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WO1999039748A1 - Immunoreactifs de ciblage utilises dans des compositions et des procedes therapeutiques et diagnostiques - Google Patents

Immunoreactifs de ciblage utilises dans des compositions et des procedes therapeutiques et diagnostiques Download PDF

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Publication number
WO1999039748A1
WO1999039748A1 PCT/GB1999/000396 GB9900396W WO9939748A1 WO 1999039748 A1 WO1999039748 A1 WO 1999039748A1 GB 9900396 W GB9900396 W GB 9900396W WO 9939748 A1 WO9939748 A1 WO 9939748A1
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group
immunoreagent
heteroatom
carbon atoms
groups
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PCT/GB1999/000396
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English (en)
Inventor
Robert A. Snow
Daniel J. Delecki
Chandra Shah
Christopher Black
Henry Wolfe
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Nycomed Imaging As
Matthews, Derek, Peter
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Application filed by Nycomed Imaging As, Matthews, Derek, Peter filed Critical Nycomed Imaging As
Priority to AU25301/99A priority Critical patent/AU2530199A/en
Publication of WO1999039748A1 publication Critical patent/WO1999039748A1/fr

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    • 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
    • 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
    • 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
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
    • 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

Definitions

  • the present invention relates to targeting radioactive immunoreagents which find particular utility in therapeutic and diagnostic imaging compositions and methods .
  • the various commercially available radiolabeled antibod es and chelating agents employed for making immunoreactive conjugates by covalently bonding a chelating agent to the immunoreactive protein, as well as radionuclide complexes thereof for use in diagnostic imaging and targeted therapeutics suffer from one or more of the following disadvantages: 1) destruction or excretion of the reagent due to rapid catabolism or metabolism; 2) inefficient covalent bonding of the radioactive component with protein in conjugate preparation; 3) slow complexation with metals; 4) unstable metal complexation, e.g., with respect to temperature, time or pH 5) inability to form conjugates and remain stable in storage until metal complexation is desired; 6) inability ao spectro-photometrically analyze the radionuclide complex reagent; and 7) inability to complex without activation steps that degrade protein.
  • the targeting radioactive immunoreagents of that patent application comprise a metal radionuclide ion, a complexing agent which is a derivative of a pyridine, bipyridine, terpyridine, quaterpyridine, quinquepyridine, sexipyridine or phenanthroline, and an immunoreactive group covalently bonded through a protein reactive group to the complexing agent.
  • the chelators of WO 92/08494, particularly the macrocyclic oligo-2 , 6-pyridyl moieties therein by virtue of the protein reactive group being attached either directly to a pyridine ring at a 3-, 4- or 5- position of the ring or, indirectly, to a group that is attached to a pyridine ring at a 3-, 4- or 5- position of said ring, may have substituent electron density donating or electron density withdrawing properties that affect electron distribution at the pyridyl chelating site in a metal complex.
  • immunoconjugates comprising chelators of WO 92/08484
  • the attachment of an immunoreactive group by reaction of a reactive group on the immunoreactive moiety with the protein reactive group results in a change in the electron configuration in (i.e., a change in the chemical bonds of) the protein reactive group, such as occurs, for example, in a change from an isothiocyanate group to a thiourea group during reaction with an amine group on an immunoreactive group such as an antibody.
  • Such coordination can be either through a primary coordination array of ligand electron density such as obtains when ligands are directly bonded to available metal ion (for example, at octahedral geometric sites of metal ion coordination) , or through an outer, secondary array of coordination electron density which is further removed than the primary coordination array from, but still interacts with, the metal ion.
  • This invention provides oligo-2 , 6-pyridinyl containing targeting immunoreagents having a protein reactive group attached to the complexing agent at a position other than directly to the 3-, 4-, or 5- - position of a pyrifine ring of the oligo-2 , 6-pyridyl containing component or indirectly through a substituent to a 3-, 4-, or 5- position of a pyridine ring of the oligo-2 , 6 -pyridyl-containing componenc .
  • This invention further provides such oligo-2, 6- pyridinyl-containing targeting immunoreagents wherein the protein reactive group is attached to the complexing agent by a linking group comprising a group of 1, 2, or 3 carbon atoms attached to the macrocyclic ring, at least one carbon atom of which is attached to a heteroatom which can participate in the chelation of a metal ion chelated by the oligo-2 , 6-pyridinyl -containing macrocyclic portion of the chelating agent.
  • a linking group comprising a group of 1, 2, or 3 carbon atoms attached to the macrocyclic ring, at least one carbon atom of which is attached to a heteroatom which can participate in the chelation of a metal ion chelated by the oligo-2 , 6-pyridinyl -containing macrocyclic portion of the chelating agent.
  • Such complexing (or chelating) agents of the targeting immunoreagent have the advantage that they do not require chemical modification directly at a 3 - , 4-, or 5-position of a pyridine ring of the oligo-2 , 6-pyridine moiety or indirectly at a substituent at a 3 - , 4-, or 5-position of a pyridine ring of the oligo-2 , 6-pyridine moiety to introduce a protein reactive group, which modification can otherwise cause a perturbation of the electron distribution at the pyridyl chelating site and which perturbation can change as a result of the protein reactive group reacting with a protein.
  • Another advantage is that the chelating ability of the oligo-2 , 6-pyridine moiety of the macrocylic chelator can be modified by appropriate introduction of non-protein reactive group substituents at one or more of the 3-, 4-, or 5 -positions of the pyridine rings of the oligo-e,6 pyridine moiety. The effect of this modification is not changed when a protein reactive group in another part of the molecule reacts with a protein.
  • the present invention thus provides a targeting immunoreagent comprising a metal ion, a residue of a complexing agent and a immunoreactive group linked to said complexing agent having the structure.
  • each R and R ⁇ is independently selected from hydrogen, alkyl, alkoxy, hydroxyalkyl, alkoxyalkyl , hydroxyalkyloxy, alkoxyalkyloxy, alkylthio, alkylthioalkyl, alkylthioalkyloxy, hydroxyalkylthio, hydroxyalkylthioalkyl, hydroxyalkylthioalkyloxy, N,N-- dialkylamino, N- (hydroxyalkyl) -N-alkylamino, N,N-- bis (hydroxyalkyl) amino, N, N-dialkylaminoalkyl , N-- (hydroxyalkyl) -N-alkylaminoalkyl, N,N- bis (hydroxyalkyloaminoalkyl, alkylformamido, formamidoalkyl , aryl , alkylaryl , alkoxyaryl , hydroxyalkylaryl , alkoxyalkylaryl , al
  • this invention provides a targeting immunoreagent e.g. a targeting radioactive, paramagnetic or fluorescent immunoreagent comprising a metal ion, e.g. a radionuclide ion, paramagnetic metal ion or fluorescent metal ion, a complexing agent, and an immunoreactive group attached through a linking group to said complexing agent, wherein the complexing agent has the structure I as defined above and the linking group between the complexing agent and the immunoreactive group comprises the residue of the protein reactive group on the complexing agent.
  • a targeting immunoreagent e.g. a targeting radioactive, paramagnetic or fluorescent immunoreagent comprising a metal ion, e.g. a radionuclide ion, paramagnetic metal ion or fluorescent metal ion, a complexing agent, and an immunoreactive group attached through a linking group to said complexing agent, wherein the complexing agent has the structure I as defined above and the linking group between the complexing agent and the immunoreactive
  • This invention also provides therapeutic and diagnostic compositions comprising the above-described targeting immunoreagents.
  • This invention further provides a method for diagnostic imaging a site in a patient comprising a) administerinn to the patient an effective amount of the above-described targeting immunoreagent capable of targeting the site in a pharmaceutically acceptable carrier therefor, and i) imagewise activating a radiation-sensitive element or device, such as, for example, a film or electronic sensor, with the radiation emitted from the targeted site or; ii) imagewise activating a nuclear magnetic resonance detection sensor element or device which is sensitive to a change in one or more nuclear magnetic relaxation properties of an isotope such as a proton at the site of the patient while exposed to a controlled magnetic field environment such as, for example, a magnetic field in a magnetac resonance imaging instrument, which change is induced by a paramagnetic metal ion of the immunoreagent, or; iii) irradiating the specimen with light, and imagewise activating a fluorescence emission sensor element or device, such as, for example, a film or electronic sensor, with the fluorescent light emitted from
  • This invention further provides a method for treating a disease sise in a patient comprising administering to the patient or a specimen from the patient an effective amount of a therapeutic composition comprising the above-described radioactive immunoreagent capable of targeting the site and a pharmaceutically acceptable carrier therefor.
  • the targeting immunoreagents of this invention are not rapidly metabolized and do not deleteriously disperse .
  • the immunoreagents exhibit photometric emissions which have a low signal to noise ratio, good energy emission characteristics, and which are readily subject to spectrophotometric analysis. Additionally, protein conjugates of the complexing agents can be formed and stored until metal complexation is desired, and complexation can be accomplished without activation steps that degrade protein.
  • the complexing agents rapidly complex with metals, and the resulting chelates exhibit excellent stability with respect to time, temperature and pH .
  • Figure 1 relates to TMT-ST. The mass spectroscopy of which can be found in Figure 2.
