Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
  • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
  • C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

• This invention provides prodrug and conjugates thereof, uses, kits and pharmaceutical compositions thereof.
• ADCs Antibody Drug conjugates
• ADCs Antibody drug conjugates
• ADCs present a unique opportunity to increase the safety of highly toxic drugs by utilizing the specificity of antibodies to obtain targeted delivery of potent drugs to specific tissues.
• Much of the success of ADCs is due to technological advances made in the design of the linkage between the antibody and the therapeutic payload.
• cleavable linkers are generally preferred to non-cleavable linkers due to their range of applicability, there is a greater potential for instability in circulation. The success of cleavable linkers therefore depends on their ability to effectively differentiate between circulatory and target-cell conditions.
• ADC linkers are not only defined by the cleavable linker technology but also the attachment site on the antibody. Conjugation to a large antibody renders linkers less accessible to chemical and enzymatic triggers, thereby increasing plasma stability and slowing target cell release rates. [005] This effect can be further amplified when site-selective bioconjugation techniques are employed to attach the linker-payload to less solvent-accessible sites.
• this invention provides a compound represented by Formula
• R 1 is a reactive moiety
• R 2 and R 3 are the same or different active pharmaceutical ingredient moiety
• R 4 is a hydrophilic moiety
• X 1 is a mono-, di-, tri-, tetra-, oligo- or polypeptide moiety, an oligo or polyethylene glycol or an oligo or polyvinylalcohol or oligo- or poly -glycerol moiety.
• this invention provides a Conjugate represented by Formula (1(a)):
• R 2 and R 3 are the same or different active pharmaceutical ingredient moiety
• R 4 is a hydrophilic moiety
• R 5 is an antibody or antigen moiety
• X 1 is a mono-, di-, tri-, tetra-, oligo- or polypeptide moiety, an oligo or polyethylene glycol or an oligo or polyvinylalcohol or oligo- or poly -glycerol moiety;
• X 2 is a linker; and n is a number between 0.01 - 10.
• this invention provides the compound or the conjugate as described hereinabove, for use in treating cancer
• this invention provides a kit comprising: i) the compound as described hereinabove; and ii) an antibody or an antigen.
• Figures 1A-1D depict cytotoxic effects of the conjugates of this invention on various cell viability assays (exposure time: 5 days).
• Figure 1A HCC1954 cell viability following 120 h (5 days) treatment with serial dilutions of the conjugates
• Figure IB MDA- MB-231 cell viability following 120 h (5 days) treatment with serial dilutions of the conjugates
• Figure 1C Table summarizing the IC50 values of the different treatments in HCC1954 cells
• Figure ID Table summarizing the IC50 values of the different treatments in MDA-MB-231 cells.
• data represent mean +SD; and is representative of 2 individual experiments.
• Figures 2A-2D depict cytotoxic effects of the conjugates of this invention on various cell viability assays (exposure time: 6 days).
• Figure 2A Viability of HCC1954
• Figure 2B Viability of JIMT-1
• Figure 2C Viability of MDA-MB-468 cells, following 144 h (6 days) treatment with serial dilutions of Trastuzumab, Exatecan or Tras- Exa-1
• Figure 2D Table summarizing the IC50 values of the different treatments, where first two lines are IC50 values, “Tras-Exa/Exa fold change” is the division of Tras-Exa divided by exa values and “+” and “++” denote her2 levels as found in the literature - not detected, low level and high level, respectively (relative levels, as in e.g.
• Figures 3A-3D show that Trastuzumab -based ADCs lead to a dose-dependent inhibition of tumor growth.
• Figure 3D Body weight change, expressed as percent change from the day of tumor cell inoculation. Data are presented as mean ⁇ s.e.m.
• Figures 4A-4D show that Trastuzumab-based ADCs prolong the survival of mice in a dose-dependent manner.
• Figure 4D Median survival and individual time-point to death. Dashed line marks the pre-defined end point of the study (day 152).
• this invention provides a compound represented by Formula
• R 1 is a reactive moiety
• R 2 and R 3 are the same or different active pharmaceutical ingredient moiety
• R 4 is a hydrophilic moiety
• X 1 is a mono-, di-, tri-, tetra-, oligo- or polypeptide moiety, an oligo- or poly-ethylene glycol or an oligo- or poly-vinylalcohol or oligo- or poly-glycerol moiety.
• R 1 is a reactive moiety.
