Paramagnetic compounds

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 magnetic resonance imaging.

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 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 by 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 use of contrast 
agents containing paramagnetic, superparamagnetic or ferromagnetic 
species. For paramagnetic contrast agents, the enhanced image contrast 
derives predominantly from the reduction in the spin reequilibration 
coefficient known as T.sub.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 agents in MRI has been widely 
advocated and a broad range of paramagnetic 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 Lauterbur et al. in "Frontiers of Biological Energetics", 
volume 1, pages 752-759, Academic Press (1978), Lauterbur in Phil. Trans. 
R. Soc. Lond. B 289: 483-487 (1980) and Doyle et al. in J. Comput. Assist. 
Tomogr. 5(2): 295-296 (1981)), Runge et al. have suggested the use of 
particulate gadolinium oxalate (see U.S. Pat. No. 4,615,879 and Radiology 
147(3): 789-791 (1983)), Schering AG have suggested the use of 
paramagnetic metal chelates, for example of aminopolycarboxylic acids such 
as nitrilotriacetic acid (NTA), N,N,N',N'-ethylenediaminetetraacetic acid 
(EDTA), N-hydroxyethyl-N,N',N'-ethylenediaminetriacetic acid (HEDTA), 
N,N,N',N",N"-diethylenetriaminepentaacetic acid (DTPA) and 
1,4,7,10-tetraazacyclododecanetetraacetic 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 
EP-A-165728). Many other paramagnetic contrast agents have been suggested 
in the literature, for example in EP-A-230893, EP-A-232751, EP-A-292689, 
EP-A-255471, EP-A-292689, EP-A-287465, U.S. Pat. No. 4,687,659 and 
WO86/02005. Besides the chelates of DOTA and DTPA, the chelates of N,N" 
(bis methyl-carbamoylmethyl) N,N',N"-diethylenetriamine triacetic acid 
(DTPA-BMA), 1-oxa-4,710-triazacyclododecane-N,N',N"-triacetic acid (OTTA) 
and 
N-[2,3-dihydroxy-N-methyl-propylcarbamoylmethyl]-1,4,7,10-tetraazacyclodod 
ecane-N',N",N'"-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 metals, 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 paramagnetic 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 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 WO85/05554, 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--(CH.sub.2).sub.n --NHCO--Y, where X is the 
macromolecule and Y is the chelating entity. One such 
chelate-linker-macromolecule compound, GdDOTA-glycine-dextran, is also 
disclosed in EP-A-186947. 
Other paramagnetic MRI contrast agents are disclosed in the literature (see 
for example WO87/02893, U.S. Pat. No. 4,639,365 and WO87/01594 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 be imaged, the fate of the compound depends on a number of 
factors. If it comprises insoluble particulate matter, it will be removed 
from the blood system by the reticuloendothelial system (RES), in 
particular by Kupffer 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 paramagnetic 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 by an ester grouping and to the chelating moiety by an amide 
grouping 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 present invention 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 
EQU HOOC--CH.sub.2 --(CHR).sub.n --NH.sub.2 (I) 
(wherein, n is an integer of from 1 to 10, and each R, which may be the 
same or different, represents a hydrogen atom or a hydroxyl, hydroxyalkyl, 
or C.sub.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 (C.sub.1-6 alkyl), especially mono- 
or poly-hydroxy (C.sub.1-4 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 the literature relating to MRI 
contrast agents discussed above (see for example EP-A-71564, EP-A-130934, 
EP-A-186947, U.S. Pat. No. 4,639,365, EP-A-230893, EP-A-232751, 
EP-A-292689, EP-A-255471, U.S. Pat. No. 4,687,659, 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 
##STR1## 
(wherein each of the groups Z is a group --CHR.sub.1 X or the groups Z are 
together a group --(CHR.sub.1).sub.2 --A'--(CHR.sub.1).sub.2, where A' is 
O, S, N--CHR.sub.1 X or N--(CHR.sub.1).sub.p --N(CHR.sub.1 X).sub.2 where 
p is 2, 3 or 4; Y is a group --(CHR.sub.1).sub.2 --N(CHR.sub.1 X).sub.2 or 
a group --CHR.sub.1 X; each X, which may be the same or different, is a 
carboxyl group or a derivative thereof such as an amide, ester or 
carboxylate salt derivative, or a group R.sub.1 ; each R.sub.1, which may 
be the same or different, is a hydrogen atom, a hydroxyalkyl group or an 
optionally hydroxylated alkoxy group; with the proviso that at least two 
nitrogens carry a --CHR.sub.1 X moiety wherein X is a carboxyl group or a 
derivative thereof, and preferably the provisos that each --CHR.sub.1 X 
moiety is other than a methyl group, and that where Y and Z are 
--CHR.sub.1 X groups at least one R.sub.1 is other than hydrogen, and 
preferably also that each nitrogen atom carrying a --CHR.sub.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.sub.2 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; DO3A; 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 group 
containing material selected from the group consisting of polymeric and 
polymerized carbohydrates and polymerized sugar alcohols and derivatives 
thereof. The term "polymeric carbohydrate" is used to designate a 
naturally occurring polymer built up of carbohydrate monomers and the term 
"polymerized carbohydrate" is used to designate a synthetic polymer 
obtained by polymerizing carbohydrate molecules, for example with the aid 
of coupling or cross-linking agents. 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 glucan, for example starch, amylose, amylopectin 
(including macromolecular dextrins thereof), glycogen, dextran and 
pullalan, or a fructan, for example inulin and levan, cyclodextrine or 
other physiologically tolerable polysaccharides of vegetable, microbial or 
animal origin. 
