NO880179L - POLYMER COMPLEX, PROCEDURE FOR THE PREPARATION OF THESE AND PHARMACEUTICAL AGENTS CONTAINING THESE. - Google Patents

Description

The invention relates to the subject matter characterized in the patent claims, i.e. new polymer complexes, agents containing these compounds, their use in diagnostics and therapy, as well as methods for producing these compounds and agents.

The use of complexing agents or complexes,

respectively their salts in medicine, have long been known. As examples should be mentioned: Complex formers as stabilizers for pharmaceutical preparations, complexes and their salts as aids for the supply of poorly soluble ions (e.g. iron), complex formers and complexes (preferably calcium or zinc), possibly as salts with inorganic and /or organic bases, as antidotes for the detoxification of heavy metals or their radioactive isotopes that have been ingested accidentally, or complex formers as aids in nuclear medicine during

99m ■ use of radioactive isotopes such as Tc for scintigraphy is known.

In patent document DE-OS 3401052, paramagnetic complex salts are proposed as diagnostics, mainly as NMR diagnostics.

These complexes or complex salts are quite well tolerated and cause extensive, complete separation of the paramagnetic ions. The disadvantage, however, is that they are only distributed non-specifically in the extracellular space and are therefore only in exceptional cases sufficient for the recognition of pathologically changed tissue.

The attempt to solve at least part of these problems by using complexing agents which are on the one hand bound by ionic bonding to the possibly suitable metal (see below) as well as on the other hand by binding to a functional group or a non-toxic and most possible organ-specific molecule that serves as a carrier molecule has so far only been of limited success.

Thus, for example, the number of paramagnetic centers in the complexes described in European patent applications no. 88,695 and 150,884 is not sufficient to

an organ-specific image rendering.

If the number of the necessary metal ions is increased by several times the introduction of complexing units in a macromolecule, this is always associated with an intolerable influence on the affinity and/or specificity of this macromolecule [J. Nucl. With. 2A, 1158 (1983)].

For many reasons, there is therefore a need for stable, well-soluble, but also better tolerable, well-available complex compounds which in the complex contain the greatest possible number of the necessary metal ions without their affinity and/or specificity being lost. It is therefore a task for the invention to provide these compounds and agents, as well as the simplest possible method for producing them. This task is solved with the help of the invention.

It was found that polymer complexes consisting of

a ligand containing a carboxylic acid, phosphonic acid and/or phenolic group, at least one ion of an element with the ordinal numbers 21 - 29, 31, 32, 38, 39, 42 - 44, 49 or 57 - 83 as well as optionally cations of inorganic and/or organic bases or amino acids or amino acid amides, surprisingly, are excellently suited for the production of NMR, X-ray and radiodiagnostics as well as radiotherapeutics, as above all they contain stably bound in the complex the number of metal ions that are necessary for this application.

As a ligand, e.g. compounds of the general formula I

where

n means the whole number 7 to 20,000,

12 3 12

X means a hydrogen atom, a CR R R - or a CR R —U residue with U meaning one

where

A<1> stands for OH or A and A" stands for H or A<1>, with A meaning a (NH)u~[B-(NH) ] -W- or OR 4 group where u and v stands for the digits 0, 1 or 2,

w for the digits 0 or 1,

R for a (CH2)m Z group,

R^ for a hydrogen atom or a (CH„), Z group,

R 3 for a hydrogen atom or a (CH„), Z group, and

41K

R for a C 1 -C 8 alkyl or benzyl residue,

whereby

m, 1 and k stand for the digits 0 to 10, and

Z stands for a hydrogen atom, a C1-C2 -alkyl-, a C00H-,

P03H2- or

where Y means a hydrogen

atom or the residue OH,

B means a C0-C2Q alkylene group, and

W means a hydrogen atom, a macromolecule, a functional group which is optionally bound over a linear, branched, saturated or unsaturated C-^-C2(_)-alkylene group or bound over this functional group a macro- or biomolecule, whereby the alkylene group optionally contains imino, phenylenoxy, phenylenimino, amide, ester group(s), oxygen, sulfur and/or nitrogen atom(s) and optionally substituted with hydroxy, mercapto and/or amino group(s), whereby the substituents X in the individual links and at the ends of the compound as well as the substituents Z in R 1, R 2 and R<3> can be the same or different, with the proviso that not all X must simultaneously mean hydrogen, that at least one Z does not mean hydrogen or C-^-C2Q-alkyl, that if m, 1 and k = 0, not all the substituents Z stand for -C00H, -P0^H2 or

and that, if desired, a part of -COOH-, -PO3H2- or as esters or amides is present, and II

where

Q means the residue of a natural amino acid containing

group(s) NX and/or NX2, and

n and X have the meanings mentioned above.

The element with the above-mentioned order number which forms the central ion in the physiologically tolerable complex salt can, for the intended purpose of use of the diagnostic agent according to the invention, naturally also be radioactive.

If the agent according to the invention is intended for use in NMR diagnostics, the central ion in the complex must

the salt be paramagnetic. These are especially the divalent and trivalent ions of the elements with ordinal numbers 21-29, 42, 44

and 57-70. Suitable ions are, for example, chromium(III), manganese(II), iron(II), cobalt(II), nickel(II), copper(II), praseodymium(III), neodymium( III), samarium(III) and ytterbium(III) ion. Due to their very strong magnetic moment, the gadolinium(III), terbium(III), dysprosium(III), holmium(III), erbium(III) and iron(III) ions are particularly preferred.

For the use of the agents according to the invention in nuclear medicine, the central ion must be radioactive. Suitable is e.g. radioisotopes of the elements copper, cobalt, gallium, germanium, yttrium, strontium, technetium, indium, ytterbium, gadolinium, samarium and iridium.

If the agent according to the invention is intended for use in X-ray diagnostics, the central ion must be derived from an element with a higher order number in order to achieve sufficient absorption of the X-rays. It was found that for this purpose, diagnostic agents containing a physiologically compatible complex salt with central ions of elements with ordinal numbers between 21-29, 42, 44, 57-83 are suitable, for example the lanthanum(III) ion and the above-mentioned ions of the lanthanide series.

The polymer complexes according to the invention contain at least one ion of an element with the above-mentioned order number. Complexes which exhibit one ion of 5-50, especially 8-20 CO0H, PO3H2 or phenolic groups are preferred.

The alkylene group contained in W and representing B can be linear, branched, cyclic, aliphatic, aromatic or arylaliphatic and have up to 20 carbon atoms. Preference is given to linear mono- to hexamethylene groups as well as C1-C1-alkylenephenyl groups.

The alkyl group contained in Z or R 4, can

be linear, branched or cyclic and exhibit up to 20 or 6 carbon atoms.. Preferred are linear C^-Cg- respectively. C, -C. residues. If the polymer complex according to the invention contains the residue CR 1R 2-U, per molecule 1 to n/10, preferably 1 to n/50 of these residues.

Preferred functional groups that stand for the substituent W are, for example, maleimidobenzoy1-, 3-sulfo-maleimidobenzoyl-, 4-(maleimidomethyl)-cyclohexylcarbonyl-, 4-[3-sulfo-(maleimidomethyl)-cyclohexy1-carbony1-, 4 -(p-maleimidophenyl)-butyryl-, 3-(2-pyridyldithio)-propionyl-, methacryloy1-(pentamethylene)-amido-, bromoacetyl-, iodoacetyl-, 3-iodopropyl-, 2-bromomethyl-, 3-mercaptopropyl- , 2-mercapto-ethyl-, phenylene isothiocyanate-, 3-aminopropyl-, benzyl ester-, ethyl ester-, t-butyl ester-, amino-, C-^-Cg-alkyl lamino-, aminocarbonyl-, hydrazino-, hydrazinocarbonyl-, maleimido -, methacrylamide-, methacryloylhydrazinocarbonyl-, maleimide-amidocarbonyl-, halogeno-, mercapto-, hydrazinotrimethylene-hydrazinocarbonyl-, aminodimethylenamidocarbonyl-, bromo-carbonyl-, phenylenediazonium-, isothiocyanate-, semicarbazide, thiosemicarbazide groups.

