WO2014058947A1 - Compositions and methods for treating cancer using pi3k inhibitor and anti-cd19 maytansinoid immunoconjugate - Google Patents

Compositions and methods for treating cancer using pi3k inhibitor and anti-cd19 maytansinoid immunoconjugate Download PDF

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Publication number
WO2014058947A1
WO2014058947A1 PCT/US2013/063983 US2013063983W WO2014058947A1 WO 2014058947 A1 WO2014058947 A1 WO 2014058947A1 US 2013063983 W US2013063983 W US 2013063983W WO 2014058947 A1 WO2014058947 A1 WO 2014058947A1
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compound
immunoconjugate
ser
cancer
pharmaceutically acceptable
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PCT/US2013/063983
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French (fr)
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Ti Cai
Tal ZAKS
Angela Romanelli
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Sanofi
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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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6871—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting an enzyme
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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

Definitions

  • an anti-CD 19 maytansinoid immunoconjugate and a phosphoinositide 3-kinase (PI3K) inhibitor which are therapeutically useful in the treatment of cancer.
  • compositions and uses thereof in the treatment of a variety of cancers are provided herein.
  • the immunoconjugate comprising a light chain and a heavy chain, said light chain having the sequence represented in SEQ ID NO. 7, and said heavy chain having the sequence represented in SEQ ID NO. 8.
  • the immunoconjugate further comprises a cytotoxic agent that is a maytansinoid such as DM4 that is covalently attached, directly or via a cleavable or non-cleavable linker, to the antibody wherein about 3.5 molecules of maytansinoid are bound through the SPDB linker to each antibody molecule.
  • a cytotoxic agent that is a maytansinoid such as DM4 that is covalently attached, directly or via a cleavable or non-cleavable linker, to the antibody wherein about 3.5 molecules of maytansinoid are bound through the SPDB linker to each antibody molecule.
  • methods of treating a subject with cancer comprise administering to the subject a therapeutically effective amount of Compound (1), or a pharmaceutically acceptable salt thereof, in combination with Immunoconjugate (1).
  • the method of treating a subject with cancer comprises administering to the subject a first dosage of a PI3K inhibitor, and a second dosage of an anti- CD 19 maytansinoid immunoconjugate wherein said PI3K inhibitor is Compound (1).
  • the method of treating a subject with cancer comprises administering to the subject a first dosage of an anti-CD 19 maytansinoid immunoconjugate, and a second dosage of a PI3K inhibitor wherein said PI3K inhibitor is Compound (1).
  • the methods involve treating cancer selected from the group consisting of lymphomas and leukemias including, but not limited to, germinal center B-cell like diffuse large B-cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia and mantle cell lymphoma.
  • lymphomas and leukemias including, but not limited to, germinal center B-cell like diffuse large B-cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia and mantle cell lymphoma.
  • compositions and methods of use described herein are in amounts (i.e., either in the composition are in an administered dosage) that provide a synergistic effect in the treatment of the subject.
  • a combination for use in treating a patient with cancer comprising a therapeutically effective amount of (A) Compound (1), or a pharmaceutically acceptable salt thereof, and (B) Immunoconjugate (1).
  • uses of a combination comprising a therapeutically effective amount of (A) Compound (1), or a pharmaceutically acceptable salt thereof, and (B) Immunoconjugate (1) are provided for the preparation of a medicament for use in treatment of lymphoma.
  • kits comprising: (A) Compound (1), or a
  • Figure 1 is a schematic showing the structure of an anti-CD 19 maytansinoid immunoconjugate.
  • Figure 2 demonstrates the enhanced apoptosis observed with Compound (1) (i.e. CPD(l)) and an anti-CD19 maytansinoid immunoconjugate (i.e. IC(1)) in the OCI-LY7 cell line.
  • Compound (1) i.e. CPD(l)
  • an anti-CD19 maytansinoid immunoconjugate i.e. IC(1)
  • Figure 3 demonstrates the enhanced anti-proliferative activity observed with
  • Compound (1) i.e. CPD(l)
  • an anti-CD19 maytansinoid immunoconjugate i.e. IC(1)
  • compositions and methods for the treatment of cancer are provided.
  • compositions and methods comprise inhibitors of the phosphoinositide 3-kinase (PI3K) pathway (Compound (1)) and an anti-CD 19 maytansinoid immunoconjugate
  • Immunoconjugate (1) Compound (1) and Immunoconjugate (1) can be administered in a single dosage form, or separate dosage forms.
  • Compound (1) 2-amino-8-ethyl-4-methyl-6-(lH-pyrazol-5-yl)pyrido[2,3- d]pyrimidin-7(8H)-one, is a selective inhibitor of class I PI3K lipid kinases.
  • Compound (1) targets both PI3K isoforms (IC 50 values in nM: PI3Ka 39, ⁇ 3 ⁇ 113, ⁇ 3 ⁇ 43, ⁇ 3 ⁇ 9) and mTOR (157 nM).
  • Compound (1) alone inhibits tumor growth in mice bearing xenografts in which PI3K signaling is activated, such as the PTEN-deficient PC-3 prostate adenocarcinoma, U87-MG gliobastoma, A2058 melanoma and WM -266-4 melanoma, or the PIK3CA mutated MCF7 mammary carcinoma.
  • Compound (1) is the subject of several clinical trials involving patients with, for example, solid tumors, lymphoma, glioblastoma or hormone receptor-positive breast cancer.
  • CD 19 is the earliest differentiation antigen of the B lymphocyte lineage, expressed on most B-cells, but not detected on plasma cells, stem cells, or on normal myeloid lineage. Therefore, CD 19 is expressed on tumor cells from all B cell-derived neoplasms (B-cell non- Hodgkin's lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia), except myeloma.
  • Immunoconjugate (1) is an antibody-drug conjugate composed of a humanized IgGl monoclonal antibody, huB4 (Roguska et al, Proc. Natl. Acad. Sci. USA, 91: 969-973, 1994), which specifically targets the CD 19 antigen, conjugated through a disulfide link to the maytansinoid derivative DM4, a potent tubulin inhibitor (US 2004/0235840).
  • the structure of Immunoconjugate (1) is disclosed in Figure 1. After binding to the CD 19 antigen,
  • Immunoconjugate (1) undergoes internalization and intracellular release of DM4.
  • the entire contents of Roguska et al, Proc. Natl. Acad. Sci. USA, 91: 969-973, 1994 are incorporated herein by reference.
  • methods for treating subjects with cancer comprise administering to the subject a therapeutically effective amount of an anti-CD 19 maytansinoid immunoconjugate referred to herein as
  • Immuno conjugate (1) and a therapeutically effective amount of a PI3K inhibitor having the structural Formula (1), as further described below.
  • Immunoconjugate (1) and Compound (1) can be in single or separate dosage forms.
  • inventive methods and compositions comprise a PI3K inhibitor having the following s
  • Compound (1) 2-amino-8-ethyl-4-methyl-6-(lH-pyrazol-5- yl)pyrido[2,3-d]pyrimidin-7(8H)-one.
  • the preparation and properties of Compound (1) are provided in, for example, International
  • Patent Publication No. WO 07/044813 particularly Example 56 therein.
  • the entire contents of WO 07/044813 are incorporated herein by reference.
  • Neutral and salt forms of the compound of Formula (1) are all considered herein.
  • the anti-CD 19 maytansinoid immunoconjugate (Immunoconjugate (1)) comprises two primary components, a cell-binding agent and a cytotoxic agent.
  • cell binding agent refers to an agent that specifically recognizes and binds the CD 19 antigen on the cell surface.
  • the cell-binding agent is an antibody which binds specifically to the CD 19 antigen.
  • the said antibody comprises six complementary determining region (CDR), said CDR having the sequences represented in SEQ ID NOs 1 to 6.
  • the antibody comprises a light chain, wherein the sequence of the said light chain has at least 60%, at least 75%, at least 85%, at least 90 %, at least 95 % or at least 99% identity with the sequence displayed in SEQ ID NO. 7.
  • the antibody comprises a heavy chain, wherein the sequence of the said heavy chain has at least 60%, at least 75%, at least 85%, at least 90 %, at least 95 % or at least 99% identity with the sequence displayed in SEQ ID NO. 8.
  • the antibody of the invention is the humanized antibody huB4 described in Roguska et al. (Proc. Natl. Acad. Sci. USA, 91 : 969-973, 1994).
