KR20000070544A - Polymers - Google Patents

Abstract

The present invention provides compounds comprising a straight chain, branched chain or water phase polymer backbone comprising a plurality of amine-containing acids and having one or more reporter moieties bound thereto. Such compounds can be bound to one or more targeting agents and are particularly useful as therapeutic and diagnostic agents in medical imaging techniques.

Description

Polymers

The polymers according to the invention are suitable for use in a variety of applications where specific delivery is required and in particular for the delivery of medicaments with biological activity. However, preferred uses of the polymers of the present invention utilize the enhanced imaging properties and site specificity of the polymers to capture images of organs, tissues, and cells of selected mammals using MR, X-ray, ultrasound, light, and nuclear imaging techniques. Strengthening in vivo. Polymers are particularly suitable for use as intravascular contrast agents and blood pool formulations in such imaging techniques. As such, they can be used for imaging blood vessels, such as magnetic resonance angiography, measurement of blood flow and blood volume, identification and characterization of lesions using differences in the distribution of blood vessels from normal tissues, imaging of the lungs for the examination of lung diseases and blood. It can be used for the study of perfusion.

Medical imaging techniques such as MRI and X-rays have become very important tools for the diagnosis and treatment of diseases. Some imaging techniques inside the body rely on the inherent properties of those parts of the body, such as bones, that distinguish them from surrounding tissue in certain types of imaging, such as X-rays. Other organs and anatomical tissues are only visible if they are specifically highlighted by special imaging techniques.

One such technique with the potential to provide images of various anatomical tissues involves biologically targeted image-enhanced metals. This method reduces the background and potential interference caused by simultaneous highlighting of unintended sites, but has the ability to generate or enhance images of specific organs and / or tumors or other such localized sites in the body.

It has been recognized for many years that chelation of various metals increases the physiological allowance of such metals to allow for in vivo use to enhance images of body parts. One chelate complex that has been the subject of numerous studies is Gd-DTPA. However, despite its satisfactory relaxation and stability, this has some disadvantages. Because of its low molecular weight, Gd-DTPA disappears rapidly in the bloodstream. This severely limits the imaging range, the optimal number of images that can be obtained after each injection, and increases the required volume and relative toxicity of the formulation. In addition, these simple metal chelate image enhancers generally do not provide any significant site specificity without further modification.

It has been widely proposed to combine metal chelates with tissue or organ target molecules such as biomolecules such as proteins for the preparation of site specific therapeutic or diagnostic agents. Many of these bifunctional chelating agents, i.e., chelating moieties, can bind metal ions useful for therapeutic or diagnostic purposes and can selectively deliver chelated metal ions to site parts of interest by site-specific molecular constituents. Formulations are known or have been proposed. However, disadvantages of combining metal chelates with protein carriers for use in MR imaging include inadequate biological distribution, toxicity and short blood half-life. Thus, their use in MR imaging is limited. In addition, proteins provide limited structure that cannot accommodate a variety of synthetic modifications.

Site-specific use of various imaging techniques is enhanced by the use of a number of suitable metals bound to site-directed macromolecules, and bifunctional polykills with an increased number of chelating moieties per site-specific macromolecule There have been numerous attempts to produce polychelants.

However, for site specific image enhancement, it is important that the tissue or organ targeting portion of this chelate of the bifunctional chelating agent is not destroyed while bound to the targeting portion of the chelating agent portion. If the bifunctional chelating agent contains only one chelating moiety this is generally not a serious problem. However, when attempting to prepare a bifunctional polychelant by combining several chelating moieties to a single site-specific macromolecule, the ratio of the maximum chelating agent: site-specific macromolecule obtainable may be relatively limited. In addition, it has been found that the site-specificity of the resulting bifunctional polychelant decreases as the proportion obtained increases.

In order to solve the problem of binding a large number of chelated moieties to site-specific macromolecules without compromising site-specificity, i.e. without interfering with their binding site, a large number of chelated moieties are bound to There have been a number of proposals for the use of backbone molecules that can form polychelants, and then one or more of them can bind site-specific macromolecules to form bifunctional polychelants.

Bifunctional polychelants have been prepared in which the chelated moiety is a residue of open chain PAPCAs such as EDTA and DTPA and the backbone molecule is a polyamine such as polylysine or polyethyleneimine.

International Publication No. 90/12050 describes techniques for the production of polychelants comprising macrocyclic chelating moieties such as polylysine-polyDOTA, and techniques for the production of corresponding bifunctional polychelants. This document also suggests the use of starburst dendritic bodies, such as the sixth produced PAMAM starburst dendritic, as the backbone of these polychelants. International Publication No. 93/06868 similarly describes polychelants comprising aquatic backbone molecules bound to a number of macrocyclic chelating moieties such as, for example, DOTA residues. They are in turn bound to site-directed molecules such as, for example, proteins. However, today Starburst recipients are rarely used for imaging.

Thus, for example, in the fields of MR, X-ray, ultrasound, light-substrate and nucleus, it contains relatively large amounts of metals per molecule and allows them to circulate in the blood for long periods of time resulting in improved biological distribution. There is still a need for other polymer counterparts with visible molecular weights.

