MXPA96003679A - Poliquelan - Google Patents

Abstract

The present invention provides polychelators, which are useful, for example, in diagnostic imaging procedures, and which are degradable in vivo to release excretable fragments. Such compounds conveniently are of the formula (I): R1 (X1R2 ((X2) pL) n) m (wherein X1 is a metabolically hydrolysable binding moiety to release fragments R1X3m and X4R2 ((X2) L) m, wherein X3 and X4 are the residues of the hydrolysis of X1; R1X3m is a biotolerable polymer, preferably a substantially monodisperse polymer, and especially one with a molecular weight below 40,000 D, particularly below 30,000 D, and especially below 20,000 D D, for example a first to sixth generation dendrimer, X4R2 ((X2) pL) n is a polychelant fragment having a molecular weight below 40,000 D, preferably below 30,000 D, especially below 20,000 D, each such portion is preferably the same, p is 0 or 1, X2, when present, is a metabolically hydrolyzable binding portion for releasing a single-stranded fragment, each L is a macrocyclic chelating moiety, wherein the macrocyclic backbone preferably it has from 9 to 25 members in the ring, and preferably it is a polyazacycloalkane optionally interrupted by oxygen or sulfur; R2 ((X2) p) n is a portion of the straight or branched chain backbone, which preferably provides a chain of up to 20 atoms between each group L and the portion X1 to which it is attached, and a chain of up to 25 atoms between each pair of L groups joined by means of this, such chains are conveniently carbon chains interrupted by nitrogen and / or oxygen and / or sulfur each n is an integer having a value of at least 2, preferably a value of 2 to 25, especially 2 to 12, and each m is an integer having a value of at least 2, preferably a value of up to 200 , especially from 3 to 100, such that the total number of L groups in the polychelant of formula (I) is at least 20, preferably 50 to 200, having a molecular weight of at least 30,000 D, preferably at least 40,000 D, and especially preferably 50,000 to 150,000D, and the metal chelates and salts of the same

Description

POINTERS DESCRIPTION OF THE HWPIC10N The present invention relates to polychelants, as well as to the corresponding bifunoional polycarboxylates (for example macromolecular conjugates directed to a specific site or site directed by the polychelants, and the chelates and salts thereof, and to their applications. In medicine, especially in the field of diagnostic imaging, polychelates are especially adapted for use to intensify images of organs, tissues, cells, and the like, in vivo selected from mammals, using Magnetic Resonance Imaging. (FIRM), X-rays, sc in igr af í gamma, and CT scanning, by virtue of their imaging properties and if i > * intensified specificity The polychelants are also particularly adapted for used as agents ie intra-vascular contrast, agents to measure blood accumulation, in these modalities of image formation. As such, they can be used in the formation of blood vessel images, for example, in magnetic resonance angiography, in the measurement of blood flow and volume, in the identification and characterization of lesions by virtue of differences in vascularity of the tissue. normal, in lung imaging, for the evaluation of pulmonary diseases, and in blood perfusion studies, -the polychelators can also be well adapted for the desotoxication of metals, therapeutic supply of radioisotope , and applications in diagnostic nuclear medicine. The modalities of imaging in medicine, such as FIRM, X-rays, gamma scintigraphy, and CT scans, have become extremely important tools in the diagnosis and treatment of diseases. Some imaging of internal parts depends on inherent attributes of those parts, such as bones, to be differentiated from the surrounding tissue in a particular type of imaging, such as X-rays. Other organs and anatomical components are only visible when they are highlighted is eclectically by particular imaging techniques. A technique with the potential to provide images of a wide variety of anomic components invokes metals that intensify the image to target bio logical targets. Such a procedure has the possibility of creating or intensifying images of specific organs and / or tjnores, or other sites located within the body, while reducing the background and potential interference created by the simultaneous enhancement of undesired sites. Researchers have recognized for many years that when chelating several metals the physiologically tolerable dose of such metals is increased, and thus their in vivo use is allowed to intensify images of body parts (see for example CD Russell and AG Spei.er, J. Nucí, Med.?: 108b (1988) and EUV Patent No. 4,647,447 (Gries et al.)). However, intensifiers of simple metal chelate images, without further modification, generally do not provide any particularly significant site specificity. The binding of metal chelate to target non-target tissues or organs, for example biomolecule such as pro-teins, to produce site-specific therapeutic or diagnostic agents has been widely suggested. Many such bifunctional chelating agents, that is, agents which by virtue of the chelating ratio are capable of strongly binding a therapeutically or diagnostically useful metal ion, and by virtue of the specific molecular component are capable of selectively supplying the chelated metal ion to the site. of the interest rate, are known or have been proposed in the literature. For example, even relatively old publications in the field of contrast agents for FIPM, such as GB-A-2169598 (Schering) and EP-A-136812 (Technicare) suggested the use as chemists of paramagnetic metal chelates of bifunctional chelants. The binding of chelating portions to specific macromolecules of a site has been carried out in a number of ways, for example, the mixed anhydride method of Krejcarek et al. (Biochemical and Biophysicai Research Communica- tions 77: 581 (1977)), the cyclic anhydride procedure of Sr. Owich et al. (see Science 220: 613 (1983) and in another part), the method of forming derivatives of the main structure of Meares et al. (see Anal. Biochem. 142: 68 (18) and elsewhere - this is a technique used by Schering on EP-A-331616 to produce specific site polychaelates for use with contrast agents for FIRM or X-rays), v the method of the binding molecule, used for example by Amersham (see WO -A-85/05554) and Nycomed (see EP-A-186 47 and elsewhere) to produce paramagnetic melting ion chelates of bi-functional chelators , to be used as contrast agents for F1RM. Thus, Krejcarek et al (above) were described as the polylactic acid polycarboxylic acid (APAPC) chelators, particularly the ADTP (diethylene tpaminepentaacetic acid) could be conjugated to a protein, such as albumin of human uero (ASH), by the reaction ie the triethylamine salt of the APAPC with chloro formate of i. so butyl (CFIB), and reacting the CFIB-APAPC adduct with the protein. His goal was to bind a radioactive metal per molecule of human serum albumin, for the purposes of measuring biological function. Specific site uses of various imaging techniques require or would be enhanced by the use of a multiplicity of the appropriate metal ion conjugated to a targeted macromolecule site. For example, it is believed that a 50% reduction in the relaxation time T. of the water pro-tones in a target tissue is a requirement for an effective treatment agent for IIRM. Considering the affinity of the antibodies for their antigens, and the concentration of these antigens in the target tissues, it has been calculated that each antibody molecule must carry a number of paramagnetic centers to originate these levels of T- reduction. (see Eckelman, et al., N'ATO ASI Series, Series A, _152_: 571 (1988)). llnger et al. in Investigative Radiology _2_0: 693 (1985) analyzed the intensification of a tum r by magnetic resonance imaging, uses ncl c an anti-CEA monoclonal antibody conjugated with Gd-ADTP. Nc found tumor enhancement when Gd atoms were attached per antibody molecule, and predicted that s would require a much larger proportion of metal atoms for macromolecule imaging to be effective. Also, Schreve and Aisen in Magn. Res. In Medicine 3: 336 (1986), concluded that the concentrations of the paramagnetic ion that could be delivered to a tumor using the described technology would result in large doses for humans, making highly limited "even close to the formation of images in u use. However, for the intensification of site-specific images it is important that the site specificity of the portion targeting the tissue or organ target of such chelates of functional chelators should not be destroyed by conjugation. of the chelating portion. When the bifunctional chelator contains only a chelating moiety, this is generally not a severe problem; However, when attempts have been made to produce bi-functional polychelants by conjugating several chelating moieties on a single site-specific macromolecule, s has not only found that the maximum achievable chelator ratio: macromole-t. The specific site may be relatively limited, but rather, when the ratio achieved increases, the specificity of the bifunctional polychaelae decreases. However, numerous attempts have been made to produce bifunctional polychelants with increased numbers of chelating portions per m site-specific chromol- ecule. Thus, Hnatowich et al. (above) used the cyclic anhydride of the ADTP chelator to bind it to a protein. This is a relatively simple step synthesis procedure, which as a result has been used by many other researchers. However, due to the presence of two cyclic anhydride groups in the starting material, extended cross-linking of the macromolecules can result in the production of conjugates that can not be easily characterized (see Hnatowic et al., J. Emmunol. 65: 147 (1983)). In addition, this process suffers from the same disadvantage as that of the Krejcarek mixed anhydride method, in that the uncontrolled addition of more than a few chelator portions destroys the site-specificity of the macromolecule to which they are attached. (See also Paik et al., J. Nucí. Med, 2_5: 1158 (1983)). To overcome the problems of joining larger numbers of chelation portions to a site-specific macromolecule without destroying its site-specificity, that is, without disturbing its binding site (s), many propositions have been made for the use of a molecule of the main structure, to which large numbers of chelating portions can be attached, to produce a polychelant, one or more of which can then be conjugated to a 1-site-specific monochrome to produce the bifunctional polychelant. So, the now conventional. conjugation technique of the cyclic anhydride of Hnatowich et al.?. (above) it has been used to produce bifunctional polychelants, in which the chelating portions are residues of open-chain PAPCs, such as the AEDT and the ADTP, and in which the main structure molecule is a polyamine, such as polylysine or polyethyleneimine Thus, for example, Manabe et al in Biochemica et Biophysi to Acta 883: 460-46? '(1986) reported the binding of up to 105 ADTP residues on a primary structure of poly iL. -1 isine, using the cyclic anhydride method, and also the polychelan binding is poly 1 isine-poly-ADTP on a monoclonal antibody and IgG, anti-HLA) using a 2-polyvinyl disulfide binder. achieving a substitution of up to about 42.5 chelators (ADTP residues) per site-specific macromolecule. Torchlin et al. in Hybri-doma _6: 229-240 (1987) also reported the binding of ADTP and AEDT to polyethylenimine and poly-lysine backbones, which were then linked to a myosin-specific monoclonal antibody, or its Fab fragment, to produce bifunctional polychelants for t, be in FIRM or in escrow. While Manabe and Torchlin have reported the production of bifunctional polychelators, the cyclic anhydride path adopted by Manabe presents reticulation, and hence the characterization oakms, and Tcrchlin et al. in their conclusion they doubted that their technique would allow the concentration of the paramagnetic element to increase sufficiently to allow the FIRM of tumors. Sieving et al. in W0-90 / 12050 he described techniques for producing polychelants comprising macrocyclic chelating portions, such as polylysine-polyADOT, and for the preparation of the corresponding bifunctional polychelants. Sieving et al. he also suggested the use of radial or star dendrimers, such as the PAMAM radial dendrimer of the sixth generation of Tomalia et al. (See US-A-4587329 and Polymer Journal J.7_: 117 (1985)), as the main structure or skeleton for such polychelants. The path towards a greater metal loading capacity in the production of polycarbonates has resulted in the production of high molecular weight products. For soluble products, this has the advantage that with administration to the circulatory system, the compounds are retained within the blood, rather than rapidly diffusing into the extracellular fluid, or being excreted by the glo- lar system. Accordingly, these compounds can serve as effective imaging agents for measuring blood accumulation. However, it is undesirable with all that the poly-chelates must remain in the body for longer than necessary, for example, after the image has been completed. The present invention resides in part in the realization that polykelet can be produced. s that are diagnostically or therapeutically effective, and are still easily metabolized to fragments that can be easily excreted, for example by glomerular filtration, following the metabolic cleavage of well-characterized pol-chelate fragments thereof. The incorporation within the total structure of the polychelant of divisible polychelant fragments has added advantages in that the number of chelating groups that can be added to each binding site on the polymer of the main structure is increased, and that polychelates can be produced by conjugating poliquelante sub-units already metalated on a main structure polymer, improving with this the metal charge ratio for the complete polychelant. Thus, viewed from one aspect, the invention provides a polychelant of formula I 1 2 (X 2) p L) n) m (I) (wherein X is a metabolically divisible binding portion for releasing RX fragments and XR ((X¿) L), in mpn where X and X are the residues of the division of X; RX is a biotoLerable polymer, preferably a polymer which is usually monodisp, and especially one with a molecular weight below 4,000 D, particularly below 30,000 D, and especially below 20,000 D, for example a dendrimer from first to sixth generation; 4 2 2 XR ((X) L) is a polychelant fragment having a molecular weight below 40,000 D, preferably by de-low of 30,000 D, especially below 20,000 D, each such portion being preferably the same; p is 0 or 1; 2 X, where present, is a portion of metabolically divisible union, to release a fragment of monochlear; each L is a macrocolic chelating portion, wherein the macrocyclic main structure preferably has from 9 to 25 members in the ring, and preferably is an optionally interrupted polyazacyl-cloalkane or oxygen or sulfur; 2 2 R ((X)) is a portion of the straight or branched chain main structure, which preferably provides a chain of up to 20 atoms between each group L and the X portion to which it is attached, and a chain up to 25 atoms between each pair of groups L joined by means of sa, such chains conveniently are carbon chains-interrupted by nitrogeny and / or oxygen / or sulfur; each n is an integer having a value of at least 2, preferably a value of 2 to 25, especially from 2 to 12; and each m is an integer having a value of at least 2, preferably a value of up to 200, especially from 3 to 100, such that the total number of L groups in the polycarbonate of formula (I) is of at least 20, preferably 50 to 200), having a molecular weight of at least 30,000 D, preferably at least 40,000 D, and especially, preferably from 50,000 to 150,000 D, and the chelates and metal salts of the is or . The term "polychelant" is used hereinafter, wherever the context permits, to designate not only the unmetalated compound, but also its fully and metallically formed forms.

