Note: Descriptions are shown in the official language in which they were submitted.
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CHELATE COMPLEX WITH HIGH CONSPICUITY FOR MAGNETIC RESONANCE
IMAGING
R~ OUn~D OF T~ ~ NY~N~I ON
Modern clinical medical practice along with advances
in industrial technology and manufacturing has created a need
for sophisticated non-invasive imagining technologies. The
response to this need has been met by the marriage of radiology
and computers. The net result has been an imaging technological
revolution.
A product of this revolution has been the technology
of magnetic resonance imagining (MRI). The origin of MRI lies
10 in nuclear magnetic resonance (NMR) technology which has been
used for years as a means of chemical analysis. This technology
has been subsequently refined and combined with computer
technology in which electromagnetic fluctuations are resolved
and presented in an imaging format. This emerging technology
15 has become known as magnetic resonance imaging in the art.
The MRI technigue has become a valuable imaging
modality in particular for ~iomedical usages. M~I in biomedical
applications relies on low energy radiation or radio waves to
probe the organs and tissues deep inside the human body with
20 high resolution as opposed to computerized tomography (CT) and
x-ray technology which rely on high energy ra~iation or
electromagnetic pulses targeted at the organ systems to be
examined. In this regard, MRI has proven more effective than CT
and x-rays in providing detailed information on both the
2~ structure and function of tissues, in that biologic tissues are
relatively transparent to x-rays.
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For example, MRI technology can distinguish different
tissue types or detect tissues whose ~iochemical environments
may have been altered due to a disease process. M~I also
provides a means to e~m;ne and diagnose with high resolution,
5 specificity, and detail, the internal structures, organs and
disease processes of the body in a dynamic format. Furthermore
the lack of definitively known hazards associated with low
levels of magnetic and radio-frequency fields permits repeated
scans along any plane, including, but not limited to,
10 transverse, coronal and sagittal sections.
MRI technology utilizes the property that each atomic
species has its own characteristic magnetic mo~nt. In the case
where there is an even number of protons or neutrons, or the
particles are paired in a given species, the magnetic potential
15 is statistically canceled out. However, nuclei of certain atoms
and isotopes which are unpaired or have an odd number of protons
or neutrons possess an intrinsic spin. A potential
magnetization occurs from the rapid spinning of the unpaired
particle. Species which satisfy this parameter, among others,
20 are phosphorus-31, carbon-13, sodium-23, fluorine-19, oxygen-17
and hydrogen-1 and can be used in MRI studies. Consequently, lH
protons and water which encompass many biochemical processes and
tissues are often the atomic species of choice to produce
revealing in-depth images of the body.
In practice in MRI technology, a magnetic field of a
given strength t~, expressed generally in units of gauss or
telsa) is applied to the target area of investigation. Thè~MRI
technician can manipulate the magnetic field environment so that
only protons at very specific sites are affected. The magnetic
30 nuclei of the various atomic species in the target area line up
or specifically orient relative to the magnetic field according
to their allowed quantum mechanic states. In the case of a
proton, the principle isotope of hydrogen, there are two
allowable states of orientation -- parallel (low energy) or
35 anti-parallel (high energy) to the magnetic field.
A radio wave pulse is also directed at the atomic
species in the target area. The atomic species or protons
within a given magnetic field have a characteristic magnetic
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m~ment. These atomic species or protons will resonate upon
absorption of a pre-designated eneryy input from a specific
characteristic radio frequency. The characteristic freguency at
which resonance occurs for an atomic specie under a given
5 maynetic field is known as the Larmor frequency. In this
instance, resonance is the act of the atomic species to absorb
and emit eneryy repeatedly and rapidly. Thus the infusion of
eneryy from the radio waves with the magnetic field causes the
atomic species to reorient in the magnetic field due to the
l0 changes in energy state and subsequently leads to oscillation
between the various eneryy states.
This oscillating activity or resonance signal of the
atomic species can be detected by a radio receiver. This
information is then integrated with the measurable period of
l' time or relaxat~on that it takes the species, often ln a.om~, .o
lose the energy that was absorbed by the directed pulses. Since
a 1H proton has predictable atomic activity, any variances in
behavior are due to the local biochemical environment of the
proton. Thus the patterns and characteristics of the resonance
20 signals can be converted into an image to reflect the
biochemical environment surrounding the target area.
One of the measurements of relaxation in this process
is the spin-lattice, thermal or longitudinal relaxation time
(Tl). This measurement reflects the characteristic time it
25 takes the excited nuclei to return to the ground state by
dissipating the excess eneryy to the surrol-n~ s or lattice
field. The dissipation process is dependent on many factors
which include but are not limited to the Larmor frequency, the
magnetic field strength, the size of the molecule, and the
30 biochemical environment. Relaxation times have been measured
for various fluids, organs and tissues in different species of
mAmmA~s. InvestigatiOnS have shown that tissues cont~in;ng more
water have longer Tl relaxation times, whereas those containing
lipid or paramagnetic species have the capacity to shorten Tl.
35 This is significant because in various instances, shorter Tl
times have resulted in hisher siynal intensities and therefore
brighter image enhAncement. Thus a skilled practitioner can
change many of the variables to enhance or improve the image of
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the voxel or volume element of the species under ex~mi~tion.
See Stark, D.D. and W.G. Bradley (eds.). 1992. Magnetic
~esonance Imaging (Second edition. Volumes One and Two. Mosby,
St. Louis), which is incorporated herein by reference.
Another of the measurements of relaxation is spin-spin
or transverse relaxation time (T2). This measurement reflects
the characteristic time it takes the nuclei in various states of
excitation to ~xchA~ge energy with each other as opposed to the
lattice which results in a loss of magnetization. The magnetic
10 decay is due to the various nuclear magnetic mnmpnts being out
of phase with each other or dephasing as a result of their
mutual interaction. This phPnomPn~ is a direct consequence of
magnetic field imperfections and resultant generated fields in
the biological system under investigation which cause the nuclei
15 to precess at slightly different rates. This loss of phase
coherence leads to a loss of magnetization. The spin-spin or
transverse relaxation time (T2) is a means to measure the loss
of magnetization. Investigations have shown that large
macromolecules and water molecules which bind to macromolecules
20 reorient or tumble more slowly than small molecules causing
efficient T2 relaxation. By contrast such molecules which
tumble at rates much slower than the Larmor frequency result in
inefficient T1 relaxation.
A simplified expression that describes MRI signal
25 intensity in terms of the parameters of relaxation is the
following:
SI = N(H)[1-e~~1]e~~2, wherein
SI = signal intensity
N(H) = the spin density, the density of resonating
spins (e.g. number of protons) in a given
volume (e.g. a discrete volume of tissue)
TR = repetition time, the time between the beginning
of one radio frequency pulse sequence and the
beg;nn;~ of the succeeding pulse sequence at a
specified tissue location
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TE = echo time delay, which is the time between the
center of the 90-degree pulse and the cen~er of
the spin echo
Tl = spin-lattice relaxation time
T2 = spin-spin relaxation time.
The above expression delineates that signal intensity
will increase when N(H) increases, Tl decreases, or T2
increases. Alternatively, the signal intensity will decrease
when N(H) decreases, Tl increases or T2 decreases. Thus Tl and
lO T2 times have reciprocal effects on image intensity. The above
parameters play an intrinsic role in the dynamics of the imaging
process.
The altering effects on Tl and T2 are critically
dependent on the magnetic entities and the concentrations
l~ thereof utilized in an imaging agent. Paramagnetic materials
reduce both Tl and T2, however the effects on Tl predominate,
particularly at low concentrations. This means that they are
best detected using Tl weighted techniques. Conversely,
ferromagnetic and superparamagnetic entities rely on T2 weighted
20 imaging.
