AU623901B2 - Paramagnetic compounds comprising chelating moiety, linker group macro molecule and paramagnetic metal - Google Patents

Description

OPI DATE 25/08/89 AOJP DATE 28/09/89 APPLN. ID 32162 89 PCT PCT NUMBER PCT/EP89/00078 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4: (11) International Publication Number: WO 89/ 06979 A61K 49/00 Al (43) International Publication Date: 10 August 1989 (10.08.89) (21) International Application Number: PCT/EP89/00078 (74) Common Representatives: COCKBAIN, Julian, M.

et al.; Frank B. Dehn Co., Imperial House, 15-19 (22) International Filing Date: 26 January 1989 (26,01,89) Kingsway, London WC2B 6UZ (GB).

(31) Priority Application Number: 8801646 (81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (Euro- (32) Priority Date: 26 January 1988 (26.01.88) pean patent), DK, FI, FR (European patent), GB, GB (European patent), HU, IT (European patent), JP, (33) Priority Country: GB LU (European patent), NL (European patent), NO, SE (European patent), SU, US.

(71) Applicant (for GB only):COCKBAIN, Julian, Roderick, Michaelson [GB/GB]; 27 Ladbroke Road, London Published W11 3PD With international search report, (71) Applicant (for all designated Slates except GB US), NY- COMED AS [NO/NO]; Nycoveien 1-2, N-0401 Oslo 4 (NO), (72) Inventors; and Inventors/Applicants (for US only) KLAVENESS, Jo [NO/NO]; Skoyen Terrasse 15, N-0276 Oslo 2 (NO), RONGVED, Pal [NO/NO]; Hovdens vei 11, N-1457 Hellvik STRANDE, Per [NO/NO]; Nils Bays vei 60, N-0855 Oslo 8 (NO), (54)Title: PARAMAGNETIC COMPOUNDS (57) Abstract There are provided paramagnetic compounds comprising a paramagnetic metal species chelated by a chelating moiety bound by an amide group to a linker group itself bound by an ester group to a macromolecule, wherein said linker group provides a carbon chain of at least 2 atoms between said amide group and said ester group. The novel compounds are particularly suitable as contrast agents, e.g. in m gnetic resonance imaging, d -11 i WO 89/06979 PCT/EP89/00078 1 PARAMAGNETIC COMPOUNDS The present invention relates to macromolecular paramagnetic compounds, to contrast agents containing such compounds and their use in magnetic resonance imaging (MRI) of human and non-human subjects, to A chelating agents for use in the manufacture of such compounds and to the use of such chelating agents and chelates and and salts thereof in therapy and diagnosis.

In MRI, the contrast in the images generated may be enhanced hy introducing into the zone being imaged an agent which affects the spin reequilibration characteristics of the nuclei (the "imaging nuclei", which are generally protons and more especially water protons) which are responsible for the resonance signals from which the images are generated. In this respect it has been found that contrast enhancement results from the u.s of contrast agents containing paramagnetic, superparamagnetic or ferromagnetic species. Fot paramagnetic contrast agents, the enhanced image contrast derives predominantly from the reduction in the spin reectuilibration coefficient known as T 1 or as the spin-lattice relaxation time, a reduction which arises from the effect on the imaging nuclei of the fields generated by the paramagnetic centres.

The use of paramagnetic compounds as contrast Sragents in MRI has been widely advocated and a broad range of paramaqnetic compounds has been suggested in this regard. Thus for example Lauterbur and others have suggested the use of manganese salts and other paramagnetic inorganic salts and complexes (see Lputerbur et al. in "Frontiers of Biological Energetics", volume 1, pages 752-759, Academic Press (1978), Lauterbur in Phil. Trans. R. Soc.

~4'V WO 89/06979 PCT/EP89/00078 2 Lond. B 289: 483-487 (1980) and Doyle et al. in J. Comput. Assist. Tomogr. 5 295-296 (1981)) Runge et al. have suggested the use of particulate gadolinium oxalate (see US-A-4615879 and 'Radiology 147(3): 789-791 (1983), Schering AG have suggested the use of paramagnetic metal chelates,- for example of aminopolycarboxylic acids such as nitriloxtriacetic acid (NTA) N,N,N' ,N'-ethylenediaminetetraacetic acig21 (EDTA) N-hydroxyethyl-N,N',N'-ethylenediaminetriacetic acid (HEDTA) N,N,N' NI I -diethylenetriami''nepentaacetic acid (DTPA) and l,4,7,l0-tetraazacyclod3odecanetetraacetic acid (DOTA) (see for example EP-A-71564, EP-A-130934 and DE-A-3401052) and Nycomed AS have suggested the use of paramagnetic metal chelates of iminodiacetic acids (see E-P-A- 165728) .Many other paramagnetic contrast agents have been suggested in, the lite~rature, for example in EP-A--230893, EP-A-232751, EP-A--292689, RP-A- 255471, EP-A-292689, EP-A-287465, US-A-4687659 and 11086/02005. Besides the chelates of DOTA and DTPA, the chelates of N,N' I (bis methyl-carbamoylmethyl) N1NN I- diethylenetriamine triacetic acid (DTPA- BMA) 1-oxa-4,710 triazacyclododecane N, NI t NO'I-triacetic acid (OTTA) and N-r2,3-dihydroxy- N -m e th y I-p r Qpyl ca rb amoY 1m e th y 1 1, 4 ,7,10 tetra a L4-lododecane- NO ,NO IN' I''-triacetic acid, etc. (DO3A) deserve particular mention.

Paramagnetic compounds in which the paramagnetic centre is bound in a chelate complex have been considered particularly desirable as otherwise toxic heavy mnetals, such as gadolinium for example, may in this way be presented in a biotolerable form. The use of chelating agents, such as EDTA, DTPA, etc., known for their efficacy as heavy metal detoxification agents has thus received particular attention (see for example Weinmann et al. in AJR 142: 619-624 (1984)).

While the toxicities of the paramaS'netic i i unmoor----,,; WO 89/06979 PCTIEP89/00078 3 chelates are generally lower than those of the inorganic salts of the same paramagnetic metal species, the efficiency of such chelate complexes in contrast enhancement is not greatly improved relative to that of the salts.

It has however been found that by*.binding the paramagnetic species to a relatively heavy carrier, for example a macromolecule, increased contrast effect can be achieved, perhaps at least in part due to the effect of the heavy carrier in slowing down tumbling motions of the paramagnetic species. This is well illustrated by Technicare Corporation in EP-A-136812. Binding macromolecules to paramagnetic compounds has also been suggested as a means by which tissue-specific paramagnetic contrast agents can be produced. Thus, for example, Schering AG in EP-A-71564, suggest binding paramagnetic chelates to biomolecules such as hormones, proteins and the like to cause the contrast agent after administration to congregate at particular body sites. Technicare Corporation, in EP-A-136812, similarly suggest binding paramagnetic ions to tissue-specific macromolecules such as, for example, antibodies.