• a “reactive moiety” in the context of this invention is defined as a chemical moiety (e.g. alkylene group) which may contain functional group or groups (identical or different), wherein at least one such functional group is able to react or interact with an antibody or an antigen in order to conjugate such moiety and the antibody or antigen.
• a thiol of Cys or an amine of Lys from the antibody or an antigen reacts with this moiety in a 1,4 (Michael) addition.
• R 1 comprises dihydropyridazine maleimide, bromoacetamide, tetrazine, alkyne or amine moiety or any combination thereof.
• R 1 comprises an alkyne moiety.
• R 1 comprises a constrained alkyne moiety.
• R 1 comprises a cycloalkyne moiety.
• cyclooctyne moieties include dibenzocyclooctyne (DBCO), bicyclo[6.1.0]nonyne (BCN) and biarylazacyclooctynone (BARAC) moieties.
• DBCO dibenzocyclooctyne
• BCN bicyclo[6.1.0]nonyne
• BARAC biarylazacyclooctynone
• R 2 of Formula (I) is an anti-cancer, immune stimulating (for immune-oncology) or immune dampening (e.g. steroids for immunology) agent.
• R 2 is an agent that is used in immunology, immune-oncology, in treating cancer-related disease and/or disorder or in treating deficiencies/malfunctions of the immune system.
• the conditions/disorders related/associated with cancer and/or deficiencies/malfunctions of the immune system include at least one of the following non limiting examples: auto-immune diseases, arthritis, ectopic dermatitis, Crohn’s disease, colitis, IBD, fibrosis, psoriasis, Lupus, Multiple Sclerosis, neuro- degenerative disease (Parkinson, Alzheimer, ALS, myasthenia gravis, Graves’ disease, Ankylosing spondylitis), and respiratory disease (COPD, Asthma).
• R 2 is an exatecan, belotecan, camptothecin, auristatin, Monomethyl auri statin E (MMAE) or a doxorubicin moiety.
• R 2 is exatecan, auristatin or belotecan.
• R 3 of Formula (I) is an anti-cancer, immune stimulating (for immune-oncology) or immune dampening (e.g. steroids for immunology) agent.
• R 3 is an agent that is used in immunology, immune-oncology, in treating cancer-related disease and/or disorder or in treating deficiencies/malfunctions of the immune system.
• the conditions/disorders related/associated with cancer and/or deficiencies/malfunctions of the immune system include at least one of the following non limiting examples: auto-immune diseases, arthritis, ectopic dermatitis, Crohn’s disease, colitis, IBD, fibrosis, psoriasis, Lupus, Multiple Sclerosis, neuro- degenerative disease (Parkinson, Alzheimer, ALS, myasthenia gravis, Graves’ disease, Ankylosing spondylitis), and respiratory disease (COPD, Asthma).
• auto-immune diseases arthritis, ectopic dermatitis, Crohn’s disease, colitis, IBD, fibrosis, psoriasis, Lupus, Multiple Sclerosis, neuro- degenerative disease (Parkinson, Alzheimer, ALS, myasthenia gravis, Graves’ disease, Ankylosing spondylitis), and respiratory disease (COPD, Asthma).
• R 3 is an exatecan, belotecan, camptothecin, auristatin, Monomethyl auristatin E (MMAE) or a doxorubicin moiety.
• R 3 is exatecan, auristatin, Monomethyl auristatin E (M MAE) or belotecan.
• R 2 and R 3 of Formula (I) are anticancer, immune stimulating (for immune-oncology) or immune dampening agents (e.g. steroids), or any agents used in immunology, immune-oncology, in treating cancer-related disease and/or disorder or in treating deficiencies/malfunctions of the immune system, where said conditions/disorders related/associated with cancer and/or deficiencies/malfunctions of the immune system are as described hereinabove (see R 2 and/or R 3 embodiments).
• R 2 and R 3 are each independently exatecan, belotecan, auristatin, Monomethyl auristatin E (MMAE) camptothecin or a doxorubicin moiety.
• R 2 and R 3 are each independently exatecan, auristatin, Monomethyl auristatin E (MMAE) or belotecan moieties. In another embodiment, R 2 and R 3 are each independently exatecan moieties. In another embodiment, R 2 and R 3 are belotecan moieties.
• R 4 of Formula (I) comprises an oligo- or poly- carboxylic acid moiety or oligo- or poly-ethylene glycol or oligo- or poly-alcohol or oligo- or poly- vinylalcohol or oligo- or poly-glycerol moiety.