The relationship between the concentration of an MRI
imaging agent or its magnetic entity is distinctly non-linear.
This phenomena is distinctly different from radiographic
contrast agents in which signal intensity depends linearly on
2~ the concentration of the material present. As a consequence,
the signal intensity or conspicuity of an MRI agent represents
the net effect of the positive and negative contributions of Tl
and T2 which are both concentration dependent. For example, in
the case of the paramagnetic specie, gadolinium, the
3D contribution of Tl pre~nm;nAtes at low concentrations, whereas
when the concentration increases, the effects of T2 become more
pronounced. Thus, as gadolinium concentration increases, an
initial threshold level is met at which imaging becomes
possible. This is followed by a continual increase in signal
3~ intensity with increasing gadolinium concentration until an
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optimal threshold is met at which point the contributions of T2
result in a ~;m;n;5hing of signal intensity or conspicuity.
A commo~ means to impro~e imaging is through the use
of contrast agents, which alter the a~ove described parameters.
These agents increase and clarify the information content of
diagnostic images. Contrast agents enhance a diagnostic image
by altering the image contrast or the difference in signal
intensity between the different biochemical en~ironments (e.g.
tissues). In NRI, the contrast agents work by altering the
10 local magnetic environment of tissues primarily by altering
tissue relaxation rates. The contribution of ~arious contrast
agents has been attributed to the interaction of unpaired
electrons of the contrast agent and the hydrogen nuclei of water
molecules. A theoretical explanation of the effects of these
1~ interactions has indicated that the distance from the center of
the paramagnetic species of the contrast agent to the center of
the hydrogen nucleus undergoing relaxation is critical. This
theoretical work indicates that relaxation times are
proportional to the distance raised to the 6th power. Thus
20 changes in signal intensity are dependent on the ability of the
paramagnetic species of the contrast agent to approach the
protons of the sample ~eing examined. See Chapter 14, nContrast
Agents n in Stark and Bradley and Chapter 6, ~MRI:New
Breakthroughs in Medical Diagnosis" in Science at the Fro~tier,
25 Volume 1 by Addison Greenwood, National Academy Press,
W~.sh;ngton, D.C. 1992, which are incorporated herein by
reference.
Historically~ magnetic materials were shown to affect
the relaxation times of resonating protons. The early work
30 concentrated on paramagnetic ferric ions in solution. This work
was later extended to a variety of paramagnetic transition
metals. Subseguently, the research led to the use of
paramagnetic ions and chelate complexes to alter relaxation
times. This line of research resulted in the first commercial
35 MRI contrast agent, Gadolinium-Diethylenetriaminepentaacetic
Acid-Dimeglumine ([NMG]2Gd-DTPA~) or Gadiopentetate Dimeglumine.
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The most recent work has focused on attempts to
associate metal chelate complexes with large molecular weight
entities such as macromolecules to improve relaxivity. These
macromolecules also serve as vehicles of transport and include
S ~ut are not limited to oligopeptides, proteins, lipids,
polysaccharides, and synthetic polymers. Furthermore, in this
regard, attempts have been made to combine the metal chelate
complexes with macromolecules such as monoclonal antibodies to
provide target-specific imaging.
Optimal relaxation enhancement, which results in
improved imaging, occurs when molecules or tissues bearing
nuclear spins have fast access to as many sites near the
paramagnetic molecule as possible. This effect can be amplified
~y increasing the concentration or number of metal ions per
1~ macromolecule by polymerization. The addition of a metal ion to
a chelating agent reduces the number of effective bonding and
interaction sites.
To be an effective contrast agent, the metal chelate
complex must be stable. Increased stability of the complex
20 results by multiple bond formation between the chelating agent
and the metal ions. Thus, the stability is a function of the
number of bonding sites of the chelating agent, the coordination
number of the metal ion, steric factors and the biochemical environment.
The stability of the metal chelate complex and its
2~ toxicity are intimately related. This is due to the fact that
excessive quantities of transition and Lanthanide metals can be
toxic. A stable metal chelate complex prevents the presence of
free metal ions and shields the toxic effects of ~onded metal
ions. ThermodynamiC stability of the complex is also important
30 in that a free metal ion and a free ligand tend to ~e more toxic
than the resulting metal complex. In addition, a
thermodynamically stable metal chelate complex will hinder metal
ion substitution in vivo and chelate dissociation. Finally, a
thermodynamically stable metal chelate complex can alter or
3~ reduce meta~olic attack which could result in the release of
toxic metabolites.
Another factor for providing effective contrast agents
is the biodistribution and pharmacokinetics of the metal chelate
~ ~=
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complexes. Since the complexes have toxic components, the
upta~e and clearance of these compounds from the targeted site
of ey~m;n~tion is critical. This is particularly true for
suscepti~le organ systems. The ~iodistri~ution and transport
5 kinetics characteristics of these compounds are also important
in that these parameters affect the time period in which
effective imaging can occur. Thus, contrast agent compounds are
often organ specific in their effectiveness.
~11 agents proposed up to now for imaging diagnosis,
10 which consist of complexes of heavy metals, are not very
satisfactory with regard to their practical use in man, or
create more or less serious problems with regard to relaxivity
and tolerance. Also, they frequently exhibit insufficient
selectivity of the ~ond with the heavy metal, insufficient
15 stability, and particularly, lack of selective targeting to
certain organs.
Another problem is the tendency of many complexes to
exchAnge the central metal ion for trace metals which are
essential to the organism, or for ions, for example, Ca~2, which
20 in ~ivo are present in relatively large amounts. In the case of
insufficient specific sta~ility of the complex, trace metals of
vital importance may, in fact, ~e extracted from the organism.
In their place, undesirable heavy metals, such as gadolinium,
may be deposited in their place, which may remain in the
25 organism for a long time. Particularly problematic is the use
of these complexes in dosages which would be suitable or
desira~le for imaging diagnosis.
With regards to synthesizing contrast agents, the
preparation of MRI contrast agents utilizing the conjugation of
30 macromolecules with chelated polymer carriers (Sieving, 1990)
includes operations with water and air-sensitive reagents. This
has ~een difficult to achieve for milligram quantities of
reagents. Moreover, these prepared conjugates have a low
stability within the living cell, which can limit their in ~ivo
35 utility for NRI.
Besides sta~ility pro~lems, previous methods have also
~een unsuccessful in bin~ a sufficiently effective num~er of
atoms of a contrast metal (paramagnetic atoms) to a chelating
-
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_ 9 _
agent to be clinically useful in an imaging technology, such as
MRI.
With respect to the above shortcomings, there
therefore exists an urgent and unfulfilled need for contrast
agents which are stable for in vivo usage. These contrast
agents would also ideally possess a sufficiently concentrated
number of bound or complexed paramagnetic atoms of a contrast
metal. This num~er of concentrated atoms advantageously exceeds
the threshold level required for visualization in MRI. Such
10 contrast agents would be ideal for clinical image. Such useful
clinical agents would then be of significant commercial value to
the medical co~mll~;ty as a diagnostic tool for differentiating
and identifying diseased tissue from normal tissue.
~urthermore, there is a need for effective contrast
15 agents which avoid the toxicity and stability problems inherent
in using yadolinium. ~urther, there is a need for new and
improved contrast agents, whether they include gadolinium, or
another paramagnetic metal in place of gadolinium.
In another respect, the combination of some metal
20 chelate complexes with macromolecules has resulted in ~;m;nished
biological activity of the macromolecules. It has been
theorized that the combination has altered the chemistry or
steric factors of the macromolecules. This alteration has
resulted in ~;m;n;shed biological activity. Thus, there is also
25 an urgent need for target-specific contrast agents which are
conjugated with macromolecules, e.g., antibodies, for specific
targeting to receptor or antigenic sites, which targeting
macromolecules advantageously retain their biological or
;mmllnological activities in vi~o.
