some embodiments of the invention. Figure 1A
depicts the situation
wherein the drug 20 provides the coordination atom to the metal ion 10 (shown
as Gd, herein) in the
chelate 5, although the coordination atom may be contributed by a linker, a
coordination site barrier, or
a cleavage site (figure 1 B). Upon exposure to the physiological target 30, a
conformational change
occurs, allowing the rapid exchange of water. Figure 1 B is similar, except a
cleavage site 40 is used,
which upon exposure to a cleavage agent cleaves off the drug 20. Figure 1 C
depicts the use of a
coordination site barrier 50, which in the absence of a cleavage agent such as
an enzyme hinders the
exchange of water, However, after cleavage, the target 30 is able to interact
with the drug 20.
Although not depicted, a targeting moiety may also be included in any of the
embodiments herein; for
example, a targeting moiety used to target the drug may attached to the drug
20 in Figure 1A, either
between the metal chelate and the drug or as a "terminal group" to the drug.
Alternatively, the
targeting moiety may be attached to the chelate 5 in another position.
Similarly, a targeting moiety
may be attached to the Figures 1 B and 1 C embodiments between the cleavage
site 40 and the drug
20, or as a terminal group.
Figures 2A, 2B and 2C depict the use of coordination site barriers 60. A
variety of conformations may
be utilized as generally described for Figure 1, including the use of
targeting groups. It should also be
noted that additional cleavage sites may be put into the system, for example
to cleave the coordination
site barrier 60 from the drug 20 in Figure 2C.
Figures 3A, 3B, 3C, 3D, 3E, 3F, and 3G depict several of the possible
conformations of the dimer
embodiments. Boxes represent chelators, with M being the paramagnetic metal
ions. Figures 3A and
3B represent two possible duplex conformations. In Figure 3A, R~~ can be a
linker, such as described
herein as R13 or Rzs, a cleavable moiety such as an enzyme substrate such as a
peptide, or a
therapeutic blocking moiety that will preferentially interact with the target
molecule. RzB, which may or
may not be present depending on R2~, is a coordination site barrier similar to
Ra3 or a therapeutic
blocking moiety. Figure 3B has Rz8 therapeutic blocking moieties or
coordination site barriers attached
via an Rz~ group to two chelators. Figure 3C is similar to Figure 3A, but at
least one of the R2, groups
must be a cleavable moiety. Figure 3D depicts the case where two therapeutic
blocking moieties or
coordination site barriers are present; if Rz, is a blocking moiety, Ra8 need
not be present. Figure 3E is
similar to 3B but the chelators need not be covalently attached. Figures 3F
(single MRI agents) and
and 3G (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. Figures 3H and
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31 depict polymer 10 as defined herein being attached to either single MRI
agents (3H) or duplex MRI
agents (31).
Figures 4A, depicts a number of different drugs that can be used as
therapeutic blocking moieties.
As will be appreciated by those in the art, and as described herein, any
number of functional groups
(either endogeneous to the structure or added exogeneously) can be used to
attach these drugs, and
their derivatives, to the chelates. Figure 4A is docetaxel; 4B is etoposide;
4C is irinotecan; 4D is
paclitaxel; 4E is tenoposide; 4F is is topotecan; 4G is vinblastine (note its
derivative, vincristine); 4H is
vindesine.
Figure 5 depicts a preferred structure comprising a DOTA chelate, complexed
with Gd+3, and
comprising a doxorubicin therapeutic blocking moiety.
Figures 6A and 6B depict preferred structures comprising a DOTA chelate,
complexed with Gd+3, and
comprising a taxol therapeutic blocking moiety attached in two separate
locations on the taxol. R1 in
this case can be a variety of linkers, including esters, amides, cleavable
linkers (particularly cleavable
peptides such as DEVD), etc. In addition, while the therapeutic blocking
moiety is shown attached to
an "arm" of the DOTA chelate, attachment at the macrocycle is also done, as is
outlined herein.
Similarly, other moieties such as targeting moieties and other R substitution
groups may be used.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides magnetic resonance imaging (MRI) contrast
agents which can detect
the delivery of therapeutically active agents as a result of an interaction of
a physiological target agent
and the MRI agents of the invention. 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, upon the delivery of the
therapeutically active agent.
The imaging of the delivery of the therapeutically active agent can occur in
two basic ways. In one
embodiment, it is the actual interaction of the therapeutically active agent
with its target that causes
the MRI agent to turn "on", as described below; that is, as a result of the
presence of the physiological
target, the MRI agent undergoes a reorganization that can include the cleavage
of the therapeutically
active agent off the remainder of MRI agent, causing an increase in signal as
a result of an increase in
the exchange of water in an inner coordination site. Alternatively, it is the
delivery event of the active
agent that is imaged; that is, the therapeutically active agent is cleaved off
of the MRI agent as a result
of exposure to a cleavage agent such as a protease, freeing the
therapeutically active agent to interact
with its target. In general, it is the former that is discussed herein,
although as will be appreciated by
those in the art, either possibility can occur.
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Viewed simplistically, this "trigger" mechanism, whereby the contrast agent is
"turned on" (i.e.
increases the relaxivity) by the delivery of the therapeutically active agent,
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 therapeutic
blocking moiety on the MRI agent. Thus, in the presence of the therapeutically
active agent, 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 therapeutic 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 therapeutic
blocking moiety is present,
there is little if any net enhancement of the imaging signal by the metal ion
complexes of the invention.
However, when the therapeutically active agent is removed (either as a result
of an interaction with its
physiological target or by cleavage), this efFectively frees 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
reversibly 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 delivery of the therapeutically active
agent.
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 display an
increase the MRI signal by
at least 2 to 5% upon delivery of the therapeutically active agent as compared
to the signal when the
therapeutically active agent is present on the MRI agent. Signal enhancement
of 2 to 90% is
preferred, and 10 to 50% is more preferred for each coordination site made
available by the delivery of
the therapeutically active agent. That is, when the therapeutic blocking
moiety occupies two or more
coordination sites, the release of the therapeutic blocking moiety can result
in a significant increase in
the signal or more as compared to a single coordination site.
It should be understood that even in the presence of the therapeutic blocking
moiety, at any particular
coordination site, there will be a dynamic equilibrium for one or more
coordination sites as between a
coordination atom of the therapeutic 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 delivery
of the therapeutically
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active agent, the therapeutic 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.
See generally U.S. Patent Nos. 5,707,605 and 5,980,862; U.S.S.N.s 09/405,046,
filed September 27,
1999; 60/287,619, filed May 26, 2000; 09/179,927, filed October 27, 1998;
60/201,817, filed May 4,
2000; 60/203,224, filed June 6, 2000; and 60/202,108, filed May 4, 2000, all
of which are expressly
incorporated by reference herein.
