WO2021170772A1 - Combination treatment with a p53 reactivator and an inhibitor of cd47 mediated signaling - Google Patents

Combination treatment with a p53 reactivator and an inhibitor of cd47 mediated signaling Download PDF

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Publication number
WO2021170772A1
WO2021170772A1 PCT/EP2021/054776 EP2021054776W WO2021170772A1 WO 2021170772 A1 WO2021170772 A1 WO 2021170772A1 EP 2021054776 W EP2021054776 W EP 2021054776W WO 2021170772 A1 WO2021170772 A1 WO 2021170772A1
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methyl
oxoquinuclidin
inhibitor
lymphoma
cancer
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PCT/EP2021/054776
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French (fr)
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Lars Abrahmsén
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Aprea Therapeutics Ab
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/439—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom the ring forming part of a bridged ring system, e.g. quinuclidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • p53 plays a critical role as a tumor suppressor and its gene TP 53 is a common target for mutations in tumors. p53 halts the cell cycle and/or triggers apoptosis in response to various stress stimuli, including DNA damage, hypoxia, and oncogene activation (Ko, L. J. & Prives, C., Genes Dev. 10, 1054-1072 (1996); Sherr, C. J. , Genes Dev. 12, 2984-2991 (1998)). [0003] Both p53-induced cell cycle arrest and apoptosis could be involved in p53- mediated tumor suppression.
  • Tumor cells are particularly sensitive to p53 reactivation, supposedly for two main reasons. First, tumor cells are sensitized to apoptosis due to oncogene activation (reviewed in Evan, G. & Littlewood, T., Science. 281, 1317-1322 (1998)). Second, mutant p53 proteins tend to accumulate at high levels in tumor cells.
  • Binding of the receptor CD47 to SIRP1 alpha found on macrophages provides a signal blocking phagocytosis of for example cancer cells cell displaying CD47 (Majeti et al., Cell 138: 286-99, 2009).
  • An inhibitor of CD47 mediated signaling removes the block for phagocytosis. This may not only enable but directly promote phagocytosis if the inhibitor of CD47 mediated signaling is an antibody or Fc fusion protein binding CD47 containing an antibody Fc portion capable of interaction with the Fc gamma receptor on macrophages.
  • a method of treating cancer in a subject comprising: (i) administering a compound that can give reactivation of a mutant p53; and (ii) administering an inhibitor of CD47 mediated signaling, wherein the cancer in the subject comprises a cell that carries a mutation in TP53.
  • a combination comprising (i) a compound that can give reactivation of a mutant p53; and (ii) an inhibitor of CD47 mediated signaling, for use in a method of treating cancer in a subject, wherein the cancer comprises a cell that carries a mutation in TP53.
  • a combination comprising (i) a compound that can give reactivation of a mutant p53; and (ii)an inhibitor of CD47 mediated signaling in the preparation of a medicament for use in a method of treating cancer in a subject, wherein the cancer comprises a cell that carries a mutation in TP53.
  • the mutation in TP53 is selected from the group consisting of R248Q, R248W, R273H, R273C, R175H, Y220C, G245S, R249S, R282W, and a combination thereof.
  • the compound that can give reactivation of the mutant p53 promotes proper folding of the mutant p53 and restores at least part of a normal p53 function.
  • the compound that can give reactivation of the mutant p53 can result in a shift of the equilibrium from unfolded towards a wild-type like p53 conformation.
  • the compound that can give reactivation of the mutant p53 interferes with aggregation of misfolded mutant p53 or reduces aggregation of the mutant p53.
  • the compound that can give reactivation of the mutant p53 or its metabolite or degradation product thereof can restore a p53 wild type function by covalent binding to the mutant p53.
  • the compound that can give reactivation of the mutant p53 can bind to thiol groups in the core domain of the mutant p53 and restore wild-type conformation.
  • the compound that can give reactivation of the mutant p53 is selected from the group consisting of: 2-(hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one;
  • the compound is 2-(hydroxymethyl)-2-(methoxymethyl) quinuclidin-3-one having the following formula: or a pharmaceutically acceptable salt thereof.
  • the compound is 2,2,2-trifluoro-N-((3-oxoquinuclidin-2- yl)methyl)acetamide having the following formula: or a pharmaceutically acceptable salt thereof.
  • the inhibitor of CD47 mediated signaling is an inhibitor of
  • the inhibitor of CD47 mediated signaling is an inhibitor of SIRPa.
  • the inhibitor of CD47 mediated signaling attenuates the interaction between CD47 and SIRPa.
  • the inhibitor of CD47 mediated signaling is an anti-CD47 antibody.
  • the inhibitor of CD47 mediated signaling is an anti- SIRPa antibody.
  • the inhibitor of CD47 mediated signaling is magrolimab.
  • the inhibitor of CD47 mediated signaling is SRF231.
  • the p53 reactivator is formulated in a first pharmaceutical composition and the inhibitor of CD47 mediated signaling is formulated in a second pharmaceutical composition.
  • the cancer is a hematological malignancy.
  • the hematological malignancy is leukemia, lymphoma, or myeloma.
  • the cancer is selected from the group consisting of: Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular center lymphoma, transformed lymphoma, lymphocytic lymphoma of intermediate differentiation, intermediate lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse small-cleaved cell lymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-cell lymphoma, mantle zone lymphoma, low grade follicular lymphoma, multiple myeloma (MM), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL)
  • the cancer is myelodysplastic syndromes (MDS). In some embodiments, the cancer is acute myeloid leukemia (AML). In other embodiments, the cancer is chronic lymphocytic leukemia (CLL). In yet other embodiments, the cancer is multiple myeloma (MM). 4. DESCRIPTION OF THE FIGURES
  • Described herein are combination treatments with a p53 reactivator (see Section 5.2.1) and an inhibitor of CD47-SIRPa signaling (see Section 5.2.2) for treatment of a cancer (see Section 5.4).
  • the cancer can comprise cells that are mutant for p53 (see Section 5.4). Manner of administration and coordination of treatment regimen are described and exemplified in Section 5.4 and Section 6 below.
  • an effective amount or “therapeutically effective amount” as used herein refers to the amount of a therapeutic compound, a combination of therapeutic compounds or pharmaceutical compositions thereof provided herein, which is sufficient to result in the desired outcome.
  • a subject is a mammal.
  • the subject is a human.
  • the subject is a mammal, e.g., a human, diagnosed with a disease or disorder.
  • the subject is a mammal, e.g., a human, at risk of developing a disease or disorder.
  • administer refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and/or any other method of physical delivery described herein or known in the art.
  • treat refers to the reduction or amelioration of the progression, severity, and/or duration of a disease or disorder resulting from the administration of one or more therapies. Treating may be determined by assessing whether there has been a decrease, alleviation and/or mitigation of one or more symptoms associated with the underlying disorder such that an improvement is observed with the patient, despite that the patient may still be afflicted with the underlying disorder.
  • treating includes both managing and ameliorating the disease.
  • prevent refers to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptom(s).
  • mutant p53 mediated disease or disorder refers to a disease or disorder that is caused or partially caused by mutation of the p53 gene ( TP53 ).
  • a mutant p53 mediated cancer means a cancer that contains a cell having a mutant TP53.
  • alkyl unless otherwise stated, means an unbranched or branched, cyclic, saturated or unsaturated (alkenyl or alkynyl) hydrocarbyl radical.
  • Cx-Cy alkyl means a straight or branched chain hydrocarbon containing from x to y carbon atoms.
  • C1-C6 alkyl means a straight or branched chain hydrocarbon containing from 2 to 6 carbon atoms.
  • alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.
  • aryl means an aromatic group, such as phenyl or naphthyl.
  • heteroaryl means a mono-, bi-, or tricyclic heteroaromatic group containing one or more heteroatom(s) preferably selected from N, O and S, such as pyridyl, pyrrolyl, quinolinyl, furanyl, thienyl, oxadiazolyl, thiadiazolyl, thiazolyl, oxazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, isothiazolyl, imidazolyl, pyrimidinyl, indolyl, pyrazinyl, indazolyl, pyrimidinyl, thiophenetyl, pyranyl, carbazolyl, acridinyl, quinolinyl, benzimid
  • non-aromatic heterocycle means a non-aromatic cyclic group containing one or more heteroatom(s) preferably selected from N, O and S, such as a pyrrolidinyl, piperidyl, piperazinyl, morpholinyl, tetrahydrofuranyl or monosaccharide.
  • halogen means a fluorine, chlorine, bromine or iodine.
  • substituted means that the concerned groups are substituted with at least one functional group, such as hydroxyl, amine, sulfide, silyl, carboxylic acid, halogen, aryl, etc.
  • pharmaceutically acceptable means being approved by a regulatory agency of the Federal or a state government, or listed in United States Pharmacopeia, European Pharmacopeia, or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.
  • Excipient means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material.
  • Excipients include, for example, encapsulating materials or additives such as absorption accelerators, antioxidants, binders, buffers, carriers, coating agents, coloring agents, diluents, disintegrating agents, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, perfumes, preservatives, propellants, releasing agents, sterilizing agents, sweeteners, solubilizers, wetting agents and mixtures thereof.
  • the term “excipient” can also refer to a diluent, adjuvant (e.g., Freunds’ adjuvant (complete or incomplete) or vehicle.
  • excipients are pharmaceutically acceptable excipients.
  • pharmaceutically acceptable excipients include buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (e.g., fewer than about 10 amino acid residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM.
  • buffers such as phosphate, citrate, and other organic
  • each component is “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable excipients are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed.
  • a pharmaceutically acceptable excipient is an aqueous pH buffered solution.
  • the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include both A and B; A or B; A (alone); and B (alone).
  • the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • One therapeutic agent in the present combination therapies is a p53 reactivator.
  • the p53 gene TP53 is a very common target for mutations in tumors. Around half of all human tumors carry mutations in TP53. p53 halts the cell cycle and/or triggers apoptosis in response to various stress stimuli, including DNA damage, hypoxia, and oncogene activation (Ko, L. J.
  • p53 Upon activation, p53 initiates the p53-dependent biological responses through transcriptional transactivation of specific target genes carrying p53 DNA binding motifs.
  • autoimmune diseases share the common feature of an imbalance between the production and destruction of various cell types including lymphocytes (SLE), synovial cells (RA), and fibroblasts (scleroderma). Genes including TP53 that regulates apoptosis are also expressed abnormally. According to the authors, specific therapies that induce apoptosis without incurring side effects should improve treatment of autoimmune disease.
  • TP53 codon 72 polymorphism contributes to a genetically determined variability in apoptotic susceptibility among old people, which has a potentially relevant role in the context of an age-related pathologic condition, such as myocardial ischaemia.
  • EAE experimental autoimmune encephalomyelitis
  • CNS central nervous system
  • the p53 reactivator directly or indirectly targets a mutant p53 protein.
  • the mutant TP53 includes a missense mutation, which is a point mutation in which a single nucleotide change results in a codon that codes for a different amino acid.
  • p53 proteins resulting from a missense mutation in the part of TP53 encoding the DNA binding domain can be broadly classified as DNA-contact mutants and structural mutants.
  • p53 DNA contact mutant contains mutations present on amino acids directly binding to DNA, such as in mutants carrying single amino acid changes R248Q, R248W, R273H, and R273C, where R248Q denotes that the wild type residue arginine in position 248 has been replaced by a glutamine.
  • p53 structural mutants have an amino acid replacement that alters the overall architecture and/or stability to abolish its DNA-binding ability, as reported in mutants carrying the R175H, Y220C, G245S, R249S, and R282W residues.
  • the mutant TP53 includes a nonsense mutation.
  • a nonsense mutation is a genetic mutation changing a codon for an amino acid into a stop codon, resulting in a shorter, unfinished protein product. Nonsense mutations are less frequent than missense mutations in TP 53, but nonetheless constitute about 10% of all TP 53 mutations in cancer. The most common TP53 nonsense mutation yields a truncated p53; R213X aka R213*.
  • the p53 reactivator reactivates or restores at least part of wild type p53 activity, for example by promoting proper folding of mutant p53 and restoring normal p53 function.
  • the p53 reactivator provided herein or a degradation product or metabolite thereof inhibits improper protein misfolding and/or promotes proper protein folding by covalent binding to the mutant p53 protein, for example, by electrophiles binding to one or more thiols in the mutant p53 DNA binding domain to stabilize a folded conformation, and restoring their transcriptional activities.
  • the p53 reactivator provided herein or a degradation product or metabolite thereof binds to the thiol of cysteine residues in the core domain and stabilizes wild type p53 conformation.
  • the p53 reactivator provided herein or a degradation product or metabolite thereof is shifting the equilibrium from unfolded towards a wild-type like p53 conformation.
  • the p53 reactivator provided herein or a degradation product or metabolite thereof binds to thiol groups in the core domain and restores wild-type conformation.
  • the p53 reactivator provided herein or a degradation product or metabolite thereof inhibits improper protein misfolding and/or promotes proper protein folding by non-covalent binding to the mutant p53 protein.
  • Such p53 reactivators include chaperones that can non-covalently stabilize mutant p53 structures.
  • the p53 reactivator provided herein or a degradation product or metabolite thereof reactivates mutant p53 by interfering with aggregation of misfolded p53 or reducing aggregation of mutant p53.
  • p53 misfolds or unfolds into an aggregation-prone stage that loses its DNA-binding capacity.
  • misfolded mutant p53 may cause accumulation of wild type p53 in a misfolded form and accelerate p53 aggregation.
  • the p53 reactivator provided herein or a degradation product or metabolite thereof may reactivate p53 by interfering with aggregation of misfolded p53.
  • the p53 reactivator provided herein or a degradation product or metabolite reduces non-folded or incorrectly folded mutant p53 that may otherwise aggregate, and thereby reducing aggregation.
  • the mutant p53 contains at least one replacement in the core domain of p53 (residues 94-292) caused by a TP53 mutation.
  • the mutant p53 contains at least one of the following amino acid replacements: V173A, S241F, R249S, R273H, R175H, R248Q, and Y220C.
  • the p53 reactivator provided herein is a compound according to formula (I)
  • R 1 is selected from H, — CH — O— R 3 , — CH 2— S— R 3 , and — CH 2— NR 3 R 4 ;
  • R 2 is selected from — CH 2— O— R 3 , — CH 2— S— R 3 , and — CH 2— NR 3 R 4 ;
  • R 3 and R 4 are the same or different and are independently selected from H; substituted or non- substituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl or Cl -CIO alkyl; substituted or non-substituted benzyl; substituted or non-substituted mono- or bicyclic aryl; substituted or non-substituted mono-, bi- or tricyclic C2-C10 heteroaryl or non-aromatic C2-C10 heterocyclyl containing one or several heteroatoms independently selected from N, O and S; or R 3 and R 4 in — CH2 — NR 3 R 4 are bonded together and form, together with the nitrogen atom to which they are bonded, a substituted or non-substituted non-aromatic C2-C10 mono- or bicyclic heterocyclyl optionally containing one or several further heteroatoms independently selected from N, O and S and optionally comprising one or several cyclic keto groups; where
  • the pharmaceutically acceptable salt of the compound of formula (I) e.g. may be an acid addition salt of an inorganic mineral acid or of an organic acid.
  • R 1 is selected from H, — CH2 — O — R 3 , — CH2 —
  • R 1 is selected from H, — CH2 — O — R 3 , and — CH2 — S — R 3 . In some embodiments, R 1 is selected from H and — CH2 — O — R 3 . In other embodiments,
  • R 1 is selected from — CH2 — O — R 3 , and — CH2 — S — R 3 . In some embodiments, R 1 is H.
  • R 2 in formula (I) is selected from — CH2 — O — R 3 , — CH2 — S — R 3 , and — CH2 — NR 3 R 4 .
  • R 2 is selected from — CH2 — O — R 3 and — CH2 — S — R 3 .
  • R 2 is — CH2 — O — R 3 .
  • R 1 is selected from H, — CH2 — O — R 3 and — CH2 — S — R 3 ; and R 2 is selected from — CH2 — O — R 3 and — CH2 — S — R 3 .
  • R 1 is H; and R 2 is selected from — CH2 — O — R 3 , — CH2 —
  • R 1 is selected from H and — CH2 — O — R 3 ; and R 2 is — CH2— O— R 3 .
  • both R 1 and R 2 are — CH2 — O — R 3 .
  • each R 3 is independently selected from H; substituted or non- substituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl and Cl -CIO alkyl, and benzyl.
  • each R 3 may be independently selected from H and Cl -CIO alkyl, e.g. from H and C1-C6 alkyl, from H and C1-C4 alkyl, or from H and C1-C3 alkyl, in particular from H and methyl.