SUMMARY OF THE ~NV~Nl~Cl~
The present invention addresses and fulfills the
a~ove-described needs. The present invention, as described
further below with more particularity, addresses and
advantageously fulfills the need in the art for stable, safe,
3~ and clinically useful contrast agents for imaging technologies,
particularly for, but not limited to, M~I.
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The present invention advantageously provides contrast
agents for imaging, especially for, but not limited to MRI, and
for additional applications in various non-imaging technologies.
These other applications include diagnostic, monitoring, marking
5 and mapping.
The contrast agent of the present invention, which
advantageously and unexpectedly enhances visualization in
imaging procedures, is comprised of a conjugate of a carrier
with a chelating agent. The conjugate is complexed with an
10 effective number of atoms of a paramagnetic metal. An effective
number of atoms is defined as that number which enhances signal
intensity or enhances the visualization of an image. The
contrast agent of the present invention unexpectedly possesses
an increase in conspicuity or enhanced contrast, which increase
15 clearly enhances signal intensity or visualization. Such visual
enhancement encompasses either a positive or negative contrast.
A further aspect then of the present invention is a
composition which is comprised of the contrast agent of the
present invention and a pharmaceutically-acceptable carrier.
Another aspect o~ the present invention is a method
for synthesizing the contrast agents of the present invention.
The method of the invention is unexpectedly simpler and more
efficient than other currently availa~le techniques. The method
is also more economical. The method unexpectedly provides a
2~ clinically useful amount of a contrast agent having a higher
concentration of ~ound paramagnetic atoms than previously
achieved. The method of the invention for synthesizing a
contrast agent comprises conjugating a carrier with a chelating
agent, and complexing the conjugate with an effective num~er of
30 atoms of a paramagnetic metal. The method therefore provides
the contrast agents of the invention having a favora~le increase c
in conspicuity or e~hAnced signal intensity.
Another aspect of the present invention advantageously
provides a method for further conjugation of the contrast agents
35 of the present invention with proteins or other macromolecules
for specific organ or tissue targetin~, without adversely
affecting the ~iological activity of these macromolecules.
.
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Another aspect of the present invention is a kit for
use ~y, e.g., a clinician or diagnostician. The kit comprises a
pac~age which contains separate containers for the contrast
agents of the present invention in a form and dosage suitable
for ~m; ni stration, and an appropriate diluent or carrier.
Alternatively, the kit may provide separate containers of
reagents for making the contrast agents of the invention by way
of the method of the present invention.
The resulting novel contrast agents have many
10 unexpected properties which make them particularly advantageous,
such as ~eneficial imaging properties, low toxicity, high
stability, advantageously selective biodistribution or targeting
characteristics, and safer degradation and excretion.
Furthermore, the contrast agents of the present
15 invention form advantageously stable, safe, and effective
ph~rm~ceutical compositions for administering or using in vi~o
in m~mm~ls, including humans, in MRI and other suitable imaging
technologies.
The application of the contrast agents is not limited
20 to imaging procedures. The contrast agents of the invention are
also advantageously suitable for application or use in a wide
variety of fields which encompass non-imaging technologies.
The contrast agents in accordance with the present
invention unexpectedly reduce or prevent the toxic effects of
25 contrast-enhancing paramagnetic metals, while enhancing signal
intensity or conspicuity. The contrast agents of the present
invention thus advantageously possess very low biological
toxicity. This is so despite having a higher concentration of
paramagnetic atoms ~ound per molecule than heretofore achieved
30 ~y any other contrast agent. In addition, the agents
unexpectedly possess substantially better relaxation properties
than other contrast agents, which permits the use of a smaller
amount of the agents of the invention to achieve the same or
similar effect.
3~ The contrast agents of the present invention
unexpectedly provide a favorable ~iochemical environment for
close and efficacious interaction of the paramagnetic species
_
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and the targeted ~ody system. This is achieved while preserving
the stability and structural integrity of the contrast agents.
The contrast agents of the present invention are
advantageously useful as MRI agents, agents for cancer
detection, diagnosis of dementia and psychiatric disease, and
for tracing of neuronal pathways and ~ody mapping.
These and other features, aspects, and advantages of
the present invention will ~ecome better understood with regard
to the following description, accompanying drawings and appended
10 claims.
BRIEF DESCRIPTION OF T~E FIGURES
Fiyure 1 shows a sample image from a Tl relaxation
experiment of multiple test tubes cont~;n;ng solutions prior to
selection for in vivo use based on signal intensity. The bottom
15 tube contains the contrast agent of the present invention, which
shows a sharp signal intensity.
Figure 2 shows a coronal image of a cat injected via
the left ventricle with the contrast agent of the present
invention conjugated with antibody to antigen 301. The white
20 material (arrows~ indicates the location of the contrast agent.
Figure 3 shows an axonal tracing (arrows) in a cat
~rain utilizing a contrast agent of the present invention which
is a wheat germ agglutinin-gadolinium conjugate.
Figure 4 shows a magnified view of the axonal tracing
25 shown in Figure 3.
Figure 5 shows a fluorescence microscopy photograph
which white material (arrows) is histologic confirmation of the
presence of the contrast agent of the present invention in the
cat ~rain.
DETAI~ED DES~RIPTION OF THE ~NV~W'~' ~ON
ABBREVIATIONS
DOTA: 1, 4, 7, 10-tetraazacyclododecane-N, N', N'', N'''-
tetraacetic acid: was prepared from cyclen (Aldrich,
X33,965-2) according to Desreux (1980);
3~ DTPA: Diethylenetriaminepentaacetic acid (Aldrich, #28,556-
O);
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EDAC: l-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide
hydrochloride (Sigma, #E6383);
EDTA: Ethylenediaminetetraacetic acid;
PL: Poly-d-lysine hydrobromide, MW 15,000-30,000, degree
S of polymerization - lO0 (Sigma, #P4408);
WGA: Wheat germ agglutinin; lectin triticum vulgaris
(Sigma, #L9640).
The contrast agent of the present invention for
P~Ancing visualization in imaging or for use in non-imaging
lO applications is a conjugate of a carrier with a chelating agent.
Additionally, the conjugate is complexed with a visually
enhancing num~er of atoms of a suitable paramagnetic metal. The
contrast agents advantageously and unexpectedly possess an
overall increase in conspicuity. Such an increase in
l~ conspicuity favorably enhances visualization of an image, for
example, in MRI. For purposes of the present invention, visual
enhancement of an image is defined as either a positive or
negative contrast. Conspicuity may be defined as the ability to
detect the area which is ~nh~ced by the contrast agents of the
20 present invention relative to the background tissue.
Conspicuity is a measure of the relative signal intensity of the
area of interest relative to the rest of the tissue, taking into
account background signal intensity due to noise. Accordingly,
conspicuity is a measure of enhanced or improved contrast of an
25 image or signal intensity o~tained by way of the contrast agents
of the present invention.
The reactants for making the contrast agents of the
in~ention are generally known compounds, and otherwise are
routinely prepared by techniques within the skill of the
30 chemist.
The group of suitable carriers for conjugation with
chelating agents for the synthesis of the contrast agents of the
present invention is broad and includes, ~ut is not limited to,
oligopeptides, proteins, lipids, polysaccharides, dextran
3~ polymers and synthetic polymers.
