AU607456B2

AU607456B2 - Physiologically tolerable metal complex salts for use in nmr diagnosis

Info

Publication number: AU607456B2
Application number: AU10184/88A
Authority: AU
Inventors: Heinz Gries; Douwe Rosenberg; Hanns-Joachim Weinmann
Original assignee: Schering AG
Current assignee: Bayer Pharma AG
Priority date: 1983-01-21
Filing date: 1988-01-11
Publication date: 1991-03-07
Anticipated expiration: 2004-01-18
Also published as: JPH04217927A; DK25484A; GR81653B; NZ219079A; IE890169L; PT77983A; IL70711A; GB2169598B; DK25484D0; FI79026C; GB2169599B; FR2590484A1; AU1018488A; DK170461B1; FI840226A; ATA19184A; DK170460B1; CH660183A5; FR2539996B1; SE9301557L

Description

1 1 8-P339/EA/MP/0067T.6

AUSTRALIA

:.£9i4 6 PATENTS ACT 1952 COMPLETE SPECIFICATION

(ORIGINAL)

This document contains the amendments made under Section 49 and is correct for printing FOR OFFICE USE Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority: S Related Art: t V TO BE COMPLETED BY APPLICANT Name of Applicant: Address of Applicant: S t Ct 4 r Actual Inventor: Address for Service: SCHERING AKTIENGESELLSCHAFT Postfach 65 03 11, D-1000, Berlin 65, Germany Heinz Gries Douwe Rosenberg Hanns-Joachim Weinmann ARTHUR S. CAVE CO.

Patent Trade Mark Attorneys Level Barrack Street SYDNEY N.S.W. 2000

AUSTRALIA

Complete Specification for the invention entitled S PHYSIOLOGICALLY TOLERABLE METAL COMPLEX SALTS FOR USE IN NMR

DIAGNOSIS.

The following statement is a full description of this invention including the best method of performing it known to me:- 1 ASC 49 i i 1la Physiologically tolerable metal complex salts for use in NMR diagnosis The invention relates to physiologically tolerable metal complex salts and the use thereof in NMR diagnosis.

Complex compounds and their salts have been used for a long time in medicine, for example as auxiliaries for the administration of sparingly soluble ions (for example iron) and as antidotes (calcium or zinc complexes being preferred in this 0 case) for detoxication in the case of inadvertent incorporation o 00 00 0 10o of heavy metals or their radioactive isotopes.

000600 o0oo The present application is a divisional application of oooo 0o00o Australian Patent Application No. 23559/84 filed 18th January, 0 0 1984.

oo* We have now found that certain physiologically tolerable o eo .o complex salts containing one or more central elements having the atomic numbers of from 21 to 29, 42, 44 and from 58 to oo00 o can be used for the manufacture of preparations that are surprisingly outstandingly suitable for use in NMR diagnostics.

0.

0e:° The present.invention provides a diagnostic preparation 020 which comprises a physiologically tolerable, non-radioactive complex salts of the general formula I

X-CH

2

CH

2

-X

V-CHR

CN-A-N

V-CHR

1 CHR -V 1 1 0918E L~c '1 -2in which X represents the radicals -COOY, -PO 3 HY or -CONHOY wherein each Y represents a hydrogen atom, a metal equivalent, the metal being selected from elements having atomic numbers of from 21 to 29, 42, 44 and from 58 to 70, or a physiologically tolerable cation of an inorganic or organic base or amino acid,

R

1 represents in each case a hydrogen atom or a 10 methyl group and in which either a) A represents the group -CHR 2

-CHR

3 -CH2-CH 2

(ZCH

2

CH

2

N(CH

2

X)

2 CH 2

-CH

2

-N(CH

2 X) 2 0 9a 0 C 1 eqg~tr €1 I a o o* a* f 0 0 C 0, C 0 C t t -CHi 2-CH-CH in which x Xand R

R

2 and R 3 014(10 1 0 114 tI.t 0 1 or 2- -CH 2

CH

2 -N-CH2-CH 2 has the meanings given above, together represent a trimethylene group or a tetramethylene group, or each represent a hydrogen atom, an alkyl radical containing up to 6 carbon atoms, a phenyl radical or a benzyl radical, represents the integer 1, 2 or 3, m and -3- -3- Z represents an oxygen atom or a sulphur atom or the group '-NCH2X or NCH CH OR 2 2 2 4 in which X has the meanings given above and

R

4 represents an alkyl radical roe S' containing up to 6 carbon atoms, and t t 10 each V has the same meaning as X or represents the group -CH2OH or -COB, in which ca 4 B represents a protein or lipid radical or an tC t

-NH(CH

2 )nX radical, in which X has the meanings given above and n represents an integer from 1 to 12, i with the proviso that at least one of the symbols V represents a -COB group, as defined above, and at least two of the substituents Y are metal equivalents Sselected from elements having atomic numbers of from 21 to 29, 42, 44 and from 58 to or b) A represents the group -CHR 2

