CA2201750A1 - Chelate complex with high conspicuity for magnetic resonance imaging - Google Patents

Abstract

Constrast agents having improved contrast characteristics for use in imaging and non-imaging technologies are provided. Methods of synthesis and administration for the contrast agents are disclosed. A kit suitable for commercial sale is also provided.

Description

W O 96110359 PCTrUS95/12693 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.

CA 022017~0 1997-04-03 W O96/10359 PCTrUS95/12693 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 CA 022017~0 1997-04-03 WO96/10359 PCT~S95/12693 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 CA 022017~0 1997-04-03 W O96/10359 PCTrUS9S/12693 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 CA 022017~0 1997-04-03 PCT~Sg5/12693 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 CA 022017~0 1997-04-03 W O9611Q359 PCTrUS95/12693 - 6 -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.

CA 022017~0 1997-04-03 W O96/10359 PCTrUS95/12693 -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 ~ ~=
CA 022017~0 1997-04-03 W O96/10359 PCTrUS95112693 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 -CA 0220l7~0 l997-04-03 PCTrUS9~/12693 _ 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.

CA 022017~0 1997-04-03 PCTrUS95/12693 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.

.
CA 022017~0 1997-04-03 W 096110359 PCTrUS95/12693 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 _ CA 022017~0 1997-04-03 W O96/10359 PCTrUS9S112693 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);

CA 022017~0 1997-04-03 WO96/10359 PCT~S95/12693 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 CA 022017~0 1997-04-03 W O96/10359 PCTrUS95/12693 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 CA 022017~0 1997-04-03 WO96/10359 PCT~S95/12693 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 CA 022017~0 1997-04-03 WO96/10359 PCT~Sg5/12693 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 CA 022017~0 1997-04-03 W O g6/10359 PCT~US9~/12693 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 CA 022017~0 1997-04-03 W O 96/10359 PCTrUS95/12693 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 CA 022017~0 1997-04-03 WO96/10359 PCT~S95/12693 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 CA 022017~0 1997-04-03 PCT~US95/12693 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 --CA 022017~0 1997-04-03 W O 96tlO359 PCTAUS95112693 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 CA 022017~0 1997-04-03 W O96/10359 PCTrUS95/12693 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.

CA 022017~0 1997-04-03 WO96/10359 PCT~S~5112693 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, CA 022017~0 1997-04-03 W 096tlO359 PCTrUS95/12693 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.

CA 022017~0 1997-04-03 WO96/10359 PCT~Sg5/12693 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:

CA 022017~0 1997-04-03 W O96/10359 PCTrUS95/12693 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, CA 022017~0 1997-04-03 WO96/10359 PCT~SgS/12693 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 CA 022017~0 1997-04-03 W O96/10359 PCTrUS9S/12693 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 ~ ~ = =
CA 0220l7~0 l997-04-03 W O96/10359 PCT~US9S/12693 -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.

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.

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 CA 022017~0 1997-04-03 W O96/10359 PCTrUS95/12693 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.

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.

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 atoms are conjugated to anti~ody without loss of its ability to bind antigen.

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.

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.

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 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.

ADMINISTRATION OF CONT~AST AGENT CONJUGATED

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.

WO 96/10359 PCT~US95/12693 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.

Claims (45)

What we claim is:

1. A contrast agent for enhancing signal intensity, which contrast agent comprises a conjugate of a carrier with a chelating agent, said conjugate being complexed with a signal intensity-enhancing number of atoms of a paramagnetic metal, said contrast agent having an increase in conspicuity, which increase enhances signal intensity.

2. The contrast agent of claim 1 wherein the paramagnetic metal is a lanthanide metal selected from the group consisting of Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm and Yb or the radioisotope thereof.

3. The contrast agent of claim 2 wherein the paramagnetic metal is Gd.

4. The contrast agent of claim 3 wherein about 150 to about 200 Gd atoms are complexed with the conjugate.

5. The contrast agent of claim 1 wherein the chelating agent is selected from the group consisting of EDTA, DTPA, DTPA-BMA, BOPTA, TTHA, NOTA. DOTA, D03A, HP-D03A and TETA.

6. The contrast agent of claim 5 wherein the chelating agent is DOTA or DTPA.

7. The contrast agent of claim 1 wherein the carrier is a polymer of a polyamino acid or a copolymer thereof.

8. The contrast agent of claim 7 wherein the polyamino acid is a basic amino acid selected from the group consisting of polylysine and polyornithine.

