WO2006135371A1 - Conjugués à effets adverses systémiques réduits - Google Patents

Conjugués à effets adverses systémiques réduits Download PDF

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Publication number
WO2006135371A1
WO2006135371A1 PCT/US2005/021418 US2005021418W WO2006135371A1 WO 2006135371 A1 WO2006135371 A1 WO 2006135371A1 US 2005021418 W US2005021418 W US 2005021418W WO 2006135371 A1 WO2006135371 A1 WO 2006135371A1
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active agent
target cell
moiety
cell
group
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PCT/US2005/021418
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English (en)
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Daniel V. Santi
Brian Hearn
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Kosan Biosciences Incorporated
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Priority claimed from US11/149,758 external-priority patent/US7541330B2/en
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Publication of WO2006135371A1 publication Critical patent/WO2006135371A1/fr

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    • 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
    • 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

Definitions

  • This invention relates to conjugates having reduced adverse systemic effects and methods for making and using the same.
  • Chemotherapy involves administering to a patient an active agent designed to modulate one or more cellular functions of a target cell associated with a disease or disorder.
  • the modulating action can range from reducing an activity, such as down- regulating the expression of a gene, to suppressing the activity entirely.
  • the target cell is a cancer cell, in which case the cellular functions modulated are those vital to cell survival, with the objective of affecting them to such an extent that the cell cannot survive and a cytocidal effect is achieved — that is, the active agent is a cytotoxin.
  • a common drawback of chemotherapy is the effect of the active agent on non-target cells as well as target cells, resulting in systemic (non-selective) adverse effects.
  • this drawback can be overcome by designing an active agent that affects only the target cell, but in practice absolute selectivity is rarely achieved.
  • An alternative approach is to covalently link the active agent via a linker moiety to a targeting moiety that has affinity for the target cell, forming a targeting moiety/linker moiety/active agent conjugate.
  • the active agent is latently active: in its conjugated form it is inactive, but when released from the conjugate by cleavage of the linker moiety, it is active.
  • the targeting moiety directs the conjugate to the target cell, after which the conjugate is internalized by endocytosis.
  • the targeting moiety can be an antibody (particularly a monoclonal antibody or "mAb") having specific affinity for a tumor-associated antigen ("TAA") characteristic of a target cancer cell and the active agent is an anti-cancer drug.
  • TAA tumor-associated antigen
  • the active agent is referred to as a "warhead,” analogizing the conjugate to a military guided missile.
  • the linker moiety is designed to be stable outside of the target cell but unstable inside it (or, at least, more stable outside than inside). Cleavage of the linker moiety in response to conditions prevalent inside the target cell releases the active agent. Intracellular conditions triggering cleavage can be varied.
  • the end destination of an endocytosed molecule is normally a lysosome inside the cell.
  • the lysosomal environment is more acidic (typically about pH 5) than blood plasma (typically about pH 7.3), so that a linker moiety that is pH sensitive can be selectively cleaved inside a target cell.
  • a lysosome contains acid hydrolases, which are peptidases active at acidic pH's.
  • a peptidic linker moiety that is a specific substrate for the acid hydrolases will be cleaved preferentially inside a lysosome.
  • a redox-potential sensitive linker moiety may be preferentially cleaved in response to a difference in redox potential.
  • a certain amount of premature cleavage of the linker moiety is virtually unavoidable.
  • a pH sensitive linker moiety will cleave in blood serum at about 1% of the lysosomal rate.
  • a peptidic linker moiety designed to be a specific substrate for a lysosomal acid hydrolase may be a non-specific substrate for a serum protease.
  • this invention provides a conjugate that has affinity for a target cell and releases inside the target a modified active agent that modulates one or more cellular functions of the target cell, having the structure
  • T is a targeting moiety that has affinity for the target cell
  • D-Z is a modified active agent comprising a cell membrane-impermeabilizing moiety Z covalently attached to an active agent D that modulates one or more cellular functions of the target cell when inside the target cell
  • L is a linker moiety covalently linking targeting moiety T and modified active agent D-Z, which linker moiety L is preferentially susceptible to cleavage inside the target cell to release modified active agent D-Z
  • m is an integer ranging from 1 to 64
  • n is an integer ranging from 1 to 12.
  • this invention provides a method of making a conjugate that has affinity for a target cell and releases inside the target cell a modified active agent that modulates one or more cellular functions of the target cell, comprising the steps of:
  • targeting moiety T has affinity for the target cell; linker moiety L is preferentially susceptible to cleavage inside the target cell to release modified active agent D-Z; m is an integer ranging from 1 to 64; and n is an integer ranging from 1 to 12.
  • the linking step (b) can be performed either before or after the attachment of cell membrane-impermeabilizing moiety Z to active agent D.
  • this invention provides a method for modulating one or more target cellular functions of a target cell in a subject (e.g., a human or an animal), comprising administering to the subject an effective amount of a conjugate that has affinity for a target cell and releases inside the target cell a modified active agent that modulates one or more cellular functions of the target cell, which conjugate has the structure
  • T is a targeting moiety that has affinity for the target cell
  • D-Z is a modified active agent comprising a cell membrane-impermeabilizing moiety Z covalently attached to an active agent D that modulates one or more cellular functions of the target cell when inside the target cell;
  • L is a linker moiety covalently linking targeting moiety T and modified active agent D-Z, which linker moiety L is preferentially susceptible to cleavage inside the target cell to release modified active agent D-Z;
  • m is an integer ranging from 1 to 64; and
  • n is an integer ranging from 1 to 12.