• R 4 is represented by:
• X 1 of Formula (I) is a mono-, di-, tri-, tetra-, oligo- or polypeptide moiety or an oligo- or poly-ethylene glycol or an oligo- or poly-vinylalcohol or oligo- or poly-glycerol moiety.
• X 1 is a mono-, di-, tri-, tetra-, oligo- or polypeptide comprising any amino acid: natural, non-natural (artificial), standard or non — standard, or combination thereof, in any order/sequence.
• X 1 is CGKRK, -Val-Cit-, -Ala-Ala-, AAN, GGFG, -Val-Arg- or -Val-Ala- or any combination thereof. In one other embodiment, X 1 is -Val-Cit-, -Ala-Ala-, AAN, GGFG, -Val-Arg- or - Val-Ala- or any combination thereof. In another embodiment, X 1 comprises a sequence (e.g. Ala-Ala) cleavable by specific proteases, non-limiting examples include Cathepsin B and Legumain. Each possibility represents a separate embodiment of this invention.
• the compound of Formula (I) is represented by the structure of Formula 1,17, 18, 19 or 20:
• TFA is trifluoroacetic acid
• the compound of Formula (I) is a prodrug which is conjugated to an antibody or an antigen via R 1 which reacts and/or interacts with the relevant moieties/portions/functional groups/amino acids of the antibody or antigen, wherein the product of said reaction is the conjugate presented herein below in the following section, e.g. by Formula (1(a), 1(a), 2(a) and/or compound 21.
• R 1 reacts with artificial amino-acid such as azido- tyrosine or with alkynes to form said conjugate.
• the antibody/antigen reacts with the compound of Formula (I), and then it interacts with some portion of said compound (e.g. some peptide recognition motif/trigger), an interaction that might trigger the compound to release the covalently -loaded drugs.
• the antibody is trastuzumab or rituximab.
• this invention provides a conjugate represented by Formula
• R 5 is an antibody or antigen moiety
• X 2 is a linker; n is a number between 0.01 - 10; and
• R 2 -R 4 and X 1 are described hereinabove.
• this invention provides a conjugate represented by Formula (1(a)), prepared by reacting the prodrug or compound of Formula (I) with an antibody or an antigen, wherein all definitions and structures of the formulae are described hereinabove and/or below.
• the bond between the antigen/antibody moiety (R 5 ) and the rest of Formula (1(a)) can be covalent or non-covalent.
• the term “non covalent bond” refers within the meaning of this invention to any non-covalent interaction/bond, such as van der waals/ interaction/London dispersion forces, hydrogen bonding, halogen bonding, pie-pie (p-p) interactions, ionic bonding (e.g. salt bridge between antibody or antigen of R 5 and the peptide moiety of X 1 ), etc., as known in the art.
• R 5 is connected in more than one position thereof to the rest of Formula (1(a)), in an identical or different interaction/bond at each position.
• R 5 is connected covalently to one or more compounds of the rest of Formula (1(a)) and also connected/interacts non-covalently with one or more such compounds, at a different position compared to the covalent attachment point.
• the “rest of Formula (1(a))” refers to
• curly bond denotes the bond to R 5 (not presented).
• X 2 of Formula (1(a)) comprises succinimide, acetamide, dihydropyridazine, atkene or amine moiety or any combination thereof.
• X 2 comprises an alkene moiety.
• X 2 comprises a constrained alkene moiety.
• X 2 is represented by
• n is between 0.01-10. In one embodiment, n is between 0.01-1. In another embodiment, n is between 0.01-2. In another embodiment, n is between 0.01-5. In another embodiment, n is between 0.05-1. In another embodiment, n is between 0.05-2. In another embodiment, n is between 0.1-1. In another embodiment, n is between 0.1-2. In another embodiment, n is between 0.5-1. In another embodiment, n is between 0.01-0.5. In another embodiment, n is between 0.1-0.5. In another embodiment, n is between 0.5-2. Each possibility represents a separate embodiment of this invention.
• R 5 of Formula (1(a)) is an antibody or an antigen.
• R 5 is an antibody targeting tumor.
• R 5 is an immune cell specific antigen.
• R 5 is an anti-EGFR or anti-CD33 antibody.
• the antibody of R 5 is a model antibody or a moiety /fragment which “simulates” an antibody, where such model/moiety/fragment is exemplified by, inter alia, the CGKRK peptide.
