Alpha-polyglutamate tetrahydrofolate and its use

Abstract

To provide methods for improving the effect of tetrahydrofolate in treating disease and polyglutamated alpha tetrahydrofolate compositions with improved therapeutic effect.SOLUTION: Disclosed is a liposome composition comprising liposomes encapsulating alpha polyglutamated tetrahydrofolate, where at least one glutamyl group of the alpha polyglutamated tetrahydrofolate has two or more alpha carboxyl group linkage.SELECTED DRAWING: None

Description

Background Art

[0001] The present disclosure generally relates to compositions of polyglutamylated alpha-tetrahydrofolate, delivery vehicles such as liposomes containing such compositions, and methods of manufacturing and using such 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. Polyglutamylated alpha-tetrahydrofolate compositions have utility in combination therapy with one or more therapeutic agents, such as chemotherapeutic agents (e.g., 5-fluorouracil), to enhance the effect of the therapeutic agent(s), or as “chemoprotective agents” to reduce toxic side effects associated with the therapeutic agent(s) (e.g., in combination with folate antagonists such as methotrexate).

[0002] 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 monoglutamate, and monoglutamate folates are the only form of folic acid that is transported across cell membranes, and monoglutamate forms of tetrahydrofolate are similarly transported across cell membranes. Once taken up into cells, intracellular tetrahydrofolate is polyglutamylated by the enzyme folylpolyglutamate synthetase (FPGS). Tetrahydrofolate polyglutamylation by FPGS serves at least two major therapeutic purposes: (1) significantly increasing the affinity of tetrahydrofolate for DHFR; and (2) facilitating the accumulation of polyglutamylated tetrahydrofolate, which, unlike tetrahydrofolate (monoglutamate), is not readily transported out of cells by cellular efflux pumps.

[0003] The provided polyglutamylated alpha-tetrahydrofolate compositions provide a strategy for improving the therapeutic effects of tetrahydrofolate.

Summary of the Invention

[0004] This disclosure generally relates to polyglutamate-oxidized alpha-tetrahydrofolate (THF) compositions, and methods for manufacturing and using these compositions to treat diseases including hyperproliferative diseases such as cancer, inflammation and immune system disorders such as rheumatoid arthritis, and infectious diseases such as HIV and malaria. Polyglutamate-oxidized alpha-tetrahydrofolate compositions also have uses in combination therapy with one or more therapeutic agents, such as chemotherapeutic agents (e.g., 5-fluorouracil), to enhance the effect of one or more therapeutic agents, or as "chemoprotective agents" to mitigate toxic side effects associated with one or more therapeutic agents (e.g., in combination with folate antimetabolites such as methotrexate).

[0005] In some embodiments, this disclosure provides: [1] A composition comprising polyglutamate alphatetrahydrofolate. [2] Polyglutamate alpha-tetrahydrofolate, (a) Polyglutamic acid 5-formyl-THF (e.g., polyglutamic acid [6S]-5-formyl-THF); (b) Polyglutamic acid 10-formyl-THF (e.g., polyglutamic acid [6R]-10-formyl-THF); (c) Polyglutamic acid 5,10-methenyl-THF (e.g., polyglutamic acid [6R]-5,10-methenyl-THF); (d) Polyglutamine oxidized 5-methyl-THF (e.g., polyglutamine oxidized [6S]-5-methyl-THF); (e) Polyglutamic acid tetrahydrofolate (e.g., polyglutamic acid [6S]-tetrahydrofolate THF); (f) Polyglutamic acid 5,10-methylene-THF (e.g., polyglutamic acid [6R]-5,10-methylene-THF); and (g) Polyglutamine oxidized 5-formimino-THF (e.g., polyglutamine oxidized [6S]-5-formimino-THF) A composition selected from the group consisting of the following, as described in item [1]. [3] The composition according to item [1] or [2], wherein the polyglutamate-oxidized alphatetrahydrofolate contains 4, 5, 2 to 10, 4 to 6, or 6 or more glutamyl groups having alpha carboxyl group bonds. [4] The composition according to any one of items [1] to [3], wherein the polyglutamate-oxidized alphatetrahydrofolate is tetraglutamate-oxidized alphatetrahydrofolate (e.g., [6R]-5,10-methenyl-THF, [6S]-5-formyl-THF, and / or [6R]-10-formyl-THF). [5] The composition according to any one of items [1] to [3], wherein the polyglutamate-oxidized alphatetrahydrofolate is pentaglutamate-oxidized alphatetrahydrofolate (e.g., [6R]-5,10-methenyl-THF, [6S]-5-formyl-THF, and / or [6R]-10-formyl-THF). [6] The composition according to any of items [1] to [3], wherein the polyglutamate-oxidized alphatetrahydrofolate is hexaglutamate-oxidized alphatetrahydrofolate (e.g., [6R]-5,10-methenyl-THF, [6S]-5-formyl-THF, and / or [6R]-10-formyl-THF). [7](a) Two or more glutamyl groups having alpha-carboxyl group bonds, (b) Each glutamyl group of tetrahydrofolate other than the glutamyl group has an alpha-carboxyl group bond; or (c) Two or more glutamyl groups having gammacarboxyl group bonds, A composition as described in any of items [1] to [6]. [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](a) At least two of the glutamyl groups of polyglutamate-oxidized alphatetrahydrofolate are L-type, (b) The glutamyl groups of the polyglutamate-oxidized alphatetrahydrofolate are each in the L-type. (c) At least one of the glutamyl groups of polyglutamate-oxidized alphatetrahydrofolate is of the D type. (d) The glutamyl groups of the polyglutamate-oxidized alphatetrahydrofolate, other than the glutamyl group of tetrahydrofolate, are each of the D type, or (e) At least two of the glutamyl groups of polyglutamate-oxidized alphatetrahydrofolate are L-type, and at least one of the glutamyl groups is D-type. A composition as described in any of items [1] to [8].

[10] A composition according to any of items [1] to [9], wherein the polyglutamic acid is linear.

[11] The composition according to any of items [1] to [9], wherein the polyglutamic acid is a branched chain.

[12] A liposome composition (Lp-αPTHF) containing polyglutamic oxidized alpha-tetrahydrofolate as described in any of items [1] to

[11] ;

[13] The LαPP composition according to item

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

[14] The Lp-αPTHF composition according to item

[12] or

[13] , wherein the glutamyl groups of the polyglutamate-oxidized alphatetrahydrofolate are each in the L form;

[15] The Lp-αPTHF composition according to item

[12] or

[13] , wherein at least one of the glutamyl groups of polyglutamate-oxidized alphatetrahydrofolate is of type D;

[16] Lp-αPTHF compositions according to any of items

[12] to

[15] , wherein the liposomes contain polyglutamic oxidized alphatetrahydrofolate having 4, 5, 2 to 10, 4 to 6, or 6 or more glutamyl groups;

[17] An Lp-αPTHF composition according to any of items

[12] to

[16] , wherein at least one of the glutamyl groups of polyglutamate-oxidized alphatetrahydrofolate has a gammacarboxyl group bond;

[18] A 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] A composition according to any of items

[12] to

[18] , comprising two, three, four, five, two to ten, four to six, or six or more glutamyl groups having both alpha-carboxyl and gamma-carboxyl group bonds;

[20] Lp-αPTHF compositions according to any of items

[12] to

[19] , wherein the liposomes contain polyglutamate-oxidized alphatetrahydrofolate, including tetraglutamate-oxidized alphatetrahydrofolate, pentaglutamate-oxidized alphatetrahydrofolate, or hexaglutamate-oxidized alphatetrahydrofolate;

[21] Lp-αPTHF compositions according to any of items

[12] to

[19] , wherein the liposomes contain polyglutamate-oxidized alphatetrahydrofolate, including tetraglutamate-oxidized alphatetrahydrofolate, pentaglutamate-oxidized alphatetrahydrofolate, or hexaglutamate-oxidized alphatetrahydrofolate;

[22] Lp-αPTHF compositions according to any of items

[12] to

[21] , wherein the polyglutamic acid is linear or branched;

[23] Lp-αPTHF compositions according to any of items

[12] to

[22] , in which the liposomes are pegylated (PαLp-αPTHF);

[24] Lp-αPTHF compositions according to any of items

[12] to

[23] , wherein the liposomes contain at least 1% by weight (w / w) of polyglutamate-oxidized alpha-tetrahydrofolate, or in the step of preparing Lp-αPTHF, at least 1% of the starting material of polyglutamate-oxidized alpha-THF is encapsulated in αPTHF;

[25] Lp-αPTHF compositions according to any of items

[12] to

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

[26] Lp-αPTHF composition according to any of items

[12] to

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

[27] Lp-αPTHF compositions according to any of items

[12] to

[26] , wherein liposomes are formed from liposomal components;

[28] The Lp-αPTHF composition according to item

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

[29] The Lp-αPTHF composition according to item

[27] or

[28] , wherein the liposome component comprises 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] Lp-αPTHF compositions according to any of items

[27] to

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

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

[27] to

[30] , further comprising one or more liposome components and a steric stabilizer;

[32] The Lp-αPTHF 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; oligoglycerin, polyethylene glycol and polypropylene oxide-containing copolymer, poloxamer 188, and polyvinyl alcohol;

[33] The Lp-αPTHF composition according to item

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

[34] Lp-αPTHF compositions according to any of items

[12] to

[33] , wherein the liposomes are anionic or neutral;

[35] Lp-αPTHF compositions according to any of items

[12] to

[33] , wherein liposomes have a zeta potential of zero or less;

[36] Lp-αPTHF compositions according to any of items

[12] to

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

[37] Lp-αPTHF compositions according to any of items

[12] to

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

[38] Lp-αPTHF compositions according to any of items

[12] to

[33] , wherein the liposomes are cationic;

[39] Lp-αPTHF compositions according to any of items

[12] to

[38] , wherein the liposomes have an internal space comprising polyglutamate-oxidized alphatetrahydrofolate and an aqueous pharmaceutically acceptable carrier;

[40] The Lp-αPTHF composition described in item

[39] , wherein the pharmaceutically acceptable carrier contains an isotonic agent such as dextrose, mannitol, glycerol, potassium chloride, or sodium chloride in a concentration of more than 1%;

[41] The Lp-αPTHF composition described in item

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

[42] The Lp-αPTHF composition described in item

[41] , comprising 5% to 20% by weight of trehalose as a pharmaceutically acceptable carrier;

[43] Lp-αPTHF compositions according to any of items

[39] to

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

[44] An Lp-αPTHF composition according to any of items

[39] to

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

[45] Lp-αPTHF compositions according to any of items

[39] to

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

[46] Lp-αPTHF compositions according to any of items

[39] to

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

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

[12] to

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

[48] ​​Lp-αPTHF compositions according to any of items

[12] to

[47] , wherein the liposomes contain less than 500,000 molecules or less than 200,000 molecules of polyglutamate-oxidized alphatetrahydrofolate;

[49] Lp-αPTHF compositions according to any of items

[12] to

[48] , wherein the liposomes contain 10 to 100,000 molecules or any range in between, of polyglutamic oxidized alphatetrahydrofolate molecules;

