Gamma polyglutamylated methotrexate and uses thereof

Abstract

To provide methods of making and using gamma polyglutamated methotrexate compositions to treat hyperproliferative disorders (e.g., cancer) and disorders of the immune system (e.g., inflammation and autoimmune diseases such as rheumatoid arthritis).SOLUTION: A liposomal composition comprises a liposome in which a gamma polyglutamated methotrexate is encapsulated. The gamma polyglutamated methotrexate comprises 2-10 glutamyl groups having gamma carboxyl group linkages. The liposome is pegylated. The liposomal composition comprises a targeting moiety with a specific affinity for a surface antigen on a target cell of interest.SELECTED DRAWING: None

Description

Background Art

[0001] The present disclosure generally relates to compositions of gamma polyglutamate oxidized methotrexate, including delivery carriers such as liposomes containing gamma polyglutamate oxidized methotrexate compositions, and methods of manufacturing and using the compositions for treating diseases including hyperproliferative diseases such as cancer, immune system disorders such as rheumatoid arthritis, and infectious diseases such as HIV and malaria.

[0002] Methotrexate has become clinically widely used worldwide as an essential element of multi-drug combination therapies for the treatment of acute lymphoblastic leukemia (ALL), lymphoma, and solid tumors. Methotrexate (MTX) is also a core drug among the most widely applied disease-modifying antirheumatic drugs (DMARDs) in the treatment of patients with rheumatoid arthritis (RA). It is used as a single agent or in combination with other DMARDs (e.g., sulfasalazine and hydroxychloroquine), and the use of MTX is essential in most treatment strategies including biological agents (e.g., anti-TNFα and anti-CD20 monoclonal antibodies). This is used in the treatment of breast cancer, advanced head and neck cancer, lung cancer, and gastric cancer, osteosarcoma, non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma, and villous adenoma. Off-label cancer uses of methotrexate include the prevention of non-leukemic meningeal carcinomatosis, soft tissue sarcoma (desmoid tumor, aggressive fibromatosis), bladder cancer, central nervous system (CNS) lymphoma, and graft-versus-host disease.

[0003] MTX is also used in non-cancerous conditions such as psoriasis and rheumatoid arthritis, inflammatory bowel disease (IBD), systemic inflammation, atherosclerosis, cardiovascular disease (CVD), coronary artery disease, and gestational trophoblastic disease. Some off-label non-cancer uses include Crohn's disease, dermatomyositis / polymyositis, ectopic pregnancy, systemic lupus erythematosus, and Takayasu arteritis.

[0004] Methotrexate is a folic acid analog different from folic acid by the substitution of the hydroxyl group at the 4-position of the pteridine ring with an amino group. This slight structural change brings about the ability of MTX to inhibit the active catalytic site of dihydrofolate reductase (DHFR), which catalyzes the production of tetrahydrofolic acid (THF) from dihydrofolic acid (DHF). As a result, methotrexate interferes with the synthesis of tetrahydrofolic acid (THF), which functions as a major one-carbon carrier for the enzymatic processes involved in the de novo synthesis of thymidylate, purine nucleotides, and the amino acids serine and methionine. Inhibition of these metabolic processes interferes with the formation of DNA, RNA, and important cytoplasmic proteins.

[0005] Folic acid is an essential cofactor that mediates the transfer of one-carbon units involved in nucleotide biosynthesis and DNA repair, the remethylation of homocysteine (Hcy), and the methylation of DNA, proteins, and lipids. The only circulating form of folic acid in the blood is monoglutamic acid, and folic acid monoglutamate is the only form of folic acid that is transported across the cell membrane. Similarly, monoglutamic acid-type polyglutaminatable folic acid antagonists such as methotrexate are also transported across the cell membrane. Once taken up into the cell, intracellular folic acid is converted to polyglutamic acid by the enzyme folylpolyglutamate synthetase (FPGS).

[0006] Methotrexate is transported into cells by the reduced folate carrier (RFC) system and folate receptors (FR) α and β, and by the proton-coupled folate transporter (PCFT), which is most active in a more acidic pH environment. RFC is the major methotrexate transporter at physiological pH and is widely expressed in normal and diseased cells. Thus, methotrexate often suffers from dose-limiting toxicity, a major obstacle in cancer chemotherapy. Once inside the cell, methotrexate is polyglutamylated by FPGS, which can add up to six L-glutamyl groups during the L-gamma carboxyl group binding to methotrexate. L-gamma polyglutamylation of methotrexate by FPGS serves at least two major therapeutic purposes: (1) it greatly enhances the affinity and inhibitory activity of methotrexate for DHFR; and (2) it facilitates the accumulation of polyglutamylated methotrexate, which, unlike methotrexate (monoglutamate), is not readily transported out of the cell by the cellular efflux pump.

[0007] Targeting folate metabolism and nucleotide biosynthesis is a well-established therapeutic strategy for cancer, but for MTX, clinical efficacy is limited by the lack of tumor selectivity and the presence of new and acquired drug resistance. Like other folate antagonists, methotrexate acts during DNA and RNA synthesis and as a result has a major toxic effect on rapidly dividing cells such as malignant and myeloid cells. Myelosuppression is usually the dose-limiting toxicity of methotrexate therapy and restricts the clinical application of methotrexate.

[0008] Resistance to methotrexate therapy is usually associated with one or more of the following: (a) increased activity of the cellular efflux pump, (b) decreased transport of MTX into the cell, (c) increased DHFR activity, (d) decreased activity of holipoly-gamma-glutamate synthase (FPGS), and (e) increased activity of gamma-glutamyl hydrolase (GGH), which cleaves the gamma-polyglutamate chain attached to folate and folate antagonists.

[0009] The problem with the long-term (>30 years) observation that higher levels of polyglutamates of various folic acid antagonists have much higher potency compared to lower levels of glutamate was that the scientific community had relied on the intracellular FPGS-mediated mechanism that converts lower levels of glutamate to their higher level forms. The present invention provides a means for directly delivering higher levels of polyglutamate forms of folic acid antagonists into cells without relying on the cell's machinery to achieve this goal.

[0010] The provided gamma-polyglutamylated methotrexate compositions provide a strategy to overcome the pharmacological issues related to dose-limiting toxicity and treatment resistance associated with methotrexate therapy. The provided methods deliver a novel gamma-polyglutamylated form of methotrexate to cancer cells while simultaneously (1) minimizing / reducing exposure to normal tissue cells, (2) optimizing / improving the cytotoxic effect of methotrexate-based agents on cancer cells, and (3) minimizing / reducing the effects of efflux pumps and other resistance mechanisms that limit the therapeutic efficacy of methotrexate. SUMMARY OF THE INVENTION

[0011] The present disclosure generally relates to gamma-polyglutamylated methotrexate (MTX) compositions, and methods of making and using the compositions for treating diseases including hyperproliferative diseases such as cancer, immune system disorders such as inflammation and rheumatoid arthritis, and infectious diseases such as HIV and malaria.

[0012] In some embodiments, the present disclosure provides the following. [1] A composition comprising gamma-polyglutamylated methotrexate; [2] The composition according to item [1], wherein the gamma-polyglutamylated methotrexate comprises 1 to 10 glutamyl groups having a gamma-carboxyl group bond. [3] The composition according to item [1] or [2], wherein the gamma-polyglutamyl methotrexate contains 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups having a gamma-carboxyl group bond; [4] The composition according to any one of items [1] to [3], wherein the gamma-polyglutamyl methotrexate is gamma-tetra-glutamyl methotrexate; [5] The composition according to any one of items [1] to [3], wherein the gamma-polyglutamyl methotrexate is gamma-penta-glutamyl methotrexate; [6] The composition according to any one of items [1] to [3], wherein the gamma-polyglutamyl methotrexate is gamma-hexa-glutamyl methotrexate; [7] The composition according to any one of items [1] to [6], which is the following composition: (a) The gamma-polyglutamyl methotrexate contains L-type glutamyl groups having two or more gamma-carboxyl group bonds, (b) Each glutamyl group of the gamma-polyglutamyl methotrexate is of the L-type and has a gamma-carboxyl group bond, (c) At least one glutamyl group of the gamma-polyglutamyl methotrexate is of the D-type and has a gamma-carboxyl group bond, (d) Each glutamyl group of the gamma-polyglutamyl methotrexate other than the glutamyl group of methotrexate is of the D-type and has a gamma-carboxyl group bond, or (e) The gamma-polyglutamyl methotrexate contains two or more L-type glutamyl groups having a gamma-carboxyl group bond and at least one D-type glutamyl group; [8] The composition according to item [4], wherein (a) each glutamyl group is of the L-type and has a gamma-carboxyl group bond, or (b) each glutamyl group other than the glutamyl group of methotrexate is of the D-type and each glutamyl group has a gamma-carboxyl group bond; [9] The composition according to item [5], wherein (a) each glutamyl group is of the L-type and has a gamma-carboxyl group bond, or (b) each glutamyl group other than the glutamyl group of methotrexate is of the D-type and each glutamyl group has a gamma-carboxyl group bond;

[10] The composition according to item [6], wherein (a) each glutamyl group is of the L-type and has a gamma-carboxyl group bond, or (b) each glutamyl group other than the glutamyl group of methotrexate is of the D-type and each glutamyl group has a gamma-carboxyl group bond;

[11] The composition according to any one of items [1] to

[10] , wherein gamma-polyglutamylated methotrexate is polyglutamylatable by FGPS under physiological conditions and / or polyglutamylated MTX has a lower hepatocyte uptake rate (<30%) than MTX;

[12] A liposomal composition (Lp-γPMTX) containing the gamma-polyglutamylated methotrexate according to any one of items [1] to

[11] ;

[13] The Lp-γPMTX composition according to item

[12] , wherein the gamma-polyglutamylated methotrexate contains two or more L-type glutamyl groups;

[14] The Lp-γPMTX composition according to item

[12] or

[13] , wherein each glutamyl group of the gamma-polyglutamylated methotrexate is of the L-type;

[15] The Lp-γPMTX composition according to item

[12] or

[13] , wherein at least one glutamyl group of the gamma-polyglutamylated methotrexate is of the D-type;

[16] The Lp-γPMTX composition according to any one of items

[12] to

[15] , wherein the liposome contains gamma-polyglutamylated methotrexate containing 1 to 10 glutamyl groups having a gamma-carboxyl group bond;

[17] The Lp-γPMTX composition according to any one of items

[12] to

[16] , wherein the liposome contains gamma-polyglutamylated methotrexate containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups;

[18] The Lp-γPMTX composition according to any one of items

[12] to

[17] , wherein the liposome contains gamma-tetraglutamylated methotrexate;

[19] The Lp-γPMTX composition according to any one of items

[12] to

[17] , wherein the liposome contains gamma-pentaglutamylated methotrexate;

[20] The Lp-γPMTX composition according to any one of items

[12] to

[17] , wherein the liposome contains gamma-hexaglutamylated methotrexate;

[21] The Lp-γPMTX composition according to any one of items

[12] to

[20] , wherein the liposome is not pegylated (PγLp-γPMTX);

[22] The Lp-γPMTX composition according to any one of items

[12] to

[20] , wherein the liposome is pegylated (PγLp-γPMTX);

[23] The Lp-γPMTX composition according to any one of items

[12] to

[22] , wherein the liposome contains at least 1% by weight of gamma-polyglutamylated methotrexate, or during the process of preparing Lp-γPMTX, at least 1% of the starting material of gamma-polyglutamylated MTX is encapsulated (enclosed) in Lp-γPMTX;

[24] The Lp-γPMTX composition according to any one of items

[12] to

[23] , wherein the liposome has a diameter in the range of 20 nm to 500 nm;

[25] The Lp-γPMTX composition according to any one of items

[12] to

[24] , wherein the liposome has a diameter in the range of 20 nm to 200 nm;

[26] The Lp-γPMTX composition according to any one of items

[12] to

[25] , wherein the liposome has a diameter in the range of 80 nm to 120 nm;

[27] The Lp-γPMTX composition according to any one of items

[12] to

[26] , wherein the liposome is formed from liposome components;

[28] The Lp-γPMTX composition according to item

[27] , wherein the liposome component contains at least one of an anionic lipid and a neutral lipid;

[29] The Lp-γPMTX composition according to item

[27] or

[28] , wherein the liposome component contains at least one selected from the group consisting of DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholesterol; cholesterol-PEG; and cholesterol-maleimide;

[30] The Lp-γPMTX composition according to any one of items

[27] to

[29] , wherein the liposome component contains at least one selected from the group consisting of DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC;

[31] The Lp-γPMTX composition according to any one of items

[27] to

[30] , wherein one or more liposome components further contain a steric stabilizer;

[32] The Lp-γPMTX composition according to item

[31] , wherein the steric stabilizer is at least one selected from the group consisting of polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinylpyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol; poly[N-(2-hydroxypropyl)methacrylamide]; amphiphilic poly-N-vinylpyrrolidone; L-amino acid-based polymer; oligoglycerol, polyethylene glycol and polypropylene oxide-containing copolymer, poloxamer 188, and polyvinyl alcohol;

[33] The Lp-γPMTX composition according to item

[32] , wherein the steric stabilizer is PEG, and PEG has a number average molecular weight (Mn) of 200 to 5000 daltons;

[34] The Lp-γPMTX composition according to any one of items

[12] to

[33] , wherein the liposome is anionic or neutral;

[35] The Lp-γPMTX composition according to any one of items

[12] to

[33] , wherein the liposome has a zeta potential of zero or less;

[36] The Lp-γPMTX composition according to any one of items

[12] to

[33] , wherein the liposome has a zeta potential of 0 to -150 mV;

[37] The Lp-γPMTX composition according to any one of items

[12] to

[33] , wherein the liposome has a zeta potential of -30 to -50 mV;

[38] The Lp-γPMTX composition according to any one of items

[12] to

[33] , wherein the liposome is cationic;

[39] The Lp-γPMTX composition according to any one of items

[12] to

[38] , wherein the liposome has an internal space containing gamma-polyglutamylated methotrexate and an aqueous pharmaceutically acceptable carrier;

[40] The Lp-γPMTX composition according to item

[39] , wherein the pharmaceutically acceptable carrier contains an isotonic agent such as dextrose, mannitol, glycerol, potassium chloride, sodium chloride, etc. at a concentration greater than 1%;

[41] The Lp-γPMTX composition according to item

[39] , wherein the aqueous pharmaceutically acceptable carrier is trehalose;

[42] The Lp-γPMTX composition according to item

[41] , wherein the pharmaceutically acceptable carrier contains 1% to 50% trehalose;

[43] The Lp-γPMTX composition according to any one of items

[39] to

[42] , wherein the pharmaceutically acceptable carrier contains a 1% to 50% dextrose solution;

[44] The Lp-γPMTX composition according to any one of items

[39] to

[43] , wherein the internal space of the liposome contains 5% dextrose suspended in HEPES buffer;

[45] The Lp-γPMTX composition according to any one of items

[39] to

[44] , wherein the pharmaceutically acceptable carrier comprises a buffer solution such as HEPES buffered saline (HBS) or the like at a concentration of 1 to 200 mM and a pH of 2 to 8;

[46] The Lp-γPMTX composition according to any one of items

[39] to

[45] , wherein the pharmaceutically acceptable carrier comprises sodium acetate and calcium acetate at a total concentration of 50 mM to 500 mM;

[47] The Lp-γPMTX composition according to any one of items

[12] to

[46] , wherein the internal space of the liposome has a pH of 5 to 8 or a pH of 6 to 7, or any range of pH therebetween;

[48] The Lp-γPMTX composition according to any one of items

[12] to

[47] , wherein the liposome contains less than 500,000 or less than 200,000 gamma-polyglutamylated methotrexate molecules;

[49] The Lp-γPMTX composition according to any one of items

[12] to

[48] , wherein the liposome contains 10 to 100,000 or any range therebetween of gamma-polyglutamylated methotrexate molecules;

[50] The Lp-γPMTX composition according to any one of items

[12] to

[49] , further comprising a targeting moiety, wherein the targeting moiety has specific affinity for a surface antigen on a target cell of interest;

[51] The Lp-γPMTX composition according to item

[50] , wherein the targeting moiety is bound to one or both of the PEG and the outer surface of the liposome, and optionally, the targeting moiety is covalently bound to one or both of the PEG and the outer surface of the liposome;

[52] The Lp-γPMTX composition according to item

[50] or

[51] , wherein the targeting moiety is a polypeptide;

[53] The Lp-γPMTX composition according to any one of items

[50] to

[52] , wherein the targeting moiety is an antibody or an antigen-binding fragment of an antibody;

[54] An Lp-γPMTX composition according to any one of items

[50] to

[53] , wherein the targeting moiety has a dissociation constant (Kd) in the range of 0.5x10 -10 ~10x10 -6 and binds to a surface antigen with an equilibrium dissociation constant (Kd) in the range of;

[55] An Lp-γPMTX composition according to any one of items

[50] to

[54] , wherein the targeting moiety specifically binds to one or more folate receptors selected from the group consisting of folate receptor alpha (FR-α), folate receptor beta (FR-β), and folate receptor delta (FR-δ);

[56] An Lp-γPMTX composition according to any one of items

[50] to

[55] , wherein the targeting moiety comprises one or more selected from the group consisting of an antibody, a humanized antibody, an antigen-binding fragment of an antibody, a single-chain antibody, a single-domain antibody, a bispecific antibody, a synthetic antibody, a pegylated antibody, and a multimeric antibody;

[57] An Lp-γPMTX composition according to any one of items

[50] to

[56] , wherein each pegylated liposome contains 1 to 1000 or 30 to 200 targeting moieties;

[58] An Lp-γPMTX composition according to any one of items

[39] to

[57] , further comprising one or more of an immunostimulant, a detectable marker, and maleimide, wherein the immunostimulant, detectable marker, or maleimide is bound to the PEG or outer surface of the liposome;

[59] The Lp-γPMTX composition according to item

[58] , wherein the immunostimulant is at least one selected from the group consisting of a protein immunostimulant, a nucleic acid immunostimulant, a chemical immunostimulant, a hapten, and an adjuvant;

[60] The Lp-γPMTX composition according to item

[58] or

[59] , wherein the immunostimulant is fluorescein, fluorescein isothiocyanate (FITC), DNP, beta-glucan, beta-1,3-glucan, beta-1,6-glucan, resorbin (e.g., D n-6DPA or D n-3DPAAt least one selected from the group consisting of resorcin D, resorcin E, or T-series resorcin (such as), and toll-like receptor (TLR) regulators such as oxidized low-density lipoprotein (e.g., OXPAC, PGPC), and erythran lipids (e.g., E5564);

[61] The Lp-γPMTX composition according to any one of items

[58] to

[60] , wherein the immunostimulant and the detectable marker are the same;

[62] The Lp-γPMTX composition according to any one of items

[58] to

[61] , further comprising a hapten;

[63] The Lp-γPMTX composition according to item

[62] , wherein the hapten comprises one or more of fluorescein or beta-1,6-glucan;

[64] The Lp-γPMTX composition according to any one of items

[12] to

[63] , further comprising at least one cryoprotective substance selected from the group consisting of mannitol, trehalose, sorbitol, and sucrose;

[65] A targeted composition comprising the composition according to any one of items [1] to

[64] ;

[66] A non-targeted composition comprising the composition according to any one of items [1] to

[49] ;

[67] The Lp-γPMTX composition according to any one of items

[12] to

[66] , further comprising carboplatin and / or pembrolizumab;

[68] A pharmaceutical composition comprising the liposomal gamma-polyglutamate oxidized methotrexate composition according to any one of items

[12] to

[67] ;

[69] A pharmaceutical composition comprising the gamma-polyglutamate oxidized methotrexate composition according to any one of items [1] to [7];

[70] The composition according to any one of items [1] to

[69] for use in the treatment of a disease;

[71] Use of the composition according to any one of items [1] to

[70] in the manufacture of a drug for the treatment of a disease;

[72] A method for treating or preventing a disease in a subject in need thereof, comprising the step of administering the composition according to any one of items [1] to

[70] to the subject;

[73] A method for treating or preventing a disease in a subject in need thereof, comprising the step of administering the liposomal gamma-polyglutamylated methotrexate composition according to any one of items

[12] to

[69] to the subject;

[74] A method for killing hyperproliferative cells, comprising the step of contacting the hyperproliferative cells with the composition according to any one of items [1] to

[69] ;

[75] A method for killing hyperproliferative cells, comprising the step of contacting the hyperproliferative cells with the liposomal gamma-polyglutamylated methotrexate composition according to any one of items

[12] to

[69] ;

[76] The method according to item

[74] or

[75] , wherein the hyperproliferative cells are cancer cells, mammalian cells, and / or human cells;

[77] A method for treating cancer, comprising the step of administering an effective amount of the composition according to any one of items [1] to

[69] to a subject having or at risk of having cancer;

[78] A method for treating cancer, comprising the step of administering an effective amount of the liposomal gamma-polyglutamylated methotrexate composition according to any one of items

[12] to

[68] to a subject having or at risk of having cancer;

[79] The method according to item

[77] or

[78] , wherein the cancer is a non-hematological tumor including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and a hematological tumor selected from the group consisting of, for example, leukemia, lymphoma and other B-cell malignancies, multiple myeloma and other plasma cell dyscrasias;

[80] The method according to item

[77] or

[78] , wherein the cancer is a member selected from the group consisting of lung cancer, breast cancer, colon cancer, pancreatic cancer, gastric cancer, bladder cancer, head and neck cancer, ovarian cancer and cervical cancer;

[81] The method according to item

[77] or

[78] , wherein the cancer is a member selected from the group consisting of colorectal cancer, lung cancer, breast cancer, head and neck cancer, and pancreatic cancer;

[82] The method according to item

[77] or

[78] , wherein the cancer is selected from the group consisting of colorectal cancer, breast cancer, ovarian cancer, lung cancer, head and neck cancer, pancreatic cancer, gastric cancer, and mesothelioma;

[83] A method for treating cancer, comprising administering an effective amount of the Lp-γPMTX composition according to any one of items

[50] to

[66] to a subject having or at risk of having cancer cells expressing folate receptor on the surface bound by a targeting moiety;

[84] Maintenance therapy for a subject undergoing or having undergone cancer therapy, comprising administering an effective amount of the composition according to any one of items [1] to

[69] to the subject;

[85] Maintenance therapy for a subject undergoing or having undergone cancer therapy, comprising administering an effective amount of the liposomal gamma-polyglutamylated methotrexate composition according to any one of items

[12] to

[69] to the subject;

[86] A method for treating an immune system disorder, comprising administering an effective amount of the composition according to any one of items [1] to

[69] to a subject having or at risk of having an immune system disorder, and optionally, the immune system disorder is selected from inflammation (e.g., acute and chronic), systemic inflammation, rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn's disease, dermatomyositis / polymyositis, systemic lupus erythematosus, and Takayasu's disease, and psoriasis;

[87] A method for treating an immune system disorder, comprising administering an effective amount of the liposomal gamma-polyglutamylated methotrexate composition according to any one of items [8] to

[69] to a subject having or at risk of having an immune system disorder, and optionally, the immune system disorder is selected from inflammation (e.g., acute and chronic), systemic inflammation, rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn's disease, dermatomyositis / polymyositis, systemic lupus erythematosus, and Takayasu's disease, and psoriasis;

[88] The following treatment methods: (a) A method for treating an infectious disease, comprising the step of administering an effective amount of the composition according to any one of items [1] to

[69] to a subject having or at risk of having an infectious disease; (b) A method for treating an infectious disease, a cardiovascular disease, or another disease, comprising the step of administering an effective amount of the composition according to any one of items [1] to

[69] to a subject having or at risk of having an infectious disease, a cardiovascular disease, or another disease, wherein the disease is a member selected from atherosclerosis, cardiovascular disease (CVD), coronary artery disease, myocardial infarction, stroke, metabolic syndrome, gestational trophoblastic disease, and ectopic pregnancy; (c) A method for treating an autoimmune disease, comprising the step of administering an effective amount of the composition according to any one of items [1] to

[69] to a subject having or at risk of having an autoimmune disease; (d) A method for treating rheumatoid arthritis, comprising the step of administering an effective amount of the composition according to any one of items [1] to

[69] to a subject having or at risk of having rheumatoid arthritis; (e) A method for treating an inflammatory condition, comprising the step of administering an effective amount of the composition according to any one of items [1] to

[69] to a subject having or at risk of having inflammation, and optionally, wherein the inflammation is acute, chronic, and / or systemic inflammation; or (f) A method for treating a skin disease, comprising the step of administering an effective amount of the composition according to any one of items [1] to

[69] to a subject having or at risk of having a skin disease, and optionally, wherein the skin disease is psoriasis;

[89] A method for treating an infectious disease, comprising the step of administering an effective amount of the liposomal gamma-polyglutamate oxidized methotrexate composition according to any one of items

[12] to

[69] to a subject having or at risk of having an infectious disease;

[90] A method for delivering gamma-polyglutamyl oxidized methotrexate to tumors expressing folate receptor on the surface, comprising administering to a subject having a tumor an Lp-γPMTX composition according to any one of items [1] to

[69] in an amount sufficient to deliver a therapeutically effective amount of gamma-polyglutamyl oxidized methotrexate to the tumor;

[91] A method for preparing a gamma-polyglutamyl oxidized methotrexate composition comprising a liposomal gamma-polyglutamyl oxidized methotrexate composition according to any one of items

[12] to

[69] , comprising forming a mixture comprising a liposomal component and a gamma-polyglutamyl oxidized folic acid antagonist in solution; homogenizing the mixture in solution to form liposomes; and treating the mixture to form liposomes comprising gamma-polyglutamyl oxidized methotrexate;

[92] A method for preparing a composition according to any one of items

[12] to

[69] , comprising forming a mixture comprising a liposomal component and gamma-polyglutamyl oxidized methotrexate in solution; homogenizing the mixture in solution to form liposomes; treating the mixture to form liposomes encapsulating and / or coating gamma-polyglutamyl oxidized methotrexate; and attaching a targeting moiety on the surface of the liposomes, wherein the targeting moiety has specific affinity for at least one of folate receptor alpha (FR-α), folate receptor beta (FR-β) and folate receptor delta (FR-δ);

[93] The method according to item

[92] , wherein the treatment step comprises one or more steps of thin film hydration, extrusion, in-line mixing, ethanol injection technique, freeze-thaw method, reverse phase evaporation method, dynamic high pressure microfluidization, microfluidic mixing, double emulsion, freeze-dried double emulsion, 3D printing, membrane contactor method, and stirring; and / or

[94] The method according to item

[92] , wherein the processing step includes one or more steps of changing the size of the liposome by one or more steps of extrusion, high-pressure microfluidization, and / or ultrasonic treatment.

[0013] In some embodiments, the present disclosure provides a gamma-polyglutamylated methotrexate (γPMTX) composition, wherein at least two glutamyl residues of the gamma-polyglutamylated methotrexate have a gamma-carboxyl group bond. In some embodiments, γPMTX contains 2 to 20, 2 to 15, 2 to 10, 2 to 5, or more than 5 glutamyl groups (including the glutamyl groups of methotrexate). In some embodiments, γPMTX contains two or more L-type glutamyl groups. In other embodiments, γPMTX contains a D-type glutamyl group. In further embodiments, γPMTX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0014] In one embodiment, the γPMTX composition contains a chain of three glutamyl groups attached to the glutamyl group in methotrexate (i.e., tetraglutamylated methotrexate). In some embodiments, tetraglutamylated MTX contains two or more L-type glutamyl groups. In other embodiments, tetraglutamylated MTX contains a D-type glutamyl group. In further embodiments, tetraglutamylated MTX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0015] In one embodiment, the γPMTX composition contains a chain of four γ-glutamyl groups attached to the glutamyl group in methotrexate (e.g., γ-pentaglutamylated methotrexate). In some embodiments, gamma-pentaglutamylated MTX contains two or more L-type glutamyl groups. In other embodiments, gamma-pentaglutamylated MTX contains a D-type glutamyl group. In further embodiments, gamma-pentaglutamylated MTX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0016] In one embodiment, the γPMTX composition comprises a chain of five γ-glutamyl groups attached to the glutamyl group in methotrexate (e.g., γ-hexaglutamyl methotrexate). In some embodiments, gamma hexaglutamyl methotrexate comprises two or more L-type glutamyl groups. In other embodiments, gamma hexaglutamyl methotrexate comprises a D-type glutamyl group. In further embodiments, gamma hexaglutamyl methotrexate comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0017] In further embodiments, the present disclosure provides compositions comprising delivery carriers such as liposomes filled (e.g., encapsulated) with and / or otherwise conjugated to gamma polyglutamyl methotrexate, and methods of making a gamma PMTX-filled / conjugated delivery carrier composition (DV-γPMTX) and using the same to deliver gamma polyglutamyl methotrexate to diseased (e.g., cancerous) cells and / or target cells. These compositions have uses including, but not limited to, the treatment of diseases including hyperproliferative diseases such as cancer, immune system disorders such as inflammation and rheumatoid arthritis, and infectious diseases such as HIV and malaria. In some embodiments, the gamma polyglutamyl methotrexate in DV-γPMTX comprises 2-20, 2-15, 2-10, 2-5, more than 5, or more than 20 glutamyl groups (including the glutamyl group of methotrexate). The DV-γPMTX-filled / conjugated delivery carrier composition provides for the selective delivery of a higher cytotoxic payload (polyglutamyl methotrexate) compared to the cytotoxicity of methotrexate (MTX) administered in the monoglutamic acid state, resulting in improved efficacy and safety of methotrexate delivery to cancer cells.

[0018] In a further embodiment, the present disclosure provides a composition (Lp-γPMTX) comprising liposomes encapsulated (filled) with gamma-polyglutamyl oxidized methotrexate. In some embodiments, the gamma-polyglutamyl oxidized methotrexate in Lp-γPMTX comprises 2 to 20, 2 to 15, 2 to 10, 2 to 5, or more than 20 glutamyl groups (including the glutamyl group of methotrexate). In some embodiments, the gamma-polyglutamyl oxidized methotrexate in Lp-γPMTX comprises 2 or more L-type glutamyl groups. In other embodiments, the gamma-polyglutamyl oxidized methotrexate in Lp-γPMTX comprises a D-type glutamyl group. In a further embodiment, the gamma-polyglutamyl oxidized methotrexate in Lp-γPMTX comprises a D-type glutamyl group and 2 or more L-type glutamyl groups.

