Alpha polyglutamated methotrexate and uses thereof

Abstract

To provide a composition for treating hyperproliferative diseases such as cancer, inflammatory diseases such as rheumatoid arthritis, and immune system disorders including autoimmune diseases, and the like.SOLUTION: Provided is a liposomal composition comprising a liposome encapsulating alpha polyglutamated methotrexate, the alpha polyglutamated methotrexate comprising 2 to 10 glutamyl groups with alpha carboxyl group linkages, the liposome being pegylated, and the composition comprising a targeting moiety having a specific affinity for a surface antigen on a target cell; and the liposome further encapsulating one or more of polyglutamatable antifolates that have not been polyglutamated, or non-polyglutamatable antifolates.SELECTED DRAWING: None

Description

Background Art

[0001] The present disclosure generally relates to alpha - polyglutamylated methotrexate compositions containing delivery carriers such as liposomes, and methods of making and using the compositions for treating diseases including hyperproliferative diseases such as cancer, immune system disorders including inflammation and autoimmune diseases 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 anti - rheumatic 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). It 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 substitution of the hydroxyl group at the 4-position of the pteridine ring with an amino group. This slight structural change results in the ability of MTX to inhibit the active catalytic site of dihydrofolate reductase (DHFR), which catalyzes the production of tetrahydrofolate (THF) from dihydrofolate (DHF). As a result, methotrexate interferes with the synthesis of tetrahydrofolate (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, remethylation of homocysteine (Hcy), and 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 lower than normal 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, which is 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 binding of the L-gamma carboxyl group to methotrexate. The 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 cellular efflux pumps.

[0007] Targeting folate metabolism and nucleotide biosynthesis is a well-established therapeutic strategy for cancer, but for MTX, clinical efficacy is limited due to 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 cellular efflux pumps, (b) decreased transport of MTX into cells, (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 folates and folate antagonists.

[0009] The problem with the long-term (>30 years) observation that higher levels of polyglutamate of various folic acid antagonists have far higher potency compared to lower levels of glutamate was that the scientific community has 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 alpha-polyglutamylated methotrexate composition provides a strategy to overcome the pharmacological issues related to dose-limiting toxicity and treatment resistance associated with methotrexate therapy. The provided method delivers a novel alpha-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 impact 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 novel alpha-polyglutamylated methotrexate (MTX) compositions, as well as methods for the manufacture and use of 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.

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

[10] A composition according to any one of items [1] to [9], wherein the polyglutamate is linear;

[11] A composition according to any one of items [1] to [9], wherein the polyglutamate is branched;

[12] A liposome composition (Lp-αPMTX) containing the alpha-polyglutamine oxidized methotrexate according to any one of items [1] to

[11] ;

[13] The LαPP composition according to item

[12] , wherein the alpha-polyglutamine oxidized methotrexate contains an L-type glutamyl group having an alpha-carboxyl group bond;

[14] The Lp-αPMTX composition according to item

[12] or

[13] , wherein each glutamyl group of the alpha-polyglutamine oxidized methotrexate is of the L type;

[15] The Lp-αPMTX composition according to item

[12] or

[13] , wherein at least one glutamyl group of the alpha-polyglutamine oxidized methotrexate is of the D type;

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

[12] to

[15] , wherein the liposome contains alpha-polyglutamine oxidized methotrexate having 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups;

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

[12] to

[16] , wherein at least one glutamyl group of the alpha-polyglutamine oxidized methotrexate has a gamma-carboxyl group bond;

[18] The composition according to any one of items

[12] to

[17] , wherein at least one glutamyl group has both an alpha-carboxyl group bond and a gamma-carboxyl group bond;

[19] The composition according to any one of items

[12] to

[18] , comprising 2, 3, 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups having both an alpha carboxyl group bond and a gamma carboxyl group bond;

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

[12] to

[19] , wherein the liposome contains alpha polyglutamate oxidized methotrexate, which contains alpha tetraglutamate oxidized methotrexate, alpha pentaglutamate oxidized methotrexate or alpha hexaglutamate oxidized methotrexate;

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

[12] to

[19] , wherein the liposome contains alpha polyglutamate oxidized methotrexate, which contains alpha tetraglutamate oxidized methotrexate, alpha pentaglutamate oxidized methotrexate or alpha hexaglutamate oxidized methotrexate;

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

[12] to

[21] , wherein the polyglutamate is linear or branched;

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

[12] to

[22] , wherein the liposome is pegylated (PαLp-αPMTX);

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

[12] to

[23] , wherein the liposome contains at least 1% by weight (w / w) of alpha polyglutamate oxidized methotrexate, or during the process of preparing Lp-αPMTX, at least 1% of the starting material of alpha polyglutamate oxidized MTX is encapsulated (enclosed) in αPMTX;

[25] The Lp-αPMTX 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 or 20 nm to 200 nm;

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

[12] to

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

[28] The Lp-αPMTX composition according to item

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

[29] The Lp-αPMTX 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-αPMTX 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-αPMTX composition according to any one of items

[27] to

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

[32] The Lp-αPMTX 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-αPMTX 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-αPMTX composition according to any one of items

[12] to

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

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

[12] to

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

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

[12] to

[33] , wherein the liposome is cationic;

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

[12] to

[38] , wherein the liposome has an internal space containing alphapolyglutamate oxidized methotrexate and an aqueous pharmaceutically acceptable carrier;

[40] The Lp-αPMTX composition of 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-αPMTX composition of item

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

[42] The Lp-αPMTX composition of item

[41] , wherein the pharmaceutically acceptable carrier contains 5% to 20% by weight of trehalose;

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

[39] to

[42] , wherein the pharmaceutically acceptable carrier contains 1% to 15% by weight of dextrose;

[44] The Lp-αPMTX 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-αPMTX 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-αPMTX 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-αPMTX 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 therebetween;

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

[12] to

[47] , wherein the liposome contains less than 500,000 or less than 200,000 alpha-polyglutamine oxidized methotrexate molecules;

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

[12] to

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

[50] The Lp-αPMTX 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-αPMTX 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-αPMTX composition according to item

[50] or

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

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

[50] to

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

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

[50] to

[55] , 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-αPMTX composition according to any one of items

[50] to

[56] , 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-αPMTX composition according to any one of items

[50] to

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

[58] An Lp-αPMTX 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-αPMTX composition of 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-αPMTX 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 resolvins D, resolvins E, or T-series resolvins, and toll-like receptor (TLR) modulators such as oxidized low-density lipoprotein (e.g., OXPAC, PGPC), and erythran lipids (e.g., E5564);

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

[58] to

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

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

[58] to

[61] , further comprising a hapten;

[63] The Lp-αPMTX composition according to item

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

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

[12] to

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

[68] A pharmaceutical composition comprising the liposomal alpha polyglutamine oxidized methotrexate composition according to any one of items

[12] to

[67] ;

[69] A pharmaceutical composition comprising the alpha polyglutamine 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, 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, comprising the step of administering the liposomal alpha-polyglutamic acid oxidized methotrexate composition according to any one of items

[12] to

[69] to the subject;

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

[69] ;

[75] A method for killing proliferating cells, comprising the step of contacting the proliferating cells with the liposomal alpha-polyglutamic acid oxidized methotrexate composition according to any one of items

[12] to

[69] ;

[76] The method according to item

[74] or

[75] , wherein the proliferating 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 alpha-polyglutamic acid oxidized 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 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;

[80] The method according to item

[77] or

[78] , wherein the cancer is a member selected from the group consisting of breast cancer, advanced 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;

[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 a sarcoma such as osteosarcoma;

[83] A method for treating cancer, comprising administering an effective amount of the Lp-αPMTX 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] A maintenance therapy for a subject who is receiving or has received cancer therapy, comprising administering an effective amount of the composition according to any one of items [1] to

[69] to the subject;

[85] A maintenance therapy for a subject who is receiving or has received cancer therapy, comprising administering an effective amount of the liposomal alpha-polyglutamate oxidized 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 disease, and psoriasis;

[87] Administering an effective amount of the liposomal alpha-polyglutamate oxidized methotrexate composition according to any one of items [8] to

[69] to a subject having or at risk of having an immune system disorder, optionally wherein 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; a method for treating an immune system disorder;

[88] The following treatment methods: (a) A method for treating an infectious disease, 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 infectious disease; (b) A method for treating an infectious disease, a cardiovascular disease, or another disease, 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 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; a method for treating an infectious disease, a cardiovascular disease, a metabolic disease, or another disease; (c) A method for treating an autoimmune disease, 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 autoimmune disease; (d) A method for treating rheumatoid arthritis, 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 rheumatoid arthritis; (e) A method for treating an inflammatory condition, 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 inflammation, optionally wherein the inflammation is acute, chronic, and / or systemic inflammation; or (f) A method for treating a skin disease, 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 a skin disease;

[89] A method for treating an infectious disease, comprising the step of administering to a subject having or at risk of having an infectious disease an effective amount of the liposomal alpha-polyglutamyl oxidized methotrexate composition according to any one of items

[12] to

[69] ;

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

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

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

[12] to

[69] , the method comprising the steps of forming a mixture comprising a liposomal component and an alpha-polyglutamyl oxidized folate antagonist in solution; homogenizing the mixture in solution to form liposomes; and treating the mixture to form liposomes comprising alpha-polyglutamyl oxidized methotrexate;

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

[12] to

[69] , the method comprising the steps of forming a mixture comprising a liposomal component and alpha-polyglutamyl oxidized methotrexate in solution; homogenizing the mixture in solution to form liposomes; treating the mixture to form liposomes encapsulating and / or coating alpha-polyglutamyl 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);

[93] The method according to item

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

[0013] In some embodiments, the present disclosure provides an alpha-polyglutamyl methotrexate (αPMTX) composition, wherein at least one glutamyl residue of alpha-polyglutamyl methotrexate is bound by its alpha-carboxyl group. In some embodiments, αPMTX includes 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, αPMTX includes two or more L-type glutamyl groups. In other embodiments, αPMTX includes a D-type glutamyl group. In further embodiments, αPMTX includes a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, αPMTX includes two or more glutamyl groups having a gamma bond. In some embodiments, at least one glutamyl group has both an alpha bond and a gamma bond.

[0014] In one embodiment, the αPMTX composition comprises a chain of three glutamyl groups attached to the glutamyl group in methotrexate (i.e., tetraglutamine oxidized methotrexate). In some embodiments, tetraglutamine oxidized MTX comprises two or more L-type glutamyl groups. In other embodiments, tetraglutamine oxidized MTX comprises a D-type glutamyl group. In further embodiments, tetraglutamine oxidized MTX comprises a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, tetraglutamine oxidized MTX comprises two or more glutamyl groups having gamma linkages.

[0015] In one embodiment, the αPMTX composition comprises a chain of four glutamyl groups attached to the glutamyl group in methotrexate (i.e., pentaglutamine oxidized methotrexate). In some embodiments, pentaglutamine oxidized MTX comprises two or more L-type glutamyl groups. In other embodiments, pentaglutamine oxidized MTX comprises a D-type glutamyl group. In further embodiments, pentaglutamine oxidized MTX comprises a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, pentaglutamine oxidized MTX comprises two or more glutamyl groups having gamma linkages.

[0016] In one embodiment, the αPMTX composition comprises a chain of five glutamyl groups attached to the glutamyl group of methotrexate (i.e., hexaglutamine oxidized methotrexate). In some embodiments, hexaglutamine oxidized MTX comprises two or more L-type glutamyl groups. In other embodiments, hexaglutamine oxidized MTX comprises a D-type glutamyl group. In further embodiments, hexaglutamine oxidized MTX comprises a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, hexaglutamine oxidized MTX comprises two or more glutamyl groups having gamma linkages.

[0017] In a further embodiment, the present disclosure provides compositions comprising delivery carriers such as liposomes filled with (i.e., encapsulated) and / or otherwise conjugated with alpha-polyglutamylated methotrexate, and methods of making the αPMTX-filled / conjugated delivery carrier compositions and methods of using the same to deliver alpha-polyglutamylated 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 rheumatoid arthritis, and infectious diseases such as HIV, malaria. The αPMTX-filled / conjugated delivery carrier compositions provide for the selective delivery of a higher cytotoxic payload (polyglutamylated methotrexate) compared to the cytotoxicity of methotrexate (MTX) administered in the monoglutamate 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-αPMTX) comprising liposomes encapsulating (filling) alpha-polyglutamyl methotrexate. In some embodiments, the alpha-polyglutamyl methotrexate in Lp-αPMTX contains 2 to 20, 2 to 15, 2 to 10, 2 to 5, or more than 20 glutamyl groups (including the glutamyl groups of methotrexate). In some embodiments, the alpha-polyglutamyl methotrexate in Lp-αPMTX contains 2 or more L-type glutamyl groups. In other embodiments, the alpha-polyglutamyl methotrexate in Lp-αPMTX contains D-type glutamyl groups. In a further embodiment, the alpha-polyglutamyl methotrexate in Lp-αPMTX contains D-type glutamyl groups and 2 or more L-type glutamyl groups. In a further embodiment, the alpha-polyglutamyl methotrexate in Lp-αPMTX contains 2 or more glutamyl groups having gamma bonds. In a further embodiment, the alpha-polyglutamyl methotrexate in Lp-αPMTX contains 1 or more glutamyl groups having both alpha and gamma bonds. In some embodiments, the alpha-polyglutamyl methotrexate in Lp-αPMTX contains 2 to 10 or any range therebetween of glutamyl groups having both alpha and gamma bonds. In some embodiments, the polyglutamate chain of alpha-polyglutamyl methotrexate is linear. In some embodiments, the polyglutamate chain of alpha-polyglutamyl methotrexate is branched.

[0019] In one embodiment, the Lp-αPMTX composition comprises alpha-polyglutamylated MTX comprising a chain of three glutamyl groups attached to the glutamyl group of methotrexate (i.e., tetraglutamylated methotrexate). In some embodiments, the tetraglutamylated MTX comprises two or more L-type glutamyl groups. In other embodiments, the tetraglutamylated MTX comprises a D-type glutamyl group. In further embodiments, the tetraglutamylated MTX comprises a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, the tetraglutamylated MTX comprises two or more glutamyl groups having a gamma bond. In some embodiments, the polyglutamate chain of the alpha-polyglutamylated methotrexate is linear. In some embodiments, the polyglutamate chain of the alpha-polyglutamylated methotrexate is branched.

[0020] In one embodiment, the Lp-αPMTX composition comprises alpha-polyglutamylated MTX comprising a chain of four glutamyl groups attached to the glutamyl group of methotrexate (i.e., pentaglutamylated methotrexate). In some embodiments, the pentaglutamylated MTX comprises two or more L-type glutamyl groups. In other embodiments, the pentaglutamylated MTX comprises a D-type glutamyl group. In further embodiments, the pentaglutamylated MTX comprises a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, the pentaglutamylated MTX comprises two or more glutamyl groups having a gamma bond. In some embodiments, the polyglutamate chain of the alpha-polyglutamylated methotrexate is linear. In some embodiments, the polyglutamate chain of the alpha-polyglutamylated methotrexate is branched.

[0021] In one embodiment, the Lp-αPMTX composition comprises alpha-polyglutamylated MTX containing a chain of five glutamyl groups attached to the glutamyl group of methotrexate (i.e., hexaglutamylated methotrexate). In some embodiments, the hexaglutamylated MTX comprises two or more L-type glutamyl groups. In other embodiments, the hexaglutamylated MTX comprises a D-type glutamyl group. In further embodiments, the hexaglutamylated MTX comprises a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, the hexaglutamylated MTX comprises two or more glutamyl groups having a gamma bond. In some embodiments, the polyglutamate chain of alpha-polyglutamylated methotrexate is linear. In some embodiments, the polyglutamate chain of alpha-polyglutamylated methotrexate is branched.

[0022] In some embodiments, the Lp-αPMTX composition is cationic. In some embodiments, the Lp-αPMTX 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-αPMTX 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-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 alpha-polyglutamylated MTX. In some embodiments, during the preparation process of Lp-αPMTX, 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 alpha-polyglutamylated MTX is encapsulated (enclosed) in the cationic Lp-αPMTX. In further embodiments, the alpha-polyglutamylated methotrexate encapsulated by the liposome is present in the HEPES buffer within the liposome.

