WO1992005804A1 - Agents chelateurs - Google Patents

Agents chelateurs Download PDF

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Publication number
WO1992005804A1
WO1992005804A1 PCT/US1991/007016 US9107016W WO9205804A1 WO 1992005804 A1 WO1992005804 A1 WO 1992005804A1 US 9107016 W US9107016 W US 9107016W WO 9205804 A1 WO9205804 A1 WO 9205804A1
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Prior art keywords
molecular component
metal ion
chelating agent
metal
group
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PCT/US1991/007016
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English (en)
Inventor
Wolfgang J. Wrasidlo
Michael H. Silveira
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Brunswick Corporation
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Priority to JP3518342A priority Critical patent/JPH06505795A/ja
Publication of WO1992005804A1 publication Critical patent/WO1992005804A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6887—Antibody-chelate conjugates using chelates for therapeutic purposes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/04—X-ray contrast preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
    • A61K51/0482—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group chelates from cyclic ligands, e.g. DOTA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2121/00—Preparations for use in therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2123/00—Preparations for testing in vivo

Definitions

  • the technical field of the present invention relates to chelatin agents, particularly bifunctional chelating agents, and to conjugates of site-specific compounds, chelating agents, and metal ions, including radioisotopes, for use as therapeutic agents in radiation therapy.
  • this invention relates to a method for treating cellular disorders, particularly cancer, which employs a chelating agent for a metal ion, such as a radioisotope, conjugated to a site-specific compound or target cell binding protein, such as a monoclonal antibody.
  • a chelating agent for a metal ion such as a radioisotope
  • it relates to the use of metal chelate conjugated site-specific compounds or target cell binding proteins, such as monoclonal antibodies, for diagnostic purposes.
  • Radiation therapy has long been used in the treatment of cancer and other localized lesions.
  • the attractiveness of radiation lies in the ability to focus the energy of the radioactive particles or energy on a specific, small target. Radiation is, therefore, a treatment which minimizes overall bodily exposure to the therapeutic agent, in contrast to the general systemic exposure of chemotherapeutic agents.
  • One drawback to radiation treatments is that the radiation passes through other tissue in the process of destroying targeted cancer cells. As a result, the effects are more general than the limited bodily exposure would indicate, so that radiation therapy can be very devastating to the patient and contraindicated for weakened individuals.
  • alpha particle treatments have not proven optimum under any administrative technique, however, because of the difficulty in developing the requisite combination of alpha particle energy, suitable half life length, as well as targeting and handling problems.
  • the alternative to external application of radiation in the treatment of cancer tumors is internal application, i.e. internal specific targeting of the radiation, such as by injection.
  • the effective penetration of the radiation is preferably short to avoid undue exposure to surrounding tissues and the half life of the radioactive molecule is preferably short to minimize the total bodily exposure to freely circulating or nonspecifically bound radioactive treatment compounds and degradation products thereof.
  • the first application of this technique was the administration of radioactive iodine for thyroid tumors. This was effective due to the specific uptake of iodine into the thyroid and the reasonably short half lives of iodine isotopes. However, this is a very limited therapy and not applicable to other types of cancer.
  • target cell binding proteins such as monoclonal antibodies, polyclonal antibodies, monoclonal antibody fragments, and binding proteins, which can be targeted to specific cells with a high degree of specificity, has made it possible to selectively deliver therapeutic agents to specific targeted cells.
  • Current internal targeted therapies therefore, center primarily around the use of such monoclonal antibodies as the targeting mechanism, to deliver the radio isotope combined therewith to the specific target.
  • U.S. Patent 4,454,106 issued to Gansow et al .
  • a radioactive metal such as bismuth-212 (Bi 212 ) chelated with a derivative of diethylene triamine pentaacetic acid (DTPA)
  • DTPA diethylene triamine pentaacetic acid
  • Radioisotope complexes for cell killing has unique problems which have not been effectively solved at this time.
  • High energy alpha, beta and gamma emitting isotopes are undesirable due to the long range of their effects.