  • this invention provides for the use of a conjugate according to the first aspect for preparation of a medicament for the treatment of a disease of cell proliferation, in particular cancer.
  • Fig. 1 shows the chemistry underlying a protease-cleavable self-immolating linker moiety.
  • Figs. 2 through 4 show schemes for the assembly of conjugates of this invention.
  • Figs. 5 and 6 show schemes for the attachment of targeting moieties to bafilomycin modified active agents.
  • Fig. 7 shows the synthesis of maytansinoid compounds usable as modified active agents in conjugates of this invention.
  • Fig. 8 shows the synthesis of leptomycin B compounds usable as modified active agents in conjugates of this invention.
  • targeting moiety is to direct the conjugate to the target cell, via its affinity for a cognate site on the surface of the target cell.
  • Preferred targeting moieties T include antibodies, growth factors, serum proteins, polysaccharides or synthetic polymers, especially those that are ligands for cell-surface receptors or antigens.
  • the cell-surface receptor or antigen is unique to the target cell or at least present in a greater amount there than in a non-target cell.
  • targeting moiety T preferentially binds to a target cell compared to a non-target cell, which is what is meant by the statement that targeting moiety T has an affinity for a target cell.
  • Monoclonal antibodies are especially preferred. General protocols for the design and use of conjugated antibodies are described in Monoclonal Antibody-Based Therapy of Cancer, Vol. 15, Michael L. Grossbard, ed. ( Marcel Deklcer 1998) (incorporated herein by reference).
  • TAAs tumor-associated antigens
  • CEA carcinoembryonic antigen
  • ⁇ -fetoprotein associated with gastro-intestinal tract and some lung and breast tumors
  • gangliosides such as L6 Ag
  • blood group carbohydrates such as Lewis y (Ley)
  • Ley the transferrin receptor
  • adenocarcinoma KS 1/4 mucins
  • glycosphingolipids selectins; integrins; other adhesion molecules; mutated forms of tumor suppressor p53; and heat shock proteins overexpressed in tumor cells.
  • the antibody is directed against a cellular receptor protein or antigen.
  • Preferred examples include but are not limited to antibodies directed against HER2/neu, epidermal growth factor receptor (EGFR), ErbB2, platelet-derived growth factor (PDGF) receptor, vascular endothelial growth factor receptor 2 (VEGFR2 or KDR), and insulin-like growth factor receptor (IGFR).
  • the antibody is directed against other clinically relevant tumor markers, including but not limited to polymorphic epithelial mucin (MUC-I), the ovarian cancer- associated antigen CA125, or against the CD33 myeloid-differentiation antigen.
  • MUC-I polymorphic epithelial mucin
  • antibodies examples include alemtuzumab, abciximab, biciromab (ReoProTM), infliximab (RemicadeTM), 11 lln-capromab pendetide; trastuzumab (HerceptinTM), rituximab (RituxanTM), CEA-Scan, sulesomab, palivizumab (SynagisTM), basiliximab (SimulectTM), daclizumab (ZenapaxTM), tositumomab, efalizumab, 99mTc-fanolesomab, omalizumab, BR96, eculizumab, MH-I, ATM-027, SC-I, bivatuzumab, BMS-188667, BMS-224818, SGN-15, CAT-213, J-695, metelimumab, CAL, MRA, MLN-2704, OncoR
  • the targeting moiety T is a cellular growth factor.
  • growth factors include but are not limited to epidermal growth factor (EGF), insulin-like growth factor (ILGF), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF).
  • targeting moiety T is a polysaccharide ligand for a cellular receptor.
  • Preferred embodiments include but are not limited to ligands for the selectin receptors, such as Lewis-x, and ligands for growth factor receptors. Examples of polysaccharide ligands that are ligands for growth factor receptors are described in Magnani et al, US 6,281,202 and 6,008,203, both incorporated herein by reference.
  • Targeting moiety T can also be a protein substantially smaller in size than an antibody, for example a protein having a molecular weight of less than about 5,000 Daltons, as described in Briesewitz et al, US 6,372,712 Bl (2002), the disclosure of which is incorporated by reference.
  • the targeting moiety After the targeting moiety has directed the conjugate to the target cell, it may be internalized by endocytosis.
  • endocytosis For more background information on endocytosis, see, e.g., Cooper, The Cell: A Molecular Approach, ASM Press (Washington DC, 1997), pp. 381- 383, 492-500; and Alberts et al, Molecular Biology of the Cell, 4th Ed., Garland Science (New York, NY 2002), pp. 739-757; the disclosures of which are incorporated herein by reference.
  • the cell membrane In endocytosis, the cell membrane invaginates and progressively occludes a volume of extracellular fluid, culminating in the invaginated section of the cell membrane pinching off as an endocytic vesicle (also referred to as a pinocytic vesicle) inside the cell, thus internalizing the occluded fluid and any molecules dissolved therein.