Accordingly, the MRI agents of the invention comprise a metal ion complex. The
metal ion complexes
of the invention comprise a paramagnetic metal ion bound to a complex
comprising a chelator and a
therapeutic 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
(Yt+3 or Yt(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 (u2
= 63BM2), a symmetric electronic ground state (S8), and its current approval
for diagnostic use in
humans.
In addition to the metal ion, the metal ion complexes of the invention
comprise a chelator and a
therapeutic 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 therapeutic
blocking moieties in
coordination sites of the chelator, which upon delivery of the therapeutically
active agent vacate 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 therapeutic
moeity. However, for some
applications, e.g. use in experimental animals, tissue samples, etc., 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
therapeutic 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
therapeutic 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.
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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).
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
therapeutic 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 therapeutic 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 therapeutic blocking moieties. Thus, alternative
embodiments utilize Gd(lll)
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 therapeutic
blocking moiety either occupying or blocking the remaining sites. It should be
appreciated that the
exact structure of the chelator and therapeutic 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
therapeutic blocking moiety
impedes the rapid exchange of water at the site.
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
Jackets, 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.
Patent 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
incorporated 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
therapeutic blocking moiety.
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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:
Structure 1
HOO ~ JCOOH
N N
'N\ /N'
HOOCI(/ ~ 1ICOOH
By "substituted DOTA" herein is meant that the DOTA may be substituted at any
of the following
positions, as shown below:
Structure 2
O R
R N N R
'N N' R
R ~i~ II~. R
HOOC R COOH
Suitable R substitution groups include a wide variety of groups, as will be
understood by those in the
art and as defined below. For example, suitable substitution groups include
substitution groups
disclosed for DOTA and DOTA-type compounds in U.S. Patent 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, halogens such as
chlorine, bromine and fluorine; amines; amides; esters; ethers; glycols,
including ethylene glycols;
hydroxy groups; aldehydes; alcohols; carboxylic acids; nitro groups; sulfonyl;
silicon moieties; sulfur
containing moieties; phosphorus containing moieties; carbonyl groups;
targeting moieties; therapeutic
blocking moieties and chemical functional groups. 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 therapeutic blocking moiety and
the other R groups are
hydrogen.
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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.
Additional suitable heterocyclic substituted rings are depicted in U.S. Patent
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.
Patent 5,358,704, expressly incorporated by reference. These ring structures
may be similarly
substituted.
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.
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 (-NHzR), secondary
(-NHR~), or tertiary (-NR3). 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.
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.
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; amides; esters;
ethers; glycols, including
ethylene glycols; hydroxy groups; aldehydes; alcohols; carboxylic acids; vitro
groups; sulfonyl; silicon
moieties; sulfur containing moieties; phosphorus containing moieties; carbonyl
and other alkyl and aryl
groups as defined herein, targeting moieties, therapeutic blocking moieties
and chemical functional
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WO 01/82795 PCT/USO1/14665
groups. Thus, arylalkyl and hydroxyalkyl groups are also suitable for use in
the invention. Preferred
substitution groups include alkyl amines and alkyl hydroxy.
By "phosphorus containing moieties" herein is meant compounds containing
phosphorus, including,
but not limited to, phosphines and phosphates, including the -PO(OH)(RZS)2
group. The phosphorus
may be an alkyl phosphorus; for example, DOTEP utilizes ethylphosphorus as a
substitution group on
DOTA. Ra5 may be alkyl, substituted alkyl, hydroxy. A preferred embodiment has
a -PO(OH)2RZs
group.
By "amino groups" or grammatical equivalents herein is meant -NH2, -NHR and -
NRz groups, with R
being as defined herein.
By "nitro group" herein is meant an -NO~ group.
By "sulfur containing moieties" herein is meant compounds containing sulfur
atoms, including but not
limited to, thia-, thio- and sulfo- compounds, thiols (-SH and -SR), and
sulfides (-RSR-).
By "silicon containing moieties" herein is meant compounds containing silicon.
By "ether" herein is meant an -O-R group. Preferred ethers include alkoxy
groups, with -O-(CH~)~CH3
and -O-(CH~)4CH3 being preferred.
By "ester" herein is meant a -COOR group.
By "halogen" herein is meant bromine, iodine, chlorine, or fluorine. Preferred
substituted alkyls are
partially or fully halogenated alkyls such as CF3, etc.
By "aldehyde" herein is meant -RCOH groups.
By "alcohol" herein is meant -OH groups, and alkyl alcohols -ROH.
By "amido" herein is meant -RCONH- or RCONR- groups.
By "ethylene glycol" or "(poly)ethylene glycol" herein is meant a -(O-CHZ
CHZ)~ group, although each
carbon atom of the ethylene group may also be singly or doubly substituted,
i.e. -(O-CRz CRZ)~ , with
R as described above. Ethylene glycol derivatives with other heteroatoms in
place of oxygen (i.e. -(N-
CHa CHZ)~ or -(S-CHI CH2)~ , or with substitution groups) are also preferred.
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By the term "targeting moiety" herein is meant a functional group that serves
to target or direct the
complex to a particular location or association, i.e. a specific binding
event. Thus, for example, a
targeting moiety may be used to target a molecule to a specific target protein
or enzyme, or to a
particular cellular location, or to a particular cell type. Suitable targeting
moieties include, but are not
limited to, polypeptides, nucleic acids, carbohydrates, lipids, hormones
including proteinaceous and
steroid hormones, growth factors, receptor ligands, antigens and antibodies,
and the like. For
example, as is more fully outlined below, a therapeutically active agent such
as the cobalt compounds
outlined below may include a targeting moiety to specifically bind a
particular protein. Alternatively, as
is more fully outlined below, the MRI agents of the invention may include a
targeting moiety to target
the agents to a specific cell type such as tumor cells, such as a transferrin
moiety, since many tumor
cells have significant transferrin receptors on their surfaces. Similarly, a
targeting moiety may include
components useful in targeting the MRI agents or the therapeutically active
agents (if released) to a
particular subcellular location. As will be appreciated by those in the art,
the localization of proteins
within a cell is a simple method for increasing effective concentration. For
example, shuttling a drug
into the nucleus confines them to a smaller space thereby increasing
concentration. Finally, the
physiological target may simply be localized to a specific compartment, and
the drugs must be
localized appropriately.