  • Radioisotopes with long half lives are not acceptable because of their long term of action, unless they can be rapidly diluted and eliminated, e.g. for tritium.
  • Such molecules which undergo rapid exchange with the surrounding molecular environment, are not likely to remain targeted long enough to be effective.
  • Alpha particle emitting isotopes, with the proper energy and half life are few in number and extremely difficult to entrap and bind to targeted molecules.
  • Another aspect of the design of such complexes is the specificity of the entrapping compound.
  • Many chelating and caging compounds may hold various ions. This can be a problem in the physiological milieu of the body which contains considerable concentrations of mono- and divalent ions of various types. If these physiological ions are bound by the entrapping molecule and the kinetics are at all favorable for exchange, the metal ion, such as a radioisotope, is likely to be rapidly freed. It is thus desirable to use entrapping compounds which have a high degree of specificity for the metal ions being delivered.
  • radioactive isotope is another critical aspect of targeted radiation therapy. External treatments generally utilize long lived isotopes for economy, regulating bodily dosage by exposure time. However, when the radioactive molecule is administered internally, degradation of the therapeutic compound is likely to occur, thus necessitating the use of one having a short half life. Ideally, the half life preferably provides just ample time for targeted cell killing. The primary problem with such short and precise half lives is that it is difficult to produce the therapeutic compound, administer it and allow time for cell killing before the radioactivity has decayed below effective levels. Since most potential candidates require a cyclotron for production, they cannot be seriously considered for regular use.
  • Bi 2 ⁇ and Pb 2 ⁇ 2 have received considerable attention because they have acceptable emission energy and half life characteristics.
  • Bi 212 is the only one that has been utilized because it is the only alpha emitter among the candidates which has been chelated with any reasonable effectiveness.
  • a much better candidate in terms of on-site generation and isolation capabilities is Pb 212 as generated from Th 228 . Lead, however, has proven to be extremely difficult to effectively bind to a targeting molecule.
  • the present invention discloses novel metal chelating agents and novel ' therapeutic or diagnostic conjugates of site-specific compounds, chelating agents, and metal ions, such as radioisotopes.
  • the invention also discloses a method for producing a radiolabeled material attached to a site-specific compound, such as a monoclonal antibody, for immunotherapy of cancers and other uses.
  • the radiolabeled material is held in an entrapping compound such as a chelating or caging agent which is bound to the site-specific compound.
  • Macrocycles A, acrobicycles B, and macrotricycles C and D are good candidates as chelating agents for metal ions.
  • Macrocycles contain two dimensional cavities lined with atoms that bind cations and have been studied extensively. Macrobicycles and macrotricycles, collectively known as macropolycycles, have been studied more recently. They contain three-dimensional intramolecular cavities lined with various binding sites, have the ability to form inclusion complexes and bind selectively spherical metallic cations. They are molecules of intermediate size, mesomolecules which may display a multitude of new properties and hence have given rise to a new field of supramolecular chemistry.
  • Cryptands lined with oxygen, sulfur, and nitrogen binding sites may be synthesized from commercially available macrocycles. Their size is governed by the length of the bridges and increases stepwise along the series 1 - 7.
  • the selectivity in binding of cryptand is based on the principle of cavity size selectivity, the preferred cation being that whose size most closely fits the cavity. Replacement of oxygen binding sites with nitrogen and sulfur atoms decreases both the stability as well as selectivity. Cryptands have been used to decorporate radioactive stroncium-85 and radium-224 from rats and hence the macropolycycles should withstand the recoil energy of alpha emitters.
  • Macrotricycles g2_ - 2 L of type C have been used for complexing alkali cations and alkaline earth cations and found to form non symmetric mononuclear as well as symmetric binuclear complexes.
  • Ligands 24 - 26 from mononuclear, binuclear as well as heteronuclear bimetallic cryptates with silver nitrate and lead nitrate.
