WO2022104010A1 - Combinations of methylene tetrahydrofolate dehydrogenase 2 (mthfd2) inhibitors and folate-depleting agents and methods using same - Google Patents

Combinations of methylene tetrahydrofolate dehydrogenase 2 (mthfd2) inhibitors and folate-depleting agents and methods using same Download PDF

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WO2022104010A1
WO2022104010A1 PCT/US2021/059069 US2021059069W WO2022104010A1 WO 2022104010 A1 WO2022104010 A1 WO 2022104010A1 US 2021059069 W US2021059069 W US 2021059069W WO 2022104010 A1 WO2022104010 A1 WO 2022104010A1
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oxo
mthfd2
chromeno
tetrahydro
carbonyl
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French (fr)
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Gulam Mohmad RATHER
Joseph R. Bertino
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Rutgers, The State University Of New Jersey
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • 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
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4813—Exopeptidases (3.4.11. to 3.4.19)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D313/00—Heterocyclic compounds containing rings of more than six members having one oxygen atom as the only ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains three hetero rings
    • C07D487/14—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems
    • C07D491/044—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
    • C07D491/052—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring the oxygen-containing ring being six-membered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/17—Metallocarboxypeptidases (3.4.17)
    • C12Y304/17011—Glutamate carboxypeptidase (3.4.17.11)

Definitions

  • MTHFD2 Methylene Tetrahydrofolate Dehydrogenase 2
  • MTHFD2 Methylene tetrahydrofolate dehydrogenase
  • MTHFD2 is a mitochondrial enzyme ordinarily expressed primarily in embryonic tissues, but is overexpressed in many cancers.
  • MTHFD2 has dehydrogenase and cyclohydrolase activities, generating 10-formyl tetrahydrofolate from 5-10-methylene tetrahydrofolate (the source of formate required for purine biosynthesis), as well as nicotinamide adenine dinucleotide phosphate (NADP/NADPH) (FIG. 1).
  • MTHFD2 is an important enzyme for the generation of the formate required for macromolecular synthesis and protection from reactive oxygen species (ROS).
  • ROS reactive oxygen species
  • MTHFD2 Inhibition of MTHFD2 results in folate deficiency, contributing to the inhibition of cancer cell growth, as methylene tetrahydrofolate (MTHF) accumulates and causes a relative deficiency of other folate coenzymes. Folate depletion of cells lacking MTHFD2 further enhances the observed cytotoxicity. However, folate deficient diets are difficult for patients to maintain and the dietary approach may take weeks or months to achieve the desired folate depletion.
  • MTHF methylene tetrahydrofolate
  • the present disclosure relates in part to a method of treating, preventing, and/or ameliorating a disease or disorder involving overexpression of methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) in a subject, the method comprising administering to the subject a pharmaceutically effective amount of a MTHFD2 inhibitor and a pharmaceutically effective amount of a folate-depleting agent.
  • MTHFD2 methylene tetrahydrofolate dehydrogenase 2
  • the disease or disorder is a proliferative disease or disorder.
  • the proliferative disease or disorder is cancer.
  • the present disclosure further relates to a composition comprising a MTHFD2 inhibitor and a folate-depleting agent, and pharmaceutical compositions thereof.
  • the present disclosure further relates to an immunoconjugate comprising an antibody, or antigen binding fragment thereof, conjugated to a MTHFD2 inhibitor.
  • the immunoconjugate selectively targets a cancer cell overexpressing MTHFD2.
  • FIG. 1 provides a schematic diagram of mitochondrial enzymes as a source for one- carbon units in the synthesis of purines and thymidylate.
  • THF tetrahydrofolate
  • CH2-THF tetrahydrofolate
  • CH- THF 5,10-methylenetetrahydrofolate
  • CH- THF 5,10- methenyltetrahydrofolate
  • CHO-THF 10-formyl tetrahydrofolate
  • CH3-THF 5-methyl tetrahydrofolate.
  • FIGs. 2A-2B provide a schematic diagram of the carboxypeptidase G2 reaction for folic acid (FIG. 2A) and methotrexate (FIG. 2B).
  • FIGs. 3A-3B show a western blot analysis of MCF7 and T47D cells after shRNA MTHFD2 knockdown. 50 pg of protein was loaded in each well of 12% SDS-PAGE.
  • FIG. 3 A MCF7-pLKO-ctrl (MCF7 cells with empty vector), MCF7-MTHFD2sh-551cl and 553cl (clones transfected with MTHFD2 knockdown plasmid).
  • FIG. 3B T47D Ctrl and T47D- shMTHFD2 (clone transfected with MTHFD2 shRNA plasmid).
  • FIGs. 3C-3D show that MTHFD2 knockdown inhibits growth of two breast cancer cell lines.
  • FIG. 3C MCF7-pLKO-ctrl, MCF7-MTHFD2sh-553cl, and MCF7-MTHFD2sh- 553cl 1.
  • FIG. 3D T47D Ctrl and T47D-shMTHFD2. The experiment was done in triplicate and values are represented by mean with standard mean deviation. * represents p ⁇ Q.Q5.
  • FIGs. 3E-3H show the results of the colony assay wherein MTHFD2 knockdown results in growth inhibition on day 8 in MCF7 cells (FIG. 3E and FIG. 3G) and T47D cells (FIG. 3F and FIG. 3H).
  • the experiment was done in triplicate and values are represented by mean with standard mean deviation. ** represents /? ⁇ 0.005; *** represents ⁇ 0.01.
  • FIG. 4A provides the results of the apoptosis check.
  • MCF7 and T47D cells (controls and MTHFD2 knockdown) were cultured in media for day 8 and then incubated with V-FITC and PI and analyzed using flow cytometry.
  • the lower left quadrant shows cells with are negative for both PI and annexin V-FITC; the upper left quadrant shows only PI positive cells, which are necrotic; the lower right quadrant shows annexin positive cells (early apoptotic); and the upper right quadrant shows annexin and PI positive cells (late apoptosis cells).
  • the percentage of necrotic, early, and late apoptotic cells are represented in each quadrant.
  • FIG. 4B provides a cell cycle phase analysis.
  • MCF7 and T47D cells controls and MTHFD2 knockdown cultured in media for 8 days were analyzed for cell cycle analysis using PI staining and flow cytometry. The percentage of cells in each cell cycle phase were analyzed using FlowJo software.
  • FIGs. 5A-5B show that MTHFD2 knockdown cells in folate depleted media enhances growth inhibition in two breast cancer cell lines.
  • FIG. 5A MCF7-pLKO-ctrl (MCF7 cells with empty vector), MCF7-MTHFD2sh-553cl (clone transfected with MTHFD2 shRNA plasmid).
  • FIG. 5B T47D Ctrl and T47D-shMTHFD2 (clone transfected with MTHFD2 shRNA plasmid). The experiment was done in triplicate and values are represented by mean with standard mean deviation. * represents /? ⁇ 0.05; ** represents /? ⁇ 0.03; *** represents ⁇ 0.01.
  • FIGs. 6A-6C show that MTHFD2 knockdown cells in the presence of CPG2 (10 units) enhances growth inhibition of two breast cancer cell lines.
  • FIG. 6A MCF7-pLKO-ctrl (MCF7 cells with empty vector), MCF7-MTHFD2sh-553cl (clone transfected with MTHFD2 shRNA plasmid).
  • FIG. 6B T47D Ctrl and T47D-shMTHFD2 (clone transfected with MTHFD2 shRNA plasmid).
  • FIG. 6C shows the remaining CPG2 activity in the media at different time points (CPG2 stability check). The experiment was done in triplicate and values are represented by mean with standard mean deviation. * represents p ⁇ Q.Q5.
  • FIGs. 7A-7B show that the combination of MTHFD2 knockdown and CPG2 enhanced anti-tumor effect against prostate cancer xenograft.
  • FIGs 8A-8B show that CPG2 has moderate anti-cancer activity against MCF7 breast cancer tumors.
  • FIGs. 9A-9B shows that CPG2 (glucarpidase) enhanced the anti-tumor effect in combination with MTHFD2 inhibitor (DS18561882) against MDA-MB-231 (TNBC) xenograft.
  • Nude mice were inoculated s.c. with 5 million MDA-MB-231 cells with Matrigel (1 : 1, v/v ratio) in the right flank. The mice were treated with the MTHFD2 inhibitor (DS18561882) dose of 250 mg/kg (200 pL), p.o. twice a day for 8 days.
  • Control groups (saline and CPG2 group) also received vehicle (10% DMSO, 40% PEG-300, 5% Tween-80, and 45% saline) (200 pL), p.o. twice a day for 8 days. Tumor size was measured and used to calculate tumor volume, wherein the values indicate average tumor volume ⁇ standard error p-values (FIG. 9A). Body weight as also measured and the mice treated with CPG2 and DS 18561882 showed weakness, as indicated by the body weight measurement (FIG. 9B).
  • Carboxypeptidase G2 (CPG2) is a dimeric zinc-dependent exopeptidase. Enzymatic depletion of folates with CPG2, a treatment that is approved to inactivate methotrexate (MTX) in case of overdose, has been shown to enhance the cytotoxicity of cells with a MTHFD2 knockdown. In vitro results demonstrated that MTHFD2 knockdown in cells caused an antiproliferative effect that was enhanced by folate depletion, when combined with folate-depleting enzyme, CPG2.
  • CPG2 Carboxypeptidase G2
  • the present disclosure relates in part to a method of treating, preventing, and/or ameliorating a disorder involving overexpression of methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of a MTHFD2 inhibitor and a pharmaceutically effective amount of a folate-depleting agent.
  • MTHFD2 methylene tetrahydrofolate dehydrogenase 2
  • the disease or disorder involving overexpression of MTHFD2 is selected from the group consisting of bipolar disorder, major depressive disorder, mitochondrial neurodegeneration, and a proliferative disease or disorder.
  • the disease or disorder involving overexpression of MTHFD2 is a proliferative disorder.
  • the proliferative disorder is cancer.
  • the cancer is at least one of brain cancer, breast cancer, colon cancer, rectal cancer, kidney cancer, prostate cancer, liver cancer, thyroid cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer, ventricular cancer, melanoma, lymphatic cancer, and acute leukemia.
  • the MTHFD2 inhibitor and the folate-depleting agent are each independently administered to the subject by at least one route selected from the group consisting of nasal, inhalational, topical, oral, buccal, rectal, pleural, peritoneal, vaginal, intramuscular, subcutaneous, transdermal, epidural, intratracheal, otic, intraocular, intrathecal, and intravenous routes.
  • the MTHFD2 inhibitor is administered to the subject orally 1 to 4 times per day.
  • the MTHFD2 inhibitor is administered to the subject 1 time per day.
  • the MTHFD2 inhibitor is administered to the subject 2 times per day.
  • the MTHFD2 inhibitor is administered to the subject 3 times per day. In certain embodiments, the MTHFD2 inhibitor is administered to the subject 4 times per day. In certain embodiments, the MTHFD2 inhibitor is administered by continuous or intermittent intravenous infusion.
  • the MTHFD2 inhibitor is (4-(3 -amino- l-hydroxy-9-oxo- 5,6,6a,7-tetrahydroimidazo[l,5-f]pteridin-8(9H)-yl)benzoyl)-L-glutamic acid, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
  • the MTHFD2 inhibitor is (3R,4R,6R,E)-8- ((2S, 3 S,7R,1 OR, 11R,E)-10,11 -dihydroxy-3, 7-dimethyl-12-oxooxacyclododec-8-en-2-yl)-3- methoxy-4,6-dimethyl-5-oxonon-7-enoic acid, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
  • the MTHFD2 inhibitor is a tricyclic coumarin selected from the group consisting of:
  • the MTHFD2 inhibitor is (S)-N-(4-(8-(3,4- dimethylpiperazin-l-yl)-7-methyl-5-oxo- 1,3,4, 5-tetrahydro-2H-chromeno[3,4-c]pyridine-3- carbonyl)-2-(trifluoromethoxy)phenyl)methanesulfonamide or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
  • the MTHFD2 inhibitor is an immunoconjugate selectively targeting a cancer cell, comprising an antibody, or antigen binding fragment thereof, conjugated to an MTHFD2 inhibitor, optionally through a linker.
  • the folate-depleting agent is an immunoconjugate selectively targeting a cancer cell, comprising an antibody, or antigen binding fragment thereof, conjugated to a carboxypeptidase, optionally through a linker.
  • the antibody, or antigen binding fragment thereof is selected from the group consisting of A5B7, F(ab’)2 A5B7, W14, MFE-23, adecatumumab, intetumumab, etaracizumab, glembatumumab vedotin, leronlimab, margetuximab, sacituzumab govitecan, and trastuzumab.
  • the folate-depleting agent is administered in advance of or contemporaneously with the administration of the MTHFD2 inhibitor. In certain embodiments, the folate-depleting agent is administered after the administration of the MTHFD2 inhibitor. In certain embodiments, the folate-depleting agent is administered by continuous or intermittent intravenous infusion.
  • the folate-depleting agent is a carboxypeptidase.
  • the carboxypeptidase is carboxypeptidase G2 (CPG2) (SEQ ID NO: 1) or a biologically active fragment or derivative thereof.
  • the subject is further administered at least one zinc supplement, which in non-limiting embodiments enhances CPG2 activity.
  • the at least one zinc supplement is zinc gluconate.
  • the carboxypeptidase shares at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology with CPG2 (SEQ ID NO: 1).
  • the carboxypeptidase is modified with at least one modification to reduce immunogenicity or increase half-life in the subject.
  • the at least one modification is selected from the group consisting of fusion with human serum albumin and/or an IgG Fc domain, alkylation of at least one amide group, acetylation and/or alkylation of the N-terminus amino group, amidation of the C- terminus carboxy group, PEGylation, and phosphocholination with phosphatidylcholine.
  • the PEGylation is introduced by site-specific or random bioconjugation techniques.
  • the subject is further administered at least one additional agent useful for treating, ameliorating, and/or preventing a proliferative disease or disorder, such as cancer.
  • the at least one additional agent comprises at least one selected from the group consisting of olaparib, rucaparib, niraparib, atrezoizumab, avelumab, pembrolizumab, cisplatin, carboplatin, doxorubicin, bevacizumab, gemcitabine, topetecan, paclitaxel, docetaxel, etoposide, and nanoparticle albumin-bound paclitaxel.
  • the at least one additional agent is co-administered with at least one of the MTHFD2 inhibitor and the folate-depleting agent. In certain embodiments, the at least one additional agent is co-formulated with at least one of the MTHFD2 inhibitor and the folate-depleting agent. In certain embodiments, the subject is a mammal. In certain embodiments, the mammal is a human.
  • the present disclosure relates in part to a pharmaceutical composition
  • a pharmaceutical composition comprising a MTHFD2 inhibitor, a folate-depleting agent, and at least one pharmaceutically acceptable carrier.
  • the MTHFD2 inhibitor is (S)-N-(4-(8-(3,4- dimethylpiperazin-l-yl)-7-methyl-5-oxo- 1,3,4, 5-tetrahydro-2H-chromeno[3,4-c]pyridine-3- carbonyl)-2-(trifluoromethoxy)phenyl)methanesulfonamide or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
  • the folate-depleting agent is a carboxypeptidase.
  • the carboxypeptidase is CPG2 or a biologically active fragment or derivative thereof.
  • the subject is administered a pharmaceutically effective amount of the composition of a pharmaceutical composition comprising a MTHFD2 inhibitor, a folate-depleting agent, and at least one pharmaceutically acceptable carrier.
  • the MTHFD2 inhibitor is (S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7- methyl-5-oxo- 1,3,4, 5-tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)-2- (trifluoromethoxy)phenyl)methanesulfonamide or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
  • the folate-depleting agent is a carboxypeptidase.
  • the carboxypeptidase is CPG2 or a biologically active fragment or derivative thereof.
  • the present disclosure relates in part to an immunoconjugate comprising an antibody, or antigen binding fragment thereof, conjugated to a MTHFD2 inhibitor, optionally through a linker.
  • the immunoconjugate selectively targets a cancer cell.
  • the cancer cell overexpresses MTHFD2.
  • the antibody, or antigen binding fragment thereof is selected from the group consisting of A5B7, F(ab’)2 A5B7, W14, MFE-23, adecatumumab, intetumumab, etaracizumab, glembatumumab vedotin, leronlimab, margetuximab, sacituzumab govitecan, and trastuzumab.
  • values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.
  • a range of "about 0.1% to about 5%” or "about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range.
  • the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.
  • bioconjugation refers to a chemical strategy to form a stable covalent bond between two molecules, wherein at least one of said molecules is a biomolecule.
  • bioconjugations include the couplings of small molecule and protein, protein and protein, antibody and small molecule, protein and antibody, antibody and enzyme, and protein and oligosaccharide.
  • a "disease” is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subject's health continues to deteriorate.
  • a disorder in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject's state of health.
  • folate-depleting agent refers to any composition which, upon administration to a subject, results in reduction or elimination of folate in the subject by any mechanism, including but not limited to chemical modification or sequestration of folate.
  • immunoconjugate refers to a conjugate of an antibody or antigen fragment component with a small molecule therapeutic or a macromolecular biologic therapeutic or diagnostic agent through a covalent linkage.
  • the therapeutic or diagnostic agent can comprise a radioactive or non-radioactive label.
  • the antibodies that are used to prepare immunoconjugates include, but are not limited to, monoclonal antibodies, chimeric antibodies, humanized antibodies, and human antibodies.
  • Linkers may be cleavable or non- cleavable. Non-limiting examples of cleavable linkers include disulfides, hydrazones, peptides, or thioethers.
  • the linker may be directly conjugated to the antibody, i.e. covalently linked directly to an exposed amino acid residue on the surface of the antibody or antigen fragment.
  • Therapeutic agents of the present disclosure comprise MTHFD2 inhibitors and folate-depleting agents.
  • immunogenicity refers to the propensity of a foreign substance to induce a humoral and/or cell-mediated immune response in the body of a human or other animal.
  • independently selected from refers to referenced groups being the same, different, or a mixture thereof, unless the context clearly indicates otherwise.
  • the phrase “X 1 , X 2 , and X 3 are independently selected from noble gases” would include the scenario where, for example, X 1 , X 2 , and X 3 are all the same, where X 1 , X 2 , and X 3 are all different, where X 1 and X 2 are the same but X 3 is different, and other analogous permutations.
  • knockdown refers to an experimental technique wherein the expression of one or more of an organism’s genes and/or translation of the corresponding RNA is reduced.
  • a “prophylactic” or “preventive” treatment is a treatment administered to a subject who does not exhibit signs of a disease or disorder or exhibits only early signs of the disease or disorder for the purpose of decreasing the risk of developing pathology associated with the disease or disorder.
  • the language “pharmaceutically effective amount,” “therapeutically effective amount,” or “effective amount” refers to a non-toxic but sufficient amount of the composition used in the practice of the invention that is effective to treat, prevent, and/or ameliorate a disease or disorder in the body of a mammal.
  • the desired treatment may be prophylactic and/or therapeutic. That result may be reduction and/or alleviation of the signs, symptoms, or causes of a disease or disorder, or any other desired alteration of a biological system.
  • An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
  • PCylation refers to phosphocholination, a process whereby the nucleophilic residues of a protein are covalently linked to a choline molecule by a phosphodiester bond.
  • PEGylated or “PEGylation” as used herein refers to the process of covalent and/or non-covalent attachment or amalgamation of polyethylene glycol polymer chains to molecules to the surface of a macromolecule (i.e. amino acids on the surface of a protein).
  • composition refers to a mixture of at least one compound useful within the invention with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition facilitates administration of the compound to a subject.
  • the term "pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound useful within the invention, and is relatively non-toxic, i.e., the material may be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • the term "pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the subject such that it may perform its intended function.