Thus, suitable targeting sequences include, but are not limited to, binding
sequences capable of
causing binding of the moiety to a predetermined molecule or class of
molecules while retaining
bioactivity of the expression product, (for example by using enzyme inhibitor
or substrate sequences to
target a class of relevant enzymes); sequences signaling selective
degradation, of itself or co-bound
proteins; and signal sequences capable of constitutively localizing the
candidate expression products
to a predetermined cellular locale, including a) subcellular locations such as
the Golgi, endoplasmic
reticulum, nucleus, nucleoli, nuclear membrane, mitochondria, chloroplast,
secretory vesicles,
lysosome, and cellular membrane; and b) extracellular locations via a
secretory signal. Particularly
preferred is localization to either subcellular locations.
In some embodiments, the targeting moiety replaces a coordination atom,
although this is not
generally preferred. 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.
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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.
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.
In a preferred embodiment, the targeting moiety is an antibody. The term
"antibody" includes antibody
fragments, as are known in the art, including Fab Fab2, 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.
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)].
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. Patent No. 4,816,567),
wherein substantially
CA 02407450 2002-10-29
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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.
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. Patent 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).
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.
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 13 May 1993, and in Traunecker et al., EMBO J.
10:3655-3659 (1991 ).
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
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WO 01/82795 PCT/USO1/14665
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).
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. Patent No.
4,676,980], and for
treatment of HIV infection [VllO 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.
Patent No. 4,676,980.
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.
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).
In one embodiment, antibodies against virus or bacteria can be used as
targeting moieties. As will be
appreciated by those in the art, antibodies to any number of viruses
(including orthomyxoviruses, (e.g.
influenza virus), paramyxoviruses (e.g respiratory syncytial virus, mumps
virus, measles virus),
adenoviruses, rhinoviruses, coronaviruses, reoviruses, togaviruses (e.g.
rubella virus), parvoviruses,
poxviruses (e.g. variola virus, vaccinia virus), enteroviruses (e.g.
poliovirus, coxsackievirus), hepatitis
viruses (including A, B and C), herpesviruses (e.g. Herpes simplex virus,
varicella-zoster virus,
cytomegalovirus, Epstein-Barr virus), rotaviruses, Norwalk viruses,
hantavirus, arenavirus, rhabdovirus
(e.g. rabies virus), retroviruses (including HIV, HTLV-I and -II),
papovaviruses (e.g. papillomavirus),
polyomaviruses, and picornaviruses, and the like), and bacteria (including a
wide variety of pathogenic
and non-pathogenic prokaryotes of interest including Bacillus; Vibrio, e.g. V.
cholerae; Escherichia,
e.g. Enterotoxigenic E. coli, Shigella, e.g. S. dysenteriae; Salmonella, e.g.
S. typhi; Mycobacterium
e.g. M. tuberculosis, M. leprae; Clostridium, e.g. C. botulinum, C. tetani, C,
difficile, C.perfringens;
Cornyebacterium, e.g. C. diphtheriae; Streptococcus, S. pyogenes, S.
pneumoniae; Staphylococcus,
e.g. S, aureus; Haemophilus, e.g. H. influenzae; Neisseria, e.g. N.
meningitidis, N. gonorrhoeae;
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Yersinia, e.g. G. IambIiaY. pestis, Pseudomonas, e.g. P. aeruginosa, P.
putida; Chlamydia, e.g. C.
trachomatis; Bordetella, e.g. 8. pertussis; Treponema, e.g. T. palladium; and
the like) may be used.
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,
IL-4, 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-a and TGF-(3), 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.
In a preferred embodiment, the targeting moiety is a carbohydrate. By
"carbohydrate" herein is meant
a compound with the general formula Cx(H20)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. Patent No.
5,554,386, hereby incorporated
by reference in its entirety.
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
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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.
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.
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.
In a preferred embodiment, the targeting moiety is al! 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); Baldin et
al., EMBO J. 9:1511
(1990); Watson et al., Biochem. Pharmacol. 58:1521 (1999); Schwarze et al.,
TIPS (2000) 21:45; and
Lindgren, TIPS 21:99 (2000); all of which are incorporated by reference.
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-f3 nuclear localization
signal (ARRRRP); NFKB p50 (EEVQRKRQKL; Ghosh et al., Cell 62:1019 (1990);
NFtcB 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.
In a preferred embodiment, targeting moieties for the hepatobiliary system are
used; see U.S. Patent
Nos. 5,573,752 and 5,582,814, both of which are hereby incorporated by
reference in their entirety.
Another class of suitable substitution groups are chemical functional groups
that are used to add the
components of the invention together, as is more fully outlined below. Thus,
in general, the
components of the invention are attached through the use of functional groups
on each that can then
19
CA 02407450 2002-10-29
WO 01/82795 PCT/USO1/14665
be used for attachment. Preferred functional groups for attachment are amino
groups, carboxy
groups, oxo groups and thiol groups. These functional groups can then be
attached, either directly or
indirectly through the use of a linker. Linkers are well known in the art; for
example, homo-or hetero-
bifunctional linkers as are well known (see 1994 Pierce Chemical Company
catalog, technical section
on cross-linkers, pages 155-200, incorporated herein by reference). Preferred
linkers include, but are
not limited to, alkyl groups (including substituted alkyl groups and alkyl
groups containing heteroatom
moieties), with short alkyl groups, esters, amide, amine, epoxy groups,
nucleic acids, peptides and
ethylene glycol and derivatives being preferred.
The substitution group may also be hydrogen or a therapeutic 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: Structure 3
HOOC~ ~COOH
HOOC~N~N~N~COOH
HOOC
By "substituted DPTA" herein is meant that the DPTA may be substituted at any
of the following
positions, as shown below:
Structure 4
R R
R R
HOOC~ ~COOH
N N\ 'COON
HOOC~ ~N
R R ~R R R
HOOC
See for example U.S. Patent No. 5,087,440.
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.
In an alternative embodiment, when the metal ion is Gd(III), a preferred
chelator is 1,4,7,10-
tetraazacyclododecane-N,N',N",N"'-tetraethylphosphorus (DOTEP) or substituted
DOTEP (see U.S.
Patent No. 5,188,816). DOTEP has the structure shown below:
CA 02407450 2002-10-29
WO 01/82795 PCT/USO1/14665
Structure 5
0 0
II II_
CH3CH2- ~ ~ ~ ~ ~ CH2CH3
N N
OH OH
O O
N N
CH3CH2 II~ ~~I-CHZCH3
IH IH
DOTEP may have similar R substitution groups as outlined above.
Other suitable Gd(III) chelators are described in Alexander, supra, Jackets,
supra, U.S. Patent 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.
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. Patent Nos. 4,885,363, 5,358,704, and
5,262,532, all which
describe chelators suitable for Fe(III).
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. Patent Nos. 4,885,363, 5,358,704, and 5,262,532.