  • Ligand 25 when treated with equimolar quantities of AgN0 3 and
  • Pb(N0 3 ) 2 forms an equilibrium mixture of two homonuclear complexes
  • chelating agents Conjugated to these cryptands and macrotricycles are chelating agents having a low K s value and the ability to undergo rapid metal exchange. These novel chelating agents which result, have the properties of rapid uptake of metal ion and high stability.
  • the present invention discloses novel metal chelate conjugates and novel therapeutic or diagnostic conjugates of site-specific compounds, chelating agents, and metal ions, such as radioisotopes.
  • Chelating compounds and other caging agents are known in the prior art for entrapping lead. There are a few classes of these compounds and any number of specific embodiments within each class. These specific embodiments are normally derivatives or modifications of known core molecules. Such chemical changes are preferred so that any entrapping molecule can meet the strict requirements for the Pb 212 delivery complex. Examples of these classes of compounds include: metal organics
  • crown ethers and crown azos such as 1,4,10,13-tetraoxa- 7,16-diazacyclooctadecane (TDCOD)
  • polythiols such as mercaptanes HOOC-R-SH-M SH '' complexes, such as diethylene-triamine- pentaacetic acid (DTPA)
  • the novelty of the present invention lies in the combination of two or more of the above chelating agents to form a novel chelating agent.
  • One of the chelating agents has a high affinity for metal ions, including radioisotopes, and a high K s (stability constant) value.
  • This component is characteristically a strongly metal binding ligand with spacing for various metal ions and forms stable interaction with the chelated metal. It may even bind more than one metal ion.
  • the thiol analogs of many of the above listed classes of chelating agents exhibit these characteristics.
  • chelating agents having a low K s but a fast uptake of metal ion such as ethylene diamine tetraacetic acid (EDTA) , diethylene triamine pentaacetic acid (DTPA), 2,3- dimercapto-1-propane sulfonic acid (2,3 DMPS) , and meso- 2,3-dimercaptosuccinic acid (2,3 DMSA) .
  • metal ion such as ethylene diamine tetraacetic acid (EDTA) , diethylene triamine pentaacetic acid (DTPA), 2,3- dimercapto-1-propane sulfonic acid (2,3 DMPS) , and meso- 2,3-dimercaptosuccinic acid (2,3 DMSA) .
  • EDTA ethylene diamine tetraacetic acid
  • DTPA diethylene triamine pentaacetic acid
  • 2,3 DMPS 2,3- dimercapto-1-propane
  • novel, bifunctional, chelating agents of the present invention are 1,4,10,13-tetraoxa-7,16- diazacyclooctadecane (TDCOD) combined with two molecules of di ercaptosuccinic acid, as seen below in Example 1 and
  • TDCOD combined with one or two molecules of DTPA.
  • the novel chelator of the present invention comprises a macrocycle or macropolycycle containing a two- or three-dimensional intramolecular cavity (the high affinity chelating agent or binding group) having one or more projecting arms (the chelating agent having the low K s value) .
  • the metal ion is chelated by the agent having the fast uptake characteristic, the arm, and is then attracted into the cavity of the agent having the high affinity cahracteristics.
  • the two or three chelating agents become folded, resulting in intramolecular interaction in the form of coordinate bonding, to form a stable chelating agent having a very high K s value.
  • the chelate containing the cavity and its one or more arms may be linked directly or via linking groups inserted to connect the two agents.
  • site specific compounds such as monoclonal antibodies
  • the monoclonal antibodies can be attached either before or after the metal ion is chelated.
  • Preferred for the practice of this invention are two groups of caging molecules, captens and cryptates to which may be attached chelating agents with lower K s values, such as EDTA, DTPA, DMPS, and DMSA.
  • the captens and cryptates hold metal ions, including radioisotopes, within a physically defined space interior to the molecule.
  • the strength of the entrapment of the metals in these compounds derives from the ionic bonds to the metal ions and also from the molecular structure of the surrounding molecule which is actually built around the metal ion during synthesis, rather than merely chelated afterwards.
  • the cryptates are the more complex of the two groups and also the more three-dimensional in terms of the molecular caging. The major advantages of these compounds is that they have the ability to provide high s values, slow exchange kinetics, and the potential for the engineering of a high degree of specificity for specific metal ions, such as Pb 212 and Bi 212 .