Any protein is considered suitable as a carrier. A
group of particularly suitable protein carriers is comprised of
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a polymer or copolymer of polyamino acids. Preferred are
polymers or copolymers of ~asic amino acids such as polylysine
and polyornithine. To protect the contrast agents of the
invention over longer periods of time from protease degradation,
such as by ubiquitin, the d-isomer forms of the polyamino acids
are particularly preferred. The d-isomer forms can not be
readily degraded in most li~ing tissues, however the d-isomer
forms can be degraded by the li~er and kidney, where d-proteases
are present.
One of skill in the art should readily appreciate that
the polyamino acid polymer or copolymer carriers may have
various amino acid substitutions, so long as such substitutions
do not have a deleterious effect on the carrier properties of
the polyamino acid polymer or copolymer. Such substitutions are
therefore considered to be within the scope of the invention.
Examples of other suitable carriers for the chelating
agent-paramagnetic metal complex include macromolecules,
emulsions, liposomes, and microspheres, which are of a size
typically less than 5 microns in diameter to avoid entrapment
within (and possible adverse effects to) the lungs. Further
examples of alternative or substitute carriers for the chelating
agent-paramagnetic metal ion complex are described in U.S.
Patent No. 5,213,788, which is incorporated herein by reference.
Another suitable protein carrier is cholera toxoid.
The length of the carrier may range from about 50 to
about 200 residues, more preferably about 100 residues. The
molecular weight (MW) may range from about 15,000 to a~out
60,000, preferably from about 15,000 to about 30,000.
The chelating agents for conjugation with the carrier
molecule, and complexing with the paramagnetic atom can be
selected from the non-toxic group of chelating agents of
polyaminopolycarboxylic acids as described in Chapter 14 of
Stark and Bradley. Particularly suitable chelating agents for
practicing the present invention are DOTA and DTPA.
Examples of other suitable alternative chelating
agents are as follows: Aquo iron, EDTA, DTPA-BMA, BOPTA, TTHA,
NOTA, DO3A, HP-DO3A, TETA, HAM, DPDP, Acetate, TPPS4, E~PG,
HBED, and Desferrioxamine B. Additional chelating agent
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derivatives which may be su~stituted in the present invention
are described in U.S. Patent No. ~,281,704, which is
incorporated herein by reference.
With regards to paramagnetic species, any atom having
5 paramagnetic properties is considered suitable in the practice
of the invention. This includes transition elements of atomic
num~ers 21-29, 42 or 44 and elements of the Lanthanide series.
These transition metals include Cr'3, Cr~2, Mn'3, Mn~2,~e~3 Fe~2,
Cu~2, Co~3, Co~2, Ni~2 and radioisotopes thereof. Preferred are
10 mem~ers of the Lanthanide series which are num~ered 59-70. This
omr~cses Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, and Yb,
and the respective radioisotopes thereof. More particularly
preferred is the Lanthanide element gadolinium (Gd). Gadolinium
is particularly suitable as the paramagnetic metal of the
1~ contrast agents of the present invention as it has a large
magnetic ~o~ent, which eficiently relaxes magnetic nuclei.
Gadolinium's strong paramagnetic properties are the result of
its seven unpaired electrons. The unexpectedly highly
concentrated gadolinium incorporated in the contrast agents of
20 the present in~ention readily passes through the ~ody and is
safely excreted without causing toxic side effects. Other
examples of alternative paramagnetic species are disclosed in
U.S. Patent No. ~,213,7~8 which is incorporated herein by
reference.
2~ Another advantageous aspect of the contrast agents of
the present invention is an embodiment which is the conjugation
of the contrast agents with ~arious macromolecules, which
macromolecules have the capability of targeting a tissue or
organ. The contrast agents of the present invention can ~e
30 coupled as conjugates with such macromolecules that are known to
target an organ or part of an organ to ~e examined.
Particularly desirable or interesting macromolecules are those
which are capable of targeting specific sites, e.g., cellular
receptors or antigens. This favora~ly provides for target-
3~ specific contrast agents.
These target-specific macromolecules include, ~ut are
not limited to, hormones such as insulin, prostaglAn~inc,
ste~oid ~o~Qnes, amino sugars, peptides, proteins, e.g., serum
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albumins, lipids, and polysaccharides. Antibodies, such as
polyclonal or, more particularly, monoclonal, are especially
suitable for conjugation with the contrast agents of the
invention for targeting specific sites of interest.
Particularly interesting examples of monoclonals are those which
are specific to tumor-associated antigens, or which exhi~it a
desired diagnostic specificity, e.g., antimyosin. These
contrast agents of the present invention provide a further
battery of useful tools for diagnostic image analysis. Non-
10 limiting examples of tumor-specific monoclonals would include:
breast, lung, and prostate cancers. Other suitable monoclonal
anti~odies may be directed to non-tumor antigens, for example,
Alzheimer's mar~er antigens, which may advantageously provide
for earlier clinical diagnosis and pharmaceutical intervention
1~ to ameliorate the disease.
The present invention ad~antageously and unexpectedly
provides target-specific contrast agents, which maintain their
target specificity in vivo. Such conjugated target-specific
contrast agents of the present invention advantageously allow
20 for selection of appropriate ~iodistribution characteristics.
These conjugates permit tissue or organ targeting, i.e.,
preferential delivery to such tissue material as, e.g., tumors.
This in turn provides for improved imaging characteristics,
e.g., contrast, better selectivity, contrast/noise ratio,
2~ imaging time, enhanced signal intensity, and the li~e, for
imaging such targets of interest.
~CETHOD OF ~N~'~S~8
A method of m~k;ng or synthesizing the contrast agents
of the present invention is carried out as described
30 hereinafter. The method of the invention provides for a
preparation of stable and non-toxic contrast agents, which
method of synthesis is simpler and more efficient than other
currently available techniques. The method uses commercially
available reagents. The method also advantageously provides
3~ flexibility in the production of small or large quantities of
the contrast agents of the invention for administration,
dep~n~in~ upon the size of the subject ~mm~l. Suitable and
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preferred materials or substituents for the contrast agents of
the present invention have been described above.
For the synthetic method, an appropriate chelating
agent and conjugating agent can be dissolved in an aqueous
solution, which solution is suitable for maintA;nin~ the linear
structure of the carrier to be added. Preferred is a saturated
urea solution. The volume of the aqueous solution may range
from about 200 microliters to about 20 ml. Any conjugating
agent is suitable. Preferred conjugating agents are EDAC or
10 glutaraldehyde. An appropriate base can be added by any
a~o~iate means to the solution and in an amount sufficient to
assure complete dissolution of the chelating agent.
The amounts of chelating agent may range from about 9
to 900 mg. The amount of conjugating agent may range from about
15 20 ~mol. to about 2 mmol, and may vary depending on the amount
of contrast agent desired.
A suitable amount of carrier is then added to the
solution for conjugation with the chelating agent. This amount
may range from about 2 mg to about 200 mg. pH is maintained at
20 less than about 5 by addition of any suitable acid. After about
one hour, a sufficient amount of water may be added to the
solution. The solution is then incubated for a suitable period
of time sufficient to allow for complete conjugation of the
carrier and complexing agent. For example, the solution may be
25 typically incubated for a period of about 12 hours at a
temperature of about 4C.
Following incubation, the solution is warmed to
approximately room temperature. A suitable paramaynetic metal
salt is then added to the solution. For example, gadolinium
30 chloride hexahydrate (Gd C13 6H2O) may be added. A suitable
amount of the paramagnetic salt may range from about 10 mg to
about 1 g. The pH of the reaction is maintained above about 7
by way of addition of an appropriate ~ase. After a time
sufficient to allow for complete conjugate formation of the
3~ carrier-chelating agent - paramagnetic metal complex, the
solution may be dialyzed. At least about 5 hours is typical to
allow for complete conjugation. Dialysis is carried out with an
appropriate chelating agent for a time sufficient for removing
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any excess paramagnetic metal. Generally suitable is dialysis
with ~DTA for about 24 hours. The dialysis may be carried out
by any method known to those skilled in the art. The solution
cont~in;ng the contrast agent of the present invention may then
=
be dialyzed against water for a period of time sufficient for
de-salting. This is typically for about 24 hours. Other known
de-salting techniques may ~e employed such as gel-filtration.