  • the antibody huB4 according to the invention comprises a light chain and a heavy chain, said light chain having the sequence represented in SEQ ID NO. 7, and said heavy chain having the sequence represented in SEQ ID NO. 8.
  • the second component of the anti-CD 19 maytansinoid immunoconjugate
  • cytotoxic agent refers to a substance that reduces or blocks the function, or growth, of cells and/or causes destruction of cells.
  • the cytotoxic agent is a maytansinoid such as DM4.
  • the cell binding agent of the present invention is covalently attached, directly or via a cleavable or non-cleavable linker, to the cytotoxic agent.
  • the said cell binding agent is conjugated to DM4 through a cleavable linker.
  • this cleavable linker is a SPDB linker.
  • DM4 and a method of conjugating DM4 to the huB4 antibody through a SPDB linker are described in US Patent Publication No. 2004/0235840, which is hereby incorporated by reference.
  • the anti-CD 19 maytansinoid immunoconjugate comprises an antibody which binds specifically to the CD 19 antigen conjugated to DM4 through SPDB wherein about 3.5 molecules of DM4 are bound through the SPDB linker to each antibody molecule.
  • the anti-CD 19 maytansinoid immunoconjugate is
  • Immunoconjugate (1) as depicted in Figure 1. It comprises a light chain and a heavy chain, said light chain having the sequence represented in SEQ ID NO. 7, and said heavy chain having the sequence represented in SEQ ID NO. 8. It further comprises a cytotoxic agent is a maytansinoid such as DM1 or DM4 that is covalently attached, directly or via a cleavable or non-cleavable linker, to the antibody wherein about 3.5 molecules of maytansinoid are bound through the SPDB linker to each antibody molecule.
  • Immunoconjugate (1) comprises a light chain and a heavy chain, said light chain having the sequence represented in SEQ ID NO.
  • the compounds described above are unsolvated.
  • one or both of the compounds used in the method are in solvated form.
  • the solvate can be any of pharmaceutically acceptable solvent, such as water, ethanol, and the like. In general, the presence of a solvate or lack thereof does not have a substantial effect on the efficacy of the PI3K inhibitor or anti-CD 19 maytansinoid immunoconjugate described above.
  • the compound in Formula (1) is depicted in its neutral form, in some embodiments, this compound is used in a pharmaceutically acceptable salt form.
  • the salt can be obtained by any of the methods well known in the art.
  • a "pharmaceutically acceptable salt” of the compound refers to a salt that is pharmaceutically acceptable and that retains pharmacological activity. It is understood that the pharmaceutically acceptable salts are nontoxic. Additional information on suitable pharmaceutically acceptable salts can be found in Remington 's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, 1985, or S. M. Berge, et al, "Pharmaceutical Salts," J. Pharm. Sci., 1977;66: 1-19, both of which are incorporated herein by reference.
  • Examples of pharmaceutically acceptable acid addition salts include those formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, as well as those salts formed with organic acids, such as acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, 3-(4-hydroxybenzoyl)benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1 ,2-ethanedisulfonic acid, 2- hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2- naphthalenesulf
  • the PI3K inhibitor of formula (1) is administered simultaneously with Immunoconjugate (1).
  • Simultaneous administration typically means that both compounds enter the subject at precisely the same time.
  • simultaneous administration also includes the possibility that the PI3K inhibitor and the anti-CD 19 maytansinoid immunoconjugate enter the subject at different times, but the difference in time is sufficiently miniscule that the first administered compound is not provided the time to take effect on the subject before entry of the second administered compound.
  • Such delayed times typically correspond to less than 1 minute, and more typically, less than 30 seconds.
  • simultaneous administration can be achieved by administering a solution containing the combination of compounds.
  • simultaneous administration of separate solutions, one of which contains the PI3K inhibitor and the other of which contains the anti-CD 19 maytansinoid
  • immunoconjugate can be employed.
  • simultaneous administration can be achieved by administering a composition containing the combination of compounds.
  • the anti-CD 19 maytansinoid
  • the anti-CD 19 maytansinoid immunoconjugate and the PI3K inhibitor are administered sequentially.
  • the anti-CD 19 maytansinoid immunoconjugate is administered first, followed immediately thereafter by administration of the PI3K inhibitor.
  • the anti-CD 19 maytansinoid immunoconjugate is administered, a time interval occurs and then the PI3K inhibitor is administered.
  • the time interval can be one or more hour(s), one or more day(s) or one or more week(s).
  • the PI3K inhibitor is administered first, followed immediately thereafter by administration of anti-CD 19 maytansinoid immunoconjugate.
  • the PI3K inhibitor is administered, a time interval occurs and then anti-CD 19 maytansinoid immunoconjugate is administered.
  • one or both of the PI3K inhibitor and anti-CD 19 maytansinoid immunoconjugate are administered in a therapeutically effective (i.e., therapeutic) amount or dosage.
  • a “therapeutically effective amount” is an amount of the PI3K inhibitor or anti-CD 19 maytansinoid immunoconjugate that, when administered to a subject by itself, effectively treats the cancer (for example, decreases the number of lymphoma cells, inhibits tumor growth, stops tumor growth, or causes tumor regression).
  • An amount that proves "therapeutically effective amount" in a given instance, for a particular subject may not be effective for 100% of subjects similarly treated for the disease or condition under consideration, even though such dosage is deemed a "therapeutically effective amount” by skilled practitioners.
  • the amount of the compound that corresponds to a therapeutically effective amount is strongly dependent on the type of cancer, stage of the cancer, the age of the subject being treated, and other facts.
  • one or both of the PI3K inhibitor and the anti-CD 19 maytansinoid immunoconjugate are administered in a sub-therapeutically effective amount or dosage.
  • a sub-therapeutically effective amount is an amount of the PI3K inhibitor or anti- CD 19 maytansinoid immunoconjugate that, when administered to a subject by itself, does not completely inhibit over time the biological activity of the intended target.
  • PI3K inhibitor and anti-CD 19 maytansinoid immunoconjugate should be effective in treating the cancer.
  • a sub -therapeutic amount of anti-CD 19 maytansinoid immunoconjugate can be an effective amount if, when combined with the PI3K inhibitor, the combination is effective in the treatment of a cancer.
  • the combination of compounds exhibits a synergistic effect
  • the combination of compounds can decrease the number of lymphoma cells, inhibit tumor growth, achieve tumor stasis, or even achieve substantial or complete tumor regression.
  • the amounts of anti-CD 19 maytansinoid immunoconjugate and PI3K inhibitor should result in the effective treatment of a cancer
  • the amounts, when combined, are preferably not excessively toxic to the subject (i.e., the amounts are preferably within toxicity limits as established by medical guidelines).
  • Examples of types of cancers to be treated include, but are not limited to, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphomas.
  • the subject considered herein is typically a human. However, the subject can be any mammal for which cancer treatment is desired. Thus, the methods described herein can be applied to both human and veterinary applications.
  • treating indicates that the method has, at the least, mitigated abnormal cellular proliferation.
  • the method can reduce the rate of tumor growth in a subject, or prevent the continued growth of a tumor, or even reduce the size of a tumor.
  • terapéuticaally effective amount refers to a sufficient amount of an agent to provide the desired biological, therapeutic, and/or prophylactic result. That result can be reduction, amelioration, palliation, lessening, delaying, and/or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.
  • an effective amount comprises an amount sufficient to cause a tumor to shrink and/or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation.
  • an effective amount is an amount sufficient to delay development.
  • an effective amount is an amount sufficient to prevent or delay recurrence.
  • An effective amount can be administered in one or more administrations. The effective amount of the drug or
  • composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer.
  • an "effective amount” for therapeutic uses is the amount of Compound (1) or a pharmaceutically acceptable salt thereof, required to provide a clinically significant decrease in non-Hodgkin lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), or germinal center B-cell like diffuse large B-cell lymphoma (GCB DLBCL).
  • methods for preventing cancer in an animal are provided.
  • prevention denotes causing the clinical symptoms of the disease not to develop in an animal that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease.
  • the methods comprise administering to the subject a PI3K inhibitor and an anti-CD 19 maytansinoid immunoconjugate, as described herein.
  • a method of preventing cancer in an animal comprises administering to the animal a compound of Formula (1), or a pharmaceutically acceptable salt thereof, in combination with Immunoconjugate (1).
