NHR CONTRAST AGENTS

Abstract: The invention provides parenterally administrable, liver-specific MR imaging contrast media comprising Mn(II) chelates of N,N'-EDTA bisamides.

Description: NHR CONTRAST AGENTS This invention relates to an improved method of magnetic resonance imaging of the liver using contrast agents not previously proposed for this purpose and to the use of such contrast agents in diagnostic imaging of the liver. The use of paramagnetic ions of transition metal and rare earth elements such as iron, manganese and gadolinium in magnetic resonance imaging (MRI) contrast agents for diagnostic purposes is well documented, enhanced contrast being achieved by the effect of the magnetic field of the paramagnetic species in increasing the nuclear spin relaxation rates of non-zero spin nuclei (generally water protons) associated with or in contact with particular biological tissue. It is furthermore well known to complex such ions with a wide range of chelating agents i order to reduce their toxicity and facilitate their administration, e.g. by confering or enhancing water solubility. One particularly effective class of chelating agents for this purpose comprises aminopolycarboxylic acids and derivatives thereof, e.g as described in US-A-2407645 (Bersworth) , EP-A-71564 (Schering) , EP-A-130934 (Schering) , EP-A-165728 (Nyσomed) , US-A-4647447 (Schering) , US-A-4826673 (Mallinckrodt) , US-A-4639365 (Sherry) , EP-A-263059 (Schering) , EP-A-230893 (Bracco) , EP-A-325762 (Bracco) , WO-A-86/06605 (Lauffer) , US-A- 4746507 (Salutar) , EP-A-290047 (Salutar) , WO-A-90/01024 (Mallinckrodt) , US-A-4687659 (Salutar) and EP-A-299795 (Nycomed) and in the documents cited in these patent publications; representatives of such chelating agents include DTPA, DOTA, BOPTA, HP-D03A, DTPA-BMA, TTHA, DPDP and EDTA. It is frequently desired that such MRI contrast agents should exhibit specific affinity for a particular biological tissue or tissue component in order to permit targetting of a particular organ. Thus the aforementioned WO-A-86/06605 discusses characteristics of chelating agents which encourage specific uptake of MRI contrast agents containing such chelants by human hepatocytes, so as to permit enhanced imaging of the liver. It is observed that chelant properties which cause preferential uptake by hepatocytes compared to reticuloendothelial cells also cause tight binding to proteins. Factors which are said to encourage such protein binding include (i) the presence in the contrast agent of hydrophobic groups, which bind to hydrophobic regions in the protein through van der aals interactions - the contrast agents thus preferably contain at least one, usually two, aryl rings and/or hydrophobic alkyl groups attached to backbone carbon atoms of the chelant; (ii) the possibility of electrostatic interaction between the contrast agent and the protein, for example as achieved by using a negatively charged contrast agent (e.g. having non- coordinated carboxylate or sulphonate groups) where the protein is known to have positively charged binding sites (e.g. as in human serum albumin) ; and (iii) the use of contrast agents with comparatively high molecular weights - a preferred minimum molecular weight of 300 is quoted, but in practice contrast agents having molecular weights of 500-1000 are normally employed. It has also been observed (see, for example, the aforementioned EP-A-290047) that use of polydentate ligands rather than monodentate or bidentate ligands in complexing metals such as manganese (II) is of advantage in that it may lead to Mn(II) chelates with a high affinity hexadentate ligand, which configuration provides a particularly stable and effective form for administration. It will be noted that existing liver-specific MRI contrast agents such as the manganese (II) chelate of N, N A -bis(pyridoxal-5-phosphate)ethylenediamine-N,N l - diacetic acid - Mn(DPDP) - and the gadolinium (III) chelate Gd(BOPTA) , where the chelant BOPTA has a DTPA structure in which one of the N 3 carboxymethyl groups is replaced by a 2-benzyloxy-l-carboxyethyl group, fulfil the above criteria of containing hydrophobic aryl moieties, having molecular weights in the range 500- 1000, and containing polydentate chelating agents which form negatively charged chelate complexes. The present invention is based on our discovery that a range of hydrophilic, non-ionic