Binding paramagnetic chelates to albumin to produce a blood pooling contrast agent has also been suggested and one such compound, Gd DTPA-albumin, is discussed by Schmiedl et al. in Radiology 162: 205 (1987). Proteins such as albumin are substances of very complicated structure and generally possess limited stability. In particular, protein bound substances are difficult to formulate into solutions and should not be subjected to heat treatment, and thus contrast agents containing such substances cannot be sterilized by the application of heat.

Furthermore, to reduce the risk of allergic response it would generally be appropriate to utilize a human-derived protein, e.g. human albumin, and -r t.+r Lsrl. r. .i rc*kia~''c1 r'fT~* TI LT"(1 I 1 thus a possible risk of viral contamination from the human source arises. Consequently, Nycomed AS, in EP-A-184899 and EP-A-186947, suggested MRI contrast agents comprising paramagnetic chelates associated with thermostable, readily characterized, biologically relatively passive macromolecules such as polysaccharides, e.g. dextrans. Thus EP- A-186947 discloses soluble macromolecular paramagnetic compounds which where they have molecular weights above the kidney threshold may function as blood pooling MRI contrast agents.

Amersham International PLC have also suggested, in W085/05554, the use of macromolecular carriers for paramagnetic chelates for use as MRI contrast agents. However, stressing the importance that the chelate complex must be stable in vivo (in particular where the paramagnetic metal ion itself is toxic) Amersham have suggested that the possibility of the macromolecule sterically hindering chelation of the paramagnetic metal species by the chelating entity may be avoided by binding the chelating entity to the macromolecule through the agency of a linker molecule, for example to produce the compound X-OCONH-(CH2) -NHCO-Y, where X is the macromolecule and Y is the chelating entity. One such chelate-linker-macromolecule compound, GdDOTAglycine-dextran, is also disclosed in EP-A-186947.

Other paramagnetic MRI contrast agents are disclosed in the literature (see for example W087/02893, US-A-4639365 and W087/01594 and the references listed in these documents) and there have been several reviews of paramagnetic MRI contrast agents (see for example AJR 141: 1209-1215 (1983), Sem.

Nucl. Med. 13: 364 (1983), Radiology 147: 781 (1983) and J.Nucl. Med. 25: 506 (1984)).

When a paramagnetic compound is administered into the cardiovascular system of a subject to Sbe imaged, the fate of the compound depends on ZQ U' .1CU;- i'JT- y" T- WO 89/06979 PCT/EP89/00078 a number of factors. If it comprises insoluble particulate matter, it will be removed from the blood stream by the reticuloendothelial system (RES), in rarticular by Yupffer cells of the liver; if it contains relatively large particles, such as liposomes, these may lodge in the lungs; and if the compound is soluble and of relatively low molecular weight it may be cleared out of the blood through the kidneys relatively rapidly (as is the case with GdDTPA-dimeglumine, an agent developed and tested by Schering AG). Thus GdDTPA-dimeglumine has a half life in the blood of about 20 minutes (see Weinmann et al. in AJR 142: 619-624 (1984)).

However for a paramagnetic MRI contrast agent to be suitable as a blood pooling agent, i.e. one which is not rapidly removed from the cardiovascular system, it is necessary that the paramaqnetic compound be soluble, that it should have a molecular weight sufficiently high as to prevent rapid excretion through the kidneys, and that it should have an in vivo stability which achieves a balance between the stability required to ensure adequate half life in the blood pool and the instability required for the compound, or more particularly the paramagnetic species contained therein, to be excretable.

We have now found that by the use of a linker moiety which is bound to the macromolecule hy an ester grouping and to the chelatinq moiety by an amide qrouping and which provides a carbon chain of at least 2 atoms in length between the ester and amide groups, it is possible to provide macromolecular paramagnetic MRI contrast agents with improved properties, in particular for the imaging of the cardiovascular system. More particularly, we have found that by the use of such linker moieties a particularly desirable balance between in vivo stability and in vivo instability is achieved.

Thus in one aspect the presen- invention I

I

0 S' 0 0 e 9 e

S

provides a paramagnetic compound comprising a paramagnetic metal species chelated by a chelating moiety bound by an amide group to a linker group itself bound by an ester group to a macromolecule, wherein said linker group provides a carbon chain of from 2 to 11 atoms between said amide group and said ester group.

The linker group in the paramagnetic compounds of the present invention is preferably the residue of an amino acid of formula I

HOOC-CH

2 -(CHR) -NH2 (wherein, n is an integer of from 1 to and each R, which may be the same or different, represents a hydrogen atom or a hydroxyl, hydroxyalkyl, or C 1 4 alkyl group, with the proviso that R on the carbon attached to the amine group does not represent a hydroxyl group).

In formula I above, n is preferably an integer of from 1 to 6, an especially preferably from 1 to 3, and R is preferably hydrogen, methyl, ethyl, hydroxyl, mono- or poly-hydroxy(Cl 6 alkyl), especially mono-or poly-hydroxy(Cl_ 4 alkyl), for example hydroxymethyl or 2,3-dihydroxy-propyl. Where R is a polyhydroxyalkyl group, the ratio of hydroxyl groups to carbon atoms is preferably up to 1:1. Residues of compounds of formula I in which n is from 1 to 10 and R is hydrogen also are preferred as the linker group in the paramagnetic compounds of the invention.

Particularly preferred identities for the linker group include the residues of beta and gamma amino acids, for example beta-alanine and 4-amino-butanoic acid.

The chelating moiety in the paramagnetic compounds of the present invention may conveniently be the residue of a conventional metal chelating agent. Suitable such agents are well known from qJ pU A4K the literature relating to MRI contrast agents discussed above (see for example EP-A-71564, EP- A-130934, EP-A-186947, US-A-4639365, EP-A-230893, EP-A-232751, EP-A- 292689, EP-A-255471, US-A-4687659, WO-86/02005 and DE-A-3401052) as well as from the literature relating to chelating agents for heavy metal detoxification.

The chelating moiety chosen should clearly be one that is stable in vivo and is capable of forming a chelate complex with the selected paramagnetic species. Preferably however, the chelating moiety will be one as described in EP-A-186947 or the residue of an aminopoly(carboxylic acid or carboxylic acid derivative) (hereinafter an APCA) or a salt thereof, for example, one of those discussed by Schering AG in EP-A-71564, EP-A-130934 and DE-A- 3401052 and by Nycomed AS in International Patent Application No. PCT/GB88/00572. This latter application discloses APCAs which carry hydrophilic groups, e.g. on the amine nitrogens or on the alkylene chains linking the amine nitrogens, for example compounds of formula II

Y

X-CHR -NZ-(CHR) 2-N-(CHR I 2 -NZ-CHRI-X (II) (wherein each of the groups Z is a group -CHRIX or the groups Z are together a group -(CHR)- A'-(CHR1) 2 where A' is O, S, N-CHR X or N-(CHR 1

N(CHR

1

X)

2 where p is 2, 3 or 4 Y is a group -(CHR 1 2

-N(CHR

1

X)

2 or a group -CHIRX; each X, which may be the same or different, is a carboxyl group or a derivative thereof or a group

R

1 each R 1 which may be the same or different, is a hydrogen atom, a hydroxyalkyl group or an optionally sluaSTITUT" "t r" "c~ WO 89/06979 PCT/EP89/00078 8 hydroxylated alkoxy group; with the proviso that at least two nitrogens carry a -CHR X moiety wherein X is a carboxyl group or a derivative thereof, and preferably the provisos that each -CHR 1 X moiety is other than a methyl group, and that where Y and Z are -CHR X groups at least one R 1 is other than hydrogen, and preferably also that each nitrogen atom carrying a -CHR 1

X

moiety wherein X is a carboxyl group or a derivative thereof carries at least one such moiety which is other than a -CH X moiety) and salts thereof.