  • Pb 212 While in the past, Pb 212 has proven extremely difficult to effectively bind to a targeting molecule, several additional considerations make Pb 212 the isotope of choice for use in radiation therapy of cancers.
  • Pb 212 provides a number of decay pathways resulting in alpha particle emissions, and can be readily produced from elements of natural origin, i.e., not needing a cyclotron for production.
  • the decay pathway from ⁇ h 228 to pb 208 is the preferred source for the isotope.
  • pb 212 has a half life of 10.6 hours before decaying into stable Pb 208 .
  • Pb 212 emits an alpha particle with an ideal energy for localized cell killing.
  • This alpha particle has about 8.8 Mev of energy which translates into a tissue penetration depth of about 80 ⁇ m, equivalent to about 4-5 tumor cell diameters.
  • the alpha particle from Pb 212 has the ability to kill cells only very locally to the site of the attachment.
  • Pb 212 lends itself to simple, efficient, and economical production and isolation for use in any desired application.
  • the novel, bifunctional, chelators of the present invention have overcome the difficulties previously encountered in binding lead to a targeting molecule and hence have made it possible to use Pb 212 as the metal ion of choice.
  • Such compounds designed to contain only lead can be attached to monoclonal antibodies in the empty state (no entrapped metal ion) and then used to entrap Pb 2 ⁇ 2 .
  • the lead may be first chelated to the entrapping compound which is then in turn bound to the monoclonal antibodies.
  • the Pb 212 is to be used as a therapeutic agent in radiation therapy, one such method pursuant to one embodiment of this invention, involves dissolving the Pb 212 in a suitable antibody-chelating complex solution to entrap the Pb 212 in the complex. For therapeutic use by internal administration, the chelating complex is thereafter filtered and purified for subsequent administration.
  • the Pb 212 antibody-chelating complex to which antibody has already been conjugated by means such as interfacial condensation or any other convenient means known to the art, is preferably first separated from free uncombined isotopes. While the solution may be centrifuged, a preferred method for effecting this operation is to filter the solution through an ion exchange column such as a column of Amberlite IRA-400, Dowex 1 or equivalent ion exchange resin, and then into a second ion exchange column such as a Sephadex G-25 or G-50 cartridge for good effective separation of the complex from free isotopes. Thereafter, the purified complex is filtered to remove any solids therefrom, and sterilized.
  • an ion exchange column such as a column of Amberlite IRA-400, Dowex 1 or equivalent ion exchange resin
  • the material After analyzing the material to verify the radioactivity, the material is then ready for injection into a patient. Because of its short half life, the Pb 212 , once generated, is preferably processed and administered without delay. While Pb 212 is preferred for use in the present invention, this invention also contemplates the use of metal alpha, beta, gamma or positron emitters. Auger electron emitters, and fluorescing lanthanides for therapeutic and diagnostic use.
  • the chelating agents of the present invention have the advantage of rapid metal ion uptake. Once picked up, the entrapping chelator folds itself so that the metal ion is then tightly bound by the highly stable chelator. The metal ion is thus bound in such a way that little exchange with extraneous metal ions will take place. The best of these compounds result in very stably bound radioisotopes with short half lives and which are rapidly removed from the body.
  • site-specific compounds or target cell binding proteins such as monoclonal antibodies, polyclonal antibodies, monoclonal antibody fragments, and binding proteins, which can be targeted to specific cells with a high degree of specificity, in combination with the novel, bifunctional, chelating agents of the present invention makes it possible to selectively deliver therapeutic agents to specific targeted cells.
  • the chelating agents combined with monoclonal antibody can be used either in vivo or in vitro for such things as diagnostic evaluations, treatment of cancers and other diseases, and other radio-immuno therapy.
  • One embodiment of the present invention contemplates the use of the bifunctional chelating agents of the present invention as detoxifying agents.
  • no monoclonal antibody or other targeting molecules are necessary.