-CHR

3 i;.i ri i i* -4in which

R

2 represents a hydrogen atom and

R

3 represents the group -(CH 2

-C

6

H

4 -W-protein in which R represents 0 or 1, W represents -NHCOCH 2 or -NHCSand t re 10 -protein represents a protein radical, +tftt and c f each V has the same meaning as X or represents the group with the proviso that at least two of the substituents Y are metal equivalents selected from elements having atomic numbers of from 21 to 29, 42, 44 and from 58 to and (ii) a physiologically tolerable carrier.

t 20 Thus, the physiologically tolerable complex salt of the general formula I may be regarded as containing a central element selected from elements having atomic numbers of from 21 to 29 inclusive, 42, 44 and from 58 to 70 inclusive, and a radical of a physiologically tolerable complex-forming acid and, if desired, a radical selected from radicals of inorganic and organic bases and amino acids.

The physiologically tolerable complex salt of the general formula I may contain more than one such central element and more than one such radical For the intended use of the diagnostic agent according to the invention, the element or elements having an atomic number mentioned above, which forms the central element or elements of the physiologically tolerable complex salt, must not, of course, be o radioactive.

000oooo o* 10 Since a preparation of the invention is to be used in NMR diagnostics (see European Patent Application 71 564), the central element of the complex salt must be paramagnetic. Such elements are especially the divalent and trivalent elements having an atomic number of from 21 to 29, 42, 44 and from 58 'c to 70. Suitable elements are, for example, t t Cchromium(III), manganese(II), iron(III), iron(II), 1 cobalt(II), nickel(II), copper(II), praseodymium(III), neodymium(III), samarium(III) and ytterbium(III).

Especially preferred, owing to their strong magnetic moment, are gadolinium(III), terbium(III), dysprosium(III), holmium(III) and erbium(III).

The complex-forming acids from which the physiologically tolerable complex salts of the general formula I may be derived are, especially, those containing carboxymethylene groups.

The complex-forming acids from which the :lr -I _i i ~i A1 -6- C Cr 'it Sitt it tt.

physiologically tolerable complex salts of the general formula I are derived can, as conjugates, be bonded to biomolecules that are known to become especially concentrated in the organ or organ part under examination. Such biomolecules are, for example, proteins or lipids. There come into consideration conjugates with protein hormones, such as insulin, and more especially conjugates with albumens, such as human serum albumen, antibodies, such as, for example, monoclonal antibodies specific to tumour-associated antigens, or antimyosin. The diagnostic preparations formed therefrom are suitable, for example, for use in tumour and infarct diagnosis. For examinations of the liver there are suitable, for example, conjugates or inclusion compounds with liposomes, which are used, for example, as unilamellar or multilamellar phosphatidylcholinecholesterol vesicles. The conjugates are formed either by way of a carboxy group of the complex-forming acid or, in the case of proteins, also by way of a -(CH 2 -C6H4-W- group as defined above under R 3 In the conjugate formation of some complexforming acids-with proteins or lipids, several acid radicals may be bonded to the macromolecular biomolecule. In that case, each complex-forming acid radical may carry one central element.

Suitable complex salts of the general formula I above are, for example, those of the general formula Ia Ii

C-(

l r; ~l .1 i i Le -7- X-CH 2 CH -X N-C HR 2

CHR

3

N

V-CHR

1

CHR

1 (I a) It q I C

IKE''

C

o'er

CI''

C

COKC It t o 0.

o a 0 ft K C I (C

II'

I I .01141 *CKCI C

C

in which X, V, Rif R 2 and R 3 have the meanings given above.

The following complex-forming acids, inter alia, are suitable for the manufacture of the complex salts of the general formula Ia:ethylenediaminetetraacetic acid, ethylenediaminetetraacetohydroxamic acid, trans-l,2-cyclohexylenediaminetetraacetic acid, DL-2,3-butylenediaminetetraacetic acid, DL-l,2-butylenediaminetetraacetic acid, DL-l,2propylenediaminetetraacetic acid, 1,2-diphenylethylenediaminetetraacetic acid, ethylenedinitrolotetrakis- (methane-phosphonic acid) and N-(2-hydroxyethyl)ethylenediaminetriacetic acid.

Other suitable complex salts of the general formula I are, for example, those of the general formula Ib -8-

X-CH

2 CH 2

-X

N-CH -CH 2 -(Z-CH -CH (Ib) m V-CHR CHR 1

-V

in which X, V, Z, R 1 and m have the meanings given above. If Z represents an oxygen atom or a sulphur atom, complex salts in which m represents 1 or 2 are preferred.