Particularly preferred as chelating moieties for the paramagnetic compounds of the present invention are the residues of the following: EDTA; DTPA; OTTA; D03A; DTPA-BMA; DOTA; desferrioxamine; and the physiologically acceptable salts thereof, especially DTPA, DOTA and salts thereof.

Where the chelating moiety in the paramagnetic compounds of the present invention has a labile counterion, that counterion should be a physiologically tolerable ion, for example the ion of an alkali metal, a non-toxic amine (for example tris(hydroxymethyl)aminomethane, ethanolamine, diethanolamine and N-methylglucamine) a halogen, or a non-toxic organic or inorganic acid.

As the macromolecule component of the paramagnetic compound of the present invention there can be used any of the macromolecules previously suggested for macromolecular paramagnetic MRI contrast agents.

Preferably, the macromolecule chosen will be one which is physiologically tolerable and which contains hydroxyl groups or which can be chemically modified to introduce hydroxyl groups or to deprotect protected hydroxyl groups.

Particularly preferably, the macromolecule will be a hydroxyl roup containing material selected from the group consisting of polymeric and polymerized WO 89/06979 PCT/EP89/00078 9 carbohydrates and polymerized sugar alcohols and derivatives thereof. The term "polymeric carbohydrate" is used to desionate a naturally occuring polymer built up of carbohydrate monomers and the term "Polymerized carbohydrate" is used to designate a synthetic~ polymer obtained by polymerizingi carbohydrate molecules, for example with the aid of coupling or cross-linking ae,7nts. Similarly the term "polymerized sugar alcohol" is used to designate a synthetic polymer obtained by polymerizing sugar alcohol molecules, for example with the aid of coupling or cross-linking agents., The macromolecule may thus conveniently be a cyclic or acyclic Polysaccharide, such as a glucant for example starch, amylose, amylopectin (including macromolecular dextr ins thereof) glycogen, dextran and pullalan, or a fructan, for example inulin and levant cyclodextrIne or other physiologically tolerable polysacchar ides of I vegetable, microbial or animal origin.

Examples of poly-merized carbohydrates or sugar aleuohols which can be used as the macromolecule include so-called polyglucoset which, is obtained by polymerization of glucose, and macromolecular products obtained. by cross-likinq carbohydrates or sugar alcohols (for example mannito. or sorbitol) with at least one bifunctona. cross-linking agentt for example enichlorohydrin, a diepoxide or a corresponding halogen hydrin or with a bifunctional acylating agent., An example of such a product which is commericatly available is VicoJll (FiColl Is a Trade m..ark of Pharmacia 'wine Chemicals AB of Uppsal.a# Sweden) which is obtained by cross-linking sucrose with the, aid of epichlorohydrin, Further examples of substances which can form the hasis for the macromolecule include physiologically tolerable derivatives of the polysacchatifts mentioned above# for example hydroxylo carboxcvalkyl, *4 ,*1*Ot44~~4* 4W~ t Me ~t*M.tM *4* WO 89/06979 PCT/EP89/00078 acyl or alkyl derivatives, for example hydroxyethyl, dihydroxypropyl, carboxymethyl, acetyl and methyl derivatives of such polysaccharides.

Water-soluble derivatives of insolubfe polysaccharides (for example of cellullose) may be considered as well as the water-soluble..macromolecules mentioned 'bove. Many such macromolecules are commercially available and/or are extensively described in the literature.

Although the paramagnetic compounds of the invention are particularly suited for use as blood pooling agents when the compounds are soluble and have molecular weights above the kidney threshold, lower molecular weight paramagnetic compounds of the invention may be used in other MRI contrast agents, e.g. agents for investigation of the kidneys, bladder or gastrointestinal tract.

The macromolecule will generally be chosen according to the intended use of the macromolecular paramagnetic chelate. If for example the chelate is to be used in investigation of body cavities having outward escape ducts, for example the gastrointestinal tract, the bladder and the uterus, the macromolinalen need not be biodegradable. Furthermore where the chelate is intended for parenteral administration, the macromolecule again need not be biodegradable as long as its molecular weight is sufficiently small as to allow its excretion into the urine.

However, where the chelate is to be used in a blood pooling agent it is desirable either to use biodegradable macromolecules whose molecular weights exceed the kidney threshold or to use macromolecular compounds for which each molecule contains more than one macromolecule, for example compounds having a macromolecule-linker-chelate-linker-macromolecule structure.

Where a biodegradable macromolecule is to be used, these may for example be macromolecules which are enzymatically degradable by hydrolyses, for example i.e if tw4~M4 1 1 WO 89/06979 PCT/EP89/00078 11 endohydrolases which hydrolyze glycosidic linkages in the macromolecule. Thus for example macromolecules degradable by alpha-amylase, for example starchbased macromolecules, may be chosen.

The macromolecules used for the paramagnetic compounds of the invention may be neutral or may have a net negative or positive charge in solution.

For parenteral use, macromolecules with no net charge or with a negative net charqe in solution are preferred. A negative net charge may be obtained for example by introducing carboxyl groups or other negatively charged groups into the macromolecules if such groups are not already present therein.

it is particularly preferred that the macromolecule in the compounds of the invention be a polysaccharide and especially preferably a dextran or a derivative thereof, particularly are having a weight average molecular weight of from 40,000 to 500,000 especially about 70,000.

mhe molecular weight of the paramagnetic compounds of the present invention can easily be selected to suit the particular end use for the compound. As indicated above, this may be done either by selection of appropriately sized macromolecules or by linking together two or more macromolecules to produce the final compound. For general diagnostic purposes, the weiqht average molecular weight of the paramagnetic compound is preferably in the ranqe of 1,000 to about 2,000,000, preferably 3,000 to about 2,000,000. For the preparation of such paramagnetic compounds, macromolecules of the desired molecular weight can be obtained by conventional methods.