  • the chelating agent without metal ion is administered either in vivo or in vitro as the case may be.
  • the chelating agent picks up unwanted metal ions. Thereafter the chelated metal and chelator are removed.
  • the present invention contemplates an in vivo diagnostic procedure which comprises introducing a metal chelate conjugated monoclonal antibody into the body, allowing sufficient time for the conjugate to localize and identifying the degree and location of localization, if any.
  • the present invention also contemplates in vitro analytical procedures employing a chelate conjugated monoclonal antibody.
  • the conjugated antibody of the present invention is substantially free of adventitiously or weakly chelated metal.
  • the metal chelate conjugated antibodies of the present invention may be administered in vivo in any suitable pharmaceutical carrier, including a physiologic normal saline solution.
  • concentration of metal chelate conjugated antibodies within the solution is preferably a matter of choice. Levels ranging from 10 to 100 mg per ml are readily attainable but the concentrations may vary depending upon the specifics of the application. Appropriate concentrations of biologically active materials in a carrier are routinely determined in the art.
  • the effective dose of radiation or metal content to be utilized for any application is likely to depend upon the particulars of that application.
  • the dose is likely to depend, inter alia , upon tumor burden, accessibility, and the like.
  • the use of metal chelate conjugated antibodies for diagnostic purposes is likely to depend, inter alia , upon the sensing apparatus employed, the location of the site to be examined, and the like.
  • the following examples are for illustrative purposes only and are not intended to limit the scope of the invention.
  • a chelating agent of the present invention which contains a two dimensional cavity having two arms attached on diagonally opposite atoms. These arms are made up of polyanionic complexing agents.
  • TDCOD 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane
  • DMSA dimercaptosuccinic acid
  • ECDI l-[3- (dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride
  • a chelating agent of the present invention which contains a two dimensional cavity having two arms attached on diagonally opposite atoms. These arms are made up of polyanionic complexing agents.
  • 71 mg (0.02 moles) of DTPA and 26 mg (0.01 moles) of TDCOD were mixed and 1 ml of DMAc was added.
  • 30 ⁇ l of triethylamine (TEA) was added and the mixture was stirred.
  • another 1 ml DMAc was added.
  • another 30 ⁇ l (30 mg) of TEA was added.
  • the mixture was heated until clear. A 100 ⁇ l aliquot was removed from the clear solution and added to 1 ml (11.9 mg) 9.2.27.
  • the part of the purified sample was mixed with 3 mg Pb0 2 for 15 minutes then centrifuged for 2 minutes at 30,000 rp .
  • the supernatant was analyzed via atomic absorption which showed that 15 ppm of Pb was taken up by the mAb-TDCOD-DTPA complex.
  • a control sample of 9.2.27 and 3 mg of Pb0 2 dissolved together showed that 0 ppm of Pb was taken up by the antibody.
  • Another part of the purified mAb-TDCOD-DTPA complex was reacted with 3 mg of Pb0 2 for 2 hours. The mixture was centrifuged and stored cold. This sample had 29 ppm Pb. After running over NAP-5 (250 ⁇ l) the sample had 6 ppm Pb.
  • PbN0 3 sample contained 67 ppm Pb.
  • PbN0 3 sample was diluted with 400 ⁇ l of PBS and eluted with 400 ⁇ l of PBS, atomic absorption indicated that 2 ppm of Pb remained. This indicated that there was a 3:1 Pb to antibody ratio.
  • EXAMPLE 9 Various compounds synthesized following the process in Example 8 can be converted into other compounds suitable for attachment of monoclonal antibodies and compounds capable of fast uptake of Pb 212 .
  • One such application is shown below.
  • the following reaction results in the formation of a chelating agent of the present invention which contains a macropolycycle containing a three dimensional cavity having an arm attached on the bridge connecting the macrocyclic segments.
  • An analogue of the same, which is not shown here, could be synthesized wherein the arm is made up of a polyanionic complexing agent .
