DK170460B1 - Diagnostic agent for use in in vivo NMR diagnostics, method of preparation thereof and complex salts suitable for use in the diagnostic agent - Google Patents

Description

DK 170460 B1 i

The invention relates to the subject matter of the claims.

Complexes or their salts have been used for a long time in medicine, e.g. as aids for the administration of heavily soluble ions (e.g., iron) and as an antidote (hereby calcium or zinc complexes are preferred) for detoxification by incorporating heavy metals or their radioactive isotopes.

GB-A-2001969 discloses complexes consisting of hafnium (atomic # 72) and tantalum (atomic # 73), which elements are not included in the present diagnostic agents, and complexing agents in the form of organic acids, amino acids and phosphonic acids, cf. page 2, lines 4-28 of the script, which complexing agents are not also referred to in the present invention. The complexes are used for radiographic examination of the gastrointestinal tract, cf. claims 15 and page 3 of the specification, which use is not the subject of the present invention.

PR-Publication No. 2322586 discloses radiodiagnostics of metal chelates with a substituted iminodiacetic acid. Neither these radiodiagnostic agents nor their uses are the subject of the present invention.

Radiology, 91, pp. 1191-1203 (1968) discloses radiodiagnostic studies with compounds which are not the subject of the present invention. Thus, outer bium-169 diethylenetriamine pentaacetic acid complex for use in brain scanning.

None of the compounds disclosed in these aforementioned scripts is bound to biomolecules.

GB-A-2060623 discloses metal chelates of · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · Aim-2060623 discloses metal chelates of biodegradable diamine treacidic acid derivatives bound to biomolecules and which are applicable to fluorescence assays

2 DK 170460 B1 In the document, the use of the compounds for in vivo NMR diagnostics is neither suggested nor suggested.

It has now been found that physiologically acceptable complex salts of the anion of a complexing acid and one or more central ions of an element having the atomic numbers (order numbers) 21-29, 42, 44 or 58-70 and optionally one or more physiologically acceptable cations of an inorganic and / or organic base or amino acid, are surprisingly excellent for the preparation of diagnostic agents suitable for use in in vivo NMR diagnostics.

Thus, the present invention relates to a diagnostic agent for use in in vivo NMR diagnostics, which is characterized in that liter contains 1 μιηοΐ - 1 mole of at least one physiologically acceptable complex salt with 15 of the general formula I

X-CH2, CH2'X

^ Jn-a-n ^ (i)

V-CHR1 CKPfV

wherein X represents the group -COOY, -PO-jHY or -CONHOY with Y in the sense a hydrogen atom, a metal ion equivalent and / or a physiologically acceptable cation of an inorganic or organic base or amino acid, and wherein R 1 represents hydrogen atoms 20 or methyl groups and wherein a) A represents the group -CHR2-CHR3-, -CH2-CH2- (ZCH2-CH2) m-, N (CH2X) 2 CH2-CH2-N (CH2X) 2 -CH2-CH-CH2- or -CH2-CH2 -N-CH 2 -CH 2 - wherein X is as defined above, R 2 and R 3 together represent a trimethylene group or a tetramethylene group or hydrogen atoms, lower alkyl groups, phenyl groups, benzyl groups, m represents the numbers 1, 2 or 3, Z represents an oxygen atom or a sulfur atom. or the group NCH2X or NCH2CH2OR4 DK 170460 B1 3 with X in the sense given above and R4 in the sense a lower alkyl group and wherein V has the same meaning as X, or the groups

-CH 2 OH or -COS

5 with B in the sense a protein or lipid residue or a -NH (CH2) nX residue with X in the above meaning, and nine meaning the digits 1-12, with the proviso that at least one substituent represents the group -COB, and at least two of the substituents Y represent metal ion equivalents of an element having atomic numbers 21-29, 42, 44 or 58-70 or b) wherein A represents the group -CHR2-CHR3-, where R2 represents a hydrogen atom and R3 represents a group * · - (CH 2) p -CgH 4 -W protein having p in the meaning of 0 or 1, W in the sense -NN-, -NHCOCH2 ~ 15 or -NHCS- and -protein in the meaning of a protein residue and wherein V has the meaning as X, or the group -ch2oh, provided that at least two of the substituents Y denote metal ion equivalents of an element with the atomic numbers 21-20 29, 42, 44 or 58-70, optionally dissolved with the usual additions in the Galenic father maci or suspended in aqueous medium.