Mn(EDTA) complexes promote good MRI contrast in the liver following parenteral administration. This liver- specificity is most surprising in view of the findings discussed above - it would conventionally be expected that such hydrophilic non-ionic chelate complexes would be extracellular agents and as such would be excreted renally rather than acting as liver contrast agents. Thus viewed from one aspect the invention provides the use of a manganese ■ compound for the manufacture of a parenteral hepatobiliary MR imaging contrast medium, said manganese compound being a complex of Mn(II) with a hydrophilic chelating agent of formula I wherein each R, which may be the same or different, is a hydrogen atom or an optionally hydroxylated C w alkyl or alkoxyalkyl group. In the chelants of formula I, the R groups are preferably selected from hydrogen, methyl and hydroxylated C,. 3 alkyl. Particularly preferably, carbon backbone R groups, i.e. in CHRCHR, are hydrogen, hydroxymethyl or 2,3-dihydroxypropyl, especially hydrogen, and amide R groups, i.e. in NRj, are independently hydrogen, methyl or hydroxylated C h alk 1. The NR j groups are conveniently the same and advantageously selected from N-methy1-2,3- dihydroxypropylamine, methylamine, dimethylamine, bis(2- hydroxyethyl)amine, N-meth 1-2-hydroxyethylamine, and 2,3-dihydroxypropylamine. The liver-imaging complexes according to the invention are Mn(II) chelates with hydrophilic bisamide derivatives of EDTA. The use of a number of chelants of this type has previously been proposed, for example in the above-mentioned EP-A-130934, which discloses a broad range of amide group-containing paramagnetic hydrophilic chelates; there is no suggestion in this specification of any advantage to be gained by complexing Mn(II) with EDTA-derived chelants of this type or that the complexes may be used directly for liver imaging purposes, it being suggested that they should be formulated as liposome inclusion compounds where it is desired to enhance liver contrast. White et al in Invest. Radiol. 25: S56-S57 (1990) describe a number of nonionic paramagnetic metal complexes as potential MRI contrast agents, including an Mn(II) complex with an EDTA bisamide derivative in which the amide groups are both N-substituted by dihydroxypropyl groups; again there is no suggestion that such a complex might exhibit liver specificity. Complexes useful in accordance with the invention include those of Mn(II) with hydrophilic chelants of formula (la) where each of R'-R 6 , which may be the same or different, is selected from hydrogen atoms, optionally monohydroxylated lower alkyl and lower alkoxyalkyl groups, and polyhydroxylated alkyl and alkoxyalkyl groups. Lower alkyl and lower alkoxyalkyl groups may, for example, contain up to six carbon atoms, e.g. as in methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec- butyl, tert-butyl, methoxymethyl and methoxyethyl groups; such groups may be substituted by one or more hydroxyl groups as in, for example, 2-hydroxypropyl, 3- hydroxypropy1, l-(hydroxymethyl)ethyl, 2,3- dihydroxypropyl, tris(hydroxymethyl) ethyl, 2,3- dihydroxy-l-(hydroxymethyl)propyl, 2,3,4,5,6- pentahydroxyhexyl, 2-hydroxyethyl, 2-hydroxy-l- (hydroxymethyl)ethyl and 2,3,4-trihydroxybutyl groups. Hydrophilic chelants of formula (la) include compounds in which R 3 and R 4 both represent hydrogen atoms and R 1 , R 2 , R 5 and R 6 are each selected from hydrogen atoms and methyl groups and compounds in which at least one of R'-R 6 is a hydrophilic hydroxylated or polyhydroxylated group, for example hydroxymethyl, 2- hydroxyethyl or 2,3-dihydroxypropyl. Useful chelants of this type include compounds of formula (la) in which R 3 and R 4 are selected from hydrogen atoms, hydroxymethyl and 2,3-dihydroxypropyl groups, R 1 and R 5 both represent hydrogen atoms and R 2 and R 6 both represent 2- hydroxyethyl or 2,3-dihydroxypropyl groups; compounds in which R 3 and R 4 are selected from hydrogen atoms, hydroxymethyl and 2,3-dihydroxypropyl groups and R 1 , R 2 , R 5 and R 6 all represent 2-hydroxyethyl groups; and compounds in which R 1 , R 2 , R 5 and R 6 are each selected from hydrogen atoms and methyl groups, preferably such that R 1 = R 5 and R 2 = R 6 , R 3 is a hydroxymethyl or 2,3- dihydroxypropyl group and R 4 is a hydrogen atom or a hydroxymethyl or 2,3-dihydroxypropyl group. The invention includes within its scope (i) use of hydrophilic, non-ionic Mn(II) chelates with bisamide derivatives of EDTA, e.g. as represented by Formula (I) , for the manufacture of liver-specific MRI contrast media for parenteral administration, (ii) the use of such chelates in diagnostic MRI studies of the hepatobiliary system of a human or non-human animal body; (iii) a method of generating enhanced images of the liver in a human or non-human animal body consisting essentially of parenterally administering such a chelate to the said body and generating a magnetic resonance image of the liver thereof; and (iv) parenterally-administrable diagnostic compositions containing such chelates for use as liver-specific MRI contrast media. The parenterally-administrable compositions are advantageously applied intravenously. It will be appreciated that the compositions should be sterile and free from physiologically unacceptable agents, and should have low osmolality to minimise irritation or other adverse effects upon administration, preferably being isotonic or slightly hypertonic. Suitable vehicles include aqueous vehicles customarily used for administering parenteral solutions, such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection and other solutions such as are described in Remington's Pharmaceutical Sciences, 15th ed., Easton: Mack Publishing Co., pp. 1405-1412 and 1461-1487 (1975) and The National Formulary XIV, 14th ed. Washington: American Pharmaceutical Association (1975) . The solutions can contain preservatives, antimicrobial agents, buffers and antioxidants conventionally used for parenteral solutions, excipients and other additives which are compatible with the chelates and which will not interfere with the manufacture, storage or use of products. The chelates may, for example, be present in such compositions at concentrations in the range 1 micromole to 1.5 mole per litre, advantageously 0.1 to 700 mM, although more concentrated solutions may be supplied for dilution prior to administration, and may conveniently be administered in amounts of from 10" 5 to 3 mmol per kilogram of body weight, e.g. 10" 4 to 1 mmol per kilogram, advantageously, 10" 3 to 0.1 mmol per kilogram. Advantages of the chelates useful according to the invention include (i) lower acute toxicity than ionic contrast agents such as Mn(EDTA) - tests in mice have shown chelates of Mn(II) with chelants of Formula (I) to exhibit LD J0 values at least 4 times higher than Mn(EDTA) ; (ii) high efficiency as liver contrast agents; (iii) substantially higher safety factors than existing liver contrast agents, as a consequence of the above points (i) and (ii) ; and (iv) low manufacturing costs, since the chelates are easily synthesised from inexpensive and readily available materials, as described in greater detail hereinafter. Chelates useful according to the invention may be prepared in conventional manner, for example using methods as described or analogous to those described for the preparation of such chelates which are already known. Thus, for example, chelating agents of the type represented in Formula (I) may be prepared by amidation of EDTA or a derivative thereof, e.g. a compound of formula HOOC.CH 2 R R \ ( / - CH 2 COOH N.CH.CH.N (II) HOOC.CH- X \ CH 2 COOH (where R is as hereinbefore defined) or an activated and/or protected derivative thereof (e.g. an acid anhydride or bisanhydride) in one or two stages to introduce the desired NR α groups or protected versions thereof (e.g. by reaction with amines RijNH or protected derivatives thereof) , followed where necessary by deprotection. Conventional protecting groups, for example such as are described by T.W. Greene in "Protective Groups in Organic Synthesis", John Wiley and Sons (1981) , and methods for their removal may be employed. Amidation reactions of this type are described in, for example, EP-A-130934 and US-A-4687659. Chelation may similarly be effected by conventional procedures, for example by dissolving or suspending a manganous salt or manganous oxide in water or a lower alkanol and adding an equivalent amount of the chelating acid in water or a lower alkanol, with stirring and/or heating as necessary; the chelate may be isolated by filtration or solvent evaporation as appropriate. The following non-limitative Examples serve to illustrate the invention:- EXAMPLE 1 Solution of the manganese chelate of 