The present invention relates to polymers useful as therapeutic and diagnostic agents and methods for their preparation. In particular, the present invention relates to biodegradable polymers based on amino acids used in the targeting of diagnostic imaging agents and therapeutic agents.

The present invention is particularly useful for diagnostic or therapeutic purposes because copolymers of amino acids having one or more reporter groups such as, for example, chelating moieties, fluorescent agents or absorbers, or bound thereto, due to the inherent homogeneity of their structure and molecular weight distribution. Based on the perception that it is appropriate. Moreover, the relatively large molecular weight allows these compounds to function as effective blood pool preparations without the need to bind site-directed biomolecules.

Thus, in one aspect, the present invention encompasses a straight, branched, or water phase polymer backbone wherein one or more reporter moieties are linked and comprise a plurality of amine-containing acids, such as, for example, amino acid residues or similar non-natural amine containing acids. To provide a compound; Provided that the polymer moiety is straight, provided that the reporter moiety comprises an iodide contrast agent, an ultrasonic contrast agent, a light-based reporter, or a metal chelating agent other than DOTA, DTPA or similar polyaminopolycarboxylic acid. If the polymer backbone is straight chain, the reporter moiety is preferably an iodide counterpart or TMT.

As used herein, the term “reporter moiety” is intended to define any atom, ion or molecule that is bound to the polymer backbone and can be sensed by any chemical, physical or biological test. Thus, the reporter moiety can be a therapeutic or diagnostic agent such as, for example, a contrast agent or a pharmacological agent. If two or more reporter moieties are bonded to a given polymer backbone, they may be the same or different. Thus, they may include any combination of diagnostic and / or therapeutic agents. The number of bound reporter moieties depends on the structure of the polymer backbone, in particular on any degree of branching, but is generally in the range of 3 to 200, preferably 100 or less, for example 50 or less.

Aqueous (or cascade) polymers are preferred as backbone moieties. They are formed from monomers that act as branching sites and form new “generations” each having a continuous branch. The dendritic backbone molecule is preferably a plurality of natural or non-natural amino acid residues, more preferably natural amino acid residues arranged to radially extend outward from the central core portion. Such amino acid residues may be linked to one or more reporter groups either directly or optionally via a linking group. On the other hand, they may be branched by the addition of additional amino acid residues. Skeletal molecules in which the central branching core itself is branched once are referred to as first generation skeletal molecules. Additional terminal branches of amino acid residues of the first generation backbone molecules provide backbones of second generation, third generation, fourth generation, and the like. With each successive branch, the number of binding points capable of binding to the reporter group increases. Depending on the nature of the central core portion, the branches therefrom may extend radially in one or more directions, producing radially asymmetric or symmetrical dendritic bodies. Preferably, the dendritic backbone molecule is radially asymmetric.

A water polymer comprising a plurality of natural or unnatural amino acid residues, preferably natural amino acid residues, forms a further aspect of the present invention. Conveniently they comprise 3 to 200 amino acid residues extending radially from the central core portion, for example 3 to 100 amino acid residues.

While the core portion itself comprises one or more amino acid residues, other core portions are contemplated. Typically, the core portion may be any molecule to which a plurality of consecutive amino acid residues are bound and may itself include a reporter portion. Suitable core moieties include H ₂ NCOCH ₂ CH ₂ CONH ₂ , and the formula below.

Wherein m is 0-4;

Y represents hydrogen or an alkyl or aryl group, for example a C _1-6 alkyl group;

X represents a -CO ₂ H, -SO ₂ Cl or -CH ₂ Br group and variants and derivatives thereof.

In one embodiment of the invention, the dendritic core may itself comprise a reporter moiety. Thus, in another aspect, the present invention provides a compound comprising an aqueous polymer backbone comprising a plurality of amino acid residues extending radially from a reporter moiety.

Preferably, the biodegradable linking group binds the reporter moiety to the polymer backbone. In this way, biodegradation of the compound at the target site results in release of the reporter moiety (eg, an ionic or nonionic counterpart at the site of interest). Examples of suitable bonding groups are amide, ether, thioether, guanidyl, acetal, ketal and phosphoester groups. The bond between the backbone and the reporter group is preferably such that the amide nitrogen is derived from the backbone molecule and the amide carbonyl group is made via an amide bond derived from the carboxyl or carboxyl derivative of the reporter group.

The advantage of a biodegradable polymer is that its rate of degradation does not accumulate at the injection site, for example during lymphatic X-rays, or at tissues such as the liver, for example during angiography, when it is adjusted for the required imaging time. . The degree of biodegradation of the compounds of the invention can be controlled by the selection of specific binders and peptide cluster compounds. In addition, if desired, the degree of biodegradation of the binder and polymer backbone can be optimized ex vivo using purified enzymes and / or biological fluids / tissues. The use of amino acids that remove themselves quickly can further aid in removal after imaging.

Preferred polymer backbones comprise 3 to 200 amino acid residues, more preferably 3 to 100 amino acid residues and have a molecular weight of 300 to 20,000 Daltons. They are preferably bound via peptide bonds, thereby ensuring biodegradation of the polymer and subsequent removal from the body. The polyamino acid may be a polymer of a single species amino acid or of two or more different species of amino acids, or may be a block copolymer. Preferably, the polyamino acid is poly-1-aspartic acid.