If desired, one or more polychelants according to the invention can be conjugated to a biodistri-buclone modifier, i.e., a portion which serves to alter the pharmacokinetics of the total molecule, for example, a hydrophilic moiety. drofílica or a molecule directed to a specific site, for example a protein or fragment of protein, to form a bifunctional polychelant. In this case, the linkage with the site-directed molecule is preferably also metabolically divisible, so that biodegration of the b-functional polychelant will release the targeted-siti-molecule (or its metabolites) as well as fragments of the polychelant. These bifunctional polychelants form a further aspect of the present invention, and can be used, for example, to intensify images and / or to deliver cytotoxic doses of radioactivity to cells, tissues, organs and / or body conducts fixed as targets. Alternatively, polychelators can be used as agents for measuring blood pooling, without being coupled to site-directing molecules. Polychelators are in and of themselves entities useful in medical diagnosis and therapy, due in part to their unique location within the body. The monomeric chelates currently used for enhanced contrast in-MR imaging (eg GdADTP2"GdADOT ~ and GdADTP-BMA) have in vivo applications limited to their specific rapid biodistribution, which causes the localization of these chelates in all the extracellular (and extravascular) spaces of the body The polychelants of the invention, which typically have molecular weights of 30 to 200 kD, especially of 40 to 150 kD, and paricularly of 50 to 120 kD, have a biodistribution To the monokelators, the liquefiers of the invention generally have extended ntravascular residence times, generally of the order of hours, however, by virtue of their biodegradability, generally they can eventually be eliminated to the extracellular fluid (ECF) and suffer renal excretion. , as these polychaelators, hereafter referred to as image enhancers, remain primarily In the intravascular system by a lymphoid of diagostically useful residence, they are suitable for a range of uses, such as measurement of blood collection and cardiac perfusion imaging, brain imaging, vessel imaging blood, in the formation of lung images, for the evaluation of pulmonary diseases, detection of tumors in the CNS, and determination of volumes and detection of thrombi and angiography. As agents for measuring blood accumulation, they are particularly suitable for use in studies of blood flow or volume, especially in relation to the detection of lesions and studies of myocardial perfusion. The contrast agents for conventional monomeric MR imaging that rapidly disperse in the extracellular / ex travascular space can not be easily used for these purposes. On the other hand, in view of their increased relaxivity, the contrast agents for MR imaging of the invention can be administered in significantly reduced doses relative to contrast agents for current monomeric MR imaging, such as the GdADTP , GdADOT, and GdADTP-BMA, thus providing a significantly improved security mrgen in its use. It is particularly preferred that all metabolic degradation products of the polychelant of the invention, in particular the polymer R X and the polychelant fragrances 4 2 2 X R ^ ((X) L), are of sufficiently low molecular weight, p n to be renally excretable n when the non-degraded polycarbonate itself can have a sufficiently high molecular weight to not be. SimilarlyIt is preferred that the polychelant (and its chelates) be water soluble, so that they can function as an agent for measuring blood pooling. For an agent for measuring blood accumulation, one requires a molecule having a size or total molecular weight large enough so that the capillary blood filtration is slow enough so that the contrast of the image of the blood pool can be obtained. By analogy with globular proteins, the "threshold of the kidney (the minimum molecular weight) can generally be considered to fall in the range of 30 to 40 kD. Molecular size and configuration are of course more important than rao weight. Total molecular weight, but these minimum molecular weights (for the non-metallated polychelant) provide a reasonably accurate guideline Thus, the invention provides a way in which one can construct an agent to measure the well-characterized blood accumulation, which it will be biodegraded in well-characterized fragments, excretable easily and usually by the renal route, thus reducing the pro-active accumulation within the body of potentially toxic diagnostic or therapeutic metal ions, with which the polycarpal is charged, the main structure (polymeric RI on which the polychelan fragments are assembled is in itself preferably substantially It is monodisperse, so that the biodistribution of the polychelant is uniform, so that a uniform and reproducible charge ratio can be achieved. By the charge ratio of me, the relationship between the number of therapeutic or diagnostic metal ions transported by the macrocyclic chelating groups and the number of polychelating molecules is understood. Virtual any biotolerable polymer having binding sites (for example reactive functional groups on the main chain or slopes, such as amines, hydroxyls, carboxyls, etc.) can be used for the fragments of the polychelant (R ((X ) L)), for example polylysine, polyethyleneimine, polysaccharides, etc. However, it is particularly preferred to use demeritic polymers, and especially the so-called radial dendrimers, since these can be produced in substantially monodisperse and well-characterized form, and since the dendrimeric structure facilitates the uniform loading of the polylayer fragments on the junction points on the main structure of the polymer, since these tend to be arranged around the periphery of the dendrimeric molecule. With the radial or star-shaped dendrimers, this is particularly in this way, and its substantial spherical shape provides the maximum access of the water to the chelated metal sheets, as well as allowing an optimum metalation efficiency, that is, a maximum metal load. The dendrimers which are particularly suitable for use according to the invention as the polymeric main structure R include the dendrimers of the first to the sixth generation. The optimal generation depends of course on the nature of the fragment of the polychelant, the route of administration and elimination proposed, etc. Suitable polymers for the main structure, including radial or star-shaped dendrimers, are discussed in detail in previous patent applications, such as WO-A-91/05762, W0-A-90/12050, and PCT / EP92 / 02308, as well as by Tomalia et al. in US-A-4587329, l S-A-4568737, US-A-4558120, US-A-4507466, WO-A-88/01178 and Angew, Chem. Int. Ed. Eng, 2_9: 138-175 (1990). Where a contrast agent is required to collect in the blood pool, then a lower generation dendrimeric main structure, for example a fourth or fifth generation dendrimeric main structure, will preferably be used. Such poly iquelant is have an intensified relaxivity, compared to contrast agents to measure blood accumulation and ECF, and thus, a lower effective dose can be administered. The binding portions X and X me a bically divisible can be or incorporate any g r u p) unional that is divided in vivo following the administration, usually parepteral administration. The degree of susceptibility to division can be selected by the appropriate selection of these binding portions, to achieve a desired half-life before rupture, for example, a residence type in The desired blood, or to ensure that the rupture occurs predominantly at particular sites in the body, such as the liver. The functionalities of ester, disulfide, amide, acetal, ketal, ether, anhydride and lactase are examples of groups that can be considered to be readable or otherwise biodegradable. Depending on the desired cleavage site, it may be desirable to incorporate portions of the polychelant fragments that are biodirected to selected targets, for example hydrophilic, lipophilic or charged, to assist in post-hydrolysis removal. It is particularly preferred that there are raeta-bolically-hydrolyzable X groups at the junction points during the synthesis of the pre-formed polylinker groups (ie, 2 2 of the R ((X) L)) portions. Thus, while it would be possible to conjugate monomeric chelating groups over a preformed main structure, it is preferable according to the invention to conjugate conjugated preformed polycarboxylic moieties onto a polymer of the main structure. For a polymer of given major structure, this synthetic route gives rise to polychelants having multiple chelating portions per site of attachment to the main structure, and as a result, they have a high charge ratio for the metallated product. This, on the other hand, has the advantage that dimeric, trimeric or oligomeric bulky fragments of the polythene will have restricted rotation at the site and join to the main structure of the polymer, thus giving rise to greater relaxivities when the total polychelant is a contrast agent for MR imaging of T .. In this regard, it is particularly preferred to use preformed dimeric polythene dimer or dendrimer fragments, which incorporate an active site for binding to a polymer of polymeric or rimeric backbone . Where the conjugation site, or conjugation, does not itself produce a metabolically hydrolyzable linkage, then a metabolically hydrolyzable functional binding moiety must be incorporated within the preformed polychelant fragment, or within the polymer of principle structure, In this respect, urea, ether, ester, double ester, carbamate, disulfide or other electrolytically separable groups can be incorporated, for example in the preformed polychelant fragment, between the main structure of the polyalant and the reactive group by which to be united to the main polymer structure the total polychelant. Alternatively, these hydrolyzable groups may be located between the monokelating portions, so that with metabolic cooling, monokelators or other small fragments of polychelan e may be released. Polychelants of this nature form a further aspect of the invention, and are of the formula II 1 1 '. 1 R1 (X1R2 ((X2) pL)) m (II) 1 2 (wherein R, R "L, p, n and m are as defined above, and X 1 and X 2 are binding portions, with the proviso that between 1 2 1-each L and R at least one of the X and X binding portions is metabolically hydrolyzable) and the metal chelates and salts thereof, as well as the polychelants - 2 ') n0 - bifunctional correspondents. Thus, in preferred aspects of the invention, the main structure R ((X)) of the polychelant fragment comprises a branched polyalkane chain, which optionally incorporates heterocyclic, saturated or unsaturated homoi rings (e.g. rings of 5 to 8 members, which incorporate 0, 1 or 2 heteroatoms, chosen from 0, M and S, for example phenyl rings), nitrogen, oxygen or sulfur atoms, or carbonyl groups, the latter preferably being chain heteroatoms adjacent. Examples of preformed chelator fragments include the following: O O -NH N NH- (B) N- -N J i » N wherein X is a group that provides an appropriate site for its attachment to the central structure of the backbone or backbone, for example a modified amine group that, D can be attached to an amine function, for example X can be a group -alk- fX, -alq- kt -C ^ CONH-al q - < ? > -X, X ° or -NHC0CH CH2SS- < 3 ^ "where X6 is N0," NCS, N *. CO, -Alk-COOH, -NHCOCHCH.-, -NHC0CH2- ^, -NHC0CH2C1 or -NHC0CH2Br, and alk is a bond or an alkylene chain having 1 to 4 carbon atoms, and ~ N) is a metalated or non-metalated macrocyclic chelating portion, preferably but not essentially bound to the remainder of the structure in the ring nitrogen of the macrocycle. The raacrocyclic chelator portions in the polychelants of the invention may be residues of any of the conventional mac rocyclic chelators, such as for example ADOT, ATET, D03A, etc. The macrocyclic backbone or backbone, as mentioned above, preferably has 9 to 25 members in the ring, and conveniently is a polyazacyl loalkane ring optionally interrupted by oxygen or sulfur. The binding site for the polynucleotide fragment 2 Lante R ((X)) is preferably a nitrogen of the ring, pn but alternatively, the linkage may be on a ring carbon, for example as described by Meares et al in US. -A-4687667. The macrocyclic chelating portion may depend only on the ring heteroatoms for its chelating capacity, and thus, may be a cyclic polyether or polylamin. However, the macromolecular chelating portions preferably have pendant groups, which participate in the chelation of the metal, for example alkyl groups of 1 to 6 carbon atoms comprising hydroxyl or amino, phosphonate groups. , or phosphinate, or more preferably carboxyl. The macrocycles derived from D03A and ADOT are substantially preferred, ie, groups of formula ...

