GB2169599A

GB2169599A - Physiologically tolerable metal complex salts for use in diagnosis

Info

Publication number: GB2169599A
Application number: GB08529903A
Authority: GB
Inventors: Heinz Gries; Douwe Rosenberg; Hanns-Joachim Weinmann
Original assignee: Schering AG
Current assignee: Bayer Pharma AG
Priority date: 1983-01-21
Filing date: 1985-12-04
Publication date: 1986-07-16
Also published as: DK89794A; ES529020A0; ES8504668A1; ZA84472B; IE56857B1; SE8400254D0; PT77983B; LU85177A1; NZ206868A; GB2137612A; IL70711A; IE840126L; NZ219079A; GB8401486D0; FR2539996B1; ATA19184A; CH660183A5; SE8400254L; JPS59139390A; DK170460B1

Abstract

Physiologically tolerable complex salts which contain (a) a central element selected from elements having atomic numbers of from 21 to 29, 42, 44 and from 57 to 83, and (b) a radical of a physiologically-tolerable complex-forming acid and, if desired, (c) a radical selected from radicals of inorganic and organic bases and amino acids, and their use in NMR, X-ray and ultra-sound diagnosis.

Description

11 GB 2169599 A 1

SPECIFICATION

Diagnostic preparations and their use in a method of diagnosis The invention relates to diagnostic preparations and the use thereof in diagnosis 5 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 case) for detoxication in the case of inadvertent incorporation of heavy metals or their radioactive isotopes.

We have now found that certain physiologically tolerable complex salts containing one or more central 10 elements having the atomic numbers of from 21 to 29,42,44 and from 57 to 83 can be used for the manufacture of preparations that are surprisingly outstandingly suitable for use in NMR, ultra-sound and X-ray diagnostics.

The present invention provides a diagnostic preparation which comprises (i) a physiologicallytolerable complex salt which contains (a) a central element selected from elements having atomic numbers of from 21 15 to 29 inclusive, 42,44 and from 57 to 83 inclusive, and (b) a radical of a physiologically tolerable complex-forming acid and, if desired, (c) a radical selected from radicals of inorganic and organic bases and amino acids, and (ii) a physiologically tolerable carrier, especially an aqueous carrier.

The physiologically tolerable complex salt (i) may contain more than one central element and more than one of the radicals (b) and (c) 20 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 radioactive.

In the case where 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 25 especially the divalent and trivalent elements having an atomic number of from 21 to 29,42,44 and from 58 to 70 Suitable elements are, for example, chromium(l Il), manganese(ll), iron(l Il), iron(ll), cobalt(ll), nickel(ll), copper(ll), praseodymium(lil), neodymium(lll), samarium(lll) and ytterbium(I) Especially preferred, owing to their strong magnetic moment, are gadolinium(lil), terbium(l Il), dysprosium(lll), holmium(l Il) and erbium(l II) 30 If the preparation of the invention is to be used in X-ray diagnostics, the central element must be one having a relatively high atomic number in order to obtain sufficient absorption of the X-rays It has been found that diagnostic preparations that comprise a physiologically tolerable complex salt containing a central element or elements having an atomic number of from 57 to 83 are suitable for this purpose; such elements are, for example, lanthanum(lll), the above-mentioned elements of the lanthanide series, gold(l Il), 35 lead(ll) or, especially, bismuth(lll) Especially suitable are physiologically tolerable complex salts in which the central element (a) has an atomic number of from 71 to 83.

The preparations of the invention that are to be used in NMR diagnostics and those that are to be used in X-ray diagnostics are also suitable for use in ultra-sound diagnostics.

Suitable complex-forming acids are those which are customarily used for complex formation of the 40 above-mentioned central elements Suitable complex-forming acids are, for example, those which contain from 3 to 12, preferably from 3 to 8, methylenephosphonic acid groups, methylenecarbohydroxamic acid groups, carboxyethylidene groups or, especially, carboxymethylene groups of which at least one, two or three are bound to a nitrogen atom supporting the complex formation If three of the acid groups are bonded to a nitrogen atom, then the complex-forming acids in question are those which form the basis of the 45 complex salts of the general formula N(CH 2 X)3 ( 11) in which 50 X represents the radicals -COOY, -PO 3 HY or -CONHOY wherein Y represents a hydrogen atom, a metal equivalent and/or a physiologically tolerable cation of an inorganic or organic base or amino acid, with the proviso that at least two of the substituents Y are metal equivalents in which the metal has an atomic number of from 21 to 29,42,44 or from 57 to 83.

If in each case only one or two of the acid groups are bonded to a nitrogen atom, then the nitrogen atom is 55 bonded to a further nitrogen atom by way of optionally substituted ethylene or by way of up to four ethylene units each of which is separated by a nitrogen, oxygen or sulphur atom supporting the complex formation.

Preferred complex-forming acids of that type are those forming the basis of complex salts of the general formula 60 X-CH 2 CH 2-X N-A-N / \ V-CHR 1 CHR 1-V 65 2 GB 2169599 A in which X represents the radicals -COOY, -PO 3 HY or -CONHOY wherein Y represents a hydrogen atom, a metal equivalent and/or a physiologically tolerable cation of an inorganic or organic base or amino acid, and in which A represents the group -CHR 2-CHR 3-, -CH 2,-CH 2-(ZCH 2-CH 2)m-, 5 N(CH 2 X)2 CH 2-CH 2-N(CH 2 X)2 I or -CH 2-CH-CH 2 CH 2 CH 2-N-CH 2-CH 2- 10 in which X has the meanings given above, R, represents in each case a hydrogen atom or methyl group, R 2 and R 3 together represent a trimethylene group or a tetramethylene group, or each represents a hydrogen atom, lower alkyl group, phenyl group or benzyl group, or 15 R 2 represents a hydrogen atom, and R 3 represents a group -(CH 2)p-C 6 H 4-W-protein in which p represents O or 1, W represents -NN-, -NHCOCH 2 or -NHCS and -protein represents a protein radical and 20 m represents the integer 1, 2 or 3, Z represents an oxygen atom or a sulphur atom orthe group NCH 2 X or -NCH 2 CH 20 R 4 25 in which X has the meanings given above and R 4 represent a lower alkyl group, and in which 30 V has the same meaning as X or represents the group -CH 20 H, -CONH(CH 2)n X or -COB in which X has the meanings given above, B represents a protein or lipid radical, and n represents the integers from 1 to 12 or if R 1, R 2 and R 3 are hydrogen atoms both V's together represent the group 35 CH 2 X CH 2 X I I -(CH 2)w N -CH 2-CH 2-N-(CH 2)w- 40 in which X has the meanings given above, and w represents the integer 1, 2 or 3, with the proviso that at least two of the substituents Y are metal equivalents in which the metal has an atomic number of from 21 to 29,42,44 orfrom 57 to 83 45 The complex-forming acids 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, hormones, such as insulin, prostaglandins, steroid hormones, amino sugars, peptides, proteins or lipids.

There come into consideration more especially conjugates with albumens, such as human serum albumen, antibodies, such as, for example, monoclonal antibodies specific to tumour-associated antigens, or 50 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 lipsomes, which are used, for example, as unilamellar or multilamellar phosphatidylcho- line-cholesterol vesicles The conjugates are formed either by way of a carboxy group of the complex- forming acid or, in the case of proteins or peptides, also by way of a (CH 2)p-C 6 H 4-W group as defined 55 above under R 3 In the conjugate formation of some complex-forming acids with proteins, peptides 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 If the complex-forming acids are not bonded to biomolecules, they carry optionally two central elements, and especially one central element.

Suitable complex salts of the general formula I above are, for example, those of the general formula la 60 4 I: GB':2 f 4959 '99 A El is manufactured in the following manner, which is an improvemento:tl method proposed by R A;l-,;ulrnan et al in Naturwissenschaften 68, ( 1981) 483; 17.85 g f(:i 50 mmol) of 1,5-bis-( 2,6-dioxomorpholino-3-azapentane-3aceti't ap d-are s tpjen Fld 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 'C for 6 hours The clear solution is concentrated- in vacuo The yellow oily rei egrq isi 5, stirred with-500 ml of water at room temperature In so doing, an almost white, voluminous solid precipitates which is suction-filtered and washed several times with water- Forfurtherp trifctiil, th F resqltirg'pr qlu-,ot u,; is introduced into 200 ml of acetone and the wholeis stirred fo:30 minutesiat rpo;m ter npe Irt Ve S;r, o ',fl ti suction-filtering and drying in vacuo at 50 C,36 9 g (= 97 % ofthetheoretical yield) of a wht qpwqr Qgf: rn 1 G:melting point 134-138 C are obtained, ,,,;, rhl Conjugation ofthe complex-forming acidswith biomoleculesis likewise effete dacordirngitometh,qgs l known perse, for example by reacting the nucleophilic groups of the biotnolecu Le, such asifo,;x eml e l amino, hydroxy, thio or imidazole groups, with an activated derivative of the complex-f ming acil,: S b Activated derivatives of the complex-forming acid which come into consideration, are -,f'r ex ampl Jeiaiaó4 l:, -:

chlorides, acid anhydrides, activated esters, nitrenes or isothiocyanates -Conversely it,is a Jse Po Qssible to;=;r 1; react an activated biomolecule with the complex-forming acid.

For conjugation with proteins, substituents of the structure -C 6 H 4 N or -C 6 H 4 NHCOCH 2 halogen;rnmay also be considered.

The manufacture of some of the complex salts is likewise known or can be carried out in:a-manner known perse by dissolving or suspending the metal oxide or metal salt (for example the nitrate, chloride or 20 sulphate) of the element having an atomic number of from 21 to 29,42,44 orfrom 57 to 83 in water;anrd/qr 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-necess ry,,, whilewarming or heating to boiling point, until the reaction is complete If the complexsaltformed is -, insoluble in the solvent used, it is isolated by filtration If it is soluble, it can be isolated by concentrpa gh er 25 solution to dryness by evaporation, for example by means of spray- drying::i' l_ If acid groups are still present in the resulting complex salt, it is often advanatageoustorenvert tbe acid complex salt into a neutral complex saltor salts by means of inorganic and/or gorganic baseso,r ami p acids,;.

thatform physiologicallytolerable cations and to isolatetheneutral-salt in mantcases thisi)ine-e d-e; s unavoidable since the dissociation ofthe complex salt is so suppressed bythe shift in the p Hyalierto neuitra, 3,9:.

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 basesor basic amino acids J,,Itp;,, -.

however, also be advantageous if the neutralisation is carried out by means of inorganic bases (hydroxides, carbonates or bicarbonates) of sodium, potassium or lithium i; For the manufacture of the neutral salts there may, for example, be added i Rthe acid complexsalts in 3 - aqueous solution or suspension as much of the desired base as is necessary toidbti:n th:-t 1 uttal point The resulting solution can subsequently be concentrated to dryness in vacuo It is frequentiy of advantage to precipitate the resulting neutral salts by adding water-miscible solvents, suchtla, for example Lower alcohols (methanol, ethanol, isopropanol, etc), lower ketones (acetone, etc), and polar ethers (tetrahydro- furan, dioxane, 1,2-dimethoxyethane, etc), and thus obtain crystallisates tl::at Sq easily isolated and readily 4,;Q 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 t,:-,,:

If the acid complex salts contain several free acid groups, it is often advantageous to produce neutral mixed salts that contain both inorganic and organic physiologically tolerable cations as ions of opposijte; charge This can be effected, for example, by reacting the complex-forming acid in aqueous suspension or 4, solution with the oxide or salt of the element supplying the central element and with half the arnm ount o fn: -; organic base required for neutralisation, isolating the complex salt formed, if desired purifyingiti:andltheqrt,,; adding to it the amount of inorganic base required for complete neutralisation The order in which the bas Ps, are added can also be reversed l 8 The manufacture of the diagnostic preparations according to the invention is likewise effected in a manne r l,: $ known perse by suspending or dissolving the complex salts in an aqueous medium, optionaly-ty w-imte,0 addition of the additives customary in galenical pharmacy, and subsequently sterilisingthe-s Qlution,-,,r, >l' i suspension Suitable additives are, for example, physiologically tolerable buffers (such as, for exrnple i::, tror'iethamine hydrochloride), small additions of complexfor noers such as forexample, diethylerte-,: -,kqrm.