Particularly preferred compounds according to the invention are those having the general formula (I)

Wherein n is an integer from 1 to 100;

R is a reporter group or a biodegradable binder-reporter addition product.

In one preferred embodiment of the invention, the reporter moiety is a chelating agent. They can chelate metal ions to a high level of stability and can be added with a suitable metal ion to react, for example to enhance MRI, gamma scintillation or X-ray imaging, or to deliver a toxic dose of radioactive material to the tumor. It can destroy undesirable cells such as Conveniently, the chelating agent is a counterpart that includes one or more paramagnetic metal ions. On the other hand, chelating agents are used either in the absence of metals or in the addition of metals up to the equivalent in order to absorb (eg, detoxify) metal ions available in vivo.

The reporter portion includes, for example, a therapeutic agent such as an antibiotic, analgesic, anti-inflammatory or other bioactive agent. The prolonged circulation in the blood of the polymers carrying these agents essentially prolongs their therapeutic effect. Proteolysis of this linker provides release of the therapeutic agent. Thus, the selection of a specific linker provides the ability to release the therapeutic agent after a certain time at the desired site of interest.

If desired, the compounds according to the invention can be combined with one or more site-directed molecules or targeting agents, for example proteins, by known methods to enhance the imaging and / or to target cells, tissues, organs and / or body tracts. To form a bifunctional polymer capable of delivering a cytotoxic amount of radioactive material. Targeting the contrast agent to the site of interest in this manner enhances the efficiency of the imaging method. Such agents accumulate at sites of interest depending on the specificity of the targeting agent. On the other hand, the polymer can be used as a blood pool formulation without being bound to the site designating molecule.

Thus, for such compounds of the invention comprising a dendritic backbone moiety, any terminal amino acid residue may be bound to the reporter or targeting agent either directly or via a biodegradable linking group. Preferably, where the core portion itself is a reporter group, each terminal amino acid residue is bound to the targeting agent via a biodegradable linking group. In this way, compounds can be provided that include one or more targeting agents. Conveniently, the number of targeting agents is 1 to 128, preferably 1 to 16, for example 1 to 4.

In an alternative embodiment of the invention, such compounds comprising an aqueous polymer backbone may comprise targeting agents or site-directed macromolecules as core portions. The resulting peptide clusters are in turn bound to one or more reporter moieties. Thus, in a further aspect, the present invention includes a waterborne polymer backbone extending radially from a targeting agent, wherein the polymer backbone comprises a plurality of amino acid residues associated with one or more reporter moieties.

As described in WO 95/11694, thermally stable STa enterotoxin obtained from Escherichia coli is particularly suitable as a core targeting agent. The accompanying Figure 1 illustrates the compounds of the present invention wherein the STa peptide is bound to a poly-1-aspartic acid cluster (Asp3) which in turn is bound to a number of TMT reporter molecules.

The polymers according to the invention are useful beings of medical diagnostics and treatments themselves, in part due to their unique localization in the body. The size of the polymer is usually 200 to 100,000 Daltons, in particular 200 to 50,000 Daltons, especially 10,000 to 40,000 Daltons, and their biological distribution changes fundamentally. The selection of specific binding groups and / or changes in the polyamino acid sequence also affect the biological distribution of the polymer and the bound reporter or targeting agent.

The compounds of the present invention can be prepared specifically according to the desired use of the compound by selecting appropriate linking groups and / or modifying the polyamino acid sequence of the backbone polymer, but generally extended time in blood vessels (e.g., Extended time of several orders of magnitude). Typically the compound is discharged into the extracellular fluid (ECF) space and undergoes renal excretion. Since the compound remains primarily in the vascular system during the useful residence time for diagnosis, it is suitable for a range of uses for blood pool and cardiac perfusion imaging, CNC tumor detection and volumetric measurements for thrombus detection and angiography. As blood pool preparations, they are particularly suitable for use in the study of blood flow and blood flow, especially related to lesion detection and myocardial perfusion studies. Conventional monomeric MRI counterparts, which are rapidly dispersed into the extracellular / extravascular space, cannot be readily used for this purpose. In addition, in view of their enhanced relaxation, the polymers according to the present invention can be administered in significantly reduced amounts compared to current MRI monomer control agents such as GdDTPA and GdDOTA, thus providing a significantly improved stability margin for use.

Accordingly, the present invention may provide for long term strengthening of the blood pool relative to MR, has specificity that accumulates in various body tissues, provides a relatively large amount of metal and has a molecular weight that produces the desired composition, molecular weight and size Provided are compounds that can be synthesized.

In addition, chelates according to the present invention may be selected so that the species to be chelated can be appropriately selected to function as X-ray preparations (eg tungsten) and MR counterparts (eg lanthanide ions). It can manufacture.

Binding of the compounds to site-directed molecules also results in increased target specificity in vivo. Site-directed molecules are preferably antibodies, antibody fragments, other proteins or other macromolecules that are delivered to a designated site in vivo to deliver chelating metals. In the present invention, the ability to deliver such site-directed macromolecules and / or bind their targets is not compromised by the addition of chelated metals. The number of chelates per molecule is sufficient to enhance the image of that particular target.