For clarification, some selected groups are listed:

whereby R and R<1> are the same or different and each represents a hydrogen atom, a saturated or unsaturated C-^-C2Q alkyl group which is optionally substituted with a phenyl group, or a phenyl group.

As examples of the amino acids contained in formula II, mention should be made of lysine, arginine, ornithine, α, Tf-diamino-butyric acid and histidine.

The remaining acidic hydrogen atoms, i.e. those that have not been substituted with the central ion, can optionally be replaced with ester or amide residues or with cations of inorganic and/or organic bases or amino acids.

Suitable inorganic cations are, for example, the lithium ion, the potassium ion, the calcium ion and especially the sodium ion. Suitable cations of organic bases are i.a. those of primary, secondary or tertiary amines, such as e.g. ethanolamine, diethanolamine, morpholine, glucamine, N,N-dimethylglucamine and especially N-methylglucamine. Suitable cations of amino acids are, for example, cations of lysine, arginine and ornithine, as well as the amides of other acidic or neutral amino acids.

Suitable alkyl radicals are above all the C 1 -C 8 alkyl radicals such as e.g. methyl, ethyl, propyl, butyl as well as the benzyl residue.

Suitable amide residues are above all those residues which

formed by reaction with C-^-Cg-mono- and dialkylamines such as e.g. dimethyl-, diethyl-, dipropyl-, cyclopropyl-, N-methyl-n-propyl, N-ethylcyclohexylamine, by reaction with e.g. benzylamine, aniline or heterocyclic amines, such as e.g. morpholine, piperidine, or by reaction with hydroxylated amines, such as e.g. 2,3-dihydroxypropylamine, N-methyl-2,3-dihydroxypropylamine, 2-hydroxy-l-(hydroxymethyl)-ethylamine, N,N-bis-(2-hydroxyethyl)-amine, N-methyl-2,3, 4,5,6-pentahydroxyhexylamine, 6-amino-2,2-dimethyl-1,3-dioxepin-5-ol, 2-hydroxyethylamine, 2-amino-1,3-propanediol, diethanolamine, ethanolamine.

The polymer complexes according to the invention contain

the large number of metal ions necessary for their use stably bound in the complex.

Thus shows e.g. equilibrium or re-complexation investigations with the gadolinium complex of the diethylene-triaminepentaacetic acid DTPA (European patent document EP 71 564) which can be considered to belong to the state of the art and is recognized in the field as a good contrast agent, that the polymer complexes according to the invention are surprisingly more stable than this over the entire molecular range as the index digits.

The compatibility of the polymer complexes is also, for example, superior to organ-specific, monomeric complexes.

The value of the size of the relaxivity, which constitutes a measure of the image formation, is surprisingly high: it is too

the polymer complexes according to the invention a factor of 10 higher than for gadolithium-DTPA, calculated on the amount of complexed metal. Take as a comparison a polymer complex whose size is to be determined with a value of e.g. 5000 for n, then occurs per molecule polymer complex an approx. 5000 - 10,000 times greater relaxivity than for gadolinium-DTPA. Due

of this, a correspondingly smaller molar amount of complex and thus of the absolute amount of gadolinium compared to the monomers is needed to achieve a certain signal strength during image formation.

As a further important advantage of the polymer complexes according to the invention, their excretion ratio can be mentioned. Thus, for example, a complex with an average molecular weight of 60,000 D and a weight proportion of gadolinium of 15% is secreted

within 4 hours 80% of the total amount through the urine. The excretion rate that is desired, depending on the purpose of use, can thereby be very targeted and easily set with the help of the molecular weight that is selected.

The polymer complexes according to the invention exhibit a surprisingly high tissue specificity. Thus, for example, already a few minutes after intravenous injection of an N-methylglucamine salt solution of gadolinium(III) complex of the polyethyleneimine-polyacetic acid in the nuclear resonance image, a clear contrast enhancement is achieved in the peripheral tumor tissue which persists for a longer time and brings a clear diagnostic benefit.

Surprisingly, with the help of the polymer complexes according to the invention, blood vessels can also be detected in vivo without the use of special pulse sequences.

The production of the polymer complexes according to the invention takes place starting from adducts containing carboxylic acid, phosphonic acid and/or phenolic groups which can be obtained in a manner known per se by alkylation (J. March, Advanced Organic Chemistry, McGraw-Hill, 2nd ed. 1977 , 377-382) of ligands bearing amino groups, e.g. with bromoacetic acid.

The subsequent reaction steps are likewise carried out using methods that are known from the literature. These are complexation with at least one ion of an element with

the above-mentioned ordinal numbers and, if compounds with CR 1 R 2U residue(s) are to be synthesized, transfer of at least part of the carboxylic acid, phosphonic acid or phenolic groups that are not necessary for the complexation, to the group(s)

CR 1 R 2U1, where U<1>has the meaning given for U, but

that the residue W instead of W stands with the definitions hydrogen-

atom or a functional group which is optionally bound via a C1-C20 alkylene group, whereby the C'R 1 R 2IT groups thus obtained can be the same or different (e.g. ester

or amide and hydrazide groups), about the desired binding of a macromolecule as well as possibly subsequent substitution of the still present, acidic hydrogen atoms with cations of inorganic and/or organic bases or amino acids.

In the synthesis of the polymer complexes containing

12 12

the group(s) -CR R -U1 or -CR R U, the stated steps of complexation, functionalization, binding to a macromolecule can be permuted, whereby, of course, when binding to a macromolecule, a functionalization of the carboxylic acid, phosphonic acid or phenolic group(s) must be carried out in advance.

The introduction of the residue A" into the carboxylic acid, phosphonic acid or phenolic group(s), i.e. of amino, hydrazino and ester groups as well as the functional group contained in W and W and which is optionally bound via a C-^ -C2Q-alkylene group, takes place using methods known from the literature (Houben-Weyl, Methoden der organischen Chemie, volume VIII, Georg Thieme forlag, Stuttgart; J. Biochem. 9_2, 1413

(1982)).

Examples of conversion of amino groups which are bound to aromatic or aliphatic residues are the reactions with a substrate of the general formula III

where Nf stands for a nucleofuge such as e.g. Cl, Br, J or CH3C6H4SO3,

L for an aliphatic, aromatic, arylaliphatic, branched, linear or cyclic hydrocarbon residue of up to 20 carbon atoms, and

Fu for the desired terminal functional group, optionally in protected form (DE-OS 34 17 413), which is carried out in anhydrous, aprotic solvents such as tetrahydrofuran, dimethoxyethane or dimethylsulfoxide in the presence of an acid scavenger, such as e.g. sodium hydroxide, sodium hydride or alkali or alkaline earth carbonates such as e.g. sodium, magnesium, potassium, calcium carbonate at temperatures between 0°C and the boiling point of the relevant solvent, but preferably between 20 and 60°C.

Examples of compounds with the general formula III should be mentioned:

Conversions of carboxy groups can, for example, be carried out using the carbodiimide method (Fieser, Reagents for Organic Syntheses 10, 142) over a mixed anhydride [Org. Prep. Pro. Int. 7, 215 (1975)] or over an activated ester (Adv. Org. Chem., part B, 472).

