WO1994029333A1 - Reactifs immunoreactifs utilisant la monoamine-oxydase - Google Patents

Reactifs immunoreactifs utilisant la monoamine-oxydase Download PDF

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Publication number
WO1994029333A1
WO1994029333A1 PCT/US1994/006424 US9406424W WO9429333A1 WO 1994029333 A1 WO1994029333 A1 WO 1994029333A1 US 9406424 W US9406424 W US 9406424W WO 9429333 A1 WO9429333 A1 WO 9429333A1
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Prior art keywords
group
reagent
residue
mao
monoamine oxidase
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PCT/US1994/006424
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English (en)
Inventor
Christopher D. V. Black
Robert Allen Snow
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The Wellcome Foundation Limited
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Priority to EP94921934A priority Critical patent/EP0710244A1/fr
Priority to AU72448/94A priority patent/AU7244894A/en
Priority to JP7502055A priority patent/JPH09501147A/ja
Publication of WO1994029333A1 publication Critical patent/WO1994029333A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
    • 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
    • A61K47/6851—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 the antibody targeting a determinant of a tumour cell
    • 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
    • A61K47/6883—Polymer-drug antibody conjugates, e.g. mitomycin-dextran-Ab; DNA-polylysine-antibody complex or conjugate used for therapy
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/021—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)n-C(=0)-, n being 5 or 6; for n > 6, classification in C07K5/06 - C07K5/10, according to the moiety having normal peptide bonds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to the therapeutic treatment and diagnostic imaging of cancer by means of a tumor targeted sequential delivery system comprising a primary non radioactive targeting immunoreagent and a secondary radioactive delivery agent.
  • radioimmunotherapy and diagnostic imaging with the various currently available radiopharmaceuticals which include radionuclide-containing immunoreactive proteins can be less than optimal because these radionuclide-containing immunoreactive proteins
  • radiopharmaceuticals may bind to non-target normal tissue. This binding can result in undesirable toxicity to normal tissue during therapeutic applications as well as in high background signals during diagnostic imaging applications; the radioactive component may then deposit in healthy tissue. Also, long plasma half-lives of currently available radionuclide-containing
  • radionuclide from the body can result in prolonged exposure of normal tissue to damaging effects of
  • the number of chelating agents that can be attached to an immunoreactive protein is also limited by the number of available groups such as, for example, amino groups suitable for use in attachment of the chelating agents and by the potential immunogenicity of the thus modified protein which, being highly derivatized, could be recognized by a host immune system as being haptenated.
  • J. S. Fowler et al. in United States Patent Application A6052921 (1989) reveal a strategy for imaging and mapping the regional distribution of enzymes in a living body by using positron emitter-labeled suicide enzyme inhibitors which bind irreversibly to the enzyme through catalysis, thereby labeling the enzyme.
  • Carbon-11 labeled clorgyline and L-deprenyl are reported as selective probes for monoamine oxidase localization and reactivity In vivo using positron emission
  • the present invention is directed to a non radioactive targeting immunoreagent (sometimes hereinafter referred to as NRTIR) comprising the residue of a monoamine oxidase, a linking group, and the residue of an immunoreactive material, which immunoreactive material can bind to sites on cells of a tissue of interest.
  • NRTIR non radioactive targeting immunoreagent
  • the present invention is also directed to a radioactive delivery agent (sometimes hereinafter referred to as RDA) comprising a ligand specific for said monoamine oxidase, a linking group, and a radioactive delivery agent (sometimes hereinafter referred to as RDA) comprising a ligand specific for said monoamine oxidase, a linking group, and a radioactive delivery agent (sometimes hereinafter referred to as RDA) comprising a ligand specific for said monoamine oxidase, a linking group, and a
  • radioactive agent that is administered to the environs of said tissue.
  • the ligand of said RDA will bind to the receptor of said NRTIR which is bound to the cells of the tissue of interest and thus provides an effective amount of radioactivity to said tissue. Unbound RDA can be removed rapidly from the environs of said tissue.
  • the present invention comprises an NRTIR comprising the residue of a receptor moiety which receptor moiety comprises the residue of a proteinaceous active site of a monoamine oxidase enzyme (sometimes hereinafter referred to as MAO), a linking group, and the residue of an NRTIR comprising the residue of a receptor moiety which receptor moiety comprises the residue of a proteinaceous active site of a monoamine oxidase enzyme (sometimes hereinafter referred to as MAO), a linking group, and the residue of an NRTIR comprising the residue of a receptor moiety which receptor moiety comprises the residue of a proteinaceous active site of a monoamine oxidase enzyme (sometimes hereinafter referred to as MAO), a linking group, and the residue of an NRTIR comprising the residue of a receptor moiety which receptor moiety comprises the residue of a proteinaceous active site of a monoamine oxidase enzyme (sometimes hereinafter referred to as MAO), a linking group, and the residue of an NRTIR comprising
  • the present invention comprises a NRTIR comprising the residue of a ligand specific for an MAO receptor moiety, a linking group, and the residue of an immunoreactive material and an RDA comprising the residue of an MAO receptor moiety, a linking group, and a radioactive agent.
  • the present invention is directed to an NRTIR comprising the proteinaceous active site of a monoamine oxidase enzyme, a linking group, and the residue of an immunoreactive material such as a tumor targeting antibody together with an RDA comprising a ligand specific for said MAO receptor moiety, a linking group, and a radioactive agent
  • the present invention is directed to a NRTIR comprising the residue of a ligand specific for an MAO receptor moiety, a linking group, and the residue of an immunoreactive material such as a tumor targeting antibody together with an RDA comprising the residue of an MAO receptor moiety, a linking group, and a radioactive agent comprising a chelating agent and a radionuclide.
  • the present invention is also directed to pharmaceutical and diagnostic compositions that contain the NRTIR and a pharmaceutically acceptable carrier, and to pharmaceutical and diagnostic compositions that contain the RDA and a pharmaceutically acceptable carrier.
  • the present invention is further directed to diagnostic imaging and therapeutic methods comprising sequentially administering an effective amount of NRTIR to a patient in need of such diagnosis or treatment, allowing the NRTIR to bind to sites on cells of a tissue of interest, cleaning unbound NRTIR from the environs of said tissue, and administering RDA to said patient.
  • the RDA contains multiple
  • chelating agents which in total are capable of binding more ions of a radiometal per molecule of immunoreactive protein than can be bound per molecule by direct
  • NRTIR with the ligand of the RDA should be as long-lasting as possible (i.e., having a high affinity for each other, preferably undergoing effectively
  • any RDA which fails to bind to the receptor of the tissue bound NRTIR is rapidly removed, for example, from the plasma preferably by excretion from the body.
  • the present invention comprises an antigen on the surface of a cell, preferably a tumor cell, to which is bound a single molecule of an
  • NRTIR non radioactive targeting immunoreagent
  • RDA radioactive delivery agent
  • Z is the residue of an immunoreactive group
  • D is the residue of a ligand that will bind to the MAO receptor
  • MAO ligand is the residue of a ligand that will bind to an MAO active site
  • CLO is the residue of a clorgyline analog
  • PARG is the residue of a pargylinyl analog
  • L 1 and L 2 are independently the residues of a linking group that may independently contain a spacing group
  • Q is the residue of a chelating group
  • M is a radionuclide
  • n and m are independently integers greater than zero.
  • the immunoreactive group, Z can be selected from a wide variety of naturally occurring or synthetically prepared materials.
  • Z preferably is an antibody or antibody fragment which recognizes and is specific for a tumor associated antigen.
  • Z can contain an immunoreactive group covalently bonded thereto through a chemical bond or a linking group derived from the residue of a protein reactive group and the residue of a reactive group on the protein.
  • immunosorbent protein which can be abbreviated by “IRP” also includes an organic compound which is capable of covalently bonding to the protein and which is found in a living organism or is useful in the diagnosis, treatment or genetic engineering of cellular material or living organisms, and which has a capacity for interaction with another component which may be found in biological fluids or associated with cells to be treated such as tumor cells.
  • the immunoreactive group can be selected from a wide variety of naturally occurring or synthetically prepared materials, including, but not limited to enzymes, amino acids, peptides, polypeptides, proteins, lipoproteins, glycoproteins, lipids, phospholipids, hormones, growth factors, steroids, vitamins,
  • polysaccharides polysaccharides, viruses, protozoa, fungi, parasites, rickettsia, molds, and components thereof, blood components, tissue and organ components,
  • haptens lectins
  • toxins nucleic acids (including oligonucleotides), antibodies (monoclonal and polyclonal), anti-antibodies, antibody fragments, antigenic materials (including proteins and
  • an immunoreactive group can be any substance which when presented to an immunocompetent host will result in the production of a specific antibody capable of binding with that substance, or the antibody so produced, which participates in an antigen-antibody reaction.