  • the anti-CD 19 maytansinoid immunoconjugate and PI3K inhibiting compound in pure form or in an appropriate pharmaceutical composition, can be administered via any accepted mode of administration or agents known in the art.
  • the compounds can be administered, for example, orally, nasally, parenterally (intravenous, intramuscular, or subcutaneous), topically, transdermally, intravaginally, intravesically, intracistemally, or rectally.
  • the dosage form can be, for example, a solid, semi-solid, lyophilized powder, or liquid dosage forms, such as for example, tablets, pills, soft elastic or hard gelatin capsules, powders, solutions, suspensions, suppositories, aerosols, or the like, preferably in unit dosage forms suitable for simple administration of precise dosages.
  • a particular route of administration is oral, particularly one in which a convenient daily dosage regimen can be adjusted according to the degree of severity of the disease to be treated.
  • compositions comprising the PI3K inhibitor shown in Formula (1) and the anti-CD 19 maytansinoid immunoconjugate referred to herein as
  • the composition is in the form of a solid (e.g., a powder or tablet) including the PI3K inhibitor in solid form, and optionally, one or more auxiliary (e.g., adjuvant) or pharmaceutically active compounds in solid form.
  • the composition further includes any one or combination of pharmaceutically acceptable carriers (i.e., vehicles or excipients) known in the art, thereby providing a liquid dosage form.
  • pharmaceutically acceptable carriers i.e., vehicles or excipients
  • the components of the combination therapy can also be administered in separate dosage forms.
  • the combination therapy provided herein includes Formula (1) in solid form (e.g., a powder or tablet) and Immunoconjugate (1) in liquid form (e.g., an injectable or intravenous form).
  • Sterile compositions for parenteral administration can be prepared by incorporating the anti-CD 19 maytansinoid immunoconjugate in the required amount in the appropriate solvent, followed by sterilization by micro filtration.
  • solvent or vehicle there may be used water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as a combination thereof.
  • isotonic agents such as sugars, polyalcohols, or sodium chloride in the composition.
  • These compositions may also contain adjuvants, in particular wetting, isotonizing, emulsifying, dispersing and stabilizing agents.
  • Sterile compositions for parenteral administration may also be prepared in the form of sterile solid compositions which may be dissolved at the time of use in sterile water or any other injectable sterile medium.
  • Auxiliary and adjuvant agents for the components of the combination therapy may include, for example, preserving, wetting, suspending, sweetening, flavoring, perfuming, emulsifying, and dispensing agents.
  • Prevention of the action of microorganisms is generally provided by various antibacterial and antifungal agents, such as, parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • Isotonic agents such as sugars, sodium chloride, and the like, may also be included.
  • Prolonged absorption of an injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • the auxiliary agents also can include wetting agents, emulsifying agents, pH buffering agents, and antioxidants, such as, for example, citric acid, sorbitan monolaurate, triethanolamine oleate, butylated hydroxytoluene, and the like.
  • Dosage forms suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or
  • fillers or extenders as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid
  • binders as for example, cellulose derivatives, starch, alignates, gelatin, polyvinylpyrrolidone, sucrose, and gum acacia
  • humectants as for example, glycerol
  • disintegrating agents as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, croscarmellose sodium, complex silicates, and sodium carbonate
  • solution retarders as for example paraffin
  • Solid dosage forms as described above can be prepared with coatings and shells, such as enteric coatings and others well-known in the art. They can contain pacifying agents and can be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedded compositions that can be used are polymeric substances and waxes. The active compounds also can be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration, whether in a single dosage form or in separate dosage forms, include pharmaceutically acceptable emulsions, solutions,
  • Such dosage forms are prepared, for example, by dissolving, dispersing, etc., the PI3K inhibitor compound described herein, and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like; solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1 ,3- butyleneglycol, dimethyl formamide; oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol,
  • a carrier such as, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like
  • Suspensions may contain suspending agents in addition to the active compounds, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • suspending agents in addition to the active compounds, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • compositions for rectal administrations are, for example, suppositories that can be prepared by mixing the compounds described herein with, for example, suitable non-irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt while in a suitable body cavity and release the active component therein.
  • suitable non-irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt while in a suitable body cavity and release the active component therein.
  • Dosage forms for topical administration may include, for example, ointments, powders, sprays, and inhalants.
  • the active component is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants as can be required.
  • Ophthalmic formulations, eye ointments, powders, and solutions also can be employed.
  • the pharmaceutically acceptable compositions will contain about 1% to about 99% by weight of the compounds described herein, or a pharmaceutically acceptable salt thereof, and 99% to 1% by weight of a pharmaceutically acceptable excipient.
  • the composition will be between about 5% and about 75%> by weight of compounds described herein, or a pharmaceutically acceptable salt thereof, with the rest being suitable pharmaceutical excipients.
  • Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art. Reference is made, for example, to Remington's Pharmaceutical Sciences, 18th Ed., (Mack Publishing Company, Easton, Pa., 1990).
  • the composition does not include one or more other anti- cancer compounds. In other embodiments, the composition includes one or more other anticancer compounds.
  • administered compositions can comprise standard of care agents for the type of tumors selected for treatment.
  • kits are provided.
  • Kits according to the invention include package(s) comprising compounds or compositions of the invention.
  • kits comprise Compound (1), or a pharmaceutically acceptable salt thereof, and
  • packaging means any vessel containing compounds or compositions presented herein.
  • the package can be a box or wrapping.
  • Packaging materials for use in packaging pharmaceutical products are well-known to those of skill in the art. Examples of pharmaceutical packaging materials include, but are not limited to, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.
  • the kit also can contain items that are not contained within the package but are attached to the outside of the package, for example, pipettes.
  • Kits can contain instructions for administering compounds or compositions of the invention to a subject. Kits also can comprise instructions for approved uses of compounds herein by regulatory agencies, such as the United States Food and Drug Administration. Kits also can contain labeling or product inserts for the inventive compounds. The package(s) and/or any product insert(s) may themselves be approved by regulatory agencies.
  • the kits can include compounds in the solid phase or in a liquid phase (such as buffers provided) in a package.
  • the kits also can include buffers for preparing solutions for conducting the methods, and pipettes for transferring liquids from one container to another.
  • Compound (1) can be synthesized as described in WO 07/044813, which is hereby incorporated in its entirety.
  • compound (a) a base and an intermediate, compound (a), are added to solution of commercially available 2-metfiyl-2-thiopseudourea sulfate in a solvent such as water and stirred overnight at room temperature. After neutralization, compound (b) is collected by filtration and dried under vacuum. Treatment of compound (b) with POCI 3 and heating at reflux for 2 hours yields compound (c) which can be concentrated under vacuum to dryness.
  • Compound (c) can be used directly in the following reaction with ethylamine carried out in a solvent such as water with heating to give compound (d). Compound (d) is then treated with iodine monochloride in a solvent such as methanol to form compound (e).
  • Compound (f) is then be treated with DBU in the presence of a base, such as DIEA, and heated at reflux for 15 hours. Upon completion of the reaction, the solvent is evaporated and the residue triturated with acetone to yield compound (g). Bromination of compound (g) can be achieved through drop-wise addition of Br 2 to compound (g) in CH 2 C1 2 , followed by stirring overnight at room temperature. Next, filtration is carried out, and triethylamine is added so that, upon washing and drying, the product, compound (h) is obtained.
  • a base such as DIEA
  • compound (i) can be converted to compound (1) of the instant invention through 1) oxidation of the methylthio group with m-CPBA, carried out at room temperature with stirring and 2) treatment of the resulting product dissolved in dioxane, with liquid ammonia.
  • a Pd- catalyst such as [1,1 -bis(diphenylphosphino)ferrocene]dichloropalladium(II) in the presence of a base to yield compound (i).
  • compound (i) can be converted to compound (1) of the instant invention through 1) oxidation of the methylthio group with m-CPBA, carried out at room temperature with stirring and 2) treatment of the resulting product dissolved in dioxane, with liquid ammonia.
  • Immunoconjugate (1) can be prepared as described in, for example, U.S. Patent No. 7,811 ,572, which is hereby incorporated by reference. Briefly, huB4 humanized monoclonal antibody is modified with either (a) a 4.9-fold molar excess of SPDB relative to antibody, or (b) a 4.8-fold molar excess of SPDB relative to antibody. In either situation, reaction proceeds in 50 mM potassium phosphate, 50 mM potassium chloride, and 2 mM EDTA (pH 6.5) in 5% ethanol for a total of 120 minutes at room temperature. The samples are purified over a column of Sephadex.TM.