The thus obtained, complex-forming ligands (as well as the complexes) can also be linked to bio- or macromolecules, which are known to be particularly enriched in the organ or organ part to be examined. Such molecules are, for example, enzymes, hormones, dextrans, porphyrins, bleomycins, insulin, prostaglandins, steroid hormones, amino sugars, amino acids, peptides such as polylysine, proteins (such as immunoglobulins), monoclonal antibodies, leetins or lipids (also in the form of liposomes). Conjugates with albumins, such as human serum albumin, antibodies, such as e.g. monoclonal antibodies specific for tumor-associated antigens,

or antimyosin. Instead of biological macromolecules, suitable synthetic polymers such as polyethylene imines, polyamides, polyurea or polythiourea can also be attached. The pharmaceutical agents formed from this are, for example, suitable for use in tumor and infarction diagnostics as well as tumor therapy. As monoclonal antibodies (eg Nature 256, 495, 1975) which have the advantages over the polyclonal antibodies that they are specific for an antigenic determinant,

have a defined binding affinity, are homogeneous (thereby their purification becomes significantly easier) and can be produced in large quantities in cell cultures, for the conjugation particularly those which are directed against mainly cell membrane antigens are relevant. As such, e.g. for tumor detection monoclonal antibodies or their fragments Fab and F(ab'>2suitable, which are, for example, specific for human tumors of the gastrointestinal tract, breast, liver, bladder, gonads and of melanomas (Cancer Treatment Repts. 6_8, 317, 1984 , Bio Sei. 3j4 , 150, 1984) or is directed against carcino-embryonic antigen (CEA), human choriogonadotropin ((3-HCG) or other tumor-associated antigens, such as glycoproteins (New Engl. J. Med. 298, 1384, 1973, US-P 4,331 .647).Among other things, anti-myosin, anti-insulin and anti-fibrin antibodies are also suitable (US patent 4,036,945).

Colon carcinomas can be detected NMR diagnostically using conjugates of polyethyleneimine-polyacetic acids that are complexed with gadolinium(III) ions, with the antibody 7 B5 Dll.

For liver examinations or suitable for tumor diagnostics are, for example, conjugates or inclusion compounds with liposomes, which are used, for example, as unilamellar or multilamellar phosphatidylcholine-cholesterol vesicles.

The binding of metals to the desired macro- or biomolecules has so far taken place using methods, which are for example described in Rev. Rome. Morphol. Embryol. Physio. , Physiologie 1981, JL8, 241 and J. Pharm. Pollock. 68,

79 (1979), for example by reaction between the nucleophilic group in a macromolecule, such as the amino, phenol, sulfhydryl, aldehyde or imidazole group and an activated derivative of the polymer complex or ligands. Examples of activated derivatives include anhydrides, acid chlorides, mixed anhydrides (see e.g. G.E. Krejcarek and K.L. Tucker, Biochem., Biophys. Res. Commun. 1977, 581), activated esters, nitrenes or isothiocyanates. Conversely, it is also possible to react an activated macromolecule with the polymer complex or the ligands. For conjugation with proteins, substituents are also offered which, for example, have the structure

C,H.N„<+>, C,H.NHCOCH„, C.-H.NHCS or C,H.OCH„CO.

64264 264 642

However, this type of binding has the disadvantage of a lack of complex stability for the conjugates or lack of specificity (eg, Diagnostic Imaging 84, 56; Science 220, 613, 1983; Cancer Drug Delivery 1, 125, 1984).

The conjugate formation according to the present invention, on the other hand, takes place with the aid of the amino or hydrazino function found in the substituents A or by means of the functional group in the substituents W described above. In this way, up to several hundred metal ions can be bound to a binding site in the macromolecule.

In the case of the antibody conjugates, the binding of the antibody to the complex or the ligands must not lead to a loss or reduction of the binding affinity and binding specificity of the antibody to the antigen. This can either take place by binding to the carbohydrate part in the Fc part of glycoproteine t, or in the Fab or F(ab')^ segments or by binding to the sulfur atom in the antibody or the antibody fragments.

In the first case, an oxidative cleavage of sugar units must first be carried out to generate linkable formyl groups. This oxidation can be carried out chemically with oxidizing agents such as e.g. periodic acid, sodium metaperiodate or potassium metaperiodate using methods known from the literature (eg J. Histochem. and Cytochem. 2_2, 1084, 1974) in aqueous solution in concentrations from 1 to 100, preferably 1 to 20 mg/ ml, and a concentration of the oxidizing agent between 0.001 to 10 mmol, preferably 1 to 10 mmol in a pH range from approx. 4 to 8 at a temperature between 0 and 37°C and a reaction duration between 15 minutes and 24 hours. The oxidation can also take place enzymatically, for example with the help of galactose oxidase in an enzyme concentration of 10-100 units/ml, a substrate concentration of 1 to 20 mg/ml, at a pH value of 5 to 8, a reaction duration of 1 to 8 hours and a temperature between

20 and 40°C (eg, J. Biol. Chem. 234, 445, 1959).

Complexes or ligands with suitable functional groups such as e.g. hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide and thiosemicarbazide by reaction between 0 - 37°C, with a reaction duration of 1 to 65 hours, a pH value between approx. 5.5 and 8, an antibody concentration from 0.5 to 20 mg/ml and a molar ratio between complexing agent and antibody aldehydes from 1:1 to 1000:1. The subsequent stabilization of the conjugate takes place by reduction of the double bond, e.g. with sodium borohydride or sodium cyanoborohydride. The reducing agent is thereby used in a 10- to 100-fold excess (e.g. J. Biol. Chem. 254, 4359, 1979).

The second possibility for the formation of antibody conjugates is based on a gentle reduction of the disulphide bridges in the immunoglobulin molecule. Hereby, the more sensitive disulphide bridges in the H chains in the antibody molecule are cleaved, while the S-S bonds in the antigen-binding region remain intact so that there is practically no reduction in the binding affinity and specificity for the antibody (Biochem. 18, 2226, 1979, Handbook of Experimental Immunology, Vol. 1, Second Edition, Blackwell Scientific Publications, London 1973, Chapter 10). These free sulfhydryl groups in the intra-H-chain regions are then reacted with suitable, functional groups of complexing agents or metal complexes at 0 to 37°C, a pH value from approx. 4 to 7, and a reaction duration of 3 to 72 hours during the formation of a covalent bond which does not affect the antigen-binding region of the antibody. Examples of suitable reactive groups are: haloalkyl, haloacetyl, p-mercuribenzoate groups as well as groups which are to be subjected to a Michael addition reaction, such as e.g. maleimides, methacrylo groups (eg, J. Amer. Chem. Soc. 101, 3097, 1979).

For connecting the antibody fragments with the polymer complexes or the ligands, there are in addition a number of suitable, often also commercially available, bifunctional or "linkers" (see e.g. Pierce, Handbook and General

Catalog 1986), which are reactive both towards the SH groups

in the fragments that also opposite amino- or the hydrazino groups in the polymer.

Examples include: m-maleimidobenzoy1-N-hydroxysuccinimide ester (MBS), m-maleimidobenzoy1-N-sulfosuccinimide ester (sulfo-MBS), N-succinimidyl-[4-(iodoacetyl)-amino]-benzoic acid ester (SIAB), succinimidyl -4(N-maleimidomethyl)-cyclohexane-1-carboxylic acid ester (SMCC), succinimidyl-4(p-maleimidophenyl)-butyric acid ester (SMPB), N-succinimidyl-3-(2-pyridyldithio)-propionic acid ester (SDPD), 4-[3-(2,5-dioxo-3-pyrrolinyl)-propionyloxy]-3-oxo-2,5-diphenyl-2,3-dihydrothiophene-1,1-dioxyd.

Bonds of a non-covalent nature can also be used for the connection, whereby both ionic as well as van der Waals and hydrogen bridge bonds in alternating proportions and strength (the key-lock principle) can contribute to the bond (e.g. avidin-biotin , antibody-antigen). Containment connections (host-guest) of smaller complexes are also possible in larger cavities of macromolecules.

