WO1994001137A1 - Procede d'aministration d'agents cytotoxiques et constituants de ceux-ci - Google Patents

Procede d'aministration d'agents cytotoxiques et constituants de ceux-ci Download PDF

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WO1994001137A1
WO1994001137A1 PCT/US1993/006324 US9306324W WO9401137A1 WO 1994001137 A1 WO1994001137 A1 WO 1994001137A1 US 9306324 W US9306324 W US 9306324W WO 9401137 A1 WO9401137 A1 WO 9401137A1
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acetyl
carboxylate
phenyl
amino
antibody
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PCT/US1993/006324
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English (en)
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Victor J. Chen
Louis N. Jungheim
Timothy A. Shepherd
Damon L. Meyer
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Hybritech Incorporated
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Priority to AU46634/93A priority Critical patent/AU4663493A/en
Publication of WO1994001137A1 publication Critical patent/WO1994001137A1/fr

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    • 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
    • A61K47/54—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 organic compound
    • A61K47/55—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 organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A61K47/552—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 organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being an antibiotic
    • 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
    • A61K47/54—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 organic compound
    • A61K47/555—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 organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells
    • 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
    • A61K47/6899—Antibody-Directed Enzyme Prodrug Therapy [ADEPT]

Definitions

  • cytotoxic drugs useful for treating neoplastic diseases have been developed.
  • the chief drawback of all of the useful drugs has been the indiscriminant toxicity of these drugs to all cells of the patient, thus limiting their dosage and consequently the effectiveness of the drugs.
  • the present invention whereby a latent form of the Cytotoxic Agent is activated at the site of the neoplastic disease, offers a marked advantage in diminishing or possibly eliminating unwanted side effects of the Agent.
  • higher concentrations of the drug at the tumor site may be brought about through
  • One aspect of the present invention is a method for the treatment of neoplastic diseases, which comprises:
  • FIG.1 depicts the biodistribution of the anti KS1/4 - - ⁇ -lactamase conjugate in nude mice as % injected dose in each organ divided by the organ weight.
  • FIG.2 depicts the same measurement of FIG.1 expressed as % injected dose in each organ.
  • FIG.3 demonstrates the inhibition of T380 tumor growth in nude mice by the combination anti-CEA - - ⁇ -lactamase conjugate and the prodrug compound of Example 11. Therapy was administered in three courses over three weeks, each course consisting of 35 ⁇ g conjugate followed after 72 hours by four daily doses of 1mg prodrug/kg of body weight.
  • FIG.4 depicts a similar inhibitory effect of the same conjugate prodrug combination as in FIG.3 on T380 tumors, except that the prodrug is administered in four daily doses of 0.25mg of prodrug/kg of body weight.
  • FIG.5 compares the inhibition of LS174T tumor growth in nude mice by the combination of either anti-KS1/4 antibody - - ⁇ -lactamase conjugate or anti-CEA - - ⁇ -lactamase conjugate and the prodrug of Example 11.
  • FIG.6 depicts the inhibitory effect of a
  • FIG.7 is a comparison of the effect of the dose of conjugate on the inhibition of T380 tumor growth by the combination of anti-CEA - - ⁇ -lactamase conjugate of this invention and the prodrug of Example 11.
  • One aspect of this invention is an Enzyme- Antibody conjugate of the formula Antibody-Enzyme I wherein :
  • Substrate - Cytotoxic Agent II which compound is described below, such that the Substrate is separated from the Cytotoxic Agent
  • the Antibody complexes with the target tissue.
  • the Antibody of the present invention complexes with one or more antigens of the target cells.
  • the target cells are a part of neoplastic tissues, whether benign or malignant. Examples of such target cells include
  • psoriasis squamous carcinoma cells, adenocarcinoma cells, small cell carcinoma cells, glyoma cells, melonoma cells, renal cell carcinoma cells, transitional cell carcinoma cells, sarcoma cells, cells of supporting tumor
  • lymphoid tumors such as leukemias and lymphomas.
  • the Antibody may be chosen from any class or subclass of immunoglobulin including IgG, IgA, IgM, IgE, and IgD.
  • the species of origin is not critical so long as the Antibody complexes with target cells.
  • a newer type of Antibody is the chimeric antibody, which is prepared in the laboratory by
  • chimeric antibodies of which one portion is derived from one species, and another portion is derived from a different species, may be
  • the origin and nature of the Antibody is not otherwise critical, so long as it targets the cell to be treated and is not, in itself, toxic to the patient. Those of ordinary skill can readily prepare enzyme conjugates with a candidate Antibody and evaluate them. How such antibodies are chosen will be discussed in detail below.
  • Antibody In the practice of this invention, fragments of antibodies, such as F(ab') 2 , F(ab') and single domain antibodies (V H regions, or dAbs) and especially
  • F(ab') fragments which recognize an antigen associated with the cell to be treated, may be just as useful as are intact antibodies.
  • dAbs are described by Ward E., et al., Nature. 341, p.544, 1989; Orlandi R., et al., Proc. Natl. Acad. Sci., (USA) 86, p.3833, 1989.
  • the necessary hybridomas for the production of such antibodies are obtainable through the ATCC or the Northern Regional Research Laboratories (NRRL) of the U.S. Department of Agriculture, Peroia, Illinois, U.S.A. and other cell line collections.
  • ATCC Northern Regional Research Laboratories
  • NRRL Northern Regional Research Laboratories
  • a number of presently known antibodies are particularly interesting for use in the present invention.
  • One preferred specific antibody is L/1C, produced by ATCC hybridoma HB9682.
  • Another preferred is produced by ATCC hybridoma HB9620, which antibody (designated CEM231.6.7) complexes with a convenient carcinoembryonic antigen expressed by several types of tumor cells.
  • CEM231.6.7 has been described in U.S. Patent Application Nos. 07/165,856 and 07/272,577, filed March 3, 1988, and November 17, 1988, respectively.
  • the Antibody is also discussed in Beidler C.B., et al., J.
  • T-101 antibody An Antibody which reacts with the MAT65 T-cell marker is the T-101 antibody.
  • the T101 antibody is produced by ATCC hybridoma #CRL8023 and is described in U.S. Patent No. 4,675,386.
  • KS1/4 Another interesting antibody is KS1/4, first disclosed by Varki et al., Cancer Research, 44, pp.681-686,
  • plasmids which comprise the coding sequences of the different regions of monoclonal antibody KS1/4 are now on deposit and can be obtained from the Northern Regional Research Laboratory.
  • the plasmids can be used by those of ordinary skill to produce chimeric antibodies by recombinant means, which antibodies bind to a cell surface antigen found in high density on
  • Plasmid CH2A5 the coding sequence of the heavy chain variable region, the coding sequence of the signal peptide associated with the heavy chain, and a sequence encoding the heavy chain constant region of human IgG1; isolated from E. coli K12 MM294/CH2A5, NRRL B-18360.
  • Plasmid CH2A5IG2 the coding sequence of the heavy chain variable region, the coding sequence of the signal peptide associated with the heavy chain, and a sequence which encodes the heavy chain constant region of human IgG2; isolated from E.coli K12 DH5/CH2A5IG2, NRRL B-18361.