Of course, the element having the aforementioned atomic number which forms the central ion (s) of the physiologically acceptable complex salt should not be radioactive for the intended use of the diagnostic agent according to the invention.

DK 170460 B1 4

Since the agent of the invention is intended for use in NMR diagnostics (see European Patent Application 71 564), the central ion of the complex salt must be paramagnetic. This is especially the di- and trivalent ions of the elements with atomic numbers 21-29, 42, 44 and 58-70. Suitable ions are e.g. chromium (III) -, manganese (II) -, iron (III) -, iron (II) -, co-bolt (II) -, nickel (II) -, copper (II) -, praseodymium (III) -, neodymium (III), samarium (III) and outer bium (III) ion. Due to their very strong magnetic moment, the gado-linium (III), terbium (III), dysprosium (III), holmium (III) and erbium (III) ion are particularly preferred.

The compositions of the invention are also suitable for use in X-ray and ultrasound diagnostics.

Suitable complexing acids are those which are usually used for the complexing of the above-mentioned central ions. Suitable complexing acids are e.g. those containing 3-12, preferably 3-8 methylene phosphonic acid groups, methylene carbohydroxamic acid groups, carboxyethylidene groups or especially carboxymethylene groups, of which at any one time one, two or three are attached to a nitrogen atom supporting the complex formation. If only one or two of the azide groups are bonded to a nitrogen atom, then the nitrogen is bonded to an additional nitrogen atom via an optionally substituted ethylene or via up to four ethylene units separated at any time by a nitrogen or oxygen or sulfur atom. , which supports complex formation.

The complexing acids can be conjugated with biomolecules as conjugates, the contents of which are known to increase especially in the organ or organ moiety to be examined.

Such biomolecules are e.g. hormones such as insulin, prostaglandins, steroid hormones, amino sugars, peptides, proteins or lipids. In particular, conjugates with albumins such as human serum albumin, antibodies such as e.g. monoclonal DK 170460 B1 antibodies specific for tumor-associated antigens, or antimyosin. The diagnostic agents resulting therefrom are suitable e.g. for use in tumor and infarction diagnostics.

For example, liver tests are suitable. conjugates or inclusion compounds with liposomes, e.g. is used as unilamellar or multilamellar phosphatidylcholine-cholesterol sterol vesicle. The conjugate formation occurs either via a carboxyl group of the complexing acid or in the case of proteins or peptides also via a group (ch2) p-c6h4-w-10 as defined in claim 1. In the conjugate formation of the complexing acids with proteins , peptides or lipids, several acid residues can be partially bound to the macro-molecular biomolecule. In this case, each complex-forming acid residue can carry a central ion. If the complex-forming acids are not bound to the biomolecule, they optionally carry two central ions, especially one central ion.

Suitable complex salts of general formula I according to claim 1 are e.g. those of the general formula la

X-CHOv CHO-X

> N-CHR2-CHR3-N3 (1a)

V-CHR ^ NsCHR1-V

wherein X, V, R 1, R 2 and R 2 are as defined in claim 1.

The preparation of the complex salts with the general for flour 1a is suitable, inter alia, the following complexing acids: ethylenediaminetetraacetic acid, ethylenediaminetetraacetic hydroxamic acid, trans-1,2-cyclohexylenediaminetetraacetic acid, DL-2,3-butylenediaminetetraacetic acid, DL-1,2-butylenediaminetetraacetic acid 25 acid, DL-1,2-propylenediamine tetraacetic acid, 1,2-diphenylethylenediamine tetraacetic acid, ethylenedinitrile tetrakis (methanephosphonic acid) and N- (2-hydroxyethyl) ethylenediaminetriacetic acid.