The chelating portions of macroses in the polychelants of this invention are preferably derived from macrocyclic chelants having a carbexyl or reactive amine group which is not essential for coordination binding to the metal. The reactive group can be one of the groups that in the free chelator can function as a group that coordinates to the metal, provided that the conjugated chelating portion retains the ability to complex metal ions. Alternately, the reactive group can be a substituent on the side chain of the chelant, or on a carbon of the main structure. More particularly, as used herein, a macrolicyclic chelator is defined as a quelan (which has a closed, continuous and joined main structure, consisting of donor atoms, such as, for example, P, B, 0, S and Ace, spaced apart by carbon atoms, for example optionally substituted methylene carbons, or cyclic groups, for example aromatics, or chains thereof, particularly preferably optionally substituted 2 to 4 carbon atoms of alkylene chains. The methylene groups or donor atoms, where permitted by the conditions of allele, may be substituted, provided that the closed chain of the macrocycle remains intact In a preferred embodiment of the invention, the macrocyclic chelants are of formula III wherein a, b, d and e are independently zero or a positive integer, for b or d preferably;, 2, 3 or 4; c and f are positive integers; the sum of all 's is at least 3, preferably 3, 4 or 5; the sum of b + - d is at least 1; each Z is independently a nitrogen, oxygen, sulfur, phosphorus, boron or arsenic, preferably at least two, especially at least 3 of these are nitrogen; Y is independently a carbocyclic or heterocyclic ring of 5 to 7 members, optionally substituted; R when present is independently hydrogen, optionally hydroxylated, optionally alkoxylated alkyl, optionally including a group CO-G, where G is OR or NR ,, and where Z is phosphorus, optionally also oxo, minus 3 3 Z (R) portions, which preferably have Z as nitroge- 3 not, a = 1 and R is a G-CO-group which is optionally substituted; R and R, which may be the same or different, each independently is hydrogen, optionally alkoxylated, optionally and hydroxylated alkyl, aryl, alkylaryl or aralkyl, or R may also represent or be substituted by a CO-G group; and NR "may also represent an optionally substituted 5- to 7-membered heterocyclic ring, attached to nitrogen, optionally containing an additional heteroatom in the oxygen, nitrogen or ao ring; and wherein, in 4 place of two groups CR R, separated in any direction by at least one group Z, optionally there may be a bridged structure of formula: where u, g, h, i, j, k, 1, w, x, q, r, syt is each independently zero or a positive integer, for u, g, i, and w preferably 1, 2, 3 or 4; and it is a positive integer; h + 1 + j + x _ 1, preferably y (h "- 1) 1, and each D is independently boron, carbon, nitrogen, phosphorus or PO.