Chelating agents suitable for binding to the polymer backbone include straight chain and macrocyclic PAPCAs. Examples of suitable PAPCAs include ethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA), 1,4,7,10- Tetraazacyclododecane-1,4,7-triacetic acid (DO3A), 1-oxa-4,7,10-triazacyclododecanetriacetic acid (DOXA), 1,4,7-triazacyclononane tree Acetic acid (NOTA) and 1,4,8,11-tetraazacyclotetradecanetetraacetic acid (TETA).

Other chelating agents suitable for binding to the polymer backbone are, for example, 4 '-(3-amino-4-methoxyphenyl) -6,6 "-bis (N', such as those described in U.S. Pat. , N'-dicarboxymethyl-N-methylhydrazino) -2,2 ': 6', 2 "-terpyridine (THT) and 4 '-(3-amino-4-methoxyphenyl) -6,6 "-Bis [N, N-di (carboxymethyl) aminomethyl] -2,2 ': 6', 2" -terpyridine (TMT).

Metals that may be included through chelation include, for example, Mg, Ca, Sc, Ti, B, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, including their isotopes and radioactive isotopes. Lanthanide metals such as Sr, Y, Zr, Tc, Ru, In, Hf, W, Re, Os, Pb and Bi and other metal ions. The choice of metal ions for chelation depends on the intended therapeutic or diagnostic field.

For use in X-ray contrast imaging, the reporter portion may include ionic or nonionic iodide monocyclic or bicyclic X-ray contrast agents. Mono and bis-cyclic mean that the contrast agent contains one or two iodide rings. In general, the iodide ring can be a di- or tri-iodinated ring, for example a tri-iodinated aryl ring, in particular a phenyl ring. Examples of iodide preparations for use according to the invention are iohexol, iopenentol, iopamidol and iodixanol. Typically, one or more iodide counterparts are combined to form an alternating copolymer that can in turn be bonded to the polymer backbone. Examples of synthesizing such copolymers from iodixanol are shown below:

Bifunctional preparations according to the present invention involve binding a compound to a site-directed molecule. Site-directed molecules can be any molecule naturally concentrated in a selected target organ, tissue, cell or cell population, or other site in vivo in a mammalian body. These may include amino acids, oligopeptides (eg, hexapeptides), molecular recognition units (of MRU), single chain antibodies (of SCA), proteins, non-peptide organic molecules, Fab fragments, and antibodies. Examples of site-directed molecules include polysaccharides (eg CCK and hexapeptide), proteins (lectins, asialopetin, polyclonal IgG, blood clotting proteins (eg hirudin), lipoproteins and glyco Protein), hormones, growth factors, and clotting factors (such as PF4). Exemplary site-directed proteins include E. coli heat stable enterotoxin STa and analogs thereof, polymerized fibrin fragments (eg, E ₁ ), serum amyloid precursor (SAP) proteins, low density lipoprotein (LDL) precursors, serum albumin, Surface proteins of intact red blood cells, receptor binding molecules such as estrogens, galactosyl-neoglycoalbumin (NGA; see Vera et al. In Radiology 151: 191 (1984)) or varying numbers of bound Liver-specific proteins such as N- (2-hydroxypropyl) methacrylamide (HMPA) polymers with galactosamine (see Duncan et al., Biochim. Biophys. Acta 880: 62 (1986)) / Polymers, allyl and 6-aminohexyl glycosides (see Wong et al., Carbo. Res. 170: 27 (1987)), and fibrinogen.

Site-directed proteins can also be antibodies. The choice of antibodies, in particular the selection of antibodies with specificity for the antigen, depends on the intended use of the conjugate. Monoclonal antibodies are preferred over polyclonal antibodies.

Human serum albumin (HSA) is the preferred protein for the study of the vascular system. HSA is commercially available from a number of suppliers including Sigma Chemical Co. The preparation of antibodies that react with the intended antigen is known. Antibody preparations are commercially available from various suppliers. Fibrin flakes E ₁ are described in Olexa et al. Biol. Chem. 254: 4925 (1979). The preparation of LDL precursors and SAP proteins is described in de Beer et al. Immunol. Methods 50:17 (1982). The articles described above are hereby incorporated by reference in their entirety.

Compounds according to the invention can be conveniently prepared by combining a straight chain, branched chain or aqueous backbone comprising a plurality of amino acid residues in one or more reporter groups in a non-reactive solvent. Coupling the reporter group to the backbone molecule can be carried out via any reactive group, and standard binding techniques are known in the art. Preferred reaction conditions (eg, temperature, solvent, etc.) depend primarily on the particular reactant and can be readily determined by one skilled in the art.

Methods of adding metals to any existing chelating agent are within the skill level of the art. The metal may be bound to the chelating agent moiety by one of three general methods: direct bonding, synthesis of the template and / or metal transfer reaction. Direct bonding is preferred.

Methods of linking polymer backbones with antibodies or other proteins are within the skill of the art. Such methods are described in Pierce 1989 Handbook and General Catalog, and references cited therein, Blatter et al., Biochem. 24: 1517 (1985) and Jue et al., Biochem. 17: 5399 (1978).

The polymer backbone itself can be synthesized according to conventional peptide synthesis techniques. Suitable methods of forming amino acid units are described, for example, in Synthesis of Optically Active α-Amino Acids, Pergamon Press, 1989. Some side chain groups such as, for example, hydroxy, primary amide groups may remain unprotected during the entire synthesis process, but in general, reactive side chain groups, such as, for example, amino, thiol and / or carboxy, may be Will be protected during binding to amino acids.