When the paramagnetic ion is Yt(III), appropriate chelators will have less
than 7 or 8 coordination
atoms, since Yt(III) is capable of binding 8 or 9 coordination atoms. Suitable
chelators for Yt(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. Patent No.
4,885,363 and others,
as outlined above.
When the paramagnetic ion is Dy+3 (Dy(III)), appropriate chelators will have
less than 7 or 8
coordination atoms, since Dylll is capable of binding 8 or 9 coordination
atoms. Suitable chelators are
known in the art, as above.
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In a preferred embodiment, the chelator and the therapeutic blocking moiety
are covalently linked; that
is, the therapeutic 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.
In an alternative embodiment, the chelator and the therapeutic blocking moiety
are not covalently
attached. In this embodiment, the therapeutic 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 therapeutic blocking moiety has a higher affinity for the
target substance than for
the metal ion. Accordingly, in the presence of either a cleavage agent or the
physiological target, the
therapeutic 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.
What is important is that the metal ion complex, comprising the metal ion, the
chelator and the
therapeutic blocking moiety, is not readily able to rapidly exchange water
molecules when the
therapeutic moeities are in the inner coordination sphere of the metal ion,
such that in the presence of
the therapeutic blocking moiety, there is less or little substantial image
enhancement.
By "therapeutic blocking moiety" or grammatical equivalents herein is meant a
moiety with several
essential functions. First, some component of the therapeutic blocking moiety
must be capable of
substantially inhibiting the exchange of water in at least one inner
coordination site of the metal ion of
the metal ion complex. Second, some component of the therapeutic blocking
moiety must be capable
of effecting a therapeutic effect, i.e. altering the function of its
physiological target substance. In
addition, a further requirement is that as a result of either the interaction
of the therapeutic blocking
moiety with the physiological target substance or as a result of the action of
a separate enzyme such
as a protease on a cleavage site present in the therapeutic blocking moiety,
the exchange of water in
at least one inner coordination site of the metal ion is increased. As is more
fully described below, this
is generally done as a result of a cleavage of some or all of the therapeutic
blocking moiety off the
chelator, although other types of interactions can be utilized as well. As is
more fully described below,
each of these functions may be accomplished by a single component, or multiple
components are
used, together forming the therapeutic blocking moiety. That is, for example,
the therapeutically active
agent may provide the coordination atom(s). Furthermore, as is more fully
outlined below, the
therapeutic blocking moiety may comprise a targeting moiety to allow targeting
of the drug moiety to a
particular target. Finally, therapeutic blocking moieties may comprise one or
more linker groups to
allow for correct spacing and attachment of the components of the therapeutic
blocking moiety as
needed.
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Accordingly, a therapeutic blocking moiety can comprise one or more several
components, as outlined
herein. At a minimum, a therapeutic blocking moiety comprises a
"therapeutically active agent" or
"drug moiety" capable ofi causing a therapeutic effect, that is, it alters a
biological function of a
physiological target substance. As outlined below, this drug moiety may or may
not provide the
coordination atoms) of the therapeutic blocking moiety. By "causing a
therapeutic effect" or
"therapeutically effective" or grammatical equivalents herein is meant that
the drug alters the biological
function of its intended physiological target in a manner sufficient to cause
a therapeutic and
phenotypic effect. By "alters" or "modulates the biological fiunction" herein
is meant that the
physiological target undergoes a change in either the quality or quantity of
its biological activity; this
includes increases or decreases in activity. Thus, therapeutically active
agents include a wide variety
of drugs, including antagonists, for example enzyme inhibitors, and agonists,
for example a
transcription factor which results in an increase in the expression of a
desirable gene product
(although as will be appreciated by those in the art, antagonistic
transcription factors may also be
used), are all included.
In a preferred embodiment, the therapeutically active agent is cleaved from
the MRI agent, as is more
fully described below. In this embodiment, as a result of cleavage of the
therapeutic blocking moiety
and the release of the therapeutically active agent, a coordination site of
the MRI agent is no longer
occupied by a coordination atom and water is free to exchange in this site,
leading to signal
enhancement. Furthermore, the drug is now free to interact with its target,
which may or may not be
the same molecule which does the cleavage; for example, the cleavage site may
comprise an enzyme
substrate, for example of an HIV protease, and the drug may comprise an
inhibitor of the same
enzyme. Accordingly, in this embodiment, the nature of the interaction is
irreversible; the coordination
atom released from the MRI agent does not reassociate to block or occupy the
coordination site. This
embodiment allows the amplification of the image enhancement since a single
cleavage agent leads
to the generation of many activated metal ion complexes, i.e. metal ion
complexes in which the
therapeutic blocking moiety is no longer occupying or blocking a coordination
site of the metal ion.
In an alternate embodiment, the therapeutically active agent need not be
cleaved from the MRI agent
to be active. Thus, for example, as is more fully outlined below, some agents
cari remain associated
with the MRI agent; what is important in this instance is that the association
of the drug with its target
causes a conformational alteration that results in a coordination site,
originally occupied by a
coordination atom from the therapeutic blocking moiety, to become vacated,
allowing an increase in
the exchange of water and thus image enhancement. That is, the affinity of the
drug for its target is
greater than the affinity of the therapeutic blocking moiety for the MRI
agent. Depending on the nature
of the interaction of the drug with its physiological target, this may or may
not be a reversible
interaction. That is, in some cases, for example in the case of certain enzyme
inhibitors, the
interaction is effectively irreversible, leading to an enzyme active site
being occupied with a drug
attached to an MRI agent. Alternatively, in some embodiments, the interaction
is reversible, and an
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equilibrium is established between having the drug associated with its target
(leading to image
enhancement) and having the therapeutic blocking moiety associated with the
MRI agent (hindering
the exchange of water and thus a loss of signal).