  • a pharmaceutically acceptable material, composition or carrier such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the subject such that it may perform its intended function.
  • Such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the invention, and not injurious to the subject.
  • materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline
  • pharmaceutically acceptable carrier also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the invention, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions.
  • the "pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound useful within the invention.
  • Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.
  • pharmaceutically acceptable salt refers to a salt of the administered compound prepared from pharmaceutically acceptable non-toxic acids and/or bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates (including hydrates) and clathrates thereof.
  • room temperature refers to a temperature of about 15 °C to about 28 °C.
  • the terms “subject” and “individual” and “patient” can be used interchangeably and may refer to a human or non-human mammal or a bird.
  • Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals.
  • the subject is human.
  • substantially refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%.
  • substantially free of' as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that the composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less.
  • substantially free of can mean having a trivial amount of, such that a composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%.
  • treating means ameliorating the effects of, or delaying, halting or reversing the progress of a disease or disorder.
  • the word encompasses reducing the severity of a symptom of a disease or disorder and/or the frequency of a symptom of a disease or disorder.
  • ubiquitinylating agent refers to any entity which facilitates the formation of a covalent linkage between ubiquitin and a biomolecule of interest.
  • the ubiquitinylating agent may be a ubiquitin ligase, or E3 enzyme, or an isoform thereof.
  • the ubiquitinylation may comprise monoubiquitinylation or polyubiquitinylation.
  • CEE-THF 5,10-methylenetetrahydrofolate
  • CH-THF 5,10-methenyltetrahydrofolate
  • CHO-THF 10-formyl tetrahydrofolate
  • CH3-THF 5-methyl tetrahydrofolate
  • CPG2 carboxypeptidase G2
  • CRISPR clustered regularly interspaced short palindromic repeats
  • FITC fluorescein isothiocyanate
  • MTHF methylenetetrahydrofolate
  • MTHFD2 methylenetetrahydrofolate dehydrogenase 2
  • MTX methotrexate
  • NADP nicotinamide adenine dinucleotide phosphate
  • PEG polyethylene glycol
  • PI propidium iodide
  • ROS reactive oxygen species
  • shRNA short hairpin RNA
  • THF tetrahydrofolate.
  • MTHFD2 examples include those disclosed in U.S. Patent No. 10,774,087 and related applications, Raze Therapeutics, Inc. Patent Application Publications US2018/0370972, WO2017/023894, and Wayne State University Patent Application Publication WO2019/046612, which are incorporated herein by reference in their entireties.
  • the MTHFD2 inhibitor is amino-l-hydroxy-9-oxo-5,6,6a,7- tetrahydroimidazo[l,5-f]pteridin-8(9H)-yl)benzoyl)-L-glutamic acid (LY345899), or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
  • the MTHFD2 inhibitor is dihydroxy-3, 7-dimethyl-12-oxooxacyclododec-8-en-2-yl)-3-methoxy-4,6-dimethyl-5- oxonon-7-enoic acid (carolacton), or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
  • the MTHFD2 inhibitor is a tricyclic coumarin.
  • the tricyclic coumarin is a l,2,3,4-tetrahydrochromeno[3,4-c]-pyridin-5-one.
  • the MTHFD2 inhibitor is a l,2,3,4-tetrahydrochromeno[3,4-c]- pyridin-5-one.
  • the MTHFD2 inhibitor is oxo- 1,3,4, 5-tetrahydro-2H-chromeno[3,4-c]pyridine-3- carbonyl)benzoic acid, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
  • the MTHFD2 inhibitor is oxo- 1,3,4, 5-tetrahydro-2H-chromeno[3,4-c]pyridine-3- carbonyl)benzoic acid, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
  • MTHFD2 inhibitor is -chloro-4-(8-(3,4-dimethylpiperazin-l-yl)-
  • the MTHFD2 inhibitor is -dimethylpiperazin-l-yl)-7-methyl-
  • the MTHFD2 inhibitor i chloro-4-(8-(3,4-dimethylpiperazin-l-yl)-7,10-dimethyl-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)phenyl)methanesulfonamide, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
  • the MTHFD2 inhibitor is -dimethylpiperazin-l-yl)-7- methyl-5-oxo- 1,3,4, 5-tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)-2- (trifluoromethoxy)phenyl)ethanesulfonamide, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
  • the MTHFD2 inhibitor (4-(8-(3 ,4-dimethylpiperazin- 1 -yl)-7, 10-dimethyl-5 -oxo- 1 , 3 ,4, 5 -tetrahy dro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)ethanesulfonamide, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
  • the MTHFD2 inhibitor is -ethyl-4-methylpiperazin-l-yl)-7- methyl-5-oxo- 1,3,4, 5-tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)-2-
  • the MTHFD2 inhibitor i methyl-5-oxo-8-(3,3,4-trimethylpiperazin-l-yl)-l,3,4,5-tetrahydro-2H-chromeno[3,4- c]pyridine-3 -carbonyl)-2-(trifluorom ethoxy )phenyl)methanesulfonamide, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
  • the MTHFD2 inhibitor is methyl-8-(3-methylpiperazin-l-yl)-5- oxo-l,3,4,5-tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)-2-
  • the MTHFD2 inhibitor i chloro-4-(7-methyl-5-oxo-8-((3R,5S)-3,4,5-trimethylpiperazin-l-yl)-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)phenyl)methanesulfonamide, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
  • the MTHFD2 inhibitor is (S)-N-(4-(8-(3,4- dimethylpiperazin-l-yl)-7-methyl-5-oxo- 1,3,4, 5-tetrahydro-2H-chromeno[3,4-c]pyridine-3- carbonyl)-2-(trifluoromethoxy)phenyl)methanesulfonamide, also referred to herein as DS18561882.
  • MTHFD2 inhibitors have a greater than 5:1 selectivity for MTHFD2 inhibition over MTHFD1 inhibition. In other embodiments, MTHFD2 inhibitors have a greater than 10: 1 selectivity for MTHFD2 inhibition over MTHFD1 inhibition. In yet other embodiments, MTHFD2 inhibitors have a greater than 50: 1 selectivity for MTHFD2 inhibition over MTHFD1 inhibition. In yet other embodiments, MTHFD2 inhibitors have a greater than 100: 1 selectivity for MTHFD2 inhibition over MTHFD1 inhibition. In certain embodiments, the MTHFD2 inhibitor is DS18561882 and the MTHFD2:MTHFD1 selectivity is greater than 90: 1.
  • the folate-depleting agent is a carboxypeptidase.
  • the carboxypeptidase is carboxypeptidase G2 (CPG2) or a biologically active fragment or derivative thereof.
  • CPG2 carboxypeptidase G2
  • other glucarpidase drugs are substituted for CPG2.
  • the amino acid sequence of the carboxypeptidase is modified to improve immunogenicity and half-life in a human.
  • the carboxypeptidase shares at least 85% homology with CPG2 (SEQ ID NO: 1). In certain embodiments, the carboxypeptidase shares at least 90% homology with CPG2 (SEQ ID NO: 1).
  • the carboxypeptidase shares at least 95% homology with CPG2 (SEQ ID NO: 1). In certain embodiments, the carboxypeptidase shares at least 99% homology with CPG2 (SEQ ID NO: 1). In certain embodiments, the carboxypeptidase is modified to improve immunogenicity and half-life in a human.
  • the carboxypeptidase may be fused (on the N-terminus and/or C-terminus) with human serum albumin and/or an IgG Fc domain, alkylated on at least one amide group, acetylated and/or alkylated on the N-terminus amino group, amidated on the C-terminus carboxy group, PEGylated, and/or phosphocholinated with phosphatidylcholine.
  • the present disclosure relates in part to immunoconjugates comprising an antibody, or an antigen binding fragment thereof, covalently conjugated to a MTHFD2 inhibitor or a folate-depleting agent through a linker.
  • An antibody fragment can be prepared by known methods, for example, as disclosed by Goldenberg, U.S. Patent Nos. 4,036,945 and 4,331,647 and references contained therein.
  • Another form of an antibody fragment is a peptide coding for a single complementary- determining region (CDR).
  • CDR is a segment of the variable region of an antibody that is complementary in structure to the epitope to which the antibody binds and is more variable than the rest of the variable region.
  • CDR peptides can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, using the polymerase chain reaction to synthesize the variable region from RNA of antibodyproducing cells.
  • the antibody or antigen is selected from the group consisting of A5B7 (P-hCG targeting), F(ab’)2 A5B7, W14 (CEA targeting), MFE-23 (CEA targeting), adecatumumab (EpCAM targeting), intetumumab (CD51 targeting), etaracizumab (integrin a v p3 targeting), glembatumumab vedotin (GPNMB targeting), leronlimab (CCR5 targeting), margetuximab (HER2 targeting), sacituzumab govitecan (TROP-2 targeting), and trastuzumab (HER2/neu targeting) (J. Natl. Cancer Inst. 1996, 88(3-4): 153-165; Eur. J. Nucl. Med. Mol. Imaging. 2004, 31(8): 1090-1096; Cancer Res. 1996, 56:3287-3292).
  • the linkers (and the therapeutic agents bound to said linkers) of the present disclosure may be directly conjugated to the antibody, i.e. covalently linked directly to the conjugated antibody or antigen fragment.
  • the covalent linkage may occur directly to one of the amino acids comprising the antibody or antigen backbone, ideally located within one of the constant domains, as opposed to within the variable domains.
  • Such amino acids may be naturally occurring (e.g. a naturally occurring lysine or cysteine residue), or the antibody or antigen may be artificially mutated (e.g. a non-naturally occurring lysine or cysteine residue) in order to provide an optimal binding site with minimal steric hindrance for the linker to bind to.
  • the linker can be bound to a chemical moiety (e.g. bound to an N-glycan) found on post-translationally modified antibodies and/or antigen fragments.
  • the covalent linkages in the present immunoconjugates may comprise a cleavable linking moiety, for example, a Val-Cit linker, which is cleavable by Cathepsin B inside the lysosome.
  • cleavable linking moieties may comprise a Phe-Lys linker, which is also cleavable by Cathespin B.
  • cleavable linking moieties include disulfide (S- S) bridges, which are cleavable in reductive (i.e. intracellular environment).
  • Immunoconjugates of the present disclosure may also utilize direct attachment of the linker to any of a number of nucleophile amino acid residue side chains, including thiols, amines, and alcohols via acylation to afford thioesters, amides, and esters, respectively.
  • nucleophile amino acid residue side chains including thiols, amines, and alcohols via acylation to afford thioesters, amides, and esters, respectively.
  • the linker comprises a cleavable linking moiety.
  • the linker is covalently bound to an exposed amino acid residue on the surface of the antibody or antigen.
  • the exposed amino acid residue comprises a cysteine.
  • the linker is covalently bound to the cysteine through sulfhydryl-maleimide coupling.
  • the exposed amino acid residue is a lysine.
  • the linker is covalently bound to the lysine through am amide linkage by acylation.
  • the linker is PEG- containing.
  • the cleavable linking moiety comprises a Val-Cit moiety.
  • the linking moiety comprises a Phe-Lys moiety.
  • the linker is non-cleavable.
  • the linker comprises a first linking component and a second linking component.
  • the first linking component is conjugated to a surface of an antibody according to any aspect of the present disclosure.
  • the first linking component is linked to the second linking component.
  • the first linking component is linked to the second linking component through click chemistry.
  • the first linking component is linked to the second linking component through a triazole moiety.
  • the first linking component is PEG-containing.
  • the second linking component is PEG-containing.
  • the first and second linking component are PEG-containing.
  • the first and/or second linking component contain between 1 and 10 PEG units.
  • the second linking component comprises a cleavage linking moiety according to any aspect of the present disclosure.
  • the second linking component comprises a therapeutic agent according to any aspect of the present disclosure.
  • the antibody or antigen binding fragment is conjugated to a carboxypeptidase. In certain embodiments, the antibody or antigen binding fragment is conjugated to CPG2.
  • the antibody or antigen binding fragment is conjugated to a MTHFD2 inhibitor.
  • the MTHFD2 inhibitor is (4-(3 -amino- 1- hydroxy-9-oxo-5,6,6a,7-tetrahydroimidazo[l,5-f]pteridin-8(9H)-yl)benzoyl)-L-glutamic acid (LY345899) or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
  • the MTHFD2 inhibitor is (3R,4R,6R,E)-8-((2S,3S,7R,10R,l 1R,E)-1O,11 -dihydroxy-3 J-dimethyl- 12- oxooxacyclododec-8-en-2-yl)-3-methoxy-4,6-dimethyl-5-oxonon-7-enoic acid (carolacton) or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
  • the MTHFD2 inhibitor is a tricyclic coumarin or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
  • the MTHFD2 inhibitor is (S)-N-(4-(8-(3,4- dimethylpiperazin-l-yl)-7-methyl-5-oxo- 1,3,4, 5-tetrahydro-2H-chromeno[3,4-c]pyridine-3- carbonyl)-2-(trifluoromethoxy)phenyl)methanesulfonamide or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
  • the MTHFD2 inhibitor is conjugated to a biomolecule suitable for the recruitment of an endogenous ubiquitinylating agent.
  • recruitment of the ubiquitinylating agent results in ubiquitinylation of MTHFD2.
  • the ubiquitinylated MTHFD2 is degraded by the proteasome.
  • Biomolecular constructs suitable for conjugation with the MTHFD2 inhibitors of the present disclosure are described by Li et al. (J. Hematol. Oncol. 2020, 13:50). Such constructs may be of particular utility in the treatment of leukemia and/or colon cancer (J. Exp. Med. 2016, 213(7): 1285-1306).
  • the combination therapy of the present invention is combined with additional therapies exploiting differences in tumor tissue.
  • the method of the present disclosure may further comprise administration of one or more PARP inhibitors, such as Lynparza (olaparib) and Rubraca (rucaparib) and Zejula (niraparib); and check point inhibitors such as Tecentriq (atezoizumab) and Bavercio (avelumab).
  • PARP inhibitors such as Lynparza (olaparib) and Rubraca (rucaparib) and Zejula (niraparib
  • check point inhibitors such as Tecentriq (atezoizumab) and Bavercio (avelumab).
  • Tecentriq atezoizumab
  • Bavercio avelumab
  • the combination therapy of the present disclosure is combined with at least one additional therapeutic agent, non-limiting examples including cisplatin, carboplatin, doxorubicin, bevacizumab, gemcitabine, topetecan, paclitaxel, docetaxel, etoposide, and nanoparticle albumin-bound paclitaxel.
  • the at least one additional therapeutic agent comprises administration of bevacizumab in combination with paclitaxel, PEGlylated liposomal doxorubicin, or topotecan.
  • the compounds of the invention may possess one or more stereocenters, and each stereocenter may exist independently in either the (R)- or ( ⁇ -configuration.
  • compounds described herein are present in optically active or racemic forms.
  • the compounds described herein encompass racemic, optically-active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein.
  • Preparation of optically active forms is achieved in any suitable manner, including by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically-active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase.
  • a mixture of one or more isomer is utilized as the therapeutic compound described herein.
  • compounds described herein contain one or more chiral centers. These compounds are prepared by any means, including stereoselective synthesis, enantioselective synthesis and/or separation of a mixture of enantiomers and/ or diastereomers. Resolution of compounds and isomers thereof is achieved by any means including, by way of non-limiting example, chemical processes, enzymatic processes, fractional crystallization, distillation, and chromatography.
  • the methods and formulations described herein include the use of N-oxides (if appropriate), crystalline forms (also known as polymorphs), solvates, amorphous phases, and/or pharmaceutically acceptable salts of compounds having the structure of any compound of the invention, as well as metabolites and active metabolites of these compounds having the same type of activity.
  • Solvates include water, ether (e.g., tetrahydrofuran, methyl tert-butyl ether) or alcohol e.g., ethanol) solvates, acetates and the like.
  • the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, and ethanol. In other embodiments, the compounds described herein exist in unsolvated form.
  • the compounds of the invention exist as tautomers. All tautomers are included within the scope of the compounds recited herein.
  • compounds described herein are prepared as prodrugs.
  • a "prodrug” is an agent converted into the parent drug in vivo.
  • a prodrug upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically active form of the compound.
  • a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically or therapeutically active form of the compound.
  • sites on, for example, the aromatic ring portion of compounds of the invention are susceptible to various metabolic reactions. Incorporation of appropriate substituents on the aromatic ring structures may reduce, minimize or eliminate this metabolic pathway. In certain embodiments, the appropriate substituent to decrease or eliminate the susceptibility of the aromatic ring to metabolic reactions is, by way of example only, a deuterium, a halogen, or an alkyl group.
  • Compounds described herein also include isotopically-labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
  • isotopes suitable for inclusion in the compounds described herein include and are not limited to 2 H, 3 H, U C, 13 C, 14 C, 36 C1, 18 F, 123 I, 125 I, 13 N, 15 N, 15 O, 17 O, 18 0, 32 P, and 35 S.
  • isotopically-labeled compounds are useful in drug and/or substrate tissue distribution studies.
  • substitution with heavier isotopes such as deuterium affords greater metabolic stability (for example, increased in vivo half-life or reduced dosage requirements).
  • substitution with positron emitting isotopes, such as n C, 18 F, 15 O and 13 N is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.
  • Isotopically-labeled compounds are prepared by any suitable method or by processes using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed.
  • the compounds described herein are labeled by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.
  • compositions described herein may form salts with acids or bases, and such salts are included in the present invention.
  • the salts are pharmaceutically acceptable salts.
  • salts embraces addition salts of free acids or free bases that are compositions of the invention.
  • pharmaceutically acceptable salt refers to salts that possess toxicity profiles within a range that affords utility in pharmaceutical applications. Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallinity, which have utility in the practice of the present invention, such as for example utility in process of synthesis, purification or formulation of compositions of the invention.
  • Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid.
  • inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids.
  • Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanes
  • Suitable pharmaceutically acceptable base addition salts of compositions of the invention include, for example, ammonium salts and metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts.
  • Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N'-dibenzylethylene- diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N- methylglucamine) and procaine.
  • Examples of pharmaceutically unacceptable base addition salts include lithium salts and cyanate salts. All of these salts may be prepared from the corresponding composition by reacting, for example, the appropriate acid or base with the composition.
  • Routes of administration of any of the compounds and/or compositions of the invention include oral, nasal, rectal, intravaginal, parenteral (e.g., IM, IV and SC), buccal, sublingual or topical.
  • the regimen of administration may affect what constitutes an effective amount. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
  • compositions of the present invention may be carried out using known procedures, at dosages and for periods of time effective to treat the disorder involving overexpression of methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) in a subject.
  • An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the subject; the age, sex, and weight of the subject; and the ability of the therapeutic compound to treat the disease or disorder in the subject.
  • Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • a non-limiting example of an effective dose range for a therapeutic compound useful within the invention is from about 1 and 5,000 mg/kg of body weight/per day.
  • One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.
  • the selected dosage level depends upon a variety of factors, including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well, known in the medical arts.
  • a medical doctor e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • physician or veterinarian may start doses of the compounds useful within the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • the carboxypeptidase is glucarpidase (CPG2) which is commercially available as VORAXAZE® from Protherics Medicine Limited/BTG Specialty Pharmaceuticals.
  • the specified dose for administration to deplete patient methotrexate (MTX) in a rescue situation is 50 units/kg.
  • the dose used to deplete folate in the subject is 50 units/kg.
  • the dose used to deplete folate in the subject is greater than 50 units/kg.
  • lower doses from 1 to 50 units/kg given over the course of days or weeks may be used to achieve folate depletion in the subject.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle.
  • the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of a disorder involving overexpression of methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) in a subject.
  • MTHFD2 methylene tetrahydrofolate dehydrogenase 2
  • compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers.