3.6-bisrN-(-2.3- dihvdroxypropyli-N-methylcarbamoylmethyl1-3.6- diazaoctanedioic acid (0.07 Mi a) 4.4'- ( 1.2-ethylene )-bis(2.6-morpholinedione ) To a stirred slurry of EDTA (150 g, 0.513 mol) in acetonitrile (400 ml) at room temperature are added acetic anhydride (157 g, 1.53 mol), triethylamine (156 g, 1.53 mol) and dimethylaminopyridine (0.62 g, 5.1 mmol, 0.1 mol percent). The reaction mixture is stirred for 4 hours, and is then filtered and washed three times with acetonitrile. The product is dried under vacuum overnight. Yield: 119.3 g (90.8%). Elemental analysis: Calc: C: 46.87%, H: 4.72%. Found: C: 46.92%, H: 4.66%. b) 3.6-bisTN- ( 2 .3-dihvdroxypropyli-N- methylcarbamoylmethyl1-3.6-diazaoctanedioic acid To a solution of methylamino-2,3-propanediol (1.64 g, 15.6 mmol) in DMF (30 ml) at 60°C is added 4,4'-(l,2- ethylene)-bis(2,6-morpholinedione) (2.0 g, 7.8 mmol, Example la) , and the mixture is stirred for 4 hours. DMF is then evaporated and the oily residue is washed with ether (2X) and acetonitrile (2X) before drying under high vacuum. Yield: 2.2 g (61%). Elemental analysis: Calc: C: 46.34%, H: 7.34%. Found: C: 46.37%, H: 6.70%. MS (FAB + , thioglycerol , TFA) : 467/468 (100/20, M+H) , 489 (M+Na) . c) Solution of the manganese chelate of 3.6-bisTN- (2-3-dihvdroxypropyli-N-methylcarbamoylmethyll-3.6- diazaoctanedioic acid fθ.07 Mi The chelant from Example lb (1.0 g, 2.14 mmol) is suspended in water (20 ml) and the pH adjusted to 5.25 with NaOH. MnCl 2 .4H 2 0 (0.424 g, 2.14 mmol) dissolved in 3 ml water is added dropwise during 15 minutes at constant pH 5.25 in a pH-stat. The mixture is stirred for an additional 15 minutes. The reaction mixture is analysed for the presence of free Mn 2+ in the following manner. 0.5 ml of sample is mixed with 10 ml water and ca 2 mg ascorbic acid is dissolved followed by 0.5 ml ammonium chloride buffer (pH 10.9) and 30 μl Eriochrome Black T. Pink colour indicates free Mn 2+ and additional chelant is added until a violet colour indicates that all metal is complexed. The chelant is added to ca. 0.1% excess (by weight) and the mixture sterile filtered through a Millex-GS 0.22 μm filter. Final pH 5.3. EXAMPLE 2 Solution of the manganese chelate of 3,6-bis (N- methylcarbamoylmethyl -3,6-diazaoctanedioic acid (0.01 ML a) 3,6-bisfN-methylcarbamoylmethyli-3.6- diazaoctanedioic acid To a solution of methylamine (42.4 g 40% sol., 546.4 mmol) cooled in an ice bath is added 4,4'-(1,2- ethylene)-bis(2,6-morpholinedione) (10.0 g, 39 mmol, Example la) during 15 minutes. The mixture is stirred for 2 hours at room temperature and evaporated to give a yellow oil which is dissolved in water. pH is adjusted to 3 with HC1, and the product is precipitated by addition of 600 ml of ethanol:isopropanol (50:50). Yield: 11.0 g (89%). MS (FAB + , thioglycerol, TFA) : 319 (5%, M+H) , 341 (100%, M+Na) . b) Solution of the manganese chelate of 3.6-bis(N- methylcarbamoylmethyl)-3.6-diazaoctanedioic acid (0.01 ML A 10 mM solution is made by careful addition of MnCl 2 solution (20 ml, 0.393 g, 2.0 mmol MnCl 2 .4H 2 O/100 ml 0.9% NaCl) to a solution of 3,6-bis(N-methylcarbamoylmethyl)- 3,6-diazaoctanedioic acid (0.164 g, 0.51 mmol, Example 2a) dissolved in 20 ml 0.9% NaCl, using a magnetic stirrer. The mixture is kept at pH 4.5-6 during the co plexing by addition of 1.0 M NaOH. The final pH is 6.7. The mixture is sterile filtered (Millex-GS, 0.22 μm) and kept in a 50 ml sterile glass flask prior to use. EXAMPLE 3 Solution of the manganese chelate of 3.6- bis (carbamoylmethyl>-3.6-diazaoctanedioic acid (0.01 M) The chelant is made as described in Example 2a except that ammonia is used instead of methyla ine. The chelate is made as described in Example 2b. EXAMPLE 4 Solution of the manganese chelate of 3 .6-bis tN .N- dimethylcarbamoylmethyl)-3.6-diazaoctanedioic acid fO.Ol ML The chelant is made as described in Example 2a except that dimethylamine is used instead of methylamine. The chelate is made as described in Example 2b. EXAMPLE 5 MR Liver imaging Tests performed on rabbits and rats rapidly injected (30 seconds) i.v. with 10 μmol/kg of the chelate solutions of Examples 2(b) , 3(b) and 4(b) , imaging with maximal T t - weighting and determining image intensity in liver and muscle by the ROI-facility of the Bruker 2.4 T machine, show that administration of these chelates results in significant increase (up to 120%) in signal intensity from the liver.