[0019] In one embodiment, the Lp-γPMTX composition comprises gamma-polyglutamyl oxidized MTX comprising a chain of 3 glutamyl groups attached to the glutamyl group of methotrexate (i.e., tetraglutamyl oxidized methotrexate). In some embodiments, the tetraglutamyl oxidized MTX comprises 2 or more L-type glutamyl groups. In other embodiments, the tetraglutamyl oxidized MTX comprises a D-type glutamyl group. In a further embodiment, the tetraglutamyl oxidized MTX comprises a D-type glutamyl group and 2 or more L-type glutamyl groups.

[0020] In one embodiment, the Lp-γPMTX composition comprises gamma-polyglutamyl oxidized MTX comprising a chain of 4 γ-glutamyl groups attached to the glutamyl group of methotrexate (e.g., γ-pentaglutamyl oxidized methotrexate). In some embodiments, the gamma-pentaglutamyl oxidized MTX comprises 2 or more L-type glutamyl groups. In other embodiments, the gamma-pentaglutamyl oxidized MTX comprises a D-type glutamyl group. In a further embodiment, the gamma-pentaglutamyl oxidized MTX comprises a D-type glutamyl group and 2 or more L-type glutamyl groups.

[0021] In one embodiment, the Lp-γPMTX composition comprises gamma polyglutamylated MTX comprising a chain of five γ-glutamyl groups attached to the glutamyl group of methotrexate (e.g., γ-hexaglutamylated methotrexate). In some embodiments, the gamma hexaglutamylated MTX comprises two or more L-type glutamyl groups. In other embodiments, the gamma hexaglutamylated MTX comprises a D-type glutamyl group. In further embodiments, the gamma hexaglutamylated MTX comprises a D-type glutamyl group and two or more L-type glutamyl groups.

[0022] In some embodiments, the Lp-γPMTX composition is cationic. In some embodiments, the Lp-γPMTX liposome is cationic and has a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therebetween. In further embodiments, the Lp-γPMTX liposome is cationic and the composition has a diameter in the range of 80 nm to 120 nm, or any range therebetween. In some embodiments, the cationic Lp-γPMTX composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w of gamma polyglutamylated MTX. In some embodiments, during the preparation process of Lp-γPMTX, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% of the starting material of gamma polyglutamylated MTX is encapsulated (enclosed) in the cationic Lp-γPMTX. In further embodiments, the gamma polyglutamylated methotrexate encapsulated by the liposome is present in the HEPES buffer within the liposome.

[0023] In other embodiments, the Lp-γPMTX composition is anionic or neutral. In some embodiments, the Lp-γPMTX liposomes are anionic or neutral and have a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therebetween. In further embodiments, the Lp-γPMTX liposomes are anionic or neutral and the composition has a diameter in the range of 80 nm to 120 nm, or any range therebetween. In some embodiments, the Lp-γPMTX liposomes are anionic and have a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therebetween. In further embodiments, the Lp-γPMTX liposomes are anionic and the composition has a diameter in the range of 80 nm to 120 nm, or any range therebetween. In some embodiments, the Lp-γPMTX liposomes are neutral and have a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therebetween. In some embodiments, the anionic or neutral Lp-γPMTX composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or greater than 75% w / w of gamma polyglutamylated MTX. In some embodiments, during the preparation process of Lp-γPMTX, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or greater than 75% of the starting material of gamma polyglutamylated MTX is encapsulated (enclosed) in the anionic or neutral Lp-γPMTX. In some embodiments, the anionic or neutral Lp-γPMTX composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or greater than 75% w / w of gamma tetraglutamylated MTX.In some embodiments, the anionic or neutral Lp-γPMTX composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w of gamma pentaglutamylated MTX. In some embodiments, the anionic or neutral Lp-γPMTX composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w of gamma hexaglutamylated MTX. In a further embodiment, the gamma polyglutamylated methotrexate encapsulated by the liposome is present in the HEPES buffer within the liposome.

[0024] In a further embodiment, the liposomal gamma polyglutamylated methotrexate composition is pegylated (PLp-γPMTX).

[0025] In some embodiments, the liposomal gamma polyglutamylated methotrexate composition is not targeted (NTLp-γPMTX). That is, the NTLp-γPMTX composition does not have a specific affinity for an epitope expressed on the surface of the target cell of interest (e.g., an epitope on a surface antigen). In a further embodiment, the non-targeted liposomal gamma polyglutamylated methotrexate composition is pegylated (NTPLp-γPMTX).

[0026] In other embodiments, the liposomal gamma-polyglutamyl methotrexate composition is targeted (TLp-γPMTX). That is, the TLp-γPMTX composition includes a targeting moiety having a specific affinity for an epitope (surface antigen) on the target cell of interest. In some embodiments, the targeting moiety of TLp-γPMTX or TPLp-γPMTX is not covalently bound to the liposome. In other embodiments, the targeting moiety of TLp-γPMTX or TPLp-γPMTX is bound to one or both of the liposome's PEG and outer surface. The targeted liposomal gamma-polyglutamyl methotrexate compositions (TLp-γPMTX and TPLp-γPMTX) provide further improvement over the efficacy and safety profiles of methotrexate by specifically delivering gamma-polyglutamylated (e.g., γ-pentaglutamylated, and / or γ-hexaglutamylated) methotrexate to target cells such as cancer cells. In some embodiments, the non-targeted liposomal gamma-polyglutamyl methotrexate composition is pegylated (TPLp-γPMTX). In some embodiments, the targeting moiety of TLp-γPMTX or TPLp-γPMTX is bound to one or both of the liposome's PEG and outer surface. In some embodiments, TLp-γPMTX or TPLp-γPMTX is covalently bound to the liposome. The functions of the targeting moiety of the TLp-γPMTX and / or TPLp-γPMTX composition include, but are not limited to, targeting the liposome to the target cell of interest in vivo or in vitro; interacting with the surface antigen to which the targeting moiety has a specific affinity; and delivering the liposome payload (γPMTX) to the cell. Suitable targeting moieties are known in the art and include, but are not limited to, antibodies, antigen-binding antibody fragments, scaffold proteins, polypeptides, and peptides. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting moiety is a polypeptide comprising at least 3, 5, 10, 15, 20, 30, 40, 50, or 100 amino acid residues.

[0027] In some embodiments, the targeting moiety of TLp-γPMTX or TPLp-γPMTX is an antibody or an antigen-binding antibody fragment. In further embodiments, the targeting moiety includes one or more of an antibody, a humanized antibody, an antigen-binding fragment of an antibody, a single-chain antibody, a single-domain antibody, a bispecific antibody, a synthetic antibody, a pegylated antibody, and a multimeric antibody. In some embodiments, the targeting moiety of TLp-γPMTX or TPLp-γPMTX has specific affinity for an epitope that is selectively expressed on target cells such as tumor cells compared to normal or non-tumor cells. In some embodiments, the targeting moiety has specific affinity for an epitope on a tumor cell surface antigen that is present on tumor cells but not present on or difficult to access on non-tumor cells. In some embodiments, the targeting moiety binds to the target epitope with an equilibrium dissociation constant (Kd) in the range of 0.5x10 -10 ~10x10 -6 as measured by BIACORE® analysis.

[0028] In certain embodiments, the targeting moiety of TLp-γPMTX or TPLp-γPMTX includes a polypeptide that specifically binds to a folate receptor. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the folate receptor to which the targeting moiety binds is specific for one or more folate receptors selected from the group consisting of folate receptor alpha (FR-α, FOLR1), folate receptor beta (FR-β, FOLR2), and folate receptor delta (FR-δ, FOLR4). In some embodiments, the folate receptor to which the targeting moiety binds is folate receptor alpha (FR-α). In some embodiments, the folate receptor to which the targeting moiety binds is folate receptor beta (FR-β). In some embodiments, the targeting moiety specifically binds to FR-α and FR-β.

[0029] In a further embodiment, the Lp-γPMTX composition comprises one or more of an immunostimulant, a detectable marker, and a maleimide disposed on at least one of the PEG of the liposome or the outer surface. In some embodiments, the liposomal γPMTX composition (e.g., Lp-γPMTX, PLp-γPMTX, NTLp-γPMTX, NTPLp-γPMTX, TLp-γPMTX, or TPLp-γPMTX) is cationic. In other embodiments, the liposomal γPMTX composition (e.g., Lp-γPMTX, PLp-γPMTX, NTLp-γPMTX, NTPLp-γPMTX, TLp-γPMTX, or TPLp-γPMTX) is anionic or neutral. In a further embodiment, the liposomes of the liposomal γPMTX composition (e.g., Lp-γPMTX, PLp-γPMTX, NTLp-γPMTX, NTPLp-γPMTX, TLp-γPMTX or TPLp-γPMTX) have a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, 50 nm to 150 nm, or any range therebetween. In some embodiments, the liposomes of the liposome-γPMTX composition have a diameter in the range of 30 nm to 175 nm or 50 nm to 150 nm, or any range therebetween. In a further embodiment, the liposomes of the liposomal γPMTX composition have a diameter in the range of 80 nm to 120 nm, or any range therebetween. In some embodiments, the liposomal γPMTX composition is pegylated (e.g., PLp-γPMTX, NTPLp-γPMTX, or TPLp-γPMTX). In some embodiments, the liposomal γPMTX composition comprises a targeting moiety (e.g., TLp-γPMTX or TPLp-γPMTX). In a further embodiment, the liposomal γPMTX composition is pegylated and targeted (e.g., TPLp-γPMTX). In some embodiments, the liposomal γPMTX composition comprises gamma polyglutamylated methotrexate containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the liposomal γPMTX composition comprises gamma tetraglutamylated methotrexate.In some embodiments, the liposomal γPMTX composition comprises gamma pentaglutamyl oxidized methotrexate. In other embodiments, the liposomal γPMTX composition comprises gamma hexaglutamyl oxidized methotrexate.

[0030] In some embodiments, the liposomal composition consists of gamma polyglutamyl oxidized methotrexate containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w of gamma polyglutamyl oxidized MTX. In some embodiments, the Lp-γPMTX composition comprises gamma polyglutamyl oxidized methotrexate containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups and 1% - 98.5% w / w of gamma polyglutamyl oxidized MTX. In some embodiments, the liposome comprises gamma polyglutamyl oxidized methotrexate containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups, and during the preparation process of Lp-γPMTX, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% of the starting material of gamma polyglutamyl oxidized folic MTX is encapsulated (enclosed) in Lp-γPMTX.

[0031] In some embodiments, the liposome composition consists of gamma-tetraglutamyl oxidized methotrexate and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w of gamma-tetraglutamyl oxidized MTX. In some embodiments, the Lp-γPMTX composition contains gamma-tetraglutamyl oxidized folic acid methotrexate and 1% - 98.5% w / w of gamma-tetraglutamyl oxidized MTX. In some embodiments, the liposome contains gamma-tetraglutamyl oxidized methotrexate, and during the preparation process of Lp-γPMTX, starting materials of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% of gamma-tetraglutamyl oxidized MTX are encapsulated (enclosed) in Lp-γPMTX.

[0032] In some embodiments, the liposome composition consists of gamma pentaglutamyl oxidized methotrexate and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w of gamma pentaglutamyl oxidized MTX. In some embodiments, the Lp-γPMTX composition contains gamma pentaglutamyl oxidized folic methotrexate and 1% - 98.5% w / w of gamma pentaglutamyl oxidized MTX. In some embodiments, the liposome contains gamma pentaglutamyl oxidized methotrexate, and during the preparation process of Lp-γPMTX, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% of the starting material of gamma pentaglutamyl oxidized MTX is encapsulated (enclosed) in Lp-γPMTX. In some embodiments, the liposome composition consists of gamma hexaglutamyl oxidized methotrexate and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w of gamma hexaglutamyl oxidized MTX. In some embodiments, the Lp-γPMTX composition contains gamma hexaglutamyl oxidized methotrexate and 1% - 98.5% w / w of gamma hexaglutamyl oxidized MTX. In some embodiments, the liposome contains gamma hexaglutamyl oxidized methotrexate, and during the preparation process of Lp-γPMTX, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% of the starting material of gamma pentaglutamyl oxidized MTX is encapsulated (enclosed) in Lp-γPMTX.

[0033] Liposome compositions comprising γPMTX-encapsulated liposomes are also provided. In some embodiments, the liposome composition comprises a pegylated γPMTX composition. In some embodiments, the liposome composition comprises a γPMTX composition linked or otherwise conjugated to a targeting moiety. In further embodiments, the liposome composition comprises a pegylated γPMTX composition linked or otherwise conjugated to a targeting moiety. In some embodiments, the liposome composition comprises γPMTX comprising 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the liposome composition comprises gamma tetraglutamyl oxidized methotrexate. In some embodiments, the liposome composition comprises gamma pentaglutamyl oxidized methotrexate. In other embodiments, the liposome composition comprises gamma hexaglutamyl oxidized methotrexate.

[0034] In some embodiments, the liposomal composition comprises liposomal γPMTX (e.g., Lp-γPMTX, PLp-γPMTX, NTLp-γPMTX, NTPLp-γPMTX, TLp-γPMTX, and TPLp-γPMTX). In some embodiments, the liposomal γPMTX is pegylated (e.g., NTPLp-γPMTX, TPLp-γPMTX). In some embodiments, the liposomal γPMTX comprises a targeting moiety having a specific affinity for an epitope of an antigen on the surface of a target cell such as a cancer cell (e.g., TLp-γPMTX or TPLp-γPMTX). In further embodiments, the liposomal composition comprises pegylated liposomal γPMTX and further comprises a targeting moiety having a specific affinity for an epitope of an antigen on the surface of a target cell such as a cancer cell (e.g., TPLp-γPMTX). In some embodiments, the liposomal composition comprises liposomal γPMTX that is cationic. In other embodiments, the liposomal composition comprises liposomal γPMTX that is anionic or neutral. In further embodiments, the liposomal composition comprises liposomal γPMTX having a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, or any range therebetween. In further embodiments, the liposomal γPMTX has a diameter in the range of 80 nm to 120 nm, or any range therebetween.

[0035] Also provided are pharmaceutical compositions comprising gamma-polyglutamylmethotrexate (γPMTX) comprising a delivery carrier such as liposomal γPMTX. In some embodiments, the pharmaceutical composition comprises a pegylated γPMTX composition. In some embodiments, the pharmaceutical composition comprises a γPMTX composition linked or otherwise conjugated to a targeting moiety. In further embodiments, the pharmaceutical composition comprises a pegylated γPMTX composition linked or otherwise conjugated to a targeting moiety. In some embodiments, the pharmaceutical composition comprises γPMTX comprising 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the pharmaceutical composition comprises gamma-tetra-glutamylmethotrexate. In some embodiments, the pharmaceutical composition comprises gamma-penta-glutamylmethotrexate. In other embodiments, the pharmaceutical composition comprises gamma-hexa-glutamylmethotrexate.

[0036] In some embodiments, the pharmaceutical composition comprises liposomal γPMTX (e.g., Lp-γPMTX, PLp-γPMTX, NTLp-γPMTX, NTPLp-γPMTX, TLp-γPMTX, and TPLp-γPMTX). In some embodiments, the liposomal γPMTX composition is pegylated (e.g., NTPLp-γPMTX, and TPLp-γPMTX). In some embodiments, liposomal γPMTX comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a target cell such as a cancer cell (e.g., TLp-γPMTX or TPLp-γPMTX). In further embodiments, the pharmaceutical composition comprises a pegylated liposomal γPMTX composition and further comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a target cell such as a cancer cell (e.g., TPLp-γPMTX). In some embodiments, the pharmaceutical composition comprises liposomal γPMTX that is cationic. In other embodiments, the pharmaceutical composition comprises liposomal γPMTX that is anionic or neutral. In further embodiments, the pharmaceutical composition comprises liposomal γPMTX having a diameter in the range of 20 nm to 500 nm or 20 nm to 500 nm, or any range therebetween. In further embodiments, the liposomal γPMTX composition has a diameter in the range of 80 nm to 120 nm, or any range therebetween.

[0037] In further embodiments, the present disclosure provides a method of killing cells, the method comprising contacting the cells with a composition comprising a gamma-polyglutamylated methotrexate (γPMTX) composition (e.g., γPMTX as disclosed herein). In some embodiments, the cells to be contacted are mammalian cells. In further embodiments, the cells to be contacted are human cells. In some embodiments, the cells to be contacted are hyperproliferative cells. In further embodiments, the hyperproliferative cells are cancer cells. In further embodiments, the cancer cells to be contacted are primary cells, or cells derived from cell lines obtained from cancers selected from the group consisting of non-hematological tumors such as lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological tumors such as leukemia, lymphoma and other B-cell malignancies, multiple myeloma and other plasma cell dyscrasias or malignancies. In further embodiments, the cancer cells to be contacted are primary cells, or cells derived from cell lines obtained from cancers selected from the group consisting of breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma) choriocarcinoma, and villous adenoma, aleukemic meningeal carcinomatosis, soft tissue sarcoma (desmoid tumor, aggressive fibromatosis), bladder cancer, and central nervous system (CNS) lymphoma. In still further embodiments, the cancer cells are primary cells, or cells derived from cell lines obtained from cancers selected from colorectal cancer, lung cancer, breast cancer, head and neck cancer, and pancreatic cancer. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, γPMTX comprises 4, 5, 2-10, 4-6, or more than 5 γ-glutamyl groups. In some embodiments, γPMTX comprises D-type γ-glutamyl groups. In some embodiments, γPMTX comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 D-type γ-glutamyl groups. In some embodiments, γPMTX comprises L-type γ-glutamyl groups.In some embodiments, γPMTX comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type γ-glutamyl groups. In some embodiments, γPMTX comprises L-type and D-type γ-glutamyl groups. In some embodiments, γPMTX comprises 2, 3, 4, 5, or more than 5 L-type γ-glutamyl groups and 1, 2, 3, 4, 5, or more than 5 D-type γ-glutamyl groups. In some embodiments, the γPMTX composition comprises gamma tetraglutamyl oxidized methotrexate. In some embodiments, the γPMTX composition comprises gamma pentaglutamyl oxidized methotrexate. In other embodiments, the γPMTX composition comprises gamma hexaglutamyl oxidized methotrexate.

[0038] In further embodiments, the present disclosure provides a method of killing cells, the method comprising contacting the cells with liposomes comprising gamma-polyglutamylated methotrexate (e.g., Lp-γPMTX such as PLp-γPMTX, NTLp-γPMTX, NTPLp-γPMTX, TLp-γPMTX or TPLp-γPMTX). In some embodiments, the cells to be contacted are mammalian cells. In further embodiments, the cells to be contacted are human cells. In some embodiments, the cells to be contacted are hyperproliferative cells. In still further embodiments, the hyperproliferative cells to be contacted are cancer cells. In further embodiments, the cancer cells are primary cells, or cells derived from cell lines obtained from cancers selected from the group consisting of non-hematological tumors including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological tumors including, for example, leukemia, lymphoma and other B cell malignancies, multiple myeloma and other plasma cell dyscrasias or cachexia. In further embodiments, the cancer cells to be contacted are primary cells, or cells derived from cell lines obtained from cancers selected from the group consisting of breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T cell lymphoma) choriocarcinoma, and villous adenoma, aleukemic meningeal carcinomatosis, soft tissue sarcoma (desmoid tumor, aggressive fibromatosis), bladder cancer, and central nervous system (CNS) lymphoma. In some embodiments, the cancer cells are primary cells, or cells derived from cell lines obtained from cancers selected from the group consisting of colorectal cancer, breast cancer, ovarian cancer, lung cancer, head and neck cancer, pancreatic cancer, gastric cancer, and mesothelioma. In still further embodiments, the cancer cells are primary cells, or cells derived from cell lines obtained from cancers selected from colorectal cancer, lung cancer, breast cancer, head and neck cancer, and pancreatic cancer. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, the liposomes comprise γPMTX comprising 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups.In some embodiments, the liposome comprises gamma-tetra-glutamyl oxidized methotrexate. In some embodiments, the liposome comprises gamma-penta-glutamyl oxidized methotrexate. In other embodiments, the liposome comprises gamma-hexa-glutamyl oxidized methotrexate.

[0039] In some embodiments, the liposome comprises γPMTX containing 4, 5, 2-10, 4-6, or more than 5 γ-glutamyl groups. In some embodiments, the liposome comprises γPMTX containing D-type γ-glutamyl groups. In some embodiments, the liposome comprises γPMTX containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 D-type γ-glutamyl groups. In some embodiments, the liposome comprises γPMTX containing L-type γ-glutamyl groups. In some embodiments, the liposome comprises γPMTX containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type γ-glutamyl groups. In some embodiments, the liposome comprises γPMTX containing both L-type and D-type γ-glutamyl groups. In some embodiments, the liposome comprises γPMTX containing 2, 3, 4, 5, or more than 5 L-type γ-glutamyl groups and 1, 2, 3, 4, 5, or more than 5 D-type γ-glutamyl groups. In some embodiments, the liposome comprises gamma-penta-glutamyl oxidized methotrexate. In other embodiments, the liposome comprises gamma-hexa-glutamyl oxidized methotrexate.

[0040] In further embodiments, the present disclosure provides a method for treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of a delivery carrier (e.g., an immune complex or liposome) comprising gamma-polyglutamyl oxidized methotrexate. In some embodiments, the delivery carrier is an antibody-containing immune complex (e.g., comprising a full-length IgG antibody, a bispecific antibody, or an scFv). In some embodiments, the delivery carrier is a liposome (e.g., an Lp-γPMTX such as PLp-γPMTX, NTLp-γPMTX, NTPLp-γPMTX, TLp-γPMTX, or TPLp-γPMTX). In some embodiments, the administered delivery carrier is pegylated. In some embodiments, the administered delivery carrier is not pegylated. In further embodiments, the administered delivery carrier comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of cancer cells.In a further embodiment, the delivery carrier comprises a targeting moiety having specific affinity for an epitope of a cell surface antigen selected from the group consisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-α, folate receptor-β or folate receptor-δ), mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV-1 (ZIP6), CGEN-15027, P cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1, EphA receptor, EphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha, PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the delivery carrier comprises a targeting moiety that specifically binds to a cell surface antigen derived from or identified as expressed in a particular subject's cancer (tumor), such as a neoantigen. In some embodiments, the targeting moiety has specific affinity for an epitope of a cell surface antigen derived from or identified as expressed in a particular subject's tumor, such as a neoantigen. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the administered delivery carrier comprises γPMTX comprising 4, 5, 2-10, 4-6, or more than 5 γ-glutamyl groups. In some embodiments, the delivery carrier comprises γPMTX comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 D-type γ-glutamyl groups. In some embodiments, the delivery carrier comprises γPMTX comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type γ-glutamyl groups. In some embodiments, the delivery carrier comprises γPMTX comprising 2, 3, 4, 5, or more than 5 L-type γ-glutamyl groups and 1, 2, 3, 4, 5, or more than 5 D-type γ-glutamyl groups. In some embodiments, the administered delivery carrier comprises gamma-tetra-glutamylated methotrexate. In some embodiments, the administered delivery carrier comprises gamma-penta-glutamylated methotrexate. In other embodiments, the administered delivery carrier comprises gamma-hexa-glutamylated methotrexate. In some embodiments, the administered delivery carrier comprises L-gamma-poly-glutamylated methotrexate. In some embodiments, the administered delivery carrier comprises D-gamma-poly-glutamylated methotrexate. In further embodiments, the administered delivery carrier comprises L- and D-gamma-poly-glutamylated methotrexate. In some embodiments, the cancer is, for example, a non-hematological tumor including, such as, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma, brain cancer, central nervous system cancer, and melanoma; and, for example, a hematological tumor selected from the group consisting of leukemia, lymphoma and other B cell malignancies, myeloma and other plasma cell dyscrasias or cachexia.In some embodiments, the cancer is selected from the group consisting of breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma, villous adenoma, aleukemic meningeal carcinomatosis, soft tissue sarcoma (desmoid tumor, aggressive fibromatosis), bladder cancer, and central nervous system (CNS) lymphoma. In some embodiments, the cancer is selected from the group consisting of colorectal cancer, breast cancer, ovarian cancer, lung cancer, head and neck cancer, pancreatic cancer, gastric cancer, and mesothelioma. In yet further embodiments, the cancer cells are primary cells or cells derived from a cell line obtained from / derived from a cancer selected from colorectal cancer, lung cancer, breast cancer, head and neck cancer, and pancreatic cancer.

[0041] In a further embodiment, the present disclosure provides a method for treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of liposomes comprising gamma-polyglutamyl oxidized methotrexate (e.g., Lp-γPMTX such as PLp-γPMTX, NTLp-γPMTX, NTPLp-γPMTX, TLp-γPMTX, or TPLp-γPMTX). In some embodiments, the liposomes are pegylated. In some embodiments, the liposomes are not pegylated. In a further embodiment, the liposomes comprise a targeting moiety having specific affinity for an epitope of an antigen on the surface of cancer cells.In a further embodiment, the liposome comprises a targeting moiety having specific affinity for an epitope of a cell surface antigen selected from the group consisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-α, folate receptor-β or folate receptor-δ), mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV-1 (ZIP6), CGEN-15027, P cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1, EphA receptor, EphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha, PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.This also includes the use of cancer stem cell targeting moieties such as those targeting CD34, CD133 and CD44, CD138, and CD15. In some embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope of a cell surface antigen derived from or identified as expressed on a particular subject's tumor, such as a neoantigen. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the liposome comprises γPMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposome of the administered liposome composition comprises γPMTX containing L-type γ-glutamyl groups. In some embodiments, the liposome of the administered liposome composition comprises γPMTX containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type γ-glutamyl groups. In some embodiments, the liposome of the administered liposome composition comprises γPMTX containing L- and D-type γ-glutamyl groups. In some embodiments, the liposome of the administered liposome composition comprises γPMTX containing 2, 3, 4, 5, or more than 5 L-type γ-glutamyl groups and 1, 2, 3, 4, 5, or more than 5 D-type γ-glutamyl groups. In some embodiments, the administered liposome composition comprises tetraglutaminated γPMTX. In some embodiments, the administered liposome composition comprises pentaglutaminated γPMTX. In some embodiments, the administered liposome composition comprises hexaglutaminated γPMTX. In some embodiments, the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, melanoma, and hematological tumors (e.g., leukemia or lymphoma). In yet further embodiments, the cancer cells are primary cells or cells derived from a cell line obtained from / derived from a cancer selected from colorectal cancer, lung cancer, breast cancer, head and neck cancer, and pancreatic cancer.

[0042] In a further embodiment, the present disclosure provides a method for treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of a liposomal composition comprising liposomes comprising gamma-polyglutamyl oxidized methotrexate and a targeting moiety having specific affinity for an epitope of an antigen on the cancer surface.In some embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope of a cell surface antigen selected from the group consisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-α, folate receptor-β or folate receptor-δ), mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV-1 (ZIP6), CGEN-15027, P cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1, EphA receptor, EphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha, PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the liposome comprises a targeting moiety having specific affinity for an epitope of a cell surface antigen derived from or identified as expressed on a particular subject's cancer (tumor), such as a neoantigen. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the liposome comprises γPMTX comprising 4, 5, 2 to 10, 4 to 6, or more than 5 γ-glutamyl groups. In some embodiments, the liposome comprises γPMTX comprising L-type γ-glutamyl groups. In some embodiments, the liposome comprises γPMTX comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type γ-glutamyl groups. In some embodiments, the liposome comprises γPMTX comprising D-type γ-glutamyl groups. In some embodiments, the liposome comprises γPMTX comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 D-type γ-glutamyl groups. In some embodiments, the liposome comprises gamma tetraglutamyl methotrexate. In some embodiments, the liposome comprises gamma pentaglutamyl methotrexate. In some embodiments, the liposome comprises gamma hexaglutamyl methotrexate.

[0043] In some embodiments, the administered liposomal composition comprises pegylated liposomes (e.g., TPLp-γPMTX). In some embodiments, the administered liposomal composition comprises non-pegylated liposomes. In some embodiments, the liposomes of the administered liposomal composition comprise γPMTX containing 4, 5, 2 to 10, 4 to 6, or more than 5 gamma glutamyl groups. In some embodiments, the liposomes of the administered liposomal composition comprise γPMTX containing D-type gamma-glutamyl groups. In some embodiments, the liposomes of the administered liposomal composition comprise γPMTX containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 D-type gamma-glutamyl groups. In some embodiments, the liposomes of the administered liposomal composition comprise γPMTX containing L-type gamma-glutamyl groups. In some embodiments, the liposomes of the administered liposomal composition comprise γPMTX containing 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type gamma-glutamyl groups. In some embodiments, the liposomes of the administered liposomal composition comprise γPMTX containing both L- and D-type gamma-glutamyl groups. In some embodiments, the liposomes of the administered liposomal composition comprise γPMTX containing 2, 3, 4, 5, or more than 5 L-type gamma-glutamyl groups and 1, 2, 3, 4, 5, or more than 5 D-type gamma-glutamyl groups. In some embodiments, the liposomes of the administered liposomal composition comprise gamma tetraglutamyl methotrexate. In some embodiments, the liposomes of the administered liposomal composition comprise gamma pentaglutamyl methotrexate. In other embodiments, the liposomes of the administered liposomal composition comprise gamma hexaglutamyl methotrexate. In some embodiments, the liposomal composition is administered for treating cancer selected from the group consisting of lung cancer (e.g., non-small cell), pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, melanoma, myeloma, leukemia and lymphoma.

[0044] In a further embodiment, the present disclosure provides a method for treating cancer, the method comprising administering to a subject having or at risk of having cancer that expresses a folate receptor on its cell surface an effective amount of a liposomal composition, the liposomal composition comprising liposomes that contain (a) gamma-polyglutamylated methotrexate (γPMTX) and (b) a targeting moiety having specific binding affinity for the folate receptor. In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-α), folate receptor beta (FR-β), and / or folate receptor delta (FR-δ). In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-α), folate receptor beta (FR-β) and / or folate receptor delta (FR-δ). In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-α) and folate receptor beta (FR-β). In some embodiments, the liposomal composition to be administered comprises pegylated liposomes (e.g., TPLp-γPMTX). In some embodiments, the liposomal composition to be administered comprises non-pegylated liposomes. In some embodiments, the liposomes of the liposomal composition to be administered comprise γPMTX that contains 4, 5, 2 to 10, 4 to 6, or more than 5 γ-glutamyl groups. In some embodiments, the liposomes of the liposomal composition to be administered comprise γPMTX that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 D-type γ-glutamyl groups. In some embodiments, the liposomes of the liposomal composition to be administered comprise γPMTX that contains 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type γ-glutamyl groups. In some embodiments, the liposomes of the liposomal composition to be administered comprise γPMTX that contains 2, 3, 4, 5, or more than 5 L-type γ-glutamyl groups and 1, 2, 3, 4, 5, or more than 5 D-type γ-glutamyl groups. In some embodiments, the liposomes of the liposomal composition to be administered comprise gamma-pentaglutamylated methotrexate.In other embodiments, the liposomes of the administered liposomal composition comprise gamma hexaglutaminyl oxidized methotrexate. In some embodiments, the liposomal composition is administered for treating cancer selected from the group consisting of non - hematological tumors such as, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological tumors such as, for example, leukemia, lymphoma and other B - cell malignancies, multiple myeloma and other plasma cell dyscrasias or malignancies. In some embodiments, the liposomal composition is administered for treating cancer selected from the group consisting of breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non - Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T - cell lymphoma), choriocarcinoma, villous adenoma, non - leukemic meningeal carcinomatosis, soft tissue sarcoma (desmoid tumor, aggressive fibromatosis), bladder cancer, and central nervous system (CNS) lymphoma. In some embodiments, the liposomal composition is administered for treating cancer selected from the group consisting of colorectal cancer, breast cancer, ovarian cancer, lung cancer, head and neck cancer, pancreatic cancer, gastric cancer, and mesothelioma.