  • pharmaceutical compositions of the invention comprise a therapeutically effective amount of a compound useful within the invention and a pharmaceutically acceptable carrier.
  • the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity may 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 dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition.
  • Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
  • compositions of the invention are administered to the subject in dosages that range from one to five times per day or more.
  • the compositions of the invention are administered to the subject in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks.
  • the frequency of administration of the various combination compositions of the invention varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors.
  • the invention should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any subject are determined by the attending physical taking all other factors about the subject into account.
  • Compounds useful within the invention for administration may be in the range of from about 1 mg to about 10,000 mg, about 20 mg to about 9,500 mg, about 40 mg to about 9,000 mg, about 75 mg to about 8,500 mg, about 150 mg to about 7,500 mg, about 200 mg to about 7,000 mg, about 3050 mg to about 6,000 mg, about 500 mg to about 5,000 mg, about 750 mg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 50 mg to about 1,000 mg, about 75 mg to about 900 mg, about 100 mg to about 800 mg, about 250 mg to about 750 mg, about 300 mg to about 600 mg, about 400 mg to about 500 mg, and any and all whole or partial increments there between.
  • the dose of a compound useful within the invention is from about 1 mg and about 2,500 mg. In other embodiments, a dose of a compound useful within the invention used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg.
  • a dose of a second compound, as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments there between.
  • the present invention is directed to a packaged pharmaceutical composition
  • a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound useful within the invention, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, and/or ameliorate a disorder involving overexpression of methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) in a subject.
  • MTHFD2 methylene tetrahydrofolate dehydrogenase 2
  • Granulating techniques are well known in the pharmaceutical art for modifying starting powders or other particulate materials of an active ingredient.
  • the powders are typically mixed with a binder material into larger permanent free-flowing agglomerates or granules referred to as a "granulation.”
  • solvent-using "wet" granulation processes are generally characterized in that the powders are combined with a binder material and moistened with water or an organic solvent under conditions resulting in the formation of a wet granulated mass from which the solvent must then be evaporated.
  • Melt granulation generally consists in the use of materials that are solid or semi-solid at room temperature (i.e. having a relatively low softening or melting point range) to promote granulation of powdered or other materials, essentially in the absence of added water or other liquid solvents.
  • the low melting solids when heated to a temperature in the melting point range, liquefy to act as a binder or granulating medium.
  • the liquefied solid spreads itself over the surface of powdered materials with which it is contacted, and on cooling, forms a solid granulated mass in which the initial materials are bound together.
  • the resulting melt granulation may then be provided to a tablet press or be encapsulated for preparing the oral dosage form.
  • Melt granulation improves the dissolution rate and bioavailability of an active (i.e. drug) by forming a solid dispersion or solid solution.
  • U.S. Patent No. 5,169,645 discloses directly compressible wax-containing granules having improved flow properties.
  • the granules are obtained when waxes are admixed in the melt with certain flow improving additives, followed by cooling and granulation of the admixture.
  • certain flow improving additives such as sodium bicarbonate
  • the present invention also includes a multilayer tablet comprising a layer providing for the delayed release of one or more compounds useful within the invention, and a further layer providing for the immediate release of a medication for a disorder involving overexpression of methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) in a subject.
  • MTHFD2 methylene tetrahydrofolate dehydrogenase 2
  • Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art.
  • the pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.
  • compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets.
  • excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate.
  • the tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.
  • the compounds for use in the invention may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
  • transdermal e.g., sublingual, lingual, (trans)buccal, (trans)urethral
  • vaginal e.g., trans- and perivaginally
  • intravesical, intrapulmonary, intraduodenal, intragastrical intrathecal
  • compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein.
  • compositions of the invention may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropylmethylcellulose); fillers (e.g., cornstarch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrates (e.g., sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropylmethylcellulose
  • fillers e.g., cornstarch, lactose, microcrystalline cellulose or calcium phosphate
  • lubricants e.g., magnesium stearate, talc, or silica
  • disintegrates e.g., sodium starch glycolate
  • Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions.
  • the liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxy benzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats
  • emulsifying agent e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters or ethyl alcohol
  • preservatives e.g., methyl or propyl p-hydroxy benzoates or sorbic acid
  • compositions of the invention may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and/or continuous infusion.
  • Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulation agents such as suspending, stabilizing and/or dispersing agents may be used.
  • Additional dosage forms of this invention include dosage forms as described in U.S. Patents Nos. 6,340,475, 6,488,962, 6,451,808, 5,972,389, 5,582,837, and 5,007,790. Additional dosage forms of this invention also include dosage forms as described in U.S. Patent Applications Nos. 2003/0147952, 2003/0104062, 2003/0104053, 2003/0044466, 2003/0039688, and 2002/0051820. Additional dosage forms of this invention also include dosage forms as described in PCT Applications Nos.
  • the formulations of the present invention may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.
  • sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period.
  • the period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.
  • the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds.
  • the compounds of the present disclosure may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.
  • the compounds useful within the invention are administered to a subject, alone or in combination with another pharmaceutical agent, using a sustained release formulation.
  • delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that may, although not necessarily, include a delay of from about 10 minutes up to about 12 hours.
  • pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.
  • immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.
  • short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.
  • rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.
  • the therapeutically effective amount or dose of a compound of the present invention will depend on the age, sex and weight of the subject, the current medical condition of the subject and the nature of the disorder involving overexpression of methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) being treated.
  • MTHFD2 methylene tetrahydrofolate dehydrogenase 2
  • a suitable dose of a compound of the present invention may be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day.
  • the dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses.
  • the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days.
  • the compounds for use in the method of the invention may be formulated in unit dosage form.
  • unit dosage form refers to physically discrete units suitable as unitary dosage for subjects undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier.
  • the unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.
  • reaction conditions including but not limited to reaction times, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing/oxi dizing agents, with art- recognized alternatives and using no more than routine experimentation, are within the scope of the present application.
  • range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 and so forth, as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.1, 5.3, 5.5, and 6. This applies regardless of the breadth of the range.
  • mice were obtained from Taconic Biosciences, Inc (Germantown, NY).
  • MCF7 and T47D breast cancer cell lines originated from an invasive ductal carcinoma primary tumor and were established from metastatic pleural effusion. Both MCF7 and T47D are classified in the epithelial luminal A subtype and are hormonal sensitive, as both express the estrogen receptor (ER). Other hormonal receptors, like progesterone receptor (PR) is expressed only in T47D. Both cell lines lack the Herceptin 2 receptor (HER2). Thus, MCF7 is ER+, PR-, HER2-; and T47D is ER+, PR+, HER2-.
  • PC3 cells represent advanced prostate cancer, are metastatic in nature, and do not respond to androgens.
  • MCF7, T47D, and PC3 cells were cultured in RPMI 1640 containing 10% fetal bovine serum in a 37 °C incubator with 5% CO2.
  • Folate free media (RPMI 1640 with no folic acid) studies were performed using the dialyzed fetal bovine serum in media.
  • 500,000 cells/well in 6-well plates were transfected with a pLKOl MTHFD2 shRNA plasmid (GE Life Sciences, oligo MTHFD2sh359, MTHFD2sh966, and MTHFD2sh969) using the transfectamine-2000 reagent (Invitrogen).
  • the medium was changed 24 h after transfection. After an additional 24-48 h, cells were subjected to selection in medium containing puromycin (Sigma Aldrich). Cells were cultured for two weeks in 0.5 pg/mL puromycin (MCF7) and 0.2 pg/mL puromycin (T47D) to select for cells expressing MTHFD2 shRNA.
  • MCF7 puromycin
  • T47D 0.2 pg/mL puromycin
  • MTHFD2 Knockdown of MTHFD2 was confirmed by western blot and cells were maintained in puromycin. The transfection was also confirmed by fluorescence microscopy by the presence of a GFP signal. Note'. MTHFD2 may enhance tumor growth, but may not be essential. Thus, inhibition or knockdown of MTHFD2 would be expected to slow tumor growth rather than completely inhibit tumor growth.
  • the cytoplasmic enzyme and the mitochondrial housekeeping enzyme MTHFD2L may provide sufficient formate for a lower proliferation rate.
  • the membrane was washed thrice in Tris buffered saline + 0.1% tween-20 and incubated for two hours at room temperature with the appropriate peroxidase-conjugated secondary antibody. Bands were visualized using an enhanced chemiluminescence kit (Pierce).
  • Anti-MTHFD2 antibody was purchased from Cell Signaling Technology; Anti-tubulin and anti-mouse secondary antibodies were purchased from Santa Cruz Biotechnology.
  • 5,000 cells/well in a 24-well plates were seeded in 2 mL complete media for different amounts of time. The cells were washed twice with IX PBS and treated with 100 pL trypsin and an additional 900 pL of fresh media was used to collect the cells from the well. Cell viability was determined using the Vi-CELLTM Series Cell Viability Analyzer (Beckman Coulter, Carlsbad, CA).
  • 10 pL FITC annexin V and 10 pL PI/RNase staining solution BD PharmingenTM
  • 400 pL of IX binding buffer was added to the solution and the staining was analyzed.
  • mice Five million PC3 cells (control cells or MTHFD2 knockdown cells) with matrigel (1 : 1 v/v ratio) were injected subcutaneously in the abdominal right flanks of 40 nude mice. Once the tumor was palpable, mice were randomized and treated into four groups. One group of mice with PC3 control cells received saline and the other group was treated with CPG2 (10 units, 100 pL) intraperitoneally 3 times per week. Similarly, a group of mice with PC3 MTHFD2 knockdown cells received saline and another group was treated with CPG2 (10 units, 100 pL) intraperitoneally 3 times per week. Tumor size and body weight was measured twice a week and tumor volume was calculated using (L)(W) A 2/2. Data was plotted and SEM was calculated.
  • CPG2 glucarpidase
  • Control groups (saline and CPG2) also received vehicle (10% DMSO, 40% PEG-300, 5% Tween-80, and 45% saline) (200 pL), p.o. twice a day for 8 days. Tumor size and body weight was measured and tumor volume was calculated.
  • Candidates for this study display a tumor which overexpresses MTHFD2 and is refractory to known effective therapies.
  • the patient is Karnofsky stage (70 or above) and has measurable disease, wherein tumor tissue is available for evaluation of MTHFD2 levels.
  • Initial doses of CPG2 are 50 mg/m 2 q (per) 2 days x 7 treatments, together with a MTHFD2 inhibitor.
  • Treatment response and toxicity is measured after 2 weeks and 4 weeks by physical exam and appropriate laboratory and imaging tests.
  • Example 1 MTHFD2 knockdown study in human breast cancer cell lines MCF-7 and T47D
  • MTHFD2 knockdown was achieved using two oligos (MTHFD2sh966 and MTHFD2sh969) in MCF7 cells and by a single oligo (MTHFD2sh966) in T47D cells.
  • the knockdown of MTHFD2 using different oligos in MCF7 and T47D cells was achieved with greater than 90% knockdown, as shown by western blot (FIGs. 3 A-3B).
  • the cell lines were established after two weeks of culturing in puromycin, a selective resistance marker present in pLKOl plasmids as well as GFP.
  • the growth rates of the two cell lines were compared with and without MTHFD2 knockdown and clearly demonstrated that the knockdown of MTHFD2 in MCF7 and T47D breast cancer cells slowed growth as compared to cell lines without knockdown (FIGs. 3C- 3D).
  • the relative decrease in growth rates observed in MTHFD2 knockdown cell lines was further confirmed by colony assay, wherein a significant decrease in colony number was observed compared to the control on day 8 (FIG. 3E-3H). Growth inhibition was attributed to an increase in early apoptosis, wherein T47D and MCF7 demonstrated a five-fold and twofold increase, respectively, compared to controls.
  • Example 2 Combination of MTHFD2 Knockdown and CPG2 administration shows enhanced anti-tumor effect against prostate cancer and breast cancer xenografts
  • Carboxypeptidase GS was found to possess significant anti-tumor activity in vivo in an animal study wherein CPG2 was administered against MTHFD2 knockdown PC3 xenograft mice. In these experiments, little to no inhibition of tumor growth was observed in mice without knockdown of MTHFD2 which were treated with CPG2. Similarly, mice with MTHFD2 knockdown, but without treatment with CPG2, demonstrated only modest tumor growth arrest. However, mice which MTHFD2 knockdown and CPG2 treatment demonstrated significant tumor growth arrest (FIGs. 7A-7B).
  • CPG2 was found to possess significant anti-tumor activity in vivo in an animal pilot study wherein CPG2 was administered against MTHFD2 overexpressed MCF7 xenograft (control) mice.
  • MTHFD2 control mice treated with CPG2 showed moderate loss of body weight after 18 days, and mice bearing MCF7 MTHFD2 knockdown cells did not grow a tumor for up to 4 months (FIGs. 8A-8B).
  • Example 3 Combination of MHTFD2 inhibitor (DS18561882) and CPG2 (glucarpidase) shows tumor growth inhibition breast cancer xenograft
  • CPG2 carboxypeptidase GS
  • DS18561882 were administered against MDA-MB-231 xenograft mice (FIGs. 9A-9B).
  • CPG2 carboxypeptidase G2
  • CPG2 carboxypeptidase G2
  • Embodiment 1 provides a method of treating, preventing, and/or ameliorating a disease or disorder involving overexpression of methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) in a subject, the method comprising administering to the subject a pharmaceutically effective amount of a MTHFD2 inhibitor and a pharmaceutically effective amount of a folate-depleting agent.
  • MTHFD2 methylene tetrahydrofolate dehydrogenase 2
  • Embodiment 2 provides the method of Embodiment 1, wherein the disease or disorder is selected from the group consisting of bipolar disorder, major depressive disorder, mitochondrial neurodegeneration, and a proliferative disease or disorder.
  • Embodiment 3 provides the method of any one of Embodiments 1-2, wherein the disease or disorder is a proliferative disease or disorder.
  • Embodiment 4 provides the method of any one of Embodiments 1-3, wherein the proliferative disease or disorder is cancer.
  • Embodiment 5 provides the method of Embodiment 4, wherein the cancer is at least one of brain cancer, breast cancer, colon cancer, rectal cancer, kidney cancer, prostate cancer, liver cancer, thyroid cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer, ventricular cancer, melanoma, lymphatic cancer, and acute leukemia.
  • the cancer is at least one of brain cancer, breast cancer, colon cancer, rectal cancer, kidney cancer, prostate cancer, liver cancer, thyroid cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer, ventricular cancer, melanoma, lymphatic cancer, and acute leukemia.
  • Embodiment 6 provides the method of any one of Embodiments 1-5, wherein the
  • MTHFD2 inhibitor and the folate-depleting agent are each independently administered to the subject by at least one route selected from the group consisting of nasal, inhalational, topical, oral, buccal, rectal, pleural, peritoneal, vaginal, intramuscular, subcutaneous, transdermal, epidural, intratracheal, otic, intraocular, intrathecal, and intravenous routes.
  • Embodiment 7 provides the method of any one of Embodiments 1-6, wherein the MTHFD2 inhibitor is administered to the subject orally 1 to 4 times per day.
  • Embodiment 8 provides the method of any one of Embodiments 1-7, wherein the MTHFD2 inhibitor is administered to the subject by continuous or intermittent intravenous infusion.
  • Embodiment 9 provides the method of any one of Embodiments 1-8, wherein the MTHFD2 inhibitor is (4-(3-amino-l-hydroxy-9-oxo-5,6,6a,7-tetrahydroimidazo[l,5- f]pteridin-8(9H)-yl)benzoyl)-L-glutamic acid, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
  • the MTHFD2 inhibitor is (4-(3-amino-l-hydroxy-9-oxo-5,6,6a,7-tetrahydroimidazo[l,5- f]pteridin-8(9H)-yl)benzoyl)-L-glutamic acid, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
  • Embodiment 10 provides the method of any one of Embodiments 1-8, wherein the MTHFD2 inhibitor is (3R,4R,6R,E)-8-((2S,3S,7R,10R,l 1R,E)-1O,1 l-dihydroxy-3,7- dimethyl-12-oxo-l-oxacyclododec-8-en-2-yl)-3-methoxy-4,6-dimethyl-5-oxonon-7-enoic acid, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
  • the MTHFD2 inhibitor is (3R,4R,6R,E)-8-((2S,3S,7R,10R,l 1R,E)-1O,1 l-dihydroxy-3,7- dimethyl-12-oxo-l-oxacyclododec-8-en-2-yl)-3-methoxy
  • Embodiment 11 provides the method of any one of Embodiments 1-8, wherein the MTHFD2 inhibitor is selected from the group consisting of: 4-(5-oxo-l,3,4,5-tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)benzoic acid; N-(2-chloro-4-(7-methyl-8-(4-methylpiperazin-l-yl)-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)phenyl)methanesulfonamide;
  • Embodiment 12 provides the method of any one of Embodiments 1-8, wherein the MTHFD2 inhibitor is (S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo-l,3,4,5- tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)-2- (trifluoromethoxy)phenyl)methanesulfonamide or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
  • the MTHFD2 inhibitor is (S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo-l,3,4,5- tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)-2- (trifluoromethoxy)phenyl)methanesulf
  • Embodiment 13 provides the method of any one of Embodiments 1-12, wherein the folate-depleting agent is administered in advance of or contemporaneously with administration of the MTHFD2 inhibitor.
  • Embodiment 14 provides the method of any one of Embodiments 1-13 wherein the folate-depleting agent is administered by continuous or intermittent intravenous infusion.
  • Embodiment 15 provides the method of any one of Embodiments 1-14, wherein the folate-depleting agent is a carboxypeptidase.
  • Embodiment 16 provides the method of Embodiment 15, wherein the carboxypeptidase is carboxypeptidase G2 (CPG2) (SEQ ID NO: 1) or a biologically active fragment or derivative thereof.
  • CPG2 carboxypeptidase G2
  • Embodiment 17 provides the method of any one of Embodiments 1-16, wherein the subject is further administered at least one zinc supplement.
  • Embodiment 18 provides the method of Embodiment 17, wherein the zinc supplement is zinc gluconate.
  • Embodiment 19 provides the method of any one of Embodiments 15-18, wherein the carboxypeptidase shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology with CPG2 (SEQ ID NO: 1).
  • Embodiment 20 provides the method of any one of Embodiments 15-19, wherein the carboxypeptidase is modified with at least one modification to reduce immunogenicity and/or increase half-life in the subject.
  • Embodiment 21 provides the method of Embodiment 20, wherein the at least one modification is selected from the group consisting of fusion with human serum albumin and/or an IgG Fc domain, alkylation of at least one amide group, acetylation and/or alkylation of the N-terminus amino group, amidation of the C-terminus carboxy group, PEGylation, and phosphocholination with phosphatidylcholine.
  • the at least one modification is selected from the group consisting of fusion with human serum albumin and/or an IgG Fc domain, alkylation of at least one amide group, acetylation and/or alkylation of the N-terminus amino group, amidation of the C-terminus carboxy group, PEGylation, and phosphocholination with phosphatidylcholine.
  • Embodiment 22 provides the method of Embodiment 21, wherein the PEGylation is introduced by a site-specific or random bioconjugation technique.
  • Embodiment 23 provides the method of any one of Embodiments 1-22, wherein the subject is further administered at least one additional agent useful for treating, ameliorating, and/or preventing the disease or disorder.
  • Embodiment 24 provides the method of Embodiment 23, wherein the at least one additional agent comprises at least one selected from the group consisting of olaparib, rucaparib, niraparib, atrezoizumab, avelumab, pembrolizumab, cisplatin, carboplatin, doxorubicin, bevacizumab, gemcitabine, topetecan, paclitaxel, docetaxel, etoposide, and nanoparticle albumin-bound paclitaxel.