'triaminepentaaceticacid)or,ifnecessary,electrolytes(suchas, forexample, sodium chlo ride): , dt brjvenoi-r,:,,:

In principle, it i' also possible to manufacturethe diagnostiqprpeparations fitheinvention e Vern;With btin i isdfati Ig-thle eomplexsalts In each case, partibular care mustibtaken to, effebt tie belkeiforatd i in,swh,sl:

a mnid nerthat the salts and the sa It solutioni accordinrg to tbe l,e,hti:aheiv a /ree 1 f:rbw e- tox 4 '- tl-actirg -metal ions Thiscan-be ensured for examplevwithlthe:aid:f<bol uwindicaót srs sohja-s; L ebi g 3 xylenol orange, by test-titratibns dbring theimranufaoturing precess - The in ibrrlsother,,er,ei_',t,,(o '-o 6 Q processes fort ihi'/cu ep Of;dl com ltsrdoftheaf e sain prartw,s cf t lb e:t a;':i-e final safigdtard', lhb i' alwag pufriificatiort of the ibsolatedzcotitkex salt; 1 lo eois:,ti' snmo;;le', ri-em If t ns'jb'foh' ft o' iexa ttsi N wat 'r hysio Iogic:li'ia I 1 tb'ionmesired 'ri il;l-or q mon e-'T administration or other purposes, a sparingly soluble complex salt is mix Jadvl t oar,'e Pripteióu Aarjiurlr;,n cus 1 lir':1 ih -:a'Tiical pharmacy lnd/or sfitf'anits and/or ariq/atic asubstal,tortast(,e,ób Orns ': o 3 "o 1 5 POOR QUALITY GB 2169 599 A 3 X-CH 2 CH 2-X /N-CHR 2-CHR 3-N\ (la) V-CHR( CHR 1-V 5 in which X, V, R 1, R 2 and R 3 have the meanings given above.

The following complex-forming acids, interalia, are suitable for the manufacture of the complex salts of the general formula la:ethylenediaminetetraacetic acid, ethylenediaminetetraacetohydroxamic acid, trans-1,2 10 cyclohexylenediaminetetraacetic acid, DL-2,3-butylenediaminetetraacetic acid, DL-1,2- butylenediaminetetraacetic acid, DL-1,2-propylenediaminetetraacetic acid, 1,2diphenylethylenediaminetetraacetic acid, ethylenedinitrolotetrakis(methane-phosphonic acid) and N-( 2hydroxyethyl)-ethylenediaminetriacetic acid.

Other suitable complex salts of the general formula I are, for example, those of the general formula lb 15 X-CH 2 CH 2-X N-CH 2-CH 2 (Z-CH 2-CH 2)-N (lb) / \ 20 V-CHR 1 CHR 1-V in which X, V, Z, R, 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 a/ia, are suitable for the manufacture of the complex salts of 25 the general formula Ib:

diethylenetriaminepentaacetic acid, triethylenetetraminehexaacetic acid, tetraethylenepentamineheptaace- tic acid, 13,23-dioxo-15,18,21 -tris(carboxymethyl)-12,15,18,21,24- pentaazapentatriacontanoic diacid, 3,9-bis- ( 1-carboxyethyl)-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-1,1,10,10 O-tetraacetic 30 acid.

Suitable complex salts of the general formula I are also those of the general formula Ic X-CH 2 \/CH 2-X \ / 35 N-CH 2-CH 2-N I I CH 2 CH 2 I l (CH 2)w (CH 2)w 40 I I (Ic) N-CH 2-CH 2 N / X-CH 2 H 2-X 45 in which X and W have the meanings given above.

The following complex-forming acids, interalia, are suitable for the manufacture of the complex salts of the general formula Ic:

1,4,8,11 -tetraazacyclotetradecanetetraacetic acid and, especially, 1,4,7, 10-tetraazacyclododecanetetraacetic acid 50 Other complex-forming acids that are suitable for the manufacture of the complex salts of the general formula I are, for example:

1,2,3-tris-lbis-(carboxymethyl)-aminol-propane and nitrolotris(ethylenenitrolo)-hexaacetic acid As an ex- ample of a complex-forming acid for the manufacture of complex salts of the general formula II there may be mentioned nitrolotriacetic acid 55 If not all of the acid hydrogen atoms of the complex-forming acid are substituted by the central element or elements, it is advantageous, forthe 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 cations are for example, lithium, potassium or, especially, sodium Suitable cations of organic bases are, interalia, those of primary, secondary or tertiary amines, such as, for example, 60 ethanolamine, diethanolamine, morpholine, glucamine, N,Ndimethylglucamine 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 diagnostic preparations of the invention are known or can be manufactured in a manner known perse.

For example, 13,23-dioxo-15,18,21 -tris(carboxymethyl)-12,15,18,21-24pentaazapentatriacontanoic diacid 65 GB 2 169599 A 5 The diagnostic preparations of the invention contain preferably from 1 lLmol 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.

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 5 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 the signal intensity They also exhibit the high activity necessary to keep to a minimum the amount of foreign substances introduced into the body and the good tolerability necessary to maintain the non-invasive character of the examination (the compounds mentioned in J.

Comput Tomography 5,6: 543-46 ( 1981), in Radiology 144,343 ( 1982) and in Brevet Special de Medicament 10 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 15 only highly stable in vitro but also exhibit a surprisingly high stability in vivo, so that the perse toxic ions that are not covalently bonded in the complexes are released or exchanged only extremely slowly over the 24 hours in which, as pharmacological studies have shown, the novel contrast agents are completely eliminated The conjugates with proteins and antibodies which are used, for example, forthe diagnosis of tumours bring about a surprisingly high intensification of the signal at such a low dosage that it is possible to 20 use in this case solutions of correspondingly low concentration.

The diagnostic preparations of the invention particularly those in which the physiologically complex salt contains an element having a relatively high atomic number that is from 57 to 83, for example 71 to 83, are also outstandingly suitable as X-ray contrast agents; it should be especially emphasise that, with these, none of the symptoms of anaphylaxy-type reactions known in the case of iodine- containing contrast agents can be 25 detected in biochemical-pharmacological tests They are especially valuable by virtue of their advantageous absorption properties in regions of relatively high tube voltages for digital substraction techniques.

Further, the diagnostic preparations of the invention are also suitable as ultra-sound diagnostics owing to their property of favourably influencing the ultra-sound speed.

In contrast to conventional X-ray diagnostics with shadow-producing X-ray contrast agents, in NMR 30 diagnostics with paramagnetic contrast agents there is no linear relationship between the signal intensification and the concentration used As control studies have shown, increasing the dose administered does not necessarily result in the signal being intensified, and, in the case of a high dose of paramagnetic contrast agent, the signal can even be extinguished It was, for that reason, surprising that some pathological processes become visible only after the administration of doses higher than those specified in EP 71 564 35 (which may be from 0 0001 mmol/kg to 5 mmol/kg) of a preparation of the invention containing a strongly paramagnetic contrast agent Thus, for example, a defective blood-brain barrier in the region of a cranial abscess can be demonstrated only after giving 0 05 to 2 5 mmol/kg, preferably 0 1 0 5 mmol/kg, of paramagnetic complex salts such as, for example, gadolinium diethylenetriaminepentaacetic acid or manganese 1,2-cyclohexylenediaminetetraacetic acid in the form of its readily water-soluble salts For a 40 dose of more than 0 1 mmol/kg, solutions of higher concentrations of up to 1 mol/1, preferably from 0 25 to 0.75 mol/1, are required since only in this way is the volume reduced and the ease of handling the injection solution ensured.

Especially low doses (under 1 mg/kg) and therewith solutions of lower concentrations ( 1 lmol/1 to 5 mmol/1) than are specified in EP 71 564 are required for organ-specific NMR diagnostics, for example for 45 detecting tumours and coronary infarcts.

The invention also provides physiologically tolerable complex salts containing (a) a central element selected from elements having atomic numbers of from 21 to 29,42,44 and from 57 to 83, for example of from 71 to 83, and (b) a radical of a physiologically tolerable complex- forming acid, and, if desired, (c) a radical selected from radicals of inorganic and organic bases and amino acids, for example a physiologically 50 tolerable complex salt of the general formula I given above, in which X, A, V and R 1 have the meanings given above, with the proviso that it contains from 3 to 12 substituents Y of which at least two are metal equivalents in which the metal has an atomic number of from 21 to 29,42, 44 or from 57 to 83 and, in addition, at least one of the substituents Y is a physiologically tolerable cation of an organic base or amino acid, any substituents Y which may remain being hydrogen atoms or cations of an inorganic base 55 The present invention further provides a method of diagnosis using NMR, X- rays or ultra-sound, wherein a preparation of the present invention is administered to a human or animal body.

The following Examples illustrate the invention:- EXAMPLE 1 60

Preparation of the gadolinium(Ill) complex of nitrolo-NNN-triacetic acid, C 6 H 6 Gd NO 6 A suspension of 36 2 g (= 100 mmol) of gadolinium oxide (Gd 2 03) and 38 2 g (= 200 mmol) of nitrolotriacetic acid in 1 2 litres of water is heated, while stirring, to 90 WC to 1000 C and is stirred at this temperature for 48 hours The undissolved material is filtered off over active carbon and the filtrate is concentrated to dryness by evaporation The amorphous residue is pulverised 65 6 GB 2169599 A Yield: 60 g ( 87 % of the theoretical yield) m.p 300 C gadolinium: calculated 45 5 %, found 44 9 %.

The iron(ll) complex of nitrolo-N,N,N-triacetic acid is obtained in analogous manner with the aid of iron(Ill) chloride, Fe CI 3 5 EXAMPLE 2

Preparation of the disodium salt of the gadolinium(ll) complex of 13,23dioxo 15,18,21-tris-(carboxymethyl)12,15,18,21,24-pentaazapentatriacontanoic diacid, C 36 H 60 Gd N 502 2 Na.

15 2 g (= 20 mmol) of 13,23-dioxo-15,18,21-tris-(carboxymethyl)-12,15,18, 21,24 10 pentaazapentatriacontanoic diacid are suspended in 400 ml of water and the suspension is heated to 95 C.

7.43 g (= 20 mmol) of gadolinium(l Il) 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 orderto neutralise the hydrochloric acid formed, 60 ml of 1 N sodium hydroxide solution are added.

When the reaction is complete (testing with xylenol orange) the precipitate obtained is filtered and washed 15 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 C are obtained.

Gadolinium(l Il) complex of 13,23-dioxo-15,18,21-tris-(carboxymethyl)-1 2, 15,18,21,24-pentaazapentatri- acontanoic diacid.