  • DMA dimethylacetamide
  • Immunoconjugate (1) was evaluated in the cell line OCI-LY7, a specific GCB-DLBCL (germinal center B-cell like diffuse large B-cell lymphoma) cell line. See, e.g., Alvero et al., "Correlation of Capase Activity and Chemo- Response in Epithelial Ovarian Cancer Cell Lines", Promega Notes, pp. 15-17 (2004).
  • the OCI-LY7 cell line was obtained from Cambridge Cell Bank (Cryostock ID 5865, original from DSMZ cell collection ACC688) and grown in IMDM medium (GIBCO cat#31980) supplemented with 20% FCS (GIBCO cat#16140). Cells cultured in flasks were maintained in the atmosphere of 37°C and 5% C02 in a NAPCO Series 8000 DH C02 incubator (Thermo Scientific).
  • Cell treatment was performed in 96-well microtitre assay plates (Costar cat#3904). On the experimental day, cells in exponential growth were harvested and re-suspended in fresh medium, then plated into 96-well plate at an optimal seeding density of 12000 cells/well in 40ul. Medium without cells was used as background control.
  • Cell plate with combination treatments was incubated in the atmosphere of 37°C and 5% C02 in a NAPCO Series 8000 DH C02 incubator (Thermo Scientific) for 24hrs. After treatment, an equal volume (60ul) of Caspase-Glo 3/7 assay solution (Promega cat#8093) was added to each well of cell plate. The cell plate was allowed to incubate at room temperature in the dark (shaking) for 30min before measurement in a luminescent reader (PekinElmer's EnVison 2104 Multilabel Reader).
  • Unprocessed data from the reader were stored in MS Excel file.
  • the first step of data processing was calculating an average background value from the medium control wells (no cells) for each plate. The average background value was then subtracted from each well.
  • the resultant average value from duplicate wells containing cells that had been incubated without compound was considered as 100% of Caspase 3/7 activity.
  • the resultant average value from duplicate wells containing cells with single or combined compounds were compared with data from untreated wells to assess the synergistic effects on cell apoptosis by the compounds.
  • Example 4 In vitro activity of Compound (1) in combination with Immunoconjugate (1) in cell proliferation assay The combination of Immunoconjugate (1) and Compound (1) was evaluated in specific GCB-DLBCL (germinal center B-cell like diffuse large B-cell lymphoma) cell lines OCI-LY7, OCI-LY19, and SU-DHL4.
  • GCB-DLBCL germ center B-cell like diffuse large B-cell lymphoma
  • Compound Dilution Compound stock solution was diluted to 10X of the final treatment concentration (Immunoconjugate (1) starts from 5 ug/ml to 0 ug/ml, Compound (1) from 10 uM to 0 uM) in culture medium with corresponding percentage of Fetal Bovine Serum. Stock compound solution was serially diluted by a factor of 2.

Abstract

Compositions and methods comprising inhibitors of the phosphoinositide 3-kinase (PI3K) pathway and anti-CD19 maytansinoid immunoconjugates are provided.

Description

COMPOSITIONS AND METHODS FOR TREATING CANCER USING PI3K INHIBITOR AND ANTI-CD 19 MAYTANSINOID IMMUNOCONJUGATE
TECHNICAL FIELD
Provided herein are combinations of an anti-CD 19 maytansinoid immunoconjugate and a phosphoinositide 3-kinase (PI3K) inhibitor which are therapeutically useful in the treatment of cancer.
BACKGROUND
There is an ongoing need in the art for more efficacious methods and compositions in the treatment of cancer. In particular, there is a need for a cancer therapy that is more effective in inhibiting cell proliferation and tumor growth while minimizing subject toxicity.
SUMMARY
Provided herein are compositions and uses thereof in the treatment of a variety of cancers.
In particular aspects, there is provided herein a composition comprising Compound
(1)
Figure imgf000002_0001
or a pharmaceutically acceptable salt thereof, and a anti-CD 19 maytansinoid
immunoconjugate comprising a light chain and a heavy chain, said light chain having the sequence represented in SEQ ID NO. 7, and said heavy chain having the sequence represented in SEQ ID NO. 8. The immunoconjugate further comprises a cytotoxic agent that is a maytansinoid such as DM4 that is covalently attached, directly or via a cleavable or non-cleavable linker, to the antibody wherein about 3.5 molecules of maytansinoid are bound through the SPDB linker to each antibody molecule.
In another aspect, methods of treating a subject with cancer are provided that comprise administering to the subject a therapeutically effective amount of Compound (1), or a pharmaceutically acceptable salt thereof, in combination with Immunoconjugate (1).
In one embodiment, the method of treating a subject with cancer comprises administering to the subject a first dosage of a PI3K inhibitor, and a second dosage of an anti- CD 19 maytansinoid immunoconjugate wherein said PI3K inhibitor is Compound (1).
In another embodiment, the method of treating a subject with cancer comprises administering to the subject a first dosage of an anti-CD 19 maytansinoid immunoconjugate, and a second dosage of a PI3K inhibitor wherein said PI3K inhibitor is Compound (1).
In some embodiments, the methods involve treating cancer selected from the group consisting of lymphomas and leukemias including, but not limited to, germinal center B-cell like diffuse large B-cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia and mantle cell lymphoma.
In some embodiments, the compositions and methods of use described herein are in amounts (i.e., either in the composition are in an administered dosage) that provide a synergistic effect in the treatment of the subject.
In another aspect, a combination for use in treating a patient with cancer is provided, the combination comprising a therapeutically effective amount of (A) Compound (1), or a pharmaceutically acceptable salt thereof, and (B) Immunoconjugate (1).
In one embodiment, uses of a combination comprising a therapeutically effective amount of (A) Compound (1), or a pharmaceutically acceptable salt thereof, and (B) Immunoconjugate (1) are provided for the preparation of a medicament for use in treatment of lymphoma.
In another aspect, kits are provided comprising: (A) Compound (1), or a
pharmaceutically acceptable salt thereof; (B) Immunoconjugate (1); and (C) instructions for use.
Other objects, features and advantages will become apparent from the following detailed description. The detailed description and specific examples are given for illustration only since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Further, the examples demonstrate the principle of the invention and cannot be expected to specifically illustrate the application of this invention to all the examples where it will be obviously useful to those skilled in the prior art.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic showing the structure of an anti-CD 19 maytansinoid immunoconjugate. Figure 2 demonstrates the enhanced apoptosis observed with Compound (1) (i.e. CPD(l)) and an anti-CD19 maytansinoid immunoconjugate (i.e. IC(1)) in the OCI-LY7 cell line.
Figure 3 demonstrates the enhanced anti-proliferative activity observed with
Compound (1) (i.e. CPD(l)) and an anti-CD19 maytansinoid immunoconjugate (i.e. IC(1)) in three separate GCB-DLBCL cell lines.
DETAILED DESCRIPTION
Compositions and methods for the treatment of cancer are provided. The
compositions and methods comprise inhibitors of the phosphoinositide 3-kinase (PI3K) pathway (Compound (1)) and an anti-CD 19 maytansinoid immunoconjugate
(Immunoconjugate (1)). Compound (1) and Immunoconjugate (1) can be administered in a single dosage form, or separate dosage forms.
Compound (1), 2-amino-8-ethyl-4-methyl-6-(lH-pyrazol-5-yl)pyrido[2,3- d]pyrimidin-7(8H)-one, is a selective inhibitor of class I PI3K lipid kinases. Compound (1) targets both PI3K isoforms (IC50 values in nM: PI3Ka 39, ΡΙ3Κβ 113, ΡΙ3Κδ 43, ΡΙ3Κγ 9) and mTOR (157 nM).
Oral administration of Compound (1) alone inhibits tumor growth in mice bearing xenografts in which PI3K signaling is activated, such as the PTEN-deficient PC-3 prostate adenocarcinoma, U87-MG gliobastoma, A2058 melanoma and WM -266-4 melanoma, or the PIK3CA mutated MCF7 mammary carcinoma. Compound (1) is the subject of several clinical trials involving patients with, for example, solid tumors, lymphoma, glioblastoma or hormone receptor-positive breast cancer.