  • E 0, NH, S, and the like
  • X CH 2 , O, S, -Z-EDTA, -Z-DTPA, -Z-DMSA, and the like
  • Z the spacer between X and the polyanionic complexing agents like EDTA or DTPA
  • EXAMPLE 10 The synthesis of a polyanionic complexing agent (EDTA) - macrocyclic (18-crown-6)-monoclonal antibody conjugate 4_6 is outlined below. The following reaction results in the formation of a chelating agent of the present invention which contains a two dimensional cavity having one arm. The arm is made up of polyanionic complexing agents.
  • EDTA polyanionic complexing agent
  • the ethyl triester of EDTA on conversion to its N- hydroxy succinimide derivative under aqueous conditions using EDCI as a coupling agent reacts with the monoprotected derivative of 18-crown-6 29 , to give compound 44.
  • Hydrolysis of the ethyl esters and the benzyl chloroformate functionality in one step with HBr (48%) followed by reaction with bromo acetyl bromide in the presence of an appropriate solvent yields compound 45 . which reacts with a monoclonal antibody to give conjugate 46 capable of complexing lead to yield a radio immunoconjugate.
  • EXAMPLE 12 The synthesis of a complexing agent in which a macrocycle unit is cyclized with a DTPA unit is shown below. The following reaction results in the formation of a macropolycyclic chelating agent of the present invention in which a macrocycle is connected at diagonally opposite atoms by a bridge made up of a polyanionic complexing agent.
  • the 18-crown-6 unit has an arm with an azido functionality which on photolysis reacts with a monoclonal antibody and the resulting immunoconjugate is capable of complexing with an alpha emitter.
  • Chelating agents having low K ⁇ values such as polyanionic complexing agents may be attached to compound 3, resulting in the formation of a chelating agent of the present invention.
  • EXAMPLE 14 The synthesis of a chelating agent that reacts with a modified monoclonal antibody is shown below. The following reaction results in the formation of a chelating agent of the present invention which contains a macropolycycle containing a three dimensional intermolecular cavity having an arm attached on the bridge -30-
  • This example involves the use of a slurry of amino hexyl sepharose bound to a cage with a structure as in
  • Example 3 Lead nitrate was added to the gel, approximately 200 ⁇ g total, with almost complete absorption onto the cage.
  • the gel after extensive washes with deionized water was suspended to 1 ml volume and Pb content was based on absorbed material versus washes. Therefore since 0 ppm was recorded in the wash water, approximately 200 ⁇ g remained in the gel.
  • a first series of tests was conducted to measure how much lead remained bound after treating the cages with water, the control, MnCl, which attempts to displace lead in the cage, and DTPA, a chelating agent which attempts to pull lead out of the cage.
  • Deionized water was used to dilute 40 ⁇ g of Pb in complex form to 1 ml final volume at 80°C.
  • Deionized water was also used to dilute 40 ⁇ g of Pb in complex form and 80 ⁇ g of MnCl to 1 ml final volume at 80°C. Finally, deionized water was used to dilute 40 ⁇ g of Pb in complex form and 80 ⁇ g of DTPA to 1 ml final volume at 80°C. Aliquots were taken at 0, 1, 3, and 24 hours. Samples were spun down to remove the cages, including the cage bound Pb, and the Pb content of the supernatant was measured in ⁇ g via atomic absorption. The results are shown below in Table 2. TABLE 2
  • the pH ranged between 7.8 and 9.2 after adjustment.
  • the second sample was reacted with excess Pb(IV) oxide for 20 minutes and centrifuged for 8 minutes at 30,000 rp .
  • the supernatant had 18 ppm Pb (8.8 x 10 ⁇ 6 moles/ml) which is equivalent to a 3:1 Pb to antibody ratio.
  • the following table represents the results when various chelating agents are used to chelate lead.
  • the chelators were ethylene diamine tetraacetate (EDTA) , diethylene triamine pentaacetic acid (DTPA), 2,3- dimercapto-1-propane sulfonic acid (2,3 DMPS) , and meso- 2,3-dimercaptosuccinic acid (2,3 DMSA). All lead levels are listed in ppm (parts per million) . Levels greater than 70 ppm are considered offscale and are not as accurate. Even at levels of 1 mg of lead oxide there is very little difference in lead concentrations, indicating that uptake of lead is determined by the chelator's reaction capability.
