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WO1991001757A1 - Couplage d'un anti-tumeur a un anticorps au moyen d'un agent antitumoral pre-active a la glutaraldehyde - Google Patents

Couplage d'un anti-tumeur a un anticorps au moyen d'un agent antitumoral pre-active a la glutaraldehyde Download PDF

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Publication number
WO1991001757A1
WO1991001757A1 PCT/CA1990/000251 CA9000251W WO9101757A1 WO 1991001757 A1 WO1991001757 A1 WO 1991001757A1 CA 9000251 W CA9000251 W CA 9000251W WO 9101757 A1 WO9101757 A1 WO 9101757A1
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WO
WIPO (PCT)
Prior art keywords
daunorubicin
glutaraldehyde
antibody
conjugate
compounds according
Prior art date
Application number
PCT/CA1990/000251
Other languages
English (en)
Inventor
Michel Page
Denis Thibeault
Original Assignee
Universite Laval
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 Universite Laval filed Critical Universite Laval
Publication of WO1991001757A1 publication Critical patent/WO1991001757A1/fr

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Classifications

    • 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
    • A61K47/6853—Carcino-embryonic antigens
    • 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
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A61K47/6809—Antibiotics, e.g. antitumor antibiotics anthracyclins, adriamycin, doxorubicin or daunomycin