  • an endocytic vesicle also referred to as a pinocytic vesicle
  • the efficiency of internalization of some materials may be enhanced by the intervention of cognate binding sites (cell-surface receptors, antigens, etc.) for the material, in a process referred to as receptor-mediated endocytosis.
  • the binding sites or receptors for an extracellular ligand accumulate at clathrin-coated pits on the cell membrane, which pits are also starting loci for invagination and endocytosis.
  • the ligands bind to the binding sites and are thus internalized at an enriched concentration, without the need for the cell to take in a correspondingly large volume of extracellular fluid. This process efficiently and selectively internalizes ligands that may be present in only minute concentrations in the extracellular fluid.
  • a conjugate according to this invention When a conjugate according to this invention is internalized by endocytosis, it makes its way to other intracellular vesicular bodies, such as early endosomes, late endosomes, and lysosomes, via a series of fusions and buddings. Cleavage of the linker moiety in response to a prevailing condition inside the one of the vesicular bodies releases modified active agent D-Z.
  • lysosomal fluid is acidic and contains a variety of acid hydrolases and is thus a desirable medium for the cleavage of pH-sensitive or enzymatically sensitive linker moieties.
  • Active agent D can be a cytotoxin used for cancer chemotherapy.
  • cytotoxins include alkylating agents, angiogenesis inhibitors, antimetabolites, DNA cleavers, DNA crosslinkers, DNA intercalators, DNA minor groove binders, enediynes, heat shock protein 90 inhibitors, histone deacetylase inhibitors, microtubule stabilizers, nucleoside (purine or pyrimidine) analogs, nuclear export inhibitors, proteasome inhibitors, topoisomerase (I or II) inhibitors, tyrosine kinase inhibitors.
  • Specific anticancer or cytotoxic agents include ⁇ -lapachone, ansamitocin P3, auristatin, bicalutamide, bleomycin, bortezomib, busulfan, calicheamycin, callistatin A, camptothecin, capecitabine, CC-1065, cisplatin, cryptophycins, daunorubicin, discodermolide, disorazole, docetaxel, doxorubicin, duocarmycin, dynemycin A, epothilones, etoposide, floxuridine, floxuridine, fludarabine, fluoruracil, gefitinib, geldanamycin, 17-allylamino- 17-demethoxygeldanamycin (17-AAG), 17-(2-dimethylaminoethyl)aminol7- demethoxygeldanamycin (17-DMAG), gemcitabine, hydroxyurea, imatinib
  • maytansinoids is used to refer to the family of structurally related compounds including maytansine and ansamitocin P3.
  • active agent D is selected from the group consisting of maytansinoids and leptomycin B.
  • the objective may be to down-regulate an overexpressed gene or partially inhibit an enzymatic activity, without killing the target cell. In other instances, it may be desired to inhibit cell growth or division.
  • Z is a cell membrane-impermeabilizing moiety
  • Z renders modified active agent D-Z substantially less capable of permeating across the cell membrane than unmodified active agent D-Z, preferably at least 10 times less permeable, more preferably at least 100 times more permeable.
  • the relative cytotoxicities of the unmodified and modified active agent is possibly an indirect indication of relative permeabilities.
  • Suitable types of cell membrane-impermeabilizing moiety Z are diverse; it can be cationic, anionic, zwitterionic, or charge-neutral.
  • Exemplary suitable cationic moieties Z are basic groups that are substantially protonated at physiological pH, such as primary, secondary, and tertiary alkyl amine groups, quaternary alkyl or alkylaryl ammonium groups, guanidinium groups, imidazolium groups, triazolium groups, tetrazolium groups, and the like.
  • Exemplary suitable anionic moieties Z are acidic groups that are substantially ionized to their conjugate base form at physiological pH, such as sulfonates, phos- phonates, and, in some instances, carboxylates.
  • Zwitterionic moieties Z include moieties that have a carboxylate and an ammonium group.
  • Neutral moieties Z include glucuronate.
  • Linker moiety L can be a pH sensitive one. While the pH of blood is typically about 7.3 to 7.4, the pH in an endosome is 5.0 to 6.5, and the pH in a lysosome is about 4.0 or even as low as 3.8 during early stages of digestion.
  • the intracellular environment inside tumor tissue has been measured to be 0.5 to 1.0 pH units lower than in normal tissue as well. This pH differential can be exploited to provide a conjugate whose linker is stable until it reaches the lower pH environment of an intracellular compartment.
  • Such 5 a linker can comprise a bond that is stable at neutral pH but is readily cleavable under ' conditions of low pH, e.g., one stable at a pH between 7 and 8 but readily cleavable at a pH between 4 and 6.
  • linkers are czs-aconityl amides and acyl hydrazones, as described in Shen et ah, US 4,631,190; US 5,144,011; and Biochem. Biophys. Res. Commun. 102, 1048-1054 (1981), the disclosures of which are incorporated o herein by reference.
  • linker moiety L is a redox-potential sensitive one, such as an alkul-alkyl or alkyl-aryl mixed disulfide wherein the aryl moiety is substituted so as to control the steric and electronic properties of the disulfide towards reaction with thiols.