Preferred identities for the Y-ring portions include: • * - * where J is CH, COH or N; R6 is CH2, CHOH, NR3, 0 or S; and L e s 0 or S. Preferred identities for hetero-cyclic portions R0 include: - H p o rm e p o n e -N - OH - N - N - OH As indicated above, the macrocyclic chelator can include a second "cycle", which is blocked by joining the branches of two or more atoms of the main structure. In the macrocyclic chelators, the alkyl and alkylene portions, unless otherwise specified, preferably contain up to 8 carbon atoms, especially preferably up to 4 carbons. The hydroxy or alkoxy substituted portions may be mono- or poly-unsubstituted, and substitution by atoms is contemplated. Any aryl portions are preferably bocyclic rings of 6 to 10 carbon atoms, or heterocyclic rings of 5 or 6 members. In the first cycle, the heteroatoms of the main structure, for example N, P, 0 and S are preferably separated by 1 to 8, especially preferably from 2 to 6 carbon atoms of the. Main structure and, as mentioned, the macrocyclic chelator preferably contains at least 3 carboxyl groups or carboxyl derivative groups. Particular preference is given to macrocyclic polycarboxylates containing at least three carboxyalkyl groups linked to ring nitrogens, especially carboxymethyl. 2, 2 The binding of the macrocyclic chelant to the R ((X)) pn portion of the main structure can be effected through any reactive group, for example, R or R, particularly preferably an R group. which contains a group CO-G. The reaction of macrocycles with protonated heteroatoms (where Hal is a halogen atom Y alq and X are as defined above), or with Hai-CH-CO-CH-,, and then with a diamine such as ethylene diamine, provides a reactive group for its union to the dendrimeric main structure. They can be used; other standard coupling techniques, and thus, the macrocyclic chelating portions in the poly ions of the invention preferably comprise the residues of a chelator of formula III (ie, groups of formula III but with one of the substituents attached to the modified or replaced ring) , to provide a junction to the dendrimer).

Conveniently, the mac-sprayable chelator is the residue of a polyazacycloalkane having 3, 4, 5 or 6 (preferably 4) nitrogens in the ring, each separated by 2, 3 or 4 (preferably 2) ring carbons. Particularly preferred skeleton or backbone structures for the maleocyclic chelating portions include the following: Particularly preferred macrocyclic chelants include those of formula IV: wherein each Z is N, 0 or S, preferably all, or all exceeds Z is N; each b is independently 2, 3 or 4, preferably 2 or 3; f is 3 or 4, preferably 4; 3 each R is independently hydrogen, alkyl of 1 to 3 carbon atoms, or a group CO-G-a L or il, optionally branched, optionally hydroxylated; and each R is independently hydrogen, or a hydroxyalkyl group. Thus, in particular, macrocyclic chelants include polyazacycloalkanepolycarboxylates, azazacrocycles (HAMs) and cryptates, including sepulchres and sarcophagi. Examples of polyazacycloalka npo licarboxylates include 1,4,7,10-tetraazacyclododecanetraacetic acid (DOTA), 1, 4, 7, 10-tetraazacic acid. ododecan-1, 4, 7-triacetic-co (D03A), l-o-a-4,7,10-tria: -aciclododecatriacetic acid (DOXA), ACID 1, 4, 7-triazaciclononant riacetic (NOTE) and 1,4,8,11-tetraazaciclotetradecantetraacetic acid (TETA). In addition, the novel tetraazacycloalkane polycarboxylates, D0TA-N (2-aminoethyl) amide and D0TA-N (4-aminophenethyl) amide are also contemplated. The preparation of the t-razazacycloalkane-polycarboxylate ligands is well known. The synthesis of DOTA is described in the U.S. Patent. No. 4,647,447 (Cries et al.), The U.S. Patent. No. 4,639,365 (Sherry) and by Desreux et al. in Inorg. Chem. 1_9: 1319 (1980). Additionally, DOTA is commercially available from Parish ChemicaL Co., Orem, UT, USA. The preparation DO3 A is written in EP-A-292689 (Squibb). Desreux, in Inorg. Chem., J_9: 1319 (1980); Bryden et al, Anal. Chem., _5_3: 1418 (1981); Delgado et al., Talanta, 29: 816 (1982, Cacheris et al., Inorg, Chem., 26: 958 (1987), Moi et al., Inorg. Chera., 26_: 3458 (1987); and Meares et al., Acc. Chem. Res., _1_7, 202 (1984) describe the properties and chemistry of the macrocyclic ligands DOTA, NOTE, TETA and their analogues derived from the main structure, including the preparation of NOTA and TETA. U.S. Patent No. 4,678,667 (Meares et al.) Teaches the preparation of a number of macrocyclic ligands derivatized in the side chain, including DOTA and TETA.The formation of DOTA derivatives to form D0TA-N (2-amipoet il) amide and DOTA-N (4-aminophenethyl) amide is described in detail below in Examples 2 and 3. The references cited above and all the other references mentioned herein are incorporated by the present by reference in its entirety. The hexaazarnacrocycles include the series of macrocyclic chelates of 6 nitrogen atoms described s in DeCola et al. in Inorg. Chem. ^ _5: 1729 (1986). That article also describes the preparation of the HAMs, / is hereby incorporated by reference in its entirety. The crypts are polycyclic ligands that include sepulchates, sarcophagnes and macrocyclic polyethers (crown ethers) and macrobicyclic ligands. Preferred macrocyclic polyether cryptates include the primary amine and carboxylate cryptates derivatized in the side chain.