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

[12] to

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

[51] The Lp-αPTHF composition according to item

[50] , wherein the targeting portion is bound to either or both the PEG and / or outer layer of the liposome, and optionally, the targeting portion is covalently bound to either or both the PEG and / or outer layer of the liposome;

[52] The Lp-αPTHF composition according to item

[50] or

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

[53] Lp-αPTHF compositions according to any of items

[50] to

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

[54] The targeting portion was 0.5 × 10 in measurements using BIACORE® analysis. -10 ~10×10 -6 Lp-αPTHF compositions described in any of items

[50] to

[53] that bind to surface antigens with an equilibrium dissociation constant (Kd) in the range of

[50] to

[53] ;

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

[50] to

[55] , wherein the targeting portion 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-αPTHF composition according to any of items

[50] to

[56] , wherein the targeting portion comprises one or more selected from the group consisting of antibodies, humanized antibodies, antigen-binding fragments of antibodies, single-chain antibodies, single-domain antibodies, bispecific antibodies, synthetic antibodies, pegylated antibodies, and multimeric antibodies;

[57] Lp-αPTHF compositions according to any of items

[50] to

[56] , wherein each pegylated liposome contains 1 to 1,000 or 30 to 200 targeting moieties;

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

[39] to

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

[59] The Lp-αPTHF composition according to item

[58] , wherein the immunostimulant is at least one selected from the group consisting of protein immunostimulants, nucleic acid immunostimulants, chemoimmunostimulants, haptens, and adjuvants;

[60] Immunostimulants include fluorescein, fluorescein isothiocyanate (FITC), DNP, beta-glucan, beta-1,3-glucan, beta-1,6-glucan; resolvin (e.g., D n-6DPA Or D n-3DPA An Lp-αPTHF composition as described in item

[58] or

[59] , which is at least one selected from the group consisting of resolvin D, resolvin E, or T-series resolvins, and Toll-like receptor (TLR) modulators such as oxidized low-density lipoproteins (e.g., OXPAC, PGPC) and erythritol lipids (e.g., E5564);

[61] Lp-αPTHF compositions according to any of items

[58] to

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

[62] Lp-αPTHF compositions according to any of items

[58] to

[61] , further comprising a hapten;

[63] The Lp-αPTHF composition according to item

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

[64] An Lp-αPTHF composition according to any of items

[12] to

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

[65] Targeted compositions comprising any of the compositions described in items [1] to

[64] ;

[66] Non-targeting compositions comprising any of the compositions described in items [1] to

[49] ;

[67] Lp-αPTHF compositions according to any of items

[12] to

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

[68] A pharmaceutical composition comprising the liposomal polyglutamate alphatetrahydrofolate composition described in any of items

[12] to

[67] ;

[69] A pharmaceutical composition comprising polyglutamate alphatetrahydrofolic acid as described in any of items [1] to [7];

[70] Compositions described in any of items [1] to

[69] , used for the treatment of a disease;

[71] Use of any of the compositions described in items [1] to

[70] for the manufacture of drugs for use in combination with one or more therapeutic agents, such as chemotherapeutic agents (e.g., 5-fluorouracil), for the treatment of a disease and / or to enhance the effect of one or more therapeutic agents, or as a “chemoprotective agent” to reduce toxic side effects associated with one or more therapeutic agents (e.g., in combination with folate antagonists such as methotrexate);

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

[70] ;

[73] A method for treating or preventing a disease of a subject requiring treatment or prevention, comprising administering to the subject a liposomal polyglutamate alphatetrahydrofolate composition described in any of items

[12] to

[69] ;

[74] A method for killing overgrown cells, comprising contacting the overgrown cells with a composition described in any of items [1] to

[69] ;

[75] A method for killing hyperproliferating cells, comprising contacting the hyperproliferating cells with a liposomal polyglutamate-oxidized alpha-tetrahydrofolate composition described in any of items

[12] to

[69] ;

[76] The method according to item

[74] or

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

[77] A method for treating cancer, comprising administering an effective amount of any of the compositions described in items [1] to

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

[78] A method for treating cancer, comprising administering an effective amount of a liposomal polyglutamate alphatetrahydrofolate composition described in any of items

[12] to

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

[79] The method according to item

[77] or

[78] , wherein cancer is selected from the group consisting of non-hematological malignancies, such as lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, stomach cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcomas (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological malignancies, such as leukemia, lymphoma and other B-cell malignancies, myeloma and other plasma cell proliferation disorders;

[80] The method according to item

[77] or

[78] , which treats or prevents cancer, 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 described in item

[77] or

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

[83] A method for treating cancer, comprising administering an effective amount of an Lp-αPTHF composition described in any of items

[50] to

[66] to a subject having or at risk of having cancer cells that express folate receptors on their surface to which a targeting moiety binds;

[84] Maintenance therapy for subjects who are currently receiving or have previously received cancer treatment, comprising administering to subjects who are currently receiving or have previously received cancer treatment an effective amount of any of the compositions described in items [1] to

[69] ;

[85] Maintenance therapy for subjects who are currently receiving or have previously received cancer treatment, comprising administering to subjects who are currently receiving or have previously received cancer treatment an effective amount of a liposomal polyglutamate oxidized alpha-tetrahydrofolate composition described in any of items

[12] to

[69] ;

[86] A method for treating an immune system disorder, comprising administering an effective amount of any of the compositions described in items [1] to

[69] to a subject having or at risk of having an immune system disorder, wherein the immune system disorder is optionally 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 arteritis, and psoriasis;

[87] A method for treating an immune system disorder, comprising administering an effective amount of a liposomal polyglutamate tetrahydrofolate composition described in any of items [8] to

[69] to a subject having or at risk of having an immune system disorder, wherein the immune system disorder is optionally 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 arteriosclerosis, and psoriasis;

[88] The following treatment methods: (a) A method for treating leukopenia, comprising administering an effective dose of any of the compositions described in items [1] to

[69] to subjects who have or are at risk of having leukopenia; (b) A method for treating an infection, comprising administering an effective dose of any of the compositions described in items [1] to

[69] to a subject who has or is at risk of having an infection; (c) A method for treating a cardiovascular or metabolic disease, comprising administering an effective amount of any of the compositions described in items [1] to

[69] to a subject who has or is at risk of having an infection, 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; (d) A method for treating an autoimmune disease, comprising administering an effective amount of any of the compositions described in items [1] to

[69] to a subject who has or is at risk of having an autoimmune disease; (e) A method for treating rheumatoid arthritis, comprising administering an effective dose of any of the compositions described in items [1] to

[69] to subjects who have or are at risk of having rheumatoid arthritis; (f) A method for treating an inflammatory condition, comprising administering an effective amount of any of the compositions described in items [1] to

[69] to a subject who has or is at risk of having inflammation, wherein the inflammation is acute, chronic, and / or systemic inflammation; or (g) A method for treating a skin disease, comprising administering an effective amount of any of the compositions described in items [1] to

[69] to a subject who has or is at risk of having a skin disease;

[89] A method for treating an infection, comprising administering an effective amount of a liposomal polyglutamate-oxidized alphatetrahydrofolate composition described in any of items

[12] to

[69] to a subject who has or is at risk of having an infection;

[90] A method for delivering polyglutamic oxyalpha-tetrahydrofolate to a tumor expressing folate receptors on its surface, comprising administering to a subject having a tumor an amount of the Lp-αPTHF composition described in any of items [1] to

[69] , in an amount that delivers a therapeutically effective amount of polyglutamic oxyalpha-tetrahydrofolate to the tumor;

[91] A method for preparing a polyglutamate alpha-tetrahydrofolate composition comprising a liposome polyglutamate alpha-tetrahydrofolate composition described in any of items

[12] to

[69] , comprising: forming a mixture in solution containing a liposome component and a polyglutamate alpha-folate antimetabolite; homogenizing the mixture in solution to form liposomes; and processing the mixture to form liposomes containing polyglutamate alpha-tetrahydrofolate;

[92] A method for preparing any of the compositions described in items

[12] to

[69] , comprising: forming a mixture in solution containing a liposome component and polyglutamate-oxidized alpha-tetrahydrofolate; homogenizing the mixture in solution to form liposomes; processing the mixture to form liposomes in which polyglutamate-oxidized alpha-tetrahydrofolate is encapsulated and / or contained; and conferring a targeting moiety to 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 steps include one or more steps from thin film hydration, extrusion, in-line mixing, ethanol injection techniques, freeze-thaw methods, reverse-phase evaporation methods, dynamic high-pressure microfluidization, microfluidic mixing, double emulsion, freeze-dried double emulsion, 3D printing, membrane contactor methods, and stirring; and / or

[94] The method according to item

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

[0006] In some embodiments, the disclosure provides polyglutamate-oxidized alpha-tetrahydrofolate (αPTHF) compositions in which at least two of the glutamyl residues of the polyglutamate-oxidized alpha-tetrahydrofolate have alpha-carboxyl group bonds. In some embodiments, the αPTHF contains 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 6 or more glutamyl groups (including the glutamyl groups in tetrahydrofolate). In some embodiments, the αPTHF contains two or more L-type glutamyl groups. In other embodiments, the αPTHF contains D-type glutamyl groups. In further embodiments, the αPTHF contains a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, the αPTHF contains two or more glutamyl groups having alpha bonds. In some embodiments, at least one glutamyl group has both alpha and gamma bonds.

[0007] In one embodiment, the αPTHF composition comprises a chain consisting of three glutamyl groups bonded to a glutamyl group of tetrahydrofolate (i.e., tetraglutamate-oxidized tetrahydrofolate). In some embodiments, the tetraglutamate-oxidized THF comprises two or more L-type glutamyl groups. In other embodiments, the tetraglutamate-oxidized THF comprises a D-type glutamyl group. In further embodiments, the tetraglutamate-oxidized THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In even further embodiments, the tetraglutamate-oxidized THF comprises two or more glutamyl groups having a gamma bond.

[0008] In one embodiment, the αPTHF composition comprises a chain consisting of four glutamyl groups bonded to a gamma-glutamyl group of tetrahydrofolate (e.g., α-pentaglutamine-oxidized tetrahydrofolate). In some embodiments, pentaglutamine-oxidized alpha-THF comprises two or more L-type glutamyl groups. In other embodiments, pentaglutamine-oxidized alpha-THF comprises a D-type glutamyl group. In further embodiments, pentaglutamine-oxidized alpha-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In even further embodiments, pentaglutamine-oxidized THF comprises two or more glutamyl groups having gamma bonds.

[0009] In one embodiment, the αPTHF composition comprises a chain consisting of five glutamyl groups bonded to a gamma-glutamyl group of tetrahydrofolate (e.g., α-hexaglutamate-oxidized tetrahydrofolate). In some embodiments, the hexaglutamate-oxidized alpha-THF comprises two or more L-type glutamyl groups. In other embodiments, the hexaglutamate-oxidized alpha-THF comprises a D-type glutamyl group. In further embodiments, the hexaglutamate-oxidized alpha-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In even further embodiments, the hexaglutamate-oxidized THF comprises two or more glutamyl groups having gamma bonds.