  • Plasmid CH2A5IG4 the coding sequence of the heavy chain variable region, the coding sequence of the signal peptide associated with the heavy chain, and a sequence encoding the heavy chain constant region of human IgG4; isolated from E.coli K12 DH5/CH2AIG4, NRRL B-18363.
  • Antibody 5E9C11 produced by an ATCC hybridoma, HB21, recognizes transferrin receptor, which is expressed by many tumors.
  • An Antibody called CC49 available from the National Cancer Institute (NCI), recognizes the TAG-72 antigen expressed by both breast and colon carcinoma
  • Antibody ZCE025 is suitable for use in this invention.
  • the antibody was first described by Jean-Pierre Mach and his group (e.g., Mach J.P., et al., Int. J.
  • the Enzyme for the present conjugate is one that recognizes a Substrate, and which will also recognize that Substrate when it is bound to the present Cytotoxic Agent. Furthermore, the Enzyme must retain its activity once conjugated to the present Antibody.
  • Enzymes that can be used in this invention catalyze one of two broad classes of reactions.
  • the first class of reaction is one in which the Substrate - Cytotoxic Agent compound is cleaved into its two component parts.
  • the activity of the Enzyme toward the Substrate must be substantially unimpaired by the presence of the Cytotoxic Agent.
  • a preferred Enzyme for this type of reaction is one that is practically insensitive to the presence of the Cytotoxic Agent, regardless of the structure of the Agent. Such an Enzyme would allow that particular conjugate to be used for a variety Substrate - Cytotoxic Agent
  • Examples of such (insensitive) Enzymes include ⁇ -galactosidase, the various isoenzymes of alkaline phosphatase, certain carboxypeptidases (such as that of Pseudomonas sp.), D-aminopeptidase (Asano Y., et al., J. Biol. Chem., 264, pp.14233-14239, 1989; Asano Y., et al. Biochem. Biophys. Res. Comm., 162, pp.470-474, 1989; Asano Y., et al., Biochem., 31, pp.2316-2328, 1992),
  • Enzymes recognize galactose residues, phosphate residues, certain simple amino acids, 1-pyroglutamate, d-amino acid amides and cephalosporin compounds,
  • penicillins, cephalosporins, cephalosporin sulfoxides, 1-oxadethiacephalosporins, and 1-carbadethiacephalosporins that are its Substrates are composed of a number of functional groups that are untouched by the Enzyme.
  • the beta-lactamase simply reacts with the ⁇ -lactam ring of these substrate compounds.
  • the 3' - substituent of the Substrate will often cleave from the rest of the cephalosporin nucleus.
  • the second class of reactions catalyzed by the present Enzymes are anabolic ones, in other words,
  • the Enzyme converts the precursor into a Cytotoxic Agent.
  • the precursor would serve as first the Substrate and then the Cytotoxic Agent.
  • Examples of such combination Substrate-Enzyme compounds are the trichothecenes, saxitonin, or amanitin.
  • the purpose of the Enzyme is the same - to generate the Cytotoxic Agent in vivo (and in high
  • the Enzyme is isolated from, L-pyroglutamate aminopeptidase, ⁇ -galactosidase, D-amino peptidase, the various isoenzymes of alkaline phosphatase, various carboxypeptidases such as Pseudomonas sp.
  • Enzymes that are most useful are those that can be inhibited specifically by substances that are both not normally found in the body and that do not inhibit other enzymes found in the body.
  • Especially useful examples of this class of Enzyme are the ⁇ -lactamases, which can be inhibited by various ⁇ -lactamase resistant penicillins and cephalosporins, such as cloxacillin.
  • penicillins and cephalosporins often have the advantage that they are already approved for use in humans.
  • they would be expected to rapidly penetrate organs where the prodrug/Antibody-Conjugate drug is causing undesired effects.
  • reagents may be used in combination.
  • reagents that crosslink between two amino groups can be used, such as DMA or DSS (Pierce Chemical Company) .
  • Certain enzymes and antibodies may have amino, sulfhydryl, or hydroxyl groups in their active sites or binding sites, respectively.
  • these reactive functional groups can first be masked by protecting groups such as citraconic anhydride, DTNB, and DNFB (according to procedures referenced in the Pierce Chemical Company Catalog and Schmuel and Shaltiel, Biochem. and Biophys Res. Comm., 29, pp.178-183, 1967) before they are conjugated, with for example, the reagents listed above.
  • protecting groups such as citraconic anhydride, DTNB, and DNFB (according to procedures referenced in the Pierce Chemical Company Catalog and Schmuel and Shaltiel, Biochem. and Biophys Res. Comm., 29, pp.178-183, 1967) before they are conjugated, with for example, the reagents listed above.
  • Substrate not be found in humans.
  • One skilled in the art could readily see that if the Enzyme or the Substrate is found in the human body, the chance for the Cytotoxic Agent to be released in an area other than the location of the target tissue is increased.
  • the preferred conjugates are ones that are as homogenous as possible given the conjugation technology. Unlike whole antibody, F(ab') fragments may be derivitized
  • conjugate metabolized in degratory organs such as the liver and the spleen, an event that could prevent sufficient conjugate from accumulating at the target tissue. Yet, on the other hand, it is desirable that the conjugate be rapidly cleared from the bloodstream after the conjugate has had the opportunity to localize to the target tissue. Both of these pharmacokinetic properties reduce the chance that Cytotoxic Agent will be generated at a site other than the target tissue. These pharmacokinetic properties are facilitated by conjugate with a low molecular weight, a property supplied by Antibody fragments, and particularly F(ab') fragments. Along the same line of thought, Enzymes with a low molecular weight (e.g., 50,000 or less) are preferred for the same reason as the Antibody fragments.
  • a low molecular weight e.g., 50,000 or less
  • a further aspect of this invention are compounds of the Formula II:
  • the Substrate is the Substrate for an Enzyme, or active fragment thereof, wherein reaction of said Enzyme with the Substrate - Cytotoxic Drug causes the chemical separation of the Substrate from the Cytotoxic Agent; and the Substrate and the Cytotoxic Agent are bonded together through an ether, thioether, ester, amide, amino,
  • the Substrate may be a precursor for a Cytotoxic Agent.
  • Cytotoxic Agent means compounds that are useful in the treatment of neoplasms, whether benign or malignant.
  • drugs include, in general, alkylating agents, antiproliferative agents, tubulin-binding agents, cytotoxins in general, and the like.
  • Preferred classes of such compounds are the nitrogen mustard agents, anti- folates, nucleoside analogs, the vinca alkaloids, the anthracyclines, the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridine family of drugs, the podophyophyllotoxins, the sulfonylureas (as described in European Patent Publication No. 222,475, published May 20, 1987), and low-molecular-weight toxins such as the
  • trichothecenes and the colchicines include, for example, doxorubicin, daunorubicin, aminopterin, methotrexate, taxol,
  • methopterin dichloromethotrexate , mitomycin C ,
  • porfiromycin 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, podophyllotoxin, etoposide, melphalan, vinblastine, vincristine, desacetylvinblastine hydrazide, leurosidine, vindesine, leurosine, trichothecene,
  • Substrate is a substrate for a ⁇ -lactamase, L-pyroglutamate aminopeptidase, ⁇ -galactosidase, or D-aminopeptidase enzymes;
  • the Substrate for a ⁇ -lactamase enzyme could be a penicillin, a penem, a carbapenem, cephalosporin, a cephalosporin sulfoxide, 1-carbadethiacephalosporin, or 1-oxadethiacephlosporin;