[0045] In further embodiments, the present disclosure provides a method for maintenance therapy of cancer, the method comprising administering to a subject undergoing or having undergone cancer therapy an effective amount of a liposomal composition comprising liposomes (Lp-γPMTX) comprising gamma-polyglutamyl methotrexate. In some embodiments, the liposomal composition to be administered is PLp-γPMTX, NTLp-γPMTX, NTPLp-γPMTX, TLp-γPMTX or TPLp-γPMTX. In some embodiments, the liposomal composition to be administered comprises pegylated liposomes (e.g., PLp-γPMTX, NTPLp-γPMTX, or TPLp-γPMTX). In some embodiments, the liposomal composition to be administered comprises targeted liposomes (e.g., TLp-γPMTX or TPLp-γPMTX). In some embodiments, the liposomal composition to be administered comprises liposomes that are pegylated and comprise a targeting moiety (e.g., TPLp-γPMTX). In some embodiments, the liposomes of the liposomal composition to be administered comprise gamma-polyglutamyl methotrexate comprising 4, 5, 2-10, 4-6, or more than 5 gamma-glutamyl groups. In some embodiments, the liposomes of the liposomal composition to be administered comprise γPMTX comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 D-type gamma-glutamyl groups. In some embodiments, the liposomes of the liposomal composition to be administered comprise γPMTX comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type gamma-glutamyl groups. In some embodiments, the liposomes of the liposomal composition to be administered comprise γPMTX comprising 2, 3, 4, 5, or more than 5 L-type gamma-glutamyl groups and 1, 2, 3, 4, 5, or more than 5 D-type gamma-glutamyl groups. In some embodiments, the liposomes of the liposomal composition to be administered comprise gamma-tetra-glutamyl methotrexate. In some embodiments, the liposomes of the liposomal composition to be administered comprise gamma-penta-glutamyl methotrexate. In other embodiments, the liposomes of the liposomal composition to be administered comprise gamma-hexa-glutamyl methotrexate.

[0046] In further embodiments, the present disclosure provides a method for treating an immune system disorder, the method comprising administering to a subject having or at risk of having an immune system disorder an effective amount of a liposomal composition comprising gamma polyglutamylated methotrexate (e.g., Lp-γPMTX, PLp-γPMTX, NTLp-γPMTX, NTPLp-γPMTX, TLp-γPMTX or TPLp-γPMTX). In some embodiments, the liposomal composition is administered to treat an autoimmune disease. In further embodiments, the liposomal composition is administered to treat rheumatoid arthritis. In another embodiment, the liposomal composition is administered to treat inflammation. In some embodiments, the liposomal composition administered comprises a pegylated liposome (e.g., PLp-γPMTX, NTPLp-γPMTX, or TPLp-γPMTX). In some embodiments, the liposomal composition administered comprises a targeted liposome (e.g., TLp-γPMTX or TPLp-γPMTX) comprising a targeting moiety having specific affinity for a surface antigen on a target cell of interest (e.g., an immune cell). In further embodiments, the liposomal composition administered comprises a liposome that is pegylated and comprises a targeting moiety (e.g., TPLp-γPMTX). In some embodiments, the liposome of the liposomal composition administered comprises gamma pentaglutamylated methotrexate comprising 4, 5, 2-10, 4-6, or more than 5 gamma-glutamyl groups. In some embodiments, the liposome of the liposomal composition administered comprises γPMTX comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 D-type gamma-glutamyl groups. In some embodiments, the liposome of the liposomal composition administered comprises γPMTX comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type gamma-glutamyl groups. In some embodiments, the liposome of the liposomal composition administered comprises γPMTX comprising 2, 3, 4, 5, or more than 5 L-type gamma-glutamyl groups and 1, 2, 3, 4, 5, or more than 5 D-type gamma-glutamyl groups.In some embodiments, the liposomes of the administered liposomal composition comprise gamma-tetra-glutamyl-methotrexate. In some embodiments, the liposomes of the administered liposomal composition comprise gamma-penta-glutamyl-methotrexate. In other embodiments, the liposomes of the administered liposomal composition comprise gamma-hexa-glutamyl-methotrexate.

[0047] The present disclosure also provides a method for delivering gamma-poly-glutamyl-methotrexate to tumors and / or cancer cells, the method comprising administering to a subject having a tumor a composition comprising gamma-poly-glutamyl-methotrexate (L-γPMTX) and a targeting moiety having specific binding affinity for an epitope on the surface antigen of a tumor cell or cancer cell. In some embodiments, the administered targeting moiety is bound to a delivery carrier. In some embodiments, the delivery carrier is an antibody or an antigen-binding fragment of an antibody. In further embodiments, the delivery carrier is a liposome. In further embodiments, the antibody, antigen-binding antibody fragment, or liposome is a pegylated liposome (e.g., TPLp-γPMTX). In some embodiments, the administered composition comprises gamma-poly-glutamyl-methotrexate having 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the administered composition comprises gamma-tetra-glutamyl-methotrexate. In some embodiments, the administered composition comprises gamma-penta-glutamyl-methotrexate. In other embodiments, the administered composition comprises gamma-hexa-glutamyl-methotrexate.

[0048] In a further embodiment, the present disclosure provides a method of making a liposomal composition comprising a liposomal gamma-polyglutamyl methotrexate (γPMTX) composition, the method comprising forming a mixture comprising liposomal components and gamma-polyglutamyl methotrexate in solution; homogenizing the mixture in solution to form liposomes; and treating the mixture to form liposomes comprising polyglutamyl methotrexate. In some embodiments, the gamma-polyglutamyl methotrexate comprises 4, 5, 2-10, 4-6, or more than 5 γ-glutamyl groups. In some embodiments, the γPMTX composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 D-type γ-glutamyl groups. In some embodiments, the γPMTX composition comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 L-type γ-glutamyl groups. In some embodiments, the γPMTX composition comprises 2, 3, 4, 5, or more than 5 L-type γ-glutamyl groups and 1, 2, 3, 4, 5, or more than 5 D-type γ-glutamyl groups. In some embodiments, the γPMTX composition comprises gamma-pentaglutamyl methotrexate. In some embodiments, the γPMTX composition comprises gamma-tetraglutamyl methotrexate. In other embodiments, the γPMTX composition comprises gamma-hexaglutamyl methotrexate.

[0049] In one embodiment, the present disclosure provides a kit comprising a gamma-polyglutamyl methotrexate composition and / or a γPMTX delivery carrier such as liposomes comprising γPMTX and γPMTX immune complexes (e.g., ADCs described herein).

Brief Description of the Drawings

[0050]

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Mode for Carrying Out the Invention

[0051] In general, the present disclosure relates to gamma - polyglutamylated methotrexate compositions. The compositions provide an advancement over prior treatments for hyperproliferative diseases such as cancer. Methods of manufacturing, delivering, and using gamma - polyglutamylated methotrexate compositions are also provided. Gamma - polyglutamylated compositions have uses including, but not limited to, the treatment or prevention of hyperproliferative diseases such as cancer, immune system disorders such as inflammation and rheumatoid arthritis, and infectious diseases such as HIV and malaria.

[0052] I. Definitions Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0053] When an embodiment is described herein with the term "comprising", other similar embodiments described with the terms "containing", "consisting of", and / or "consisting essentially of" are always provided as well. However, when used as a transitional phrase in the claims, each should be interpreted separately and in the appropriate legal and factual context (e.g., in the claims, the transitional phrase "comprising" is considered a more open-ended phrase, while the transitional phrase "consisting of" is considered more exclusive, and "consisting essentially of" is considered intermediate between these).

[0054] As used herein, the singular forms "a", "an", and "the" include plural referents unless specifically stated otherwise or unless it is clear from the context that only a single referent is intended.

[0055] The term "and / or" as used in expressions such as "A and / or B" is intended herein to include both A and B; A or B; A alone; and B alone. Similarly, the term "and / or" as used in expressions such as "A, B, and / or C" encompasses each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0056] Headings and subheadings are used for convenience and / or only for compliance with official rules, and do not limit the subject technology nor are they referred to in relation to the interpretation of the description of the subject technology. Features described under one heading or one subheading of the subject disclosure may be combined with features described under other headings or subheadings in various embodiments. Further, not all features under a single heading or a single subheading are necessarily used together in some embodiments.

[0057] Unless otherwise indicated, the terms "methotrexate" and "MTX" are used interchangeably and include salts, acids and / or free base forms of methotrexate (e.g., methotrexate disodium). Compositions containing MTX salts may further contain any of various cations, such as Na + Mg 2+ K + NH 4 + and / or Ca 2+ In certain embodiments, typically, the salt is a pharmaceutically acceptable salt. In further particular embodiments, the MTX salt contains Na + Methotrexate usually contains one L-gamma-glutamyl group and thus, for the purposes of the present disclosure, is considered to be monoglutaminated.

[0058] The terms "polyglutamylated methotrexate", "polyglutamylated MTX", "MTX-PG", and "PMTX" are used interchangeably herein and refer to a methotrexate composition that contains at least one glutamyl group in addition to the glutamyl group in methotrexate (i.e., MTX-PGn, n≧1). References herein to the number of glutamyl groups in γPMTX (MTX-PG) include the glutamyl group in methotrexate. For example, an MTX-PG composition that contains five glutamyl residues in addition to the glutamyl group of MTX is referred to herein as hexaglutamylated methotrexate or methotrexate hexaglutamate. The polyglutamate chain contains an N-terminal glutamyl group and one or more C-terminal glutamyl groups. The N-terminal glutamyl group of the polyglutamate chain does not bind to another glutamyl group via its amino group, but binds to one or more glutamyl groups via its carboxylic acid group. In some embodiments, the N-terminal glutamyl group of polyglutamylated methotrexate is the glutamyl group of methotrexate. The C-terminal glutamyl group(s) of the polyglutamate chain bind to another glutamyl group via their amino groups, but do not bind to another glutamyl group via their carboxylic acid groups.

[0059] The terms "gamma-glutamyl group", "gamma-glutamyl group", and "gamma bond", when they relate to the bonding of glutamyl groups, mean a glutamyl group including a gamma-carboxyl group bond. The gamma bond is not between a glutamyl group and the glutamyl group in methotrexate, or between a glutamyl group and what is present in methotrexate (e.g., a glutamyl group within a polyglutamate chain bound to methotrexate), but can be a bond between a glutamyl group and a second glutamyl group such as a glutamyl group within a polyglutamate chain bound to methotrexate. In some embodiments, the gamma bond is an amide bond between the gamma-carboxyl group of one glutamyl group and a second glutamyl group. In some embodiments, the gamma bond means an amide bond of the glutamyl group of methotrexate. In some embodiments, the gamma bond is an amide bond between the gamma-carboxyl group of one glutamyl group and a second glutamyl group. A reference to a gamma bond includes, unless otherwise specified or clearly apparent from the context that it is not intended, the gamma bond of the glutamyl group of methotrexate. In some embodiments, the gamma-glutamyl group is of the L-type. In some embodiments, the gamma-glutamyl group is of the D-type. As discussed herein, during methotrexate therapy, methotrexate enters cells and is polyglutamylated by the enzyme folylpolyglutamate synthase (FPGS), and FPGS sequentially adds L-glutamyl groups to the gamma-carboxyl group of glutamate within the methotrexate glutamyl group of methotrexate. Thus, D-gamma-polyglutamylated methotrexate compositions are not formed intracellularly during methotrexate therapy.

[0060] The terms "gamma-polyglutamylated methotrexate", "γ-polyglutamylated methotrexate", "γPMTX", "gamma-polyglutamylated methotrexate", "polyglutamylated MTX", "γMTX-PG", and iterations thereof are used interchangeably herein and mean a methotrexate composition that includes at least one gamma-glutamyl group having a gamma-carboxyl group bond in addition to the gamma-glutamyl groups in methotrexate (i.e., MTX-PGn, where n ≧ 1 gamma-glutamyl groups). References herein to the number of glutamyl groups in γPMTX (γMTX-PG) include the glutamyl groups in methotrexate. For example, a γMTX-PG composition that includes five γ-glutamyl groups in addition to the glutamyl groups of MTX is referred to herein as gamma-hexaglutamylated methotrexate or gamma-methotrexate hexaglutamate.

[0061] The terms "alpha-glutamyl group", "α-glutamyl group", and "alpha bond", when referring to the bonding of glutamyl groups, mean a glutamyl group that includes an alpha-carboxyl group bond.

[0062] As used herein, the term "isolated" means a composition in a form not found in nature. Isolated gamma polyglutamine oxidized compositions include those that are purified to the extent that they are no longer in the form found in nature. In some embodiments, the isolated gamma polyglutamine oxidized methotrexate is substantially pure. An isolated composition is free or substantially free of naturally incorporated substances such as other cellular components such as proteins and nucleic acids that may potentially be found in nature or in the environment in which they are produced (e.g., cell culture). Gamma polyglutamine oxidized compositions can be formulated with diluents or adjuvants and further isolated for practical purposes - for example, when used in a diagnostic or therapeutic, the gamma polyglutamine oxidized composition is usually mixed with a pharmaceutically acceptable carrier or diluent. In some embodiments, the isolated gamma polyglutamine oxidized composition (e.g., delivery carriers such as gamma polyglutamate and liposomes containing gamma polyglutamate) contains less than 1% or less than 0.1% of undesirable DNA or protein contaminants. In some embodiments, the gamma polyglutamate composition (e.g., delivery carriers such as gamma polyglutamate and liposomes containing gamma polyglutamate) is "isolated".

[0063] As used herein, the term "targeting moiety" means a molecule that confers enhanced affinity for a selected target, such as a cell, cell type, tissue, organ, body region, or compartment, e.g., a compartment of a cell, tissue or organ. The targeting moiety can include a wide variety of substances. The targeting moiety includes natural molecules, or recombinant or synthetic molecules. In some embodiments, the targeting moiety is an antibody antigen-binding antibody fragment, bispecific antibody or other antibody-based molecule or compound. In some embodiments, the targeting moiety is an aptamer, avimer, receptor-binding ligand, nucleic acid, biotin-avidin binding pair, peptide, protein, carbohydrate, lipid, vitamin, toxin, microbial component, hormone, receptor ligand or any derivative thereof. Other targeting moieties are known in the art and are encompassed by the present disclosure.

[0064] The terms "specific affinity," "specifically binds," and "enhanced affinity" mean that a targeting moiety, such as an antibody or an antigen-binding antibody fragment, reacts with or binds to an epitope, protein, or target molecule with a higher frequency, more rapidly, for a longer period of time, with a greater affinity, or in some combination of these, than it does to another substance that includes a protein unrelated to the antigen that includes the target epitope. Due to sequence identity between homologous proteins in different species, in some embodiments, a particular affinity includes a binding substance that recognizes an epitope and / or a target molecule on a protein in two or more species. Similarly, due to homology within a particular region of the polypeptide sequences of different proteins, the terms "specific affinity" or "specific binding" may include a binding substance that recognizes an epitope present on two or more proteins and / or target molecules. In certain embodiments, it is understood that a targeting moiety that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific affinity" does not necessarily (although it can) require exclusive binding, e.g., binding to only one epitope on one target. Thus, in certain embodiments, a targeting moiety may specifically bind to an epitope present on two or more targets. In certain embodiments, multiple targets may be bound by the same targeting moiety that specifically binds to epitopes present on the multiple targets.

[0065] The term "epitope" means a part of an antigen that can be recognized and specifically bound by a targeting moiety (i.e., a binding moiety), such as an antibody. When the antigen is a polypeptide, the epitope can be formed from both contiguous and non-contiguous amino acids juxtaposed by the tertiary folding of the protein. Epitopes formed from contiguous amino acids are usually retained upon protein denaturation, whereas epitopes formed by tertiary folding are usually lost upon protein denaturation. An epitope typically includes at least three amino acids, more generally at least five or eight to ten amino acids, in a unique spatial higher-order structure.

[0066] Expressions such as "binding affinity for a target", "binding to a target", "enhanced affinity" that are known in the art, and similar expressions, refer to properties of a targeting moiety that can be directly measured by determining an affinity constant, e.g., the amount of targeting moiety that binds and dissociates at a given antigen concentration. Without limitation, other methods such as competitive analysis, equilibrium analysis and microcalorimetry, and real-time interaction analysis based on surface plasmon resonance interactions (e.g., using a BIACORE® device) can be used to characterize intermolecular interactions. These methods are well known to those skilled in the art and are described, for example, in Neri et al., Tibtech 14:465-470 (1996), and Jansson et al., J. Biol. Chem. 272:8189-8197 (1997).

[0067] The term "delivery carrier" generally means any composition that acts to assist, facilitate or ease the entry of gamma-polyglutamyl-methotrexate into cells. Such delivery carriers are known in the art and include, without limitation, liposomes, lipospheres, polymers (e.g., polymer conjugates), peptides, proteins such as antibodies (e.g., immunoconjugates such as antibody-drug conjugates (ADCs) and antigen-binding antibody fragments and derivatives thereof), cell components, cyclic oligosaccharides (e.g., cyclodextrins), micelles, microparticles (e.g., microspheres), nanoparticles (e.g., lipid nanoparticles, biodegradable nanoparticles, and core-shell nanoparticles), hydrogels, lipoprotein particles, viral sequences, viral substances, or lipid or liposome formulations, and combinations thereof. The delivery carrier can be bound directly or indirectly to the targeting moiety. In some embodiments, the targeting moiety is selected from a polymer, protein, peptide, monoclonal antibody or fatty acid lipid.

[0068] "Subject" means a human or, without limitation, a vertebrate mammal including a dog, cat, horse, goat, and primates such as a monkey. Thus, the present invention can also be used to treat diseases or conditions in non-human subjects. For example, cancer is one of the leading causes of death in companion animals (e.g., cats and dogs). In some embodiments of the present invention, the subject is a human. In the present disclosure, the terms "subject" and "patient" are used interchangeably and have the same meaning. Generally, the maximum dose, i.e., the maximum safe dose according to sound medical judgment, is preferably used.

[0069] As used herein, "effective amount" means an amount of a drug sufficient to produce a medically desired result. The effective amount can vary depending on the desired outcome, the particular condition to be treated or prevented, the age and health of the subject being treated, the severity of the condition, the duration of the treatment, the nature of concurrent or concomitant therapies (if any), the specific route of administration, and similar factors within the knowledge and expertise of the health care practitioner. The "effective amount" can be determined experimentally or routinely in relation to the indicated purpose. In the case of cancer, an effective amount of a drug reduces the number of cancer cells; reduces the size of the tumor; inhibits the invasion of cancer cells into surrounding organs (i.e., slows down and preferably stops to some extent); inhibits the metastasis of the tumor (i.e., slows down and preferably stops to some extent); inhibits the growth of the tumor to some extent; and / or alleviates one or more of the symptoms associated with the disorder to some extent. Depending on the extent to which the drug can prevent and / or kill the growth of existing cancer cells, the drug can be cytostatic and / or cytotoxic. For cancer therapy, in vivo efficacy can be measured, for example, by evaluating survival time, progression-free survival (PFS) period, response rate (RR), response duration, and / or quality of life.

[0070] The terms "hyperproliferative disorder", "proliferative disease", and "proliferative disorder" are used interchangeably herein and relate to unwanted or abnormal cell growth, such as neoplastic or hyperplastic growth, of unwanted or uncontrolled cells, whether in vitro or in vivo. In some embodiments, a proliferative disease is a cancer, tumor, and / or tumor metastasis, regardless of the location of the cancer, tumor, and / or tumor metastasis, and includes cancer or tumor diseases (including benign or cancerous) and / or any tumor metastasis. In some embodiments, a proliferative disease is a benign or malignant tumor. In some embodiments, a proliferative disease is a non-cancerous disease. In some embodiments, a proliferative disease is a hyperproliferative condition such as hyperplasia, fibrosis (particularly pulmonary, but also other types such as renal fibrosis), angiogenesis, psoriasis, atherosclerosis, and smooth muscle proliferation in blood vessels such as stenosis or restenosis after angioplasty.

[0071] "Cancer", "tumor", or "malignant tumor" are used as synonymous terms and mean any of a number of cell types or diseases characterized by uncontrolled, abnormal growth of cells, local or spread (metastasis) of the infected cells to other parts of the body via the bloodstream and lymphatic system, and / or a number of characteristic structures and / or molecular features known to be associated with these cell types or diseases. As used herein, "tumor" means all neoplastic cell growth and proliferation, and all precancerous and cancerous cells and tissues, regardless of malignancy or benignity. "Cancerous tumor", or "malignant cells" are understood to be cells with specific structural characteristics, lacking differentiation, and capable of invasion and metastasis. Cancers that can be treated with the γPMTX compositions provided herein include, but are not limited to, non-hematological tumors such as lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological tumors such as leukemia, lymphoma and other B cell malignancies, myeloma and other plasma cell dyscrasias or cachexia. In some embodiments, the cancer is selected from the group consisting of colorectal cancer, breast cancer, ovarian cancer, lung cancer, head and neck cancer, pancreatic cancer, gastric cancer, and mesothelioma.

[0072] Other types of cancers and tumors that can be treated with the γPMTX composition are described herein or are known in the art. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.

[0073] The terms "treating," "treatment," or "treat" mean both (a) therapeutic means that cure, slow, mitigate, and / or halt the progression of the symptoms of a diagnosed medical condition or disorder and (b) prophylactic or preventive means that prevent and / or delay the occurrence of a targeted disease or condition. Thus, subjects in need of treatment include subjects already having the cancer, disorder, or disease, subjects at risk of becoming a cancer or condition, and subjects in whom an infection or condition is to be prevented. A subject is identified as "at risk of having" a cancer, infectious disease, immune system disorder, hyperproliferative disease, or another disease or disorder mentioned herein using well-known medical and diagnostic techniques. In certain embodiments, a subject is "being treated successfully" by the methods provided herein if the subject exhibits, for example, an overall, partial, or temporary remission or alleviation of symptoms associated with a disease or condition (e.g., cancer, inflammation, and rheumatoid arthritis). In certain embodiments, the term "treating" or "treatment" or "treat" means an improvement in at least one measurable physical parameter of a proliferative disorder, such as tumor growth, which may not be distinguishable by the patient. In other embodiments, the term "treating" or "treatment" or "treat" means inhibiting the progression of a proliferative disorder, for example, physically by stabilization of distinguishable symptoms or physiologically by stabilization of physical parameters, or both. In other embodiments, the term "treating" or "treatment" or "treat" means a decrease or stabilization in size, tumor cell proliferation or survival, or cancer cell number. The treatment can use the γ-PMTX composition alone or in combination with additional therapeutic agents.

[0074] "Subject", "patient", and "animal" are used interchangeably and mean mammalian patients such as human patients and non-human primates, as well as laboratory animals such as rabbits, rats, mice, and other animals. Animals include all vertebrates, for example, mammals and non-mammals such as chickens, amphibians, and reptiles. As used herein, "mammal" includes, but is not limited to, humans and non-human primate animals such as chimpanzees and other apes and monkey species; livestock animals such as cows, sheep, pigs, goats, and horses; pet mammals such as dogs and cats; laboratory animals such as rodents such as mice, rats, guinea pigs, and other members of the mammalian class known in the art. In certain embodiments, the subject is human.

[0075] As used herein, "treatment of a proliferative disorder" includes maintaining or reducing the tumor size of a subject with a proliferative disorder, inducing tumor shrinkage (partial or complete), suppressing tumor growth, and / or extending lifespan. In one embodiment, the proliferative disorder is a solid tumor. Such tumors include, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma. In one embodiment, the proliferative disorder is a hematological tumor. Such hematological tumors include, for example, leukemia, lymphoma and other B-cell malignancies, myeloma and other plasma cell dyscrasias or malignancies.

[0076] As used herein, the term "autoimmune disease" is defined as a disorder resulting from an autoimmune reaction. Autoimmune diseases are the result of an inappropriate and excessive reaction against self-antigens. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis are specifically mentioned.

[0077] As used herein, the term "therapeutic agent" means an agent or its derivative that can interact with hyperproliferative cells such as cancer cells or immune cells, thereby reducing the proliferative state of the cells and / or killing the cells. Examples of therapeutic agents include, but are not limited to, chemotherapeutic agents, cytotoxic agents, platinum-based agents (e.g., cisplatin, carboplatin, oxaliplatin), taxanes (e.g., paclitaxel), etoposide, alkylating agents (e.g., cyclophosphamide, ifosfamide), antimetabolites (e.g., methotrexate (MTX)), 5-fluorouracil, gemcitabine, or their derivatives), antitumor antibiotics (e.g., mitomycin, doxorubicin), plant-derived antitumor agents (e.g., vincristine, vindesine, paclitaxel). Such agents further include, but are not limited to, the anticancer agents trimethoprim, temozolomide, methotrexate, S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benzylguanidine (6-BG), bis-chloronitrosourea (BCNU) and camptothecin, or any of their therapeutic derivatives. Further examples of therapeutic agents that may be suitable for use by the methods of the present disclosure include, but are not limited to, anti-restenosis agents, pro-proliferative or anti-proliferative agents, anti-inflammatory agents, anti-neoplastic agents, anti-mitotic agents, antiplatelet agents, anticoagulants, antifibrin agents, antithrombin agents, cell growth inhibitory agents, antibiotics and other anti-infective agents, anti-enzyme agents, antimetabolites, angiogenesis agents, cytoprotective agents, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists and / or cardioprotective agents. "Therapeutic agent" also means salts, acids, and free base forms of the above agents.

[0078] As used herein, the term "chemotherapeutic agent", when used in the context of cancer therapy, means any agent that causes the death of cancer cells or inhibits the growth or spread of cancer cells. Examples of such chemotherapeutic agents include alkylating agents, antibiotics, antimetabolites, plant-derived drugs, and hormones. In some embodiments, the chemotherapeutic agent is cisplatin. In some embodiments, the chemotherapeutic agent is carboplatin. In some embodiments, the chemotherapeutic agent is oxaliplatin. In other embodiments, the chemotherapeutic agent is gemcitabine. In other embodiments, the chemotherapeutic agent is doxorubicin.

[0079] As used herein, the term "antimetabolite" means a therapeutic agent that inhibits the utilization of a metabolite or its prodrug. Examples of antimetabolites include methotrexate, 5-fluorouracil such as 5-fluorouracil prodrugs like capecitabine, 5-fluorodeoxyuridine monophosphate, cytarabine, cytarabine prodrugs like nelarabine, 5-azacitidine, gemcitabine, mercaptopurine, thioguanine, azathioprine, adenosine, pentostatin, erythrohydroxynonyladenine, and cladribine. Antimetabolites useful for practicing the disclosed methods include nucleoside analogs, including purine or pyrimidine analogs. In some embodiments, the gamma-polyglutamylated methotrexate composition is used in combination with an antimetabolite selected from the group consisting of fluoropyrimidines, 5-fluorouracil, 5-fluoro-2'-deoxycytidine, cytarabine, gemcitabine, troxacitabine, decitabine, azacitidine, pseudoisocytidine, zebularine, ancitabine, fazarabine, 6-azacitidine, capecitabine, N4-octadecylcytarabine, elaidic acid cytarabine, fludarabine, cladribine, clofarabine, nelarabine, foladesine, and pentostatin, or derivatives thereof. In one example, the nucleoside analog is a substrate of a nucleoside deaminase, which is adenosine deaminase or cytidine deaminase. In some examples, the nucleoside analog is selected from fludarabine, cytarabine, gemcitabine, decitabine, and azacitidine or derivatives thereof. In certain embodiments, the antimetabolite is 5-fluorouracil.

[0080] As used herein, "taxane" is an anti-cancer agent that interferes with or disrupts microtubule stability, formation, and / or function. Taxane agents include paclitaxel and docetaxel and their derivatives, which function in the same mode of action with respect to microtubules as the taxanes from which they are derived. In certain embodiments, the taxane is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel. In certain embodiments, the taxane is paclitaxel (Taxol®), docetaxel (Taxotere®), albumin-bound paclitaxel (nab-paclitaxel; Abraxane®), DHA-paclitaxel, or PG-paclitaxel.

[0081] The term "pharmaceutically acceptable carrier" means a non-toxic component other than the active ingredient in a pharmaceutical formulation for a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, carriers, excipients, stabilizers, diluents, or preservatives. Pharmaceutically acceptable carriers include, for example, one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans or other subjects.

[0082] The present disclosure generally relates to compositions of gamma-polyglutamylated methotrexate (MTX), and methods of making and using the compositions for treating diseases including hyperproliferative diseases such as cancer, immune system disorders such as rheumatoid arthritis, and infectious diseases such as HIV and malaria.

