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EP0538268A1 - Protection ciblee contre les cytotoxines - Google Patents

Protection ciblee contre les cytotoxines

Info

Publication number
EP0538268A1
EP0538268A1 EP91910021A EP91910021A EP0538268A1 EP 0538268 A1 EP0538268 A1 EP 0538268A1 EP 91910021 A EP91910021 A EP 91910021A EP 91910021 A EP91910021 A EP 91910021A EP 0538268 A1 EP0538268 A1 EP 0538268A1
Authority
EP
European Patent Office
Prior art keywords
antagonist
conjugate
cytotoxin
binding agent
tumor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP91910021A
Other languages
German (de)
English (en)
Other versions
EP0538268A4 (en
Inventor
George Y. Wu
Catherine H. Wu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Connecticut
Original Assignee
University of Connecticut
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Connecticut filed Critical University of Connecticut
Publication of EP0538268A1 publication Critical patent/EP0538268A1/fr
Publication of EP0538268A4 publication Critical patent/EP0538268A4/en
Withdrawn legal-status Critical Current

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/66—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 a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • A61K47/67—Enzyme prodrug therapy, e.g. gene directed enzyme drug therapy [GDEPT] or VDEPT
    • 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
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A61K47/6895—Rescue therapy; Agonist-antagonist; Antidotes; Targeted rescue or protection, e.g. by folic acid-folinic acid or conjugated to antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • Chemotherapeutic agents currently available for treatment of tumors can be unsuccessful because they lack tumor specificity.
  • the use of galactosamine has been explored in the treatment of primary liver cancer (hepatocellular carcinoma) because it is a highly selective liver toxin in vitro and in vivo.
  • the selectivity is due to elevated intrahepatic levels of two enzymes of the galactose metabolic pathway, galactokinase, and UDP-glucose:galactose-l-P- uridyltransferase (Bertoli, D. and Segal, S. (1966) J. Biol. Chem. 24.1:4023 and Cuatrecasas, P. and Segal, S. (1965) J. Biol. Chem.
  • This invention pertains to a method of selective ⁇ ly protecting normal cells from the cytotoxic effects of a chemotherapeutic cytotoxin directed against diseased cells such as tumor cells.
  • the chemotherapeutic cytotoxin is administered in conjunction with, or subsequent to, administration of an antagonist-conjugate.
  • the antagonist-conjugate comprises an antagonist of the cytotoxin coupled to a cell-specific binding agent which binds to a cellular surface component present on normal, but not on diseased cells.
  • the cellular surface component is typically a receptor which mediates internalization of bound ligands by endocytosis, such as the asialo- glycoprotein receptor of hepatocytes.
  • the cell-specific binding agent can be a natural or synthetic ligand (for example, a protein, polypeptide, glycoprotein, etc.) or it can be an antibody, or an analogue thereof, which specifically binds a cellular surface structure which then mediates internalization of the bound complex.
  • the antagonist can be complexed with the cell-specific binding agent via an antagonist-binding agent, such as a polycation.
  • the antagonist-conjugate is administered in vivo where it is selectively taken up by normal cells via the surface-structure-mediated endocytotic pathway.
  • the conjugate is administered in an amount sufficient to protect normal cells from the cytotoxic effects of the cytotoxin.
  • Diseased cells which lack the cellular surface component do not take up significant amounts of the antagonist-conjugate and are unprotected from the cytotoxin.
  • the method provides for a more effective use of higher doses of cytotoxins against tumor and against other diseases by alleviating or eliminating the toxicity to normal cells usually associated with such therapy.
  • Figure 1 shows the organ distribution of radiolabeled galactosamine antagonist-conjugate.
  • Figure 2 shows the effect of galactosamine antagonist-conjugate pretreatment on galactosamine toxicity.
  • This invention pertains to a method of selectively targeting an antagonist of a cytotoxin to normal mammalian cells to protect against the adverse effects of a therapeutic cytotoxin.