  • a cephalosporin of the formula wherein zz is 0 or 1, R 1 is an acyl group derived from a C 1 to C 30 alkyl group, R 2 is hydrogen, an organic or inorganic cation, a carboxy-protecting group, or a non-toxic, metabolically-labile ester-forming group;
  • D-amino acid especially D-alanine C.
  • D-amino acid especially D-alanine C.
  • each of R 5 , R 6 , and R 7 is, independently, hydrogen, halo, hydroxy, protected hydroxy, nitro, amino, protected amino, an amine salt, cyano, trifluoromethyl, aminomethyl, protected aminomethyl, N- (methyl- or ethyl-sulfonyl)amion, C 1 to C 6 alkyl or C 1 to C 4 alkoxy, R 4 is hydroxy, protected hydroxy, formyloxy, amion, protected amino, an amine salt, carboxy, a carboxylate salt, protected carboxy, phenyl carboxylate, (5-indanyl)-carboxylate, sulfonic acid, a sulfonate salt, azido, halo or C 1 to C 6 alkyl; R 6 is C 1 to C 4 , alkoxy; Z is oxygen or sulfur; n is 0, 1, 2, or 3; and m is 0 or 1;
  • R 9 is a heterocyclic ring
  • R 10 is hydroxy
  • n 0, 1, 2, or 3; and Z is oxygen or sulfur.
  • R 11 is hydrogen or hydroxy
  • R 12 is amino or hydroxy
  • R 13 is hydrogen or methyl
  • R 14 is hydrogen, fluoro, chloro, bromo, iodo
  • R 15 is hydroxy or a moiety which completes a salt of the carboxylic acid; wherein R 16 is hydrogen or methyl;
  • R 17 is amino, C 1 -C 3 alkylamino, di-(C 1 -C 3 -alkyl) amino, or C 4 -C 6 polymethylene amino;
  • R 18 moieties is a bond and the others are hydrogen
  • R 19 is hydrogen or methyl
  • R 20 is methyl or thienyl
  • R 21 is H, CH 3 or CHO; when R 23 and R 24 are taken singly, R 24 is H, and one of R 22 and R 23 is ethyl and the other is H or OH; when R 23 and R 24 are taken together with the carbons to which they are attached, they form an oxirane ring in which case R 22 is ethyl; R 25 is hydrogen, (C 1 -C 3 alkyl)-CO-, or chloro-substituted (C 1 -C 3 alkyl)-CO;
  • p is 0 or 1;
  • R 28 is a bond, -(C 2 -C 4 alkyl)-X, or a group that requires that p is 1 and which is in turn bonded to a carbonyloxy group;
  • X is -O-, -S-, or -NH-;
  • R 27 is a base of one of the formulae:
  • R 28 is hydrogen, methyl, bromo, fluoro, chloro, or iodo
  • R 29 is -OR18 or -NHR 18 ;
  • R 30 is hydrogen, bromo, chloro, or iodo; wherein A is -O-, -NCH 3 -, -CH 2 -, -CH 2 CH 2 -, or -CH 2 O-;
  • D is -CH 2 - or -O-;
  • R 31 is hydrogen or halo
  • R 32 is halo or trifluoromethyl
  • compounds of Formula III represent the anthrocyclines of compounds;
  • Formula IV represents the methotrexate group of compounds;
  • Formula V represents the mitomycins;
  • Formula VI represents the bleomycins;
  • Formula VII represents melphalan;
  • Formula VIII represents 6-mercaptopurine;
  • Formula IX represents cytosine arabinoside;
  • Formula X represents the
  • Formula XI represents the vinca drugs
  • Formula XII represents the difluoronucleosides
  • Formula XIII and Formula XIV represents the sulfunoylurea
  • R 1 is acetyl, propionyl, isopropionyl, pivaloyl, butanoyl, methoxymethylacetyl, phenylacetyl, phenoxyacetyl, 2-(aminomethyl)phenylacetyl, 2-phenyl-2-hydroxyacetyl, 2-phenyl-2-(sodium sulfonato)-acetyl, 2-phenyl-2-2-carboxyacetyl, 2-(4-hydroxyphenyl)-2-carboxyacetyl, 2-phenyl-2-aminoacetyl, 2-(4-hydroxyphenyl)-2-aminoacetal, 2- (3-(N-(methylsulfonylamino))phenyl)-2-aminoacetyl, 2-phenyl-2-(5-indanyl carboxylate)acetyl, 2-phenyl-2-(phenyl carboxylate)acet
  • Cytotoxic Agent is methotrexate, 5-fluorouracil,
  • a compound of embodiment H, wherein the Cytotoxic Agent is desacetylvinblastine aminoethanethiol
  • a compound of embodiment Q, wherein the Cytotoxic Agent is desacetylvinblastine aminoethanethiol
  • the compounds of Formula II may also exist as solvates and hydrates.
  • these compounds may crystallize with, for example, waters of hydration, or one, or a number of, or any fraction thereof of molecules of the mother liquor solvent.
  • the solvates and hydrates are included within the scope of this invention.
  • the compounds of Formula II can exist as pharmaceutically-acceptable salts.
  • the compounds include salts of both the acidic functions, such as carboxy and sulfonate groups, and the basic functional groups, such as amino groups.
  • Such salts include the organic and inorganic cations discussed below, plus the salts formed from acid-base reactions of basic groups with acids such as hydrochloric, sulfuric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, palmitic, cholic, pamoic, mucic, D-glutamic, d-camphoric, glutaric, phthalic,
  • acyl group derived from a C 1 to C 30 carboxylic acid respresented by R 1 refers to the acyl moieties which have been bonded to the C-6 amino group of penicillins, the C-7 amino group of cephalosporins, 1-sulfoxide cephalosporin, 1-oxadethiacephalosporins or 1-carbacephalosporins and the C-3 amino of monocyclic ⁇ -lactams (such as the azthreonam series).
  • the "acyl group derived from a C 1 to C 30 refers to the acyl moieties which have been bonded to the C-6 amino group of penicillins, the C-7 amino group of cephalosporins, 1-sulfoxide cephalosporin, 1-oxadethiacephalosporins or 1-carbacephalosporins and the C-3 amino of monocyclic ⁇ -lactams (such as the azthreonam series).
  • carboxylic acid can be optionally interrupted by
  • acyl groups can be found in references such as "Cephalosporins and Penicillins,
  • acyl groups at R 1 can also be found in Yoshioka M., et al., U.S. Patent No. 4,478,997, issued
  • Patent No. 4,472,300 issued September 18, 1984,
  • organic or inorganic cation refers to counter-ions for the carboxylate anion of a carboxylate salt.
  • the counter-ions are chosen from the alkali and alkaline earth metals, such as lithium, sodium, potassium, barium and calcium; ammonium; and organic cations such as dibenzylammonium, benzylammonium, 2-hydroxyethylammonium, bis(2-hydroxyethyl)ammonium, phenylethylbenzylammonium, dibenzylethylenediammonium and like cations.
  • cations encompassed by the above term include the protonated form of procaine, quinine and N-methylglucosamine, and the protonated forms of basic amino acids such as glycine, ornithine, histidine, phenylglycine, lysine and arginine. Furthermore, any zwitterionic form of the instant compounds formed by a carboxylic acid and an amino group is referred to by this term.
  • carboxy-protecting group refers to one of the ester derivatives of the carboxylic acid group commonly employed to block or protect the carboxylic acid group while reactions are carried out on other functional groups on the compound.
  • carboxylic acid protecting groups include 2-nitrobenzyl, 4-nitrobenzyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl, 2,4-dimethoxybenzyl, 2,4,6-trimethoxybenzyl, 2,4,6-trimethylbenzyl, pentamethylbenzyl, 3,4-methylenedioxybenzyl, benzhydryl, 4,4'-dimethoxybenzhydryl, 2,2',4,4'-tetramethoxybenzhydryl, t-butyl, t-amyl, trityl, 4-methoxytrityl, 4,4'-dimethoxytrityl, 4,4',4''-trimethoxytrityl, 2-phenylprop-2-
  • carboxyl-protecting group employed is not critical so long as the derivatized carboxylic acid is stable to the condition of subsequent reaction (s) on other positions of the cephalosporin molecule and can be removed at the appropriate point without disrupting the remainder of the molecule.
  • a preferred carboxylic acid protecting group is the allyl group.
  • Similar carboxy-protecting groups used in the cephalosporin, penicillin and peptide arts can also be used to protect carboxy group substituents of the instant cephalosporin substrate. Further examples of these groups are found in Haslam E., "Protective Groups in Organic Chemistry", McOmie H.G.W., Ed., Plenum Press,
  • protected carboxy which refers to a carboxy group substituted with one of the above carboxy-protecting groups.
  • non-toxic, metabollically-labile, ester-forming group refers to those biologically active ester forms which induce increased blood levels and prolong the efficacy of the corresponding non-esterified forms of the compounds.
  • ester groups include the lower
  • alkoxymethyl groups for example, methoxymethyl