The sepulchates include the octaaza-macrobicyclic system derivatives, such as 1, 3, 6, 8, 1, 13, 16, 19-oc taazabi-c [6,6,6] eicosane. The primary amine and carbo-xylate derivatives of these chelates are especially preferred. The synthesis of chelates, such as cobalt complexes, is described in J. Amer. Chem. Soc. 104: 6016 (1982). Sarco-phages include those derived from the hexaazamacrobicyclic system, such as 3, 6, 10, i 3, 16, 19-hexaazabicyclic 6,6,6] eico-healthy. The synthesis of sepulchates and sarcophagins are described by Creaser et al. in J. Amer. Chem. Soc. 104: 6016 (1982) and Geue et al. in J. Amer. Chem. Soc. 106: 5478 (1984), respectively. Izatt and Christensen, Eds., Synthe-tic Multidentate Compounds, Academic Press (1978) and Lehn et al, Acc. Chem. Res. H_: 49 (1978) describe the synthesis of cryptats. Cotton & ilkinson in "Advanced Inorganic Chemistry" describes a general method of synthesis model of crown ethers, to prepare nitrogen-containing macrocycles for encapsulation. These references are incorporated herein by reference in their entirety. Metal ions are selected for chelation by image enhancers because of their ability to perform their diagnostic or therapeutic role. These papers include, but are not limited to, intensifying imaging in MRI imaging, scintigating gamma affectation or CT scanning, or X-rays, or supplying cytotoxic agents to kill undesirable cells, such as in tumors. For use with radionuclides, such as in nuclear medicine, this invention provides the advantage of a close binding of the radionuclides by the macrocyclic chelators. This allows a more specific image, due to lower background levels of the metals. By suitable selection of the chelated species, chelates according to the invention can be produced which are capable of functioning as X-ray agents (for example by selecting tungsten) or as both, contrast agents for MRI and X-rays, selecting an ion metallic lanthanide appropriate. For X-ray applications, to extend the range of photonic energy over which the polychelates of the invention are optimally effective, the polychelates used may be of two or more different metals, either as mixtures of homopolykelates or as a heteropoliquelate. . The metals that can be inorganized, through chelation, include lanthides and other metal ions, including isotopes and radioisotopes thereof, such as, for example, Mg, Ca, Se, Ti, B, V, C'r, Mn, Fe, Co, Ni, Cu, Zn, Ga, Sr, Y, Zr, Te, Ru, In, Hf, Re, Os, Pb and Bi. The radioisotopes particularly referred to some of the anterio-. , 153c 64p 67 68"89" 88v 90v 99mt res include Sm, Cu, Ga, Ga, r, Y, Y, le, 97Ru, 10 RUj lllIn > 186Rfi (188Re> 203pb> 211B ± # 212Bi> 213 ^ 214 and Bi. The selection of the metal ion for chelation by the poly- chelators of the in tion will be determined by the therapeutic or diagnostic application des- aala. the selection of metal ions to be chelated by the polychelants of the invention depends on the diagnostic or therapeutic technique for which the resulting polychelate is to be used.For MR imaging, the metal ions must be paramagnetic, and preferably not For X-ray and ultrasound imaging, heavy metal ions should be used, for example with atomic numbers ie at least 37, preferably at least 0, again, preferably non-radioactive species.For scintigraphy or radiotherapy, Metal ions must, of course, be dioxide isotopes, and methods for complexing metal ions with chelating agents and polythenes. before they are within the level of a person skilled in the art. Each of the heavy metals can be incorporated into a macro-chelating portion by one of three general methods: incorporation will say: - a, model synthesis and / or transmetalation. Direct incorporation is preferred. The metal ions Fe (III), Cr (i [I), Mn (II), Hg (II), Pb (II), Bi (III) and the lant. Anides can be incorporated directly into the polyopolicarboxy polias by the following general procedure. A water-soluble form of the metal, generally an inorganic salt, is dissolved in an appropriate volume of distilled and deionized water. The pH of the solution will be less than 7. An aqueous solution containing an equimolar amount of the polychelant is added to the metal solution at room temperature while stirring. The pH of the mixture is slowly raised by addition of base, typically 0.1 M NaOH, until the donor groups of the polychelant are deprotonated. generally in e- range and pH from 7 to 9, depending on the chelating portions. Particular care must be taken with 1anthian ions to keep the pH below 8 to avoid precipitation of the metal hydroxide. The incorporation of the metal into the acrocyclic chelator portions derived or related to DOTA will normally be a slow process, as described in the references cited below. Specific examples of the process are contained in Examples e, and in the following references. Choppin et al, J. Inorg. Nucí Chera., 3_3: 127 (1971), Margeru, Rec. Chem. Progr. , 2? _: 237 (1973) and D'Olieslager et al., J. Inorg. Nucí Chem., 3¿: 4255 (1973) describe the direct incorporation of lanthanides to polyaminopoly-carboxylates. Margerstadt, in Magn. Res. Med., 3_: 808 (1986) v W0-A-87/06229 describe the incorporation of Gd (III) into DOTA. A method for preparing DOTA Bi and Pb complexes is described by Kumar et al., In J. Chem. Soc. Chem. Commun., 3: 145 (1989). The apiba references are incorporated herein by reference in their entirety. The direct incorporation of Hf, Zr, W, Hg and Ta can be carried out according to well-known methods. See, for example, the U.S. Patent. No. 4,176,173 (Winchell). The transmetalation is useful when the metal ion needs to be reduced to a more appropriate oxidation state, so that the donor atoms of the chelating moiety join. D. ,. 99mt 186 / 188D. ., For example, to incorporate Te or Re, the metaXLCO ion must be reduced to T(V) or Re (V) by the use of reducing agents such as SnC1"or cysteine, by well-established methods. This method requires the formation of a complex n-99m termediary. A typical example is the reduction of Te with S a in the presence of a weakly coordinating ligand, such as glucoheptonate, prior to comelination with chelators such as DOTA. These methods are known in the radiopharmaceutical technique. Cu utilizes tetraamine chelates such as tet A or tet B (see Bhardaredj et al., JACS, 108: 1351 (1986) to stabilize (u (II) for the reaction with strongly binding chelators. The model synthesis can be performed by the method described by Smith et al in Inorg. Chem., 2 _: 3469 (1985) and 1Z; 4154 (1988) • In the case of HAM systems, the intalic is incorporated into the macrocyclic chelator constructs the chelator around the metal ion via model synthesis Model synthesis methods well known to Smith et al. (above) are described for model synthesis for lanthanides Macrob: cyclic sepulchral chelators and sarcophagina can be prepared similarly by a model syn- thesis around the Co. The Co is eliminated by reduction to Co (II) and extraction with HBr 15 M. The metal-free chelator can then be metalated via the reaction with a salt simple metal, reflowing it in methanol, or by transmetallation of a donor complex such as the salts of glyco-heptonate, ascorbate, acetate or cyanide. The use of the triflate and / or perchlorate salts is preferred. The broad class of crown ethers and cryptats, especially those containing N, 0, and S, can be metalated in a similar manner, using one or more of the methods "> two described above. The metal chelates of the oliquelants of the invention, especially the bifunctional polychelants, but optionally also the image enhancing polychaelates, can be administered to patients to image in sufficient quantities to provide the desired contrast with the technique of formation of particular images. Generally, doses from 0.001 to 5.0 mmol of metal ion that are layered to form images per kilogram of patient's body weight are effective in achieving adequate contrast intensifications. For most FIRM applications the preferred doses of metal ion for imaging will be in the range from 0.005 to 1.2, for example 0.02 to 1.0, grams / kg of body weight, while for X-ray applications, the doses from 0.5 to 1.5 mmoles / kg are generally effective to achieve an X-ray attenuation. The preferred doses for most X-ray applications are from 0.8 to 1.2 mmoles of lanthanide or heavy metal / kg body weight. . For X-ray applications, in order to extend the photonic energy range over which the polychelants of the invention are optimally effective, the polychelants used can be of two or more different metals, either as homopolykelate mixtures or as a heteropoliquelate. The binding of the image enhancer to a site-directed molecule results in even greater target specificity in vivo. The molecule is preferably an antibody, antibody fragment, another protein L otr macromolecule, which will flow in vivo to that site to release the chelated metals. In the present invention, the ability of this site-directed raacro-molecule to travel and / or bind to its target is not compromised by the addition of the chelated atoms. The number of chelates per molecule is sufficient to intensify the image of that particular objective. The resulting bifunctional polychelates are isinated, and are desirably substantially non-crosslinked.

Preferably, the incorporation of metals into the bifunctional polychelants is effected prior to attachment of the image enhancer (s) to a targeted-site molecule. The metal is titled from sub-estequ levels:? metric until total incorporation, thus eliminating the need for dialysis and extensive chromatographic purification. In this way significant losses are avoided, as well as dilution. Non-specific binding of metal ions to site-directed molecules is also prevented. However, the application of the invention to radionuclides with short half-lives may require. Metalation of the bifunctional polychelant as a final step, is effected by a rapid and simple purification (for example gel filtration) to eliminate excess free radionuclide. In the bifunctional polychelant, preferably one or two molecules of the main structure are attached to the site-directed molecule. By limiting the number of image enhancers attached to the site-directed molecule, it would be expected that the pharmacological behavior of the bifunctional protein will show high specificity for the target and low non-specific binding. The bifunctional polychelators are capable of containing a large number of chelating to rociclic portions. This allows the formation of images if 11 or -specific intensifies beyond the levels previously available.

The bifunctional polychelants of the invention involve the coupling of the image enhancer to a site-directed molecule. The site-directed molecules can be any of the molecules that naturally concentrate in an organ, tissue, cell or group of selected target cells, or another site in the body of a mammal, in vi. These may include amino acids, oligopeptides (eg hexa-peptides), molecular recognition units (RMU's), single-chain antibodies (ACU's), proteins, Fab fragments, and antibodies. Examples of site-directed molecules include polysaccharides (e.g. CCK and hexapeptides), proteins (such as lectins, asialoyetuin, polyclonal IgG, blood coagulation proteins (e.g. hiru-dina), L ipoproteins and clicoproteins) , hormones, growth factors, and coagulation factors (such as PF4). Examples of site-directed proteins include polymerized fibrin fragments (eg E.), serum arailiode precursor (PAS) proteins, low density lipoprotein precursors (LBD, serum albumin, pro-proteins of the surface of intact red blood cells, receptor-binding molecules, such as estrogens, liver-specific proteins / polymers, such as galac-tosyl-glucoalbumin (NGA) (see Vera et al., Radiology 151: 191 (1984)) ), N- (2-hydroxypropyl) meta-crilamide copolymers (HMPA) with variable numbers of bound galactose-nines 1 (see Duncan et al., Biochim Biophys, Acta 880: 62 (1986)), and allyl and 6 -amyr? ohexyl glycosyls (see Wong et al., Carbo Res. 170: 27 (1987)), and f ibr i ud ^ ene The site-directed protein can also be an antibody. In particular, the antigen specificity of the anti-tumor will depend on the desired use of the antigen. The conjugated antibodies are preferred over the polyclonal antibodies. Human serum albumin (A3'l) is a pre-L protein ferida for the study of the vasculir system. ASH is commercially available from a number of sources, including Sigma Chemical Co. The preparation of antibodies that react with a desired antigen is well known. Antibody preparations are commercially available from a L 5 variety of sources. The E-fibrin fragment can be prepared as described by Olexa et al. ^ n J. Biol. Chem. 254: 4925 (1979). The preparation of LBD precursors and PAS proteins is described by d Beer et al. in J. Immunol. Methods 5_0: 17 (1982). The articles described above are hereby incorporated by reference in their entirety. The methods for linking the main structure polymers to antibodies and other proteins are within the skill level in the art. Such methods are described in the Pierce 1989 Handbook and General and the references cited therein., in Blatter et al., Siochem., 2j4: 17 (1985) and in Jue et al., Biochem., L7: 5399 (1978). The references cited above are incorporated herein by full reference. In general, polynallants are bi-functional and are synthesized by constructing the polychelant before conjugating the main structure polymer to the site-directed macromolecule. In most cases, the reaction conditions used to attach the chelators to the main structure denatures the proteins. Therefore, in order to preserve its tertiary structure and biological function, an antibody or other stable protein will usually not be bound to a molecule of major structure before the chelating groups have been loaded onto the molecule. main structure molecule, unless of course this can be l? e >; * ho without denaturing the protein. Alkalic ions can be added to form the metal complex of the polychelants before or following the conjugation of the image enhancement to the site-directed macromolecule. Preferably, the metal will be added before conjugation of the polychelant to most of the proteins, particularly antibodies, in particular to prevent adventitious binding of the metal to the protein. However, for some metal ions such as ionucleides with a short half-life, metalation will preferably be performed following conjugation, immediately before use.