Examples of polymerized carbohydrates or sugar alcohols which can be used 
as the macromolecule include so-called polyglucose, which is obtained by 
polymerization of glucose, and macromolecular products obtained by 
cross-linking carbohydrates or sugar alcohols (for example mannitol or 
sorbitol) with at least one bifunctional cross-linking agent, for example 
epichlorohydrin, a diepoxide or a corresponding halogen hydrin or with a 
bifunctional acylating agent. An example of such a product which is 
commercially available is Ficoll (Ficoll is a Trade Mark of Pharmacia Fine 
Chemicals AB of Uppsala, Sweden) which is obtained by cross-linking 
sucrose with the aid of epichlorohydrin. 
Further examples of substances which can form the basis for the 
macromolecule include physiologically tolerable derivatives of the 
polysaccharides mentioned above, for example hydroxyl, carboxyalkyl, acyl 
or alkyl derivatives, for example hydroxyethyl, dihydroxypropyl, 
carboxymethyl, acetyl and methyl derivatives of such polysaccharides. 
Water-soluble derivatives of insoluble polysaccharides (for example of 
cellullose) may be considered as well as the water-soluble macromolecules 
mentioned above. 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 
macromolecule 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 endohydrolases which hydrolyze glycosidic linkages in the 
macromolecule. Thus for example macromolecules degradable by 
alpha-amylase, for example starch-based 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 
charge 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. 
The 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 weight 
average molecular weight of the paramagnetic compound is preferably in the 
range 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, for example less than 30,000 or more 
particularly less than 20,000. Where however, 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 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(III), Mn(II), Fe(III), Dy(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 
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. 
The macromolecular chelating agent mentioned 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 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 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 be 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, 
optionally, converting 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 MRI 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 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 (e.g. 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 for 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, dispersion, syrup, 
suppository, etc; however, solutions, suspensions and dispersions in 
physiologically 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 mol %, 
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 1 micromole to 
1.5 mole per liter, 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 10.sup.-4 to 3 mmol e.g. 10.sup.-3 to 1 
mmol of the paramagnetic metal species per kilogram of body weight, e.g. 
about 1 mmol Dy/Kg bodyweight. 
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 aspect, the present invention also provides a method of 
diagnosis practiced on the human or non-human animal body, which method 
comprises administering to said body a macromolecular 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 practiced 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 invention also provides a method of 
radiotherapy practiced 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 practiced 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 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 about 6 hours and has a distribution volume of 0.05 l/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.sup.3 daltons (such dextrans 
are available from Sigma Chemicals) 
DMSO-A: dimethylsulfoxide 
DTPA-A: diethylenetriamine pentaacetic acid bisanhydride 
ECDI: N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide 
FMOC-BA: fluorenylmethyloxycarbonyl-beta-alanine PP: 4-pyrrolidinopyridine 
Water: water deionized by reverse osmosis 
EXAMPLE 1 
GdDTPA-beta-alanine-dextran (Molecular Weight 70,000) 
To a solution of 15.9 g of Dextran 70 in 650 ml of dry DMSO was added 20.3 
g of FMOC-BA, 13.7 g of ECDI and 968 mg of PP dissolved in 350 ml of dry 
DMSO. The reaction mixture was stirred at ambient temperature for 18 hours 
and 43.1 g of piperidine was added. After 70 minutes, 7.3 ml of 
concentrated hydrochloric acid was added dropwise, and cooling on an 
ice/water bath and dropwise addition of 1.7 l 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 1400 ml 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-methylmorpholine and 
29.18 g 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, 43.78 g of citric acid dissolved in 47.4 ml of 10N NaOH was 
added, and the pH was adjusted to 6.0 with concentrated hydrochloric acid. 