The following complex-forming acids, inter alia, are suitable for the manufacture of the complex salts of the general formula Ib: ddiethylenetriaminepentaacetic acid, triethylenetetraminehexaacetic acid, tetraethylenepentaminehepta-1 acetic acid, 13,23-dioxo-15,18,21-tris-(carboxymethyl)- 12,15,18,21,24-pentaazapentatriacontanoic diacid, 3,9bis- (l-carboxyethyl)-6-carboxymethyl-3 6 ,9-triazaundecanoic diacid, diethylenetriaminepentakis- (methylenephosphonic acid), 1,10-diaza-4,7-dioxadecane- 1,1,10,10-tetraacetic acid and 1,10-diaza-4,7dithiadecane-1,1,10,l0-tetraacetic acid.

Other complex-forming acids that are suitable for the manufacture of the complex salts of the general formula I are, for example: i

,I

I'

0', i- -9ooe tI.

or C

C(C

SE

CC

1,2,3-tris-[bis-(carboxymethyl)-amino]-propane and nitrolotris-(ethylenenitrolo)-hexaacetic acid.

If not all of the acid hydrogen atoms of the complex-forming acid are substituted by the central element or elements, it is advantageous, for the purpose of increasing the solubility of the complex salt, to substitute the remaining hydrogen atoms by physiologically tolerable cations of inorganic and/or organic bases or amino acids. Suitable inorganic 10 cations are for example, lithium, potassium or, especially, sodium. Suitable cations of organic bases are, inter alia, those of primary, secondary or tertiary amines, such as, for example, ethanolamine, diethanolamine, morpholine, glucamine, N,N-dimethylglucamine or, especially, N-methylglucamine. Suitable cations of amino acids are, for example, those of lysine, arginine or ornithine.

The complex-forming acids required for the formation of the complex salts of the general formula I are known or can be manufactured in a manner known per se.

For example, 13,23-dioxo-15,18,21-tris-(carboxymethyl)-12,15,18,21-24-pentaazapentatriacontanoic diacid is manufactured in the following manner, which is an improvement to the method proposed by R. A. Bulman et al. in Naturwissenschaften 68, (1981) 483: t 17.85 g 50 mmol) of 1,5-bis-(2,6-dioxomorpholino)-3-azapentane-3-acetic acid are suspended in 400 ml of dry dimethylformamide and, after the addition of 20.13 g 100 mmol) of 11-aminoundecanoic acid, the whole is heated at 70 0 C for 6 hours. The clear solution is concentrated in vacuo. The yellow oily residue is stirred with 500 ml of water at room temperature. In so doing, an almost white, voluminous solid precipitates which is suction-filtered and washed .10 several times with water. For further purification, the resulting product is introduced into 200 ml of acetone and the whole is stirred for 30 minutes at room temperature. After suction-filtering and drying in vacuo at 50 0 C, 36.9 g 97 of the theoretical 4 15 yield) of a white powder of melting point 134-138 0

C

are obtained.

Conjugation of the complex-forming acids with biomolecules is likewise effected according to methods known per se, for example by reacting the nucleophilic groups of the biomolecule, such as, for example, amino, hydroxy, thio or imidazole groups, with an activated derivative of the complex-forming acid.

Activated derivatives of the complex-forming acid which come into consideration are, for example, acid chlorides, acid anhydrides, activated esters, nitrenes or isothiocyanates. Conversely, it is also possible to i react an activated biomolecule with the complex-forming L, i_ iv i ni i i ii iI_. i' -11acid.

a i:-

I:

/(i i ri': 1

F

i: ii rl

S

B

1; 6

I

r i i 3 r,

I

*r 4 For conjugation with proteins, substituents of the structure -C 6

H

4

N

2 or -C 6

H

4

NHCOCH

2 -halogen may also be considered.

The manufacture of the complex salts of the general formula I can likewise be carried out in a manner known per se by dissolving or suspending the metal oxide or metal salt (for example the nitrate, chloride or sulphate) of the element having an atomic number of from 21 to 29, 42, 44 or from 58 to 70 in water and/or a lower alcohol (such as methanol, ethanol or isopropanol) and adding a solution or suspension of the equivalent amount of the complex-forming acid in water and/or a lower alcohol, and stirring, if 15 necessary while warming or heating to boiling point, until the reaction is complete. If the complex salt formed is insoluble in the solvent used, it is isolated by filtration. If it is soluble, it can be isolated by concentrating the solution to dryness by evaporation, 20 for example by means of spray-drying.

If acid groups are still present in the resulting complex salt, it is often advantageous to convert the acid complex salt into a neutral complex salt or salts by means of inorganic and/or organic bases or amino acids that form physiologically tolerable cations and to isolate the neutral salt. In many cases, this is indeed unavoidable since the dissociation of the .1 u i -12complex salt is so suppressed by the shift in the pH value to neutral that only in that manner can uniform products be at all isolated or at least purified.

The manufacture is advantageously carried out with the aid of organic bases or basic amino acids.