  • R 1 is selected from H and — CFb — O — R 3 , and R 2 is —
  • each R 3 is independently selected from H; substituted or non-substituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl and C1-C10 alkyl, and benzyl, in particular from H and Cl -CIO alkyl, e.g. from H and C1-C6 alkyl, from H and Cl- C4 alkyl, or from H and C1-C3 alkyl, in particular from H and methyl.
  • R 1 and R 2 are both — CFb — O — R 3 , and each R 3 is independently selected from H; substituted or non-substituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl and C1-C10 alkyl; in particular from H and C1-C10 alkyl; e.g. from H and C1-C6 alkyl, from H and C1-C4 alkyl, or from H and C1-C3 alkyl, in particular from H and methyl.
  • any Cl -CIO alkyl e.g. may be a C1-C6 alkyl, or a C1-C4 alkyl, e.g. methyl, ethyl, propyl or butyl.
  • Any C3-C12 cycloalkyl may be e.g. a C3-C8 cycloalkyl, or a C3-C6 cycloalkyl.
  • Any mono- or bicyclic aryl may be e.g. a monocyclic aryl, such as phenyl.
  • Any mono-, bi- or tricyclic C2-C10 heteroaryl may be e.g.
  • a monocyclic or bicyclic C2-C5 heteroaryl e.g. a 5- or 6-membered monocyclic or a 9- membered bicyclic C2-C5 heteroaryl.
  • Any mono-, bi- or tricyclic non-aromatic C2-C10 heterocyclyl may be e.g. a monocyclic or bicyclic C2-C5 heterocyclyl, e.g. a 5- or 6-membered monocyclic or 9- or 10-membered bicyclic C2-C5 heterocyclyl.
  • Any halogen may be selected from F, Cl, Br and I, preferably from F and Cl.
  • Any heterocycle, aromatic or not, containing one or several heteroatoms independently selected from N, O and S e.g. may contain 1-5 heteroatoms, e.g. independently selected from N and O.
  • any substituted or non-substituted C3-C12 cycloalkyl or Cl -CIO alkyl is non-substituted.
  • any substituted or non-substituted benzyl is non-substituted.
  • any substituted or non-substituted mono- or bicyclic aryl is non-substituted.
  • any substituted or non-substituted mono-, bi- or tricyclic C2- C10 heteroaryl or non-aromatic C2-C10 heterocyclyl is non-substituted.
  • each substituent is selected from Cl -CIO alkyl, e.g. C1-C6 alkyl, C1-C4 alkyl, or C1-C3 alkyl, such as methyl; halogen, e.g. Cl; halogen-substituted Cl -CIO alkyl, e.g. trifluoromethyl; monocyclic C2-C5 heteroaryl, e.g. pyridyl; C1-C10 alkoxy, e.g. C1-C6 alkoxy, C1-C4 alkoxy, or C1-C3 alkoxy, such as methoxy; and amino.
  • the number of substituents on each substituted group is 1, 2 or 3.
  • the compound provided herein is selected from those exemplified in the prior art documents referred to herein above, e.g. W005/090341, WO04/084893, WO02/024692, W003/070250 and W02020/058458.
  • the compound of formula (I) is selected from 2- (hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one and 2,2- bis(hydroxymethyl)quinuclidin-3-one, and pharmaceutically acceptable salts of these compounds.
  • the compound of formula (I) is 2-(hydroxymethyl)-2- (methoxymethyl)quinuclidin-3-one (APR-246) or a pharmaceutically acceptable salt thereof.
  • the compound of formula (I) is 2,2- bis(hydroxymethyl)quinuclidin-3-one or a pharmaceutically acceptable salt thereof.
  • the p53 reactivator provided herein is selected from the group consisting of:
  • the p53 reactivator provided herein is 2,2,2-trifluoro-N- ((3-oxoquinuclidin-2-yl)methyl)acetamide having the following formula: or a pharmaceutically acceptable salt thereof.
  • the p53 reactivator provided herein is selected from the group consisting of the compounds in the table below.
  • the inhibitor of CD47 mediated signaling attenuates the interaction between CD47 and SIRPa.
  • the inhibitor of CD47 mediated signaling blocks the function of CD47.
  • the inhibitor of CD47 mediated signaling blocks the function of SIRPa.
  • the inhibitor of CD47 mediated signaling is an anti-CD47 antibody.
  • the inhibitor of CD47 mediated signaling is an anti-SIRPa antibody.
  • the inhibitor of CD47 mediated signaling is an antisense oligonucleotide against CD47 or against SIRPa.
  • the inhibitor of CD47 mediated signaling is an Fc fusion to a portion of the receptor SIRPa providing CD47 binding.
  • the Fc portion of the fusion protein or the anti-CD47 antibody has been inactivated by amino acid replacements eliminating binding to complement Clq protein and human Fc-gamma receptors, except for the neonatal Fc receptor (FcRn).
  • the inhibitor of CD47 mediated signaling is selected from the group consisting of SGN-CD47M (Seattle Genetics), ZL1201 (Zai Lab (Shanghai) Co., Ltd.), IBI188 (Innovent Biologies, Inc.), magrolimab (5F9) (Forty Seven, Inc.), T ⁇ -621 and TTI-622 (Trillium Therapeutics Inc.), AO-176 (Arch Oncology), FSI-189 (Forty Seven, Inc.), SHR-1603 (Jiangsu HengRui Medicine), ALX148 (ALX Oncology), SL-172154 (Shattuck Labs) and SRF231 (Surface Oncology).
  • the inhibitor of CD47 mediated signaling is an anti-CD47 antibody described in US 9,017,675, which disclosure is incorporated herein by reference.
  • the anti-CD47 antibody is selected from the group consisting of murine antibody 5F9 and humanized versions thereof, including magrolimab, and antibodies sharing the same complementarity determining region (CDR) sequences.
  • the inhibitor of CD47 mediated signaling is an anti-CD47 antibody or an antigen binding portion thereof, which comprises the following CDR sequences (i.e. the CDR sequences of 5F9 and humanized versions thereof, including magrolimab): VH-CDR1: NYNMH (SEQ ID NO:l)
  • VH-CDR2 TIYPGNDDTSYNQKFKD (SEQ ID NO:2)
  • VH-CDR3 GGYRAMDY (SEQ ID NO: 3)
  • VL-CDR1 RSSQSIVYSNGNTYLG (SEQ ID NO: 4)
  • VL-CDR2 KVSNRFS (SEQ ID NO: 5)
  • VL-CDR3 FQGSHVPYT (SEQ ID NO: 6)
  • the inhibitor of CD47 mediated signaling is an anti- CD47 antibody or an antigen binding portion thereof, which comprises a heavy chain variable domain sequence selected from the group consisting of o a sequence selected from the group consisting of SEQ ID NO:7-10 (VH sequences of 5F9 and humanized versions thereof, including magrolimab), and o sequences having at least 70 % identity to a sequence selected from the group consisting of SEQ ID NO:7-10, provided that the CDR sequences in such sequence are as defined in SEQ ID NO: 1-3; and a light chain variable domain sequence selected from the group consisting of o a sequence selected from the group consisting of SEQ ID NO: 11-14 (VL sequences of 5F9 and humanized versions thereof, including magrolimab), and o sequences having at least 70 % identity to a sequence selected from the group consisting of SEQ ID NO: 11-14, provided that the CDR sequences in such sequence are as defined in SEQ ID NO:4-6.
  • a heavy chain variable domain sequence selected from the
  • the VH and VL sequences of the antibody or antigen binding portion thereof are the listed sequences and sequences having at least 75 %, such as at least 80 %, such as at least 85 %, such as at least 90 %, such as at least 95 %, such as at least 98 %, such as at least 100 % identity thereto.
  • the inhibitor of CD47 mediated signaling is an anti- CD47 antibody or an antigen binding portion thereof, which comprises the heavy chain variable domain sequence SEQ ID NO: 7 (VH sequence of magrolimab) and the light chain variable domain sequence SEQ ID NO: 11 (VL sequence of magrolimab).
  • the inhibitor of CD47 mediated signaling is magrolimab.
  • the inhibitor of CD47 mediated signaling is a fusion protein between the CD47 binding domain of human SIRPa and an IgG. In some embodiments, the inhibitor of CD47 mediated signaling is a fusion protein between the CD47 binding domain of human SIRPa and a human IgG. In some embodiments, the inhibitor of CD47 mediated signaling is a fusion protein between the CD47 binding domain of human SIRPa and Fc domain of a human IgG.
  • the inhibitor of CD47 mediated signaling is ALX148, as described e.g. in Kauder et al. (2016), PLoS One 13(8):e0201832, which is incorporated herein by reference.
  • ALX148 is a molecule constructed by genetic fusion of an engineered version of the CD47-binding domain from human SIRPa to an inactive human IgGl Fc domain.
  • ALX148 is a 78-kDa disulfide-linked homodimer that has no glycosylation sites, and binds human, cynomolgus monkey, mouse, and rat CD47 with high affinity.
  • ALX148 blocks the CD47-SIRPa interaction and induces antitumor immunity by bridging innate and adaptive immune responses.
  • a p53 reactivator e.g., APR-246
  • an inhibitor of CD47 mediated signaling e.g., magrolimab
  • a combination therapy comprising a first pharmaceutical composition comprising a p53 reactivator provided herein and a first pharmaceutically acceptable excipient, and a second pharmaceutical composition comprising an inhibitor of CD47 mediated signaling (e.g., magrolimab) provided herein and a second pharmaceutically acceptable excipient.
  • the first and the second pharmaceutically acceptable excipients can be the same or different.
  • a p53 reactivator provided herein and an inhibitor of CD47 mediated signaling (e.g., magrolimab) provided herein are formulated together in a single pharmaceutical composition.
  • a pharmaceutical composition comprising a p53 reactivator provided herein, an inhibitor of CD47 mediated signaling (e.g., magrolimab) provided herein, and one or more pharmaceutically acceptable excipient.
  • the p53 reactivator and/or the inhibitor of CD47 mediated signaling can be formulated into suitable pharmaceutical compositions for different routes of administration, such as injection (subcutaneous, intramuscular, intravenous, intraperitoneal, intraosseous, intracardiac, intraarticular, and intracavernous), sublingual and buccal, rectal, vaginal, ocular, otic, nasal, inhalation, nebulization, cutaneous, or transdermal.
  • injection subcutaneous, intramuscular, intravenous, intraperitoneal, intraosseous, intracardiac, intraarticular, and intracavernous
  • sublingual and buccal rectal, vaginal, ocular, otic, nasal, inhalation, nebulization, cutaneous, or transdermal.
  • the compounds described above may be formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel, Introduction to Pharmaceutical Dosage Forms, (7th ed. 1999)).
  • compositions effective concentrations of one or more compounds (i.e., p53 reactivators or inhibitors of CD47 mediated signaling (e.g., magrolimab)) provided herein or pharmaceutically acceptable salts are mixed with a suitable pharmaceutical excipient.
  • concentrations of the compounds in the compositions are effective for delivery of an amount, upon administration, that treats, prevents, or ameliorates one or more of the symptoms and/or progression of a disease or disorder provided herein (e.g., cancer, including solid cancer and blood borne cancer).
  • the active compound is in an amount sufficient to exert a therapeutically useful effect on the patient treated.
  • the therapeutically effective concentration may be determined empirically by testing the compounds in in vitro and in vivo systems and then extrapolated therefrom for dosages for humans.
  • the concentration of active compound in the pharmaceutical composition will depend on absorption, tissue distribution, inactivation, and excretion rates of the active compound, the physicochemical characteristics of the compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.
  • the p53 reactivators e.g, APR-246 or inhibitors of CD47 mediated signaling (e.g., magrolimab) provided herein, or pharmaceutically acceptable salts thereof, may also be formulated to target a particular tissue, receptor, or other area of the body of the subject to be treated. Many such targeting methods are well known to those of skill in the art. All such targeting methods are contemplated herein for use in the instant compositions.
  • liposomal suspensions, including tissue-targeted liposomes, such as tumor- targeted liposomes may also be suitable as pharmaceutically acceptable excipients. These may be prepared according to methods known to those skilled in the art.
  • a Liquid Composition Comprising A P53 Reactivator e.g., APR-246 or Compound A
  • the p53 reactivator provided here is formulated in an aqueous solution, as described in US patent no. 9,061,016, which is incorporated herein by reference, and as described in more detail below.
  • the formulation comprising the p53 reactivator is a stock solution and preferably is a pharmaceutical formulation in the form of a concentrated stock solution.
  • the formulation preferably is sterile, and this may be achieved by known sterilization methods such as filtration, allowing for long term storage.
  • the formulation provided herein can be used, e.g. for administration to a patient in need thereof by direct injection or preferentially diluted with appropriate injectable solutions for i.v. infusion.
  • the formulation provided herein is an aqueous solution of the p53 reactivator provided herein (e.g., APR-246 or Compound A), wherein the p53 reactivator is present at a concentration within a range of from about 10 mg/mL to about 250 mg/mL, a range of from about 50 mg/mL to about 200 mg/mL, or a range of from about 75 mg/mL to about 150 mg/mL of the formulation.
  • the p53 reactivator provided herein (e.g., APR-246 or Compound A)
  • the p53 reactivator is present at a concentration within a range of from about 10 mg/mL to about 250 mg/mL, a range of from about 50 mg/mL to about 200 mg/mL, or a range of from about 75 mg/mL to about 150 mg/mL of the formulation.
  • the formulation may be diluted prior to use, e.g., administration to a patient.
  • the dilution factor depends on the concentration of the p53 reactivator in the formulation and the required amount of the compound needed, e.g., to meet the therapeutically effective dose.
  • the final diluted product in case of parenteral administration, has a pH within the range of from about pH 4 to about pH 6.
  • the final diluted product for parenteral administration has a pH within the range of from about pH 4.2 to about pH 5.5.
  • the liquid formulation may contain sodium chloride at a concentration of between 0% and 3%, a concentration of between 0.5% and 1.5%, or a concentration of between 0.8% and 1% weight by volume of the formulation.
  • the p53 reactivator e.g., APR-246 or Compound A
  • the liquid formulation in the form of an acid addition salt with one or several different pharmaceutically acceptable acids.
  • the pharmaceutically acceptable acid may be a mineral acid, e.g., selected from the group consisting of hydrochloric acid, hydrogen bromide, hydrogen iodide, sulphuric acid, nitric acid, phosphoric acid and the like.
  • the pharmaceutically acceptable acid may be an organic acid, e.g., a sulfonic or carboxylic acid, particularly an alkyl or aryl sulfonic acid or an alkyl or aryl carboxylic acid, such as selected from the group consisting of methanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, acetic acid, tartaric acid, maleic acid, citric acid, benzoic acid, salicylic acid, ascorbic acid and the like.
  • a sulfonic or carboxylic acid particularly an alkyl or aryl sulfonic acid or an alkyl or aryl carboxylic acid, such as selected from the group consisting of methanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, acetic acid, tartaric acid, maleic acid, citric acid, benzoic acid, salicylic acid, ascorbic acid
  • the composition provided herein contains a pH regulating agent.
  • pH regulating agent means at least one pharmaceutically acceptable organic or inorganic (mineral) acid, or at least one pharmaceutically acceptable acid buffer or a mixture of any of these.
  • the pH regulating agent may be any such acid or buffer, or a mixture of acids or buffers, or a mixture of acid(s) and buffer(s). Examples of useful acids and buffers are as indicated herein.
  • the composition may contain at least one pharmaceutically acceptable acid.
  • the acid may be an inorganic mineral acid, e.g., selected from the group consisting of hydrochloric acid, hydrobromic acid, hydroiodic acid, sulphuric acid, nitric acid, phosphoric acid or the like, or an organic acid, e.g., selected from the group consisting of acetic acid, succinic acid, tartaric acid, maleic acid, ascorbic acid, citric acid, glutamic acid, benzoic acid, ascorbic acid, methanesulfonic acid, ethanesulfonic acid and the like.
  • the composition may contain one or several acids, selected from inorganic and organic acids.
  • the required pH of the formulation is achieved by addition of hydrochloric acid.
  • composition provided herein also may comprise at least one pharmaceutically acceptable buffer, particularly selected from the group of citric buffer, acetate buffer, phosphate buffer and the like, separately or as a mixture thereof, as well as in combination with any pharmaceutically acceptable acid, as defined herein, e.g., hydrochloric acid.
  • pharmaceutically acceptable buffer particularly selected from the group of citric buffer, acetate buffer, phosphate buffer and the like, separately or as a mixture thereof, as well as in combination with any pharmaceutically acceptable acid, as defined herein, e.g., hydrochloric acid.
  • the liquid composition provided herein is aqueous, which means that it contains water.
  • the aqueous solution and the aqueous phase used to prepare the composition also may contain other pharmaceutically acceptable liquids as a solvent phase, e.g., polyethylene glycol (PEG) and alcohols, e.g., ethanol.