The nature of the therapeutic effect between the therapeutically active moiety
and the physiological
target substance will depend on the both the physiological target substance
and the nature of the
effect. In general, suitable physiological target substances include, but are
not limited to, proteins
(including peptides and oligopeptides) including ion channels and enzymes;
nucleic acids; ions such
as Ca+2, Mg+2, Zn+2, K+, CI-, Na+, and toxic ions including those of Fe, Pb,
Hg and Se; cAMP;
receptors including G-protein coupled receptors and cell-surface receptors and
ligands; hormones;
antigens; antibodies; ATP; NADH; NADPH; FADHz; FNNH2; coenzyme A (acyl CoA and
acetyl CoA);
and biotin, among others. Physiological target substances include enzymes and
proteins associated
with a wide variety of viruses including orthomyxoviruses, (e.g. influenza
virus), paramyxoviruses (e.g
respiratory syncytial virus, mumps virus, measles virus), adenoviruses,
rhinoviruses, coronaviruses,
reoviruses, togaviruses (e.g. rubella virus), parvoviruses, poxviruses (e.g.
variola virus, vaccinia
virus), enteroviruses (e.g. poliovirus, coxsackievirus), hepatitis viruses
(including A, B and C),
herpesviruses (e.g. Herpes simplex virus, varicella-zoster virus,
cytomegalovirus, Epstein-Barr virus),
rotaviruses, Norwalk viruses, hantavirus, arenavirus, rhabdovirus (e.g. rabies
virus), retroviruses
(including HIV, HTLV-I and -II), papovaviruses (e.g. papillomavirus),
polyomaviruses, and
picornaviruses, and the like. Similarly, bacterial targets can come from a
wide variety of pathogenic
and non-pathogenic prokaryotes of interest including Bacillus; Vibrio, e.g. V.
cholerae; Escherichia,
e.g. Enterotoxigenic E. coli, Shigella, e.g. S. dysenteriae; Salmonella, e.g.
S. typhi; Mycobacterium
e.g. M. tuberculosis, M. leprae; Clostridium, e.g. C. botulinum, C. tetani, C.
difficile, C.perfringens;
Cornyebacterium, e.g. C, diphtheriae; Streptococcus, S. pyogenes, S.
pneumoniae; Staphylococcus,
e.g. S. aureus; Haemophilus, e.g. H. influenzae; Neisseria, e.g. N.
meningitides, N. gonorrhoeae;
Yersinia, e.g. G. IambIiaY. pestis, Pseudomonas, e.g. P. aeruginosa, P.
putida; Chlamydia, e.g. C.
trachomatis; Bordetella, e.g. 8, pertussis; Treponema, e.g. T. palladium; and
the like.
Once the physiological target substance has been identified, a corresponding
therapeutically active
agent is chosen. These agents will be any of a wide variety of drugs,
including, but not limited to,
enzyme inhibitors, hormones, cytokines, growth factors, receptor ligands,
antibodies, antigens, ion
binding compounds including crown ethers and other chelators, substantially
complementary nucleic
acids, nucleic acid binding proteins including transcription factors, toxins,
etc. Suitable drugs include
cytokines such as erythropoietin (EPO), thrombopoietin (TPO), the interleukins
(including IL-1 through
IL-17), insulin, insulin-like growth factors (including IGF-1 and -2),
epidermal growth factor (EGF),
transforming growth factors (including TGF-a and TGF-[3), human growth
hormone, transferrin,
epidermal growth factor (EGF), low density lipoprotein, high density
lipoprotein, leptin, VEGF, PDGF,
ciliary neurotrophic factor, prolactin, adrenocorticotropic hormone (ACTH),
calcitonin, human chorionic
gonadotropin, cotrisol, estradiol, follicle stimulating hormone (FSH), thyroid-
stimulating hormone
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(TSH), leutinzing hormone (LH), progeterone, testosterone, toxins,including
ricin, and any drugs as
outlined in the Physician's Desk Reference, Medical Economics Data Production
Company, Montvale,
NJ, 1998 and the Merck Index, 11th Edition (especially pages Ther-1 to Ther-
29), both of which are
expressly incorporated by reference.
In a preferred embodiment, the therapeutically active compound is a drug used
to treat cancer.
Suitable cancer drugs include, but are not limited to, antineoplastic drugs,
including alkylating agents
such as alkyl sulfonates (busulfan, improsulfan, piposulfan); aziridines
(benzodepa, carboquone,
meturedepa, uredepa); ethylenimines and methylmelamines (altretamine,
triethylenemelamine,
triethylenephosphoramide, triethylenethiophosphoramide, trimethylolmelamine);
nitrogen mustards
(chlorambucil, chlornaphazine, cyclophosphamide, estramustine, ifosfamide,
mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine,
prednimustine,
trofosfamide, uracil mustard); nitrosoureas (carmustine, chlorozotocin,
fotenmustine, lomustine,
nimustine, ranimustine); dacarbazine, mannomustine, mitobranitol, mitolactol;
pipobroman;
doxorubicin, and cisplatin (including derivatives).
In a preferred embodiment, the therapeutically active compound is an antiviral
or antibacterial drug,
including aclacinomycins, actinomycin, anthramycin, azaserine, bleomycins,
cuctinomycin, carubicin,
carzinophilin, chromomycins, ductinomycin, daunorubicin, 6-diazo-5-oxn-I-
norieucine, duxorubicin,
epirubicin, mitomycins, mycophenolic acid, nogalumycin, olivomycins,
peplomycin, plicamycin,
portiromycin, puromyciri, streptonigrin, streptozocin, tubercidin, ubenimex,
zinostatin, zorubicin;
aminoglycosides and polyene and macrolide antibiotics.
In a preferred embodiment, the therapeutically active compound is a radio-
sensitizer drug.
In a preferred embodiment, the therapeutically active compound is an anti-
inflammatory drug (either
steroidal or non-steroidal).
In a preferred embodiment, the therapeutically active compound is involved in
angiogenesis. Suitable
guarding moieties include, but are not limited to, endostatin, angiostatin,
interferons, platelet factor 4
(PF4), thrombospondin, transforming growth factor beta, tissue inhibitors of
metalloproteinase -1, -2
and -3 (TIMP-1, -2 and -3), TNP-470, Marimastat, Neovastat, BMS-275291, COL-3,
AG3340,
Thalidomide, Squalamine, Combrestastatin, SU5416, SU6668, IFN-a, EMD121974,
CAI, IL-12 abnd
IM862.
In a preferred embodiment, the physiological target is a protein that contains
a histidine residue that is
important for the protein's bioactivity. In this case, the therapeutically
active agent can be a metal ion
complex (not to be confused with the metal ion complex of the MRI agent), such
as is generally
described in PCT US95/16377, PCT US95/16377, PCT US96/19900, PCT US96/15527,
and
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WO 01/82795 PCT/USO1/14665
references cited within, all of which are expressly incorporated by reference.
These complexes take
on the general structure outlined below, and have been shown to be efficacious
in decreasing the
bioactivity of proteins, particularly enzymes, with a biologically important
histidine residue. These
cobalt complexes appear to derive their biological activity by the
substitution or addition of ligands in
the axial positions. The biological activity of these compounds results from
the binding of a new axial
ligand, most preferably the nitrogen atom of imidazole of the side chain of
histidine which is required
by the target protein for its biological activity. Thus, proteins such as
enzymes that utilize a histidine in
the active site, or proteins that use histidine, for example, to bind
essential metal ions, can be
inactivated by the binding of the histidine in an axial ligand position of the
cobalt compound, thus
preventing the histidine from participating in its normal biological function.