[0023] In other embodiments, the Lp-αPMTX composition is anionic or neutral. In some embodiments, the Lp-αPMTX composition is cationic. In some embodiments, the Lp-αPMTX 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-αPMTX 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-αPMTX 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-αPMTX 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-αPMTX 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 further embodiments, the Lp-αPMTX liposomes are neutral and the composition has a diameter in the range of 80 nm to 120 nm, or any range therebetween. In some embodiments, the anionic or neutral 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 alpha-polyglutamate oxidized MTX. In some embodiments, during the preparation process of Lp-αPMTX, 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 alpha-polyglutamate oxidized MTX is encapsulated (enclosed) in the anionic or neutral Lp-αPMTX.In some embodiments, the anionic or neutral 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 alpha-tetra-glutamylated MTX. In some embodiments, the anionic or neutral 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 alpha-penta-glutamylated MTX. In some embodiments, the anionic or neutral 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 alpha-hexa-glutamylated MTX. In further embodiments, the alpha-poly-glutamylated methotrexate encapsulated by the liposome is present in the HEPES buffer within the liposome.

[0024] In further embodiments, the liposomal alpha-poly-glutamylated methotrexate composition is pegylated (PLp-αPMTX).

[0025] In some embodiments, the liposomal alpha-poly-glutamylated methotrexate composition is not targeted (NTLp-αPMTX). That is, the NTLp-αPMTX 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 further embodiments, the non-targeted liposomal alpha-poly-glutamylated methotrexate composition is pegylated (NTPLp-αPMTX).

[0026] In other embodiments, the liposomal alpha-polyglutamylated methotrexate compositions are targeted (TLp-αPMTX). That is, the TLp-αPMTX compositions include a targeting moiety having specific affinity for an epitope (surface antigen) on the target cell of interest. In some embodiments, the targeting moiety of TLp-αPMTX or TPLp-αPMTX is not covalently bound to the liposome. In other embodiments, the targeting moiety of TLp-αPMTX or TPLp-αPMTX is bound to one or both of the PEG and the outer surface of the liposome. The targeted liposomal alpha-polyglutamylated methotrexate compositions (TLp-αPMTX and TPLp-αPMTX) provide further improvement over the efficacy and safety profile of methotrexate by specifically delivering alpha-polyglutamylated (e.g., tetraglutamylated, pentaglutamylated, and hexaglutamylated) methotrexate to target cells such as cancer cells. In further embodiments, the targeted liposomal alpha-polyglutamylated methotrexate compositions are pegylated (TPLp-αPMTX). The functions of the targeting moiety of the TLp-αPMTX and / or TPLp-αPMTX compositions 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 specific affinity; and delivering the liposomal payload (αPMTX) to the cell.

[0027] 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. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In further embodiments, the targeting moiety comprises 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 has specific affinity for an epitope that is selectively expressed on target cells such as tumor cells as 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 for the target epitope as measured by BIACORE® analysis.

[0028] In certain embodiments, the targeting moiety comprises a polypeptide that specifically binds to the folate receptor. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the folate receptor bound by the targeting moiety is 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 bound by the targeting moiety is folate receptor alpha (FR-α). In some embodiments, the folate receptor bound by the targeting moiety is folate receptor beta (FR-β). In some embodiments, the targeting moiety specifically binds to FR-α and FR-β.

[0029] In further embodiments, the αPMTX 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 αPMTX composition (e.g., Lp-αPMTX, PLp-αPMTX, NTLp-αPMTX, NTPLp-αPMTX, TLp-αPMTX, or TPLp-αPMTX) is cationic. In other embodiments, the liposomal αPMTX composition (e.g., Lp-αPMTX, PLp-αPMTX, NTLp-αPMTX, NTPLp-αPMTX, TLp-αPMTX, or TPLp-αPMTX) is anionic or neutral. In further embodiments, the liposomes of the liposomal αPMTX composition (e.g., Lp-αPMTX, PLp-αPMTX, NTLp-αPMTX, NTPLp-αPMTX, TLp-αPMTX or TPLp-αPMTX) have a diameter in the range of 20 nm to 500 nm, or any range therebetween. In further embodiments, the liposomes of the liposomal αPMTX composition have a diameter in the range of 80 nm to 120 nm, or any range therebetween. In some embodiments, the liposomal αPMTX composition is pegylated (e.g., PLp-αPMTX, NTPLp-αPMTX, or TPLp-αPMTX). In some embodiments, the liposomal αPMTX composition is targeted (e.g., TLp-αPMTX or TPLp-αPMTX). In further embodiments, the liposomal αPMTX composition is pegylated and targeted (e.g., TPLp-αPMTX). In some embodiments, the liposomal αPMTX composition comprises alpha-polyglutamyl methotrexate containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the liposomal αPMTX composition comprises alpha-tetraglutamyl methotrexate. In some embodiments, the liposomal αPMTX composition comprises alpha-pentaglutamyl methotrexate. In other embodiments, the liposomal αPMTX composition comprises alpha-hexaglutamyl methotrexate.

[0030] In some embodiments, the liposome composition consists of alpha-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 alpha-polyglutamyl oxidized MTX. In some embodiments, the Lp-αPMTX composition contains alpha-polyglutamyl oxidized methotrexate containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups and 1% to 98.5% w / w of alpha-polyglutamyl oxidized MTX. In some embodiments, the liposome contains alpha-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-αPMTX, 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 alpha-polyglutamyl oxidized folic acid MTX is encapsulated (enclosed) in Lp-αPMTX.

[0031] In some embodiments, the liposome composition consists of alpha-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 alpha-tetraglutamyl oxidized MTX. In some embodiments, the Lp-αPMTX composition contains alpha-tetraglutamyl oxidized folic acid methotrexate and 1% to 98.5% w / w of alpha-tetraglutamyl oxidized MTX. In some embodiments, the liposome contains alpha-tetraglutamyl oxidized methotrexate, and during the preparation process of Lp-αPMTX, 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 alpha-tetraglutamyl oxidized MTX is encapsulated (enclosed) in Lp-αPMTX.

[0032] In some embodiments, the liposome composition consists of alphapentaglutamyl oxidized methotrexate and contains 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 alphapentaglutamyl oxidized MTX. In some embodiments, the Lp-αPMTX composition contains alphapentaglutamyl oxidized methotrexate and 1% - 98.5% w / w of alphapentaglutamyl oxidized MTX. In some embodiments, the liposome contains alphapentaglutamyl oxidized methotrexate, and during the preparation process of Lp-αPMTX, 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 alphapentaglutamyl oxidized MTX are encapsulated (enclosed) in Lp-αPMTX. In some embodiments, the liposome composition consists of alphahexaglutamyl oxidized methotrexate and contains 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 alphahexaglutamyl oxidized MTX. In some embodiments, the Lp-αPMTX composition contains alphahexaglutamyl oxidized methotrexate and 1% - 98.5% w / w of alphahexaglutamyl oxidized MTX. In some embodiments, the liposome contains alphahexaglutamyl oxidized methotrexate, and during the preparation process of Lp-αPMTX, 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 alphapentaglutamyl oxidized MTX are encapsulated (enclosed) in Lp-αPMTX.

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

[0034] In some embodiments, the liposomal composition comprises liposomal αPMTX (e.g., Lp-αPMTX, PLp-αPMTX, NTLp-αPMTX, NTPLp-αPMTX, TLp-αPMTX, and TPLp-αPMTX). In some embodiments, the liposomal αPMTX is pegylated (e.g., NTPLp-αPMTX, and TPLp-αPMTX). In some embodiments, the liposomal αPMTX 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-αPMTX or TPLp-αPMTX). In further embodiments, the liposomal composition comprises pegylated liposomal αPMTX 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-αPMTX). In some embodiments, the liposomal composition comprises liposomal αPMTX that is cationic. In other embodiments, the liposomal composition comprises liposomal αPMTX that is anionic or neutral. In further embodiments, the liposomal composition comprises liposomal αPMTX 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 αPMTX has a diameter in the range of 80 nm to 120 nm, or any range therebetween.

[0035] There is also provided a pharmaceutical composition comprising an alpha-polyglytamylated methotrexate (αPMTX) including a delivery carrier such as liposomal αPMTX. In some embodiments, the pharmaceutical composition comprises a pegylated αPMTX composition. In some embodiments, the pharmaceutical composition comprises an αPMTX composition linked to or otherwise conjugated to a targeting moiety. In further embodiments, the pharmaceutical composition comprises a pegylated αPMTX composition linked to or otherwise conjugated to a targeting moiety. In some embodiments, the pharmaceutical composition comprises an αPMTX comprising 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the pharmaceutical composition comprises alpha-tetraglytamylated methotrexate. In some embodiments, the pharmaceutical composition comprises alpha-pentaglytamylated methotrexate. In other embodiments, the pharmaceutical composition comprises alpha-hexaglytamylated methotrexate.

[0036] In some embodiments, the pharmaceutical composition comprises liposomal αPMTX (e.g., Lp-αPMTX, PLp-αPMTX, NTLp-αPMTX, NTPLp-αPMTX, TLp-αPMTX, and TPLp-αPMTX). In some embodiments, the liposomal αPMTX composition is pegylated (e.g., NTPLp-αPMTX, and TPLp-αPMTX). In some embodiments, the liposomal αPMTX 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-αPMTX or TPLp-αPMTX). In further embodiments, the pharmaceutical composition comprises a pegylated liposomal αPMTX composition 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-αPMTX). In some embodiments, the pharmaceutical composition comprises liposomal αPMTX that is cationic. In other embodiments, the pharmaceutical composition comprises liposomal αPMTX that is anionic or neutral. In further embodiments, the pharmaceutical composition comprises liposomal αPMTX 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 αPMTX 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 for regulating cell activation, chemokine production, or metabolic activity, the method comprising contacting a cell with a composition comprising an alpha-polyglutamylated methotrexate (αPMTX) composition. In some embodiments, the cell to be contacted is a mammalian cell. In further embodiments, the cell to be contacted is a human cell. In some embodiments, the cell to be contacted is a hyperproliferative cell. In further embodiments, the cell is an immune cell. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, αPMTX comprises 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the αPMTX composition comprises alpha-tetraglutamylated methotrexate. In some embodiments, the αPMTX composition comprises alpha-pentaglutamylated methotrexate. In other embodiments, the αPMTX composition comprises alpha-hexaglutamylated methotrexate.

[0038] In further embodiments, the present disclosure provides a method for regulating cell activation, chemokine production, or metabolic activity, the method comprising contacting a cell with a liposome comprising an alpha-polyglutamylated methotrexate (αPMTX) composition. In some embodiments, the cell to be contacted is a mammalian cell. In further embodiments, the cell to be contacted is a human cell. In some embodiments, the cell to be contacted is a hyperproliferative cell. In further embodiments, the cell is an immune cell. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, αPMTX comprises 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the αPMTX composition comprises alpha-tetraglutamylated methotrexate. In some embodiments, the αPMTX composition comprises alpha-pentaglutamylated methotrexate. In other embodiments, the αPMTX composition comprises alpha-hexaglutamylated methotrexate.

[0039] In further embodiments, the present disclosure provides a method of killing cells, the method comprising contacting the cells with a composition comprising an alphapolyglutamylated methotrexate (αPMTX) composition. 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 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 malignancies. In yet further embodiments, the cancer cells are primary cells or cells derived from cell lines obtained from cancers selected from 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 method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, αPMTX contains 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the αPMTX composition comprises alphatetraglutamylated methotrexate. In some embodiments, the αPMTX composition comprises alphapentaglutamylated methotrexate. In other embodiments, the αPMTX composition comprises alphahexaglutamylated methotrexate.

[0040] In further embodiments, the present disclosure provides a method of killing cells, the method comprising contacting the cells with liposomes comprising alpha-polyglutamylated methotrexate (e.g., Lp-αPMTX such as PLp-αPMTX, NTLp-αPMTX, NTPLp-αPMTX, TLp-αPMTX or TPLp-αPMTX). 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 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 malignancies. In still further embodiments, the cancer cells are primary cells or cells derived from cell lines obtained from cancers selected from 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, non-leukemic meningeal carcinomatosis, soft tissue sarcoma (desmoid tumor, aggressive fibromatosis), bladder cancer, and central nervous system (CNS) lymphoma. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, the liposomes comprise αPMTX having 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes comprise alpha-tetraglutamylated methotrexate. In some embodiments, the liposomes comprise alpha-pentaglutamylated methotrexate. In other embodiments, the liposomes comprise alpha-hexaglutamylated methotrexate.

[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 a delivery carrier (e.g., an immune complex or liposome) comprising alpha-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., Lp-αPMTX such as PLp-αPMTX, NTLp-αPMTX, NTPLp-αPMTX, TLp-αPMTX, or TPLp-αPMTX). In some embodiments, the administered delivery carrier is pegylated. In some embodiments, the administered delivery carrier is not pegylated. In a further embodiment, 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 that specifically binds to 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, PDGFRα, PDGFRβ, 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 that is derived from or identified as being expressed on a particular subject's cancer (tumor), such as a neoantigen. In some embodiments, the targeting moiety specifically binds to a cell surface antigen that is derived from or identified as being expressed on a particular tumor of the subject, 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 αPMTX comprising 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the administered delivery carrier comprises alphatetraglutamylmethotrexate. In some embodiments, the administered delivery carrier comprises alphapentaglutamylmethotrexate. In other embodiments, the administered delivery carrier comprises alphahexaglutamylmethotrexate. In some embodiments, the administered delivery carrier comprises L-alpha-polyglutamylmethotrexate. In some embodiments, the administered delivery carrier comprises D-alpha-polyglutamylmethotrexate. In some embodiments, the administered delivery carrier comprises L and D-alpha-polyglutamylmethotrexate. In some embodiments, the cancer is 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, myeloma and other plasma cell dyscrasias or malignancies. In yet further embodiments, the cancer cells are cells obtained from / derived from a cell line of a primary cell or a cancer selected from 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, non-leukemic meningeal carcinomatosis, soft tissue sarcoma (desmoid tumor, aggressive fibromatosis), bladder cancer, and central nervous system (CNS) lymphoma.

[0042] In a further embodiment, the present disclosure provides a method of treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of liposomes comprising alpha-polyglutamyl oxidized methotrexate (e.g., Lp-αPMTX such as PLp-αPMTX, NTLp-αPMTX, NTPLp-αPMTX, TLp-αPMTX, or TPLp-αPMTX). 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 that specifically binds to 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, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA1, 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, PDGFRα, PDGFRβ, 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 includes a targeting moiety that specifically binds to a cell surface antigen derived from or determined to be 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 includes αPMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposome includes alphatetraglutamylmethotrexate. In some embodiments, the liposome includes alphapentaglutamylmethotrexate. In other embodiments, the liposome includes alphahexaglutamylmethotrexate. In some embodiments, the liposome includes L alphapolyglutamylmethotrexate. In some embodiments, the liposome includes D alphapolyglutamylmethotrexate. In some embodiments, the liposome includes L and D alphapolyglutamylmethotrexate. 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).

[0043] In a further embodiment, the present disclosure provides a method of 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 alpha-polyglutamylated 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 that specifically binds to 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, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA1, 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, PDGFRα, PDGFRβ, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the liposomes to be administered include a targeting moiety that specifically binds to cell surface antigens derived from or determined to be expressed on a particular subject's tumor, such as neoantigens. In some embodiments, the liposome composition to be administered includes pegylated liposomes (e.g., TPLp-αPMTX). In some embodiments, the liposome composition to be administered includes non-pegylated liposomes. In some embodiments, the liposomes of the liposome composition to be administered include αPMTX containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the liposome composition to be administered include alpha-tetraglutamyl-methotrexate. In some embodiments, the liposomes of the liposome composition to be administered include alpha-pentaglutamyl-methotrexate. In other embodiments, the liposomes of the liposome composition to be administered include alpha-hexaglutamyl-methotrexate. In some embodiments, the liposome composition is administered for treating cancer 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, myeloma and other plasma cell dyscrasias or cachexia, and leukemia, lymphoma and other B-cell malignancies. In yet further embodiments, the cancer cells are cells derived from a cell line obtained from / derived from cancer selected from primary cells, or 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, non-leukemic meningeal carcinomatosis, soft tissue sarcoma (desmoid tumor, aggressive fibromatosis), bladder cancer, and central nervous system (CNS) lymphoma.

[0044] In further embodiments, the present disclosure provides a method of 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) alpha-polyglutamylated methotrexate (αPMTX) 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) and folate receptor beta (FR-β). In some embodiments, the liposomal composition administered comprises pegylated liposomes (e.g., TPLp-αPMTX). In some embodiments, the liposomal composition administered comprises non-pegylated liposomes. In some embodiments, the liposomes of the liposomal composition administered contain αPMTX having 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the liposomal composition administered contain alpha-tetraglutamylated methotrexate. In some embodiments, the liposomes of the liposomal composition administered contain alpha-pentaglutamylated methotrexate. In other embodiments, the liposomes of the liposomal composition administered contain alpha-hexaglutamylated 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, myeloma and other plasma cell dyscrasias or malignancies.In yet further embodiments, the cancer cells are cells derived from a cell line obtained from / derived from primary cells or cancer, such as 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.