  • a linker provides a means of attenuating the rate of thiol-disulfide interchange such 5 that the linkage is stable in an environment of low reductive potential, e.g., in the extracellular environment having low thiol or glutathione concentration, but is cleaved in conditions of high reduction potential (e.g., an intracellular environment having high thiol or glutathione concentration).
  • linker L comprises a moiety that is readily cleaved 0 in the presence of an enzyme, such as a peptide sequence that is a recognition sequence for an endosomal or lysosomal peptidase (e.g., a cathepsin, especially cathepsin B or D), with the result that the enzyme recognizes and cleaves the linker at or adjacent to the recognition sequence.
  • an enzyme such as a peptide sequence that is a recognition sequence for an endosomal or lysosomal peptidase (e.g., a cathepsin, especially cathepsin B or D), with the result that the enzyme recognizes and cleaves the linker at or adjacent to the recognition sequence.
  • linker L is one having a bond that is readily cleaved 5 upon exposure to radiation, for example a 2-nitrobenzyl ether cleavable upon exposure to light.
  • linker moiety L is a self-immolating linker.
  • self- immolating linker moieties comprise a peptide segment that is a substrate (preferably, a specific substrate) for a protease found inside the target cell and a self -immolating segment that "unzips" or decomposes when the peptide segment is cleaved by the protease, releasing the drug moiety D-Z.
  • the self -immolating segment comprises a p-aminobenzyloxycarbonyl (PABC) group, which can unzip as illustrated in Fig. 1. See Carl et al, J. Med. Chem. 24 (3), 479-480 (1981), "A Novel Connector Linkage Applicable in Prodrug Design”; and Carl et al, WO 81/01145 (1981); the disclosures of which are incorporated herein by reference.
  • PABC p-aminobenzyloxycarbonyl
  • linker moiety L comprises a PABC group, a dipeptide residue, and a residue L 1 forming the balance of the linker L moiety.
  • modified active agent D-Z has an amino group via which it is attached to linker moiety L.
  • the PABC group serves as a spacer, to prevent modified active agent D-Z from sterically or electronically interfering with cleavage of the dipeptide residue by the protease.
  • the dipeptide residue has amino acid side chain groups AA 1 and AA 2 , which make it a specific substrate for the protease.
  • the PABC group After cleavage of the dipeptide by the protease as indicated by the dotted line, the PABC group "unzips" in a 1,6-fragmentation reaction to release free modified active agent, in the form of its free amine H 2 N-D-Z.
  • the linkage site of drug moiety H 2 N-D-Z in Fig. 1 is an amino group, which in turn is connected to the PABC group, forming a carbamate linkage.
  • the linkage site of the drug moiety can be a hydroxyl group, as in HO-D-Z, with the corresponding conjugate having the structure shown below and the self-immolation chemistry proceeding generally analogously in a 1,6-fragmentation reaction upon cleavage of the dipeptide residue. See Told et al, J. Org. Chem. 67, 1866-1872 (2002), the disclosure of which is incorporated herein by reference.
  • linker moiety L is designed to be cleaved by a protease
  • the protease is preferably a lysosomal acid hydrolase, especially cathepsin B.
  • Cathepsin B preferentially cleaves peptides where AA 1 is a basic or strongly hydrogen bonding amino acid residue (as in lysine, arginine, or citrulline) and AA 2 is a hydrophobic residue (as in phenylalanine, valine, alanine, leucine, or isoleucine). See Dubowchik et al, Biorg. Med. Chem. Lett.
  • a preferred linker moiety L comprises the structure
  • AA 1 is a lysine, arginine, or citrulline amino acid side chain residue
  • AA 2 is a phenylalanine, valine, alanine, leucine, or isoleucine amino acid side chain residue.
  • a linker moiety L is preferentially cleaved inside a target cell if its cleavage rate inside thereof is substantially greater than in blood plasma.
  • the cleavage rate inside the target cell is at least 10x, more preferably at least 10Ox greater than in blood plasma.
  • targeting moiety T is an antibody (preferably a monoclonal antibody, or mAb)
  • a preferred site for bonding a linker moiety L is an ⁇ -amino groups of a lysine residue (lysine residues are found throughout the light and heavy chains of the antibody).
  • Linkage of targeting moiety T and linker moiety L can be effected via an amide link by reaction with an activated acyl group, via an urea link by reaction with an isocyanate group, or via a thiourea link by reaction with a thioisocyanate group or a thioisocyanate equivalent, as shown in Fig. 2. (In Fig.
  • L 1 has the same meaning as ascribed previously, i.e., a generalized representation of the balance of linker moiety L remaining outside of the atoms or groups thereof specifically depicted.
  • X 1 is a leaving group such as OH, 0-iV-succinimide, 0-4-nitrophenyl, O-penta- or O-tetrafluorophenyl, F, Cl, Br, I, and the like.
  • Another bonding site on targeting moiety T is a sugar residue in the hinge region of the antibody, its location away from the antibody binding site making it an attractive bonding site.
  • the sugars can be oxidized (e.g., with periodate) to provide aldehyde groups that can then be used in coupling reactions, via imine groups, hydrazone or hydrazone-equivalent groups, or oximino groups, as shown in Fig. 3.