CD 19 is the earliest differentiation antigen of the B lymphocyte lineage, expressed on most B-cells, but not detected on plasma cells, stem cells, or on normal myeloid lineage. Therefore, CD 19 is expressed on tumor cells from all B cell-derived neoplasms (B-cell non- Hodgkin's lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia), except myeloma.
Immunoconjugate (1) is an antibody-drug conjugate composed of a humanized IgGl monoclonal antibody, huB4 (Roguska et al, Proc. Natl. Acad. Sci. USA, 91: 969-973, 1994), which specifically targets the CD 19 antigen, conjugated through a disulfide link to the maytansinoid derivative DM4, a potent tubulin inhibitor (US 2004/0235840). The structure of Immunoconjugate (1) is disclosed in Figure 1. After binding to the CD 19 antigen,
Immunoconjugate (1) undergoes internalization and intracellular release of DM4. The entire contents of Roguska et al, Proc. Natl. Acad. Sci. USA, 91: 969-973, 1994 are incorporated herein by reference.
In one aspect, methods for treating subjects with cancer are provided. In one embodiment, the methods comprise administering to the subject a therapeutically effective amount of an anti-CD 19 maytansinoid immunoconjugate referred to herein as
"Immuno conjugate (1)" and a therapeutically effective amount of a PI3K inhibitor having the structural Formula (1), as further described below.
Immunoconjugate (1) and Compound (1) can be in single or separate dosage forms.
In one embodiment, the inventive methods and compositions comprise a PI3K inhibitor having the following s
Figure imgf000005_0001
and pharmaceutically acceptable salts thereof.
The compound according to formula (1), 2-amino-8-ethyl-4-methyl-6-(lH-pyrazol-5- yl)pyrido[2,3-d]pyrimidin-7(8H)-one, is referred to herein as "Compound (1)". The preparation and properties of Compound (1) are provided in, for example, International
Patent Publication No. WO 07/044813, particularly Example 56 therein. The entire contents of WO 07/044813 are incorporated herein by reference. Neutral and salt forms of the compound of Formula (1) are all considered herein.
The anti-CD 19 maytansinoid immunoconjugate (Immunoconjugate (1)) comprises two primary components, a cell-binding agent and a cytotoxic agent.
As used herein, the term "cell binding agent" refers to an agent that specifically recognizes and binds the CD 19 antigen on the cell surface.
In an embodiment, the cell-binding agent is an antibody which binds specifically to the CD 19 antigen.
In an embodiment, the said antibody comprises six complementary determining region (CDR), said CDR having the sequences represented in SEQ ID NOs 1 to 6. In another embodiment, the antibody comprises a light chain, wherein the sequence of the said light chain has at least 60%, at least 75%, at least 85%, at least 90 %, at least 95 % or at least 99% identity with the sequence displayed in SEQ ID NO. 7.
In yet another particular embodiment, the antibody comprises a heavy chain, wherein the sequence of the said heavy chain has at least 60%, at least 75%, at least 85%, at least 90 %, at least 95 % or at least 99% identity with the sequence displayed in SEQ ID NO. 8.
In an embodiment, the antibody of the invention is the humanized antibody huB4 described in Roguska et al. (Proc. Natl. Acad. Sci. USA, 91 : 969-973, 1994). The antibody huB4 according to the invention comprises a light chain and a heavy chain, said light chain having the sequence represented in SEQ ID NO. 7, and said heavy chain having the sequence represented in SEQ ID NO. 8.
The second component of the anti-CD 19 maytansinoid immunoconjugate
(Immunoconjugate (1)) is a cytotoxic agent. The term "cytotoxic agent" as used herein refers to a substance that reduces or blocks the function, or growth, of cells and/or causes destruction of cells. In an embodiment, the cytotoxic agent is a maytansinoid such as DM4. In another embodiment, the cell binding agent of the present invention is covalently attached, directly or via a cleavable or non-cleavable linker, to the cytotoxic agent. In another embodiment, the said cell binding agent is conjugated to DM4 through a cleavable linker. In an embodiment this cleavable linker is a SPDB linker. DM4 and a method of conjugating DM4 to the huB4 antibody through a SPDB linker are described in US Patent Publication No. 2004/0235840, which is hereby incorporated by reference.
In an embodiment of this method the anti-CD 19 maytansinoid immunoconjugate comprises an antibody which binds specifically to the CD 19 antigen conjugated to DM4 through SPDB wherein about 3.5 molecules of DM4 are bound through the SPDB linker to each antibody molecule.
In one embodiment, the anti-CD 19 maytansinoid immunoconjugate is
"Immunoconjugate (1)" as depicted in Figure 1. It comprises a light chain and a heavy chain, said light chain having the sequence represented in SEQ ID NO. 7, and said heavy chain having the sequence represented in SEQ ID NO. 8. It further comprises a cytotoxic agent is a maytansinoid such as DM1 or DM4 that is covalently attached, directly or via a cleavable or non-cleavable linker, to the antibody wherein about 3.5 molecules of maytansinoid are bound through the SPDB linker to each antibody molecule. In a one aspect, Immunoconjugate (1) comprises a light chain and a heavy chain, said light chain having the sequence represented in SEQ ID NO. 7, and said heavy chain having the sequence represented in SEQ ID NO. 8 wherein the antibody is conjugated to DM4 through SPDB wherein about 3.5 molecules of DM4 are bound through the SPDB linker to each antibody molecule. The preparation and properties of "Immunoconjugate (1)" are provided in, for example, United States Patent Application Publication No. US2004/0235840A1, particularly example 12 therein. The entire contents of US2004/0235840A1 are incorporated herein by reference.
In some embodiments, the compounds described above are unsolvated. In other embodiments, one or both of the compounds used in the method are in solvated form. As known in the art, the solvate can be any of pharmaceutically acceptable solvent, such as water, ethanol, and the like. In general, the presence of a solvate or lack thereof does not have a substantial effect on the efficacy of the PI3K inhibitor or anti-CD 19 maytansinoid immunoconjugate described above.
Although the compound in Formula (1) is depicted in its neutral form, in some embodiments, this compound is used in a pharmaceutically acceptable salt form. The salt can be obtained by any of the methods well known in the art. A "pharmaceutically acceptable salt" of the compound refers to a salt that is pharmaceutically acceptable and that retains pharmacological activity. It is understood that the pharmaceutically acceptable salts are nontoxic. Additional information on suitable pharmaceutically acceptable salts can be found in Remington 's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, 1985, or S. M. Berge, et al, "Pharmaceutical Salts," J. Pharm. Sci., 1977;66: 1-19, both of which are incorporated herein by reference.
Examples of pharmaceutically acceptable acid addition salts include those formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, as well as those salts formed with organic acids, such as acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, 3-(4-hydroxybenzoyl)benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1 ,2-ethanedisulfonic acid, 2- hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2- naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, 4,4'-methylenebis-(3-hydroxy-2-ene-l-carboxylic acid), 3-phenylpropionic acid,
trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, p-toluenesulfonic acid, and salicylic acid. In a first set of embodiments, the PI3K inhibitor of formula (1) is administered simultaneously with Immunoconjugate (1). Simultaneous administration typically means that both compounds enter the subject at precisely the same time. However, simultaneous administration also includes the possibility that the PI3K inhibitor and the anti-CD 19 maytansinoid immunoconjugate enter the subject at different times, but the difference in time is sufficiently miniscule that the first administered compound is not provided the time to take effect on the subject before entry of the second administered compound. Such delayed times typically correspond to less than 1 minute, and more typically, less than 30 seconds.
In one example, wherein the compounds are in solution, simultaneous administration can be achieved by administering a solution containing the combination of compounds. In another example, simultaneous administration of separate solutions, one of which contains the PI3K inhibitor and the other of which contains the anti-CD 19 maytansinoid
immunoconjugate, can be employed. In one example wherein the compounds are in solvated form, simultaneous administration can be achieved by administering a composition containing the combination of compounds.