• R 5 is any one of the CEACAM family, Folate receptor, ER, PR, Notch receptors, Notch ligands, PSMA, Glypican-3, Mesothelin, MET, EGFR, Erb2, EpCAM, NCAM, EphrinA4, IGF-1R, FAP, Ly6E, Cadherins family, VEGFR-2, RNF43, MUC family, PD-1, PD-L1, FAK, CCR2, CCR4, CXCR1, CXCR2, Nectin-4, Transferrin, TIM-1, LAG-3, Axl, CTLA-4, 4- IBB, MART-1, TIGIT, SLAMF6, 0X40, trastuzumab or rituximab.
• R 5 is trastuzumab or rituximab or CGKRK moiety. In another embodiment, R 5 is trastuzumab moiety. In another embodiment, R 5 is rituximab moiety. In another embodiment, R 5 is CGKRK moiety.
• the compound of Formula (1(a)) is represented by the structure of Formula 1(a) or 2(a):
• R 5 is as described hereinabove.
• the compound of Formula (1(a)) is represented by the structure of compound 21:
• the compound represented by the structure of e.g. Formula (I) or 1, 17, 18, 19 or 20 is also termed “prodrug” and it can be conjugated with an antibody or an antigen to form the conjugate.
• conjugate in the context of this invention, can be interchangeably termed “antibody-drug conjugate” (ADC).
• the drugs are released from the covalent conjugate via reaction with a protease and/or hydrolase and/or other enzyme which leads subsequently to additional cascade of reactions.
• a protease and/or hydrolase and/or other enzyme which leads subsequently to additional cascade of reactions.
• an example of said mechanism/cascade is presented in the following below (cathepsin B is the protease presented):
• the peptide in the above scheme or any other feasible antibody/antigen of R 5 affords targeted delivery of the conjugate to the desired (biological) site, in which it is activated by some trigger, e.g. reaction with a hydrolase/protease as exemplified above.
• compositions comprising the prodrug, antibody or antigen and/or the conjugate thereof
• this invention provides a pharmaceutical composition
• a pharmaceutical composition comprising a therapeutically effective amount of the prodrug or the conjugate thereof as described hereinabove and optionally at least one pharmaceutically acceptable carrier, diluent, vehicle or excipient.
• the composition comprises a combination of the prodrug and an antibody or an antigen, and at least one pharmaceutically acceptable carrier, diluent, vehicle or excipient.
• the pharmaceutical composition is in a form selected from the group consisting of tablets (including e.g.
• the composition is a solid state composition (e.g. tablet, pill, capsule, pellet, granule, powder etc.). Each possibility represents a separate embodiment of this invention.
• pharmacologically acceptable carriers, diluents, vehicles or excipients that may be used in the context of this invention include, but are not limited to, surfactants, lubricants, binders, fillers, compression aids, disintegrants, water-soluble polymers, inorganic salts, preservatives, antioxidants, coloring agents, sweetening agents, souring agents, bubbling agents and flavorings.
• surfactants lubricants
• binders fillers
• compression aids disintegrants
• water-soluble polymers inorganic salts
• preservatives antioxidants
• coloring agents coloring agents
• sweetening agents souring agents
• bubbling agents and flavorings are examples of this invention.
• Suitable carriers, diluents, vehicles or excipients within this invention include e.g. lactose, D-mannitol, starch, cornstarch, crystalline cellulose, light silicic anhydride and titanium oxide.
• Suitable surfactants include e.g. lecithin and phosphatidylcholine.
• Suitable lubricants include e.g. magnesium stearate, sucrose fatty acid esters, polyethylene glycol, talc and stearic acid.
• Suitable binders include e.g.
• Suitable disintegrants include e.g. crosslinked povidone (any crosslinked l-ethenyl-2-pyrrolidinone homopolymer including polyvinylpyrrolidone (PVPP) and 1 -vinyl-2 -pyrrolidinone homopolymer), crosslinked carmellose sodium, carmellose calcium, carboxymethyl starch sodium, low- substituted hydroxypropyl cellulose, cornstarch and the like.
• Suitable water-soluble polymers include e.g. cellulose derivatives such as hydroxypropyl cellulose, polyvinylpyrrolidone, hydroxypropylmethyl cellulose, methyl cellulose and carboxymethyl cellulose sodium, sodium polyacrylate, polyvinyl alcohol, sodium alginate, guar gum, and the like.
• Suitable inorganic salts include e.g. basic inorganic salts of sodium, potassium, magnesium and/or calcium.