  • Preferred immunoreactive groups are antibodies and various immunoreactive fragments thereof, as long as they contain at least one reactive site for reaction with a protein reactive group as described herein. That site can be inherent to the immunoreactive species or it can be introduced through appropriate chemical
  • the immunoreactive group does not bind in an immunoreactive sense to the residue of a monoamine oxidase active site or to the residue of a ligand that has an affinity for a monoamine oxidase active site so as to inhibit binding between the two species in Systems A and B.
  • antibody fragment refers to an immunoreactive material which comprises a residue of an antibody, which antibody characteristically exhibits an affinity for binding to an antigen.
  • affinity for binding to an antigen refers to the thermodynamic expression of the strength of interaction or binding between an antibody combining site and an antigenic determinant and, thus, of the stereochemical compatibility between them. As such, it is the expression of the equilibrium or association constant for the antibody-antigen interaction.
  • affinity as used herein also refers to the
  • thermodynamic expression of the strength of interaction or binding between a ligand and a receptor and, thus, of the stereochemical compatibility between them. As such, it is the expression of the equilibrium or association constant for the ligand-receptor interaction.
  • antibody fragments exhibit a percentage of said affinity for binding to said antigen, that percentage being in the range of 0.001 percent to 1,000 percent, preferably 0.01 percent to 1,000 percent, more preferably 0.1 percent to 1,000 percent, and most preferably 1.0 percent to 100 percent, of the relative affinity of said antibody for binding to said antigen.
  • An antibody fragment can be produced from an antibody by a chemical reaction comprising one or more chemical bond cleaving reactions; by a chemical reaction comprising of one or more chemical bond forming
  • reactions employing as reactants one or more chemical components selected from a group comprising amino acids, peptides, carbohydrates, linking groups as defined herein, spacing groups as defined herein, protein reactive groups as defined herein, and antibody
  • fragments such as are produced as described herein and by a molecular biological process, a bacterial process, or by a process comprising or resulting from the genetic engineering of antibody genes.
  • An antibody fragment can be derived from an antibody by a chemical reaction comprising one or more of the following reactions: (a) cleavage of one or more chemical bonds of which an antibody is comprised, said bonds being selected from, for example, carbon-nitrogen bonds, sulfur-sulfur bonds, carbon-carbon bonds, carbon-sulfur bonds, and carbon-oxygen bonds, and wherein the method of said cleavage is selected from:
  • a catalysed chemical reaction comprising the action of a biochemical catalyst such as an enzyme such as papain or pepsin which enzymes to those skilled in the art are known to produce antibody fragments commonly referred to as Fab and Fab'2, respectively;
  • a biochemical catalyst such as an enzyme such as papain or pepsin which enzymes to those skilled in the art are known to produce antibody fragments commonly referred to as Fab and Fab'2, respectively;
  • a catalysed chemical reaction comprising the action of an electrophilic chemical catalyst such as a hydronium ion which, for example, favorably occurs at a pH equal to or less than 7;
  • a catalysed chemical reaction comprising the action of a nucleophilic catalyst such as a hydroxide ion which, for example, favorably occurs at a pH equal to or greater than 7;
  • a nucleophilic catalyst such as a hydroxide ion which, for example, favorably occurs at a pH equal to or greater than 7;
  • a chemical reaction comprising an oxidation reaction such as the oxidation of a carbon-oxygen bond of a hydroxyl group or the oxidation of a carbon-carbon bond of a vicinal diol group such as occurs in a carbohydrate moiety; or
  • an antibody fragment can be derived by
  • non-covalent bonds comprise hydrophobic interactions such as occur in an aqueous medium between chemical species that independently comprise mutually accessible regions of low polarity such as regions comprising aliphatic and carbocyclic groups, and of hydrogen bond interactions such as occur in the binding of an oligonucleotide with a
  • an antibody fragment can be produced as a result of the methods of molecular biology or by genetic engineering of antibody genes, for example, in the genetic engineering of a single chain immunoreactive group or a Fv fragment.
  • An antibody fragment can be produced as a result of a combination of one or more of the above methods.
  • the immunoreactive group can be an enzyme which has a reactive group for
  • Representative enzymes include, but are not limited to, aspartate aminotransaminase, alanine
  • the immunoreactive group can be modified or chemically altered to provide a reactive group for use in the attachment to the residue of a monoamine oxidase active site in System A or to the residue of a ligand that has an affinity for binding to such a site in System B through a linking group as described below 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. Patent No. 4,719,182.
  • compositions of this invention are for the diagnostic imaging of tumors and the radiological treatment of tumors.
  • Preferred immunological groups therefore include antibodies to tumor-associated antigens.
  • An antibody is sometimes hereinafter referred to as Ab.
  • Specific non-limiting examples of antibodies include B72.3 and related antibodies (described in U.S. Patent Nos. 4,522,918 and 4,612,282) which recognize colorectal tumors; 9.2.27 and related anti-melanoma antibodies; D612 and related antibodies which recognize colorectal tumors; UJ13A and related antibodies which recognize small cell lung carcinomas; NRLU-10, NRCO-02 and related antibodies which recognize small cell lung carcinomas and
  • colorectal tumors Pan-carcinoma
  • 7E11C5 and related antibodies which recognize prostate tumors
  • CC49 and related antibodies which recognize colorectal tumors
  • TNT and related antibodies which recognize necrotic tissue
  • PR1A3 and related antibodies which recognize colon carcinoma
  • ING-1 and related antibodies which are described in International Patent Publication WO-A-90/02569
  • B174, C174 and related antibodies which recognize squamous cell carcinomas
  • B43 and related antibodies which are reactive with certain lymphomas and leukemias
  • An especially preferred antibody is ING-1.
  • Preferred receptors comprise the residue of a monoamine oxidase (MAO; monoamine:oxygen oxidoreductase, EC 1.4.3.4) active site.
  • the MAO active site can comprise any MAO enzyme, in whole or in part, isolated from any source or modified by well known techniques of molecular. biology as long as it maintains MAO activity.
  • MAO exists as two isozymes, MAO-A and MAO-B. The tissue distribution of these isozymes is different, with MAO-A being found in its purest form in the human placenta, and MAO-B found in essentially pure form in human blood platelets and predominantly in the brain.
  • the MAO isozyme chosen for use in the compositions of the present invention depends on the degrees of
  • the MAO is a recombinant human enzyme.
  • the active site of the MAO is genetically engineered into a recombinant human matrix form while the specificity of the enzyme active site for MAO substrate analogs is maintained.
  • the MAO is covalently coupled, i.e., conjugated, to an immunoreactive group, preferably an antibody or an antibody fragment, most preferably to ING-1, to form the NRTIR (i.e., Z-L 1 -Rec) of the system.
  • an immunoreactive group preferably an antibody or an antibody fragment, most preferably to ING-1
  • the MAO as a component of a radioactive delivery agent i.e., an RDA, Rec- (L 2 -Q-M) m
  • a radioactive delivery agent i.e., an RDA, Rec- (L 2 -Q-M) m
  • the chelating group is associated with a radionuclide.
  • the chelating group is TMT
  • the linking group is as described below
  • the radionuclide is 90 y.
  • the RDA comprises an MAO that contains one or more radionuclides that are covalently attached, either directly to one or more components of the MAO or to one or more components that are attached by a linking group as described below to the MAO.
  • said covalently attached radionuclide is a radioisotope of iodine attached to an aromatic ring containing moiety.
  • activated groups such as activated ethylene groups (e.g., activated ethylene groups
  • SMCC succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, which are commonly commercially available, for example, from Pierce Chemical Company are included as non-limiting examples.
  • chemical conjugation is otherwise achieved by using a linking group (L 1 and L 2 , respectively) which is introduced through mild reduction of a disulfide bond in the MAO (or in the MAO modified by reagents which contain disulfide bonds, one non-limiting example of which is succinimidyl 3-(2-pyridyldithio)propionate, SPDP, available from Pierce Chemical Company) with a reducing reagent such as dithiothreitol to produce sulfhydryl (SH) sites in the reduced MAO protein moiety.
  • a linking group L 1 and L 2 , respectively
  • Suitable reactive sites on the immunoreactive material and on the receptor moiety include:
  • activated carbon-hydrogen and carbon-carbon bonds which can react through insertion via free radical reaction or nitrene or carbene reaction of a so activated residue; sites of oxidation;
  • aromatic sites such as tyrosine
  • the ratio of MAO to immunoreactive group such as an antibody can vary widely from about 0.5 to 10 or more.
  • mixtures comprised of
  • immunoreactive groups which are modified with MAO are also suitable.
  • Such mixtures can have a bulk ratio of MAO to immunoreactive group of from about 0.1 to about 10.
  • the mole ratio of MAO to immunoreactive group is from about 1:1 to about 6:1. It is specifically contemplated that with knowledge of the DNA sequence that encodes MAO,
  • a fusion protein can be made between the antibody and the MAO, or portions thereof, through the use of genetic engineering techniques. It is specifically contemplated that in all of these compositions of MAO bound to antibody, MAO retains a capacity to bind to the ligands described in the invention.