Where it is desired that the paramagnetic compound should be excretable into the urine without prior degradation, the molecular weight is preferably less than 40,000, fot example less than 30,000 or more particularly less than 20,000. Where however I I 1 W, 4 *ne. P- I WO 89/06979 PCT/EP89/00078 12 the paramagnetic compounds of the present invention are to be used as blood pooling agents, the use to which they are particularly well adapted, the molecular weight of the paramagnetic compound should preferably lie in the range 40,000 to 2,000,000, more preferably 40,000 to 150,000, and,especially preferably 60,000 to 100,000. Where the paramagnetic compound comprises a single macromolecule residue, the molecular weight range limits listed above may be considered to be the appropriate range limits for the molecular weight of the macromolecule also.

In the paramagnetic compounds of the present invention, the paramagnetic metal species, i.e.

a paramagnetic metal atom or ion, is preferably non-radioactive and is particularly preferably selected from the group of elements having atomic numbers 21-29, 42, 44 and 57-71, the elements having atomic numbers 24-29 or 62-69 being specially preferred.

Examples of'suitable lanthanides include gadolinium, europium, dysprosium, holmium, and erbium and examples of other suitable elements include manganese, iron, nickel, chromium and copper. The particularly preferred paramagnetic metal species include Cr (II), Mn(II) r e(II), y (III and Gd(III), especially Gd and Dy and Cr.

In a further aspect, the present invention provides a process for the preparation of the macromolecular paramagnetic compounds of the present invention, which process comprises admixing in a solvent an at least sparingly soluble paramagnetic metal compound, for example a chloride, oxide or carbonate, together with a macromolecular chelating agent comprisig a chelating moiety bound by an amide gYoup to a linker group itself bound by an ester group to a macromolecule, wherein said linker group provides a carbon chain of at least two atoms between said amide qroup and said ester group.

The macromolecular chelating agent mentioned

L

I I I n^ 0Aon 7nno WO 89/06979 -/Lo/uo 13 in the previous paragraph itself represents a further aspect of the present invention.

Thus in a still further aspect the present invention provides a macromolecular chelai'ng compound comprising a chelating moiety bound by an amide group to a linker group itself bound by*-an ester group to a macromolecule, wherein said linker group provides a carbon chain of at least two atoms between said amide group and said ester group, or a salt or metal chelate thereof.

The macromolecular chelating agent can itself he prepared by condensing a hydroxyl group containing macromolecule with an amino acid or a salt thereof and reacting the product so obtained with a carboxyl group-, or reactive carboxyl derivative-, containing chelating agent. Thus in a yet still further aspect the present invention provides a process for the preparation of a macromolecular chelating agent according to the present invention which process comprises: reacting a hydroxyl group containing macromolecule with an amino acid or a salt thereof, said amino acid having a carbon chain of at least two atoms between its carboxyl and amine groups, and conveniently being an amino acid of formula I as defined above; reacting the product so obtained with a carboxyl group-, or reactive carboxyl derivative-, containing chelating agent; and, optionallyconverting the product so obtained into a salt or metal chelate thereof.

Where the paramagnetic compounds of the present invention are to be administered to the human or non-human animal body as M.I contrast agents, they will conveniently be formulated together with one or more pharmaceutical carriers or excipients.

Thus in a further aspect of the present invention provides a diagnostic contrast medium comprising a macromolecular paramagnetic compound according l l r' l|M^ 14* a. to the present invention together with at least one pharmaceutical carrier or excipient.

The chelating agents and the salts and chelates according to the invention are also useful in other fields in which chelating agents and chelates have been used, for example as stabilizers for pharmaceutical preparation, as antidotes for poisonous heavy metal species and as diagnostic agents for the administration of metal species atoms or ions) for radiotherapy or for diagnostic techniques such as X-ray, and ultrasound imaging or scintigraphy. In addition the paramagnetic compounds may also be useful in techniques such as lymph angiography. In a further aspect therefore the present invention provides a diagnostic or therapeutic composition comprising at least one pharmaceutical carrier or excipient together with a metal chelate whereof the chelating moiety is the residue of a chelating compound according to the invention.

In a still further aspect the present invention also provides a detoxification agent comprising a chelating compound according to the invention, optionally in the form of a salt or chelate with a physiologically acceptable counterion, together with at least one pharmaceutical carrier or excipient.

The compositions, e.g. contrast media of the present invention may include conventional formulation aids, for example stabilisers, antioxidants, osmolality adjusting agents, buffers, pH adjusting agents, etc. and may be in forms suitable 4or parenteral or enteral administration, for example injection or infusion or administration directly into a body cavity having an external escape duct, for example the gastrointestinal tract, the bladder or the uterus. Thus the compositions of the present invention may be in a conventional pharmaceutically administration form such as a tablet, capsule, powder, solution, suspension, 1 A U Y^ m ili ,ll ri t I WO 89/06979 PCT/EP89/00078 dispersion, syrup, suppository, etc; however, solutions, suspensions and dispersions in physic&a .oally acceptable carrier media, for example water for injections, will generally be preferred.

Where the compositions of the invention contain a chelate of a toxic metal species e.g..a heavy metal or radioactive metal ion, it may be desirable to include within the composition a slight excess, e.g. 0.5 to 20 mo preferably 1 to 10 mol %,of the chelating compound or of a weaker chelate thereof with a physiologically tolerable counterion, e.g.

as discussed by Schering AG in DE-A-3640708(and AU-A-81889/87) Where the composition is formulated for parenteral administration, for example where a contrast medium is to be used as a blood pooling agent, a solution in a sterile physiologically acceptable medium, for example an isotonic or somewhat hypertonic aqueous solution would be preferred.

For MRI examination, the contrast medium of the present invention, if in solution, suspension or dispersion form, will generally contain the paramagnetic metal species at a concentration in the range I micromole to 1.5 mole per litre, preferably 0.1 to 700 mM. The contrast medium may however be supplied in a more concentrated form for dilution prior to administration. The contrast medium of the invention may conveniently be administered in amounts of from 0 4 to 3 mmol e.g. 10 3 to 1 mmol of the paramagnetic metal species per kilogram of body weight, e.g. about 1 mmol Dy/Kg bodvweiqht.

For X-ray examination the dose of the contrast agent should generally be higher and for scintigraphic examination the dose should generally be lower than for MR examination. For radiotherapy and detoxification conventional doses may be used.

In a yet further asoect, the present invention also Provides a method of diagnosis practised on the hudan or non-human animal body, which method comprises administering to said body a macromolecular iI ll-C.L* .I r. I 2I LI I- WO 89/06979 PCT/EP89/00078 16 metal chelate, preferably a paramagnetic compound, according to the present invention and generating an X-ray, magnetic resonance, ultrasound or scintigraphic image of at least part of said body.

In a still further aspect the invention provides a method of heavy metal detoxification-practised on the human or non-human animal body, which method comprises administering to said body a chelating compound according to the invention, optionally in the form of a salt or chelate with a physiologically acceptable counterion.

In a yet still further aspect the invetnion also provides a method of radiotherapy practised on the human or non-human animal body, which method comprises administering to said body a chelate of a radioactive metal species with a chelating compound according to the invention.