Definitions

  • Chemotherapeutic agents currently used for antitumour therapy are selected for their toxicity towards rapidly proliferating cells. Most of them cause undesirable systemic effects such as cardiac or renal toxicity, marrow aplasia, alopecia, nausea and vomiting. During the last few years, many authors have tried to eliminate these side effects by increasing the availability of the drug to the tumour site. Enzymes, radioisotopes, DNA, toxins, various macromolecules, and antibodies against fibrin or against tumour-specific surface antigens were bound to drugs in an attempt to increase the selectivity of the chemotherapeutic agents, or to decrease their toxic effects on normal cells (Rubens R.D., Lancet, 1, 1974, pp.498-499; Gregoriadis G. et al., Res. Commun.Chem. Pathol. Pharm., 10, 1977, pp.351-362).
  • Dactinomycin, doxorubicin and daunorubicin are all given rapidly intravenously and all cause tissue necrosis if extravasation
  • SUBSTITUTE SHEET occurs.
  • doxorubicin and daunorubicin are given rapidly intravenously, there is rapid dispersement throughout tissues and plasma.
  • the ⁇ tl/2 is 30 min, with detectable plasma levels of doxorubicin up to 15 h.
  • Both doxorubicin and daunorubicin are extensively metabolized by the liver, yielding active and inactive metabolites.
  • Dactinomycin, doxorubicin and daunorubicin have limited antitumor activity.
  • Dactinomycin is effective in testicular carcinoma and sarcomas.
  • Daunorubicin is effective in treating acute leukemia.
  • doxorubicin is one of the most active antineoplastics ever identified. In fact it is used to treat acute leukemia, Hodgkin's disease and non-Hodgkin's lymphomas, small cell and non-small cell lung cancer, cancers? of the breast, ovaries, stomach, thyroid, and bladder, osteogenic and soft tissue sarcomas, and malignant melanoma.
  • the side effects include nausea, vomiting, alopecia, myelosuppression, and dose-dependent cardiotoxicity (>550 mg/m 2 ).
  • This method is not readily reproducible and give an unstable conjugate product.
  • this autopolymerized antitumor agent has the disadvantage of being insoluble in water and thus looses its specific activity against tumor cells.
  • This insoluble product can not be used intravenously for a systemic treatment since it is taken up by phagocytic cells such as monocytes, macrophage or cells. This product is not very stable and do not have a very long shelf life.
  • antitumor agents which overcome the drawback of the prior art.
  • the already reported techniques for coupling anthracycline d ⁇ ugs to antibody either cause polymerization or yield a product which is considerably less active than the free drug.
  • Using the method of the present invention a wide variety of monoclonal antibodies specific for various tumors are conjugated to other carriers that could be used for drug targeting.
  • - M is selected from the following group consisting of an hydrogen atom , a peptide residue and a protein residue linked to the carbon atom via the amino residue of £-lysine present therein, and
  • - R is an antitumor agent residue such as daunorubicin, doxorubicin, or epirubicin.
  • M When M is a protein, it can be an antibody which is used to target the antitumor agent to the malignant cells and thereby improving the conditions o •_f• such anti-cancer treatments.
  • the compounds of the present invention are easily produced and are devoid of significant polymerization since they are substantially pure.
  • the compounds of the present invention have the ability to
  • B TITUTE SHEET provide the full pharmacological activity of the antitumor agent without the disadvantage normally associated with said antitumor agent.
  • the improved coupling procedure of the present invention involved in the production of these compounds of formula I is readily reproducible and the resulting compounds are substiantially stable at 25 0 C.
  • the products of the present invention are prepared as follows:
  • An antitumor agent R is first reacted with an excess of glutaraldehyde, which gives an intermediate R-glutaraldehyde of formula (I), wherein M is an hydrogen atom.
  • This intermediate reaction product has a terminal aldehyde group.
  • Results- obtained with activated daunorubicin using this new procedure show that the pharmacological activity of the drug could be saved while limiting the undesirable polymerization of the antibody normally encountered with bivalent coupling agents.
  • This procedure is easy and reproducible and reagents are readily available commercially.
  • the activation of the drug can be accomplished in less than 2 hours and the activated drug remains active for a week at room temperature.
  • the activated drug can retain its activity for many months if stored in liquid nitrogen.
  • the cell lines were only used as targets to show that the conjugate can be directed to the desired sites.
  • cell lines there may be used : human embryonic intestine cells (CCL-6), human amnion cells (CCL-25), human osteosarcoma cells (CRL-1427), human ovarian carcinoma (CRL-1572), human hepatoma cells (HS-703-T), Mouse melanoma (CRL-6323) and LoVo human adenocarcinoma cells (CCL- 229).
  • CCL-6 human embryonic intestine cells
  • CCL-25 human amnion cells
  • CL-1427 human osteosarcoma cells
  • human ovarian carcinoma CL-1572
  • human hepatoma cells HS-703-T
  • Mouse melanoma CL-6323
  • LoVo human adenocarcinoma cells CL- 229).
  • the LoVo cells produce carcinoembryonic antigen in culture.
  • the human ovarian carcinoma cells produce alphafoetoprotein. All cell lines are routinely cultured in RPMI-1640® medium supplemented with 10% foetal bovine serum and 100 ug per ml of streptomycin and
  • the antibodies and peptides were only used in order to direct the conjugates to the desired cell lines used.
  • the antibodies were obtained through standard monoclonal antibody production procedures using the above-mentioned cell lines.
  • antibodies there may be used anti-carcinoembryonic monoclonal, anti-carcinoembryonic polyclonal ' antibody, anti-alphafetoprotein monoclonal, anti-alphafetoprotein polyclonal antibody, anti-embryonic pre-albumine monoclonal antibody.
  • a peptide there may be used: human transferin and lys- bombesin.
  • the conjugate solution is adjusted to 2% bovine serum albumin in 0,05 M ammonium acetate buffer.
  • the solution is then freeze dried and gamma radiated wtih 16 000 rads.
  • the dry conjugate is taken up in Dulbecco® phosphate buffer saline and added at various concentrations to culture medium.
  • cytotoxic activity of daunorubicin and antiCEA conjugate on the various cell lines is evaluated by inhibition of colony formation as described in Emond et al., Anthracyclines, 1983, Ed. G. Mathe, Masson Publish N.Y., U.S.A., 105. Briefly, 2,500 cells are added to 1 ml of RPMI 1640® medium supplemented with 10% foetal bovine serum in 24 well plates. Cells are allowed to attach for 24 hours, medium is removed and replaced by various test compounds diluted in growth medium. The tested drugs are incubated with the cells for four days in complete growth medium. Each assay is performed in quadruplicate.
  • the LD 50 lethal dose to kill 50% of the malignant cells
  • Table 1 The LD 50 (lethal dose to kill 50% of the malignant cells) of free and bound antitumor agent for the various cell lines is reported in Table 1 below.
  • the dosage required to inhibit 50% of the malignant cells for the compounds of the present invention is much lower than for the free drug itself.
  • the LD 50 % decrease is found between 14% to 68% depending on the drug or the cell line used. Compounds with such an ability to target antitumor agents without substantially lowering their pharmaceutical activity were long waited for.
  • the method of the present invention for coupling an anti ⁇ tumor agent to an antibody provides molar ratios of anti-tumor to antibody varying from 0.5:1 to 13:1 as desired.
  • the preferred molar ratios for coupling anti-tumor agent to antibody being 5:1 to 7:1.
  • the glutaraldehyde being used is a 25% aqueous solution of glutaraldehyde in a stoichiometric amount.
  • daunorubicin hydrochloride is dissolved in 4 ml of 0.05 M phosphate buffer at pH 7.5 and 60 ul of 25% glutaraldehyde is added (grade II, Sigma Chemicals, St. Louis, USA). The mixture is stirred for 15 minutes at room temperature and 1 ml of distilled water is added. The mixture is extracted twice with 5 ml of dichloromethane; the organic phases are pooled and treated four times with an equal volume of 5% NaHC03 solution containing 15% glycine. The organic phase is dried with anhydrous sodium sulphate, filtered, and the solvent is evaporated under a stream of nitrogen at room temperature. The dried product is taken in a minimum volume of dimethylsulfoxide