It can, however, also be advantageous if the neutralisation is carried out by means of inorganic bases (hydroxides, carbonates or bicarbonates) of sodium, potassium or lithium.

S 10 For the manufacture of the neutral salts there imay, for example, be added to the acid complex salts o'e in aqueous solution or suspension as much of the 'desired base as is necessary to obtain the neutral point. The resulting solution can subsequently be a 0 o o o 15 concentrated to dryness in vacuo. It is frequently of advantage to precipitate the resulting neutral salts by adding water-miscible solvents, such as, for example, lower alcohols (methanol, ethanol, isopropanol, etc.), lower ketones (acetone, etc.), and polar ethers (tetrat 20 hydrofuran, dioxane, 1,2-dimethoxyethane, etc.), and thus obtain crystallisates that are easily isolated and readily purified. It has been found especially advantageous to add the desired base to the reaction mixture during the complex formation and thereby dispense with one process step.

If the acid complex salts contain several free acid groups, it is often advantageous to produce

*I

A

-13- 9 dlr.

I

o 04 0 04 1

C

4*44 49a neutral mixed salts that contain both inorganic and organic physiologically tolerable cations as ions of opposite charge. This can be effected, for example, by reacting the complex-forming acid in aqueous suspension or solution with the oxide or salt of the element supplying the central element and with half the amount of organic base required for neutralisation, isolating the complex salt formed, if desired purifying it, and then adding to it the amount of inorganic base required 10 for complete neutralisation. The order in which the bases are added can also be reversed.

The manufacture of the diagnostic preparations according to the invention is likewise effected in a manner known per se by suspending or dissolving the 15 complex salts in an aqueous medium, for example water or physiological salt solution, optionally with the addition of the additives customary in galenical pharmacy, and subsequently sterilising the solution or suspension. Suitable additives are, for example, physiologically tolerable buffers (such as, for example, tromethamine hydrochloride), small additions of complex formers (such as, for example, diethylenetriaminepentaacetic acid) or, if necessary, electrolytes (such as, for example, sodium chloride).

In principle, it is also possible to manufacture the diagnostic preparations of the invention even without isolating the complex salts. In each case,

I:

k t i--I i. t w 0000 0 00 0 a 9 a 0 0 0 0 0 V 0 0 o 4 -14particular care must be taken to effect the chelate formation in such a manner that the salts and salt solutions according to the invention are virtually free of non-complexed toxically acting metal ions. This can be ensured, for example, with the aid of colour indicators, such as xylenol orange, by test titrations during the manufacturing process. The invention also therefore provides processes for the manufacture of the complex salts and of the aforesaid preparations containing them. As a final safeguard, there is always purification of the isolated complex salt.

If suspensions of the complex salts in water or physiological salt solution are desired for oral administration or other purposes, a sparingly soluble complex salt is mixed with one or more auxiliaries customary in galenical pharmacy and/or surfactants and/or aromatic substances for taste correction.

The diagnostic preparations of the invention contain preferably from 1 Vmol to 1 mol per litre of the complex salt and are, as a rule, administered in doses of from 0.001 to 5 mmol/kg. They are intended for oral, and especially parenteral, administration.

If desired, they may be in a dosage form suitable for administration orally, neurally or intravasally. When the preparations are in a form suitable for injections, they may be contained in ampoules.

The diagnostic preparations of the invention meet the many requirements for suitability as contrast agents for nuclear spin tomography. For example, after oral or parenteral administration, they are outstandingly suitable for improving the information that can be provided by the image obtained with the aid of nuclear spin tomography, as a result of increasing n the signal intensity. They also exhibit the high S.activity necessary to keep to a minimum the amount of I foreign substances introduced into the body and the good tolerability necessary to maintain the noninvasive character of the examination (the compounds mentioned in J. Comput. Tomography 5,6: 543-46 (1981), i, in Radiology 144, 343 (1982) and in Brevet Special de I Medicament No. 484 M (1960) are, for example, too toxic). The ready water-solubility of the complex salts used in the preparations of the invention enables the preparation of highly concentrated solutions, so that the volume introduced into the circulation can be kept within reasonable limits and the dilution by body fluid can be compensated, that is to say the NMR diagnostic preparations must be 100 to 1000 times more water-soluble than is necessary for NMR spectroscopy.

Furthermore, the diagnostic preparations of the invention are not only highly stable in vitro but also exhibit a surprisingly high stability in vivo, so that the per se toxic ions that are not covalently bonded in the complexes are released or exchanged only .i-.i3-.lli LL -16extremely slowly. The conjugates with proteins and antibodies which are used, for example, for the diagnosis of tumours bring about a surprisingly high intensification of the signal at such a low dosage that it is possible to use in this case solutions of correspondingly low concentration.