  • the at least one additional agent comprises at least one selected from the group consisting of olaparib, rucaparib, niraparib, atrezoizumab, avelumab, pembrolizumab, cisplatin, carboplatin, doxorubicin, bevacizumab, gemcitabine, topetecan, paclitaxel, docetaxel, etoposide, and nanoparticle albumin
  • Embodiment 25 provides the method of any one of Embodiments 23-24, wherein the at least one additional agent is co-administered with at least one of the MTHFD2 inhibitor and the folate-depleting agent.
  • Embodiment 26 provides the method of any one of Embodiments 23-25, wherein the at least one additional agent is co-formulated with at least one of the MTHFD2 inhibitor and the folate-depleting agent.
  • Embodiment 27 provides the method of any one of Embodiments 1-26, wherein the subject is administered a composition comprising the MTHFD2 inhibitor and the folate- depleting agent.
  • Embodiment 28 provides the method of Embodiment 27, wherein the MTHFD2 inhibitor is (S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)methanesulfonamide or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
  • the MTHFD2 inhibitor is (S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)methanesulfonamide or a salt,
  • Embodiment 29 provides the method of Embodiment 27, wherein the folate-depleting agent is CPG2 or a biologically active fragment or derivative thereof.
  • Embodiment 30 provides the method of any one of Embodiments 1-29, wherein the MTHFD2 inhibitor is conjugated to an antibody, or antigen binding fragment thereof, and wherein the conjugate selectively targets a cancer cell.
  • Embodiment 31 provides the method of Embodiment 30, wherein the antibody, or antigen binding fragment thereof, is selected from the group consisting of A5B7, F(ab’)2 A5B7, W14, MFE-23, adecatumumab, intetumumab, etaracizumab, glembatumumab vedotin, leronlimab, margetuximab, sacituzumab govitecan, and trastuzumab.
  • the antibody, or antigen binding fragment thereof is selected from the group consisting of A5B7, F(ab’)2 A5B7, W14, MFE-23, adecatumumab, intetumumab, etaracizumab, glembatumumab vedotin, leronlimab, margetuximab, sacituzumab govitecan, and trastuzumab.
  • Embodiment 32 provides the method of Embodiment 30, wherein the MTHFD2 inhibitor is conjugated to a biomolecule suitable for the recruitment of an endogenous ubiquitinylating agent.
  • Embodiment 33 provides the method of Embodiment 32, wherein the MTHFD2 is targeted for proteasome degradation.
  • Embodiment 34 provides the method of any one of Embodiments 1-29, wherein the folate-depleting agent is conjugated to an antibody, or antigen binding fragment thereof, wherein the conjugate selectively targets a cancer cell.
  • Embodiment 35 provides the method of Embodiment 34, wherein the folate-depleting agent is a carboxypeptidase.
  • Embodiment 36 provides the method of Embodiment 34, wherein the carboxypeptidase is CPG2 or a biologically active fragment or derivative thereof.
  • Embodiment 37 provides the method of any one of Embodiments 34-36, wherein the antibody, or antigen binding fragment thereof, is selected from the group consisting of A5B7, F(ab’)2 A5B7, W14, MFE-23, adecatumumab, intetumumab, etaracizumab, glembatumumab vedotin, leronlimab, margetuximab, sacituzumab govitecan, and trastuzumab.
  • the antibody, or antigen binding fragment thereof is selected from the group consisting of A5B7, F(ab’)2 A5B7, W14, MFE-23, adecatumumab, intetumumab, etaracizumab, glembatumumab vedotin, leronlimab, margetuximab, sacituzumab govitecan, and trastuzumab
  • Embodiment 38 provides the method of any one of Embodiments 1-37, wherein the subject is a mammal.
  • Embodiment 39 provides the method of Embodiment 38, wherein the mammal is a human.
  • Embodiment 40 provides a composition comprising a MTHFD2 inhibitor and a folate-depleting agent.
  • Embodiment 41 provides the composition of Embodiment 40, wherein the MTHFD2 inhibitor is (S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)methanesulfonamide or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
  • the MTHFD2 inhibitor is (S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)methanesulfonamide or a salt
  • Embodiment 42 provides the composition of Embodiment 40, wherein the folate- depleting agent is CPG2 or a biologically active fragment or derivative thereof.
  • Embodiment 43 provides a pharmaceutical composition comprising the composition of any one of Embodiments 40-42 and at least one pharmaceutically acceptable carrier.
  • Embodiment 44 provides an immunoconjugate selectively targeting a cancer cell, comprising an antibody, or antigen binding fragment thereof, conjugated to a MTHFD2 inhibitor.
  • Embodiment 45 provides the immunoconjugate of Embodiment 44, which selectively targets a cancer cell which overexpresses MTHFD2.
  • Embodiment 46 provides the immunoconjugate of any one of Embodiments 44-45, wherein the antibody, or antigen binding fragment thereof, is selected from the group consisting of A5B7, F(ab’)2 A5B7, W14, MFE-23, adecatumumab, intetumumab, etaracizumab, glembatumumab vedotin, leronlimab, margetuximab, sacituzumab govitecan, and trastuzumab.
  • the antibody, or antigen binding fragment thereof is selected from the group consisting of A5B7, F(ab’)2 A5B7, W14, MFE-23, adecatumumab, intetumumab, etaracizumab, glembatumumab vedotin, leronlimab, margetuximab, sacituzumab govitecan, and trastu
  • Embodiment 47 provides the immunoconjugate of any one of Embodiments 44-46, wherein the MTHFD2 inhibitor is:
  • Embodiment 48 provides the immunoconjugate of any one of Embodiments 44-47, wherein the MTHFD2 inhibitor is (S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo- l,3,4,5-tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)-2- (trifluoromethoxy)phenyl)methanesulfonamide or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
  • the MTHFD2 inhibitor is (S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo- l,3,4,5-tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)-2- (trifluoromethoxy)phenyl)methan

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Abstract

The present disclosure relates in part to methods of treating, preventing, and/or ameliorating a disease or disorder involving overexpression of methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) in a subject in need thereof, comprising administering to the subject a MTHFD2 inhibitor and a folate-depleting agent. In certain embodiments, the folate-depleting agent is a carboxypeptidase G2 (CPG2). In certain embodiments, the disease or disorder is a proliferative disease or disorder comprising a number of cancers.

Description

TITLE OF THE INVENTION
Combinations of Methylene Tetrahydrofolate Dehydrogenase 2 (MTHFD2) Inhibitors and Folate-Depleting Agents and Methods Using Same
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63/113,602, filed November 13, 2020, which is hereby incorporated by reference in its entirety herein.
SEQUENCE LISTING
The ASCII text file named "370602-7044W01_Sequence_Listing" created on November 12, 2021, comprising 3.80 Kbytes, is hereby incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
Methylene tetrahydrofolate dehydrogenase (MTHFD), which is a key bi-functional enzyme for the regulation of one-carbon folate metabolism, is present in both the mitochondria and the cytoplasm. MTHFD2 is a mitochondrial enzyme ordinarily expressed primarily in embryonic tissues, but is overexpressed in many cancers. Several reports have identified the key role of mitochondrial folate enzymes, and MTHFD2 in particular, in proliferation of tumor cells. MTHFD2 has dehydrogenase and cyclohydrolase activities, generating 10-formyl tetrahydrofolate from 5-10-methylene tetrahydrofolate (the source of formate required for purine biosynthesis), as well as nicotinamide adenine dinucleotide phosphate (NADP/NADPH) (FIG. 1). Thus, MTHFD2 is an important enzyme for the generation of the formate required for macromolecular synthesis and protection from reactive oxygen species (ROS). Knockdown studies of MTHFD2 by shRNA and CRISPR have shown that MTHFD2 may be a selective anti-tumor target.
Inhibition of MTHFD2 results in folate deficiency, contributing to the inhibition of cancer cell growth, as methylene tetrahydrofolate (MTHF) accumulates and causes a relative deficiency of other folate coenzymes. Folate depletion of cells lacking MTHFD2 further enhances the observed cytotoxicity. However, folate deficient diets are difficult for patients to maintain and the dietary approach may take weeks or months to achieve the desired folate depletion.
Thus, there is a need in the art for a method of treating, preventing, and/or ameliorating disorders involving overexpression of MTHFD2. The present disclosure addresses this need.
BRIEF SUMMARY OF THE INVENTION
The present disclosure relates in part to a method of treating, preventing, and/or ameliorating a disease or disorder involving overexpression of methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) in a subject, the method comprising administering to the subject a pharmaceutically effective amount of a MTHFD2 inhibitor and a pharmaceutically effective amount of a folate-depleting agent. In certain embodiments, the disease or disorder is a proliferative disease or disorder. In certain embodiments, the proliferative disease or disorder is cancer.
The present disclosure further relates to a composition comprising a MTHFD2 inhibitor and a folate-depleting agent, and pharmaceutical compositions thereof.
The present disclosure further relates to an immunoconjugate comprising an antibody, or antigen binding fragment thereof, conjugated to a MTHFD2 inhibitor. In certain embodiments, the immunoconjugate selectively targets a cancer cell overexpressing MTHFD2.
BRIEF DESCRIPTION OF THE FIGURES
The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments of the present application.
FIG. 1 provides a schematic diagram of mitochondrial enzymes as a source for one- carbon units in the synthesis of purines and thymidylate. Abbreviations: THF, tetrahydrofolate, CH2-THF 5,10-methylenetetrahydrofolate; CH- THF, 5,10- methenyltetrahydrofolate; CHO-THF, 10-formyl tetrahydrofolate; CH3-THF, 5-methyl tetrahydrofolate.
FIGs. 2A-2B provide a schematic diagram of the carboxypeptidase G2 reaction for folic acid (FIG. 2A) and methotrexate (FIG. 2B).
FIGs. 3A-3B show a western blot analysis of MCF7 and T47D cells after shRNA MTHFD2 knockdown. 50 pg of protein was loaded in each well of 12% SDS-PAGE. FIG. 3 A: MCF7-pLKO-ctrl (MCF7 cells with empty vector), MCF7-MTHFD2sh-551cl and 553cl (clones transfected with MTHFD2 knockdown plasmid). FIG. 3B: T47D Ctrl and T47D- shMTHFD2 (clone transfected with MTHFD2 shRNA plasmid). FIGs. 3C-3D show that MTHFD2 knockdown inhibits growth of two breast cancer cell lines. FIG. 3C: MCF7-pLKO-ctrl, MCF7-MTHFD2sh-553cl, and MCF7-MTHFD2sh- 553cl 1. FIG. 3D: T47D Ctrl and T47D-shMTHFD2. The experiment was done in triplicate and values are represented by mean with standard mean deviation. * represents p<Q.Q5.
FIGs. 3E-3H show the results of the colony assay wherein MTHFD2 knockdown results in growth inhibition on day 8 in MCF7 cells (FIG. 3E and FIG. 3G) and T47D cells (FIG. 3F and FIG. 3H). The experiment was done in triplicate and values are represented by mean with standard mean deviation. ** represents /?<0.005; *** represents <0.01.
FIG. 4A provides the results of the apoptosis check. MCF7 and T47D cells (controls and MTHFD2 knockdown) were cultured in media for day 8 and then incubated with V-FITC and PI and analyzed using flow cytometry. The lower left quadrant shows cells with are negative for both PI and annexin V-FITC; the upper left quadrant shows only PI positive cells, which are necrotic; the lower right quadrant shows annexin positive cells (early apoptotic); and the upper right quadrant shows annexin and PI positive cells (late apoptosis cells). The percentage of necrotic, early, and late apoptotic cells are represented in each quadrant.
FIG. 4B provides a cell cycle phase analysis. MCF7 and T47D cells (controls and MTHFD2 knockdown) cultured in media for 8 days were analyzed for cell cycle analysis using PI staining and flow cytometry. The percentage of cells in each cell cycle phase were analyzed using FlowJo software.
FIGs. 5A-5B show that MTHFD2 knockdown cells in folate depleted media enhances growth inhibition in two breast cancer cell lines. FIG. 5A: MCF7-pLKO-ctrl (MCF7 cells with empty vector), MCF7-MTHFD2sh-553cl (clone transfected with MTHFD2 shRNA plasmid). FIG. 5B: T47D Ctrl and T47D-shMTHFD2 (clone transfected with MTHFD2 shRNA plasmid). The experiment was done in triplicate and values are represented by mean with standard mean deviation. * represents /?<0.05; ** represents /?<0.03; *** represents <0.01.
FIGs. 6A-6C show that MTHFD2 knockdown cells in the presence of CPG2 (10 units) enhances growth inhibition of two breast cancer cell lines. FIG. 6A: MCF7-pLKO-ctrl (MCF7 cells with empty vector), MCF7-MTHFD2sh-553cl (clone transfected with MTHFD2 shRNA plasmid). FIG. 6B: T47D Ctrl and T47D-shMTHFD2 (clone transfected with MTHFD2 shRNA plasmid). FIG. 6C shows the remaining CPG2 activity in the media at different time points (CPG2 stability check). The experiment was done in triplicate and values are represented by mean with standard mean deviation. * represents p<Q.Q5. FIGs. 7A-7B show that the combination of MTHFD2 knockdown and CPG2 enhanced anti-tumor effect against prostate cancer xenograft. Nude mice were inoculated subcutaneously with 5-million PC3 cells (control or MTHFD2 knockdown cells) with matrigel (1 : 1, v/v ratio) in the right flank. After tumors were palpable (100-200 mm3), animals were randomized into four groups (n=10) and treatments were initiated. The mice were administered the CPG2 (glucarpidase) intraperitoneally thrice per week for 8 weeks (10 units in 100 pL). Tumor size and body weight was measured twice a week and tumor volume was calculated using (L)(W)A2/2 (FIG. 7A). MTHFD2 knockdown mice treated with CPG2 showed weakness as shown by body weight measurement (FIG. 7B). The values indicate average tumor volume ± standard error, /?-values below 0.05 are considered significant.
FIGs 8A-8B show that CPG2 has moderate anti-cancer activity against MCF7 breast cancer tumors. 4 xlO6 MCF7 cells were injected subcutaneously in the abdominal flanks of nude female mice. Once the tumor was palpable, mice were randomized into 4 groups (n=5). Mice were injected then with CPG2 (10 units in 100 pL) twice a week. Control mice received no treatment. Tumor size was measured twice a week, tumor volume was measured using (L)(W)A2/2 (FIG. 8 A) p=0.188 (on the last day of treatment). Data was plotted and SEM was calculated. Mice treated with CPG showed moderate loss of body weight after 18 days of treatment (FIG. 8B). Mice bearing MCF7 MTHFD2 knockdown cells didn't grow any tumor for up to 4 months.
FIGs. 9A-9B shows that CPG2 (glucarpidase) enhanced the anti-tumor effect in combination with MTHFD2 inhibitor (DS18561882) against MDA-MB-231 (TNBC) xenograft. Nude mice were inoculated s.c. with 5 million MDA-MB-231 cells with Matrigel (1 : 1, v/v ratio) in the right flank. The mice were treated with the MTHFD2 inhibitor (DS18561882) dose of 250 mg/kg (200 pL), p.o. twice a day for 8 days. Control groups (saline and CPG2 group) also received vehicle (10% DMSO, 40% PEG-300, 5% Tween-80, and 45% saline) (200 pL), p.o. twice a day for 8 days. Tumor size was measured and used to calculate tumor volume, wherein the values indicate average tumor volume ± standard error p-values (FIG. 9A). Body weight as also measured and the mice treated with CPG2 and DS 18561882 showed weakness, as indicated by the body weight measurement (FIG. 9B).
DETAILED DESCRIPTION OF THE INVENTION
Reference will now be made in detail to certain embodiments of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.
Carboxypeptidase G2 (CPG2) is a dimeric zinc-dependent exopeptidase. Enzymatic depletion of folates with CPG2, a treatment that is approved to inactivate methotrexate (MTX) in case of overdose, has been shown to enhance the cytotoxicity of cells with a MTHFD2 knockdown. In vitro results demonstrated that MTHFD2 knockdown in cells caused an antiproliferative effect that was enhanced by folate depletion, when combined with folate-depleting enzyme, CPG2.
The present disclosure relates in part to a method of treating, preventing, and/or ameliorating a disorder involving overexpression of methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of a MTHFD2 inhibitor and a pharmaceutically effective amount of a folate-depleting agent.
In certain embodiments, the disease or disorder involving overexpression of MTHFD2 is selected from the group consisting of bipolar disorder, major depressive disorder, mitochondrial neurodegeneration, and a proliferative disease or disorder. In certain embodiments, the disease or disorder involving overexpression of MTHFD2 is a proliferative disorder. In certain embodiments, the proliferative disorder is cancer. In certain embodiments, the cancer is at least one of brain cancer, breast cancer, colon cancer, rectal cancer, kidney cancer, prostate cancer, liver cancer, thyroid cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer, ventricular cancer, melanoma, lymphatic cancer, and acute leukemia.
In certain embodiments, the MTHFD2 inhibitor and the folate-depleting agent are each independently administered to the subject by at least one route selected from the group consisting of nasal, inhalational, topical, oral, buccal, rectal, pleural, peritoneal, vaginal, intramuscular, subcutaneous, transdermal, epidural, intratracheal, otic, intraocular, intrathecal, and intravenous routes. In certain embodiments, the MTHFD2 inhibitor is administered to the subject orally 1 to 4 times per day. In certain embodiments, the MTHFD2 inhibitor is administered to the subject 1 time per day. In certain embodiments, the MTHFD2 inhibitor is administered to the subject 2 times per day. In certain embodiments, the MTHFD2 inhibitor is administered to the subject 3 times per day. In certain embodiments, the MTHFD2 inhibitor is administered to the subject 4 times per day. In certain embodiments, the MTHFD2 inhibitor is administered by continuous or intermittent intravenous infusion.
In certain embodiments, the MTHFD2 inhibitor is (4-(3 -amino- l-hydroxy-9-oxo- 5,6,6a,7-tetrahydroimidazo[l,5-f]pteridin-8(9H)-yl)benzoyl)-L-glutamic acid, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof. The method of claim 1, wherein the MTHFD2 inhibitor is (3R,4R,6R,E)-8- ((2S, 3 S,7R,1 OR, 11R,E)-10,11 -dihydroxy-3, 7-dimethyl-12-oxooxacyclododec-8-en-2-yl)-3- methoxy-4,6-dimethyl-5-oxonon-7-enoic acid, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
In certain embodiments, the MTHFD2 inhibitor is a tricyclic coumarin selected from the group consisting of:
4-(5-oxo-l,3,4,5-tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)benzoic acid; N-(2-chloro-4-(7-methyl-8-(4-methylpiperazin-l-yl)-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)phenyl)methanesulfonamide;
N-(2-chloro-4-(7-methyl-8-(4-methylpiperazin-l-yl)-5-oxo- 1,3,4, 5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)phenyl)cyclopropanesulfonamide;
N-(4-(7-methyl-8-(4-methylpiperazin-l-yl)-5-oxo-l,3,4,5-tetrahydro-2H-chromeno[3,4- c]pyridine-3-carbonyl)-2-(tri fluoromethoxy )phenyl)methanesulfonamide;
(S)-N-(2-chloro-4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo- 1,3,4, 5-tetrahydro- 2H-chromeno[3,4-c]pyridine-3-carbonyl)phenyl)methanesulfonamide;
(S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)m ethanesulfonamide;
(S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo- 1,3,4, 5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethyl)phenyl)methanesulfonamide;
(S)-N-(2-chl oro-4-(8-(3,4-dimethylpiperazin-l-yl)-7,10-dimethyl-5-oxo-l, 3,4,5- tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)phenyl)methanesulfonamide;
(S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7,10-dimethyl-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)m ethanesulfonamide;
(S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo- 1,3,4, 5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)ethanesulfonamide;
(S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7,10-dimethyl-5-oxo- 1,3,4, 5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)ethanesulfonamide;
(S)-N-(4-(8-(3-ethyl-4-methylpiperazin-l-yl)-7-methyl-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)m ethanesulfonamide;
N-(4-(7-methyl-5-oxo-8-(3,3,4-trimethylpiperazin-l-yl)-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)m ethanesulfonamide; (S)-N-(4-(7-methyl-8-(3-methylpiperazin-l-yl)-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)methanesulfonamide; and N-(2-chloro-4-(7-methyl-5-oxo-8-((3R,5S)-3,4,5-trimethylpiperazin-l-yl)-l,3,4,5- tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)phenyl)methanesulfonamide; or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
In certain embodiments, the MTHFD2 inhibitor is (S)-N-(4-(8-(3,4- dimethylpiperazin-l-yl)-7-methyl-5-oxo- 1,3,4, 5-tetrahydro-2H-chromeno[3,4-c]pyridine-3- carbonyl)-2-(trifluoromethoxy)phenyl)methanesulfonamide or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
In certain embodiments, the MTHFD2 inhibitor is an immunoconjugate selectively targeting a cancer cell, comprising an antibody, or antigen binding fragment thereof, conjugated to an MTHFD2 inhibitor, optionally through a linker. In certain embodiments, the folate-depleting agent is an immunoconjugate selectively targeting a cancer cell, comprising an antibody, or antigen binding fragment thereof, conjugated to a carboxypeptidase, optionally through a linker. In certain embodiments, the antibody, or antigen binding fragment thereof, is selected from the group consisting of A5B7, F(ab’)2 A5B7, W14, MFE-23, adecatumumab, intetumumab, etaracizumab, glembatumumab vedotin, leronlimab, margetuximab, sacituzumab govitecan, and trastuzumab.