In general, known methods can be used to bind the macrocyclic chelants to the main structure molecules. While for the preferred macrocyclic chelators, such as DOTA, the techniques of conjugation of the mixed anhydride and conventional cyclic anhydride are ineffective, it has been found that by modifying the mixed anhydride process by reacting a polycarboxylic acrocyclic chelator in an anhydrous medium z? N an amine base of sufficient strength to abstract all protons from the carboxyl (ie, a sufficiently high pKa) produces an amine salt, which can react with an alkyl haloformate (eg, isobutyl chloroformate), to produce an activated anhydride capable of being conjugated to the amine groups of the main structure molecule without causing the undesired crosslinking associated with the bifunctional polychelants of the prior art. For most macroclic chelators, tetramethylguanidine or an amine base of similar strength will be the preferred base. Instead, more complex conjugation techniques can be used, which involve, for example, the use of derivatized macrocyclic chelators. the main structure, in a manner analogous to that of Meares et: al. ( above ). Similarly, the chelators can be linked to the main structure molecule by a haloacetylhalfide method, a phosgene or a thiophos, depending on the reactive group available on the chelating agent. For ratocrocycles with a pending carboxylate, which include but are not limited to DOTA, TETA, TRITA (1, 4, 7, 10-tetraazacylcytidecarotriatic acid) and NOTE, one of the carboxylates may form an entity that can react with a Primary amine group of the main structure molecule. Methods for forming a reactive entity of a carboxylate group include the reaction of the modified mixed anhydride, for example using isobutyl chloroform (CFIB), or the formation of an "activated ester" using a carbodiimide (DCC or EDAC, see Pierce Catalog ( 1988 l pages 252 and 253.) Both reaction sequences give origin to a polymer with a multiple-substituted substituted backbone, with the macrocyclic chelating portions through stable amide linkages, however, the modified mixed hybrid method is the Preferred method to use to bind the carbocyclic containing ionic chelators to the main structure molecule The reaction of the modified mixed anhydride is carried out in an anhydrous solvent, preferably with a melting point below 5 ° C, cooled to a temperature not lower than 5o C or greater than about 55 ° C above its freezing point .The solubilization of the chelant in the dissolven The appropriate one is conveniently carried out by the preparation of the amine salt of the chelant, using the amine base in situ.

The selection of b is determined by the pKa of the relevant carboxylates. For most macrocycles, tetramethylguanidine (TMG) is especially preferred. In general, bases will be conveniently selected from those bases whose pKa value exceeds the highest Ka of the acrocyclic chelator by at least 0.5, preferably 0.8, especially preferably at least 1.3. Particularly preferred are amine bases having pKa's of at least 11, especially at least L1.3, particularly at least 12, and in addition to the TMG, particular mention may be made of the piperazine, quinuclidine and N-ethylpiperidine , and more especially of DBU (1, 8-diazab iciclo [5.4.0] undec - 7- no) and DBN (1,5-d? azabic? clo [4.3.0] non-5-ene). Additional bases are listed by Martell and Smith in "Critical Stimulation Constants" Vol. 5, first supplement, Plenum Press, \ 'Y 1982. The appropriate amount of unblended (cooled) alkyl haloformate is now added with agitation, and the temperature The original solvent is maintained by cooling, p r adding a cooler, if required. Specifically preferred is isobutyl chloroformate. The activated anhydride resulting from the macrocyclic chelator can be reacted with an amine-containing main structure molecule to form a polychelant or polychelant fragment. The polychelant, for most applications, is metalized at this point, and purified by chromatography or by crystallization, to remove excess metal ions and metal complexes of lower molecular weight. For use with specific molecules for a purpose, the imaging enhancer polychelant, or the at least partially metalated form thereof, which still contains at least one free amine, is conjugated to the molecula directed to selected targets, for example by reaction with One of the many coupling agents has been known to work well. In situations where the goal is not appropriate, such as with radionucl ion ions, those with short half-lives, the bifunctional polycarbonate can be prepared using a metal-free image enhancer, and coupling as describes above, followed by me ta 1ac i <; "> n (see below) and a rapid and simple final purification, by chromatography or filtration.Racrocroccal chelators can also be attached to a major structure molecule through a primary amine group that is not coordinated. Macrocyclic chelators that have a primary uncoordinated primary arrine group include the DOTA macrocycles derived in the side chain with primary amine, D03A derivatized with primary amine, and macrocycles and macroazikes of hepaza and octaza derivatized with primary amine. (HAMs, sepulchres and sarco-phaginas), as well as the wide range of derivatized crown ether cryptats.