The linking principle consists of first producing a bifunctional macromolecule, whereby either an antibody hybridoma that is directed against a tumor antigen is fused with a second antibody hybridoma that is directed against a complex according to the invention, or the two antibodies are chemically connected to each other by means of a linker (for example, in the manner indicated in J. Amer. Chem. Soc. 101, 3097 (1979)) or the antibody directed against the tumor antigen is bound to avidin (or biotin) via a linker [D.J. Hnatowich et al., J. Nucl. With. 28, 1294 (1987)]. Instead of the antibody, its corresponding F(ab)- or F(ab<1>)2 fragments are used. For pharmaceutical use, first the bifunctional macromolecule that is enriched at the target site is injected, and then at a time interval the complex compound according to the invention [possibly bound to biotin (or avidin)], which is connected in vivo at the target site and can unfold its diagnostic or therapeutic effect there. Other connection methods can also be used, e.g. the "Reversible Radiolabeling" described in Protein Tailoring Food Med. Uses [Am. Chem. Soc. Symp.] (1985), 349.

A particularly simple method for producing antibody conjugates or antibody-fragment conjugates, is the so-called solid phase coupling: the antibody is coupled to a stationary phase (e.g. an ion exchanger) which is, for example, located in a glass column. By successive flushing of the column with a solution suitable for the generation of aldehyde groups, washing, flushing with a solution of the functionalized complex (or the ligands), washing (if a ligand is used, a flushing with a solution containing the metal salt is carried out , followed by another rinse) and finally elution of the conjugate, very high conjugate yields are obtained.

This method enables an automatic and continuous production of desired quantities of conjugates.

Other connection steps can also be carried out in this way.

Thus, it can e.g. by the sequence papain reduction/bifunctional linker/functionalized complex or ligand, fragment conjugates are produced.

The thus produced compounds are then preferably purified chromatographically using ion exchangers in a Fast-Protein-Liquid-Chromatography facility.

The production of the metal complexes according to the invention takes place in the manner shown in DE-OS 34.01.052, the metal oxide or a metal salt (for example the nitrate, acetate, carbonate, chloride or sulphate) of elements with the ordinal numbers 21-29, 31, 32, 38 , 39, 42-44, 49, 57-83 are dissolved or suspended in water and/or a lower alcohol (such as methanol, ethanol or isopropanol) and reacted with the solution or suspension of the equivalent amount of the complexing ligand and then, if desired , present acidic hydrogen atoms are substituted in acid or the phenol groups with cations of inorganic and/or organic bases or amino acids.

The neutralization thereby takes place with the help of inorganic bases (e.g. hydroxides, carbonates or bicarbonates) of e.g. sodium, potassium or lithium and/or organic bases such as primary, secondary and tertiary amines, such as ethanolamine, morpholine, glucamine, N-methyl- and N,N-dimethylglucamine, as well as basic amino acids, such as e.g. lysine, arginine and ornithine or of amides of originally neutral or acidic amino acids.

For the preparation of the neutral complex compounds, for example, the acidic complex salts in aqueous solution or suspension can be added in such a quantity of the desired bases that the neutral point is achieved. The solution obtained can then be evaporated to dryness in a vacuum. It is often an advantage to precipitate the formed neutral salts by adding solvents that are miscible with water, such as e.g. lower alcohols (methanol, ethanol, isopropanol and others), lower ketones (acetone and others), polar ethers (tetrahydrofuran, dioxane, 1,2-dimethoxyethane and others) and thus obtain crystallisates that are easy to isolate and to purify. It has been found to be particularly advantageous to add the desired base to the reaction mixture during the complex formation, thereby saving a process step.

If the acidic complex compounds contain several free, acidic groups, it is often appropriate to prepare neutral mixed salts that contain inorganic as well as organic cations such as counterions.

This can take place, for example, by reacting the complex-forming ligands in aqueous suspension or solution with the oxide or salt of the element that supplies the central ion and half the amount of an organic base that is necessary for the neutralization, the formed complex salt is isolated, it is purified if desired and added then the necessary amount of inorganic base for complete neutralization. The order of base addition can also be reversed.

The conjugates of antibody and complex are dialysed before the use in vivo after incubation with a weak complex former, such as e.g. sodium citrate, sodium-ethylene-diamine-tetraacetic acid, to remove weakly bound metal atoms.

If complex compounds containing radioisotopes are used, their preparation can be carried out using the method described in "Radiotracers for Medical Applications", vol. 1, CRC-Press, Boca Raton, Florida.

The production of the pharmaceutical agents according to the invention likewise takes place in a manner known per se, as the complex compounds according to the invention, optionally with the addition of common galenic additives, are suspended or dissolved in an aqueous medium, and the suspension or solution is then optionally sterilized. Suitable additions are, for example, physiologically harmless buffers (such as tromethamine), small additions of complex formers (such as diethylenetriaminepentaacetic acid) or, if necessary, electrolytes such as sodium chloride or, if necessary, antioxidants such as ascorbic acid.

If suspensions or solutions of the agents according to the invention in water or physiological salt solution are desired for enteral administration or other purposes, they are mixed with one or more of the usual galenic excipients (e.g. methylcellulose, lactose, mannitol) and/or surfactants (e.g. lecithin, "Tween", "Myrj") and/or flavoring agents for flavor correction (e.g. essential oils).

In principle, it is also possible to prepare the pharmaceutical agents according to the invention also without isolating the complex salts. In all cases, particular care must be taken when carrying out the chelation so that the salts and salt solutions according to the invention are practically free of non-complexed, toxic metal ions.

This can, for example, be achieved with the help of color indicators such as xylenol orange in control titrations during the manufacturing process. The invention therefore also applies to methods for producing the complex compounds and their salts. As a last resort, a purification of the isolated complex salt remains.

The pharmaceutical agents according to the invention preferably contain 1 umol - 1 mol/l of the metal in the form of its complex salt and are usually dosed in amounts from 0.001 to 5 mmol metal/kg. They are intended for enteral and parenteral application.

The complex compounds according to the invention are used 1. for NMR, X-ray and ultrasound diagnostics in the form of their complexes with the ions of the elements with the ordinal numbers 21-29, 42, 44 and 57-83, 2. for radiodiagnostics and radiotherapy in the form of their complexes with the radioisotopes of the elements with the order numbers 27, 29, 31, 32, 38, 39, 43, 49, 62, 64, 70 and 77.

The agents according to the invention fulfill the various prerequisites for being suitable as a contrast agent for nuclear spin tomography. Thus, they are eminently suitable for, after oral or parenteral administration by increasing the signal intensity, to improve the image obtained by means of the nuclear spin tomograph in its expressive power. Furthermore, they exhibit the high activity that is necessary to burden the body with the least possible amount of foreign substances, and the good compatibility that is necessary to maintain the non-invasive nature of the examinations.

The good water solubility of the agents according to the invention makes it possible to prepare highly concentrated solutions so that the volume load on the circuit is kept within tolerable limits and the dilution with the body fluid is equalised, i.e. that NMR diagnostics must be 100 - 1000 times better water soluble than for the NMR spectroscopy. Furthermore, the agents according to the invention not only show a high stability in vitro, but also a surprisingly high stability in vivo, so that only an extremely slow release or exchange of the inherently toxic ions that are not covalently bound in the complexes takes place during which time the new contrast agents are again completely excreted.

Usually, the agents according to the invention are dosed for use as NMR diagnostics in quantities of 0.001 - 5 mmol metal/kg, preferably 0.005 - 0.5 mmol metal/kg. Details of the application are discussed, for example, in H.J. Weinmann et al., Am. J. of Roentgenology 142, 619 (1984).

Particularly low doses (below 1 mg/kg) of organ-specific NMR diagnostics can, for example, be used to detect tumors and heart attacks.

Furthermore, the complex compounds according to the invention can advantageously be used as shift reagents.

The agents according to the invention are, due to their favorable radioactive properties and the good stability of the complex compounds contained in them, also suitable as radiodiagnostics. Details for their application and dosage are described, e.g. in "Radiotracers for Medical Applications", CRC-Press, Boca Raton, Florida.

A further imaging method with radioisotopes is positron emission tomography, which uses positron-43 44 52 55 68 emitting isotopes such as Sc, Sc, Fe, Co, Ga and Rb.