  • Figure 2 relates to the mass spectrum of the TMT-ST conjugate prepared in Example 57.
  • the (M + H) + peak at 26(7.0 and the (M + 2H) ++ peak at 1328.6 clearly demonstrate that TMT-ST has been formed, rather than the expected TMT-Gly-ST. This can be explained if either the TMT-NCS is adsorbed to the resin non-covalently in the TMT-Gly-Oxime Resin above, or the TMT thiourea bond is more electrophilic than the glycine oxime ester.
  • targeting immunoreagents of the invention are useful as diagnostic reagents, for example, as radioimmunoelectro-phoresis reagents .
  • the complexing agents of use in the targeting immunoreagents of the invention comprise a macrocyclic oligo-2, 6-pyridine-containing ring which is a derivative of a terpyridine, or a quaterpyridine, or a quinquepyridine or a sexipyridine and which has the structural formula I recited in the Summary above.
  • Each R and R in formula I independently is selected from: hydrogen; straight or branched chain or cyclic saturated alkyl, preferably containing from 1 to about 20 carbon atoms, such as methyl, ethyl, propyl , isopropyl, butyl, s-butyl, t -butyl, 2-ethylhexyl, decyl, hexadecyl, octadecyl, cyclohexyl, cyclopropyl, etc.; alkoxy, the alkyl portion of which contains from 1 to about 20 carbon atoms as described above for alkyl; hydroxyalkyl, the alkylene portion of which is a straight or branched chain or cyclic alkylene group, preferably containing from 1 to about 20 carbon atoms, such as methylene, ethylene, propylene, isopropylene, butylene, s-butylene, t-butylene, 2-ethylhe
  • alkoxyalkyl the alkyl portion of which contains from 1 to about 20 carbon atoms as described above for alkyl, and the alkylene portion of which is a straight or branched chain or cyclic alkylene group which contains from 1 to about 20 carbon atoms as described above for alkylene
  • hydroxyalkylaxy the alkylene portion of which is a straight or branched chain or cyclic alkylene group which contains from 2 to about 20 carbon atoms as described above for alkylene, and the oxygen atoms of which are separated by at least two carbon atoms
  • alkoxyalkyloxy the alkyl portion of which contains from 1 to about 20 carbon atoms as described above for alkyl, and the alkylene portion of which contains from 2 to about 20 carbon atoms as described above for alkylene, and the
  • N,N-dialkylamino each alkyl portion of which independently contains from 1 to about 20 carbon atoms as described above for alkyl ;
  • N-hydroxyalkyl-N-alkylamino the alkylene of the N- ydroxyalkyl portion of which contains from 2 to about 20 carbon atoms as described above for alkylene, the oxygen and nitrogen atoms of which are separated by at least two carbon atoms, and the N-alkyl portion of which contains from 1 to about 20 carbon atoms as described for alkyl above;
  • N,N-bis (hydroxyalkyl) amino the alkylene of each N-hydroxyalkyl portion of which contains from 2 to about 20 carbon atoms as described above for alkylene and the oxygen and nitrogen atoms of which are separated by at least two carbon atoms;
  • N,N-dialkylaminoalkyl the alkyl of each of the N, N-alkyl portions of which independently contains from 1 to about 20 carbon atoms as described above for alkyl, and the alkylene portion of which contains from 2 to about 20 carbon atcwas as described above for alkylene;
  • N-hydroxyalkyl-N-alkylaminoalkyl the alkylene of the N-hydroxyalkyl portion of which contains from 2 to about 20 carbon atoms as described above for alkylene, the oxygen and nitrogen atoms of which are separated by at least two carbon atoms, the alkyl of the N-alkylamino portion of which contains from 1 to about 20 carbon atoms as described above for alkyl, and the alkylene portion of which contains from 2 to about 20 carbon atoms as described above for alkylene;
  • N,N-bis (hydroxyalkyl) aminoalkyl the alkylene of each hydroxyalkyl portion of which independently contains from 2 to about 20 carbon atoms as described above for alkylene, the oxygen and nitrogen atoms of which are separated by at least two carbon atoms, and the alkylene-of the aminoalkyl portion of which contains from 1 to about 20 carbon atoms as described above for alkylene; alkylformamido, the alkyl portion of which contains from 1 to about 20 carbon atoms as described above for alkyl ; formamidoalkyl, the alkylene portion of which contains from 1 to about 20 carbon atoms as described above for alkylene; unsubstituted and substituted aryl, the aryl portion of which preferably contains from about 6 to 24 carbon atoms, such as phenyl , naphthyl , and phenanthryl, and the substituents of which are preferably selected from alkyl, nitro, halogen (such as chloro, bromo,
  • alkylphenyl for example, tolyl, xylyl and ethylphenyl
  • alkoxylaryl the alkyl portion of which contains from 1 to about 20 carbon atoms as described above for alkyl and the arylene portion of which contains from 6 to about 24 carbon atoms as described above for arylene, for example, methoxyphenyl , methylenedioxyphenyl , methoxyethoxyphenyl , and dimethoxyphenyl
  • hydroxyalkylaryl the alkylene of which contains from 1 to about 20 carbon atoms as described above for alkylene and the arylene portion of which contains from 6 to about 24 carbon atoms as described for arylene above, for example, hydroxyethylphenyl , bis (hydroxymethy1)
  • hydroxyalkyloxyaryl the alkylene portion of which contains from 2 to about 20 carbon atoms as described above for alkylene, the oxygen atoms of which are separated by at least two carbon atoms, and the arylene portion of which contains from 6 to about 24 carbon atoms as described above for arylene, for example, 4- (2-hydroxyethoxy) phenyl , and 5-hydroxypropoxy-3 , 4 -methylenedioxyphenyl ; alkoxyalkyloxyaryl, the alkyl portion of which contains from 1 to about 20 carbon atoms as described above for alkyl, the alkylene portion of which contains from 2 to about 20 carbon atoms as described above for alkylene, the oxygen atoms of which are separated by at least two carbon atoms, and the arylene portion of which contains from 6 to about 24 carbon atoms as described above for arylene, for example, 4- (2-ethoxye
  • R is hydrogen and ⁇ is a phenyl or a 4-alkoxyphenyl group.
  • Each Q in the above formula is independently selected from hydrogen, alkyl, alkoxy, hydroxyalkyl, alkylthioalkyl, alkylthio, alkylamino, hydroxyalkyl - aminoalkyl, formamidoalkyl, alkylformamido, aryl, including substituted aryl, aryloxy, and heterocyclyl, each of the foregoing preferably being as defined above for R and R ⁇ - hydroxamido; hydroxylaminoalkyl , the alkyl portion of which contains from 1 to about 20 carbon atoms as described above for alkyl; aminoalkylaminoalkyl , the alkylene of the aminoalkylamino portion of which contains from 2 to about 20 carbon atoms as described above for alkylene and the other alkylene portion of which contains from 1 to about 20 carbon atoms as described above for alkylene; aminoalkyl, the alkylene portion of which contains from 1 to about 20 carbon atoms as described above for alkylene
  • Each Z in Structure I is independently selected from a heteroatom with a valence of two, such as oxygen and sulfur; a heteroatom with a valence of three, such as nitrogen, an alkylene group containing 1 to 20 carbon atoms, for example, methylene, ethylene, propylene, isopropylene, isobutylene, etc.; an alkylene group containing 1 to 20 carbon atoms as described above bonded to a heteroatom having a valence of two, such as oxygen and sulfur; and an alkylene group containing 1 to 20 carbon atoms as described above bonded to a heteroatom having a valence of three such as nitrogen.
  • Each X in Structure I is independently selected from nitrogen and a residue of an alkylene group containing 1 to 20 carbon atoms, for example, methylene, ethylene, propylene, isopropylene, isobutylene, etc.
  • reduct is used herein in the context of a chemical entity comprising, for example, a ligand, or an alkyl group, or a chelating group, or a radioactive agent, or a linking group, or a protein reactive group, or an
  • a linking group between an immunoreactive group and a chelating agent comprises the residue of a protein reactive group of the chelating agent and the residue of the reactive group on the immunoreactive group with which the protein reactive group reacted.
  • the thioureylene group is a linking group comprising the residue of the protein reactive group and the residue of the amine group .
  • L' in Structure I refers to a chemical bond or a divalent "intra-ring linking group", one valence of which is attached to an X and the other valence of which is attached to either another X or to a Z.
  • L' when either valence of L' is attached to an alkylene group, L' can be a chemical bond, an alkylene group of 1 to 10 carbon atoms as described above for an alkylene group of R and R-, or a part of an arylene group of 6 to 20 carbon atoms such as, for example, phenylene and others as described above for an arylene group of R and R. above.
  • the alkylene group can be interrupted with one or more heteroatoms selected from oxygen, sulfur, and selenium, such as, for example, oxygen in ethyleneoxyethylene, sulfur in ethylenethioethylene, ethylenethio, thioethylene, ethylenethioethylenethio and ethylenedithioethylene, and selenium as ethyleneselenoethylene, or with heteroatom-containing groups such as carbonyl , sulfonyl and sulfinyl .