Especially low doses (under 1 mg/kg) and therewith solutions of lower concentrations (1 pmol/l to 5 mmol/l) than are specified in EP 71 564 are required for organ-specific NMR diagnostics, for example for detecting tumours and coronary infarcts; o" The invention also provides physiologically tolerable, non-radioactive complex salts of the general i 1 formula I, as defined above. In those complex salts of the general formula I containing more than two of the S' 4 radicals represented by X, it may be advantageous for at least one of the substituents Y to be a physiologically tolerable cation of an organic base or amino acid.

The present invention further provides the use of a physiologically tolerable, non-radioactive complex salt of the general formula I, as defined above, for the manufacture of a preparation for a method of NMR diagnosis of the human or animal body.

-s 4- Ifl -17- The following Examples illustrate the invention:- Example 1 Preparation of the disodium salt of the gadolinium(III) complex of 13,23-dioxo-15,18,21-tris-(carboxymethyl)- 5 12,15,18,21,24-pentaazapentatriacontanoic diacid,

C

36

H

60 GdN5O 2 2 Na.

15.2 g 20 mm6l) of 13,23-dioxo-15,18,21-trisdiacid are suspended in 400 ml of water and the suspension is heated to 95 0 C. 7.43 g 20 mmol) of gadolinium(III) chloride hexahydrate, dissolved in 60 ml of water, are slowly added dropwise. The whole is maintained at this temperature for 2 hours and then, in order to neutralise the hydrochloric acid formed, 60 ml of 1N sodium hydroxide solution are added.

When the reaction is complete (testing with xylenol orange) the precipitate obtained is filtered and washed free of sodium chloride with water. 17.60 g (96 of the theoretical yield) of a water-insoluble, white powder of melting point 290-292 0 C are obtained.

i.

1 ii. l;lI;i ;.ri :li: -18- Gadolinium(III) complex of 1 3 2 3-dioxo-15,18,21-tris- (carboxymethyl)-1 2 ,15,18,21,24-pentaazapentatriacontanoic diacid.

Analysis: (calculated) (found) C 47.30 C 47.13 H 6.84 H 6.83 N 7.66 N 7.60 Gd 17.20 Gd 17.06 1 44r 4 I 4 I ~r

I

r r rr t 4' C~ C 44 4.

CC 4t 4*( 4.4r 1t 14.6 g 16 mmol) of the gadolinium(III) complex so obtained are suspended in 200 ml of water, and 31.4 1 6f lN sodium hydroxide solution are added 10 dropwise thereto. After 1 hour a clear solution is obtained which is filtered and then concentrated in vacuo. After drying in vacuo at 80 0 C 13.2 g (87 of the theoretical yield) of a readily water-soluble, white powder of melting point 279-285 0 C are obtained.

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 In analogous manner there is obtained, using N-methylglucamine in place of sodium hydroxide solution, the di-N-methylglucamine salt of the gadolinium(III) /4 c Li lr. -19complex of 13,23-dioxo-15,18,21-tris-(carboxymethyl)-12, 15,18,21,24-pentaazapentatriacontanoic diacid, C 5 0

H

96 GdN 7 O0 22 Example 2 Preparation of a solution of the disodium salt of the gadolinium(TII) complex of 13,23-dioxo-15,18,21-tris- (carboxymethyl) -12, 15, 18, 21, 24-pentaazapentatriacontanoic diacid 01T 392.0) g C 400 mmol) of the salt described in 44 10 Example 1 are made into a paste in 500 ml of water 000 p-i- (pro injectione) and dissolved by making up the volume to 1000 ml with water pji. while heating slightly. The solution is placed in bottles and hea-t-sterilised.

Example 3 Preparation of a solution of the di-N-methylglucamine salt of the gadolinium(TII) complex of 13,23-dioxo-iS, 18, 21-tris- (carboxymethyl) -12,15,18,21, 24-pentaazapentatriacontanoic diacid 130.4 g C=100 mmol) of the di-N--methylglucamine complex salt listed in Example 1 are made into a paste[ in 250 ml of water p.i. and dissolved while heating.

The solution is made up to 500 ml with water p.i., introduced into ampoules and heat-sterilised.

Example 4 Preparation of a solution of the gadolinium(II) complex of the conjugate of diethylenetriaminepentaacetic acid with human serum albumen a a 0o 4 0 G of a solution of 3 mg of the protein in a 0.05 molar sodium bicarbonate buffer (pH The whole is stirred for 30 minutes at room temperature and is then dialysed against a 0.3 molar sodium phosphate buffer.

15 50 mg of gadolinium(III) acetate are then added and purification is effected by gel chromatography over a Sephadex G25 column; Sephadex is a Registered Trade Mark. The fraction obtained is sterile-filtered and placed in multi-dose phials. Freeze-drying produces a dry preparation that can be stored.

In an analogous manner, there is obtained with immunoglobulin a solution of the corresponding complex conjugate.