The compounds according to the invention can also be used

in radioimmunotherapy. This differs from the corresponding diagnostics only in the quantity and nature of the radioactive isotope used. The goal is thereby the destruction of tumor cells using energy-rich, short-wave radiation with the smallest possible range. The specificity of the carrier used (e.g. chelate antibody) is therefore of decisive importance as non-specifically localized antibody conjugates lead to the destruction of healthy tissue.

The antibody or the antibody fragment in the antibody-metal complex according to the invention serves to transport the complex immunospecifically for the relevant antigen to the target organ where, due to its cell-killing properties, the selected metal ion can emit rays that damage

46 cells lethal. Suitable (3-emitting ions are, for example, Sc, Sc, Sc, Ga and Ga. Suitable oc-emitting ions exhibiting low half-lives are, for example, Bi, Bi, Bi

214 212

and Bi, whereby Bi is preferred.

In the in vivo use of the therapeutic agents according to the invention, these can be supplied with a suitable carrier such as e.g. serum or physiological saline solution and together with another protein such as e.g. human serum albumin. The dosage is therefore dependent on the nature of the cellular disorder and the metal ion used.

The therapeutic agents according to the invention are administered parenterally, preferably i.v.

Details of the application of radiotherapeutics are discussed, e.g. in R.W. Kozak et al., TIBTEC, October 1986, 262.

The agents according to the invention are likewise suitable as X-ray contrast agents, whereby it should be particularly emphasized that they can demonstrate significantly more useful pharmaco-kinetics for diagnostics, even compared to the hitherto common iodine-containing contrast agents. They are also particularly valuable because of the favorable absorption properties in areas of higher tube voltages for digital subtraction techniques.

In general, the agents according to the invention are dosed for use as X-ray contrast agents in analogy with e.g.

meglumine diatrizoate in amounts of 0.1 - 5 mmol metal/kg, preferably 0.25 - 1 mmol metal/kg.

Details of the use of X-ray contrast agents are discussed, for example, in Barke, Rontgenkontrastmittel,

G. Thieme, Leipzig (1970) and P. Thurn, E. Biicheler - "Ein-fiihrung in die Rontgendiagnostik", G. Thieme, Stuttgart,

New York (1977).

The agents according to the invention are, as their acoustic impedance is higher than the impedance of body fluids and tissues, also suitable as contrast agents for ultrasound diagnostics, particularly in the form of suspensions. They are generally dosed in amounts from 0.1 to 5 mmol/kg, preferably from 0.25 to 1 mmol/kg.

Details of the application of ultrasound diagnostics are described, e.g. in T.B. Tyler et al., Ultrasonic Imaging 3, 323

(1981), J.I. Haft, "Clinical Echocardiography", Futura,

Mount Kisco, New York 1978 and G. Stefan "Echokardiographie",

G. Thieme/Stuttgart/New York 1981.

All in all, it has been successful in synthesizing new polymer complexes that offer new opportunities in diagnostic and therapeutic medicine. Above all, the development of new imaging procedures in medical diagnostics has shown how desirable this development has been.

The following examples serve to further explain the object of the invention.

Example 1

a) Gadolinium complex of polyethyleneimine polyacetic acid

37.0 g of polyethyleneimine polyacetic acid, prepd

according to DE 2,303,081, dissolve in 500 ml of double-distilled water and heat with 10 g of gadolinium carbonate (hydrated,

59% by weight Gd) with stirring for 2 hours at 80°C.

After ultrafiltration and freeze-drying, 41.8 g of white, voluminous material is obtained which, according to elemental analysis, contains 13.55% by weight of gadolinium.

Analogous to regulation la obtained from polyethyleneimine polyacetic acid and b) manganese-(II)-carbonate the manganese complex with 5.19% by weight of manganese

c) cobalt carbonate the cobalt complex with 5.45% by weight of cobalt

d) basic copper carbonate the copper complex with 5.74 wt% copper e) dysprosium carbonate the dysprosium complex with 15.17 wt% dysprosium

f) the lanthanum carbonate lanthanum complex with 15.08% by weight of lanthanum

g) yttrium carbonate yttrium complex with 8.14% by weight yttrium

h) samarium carbonate the samarium complex with 16.33% by weight

samarium

i) holmium carbonate the holmium complex with 14.12% by weight holmium j) ytterbium carbonate the ytterbium complex with 17.03% by weight

ytterbium

k) bismuth oxide bismuth complex with 17.39 wt% bismuth 1) iron (III) oxide iron III complex with 5.27 wt% iron

m) lead carbonate lead complex with 19.08% lead by weight.

Example 2

a) Sodium salt of the gadolinium complex of polyethyleneimine polyacetic acid

12.36 g of the complex prepared as described under la), was dissolved in 50 ml of water and 78.7 ml of 1 N caustic soda was added. With vigorous stirring, 2 liters of acetone are poured into the solution, the precipitate is filtered off and dried in a vacuum to constant weight. 13.0 g of a white granular product containing 11.83% by weight of gadolinium and 12.84% by weight of sodium are obtained.

b) Calcium salt of the gadolinium complex of polyethyleneimine polyacetic acid

6.38 g of the complex prepared as described under la), is dissolved in 100 ml of water and stirred with 2.1 g of calcium carbonate for 5 hours at 80°C. After ultrafiltration and freeze-drying, 7.03 g is obtained as a white powder containing 12.08% by weight of gadolinium and 10.81% by weight of calcium.

c) N-methyl-D-glucamine salt of the gadolinium complex of polyethyleneimine polyacetic acid

21.35 g of the complex prepared as described under la), is dissolved in 200 ml of water and a solution containing 1 mol of N-methyl-D-glucamine per litres, with constant stirring at 30°C for as long as the pH value is at 6.85. After freeze-drying, 47.48 g are obtained as white powder containing 6.19% by weight of gadolinium.

d) Ethanolamine salt of the gadolinium complex of polyethyleneimine polyacetic acid

3.75 g of the gadolinium complex that has been prepared

as described under la), dissolve in 50 ml of water and add a 10% aqueous solution of ethanolamine, until the pH value reaches 6.85. After freeze-drying, 5.2 g of white substance with a gadolinium content of 9.84% by weight is obtained.

e) L-lysine salt of the gadolinium complex of polyethyleneimine polyacetic acid

2.58 g of the gadolinium complex prepared as described under 1a) are dissolved in 50 ml of water and a 10% aqueous L-lysine solution is added until the pH value is 6.85. After freeze-drying, the product returns as a volumeless, white powder. The gadolinium content amounts to 7.16% by weight.

f) Morpholine salt of the gadolinium complex of polyethyleneimine polyacetic acid

To a solution of 3.3 g of the gadolinium complex prepared as described under la), a 10% aqueous morpholine solution is added until the pH value reaches 6.85. Freeze-drying yields 5.1 g of white, slightly hygroscopic, solid substance with 8.87% by weight of gadolinium.

Analogously, the corresponding salts of the complexes described in examples 1b) to 1m) are obtained.

Example 3

a) Polyethyleneimine polyacetic acid polyhydrazide

10.37 g of polyethyleneimine polyacetic acid is heated with

600 mg of finely powdered potassium hydroxide in 150 ml of dry dimethylsulfoxide with stirring for 20 minutes at 60°C. 0.5 ml of dimethylsulphate is then added, and the mixture is cooled with stirring in a heating bath to room temperature. After 3 hours, cool to 10°C, and 1 ml of hydrazine monohydrate is added dropwise. Then heat for another 1 hour in a steam bath. It is diluted with twice the volume of water and dialysed, until hydrazine can no longer be detected in the filtrate. After freeze-drying, 8.12 g of colorless crystals are obtained.