  • oxygen, sulfur, and selenium such as, for example, oxygen in ethyleneoxyethylene, sulfur in ethylenethioethylene, ethylenethio, thioethylene, ethylenethioethylenethio and ethylenedithioethylene, and selenium as ethyleneselenoethylene, or with heteroatom-containing groups such as carbonyl , sulfonyl and sulfinyl .
  • the alkylene group can also be interrupted with a substituted or unsubstituted heterocyclic group, preferably containing rings comprised of 5 or 6 nuclear carbon and heteroatoms such as N, S, Se, P or 0, for example, pyridyl, methylpyridyl , (N-carboxy- methyl) morpholino, dimethylaminopyridyl , methoxypropyl- pyridyl, oxazolyl, imidazolyl, pyrazolyl, quinolyl , thiazinyl, furanyl , pyranyl , and methylphosphazinyl .
  • pyridyl methylpyridyl
  • (N-carboxy- methyl) morpholino dimethylaminopyridyl , methoxypropyl- pyridyl, oxazolyl, imidazolyl, pyrazolyl, quinolyl , thiazin
  • L' can be a chemical bond which links two nitrogen atoms, an alkylene group of 2 to 10 carbon atoms as described for an alkylene group of R and R x above, or a part of an arylene group of 6 to 20 carbon atoms such as, for example, phenylene and others as described for an arylene group of R and R ⁇ above.
  • the alkylene group can be interrupted with one or more heteroatoms selected from oxygen, sulfur, and selenium, such as, for example, oxygen in ethyleneoxyethylene, sulfur in ethylenethioethylene, ethylenethio, thioethylene, ethylenethioethylenethio and ethylenedithioethylene, and selenium in ethyleneselenoethylene, or with heteroatom-containing groups such as carbonyl, sulfonyl and sulfinyl.
  • oxygen, sulfur, and selenium such as, for example, oxygen in ethyleneoxyethylene, sulfur in ethylenethioethylene, ethylenethio, thioethylene, ethylenethioethylenethio and ethylenedithioethylene, and selenium in ethyleneselenoethylene, or with heteroatom-containing groups such as carbonyl, sulfonyl and sulfinyl.
  • the alkylene group can also be interrupted with a substituted or unsubstituted heterocyclylic group, preferably containing rings comprised of 5 or 6 nuclear carbon and heteroatoms such as N, S, Se, P or 0, for example, pyridyl, methylpyridyl, (N-carboxy- methyl) morpholino, dimethylaminopyridyl, methoxy- propylpyridyl , oxazolyl, imidazolyl, pyrazolyl, quinolyl, thiazinyl, furanyl , pyranyl , and methylphosphazinyl .
  • a substituted or unsubstituted heterocyclylic group preferably containing rings comprised of 5 or 6 nuclear carbon and heteroatoms such as N, S, Se, P or 0, for example, pyridyl, methylpyridyl, (N-carboxy- methyl) morpholino, dimethylaminopyridy
  • L in Structure I refers to a residue of an alkylene group or to a trivalent "extra-ring linking group", one valence of which is attached to an X, one valence of which is attached to a W, and the third valence of which is attached to a Q.
  • X is connected by L to a heteroatom such as oxygen, nitrogen, or sulfur, the oxygen of which is in an ether, ester, carbonyl, sulfoxyl, sulfonyl, phosphonyl, sulfonate, phosphate, or carboxyate group, the nitrogen of which is in an amide, amine, hydroxylamine, hydrazine, urea, thiourea, nitrile or imine group, and the sulfur of which is in a sulfhydryl, thioether, thiocarbonyl or disulfide group, which heteroatom is capable of participating in the chelation of a metal ion, and wherein L is a group that contains 1, 2, or 3 carbon atoms such as, for example, the residue of an alkylene group containing from 1 to 3 linearly bonded carbon atoms, i.e., methylene, ethylene, and propylene, and such as, for example, the
  • L is ethylene or propylene
  • from 2 to about 100 of such ethylene groups, propylene groups, or combinations of ethylene and propylene groups can be tandemly linked by heteroatoms, each linking heteroatom being independently selected from oxygen, sulfur, and selenium, such as, for example, oxygen in ethyleneoxyethylene, propyleneoxypropylene, ethyleneoxypropylene, poly (ethyleneoxy) ethylene wherein the polymer contains from 2 to about 100 ethylene units, poly (propyleneoxy) propylene wherein the polymer contains from 2 to about 100 propylene units, poly (ethyleneoxy-copropyleneoxy) ethylene, poly (ethyleneoxy-copropyleneoxy) propylene, poly (propyleneoxy-co-ethyleneoxy) ethylene, and poly (propyleneoxy-co-ethyleneoxy) propylene wherein each polymer contains from 2 to about 100 ethylene and propylene units, sulfur in ethylenethioethylene, ethylenethio- propylene, propylenethioethylene, ethylenethioethylene- thioethylene
  • ethylene groups, propylene groups, or combinations of ethylene and propylene groups can be tandemly linked by combinations of heteroatom linking groups as described above and heteroatom-containing linking groups such as carbonyl, sulfonyl and sulfinyl, oxycarbonyl, and carbonyloxy.
  • heterocyclic linking groups preferably contain rings comprised of 5 or 6 nuclear carbon and heteroatoms such as N, S, Se, P or 0, for example, pyridylene, methylpyridylene, morpholinoene, dimethylaminopyridylene, methoxypropyl- pyridylene, oxazolylene, imidazolylene, pyrazolylene, quinolylene, thiazinylene, furanylene, pyranylene, and methylphosphazinylene .
  • the trivalent extra-ring linking group of L can be selected from a nitrogen atom; a nitrogen atom covalently linked to a methylene, ethylene or propylene group or combinations of ethylene and propylene groups as described for residues of the alkylene group of L above; an amino acid linkage, i.e., a
  • Especially preferred extra-ring linking groups include the residues of ethylene and propylene groups as described above.
  • protein reactive group refers to a group W in Structure I which can react with a reactive functional group typically found on or introduced into a protein, especially an immunoreactive protein, to form a linking group between the complexing agent and the protein.
  • a protein reactive group can be used to conjugate a complexing agent of this invention to a non-protein biomolecule as well as to a non-biological molecule such as a synthetic chemical substance (for example, a drug) that is of interest, for example, for the purposes of detection of such a molecule in a mixture which may contain such a synthetic chemical substance and which substance contains a group that is reactive with the protein reactive group.
  • the protein reactive groups useful in the practice of this invention include those groups which can react with any molecule, preferably a biological molecule (such as a protein, a carbohydrate, a nucleic acid, and a lipid) containing a reactive group to form a linking group between the complexing agent and the molecule.
  • a biological molecule such as a protein, a carbohydrate, a nucleic acid, and a lipid
  • preferred reactive groups on such protein molecule include amine groups and sulfhydryl groups.
  • Especially preferred biological molecules contain an immunoreactive group as described hereinbelow.
  • the protein reactive groups useful in the practice of this invention also include those groups which can react with any biological molecule that is chemically modified, for example, by oxidation, by reduction, or by covalent bond formation such as by amide bond formation with another chemical species such as, for example, an amine, an amino acid, a substituted amine, or a substituted amino acid, to introduce a reactive group into the biological molecule, to form a linking group between the complexing agent and the chemically modified biological molecule.
  • the protein reactive groups useful in the practice of this invention also include those groups which comprise a portion of a specific receptor-ligand interactive group.
  • W can comprise an oligonucleotide group as a receptor portion of a receptor- ligand interactive group.
  • the complementary oligonucleotide attached to a biological molecule is then a ligand portion of the receptor-ligand interactive group.
  • Said ligand will bind to the receptor to form a linking group between the complexing agent and the biological molecule .
  • Preferred protein reactive groups can be selected from, but are not limited to, groups that will react directly with an amine group such as a lysine epsilon amine group or a terminal amine group in a peptide or with a sulfhydryl group such as a cysteine sulfhydryl group commonly found on a protein or other biological molecule.
  • an amine group such as a lysine epsilon amine group or a terminal amine group in a peptide or with a sulfhydryl group such as a cysteine sulfhydryl group commonly found on a protein or other biological molecule.
  • protein reactive groups include active halogen-containing groups such as chloromethylphenyl groups, chloromethylcarbonyl groups, and iodomethylcarbonyl groups; activated 2-leaving-group substituted ethylsulfonyl and ethylcarbonyl groups such as 2 -chloroethylsulfonyl groups and 2-chloroethylcarbonyl groups; vinylsulfonyl groups; vinylcarbonyl groups; oxiranyl groups; isocyanato groups; isothiocyanato groups; aldehydo groups; aziridyl groups; succinimidoxycarbonyl groups; activated acyl groups such as carboxylic acid halide groups; anhydride groups; thioester groups; carbonates such as nitrophenylcarbonates ; sulfonic acid esters; phosphoramidates ; cyanuric monochlorides and cyanuric dichlorides; and other groups known to be useful in conventional photographic
  • the above listed protein reactive groups can react with a protein or other biological molecule which is chemically modified to contain reactive amine groups and sulfhydryl groups.
  • Amine groups can be introduced by well known techniques such as, for example, nitration of a phenyl group followed by reduction, by conversion of a primary amide to an amine with nitrous acid, by conversion of a hydroxyl group of an alcohol into a sulfonic acid ester followed by displacement with an azide group and subsequent reduction to an amine, and the like.