Structure 6
R4 R5
Rs ~~ Rs
N'; ,,,.N
R2 ~ . C o ' ~ R7
O
R~ R$
In Structure 6, the metal ion is shown as Co, which may be either Co(II) or
Co(III) or other tetradentate
metal ions, including Fe, Au and Cr (see PCT US96/15527). Each of R~, R2, R3,
R4, R5, R6, R, and R8
is a substitution group as defined herein, with hydrogen, alkyl, aryl, or
targeting moieties being
preferred. In general, at least one of the R groups will include a chemical
functional group for the
attachment of the cobalt complex to the remainder of the MRI agent.
In a preferred embodiment, the physiological target protein is an enzyme. As
will be appreciated by
those skilled in the art, the possible enzyme target substances are quite
broad. 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. Enzymes associated with the generation or maintenance
of
arterioschlerotic plaques and lesions within the circulatory system,
inflammation, wounds, immune
response, tumors, apoptosis, exocytosis, etc. may all be treated using the
present invention. Enzymes
such as lactase, maltase, sucrase or invertase, cellulase, a-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; and caspases, such as caspase-3, -5, -8 and other caspases of the
apoptotic pathway, such
as interleukin-converting enzyme (ICE). Similarly, bacterial and viral
infections may be detected via
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characteristic bacterial and viral enzymes. As will be appreciated in the art,
this list is not meant to be
limiting.
Once the target enzyme is identified or chosen, enzyme inhibitor
therapeutically active agents can be
designed using well known parameters of enzyme substrate specificities. As
outlined above, the
inhibitor may be another metal ion complex such as the cobalt complexes
described above. Other
suitable enzyme inhibitors include, but are not limited to, the cysteine
protease inhibitors described in
PCT US95/02252, PCT/US96/03844 and PCT/US96/08559, and known protease
inhibitors that are
used as drugs such as inhibitors of HIV proteases.
In one embodiment, the therapeutically active agent is a nucleic acid, for
example to do gene therapy
or antisense therapy. By "nucleic acid" or "oligonucleotide" or grammatical
equivalents herein means
at least two nucleotides covalently linked together. A nucleic acid of the
present invention will
generally contain phosphodiester bonds, although in some cases, as outlined
below, nucleic acid
analogs are included that may have alternate backbones, comprising, for
example, phosphoramide
(Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein;
Letsinger, J. Org. Chem.
35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:579 (1977); Letsinger et
al., Nucl. Acids Res.
14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am.
Chem. Soc. 110:4470
(1988); and Pauwels et al., Chemica Scripts 26:141 91986)), phosphorothioate
(Mag et al., Nucleic
Acids Res. 19:1437 (1991 ); and U.S. Patent No. 5,644,048), phosphorodithioate
(Briu et al., J. Am.
Chem. Soc. 111:2321 (1989), O-methylphophoroamidite linkages (see Eckstein,
Oligonucleotides and
Analogues: A Practical Approach, Oxford University Press), and peptide nucleic
acid backbones and
linkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem.
Int. Ed. Engl. 31:1008
(1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature 380:207
(1996), all of which are
incorporated by reference). Other analog nucleic acids include those with
positive backbones (Denpcy
et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones (U.S.
Patent Nos. 5,386,023,
5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem.
Intl. Ed. English
30:423 (1991 ); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger
et al., Nucleoside &
Nucleotide 13:1597 (1994); Chapters 2 and 3, ASC Symposium Series 580,
"Carbohydrate
Modifications in Antisense Research", Ed. Y.S. Sanghui and P. Dan Cook;
Mesmaeker et al.,
Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J. Biomolecular
NMR 34:17 (1994);
Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, including those
described in U.S. Patent
Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,
"Carbohydrate
Modifications in Antisense Research", Ed. Y.S. Sanghui and P. Dan Cook.
Nucleic acids containing
one or more carbocyclic sugars are also included within the definition of
nucleic acids (see Jenkins et
al., Chem. Soc. Rev. (1995) pp 169-176). Several nucleic acid analogs are
described in Rawls, C &
E News June 2, 1997 page 35. All of these references are hereby expressly
incorporated by
reference. These modifications of the ribose-phosphate backbone may be done to
increase the
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stability and half life of such molecules in physiological environments; for
example, PNA antisense
embodiments are particularly preferred.
As will be appreciated by those in the art, all of these nucleic acid analogs
may find use in the present
invention. In addition, mixtures of naturally occurring nucleic acids and
analogs can be made or
mixtures of different nucleic acid analogs.
The nucleic acid may be single-stranded or double stranded. The physiological
target molecule can be
a substantially complementary nucleic acid or a nucleic acid binding moiety,
such as a protein.
In a preferred embodiment, the physiological target substance is a
physiologically active ion, and the
therapeutically active agent is an ion binding ligand or chelate. For example,
toxic metal ions could be
chelated to decrease toxicity, using a wide variety of known chelators
including, for example, crown
ethers.
In addition to the therapeutically active agent, a therapeutic blocking moiety
may include other
components. As outlined herein, a preferred embodiment provides that
coordination atoms) for the
metal ion are provided by the therapeutically active agent. However, if the
therapeutically active agent
does not contribute a coordination atom, the therapeutic blocking moiety
further comprises a
"coordination site barrier" which is covalently tethered to the complex in
such a manner as to allow
disassociation upon delivery of the therapeutically active agent. For example,
it may be tethered by
one or more enzyme substrate cleavage site's. 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
substance. Coordination site barriers are used when coordination atoms are not
provided by the other
components of the MRI agents, i.e. the therapeutically active agents, the
cleavage site(s), the
targeting moiety, linkers, etc. The other components of the therapeutic
blocking moiety such as an
enzyme cleavage site serves as the tether, covalently linking the coordination
site barrier to the metal
ion complex. As a result of either a cleavage or a conformation change due to
the interaction of the
therapeutically active agent with its physiological target, 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.
In one embodiment, the coordination site barrier is attached to the metal ion
complex at one end, as is
depicted in Figures 2A and 2B. Figure 2A depicts the case where an enzyme
cleaves at the cleavage
point, thereby releasing the coordination site barrier. Figure 2B depicts the
case wherein it is a
conformational change as a result of the interaction of the drug with its
physiological target that leads
to the removal of the coordination site barrier. In another embodiment, the
coordination site barrier is
attached to the metal ion complex with more than one cleavage site, as is
depicted in Figure 2C for
two attachments. The enzyme target may cleave only one side, thus removing the
coordination site
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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.
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 considered a
linker group.
In an alternative embodiment, the coordination site barrier does not directly
occupy a coordination site,
but instead blocks the site sterically. This may also be true for the
therapeutically active agents. 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.