• Particular embodiments include the basic inorganic salts of magnesium and/or calcium.
• Basic inorganic salts of sodium include, for example, sodium carbonate, sodium hydrogen carbonate, disodiumhydrogenphosphate, and the like. Each possibility represents a separate embodiment of this invention.
• Basic inorganic salts of potassium include, for example, potassium carbonate, potassium hydrogen carbonate, and the like. Each possibility represents a separate embodiment of this invention.
• Basic inorganic salts of magnesium include, for example, heavy magnesium carbonate, magnesium carbonate, magnesium oxide, magnesium hydroxide, magnesium metasilicate aluminate, magnesium silicate, magnesium aluminate, synthetic hydrotalcite, aluminahydroxidemagnesium, and the like. Each possibility represents a separate embodiment of this invention.
• Basic inorganic salts of calcium include, for example, precipitated calcium carbonate, calcium hydroxide, and the like. Each possibility represents a separate embodiment of this invention.
• Suitable preservatives include e.g. sodium benzoate, benzoic acid, and sorbic acid. Each possibility represents a separate embodiment of this invention.
• Suitable antioxidants include e.g. sulfites, ascorbic acid and a-tocopherol. Each possibility represents a separate embodiment of this invention.
• Suitable coloring agents include e.g. food colors such as Food Color Yellow No. 5, Food Color Red No. 2 and Food Color Blue No. 2, and the like. Each possibility represents a separate embodiment of this invention.
• Suitable sweetening agents include e.g. dipotassium glycyrrhetinate, aspartame, stevia and thaumatin. Each possibility represents a separate embodiment of this invention.
• Suitable souring agents include e.g. citric acid (citric anhydride), tartaric acid and malic acid. Each possibility represents a separate embodiment of this invention.
• Suitable bubbling agents include e.g. sodium bicarbonate.
• Suitable flavorings include synthetic substances or naturally occurring substances, including e.g. lemon, lime, orange, menthol and strawberry. Each possibility represents a separate embodiment of this invention.
• the compounds (prodrug and/or conjugate thereof) of this invention are useful as pharmaceuticals for medical treatment.
• This invention thus provides pharmaceutical compositions comprising the prodrug or the conjugate thereof disclosed herein, or a combination of the prodrug and an antibody or an antigen, and at least one pharmaceutically acceptable carrier, diluent, vehicle or excipient.
• the compounds of this invention can be safely administered orally or non-orally. Routes of administration include, but are not limited to, oral, topical, subcutaneous, intraperitoneal, rectal, intravenous, intra arterial, transdermal, intramuscular, topical, and intranasal. Each possibility represents a separate embodiment of this invention.
• Additional routes of administration include, but are not limited to, mucosal, nasal, parenteral, gastrointestinal, intraspinal, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intracerebroventricular, intracerebral, ophthalmic, buccal, epidural and sublingual. Each possibility represents a separate embodiment of this invention.
• the tablets and other solid dosage forms of the pharmaceutical compositions described herein may optionally be stored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices and the like.
• the active ingredient can also be in micro -encapsulated form, if appropriate, with one or more of the above-described excipients. Each possibility represents a separate embodiment of this invention.
• this invention provides a method of treating cancer or related disorders/conditions, comprising the step of administering to a subject in need thereof the prodrug (e.g. compound of Formula (I)), conjugate thereof (e.g. compound of Formula (1(a))) or a combination of the prodrug and an antibody or an antigen or a pharmaceutical composition comprising the prodrug/conjugate/combination and at least one pharmaceutically acceptable carrier, diluent, vehicle or excipient.
• this invention provides a method of treating cancer or related disorders/conditions, comprising the step of administering to a subject in need thereof the conjugate (e.g.
• the method is an immunological method.
• the method is an immune -oncological method.
• this invention provides a method of treating conditions and/or diseases associated with deficiencies/malfunctions of the immune system, comprising the step of administering to a subject in need thereof the prodrug (e.g. compound of Formula (I), conjugate thereof (e.g. compound of Formula (1(a)) or a combination of the prodrug and an antibody or an antigen or a pharmaceutical composition comprising the prodrug/conjugate/combination and at least one pharmaceutically acceptable carrier, diluent, vehicle or excipient.
• this invention provides a method of treating cancer or related disorders/conditions, comprising the step of administering to a subject in need thereof the conjugate (e.g.
• the method is an immunological method.
• the method is an immune- oncological method.