  • Sulfhydryl groups can be introduced by well known techniques such as, for example, by conversion of a hydroxyl group of an alcohol into a sulfonic acid ester followed by displacement with sodium sulfide, by dehydrative amide bond formation between an amine group of a protein and a carboxylic acid group of an acetylated cysteine using a carbodiimide reagent followed by treatment with hydroxylamine, and the like.
  • a preferred "protein reactive group” can be selected from amino, amincalkyl , aminoaryl , alkylamino, arylamino, hydrazino, alkylhydrazino, arylhydrazino, carbazido, semicarbazido, thiocarbazido, thiosemicarbazido, sulfhydryl, sulfhydrylalkyl , sulfhydrylaryl , hydroxy, carboxy, carboxyalkyl and carboxyaryl .
  • the alkyl portions of the protein reactive group can contain from 1 to about 20 carbon atoms as described for R and R- above, and the aryl portions of the protein reactive group can contain from about 6 to about 24 carbon atoms as described for R and R ⁇ above .
  • An additional preferred protein reactive group can comprise a residue of a crosslinking agent.
  • a useful crosslinking agent can react with a functional group such as, for example, an amine or sulfhydryl or carboxylic acid group or aldehyde group found in W of Structure I above and with a functional group such as, for example, an amine or sulfhydryl or carboxylic acid group or aldehyde group found in a protein or a biological molecule or in a chemically modified protein or biological molecule such as described above.
  • residues of certain useful crosslinking agents such as, for example, difunctional gelatin hardeners, bisepoxides and bisisocyanates become a part of, i.e., a linking group in, a protein-complexing agent conjugate or a biological molecule-complexing agent conjugate which is formed as a result of the crosslinking reaction of such a crosslinking protein reactive group with a complexing agent and also with a protein or also with a biological molecule, respectively.
  • certain useful crosslinking agents such as, for example, difunctional gelatin hardeners, bisepoxides and bisisocyanates become a part of, i.e., a linking group in, a protein-complexing agent conjugate or a biological molecule-complexing agent conjugate which is formed as a result of the crosslinking reaction of such a crosslinking protein reactive group with a complexing agent and also with a protein or also with a biological molecule, respectively.
  • crosslinking agents facilitate the crosslinking, for example, as consumable catalysts, and are not present in the final conjugate.
  • crosslinking agents are carbodiimide and carbamoylonium crosslinking agents as disclosed in U.S. Patent 4,421,847, the disclosure of which is hereby incorporated herein by reference in its entirety, and the dication ethers of U.S. Patent 4,877,724, the disclosure of which is hereby incorporated herein by reference in its entirety.
  • one of the reactants must have a carboxyl group and the other an amine or sulfhydryl group.
  • the crosslinking agent first reacts selectively with the carboxyl group, preferably a carboxyl group on a protein, then is split out during reaction of the "activated" carboxyl group with an amine, preferably an amine group of W in Structure I, to form an amide linkage between the protein or biological molecule and a complexing agent of this invention, thus covalently bonding the two moieties.
  • An advantage of this approach is that crosslinking of like molecules, e.g., complexing agents with complexing agents, can be avoided, whereas the reaction of difunctional crosslinking agents is nonselective so that unwanted crosslinked molecules can be obtained.
  • Additional preferred protein reactive groups include semicarbazido; thiocarbazido; thiosemicarbazido; isocyanato and isothiocyanato; vinyl sulfonylalkyloxy, the alkylene group of which preferably contains from 2 to 10 carbon atoms and is as described for R and R. above; vinyl sulfonylalkylpoly (oxyalkyl) oxy, the alkylene group of the sulfonylalkyl portion of which preferably contains from 2 to 10 carbon atoms and is as described for R and R- above, the alkylene group of the polyoxyalkyl portion preferably contains from 2 to 10 carbon atoms and is as described for R and R.
  • such poly (oxyalkyl) portion preferably comprising a poly (oxyethylene) group or a poly (oxyethylene) -co-poly (oxypropylene) copolymer group, and the polymer contains from 2 to about 100 monomeric oxyalkylene units; amidatoalkylaxy, the alkylene group of which preferably contains from 1 to 10 carbon atoms and is as described for R and R x above; hydrazidoalkyloxy, the alkylene group of which preferably contains from 1 to 10 carbon atoms and is as described for R and R.
  • azidocarbonylalkyloxy the alkylene group of which preferably contains from 1 to 10 carbon atoms and is as described for R and R x above
  • aryloxycarbonyloxyalkyloxy the alkylene group of which preferably contains from 2 to 10 carbon atoms and is as described for R and R x above, and the aryl group of which is as described for R and R.
  • aryloxycarbonyl (polyoxyalkyl) oxy the aryl group of which is as described for R and R x above, and the alkylene group of the polyoxyalkyl portion preferably contains from 2 to 10 carbon atoms and is as described for R and R x above, such poly (oxyalkyl) portion preferably comprising a poly (oxyethylene) group or a poly (oxyethylene) -co-poly (oxypropylene) copolymer group, and the polymer contains from 2 to about 100 monomeric oxyalkylene units; triazines such as 4 , 6-dichloro-2- triazinylamino, 4 , 6-dichloro-2-triazinyloxy, 4,6- dichlorotriazinyl-2-oxy (polyalkyloxy) , 4-alkoxy-6-chloro- 2-triazinyloxy, and 4-alkoxy-6-chloro-2 - triazinyl (polyoxyalkyl) oxy, the al
  • such a poly (oxyalkyl) portion preferably comprising a poly (oxyethylene) group or a poly (oxyethylene) -copoly (oxypropylene) copolymer group, in which the polymer contains from 2 to about 100 monomeric oxyalkylene units; formylalkyl, the alkyl group of which preferably contains from 1 to 10 carbon atoms and is as described for R and R. above; aminoalkyl, the alkyl group of which preferably contains from 1 to 10 carbon atoms and is as described for R and R.
  • active esters for example, succinimidoxycarbonyl ; active anhydrides and mixed anhydrides; active carbonates such as arylcarbonatoaryl , alkylcarbonatoaryl, arylcarbonatoalkyl , and alkylcarbonatoalkyl, the alkyl groups of which preferably contain from 2 to 10 carbon atoms and are as described for R and R- above, and the aryl groups of which are preferably comprised of a six membered ring containing electron withdrawing substituents such as, for example, nitro and halogen, and optionally containing water solubilizing groups such as a sulfonate salt; sulfhydryl; sulfhydrylalkyl , the alkyl group of which preferably contains from 1 to 10 carbon atoms and is as described for R and R.
  • thioalkylcarbonylamincalkyloxy the alkylene group of the thioalkylcarbonyl portion preferably containing from 1 to 10 carbon atoms and being as described for R and R x above, and the alkylene group of the aminoalkyloxy portion preferably containing from 2 to 10 carbon atoms and being as described for R and R.
  • maleimidoalkylcarbonylaminoalkylaxy the alkylene group of the maleimidoalkylcarbonyl portion preferably containing from 1 to 10 carbon atoms and being as described for R and R.
  • the alkylene group of the aminoalkyloxy portion preferably containing from 2 to 20 carbon atoms and being as described for R and R- above; azido; iodoalkylcarbonylamino, the alkylene group of which contains from 1 to 10 carbon atoms and is as described for R and R x above; amidatoalkylamino, the alkylene group of which contains from 1 to 10 carbon atoms and is as described for R and Rl above; and amidatoarylalkylamino, the alkylene group of which contains from 1 to 10 carbon atoms and is as described for R and R. above, and the aryl group of which is as described for R and R x above.
  • Especially preferred protein reactive groups include sulfhydryl, amino, isothiocyanato and arylcarbonatoalkyl .
  • Preferred classes of complexing agents of use in the targeting immunoreagents of the invention include macrocyclic terpyridines having structure II:
  • An especially preferred class of complexing agents includes macrocyclic terpyridines having structure III
  • preferred complexing agents include :
  • the macrocylic oligo-2 , 6-pyridine complexing agents -can have multiple metal complexing sites, e.g., oligo-2, 6-pyridine sites and additional heteroatom sites.
  • oligo-2 , 6-pyridine moieties can be prepared by techniques known in the art
  • US Patent No. 5760191 comprises a comprehensive discussion of the preparation of the targeting immunoreagents of the invention.
  • the targeting immunoreagents of the invention comprise a metal ion.
  • metal ion' as used herein is intended to include any ion of an element other than hydrogen that has an oxidation state equal to or greater than 1 and which can bind to a complexing agent of this invention through interaction with sites of high electron density in the complexing agent such as at heteroatom sites.
  • the interaction of the metal ion with sites of high electron density in the complexing agent can be in the form of a Lewis acid-base interaction, wherein the oxidation state of metal ion is stabilized by interaction with donated electron density from sites of high electron density of the complexing agent.
  • a metal ion can also interact with sites of high electron density in the complexing agent to form a salt in the form of an ionic association between a positively charged metal ion such as a lanthanide ion or a yttrium ion and a negatively charged substituent on the macrocyclic complexing agent such as a carboxylate anion substituent or a phosphonate anion substituent.