DK 170460 B1 6

Further suitable complex salts of the general formula I according to claim 1 are e.g. those of the general formula Ib

X-CH2 ^ CH2 "X

N-CH2-CH2 - (Z-CH2-CH2) mNv (Ib)

V-CHR1 CHR ^ ”V

wherein X, V, Z, and m are as defined in claim 1.

If Z is an oxygen atom or a sulfur atom, complex salts with m in the meaning of 1 or 2 are preferred.

Suitable for the preparation of the complex salts of general formula Ib include the following complexing acids: Diethylenetriamine pentaacetic acid, triethylenetetramine hexaacetic acid, tetraethylenepentamine heptaacetic acid, 13,23-dioxo-15,18,21-10 tris (carboxymethyl) -12,15,18 , 21,24-pentaazapentatriacontanic acid, 3,9-bis- (1-carboxyethyl) -3,6,9-triaza-decanedioic acid, diethylenetriamine pentakis (methylene phosphonic acid), 1,10-diaza-4,7-dioxadecan-1,1, 10,10-tetraacetic acid and 1,10-diaza-4,7-dithiadecane-1,1,10,1O-tetraacetic acid.

Additional complexing acids suitable for the preparation of the complex salts of general formula I are, for example: 1,2,3-tris- [bis- (carboxymethyl) -amino] -propane and nitrilotris- (ethylene nitrilo) -hexaeddikesyre.

When not all azide hydrogen atoms of the complexing acid are substituted with the central ion or central ions, it is convenient to increase the solubility of the complex salt to substitute the remaining hydrogen atoms with physiologically acceptable cations of inorganic and / or organic bases or amino acids. Suitable inorganic 25 * cations are e.g. the lithium ion, potassium ion or especially the sodium ion. Suitable cations of organic bases include those of primary, secondary or tertiary amines, such as e.g. ethanolamine, diethanolamine, morpholine, glucamine, N, N-dimethylglucamine or especially N-methylglucamine. Suitable cations of amino acids are e.g. cations of lysines, arginines and ornithins.

The complexing acids required for the composition according to the invention are known or can be prepared in a manner known per se.

Thus, the invention also relates to novel complex salts which are characterized by the general formula I

2> N-A-MCr 2 U) V-CHR1 CHSj-v wherein X, A, V and R1 have the meaning set forth in claim 1 with the proviso that they contain 3-7 substituents Y, hvcraj. at least two are a metal ion equivalent of an element having atomic numbers 21-29, 42, 44 or 58-70 and further at least one of the substituents Y is the physiologically acceptable cation of an organic base or amino acid, where appropriate residual Y substituents Y represent hydrogen atoms or cations of an inorganic base.

Thus, e.g. the preparation of 13,23-dioxo-15,18,21-tris (carboxymethyl) -12,15,18,21,24-pentaazapentatriacontanoic acid to improve that of R. A. Bulman et. eel. In the Natur-Wissenschaften 68 (1981) 483 proposed manner: 17.85 g (= 50 mmol) of 1,5-bis- (2,6-dioxomorpholine) -3-azapentane-3-acetic acid are suspended in 400 ml of dry dimethylformamide or after addition of 20.13 g (= 100 mmol) of 11-aminoundecanoic acid is heated to 70 ° C for 6 hours. The clear solution is evaporated in vacuo. The yellow oily residue is stirred at room temperature with 500 ml of water. Thereby a solid white bulky solid precipitates, which is sucked off and washed several times with water. The obtained product is transferred to further purification in 200 ml of acetone and stirred for 30 minutes at room temperature. After 30 DEG extraction and drying in vacuo at 50 ° C, 36.9 g (= 97% of theoretical yield) of white powder, mp 134-138 ° C, are obtained.

in DK 170460 B1 8

The conjugation of the complexing acids with biomolecules is also effected by methods known per se, e.g. by reaction of nucleophilic groups of the biomolecule, e.g. amino, hydroxy, thio or imidazole groups, with an activated derivative of the complexing acid.

v

As activated derivatives of complexing acids, e.g. acid chlorides, acid anhydrides, activated esters, nitrenes or isothiocyanates under consideration. Conversely, it is also possible to react with an activated biomolecule with the complexing acid.