[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 containing alpha-polyglutamylated methotrexate (Lp-αPMTX). In some embodiments, the liposomal composition to be administered is PLp-αPMTX, NTLp-αPMTX, NTPLp-αPMTX, TLp-αPMTX or TPLp-αPMTX. In some embodiments, the liposomal composition to be administered comprises pegylated liposomes (e.g., PLp-αPMTX, NTPLp-αPMTX, or TPLp-αPMTX). In some embodiments, the liposomal composition to be administered comprises targeted liposomes (e.g., TLp-αPMTX or TPLp-αPMTX). In some embodiments, the liposomal composition to be administered comprises pegylated and targeted liposomes (e.g., TPLp-αPMTX). In some embodiments, the liposomes of the liposomal composition to be administered comprise alpha-polyglutamylated methotrexate containing 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the liposomal composition to be administered comprise alpha-tetraglutamylated methotrexate. In some embodiments, the liposomes of the liposomal composition to be administered comprise alpha-pentaglutamylated methotrexate. In other embodiments, the liposomes of the liposomal composition to be administered comprise alpha-hexaglutamylated methotrexate.

[0046] In further embodiments, the present disclosure provides a method of treating a disorder of the immune system, the method comprising administering to a subject having or at risk of having a disorder of the immune system an effective amount of a liposomal composition comprising alphapolyglutamyl methotrexate (e.g., Lp-αPMTX, PLp-αPMTX, NTLp-αPMTX, NTPLp-αPMTX, TLp-αPMTX or TPLp-αPMTX). 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 some embodiments, the liposomal composition administered comprises a pegylated liposome (e.g., PLp-αPMTX, NTPLp-αPMTX, or TPLp-αPMTX). In some embodiments, the liposomal composition administered comprises a targeted liposome (e.g., TLp-αPMTX or TPLp-αPMTX) 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 pegylated and targeted liposome (e.g., TPLp-αPMTX). In some embodiments, the liposomes of the liposomal composition administered comprise alphapentaglutamyl methotrexate comprising 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the liposomal composition administered comprise alphatetraglutamyl methotrexate. In some embodiments, the liposomes of the liposomal composition administered comprise alphapentaglutamyl methotrexate. In other embodiments, the liposomes of the liposomal composition administered comprise alphapentaglutamyl methotrexate. In some embodiments, the disorder of the immune system 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.

[0047] In a further embodiment, the present disclosure provides a method for treating an autoimmune disease, the method comprising administering to a subject having or at risk of having an inflammatory disease an effective amount of a liposomal composition comprising alpha-polyglutamyl methotrexate (e.g., Lp-αPMTX, PLp-αPMTX, NTLp-αPMTX, NTPLp-αPMTX, TLp-αPMTX or TPLp-αPMTX). In some embodiments, the liposomal composition to be administered comprises a pegylated liposome (e.g., PLp-αPMTX, NTPLp-αPMTX, or TPLp-αPMTX). In some embodiments, the liposomal composition to be administered comprises a targeted liposome (e.g., TLp-αPMTX or TPLp-αPMTX) comprising a targeting moiety having specific affinity for a surface antigen on a target cell of interest (e.g., an immune cell). In a further embodiment, the liposomal composition to be administered comprises a pegylated and targeted liposome (e.g., TPLp-αPMTX). In some embodiments, the liposome of the liposomal composition to be administered comprises alpha-pentaglutamyl methotrexate comprising 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the liposome of the liposomal composition to be administered comprises alpha-tetraglutamyl methotrexate. In some embodiments, the liposome of the liposomal composition to be administered comprises alpha-pentaglutamyl methotrexate. In other embodiments, the liposome of the liposomal composition to be administered comprises alpha-hexaglutamyl methotrexate. In some embodiments, the autoimmune disorder is selected from rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn's disease, systemic lupus erythematosus, and psoriasis.

[0048] In a further embodiment, the present disclosure provides a method of treating an inflammatory disease, the method comprising administering to a subject having or at risk of having an inflammatory disease an effective amount of a liposomal composition comprising alpha-polyglutamylated methotrexate (e.g., Lp-αPMTX, PLp-αPMTX, NTLp-αPMTX, NTPLp-αPMTX, TLp-αPMTX or TPLp-αPMTX). In some embodiments, the liposomal composition to be administered comprises pegylated liposomes (e.g., PLp-αPMTX, NTPLp-αPMTX, or TPLp-αPMTX). In some embodiments, the liposomal composition to be administered comprises targeted liposomes (e.g., TLp-αPMTX or TPLp-αPMTX) comprising a targeting moiety having specific affinity for a surface antigen on a target cell of interest (e.g., an immune cell). In a further embodiment, the liposomal composition to be administered comprises pegylated and targeted liposomes (e.g., TPLp-αPMTX). In some embodiments, the liposomes of the liposomal composition to be administered comprise alpha-pentaglutamylated methotrexate comprising 4, 5, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the liposomal composition to be administered comprise alpha-tetraglutamylated methotrexate. In some embodiments, the liposomes of the liposomal composition to be administered comprise alpha-pentaglutamylated methotrexate. In other embodiments, the liposomes of the liposomal composition to be administered comprise alpha-hexaglutamylated methotrexate. In some embodiments, the inflammatory disease is selected from acute inflammation, chronic inflammation, systemic inflammation, rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn's disease, dermatomyositis / polymyositis, and systemic lupus erythematosus.

[0049] The present disclosure also provides a method for delivering alpha-polyglutamylated methotrexate to an inflamed site of a subject, the method comprising administering to a subject having inflammation a composition comprising alpha-polyglutamylated methotrexate (L-αPMTX) and a targeting moiety having specific binding affinity for an epitope on the surface antigen of a cell that is at the site of inflammation or that otherwise affects inflammation (e.g., affects through promoting pro-inflammatory cytokine production). In some embodiments, the targeting moiety being administered 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-αPMTX). In some embodiments, the composition being administered comprises alpha-polyglutamylated methotrexate having 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the composition being administered comprises alpha-tetra-glutamylated methotrexate. In some embodiments, the composition being administered comprises alpha-penta-glutamylated methotrexate. In other embodiments, the composition being administered comprises alpha-hexa-glutamylated methotrexate.

[0050] The present disclosure also provides a method for delivering alpha-polyglutamyl oxidized methotrexate to tumor and cancer cells, the method comprising administering to a subject having a tumor a composition comprising alpha-polyglutamyl oxidized methotrexate (L-αPMTX) and a targeting moiety having specific binding affinity for an epitope on the surface antigen of the tumor cell or cancer cell. In some embodiments, the targeting moiety to be administered 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-αPMTX). In some embodiments, the composition to be administered comprises alpha-polyglutamyl oxidized methotrexate containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the composition to be administered comprises alpha-tetraglutamyl oxidized methotrexate. In some embodiments, the composition to be administered comprises alpha-pentaglutamyl oxidized methotrexate. In other embodiments, the composition to be administered comprises alpha-hexaglutamyl oxidized methotrexate.

[0051] In a further embodiment, the present disclosure provides a method of making a liposomal composition comprising a liposomal alpha-polyglutamyl methotrexate (αPMTX) composition, the method comprising: forming a mixture comprising liposomal components and alpha-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 alpha-polyglutamyl methotrexate comprises 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the polyglutamyl methotrexate composition comprises alpha-tetraglutamyl methotrexate. In some embodiments, the polyglutamyl methotrexate composition comprises alpha-pentaglutamyl methotrexate. In other embodiments, the polyglutamyl methotrexate composition comprises alpha-hexaglutamyl methotrexate.

[0052] In one embodiment, the present disclosure provides a kit comprising an alpha-polyglutamyl methotrexate composition and / or an αPMTX delivery carrier such as liposomes comprising αPMTX and αPMTX immune complexes (e.g., ADCs as described herein). BRIEF DESCRIPTION OF THE DRAWINGS

[0053]

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Modes for Carrying Out the Invention

[0054] In general, the present disclosure relates to novel alpha - polyglutamylated methotrexate compositions. The compositions provide an advancement over prior treatments for hyperproliferative diseases such as cancer. Methods for the manufacture, delivery, and use of alpha - polyglutamylated methotrexate compositions are also provided. Alpha - polyglutamylated compositions have uses including, but not limited to, the treatment or prevention of hyperproliferative diseases such as cancer, immune system disorders such as rheumatoid arthritis, and infectious diseases such as HIV and malaria.

[0055] 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.

[0056] When an embodiment is described herein using the term "comprising", other similar embodiments described with the terms "containing", "consisting of", and / or "consisting essentially of" are provided as well. However, when used as a transitional phrase in the claims, each should be interpreted separately and in an appropriate legal and factual context (e.g., in the claims, the transitional phrase "comprising" is considered a more open-ended term, "consisting of" is considered more exclusive, and "consisting essentially of" is considered intermediate between these).

[0057] 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.

[0058] 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.

[0059] 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 various cations, such as Na + , Mg 2+ , K + , NH 4+ , and / or Ca 2+may include any of the following. In certain embodiments, typically, the salt is a pharmaceutically acceptable salt. In a further particular embodiment, the MTX salt is Na + and contains. Methotrexate usually contains one L-gamma-glutamyl group and thus, for the purposes of the present disclosure, is considered to be monoglutaminated.

[0060] Headings and subheadings are used for convenience and / or only for compliance with official rules, and neither limit the subject matter nor are they referred to in connection with the interpretation of the description of the subject matter. 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.

[0061] The term "polyg lutamate", "polyg lutamination", or variations thereof, refers to a composition containing at least one chain of two or more linked glutamyl groups. The polyglutamate chain can be linear or branched. A linear polyglutamate chain can include, for example, glutamyl groups containing an alpha carboxyl group or a gamma carboxyl group linkage. A branched polyglutamate chain includes, for example, one or more glutamyl groups containing both an alpha carboxyl group and a gamma carboxyl group linkage to another glutamyl group, thereby providing a branch point for the polyglutamate. Representative branched polyglutamates are shown in FIGS. 1R-1U. The polyglutamate chain includes 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 polyglutaminated 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.

[0062] The terms "polyg lutaminated methotrexate", "polyg lutaminated MTX", "MTX-PG", "PMTX" are used interchangeably herein and refer to a methotrexate composition containing at least one glutamyl group in addition to the glutamyl groups in methotrexate (i.e., MTX-PG n , n≧1). References herein to the number of glutamyl groups in αPMTX (MTX-PG) include the glutamyl groups in methotrexate. For example, an MTX-PG composition containing five glutamyl residues in addition to the glutamyl groups of MTX is referred to herein as hexag lutaminated methotrexate or methotrexate hexag lutamate.

[0063] The terms "alpha-glutamyl group", "alpha-glutamate", and "alpha bond", when they relate to the bonding of glutamyl groups, mean a glutamyl group that includes an alpha-carboxyl group bond. In some embodiments, the alpha bond is an amide bond between the alpha-carboxyl group of one glutamyl group and a second glutamyl group. The alpha bond can be a bond between a glutamyl group and a glutamyl group in methotrexate, or between a glutamyl group and a second glutamyl group such as a glutamyl group within a polyglutamate chain attached to methotrexate and not present in methotrexate.

[0064] The terms "gamma-glutamyl group", "gamma-glutamate", and "gamma bond", when they relate to the bonding of glutamyl groups, mean a glutamyl group that includes a gamma-carboxyl group bond. As discussed herein, when methotrexate enters a cell, it is polyglutamylated by the enzyme folylpolyglutamate synthase (FPGS), which sequentially adds L-glutamyl groups to the gamma-carboxyl group of the glutamate in methotrexate. Thus, alpha-polyglutamylated methotrexate compositions are not formed intracellularly during methotrexate therapy. In some embodiments, the gamma bond is an amide bond between the gamma-carboxyl group of one glutamyl group and a second glutamyl group. The gamma bond can be a bond between a glutamyl group and a glutamyl group in methotrexate, or between a glutamyl group and a second glutamyl group such as a glutamyl group within a polyglutamate chain attached to methotrexate and not present in methotrexate. In some embodiments, the gamma bond means an amide bond of the glutamyl group of methotrexate. References to gamma bonds include, unless specifically stated otherwise or clearly apparent from the context that it is not intended, the gamma bond of the glutamyl group of methotrexate.

[0065] Unless otherwise indicated, the terms "alpha-polyglutamylated methotrexate," "αPMTX," "alpha MTX-PG," and iterations thereof are used interchangeably herein and mean a polyglutamylated methotrexate composition comprising at least one glutamyl group with an alpha linkage. For example, a pentaglutamylated MTX composition in which the second glutamyl group has an alpha linkage but each of the other glutamyl groups has a gamma linkage is considered an alpha MTX-PG in the present disclosure. In some embodiments, each glutamyl group of MTX-PG other than the glutamyl groups of MTX has an alpha linkage (e.g., when n = 5 and each of G 1 、G 2 、G 3 、G 4 、and G 5 has an alpha linkage, MTX-PG n ). In some embodiments, the C-terminal glutamyl group(s) or each glutamyl group of MTX-PG other than the glutamyl groups of MTX has an alpha linkage (e.g., when n = 5 and each of G 1 、G 2 、G 3 、alpha G 4 has an alpha linkage, MTX-PG n ). In some embodiments, each glutamyl group of MTX-PG other than the C-terminal glutamyl group(s) has an alpha linkage (e.g., when n = 5 and each of MTX and G 1 、G 2 、G 3 、and G 4 has an alpha linkage, MTX-PG n ).

[0066] As used herein, the term "isolated" means a composition in a form not found in nature. Isolated alpha-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 alpha-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). Alpha-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 agent, the alpha-polyglutamine oxidized composition is usually mixed with a pharmaceutically acceptable carrier or diluent. In some embodiments, the isolated alpha-polyglutamine oxidized composition (e.g., delivery carriers such as alpha-polyglutamate and liposomes containing alpha-polyglutamate) contains less than 1% or less than 0.1% of unwanted DNA or protein contaminants. In some embodiments, the alpha-polyglutamate composition (e.g., delivery carriers such as alpha-polyglutamate and liposomes containing alpha-polyglutamate) is "isolated".

[0067] 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, such as a compartment of a cell, tissue or organ. A targeting moiety can include a wide variety of substances. Targeting moieties include 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.

[0068] The terms "specific affinity" or "specifically binds" 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, with greater affinity, or in some combination of these, than it does to another substance, including a protein unrelated to the target epitope. Due to sequence identity between homologous proteins in different species, in some embodiments, a specific affinity includes a binding substance that recognizes a protein or target in two or more species. Similarly, due to homology within a specific region of the polypeptide sequences of different proteins, the terms "specific affinity" or "specific binding" can include a binding substance that recognizes two or more proteins or targets. 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 target. Thus, in certain embodiments, a targeting moiety can specifically bind to two or more targets. In certain embodiments, multiple targets can be bound by the same targeting moiety.

[0069] The term "epitope" means a portion of an antigen that is recognized by and can be specifically bound by a targeting moiety (i.e., a binding portion), 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 contains at least 3 amino acids, more commonly at least 5 or 8 - 10 amino acids, in a unique spatial higher-order structure.

[0070] Expressions such as "binding affinity for a target" and "binding to a target" 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, intermolecular interactions can be characterized using 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® instrument). 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).

[0071] The term "delivery carrier" generally means any composition that acts to assist, facilitate, or ease the entry of alpha-polyglutamyl oxidized 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., antibody-drug conjugates (ADCs)), immunoconjugates 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.

[0072] "Subject" means a vertebrate mammal, including but not limited to humans, dogs, cats, horses, goats, and primates such as monkeys. 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 (i.e., 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 in accordance with sound medical judgment, is preferably used.

[0073] 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 status of the subject being treated, the severity of the condition, the duration of treatment, the nature of concurrent or concomitant therapies (if any), the specific route of administration, and similar factors within the knowledge and professional opinion of the health care provider. 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 to some extent and preferably stops); inhibits the metastasis of the tumor (i.e., slows down to some extent and preferably stops); 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.

[0074] The terms "hyperproliferative disorder", "proliferative disease", and "proliferative disorder" are used interchangeably herein and relate to unwanted or uncontrolled cell growth of undesirable or abnormal cells, such as neoplastic or hyperplastic growth, whether in vitro or in vivo. In some embodiments, the proliferative disease is a cancer or tumor disease (including benign or cancerous) and / or any tumor metastasis, regardless of the location of the cancer, tumor, and / or tumor metastasis. In some embodiments, the proliferative disease is a benign or malignant tumor. In some embodiments, the proliferative disease is a non-cancerous disease. In some embodiments, the 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.

