Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
  • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
  • A61K49/10—Organic compounds
  • A61K49/14—Peptides, e.g. proteins
  • A61K49/16—Antibodies; Immunoglobulins; Fragments thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
  • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
  • A61K49/085—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier conjugated systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
  • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
  • A61K49/10—Organic compounds
  • A61K49/101—Organic compounds the carrier being a complex-forming compound able to form MRI-active complexes with paramagnetic metals
  • A61K49/106—Organic compounds the carrier being a complex-forming compound able to form MRI-active complexes with paramagnetic metals the complex-forming compound being cyclic, e.g. DOTA

Definitions

• Contra enhancement agents Paramagnetic metal chelates used in this manner are referred to as contrast enhancement agents or contrast agents.
• paramagnetic metal ions there are a number of paramagnetic metal ions which can be considered when undertaking the design of an MRI contrast agent.
• the most useful paramagnetic metal ions are gadolinium (Gd +3 ), iron (Fe +3 ), manganese (Mn +2 ) and (Mn +3 ), and chromium (Cr +3 ), because these ions exert the greatest effect on water protons by virtue of their large magnetic moments.
• GdCl 3 non-complexed form
• these metal ions are toxic to an animal, thereby precluding their use in the simple salt form. Therefore, a fundamental role of the organic chelating agent (also referred to as a ligand) is to render the paramagnetic metal non-toxic to the animal while preserving its desirable influence on T1 and T2 relaxation rates of the surrounding water protons.
• U.S. Patent 4,899,755 discloses a method of alternating the proton NMR relaxation times in the liver or bile duct of an animal using Fe +3 -ethylene-bis(2-hydroxyphenylgiycine) complexes and its derivatives, and suggests among various other compounds the possible use of a pyridine macrocyclomethylenecarboxylic acid.
• U.S. Patent 4,880,008 (a CIP of U.S. Patent 4,899,755) discloses additional imaging data for liver tissue of rats, but without any additional complexes being shown.
• Patent 4,980, 148 disclose gadolinium complexes for MRI which are non-cyclic compounds.
• C. J. Broan et al., J. Chem. Soc, Chem. Commun., 1739-1741 (1990) describe some bifunctional macrocyclic phosphinic acid compounds.
• C. J. Broan et al., J. Chem. Soc, Chem. Commun., 1738-1739 (1990) describe compounds that are triazabicyclo compounds.
• I. K. Adzamli et al., J. Med. Chem. 32, 139-144 (1989) describes acyclic phosphonate derivatives of gadolinium complexes for NMR imaging.
• MAGNEVISTTM gadolinium with diethylenetriaminepentaacetic acid
• MAGNEVISTTM gadolinium with diethylenetriaminepentaacetic acid
• MAGNEVISTTM is considered as a non-specific/perfusion agent since it freely distributes in extracellular fluid followed by efficient elimination through the renal system.
• MAGNEVISTTM has proven to be extremely valuable in the diagnosis of brain lesions since the accompanying breakdown of the blood/brain barrier allows perfusion of the contrast agent into the affected regions.
• Guerbet is commercially marketing a macrocyclic perfusion agent (DOTAREMTM) which presently is only available in Europe.
• a number of other potential contrast agents are in various stages of development.
• contrast agents could have site specificity for the tissue desired to be imaged, rather than non-specific/perfusion agents.
• the present invention is directed to just such novel complexes comprising a ligand that is a bicyclopolyazamacrocyclocarboxylic acid of the formula BICYCLOPOLYAZAMACROCYCLOCARBOXYLIC ACID COMPLEXES, THEIR CONJUGATES, PROCESSES FOR THEIR PREPARATION, AND USE AS CONTRAST AGENTS
• This invention concerns complexes that contain as the ligand bicyclopolyazamacrocyclocarboxylic acids, and conjugates thereof, for use as contrast agents in magnetic resonance imaging (MRI). Processes for preparing these complexes and conjugates are also provided. To better understand this invention, a brief background on MRI is provided in the following section.
• MRI is a non-invasive diagnostic technique which produces well resolved cross-sectional images of soft tissue within an animal body, preferably a human body.
• This technique is based upon the property of certain atomic nuclei (e.g. water protons) which possess a magnetic moment [as defined by mathematical equations; see G. M. Barrow, Physical
• the relaxation time consists of two parameters known as spin-lattice (T1) and spin-spin (T2) relaxation and it is these relaxation measurements which give information on the degree of molecular organization and interaction of protons with the surrounding environment.