Analysis: 20 (calculated) 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 gadolinium(lli) complex so obtained are suspended in 200 ml of water, and 31 41 of 1 N sodium hydroxide solution are added dropwise thereto After 1 hour a clear solution is obtained which 25 is filtered and then concentrated in vacuo After drying in vacuo at 80 C 13 2 g ( 87 % of the theoretical yield) of a readily water-soluble, white powder of melting point 279-285 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 30 In analogous manner there is obtained, using N-methylglucamine in place of sodium hydroxide solution, the di-n-methylglucamine salt of the gadolinium(l Il) complex of 13,23dioxo-15,18,21-tris(carboxymethyl)12,15,18,21,24-pentaazapentatriacontanoic diacid, C 50 H 96 Gd N 7022.

35 EXAMPLE 3

Preparation of the disodium salt of the gadolinium(Ill) complex of 3,9bis( 1-carboxyethyl)-6-carboxymethyl- 3,6,9-triazaundecanoic diacid, C 16 H 22 Gd N 3010 2 Na 36.2 g (= 0 1 mol) of gadolinium(l Il) oxide and 84 2 g (= 0 2 mol) of 3, 9-bis( 1-carboxyethyl)-6carboxymethyl-3,6,9-triazaundecanoic diacid are suspended in 250 ml of water and the whole is refluxed for 40 1 hour The small amount of undissolved material is filtered off and the solution is concentrated to dryness in vacuo The residue is pulverised and dried in vacuo at 60 C 112 8 g (= 98 % of the theoretical yield) of the complex salt (chelate) is obtained in the form of a white powder.

Analysis: C 16 H 24 Gd N 3010 (calculated) C 33 39 H 4 20 Gd 27 32 N 7 30 45 (found) C 47 13 H 6 63 Gd 27 42 N 7 21 57.6 g (= 0 1 mol) of the complex salt are introduced into a solution of 0 1 mol of caustic soda in 100 ml of water By adding a further 0 1 ml of caustic soda powder a p H of 7 5 is established in the solution, the solution is heated to boiling point and ethanol is added dropwise until the reaction mixture remains turbid 50 After stirring for several hours in an ice bath, the crystallisate is suction-filtered, washed with ethanol and dried in vacuo The disodium salt is obtained in quantitative yield in the form of a white powder.

Analysis:

(calculated C 31 02 H 3 58 Gd 25 38 N 6 78 (found) C 31 10 H 3 71 Gd 25 50 N 6 61 55 EXAMPLE 4

Preparation of the dimorpholine salt of the gadolinium(Nll) complex of 3, 9-bis-( 1-carboxyethylj-6- carboxymethyl-3,6,9-triazaundecanoic diacid, C 24 H 42 Gd N 5072 17 4 g (= 0 2 mol) of morpholine are dissolved in 50 ml ofwater 42 1 g (= O 1 mol) of 60 3,9-bis(1-carboxyethyl)-6-carboxymethyl-3,6,9-triazaundecanoic diacid and then 18 2 g (= 0 05 mol) of gadolinium(l Il) oxide are added and the whole is maintained at refluxtemperature until a clear solution has appeared Acetone is then added dropwise until the reaction mixture remains turbid After stirring for several hours in an ice bath, the crystallisate is suction-filtered, washed with acetone and dried in vacuo The dimorpholine salt is obtained in quantitative yield in the form of a white powder 65 GB 2 169599 A 7 Analysis:

(calculated) C 38 44 H 5 65 Gd 20 97 N 9 34 (found) C 38 31 H 5 72 Gd 20 76 N 9 32 EXAMPLE 55

Preparation of the di-N-methylglucamine salt of the gadolinium(Ill) complex of diethylenetriamine- N,N,N',N',N"-pentaacetic acid, C 28 H 54 Gd N 5020.

39.3 g (= 100 mmol) of diethylenetriamine-N,N,N',N",N"-pentaacetic acid are suspended in 200 ml of water, and 19 5 g (= 100 mmol) of N-methylglucamine are added 18 12 g (= 50 mmol) of gadolinium(l Il) oxide, Gd 203, are then added in portions and the resulting suspension is heated to 95 C After approximately 10 1 hour, a further 19 5 g (= 100 mmol) of N-methylglucamine are added and, after heating for a further 2 hours, a clear solution is obtained When the reaction is complete (testing with xylenol orange), the small amount of undissolved material is filtered off and the filtrate is concentrated to dryness in vacuo The residue is again dissolved in 100 ml of water and stirred into 250 ml of ethanol After cooling for several hours, the crystallisate is suction-filtered, washed with cold ethanol and dried at 60 C in vacuo 92 7 g ( 99 % of the 15 theoretical yield) of a white powder of indeterminate melting point is obtained.

Analysis:

(calculated) C 35 85 H 5 80 N 7 47 Gd 16 77 (found) C 35 50 H 5 72 N 7 20 Gd 67 54 20 For purification of the complex salt, it is possible to use, in place of ethanol, also acetone, propanol or isopropanol.

In analogous manner, there are obtained: with dysprosium( 111) oxide, Dy 203, the di-N-methylglucamine salt of the dysprosium( 111) complex of diethylenetriamine-N,N,N',N",N"- pentaacetic acid, C 28 H 54 Dy N 5020; 25 with lanthanum(l Il) oxide, La 203, the di-N-methylglucamine salt of the lanthanum(l Il) complex of diethylenetriamine-N,N,N',N",N"-pentaacetic acid, C 24 H 54 La N 5020; with ytterbium(l Ill) oxide, Yb 203, the di-N-methylglucamine salt of the ytterbium(lll) complex of diethylenetriamine N,N,N',N",N"-pentaacetic acid, C 28 H 54 Yb N 5020; with samarium(ll) oxide, Sm 203, the di-N- methylglucamine salt of the samarium(Ill) complex of diethylenetriamine-N,N,N',N",N"-pentaacetic acid, C 28 H 54 Sm N 5020; with holmium(l Il) oxide, 30 Ho 203, the di-N-methylglucamine salt of the holmium(ll) complex of diethylenetriamine-N,N,N',N",N"- pentaacetic acid, C 28 H 54 Ho N 5020; with bismuth(lll) oxide, Bi 203, the di-N-methylglucamine salt of the bismuth(Ill) complex of diethylenetriamine-N,N,N',N",N"-pentaacetic acid, C 28 H 54 Bi N 5020; with gadoli- nium(l Il) oxide, Gd 203, the tri-N-methylglucamine salt of the gadolinium(l Il) complex of triethylenetetramine- N,N,N',N",N"',N"'-hexaacetic acid, C 39 H 78 Gd N 7027 35 There are also obtained in analogous manner: with holmium(lll) oxide, Ho 203, and ethanolamine in place of N-methylglucamine, the diethanolamine salt of the holmium(lll) complex of diethylenetriamineN,N,N',N",N"-pentaacetic acid, C 18 H 34 Ho N 5 012; with gadolinium(l Il) oxide, Gd 203, and lysine in place of N-methylglucamine, the dilysine salt of the gadolinium(l Il) complex of diethylenetriamine-N,N,N',N",N"pentaacetic acid, C 22 H 42 Ho N 5014 40 The salts are obtained in the form of a white powder of indeterminate melting point They are very readily soluble in water.

EXAMPLE 6

Manufacture of the disodium salt of the gadolinium( 111) complex of diethylenetriamine-N,N,N'-N';N" 45 pentaacetic acid, C 14 H 18 Gd N 301 o 2 Na 18.2 g ( = 0 05 mol) of gadolinium (Ill) oxide and 39 3 g (= 0 1 mol) of diethylenetriaminepentaacetic acid are suspended in 110 ml of water and refluxed for 1 hour The clear solution is cooled and adjusted to p H 7 5 by the addition of approximately 80 ml of 5 N sodium hydroxide solution The solution is again heated to 50 boiling point and 250 ml of ethanol are added dropwise After stirring for several hours in an ice bath, the crystallisate is suction-filtered, washed with ice-cold ethanol and dried at 60 C in vacuo There is obtained in quantitative yield a white powder which does not melt until 300 C.

Analysis:

(calculated) C 28 43 H 3 07 N 7 10 Gd 26 58 55 (found) C 28 35 H 2 95 N 7 05 Gd 26 37 In analogous manner, there are obtained: with dysprosium (I 111) oxide, Dy 203, the disodium salt of the dysprosium (I 111) complex of diethylenetriamine-N,N,N',N",N"-pentaacetic acid, C 14 H 18 Dy N 3 Oo 2 Na; with lanthanum (Ill) oxide, La 203, the disodium salt of the lanthanum (I 111) complex of diethylenetriamine 60 N,N,N',N",N"-pentaacetic acid, C 14 H 18 La N 3,01 2 Na; with holmium (Ill) oxide, Ho 203, the disodium salt of the holmium (I 111) complex of diethylenetriamine-N,N,N',N",N"-pentaacetic acid C 14 H,8 Ho N 3010 2 Na; with ytterbium (Ill) oxide, Yb 203, the disodium salt of the ytterbium (Ill) complex of diethylenetriamine- N,N,N',N',N"-pentaacetic acid, C 14 H 18 Yb N 3 010 2 Na; with samarium (Ill) oxide, Sm 203, the disodium salt of the samarium(lll) complex of diethylenetriamine-N,N,N',N",N"-pentaacetic acid, C 14 H 18 Sm N 3,0 2 Na; with 65 8 GB 2169599 A erbium(ll) oxide, Eb 203, the disodium salt of the erbium (I 111) complex of diethylenetriamine-N,N,N',N",N"- pentaacetic acid, C 14 H 18 Eb N 3 0010 2 Na; with gadolinium (Ill) oxide, Gd 203, the sodium salt of the digadolinium(l Il) complex of tetraethylenepentamine-N,N,N',N",N'", N'V, N'v heptaacetic acid, C 22 H 30 Gd 2 N 5014 À Na.

These salts are obtained as a white powder of indeterminate melting point and are very readily soluble in 5 water.

EXAMPLE 7

Manufacture of the N-methylglucamine salt of the iron(Ill) complex of diethylenetriaminepentaacetic acid, C 21 H 37 Fe N 4015 10 35.4 ( = 90 mmol) of diethylenetriaminepentaacetic acid are suspended in 100 ml of water, and 24 3 g ( = mmol) of iron(ll) chloride hexahydrate (Fe CI 3 6 H 20), dissolved in 100 ml of water, are added thereto.

The initially dark brown suspension is heated to 95 C After approximately 1 hour, the colour changes to a light yellow 270 ml of 1 N sodium hydroxide solution are added to neutralise the hydrochloric acid formed and the whole is heated for a further 3 hours at 95 C The resulting light yellow precipitate is suction-filtered, 15 washed free of chloride with water and dried at 60 C in vacuo 17 85 g ( 45 % of the theoretical yield) of a light yellow powder is obtained the melting point of which is > 300 .

17.85 g ( = 40 mmol) of the iron(Ill) complex salt obtained are suspended in 200 ml of water, and 7 8 g (= mmol) of solid N-methylglucamine are added in portions The whole is heated for approximately 3 hours at 50 C and an almost clear, red-brown solution is obtained which is filtered and then concentrated to 20 dryness in vacuo The residue is dried at 50 C in vacuo 24 3 g ( 95 % of the theoretical yield) of a red-brown powder of melting point 131-133 C are obtained.

Analysis:

(calculated) C 39 82 H 5 89 N 8 85 Fe 8 81 (found) C 39 70 H 6 00 N 8 65 Fe 9 01 25 By using sodium hydroxide solution in place of the N-methylglucamine there are obtained in analogous manner: the sodium salt of the iron(li) complex of ethylenediaminetetraacetic acid, Cjo H 12 Fe N 208 - Na; the sodium salt of the iron(Ill) complex of trans-1,2cyclohexylenediaminetetraacetic acid, C 14 H 18 Fe N 208- Na; the disodium salt of the iron(lll) complex of diethylenetrinitrolopenta (methanephosphonic acid), 30 C 9 H 23 Fe N 3 015 P 5 2 Na; the sodium salt of the iron(lil) complex of 1,10-diaza-4,7-dioxadecane-1,1,10,10tetraacetic acid, C 14 H 20 Fe N 2010 Na; the sodium salt of the iron(lll) complex of ethylenediaminetetraacetohy- droxamic acid, Co 10 H 16 Fe N 608 Na.