[0083] In some embodiments, the present disclosure provides the following. [1] A composition comprising gamma-polyglutamylated methotrexate; [2] The composition of item [1], wherein the gamma-polyglutamylated methotrexate comprises 1 to 10 glutamyl groups having gamma-carboxyl group linkages. [3] The composition according to item [1] or [2], wherein the gamma-polyglutamyl oxidized methotrexate contains 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups having a gamma-carboxyl group bond; [4] The composition according to any one of items [1] to [3], wherein the gamma-polyglutamyl oxidized methotrexate is gamma-tetra-glutamyl oxidized methotrexate; [5] The composition according to any one of items [1] to [3], wherein the gamma-polyglutamyl oxidized methotrexate is gamma-penta-glutamyl oxidized methotrexate; [6] The composition according to any one of items [1] to [3], wherein the gamma-polyglutamyl oxidized methotrexate is gamma-hexa-glutamyl oxidized methotrexate; [7] The composition according to any one of items [1] to [6], which is the following composition: (a) The gamma-polyglutamyl oxidized methotrexate contains L-type glutamyl groups having two or more gamma-carboxyl group bonds, (b) Each glutamyl group of the gamma-polyglutamyl oxidized methotrexate is of the L-type and has a gamma-carboxyl group bond, (c) At least one glutamyl group of the gamma-polyglutamyl oxidized methotrexate is of the D-type and has a gamma-carboxyl group bond, (d) Each glutamyl group of the gamma-polyglutamyl oxidized methotrexate other than the glutamyl group of methotrexate is of the D-type and has a gamma-carboxyl group bond, or (e) The gamma-polyglutamyl oxidized methotrexate contains two or more L-type glutamyl groups having a gamma-carboxyl group bond and at least one D-type glutamyl group; [8] The composition according to item [4], wherein (a) each glutamyl group is of the L-type and has a gamma-carboxyl group bond, or (b) each glutamyl group other than the glutamyl group of methotrexate is of the D-type and each glutamyl group has a gamma-carboxyl group bond; [9] The composition according to item [5], wherein (a) each glutamyl group is of the L-form and has a gamma-carboxyl group bond, or (b) each glutamyl group other than the glutamyl group of methotrexate is of the D-form and each glutamyl group has a gamma-carboxyl group bond;

[10] The composition according to item [6], wherein (a) each glutamyl group is of the L-form and has a gamma-carboxyl group bond, or (b) each glutamyl group other than the glutamyl group of methotrexate is of the D-form and each glutamyl group has a gamma-carboxyl group bond;

[11] The composition according to any one of items [1] to

[10] , wherein gamma-polyglutamylated methotrexate is polyglutamylatable by FGPS under normal physiological conditions, and / or polyglutamylated MTX has a lower hepatocyte uptake rate (<30%) than MTX;

[12] A liposomal composition (Lp-γPMTX) comprising the gamma-polyglutamylated methotrexate according to any one of items [1] to

[11] ;

[13] The Lp-γPMTX composition according to item

[12] , wherein the gamma-polyglutamylated methotrexate contains two or more L-form glutamyl groups;

[14] The Lp-γPMTX composition according to item

[12] or

[13] , wherein each glutamyl group of the gamma-polyglutamylated methotrexate is of the L-form;

[15] The Lp-γPMTX composition according to item

[12] or

[13] , wherein at least one glutamyl group of the gamma-polyglutamylated methotrexate is of the D-form;

[16] The Lp-γPMTX composition according to any one of items

[12] to

[15] , wherein the liposome contains gamma-polyglutamylated methotrexate having 1 to 10 glutamyl groups having a gamma-carboxyl group bond;

[17] The Lp-γPMTX composition according to any one of items

[12] to

[16] , wherein the liposome contains gamma polyglutamylated methotrexate containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups;

[18] The Lp-γPMTX composition according to any one of items

[12] to

[17] , wherein the liposome contains gamma tetraglutamylated methotrexate;

[19] The Lp-γPMTX composition according to any one of items

[12] to

[17] , wherein the liposome contains gamma pentaglutamylated methotrexate;

[20] The Lp-γPMTX composition according to any one of items

[12] to

[17] , wherein the liposome contains gamma hexaglutamylated methotrexate;

[21] The Lp-γPMTX composition according to any one of items

[12] to

[20] , wherein the liposome is not pegylated (PγLp-γPMTX);

[22] The Lp-γPMTX composition according to any one of items

[12] to

[20] , wherein the liposome is pegylated (PγLp-γPMTX);

[23] The Lp-γPMTX composition according to any one of items

[12] to

[22] , wherein the liposome contains at least 1% by weight (w / w) of gamma polyglutamylated methotrexate, or during the process of preparing Lp-γPMTX, at least 1% of the starting material of gamma polyglutamylated MTX is encapsulated (enclosed) in Lp-γPMTX;

[24] The Lp-γPMTX composition according to any one of items

[12] to

[24] , wherein the liposome has a diameter in the range of 20 nm to 500 nm;

[25] The Lp-γPMTX composition according to any one of items

[12] to

[24] , wherein the liposome has a diameter in the range of 20 nm to 200 nm;

[26] The Lp-γPMTX composition according to any one of items

[12] to

[25] , wherein the liposome has a diameter in the range of 80 nm to 120 nm;

[27] The Lp-γPMTX composition according to any one of items

[12] to

[26] , wherein the liposome is formed from liposome components;

[28] The Lp-γPMTX composition according to item

[27] , wherein the liposome components include at least one of an anionic lipid and a neutral lipid;

[29] The Lp-γPMTX composition according to item

[27] or

[28] , wherein the liposome components include at least one selected from the group consisting of DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholesterol; cholesterol-PEG; and cholesterol-maleimide;

[30] The Lp-γPMTX composition according to any one of items

[27] to

[29] , wherein the liposome components include at least one selected from the group consisting of DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC;

[31] The Lp-γPMTX composition according to any one of items

[27] to

[30] , wherein one or more liposome components further include a steric stabilizer;

[32] The Lp-γPMTX composition according to item

[31] , wherein the steric stabilizer is at least one selected from the group consisting of polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinylpyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol; poly[N-(2-hydroxypropyl)methacrylamide]; amphiphilic poly-N-vinylpyrrolidone; L-amino acid-based polymer; oligoglycerol, polyethylene glycol and polypropylene oxide-containing copolymer, poloxamer 188, and polyvinyl alcohol;

[33] The Lp-γPMTX composition according to item

[32] , wherein the steric stabilizer is PEG, and PEG has a number average molecular weight (Mn) of 200 to 5000 daltons;

[34] The Lp-γPMTX composition according to any one of items

[12] to

[33] , wherein the liposome is anionic or neutral;

[35] The Lp-γPMTX composition according to any one of items

[12] to

[33] , wherein the liposome has a zeta potential of zero or less;

[36] The Lp-γPMTX composition according to any one of items

[12] to

[33] , wherein the liposome has a zeta potential of 0 to -150 mV;

[37] The Lp-γPMTX composition according to any one of items

[12] to

[33] , wherein the liposome has a zeta potential of -30 to -50 mV;

[38] The Lp-γPMTX composition according to any one of items

[12] to

[33] , wherein the liposome is cationic;

[39] The Lp-γPMTX composition according to any one of items

[12] to

[38] , wherein the liposome has an internal space containing γ-polyglutamylated methotrexate and an aqueous pharmaceutically acceptable carrier;

[40] The Lp-γPMTX composition according to item

[39] , wherein the pharmaceutically acceptable carrier contains an isotonic agent such as dextrose, mannitol, glycerol, potassium chloride, sodium chloride, etc. at a concentration greater than 1%;

[41] The Lp-γPMTX composition according to item

[39] , wherein the aqueous pharmaceutically acceptable carrier is trehalose;

[42] The Lp-γPMTX composition according to item

[41] , wherein the pharmaceutically acceptable carrier contains 1% to 20% trehalose;

[43] The Lp-γPMTX composition according to any one of items

[39] to

[42] , wherein the pharmaceutically acceptable carrier contains 1% to 50% dextrose;

[44] The Lp-γPMTX composition according to any one of items

[39] to

[43] , wherein the internal space of the liposome contains 5% dextrose suspended in HEPES buffer;

[45] An Lp-γPMTX composition according to any one of items

[39] to

[44] , wherein the pharmaceutically acceptable carrier comprises a buffer such as HEPES buffered saline (HBS) or the like at a concentration of 1 to 200 mM and a pH of 2 to 8;

[46] An Lp-γPMTX composition according to any one of items

[39] to

[45] , wherein the pharmaceutically acceptable carrier comprises sodium acetate and calcium acetate at a total concentration of 50 mM to 500 mM;

[47] An Lp-γPMTX composition according to any one of items

[12] to

[46] , wherein the internal space of the liposome has a pH of 5 to 8 or a pH of 6 to 7, or any range of pH therebetween;

[48] An Lp-γPMTX composition according to any one of items

[12] to

[47] , wherein the liposome contains less than 500,000 or less than 200,000 gamma-polyglutamylated methotrexate molecules;

[49] An Lp-γPMTX composition according to any one of items

[12] to

[48] , wherein the liposome contains 10 to 100,000 or any range therebetween of gamma-polyglutamylated methotrexate molecules;

[50] An Lp-γPMTX composition according to any one of items

[12] to

[49] , further comprising a targeting moiety, wherein the targeting moiety has specific affinity for a surface antigen on a target cell of interest;

[51] The Lp-γPMTX composition according to item

[50] , wherein the targeting moiety is bound to one or both of the PEG and the outer surface of the liposome, and optionally, the targeting moiety is covalently bound to one or both of the PEG and the outer surface of the liposome;

[52] The Lp-γPMTX composition according to item

[50] or

[51] , wherein the targeting moiety is a polypeptide;

[53] The Lp-γPMTX composition according to any one of items

[50] to

[52] , wherein the targeting moiety is an antibody or an antigen-binding fragment of an antibody;

[54] The Lp-γPMTX composition according to any one of items

[50] to

[53] , wherein the targeting moiety binds to the surface antigen with an equilibrium dissociation constant (Kd) in the range of 0.5x10 -10 ~10x10 -6 as measured by BIACORE® analysis;

[55] The Lp-γPMTX composition according to any one of items

[50] to

[54] , wherein the targeting moiety specifically binds to one or more folate receptors selected from the group consisting of folate receptor alpha (FR-α), folate receptor beta (FR-β), and folate receptor delta (FR-δ);

[56] The Lp-γPMTX composition according to any one of items

[50] to

[55] , wherein the targeting moiety comprises one or more selected from the group consisting of an antibody, a humanized antibody, an antigen-binding fragment of an antibody, a single-chain antibody, a single-domain antibody, a bispecific antibody, a synthetic antibody, a pegylated antibody, and a multimeric antibody;

[57] The Lp-γPMTX composition according to any one of items

[50] to

[56] , wherein each pegylated liposome contains 1 to 1000 or 30 to 200 targeting moieties;

[58] The Lp-γPMTX composition according to any one of items

[39] to

[57] , further comprising one or more of an immunostimulant, a detectable marker, and maleimide, wherein the immunostimulant, detectable marker, or maleimide is bound to the PEG or outer surface of the liposome;

[59] The Lp-γPMTX composition according to item

[58] , wherein the immunostimulant is at least one selected from the group consisting of a protein immunostimulant, a nucleic acid immunostimulant, a chemical immunostimulant, a hapten, and an adjuvant;

[60] The Lp-γPMTX composition according to item

[58] or

[59] , wherein the immunostimulant is fluorescein, fluorescein isothiocyanate (FITC), DNP, beta-glucan, beta-1,3-glucan, beta-1,6-glucan, resorbin (e.g., D n-6DPA or D n-3DPAAt least one selected from the group consisting of resorcin D, resorcin E, or T-series resorcin (such as), oxidized low-density lipoprotein (for example, OXPAC, PGPC), and toll-like receptor (TLR) regulators such as erythran lipids (for example, E5564);

[61] The Lp-γPMTX composition according to any one of items

[58] to

[60] , wherein the immunostimulant and the detectable marker are the same;

[62] The Lp-γPMTX composition according to any one of items

[58] to

[61] , further comprising a hapten;

[63] The Lp-γPMTX composition according to item

[62] , wherein the hapten comprises one or more of fluorescein or beta-1,6-glucan;

[64] The Lp-γPMTX composition according to any one of items

[12] to

[63] , further comprising at least one cryoprotective substance selected from the group consisting of mannitol, trehalose, sorbitol, and sucrose;

[65] A targeted composition comprising the composition according to any one of items [1] to

[64] ;

[66] An untargeted composition comprising the composition according to any one of items [1] to

[49] ;

[67] The Lp-γPMTX composition according to any one of items

[12] to

[66] , further comprising carboplatin and / or pembrolizumab;

[68] A pharmaceutical composition comprising the liposomal gamma-polyglutamate oxidized methotrexate composition according to any one of items

[12] to

[67] ;

[69] A pharmaceutical composition comprising the gamma-polyglutamate oxidized methotrexate composition according to any one of items [1] to [7];

[70] The composition according to any one of items [1] to

[69] for use in the treatment of a disease;

[71] Use of the composition according to any one of items [1] to

[70] in the manufacture of a drug for the treatment of a disease;

[72] A method for treating or preventing a disease in a subject in need of treatment or prevention, the method comprising the step of administering the composition according to any one of items [1] to

[70] to the subject;

[73] A method for treating or preventing a disease in a subject in need of treatment or prevention, the method comprising the step of administering the liposomal gamma-polyglutamylated methotrexate composition according to any one of items

[12] to

[69] to the subject;

[74] A method for killing hyperproliferative cells, the method comprising the step of contacting the hyperproliferative cells with the composition according to any one of items [1] to

[69] ;

[75] A method for killing hyperproliferative cells, the method comprising the step of contacting the hyperproliferative cells with the liposomal gamma-polyglutamylated methotrexate composition according to any one of items

[12] to

[69] ;

[76] The method according to item

[74] or

[75] , wherein the hyperproliferative cells are cancer cells, mammalian cells, and / or human cells;

[77] A method for treating cancer, the method comprising the step of administering an effective amount of the composition according to any one of items [1] to

[69] to a subject having or at risk of having cancer;

[78] A method for treating cancer, the method comprising the step of administering an effective amount of the liposomal gamma-polyglutamylated methotrexate composition according to any one of items

[12] to

[68] to a subject having or at risk of having cancer;

[79] The method according to item

[77] or

[78] , wherein the cancer is a non-hematological tumor including, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and a hematological tumor including, for example, leukemia, lymphoma and other B-cell malignancies, multiple myeloma and other plasma cell dyscrasias, and is selected from the group consisting of;

[80] The method according to item

[77] or

[78] , wherein the cancer is a member selected from the group consisting of lung cancer, breast cancer, colon cancer, pancreatic cancer, gastric cancer, bladder cancer, head and neck cancer, ovarian cancer and cervical cancer;

[81] The method according to item

[77] or

[78] , wherein the cancer is a member selected from the group consisting of colorectal cancer, lung cancer, breast cancer, head and neck cancer, and pancreatic cancer;

[82] The method according to any one of items

[77] to

[78] , wherein the cancer is a sarcoma such as osteosarcoma;

[83] A method for treating cancer, comprising administering an effective amount of the Lp-γPMTX composition according to any one of items

[50] to

[66] to a subject having or at risk of having cancer cells expressing folate receptor on the surface bound by a targeting moiety;

[84] Maintenance therapy for a subject who is or has been receiving cancer therapy, comprising administering an effective amount of the composition according to any one of items [1] to

[69] to the subject who is or has been receiving cancer therapy;

[85] Maintenance therapy for a subject who is or has been receiving cancer therapy, comprising administering an effective amount of the liposomal gamma-polyglutamylated methotrexate composition according to any one of items

[12] to

[69] to the subject who is or has been receiving cancer therapy;

[86] A method for treating an immune system disorder, comprising administering an effective amount of the composition according to any one of items [1] to

[69] to a subject having or at risk of having an immune system disorder, and optionally, the immune system disorder is selected from inflammation (e.g., acute and chronic), systemic inflammation, rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn's disease, dermatomyositis / polymyositis, systemic lupus erythematosus, and Takayasu disease, and psoriasis;

[87] A method for treating an immune system disorder, comprising administering an effective amount of the liposomal gamma-polyglutamylated methotrexate composition according to any one of items [8] to

[69] to a subject having or at risk of having an immune system disorder, and optionally, the immune system disorder is selected from inflammation (e.g., acute and chronic), systemic inflammation, rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn's disease, dermatomyositis / polymyositis, systemic lupus erythematosus, and Takayasu disease, and psoriasis;

[88] The following treatment method: (a) A method for treating an infectious disease, comprising the step of administering an effective amount of the composition according to any one of items [1] to

[69] to a subject having or at risk of having an infectious disease; (b) A method for treating an infectious disease, a cardiovascular disease, or another disease, comprising the step of administering an effective amount of the composition according to any one of items [1] to

[69] to a subject having or at risk of having an infectious disease, a cardiovascular disease, or another disease, wherein the disease is a member selected from atherosclerosis, cardiovascular disease (CVD), coronary artery disease, myocardial infarction, stroke, metabolic syndrome, gestational trophoblastic disease, and ectopic pregnancy; (c) A method for treating an autoimmune disease, comprising the step of administering an effective amount of the composition according to any one of items [1] to

[69] to a subject having or at risk of having an autoimmune disease; (d) A method for treating rheumatoid arthritis, comprising the step of administering an effective amount of the composition according to any one of items [1] to

[69] to a subject having or at risk of having rheumatoid arthritis; (e) A method for treating an inflammatory condition, comprising the step of administering an effective amount of the composition according to any one of items [1] to

[69] to a subject having or at risk of having inflammation, optionally wherein the inflammation is acute, chronic, and / or systemic inflammation; or (f) A method for treating a skin disease, comprising the step of administering an effective amount of the composition according to any one of items [1] to

[69] to a subject having or at risk of having a skin disease, optionally wherein the skin disease is psoriasis;

[89] A method for treating an infectious disease, comprising the step of administering an effective amount of the liposomal gamma-polyglutamyl oxidized methotrexate composition according to any one of items

[12] to

[69] to a subject having or at risk of having an infectious disease;

[90] A method for delivering gamma-polyglutamyl oxidized methotrexate to a tumor expressing a folate receptor on its surface, the method comprising administering to a subject having a tumor an amount of the Lp-γPMTX composition according to any one of items [1] to

[69] that delivers a therapeutically effective amount of gamma-polyglutamyl oxidized methotrexate to the tumor.

[91] A method for producing a gamma-polyglutamylmethotrexate composition comprising the liposomal gamma-polyglutamylmethotrexate composition according to any one of items

[12] to

[69] , the method comprising: forming a mixture comprising a liposomal component and a gamma-polyglutamylmethotrexate in a solution; homogenizing the mixture in the solution to form liposomes; and treating the mixture to form liposomes containing gamma-polyglutamylmethotrexate.

[92] A method for producing a gamma-polyglutamylmethotrexate composition comprising the liposomal gamma-polyglutamylmethotrexate composition according to any one of items

[12] to

[69] , the method comprising: forming a mixture comprising a liposomal component and a gamma-polyglutamylmethotrexate in a solution; and treating the mixture to form liposomes containing gamma-polyglutamylmethotrexate.

[93] The method according to item

[92] , wherein the step of treating the mixture comprises homogenizing the mixture in a solution to form liposomes.

[94] A method for producing a composition according to any one of items

[50] to

[69] , the method comprising: forming a mixture comprising a liposomal component and a gamma-polyglutamylmethotrexate in a solution; homogenizing the mixture in the solution to form liposomes; treating the mixture to form liposomes encapsulating and / or coating gamma-polyglutamylmethotrexate; and imparting a targeting moiety on the surface of the liposomes, wherein the targeting moiety has specific affinity for at least one of folate receptor alpha (FR-α), folate receptor beta (FR-β), and folate receptor delta (FR-δ).

[95] A method for preparing the composition according to any one of items

[50] to

[69] , comprising the steps of: forming a mixture containing liposome components and gamma-polyglutamate oxidized methotrexate in a solution; treating the mixture to form liposomes encapsulating and / or coating gamma-polyglutamate oxidized methotrexate; and imparting a targeting moiety on the surface of the liposomes, wherein the targeting moiety has specific affinity for at least one of folate receptor alpha (FR-α), folate receptor beta (FR-β), and folate receptor delta (FR-δ);

[96] The method according to item

[95] , wherein the treating step comprises homogenizing the mixture in a solution to form liposomes;

[97] The method according to item

[92] , wherein the treating step comprises one or more steps of thin film hydration, extrusion, in-line mixing, ethanol injection technique, freeze-thaw method, reverse phase evaporation method, dynamic high pressure microfluidization, microfluidic mixing, double emulsion, freeze-drying double emulsion, 3D printing, membrane contactor method, and stirring; and / or

[98] The method according to any one of items

[95] to

[97] , wherein the treating step comprises one or more steps of changing the size of the liposomes by one or more steps of extrusion, high pressure microfluidization, and / or sonication; and / or

[99] The method according to any one of items

[91] to

[98] , wherein at least 1% of the starting material of gamma-polyglutamate oxidized methotrexate is encapsulated or enclosed in the liposomes.

[0084] II. Gamma-polyglutamate oxidized methotrexate (γPMTX) Generally, the present disclosure relates to gamma-polyglutamylated methotrexate (γPMTX) compositions. The γPMTX compositions include at least one glutamyl group having a gamma-carboxyl group bond. These are structurally different from L-gamma-polyglutamylated methotrexate (Lγ1PMTX) produced by the enzyme folylpolyglutamate synthase (FPGS) in cells during methotrexate therapy.

[0085] In some embodiments, the γPMTX composition includes 2 to 20, 2 to 15, 2 to 10, 2 to 5, or more than 5 glutamyl groups (including the glutamyl group of methotrexate). In some embodiments, each glutamyl group in γPMTX other than the glutamyl group of methotrexate has a gamma bond. In some embodiments, two or more glutamyl groups in γPMTX have a gamma bond. In some embodiments, each glutamyl group in γPMTX is of the L-type. In some embodiments, each glutamyl group in γPMTX other than the glutamyl group of methotrexate is of the D-type. In some embodiments, γPMTX includes two or more L-type glutamyl groups and one or more D-type glutamyl groups.

[0086] In some embodiments, the gamma-polyglutamylated methotrexate is diglutamylated. That is, the gamma-polyglutamylated methotrexate includes one gamma-glutamyl group in addition to the glutamyl group of methotrexate (γMTX-PG 1 ). In some embodiments, each glutamyl group of gamma-diglutamylated methotrexate is of the L-type. In other embodiments, gamma-diglutamylated MTX includes a D-type glutamyl group.

[0087] In some embodiments, the gamma-polyglutamylated methotrexate is triglutamylated. That is, the gamma-polyglutamylated methotrexate includes two gamma-glutamyl groups in addition to the glutamyl group of methotrexate (γMTX-PG 2)。In some embodiments, each glutamyl group of gamma-glutamyl oxidized methotrexate is of the L-type. In other embodiments, gamma-glutamyl oxidized MTX contains a D-type glutamyl group. In further embodiments, each glutamyl group of gamma-triglutamyl oxidized methotrexate other than the glutamyl group of methotrexate is of the D-type. In further embodiments, gamma-triglutamyl oxidized MTX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0088] In some embodiments, gamma-polyglutamyl oxidized methotrexate is tetraglutamylated and thus contains three gamma-glutamyl groups in addition to the gamma-glutamyl group in methotrexate (γMTX-PG 3 )。In some embodiments, gamma-tetraglutamyl oxidized MTX contains two or more L-type gamma-glutamyl groups. In further embodiments, each gamma-glutamyl group of gamma-tetraglutamyl oxidized methotrexate is of the L-type. In other embodiments, gamma-tetraglutamyl oxidized MTX contains a D-type gamma-glutamyl group. In some embodiments, gamma-tetraglutamyl oxidized MTX contains two D-type gamma-glutamyl groups. In some embodiments, each glutamyl group of gamma-tetraglutamyl oxidized methotrexate other than the glutamyl group of methotrexate is of the D-type. In further embodiments, tetraglutamyl oxidized MTX contains a D-type gamma-glutamyl group and two or more L-type gamma-glutamyl groups.

[0089] In some embodiments, gamma-polyglutamyl oxidized methotrexate is pentaglutamylated (γMTX-PG 4) It contains a chain of 4 γ-glutamyl groups attached to the glutamyl group of methotrexate. In some embodiments, gamma pentaglutamyl oxidized MTX contains 2 or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma pentaglutamyl oxidized methotrexate is of the L-type. In other embodiments, gamma pentaglutamyl oxidized MTX contains a D-type glutamyl group. In some embodiments, gamma tetraglutamyl oxidized MTX contains 2 or 3 D-type γ-glutamyl groups. In further embodiments, each γ-glutamyl group of gamma pentaglutamyl oxidized methotrexate other than the glutamyl group of methotrexate is of the D-type. In further embodiments, pentaglutamyl oxidized MTX contains a D-type γ-glutamyl group and 2 or more L-type γ-glutamyl groups.

[0090] In some embodiments, gamma polyglutamyl oxidized methotrexate is hexaglutamylated (γMTX-PG 5 ) It contains a chain of 5 γ-glutamyl groups attached to the glutamyl group of methotrexate. In some embodiments, gamma hexaglutamyl oxidized MTX contains 2 or more L-type γ-glutamyl groups. In further embodiments, each glutamyl group of gamma hexaglutamyl oxidized methotrexate is of the L-type. In other embodiments, gamma hexaglutamyl oxidized MTX contains a D-type γ-glutamyl group. In some embodiments, gamma tetraglutamyl oxidized MTX contains 2, 3, 4, or 5 D-type γ-glutamyl groups. In further embodiments, each glutamyl group of gamma hexaglutamyl oxidized methotrexate other than the glutamyl group of methotrexate is of the D-type. In further embodiments, hexaglutamyl oxidized MTX contains a D-type γ-glutamyl group and 2 or more L-type γ-glutamyl groups.

[0091] In some embodiments, gamma polyglutamyl oxidized methotrexate is heptaglutamylated (γMTX-PG 6) It contains a chain of six γ-glutamyl groups attached to the glutamyl group of methotrexate. In some embodiments, gamma heptaglutamyl oxidized MTX contains two or more L-type γ-glutamyl groups. In further embodiments, each γ-glutamyl group of gamma heptaglutamyl oxidized methotrexate is of the L-type. In other embodiments, gamma heptaglutamyl oxidized MTX contains a D-type γ-glutamyl group. In some embodiments, gamma tetraglutamyl oxidized MTX contains two, three, four, five, or six D-type γ-glutamyl groups. In further embodiments, each γ-glutamyl group of gamma heptaglutamyl oxidized methotrexate other than the glutamyl group of methotrexate is of the D-type. In further embodiments, heptaglutamyl oxidized MTX contains a D-type γ-glutamyl group and two or more L-type γ-glutamyl groups.

[0092] In some embodiments, gamma polyglutamyl oxidized methotrexate is octaglutamylated (γMTX-PG 7 ) and thus contains a chain of seven γ-glutamyl groups attached to the glutamyl group of methotrexate. In some embodiments, gamma octaglutamyl oxidized MTX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma octaglutamyl oxidized methotrexate is of the L-type. In other embodiments, gamma octaglutamyl oxidized MTX contains a D-type glutamyl group. In some embodiments, gamma octaglutamyl oxidized MTX contains two, three, four, five, six, or seven D-type γ-glutamyl groups. In further embodiments, each glutamyl group of gamma octaglutamyl oxidized methotrexate other than the glutamyl group of methotrexate is of the D-type. In further embodiments, octaglutamyl oxidized MTX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0093] In some embodiments, gamma polyglutamyl oxidized methotrexate is nonaglutamylated (γMTX-PG 8) It contains a chain of 8 γ-glutamyl groups attached to the glutamyl group of methotrexate. In some embodiments, gamma nonaglutaminyl oxidized MTX contains 2 or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma nonaglutaminyl oxidized methotrexate is of the L-type. In other embodiments, gamma nonaglutaminyl oxidized MTX contains D-type glutamyl groups. In further embodiments, each glutamyl group of gamma nonaglutaminyl oxidized methotrexate other than the glutamyl group of methotrexate is of the D-type. In further embodiments, nonaglutaminyl oxidized MTX contains D-type γ-glutamyl groups and 2 or more L-type γ-glutamyl groups.

[0094] In some embodiments, gamma polyglutamyl oxidized methotrexate is decaglutaminylated (γMTX-PG 9 ) It contains a chain of 9 γ-glutamyl groups attached to the glutamyl group of methotrexate. In some embodiments, gamma decaglutaminyl oxidized MTX contains 2 or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma decaglutaminyl oxidized methotrexate is of the L-type. In other embodiments, gamma decaglutaminyl oxidized MTX contains D-type glutamyl groups. In further embodiments, each glutamyl group of gamma decaglutaminyl oxidized methotrexate other than the glutamyl group of methotrexate is of the D-type. In further embodiments, decaglutaminyl oxidized MTX contains D-type glutamyl groups and 2 or more L-type glutamyl groups.

[0095] In some embodiments, gamma polyglutamyl oxidized methotrexate is undecaglutaminylated (γMTX-PG 10) It contains a chain of 10 γ-glutamyl groups attached to the glutamyl group of methotrexate. In some embodiments, gamma undecaglutamyl oxidized MTX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma undecaglutamyl oxidized methotrexate is of the L-type. In other embodiments, gamma undecaglutamyl oxidized MTX contains a D-type glutamyl group. In further embodiments, each glutamyl group of gamma undecaglutamyl oxidized methotrexate other than the glutamyl group of methotrexate is of the D-type. In further embodiments, undecaglutamyl oxidized MTX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0096] In some embodiments, gamma polyglutamyl oxidized methotrexate is dodecaglutamyl oxidized (γMTX-PG 11 ) It contains a chain of 11 γ-glutamyl groups attached to the glutamyl group of methotrexate. In some embodiments, gamma dodecaglutamyl oxidized MTX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma dodecaglutamyl oxidized methotrexate is of the L-type. In other embodiments, gamma dodecaglutamyl oxidized MTX contains a D-type glutamyl group. In further embodiments, each glutamyl group of gamma dodecaglutamyl oxidized methotrexate other than the glutamyl group of methotrexate is of the D-type. In further embodiments, dodecaglutamyl oxidized MTX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0097] In some embodiments, gamma polyglutamyl oxidized methotrexate is tridecaglutamyl oxidized (γMTX-PG 12) It contains a chain of 12 γ-glutamyl groups attached to the glutamyl group of methotrexate. In some embodiments, gamma-triskadecakaglutaminated MTX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma-triskadecakaglutaminated methotrexate is of the L-type. In other embodiments, gamma-triskadecakaglutaminated MTX contains D-type glutamyl groups. In further embodiments, each glutamyl group of gamma-triskadecakaglutaminated methotrexate other than the glutamyl group of methotrexate is of the D-type. In further embodiments, triskadecakaglutaminated MTX contains D-type glutamyl groups and two or more L-type glutamyl groups.

[0098] In some embodiments, gamma-polyglutaminated methotrexate is tetradecakaglutaminated (γMTX-PG 13 ) It contains a chain of 13 γ-glutamyl groups attached to the glutamyl group of methotrexate. In some embodiments, gamma-tetradecakaglutaminated MTX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma-tetradecakaglutaminated methotrexate is of the L-type. In other embodiments, gamma-tetradecakaglutaminated MTX contains D-type glutamyl groups. In further embodiments, each glutamyl group of gamma-tetradecakaglutaminated methotrexate other than the glutamyl group of methotrexate is of the D-type. In further embodiments, tetradecakaglutaminated MTX contains D-type glutamyl groups and two or more L-type glutamyl groups.