[0010] In further embodiments, the Disclosure provides compositions comprising a delivery medium such as liposomes in which polyglutamate-oxidized alpha-tetrahydrofolate is filled (e.g., encapsulated) and / or otherwise associated, as well as methods for producing and using αPTHF-filled / associated delivery medium compositions (DV-αPTHF) for delivering polyglutamate-oxidized alpha-tetrahydrofolate to diseased (e.g., cancerous) and / or target cells. These compositions have applications including, but are not particularly limited, treating (e.g., treating or preventing) diseases including, for example, hyperproliferative diseases such as cancer, immune system disorders such as inflammation and rheumatoid arthritis, and infectious diseases such as HIV and malaria. In some embodiments, the polyglutamate-oxidized alpha-tetrahydrofolate in DV-αPTHF contains 2 to 20, 2 to 15, 2 to 10, 2 to 5, 6 or more, or 21 or more glutamyl groups (including glutamyl groups in tetrahydrofolate). The DV-αPTHF-filled / accompanied delivery medium composition improves the efficacy and safety of delivering tetrahydrofolate (THF) to cancer cells by preferentially delivering a payload (e.g., polyglutamate-oxidized tetrahydrofolate) that is more cytotoxic than tetrahydrofolate (THF) administered in monoglutamate form.

[0011] In some embodiments, the Disclosure provides for the use of compositions comprising a delivery medium, such as liposomes, in which polyglutamate-oxidized alpha-tetrahydrofolate is filled (e.g., encapsulated) and / or otherwise associated, in combination therapy with one or more therapeutic agents, such as chemotherapeutic agents (e.g., 5-fluorouracil), to enhance the effect of one or more therapeutic agents, or as a “chemoprotective agent” to mitigate toxic side effects associated with one or more therapeutic agents (e.g., in combination with folate antimetabolites such as methotrexate). In some embodiments, the polyglutamate-oxidized alpha-tetrahydrofolate in DV-αPTHF contains 2 to 20, 2 to 15, 2 to 10, 2 to 5, 6 or more, or 21 or more glutamyl groups (including glutamyl groups in tetrahydrofolate). The DV-αPTHF-filled / accompanied delivery medium composition improves the efficacy and safety of delivering tetrahydrofolate (THF) to cancer cells by preferentially delivering a payload (e.g., polyglutamate-oxidized tetrahydrofolate) that is more cytotoxic than tetrahydrofolate (THF) administered in monoglutamate form.

[0012] In further embodiments, the disclosure provides compositions comprising polyglutamate-oxidized alpha-tetrahydrofolate (αPTHF).

[0013] In some embodiments, the disclosure provides compositions comprising polyglutamate alpha-5-formyl-THF. In some embodiments, polyglutamate alpha-5-formyl-THF is obtained by polyglutamate oxidizing polyglutamate alpha[6S]-5-formyl-THF. In some embodiments, the composition comprises polyglutamate alpha[6R,S]-5-formyl-THF. In some embodiments, the composition comprises polyglutamate alpha[6R]-5-formyl-THF. In some embodiments, the composition comprises polyglutamate alpha-5-formyl-THF having 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 21 or more glutamyl groups (including the glutamyl groups in 5-formyl-THF). In some embodiments, polyglutamate alpha-5-formyl-THF contains 1, 2, 3, or 4 or more glutamyl groups having alpha bonds. In some embodiments, the composition comprises polyglutamate alpha-5-formyl-THF having two or more L-type glutamyl groups. In other embodiments, the composition comprises polyglutamate alpha-5-formyl-THF having D-type glutamyl groups. In further embodiments, the composition comprises polyglutamate alpha-5-formyl-THF having a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, the polyglutamate alpha-tetrahydrofolate in Lp-αPTHF comprises two or more glutamyl groups having alpha bonds. In further embodiments, the polyglutamate alpha-tetrahydrofolate in Lp-αPTHF comprises one or more glutamyl groups having both alpha and gamma bonds. In some embodiments, the polyglutamate alpha-tetrahydrofolate in Lp-αPTHF comprises 2 to 10, or any number in between, glutamyl groups having both alpha and gamma bonds. In some embodiments, the polyglutamate chain of polyglutamate alpha-5-formyl-THF is linear. In some embodiments, the polyglutamate chain of polyglutamate alpha-5-formyl-THF is branched.

[0014] In some embodiments, the Disclosure provides compositions comprising polyglutamate alpha-5-formyl-THF (i.e., tetraglutamate alpha-5-formyl-THF) comprising a chain of three glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the composition comprises tetraglutamate alpha[6S]-5-formyl-THF. In some embodiments, the composition comprises tetraglutamate alpha[6R,S]-5-formyl-THF. In some embodiments, the composition comprises tetraglutamate alpha[6R]-5-formyl-THF. In some embodiments, tetraglutamate alpha-5-formyl-THF comprises one, two, or three glutamyl groups having alpha bonds. In some embodiments, tetraglutamate alpha-5-formyl-THF comprises two or more L-type glutamyl groups. In other embodiments, tetraglutamate alpha-5-formyl-THF comprises D-type glutamyl groups. In further embodiments, tetraglutamate alpha-5-formyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of tetraglutamate alpha-5-formyl-THF is linear. In some embodiments, the polyglutamate chain of tetraglutamate alpha-5-formyl-THF is branched.

[0015] In some embodiments, the Disclosure provides compositions comprising polyglutamine oxidized alpha-5-formyl-THF (i.e., pentaglutamine oxidized 5-formyl-THF) comprising a chain of four glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the composition comprises pentaglutamine oxidized alpha[6S]-5-formyl-THF. In some embodiments, the composition comprises pentaglutamine oxidized alpha[6R,S]-5-formyl-THF. In some embodiments, the composition comprises pentaglutamine oxidized alpha[6R]-5-formyl-THF. In some embodiments, pentaglutamine oxidized alpha-5-formyl-THF comprises one, two, three, or four glutamyl groups having alpha bonds. In some embodiments, pentaglutamine oxidized 5-formyl-THF comprises two or more L-type glutamyl groups. In other embodiments, pentaglutamine oxidized 5-formyl-THF comprises D-type glutamyl groups. In further embodiments, pentaglutamine-oxidized 5-formyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of pentaglutamine-oxidized alpha-5-formyl-THF is linear. In some embodiments, the polyglutamate chain of pentaglutamine-oxidized alpha-5-formyl-THF is branched.

[0016] In some embodiments, the Disclosure provides compositions comprising polyglutamic acid alpha-5-formyl-THF (i.e., hexaglutamic acid 5-formyl-THF) comprising a chain of five glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the composition comprises hexaglutamic acid alpha[6S]-5-formyl-THF. In some embodiments, the composition comprises hexaglutamic acid alpha[6R,S]-5-formyl-THF. In some embodiments, the composition comprises hexaglutamic acid alpha[6R]-5-formyl-THF. In some embodiments, hexaglutamic acid alpha-5-formyl-THF comprises one, two, three, four, or five glutamyl groups having alpha bonds. In some embodiments, hexaglutamic acid alpha-5-formyl-THF comprises two or more L-type glutamyl groups. In other embodiments, hexaglutamic acid THF comprises D-type glutamyl groups. In further embodiments, hexaglutamate alpha-5-formyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of hexaglutamate alpha-5-formyl-THF is linear. In some embodiments, the polyglutamate chain of hexaglutamate alpha-5-formyl-THF is branched.

[0017] In some embodiments, the Disclosure provides compositions comprising polyglutamic alpha-5,10-methenyl-THF. In some embodiments, the composition comprises polyglutamic alpha-[6R]-5,10-methenyl-THF. In some embodiments, the composition comprises polyglutamic alpha-[6R,S]-5,10-methenyl-THF. In some embodiments, the composition comprises polyglutamic alpha-[6S]-5,10-methenyl-THF. In some embodiments, the composition comprises polyglutamic alpha-5,10-methenyl-THF having 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 21 or more glutamyl groups (including the glutamyl groups in 5,10-methenyl-THF). In some embodiments, the polyglutamic alpha-5,10-methenyl-THF contains 1, 2, 3, or 4 or more glutamyl groups having alpha bonds. In some embodiments, the composition comprises polyglutamic acid alpha-5-formyl-THF having two or more L-type glutamyl groups. In other embodiments, the composition comprises polyglutamic acid alpha-5,10-methenyl-THF having a D-type glutamyl group. In further embodiments, the composition comprises polyglutamic acid alpha-5,10-methenyl-THF having a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of polyglutamic acid alpha-5,10-methenyl-THF is linear. In some embodiments, the polyglutamic acid chain of polyglutamic acid alpha-5,10-methenyl-THF is branched.

[0018] In some embodiments, the Disclosure provides compositions comprising polyglutamate alpha-5,10-methenyl-THF (i.e., tetraglutamate 5,10-methenyl-THF) comprising a chain of three glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the composition comprises tetraglutamate alpha[6R]-5,10-methenyl-THF. In some embodiments, the composition comprises tetraglutamate alpha[6R,S]-5,10-methenyl-THF. In some embodiments, the composition comprises tetraglutamate alpha[6S]-5,10-methenyl-THF. In some embodiments, tetraglutamate alpha-5,10-methenyl-THF comprises one, two, or three glutamyl groups having alpha bonds. In some embodiments, tetraglutamate alpha-5,10-methenyl-THF comprises two or more L-type glutamyl groups. In other embodiments, tetraglutamate alpha-5,10-methenyl-THF contains a D-type glutamyl group. In further embodiments, tetraglutamate alpha-5,10-methenyl-THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of 5,10-methenyl-THF tetrahydrofolate is linear. In some embodiments, the polyglutamate chain of tetraglutamate alpha-5,10-methenyl-THF is branched.

[0019] In some embodiments, the Disclosure provides compositions comprising polyglutamine oxidized alpha-5,10-methenyl-THF (i.e., pentaglutamine oxidized 5,10-methenyl-THF) comprising a chain of four glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the composition comprises pentaglutamine oxidized alpha[6R]-5,10-methenyl-THF. In some embodiments, the composition comprises pentaglutamine oxidized alpha[6R,S]-5,10-methenyl-THF. In some embodiments, the composition comprises pentaglutamine oxidized alpha[6S]-5,10-methenyl-THF. In some embodiments, pentaglutamine oxidized alpha-5,10-methenyl-THF comprises one, two, three, or four glutamyl groups having alpha bonds. In some embodiments, pentaglutamine oxidized alpha-5,10-methenyl-THF comprises two or more L-type glutamyl groups. In other embodiments, pentaglutamine alpha-5,10-methenyl-THF contains a D-type glutamyl group. In further embodiments, pentaglutamine alpha-5,10-methenyl-THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of pentaglutamine alpha-5,10-methenyl-THF is linear. In some embodiments, the polyglutamate chain of pentaglutamine alpha-5,10-methenyl-THF is branched.

[0020] In some embodiments, the Disclosure provides compositions comprising polyglutamic acid alpha-5,10-methenyl-THF (i.e., hexaglutamic acid alpha-5,10-methenyl-THF) comprising a chain of five glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the composition comprises hexaglutamic acid alpha[6R]-5,10-methenyl-THF. In some embodiments, the composition comprises hexaglutamic acid alpha[6R,S]-5,10-methenyl-THF. In some embodiments, the composition comprises hexaglutamic acid alpha[6S]-5,10-methenyl-THF. In some embodiments, hexaglutamic acid alpha-5,10-methenyl-THF comprises one, two, three, four, or five glutamyl groups having alpha bonds. In some embodiments, alpha-5,10-methenyl-THF hexaglutamate contains two or more L-type glutamyl groups. In other embodiments, alpha-5,10-methenyl-THF hexaglutamate contains a D-type glutamyl group. In further embodiments, alpha-5,10-methenyl-THF hexaglutamate contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of alpha-5,10-methenyl-THF hexaglutamate is linear. In some embodiments, the polyglutamate chain of alpha-5,10-methenyl-THF hexaglutamate is branched.