30.37 g of gadolinium chloride hexahydrate dissolved in 200 ml of 
distilled water was added quickly and the pH was adjusted to 5.5 using 10N 
NaOH. The solution was dialyzed against distilled water until the 
relaxation time T.sub.1 (determined using a NMR Proton Spin Analyzer, RADX 
Corporation, Houston, Tex., USA, at 10 MHz and 37.degree. C.) was above 
2000 ms. Lyophilization of the solution yielded 15.3 g of a light yellow 
coloured powder. 
ANALYSIS 
Elemental analysis: Gd 4.6%; N 2.15%; Na 0.16%; Cl less than 0.01%. Free Gd 
(xylene orange 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.sub.1) enhancement (SRRE) (measured in an 
NMR Proton Spin Analyzer RADX Corp. Houston, Tex., USA at 10 MHz and 
37.degree. C.) in distilled water was 9.6 s.sup.-1 mM.sup.-1 Gd. 
EXAMPLE 2 
Injection Solution 
78.6 mg of gadolinium (III) DTPA-beta-alanine-dextran (molecular weight 
70,000) were prepared in accordance with Example 1 and dissolved in 10 ml 
of distilled water. The solution was sterile filtered and filled into a 10 
ml 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 0.05 mmol Gd/kg body weight. Three other rabbits received 
gadolinium 
(III) DTPA-dimeglumine salt intravenously at a dose of 0.05 mmol Gd/kg body 
weight. 
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 and the relaxation times T.sub.1 
and T.sub.2 were determined in an RADX NMR spectrometer (37.degree. C., 10 
MHz). The gadolinium concentration in the serum samples were determined by 
ICP (inductive coupled plasma). The apparent volume of distribution 
(V.sub.D) and the biological half-life (t.sub.1/2) were determined using 
the two-compartment model. The results are as set forth in the Table 
below: 
TABLE 
______________________________________ 
V.sub.D t.sub.1/2 
SAMPLE (l/kg) (hours) 
______________________________________ 
Gd(III)DTPA-beta- 0.05 .+-. 0.003 
6.7 .+-. 0.18 
alanine-dextran 70 
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 and no serum gadolinium were 
observed in serum 48 hours after injection. The observed apparent volume 
of distribution of 0.05 confirms its blood pooling property. 
EXAMPLE 4 
Dextran 70-beta-alanine-DTPA 
10.0 g of Dextran 70 was reacted with 12.8 g of FMOC-BA, 8.7 g of ECDI, 
0.61 g of PP and 31.5 ml of piperidine in 600 ml of dry DMSO and then with 
22 g 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 and 6M HCl and the 
solution was dialyzed against 4 l of water. The solution was lyophilized 
to give 5.5 g of a light yellow solid. 
Elemental analysis: N 2.82%, C 42.52%, H 6.74%. 
EXAMPLE 5 
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 10N NaOH and 417 mg of 
FeCl.sub.3 dissolved in 10 ml of water. The pH was adjusted to 5 with 10N 
NaOH and, after reaction overnight, the solution was dialyzed against 
water until T.sub.1 in the filtrate was above 2000 ms. Lyophilization gave 
0.47 g of a light brown solid, 5.5% Fe, relaxivity 0.8 s.sup.-1 mM.sup.-1. 
EXAMPLE 6 
Dextran 70-beta-alanine-DTPA-Dy 
0.5 g of the product of Example 4 was complexed with 1.1 g of DyCl.sub.3 
and isolated as described in Example 5. Yield 0.57 g of a white solid, 
9.8% Dy, relaxivity 0.2 s.sup.-1 mM.sup.-1. 
EXAMPLE 7 
Dextran 70-beta-alanine-DTPA-Yb 
0.5 g of the product of Example 4 was complexed with 1.15 g of 
Yb(NO.sub.3).sub.3 and isolated as described in Example 5. Yield 0.5 g of 
a yellowish solid, 3.5% Yb, relaxivity 0.03 s.sup.-1 mM.sup.-1. 