[0075] "Cancer", "tumor", or "malignant tumor" are used as synonymous terms and mean any of a number of 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, as well as a number of characteristic structures and / or molecular features. 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 that have specific structural characteristics, lack differentiation, and are capable of invasion and metastasis. Cancers that can be treated with the αPMTX compositions provided herein include, but are not limited to, for example, non-blood system 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, for example, blood system tumors such as leukemia, lymphoma and other B cell malignancies, myeloma and other plasma cell dyscrasias or cachexia. Other types of cancers and tumors that can be treated with the αPMTX compositions 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.

[0076] The terms "treating", or "treatment", or "treat" mean both (a) therapeutic means that cure, slow down, reduce the symptoms of, and / or halt the progression 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 which 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, 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 αPMTX composition can be used alone or in combination with additional therapeutic agents for treatment.

[0077] "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 primates, 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 any other member of the class Mammalia known in the art. In certain embodiments, the patient is human.

[0078] 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, multiple 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, non-leukemic meningeal carcinomatosis, soft tissue sarcoma (desmoid tumor, aggressive fibromatosis, bladder cancer, and central nervous system (CNS) lymphoma.

[0079] 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 to 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, 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, ulcerative colitis.

[0080] 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., Taxol®), 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, Taxol®). Such agents further include, but are not limited to, the anticancer agents trimethoprim, temozolomide, raltitrexed, S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benzylguanidine (6-BG), bis-chloronitrosourea (BCNU) and camptothecin (trademark), or any of their therapeutic derivatives. Further examples of therapeutic agents that may be suitable for use in 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, anti-metabolites, 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.

[0081] As used herein, the term "chemotherapeutic agent", when used in the context of cancer therapy, means any agent that causes 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.

[0082] 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, methotrexate, 5-fluorouracil, 5-fluorouracil prodrugs such as capecitabine, 5-fluorodeoxyuridine monophosphate, cytarabine, cytarabine prodrugs such as 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 alpha-polyglutamylated methotrexate composition is a fluoropyrimidine, 5-fluorouracil, 5-fluoro-2'-deoxycytidine, cytarabine, gemcitabine, troxacitabine, decitabine, azacitidine, pseudoisocytidine, zebularine, ancitabine, fazarabine, 6-azacitidine, capecitabine, N 4- It is used in combination with an antimetabolite selected from the group consisting of octadecyl cytarabine, elaidic acid cytarabine, fludarabine, cladribine, clofarabine, nelarabine, forodesine, and pentostatin, or derivatives thereof. In one example, the nucleoside analog is a substrate for a nucleoside deaminase that 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.

[0083] 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, and the derivatives 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.

[0084] The terms "pharmaceutically-acceptable carrier" and "pharmaceutically acceptable carrier" mean components other than the active ingredient in a pharmaceutical formulation that are non-toxic to the 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 a human or other subject.

[0085] The present disclosure generally relates to novel alpha-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 rheumatoid arthritis, and infectious diseases such as HIV and malaria.

[0086] In some embodiments, the present disclosure provides the following. [1] A composition comprising alpha-polyglutamylated methotrexate, wherein at least one glutamyl group has an alpha-carboxyl group linkage; [2] The composition of item [1], wherein the alpha-polyglutamylated methotrexate comprises 1 to 10 glutamyl groups having an alpha-carboxyl group linkage; [3] The composition of item [1] or [2], wherein the alpha-polyglutamylated methotrexate comprises 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups; [4] The composition of any one of items [1] to [3], comprising alpha-tetraglutamylated methotrexate; [5] The composition of any one of items [1] to [3], comprising alpha-pentaglutamylated methotrexate; [6] The composition of any one of items [1] to [3], comprising alpha-hexaglutamylated methotrexate; [7] The composition of any one of items [1] to [6], which is the following composition: (a) Two or more glutamyl groups have an alpha-carboxyl group linkage, (b) Each glutamyl group other than the glutamyl group of methotrexate has an alpha-carboxyl group linkage, or (c) Two or more glutamyl groups have a gamma-carboxyl group linkage; [8] The composition of any one of items [1] to [6], which is the following composition: (a) Each glutamyl group other than the C-terminal glutamyl group(s) and the glutamyl group of methotrexate has an alpha-carboxyl group bond; or (b) A composition in which each glutamyl group other than the C-terminal glutamyl group(s) has an alpha-carboxyl group bond; [9] The composition according to any one of items [1] to [8], wherein at least one glutamyl group has both an alpha-carboxyl group bond and a gamma-carboxyl group bond;

[10] The composition according to any one of items [1] to [9], which is the following composition: (a) At least two glutamyl groups of alpha-polyglutamylated methotrexate are of the L-form, (b) Each glutamyl group of alpha-polyglutamylated methotrexate is of the L-form, (c) At least one glutamyl group of alpha-polyglutamylated methotrexate is of the D-form, (d) Each glutamyl group of alpha-polyglutamylated methotrexate other than the glutamyl group of methotrexate is of the D-form, or (e) At least two glutamyl groups of alpha-polyglutamylated methotrexate are of the L-form and at least one glutamyl group is of the D-form;

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

[10] , wherein the polyglutamate is linear;

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

[10] , wherein the polyglutamate is branched;

[13] A liposomal composition (Lp-αPMTX) containing the alpha-polyglutamylated methotrexate according to any one of items [1] to

[12] ;

[14] The LαPP composition according to item

[13] , wherein the alpha-polyglutamylated methotrexate contains an L-form glutamyl group having an alpha-carboxyl group bond;

[15] The Lp-αPMTX composition according to item

[13] or

[14] , wherein each glutamyl group of alpha-polyglutamate oxidized methotrexate is of the L type;

[16] The Lp-αPMTX composition according to item

[13] or

[14] , wherein at least one glutamyl group of alpha-polyglutamate oxidized methotrexate is of the D type;

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

[13] to

[16] , wherein the liposome contains alpha-polyglutamate oxidized methotrexate having 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups;

[18] The Lp-αPMTX composition according to items

[13] to

[17] , wherein at least one glutamyl group of alpha-polyglutamate oxidized methotrexate has a gamma-carboxyl group bond;

[19] The composition according to any one of items

[13] to

[18] , wherein at least one glutamyl group has both an alpha-carboxyl group bond and a gamma-carboxyl group bond;

[20] The composition according to any one of items

[13] to

[19] , comprising 2, 3, 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups having both an alpha-carboxyl group bond and a gamma-carboxyl group bond;

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

[13] to

[20] , wherein the liposome contains alpha-polyglutamate oxidized methotrexate, and the alpha-polyglutamate oxidized methotrexate contains alpha-tetraglutamate oxidized methotrexate, alpha-pentaglutamate oxidized methotrexate, or alpha-hexaglutamate oxidized methotrexate;

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

[13] to

[21] , wherein the polyglutamate is linear or branched;

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

[13] to

[22] , wherein the liposome is pegylated (PαLp-αPMTX);

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

[13] to

[23] , wherein the liposome contains at least 1% by weight of alpha-polyglutamate oxidized methotrexate, or during the process of preparing Lp-αPMTX, at least 1% of the starting material of alpha-polyglutamate oxidized MTX is encapsulated (enclosed) in αPMTX;

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

[13] to

[24] , wherein the liposome has a diameter in the range of 20 nm to 500 nm or 20 nm to 200 nm;

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

[13] to

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

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

[13] to

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

[28] The Lp-αPMTX composition according to item

[27] , wherein the liposome components contain at least one anionic lipid and neutral lipid;

[29] The Lp-αPMTX composition according to item

[27] or

[28] , wherein the liposome components contain 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-αPMTX composition according to any one of items

[27] to

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

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

[27] to

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

[32] The Lp-αPMTX composition according to item

[31] , wherein the steric stabilizer is 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, a copolymer containing polyethylene glycol and polypropylene oxide, poloxamer 188, and polyvinyl alcohol, and the composition is at least one selected from the group consisting of;

[33] The Lp-αPMTX 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-αPMTX composition according to any one of items

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

[38] , wherein the liposome has an internal space containing alphapolyg lutamyl methotrexate and an aqueous pharmaceutically acceptable carrier;

[40] The Lp-αPMTX composition of 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-αPMTX composition of item

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

[42] The Lp-αPMTX composition of item

[41] , wherein the pharmaceutically acceptable carrier contains 5 wt% to 20 wt% of trehalose;

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

[39] to

[42] , wherein the pharmaceutically acceptable carrier contains 1 wt% to 15 wt% of dextrose;

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

[39] to

[44] , wherein the pharmaceutically acceptable carrier contains 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] The Lp-αPMTX composition according to any one of items

[39] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

[48] , wherein the liposome contains 10 to 100,000 or any range therebetween of alpha polyglutamate oxidized methotrexate molecules;

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

[13] 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-αPMTX 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-αPMTX composition according to item

[50] or

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

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

[50] to

[55] , 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-αPMTX composition according to any one of items

[50] to

[56] , 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-αPMTX 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-αPMTX 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-αPMTX composition of 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-αPMTX composition according to item

[58] or

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

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

[58] to

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

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

[58] to

[61] , further comprising a hapten;

[63] The Lp-αPMTX composition of item

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

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

[13] to

[63] , further comprising at least one cryoprotective substance selected from the group consisting of mannitol, trehalose, sorbitol, and sucrose in the internal space, external space, or both;

[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-αPMTX composition according to any one of items

[13] to

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

[68] A pharmaceutical composition comprising the liposomal alpha polyglutamine oxidized methotrexate composition according to any one of items

[13] to

[67] ;

[69] A pharmaceutical composition comprising the alpha polyglutamine oxidized methotrexate composition according to any one of items [1] to [8];

[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, 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, comprising the step of administering the liposomal alpha polyglutamine oxidized methotrexate composition according to any one of items

[13] to

[69] to the subject;

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

[69] ;

[75] A method for killing proliferating cells, comprising the step of contacting the proliferating cells with the liposomal alpha polyglutamine oxidized methotrexate composition according to any one of items

[13] 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 alpha-polyglutamylated methotrexate composition according to any one of items

[13] 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 mesothelioma or non-small cell lung cancer (NSCLC);

[82] The method according to item

[77] or

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

[83] A method for treating cancer, comprising the step of administering an effective amount of the Lp-αPMTX 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 the targeting moiety;

[84] A maintenance therapy for a subject who has received or has ever received cancer therapy, comprising the step of administering an effective amount of the composition according to any one of items [1] to

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

[85] A maintenance therapy comprising the step of administering an effective amount of the liposomal alpha-polyglutamate oxidized methotrexate composition according to any one of items

[13] to

[69] to a subject who has received or has ever received cancer therapy;

[86] A method for treating an immune system disorder, 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 immune system disorder;

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

[69] to a subject having or at risk of having an immune system disorder;

[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 administering to a subject having or at risk of having inflammation an effective amount of the composition according to any one of items [1] to

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

[69] ;

[89] A method for treating an infectious disease, comprising administering to a subject having or at risk of having an infectious disease an effective amount of the liposomal alpha-polyglutamate oxidized methotrexate composition according to any one of items

[13] to

[69] ;

[90] A method for delivering alpha-polyglutamate 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-αPMTX composition according to any one of items [1] to

[69] effective to deliver a therapeutically effective amount of alpha-polyglutamate oxidized methotrexate to the tumor;

[91] A method for preparing an alpha-polyglutamate oxidized methotrexate composition comprising the liposomal alpha-polyglutamate oxidized methotrexate composition according to any one of items

[13] to

[69] , the method comprising forming a mixture comprising a liposomal component and an alpha-polyglutamate oxidized folate antagonist in solution; homogenizing the mixture in solution to form liposomes; and treating the mixture to form liposomes comprising alpha-polyglutamate oxidized methotrexate;

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

[13] to

[69] , comprising the steps of: forming a mixture containing liposome components and alpha-polyglutamic acid oxidized methotrexate in a solution; homogenizing the mixture in the solution to form liposomes; treating the mixture to form liposomes encapsulating and / or coating alpha-polyglutamic acid 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);

[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;

[94] 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 alpha-polyglutamic acid oxidized methotrexate in a solution; homogenizing the mixture in the solution to form liposomes; treating the mixture to form liposomes encapsulating and / or coating alpha-polyglutamic acid 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);

[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 alpha polyglutamine oxidized methotrexate in a solution; treating the mixture to form liposomes encapsulating and / or coating alpha polyglutamine 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);

[96] The method according to item

[95] , wherein the treating step comprises the step of 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 selected from 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

[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 selected from 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 alpha polyglutamine oxidized methotrexate is encapsulated or enclosed in the liposomes.

[0087] II. Alpha polyglutamine oxidized methotrexate (αPMTX) Generally, the present disclosure relates to alpha-polyglutamyl methotrexate (αPMTX) compositions. The αPMTX compositions include at least one glutamyl group having an alpha bond. These compositions are structurally different from L-gamma-polyglutamylated methotrexate (LαPMTX) produced by the enzyme folylpoly-gamma-glutamate synthase (FPGS) in cells during methotrexate therapy.

[0088] In some embodiments, the αPMTX composition includes 2 to 20, 2 to 15, 2 to 10, 2 to 5, 2 to 6, or more than 5 glutamyl groups (including the glutamyl group of methotrexate). In some embodiments, each glutamyl group in αPMTX other than the glutamyl group of methotrexate has an alpha bond. In some embodiments, the C-terminal glutamyl group(s) and each glutamyl group in αPMTX other than the glutamyl group of methotrexate have an alpha bond. In some embodiments, each of the glutamyl groups in αPMTX other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, two or more glutamyl groups in αPMTX have a gamma bond. In some embodiments, at least one glutamyl group of alpha-polyglutamyl methotrexate has an alpha carboxyl group bond and a gamma carboxyl group bond. In some embodiments, each glutamyl group in αPMTX is of the L-type. In some embodiments, each glutamyl group in αPMTX other than the glutamyl group of methotrexate is of the D-type. In some embodiments, αPMTX includes two or more L-type glutamyl groups and one or more D-type glutamyl groups. In some embodiments, the polyglutamate chain of αPMTX is linear (not branched). In some embodiments, the polyglutamate chain of αPMTX is branched.

[0089] In some embodiments, alpha-polyglutamylated methotrexate is diglutamylated. That is, alpha-polyglutamylated methotrexate contains one additional glutamyl group in addition to the glutamyl group of methotrexate (αMTX-PG 1 ), and the additional glutamyl group is attached to the glutamyl group in methotrexate via an alpha bond. In some embodiments, each glutamyl group of alpha-diglutamylated methotrexate is of the L-type. In other embodiments, alpha-diglutamylated MTX contains a D-type glutamyl group.