The non-coordinating primary amine group on the chelating agents can be reacted with a haloacetyl halide under well-known conditions to form a haloacetamide. The haloacetamide may react with a primary amine of the parent structure molecule to form a stable amide bond between the chelant and the backbone. The haloacetylhalur method described in De Riemer et al., In J. Labelled Compd. Radiophar 1: 8: 1517 (1981) can be used to bind amine-containing chelators to the main structure. The amine groups found in a macrocyclic chelator can also be reacted with phosgene, to generate a reactive isocyanate group, or with thiophosgene to generate a reactive isothiocyanate group. These groups can react with a primary amine of the main structure, to form a urea or more stable thiourea link respectively, between the ligand and the main structure olimer. Gansow, in Inorg. Chimica Acta _9] _: 213 (1984) and Moi et al., In J. Amer. Chem. Soc. 110: 6266 (1988) describe methods for linking chelants to proteins having an amine group, through the formation of the isocyanate or isothiocyanate portions, using the phosgene or thiophosgene methods, respectively, see also Desreux , in Inorg, Chem. _1_9: 1319 (1980), Bryden et al., in Anal. Chem. 5_3: 1418 (1981), Delgado et al., in Talanta 29: 815 (1982), Cacheris et al., in Enorg, Chem. _ 26, 958 (1987), MOL et al., In Inorg. Chem. 26: 3458 (1987), and Meares et al., Cc Chem. Res. J_7: 202 (1984). the main structure to which the chelants are conjugated in this way can be formed in whole, ie, it can comprise a portion R (XR), alternatively and preferably, the polychelant fragments can be conjugated on A pre-formed polymeric main structure R. This forms a main aspect of the invention Thus, in this way, each bonding site on the ur structure The polymer can be charged with a plurality of chelating groups. On the other hand, if the polylayer fragments are already metalated, the metal charge of the final polychaelating species can be either minimized, since the chelating groups that are not located near the periphery of the molecule can be inefficiently metalated in another way. While for the compounds of? It is preferred that the present invention be X 'metabolically hydrolysable, the conjugation of the preformed polycarbon subunits offers a synthetic approach to other chelating agents, and in particular to those in which X more than X is hydrolyzable, and these new compounds also form an aspect of the invention. Accordingly, in a further aspect, the invention provides a method for preparing polychelants of formula II (in which between each group L and R at least one of the 1 2 X and X portions involved is metabolically hydrolysable), the method comprises conjugating compound of formula with a polychelant fragment molecule of formula VI 8"> 2 XßR '- ((XZ) pL) n (VI) 7 8 (wherein X and X comprise reactive groups which can be conjugated to form a group X). The polychelant fragments of formula VI are in themselves novel, and they, their chelates and their salts also form an addition aspect of the present invention, It is especially preferred to carry out this method according to the invention using metallated polychelant fragments. of formula VI.In the compounds, the chelated metal is firmly retained by the macrocyclic gruí or chelator, and thus an optimum metal loading of the final polythelant can be achieved.However, the chelating portions can of course be metalated or transmuted. cuttings after the polychelant has been formed, or a bifunctional polycarbonate derivative thereof, and such metallization or metallization processes form an additional aspect of the present invention. of the polychelant fragments of the present invention can be prepared by reaction of monokelating species with a binding molecule, followed if necessary by the activation or introduction of the reactive group X, through which the molecule of the poly-chelator fragment is bound in due time to the main structure of the polymer. The polychelant fragments can be dimers, trimers or higher oligomers, but in a preferred embodiment of the present invention, they are based on low-generation den-drimeric polychelators, that is, from zero to sixth generation, for example dermateric polychelators such as those described in International Patent Application No. PCT / EP92 / 02308. By using such a molecule of fragments of den-drimeric polychelants, poly (poly-Dendritic elastants) can be produced. This can be done by reacting a dendrimer with a bifunctional linking agent, to produce a monoderivatized dendrimer, which can then be loaded with monokelating groups, to produce the molecule of the polylinker fragment. This molecule of the dendritic lichen fragment p can be conjugated to a linear or branched polymer, but preferably it will be conjugated to a dendrimeric backbone. Alternatively, the polychelant fragments themselves can be polymerized to produce linear or branched poly (chelator) molecules. Such poly (polysiloxanes) form a further aspect of the invention, wherein they incorporate metabolically and hydrolyzable linking moieties, to release fragments below 40, 0D with the hydrolysis, as well as their chelates and salts, and the corresponding bifunctional polychelants. Such non-hydrolysable binding moieties may be between or within the polychelant fragments, preferably in both positions when the fragments cise more than 4 patent chelating portions, for example when they are dendrimeric. Such poly (pol-chelants) can be represented by formula VII: [R2 ((X2) p KxM (VII) nqm where R, L, p, n and m are as defined for formula 1. q is a positive integer , preferably 1, 2 or 3, X is 2 2 a binding portion joining two R os, X is a binding portion 1 2, and X and / or X are metabolically hydrolyzable, to provide hydrolysis products having molecular weights below 40,000 D, preferably below 0.000 D, especially below 2 O. U00 D. Thus, for example, when the polychelant fragment is a star-shaped dendrimer of μiamine of G, n of the type described by Tomalia et al (top) loaded in 22 of its 24 terminal amino groups with GdD03A groups, then only about 4 of the fragments need to be linked with one another to produce an imaging agent to measure the accumulation of blood. Therefore, viewed from another aspect, the invention also provides ona polychelant cunds of formula VIII (where each p is 0 or 1; n and m are each positive integers, which have values of at least 2, and are such that the total number of portions L is at least 20; q is a positive integer, example from 1 to 100, X 1 and where X2 is present are metabolically hydrolysable portions, each X serves 2 2 1 to join a portion R ((?) L) to one portion R, or to another 2 2 portion R ((X)); each L is a macrocyclic chelating moiety?; R when present is a linear or branched polymer portion; and each R is a portion of the linear or branched main structure; the compound in its non-metalated form has a molecular weight of at least 30,000, and the fragments resulting from the metabolic hydrolysis of the X-portions and wherein this pre-X, have molecular weights below 30,000. D in its non-neat forms), and those which, salts and conjugates thereof with site-directed molecules. Examples of suitable reaction schemes for the preparation of polychelant molecules and polychelant fragments according to the invention include the following: < TO) , NH2 Gd OTA? Í? Ydri-; + H2N * NH2 (branched triamine monoprotegi a) Gd - -NH • N * H2 1) NH deprotection Gd N- O 23 C on version (Where X - NCS, NCO, NHCOC ^ O = Main structure, for example alkylene chain = protected amine • GdD03A residue bound to N) (B) activated Aivación or deprotection GdN-, optional, where is V -NH? Jz appropriate // O (Where z = 0 to 4 and A-NC-, CI ^. COOH, Nl ^ or OH) N ^ CHICHOHCHÍNHJ Ns ClCOC ^ Cl (11 • pp? .rpNHpj.p. (where Lv = leaving group, for example halide, OTs) CD) It has HO-. Í4) -O HO-- -NO. iS (Where Lv '= OTs, OMs, ^ r, etc. and R "= 0, CONH, NHCO, _-c) (E > (Where N * H represents a 1, 4, 7, 10-t etraazacyclododecane protected in ngens 4, 7 and 10) (P) BocNH (7) OC CONH N NHCOCH, at 2 > aciicoa (8) (10) GdD03A activated NH | 0 *? 'r ~ ís afiB, oo Poly (dendrimeric polychloride) (H) Dendrimer formation HiN [COOH] » (aminated carboxylic acid 1) CI% CHOQCHj long chain, protected 2) NHj C3iCH1NHJ in the carboxy group, for 3), 4) Repeat 1), 2), etc., example protected 8-aminocaprilic acid) Quelate binding (As in Scheme B above CD or Dendrimeric poly chelate (where SSS = solid state support, for example Merrifield resin, X = SSS binding site, X = X hydrolysis residue, activated GdD03A = eg GdD03A with NH of the substituted ring, for example M ^ In the schemes outlined above, (Gd N- is indicated as representing a D03? Residue loaded with gadolinium.) However, it can also represent any other necrotic or non-charged macrocyclic chelator residue bound to a ring heteroatom or A carbon of the ring In the case that non-metallated chelating portions are used, the metallation can be carried out in an intermediate step, during the preparation of the molecule of the polychelant fragment, after the preparation of the fragment molecule. of polychelant, or even after the preparation of the total polycarbonate In the method of scheme I, the fragments of the polychelant are constructed on a support in the solid state.This can be done using processes analogous to those described for the synthesis of Peptides by Stewart and Young in "Solid State Peptide Synthesis", 2nd Edition, 1984, Pierce Chemical.

Where, as in schemes 0, B, the polychelant fragments are dendrimeric polychelators, it is preferred that they have molecular weights in the unmetalated state in the range of 5 to 25 kD, and that poly (polychelators) constructed with these should contain from 3 to 10, preferably from 3 to 6, of such fragments. In addition to the methods discussed above, other methods can be used to join fragments of dendrimeric polychelant with one another. In this respect, attention is directed to the techniques discussed by Torchilin et al. in Criti-cal Revie s in Therapeutic Drug Carrier Systems 7: 275-308 (1991), US-A-4737550 (Tomalia et al.) and Brinkley, Bioconjugate Chem. 3: 2-13 (1991). The compounds of the present invention can be formulated with conventional pharmaceutical or veterinary auxiliaries, for example, emulsifiers, fatty acid esters, gelling agents, stabilizers, antioxidants, osmolality adjusting agents, buffers, pH adjusting agents, etc. ., and may be in a form suitable for parenteral or enteral administration, for example, injection or infusion or administration directly to a body cavity having an external vent, for example the gastrointestinal tract, the bladder or the uterus. Thus, the compounds of the present invention may be in conventional pharmaceutical administration forms, such as tablets, capsules, powders, solutions, uspensions, dispersions, syrups, suppositories, etc.; however, solutions, suspensions and dispersions in a physiologically acceptable carrier medium, for example water for injections, will generally be preferred. The compounds according to the invention can therefore be formulated for administration using physiologically acceptable carriers or excipients, in a manner fully within the skill of the art. For example, the compounds, optionally with the addition of pharmaceutically acceptable excipients, can be suspended or dissolved in an aqueous medium, and the resulting solution or suspension is then sterilized. Suitable additives include, for example, physiologically compatible buffers (such as for example tromethamine hydrochloride), additions (for example, from 0.01 to 10 mole percent) of chelants (such as, for example, DTPA, DTPA-bisa ida or non-complexed image enhancer polychelant) or calcium qaelate complexes (such as for example calcium DTPA, CaNaDTPA-b and amide, calcium image enhancer polychelant, or CaNa salts of image enhancer polychelants), optionally, additions (for example 1 to 50 moles per scientist) of calcium or sodium salts (eg, calcium chloride), calcium ascorbate, calcium gluconate or calcium lactate combined with metal chelate complexes or ligands of image enhancer, and the like). If the compounds are to be formulated in suspension form, for example in water or physiological saline for oral administration, a small amount of soluble chelate can be mixed with one or more of the inactive ingredients traditionally present in the oral solutions and / or surfactants and / or aromatic compounds, to aromatize it.