The solution may then be stored in liquid or lyophilized form,
if desired, for later use.
If one desires to analyze with the aid of
fluorescence, a suitable fluorescing agent may be added along
with the addition of the paramagnetic metal. Typically, the
molar ratio of paramagnetic metal to fluorescing agent ranges
from about 2:1. An example of a suitable fluorescing agent is
1~ TbCl3.
The degree of conjugation of carrier and chelating
agent after the first conjugation reaction of the method of the
invention is typically less than about 35~. This value is
independent of an excess of chelating reagent: a double excess
20 of, for example, DOTA is required due to the possibility of its
precipitation at low pH. The degree of conjugation has been
noted to decrease with higher pH. While wishing not to be
limited or bound by any particular theory, the relatively low
efficiency of the first conjugation may be due to the
25 interaction between conjugated chelating agent and carrier. For
the complexing agent, DOTA, it has a pK~=4.41; pK2=4.54; pK3=9.73
(Stetter, 1976). These values can further decrease for the
conjugated molecule, which has a modified carboxyl. This
probably means that two carboxyls of DOTA have a negative charge
30 at a pH of about ~. At the same time, most of the amino groups
of the polyaminoacid carrier, such as polylysine, are charged
positively. As a result of electrostatic interaction, every
conjugated molecule of DOTA is able to block at least two
unbound amino groups of polylysine preventing their further
3~ participation in the reaction. Complexing with Gd3~ compensates
for the negative charges of DOTA. This favorably releases amino
groups fo further conjugation, if desired by one of skill in
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the art, to advantageously increase the efficiency of
conjugation of the method of the invention.
The method of the invention advantageously provides a
second conjugation step for increasing the efficiency of
~ 5 conjugation, which utilizes and subjects the conjugate
synthesized in the first conjugation with all the steps
descri~ed above. In the second conjugation, the conjugate
replaces the carrier used in the first conjugation. The second
conjugation reaction unexpectedly and advantageously results in
10 an increased concentration of paramagnetic atom bound to the
conjugate. ~ollowing the second conjugation, the concentration
of paramagnetic atom shows an increased efficiency of
conjugation in a range from about 50 to about 55~. The
increased concentration of ~ound paramagnetic atom (and hence
15 increase in efficiency of conjugation) is demonstrated by
comparing relaxation times for the respective conjugations. The
first conjugation reaction shows that the relaxation time, Tl,
is equal to 0.15 seconds. The value of T1 for the second
conjugation is advantageously and unexpectedly shortened to 0.08
20 seconds for a 1.5 ml solution, which is equal to 3 and 5 ~mol
Gd. DOTA, respectively, for relaxivity of Gd. DOTA (3.4 mM~ls~l).
The increased concentration of ~ound paramagnetic atom
has been quantitated to advantageously and unexpectedly
represent a number ranging ~etween about 150 to 200 atoms. This
25 is a concentration not heretofore reported.
The amount or yield of contrast agent synthesized by
way of the present invention typically ranges from about 2 mg to
about 200 mg of product, dep~n~; n~ on the amount of reactants
utilized.
The contrast agents of the invention may be further
advantageously protected from ~iodegradation, which otherwise
may occur during in vivo use. Any treatment with which one of
skill in the art is familiar may ~e suitable, so long as the
treatment does not disrupt the integrity of the complex.
35 Preferred is acetylation. Other examples are treatment with
succinic or propionic anhydride.
The contrast enhancing agents of the present invention
are readily usa~le in any detection or imaging system involving
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administration of paramagnetic marker or tracer ions~ The
appropriate paramagnetic metal may be added to the carrier-
chelate complex at a suitable pH consistent with stable
chelation ~i n~; ngS .
Another em~odiment of the present invention provides a
method for producing target-specific contrast agents for
imaging, particularly for, but not limited exclusively to MRI
applications. These contrast agents of the present invention
are suitable for use both as diagnostic pharmaceuticals in
10 clinical medicine and as ~iologic probes. Target-specific
contrast agents are particularly useful, because they aid the
medical practitioner in locating specific tissues or organs of
interest in a patient for detection of abnormal or diseased
tissue. The specificity of location is provided by the contrast
1~ agents' specific bonding to the tissue or organ to be examined.
The tissue of interest to ~e examined is thus bound by the
target-specific contrast agents of the present in~ention. The
target tissue, when diseased, effectively contrasts with the
normal surrounding tissues during an imaging procedure. The
20 target-specific contrast agents of the invention having
enhanced conspicuity or signal intensity advantageously provide
a visually-enhanced image of a particular tissue of interest.
The ad~antages of such are readily apparent.
With regards to synthesizing target-specific contrast
25 agents, any macromolecule having target-specific capability is
considered suita~le for conjugation with the contrast agents of
the present invention.
Any protein, or peptide fragments thereof, is suitable
for linking or conjugating to the contrast agents of the
30 invention for the purpose of targeting a specific tissue. Non-
limiting examples of such suitable targeting proteins are
hormones, antibodies, polyclonal or particularly monoclonal, or
the Fa~ fragments thereof, and lectins, such as wheat germ
agglutinin. For example, a contrast agent-antibody com~ination
3~ may ~e used to locate specific diseased tissues, such as breast,
lung, ~rain, and prostate tumors, which possess antigenic
det~rm;nAnts specific to the antibody conjugated to the contrast
agents of the invention. Alternatively, non-tumor sites of
-
-
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interest may also be targeted by way of a suitable antibody,
e.g., a marker antibody for Alzheimer's.
The contrast agent-wheat germ agglutinin combination
may be advantageously used as a means to locate and target
5 particular neural connections of interest within the m~mm~l ian
~rain as a means of ~rain mapping.
Other macromolecules which are suitable for targeting
include prostaglA~;n~, zmino sugars, polysaccharides and
lipids. Hence, these macromolecules may also be utilized in the
10 synthesis of the target-specific contrast agents of the present
inventlon.
With regards to synthesizing the ~arget-specific
contrast agents of the present invention, the method provides
for the conjugation of the macromolecule with the contrast agent
15 of the invention. For example, the contrast agent may be
provided in a suitable salt solution. The macromolecule can
then be added to the solution along with an appropriate
conjugating agent. The reactants are incubated for a period of
time sufficient for conjugation of the macromolecule with the
20 contrast agent. Typically, a period of about 24 hours, in which
the target-specific conjugate is maintained at a temperature of
about ~C, is suitable. The target-specific contrast agent can
then ~e separated from excess conjugating agent by any
appropriate method known to one of skill in the art. One
25 suitable method is gel filtration. Other methods are readily
known to one of skill in the art. The contrast agents can be
stored as described as above.
We have ~em~n~trated that advantageously and
unexpectedly between about 1~0 to 200 paramagnetic ions are
30 attached per macromolecule of the conjugate. This is a greater
number of attached or bound paramagnetic ions than has
heretofore been reported.
~CET~ODS OF ADMINIST~U~TION
Another aspect of the present invention is directed to
35 a method for clinical or diagnostic analysis by administering
the contrast agents of the present in~ention to a host,
preferably a m~m~liarl host, in an amount s~lffi~ient to effect
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the desired enhanced contrast (or shift). The host may then be
subjected to diagnostic analysis. Preferably, diagnostic
analysis is MRI or NMR imaging analysis. Further, the contrast
agents are useful in x-ray image or ultrasonic analysis. While
5 described primarily as contrast enhancing agents, the contrast
agents of the invention may also act as NMR shift reagents, and
such use is contemplated to be within the scope of the
invention.