In another embodiment of the invention, the anti-CD 19 maytansinoid
immunoconjugate and the PI3K inhibitor are administered sequentially. According to an embodiment of the invention, the anti-CD 19 maytansinoid immunoconjugate is administered first, followed immediately thereafter by administration of the PI3K inhibitor. In another embodiment, the anti-CD 19 maytansinoid immunoconjugate is administered, a time interval occurs and then the PI3K inhibitor is administered. The time interval can be one or more hour(s), one or more day(s) or one or more week(s). In an embodiment of the invention, the PI3K inhibitor is administered first, followed immediately thereafter by administration of anti-CD 19 maytansinoid immunoconjugate. In another embodiment, the PI3K inhibitor is administered, a time interval occurs and then anti-CD 19 maytansinoid immunoconjugate is administered.
In one set of embodiments, one or both of the PI3K inhibitor and anti-CD 19 maytansinoid immunoconjugate are administered in a therapeutically effective (i.e., therapeutic) amount or dosage. A "therapeutically effective amount" is an amount of the PI3K inhibitor or anti-CD 19 maytansinoid immunoconjugate that, when administered to a subject by itself, effectively treats the cancer (for example, decreases the number of lymphoma cells, inhibits tumor growth, stops tumor growth, or causes tumor regression). An amount that proves "therapeutically effective amount" in a given instance, for a particular subject, may not be effective for 100% of subjects similarly treated for the disease or condition under consideration, even though such dosage is deemed a "therapeutically effective amount" by skilled practitioners. The amount of the compound that corresponds to a therapeutically effective amount is strongly dependent on the type of cancer, stage of the cancer, the age of the subject being treated, and other facts.
In other embodiments, one or both of the PI3K inhibitor and the anti-CD 19 maytansinoid immunoconjugate are administered in a sub-therapeutically effective amount or dosage. A sub-therapeutically effective amount is an amount of the PI3K inhibitor or anti- CD 19 maytansinoid immunoconjugate that, when administered to a subject by itself, does not completely inhibit over time the biological activity of the intended target.
Whether administered in therapeutic or sub-therapeutic amounts, the combination of
PI3K inhibitor and anti-CD 19 maytansinoid immunoconjugate should be effective in treating the cancer. A sub -therapeutic amount of anti-CD 19 maytansinoid immunoconjugate can be an effective amount if, when combined with the PI3K inhibitor, the combination is effective in the treatment of a cancer.
In some embodiments, the combination of compounds exhibits a synergistic effect
(i.e., greater than additive effect) in treating the cancer. In different embodiments, depending on the combination and the effective amounts used, the combination of compounds can decrease the number of lymphoma cells, inhibit tumor growth, achieve tumor stasis, or even achieve substantial or complete tumor regression.
While the amounts of anti-CD 19 maytansinoid immunoconjugate and PI3K inhibitor should result in the effective treatment of a cancer, the amounts, when combined, are preferably not excessively toxic to the subject (i.e., the amounts are preferably within toxicity limits as established by medical guidelines).
Examples of types of cancers to be treated include, but are not limited to, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphomas.
The subject considered herein is typically a human. However, the subject can be any mammal for which cancer treatment is desired. Thus, the methods described herein can be applied to both human and veterinary applications.
The term "treating" or "treatment", as used herein, indicates that the method has, at the least, mitigated abnormal cellular proliferation. For example, the method can reduce the rate of tumor growth in a subject, or prevent the continued growth of a tumor, or even reduce the size of a tumor.
The terms "effective amount" or "pharmaceutically effective amount" or
"therapeutically effective amount" refer to a sufficient amount of an agent to provide the desired biological, therapeutic, and/or prophylactic result. That result can be reduction, amelioration, palliation, lessening, delaying, and/or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. In reference to cancer, an effective amount comprises an amount sufficient to cause a tumor to shrink and/or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay development. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more administrations. The effective amount of the drug or
composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer. For example, an "effective amount" for therapeutic uses is the amount of Compound (1) or a pharmaceutically acceptable salt thereof, required to provide a clinically significant decrease in non-Hodgkin lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), or germinal center B-cell like diffuse large B-cell lymphoma (GCB DLBCL).
In another aspect, methods for preventing cancer in an animal are provided. In this regard, prevention denotes causing the clinical symptoms of the disease not to develop in an animal that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease. The methods comprise administering to the subject a PI3K inhibitor and an anti-CD 19 maytansinoid immunoconjugate, as described herein. In one example, a method of preventing cancer in an animal comprises administering to the animal a compound of Formula (1), or a pharmaceutically acceptable salt thereof, in combination with Immunoconjugate (1).
The anti-CD 19 maytansinoid immunoconjugate and PI3K inhibiting compound, in pure form or in an appropriate pharmaceutical composition, can be administered via any accepted mode of administration or agents known in the art. The compounds can be administered, for example, orally, nasally, parenterally (intravenous, intramuscular, or subcutaneous), topically, transdermally, intravaginally, intravesically, intracistemally, or rectally. The dosage form can be, for example, a solid, semi-solid, lyophilized powder, or liquid dosage forms, such as for example, tablets, pills, soft elastic or hard gelatin capsules, powders, solutions, suspensions, suppositories, aerosols, or the like, preferably in unit dosage forms suitable for simple administration of precise dosages. A particular route of administration is oral, particularly one in which a convenient daily dosage regimen can be adjusted according to the degree of severity of the disease to be treated.
In another aspect, compositions are provided comprising the PI3K inhibitor shown in Formula (1) and the anti-CD 19 maytansinoid immunoconjugate referred to herein as
Immunoconjugate (1). In some embodiments, the composition is in the form of a solid (e.g., a powder or tablet) including the PI3K inhibitor in solid form, and optionally, one or more auxiliary (e.g., adjuvant) or pharmaceutically active compounds in solid form. In other embodiments, the composition further includes any one or combination of pharmaceutically acceptable carriers (i.e., vehicles or excipients) known in the art, thereby providing a liquid dosage form. The components of the combination therapy can also be administered in separate dosage forms. For example, in an embodiment, the combination therapy provided herein includes Formula (1) in solid form (e.g., a powder or tablet) and Immunoconjugate (1) in liquid form (e.g., an injectable or intravenous form).
Sterile compositions for parenteral administration can be prepared by incorporating the anti-CD 19 maytansinoid immunoconjugate in the required amount in the appropriate solvent, followed by sterilization by micro filtration. As solvent or vehicle, there may be used water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as a combination thereof. In many cases, it will be advantageous to include isotonic agents, such as sugars, polyalcohols, or sodium chloride in the composition. These compositions may also contain adjuvants, in particular wetting, isotonizing, emulsifying, dispersing and stabilizing agents. Sterile compositions for parenteral administration may also be prepared in the form of sterile solid compositions which may be dissolved at the time of use in sterile water or any other injectable sterile medium.
Auxiliary and adjuvant agents for the components of the combination therapy, whether in a single dosage form or in separate dosage forms, may include, for example, preserving, wetting, suspending, sweetening, flavoring, perfuming, emulsifying, and dispensing agents. Prevention of the action of microorganisms is generally provided by various antibacterial and antifungal agents, such as, parabens, chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents, such as sugars, sodium chloride, and the like, may also be included. Prolonged absorption of an injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. The auxiliary agents also can include wetting agents, emulsifying agents, pH buffering agents, and antioxidants, such as, for example, citric acid, sorbitan monolaurate, triethanolamine oleate, butylated hydroxytoluene, and the like.
Dosage forms suitable for parenteral injection, whether in a single dosage form or in separate dosage forms, may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
Solid dosage forms for oral administration, whether in a single dosage form or in separate dosage forms, include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, as for example, cellulose derivatives, starch, alignates, gelatin, polyvinylpyrrolidone, sucrose, and gum acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, croscarmellose sodium, complex silicates, and sodium carbonate, (e) solution retarders, as for example paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol, and glycerol monostearate, magnesium stearate and the like (h) adsorbents, as for example, kaolin and bentonite, and (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms also may comprise buffering agents.
Solid dosage forms as described above can be prepared with coatings and shells, such as enteric coatings and others well-known in the art. They can contain pacifying agents and can be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedded compositions that can be used are polymeric substances and waxes. The active compounds also can be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients. Liquid dosage forms for oral administration, whether in a single dosage form or in separate dosage forms, include pharmaceutically acceptable emulsions, solutions,
suspensions, syrups, and elixirs. Such dosage forms are prepared, for example, by dissolving, dispersing, etc., the PI3K inhibitor compound described herein, and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like; solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1 ,3- butyleneglycol, dimethyl formamide; oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol,
polyethyleneglycols and fatty acid esters of sorbitan; or mixtures of these substances, and the like, to thereby form a solution or suspension.