  • ethoxymethyl iso-propoxymethyl and the like;
  • ⁇ -(C 1 to C 4 ) alkoxyethyl groups for example, methoxyethyl
  • the 2-oxo-1,3-dioxolen-4-ylmethyl groups such as 5-methyl- 2-oxo-1,3-dioxolen-4-ylmethyl, 5-phenyl-2-oxo-1,3-dioxolen-4-ylmethyl, and the like
  • the C 1 to C 3 alkylthiomethyl groups for example methylthiomethyl, ethylthiomethyl, iso-propylthiomethyl, and the like
  • the acyloxymethyl groups for example, pivaloyloxymethyl, ⁇ -acetoxymethyl, and the like
  • the ethoxycarbonyl-1-methyl group the ⁇ -acyloxy- ⁇ -substituted methyl groups, for example ⁇ -acetoxyethyl
  • C 1 to C 6 alkyl denotes such radicals as methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, amyl, tert-amyl, hexyl and the like.
  • C 1 to C 6 substituted alkyl denotes the above C 1 to C 6 alkyl groups that are substituted by one or two halogen, hydroxy, protected hydroxy, amino,
  • substituted alkyl groups may be substituted once or twice with the same or with different substituents.
  • blocking groups such as the tert-butoxycarbonyl group (t-BOC), the benzyloxycarbonyl group, the 4-methoxybenzyloxycarbonyl group, the 2,2,2-trichloroethoxycarbonyl group, the trimethylsilyl group, and like amino protecting groups.
  • t-BOC tert-butoxycarbonyl group
  • benzyloxycarbonyl group the 4-methoxybenzyloxycarbonyl group
  • 2,2,2-trichloroethoxycarbonyl group the trimethylsilyl group
  • amino protecting groups such as the tert-butoxycarbonyl group (t-BOC), the benzyloxycarbonyl group, the 4-methoxybenzyloxycarbonyl group, the 2,2,2-trichloroethoxycarbonyl group, the trimethylsilyl group, and like amino protecting groups.
  • the nature of such amino protecting groups is not critical so long as the protected amino functionality is stable under the reaction conditions described hereinafter.
  • protected hydroxy has reference to any group stable under the reaction conditions of the subsequent step in the synthesis of the cephalosporin substrate compounds, but readily cleavable thereafter.
  • Such groups include the formyloxy group, the chloroacetoxy group, the benzhydryloxy group, the trityloxy group, the trimethylsilyl group, and the like.
  • hydroxy, amino, and carboxy protecting groups are not exhaustively defined.
  • the function of such groups is to protect the reactive functional groups during the preparation of the desired products and then to be removed without disrupting the remainder of the molecule.
  • Many such protecting groups are well known in the art and the use of other groups equally applicable to the process and compounds of the present invention, such as those described in McOmie J.F.W.,
  • heterocyclic ring when used in conjunction with the term “acyl group derived from a C 1 to C 30 carboxylic acid”, refers to the rings in Durckheimer W., et al., U.S. Patent No. 4,278,793. Particular examples are unsubstituted or substituted rings such as tetrazolyl, oxazolyl, isoxazolyl, thienyl, furyl, thiazolyl,
  • a third aspect of this invention is a method of treatment for the treatment of neoplastic diseases which comprises:
  • the term "therapeutically-effective", as it relates to the Substrate - Cytotoxic Agent will be an amount sufficient to cause some target neoplastic cell death, and may further refer to the use of this drug to keep a neoplastic disease in remission, or, in general, in an amount sufficient for prophylaxis.
  • the term "therapeutically effective" means at least the same molar equivalent amount of the Substrate - Cytotoxic Agent be given for the
  • Cytotoxic Agent alone for the particular purpose at hand.
  • Larger amounts of the Substrate -Cytotoxic Agent may be given in the instant invention because the toxicity of the Cytoxic Agent is initially masked by the attached Substrate but subsequently unmasked upon reaction with the Antibody-Enzyme conjugate at its point of delivery, thus relieving the patient of the side effects normally associated with that amount of Cytotoxic Agent alone.
  • a therapeutically effective dose of the Antibody-Enzyme conjugate is the amount that delivers between 1 through 500, and preferrably, 1 through 50, milligrams of Antibody (or fragments thereof) to the affected host.
  • Examples of the preferred embodiments of the present method include:
  • a method wherein the Substrate is a substrate for a ⁇ -lactamase, pyroglutamate aminopeptidase, ⁇ -galactosidase, or D-aminopeptidase, and the Antibody-Enzyme conjugate compound wherein the Enzyme is a ⁇ -lactamase, pyroglutamate aminopeptidase, ⁇ -galactosidase, or D-aminopeptidase;
  • Substrate is a cephalosporin of the formula:
  • R 1 is an acyl group derived from a C 1 to C 30 alkyl group
  • R 2 is hydrogen, an organic or inorganic cation, a carboxy-protecting group, or a non-toxic, metabolically-labile ester-forming group; as those terms are defined regarding preferred embodiment B of the Substrate-Cytotoxic Agent aspect of this invention
  • Antibody complexes with carcinoembryonic antigen
  • Cytotoxic Agent is a compound of the formula listed in the preferred embodiment E of the compounds of Formula II;
  • Cytotoxic Agent is methotrexate, 5-fluorouracil,
  • Antibody is designated CEM 231.6.7;
  • Cytotoxic Agent is methotrexate
  • Cytotoxic Agent is 5-fluorouracil
  • Cytotoxic Agent is desacetylvinblastine aminoethanethiol
  • HH A method of treatment of embodiment GG, wherein doxorubicin is bonded through nitrogen of the aminosaccharide moiety to the cephalosporin Substrate.
  • the Substrate - Cytotoxic Agent compounds of the instant invention can be synthesized by a variety of methods that are known. Thus, a nucleophilic group of the
  • Substrate could be used to displace a leaving group at an electrophilic center of the Cytotoxic Agent under S n 1 or
  • Substrate as is effected by the Enzyme, and that the derivatization of the Substrate with Cytotoxic Agent does not significantly decrease the reactivity of the Substrate with Enzyme. It is preferred that the Substrate -
  • Cytotoxic Agent compound mask the toxicity of the Cytotoxic
  • cytotoxicity be no more than 70% of the cytotoxicity of the unsubstituted Cytotoxic Agent.
  • Enzyme have a reasonable catalytic rate.
  • the meager concentrations of Substrate in the form of the Substrate - Cytotoxic Agent compounds
  • Enzyme will encounter, it is especially important that the enzyme-substrate combination have a high ratio of k cat /K m .
  • an Enzyme that has a k cat /K m ratio of at least 1 x 10° M -1 S -1 . This same ratio is desirable in the Enzyme once it is conjugated to the Antibody.
  • the Antibody it is preferred for the Antibody to have high affinity for the antigen on the target neoplastic cell, preferably about 1 ⁇ 10 6 (liter/mole) or above. It is also desirable that the conjugated Antibody retain about 80% of its unconjugated activity, thus desirably exhibiting a 60% immunoreactivity once conjugated.
  • Conjugating an Enzyme to an Antibody is accomplished by methods standard in the protein chemistry art. Conjugates could also be formed by Offord and Rose methodology described above, or by gene fusion to produce fusion protein (European Patent Application No. 392 745, published October 17, 1990; Neuberger M.S., et al., Nature. 312, p.604, 1984); Seehaus T., et al., Gene, 114, p.1, 1992). Heterobifunctional and homobifunctional reagents can be used.
  • the present conjugates can be formed using MBS, Sulfo-MBS, SMCC, Sulfo-SMCC, SIAB, SPDP, Sulfo-SMPB, DIDS, DFDNB, BMH, (which are commercially available) and the like. Purification of the resulting conjugate may be accomplished by appropriate, well-known chromatographic methods such as gel permeation or ion exchange. Thus, nothing is novel in the instant invention in the way the Enzyme and the Antibody are conjugated. It is preferred that the stoichiometry and other conditions of the
  • conjugation reaction and purification
  • conjugation reaction and purification