In analogous manner, there are obtained with N-methylglucamine: the di-Nmethylglucamine salt of the iron(i I) complex of diethylenetriamine-N,N,N',N",N"-pentaacetic acid, C 28 H 54 Fe N 5020; the N 35 methylglucamine salt of the iron(lll) complex of trans-1,2cyclohexylenediamine-N,N,N',N'-tetraacetic acid, C 21 H 36 Fe N 3013; the N-methylglucamine salt of the iron(lll) complex of ethylenediamine-N,N,N',N'-tetraacetic acid, C 17 H 30 Fe 3 013; the tri-N-methylglucamine salt of the iron(lll) complex of triethylenetetramine- N,N,N',N",N"',N"'-hexaacetic acid, C 39 H 78 Fe N 7027.

By using diethanolamine in place of N-methylglucamine there is obtained in analogous manner: the 40 di-diethanolamine salt of the iron(i) complex of diethylenetriamine-N,N, N"N",N"-pentaacetic acid, C 22 H 42 Fe N 5014.

EXAMPLE 8

Manufacture of the N-methylglucamine salt of the gadolinium(Ill) complex of trans 1,2-cyclohexylene 45 diamine-N,N,N',N'-tetraacetic acid, C 21 H 36 Gd N 3013 20.78 g ( = 60 mmol) of trans-1,2-cyclohexylenediamine-N,N,N',N'- tetraacetic acid are suspended in 150 ml of water After the addition of 11 7 g ( = 60 mmol) of Nmethylglucamine, an almost clear solution is obtained to which 10 88 g ( = 30 mmol) of gadolinium oxide (Gd 203) are added The suspension again obtained is heated for 6 hours at 95 C The small amount of undissolved material is filtered off and the filtrate 50 is concentrated to dryness in vacuo The residue is dried in vacuo at 60 C and pulverised 38 6 g ( 92 % of the theoretical yield) of a white powder of melting point 258-261 C are obtained.

Analysis:

(calculated) C 36 25 H 5 22 N 6 04 Gd 22 60 (found) C 36 40 H 5 50 N 5 98 Gd 22 52 55 In analogous manner, by using sodium hydroxide solution in place of N- methylglucamine, the sodium salt of the gadolinium(l Il) complex of trans-1,2-cyclohexylenediamine-N,N,N', N'-tetraacetic acid, C 14 H 18 Gd N 208.

Na, is obtained.

By using freshly precipitated chromium(lll) hydroxide, Cr(OH)3, the sodium salt of the chromium(lll) 60 complex of ethylenediamine-N,N,N',N'-tetraacetic acid, Cjo H 12 Cr N 208 Na, is obtained.

GB 2169599 A 9 EXAMPLE 9

Preparation of the disodium salt of the manganese(ll) complex of trans 1, 2-cyclohexylenediamine-N,N,N', N'-tetraacetic acid, C 14 H 16 Mn N 208 2 Na Under nitrogen, 34 6 g ( = 100 mmol) of trans-1,2-cyclohexylenediamine-N, N,N',N'-tetraacetic acid are suspended in 100 ml of water, and 11 5 g ( = 100 mmol) of manganese( 11) carbonate, Mn CO 3, are added The 5 whole is heated to 95 C and 200 ml of 1 N sodium hydroxide solution are added dropwise thereto The clear solution is concentrated in vacuo and the residue is dried in vacuo at 60 C 40 8 g ( 92 % of the theoretical yield) of a pink-coloured powder are obtained.

Analysis:

(calculated) C 37 94 H 4 09 N 6 32 Mn 12 40 10 (found) C 37 78 H 4 12 N 6 20 Mn 12 31 In analogous manner, there are obtained: from copper(ll) carbonate the disodium salt of the copper( 11) complex of trans-1,2-cyclohexylenediaminetetraacetic acid, C 14 H 18 Cu N 208 À 2 Na; from cobalt(ll) carbonate thedisodiumsaltofthecobalt(ll)complexoftrans-1,2cyclohexylenediaminetetraaceticacid,C 14 H 18 Co N 208 15 2 Na; from nickel( 11) carbonate the disodium salt of the nickel(ll) complex of trans-1,2- cyclohexylenediaminetetraacetic acid, C 14 H 18 Ni N 208 À 2 Na.

By using N-methylglucamine in place of sodium hydroxide solution there are obtained: the di-N- methylglucamine salt of the manganese(ll) complex of trans-1,2- cyclohexylenediaminetetraacetic acid, C 28 H 54 Mn N 4018; the di-N-methylglucamine salt of the manganese(ll) complex of DL-2,3 20 butylenediaminetetraacetic acid, C 26 H 52 Mn N 4018; the di-Nmethylglucamine salt of the manganese( 11) complex of ethylenediamine-N,N,N',N'-tetraacetic acid, C 24 H 48 Mn N 4018; the di-N-methylglucamine salt of the manganese (II) complex of DL-1,2-butylenediamine-N,N,N',N'- tetraacetic acid, C 26 H 52 Mn M 4018; the di-N-methylglucamine salt of the manganese ( 11) complex of DL-1,2propylenediamine-N,N,N',N'-tetraacetic acid, C 25 H 50 Mn N 4018; the tri-N-methylglucamine salt of the manganese( 11) complex of diethylenet 25 riaminepeptaacetic acid, C 35 H 72 Mn N 6025; with nickel(ll) carbonate, Ni CO 3, there is obtained: the di-N- methylglucamine salt of the nickel( 11) comple of ethylenediamine-N,N,N', N'-tetraacetic acid, C 24 H 48 Ni N 4018; with cobalt(ll) carbonate, COCO 3, there is obtained: the diethanolamine salt of the cobalt(ll) complex of ethylenediamine-N,N,N',N'-tetraacetic acid, C 14 H 28 Co N 4010; with copper( 11) carbonate, Cu CO 3, and ethanola- mine there is obtained: the diethanolamine salt of the copper(l) complex of ethylenediamine-N,N,N',N' 30 tetraacetic acid, C 14 H 28 Cu N 4010; with manganese(ll) carbonate, Mn CO 3, and diethanolamine there is obtained:

the tri-diethanolamine salt of the manganese( 11) complex of diethylenetriamine-N,N,N',N",N"-pentaacetic acid, C 26 H 54 Mn N 6016; with manganese(ll) carbonate, Mn CO 3, and morpholine there is obtained:

the dimorpholine salt of the manganese(ll) complex of ethylenediamine-N,N, N",N"-tetraacetic acid, 35 C 18 H 32 Mn N 4010.

EXAMPLE 10

Preparation of the N-methylglucamine salt of the gadolinium ( 1 i 1) complex of ethylenediamine-N,N,N',N'- tetraacetic acid, C 17 H 30 Gd N 3013 40 29.2 g ( = 100 mmol) of ethylenediamine-N,N,N',N'-tetraacetic acid are suspended in 100 ml of water and heated to 950 C with 18 1 g ( = 50 mmol) of gadolinium( 111) oxide, Gd 203 During the heating-up process, 19 5 g ( = 100 mmol) of N-methylglucamine are added in portions After approximately 3 hours, a clear solution is obtained which is filtered and concentrated to dryness in vacuo The residue is dried at 60 C in vacuo 61 3 g ( 95 % of the theoretical yield) of a white powder having an indeterminate melting point are obtained 45 Analysis:

(calculated) C 31 82 H 4 71 N 6 55 Gd 24 51 (found) C 31 65 H 4 59 N 6 52 Gd 24 56 In analogous manner, there are obtained: with dysprosium( 11) oxide Dy 203: the N-methylglucamine salt of 50 the dysprosium(lll) complex of ethylenediamine-N,N,N',N'-tetraacetic acid, C 17 H 30 Dy N 3013.

By using 1,10-diaza-4,7-dioxadecane-1,1,10,10-tetraacetic acid in place of ethylenediamine-N,N,N',N'- tetraacetic acid there is obtained: the N-methylglucamine salt of the gadolinium(lll) complex of 1,10-diaza-4,7-dioxadecane-1,1,10,1 0-tetraacetic acid, C 21 H 38 Gd N 3015; Similarly, by using 1,2-diphenylethylenediaminetetraacetic acid there is obtained: 55 the N-methylglucamine salt of the gadolinium(l Il) complex of 1,2- diphenylethylenediaminetetraacetic acid, C 29 H 38 N 3013 Gd; By using lead( 11) oxide, Pb O, and sodium chloride, there is obtained: the disodium salt of the lead(ll) complex of ethylenediaminetetraacetic acid, Co O H 12 N 208 Pb À Na; By using freshly precipitated chromium(lll) hydroxide, Cr(OH)3, there is obtained: the sodium salt of the 60 chromium(lll) complex of ethylenediaminetetraacetic acid, C O o H 12 Cr N 208 Na; and analogously: the sodium salt of the gadolinium(Ill) complex of ethylenediaminetetraacetohydroxamic acid, C 10 H 16 Gd N 608 Na; and the sodium salt of the gadolinium(lll) complex of ethylenediamine-N,N,N', N'-tetraacetic acid, Cj O H 12 Gd N 208 Na.

GB 2 169 599 A 10 EXAMPLE 11

Preparation of the sodium salt of the gadolinium(III) complex of 1,4,7,10tetraazacyclododecane-N,N',N",NV"- tetraacetic acid, C 16 H 24 Gd N 408 Na 4.0 g ( = 10 mmol) of 1,4,7,10-tetraazacyclododecane-N,N',N",N"'tetraacetic acid are suspended in 20 ml of water, and 10 ml of 1 N sodium hydroxide solution are added 1 8 g ( = 5 mmol) of gadolinium(lll) oxide, 5 Gd 203, are added and the suspension is heated for 2 hours at 50 C The clear solution is filtered and concentrated in vacuo The residue is dried and pulverised 5 5 g ( 95 % of the theoretical yield) of a white powder are obtained.

Analysis:

(calculated) C 33 10 H 4 17 N 9 65 Gd 27 08 10 (found) C 33 01 H 4 20 N 9 57 Gd 27 16 In analogous mannerthere are obtained: the N-methylglucamine salt of the gadolinium(l Il) complex of 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid, C 23 H 42 Gd N 5013; the sodium salt of the gadoli- nium(Ill) complex of 1,4,8,11 -tetraazacyclotetradecane-N,N',N",N"'- tetraacetic acid, C 18 H 28 Gd N 408 Na 15 EXAMPLE 12

Preparation of the tetra-N-methylglucamine salt of the gadolinium( 11 i) complex of ethylenedinitrolotetrakis- (methanephosphonic acid), C 34 H 85 Gd N 6032 9 11 g ( = 20 mmol) of ethylenedinitrolotetrakis(methanephosphonic acid) are suspended in 150 ml of 20 water and the suspension is adjusted to a p H of 5 with the corresponding amount of N-methylglucamine 3 6 g ( = 10 mmol) of gadolinium(l Il) oxide, Gd 203, are added thereto and the whole is heated to 70 C After approximately 1 hour, a clear solution is obtained to which there is added the remaining portion of the N-methylglucamine Atotal of 15 6 g ( = 80 mmol) of N-methylglucamine is used The solution is concentrated to dryness in vacuo and the gel-like residue remaining is introduced into 200 ml of acetonitrile 25 The mixture is stirred for approximately 20 hours at 30 and the resulting fine precipitate is suction-filtered.