In certain embodiments, the folate-depleting agent is administered in advance of or contemporaneously with the administration of the MTHFD2 inhibitor. In certain embodiments, the folate-depleting agent is administered after the administration of the MTHFD2 inhibitor. In certain embodiments, the folate-depleting agent is administered by continuous or intermittent intravenous infusion.
In certain embodiments, the folate-depleting agent is a carboxypeptidase. In certain embodiments, the carboxypeptidase is carboxypeptidase G2 (CPG2) (SEQ ID NO: 1) or a biologically active fragment or derivative thereof. In certain embodiments, the subject is further administered at least one zinc supplement, which in non-limiting embodiments enhances CPG2 activity. In certain embodiments, the at least one zinc supplement is zinc gluconate. In certain embodiments, the carboxypeptidase shares at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology with CPG2 (SEQ ID NO: 1). In certain embodiments, the carboxypeptidase is modified with at least one modification to reduce immunogenicity or increase half-life in the subject. In certain embodiments, the at least one modification is selected from the group consisting of fusion with human serum albumin and/or an IgG Fc domain, alkylation of at least one amide group, acetylation and/or alkylation of the N-terminus amino group, amidation of the C- terminus carboxy group, PEGylation, and phosphocholination with phosphatidylcholine. In certain embodiments, the PEGylation is introduced by site-specific or random bioconjugation techniques.
In certain embodiments, the subject is further administered at least one additional agent useful for treating, ameliorating, and/or preventing a proliferative disease or disorder, such as cancer. In certain embodiments, the at least one additional agent comprises at least one selected from the group consisting of olaparib, rucaparib, niraparib, atrezoizumab, avelumab, pembrolizumab, cisplatin, carboplatin, doxorubicin, bevacizumab, gemcitabine, topetecan, paclitaxel, docetaxel, etoposide, and nanoparticle albumin-bound paclitaxel. In certain embodiments, the at least one additional agent is co-administered with at least one of the MTHFD2 inhibitor and the folate-depleting agent. In certain embodiments, the at least one additional agent is co-formulated with at least one of the MTHFD2 inhibitor and the folate-depleting agent. In certain embodiments, the subject is a mammal. In certain embodiments, the mammal is a human.
The present disclosure relates in part to a pharmaceutical composition comprising a MTHFD2 inhibitor, a folate-depleting agent, and at least one pharmaceutically acceptable carrier. In certain embodiments, the MTHFD2 inhibitor is (S)-N-(4-(8-(3,4- dimethylpiperazin-l-yl)-7-methyl-5-oxo- 1,3,4, 5-tetrahydro-2H-chromeno[3,4-c]pyridine-3- carbonyl)-2-(trifluoromethoxy)phenyl)methanesulfonamide or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof. In certain embodiments, the folate-depleting agent is a carboxypeptidase. In certain embodiments, the carboxypeptidase is CPG2 or a biologically active fragment or derivative thereof.
In certain embodiments, the subject is administered a pharmaceutically effective amount of the composition of a pharmaceutical composition comprising a MTHFD2 inhibitor, a folate-depleting agent, and at least one pharmaceutically acceptable carrier. In certain embodiments, the MTHFD2 inhibitor is (S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7- methyl-5-oxo- 1,3,4, 5-tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)-2- (trifluoromethoxy)phenyl)methanesulfonamide or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof. In certain embodiments, the folate-depleting agent is a carboxypeptidase. In certain embodiments, the carboxypeptidase is CPG2 or a biologically active fragment or derivative thereof.
The present disclosure relates in part to an immunoconjugate comprising an antibody, or antigen binding fragment thereof, conjugated to a MTHFD2 inhibitor, optionally through a linker. In certain embodiments, the immunoconjugate selectively targets a cancer cell. In certain embodiments, the cancer cell overexpresses MTHFD2. In certain embodiments, the antibody, or antigen binding fragment thereof, is selected from the group consisting of A5B7, F(ab’)2 A5B7, W14, MFE-23, adecatumumab, intetumumab, etaracizumab, glembatumumab vedotin, leronlimab, margetuximab, sacituzumab govitecan, and trastuzumab.
Throughout this document, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not just about 0.1% to about 5%, but also the individual values e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement "about X to Y" has the same meaning as "about X to about Y," unless indicated otherwise. Likewise, the statement "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z," unless indicated otherwise.
In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.
Definitions
In this document, the terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. The statement "at least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B." In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference.
The term "about" as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range.
The term "bioconjugation" as used herein refers to a chemical strategy to form a stable covalent bond between two molecules, wherein at least one of said molecules is a biomolecule. Non-limiting examples of bioconjugations include the couplings of small molecule and protein, protein and protein, antibody and small molecule, protein and antibody, antibody and enzyme, and protein and oligosaccharide.
As used herein, a "disease" is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subject's health continues to deteriorate.
As used herein, a "disorder" in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject's state of health.
The term "folate-depleting agent" as used herein refers to any composition which, upon administration to a subject, results in reduction or elimination of folate in the subject by any mechanism, including but not limited to chemical modification or sequestration of folate.
The term "immunoconjugate" as used herein refers to a conjugate of an antibody or antigen fragment component with a small molecule therapeutic or a macromolecular biologic therapeutic or diagnostic agent through a covalent linkage. The therapeutic or diagnostic agent can comprise a radioactive or non-radioactive label. The antibodies that are used to prepare immunoconjugates include, but are not limited to, monoclonal antibodies, chimeric antibodies, humanized antibodies, and human antibodies. Linkers may be cleavable or non- cleavable. Non-limiting examples of cleavable linkers include disulfides, hydrazones, peptides, or thioethers. The linker may be directly conjugated to the antibody, i.e. covalently linked directly to an exposed amino acid residue on the surface of the antibody or antigen fragment. Therapeutic agents of the present disclosure comprise MTHFD2 inhibitors and folate-depleting agents.
The term "immunogenicity" as used herein refers to the propensity of a foreign substance to induce a humoral and/or cell-mediated immune response in the body of a human or other animal. The term "independently selected from" as used herein refers to referenced groups being the same, different, or a mixture thereof, unless the context clearly indicates otherwise. Thus, under this definition, the phrase "X1, X2, and X3 are independently selected from noble gases" would include the scenario where, for example, X1, X2, and X3 are all the same, where X1, X2, and X3 are all different, where X1 and X2 are the same but X3 is different, and other analogous permutations.
The term "knockdown" or "KD" as used herein refers to an experimental technique wherein the expression of one or more of an organism’s genes and/or translation of the corresponding RNA is reduced.
As used herein, a "prophylactic" or "preventive" treatment is a treatment administered to a subject who does not exhibit signs of a disease or disorder or exhibits only early signs of the disease or disorder for the purpose of decreasing the risk of developing pathology associated with the disease or disorder.
As used herein, the language "pharmaceutically effective amount," "therapeutically effective amount," or "effective amount" refers to a non-toxic but sufficient amount of the composition used in the practice of the invention that is effective to treat, prevent, and/or ameliorate a disease or disorder in the body of a mammal. The desired treatment may be prophylactic and/or therapeutic. That result may be reduction and/or alleviation of the signs, symptoms, or causes of a disease or disorder, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
The term "PCylation" as used herein refers to phosphocholination, a process whereby the nucleophilic residues of a protein are covalently linked to a choline molecule by a phosphodiester bond.
The term "PEGylated" or "PEGylation" as used herein refers to the process of covalent and/or non-covalent attachment or amalgamation of polyethylene glycol polymer chains to molecules to the surface of a macromolecule (i.e. amino acids on the surface of a protein).
As used herein, the term "pharmaceutical composition" or "composition" refers to a mixture of at least one compound useful within the invention with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a subject.
As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound useful within the invention, and is relatively non-toxic, i.e., the material may be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the subject such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the invention, and not injurious to the subject. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the invention, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions. The "pharmaceutically acceptable carrier" may further include a pharmaceutically acceptable salt of the compound useful within the invention. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.
As used herein, the language "pharmaceutically acceptable salt" refers to a salt of the administered compound prepared from pharmaceutically acceptable non-toxic acids and/or bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates (including hydrates) and clathrates thereof.
The term "room temperature" as used herein refers to a temperature of about 15 °C to about 28 °C.
As used herein, the terms "subject" and "individual" and "patient" can be used interchangeably and may refer to a human or non-human mammal or a bird. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. In certain embodiments, the subject is human.
The term "substantially" as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. The term "substantially free of' as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that the composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less. The term "substantially free of can mean having a trivial amount of, such that a composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%.
As used herein, the term "treating" means ameliorating the effects of, or delaying, halting or reversing the progress of a disease or disorder. The word encompasses reducing the severity of a symptom of a disease or disorder and/or the frequency of a symptom of a disease or disorder.
The term “ubiquitinylating agent” as used herein refers to any entity which facilitates the formation of a covalent linkage between ubiquitin and a biomolecule of interest. The ubiquitinylating agent may be a ubiquitin ligase, or E3 enzyme, or an isoform thereof. The ubiquitinylation may comprise monoubiquitinylation or polyubiquitinylation.
Certain abbreviations used herein follow: CEE-THF, 5,10-methylenetetrahydrofolate; CH-THF, 5,10-methenyltetrahydrofolate; CHO-THF, 10-formyl tetrahydrofolate; CH3-THF, 5-methyl tetrahydrofolate; CPG2, carboxypeptidase G2; CRISPR, clustered regularly interspaced short palindromic repeats; FITC, fluorescein isothiocyanate; MTHF, methylenetetrahydrofolate; MTHFD2, methylenetetrahydrofolate dehydrogenase 2; MTX, methotrexate; NADP, nicotinamide adenine dinucleotide phosphate; PEG, polyethylene glycol; PI, propidium iodide; ROS, reactive oxygen species; shRNA, short hairpin RNA; THF, tetrahydrofolate.
Compounds and Compositions
Small molecules
Examples of suitable MTHFD2 include those disclosed in U.S. Patent No. 10,774,087 and related applications, Raze Therapeutics, Inc. Patent Application Publications US2018/0370972, WO2017/023894, and Wayne State University Patent Application Publication WO2019/046612, which are incorporated herein by reference in their entireties.
In certain embodiments, the MTHFD2 inhibitor is
Figure imgf000016_0001
amino-l-hydroxy-9-oxo-5,6,6a,7- tetrahydroimidazo[l,5-f]pteridin-8(9H)-yl)benzoyl)-L-glutamic acid (LY345899), or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
In certain embodiments, the MTHFD2 inhibitor is
Figure imgf000016_0002
dihydroxy-3, 7-dimethyl-12-oxooxacyclododec-8-en-2-yl)-3-methoxy-4,6-dimethyl-5- oxonon-7-enoic acid (carolacton), or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
In certain embodiments, the MTHFD2 inhibitor is a tricyclic coumarin. In certain embodiments, the tricyclic coumarin is a l,2,3,4-tetrahydrochromeno[3,4-c]-pyridin-5-one. In certain embodiments, the MTHFD2 inhibitor is a l,2,3,4-tetrahydrochromeno[3,4-c]- pyridin-5-one. In certain embodiments the MTHFD2 inhibitor is
Figure imgf000016_0003
oxo- 1,3,4, 5-tetrahydro-2H-chromeno[3,4-c]pyridine-3- carbonyl)benzoic acid, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof. In certain embodiments the
MTHFD2 inhibitor i
Figure imgf000017_0001
(4-methylpiperazin-l-yl)-5-oxo-l,3,4,5-tetrahydro-2H-chromeno[3,4-c]pyridine-3- carbonyl)phenyl)methanesulfonamide, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof. In certain embodiments the tricyclic coumarin
Figure imgf000017_0002
chloro-4-(7-methyl-8-(4-methylpiperazin-l-yl)-5-oxo-l,3,4,5-tetrahydro-2H-chromeno[3,4- c]pyridine-3-carbonyl)phenyl)cyclopropanesulfonamide, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof. In certain embodiments the MTHFD2 inhibitor i
Figure imgf000017_0003
(4-(7-methyl-8-(4-methylpiperazin-l-yl)-5-oxo-l,3,4,5-tetrahydro-2H-chromeno[3,4- c]pyridine-3 -carbonyl)-2-(trifluorom ethoxy )phenyl)methanesulfonamide, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof. In certain embodiments the MTHFD2 inhibitor is
Figure imgf000017_0004
-chloro-4-(8-(3,4-dimethylpiperazin-l-yl)-
7-methyl-5-oxo-l,3,4,5-tetrahydro-2H-chromeno[3,4-c]pyridine-3- carbonyl)phenyl)methanesulfonamide, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof. In certain embodiments the MTHFD2 inhibitor
Figure imgf000018_0001
(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo-l,3,4,5-tetrahydro-2H-chromeno[3,4- c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)methanesulfonamide (z.e., DS 18561882), or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof. In certain embodiments, the MTHFD2 inhibitor is
Figure imgf000018_0002
-dimethylpiperazin-l-yl)-7-methyl-
5-oxo-l,3,4,5-tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)-2-
(trifluoromethyl)phenyl)methanesulfonamide, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof. In certain embodiments, the MTHFD2 inhibitor i
Figure imgf000018_0003
chloro-4-(8-(3,4-dimethylpiperazin-l-yl)-7,10-dimethyl-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)phenyl)methanesulfonamide, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof. In certain embodiments, the MTHFD2 inhibitor
Figure imgf000018_0004
(S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7,10-dimethyl-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)methanesulfonamide, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof. In certain embodiments, the MTHFD2 inhibitor is
Figure imgf000019_0001
-dimethylpiperazin-l-yl)-7- methyl-5-oxo- 1,3,4, 5-tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)-2- (trifluoromethoxy)phenyl)ethanesulfonamide, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof. In certain embodiments, the MTHFD2 inhibitor
Figure imgf000019_0002
(4-(8-(3 ,4-dimethylpiperazin- 1 -yl)-7, 10-dimethyl-5 -oxo- 1 , 3 ,4, 5 -tetrahy dro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)ethanesulfonamide, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof. In certain embodiments, the MTHFD2 inhibitor is
Figure imgf000019_0003
-ethyl-4-methylpiperazin-l-yl)-7- methyl-5-oxo- 1,3,4, 5-tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)-2-
(trifluoromethoxy)phenyl)methanesulfonamide, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof. In certain embodiments, the MTHFD2 inhibitor i
Figure imgf000020_0001
methyl-5-oxo-8-(3,3,4-trimethylpiperazin-l-yl)-l,3,4,5-tetrahydro-2H-chromeno[3,4- c]pyridine-3 -carbonyl)-2-(trifluorom ethoxy )phenyl)methanesulfonamide, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof. In certain embodiments, the MTHFD2 inhibitor is
Figure imgf000020_0002
methyl-8-(3-methylpiperazin-l-yl)-5- oxo-l,3,4,5-tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)-2-
(trifluoromethoxy)phenyl)methanesulfonamide, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof. In certain embodiments, the MTHFD2 inhibitor i
Figure imgf000020_0003
chloro-4-(7-methyl-5-oxo-8-((3R,5S)-3,4,5-trimethylpiperazin-l-yl)-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)phenyl)methanesulfonamide, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
In certain embodiments, the MTHFD2 inhibitor is (S)-N-(4-(8-(3,4- dimethylpiperazin-l-yl)-7-methyl-5-oxo- 1,3,4, 5-tetrahydro-2H-chromeno[3,4-c]pyridine-3- carbonyl)-2-(trifluoromethoxy)phenyl)methanesulfonamide, also referred to herein as DS18561882.
Methods suitable for the synthesis of DS18561882, and analogs thereof including tricyclic coumarins contemplated herein, are described in Daiichi and Kawai et al. (J. Med. Chem. 2019, 62:10204-10220). A compound with greater potency than DS18561882, but with reduced growth inhibition activity as compared to DS 18561882, has been reported in Kawai et al. (ACS Med. Chem. Lett. 2019, 10:893-898). Further MTHFD2 inhibitors may be identified by employing the methods described in Jain (W02014/15068).
In certain embodiments, MTHFD2 inhibitors have a greater than 5:1 selectivity for MTHFD2 inhibition over MTHFD1 inhibition. In other embodiments, MTHFD2 inhibitors have a greater than 10: 1 selectivity for MTHFD2 inhibition over MTHFD1 inhibition. In yet other embodiments, MTHFD2 inhibitors have a greater than 50: 1 selectivity for MTHFD2 inhibition over MTHFD1 inhibition. In yet other embodiments, MTHFD2 inhibitors have a greater than 100: 1 selectivity for MTHFD2 inhibition over MTHFD1 inhibition. In certain embodiments, the MTHFD2 inhibitor is DS18561882 and the MTHFD2:MTHFD1 selectivity is greater than 90: 1.
Enzymes
In certain embodiments the folate-depleting agent is a carboxypeptidase. In certain embodiments, the carboxypeptidase is carboxypeptidase G2 (CPG2) or a biologically active fragment or derivative thereof. In certain embodiments, other glucarpidase drugs are substituted for CPG2. In certain embodiments, the amino acid sequence of the carboxypeptidase is modified to improve immunogenicity and half-life in a human. In certain embodiments, the carboxypeptidase shares at least 85% homology with CPG2 (SEQ ID NO: 1). In certain embodiments, the carboxypeptidase shares at least 90% homology with CPG2 (SEQ ID NO: 1). In certain embodiments, the carboxypeptidase shares at least 95% homology with CPG2 (SEQ ID NO: 1). In certain embodiments, the carboxypeptidase shares at least 99% homology with CPG2 (SEQ ID NO: 1). In certain embodiments, the carboxypeptidase is modified to improve immunogenicity and half-life in a human. In other embodiments, the carboxypeptidase may be fused (on the N-terminus and/or C-terminus) with human serum albumin and/or an IgG Fc domain, alkylated on at least one amide group, acetylated and/or alkylated on the N-terminus amino group, amidated on the C-terminus carboxy group, PEGylated, and/or phosphocholinated with phosphatidylcholine.
Bioconjugates
The present disclosure relates in part to immunoconjugates comprising an antibody, or an antigen binding fragment thereof, covalently conjugated to a MTHFD2 inhibitor or a folate-depleting agent through a linker.