-21- Example Preparation of a solution of the gadolinium(III) complex of the conjugate of diethylenetriaminepentaacetic acid (DTPA) with a monoclonal antibody 1 mg of a mixed DTPA anhydride (obtained, for example, from DTPA and isobutyl chloroformate) is added to 20 pl of a solution of 0.3 mg of a monoclonal antibody in a 0.05 molar sodium bicarbonate buffer (pH 7-8) and the whole is stirred for 30 minutes at room temperature. Dialysis is carried out against a 0.3 molar sodium phosphate buffer, and 2 mg of the O "0 gadolinium(III) complex of ethylenediaminetetraacetic acid (EDTA) are added to the antibody fraction Sobtained. After purification by gel chromatography over Sephadex G25, the sterile-filtered solution is placed in multi-dose phials and freeze-dried.

Using the mixed anhydride of trans-1,2-diaminocyclohexanetetraacetic acid (CDTA) there is obtained, in analogous manner, a solution of the corresponding gadolinium(III) complex of the CDTA antibody.

Using the manganese(II) complex of ethylenediaminetetraacetic acid there is obtained in an analogous manner the manganese(II) complexes of the antibodies coupled with DTPA or CDTA.

i t -22- Example 6 Preparation of a solution of the gadolinium(III) complex of the conjugate of 1-phenyl-ethylenediaminetetraacetic acid with immunoglobulin Chem. 1974, vol. 17, p. 1307, a 2 solution of the protein in a 0.12 molar sodium bicarbonate solution containing 0.01 mol of ethylenediaminetetraacetic acid aminophenyl)-ethylenediaminetetraacetic acd. The is cooled to +4 0 C and there is added dropwise the proportion, equivalent to the protein, of a freshly prepared ice-cold diazonium salt solution of 1-(paminophenyl)-ethylenediaminetetraacetic ac.d. The whole is stirred overnight (pH 8.1) at +4 0 C and is Sthen dialysed against a 0.1 molar sodium citrate solution. When dialysis is complete, an excess of gadolinium(III) chloride is added to the solution of the conjugate and ultra-filtration is carried out to remove ions. Finally, the sterile-filtered solution is placed in multi-dose phials and freeze-dried.

f-i thn dilysd agins a 01 mlar odim cirat I~LC -23- Example 7 Preparation of a colloidal dispersion of a Mn(II)-CDTAlipid conjugate 0.1 mmol of distearoylphosphatidylethanolamine and 0.1 mmol of the bisanhydride of trans-1,2-diaminocyclohexanetetraacetic acid in 50 ml of water are stirred at room temperature for 24 hours. 0.1 mmol of manganese(II) carbonate is added and stirring is again carried out at room temperature for 6 hours. After purification over a Sephadex G50 column, the sterilefiltered solution is placed in multi-dose phials and 0 0 2 S freeze-dried.

0 4 I A colloidal dispersion of the gadolinium-DTPAr lipid conjugate can be obtained analogously with gadolinium(III) oxide.

Example 8 Preparation of liposomes charged with gadolinium(III)-

DTPA

According to the procedure described in Proc.

Natl. Acad. Sci. U.S.A. 75, 4194, a lipid mixture comprising 75 mol of egg phosphatidylcholine and mol of cholesterol is prepared as a dry substance.

I S° I o

H:

-24- 500 mg thereof are dissolved in 30 ml of diethyl ether and, in an ultasonic bath, 3 ml of a 0.1 molar solution of the di-N-methylglucamine salt of the gadolinium(III) complex of diethylenetriaminepentaacetic acid in water p.i. are added dropwise thereto. When the addition of the solution is complete, the exposure to ultrasonic waves is continued for 10 minutes and then concentration is carried out in o t I-24 a rotary evaporator. The gel-like residue is suspended in a 0.125 molar sodium chloride solution and, at

S

0 C, repeatedly freed of non-encapsulated contrast agent portions by centrifugation (20000 g/29 minutes).

Finally, the liposomes so obtained are freeze-dried in multi-dose phials. The preparation is administered as a colloidal dispersion in a 0.9 by weight sodium chloride solution.

i i '0 °C r ep at ed y f e ed f n o -e n ap s u at e c o n r a s i -1

Claims

1. A diagnostic preparation which comprises a physiologically tolerable, non-radioactive complex salt of the general formula I o 100 0 50 O Gets o 0 0 CO GO 4O 4 Ittl X-CH