[0021] In some embodiments, the disclosure provides compositions comprising polyglutamate alpha-5-methyl-THF. In some embodiments, the composition comprises polyglutamate alpha[6S]-5-methyl-THF. In some embodiments, the composition comprises polyglutamate alpha[6R,S]-5-methyl-THF. In some embodiments, the composition comprises polyglutamate alpha[6R]-5-methyl-THF. In some embodiments, the composition comprises polyglutamate alpha-5-methyl-THF having 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 21 or more glutamyl groups (including the glutamyl groups in 5-methyl-THF). In some embodiments, the polyglutamate alpha-5-methyl-THF contains 1, 2, 3, or 4 or more glutamyl groups having alpha bonds. In some embodiments, the composition comprises polyglutamate alpha-5-methyl-THF having 2 or more L-type glutamyl groups. In other embodiments, the composition comprises polyglutamate alpha-5-methyl-THF having a D-type glutamyl group. In further embodiments, the composition comprises polyglutamate alpha-5-methyl-THF having a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of polyglutamate alpha-5-methyl-THF is linear. In some embodiments, the polyglutamate chain of polyglutamate alpha-5-methyl-THF is branched.

[0022] In some embodiments, the Disclosure provides compositions comprising polyglutamate alpha-5-methyl-THF (i.e., tetraglutamate alpha-5-methyl-THF) comprising a chain of three glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the composition comprises tetraglutamate alpha[6S]-5-methyl-THF. In some embodiments, the composition comprises tetraglutamate alpha[6R,S]-5-methyl-THF. In some embodiments, the composition comprises tetraglutamate alpha[6R]-5-methyl-THF. In some embodiments, tetraglutamate alpha-5-methyl-THF comprises one, two, or three glutamyl groups having gamma bonds. In some embodiments, tetraglutamate alpha-5-methyl-THF comprises two or more L-type glutamyl groups. In other embodiments, tetraglutamate alpha-5-methyl-THF comprises D-type glutamyl groups. In further embodiments, tetraglutamate alpha-5-methyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of tetraglutamate alpha-5-methyl-THF is linear. In some embodiments, the polyglutamate chain of tetraglutamate alpha-5-methyl-THF is branched.

[0023] In some embodiments, the Disclosure provides compositions comprising polyglutamine alpha-5-methyl-THF (i.e., pentaglutamine alpha-5-methyl-THF) comprising a chain of four glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the composition comprises pentaglutamine alpha-[6S]-5-methyl-THF. In some embodiments, the composition comprises pentaglutamine alpha-[6R,S]-5-methyl-THF. In some embodiments, the composition comprises pentaglutamine alpha-[6R]-5-methyl-THF. In some embodiments, pentaglutamine alpha-5-methyl-THF comprises one, two, three, or four glutamyl groups having gamma bonds. In some embodiments, pentaglutamine alpha-5-methyl-THF comprises two or more L-type glutamyl groups. In other embodiments, pentaglutamine alpha-5-methyl-THF comprises D-type glutamyl groups. In further embodiments, pentaglutamine alpha-5-methyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of pentaglutamine alpha-5-methyl-THF is linear. In some embodiments, the polyglutamate chain of pentaglutamine alpha-5-methyl-THF is branched.

[0024] In some embodiments, the Disclosure provides compositions comprising polyglutamate alpha-5-methyl-THF (i.e., hexaglutamate 5-methyl-THF) comprising a chain of five glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the composition comprises hexaglutamate alpha[6S]-5-methyl-THF. In some embodiments, the composition comprises hexaglutamate alpha[6R,S]-5-methyl-THF. In some embodiments, the composition comprises hexaglutamate alpha[6R]-5-methyl-THF. In some embodiments, hexaglutamate alpha-5-methyl-THF comprises one, two, three, four, or five glutamyl groups having alpha bonds. In some embodiments, hexaglutamate alpha-5-methyl-THF comprises two or more L-type glutamyl groups. In other embodiments, hexaglutamate alpha-5-methyl-THF comprises D-type glutamyl groups. In further embodiments, hexaglutamate alpha-5-methyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of hexaglutamate alpha-5-methyl-THF is linear. In some embodiments, the polyglutamate chain of hexaglutamate alpha-5-methyl-THF is branched.

[0025] In some embodiments, the disclosure provides compositions comprising polyglutamate-oxidized alpha-tetrahydrofolate THF. In some embodiments, the composition comprises polyglutamate-oxidized alpha[6S]-tetrahydrofolate THF. In some embodiments, the composition comprises polyglutamate-oxidized alpha[6R,S]-tetrahydrofolate THF. In some embodiments, the composition comprises polyglutamate-oxidized alpha[6R]-tetrahydrofolate THF. In some embodiments, the composition comprises polyglutamate-oxidized alpha-tetrahydrofolate THF having 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 21 or more glutamyl groups (including the glutamyl groups in tetrahydrofolate THF). In some embodiments, the alpha-polyglutamate-oxidized tetrahydrofolate THF comprises 1, 2, 3, or 4 or more glutamyl groups having alpha bonds. In some embodiments, the composition comprises polyglutamate-oxidized alpha-tetrahydrofolate THF having 2 or more L-type glutamyl groups. In other embodiments, the composition comprises polyglutamic acid alpha-tetrahydrofolate THF having a D-type glutamyl group. In further embodiments, the composition comprises polyglutamic acid alpha-tetrahydrofolate THF having a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of polyglutamic acid alpha-tetrahydrofolate THF is linear. In some embodiments, the polyglutamic acid chain of polyglutamic acid alpha-tetrahydrofolate THF is branched.

[0026] In some embodiments, the Disclosure provides compositions comprising polyglutamate-oxidized alpha-tetrahydrofolate THF (i.e., tetraglutamate-oxidized tetrahydrofolate THF) comprising a chain of three glutamyl groups bonded to a glutamyl group of tetrahydrofolate THF. In some embodiments, the composition comprises tetraglutamate-oxidized alpha[6S]tetrahydrofolate THF. In some embodiments, the composition comprises tetraglutamate-oxidized alpha[6R,S]-tetrahydrofolate THF. In some embodiments, the composition comprises tetraglutamate-oxidized alpha[6R]-tetrahydrofolate THF. In some embodiments, tetraglutamate-oxidized alpha-tetrahydrofolate THF comprises one, two, or three glutamyl groups having alpha bonds. In some embodiments, tetraglutamate-oxidized alpha-tetrahydrofolate THF comprises two or more L-type glutamyl groups. In other embodiments, tetraglutamate-oxidized alpha-tetrahydrofolate THF comprises D-type glutamyl groups. In further embodiments, tetraglutamate-oxidized alpha-tetrahydrofolate THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of tetraglutamate-oxidized alpha-tetrahydrofolate THF is linear. In some embodiments, the polyglutamate chain of tetraglutamate-oxidized alpha-tetrahydrofolate THF is branched.

[0027] In some embodiments, the Disclosure provides compositions comprising polyglutamate-oxidized alpha-tetrahydrofolate THF (i.e., pentaglutamine-oxidized tetrahydrofolate THF) comprising a chain of four glutamyl groups bonded to a glutamyl group of THF. In some embodiments, the composition comprises pentaglutamine-oxidized alpha[6S]tetrahydrofolate THF. In some embodiments, the composition comprises pentaglutamine-oxidized alpha[6R,S]-tetrahydrofolate THF. In some embodiments, the composition comprises pentaglutamine-oxidized alpha[6R]-tetrahydrofolate THF. In some embodiments, pentaglutamine-oxidized alpha-tetrahydrofolate THF comprises one, two, three, or four glutamyl groups having alpha bonds. In some embodiments, pentaglutamine-oxidized alpha-THF comprises two or more L-type glutamyl groups. In other embodiments, pentaglutamine-oxidized alpha-tetrahydrofolate THF comprises D-type glutamyl groups. In further embodiments, pentaglutamine-oxidized alpha-tetrahydrofolate THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of pentaglutamine-oxidized alpha-tetrahydrofolate THF is linear. In some embodiments, the polyglutamate chain of pentaglutamine-oxidized alpha-THF is branched.

[0028] In some embodiments, the Disclosure provides compositions comprising polyglutamate-oxidized alpha-tetrahydrofolate THF (i.e., hexaglutamate-oxidized tetrahydrofolate THF) comprising a chain of five glutamyl groups bonded to a glutamyl group of tetrahydrofolate THF. In some embodiments, the composition comprises hexaglutamate-oxidized alpha[6S]tetrahydrofolate THF. In some embodiments, the composition comprises hexaglutamate-oxidized alpha[6R,S]-tetrahydrofolate THF. In some embodiments, the composition comprises hexaglutamate-oxidized alpha[6R]-tetrahydrofolate THF. In some embodiments, hexaglutamate-oxidized alpha-tetrahydrofolate THF comprises one, two, three, four, or five glutamyl groups having alpha bonds. In some embodiments, hexaglutamate-oxidized alpha-tetrahydrofolate THF comprises two or more L-type glutamyl groups. In other embodiments, hexaglutamate-oxidized tetrahydrofolate THF contains a D-type glutamyl group. In further embodiments, hexaglutamate-oxidized alphatetrahydrofolate THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of hexaglutamate-oxidized alphatetrahydrofolate THF is linear. In some embodiments, the polyglutamate chain of hexaglutamate-oxidized alphatetrahydrofolate THF is branched.

[0029] In some embodiments, the disclosure provides compositions comprising polyglutamic acid alpha-5,10-methylene-THF. In some embodiments, the composition comprises polyglutamic acid alpha[6R]-5,10-methylene-THF. In some embodiments, the composition comprises polyglutamic acid alpha[6R,S]-5,10-methylene-THF. In some embodiments, the composition comprises polyglutamic acid alpha[6S]-5,10-methylene-THF. In some embodiments, the composition comprises polyglutamic acid alpha-5,10-methylene-THF having 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 21 or more glutamyl groups (including the glutamyl groups in 5,10-methylene-THF). In some embodiments, the polyglutamic acid alpha-5,10-methylene-THF contains 1, 2, 3, or 4 or more glutamyl groups having alpha bonds. In some embodiments, the composition comprises polyglutamic acid alpha-5,10-methylene-THF having two or more L-type glutamyl groups. In other embodiments, the composition comprises polyglutamic acid alpha-5,10-methylene-THF having D-type glutamyl groups. In further embodiments, the composition comprises polyglutamic acid alpha-5,10-methylene-THF having a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of polyglutamic acid alpha-5,10-methylene-THF is linear. In some embodiments, the polyglutamic acid chain of polyglutamic acid alpha-5,10-methylene-THF is branched.