  • An antagonist- conjugate targetable to normal mammalian cells is used to selectively deliver an antagonist to the cells in vivo.
  • the antagonist-conjugate comprises an antagonist of the cytotoxin complexed with a cell-specific binding agent which binds a cellular surface component present on normal, but not diseased cells.
  • the antagonist-conjugate is selectively taken up by the normal mammalian cells and the antagonist is released into the cell in functional form to provide protection against the effects of the cytotoxin.
  • the cell-specific binding agent specifically binds a cellular surface component which mediates internalization by, for example, the process of endocytosis.
  • the surface component can be a protein, polypeptide, carbohydrate, lipid or combination thereof. It is typically a surface receptor which mediates endocytosis of a ligand.
  • the surface component can be a natural or synthetic ligand which binds the receptor.
  • the ligand can be a protein, polypeptide, glycoprotein or glycopeptide which has functional groups that are exposed sufficiently to be recognized by the cell surface structure. It can also be a component of a biological organism such as a virus, cells (e.g., mammalian, bacterial, protozoan) or artificial carriers such as liposomes.
  • the cell-specific binding agent can also be an antibody, or an analogue of an antibody such as a single chain antibody which binds the cellular surface component.
  • Ligands useful in forming the antagonist- conjugate will vary according to the particular cell to be targeted. For targeting hepatocytes, glyco- proteins having exposed terminal carbohydrate groups such as asialoglycoprotein (galactose-terminal) can be used, although other ligands such as polypeptide hormones may also be employed. Examples of asialoglycoproteins include asialoorosomucoid, asialofetuin and desialylated vesicular stomatitis virus.
  • Such ligands can be formed by chemical or enzymatic desialylation of glycoproteins that possess terminal sialic acid and penultimate galactose residues.
  • asialoglycoprotein ligands can be formed by coupling galactose terminal carbo ⁇ hydrates such as lactose or arabinogalactan to non-galactose bearing proteins by reductive amination.
  • other types of ligands can be used, such as mannose for macrophages, mannose-6-phosphate glycoproteins for fibroblasts, intrinsic factor-vitamin B12 for enterocytes and insulin for fat cells.
  • the cell-specifc binding agent can be a receptor or receptor-like molecule, such as an antibody which binds a ligand (e.g., antigen) on the cell surface.
  • a ligand e.g., antigen
  • the antagonist-conjugate can be made by binding the antagonist directly to the ligand or by binding it with the ligand through an antagonist-binding agent.
  • the antagonist-binding agent complexes the antagonist to be delivered. Complexation with the antagonist must be sufficiently stable in vivo to prevent significant uncoupling of the antagonist extracellu- larly prior to internalization by the cell. However, the complex is cleavable under appropriate conditions within the cell so that the antagonist is released in functional form.
  • the complex can be labile in the acidic and enzyme rich environment of lysosomes.
  • a noncovalent bond based on electrostatic attraction between the antagonist-binding agent and the antagonist provides extracellular stability and is releasable under intracellular conditions.
  • Preferred antagonist-binding agents are polycations which provide multiple binding sites for antagonists. Examples of polycations include polylysine, polyornithine or histones.
  • the antagonist-binding component can be covalently bonded to the ligand.
  • a preferred linkage is a peptide bond. This can be formed with a water soluble carbodiimide as described by Jung, G., e_fe al. (1981) Biochem. Biophys. Res. Commun. 101:599-606.
  • An alternative linkage is a disulfide bond.
  • the linkage reaction can be optimized for the particular antagonist-binding agent and ligand used to form the conjugate. Reaction conditions can be designed to maximize linkage formation but to minimize the formation of aggregates of the conjugate components. The optimal ratio of antagonist-binding agent to ligand can be determined empirically. Uncoupled components and aggregates can be separated from the conjugate by molecular sieve chromatography.