[0099] In some embodiments, gamma-polyglutaminated methotrexate is pentadecakaglutaminated (γMTX-PG 14) It contains a chain of 14 γ-glutamyl groups attached to the glutamyl group of methotrexate. In some embodiments, gamma pentadeca-glutaminyl oxidized MTX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma pentadeca-glutaminyl oxidized methotrexate is of the L-type. In other embodiments, gamma pentadeca-glutaminyl oxidized MTX contains a D-type glutamyl group. In further embodiments, each glutamyl group of gamma pentadeca-glutaminyl oxidized methotrexate other than the glutamyl group of methotrexate is of the D-type. In further embodiments, pentadeca-glutaminyl oxidized MTX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0100] In some embodiments, gamma polyglutamyl oxidized methotrexate is hexadeca-glutaminyl oxidized (γMTX-PG 15 ) It contains a chain of 15 γ-glutamyl groups attached to the glutamyl group of methotrexate. In some embodiments, gamma hexadeca-glutaminyl oxidized MTX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma hexadeca-glutaminyl oxidized methotrexate is of the L-type. In other embodiments, gamma hexadeca-glutaminyl oxidized MTX contains a D-type glutamyl group. In further embodiments, each glutamyl group of gamma hexadeca-glutaminyl oxidized methotrexate other than the glutamyl group of methotrexate is of the D-type. In further embodiments, hexadeca-glutaminyl oxidized MTX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0101] In other embodiments, gamma polyglutamyl oxidized methotrexate is heptadeca-glutaminyl oxidized (γMTX-PG 16) It contains a chain of 16 γ-glutamyl groups attached to the glutamyl group of methotrexate. In some embodiments, gamma heptadeca-glutamylated MTX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma heptadeca-glutamylated methotrexate is of the L-type. In other embodiments, gamma heptadeca-glutamylated MTX contains a D-type glutamyl group. In further embodiments, each glutamyl group of gamma heptadeca-glutamylated methotrexate other than the glutamyl group of methotrexate is of the D-type. In further embodiments, heptadeca-glutamylated MTX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0102] In some embodiments, gamma polyglutamylated methotrexate is octadeca-glutamylated (γMTX-PG 17 ) It contains a chain of 17 γ-glutamyl groups attached to the glutamyl group of methotrexate. In some embodiments, gamma octadeca-glutamylated MTX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma octadeca-glutamylated methotrexate is of the L-type. In other embodiments, gamma octadeca-glutamylated MTX contains a D-type glutamyl group. In further embodiments, each glutamyl group of gamma octadeca-glutamylated methotrexate other than the glutamyl group of methotrexate is of the D-type. In further embodiments, octadeca-glutamylated MTX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0103] In some embodiments, gamma polyglutamylated methotrexate is enneadeca-glutamylated (γMTX-PG 18) It contains a chain of 18 γ-glutamyl groups attached to the glutamyl group of methotrexate. In some embodiments, gamma enniadeca glutaminated MTX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma enniadeca glutaminated methotrexate is of the L-type. In other embodiments, gamma enniadeca glutaminated MTX contains a D-type glutamyl group. In further embodiments, each glutamyl group of gamma enniadeca glutaminated methotrexate other than the glutamyl group of methotrexate is of the D-type. In further embodiments, enniadeca glutaminated MTX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0104] In some embodiments, gamma polyglutaminated methotrexate is eicosaglutaminated (γMTX-PG 19 ) It contains a chain of 19 γ-glutamyl groups attached to the glutamyl group of methotrexate. In some embodiments, gamma eicosaglutaminated MTX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma eicosaglutaminated methotrexate is of the L-type. In other embodiments, gamma eicosaglutaminated MTX contains a D-type glutamyl group. In further embodiments, each glutamyl group of gamma eicosaglutaminated methotrexate other than the glutamyl group of methotrexate is of the D-type. In further embodiments, eicosaglutaminated MTX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0105] In some embodiments, gamma polyglutaminated methotrexate is eicosakaihenaglutaminated (γMTX-PG 20) It contains a chain of 20 γ-glutamyl groups attached to the glutamyl group of methotrexate. In some embodiments, gamma mycocaihena glutamine oxidized MTX contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of gamma mycocaihena glutamine oxidized methotrexate is of the L-type. In other embodiments, gamma mycocaihena glutamine oxidized MTX contains a D-type glutamyl group. In further embodiments, each glutamyl group of gamma mycocaihena glutamine oxidized methotrexate other than the glutamyl group of methotrexate is of the D-type. In further embodiments, icosaihena glutamine oxidized MTX contains a D-type glutamyl group and two or more L-type glutamyl groups.

[0106] In some embodiments, gamma polyglutamine oxidized methotrexate contains 4 to 7 glutamyl groups attached to methotrexate (i.e., γMTX-PGn, n = 4 to 7), and each of the 4 to 7 attached glutamyl groups has a gamma bond. In some embodiments, each of the 4 to 7 attached glutamyl groups is of the L-type. In other embodiments, each of the 4 to 7 attached glutamyl groups is of the D-type. In other embodiments, the 4 to 7 attached glutamyl groups are of the L-type and the D-type.

[0107] In one embodiment, gamma polyglutamine oxidized methotrexate is tetra-glutamine oxidized, and each of the three glutamyl groups in the polyglutamate chain attached to methotrexate contains a gamma bond. In some embodiments, each of the 4 glutamyl groups is of the L-type. In some embodiments, each glutamyl group of gamma tetra-glutamine oxidized methotrexate other than the glutamyl group of methotrexate is of the D-type. In other embodiments, at least two of the glutamyl groups in gamma tetra-glutamate methotrexate are of the L-type and at least one glutamyl group is of the D-type.

[0108] In one embodiment, gamma-polyglutamyl methotrexate is pentaglutamylated, and each of the four glutamyl groups in the polyglutamate chain bound to methotrexate contains a gamma bond. In some embodiments, each of the four glutamyl groups is of the L-type. In some embodiments, each glutamyl group of gamma-pentaglutamyl methotrexate other than the glutamyl group of methotrexate is of the D-type. In other embodiments, at least two glutamyl groups in gamma-pentaglutamyl methotrexate are of the L-type and at least one glutamyl group is of the D-type.

[0109] In one embodiment, gamma-polyglutamyl methotrexate is hexaglutamylated. In some embodiments, each of the five glutamyl groups is of the L-type. In some embodiments, each glutamyl group of gamma-hexaglutamyl methotrexate other than the glutamyl group of methotrexate is of the D-type. In other embodiments, at least two glutamyl groups in gamma-hexaglutamyl methotrexate are of the L-type and at least one glutamyl group is of the D-type.

[0110] In another embodiment, gamma-polyglutamyl methotrexate is heptaglutamylated. In some embodiments, each of the six glutamyl groups is of the L-type. In some embodiments, each glutamyl group of gamma-heptaglutamyl methotrexate other than the glutamyl group of methotrexate is of the D-type. In other embodiments, at least two glutamyl groups in gamma-heptaglutamyl methotrexate are of the L-type and at least one glutamyl group is of the D-type.

[0111] In some embodiments, gamma-polyglutamyl methotrexate (γPMTX) contains a total of 1 to 15, 1 to 10, 2 to 15, 2 to 10, 3 to 15, 3 to 10, 3 to 6, 3 to 5, 4 to 10, 4 to 7, or 4 to 6 glutamyl groups, including the glutamyl group of methotrexate, or any range therebetween. In some embodiments, each glutamyl group in γPMTX other than the glutamyl group of methotrexate has a gamma bond. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the glutamyl groups in γPMTX have a gamma bond. In some embodiments, γPMTX contains L-type and D-type γ-glutamyl groups. In some embodiments, each glutamyl group in the polyglutamate structure of polyglutamyl methotrexate is of the L-type. In some embodiments, each glutamyl group in γPMTX other than the glutamyl group of methotrexate is of the D-type. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the glutamyl groups in γPMTX are of the L-type. In another embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the glutamyl groups in γPMTX are of the D-type.

[0112] In a further embodiment, gamma-polyglutamyl methotrexate contains 20 to 100, 20 to 75, 20 to 50, 20 to 40, 20 to 30, 20 to 25, or more than 100 gamma-glutamyl groups, or any range therebetween. In some embodiments, each glutamyl group of gamma-polyglutamyl methotrexate is of the L-type. In other embodiments, each glutamyl group of gamma-polyglutamyl methotrexate other than the glutamyl group of methotrexate is of the D-type. In an alternative embodiment, at least 2 of the glutamyl groups in gamma-polyglutamyl methotrexate are of the L-type and at least 1 of the glutamyl groups in gamma-polyglutamyl methotrexate is of the D-type.

[0113] In further embodiments, the provided composition comprises gamma-polyglutamylated methotrexate having 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 gamma-linked glutamyl groups. In some embodiments, the gamma-polyglutamylated methotrexate comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 L-type glutamyl groups. In some embodiments, the gamma-polyglutamylated methotrexate comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 D-type glutamyl groups. In some embodiments, the gamma-polyglutamylated methotrexate comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 L-type glutamyl groups and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 D-type glutamyl groups.

[0114] In some embodiments, the gamma-polyglutamylated methotrexate composition provided herein can accept one or more additional glutamyl groups, i.e., the composition can serve as a substrate for FPGS (folylpolyglutamate synthetase). Reagents and assays for measuring the ability of the gamma-polyglutamylated methotrexate composition to act as a substrate for FPGS (e.g., human FPGS, or rat liver FPGS) are readily available and can be performed as routine business.

[0115] In some embodiments, the uptake rate of the naked gamma-PPMX composition (e.g., gamma-PMTX not bound to a delivery carrier) disclosed herein by liver cells is significantly reduced compared to the uptake rate of methotrexate under the same physiological conditions. In some embodiments, the liver cell uptake rate of the naked gamma-PMTX composition is less than 30%, 20%, 15%, or 10% compared to the uptake rate of methotrexate. In further embodiments, the efflux (transport) ratio of the gamma-PMTX composition disclosed herein from liver cells is significantly reduced (less than 30%, 20%, 15%, or 10%) compared to methotrexate (MTX).

[0116] In some embodiments, the gamma-polyglutamyl oxidized methotrexate compositions provided herein have higher cytotoxicity against hyperproliferative cells than methotrexate. In some embodiments, the hyperproliferative cells are cancer cells. In some embodiments, the hyperproliferative cells are colorectal cancer cells, colon cancer cells, breast cancer cells, or ovarian cancer cells. In some embodiments, the cancer cells are mesothelioma cells or non-small cell lung cancer cells. In some embodiments, the cytotoxicity is measured in an in vitro assay. In some embodiments, the gamma-polyglutamyl oxidized methotrexate is hexaglutamyl oxidized methotrexate.

[0117] In some embodiments, the gamma-polyglutamyl oxidized methotrexate compositions provided herein have lower toxic side effects than methotrexate. In some embodiments, the gamma-polyglutamyl oxidized methotrexate compositions provided herein have less toxicity against non-hyperproliferative cells than methotrexate. In some embodiments, the gamma-polyglutamyl oxidized methotrexate compositions provided herein have less toxicity against neutrophils, liver cells, or colon epithelial cells than methotrexate. In some embodiments, the neutrophils are human neutrophils, differentiated human neutrophils, or neutrophils differentiated from CD34+ cells. In some embodiments, the liver cells are AML12 liver cells. In some embodiments, the colon epithelial cells are CCD841 colon epithelial cells. In some embodiments, the toxicity is measured in an in vitro assay. In some embodiments, the gamma-polyglutamyl oxidized methotrexate is hexaglutamyl oxidized methotrexate.

[0118] In some embodiments, the gamma polyglutamyl oxidized methotrexate compositions provided herein have lower toxic side effects than methotrexate. In some embodiments, the gamma polyglutamyl oxidized methotrexate compositions provided herein result in fewer or less severe toxic side effects than methotrexate in in vivo assays. In some embodiments, the in vivo assay is performed in an in vivo mouse model. In some embodiments, the gamma polyglutamyl oxidized methotrexate compositions provided herein result in fewer or less severe hematological or liver toxic side effects than methotrexate. In some embodiments, the hematological side effects are evaluated by mean neutrophils, mean white blood cells or mean platelet counts. In some embodiments, the liver toxic side effects are evaluated by measuring serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), and / or serum albumin levels. In some embodiments, the in vivo assay comprises administering a gamma polyglutamyl oxidized methotrexate composition at 40 mg / kg or 80 mg / kg once a week for 4 weeks. In some embodiments, the gamma polyglutamyl oxidized methotrexate is hexaglutamyl oxidized methotrexate.

[0119] In some embodiments, treatment with the gamma-polyglutamyl methotrexate compositions provided herein does not induce significant hematological or liver toxic side effects in an in vivo mouse model. In some embodiments, hematological side effects are evaluated by mean neutrophils, mean white blood cells, or mean platelet counts. In some embodiments, liver toxic side effects are evaluated by measuring serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), and / or serum albumin levels. In some embodiments, the gamma-polyglutamyl methotrexate compositions provided herein do not significantly decrease mean neutrophils, mean white blood cells, or mean platelet counts. In some embodiments, the gamma-polyglutamyl methotrexate compositions provided herein do not significantly increase serum aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT) levels. In some embodiments, the gamma-polyglutamyl methotrexate compositions provided herein do not significantly reduce serum albumin levels. In some embodiments, the in vivo assay comprises administering the gamma-polyglutamyl methotrexate composition at 40 mg / kg or 80 mg / kg once a week for 4 weeks. In some embodiments, the gamma-polyglutamyl methotrexate is hexaglutamyl methotrexate.

[0120] In some embodiments, the gamma-polyglutamyl methotrexate composition does not contain a fluorine atom. In some embodiments, the gamma-polyglutamyl methotrexate composition does not contain a 4-fluoroglutamyl group.

[0121] The gamma-polyglutamylated methotrexate (γPMTX) compositions and their uses are further described in each of U.S. Patent Application Nos. 62 / 374,458, 15 / 675,695, 15 / 675,701, and 62 / 583,432, as well as International Application Nos. PCT / US2017 / 046666 and PCT / US2017 / 046667. The disclosure of each of these is hereby incorporated by reference in its entirety.

[0122] A. Polyglutamylated Methotrexate Analogs and Derivatives The disclosure also encompasses gamma-polyglutamylated methotrexate derivatives and analogs. The compositions and methods disclosed herein are contemplated for application to any and all known derivatives or analogs of polyglutamylated methotrexate. In some embodiments, polyglutamylated methotrexate analog or derivative compositions made and used in accordance with the disclosed compositions and methods are shown in FIGS. 1I and 1J. In some embodiments, the analog corresponds to a modified form of methotrexate, in which case the glutamyl group of methotrexate does not bind to the remainder of the methotrexate molecule via a gamma peptide bond. In some embodiments, the analog is a variant of methotrexate, in which case the glutamyl group in methotrexate is of the D-form. In some embodiments, the polyglutamylated form of methotrexate, or polyglutamylated methotrexate analog or derivative, is not fluorinated.

[0123] In some embodiments, the polyglutamylated methotrexate analogs or derivatives encompassed by the present disclosure are indoline ring and modified ornithine or glutamate-containing methotrexate derivatives. In some embodiments, the polyglutamylated methotrexate analogs or derivatives encompassed by the present disclosure are indoline moiety-containing methotrexate derivatives, lipophilic amide methotrexate derivatives, alkyl-substituted benzene ring C-containing methotrexate derivatives, polymeric platinol methotrexate derivatives, N-(L-α-aminoacyl) methotrexate derivatives, halogenated methotrexate derivatives, 7-methylmethotrexate derivatives, N-(ac-aminoacyl) methotrexate derivatives, biotin methotrexate derivatives, dichloromethotrexate, lipophilic methotrexate derivatives, benzoxazine or benzothiazine moiety-containing methotrexate derivatives, and members selected from the group consisting of N delta-acyl-N alpha-(4-amino-4-deoxypteroyl)-L-ornithine derivatives.

[0124] In some embodiments, the polyglutamine oxidized methotrexate analogs or derivatives encompassed by the present disclosure are deoxyuridylate methotrexate, 10-deazaaminopterin analogs, 5-deazaaminopterin or 10-deazaaminopterin (10-EDAM) analogs, 5,10-dideazaaminopterin methotrexate analogs, 8-alkyl-7,8-dihydro analogs, L-threo-(2S,4S)-4-fluoroglutamic acid or DL-3,3-difluoroglutamic acid-containing methotrexate analogs, methotrexate tetrahydroquinazoline analogs, D-glutamic acid, D-erythro, threo-4-fluoroglutamic acid methotrexate analogs, βγ-methanomethotrexate analogs, γ-tetrazole methotrexate analogs, orthoisomers of aminopterin, hydroxymethylmethotrexate, γ-fluoromethotrexate, gem-diphosphonate methotrexate analogs, α- and / or γ-substituted methotrexate analogs, 5-methyl-5-deazamethotrexate analogs, 8-deazamethotrexate analogs, ashibicin methotrexate analogs, phosphonoglutamic acid analogs, poly(L-lysine) methotrexate complexes, disilyne or trilysin methotrexate derivatives, methotrexate-γ-dimyristoylphosphatidylethanolamine, iodoacetyllysine methotrexate analogs, 2,ω-diaminoalkanoic acid-containing methotrexate analogs, -methyl-5-deaza analogs, quinazoline methotrexate analogs, pyrazine methotrexate analogs, cysteic acid or homocysteic acid methotrexate analogs, γ-tert-butyl methotrexate ester, fluorinated methotrexate analogs, folic acid methotrexate analogs, 7-hydroxymethotrexate, poly-γ-glutamyl methotrexate analogs, 3’,5’-dichloromethotrexate, diazoketone and chloromethyl ketone methotrexate analogs, 10-propylgylaminopterin or alkyl methotrexate homologs, lectin derivatives of methotrexate, 3’,5’-dichloromethotrexate, deazaamethopterin analogs, cysteic acid and homocysteic acid methotrexate analogs, and members selected from the group consisting of MX068.

[0125] In further embodiments, the gamma-polyglutamylated methotrexate derivatives or analogs have variant polyglutamate chains. In some embodiments, the polyglutamate chain contains one or more natural or synthetic residues other than glutamate. In some embodiments, the polyglutamate chain contains one or more glutamyl groups that do not contain amide bonds. In other embodiments, one or more glutamyl groups of the polyglutamate chain are derivatized.

[0126] B. MTX-PG Synthesis The methotrexate polyglutamate compositions provided herein are obtained by the following synthetic methods known in the art. Procedures for synthesizing methotrexate (including different pharmaceutically acceptable salts or acids (e.g., methotrexate disodium) and crystalline and amorphous forms) and intermediates for synthesizing methotrexate include, but are not limited to, U.S. Patent Nos. 2,512,572; 3,892,801; 3,989,703; 4,057,548; 4,067,867; 4,079,056; 4,080,325; 4,106,488; 4,136,101; 4,224,446; 4,306,064; 4,374,987; 4,421,913; 4,558,690; 4,662,359; and 4,767,859; and those described in Calvert, Semin. Oncol. 26:3-10 (1999).

[0127] The addition of methotrexate to the glutamyl residue can be carried out using synthetic methods known in the art. In some embodiments, the glutamyl residue is sequentially added to the glutamyl residue of methotrexate. In further embodiments, the polyglutamate is added to the glutamyl residue of methotrexate using "click chemistry" methods or other bioconjugate chemistries known to those skilled in the art. Alternatively, a peptide of the desired length of the glutamyl residue can be generated and added to a precursor without the glutamyl residue of methotrexate. The peptide can be prepared using methods known in the art. In some embodiments, the initial glutamyl residue is attached to the Wang resin, and additional glutamyl residues are sequentially added by solid-phase peptide synthesis using F-moc chemistry. After the last glutamyl residue is added, the methotrexate precursor is attached to the peptide, and the molecule is cleaved from the resin.

[0128] C. Methotrexate-PG Complex Surprisingly, the inventors have found that polyglutamine oxidized folate antagonists such as methotrexate (γPMTX) can form complexes with other compositions including therapeutic agents, including cytotoxic compounds such as platinum-based compounds. Thus, in some embodiments, the present disclosure provides a complex of γPMTX (e.g., γPMTX disclosed herein) with a therapeutic agent or a salt or acid thereof.

[0129] In some embodiments, the γPMTX / complex comprises γPMTX and a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic compound such as a chemotherapeutic agent. In further embodiments, the γPMTX / complex comprises a platinum-based drug such as a platinum-based chemotherapeutic agent (e.g., cisplatin, carboplatin, and oxaliplatin). In other embodiments, the αPMTX / complex comprises a taxane-based chemotherapeutic agent (e.g., paclitaxel and docetaxel). In other embodiments, the γPMTX / complex comprises cyclodextrin. In further embodiments, the γPMTX / complex is encapsulated in liposomes.

[0130] In some embodiments, the present disclosure provides a composition comprising a complex of γPMTX and a therapeutic agent or a salt or acid thereof. In further embodiments, the γPMTX / therapeutic agent complex comprises one or more γPMTXs comprising 2 to 150, 2 to 100, 2 to 75, 2 to 50, 2 to 24, 2 to 30, 2 to 20, 2 to 19, 2 to 15, 2 to 10, or 2 to 5 glutamyl groups. In some embodiments, the γPMTX / therapeutic agent complex comprises one or more γPMTXs comprising 3 to 10, 3 to 9, 3 to 8, or 3 to 7 glutamyl groups, or any range therebetween. In other embodiments, the γPMTX / therapeutic agent complex comprises one or more γPMTXs comprising 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, or 4 to 5 glutamyl groups, or any range therebetween. In a particular embodiment, the complex comprises one or more γPMTXs comprising 3 to 10 glutamyl groups. In further embodiments, the γPMTX / therapeutic agent complex comprises one or more γPMTXs comprising 3 to 7 glutamyl groups. In another embodiment, the γPMTX / therapeutic agent complex comprises one or more γPMTXs comprising 5 glutamyl groups. In another embodiment, the γPMTX / therapeutic agent complex comprises one or more γPMTXs comprising 6 glutamyl groups. In some embodiments, the therapeutic agent is a cytotoxic compound or a salt or acid thereof. In further embodiments, the therapeutic agent is a chemotherapeutic agent or a salt or acid thereof. In another embodiment, the chemotherapeutic agent is a platinum-based drug. In another embodiment, the chemotherapeutic agent is a taxane-based drug. In further embodiments, the molar ratio of γPMTX / therapeutic agent in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of γPMTX / therapeutic agent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In other embodiments, the molar ratio of γPMTX / cyclodextrin in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween.In some embodiments, the molar ratio of γPMTX / to the therapeutic agent is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 - 50), or 1:>50. In some embodiments, the γPMTX / therapeutic agent complex is encapsulated in liposomes (e.g., in the manner described herein or by another method known in the art).

[0131] In alternative embodiments, the γPMTX complex comprises γPMTX and cyclodextrin. In some embodiments, the molar ratio of γPMTX (e.g., γPMTX salt) / cyclodextrin in the complex ranges from 1 - 20:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / cyclodextrin in the complex ranges from 1 - 10:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / cyclodextrin in the complex ranges from 2 - 8:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / cyclodextrin in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 - 50):1, or >50:1. In other embodiments, the molar ratio of γPMTX / cyclodextrin in the complex ranges from 1:1 - 20, 1:1 - 10, or 1:2 - 8, or any range therebetween. In some embodiments, the molar ratio of γPMTX / cyclodextrin is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 - 50), or 1:>50. In some embodiments, the γPMTX / cyclodextrin complex is encapsulated in liposomes (e.g., in the manner described herein or by another method known in the art).

[0132] In some embodiments, the present disclosure provides a composition comprising a γPMTX / platinum-based chemotherapeutic agent complex. In some embodiments, the platinum-based chemotherapeutic agent is selected from the group consisting of cisplatin, carboplatin, and oxaliplatin, or salts or acids thereof. In other embodiments, the γPMTX / platinum-based chemotherapeutic agent complex comprises an analogue of cisplatin, carboplatin, oxaliplatin, or salts or acids thereof. In some embodiments, the molar ratio of γPMTX / platinum-based agent in the complex ranges from 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / platinum-based agent in the complex ranges from 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / platinum-based agent in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / platinum-based agent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In other embodiments, the molar ratio of γPMTX / platinum-based agent in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of γPMTX / platinum-based agent is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50. In further embodiments, the γPMTX / platinum-based agent complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).

[0133] In a further embodiment, the γPMTX / platinum-based chemotherapeutic agent complex comprises cisplatin, carboplatin, an oxaliplatin analog, or a salt or acid thereof. In some embodiments, the molar ratio of γPMTX / platinum-based analog in the complex ranges from 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / platinum-based analog in the complex ranges from 1 to 10:1, or any range therebetween. In a further embodiment, the molar ratio of γPMTX / platinum-based agent in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / platinum-based analog in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In other embodiments, the molar ratio of γPMTX / platinum-based agent in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of γPMTX / platinum-based analog is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50. In a further embodiment, the γPMTX / platinum-based analog complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).

[0134] In a further embodiment, the present disclosure provides a complex comprising γPMTX and cisplatin or a salt or acid thereof. In some embodiments, the molar ratio of γPMTX / cisplatin (or a salt or acid of cisplatin) in the complex ranges from 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / cisplatin (or a salt or acid of cisplatin) in the complex ranges from 1 to 10:1, or any range therebetween. In a further embodiment, the molar ratio of γPMTX / cisplatin (or a salt or acid of cisplatin) in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / cisplatin (or a salt or acid of cisplatin) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In other embodiments, the molar ratio of γPMTX / cisplatin (or a salt or acid of cisplatin) in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of γPMTX / cisplatin (or a salt or acid of cisplatin) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50. In a further embodiment, the γPMTX / cisplatin (or a salt or acid of cisplatin) complex is encapsulated in liposomes (e.g., in the manner described herein or by another method known in the art).

[0135] In another embodiment, the present disclosure provides a complex comprising γPMTX and carboplatin or a salt or acid thereof. In some embodiments, the molar ratio of γPMTX / carboplatin (or a salt or acid of carboplatin) in the complex ranges from 1 to 20:1, or any range therebetween. In a further embodiment, the molar ratio of γPMTX / carboplatin (or a salt or acid of carboplatin) in the complex ranges from 1 to 10:1, or any range therebetween. In a further embodiment, the molar ratio of γPMTX / carboplatin (or a salt or acid of carboplatin) in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / carboplatin (or a salt or acid of carboplatin) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In other embodiments, the molar ratio of γPMTX / carboplatin (or a salt or acid of carboplatin) in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of γPMTX / carboplatin (or a salt or acid of carboplatin) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50. In a further embodiment, the γPMTX / carboplatin (or a salt or acid of carboplatin) complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).

[0136] In another embodiment, the present disclosure provides a complex comprising γPMTX and oxaliplatin or a salt or acid thereof. In some embodiments, the molar ratio of γPMTX / oxaliplatin (or a salt or acid of oxaliplatin) in the complex ranges from 1 to 20:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / oxaliplatin (or a salt or acid of oxaliplatin) in the complex ranges from 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / oxaliplatin (or a salt or acid of oxaliplatin) in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / oxaliplatin (or a salt or acid of oxaliplatin) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In other embodiments, the molar ratio of γPMTX / oxaliplatin (or a salt or acid of oxaliplatin) in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of γPMTX / oxaliplatin (or a salt or acid of oxaliplatin) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50. In further embodiments, the γPMTX / oxaliplatin (or a salt or acid of oxaliplatin) complex is encapsulated in liposomes (e.g., in the manner described herein or by another method known in the art).

[0137] In a further embodiment, the present disclosure provides a complex comprising γPMTX and a platinum-based chemotherapeutic agent ( "platinum") selected from the group consisting of nedaplatin, heptaplatin, lobaplatin, satraplatin, paraplatin, platinol, cycloplatin, dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iplatin, ormaplatin, zeniplatin, platinum-triamine, satraplatin, enroplatin, JM216, NK121, CI973, DWA2114R, NDDP, and nedaplatin, or salts or acids thereof. In other embodiments, the γPMTX / platinum-based chemotherapeutic agent complex comprises nedaplatin, heptaplatin, lobaplatin, satraplatin, paraplatin, platinol, cycloplatin, dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iplatin, ormaplatin, zeniplatin, platinum-triamine, satraplatin, enroplatin, JM216, NK121, CI973, DWA2114R, NDDP, or an analogue of nedaplatin, or salts or acids thereof. In some embodiments, the molar ratio of γPMTX / platinum (or a salt or acid of platinum) in the complex ranges from 1 to 20:1, or any range therebetween. In a further embodiment, the molar ratio of γPMTX / platinum (or a salt or acid of platinum) in the complex ranges from 1 to 10:1, or any range therebetween. In a further embodiment, the molar ratio of γPMTX / platinum (or a salt or acid of platinum) in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / platinum (or a salt or acid of platinum) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In other embodiments, the molar ratio of γPMTX / platinum (or a salt or acid of platinum) in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween.In some embodiments, the molar ratio of γPMTX / platinum (or a salt or acid of platinum) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 - 50), or 1:>50. In further embodiments, the γPMTX / platinum (or its salt or acid or analog) complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).

[0138] In some embodiments, the present disclosure provides a composition comprising a γPMTX / taxane chemotherapeutic agent (taxane) complex. In some embodiments, the taxane chemotherapeutic agent is selected from the group consisting of paclitaxel (PTX), docetaxel (DTX), larotaxel (LTX), and cabazitaxel (CTX), or salts or acids thereof. In some embodiments, the molar ratio of γPMTX / taxane agent in the complex ranges from 1 to 20:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / taxane (or salt or acid of taxane) in the complex ranges from 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / taxane (or salt or acid of taxane) in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / taxane (or salt or acid of taxane) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 - 50):1, or >50:1. In other embodiments, the molar ratio of γPMTX / taxane (or salt or acid of taxane) in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of γPMTX / taxane (or salt or acid of taxane) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 - 50), or 1:>50. In further embodiments, the γPMTX / taxane agent complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).