[0090] In some embodiments, alpha-polyglutamylated methotrexate is triglutamylated. That is, alpha-polyglutamylated methotrexate contains two additional glutamyl groups in addition to the glutamyl group of methotrexate (αMTX-PG 2)。In some embodiments, each of the two additional glutamyl groups has an alpha bond. In other embodiments, one of the two additional glutamyl groups has an alpha bond and the other glutamyl group has a gamma bond. In some embodiments, one of the two additional glutamyl groups has an alpha bond. In some embodiments, one of the two additional glutamyl groups has a gamma bond. In some embodiments, two of the three glutamyl groups have an alpha bond. In other embodiments, one of the three glutamyl groups has an alpha bond and another glutamyl group has a gamma bond. In some embodiments, one glutamyl group has both an alpha bond and a gamma bond. In some embodiments, each glutamyl group of alpha-triglutamyl oxidized methotrexate is of the L-type. In other embodiments, alpha-triglutamyl oxidized MTX contains a D-type glutamyl group. In further embodiments, each glutamyl group of alpha-triglutamyl oxidized methotrexate other than the glutamyl group of methotrexate is of the D-type. In further embodiments, triglutamyl oxidized MTX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0091] In some embodiments, alpha-polyglutamyl oxidized methotrexate is tetraglutamylated and thus contains three additional glutamyl groups in addition to the glutamyl groups in methotrexate (αMTX-PG 3)。In some embodiments, each of the three glutamyl groups has an alpha bond. In other embodiments, one or two of the three additional glutamyl groups have an alpha bond, and the remaining two or one glutamyl groups each have a gamma bond. In some embodiments, two of the three additional glutamyl groups have an alpha bond. In other embodiments, one of the three glutamyl groups has an alpha bond and another additional glutamyl group has a gamma bond. In other embodiments, one of the three additional glutamyl groups has both an alpha bond and a gamma bond. In other embodiments, three of the four glutamyl groups have an alpha bond. In some embodiments, at least one glutamyl group has both an alpha bond and a gamma bond. In some embodiments, alpha-tetraglutamine oxidized MTX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alpha-tetraglutamine oxidized methotrexate is of the L-type. In other embodiments, alpha-tetraglutamine oxidized MTX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alpha-triglutamine oxidized methotrexate other than the glutamyl group of methotrexate is of the D-type. In a further embodiment, tetraglutamine oxidized MTX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0092] In some embodiments, alpha-polyglutamine oxidized methotrexate is pentaglutamine oxidized (αMTX-PG 4) It contains a chain of 4 additional glutamyl groups attached to the glutamyl group of methotrexate. In some embodiments, each of the 4 additional glutamyl groups in the chain has an alpha bond. In some embodiments, each of the 4 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In other embodiments, 1, 2, or 3 of the 4 additional glutamyl groups have an alpha bond, and the remaining 3, 2, or 1 glutamyl groups are each attached to the glutamyl group of the molecule via a gamma bond. In other embodiments, 1 or 2 of the 4 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least 1 additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least 1 of the 5 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 5 glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, alpha pentaglutamyl oxidized MTX contains 2 or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alpha pentaglutamyl oxidized methotrexate is of the L-type. In other embodiments, alpha pentaglutamyl oxidized MTX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alpha pentaglutamyl oxidized methotrexate other than the glutamyl group of methotrexate is of the D-type. In a further embodiment, pentaglutamyl oxidized MTX contains a D-type glutamyl group and 2 or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0093] In some embodiments, alpha polyglutamyl oxidized methotrexate is hexaglutamyl oxidized (αMTX-PG 5) It contains a chain of 5 additional glutamyl groups attached to the glutamyl group of methotrexate. In some embodiments, each of the 5 additional glutamyl groups in the chain has an alpha bond. In some embodiments, each of the 5 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 4 of the 5 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, or 4 of the 5 additional glutamyl groups are attached to the glutamyl group of the molecule via an alpha bond, and the remaining 4, 3, 2, or 1 glutamyl groups are each attached to the glutamyl group of the molecule via a gamma bond. In other embodiments, 1, 2, 3, or 4 of the 5 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least 1 additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least 1 of the 6 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 6 glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 5 of the 6 glutamyl groups have an alpha bond. In some embodiments, alpha-hexaglutaminyl oxidized MTX contains 2 or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alpha-hexaglutaminyl oxidized methotrexate is of the L-type. In other embodiments, alpha-hexaglutaminyl oxidized MTX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alpha-hexaglutaminyl oxidized methotrexate other than the glutamyl group of methotrexate is of the D-type. In a further embodiment, hexaglutaminyl oxidized MTX contains a D-type glutamyl group and 2 or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0094] In some embodiments, alpha-polyglutamyl methotrexate is heptaglutamylated (αMTX-PG 6 ), and includes a chain of six additional glutamyl groups attached to the glutamyl group of methotrexate. In some embodiments, each of the six additional glutamyl groups has an alpha bond. In some embodiments, each of the six additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, five of the six additional glutamyl groups in the chain have an alpha bond. In other embodiments, one, two, three, four, or five of the six additional glutamyl groups have an alpha bond, and the remaining five, four, three, two, or one glutamyl group has a gamma bond, respectively. In other embodiments, one, two, three, four, or five of the six additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the seven glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the seven glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, six of the seven glutamyl groups have an alpha bond. In some embodiments, alpha-heptaglutamylated MTX includes two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alpha-heptaglutamylated methotrexate is of the L-type. In other embodiments, alpha-heptaglutamylated MTX includes a D-type glutamyl group. In a further embodiment, each glutamyl group of alpha-heptaglutamylated methotrexate other than the glutamyl group of methotrexate is of the D-type. In a further embodiment, heptaglutamylated MTX includes a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0095] In some embodiments, alpha-polyglutamylated methotrexate is octaglutamylated (αMTX-PG 7 ) and contains a chain of seven additional glutamyl groups attached to the glutamyl group of methotrexate. In some embodiments, each of the seven additional glutamyl groups in the chain, other than the C-terminal glutamyl group(s), has an alpha bond. In some embodiments, six of the seven additional glutamyl groups in the chain have an alpha bond. In some embodiments, each of the seven additional glutamyl groups has an alpha bond. In other embodiments, one, two, three, four, five, or six of the seven additional glutamyl groups have an alpha bond, and the remaining six, five, four, three, two, or one glutamyl group has a gamma bond, respectively. In other embodiments, one, two, three, four, five, or six of the seven additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the eight glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the eight glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, seven of the eight glutamyl groups have an alpha bond. In some embodiments, alpha-octaglutamylated MTX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alpha-octaglutamylated methotrexate is of the L-type. In other embodiments, alpha-octaglutamylated MTX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alpha-octaglutamylated methotrexate other than the glutamyl group of methotrexate is of the D-type. In a further embodiment, octaglutamylated MTX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0096] In some embodiments, alpha-polyglutamylated methotrexate is nona-glutamylated (αMTX-PG 8 ) and contains a chain of eight additional glutamyl groups attached to the glutamyl group of methotrexate. In some embodiments, each of the eight additional glutamyl groups in the chain, other than the C-terminal glutamyl group(s), has an alpha bond. In some embodiments, seven of the eight additional glutamyl groups in the chain have an alpha bond. In some embodiments, each of the eight additional glutamyl groups has an alpha bond. In other embodiments, one, two, three, four, five, six, or seven of the eight additional glutamyl groups have an alpha bond, and the remaining seven, six, five, four, three, two, or one glutamyl group has a gamma bond, respectively. In other embodiments, one, two, three, four, five, six, or seven of the eight additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the nine glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the nine glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, eight of the nine glutamyl groups have an alpha bond. In some embodiments, alpha-nona-glutamylated MTX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alpha-nona-glutamylated methotrexate is of the L-type. In other embodiments, alpha-nona-glutamylated MTX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alpha-nona-glutamylated methotrexate other than the glutamyl group of methotrexate is of the D-type. In a further embodiment, nona-glutamylated MTX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0097] In some embodiments, alpha-polyglutamylated methotrexate is decaglutamylated (αMTX-PG 9) It contains a chain of 9 additional glutamyl groups attached to the glutamyl group of methotrexate. In some embodiments, each of the 9 additional glutamyl groups has an alpha bond. In some embodiments, each of the 9 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 8 of the 9 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 of the 9 additional glutamyl groups have an alpha bond, and the remaining 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group has a gamma bond, respectively. In other embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 of the 9 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 10 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 10 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 9 of the 10 glutamyl groups have an alpha bond. In some embodiments, alphadecaglutamylated MTX contains 2 or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alphadecaglutamylated methotrexate is of the L-type. In other embodiments, alphadecaglutamylated MTX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alphadecaglutamylated methotrexate other than the glutamyl group of methotrexate is of the D-type. In a further embodiment, decaglutamylated MTX contains a D-type glutamyl group and 2 or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0098] In some embodiments, alpha-polyglutamylated methotrexate is undecaglutamylated (αMTX-PG 10 ). In some embodiments, each of the 10 additional glutamyl groups has an alpha bond. In some embodiments, each of the 10 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 9 of the 10 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the 10 additional glutamyl groups have an alpha bond, and the remaining 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group has a gamma bond, respectively. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the 10 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 11 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 11 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 10 of the 11 glutamyl groups have an alpha bond. In some embodiments, alpha-undecaglutamylated MTX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alpha-undecaglutamylated methotrexate is of the L-type. In other embodiments, alpha-undecaglutamylated MTX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alpha-undecaglutamylated methotrexate other than the glutamyl group of methotrexate is of the D-type. In a further embodiment, undecaglutamylated MTX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0099] In some embodiments, alpha-polyglutamylated methotrexate is dodecaglutamylated (αMTX-PG 11 ). In some embodiments, each of the 11 additional glutamyl groups has an alpha bond. In some embodiments, each of the 11 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 10 of the 11 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the 11 additional glutamyl groups have an alpha bond, and the remaining 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the 11 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 12 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 12 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 11 of the 12 glutamyl groups have an alpha bond. In some embodiments, alpha-dodecaglutamylated MTX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alpha-dodecaglutamylated methotrexate is of the L-type. In other embodiments, alpha-dodecaglutamylated MTX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alpha-dodecaglutamylated methotrexate other than the glutamyl groups of methotrexate is of the D-type. In a further embodiment, dodecaglutamylated MTX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0100] In some embodiments, alpha-polyglutamylated methotrexate is tridecaglutamylated (αMTX-PG 12)。In some embodiments, each of the 12 additional glutamyl groups has an alpha bond. In some embodiments, each of the 12 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 11 of the 12 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the 12 additional glutamyl groups have an alpha bond, and the remaining 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the 12 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 13 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 13 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 12 of the 13 glutamyl groups have an alpha bond. In some embodiments, alpha-triskadecakaglutaminyl oxidized MTX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alpha-triskadecakaglutaminyl oxidized methotrexate is of the L-type. In other embodiments, alpha-triskadecakaglutaminyl oxidized MTX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alpha-triskadecakaglutaminyl oxidized methotrexate other than the glutamyl group of methotrexate is of the D-type. In a further embodiment, triskadecakaglutaminyl oxidized MTX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0101] In some embodiments, alpha-polyglutamylated methotrexate is tetradeca-glutamylated (αMTX-PG) 13 13)。In some embodiments, each of the 13 additional glutamyl groups has an alpha bond. In some embodiments, each of the 13 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 12 of the 13 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the 13 additional glutamyl groups have an alpha bond, and the remaining 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group(s) each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the 13 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 14 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 14 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 13 of the 14 glutamyl groups have an alpha bond. In some embodiments, alpha-tetradecaglutamyl oxidized MTX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alpha-tetradecaglutamyl oxidized methotrexate is of the L-type. In other embodiments, alpha-tetradecaglutamyl oxidized MTX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alpha-tetradecaglutamyl oxidized methotrexate other than the glutamyl group of methotrexate is of the D-type. In a further embodiment, tetradecaglutamyl oxidized MTX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0102] In some embodiments, alpha-polyglutamylated methotrexate is pentadeca-glutamylated (αMTX-PG). 14)。In some embodiments, each of the 14 additional glutamyl groups has an alpha bond. In some embodiments, each of the 14 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 13 of the 14 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the 14 additional glutamyl groups have an alpha bond, and the remaining 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group each has a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the 14 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 15 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 15 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 14 of the 15 glutamyl groups have an alpha bond. In some embodiments, alpha-pentadeca-glutamylated MTX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alpha-pentadeca-glutamylated methotrexate is of the L-type. In other embodiments, alpha-pentadeca-glutamylated MTX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alpha-pentadeca-glutamylated methotrexate other than the glutamyl group of methotrexate is of the D-type. In a further embodiment, pentadeca-glutamylated MTX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0103] In some embodiments, alpha-polyglutamated methotrexate is hexadeca-glutamated (αMTX-PG 15)。In some embodiments, each of the 15 additional glutamyl groups has an alpha bond. In some embodiments, each of the 15 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 14 of the 15 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the 15 additional glutamyl groups have an alpha bond, and the remaining 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group has a gamma bond, respectively. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the 15 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 16 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 16 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 15 of the 16 glutamyl groups have an alpha bond. In some embodiments, alpha-hexadeca glutaminyl oxidized MTX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alpha-hexadeca glutaminyl oxidized methotrexate is of the L-type. In other embodiments, alpha-hexadeca glutaminyl oxidized MTX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alpha-hexadeca glutaminyl oxidized methotrexate other than the glutamyl group of methotrexate is of the D-type. In a further embodiment, hexadeca glutaminyl oxidized MTX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0104] In other embodiments, alpha-polyglutamylated methotrexate is heptadeca-glutamylated (αMTX-PG 16)。In some embodiments, each of the 16 additional glutamyl groups has an alpha bond. In some embodiments, each of the 16 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 15 of the 16 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the 16 additional glutamyl groups have an alpha bond, and the remaining 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group each has a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the 16 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 17 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 17 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 16 of the 17 glutamyl groups have an alpha bond. In some embodiments, alpha-heptadeca glutamyl oxidized MTX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alpha-heptadeca glutamyl oxidized methotrexate is of the L-type. In other embodiments, alpha-heptadeca glutamyl oxidized MTX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alpha-heptadeca glutamyl oxidized methotrexate other than the glutamyl group of methotrexate is of the D-type. In a further embodiment, heptadeca glutamyl oxidized MTX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0105] In some embodiments, alpha-polyglutamylated methotrexate is octadeca-glutamylated (αMTX-PG 17)。In some embodiments, each of the 17 additional glutamyl groups has an alpha bond. In some embodiments, each of the 17 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 16 of the 17 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the 17 additional glutamyl groups have an alpha bond, and the remaining 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group each has a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the 17 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 18 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 18 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 17 of the 18 glutamyl groups have an alpha bond. In some embodiments, alpha-octadeca-glutamylated MTX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alpha-octadeca-glutamylated methotrexate is of the L-type. In other embodiments, alpha-octadeca-glutamylated MTX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alpha-octadeca-glutamylated methotrexate other than the glutamyl group of methotrexate is of the D-type. In a further embodiment, octadeca-glutamylated MTX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0106] In some embodiments, alpha-polyglutamylated methotrexate is eicosaglutamylated (αMTX-PG 18)。In some embodiments, each of the 18 additional glutamyl groups has an alpha bond. In some embodiments, each of the 18 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 17 of the 18 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of the 18 additional glutamyl groups have an alpha bond, and the remaining 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group each has a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of the 18 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 19 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 19 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 18 of the 19 glutamyl groups have an alpha bond. In some embodiments, alpha-enniadecakoglutamine oxidized MTX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alpha-enniadecakoglutamine oxidized methotrexate is of the L-type. In other embodiments, alpha-enniadecakoglutamine oxidized MTX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alpha-enniadecakoglutamine oxidized methotrexate other than the glutamyl group of methotrexate is of the D-type. In a further embodiment, enniadecakoglutamine oxidized MTX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0107] In some embodiments, alpha-polyglutamylated methotrexate is eicosaglutamylated (αMTX-PG 19)。In some embodiments, each of the 19 additional glutamyl groups has an alpha bond. In some embodiments, each of the 19 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 18 of the 19 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 19 additional glutamyl groups have an alpha bond, and the remaining 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group each has a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 19 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are bonded to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 20 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 20 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 19 of the 20 glutamyl groups have an alpha bond. In some embodiments, alphaiCOS glutaminated MTX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alphaiCOS glutaminated methotrexate is of the L-type. In other embodiments, alphaiCOS glutaminated MTX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alphaiCOS glutaminated methotrexate other than the glutamyl group of methotrexate is of the D-type. In a further embodiment, iCOS glutaminated MTX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0108] In some embodiments, alpha-polyglutamylated methotrexate is eicosakainic acid-glutamylated (αMTX-PG). 20)。In some embodiments, each of the 20 additional glutamyl groups has an alpha bond. In some embodiments, each of the 20 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 19 of the 20 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the 20 additional glutamyl groups have an alpha bond, and the remaining 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group each has a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the 20 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 21 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 21 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 20 of the 21 glutamyl groups have an alpha bond. In some embodiments, alphaiocosikahenaglutamine oxidized MTX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alphaiocosikahenaglutamine oxidized methotrexate is of the L-type. In other embodiments, alphaiocosikahenaglutamine oxidized MTX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alphaiocosikahenaglutamine oxidized methotrexate other than the glutamyl group of methotrexate is of the D-type. In a further embodiment, icosikahenaglutamine oxidized MTX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear.In other embodiments, the polyglutamate chain is a branched chain.

[0109] In some embodiments, alpha-polyglutamylated methotrexate contains 4 to 7 glutamyl groups attached to methotrexate (i.e., αMTX-PGn, n = 4 to 7), and each of the 4 to 7 attached glutamyl groups has an alpha bond. In some embodiments, alpha-polyglutamylated folylmethotrexate contains 4 to 7 glutamyl groups attached to methotrexate (i.e., αMTX-PGn, n = 4 to 7), and each of the 4 to 7 attached glutamyl groups, other than the C-terminal glutamyl group(s), has an alpha 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 D-type. In some embodiments, the polyglutamate chain is a straight chain. In other embodiments, the polyglutamate chain is a branched chain.

[0110] In one embodiment, alpha-polyglutamylated methotrexate is tetraglutamylated, and each of the 3 glutamyl groups in the polyglutamate chain attached to methotrexate contains an alpha bond. In one embodiment, alpha-polyglutamylated methotrexate is tetraglutamylated, and each of the 3 glutamyl groups in the polyglutamate chain attached to methotrexate, other than the C-terminal glutamyl group(s), contains an alpha bond. In some embodiments, each of the 4 glutamyl groups is of the L-type. In some embodiments, each glutamyl group of alpha-tetraglutamylated methotrexate, other than the glutamyl groups of methotrexate, is of the D-type. In other embodiments, at least 2 of the glutamyl groups in alpha-tetraglutamate methotrexate are of the L-type and at least 1 glutamyl group is of the D-type. In some embodiments, the polyglutamate chain is a straight chain. In other embodiments, the polyglutamate chain is a branched chain.