  • conjugation reaction be adjusted such that a 1:1 Antibobdy : Enzyme conjugate results, although conjugates with disproportionately larger amounts of either moiety in the conjugate are encompassed in the present invention.
  • HPLC high pressure liquid chromatography
  • the activity of the Enzyme of the conjugate is again measured, this time employing Substrate bound to a molecule that imparts to the Substrate (or the molecule itself) a measurable spectrophotometric change upon
  • the next parameter to be measured is the immunoreactivity of the conjugate in vitro. This measurement is usually made by first
  • the conjugate (with, e.g., 125 I) coating a solid surface (e.g., small plastic beads or microtiter plate wells) with the target antigen.
  • This measurement will detect if the Antibody portion of the conjugate has been unacceptably altered in the conjugation step.
  • the first step toward seeing if the components of the instant method function in the presence of the target antigen comes when the k cat and the K M of the conjugate are measured with the Substrate - Cytotoxic Agent. (That free Cytotoxic Agent is released by the Enzyme can be shown chromatographically.)
  • the conjugate is then studied to assess if it will localize to tumors in vivo.
  • target-antigen expressing cells are suspended and incubated either with conjugate or, for a control, Enzyme only.
  • the cells so treated are resuspended in another amount of buffer, then incubated with a
  • Substrate that is coupled with a compound such that the pair absorb at different wavelengths in the visible
  • the serum kinetics of the conjugate in a test animal such as mice should be measured to determine if and when the Substrate - Cytotoxic Agent can be administered to the affected host after administration of the conjugate. It is desirable that the conjugate demonstrate a short serum half-life, so that the Substrate - Cytotoxic Agent molecule can be administered within a reasonable time (e.g., 72 hours) after
  • mice such as the mice.
  • the conjugate is administered to the target-tumor-bearing mice, and after sufficient time has passed for the conjugate to localize, the mice are sacrificed and their tumors are excised. The tumor is minced and incubated with a chromogenic substrate. The supernatants of these suspensions are measured for signs of enzymatic activity.
  • Target cells are then suspended and incubated with conjugate.
  • the treated cells are then resuspended and incubated with Substrate - Cytotoxic Agent.
  • Cell growth is monitored by the uptake of radiolabeled nutrients
  • the final step in evaluating the present method is to evaluate the efficacy of the components in a nude mouse tumor model study.
  • a further aspect of this invention is the pharmaceutical compositions of the Antibody-Enzyme
  • compositions are useful for the treatment of neoplastic diseases of the present invention and comprise a suitable vehicle and a therapeutically effective amount of either the conjugates of Formula I or the compounds of Formula II.
  • compositions for oral are provided.
  • suitable vehicle means common excipients such as binding agents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidine (Povidone),
  • methylcellulose, ethylcellulose, sodium carboxymethyl-cellulose, hydroxypropylmethylcellulose, surcrose and starch fillers and carriers, for example corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium, phosphate, sodium chloride and alginic acid; disintegrators such as croscarmellose sodium, microcrystalline cellulose, corn starch, sodium starch glycolate, alginic acid and mutable wetting agents such as sodium lauryl sulfate; and lubricants such as magnesium stearate and other metallic stearates, stearic acid, silicone fluid, talc, waxes oils and colloidal silica.
  • fillers and carriers for example corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium, phosphate, sodium chloride and alginic acid
  • disintegrators such as croscarmellose sodium,
  • Flavoring agents such as peppermint, oil of wintergreen, cherry flavoring or the like can also be used. It may be desirable to add a coloring agent to make the dosage form more aesthetically pleasing in appearance or to help identify the product.
  • the tablets may also be coated by methods well known in the art .
  • compositions of the present invention may also be in the form of oral liquid
  • suitable vehicle means conventional additives such as suspending agents, for example, sorbitol, syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminum stearate gel or hydrogenated edible oils, for example almond oil, fractionated coconut oil, oily esters, propylene glycol or ethyl alcohol; and preservatives such as methyl or propyl p-hydroxybenzoates or sorbic acid.
  • suspending agents for example, sorbitol, syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminum stearate gel or hydrogenated edible oils, for example almond oil, fractionated coconut oil, oily esters, propylene glycol or ethyl alcohol
  • preservatives such as methyl or propyl p-hydroxybenzoates or sorbic acid.
  • the pharmaceutical composition can also be for intravenous (IV) use .
  • a water soluble form of the conjugate or Substrate - Cytotoxic Agent can be dissolved in one of the commonly used intravenous fluids and administrated by infusion.
  • suitable vehicle means such fluids as physiological saline, Ringer's solution or 5% dextrose solution.
  • a suitable salt form of the conjugate or Substrate - Cytotoxic Agent compound for example, the hydrochloride salt or sodium salt
  • a suitable vehicle for example, the hydrochloride salt or sodium salt
  • diluent e.g., Water-for-Injection
  • Topical compositions can be formulated with "suitable vehicles” such as hydrophobic or hydrophilic bases.
  • bases include ointments, creams or lotions.
  • Veterinary pharmaceutical compositions of the conjugates and the Substrate - Cytotoxic Agent compounds may be administered in the feed or the drinking water of farm animals.
  • the conjugate or Substrate - Cytotoxic Agent compounds can be formulated as intramammary preparations with "suitable vehicles" such as long-or quick-release bases.
  • the Antibody-Enzyme conjugate of Formula I and the Substrate - Cytotoxic Agent compounds of Formula II can also be formulated in unit dosage form in sterile vials sterile plastic pouches containing a port with a septum, or sterile, hermetically sealed ampoules.
  • the conjugate or compound (or the corresponding pharmaceutically-acceptable salt) may be a dry powder or in crystalline or lyophylized form.
  • the amount of the conjugate or Substrate - Cytotoxic Agent compound per unit dosage may vary from about 5 milligrams to about 10 grams.
  • n.m.r. spectra were taken on General Electric G.E.-300 instrument.
  • the infrared spectra were obtained on a Perkin-Elmer 281 instrument.
  • Ultraviolet spectra were obtained on a Cary 118 instrument (except for Example 7 and Procedures 6 through 13, where the U.V. spectra were obtained on a Hewlett-Packard 8451A instrument.)
  • Fast atom bombardment mass spectra were obtained on a VG ZAB-3 instrument.
  • reaction mixture was diluted with ethyl acetate and then 0.1N hydrochloric acid.
  • organic layer was separated and dried over magnesium sulfate, filtered, and concentrated in vacuo to give an orange oil.
  • the oil was flash chromatographed on a 3 inch column with a 100-200 mesh activated silica support eluted with ethyl acetate.
  • methotrexate-gamma-ester (4.6g, 9.1mmol, Sigma), DMF (50ml), and sodium bicarbonate (1.54g, 18.25mmol) were combined.
  • the solution was stirred at room temperature for 18 hours then added dropwise to a solution of acetic acid (4ml) in water (400ml).
  • the resultant precipatate was collected and dried in vacuo to yield approximately 7g of powder.
  • the powder was flash chromatographed over a silica gel column eluted with a solution of 15% methanol : 2% acetic acid in chloroform, to give approximately 2. lg of yellow-orange solid.