• paramagnetic chelates be administered to animals in order to increase the diagnostic information obtained by MRI [Frontiers of Biol. Energetics 1, 752-759 (1978); J. Nucl. Med. 25, 506-513 (1984); Proc. of NMR Imaging Symp. (Oct. 26-27, 1980); F. A. Cotton et al., Adv. Inorg.
• R is hydrogen, , or
• X and Y are independently H, OH, C 1 -C 3 alkyl or COOH;
• R 7 is H or OH
• R 4 is H, NO 2 , NH 2 , isothiocyanato, semicarbazido, thiosemicarbazido, maleimido, bromoacetamido or carboxyl;
• A CH, N, C-Br, C-CI, C-OR 1 , C-OR 2 , N + -R 3 X , or ;
• R 1 H, C 1 -C 5 alkyl, benzyl, or benzyl substituted with at least one R 4 ;
• R 2 is C 1 -C 16 alkylamino
• R 3 is C 1 -C 16 alkyl, benzyl, or benzyl substituted with at least one R 4 ;
• R 4 is defined as before
• X is CI-, Br-, l- or H 3 CCO 2 ;
• Q and Z independently are CH, N, N + -R 3 X-, C-CH 2 -OR 1 or C-C(O)-R 5 ;
• R 1 and R 3 a re defined as above;
• R 5 is-O-(C 1 -C 3 alkyl), OH or NHR 6 ;
• R 6 is C 1 -C 5 alkyl or a biologically active material
• Bifunctional complexes of Formula (I) are desirable to prepare the conjugates of this invention.
• Such ligands must have:
• R 4 and R 7 are defined as above;
• A is C-OR 1 , C-OR 2 , where R 1 and R 2 are defined as above or ;
• R 4 is defined as above;
• A is CH, and one of Q or Z is CH and the other is C-C(O)-R 5 or C-CH 2 -OR 1 , where R 1 and R 5 are defined as above;
• the ligands of Formula (I) are complexed with various metal ions, such as gadolinium (Gd +3 ), iron (Fe + 3 ), and manganese (Mn +2 ), and Gd +3 being preferred.
• the complexes so formed can be used by themselves or can be attached, by being covalently bonded, to a larger molecule, such as a dextran, a polypeptide or a biologically active molecule, including an antibody or fragment thereof, and used for diagnostic purposes. Such conjugates and complexes are useful as contrast agents.
• the complexes and conjugates of this invention can be modified to provide a specific overall charge.
• the metal ion is + 3 the following can be obtained:
• one of A, Q or Z is N + -R 3 X-, where R 3 and X- are defined as above; and the three R terms are
• Both the complexes and conjugates may be formulated to be in a
• the complex has the ligand of Formula (I) numbered for nomenclature purposes as follows:
• the present invention concerns development of contrast agents having a neutral or + 1 charge which enables site specific delivery of the contrast agent to a desired tissue.
• the advantage being increased contrast in the areas of interest based upon tissue affinity as opposed to contrast arising from non-specific perfusion which may or may not be apparent with an extracellular agent.
• the specificity of the ligand of Formula (I) may be controlled by adjusting the total charge and lipophilic character of the complex.
• the overall range of the charge of the complex is from + 1 to 0. For example, for a complex having a + 1 overall charge has heart and/or bone uptake expected; whereas when the overall charge of the complex is 0 (thus neutral), the complex may have the ability to cross the blood brain barrier and normal brain uptake may be possible.
• Tissue specificity may also be realized by ionic or covalent attachment of the chelate to a naturally occurring or synthetic macromolecule having specificity for a desired target tissue.
• a paramagnetic chelate to a macromolecule can further increase the contrast agent efficiency resulting in improved contrast relative to the unbound chelate.
• Lauffer U.S. Patents 4,880,008 and 4,899,755 has demonstrated that variations in lipophilicity can result in tissue ⁇ specific agents and that increased lipophilic character favors non-covalent interactions with blood proteins resulting in enhancement of relaxivity.
• the present contrast agents of Formula (I) which are neutral in charge are particularly preferred for forming the conjugates of this invention since undesirable ionic interactions between the chelate and protein are minimized which preserves the antibody immunoreactivity. Also the present neutral complexes reduce the osmolarity relative to DTPA-Gd +3 , which may alleviate the discomfort of injection.