For the conjugation with proteins, substitutions of the structure or C

The preparation of the complex salts is also known in part or may be carried out in a manner known per se, by dissolving the metal oxide or metal salt (e.g., the nitrate, chloride or sulfate) of the element having atomic numbers 21-29, 42, 44 or 58-70 in water and / or a lower alcohol (such as methanol, ethanol or isopropanol) suspend and treat with the solution or suspension the equivalent amount of the complexing acid in water and / or a lower alcohol and stir, if necessary, during heating. to the boiling point until the turnover is completed. When the complex salt formed is insoluble in the solvent used, it is isolated by filtration. If it is soluble, it can e.g.

25 is isolated by evaporation of the solution to dryness, e.g. using spray drying.

If azide groups are still present in the obtained complex salt, it is often convenient to transfer the acidic complex into neutral complex by means of inorganic and / or organic bases or amino acids which form physiologically acceptable cations. salts to isolate these. In many cases it is even indispensable that the dissociation of the complex salt by the shifting of the pH to the neutral is reduced to such an extent that only the isolation of homogeneous products or at least their purification is allowed.

The preparation is conveniently carried out by means of organic bases or basic amino acids. However, it may also be advantageous to neutralize by means of inorganic bases (hydroxides, carbonates or bicarbonates) of sodium, potassium or lithium.

For the preparation of the neutral salts, for example, add to the acidic complex salts in aqueous solution or suspension so much of the desired base as to reach the neulyral point. The solution obtained can then be evaporated to dryness in vacuo. Frequently, it is advantageous to precipitate the neutral salts formed by the addition of water-miscible solvents, e.g. lower alcohols (methanol, ethanol, isopropanol, etc.), lower ketones (acetone, etc.), polar ethers (tetrahydrofuran, dioxane, 1,2-dimethoxyethane, etc.) and thus obtain crystallisers which are readily isolated and are good to clean. It has proved particularly advantageous to add the desired base already during the complexing of the reaction mixture, thereby saving a production step.

If the acidic complex salts contain several free azide groups, it is often convenient to prepare neutral mixture salts containing both inorganic and organic physiologically acceptable cations as counterions. This can be done, for example. by reacting the complexing acid in aqueous suspension or solution with the oxide or salt of the element providing the central ion and half of the amount of organic base needed for neutralization isolates the complexed salt formed, if desired the detergent and 10 DK 170460 B1 thereafter for complete neutralization treat with the required amount of inorganic base. The base addition can be done in reverse order.

The preparation of diagnostic agents according to the invention also occurs in a manner known per se, by suspending or dissolving the complex salts in aqueous medium, optionally with the addition of the usual additives in the galenic pharmacy and then sterilizing the solution or suspension. Suitable additives are e.g. physiologically acceptable buffers (such as tromethamine hydrochloride), poor additions of complexing agents (such as diethylenetriamine pentaacetic acid) or, if necessary, electrolytes (such as sodium chloride).

In principle, it is also possible to prepare the diagnostic agents of the invention without isolating the complex salts.

In each case, special care must be taken to ensure that the chelate formation takes place such that the salts and salt solutions of the invention are practically free of non-complexed toxic metal ions. This can be done, for example. is assured by color indicators such as xylenol orange in control titrations during the manufacturing process. As a final security, a purification of the isolated complex salt is performed.