[0030] In some embodiments, the Disclosure provides compositions comprising polyglutamate alpha-5,10-methylene-THF (i.e., tetraglutamate 5,10-methylene-THF) comprising a chain of three glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the composition comprises tetraglutamate alpha[6R]-5,10-methylene-THF. In some embodiments, the composition comprises tetraglutamate alpha[6R,S]-5,10-methylene-THF. In some embodiments, the composition comprises tetraglutamate alpha[6S]-5,10-methylene-THF. In some embodiments, tetraglutamate alpha-5,10-methylene-THF comprises one, two, or three glutamyl groups having alpha bonds. In some embodiments, tetraglutamate alpha-5,10-methylene-THF comprises two or more L-type glutamyl groups. In other embodiments, tetraglutamate alpha 5,10-methylene-THF contains a D-type glutamyl group. In further embodiments, tetraglutamate alpha 5,10-methylene-THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of tetraglutamate alpha 5,10-methylene-THF is linear. In some embodiments, the polyglutamate chain of tetraglutamate alpha 5,10-methylene-THF is branched.

[0031] In some embodiments, the Disclosure provides compositions comprising polyglutamate alpha-5,10-methylene-THF (i.e., pentaglutamate 10-methylene-THF) comprising a chain of four glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the composition comprises pentaglutamate alpha[6R]5,10-methylene-THF. In some embodiments, the composition comprises pentaglutamate alpha[6R,S]-5,10-methylene-THF. In some embodiments, the composition comprises pentaglutamate alpha[6S]-5,10-methylene-THF. In some embodiments, pentaglutamate alpha-5,10-methylene-THF comprises one, two, three, or four glutamyl groups having alpha bonds. In some embodiments, pentaglutamate alpha-5,10-methylene-THF comprises two or more L-type glutamyl groups. In other embodiments, pentaglutamine alpha-5,10-methylene-THF contains a D-type glutamyl group. In further embodiments, pentaglutamine alpha-5,10-methylene-THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of pentaglutamine alpha-5,10-methylene-THF is linear. In some embodiments, the polyglutamate chain of pentaglutamine alpha-5,10-methylene-THF is branched.

[0032] In some embodiments, the Disclosure provides compositions comprising polyglutamate alpha-5,10-methylene-THF (i.e., hexaglutamate 5,10-methylene-THF) comprising a chain of five glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the composition comprises hexaglutamate alpha[6R]5,10-methylene-THF. In some embodiments, the composition comprises hexaglutamate alpha[6R]5,10-methylene-THF. In some embodiments, the composition comprises hexaglutamate alpha[6R,S]-5,10-methylene-THF. In some embodiments, the composition comprises hexaglutamate alpha[6S]-5,10-methylene-THF. In some embodiments, alpha-hexaglutamate 5,10-methylene-THF comprises one, two, three, four, or five glutamyl groups having alpha bonds. In some embodiments, alpha-5,10-methylene-THF hexaglutamate contains two or more L-type glutamyl groups. In other embodiments, alpha-5,10-methylene-THF hexaglutamate contains a D-type glutamyl group. In further embodiments, alpha-5,10-methylene-THF hexaglutamate contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of alpha-5,10-methylene-THF hexaglutamate is linear. In some embodiments, the polyglutamate chain of alpha-5,10-methylene-THF hexaglutamate is branched.

[0033] In some embodiments, the disclosure provides compositions comprising polyglutamate alpha-5-formimino-THF. In some embodiments, the composition comprises polyglutamate alpha[6S]-5-formimino-THF. In some embodiments, the composition comprises polyglutamate alpha[6R,S]-5-formimino-THF. In some embodiments, the composition comprises polyglutamate alpha[6R]-5-formimino-THF. In some embodiments, the composition comprises polyglutamate alpha-5-formimino-THF having 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 21 or more glutamyl groups (including the glutamyl groups in 5-formimino-THF). In some embodiments, the polyglutamate alpha-5-formimino-THF contains 1, 2, 3, or 4 or more glutamyl groups having alpha bonds. In some embodiments, the composition comprises polyglutamate alpha-5-formimino-THF having two or more L-type glutamyl groups. In other embodiments, the composition comprises polyglutamate alpha-5-formimino-THF having D-type glutamyl groups. In further embodiments, the composition comprises polyglutamate alpha-5-formimino-THF having a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of polyglutamate alpha-5-formimino-THF is linear. In some embodiments, the polyglutamate chain of polyglutamate alpha-5-formimino-THF is branched.

[0034] In one embodiment, the composition comprises polyglutamate alpha-5-formimino-THF (i.e., tetraglutamate alpha-5-formimino-THF) containing a chain of three glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the composition comprises tetraglutamate alpha[6S]-5-formimino-THF. In some embodiments, the composition comprises tetraglutamate alpha[6R,S]-5-formimino-THF. In some embodiments, the composition comprises tetraglutamate alpha[6R]5-formimino-THF. In some embodiments, tetraglutamate alpha-5-formimino-THF contains one, two, or three glutamyl groups having alpha bonds. In some embodiments, tetraglutamate alpha-5-formimino-THF contains two or more L-type glutamyl groups. In other embodiments, tetraglutamate alpha-5-formimino-THF contains a D-type glutamyl group. In further embodiments, tetraglutamate alpha-5-formimino-THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of tetraglutamate alpha-5-formimino-THF is linear. In some embodiments, the polyglutamate chain of tetraglutamate alpha-5-formimino-THF is branched.

[0035] In one embodiment, the composition comprises polyglutamine oxidized alpha-5-formimino-THF (i.e., pentaglutamine oxidized 5-formimino-THF) containing a chain of four glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the composition comprises pentaglutamine oxidized alpha[6S]-5-formimino-THF. In some embodiments, the composition comprises pentaglutamine oxidized alpha[6R,S]-5-formimino-THF. In some embodiments, the composition comprises pentaglutamine oxidized alpha[6R]5-formimino-THF. In some embodiments, pentaglutamine oxidized alpha-5-formimino-THF contains one, two, three, or four glutamyl groups having alpha bonds. In some embodiments, pentaglutamine oxidized alpha-5-formimino-THF contains two or more L-type glutamyl groups. In other embodiments, pentaglutamine oxy-alpha 5-formimino-THF contains a D-type glutamyl group. In further embodiments, pentaglutamine oxy-alpha 5-formimino-THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of pentaglutamine oxy-alpha 5-formimino-THF is linear. In some embodiments, the polyglutamate chain of pentaglutamine oxy-alpha 5-formimino-THF is branched.

[0036] In one embodiment, the composition comprises polyglutamate alpha-5-formimino-THF (i.e., hexaglutamate 5-formimino-THF) containing a chain of five glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the composition comprises hexaglutamate alpha[6S]-5-formimino-THF. In some embodiments, the composition comprises hexaglutamate alpha[6R,S]-5-formimino-THF. In some embodiments, the composition comprises hexaglutamate alpha[6R]5-formimino-THF. In some embodiments, hexaglutamate alpha-5-formimino-THF contains one, two, three, four, or five glutamyl groups having alpha bonds. In some embodiments, hexaglutamate alpha-5-formimino-THF contains two or more L-type glutamyl groups. In other embodiments, hexaglutamate alpha-5-formimino-THF contains a D-type glutamyl group. In further embodiments, hexaglutamate alpha-5-formimino-THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of hexaglutamate alpha-5-formimino-THF is linear. In some embodiments, the polyglutamate chain of hexaglutamate alpha-5-formimino-THF is branched.

[0037] In further embodiments, the disclosure provides a composition comprising liposomes (Lp-αPTHF) encapsulated (filled) with polyglutamate-oxidized alpha-tetrahydrofolate.

[0038] In some embodiments, the Disclosure provides compositions comprising liposomes encapsulated (filled) with polyglutamic acid alpha-5-formyl-THF. In some embodiments, the liposomes comprise polyglutamic acid alpha[6S]-5-formyl-THF. In some embodiments, the liposomes comprise polyglutamic acid alpha[6R,S]-5-formyl-THF. In some embodiments, the liposomes comprise polyglutamic acid alpha[6R]-5-formyl-THF. In some embodiments, the liposomes comprise polyglutamic acid alpha-5-formyl-THF having 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 21 or more glutamyl groups (including the glutamyl groups in 5-formyl-THF). In some embodiments, the polyglutamic acid alpha-5-formyl-THF comprises 1, 2, 3, or 4 or more glutamyl groups having alpha bonds. In some embodiments, the liposomes contain polyglutamate alpha-5-formyl-THF having two or more L-type glutamyl groups. In other embodiments, the liposomes contain polyglutamate alpha-5-formyl-THF having D-type glutamyl groups. In further embodiments, the liposomes contain polyglutamate alpha-5-formyl-THF having a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of polyglutamate alpha-5-formyl-THF is linear. In some embodiments, the polyglutamate chain of polyglutamate alpha-5-formyl-THF is branched.

[0039] In one embodiment, the Lp-αPTHF composition comprises polyglutamate alpha-5-formyl-THF (i.e., tetraglutamate alpha-5-formyl-THF) containing a chain of three glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the liposomes contain tetraglutamate alpha[6S]-5-formyl-THF. In some embodiments, the liposomes contain tetraglutamate alpha[6R,S]-5-formyl-THF. In some embodiments, the liposomes contain tetraglutamate alpha[6R]-5-formyl-THF. In some embodiments, the tetraglutamate alpha-5-formyl-THF contains one, two, or three glutamyl groups having gamma bonds. In some embodiments, the tetraglutamate alpha-5-formyl-THF contains two or more L-type glutamyl groups. In other embodiments, the tetraglutamate alpha-5-formyl-THF contains D-type glutamyl groups. In further embodiments, tetraglutamate alpha-5-formyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of tetraglutamate alpha-5-formyl-THF is linear. In some embodiments, the polyglutamate chain of tetraglutamate alpha-5-formyl-THF is branched.

[0040] In one embodiment, the Lp-αPTHF composition comprises polyglutamine oxidized alpha-5-formyl-THF (i.e., pentaglutamine oxidized 5-formyl-THF) containing a chain of four glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the liposomes contain pentaglutamine oxidized alpha[6S]-5-formyl-THF. In some embodiments, the liposomes contain pentaglutamine oxidized alpha[6R,S]-5-formyl-THF. In some embodiments, the liposomes contain pentaglutamine oxidized alpha[6R]-5-formyl-THF. In some embodiments, the pentaglutamine oxidized alpha-5-formyl-THF contains one, two, three, or four glutamyl groups having alpha bonds. In some embodiments, the pentaglutamine oxidized 5-formyl-THF contains two or more L-type glutamyl groups. In other embodiments, pentaglutamine-oxidized 5-formyl-THF contains a D-type glutamyl group. In further embodiments, pentaglutamine-oxidized 5-formyl-THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of pentaglutamine-oxidized alpha-5-formyl-THF is linear. In some embodiments, the polyglutamate chain of pentaglutamine-oxidized alpha-5-formyl-THF is branched.