[0111] In one embodiment, alpha-polyglutamyl methotrexate is pentaglutamylated, and each of the four glutamyl groups in the polyglutamate chain attached to methotrexate contains an alpha bond. In one embodiment, alpha-polyglutamyl methotrexate is pentaglutamylated, and each of the four glutamyl groups in the polyglutamate chain attached to methotrexate other than the C-terminal glutamyl group(s) contains an alpha bond. In some embodiments, each of the four glutamyl groups is of the L-type. In some embodiments, each glutamyl group in alpha-pentaglutamyl methotrexate other than the glutamyl group of methotrexate is of the D-type. In other embodiments, at least two of the glutamyl groups in alpha-pentaglutamyl methotrexate are of the L-type and at least one glutamyl group is of the D-type. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0112] In one embodiment, alpha-polyglutamyl methotrexate is hexaglutamylated, and each of the five glutamyl groups in the polyglutamate chain attached to methotrexate contains an alpha bond. In one embodiment, alpha-polyglutamyl methotrexate is hexaglutamylated, and each of the five glutamyl groups in the polyglutamate chain attached to methotrexate other than the C-terminal glutamyl group(s) contains an alpha bond. In some embodiments, each of the five glutamyl groups is of the L-type. In some embodiments, each glutamyl group in alpha-hexaglutamyl methotrexate other than the glutamyl group of methotrexate is of the D-type. In other embodiments, at least two of the glutamyl groups in alpha-hexaglutamyl methotrexate are of the L-type and at least one glutamyl group is of the D-type. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0113] In another embodiment, alpha-polyglutamyl methotrexate is heptaglutamylated and each of the six glutamyl groups in the polyglutamate chain attached to methotrexate contains an alpha bond. In another embodiment, alpha-polyglutamyl methotrexate is heptaglutamylated and each of the six glutamyl groups in the polyglutamate chain attached to methotrexate other than the C-terminal glutamyl group(s) contains an alpha bond. In some embodiments, each of the six glutamyl groups is of the L-type. In some embodiments, each glutamyl group in alpha-heptaglutamyl methotrexate other than the glutamyl group of methotrexate is of the D-type. In other embodiments, at least two of the glutamyl groups in alpha-heptaglutamyl methotrexate are of the L-type and at least one glutamyl group is of the D-type. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0114] In some embodiments, alpha-polyglutamylated methotrexate (αPMTX) 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 αPMTX other than the glutamyl group of methotrexate has an alpha bond. In some embodiments, the C-terminal glutamyl group(s) and each glutamyl group in αPMTX other than the glutamyl group of methotrexate have an alpha bond. In some embodiments, each of the glutamyl groups in αPMTX other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the glutamyl groups in αPMTX have an alpha bond. In some embodiments, αPMTX contains L- and D-type glutamyl groups. In further embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the glutamyl groups in αPMTX have an alpha bond and 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 of the glutamyl groups each have a gamma bond. In some embodiments, each of the glutamyl groups in the polyglutamate structure of polyglutamylated methotrexate is of the L-type. In some embodiments, each of the glutamyl groups in αPMTX 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 αPMTX 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 αPMTX are of the D-type. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0115] In some embodiments, alpha-polyglutamyl methotrexate (αPMTX) contains a total of 2 to 20, 2 to 15, 2 to 10, 2 to 5, or any range therebetween glutamyl groups, including the glutamyl group of methotrexate. In some embodiments, each glutamyl group in αPMTX other than the glutamyl group of methotrexate has an alpha bond. In some embodiments, each glutamyl group in αPMTX other than the C-terminal glutamyl group(s) and the glutamyl group of methotrexate has an alpha bond. In some embodiments, each of the glutamyl groups in αPMTX other than the C-terminal glutamyl group(s) has an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 glutamyl groups have an alpha bond. In some embodiments, αPMTX contains two or more glutamyl groups having a gamma bond. In a further embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 glutamyl groups in αPMTX other than the glutamyl group of methotrexate have an alpha bond, and 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 glutamyl groups each have a gamma bond. In some embodiments, each glutamyl group in αPMTX is of the L-type. In some embodiments, each glutamyl group in αPMTX other than the glutamyl group of methotrexate is of the D-type. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the glutamyl groups in αPMTX are of the L-type. In another embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the glutamyl groups in αPMTX are of the D-type.

[0116] In some embodiments, alpha-polyglutamyl methotrexate contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 glutamyl groups in addition to the glutamyl group of methotrexate. In further embodiments, the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additional glutamyl groups have alpha linkages. In further embodiments, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group in alpha-polyglutamyl methotrexate has a gamma linkage. In some embodiments, at least one glutamyl group has both alpha and gamma linkages. In some embodiments, the glutamyl group in methotrexate has an alpha linkage. In some embodiments, the glutamyl group in methotrexate has both alpha and gamma linkages.

[0117] In some embodiments, the total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 glutamyl groups in alpha-polyglutamyl methotrexate are of the L-form, D-form, or both L-form and D-form. In some embodiments, each glutamyl group of alpha-polyglutamyl methotrexate is of the L-form. In other embodiments, each glutamyl group of alpha-polyglutamyl methotrexate other than the glutamyl group of methotrexate is of the D-form. In alternative embodiments, at least two of the glutamyl groups in alpha-polyglutamyl methotrexate are of the L-form and at least one of the glutamyl groups in alpha-polyglutamyl methotrexate is of the D-form. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 glutamyl groups in alpha-polyglutamyl methotrexate are of the L-form. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 glutamyl groups in alpha-polyglutamyl methotrexate are of the D-form.

[0118] In further embodiments, alpha-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 glutamyl groups, or any range therebetween. In some embodiments, each glutamyl group of alpha-polyglutamyl-methotrexate is of the L-type. In other embodiments, each glutamyl group of alpha-polyglutamyl-methotrexate other than the glutamyl groups of methotrexate is of the D-type. In alternative embodiments, at least two of the glutamyl groups in alpha-polyglutamyl-methotrexate are of the L-type and at least one of the glutamyl groups in alpha-polyglutamyl-methotrexate is of the D-type.

[0119] In further embodiments, the provided composition contains alpha-polyglutamyl-methotrexate having 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 to 10, or 1 to 20 alpha-linked glutamyl groups. In some embodiments, alpha-polyglutamyl-methotrexate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 to 10, or 1 to 20 L-type glutamyl groups. In some embodiments, alpha-polyglutamyl-methotrexate contains 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 to 10, or 1 to 20 D-type glutamyl groups. In some embodiments, alpha-polyglutamyl-methotrexate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 to 10, or 1 to 20 L-type glutamyl groups and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 to 10, or 1 to 20 D-type glutamyl groups. In other embodiments, alpha-polyglutamyl-methotrexate contains at least one glutamyl group having both alpha- and gamma-bonds. In some embodiments, alpha-polyglutamyl-methotrexate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 to 10, or more than 10 glutamyl groups having both alpha- and gamma-bonds.

[0120] In some embodiments, alpha-polyglutamylated methotrexate contains at least 1 glutamyl group having an alpha bond and contains 2, 3, 4, 5, 6, 7, 8, 9, 1-10, 1-20, or more glutamyl groups having a gamma bond. For example, in some embodiments, alpha-polyglutamylated methotrexate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10 L-alpha-glutamyl group bonds and further contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 L-gamma-glutamyl group bonds. In some further embodiments, alpha-polyglutamylated methotrexate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 L-alpha-glutamyl group bonds and further contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 D-gamma-glutamyl group bonds. In additional further embodiments, alpha-polyglutamylated methotrexate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 D-alpha-glutamyl group bonds and further contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 D-gamma-glutamyl group bonds. In other further embodiments, alpha-polyglutamylated methotrexate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1-10 D-gamma-glutamyl group bonds and further contains 1, 2, 3, 4, 5, 6, or 1-10 L-gamma-glutamyl group bonds. In other embodiments, alpha-polyglutamylated methotrexate contains at least 1 glutamyl group having both an alpha bond and a gamma bond. In some embodiments, alpha-polyglutamylated methotrexate contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or more than 10 glutamyl groups having both an alpha bond and a gamma bond.

[0121] In some embodiments, the alpha-polyglutamylated methotrexate compositions provided herein can accept one or more additional glutamyl groups, i.e., the compositions can serve as substrates for FPGS (folylpolyglutamate synthetase). Reagents and assays for measuring the ability of an alpha-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.

[0122] In some embodiments, the rate of uptake of the naked alpha-PMTX compositions (e.g., alpha-PMTX not conjugated to a delivery carrier) disclosed herein by liver cells is significantly reduced compared to the rate of uptake of methotrexate under physiological conditions. In some embodiments, the rate of liver cell uptake of the naked alpha-PMTX composition is less than 30%, 20%, 15%, or 10% compared to the rate of methotrexate. In further embodiments, the rate of efflux (transport) of the alpha-PMTX compositions disclosed herein from liver cells occurs at a significantly slower rate (less than 30%, 20%, 15%, or 10%) compared to methotrexate.

[0123] In some embodiments, the alpha-polyglutamylated 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 alpha-polyglutamylated methotrexate is hexaglutamylated methotrexate.

[0124] In some embodiments, the alpha-polyglutamylated methotrexate compositions provided herein have lower toxic side effects than methotrexate. In some embodiments, the alpha-polyglutamylated methotrexate compositions provided herein are less toxic to non-proliferating cells than methotrexate. In some embodiments, the alpha-polyglutamylated methotrexate compositions provided herein are less toxic to 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 alpha-polyglutamylated methotrexate is hexaglutamylated methotrexate.

[0125] In some embodiments, the alpha-polyglutamylated methotrexate compositions provided herein have lower toxic side effects than methotrexate. In some embodiments, the alpha-polyglutamylated 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 assays are performed in an in vivo mouse model. In some embodiments, the alpha-polyglutamylated methotrexate compositions provided herein result in fewer or less severe hematological or hepatic 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 hepatic 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 the alpha-polyglutamylated methotrexate composition at 40 mg / kg or 80 mg / kg once a week for 4 weeks. In some embodiments, the alpha-polyglutamylated methotrexate is hexaglutamylated methotrexate.

[0126] In some embodiments, treatment with the alpha-polyglutamylated 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 alpha-polyglutamylated methotrexate compositions provided herein do not significantly decrease mean neutrophils, mean white blood cells, or mean platelet counts. In some embodiments, the alpha-polyglutamylated methotrexate compositions provided herein do not significantly increase serum aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT) levels. In some embodiments, the alpha-polyglutamylated methotrexate compositions provided herein do not significantly reduce serum albumin levels. In some embodiments, the in vivo assay comprises administering the alpha-polyglutamylated methotrexate composition at 40 mg / kg or 80 mg / kg once a week for 4 weeks. In some embodiments, the alpha-polyglutamylated methotrexate is hexaglutamylated methotrexate.

[0127] In some embodiments, the alpha-polyglutamylated methotrexate composition does not contain a fluorine atom. In some embodiments, the alpha-polyglutamylated methotrexate composition does not contain a 4-fluoroglutamyl group.

[0128] Alpha-polyglutamylated methotrexate (α PMTX) compositions and their uses are further described in each of U.S. Patent Application No. 62 / 374,458, International Application No. PCT / US2017 / 046666, and International Application No. PCT / US2017 / 046667. The respective disclosures of each of these are hereby incorporated by reference in their entirety.

[0129] A. Polyglutamylated methotrexate analogs and derivatives The present disclosure also encompasses alpha 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 is not attached 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 the polyglutamylated methotrexate analog or derivative, is not fluorinated.

[0130] In some embodiments, the polyglutaminated methotrexate analogs or derivatives encompassed by the present disclosure are indoline ring and modified ornithine or glutamate-containing methotrexate derivatives. In some embodiments, the polyglutaminated 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 platinumol 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.

[0131] In some embodiments, the polyglutamate 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, ortho isomers of aminopterin, hydroxymethylmethotrexate, γ-fluoromethotrexate, gem-diphosphonate methotrexate analogs, α- or / and 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, 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’,It is a member selected from the group consisting of 5'-dichloromethotrexate, deazaaminopterin analogs, cysteic acid and homocysteic acid methotrexate analogs, and MX068.,

[0132] In further embodiments, the alpha-polyglutamyl oxidized methotrexate derivative or analog has a variant polyglutamate chain. 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 an amide bond. In other embodiments, one or more glutamyl groups of the polyglutamate chain are derivatized.,

[0133] 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).

[0134] The addition of the glutamyl residue 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 a desired length of 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 bound to 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 bound to the peptide, and the molecule is cleaved from the resin.

[0135] C. Methotrexate-PG Complex Surprisingly, the inventors have found that polyglutamine oxidized metabolic antagonists such as polyglutamine oxidized methotrexate (αPMTX) can form complexes with other compositions including therapeutic agents containing cytotoxic compounds such as platinum-based compounds. Accordingly, in some embodiments, the present disclosure provides a complex of αPMTX (e.g., αPMTX disclosed herein) with a therapeutic agent or a salt or acid thereof.

[0136] In some embodiments, the αPMTX / complex comprises αPMTX and a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic compound such as a chemotherapeutic agent. In further embodiments, the αPMTX / 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 αPMTX / complex comprises cyclodextrin. In further embodiments, the αPMTX / complex is encapsulated in liposomes.

[0137] In some embodiments, the present disclosure provides a composition comprising a complex of αPMTX and a therapeutic agent or a salt or acid thereof. In further embodiments, the αPMTX / therapeutic agent complex comprises one or more αPMTXs 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 αPMTX / therapeutic agent complex comprises one or more αPMTXs comprising 3 to 10, 3 to 9, 3 to 8, or 3 to 7 glutamyl groups. In other embodiments, the αPMTX / therapeutic agent complex comprises one or more αPMTXs comprising 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, or 4 to 5 glutamyl groups. In one embodiment, the complex comprises one or more αPMTXs comprising 3 to 10 glutamyl groups. In further embodiments, the αPMTX / therapeutic agent complex comprises one or more αPMTXs comprising 3 to 7 glutamyl groups. In another embodiment, the αPMTX / therapeutic agent complex comprises one or more αPMTXs comprising 5 glutamyl groups. In another embodiment, the αPMTX / therapeutic agent complex comprises one or more αPMTXs 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 αPMTX / therapeutic agent in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of αPMTX / therapeutic agent in the complex 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 some embodiments, the molar ratio of αPMTX / 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 some embodiments, the αPMTX / therapeutic agent complex is encapsulated in liposomes (e.g., in the manner described herein or by another method known in the art).

[0138] In alternative embodiments, the αPMTX complex comprises αPMTX and cyclodextrin. In some embodiments, the molar ratio of αPMTX (e.g., αPMTX salt) / cyclodextrin in the complex ranges from 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of αPMTX / cyclodextrin in the complex ranges from 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of αPMTX / cyclodextrin in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of αPMTX / 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 αPMTX / 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 αPMTX / cyclodextrin in the complex 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 αPMTX / cyclodextrin complex is encapsulated in liposomes (e.g., in the manner described herein or by another method known in the art).

[0139] In some embodiments, the present disclosure provides a composition comprising an αPMTX / 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 αPMTX / platinum-based chemotherapeutic agent complex comprises an analog of cisplatin, carboplatin, oxaliplatin, or salts or acids thereof. In some embodiments, the molar ratio of αPMTX / platinum-based agent in the complex ranges from 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of αPMTX / platinum-based agent in the complex ranges from 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of αPMTX / platinum-based agent in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of αPMTX / 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 αPMTX / platinum-based chemotherapeutic 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 αPMTX / platinum-based agent in the complex is 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 αPMTX / taxane-based agent complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).

[0140] In a further embodiment, the αPMTX / platinum-based chemotherapeutic agent complex comprises cisplatin, carboplatin, an oxaliplatin analog, or a salt or acid thereof. In some embodiments, the molar ratio of αPMTX / platinum-based analog in the complex ranges from 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of αPMTX / platinum-based analog in the complex ranges from 1 to 10:1, or any range therebetween. In a further embodiment, the molar ratio of αPMTX / platinum-based agent in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of αPMTX / 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 some embodiments, the molar ratio of αPMTX / platinum-based agent in the complex is 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 αPMTX / platinum-based analog complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).

[0141] In a further embodiment, the present disclosure provides a complex comprising αPMTX and cisplatin or a salt or acid thereof. In some embodiments, the molar ratio of αPMTX / 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 αPMTX / 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 αPMTX / 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 αPMTX / 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 some embodiments, the molar ratio of αPMTX / cisplatin (or a salt or acid of cisplatin) in the complex is 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 αPMTX / 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).