  • the ratio of ligand to immunoreactive group such as an antibody can vary widely from about 0.5 to 10 or more.
  • mixtures comprised of
  • immunoreactive groups which are modified with ligand are also suitable.
  • Such mixtures can have a bulk ratio of ligand to immunoreactive group of from about 0.1 to about 10.
  • the mole ratio of ligand to immunoreactive group is from about 1:1 to about 6:1.
  • the conjugate is purified by passage of the material through a gel permeation column such as Superose 6 using an appropriate elution buffer or by elution from a HPLC column such as a Shodex WS-803F size exclusion column. Both these methods separate the applied materials by molecular size resulting in the elution of the antibody/MAO conjugate in a different fraction from any residual non-conjugated MAO.
  • the concentrations of the antibody in the conjugate solutions are determined by the BioRad protein assay using bovine immunoglobulin as the protein standard.
  • the conjugate is purified by passage of the material through a gel permeation column such as Superose 6 using an appropriate elution buffer or by elution from a HPLC column such as a Shodex WS-803F size exclusion column. Both these methods separate the applied materials by molecular size resulting in the elution of the
  • the concentrations of the antibody in the conjugate solutions are determined by the BioRad protein assay using bovine immunoglobulin as the protein standard.
  • the ability of the antibody to bind to its target antigen following conjugation to MAO can be assayed by ELISA or flow cytometry.
  • a 30 cm ⁇ 7.5 mm TSK-G3000SW size-exclusion HPLC column (Supelco) fitted with a guard column of the same material can be used to determine the amount of aggregation in the final conjugate.
  • the monoamine oxidase enzymic activity of the antibody-associated MAO can be assayed by
  • the monoamine oxidase enzymic activity of the chelating agent-associated MAO can be assayed by following the rate of oxidation of kynurine to 4-hydroxyquinoline as described by Weyler, W. and Salach, J.I. (J. Biological Chemistry, 260:13199 - 13207
  • This method can also be used to assay the MAO inhibitory effects of the novel ligands which have an affinity for binding to MAO and which are linked to immunoreactive groups as described in this invention. c) Linking Group
  • 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 reactive group refers to any group which can react with
  • linking groups useful in the practice of this invention derive from those groups which can react with any relevant molecule "Z" or "Rec” as described above containing a reactive group, whether or not such
  • linking group L 1 , between the
  • linking groups are derived from protein reactive groups selected from but not limited to:
  • (1) a group that will react directly with amine, alcohol, or sulfhydryl groups on the immunoreactive protein or biological molecule containing the reactive group, for example, active halogen containing groups including, for example, chloromethylphenyl groups and chloroacetyl [ClCH 2 C( O)-] groups, activated 2-(leaving group substituted)-ethylsulfonyl and ethylcarbonyl groups such as 2-chloroethylsulfonyl and 2-chloroethylcarbonyl; vinylsulfonyl; vinylcarbonyl;
  • the immunoreactive group i.e., proteins or biological molecules containing the immunoreactive group modified to contain reactive groups such as those mentioned in (1) above, for example, by oxidation of the protein to an aldehyde or a carboxylic acid, in which case 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 said linking groups can contain from 1 to about 20 carbon atoms.
  • the aryl portions of said linking groups can contain from about 6 to about 20 carbon atoms; and
  • hardeners, bisepoxides, and bisisocyanates can become a part of, i.e., a linking group in, for example, the protein-(MAO active site-containing species) conjugate in System A 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. Patent No. 4,877,724.
  • one of the reactants such as the immunoreactive group must have a carboxyl group and the other such as the oligonucleotide containing species must have a reactive amine, alcohol, or sulfhydryl group.
  • the crosslinking agent first reacts selectively with the carboxyl group, then is split out during reaction of the thus "activated" carboxyl group with an amine to form an amide linkage between, for example, the protein and MAO active site containing species, thus covalently bonding the two moieties.
  • An advantage of this approach is that crosslinking of like molecules, e.g., proteins with proteins or MAO active site containing species with themselves is avoided, whereas the reaction of, for example, homo-bifunctional crosslinking agents is nonselective and unwanted crosslinked molecules are obtained.
  • Preferred useful linking groups are derived from various heterobifunctional cross-linking reagents such as those listed in the Pierce Chemical Company
  • Sulfo-SMCC Sulfosuccinimidyl 4-(N- maleimidomethyl)cyclohexane-1- carboxylate.
  • Sulfo-SIAB Sulfosuccinimidyl (4- iodoacetyl)aminobenzoate.
  • Sulfo-SMPB Sulfosuccinimidyl 4-(p- maleimidophenyl)butyrate.
  • linking groups in whole or in part, can also comprise and be 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 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
  • conjugate one sequence to the immune reactive agent and the complementary oligonucleotide sequence to the MAO active site containing moiety.
  • the hybrid formed between the two complementary oligonucleotide sequences then comprises the linking group between the immune reactive agent and the MAO active site containing moiety.
  • the complementary oligonucleotide sequences are attached to two components of the conjugate, one sequence to the residue comprising one or more chelating agents and the complementary
  • the hybrid formed between the two complementary oligonucleotide sequences then comprises the linking group between the MAO active site containing moiety and the chelating agent (s).
  • two or more copies of the same oligonucleotide sequence can be linked, for example, in tandem to one MAO active site containing moiety and a complementary oligonucleotide sequence comprising multiple chelating agents can be added.
  • the multiple hybrids formed between the two complementary oligonucleotide sequences then comprise the linking group between the MAO active site containing moiety and multiple chelating agents.
  • one or more MAO active site binding ligands can be attached to the immunoreactive group using complementary oligonucleotide hybrids as described above.
  • multiple MAO sequences can be attached to the immunoreactive protein.
  • one or more MAO active site binding ligands can be attached to multiple chelating agents using complementary oligonucleotide hybrids as described above. d) Residues of Chelating groups
  • a chelating agent is a compound containing donor atoms that can combine by coordinate bonding with a metal atom to form a cyclic structure called a chelation complex or chelate. This class of compounds is described in the
  • residues of suitable chelating agents can be independently selected from polyphosphates, such as sodium tripolyphosphate and hexametaphosphoric acid; aminocarboxylic acids, such as
  • nitrilotriacetic acid N,N-di(2-hydroxyethyl)glycine, ethylenebis(hydroxyphenylglycine) and diethylenetriamine pentacetic acid
  • 1,3-diketones such as acetylacetone, trifluoroacetylacetone, and thenoyltrifluoroacetone
  • hydroxycarboxylic acids such as tartaric acid, citric acid, gluconic acid, and 5-sulfosalicylic acid
  • polyamines such as ethylenediamine, diethylenetriamine, triethylenetetramine, and triaminotriethylamine;
  • aminoalcohols such as triethanolamine and N-(2- hydroxyethyl)ethylenediamine
  • aromatic heterocyclic bases such as 2,2'-dipyridyl, 2,2'-diimidazole, dipicoline amine and 1, 10-phenanthroline
  • phenols such as salicylaldehyde, disulfopyrocatechol, and
  • chromotropic acid aminophenols, such as 8-hydroxyquinoline and oximesulfonic acid; oximes, such as dimethylglyoxime and salicylaldoxime; peptides
  • proximal chelating functionality such as polycysteine, polyhistidine, polyaspartic acid
  • polyglutamic acid or combinations of such amino acids; Schiff bases, such as disalicylaldehyde 1,2-propylenediimine; tetrapyrroles, such as
  • sulfur compounds such as toluenedithiol, meso-2,3-dimercaptosuccinic acid, dimercaptopropanol, thioglycolic acid, potassium ethyl xanthate, sodium diethyldithiocarbamate,
  • Preferred residues of chelating agents contain polycarboxylic acid groups and include: ethylenediamine-N, N, N',N'-tetraacetic acid (EDTA); N,N,N',N",N"- diethylene-triaminepentaacetic acid (DTPA); 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA); 1,4,7,10-tetraazacyclododecane-N,N',N"-triacetic acid (DO3A); 1-oxa-4,7,10-triazacyclododecane-N,N',N"-triacetic acid (OTTA); and trans (1,2)-cyclohexanodiethylenetriamine pentaacetic acid (CDTPA).
  • EDTA ethylenediamine-N, N, N',N'-tetraacetic acid
  • DTPA diethylene-triaminepentaacetic acid
  • Preferred residues of chelating agents contain polycarboxylic acid groups and include: B4A, P4A, TMT, DCDTPA, PheMT, macroPheMT, and macroTMT;
  • Suitable residues of chelating agents comprise proteins modified for the chelation of metals such as technetium and rhenium as described in U.S. Patent No. 5,078,985, the disclosure of which is hereby incorporated by reference.
  • suitable residues of chelating agents are derived from N 3 S and N 2 S 2 containing
  • linking groups 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.,
  • heterocyclylalkyl the heterocyclyl and alkyl portions of which preferably are described above; or a peptide linkage, i.e., a
  • linking groups can be used, such as, for example, alkyleneimino and
  • 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 for L 1 or L 2 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 Q and the other components of this invention.