  • a third binding site for targeting moiety T is as thiol group, by addition across a maleimide group, by displacement of a substituent alpha to a carbonyl group, or formation of a disulfide with a thiol group in the linker in a disulfide exchange reaction, as shown in Fig. 4.
  • X 2 is a nucleophilically displaceable group such as OSO 2 (alkyl), Cl, Br, I, and the like
  • Y 1 is a divalent moiety such as CH 2 , O, NH, N(alkyl), and the like.
  • linker moieties can be used in the conjugates of this invention. Additional disclosures relating to linker moieties that can be used include: Senter et al. , US 4,952,394 (1990); Kaneko et al., US 5,137,877 (1992); Chad et al, US 5,416,064 (1995); Willner et al, US 5,708,146 (1998); King et al, US 5,824,805 (1998); Chari et al, US 5,846,545 (1998); Hellstrom et al, US 5,869,045 (1999); Hellstrom et al, US 5,980,896 (1999); Chari et al, US 6,333,410 Bl (2001); Chari et al, US 6,436,931 Bl (2002); Chari et al, US 6,441,163 Bl (2002); Chari et al, US 2003/0055226 Al (2003); Firestone, WO
  • Assembly of a conjugate of this invention can be effected either by covalently linking together targeting moiety T and linker moiety L, followed by covalent attachment of active agent D-Z.
  • the order of the steps can be reversed, with the linker moiety L-active agent D-Z attachment formed first.
  • a conjugated moiety T-L-(D) n is formed first, and then cell membrane impermeabilizing group Z is attached to D.
  • active agent D can be covalently linked to linker moiety L before cell membrane-impermeabilizing group Z is attached thereto.
  • a linker moiety L comprising a dendrimer can be used for the attachment of more than one modified active agent D-Z to each linker moiety L (i.e., m > 1).
  • mAb is a monoclonal antibody
  • L in formulae XIII and XIV is a linker moiety as defined hereinabove;
  • AA 1 is a lysine, arginine, or citrulline amino acid side chain residue
  • X ⁇ in formulae IV to VI and X to XII is a pharmaceutically acceptable counteranion (chloride, acetate, citrate, fumarate, maleate, succinate, benzoate, sulfate, tartrate, and the like).
  • Conjugates I to VI have a maytansinoid (an anti-cancer cyto toxin) compound as the active agent D.
  • Linkage to the targeting moiety mAb is variously via maleimide, amide, or imine linkages as shown.
  • the cell membrane-impermeabilizing moiety Z is anionic (phosphonate, conjugates I to III) or cationic (quaternary ammonium; conjugates IV to VI).
  • the linker moiety includes a self -immolating moiety and a protease susceptible dipeptide moiety.
  • Conjugates VII to XII have leptomycin B (a cyto toxin that acts as a nuclear export inhibitor) as the active agent D.
  • Linkage to the targeting moiety mAb is variously via maleimide, amide, or imine linkages as shown.
  • the cell membrane-impermeabilizing moiety Z is anionic (phosphonate, conjugates VII to IX) or cationic (quaternary ammonium; conjugates X to XII).
  • the linker moiety includes a self-immolating moiety and a protease susceptible dipeptide moiety.
  • stereoisomers are specifically indicated (e.g., by a bolded or dashed bond at a relevant stereocenter in a structural formula, by depiction of a double bond as having E or Z configuration in a structural formula, or by use stereochemistry- designating nomenclature), all stereoisomers are included within the scope of the invention, as pure compounds as well as mixtures thereof. Unless otherwise indicated, individual enantiomers, diastereomers, geometrical isomers, and combinations and mixtures thereof are all encompassed by the present invention. Polymorphic crystalline forms and solvates are also encompassed within the scope of this invention.
  • Diseases treatable by conjugates of this invention include, but are not limited to, hyperproliferative diseases, including: cancers of the head and neck which include tumors of the head, neck, nasal cavity, paranasal sinuses, nasopharynx, oral cavity, oropharynx, larynx, hypopharynx, salivary glands, and paragangliomas; cancers of the liver and biliary tree, particularly hepatocellular carcinoma; intestinal cancers, particularly colorectal cancer; treat ovarian cancer; small cell and non-small cell lung cancer; breast cancer sarcomas, such as fibrosarcoma, malignant fibrous histiocytoma, embryonal rhabdomysocarcoma, leiomysosarcoma, neurofibrosarcoma, osteosarcoma, synovial sarcoma, liposarcoma, and alveolar soft part sarcoma; neoplasms of the central nervous systems, particularly brain cancer
  • non-cancer disorders that are characterized by cellular hyperproliferation are treated.