Hydrazine (colorimetric): 1.34 mol%

b) Gadolinium complex of polyethyleneimine polyacetic acid polyhydrazide

3.98 g of the hydrazide described above are dissolved in

50 ml of water and half-concentrated hydrochloric acid is added, until the sediment that has formed in the meantime is completely dissolved again. To this is added 4 ml of a lm gadolinium chloride solution in 1 N hydrochloric acid.

By adding solid sodium hydroxide, the pH value is raised to 9 with vigorous stirring, and the solution is dialysed. After freeze-drying, 4.51 g of white substance with a gadolinium content of 13.3% by weight is obtained.

This was divided into fractions with different molecular weights over a column with "Sephacryl" S300. Of these, the following relaxivities were measured:

Alternative preparation of the gadolinium complex of polyethylenimine polyacetic acid polyhydrazide

11.35 g of the gadolinium complex salt of polyethyleneimine polyacetic acid described in example 2a), add 0.5 ml of dimethylsulphate to 250 ml of aqueous methanol and stir for 6 hours. 20 ml of hydrazine monohydrate is then added, stirred overnight, and the solution is dialysed until free hydrazine can no longer be detected. After freeze-drying, 10.3 g of colorless crystals with a gadolinium content of 11.7% by weight are obtained.

Hydrazine (colorimetric after hydrolysis): 1.28 mol%

Example 4

a) Polyethyleneimine polymalonic acid

Dissolve 26.8 g (146.5 mmol) of bromomalonic acid in 250 ml of water and add carefully and while cooling 39.1 g (466 mmol) of sodium bicarbonate. A solution of 4.2 g of polyethyleneimine in 110 ml of water is added dropwise, whereby the temperature is kept at 40-50°C. After 14 hours of stirring at this temperature, 30 ml of concentrated hydrochloric acid are added while cooling, and the mixture is poured into 3 1 of methanol. The precipitate is dissolved again with diluted caustic soda and dialyzed against deionized water until the dialysate no longer precipitates with silver nitrate solution. After freeze-drying, a yellowish crystal mass remains.

Yield: 9.6 3 g.

The titrimetric determination of acid groups shows that 22% of the nitrogen atoms are alkylated.

b) The gadolinium complex is prepared using the method described in example la), and contains

7.38 wt% gadolinium.

Example 5

Gadolinium complex of polyethyleneimine polymalonic acid polyacetic acid a) 13.8 g of the polyacid described in example 4a) are reacted with 20 g bromoacetic acid and 23 g sodium bicarbonate analogously to the method described in 4a). After dialysis and freeze-drying, 17.35 g of colourless, solid substance is obtained.

Titration of the acid groups shows that 95% of the nitrogen atoms are alkylated. b) The gadolinium complex is obtained analogously to example la). The content of gadolinium amounts to 13.47% by weight.

Example 6

a) Polylysine polyacetic acid

A solution of 13.9 g (100 mmol) bromoacetic acid i

26.8 g (319 mmol) of sodium bicarbonate are added in portions to 150 ml of water under ice-cooling. A solution of 2.5 g of polylysine (average molecular weight 30,000) in 50 ml of water is then added drop by drop, whereby the temperature is kept at 40 - 50°C. The mixture

is stirred overnight at this temperature, 12 ml of concentrated hydrochloric acid are added under ice-cooling, and the mixture is poured into 1.5 1 methanol while stirring. The separated precipitate is suctioned off, dissolved moist in diluted caustic soda and dialyzed against deionized water until the dialysate is chloride-free. Freeze-drying yields 4.8 g as a white powder. The elementary analysis shows that approx. 90% of the free amino groups are alkylated.

b) Gadolinium complex of polylysine polyacetic acid

3 g of the compound obtained under a), is heated in 50 ml of water with 1.5 g of gadolinium carbonate,

Gd2(CO3)3' with stirring at 80°C. After membrane filtration and freeze-drying of the solution, 3.8 g are obtained as white powder. The content of gadolinium amounts to 10.5%, calculated on anhydrous substance.

Example 7

Gadolinium complex of the conjugate of the monoclonal antibody MARGll with polyethyleneimine polyacetic acid polyhydrazide a) 4 ml of a 50 n molar solution of the monoclonal antibody MARGll (subclass IgG/1, Camon Laborservice GmbH, Wiesbaden) in 0.1 M acetate buffer (pH 4.5) is added the same volume of buffered 50 mM sodium periodate solution and shake for 30 minutes at room temperature under the exclusion of light. Excess periodate is then destroyed with ethylene glycol, and the solution is dialysed. A solution of 216 mg of the polyethyleneimine polyacetic acid polyhydrazide described in example 3 is then added in 8 ml of acetate buffer and 6 mg of sodium borohydride and incubated for 40 hours at 4°C with shaking. The solution is purified over a cation exchanger, dialyzed against a 0.1 m ammonium acetate buffer and lyophilized. 47 mg of a white powder is obtained.

b) The conjugate can also be prepared in the following way:

30 nmol of the antibody is bound to a macroporous, strongly acidic

cation exchanger which has previously been brought into equilibrium with a

0.1 m sodium acetate buffer (pH 5). The exchanger is then flushed with a 0.03 m sodium metaperiodate solution in acetate buffer. If periodate appears in the eluate, the flow is interrupted for 1 30 minutes. The solid phase is then washed with acetate buffer and in the next step a 0.03 M polyethyleneimine polyacetic acid polyhydrazide solution in acetate buffer is applied. After the appearance of the complex former in the eluate, the flow is interrupted for 2 hours. Then uncoupled complex formers are eluted with acetate buffer. The elution of the antibody conjugate from the solid phase is carried out using a saline gradient in acetate buffer. The isolation of the conjugate takes place by freeze-drying the desalted solution. c) The complexation of the antibody conjugate obtained according to a) or b) takes place as follows: 20 mg of the conjugate is dissolved in 4 ml of a 50 mmol sodium acetate/150 mmol sodium chloride buffer (pH 6.0). To this is added a solution of 6.7 umol of gadolinium(III) chloride in the same buffer, and it is incubated for 4 hours at 2°C. After adding 6.7 µmol of sodium citrate, stir for 30 minutes and dialyze exhaustively against sodium acetate buffer. By freeze-drying the solution, a complex conjugate is obtained as a white powder, which bound contains 85-3 gadolinium ions per antibody conjugate.

Example 8

Indium complex of the conjugate of polyethyleneimine polyacetic acid and the monoclonal antibody MARGll 12 mg of polyethyleneimine polyacetic acid and 20 ul of triethylamine are dissolved in 0.5 ml of acetonitrile and stirred with 2 mg of N-hydroxysuccinimide and 2.5 mg of diisopropylcarbodiimide for 3 hours.

At the same time, a solution of 2 mg of the monoclonal antibody MARG11 is prepared in 1 ml of aqueous buffer (0.1 M sodium bicarbonate and 0.2 M ethylenediaminetetraacetic acid, pH 7). To this is added 100 ul of the above solution of the activated ester and allowed to stand for 1 hour with random shaking.

After purification over a cation exchanger, the complexation takes place with<111>InCl3. After dialysis and freeze-drying, the specific activity amounts to 560 m Ci/mg.

Example 9

Conjugate of the gadolinium complex of the polyethyleneimine polyacetic acid polyhydrazide with Fab fragments of the monoclonal antibody MARG11.

a) Preparation of F(ab')^- fragments:

16 mg (100 nmol) of the antibody MARG2b8 (subclass

IgG2b, Camon Laborservice GmbH, Wiesbaden) is dissolved in 1 ml of a mixture of 0.1 M acetate buffer (pH 4.5) and 0.1 M saline and incubated after the addition of 0.3 mg pepsin for 20 hours at 37°C . After purification over "Ultragel" AcA 44 (company LKB) at pH 7.0 and after freeze-drying, 6.3 mg (63% of the theoretical) of the desired fragments are obtained.

b) Preparation and ligation of Fab fragments:

15 mg (150 nmol) of the fragments obtained

according to a), dissolve in 14.5 ml of 0.1 M phosphate buffer (pH 6.0) and dissolve with 0.15 ml of a 0.1 M mercaptoethylamine solution in 0.1 M phosphate buffer (pH 6.0) under addition of 15 mmol of ethylenediaminetetraacetic acid. After 2 hours

incubation at 37°C is partitioned under argon protection over a "Sephadex" G 25 column. A determination of the sulfhydryl groups gives 238 nmol of SH groups in the reaction mixture.