  • the conjugate prepared in Example 3 is evaluated for in vitro cytotoxic activity using the following procedure.
  • thymidine uptake After 24 hours, 10 ⁇ l of 1 ⁇ Ci 3 H-thymidine containing medium is added in order to measure thymidine uptake. Thymidine is incorporated into DNA and thymidine uptake is used to measure DNA synthesis which relates to cell viability. After another day of growth the plates are shock frozen, then thawed and the individual well contents passed through glass fiber filters. The radioactivity is determined and taken as a measure of cell viability.
  • the results of the in vitro assay show a high level of cytotoxicity activity for the conjugate.
  • EXAMPLE 20 Assay for in vivo binding specificity and affinity The conjugate prepared in Example 3 is evaluated for in vivo binding specificity and affinity by the following procedure.
  • Thy us deficient BALBc (nude/nude) mice are subcutaneously injected with 2xl0 6 M21-UCLA melanoma cells. After two weeks, 35 ⁇ g of metal chelate immunoconjugate is injected into the tail vein. After 48 hours the animals are sacrificed and the radioactivity in individual organs is determined. The metal chelate immunoconjugate is formed using TDCOD-DTPA, Pb0 2 , and 9.2.27 monoclonal antibody, following the procedures discussed in Example 3.
  • the in vivo biodistribution data obtained with tumor bearing nude mice also shows that the conjugate has a high degree of binding specificity and affinity. Further, the data indicates that any unbound conjugate is cleared from the body as indicated by the low levels of conjugate found in the blood, liver, kidney, spleen and intestine.
  • the bifunctional chelating agents of the present invention are useful as therapeutic agents in radiation therapy.
  • chelating agents for a metal ion such as a radioisotope
  • a site-specific compound or target cell binding protein such as a monoclonal antibody.
  • a chelating agent for a metal ion such as a radioisotope
  • a site-specific compound or target cell binding protein such as a monoclonal antibody.

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Abstract

Sont décrits des chélates de métaux ainsi que des conjugués thérapeutiques ou diagnostiques de composés spécifiques du site, des chélates de métaux, et des ions métal. Les chélates de métaux se composent d'au moins deux constituants moléculaires liés ensemble. Un constituant moléculaire est un chélate présentant une affinité élevée pour les ions métal et une valeur Ks élevée. L'autre constituant moléculaire est un chélate ayant une faible valeur Ks et la faculté de subir un échange rapide de métaux.
PCT/US1991/007016 1990-09-27 1991-09-26 Agents chelateurs WO1992005804A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995018156A1 (fr) * 1993-12-30 1995-07-06 Abbott Laboratories Complexes metalliques de coordination ion-ligand, anticorps diriges contre ceux-ci, et dosages effectues avec ces anticorps
US5695737A (en) * 1994-08-08 1997-12-09 Schering Aktiengesellschaft Dimeric DTPA derivatives, their metal complexes and pharmaceutical agents containing these complexes
WO2002085413A1 (fr) * 2001-04-24 2002-10-31 Ts Corporation Catalyseur synthetique pour le clivage selectif d'une proteine et procede de clivage selectif d'une proteine faisant appel a ce catalyseur
DE102004022461A1 (de) * 2004-05-06 2005-12-01 Forschungszentrum Rossendorf E.V. Metallkomplexe auf der Basis von Tetrathiol-Liganden und deren Anwendung in der nuklearmedizinischen Diagnostik und Endoradionuklidtherapie sowie Verfahren zur Herstellung der Metallkomplexe
EP3000811A1 (fr) * 2014-09-24 2016-03-30 Friedrich-Alexander-Universität Erlangen-Nürnberg Complexes de éther couronne et des méthodes pour les produire