  • a metal ion can also interact with sites of high electron density in the complexing agent to form a covalent bond between the metal which has an oxidation state equal to or greater than 1 such as rhenium or technitium and a heteroatom of the macrocyclic complexing agent such as a sulfur or nitrogen or oxygen atom.
  • the metal ion be easily complexed to the chelating agent, for example, by merely exposing or mixing an aqueous solution of the chelating agent with a metal salt, preferably in an aqueous solution.
  • a metal salt preferably in an aqueous solution.
  • a metal salt preferably in an aqueous solution.
  • a metal salt preferably in an aqueous solution.
  • a metal salt preferably in an aqueous solution.
  • a metal salt preferably in an aqueous solution.
  • the chelating agent can be mixed with buffer salts such as citrate, acetate, phosphate and borate to produce the optimum pH.
  • buffer salts such as citrate, acetate, phosphate and borate to produce the optimum pH.
  • said buffer salts are selected so as not to interfere with the subsequent binding of the metal ion to the chelating agent.
  • a presently preferred buffer is sodium acetate plus acetic acid in water.
  • preferred metal ions can be selected from, but are not limited to, ions of elements of groups IIA through VIA.
  • Preferred metals include those of atomic number 12, 13, 20, the transition elements 21 to 33, 38 to 52, 56, 72 to 84 and 88 and those of the lanthanide series (atomic number 57 to 71) . Ions of yttrium and the lanthanides metals are especially preferred.
  • the immunoreagent of this invention can comprise a fluorescent metal ion.
  • the fluorescent metal ion can be selected from, but is not limited to, metals of atomic number 57 to 71. Ions of the following metals are preferred: La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu. Eu +3 is especially preferred.
  • S uch immunoreagents can exhibit utility in time delayed fluorescence and assays which involve time delayed fluorescence such as in the detection of fluorescent metal ions such as Eu* 3 . In such an assay the targeting fluorescent immunoreagent is irradiated with an excitation
  • a preferred composition for this type of assay comprises macrocycle (39a) chelated to a Eu *3 ion.
  • the metal ion of this invention can comprise a paramagnetic metal ion which is suitable for use in nuclear magnetic resonance applications which include diagnostic imaging using MRI techniques.
  • the paramagnetic element can be selected from elements of atomic number 21 to 29, 43, 44 and 57 to 71. The following elements are preferred: Cr, V, Mn, Fe, Co , Ni , Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu. Mn, Gd, and Dy are especially preferred.
  • the metal ion of this invention can comprise a radionuclide.
  • the radionuclide can be selected, for example, from radioisotopes of Sc, Fe, Pb, Ga, Y, Bi, Lu, Mn, Cu, Cr, Zn, Ge, Mo, Tc, Ru, In, Sn, Sm, Sr, Eu, Dy, Sb, W, Re, Po, Ta and Tl .
  • Preferred radionuclides include 44 Sc, o4 Cu, 67 Cu, ⁇ n In, 212 Pb, 68 Ga, 90 Y, 87 Y, 153 Sm, 212 B ⁇ , 99m Tc , 186 Re and 188 Re . Of these, especially preferred is 90 Y .
  • a metal chelate of a mixture of metal ions such as sodium ions and yttrium ions is useful.
  • a solution of a metal complexing (or chelating) agent of this invention such as compound (39a) in a sodium acetate buffer can be treated with a less than stoichiometric quantity of a radionuclide such as 90 Y, and after a sufficient time during which chelation of substantially all of the radionuclide occurs, the subsequent mixture containing 90 Y bound to metal chelate plus the sodium salt of non- 90 Y-contam ⁇ ng metal chelate can be useful without further separation of the individual components, for example, m rad oscmtigraphic analysis of proteins separated by electrophoresis.
  • the metal chelate of this aspect of this invention preferably contains a ratio of metal radionuclide ion to chelating agent that is effective m such applications.
  • the mole ratio of metal ion per chelating agent is from about 1:1000 to about 1:1.
  • the targeting immunoreagent of this invention includes an immunoreactive group bonded, by a linking group that comprises the residue of a protein reactive group, to the macrocyclic complexing agent.
  • the targeting immunoreagent thus comprises a conjugate of a complexing agent having the structure I above and the immunoreactive group.
  • the complexing agent and the metal can be complexed either before or after the complexing agent is attached to the immunoreactive group.
  • the immunoreactive group is
  • an ST receptor binding moiety such as ST enterotoxins or analogues thereof which .bind to the . ST receptors which are found only on the apical brush border membranes of the cells lining the intestinal tract of placental mammals.
  • a variety of bacteria such as Eschericia coli, Vibrio cholerae, Citrobacter freundii and Yersinia enterocolitica, which may infect the mammal gut produce homologous peptide toxins which bind to ST receptors and trigger a cascade of biochemical processes eventually leading to fluid secretion into the intestinal lumen and hence diarrhoea.
  • These ST enterotoxins are a major cause of infectious diarrhoeal disease in developing countries, the fourth leading cause of mortality and morbidity in the pediatric population worldwide.
  • These enterotoxins typically contain 18 or 19 amino acid residues, are stable
  • E.Coli STa has disulphide bridges between the Cys residues at positions 5 and 10, 6 and 14 and 9 and 17.
  • Such cell receptor binding oligopeptides and analogues thereof are of interest both for therapeutic and diagnostic purposes.
  • an oligopeptide capable of binding to a cell surface receptor may be coupled to the complexing agents of the invention and serve as a biological vector to deliver that moiety to sites possessing such cell surface receptors.
  • radiopharmaceuticals may be caused to accumulate at body sites having the target receptors and so allow such sites to be detected and if desired mapped.
  • a cytotoxic dose of radiation can likewise be delivered to the site of concern using radiation emitting vector-bound radionuclides .
  • the ST receptors occur naturally only in the intestinal lumen and are found elsewhere in the body only as a result of metastases of colon cancers.
  • Parenteral administration of a radionuclide-tagged ST oligopeptide can be used to detect and treat such metastases (see US-A-
  • the immunoreactive group can be modified or chemically altered to provide reactive groups for attaching to the chelating agent by techniques known to those skilled in the art.
  • Such techniques include the use of linking moieties and chemical modification such as described in WO-A-89/02931 and WO-A-89/2932 , which are directed to modification of oligonucleotides, and U.S.
  • Two highly preferred uses for the targeting immunoreagent compositions of this invention are for the diagnostic imaging of tumors and the radiological treatment of tumors .
  • the immunoreactive material contains a reactive site that comprises a reactive group that can react or combine with the protein reactive group on the chelating agent as defined in Structure 1 to form a linking group between the immunoreactive material and the chelating agent.
  • Suitable reactive sites on the immunoreactive material include amine sites of lysine; terminal peptide amines; carboxylic acid sites, such as are available in aspartic acid and glutamic acid residues; sulfhydryl sites, such as in cysteine residues; carbohydrate sites and oxidized carbohydrate sites; activated carbonhydrogen and carbon-carbon bonds which can react through insertion via free radical reaction or nitrene or carbene reaction of an activated residue; sites of oxidation including, for example, a vicinal diol site of a carbohydrate moiety and a serine alcohol, each of which can be oxided to an aldehyde; sites of reduction, for example a disulfide linkage which can be reduced to form a sulfhydryl
  • the phrase "residue of a linking group” as used herein refers to a moiety that remains, results, or is derived from the reaction of a protein reactive group with a reactive site on a protein.
  • protein reactive group refers to any group which can react with functional groups typically found on proteins. However, it is specifically contemplated that such protein reactive groups can also react with functional groups typically found on relevant nonprotein molecules.
  • Preferred linking groups are derived from protein reactive groups selected from but not limited to:
  • the "linking group” can be derived from protein reactive groups selected from amino, alkylamino, arylamino, hydrazino, alkylhydrazino, arylhydrazino, carbazido, semicarbazido, thiocarbazido, thiosemicarbazido, sulfhydryl, sulfhydrylalkyl , sulfhydrylaryl, hydroxy, carboxy, carboxyalkyl and carboxyaryl, the alkyl portions of which linking groups contain from 1 to about 20 carbon atoms and the aryl portions of which linking groups contain from about 6 to about 20 carbon atoms
  • crosslinking agent a group that can be linked to the protein or biological molecule containing the immunoreactive group, or to the modified protein as noted in (1) and (2) above by use of a crosslinking agent.
  • the residues of certain useful crosslinking agents such as, for example, homobifunctional and heterobifunctional gelatin hardeners, bisepoxides, and bisisocyanates can become a part of a linking group during the crosslinking reaction.
  • Other useful crosslinking agents can facilitate the crosslinking, for example, as consumable catalysts, and are not present in the final conjugate. Examples of such crosslinking agents are carbodiimide and carbamoylonium crosslinking agents as disclosed in U.S. Patent No. 4,421,847 and the ethers of U.S.
  • Useful linking groups are derived from various heterobifunctional cross-linking reagents such as those listed in the Pierce Chemical Company Immunotechnology Catalog - Protein Modification Section, (1991 and 1992) .