[0139] In further embodiments, the present disclosure provides a complex comprising γPMTX and paclitaxel (PTX) or a salt or acid thereof. In other embodiments, the γPMTX / taxane-based chemotherapeutic agent complex comprises an analog of paclitaxel (PTX), or a salt or acid thereof. In some embodiments, the molar ratio of γPMTX / paclitaxel (or a salt or acid of paclitaxel) in the complex ranges from 1 to 20:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / paclitaxel (or a salt or acid of paclitaxel) in the complex ranges from 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / paclitaxel (or a salt or acid of paclitaxel) in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / paclitaxel (or a salt or acid of paclitaxel) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPMTX / paclitaxel (or a salt or acid of paclitaxel) in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of γPMTX / paclitaxel (or a salt or acid of paclitaxel) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the γPMTX / paclitaxel (or a salt or acid of paclitaxel) complex is encapsulated in liposomes (e.g., in the manner described herein or by another method known in the art).

[0140] In further embodiments, the present disclosure provides a complex comprising γPMTX and docetaxel (DTX) or a salt or acid thereof. In other embodiments, the γPMTX / taxane-based chemotherapeutic agent complex comprises an analog of docetaxel (DTX), or a salt or acid thereof. In some embodiments, the molar ratio of γPMTX / docetaxel (or a salt or acid of docetaxel) in the complex ranges from 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / docetaxel (or a salt or acid of docetaxel) in the complex ranges from 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / docetaxel (or a salt or acid of docetaxel) in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / docetaxel (or a salt or acid of docetaxel) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of γPMTX / docetaxel (or a salt or acid of docetaxel) in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of γPMTX / docetaxel (or a salt or acid of docetaxel) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the γPMTX / docetaxel (or a salt or acid of docetaxel) complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).

[0141] In further embodiments, the present disclosure provides a complex comprising γPMTX and larotaxel (LTX) or a salt or acid thereof. In other embodiments, the γPMTX / taxane-based chemotherapeutic agent complex comprises an analog of larotaxel (LTX), or a salt or acid thereof. In some embodiments, the molar ratio of γPMTX / larotaxel (or a salt or acid of larotaxel) in the complex ranges from 1 to 20:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / larotaxel (or a salt or acid of larotaxel) in the complex ranges from 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / larotaxel (or a salt or acid of larotaxel) in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / larotaxel (or a salt or acid of larotaxel) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 - 50):1, or >50:1. In other embodiments, the molar ratio of γPMTX / larotaxel (or a salt or acid of larotaxel) in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of γPMTX / larotaxel (or a salt or acid of larotaxel) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 - 50), or 1:>50. In further embodiments, the γPMTX / larotaxel (or a salt or acid of larotaxel) complex is encapsulated in liposomes (e.g., in the manner described herein or by another method known in the art).

[0142] In further embodiments, the present disclosure provides a complex comprising γPMTX and cabazitaxel (CTX) or a salt or acid thereof. In other embodiments, the γPMTX / taxane-based chemotherapeutic agent complex comprises an analog of cabazitaxel (CTX), or a salt or acid thereof. In some embodiments, the molar ratio of γPMTX / cabazitaxel (or a salt or acid of cabazitaxel) in the complex ranges from 1 to 20:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / cabazitaxel (or a salt or acid of cabazitaxel) in the complex ranges from 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / cabazitaxel (or a salt or acid of cabazitaxel) in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / cabazitaxel (or a salt or acid of cabazitaxel) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In other embodiments, the molar ratio of γPMTX / cabazitaxel (or a salt or acid of cabazitaxel) in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of γPMTX / cabazitaxel (or a salt or acid of cabazitaxel) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50. In further embodiments, the γPMTX / cabazitaxel (or a salt or acid of cabazitaxel) complex is encapsulated in liposomes (e.g., in the manner described herein or by another method known in the art).

[0143] In further embodiments, the present disclosure provides a complex comprising γPMTX and another antimetabolite, or a salt or acid thereof. An antimetabolite is a chemical substance that is similar to a metabolite required for normal biochemical reactions but has a structure with sufficient differences to interfere with the normal functions of one or more cells, such as cell division. In some embodiments, the present disclosure provides a complex comprising γPMTX and methotrexate (MTX), or a salt or acid thereof. In some embodiments, the present disclosure provides a complex comprising γPMTX and an antimetabolite selected from the group consisting of gemcitabine, fluorouracil, capecitabine, antifolates (e.g., methotrexate, methotrexate), tegafur, cytarabine, thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, and any pharmaceutically acceptable salt or acid (s) or derivative thereof. In some embodiments, the molar ratio of γPMTX / antimetabolite (or salt or acid of antimetabolite) in the complex ranges from 1 to 20:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / antimetabolite (or salt or acid of antimetabolite) in the complex ranges from 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of γPMTX / antimetabolite (or salt or acid of antimetabolite) in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of γPMTX / antimetabolite (or salt or acid of antimetabolite) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 - 50):1, or >50:1. In other embodiments, the molar ratio of γPMTX / antimetabolite (or salt or acid of antimetabolite) in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween.In some embodiments, the molar ratio of γPMTX / antimetabolite (or a salt or acid of the antimetabolite) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 - 50), or 1:>50. In further embodiments, the γPMTX / antimetabolite (or a salt or acid of the antimetabolite) complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).

[0144] In further embodiments, the present disclosure provides a complex of γPMTX (e.g., γPMTX disclosed herein) and cyclodextrin. Cyclodextrin (CD) is a group of cyclic oligosaccharides that have been shown to improve the physicochemical properties of many drugs through complex formation. CD is a cyclic oligosaccharide composed of several D-glucose units linked by α-(1,4) bonds. This cyclic structure provides a hydrophobic internal cavity and gives CD a truncated cone shape. Many hydroxyl groups are located on the ends of the ring, which makes CD both lipophilic and water-soluble. As a result, CD can form complexes with a wide variety of hydrophobic agents, thereby changing the physicochemical properties of these complexed agents.

[0145] The term "cyclodextrin" or "CD" generally means a parent or derivatized cyclic oligosaccharide that can form a complex with methotrexate-PG and contains a variable number of (α-1,4)-linked D-glucopyranoside units, unless otherwise specified. Each cyclodextrin ring glucopyranoside subunit has secondary hydroxyl groups at the 2 and 3 positions and a primary hydroxyl group at the 6 position. The terms "parent," "underdivatized," or "inactive" cyclodextrin refer to the basic formula C 6 H 12 O 6And means cyclodextrin having a glucose structure and no additional chemical substituents (e.g., α-cyclodextrin consisting of 6 D-glucopyranoside units, β-cyclodextrin consisting of 7 D-glucopyranoside units, and γ-cyclodextrin consisting of 8 D-glucopyranoside units). The physical and chemical properties of the parent cyclodextrin can be modified by derivatizing the hydroxyl groups with other functional groups. Any substance located in the inner phase of cyclodextrin is said to be "complexed" with cyclodextrin or to form a complex (inclusion complex) with cyclodextrin.

[0146] As used herein, there are no special restrictions on the cyclodextrin component of the γPMTX / cyclodextrin complex as long as cyclodextrin can form a complex with γPMTX. In certain embodiments, the cyclodextrin is derivatized to have ionizable (e.g., weakly basic and / or weakly acidic) functional groups to facilitate complex formation with γPMTX and / or liposomal encapsulation.

[0147] Modification of hydroxyl groups of cyclodextrin, such as hydroxyl groups directed from the inner phase of cyclodextrin to the opposite side using ionizable chemical groups, is known to facilitate the addition of cyclodextrin and therapeutic agents complexed with cyclodextrin. In some embodiments, the cyclodextrin of the γPMTX / cyclodextrin complex has hydroxyl groups substituted with at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 ionizable chemical groups. The term "charged cyclodextrin" means cyclodextrin having hydroxyl groups substituted with one or more of its charged moieties. Such moieties can include a charged group per se or an organic moiety substituted with one or more charged moieties (e.g., C 1 -C 6 alkyl or C 1 -C 6 alkyl ether moiety).

[0148] In some embodiments, the "ionizable" or "charged" portion of the CD derivative is weakly ionizable. The weakly ionizable portion is a weakly basic or weakly acidic portion. The weakly basic functional group (W) has a pKa by CH3-W in the range of about 6.0 - 9.0, 6.5 - 8.5, 7.0 - 8.0, 7.5 - 8.0, and any range therebetween (including endpoints). Similarly, the weakly acidic functional group (X) has a logarithm of the dissociation constant (pKa) by CH3-X in the range of about 3.0 - 7.0, 4.0 - 6.5, 4.5 - 6.5, 5.0 - 6.0, 5.0 - 5.5, and any range therebetween (including endpoints). Representative anionic portions include, but are not limited to, carboxylate, carboxymethyl, succinyl, sulfonyl, phosphate, sulfoalkyl ether, sulfate carbonate, thiocarbonate, dithiocarbonate, phosphate, phosphonate, sulfonate, nitrate, and borate groups. Representative cationic portions include, but are not limited to, amino, guanidine, and quaternary ammonium groups.

[0149] In another embodiment, the derivatized cyclodextrin is a "polyanion" or a "polycat ion". A polyanion is a derivatized cyclodextrin having two or more negatively charged groups, resulting in a net negative ionic charge of three or more units. A polycation is a derivatized cyclodextrin having two or more positively charged groups, resulting in a net positive ionic charge of three or more units.

[0150] In another embodiment, the derivatized cyclodextrin is a "chargeable amphiphile". "Chargeable" means that the amphiphile has a pK in the range of pH 4 to pH 8 or 8.5. The chargeable amphiphile can thus be a weak acid or base. "Amphoteric" as used herein means a derivatized cyclodextrin having ionizable groups with both anionic and cationic characteristics, (a) at least one, optionally both, of the cationic and anionic amphiphiles being chargeable and having at least one charge group with a pK between 4 and 8 - 8.5, (b) the cationic charge being dominant at pH 4, and (c) the anionic charge being dominant at pH 8 - 8.5.

[0151] In some embodiments, the "ionizable" or "charged" derivatized cyclodextrins are generally polyionic, amphiphilic, or otherwise, weakly ionizable (e.g., having a pKai of about 4.0 - 8.5, 4.5 - 8.0, 5.0 - 7.5, 5.5 - 7.0, 6.0 - 6.5, and any range (including both ends) therebetween).

[0152] Any one, some, or all of the hydroxyl groups of any one, some, or all of the α-D-glucopyranoside units of any cyclodextrin can be modified to an ionizable chemical group as described herein. Since each cyclodextrin hydroxyl group has different chemical reactivity, the reaction with the modifying moiety can produce a mixture of positional and optical isomers. Alternatively, with specific chemistries, it can be reacted to form a homogeneous product of pre-modified α-D-glucopyranoside units.

[0153] Aggregate substitution occurring in cyclodextrin derivatives in a mixture is described by a term called degree of substitution. For example, 6-ethylenediamino-β-cyclodextrin having a degree of substitution of 7 would be composed of the distribution of isomers of 6-ethylenediamino-β-cyclodextrin where the number of ethylenediamino groups per 6-ethylenediamino-β-cyclodextrin molecule is 7. The degree of substitution of a cyclodextrin derivative mixture can be routinely measured using mass spectrometry or nuclear magnetic resonance spectroscopy.

[0154] In one embodiment, at least one hydroxyl moiety oriented in the opposite direction from within the cyclodextrin is replaced by an ionizable chemical group. For example, at least one of the γ-D-glucopyranoside units of C2, C3, C6, C2 and C3, C2 and C6, C3 and C6, and all three of C2-C3-C6 hydroxyls is replaced by an ionizable chemical group. Any such combination of hydroxyls can be combined with any of the degrees of substitution described herein, in addition to being able to similarly combine at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, up to all of the alpha-D-glucopyranoside units in the modified cyclodextrin. One such derivative is sulfalkyl ether cyclodextrin (SAE-CD). The sulfobutyl ether derivative of beta-cyclodextrin (SBE-β-CD) has been shown to have significantly improved water solubility compared to the parent cyclodextrin.

[0155] Additional cyclodextrin derivatives that can complex with a therapeutic agent in the disclosed liposome compositions include sugammadex or Org-25969, in which case the 6-hydroxy group on γ-CD is replaced by a carboxythioacetate ether bond and hydroxybutenyl-β-CD. Alternative forms of cyclodextrin include 2,6-di-O-methyl-β-CD (DIMEB), 2-hydroxylpropyl-3-cyclodextrin (HP-β-CD), randomly methylated-β-cyclodextrin (RAMEB), sulfobutyl ether β-cyclodextrin (SBE-β-CD), and sulfobutyl ether-γ-cyclodextrin (SBEγCD), sulfobutylated beta cyclodextrin sodium salt, (2-hydroxypropyl)-gamma-cyclodextrin, (2-hydroxypropyl)-beta-cyclodextrin, (2-hydroxypropyl)-γ-cyclodextrin, 2,6-di-O-methyl)-beta-cyclodextrin (DIMEB-50 heptakis), 2,3,6-tri-O-methyl)-beta-cyclodextrin (TRIMEB heptakis), methyl-beta-cyclodextrin, octakis(6-deoxy-6-iodo)-γ-cyclodextrin, and octakis(6-deoxy-6-bromo)-gamma-cyclodextrin.

[0156] In some embodiments, the cyclodextrin has high solubility in water to facilitate greater capture of the cyclodextrin in the liposome inner phase. In some embodiments, the aqueous solubility of the cyclodextrin is at least 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, or more. In some embodiments, the water solubility of the cyclodextrin is in the range of 10-150 mg / mL, 20-100 mg / mL, 20-75 mg / mL, and within any range (including both ends) therebetween.

[0157] In some embodiments, a large binding constant between cyclodextrin and γPMTX and / or other therapeutic agents complexed with cyclodextrin is preferred and can be obtained by selecting the number of glucose units in cyclodextrin based on the size of the therapeutic agent (see, for example, Albers et al., Crit. Rev. Therap. Drug Carrier Syst. 12:311 - 337 (1995); Stella et al., Toxicol. Pathol. 36:30 - 42 (2008)). When the binding constant is pH - dependent, cyclodextrin can be selected such that the binding constant is large at the pH of the inner phase of the liposome. As a result, the solubility (apparent solubility) of the therapeutic agent in the presence of cyclodextrin can be further improved. In some embodiments, the binding constant between cyclodextrin and the therapeutic agent is 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more. In some embodiments, the binding constant between cyclodextrin and the therapeutic agent is in the range of 100 - 1,200, 200 - 1,000, 300 - 750, and any range therebetween.

[0158] In some embodiments, the cyclodextrin of the γPMTX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is non - derivatized.

[0159] In some embodiments, the cyclodextrin of the γPMTX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is derivatized. In a further embodiment, the cyclodextrin derivative of the complex has the structure of Formula I:

Chemical Formula

[0160] In some embodiments, the cyclodextrin derivative of the γPMTX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex has the formula II:

Chemical formula

[0161] In some embodiments, the cyclodextrin derivatives of the γPMTX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex are cyclodextrins disclosed in U.S. Patent Nos. 6,133,248, 5,874,418, 6,046,177, 5,376,645, 5,134,127, 7,034,013, 6,869,939; and International Publication No. 02005 / 117911. The content of each of these patent documents is hereby incorporated by reference into this specification in preference.

[0162] In some embodiments, the cyclodextrin derivatives of the γPMTX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex are sulfoalkyl ether cyclodextrins. In some embodiments, the cyclodextrin derivative of the complex is a sulfobutyl ether - 3 - cyclodextrin such as CAPTISOL® (CyDex Pharma.Inc., Lenexa, Kansas). Methods for preparing sulfobutyl ether - 3 - cyclodextrin and other sulfoalkyl ether cyclodextrins are known in the art.

[0163] In some embodiments, the cyclodextrin derivatives of the γPMTX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex are of Formula III:

Chemical formula

[0164] In a further embodiment, the γPMTX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).

[0165] III. γPMTX Delivery Carrier In an alternative embodiment, the present disclosure provides γPMTX delivery systems and their use for delivering the payload of γPMTX to cells (single or plural) in vitro or in vivo. In some embodiments, γPMTX is complexed with or incorporated into a delivery carrier. Such delivery carriers are known in the art and include, but are not limited to, liposomes, lipospheres, polymers, peptides, proteins, antibodies (e.g., ADCs such as antibody-γPMTX complexes), cell components, cyclic oligosaccharides (e.g., cyclodextrins), nanoparticles (e.g., lipid nanoparticles, biodegradable nanoparticles, and core-shell nanoparticles), lipoprotein particles, and combinations thereof. In certain embodiments, the delivery carrier is a liposome. In other certain embodiments, the delivery carrier is an antibody or an antigen-binding antibody fragment.

[0166] A. Liposomes In some embodiments, the present disclosure provides a liposomal composition comprising liposomes encapsulating (i.e., filled with) gamma-polyglutamyl oxidized methotrexate (e.g., γPMTX as disclosed herein). In some embodiments, the liposomes in the liposomal composition comprise γPMTX comprising 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups (including the glutamyl groups of methotrexate). In some embodiments, the gamma-polyglutamyl oxidized methotrexate in Lp-γPMTX comprises two or more L-type glutamyl groups. In other embodiments, the gamma-polyglutamyl oxidized methotrexate in Lp-γPMTX comprises a D-type glutamyl group. In further embodiments, the gamma-polyglutamyl oxidized methotrexate in Lp-γPMTX comprises a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, the gamma-polyglutamyl oxidized methotrexate in Lp-γPMTX comprises two or more glutamyl groups having gamma-carboxyl bonds. In some embodiments, the liposomal composition comprises liposomes comprising γ-pentaglutamyl oxidized MTX. In further embodiments, the liposomes comprise L-γ-pentaglutamyl oxidized MTX, D-γ-pentaglutamyl oxidized MTX, or L- and D-γ-pentaglutamyl oxidized MTX. In some embodiments, the liposomal composition comprises liposomes comprising γ-hexaglutamyl oxidized MTX (Lp-γPMTX). In further embodiments, the liposomes comprise L-γ-hexaglutamyl oxidized MTX, D-γ-hexaglutamyl oxidized MTX, or L- and D-γ-hexaglutamyl oxidized MTX. In some embodiments, the liposomal composition comprises liposomes that are anionic or neutral. In some embodiments, the liposomal composition comprises liposomes that are cationic. In some embodiments, the Lp-γPMTX composition is not pegylated. In some embodiments, the Lp-γPMTX composition is not targeted (NTLp-γPMTX). In other embodiments, the Lp-γPMTX composition is targeted (TLp-γPMTX). In some embodiments, the liposomal composition comprises liposomes having a diameter in the range of 20 nm to 500 nm, or any range therebetween.In some embodiments, the liposome composition comprises liposomes having a diameter in the range of 20 nm to 400 nm, or any range therebetween. In some embodiments, the liposome composition comprises liposomes having a diameter in the range of 20 nm to 300 nm, or any range therebetween. In some embodiments, the liposome composition comprises liposomes having a diameter in the range of 20 nm to 200 nm, or any range therebetween. In further embodiments, the liposome composition comprises liposomes having a diameter in the range of 20 nm to 150 nm, or any range therebetween. In further embodiments, the liposome composition comprises liposomes having a diameter in the range of 80 nm to 120 nm, or any range therebetween. In further embodiments, 30 to 70%, 30 to 60%, or 30 to 50% w / w, or any range therebetween of gamma-polyglutamylated methotrexate is encapsulated (enclosed) in Lp-γPMTX during the liposome preparation process. In some embodiments, the Lp-αPMTX composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w of gamma-polyglutamylated MTX. In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% of gamma-polyglutamylated methotrexate is encapsulated in Lp-γPMTX during the liposome preparation process.

[0167] In some embodiments, the provided liposomes further comprise an immunostimulant, a detectable marker, or both, disposed on the outer surface of the liposomes. The immunostimulant or detectable marker can be ionically or covalently bound to the outer surface of the liposome, optionally including, for example, binding to the steric stabilizer component of the liposome.

[0168] The term "immunostimulatory agent" is also known as "immunostimulant" and "immunostimulator", and refers to a substance that stimulates immunity (including existing immune responses) by inducing the activation or increased activity of any component of the immune system. These immunostimulatory agents include one or more of haptens, adjuvants, protein immunostimulatory agents, nucleic acid immunostimulatory agents, and chemical immunostimulatory agents. Many adjuvants contain substances designed to stimulate immune responses, such as lipid A, proteins derived from Bordetella pertussis or Mycobacterium tuberculosis. Specific adjuvants include, for example, Freund's incomplete adjuvant and complete adjuvant (Difco Laboratories, Detroit, Mich.); Merck adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, PA.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron, or zinc; insoluble suspensions of acylated tyrosine; acylated saccharides; polysaccharides derivatized cationically or anionicly; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A; IFN alpha, IFN gamma, FLT3 ligand; and immunostimulatory antibodies (e.g., anti-CTLA-4, anti-CD28, anti-CD3) are commercially available. Cytokines such as GM-CSF, interleukin 2, 7, 12, and 15, and other similar growth factors can also be used as adjuvants. In a preferred embodiment, the immunostimulant can be at least one selected from the group consisting of fluorescein, DNP, beta glucan, beta-1,3-glucan, beta-1,6-glucan. In a further preferred embodiment, the immunostimulant is a toll-like receptor (TLR) regulator. In a further embodiment, the toll-like receptor (TLR) regulator is one or more of oxidized low density lipoprotein (e.g., OXPAC, PGPC), eritoran lipid (e.g., E5564), and resolvin.In some embodiments, the liposome contains fluorescein isothiocyanate (FITC), which, based on our experiments, surprisingly functions as both an immunostimulant and a detectable marker.

[0169] In some embodiments, the liposome contains a detectable marker. Detectable markers can include, for example, any suitable means known in the art, such as, at least, radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelating agents, enzymes, dyes, inks, magnetic compounds, biocatalysts, or pigments that are detectable by magnetic resonance imaging (MRI), optical imaging, fluorescence / luminescence imaging, and / or nuclear imaging techniques.

[0170] In some embodiments, the immunostimulant and / or detectable marker is bound to the outer surface by co-incubation with the liposome. For example, the immunostimulant and / or detectable marker can be bound to the liposome membrane by hydrophobic interactions or ionic bonds such as avidin / biotin binding or metal chelate binding (e.g., Ni-NTA). Alternatively, the immunostimulant or detectable marker can be covalently bound to the outer surface of the liposome, for example, by covalently binding to a liposome component or to a steric stabilizer that is PEG.

[0171] In some embodiments, the liposome further contains an agent that increases the uptake of the liposome into the intracellular compartment of the target cell containing the cytosol.

[0172] In some embodiments, the liposome comprises a mitochondrial targeting agent. In some embodiments, the liposome comprises triphenylphosphonium (TPP). Methods and mechanisms for surface functionalization of liposomes using TPP are known in the art (e.g., binding to a lipid anchor via a peg spacer group and modifying TPP with a stearyl group (stearyltriphenylphosphonium (STPP))). In some embodiments, the liposome comprises high-density octaarginine. In some embodiments, the liposome comprises sphingomyelin and / or sphingomyelin metabolites. Sphingomyelin metabolites used to formulate the liposomes of the present invention can include, for example, ceramide, sphingosine or sphingosine 1-phosphate. In some embodiments, the liposome comprises rhodamine 123. In some embodiments, the liposome comprises a mitochondrial permeability peptide. In some embodiments, the liposome comprises a mitofusin peptide, a mitochondrial targeting signal peptide, and an antennapedia helix III homeodomain cell membrane permeable peptide (ANT) (e.g., RQIKIWFQNRRMKWKKRKKRRQRRR (SEQ ID NO: 1), RKKRRXRRRGC, wherein X is any natural or non-natural amino acid (SEQ ID NO: 2), CCGCCAAGAAGCG (SEQ ID NO: 3), GCGTGCACACGCGCGTAGACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAAGAAGCGACCCCTCCGGGGCGAGCTGAGCGGCGTGGCGCGGGGGCGTCAT (SEQ ID NO: 4), ACGTGCATACGCACGTAGACATTCCCCGCTTCCCACTCCAAAGTCCGCCAAGAAGCGTATCCCGCTGAGCGGCGTGGCGCGGGGGCGTCATCCGTCAGCTC (SEQ ID NO: 5), or ACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAAGAAGCGACCCCTCCGGGGCGAGCTG (SEQ ID NO: 6)), or a mitochondrial permeabilizing agent selected from the group consisting of mitochondrial permeable fragments thereof).

[0173] In some embodiments, the liposomes in the provided liposome composition include a mitochondrial permeabilizing agent selected from guanidine-rich peptides, tetraguanidium, triguanidium, diguanidium, monoguanidium, guanidine-rich polycarbamate, beta-oligoarginine, proline-rich dendrimers, and phosphonium salts (e.g., methyltriphenylphosphonium and / or tetraphenylphosphonium).

[0174] In some embodiments, the liposomes in the provided liposome composition include sphingomyelin and / or stearyl octaarginine. In some embodiments, the liposomes include sphingomyelin and / or stearyl octaarginine. In some embodiments, the liposomes include DOPE, sphingomyelin, stearyl octaarginine sphingomyelin and stearyl octaarginine. In some embodiments, the liposomes include DOPE, sphingomyelin, stearyl octaarginine sphingomyelin and stearyl octaarginine in a molar ratio of 9:2:1. In some embodiments, the liposomes include the MITO-Porter® system or a variant thereof.

[0175] In some embodiments, the liposomes in the provided liposome composition include agents such as membrane permeabilizing agents that facilitate the delivery of the liposomes across the cell membrane and confer the ability to bypass the harsh environment of the endocytic pathway and lysosomes to the liposomes. Membrane permeabilizing agents are known in the art and can be routinely used and applied to the manufacture and use of the provided liposome composition. In some embodiments, the membrane penetration agent / lysosome bypass agent is chloroquine. In some embodiments, the membrane permeabilizing agent is a cell-permeable peptide.In some embodiments, the liposomes in the provided liposome composition comprise a membrane permeabilizing agent selected from the following group: RKKRRQRRR (SEQ ID NO: 7), GRKKRRQRRRTPQ (SEQ ID NO: 8), YGRKKRRQRRR (SEQ ID NO: 9), AAVALLPAVLLALLA (SEQ ID NO: 10), MGLGLHLLVLAAALQ (SEQ ID NO: 11), GALFLGFLGAAGSTM (SEQ ID NO: 12), AGYLLGKINLKALAALAKKIL (SEQ ID NO: 13), RVIRVWFQNKRCKDKK (SEQ ID NO: 14), RQIKIWFQNRRMKWKK (SEQ ID NO: 15), GLFEAIAGFIENGWEGMIDG (SEQ ID NO: 16), GWTLNSAGYLLGKIN (SEQ ID NO: 17), RSQSRSRYYRQRQRS (SEQ ID NO: 18), LAIPEQEY (SEQ ID NO: 19), LGIAEQEY (SEQ ID NO: 20), LGIPAQEY (SEQ ID NO: 21), LGIPEAEY (SEQ ID NO: 22), LGIPEQAY (SEQ ID NO: 23), LGIAEAEY (SEQ ID NO: 24), LGIPEAAY (SEQ ID NO: 25), LGIAEQAY (SEQ ID NO: 26), LGIAEAAY (SEQ ID NO: 27), LLIILRRRIRKQAHAHSK (SEQ ID NO: 28), LKALAALAKKIL (SEQ ID NO: 29), KLALKLALKALKAALKLA (SEQ ID NO: 30), KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 31), DHQLNPAF (SEQ ID NO: 32), DPKGDPKG (SEQ ID NO: 33), VTVTVTVTVTGKGDPKPD (SEQ ID NO: 34), RQIKIWFQNRRMKWKK (SEQ ID NO: 35), GRKKRRQRRRPPQ (SEQ ID NO: 36), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 37), GRKKRRQRRR (SEQ ID NO: 38), RRRRRRR (SEQ ID NO: 39), RRRRRRRR (SEQ ID NO: 40), RRRRRRRRR (SEQ ID NO: 41), RRRRRRRRRR (SEQ ID NO: 42), RRRRRRRRRRR (SEQ ID NO: 43), and YTIWMPENPRPGTPCDIFTNSRGKRASNGGGG(R)n (wherein n = 2 to 15 R in L- and / or D-form) (SEQ ID NO: 44), or cell permeable fragments thereof.

[0176] As discussed above, liposomes can include steric stabilizers that can extend their lifetime in circulation. For these embodiments incorporating a steric stabilizer, the steric stabilizer can be at least one member selected from the group consisting of polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinylpyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol; poly[N-(2-hydroxypropyl)methacrylamide]; amphiphilic poly-N-vinylpyrrolidone; L-amino acid-based polymers; oligoglycerin, polyethylene glycol and polypropylene oxide-containing copolymers, poloxamer 188, and polyvinyl alcohol. In some embodiments, the steric stabilizer or group of steric stabilizers is PEG. In one embodiment, the steric stabilizer is PEG. In a further embodiment, the PEG has a number average molecular weight (Mn) of 200 to 5000 daltons. These PEGs can have any structure such as a linear, branched, star or comb structure and are commercially available.

[0177] In some embodiments, the liposomal composition comprises pegylated liposomes (PLp-γPMTX). In some embodiments, the pegylated liposomes in the liposomal composition comprise γPMTX containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the gamma-polyglutamylated methotrexate in Lp-γPMTX comprises 2 or more L-type glutamyl groups. In other embodiments, the gamma-polyglutamylated methotrexate in Lp-γPMTX comprises a D-type glutamyl group. In further embodiments, the gamma-polyglutamylated methotrexate in Lp-γPMTX comprises a D-type glutamyl group and 2 or more L-type glutamyl groups. In some embodiments, the liposomal composition comprises pegylated liposomes containing γ-pentaglutamylated MTX. In further embodiments, the liposomes comprise L-γ-pentaglutamylated MTX, D-γ-pentaglutamylated MTX, or L- and D-γ-pentaglutamylated MTX. In some embodiments, the liposomal composition comprises pegylated liposomes containing γ-hexaglutamylated MTX. In further embodiments, the liposomes comprise L-γ-hexaglutamylated MTX, D-γ-hexaglutamylated MTX, or L- and D-γ-hexaglutamylated MTX. In some embodiments, the liposomal composition comprises pegylated liposomes that are anionic or neutral. In some embodiments, the liposomal composition comprises pegylated liposomes that are cationic. In some embodiments, the PLp-γPMTX composition is not targeted (NTPLp-γPMTX). In other embodiments, the PLp-γPMTX composition is targeted (TPLp-γPMTX). In some embodiments, the liposomal composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w of gamma-polyglutamylated methotrexate.In some embodiments, a liposomal composition comprising pegylated liposomes containing at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% of gamma-polyglutamylated methotrexate is encapsulated (enclosed) in PLp-γPMTX during the process of preparing the liposomes. In some embodiments, the liposomal composition comprises pegylated liposomes having a diameter in the range of 20 nm to 500 nm. In some embodiments, the liposomal composition comprises pegylated liposomes having a diameter in the range of 20 nm to 400 nm. In some embodiments, the liposomal composition comprises pegylated liposomes having a diameter in the range of 20 nm to 300 nm. In some embodiments, the liposomal composition comprises pegylated liposomes having a diameter in the range of 20 nm to 200 nm. In a further embodiment, the liposomal composition comprises pegylated liposomes having a diameter in the range of 80 nm to 120 nm.