Claims: Claims : 1. The use of a manganese compound for the manufacture of a parenteral hepatobiliary MR imaging contrast medium, said manganese compound being a complex of Mn(II) with a hydrophilic chelating agent of formula I RjNCOCH 2 NCHRCH-_rcH 2 CONR 2 (I) HOOCC IH 2 ICH 2 COOH wherein each R, which may be the same or different, is a hydrogen atom or an optionally hydroxylated C M alkyl or alkoxyalkyl group. 2. Use as claimed in claim 1 of a said compound wherein in the chelating agent of formula I the R groups are independently selected from hydrogen, methyl and hydroxylated C,_ 3 alkyl. 3. Use as claimed in claim 1 of a said compound wherein in the chelating agent of formula I the R groups in the CHRCHR moiety are independently selected from hydrogen, hydroxymethyl and 2,3-dihydroxypropyl and the R groups in the NR 2 moieties are independently selected from methyl, 2-hydroxyethyl and 2,3-dihydroxypropyl. 4. Use as claimed in claim 2 of a said compound wherein in the chelating agent of formula I the R groups in the CHRCHR moiety are hydrogen and the NR 2 moieties are both N-methyl-2,3-dihydroxypropyl-amine groups. 5. Use as claimed in claim 2 of a said compound wherein in the chelating agent of formula I the R groups in the CHRCHR moiety are hydrogen and the R groups in the NR 2 moieties are independently selected from hydrogen and methyl. 6. Use as claimed in claim 2 of a said compound wherein in the chelating agent of formula I the R groups in the CHRCHR moiety are hydrogen and the NR 2 moieties are both methylamine groups. 7. A method of generating enhanced images of the liver of a human or non-human animal body, comprising parenterally administering to said body a manganese compound as defined in any one of claims 1 to 6 and generating an image of the liver of said body. 8. Use of a manganese compound as defined in any one of claims 1 to 6 in diagnostic MR imaging studies of the hepatobiliary system of a human or non-human animal body of a diagnostic composition for use in magnetic resonance imaging. 9. A liver specific, parenterally administrable, diagnostic MR imaging contrast enhancing composition comprising a manganese compound as defined in any one of claims 1 to 6 together with a physiologically acceptable carrier or excipient. MENDED CLAIMS [received by the International Bureau on 15 October 1993 (15.10.93) ; original claims 1-9 replaced by amended claims 1-9 (2 pages) ] 1. The use of a manganese compound for the manufacture of a parenteral hepatobiliary MR imaging contrast medium, said manganese compound being a complex of Mn (II) with a hydrophilic chelating agent of formula I R 2 NCOCH 2 NCHRCHRNCH 2 CONR 2 ( I) I I HOOCCH 2 CH 2 C00H wherein each R, which may be the same or different, is a hydrogen atom or an optionally hydroxylated C w alkyl or alkoxyalkyl group; with the proviso that the groups NR 2 represent other than NH(hydroxylated-C,__alkyl) groups. 2. Use as claimed in claim 1 of a said compound wherein in the chelating agent of formula I the R groups are independently selected from hydrogen, methyl and hydroxylated C 3 alkyl. 3. Use as claimed in claim 1 of a said compound wherein in the chelating agent of formula I the R groups in the CHRCHR moiety are independently selected from hydrogen, hydroxymethyl and 2,3-dihydroxypropyl and the R groups in the NR 2 moieties are independently selected from methyl, 2-hydroxyethyl and 2,3-dihydroxypropyl. 4. Use as claimed in claim 2 of a said compound wherein in the chelating agent of formula I the R groups in the CHRCHR moiety are hydrogen and the NR 2 moieties are both N-methyl-2,3-dihydroxypropyl-amine groups. 5. Use as claimed in claim 2 of a said compound wherein in the chelating agent of formula I the R groups in the CHRCHR moiety are hydrogen and the R groups in the NR 2 moieties are independently selected from hydrogen and methyl. 6. Use as claimed in claim 2 of a said compound wherein in the chelating agent of formula I the R groups in the CHRCHR moiety are hydrogen and the NR 2 moieties are both methylamine groups. 7. A method of generating enhanced images of the liver of a human or non-human animal body, comprising parenterally administering to said body a manganese compound as defined in any one of claims 1 to 6 and generating an image of the liver of said body. 8. Use of a manganese compound as defined in any one of claims 1 to 6 in diagnostic MR imaging studies of the hepatobiliary system of a human or non-human animal body of a diagnostic composition for use in magnetic resonance imaging. 9. A liver specific, parenterally administrable, diagnostic MR imaging contrast enhancing composition comprising a manganese compound as defined in any one of claims 1 to 6 together with a physiologically acceptable carrier or excipient.

Inventors: BERG ARNE, KLAVENESS JO, BERG, ARNE, KLAVENESS, JO
Applicants: BERG ARNE, COCKBAIN JULIAN, KLAVENESS JO, NYCOMED IMAGING AS, COCKBAIN, JULIAN
Application year: 1993
Main classification: A61K49
IPC codes: A61K49