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Cell Biology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Oncology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Saccharide Compounds (AREA)

Abstract

L'invention se rapporte a des composés antitumoraux représentés par la formule (I) où M est chosi dans le groupe composé d'un atome d'hydrogène, d'un résidu peptidique et d'un résidu de protéine lié à l'atome de carbone par l'intermédiaire du résidu amino de ε-lysine présent qui peut être un anticorps utilisé pour cibler l'agent antitumoral sur les cellules malignes, et R peut représenter un agent antitumoral tel que la daunorubicin, doxorubicin ou un dérivé d'épirubicine.
PCT/CA1990/000251 1989-08-10 1990-08-09 Couplage d'un anti-tumeur a un anticorps au moyen d'un agent antitumoral pre-active a la glutaraldehyde WO1991001757A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US39192889A 1989-08-10 1989-08-10
US391,928 1989-08-10

Publications (1)

Publication Number Publication Date
WO1991001757A1 true WO1991001757A1 (fr) 1991-02-21

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PCT/CA1990/000251 WO1991001757A1 (fr) 1989-08-10 1990-08-09 Couplage d'un anti-tumeur a un anticorps au moyen d'un agent antitumoral pre-active a la glutaraldehyde

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CA (1) CA2021942C (fr)
WO (1) WO1991001757A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6310039B1 (en) 1996-09-11 2001-10-30 Felix Kratz Antineoplastic conjugates of transferrin, albumin and polyethylene glycol

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0074279A2 (fr) * 1981-09-08 1983-03-16 Suntory Limited Agents anti-tumeur sélectifs
GB2116979A (en) * 1982-02-25 1983-10-05 Ward Page Faulk Conjugates of proteins with anti- tumour agents
EP0114685A2 (fr) * 1983-01-21 1984-08-01 The Green Cross Corporation Complexes de fibronectine
EP0122132A2 (fr) * 1983-04-08 1984-10-17 Kureha Kagaku Kogyo Kabushiki Kaisha Substance anti-tumorale
US4650675A (en) * 1983-08-18 1987-03-17 The Children's Medical Center Corporation Oligonucleotide conjugates

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0074279A2 (fr) * 1981-09-08 1983-03-16 Suntory Limited Agents anti-tumeur sélectifs
GB2116979A (en) * 1982-02-25 1983-10-05 Ward Page Faulk Conjugates of proteins with anti- tumour agents
EP0114685A2 (fr) * 1983-01-21 1984-08-01 The Green Cross Corporation Complexes de fibronectine
EP0122132A2 (fr) * 1983-04-08 1984-10-17 Kureha Kagaku Kogyo Kabushiki Kaisha Substance anti-tumorale
US4650675A (en) * 1983-08-18 1987-03-17 The Children's Medical Center Corporation Oligonucleotide conjugates

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Anticancer Research, Volume 10, No. 2A, March-April 1990, M. PAGE et al.: "Coupling a Preactivated Daunorubicin Derivative to Antibody. A New Approach", pages 353-357 see the whole article *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6310039B1 (en) 1996-09-11 2001-10-30 Felix Kratz Antineoplastic conjugates of transferrin, albumin and polyethylene glycol
US6709679B2 (en) 1996-09-11 2004-03-23 Felix Kratz Antineoplastic conjugates of transferin, albumin and polyethylene glycol

Also Published As

Publication number Publication date
CA2021942A1 (fr) 1991-02-11
CA2021942C (fr) 2001-04-10

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