[0041] In one embodiment, the Lp-αPTHF composition contains polyglutamate alpha-5-formyl-THF (i.e., hexaglutamate 5-formyl-THF) comprising a chain of five glutamyl groups bonded to the glutamyl group of tetrahydrofolate. In some embodiments, the liposomes contain hexaglutamate alpha[6S]-5-formyl-THF. In some embodiments, the liposomes contain hexaglutamate alpha[6R,S]-5-formyl-THF. In some embodiments, the liposomes contain hexaglutamate alpha[6R]-5-formyl-THF. In some embodiments, the hexaglutamate alpha-5-formyl-THF contains one, two, three, four, or five glutamyl groups having alpha bonds. In some embodiments, the hexaglutamate alpha-5-formyl-THF contains two or more L-type glutamyl groups. In other embodiments, hexaglutamate-oxidized THF contains a D-type glutamyl group. In further embodiments, hexaglutamate-oxidized alpha-5-formyl-THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of hexaglutamate-oxidized alpha-5-formyl-THF is linear. In some embodiments, the polyglutamate chain of hexaglutamate-oxidized alpha-5-formyl-THF is branched.

[0042] In some embodiments, the Disclosure provides compositions comprising liposomes encapsulated (filled) with polyglutamic acid alpha-5,10-methenyl-THF. In some embodiments, the liposomes comprise polyglutamic acid alpha[6R]-5,10-methenyl-THF. In some embodiments, the liposomes comprise polyglutamic acid alpha[6R,S]-5,10-methenyl-THF. In some embodiments, the liposomes comprise polyglutamic acid alpha[6S]-5,10-methenyl-THF. In some embodiments, the liposomes comprise polyglutamic acid alpha-5,10-methenyl-THF having 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 21 or more glutamyl groups (including the glutamyl groups in 5,10-methenyl-THF). In some embodiments, polyglutamate alpha-5,10-methenyl-THF contains one, two, three, or four or more glutamyl groups having alpha bonds. In some embodiments, the liposome contains polyglutamate alpha-5-formyl-THF having two or more L-type glutamyl groups. In other embodiments, the liposome contains polyglutamate alpha-5,10-methenyl-THF having D-type glutamyl groups. In further embodiments, the liposome contains polyglutamate alpha-5,10-methenyl-THF having a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of polyglutamate alpha-5,10-methenyl-THF is linear. In some embodiments, the polyglutamate chain of polyglutamate alpha-5,10-methenyl-THF is branched.

[0043] In one embodiment, the Lp-αPTHF composition contains polyglutamate alpha-5,10-methenyl-THF (i.e., tetraglutamate 5,10-methenyl-THF) comprising a chain of three glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the liposomes contain tetraglutamate alpha[6R]-5,10-methenyl-THF. In some embodiments, the liposomes contain tetraglutamate alpha[6R,S]-5,10-methenyl-THF. In some embodiments, the liposomes contain tetraglutamate alpha[6S]-5,10-methenyl-THF. In some embodiments, the tetraglutamate alpha-5,10-methenyl-THF contains one, two, or three glutamyl groups having alpha bonds. In some embodiments, the tetraglutamate alpha-5,10-methenyl-THF contains two or more L-type glutamyl groups. In other embodiments, tetraglutamate alpha-5,10-methenyl-THF contains a D-type glutamyl group. In further embodiments, tetraglutamate alpha-5,10-methenyl-THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of 5,10-methenyl-THF tetrahydrofolate is linear. In some embodiments, the polyglutamate chain of tetraglutamate alpha-5,10-methenyl-THF is branched.

[0044] In one embodiment, the Lp-αPTHF composition comprises polyglutamate alpha-5,10-methenyl-THF (i.e., pentaglutamine 5,10-methenyl-THF) containing a chain of four glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the liposomes contain pentaglutamine alpha[6R]-5,10-methenyl-THF. In some embodiments, the liposomes contain pentaglutamine alpha[6R,S]-5,10-methenyl-THF. In some embodiments, the liposomes contain pentaglutamine alpha[6S]-5,10-methenyl-THF. In some embodiments, the pentaglutamine alpha-5,10-methenyl-THF contains one, two, three, or four glutamyl groups having alpha bonds. In some embodiments, pentaglutamine alpha-5,10-methenyl-THF contains two or more L-type glutamyl groups. In other embodiments, pentaglutamine alpha-5,10-methenyl-THF contains a D-type glutamyl group. In further embodiments, pentaglutamine alpha-5,10-methenyl-THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of pentaglutamine alpha-5,10-methenyl-THF is linear. In some embodiments, the polyglutamate chain of pentaglutamine alpha-5,10-methenyl-THF is branched.

[0045] In one embodiment, the Lp-αPTHF composition comprises polyglutamate alpha-5,10-methenyl-THF (i.e., hexaglutamate 5,10-methenyl-THF) containing a chain of five glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the liposomes contain hexaglutamate alpha[6R]-5,10-methenyl-THF. In some embodiments, the liposomes contain hexaglutamate alpha[6R,S]-5,10-methenyl-THF. In some embodiments, the liposomes contain hexaglutamate alpha[6S]-5,10-methenyl-THF. In some embodiments, the hexaglutamate alpha-5,10-methenyl-THF contains one, two, three, four, or five glutamyl groups having alpha bonds. In some embodiments, alpha-5,10-methenyl-THF hexaglutamate contains two or more L-type glutamyl groups. In other embodiments, alpha-5,10-methenyl-THF hexaglutamate contains a D-type glutamyl group. In further embodiments, alpha-5,10-methenyl-THF hexaglutamate contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of alpha-5,10-methenyl-THF hexaglutamate is linear. In some embodiments, the polyglutamate chain of alpha-5,10-methenyl-THF hexaglutamate is branched.

[0046] In some embodiments, the Disclosure provides compositions comprising liposomes encapsulated (filled) with polyglutamate alpha-5-methyl-THF. In some embodiments, the liposomes comprise polyglutamate alpha[6S]-5-methyl-THF. In some embodiments, the liposomes comprise polyglutamate alpha[6R,S]-5-methyl-THF. In some embodiments, the liposomes comprise polyglutamate alpha[6R]-5-methyl-THF. In some embodiments, the liposomes comprise polyglutamate alpha-5-methyl-THF having 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 21 or more glutamyl groups (including the glutamyl groups in 5-methyl-THF). In some embodiments, the polyglutamate alpha-5-methyl-THF comprises 1, 2, 3, or 4 or more glutamyl groups having alpha bonds. In some embodiments, the liposomes contain polyglutamate alpha-5-methyl-THF having two or more L-type glutamyl groups. In other embodiments, the liposomes contain polyglutamate alpha-5-methyl-THF having D-type glutamyl groups. In further embodiments, the liposomes contain polyglutamate alpha-5-methyl-THF having a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of polyglutamate alpha-5-methyl-THF is linear. In some embodiments, the polyglutamate chain of polyglutamate alpha-5-methyl-THF is branched.

[0047] In one embodiment, the Lp-αPTHF composition comprises polyglutamate alpha-5-methyl-THF (i.e., tetraglutamate 5-methyl-THF) containing a chain of three glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the liposomes contain tetraglutamate alpha[6S]-5-methyl-THF. In some embodiments, the liposomes contain tetraglutamate alpha[6R,S]-5-methyl-THF. In some embodiments, the liposomes contain tetraglutamate alpha[6R]-5-methyl-THF. In some embodiments, the tetraglutamate alpha-5-methyl-THF contains one, two, or three glutamyl groups having alpha bonds. In some embodiments, the tetraglutamate alpha-5-methyl-THF contains two or more L-type glutamyl groups. In other embodiments, the tetraglutamate alpha-5-methyl-THF contains D-type glutamyl groups. In further embodiments, tetraglutamate alpha-5-methyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of tetraglutamate alpha-5-methyl-THF is linear. In some embodiments, the polyglutamate chain of tetraglutamate alpha-5-methyl-THF is branched.

[0048] In one embodiment, the Lp-αPTHF composition comprises polyglutamate alpha-5-methyl-THF (i.e., pentaglutamine 5-methyl-THF) containing a chain of four glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the liposomes contain pentaglutamine alpha[6S]-5-methyl-THF. In some embodiments, the liposomes contain pentaglutamine alpha[6R,S]-5-methyl-THF. In some embodiments, the liposomes contain pentaglutamine alpha[6R]-5-methyl-THF. In some embodiments, the pentaglutamine alpha-5-methyl-THF contains one, two, three, or four glutamyl groups having alpha bonds. In some embodiments, the pentaglutamine alpha-5-methyl-THF contains two or more L-type glutamyl groups. In other embodiments, the pentaglutamine alpha-5-methyl-THF contains D-type glutamyl groups. In further embodiments, pentaglutamine alpha-5-methyl-THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of pentaglutamine alpha-5-methyl-THF is linear. In some embodiments, the polyglutamate chain of pentaglutamine alpha-5-methyl-THF is branched.

[0049] In one embodiment, the Lp-αPTHF composition comprises polyglutamate alpha-5-methyl-THF (i.e., hexaglutamate 5-methyl-THF) containing a chain of five glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the liposomes contain hexaglutamate alpha[6S]-5-methyl-THF. In some embodiments, the liposomes contain hexaglutamate alpha[6R,S]-5-methyl-THF. In some embodiments, the liposomes contain hexaglutamate alpha[6R]-5-methyl-THF. In some embodiments, the hexaglutamate alpha-5-methyl-THF contains one, two, three, four, or five glutamyl groups having alpha bonds. In some embodiments, the hexaglutamate alpha-5-methyl-THF contains two or more L-type glutamyl groups. In other embodiments, hexaglutamate alpha-5-methyl-THF contains a D-type glutamyl group. In further embodiments, hexaglutamate alpha-5-methyl-THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of hexaglutamate alpha-5-methyl-THF is linear. In some embodiments, the polyglutamate chain of hexaglutamate alpha-5-methyl-THF is branched.

[0050] In some embodiments, the Disclosure provides compositions comprising liposomes encapsulated (filled) with polyglutamate-oxidized alpha[6S]-tetrahydrofolate THF. In some embodiments, the liposomes comprise polyglutamate-oxidized alpha[6R,S]-tetrahydrofolate THF. In some embodiments, the liposomes comprise polyglutamate-oxidized alpha[6R]-tetrahydrofolate THF. In some embodiments, the liposomes comprise polyglutamate-oxidized alpha[6R]-tetrahydrofolate THF. In some embodiments, the liposomes comprise polyglutamate-oxidized alpha[6R]-tetrahydrofolate THF having 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 21 or more glutamyl groups (including glutamyl groups in tetrahydrofolate THF). In some embodiments, the polyglutamate-oxidized alphatetrahydrofolate THF comprises 1, 2, 3, or 4 or more glutamyl groups having alpha bonds. In some embodiments, the liposomes comprise polyglutamic alpha-tetrahydrofolate THF having two or more L-type glutamyl groups. In other embodiments, the liposomes comprise polyglutamic alpha-tetrahydrofolate THF having D-type glutamyl groups. In further embodiments, the liposomes comprise polyglutamic alpha-tetrahydrofolate THF having a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of polyglutamic alpha-tetrahydrofolate THF is linear. In some embodiments, the polyglutamic acid chain of polyglutamic alpha-tetrahydrofolate THF is branched.