  • 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
  • aryl preferably containing from 6 to about 20, more preferably 6 to 10 carbon atoms such as phenyl, naphthyl, hydroxyphenyl, iodophenyl,
  • 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 alkyl above; alkoxyaralkyl, such as ethoxybenzyl; substituted or unsubstituted heterocyclyl, preferably containing from 5 to 7 nuclear carbon and heteroatoms such as S, N, P or O, examples of preferred heterocyclyl groups being pyridyl, quinolyl, imidazolyl and thienyl; a carboxyl group; a carboxyalkyl group, the alkyl portion of which preferably contains from 1 to 8 carbon atoms; or the residue of a chelating group.
  • MAO substrate analogs are those which will fit the active site of the enzyme and include, for example, those with an aromatic ring and a side chain with an amino group located in the ring's plane.
  • the distance between the center of the nitrogen and the ring should be 0.5 to 0.55 nanometers.
  • specific examples of the MAO active site binding ligands useful in this invention include propargyl amine derivatives such as derivatives of clorgyline, a suicide inhibitor of MAO-A, or of
  • one of such groups represents a possible site for the attachment by a linking group L 2 to a chelating group in the RDA of System A or by a linking group L 1 to an immunoreactive group in the NRTIR or System B.
  • one of the groups X' and Y' is selected from the group consisting of H, a halogen such as F, Cl, Br, and I, an alkyl group of 1 to 6 carbons, a carboxylic acid group, a carboxylic amide group, and an alkyl ether group wherein the alkyl group is as defined above
  • the other of the groups X 1 and Y' comprises a group selected from the group consisting of a suitably substituted linear alkylene group containing from 1 to 12 carbons, a branched alkylene group containing from 2 to 12 carbons, a cyclic alkylene group of from 3 to 12 carbons, an ether group linked to an alkylene group where alkylene is defined above, an alkylene group as defined above
  • alkyl and alkylene groups may also contain one or more sites of
  • Preferred analogs include those modified in such a way so as to permit or facilitate binding of one portion of the ligand to the immunoreactive species in system B by means of a linking group L 1 and to the chelating species in system A by means of a linking group L 2 .
  • MAO inhibitory compounds which could be similarly derivatized so as to act as MAO substrate analogs include N-cyclopropyl-N-arylalkyl amines and drugs of the type exemplified by 3-[4-(3-cyanophenylmethoxy)phenyl]-5-(methoxymethyl)-2-oxazolidinone.
  • An especially preferred class of compounds that will form irreversible, covalent attachments with the elements of the active site of MAO are derivatives of pargyline.
  • the group X' is as defined above and represents a site for the attachment by a linking group L 2 to a chelating group in the RDA of System A or by a linking group L 1 to an immunoreactive group in the NRTIR or System B. Further substitution may also be possible on the groups adjacent to the amino group as long as the derivative falls within the description outlined above.
  • the preparation of a preferred RDA in system A is as defined above and represents a site for the attachment by a linking group L 2 to a chelating group in the RDA of System A or by a linking group L 1 to an immunoreactive group in the NRTIR or System B.
  • linking groups between the pargyline aromatic ring and a chelating group comprise the residue of a 6-methylenecarbonylaminohexanoic acid amide with the N-terminal amine of a peptide such as H 2 N-(Ala) 4 -Lys-Lys-OH (SEQ ID NO:1) as well as the residue of a 7-methylenecarbonylaminoheptanoic acid amide with the N-terminal amine of a peptide such as H 2 N-(Ala) 4 -Lys-Lys-OH (SEQ ID NO:1).
  • the number of alanine (Ala) residues is preferably in the range of from 0 to 12
  • the number of lysine (Lys) residues is preferably in the range from 2 to 20.
  • the lysines can be contiguous or can be separated by spacer groups such as the residue of amino acids such as 6-aminohexanoic acid, 7-aminoheptanoic acid, alanine, glycine, valine, glutaminic acid, aspartic acid, phenylalanine, serine, threonine, leucine, isoleucine, and other amino acids that will not interfere with the binding of the
  • the spacing group between the lysines is selected from the group consisting of one or two 6-aminohexanoic acid residues, one or two 7-aminoheptanoic acid
  • the t-butoxycarbonyl (t-Boc) epsilon amine blocked Lys-Lys (structure 5) is prepared using a dehydrative coupling method, for example, using DCC (dicyclohexylcarbodiimide) and two epsilon amine
  • a reagent such as benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) can be employed to react with a carboxylic acid to form a benzotriazoyloxy (BTAZ) ester which will react with the unblocked, N-terminal amine of an amino acid
  • the FMOC (9-fluorenylmethoxycarbonyl) group of resin linked Lys-Lys is then removed via base treatment, and 4 units of alanine are introduced as shown in SCHEME 2 to afford the resin bound bis (t-BOC) FMOC-blocked Ala-Ala-Ala-Ala-Lys-Lys-COO-resin (SEQ ID NO:2; structure 7).
  • the acidic removal of the t-BOC groups and acidic removal of the peptide from the resin affords the FMOC-blocked peptide, FMOC-NH-(Ala) 4 -(Lys) 2 -COOH (SEQ ID NO:3;
  • This peptide is then coupled to 6- (FMOC-amino)-hexanoic acid or to 7-(FMOC-amino)-heptanoic acid by means of an activated ester such as an N-hydroxysuccinimide (NHS) ester which is formed by the reaction of N-hydroxysuccinimide with the adduct of dicyclohexylcarbodiimide and 6-(FMOC-amino)-hexanoic acid or which is formed by the reaction of N-hydroxysuccinimide with the adduct of
  • NHS N-hydroxysuccinimide
  • BTAZ benzotriazoyl-1-oxy
  • This material can be purified and isolated using reverse phase and size exclusion high pressure liquid
  • a solution containing a desired number of mCi of 90 y +3 (as 90 YCl 3 in hydrochloric acid) is preferably added at room
  • Enantiomerically pure L- and pure D-amino acid derivatives as well as racemic mixtures of D- and L- amino acid enantiomers of the above described pargyline derivatives are also useful in this invention.
  • Additional chelating agents and radionuclides bound to chelating agents are incorporated by preparing, for example, analogous peptides comprised of additional Lys- TMT and Lys-TMT-radionuclide groups.
  • the number of such Lys-TMT and Lys-TMT-radionuclide residues is from 1 to about 20, and more preferably from 2 to about 10.
  • the NRTIR is comprised of one or more ligands that have an affinity for binding to a MAO active site each with a suitably substituted linking group (L 1 ) conjugated to the immunoreactive group (Z).
  • said ligand that has an affinity for binding to a MAO active site is comprised of a 4-substituted pargyline residue linked to Z by a linking group (L 1 ) as defined above.
  • the NRTIR preferably contains 2 to about 10 of such groups, more preferably 2 to about 4.
  • This pargyline derivative is then converted to an activated ester for coupling to a protein by reaction of the carboxylic acid group with NHS and DCC or with BOP as described above.
  • the NRTIR of System B comprises from one to about 20 of such pargyline containing groups, preferably from one to about 6.
  • the salt can be any water soluble salt of the metal such as a halogen salt, but preferably such salts are selected so as not to interfere with the binding of the metal ion with the chelating agent.
  • the chelating agent-containing moiety is preferably in aqueous solution at a pH of between about 5 and about 9, more preferably between pH about 6 to about 8.
  • the chelating agent-containing moiety optionally is mixed with buffers such as citrate, acetate, phosphate and borate to produce the optimum pH.
  • the buffer salts are selected so as not to interfere with the subsequent binding of the metal ion to the chelating agent.
  • the radioactive chelating agent-containing moiety of this invention can contain any ratio of metal
  • radionuclide ion to chelating agent that is effective in therapeutic and diagnostic imaging applications.
  • the mole ratio of metal ion per chelating agent is from about 1:10.0 to about 1:1.
  • the mole ratio of metal ion per chelating agent is from about 1:1,000 to about 1:1.
  • the metal ion of this invention can comprise a non radioisotope.
  • the metal ions can be selected from, but are not limited to, elements of groups IIA through VIA. Preferred metals include those of atomic number 12, 13, 20, the
  • 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, Mn, Cu, Cr, Zn, Ge, Mo, Tc, Ru, In, Sn, Sr, Sm, Lu, Sb, W, Re, Po, Ta and TI.
  • Preferred radionuclides include 44 Sc, 64 Cu, 67 Cu, 111 In, 212 Pb, 68 Ga, 90 Y, 153 Sm, 212 Bi, 99m Tc, 186 Re and 188 Re. Of these, especially preferred is 90 Y.
  • These metals can be atomic or preferably ionic. Fluorescent metals:
  • the metal ion 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 is especially preferred. Paramagnetic metals:
  • the metal ion of this invention can comprise one or more paramagnetic elements which are suitable for the use in MRI applications.
  • 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.