In a still further aspect, the present invention also provides the use of a macromolecular compound or salt or chelate thereof according to the invention for the manufacture of a diagnostic agent for use in methods of image generation, detoxification or therapy practised on the human or non-human animal body.

As mentioned above, as a result of the use of the particular linker groups, the paramagnetic compounds of the present invention have properties which are particularly improved relative to those of the prior art compounds.

Thus where the paramagnetic chelate GdDTPA is bound directly to dextran, the resulting compound is not stable either in vivo or in vitro. On administration of such a compound, GdDTPA-dextran (molecular weight 70,000) to rabbits, no blood pooling effect was observed and the rapid elimination of the gadolinium into the urine that was observed was very similar to that which is observed for GdDTPA or its salts.

In contrast, GdDTPA linked by beta-alanine to dextran I WO 89/06979 PCT/EP89/00078 17 of molecular weight 70,000, a compound according to the present invention, is stable in vitro and has almost ideal blood pooling properties insofar as it exhibits a half life in the blood of "bout 6 hours and has a distribution volume of 0.05 1/kg, a distribution volume which indicates that at least until degradation the distribution of the compound is essentially only within the blood pool.

Nevertheless, the increased blood pooling effect achieved using the amino acid residue linker is not obtained at the expense of ready excretability of the paramagnetic species due to the presence in the paramagnetic compound between the macromolecule and the linker of an ester bond which, unlike the essentially non-biodegradable amide bonds in the macromolecule-linker-chelate compounds of WO-85/05554, is biodegradable.

The disclosures of all of the documents mentioned herein are incorporated by reference.

The following Examples are provide to illustrate the present invention in a non-limiting manner.

The products of Examples 1 and 14 are however particularly preferred. The following abbreviations are used herein: Dextran X: Dextran with molecular weight X. 10 daltons (such dextrans are available from Sigma Chemicals) DMSO-A: dimethylsulfoxide DTPA-A: diethylenetriamine pentaacetic acid bisanhydride ECDI: N-ethyl-N'-(3-dimethylaminopr'opyl) carbodiiinide FMOO-BA: fluorenylmethyloxycarbonyl-beta-iaanine PP: 4-pyrrolidinopyridine Water: water deionized by reverse osmosis WO 89/06979 PCT/EP89/00078 18 Example 1 GdDTPA-beta-alanine-dextran (Molecular Weight 70,000) To a solution of 15.9 g of Dextran 70 in 650ml of dry DMSO was added 20.3q of FMOC-BA,-,13.7g of ECDI and 968mg of PP dissolved in 350ml of dry DMSO. The reaction mixture was stirred at ambient temperature for 18 hours and 43.1g of piperidine was added. After 70 minutes, 7.Jml of concentrated hydrochloric acid was added dropwise, and cooling on an ice/water bath and dropwise addition of 1.7 1 of an ether/chloroform mixture (7:3 w/w) yielded a yellow oil. After decantation, the oil was dissolved in distilled water and the pH was adjusted to 4. Sodium chloride was added until the salt concentration was 0.9% in 1400ml of solution, and the product was dialyzed against 0.9% sodium chloride in water at pH 4 in a hollow fibre cartridge (Amicon HP 10-20) for 24 hours. The solution was then concentrated using the same equipment against distilled water to a volume of 1150 ml, the pH was adjusted to 9 with N-Tmtthylmorpholine and 29.18g of DTPA-A was added while the pH was kept at 8 using the same base. When the solution became clear, the reaction mixture was stirred for 2 hours, 43.78g of citric acid dissolved in 47.4ml of 10 N NaCO was added, and the pH was adjusted to 6.0 with concentrated hydrochloric acid. 30.37g of gadolinium chloride hexahydrate dissolved in 200ml of distilled water was added quickly and the pH was adjusted to 5.5 using 10 N NaOH. The solution was dialyzed against distilled water until the relaxation time T 1 (determined using a NMR Proton Spin Analyzer, RADX Corporation, Houston, Texas, USA, at 10 MHz and 37 0 C) was above 2000 ms. Lyophilization of the solution yielded 15.3g of a light yellow coloured powder.

WO 89/06979 PCT/EP89/00078 19

ANALYSIS

Elemental analysis: Gd N 2.15%; Na 0.16%; Cl less than 0. b1%.

Free Gd (xylene oranqe titration), DTPA,, GdDTPA, citric acid, or DMSO (HPLC): less than 0.01% (The percentages in the analysis results are by weight).

The specific relaxation rate (T 1 enhancement (SRRE) (measured in an NMR Proton Spin Analyzer RADX Corp.

Houston, Texas, USA at 10 MHz and 37 0 C) in distilled water w4s 9.6 s mM Gd.

Example 2 Iniection Solution 78.6mg of gadolinium (III) DTPA-beta-alanine-dextran (molecular weight 70,000) were prepared in accordance with Example 1 and dissolved in 10ml of distilled water. The solution was sterile filtered and filled into a 10ml vial. The solution contained 0.05 mmol Gd/ml.

Example 3 Pharmacokinetics in Rabbits The solution of Example 2 was injected intravenously into three rabbits at a dose of 0.05mmol Gd/kg body weight. Three other rabbits received gadolinium (III)DTPA-dimeglumine salt intravenously at a dose of 0.05 mmol rd/kg body weiqht.

e WO 89/06979 PCT/EP89/00078 Blood samples were drawn from an ear vein before injection and at 1, 5, 10, 15, 30, 120, 180 and 300 minutes and 24 and 48 hours after injection.

Serum was prepared from the blood samples a~id "the relaxation times T 1 and T2 were determined in an RADX NMR. spectrometer (37 0 C, 10 MHz). The gadolinium concentration in the serum samples were determined by TCP (inductive coupled plasma). The apparent volume of distribution (VD) and the biological half-life (tI) were determined using the two-compartment model. The results are as set forth in the Table below:

TABLE

SAMPLE V tI (1/kg) (hours) Gd III) DTPA-betaalanine-dextran 70 0.05 0.003 6.7 0.18 Gd(III)DTPA-dimeglumine 0.26 0.037 0.72 0.11 The results presented above show the compound of Example 1 to have a considerably longer half-life than GdDTPA and yet still to be biodegradable as no relaxation effects an8 no serum gadolinium were observed in serum 48 hours after iniection. The observed apparent volume of distribution of 0.05 confirms its blood pooling property.

21 Example 4 Dextran 10.0 g of Dextran 70 was reacted with 12.8 g of FMOC-BA, 8.7 g of ECDI, 0.61g of PP and 31.5 ml of piperidine in 600 ml of dry DMSO and then with 22g of DTPA-A as described in Example 1 to the point where the citric acid buffer was added. The pH was adjusted to 5.1 with 6M HCl and the solution was dialyzed against 4 8 of water. The i solution was lyophilized to give 5.5 g of a light yellow solid.