  • the aqueous phase mainly comprises water as a solvent.
  • the solvent phase is comprised of from 50 to 100% water, at least 80% water, at least 90% water, at least 95% water, at least 98% water or 100% water.
  • the composition described herein is provided as a stable stock solution, particularly as a concentrated stock solution for long term storage at a temperature range of 2-8 °C., in a container, for example, a sealed and sterilized container.
  • the composition may comprise a stable aqueous WFI (water for injection) solution of the p53 reactivator as an acid addition salt, in particular a hydrochloride addition salt, in a concentration of from about 10 mg/mL to about 250 mg/mL, from about 50 mg/mL to about 200 mg/mL, or from about 75 mg/mL to about 150 mg/mL, and a pH regulating agent in such an amount as to provide a pH in the solution in the range between pH 3.0 and pH 5.0, between pH 3.2 and pH 4.7, between pH 3.5 and pH 4.5, or between pH 3.8 and pH 4.2, e.g., approximately 4.0.
  • the pH of the stock solution may have a lower limit selected from a pH of about 3.0, or about 3.2, e.g. about 3.4, such as about 3.6 or about 3.8, and an upper limit of about 5.0, or about 4.7, or about 4.5, or about 4.2, e.g. about 4.0.
  • compositions also may be added to or present in the aqueous phase, such as pharmaceutically acceptable inorganic salts, e.g., NaCl, preservatives, or further pharmaceutically acceptable compounds, e.g., further therapeutically active ingredients, such as cytostatics, particularly cisplatin, daunorubicin, cerubidine, cytarabine and fludarabine.
  • pharmaceutically acceptable inorganic salts e.g., NaCl
  • preservatives e.g., cytostatics, particularly cisplatin, daunorubicin, cerubidine, cytarabine and fludarabine.
  • further therapeutically active ingredients such as cytostatics, particularly cisplatin, daunorubicin, cerubidine, cytarabine and fludarabine.
  • NaCl is added to the aqueous phase in an amount so as to provide a final liquid composition as defined herein above, containing NaCl at a concentration of between 0% and 3%, between 0.5% and 1.5%, or between 0.8% and 1% weight by volume of the formulation.
  • the composition is a sterile formulation.
  • sterilization of the composition may be accomplished by passing the formulation, e.g., a formulated stock solution, through a sterile filter with a nominal pore size of 0.2 pm into a cleaned and sterilized container.
  • the composition may be provided as a ready -to-use injection solution, wherein a liquid formulation, e.g., a stock solution, is brought to the desired volume by addition of one or more pharmaceutically acceptable solvents, such as selected from the group consisting of WFI, a glucose solution, electrolyte solution containing amino acids, lipids, vitamins, and other minerals, Ringer's solution, Hartmann's solution, or a sodium chloride solution in the form of an isotonic, hypotonic or hypertonic solution.
  • a pharmaceutically acceptable solvents such as selected from the group consisting of WFI, a glucose solution, electrolyte solution containing amino acids, lipids, vitamins, and other minerals, Ringer's solution, Hartmann's solution, or a sodium chloride solution in the form of an isotonic, hypotonic or hypertonic solution.
  • a pharmaceutically acceptable solvents such as selected from the group consisting of WFI, a glucose solution, electrolyte solution containing amino acids, lipids, vitamins,
  • the p53 reactivator is APR-246, which is formulated in liquid formulation, which comprises at least one pH regulating agent in an amount such as to provide a pH in the aqueous solution of from about 3.0 to about 5.0.
  • APR-246 is present in the aqueous solution at a concentration of from 10 mg/mL to 250 mg/mL.
  • the aqueous solution comprises NaCl at a concentration of between 0% and 3% weight by volume.
  • the p53 reactivator is Compound A.
  • the p53 reactivator provided here (e.g., Compound A) is formulated in a composition for oral administration.
  • the oral dosage form is a solid form.
  • compositions that are suitable for oral administration can be presented as discrete dosage forms, such as, but are not limited to, tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flavored syrups).
  • dosage forms contain predetermined amounts of active ingredients, and may be prepared by methods of pharmacy well known to those skilled in the art.
  • Typical oral dosage forms are prepared by combining the active ingredients in an intimate admixture with at least one excipient according to conventional pharmaceutical compounding techniques.
  • Excipients can take a wide variety of forms depending on the form of preparation desired for administration.
  • excipients suitable for use in solid oral dosage forms include, but are not limited to, starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents.
  • tablets can be coated by standard aqueous or nonaqueous techniques.
  • dosage forms can be prepared by any of the methods of pharmacy.
  • pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredients with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary.
  • a tablet can be prepared by compression or molding.
  • Compressed tablets can be prepared by compressing in a suitable machine the active ingredients in a free- flowing form such as powder or granules, optionally mixed with an excipient.
  • Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • excipients that can be used in oral dosage forms provided herein include, but are not limited to, binders, fillers, disintegrants, and lubricants.
  • Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives ( e.g ., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose, microcrystalline cellulose, and mixtures thereof.
  • Suitable forms of microcrystalline cellulose include, but are not limited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICEL RC-581, AVICEL-PH-105, and mixtures thereof.
  • Suitable anhydrous or low moisture excipients or additives include AVICEL-PH-103TM and Starch 1500 LM.
  • fillers suitable for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.
  • Disintegrants are used in compositions to provide tablets that disintegrate when exposed to an aqueous environment.
  • the amount of disintegrant used varies based upon the type of formulation, and is readily discernible to those of ordinary skill in the art.
  • Disintegrants that can be used in pharmaceutical compositions and dosage forms include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof.
  • Lubricants that can be used in pharmaceutical compositions and dosage forms include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil, zinc stearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof.
  • Additional lubricants include, for example, a syloid silica gel, a coagulated aerosol of synthetic silica, and mixtures thereof.
  • the inhibitor of CD47 mediated signaling (e.g., magrolimab) provided herein is supplied in a sterile form for reconstitution as a suspension for subcutaneous injection or reconstitution as a solution with further dilution for intravenous infusion.
  • the inhibitor of CD47 mediated signaling is formulated for parenteral administration.
  • Parenteral administration of the compositions includes intravenous, subcutaneous, and intramuscular administrations.
  • Compositions for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, sterile suspensions ready for injection, and sterile emulsions.
  • the solutions may be either aqueous or nonaqueous.
  • the unit dose parenteral preparations can be packaged in an ampoule, a vial or a syringe with a needle.
  • pharmaceutically acceptable excipients used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharmaceutically acceptable substances.
  • the inhibitor of CD47 mediated signaling e.g., magrolimab
  • Lyophilized powders can be reconstituted for administration as solutions, emulsions, and other mixtures.
  • the sterile, lyophilized powder can be prepared by dissolving an inhibitor of CD47 mediated signaling (e.g., magrolimab) provided herein, or a pharmaceutically acceptable salt thereof, in a suitable solvent.
  • the solvent may contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose.
  • the solvent may also contain a buffer, such as citrate, phosphate, or other buffers known to those of skill in the art.
  • the lyophilized formulations are suitable for reconstitution with a suitable diluent to the appropriate concentration prior to administration.
  • the lyophilized formulation is suitable for reconstitution with an aqueous solution for intravenous administrations.
  • the lyophilized formulation provided herein is suitable for reconstitution with water.
  • the lyophilized formulations comprise the inhibitor of CD47 mediated signaling (e.g., magrolimab) provided herein, a buffer and a bulking agent.
  • a lyophilized formulation comprises an inhibitor of CD47 mediated signaling (e.g., magrolimab) provided herein, in about 0.1 to about 60% based on the total weight of the lyophilized formulation.
  • the lyophilized formulation can be reconstituted for parenteral administration to a patient using any pharmaceutically acceptable diluent.
  • diluents include, but are not limited to Sterile Water for Injection (SWFI), Dextrose 5% in Water (D5W), or a cosolvent system.
  • the inhibitor of CD47 mediated signaling (e.g., magrolimab) is formulated as lyophilized formulation containing azacitidine and mannitol (e.g., 100 mg of azacitidine and 100 mg mannitol each vial).
  • compositions for use in the prevention and/or treatment of a disease or condition comprising the p53 reactivator (e.g., APR-246) and the inhibitor of CD47 mediated signaling (e.g., magrolimab).
  • a method comprising administering an effective amount of the p53 reactivator and the inhibitor of CD47 mediated signaling (e.g., magrolimab) to a subject.
  • the subject is a human.
  • the disease or disorder is a neoplastic disease.
  • the disease or disorder is a cancer.
  • the disease or disorder is a hematogical malignancy.
  • the neoplastic, cancerous, or malignant cell in a subject to be treated with a method provided herein comprises a mutation in TP53.
  • provided herein is a method to treat or prevent a hematological cancer with a mutation in the p53 gene.
  • the cancer to be treated with a method provided herein comprises cancer cells that carry a mutation in TP53.
  • the mutation in TP53 is R248Q, R248W, R273H, R273C, R175H, Y220C, G245S, R249S, R282W, VI 73 A, S241F, R249S or a combination thereof.
  • the mutant p53 contains at least one replacement in the core domain of p53 (residues 94-292) caused by a TP53 mutation.
  • the mutant TP53 includes a nonsense mutation.
  • a nonsense mutation is a genetic mutation changing a codon for an amino acid into a stop codon, resulting in a shorter, unfinished protein product.
  • Nonsense mutations are less frequent than missense mutations in TP53, but nonetheless constitute about 10% of all TP 53 mutations in cancer.
  • the most common TP 53 nonsense mutation yields a truncated p53; R213X aka R213*.
  • the cancer to be treated does not comprise any cells carrying a mutation in the TP53 gene.
  • the disease or disorder is a disease of abnormal cell growth and/or dysregulated apoptosis.
  • diseases include, but are not limited to, cancer, mesothelioma, bladder cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, ovarian cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, bone cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, gastrointestinal (gastric, colorectal and/or duodenal) cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, esophageal cancer, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the ure
  • the disease or disorder is selected from the group consisting of bladder cancer, brain cancer, breast cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small- cell lung cancer, prostate cancer, small-cell lung cancer and spleen cancer.
  • the disease or disorder is a tumor of the hematopoietic or lymphoid tissue.
  • the disease or disorder is a hematological cancer, such as leukemia, lymphoma, or myeloma.
  • the cancer is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular center lymphoma, transformed lymphoma, lymphocytic lymphoma of intermediate differentiation, intermediate lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse small- cleaved cell lymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-cell lymphoma, mantle zone lymphoma, low grade follicular lymphoma
  • NHL non-Hodgkin's
  • the disease or disorder is myelodysplastic syndromes (MDS).
  • MDS myelodysplastic syndromes
  • AML acute myeloid leukemia
  • CLL chronic lymphocytic leukemia
  • MM multiple myeloma
  • the disease or disorder is a solid tumor cancer.
  • the solid tumor cancer is selected from the group consisting of a carcinoma, an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a ductal cell carcinoma, a lung carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, a non-melanoma skin carcinoma, and a lung cancer.
  • a prophylactic or therapeutic agent the p53 reactivator and the inhibitor of CD47 mediated signaling (e.g., magrolimab) provided herein
  • a composition provided herein that will be effective in the prevention and/or treatment of a disease or condition can be determined by standard clinical techniques.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of a disease or condition, and in some embodiments, should be decided according to the judgment of the practitioner and each patient’s circumstances.
  • the dose administered to a subject in the context of the present disclosure should be sufficient to effect a therapeutic response.
  • dosage will depend upon a variety of factors including the potency of the specific compound, the age, condition and body weight of the patient, as well as the stage/severity of the disease.
  • the dose will also be determined by the route (administration form) timing and frequency of administration.
  • the p53 reactivator (e.g., APR-246) and the inhibitor of CD47 mediated signaling (e.g., magrolimab) can be formulated in different pharmaceutical compositions and administered separately to the subject in need thereof.
  • the p53 reactivator (e.g., APR-246) and the inhibitor of CD47 mediated signaling (e.g., magrolimab) are administered together in the same pharmaceutical composition.
  • the p53 reactivator (e.g., APR-246) and the inhibitor of CD47 mediated signaling (e.g., magrolimab) are administered simultaneously.
  • the term “simultaneously” means at the same time or within a short period of time, for example, less than 1 hour, less than 2 hours, less than 3 hours, less than 4 hours, or less than 12 hours.
  • the p53 reactivator (e.g., APR-246) and the inhibitor of CD47 mediated signaling (e.g., magrolimab) are not administered simultaneously, and instead the two compounds are administered at different times.
  • the p53 reactivator (e.g., APR-246) and the inhibitor of CD47 mediated signaling (e.g., magrolimab) are administered at least once during a dosing period.
  • a dosing period as used herein is meant a period of time, during which each therapeutic agent has been administered at least once.
  • a dosing cycle can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.
  • a dosing cycle is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks.
  • a dosing period is a dosing cycle.
  • the prophylactic or therapeutic agent (the p53 reactivator and/or the inhibitor of CD47 mediated signaling provided herein) can be delivered as a single dose (e.g., a single bolus injection), or over time (e.g., continuous infusion over time or divided bolus doses over time).
  • the agent can be administered repeatedly if necessary, for example, until the patient experiences stable disease or regression, or until the patient experiences disease progression or unacceptable toxicity.
  • Stable disease or lack is determined by methods known in the art such as evaluation of patient symptoms, physical examination, and visualization of the tumor that has been imaged using X-ray, CAT, PET, MRI scan, or other commonly accepted evaluation modalities.
  • the prophylactic or therapeutic agent can be administered once daily (QD) or divided into multiple daily doses such as twice daily (BID), three times daily (TID), and four times daily (QID).
  • the administration can be continuous (i.e., daily for consecutive days or every day) or intermittent, e.g, in cycles (i.e., including days, weeks, or months of rest without drug).
  • the term “daily” is intended to mean that a therapeutic compound is administered once or more than once each day, for example, for a period of time.
  • continuous is intended to mean that a therapeutic compound is administered daily for an uninterrupted period of, e.g., at least 10 days.
  • intermittent or “intermittently” as used herein is intended to mean stopping and starting at either regular or irregular intervals.
  • intermittent administration of the compound is administration for one to six days per week, administration in cycles (e.g., daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week), or administration on alternate days.
  • the frequency of administration is in the range of about a daily dose to about a monthly dose.
  • administration is once a day, twice a day, three times a day, four times a day, once every other day, twice a week, once every week, once every two weeks, once every three weeks, or once every four weeks.
  • the compound is administered once per day from one day to six months, from one week to three months, from one week to four weeks, from one week to three weeks, or from one week to two weeks.
  • APR-246 is administered at a dose of less than 150 mg/kg. In some embodiments, APR-246 is administered at a dose of less than 100 mg/kg. In some embodiments, APR-246 is administered at a dose of betweenlOO mg/kg and 25 mg/kg. In other embodiments, APR-246 is administered at dose of less than 75 mg/kg. In yet other embodiments, the APR-246 is administered at a dose of less than 65 mg/kg. In yet other embodiments, the APR-246 is administered at a dose of less than 50 mg/kg.
  • APR-246 is administered at a fixed dose within the interval 2.7-7.5 g. In some embodiments, the fixed dose of APR-246 is no more than 4.5 g. In some embodiments, the fixed dose of APR-246 is no more than 3.7 g. In some embodiments, APR-246 is administered at a fixed dose during a period of about 3 to 7 hours. In some embodiments, APR-246 is administered at a fixed dose during a period of about 6 or 4 hours.
  • a model system for a hematological malignancy may be treated with a p53 reactivator, an inhibitor of CD47 mediated signaling, or a combination of the p53 reactivator and the inhibitor of CD47 mediated signaling, and the effects of the combination treatment are analyzed and compared to the monotherapies.
  • the analytical tool is COMBENEFIT.
  • Combenefit (Di Veroli. C, Bioinformatics, 32(18), 2016, 2866-2868) is an interactive platform for the analysis and visualization of drug combinations and only requires the user to save the data in the predefined .xls template files. Combenefit performs combination analyses using the standard Loewe, Bliss and HSA methods (see Ianevski et al., Bioinformatics, 2017, 33(15): 2413-2415).
  • a “combination index” (Cl) is then calculated as the measured cell viability of the sample with two co-incubated substances divided by the predicted cell viability.
  • the following classifications are used in this example:
  • the measured cell viability for a combination of two substances is higher than the cell viability for one or both substances, the effect is considered antagonistic. If the predicted viability is very low, the quote “measured viability / predicted viability” may give false Cl values. Thus, a lower limit of ⁇ 5% of the predicted viability may be set.
  • This experiment can be used to assess the enhancement of phagocytosis facilitated by an inhibitor of CD47 mediated signaling (e.g., magrolimab) by pretreatment with a p53 reactivator (e.g., APR-246).