  • a therapeutically effective dose of a radionuclide such as 90 ⁇ +3 together in the same chelating agent containing moiety sample with a diagnostic imaging effective dose of a paramagnetic ion such as Gd +3 , the molar concentration of the latter ion being typically in excess with respect to that of the former ion in the conjugated complex, would permit the simultaneous magnetic resonance imaging of at least a portion of the tissue of a host patient during
  • radioisotopes of iodine is specifically contemplated.
  • the RDA of System A or of System B comprises substituents that can be chemically
  • substituents can be labeled by methods well known in the art with a radioisotope of iodine.
  • the thus covalently linked iodine species can be used in the aforementioned fashion in therapeutic and diagnostic imaging
  • radioisotope can be delivered to a diseased tissue site
  • radioisotope is site specific; delivery of radionuclide to a diseased tissue site can be achieved in amplification over that which can be achieved with a single stage delivery system;
  • the system will reduce the exposure of non-target tissues to damage from radiation
  • the binding of the ligand to the receptor is essentially irreversible and selective
  • the system can be used in both therapeutic and diagnostic imaging applications
  • the above-described NRTIR can accumulate at a tumor site in vivo while it is not accumulated at normal tissue sites;
  • the in vivo residence half life of the above-described RDA is shorter than the residence half life of the above-described NRTIR;
  • radionuclide or a chelate containing a radionuclide an amplification of the number of radionuclides per site of modification per targeting immune reagent can be
  • the above-described NRTIR can comprise a wide variety of immune reactive groups, linking groups, and MAO active site residues in system A or MAO active site binding ligand residues in system B;
  • RDA can comprise a wide variety of spacing, linking and chelating groups, radionuclides, and MAO active site binding ligand residues in system A or MAO active site residues in system B;
  • compositions, size and molecular weight can be prepared in accordance with this invention.
  • an effective dose of an RDA of System A or System B as described above in a pharmaceutically acceptable medium is prepared by exposing a composition of a precursor of an RDA (said precursor comprising a residue of a ligand that has an ability to covalently bind to a MAO active site, a linking group, and a residue of a chelating agent in System A and of a residue of a MAO active site, a linking group, and a residue of a chelating agent in System B) to a composition containing a radioactive metal ion such that the molar amount of said
  • radionuclide metal ion is less than the molar amount of the chelating groups comprising the RDA, said duration of exposure lasting an effective time to permit uptake of said metal ion into said RDA.
  • an effective dose of a NRTIR of System A or System B as described above in a pharmaceutically acceptable medium is administered to a patient and said NRTIR is allowed to accumulate at the target site such as at a tumor site in said patient. Subsequently, at an effective time, an effective dose of a RDA as described above in a pharmaceutically
  • the present invention includes one or more NRTIR as described above and one or more RDA 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
  • compositions can be administered to humans and animals either orally, rectally, parenterally (intravenous, by intramuscularly or subcutaneously), intracisternally, intravaginally, intraperitoneally, intravesically, locally (powders, ointments or drops), or as a buccal or nasal spray.
  • compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
  • compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents.
  • adjuvants such as preserving, wetting, emulsifying, and dispensing agents.
  • Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • isotonic agents for example sugars, sodium chloride and the like.
  • Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active compound is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or
  • fillers or extenders as for example, starches, lactose, sucrose, glucose, mannitol and silicic acid
  • binders as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose and acacia
  • humectants as for example, glycerol
  • disintegrating agents as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates and sodium carbonate
  • solution retarders as for example paraffin
  • absorption accelerators as for example, quaternary ammonium compounds
  • wetting agents as for example
  • lubricants as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate or mixtures thereof.
  • the dosage forms may also comprise buffering agents.
  • compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin NOT FURNISHED UPON FILING
  • compositions of the present invention may be varied so as to obtain an amount of active ingredient that is effective to obtain a desired therapeutic response for a particular composition and method of administration.
  • the selected dosage level therefore depends upon the desired therapeutic effect, on the route of
  • the total daily dose of the compounds of this invention administered to a host in single of 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 of 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 present invention is directed to a method of diagnosis and comprises the administration of a contrast effective amount of the compositions of the present invention to a mammal in need of such diagnosis.
  • a method for diagnostic imaging for use in medical procedures in accordance with this invention comprises administering to the body of a test subject in need of a diagnostic image an effective contrast producing amount of the above-described NRTIR containing composition.
  • the NRTIR is allowed to bind to sites on cells of a tissue of interest, the unbound NRTIR is cleared from the environs of the tissue and an RDA containing composition as described above in a pharmaceutically acceptable medium is administered to the subject.
  • the radioactive targeting reagent is allowed to accumulate at the target site, said target site being the non-radioactive targeting immunoreagent accumulated at the sites on cells of a tissue of interest in said subject.
  • the image pattern can then be visualized, for example, by radioscintigraphy, by a radiation sensitive detector and signal amplifier.
  • test subject can include mammalian species such as rabbits, dogs, cats, monkeys, sheep, pigs, horses, bovine animals and the like.
  • the NRTIR may be reacted with a diagnostic imaging effective amount of a reagent comprised of a radionuclide prior to administration to the environs of a tissue of interest of a patient undergoing such diagnostic imaging, waiting for an effective period of time during which time said NRTIR will bind to sites on cells of said tissue of interest and during which time unbound NRTIR will be removed from the environs of said tissue and then obtaining an image as a function of time of all or part of said tissue of interest.
  • a diagnostic imaging or a therapeutically effective amount of RDA containing the same or a different radionuclide as that employed on the NRTIR is administered to said tissue of interest of said patient.
  • At least a portion of the body containing the administered contrast agent is exposed to x-rays or to a magnetic field to produce an x-ray or magnetic resonance image pattern corresponding to the presence of heavy elements such as iodine and heavy metal ions in the contrast agent.
  • the image pattern can then be visualized.
  • transmitted radiation is used to produce a radiograph based upon overall tissue
  • X-rays pass through various tissues and are attenuated by scattering, i.e., reflection or refraction or energy absorption.
  • scattering i.e., reflection or refraction or energy absorption.
  • certain body organs, vessels and anatomical sites exhibit so little absorption of x-ray radiation that radiographs of these body portions are difficult to obtain.
  • an x-ray absorbing medium containing a contrast agent routinely introduce an x-ray absorbing medium containing a contrast agent into such body organs, vessels and anatomical sites.
  • Any x-ray visualization technique preferably, a high contrast technique such as computed tomography, can be applied in a conventional manner.
  • the image pattern can be observed directly on an x-ray sensitive phosphor screen-silver halide photographic film combination.
  • Magnetic imaging systems such as a General Electric 1.5 T Signa imaging system [1H resonant frequency 63.9 megahertz (MHz)].
  • Commercially available magnetic resonance imaging systems are typically characterized by the magnetic field strength used, with a field strength of 2.0 Tesla as the current maximum and 0.2 Tesla as the current minimum.
  • each detected nucleus has a characteristic frequency.
  • the resonance frequency for hydrogen is 42.57 MHz; for phosphorus-31 it is 17.24 MHz; and for sodium-23 it is 11.26 MHz.
  • a contrast effective amount of the compositions of the present invention is that amount necessary to provide tissue visualization with, for example, magnetic resonance imaging or x-ray imaging.
  • determining a contrast effective amount in a particular subject will depend, as is well known in the art, on the nature of the magnetically reactive material used, the mass of the subject being imaged, the sensitivity of the magnetic resonance or x-ray imaging system and the like.
  • compositions of the present invention After administration of the compositions of the present invention, the subject mammal is maintained for a time period sufficient for the administered
  • compositions to be distributed throughout the subject and enter the tissues of the mammal Typically, a sufficient time period is from about 20 minutes to about 2 weeks or more and, preferably from about 20 minutes to about 1 week.
  • Z is the residue of an immunoreactive group, preferably an antibody
  • Rec is the residue of a receptor, preferably a monoamine oxidase (MAO);
  • MAO monoamine oxidase
  • D is the residue of a ligand that has an affinity for covalent binding to the receptor, and preferably said ligand is pargyline, an inhibitor of monoamine oxidase;
  • L 1 and L 2 are each independently the residue of a linking group that may contain a spacing group;
  • Q is the residue of a chelating group such as TMT, above;
  • M is a radionuclide, preferably 90 Y; and n and m are each independently an integer greater than zero.
  • the peptide is removed from the resin and t-BOC groups are removed from the lysine amines by treatment of the peptide-resin with 15 mL of a 95:5 solution of
  • the mixture is then filtered using a scintered glass funnel.
  • the filtrate volume is then reduced to about 3 mL by rotoevaporation, and the peptide is precipitated by dropping the oil into a centrifuge tube containing 50 mL of ether.