Elemental analysis S* N 2.82 C 42.52 H 6.74 Example Dextran 70-beta-alanine-DTPA-Fe (III) 0.5 g of the product of Example 4 was dissolved in 70 ml of water and to this were added 1.38 g of citric acid, 1.49 ml of 10 N NaOH and 417 mg of FeCl 3 dissolved in 10 ml of water. The pH was adjusted to 5 With 10 N NaO and, after reaction overnight, the solution was dialyzed against water until T 1 in the filtrate was above 2000 ms.

Lyophilization gave 0.47 g of a light brown solid, Fe, relaxivity 0.8 s" 1 mM" i Example 6 Dextran g of the product of Example 4 was complexed with 1.1 g ,fA of DyC1 3 and isolated as described in Example 5. Yield of S 0.57 g of a white solid, S98 Oy, relaxivity 0.2 smM' 1

T;

b Example 7 -2 Dextran g of the product of Example 4 was complexed with 1.15 g of Yb(N03) 3 and isolated as described in Example Yield 0.5 g of a yellowish solid, 3.5% Yb, relaxivity Examnle 8 Dextran V g of the product of Example 4 was complexed with 642 mg of CuS0 4 and isolated as described in Example Yield 0.55 g of a liqht blue solid, 1,5 Cut relaxivity 4 0.3 s*NIn14".

d xample 9 Dextran 4 2,0 g of Dextran 40 was reacted with 2.6 g of FMOC-BA,j 1.73 q of ECDX, 122: mq of PP and 6.3 ml, of piperidine in dry OMSO as described in Examnple 1. The product was reacted further with 3.77 qj of DTPA-A as described herein, and after comnplexation in oitrate buffer with 3.82 g of GdCl 3 .6 1420 the product was dialysed and. lyophilized to yield 1.05 g of a white. solid, 5,1% Gd, rela~ivity tHyg.vthvl stp rch.=beta -a Ia n inhe TPA -C!4 q of hydroxyethylstarch (prepared by hycftoxyethylation of waky starch with ethylene ocide according to the method, -23 weight 131000 and degree of substitution 0.52, was dissolved in 120 ml of dry DMSO. it was reacted with the same reagents in the same quantities and isolated as described in Example 9. Yield 2.3 g of white solid, 5.1% Gd, relaxivity 6.0 s"MM- 1 Example 11 Dextran g of Dextran 40 was reacted as described in Example 9 GO% Up to the point where the Dextran 40-beta-alanine water :~:solution had been dialysed, at pH- 4,2. 2,65 g of EDTA-bisanhydride (prepared Using the method of EckeJlman etal J Pharm. Sci. 6- (1975) 704) was reacted with the Dextranderivative, copleXed With 2, 74 g of Cr01 39 f product was isolated as described In Excampl.e 9, Yield 2.9 g off a purple Solid, Cr, relaXiVity 1,1 S" 1 mwl.

EXamP1e 12

GOP*

De ora D0-etan 5alaWni ectedasdsciediBiapl 6*6 .0go D~rw 0 as reactedy isolig .8 af deibe in E00 mpl of 9 ecepat tht pH4 ofW- as used.te to7ih ratidaoni Iinr maerymorholiseine Th 1IWas henifugeded toe wiprnthnt was decanted oft, The precipitate Was resuspended and cerhtriruged twice and the white jelly-iliko precipitate Was WO 89/06979 PCT(EP89100078 24 added to the buffer solution of the Dextran. The pH was 5.0 and, after reaction overnight, the clear solution was dialysed against 12 1 of water and lyophilization yielded 0.8 g of a white s611.d, 9,4 Bi.

Example 13 Dextran 2000-.beta-ala."ire-H~tDTPA-Gd The DTPA-derivative 3f6?9-tris-carboxymethyl- 4-(2-hydroxyethyl)- 3,6f9-triazaundecane diacid (EtEtDTPA) was synthesized according to the method of PCT/G888/00572. H~ftDTPA trihydrochioride was prepared by loading HEtQTPA on a strong anion ion exhanger and eluting with 1 11 UC, followed by evaporation.

The product was a white solid, inp. qreater than 350 0 C (decomp.).

Elemental Analysis Calc,; C 35i 14% H 5.54% N'.7.69% C1 l9,45t Found; Q 34.76% H 5.40% N 7.74% C1 19.56% 2.0 ci of DeXtran 2000 was reacted as in Example 9 to the point before reaction O)TPA-A. The solution was lyophiliz~ed to yield 1.5g of a white solid.

The product was dissolved in 200 ml of dry DMSO and there were added 2,2,4g of flEtD'rPA trihydrochloride# 0.86 9 of ECDI and 35 mg of PP'. After stirring for 24 hours the solution was added to a miXture of 300 nml of ether and 1,25 ml, of CHCI 3 The product was isolated by decantation of the supernatant.

The product was dissolved In 1,20 ml of water and the PHl was adjusted to 5 ;W.ith 10 NU NaOrI. To the solution was added a buffer, containing q of citric acid and 5.96~ ml of 1.0 N Na(CI# and then 1.53 a of GdCl 3 6Rl 2 0 dissolved in 10 ml of Water.

TXhe PH was adjusted to 5 with 10 N NaOR and after WO 89/06979 PCT/EP89/00078 3 hours the product was dialyzed and isolated as described in Example 9. Yield 2.5 g of a light brown solid, 5.7% Gd, relaxivity 1.4 s~ mm- Example 14 Dextran N',N -Tetracar boxymethyl-1, 4 7,10tetraazacyclododecane (DOTA) 5.26 g of 1,4,7,10-tetraazacyclododecane (prepared as described by Stetter et al. Tetrahedron, 37(1981)767) was dissolved in 50 ml of water, The p-I was adjusted to I0 with conc. ffBr, 20.16 g of bromoacetic acid was dissolved in 7 ml of water and a solution of LiOH carefully added with cooling in an ice/water bath. The br omoacetic acid lithium, solutlon was added to the 1,4,7,10 -tetraazacyclododecan-e solution in one portion, The pH was kept between 8 and with 4 N LIQfl while the temperature was gradually increased to 80 *C during 4 hours. After cooling, the solution was mixed with 494 ml1 of wet Dowex 4 acidic ion eXchange resin, in 1.5 1 of water and stirred for 1 hour, After thorough washing with water, the gel was washed with 2x7S0 tl of saturated ammonia. The filtrate was evaporated to yield 1.9 9 of a white solid, mp greater than 350 0 Qt FAB-ms M+1 411 and 417 -mono- and di-lithlum salt. ~C-an1 Hf-NMR contirmed the structure.

8.72 g of the solid was dissolved in J.6 ml of water and the pH was adjusted to 2.5 wtth conc. IfCl.

The white solid was filtered off and the process repeated with the, evaporated filtrate. The collected solids were dr~ied to yield 4.5 g of, a white solid, nip greater than 350 *c (decompl mannummmmmm 26 b) Dextran 70-beca -alanine-D TAGd 9* 9.