  • CD47 mediated signaling e.g., magrolimab
  • a p53 reactivator e.g., APR-246
  • CD 14+ monocytes are prepared from blood according to standard protocols, and subsequently matured into macrophages on culture dishes in growth medium supplemented with 10% human AB serum. After 7-11 days, the macrophages are detached from culture plates by trypsination.
  • the target cells e.g., SKM-1 and NCI-1688
  • a p53 reactivator e.g., APR- 246
  • Calcein acetoxymethyl e.g., Calcein acetoxymethyl
  • Flow cytometry is used to measure phagocytosis, evaluated by the percentage of macrophages engulfing calcein acetoxymethyl-labeled tumor cells.
  • This experiment can be used to assess the effects of an inhibitor of CD47 mediated signaling (e.g., magrolimab) and/or a p53 reactivator (e.g., APR-246) on AML.
  • This experiment is done twice, first at pilot scale to titrate doses and incubation times and second as a full-scale experiment.
  • 1-2.5 x 10 5 FACS purified leukemic stem cells are transplanted into NOG juvenile mice. Eight to twelve weeks later, the presence of human AML engraftment (hCD45 + CD33 + cells) is assessed in the blood and the femur bone marrow.
  • mice are treated with intra peritoneal injections of an inhibitor of CD47 mediated signaling (e.g ., magrolimab) and/or a p53 reactivator (e.g., APR-246), at a dose of 1-5 mg/kg daily and 25-100 mg/kg twice daily, respectively.
  • an inhibitor of CD47 mediated signaling e.g ., magrolimab
  • a p53 reactivator e.g., APR-246
  • This in vivo experiment can also be performed with the inhibitor of CD47 mediated signaling denoted ALX148, in which case the administration is done less frequently and at a higher dose; once weekly and at a dose of 30 mg/kg.
  • Mce are randomized into treatment groups when tumors have reached an average size of 80 mm 3 .
  • Mce are injected intraperitoneally with 5 mg/kg anti -PD- 1 mAh every 5 days, an inhibitor of CD47 mediated signaling (e.g., magrolimab) at 1-5 mg/kg once daily, and/or or a p53 reactivator (e.g., APR- 246) at 25-100 mg/kg twice daily.
  • an inhibitor of CD47 mediated signaling e.g., magrolimab
  • a p53 reactivator e.g., APR- 246
  • One group gets the inhibitor of CD47 mediated signaling
  • a second group gets the p53 reactivator
  • a third group get the inhibitor of CD47 mediated signaling and the p53 reactivator
  • a fourth group gets the inhibitor of CD47 mediated signaling and anti-PD-1
  • a fifth group all three treatments.
  • the control group is injected with an irrelevant mouse IgGl every 5 days at a dose of 5 mg/kg.
  • the tumor volumes are estimated from caliper measurements every second day, until a maximum volume of 2000 mm 3 is reached, at which point the mice are euthanized.
  • This in vivo experiment can also be performed with the inhibitor of CD47 mediated signaling denoted ALX148, in which case the administration is done less frequently and at a higher dose; once weekly and at a dose of 30 mg/kg. 6.4
  • B16.H12 a mouse monoclonal antibody (mAh) binding murine CD47 (Bio X).
  • APR-246 were intraperitoneally administered alone and in combination to NOG mice (NOD. Cg-Prkdc scid I l2rg tm 15ug /J icTac) . These T, B and NK cell deficient mice had previously been injected with the human AML cell line SKM-1. About 10 million cells were intravenously injected into each mouse. A 3 -week treatment regime was initiated six days after SKM-1 cell injection. The mice were divided into four groups of six mice: control, anti-CD47 mAh, APR-246 and combination. APR-246 was administered twice daily at 100 mg/kg for 5 days per week, and the mAh at 5 mg/kg twice per week.
  • the fraction of human CD45+ cells was determined by flow cytometry using a pre-labeled mAh designated APC Mouse Anti- Human CD45 (HI30; BD Biosciences). Negative control blood was obtained from mice not injected with tumor cells to identify CD45 + human cells in inoculated mice samples. The results after 2 and 3 weeks shows a beneficial effect of the combination in comparison to the individual treatments ( Figure 1).

Abstract

A method of treating cancer in a subject, comprising (i) administering a compound that can give reactivation of a mutant p53; and (ii) administering an inhibitor of CD47 mediated signaling, wherein the cancer in the subject comprises a cell that carries a mutation in TP53.

Description

COMBINATION TREATMENT WITH A P53 REACTIVATOR AND
AN INHIBITOR OF CD47 MEDIATED SIGNALING 1. FIELD
[0001] Provided herein are combination therapies using a p53 reactivator in combination with an inhibitor of CD47-SIRPa signaling for treating a disease or disorder such as cancer. 2. BACKGROUND
[0002] p53 plays a critical role as a tumor suppressor and its gene TP 53 is a common target for mutations in tumors. p53 halts the cell cycle and/or triggers apoptosis in response to various stress stimuli, including DNA damage, hypoxia, and oncogene activation (Ko, L. J. & Prives, C., Genes Dev. 10, 1054-1072 (1996); Sherr, C. J. , Genes Dev. 12, 2984-2991 (1998)). [0003] Both p53-induced cell cycle arrest and apoptosis could be involved in p53- mediated tumor suppression. A significant proportion of human tumors express a “mutant” p53 protein due to a TP53 mutation, making it highly desirable to restore the wild type p53 activity to yield growth suppression to tumors. Tumor cells are particularly sensitive to p53 reactivation, supposedly for two main reasons. First, tumor cells are sensitized to apoptosis due to oncogene activation (reviewed in Evan, G. & Littlewood, T., Science. 281, 1317-1322 (1998)). Second, mutant p53 proteins tend to accumulate at high levels in tumor cells. Therefore, restoration of the wild type function to the abundant and presumably “activated” mutant p53 should trigger a massive apoptotic response in already sensitized tumor cells, whereas normal cells that harbour low or undetectable levels of p53 should not be affected. [0004] Therapeutically useful compounds have previously been generated based on showing mutant p53 dependent anti-proliferative activity in a cellular assay, including the compound PRIMA-1 (i.e. 2,2-bis(hydroxymethyl)-l-azabicyclo[2.2.2]octan-3-one) (disclosed in WO 02/24692), and its analogs (such as those disclosed in W003/070250, W02004/084893, W02005/090341 and W02020/058458). Nonetheless, there still remains a general need of effective combination therapies using these compounds in combination with a second therapeutic agent for treating cancer and other diseases or disorders.
[0005] Binding of the receptor CD47 to SIRP1 alpha found on macrophages provides a signal blocking phagocytosis of for example cancer cells cell displaying CD47 (Majeti et al., Cell 138: 286-99, 2009). An inhibitor of CD47 mediated signaling removes the block for phagocytosis. This may not only enable but directly promote phagocytosis if the inhibitor of CD47 mediated signaling is an antibody or Fc fusion protein binding CD47 containing an antibody Fc portion capable of interaction with the Fc gamma receptor on macrophages. In some presented inhibitors of CD47 mediated signaling such active induction of phagocytosis has been avoided since it is associated with phagocytosis of blood cells displaying CD47 (Kauder et al., PLOS One August 22, 2018). Depending on the biological context, the absence of an active Fc portion may require a relevant concomitant treatment leading to antibodies displaying a functional Fc gamma portion binding to the tumor cells, to promote phagocytosis by macrophages. 3. SUMMARY
[0006] In one aspect, provided herein is a method of treating cancer in a subject, comprising: (i) administering a compound that can give reactivation of a mutant p53; and (ii) administering an inhibitor of CD47 mediated signaling, wherein the cancer in the subject comprises a cell that carries a mutation in TP53.
[0007] In another aspect, provided herein is a combination comprising (i) a compound that can give reactivation of a mutant p53; and (ii) an inhibitor of CD47 mediated signaling, for use in a method of treating cancer in a subject, wherein the cancer comprises a cell that carries a mutation in TP53.
[0008] In another aspect, provided herein is a use of a combination comprising (i) a compound that can give reactivation of a mutant p53; and (ii)an inhibitor of CD47 mediated signaling in the preparation of a medicament for use in a method of treating cancer in a subject, wherein the cancer comprises a cell that carries a mutation in TP53.
[0009] In some embodiments, the mutation in TP53 is selected from the group consisting of R248Q, R248W, R273H, R273C, R175H, Y220C, G245S, R249S, R282W, and a combination thereof.
[0010] In some embodiments, the compound that can give reactivation of the mutant p53 promotes proper folding of the mutant p53 and restores at least part of a normal p53 function. [0011] In some embodiments, the compound that can give reactivation of the mutant p53 can result in a shift of the equilibrium from unfolded towards a wild-type like p53 conformation. [0012] In some embodiments, the compound that can give reactivation of the mutant p53 interferes with aggregation of misfolded mutant p53 or reduces aggregation of the mutant p53. [0013] In some embodiments, the compound that can give reactivation of the mutant p53 or its metabolite or degradation product thereof can restore a p53 wild type function by covalent binding to the mutant p53.
[0014] In other embodiments, the compound that can give reactivation of the mutant p53 can bind to thiol groups in the core domain of the mutant p53 and restore wild-type conformation.
[0015] In some embodiments, the compound that can give reactivation of the mutant p53 is selected from the group consisting of: 2-(hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one;
2.2-bis(hydroxymethyl)quinuclidin-3-one;
2.2.2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide;
2.2.2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide;
2.2.2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide;
2.2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide, N-((3-oxoquinuclidin-2-yl)methyl)pyridine-3-sulfonamide; 4-fluoro-N-((3-oxoquinuclidin-2-yl)methyl)benzenesulfonamide; N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)benzenesulfonamide; 2-(N-((3-oxoquinuclidin-2-yl)methyl)methylsulfonamido)acetamide; N-(methylsulfonyl)-N-((3-oxoquinuclidin-2-yl)methyl)glycine; N-((3-oxoquinuclidin-2-yl)methyl)pyridine-4-sulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)pyridine-2-sulfonamide; N-ethyl-1,1,1-trifluoro-N-((3-oxoquinuclidin-2-yl)-methyl)methanesulfonamide;
1,1,1 -trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N,N-bis((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)propane-2-sulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)cyclopropanesulfonamide; 1-methyl-N-((3-oxoquinuclidin-2-yl)methyl)cyclopropane-1-sulfonamide; N-cyclopropyl-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)-N-phenylmethanesulfonamide;
1 -((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione;
5-methyl- 1 -((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione; tert- butyl 5-methyl-2,6-dioxo-3-((3-oxoquinuclidin-2-yl)methyl)-3,6-dihydropyrimidine- 1 (2 H)-carboxylate;
5-methyl-1,3-bis((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(1H,3H)-dione;
/V-methyl- 1 -((3-oxoquinuclidin-2-yl)methyl)- 1 H- 1 ,2,4-triazole-3-carboxamide;
2-((3 -chloro-1H- 1 ,2,4-triazol- 1 -yl)methyl)quinuclidin-3 -one;
/V,/V-dimethyl- 1 -((3-oxoquinuclidin-2-yl)methyl)- 1 H-1 ,2,4-triazole-3-carboxamide;
2-(( 1H- 1 ,2,4-triazol- 1 -yl)methyl)quinuclidin-3 -one;
1 -((3-oxoquinuclidin-2-yl)methyl)-1H- 1 ,2,4-triazole-3-carbonitrile; and 1 -((3-oxoquinuclidin-2-yl)methyl)-1H- 1 ,2,4-triazole-3-carboxamide, or a pharmaceutically acceptable salt thereof.
[0016] In other embodiments, the compound is 2-(hydroxymethyl)-2-(methoxymethyl) quinuclidin-3-one having the following formula:
Figure imgf000006_0001
or a pharmaceutically acceptable salt thereof.
[0017] In other embodiments, the compound is 2,2,2-trifluoro-N-((3-oxoquinuclidin-2- yl)methyl)acetamide having the following formula:
Figure imgf000006_0002
or a pharmaceutically acceptable salt thereof.
[0018] In some embodiments, the inhibitor of CD47 mediated signaling is an inhibitor of
CD47. [0019] In other embodiments, the inhibitor of CD47 mediated signaling is an inhibitor of SIRPa.
[0020] In some embodiments, the inhibitor of CD47 mediated signaling attenuates the interaction between CD47 and SIRPa.
[0021] In some embodiments, the inhibitor of CD47 mediated signaling is an anti-CD47 antibody.
[0022] In other embodiments, the inhibitor of CD47 mediated signaling is an anti- SIRPa antibody.
[0023] In some embodiments, the inhibitor of CD47 mediated signaling is magrolimab.
[0024] In some embodiments, the inhibitor of CD47 mediated signaling is SRF231.
[0025] In some embodiments, the p53 reactivator is formulated in a first pharmaceutical composition and the inhibitor of CD47 mediated signaling is formulated in a second pharmaceutical composition.
[0026] In some embodiments, the cancer is a hematological malignancy. In some embodiments, the hematological malignancy is leukemia, lymphoma, or myeloma.
[0027] In some embodiments, the cancer is selected from the group consisting of: Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular center lymphoma, transformed lymphoma, lymphocytic lymphoma of intermediate differentiation, intermediate lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse small-cleaved cell lymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-cell lymphoma, mantle zone lymphoma, low grade follicular lymphoma, multiple myeloma (MM), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), myelodysplastic syndrome (MDS), acute T cell leukemia, acute myeloid leukemia (AML), acute promyelocytic leukemia, acute myeloblastic leukemia, acute megakaryoblastic leukemia, precursor B acute lymphoblastic leukemia, precursor T acute lymphoblastic leukemia, Burkitt’s leukemia (Burkitt’s lymphoma), acute biphenotypic leukemia, chronic myeloid lymphoma, chronic myelogenous leukemia (CML), and chronic monocytic leukemia.
[0028] In some embodiments, the cancer is myelodysplastic syndromes (MDS). In some embodiments, the cancer is acute myeloid leukemia (AML). In other embodiments, the cancer is chronic lymphocytic leukemia (CLL). In yet other embodiments, the cancer is multiple myeloma (MM). 4. DESCRIPTION OF THE FIGURES
[0029] Figure 1 is a diagram showing the average (n=6) percentages of human CD45+ cells in NOG mice previously injected with human AML cells, following 2 and 3 weeks of treatment with anti-CD47 mAh, APR-246 or a combination of anti-CD47 mAh and APR-246, or not treated (control), as indicated. 5. DETAILED DESCRIPTION
[0030] Described herein are combination treatments with a p53 reactivator (see Section 5.2.1) and an inhibitor of CD47-SIRPa signaling (see Section 5.2.2) for treatment of a cancer (see Section 5.4). The cancer can comprise cells that are mutant for p53 (see Section 5.4). Manner of administration and coordination of treatment regimen are described and exemplified in Section 5.4 and Section 6 below.
5.1 Definitions
[0031] Techniques and procedures described or referenced herein include those that are generally well understood and/or commonly employed using conventional methodology by those skilled in the art. Unless otherwise defined herein, technical and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any description of a term set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall control.
[0032] The term “effective amount” or “therapeutically effective amount” as used herein refers to the amount of a therapeutic compound, a combination of therapeutic compounds or pharmaceutical compositions thereof provided herein, which is sufficient to result in the desired outcome.
[0033] The terms “subject” and “patient” may be used interchangeably. As used herein, in certain embodiments, a subject is a mammal. In specific embodiments, the subject is a human. In one embodiment, the subject is a mammal, e.g., a human, diagnosed with a disease or disorder. In another embodiment, the subject is a mammal, e.g., a human, at risk of developing a disease or disorder.
[0034] “Administer” or “administration” refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and/or any other method of physical delivery described herein or known in the art.
[0035] As used herein, the terms “treat,” “treatment” and “treating” refer to the reduction or amelioration of the progression, severity, and/or duration of a disease or disorder resulting from the administration of one or more therapies. Treating may be determined by assessing whether there has been a decrease, alleviation and/or mitigation of one or more symptoms associated with the underlying disorder such that an improvement is observed with the patient, despite that the patient may still be afflicted with the underlying disorder. The term “treating” includes both managing and ameliorating the disease.
[0036] The terms “prevent,” “preventing,” and “prevention” refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptom(s).
[0037] The term “a mutant p53 mediated disease or disorder” as used herein refers to a disease or disorder that is caused or partially caused by mutation of the p53 gene ( TP53 ). For example, a mutant p53 mediated cancer means a cancer that contains a cell having a mutant TP53.
[0038] As used herein, the term “alkyl” unless otherwise stated, means an unbranched or branched, cyclic, saturated or unsaturated (alkenyl or alkynyl) hydrocarbyl radical. The term “Cx-Cy alkyl” means a straight or branched chain hydrocarbon containing from x to y carbon atoms. For example, “C1-C6 alkyl” means a straight or branched chain hydrocarbon containing from 2 to 6 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.