After drying in vacuo at 40 C, 23 4 g ( 85 % of the theoretical yield) of a white powder of melting point 115-118 C are obtained.

Analysis:

(calculated) C 29 78 H 6 25 N 6 13 P 9 04 Gd 11 47 30 (found) C 29 85 H 6 57 N 5 98 P 8 78 Gd 11 26 In analogous mannerthere are obtained: the hepta-N-methylglucamine saltof the gadolinium(l Il) complex of diethylenetriamine-N,N,N',N",N"-penta(methanephosphonic acid), C 58 H 144 Gd N 10050 P 5, and, by using sodium hydroxide solution in place of N-methylglucamine the disodium salt of the gadolinium(lll) complex 35 of diethylene-trinitrolo-penta (methanephosphonic acid), Cg H 23 Gd N 3015 P 5 2 Na.

EXAMPLE 13

Preparation of the disodium salt of the manganese(l) complex of ethylenedinitrolo-tetra(acetohydroxamic acid), C 1 o H 16 Mn N 608 2 Na 40 2.30 g of manganese(l) carbonate and 7 05 g of ethylenedinitrolotetra(acetohydroxamic acid) are refluxed in 18 ml of water for 3 hours The p H is then adjusted to 7 by the addition of dilute sodium hydroxide solution and 40 ml of acetone are added dropwise After stirring for several hours in an ice bath, the crystallisate which has separated is suction-filtered, washed with acetone and dried at 50 C in vacuo A dihydrate is obtained in quantitative yield in the form of a white powder having a melting point above 300 C 45 Mn: (calculated) 11 30 (found) 11 12 EXAMPLE 14

Preparation of a mixed saltsolution-comprising the sodium and the Nmethylglucamine salt of the 50 gadolinium ( 11) complex of diethylenetriaminepentaacetic acid a) preparation of the mono-N-methylglucamine salt of the complex, C 21 H 37 Gd N 4015 2 g ( 1 mol) of N-methylglucamine are dissolved in 7 litres of water 393 3 g ( 1 mol) of diethylenetriaminepentaacetic acid and 181 3 g ( 0 5 mol) of gadolinium(lll) oxide, Gd 203, are added and whole is refluxed for 2 hours The filtered clear solution is spray-dried A white crystalline powder having a 55 water content of 2 6 %, which sinters at 133 C and melts, with foaming, at 190 C is obtained.

Gd: (calculated) 21 17 (found) 21 34 b) preparation of the neutral mixed salt solution 730 8 g ( = 1 mol) of the salt obtained in a) are suspended in 630 ml ofwaterp i (pro injectione), and 40 g 60 ( = 1 mol) of caustic soda powder are added in portions The neutral solution is made up to 1000 ml with waterp i, introduced into bottles over a pyrogen filter and heatsterilised This 1 molar solution contains 753 8 g of the mixed salt per litre.

GB 2 169 599 A 11 EXAMPLE 15

Preparation of a solution of the di-N-methylglucamine salt of the gadolinium(ll) complex of diethylene- triaminepentaacetic acid 0 g ( = 730 mmol) of the salt described in Example 5 are made into a paste in 500 ml of waterp i and dissolved by adding 142 4 g ( = 730 mmol) of N-methylglucamine at p H 7 2 The solution is then made up to 5 1000 ml with waterp i, is introduced into ampoules and heat-sterilised.

EXAMPLE 16

Preparation of a solution of the disodium salt of the gadolinium(ill) complex of diethylenetriaminepentaace- tic acid 10 485 1 g = 820 mmol) of the disodium salt obtained in Example 6 are made into a paste in 500 ml of water p.i The volume is then made up to 1000 ml with waterp i and the solution is introduced into ampoules and heat-sterilised.

EXAMPLE 17 15

Preparation of a solution of the disodium salt of the gadolinium(ill) complex of 13,23-dioxo 15,18,21-tris- (carboxymethyl) 12, 15,18,21,28-pentaazapentatriacontanoic diacid 392 0 g ( = 400 mmol) of the salt described in Example 2 are made into a paste in 500 ml of waterp i and dissolved by making up the volume to 1000 ml with water p i while heating slightly The solution is placed in bottles and heat-sterilised 20 EXAMPLE 18

Preparation of a solution of the N-methylglucamine salt of the gadolinium(Ill) complex of 1,4,7,10- tetraazacyclododecanetetraacetic acid 370 9 g ( = 500 mmol) of the complex salt described in Example 11 are made into a paste in 500 ml of water 25 p.i and dissolved by making up the volume to 1000 ml with waterp i The solution is introduced into ampoules and heat-sterilised.

EXAMPLE 19

Preparation of a solution of the di-N-methylglucamine salt of the manganese( 11) complex of trans 1,2 30 cyclohexylenediaminetetraacetic acid 395 9 g ( = 500 mmol) of the complex salt described in Example 9 are suspended in 500 ml of water p i 1 3 g of ascorbic acid are added and the suspension is dissolved by making up the volume to 1000 ml with water p.i The solution is sterile-filtered and placed in ampoules.

35 EXAMPLE 20

Preparation of a solution of the tri-N-methylglucamine salt of the manganese( 11) complex of diethylenet- riaminepentaacetic acid 514 4 g ( = 500 mmol) of the complex salt described in Example 9 are suspended in 600 ml of waterp i 1 3 g of ascorbic acid are added and the solid matter is dissolved by making up the volume to 1000 ml with water 40 p.i After being sterile-filtered, the solution is placed in ampoules.

EXAMPLE 21

Preparation of a solution of the di-N-methylglucamine salt of the iron(Ill) complex of diethylenetriaminepen- taacetic acid 45 44.6 g ( = 0 1 mol) of the iron(ll) complex of diethylenetriaminepentaacetic acid obtained in Example 7 are suspended in 40 ml of waterp i After the addition of 0 18 g of tromethamine hydrochloride and 39 1 g ( = 0.2 mol) of N-methylglucamine, the solid matter is dissolved at the neutral point, the solution is made up to ml with waterp i, introduced into ampoules and heat-sterilised.

50 EXAMPLE 22

Preparation of a solution of the gadolinium( 111) complex of nitrolotriacetic acid 1.9 g ( = 10 mmol)m of nitrolotriacetic acid and 1 8 g ( = 5 mmol) of gadolinium(Ill) oxide are dissolved in ml of water p i while heating The solution is introduced into ampoules and heat-sterilised.

55 EXAMPLE 23

Preparation of a solution of the N-methylglucamine salt of the gadolinium( 11 i) complex of ethylene- diaminetetraacetic acid 38.52 g ( = 60 mmol) of the substance described in Example 10 are dissolved in 70 ml of water p i Afterthe addition of 0 12 g of tromethamine, the solution is made up to 100 ml with water p i, placed in ampoules and 60 heat-sterilised.

EXAMPLE 24

Preparation of a solution of the di-N-methylglucamine salt of the dysprosium(ill) complex of diethylene- triaminepentaacetic acid 65 12 GB 2 169599 A 35.7 g ( = 60 mmol) of the dysprosium(lll) complex of diethylenetriaminepentaacetic acid (water content 8.0 %) are suspended in 70 ml of waterp i and dissolved at p H 7 5 by adding 21 2 g ( = 120 mmol) of N-methylglucamine The solution is then made up to 100 ml with waterp i, placed in ampoules and heat-sterilised.

5 EXAMPLE 25

Preparation of a solution of the N-methylglucamine salt of the gadolinium(ill) complex of trans-1,2- cyclohexylenediaminetetraacetic acid 555 8 g ( = 0 8 mol) of the salt described in Example 8 are dissolved in water p i to a volume of 1000 ml.

After filtration over a pyrogen filter, the solution is placed in ampoules and heat-sterilised 10 EXAMPLE 26

Preparation of a solution of the N-methylglucamine salt of the ruthenium(Ill) complex of 1,10-diaza-4,7- dithiadecane 1,1,10,10-tetraacetic acid 15 6 g ( = 0 03 mmol) of the ruthenium( 11 l) complex of 1,10-diaza-4,7- dithiadecane-1,1,10,10-tetraacetic 15 acid are suspended in 50 ml ofwaterp i and dissolved at p H 7 5 by adding 5 9 g ( = 0 03 mol) of N-methylglucamine The solution is then made up to 1000 ml with waterp i, placed in ampoules and heat-sterilised.

EXAMPLE 27 20

Preparation of a solution of the dilysine salt of the gadolinium(ill) complex of diethylenetriaminepentaacetic acid 273 8 g ( = 0 5 mol) of the gadolinium(lll) complex of diethylenetriaminepentaacetic acid are suspended in 500 ml of waterp i 292 4 g ( = 1 mol) of lysine are added, the whole is stirred for several hours while heating gently and the volume is then made up to 1000 ml with waterp i The solution is placed in bottles and 25 heat-sterilised.

EXAMPLE 28

Preparation of a solution of the tri-N-methylglucamine salt of the molybdenum(VI) complex of diethylene- triaminepentaacetic acid 30 18.8 g ( = 0 28 mol) of the complex H 3 lMo 202 (OH)4 À C 14 H 23 N 3010 l are suspended in 50 ml of waterp i and dissolved at the neutral point by adding 16 4 g ( = 0 84 mol) of N- methylglucamine 0 15 g of tromethamine is added, the solution is made up to 100 ml with waterp i, subjected to sterile filtration and placed in ampoules.

35 EXAMPLE 29

Preparation of a solution of the disodium salt of the manganese(//) complex of ethylenediaminetetraacetic acid 343,2 g ( = 1 mol) of the manganese( 1) complex of ethylenediaminetetraacetic acid are suspended in 500 ml of waterp i and dissolved atthe neutral point by adding, in portions, 80 g ( = 2 mol) of caustic soda After 40 the addition of 1 5 g of tromethamine, the solution is made up to 1000 ml with waterp i, placed in bottles and heat-sterilised.

EXAMPLE 30

Preparation of a solution of the sodium salt of the iron (l/l) complex of ethylenediaminetetraacetic acid 45 345 7 g ( = 1 mol) of the iron(Ill) complex of the iron(lll) complex of ethylenediaminetetraacetic acid are suspended in 500 ml of waterp i and dissolved at the neutral point by adding, in portions, 40 g ( = 1 mol) of caustic soda After the addition of 1 5 g of tromethamine, the solution is made up to 1000 ml with waterp i, placed in bottles and heat-sterilised.

50 EXAMPLE 31

Preparation of a solution of the disodium salt of the iron(ll) complex of diethylenetriaminepentaacetic acid 334 6 g ( = 0 75 mol) of the iron(lll) complex of diethylenetriaminepentaacetic acid are suspended in 500 ml of waterp i and dissolved at the neutral point by adding, in portions, 60 g ( = 1 5 mol) of caustic soda The solution is made up to 1000 ml with waterp i, placed in bottles and heat- sterilised 55 EXAMPLE 32

Preparation of a solution of the sodium salt of the gadolinium(il) complex of trans 1,2- cyclohexylenediaminetetraacetic acid 558 6 g ( = 1 mol) of the sodium salt of the complex salt listed in Example 8 are dissolved in waterp i and 60 made up to 1000 ml The solution is placed in bottles and heat-sterilised.