A detailed discussion of the theoretical
10 considerations in selecting the appropriate parameters for MRI
and NMR diagnostic analysis is disclosed in U.S. Patent
No. 4,749,560 which is incorporated herein by reference. CAT
scans, x-ray image analysis and ultrasonic diagnosis are carried
out in accordance with well-established techniques.
The contrast agents of the invention may be
administered to a mAmm~l, including a human patient, in the form
of a pharmaceutical composition in a contrast-, or visually-
enhancing amount, together with a pharmaceutically acceptable
carrier .
The contrast enhancing agents of the invention are
administered in an amount clinically or diagnostically
sufficient to effect the desired ~nh~nced contrast. For MRI,
this amount is an MRI signal-affecting amount, i.e., an amount
that will alter the spin-lattice, spin-spin or spin-echo
25 relaxation times of an MRI signal. This alteration is affected
so as to ~nh~nce the signals received from the patient under
analysis by reducing the aforementioned relaxation times with
respect to an area of the patient.
In another embodiment, the MRI signal affecting amount
30 is that amount, which in addition to altering the relaxation
times of the NRI signals in the patient, will also
advantageously sufficiently alter such relaxation times, so that
the desired le~el of differentiation can be achieved. This
provides a visually-enhanced differentiation between those parts
35 of the patient that have and have not ta~en up the contrast
agents of the present invention.
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The enhanced visualization of an image by way of the
present invention favorably results from the increased
concentration of bound paramagnetic atom, as described above.
With regards to administration, the compositions of
5 the present invention are A~m;n;stered in doses effective to
achieve the desired enhancement or improved contrast. Such
doses may vary, dep~n~; n~ upon the particular paramagnetic ion
complex employed, the organs or tissues targeted, MRI equipment
and the like. Effective amounts typically may range from about
lO ~ to about 500 micromoles of the paramagnetic ion complex per
liter. The doses ~;n;stered orally or parenterally may range
from about l to about lO0 micromoles per kilogram of body
weight, which corresponds to about l to about 20 mmol for an
adult human patient. For smaller patients or An;m~ls, the
l~ dosage may be varied accordingly.
The particular paramagnetic atom employed and organ to
~e imaged will determine the waiting period ~etween
administration and imaging. It will generally be at least about
15 minutes but typically less than about an hour.
Compositions are provided having effective dosages of
contrast agents in the range of about 0.OOl-~mmol per kg for NMR
diagnostics, preferably about 0.005-O.~mmol per kg; in the range
of about 0.l-~mmol per kg for x-ray diagnostics; and in the
range of about 0.l-~mmol per kg for ultrasound diagnostics.
The compositions of the present invention can be
~dm; n; stered by any number of well-known routes. These include
intravenous, intraarterial, intrathecal, intraperitoneal,
parenteral, enteral, oral, intrapleural, subcutaneous, by
infusion through a catheter, or by direct intralesional
30 injection.
While one of skill in the art may readily ascertain an
effective route of A~m;n;s ration of the contrast agents of the
invention, the following guidelines are provided. Intravenous,
intraarterial or intrapleural administration is generally
3~ suitable for use for lung, breast, and leukemic tumors.
Intraperitoneal administration is suita~le for ovarian tumors.
Intrathecal administration is suita~le for brain tumors.
Subcutaneous ~ ;stration is suitable for Hodgkins disease,
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lymrhom~ and breast carcinoma. Catheter infusion is useful for
metastatic lung, breast or germ cell carc;nom~s of the liver.
Intralesional administration is useful for lung and breast
lesions. Depending on the route of A~m; n;stration~ the
5 phArmAceutical compositions may require protective coatings,
which are known in the art.
For parenteral A~m; n; stration, the compositions may be
injected directly or mixed with a volume of carrier sufficient
for systemic ~m; n; stration. Formulations for enteral
10 administration may vary widely, as is well-known in the art. In
general, such formulations include an effective amount of the
contrast agent of the invention in aqueous solution or
suspension. Such enteral compositions may optionally include
buffers, surfactants, thixotropic agents, and the like.
15 Compositions for oral administration may also contain flavoring
agents and other ingredients for enhancing their organoleptic
qualities.
The pharmaceutical compositions of the present
invention cont~;n;ng the contrast agents of the present
20 invention, which contrast agents are essentially neutral, may be
provided for injectable use in sterile solutions or dispersions,
or in sterile powders for the extemporaneous preparation of
sterile injectable solutions or dispersions. The composition is
preferably sterile and fluid.
Sterilization can be achieved by any art recognized
technique, including but not limited to, addition of
antibacterial or antifungal preservatives, for example, paraben,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like, so
long as the integrity of the contrast agent of the composition
30 is not adversely affected. ~urther, isotonic agents, such as
sugars or sodium chloride may be incorporatedinto the present compositions.
Production of sterile injectable solutions cont~;n;n~
the contrast agents of the present invention is accomplished by
incorporating the contrast agents in an appropriate solvent with
35 various ingredients enumerated above. Sterilization may then be
carried out, for example, by filter sterilization. To obtain a
sterile powder, the above solutions may be vacuum-, or
freeze-dried as necessary.
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The contrast agents of the invention are thus
compounded for convenient and effective administration in
ph~rm~ceutically effective amounts with a suitable
phArm~ceutically accepta~le carrier in a dosage form, which
composition favorably affects contrast enhancement or
conspicuity in a manner which heretofore has not been
accomplished.
As used herein, a phArmAceutically acceptable carrier
includes any and all solvents, dispersion media, coatings,
lO preservatives, antibacterial and antifungal agents, isotonic
agents, and the like. The use of such materials is well-known
in the art.
Typical pharmaceutically acceptable carriers are well-
known and include a solvent or dispersion medium containing, for
1~ example, water, buffered a~ueous solutions (i.e., biocompatible
buffers), ethanol, polyol (glycerol, propylene glycol,
polyethylene glycol and the like), suitable mixtures thereof,
surfactants or vegetable oils.
IT
Another embodiment of the invention is a kit
cont~;n;ng the contrast ayents of the present invention suitable
for commercial sale.
With regards to the kit, a solution of the contrast
agents may be sterilized and made up into containers of ampules
2~ or vials, or may be lyophilized to a powder for dissolution when
ready to be used. The contrast agents may be mixed with a
carrier, as discussed above. If the contrast agent is provided
in lyophilized form, the carrier may also ~e provided in
appropriate vials or ampules for m;~; n~ with the lyophilized
30 powder. If desired, ampules may contain lyophilized powder of
the contrast agent in one compartment and a carrier in another,
the compartments being separated by a frangible barrier. When
ready to use, the barrier is broken and the ampule is shaken to
form a solution suitable for administration.
3~ Prior to A~m; n; stration of the contrast agent of the
present invention, the reconstituted contrast agent may be
~urthér diluted ~y addition of a suitable diluent such as:
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Ringers Injection, USP
Sodium Chloride Injection, USP
Dextrose Injection, USP (5% dextrose in sterile
water)
Dextrose Sodium Chloride Injection, USP (5
dextrose in sodium chloride)
Lactated Ringers Injection, USP
Protein Hydrolysate Injection Low Sodium, USP
Water for Injection, USP, preferably of a
suitable osmolality.
The amounts of the contrast agents in solution or
lyophilized powder form may be provided in any suita~le amount
for commercial use in the appropriate clinical setting.
Alternatively, reagents for making the contrast agents
15 of the invention may be pro~ided in separate ampules or vials
for the purpose of the buyer or user synthesizing the contrast
agent by way of the present invention, if so desired.