[0039] As used herein, the term “aryl” means an aromatic group, such as phenyl or naphthyl. [0040] As used herein, the term “heteroaryl” means a mono-, bi-, or tricyclic heteroaromatic group containing one or more heteroatom(s) preferably selected from N, O and S, such as pyridyl, pyrrolyl, quinolinyl, furanyl, thienyl, oxadiazolyl, thiadiazolyl, thiazolyl, oxazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, isothiazolyl, imidazolyl, pyrimidinyl, indolyl, pyrazinyl, indazolyl, pyrimidinyl, thiophenetyl, pyranyl, carbazolyl, acridinyl, quinolinyl, benzimidazolyl, benzthiazolyl, purinyl, cinnolinyl and pteridinyl.
[0041] As used herein, the term “non-aromatic heterocycle” means a non-aromatic cyclic group containing one or more heteroatom(s) preferably selected from N, O and S, such as a pyrrolidinyl, piperidyl, piperazinyl, morpholinyl, tetrahydrofuranyl or monosaccharide.
[0042] As used herein, the term “halogen” means a fluorine, chlorine, bromine or iodine.
[0043] As used herein, and unless specified otherwise, the term “substituted” means that the concerned groups are substituted with at least one functional group, such as hydroxyl, amine, sulfide, silyl, carboxylic acid, halogen, aryl, etc.
[0044] The term “pharmaceutically acceptable” as used herein means being approved by a regulatory agency of the Federal or a state government, or listed in United States Pharmacopeia, European Pharmacopeia, or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.
[0045] “Excipient” means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients include, for example, encapsulating materials or additives such as absorption accelerators, antioxidants, binders, buffers, carriers, coating agents, coloring agents, diluents, disintegrating agents, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, perfumes, preservatives, propellants, releasing agents, sterilizing agents, sweeteners, solubilizers, wetting agents and mixtures thereof. The term “excipient” can also refer to a diluent, adjuvant (e.g., Freunds’ adjuvant (complete or incomplete) or vehicle.
[0046] In some embodiments, excipients are pharmaceutically acceptable excipients. Examples of pharmaceutically acceptable excipients include buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (e.g., fewer than about 10 amino acid residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington’s Pharmaceutical Sciences (18th ed. 1990).
[0047] In one embodiment, each component is “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio. See, e.g., Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al, Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, pharmaceutically acceptable excipients are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. In some embodiments, a pharmaceutically acceptable excipient is an aqueous pH buffered solution.
[0048] The terms “about” and “approximately” mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.
[0049] As used in the present disclosure and claims, the singular forms “a”, “an” and “the” include plural forms unless the context clearly dictates otherwise.
[0050] It is understood that wherever embodiments are described herein with the term “comprising” otherwise analogous embodiments described in terms of “consisting of’ and/or “consisting essentially of’ are also provided. It is also understood that wherever embodiments are described herein with the phrase “consisting essentially of’ otherwise analogous embodiments described in terms of “consisting of’ are also provided.
[0051] The term “between” as used in a phrase as such “between A and B” or “between A-B” refers to a range including both A and B. [0052] As used herein, numerical values are often presented in a range format throughout this document. The use of a range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention unless the context clearly indicates otherwise. Accordingly, the use of a range expressly includes all possible subranges, all individual numerical values within that range, and all numerical values or numerical ranges including integers within such ranges and fractions of the values or the integers within ranges unless the context clearly indicates otherwise. This construction applies regardless of the breadth of the range and in all contexts throughout this patent document.
[0053] The term “and/or” as used in a phrase such as “A and/or B” herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
5.2 Agents To Be Used With The Methods Provided
[0054] Provided herein a methods and compositions relating to the combination of a p53 reactivator and inhibitors of CD47-mediated signaling. Agents that can be used as p53 reactivators are set forth in Section 5.2.1. Agents that can be used as inhibitors of CD47- mediated signaling are set forth in Section 5.2.2.
5.2.1 P53 Reactivators
[0055] One therapeutic agent in the present combination therapies is a p53 reactivator. The p53 gene TP53 is a very common target for mutations in tumors. Around half of all human tumors carry mutations in TP53. p53 halts the cell cycle and/or triggers apoptosis in response to various stress stimuli, including DNA damage, hypoxia, and oncogene activation (Ko, L. J.
& Prives, C., Genes Dev. 10, 1054-1072 (1996); Sherr, C. J., Genes Dev. 12, 2984-2991 (1998)). Upon activation, p53 initiates the p53-dependent biological responses through transcriptional transactivation of specific target genes carrying p53 DNA binding motifs.
[0056] Analyses of a large number of mutant p53 genes in human tumors have revealed a strong selection for mutations that inactivate the DNA binding function of p53; most mutations in tumors are point mutations clustered in the part encoding the core domain of p53 (residues 94-292) that harbors the DNA binding activity (Beroud, C. & Soussi, T., Nucl. Acids Res. 26, 200-204 (1998)). [0057] Both p53-induced cell cycle arrest and apoptosis could be involved in p53- mediated tumor suppression. While p53-induced cell cycle arrest could conceivably be reversed in different ways, p53-induced cell death would have advantage of being irreversible. There is indeed evidence from animal in vivo models (Symonds et al., Cell 78, 703-711 (1994)) and human tumors (Bardeesy et al, Cancer Res 55, 215-9 (1995)) indicating that p53-dependent apoptosis plays a major role in the elimination of emerging tumors, particularly in response to oncogenic signaling. Moreover, the ability of p53 to induce apoptosis often determines the efficacy of cancer therapy (Lowe et al., 1994 Science 266, 807-10 (1994)).
[0058] In addition to hyperproliferative diseases, such as cancer, it is also known in the art that deficient p53 function is involved in a number of other disease states, e.g. autoimmune diseases and cardiac diseases.
[0059] For example, as shown in Mountz et al., Immunology, 6: 27-37 (1994), human autoimmune diseases share the common feature of an imbalance between the production and destruction of various cell types including lymphocytes (SLE), synovial cells (RA), and fibroblasts (scleroderma). Genes including TP53 that regulates apoptosis are also expressed abnormally. According to the authors, specific therapies that induce apoptosis without incurring side effects should improve treatment of autoimmune disease.
[0060] For another example, Bonafe et al., Cell Death and Differentiation, 11 : 962-973 (2004) suggest that TP53 codon 72 polymorphism contributes to a genetically determined variability in apoptotic susceptibility among old people, which has a potentially relevant role in the context of an age-related pathologic condition, such as myocardial ischaemia.
[0061] Okuda et al., Journal of Neuroimmunology, 135: 29-37 (2003) suggest that p53 may be involved in the regulatory process of experimental autoimmune encephalomyelitis (EAE) through the control of cytokine production and/or the apoptotic elimination of inflammatory cells. EAE as a model for autoimmune inflammatory diseases of the central nervous system (CNS) is a widely used model for the human disease multiple sclerosis.
[0062] These results suggest that pharmacological restoration of p53 function would be beneficial in a number of disorders and diseases.
[0063] In some embodiments of the combination therapies provided herein, the p53 reactivator directly or indirectly targets a mutant p53 protein. [0064] In some embodiments, the mutant TP53 includes a missense mutation, which is a point mutation in which a single nucleotide change results in a codon that codes for a different amino acid. p53 proteins resulting from a missense mutation in the part of TP53 encoding the DNA binding domain can be broadly classified as DNA-contact mutants and structural mutants. p53 DNA contact mutant contains mutations present on amino acids directly binding to DNA, such as in mutants carrying single amino acid changes R248Q, R248W, R273H, and R273C, where R248Q denotes that the wild type residue arginine in position 248 has been replaced by a glutamine. p53 structural mutants have an amino acid replacement that alters the overall architecture and/or stability to abolish its DNA-binding ability, as reported in mutants carrying the R175H, Y220C, G245S, R249S, and R282W residues.
[0065] In other embodiments, the mutant TP53 includes a nonsense mutation. A nonsense mutation is a genetic mutation changing a codon for an amino acid into a stop codon, resulting in a shorter, unfinished protein product. Nonsense mutations are less frequent than missense mutations in TP 53, but nonetheless constitute about 10% of all TP 53 mutations in cancer. The most common TP53 nonsense mutation yields a truncated p53; R213X aka R213*. [0066] In some embodiments, the p53 reactivator reactivates or restores at least part of wild type p53 activity, for example by promoting proper folding of mutant p53 and restoring normal p53 function.
[0067] In some more specific embodiments, the p53 reactivator provided herein or a degradation product or metabolite thereof inhibits improper protein misfolding and/or promotes proper protein folding by covalent binding to the mutant p53 protein, for example, by electrophiles binding to one or more thiols in the mutant p53 DNA binding domain to stabilize a folded conformation, and restoring their transcriptional activities. In some embodiments, the p53 reactivator provided herein or a degradation product or metabolite thereof binds to the thiol of cysteine residues in the core domain and stabilizes wild type p53 conformation. In other embodiments, the p53 reactivator provided herein or a degradation product or metabolite thereof is shifting the equilibrium from unfolded towards a wild-type like p53 conformation. In yet other embodiments, the p53 reactivator provided herein or a degradation product or metabolite thereof binds to thiol groups in the core domain and restores wild-type conformation. [0068] In other more specific embodiments, the p53 reactivator provided herein or a degradation product or metabolite thereof inhibits improper protein misfolding and/or promotes proper protein folding by non-covalent binding to the mutant p53 protein. Such p53 reactivators include chaperones that can non-covalently stabilize mutant p53 structures.
[0069] In other embodiments, the p53 reactivator provided herein or a degradation product or metabolite thereof reactivates mutant p53 by interfering with aggregation of misfolded p53 or reducing aggregation of mutant p53. Sometimes, p53 misfolds or unfolds into an aggregation-prone stage that loses its DNA-binding capacity. Similarly, misfolded mutant p53 may cause accumulation of wild type p53 in a misfolded form and accelerate p53 aggregation. Thus, in some embodiments, the p53 reactivator provided herein or a degradation product or metabolite thereof may reactivate p53 by interfering with aggregation of misfolded p53. In other embodiments, the p53 reactivator provided herein or a degradation product or metabolite reduces non-folded or incorrectly folded mutant p53 that may otherwise aggregate, and thereby reducing aggregation.
[0070] In some embodiments, the mutant p53 contains at least one replacement in the core domain of p53 (residues 94-292) caused by a TP53 mutation. In some embodiments, the mutant p53 contains at least one of the following amino acid replacements: V173A, S241F, R249S, R273H, R175H, R248Q, and Y220C.
[0071] In some embodiments, the p53 reactivator provided herein is a compound according to formula (I)
Figure imgf000015_0001
I wherein:
R1 is selected from H, — CH — O— R3, — CH2— S— R3, and — CH2— NR3R4;
R2 is selected from — CH2— O— R3, — CH2— S— R3, and — CH2— NR3R4;
R3 and R4 are the same or different and are independently selected from H; substituted or non- substituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl or Cl -CIO alkyl; substituted or non-substituted benzyl; substituted or non-substituted mono- or bicyclic aryl; substituted or non-substituted mono-, bi- or tricyclic C2-C10 heteroaryl or non-aromatic C2-C10 heterocyclyl containing one or several heteroatoms independently selected from N, O and S; or R3 and R4 in — CH2 — NR3R4 are bonded together and form, together with the nitrogen atom to which they are bonded, a substituted or non-substituted non-aromatic C2-C10 mono- or bicyclic heterocyclyl optionally containing one or several further heteroatoms independently selected from N, O and S and optionally comprising one or several cyclic keto groups; wherein the substituents of the substituted groups are independently selected from unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl or Cl -CIO alkyl; halogen; halogen- substituted C1-C10 alkyl, mono- or bicyclic aryl; mono-, bi- or tricyclic C2-C10 heteroaryl or non-aromatic C2-C10 heterocyclyl containing one or several heteroatoms independently selected from N, O and S; Cl -CIO alkoxy; amino; and Cl -CIO alkylamino; or a pharmaceutically acceptable salts thereof.
[0072] The pharmaceutically acceptable salt of the compound of formula (I) e.g. may be an acid addition salt of an inorganic mineral acid or of an organic acid.
[0073] In a compound of formula (I), R1 is selected from H, — CH2 — O — R3, — CH2 —
S— R3, and — CH2— NR3R4.
[0074] In some embodiments, R1 is selected from H, — CH2 — O — R3, and — CH2 — S — R3. In some embodiments, R1 is selected from H and — CH2 — O — R3. In other embodiments,
R1 is selected from — CH2 — O — R3, and — CH2 — S — R3. In some embodiments, R1 is H.
[0075] R2 in formula (I) is selected from — CH2 — O — R3, — CH2 — S — R3, and — CH2 — NR3R4. In some embodiments, R2 is selected from — CH2 — O — R3 and — CH2 — S — R3. In still other embodiments, R2 is — CH2 — O — R3.
[0076] In one embodiment, R1 is selected from H, — CH2 — O — R3 and — CH2 — S — R3; and R2 is selected from — CH2 — O — R3 and — CH2 — S — R3.
[0077] In one embodiment, R1 is H; and R2 is selected from — CH2 — O — R3, — CH2 —
S — R3 and — CH2 — NR3R4; e.g. from — CH2 — O — R3 and — CH2 — S — R3, and in particular is — CH2— O— R3.
[0078] In one embodiment, R1 is selected from H and — CH2 — O — R3; and R2 is — CH2— O— R3.
[0079] In one embodiment, both R1 and R2 are — CH2 — O — R3. [0080] In one embodiment, each R3 is independently selected from H; substituted or non- substituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl and Cl -CIO alkyl, and benzyl. For example, each R3 may be independently selected from H and Cl -CIO alkyl, e.g. from H and C1-C6 alkyl, from H and C1-C4 alkyl, or from H and C1-C3 alkyl, in particular from H and methyl.
[0081] In one embodiment, R1 is selected from H and — CFb — O — R3, and R2 is —
CFh — O — R3, and each R3 is independently selected from H; substituted or non-substituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl and C1-C10 alkyl, and benzyl, in particular from H and Cl -CIO alkyl, e.g. from H and C1-C6 alkyl, from H and Cl- C4 alkyl, or from H and C1-C3 alkyl, in particular from H and methyl.
[0082] In one embodiment, R1 and R2 are both — CFb — O — R3, and each R3 is independently selected from H; substituted or non-substituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl and C1-C10 alkyl; in particular from H and C1-C10 alkyl; e.g. from H and C1-C6 alkyl, from H and C1-C4 alkyl, or from H and C1-C3 alkyl, in particular from H and methyl.
[0083] In a compound of formula (I), as defined herein above, any Cl -CIO alkyl e.g. may be a C1-C6 alkyl, or a C1-C4 alkyl, e.g. methyl, ethyl, propyl or butyl. Any C3-C12 cycloalkyl may be e.g. a C3-C8 cycloalkyl, or a C3-C6 cycloalkyl. Any mono- or bicyclic aryl may be e.g. a monocyclic aryl, such as phenyl. Any mono-, bi- or tricyclic C2-C10 heteroaryl may be e.g. a monocyclic or bicyclic C2-C5 heteroaryl, e.g. a 5- or 6-membered monocyclic or a 9- membered bicyclic C2-C5 heteroaryl. Any mono-, bi- or tricyclic non-aromatic C2-C10 heterocyclyl may be e.g. a monocyclic or bicyclic C2-C5 heterocyclyl, e.g. a 5- or 6-membered monocyclic or 9- or 10-membered bicyclic C2-C5 heterocyclyl. Any halogen may be selected from F, Cl, Br and I, preferably from F and Cl. Any heterocycle, aromatic or not, containing one or several heteroatoms independently selected from N, O and S, e.g. may contain 1-5 heteroatoms, e.g. independently selected from N and O.
[0084] In one embodiment, in a compound of formula (I) as defined herein above, any substituted or non-substituted C3-C12 cycloalkyl or Cl -CIO alkyl is non-substituted.
[0085] In one embodiment, any substituted or non-substituted benzyl is non-substituted.
[0086] In one embodiment, any substituted or non-substituted mono- or bicyclic aryl is non-substituted. [0087] In one embodiment, any substituted or non-substituted mono-, bi- or tricyclic C2- C10 heteroaryl or non-aromatic C2-C10 heterocyclyl is non-substituted.
[0088] In one embodiment, when any of the above groups is substituted, each substituent is selected from Cl -CIO alkyl, e.g. C1-C6 alkyl, C1-C4 alkyl, or C1-C3 alkyl, such as methyl; halogen, e.g. Cl; halogen-substituted Cl -CIO alkyl, e.g. trifluoromethyl; monocyclic C2-C5 heteroaryl, e.g. pyridyl; C1-C10 alkoxy, e.g. C1-C6 alkoxy, C1-C4 alkoxy, or C1-C3 alkoxy, such as methoxy; and amino.