  • Useful non-limiting examples of such reagents include: Sulfo-SMCC, i.e., Sulfosuccinimidyl 4- (N- maleimidomethyl) cyclohexane-1-carboxylate ; Sulfo-SIAB, i.e., Sulfosuccinimidyl (4-iodoacetyl) aminobenzoate ; Sulfo-SMPB, i.e., Sulfosuccinimidyl 4- (pmaleimidophenyl) butyrate ; 2-IT, i.e., 2-Iminothiolane; and SATA, i.e., N-Succinimidyl S-acetylthioacetate .
  • linking groups in whole or in part, can also be comprised of and derived from complementary sequences of nucleotides and residues of nucleotides, both naturally occurring and modified, preferably non-self-associating oligonucleotide sequences.
  • Particularly useful, non-limiting examples of reagents for incorporation of modified nucleotide moieties containing reactive functional groups, such as amine and sulfhydryl groups, into an oligonucleotide sequence are commercially available from, for example, Clontech Laboratories Inc.
  • linking groups of this invention are derived from the reaction of a reactive- functional group such as an amine or sulfhydryl group as are available in the above Clontech reagents, one or more of which has been incorporated into an oligonucleotide sequence, with, for example, one or more of the previously described protein reactive groups such as heterobifunctional protein reactive groups, one or more of which has been incorporated into, for example, an immunoreactive material as described above.
  • a reactive- functional group such as an amine or sulfhydryl group as are available in the above Clontech reagents, one or more of which has been incorporated into an oligonucleotide sequence
  • protein reactive groups such as heterobifunctional protein reactive groups
  • Respectively complementary individual oligonucleotide sequences are attached to the two components of the conjugate, one sequence to the immunoreactive material and the complementary oligonucleotide sequence to the chelating agent.
  • the hybrid formed between the two complementary oligonucleotide sequences then comprises the linking group between the immunoreactive material and the chelating agent.
  • two or more copies of the same oligonucleotide sequence can be linked, for example, in tandem to one immunoreactive material, and a complementary oligonucleotide sequence comprised of multiple chelating agents can be added.
  • the multiple hybrids formed between the two complementary oligonucleotide sequences then comprises the linking group between the immunoreac ive group and multiple chelating agents.
  • Preferred linking groups also include nitrogen atoms in groups such as amino, imido, nitrilo and imino groups; alkylene, preferably containing from 1 to 18 carbon atoms such as methylene, ethylene, propylene, butylene and hexylene, such alkylene optionally being interrupted by 1 or more heteroatoms such as oxygen, nitrogen and sulfur or heteroatom-containing groups; carbonyl; sulfonyl; sulfinyl; ether; thioether; ester, i.e., carbonyloxy and oxycarbonyl ; thioester, i.e., carbonylthio, thiocarbonyl, thiocarbonyloxy, and oxythiocarboxy; amide, i.e., iminocarbonyl and carbonylimino; thioamide, i.e., iminothiocarbonyl and thiocarbonylimino; thio; dithio; phosphate;
  • linking groups can be used, such as, for example, alkyleneimino and iminoalkylene . It is contemplated that other linking groups may be suitable for use herein, such as linking groups commonly used in protein heterobifunctional and homobifunctional conjugation and crosslinking chemistry as described above. Especially preferred linking groups include amino groups which, when linked to the residue of a chelating agent via an isothiocyanate group on the chelating agent, form thiourea groups.
  • the linking groups can contain various substituents which do not interfere with the coupling reaction between the chelating agent of this invention and the immunoreactive group.
  • the linking groups can also contain substituents which can otherwise interfere with such reaction, but which during the coupling reaction, are prevented from so doing with suitable protecting groups commonly known in the art and which substituents are regenerated after the coupling reaction by suitable deprotection.
  • the linking groups can also contain substituents that are introduced after the coupling reaction.
  • the linking group can be substituted with substituents such as halogen, such as F, Cl, Br or I; an ester group; an amide group; alkyl, preferably containing from 1 to about 18, more preferably, 1 to 4 carbon atoms such as methyl, ethyl, propyl , i-propyl, butyl, and the like; substituted or unsubstituted aryl, preferably containing from 6 to about 20, more preferably 6 to 10 carbon atoms, such as phenyl, naphthyl , hydroxyphenyl , iodophenyl, hydroxyiodophenyl , fluorophenyl and methoxyphenyl ; substituted or unsubstituted aralkyl , preferably containing from 7 to about 12 carbon atoms, such as benzyl and phenylethyl ; alkoxy, the alkyl portion of which preferably contains from 1 to 18 carbon atoms as described for
  • the products of the reaction of any of these protein reactive group containing chelating agents with immunoreactive materials, preferably with proteins, can be purified by conventional techniques such as diaflltration, HPLC, electrophoresis, and the like.
  • the immunoreac ive materials may be subsequently modified with agents such as PEG (polyethylene glycol) reagents as is well known in the art to impart reduced immunogenicity to the modified proteins .
  • Techniques for performing the covalent binding of the immunoreactive group to the metal complexing agents include simply mixing the materials together, preferably in aqueous solution in the presence of a buffer salt such as sodium borate or sodium phophate, or sodium acetate at a pH of about 4 to about 11, preferably from about 7 to about 10.
  • a buffer salt such as sodium borate or sodium phophate, or sodium acetate at a pH of about 4 to about 11, preferably from about 7 to about 10.
  • the ratio of the complexing agent to the immunoreactive group can vary widely from about 0.5:1 to 10:1 or more. In some embodiments, the mole ratio of complexing agent to immunoreactive groups is from about 1:1 to about 6:1. In some uses of the immunoconjugates of this invention, the bulk ratio of the chelating agent to the immunoreactive group can be an apparent fraction because the immunoconjugate can be used in the presence of unmodified immunoreactive material. Tne immunoreagents of this invention can contain a wide range of ratios of metal ion to complexing agent. In preferred embodiments, the mole ratio of metal ion to complexing agent is from about 1:1000 to about 1:1.
  • the ratio of the complexing agent to the immunoreactive group can vary widely from about 0.5:1 to 10:1 or more. In some embodiments, the mole ratio of complexing agent to immunoreactive groups is from about 1:1 to about 6:1.
  • the targeting immunoreagent of this invention comprising a radioisotope of a metal ion such as 90 Y *3 (as a non-limiting example) can be used for the therapeutic treatment of tumors, particularly if the immunoreagent is a tumor antigen specific antibody or a fragment of such antibody.
  • the targeting immunoreagent of this invention preferably contains a ratio of metal radionuclide ion to chelating agent that is effective in such therapeutic applications.
  • the mole ratio of metal ion per chelating agent is from about 1:100 to about 1:1.
  • the targeting immunoreagent of this invention comprising a radioisotope of a metal ion such as 311 In +3 or i87 ⁇ +3 ( as non _limiting examples) can be used for the diagnostic imaging of tumors in cancer patients, particularly if the immunoreagent is a tumor antigen specific antibody or a fragment of such antibody.
  • the targeting immunoreagent of this invention preferably contains a ratio of metal radionuclide ion to chelating agent that is effective in such diagnostic imaging applications.
  • the mole ratio of metal ion per chelating agent is from about 1:10,000 to about 1:1.
  • a targeting immunoreagent as described above comprising at least two metal ions in combination with one another in the same formulation is specifically contemplated. For example,
  • a therapeutically effective dose of a radionuclide such as 90 Y +3 together with a diagnostic imaging effective dose of a paramagnetic ion such as Gd +3
  • the ratio of the molar concentration of the diagnostic imaging effective ion to the molar concentration of the radionuclide ion being typically greater than one, in a pharmaceutically effective formulation of said targeting immunoreagent, permits the simultaneous magnetic resonance imaging of at least a portion of the tissue of a host patient during therapeutic treatment of said patient.
  • radioisotopes of iodine is specifically contemplated.
  • the targeting immunoreagent comprises a substituent that can be chemically substituted by iodine in a covalent bond forming reaction, such as, for example, a substituent containing hydroxyphenyl functionality, such a substituent can be labeled by methods well known in the art with a radioisotope of iodine.
  • the thus covalently linked radioactive iodine species can be used in therapeutic and diagnostic imaging applications as described herein.
  • an effective dose of a targeting radioactive immunoreagent as described above in a pharmaceutically acceptable medium is prepared by exposing a composition comprising a residue of an immunoreactive group as described above and a residue of a chelating agent having Structure I as described above linked to the immunoreactive group by a linking group as described above to a composition containing a radioactive metal ion as described above such that the molar amount of said radioactive metal ion is less than the molar amount of the chelating group comprising the targeting immunoreagent in said composition, the duration of the exposure lasting an effective time to permit uptake of said radioactive metal ion into said targeting immunoreagent .
  • an effective dose of a targeting immunoreagent as described above in a pharmaceutically acceptable medium is administered to a patient and said targeting immunoreagent is allowed to accumulate at the target site such as at a tumor site in said patient.
  • a therapeutically effective dose of a targeting radioactive immunoreagent as described above in a pharmaceutically acceptable medium is administered to a patient or to a tissue from a patient and said targeting radioactive immunoreagent is allowed to accumulate at the target site such as at a tumor site in said patient.
  • the present invention also comprises one or more targeting immunoreagents as described above formulated into compositions together with one or more non- toxic physiologically acceptable carriers, adjuvants or vehicles which are collectively referred to herein as carriers, for parenteral injection, for oral administration in solid or liquid form, for rectal or topical administration, or the Like.