2 CH -X -A-N V-CR CH V-CHR CHR1 V in which X represents the radicals -COOY, -PO

3 HY or -CONHOY wherein each Y represents a hydrogen atom, a metal equivalent, the metal being selected from elements having atomic numbers of from 21 to 29, 42, 44 and from 58 to 70, or a physiologically tolerable cation of an inorganic or organic base or amino acid, R 1 represents in each case a hydrogen atom or a methyl group and in which either i iii -26- a) A represents the group -CHR2-CHR -CH 2 -CH 2 (ZC H 2 -C H 2 m N(CH2X) 2 CH2 or -CH2-CH-CH 2 -CH CH2-N-C 2 2 2 -CH2-N(CH2X)2 H -CH 2 2 2 in which X R 2 and R 3 m and Z has the meanings given above, together represent a trimethylene group or a tetramethylene group, or each represent a hydrogen atom, an alkyl radical containing up to 6 carbon atoms, a phenyl radical or a benzyl radical, represents the integer 1, 2 or 3, represents an oxygen atom or a sulphur atom or the group 44 4 4 4 14 NCH2X o2 or NCHCH OR 2 4 in which X has the meanings given above and R 4 represents an alkyl radical containing up to 6 carbon atoms, and -27- each V has the same meaning as X or represents the group -CH2OH or -COB, in which B represents a protein or lipid radical or an -NH(CH 2 )nX radical, in which i X has the meanings given above and n represents an integer from 1 to 12, with the proviso that at least one of the symbols V represents a -COB group, as defined above, and at least two of the substituents Y are metal equivalents selected from elements having atomic i numbers of from 21 to 29, 42, 44 and from 58 i to or i b) A represents the group -CHR 2 -CHR 3 in which R 2 represents a hydrogen atom and R 3 represents the group -(CH 2 -C 6 H 4 -W-protein in which 1P_ represents 0 or 1, W represents -NHCOCH 2 or -NHCS- and -protein represents a protein radical, i L ~L -1 L-Z-i- I- F -28- and each V has the same meaning as X or represents the group with the proviso that at least two of the substituents Y are metal equivalents selected from elements having atomic numbers of from 21 to 29, 42, 44 and from 58 to and (ii) a physiologically tolerable carrier. "it 2. A diagnostic preparation as claimed in claim 1, wherein the carrier (ii) is an aqueous carrier. 4 °0 3. A diagnostic preparation as claimed in claim 2, 0 4 0 wherein the carrier (ii) is water or physiological salt solution, and the complex salt is dissolved or suspended in it.

4. A diagnostic preparation as claimed in claim 2 or claim 3, wherein the complex salt is present in a concentration of from 1 pmol to 1 mol per litre. I

5. A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt is derived from diethylenetriaminepentaacetic acid.

6. A diagnostic preparation as claimed in any one of -ii- -29- claims 1 to 4, wherein the complex salt is derived from ethylenediaminetetraacetic acid.

7. A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt is derived from trans-1,2-cyclohexylenediaminetetraacetic acid or 13,23-dioxo-15,18,21-tris-(carboxymethyl)- 12,15,18,21,24-pentaazapentatriacontanoic diacid. ft

8. .A diagnostic preparation as claimed in any one of claims 1 to 7, wherein the complex salt is derived from a complex-forming acid linked as a conjugate with a biomolecule. i

9. A diagnostic preparation as claimed in claim 8, wherein the biomolecule is a lipid.

A diagnostic preparation as claimed in claim 8, wherein the biomolecule is a protein.

11. A diagnostic preparation as claimed in claim wherein the protein is insulin.

12. A diagnostic preparation as claimed in claim wherein the protein is an albumen.

13. A diagnostic preparation as claimed in claim I- -i~r rrr LL_ I-II~* wherein the protein is a monoclonal antibody.

14. A diagnostic preparation as claimed in claim 13, wherein the monoclonal antibody is specific to tumour- associated antigens.

A diagnostic preparation as claimed in any one of claims 1 to 7, wherein the complex salt is derived ot* from a complex-forming acid forming a conjugate or inclusion compound with a liposome.

'16. A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt is the S\0' di-N-methylglucamine salt of the gadolinium(III) Ol' complex of 13,23-dioxo-15,18,21-tris-(carboxymethyl)- 12,15,18,21,24-pentaazapentatriacontanoic diacid.

17. A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt is the gadolinium(III) complex of the conjugate of diethylenetriaminepetaacetic acid with immunoglobulin.

18. A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt is the gadolinium(III) complex of the conjugate of diethylenetriaminepentaacetic acid with human serum albumen. -31-

19. A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt is the gadolinium(III) complex of the conjugate of diethylenetriaminepentaacetic acid with a monoclonal antibody.

A diagnostic preparation 'as claimed in any one of claims 1 to 4, wherein the complex salt is the manganese(II) complex of the conjugate of trans-1,2- cyclohexylenediaminetetraacetic acid with a monoclonal antibody. 44

21. A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt is the manganese(II) complex of a lipid conjugate of trans-1,2-cyclohexylenediaminetetraacetic acid.

'22. A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt is the gadolinium(III) complex of an inclusion compound of diethylenetriaminepentaacetic acid with a liposome.