The final step in the synthesis of the compounds according to the invention is the deprotection of all or part of the protected derivatives of such compounds, which process also forms part of the invention. Accordingly, the present invention provides a method comprising deprotecting a fully or partially protected derivative of a compound, which produces the compound as mentioned above.

In preparing peptide chains it is possible in principle to start at the C-terminus or the N-terminus. However, only processes starting at the C-terminus are generally used. This includes difficulties involving high levels of racemization (see Konig & Geiger, Chemische Berichte 103: 2024-2033, 1970), which are unacceptable when synthesized in the N to C direction. Because it suffers.

As expected, peptide compounds for use according to the invention can be prepared in good yield and high purity (<0.1% racemization per step) when synthesized in the amino to carboxy direction. It has been found that this synthetic method is particularly effective for preparing the aqueous polymer backbone. In particular, they have been found to be more stable compared to the more common Michael addition reactions. It has also been found that the synthesis of the polymer backbone from amino to carboxy direction results in isolated polymers which are generally non-crosslinkable and have particularly low levels of racemate impurities.

Thus, in another aspect, the present invention provides a process for the preparation of a compound comprising a straight, branched or watery polymer backbone comprising one or more reporter moieties bound thereto and comprising a plurality of amino acid residues. The preparation method comprises the steps of: (a) stepwise joining successively protected amino acid residues from amino to carboxy to form a polymer backbone;

(b) optionally joining the polymer backbone and one or more reporter moieties through a linking group;

(c) deprotecting any protected group

It includes.

Thus, for example, at the N-terminus, an appropriately protected aspartic acid derivative can be initiated by reacting with a second protected aspartic acid derivative molecule. The first aspartic acid derivative will have protected amino and free carboxyl groups while other reactants have free or activated α-amino groups and protected carboxyl groups. After the binding is performed, the intermediate will be purified, for example by chromatography, and then optionally deprotected so that additional amino acid residues can be added. This process continues until the required amino acid sequence is complete.

A wide variety of protecting groups for amino acids are known. Suitable amine protecting groups are carbenzoxy (z- or Cbz), t-butoxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc-). Carboxyl protecting groups that may be used include benzyl (-Bzl) and t-butyl (-tBu).

Various methods exist for removing amine- and carboxyl-protecting groups. Amine protecting groups such as Boc and carboxyl protecting groups such as -tBu can be removed simultaneously by treatment with an acid such as, for example, trifluoroacetic acid.

Bonding of the free amino group and the carboxyl group can be carried out using, for example, N, N'-dicyclohexylcarbodiimide (DCC). Other binders that can be used include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and 2- (11-H-benzotriazol-1-yl) -1,1,3-tetra Methyluranium tetrafluoroborate (TBTU).

The coupling reaction can be carried out at room temperature, conveniently in a suitable solvent system such as, for example, tetrahydrofuran, dimethylformamide, dimethylsulfoxide or mixtures of these solvents.

Peptide synthesis may be conveniently performed on a solid phase resin support. Amino acids are added stepwise to the growing peptide chains bound to insoluble matrices such as polystyrene beads. One advantage of this solid phase process is the fact that the product of each step is bound to the beads which can be quickly filtered and washed so that the intermediate does not need to be purified. Numerous solid phase supports are known in the art (eg, 4-hydroxy benzyl alcohol resins which can be modified with succinic anhydride to form esters).

Compounds of the invention (particularly bifunctional polymers) can be administered to a patient for imaging in an amount sufficient to achieve the intended contrast in a particular imaging technique. General doses of 0.001 to 5.0 mmole of chelate imaging metal ions per kilogram of body weight of the patient can be used to perform appropriate contrast enhancement. While amounts of 0.5 to 1.5 mmole / kg are generally effective for X-ray attenuation for X-ray applications, for most MRI applications, the preferred amount of imaging metal ions is 0.02 to 1.2 mmole / kg body weight. Will be in the range of. Preferred doses for most X-ray applications are 0.8-1.2 mmole lanthanide metals or heavy metals per kilogram of body weight.

The amount of compound of the invention for treatment will vary depending on the conditions of treatment, but will generally be on the order of 1 pmol / kg to 1 mmol / kg body weight.

The compounds of the present invention may be formulated with conventional pharmaceutical or veterinary adjuvants such as, for example, emulsifiers, fatty acid esters, gelling agents, stabilizers, antioxidants, osmotic pressure regulators, buffers, pH regulators, and the like. It may be in a form suitable for parenteral or digestive tract administration such as, for example, direct injection or infusion or administration to a body space having an external discharge duct such as the gastrointestinal tract, bladder or uterus. Thus, the compounds of the present invention may be conventional pharmaceutical dosage forms such as tablets, capsules, powders, solutions, suspensions, dispersants, syrups, suppositories, and the like. However, solutions, suspensions and dispersants in physiologically acceptable carrier media, for example water for injection, will generally be preferred.