• the conditions/disorders related/associated with cancer and/or deficiencies/malfunctions of the immune system include at least one of the following non limiting examples: auto-immune diseases, arthritis, ectopic dermatitis, Crohn’s disease, colitis, IBD, fibrosis, psoriasis, Fupus, Multiple Sclerosis, neuro-degenerative disease (Parkinson, Alzheimer, AFS, myasthenia gravis, Graves’ disease, Ankylosing spondylitis), and respiratory disease (COPD, Asthma).
• this invention provides a method of treating cancer, comprising administering: a) the prodrug (e.g. compound of Formula (I); and b) an antibody or an antigen; or administering the conjugate of the prodrug and the antibody or antigen, to a subject in need thereof.
• the prodrug e.g. compound of Formula (I)
• an antibody or an antigen an antibody or an antigen
• the cancer is a breast, kidney, brain, skin, ovary, lung, pancreas, colon, head/neck, prostate, endometrium, liver, blood, stomach, fibrosarcoma, bone neoplasms/osteosarcoma or duodenum cancer.
• the cancer is a breast cancer.
• the administration of the prodrug (e.g. compound of Formula (I) and the antibody or antigen is subsequent or in conjunction. Each possibility represents a separate embodiment of this invention.
• the subject is a mammal, preferably a human.
• this invention provides the prodrug (e.g. compound of Formula (I), conjugate thereof (e.g. compound of Formula (1(a)) or a combination of the prodrug and an antibody or an antigen or a pharmaceutical composition comprising the prodrug/conjugate/combination and at least one pharmaceutically acceptable carrier, diluent, vehicle or excipient - for use in the treatment of cancer or the conditions/disorders related/associated with cancer and/or of deficiencies/malfunctions of the immune system, as described within the above method embodiments.
• the prodrug e.g. compound of Formula (I)
• conjugate thereof e.g. compound of Formula (1(a)
• an antibody or an antigen or a pharmaceutical composition comprising the prodrug/conjugate/combination and at least one pharmaceutically acceptable carrier, diluent, vehicle or excipient - for use in the treatment of cancer or the conditions/disorders related/associated with cancer and/or of deficiencies/malfunctions of the immune system, as described within
• this invention provides a kit comprising: i) the prodrug as described hereinabove (e.g. compound of Formula (I); and ii) an antibody or an antigen.
• kit further comprises instructions for use.
• the term "therapeutically effective amount” as used herein refers to an amount of an agent which is effective, upon single or multiple dose administration to the subject in providing a therapeutic benefit to the subject.
• the crystalline forms of the present invention are used for the preparation of a medicament for treating the aforementioned diseases or disorders.
• treating in regards to medical treatment, as used herein - refers to medical care of a medical condition, disease or disorder following its appearance/identification in a subject, or alleviation of the same or inhibition or prevention of the same.
• DAR drug-Antibody Ratio
• DAR is between 0.5 and 200. In another embodiment, DAR is between 0.5 and 50. In another embodiment, DAR is between 0.5 and 20. In another embodiment, DAR is between 0.5 and 10. In another embodiment, DAR is between 0.5 and 5. In another embodiment, DAR is between 1 and 10. In another embodiment, DAR is between 1 and 20. In another embodiment, DAR is between 1 and 50. In another embodiment, DAR is between 1 and
• DAR is between 1 and 200. In another embodiment, DAR is between 5 and 10. In another embodiment, DAR is between 5 and 20. In another embodiment, DAR is between 5 and 50. In another embodiment, DAR is between 10 and 20. In another embodiment, DAR is between 20 and 50. In another embodiment, DAR is between 50 and 100. In another embodiment, DAR is between 50 and 200. In another embodiment, DAR is between 100 and 200. In some embodiments, DAR is explicitly mentioned (e.g. Table 1 within Example 4). In other embodiments, the DAR can be calculated via “n” within the meaning of Formula (1(a)). Few examples for said calculation are given below, where it is known that for the compounds/conjugates of this invention, i.e.
• Compound 4 Compound 2 (see E. Danieli, D. Shabat, Bioorg. Med. Chem. 2007, 15, 7318-7324; R. J. Amir, E. Danieli, D. Shabat, Chem. A Eur. J. 2007, 13, 812-821; and A. Gopin, S. Ebner, B. Attali, D. Shabat, Bioconjug. Chem. 2006, 17, 1432-1440) (1.07 g, 2.32 mmol) was dissolved in dry DMF (5 mL) under argon atmosphere and cooled to 0 °C. Sodium hydride (97 mg, 2.44 mmol) was added and the reaction was allowed to warm to room temperature. After stirring for 15 minutes at room temperature, compound 3 (M. E.