For MRI and X-ray imaging of some portions of the body, the most preferred way to administer metal chelates as contrast agents is parenteral administration, for example intravenous. Parenterally administrable forms, for example intravenous solutions, must be sterile and free of physiologically unacceptable agents, and must have low osmolality, to minimize irritation or other adverse effects of administration, and thus, the medium of contraction. This should preferably be isotonic or slightly hypertonic. Suitable carriers include aqueous vehicles, ordinarily used to administer parenteral solutions such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Throat Injection and Sodium Chloride, Lactated Ringer's Injection and other solutions, such as or those described in Reming-ton's Pharmaceutical Sciences, 15th d. Easton: Mack Publishing Co., pages 1405-1412 and 1461 -? 87 (1975) and The National Formulary XIV, 14th ed. Washington :: American Pharmaceutical Association (1975). The solutions may contain preservatives, anti-microbial agents, buffers and antioxidants conventionally used for surface solutions, excipients and other additives which are compatible with the chelates, and which will not interfere with the manufacture, storage or use of the products. Viewed from a further aspect, the invention provides an imaging or therapeutic enhancement composition, comprising a metal chelate of a pendant of the invention, or a salt thereof, together with at least one pharmaceutical carrier or excipient. Viewed from a still further aspect, the invention provides for the use of a lip ballast according to the invention, or a chela or salt thereof, for the manufacture of an image enhancing contrast medium, or a therapeutic composition. Seen from another aspect, the invention provides a method for generating an image of a body of a human or non-human animal, especially a mammal, the method comprising administering to the body an image enhancing amount of a polychelate according to the invention, or a salt thereof, and after that, generate an image, for example an image by RM, r yos X, ultrasound or graphic scintigraphy, of at least a part of the body. Viewed from a still further aspect, the invention provides a method of radiotherapy of the human or animal body, the method comprising administering to the body a therapeutically effective amount of a radioactive metal chelate of a polychelant according to the invention. Viewed from another aspect, the invention provides a composition for detoxification, comprising a polychelant according to the invention, or a weak complex of chelate, or a salt thereof, with physiologically tolerable counterions, together with a carrier or excipient. pharmacist.

Viewed from a still further aspect, the invention provides a method of metal stoxification, comprising administering to a human or non-human animal a detoxifying amount of a polychelant according to the invention, or a weak complex of chelate or a salt of it, with physiologically tolerable counterions.

It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present disclosure of the invention.

Having described the invention as above, property is claimed as contained in the following:

Claims (9)

1. CLAIMS 1. A compound of formula I R1 (X1R2 ((X2) pL) n) m (I) (characterized in that X is a hydrolyzable, hydrolyzable binding moiety, to release RX v XR fragments ((X) L) m 'pn where X 3 and .X4 are the hydrolysis residues of X 1; 1 3 RX is a biotolerable polymer; m XR "((X) L) is a fragment of pol iq uelante that has a molecular weight below 40,000 D, p is 0 or 1; 2 X, when present is a hydrolyzable metabolically binding portion to release a monokelan fragment, each L is a portion of a rocyclic raa chelator, wherein the macrocyclic backbone preferably has from 9 to 25 members in the ring, 2 2 R ((X)) is a portion of the straight or branched chain main structure, each n is an integer that has a value of at least 2, and each m is a whole having a value of at least 2, such that the total number of L groups in the polychelant of formula I is at least 20), which has a molecular weight of at least 30,000 D, and metal chelates and salts thereof.
2. 2. A compound according to claim 1 3, characterized in that the R X fragment of hydrolysis is a monodisperse oligomer having a molecular weight below 40 kD.
3. 3. A compound according to any of the rei indications 1 and 2, characterized in that the fragment "i R ((X) L) has a molecular weight below 30 k D. p n J
4. 4. A compound according to the claim 3, characterized by the fact that the X R ((X) L) fragments of hi-P n d rol i sis are monodisperse.
5. 5. A compound according to any of the indications 1 to 4, characterized in that the fragment 1 Xm ~ of hydrolysis is a dendrimer from the first to sixth administration. 6. A compound according to any of the rei indications 1 to 5, characterized in that it has a molecular weight in the range of 50 to 150 kD. 7. A compound of formula II R1 (X1R2 ((X2) npL) nn) mm (II) 1 2 (characterized in that R, R, L, p, rt and m are as defined 1 and nio in claim 1, and X and X are linking portions, with the proviso that between each LR at least one of the 2 1 X and X binding portions is metabolically hydrolysable) and the metal chelates and salts thereof, as well as the corresponding bifunctional polychelants. 8. A polychelant fragment compound of formula X5R2 ((X2) L) p n
6. 6. 9 - (characterized in that R 2, X 2, p, L and n are as defined in claim 1, and X is a functional group that provides a c-valent binding site for the fragment compound). 9. A compound according to claim 8, of formula (A) to (G) - or ÍD) -or N fifteen 25 (characterized in that -N) is a macrocyclic chelator bound to nitrogen, and (G) is a dendrimer terminated in (N-). 10. A polymeric polychelant compound of formula VII: ## STR4 ## (R2 ((X2) PL) nX1 q] Jm (VII) characterized in that R, L, p, n and m are as defined for formula II in claim 7, that is a positive integer, X 1 is a linking portion joining two groups R2, X2 is a linking portion, and X and / or X are metabolically hydrolysable, to give products and hydrolysis having molecular weights below 40,000D. 11. A compound of polychelant of formula VIII: (R1) p [R2 ((X2) pL) nX] q] i (VIII) (characterized in that ca ap is 0 or 1; n and m are each positive integers having a value of at least 2, and are such that the total number of portions L is at least 20, q is 1 2 a positive integer, X and when X is present are hydrolysable metabolic portions 1, each X serves to join a 2 2 1 2 2 portion R ((X) L) au portion R oot ra tion R ((X)); pnpn each L is a macrocyclic chelating portion, R when present is a linear or branched polymer portion; and every 2 R is a portion of the linear or branched main structure; the compound in its non-metallated form has a molecular weight of at least 30,000 D, and the fragments resulting from the metabolic hydrolysis of the X portions and when X is present having molecular weights below 30,000 D in its non-metallated forms) and the chelates, salts and conjugates thereof with site-direct molecules. 12. A compound according to any of claims 1 to 11, characterized in that it comprises L-portions of metallated chelators. 13. A compound in accordance with the claim 12, characterized in that the chelator portions are metalated with paramagnetic metal or heavy metal ions, or with radionuclides. 14. A compound in accordance with the claim 13, characterized in that the chelating portions are metalated with paramagnetic lanthanide ions. 15. A compound according to any of claims 1 to 14, characterized in that it is conjugated to an agent that modifies the biodistribution. 16. A pharmaceutical composition characterized in that it comprises a compound according to any of claims 1 to 15, with at least one physiologically tolerable carrier or excipient. 1
7. 7. A process for the preparation of a chelate of a chelating compound of conf rity in any of claims 1 to 15, the process comprises metalating chelating groups L which are therein. 1
8. 8. A process for the preparation of a compound according to claim 1, the process is characterized in that it comprises conjugating to a polyfunctional main structure compound a plurality of polychelant fragment compounds defined in claim 8, optionally in the metalated form. 1
9. 9. A method for generating an image of a human or non-human animal body, the method is characterized in that it comprises administering to the body an image enhancing amount of a polychelate of a compound according to claim 1, or a salt thereof, and after that, generate an image of at least one part of the body. 20. The use of a polychelant compound according to claim 1, or a chelate or salt thereof, the use is characterized in that it is for the manufacture of a contrast agent that improves the image, or a therapeutic composition. SUMMARY OF THE INVENTION The present invention provides polychelant compounds, which are useful for example in diagnostic imaging procedures, and which are degradable in vivo to release excretable fragments. Such compounds 1 1 2 2 conveniently are of the formula (I): R (X R ((X) L)) p n m (wherein X is a metabolically hydrolyzable binding moiety for releasing RX and R) fragments ((X) L), where mpm X and X are the hydrolysis residues of X; RX is a biotolerable lime pore, preferably a substantially monodisperse polymer, and especially an anus with a molecular weight below 40,000 D, particularly below 30,000 D, and especially below 20,000 D, for example a first to sixth generation dendrimer; X. F ((X) L) is a polychelant fragment having a molecular weight below 40,000 D, preferably below 30,000 D, especially below 20,000 D, each such portion preferably being the same, p is 0 or 1;>, when present, is a metabolic and hydrolyzable binding portion to release a monochelant fragment, each L is a macrocyclic chelating moiety, wherein the macrocyclic backbone preferably has 9 to 25 members in the ring, and preferably is a polyazacycloalcium optionally interrupted by oxygen or sulfur; R ((X ^)) is a portion of the straight or branched chain main structure, which preferably provides a chain of up to 20 atoms between each group L and the X portion to which it is attached, and a chain of up to 25. atoms between each pair of L groups bonded thereto, such chains conveniently are carbon chains interrupted by nitrogen and / or oxygen and / or sulfur; each n is an integer having a value of at least 2, preferably a value of 2 to 25, especially from 2 to 12; and each m is an integer having a value of at least 2, preferably a value of up to 200, especially from 3 to 100, - such that the total number of L groups in the polychelant of formula (I) is at least 20. , preferably from 50 to 200), having a molecular weight of at least 30.D00D, preferably at least 40,000D, and especially preferably 50,000 to 150,000D, and metal chelates and salts thereof.