A~T~NATIVE ~ OGI~S
It is contemplated that the contrast agents of the
20 present application can be employed in a wide variety of
applications.
Recent advances in MRI and related technologies have
resulted in new and potential applications of the contrast
agents of the present invention. The development of
25 superco~flllcting quantum interference devices (SQUIDS) has led to
exceedingly sensitive detectors of magnetic fields which can be
utilized to measure relaxation ~he~om~n~, as described in
Scientific American, August, 1994, which is incorporated herein
~y reference. The sensitivity of SQUIDS in detecting changes in
30 magnetic flux is 100 times more sensitive than the amount of
mechanical energy to raise a single electron one millimeter in
the earth's gravitational field or I0-32 Joules. These devices
approach the quantum f~ln~mPntal boundaries as set by
Heisenberg's uncertainty principle. SQUID systems are
35 increasingly being utilized for biomedical applications. Thus,
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the contrast agents of the present invention may play a role in
this evol~ing technology.
The contrast agents of the present invention can also
~e utilized in conjunction with other types of imaging
5 technology including, but not limited to, x-ray, ultrasound and
acoustic imaging. In particular, the radioactive metal species
and complexes of the contrast agents of the invention can be
utilized as diagnostic, monitoring and therapeutic agents. With
regards to particularly interesting medical applications, the
l0 contrast agents of the invention can be utilized in a ~ariety of
radiographic procedures including, but not limited to, those
invol~ing cardiography, coronary arteriography, aortography,
cerebral and peripheral angiography, arthrography, intravenous
pyelography and urography.
In addition, the contrast agents can be advantageously
utilized as carriers to deliver pharmaceuticals, including
radioph~rm~ceuticals, to various body sites with or without
target specificity. The advantage of this technique is that
drug deli~ery can be monitored in real time with specificity to
20 the organ system or tissue under e~m;n~tion. Furthermore, the
ph~r~ceutic can be targeted to a given site ~e.g., a tumor or
site of infection) and/or release its therapeutic agent at the
target site.
Contrast agent utilization is not limited to medical
25 applications. As magnetic resonance and other imaging and
monitoring technologies proliferate, more industrial
applications are evolving. For example, magnetic resonance
technology has been applied to oil exploration. The ad~ent of
SQUIDS has made it possible to map the earth's crust to
30 determine the whereabouts of oil or geothermal energy sources.
In this context, contrast agents can be utilized to map, track,
and monitor oil deposits and their flow rates. In addition,
contrast agents can ~e used to diaynose flow rate and/or leaks
in oil pipeline delivery systems.
Other types of industrial usage include monitoring and
feed~ack systems in productior, or manufacturing processes as
disclosed in U.S. patent 5,015,954, herein incorporated by
~eference. The contrast agents of the present in~ention can ~e
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applied to such processes to assist in the means of monitoring,
analysis and quality control.
AD~ANTAGES
The present invention represents a significant
5 improvement in the state of the art with regards to contrast
agents and method of synthesis of the agents. Conjugating
anti~odies with gadolinium has been known for several years, but
the present method of conjugation produces a more clinically and
commercially useful contrast agent. The method of the invention
10 provides contrast agents having a greater concentration of bound
paramagnetic atom than heretofore accomplished. This
advantageously and unexpectedly provides for enhanced signal
intensity or conspicuity. The contrast agents of the invention
thus provide a superior MRI contrast, as compared with other
15 contrast agents.
Further in this regard, the present invention
advantageously provides contrast agents having a significantly
higher concentration of a paramagnetic metal, such as
gadolinium, conjugated to a targeting molecule than has
20 heretofore been achieved. The heavier-loaded targeting molecule
surprisingly better retains its biological activity than in
other reported methods.
The contrast agents of the present invention
unexpectedly provide for shorter imaging time at a given level
25 of point resolution. Shorter imaging times are achieved because
of the greater signal ~n~ncement and image contrast produced
per unit utilized of the contrast agents of the present
invention.
The method of the present invention for making the
30 contrast agents is also advantageously and unexpectedly simpler
and easier than other reported methods.
Given the tr~m~ous growth in the diffusion of
clinical NRI technology over the past decade, and the current
healthcare environment with concerns over cost and duplication
35 of diagnostic tests, the present invention advantageously
represents a significant potential for com~ining the
~ ~ = =
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- 29 -
technological strengths of MRI with target-specific imaging
techniques, such as nuclear medicine.
While the preferred aspects and em-hodiments of the
in~ention have been described in detail a-hove to allow one of
skill in the art to carry out the invention, it should be
appreciated that various su~stitutions may be made, if one of
skill in the art is satisfied with less than preferred or
optimal contrast agents.
The invention is further illustrated by the following
10 examples, which should not he construed in any fashion as
limiting the spirit or scope of the invention.
EXAMPLE 1
PREPARATION OF PL-Gd-DOTA BY TWO-STEP CARBODIIMIDE CONJUGATION
In a typical preparation 9 mg of DOTA (22 umol) and 3.84 mg
1~ of EDAC (20 umol) are dissolved in 200 uL of saturated urea
solution. Addition of lN NaOH can be added to assure complete
dissolving of the DOTA, 2.1 mg of polylysine PL (10 umol lysine,
100 nmol PL) is added. p~ is maintained less at than 5 ~y lN
HC1. After 1 hr, 200 uL water is added and then the solution is
20 incubated 12 hrs at 4C.
The solution is warmed to room temperature and 10 mg of
GdC13-6H2O (27 umol) is added; pH is maintained above 7.0 ~y lN
NaOH. If experiments include analysis of fluorescence, an
equivalent quantity of GdC13 and ThC13 mixture with molar ratio
25 2:1 is used. After 5 hrs., the solution is dialyzed (MWCO
12,000-14,000) 24 hrs against 8 mM trisodium EDTA and 24 hrs
against water and lyophilized. The yield of PL-Gd-DOTA after
lyophilization is typically 2.2-2.5 mg.
In the second step of conjugation all the procedures are
30 performed in the same way using prepared PL-Gd-DOTA instead of
PL. Another chelating agent, DTPA, may be used in the reaction
instead of DOTA. In this case, all the operations and
quantities of reagents are the same as in the first conjugation.
EXAMPLE 2
3~ PRO~ ON OF PL-Gd-DOTA FROM BIODEGRADATION
Dissociation of conjugated polylysine within the cells
creates a serious problem for in vivo use. There are several
mechanisms by which cells may degrade exogenous proteins. The
most important is h;~;ng of uhiquitin to the amino group of
40 lysine with the further removal of bound amino acid from the
protein chain. This process can he prevented by acetylation of
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- 30 -
residual amino groups of PL-Gd-DOTA making binding between them
and ubiquitin impossible.
After the second step of conjugation, an equal volume of
saturated sodium acetate is added to the dialyzed solution. The
5 mixture is cooled on ice and 30 ,uL of acetic anhydride is added
in 5 ,uL portions at 30 minute intervals. The pH is checked
before each addition and maintained at 9 by lN ~aOH to prevent
the dissociation of Gd-DOTA in acid ~; A . The solution is then
incubated for 12 hrs at 4C and desalted by dialysis and
10 gel-filtration (Sephadex G-25, water).
Acetylation insures protection of the polymer from rapid
destruction by ubiquitin. In order to protect the contrast
agent over longer periods of time from protease degradation,
poly-d-lysine (or poly-d-ornithine) instead of biological
15 l-isomer is used. This polymer cannot be easily degraded in
most living tissues, however it can be utilized by the liver and
kidney, where d-proteases are present.