• Bioly active material refers to, for example, a dextran, peptide, or molecules that have specific affinity for a receptor, or preferably antibodies or antibody fragments.
• Antibody refers to any polyclonal, monoclonal, chimeric antibody or heteroa ⁇ tibody, preferably a monoclonal antibody; "antibody fragment” includes Fab fragments and F(ab') 2 fragments, and any portion of an antibody having specificity toward a desired epitope or epitopes.
• antibody fragment includes Fab fragments and F(ab') 2 fragments, and any portion of an antibody having specificity toward a desired epitope or epitopes.
• Possible antibodies are 1116-NS-19-9 (anti-colorectal carcinoma), 1116-NS-3d (anti-CEA), 703D4 (anti-human lung cancer), 704A1 (anti-human lung cancer), CC49 (anti-TAG-72), CC83 (anti-TAG-72) and B72.3.
• the hybridoma cell lines 1116-NS-19-9, 1116-NS-3d, 703D4, 704A1 , CC49, CC83 and B72.3 are deposited with the American Type Culture Collection, having the accession numbers ATCC HB 8059, ATCC CRL8019, ATCC HB 8301 , ATCC HB 8302, ATCC HB 9459, ATCC HB 9453 and ATCC HB 8108, respectively.
• complex refers to a complex of the compound of the invention, e.g. Formula (I), complexed with a metal ion, where at least one metal atom is chelated or sequestered;
• conjuggate refers to a metal ion chelate that is covalently attached to an antibody or antibody fragment.
• bifunctional coordinator e.g., bifunctional chelating agent
• functionalized chelant are used interchangeably and referto compounds that have a chelant moiety capable of chelating a metal ion and a moiety covalently bonded to the chelant moiety that is capable of serving as a means to covalently attach to an antibody or antibody fragment.
• the bifunctional chelating agents described herein can be used to chelate or sequester the metal ions so as to form metal ion chelates (also referred to herein as "complexes").
• the complexes because of the presence of the functionalizing moiety (represented by R 2 , R 4 or R 6 in Formula I), can be covalently attached to biologically active materials, such as dextran, molecules that have specific affinity for a receptor, or preferably covalently attached to antibodies or antibody fragments.
• biologically active materials such as dextran, molecules that have specific affinity for a receptor, or preferably covalently attached to antibodies or antibody fragments.
• conjugates are referred to herein as "conjugates”.
• salts means any salt or mixtures of salts of a complex or conjugate of formula (I) which is sufficiently non-toxic to be useful in therapy or diagnosis of animals, preferably mammals. Thus, the salts are useful in accordance with this invention.
• salts formed by standard reactions from both organic and inorganic sources include, for example, sulfuric, hydrochloric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, palmitic, cholic, palmoic, mucic, glutamic, glu ⁇ nic, d- camphoric, glutaric, glycolic, phthalic, tartaric, formic, lauric, steric, salicylic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic, cinnamic acids and other suitable acids.
• salts formed by standard reactions from both organic and inorganic sources such as ammonium or 1-deoxy-1-(methylamino)-D-glucitol, alkali metal ions, alkaline earth metal ions, and other similar ions.
• Particularly preferred are the salts of the complexes or conjugates of formula (I) where the salt is potassium, sodium or ammonium.
• mixtures of the above salts are also included.
• the complexes or conjugates of the present invention contain a ligand of Formula
• the ligands are prepared by various processes. Typical general synthetic approaches to such processes are provided by the reaction schemes given below.
• R 4 NO 2 or NH 2 ;
• Scheme 1 1 prepares the compounds of Formula (I) wherein the R term at the 9 position is
• the synthetic Scheme 1 begins with a halogenation of commercially available bis-pyridyl alcohol (1) using thionyl chloride. Similar procedures for converting an alcohol to an eiectrophilic substrate, such as treatment with toluenesulfonyl chloride, HBr or HCI, should also result in a similarly reactive product which would work well in subsequent ring closure reactions. Macrocyclization procedures are numerous in the literature and the desired tetraazamacrocycle (3) was prepared according to the method of Stetter et al., Tetrahedron 37, 767-772 (1981). More general procedures have since been published which give good yields of similar macrocycles using milder conditions [A. D. Sherry et al., J. Org. Chem.
• Schemes 10, 11 and 12 delineate a synthetic approach which introduces an aromatic nitrobenzyl substituent at one of the macrocyclic nitrogen positions.