In this embodiment, the coordination site barrier is preferably 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 Figure
2C.
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 moiety,
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 therapeutic 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.
In addition to the therapeutically active agents and coordination site
barriers, a therapeutic blocking
moiety may also comprise a cleavage site. As described herein, one way of
delivering the
therapeutically active agent is to cleave it off the MRI agent. It is also
possible to configure the MRI
agents of the invention such that a coordination site barrier is cleaved off,
leaving the therapeutically
active agent attached to the MRI agent, but in a conformation wherein the drug
is now able to interact
with its target in a way it was not able to prior to cleavage as is generally
depicted in Figure 1 C. In
some embodiments, the coordination atoms) that hinder the rapid exchange of
water at a
coordination site may be contributed by the cleavage site. Thus for example,
when a proteolytic
cleavage site is used, coordination atoms may be provided by an atom of the
peptide chain.
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In a preferred embodiment, the physiological target is an enzyme and the
cleavage site corresponds to
that enzyme. Alternatively, the cleavage site is unrelated to the
physiological target. In this
embodiment, the cleavage site may be either specific to a disease condition,
for example the cleavage
site may be an HIV protease site and the therapeutically active agent is
chosen to interfere with a
different viral function (for example viral replication), or it may be non-
specific, relying instead on a
different mechanism for specificity, if desired. For example, the cleavage
site may be a "generic"
intracellular protease site, and specificity can be provided by a targeting
moiety attached to the MRI
agent; for example, a cell-specific ligand could be used to target a specific
set of cells such as tumor
cells.
Thus, for example, a proteolytic cleavage site may be used for cleavage by
proteases. The cleavage
site thus comprises a peptide or polypeptide that is capable of being cleaved
by a defined 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.
Similarly, when the enzyme is a carbohydrase, the cleavage site will be a
carbohydrate group which is
capable of being cleaved by the target carbohydrase. For example, when the
enzyme is lactase or f3-
galactosidase, the cleavage site is lactose or galactose. Similar pairs
include sucrase/sucrose,
maltase/maltose, and a-amylase/amylose.
In a preferred embodiment, the cleavage site is a phosphorus moiety, as
defined above, such as -
(OPO(OR~))~, 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 cleavage
enzyme is alkaline phosphatase or a phosphodiesterase, or other enzymes known
to cleave
phosphorus containing moieties such as these.
In a preferred embodiment, the cleavage site utilizes a photocleavable moiety.
That is, upon exposure
to a certain wavelength of light, cleavage occurs, 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
therapeutic blocking moiety
via an ether, thioether, ester (including phosphate esters), amine or similar
linkage to a heteroatom
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(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.
In a preferred embodiment, a photocleavable site has a structure depicted
below in Structure 7, 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:
Structure 7
Xi Rzs
./
R ~N.R~p~N/Rzs
~\ NOz
s ~ R3
R~N N~Ra
C
Rtz~ ~ ~ ~Ra
Rs Rs
Xp X3
Structure 7 depicts a DOTA-type chelator, although as will be appreciated by
those in the art, other
chelators may be used as well. R26 is a linker as defined herein. The XZ group
includes the other
components of the therapeutic blocking moiety, including a therapeutically
active agent and an
optional coordination site barrier. Similarly, there may be substitutent
groups on the aromatic ring, as
is known in the art.
In addition to the components outlined above, it should be appreciated that
the therapeutic blocking
moieties of the present invention may further comprise a linker group as well
as a functional
therapeutic blocking moiety. Again, as outlined herein for the therapeutically
active agents and the
cleavage sites, a coordination atom may actually be provided by the linker.
Linker groups (sometimes depicted herein as RZ6) will be used to optimize the
steric considerations of
the metal ion complex. That is, in order to optimize the interaction of the
therapeutic blocking moiety
with the metal ion, linkers may be introduced to allow the coordination site
to be blocked. In general,
the linker group is chosen to allow a degree of structural flexibility. For
example, when a
therapeutically active agent interacts with its physiological target in a
manner that does not result in the
therapeutically active agent being cleaved from the complex, the linker must
allow some movement of
the therapeutically active agent away from the complex, such that the exchange
of water at at least
one coordination site is increased.
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,
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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, the length of this linker may be very important
in order to achieve optimal
results.
Accordingly, the therapeutic blocking moieties of the invention include a
therapeutically active agent
and optional cleavage sites, coordination site barriers, and tinkers, if
required.
In some embodiments, the metal ion complexes of the invention have a single
associated or bound
therapeutic blocking moiety. In such embodiments, the single therapeutic
blocking moiety impedes
the exchange of water molecules in at least one coordination site.
Alternatively, as is outlined below, a
single therapeutic blocking moiety may hinder the exchange of water molecules
in more than one
coordination site.
In alternative embodiments, two or more therapeutic blocking moieties are
associated with a single
metal ion complex, to implede the exchange of water in at least one or more
coordination sites.
The therapeutic blocking moiety is attached to the metal ion complex in a
variety of ways, a small
number of which are depicted in the Figures. In a preferred embodiment, as
noted above, the
therapeutic blocking moiety is attached to the metal ion complex via a linker
group. Alternatively, the
therapeutic blocking moiety is attached directly to the metal ion complex; for
example, as outlined
below, the therapeutic blocking moiety may be a substituent group on the
chelator.
In a preferred embodiment at least one of the R groups attached to the "arms"
of the chelator, for
example R9, R,o, R~1 or R~2 of the DOTA structures, or R~3, R~4, R~~, R2o or
R~, 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 MRl contrast agents. Preferred groups include the C1
through C6 alkyl
groups with methyl being particularly preferred.
This is particularly preferred when the therapeutic blocking moiety is
attached via one of the "arms",
for example when a therapeutic blocking moiety is at position X, to X4
(Structure 8), or position H, I, J
or K of Structure 9.
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However the inclusion of too many groups may drive the equilibrium in the
other direction effectively
locking the coordination atom out of position. 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.
The therapeutic blocking moieties are chosen and designed using a variety of
parameters. In the
embodiment which uses a coordination site barrier and the coordination site
barrier is fastened or
secured on two sides, the affinity of the coordination site barrier of the
therapeutic 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 cleavage agent. However, in the
embodiment where the
therapeutic blocking moiety is linked to the complex in such a manner as to
allow some rotation or
flexibility of the therapeutic blocking moiety, for example, it is linked on
one side only, the therapeutic
blocking moiety should be designed such that it occupies the coordination site
a majority of the time.