An antibody fragment can be prepared by known methods, for example, as disclosed by Goldenberg, U.S. Patent Nos. 4,036,945 and 4,331,647 and references contained therein. Another form of an antibody fragment is a peptide coding for a single complementary- determining region (CDR). A CDR is a segment of the variable region of an antibody that is complementary in structure to the epitope to which the antibody binds and is more variable than the rest of the variable region. CDR peptides can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, using the polymerase chain reaction to synthesize the variable region from RNA of antibodyproducing cells.
In certain embodiments the antibody or antigen is selected from the group consisting of A5B7 (P-hCG targeting), F(ab’)2 A5B7, W14 (CEA targeting), MFE-23 (CEA targeting), adecatumumab (EpCAM targeting), intetumumab (CD51 targeting), etaracizumab (integrin avp3 targeting), glembatumumab vedotin (GPNMB targeting), leronlimab (CCR5 targeting), margetuximab (HER2 targeting), sacituzumab govitecan (TROP-2 targeting), and trastuzumab (HER2/neu targeting) (J. Natl. Cancer Inst. 1996, 88(3-4): 153-165; Eur. J. Nucl. Med. Mol. Imaging. 2004, 31(8): 1090-1096; Cancer Res. 1996, 56:3287-3292).
The linkers (and the therapeutic agents bound to said linkers) of the present disclosure may be directly conjugated to the antibody, i.e. covalently linked directly to the conjugated antibody or antigen fragment. The covalent linkage may occur directly to one of the amino acids comprising the antibody or antigen backbone, ideally located within one of the constant domains, as opposed to within the variable domains. Such amino acids may be naturally occurring (e.g. a naturally occurring lysine or cysteine residue), or the antibody or antigen may be artificially mutated (e.g. a non-naturally occurring lysine or cysteine residue) in order to provide an optimal binding site with minimal steric hindrance for the linker to bind to. Alternatively, the linker can be bound to a chemical moiety (e.g. bound to an N-glycan) found on post-translationally modified antibodies and/or antigen fragments.
The covalent linkages in the present immunoconjugates may comprise a cleavable linking moiety, for example, a Val-Cit linker, which is cleavable by Cathepsin B inside the lysosome. Other cleavable linking moieties may comprise a Phe-Lys linker, which is also cleavable by Cathespin B. Other examples of cleavable linking moieties include disulfide (S- S) bridges, which are cleavable in reductive (i.e. intracellular environment). Immunoconjugates of the present disclosure may also utilize direct attachment of the linker to any of a number of nucleophile amino acid residue side chains, including thiols, amines, and alcohols via acylation to afford thioesters, amides, and esters, respectively. An overview of cleavable linking moieties which may be suitable in the context of the present disclosure are provided in Leriche et al. (Bioorg. Med. Chem. 2012, 20(2):571-582), which is incorporated herein by reference in its entirety.
In certain embodiments, the linker comprises a cleavable linking moiety. In certain embodiments, the linker is covalently bound to an exposed amino acid residue on the surface of the antibody or antigen. In certain embodiments, the exposed amino acid residue comprises a cysteine. In certain embodiments, the linker is covalently bound to the cysteine through sulfhydryl-maleimide coupling. In certain embodiments, the exposed amino acid residue is a lysine. In certain embodiments, the linker is covalently bound to the lysine through am amide linkage by acylation. In certain embodiments, the linker is PEG- containing. In certain embodiments, the cleavable linking moiety comprises a Val-Cit moiety. In certain embodiments, the linking moiety comprises a Phe-Lys moiety. In certain embodiments the linker is non-cleavable.
In certain embodiments, the linker comprises a first linking component and a second linking component. In certain embodiments, the first linking component is conjugated to a surface of an antibody according to any aspect of the present disclosure. In certain embodiments, the first linking component is linked to the second linking component. In certain embodiments, the first linking component is linked to the second linking component through click chemistry. In certain embodiments, the first linking component is linked to the second linking component through a triazole moiety. In certain embodiments, the first linking component is PEG-containing. In certain embodiments, the second linking component is PEG-containing. In certain embodiments, the first and second linking component are PEG-containing. In certain embodiments, the first and/or second linking component contain between 1 and 10 PEG units. In certain embodiments, the second linking component comprises a cleavage linking moiety according to any aspect of the present disclosure. In certain embodiments, the second linking component comprises a therapeutic agent according to any aspect of the present disclosure.
In certain embodiments, the antibody or antigen binding fragment is conjugated to a carboxypeptidase. In certain embodiments, the antibody or antigen binding fragment is conjugated to CPG2.
In certain embodiments, the antibody or antigen binding fragment is conjugated to a MTHFD2 inhibitor. In certain embodiments, the MTHFD2 inhibitor is (4-(3 -amino- 1- hydroxy-9-oxo-5,6,6a,7-tetrahydroimidazo[l,5-f]pteridin-8(9H)-yl)benzoyl)-L-glutamic acid (LY345899) or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof. In certain embodiments, the MTHFD2 inhibitor is (3R,4R,6R,E)-8-((2S,3S,7R,10R,l 1R,E)-1O,11 -dihydroxy-3 J-dimethyl- 12- oxooxacyclododec-8-en-2-yl)-3-methoxy-4,6-dimethyl-5-oxonon-7-enoic acid (carolacton) or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof. In certain embodiments, the MTHFD2 inhibitor is a tricyclic coumarin or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof. In certain embodiments, the MTHFD2 inhibitor is (S)-N-(4-(8-(3,4- dimethylpiperazin-l-yl)-7-methyl-5-oxo- 1,3,4, 5-tetrahydro-2H-chromeno[3,4-c]pyridine-3- carbonyl)-2-(trifluoromethoxy)phenyl)methanesulfonamide or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
In certain embodiments, the MTHFD2 inhibitor is conjugated to a biomolecule suitable for the recruitment of an endogenous ubiquitinylating agent. In certain embodiments, recruitment of the ubiquitinylating agent results in ubiquitinylation of MTHFD2. In certain embodiments, the ubiquitinylated MTHFD2 is degraded by the proteasome. Biomolecular constructs suitable for conjugation with the MTHFD2 inhibitors of the present disclosure are described by Li et al. (J. Hematol. Oncol. 2020, 13:50). Such constructs may be of particular utility in the treatment of leukemia and/or colon cancer (J. Exp. Med. 2016, 213(7): 1285-1306).
Additional therapeutics
In certain embodiments, the combination therapy of the present invention is combined with additional therapies exploiting differences in tumor tissue. In certain embodiments, the method of the present disclosure may further comprise administration of one or more PARP inhibitors, such as Lynparza (olaparib) and Rubraca (rucaparib) and Zejula (niraparib); and check point inhibitors such as Tecentriq (atezoizumab) and Bavercio (avelumab). Keytruda (pembrolizumab) has also been found to have some efficacy where high levels of PD-1 are found on T-lymphocytes and tumors.
In certain embodiments, the combination therapy of the present disclosure is combined with at least one additional therapeutic agent, non-limiting examples including cisplatin, carboplatin, doxorubicin, bevacizumab, gemcitabine, topetecan, paclitaxel, docetaxel, etoposide, and nanoparticle albumin-bound paclitaxel. In certain embodiments, the at least one additional therapeutic agent comprises administration of bevacizumab in combination with paclitaxel, PEGlylated liposomal doxorubicin, or topotecan.
The compounds of the invention may possess one or more stereocenters, and each stereocenter may exist independently in either the (R)- or (^-configuration. In certain embodiments, compounds described herein are present in optically active or racemic forms. The compounds described herein encompass racemic, optically-active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein. Preparation of optically active forms is achieved in any suitable manner, including by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically-active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase. In certain embodiments, a mixture of one or more isomer is utilized as the therapeutic compound described herein. In other embodiments, compounds described herein contain one or more chiral centers. These compounds are prepared by any means, including stereoselective synthesis, enantioselective synthesis and/or separation of a mixture of enantiomers and/ or diastereomers. Resolution of compounds and isomers thereof is achieved by any means including, by way of non-limiting example, chemical processes, enzymatic processes, fractional crystallization, distillation, and chromatography.
The methods and formulations described herein include the use of N-oxides (if appropriate), crystalline forms (also known as polymorphs), solvates, amorphous phases, and/or pharmaceutically acceptable salts of compounds having the structure of any compound of the invention, as well as metabolites and active metabolites of these compounds having the same type of activity. Solvates include water, ether (e.g., tetrahydrofuran, methyl tert-butyl ether) or alcohol e.g., ethanol) solvates, acetates and the like. In certain embodiments, the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, and ethanol. In other embodiments, the compounds described herein exist in unsolvated form.
In certain embodiments, the compounds of the invention exist as tautomers. All tautomers are included within the scope of the compounds recited herein.
In certain embodiments, compounds described herein are prepared as prodrugs. A "prodrug" is an agent converted into the parent drug in vivo. In certain embodiments, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically active form of the compound. In other embodiments, a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically or therapeutically active form of the compound.
In certain embodiments, sites on, for example, the aromatic ring portion of compounds of the invention are susceptible to various metabolic reactions. Incorporation of appropriate substituents on the aromatic ring structures may reduce, minimize or eliminate this metabolic pathway. In certain embodiments, the appropriate substituent to decrease or eliminate the susceptibility of the aromatic ring to metabolic reactions is, by way of example only, a deuterium, a halogen, or an alkyl group.
Compounds described herein also include isotopically-labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds described herein include and are not limited to 2H, 3H, UC, 13C, 14C, 36C1, 18F, 123I, 125I, 13N, 15N, 15O, 17O, 180, 32P, and 35 S. In certain embodiments, isotopically-labeled compounds are useful in drug and/or substrate tissue distribution studies. In other embodiments, substitution with heavier isotopes such as deuterium affords greater metabolic stability (for example, increased in vivo half-life or reduced dosage requirements). In yet other embodiments, substitution with positron emitting isotopes, such as nC, 18F, 15O and 13N, is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically-labeled compounds are prepared by any suitable method or by processes using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed.
In certain embodiments, the compounds described herein are labeled by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.
Salts
The compositions described herein may form salts with acids or bases, and such salts are included in the present invention. In certain embodiments, the salts are pharmaceutically acceptable salts. The term "salts" embraces addition salts of free acids or free bases that are compositions of the invention. The term "pharmaceutically acceptable salt" refers to salts that possess toxicity profiles within a range that affords utility in pharmaceutical applications. Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallinity, which have utility in the practice of the present invention, such as for example utility in process of synthesis, purification or formulation of compositions of the invention.
Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p- toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, P-hydroxybutyric, salicylic, galactaric and galacturonic acid.
Suitable pharmaceutically acceptable base addition salts of compositions of the invention include, for example, ammonium salts and metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N'-dibenzylethylene- diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N- methylglucamine) and procaine. Examples of pharmaceutically unacceptable base addition salts include lithium salts and cyanate salts. All of these salts may be prepared from the corresponding composition by reacting, for example, the appropriate acid or base with the composition.
Administration/Dosage/Formulations
Routes of administration of any of the compounds and/or compositions of the invention include oral, nasal, rectal, intravaginal, parenteral (e.g., IM, IV and SC), buccal, sublingual or topical. The regimen of administration may affect what constitutes an effective amount. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
Administration of the compositions of the present invention to a subject, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to treat the disorder involving overexpression of methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) in a subject. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the subject; the age, sex, and weight of the subject; and the ability of the therapeutic compound to treat the disease or disorder in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound useful within the invention is from about 1 and 5,000 mg/kg of body weight/per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.
Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.
In particular, the selected dosage level depends upon a variety of factors, including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well, known in the medical arts.
A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian may start doses of the compounds useful within the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
In certain embodiments, the carboxypeptidase is glucarpidase (CPG2) which is commercially available as VORAXAZE® from Protherics Medicine Limited/BTG Specialty Pharmaceuticals. The specified dose for administration to deplete patient methotrexate (MTX) in a rescue situation is 50 units/kg. In certain embodiments, the dose used to deplete folate in the subject is 50 units/kg. In other embodiments, the dose used to deplete folate in the subject is greater than 50 units/kg. In yet other embodiments, lower doses from 1 to 50 units/kg given over the course of days or weeks may be used to achieve folate depletion in the subject. A non-limiting example including CPG2 administration by injection/intravenous drop several times a week, for example 2, 3, or 4 times a week for 1 to 8 weeks.
In certain embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of a disorder involving overexpression of methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) in a subject.
In certain embodiments, the compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the invention comprise a therapeutically effective amount of a compound useful within the invention and a pharmaceutically acceptable carrier.
The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may 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 dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
In certain embodiments, the compositions of the invention are administered to the subject in dosages that range from one to five times per day or more. In other embodiments, the compositions of the invention are administered to the subject in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the invention varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the invention should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any subject are determined by the attending physical taking all other factors about the subject into account.
Compounds useful within the invention for administration may be in the range of from about 1 mg to about 10,000 mg, about 20 mg to about 9,500 mg, about 40 mg to about 9,000 mg, about 75 mg to about 8,500 mg, about 150 mg to about 7,500 mg, about 200 mg to about 7,000 mg, about 3050 mg to about 6,000 mg, about 500 mg to about 5,000 mg, about 750 mg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 50 mg to about 1,000 mg, about 75 mg to about 900 mg, about 100 mg to about 800 mg, about 250 mg to about 750 mg, about 300 mg to about 600 mg, about 400 mg to about 500 mg, and any and all whole or partial increments there between.
In certain embodiments, the dose of a compound useful within the invention is from about 1 mg and about 2,500 mg. In other embodiments, a dose of a compound useful within the invention used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in certain embodiments, a dose of a second compound, as described herein, is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments there between.
In certain embodiments, the present invention is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound useful within the invention, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, and/or ameliorate a disorder involving overexpression of methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) in a subject.
Granulating techniques are well known in the pharmaceutical art for modifying starting powders or other particulate materials of an active ingredient. The powders are typically mixed with a binder material into larger permanent free-flowing agglomerates or granules referred to as a "granulation." For example, solvent-using "wet" granulation processes are generally characterized in that the powders are combined with a binder material and moistened with water or an organic solvent under conditions resulting in the formation of a wet granulated mass from which the solvent must then be evaporated.
Melt granulation generally consists in the use of materials that are solid or semi-solid at room temperature (i.e. having a relatively low softening or melting point range) to promote granulation of powdered or other materials, essentially in the absence of added water or other liquid solvents. The low melting solids, when heated to a temperature in the melting point range, liquefy to act as a binder or granulating medium. The liquefied solid spreads itself over the surface of powdered materials with which it is contacted, and on cooling, forms a solid granulated mass in which the initial materials are bound together. The resulting melt granulation may then be provided to a tablet press or be encapsulated for preparing the oral dosage form. Melt granulation improves the dissolution rate and bioavailability of an active (i.e. drug) by forming a solid dispersion or solid solution.
U.S. Patent No. 5,169,645 discloses directly compressible wax-containing granules having improved flow properties. The granules are obtained when waxes are admixed in the melt with certain flow improving additives, followed by cooling and granulation of the admixture. In certain embodiments, only the wax itself melts in the melt combination of the wax(es) and additives(s), and in other cases both the wax(es) and the additives(s) will melt.
The present invention also includes a multilayer tablet comprising a layer providing for the delayed release of one or more compounds useful within the invention, and a further layer providing for the immediate release of a medication for a disorder involving overexpression of methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) in a subject. Using a wax/pH-sensitive polymer mix, a gastric insoluble composition may be obtained in which the active ingredient is entrapped, ensuring its delayed release.
Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents. For oral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gelcaps. The compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate. The tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients. Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.
The compounds for use in the invention may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein.
Oral Administration
For oral administration, the compositions of the invention may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropylmethylcellulose); fillers (e.g., cornstarch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrates (e.g., sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). If desired, the tablets may be coated using suitable methods and coating materials such as OP ADR Y™ film coating systems available from Colorcon, West Point, Pa. (e.g., OP ADR Y™ OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and OP ADR Y™ White, 32K18400). Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions. The liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxy benzoates or sorbic acid).
Parenteral Administration
For parenteral administration, the compositions of the invention may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and/or continuous infusion. Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulation agents such as suspending, stabilizing and/or dispersing agents may be used.
Additional Administration Forms
Additional dosage forms of this invention include dosage forms as described in U.S. Patents Nos. 6,340,475, 6,488,962, 6,451,808, 5,972,389, 5,582,837, and 5,007,790. Additional dosage forms of this invention also include dosage forms as described in U.S. Patent Applications Nos. 2003/0147952, 2003/0104062, 2003/0104053, 2003/0044466, 2003/0039688, and 2002/0051820. Additional dosage forms of this invention also include dosage forms as described in PCT Applications Nos. WO 03/35041, WO 03/35040, WO 03/35029, WO 03/35177, WO 03/35039, WO 02/96404, WO 02/32416, WO 01/97783, WO 01/56544, WO 01/32217, WO 98/55107, WO 98/11879, WO 97/47285, WO 93/18755, and WO 90/11757.
Controlled Release Formulations and Drug Delivery Systems
In certain embodiments, the formulations of the present invention may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.
The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.
For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds. As such, the compounds of the present disclosure may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.
In a preferred embodiment of the invention, the compounds useful within the invention are administered to a subject, alone or in combination with another pharmaceutical agent, using a sustained release formulation.
The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that may, although not necessarily, include a delay of from about 10 minutes up to about 12 hours.
The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.
The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.
As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.
As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.
Dosing
The therapeutically effective amount or dose of a compound of the present invention will depend on the age, sex and weight of the subject, the current medical condition of the subject and the nature of the disorder involving overexpression of methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) being treated. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
A suitable dose of a compound of the present invention may be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day. The dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses.
It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days.
The compounds for use in the method of the invention may be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosage for subjects undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of this invention and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction times, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing/oxi dizing agents, with art- recognized alternatives and using no more than routine experimentation, are within the scope of the present application.
It is to be understood that, wherever values and ranges are provided herein, the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, all values and ranges encompassed by these values and ranges are meant to be encompassed within the scope of the present invention. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application. The description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range and, when appropriate, partial integers of the numerical values within ranges. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 and so forth, as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.1, 5.3, 5.5, and 6. This applies regardless of the breadth of the range.
EXAMPLES The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the invention is not limited to these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein.
Materials and Methods
Animals
Nude mice were obtained from Taconic Biosciences, Inc (Germantown, NY).
Cell culture
MCF7 and T47D breast cancer cell lines originated from an invasive ductal carcinoma primary tumor and were established from metastatic pleural effusion. Both MCF7 and T47D are classified in the epithelial luminal A subtype and are hormonal sensitive, as both express the estrogen receptor (ER). Other hormonal receptors, like progesterone receptor (PR) is expressed only in T47D. Both cell lines lack the Herceptin 2 receptor (HER2). Thus, MCF7 is ER+, PR-, HER2-; and T47D is ER+, PR+, HER2-. PC3 cells represent advanced prostate cancer, are metastatic in nature, and do not respond to androgens.
MCF7, T47D, and PC3 cells were cultured in RPMI 1640 containing 10% fetal bovine serum in a 37 °C incubator with 5% CO2. Folate free media (RPMI 1640 with no folic acid) studies were performed using the dialyzed fetal bovine serum in media.