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US4867951A (en) * 1988-03-31 1989-09-19 The United States Of America As Represented By The United States Department Of Energy Separation of actinides from lanthanides
US4880008A (en) * 1985-05-08 1989-11-14 The General Hospital Corporation Vivo enhancement of NMR relaxivity
EP0355045A2 (fr) * 1988-08-15 1990-02-21 Zeneca Limited Composition comprenant un composé macrocyclique et un acide organique et son utilisation pour l'extraction des métaux
US4927923A (en) * 1984-09-26 1990-05-22 Compagnie Oris Industries Macropolycyclic rare earth complexes and application as fluorescent tracers
US5006643A (en) * 1987-06-24 1991-04-09 The Dow Chemical Company Process for preparing isothiocyanato functionalized metal complexes

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US3847949A (en) * 1970-05-12 1974-11-12 Du Pont Macrocyclic hetero imine complexing agents
HU210667B (hu) * 1990-01-16 1998-03-30 NUCLETECH Kutató-Fejlesztő, Egészségügyi és Kereskedelmi Korlátolt Felelősségű Társaság Eljárás N,N'-bisz(dikarboxi-metil)-1,4,10,13-tetraoxa-7,16-diaza-ciklooktadekán-származékok sói és komplexei és a vegyületeket tartalmazó gyógyászati készítmények előállítására

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US4927923A (en) * 1984-09-26 1990-05-22 Compagnie Oris Industries Macropolycyclic rare earth complexes and application as fluorescent tracers
US4880008A (en) * 1985-05-08 1989-11-14 The General Hospital Corporation Vivo enhancement of NMR relaxivity
US5006643A (en) * 1987-06-24 1991-04-09 The Dow Chemical Company Process for preparing isothiocyanato functionalized metal complexes
US4867951A (en) * 1988-03-31 1989-09-19 The United States Of America As Represented By The United States Department Of Energy Separation of actinides from lanthanides
EP0355045A2 (fr) * 1988-08-15 1990-02-21 Zeneca Limited Composition comprenant un composé macrocyclique et un acide organique et son utilisation pour l'extraction des métaux

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995018156A1 (fr) * 1993-12-30 1995-07-06 Abbott Laboratories Complexes metalliques de coordination ion-ligand, anticorps diriges contre ceux-ci, et dosages effectues avec ces anticorps
US5695737A (en) * 1994-08-08 1997-12-09 Schering Aktiengesellschaft Dimeric DTPA derivatives, their metal complexes and pharmaceutical agents containing these complexes
WO2002085413A1 (fr) * 2001-04-24 2002-10-31 Ts Corporation Catalyseur synthetique pour le clivage selectif d'une proteine et procede de clivage selectif d'une proteine faisant appel a ce catalyseur
DE102004022461A1 (de) * 2004-05-06 2005-12-01 Forschungszentrum Rossendorf E.V. Metallkomplexe auf der Basis von Tetrathiol-Liganden und deren Anwendung in der nuklearmedizinischen Diagnostik und Endoradionuklidtherapie sowie Verfahren zur Herstellung der Metallkomplexe
DE102004022461B4 (de) * 2004-05-06 2006-07-06 Forschungszentrum Rossendorf E.V. Metallkomplexe auf der Basis von Tetrathiol-Liganden und deren Anwendung in der nuklearmedizinischen Diagnostik und Endoradionuklidtherapie sowie Verfahren zur Herstellung der Metallkomplexe
US7731936B2 (en) 2004-05-06 2010-06-08 Forschungszentrum Rossendorf E,V Metal complexes based on tetrathiol ligands and their use in nuclear medical diagnostics and endoradionuclide therapy and method for producing said metal complexes
EP3000811A1 (fr) * 2014-09-24 2016-03-30 Friedrich-Alexander-Universität Erlangen-Nürnberg Complexes de éther couronne et des méthodes pour les produire

Also Published As

Publication number Publication date
JPH06505795A (ja) 1994-06-30
EP0554358A1 (fr) 1993-08-11
EP0554358A4 (en) 1995-09-06
CA2092434A1 (fr) 1992-03-28

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