[0142] In another embodiment, the present disclosure provides a complex comprising αPMTX and carboplatin or a salt or acid thereof. In some embodiments, the molar ratio of αPMTX / carboplatin (or a salt or acid of carboplatin) in the complex ranges from 1 to 20:1, or any range therebetween. In further embodiments, the molar ratio of αPMTX / carboplatin (or a salt or acid of carboplatin) in the complex ranges from 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of αPMTX / 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 αPMTX / 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 - 50):1, or >50:1. In some embodiments, the molar ratio of αPMTX / cyclodextrin in the complex is 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 αPMTX / carboplatin (or a salt or acid of carboplatin) complex is encapsulated in liposomes (e.g., in the manner described herein or by another method known in the art).

[0143] In another embodiment, the present disclosure provides a complex comprising αPMTX and carboplatin or a salt or acid thereof. In some embodiments, the molar ratio of αPMTX / oxaliplatin (or a salt or acid of oxaliplatin) in the complex ranges from 1 to 20:1, or any range therebetween. In a further embodiment, the molar ratio of αPMTX / oxaliplatin (or a salt or acid of oxaliplatin) in the complex ranges from 1 to 10:1, or any range therebetween. In a further embodiment, the molar ratio of αPMTX / 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 αPMTX / 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 some embodiments, the molar ratio of αPMTX / oxaliplatin (or a salt or acid of oxaliplatin) in the complex is 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 αPMTX / oxaliplatin (or a salt or acid of oxaliplatin) complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).

[0144] In a further embodiment, the present disclosure provides a complex comprising αPMTX and a platinum-based chemotherapeutic agent ( "platinum") selected from the group consisting of nedaplatin, heptaplatin, lobaplatin, satraplatin, paraplatin, platino, 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 αPMTX / platinum-based chemotherapeutic agent complex comprises nedaplatin, heptaplatin, lobaplatin, satraplatin, paraplatin, platino, 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 αPMTX / platinum (or platinum salt or acid) in the complex ranges from 1 to 20:1, or any range therebetween. In a further embodiment, the molar ratio of αPMTX / platinum (or platinum salt or acid) in the complex ranges from 1 to 10:1, or any range therebetween. In a further embodiment, the molar ratio of αPMTX / platinum (or platinum salt or acid) in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of αPMTX / platinum (or platinum salt or acid) 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 some embodiments, the molar ratio of αPMTX / platinum (or a salt or acid of platinum) in the complex is 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 αPMTX / 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).

[0145] In some embodiments, the present disclosure provides a composition comprising an αPMTX / 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 αPMTX / taxane agent in the complex ranges from 1 to 20:1, or any range therebetween. In further embodiments, the molar ratio of αPMTX / taxane (or a salt or acid of the taxane) in the complex ranges from 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of αPMTX / taxane (or a salt or acid of the taxane) in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of αPMTX / taxane (or a salt or acid of the 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 some embodiments, the molar ratio of αPMTX / taxane (or a salt or acid of the taxane) in the complex is 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 αPMTX / taxane agent complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).

[0146] In further embodiments, the present disclosure provides a complex comprising αPMTX and paclitaxel (PTX) or a salt or acid thereof. In other embodiments, the αPMTX / taxane-based chemotherapeutic agent complex comprises an analogue of paclitaxel (PTX), or a salt or acid thereof. In some embodiments, the molar ratio of αPMTX / 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 αPMTX / 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 αPMTX / 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 αPMTX / 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 to 50):1, or >50:1. In some embodiments, the molar ratio of αPMTX / paclitaxel (or a salt or acid of paclitaxel) in the complex is 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 αPMTX / paclitaxel (or a salt or acid of paclitaxel) complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).

[0147] In a further embodiment, the present disclosure provides a complex comprising αPMTX and docetaxel (DTX) or a salt or acid thereof. In other embodiments, the αPMTX / taxane-based chemotherapeutic agent complex comprises an analogue of docetaxel (DTX), or a salt or acid thereof. In some embodiments, the molar ratio of αPMTX / 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 αPMTX / docetaxel (or a salt or acid of docetaxel) in the complex ranges from 1 to 10:1, or any range therebetween. In a further embodiment, the molar ratio of αPMTX / 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 αPMTX / 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 to 50):1, or >50:1. In some embodiments, the molar ratio of αPMTX / docetaxel (or a salt or acid of docetaxel) in the complex is 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 αPMTX / docetaxel (or a salt or acid of docetaxel) complex is encapsulated in liposomes (e.g., in the manner described herein or by another method known in the art).

[0148] In a further embodiment, the present disclosure provides a complex comprising αPMTX and larotaxel (LTX) or a salt or acid thereof. In other embodiments, the αPMTX / taxane-based chemotherapeutic agent complex comprises an analogue of larotaxel (LTX), or a salt or acid thereof. In some embodiments, the molar ratio of αPMTX / larotaxel (or a salt or acid of larotaxel) in the complex ranges from 1 to 20:1, or any range therebetween. In a further embodiment, the molar ratio of αPMTX / larotaxel (or a salt or acid of larotaxel) in the complex ranges from 1 to 10:1, or any range therebetween. In a further embodiment, the molar ratio of αPMTX / 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 ααPMTX / 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 to 50):1, or >50:1. In some embodiments, the molar ratio of αPMTX / larotaxel (or a salt or acid of larotaxel) in the complex is 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 αPMTX / 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).

[0149] In further embodiments, the present disclosure provides a complex comprising αPMTX and cabazitaxel (CTX) or a salt or acid thereof. In other embodiments, the αPMTX / taxane-based chemotherapeutic agent complex comprises an analogue of cabazitaxel (CTX), or a salt or acid thereof. In some embodiments, the molar ratio of αPMTX / 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 αPMTX / 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 αPMTX / 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 αPMTX / 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 some embodiments, the molar ratio of αPMTX / cabazitaxel (or a salt or acid of cabazitaxel) in the complex is 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 αPMTX / 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).

[0150] In a further embodiment, the present disclosure provides a complex comprising αPMTX 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 αPMTX and methotrexate (MTX), or a salt or acid thereof. In some embodiments, the present disclosure provides a complex comprising αPMTX and an antimetabolite selected from the group consisting of gemcitabine, fluorouracil, capecitabine, antifolates (e.g., methotrexate, methotrexate), tegafur, cytarabine, thioguanine, 5-azacitidine, 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 αPMTX / antimetabolite (or salt or acid of the antimetabolite) in the complex ranges from 1 to 20:1, or any range therebetween. In a further embodiment, the molar ratio of αPMTX / antimetabolite (or salt or acid of the antimetabolite) in the complex ranges from 1 to 10:1, or any range therebetween. In a further embodiment, the molar ratio of αPMTX / antimetabolite (or salt or acid of the antimetabolite) in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of αPMTX / antimetabolite (or salt or acid of the 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 to 50):1, or >50:1. In some embodiments, the molar ratio of αPMTX / antimetabolite (or salt or acid of the antimetabolite) in the complex is 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 αPMTX / antimetabolite (or a salt or acid of an antimetabolite) complex is encapsulated in liposomes (e.g., in the manner described herein or by another method known in the art).

[0151] In a further embodiment, the present disclosure provides a complex of αPMTX (e.g., αPMTX as 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.

[0152] 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", "undenivatized", 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 the cyclodextrin is said to be "complexed" with the cyclodextrin or to form a complex (inclusion complex) with the cyclodextrin.

[0153] As used herein, there are no special restrictions on the cyclodextrin component of the αPMTX / cyclodextrin complex as long as the cyclodextrin can form a complex with αPMTX. 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 αPMTX and / or liposomal encapsulation.

[0154] 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 a therapeutic agent complexed with cyclodextrin. In some embodiments, the cyclodextrin of the αPMTX / 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 a cyclodextrin having hydroxyl groups substituted with one or more of its charged moieties. Such moieties can be charged groups themselves or organic moieties substituted with one or more charged moieties (e.g., C 1 -C 6 alkyl or C 1 -C 6 alkyl ether moieties).

[0155] 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 to 9.0, 6.5 to 8.5, 7.0 to 8.0, 7.5 to 8.0, and any range therebetween (including the 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 to 7.0, 4.0 to 6.5, 4.5 to 6.5, 5.0 to 6.0, 5.0 to 5.5, and any range therebetween (including the 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.

[0156] 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.

[0157] 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 to 8.5, (b) the cationic charge being dominant at pH 4, and (c) the anionic charge being dominant at pH 8 to 8.5.

[0158] In some embodiments, the "ionizable" or "charged" derivatized cyclodextrins are generally polyionic, amphiphilic, or otherwise, weakly ionizable (i.e., 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 therebetween (including both ends)).

[0159] 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 chemistry, the reaction can be made to form a homogeneous product of pre-modified α-D-glucopyranoside units.

[0160] 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 a 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.

[0161] In one embodiment, at least one hydroxyl moiety oriented in the opposite direction from within the cyclodextrin is substituted by an ionizable chemical group. For example, at least one of the α-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 substituted with 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 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 in a similar manner. 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.

[0162] 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 substituted 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-β-cyclodextrin sodium salt, (2-hydroxypropyl)-alpha-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, (2-hydroxypropyl)-γ-cyclodextrin, 2,6-di-O-methyl)-β-cyclodextrin (DIMEB-50 heptakis), 2,3,6-tri-O-methyl)-β-cyclodextrin (TRIMEB heptakis), methyl-β-cyclodextrin, octakis(6-deoxy-6-iodo)-γ-cyclodextrin, and octakis(6-deoxy-6-bromo)-γ-cyclodextrin.

[0163] In some embodiments, the cyclodextrin has high solubility in water to facilitate capture of a greater amount 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.

[0164] In some embodiments, a large binding constant between cyclodextrin and αPMTX 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, e.g., 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.

[0165] In some embodiments, the cyclodextrin of the αPMTX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is non - derivatized.

[0166] In some embodiments, the cyclodextrin of the αPMTX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is derivatized. In further embodiments, the cyclodextrin derivative of the complex has the structure of Formula I:

Chemical formula

[0167] In some embodiments, the cyclodextrin derivatization of the αPMTX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is of formula II: [Chemical formula] having the structure of, where n is 4, 5, or 6; R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are each independently -O- or -O-(C 2 -C 6 alkylene)-SO 3 - group; at least one of R 1 and R 2 is independently -O-(C 2 -C 6 alkylene)-SO 3 - group; S 1 , S 2 , S 3 , S 4, S 5 , S 6 , S 7 , S 8 , and S 9 are each independently a pharmaceutically acceptable cation. In a further embodiment, the pharmaceutically acceptable cation is Li + , Na + , or K + such as an alkali metal; Ca 2+ , or Mg 2+ such as an alkaline earth metal and an ammonium ion and an amine cation such as a cation of (C1-C6)-alkylamine, piperidine, pyrazine, (C1-C6)-alkanolamine and (C4-C8)-cycloalkanolamine. In some embodiments, at least one of R1 and R2 is independently an -O-(CH2) m SO3- group, an -O-(C2-C6 alkylene)-SO3- group, where m is 2-6, preferably 2-4 (for example, -O-CH2CH2CH2S03- or -O-CH2CH2CH2CH2S03-); and S 1 , S 2 , S 3 , S 4 , S 5 , S 6 , S 7 , S 8 , and S 9 are each independently H or a pharmaceutically acceptable cation, which includes, for example, an alkali metal (e.g., Li + , Na + , K + ), an alkaline earth metal (e.g., Ca 2+ , Mg 2+ ), an ammonium ion and an amine cation such as a cation of (C1-C6)-alkylamine, piperidine, pyrazine, (C 1 -C 6 ), (C 4 -C 8 )-alkanolamine and (C

[0168] In some embodiments, the cyclodextrin derivatization of the αPMTX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is the cyclodextrin 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 preferentially.

[0169] In some embodiments, the cyclodextrin derivative of the αPMTX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is a sulfoalkyl ether cyclodextrin. 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 making sulfobutyl ether-3-cyclodextrin and other sulfoalkyl ether cyclodextrins are known in the art.

[0170] In some embodiments, the cyclodextrin derivative of the αPMTX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is of Formula III:

Chemical Formula

[0171] In a further embodiment, the αPMTX / 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).

[0172] III. αPMTX Delivery Carrier In an alternative embodiment, the present disclosure provides an αPMTX delivery system and their use for delivering the payload of αPMTX to cells (single or plural) in vitro or in vivo. In some embodiments, αPMTX 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-αPMTX 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.

[0173] A. Liposomes In some embodiments, the present disclosure provides a liposomal composition comprising liposomes encapsulating (filling) alpha-polyglutamylated methotrexate (e.g., αPMTX disclosed herein). In some embodiments, the liposomes in the liposomal composition comprise αPMTX comprising 4, 5, 2-10, 4-6, or more than 5 glutamyl groups (including the glutamyl group of methotrexate). In some embodiments, the alpha-polyglutamylated methotrexate in Lp-αPMTX comprises two or more L-type glutamyl groups. In other embodiments, the alpha-polyglutamylated methotrexate in Lp-αPMTX comprises a D-type glutamyl group. In further embodiments, the alpha-polyglutamylated methotrexate in Lp-αPMTX comprises a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, the alpha-polyglutamylated methotrexate in Lp-αPMTX comprises two or more glutamyl groups having a gamma-carboxyl bond. In some embodiments, the alpha-polyglutamylated methotrexate in Lp-αPMTX comprises at least one glutamyl group having both an alpha-carboxyl bond and a gamma-carboxyl bond. In some embodiments, the liposomal composition comprises liposomes comprising pentaglutamylated MTX. In further embodiments, the liposomes comprise L-α-pentaglutamylated MTX, D-α-pentaglutamylated MTX, or L- and D-α-pentaglutamylated MTX. In some embodiments, the liposomal composition comprises liposomes comprising hexaglutamylated MTX (Lp-αPMTX). In further embodiments, the liposomes comprise L-α-hexaglutamylated MTX, D-α-hexaglutamylated MTX, or L- and D-α-hexaglutamylated 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-αPMTX composition is not bevacizumabylated. In some embodiments, the Lp-αPMTX composition is not targeted (NTLp-αPMTX).In other embodiments, the Lp-αPMTX composition is targeted (TLp-αPMTX). 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 liposomal composition comprises liposomes having a diameter in the range of 20 nm to 400 nm, or any range therebetween. In some embodiments, the liposomal composition comprises liposomes having a diameter in the range of 20 nm to 300 nm, or any range therebetween. In some embodiments, the liposomal composition comprises liposomes having a diameter in the range of 20 nm to 200 nm, or any range therebetween. In further embodiments, the liposomal composition comprises liposomes having a diameter in the range of 20 nm to 150 nm, or any range therebetween. In further embodiments, the liposomal composition comprises liposomes having a diameter in the range of 80 nm to 120 nm, or any range therebetween. In further embodiments, 30 - 70%, 30 - 60%, or 30 - 50% w / w, or any range therebetween of alpha-polyglutamyl methotrexate is encapsulated (enclosed) in the Lp-αPMTX. In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or greater than 75% of alpha-polyglutamyl methotrexate is encapsulated in the Lp-αPMTX during the liposome preparation process.

[0174] In some embodiments, the liposomes provided further comprise an immunostimulant, a detectable marker, or both, disposed on the outer surface of the liposome. The immunostimulant or detectable marker can be ionically or covalently bound to the outer surface of the liposome, optionally including binding to the steric stabilizer component of the liposome.

[0175] 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 gamma, IFN alpha, FLT3 ligand; and immunostimulatory antibodies (e.g., anti-CTLA-4, anti-CD28, anti-CD3) and 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), erythran 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.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] In some embodiments, the liposomes contain a mitochondrial targeting agent. In some embodiments, the liposomes contain triphenylphosphonium (TPP). Methods and mechanisms for surface functionalization of liposomes using TPP are known in the art (e.g., binding TPP to a lipid anchor via a peg spacer group and modifying TPP with a stearyl group (stearyltriphenylphosphonium (STPP))). In some embodiments, the liposomes contain high density octaarginine. In some embodiments, the liposomes contain 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 liposomes contain rhodamine 123. In some embodiments, the liposomes contain a mitochondrial permeable peptide. In some embodiments, the liposomes contain 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).

[0180] In some embodiments, the liposomes in the provided liposomal composition comprise a mitochondrial permeabilizing agent selected from guanidine-rich peptides, tetraguanidium, triguanidium, diguanidium, monoguanidium, guanidine-rich polycarbamate, beta-oligoarginine, proline-rich dendrimer, and phosphonium salts (e.g., methyltriphenylphosphonium and / or tetraphenylphosphonium).

[0181] In some embodiments, the liposomes in the provided liposomal composition comprise sphingomyelin and / or stearyl octaarginine. In some embodiments, the liposomes comprise sphingomyelin and / or stearyl octaarginine. In some embodiments, the liposomes comprise DOPE, sphingomyelin, stearyl octaarginine sphingomyelin and stearyl octaarginine. In some embodiments, the liposomes comprise DOPE, sphingomyelin, stearyl octaarginine sphingomyelin and stearyl octaarginine in a molar ratio of 9:2:1. In some embodiments, the liposomes comprise a MITO porter system or a variant thereof.