[0089] In one embodiment, when any of the above groups is substituted, the number of substituents on each substituted group is 1, 2 or 3.
[0090] In another embodiment, the compound provided herein is selected from those exemplified in the prior art documents referred to herein above, e.g. W005/090341, WO04/084893, WO02/024692, W003/070250 and W02020/058458.
[0091] In one embodiment, the compound of formula (I) is selected from 2- (hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one and 2,2- bis(hydroxymethyl)quinuclidin-3-one, and pharmaceutically acceptable salts of these compounds.
[0092] In one embodiment, the compound of formula (I) is 2-(hydroxymethyl)-2- (methoxymethyl)quinuclidin-3-one (APR-246) or a pharmaceutically acceptable salt thereof. [0093] In another embodiment, the compound of formula (I) is 2,2- bis(hydroxymethyl)quinuclidin-3-one or a pharmaceutically acceptable salt thereof.
[0094] In yet other embodiments, the p53 reactivator provided herein is selected from the group consisting of:
2.2.2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide;
2.2.2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide;
2.2.2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide;
2.2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide, N-((3-oxoquinuclidin-2-yl)methyl)pyridine-3-sulfonamide; 4-fluoro-N-((3-oxoquinuclidin-2-yl)methyl)benzenesulfonamide; N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)benzenesulfonamide;
2-(N-((3-oxoquinuclidin-2-yl)methyl)methylsulfonamido)acetamide;
N-(methylsulfonyl)-N-((3-oxoquinuclidin-2-yl)methyl)glycine;
N-((3-oxoquinuclidin-2-yl)methyl)pyridine-4-sulfonamide;
N-((3-oxoquinuclidin-2-yl)methyl)pyridine-2-sulfonamide;
N-ethyl-1,1,1-trifluoro-N-((3-oxoquinuclidin-2-yl)-methyl)methanesulfonamide;
1,1,1 -trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N,N-bis((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)propane-2-sulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)cyclopropanesulfonamide;
1 -methyl-N-((3 -oxoquinuclidin-2-yl)methyl)cyclopropan-1-sulfonamide;
N-cyclopropyl-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide;
N-((3-oxoquinuclidin-2-yl)methyl)-N-phenylmethanesulfonamide;
1 -((3 -oxoquinucl idin-2-yl (methyl )pyrimidine-2, 4(1 H,3H)-dione;
5-methyl- 1((3-oxoquinuclidin-2-yl (methyl )pyrimidine-2, 4(1 H,3H)-dione; tert- butyl 5-methyl-2,6-dioxo-3-((3-oxoquinuclidin-2-yl)methyl)-3,6-dihydropyrimidine- 1 (2H)-carboxylate;
5 -methyl- 1 ,3 -bis((3 -oxoquinucl idin-2-yl (methyl )pyrimidine-2, 4(1 H, 3H)-dione;
/V-methyl- 1 -((3 -oxoquinuclidin-2-yl)methyl)- 1 H- 1 ,2,4-triazole-3 -carboxamide; 2-((3-chloro-1H-1 ,2,4-triazol-1 -yl)methyl)quinuclidin-3-one;
/V,/V-dimethyl- 1 -((3 -oxoquinuclidin-2-yl)methyl)- 1 H- 1 ,2,4-triazole-3 -carboxamide;
2-((1H-1 ,2,4-triazol-1 -yl)methyl)quinuclidin-3-one;
1 -((3-oxoquinuclidin-2-yl)methyl)-1H- 1 ,2,4-triazole-3-carbonitrile; and 1 -((3-oxoquinuclidin-2-yl)methyl)-1H- 1 ,2,4-triazole-3-carboxamide, or a pharmaceutically acceptable salt thereof.
[0095] In a specific embodiment, the p53 reactivator provided herein is 2,2,2-trifluoro-N- ((3-oxoquinuclidin-2-yl)methyl)acetamide having the following formula:
Figure imgf000019_0001
or a pharmaceutically acceptable salt thereof.
[0096] In some embodiments, the p53 reactivator provided herein is selected from the group consisting of the compounds in the table below.
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
5.2.2 Inhibitors of CD47 Mediated Signaling
[0097] In certain embodiments, the inhibitor of CD47 mediated signaling provided herein attenuates the interaction between CD47 and SIRPa. In some embodiments, the inhibitor of CD47 mediated signaling blocks the function of CD47. In other embodiments, the inhibitor of CD47 mediated signaling blocks the function of SIRPa. In some embodiments, the inhibitor of CD47 mediated signaling is an anti-CD47 antibody. In other embodiments, the inhibitor of CD47 mediated signaling is an anti-SIRPa antibody. In certain embodiments, the inhibitor of CD47 mediated signaling is an antisense oligonucleotide against CD47 or against SIRPa. [0098] In some embodients the inhibitor of CD47 mediated signaling is an Fc fusion to a portion of the receptor SIRPa providing CD47 binding.
[0099] In certain embodiments the Fc portion of the fusion protein or the anti-CD47 antibody has been inactivated by amino acid replacements eliminating binding to complement Clq protein and human Fc-gamma receptors, except for the neonatal Fc receptor (FcRn). [00100] In some embodiments, the inhibitor of CD47 mediated signaling is selected from the group consisting of SGN-CD47M (Seattle Genetics), ZL1201 (Zai Lab (Shanghai) Co., Ltd.), IBI188 (Innovent Biologies, Inc.), magrolimab (5F9) (Forty Seven, Inc.), TΉ-621 and TTI-622 (Trillium Therapeutics Inc.), AO-176 (Arch Oncology), FSI-189 (Forty Seven, Inc.), SHR-1603 (Jiangsu HengRui Medicine), ALX148 (ALX Oncology), SL-172154 (Shattuck Labs) and SRF231 (Surface Oncology).
[00101] In some embodiments, the inhibitor of CD47 mediated signaling is an anti-CD47 antibody described in US 9,017,675, which disclosure is incorporated herein by reference. In some such embodiments, the anti-CD47 antibody is selected from the group consisting of murine antibody 5F9 and humanized versions thereof, including magrolimab, and antibodies sharing the same complementarity determining region (CDR) sequences.
[00102] In one embodiment, the inhibitor of CD47 mediated signaling is an anti-CD47 antibody or an antigen binding portion thereof, which comprises the following CDR sequences (i.e. the CDR sequences of 5F9 and humanized versions thereof, including magrolimab): VH-CDR1: NYNMH (SEQ ID NO:l)
VH-CDR2: TIYPGNDDTSYNQKFKD (SEQ ID NO:2)
VH-CDR3: GGYRAMDY (SEQ ID NO: 3)
VL-CDR1 : RSSQSIVYSNGNTYLG (SEQ ID NO: 4)
VL-CDR2: KVSNRFS (SEQ ID NO: 5)
VL-CDR3: FQGSHVPYT (SEQ ID NO: 6)
[00103] In one such embodiment, the inhibitor of CD47 mediated signaling is an anti- CD47 antibody or an antigen binding portion thereof, which comprises a heavy chain variable domain sequence selected from the group consisting of o a sequence selected from the group consisting of SEQ ID NO:7-10 (VH sequences of 5F9 and humanized versions thereof, including magrolimab), and o sequences having at least 70 % identity to a sequence selected from the group consisting of SEQ ID NO:7-10, provided that the CDR sequences in such sequence are as defined in SEQ ID NO: 1-3; and a light chain variable domain sequence selected from the group consisting of o a sequence selected from the group consisting of SEQ ID NO: 11-14 (VL sequences of 5F9 and humanized versions thereof, including magrolimab), and o sequences having at least 70 % identity to a sequence selected from the group consisting of SEQ ID NO: 11-14, provided that the CDR sequences in such sequence are as defined in SEQ ID NO:4-6.
[00104] In embodiments of such an anti-CD47 antibody or an antigen binding portion thereof, the VH and VL sequences of the antibody or antigen binding portion thereof are the listed sequences and sequences having at least 75 %, such as at least 80 %, such as at least 85 %, such as at least 90 %, such as at least 95 %, such as at least 98 %, such as at least 100 % identity thereto.
[00105] In a specific embodiment, the inhibitor of CD47 mediated signaling is an anti- CD47 antibody or an antigen binding portion thereof, which comprises the heavy chain variable domain sequence SEQ ID NO: 7 (VH sequence of magrolimab) and the light chain variable domain sequence SEQ ID NO: 11 (VL sequence of magrolimab).
[00106] In a specific embodiment, the inhibitor of CD47 mediated signaling is magrolimab.
[00107] In some embodiments, the inhibitor of CD47 mediated signaling is a fusion protein between the CD47 binding domain of human SIRPa and an IgG. In some embodiments, the inhibitor of CD47 mediated signaling is a fusion protein between the CD47 binding domain of human SIRPa and a human IgG. In some embodiments, the inhibitor of CD47 mediated signaling is a fusion protein between the CD47 binding domain of human SIRPa and Fc domain of a human IgG.
[00108] In another specific embodiment, the inhibitor of CD47 mediated signaling is ALX148, as described e.g. in Kauder et al. (2018), PLoS One 13(8):e0201832, which is incorporated herein by reference. ALX148 is a molecule constructed by genetic fusion of an engineered version of the CD47-binding domain from human SIRPa to an inactive human IgGl Fc domain. ALX148 is a 78-kDa disulfide-linked homodimer that has no glycosylation sites, and binds human, cynomolgus monkey, mouse, and rat CD47 with high affinity. ALX148 blocks the CD47-SIRPa interaction and induces antitumor immunity by bridging innate and adaptive immune responses.
5.3 Pharmaceutical Compositions
[00109] A p53 reactivator (e.g., APR-246) can be formulated with one or more pharmaceutically acceptable excipient. Similarly, an inhibitor of CD47 mediated signaling (e.g., magrolimab) can be formulated in a pharmaceutical composition with one or more pharmaceutically acceptable excipients. In some embodiments, provided herein is a combination therapy comprising a first pharmaceutical composition comprising a p53 reactivator provided herein and a first pharmaceutically acceptable excipient, and a second pharmaceutical composition comprising an inhibitor of CD47 mediated signaling (e.g., magrolimab) provided herein and a second pharmaceutically acceptable excipient. The first and the second pharmaceutically acceptable excipients can be the same or different. In some embodiments, a p53 reactivator provided herein and an inhibitor of CD47 mediated signaling (e.g., magrolimab) provided herein are formulated together in a single pharmaceutical composition. In other embodiments, provided herein is a pharmaceutical composition comprising a p53 reactivator provided herein, an inhibitor of CD47 mediated signaling (e.g., magrolimab) provided herein, and one or more pharmaceutically acceptable excipient.
[00110] The p53 reactivator and/or the inhibitor of CD47 mediated signaling (e.g., magrolimab) provided herein can be formulated into suitable pharmaceutical compositions for different routes of administration, such as injection (subcutaneous, intramuscular, intravenous, intraperitoneal, intraosseous, intracardiac, intraarticular, and intracavernous), sublingual and buccal, rectal, vaginal, ocular, otic, nasal, inhalation, nebulization, cutaneous, or transdermal. The compounds described above may be formulated into pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel, Introduction to Pharmaceutical Dosage Forms, (7th ed. 1999)).
[00111] In the compositions, effective concentrations of one or more compounds (i.e., p53 reactivators or inhibitors of CD47 mediated signaling (e.g., magrolimab)) provided herein or pharmaceutically acceptable salts are mixed with a suitable pharmaceutical excipient. In certain embodiments, the concentrations of the compounds in the compositions are effective for delivery of an amount, upon administration, that treats, prevents, or ameliorates one or more of the symptoms and/or progression of a disease or disorder provided herein (e.g., cancer, including solid cancer and blood borne cancer).
[00112] The active compound is in an amount sufficient to exert a therapeutically useful effect on the patient treated. The therapeutically effective concentration may be determined empirically by testing the compounds in in vitro and in vivo systems and then extrapolated therefrom for dosages for humans. The concentration of active compound in the pharmaceutical composition will depend on absorption, tissue distribution, inactivation, and excretion rates of the active compound, the physicochemical characteristics of the compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.
[00113] It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.
[00114] The p53 reactivators (e.g, APR-246) or inhibitors of CD47 mediated signaling (e.g., magrolimab) provided herein, or pharmaceutically acceptable salts thereof, may also be formulated to target a particular tissue, receptor, or other area of the body of the subject to be treated. Many such targeting methods are well known to those of skill in the art. All such targeting methods are contemplated herein for use in the instant compositions. In one embodiment, liposomal suspensions, including tissue-targeted liposomes, such as tumor- targeted liposomes, may also be suitable as pharmaceutically acceptable excipients. These may be prepared according to methods known to those skilled in the art.
5.3.1 A Liquid Composition Comprising A P53 Reactivator [00115] In some specific embodiments, the p53 reactivator provided here (e.g., APR-246 or Compound A) is formulated in an aqueous solution, as described in US patent no. 9,061,016, which is incorporated herein by reference, and as described in more detail below.
[00116] In one embodiment, the formulation comprising the p53 reactivator is a stock solution and preferably is a pharmaceutical formulation in the form of a concentrated stock solution. The formulation preferably is sterile, and this may be achieved by known sterilization methods such as filtration, allowing for long term storage.
[00117] The formulation provided herein can be used, e.g. for administration to a patient in need thereof by direct injection or preferentially diluted with appropriate injectable solutions for i.v. infusion.
[00118] In one embodiment, the formulation provided herein is an aqueous solution of the p53 reactivator provided herein (e.g., APR-246 or Compound A), wherein the p53 reactivator is present at a concentration within a range of from about 10 mg/mL to about 250 mg/mL, a range of from about 50 mg/mL to about 200 mg/mL, or a range of from about 75 mg/mL to about 150 mg/mL of the formulation.
[00119] The formulation may be diluted prior to use, e.g., administration to a patient. The dilution factor depends on the concentration of the p53 reactivator in the formulation and the required amount of the compound needed, e.g., to meet the therapeutically effective dose. In some embodiments, in case of parenteral administration, the final diluted product has a pH within the range of from about pH 4 to about pH 6. In some embodiments, the final diluted product for parenteral administration has a pH within the range of from about pH 4.2 to about pH 5.5.
[00120] The liquid formulation may contain sodium chloride at a concentration of between 0% and 3%, a concentration of between 0.5% and 1.5%, or a concentration of between 0.8% and 1% weight by volume of the formulation.
[00121] In one embodiment, the p53 reactivator (e.g., APR-246 or Compound A) is present in the liquid formulation in the form of an acid addition salt with one or several different pharmaceutically acceptable acids. The pharmaceutically acceptable acid may be a mineral acid, e.g., selected from the group consisting of hydrochloric acid, hydrogen bromide, hydrogen iodide, sulphuric acid, nitric acid, phosphoric acid and the like. As an alternative, the pharmaceutically acceptable acid may be an organic acid, e.g., a sulfonic or carboxylic acid, particularly an alkyl or aryl sulfonic acid or an alkyl or aryl carboxylic acid, such as selected from the group consisting of methanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, acetic acid, tartaric acid, maleic acid, citric acid, benzoic acid, salicylic acid, ascorbic acid and the like.
[00122] In some embodiments, to be at the required pH, the composition provided herein contains a pH regulating agent. The term “pH regulating agent,” as used herein, means at least one pharmaceutically acceptable organic or inorganic (mineral) acid, or at least one pharmaceutically acceptable acid buffer or a mixture of any of these. Thus, the pH regulating agent may be any such acid or buffer, or a mixture of acids or buffers, or a mixture of acid(s) and buffer(s). Examples of useful acids and buffers are as indicated herein.
[00123] For example, the composition may contain at least one pharmaceutically acceptable acid. The acid may be an inorganic mineral acid, e.g., selected from the group consisting of hydrochloric acid, hydrobromic acid, hydroiodic acid, sulphuric acid, nitric acid, phosphoric acid or the like, or an organic acid, e.g., selected from the group consisting of acetic acid, succinic acid, tartaric acid, maleic acid, ascorbic acid, citric acid, glutamic acid, benzoic acid, ascorbic acid, methanesulfonic acid, ethanesulfonic acid and the like. It is contemplated that the composition may contain one or several acids, selected from inorganic and organic acids. In one embodiment, the required pH of the formulation is achieved by addition of hydrochloric acid.
[00124] The composition provided herein also may comprise at least one pharmaceutically acceptable buffer, particularly selected from the group of citric buffer, acetate buffer, phosphate buffer and the like, separately or as a mixture thereof, as well as in combination with any pharmaceutically acceptable acid, as defined herein, e.g., hydrochloric acid.