  • the peptide is separated by
  • N-alpha-Fmoc-protected peptide, Fmoc-HN-Ala-Ala-Ala-Ala-Lys-Lys-OH (SEQ ID NO: 3), from Example 1 (20 mM) is dissolved in a saturated sodium bicarbonate solution (pH 9) in deionized water (50 mL) containing
  • Example 3 dimethylsulfoxide (DMSO, 5 mL). This solution is treated with TMT-NCS (25 mM), and the reaction is allowed to continue for 12 hours at room temperature. This solution is then treated with more TMT-NCS (25 mM), and the reaction is allowed to continue for 24 hours at room temperature. The reaction is followed by size exclusion HPLC until all the TMT-NCS and all of the peptide are reacted.
  • the desired lysyl-TMT-containing peptide is isolated by chromatography on a Shodex WS-803F size exclusion column using a UV-visible detector monitoring the absorption of TMT. A solid sample can be isolated from solution by lyophilization.
  • DMSO dimethylsulfoxide
  • N-alpha-Fmoc-protected peptide, Fmoc-HN-Ala-Ala-Ala-(Lys-TMT)-(Lys-TMT)-OH (SEQ ID NO:5)
  • Example 2 10 mM
  • the reaction vessel is sealed and warmed to 40 °C for 12 hours.
  • the reaction is cooled, and the desired product is isolated by HPLC using a Shodex WS-803F size exclusion column and a UV-visible detector monitoring the absorption of TMT.
  • a solid sample is isolated from solution by lyophilization.
  • the crude reaction product from Example 6 is treated with diethylamine according to the procedure of Example 3.
  • the desired product is purified by HPLC using a Shodex WS-803F size exclusion column and a UV-visible detector monitoring the absorption of TMT.
  • a solid sample is isolated from solution by lyophilization.
  • Example 10 To the crude reaction product of Example 8 is added 10 mL of N-methylpropargylamine (Aldrich), and the reaction mixture is stirred for 6 hours at room temperature. The desired product is isolated by HPLC using a Shodex WS-803F size exclusion column and a UV-visible detector monitoring the absorption of TMT. A solid sample is isolated from solution by lyophilization.
  • N-methylpropargylamine Aldrich
  • Radionuclide, 90 Y +3 chelated to H-CC-CH 2 -N(CH 3 )-CH 2 -C 6 H 6 -CH 2 -CO-HN-(CH 2 ) 5 -CO-HN-(Ala) 4 -(Lys-TMT) 2 -OH (SEQ ID NO: 9) (formation of D-(L 2 -Q-M) m )
  • hydrochloric acid at a specific activity of >500 Ci/mg; Amersham-Mediphysics) is neutralized using two volumes of 0.5 M sodium acetate pH 6.0 and added to a solution of H-CC-CH 2 -N(CH 3 )-CH 2 -C 6 H 6 -CH 2 -CO-HN-(CH 2 ) 5 -CO-HN- (Ala) 4 -(Lys-TMT) 2 -OH (SEQ ID NO: 9) from Example 9 in deionized water buffered with 0.5 M sodium acetate at pH 6.0 at room temperature. The molar ratio of TMT to 90 Y is greater than one at all times. The chelation of the 90 Y is allowed to proceed for one hour.
  • the labeling efficiency is determined by removing 1.0 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
  • ING-1 (a chimeric IgG 1 antibody) is a non-limiting example of such an antibody; other antibodies such as those described herein are useful.
  • MAOs referred to hereinbelow are of human origin.
  • Purified monoamine oxidase A (MAO-A) is isolated from the membranes of human placental
  • the cDNAs encoding human MAO-A and MAO-B are obtained according to published methods (Bach A.W., Lan N.C., Johnson D.L. et al [1988]; Proc. Natl . Acad. Sci . USA . 85: 4934-4934) and expressed in a CosII cell line (Lan N.C., Heinzmann C, Klisak I., et al [1988]; Abst . Soc. Neurosci . 14.:317) or is produced as a recombinant product from the cloned MAO genes which are overexpresed in Escherichia coli (E.coli). Purification from these sources is carried out as described above.
  • a sulfo-SMCC solution (36 nmoles; Pierce Chemical Co.) in phosphate buffered saline (PBS) is added to a solution containing of a chimeric antibody (ING-1; 6 nmoles) in phosphate buffer ( at pH 7).
  • PBS phosphate buffered saline
  • ING-1 6 nmoles
  • phosphate buffer pH 7
  • the resulting reaction mixture is allowed to stand for 30 minutes with occasional mixing at room temperature.
  • the reaction is stopped with 60 nmoles of basic tris buffer.
  • reaction mixture is diluted with phosphate buffered saline, added to a prewashed PD-10 column, and eluted with PBS to afford ING-1-maleimide. This material is stored on ice until use.
  • a solution containing a chimeric antibody (ING-1; 6 nmoles) in 0.1 M carbonate buffer (pH 8.8) is mixed with 200 nmoles of an aqueous solution of 2-iminothiolane.
  • the resulting mixture is allowed to stand for 30 minutes with occasional mixing at room temperature.
  • the reaction mixture is diluted with phosphate buffed saline, added directly to a prewashed PD-10 column, and eluted with PBS to afford mercaptoalkyl-ING-1. This material is stored on ice until use.
  • This S-acetyl thioacetylated antibody is deacylated by the addition of 30 mL of a pH 7.5 solution containing 100 mM sodium phosphate, 25 mM EDTA, and 50 mM NH 2 OH. The reaction proceeds for two hours at room temperature after which the material is passed down a PD-10 column eluting with PBS. The final product, ING-1(NH)-CO-CH 2 -SH, is used immediately. (11d) Radioisotopic labeling of ING-1 with 125 I; the formation of 125 I-labeled ING-1
  • Example 12 (12a) The Reaction of MAO using SATA; Preparation of a mercapto-MAO (formation of (L 1 -Rec))
  • a solution containing 50 nmoles of MAO in PBS is vortexed while 500 nmoles of SATA (in DMSO) are added. After mixing and standing at room temperature for 60 min, the reaction mixture is diluted with PBS, and eluted from a PD-10 column with PBS to afford an S-acetyl thioacetylated MAO, MAO(NH)-CO-CH 2 -S-CO-CH 3 .
  • the S-acetyl thioacetylated MAO is deacylated by the
  • Example 13 General method for the conjugation of a MAO to an Antibody employing the reaction of a sulfhydryl-containing species with a maleimide-containing species, (formation of Z-(L 1 -Rec) n )
  • the following procedure is generally applicable to the conjugation of sulfhydryl-containing MAO species to maleimide-containing antibody species as well as to the conjugation of sulfhydryl-containing antibody species to maleimide-containing MAO species.
  • the following procedure is applicable to the conjugation of ING-1-Maleimide of Example 11a to mercapto-MAO of
  • maleimide-containing reactant species for example, ING-1-Maleimide of Example 11a and MAO-Maleimide of Example 12c
  • a freshly prepared sample of a sulfhydryl-containing reactant species as described above (50 nmoles) is eluted off a PD-10 column directly into a solution of maleimide-containing species as described above (5 nmoles). After a brief mixing the solution is rapidly concentrated by centrifugation in a Centricon-30® device to a concentration of approximately 3.0 mg/mL protein. The reaction then is allowed to proceed for 4 hours at room temperature. The thus prepared antibody-MAO conjugate is transferred to an Amicon stirred cell fitted with a YM-100 membrane filter, the sample is diluted to 10 mL with PBS and then concentrated, under a nitrogen pressure of 5 kg/cm 2 , to a volume of about 500 microliters.
  • the retentate material is again diluted with PBS to 10 mL and reconcentrated to 1.0 mL. This procedure, which separates unconjugated MAO and other low molecular weight species from the retained antibody-MAO conjugate and unconjugated antibody, is repeated 4 times or until spectrophotometric monitoring of the diafiltrate at 280 nm shows that no further protein is present in the diafiltrate.
  • the retentate material is then concentrated to approximately 1.0 mg of antibody-MAO conjugate per milliliter solution. This solution is then applied to a 2.6 ⁇ 60 cm Sephacryl S-200
  • Centricon-30 device to a concentration of approximately 1.0 mg of antibody-MAO conjugate per milliliter of solution.
  • the solution of the conjugate is sterile filtered through a 0.22 m filter and stored at 4°C until used.
  • Z is the residue of an immunoreactive group, preferably an antibody
  • Rec is the residue of a receptor, preferably a MAO receptor
  • D is the residue of a ligand, preferably a pargyline-containing ligand, that has an affinity for covalent binding to the receptor, preferably to a MAO receptor
  • L 1 and L 2 are each independently the residue of a linking group that may contain a spacing group
  • Q is the residue of a chelating group, preferably TMT;
  • M is a radionuclide, preferably 90 Y;
  • n and m are each independently an integer greater than zero.
  • Fmoc-HN-Ala-Ala-Ala-OH (SEQ ID NO: 10)
  • the linear N-alpha-Fmoc-protected peptide, Fmoc-HN- Ala-Ala-Ala-OH (SEQ ID NO: 10)
  • the solid support used in the synthesis is a 4-alkoxybenzyl alcohol polystyrene resin (Wang resin).