If, for oral administration or other purposes, suspensions of the complex salts in water or physiological saline are desired, a small amount of soluble complex salt is mixed with one or more of the usual auxiliary substances and / or surfactants and / or flavor correction agents. .

The diagnostic agents of the invention contain *, 1 μιηοΐ - 1 mole per liter of complex salt and is usually dosed in amounts of 0.001-5 mmol / kg. They are intended for oral and especially parenteral application.

3 5 11 DK 170460 B1

The agents of the invention fulfill the many conditions of suitability as a contrast agent for the core spider tomography. Thus, they are particularly well suited for, after oral or parenteral application, by enhancing the signal intensity to improve the image obtained by the nuclear spin tomograph in its pronunciation power.

5 Further, they show the high efficacy required to load the body with the least possible amount of foreign matter and the good tolerability needed to maintain the non-invasive nature of the study (those in J. Comput. Tomography 5, 6: 543-46 (1981), in Radiology 144, 343 10 (1982) and in the Letter Special De Medicament No. 484M (1960), the compounds cited are, for example, toxic). The good water solubility of the compositions of the invention allows high concentration solutions to be prepared so as to keep the volume load of the circuit within reasonable limits and smooth the dilution with the body fluids, i.e., # NMR diagnostics should be 100- 1000 times more water soluble than for NMR spectroscopy. Further, the agents of the invention exhibit not only a high stability in vitro, but also a surprisingly high stability in vivo, such that a release 20 or a substitution of the non-covalently bound - per se toxic ions in the complexes over 24 hours, in which, as pharmacological studies have shown, the new contrast agents are completely excreted again only very slowly. They e.g. 25 conjugates of proteins and antibodies used for tumor diagnosis already produce a surprisingly high signal amplification at such a low dosage that solutions of similarly low concentration can be used here.

The agents of the invention are furthermore useful as X-ray contrast agents, where it should be particularly emphasized that the anaphylaxis-like reactions known with the iodine-containing contrast agents cannot be seen in biochemical-pharmacological studies. Particularly valuable are their absorption properties in areas with higher pipe voltages for digital subtraction techniques because of the favorable 35 DK 170460 B1 12 absorption characteristics. in

The agents of the invention are also suitable as ultrasound diagnostics.

nostika.

5 Contrary to conventional X-ray diagnostics with shadow-giving X-ray contrast agents, there is no linear dependence on the signal amplification of the concentration used for NMR diagnostics with paramagnetic contrast agents.

10 As control studies show, an increase in the dose administered does not necessarily lead to a signal amplification, and it can even lead to a dissolution of the signal at a high dose of paramagnetic contrast agent. For this reason, it was surprising that some pathological processes became visible only after the application of higher doses of a potent paramagnetic contrast agent of the invention than the doses specified in EP 71564 (which may amount to 0.001 mmol / kg to 5 mmol / kg). Thus, e.g. the detection of a defective blood-brain boundary in a cranial abscess region is carried out only after administration of 0.05-2.5 mmol / kg, preferably 0.1-0.5 mmol / kg of paramagnetic complex salts, e.g. gadolinium diethylenetriamine pentaacetic acid or manganese 1,2-cyclohexylenediaminotetraacetic acid in the form of their water-soluble salts. At a dose greater than 0.1 mmol / kg, solutions of higher concentrations up to 1 mol / l are needed, preferably 0.25-0.75 mol / l, since the volume load is thus only reduced and the handling of the injection solution is ensured. .

Particularly low doses (less than 1 mg / kg) and thus less concentrated solutions (lpmol / l-5 mmol / l) than those specified in EP 71564 are required for the organ-specific NMR diagnostics, e.g. ^ for the detection of tumors and heart attacks.

The following embodiments serve to further illustrate the invention.

13 DK 170460 B1

Example 2.