9 a 9 9 0 9* I 9r 9 9*

I

S

4*,1j g of Dextran 70 was reacted as described in Example 9 to the point before DTPA-A was reacted. The product was lyophilized and dissolved in 100 ml of dry DMSQ.

1.66 g of the precipitated DOTA, 0.86g of ECDI and 62 mg of PP were added and the reaction mixture was stirred overnight at ambient temperature. To the reaction mixture was added a mixture of 150 m! of ether and 62 ml of CHC1 3 the white precipitate was isolated by decantation and washing with ether and then dissolved in 80 ml of water. The pH was adjusted to 5 with 10 N NaOH and there was added a mixture of 5.5 g of citric acid and 5.96 ml of 10 N NaQOH and then 0.766 g QdC13,6H a0.

The reaction mixture was stirred for 50 hours and the product was isolated by dialysis and lyophilization.

Yield 2.4 g of a white solid, 7.4 Gd, relaxivity 11. 7 s'IMM 1 Example Dextran70-5-aminopentanoic acid-DTPA-Gd 5.65 g of acid (prepared from 5-aMiio-valeric acid and 9-fluorenylmethyl ohlorformate as dcscribed by Carpino et al.

J. Org. Chem., 27 (197w) 34041 was reacted with 2.0 q of Dextran 70, 3.5 g of EcDJ and 0.5 q of PP as described in Example 9. The reaction mixture was treated with 12.75 mi of piperidine, the product was tsolated and dissolved in water and reacted with 7.44 g of DTPA-A as described herein. The product was complexed with 7.74 g of GdC, 3 6;40 in citrate buffer and isolated by dialysis and lyophilization as described abovG. Yield 9 66y of a light brown solid, 11.4 k Gd, relaxivity 5.6 st M41* 7 :i.fr i/ -27 Example 16 Glvcogfen-beta-a lanine-DTPA-Gd g of bovine liver glycogen (Sigma Chemicals) was reacted and isolated as described in Example 9. Yield 2.7 g of a white solid, 7.5% Gd, relaxivity 6.8 s- im1.

Example 17 Vial containing~ A vial is filled with 20 mg of DTPA (Example 4) and 0.2 mg of Sn(II)C1 2 as a dry solid, .9 A solution of 99 '"Tc as pertechnetate in 0.9%strl *sodium chloride should be added before use. The technetium chelate with Dextran7o-beta--alanine-DTPA is 0 0 for intravenous or sub~cutaneous administration and is a 996* contrast agent for the vascular system or for S Lymphangiography.

0 0.

Example 18 DextranOrbeta-alanine-DTPA-Gd and the Calcium-disodium salt of 760 mg of DeXtran 70-beta--alanine-DTPA (Example 4) was d~issolved in 2.0 Ml water and 28 mg of Ca(OH) 2 Were added.

The pH was adjusted with NaO1i under ambient conditions.

I. ml of the resulting solution Was added to a solution of 1.0 g of Dextran 70-beta-a.anIne-TPA-Gd (Example 1) in 9 ml of water, and the resultant solution was sterile filtered, filled into a 20 ml vial and lyophilized.

tl 0 WO 89/06979 PC-T/EP89/00078 28 Example 19 -beta- alanine-DrPA-Gd and the ca1lciumtrisodium salt of DTPA To a solution of 1.0 g of Dextrzan7O-beta-alanine- DTPA-Gd (Example 1) in 10 ml of water was added 17 mg of the calciuim-trisodium 6alt of £rTPA (Fluka).

The solution was sterile filtered, filled into ml vial anO lyophilized.

Claims (22)

1. A paramagnetic compound comprising a paramagnetic metal species chelated by a chelating moiety bound by an amide group to a linker group itself bound by an ester group to a macromolecule, wherein said linker group provides a carbon chain of from 2 to 11 atoms between said amide group and said ester group.

2. A compound as claimed in claim 1 wherein said linker group is a residue of an amino acid of formula I HOOC-CH 2 (CHR) n-NH2 (I) (wherein, n is an integer of from 1 to and each R, which may be the same or different, represents a hydrogen atom or a hydroxyl, C1-6 hydroxyalkyl, or CI_ 4 alkyl group, with the proviso that R on the carbon attached to the amine group does not represent a hydroxyl group).

3. A compound as claimed inclaim 2 wherein in formula I n is an integer of from 1 to 10 and R represents a hydrogen atom.

4. A compound as claimed in claim 1 wherein said linker group is a residue of a beta or gamma amino acid. A compound as claimed in claim 1 wherein said linker group is a residue of beta-alanine.

6. A compound as claimed in any one of claims 1 to 5 wherein said chelating moiety is a residue of an amino poly(carboxylic acid or carboxylic .1 j 0 4 a S- acid derivative).

7. A compound as claimed in any one of claims 1 to 6 wherein said chelating moiety is a residue of a compound of formula II Y X-CHR 1 -NZ-(CHRI) 2 -N-(CHR 1 2 -NZ-CHRI-X (II) (wherein each of the groups Z is a group -CHR 1 X or the groups Z are together a group -(CHR 2 A'-(CHR1) 2 where A' is 0, S, N-CHR X or N-(CHR)p N(CHRIX) 2 where p is 2, 3 or 4 Y is a group (CHR 1 2 -N(CHR X) 2 or a group -CHR 1 X; each X which may be the same or different, is a carboxyl group or a derivative thereof or a group R 1 Each RI, which may be the same or different, is a hydrogen atom, a hydroxy&1ikyl group or an optionally hy? ylated alkoxy group; with the proviso that at least two nitrogens carry a -CHR X moiety wherein X is a carboxyl group or a derivative thereof) or a salt thereof.

8. A compound as claimed in any one of claims 1 to 7 wherein said chelating moiety is a residue of a compound selected from DTPA, DOTA, and salts thereof.

9. A compound as claimed in any one of claims 1 to 8 wherein said macromolecule is a hydroxyl group containing material selected from the group consisting of polymeric and pol.merized carbohydrates and polymerized sugar alcohols and derivatives thereof. UBSTITUTE SHEET 31 A compound as claimed in claim 9 wherein said macromolecule is a polysaccharide.

11. A compound as claimed in claim 9 wherein said macromolecule is a dextran or a derivative thereof.

12. A compound as claimed in claim 11 wherein said macromolecule has a weight average molecular weight of from 40,000 to 500,000.

13. A compound as claimed in any one of claims 1 to 12 wherein said paramagnetic metal is of atomic number 21-29, 42, 44 or 57-71.

14. A compound as claimed in any one of claims 1 to 13 wherein said paramnagnetic metal is Gd, Dy or Cr. A di,~nostic contrast medium comprising a macromolecular paramnagnetic compound as claimed in any one of claims 1 to 14 together with at least one pharmaceutical carrier of excipient.