MTHFD2 knockdown by shRNA
500,000 cells/well in 6-well plates were transfected with a pLKOl MTHFD2 shRNA plasmid (GE Life Sciences, oligo MTHFD2sh359, MTHFD2sh966, and MTHFD2sh969) using the transfectamine-2000 reagent (Invitrogen). The medium was changed 24 h after transfection. After an additional 24-48 h, cells were subjected to selection in medium containing puromycin (Sigma Aldrich). Cells were cultured for two weeks in 0.5 pg/mL puromycin (MCF7) and 0.2 pg/mL puromycin (T47D) to select for cells expressing MTHFD2 shRNA. Knockdown of MTHFD2 was confirmed by western blot and cells were maintained in puromycin. The transfection was also confirmed by fluorescence microscopy by the presence of a GFP signal. Note'. MTHFD2 may enhance tumor growth, but may not be essential. Thus, inhibition or knockdown of MTHFD2 would be expected to slow tumor growth rather than completely inhibit tumor growth. The cytoplasmic enzyme and the mitochondrial housekeeping enzyme MTHFD2L may provide sufficient formate for a lower proliferation rate.
Western blotting
Cells were scraped into a microcentrifuge tube. After brief centrifugation, cell pellets were lysed in RIPA buffer containing a commercial protease inhibitor cocktail (Roche). After protein quantification by the Bradford assay (Bio-Rad Laboratories), proteins were resolved by 12% SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-Rad Laboratories). After blocking the membrane with 5% non-fat dry milk prepared in Tris buffered saline + 0.1% tween 20, the membrane was incubated with the desired primary antibody according to the manufacturer's directions at 4 °C overnight. The membrane was washed thrice in Tris buffered saline + 0.1% tween-20 and incubated for two hours at room temperature with the appropriate peroxidase-conjugated secondary antibody. Bands were visualized using an enhanced chemiluminescence kit (Pierce). Anti-MTHFD2 antibody was purchased from Cell Signaling Technology; Anti-tubulin and anti-mouse secondary antibodies were purchased from Santa Cruz Biotechnology.
Proliferation and colony assays
5,000 cells/well in a 24-well plates were seeded in 2 mL complete media for different amounts of time. The cells were washed twice with IX PBS and treated with 100 pL trypsin and an additional 900 pL of fresh media was used to collect the cells from the well. Cell viability was determined using the Vi-CELL™ Series Cell Viability Analyzer (Beckman Coulter, Carlsbad, CA).
10,000 cells/well in a 12-well plate were seeded in 2 mL complete media, without folate (RPMI 1640 without folic acid), having dialyzed fetal bovine serum for different amounts of time. The cells were washed twice with IX PBS and treated with 100 pL trypsin and an additional 900 pL fresh complete media (without folate) was used to collect the cells from the well. Cell viability was determined using the Vi-CELL™ Series Cell Viability Analyzer (Beckman Coulter, Carlsbad, CA).
500 MCF7 cells and 1500 T47D cells were plated with uniform distribution in a 6- well plate in 2 mL of media for 8 days. The cells were washed twice with IX PBS buffer, then stained with crystal violet blue containing fixing solution for 15-20 min. The excess dye was washed with water and air dried. Cells were observed using bright field microscopy and analyzed using ImageJ software to determine the number of colonies. Cell cycle analysis and apoptosis measurement
For cell cycle analysis, 106 cells were fixed in 1 mL of cold 70% ethanol. Cells were then washed with 3 mL of cold PBS, suspended in 100 pL PBS, and stained using 100 pL of PI/RNase staining solution (BD Pharmingen™). Cells were stained for 30 minutes at room temperature, then 300 pL PBS was added to the solution before analysis. In each cell, the suspension of cells was homogenous, and staining was analyzed at a low flow rate by flow cytometry with a FACSCalibur (BD Biosciences) instrument. At least 10,000 single cell events were acquired. FlowJo software (version 8.8.2; Watson algorithm) was used to estimate the reaction of cells in Gi, S, and G2/M phases.
For the measurement of apoptosis, 106 cells in 1 mL were washed twice with 3 mL of cold IX PBS. Cells were suspended in 100 pL IX Annexin V binding buffer (10X binding buffer: 0.1 M Hepes/NaOH, pH = 7.4; 1.4 M NaCl; 22 mM CaCh) and stained with 10 pL FITC annexin V and 10 pL PI/RNase staining solution (BD Pharmingen™) for 15 minutes at room temperature (in the dark). Next, 400 pL of IX binding buffer was added to the solution and the staining was analyzed. In each cell, the suspension of cells was homogenous, and staining was analyzed at a low flow rate by flow cytometry with FACSCalibur (BD Biosciences), 20,000-30,000 single cell events were acquired. BD FACSArray™ software was used to estimate the fraction of cells in different stages as shown in quadrants.
Enzyme activity
The CPG2 enzyme assay was performed as described in the literature (J. Bio. Chem. 1971, 246(23):7207-7213). Standard enzyme assay conditions: volume (1 mL), MTX (60 nmol), Tris-HCl buffer (pH = 7.3, 50 pmol), and ZnCh (100 nmol). Enzyme activity was assayed at 37 °C with a Beckman spectrophotometer. One unit of CPG2 enzyme activity is defined herein as the amount of enzyme sufficient to catalyze 1 pmol/min MTX under the assay conditions described herein.
Xenograft Studies (PC 3 cells)
Five million PC3 cells (control cells or MTHFD2 knockdown cells) with matrigel (1 : 1 v/v ratio) were injected subcutaneously in the abdominal right flanks of 40 nude mice. Once the tumor was palpable, mice were randomized and treated into four groups. One group of mice with PC3 control cells received saline and the other group was treated with CPG2 (10 units, 100 pL) intraperitoneally 3 times per week. Similarly, a group of mice with PC3 MTHFD2 knockdown cells received saline and another group was treated with CPG2 (10 units, 100 pL) intraperitoneally 3 times per week. Tumor size and body weight was measured twice a week and tumor volume was calculated using (L)(W)A2/2. Data was plotted and SEM was calculated.
Xenograft Studies (MCF7 cells)
4 million MCF7 cells were injected subcutaneously in the abdominal flanks of nude female mice. Once the tumor was palpable, mice were randomized into 4 groups (n=5). Mice were injected then with CPG2 (10 units in 100 pL) twice a week. Control mice received no treatment. Tumor size and body weight was measured twice a week and tumor volume was calculated using (L)(W)A2/2. Data was plotted and SEM was calculated.
Xenograft Studies (MDA-MB-231 cells)
5 million MDA-MB-231 cells were injected subcutaneously with matrigel (1 : 1, v/v ratio) in the right flank of nude mice. After tumors were palpable, animals were randomized into four groups (n=7-8) and treatments were initiated. The mice were administered the CPG2 (glucarpidase) intraperitoneally thrice a week at 10U in 100 pL or saline (100 pL). The mice were treated with the MTHFD2 inhibitor (DS 18561882) dose of 250 mg/kg (200 pL), p.o. twice a day for 8 days. Control groups (saline and CPG2) also received vehicle (10% DMSO, 40% PEG-300, 5% Tween-80, and 45% saline) (200 pL), p.o. twice a day for 8 days. Tumor size and body weight was measured and tumor volume was calculated.
Statistical analysis
All in vitro experiments were performed three times, and each experiment was done in triplicate. Statistical analysis was performed using Prism software (GraphPad). In all cases, ANOVA followed by two-tailed, unpaired Student t-tests were performed to analyze statistical differences between groups. P values of <0.05 were considered statistically significant.
Cancer treatment
Candidates for this study display a tumor which overexpresses MTHFD2 and is refractory to known effective therapies. The patient is Karnofsky stage (70 or above) and has measurable disease, wherein tumor tissue is available for evaluation of MTHFD2 levels. Initial doses of CPG2 are 50 mg/m2 q (per) 2 days x 7 treatments, together with a MTHFD2 inhibitor. Treatment response and toxicity is measured after 2 weeks and 4 weeks by physical exam and appropriate laboratory and imaging tests.
Example 1: MTHFD2 knockdown study in human breast cancer cell lines MCF-7 and T47D
Two human breast cancer cell lines, MCF-7 and T47D, which overexpress MTHFD2 were used in the study described herein. MTHFD2 knockdown was achieved using two oligos (MTHFD2sh966 and MTHFD2sh969) in MCF7 cells and by a single oligo (MTHFD2sh966) in T47D cells. The knockdown of MTHFD2 using different oligos in MCF7 and T47D cells was achieved with greater than 90% knockdown, as shown by western blot (FIGs. 3 A-3B). Following knockdown, the cell lines were established after two weeks of culturing in puromycin, a selective resistance marker present in pLKOl plasmids as well as GFP.
The growth rates of the two cell lines were compared with and without MTHFD2 knockdown and clearly demonstrated that the knockdown of MTHFD2 in MCF7 and T47D breast cancer cells slowed growth as compared to cell lines without knockdown (FIGs. 3C- 3D). The relative decrease in growth rates observed in MTHFD2 knockdown cell lines was further confirmed by colony assay, wherein a significant decrease in colony number was observed compared to the control on day 8 (FIG. 3E-3H). Growth inhibition was attributed to an increase in early apoptosis, wherein T47D and MCF7 demonstrated a five-fold and twofold increase, respectively, compared to controls.
Cell cycle analysis showed that, compared to the control, T47D cells with the MTHFD2 knockdown had no change in their cell cycle. In contrast, a S-G2 block was observed in the case of MCF7 cells with the MTHFD2 knockdown (FIGs. 4A-4B).
Folate depletion in media (used with dialyzed fetal bovine serum) showed significant growth inhibition in both MCF7 and T47D knockdown breast cancer cell lines (FIGs. 5 A- 5B). At a concentration of 10 units/mL, CPG2 alone had no effect on the growth of MCF7 cells and had a minimal effect on T47D cells. Cells with the MTHFD2 knockdown experienced growth inhibition to a greater extent in both MCF7 and T47D cell lines, as compared to controls, however the magnitude of growth inhibition was greater for MCF7 cells than T47D cells (FIGs. 6A-6C). Without wishing to be bound by theory, the greater effect of the combination in MCF7 cells, as compared to T47D cells, may be a result of the higher rate of proliferation (doubling time) of the MCF7 cells as compared to T47D cells. Example 2: Combination of MTHFD2 Knockdown and CPG2 administration shows enhanced anti-tumor effect against prostate cancer and breast cancer xenografts
Carboxypeptidase GS (CPG2) was found to possess significant anti-tumor activity in vivo in an animal study wherein CPG2 was administered against MTHFD2 knockdown PC3 xenograft mice. In these experiments, little to no inhibition of tumor growth was observed in mice without knockdown of MTHFD2 which were treated with CPG2. Similarly, mice with MTHFD2 knockdown, but without treatment with CPG2, demonstrated only modest tumor growth arrest. However, mice which MTHFD2 knockdown and CPG2 treatment demonstrated significant tumor growth arrest (FIGs. 7A-7B).
CPG2 was found to possess significant anti-tumor activity in vivo in an animal pilot study wherein CPG2 was administered against MTHFD2 overexpressed MCF7 xenograft (control) mice. In these experiments, MTHFD2 control mice treated with CPG2 showed moderate loss of body weight after 18 days, and mice bearing MCF7 MTHFD2 knockdown cells did not grow a tumor for up to 4 months (FIGs. 8A-8B).
Example 3: Combination of MHTFD2 inhibitor (DS18561882) and CPG2 (glucarpidase) shows tumor growth inhibition breast cancer xenograft
The combination of carboxypeptidase GS (CPG2) and DS18561882 was found to possess significant anti-tumor activity in vivo in an animal model wherein both CPG2 and DS18561882 were administered against MDA-MB-231 xenograft mice (FIGs. 9A-9B).
The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.
The terms and expressions employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the embodiments of the present application. Thus, it should be understood that although the present application describes specific embodiments and optional features, modification and variation of the compositions, methods, and concepts herein disclosed may be resorted to by those of ordinary skill in the art, and that such modifications and variations are considered to be within the scope of embodiments of the present application.
Sequence Listings:
SEQ ID NO : 1 carboxypeptidase G2 (CPG2 ) ALAQKRDNVLFQAATDEQPAVIKTLEKLVNIETGTGDAEGIAAAGNFLEAELKNLGFTVT RSKSAGLWGDNIVGKIKGRGGKNLLLMSHMDTVYLKGILAKAPFRVEGDKAYGPGIADD KGGNAVI LHTLKLLKE YGVRDYGT I TVLFNTDEEKGS FGSRDL I QEEAKLADYVLS FEPT SAGDEKLSLGTSGIAYVQVNITGKASHAGAAPELGVNALVEASDLVLRTMNIDDKAKNLR FNWT IAKAGNVSNI I PASATLNADVRYARNEDFDAAMKTLEERAQQKKLPEADVKVI VTR
GRPAFNAGEGGKKLVDKAVAYYKEAGGTLGVEERTGGGTDAAYAALSGKPVIESLGLPGF GYHSDKAEYVDISAIPRRLYMAARLIMDLGAGK
Enumerated Embodiments
The following exemplary embodiments are provided, the numbering of which is not to be construed as designating levels of importance:
Embodiment 1 provides a method of treating, preventing, and/or ameliorating a disease or disorder involving overexpression of methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) in a subject, the method comprising administering to the subject a pharmaceutically effective amount of a MTHFD2 inhibitor and a pharmaceutically effective amount of a folate-depleting agent.
Embodiment 2 provides the method of Embodiment 1, wherein the disease or disorder is selected from the group consisting of bipolar disorder, major depressive disorder, mitochondrial neurodegeneration, and a proliferative disease or disorder.
Embodiment 3 provides the method of any one of Embodiments 1-2, wherein the disease or disorder is a proliferative disease or disorder.
Embodiment 4 provides the method of any one of Embodiments 1-3, wherein the proliferative disease or disorder is cancer.
Embodiment 5 provides the method of Embodiment 4, wherein the cancer is at least one of brain cancer, breast cancer, colon cancer, rectal cancer, kidney cancer, prostate cancer, liver cancer, thyroid cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer, ventricular cancer, melanoma, lymphatic cancer, and acute leukemia.
Embodiment 6 provides the method of any one of Embodiments 1-5, wherein the
MTHFD2 inhibitor and the folate-depleting agent are each independently administered to the subject by at least one route selected from the group consisting of nasal, inhalational, topical, oral, buccal, rectal, pleural, peritoneal, vaginal, intramuscular, subcutaneous, transdermal, epidural, intratracheal, otic, intraocular, intrathecal, and intravenous routes.
Embodiment 7 provides the method of any one of Embodiments 1-6, wherein the MTHFD2 inhibitor is administered to the subject orally 1 to 4 times per day. Embodiment 8 provides the method of any one of Embodiments 1-7, wherein the MTHFD2 inhibitor is administered to the subject by continuous or intermittent intravenous infusion.
Embodiment 9 provides the method of any one of Embodiments 1-8, wherein the MTHFD2 inhibitor is (4-(3-amino-l-hydroxy-9-oxo-5,6,6a,7-tetrahydroimidazo[l,5- f]pteridin-8(9H)-yl)benzoyl)-L-glutamic acid, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
Embodiment 10 provides the method of any one of Embodiments 1-8, wherein the MTHFD2 inhibitor is (3R,4R,6R,E)-8-((2S,3S,7R,10R,l 1R,E)-1O,1 l-dihydroxy-3,7- dimethyl-12-oxo-l-oxacyclododec-8-en-2-yl)-3-methoxy-4,6-dimethyl-5-oxonon-7-enoic acid, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
Embodiment 11 provides the method of any one of Embodiments 1-8, wherein the MTHFD2 inhibitor is selected from the group consisting of: 4-(5-oxo-l,3,4,5-tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)benzoic acid; N-(2-chloro-4-(7-methyl-8-(4-methylpiperazin-l-yl)-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)phenyl)methanesulfonamide;
N-(2-chloro-4-(7-methyl-8-(4-methylpiperazin-l-yl)-5-oxo- 1,3,4, 5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)phenyl)cyclopropanesulfonamide;
N-(4-(7-methyl-8-(4-methylpiperazin-l-yl)-5-oxo-l,3,4,5-tetrahydro-2H-chromeno[3,4- c]pyridine-3-carbonyl)-2-(tri fluoromethoxy )phenyl)methanesulfonamide;
(S)-N-(2-chloro-4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo- 1,3,4, 5-tetrahydro- 2H-chromeno[3,4-c]pyridine-3-carbonyl)phenyl)methanesulfonamide;
(S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)m ethanesulfonamide;
(S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo- 1,3,4, 5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethyl)phenyl)methanesulfonamide;
(S)-N-(2-chl oro-4-(8-(3,4-dimethylpiperazin-l-yl)-7,10-dimethyl-5-oxo-l, 3,4,5- tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)phenyl)methanesulfonamide;
(S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7,10-dimethyl-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)m ethanesulfonamide;
(S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo- 1,3,4, 5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)ethanesulfonamide;
(S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7,10-dimethyl-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)ethanesulfonamide;
(S)-N-(4-(8-(3-ethyl-4-methylpiperazin-l-yl)-7-methyl-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)m ethanesulfonamide;
N-(4-(7-methyl-5-oxo-8-(3,3,4-trimethylpiperazin-l-yl)-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)m ethanesulfonamide;
(S)-N-(4-(7-methyl-8-(3-methylpiperazin-l-yl)-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)methanesulfonamide; and N-(2-chloro-4-(7-methyl-5-oxo-8-((3R,5S)-3,4,5-trimethylpiperazin-l-yl)-l,3,4,5- tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)phenyl)methanesulfonamide; or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
Embodiment 12 provides the method of any one of Embodiments 1-8, wherein the MTHFD2 inhibitor is (S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo-l,3,4,5- tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)-2- (trifluoromethoxy)phenyl)methanesulfonamide or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
Embodiment 13 provides the method of any one of Embodiments 1-12, wherein the folate-depleting agent is administered in advance of or contemporaneously with administration of the MTHFD2 inhibitor.
Embodiment 14 provides the method of any one of Embodiments 1-13 wherein the folate-depleting agent is administered by continuous or intermittent intravenous infusion.
Embodiment 15 provides the method of any one of Embodiments 1-14, wherein the folate-depleting agent is a carboxypeptidase.
Embodiment 16 provides the method of Embodiment 15, wherein the carboxypeptidase is carboxypeptidase G2 (CPG2) (SEQ ID NO: 1) or a biologically active fragment or derivative thereof.
Embodiment 17 provides the method of any one of Embodiments 1-16, wherein the subject is further administered at least one zinc supplement.
Embodiment 18 provides the method of Embodiment 17, wherein the zinc supplement is zinc gluconate.
Embodiment 19 provides the method of any one of Embodiments 15-18, wherein the carboxypeptidase shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology with CPG2 (SEQ ID NO: 1).
Embodiment 20 provides the method of any one of Embodiments 15-19, wherein the carboxypeptidase is modified with at least one modification to reduce immunogenicity and/or increase half-life in the subject.
Embodiment 21 provides the method of Embodiment 20, wherein the at least one modification is selected from the group consisting of fusion with human serum albumin and/or an IgG Fc domain, alkylation of at least one amide group, acetylation and/or alkylation of the N-terminus amino group, amidation of the C-terminus carboxy group, PEGylation, and phosphocholination with phosphatidylcholine.
Embodiment 22 provides the method of Embodiment 21, wherein the PEGylation is introduced by a site-specific or random bioconjugation technique.
Embodiment 23 provides the method of any one of Embodiments 1-22, wherein the subject is further administered at least one additional agent useful for treating, ameliorating, and/or preventing the disease or disorder.
Embodiment 24 provides the method of Embodiment 23, wherein the at least one additional agent comprises at least one selected from the group consisting of olaparib, rucaparib, niraparib, atrezoizumab, avelumab, pembrolizumab, cisplatin, carboplatin, doxorubicin, bevacizumab, gemcitabine, topetecan, paclitaxel, docetaxel, etoposide, and nanoparticle albumin-bound paclitaxel.
Embodiment 25 provides the method of any one of Embodiments 23-24, wherein the at least one additional agent is co-administered with at least one of the MTHFD2 inhibitor and the folate-depleting agent.
Embodiment 26 provides the method of any one of Embodiments 23-25, wherein the at least one additional agent is co-formulated with at least one of the MTHFD2 inhibitor and the folate-depleting agent.