Suspensions, whether in a single dosage form or in separate dosage forms, may contain suspending agents in addition to the active compounds, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
Compositions for rectal administrations, whether in a single dosage form or in separate dosage forms are, for example, suppositories that can be prepared by mixing the compounds described herein with, for example, suitable non-irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt while in a suitable body cavity and release the active component therein.
Dosage forms for topical administration , whether in a single dosage form or in separate dosage forms, may include, for example, ointments, powders, sprays, and inhalants. The active component is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants as can be required. Ophthalmic formulations, eye ointments, powders, and solutions also can be employed.
Generally, depending on the intended mode of administration, the pharmaceutically acceptable compositions will contain about 1% to about 99% by weight of the compounds described herein, or a pharmaceutically acceptable salt thereof, and 99% to 1% by weight of a pharmaceutically acceptable excipient. In one example, the composition will be between about 5% and about 75%> by weight of compounds described herein, or a pharmaceutically acceptable salt thereof, with the rest being suitable pharmaceutical excipients. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art. Reference is made, for example, to Remington's Pharmaceutical Sciences, 18th Ed., (Mack Publishing Company, Easton, Pa., 1990).
In some embodiments, the composition does not include one or more other anti- cancer compounds. In other embodiments, the composition includes one or more other anticancer compounds. For example, administered compositions can comprise standard of care agents for the type of tumors selected for treatment.
In another aspect, kits are provided. Kits according to the invention include package(s) comprising compounds or compositions of the invention. In one embodiment, kits comprise Compound (1), or a pharmaceutically acceptable salt thereof, and
Immunoconjugate (1).
The phrase "package" means any vessel containing compounds or compositions presented herein. In some embodiments, the package can be a box or wrapping. Packaging materials for use in packaging pharmaceutical products are well-known to those of skill in the art. Examples of pharmaceutical packaging materials include, but are not limited to, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.
The kit also can contain items that are not contained within the package but are attached to the outside of the package, for example, pipettes.
Kits can contain instructions for administering compounds or compositions of the invention to a subject. Kits also can comprise instructions for approved uses of compounds herein by regulatory agencies, such as the United States Food and Drug Administration. Kits also can contain labeling or product inserts for the inventive compounds. The package(s) and/or any product insert(s) may themselves be approved by regulatory agencies. The kits can include compounds in the solid phase or in a liquid phase (such as buffers provided) in a package. The kits also can include buffers for preparing solutions for conducting the methods, and pipettes for transferring liquids from one container to another.
Examples have been set forth below for the purpose of illustration and to describe certain specific embodiments of the invention. However, the scope of the claims is not to be in any way limited by the examples set forth herein.
Example 1. Synthesis of Compound (1)
Compound (1) can be synthesized as described in WO 07/044813, which is hereby incorporated in its entirety.
Figure imgf000015_0001
Briefly, a base and an intermediate, compound (a), are added to solution of commercially available 2-metfiyl-2-thiopseudourea sulfate in a solvent such as water and stirred overnight at room temperature. After neutralization, compound (b) is collected by filtration and dried under vacuum. Treatment of compound (b) with POCI3 and heating at reflux for 2 hours yields compound (c) which can be concentrated under vacuum to dryness. Compound (c) can be used directly in the following reaction with ethylamine carried out in a solvent such as water with heating to give compound (d). Compound (d) is then treated with iodine monochloride in a solvent such as methanol to form compound (e). Compound (e) is then dissolved in DMA, to which ethyl acrylate, Pd(OAc)2 and a base are added. This reaction mixture is heated and reacted overnight until completion of the reaction to give compound (f), which can be purified via column chromatography.
Compound (f) is then be treated with DBU in the presence of a base, such as DIEA, and heated at reflux for 15 hours. Upon completion of the reaction, the solvent is evaporated and the residue triturated with acetone to yield compound (g). Bromination of compound (g) can be achieved through drop-wise addition of Br2 to compound (g) in CH2C12, followed by stirring overnight at room temperature. Next, filtration is carried out, and triethylamine is added so that, upon washing and drying, the product, compound (h) is obtained. A Suzuki coupling between compound (h) and lH-pyrazol-5-yl boronic acid is carried out using a Pd- catalyst such as [1,1 -bis(diphenylphosphino)ferrocene]dichloropalladium(II) in the presence of a base to yield compound (i). Finally, compound (i) can be converted to compound (1) of the instant invention through 1) oxidation of the methylthio group with m-CPBA, carried out at room temperature with stirring and 2) treatment of the resulting product dissolved in dioxane, with liquid ammonia. Stirring at room temperature overnight followed by purification by column chromatography gives the desired product, 2-amino-8-ethyl-4-methyl- 6-(lH-pyrazol-5-yl)pyrido[2,3-d]pyrimidin-7(8H)-one, compound (1).
Example 2. Preparation of Immunoconjugate (1)
Immunoconjugate (1) can be prepared as described in, for example, U.S. Patent No. 7,811 ,572, which is hereby incorporated by reference. Briefly, huB4 humanized monoclonal antibody is modified with either (a) a 4.9-fold molar excess of SPDB relative to antibody, or (b) a 4.8-fold molar excess of SPDB relative to antibody. In either situation, reaction proceeds in 50 mM potassium phosphate, 50 mM potassium chloride, and 2 mM EDTA (pH 6.5) in 5% ethanol for a total of 120 minutes at room temperature. The samples are purified over a column of Sephadex.TM. G25F resin equilibrated in 50 mM potassium phosphate, 50 mM sodium chloride, and 2 mM EDTA at pH 7.5. Samples are conjugated with DM4 (1.7 fold molar excess over bound linker) for 18 hours at room temperature in a final
concentration of dimethylacetamide (DMA) of 3%.
Example 3. In vitro activity of Compound (1) in combination with Immunoconjugate (1) in Apoptosis Assay
The combination of Immunoconjugate (1) and Compound (1) was evaluated in the cell line OCI-LY7, a specific GCB-DLBCL (germinal center B-cell like diffuse large B-cell lymphoma) cell line. See, e.g., Alvero et al., "Correlation of Capase Activity and Chemo- Response in Epithelial Ovarian Cancer Cell Lines", Promega Notes, pp. 15-17 (2004).
Materials and methods
The OCI-LY7 cell line was obtained from Cambridge Cell Bank (Cryostock ID 5865, original from DSMZ cell collection ACC688) and grown in IMDM medium (GIBCO cat#31980) supplemented with 20% FCS (GIBCO cat#16140). Cells cultured in flasks were maintained in the atmosphere of 37°C and 5% C02 in a NAPCO Series 8000 DH C02 incubator (Thermo Scientific).
Cell treatment was performed in 96-well microtitre assay plates (Costar cat#3904). On the experimental day, cells in exponential growth were harvested and re-suspended in fresh medium, then plated into 96-well plate at an optimal seeding density of 12000 cells/well in 40ul. Medium without cells was used as background control.
Stock solution of Immunoconjugate (1) (5mg/ml or 33uM) was stored at 4°C. On the experiment day, a 6x working solution of Immunoconjugate (1) (99nM) was prepared with cell culture medium. This solution was then further serially diluted 1 :2 in medium
(concentration = 0, 3.1, 6.2, 12.3, 25, 50, 99nM) as 6x working solutions. For combination treatments, lOul of each 6x Immunoconjugate (l)working solution or medium was added to cell plate in duplicate wells (resulting in final Immunoconjugate (1) concentrations of 0, 0.52, 1.0, 2.1, 4.1, 8.3, 16.5nM).
Serial dilutions of Compound (1) were prepared with DMSO in PCR tubes
(concentration = 0, 0.94, 1.9, 3.8, 7.5, 15, 30uM) and stored at -20°C. On the experimental day, serial diluted compound stocks were thawed and further diluted 1 :300 with cell culture medium as 6x working solutions. For combination treatments, lOul of each 6x Compound (1) working solution or medium was added to cell plate in duplicate wells (resulting in a final DMSO concentration of 0.06%, Compound (1) at 0, 0.16, 0.31, 0.63, 1.25, 2.5, 5uM).