• Compound 11 Compound 10 (52 mg, 0.025 mmol) was dissolved in DMF (2 mL) under argon followed by the addition of Pd(PPh 3 ) 4 (15 mg, 0.012) and 1,3-dimethyl barbituric acid (8.0 mg, 0.05 mmol). The mixture was stirred at 45 °C under argon and the progress of the reaction was monitored by RP-HPLC. After 30 min the HPLC chromatogram shows that about 50% conversion has occurred. Thereby another batches of Pd(PPli3)4 and 1,3-dimethyl barbituric acid were added.
• Compound 16 is prepared according to the literature procedure (E. Roussakis, Z. Li, N. H. Nowell, A. J. Nichols, C. L. Evans, Angew. Chemie Int. Ed. 2015, 54, 14728-14731).
• the L-azidoglutamic acid 14 (J. Bachl, J. Mayr, F. J. Sayago, C. Cativiela, D. Diaz Diaz, Chem. Commun. 2015, 51, 5294-5297) (222 mg, 1.3 mmol) was dissolved in dry DMF (10 mL) under an argon atmosphere.
• HBTU (1.23 g, 3.25 mmol) was added to the solution and the mixture was stirred for 5 min at room temperature.
• Compound 20 was synthesized according to the above scheme.
• Compound 21 was synthesized according to the above scheme and was confirmed by mass spectrometry.
• HCC1954 and MDA-MB-468 human mammary adenocarcinoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). HCC1954 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml Penicillin, 100 pg/ml Streptomycin, 12.5 U/ml Nystatin, 2 mM L-glutamine and 100 pg/ml sodium pyruvate.
• DMEM Dulbecco’s modified Eagle’s medium
• FBS fetal bovine serum
• MDA-MD-468 cells were cultured in RPMI supplemented with 10% fetal bovine serum (FBS), 100 IU/ml Penicillin, 100 ⁇ g/rnl Streptomycin, 12.5 U/ml Nystatin, 2 mM L- glutamine. Cells were grown at 37°C in 5% CO2.
• FBS fetal bovine serum
• Penicillin 100 IU/ml Penicillin
• 100 ⁇ g/rnl Streptomycin 100 ⁇ g/rnl Streptomycin
• 12.5 U/ml Nystatin 12.5 U/ml Nystatin
• 2 mM L- glutamine 2 mM L- glutamine
• Human mammary HCC1954 and MDA-MB231 cells were plated onto 24-well culture plates (5,000 cells/well and 10,000 cells/well, respectively) and incubated for 24 h. Cells were then exposed to serial dilutions of Exatecan or Belotecan, free or conjugated to trastuzumab. Cell viability was evaluated following 5-6 days incubation using MTT (3 pg/ml, Sigma). MTT absorbance was measured at 570 nm using SpectraMax M5e multidetection reader.
• trastuzumab-Exatecan/Belotecan conjugates inhibit the proliferation of HER2- positive HCC1954 cells
• trastuzumab conjugated to exatecan or belotecan were obtained from ITL (Table 1). The different conjugates were evaluated for their inhibitory effect on the proliferation of two human mammary adenocarcinoma cells lines, HCC1954 (HER2- positive) and MDA-MB-468 cells (HER2-negative), compared to the free drugs. Belotecan and Exatecan exhibited similar IC50 values in both cell lines tested (7 and 10 nM in HCC1954; 5 and 2.5 nM in MDA-MB-468).
• Tras-Exa-1 was selected for further evaluation due to its enhanced cytotoxicity. To evaluate whether increased exposure time of the cells to the conjugates will result in enhanced activity compared to free Exatecan, cells were exposed to the drug for an additional 24 h (6 days total). Moreover, to further demonstrate the advantage of the conjugate in HER2 positive cells, another HER2+ breast cancer cell line, JIMT-1, was evaluated. In correlation with the previous experiment, HCC1954 cells were significantly more sensitive to Tras-Exa-1 compared to MDA-MB-468. Tras-Exa-1 did not show any advantage compared to free Exatecan in JIMT-1 cells ( Figures 2A-2D).