[00125] The liquid composition provided herein is aqueous, which means that it contains water. However, it is contemplated that the aqueous solution and the aqueous phase used to prepare the composition also may contain other pharmaceutically acceptable liquids as a solvent phase, e.g., polyethylene glycol (PEG) and alcohols, e.g., ethanol. In some embodiments, the aqueous phase mainly comprises water as a solvent. For example, the solvent phase is comprised of from 50 to 100% water, at least 80% water, at least 90% water, at least 95% water, at least 98% water or 100% water. [00126] In one embodiment, the composition described herein is provided as a stable stock solution, particularly as a concentrated stock solution for long term storage at a temperature range of 2-8 °C., in a container, for example, a sealed and sterilized container. For example, the composition may comprise a stable aqueous WFI (water for injection) solution of the p53 reactivator as an acid addition salt, in particular a hydrochloride addition salt, in a concentration of from about 10 mg/mL to about 250 mg/mL, from about 50 mg/mL to about 200 mg/mL, or from about 75 mg/mL to about 150 mg/mL, and a pH regulating agent in such an amount as to provide a pH in the solution in the range between pH 3.0 and pH 5.0, between pH 3.2 and pH 4.7, between pH 3.5 and pH 4.5, or between pH 3.8 and pH 4.2, e.g., approximately 4.0. For example, the pH of the stock solution may have a lower limit selected from a pH of about 3.0, or about 3.2, e.g. about 3.4, such as about 3.6 or about 3.8, and an upper limit of about 5.0, or about 4.7, or about 4.5, or about 4.2, e.g. about 4.0.
[00127] Other components also may be added to or present in the aqueous phase, such as pharmaceutically acceptable inorganic salts, e.g., NaCl, preservatives, or further pharmaceutically acceptable compounds, e.g., further therapeutically active ingredients, such as cytostatics, particularly cisplatin, daunorubicin, cerubidine, cytarabine and fludarabine.
[00128] In one embodiment, NaCl is added to the aqueous phase in an amount so as to provide a final liquid composition as defined herein above, containing NaCl at a concentration of between 0% and 3%, between 0.5% and 1.5%, or between 0.8% and 1% weight by volume of the formulation.
[00129] In one embodiment, the composition is a sterile formulation. In this case, sterilization of the composition may be accomplished by passing the formulation, e.g., a formulated stock solution, through a sterile filter with a nominal pore size of 0.2 pm into a cleaned and sterilized container.
[00130] The composition may be provided as a ready -to-use injection solution, wherein a liquid formulation, e.g., a stock solution, is brought to the desired volume by addition of one or more pharmaceutically acceptable solvents, such as selected from the group consisting of WFI, a glucose solution, electrolyte solution containing amino acids, lipids, vitamins, and other minerals, Ringer's solution, Hartmann's solution, or a sodium chloride solution in the form of an isotonic, hypotonic or hypertonic solution. An example of such pharmaceutically acceptable solution is Baxter Viaflo 9 mg/mL. [00131] In a specific embodiment, the p53 reactivator is APR-246, which is formulated in liquid formulation, which comprises at least one pH regulating agent in an amount such as to provide a pH in the aqueous solution of from about 3.0 to about 5.0. In some embodiments, APR-246 is present in the aqueous solution at a concentration of from 10 mg/mL to 250 mg/mL. In some embodiments, the aqueous solution comprises NaCl at a concentration of between 0% and 3% weight by volume. In another specific embodiment, the p53 reactivator is Compound A.
5.3.2 An Oral Dosage Form Comprising A P53 Reactivator
[00132] In some specific embodiments, the p53 reactivator provided here (e.g., Compound A) is formulated in a composition for oral administration. In a specific embodiment, the oral dosage form is a solid form.
[00133] Pharmaceutical compositions that are suitable for oral administration can be presented as discrete dosage forms, such as, but are not limited to, tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flavored syrups). Such dosage forms contain predetermined amounts of active ingredients, and may be prepared by methods of pharmacy well known to those skilled in the art.
[00134] Typical oral dosage forms are prepared by combining the active ingredients in an intimate admixture with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients can take a wide variety of forms depending on the form of preparation desired for administration. Examples of excipients suitable for use in solid oral dosage forms (e.g., powders, tablets, capsules, and caplets) include, but are not limited to, starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents.
[00135] If desired, tablets can be coated by standard aqueous or nonaqueous techniques. Such dosage forms can be prepared by any of the methods of pharmacy. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredients with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary.
[00136] For example, a tablet can be prepared by compression or molding. Compressed tablets can be prepared by compressing in a suitable machine the active ingredients in a free- flowing form such as powder or granules, optionally mixed with an excipient. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
[00137] Examples of excipients that can be used in oral dosage forms provided herein include, but are not limited to, binders, fillers, disintegrants, and lubricants. Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives ( e.g ., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose, microcrystalline cellulose, and mixtures thereof.
[00138] Suitable forms of microcrystalline cellulose include, but are not limited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICEL RC-581, AVICEL-PH-105, and mixtures thereof. Suitable anhydrous or low moisture excipients or additives include AVICEL-PH-103™ and Starch 1500 LM.
[00139] Examples of fillers suitable for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.
[00140] Disintegrants are used in compositions to provide tablets that disintegrate when exposed to an aqueous environment. The amount of disintegrant used varies based upon the type of formulation, and is readily discernible to those of ordinary skill in the art. Disintegrants that can be used in pharmaceutical compositions and dosage forms include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof.
[00141] Lubricants that can be used in pharmaceutical compositions and dosage forms include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil, zinc stearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof. Additional lubricants include, for example, a syloid silica gel, a coagulated aerosol of synthetic silica, and mixtures thereof.
5.3.3 A Lyophilized Composition Comprising An Inhibitor of CD47 Mediated Signaling
[00142] In some embodiments, the inhibitor of CD47 mediated signaling (e.g., magrolimab) provided herein is supplied in a sterile form for reconstitution as a suspension for subcutaneous injection or reconstitution as a solution with further dilution for intravenous infusion.
[00143] In some embodiments, the inhibitor of CD47 mediated signaling (e.g., magrolimab) is formulated for parenteral administration. Parenteral administration of the compositions includes intravenous, subcutaneous, and intramuscular administrations. Compositions for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, sterile suspensions ready for injection, and sterile emulsions. The solutions may be either aqueous or nonaqueous. The unit dose parenteral preparations can be packaged in an ampoule, a vial or a syringe with a needle.
[00144] As described above, pharmaceutically acceptable excipients used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharmaceutically acceptable substances. [00145] In a specific embodiment, the inhibitor of CD47 mediated signaling (e.g., magrolimab) is formulated as lyophilized powder for reconstitution for parenteral administration. Lyophilized powders can be reconstituted for administration as solutions, emulsions, and other mixtures.
[00146] The sterile, lyophilized powder can be prepared by dissolving an inhibitor of CD47 mediated signaling (e.g., magrolimab) provided herein, or a pharmaceutically acceptable salt thereof, in a suitable solvent. The solvent may contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose. The solvent may also contain a buffer, such as citrate, phosphate, or other buffers known to those of skill in the art. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation. Generally, the resulting solution will be apportioned into vials for lyophilization. Each vial will contain a single dosage or multiple dosages of the inhibitor of CD47 mediated signaling (e.g., magrolimab). The lyophilized powder can be stored under appropriate conditions, such as at about 4 °C to room temperature. [00147] In one aspect, the lyophilized formulations are suitable for reconstitution with a suitable diluent to the appropriate concentration prior to administration. In some embodiments, the lyophilized formulation is suitable for reconstitution with an aqueous solution for intravenous administrations. In certain embodiments, the lyophilized formulation provided herein is suitable for reconstitution with water.
[00148] In certain embodiment, the lyophilized formulations comprise the inhibitor of CD47 mediated signaling (e.g., magrolimab) provided herein, a buffer and a bulking agent. [00149] In certain embodiments, a lyophilized formulation comprises an inhibitor of CD47 mediated signaling (e.g., magrolimab) provided herein, in about 0.1 to about 60% based on the total weight of the lyophilized formulation.
[00150] The lyophilized formulation can be reconstituted for parenteral administration to a patient using any pharmaceutically acceptable diluent. Such diluents include, but are not limited to Sterile Water for Injection (SWFI), Dextrose 5% in Water (D5W), or a cosolvent system.
[00151] In a specific embodiment, the inhibitor of CD47 mediated signaling (e.g., magrolimab) is formulated as lyophilized formulation containing azacitidine and mannitol (e.g., 100 mg of azacitidine and 100 mg mannitol each vial).
5.4 Uses for Treatment of A Disease or Disorder and Dosing
[00152] In some embodiments, provided herein is a composition or a combination of compositions for use in the prevention and/or treatment of a disease or condition comprising the p53 reactivator (e.g., APR-246) and the inhibitor of CD47 mediated signaling (e.g., magrolimab). Such uses relate to a method comprising administering an effective amount of the p53 reactivator and the inhibitor of CD47 mediated signaling (e.g., magrolimab) to a subject. In one embodiment, the subject is a human.
[00153] In some embodiments, the disease or disorder is a neoplastic disease. In certain embodiments, the disease or disorder is a cancer. In certain embodiments, the disease or disorder is a hematogical malignancy. In certain embodiments, the neoplastic, cancerous, or malignant cell in a subject to be treated with a method provided herein comprises a mutation in TP53. In a specific embodiment, provided herein is a method to treat or prevent a hematological cancer with a mutation in the p53 gene. In certain embodiments, the cancer to be treated with a method provided herein comprises cancer cells that carry a mutation in TP53. In some embodiments, the mutation in TP53 is R248Q, R248W, R273H, R273C, R175H, Y220C, G245S, R249S, R282W, VI 73 A, S241F, R249S or a combination thereof. In some embodiments, the mutant p53 contains at least one replacement in the core domain of p53 (residues 94-292) caused by a TP53 mutation. In other embodiments, the mutant TP53 includes a nonsense mutation. A nonsense mutation is a genetic mutation changing a codon for an amino acid into a stop codon, resulting in a shorter, unfinished protein product. Nonsense mutations are less frequent than missense mutations in TP53, but nonetheless constitute about 10% of all TP 53 mutations in cancer. The most common TP 53 nonsense mutation yields a truncated p53; R213X aka R213*.
[00154] In other embodiments, the cancer to be treated does not comprise any cells carrying a mutation in the TP53 gene.
[00155] In some embodiments, the disease or disorder is a disease of abnormal cell growth and/or dysregulated apoptosis. Examples of such diseases include, but are not limited to, cancer, mesothelioma, bladder cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, ovarian cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, bone cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, gastrointestinal (gastric, colorectal and/or duodenal) cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, esophageal cancer, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, testicular cancer, hepatocellular (hepatic and/or biliary duct) cancer, primary or secondary central nervous system tumor, primary or secondary brain tumor, Hodgkin's disease, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphoma, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, multiple myeloma, oral cancer, non-small-cell lung cancer, prostate cancer, small-cell lung cancer, cancer of the kidney and/or ureter, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system, primary central nervous system lymphoma, non-Hodgkin's lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma, adrenocortical cancer, gall bladder cancer, cancer of the spleen, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma or a combination thereof.
[00156] In some embodiments, the disease or disorder is selected from the group consisting of bladder cancer, brain cancer, breast cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small- cell lung cancer, prostate cancer, small-cell lung cancer and spleen cancer.
[00157] In some embodiments, the disease or disorder is a tumor of the hematopoietic or lymphoid tissue.
[00158] In some embodiments, the disease or disorder is a hematological cancer, such as leukemia, lymphoma, or myeloma. In some embodiments, the cancer is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular center lymphoma, transformed lymphoma, lymphocytic lymphoma of intermediate differentiation, intermediate lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse small- cleaved cell lymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-cell lymphoma, mantle zone lymphoma, low grade follicular lymphoma, multiple myeloma (MM), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), myelodysplastic syndrome (MDS), acute T cell leukemia, acute myeloid leukemia (AML), acute promyelocytic leukemia, acute myeloblastic leukemia, acute megakaryoblastic leukemia, precursor B acute lymphoblastic leukemia, precursor T acute lymphoblastic leukemia, Burkitt’s leukemia (Burkitt’s lymphoma), acute biphenotypic leukemia, chronic myeloid lymphoma, chronic myelogenous leukemia (CML), and chronic monocytic leukemia. In a specific embodiment, the disease or disorder is myelodysplastic syndromes (MDS). In another specific embodiment, the disease or disorder is acute myeloid leukemia (AML). In another specific embodiment, the disease or disorder is chronic lymphocytic leukemia (CLL). In yet another specific embodiment, the disease or disorder is multiple myeloma (MM).
[00159] In other embodiments, the disease or disorder is a solid tumor cancer. In some embodiments, the solid tumor cancer is selected from the group consisting of a carcinoma, an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a ductal cell carcinoma, a lung carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, a non-melanoma skin carcinoma, and a lung cancer.
[00160] The amount of a prophylactic or therapeutic agent (the p53 reactivator and the inhibitor of CD47 mediated signaling (e.g., magrolimab) provided herein), or a composition provided herein that will be effective in the prevention and/or treatment of a disease or condition can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of a disease or condition, and in some embodiments, should be decided according to the judgment of the practitioner and each patient’s circumstances.
[00161] The dose administered to a subject in the context of the present disclosure should be sufficient to effect a therapeutic response. One skilled in the art will recognize that dosage will depend upon a variety of factors including the potency of the specific compound, the age, condition and body weight of the patient, as well as the stage/severity of the disease. The dose will also be determined by the route (administration form) timing and frequency of administration.
[00162] The p53 reactivator (e.g., APR-246) and the inhibitor of CD47 mediated signaling (e.g., magrolimab) can be formulated in different pharmaceutical compositions and administered separately to the subject in need thereof. Alternatively, the p53 reactivator (e.g., APR-246) and the inhibitor of CD47 mediated signaling (e.g., magrolimab) are administered together in the same pharmaceutical composition.
[00163] In some embodiments, the p53 reactivator (e.g., APR-246) and the inhibitor of CD47 mediated signaling (e.g., magrolimab) are administered simultaneously. The term “simultaneously” means at the same time or within a short period of time, for example, less than 1 hour, less than 2 hours, less than 3 hours, less than 4 hours, or less than 12 hours. [00164] In some embodiments, the p53 reactivator (e.g., APR-246) and the inhibitor of CD47 mediated signaling (e.g., magrolimab) are not administered simultaneously, and instead the two compounds are administered at different times. In some embodiments, the p53 reactivator (e.g., APR-246) and the inhibitor of CD47 mediated signaling (e.g., magrolimab) are administered at least once during a dosing period. A dosing period as used herein is meant a period of time, during which each therapeutic agent has been administered at least once. A dosing cycle can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. In some embodiments, a dosing cycle is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. In certain embodiments, a dosing period is a dosing cycle.
[00165] The prophylactic or therapeutic agent (the p53 reactivator and/or the inhibitor of CD47 mediated signaling provided herein) can be delivered as a single dose (e.g., a single bolus injection), or over time (e.g., continuous infusion over time or divided bolus doses over time). The agent can be administered repeatedly if necessary, for example, until the patient experiences stable disease or regression, or until the patient experiences disease progression or unacceptable toxicity. Stable disease or lack is determined by methods known in the art such as evaluation of patient symptoms, physical examination, and visualization of the tumor that has been imaged using X-ray, CAT, PET, MRI scan, or other commonly accepted evaluation modalities.
[00166] The prophylactic or therapeutic agent (the p53 reactivator and/or the inhibitor of CD47 mediated signaling provided herein) can be administered once daily (QD) or divided into multiple daily doses such as twice daily (BID), three times daily (TID), and four times daily (QID). In addition, the administration can be continuous (i.e., daily for consecutive days or every day) or intermittent, e.g, in cycles (i.e., including days, weeks, or months of rest without drug). As used herein, the term “daily” is intended to mean that a therapeutic compound is administered once or more than once each day, for example, for a period of time. The term “continuous” is intended to mean that a therapeutic compound is administered daily for an uninterrupted period of, e.g., at least 10 days. The term “intermittent” or “intermittently” as used herein is intended to mean stopping and starting at either regular or irregular intervals.
For example, intermittent administration of the compound is administration for one to six days per week, administration in cycles (e.g., daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week), or administration on alternate days.
[00167] In some embodiments, the frequency of administration is in the range of about a daily dose to about a monthly dose. In certain embodiments, administration is once a day, twice a day, three times a day, four times a day, once every other day, twice a week, once every week, once every two weeks, once every three weeks, or once every four weeks.
[00168] In certain embodiments, the compound is administered once per day from one day to six months, from one week to three months, from one week to four weeks, from one week to three weeks, or from one week to two weeks.