Thus, the compounds according to the invention can be formulated for administration using physiologically acceptable carriers or excipients which are completely within the skill of the art. For example, a compound to which optionally a pharmaceutically acceptable excipient may be added may be suspended or dissolved in an aqueous medium, and the resulting solution or suspension is then sterilized.

For MRI and X-ray imaging of a portion of the body, the most preferred method of administering metal chelates as a contrast agent is parenteral administration, for example intravenous administration. For example, parenterally administrable forms, such as intravenous solutions, must be sterile and free of physiologically unacceptable preparations, have a low osmotic pressure to minimize dosing wounds and other detrimental effects, and thus contrast media Is preferably isotonic or slightly hypertonic. Suitable carriers are conventionally used for the administration of parenteral solutions, including sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection and Remington's Pharmaceutical Sciences, 15th. ed., Easton: Mack Publishing Co., pp. 1405-1412 and 1461-1487 (1975) and The National Formualry XIV, 14th ed. Aqueous carriers such as those described in Washington: American Pharmaceutical Association (1975). The solution may contain preservatives, antimicrobial agents, buffers, and antioxidants that have been used as parenteral solutions, excipients, and other additives that are typically compatible with chelates and do not interfere with the manufacture, storage, or use of the product.

In a further aspect, the present invention provides pharmaceutical compositions, such as, for example, image enhancing or therapeutic compositions, comprising a compound of the present invention and one or more pharmaceutical carriers or excipients.

In another further aspect, the present invention provides a compound according to the present invention or a chelate thereof for the preparation of an imaging enhancement contrast medium or therapeutic composition.

In yet a further aspect, the present invention administers to the body an amount of a compound according to the present invention that can enhance an image, followed by imaging of at least a portion of the body, such as, for example, MR, X-ray, ultrasound or scintillation images. A method is provided for generating an image of a human or non-human animal (especially a mammal), including generating.

The invention is further illustrated by the following non-limiting method. Unless otherwise indicated, all percentages are by weight.

Example 1 Asymmetric Peptide Clusters

Z- [Asp (α, γ-Asp ₂ (α, γ-Asp ₄ (α, γ-Asp ₈ (α, γ-Lys ₁₆ (α-reporter ₁₆ )])

(a) bis-α, γ- (α, γ- (t-butyl) -aspartyl) -N-Cbz-aspartamide

"Asp3 Cluster" (Compound I)

In a round bottom 500 mL flask, 8.5 mmole N-Cbz-L-aspartic acid, 10.2 mmole N-hydroxybenzotriazole, 25 mL THF: DMF (2: 1, volume / volume), and 10.2 mmole EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) was added. After stirring for 45 min at room temperature, 20.4 mmole of α, γ- (t-butyl) -aspartyl) -L-aspartic acid and 25 mmole of N, N'-diisopropylethylamine were added with stirring. After 4 hours, an additional 10.2 mmole of ECC was added and the reaction continued for 3 days as above. This slurry was purified by aqueous extraction.

Purity: The identity of a single point on TLC was confirmed by MS and NMR. Yield: 4 4.5%

(b) N-Cbz-aspartamide-((α, γ-aspartyl- (α, γ- (t-butyl) -aspartyl)) “Asp7 cluster” (Compound II)

In a round bottom 500 mL flask, 10 mmole of compound I, 95 mL of chloroform: THF: acetonitrile (2.5: 7: 7), 36.4 mmole of N-hydroxybenzotriazole, and 36.5 mmole of DCC (N, N'-dicyclosecsilcarbodiim) was added. After stirring for 20 minutes at room temperature, 40 mmole of α, γ- (t-butyl) -L-aspartic acid and N, N'-diisopropylethylamine were added until the pH was approximately 7. After stirring for 16 hours at room temperature, the reaction was purified by aqueous extraction.

Purity: The identity of a single point on TLC was confirmed by MS and NMR. Yield: 12.1%

(b) N-Cbz-aspartamide-((α, γ-aspartyl- (α, γ- (t-butyl) -aspartyl))) “Asp15 cluster” (Compound III)

Step 1:

0.85 mmole of compound II was stirred at room temperature in 200 mL of 95% trifluoroacetic acid (aqueous solution) for 8 hours. The reaction was dried by distillation under reduced pressure at 40 ° C., followed by re-pressure distillation from 200 mL of toluene, followed by distillation under reduced pressure from THF.

Step 2:

In a round bottomed 250 mL flask, 0.85 mmole of the product of step 1, 90 mL of DMF: THF (1: 1, volume / volume), 8.12 mmole of N-hydroxybenzotriazole, and 8.12 mmole of EDC ( 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) was added. After stirring for 20 minutes at room temperature, 16.24 mmole of α, γ- (t-butyl) -L-aspartic acid and 19.92 mmole of N, N'-diisopropylethylamine were added. After stirring for 16 hours at room temperature, the reaction was purified by aqueous extraction and ion exchange chromatography.

Purity: The identity of a single point on TLC was confirmed by MS and NMR. Yield: 82.2%

(d) N-Cbz-aspartamide-((α, γ-aspartyl- (α, γ-aspartyl- (aspartylα, γ-lysyl (α-methoxyethylamide, ε-amine) ))))) "Asp15 Lysyl16 Cluster" (Compound IV)

Step 1:

0.7 mmole of compound III at room temperature was stirred for 8 h in 200 mL of 95% trifluoroacetic acid (aqueous solution). The reaction was dried by distillation under reduced pressure at 40 ° C., followed by re-pressure distillation from 200 mL of toluene, followed by distillation under reduced pressure from THF.