Preparation of the sodium salt of the gadolinium III grain peak of 13,23-dioxo-15,18,21-tris (carboxymethyl) -12,15,18,21,24-pentaazapentatriacontanoic acid, * 2 Na 5 15.2 g (= 20 mmol) of 13,23-dioxo-15,18,21-tris (carboxymethyl) -12,15,18,21,24-pentaazapentatriacontanoic acid is suspended in 400 ml of water and heated to 95 ° C. 7.43 g (= 20 mmol) of gadolinium III chloride hexahydrate dissolved in 60 ml of water are slowly added dropwise. This temperature is maintained for 2 hours and then treated with 60 ml of 1N sodium hydroxide to neutralize the resulting hydrochloric acid.

After complete reaction (examined with xylenol orange), the precipitate obtained is filtered off and washed chloride-free with water. 17.60 g (96% of theoretical yield) of a 15 q in water insoluble white powder having a melting point 290-292 ° C is obtained.

Gadolinium III complex of 13,23-dioxo-15,18,21-tris (carboxy-methyl) -12,15,18,21,24-pentaazapentatriacontanedioic acid.

Analysis: 20 (Calcd) C 47.30, H 6.84, N 7.66, Gd 17.20 (found) C 47.13, H 6.83, N 7.60, Gd 17.06 14.6 g (= 16 mmol) of the thus obtained gadolinium-III complex is suspended in 200 ml of water and treated dropwise with 31.4 ml of 1 N sodium hydroxide. After 1 hour, a clear solution is obtained which is filtered and then evaporated in vacuo. After drying in vacuo at 80 ° C, 13.2 g (87% of theoretical yield) of a water-soluble white powder, mp 279-285 ° C, is obtained.

14 DK 170460 B1

Analysis: (calculated) C 45.13, H 6.31, N 7.31, Gd 16.41, Na 4.80 (found) C 45.20, H 6.12, N 7.28, Gd 16, 26, Na 4.75 is similarly obtained with N-methylglucamine instead of the sodium 5 hydroxide> di-N-methylglucamine salt of the gadolinium-III complex of 13.2 3-dioxo-15,18,21-tris (carboxymethyl) ) -12,15,18,21,2 4-penetazapentatriacontanedioic acid, c50H96GdN7 ° 22 *

Example 17

Preparation of a solution of the sodium salt of the gadolinium-III complex of 13,23-dioxo-15,18,21-tris (carboxymethyl) -12,15, L8,21,24-pentaazapentatriacontanedioic. 392 0 g (= 400 mmol) of the salt described in Example 2 is slurried in 500 ml of water pi and after filling with water 15 pi dissolve to 1000 ml under gentle heating. The solution is filled into bottles and heat sterilized.

Example 45

Preparation of a solution of the di-N-methylglucamine salt 20 gadolinium-III complex of 13,23-dioxc-15,18,21-tris- (carboxymethyl) -12,15,18,21,24-pentaazapentatriacontanedioic acid.

130.4 g (= 100 mmol) of the salt listed in Example 2 are suspended in 250 ml of water p.i. and dissolves during heating. Water is then refilled with p.i. to 500 ml, then the solution is filled into ampoules and heat sterilized.

DK 170460 B1 15

Example 50

Preparation of a solution of the gadolinium-III complex of the conjugate of diethylenetriamine pentaacetic acid with human serum albumin. 5 To 20 ml of a solution of 3 mg of the protein in 0.05 molar sodium bicarbonate buffer (pH 7-8) is added 10 µl, 5-bis (2,6-dioxomorfolin) -3-azapentane-3-acetic acid. Stir for 30 minutes at room temperature and then dialyze against a 0.3 molar sodium phosphate buffer. Then 50 mg of gadolinium β-acetate is added and purified by gel chromatography on a Sephadex G25 column. The obtained fraction is sterile filtered and filled into multivials. Freeze-drying gives a storage-able dry preparation.

Similarly, with the immunoglobulin solution of the corresponding complex conjugate is obtained.

Example 51.