236 mg of the gadolinium complex described in example 3 is dissolved in 10 ml of 0.1 m phosphate buffer (pH 6.0). To this is added at 10°C 0.5 ml of a 0.1 M solution of sulfonsuccinimidyl-4-(4-maleimidophenyl)-butyrate ("Sulfo-SMPB", company Pierce Chemical) in the same buffer and stirred for 2 hours at room temperature.

The solution of the fragment is then combined with the solution of the activated complex and allowed to react overnight with gentle shaking. The subsequent work-up over a cation exchanger takes place using the procedure described in example 7. By freeze-drying the purified solution, 24.1 mg of a white powder with a content of 8.89% by weight of gadolinium is obtained.

Example 10

Gadolinium complex of polyethyleneiminepolymethylenephosphonic acid

21.17 g of polyethyleneimine polymethylenephosphonic acid, prepared according to US patent 2,599,807, is dissolved in 300 ml of double-distilled water and heated with 4.3 g of gadolinium carbonate (hydrated, 59% by weight Gd) with stirring for 2 hours at 80°C.

After ultrafiltration and freeze-drying, 23.54 g of white, voluminous material is obtained which, according to elemental analysis, contains 10.29% by weight of gadolinium.

Example 11

Biotinylated gadolinium complex of polyethyleneimine polyacetic acid polyhydrazide

1.31 g of the Gd complex of polyethyleneimine polyacetic acid polyhydrazide described in Example 3b), is added to 20 ml of water, 133 mg of "Sulfo-NHS-Biotin" (Pierce Chemical Company) and maintained by the addition of NaHCO^ at

pH 8-9. After stirring for several hours at room temperature, dilute with water and dialyze until biotin can no longer be detected in the filtrate. After freeze-drying, 1.08 g of colorless substance is obtained.

Biotin content: 1.3 mol%

Example 12

Sodium salt of polyethyleneimine polysuccinic acid

To a solution of 36.15 g of polyethyleneimine

("Polymin" P) in 320 ml of methanol is added, possibly under gentle heating, a solution of 181.75 g of maleic acid dimethyl ester in 200 ml of methanol in such a way that the temperature does not exceed 40°C.

It is then stirred for a further 4 hours at 50-60°C, and 53.8 g of sodium hydroxide beads are then added, whereby the temperature during stirring is kept at a maximum temperature of 40°C. 200 ml of ice water is added, and the mixture is evaporated on a rotary evaporator. The residue is dissolved in 500 ml of 0.1 m ammonium acetate buffer and dialysed. After freeze-drying, 116.39 g of colourless, voluminous crystals are obtained which decompose above 300°C during fading.

Gadolinium Complex:

The gadolinium complex is prepared as described in example la), and contains 12.49% by weight of gadolinium.

Example 13

Gadolinium complex of polyethyleneimine polyacetic acid polymethyl ester polyhydrazide

11.35 g of the gadolinium complex salt of polyethyleneimine polyacetic acid described in example 2a), 250 ml of aqueous methanol with 5.9 ml of dimethylsulphate are added, stirred for 6 hours at room temperature and then heated in a heating bath at 60°C. It is then cooled to room temperature, and hydrazine hydrate is added and then heated for a further 4 hours on a steam bath. The solution thus obtained is dialysed and freeze-dried. 11.5 g of colorless crystals with a gadolinium content of 10.6% by weight are obtained. The determination of methyl ester groups gives an esterification of 65% of all carboxy groups.

Hydrazine (colorimetric after hydrolysis): 1.17 mol%

Examples of the production of supply forms

Example 14

Solution of gadolinium complex of polylysine polyacetic acid

5.98 g (corresponding to 4 mmol Gd"^ + ) of the gadolinium complex prepared according to example 6 is brought into solution in 800 ml of water pro injectione (p.i.) by adding N-methylglucamine at pH 7.2. After filling with water p.i. to 1000 ml, the solution is sterile filtered, filled in 10 ml aliquots in multi-medicin glasses and lyophilized. The remainder each corresponds to a single dose of 0.04 mmol Gd"^ + /kg body weight.

Example 15

Solution of the sodium salt of indium-lll complex of polyethyleneimine polyacetic acid

A solution of 100 ug of a polyethyleneimine polyacetic acid with an average molecular weight of 100,000 in 5 ml of a mixture of a 150 mmolar sodium acetate solution and a 150 mmolar sodium acetate solution (pH 5.8) is added to a solution of 5 mCi indium-111 chloride in 1 ml n-hydrochloric acid. By adding 1 n caustic soda, the mixture is brought to pH 6 and dialyzed against sodium acetate solution, then against sodium chloride solution. The mixture is then brought to pH 7.2 by the addition of 0.1 n caustic soda, the sterile-filtered solution is filled into a multi-purpose glass and lyophilized. The rest is taken up in physiological saline solution, and a preparation suitable for radiodiagnostics is then prepared.

In an analogous manner, a preparation suitable for radiotherapy is obtained from 5 mg of polyethyleneimine polyacetic acid and a solution of 500 mCi of yttrium-90 chloride.

Example 16

Resolution of the In-lll complex of the conjugate of polyethylene-imine polyacetic acid hydrazide with the monoclonal antibody MARGll

A solution of 200 µg of the complexing agent described in example 7, in 5 ml of a mixture of a 150 mmolar sodium acetate solution and a 150 mmolar sodium acetate solution (pH 5.8) is added to a solution adjusted to pH 5 of 5 mCi indium-111 chloride in dilute hydrochloric acid. The preparation corresponding to example 11 gives a preparation which is suitable for radiodiagnostics.

In an analogous manner, a preparation suitable for radiotherapy is obtained from 10 mg of the complex former described in example 7 and a solution of 500 mCi of yttrium-90 chloride (pH 5).

Example 17

Injection solution of the gadolinium complex of the conjugate of the monoclonal antibody MARG11 with polyethyleneimine polyacetic acid polyhydrazide

1.08 of the antibody-gadolinium complex conjugate which

is described in example 7c), dissolve in 80 ml of water p.i. and add 50 mg of the calcium-di-sodium complex of DTPA and make up to 100 ml. After sterile filtration, it is filled into 5 ml multi-medicine vials and lyophilized.

When dissolved in 5 ml of physiological saline solution, a preparation for parenteral use is obtained.

Example 18

Injection solution of the N-methyl-1-D-glucamine salt of the gadolinium complex of polyethyleneimine polyacetic acid

127 g (= 50 mmol Gd^ + ) of the N-methyl 1-D-glucamine salt of the Gd complex of polyethyleneimine polyacetic acid which is described in example 2c), dissolve in 800 ml of water p.i., add 1.2 g of calcium -di-sodium salt of DTPA and made up to 1 liter of water p.i.

The solution is sterile filtered, filled in portions of 5 ml each into multi-dose vials and lyophilized. When dissolved in 5 ml of physiological saline solution, a preparation for parenteral use is obtained.

Example 19

Preparation of a solution with biotinylated gadolinium complex of polyethyleneimine polyacetic acid polyhydrazide for in vivo use

50 mg of a biotinylated gadolinium complex of polyethyleneimine polyacetic acid polyhydrazide (biotin content 1.3 mol%)

(example 11) is dissolved in 10 ml of physiological saline solution.

The pH is adjusted to pH 7.2 with acetic acid, and the solution is sterile filtered with a 0.2 µm filter. 0.2 ml of this solution with a Gd content of 0.9 umol is applied i.v. 48 hours after application of a tumor-specific, monoclonal antibody-avidin conjugate.