[0178] In some embodiments, more than 70%, 80% or 90% of the polyglutamylated methotrexate in the provided liposomal composition is pentaglutamylated. In some embodiments, more than 70%, 80% or 90% of the polyglutamylated methotrexate in the provided liposomal composition is hexaglutamylated. In some embodiments, more than 70%, 80% or 90% of the polyglutamylated methotrexate in the composition has 4 to 10, 4 to 6, or more than 5 γ-glutamyl groups.

[0179] In some embodiments, a gamma-polyglutamylated methotrexate composition (e.g., a delivery carrier such as a polyglutamate and a liposome containing a polyglutamate) is in an aqueous solution. In some embodiments, the γPMTX composition, as a liposomal composition, is administered at a dose of about 0.005 to about 5000 mg of γPMTX per square meter (m 2 ) of body surface area, or any range therebetween. In a further embodiment, the γPMTX composition, as a liposomal composition, is per square meter (m2 ) is administered at a dose of about 0.1 to about 1000 mg of γPMTX per body surface area, or any range therebetween.

[0180] (1) Liposomal composition The lipids and other components of the liposomes contained in the liposomal composition can be any lipid, combination and ratio of lipids, or combination of lipids and other liposomal components and their respective ratios known in the art. However, without limitation, it will be understood by those skilled in the art that the liposomal encapsulation of any particular drug, such as gamma polyglutamylated MTX discussed herein, may involve substantially routine experimentation to obtain a useful and functional liposomal formulation. Generally, the liposomes provided can have any liposomal structure, for example, a structure having an inner space isolated from the outer medium by one or more lipid bilayers, or any microcapsule structure having a semipermeable membrane with a lipophilic central portion where the membrane isolates the interior. The lipid bilayer can be any amphiphilic molecule having a hydrophilic moiety (hydrophilic moiety hydrophilic moiety) and a hydrophobic moiety (hydrophobic moiety). Usually, the amphiphilic molecules in the bilayer are arranged in a two-dimensional sheet where the hydrophobic moieties face the inside of the sheet while the hydrophilic moieties face the outside. The amphiphilic molecules forming the liposomes provided can be any known or hereafter discovered amphiphilic molecules (e.g., synthetic or natural origin lipids or biocompatible lipids). Liposomes can be formed by amphiphilic polymers and surfactants, such as polymersomes and niosomes. In the present disclosure, without limitation, these liposome-forming materials are also referred to as "lipids".

[0181] The liposomal composition formulations provided herein can be in liquid or dry forms such as dry powder or dry cake. The dry powder or dry cake can be, for example, subjected to primary drying under lyophilization conditions, or can be subjected to only primary drying or both primary and secondary drying. In the dry form, the powder or cake can have, for example, 1% to 6% moisture, such as 2% to 5% moisture or 2% to 4% moisture. An example of the drying method is lyophilization (also called freeze-drying or cryodessication). Any of the compositions and methods of the present disclosure can include liposomes, lyophilized liposomes, or liposomes reconstituted from lyophilized liposomes. In some embodiments, the disclosed compositions and methods include one or more lyoprotectants or cryoprotective substances. These protectants are typically polyhydroxy compounds such as saccharides (monosaccharides, disaccharides, and polysaccharides), polyhydric alcohols, and their derivatives, glycerol or polyethylene glycol, trehalose, maltose, sucrose, glucose, lactose, dextran, glycerol, or aminoglycosides. In further embodiments, the lyoprotectant or cryoprotective substance comprises up to 10% or up to 20% of the solution outside, inside, or both outside and inside the liposome.

[0182] In some embodiments, the liposomes contain steric stabilizers that extend their lifespan in circulation. One or more steric stabilizers, such as hydrophilic polymers (polyethylene glycol (PEG)), glycolipids (monosialoganglioside (GM1)), or others, occupy the space directly adjacent to the liposome surface and exclude other polymers from this space. As a result, access and binding of plasma opsonins to the liposome surface are hindered, thus suppressing the interaction of such liposomes with macrophages or any other removal mechanism, and extending the lifespan of the liposomes in circulation. In some embodiments, the steric stabilizer or group of steric stabilizers is PEG or a combination containing PEG. In further embodiments, the steric stabilizer is PEG or a combination containing PEG with a number average molecular weight (Mn) in the range of 200 to 5000 daltons. These PEGs can have any structure, such as linear, branched, star-shaped, or comb-shaped structures, and are commercially available.

[0183] The diameter of the disclosed liposomes is not particularly limited. In some embodiments, the liposomes have a diameter in the range of, for example, 30 nm to 150 nm (nanometers). In other embodiments, the liposomes have a diameter in the range of 40 nm to 70 nm.

[0184] The properties of the liposomes are affected by the nature of the lipids used to produce the liposomes. A wide variety of lipids have been used to produce liposomes. These include cationic, anionic, and neutral lipids. In some embodiments, the liposomes containing gamma-polyglutamylated methotrexate are anionic or neutral. In other embodiments, the provided liposomes are cationic. The determination of the charge (e.g., anionic, neutral, or cationic) can be determined by routine work by measuring the zeta potential of the liposomes. The zeta potential of the liposomes can be positive, zero, or negative. In some embodiments, the zeta potential of the liposomes is below zero. In some embodiments, the zeta potential of the liposomes is in the range of 0 to -150 mV. In another embodiment, the zeta potential of the liposomes is in the range of -30 to -50 mV.

[0185] In some embodiments, cationic lipids are used to create cationic liposomes, which are commonly used as gene delivery agents. The positive charges on the cationic liposomes enable their interaction with the negative charges on the cell surface. After the cationic liposomes bind to the cells, the liposomes are transported into the cells by endocytosis.

[0186] In some preferred embodiments, neutral to anionic liposomes are used. In a preferred embodiment, anionic liposomes are used. For example, by using a mixture of neutral lipids such as HSPC and anionic lipids such as PEG-DSPE, anionic liposomes are formed, which have a low likelihood of non-specific binding to normal cells. Specific binding to tumor cells can be achieved using tumor targeting antibodies such as folate receptor antibodies including, for example, folate receptor alpha antibody, folate receptor beta antibody, and / or folate receptor delta antibody.

[0187] As an example, at least one (or several) lipids are amphiphilic lipids defined as having hydrophilic and hydrophobic moieties (usually a hydrophilic head and a hydrophobic tail). The hydrophobic moiety usually faces the hydrophobic phase (e.g., within the bilayer), while the hydrophilic moiety usually faces the aqueous phase (e.g., outside the bilayer). The hydrophilic moiety can include polar or charged groups such as carbohydrates, phosphates, carboxylic acids, sulfates, aminos, sulfhydryls, nitros, hydroxyls, and other similar groups. The hydrophobic moiety can include nonpolar groups including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and groups substituted by one or more aromatic, alicyclic, or heterocyclic groups. Examples of amphiphilic compounds include, but are not limited to, phospholipids, amino lipids, and sphingolipids.

[0188] Typically, for example, the lipid is a phospholipid. Phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and the like. It should be understood that other lipid membrane components such as cholesterol, sphingomyelin, and cardiolipin can also be used.

[0189] The lipids including liposomes provided herein can be anionic and neutral (including zwitterionic and polar) lipids including anionic and neutral phospholipids. Neutral lipids exist in an uncharged or neutral zwitterionic form at the selected pH. At physiological pH, such lipids include, for example, dioleoylphosphatidylglycerol (DOPG), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebroside, and diacylglycerol. Examples of zwitterionic lipids include, but are not limited to, dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), and dioleoylphosphatidylserine (DOPS). Anionic lipids are negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and anionic modifications attached to neutral lipids.

[0190] In this specification, anionic and neutral lipids are collectively referred to as non-cationic lipids. Such lipids may contain phosphorus, but they are not so limited. Examples of non-cationic lipids include lecithin, lysophosphatidylcholine, phosphatidylethanolamine, lysophosphatidylethanolamine, dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleyoylphosphatidylglycerol (POPG), 16-0-monomethyl PE, 16-0-dimethyl PE, 18-1-trans PE, palmitoyloleoylphosphatidylethanolamine (POPE), 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, and cholesterol.

[0191] Liposomes can be constructed using any liposome assembly method and liposomal components (also referred to as liposome components) known in the art. Liposomal components include, for example, lipids such as DSPE, HSPC, cholesterol, and derivatives of these components. Other suitable lipids are commercially available, for example, from Avanti Polar Lipids, Inc. (Alabaster, Alabama, USA). A partial list of available negatively or neutrally charged lipids suitable for the preparation of anionic liposomes can be, for example, at least one of the following: DLPC, DMPC, DPPC, DSPC, DOPC, DMPE, DPPE, DOPE, DMPA·Na, DPPA·Na, DOPA·Na, DMPG·Na, DPPG·Na, DOPG·Na, DMPS·Na, DPPS·Na, DOPS·Na, DOPE-glutaryl·(Na)2, tetramyristoyl cardiolipin·(Na)2, DSPE-mPEG-2000·Na, DSPE-mPEG-5000·Na, and DSPE-maleimide PEG-2000·Na.

[0192] In some embodiments, the γPMTX compositions provided herein are formulated in liposomes comprising a cationic lipid. In one embodiment, but not limited thereto, the cationic lipid is selected from the cationic lipids described in International Publication Nos. WO 2012 / 040184, WO 2011 / 153120, WO 2011 / 149733, WO 2011 / 090965, WO 2011 / 043913, WO 2011 / 022460, WO 2012 / 061259, WO 2012 / 054365, WO 2012 / 044638, WO 2010 / 080724, WO 2010 / 21865, and WO 2008 / 103276, U.S. Pat. Nos. 7,893,302, 7,404,969, 8,283,333, U.S. Patent Application Publication Nos. 20100036115, and 20120202871. Each of these patent documents is hereby incorporated by reference in its entirety. In another embodiment, the cationic lipid can be selected from Formula A described in International Publication Nos. WO 2012 / 040184, WO 2011 / 153120, WO 2011 / 149733, WO 2011 / 090965, WO 2011 / 043913, WO 2011 / 022460, WO 2012 / 061259, WO 2012 / 054365, and WO 2012 / 044638, but not limited thereto. Each of these patent documents is hereby incorporated by reference in its entirety. In yet another embodiment, the cationic lipid can be selected from Formulas CLI - CLXXIX of International Publication No. WO 2008 / 103276, Formulas CLI - CLXXIX of U.S. Pat. No. 7,893,302, Formulas CLI - CLXXXXII of U.S. Pat. No. 7,404,969, and Formulas I - VI of U.S. Patent Application Publication No. 20100036115, but not limited thereto. Each of these respective patent documents is hereby incorporated by reference in its entirety. By way of non-limiting example, the cationic lipid can be selected from the following: (20Z,23Z)-N,N-dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z)-N,N-dimethyl-hexacosa-17,20-dien-9-amine, (1Z,19Z)-N5N-dimethylpentacosa-16,19-dien-8-amine, (13Z,16Z)-N,N-dimethyldocosa-13,16-dien-5-amine, (12Z,(15Z)-N,N-Dimethylheneicos-12,15-dien-4-amine, (14Z,17Z)-N,N-dimethyltricos-14,17-dien-6-amine, (15Z,18Z)-N,N-dimethyltetracos-15,18-dien-7-amine, (18Z,21Z)-N,N-dimethylheptacos-18,21-dien-10-amine, (15Z,18Z)-N,N-dimethyltetracos-15,18-dien-5-amine, (14Z,17Z)-N,N-dimethyltricos-14,17-dien-4-amine, (19Z,22Z)-N,N-dimethylocatacos-19,22-dien-9-amine, (18Z,21Z)-N,N-dimethylheptacos-18,21-dien-8-amine, (17Z,20Z)-N,N-dimethylhexacos-17,20-dien-7-amine, (16Z,19Z)-N,N-dimethylpentacos-16,19-dien-6-amine, (22Z,25Z)-N,N-dimethylhentriaconta-22,25-dien-10-amine, (21Z,24Z)-N,N-dimethyltriaconta-21,24-dien-9-amine, (18Z)-N,N-dimethylheptacos-18-en-10-amine, (17Z)-N,N-dimethylhexacos-17-en-9-amine, (19Z,22Z)-N,N-dimethyloctacos-19,22-dien-7-amine, N,N-dimethylheptacosan-10-amine, (20Z,23Z)-N-ethyl-N-methylnonacos-20,23-dien-10-amine, 1-[(11Z,14Z)-1-nonylicos-11,14-dien-1-yl]pyrrolidine, (20Z)-N,N-dimethylheptacos-20-en-10-amine, (15Z)-N,N-dimethylheptacos-15-en-10-amine, (14Z)-N,N-dimethylnonacos-14-en-10-amine, (17Z)-N,N-dimethylnonacos-17-en-10-amine, (24Z)-N,N-dimethyltritriaconta-24-en-10-amine, (20Z)-N,N-dimethylnonacos-20-en-10-amine, (22Z)-N,N-dimethylhentriaconta-22-en-10-amine, (16Z)-N,N-dimethylpentacos-16-en-8-amine, (12Z,15Z)-N,N-dimethyl-2-nonylheneicos-12,15-dien-1-amine, (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecane-8-amine, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecane-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecane-10-amine, N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]heneicosane-10-amine, N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecane-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecane-8-amine, N,N-dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradecane-5-amine, N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecane-1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecane-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecane-6-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecane-8-amine, R--N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, S--N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propane-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z-)-oct-5-en-1-yloxy]propane-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine, (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,(12Z)-Octadeca-9,12-dien-1-yloxy]propan-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-[(9Z)-octadeca-9-en-1-yloxy]-3-(octyloxy)propan-2-amine; (2S)-N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-triene-1-yloxy]-3-(octyloxy)propan-2-amine, (2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine, 1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl 1-3-(octyloxy)propan-2-amine, 1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethyl-propan-2-amine, (2S)-1-[(13Z)-docosa-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, 1-[(13Z)-docosa-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(9Z)-hexadeca-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2R)-N,N-dimethyl-H(1-methyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-R9Z,12Z)-octadeca-9,12-Dien-1-yloxypropan-2-amine, N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine, N,N-dimethyl-1-{[-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine and (11E,20Z,23Z)-N,N-dimethylnonacosa-11,20,23-triene-10-amine or a pharmaceutically acceptable salt or acid or stereoisomer thereof.,

[0193] In one embodiment, the lipid can be a cleavable lipid such as those described in WO 2012 / 170889, which is hereby incorporated by reference in its entirety.,

[0194] The cationic lipid can be synthesized routinely using methods known in the art and / or as described in WO 2012 / 040184, WO 2011 / 153120, WO 2011 / 149733, WO 2011 / 090965, WO 2011 / 1043913, WO 2011 / 022460, WO 2012 / 061259, WO 2012 / 054365, WO 2012 / 044638, WO 2010 / 080724 and WO 2010 / 21865, which are hereby incorporated by reference in their entirety.,

[0195] The lipid derivative can include, for example, at least one or more steric stabilizers and / or the attachment (preferably covalent attachment) of a functional group to the liposome component, after which the steric stabilizer and / or the functional group should be regarded as part of the liposome component. The functional group includes groups that can be used to attach the liposome component to another moiety such as a protein. Such functional groups include at least maleimide. These steric stabilizers include at least one selected from the group consisting of polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinylpyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol; poly[N-(2-hydroxypropyl)methacrylamide]; amphiphilic poly-N-vinylpyrrolidone; L-amino acid-based polymers; and polyvinyl alcohol.

[0196] In some embodiments, the γPMTX composition is formulated in a lipid-polycation complex. The formation of the lipid-polycation complex can be achieved using methods known in the art and / or as described in U.S. Patent Application Publication No. 20120178702, which is hereby incorporated by reference in its entirety. As a non-limiting example, polycations include, but are not limited to, cationic peptides or polypeptides such as polylysine, polyornithine, and / or polyarginine, and the cationic peptides described in International Publication No. 2012 / 013326, which is hereby incorporated by reference in its entirety. In another embodiment, γPMTX is formulated in a lipid-polycation complex, which further includes neutral lipids such as cholesterol or dioleoylphosphatidylethanolamine (DOPE), among others.

[0197] The components of the liposome can include any molecule that binds thereto (e.g., chemical / drug / reagent / protein), and in some embodiments, the components of the provided liposome include at least members selected from the group DSPE, DSPE-PEG, DSPE-maleimide, HSPC; HSPC-PEG; HSPC-maleimide; cholesterol; cholesterol-PEG; and cholesterol-maleimide. In some embodiments, the components of the provided liposome include DSPE, DSPE-PEG, DSPE-maleimide, HSPC; HSPC-PEG; HSPC-maleimide; cholesterol; cholesterol-PEG; and cholesterol-maleimide. In a preferred embodiment, the liposome components constituting the liposome include DSPE; DSPE-FITC; DSPE-maleimide; cholesterol; and HSPC.

[0198] In a further embodiment, the liposomes of the liposome compositions provided herein include oxidized phospholipids. In some embodiments, the liposome includes an oxidized phospholipid that is a member selected from the group consisting of phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, and 1-palmitoyl-2-arachidonoyl-sn-glycero-2-phosphate. In some embodiments, the phospholipid has an unsaturated bond. In some embodiments, the phospholipid is arachidonic acid containing the phospholipid. In a further embodiment, the phospholipid is sn-2-oxygenated. In a further embodiment, the phospholipid is not fragmented.

[0199] In some embodiments, the liposomes of the disclosed liposome compositions comprise oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC). As used herein, the term "oxPAPC" means the lipid produced by the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), which results in a mixture of oxidized phospholipids containing fragmented or full-length oxygenated sn-2 residues. Characteristic oxidized fragmentation species contain 5-carbon sn-2 residues with omega aldehyde or omega carboxyl groups. Oxidation of the arachidonic acid residue also produces phospholipids containing esterified isoprostanes. OxPAPC includes, among many of the oxidized products present in oxPAPC, in particular, the HOdiA-PC, KOdiA-PC, HOOA-PC and KOOA-PC species. In further embodiments, oxPAPC is an epoxyisoprostane-containing phospholipid. In further embodiments, oxPAPC is 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine (5,6-PEIPC), 1-palmitoyl-2-(epoxycyclopentenone)-sn-glycero-3-phosphocholine (PECPC) and / or 1-palmitoyl-2-(epoxy-isoprostane E2)-sn-glycero-4-phosphocholine (PEIPC). In some embodiments, the phospholipid has an unsaturated bond. In some embodiments, the phospholipid is arachidonic acid containing the phospholipid. In further embodiments, the phospholipid is sn-2-oxygenated. In further embodiments, the phospholipid is not fragmented.

[0200] In some embodiments, the liposomal gamma-polyglutamyl oxidized methotrexate composition is pegylated (i.e., pegylated liposomal gamma-polyglutamyl oxidized (e.g., pentaglutamyl oxidized or hexaglutamyl oxidized) folate antagonist (PLp-γPMTX or TPLp-γPMTX)). In some embodiments, PLp-γPMTX or TPLp-γPMTX is water-soluble. That is, PLp-γPMTX or TPLp-γPMTX is in the form of an aqueous solution.

[0201] In some embodiments, the liposomes of the disclosed liposome compositions comprise lipids selected from the following: 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC); 1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphocholine; 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-hexadecyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine; and 1-palmitoyl-2-acetyl-sn-glycero-3-phosphocholine. In further embodiments, the liposomes comprise PGPC.

[0202] In some embodiments, the pH of the solution comprising the liposome composition is from pH 5 to 8, or from pH 2 to 6, pH 2 to 8, or any range therebetween. In some embodiments, the pH of the solution comprising the liposome composition is from pH 5 to 8, or any range therebetween. In some embodiments, the pH of the solution comprising the liposome composition is from pH 6 to 7, or any range therebetween. In some embodiments, the pH of the solution comprising the liposome composition is from 6 to 7.5, 6.5 to 7.5, 6.7 to 7.5, or 6.3 to 7.0, or any range therebetween.

[0203] In some embodiments, at least one component of the liposomal lipid bilayer is functionalized (or reactive). As used herein, a functionalized component is a component that includes a reactive group that can be used to crosslink reagents and moieties to the lipid. When a lipid is functionalized, any liposomes it forms are also functionalized. In some embodiments, the reactive group is one that reacts with a crosslinking agent (or other moiety) to form a crosslink. The reactive groups in the liposomal lipid bilayer are positioned somewhere on the lipid such that upon contact with a crosslinking agent, crosslinking with another moiety (e.g., a steric stabilizer or targeting moiety) is enabled. In some embodiments, the reactive group is in the head group of the lipid, such as a phospholipid. In some embodiments, the reactive group is a maleimide group. Maleimide groups can crosslink with each other in the presence of a dithiol crosslinking agent such as, but not limited to, dithiothreitol (DTT).

[0204] It should be understood that the use of other functionalized lipids, other reactive groups, and other crosslinking agents beyond those described above is further contemplated. In addition to maleimide groups, other examples of reactive groups contemplated include, but are not limited to, other thiol-reactive groups, amino groups such as primary or secondary amines, carboxyl groups, hydroxyl groups, aldehyde groups, alkyne groups, azide groups, carbonyl groups, haloacetyl (e.g., iodoacetyl) groups, imidoester groups, N-hydroxysuccinimide esters, sulfhydryl groups, and pyridyl disulfide groups.

[0205] Functionalized and non-functionalized lipids are available from many commercial sources such as Avanti Polar Lipids (Alabaster, AL) and Lipoid LLC (Newark, NJ).

[0206] (2) Liposomal interior space In further non-limiting embodiments, the liposomes provided include an interior space. In some embodiments, the internal space, although not limited, contains an aqueous solution. In some embodiments, the internal space contains the gamma-polyglutamyl oxidized methotrexate provided herein. In further embodiments, the internal space of the liposome contains an isotonic agent. In some embodiments. In some embodiments, the concentration (wt%) of the isotonic agent is 0.1-20%, 1-20%, 0.5-15%, 1-15% or 1-50%, or any range therebetween. In some embodiments, the internal space of the liposome contains a sugar (e.g., trehalose, maltose, sucrose, lactose, mannose, mannitol, glycerol, dextrose, fructose, etc.). In further embodiments, the concentration (wt%) of the sugar is 0.1-20%, 1-20%, 0.5-15%, 1-15% or 1-50%, or any range therebetween. In some embodiments, the pH of the internal space of the liposome is pH 2-8, or any range therebetween. In some embodiments, the pH of the solution containing the liposome composition is pH 5-8, or any range therebetween. In some embodiments, the pH of the solution containing the liposome composition is pH 6-7, or any range therebetween. In some embodiments, the pH of the solution containing the liposome composition is 6-7.5, 6.5-7.5, 6.7-7.5, or 6.3-7.0, or any range therebetween. In some embodiments, the internal space contains a buffer. In further embodiments, the buffer is a buffer selected from HEPES, citrate, or sodium phosphate (e.g., monosodium phosphate and / or disodium phosphate). In some embodiments, the buffer is HEPES. In some embodiments, the buffer is citrate. In some embodiments, the buffer is sodium phosphate (e.g., monosodium phosphate and / or disodium phosphate). In some embodiments, the buffer is at a concentration of 15-200 mM, or any range therebetween. In still further embodiments, the buffer is at a concentration of 5-200 mM, 15-200 mM, 5-100 mM, 15-100 mM, 5-50 mM, 15-50 mM, 5-25 mM, 5-20 mM, 5-15 mM, or any range therebetween.In some embodiments, the buffer is HEPES at a concentration of 15 - 200 mM, or any range therebetween. In some embodiments, the buffer is citrate at a concentration of 15 - 200 mM, or any range therebetween. In some embodiments, the buffer is sodium phosphate at a concentration of 15 - 200 mM, or any range therebetween. In some embodiments, the internal space of the liposome contains a total concentration of sodium acetate and calcium acetate of 5 mM - 500 mM, or 50 mM - 500 mM, or any range therebetween.

[0207] In some embodiments, the internal space of the liposome contains trehalose. In further embodiments, the concentration (wt%) of trehalose is 0.1 - 20%, 1 - 20%, 0.5 - 15%, 1 - 15%, or 5 - 20%, or any range therebetween. In still further embodiments, the concentration (wt%) of trehalose is 1 - 15%, or any range therebetween. In additional embodiments, trehalose is at about 5% - 20% (wt) trehalose, or any combination of one or more lyoprotectants or cryoprotective substances is present at a total concentration of 5% - 20%. In some embodiments, the pH of the solution containing the liposome composition is 6 - 7.5, 6.5 - 7.5, 6.7 - 7.5, or 6.3 - 7.0, or any range therebetween. In some embodiments, the internal space contains a buffer solution. In some embodiments, the buffer solution is selected from HEPES, citrate, or sodium phosphate (e.g., monosodium phosphate and / or disodium phosphate). In some embodiments, the buffer solution is HEPES. In some embodiments, the buffer solution is citrate. In some embodiments, the buffer solution is sodium phosphate (e.g., monosodium phosphate and / or disodium phosphate). In some embodiments, the buffer solution is at a concentration of 15 - 200 mM, or any range therebetween. In still further embodiments, the buffer solution is at a concentration of 5 - 200 mM, 15 - 200 mM, 5 - 100 mM, 15 - 100 mM, 5 - 50 mM, 15 - 50 mM, 5 - 25 mM, 5 - 20 mM, 5 - 15 mM, or any range therebetween. In some embodiments, the buffer solution is HEPES at a concentration of 15 - 200 mM, or any range therebetween. In some embodiments, the buffer solution is citrate at a concentration of 15 - 200 mM, or any range therebetween. In some embodiments, the buffer solution is sodium phosphate at a concentration of 15 - 200 mM, or any range therebetween. In further embodiments, the internal space of the liposome contains sodium acetate and / or calcium acetate.In some embodiments, the internal space of the liposome contains a total concentration of sodium acetate and calcium acetate in the range of 5 mM to 500 mM, or 50 mM to 500 mM, or any range therebetween.

[0208] In some embodiments, the internal space of the liposome contains dextrose. In further embodiments, the concentration (weight %) of dextrose is 0.1 - 20%, 1 - 20%, 0.5 - 15%, 1 - 15%, or 5 - 20%, or any range therebetween. In still further embodiments, the concentration (weight %) of dextrose is 1 - 15%, or any range therebetween. In additional embodiments, dextrose is present at a dextrose concentration of about 5% - 20% (weight %), or any combination of one or more cryoprotectants or cryoprotective substances is present at a total concentration of 5% - 20%. In some embodiments, the pH of the solution containing the liposome composition is 6 - 7.5, 6.5 - 7.5, 6.7 - 7.5, or 6.3 - 7.0, or any range therebetween. In some embodiments, the internal space contains a buffer solution. In some embodiments, the buffer solution is selected from HEPES, citrate, or sodium phosphate (e.g., monosodium phosphate and / or disodium phosphate). In some embodiments, the buffer solution is HEPES. In some embodiments, the buffer solution is citrate. In some embodiments, the buffer solution is sodium phosphate (e.g., monosodium phosphate and / or disodium phosphate). In some embodiments, the buffer solution is at a concentration of 15 - 200 mM, or any range therebetween. In still further embodiments, the buffer solution is at a concentration of 5 - 200 mM, 15 - 200 mM, 5 - 100 mM, 15 - 100 mM, 5 - 50 mM, 15 - 50 mM, 5 - 25 mM, 5 - 20 mM, 5 - 15 mM, or any range therebetween. In some embodiments, the buffer solution is HEPES at a concentration of 15 - 200 mM, or any range therebetween. In some embodiments, the buffer solution is citrate at a concentration of 15 - 200 mM, or any range therebetween. In some embodiments, the buffer solution is sodium phosphate at a concentration of 15 - 200 mM, or any range therebetween. In further embodiments, the internal space of the liposome contains sodium acetate and / or calcium acetate.In some embodiments, the internal space of the liposome contains a total concentration of sodium acetate and calcium acetate in the range of 5 mM to 500 mM, or 50 mM to 500 mM, or any range therebetween.

[0209] In further embodiments, the present disclosure provides a liposomal composition comprising liposomes encapsulating (filling) gamma-polyglutamylated methotrexate (e.g., γPMTX disclosed herein). In some embodiments, the liposomes in the liposomal composition contain γPMTX containing 4, 5, 2 to 10, 4 to 6, or more than 5 γ-glutamyl groups (including the glutamyl groups of methotrexate). In some embodiments, the gamma-polyglutamylated methotrexate in Lp-γPMTX contains two or more L-type glutamyl groups. In other embodiments, the gamma-polyglutamylated methotrexate in Lp-γPMTX contains a D-type glutamyl group. In further embodiments, the gamma-polyglutamylated methotrexate in Lp-γPMTX contains a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, the gamma-polyglutamylated methotrexate in Lp-γPMTX contains two or more glutamyl groups having gamma-carboxyl bonds. In some embodiments, the liposomal composition comprises liposomes containing γ-pentaglutamylated MTX. In further embodiments, the liposomes contain L-γ-pentaglutamylated MTX, D-γ-pentaglutamylated MTX, or L- and D-γ-pentaglutamylated MTX. In some embodiments, the liposomal composition comprises liposomes (Lp-γPMTX) containing γ-hexaglutamylated MTX. In further embodiments, the liposomes contain L-γ-hexaglutamylated MTX, D-γ-hexaglutamylated MTX, or L- and D-γ-hexaglutamylated MTX.

[0210] In some embodiments, the targeted pegylated liposome gamma-polyglutamylated (e.g., pentaglutamylated or hexaglutamylated) methotrexate comprises a medium comprising a liposome comprising an internal space; aqueous gamma-polyglutamylated methotrexate disposed within the internal space; and a targeting moiety comprising a protein having specific affinity for at least one folate receptor, wherein the targeting moiety is disposed on the outer surface of the liposome. In some embodiments, the medium is an aqueous solution. In some embodiments, the internal space, the external space (e.g., the medium), or both the internal space and the medium comprise one or more of the cryoprotectants or cryoprotective substances listed above. In some embodiments, the cryoprotective substance is mannitol, trehalose, sorbitol, or sucrose.