EXAMPLE 33

Preparation of a solution of the N-methylglucamine salt of the gadoliniumr(Ill) complex of 1,2- diphenylethylenediaminetetraacetic acid 65 GB 2 169 599 A 13 396 9 g ( = 500 mmol) of the N-methylglucamine salt of the complex salt containing gadolinium and the 1,2-diphenylethylenediaminetetraacetic acid radical listed in Example 10 are made into a paste in 600 ml of water p i and dissolved by making up the volume to 1000 ml The solution is placed in ampoules and heat-sterilised.

5 EXAMPLE 34

Preparation of a solution of the sodium salt of the iron(Ill) complex of ethylenediaminetetraacetic acid 183 5 g ( = 500 mmol) of the sodium saltof the complex salt of iron and ethylenediaminetetraacetic acid listed in Example 7 are made into a paste in 500 ml of waterp i 1 0 g of tromethamine are added, the volume is made up to 1000 ml with waterp i, and the solution is placed in ampoules and heat-sterilised 10 EXAMPLE 35

Preparation of a solution of the di-N-methylglucamine salt of the lanthanum(ll J) complex of diethylene- triaminepentaacetic acid 459 8 g ( = 500 mmol) of the di-N-methylglucamine salt of the complex salt containing lanthanum and the 15 diethylenetriaminepentaacetic acid radical listed in Example 5 are made into a paste in 650 ml of water p i.

and dissolved by making up the volume to 1000 ml with waterp i The solution is placed in ampoules and heat-sterilised.

EXAMPLE 36 20

Preparation of a solution of the di-N-methylglucamine salt of the bismuth(ll) complex of diethylene- triaminepentaacetic acid 692 8 g ( = 700 ml) of the di-N-methylglucamine salt of the complex salt containing bismuth and the diethylenetriaminepentaacetic acid radical listed in Example 5 are made into a paste in 600 ml of water p i.

and, after the addition of 1 8 g of tromethamine, dissolved by making up the volume to 1000 ml with water 25 p.i while heating slightly The solution is placed in ampoules and heat- sterilised.

EXAMPLE 37

Preparation of a solution of the di-N-methylglucamine salt of the holmium(ll) complex of diethylene- triaminepentaacetic acid 30 662 0 g ( = 700 mmol) of the di-N-methylglucamine salt of the complex salt containing holmium and the diethylenetriaminepentaacetic acid radical listed in Example 5 are made into a paste with 600 ml of waterp i.

and, after the addition of 1 8 g of tromethamine, dissolved by making up the volume to 1000 ml with water p.i while heating slightly The solution is placed in ampoules and heat- sterilised.

35 EXAMPLE 38

Preparation of a solution of the di-N-methylglucamine salt of the ytterbium(Ill) complex of diethylene- triaminepentaacetic acid 476 9 g ( = 500 ml) of the di-N-methylglucamine salt of the complex salt containing ytterbium and the diethylenetriaminepentaacetic acid radical listed in Example 5 are made into a paste with 650 ml of water p i 40 and, after the addition of 1 5 g of tromethamine, dissolved by making up the volume to 1000 ml with water p.i The solution is placed in ampoules and heat-sterilised.

EXAMPLE 39

Preparation of a solution of the disodium salt of the lanthanum(ll) complex of diethylenetriaminepentaacetic 45 acid 573 2 g ( = 1000 mol) of the disodium salt of the complex salt containing lanthanum and diethylenetriaminepentaacetic acid radical listed in Example 6 are made into a paste in 650 ml of water p i.

and dissolved by making up the volume to 1000 ml with waterp i The solution is placed in ampoules and heat-sterilised 50 EXAMPLE 40

Preparation of a solution of the disodium salt of the dysprosium(ill) comples of diethylenetriaminepentaace- tic acid 477 4 g (= 800 mmol) of the disodium salt of the complex salt containing dysprosium and the 55 diethylenetriaminepentaacetic acid radical listed in Example 6 are made into a paste in 600 ml of waterp i.

and dissolved by making up the volume to 1000 ml with water p i The solution is placed in ampoules and heat-sterilised.

EXAMPLE 41 60

Preparation of a solution of the disodium salt of the holmium(Ill) complex of diethylenetriaminepentaacetic acid 299 6 g ( = 500 mmol) of the disodium salt of the complex salt containing holmium and the diethylenetriaminepentaacetic acid radical listed in Example 6 are made into a paste in 500 ml of water p i.

and dissolved by making up the volume to 1000 ml with waterp i The solution is placed in ampoules and 65 14 GB 2 169 599 A heat-sterilised.

EXAMPLE 42

Preparation of a solution of the disodium salt of the ytterbium(ill) complex of diethylenetriaminepentaacetic acid 5 303 5 g (= 500 mmol) of the complex salt containing ytterbium listed in Example 6 are made into a paste in 500 ml of waterp i and dissolved by making up the volume to 1000 ml with waterp i The solution is placed in ampoules and heat-sterilised.

EXAMPLE 43 10

Preparation of a solution of the tetra-N-methylglucamine salt of the gadolinium( 111) complex of ethylenedini- trolo-tetrakis(methanephosphonic acid) 137 1 g ( = 100 mmol) of the complex salt described in Example 12 are made into a paste in 500 ml of water p.i and, after the addition of 0 8 g of tromethamine, dissolved by making up the volume to 1000 ml with water p i The solution is placed in ampoules and heat-sterilised 15 EXAMPLE 44

Preparation of a solution of the gadolinium(ill) complex of N'-( 2hydroxyethyl)-ethylenediamine-N,N,N'- triacetic acid 1 9 g ( = 6 7 mmol) of N'-( 2-hydroxyethyl)-ethylenediamine-N,N,N'- triacetic acid and 1 2 g ( = 3 35 mol) of 20 gadolinium(lll) oxide are dissolved in 6 ml of waterp i while heating The solution is placed in ampoules and heat-sterilised.

EXAMPLE 45

Preparation of a solution of the disodium salt of the manganese(I) complex of trans-1,2 25 cyclohexylenediaminetetraacetic acid Under nitrogen, 44 3 g ( = 100 mmol) of the complex salt described in Example 9 are made into a paste in ml of waterp i and dissolved by making up the volume to 100 ml with water p i The solution is placed in ampoules and heat-sterilised.

30 EXAMPLE 46

Preparation of a solution of the sodium salt of the gadolinium(ill) complex of 1,4,8, 1- tetraazacyclotetradecane-N,N',N",N"'-tetraacetic acid 552 6 g ( = 1 mol) of the complex salt containing gadolinium and the 1,4, 8,11- tetraazacyclotetradecanetetraacetic acid radical listed in Example 11 are dissolved in waterp i and made up 35 to 1000 ml The solution is placed in bottles and heat-sterilised.

EXAMPLE 47

Preparation of a solution of the disodium salt of the bismuth ( 111) complex of diethylenetriaminepentaacetic acid 40 23.4 g ( = 50 mmol) of bismuth (I 111) oxide are suspended in 50 ml of waterp i After the addition of 39 3 g (= 100 mmol) of diethylenetriaminepentaacetic acid and 4 0 g (= 50 mmol) of caustic soda, the whole is refluxed until a clear solution is obtained The solution, cooled to room temperature, is neutralised by adding 4.0 g of caustic soda and made up to 100 ml with waterp i The solution is introduced into ampoules and heat-sterilised 45 EXAMPLE 48

Preparation of a solution of the disodium salt of the samarium(Ill) complex of diethylenetriaminepentaacetic acid 58 5 g ( = 100 mmol) ofthe complex salt containing samarium listed in Example 6 are dissolved in 65 ml of 50 waterp i while heating Waterp i is added to make a total volume of 100 ml, and the solution is introduced into ampoules and heat-sterilised.

EXAMPLE 49

Preparation of a solution of the di-N-methylglucamine salt of the gadolinium(ll J) complex of 13,23-dioxo 55 15,18,2 1-tris(carboxymethyl) 12,15,18,21,24-pentaazapentatriacontanoic diacid 4 g ( = 100 mmol) of the di-N-methylglucamine complex salt listed in Example 2 are made into a paste in 250 ml of waterp i and dissolved while heating The solution is made up to 500 ml with waterp i, introduced into ampoules and heat-sterilised.

60 EXAMPLE 50

Preparation of a solution of the di-N-methylglucamine salt of the manganese(l/) complex of ethylene- diaminetetraacetic acid 3.68 g ( = mmol) of the complex salt containing manganese and the ethylenediaminetetraacetic acid radical listed in Example 9 are dissolved in 70 ml of waterp i, and 0 4 g of sodium chloride is added to the 65 GB 2 169599 A 15 solution The solution is then made up to 100 ml with water p i and introduced into ampoulesthrough a sterile filter The solution is at 280 m Osm isotonic with blood.

EXAMPLE 51

Preparation of a solution of the disodium salt of the gadolinium(III) complex of diethylenetrinitrolo-penta 5 (methanephosphonic acid) 38.57 g ( = 50 mmol) of the disodium salt of the complex containing gadolinium and the diethylenetrinitrolo-penta(methanephosphonic acid) listed in Example 12 are made into a paste with 50 ml of waterp i The p H is adjusted to 7 2 by adding caustic soda powder and the volume is made up to 100 ml with waterp i The solution is introduced into ampoules and heat-sterilised 10 EXAMPLE 52

Preparation of a solution of the trisodium salt of the manganese( 11) complex of diethylenetriaminepentaace- tic acid Under nitrogen, 39 3 g ( = 100 mmol) of diethylenetriaminepentaacetic acid are suspended in 100 ml of 15 waterp i, and 11 5 g of manganese( 11) carbonate are added The whole is heated to 95 C and 300 ml of 1 N sodium hydroxide solution are added dropwise The neutral solution is sterile-filtered and introduced into ampoules.

EXAMPLE 53 20

Composition of a powder for the preparation of a suspension 4.000 g gadolinium(ill) complex of diethylenetriaminepentaacetic acid (water content 8 0 %) 3 895 g saccharose 25 0.100 g polyoxyethylenepolyoxypropylene polymer 0.005 g aromatic substance 8.000 g 30 EXAMPLE 54

Preparation of a solution of the gadolinium(Ill) complex of the conjugate of diethylenetriaminepentaacetic acid with human serum albumen 10 mg of 1,5-bis-( 2,6-dioxomorpholino-3-azapentane-3-acetic acid are added to 20 ml of a solution of 3 mg 35 of the protein in a 0 05 molar sodium bicarbonate buffer (p H 7-8) The whole is stirred for 30 minutes at room temperature and is then dialysed against a 0 3 molar sodium phosphate buffer 50 mg of gadolinium (Ill) acetate are then added and purification is effected by gel chromatrography over a Sephadex G 25 column.

The fraction obtained is sterile-filtered and placed in multi-dose phials Freeze-drying produces a dry 40 preparation that can be stored 40 In an analogous manner, there is obtained with immunoglobulin a solution of the corresponding complex conjugate.

EXAMPLE 55

Preparation of a solution of the gadolinium (III) complex of the conjugate of diethylenetriaminepentaacetic 45 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 Ei of a solution of 0 3 mg of a monoclonal antibody in a 0 05 molar sodium bicarbonate buffer (p H 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 gadolinium (III) complex of ethylenediaminetetraacetic acid 50 (EDTA) are added to the antibody fraction obtained After purification by gel chromatography over Sephadex G 25, 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(Ill) complex of the CDTA antibody.

Using the manganese( 11) complex of ethylenediaminetetraacetic acid there is obtained in an analogous 55 manner the manganese( 11) complexes of the antibodies coupled with DTPA or CDTA.