EXAMPLE 8 
Dextran 70-beta-alanine-DTPA-Cu 
0.5 g of the product of Example 4 was complexed with 642 mg of CuSO.sub.4 
and isolated as described in Example 5. Yield 0.55 g of a light blue 
solid, 1.5% Cu, relaxivity 0.3 s.sup.-1 mM.sup.-1. 
EXAMPLE 9 
Dextran 40-beta-alanine-DTPA-Gd 
2.0 g of Dextran 40 was reacted with 2.6 g of FMOC-BA, 1.73 g of ECDI, 122 
mg of PP and 6.3 ml of piperidine in dry DMSO as described in Example 1. 
The product was reacted further with 3.77 g of DTPA-A as described herein, 
and after complexation in citrate buffer with 3.82 g of GdCl.sub.3. 6 
H.sub.2 O the product was dialysed and lyophilized to yield 1.05 g of a 
white solid, 5.1% Gd, relaxivity 5.1 s.sup.1 mM.sup.-1. 
EXAMPLE 10 
Hydroxyethylstarch-beta-alanine-DTPA-Gd 
2.0 g of hydroxyethylstarch (prepared by hydroxyethylation of waxy starch 
with ethylene oxide according to the method described in U.S. Pat. No. 
2,516,634) molecular weight 131,000 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.sup.1 mM.sup.-1. 
EXAMPLE 11 
Dextran 40-beta-alanine-EDTA-Cr 
2.0 g of Dextran 40 was reacted as described in Example 9 up to the point 
where the Dextran 40-beta-alanine water solution had been dialysed at pH 
4.2.2.65 g of EDTA-bis-anhydride (prepared using the method of Eckelman et 
al, J Pharm. Sci., 64 (1975) 704) was reacted with the Dextran-derivative, 
complexed with 2.74 g of CrCl.sub.3. 6 H.sub.2 O and the product was 
isolated as described in Example 9. Yield 2.9 g of a purple solid, 3.1% 
Cr, relaxivity 1.1 s.sup.-1 mM.sup.-1. 
EXAMPLE 12 
Dextran 500-beta-alanine-DTPA-Bi 
2.0 g of Dextran 500 was reacted as described in Example 9 except that 4.4 
g of DTPA-A was used. The reaction mixture was stirred for 3 hours while 
the pH was kept at 8 with N-methylmorpholine. The pH was then adjusted to 
5 with 6N HCl and a buffer solution containing 5.5 g of citric acid and 
5.96 ml of 10N NaOH was added. A solution of Bi(III) was prepared by 
dissolving 3.89 g of BiCl.sub.3 in 100 ml of 1M HCl and the pH was 
adjusted to 7 with saturated ammonia in water. The suspension was 
centrifuged and the supernatant was decanted off. The precipitate was 
resuspended and centrifuged twice and the white jelly-like precipitate was 
added to the buffer solution of the Dextran. The pH was 5.0 and, after 
reaction overnight, the clear solution was dialysed against 12 l of water 
and lyophilization yielded 0.8 g of a white solid, 9.4% Bi. 
EXAMPLE 13 
Dextran 2000-beta-alanine-HEtDTPA-Gd 
(a) The DTPA-derivative 
3,6,9-tris-carboxymethyl-4-(2-hydroxyethyl)-3,6,9-triazaundecane diacid 
(HEtDTPA) was synthesized according to the method of PCT/GB88/00572. 
HEtDTPA trihydrochloride was prepared by loading HEtDTPA on a strong anion 
ion exhanger and eluting with 1M HCl followed by evaporation. The product 
was a white solid, mp. greater than 350.degree. C. (decomp.). 
Elemental Analysis: Calc.: C 35.14%, H 5.54%, N. 7.69%, Cl 19.45%. Found: C 
34.76%, H 5.46%, N 7.74%, Cl 19.56%. 
(b) 2.0 g of Dextran 2000 was reacted as in Example 9 to the point before 
reaction DTPA-A. The solution was lyophilized to yield 1.9 g of a white 
solid. The product was dissolved in 200 ml of dry DMSO and there were 
added 2.24 g of HEtDTPA trihydrochloride, 0.86 g of ECDI and 35 mg of PP. 
After stirring for 24 hours the solution was added to a mixture of 300 ml 
of ether and 125 ml of CHCl.sub.3. The product was isolated by decantation 
of the supernatant. The product was dissolved in 120 ml of water and the 
pH was adjusted to 5 with 10N NaOH. To the solution was added a buffer, 
containing 5.5 g of citric acid and 5.96 ml of 10N NaOH, and then 1.53 g 
of GdCl.sub.3. 6 H.sub.2 O dissolved in 10 ml of water. The pH was 
adjusted to 5 with 10N NaOH and after 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.sup.-1 mM.sup.-1. 