  • Illustrative examples of such disorders include but are not limited to: atrophic gastritis, inflammatory hemolytic anemia, graft rejection, inflammatory neutropenia, bullous pemphigoid, coeliac disease, demyelinating neuropathies, dermatomyositis, inflammatory bowel disease (ulcerative colitis and Crohn's disease), multiple sclerosis, myocarditis, myositis, nasal polyps, chronic sinusitis, pemphigus vulgaris, primary glomerulonephritis, psoriasis, surgical adhesions, stenosis or restenosis, scleritis, scleroderma, eczema (including atopic dermatitis, irritant dermatitis, allergic dermatitis), periodontal disease (i.e., periodontitis), polycystic kidney disease,
  • vasculitis e.g., Giant cell arteritis (temporal arteritis, Takayasu's arteritis), polyarteritis nodosa, allergic angiitis and granulomatosis (Churg-Strauss disease), polyangitis overlap syndrome, hypersensitivity vasculitis (Henoch-Schonlein purpura), serum sickness, drug- induced vasculitis, infectious vasculitis, neoplastic vasculitis, vasculitis associated with connective tissue disorders, vasculitis associated with congenital deficiencies of the complement system, Wegener's granulomatosis, Kawasaki's disease, vasculitis of the central nervous system, Buerger's disease and systemic sclerosis); gastrointestinal tract diseases (e.g., pancreatitis, Crohn's disease, ulcerative colitis, ulcerative proctitis, primary sclerosing cholangitis, benign strictures of any cause including ideopathic (e.g.,
  • Bone matrix is a material in a continuous state of remodeling, with specialized cells
  • osteoblasts continuously depositing bone matrix and other specialized cells (osteoclasts) continuously eroding it.
  • osteoclasts specialized cells
  • Osteoclast-mediated bone erosion proceeds via the secretion of acid onto the bone surface by vacuolar H + -ATPaSe located in the osteoclast membrane.
  • Bafilomycin A 1 is a macrolide cytotoxin that was originally identified as an antibacterial and antifungal agent (Hagenmaier et al, US 4,558,139 (1985)) but has since been shown to be also a potent and specific inhibitor of vacuolar H + -ATPaSe. The bone resorption process can be blocked by inhibition of osteoclastic vacuolar H + -ATPaSe by bafilomycin A 1 .
  • Sundquist et al Biochem. Biophys. Res. Commun., 168 (1), 309-313 (1990); Mattsson et al, J. Biol. Chem. 269 (40), 24979-24982 (1994).
  • conjugates according to this invention where the active agent D is bafilomycin A 1 or a related compound can be used to treat osteoporosis.
  • Position 21 of bafilomycin A 1 is a suitable position both for modification of bafilomycin A 1 with a cell membrane-impermeabilizing group Z and for attachment to a linker moiety L (and, via linker moiety L, a targeting moiety T).
  • a linker moiety L and, via linker moiety L, a targeting moiety T.
  • the targeting moiety T wherein the target cell is an osteoclast can be RANKL (receptor activator or NF- ⁇ ligand) or TNF- ⁇ , both of which are implicated in osteoclastogenesis.
  • RANKL receptor activator or NF- ⁇ ligand
  • TNF- ⁇ TNF- ⁇
  • bafilomycin A 1 also inhibits acidification of lysosomes (Yoshimori et al, J. Biol. Chem. 266 (26), 17707-17712 (1991), for which reason it may be desirable to avoid pH sensitive or acid hydrolase- cleavable linker moieties L in this particular embodiment and use instead other types of linker moieties L.
  • the method of treating the aforementioned diseases comprises administering a therapeutically effective amount of an inventive combination to a subject.
  • the method may be repeated as necessary.
  • the practice of this invention can be further understood by reference to the following examples, which are provided by way of illustration and not of limitation.
  • Fig. 7 depicts the synthesis of maytansinoid compounds usable as modified active agents in conjugates of this invention, as detailed in this example.
  • the preparation of maytansinoid conjugates is also described in Santi et al, US 2003/0109682 Al (2003), the disclosure of which is incorporated herein by reference.
  • Maytansinol-3-bromoacetate (2 ).
  • Maytansinol (1) is obtained by reduction of ansamitocin P3 (AP3), as described in Kupchan et al, J. Am. Chem. Soc, 97, 5294-5295 (1975), and is then acylated according to the procedure of Kawai et al, Chem. Pharm. Bull, 32, 1001-1002 (1984), the disclosure of both documents being incorporated herein by reference.
  • AP3 ansamitocin P3
  • 3-Bromoacetyl-20-desmethyl-maytansine (3 ).
  • Treatment of maytansinol 3- bromoacetate (2) with Bacillus megaterium results in C20 demethylation to generate 3- bromoacetyl-20-desmethyl-maytansine, as described in Isawa et al, J. Antibiotics, 34 (12), 1587-1590 (1981), incorporated herein by reference.
  • Trimethylammoniumacetyl-20-desmethylmaytansine 5
  • ethanol 3-5 eq.
  • aniline 2-4 eq.
  • Trimethylammoniumacetyl-20-desmethylmaytansine 5
  • To a solution of 3- bromoacetyl-20-desmethyl-maytansine (3) (1 eq) in anhydrous THF is added a solution of trimethylamine (1.1 eq) in anhydrous THF. The reaction is stirred at room temperature until bromoacetate (3) no longer persists by TLC analysis. The product is isolated by concentration under reduced pressure.
  • Trimethylammonium compound 5 is usable in conjugates such as those shown in formulae IV, V, and VI.
  • Fig. 8 shows the synthesis of leptomycin B compounds usable as modified active agents in conjugates of this invention, as detailed in this example.