  • the conjugate can contain more than one antagonist molecule or one or more different antagonist molecules. Preferably, from about 10-15 antagonist molecules per conjugate. The number may vary, depending upon factors such as the effect on solubility or capillary permeability of the conjugate.
  • the cytotoxin and antagonist can be selected from any of those effective in treatment of the disease. For tumor therapy, various antitumor agents for which antagonists are available can be used.
  • antitumor cytotoxins and corresponding antagonists examples include methotrexate/folinic acid, acetaminophen/ N-acetyl cysteine, l,3-bis(2-chloroethyl)-l- nitrosourea (BCNU)/N-acetyl cysteine, glutathione or WR2721 and galactosamine/uridine monophosphate or orotic acid.
  • BCNU 2-chloroethyl)-l- nitrosourea
  • glutathione or WR2721 examples of antitumor cytotoxins and corresponding antagonists
  • combinations of two different cytotoxins and respective antagonists can be used to reduce selection of resistant cells.
  • the cytotoxin is specific for the diseased organ or tissue. This helps minimize toxicity of uninvolved organs.
  • galactosamine is a highly selective hepatotoxin and therefore, is preferred for treatment of primary liver cancer such as hepatocellular carcinoma.
  • the antagonist- conjugate is soluble in physiological fluids.
  • the antagonist-conjugate is generally administered parenterally in a physiologically acceptable vehicle in an amount sufficient to protect normal cells against the toxic effects of a cytotoxin.
  • the asialoglycoprotein, asialofetuin (AsF) was prepared by desialylation of bovine fetuin (GIBCO, Grand Island, New York), using neuraminidase (Sigma Chemical Co., St. Louis, Missouri) to expose terminal galactose residues by a modification of the method of Oka and Weigel (Oka, J.A. and eigel P.H. (1983) J. Biol. Chem. 258:10253). Analysis of residual protein- bound sialic acid by the method of Warren (Warren, L. (1959) J. Biol. Chem. 234:1971) determined the fetuin to be 94% desialylated.
  • AsF-PL-UMP (based on AsF content) .
  • AsF-PL-UMP (based on AsF content) .
  • a control rat was given 1 ⁇ g 125I_ASF-PL-UMP plus an excess, 10 mg, of unlabeled asialoorosomucoid (AsOR) to compete for hepatic asialoglycoprotein receptors.
  • the minimum amount of conjugate required to protect hepatocytes was determined by i.v. injection of varying doses of conjugate. Using the conjugate dose thus determined optimal (34 mg/kg), the ability of this antagonist conjugate to prevent galactosamine toxicity was evaluated relative to controls receiving i.v. injected pretreatments of equal volumes of sterile saline, or saline containing AsF or UMP in molar amounts equivalent to that provided by the conjugate. Blood was withdrawn from the retro-orbital plexus at 24, 42, 48 and 72 h after galactosamine injection. Hepatotoxicity was evaluated by measurement of serum alanine aminotransferase (ALT) levels (Sigma assay kit) according to the manufacturer. All assays were performed in duplicate and expressed as international units per liter (IU/1). Addition of conjugate to ALT standards as well as serum samples demonstrated that the conjugate had no effect on the ALT assays.
  • ALT serum alanine aminotransferase
  • AsF-PL-UMP conjugate was achieved by i.v. infusion of the AsOR-PL-UMP conjugate over a 4 h period (at the saturation rate of hepatic asialoglycoprotein receptors).
  • ALT median peak alanine aminotransferase
  • conjugate-pretreated animals Protection of conjugate-pretreated animals was increased by administering uridine (1.2 g/kg) 5 h after galactosamine (a point when galactosamine damage in saline controls should be irreversible) .
  • uridine 1.2 g/kg
  • galactosamine a point when galactosamine damage in saline controls should be irreversible
  • conjugate (+ uridine) treated animals experienced median peak ALT levels of 258, compared to 729 for saline (+ uridine) treated controls.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Molecular Biology (AREA)
  • Nanotechnology (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Cell Biology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medical Informatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Production Of Liquid Hydrocarbon Mixture For Refining Petroleum (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Measurement And Recording Of Electrical Phenomena And Electrical Characteristics Of The Living Body (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Butt Welding And Welding Of Specific Article (AREA)