23. A diagnostic preparation as claimed in any one of claims'l to 4, wherein the complex salt is the disodium salt of the gadolinium(III) complex of 13,23-dioxo-15,18,21-tris-(carboxymethyl)-12,15,18,21,

24-pentaazapentatriacontanoic diacid. -32- 24. A diagnostic preparation as claimed in claim 1, substantially as described in any one of Examples 2 to 8 herein. A diagnostic preparation as claimed in any one of claims 1 to 23, which is in a dosage form suitable for administration orally, neurally or intravasally.

OC,, 0 00 S°o

26. An ampoule containing a diagnostic preparation as claimed in any one of claims 1 to 23, in a form o I suitable for injection. C I

27. A process for the manufacture of a diagnostic preparation as claimed in any one of claims 1 to 23, wherein the complex salt is dissolved or suspended in water or physiological salt solution, and is made up, if desired with the incorporation of one or.more physiologically tolerable adjuncts, in a form that is 0 suitable for intravasal or oral administration. I 3

28. A process for the manufacture of a diagnostic preparation as claimed in claim 1, conducted II substantially as described in any one of Examples 2 to 8 herein.

29. A physiologically tolerable, non-radioactive complex salt of the general formula I ,4 -33- X-CH 2 CH2-X N-A-N V- HR 1 1 CHR -V 41r 9444 4, I so o A 99r 4 in which X represents the radicals -COOY, -PO 3 HY or -CONHOY wherein each Y represents a hydrogen atom, a metal equivalent, the metal being selected from elements having atomic numbers of from 21 to 29, 42, 44 and from 58 to 70, or a physiologically tolerable cation of an inorganic or organic base or amino acid, R 1 represents in each case a hydrogen atom or a methyl group and in which either a) A represents the group -CHR 2 -CHR 3 -CH 2 -CH 2 (ZCH 2 -CH )m- N(CH2X) 2 CH2-CH2 N(CH 2 X) 2 or -CH 2 -CH-CH 2 -CH2CH2-N-CH2-CH- '2 ZC2 2 2 I:: lr, .1 r '-B L -34- in which X R 2 and R 3 m and Z has the meanings given above, together represent a trimethylene group or a tetramethylene group, or each represent a hydrogen atom, an alkyl radical containing up to 6 carbon atoms, a phenyl radical or a benzyl radical, represents the integer 1, 2 or 3, represents an oxyged atom or a sulphur atom or the group 4 4 44 4 4* 4r 4 4 441 NCH2X or NCH2CH2OR4 2 2 4 in which X has the meanings given above and R represents an alkyl radical containing up to 6 carbon atoms, and each V has the same meaning as X or represents the group -CH2OH or -COB, in which B represents a protein or lipid radical or an -NH(CH 2 )nX radical, L- 1~ i in which X has the meanings given above and n represents an integer from 1 to 12, with the proviso that at least one of the symbols V represents a -COB group, as defined above, and at least two of the substituents Y are metal equivalents selected from elements having atomic numbers of from 21 to 29, 42, 44 and from 58 to or b) A represents the group -CHR 2 -CHR 3 in which R 2 represents a hydrogen atom and R 3 represents the group -(CH2) -C64-W-protein in which n represents 0 or 1, W represents -NHCOCH 2 or -NHCS- and -protein represents a protein radical, and each V has the same meaning as X or represents the group -CH 2 0H, with the proviso that at least two of the substituents Y are metal equivalents selected from elements having -36- atomic numbers of from 21 to 29, 42, 44 and from 58 to A physiologically tolerable complex salt as claimed in claim 29, which contains more than two of the radicals represented by X and in which at least one of the substituents Y is a physiologically tolerable tcation of an organic base or amino acid.

It

31. Any one of the physiologically tolerable complex salts as claimed in claim 29, specified in any one of *;the Examples herein.

32. A process for the manufacture of a physiologically tolerable complex salt as claimed in any one of claims 29 to 31, substantially as described herein. II

33. A physiologically tolerable complex salt as claimed in claim 29, for use in a method of NMR diagnosis of the human or animal body.

34. A physiologically tolerable complex salt as claimed in claim 30, for use in a method of NMR diagnosis of the human or animal body.

A physiologically tolerable complex salt asJ claimed in claim 31, for use in a method of NMR I .L S-37- -37- diagnosis of the human or animal body.

36. The use of a physiologically tolerable complex salt as claimed in claim 29, for the manufacture of a preparation for a method of NMR diagnosis of the human or animal body. jB

37. The use of a physiologically tolerable complex S salt as claimed in claim 30, for the manufacture *of a preparation for a method of NMR diagnosis of the ll .human or animal body.

38. The use of a physiologically tolerable complex o salt as claimed in claim 31. for the manufacture of a preparation for a method of NMR diagnosis of the Ij human or animal body. II DATED this 5th day of January, 1988. SCHERING AKTIENGESELLSCHAFT By Its Patent Attorneys ARTHUR S. CAVE CO.