The implementation of an NMR diagnostic examination using a polymer complex according to the invention is explained in more detail with the help of the following example: Detection of blood vessels in the liver after application of 0.02 mmol of gadolinium as a gadolinium complex of polyethyleneimine polyacetic acid (example 1)

A mouse (NMRI, SPF Schering) is anesthetized with nembutal i.p. The animal is placed in a 2 Tesla nuclear spin tomograph (General Electric). Recordings are made in the area of the liver (transverse section) in a spin-echo sequence with TD = 400 msec and T„ = 30 msec (illustration 1).

Then applied i.v. 0.02 mmol of gadolinium in the form of the gadolinium complex of polyethyleneimine polyacetic acid (Example 1) dissolved in 0.9% physiological saline solution (200 ul). Two minutes after application, recordings are again made in a spin-echo sequence (T_ = 400 msec, T„ - 30 msec) in the same section plane as described above (illustration 2).

Claims (14)

1. Polymer complexes, characterized in that they consist of a ligand with alkylated amine units containing carboxylic acid, phosphonic acid and/or phenolic groups, at least one ion of an element with ordinal numbers 21 - 29, 31, 32, 38, 39, 42 - 44, 49 or 57 - 83, as well as possibly cations of inorganic and/or organic bases, amino acids or amino acid amides. 2. Polymer complexes according to claim 1, characterized in that they contain a compound of the general formula I as a ligand where n means the whole number 7 to 20,000.12 3 12 X means a hydrogen atom, a CR R R - or a CR R —U residue with U meaning one where A<1> stands for OH or A and A" stands for H or A', with A meaning a (NH)-[B-(NH)v]^-W- or OR 4 group where u and v stand for the digits 0, 1 or 2, w for the digits 0 or 1, R 1 for a (CH„) Z group, ~ 2 m R for a hydrogen atom or a (CH ), Z group, R 3 for a hydrogen atom or a (CH„), Z group, and R 4 for a C^ -C^ alkyl or benzyl residue, whereby m, 1 and k stand for the digits 0 to 10, and Z stands for a hydrogen atom, a C-^ -C^ -alkyl-, a -C00H-,-PO3 H2 - or where Y means a hydrogen atom or the residue OH, B means a C0 -C20 alkylene group, and W means a hydrogen atom, a macromolecule, a functional group which is optionally bound over a linear, branched, saturated or unsaturated C^ -C2Q -alkylene group or bound over this functional group a macro- or biomolecule, whereby the alkylene group optionally contains an imino-, phenylenoxy-, phenylenimino-, amide, ester group ( r), oxygen, sulfur and/or nitrogen atom(s) and optionally substituted with hydroxy, mercapto and/or amino group(s), whereby the substituents X in the individual links and at the ends 12 3 of the compound as well as the substituents Z in R , R and R can be the same or different, with the proviso that not all X must simultaneously mean hydrogen, that at least one Z does not mean hydrogen or C-^ -C^Q -alkyl, that if m, 1 and k = 0, not all of the substituents Z stand for -COOH, -PO^ H» or and that, if desired, part of -COOH-,-PO3 H2 - or -groups exist as esters or amides. 3. Polymer complexes according to claim 2, characterized in that they contain between 1 ? 12 1 and n/10 CR R — U residues, where n, R , R and U have the above-mentioned meaning. 4. Polymer complexes according to claim 1, characterized in that they contain as ligand where Q means the residue of a natural amino acid containing the groups NX and/or NX2 , and n and X have the meanings mentioned in claim 2. 5. Polymer complexes according to claim 1, characterized in that they contain at least one ion of an element with the ordinal numbers 21 - 29, 42, 44 or 57 - 83. 6. Polymer complexes according to claim 1, characterized in that they contain at least one radionuclide of an element with the ordinal numbers 27, 29, 31, 32, 38, 39, 43, 49, 62, 64, 70 or 77. 7. Polymer complexes according to claims 2 to 4, characterized in that the functional group W means 8. Polymer complexes according to claims 2 to 4, characterized in that the macromolecule contained in W is an antibody or an antibody fragment. 9. Polymer complexes according to claims 2 to 4, characterized in that the macromolecule contained in W is an avidin-biotin antibody or avidin-biotin-antibody fragment. 10. Use of at least one polymer complex according to claim 5 for the production of agents for NMR, X-ray or ultrasound diagnostics. 11. Use of at least one polymer complex according to claim 6 for the production of agents for radiodiagnostics or radiotherapy. 12. Use of at least one polymer complex with the general formulas I <1> and II <1>, where W stands for a hydrogen atom or a functional group, according to claims 2 to 4 as the hapten for the production of antibodies. 13. Process for producing polymer complexes according to claims 1 to 9, characterized in that a ligand containing carboxylic acid, phosphonic acid and/or phenolic groups obtained in a manner known per se by N-alkylation, in a manner known per se a) is reacted with at least one metal oxide or metal salt of an element with the ordinal numbers 21 - 29, 31, 32, 38, 39, 42 - 44, 49 or 57 - 83, or b) is reacted with at least one metal oxide or metal salt of an element with the ordinal numbers 21 - 29, 31, 32, 38, 39, 42 - 44,49 or 57 - 83, where at least part of the carboxylic acid, phosphonic acid or phenolic groups which are not necessary for the complexation of the thus obtained metal complex, are converted into the groups CR <1> R <2-> UI with U<1> in the sense of one when 41 5' 41 A stands for OH or A" <1> , and A stands for H or A , with A"' in the sense of a (NH)u~[ B-(NH) ]w~W' or OR4 group where u and v stand for the digits 0, 1 or 2, w for the digits 0 or 1, R <1> for a (CH2)Z group, R <2> for a hydrogen atom or a (CH,,)^ Z group with m and 1 in the sense of the digits 0 to 10 and Z in the sense of a hydrogen atom, a C-^-C-^Q-alkyl group, a COOH-, PO^H,,- or where Y means a hydrogen atom or the residue OH, R 4 for a C -C -C -alkyl radical or benzyl radical, B for a C0 -C2Q -alkylene group, and W for a hydrogen atom or a functional group which is optionally bound over a linear, branched, saturated or unsaturated C 1 -C 2 Q -alkylene group, whereby the alkylene group optionally contains imino-, phenylenoxy-, phenylenimino-, amide, ester group(s), oxygen, sulfur and/or nitrogen atom(s) and optionally substituted with hydroxy, mercapto and/or amino group(s), 12 .. whereby the CR R U1 groups may be the same or different, and then, if desired, attached directly or above the functional group containing the above-mentioned residue, to a macro- or bio-molecule, or c) is transferred to a derivative containing at least one CR 1 R 2—U1 group, whereby R 1 , R 2 and U <1> have the meanings mentioned above, this is then reacted with at least one metal oxide or metal salt of an element with the ordinal numbers 21 - 29, 31, 32, 38, 39, 42 - 44, 49 or 57 - 83 and then, if desired, bound directly or via the functional group containing the above-mentioned residue, to a macro- or bio-molecule or d) is transferred to a derivative that contains at least one CR 1 R 2-U' group, whereby R 1 , R 2 and U <1> have the meanings mentioned above, this is then bound, if W is to contain a macro- or bio- molecule, directly or via the functional group containing the above-mentioned residue, into a macromolecule and is then reacted with at least one metal oxide or metal salt of an element with ordinal numbers 21 - 29, 31, 32, 38, 39, 42 - 44, 49 or 57 - 83, and optionally the acidic hydrogen atoms still present in the polymer complexes obtained according to a), b), c) or d) are then substituted with cations of inorganic and/or organic bases, amino acids or amino acid amides. 14. Process for producing polymer complexes according to claim 14, characterized in that the coupling of the macro- or bio-molecule to the functionalized polymer complex or the ligands as well as, in the case of coupling to the ligands, the subsequent complexation is carried out with the desired metal ion(s) in a stationary phase.