EXAMPLE 56

Preparation of a solution of the gadolinium(I 11) complex of the conjugate of 1-phenyl 60 ethylenediaminetetraacetic acid with immunoglobulin 60 According to the procedure described in J Med 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 is cooled to + 4 C and there is added dropwise the proportion, equivalent to the protein, of a freshly prepared ice-cold diazonium salt solution of 1 -(p-aminophenyl)-ethylenediaminetetraacetic acid The whole is stirred overnight (p H 8 1) at + 4 C and is then dialysed against a 0 1 molar sodium citrate solution When dialysis is 65 16 GB 2 169 599 A complete, an excess of gadolinium(lll) 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.

EXAMPLE 57

Preparation of a colloidal dispersion of a Mn(ll)-CDTA-lipid conjugate 5 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 G 50 column, the sterile-filtered solution is placed in multi-dose phials and freeze-dried 10 A colloidal dispersion of the gadolinium-DTPA-lipid conjugate can be obtained analogously with gadolinium(ill) oxide.

EXAMPLE 58

Preparation of lipsomes charged with gadolinium(ill)-DTPA 15 According to the procedure described in Proc Natl Acad Sci U S A 75,4194, a lipid mixture comprising mol % of egg phosphatidylchloline and 25 mol % of cholesterol is prepared as a dry substance 500 mg thereof are dissolved in 30 ml of diethyl ether and, in an ultrasonic bath, 3 ml of a 0 1 molar solution of the di-N-methylglucamine salt of the gadolinium(l Il) complex of diethylenetriaminepentaacetic acid in waterp i are added dropwise thereto When the addition of the solution is complete, the exposure to ultrasonic waves 20 is continued for 10 minutes and then concentration is carried out in a rotary evaporator The gel-like residue is suspended in a O 125 molar sodium chloride solution and, at O OC, repeatedly freed of non-encapsulated contrast agent portions by centrifugation ( 20000 g/29 minutes) Finally, the lipsomes 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 25

Claims

1 A diagnostic preparation which comprises (i) a physiologically tolerable complex salt which contains (a) a central element selected from elements having atomic numbers of from 21 to 29, 42,44 and from 57 to 30 83, and (b) a radical of a physiologically-tolerable complex-forming acid and, if desired, (c) a radical selected from radicals of inorganic and organic bases and amino acids, and (ii) a physiologically tolerable carrier.

2 A diagnostic preparation as claimed in claim 1 wherein the carrier is an aqueous carrier.

3 A diagnostic preparation as claimed in claim 2, wherein the carrier is water or physiological salt solution, and the complex salt is dissolved or suspended in it 35 4 A diagnostic preparation as claimed in claim 2 or claim 3, wherein the complex salt is present in a concentration of from 1 pimol to 1 mol per litre.

A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the physiologically tolerable complex salt is a compound of the general formula 40 X-CH 2 CH 2-X \(I) /A-N V-CHR, CHR 1-V 45 or N(CH 2 X)3 ( 11) 50 in which X represents the radicals -COOY, -PO 3 HY or -CONHOY wherein Y represents a hydrogen atom, a metal equivalent and/or a physiologically tolerable cation of an inorganic or organic base or amino acid.

and in which A represents the group -CHR 2-CHR 3-, -CH 2, -CH 2-(ZCH 2-CH 2)m-, 55 N(CH 2 X)2 CH 2-CH 2-N(CH 2 X)2 t or I -CH 2-CH-CH 2 CH 2 CH 2-N-CH 2-CH 2- 60 in which X has the meanings given above, R 1 represents in each case a hydrogen atom or methyl group, R 2 and R 3 together represent a trimethylene group or a tetramethylene group, or each represent a hydrogen atom, lower alkyl radical, phenyl radical or benzyl radical, or 65 GB 2 169599 A 17 R 2 represents a hydrogen atom, and R 3 represents a group -(CH 2)2 P-C 6 H 4-W-protein in which p represents 0 or 1, W represents -NN or NHCOCH 2-, and -protein represents a protein radical, and 5 m represents the integer 1,2 or 3, Z represents an oxygen atom or a sulphur atom orthe group NCH 2 X or NCH 2 CH 2 OR 4 10 in which X has the meanings given above, and R 4 represent a lower alkyl radical, and in which 15 V has the same meaning as X or represents the group -CH 2 OH, -CONH(CH 2) X or -COB in which X has the meanings given above, B represents a protein or lipid radical, and n represents the integers from 1 to 12 or if R 1, R 2 and R 3 are hydrogen atoms both V's together represent the group 20 CH 2 X CH 2 X I I -(CH 2)w N-CH 2-CH 2-N-(CH 2),25 in which X has the meanings given above, and w represents the integer 1, 2 or 3, with the proviso that at least two of the substituents Y are metal equivalents in which the metal has an atomic number of from 21 to 29,42,44 or from 57 to 83 30 6 A diagnostic preparation as claimed in any one of claims 1 to 5, wherein the complex-forming acid is diethylenetriaminepentaacetic acid.

7 A diagnostic preparation as claimed in any one of claims 1 to 5, wherein the complex-forming acid is ethylenediaminetetraacetic acid.

8 A diagnostic preparation as claimed in any one of claims 1 to 5, wherein the complex-forming acid is 35 trans-1,2-cyclohexylenediaminetetraacetic acid or 1,4,7,10- tetraazacyclododecanetetraacetic acid.

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

A diagnostic preparation as claimed in claim 9, wherein the biomolecule is insuline or a prostaglandin, steroid hormone, amino sugar, peptide, protein or lipid 40 11 A diagnostic preparation as claimed in claim 9, wherein the biomolecule is an albumen.

12 A diagnostic preparation as claimed in claim 9, wherein the biomolecule is a monoclonal antibody.

13 Adiagnostic preparation as claimed in claim 12,wherein the monoclonal antibody is specificto tumour-associated antigens.

14 A diagnostic preparation as claimed in claim 9, wherein the complexforming acid forms a conjugate 45 or inclusion compound with a lipsome.

A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is the di-N-methylglucamine salt of the manganese(ll) complex of ethylenediaminetetraacetic acid.

16 A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is the di-N-methylglucamine salt of the gadolinium(Ill) complex of diethylenetriaminepentaacetic acid 50 17 A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is the di-N-methylglucamine salt of the dysprosium(lll) complex of diethylenetriaminepentaacetic acid.

18 A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is the monosodium/mono-N-methylglucamine mixed salt of the gadolinium(lll) complex of diethylenetriaminepentacetic acid 55 19 A diagnostic preparation as claimed in any one of claims I to 4, wherein the complex salt (i) is the dilysine salt of the gadolinium(Ill) complex of diethylenetriaminepentaacetic acid.

A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is the disodium salt of the gadolinium(Ill) complex of diethylenetriaminepentaacetic acid.

21 A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is the 60 iron(ll) complex of diethylenetriaminepentaacetic acid.

22 A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is the disodium salt of the iron(ll) complex of diethylenetriaminepentaacetic acid.

23 A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is the disodium salt of the manganese(ll) complex of diethylenetriaminepentaacetic acid 65 18 GB 2 169599 A 24 A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is the di-N-methylglucamine salt of the holmium(lll) complex of diethylenetriaminepentaacetic acid.

A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is the disodium salt of the manganese( 11) complex of ethylenediaminetetraacetic acid.

26 A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is the 5 di-N-methylglucamine salt of the bismuth(lll) complex of diethylenetriaminepentaacetic acid.

27 A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is the di-N-methylglucamine salt of the manganese( 11) complex of trans-1,2- cyclohexylenediaminetetraacetic acid.

28 A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is the disodium salt oftheytterbium(l Il) complex of diethylenetriaminepentaacetic acid 10 29 A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is the N-methylglucamine salt of the gadolinium(l Il) complex of 1,4,7,10- tetraazacyclododecanetetraacetic acid.

A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is the disodium salt of the manganese( 11) complex of trans-1,2- cyclohexylenediaminetetraacetic acid.

31 A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) isthe 15 disodium salt of the bismuth(Ill) complex of diethylenetriaminepentaacetic acid.

32 A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is the di-N-methylglucamine salt of the gadolinium(Ill) complex of 13,23-dioxo- 15,18,21-tris-(carboxymethyl)12,15,18,21,24-pentaazapentatriacontanoic diacid.

33 A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is the 20 sodium salt of the gadolinium(lll) complex of 1,4,7,10- tetraazacyclododecanetetraacetic acid.

34 A diagnostic preparation as claimed in any one of claims i to 4, wherein the complex salt (i) is the gadolinium(l Il) complex of the conjugate of diethylenetriaminepentaacetic acid with immunoglobulin.

A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is the gadolinium(lil) complex of the conjugate of diethylenetriaminepentaacetic acid with human serum albumen 25 36 A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is the gadolinium(lll) complex of the conjugate of diethylenetriaminepentaacetic acid with a monoclonal antibody.

37 A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is the manganese(ll) complex of the conjugate oftrans-1,2- cyclohexylenediaminetetraacetic acid with a monoclon- al antibody 30 38 A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is the manganese(ll) complex of the lipid conjugate of trans-1,2- cyclohexylenediaminetetraacetic acid.

39 A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is the gadolinium(lll) complex of diethylenetriaminepentaacetic acid conjugated with a lipsome.

40 A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is the 35 disodium salt of the holmium(lll) complex of diethylenetriaminepentaacetic acid.

41 A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is the disodium salt of the lanthanum( 11) complex of diethylenetriaminepentaacetic acid.

42 A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is the di-N-methylglucamine salt of the ytterbium(ll) complex of diethylenetriaminepentaacetic acid 40 43 A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is the disodium salt of the samarium(ll) complex of diethylenetriaminepentaacetic acid.

44 A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is the disodium salt of the gadolinium( 1 l) complex of 1 3,23-dioxo-15,18,21- tris-(carboxymethyl)-12,15,18,21,24pentaazapentatriacontanoic diacid 45 A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the physiologically tolerable complex salt contains a central element selected from elements having an atomic number of from 71 to 83.

46 A diagnostic preparation as claimed in claim 1, substantially as described in any one of Examples 14 to 58 herein.

47 A diagnostic preparation as claimed in any one of claims 1 to 45, which is in a dosage form suitable 50 for administration orally, neurally or intravasally.

48 An ampoule containing a diagnostic preparation as claimed in any one of claims 1 to 45, in a form suitable for injection.

49 A process forthe manufacture of a diagnostic preparation as claimed in any one of claims 1 to 45, wherein the complex salt (i) is dissolved or suspended in water or physiological salt solution, and is made 55 up, if desired with the incorporation of one or more physiologically tolerable adjuncts, in a form that is suitable for intravasal or oral administration.

A physiologically tolerable complex salt which contains (a) a central element selected from elements having atomic numbers of from 21 to 29,42,44 and from 57 to 83, and (b) a radical of a physiologically tolerable complex-forming acid and, if desired, (c) a radical selected from radicals of inorganic and organic 60 bases and amino acids.

51 A physiologicallytolerablecomplexsaltas claimed in claim 50,whereinthe central element (a) is one selected from elements having atomic numbers of from 71 to 83.

GB 2 169 599 A 19 52 A physiologically tolerable complex salt of the general formula X-CH 2 CH 2-X Y /N-A-N (I) 5 V-CHR 1 CHR,-V in which X, A, V and R, have the meanings give in claim 5, with the proviso that it contains from 3 to 12 substituents Y of which at least two are metal equivalents in which the metal has an atomic number of from 10 21 to 29, 42,44 or from 57 to 83 and, in addition, at least one of the substituents Y is a physiologically tolerable cation of an organic base or amino acid, any substituents Y which may remain being hydrogen atoms or cations of an inorganic base.