  • the N-alpha-Fmoc protecting group is used throughout the synthesis.
  • the peptide chain is assembled using the ABI FastMocTM software protocols for Fmoc-chemistry (0.25 mmole scale, HBTU activated couplings, 4 fold excess of amino acid, 1 hour).
  • the peptide is removed from the resin by treatment of the peptide-resin with 15 mL of a 95:5 solution of trifluoroacetic acid in water in a sealed vessel followed by shaking at room temperature for 2 hours.
  • the mixture is then filtered using a scintered glass funnel.
  • the filtrate volume is then reduced to about 3 mL by rotoevaporation, and the peptide is precipitated by dropping the oil into a centrifuge tube containing 50 mL of ether.
  • the peptide is separated by centrifugation, the ether is decanted, the solid is washed with more ether and then allowed to air dry.
  • N-alpha-Fmoc-protected peptide, Fmoc-HN-Ala-Ala-Ala-OH (SEQ ID NO: 10), from Example 14 (10 mM) is dissolved in 50 mL of deionized water and 50 mL of dimethylsulfoxide, and then treated with 25 mM of N,N-diethylamine.
  • the reaction vessel is sealed and warmed to 40 °C for 12 hours.
  • the reaction is cooled, and the desired product is isolated by HPLC using a Shodex WS-803F size exclusion column and a UV-visible detector monitoring the absorption of the peptide.
  • a solid sample is isolated from solution by lyophilization.
  • Example 5 The crude reaction product from Example 5 is treated with 10 mM of H 2 N-Ala-Ala-Ala-OH (SEQ ID NO: 11) from Example 15 in 50 mL of deionized water and 50 mL of dimethylsulfoxide containing 1 mL of pyridine. The reaction is allowed to proceed overnight at room temperature.
  • Example 16 The crude reaction product from Example 16 is treated with diethylamine according to the procedure of Example 3.
  • the desired product is purified by HPLC using a
  • Example 18 To the crude reaction product of Example 18 is added 10 mL of N-methylpropargylamine (Aldrich), and the reaction mixture is stirred for 6 hours at room temperature.
  • the desired product is isolated by HPLC using a Shodex WS-803F size exclusion column and a UV-visible detector monitoring the absorption of the aromatic species .
  • a solid sample is isolated from solution by
  • Example 19 The product of Example 19 (12 nmoles) in 2.5 mL of 50% DMSO in 50 mM sodium phosphate buffer at pH 7.2 is mixed with a solution of EDC (1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide) in DMSO to give a final EDC concentration of 50 micromolar. After 1 hour at room temperature, the O-acylisourea derivative of Example 19 is isolated by elution from a PD-10
  • the pargylinyl tetrapeptide-O-acylisourea obtained from Example 20 (10 nmoles) is eluted directly into a solution of 10 nmoles of ING-1 antibody in 200 mM acetate buffer (pH 5.0). The reaction mixture is slowly stirred overnight at room temperature. The thus
  • pargylinyl tetrapeptide-ING-1 conjugate is separated from unconjugated pargylinyl tetrapeptide-O-acylisourea and other low molecular weight products using a Superose 6 HPLC column equilibrated in and eluted with 50 mM sodium phosphate buffer at pH 7.2 supplemented with 150 mM sodium chloride. The eluate is concentrated using a Centricon-30 device to a
  • TMT-isothiocyante formation of REC-L 2 -Q, a MAO-to-TMT conjugate
  • a solution of monoamine oxidase (50 nmoles) in 1 mL of 1.0 M carbonate buffer containing 150 mM sodium chloride at pH 9 .3 in an acid washed, conical, glass reaction vial is treated with 250 nmoles of the chelating agent, tetrasodium 4 ' - (3-isothiocyanato-4-methoxyphenyl) -6, 6"-bis [N,N-di (carboxymethyl) aminomethyl] -2, 2 ' : 6 ' , 2"-terpyridine, hereinafter referred to as TMT-isothiocyanate which is disclosed in WO 92/08494 (PCT/US91/08253) .
  • the reaction mixture is stirred briefly to mix the reactants and then left in the dark at room temperature. After 16 hours, the therein produced MAO-to-TMT conjugate is separated from unconjugated TMT-isothiocyanate by applying the reaction mixture to a PD-10 chromatography column which has been pre-washed and equilibrated with 50 mM sodium acetate buffer containing 150 mM sodium chloride at pH 5.6. The conjugate is eluted off the column with 2.5 mL of that same buffer, and concentrated on a Centricon-10 ® concentration device.
  • Radioisotopic labeling of MAO-to-TMT conjugate with 90 Y (formation of Rec- (L2-Q-M) m )
  • a volume of radioactive yttrium chloride ( 90 Y in 0.04 M hydrochloric acid at a specific activity of >500 Ci/g: Amersham-Mediphysics) is neutralized using two volumes of 0.5 M sodium acetate pH 6.0.
  • the neutralized 90 Y solution (1.0 mCi) is added to 1.0 mL of MAO-to-TMT conjugate (1 mg/mL) in 50 mM sodium acetate buffer containing 150 mM sodium chloride at pH 5.6.
  • the reaction mixture is loaded on to a PD-10 chromatography column which has been pre-washed with and equilibrated in a pH 7.4 phosphate buffer containing 50 mM sodium phosphate and 150 mM sodium chloride (PBS).
  • the sample is eluted from the column with 1.5 mL of PBS.
  • Fractions of radioisotopically labeled MAO-to-TMT conjugate (0.5 mL) are collected, assayed for radioactivity, and pooled.
  • the labeling efficiency is determined by removing 1.0 ⁇ L 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 is optimized for 90 Y and controlled by a Compaq 386/20e computer.
  • the concentrations of ING-1 and MAO for use in the conjugate reactions are determined by the BioRad protein assay using bovine immunoglobulin as the protein
  • MAO labeled with other materials such as TMT-isothiocyante which is then chelated to 90 Y as described above can be used.
  • TMT-isothiocyante which is then chelated to 90 Y as described above.
  • radioactivity can be measure as above.
  • MAO labeled with a chelating agent such as TMT-isothiocyante as described above can be chelated to europium ion for use in fluorescence detection assays.
  • Other known assays for quantifying concentrations of proteins such as those involving biotinylating agents such as those described in the Pierce Chemical Company 1992 catalog, or assays involving fluoroscein
  • isothiocyanate are useful to detect and quantify the amount of MAO or MAO conjugated to another protein and present in a solution.
  • Such species include MAO conjugated to an antibody, sometimes hereinafter referred to as antibody-MAO conjugates.
  • adenocarcinoma cell line ATCC
  • ATCC adenocarcinoma cell line
  • the cells are resuspended in 100 mL flow buffer and incubated at 4°C for 1 hour with goat-anti-human antibody labelled with fluoroscein isothiocyanate (FITC). After further washing in flow buffer the samples are analyzed by flow cytometry on a Coulter EPICS 753 flow cytometer.
  • FITC fluoroscein isothiocyanate
  • Fluoroscein isothiocyanate (FITC) and propidium iodide (PI) are excited using the 488 nm emission line of an argon laser. The output is set at 500 mw in light regulation mode. Single cells are identified by 90 degree and forward angle light scatter. Analysis windows are applied to these parameters to separate single cells from aggregates and cell debris. Fluorescence from FITC and propidium are separated with a 550nm long pass dichroic filter and collected through a 530 nm band pass filter (for FITC), and a 635 nm band pass filter (for PI). Light scatter parameters are collected as
  • the antigen to which the antibody, ING-1, binds is prepared from LS174T or HT 29 cells (available from ATTC) by scraping confluent monolayers of cells from the walls of culture flasks with a cell scraper. The cells from many flasks are combined and a sample is taken and counted to estimate the total number of cells harvested. At all times the cells are kept on ice.
  • the cells are washed once in 25 mL ice-cold 50 mM sodium phosphate buffer, pH 7.4 supplemented with 150 mM sodium chloride (PBS), pelleted under the same conditions and transferred in 10 mL PBS to an ice-cold glass mortar.
  • the cells are homogenized at 4°C using a motor-driven pestle and then centrifuged at 3000 ⁇ g for 5 minutes. The antigen-rich supernatant is removed from the other cell debris and subjected to further
  • the pellet (antigen fraction) from this final step is suspended in 100 mL of PBS for every million cells harvested. Following an estimate of the protein
  • the antigen is stored at at -20°C until use.
  • Each well of a 96-well Costar microtiter plates is coated with antigen by adding 100 mL/well of cell lysate (10 mg/ml) prepared as above.
  • the microtiter plates are allowed to dry overnight in a 37°C incubator. After washing the plate five times with 0.05% Tween-20 (Sigma) they were blotted dry.