  • Phosphono leptomycin B (10).
  • An Arbuzov type reaction is employed for the synthesis of phosphonoacetamide leptomycins, as described above in Example 1.
  • a solution of bromoacetamide LMB derivative 9 (1 eq) in anhydrous THF is added a solution of tris(trimethylsilyl) phosphite (2 eq) in anhydrous THF.
  • the reaction is stirred at rt until the bromoacetamide no longer persists by TLC analysis.
  • phosphonic acid 10 which is usable in conjugates of this invention having structures according to formulae VII, VIH, and IX.
  • Trimetl ⁇ ylammonium acetamide leptomycin B (11 ). To a solution of bromoacetamide leptomycin B 8 (1 eq) in anhydrous THF is added a solution of trimethylamine (1.5 eq) in anhydrous THF. The reaction is stirred at room temperature until the bromoacetamide no longer persists by TLC analysis. The product is isolated by concentration under reduced pressure, to yield an intermediate usable for making conjugates such as X, XI, and XTT.
  • This example describes the preparation of maleimide linked conjugates.
  • MC- VaI-Ci t-P AB -OH is prepared as described in Dubowchik et al, Bioconjugate Chem., 13, 855-869 (2002) ("Dubowchik et al ", incorporated herein by reference).
  • MC stands for a maleimidocaproyl moiety
  • VaI stands for a valyl moiety
  • Cit stands for a citrullinyl moiety
  • PAB stands for p-aminobenzyl.
  • active agents or modified active agents can be connected to linker moieties via an ether linkage according to the procedure of Told et al, J. Org. Chem., 67, 1866-1872 (2002) ("Told et al", incorporated herein by reference).
  • a two-step sequence involving Fmoc deprotection and acylation with MC-OSu (OSu denoting an N-hydroxysuccinate group; Dubowchik et al.) is used to provide the desired active agent (or modified active agent) maleimide linker moiety intermediates.
  • active agents or modified active agents can be connected to linker moieties via a carbamate linkage according to the procedure of Dubowchik et al.
  • MC-Val-Cit-PABOH (1 eq) and bis-p-nitrophenyl carbonate (bis-PNP carbonate) (5 eq) are dissolved in anhydrous dichloromethane at room temperature under nitrogen.
  • DIPEA N-ethyldiisopropylamine
  • This example describes the preparation of amide linked conjugates.
  • Fmoc-Val-Cit-PABOH was prepared according to the procedure of Dubowchik et al
  • Ether linked intermediates are prepared according to the procedure of Toki et al. , with the following detailed procedure being representative.
  • triphenylphosphine (1.1 eq) and the phenolic drug (1.1 eq) are dissolved in DMF:toluene (1:1) and evaporated to dryness under high vacuum. The residue is dissolved in anhydrous DMF under nitrogen. After cooling the reaction vessel in a 0 0 C ice bath, neat DIAD (1.1 eq) is added dropwise over a 1 min period.
  • Carbamate linked intermediates are prepared according to the procedure of Dubowchik et al. Fmoc-Val-Cit-PABOH (1 eq) and bis- PNP carbonate (5 eq) are dissolved in anhydrous dichloromethane at room temperature under nitrogen. To this solution is added DIPEA (3 eq), and the reaction is stirred at room temperature for 3 days. The solution is then concentrated to near dryness, and the resulting residue is diluted with ethyl acetate and washed with phosphate buffer (pH 5), water and brine. The organic phase is then separated, dried over magnesium sulfate, filtered and concentrated under reduced pressure.
  • Antibodies or protein carriers (5-100 mg/mL) are activated according to the procedure of Trouet et al, Proc. Nat'lAcad. Sci. USA, 79, 626-629 (1982), incorporated by reference. Antibodies are dissolved in water (pH 7.5) and to this solution is added succinic (or glutaric) anhydride stepwise while maintaining the pH at 7.5. The succinylated protein is then extensively dialyzed against PBS, sterilized by filtration and maintained at 4 0 C.
  • active agent (or modified active agent)-linker moiety intermediate (20 ⁇ mol) is added to the succinylated protein (50 mg, 5 mL of saline at 10 mg/mL).
  • EDC 7.5 mg
  • Additional EDC is added, and the solution is maintained at room temperature overnight.
  • the solution is then concentrated by ultrafiltration, and gel filtration is used to remove any low molecular weight impurities. This general procedure can be used for the synthesis of conjugates such as II, V, VIII and XI.

Abstract

L’invention concerne le conjugué d'un principe actif et d’une fraction de ciblage montrant de l'affinité pour une cellule cible, dans lequel le principe actif a été modifié par fixation d'un groupe à fonction imperméabilisante de membrane cellulaire, afin que, si le principe actif ainsi modifié est clivé du conjugué dans le plasma sanguin au lieu d'à l'intérieur de la cellule cible, le groupe à fonction imperméabilisante de membrane cellulaire empêchera ou limitera l’entrée du principe actif modifié dans les cellules, réduisant ainsi ses effets adverses systémiques ou non spécifiques, y compris toxiques.