  • the concentrate was diluted with water (15ml) and the pH of the solution was adjusted to 7 by the addition of NaHCO 3 .
  • the resultant solution was layered with ethyl acetate and the mixture was stirred for 1 hour.
  • the mixture was filtered, the phases were separated and the aqueous phase was extracted with additional ethyl acetate then freeze dried.
  • the freeze-dried solid was subjected to medium pressure liquid chromatography on a C 18 reverse phase column.
  • the column was first equilibrated with a solution of 15% acetonitrile in water. Then the freeze dried material was loaded on the column and eluted first with 30% acetonitrile : 1% acetic acid in water (600ml); then 40% acetonitrile : 1% acetic acid in water.
  • Tetrakis [triphenylphosphine] palladium [O] (8mg, 0.006mmol), and triphenylphosphine (3mg, 0.01mmol) were combined in a 1:1 mixture of methylene chloride and ethyl acetate (2ml). Allyl 7- ⁇ -(2-(Thien-2-yl)acetamido)-3-((N- 1(5-fluorouracil)yl)methylene)-3-cephem-4-carboxylate
  • 2-Aminoethanethiol hydrochloride (9.9g, 0.08mol) was suspended in acetonitrile (approximately 25ml) under a nitrogen atmosphere, di(Isopropyl)amine (14.9ml) was added as the mixture was kept at 0°C during the addition. After 10 minutes at 0°, di(t-butyl)dicarbonate (20.0ml) was added and the resultant mixture was stirred at 0° for 15 minutes then at room temperature for another 45 minutes. The reaction mixture was concentrated in vacuo and a 40:60 mixture of ethyl acetate:hexane was added. The white solid thus formed was removed by filtration.
  • the deprotected amino compound was dissolved in methylene chloride (3ml) and the solution was cooled to 0°C.
  • N-Methylmorpholine (0.3ml) was added to the mixture followed by the slow addition of a methylene chloride solution (5ml) of desacetylvinblastine azide (preparation described in U.S. Patent No. 4,203,898, Example 5, Column 20) (0.47g, 0.543mmol).
  • the resultant mixture was stirred at 0°C for 30 minutes, the reaction mixture was
  • ⁇ -lactamase ( ⁇ -L) from the P99 strain of
  • CentiprepTM concentrator Analog tomicon
  • Nonidet P-40 Sigma Chemical Company
  • a volume of Sulfo-SMCC solution containing 2.0 molar equivalents ( ⁇ 35 ⁇ L) was added to the ⁇ -L solution, and the pH was adjusted to 8.0 with 0.3M Na 2 CO 3 .
  • the unstirred solution was left at room temperature for 1 hour.
  • the reaction mixture was then eluted through a 50mL P6DG (Bio-Rad Laboratories, Richmond, California) column with 50mM ammonium citrate, 1mM DTPA, 100mM NaCl, pH 6.2.
  • the protein containing peak (monitored at 280nm) was collected in a single fraction, and its concentration determined by measurement of the Absorbance at 280nm of an aliquot of the solution.
  • the maleimide content of the protein solution was determined by reaction of an aliquot with a twofold excess of cysteine (10 min., room).
  • F(ab') fragments of the anti-carcinoembryonic antigen antibody, CEM231 were prepared by pepsin digestion followed by cysteine reduction according methods known to the art and described by Breman, et al., and Glennie, et al. cited above.
  • reaction mixture was then applied directly to a 500mL Sephadex G-150 Superfine column and eluted with BBS buffer.
  • Product fractions were those determined by PAGE to contain pure material of molecular weight ⁇ 90,000. Pooled product fractions were then concentrated to
  • substrate at an assay concentration sufficient to give an absorbance of 0.5-1.0 at the wavelength of interest, (e.g., 50 ⁇ M), was added to a stirred, referenced solution of -5nM P-99 E. cloacae ⁇ -lactamase Enzyme (" ⁇ -L") in an appropriate buffer (PBS,
  • pencillinase can be obtained commercially (e.g., Sigma Chemical Company) and from procedures in the literature.) The change in absorbance was then determined by the spectrophotometer at intervals of 5 or more seconds.
  • Part B demonstrates the determination of enyzmatic activity of ⁇ -L-CEM 231 conjugate on the compound of Example 11.
  • a stock solution of cephalothin was prepared by dissolving 5.5mg in 1.0mL 10mM sodium phosphate, 150mM sodium chloride, pH 7.2 (PBS): 13mM.
  • a stock solution of P-99 E. cloacae ⁇ -lactamase (" ⁇ -L") enzyme was determined by A 280 to be 1.3 ⁇ M. ⁇ -L solution, 7.7 ⁇ L, was added to a cuvette containing stirred, temperature equilibrated PBS, such that a final volume of 2.0mL would be obtained, and the spectrophotometer was referenced. Substrate stock solution was then added to obtain assay concentrations between 5 and 130 ⁇ M. The spectrophotometer was instructed to wait 5 seconds for initial substrate mixing, then record the absorbance at 264nm every 10 seconds for 2 minutes.
  • cephalosporin Prodrug was determined by averaging duplicate HPLC injections of the quenched sample on a 0.46 ⁇ 15cm C-18 reversed phase column monitored at 266nm. The flow rate was 1ml per minute, run isocratically with the 34%
  • the mixture was purified on a 5mL Sephadex G-25 column equilibrated with PBS, 0.1% sodium azide, 0.1% gelatin solution, and eluted with PBS containing 0.1% sodium azide, pH 7.5, into tubes containing 100 ⁇ L of a 0.1% gelatin solution in 0.5mL fractions.
  • the fractions were counted for radioactivity in an ISODATA 20/20 Series Gamma Counter (RIA Data Systems, Rolling Meadows, Illinois), and the first peak of radioactivity was the labeled conjugate.
  • Immunoreactivity Polystyrene beads were prepared with carcinoembryonic antigen (CEA) or with irrelevant protein (for non-specific binding control) by soaking the beads in a solution of the desired protein at a concentration of 150ng/bead in 10% horse serum. Iodinated conjugate was diluted to about 200cpm/ ⁇ L in 10% horse serum. Three each antigen positive and antigen negative beads were placed in individual gamma counting tubes, washed, and treated with 200 ⁇ L labeled conjugate. The beads were incubated at 37°C overnight. Total counts were measured in each tube; supernatants were transferred to tubes containing a second bead of the same type. Finally, all beads were washed, and the fraction of counts on each bead was determined. Immunoreactivity was calculated as: A + + B + (1-A + ),
  • a + and B + are the fractions bound on the antigen positive beads in the first and second rounds
  • Immunoreactivity for the ⁇ -L-CEM231 conjugate was found to be about 80%, which compares well with values obtained for unmodified F(ab') fragments of CEM231. Non-specific binding had also not increased by conjugation with ⁇ -lactamase.
  • the Enzyme can be stopped at the desired time by addition of an inhibitor such as cloxacillin (Sigma Chemical Company) ) at its effective concentration: 5 ⁇ M for cloxacillin.)
  • an inhibitor such as cloxacillin (Sigma Chemical Company) ) at its effective concentration: 5 ⁇ M for cloxacillin.
  • a model system for the present method employs the expulsion of aminoethanethiol from 7- ⁇ - (2-(thien-2-yl)acetamido)-3-((2-aminoethyl-1-sulfide)methylene)-3-cephem-4-carboxylic acid.
  • a typical enzyme assay was performed (following the 264nm
  • cephalosporin a stock solution of the cephalosporin was prepared (1mg of cephalosporin in 2ml of 20% N,N-dimethylacetamide in pH 7.0 HEPES buffer, final
  • the Enzyme caused conversion of the cephalosporin - (5-fluorouracil) Substrate - Cytotoxic Agent to the Cytotoxic Agent.
  • Conjugate was labeled with 125 I as described for the immunoreactivity assay. Twelve nude mice in which LS174T tumor cells had been injected subcutaneously and allowed to reach a size of about 0.4g were injected in the tail vein with 50 ⁇ g of the labeled conjugate in 100 ⁇ L BBS.
  • mice were sacrificed, dissected, and the weight and amount of radioactivity in each organ was quantitated. The results are reported as % of injected dose/gm tissue.
  • CEA expressing LS174T cells and CEA negative MOLT4 cells were prepared as single cell suspensions in Autopow media (GIBCO, Grand Island, New York). Both suspensions were at a density of 2 ⁇ 10 7 cells/mL; LS174T were 23% viable, MOLT4 80% viable. Suspensions were maintained on ice until use.