Preparation of a solution of the gadolinium-III complex of the conjugate of diethylenetriamine-pentaacetic acid (DTPA) with monoclonal antibodies.

To 20 μΐ of a solution of 0.3 mg monoclonal antibodies in 0.05 molar sodium bicarbonate buffer (pH 7-8) is added 1 mg of a mixed DTPA anhydride (obtained, for example, by DTPA and iso-butyl chloroformate) and there stir for 30 minutes at room temperature. It is dialyzed against 0.3 molar sodium phosphate buffer and the resulting antibody fraction is treated with 2 mg of the gadolinium-III complex of ethylenediamine tetraacetic acid (EDTA). After purification by gel chromatography over Sephadex G25, the sterile filtered solution is filled into multivials and lyophilized.

DK 170460 B1 16

Similarly, using the mixed anhydride of trans-1,2-di-aminocyclohexane-tetraacetic acid (CDTA), a solution of the corresponding gadolinium-III complex of the CDTA antibody is obtained. 5 Using the manganese-II complex of ethylenediamine-tetraacetic acid is similarly obtained by the manganese-II complex of the. with DTPA or CDTA coupled antibodies.

Example 52

Preparation of a solution of the gadolinium-III complex of the conjugate of 1-phenylethylenediamine tetraacetic acid with immunoglobulin.

Using it in J. Med. Chem. 1974, Vol. 17, p. 1307, a 2% solution of the protein is cooled in 0.12 molar sodium bicarbonate solution containing 0.01 mol of ethylenediamine tetraacetic acid to 4 ° C and then treated dropwise with the equivalent of the protein to a freshly prepared ice-cold diazonium salt solution of 1- (p-aminophenyl) -ethylenediamine-tetraacetic acid. Stir overnight at 4 ° C (pH 8.1) and then dialyze with a 0.1 molar sodium citrate solution. At the end of the dialysis, the solution of the conjugate is treated with an excess of gadolinium-III chloride and ultrafiltered to remove ions. Then the sterile filtered solution is filled into multivials and lyophilized.

Example 53.

Preparation of a colloidal dispersion of a Mn-CDTA lipid conjugate. 0.1 mmol of distearoylphosphatidylethanolamine and 0.1 mmol of bis anhydride of trans-1,2-diaminocyclohexane tetraacetic acid is stirred in 50 ml of water. for 24 hours at room temperature. 0.1 mmol of manganese II carbonate was added and stirred again for 6 hours at room temperature. After purification over a Sephadex G50 column, the sterile filtered solution is filled into multivials and lyophilized.

Similarly, with gadolinium-III oxide, a colloidal dispersion of the gadolinium-DTPA-lipid conjugate can be obtained.

Example .54, 10

Preparation of gadolinium-DTPA-loaded liposomes.

Using the one in Proc. Natl. Acad. Sci. U.S.A. 75, 4194 disclose a lipid mixture of 75 mole% EI-phosphatidylcholine and 25 mole% cholesterol as a dry substance. Of this, 500 mg is dissolved in 30 ml of diethyl ether and treated dropwise with 3 ml of a 0.1 molar solution of the di- - N-methylglucamine salt of the gadolinium-III complex of diethylenetriamine-pentaacetic acid in water pi. of the solution, the ultrasonic treatment is continued for another 10 minutes and then evaporated in a rotavapor. The gel-like residue is suspended in 0.125 molar sodium chloride solution and freed at 0 ° C for non-encapsulated contrast media by repeated centrifugations (20,000 g / 20 minutes). Then the liposomes thus obtained are lyophilized in the multivial.

2 ^. The application is carried out as colloidal dispersion in 0.9% by weight boiling salt solution.