Cell plate with combination treatments was incubated in the atmosphere of 37°C and 5% C02 in a NAPCO Series 8000 DH C02 incubator (Thermo Scientific) for 24hrs. After treatment, an equal volume (60ul) of Caspase-Glo 3/7 assay solution (Promega cat#8093) was added to each well of cell plate. The cell plate was allowed to incubate at room temperature in the dark (shaking) for 30min before measurement in a luminescent reader (PekinElmer's EnVison 2104 Multilabel Reader).
Unprocessed data from the reader were stored in MS Excel file. The first step of data processing was calculating an average background value from the medium control wells (no cells) for each plate. The average background value was then subtracted from each well. The resultant average value from duplicate wells containing cells that had been incubated without compound was considered as 100% of Caspase 3/7 activity. The resultant average value from duplicate wells containing cells with single or combined compounds were compared with data from untreated wells to assess the synergistic effects on cell apoptosis by the compounds.
Summary of in vitro results
The results of this assay, shown in Figure 2, demonstrate enhanced apoptosis in the OCI-LY7 cell line when treated with a combination of Compound (1) and Immunoconjugate (1) as compared to treatments using either substance individually.
Example 4. In vitro activity of Compound (1) in combination with Immunoconjugate (1) in cell proliferation assay The combination of Immunoconjugate (1) and Compound (1) was evaluated in specific GCB-DLBCL (germinal center B-cell like diffuse large B-cell lymphoma) cell lines OCI-LY7, OCI-LY19, and SU-DHL4.
Materials and methods
Cell Line and Culture Condition: (a) OCI-LY7, IMDM (GIBCO, 31980-030) with 20% Fetal Bovine Serum (GIBCO,10082-147) (b) OCI-LY19, MEM alpha (GIBCO, 12571-063) with 20% Fetal Bovine Serum (c) SU-DHL4, RPMI1640 (GIBCO, 72400-047) with 20% Fetal Bovine Serum. Experiment Plate: 384 well black with clear bottom assay plate, CORNING, 3712.
CellTiter-Glo Luminescent: Promega , G7573.
Compound Dilution: Compound stock solution was diluted to 10X of the final treatment concentration (Immunoconjugate (1) starts from 5 ug/ml to 0 ug/ml, Compound (1) from 10 uM to 0 uM) in culture medium with corresponding percentage of Fetal Bovine Serum. Stock compound solution was serially diluted by a factor of 2.
Proliferation Assay:
1. Log growth phase cells were suspended in corresponding fresh culture medium in
advance of one day before treatment.
2. Seed appropriate number of cells in each well on experiment plate with 20 micro litter volume, OCI-LY7 4500 cells/well; OCI-LY19 4500 cells/well; SU-DHL4 3000 cells/well. Prepare three plates for each cell line to get triplicate value for each treatment.
3. Place the experiment plate in C02 incubator at 37°C overnight.
4. Treat cells with 2.5 microliter Immunoconjugate (1) plus 2.5 micro litter Compound (1) for each well.
5. Place the experiment plate in C02 incubator at 37°C for 72 hours.
6. Take out experiment plates and leave at room temperature for 10 minutes.
7. Add 25 microliter CellTiter-Glo in each well and incubate for 10 minutes at room
temperature with shaking.
8. Read luminescence in PerkinElmer Envision plate reader.
Results
The results are shown in Figure 3. The data demonstrate that the combination of Compound (1) and Immunoconjugate (1) produced enhanced anti -proliferative activity, as compared to either individual treatment, in the OCI-LY7, OC-LY19, and SUDHL4 cell lines.
Table of Sequences
SEQ ID1 : CDR LI sequence:
Ser Ala Ser Ser Gly Val Asn Tyr Met His
1 5 10
SEQ ID 2: CDR L2 sequence:
Asp Thr Ser Lys Leu Ala Ser
1 5
SEQ ID 3: CDR L3 sequence:
His Gin Arg Gly Ser Tyr Thr
1 5 SEQ ID 4: CDR HI sequence:
Ser Asn Trp Met His
1 5
SEQ ID 5: CDR H2 sequence:
Glu He Asp Pro Ser Asp Ser Tyr Thr Asn
1 5 10
SEQ ID 6: CDR H3 sequence:
Gly Ser Asn Pro Tyr Tyr Tyr Ala Met Asp Tyr
1 5 10
SEQ ID 7: huB4 Light Chain Sequence:
Glu He Val Leu Thr Gin Ser Pro Ala He Met Ser Ala Ser Pro Gly 1 10 15
Glu Arg Val Thr Met Thr Cys Ser Ala Ser Ser Gly Val Asn Tyr Met 20 25 30
His Trp Tyr Gin Gin Lys Pro Gly Thr Ser Pro Arg Arg Trp He Tyr 35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Ser Leu Thr He Ser Ser Met Glu Pro Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys His Gin Arg Gly Ser Tyr Thr Phe Gly
85 90 95
Gly Gly Thr Lys Leu Glu He Lys Arg Thr Val Ala Ala Pro Ser Val 100 105 110
Phe He Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly Thr Ala Ser 115 120 125
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gin 130 135 140
Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin Glu Ser Val 145 150 155 160
Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu
165 170 175
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu 180 185 190
Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg 195 200 205
Gly Glu Cys
210
SEQ ID 8:huB4 Heavy Chain Sequence:
Gin Val Gin Leu Val Gin Pro Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15
Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Ser Asn 20 25 30
Trp Met His Trp Val Lys Gin Ala Pro Gly Gin Gly Leu Glu Trp He 35 40 45
Gly Glu He Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gin Asn Phe 50 55 60
Gin Gly Lys Ala Lys Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80
Met Glu Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Ser Asn Pro Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gin 100 105 110
Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190
Ser Ser Ser Leu Gly Thr Gin Thr Tyr He Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met He
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg 290 295 300
Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys 305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro He Glu
325 330 335
Lys Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr 340 345 350
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu 355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala Val Glu Trp 370 375 380
Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430
Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro 435 440 445
Gly Lys

Claims

We claim:
1. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of Compound (1):
Figure imgf000025_0001
or a pharmaceutically acceptable salt thereof, in combination with Immunoconjugate (1).
2. The method of claim 1, wherein Compound (1) and/or Immunoconjugate (1) are
administered with a pharmaceutically acceptable carrier.
3. The method of claim 1, wherein Compound (1) and Immunoconjugate (1) are
administered separately.
4. The method of claim 1, wherein Compound (1) and/or Immunoconjugate (1) are
administered at substantially the same time.
The method of claim 1, wherein Compound (1) and/or Immunoconjugate (1)
administered at different times.
The method of claim 1, wherein Compound (1) is administered to the subject prior to administration of Immunoconjugate (1).
The method of claim 1, wherein Immunoconjugate (1) is administered to the subject prior to administration of Compound (1).
8. The method of claim 1 , wherein the cancer is a non-Hodgkin's lymphoma.
9. The method of claim 1, wherein the cancer is a B-cell lymphoma.
10. The method of claim 1, wherein the cancer is a diffuse large B-cell lymphoma (DLBCL).
11. The method of claim 1 , wherein the cancer is a germinal center B-cell like diffuse large B-cell lymphoma (GCB DLBCL).
12. The method of claim 1, wherein the cancer is a leukemia.
13. The method of claim 12, wherein the leukemia is acute lymphoblastic leukemia.
14. The method of claim 12, wherein the leukemia is chronic lymphocytic leukemia.
15. The method of any one of claims 1-14, wherein the effective amount provides a
synergistic effect in the treatment of the subject.
16. A composition comprising a compound having the following structural formula:
Figure imgf000026_0001
or a pharmaceutically acceptable salt thereof, and Immunoconjugate (1).
17. The composition of claim 16, further comprising a pharmaceutically acceptable carrier. The composition of claim 16, wherein said compound according to Formula (1) and said Immunoconjugate (1) are in amounts that produce a synergistic effect in the treatment of lymphoma.
A kit comprising: (A) Compound (1), or a pharmaceutically acceptable salt thereof; (B) Immunoconjugate (1); and (C) instructions for use.
A method of treating a GCB DLBCL in a subject in need thereof, comprising
administering to the subject a therapeutically effective amount of a compound of Formula (1):
Figure imgf000027_0001
or a pharmaceutically acceptable salt thereof, in combination with Immunoconjugate (1), and a pharmaceutically acceptable carrier.
PCT/US2013/063983 2012-10-12 2013-10-09 Compositions and methods for treating cancer using pi3k inhibitor and anti-cd19 maytansinoid immunoconjugate WO2014058947A1 (en)

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