  • Results are reported as % of original absorbance remaining after incubation at the PADAC peak absorbance wavelength of 570nm.
  • ⁇ -L-CEM231 conjugate 50 ⁇ g in 50mM sodium borate, 100mM NaCl (BBS) was injected into the tail vein of each of 18 tumor- free nude mice.
  • BBS 100mM NaCl
  • Target cells (antigen positive or antigen
  • the cells were then rinsed and resuspended in media
  • the free base of desacetylvinblastine hydrazide sulfate was made by dissolving the sulfate salt (900mg) in aqueous sodium bicarbonate solution then extracting the solution with methylene chloride. The organic layer was dried over sodium sulfate, filtered and concentrated in vacuo to give a white solid.
  • the free base was dissolved in dry pyridine (6ml) and added to t-butyl 7- ⁇ -(2- (phenoxy)acetamido)-3-(O-(p-nitrophenyl)carbonato)
  • the free base of desacetylvinblastinehydrazide sulfate (0.50g) was made as described above in Example 8.
  • the free base was dissolved in dry pyridine (10 ml) and added to benzhydryl 7- ⁇ -(2-(thien-2-yl)acetamido)-3-(O-(p-nitrophenyl)carbonato)methylene)-3-cephem-1- ⁇ -sulfoxide (0.32g, 0.456mmol).
  • the mixture was allowed to stir at room temperature under nitrogen after N,N-diisopropylethylamine (2 drops) was added.
  • the resultant reaction mixture was stirred overnight at room temperature under nitrogen, then diluted with ethyl acetate and
  • Example 5 The procedure of Example 5 was repeated, substituting the cephalosporin-1- ⁇ -sulfoxide starting material of Procedure 18 (0.34g, 0.598mmol) for the
  • Example 5 The product was flash chromatographed over silica gel eluted with a gradient of 95/5 to 90/10
  • Desacetylcolchicine (70mg, 0.196mmol) was made according to the procedure described in Raffholz, J. Amer.
  • Doxorubicin-HCl (0.87g, 1.5mmoles) was slurried in (5.0ml, Aldrich Sure-Seal) DMF. Disopropylethylamine (0.3ml, dried over KOH pellets, 0.25mmoles) was added to the slurry, then allyl-7- ⁇ -(2-(thien-2-yl)acetamido)-3-((p- nitrophenylcarbonato) methylene-3-cephem-1- ⁇ -sulfoxide-4-carboxylate (0.87g, 1.51mmoles) was added to the resultant mixture and the reaction was shielded from light and stirred for 2.5 hours. Ethyl ether was added to the reaction to precipitate 1.29g of a red solid. This
  • Example 21 Materials may be used without purification in Example 21.
  • the purpose of the following procedure was to compare the toxicity of the Substrate-Cytotoxic Agent to the toxicity of Cytotoxic Agent alone.
  • GROUP A 5 rats + vehicle control: 0.5ml, 10mM HEPES, 150 NaCl, pH7.1 buffer containing 10% ethanol.
  • GROUP B 5 rats + 1mg/kg 4-desacetylvinblastine -3-carboxhydrazide (desacetylvmblastmehydrazide). Dissolved 1mg desacetylvmblastmehydrazide in 0.5ml absolute ethanol and added 4.5ml HEPES-NaCl buffer. Injected each rat with 0.5ml.
  • GROUP C 5 rats + 1mg/kg LY262758 (7- ⁇ -2- (phenoxyacetamido)-3- (((desacetylvinblastinehydrazido)carbonyloxy)methylene)-3-cephem-1- ⁇ -sulfoxide-4-carboxybiacid, trifluoroacetic acid salt) (vinca content, 58% vinca). Dissolved 2mg as above and injected 0.5ml.
  • GROUP D 5 rats + 1mg/kg LY266494, (7- ⁇ -(2-(thien-2-yl)acetamido-3- (((desacetylvinblastinehydrazido)carbonyloxy)methylene)-3-cephem-4-carboxylic acid, hydrochloride salt (55.8% vinca). Dissolved 2mg and injected 0.5 ml.
  • GROUP E 5 rats + 1mg/kg compound of Example 17 7- ⁇ -(2- (thien-2-yl)acetamido-3-(((desacetylvinblastinehydrazido)carbonyloxy)methylene)-3-cephem-1- ⁇ -sulfoxide-4-carboxylic acid, trifluoroacetic acid salt. Dissolved 2mg and injected 0.5ml.
  • the desacetylvinblastinehydrazide-treated animals exhibited a significant WBC suppression on day 3 compared to all other groups.
  • the WBC values of the groups were indistinguishable at day 7 (and lower than the prebleed which may have been due to using a different
  • rat #2 12.4 16.4 9.3 rat #3 13.1 13.7 6.8 rat #4 10.4 10.3 10.1 rat #5 10.1 13.1
  • DAY 0 Implant 30 Nude Mice (CHARLES RIVER, 25G) WITH 1X10 7 LS174T Tumor Cells (ATCC) subcutaneously;
  • GROUP A Inject with 0.25ml saline per mouse
  • GROUP B Placed 1.7Ml stock solution in vial, injecte
  • GROUP C 0.625Ml stock solution + 0.775ml saline
  • GROUP D 0.31Ml stock solution + 1.09ml saline
  • GROUP E 0.15Ml stock solution + 1.25ml saline
  • GROUP F 0.075 Ml stock solution + 1.325 ml Saline
  • GROUP A 5 mice injected with 0.25ml saline
  • GROUP B 5 mice injected with 20mg/kg ceph-vinca PRODRUG
  • GROUP C 5 mice injected with 10MG/KG PRODRUG
  • GROUP D 5 mice injected with 5MG/KG PRODRUG
  • GROUP E 5 mice injected with 2.5MG/KG PRODRUG
  • GROUP F 5 mice injected with 1.25MG/KG PRODRUG
  • the Group B dosage was toxic.
  • the Group C dosage while not toxic, showed that Prodrug alone had anti-tumor activity at that level. It is speculated from these results that a maximum effective dose would be somewhere in the range of 1 to 8mg/kg.
  • the biodistribution of the anti-KS1/4(i.e.007B) - - ⁇ -lactamase conjugate was measured by labeling the conjugate with 111 Indium.
  • the conjugate was first reacted with isothiocyanatobenzyl DTPA, then labeled according to established procedures (Meares C., et al., Anal. Biochem., 142, pp.68-78, 1984).
  • the labeled conjugate (20 ⁇ g and lO ⁇ Ci per animal) was injected into the tail vein of nude mice with LS174T tumors. The animals were separated into groups of six, and groups were sacrificed at 4, 24, 48, 72 and 120 hours after conjugate injection.
  • mice Treatment groups of 8 mice each were injected with either the anti-CEA (i.e., CEM231) - - ⁇ -lactamase (35 ⁇ g) conjugate, the irrelevant-antibody (i.e., CHA255) - - ⁇ -lactamase (35 ⁇ g) conjugate, or saline on days 14, 21 and 28. These injections were followed by four daily
  • the prodrug injections were substituted with either saline, or 0.6mg/kg (FIG.3) or 0.15mg/kg (FIG.4) desacetylvmblastmehydrazide, the molar equivalent of the prodrug doses.
  • saline/prodrug open squares: saline/drug; open diamonds: irrelevant antibody - - ⁇ -lactamase conjugate/prodrug; open arrows: conjugate or saline injection; filled arrows:
  • control arms showed only tumor growth inhibition, no regressions, and long-term disease
  • LS174T tumors were implanted as in Procedure 24 and measured as in Procedure 25.
  • Animals (ten in the saline control group; five in the treatment groups) were treated with 35 ⁇ g each of either the anti-KS1/4 (007B) - - ⁇ -lactamase conjugate, the anti-CEA (CEM231) - - ⁇ -lactamase conjugate , the irrelevant antibody (CHA255 ) - - ⁇ -lactamase conjugate, or saline on days 9, 16 and 23.
  • Tumor volumes were measured and are depicted in FIG.5. As with T380, tumors shown in FIG.3, tumor regressions were observed only in the tumor-specific treatment groups, not in any of the control groups.
  • Procedure 26 except that the tumors were allowed to reach a larger size before treatment was started, and that the conjugate used is anti-TAG-72 (CC49) - - ⁇ -lactamase. Even with large tumors and TAG-72 as target antigens, tumor regressions resulted from, tumor specific treatment, but not from any control treatment.
  • mice received either 35 ⁇ g (filled squares), 14_g (filled circles), or 3.5 ⁇ g (filled triangles) of the anti-CEA(CEM231) - - ⁇ -lactamase conjugate on days 14, 21, and 28 followed after 72 hours by prodrug at 1mg/Kg/day for four days.
  • a fourth experimental group received 35 ⁇ g of the anti-CEA - - ⁇ -lactamase conjugate on day 14 only (filled diamonds) followed by prodrug on the same schedule as the other mice.
  • the data points in FIG.5 represent the following results for this procedure: open circle:
  • conjugate or saline injection filled arrows: first of 4 days of prodrug, drug, or saline injections.
  • maximal efficecy may be duration, not magnitude, of therapeutic effect depended on conjugate dose.
  • conjugate in each course of therpy contributes to the duration of response.