  • compositions can be administered to humans and animals either orally, rectally, parenterally (intravenously, by intramuscularly or subcutaneously) , intracisternally, intravaginally, intraperitoneally, intravesically, locally (powders, ointments or drops), or as a buccal or nasal spray
  • a targeting paramagnetic immunoreagent as described above and targeting radioactive immunoreagent as described above can be administered by the same route.
  • a paramagnetic immunoreagent as described above can be administered by a route different from that of a targeting radioactive immunoreagent as described above.
  • compositions suitable for oral, rectal, parenteral, intercisternal , intravaginal , intraperitoneal , intravesical, topical, buccal or nasal is well known in the art and is comprehensively described in US Patent No. 5760191.
  • compositions of the present invention may be varied so as to obtain an amount of active ingredient that is effective for imaging or to obtain a desired therapeutic response for a particular composition and method of administration.
  • the selected dosage level therefore depends upon the desired imaging or therapeutic effect, on the route of administration, on the desired duration of treatment and other factors .
  • the total daily dose of the compounds of this invention administered to a host in single or divided dose may be in amounts, for example, of from about 1 nanomol to about 5 micromols per kilogram of body weight.
  • Dosage unit compositions may contain such amounts or such submultiples thereof as may be used to make up the daily dose. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the body weight, general health, sex, diet, time and route of administration, rates of absorption and excretion, combination with other drugs and the severity of the particular disease being treated.
  • the subject mammal After administration of a composition of the present invention, the subject mammal is maintained for a time period sufficient for the administered composition to be distributed throughout the subject and enter the tissues of the mammal.
  • a sufficient time period is generally from about 1 hour to about 2 weeks or more and, preferably from about 2 hours to about 1 week.
  • Boc-Gly-N- hydroxybenzotriazole (HOBT) ester was made by mixing HOBT and diisopropyl-carbodiimide (1:1) with Boc-Gly, and added to CHC1 3 immediately prior to mixing with the oxime resin.
  • the mixture was stirred overnight, washed three times each with dimethylformamide (DMF) and
  • Example 3,3 9-Bis (t-butoxycarbonvmethyl) -18- (4-methoxyphenyl) -6- f3- (4-nitrophenyl) -oxycarbonyl- oxypropyll -3,6,9,25,26, 27-hexaazatetracvclo- f19.3.1.
  • I 11 is _ -L 16 . 2Q -) -heptacosa-11 , 13 , 15 27 , 16 26 , 17 , 19 , 21 25 , 22 , 24-nonaene .
  • the vial is then silanized by treatment with trimethylchlorosilane and then dried at 110 'C.
  • Example 5 General method for radiolabeling of a chelating agent of this invention as a conjugate of STa with radionuclide ion illustrated using the STa-chelateenfin and 90 Y.
  • a volume of radioactive yttrium chloride ( 90 YC1 3 in 0.04 M hydrochloric acid at a specific activity of >500 Ci/mg;
  • the labelling efficiency is determined by removing 0.5 mL of the sample and spotting it on to a Gelman ITLC-SG strip.
  • the strip is developed in a glass beaker containing 0.1 M sodium citrate, pH 6.0, for a few minutes until the solvent front has reached three quarters of the way to the top of the paper.
  • the strip is inserted into a System 200 Imaging scanner (Bioscan) which has been optimized for 90 Y . In this system, free 90 Y migrates at the solvent front while the Sta-chelate 5.8p " - Y does not
  • Example S The method of Example S is repeated using m InCl 3 in 0.04 M hydrochloric acid (Amersham-Mediphysics) in place
  • Example 55 of US Patent No. 5760191 is repeated using STa- chelate 58 conjugate. Progress , of the reaction is followed spectroscopically .
  • Example 8 _ (a) . Preparation of Escherichia Coli Heat-Stable Enterotoxin (STa) conjugate with ⁇ -amino -a-carboxylpoly (ethylene glycol)-, ⁇ nn (H,N-PEG, ⁇ nn -CO-STa) .
  • the reaction mixture is stirred at 40 ' C.
  • the extent of reaction was followed by SE-HPLC.
  • the conjugate (Sta-PEG- chelate 63 ) can be isolated by preparative SE-HPLC.
  • Example 9 . 3 9-Bis (t-butoxy- carbonylmethyl ) -18- (4-methoxy- henyl) -6- (3-aminopropyl) -3, 6, 9, 25, 26,27- hexaazatetracvclo f19.3.1. l 11 - 15 . ⁇ ----°] - heptacosa-11, 13, 15 27 , 16 26 , 17, 19 , 21 2S , 22.24-nonaene
  • Example 2 In a 10 ml Reacti-Vial silanized as in Example 2 , a solution of 1 part of Escherichia Coli heat-stable enterotoxin (STa) and 1 part of the compound prepared in
  • Example 9 in 5 parts of anhydrous DMF is treated with 1 part of 1, 1' -carbonyldiimidazole.
  • the symmetrical products of the reaction are separated by preparative HPLC from the desired unsymmetrical conjugate which is treated with trifluoroacetic acid to remove the t-butyl groups.
  • the desired product, STa-chelate 65 can be isolated by preparative HPLC.
  • a solution containing one part of the compound prepared in Example 9 and one part of 9-fluorenyl- methoxycarbonyl -L-glutamic acid- ⁇ -t-butyl ester (Peptides International, Inc.; product Fmoc-Glu (OtBu) ) in 10 parts of 50:50 DMF acetonitrile is treated with one part of benzotriazol-1-yl-oly-tris (dimethylamino) phosphonium hexafluorophosphate (BOP) and two parts of triethylamine to produce the Fmoc blocked glutamic acid amide. Progress of the reaction can be followed by HPLC. The Fmoc group on the product can be removed by the addition of a solution of piperidine in DMF, and the desired material can be isolated by preparative HPLC.
  • Example 11 in 10 parts of DMF is treated with one part of
  • reaction mixture is added to a solution of nitrilotriacetic anhydride (1 part; CAS Registry number
  • N- (3-methylimidazolium) carbonylmethyliminodiacetic anhydride trifluoromethane sulfonate which can be used directly.
  • the crude reaction product is isolated and purified by chromatography on silica gel using step gradients comprising increasing amounts of methanol in methylene chloride and subsequent elution with ammonium hydroxide in methanolic methylene chloride. Fractions containing the desired compound are combined and the solvent is evaporated. The residue is taken up in anhydrous DMF and treated with acetic anhydride. The solvents are removed under high vacuum, and the residue is triturated with cold anhydrous ether and filtered.
  • Example 14 in 50 parts of anhydrous DMF is heated at 50 "C.
  • Example 5 The method of Example 5 is repeated using STa- chelate 58 prepared in Example 4 and 67 CuCl 2 in 0. 04 M hydrochloric acid (University of Missouri) in place of 90 YC1 3 .
  • Example 17 Labeling of the conjugate of STa- chelate -- with gadolinium ion.
  • Example 55 of US Patent No. 5760191 is repeated using the STa-chelate 70 prepared in Example 15 and GdCl 3 in place of EuCl 3 . Progress of the rection is followed spectroscopically .
  • Example 55 of US Patent No. 5760191 is repeated using the STa-chelate 70 prepared in Example 15 and DyCl 3 , in place of EuCl 3 . Progress of the rection is followed spectroscopically .

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Abstract

La présente invention concerne un immunoréactif de ciblage comprenant un ion métallique, un reste d'un agent de complexion et un groupe immunoréactif lié audit agent de complexion, représenté par la formule (I), dans laquelle chaque R et R1 est individuellement sélectionné à partir d'hydrogène, alkyle, alcoxy, hydroxyalkyle, alcoxyalkyle, hydroxyalkyloxy, alcoxyalkyloxy, alkylthio, alkylthioalkyle, alkylthioalkyloxy, hydroxyalkylthio, hydroxyalkylthioalkyle, hydroxyalkylthioalkyloxy, N, N--dialkylamino, N-(hydroxyalkyle)-N-alkylamino, N, N--bis(hydroxyalkyl)amino, N, N-dialcoylaminoalkyle, N--(hydroxyalkyl)-N-alkylaminoalkyle, N, N-bis(hydroxyalkyloaminoalkyle, alkylformamido, formamidoalkyle, aryle, alkylaryle, alcoxyaryle, hydroxyaryle, alcoxyalkylaryle, hydroxyalkyloxyaryle, alcoxyalkyloxyaryle, alkylthioaryle, hydroxyalkylthioarayle, hydroxyalkylethioalkylaryle, hydroxyalkylthioalkyloxyaryle, aralkyle, aralkyloxy, alcoxyaralkyle, alcoxyaralkyloxy, aryloxy, alkylarylaxy, alcoxyaryloxy, et hétérocyclyle; chaque Q est individuellement sélectionné à partir d'hydrogène, alkyle, hydroxyle, carboxyle, carboxyalkyle, hydroxyalkyle, alkylthioalkyle, sulfhydryle, thioalkyle, alcoxy, alkylthio, alkylamino, aminoalkyle, aminoalkylaminoalkyle, hydroxy-alkylaminoalkyle, hydroxylaminoalkyle, hydroxamido, formamidoalkyle, alkylformamido, aryle, comprenant aryle substitué, aryloxy, hétérocylcyle, acide carbonyliminodiacétique, acide méthyle eiminodiacétique, acide méthylènethioéthylène-iminodiacétique, carboxyalkylthioalkyle, un reste d'acide éthylènediaminetétraacétique (EDTA), un reste d'acide diéthylènetriaminepentaacétique (DTPA), un acide hydrazinylidenediacétique et un sel de l'un des acides précités; chaque Z est individuellement sélectionné à partir d'un hétéroatome de valence deux, un hétéroatome de valence trois, un groupe alkylène, un groupe alkylène lié à un hétéroatome de valence deux, ainsi qu'un groupe alkylène lié à un hétéroatome de valence trois; chaque X est individuellement sélectionné à partir de l'azote et d'un reste d'un groupe alkylène; chaque W est individuellement sélectionné à partir d'hydrogène et d'un substituant comprenant un groupe réactif à une protéine; chaque L' est individuellement sélectionné à partir d'une liaison chimique et d'un groupe de liaison intra-cyclique; chaque L est individuellement sélectionné à partir d'un reste d'un groupe alkylène et d'un groupe de liaison extra-cyclique; n représente 1, 2, 3 ou 4; et chaque p représente individuellement 0, 1, 2, 3 ou 4; à condition qu'un seul W soit un groupe réactif à une protéine, le L lié à W qui est un groupe réactif à une protéine contienne 1, 2 ou 3 atomes de carbone et relie X à un hétéroatome capable de participer à la chélation d'un ion métallique; et lorsque X représente un azote et un hétéroatome de Z est lié à X, l'hétéroatome de Z représente également l'azote; et enfin le groupe immunoréactif est une fraction de liaison du récepteur ST.