Abstract

Procédé destiné à sauver, de manière ciblée, des cellules contre l'effet d'une cytotoxine chimiothérapique. Une cytotoxine dirigée contre des cellules malades est administrée en même temps qu'un conjugué d'antagoniste ayant pour cible des cellules normales. Le conjugué d'antagoniste est composé d'un antagoniste de la cytotoxine associée à un agent de liaison spécifique à certaines cellules qui se fixe spécifiquement sur un élément de surface cellulaire présent sur les cellules normales, mais non sur les cellules malades. L'élément de surface cellulaire est typiquement un récepteur de surface de la cellule qui participe à l'endocytose. Le conjugué d'antagoniste est spécifiquement absorbé par les cellules normales de manière à fournir une protection contre les effets toxiques de la cytotoxine. Les cellules malades qui sont exemptes du récepteur de surface n'absorbent pas des quantités efficaces d'antagoniste et sont donc sans protection.
EP19910910021 1990-05-11 1991-05-10 Targeted protection from cytotoxins Withdrawn EP0538268A4 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US52265390A 1990-05-11 1990-05-11
US522653 1990-05-11

Publications (2)

Publication Number Publication Date
EP0538268A1 true EP0538268A1 (fr) 1993-04-28
EP0538268A4 EP0538268A4 (en) 1993-05-12

Family

ID=24081756

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19910910021 Withdrawn EP0538268A4 (en) 1990-05-11 1991-05-10 Targeted protection from cytotoxins

Country Status (5)

Country Link
EP (1) EP0538268A4 (fr)
JP (1) JPH05508398A (fr)
AU (1) AU652939B2 (fr)
CA (1) CA2082507A1 (fr)
WO (1) WO1991017761A1 (fr)

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5490991A (en) * 1986-07-03 1996-02-13 Advanced Magnetics, Inc. Directed delivery of radioprotectants using a receptor specific carrier
WO1993018750A1 (fr) * 1992-03-23 1993-09-30 Kenneth Naoyuki Matsumura Procede permettant de reduire les effets secondaires d'un medicament
US5449720A (en) * 1993-05-24 1995-09-12 Biotech Australia Pty Limited Amplification of the VB12 uptake system using polymers
AU1679195A (en) * 1994-01-11 1995-08-01 Targetech, Incorporated, A Wholly Owned Subsidiary Of The Immune Response Corporation Hepatocyte-targeted drug conjugates
US5830686A (en) * 1994-01-13 1998-11-03 Calydon Tissue-specific enhancer active in prostate
US6057299A (en) * 1994-01-13 2000-05-02 Calydon, Inc. Tissue-specific enhancer active in prostate
EP1002103B1 (fr) 1997-08-04 2007-10-17 Cell Genesys, Inc. Sequence stimulatrice de la kallicreine glandulaire chez l'homme, vecteurs comprenant cette sequence et procedes d'utilisation de ces derniers
US6919076B1 (en) 1998-01-20 2005-07-19 Pericor Science, Inc. Conjugates of agents and transglutaminase substrate linking molecules
US6958148B1 (en) 1998-01-20 2005-10-25 Pericor Science, Inc. Linkage of agents to body tissue using microparticles and transglutaminase
GB0916749D0 (en) 2009-09-23 2009-11-04 Mologic Ltd Peptide cleaning agents

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2579497B2 (ja) * 1987-09-07 1997-02-05 オリエンタル酵母工業株式会社 肝臓疾患診断剤
CA2092319A1 (fr) * 1990-09-25 1992-03-26 George Y. Wu Expression prolongee de polynucleotides introduits dans une cellule

Also Published As

Publication number Publication date
JPH05508398A (ja) 1993-11-25
WO1991017761A1 (fr) 1991-11-28
AU7902691A (en) 1991-12-10
CA2082507A1 (fr) 1991-11-12
EP0538268A4 (en) 1993-05-12
AU652939B2 (en) 1994-09-15

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