Step 2:

In a round bottomed 250 mL flask, 0.7 mmole of the product of step 1, 90 mL of DMF: THF (1: 1, volume / volume), 8.12 mmole of N-hydroxybenzotriazole, and 8.12 mmole of EDC ( 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) was added. After stirring for 20 minutes at room temperature, 16.24 mmole of α, γ- (t-butyl) -L-aspartic acid and 19.92 mmole of N, N'-diisopropylethylamine were added. After stirring for 16 hours at room temperature, the reaction was purified by aqueous extraction and ion exchange chromatography.

Purity: The identity of a single point on TLC was confirmed by MS and NMR. Yield: 99.2%

Step 3:

In a round bottomed 250 mL flask, 0.7 mmole of the product of step 2, 100 mL of DMSO: DMF: THF (1.5: 3.5: 5, volume / volume), 27.5 mmole of N-hydroxybenzotriazole, and 27.5 mmole EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) was added. After stirring for 20 minutes at room temperature, 55 mmole of α-Boc-L-lysine and 68.7 mmole of N, N'-diisopropylethylamine were added. After stirring for 16 hours at room temperature, the reaction was purified by aqueous extraction and gel permeation chromatography.

Purity: Single point on TLC.

Step 4:

In a round bottom 250 mL flask, the product of step 3, 40 mL of DMF: DCM (2: 2, volume / volume), 23.5 mmole of N-hydroxybenzotriazole, and 23.5 mmole of EDC (1-ethyl -3- (3-dimethylaminopropyl) carbodiimide) was added. After stirring for 30 minutes at room temperature, 75 mmole of 2-methoxyethanolamine was added. After stirring overnight at room temperature, the reaction was purified by aqueous extraction and ion exchange chromatography.

Purity: Single point on TLC. Yield: 90%.

(e) N-Cbz-aspartamide-((α, γ-aspartyl- (α, γ-aspartyl- (aspartylα, γ-lysyl (α-methoxyethylamide, ε-TMT) )))) "Asp15 Lysyl16TMT16 Cluster" (Compound V)

To a round bottom 250 mL flask was added Compound IV, 1.1 molar equivalents of TMT-NCS and 100 mL of 50 mM pH 9.0 sodium borate. After stirring for 48 hours at room temperature, the reaction was purified by aqueous extraction and diafiltration (2000 MW cutoff).

Purity: 80% (RP-HPLC)

Example 2: Symmetric Aspartic Acid Cluster

(a) Bis- (α, γ- (tbutyl) -aspartyl) succinamide (Compound I)

Reaction Path A:

20 mmole succinic acid, 26 mmole EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide), 24 mmole triethylamine, 12 mmole TBTU (2- ( 1-H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate), and 150 mL of THF: DMF (2: 1, volume: volume) were added 20 mmole of α, γ- (tbutyl) -L-aspartic acid was then added. The slurry was reacted at room temperature for 4 days and then purified by aqueous extraction.

Purity: The identity of a single point on TLC was confirmed by MS and NMR. Yield: 23.2%

Reaction Path B:

10 mmole succinic acid, 100 mL THF: DMF (2: 1, volume: volume), 60 mmole triethylamine and 20 mmole TBTU (2- (1-H-benzotriazolyl) in a round 2 liter flask -1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate) was added. After stirring for 15 minutes, 22 mmole of α, γ- (tbutyl) -L-aspartic acid was added. The slurry was reacted at room temperature for 21 hours and then purified by aqueous extraction.

Purity: The identity of a single point on TLC was confirmed by MS and NMR. Yield: 64.7%

(b) (bis-α, γ-aspartyl- (α, γ- (tbutyl) -aspartyl)) succinamide (Compound II)

Step 1:

At room temperature 4.6 mmole of compound I was stirred in 100 mL of trifluoroacetic acid / dichloromethane (1: 1, volume: volume) for 45 minutes. The reaction was dried by distillation under reduced pressure at 30 ° C. followed by five successive re-pressure distillations from 200 mL volume of chloroform each.

Step 2:

The product of step 1 above was dissolved in 250 mL of DMF: THF (1: 1, volume / volume) with 60 mmole triethylamine and 40 mmole L-aspartic acid- (α, γ- (tbutyl) ester. TBTU 60 mmole was added to this solution After 16 hours, an additional 20 mmole of L-aspartic acid- (α, γ- (tbutyl) ester was added and the reaction was continued overnight.

Purification by aqueous purification and ion-exchange chromatography gave a single principal point identified on MS by N and NMR as the intended compound on TLC. Yield: 90%

Example 3: X-ray contrast agent

(a) Synthesis of Iodide Monomer (Compound I)

(b) Compound I can be combined with one of the Asp _x clusters described in Examples 1 and 2 to form an iodinated X-ray contrast agent.