PCT/GB1999/000396 1998-02-06 1999-02-08 Immunoreactifs de ciblage utilises dans des compositions et des procedes therapeutiques et diagnostiques WO1999039748A1 (fr)

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001025266A1 (fr) * 1999-10-06 2001-04-12 Pharmacia Corporation Uroguanyline, anticancereux intestinal
FR2805994A1 (fr) * 2000-03-10 2001-09-14 Agronomique Inst Nat Rech Methode de detection de la guanylyl cyclase-c et son utilisation pour le diagnostic des tumeurs colorectales metastatiques
WO2002070018A2 (fr) 2001-03-02 2002-09-12 Amersham Plc Conjugues peptide-chelate ameliores
WO2002087498A2 (fr) * 2001-04-26 2002-11-07 Board Of Regents, The University Of Texas System Compositions d'imagerie diagnostique, leurs methodes de synthese et utilisation
US7067111B1 (en) 1999-10-25 2006-06-27 Board Of Regents, University Of Texas System Ethylenedicysteine (EC)-drug conjugates, compositions and methods for tissue specific disease imaging
US7261875B2 (en) 2001-12-21 2007-08-28 Board Of Regents, The University Of Texas System Dendritic poly (amino acid) carriers and methods of use
US7615208B2 (en) 1999-10-25 2009-11-10 Board Of Regents, The University Of Texas System Metal ion-labeled bis-aminoethanethiol-targeting ligand conjugates, compositions, and methods for tissue-specific disease imaging
WO2011071927A3 (fr) * 2009-12-07 2011-10-13 Ironwood Pharmaceuticals, Inc. Traitement des troubles gastro-intestinaux
US9050378B2 (en) 2003-12-10 2015-06-09 Board Of Regents, The University Of Texas System N2S2 chelate-targeting ligand conjugates
US9527887B2 (en) 2011-06-08 2016-12-27 Ironwood Pharmaceutical, Inc. Treatments for gastrointestinal disorders
US9617305B2 (en) 2011-06-08 2017-04-11 Ironwood Pharmaceuticals, Inc. Treatments for gastrointestinal disorders
CN109535026A (zh) * 2017-09-22 2019-03-29 国立研究开发法人日本原子力研究开发机构 合成四烷基次氮基乙酸二乙酰胺化合物的方法
US10814013B2 (en) 2006-10-05 2020-10-27 The Board Of Regents Of The University Of Texas System Efficient synthesis of chelators for nuclear imaging and radiotherapy: compositions and applications

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992008494A2 (fr) * 1990-11-08 1992-05-29 Sterling Winthrop Inc. Immunoreactifs de ciblage radioactifs
WO1995011694A1 (fr) * 1993-10-26 1995-05-04 Thomas Jefferson University Compositions se fixant specifiquement a des cellules cancereuses colo-rectales et procedes d'utilisation
WO1999021587A1 (fr) * 1997-10-15 1999-05-06 Nycomed Imaging As Complexants et immunoreactifs de ciblage

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992008494A2 (fr) * 1990-11-08 1992-05-29 Sterling Winthrop Inc. Immunoreactifs de ciblage radioactifs
WO1995011694A1 (fr) * 1993-10-26 1995-05-04 Thomas Jefferson University Compositions se fixant specifiquement a des cellules cancereuses colo-rectales et procedes d'utilisation
WO1999021587A1 (fr) * 1997-10-15 1999-05-06 Nycomed Imaging As Complexants et immunoreactifs de ciblage

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001025266A1 (fr) * 1999-10-06 2001-04-12 Pharmacia Corporation Uroguanyline, anticancereux intestinal
US7582281B2 (en) 1999-10-25 2009-09-01 Board Of Regents, The University Of Texas System Ethylenedicysteine (EC)-drug conjugates compositions and methods for tissue specific disease imaging
US7632484B2 (en) 1999-10-25 2009-12-15 Board Of Regents, The University Of Texas System Ethylenedicysteine (EC)-drug conjugates, compositions and methods for tissue specific disease imaging
US7067111B1 (en) 1999-10-25 2006-06-27 Board Of Regents, University Of Texas System Ethylenedicysteine (EC)-drug conjugates, compositions and methods for tissue specific disease imaging
US7223380B2 (en) 1999-10-25 2007-05-29 Board Of Regents, The University Of Texas System Ethylenedicysteine (EC)-drug conjugates, compositions and methods for tissue specific disease imaging
US7229604B2 (en) 1999-10-25 2007-06-12 Board Of Regents, The University Of Texas System Ethylenedicysteine (EC)-drug conjugates, compositions and methods for tissue specific disease imaging
US7615208B2 (en) 1999-10-25 2009-11-10 Board Of Regents, The University Of Texas System Metal ion-labeled bis-aminoethanethiol-targeting ligand conjugates, compositions, and methods for tissue-specific disease imaging
FR2805994A1 (fr) * 2000-03-10 2001-09-14 Agronomique Inst Nat Rech Methode de detection de la guanylyl cyclase-c et son utilisation pour le diagnostic des tumeurs colorectales metastatiques
WO2001069259A1 (fr) * 2000-03-10 2001-09-20 Institut National De La Recherche Agronomique (I.N.R.A.) Methode de detection de la guanylyl cyclase-c et son utilisation pour le diagnostic des tumeurs colorectales metastatiques
WO2002070018A2 (fr) 2001-03-02 2002-09-12 Amersham Plc Conjugues peptide-chelate ameliores
WO2002087498A3 (fr) * 2001-04-26 2003-10-30 Univ Texas Compositions d'imagerie diagnostique, leurs methodes de synthese et utilisation
WO2002087498A2 (fr) * 2001-04-26 2002-11-07 Board Of Regents, The University Of Texas System Compositions d'imagerie diagnostique, leurs methodes de synthese et utilisation
US7261875B2 (en) 2001-12-21 2007-08-28 Board Of Regents, The University Of Texas System Dendritic poly (amino acid) carriers and methods of use
US9050378B2 (en) 2003-12-10 2015-06-09 Board Of Regents, The University Of Texas System N2S2 chelate-targeting ligand conjugates
US10814013B2 (en) 2006-10-05 2020-10-27 The Board Of Regents Of The University Of Texas System Efficient synthesis of chelators for nuclear imaging and radiotherapy: compositions and applications
US10925977B2 (en) 2006-10-05 2021-02-23 Ceil>Point, LLC Efficient synthesis of chelators for nuclear imaging and radiotherapy: compositions and applications
WO2011071927A3 (fr) * 2009-12-07 2011-10-13 Ironwood Pharmaceuticals, Inc. Traitement des troubles gastro-intestinaux
US8735360B2 (en) 2009-12-07 2014-05-27 Ironwood Pharmaceuticals, Inc. Treatments for gastrointestinal disorders
US9527887B2 (en) 2011-06-08 2016-12-27 Ironwood Pharmaceutical, Inc. Treatments for gastrointestinal disorders
US9617305B2 (en) 2011-06-08 2017-04-11 Ironwood Pharmaceuticals, Inc. Treatments for gastrointestinal disorders
CN109535026A (zh) * 2017-09-22 2019-03-29 国立研究开发法人日本原子力研究开发机构 合成四烷基次氮基乙酸二乙酰胺化合物的方法
CN109535026B (zh) * 2017-09-22 2021-10-29 国立研究开发法人日本原子力研究开发机构 合成四烷基次氮基乙酸二乙酰胺化合物的方法

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