9. The contrast agent of claim 8 wherein the polyamino acid is in the d-isomer form.

10. The contrast agent of claim 7 wherein the carrier is of a MW ranging from about 15,000 to about 60,000.

11. The contrast agent of claim 1 which further comprises a conjugated macromolecule.

12. The contrast agent of claim 11 wherein the macromolecule is a protein.

13. The contrast agent of claim 12 wherein about 150 to about 200 paramagnetic atoms are conjugated to the protein.

14. The contrast agent of claim 13 wherein the protein is an antibody or an Fab fragment thereof.

15. The contrast agent of claim 14 wherein the antibody is monoclonal or polyclonal.

16. A method of synthesizing a contrast agent for enhancing signal intensity, which method comprises conjugating a carrier with a chelating agent, and complexing the conjugate with a signal intensity-enhancing number of atoms of a paramagnetic metal, thereby forming the contrast agent having an increase in conspicuity, which increase enhances signal intensity.

17. The method of claim 16 which further comprises a second conjugating and complexing of the contrast agent with the chelating agent and the paramagnetic metal, respectively.

18. A contrast agent obtainable by the method of claim 17.

19. The method of claim 17 which further comprises conjugating the contrast agent with a macromolecule.

20. A contrast agent obtainable by the method of claim 19.

21. The method of claim 16 wherein the paramagnetic metal is selected from the group of lanthanide metals consisting of Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm and Yb or the radioisotope thereof.

22. The method of claim 21 wherein the paramagnetic metal is Gd.

23. The method of claim 22 wherein about 150 to about 200 Gd atoms are complexed with the conjugate.

24. The method of claim 16 wherein the chelating agent is selected from the group consisting of EDTA, DTPA, DTPA-BMA, BOPTA, TTHA, NOTA, DOTA, DO3A, HP-DO3A and TETA.

25. The method of claim 24 wherein the chelating agent is DOTA
or DTPA.

26. The method of claim 16 wherein the carrier is a polymer of a polyamino acid or a copolymer thereof.

27. The method of claim 26 wherein the polyamino acid is a basic amino acid selected from the group consisting of polylysine and polyornithine.

28. The method of claim 27 wherein the polyamino acid is in the d-isomer form.

29. The method of claim 26 wherein the carrier is of a MW
ranging from about 15,000 to about 60,000.

30. The method of claim 19 wherein the macromolecule is a protein.

31. The method of claim 30 wherein about 150 to about 200 paramagnetic atoms are conjugated to the protein.

32. The method of claim 31 wherein the protein is an antibody or an Fab fragment thereof.

33. The method of claim 32 wherein the antibody is monoclonal or polyclonal.

34. A method of using a contrast agent for enhancing visualization of an image, which method comprises administering to a mamma1 a pharmaceutical composition which comprises a pharmaceutically acceptable carrier and clinically effective amount of the contrast agent, which contrast agent comprises a conjugate of a carrier with a chelating agent, said conjugate being complexed with a visually-enhancing number of atoms of a paramagnetic metal, said contrast agent providing an increase in conspicuity, which increase enhances visualization of the image.

35. The method of claim 34 which further comprises exposing the mammal to an imaging procedure, thereby enhancing the image of at least a portion of the body of the mammal.

36. A composition which comprises a pharmaceutically acceptable carrier and a signal intensity-enhancing amount of a contrast agent, which contrast agent comprises a conjugate of a carrier with a chelating agent, said conjugate being complexed with a signal intensity-enhancing number of atoms of a paramagnetic metal, said contrast agent having an increase in conspicuity, which increase enhances signal intensity.

37. The composition of claim 36 wherein the paramagnetic metal is a lanthanide metal selected from the group consisting of Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm and Yb or the radioisotope thereof.

38. The composition of claim 37 wherein the paramagnetic metal is Gd.

39. The composition of claim 38 wherein about 150 to about 200 Gd atoms are complexed with the conjugate.

40. The composition of claim 36 which further comprises a conjugated macromolecule.

41. The composition of claim 40 wherein the macromolecule is a protein.

42. The composition of claim 41 wherein about 150 to about 200 atoms of the paramagnetic metal are conjugated to the protein.

43. The composition of claim 42 wherein the protein is an antibody or an Fab fragment thereof.

44. The composition of claim 43 wherein the antibody is monoclonal or polyclonal.

45. A kit for use of a contrast image for enhancing signal intensity which comprises:

(a) a container containing the contrast agent of claim 1, and (b) a container containing a pharmaceutically acceptable carrier for the contrast agent.