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Abstract

L'invention concerne un procédé destiné à traiter les maladies néoplasiques et qui consiste: a) à administrer une quantité thérapeutiquement efficace d'un conjugué anticorps/enzyme à l'hôte affecté; et ensuite b) à administrer une quantité thérapeutiquement efficace d'un substrat/agent cytotoxique à l'hôte affecté, le substrat étant un substrat destiné à l'enzyme. L'invention se rapporte également à des conjugués anticoprs/enzyme et à des composés substrat/agent cytotoxique.
PCT/US1993/006324 1992-07-06 1993-07-02 Procede d'aministration d'agents cytotoxiques et constituants de ceux-ci WO1994001137A1 (fr)

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AU46634/93A AU4663493A (en) 1992-07-06 1993-07-02 Method for delivery of cytotoxic agents and components thereof

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US07/909,924 1992-07-06

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994025061A1 (fr) * 1993-04-23 1994-11-10 Glyko, Inc. Procedes et compositions permettant de traiter des maladies au moyen d'enzymes de modification d'hydrates de carbone
US5928888A (en) * 1996-09-26 1999-07-27 Aurora Biosciences Corporation Methods and compositions for sensitive and rapid, functional identification of genomic polynucleotides and secondary screening capabilities
EP2514763A1 (fr) 2004-02-20 2012-10-24 The Ludwig Institute for Cancer Research Peptides épitopes du récepteur de l'EGF et leurs utilisations

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WO1990007929A1 (fr) * 1989-01-23 1990-07-26 Akzo N.V. Activation in vivo specifique a un site de medicaments therapeutiques
JPH02247164A (ja) * 1989-02-02 1990-10-02 Eli Lilly & Co 細胞毒性剤の導入方法
US4975278A (en) * 1988-02-26 1990-12-04 Bristol-Myers Company Antibody-enzyme conjugates in combination with prodrugs for the delivery of cytotoxic agents to tumor cells
EP0484870A2 (fr) * 1990-11-06 1992-05-13 Bristol-Myers Squibb Company Prodrogues pour bêta-lactamase et leurs utilisations

Patent Citations (4)

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Publication number Priority date Publication date Assignee Title
US4975278A (en) * 1988-02-26 1990-12-04 Bristol-Myers Company Antibody-enzyme conjugates in combination with prodrugs for the delivery of cytotoxic agents to tumor cells
WO1990007929A1 (fr) * 1989-01-23 1990-07-26 Akzo N.V. Activation in vivo specifique a un site de medicaments therapeutiques
JPH02247164A (ja) * 1989-02-02 1990-10-02 Eli Lilly & Co 細胞毒性剤の導入方法
EP0484870A2 (fr) * 1990-11-06 1992-05-13 Bristol-Myers Squibb Company Prodrogues pour bêta-lactamase et leurs utilisations

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Title
CHEMICAL ABSTRACTS, Vol. 115, issued 1990, Abstract No. 49250P, "Cephalosporin-Cytotoxic Agent--to Tumor Cell", LILLY. ELI AND CO.; & JP,A,02 247 164 (02 October 1990). *
CHEMICAL ABSTRACTS, Vol. 117, issued 1992, KADOW et al., Abstract No. 55985q, "Cephalosporin-Cytotoxic Agent... and Uses Thereof", (Bristol-Myers); & EP,A,484 870 (13 May 1992). *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994025061A1 (fr) * 1993-04-23 1994-11-10 Glyko, Inc. Procedes et compositions permettant de traiter des maladies au moyen d'enzymes de modification d'hydrates de carbone
US5928888A (en) * 1996-09-26 1999-07-27 Aurora Biosciences Corporation Methods and compositions for sensitive and rapid, functional identification of genomic polynucleotides and secondary screening capabilities
EP2514763A1 (fr) 2004-02-20 2012-10-24 The Ludwig Institute for Cancer Research Peptides épitopes du récepteur de l'EGF et leurs utilisations

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CN1087642A (zh) 1994-06-08
IL106228A0 (en) 1993-11-15
AU4663493A (en) 1994-01-31
ZA934825B (en) 1995-01-05
MX9304056A (es) 1994-05-31

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