[00169] In some embodiments, APR-246 is administered at a dose of less than 150 mg/kg. In some embodiments, APR-246 is administered at a dose of less than 100 mg/kg. In some embodiments, APR-246 is administered at a dose of betweenlOO mg/kg and 25 mg/kg. In other embodiments, APR-246 is administered at dose of less than 75 mg/kg. In yet other embodiments, the APR-246 is administered at a dose of less than 65 mg/kg. In yet other embodiments, the APR-246 is administered at a dose of less than 50 mg/kg.
[00170] In other embodiments, APR-246 is administered at a fixed dose within the interval 2.7-7.5 g. In some embodiments, the fixed dose of APR-246 is no more than 4.5 g. In some embodiments, the fixed dose of APR-246 is no more than 3.7 g. In some embodiments, APR-246 is administered at a fixed dose during a period of about 3 to 7 hours. In some embodiments, APR-246 is administered at a fixed dose during a period of about 6 or 4 hours.
5.5 Assays for Demonstrating Synergistic Effects of a P53 Reactivator and an Inhibitor of CD47 Mediated Signaling
[00171] Also provided herein are assays for demonstrating the effects of the combination treatment with a p53 reactivator and an inhibitor of CD47 mediated signaling.
[00172] A model system for a hematological malignancy (e.g, an in vivo cancer model such as a mouse model) may be treated with a p53 reactivator, an inhibitor of CD47 mediated signaling, or a combination of the p53 reactivator and the inhibitor of CD47 mediated signaling, and the effects of the combination treatment are analyzed and compared to the monotherapies.
[00173] Synergistic effects of a P53 Reactivator and an inhibitor of CD47 mediated signaling can then be analyzed using well-known analytical tools. [00174] In one embodiment, the analytical tool is COMBENEFIT. Combenefit (Di Veroli. C, Bioinformatics, 32(18), 2016, 2866-2868) is an interactive platform for the analysis and visualization of drug combinations and only requires the user to save the data in the predefined .xls template files. Combenefit performs combination analyses using the standard Loewe, Bliss and HSA methods (see Ianevski et al., Bioinformatics, 2017, 33(15): 2413-2415). [00175] Combination effects may also be analyzed using the Additive model (see Valeriote et al., Cancer Chemother Rep. 1975, 59:895-900; Lepri et al., Hematol Oncol. 1991, 9:79-86; and Jonsson E et al., EurJ Clin Pharmacol. 1998, 54:509-14.) In samples with two co-incubated substances, a predicted cell viability (%) is calculated according to the following formula: Predicted cell viability (%) = cell viability of substance 1 (%) x cell viability of substance 2 (%) x 0.01.
[00176] A “combination index” (Cl) is then calculated as the measured cell viability of the sample with two co-incubated substances divided by the predicted cell viability. The following classifications are used in this example:
Cl > 1.2 sub-additive effect Cl = 0.8-1.2 additive effect Cl < 0.8 synergistic effect Cl < 0.5 strong synergistic effect
[00177] If the measured cell viability for a combination of two substances is higher than the cell viability for one or both substances, the effect is considered antagonistic. If the predicted viability is very low, the quote “measured viability / predicted viability” may give false Cl values. Thus, a lower limit of < 5% of the predicted viability may be set.
[00178] The invention is generally disclosed herein using affirmative language to describe the numerous embodiments. The invention also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, procedures, assays or analysis. Thus, even though the invention is generally not expressed herein in terms of what the invention does not include, aspects that are not expressly included in the invention are nevertheless disclosed herein. [00179] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the following examples are intended to illustrate but not limit the scope of invention described in the claims. . EXAMPLES
[00180] The following is a description of various methods and materials used in the studies, and are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below were performed and are all of the experiments that may be performed. It is to be understood that exemplary descriptions written in the present tense were not necessarily performed, but rather that the descriptions can be performed to generate the data and the like associated with the teachings of the present invention. Efforts have been made to ensure accuracy with respect to numbers used ( e.g ., amounts, percentages, etc.), but some experimental errors and deviations should be accounted for.
6.1 Example 1 - In vitro Experiment Assessing the Enhancement of Inhibitor of CD47 Facilitated Phagocytosis
[00181] This experiment can be used to assess the enhancement of phagocytosis facilitated by an inhibitor of CD47 mediated signaling (e.g., magrolimab) by pretreatment with a p53 reactivator (e.g., APR-246). In preparation for a phagocytosis assay, CD 14+ monocytes are prepared from blood according to standard protocols, and subsequently matured into macrophages on culture dishes in growth medium supplemented with 10% human AB serum. After 7-11 days, the macrophages are detached from culture plates by trypsination. The target cells (e.g., SKM-1 and NCI-1688) are pretreated for 12 hours with a p53 reactivator (e.g., APR- 246), or not as a control, and labeled with Calcein acetoxymethyl. Phagocytosis reactions are carried out using 50,000 macrophages and 100,000 target cells, incubated together at 37 °C for 2 hours.
[00182] Flow cytometry is used to measure phagocytosis, evaluated by the percentage of macrophages engulfing calcein acetoxymethyl-labeled tumor cells.
6.2 Example 2 - In vivo Experiments in AML Model
[00183] This experiment can be used to assess the effects of an inhibitor of CD47 mediated signaling (e.g., magrolimab) and/or a p53 reactivator (e.g., APR-246) on AML. This experiment is done twice, first at pilot scale to titrate doses and incubation times and second as a full-scale experiment. 1-2.5 x 105 FACS purified leukemic stem cells are transplanted into NOG juvenile mice. Eight to twelve weeks later, the presence of human AML engraftment (hCD45+CD33+ cells) is assessed in the blood and the femur bone marrow. Subsequently the mice are treated with intra peritoneal injections of an inhibitor of CD47 mediated signaling ( e.g ., magrolimab) and/or a p53 reactivator (e.g., APR-246), at a dose of 1-5 mg/kg daily and 25-100 mg/kg twice daily, respectively. The decrease in the number of human CD34+ cells in the bone marrow following 1 -3 weeks of treatment is determined by flow cytometry.
[00184] This in vivo experiment can also be performed with the inhibitor of CD47 mediated signaling denoted ALX148, in which case the administration is done less frequently and at a higher dose; once weekly and at a dose of 30 mg/kg.
6.3 Example 3 -In vivo Experiments in Solid Tumor Model [00185] This experiment can be used to assess the effects of an inhibitor of CD47 mediated signaling (e.g., magrolimab) and/or a p53 reactivator (e.g., APR-246) on solid tumors. This experiment is done twice, first at pilot scale to titrate doses and incubation times and second as a full-scale experiment. Experiments are made in syngeneic mice to enable a complete immune response. 0.5 x 106 CT26 colon cancer cells are resuspended in 100 mΐ PBS and implanted subcutaneously into the flank of female BALB/c mice. Mice are randomized into treatment groups when tumors have reached an average size of 80 mm3. Mce are injected intraperitoneally with 5 mg/kg anti -PD- 1 mAh every 5 days, an inhibitor of CD47 mediated signaling (e.g., magrolimab) at 1-5 mg/kg once daily, and/or or a p53 reactivator (e.g., APR- 246) at 25-100 mg/kg twice daily. One group gets the inhibitor of CD47 mediated signaling, a second group gets the p53 reactivator, a third group get the inhibitor of CD47 mediated signaling and the p53 reactivator, a fourth group gets the inhibitor of CD47 mediated signaling and anti-PD-1, and a fifth group all three treatments. The control group is injected with an irrelevant mouse IgGl every 5 days at a dose of 5 mg/kg.
[00186] The tumor volumes are estimated from caliper measurements every second day, until a maximum volume of 2000 mm3 is reached, at which point the mice are euthanized. [00187] This in vivo experiment can also be performed with the inhibitor of CD47 mediated signaling denoted ALX148, in which case the administration is done less frequently and at a higher dose; once weekly and at a dose of 30 mg/kg. 6.4 Example 4 -In vivo Experiment
[00188] A pilot scale experiment was performed to assess in vivo the effects of combining a p53 reactivator with an inhibitor of CD47 mediated signaling.
[00189] B16.H12, a mouse monoclonal antibody (mAh) binding murine CD47 (Bio X
Cell), and APR-246 were intraperitoneally administered alone and in combination to NOG mice (NOD. Cg-PrkdcscidI l2rgtm 15ug/J icTac) . These T, B and NK cell deficient mice had previously been injected with the human AML cell line SKM-1. About 10 million cells were intravenously injected into each mouse. A 3 -week treatment regime was initiated six days after SKM-1 cell injection. The mice were divided into four groups of six mice: control, anti-CD47 mAh, APR-246 and combination. APR-246 was administered twice daily at 100 mg/kg for 5 days per week, and the mAh at 5 mg/kg twice per week. The fraction of human CD45+ cells was determined by flow cytometry using a pre-labeled mAh designated APC Mouse Anti- Human CD45 (HI30; BD Biosciences). Negative control blood was obtained from mice not injected with tumor cells to identify CD45+ human cells in inoculated mice samples. The results after 2 and 3 weeks shows a beneficial effect of the combination in comparison to the individual treatments (Figure 1).
[00190] From the foregoing, it will be appreciated that, although specific embodiments have been described herein for the purpose of illustration, various modifications may be made without deviating from the spirit and scope of what is provided herein. All of the references referred to above are incorporated herein by reference in their entireties.

Claims

1. A method of treating cancer in a subject, comprising:
(i) administering a compound that can give reactivation of a mutant p53; and
(ii) administering an inhibitor of CD47 mediated signaling, wherein the cancer in the subject comprises a cell that carries a mutation in TP53.
2. The method of claim 1 , wherein the mutation in TP 53 is selected from the group consisting of R248Q, R248W, R273H, R273C, R175H, Y220C, G245S, R249S, R282W, and a combination thereof.
3. The method of claim 1 or claim 2, wherein the compound that can give reactivation of the mutant p53 promotes proper folding of the mutant p53 and restores at least part of a normal p53 function.
4. The method of claim 1 or claim 2, wherein the compound that can give reactivation of the mutant p53 can result in a shift of the equilibrium from unfolded towards a wild-type like p53 conformation.
5. The method of claim 1 or claim 2, wherein the compound that can give reactivation of the mutant p53 interferes with aggregation of misfolded mutant p53 or reduces aggregation of the mutant p53.
6. The method of claim 1 or claim 2, wherein the compound that can give reactivation of the mutant p53 or its metabolite or degradation product thereof can restore a p53 wild type function by covalent binding to the mutant p53.
7. The method of claim 6, wherein the compound that can give reactivation of the mutant p53 can bind to thiol groups in the core domain of the mutant p53 and restore wild-type conformation.
8. The method of claim 1 , wherein the compound that can give reactivation of the mutant p53 is selected from the group consisting of: 2-(hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one;
2.2-bis(hydroxymethyl)quinuclidin-3-one;
2.2.2-trichloro-N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)acetamide;
2.2.2-trichloro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide; N-ethyl-2,2,2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide;
2.2.2-trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide;
2.2-difluoro-N-((3-oxoquinuclidin-2-yl)methyl)acetamide, N-((3-oxoquinuclidin-2-yl)methyl)pyridine-3-sulfonamide;
4-fluoro-N-((3-oxoquinuclidin-2-yl)methyl)benzenesulfonamide; N-ethyl-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)benzenesulfonamide; 2-(N-((3-oxoquinuclidin-2-yl)methyl)methylsulfonamido)acetamide; N-(methylsulfonyl)-N-((3-oxoquinuclidin-2-yl)methyl)glycine; N-((3-oxoquinuclidin-2-yl)methyl)pyridine-4-sulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)pyridine-2-sulfonamide; N-ethyl-1,1,1-trifluoro-N-((3-oxoquinuclidin-2-yl)-methyl)methanesulfonamide;
1,1,1 -trifluoro-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N,N-bis((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)propane-2-sulfonamide; N-((3-oxoquinuclidin-2-yl)methyl)cyclopropanesulfonamide;
1 -methyl-N -((3 -oxoquinuclidin-2-yl)methyl)cyclopropane- 1 -sulfonamide;
N-cyclopropyl-N-((3-oxoquinuclidin-2-yl)methyl)methanesulfonamide;
N-((3-oxoquinuclidin-2-yl)methyl)-N-phenylmethanesulfonamide;
1 -((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4( 1 H,3H)-dione;
5-methyl- 1 -((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4( 1 H,3H)-dione; tert- butyl 5-methyl-2,6-dioxo-3-((3-oxoquinuclidin-2-yl)methyl)-3,6-dihydropyrimidine- 1 (2 H)- carboxylate;
5-methyl-l,3-bis((3-oxoquinuclidin-2-yl)methyl)pyrimidine-2,4(liT,3i/)-dione; /V-methyl- 1 -((3-oxoquinuclidin-2-yl)methyl)-1 H- 1 ,2,4-triazole-3-carboxamide;
2-((3 -chloro- 1H- 1 ,2,4-triazol- 1 -yl)methyl)quinuclidin-3 -one; /V,/V-dimethyl-l-((3-oxoquinuclidin-2-yl)methyl)-1 H-1,2,4-triazole-3-carboxamide;
2-(( 1H- 1 ,2,4-triazol- 1 -yl)methyl)quinuclidin-3 -one;
1 -((3-oxoquinuclidin-2-yl)methyl)-1 H-1 ,2,4-triazole-3-carbonitrile; and 1 -((3-oxoquinuclidin-2-yl)methyl)-1 H-1 ,2,4-triazole-3-carboxamide, or a pharmaceutically acceptable salt thereof.
9. The method of claim 8, wherein the compound is 2-(hydroxymethyl)-2-(methoxymethyl) quinuclidin-3-one having the following formula:
Figure imgf000045_0001
or a pharmaceutically acceptable salt thereof.
10. The method of claim 8, wherein the compound is 2,2,2-trifluoro-N-((3-oxoquinuclidin-2- yl)methyl)acetamide having the following formula:
Figure imgf000045_0002
or a pharmaceutically acceptable salt thereof.
11. The method of any one of claims 1-10, wherein the inhibitor of CD47 mediated signaling attenuates the interaction between CD47 and SIRPa.
12. The method of any one of claims 1-11, wherein the inhibitor of CD47 mediated signaling is an inhibitor of CD47.
13. The method of claim 12, wherein the inhibitor of CD47 is an anti-CD47 antibody.
14. The method of claim 13, wherein the anti-CD47 antibody is magrolimab.
15. The method of claim 13, wherein the anti-CD47 antibody is SRF231.
16. The method of any one of claims 1-11, wherein the inhibitor of CD47 mediated signaling is an inhibitor of SIRPa.
17. The method of claim 16, wherein the inhibitor of SIRPa is an anti-SIRPa antibody.
18. The method of any one of claims 1-17, wherein the p53 reactivator is formulated in a first pharmaceutical composition and the inhibitor of CD47 mediated signaling is formulated in a second pharmaceutical composition.
19. The method of any one of claims 1-18, wherein the cancer is a hematological malignancy.
20. The method of claim 19, wherein the hematological malignancy is selected from the group consisting of leukemia, lymphoma, and myeloma.
21. The method of any one of claims 1-20, wherein the cancer is selected from the group consisting of: Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular center lymphoma, transformed lymphoma, lymphocytic lymphoma of intermediate differentiation, intermediate lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse small-cleaved cell lymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-Cell lymphoma, mantle zone lymphoma, low grade follicular lymphoma, multiple myeloma (MM), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), myelodysplastic syndrome (MDS), acute T cell leukemia, acute myeloid leukemia (AML), acute promyelocytic leukemia, acute myeloblastic leukemia, acute megakaryoblastic leukemia, precursor B acute lymphoblastic leukemia, precursor T acute lymphoblastic leukemia, Burkitt’s leukemia (Burkitt’s lymphoma), acute biphenotypic leukemia, chronic myeloid lymphoma, chronic myelogenous leukemia (CML), and chronic monocytic leukemia.
22. The method of claim 21, wherein the cancer is myelodysplastic syndromes (MDS).
23. The method of claim 21, wherein the cancer is acute myeloid leukemia (AML).
24. The method of claim 21, wherein the cancer is chronic lymphocytic leukemia (CLL).
25. The method of claim 21, wherein the cancer is multiple myeloma (MM).
26. A combination comprising:
(i) a compound that can give reactivation of a mutant p53; and
(ii) an inhibitor of CD47 mediated signaling, for use in a method of treating cancer in a subject, wherein the cancer comprises a cell that carries a mutation in TP53.
27. The combination for use according to claim 26, wherein said method is as disclosed in any one of claims 2-25.
28. Use of a combination comprising:
(i) a compound that can give reactivation of a mutant p53; and
(ii) an inhibitor of CD47 mediated signaling, in the preparation of a medicament for use in a method of treating cancer in a subject, wherein the cancer comprises a cell that carries a mutation in TP53.
29. Use according to claim 28, wherein said method is as disclosed in any one of claims 2-25.
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