  • the wells of each plate were blocked by adding 125 mL/well of a 1% BSA (bovine serum albumin, Sigma A-7906) solution in PBS and incubated for 1 hour at room temperature. The plates were washed five times with 0.05% Tween-20. Samples (50 mL/well in duplicate) of ING-MAO conjugates and standard ING-1 antibody solutions were prepared at a range of
  • Biotinylated ING-1 1.0 mg/mL in 0.1% BSA
  • Biotinylated ING-1 1.0 mg/mL in 0.1% BSA
  • Tween-20 the plates are blotted dry and incubated at room temperature for one hour with dilute (1:2000 in 0.1% BSA) streptavidin-alkaline phosphatase (Tago;
  • a 30 cm ⁇ 7.5 mm TSK-G3000SW size-exclusion HPLC column (Supelco) fitted with a guard column of the same material is equilibrated with 12 column volumes of 10 mM sodium phosphate buffer pH 6.0 supplemented with 150 mM sodium chloride using a Waters 600E HPLC system with a flow rate of 1.0 mL per minute at 400-600 PSI.
  • a sample (25 mL) of BioRad gel filtration protein standards is injected on to the column. The retention time of each standard is monitored by a Waters 490 UV detector set at 280 nm.
  • microgram/mL are injected on to the column and their respective retention times are recorded on a linear response strip chart recorder which displays the elution of the samples as a function of time and which provides a linear correlation recording of sample elution profile as peaks on the chart. From the areas of the peaks and the retention time measured at peak maxima, the amount of aggregated material in the samples is calculated.
  • conjugation is assayed to ensure that the process of conjugation of MAO to another species as described above does not inhibit the enzymatic activity of MAO.
  • the enzymatic activity of MAO is used to monitor inhibitory effect of drugs such as clorgyline, pargyline and their analogs as described above in Examples 1 - 9 on unconjugated MAO and on antibody-MAO conjugates.
  • the enzymatic activity of MAO in antibody-MAO conjugates as described above is used as a measure of efficacy of new drugs designed to inhibit MAO activity. It is also used as a measurement of the effect of clorgyline-derived or pargyline-derived TMT delivery systems, D-(L 2 -Q) m as described above, such as H-CC-CH 2 -N(CH 3 )-CH 2 -C 6 H 6 -CH 2 -CO-HN-(CH 2 ) 5 -CO-HN-(Ala) 4 -(Lys-TMT) 2 -OH (SEQ ID NO:7) of
  • Example 9 as well as those systems that contain a chelated metal ion, D-(L 2 -Q-M) m , such as described above for 90 Y.
  • the enzymatic activity of MAO is used to as a measure of the relative amount of MAO in a solution.
  • 1 unit of MAO activity is defined as the amount of material needed to convert 1.0 micromole of kynurine to 4-hydroxyquinoline per minute at pH 7.2 and 25°C. The activity of the enzyme is measured
  • a sample of purified MAO enzyme (approximately 1.0 mg) or a sample of ING-1-MAO conjugate (Example 13) or a sample of MAO-to-TMT conjugate (Example 22) is added to a 1.0 mM solution of kynurine dissolved in a 50 mM phosphate buffer (pH 7.2) containing 0.2% Triton X-100 at 30°C to give a final volume of 1.0 mL.
  • the increase in absorbance at 314 nm is measured over a 10 minute period and the activity (units per mL) is calculated from the slope of the optical density vs time plot (Weyler, W. and Salach, J.I [1985]; J. Biological Chemistry, 260:13199 - 13207).
  • inhibitors e.g. Examples 9, 19 or 21
  • increasing concentrations of the inhibitor are added into the basic MAO enzyme assay, described above, without changing the volume or concentration of the reactants.
  • concentration of inhibitor required to reduce the amount of 4-hydroxyquinoline produced by 50% is calculated and compared with known concentrations of pargyline.

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Abstract

Dans une variante, cette invention concerne un immunoréactif de ciblage non radioactif comprenant le résidu d'un site actif protéique d'une enzyme monoamine-oxydase (MAO), un groupe de liaison, et le résidu d'un matériau immunoréactif ainsi qu'un agent d'administration radioactif comprenant un liant spécifique à cette fraction du récepteur de MAO, un groupe de liaison et un agent radioactif. Dans une autre variante, cette invention concerne un immunoréactif de ciblage non radioactif comprenant le résidu d'un liant spécifique à une fraction réceptrice du site actif protéique de MAO, un groupe de liaison, et le résidu d'un matériau immunoréactif ainsi qu'un agent d'administration radioactif comprenant le résidu d'une fraction réceptrice du site actif protéique de MAO, un groupe de liaison et un agent radioactif. Ces compositions comprennent des systèmes utiles dans la production d'une amplification de l'administration de l'agent radioactif sur des sites tumoraux dans l'imagerie thérapeutique et de diagnostic du cancer.
PCT/US1994/006424 1993-06-07 1994-06-07 Reactifs immunoreactifs utilisant la monoamine-oxydase WO1994029333A1 (fr)

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JP7502055A JPH09501147A (ja) 1993-06-07 1994-06-07 モノアミンオキシダーゼを使用する免疫反応性試薬

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WO2007088051A2 (fr) 2006-01-31 2007-08-09 Bayer Schering Pharma Aktiengesellschaft Modulation de l'activite mdl-1 pour le traitement de maladies inflammatoires
EP2072060A1 (fr) 2007-12-18 2009-06-24 Institut Curie Procédés et compositions pour la préparation et l'utilisation de conjugués de toxines
EP2514442A2 (fr) 2007-06-22 2012-10-24 mivenion GmbH Diagnostics d'imagerie par combinaison d'agents de contraste
WO2013151584A1 (fr) * 2011-10-31 2013-10-10 The Methodist Hospital Research Institute Composé comprenant un fragment de ciblage/recherche de mao pour le traitement de gliomes humains

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US4913891A (en) * 1986-04-17 1990-04-03 The United States Of America As Represented By The United States Department Of Energy Positron emitter labeled enzyme inhibitors
US4863713A (en) * 1986-06-23 1989-09-05 The Board Of Trustees Of Leland Stanford Jr. Univ. Method and system for administering therapeutic and diagnostic agents
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Cited By (15)

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Publication number Priority date Publication date Assignee Title
WO2003014145A3 (fr) * 2001-08-10 2003-12-04 Novartis Ag Peptides se liant avec les lesions atherosclereuses
WO2003014145A2 (fr) * 2001-08-10 2003-02-20 Novartis Ag Peptides se liant avec les lesions atherosclereuses
WO2007088051A2 (fr) 2006-01-31 2007-08-09 Bayer Schering Pharma Aktiengesellschaft Modulation de l'activite mdl-1 pour le traitement de maladies inflammatoires
EP2907526A1 (fr) 2007-06-22 2015-08-19 mivenion GmbH Diagnostics d'imagerie par combinaison d'agents de contraste
EP2514442A2 (fr) 2007-06-22 2012-10-24 mivenion GmbH Diagnostics d'imagerie par combinaison d'agents de contraste
EP2572735A1 (fr) 2007-06-22 2013-03-27 mivenion GmbH Diagnostics d'imagerie par combinaison d'agents de contraste
EP2647392A2 (fr) 2007-06-22 2013-10-09 mivenion GmbH Diagnostics d'imagerie par combinaison d'agents de contraste
EP2072060A1 (fr) 2007-12-18 2009-06-24 Institut Curie Procédés et compositions pour la préparation et l'utilisation de conjugués de toxines
CN104136026A (zh) * 2011-10-31 2014-11-05 卫理公会医院研究所 包含mao靶向/探寻器部分的人神经胶质瘤治疗用化合物
KR20140128943A (ko) * 2011-10-31 2014-11-06 더 메서디스트 하스피틀 리서치 인스티튜트 인간의 신경교종을 치료하기 위한 모노아마이드 옥시다아제(Monoamide oxidase,MAO) 타겟팅/시커(seeker) 부분(moiety)을 포함하는 조성물
WO2013151584A1 (fr) * 2011-10-31 2013-10-10 The Methodist Hospital Research Institute Composé comprenant un fragment de ciblage/recherche de mao pour le traitement de gliomes humains
CN104136026B (zh) * 2011-10-31 2018-03-02 卫理公会医院研究所 包含mao靶向/探寻器部分的人神经胶质瘤治疗用化合物
US10555936B2 (en) 2011-10-31 2020-02-11 The Methodist Hospital Chemotherapeutic compositions and methods for treating human gliomas
KR102110794B1 (ko) 2011-10-31 2020-05-15 더 메서디스트 하스피틀 리서치 인스티튜트 인간의 신경교종을 치료하기 위한 모노아마이드 옥시다아제(Monoamide oxidase,MAO) 타겟팅/시커(seeker) 부분(moiety)을 포함하는 조성물
EP3815685A3 (fr) * 2011-10-31 2021-10-13 The Methodist Hospital Research Institute Composé comprenant un fragment de ciblage/recherche de mao pour le traitement de gliomes humains

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EP0710244A1 (fr) 1996-05-08
JPH09501147A (ja) 1997-02-04
AU7244894A (en) 1995-01-03
CA2164518A1 (fr) 1994-12-22

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