[0211] In some embodiments, gamma-polyglutamyl methotrexate (e.g., Lp-γPMTX including PLp-γPMTX, TPLp-γPMTX, TLp-γPMTX, and NTLp-γPMTX) encapsulated liposomes have an internal space containing less than 500,000 or less than 200,000 gamma-polyglutamyl methotrexate molecules. In some embodiments, the liposome internal space contains from 10 to 100,000 or any range in between gamma-polyglutamyl methotrexate molecules. In some embodiments, the liposome internal space contains from 10,000 to 100,000 or any range in between gamma-polyglutamyl methotrexate molecules. In some embodiments, the liposome is not pegylated and has an internal space containing less than 500,000 or less than 200,000 gamma-polyglutamyl methotrexate molecules. In some embodiments, the liposome is not pegylated and the internal space of the liposome contains from 10 to 100,000 or any range in between gamma-polyglutamyl methotrexate molecules. In a further embodiment, the liposome is not pegylated and the internal space of the liposome contains from 10,000 to 100,000 or any range in between gamma-polyglutamyl methotrexate molecules. In some embodiments, the liposome is targeted and not pegylated (TLp-γPMTX) and has an internal space containing less than 500,000 or less than 200,000 gamma-polyglutamyl methotrexate molecules. In some embodiments, the liposome is targeted and not pegylated and the internal space of the liposome contains from 10 to 100,000 or any range in between gamma-polyglutamyl methotrexate molecules. In a further embodiment, the liposome is targeted and not pegylated and the internal space of the liposome contains from 10,000 to 100,000 or any range in between gamma-polyglutamyl methotrexate molecules.In some embodiments, the liposomes are not targeted and not PEGylated (NTLp-γPMTX), and have an internal space containing less than 500,000 or less than 200,000 gamma-polyglutamylated methotrexate molecules. In some embodiments, the liposomes are not targeted and not PEGylated, and the internal space of the liposomes contains from 10 to 100,000 or any range therebetween of gamma-polyglutamylated methotrexate molecules. In further embodiments, the liposomes are not targeted and not PEGylated, and the internal space of the liposomes contains from 10,000 to 100,000 or any range therebetween of gamma-polyglutamylated methotrexate molecules.

[0212] In some embodiments, the liposome encapsulates gamma-polyglutamylated methotrexate containing 2 to 10 glutamyl groups (i.e., Lp-γPMTX including PLp-γPMTX, TPLp-γPMTX, TLp-γPMTX, and NTLp-γPMTX), and has an internal space containing less than 500,000, or less than 200,000, gamma-polyglutamylated methotrexate molecules containing 2 to 10 glutamyl groups. In some embodiments, the internal space of the liposome contains 10 to 100,000 or any range therebetween of gamma-polyglutamylated methotrexate molecules containing 2 to 10 glutamyl groups. In further embodiments, the internal space of the liposome contains 10,000 to 100,000 or any range therebetween of gamma-polyglutamylated methotrexate molecules containing 2 to 10 glutamyl groups. In some embodiments, the liposome is not pegylated and has an internal space containing less than 500,000 or less than 200,000 gamma-polyglutamylated methotrexate molecules containing 2 to 10 glutamyl groups. In some embodiments, the liposome is not pegylated, and the internal space of the liposome contains 10 to 100,000 or any range therebetween of gamma-polyglutamylated methotrexate molecules containing 2 to 10 glutamyl groups. In further embodiments, the liposome is not pegylated, and the internal space of the liposome contains 10,000 to 100,000 or any range therebetween of gamma-polyglutamylated methotrexate molecules containing 2 to 10 glutamyl groups. In some embodiments, the liposome is targeted and not pegylated (TLp-γPMTX) and has an internal space containing less than 500,000 or less than 200,000 gamma-polyglutamylated methotrexate molecules containing 2 to 10 glutamyl groups. In some embodiments, the liposome is targeted and not pegylated, and the internal space of the liposome contains 10 to 100,000 or any range therebetween of gamma-polyglutamylated methotrexate molecules containing 2 to 10 glutamyl groups.In a further embodiment, the liposome is targeted and not pegylated, and the internal space of the liposome contains gamma-polyglutamylated methotrexate molecules containing 2 to 10 glutamyl groups in an amount of 10,000 to 100,000 or any range therebetween. In some embodiments, the liposome is not targeted and not pegylated (NTLp-γPMTX) and has an internal space containing gamma-polyglutamylated methotrexate molecules containing 2 to 10 glutamyl groups in an amount less than 500,000 or less than 200,000. In some embodiments, the liposome is not targeted and not pegylated, and the internal space of the liposome contains gamma-polyglutamylated methotrexate molecules containing 2 to 10 glutamyl groups in an amount of 10 to 100,000 or any range therebetween. In a further embodiment, the liposome is not targeted and not pegylated, and the internal space of the liposome contains gamma-polyglutamylated methotrexate molecules containing 2 to 10 glutamyl groups in an amount of 10,000 to 100,000 or any range therebetween.

[0213] In some embodiments, gamma-polyglutamyl oxidized methotrexate (i.e., Lp-γPMTX including PLp-γPMTX, TPLp-γPMTX, TLp-γPMTX, and NTLp-γPMTX) encapsulated liposomes have an internal space containing less than 500,000 or less than 200,000 gamma-tetraglutamyl oxidized methotrexate molecules. In some embodiments, the liposome internal space contains 10 to 100,000 or any range therebetween of gamma-tetraglutamyl oxidized methotrexate molecules. In some embodiments, the liposome internal space contains 10,000 to 100,000 or any range therebetween of gamma-tetraglutamyl oxidized methotrexate molecules. In some embodiments, the liposomes are not pegylated and have an internal space containing less than 500,000 or less than 200,000 gamma-tetraglutamyl oxidized methotrexate molecules. In some embodiments, the liposomes are not pegylated and the internal space of the liposomes contains 10 to 100,000 or any range therebetween of gamma-tetraglutamyl oxidized methotrexate molecules. In a further embodiment, the liposomes are not pegylated and the internal space of the liposomes contains 10,000 to 100,000 or any range therebetween of gamma-tetraglutamyl oxidized methotrexate molecules. In some embodiments, the liposomes are targeted and not pegylated (TLp-γPMTX) and have an internal space containing less than 500,000 or less than 200,000 gamma-tetraglutamyl oxidized methotrexate molecules. In some embodiments, the liposomes are targeted and not pegylated and the internal space of the liposomes contains 10 to 100,000 or any range therebetween of gamma-tetraglutamyl oxidized methotrexate molecules. In a further embodiment, the liposomes are targeted and not pegylated and the internal space of the liposomes contains 10,000 to 100,000 or any range therebetween of gamma-tetraglutamyl oxidized methotrexate molecules.In some embodiments, the liposome is not targeted and not PEGylated (NTLp-γPMTX) and has an internal space containing less than 500,000 or less than 200,000 gamma-tetraglutamylated methotrexate molecules. In some embodiments, the liposome is not targeted and not PEGylated, and the internal space of the liposome contains from 10 to 100,000 or any range therebetween of gamma-tetraglutamylated methotrexate molecules. In further embodiments, the liposome is not targeted and not PEGylated, and the internal space of the liposome contains from 10,000 to 100,000 or any range therebetween of gamma-tetraglutamylated methotrexate molecules.

[0214] In some embodiments, gamma-polyglutamyl methotrexate (i.e., Lp-γPMTX including PLp-γPMTX, TPLp-γPMTX, TLp-γPMTX, and NTLp-γPMTX) encapsulated liposomes have an internal space containing less than 500,000 or less than 200,000 gamma-pentaglutamyl methotrexate molecules. In some embodiments, the liposome internal space contains from 10 to 100,000 or any range therebetween of gamma-pentaglutamyl methotrexate molecules. In some embodiments, the liposome internal space contains from 10,000 to 100,000 or any range therebetween of gamma-pentaglutamyl methotrexate molecules. In some embodiments, the liposome is not pegylated and has an internal space containing less than 500,000 or less than 200,000 gamma-pentaglutamyl methotrexate molecules. In some embodiments, the liposome is not pegylated and the internal space of the liposome contains from 10 to 100,000 or any range therebetween of gamma-pentaglutamyl methotrexate molecules. In further embodiments, the liposome is not pegylated and the internal space of the liposome contains from 10,000 to 100,000 or any range therebetween of gamma-pentaglutamyl methotrexate molecules. In some embodiments, the liposome is targeted and not pegylated (TLp-γPMTX) and has an internal space containing less than 500,000 or less than 200,000 gamma-pentaglutamyl methotrexate molecules. In some embodiments, the liposome is targeted and not pegylated and the internal space of the liposome contains from 10 to 100,000 or any range therebetween of gamma-pentaglutamyl methotrexate molecules. In further embodiments, the liposome is targeted and not pegylated and the internal space of the liposome contains from 10,000 to 100,000 or any range therebetween of gamma-pentaglutamyl methotrexate molecules.In some embodiments, the liposome is not targeted and not PEGylated (NTLp-γPMTX) and has an internal space containing less than 500,000 or less than 200,000 gamma pentaglutamylated methotrexate molecules. In some embodiments, the liposome is not targeted and not PEGylated, and the internal space of the liposome contains from 10 to 100,000 or any range therebetween of gamma pentaglutamylated methotrexate molecules. In further embodiments, the liposome is not targeted and not PEGylated, and the internal space of the liposome contains from 10,000 to 100,000 or any range therebetween of gamma pentaglutamylated methotrexate molecules.

[0215] In some embodiments, the gamma-hexaglutamyl oxidized methotrexate (i.e., Lp-γPMTX including PLp-γPMTX, TPLp-γPMTX, TLp-γPMTX, and NTLp-γPMTX) encapsulated liposomes have an internal space containing less than 500,000 or less than 200,000 gamma-hexaglutamyl oxidized methotrexate molecules. In some embodiments, the liposome internal space contains from 10 to 100,000 or any range therebetween gamma-hexaglutamyl oxidized methotrexate molecules. In further embodiments, the liposome internal space contains from 10,000 to 100,000 or any range therebetween gamma-hexaglutamyl oxidized methotrexate molecules. In some embodiments, the liposomes are not pegylated and have an internal space containing less than 500,000 or less than 200,000 gamma-hexaglutamyl oxidized methotrexate molecules. In some embodiments, the liposomes are not pegylated and the internal space of the liposome contains from 10 to 100,000 or any range therebetween gamma-hexaglutamyl oxidized methotrexate molecules. In further embodiments, the liposomes are not pegylated and the internal space of the liposome contains from 10,000 to 100,000 or any range therebetween gamma-hexaglutamyl oxidized methotrexate molecules. In some embodiments, the liposomes are targeted and not pegylated (TLp-γPMTX) and have an internal space containing less than 500,000 or less than 200,000 gamma-hexaglutamyl oxidized methotrexate molecules. In some embodiments, the liposomes are targeted and not pegylated and the internal space of the liposome contains from 10 to 100,000 or any range therebetween gamma-hexaglutamyl oxidized methotrexate molecules. In further embodiments, the liposomes are targeted and not pegylated and the internal space of the liposome contains from 10,000 to 100,000 or any range therebetween gamma-hexaglutamyl oxidized methotrexate molecules.In some embodiments, the liposome is not targeted and not PEGylated (NTLp-γPMTX) and has an internal space containing less than 500,000 or less than 200,000 gamma-hexaglutamylated methotrexate molecules. In some embodiments, the liposome is not targeted and not PEGylated, and the internal space of the liposome contains from 10 to 100,000 or any range therebetween of gamma-hexaglutamylated methotrexate molecules. In further embodiments, the liposome is not targeted and not PEGylated, and the internal space of the liposome contains from 10,000 to 100,000 or any range therebetween of gamma-hexaglutamylated methotrexate molecules.

[0216] In some embodiments, the present disclosure provides a liposomal gamma-polyglutamylated methotrexate composition, wherein the liposome encapsulates gamma-polyglutamylated methotrexate or a salt or acid thereof, and one or more aqueous pharmaceutically acceptable carriers. In some embodiments, the internal space of the liposome contains trehalose. In some embodiments, the internal space of the liposome contains 5% to 20% (w / w) trehalose. In some embodiments, the internal space of the liposome contains HBS at a concentration of 1 to 200 mM and a pH of 2 to 8. In some embodiments, the internal space of the liposome has a pH of 5 to 8, or any range therebetween. In some embodiments, the internal space of the liposome has a pH of 6 to 7, or any range therebetween. In some embodiments, the internal space of the liposome contains a total concentration of sodium acetate and calcium acetate in the range of 50 mM to 500 mM, or any range therebetween.

[0217] A polyglutamylated non-polyglutamylatable folate antagonist In some embodiments, liposomal gamma-polyglutamylated methotrexate (e.g., Lp-γPMTX including PLp-γPMTX, TPLp-γPMTX, TLp-γPMTX, and NTLp-γPMTX) compositions comprise gamma-polyglutamylated methotrexate (e.g., γPMTX disclosed herein) and one or more non-polyglutamylated polyglutamylatable folate antimetabolite compositions.

[0218] In some embodiments, Lp-γPMTX (e.g., PLp-γPMTX, TPLp-γPMTX, TLp-γPMTX, and NTLp-γPMTX) comprises gamma-polyglutamylated methotrexate (e.g., γPMTX disclosed herein) and methotrexate (MTX). In some embodiments, Lp-γPMTX (i.e., liposomal gamma-polyglutamylated methotrexate) comprises gamma-polyglutamylated methotrexate, and a polyglutamylatable folate antagonist selected from the group consisting of methotrexate (MTX), pemetrexed (PMX), raltitrexed (RTX), pralatrexate, AG2034, GW1843, aminopterin, and LY309887. In some embodiments, Lp-γPMTX comprises gamma-polyglutamylated methotrexate and raltitrexed. In some embodiments, Lp-γPMTX comprises gamma-polyglutamylated methotrexate and pemetrexed. In some embodiments, Lp-γPMTX comprises gamma-polyglutamylated methotrexate and leucovorin. In some embodiments, Lp-γPMTX comprises gamma-polyglutamylated methotrexate and a triazine folate antagonist derivative (e.g., sulfonylurilidotriazine such as NSC127755). In some embodiments, Lp-γPMTX comprises gamma-polyglutamylated methotrexate and a serine hydroxymethyltransferase (SHMT2) inhibitor. In some embodiments, the SHMT2 inhibitor is a folate antagonist (e.g., a polyglutamylatable or non-polyglutamylatable folate antagonist). In some embodiments, the SHMT2 inhibitor is a folate antagonist.

[0219] B Non-polyglutamylatable folate antagonist In some embodiments, Lp-γPMTX (e.g., PLp-γPMTX, TPLp-γPMTX, TLp-γPMTX, and NTLp-γPMTX) comprises gamma-polyglutamylated methotrexate (e.g., γPMTX as disclosed herein) and a so-called "non-polyglutamylatable" folate antagonist. In some embodiments, the liposome comprises gamma-polyglutamylated methotrexate and a non-polyglutamylatable folate antagonist that inhibits one or more enzymes in the folate cycle metabolic pathway. In further embodiments, the non-polyglutamylatable folate antagonist inhibits one or more enzymes selected from thymidylate synthase (TS), dihydrofolate reductase (DHFR), glycineamide ribonucleotide (GAR) transformylase, and aminoimidazole carboxamide ribonucleotide (AICAR) transformylase. In some embodiments, the liposome comprises gamma-polyglutamylated methotrexate and a non-polyglutamylatable folate antagonist that inhibits DHFR. In some embodiments, the liposome comprises gamma-polyglutamylated methotrexate and a non-polyglutamylatable folate antagonist that inhibits TS. In some embodiments, the liposome comprises gamma-polyglutamylated methotrexate and a non-polyglutamylatable folate antagonist that inhibits GAR or AICAR transformylase. In further embodiments, the non-polyglutamylatable folate antagonist is selected from the group consisting of trimethoprim (TMQ), piritrexim (BW301U), and talotrexin (PT523). In further embodiments, the non-polyglutamylatable folate antagonist is selected from the group consisting of nolatrexed (AG337), premetrexed (ZD9331, BGC9331), and BGC945 (ONX0801).

[0220] C Platinum In some embodiments, liposomes containing gamma-polyglutamyl methotrexate (e.g., Lp-γPMTX such as PLp-γPMTX, TPLp-γPMTX, TLp-γPMTX, and NTLp-γPMTX) contain gamma-polyglutamyl methotrexate (e.g., γPMTX disclosed herein) and a platinum-based chemotherapeutic agent or a salt or acid thereof. In some embodiments, the liposomes contain a gamma-polyglutamyl methotrexate / platinum-based drug complex (e.g., as described in Section IIC).

[0221] In some embodiments, Lp-γPMTX contains a platinum-based chemotherapeutic agent selected from the group consisting of cisplatin, carboplatin, and oxaliplatin, or a salt or acid thereof. In other embodiments, Lp-γPMTX contains an analog of a platinum-based chemotherapeutic agent selected from the group consisting of cisplatin, carboplatin, and oxaliplatin, or a salt or acid thereof.

[0222] In some embodiments, Lp-γPMTX contains gamma-polyglutamyl methotrexate and cisplatin or a salt or acid thereof. In some embodiments, Lp-γPMTX contains gamma-polyglutamyl methotrexate and a cisplatin analog or a salt or acid thereof.

[0223] In some embodiments, Lp-γPMTX contains gamma-polyglutamyl methotrexate and carboplatin or a salt or acid thereof. In some embodiments, the liposomes contain gamma-polyglutamyl methotrexate and a carboplatin analog or a salt or acid thereof.

[0224] In some embodiments, Lp-γPMTX contains gamma-polyglutamyl methotrexate and oxaliplatin or a salt or acid thereof. In some embodiments, the liposomes contain gamma-polyglutamyl methotrexate and an oxaliplatin analog or a salt or acid thereof.

[0225] In some embodiments, the liposome comprises a platinum-based chemotherapeutic agent selected from the group consisting of gamma polyglutamyl oxidized methotrexate (e.g., γPMTX as disclosed herein) and nedaplatin, heptaplatin, and lobaplatin, nedaplatin, heptaplatin and lobaplatin or salts or acids thereof. In some embodiments, Lp-γPMTX comprises a gamma polyglutamyl oxidized methotrexate and an analog of a platinum-based chemotherapeutic agent selected from the group consisting of nedaplatin, heptaplatin, and lobaplatin, or salts or acids thereof.

[0226] In some embodiments, Lp-γPMTX comprises a gamma polyglutamyl oxidized methotrexate and a platinum-based chemotherapeutic agent selected from the group consisting of satraplatin, paraplatin, platinol, cisplatin, dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iplatin, ormaplatin, zeniplatin, platinum-triamine, satraplatin, enroplatin, JM216, 254-S, NK121, CI973, DWA2114R, NDDP, and nedaplatin, or salts or acids thereof. In some embodiments, Lp-γPMTX comprises a gamma polyglutamyl oxidized methotrexate and an analog of a platinum-based chemotherapeutic agent selected from the group consisting of satraplatin, paraplatin, platinol, cisplatin, dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iplatin, ormaplatin, zeniplatin, platinum-triamine, satraplatin, enroplatin, JM216, 254-S, NK121, CI973, DWA2114R, NDDP, and nedaplatin, or salts or acids thereof.

[0227] In some embodiments, the liposome composition comprises liposomes further comprising one or more of an immunostimulant, a detectable marker, and a maleimide disposed in at least one of the PEG of the liposome or the outer surface.

[0228] D-Cyclodextrin In further embodiments, the γPMTX liposomes comprise γPMTX (e.g., γPMTX disclosed herein) and cyclodextrin (e.g., the cyclodextrin in Section IIC herein).

[0229] In some embodiments, the γPMTX liposomes comprise a complex formed by cyclodextrin and a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic compound or a salt or acid thereof. In further embodiments, the therapeutic agent is a chemotherapeutic agent or a salt or acid thereof. In another embodiment, the chemotherapeutic agent is a platinum-based drug. In another embodiment, the chemotherapeutic agent is a taxane-based drug. In further embodiments, the therapeutic agent of the cyclodextrin / therapeutic agent complex is a member selected from the group consisting of gemcitabine, gemcitabine-based therapeutic agents, doxorubicin, folic acid antagonists, folic acid antagonist-based chemotherapeutic agents, or salts or acids thereof, acid form or free base form. In further embodiments, the molar ratio of cyclodextrin / therapeutic agent in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin / therapeutic agent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin / therapeutic agent in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin / therapeutic agent is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50.

[0230] In some embodiments, the γPMTX liposomes contain γPMTX and a cyclodextrin / platinum-based chemotherapeutic agent complex. In some embodiments, the platinum-based chemotherapeutic agent is selected from the group consisting of cisplatin, carboplatin, and oxaliplatin, or salts or acids thereof. In other embodiments, the cyclodextrin / platinum-based chemotherapeutic agent complex contains cisplatin, carboplatin, oxaliplatin, or analogs of salts or acids thereof. In some embodiments, the molar ratio of cyclodextrin / platinum-based drug in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin / platinum-based drug in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin / platinum-based drug in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin / platinum-based drug is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50.

[0231] In some embodiments, the platinum-based chemotherapeutic agent is selected from the group consisting of cisplatin, carboplatin, and oxaliplatin, or salts or acids thereof. In other embodiments, the cyclodextrin / platinum-based chemotherapeutic agent complex comprises cisplatin, carboplatin, oxaliplatin, or analogs of salts or acids thereof. In some embodiments, the molar ratio of cyclodextrin / platinum-based agent in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin / platinum-based agent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin / platinum-based agent in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin / platinum-based agent is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50. In further embodiments, the cyclodextrin / platinum-based agent complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).

[0232] In a further embodiment, the present disclosure provides a complex comprising cyclodextrin and cisplatin or a salt or acid thereof. In some embodiments, the molar ratio of cyclodextrin / cisplatin (or a salt or acid of cisplatin) in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin / cisplatin (or a salt or acid of cisplatin) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin / cisplatin (or a salt or acid of cisplatin) in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin / cisplatin (or a salt or acid of cisplatin) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50. In a further embodiment, the cyclodextrin / cisplatin (or a salt or acid of cisplatin) complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).

[0233] In another embodiment, the present disclosure provides a complex comprising cyclodextrin and carboplatin or a salt or acid thereof. In some embodiments, the molar ratio of cyclodextrin / carboplatin (or a salt or acid of carboplatin) in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin / carboplatin (or a salt or acid of carboplatin) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin / carboplatin (or a salt or acid of carboplatin) in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin / carboplatin (or a salt or acid of carboplatin) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50. In a further embodiment, the cyclodextrin / carboplatin (or a salt or acid of carboplatin) complex is encapsulated within liposomes (e.g., in the manner described herein or by another method known in the art).

[0234] In another embodiment, the present disclosure provides a complex comprising cyclodextrin and oxaliplatin or a salt or acid thereof. In some embodiments, the molar ratio of cyclodextrin / oxaliplatin (or a salt or acid of oxaliplatin) in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of cyclodextrin / oxaliplatin (or a salt or acid of oxaliplatin) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In other embodiments, the molar ratio of cyclodextrin / oxaliplatin (or a salt or acid of oxaliplatin) in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of cyclodextrin / oxaliplatin (or a salt or acid of oxaliplatin) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50. In a further embodiment, the cyclodextrin / oxaliplatin (or a salt or acid of oxaliplatin) complex is encapsulated in liposomes (e.g., in the manner described herein or by another method known in the art).

[0235] In a further embodiment, the present disclosure provides a complex comprising a cyclodextrin and a platinum-based chemotherapeutic agent selected from the group consisting of nedaplatin, heptaplatin, lobaplatin, satraplatin, paraplatin, platinol, cycloplatin, dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iplatin, ormaplatin, zeniplatin, platinum-triamine, satraplatin, enroplatin, JM216, NK121, CI973, DWA2114R, NDDP, and nedaplatin, or salts or acids thereof. In other embodiments, the cyclodextrin / platinum-based chemotherapeutic agent complex comprises nedaplatin, heptaplatin, lobaplatin, satraplatin, paraplatin, platinol, cycloplatin, dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iplatin, ormaplatin, zeniplatin, platinum-triamine, satraplatin, enroplatin, JM216, NK121, CI973, DWA2114R, NDDP, or nedaplatin, or analogs of salts or acids thereof. In some embodiments, the molar ratio of cyclodextrin / oxaliplatin (or a salt or acid of oxaliplatin) in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of ...

Claims

1. A liposome composition comprising a liposome encapsulating gamma polyglutamated methotrexate, said gamma polyglutamated methotrexate comprising 2-10 glutamyl groups with gamma carboxyl group linkages, said liposome being pegylated and comprising a targeting moiety having specific affinity for a surface antigen on a target cell of interest; (a) the gamma polyglutamylated methotrexate contains an L-type glutamyl group having two or more gamma carboxyl group bonds; (b) each glutamyl group of the gamma polyglutamylated methotrexate is in the L-configuration and has a gamma carboxyl group bond; (c) at least one glutamyl group of the gamma polyglutamylated methotrexate is in the D-form and has a gamma carboxyl group linkage; (d) each glutamyl group of said gamma polyglutamylated methotrexate other than the glutamyl group of methotrexate is in the D-form and has a gamma carboxyl group linkage; or (e) the gamma polyglutamylated methotrexate contains two or more L-glutamyl groups having gamma carboxyl group bonds and at least one D-glutamyl group; Liposomal compositions.

2. The liposome composition of claim 1 , wherein the gamma polyglutamated methotrexate is gamma tetraglutamated methotrexate or gamma pentaglutamated methotrexate.

3. The liposome composition of claim 1 , wherein the gamma polyglutamated methotrexate is gamma hexaglutamated methotrexate.

4. The liposome composition of claim 1 , wherein the targeting moiety is covalently attached to one or both of the PEG and the exterior surface of the liposome.

5. 2. The liposome composition of claim 1, wherein the targeting moiety is one or more polypeptides selected from the group consisting of an antibody, a humanized antibody, an antigen-binding fragment of an antibody, a single-chain antibody, a single-domain antibody, a bispecific antibody, a synthetic antibody, a pegylated antibody, and a multimeric antibody.

6. The liposome composition of claim 1, wherein the liposome comprises 30 to 200 targeting moieties.

7. 2. The liposome composition of claim 1, wherein the liposomes have a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, or 80 nm to 120 nm.

8. The liposomes are formed from liposome components comprising at least one of anionic lipids and neutral lipids, at least one selected from the group consisting of DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholesterol; cholesterol-PEG; and cholesterol-maleimide, optionally wherein the liposome components comprise at least one selected from the group consisting of DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC, optionally wherein one or more liposome components further comprise a steric stabilizer, optionally wherein the steric stabilizer is polyethylene glycol (PEG); poly-L-lysine (PLL); 2. The liposome composition of claim 1, wherein the steric stabilizer is at least one selected from the group consisting of monosialoganglioside (GM1); poly(vinylpyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidylpolyglycerol; poly[N-(2-hydroxypropyl)methacrylamide]; amphiphilic poly-N-vinylpyrrolidone; L-amino acid based polymers; oligoglycerin, polyethylene glycol and polypropylene oxide containing copolymers, poloxamer 188, and polyvinyl alcohol, and optionally the steric stabilizer is PEG, the PEG having a number average molecular weight (Mn) of 200 to 5000 Daltons.

9. The liposome is anionic. the liposome is cationic; The liposome is neutral. the liposome has a zeta potential of zero or less; the liposome has a zeta potential of 0 to -150 mV; or The liposome has a zeta potential of -30 to -50 mV. The liposome composition of claim 1.

10. 2. The liposome composition of claim 1, wherein the liposome comprises gamma polyglutamated methotrexate and an aqueous pharma- ceutically acceptable carrier, and optionally has an interior space comprising an isotonicity agent such as dextrose, mannitol, glycerol, potassium chloride, sodium chloride at a concentration greater than 1%, 1%-50% trehalose, 5% dextrose suspended in HEPES buffer, or sodium acetate and calcium acetate at a total concentration of 50 mM-500 mM, and optionally the interior space of the liposome has a pH of 5-8 or a pH of 6-7, or any range therebetween, and optionally the liposome comprises less than 500,000 or less than 200,000 gamma polyglutamated methotrexate molecules, or 10-100,000 gamma polyglutamated methotrexate molecules, or any range therebetween.

11. The liposome further comprises one or more of an immunostimulant, a detectable marker, and a maleimide, wherein the immunostimulant, the detectable marker, or the maleimide is attached to PEG or to the outer surface of the liposome, and the immunostimulant is selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), DNP, beta-glucan, beta-1,3-glucan, beta-1,6-glucan, a resolvin (e.g., resolvin D, such as Dn-6DPA or Dn-3DPA, resolvin E, or T-silicate).

2. The liposome composition of claim 1, wherein the liposome is at least one selected from the group consisting of liposome inhibitors such as erythropoietin lipids (e.g., erythropoietin resolvins), and toll-like receptor (TLR) modulators such as oxidized low density lipoproteins (e.g., OXPAC, PGPC), and erythropoietin lipids (e.g., E5564), optionally further comprising at least one cryoprotectant selected from the group consisting of mannitol, trehalose, sorbitol, and sucrose, or further comprising carboplatin and / or pembrolizumab.

12. A pharmaceutical composition comprising the liposome composition of claim 1.

13. A composition for use in the treatment of cancer, immune system disorders, or infectious diseases, comprising a liposome composition according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 12.

14. 14. The composition of claim 13 for use in treating cancer or an immune system disorder, wherein the cancer is a member selected from the group consisting of lung cancer, breast cancer, colon cancer, pancreatic cancer, gastric cancer, bladder cancer, head and neck cancer, ovarian cancer and cervical cancer, or is non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma) choriocarcinoma and villous adenoma, non-leukemic meningeal carcinomatosis, soft tissue sarcoma, desmoid tumor, aggressive fibromatosis, bladder cancer, central nervous system (CNS) lymphoma, and the immune system disorder is an autoimmune disease, rheumatoid arthritis, or an inflammatory condition.

15. 12. A method of making a gamma polyglutamated methotrexate composition comprising the liposome composition of any one of claims 1 to 11, the method comprising the steps of forming a mixture comprising liposome components and gamma polyglutamated methotrexate in a solution; homogenizing the mixture in the solution to form liposomes; and processing the mixture to form liposomes comprising gamma polyglutamated methotrexate; optionally, the processing step comprises one or more steps of thin film hydration, extrusion, in-line mixing, ethanol injection technique, freeze-thaw method, reverse phase evaporation, dynamic high pressure microfluidization, microfluidic mixing, double emulsion, freeze-dried double emulsion, 3D printing, membrane contactor method, and stirring.

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