[0051] In one embodiment, the Lp-αP tetrahydrofolate THF composition comprises polyglutamate-oxidized alpha-tetrahydrofolate (i.e., tetraglutamate-oxidized tetrahydrofolate) containing a chain of three glutamyl groups bonded to the glutamyl group of tetrahydrofolate THF. In some embodiments, the liposomes contain tetraglutamate-oxidized alpha[6S]tetrahydrofolate. In some embodiments, the liposomes contain tetraglutamate-oxidized alpha[6R,S]-tetrahydrofolate THF. In some embodiments, the liposomes contain tetraglutamate-oxidized alpha[6R]-tetrahydrofolate THF. In some embodiments, the tetraglutamate-oxidized alpha-tetrahydrofolate THF contains one, two, or three glutamyl groups having alpha bonds. In some embodiments, the tetraglutamate-oxidized alpha-tetrahydrofolate THF contains two or more L-type glutamyl groups. In other embodiments, the tetraglutamate-oxidized alpha-tetrahydrofolate THF contains D-type glutamyl groups. In further embodiments, tetraglutamate-oxidized alpha-tetrahydrofolate THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of tetraglutamate-oxidized alpha-tetrahydrofolate is linear. In some embodiments, the polyglutamate chain of tetraglutamate-oxidized alpha-tetrahydrofolate is branched.

[0052] In one embodiment, the Lp-αP tetrahydrofolate THF composition comprises polyglutamate-oxidized alpha-tetrahydrofolate (i.e., pentaglutamine-oxidized tetrahydrofolate) containing a chain of four glutamyl groups bonded to the glutamyl group of tetrahydrofolate THF. In some embodiments, the liposomes contain pentaglutamine-oxidized alpha[6S]tetrahydrofolate. In some embodiments, the liposomes contain pentaglutamine-oxidized alpha[6R,S]-tetrahydrofolate THF. In some embodiments, the liposomes contain pentaglutamine-oxidized alpha[6R]-tetrahydrofolate THF. In some embodiments, the pentaglutamine-oxidized alpha-tetrahydrofolate THF contains one, two, three, or four glutamyl groups having alpha bonds. In some embodiments, the pentaglutamine-oxidized alpha-tetrahydrofolate THF contains two or more L-type glutamyl groups. In other embodiments, the pentaglutamine-oxidized alpha-tetrahydrofolate THF contains D-type glutamyl groups. In further embodiments, pentaglutamic acid alpha-tetrahydrofolate THF comprises a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamic acid chain of pentaglutamic acid alpha-tetrahydrofolate THF is linear. In some embodiments, the polyglutamic acid chain of pentaglutamic acid alpha-tetrahydrofolate THF is branched.

[0053] In one embodiment, the Lp-αP tetrahydrofolate THF composition comprises polyglutamate-oxidized alpha-tetrahydrofolate (i.e., hexaglutamate-oxidized tetrahydrofolate) containing a chain of five glutamyl groups bonded to the glutamyl group of tetrahydrofolate. In some embodiments, the liposomes contain hexaglutamate-oxidized alpha[6S]tetrahydrofolate. In some embodiments, the liposomes contain hexaglutamate-oxidized alpha[6R,S]-tetrahydrofolate THF. In some embodiments, the liposomes contain hexaglutamate-oxidized alpha[6R]-tetrahydrofolate THF. In some embodiments, the hexaglutamate-oxidized alpha-tetrahydrofolate THF contains one, two, three, four, or five glutamyl groups having alpha bonds. In some embodiments, the hexaglutamate-oxidized alpha-tetrahydrofolate THF contains two or more L-type glutamyl groups. In other embodiments, hexaglutamate-oxidized tetrahydrofolate THF contains a D-type glutamyl group. In further embodiments, hexaglutamate-oxidized alphatetrahydrofolate THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of hexaglutamate-oxidized alphatetrahydrofolate is linear. In some embodiments, the polyglutamate chain of hexaglutamate-oxidized alphatetrahydrofolate is branched.

[0054] In some embodiments, the Disclosure provides compositions comprising liposomes encapsulated (filled) with polyglutamic acid alpha-5,10-methylene-THF. In some embodiments, the liposomes comprise polyglutamic acid alpha[6R]-5,10-methylene-THF. In some embodiments, the liposomes comprise polyglutamic acid alpha[6R,S]-5,10-methylene-THF. In some embodiments, the liposomes comprise polyglutamic acid alpha[6S]-5,10-methylene-THF. In some embodiments, the liposomes comprise polyglutamic acid alpha-5,10-methylene-THF having 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 21 or more glutamyl groups (including the glutamyl groups in 5,10-methylene-THF). In some embodiments, polyglutamate alpha-5,10-methylene-THF contains one, two, three, or four or more glutamyl groups having alpha bonds. In some embodiments, the liposome contains polyglutamate alpha-5,10-methylene-THF having two or more L-type glutamyl groups. In other embodiments, the liposome contains polyglutamate alpha-5,10-methylene-THF having D-type glutamyl groups. In further embodiments, the liposome contains polyglutamate alpha-5,10-methylene-THF having a D-type glutamyl group and two or more glutamyl molecules. In some embodiments, the polyglutamate chain of polyglutamate alpha-5,10-methylene-THF is linear. In some embodiments, the polyglutamate chain of polyglutamate alpha-5,10-methylene-THF is branched.

[0055] In one embodiment, the Lp-αPTHF composition contains polyglutamate alpha-5,10-methylene-THF (i.e., tetraglutamate 5,10-methylene-THF) comprising a chain of three glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the liposomes contain tetraglutamate alpha[6R]-5,10-methylene-THF. In some embodiments, the liposomes contain tetraglutamate alpha[6R,S]-5,10-methylene-THF. In some embodiments, the liposomes contain tetraglutamate alpha[6S]-5,10-methylene-THF. In some embodiments, the tetraglutamate alpha-5,10-methylene-THF contains one, two, or three glutamyl groups having alpha bonds. In some embodiments, the tetraglutamate alpha-5,10-methylene-THF contains two or more L-type glutamyl groups. In other embodiments, tetraglutamate alpha 5,10-methylene-THF contains a D-type glutamyl group. In further embodiments, tetraglutamate alpha 5,10-methylene-THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of tetraglutamate alpha 5,10-methylene-THF is linear. In some embodiments, the polyglutamate chain of tetraglutamate alpha 5,10-methylene-THF is branched.

[0056] In one embodiment, the Lp-αPTHF composition comprises polyglutamate alpha-5,10-methylene-THF (i.e., pentaglutamine 10-methylene-THF) containing a chain of four glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the liposomes contain pentaglutamine alpha[6R]5,10-methylene-THF. In some embodiments, the liposomes contain pentaglutamine alpha[6R,S]-5,10-methylene-THF. In some embodiments, the liposomes contain pentaglutamine alpha[6S]-5,10-methylene-THF. In some embodiments, the pentaglutamine alpha-5,10-methylene-THF contains one, two, three, or four glutamyl groups having alpha bonds. In some embodiments, pentaglutamine alpha-5,10-methylene-THF contains two or more L-type glutamyl groups. In other embodiments, pentaglutamine alpha-5,10-methylene-THF contains a D-type glutamyl group. In further embodiments, pentaglutamine alpha-5,10-methylene-THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of pentaglutamine alpha-5,10-methylene-THF is linear. In some embodiments, the polyglutamate chain of pentaglutamine alpha-5,10-methylene-THF is branched.

[0057] In one embodiment, the Lp-αPTHF composition comprises polyglutamate alpha-5,10-methylene-THF (i.e., hexaglutamate 5,10-methylene-THF) containing a chain of five glutamyl groups bonded to the glutamyl group of tetrahydrofolate. In some embodiments, the liposomes contain hexaglutamate alpha[6R]5,10-methylene-THF. In some embodiments, the liposomes contain hexaglutamate alpha[6R]5,10-methylene-THF. In some embodiments, the liposomes contain hexaglutamate alpha[6R,S]-5,10-methylene-THF. In some embodiments, the liposomes contain hexaglutamate alpha[6S]-5,10-methylene-THF. In some embodiments, hexaglutamate alpha 5,10-methylene-THF contains one, two, three, four, or five glutamyl groups having alpha bonds. In some embodiments, hexaglutamate alpha 5,10-methylene-THF contains two or more L-type glutamyl groups. In other embodiments, hexaglutamate alpha 5,10-methylene-THF contains a D-type glutamyl group. In further embodiments, hexaglutamate alpha 5,10-methylene-THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of hexaglutamate alpha 5,10-methylene-THF is linear. In some embodiments, the polyglutamate chain of hexaglutamate alpha 5,10-methylene-THF is branched.

[0058] In some embodiments, the Disclosure provides compositions comprising liposomes encapsulated (filled) with polyglutamic acid alpha-5-formimino-THF. In some embodiments, the liposomes comprise polyglutamic acid alpha[6S]-5-formimino-THF. In some embodiments, the liposomes comprise polyglutamic acid alpha[6R,S]-5-formimino-THF. In some embodiments, the liposomes comprise polyglutamic acid alpha[6R]-5-formimino-THF. In some embodiments, the liposomes comprise polyglutamic acid alpha-5-formimino-THF having 2 to 20, 2 to 15, 2 to 10, 2 to 5, or 21 or more glutamyl groups (including the glutamyl groups in 5-formimino-THF). In some embodiments, polyglutamate alpha-5-formimino-THF contains one, two, three, or four or more glutamyl groups having alpha bonds. In some embodiments, the liposome contains polyglutamate alpha-5-formimino-THF having two or more L-type glutamyl groups. In other embodiments, the liposome contains polyglutamate alpha-5-formimino-THF having D-type glutamyl groups. In further embodiments, the liposome contains polyglutamate alpha-5-formimino-THF having a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of polyglutamate alpha-5-formimino-THF is linear. In some embodiments, the polyglutamate chain of polyglutamate alpha-5-formimino-THF is branched.

[0059] In one embodiment, the Lp-αPTHF composition comprises polyglutamate alpha-5-formimino-THF (i.e., tetraglutamate alpha-5-formimino-THF) containing a chain of three glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the liposomes contain tetraglutamate alpha[6S]-5-formimino-THF. In some embodiments, the liposomes contain tetraglutamate alpha[6R,S]-5-formimino-THF. In some embodiments, the liposomes contain tetraglutamate alpha[6R]5-formimino-THF. In some embodiments, the tetraglutamate alpha-5-formimino-THF contains one, two, or three glutamyl groups having alpha bonds. In some embodiments, the tetraglutamate alpha-5-formimino-THF contains two or more L-type glutamyl groups. In other embodiments, tetraglutamate alpha-5-formimino-THF contains a D-type glutamyl group. In further embodiments, tetraglutamate alpha-5-formimino-THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of tetraglutamate alpha-5-formimino-THF is linear. In some embodiments, the polyglutamate chain of tetraglutamate alpha-5-formimino-THF is branched.