Claims (34)

One or more reporter moieties comprising a straight, branched, or water phase polymer backbone to which the one or more reporter moieties are bound and comprising a plurality of amine-containing acids. If the polymer backbone is straight chain, the at least one reporter moiety comprises an iodide contrast agent, an ultrasonic contrast agent, a light-based reporter or a metal chelating agent that is not a DOTA or DTPA-based polyaminocarboxylic acid. The compound of claim 1, wherein the polymer backbone comprises a plurality of natural or unnatural amino acid residues. The compound of claim 2, wherein the polymer backbone comprises 3 to 200 amino acid residues. The compound according to any one of claims 1 to 3, wherein the polymer backbone comprises an aqueous phase. The compound of claim 4, wherein the dendritic body comprises 3 to 200 amino acid residues extending radially from the central core portion. The compound of claim 5, wherein the core portion is selected from H ₂ NCOCH ₂ CH ₂ CONH ₂ , and compounds having the formula: and derivatives thereof. Wherein m is 0-4; Each Y independently represents hydrogen or an alkyl or aryl group; Each X independently represents a -CO ₂ H, -SO ₂ Cl or -CH ₂ Br group. The compound of claim 5, wherein the core portion comprises a reporter portion. The compound of claim 5, wherein the core portion comprises a targeting agent capable of being delivered to or specifically bound to a target cell, tissue, organ, or other site of the mammalian body. The compound of any one of claims 4-8, wherein the dendritic body is radially asymmetric. The compound of any one of the preceding claims, wherein the polymer backbone has a molecular weight of 300 to 20,000 Daltons. The compound of claim 2, wherein the polymer backbone is a polymer or block copolymer of amino acids of a single species or two or more different species. 12. The compound of claim 11, wherein said polymer backbone is poly-1-aspartic acid. The compound of any one of the preceding claims, wherein the compound comprises 3 to 200 reporter moieties. The compound of any one of the preceding claims, wherein each reporter moiety is bound to the polymer backbone via a biodegradable linking group. 15. The compound of claim 14, wherein said linking group is selected from amide, ether, thioether, guanidyl, acetal, ketal and phosphoester groups. 15. The compound of claim 14, wherein the linking group comprises an amide bond, an amide nitrogen derived from a backbone molecule and an amide carbonyl derived from a carboxyl or carboxyl derivative of a reporter group. The compound of claim 1 having the formula (I) Wherein n is an integer from 1 to 100; R represents a reporter moiety that is optionally bonded to the polymer backbone via a biodegradable linking group. The compound of any one of the preceding claims, wherein the one or more reporter moieties comprise a diagnostic or therapeutic agent. The compound of claim 18, wherein said agent comprises a residue of a chelating agent or a metal chelate thereof. 20. The compound of claim 19, wherein said chelating agent is a counterpart comprising at least one paramagnetic metal ion. The method of claim 20, wherein the metal ions are lanthanide metal ions, Mg, Ca, Sc, Ti, B, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Sr, Y, Zr, Tc , Ru, In, Hf, W, Re, Os, Pb and Bi. The method according to claim 19 or 20, wherein the chelating agent is ethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA), 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A), 1-oxa-4,7,10-triazacyclododecanetriacetic acid (DOXA), 1,4 , 7-triazacyclononanetriacetic acid (NOTA) and 1,4,8,11-tetraazacyclotetradecanetetraacetic acid (TETA). 21. The chelating agent of claim 19 or 20, wherein the chelating agent is 4 '-(3-amino-4-methoxyphenyl) -6,6 "-bis (N', N'-dicarboxymethyl-N-methylhydrazino) ) -2,2 ': 6', 2 "-terpyridine (THT) and 4 '-(3-amino-4-methoxyphenyl) -6,6" -bis [N, N-di (carboxymethyl) Aminomethyl] -2,2 ': 6', 2 "-terpyridine (TMT). 19. The compound of claim 18, wherein said agent is an ionic or nonionic iodide monocyclic or bi-cyclic X-ray contrast agent. The compound of any one of the preceding claims, wherein the compound is bound to a targeting agent capable of being delivered to or associated with a target cell, tissue, organ, or other site of the mammalian body. The compound of claim 8 or 25, wherein the targeting agent is E. coli heat stable enterotoxin STa or an analog thereof. A water phase polymer comprising a plurality of natural or unnatural amino acid residues extending radially from a central core portion. 28. The water phase polymer of claim 27, wherein said core portion is as defined in any of claims 6-8. 27. A process for the preparation of a compound of any one of claims 1 to 26 comprising coupling one or more reporter moieties to a straight, branched or water polymer backbone comprising a plurality of amino acid residues. 27. A process for the preparation of a compound of any one of claims 1 to 26, comprising deprotecting a partially or fully protected derivative. (a) stepwise joining sequentially protected amino acids in amino to carboxy direction to form a polymer backbone; (b) binding the polymer backbone to one or more reporter moieties, optionally through a linking group; And (c) deprotecting any protected group A method for preparing a compound comprising a straight chain, branched chain or water phase polymer backbone comprising a plurality of amino acids, wherein one or more reporter moieties are bound. A pharmaceutical composition comprising a compound of any one of claims 1-26 together with at least one pharmaceutical carrier or excipient. Use of a compound of any one of claims 1 to 26 in the preparation of a medium or therapeutic composition for contrast enhancement. 27. A method of generating an image of the body, comprising administering a compound of any one of claims 1 to 26 to a human or non-human animal body and then generating an image of at least a portion of the body.