EXAMPLE 14 
Dextran 70-beta-alanine-DOTA-Gd 
(a) N',N",N"N'"-Tetracarboxymethyl-1,4,7,10-tetraazacyclododecane (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 pH 
was adjusted to 10 with conc. HBr, 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 bromoacetic acid lithium solution was 
added to the 1,4,7,10-tetraazacyclododecane solution in one portion. The 
pH was kept between 8 and 9.5 with 4N LiOH while the temperature was 
gradually increased to 80.degree. C. during 4 hours. After cooling, the 
solution was mixed with 494 ml of wet Dowex 50WX 4 acidic ion exchange 
resin in 1.5 l of water and stirred for 1 hour. After thorough washing 
with water, the gel was washed with 2.times.750 ml of saturated ammonia. 
The filtrate was evaporated to yield 10.9 g of a white solid, mp greater 
than 350.degree. C., FAB-ms M+1 411 and 417 -mono- and di-lithium salt. 
.sup.13 C-and .sup.1 H-NMR confirmed the structure. 8.72 g of the solid 
was dissolved in 16 ml of water and the pH was adjusted to 2.5 with conc. 
HCl. The white solid was filtered off and the process repeated with the 
evaporated filtrate. The collected solids were dried to yield 4.5 g of a 
white solid, mp greater than 350.degree. C. (decomp). 
(b) Dextran 70-beta-alanine-DOTA-Gd 
2.0 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 DMSO. 1.66 g of the precipitated DOTA, 0.86 g 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 ml 
of ether and 62 ml of CHCl.sub.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 10N NaOH and there was added a mixture of 
5.5 g of citric acid and 5.96 ml of 10N NaOH and then 0.766 g 
GdCl.sub.3.6H.sub.2 O. 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.sup.-1 mM.sup.-1. 
EXAMPLE 15 
Dextran70-5-aminopentanoic acid-DTPA-Gd 
5.65 g of 9-fluorenylmethyloxycarbonyl-5-amino-valeric acid (prepared from 
5-amino-valeric acid and 9-fluorenylmethyl chloroformate as described by 
Carpino et al. J. Org. Chem., 37 (1972)3404) was reacted with 2.0 g of 
Dextran 70, 3.5 g of ECDI and 0.25 g of PP as described in Example 9. The 
reaction mixture was treated with 12.75 ml of piperidine, the product was 
isolated 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 
GdCl.sub.3.6H.sub.2 O in citrate buffer and isolated by dialysis and 
lyophilization as described above. Yield 6.6 g of a light brown solid, 
11.4% Gd, relaxivity 5.6 s.sup.-1 mM.sup.-1. 
EXAMPLE 16 
Glycogen-beta-alanine-DTPA-Gd 
2.0 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.sup.-1 mM.sup.-1. 
EXAMPLE 17 
Vial containing Dextran70-beta-alanine-DTPA 
A vial is filled with 20 mg of Dextran70-beta-alanine-DTPA (Example 4) and 
0.2 mg of Sn(II)Cl.sub.2 as a dry solid. 
A solution of .sup.99m Tc as pertechnetate is 0.9% sterile sodium chloride 
should be added before use. The technetium chelate with 
Dextran70-beta-alanine-DTPA is for intravenous or subcutaneous 
administration and is a contrast agent for the vascular system or for 
lymphangiography. 
EXAMPLE 18 
Dextran70-beta-alanine-DTPA-Gd and the Calcium-disodium salt of 
Dextran70-beta-alanine-DTPA 
760 mg of Dextran 70-beta-alanine-DTPA (Example 4) was dissolved in 10 ml 
water and 28 mg of Ca(OH).sub.2 were added. The pH was adjusted with NaOH 
under ambient conditions. 1 ml of the resulting solution was added to a 
solution of 1.0 g of Dextran 70-beta-alanine-DTPA-Gd (Example 1) in 9 ml 
of water, and the resultant solution was sterile filtered, filled into a 
20 ml vial and lyophilized. 
EXAMPLE 19 
Dextran70-beta-alanine-DTPA-Gd and the calcium-trisodium salt of DTPA 
To a solution of 1.0 g of Dextran70-beta-alanine-DTPA-Gd (Example 1) in 10 
ml of water was added 17 mg of the calcium-trisodium salt of DTPA (Fluka). 
The solution was sterile filtered, filled into a 20 ml vial and 
lyophilized.