16. A process for the preparation of a macromolecular paramnagnetic compound as claimed in any one of claims 1 to 14, which process comprises admixing in a solvent an at least sparingly soluble paramagnetic metal compound together with a macromolecular chelating agent comprising a chelating moiety bound by an amide group to a linker group itself bound by an ester group to a macromolecule, wherein said linker group provides a carbon chain of from 2 to 11 atoms between said amide group and said ester group.

17. A process for the preparation of a diagnostic medium as claimed in claim 15, said process comprising admixing a macromolecular paramagnetic compound ti H 9. 9, i r 9 as claimed in any one of claims 1 to 14 together with at least one pharmaceutical carrier or excipient.

18. A macromolecular chelating compound comprising a chelating moiety bound by an amide group to a linker group itself bound by an ester group to a macromolecule, wherein said linker group provides a carbon chain of from 2 to 11 atoms between said amide group and said ester group, or a salt or metal chelate thereof.

19. A chelating compound as claimed in claim 18 wherein at least one of said chelating moiety, said linker group and said macromolecule are as defined in any one of claims 2 to 12. A process for the preparation of a chelating compound as claimed in any one of claims 18 and 19 which process comprises reacting a hydroxyl group containing macromolecule with an amino acid or a salt thereof, said amiio acid having a carbon chain of at least two atoms between its carboxyl and amine groups; reacting the product so obtained with a carboxyl group-, or reactive carboxyl derivative- containing chelating agent; and, optionally, converting the product so obtained into a salt or metal chelate thereof.

21. A diagnostic or therapeutic composition comprising at least one pharmaceutical carrier or excipient together with a metal chelate whereof the chelating entity is the residue of a chelating compound as claimed in either one of claims 18 and 19.

22. A detoxification agent comprising a chelating compound as claimed in either one of claims 18 and 19, optionally in the form of a salt or chelate SUBSTITUTE SHEf II 33 with a physiologically acceptable counterion, together with at least one pharmaceutical carrier or excipient.

23. The use of a macromolecular compound or a salt or chelate thereof as claimed in any one of claims 1 to 14, 18 and 19 for the manufacture of a diagnostic or therapeutic agent for use in a method of image generation, detoxification, or therapy practised on the human or non-human animal body.

24. A method of image generation, which method involves administering to a human or non-human animal body a macromolecular metal chelate as claimed in any one of *1 claims 1 to 14, 18 and 19 and generating an X-ray, magnetic resonance, ultrasound or scintigraphic image of at least part of said body. S: 25. A method of heavy metal detoxification practised on the human or non-human animal body, which method comprises administering to said body a chelating I acompound as claimed in either of claims 18 and 19, optionally in the form of a salt or chelate with a physiologically acceptable counterion.

26. A method of radiotherapy practised on the human or non-human animal body, which method comprises administering to said body a chelate of a radioactive metal species as claimed in either of claims 18 and 19.

27. Macromolecular chelates or chelating agents substantially as hereinbefore described in any one of the Examples. nATED this 27th day of February 1992. NYCONED AS By Their Patent Attorneys 44 ji i I f INTERNATIONAL SEARCH REPORT International Application No PCT/EP 89/00078 I. CLASSIFICATION OF SUBJECT MATTER (it several classiinc~tion symbols apply, Indicate all) According to International Patent Classification (IPC) or to both National Classification and IPC IPC 4:A 61 K 49/00 I1, FIELDS SEARtCHED Minimum Documentation Searched Clasairication System jClasalIncatlon Symbols IPC 4A 61 K Documeint-i'1fl Searched other than Minimum Documentation to the Extent that auch Documenta are Included In the Fields Searchedi I Ill, DOCUMENTS CONSIDERED TO 8E RELIVANT' Category *I Citation ot Document, 11 with Indication, where appropriate, of the relevant passages 12 Relevant to Clalm No. 11 A WO, A, 87/02893 (UNIVERSITY OF TEXAS) 1-28 21 May 1987 see page 21 A EP, At 0184899 (NYEGAARD) 1-28 18 June 1986 see pages 5-6 A WO, A, 85/05554 (AMERSHAM4, 1-28 19 December 1985 see page 4; claims 1-9 cited in thie application A WO, At 86/02352 (UNIVERSXTY OF TEXAS) 1-28 24 April. 1986 see page 3, A US, A, 4124705 (RQTHMAN tJ.S.E.) 1-28 7 November 1978 see col~umn 3 0Special categories of Cited documentalis 'aT" tater document Published after the Internationat Miing dtate document defninrg the generat state of the ant which ts not or priority date and not In Conflict with the application but considered to be o1 particular relevance Cited to Understand the principle or theory undterlying the 49" earlier document but published on or aftear the International oueto atclrrtvne h tle neto .iligdt cannot be Considered novel or cannot, be consideredt to "I'document which may throw doubts on prtority ;Ioim(s) or Involve art Inventive step which ts cited to establish the publication date of the NY oun fPriua envne, h lie neto citaionor oherspeial easn (s spcifed)Cannot be Considered to Involve an Inventive stop when the document roefering to a n oral disclosure, Use, exhibition or document is combined with one or more other such docu. other means monts, such combination being obvious to a person skilied 1110 document published prior to the International filing date but In the art, later than the priority date claimed document member of the same patent family IV. CERTIFICATION Dale of the Actual Completion of the International Search, Date of W'Mtng of this International Search Report 3rd April 1989 0 3. 05.89 International Sarchig Authority 31igneturs al-Althorted31flCe EUJROPEAN PATENT OFFICE oq Form PCT1ISAI210 feecond shoet) (Januay 1046) L 14 t4 0 ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. EP 8900078 SA 26492 This annex lists the patent family members relating to the patent documents cited in the above-mentioned international search report, The memher,. are as contained in the European Ptetnt Office EDP file on '.6/04/89 Thc European Patent office is in no way liable for these particulars nhich are merely giveni for the purpose of Information. WO-A- 8102893 21-05-87 AU-A- 6621586 04-06-87 EP-A- 0247156 02-12-87 JP-T- 63501798 21-07-88 EP-A- 0184899 18-06-86 SE-A- 8405500 02-05-86 JP-A- 61155338 15-07-86 SE-A- 8405501 02-05-86 Wa-A- 8505554 19-12-85 EP-A- 0183760 11-06-86 JP-T- 61501571 31-07-86 WO-A- 8602352 24-04-86 AU-A- 4869185 02-05-86 EP-A- 0198051 22-10-86 US-A- 4639365 27-Q1-87 JP-T- 62501070 30-04-87 AU-B- 579411 24-11-88 US-A- 4124705 07-11-78 NL-A- 7506757 09-12-75 FR-A,B 2273555 02-01-76 DE-A- 2524279 18-12-75 AU-A- 8164775 02-12-76 GB-A- 1518121 19-07-78 CA-A- 1037950 05-09-78 UP-A- 51007115 21-01-76 SE-B- 420565 19-10-81 SE-A- 7407462 08-14-75 SFor morq details about this annex see Official Journal of the European Patent Office, No. 12/81