[0182] 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.

[0183] In some embodiments, the liposome comprises a mitochondrial permeabilizer selected from the group consisting of guanidine-rich peptides, tetraguanidium, triguanidium, diguanidium, monoguanidium, guanidine-rich polycarbamate, beta-oligoarginine, proline-rich dendrimers, and phosphonium salts (e.g., methyltriphenylphosphonium and / or tetraphenylphosphonium).

[0184] In some embodiments, the liposome comprises sphingomyelin and / or stearyl octaarginine. In some embodiments, the liposome comprises sphingomyelin and / or stearyl octaarginine. In some embodiments, the liposome comprises DOPE, sphingomyelin, stearyl octaarginine sphingomyelin and stearyl octaarginine. In some embodiments, the liposome comprises DOPE, sphingomyelin, stearyl octaarginine sphingomyelin and stearyl octaarginine in a molar ratio of 9:2:1. In some embodiments, the liposome comprises a MITO porter system or a variant thereof.

[0185] In some embodiments, the liposomes include agents such as membrane permeabilizing agents that facilitate delivery of the liposomes across cell membranes 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 are routinely used and applicable to the manufacture and use of αPMTX compositions. In some embodiments, the membrane penetration agent / lysosome bypass agent is chloroquine. In some embodiments, the membrane permeabilizing agent is a cell-penetrating peptide. In some embodiments, the liposomes include a membrane permeabilizing agent selected from the group consisting of: 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 permeability fragment thereof.

[0186] In some embodiments, the liposomes in the provided liposome composition include a mitochondrial permeability agent selected from guanidine-rich peptoids, tetraguanidinium, triguanidinium, diguanidinium, monoguanidinium, guanidine-rich polycarbamates, beta-oligoarginine, proline-rich dendrimers, and phosphonium salts (e.g., methyltriphenylphosphonium and / or tetraphenylphosphonium).

[0187] In some embodiments, the liposomes in the provided liposome composition comprise sphingomyelin and / or stearyl octaarginine. In some embodiments, the liposomes comprise sphingomyelin and / or stearyl octaarginine. In some embodiments, the liposomes comprise DOPE, sphingomyelin, stearyl octaarginine sphingomyelin and stearyl octaarginine. In some embodiments, the liposomes comprise DOPE, sphingomyelin, stearyl octaarginine sphingomyelin and stearyl octaarginine in a molar ratio of 9:2:1. In some embodiments, the liposomes comprise the MITO-Porter® system or a variant thereof.

[0188] 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 are routinely used and applicable 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 liposomal 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 a cell permeable fragment thereof.

[0189] As discussed above, liposomes can include steric stabilizers that can extend their lifespan in circulation. For these embodiments incorporating steric stabilizers, 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 linear, branched, star or comb-shaped structures and are commercially available.

[0190] In some embodiments, the liposomal composition comprises pegylated liposomes (PLp-αPMTX). In some embodiments, the pegylated liposomes in the liposomal composition comprise αPMTX containing 4, 5, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the alpha-polyglutamylated methotrexate in Lp-αPMTX comprises two or more L-type glutamyl groups. In other embodiments, the alpha-polyglutamylated methotrexate in Lp-αPMTX comprises a D-type glutamyl group. In further embodiments, the alpha-polyglutamylated methotrexate in Lp-αPMTX comprises a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, the alpha-polyglutamylated methotrexate in Lp-αPMTX comprises two or more glutamyl groups having a gamma bond. In some embodiments, at least one glutamyl group has both an alpha bond and a gamma bond. In some embodiments, the liposomal composition comprises pegylated liposomes containing α-pentaglutamylated MTX. In further embodiments, the liposomes comprise L-α-pentaglutamylated MTX, D-α-pentaglutamylated MTX, or L- and D-α-pentaglutamylated MTX. In some embodiments, the liposomal composition comprises pegylated liposomes containing α-hexaglutamylated MTX. In further embodiments, the liposomes comprise L-α-hexaglutamylated MTX, D-α-hexaglutamylated MTX, or L- and D-α-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-αPMTX composition is not targeted (NTPLp-αPMTX). In other embodiments, the PLp-αPMTX composition is targeted (TPLp-αPMTX). In further embodiments, the liposomal composition comprises pegylated liposomes containing 30 to 70%, 30 to 60%, or 30 to 50%, or any range therebetween of liposome-encapsulated alpha-polyglutamylated methotrexate.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% (w / w) of alpha-polyglutamyl methotrexate is encapsulated (enclosed) in PLp-αPMTX. 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 200 nm. In a further embodiment, the liposomal composition comprises pegylated liposomes having a diameter in the range of 80 nm to 120 nm.

[0191] In some embodiments, more than 30%, 40%, 50%, 60%, 70%, 80% or 90% of the polyglutamyl methotrexate in the composition has 4 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, more than 30%, 40%, 50%, 60%, 70%, 80% or 90% of the polyglutamyl methotrexate in the provided liposomal composition is tetraglutamylated. In some embodiments, more than 30%, 40%, 50%, 60%, 70%, 80% or 90% of the polyglutamyl methotrexate in the provided liposomal composition is pentaglutamylated. In some embodiments, more than 30%, 40%, 50%, 60%, 70%, 80% or 90% of the polyglutamyl methotrexate in the provided liposomal composition is hexaglutamylated.

[0192] In some embodiments, the alpha-polyglutamyl methotrexate composition (for example, a delivery carrier such as a liposome containing alpha-polyglutamate and polyglutamate) is in an aqueous solution. In some embodiments, the αPMTX composition is in the form of a liposomal composition, 1 square meter (m 2) is administered at a dosage of about 0.005 to about 5000 mg of αPMTX per square meter of body surface area, or any range therebetween. In a further embodiment, the αPMTX composition is administered as a liposomal composition at a dosage of about 0.1 to about 1000 mg of αPMTX per square meter of body surface area, or any range therebetween.

[0193] (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 alpha polyglutamine oxidized 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 that 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 molecule (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".

[0194] The liposome 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 subjected to primary drying, for example, 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 saccharides (monosaccharides, disaccharides, and polysaccharides), polyhydric alcohols, and their derivatives, polyhydroxy compounds such as glycerol or polyethylene glycol, trehalose, maltose, sucrose, glucose, lactose, dextran, glycerol, or aminoglycoside. In further embodiments, the lyoprotectant or cryoprotective substance comprises up to 10% or up to 20% of the solution outside the liposome, inside the liposome, or both outside and inside the liposome.

[0195] 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 immediately 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 clearance 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, or comb-shaped structures, and are commercially available.

[0196] 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.

[0197] 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 methotrexate alphapolygultamate 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.

[0198] 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 interaction with the negative charges on the cell surface. After binding of the cationic liposomes to the cells, the liposomes are transported into the interior of the cells by endocytosis.

[0199] 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 potential to bind non-specifically 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.

[0200] As an example, at least one (or some) lipid is an amphiphilic lipid 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.

[0201] 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.

[0202] The lipids, including liposomes, provided herein can be anionic and neutral (including zwitterionic and polar) lipids, including anionic and neutral phospholipids. Neutral lipids are present in an uncharged or neutral zwitterionic form at a 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 modifying groups attached to neutral lipids.

[0203] 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.

[0204] Liposomes can be constructed using liposomal components (also referred to as liposome components) known in the art using any liposome assembly method. 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.

[0205] In some embodiments, the αPMTX compositions provided herein are formulated in liposomes comprising a cationic lipid. In one embodiment, but not limited to, 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. Patent 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 incorporated herein by reference in its entirety. In another embodiment, the cationic lipid can be selected from, but not limited to, Formula A described in International Publication Nos. WO 2012 / 040184, WO 2011 / 153120, WO 2011 / 1149733, WO 2011 / 090965, WO 2011 / 043913, WO 2011 / 022460, WO 2012 / 061259, WO 2012 / 054365, and WO 2012 / 044638. Each of these patent documents is incorporated herein by reference in its entirety. In yet another embodiment, the cationic lipid can be selected from, but not limited to, Formulas CLI - CLXXIX of International Publication No. WO 2008103276, Formulas CLI - CLXXIX of U.S. Patent No. 7,893,302, Formulas CLI - CLXXXXII of U.S. Patent No. 7,404,969, and Formulas I - VI of U.S. Patent Application Publication No. 20100036115. Each of these respective patent documents is incorporated herein 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-dimethyleptacos-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-undecyl-cyclopropyl]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-dimethyl-pentadecane-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)propane-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.,

[0206] In one embodiment, the lipid can be a cleavable lipid such as those described in International Publication No. WO 2012 / 170889, which is hereby incorporated by reference in its entirety.,

[0207] The cationic lipid can be synthesized routinely using methods known in the art and / or as described in International Publication Nos. 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.,

[0208] The lipid derivative can include, for example, at least one or more steric stabilizers and / or the binding (preferably covalent binding) 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 a group that can be used to bind the liposome component to another moiety such as a protein. Such functional groups include at least maleimide. These steric stabilizers include 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 at least one selected from the group consisting of polyvinyl alcohol.

[0209] In some embodiments, the αPMTX 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 US 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, αPMTX is formulated in a lipid-polycation complex, which further includes neutral lipids such as cholesterol or dioleoylphosphatidylethanolamine (DOPE), among others.

[0210] The components of the liposome can include any molecule that binds thereto (i.e., chemical / drug / reagent / protein), and in some embodiments, the components of the provided liposome include at least members selected from the group consisting of 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 preferred embodiments, the liposome components constituting the liposome include DSPE; DSPE-FITC; DSPE-maleimide; cholesterol; and HSPC.

[0211] In further embodiments, 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 further embodiments, the phospholipid is sn-2-oxygenated. In further embodiments, the phospholipid is not fragmented.

[0212] 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 a lipid produced by the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), thereby resulting 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 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.

[0213] In some embodiments, the liposomal alpha-polyglutamate oxidized methotrexate composition is pegylated (i.e., pegylated liposomal alpha-polyglutamate oxidized (e.g., pentaglutamate oxidized or hexaglutamate oxidized) folic acid antagonist (PLp-αPMTX or PLp-αPMTX)). In some embodiments, PLp-αPMTX or PLp-αPMTX is water-soluble. That is, PLp-αPMTX or PLp-αPMTX is in the form of an aqueous solution.

[0214] In some embodiments, the liposomes of the disclosed liposome composition comprise a lipid 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.

[0215] In some embodiments, the pH of the solution comprising the liposome composition is from 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, from 6.5 to 7.5, from 6.7 to 7.5, or from 6.3 to 7.0, or any range therebetween.

[0216] 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 group in the liposomal lipid bilayer is 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).

[0217] 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.

[0218] Functionalized and non-functionalized lipids are available from many commercial sources such as Avanti Polar Lipids (Alabaster, AL) and Lipoid LLC (Newark, NJ).

[0219] (2) Liposomal interior space In further non-limiting embodiments, the provided liposomes contain an internal space. In some embodiments, the internal space contains, without limitation, an aqueous solution. In some embodiments, the internal space contains the alpha polyglutamine 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 yet 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.

[0220] In some embodiments, the internal space of the liposome contains trehalose. In further embodiments, the concentration (wt...

Claims

1. 1. A liposome composition comprising liposomes encapsulating alpha polyglutamated methotrexate, the alpha polyglutamated methotrexate contains 2 to 10 glutamyl groups with alpha carboxyl linkages; A liposome composition, wherein the liposome is PEGylated and contains a targeting moiety having specific affinity for a surface antigen on a target cell; and the liposome further encapsulates one or more non-polyglutamylatable or non-polyglutamylatable antifolates.

2. 2. The liposome composition of claim 1, wherein the alpha polyglutamylated methotrexate contains 4 to 6 glutamyl groups with alpha carboxyl group linkages.

3. 2. The liposomal composition of claim 1, wherein the non-polyglutamylatable antifolate is selected from the group consisting of methotrexate (MTX), pemetrexed (PMX), lometrexol (LMX), raltitrexed (RTX), pralatrexate, AG2034, GW1843, aminopterin, and LY309887; or the non-polyglutamylatable antifolate is selected from the group consisting of trimetrexate (TMQ), piritrexim (BW301U), tarotrexin (PT523), nolatrexed (AG337), previtrexed (ZD9331, BGC9331), and BGC945 (ONX0801).

4. 2. The liposome composition of claim 1 comprising alpha tetraglutamated methotrexate, alpha pentaglutamated methotrexate or alpha hexaglutamated methotrexate.

5. (a) two or more glutamyl groups of the alpha polyglutamated methotrexate have an alpha carboxyl group linkage; (b) each of the glutamyl groups of the alpha polyglutamated methotrexate has an alpha carboxyl group linkage; (c) two or more glutamyl groups of the alpha polyglutamated methotrexate have a gamma carboxyl group linkage; (d) at least one of the glutamyl groups of the alpha polyglutamated methotrexate has both an alpha carboxyl group linkage and a gamma carboxyl group linkage; (e) at least two of the glutamyl groups of the alpha polyglutamated methotrexate are in the L-form; (f) each of the glutamyl groups of the alpha polyglutamated methotrexate is in the L-form; (g) at least one of the glutamyl groups of the alpha polyglutamated methotrexate is in the D form; (h) each of the glutamyl groups of the alpha polyglutamylated methotrexate other than the glutamyl groups of methotrexate is in the D form; or (i) at least two of the glutamyl groups of the alpha polyglutamated methotrexate are in the L-form and at least one of the glutamyl groups is in the D-form; The liposome composition of claim 1.

6. 2. The liposome composition of claim 1, wherein the liposome comprises an alpha polyglutamated methotrexate, including alpha tetraglutamated methotrexate, alpha pentaglutamated methotrexate, or alpha hexaglutamated methotrexate.

7. The liposome composition of claim 1 , wherein the polyglutamate is linear or branched.

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

9. 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.

10. 2. The liposome composition of claim 1, wherein the targeting moiety is one or more selected from the group consisting of a polypeptide or 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.

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

12. 2. The liposome composition of claim 1, wherein the liposomes are formed from liposome components, the liposome components comprising at least one of anionic lipids and neutral lipids, and at least one selected from the group consisting of DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholesterol; cholesterol-PEG; and cholesterol-maleimide, The liposome component may comprise at least one selected from the group consisting of DSPE; DSPE-PEG; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC; One or more of the liposome components may further comprise a steric stabilizer; The steric stabilizer may be 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, and the steric stabilizer may be PEG and the PEG may have a number average molecular weight (Mn) of 200 to 5000 Daltons. Liposomal compositions.

13. 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.

14. 2. The liposome composition of claim 1, wherein the liposome has an interior space containing the alpha polyglutamated methotrexate and an aqueous pharma- ceutically acceptable carrier, The pharma- ceutically acceptable carrier may include isotonicity agents such as dextrose, mannitol, glycerol, potassium chloride, sodium chloride at concentrations greater than 1%, 1%-50% trehalose, 1%-50% dextrose, 5% dextrose suspended in HEPES buffer, or sodium acetate and calcium acetate at a total concentration of 50 mM-500 mM; The interior space of the liposome may have a pH of 5 to 8 or a pH of 6 to 7, or any range therebetween; The liposome may contain less than 500,000 or less than 200,000 alpha polyglutamated methotrexate molecules or between 10 and 100,000 or any range therebetween. Liposomal compositions.

15. 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 the PEG or 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, resolvin (e.g., DNP, ｎ－６ＤＰＡ Or D ｎ－３ＤＰＡ 2. The liposome composition of claim 1, wherein the liposome is at least one selected from the group consisting of resolvin D, resolvin E, or T series resolvins such as erythropoietin (E5564), oxidized low density lipoprotein (E5564), and a toll-like receptor (TLR) modulator such as erythropoietin lipid (E5564), may further comprise at least one cryoprotectant selected from the group consisting of mannitol, trehalose, sorbitol, and sucrose, or may further comprise carboplatin and / or pembrolizumab; Liposomal compositions.

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

17. A liposome composition according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 16 for use in the treatment of a disease, comprising The disease may be cancer, a disorder of the immune system, or an infectious disease, and the disease may be an autoimmune disease, rheumatoid arthritis, or an inflammatory condition. The liposome composition according to any one of claims 1 to 12 or the pharmaceutical composition according to claim 13.

18. 16. A method of making an alpha polyglutamated methotrexate composition comprising the liposome composition of any one of claims 1-15, comprising forming a mixture in solution comprising liposome components and alpha polyglutamated methotrexate; homogenizing the mixture in solution to form liposomes; and treating the mixture to form liposomes comprising alpha polyglutamated methotrexate. The method, wherein the processing steps may include 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.

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