EXAMPLE 3
CONJUGATION OF PL-Gd-DOTA WITH PROTEINS
Essentially desalted PL-Gd-DOTA solution is lyophilized and
dissolved in 100 u~L of 10 mM KH2PO4 followed by the addition of 1
mg of EDAC and 0.5 mg of WGA (12 nmol). After 24 hrs at 4C,
the conjugate is separated from excess of glutaraldehyde by
gel-filtration (Sephadex G-50, water). The M~I-analysis shows
25 that 150-200 Gd atoms (or 3-4 polymer r~ ) are attached to 1
molecule of WGA. According to Wright (1984), WGA has a dimer
structure; each monomer has 6 lysine residues, which are
probably involved in the conjugation with carboxyl groups of
PL-DOTA complex. Prepared contrast agent is used for direct MRI
30 investigation of axonal transport in the cat brain and (with
addition 30% TbCl3) for its fluorescence visualization.
EXAMPLE 4
CONJUGATION OF PL-Gd- DOTA WIT~ ANTIBODY
The same method of conjugation is used for preparation of
35 the ~RI contrast agent with monoclonal IgGl anti-vitamin Bl2
antibody (Sigma, #V9505, clone #CD-29). Antibody solution is
dialyzed against water and 30 ~L (5 nmol of protein) is
conjugated with PL-Gd-DOTA obtained from 1 mg of PL. Antigen is
then added and the mixture is gel-filtered through SephA~ex
40 G-200 with elution by water. The first elution peak with
absorption at 280 nm has also 360 nm and 548 nm absorption of
vitamin Bl2, which means the presence of the antibody-antigen
complex. This peak is separated from the major peaks of
excessive polylysine and antigen. Eluted solution of antibody-
45 antigen complex has a concentration of antibody of 0.6 ~M,antigen of 1.7 ~M (measured spectrometrically) and Gd-DOTA 130
~M (measured by MRI; T~=1.5s). This means that up to 200 Gd
CA 022017~0 1997-04-03
WO96110359 PCT~S95112693
- 31 -
atoms are conjugated to anti~ody without loss of its ability to
bind antigen.
EXAMPLE 5
CONJUGATION WITH GLUTARALDEHYDE
5If conditions of carbodiimide reaction are not suitable for
protein survivability, conjugation with PL-Gd-DOTA can be
provided ~y glutaraldehyde. A molecule of acetylated polymer
still has several free amino groups, which are able to react
with glutaraldehyde. In a typical preparation, a solution of
l0 PL-Gd-DOTA in 300 ~uL l00mN phosphate buffer (pH=7) is added in
30 ~L portion to ice-cooled 200 uL 2~ glutaraldehyde. After 12
hrs, the incu~ation mixture is gel-filtered (Sephadex-25, P8S).
The fraction with absorption at 280 nm is collected and added to
the protein solution. The mixture is incubated 24 hrs at 4C
l~ and gel-filtered through the gel, which is suita~le for used
protein. This method of conjugation has an efficiency
compara~le to carbodiimide reaction and is suitable for
preparation of the MRI contrast agents conjugated with
antibodies and other macromolecules.
EXAMPLE 6
IN VITRO TESTING FOR CONTRAST CONSPICUITY AND RELAXIVIT~
Adequate potential for signal enhancement was confirmed by
eX~m;n;ng vials filled with sample solutions in a 1.5 Tesla
clinical M~ imaging machine (Signa Systems, GE, Milwaukee, USA).
2~ Qualitative evaluation of signal intensity relative to water
vials and gel phantoms was performed as was a more rigorous
quantitative evaluation. For quantitative measurement, multiple
imaging sessions were performed using a linear extremity coil
with varying TR for a fixed TE ~alue with other parameters held
30 constant. The signal intensity was measured by two observers in
consensus fashion at the imaging system console using a region
of interest cursor. This was performed ~or each vial after each
TR increment. The TR was varied from 1600 ms to less than l00
ms and the signal intensity alteration was plotted versus the
3~ repetition time in order to enable a calculation of the
approximate Tl relaxation rate constant. Figure l shows a
sample image from a Tl relaxation experiment.
EXAMPLE 7
ADNINISTRATION OF CONTRAST AGENT CONJUGATED
40WITH WHEAT GERM AGGL~ (WGA)
Using a stereotaxic frame, an anesthetized cat was
maintained in the Horsley-Clark plane. Under aseptic conditions
and in compliance with all Federal, University and local
regulations a craniotomy was performed. The visual cortex was
4~ ex~osed and a Hamilton 30 gauge microliter syringe was preloaded
with the contrast agent cont~;n;ns gadolinium conjugated with
WGA for injection. This was guided to 2 mm ~elow the cortical
CA 022017~0 1997-04-03
W O96/10359 PCTrUS95/12693
- 32 -
surface and 0.2-0.4 microliter amounts of the conjugate at a
concentration of 20 mg/ml was injected with greater than 1
millimeter of surface separation. The craniotomy was closed
after the injections were completed and the ~;m~l was allowed
5 to recover. Axonal tracing was carried out as shown in Figures
3 and 4. Fluorescence microscopy was carried out using TbCl3 to
confirm the presence of the contrast agent, as shown in Figure
5.
EXAMPLE 8
ADMINISTRATION OF CONT~AST AGENT CONJUGATED
WITH ANTIBODY TO ANTIGEN 301
Using a stereotaxic frame, the anesthetized cat was
maintained in the Horsley-Clark plane. Under aseptic conditions
and in compliance with all Federal, University and local
15 regulations a craniotomy was performed. The visual cortex was
exposed and a 25 gauge 3.5 inch needle was mounted vertically in
a stereotactic carrier. Using an atlas for guidance, the
ventricle was entered and a small quantify of CSF-approximately
the same volume as the injectate-was removed. The material
20 (0.33 ml) of cat 301 conjugated with the gadolinium contrast
agent of the present invention at a concentration of 3 mg/ml was
then infused into the left ventricle and the craniotomy was
closed. Figure 2 shows a coronal image of the contrast agent,
conjugated with antibody directed to antigen 301, (white
25 material shown by arrows) localized in the cat brain at the site
of the 301 antigen. This demonstrates that antibody conjugated
with the contrast agent of the present invention advantageously
retains its ability to bind in vivo its target antigen.
It is to be understood that the foregoing detailed
30 description and accompanying examples are merely illustrative
and are not to be taken as limitations upon the scope of the
invention, which is defined solely by the appended claims and
their e~uivalents. Various changes and modifications, including
without limitation those relating to the substituents,
35 derivatives, syntheses, formulations and/or methods of use of
the invention, may be made without departing from the spirit and
scope thereof.
CA 02201750 1997-04-03
WO 96/10359 PCT~US95/12693
-- 33 --
PUBLICATIONS
LITERATURE:
Desreux J.F. (1980). Nuclear magnetic spectroscopy of
lanthanide complexes with tetraaza macrocycle. Unusual
conformation properties. Inorg. Chem., 19, 1319-1324.
Sie~ing P.F. Watson A.D. and ~ocklage S.M. (1990). Preparation
and characterization of paramagnetic polychelates and their
protein conjugates. Bioconjugate Chem., 1(1), 6~-71.
Stetter H. and Wolfram F. (1976). Complex formation with
10 tetraazacycloalcane-N,N',N",N"'-tetraacetic acids as a function
of ring size. Angew. Chem., Int. Ed. Engl., 15(11), 686.
Wright C.S., Ga~ilanes F. and Peterson D.L. (1984). Primary
structure of wheat germ agglutinin isolectin 2. Peptide order
deduced from X-ray structure. Biochemistry, 23, 280-287.
15 Clarke J. (August, 1994) SQUIDS Scientific American, pages 46-
~3.
All publications are incorporated herein by reference.