53 Any one of the physiologically tolerable complex salts specified in any one of the Examples herein.

54 The N-methylglucamine salt of the gadolinium(ll) complex of ethylenediaminetetraacetic acid 15 The di-N-methylglucamine salt of the gadolinium(ll) complex of diethylenetriaminepentaacetic acid.

56 The di-N-methylglucamine salt of the iron(lll) complex of diethylenetriaminepentaacetic acid.

57 The di-N-methylglucamine salt of the manganese(Il) complex of ethylenediaminetetraacetic acid.

58 The disodium salt of the gadolinium(l Il) complex of diethylenetriaminepentaacetic acid.

59 The tri-N-methylglucamine salt of the manganese(ll) complex of diethylenetriaminepentaacetic acid 20 The N-methylglucamine salt of the dysprosium(ll) complex of ethylenediaminetetraacetic acid.

61 The di-N-methylglucamine salt of the holmiunm(Ill) complex of diethylenetriaminepentaacetic acid.

62 The dilysine salt of the gadolinium(ll) complex of diethylenetriaminepentaacetic acid.

63 The di-N-methylglucamine salt of the manganese( 11) complex of trans-i, 2-cyclohexylenetetraacetic acid 25 64 The di-N-methylglucamine salt of the bismuth(ll) complex of diethylenetriaminepentaacetic acid.

The disodium salt of the ytterbium(lll)complex of diethylenetriaminepentaacetic acid.

66 The N-methylglucamine salt of the gadolinium(l 11) complex of 1,4,7,10tetraazacyclododecanetetraacetic acid.

67 The N-methylglucamine-sodium-mixed salt of the gadolinium(l Il) complex of diethylenetriaminepen 30 taacetic acid.

68 A diagnostic preparation which comprises a physiologically tolerable complex salt of the general formula I given in claim 5, with the exception of preparations for use in NMR diagnostics containing from 5 to 250 mmol per litre of a neutral N-methylglucamine salt of the manganese( 11) complex, nickel( 11) complex, gadolinium(l Il) complex, dysprosium(Ill) complex or holmium(ll) complex of ethylenediaminetetraacetic 35 acid or diethylenetriaminepentaacetic acid, or a neutral lysine salt of the gadolinium(ll) complex of diethylenetriaminepentaacetic acid, or a neutral sodium or morpholine salt of the manganese( 11) complex of ethylenediaminetetraacetic acid, or a neutral diethanolamine salt of the copper( 11) complex or cobalt(ll) complex of ethylenediaminetetraacetic acid.

69 A process for the manufacture of a physiologically tolerable complex salt as claimed in any one of 40 claims 50 to 67, substantially as described herein.

A method of diagnosis using NMR, wherein a preparation as claimed in any one of claims I to 4 in which the complex salt (i) contains an element having an atomic number of from 21 to 29,42,44 and from 58 to 70 is administered to a human or animal body.

71 A method of diagnosis using X-rays, wherein a preparation as claimed in any one of claims 1 to 4 and 45 in which the complex salt (i) contains an element having an atomic number of from 57 to 83 is administered to a human or animal body.

72 A method as claimed in claim 71, wherein the complex salt (i) contains an element having an atomic number of from 71 to 83.

73 A method of diagnosis using ultra-sound, wherein a preparation as claimed in any one of claims 1 to 50 4 is administered to a human or animal body.

74 A physiologically tolerable complex salt containing (a) a central element selected from elements having atomic numbers of from 21 to 29,42,44 and from 57 to 83, and (b) a radical of a physiologically tolerabe complex-forming acid and, if desired, (c) a radical selected from radicals of inorganic and organic bases and amino acids, for use in a method of diagnosis of the human or animal body by NMR diagnosis, 55 X-ray diagnosis or ultra-sound diagnosis.

A physiologicallytolerable complex salt as claimed in claim 74, wherein the central element (a) is one selected from elements having atomic numbers of from 71 to 83.

76 A physiologicallytolerable complex salt as claimed in any one of claims 52 to 67, for use in a method of diagnosis of the human or animal body by NMR diagnosis, X-ray diagnosis or ultra-sound diagnosis 60 77 A process forthe manufacture of a diagnostic preparation as claimed in claim 1, conducted substantially as described in any one of Examples 14 to 58 herein.

Amendments to the claims have been filed, and have the following effect:(a) Claims 1-77 above have been deleted 65 GB 2 169 599 A (b)New or textually amended claims have been filed as follows:- 1 A diagnostic preparation which comprises (i) a physiologicallytolerable, non-radioactive complex salt of the general formula I 5 X-CH 2 CH 2-X / N-A-N (I) /\ V-CHR, CHR 1-V 10 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 57 to 83, or a physiologically tolerable cation of an inorganic or organic base or amino acid, 15 and in which either a) Rl represents in each case a hydrogen atom or methyl group A represents the group -CHR 2-CHR 3 or -CH 2-CH 2-(ZCH 2-CH 2)m-, inwhich 20 R 2 and R 3 together represent a trimethylene group or a tetramethylene group, or each represents a phenyl radical or benzyl radical, m represents the integer 1,2 or 3, and Z represents an oxygen atom or a sulphur atom, and each V has the same meaning as X, 25 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 57 to 83, or b) R, represents in each case a hydrogen atom, A represents the group -CH 2-CH 2 and both V'stogether representthe group 30 CH 2 X CH 2 X l I -(CH 2)_N-CH 2-CH 2-N-(CH 2)W- 35 in which X has the meanings given above and w represents the integer 1,2 or 3, with the proviso that at leasttwo of the substituents Y are metal equivalents selected from elements having atomic numbers of from 21 to 29,42,44 and from 57 to 83, and 40 (ii) a physiologically tolerable carrier.

2 A diagnostic preparation as claimed in claim 1, wherein the carrier (ii) is an aqueous carrier.

3 A diagnostic preparation as claimed in claim 2, wherein the carrier (ii) is water or physiological salt solution, and the complex salt (i) is dissolved or suspended in it.

4 A diagnostic preparation as claimed in claim 2 or claim 3, wherein the complex salt (i) is present in a 45 concentration of from 1 p Lmol to 1 mol per litre.

A diagnostic preparation as claimed in any one of claims 1 to 4, wherein the complex salt (i) is derived from trans-1,2-cyclohexylenediaminetetraacetic acid, 1,4,7,10tetraazacyclododecanetetraacetic acid or 1,4,8,11 -tetraazacyclotetradecanetetraacetic acid.

6 A diagnostic preparation as claimed in any one of claims 1 to 5, wherein the complex salt (i) is the 50 di-N-methylglucamine salt of the manganese (II) complex of trans-1,2- cyclohexylenediaminetetraacetic acid.

7 A diagnostic preparation as claimed in any one of claims 1 to 5, wherein the complex salt (i) is the N-methylglucamine salt of the gadolinium (III) complex of 1,4,7,10- tetraazacyclododecanetetraacetic acid.

8 A diagnostic preparation as claimed in any one of claims 1 to 5, wherein the complex salt (i) is the disodium salt of the manganese (II) complex of trans-1,2- cyclohexylenediaminetetraacetic acid 55 9 A diagnostic preparation as claimed in any one of claims 1 to 5, wherein the complex salt (i) is the sodium salt of the gadolinium ( 111) complex of 1,4,7,10tetraazacyclododecanetetraacetic acid.

A diagnostic preparation as claimed in any one of claims 1 to 5, wherein the complex salt (i) is the sodium salt of the gadolinium (III) complex of 1,4,8,11- tetraazacyclotetradecanetetraacetic acid.

11 A diagnostic preparation as claimed in claim 1, substantiallyas described in any one of Examples 6 to 60 13 herein.

12 A diagnostic preparation asclaimed in anyone of claims 1 to 10,which is in a dosageform suitable for administration orally, neurally or intravasally.

13 An ampoule containing a diagnostic preparation as claimed in any one of claims 1 to 10, in a form suitable for injection 65 GB 2 169599 A 21 14 A processforthe manufacture of a diagnostic preparation as claimed in any one of claims 1 to 10, wherein the complex salt (i) 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 suitable for intravasal or oral administration.

15 A process forthe manufacture of a diagnostic preparation as claimed in claim 1, conducted 5 substantially as described in any one of Examples 6 to 13 herein.

16 A physiologically tolerable, non-radioactive complex salt of the general formula I X-CH 2 CH 2-X 10 N-A-N (I) V-CHR 1 CHR 1-V in which 15 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 57 to 83, or a physiologically tolerable cation of an inorganic or organic base or amino acid, and in which either a) R, represents in each case a hydrogen atom or methyl group, 20 epresents the group -CHR 2-CHR 3 or -CH 2-CH 2-(ZCH 2-CH 2)m-, in which R 2 and R 3 together represent a trimethylene group or a tetramethylene group, or each represents a phenyl radical or benzyl radical, m represents the integer 1,2, or 3, and 25 Z represents an oxygen atom or a sulphur atom, and each V has the same meaning as X, 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 57 to 83, or b) R 1 represents in each case a hydrogen atom, 30 A represents the group -CH 2-CH 2 and both V's together represent the group CH 2 X CH 2 X 1 1 35 -(CH 2)w N-CH 2-CH 2-N-(CH 2)win which X has the meanings given above and W represents the integer 1,2 or 3, 40 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 57 to 83.

17 A physiologically tolerable complex salt as claimed in claim 16, wherein at least one ofthe substituents Y is a physiologicallytolerable cation of an organic base or amino acid.

18 Any one of the physiologically tolerable complex salts specified in any one ofthe Examples herein 45 19 The di-N-methylglucamine salt of the manganese (II) complex of trans-1, 2-cyclohexylenediamine- tetraacetic acid.

The N-methylglucamine salt of the gadolinium (Ill) complex of 1,4,7,10tetraazacyclododecanetetra- acetic acid.

21 The sodium salt of the gadolinium (III) complex of 1,4,7,10tetraazacyclododecanetetraacetic acid 50 22 The sodium salt of the gadolinium (III) complex of 1,4,8,11- tetraazacyclotetradecanetetraacetic acid.

23 A process forthe manufacture of a physiologically tolerable complex salt as claimed in any one of claims 16 to 22, substantially as described herein.

24 A physiologically tolerable complex salt as claimed in claim 16, for use in a method of diagnosis of the human or animal body by NMR diagnosis, X-ray diagnosis or ultra-sound diagnosis 55 A physiologically tolerable complex salt as claimed in claim 17, for use in a method of diagnosis of the human or animal body by NMR diagnosis, X-ray diagnosis or ultra-sound diagnosis.

26 A physiologically tolerable complex salt as claimed in any one of claims 18 to 22, for use in a method of diagnosis of the human or animal body by NMR diagnosis, X-ray diagnosis or ultra-sound diagnosis.

27 The use of a physiologicallytolerable complex saltas claimed in claim 16,forthe manufacture of a 60 preparation for a method of diagnosis of the human or animal body by NMR diagnosis, X-ray diagnosis or ultra-sound diagnosis.

28 The use of a physiologicallytolerablecomplexsaltasclaimed in claim 17, forthe manufactureof a preparation for a method of diagnosis of the human or animal body by NMR diagnosis, X-ray diagnosis or ultra-sound diagnosis 65 22 GB 2 169 599 A 22 29 The use of a physiologically tolerable complex salt as claimed in any one of claims 18 to 22, for the manufacture of a preparation for a method of diagnosis of the human or animal body by NMR diagnosis, X-ray diagnosis or ultra-sound diagnosis.

Printed in the UK for HMSO, D 8818935, 5/88, 7102.

Published by The Patent Office, 25 Southampton Buildings, London, WC 2 A l AY, from which copies may be obtained.