• Example 5 Trastuzumab-Exatecan/Belotecan conjugates tumor growth compared to commercial Enhertu®
• HCC1954 human mammary adenocarcinoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml Penicillin, 100 ⁇ g/ml Streptomycin, 12.5 U/ml Nystatin, 2 mM L-glutamine and 100 pg/ml sodium pyruvate. Cells were grown at 37°C in 5% CO2.
• DMEM Dulbecco’s modified Eagle’s medium
• FBS fetal bovine serum
• Penicillin 100 IU/ml
• Penicillin 100 ⁇ g/ml Streptomycin
• 12.5 U/ml Nystatin 12.5 U/ml Nystatin
• 2 mM L-glutamine 2 mM L-glutamine
• 100 pg/ml sodium pyruvate Cells
• Endu® Endotu®
• DAR Trastuzumab-Exatecan
• ADCs antibody-drug conjugates
• Tras-Exa linked to exatecan
• belotecan Tras-Bel
• mice bearing HCC1954 (HER2+) human mammary adenocarcinoma xenografts were treated with a single dose of Tras-Exa or Tras-Bel at three concentrations (1, 3 and 10 mg/kg) and monitored for tumor growth and survival.
• mice of all experimental groups including the vehicle and isotype control, exhibited transient body weight loss, which was recovered after 5 days (Figure 3D).
• our trastuzumb-based ADCs as well as Enhertu® were well tolerated in all concentrations evaluated, similarly to vehicle or isotype control, with net positive weight gain over the course of the study (Figure 3D).
• transglutaminase Linkage by transglutaminase was carried out with wild type microbial transglutaminase (WT Transglutaminase purchased from Zedira, Germany) to the light chain of trastuzumab-T by standard methods, following a procedure that is also described in Dickgiesser et al., Bioconjugate Chem. (2020), vol. 31(4), p. 1070-1076.
• 1 eq antibody in a buffer with 150 mM NaCl, 25 mM Tris (pH 8.0) was mixed with a solution of the respective payload (lOx or 20x surplus to the concentration of the antibody, depending on the number of linkage sites) and 6 U/ml of transglutaminase. The mixture was incubated for 16 h in a thermomixer at 37 °C and 450 rpm.
• TCEP was used as 2 mM stock, pH 7.0) or in some cases with DTT (dithiothreitol; 20 mM). After incubation for 0.5-2 hours (depending on the number of cysteines to be activated) in a 37 °C water bath, the reaction was allowed to cool down to room temperature. The solution was desalted to conjugation buffer (10 mM sodium phosphate, pH 6.0, 2 Mm EDTA, N2 degassed) on a Sephadex G25 column and adjusted to an antibody concentration of 0.2 mg/mL in conjugation buffer.
• conjugation buffer (10 mM sodium phosphate, pH 6.0, 2 Mm EDTA, N2 degassed
• the reaction was incubated for 1 h at 22 °C with slow rocking, then the conjugation was checked by LCMS. If necessary, the reaction was continued until the desired DAR was reached.
• the conjugated sample was purified by hydrophobic interaction chromatography (HIC) on a 15 PHE (Phenyl) column (GE Healthcare) or HiTrap HP or FF Butyl Sepharose column (GE Healthcare). Elution fractions were concentrated, and buffer exchanged to PBS pH 7.4 or 10 mM potassium phosphate, 200 mM NaCl, 10 mM histidine, 50 mM trehalose, pH 7.0. The identity and purity of each prepared ADC was confirmed by LCMS and SDS-PAGE. The DAR for each prepared ADC was calculated and confirmed from HIC (hydrophobic interaction chromatography) data and mass spectrometry data. The stability of each prepared ADC was tested by a freeze-thawing experiment.
• HIC hydrophobic interaction chromatography
• the ADC in histidine buffer was shock-frozen in liquid nitrogen to -80°C and stored at this temperature for several weeks up to a few months.
• the sample was subjected to SE-HPLC (size exclusion-high performance liquid chromatography) analysis to test for compound degradation, and the activity of the thawed ADC was examined.
• target antigen binding was examined by Octet binding assay and payload- mediated cytotoxicity was tested by cell titer glow assay on positive and negative cell lines.
• the results for the freeze-thawed ADC was compared to the corresponding ADC without freeze-thawing. In each case, the ADC with solubility tag was found to be stable in this freeze-thawing procedure.
• Endotoxin was determined by the PTS (Portable Test System) cartridge method (Nexgen) according to the manufacturer's instructions under standard conditions. For each prepared ADC, it was found that the endotoxin level was ⁇ 5.0 endotoxin units (EU)/mg.

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