• the macrocyclic amine is mono-N-functionalized in an organic solvent such as acetonitrile or DMF at room temperature using a non-nucleophilic base such as potassium carbonate. Additional functionalization of the remaining nitrogen positions is then preformed by methodsand conditions described in previous Schemes.
• the nitro group is reduced using platinum oxide and hydrogen in water.
• the chelating agent is compatible with conjugation techniques which will enable attachment to larger synthetic or natural molecules.
• the metal ions used to form the complexes of this invention are Gd +3 , Mn + 2 , Fe +3 and available commercially, e.g. from Aldrich Chemical Company.
• the anion present is halide, preferably chloride, or salt free (metal oxide).
• a "paramagnetic nuclide” of this invention means a metal ion which displays spin angular momentum and/or orbital angular momentum.
• the two types of momentum combine to give the observed paramagnetic moment in a manner that depends largely on the atoms bearing the unpaired electron and, to a lesser extent, upon the environment of such atoms.
• the paramagnetic nuclides found to be useful in the practice of the invention are gadolinium (Gd + 3 ), iron (Fe +3 ) and manganese (Mn +2 ), with Gd + 3 being preferred.
• the complexes are prepared by methods well known in the art. Thus, for example, see Chelating Agents and Metal Chelates, Dwyer & Mellor, Academic Press (1964), Chapter 7. See also methods for making amino acids in Synthetic Production and Utilization of Amino Acids, (edited by Kameko, et al.) John Wiley & Sons (1974).
• An example of the preparation of a complex involves reacting a bicyclopoiyazamacrocyclophosphonic acid with the metal ion under aqueous conditions at a pH from 5 to 7.
• the complex formed is by a chemical bond and results in a stable paramagnetic nuclide composition, e.g. stableto the disassociation of the paramagnetic nuclide from the ligand.
• the complexes of the present invention are administered at a ligand to metal molar ratio of at least about 1 : 1, preferably from 1 : 1 to 3: 1 , more preferably from 1 : 1 to 1.5: 1.
• a large excess of ligand is undesirable since uncomplexed ligand may be toxic to the animal or may result in cardiac arrest or hypocalcemic convulsions.
• the antibodies or antibody fragments which may be used in the conjugates described herein can be prepared by techniques well known in the art. Highly specific monoclonal antibodies can be produced by hybridization techniques well known in the art, see for example, Kohler and Milstein [Nature, 256, 495-497 (1975); and Eur. J. Immunol., 6, 511-519
• antibodies directed against any desired antigen or hapten may be used.
• the antibodies which are used in the conjugates are monoclonal antibodies, or fragments thereof having high specificity for a desired epitope(s).
• Antibodies used in the present invention may be directed against, for example, tumors, bacteria, fungi, viruses, parasites, mycoplasma, differentiation and other cell membrane antigens, pathogen surface antigens, toxins, enzymes, allergens, drugs and any biologically active molecules.
• Some examples of antibodies or antibody fragments are 1116-NS-19-9, 1116-NS-3d, 703D4, 704A1 , CC49, CC83 and B72.3. All of these antibodies have been deposited in ATCC. A more complete list of antigens can be found in U.S. Patent 4, 193,983.
• the conjugates of the present invention are particularly preferred for the diagnosis of various cancers.
• This invention is used with a physiologically acceptable carrier, excipient or vehicle therefor.
• a physiologically acceptable carrier excipient or vehicle therefor.
• the methods for preparing such formulations are well known.
• the formulations may be in the form of a suspension, injectable solution or other suitable formulations.
• Physiologically acceptable suspending media, with or without adjuvants, may be used.
• an "effective amount" of the formulation is used for diagnosis.
• the dose will vary depending on the disease and physical parameters of the animal, such as weight.
• In vivo diagnostics are also contemplated using formulations of this invention.
• chelants of the present invention may include the removal of undesirable metals (i.e. iron) from the body, attachment to polymeric supports for various purposes, e.g. as diagnostic agents, and removal of metal ion by selective extraction.
• undesirable metals i.e. iron
• the ligands of Formula (I) having in at least two R terms T equal to P(O)R 1 OH may be used for metal ion control as scale inhibitors. It is likely that these ligands could be used in less than stoichiometric amounts. Similar uses are known for compounds described in U.S. Patents
• ICP inductively coupled plasma
• a stock 159 GdCI 3 or 153 SmCI 3 solution was prepared by adding 2 ⁇ L of 3 ⁇ 10 -4 M 159 GdCI 3 in 0.1 N HCI to 2 mL of a 3 ⁇ 10 -4 M GdCI 3 carrier solution.