Claims (5)

1. Diagnostic agent for use in in vivo NMR diagnostic connectors, characterized in that liter contains $ 5 1 μτηοΐ - 1 mole of at least one physiologically acceptable complex salt of the general formula I X-CH0 CH0-X 2 \ / 2 ^ NAN ^ (I) V-CEP1 XHP-jV wherein X represents the group -C00Y, -PO-HY or -CONHOY with Y in the meaning of a hydrogen atom, a metal ion equivalent and / or a physiologically acceptable cation of an inorganic or organic base or amino acid, and wherein ^ represents hydrogen atoms or methyl groups and wherein a) A represents the group · - CHR2-CHR3- / -CH2-CH2- (ZCH2-CH2) m-, n (ch2x) 2ch2-ch2-n (ch2x) 2 -CH2-CH-CH2 ~ or -CH2-CH2-N-CH2-CH2 - wherein X is as defined above, R2 and R3 together represent a trimethylene group or a tetramethylene group or hydrogen atoms, lower alkyl groups, phenyl groups, benzyl groups, m represents the numbers 1, 2 or 3, Z represents an oxygen atom or a sulfur atom or the group NCH2X or NCH2CH20R4 with X in the meaning given above, and R4 in the sense a lower alkyl group and wherein V has the same meaning as X, 20 or the groups -CH20H or -COB DK 170460 B1 with B in the sense a protein or lipid residue or a -NH (CH2) nX residue with X in the above meaning, and nine meaning the digits 1-12, with the proviso that at least one substituent represents the group -COB, and at least two of the substituents Y denote metal ion equivalents of an element having the atomic numbers 21-29, 42, 44 or 58-70 or b) wherein A represents the group -CHR2-CKR3- wherein R 2 represents a hydrogen atom and R 3 represents a group of - (CH 2) p -C 6 H 4 -W protein 10 with p in the meaning of 0 or 1, W in the sense -NN-, -NHCOCH 2 - or -NHCS and wherein V has the same meaning as X, or the group -ch2oh, provided that at least two of the substituents Y denote metal ion equivalents of an element having the atomic numbers 21-29, 42, 44 or 58-70, optionally with the in the usual additions of the galenic pharmacy, dissolved or suspended in aqueous medium. Diagnostic agent according to claim 1, characterized by a content of the di-N-methylglucamine salt of the gadolinium (III) complex of 13,23-dioxo-15,18,21-tris- (carboxymethyl) -12,15, 18,21,24-pen-taazapentatriacontandisyre; gadolinium (III) complex of the diethylenetriamine pen-25 conjugate. acetic acid with immunoglobulin; gadolinium (III) complex of the diethylenetriamine pentaacetic acid conjugate with human serum albumin; DK 170460 B1 gadolinium (III) complex of the conjugate of diethylenetriamine pentaacetic acid with monoclonal antibodies; , manganese (II) complex of the conjugate of trans-1,2-cyclohexylene-ψ diamine-tetraacetic acid with monoclonal antibodies; 5 manganese (II) complex of the lipid conjugate of trans-1,2-cyclohexylenediamine tetraacetic acid; the gadolinium (III) complex of diethylenetriamine-penta-diacetic acid-loaded liposomes; di-sodium salt of the gadolinium (III) complex of 13,23-dioxo-15,18,21-tris- (carboxymethyl) -12,15,18,21,24-pentaazapentatriacontanedioic acid. Process for the preparation of the diagnostic agent according to claim 1, characterized in that it is dissolved or suspended in water or physiologically saline complex salt with the usual additions in the galenic pharmacy to one for intravasal or oral. application suitable form. 4. Complex salts, characterized by the general formula I x -CH2 \ ^ CH, -X 20. NAN CT 2 (1) V ~ CHR1 CHRx-v wherein X, A, V and R-j_ have the meaning set forth in claim 1, provided that they contain 3-7 substituents Y, of which at least two are a metal ion equivalent of an element having atomic numbers 21-29, 42, 44 or 58-70 and further at least one of the 25 substituents Y is the physiologically acceptable cation of an organic base or amino acid, wherein the residual substituents Y, if any, represent hydrogen atoms or cations of a inorganic base.