Embodiment 27 provides the method of any one of Embodiments 1-26, wherein the subject is administered a composition comprising the MTHFD2 inhibitor and the folate- depleting agent.
Embodiment 28 provides the method of Embodiment 27, wherein the MTHFD2 inhibitor is (S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)methanesulfonamide or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
Embodiment 29 provides the method of Embodiment 27, wherein the folate-depleting agent is CPG2 or a biologically active fragment or derivative thereof.
Embodiment 30 provides the method of any one of Embodiments 1-29, wherein the MTHFD2 inhibitor is conjugated to an antibody, or antigen binding fragment thereof, and wherein the conjugate selectively targets a cancer cell.
Embodiment 31 provides the method of Embodiment 30, wherein the antibody, or antigen binding fragment thereof, is selected from the group consisting of A5B7, F(ab’)2 A5B7, W14, MFE-23, adecatumumab, intetumumab, etaracizumab, glembatumumab vedotin, leronlimab, margetuximab, sacituzumab govitecan, and trastuzumab.
Embodiment 32 provides the method of Embodiment 30, wherein the MTHFD2 inhibitor is conjugated to a biomolecule suitable for the recruitment of an endogenous ubiquitinylating agent.
Embodiment 33 provides the method of Embodiment 32, wherein the MTHFD2 is targeted for proteasome degradation.
Embodiment 34 provides the method of any one of Embodiments 1-29, wherein the folate-depleting agent is conjugated to an antibody, or antigen binding fragment thereof, wherein the conjugate selectively targets a cancer cell.
Embodiment 35 provides the method of Embodiment 34, wherein the folate-depleting agent is a carboxypeptidase.
Embodiment 36 provides the method of Embodiment 34, wherein the carboxypeptidase is CPG2 or a biologically active fragment or derivative thereof.
Embodiment 37 provides the method of any one of Embodiments 34-36, wherein the antibody, or antigen binding fragment thereof, is selected from the group consisting of A5B7, F(ab’)2 A5B7, W14, MFE-23, adecatumumab, intetumumab, etaracizumab, glembatumumab vedotin, leronlimab, margetuximab, sacituzumab govitecan, and trastuzumab.
Embodiment 38 provides the method of any one of Embodiments 1-37, wherein the subject is a mammal.
Embodiment 39 provides the method of Embodiment 38, wherein the mammal is a human.
Embodiment 40 provides a composition comprising a MTHFD2 inhibitor and a folate-depleting agent.
Embodiment 41 provides the composition of Embodiment 40, wherein the MTHFD2 inhibitor is (S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)methanesulfonamide or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
Embodiment 42 provides the composition of Embodiment 40, wherein the folate- depleting agent is CPG2 or a biologically active fragment or derivative thereof.
Embodiment 43 provides a pharmaceutical composition comprising the composition of any one of Embodiments 40-42 and at least one pharmaceutically acceptable carrier.
Embodiment 44 provides an immunoconjugate selectively targeting a cancer cell, comprising an antibody, or antigen binding fragment thereof, conjugated to a MTHFD2 inhibitor.
Embodiment 45 provides the immunoconjugate of Embodiment 44, which selectively targets a cancer cell which overexpresses MTHFD2.
Embodiment 46 provides the immunoconjugate of any one of Embodiments 44-45, wherein the antibody, or antigen binding fragment thereof, is selected from the group consisting of A5B7, F(ab’)2 A5B7, W14, MFE-23, adecatumumab, intetumumab, etaracizumab, glembatumumab vedotin, leronlimab, margetuximab, sacituzumab govitecan, and trastuzumab.
Embodiment 47 provides the immunoconjugate of any one of Embodiments 44-46, wherein the MTHFD2 inhibitor is:
(4-(3-amino-l-hydroxy-9-oxo-5,6,6a,7-tetrahydroimidazo[l,5-f]pteridin-8(9H)- yl)benzoyl)-L-glutamic acid;
(3R,4R,6R,E)-8-((2S,3S,7R,10R,l lR,E)-10,l l-dihydroxy-3,7-dimethyl-12- oxooxacyclododec-8-en-2-yl)-3-methoxy-4,6-dimethyl-5-oxonon-7-enoic acid;
4-(5-oxo-l,3,4,5-tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)benzoic acid; N-(2-chloro-4-(7-methyl-8-(4-methylpiperazin-l-yl)-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)phenyl)methanesulfonamide;
N-(2-chloro-4-(7-methyl-8-(4-methylpiperazin-l-yl)-5-oxo- 1,3,4, 5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)phenyl)cyclopropanesulfonamide;
N-(4-(7-methyl-8-(4-methylpiperazin-l-yl)-5-oxo-l,3,4,5-tetrahydro-2H-chromeno[3,4- c]pyridine-3-carbonyl)-2-(tri fluoromethoxy )phenyl)methanesulfonamide;
(S)-N-(2-chloro-4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo- 1,3,4, 5-tetrahydro- 2H-chromeno[3,4-c]pyridine-3-carbonyl)phenyl)methanesulfonamide;
(S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)m ethanesulfonamide;
(S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo- 1,3,4, 5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethyl)phenyl)methanesulfonamide;
(S)-N-(2-chl oro-4-(8-(3,4-dimethylpiperazin-l-yl)-7,10-dimethyl-5-oxo-l, 3,4,5- tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)phenyl)methanesulfonamide; (S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7,10-dimethyl-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)m ethanesulfonamide;
(S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo- 1,3,4, 5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)ethanesulfonamide;
(S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7,10-dimethyl-5-oxo- 1,3,4, 5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)ethanesulfonamide;
(S)-N-(4-(8-(3-ethyl-4-methylpiperazin-l-yl)-7-methyl-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)m ethanesulfonamide;
N-(4-(7-methyl-5-oxo-8-(3,3,4-trimethylpiperazin-l-yl)-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)m ethanesulfonamide;
(S)-N-(4-(7-methyl-8-(3-methylpiperazin-l-yl)-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)methanesulfonamide; or N-(2-chloro-4-(7-methyl-5-oxo-8-((3R,5S)-3,4,5-trimethylpiperazin-l-yl)-l,3,4,5- tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)phenyl)methanesulfonamide; or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
Embodiment 48 provides the immunoconjugate of any one of Embodiments 44-47, wherein the MTHFD2 inhibitor is (S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo- l,3,4,5-tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)-2- (trifluoromethoxy)phenyl)methanesulfonamide or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.

Claims

CLAIMS What is claimed is:
1. A method of treating, preventing, and/or ameliorating a disease or disorder involving overexpression of methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) in a subject, the method comprising administering to the subject a pharmaceutically effective amount of a MTHFD2 inhibitor and a pharmaceutically effective amount of a folate-depleting agent.
2. The method of claim 1, wherein the disease or disorder is selected from the group consisting of bipolar disorder, major depressive disorder, mitochondrial neurodegeneration, and a proliferative disease or disorder.
3. The method of claim 2, wherein the disease or disorder is a proliferative disease or disorder.
4. The method of claim 3, wherein the proliferative disease or disorder is cancer.
5. The method of claim 4, wherein the cancer is at least one of brain cancer, breast cancer, colon cancer, rectal cancer, kidney cancer, prostate cancer, liver cancer, thyroid cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer, ventricular cancer, melanoma, lymphatic cancer, and acute leukemia.
6. The method of claim 1, wherein the MTHFD2 inhibitor and the folate-depleting agent are each independently administered to the subject by at least one route selected from the group consisting of nasal, inhalational, topical, oral, buccal, rectal, pleural, peritoneal, vaginal, intramuscular, subcutaneous, transdermal, epidural, intratracheal, otic, intraocular, intrathecal, and intravenous routes.
7. The method of claim 6, wherein the MTHFD2 inhibitor is administered to the subject orally 1 to 4 times per day.
8. The method of claim 6, wherein the MTHFD2 inhibitor is administered to the subject by continuous or intermittent intravenous infusion.
9. The method of claim 1, wherein the MTHFD2 inhibitor is (4-(3 -amino- 1 -hydroxy-9-
- 47 - oxo-5,6,6a,7-tetrahydroimidazo[l,5-f]pteridin-8(9H)-yl)benzoyl)-L-glutamic acid, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
10. The method of claim 1, wherein the MTHFD2 inhibitor is (3R,4R,6R,E)-8-
((2S, 3 S,7R,1 OR, 11R,E)-10,11 -dihydroxy-3, 7-dimethyl-12-oxo- 1-oxacy clododec-8-en-2 -yl)- 3-methoxy-4,6-dimethyl-5-oxonon-7-enoic acid, or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
11. The method of claim 1, wherein the MTHFD2 inhibitor is selected from the group consisting of:
4-(5-oxo-l,3,4,5-tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)benzoic acid;
N-(2-chloro-4-(7-methyl-8-(4-methylpiperazin-l-yl)-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)phenyl)methanesulfonamide;
N-(2-chloro-4-(7-methyl-8-(4-methylpiperazin-l-yl)-5-oxo- 1,3,4, 5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)phenyl)cyclopropanesulfonamide;
N-(4-(7-methyl-8-(4-methylpiperazin-l-yl)-5-oxo-l,3,4,5-tetrahydro-2H-chromeno[3,4- c]pyridine-3-carbonyl)-2-(tri fluoromethoxy )phenyl)methanesulfonamide;
(S)-N-(2-chloro-4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo- 1,3,4, 5-tetrahydro- 2H-chromeno[3,4-c]pyridine-3-carbonyl)phenyl)methanesulfonamide;
(S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)m ethanesulfonamide;
(S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo- 1,3,4, 5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethyl)phenyl)methanesulfonamide;
(S)-N-(2-chl oro-4-(8-(3,4-dimethylpiperazin-l-yl)-7,10-dimethyl-5-oxo-l, 3,4,5- tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)phenyl)methanesulfonamide;
(S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7,10-dimethyl-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)m ethanesulfonamide;
(S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo- 1,3,4, 5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)ethanesulfonamide;
(S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7,10-dimethyl-5-oxo- 1,3,4, 5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)ethanesulfonamide;
(S)-N-(4-(8-(3-ethyl-4-methylpiperazin-l-yl)-7-methyl-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)m ethanesulfonamide;
- 48 - N-(4-(7-methyl-5-oxo-8-(3,3,4-trimethylpiperazin-l-yl)-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)m ethanesulfonamide;
(S)-N-(4-(7-methyl-8-(3-methylpiperazin-l-yl)-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)methanesulfonamide; and
N-(2-chloro-4-(7-methyl-5-oxo-8-((3R,5S)-3,4,5-trimethylpiperazin-l-yl)-l,3,4,5- tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)phenyl)methanesulfonamide; or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
12. The method of claim 11, wherein the MTHFD2 inhibitor is (S)-N-(4-(8-(3,4- dimethylpiperazin-l-yl)-7-methyl-5-oxo- 1,3,4, 5-tetrahydro-2H-chromeno[3,4-c]pyridine-3- carbonyl)-2-(trifluoromethoxy)phenyl)methanesulfonamide or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
13. The method of claim 1, wherein the folate-depleting agent is administered in advance of or contemporaneously with administration of the MTHFD2 inhibitor.
14. The method of claim 6, wherein the folate-depleting agent is administered by continuous or intermittent intravenous infusion.
15. The method of claim 1, wherein the folate-depleting agent is a carboxypeptidase.
16. The method of claim 15, wherein the carboxypeptidase is carboxypeptidase G2 (CPG2) (SEQ ID NO: 1) or a biologically active fragment or derivative thereof.
17. The method of claim 16, wherein the subject is further administered at least one zinc supplement.
18. The method of claim 17, wherein the zinc supplement is zinc gluconate.
19. The method of claim 15, wherein the carboxypeptidase shares at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology with CPG2 (SEQ ID NO:1).
20. The method of claim 15, wherein the carboxypeptidase is modified with at least one
- 49 - modification to reduce immunogenicity and/or increase half-life in the subject.
21. The method of claim 20, wherein the at least one modification is selected from the group consisting of fusion with human serum albumin and/or an IgG Fc domain, alkylation of at least one amide group, acetylation and/or alkylation of the N-terminus amino group, amidation of the C-terminus carboxy group, PEGylation, and phosphocholination with phosphatidylcholine.
22. The method of claim 21, wherein the PEGylation is introduced by a site-specific or random bioconjugation technique.
23. The method of claim 1, wherein the subject is further administered at least one additional agent useful for treating, ameliorating, and/or preventing the disease or disorder.
24. The method of claim 23, wherein the at least one additional agent comprises at least one selected from the group consisting of olaparib, rucaparib, niraparib, atrezoizumab, avelumab, pembrolizumab, cisplatin, carboplatin, doxorubicin, bevacizumab, gemcitabine, topetecan, paclitaxel, docetaxel, etoposide, and nanoparticle albumin-bound paclitaxel.
25. The method of claim 23, wherein the at least one additional agent is co-administered with at least one of the MTHFD2 inhibitor and the folate-depleting agent.
26. The method of claim 23, wherein the at least one additional agent is co-formulated with at least one of the MTHFD2 inhibitor and the folate-depleting agent.
27. The method of claim 1, wherein the subject is administered a composition comprising the MTHFD2 inhibitor and the folate-depleting agent.
28. The method of claim 27, wherein the MTHFD2 inhibitor is (S)-N-(4-(8-(3,4- dimethylpiperazin-l-yl)-7-methyl-5-oxo- 1,3,4, 5-tetrahydro-2H-chromeno[3,4-c]pyridine-3- carbonyl)-2-(trifluoromethoxy)phenyl)methanesulfonamide or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
29. The method of claim 27, wherein the folate-depleting agent is CPG2 or a biologically
- 50 - active fragment or derivative thereof.
30. The method of claim 1, wherein the MTHFD2 inhibitor is conjugated to an antibody, or antigen binding fragment thereof, and wherein the conjugate selectively targets a cancer cell.
31. The method of claim 30, wherein the antibody, or antigen binding fragment thereof, is selected from the group consisting of A5B7, F(ab’)2 A5B7, W14, MFE-23, adecatumumab, intetumumab, etaracizumab, glembatumumab vedotin, leronlimab, margetuximab, sacituzumab govitecan, and trastuzumab.
32. The method of claim 1, wherein the MTHFD2 inhibitor is conjugated to a biomolecule suitable for the recruitment of an endogenous ubiquitinylating agent.
33. The method of claim 32, wherein MTHFD2 is targeted for proteasome degradation.
34. The method of claim 1, wherein the folate-depleting agent is conjugated to an antibody, or antigen binding fragment thereof, wherein the conjugate selectively targets a cancer cell.
35. The method of claim 34, wherein the folate-depleting agent is a carboxypeptidase.
36. The method of claim 35, wherein the carboxypeptidase is CPG2 or a biologically active fragment or derivative thereof.
37. The method of claim 34, wherein the antibody, or antigen binding fragment thereof, is selected from the group consisting of A5B7, F(ab’)2 A5B7, W14, MFE-23, adecatumumab, intetumumab, etaracizumab, glembatumumab vedotin, leronlimab, margetuximab, sacituzumab govitecan, and trastuzumab.
38. The method of claim 1, wherein the subject is a mammal.
39. The method of claim 38, wherein the mammal is a human.
40. A composition comprising a MTHFD2 inhibitor and a folate-depleting agent.
41. The composition of claim 40, wherein the MTHFD2 inhibitor is (S)-N-(4-(8-(3,4- dimethylpiperazin-l-yl)-7-methyl-5-oxo- 1,3,4, 5-tetrahydro-2H-chromeno[3,4-c]pyridine-3- carbonyl)-2-(trifluoromethoxy)phenyl)methanesulfonamide or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
42. The composition of claim 40, wherein the folate-depleting agent is CPG2 or a biologically active fragment or derivative thereof.
43. A pharmaceutical composition comprising the composition of any one of claims 40- 42 and at least one pharmaceutically acceptable carrier.
44. An immunoconjugate comprising an antibody, or antigen binding fragment thereof, conjugated to a MTHFD2 inhibitor.
45. The immunoconjugate of claim 44, which selectively targets a cancer cell which overexpresses MTHFD2.
46. The immunoconjugate of claim 44, wherein the antibody, or antigen binding fragment thereof, is selected from the group consisting of A5B7, F(ab’)2 A5B7, W14, MFE-23, adecatumumab, intetumumab, etaracizumab, glembatumumab vedotin, leronlimab, margetuximab, sacituzumab govitecan, and trastuzumab.
47. The immunoconjugate of claim 44, wherein the MTHFD2 inhibitor is: (4-(3-amino-l-hydroxy-9-oxo-5,6,6a,7-tetrahydroimidazo[l,5-f]pteridin-8(9H)- yl)benzoyl)-L-glutamic acid;
(3R,4R,6R,E)-8-((2S,3S,7R,10R,l lR,E)-10,l l-dihydroxy-3,7-dimethyl-12- oxooxacyclododec-8-en-2-yl)-3-methoxy-4,6-dimethyl-5-oxonon-7-enoic acid;
4-(5-oxo-l,3,4,5-tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)benzoic acid;
N-(2-chloro-4-(7-methyl-8-(4-methylpiperazin-l-yl)-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)phenyl)methanesulfonamide;
N-(2-chloro-4-(7-methyl-8-(4-methylpiperazin-l-yl)-5-oxo- 1,3,4, 5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)phenyl)cyclopropanesulfonamide; N-(4-(7-methyl-8-(4-methylpiperazin-l-yl)-5-oxo-l,3,4,5-tetrahydro-2H-chromeno[3,4- c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)methanesulfonamide;
(S)-N-(2-chloro-4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo- 1,3,4, 5-tetrahydro- 2H-chromeno[3,4-c]pyridine-3-carbonyl)phenyl)methanesulfonamide;
(S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo- 1,3,4, 5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)m ethanesulfonamide;
(S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo- 1,3,4, 5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethyl)phenyl)methanesulfonamide;
(S)-N-(2-chl oro-4-(8-(3,4-dimethylpiperazin-l-yl)-7,10-dimethyl-5-oxo-l, 3,4,5- tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)phenyl)methanesulfonamide;
(S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7,10-dimethyl-5-oxo- 1,3,4, 5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)m ethanesulfonamide;
(S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo- 1,3,4, 5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)ethanesulfonamide;
(S)-N-(4-(8-(3,4-dimethylpiperazin-l-yl)-7,10-dimethyl-5-oxo- 1,3,4, 5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)ethanesulfonamide;
(S)-N-(4-(8-(3-ethyl-4-methylpiperazin-l-yl)-7-methyl-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)m ethanesulfonamide;
N-(4-(7-methyl-5-oxo-8-(3,3,4-trimethylpiperazin-l-yl)-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)m ethanesulfonamide;
(S)-N-(4-(7-methyl-8-(3-methylpiperazin-l-yl)-5-oxo-l,3,4,5-tetrahydro-2H- chromeno[3,4-c]pyridine-3-carbonyl)-2-(trifluoromethoxy)phenyl)methanesulfonamide; or
N-(2-chloro-4-(7-methyl-5-oxo-8-((3R,5S)-3,4,5-trimethylpiperazin-l-yl)-l,3,4,5- tetrahydro-2H-chromeno[3,4-c]pyridine-3-carbonyl)phenyl)methanesulfonamide; or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
48. The immunoconjugate of claim 47, wherein the MTHFD2 inhibitor is (S)-N-(4-(8- (3,4-dimethylpiperazin-l-yl)-7-methyl-5-oxo-l,3,4,5-tetrahydro-2H-chromeno[3,4- c]pyridine-3 -carbonyl)-2-(trifluorom ethoxy )phenyl)methanesulfonamide or a salt, solvate, prodrug, isotopically labelled derivative, stereoisomer, or tautomer thereof, or any mixtures thereof.
- 53 -
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