• Appropriate ligand solutions were then prepared in deionized water.
• the percent metal as a complex was then determined by passing a sample of the complex solution through a SephadexTM G-50 column, eluting with 4: 1 saline (85% NaCI/NH 4 OH) and collecting 2 ⁇ 3 mL fractions. The amount of radioactivity in the combined elutions was then compared with that left on the resin (non-complexed metal is retained on the resin).
• the pH stability profile was generated by adjusting the pH of an aliquet of the complex solution using 1M NaOH or 1M HCI and determining the percent of the metal existing as a complex using the ion exchange method described above. The Sm results are known by expermintal comparison to be identical for complexation and biodistribution of the ligands of this invention.
• a solution of HBr and AcOH was prepared by mixing 48% HBr and glacial AcOH in a 64:35 ratio.
• To 112 mL of the HBr/AcOH mixture was added 5.5 g (8.2 mmol) of 3,6,9-tris(p-tolylsulfonyl)-3,6,9,15-tetraazabicyclo[9.3.1 ]pentadeca-1(15),11,13-triene (prepared by the procedure of Example B) and the reaction mixture was heated at mild reflux with constant stirring for 72 hrs. The reaction mixture was then cooled to room temperature and
• Example G was stirred for 12 hrs at reflux in 6N HCI. The solution was then cooled and concentrated in vacuo. The residue was then dissolved in water and lyophilized to give the desired product.
• PC2A PC2A
• a concentrated aqueous solution of 30 mL of HCI (37%) and mg (2.5 mmol) of 3,9-bis(methylenenitrile)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene(prepared by the procedure of Example F) was heated at reflux for 2 hrs. After cooling, the aqueous solution was evaporated to dryness, followed by coevaporation with deionized water (2 ⁇ 10 mL) to eliminate excess HCI. The pH of the reaction mixture was adjusted to 7 with
• a 1 : 1 ligand/metal complex was then prepared by combining 40 ⁇ L of the ligand solution with 2 mL of aqueous SmCl 3 ⁇ H 2 0 (3 ⁇ 10 -4 M in 0.01 N HCI) containing tracer 153 SmCI 3 .
• the percent metal as a complex was determined by passing a sample of the complex solution through a SephadexTM column, eluting with 4: 1 saline (0.85% NaCI/NH 4 OH), and collecting 2 ⁇ 3 mL fractions.
• Sprague Dawley rats were allowed to acclimate for five days then injected with 100 ⁇ L of the complex solution via a tail vein. The rats weighed between 150 and 200 g at the time of injection. After 30 min. the rats were killed by cervical dislocation and dissected. The amount of radioactivity in each tissue was determined by counting in a Nal scintillation counter coupled to a multichannel analyzer. The counts were compared to the counts in 100 ⁇ L standards in order to determine the percentage of the dose in each tissue or organ.
• the percent dose in blood was estimated assuming blood to be 7% of the body weight.
• the percent dose in bone was estimated by multipuling the percent dose in the femur by 25.
• the percent dose in muscle was estimated assuming muscle to be 43% of the body weight.
• chelates of the compounds of Formula (I) were evaluated for efficiency of bone localization since phosphonates are known for their ability to bind to hydroxyapatite.
• the percent of the injected dose of complex of of of Example 1 ( 153 Sm-PCTA) in several tissues are iven in Table I.
• the numbers re resent the average of 5 rats per data point.
• the bone to blood ratio (% dose) was 7.
• the bone to liver ratio was 3.5.
• the bone to muscle ratio was 4.8.
• injectable solutions were first prepared (0.5M) by dissolving the appropriate amount of each complex in 2 mL of deionized water. The pH of the solutions were then adjusted to 7.4 using 1M HCI or NaOH as needed. The total Gd content of each solution was then determined by ICP analysis.
• the Gd-PCTA complex (prepared in Example 1 ) was rapidly taken up by the renal system with brilliant enhancement of the kidney cortex as well as peripheral kidney tissue.
• Other embodiments of the invention will be apparent to those skilled in the art from a consideration of this specification or practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

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• 1993
  • 1993-05-06 WO PCT/US1993/004322 patent/WO1994026313A1/en active Application Filing
  • 1993-08-09 FI FI933506A patent/FI933506A/fi not_active Application Discontinuation

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