PCT/US2005/021418 2005-06-09 2005-06-16 Conjugués à effets adverses systémiques réduits WO2006135371A1 (fr)

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WO2008028934A1 (fr) * 2006-09-06 2008-03-13 Aeterna Zentaris Gmbh Conjugués de disorazoles et leurs dérivés avec des molécules se fixant à des cellules, nouveaux dérivés de disorazole et procédés de fabrication et d'utilisation de ces conjugués et dérivés
CN101578287B (zh) * 2006-09-06 2012-09-05 阿特纳赞塔里斯有限公司 地索拉唑及其衍生物与细胞结合分子的轭合物，新的地索拉唑衍生物，其制备方法及其应用
CN101466409B (zh) * 2006-06-09 2012-11-21 赛诺菲-安万特 细霉素衍生物
EP2635301A4 (fr) * 2010-11-03 2016-06-01 Immunogen Inc Agents cytotoxiques comprenant de nouveaux dérivés d'ansamitocine
WO2018159582A1 (fr) 2017-02-28 2018-09-07 学校法人近畿大学 Méthode de traitement du cancer du poumon non à petites cellules résistant à l'egfr-tki par administration d'un conjugué anticorps anti-her3-médicament
WO2019094395A3 (fr) * 2017-11-07 2019-08-22 Regeneron Pharmaceuticals, Inc. Lieurs hydrophiles pour conjugués anticorps-médicament
WO2020059772A1 (fr) 2018-09-20 2020-03-26 第一三共株式会社 Traitement d'un cancer à her3 mutant par l'administration d'un conjugué anticorps anti-her3-médicament
US11377502B2 (en) 2018-05-09 2022-07-05 Regeneron Pharmaceuticals, Inc. Anti-MSR1 antibodies and methods of use thereof
US11491237B2 (en) 2017-05-18 2022-11-08 Regeneron Pharmaceuticals, Inc. Cyclodextrin protein drug conjugates
US11760775B2 (en) 2016-11-08 2023-09-19 Regeneron Pharmaceuticals, Inc. Steroids and protein-conjugates thereof

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US6759509B1 (en) * 1996-11-05 2004-07-06 Bristol-Myers Squibb Company Branched peptide linkers

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US6214345B1 (en) * 1993-05-14 2001-04-10 Bristol-Myers Squibb Co. Lysosomal enzyme-cleavable antitumor drug conjugates
US6759509B1 (en) * 1996-11-05 2004-07-06 Bristol-Myers Squibb Company Branched peptide linkers
US20030096743A1 (en) * 2001-09-24 2003-05-22 Seattle Genetics, Inc. p-Amidobenzylethers in drug delivery agents

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101466409B (zh) * 2006-06-09 2012-11-21 赛诺菲-安万特 细霉素衍生物
WO2008028934A1 (fr) * 2006-09-06 2008-03-13 Aeterna Zentaris Gmbh Conjugués de disorazoles et leurs dérivés avec des molécules se fixant à des cellules, nouveaux dérivés de disorazole et procédés de fabrication et d'utilisation de ces conjugués et dérivés
CN101578287B (zh) * 2006-09-06 2012-09-05 阿特纳赞塔里斯有限公司 地索拉唑及其衍生物与细胞结合分子的轭合物，新的地索拉唑衍生物，其制备方法及其应用
US8470776B2 (en) 2006-09-06 2013-06-25 Aeterna Zentaris Gmbh Conjugates of disorazoles and their derivatives with cell-binding molecules, novel disorazole derivatives, processes of manufacturing and uses thereof
EP1900742A1 (fr) * 2006-09-07 2008-03-19 AEterna Zentaris GmbH Conjugues des disorazoles et leurs dérives avec des molécules de liaison cellulaire, nouveaux dérives de disorazole, leurs modes de préparation et utilisation
CN106349254A (zh) * 2010-11-03 2017-01-25 伊缪诺金公司 包含新型安丝菌素衍生物的细胞毒性剂
EP2635301A4 (fr) * 2010-11-03 2016-06-01 Immunogen Inc Agents cytotoxiques comprenant de nouveaux dérivés d'ansamitocine
US11760775B2 (en) 2016-11-08 2023-09-19 Regeneron Pharmaceuticals, Inc. Steroids and protein-conjugates thereof
WO2018159582A1 (fr) 2017-02-28 2018-09-07 学校法人近畿大学 Méthode de traitement du cancer du poumon non à petites cellules résistant à l'egfr-tki par administration d'un conjugué anticorps anti-her3-médicament
US11491237B2 (en) 2017-05-18 2022-11-08 Regeneron Pharmaceuticals, Inc. Cyclodextrin protein drug conjugates
WO2019094395A3 (fr) * 2017-11-07 2019-08-22 Regeneron Pharmaceuticals, Inc. Lieurs hydrophiles pour conjugués anticorps-médicament
US11377502B2 (en) 2018-05-09 2022-07-05 Regeneron Pharmaceuticals, Inc. Anti-MSR1 antibodies and methods of use thereof
WO2020059772A1 (fr) 2018-09-20 2020-03-26 第一三共株式会社 Traitement d'un cancer à her3 mutant par l'administration d'un conjugué anticorps anti-her3-médicament

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