When the therapeutic blocking moiety is not covalently tethered on two sides,
it should be understood
that the components of the therapeutic blocking moieties are chosen to
maximize three basic
interactions that allow the therapeutic 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 therapeutic blocking moiety and the
metal ion, to allow the
therapeutic 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 therapeutic 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 therapeutic blocking moiety. Similarly, removing degrees
of freedom 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 R9 to
R~~ positions of Structure 6
when the therapeutic blocking moiety is attached at an "X" position can lead
the therapeutic 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.
Furthermore, effective "tethering" of the therapeutic 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
therapeutic blocking moiety to the chelator complex, as is depicted below.
Potential therapeutic 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
therapeutic blocking
moieties and then compared with the chelator without the therapeutic blocking
moiety in imaging
experiments. Once it is shown that the therapeutic blocking moiety is a
sufficient "blocker", the
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experiments are repeated in the presence of either the physiological target
(when it is the interaction of
the target and the drug which causes either cleavage or a conformational
change resulting in opening
up of a coordination site) or the cleavage agent, to show an increase in the
exchange of water and
thus enhancement of the image.
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 therapeutic blocking moiety. In
a preferrred
embodiment, the metal ion complexes have the formula shown in Structure 8:
Structure 8
xt x
R N A~N~Rto
\/R
M
R~N N~~
C
Rt2 ~ ~ Rtt
R6 RS
x4 x3
In Structure 8, M is a paramagnetic metal ion selected from the group
consisting of Gd(III), Fe(III),
Mn(ll), Yt(III), and Dy(III). A, B, C and D are each either single or double
bonds. The R~ through R,2
groups are substitution groups as defined above, including therapeutic
blocking moieties and targeting
moieties. X, through X4 are -OH, -COO-, -(CH2)~OH (with -CHZOH being
preferred), -(CH2)~COO-
(with CHaC00- being preferred), or a substitution group, including therapeutic
blocking moieties and
targeting moieties. n is from 1 to 10, with from 1 to 5 being preferred. At
least one of R~ to R~2 and X,
to X4 is a therapeutic blocking moiety.
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~ and a single RZ group attached to
the respective carbons;
in a preferred embodiment, as described below, the R~ and R2 groups are
hydrogen. In a preferred
embodiment, A is a single bond, and it is possible to have two R~ groups and
two RZ groups on the
respective carbons. In a preferred embodiment, these groups are all hydrogen
with the exception of a
single therapeutic 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 therapeutic group
attached in the R~
position, and two hydrogens, two alkyl groups, or a hydrogen and an alkyl
group in the RZ positions.
It is to be understood that the exact composition of the X~-X4 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, -(CHZ)~OH, or
(CH~)~COOH; however, when the metal is present, the groups may be -OH, -COO-, -
(CH2)~O-, or
(CHa)"COO-.
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A further embodiment utilizes metal ion complexes having the formula shown in
Structure 9:
Structure 9
M
~zo
R» R~s
K
N N
\N
Ris ~RI~ 1g Rzi
J
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, -(CH2)nOH, or (CH2)~COOH; however, when the metal is present, the groups
are -OH, -COO-,
-(CH~)~OH, Or (CHZ)~COO-.
In this embodiment, R,3 through R2, are substitution groups as defined above.
In a preferred
embodiment, R,~ to R2~ are hydrogen. At least one of R,3 - Rz~, H, I, J, K or
L is a therapeutic blocking
moiety, as defined above.
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 or therapeutic blocking moiety, etc.), although in a preferred
embodiment the targeting moiety
does not replace a coordination atom.
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).
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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.
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; Jackets (1990), supra, U.S. Patent Nos. 5,155,215,
5,087,440, 5,219,553,
5,188,816, 4,885,363, 5,358,704, 5,262,532 and 5,707,605; Meyer et al.,
(1990), supra, Moi et al.,
(1988), and McMurray et al., Bioconjugate Chem. 3(2):108-117 (1992)); see also
PCT US95/16377,
PCT US96/19900, and PCT US96/15527 all of which are expressly incorporated by
reference.
For DOTA derivatives, the synthesis depends on whether nitrogen substitution
or carbon substitution
of the cyclen ring backbone is desired. For nitrogen substitution, the
synthesis begins with cyclen or
cyclen derivatives, as is well known in the art; see for example U.S. Patent
Nos. 4,885,363 and
5,358,704.
For carbon substitution, well known techniques are used. See for example Moi
et al., supra, and
Gansow, supra.
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°C may be required.
The complexes of the invention can be isolated and purified, for example using
HPLC systems.
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.
In addition, the MRI agents of the invention can be added to polymers, using
techniques generally
outlined in PCT US95/14621 and U.S.S.N. 08/690,612 (allowed), both of which
are expressly
incorporated by reference. Briefly, chemical functional groups are added to
the MRI agents to allow
the chemical attachment of a plurality of MRI agents to polymers. A "polymer"
comprises at least two
or three subunits, which are covalently attached. At least some portion of the
monomeric subunits
contain functional groups for the covalent attachment of the MRI contrast
agents. In some
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embodiments coupling moieties are used to covalently link the subunits with
the MRI agents. As will be
appreciated by those in the art, a wide variety of polymers are possible.
Suitable polymers include
functionalized styrenes, such as amino styrene, functionalized dextrans, and
polyamino acids.
Preferred polymers are polyamino acids (both poly-D-amino acids and poly-L-
amino acids), such as
polylysine, and polymers containing lysine and other amino acids being
particularly preferred. Other
suitable polyamino acids are polyglutamic acid, polyaspartic acid, co-polymers
of lysine and glutamic
or aspartic acid, co-polymers of lysine with alanine, tyrosine, phenylalanine,
serine, tryptophan, and/or
proline.
Similarly, it is also possible to create "multimers" of MRI agents, by either
direct attachment or through
the use of linkers as is generally described in U.S.S.N. 08/690,612 (allowed),
incorporated by
reference.
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, including the non-invasive imaging of drug delivery, imaging
the interaction of the drug
with its physiological target, monitoring gene therapy, in vivo gene
expression (antisense),
transfection, changes in intracellular messengers as a result of drug
delivery, etc.
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. Patent No. 5,155,215;
U.S. Patent 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).
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. Patents
4,885,363 and 5,358,704.
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. Patent No.
5,155,215).
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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. Patent 5,155,215). Thus, one embodiment utilizes
polysaccharides as
substitution R groups on the compositions of the invention.
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 therapeutic
blocking moieties to treat
disorders of the brain.
In a preferred embodiment, the therapeutic moiety is attached to the chelate
using a photocleavable
moiety as defined on the next page. Also included in the photocleavable
moieties are guinone
derivatives. Also included are W099/25389, PCT US/9822743, hereby incorporated
by reference.
The references cited herein are expressly incorporated by reference in their
entirety.
38