[0060] In one embodiment, the Lp-αPTHF composition comprises polyglutamine oxidized alpha-5-formimino-THF (i.e., pentaglutamine oxidized 5-formimino-THF) containing a chain of four glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the liposomes contain pentaglutamine oxidized alpha[6S]-5-formimino-THF. In some embodiments, the liposomes contain pentaglutamine oxidized alpha[6R,S]-5-formimino-THF. In some embodiments, the liposomes contain pentaglutamine oxidized alpha[6R]5-formimino-THF. In some embodiments, the pentaglutamine oxidized alpha-5-formimino-THF contains one, two, three, or four glutamyl groups having alpha bonds. In some embodiments, the pentaglutamine oxidized alpha-5-formimino-THF contains two or more L-type glutamyl groups. In other embodiments, pentaglutamine oxy-alpha 5-formimino-THF contains a D-type glutamyl group. In further embodiments, pentaglutamine oxy-alpha 5-formimino-THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of pentaglutamine oxy-alpha 5-formimino-THF is linear. In some embodiments, the polyglutamate chain of pentaglutamine oxy-alpha 5-formimino-THF is branched.

[0061] In one embodiment, the Lp-αPTHF composition comprises polyglutamate alpha-5-formimino-THF (i.e., hexaglutamate 5-formimino-THF) containing a chain of five glutamyl groups bonded to a glutamyl group of tetrahydrofolate. In some embodiments, the liposomes contain hexaglutamate alpha[6S]-5-formimino-THF. In some embodiments, the liposomes contain hexaglutamate alpha[6R,S]-5-formimino-THF. In some embodiments, the liposomes contain hexaglutamate alpha[6R]5-formimino-THF. In some embodiments, the hexaglutamate alpha-5-formimino-THF contains one, two, three, four, or five glutamyl groups having alpha bonds. In some embodiments, hexaglutamate alpha-5-formimino-THF contains two or more L-type glutamyl groups. In other embodiments, hexaglutamate alpha-5-formimino-THF contains a D-type glutamyl group. In further embodiments, hexaglutamate alpha-5-formimino-THF contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain of hexaglutamate alpha-5-formimino-THF is linear. In some embodiments, the polyglutamate chain of hexaglutamate alpha-5-formimino-THF is branched.

[0062] In some embodiments, the Lp-αPTHF composition is cationic. In some embodiments, the Lp-αPTHF liposomes are cationic 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 in between. In further embodiments, the Lp-αPTHF liposomes are cationic and the composition has a diameter in the range of 80 nm to 120 nm, or any range in between. In some embodiments, the cationic Lp-αPTHF composition contains 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 polyglutamic oxidized alpha-THF. In some embodiments, during the preparation of Lp-αPTHF, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% of polyglutamic oxidized alpha-THF starting material is encapsulated in cationic Lp-αPTHF. In further embodiments, the polyglutamic oxidized alpha-tetrahydrofolate encapsulated by liposomes is present in HEPES buffer within the liposomes.

[0063] In other embodiments, the Lp-αPTHF composition is anionic or neutral. In some embodiments, the Lp-αPTHF 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 in between. In further embodiments, the Lp-αPTHF liposomes are anionic or neutral and the composition has a diameter in the range of 80 nm to 120 nm, or any range in between. In some embodiments, the Lp-αPTHF 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 in between. In further embodiments, the Lp-αPTHF liposomes are anionic and the composition has a diameter in the range of 80 nm to 120 nm, or any range in between. In some embodiments, the Lp-αPTHF liposomes are neutral and have diameters 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 in between. In some embodiments, the anionic or neutral Lp-αPTHF composition contains 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 polyglutamic oxidized alpha-THF. In some embodiments, during the process of preparing Lp-αPTHF, 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 the polyglutamate oxidized alpha-THF starting material is encapsulated in an anionic or neutral Lp-αPTHF. In some embodiments, the anionic or neutral Lp-αPTHF composition contains 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 tetraglutamate oxidized alpha-THF.In some embodiments, the anionic or neutral Lp-αPTHF composition contains 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 pentaglutamine-oxidized alpha-THF. In some embodiments, the anionic or neutral Lp-αPTHF composition contains 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 hexaglutamine-oxidized alpha-THF. In further embodiments, polyglutamate-oxidized alpha-tetrahydrofolate encapsulated by liposomes is present in a HEPES buffer solution within the liposomes.

[0064] In further embodiments, the liposomal polyglutamate-oxidized alpha-tetrahydrofolate composition is pegylated (PLp-αPTHF).

[0065] In some embodiments, the liposomal polyglutamate-oxidized alpha-tetrahydrofolate composition is untargeted (NTLp-αPTHF). That is, the NTLp-αPTHF composition does not have specific affinity for epitopes expressed on the surface of target cells of interest (e.g., epitopes of surface antigens). In further embodiments, the untargeted liposomal polyglutamate-oxidized alpha-tetrahydrofolate composition is pegylated (NTPLp-αPTHF).

[0066] In other embodiments, liposomal polyglutamic acid alpha-tetrahydrofolate compositions are targeted (TLp-αPTHF). That is, the TLp-αPTHF composition includes a targeting moiety that has specific affinity for an epitope (surface antigen) on a target cell of interest. In some embodiments, the targeting moiety of TLp-αPTHF or TPLp-αPTHF is conjugated to the liposome via covalent bonding. In other embodiments, the targeting moiety of TLp-αPTHF or TPLp-αPTHF is conjugated to either or both the PEG and / or outer layer of the liposome. Targeted liposomal polyglutamic acid alpha-tetrahydrofolate compositions (TLp-αPTHF and TPLp-αPTHF) offer further improvements to the efficacy and safety profile of tetrahydrofolate by specifically delivering polyglutamic acid alpha-tetrahydrofolate (e.g., α-pentaglutamine-oxidized and / or α-hexaglutamine-oxidized) tetrahydrofolate to target cells such as cancer cells. In some embodiments, the targeted liposome polyglutamate-oxidized alpha-tetrahydrofolate composition is pegylated (TPLp-αPTHF). In some embodiments, the targeting portion of TLp-αPTHF or TPLp-αPTHF is bound to either or both the PEG and / or outer layer of the liposome. In some embodiments, the targeting portion of TLp-αPTHF or TPLp-αPTHF is bound to the liposome via covalent bonding. The functions of the targeting portion of the TLp-αPTHF and / or TPLp-αPTHF composition include, but are not limited to, targeting the liposome to a target cell of interest in vivo or in vitro; interacting with a surface antigen with which the targeting portion has specific affinity; and delivering the liposome payload (αPTHF) to the cell. Preferred targeting portions are known in the art and are not limited to, antibodies, antigen-binding antibody fragments, scaffold proteins, polypeptides, and peptides. In some embodiments, the targeting portion is a polypeptide.In further embodiments, the targeting portion is a polypeptide containing at least 3, 5, 10, 15, 20, 30, 40, 50, or 100 amino acid residues.

[0067] In some embodiments, the targeting portion of TLp-αPTHF or TPLp-αPTHF is an antibody or an antigen-binding antibody fragment. In further embodiments, the targeting portion includes one or more of the following: 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 portion of TLp-αPTHF or TPLp-αPTHF has specific affinity for epitopes that are selectively expressed on target cells, such as tumor cells, compared to normal cells or non-tumor cells. In some embodiments, the targeting portion has specific affinity for epitopes on tumor cell surface antigens that are present on tumor cells but not present on or difficult to access on non-tumor cells. In some embodiments, the targeting portion has specific affinity for 0.5 × 10⁻⁶ epitopes as measured using BIACORE® analysis. -10 ~10×10 -6 It binds to the target epitope with an equilibrium dissociation constant (Kd) within the range of [specify range].

[0068] In certain embodiments, the TLp-αPTHF or TPLp-αPTHF targeting moiety comprises a polypeptide that specifically binds to a folate receptor. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the folate receptor to which the targeting moiety binds specifically binds to one or more folate receptors selected from the group consisting of folate receptor alpha (FR-α, FOLR1), folate receptor beta (FR-β, FOLR2), and folate receptor delta (FR-δ, FOLR4). In some embodiments, the folate receptor to which the targeting moiety binds is folate receptor alpha (FR-α). In some embodiments, the folate receptor to which the targeting moiety binds is folate receptor beta (FR-β). In some embodiments, the targeting moiety specifically binds to both FR-α and FR-β.

[0069] In further embodiments, the Lp-αPTHF compositions include one or more immunostimulants, detectable markers, and maleimides, which are located on at least one of the PEG and outer surfaces of the liposome. In some embodiments, the liposomal αPTHF composition (e.g., Lp-αPTHF, PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF) is cationic. In other embodiments, the liposomal αPTHF composition (e.g., Lp-αPTHF, PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF) is anionic or neutral. In further embodiments, the liposomes of the liposome-αPTHF composition (e.g., Lp-αPTHF, PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF) have diameters in the ranges of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, 50 nm to 150 nm, or any range in between. In some embodiments, the liposomes of the liposome-αPTHF composition have diameters in the ranges of 30 nm to 175 nm or 50 nm to 150 nm, or any range in between. In further embodiments, the liposomes of the liposome-αPTHF composition have diameters in the ranges of 80 nm to 120 nm, or any range in between. In some embodiments, the liposome-αPTHF composition is pegylated (e.g., PLp-αPTHF, NTPLp-αPTHF, or TPLp-αPTHF). In some embodiments, the liposomal αPTHF composition includes a targeting moiety (e.g., TLp-αPTHF or TPLp-αPTHF). In further embodiments, the liposomal αPTHF composition is pegylated and targeted (e.g., TPLp-αPTHF). In some embodiments, the liposomal αPTHF composition includes polyglutamic alpha-tetrahydrofolate containing 4, 5, 2-10, 4-6, or 6 or more glutamyl groups. In some embodiments, the liposomal αPTHF composition includes tetraglutamic alpha-tetrahydrofolate.In some embodiments, the liposomal αPTHF composition comprises pentaglutamine-oxidized alpha-tetrahydrofolate. In other embodiments, the liposomal αPTHF composition comprises hexaglutamine-oxidized alpha-tetrahydrofolate.

[0070] In some embodiments, the liposome composition comprises polyglutamic oxidized alpha-tetrahydrofolate containing 4, 5, 2-10, 4-6, or 6 or more glutamyl groups, and polyglutamic oxidized alpha-THF in amounts of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more than 75% w / w. In some embodiments, the Lp-αPTHF composition comprises polyglutamic oxidized alpha-tetrahydrofolate containing 4, 5, 2-10, 4-6, or 6 or more glutamyl groups, and polyglutamic oxidized alpha-THF in amounts of 1% to 98.5% w / w. In some embodiments, the liposomes contain polyglutamic oxidized alpha-tetrahydrofolate containing 4, 5, 2-10, 4-6, or 6 or more glutamyl groups, and in the process of preparing Lp-αPTHF, 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 the starting material of polyglutamic oxidized alpha-THF is encapsulated in the Lp-αPTHF.

[0071] In some embodiments, the liposome composition comprises tetraglutamate-oxidized alpha-tetrahydrofolate and 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 tetraglutamate-oxidized alpha-THF. In some embodiments, the Lp-αPTHF composition comprises tetraglutamate-oxidized alpha-tetrahydrofolate and 1% to 98.5% w / w of tetraglutamate-oxidized alpha-THF. In some embodiments, the liposomes contain tetraglutamate-oxidized alpha-tetrahydrofolate, and in the process of preparing Lp-αPTHF, 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 the tetraglutamate-oxidized alpha-THF starting material is encapsulated in the Lp-αPTHF.

[0072] In some embodiments, the liposome composition comprises pentaglutamine-oxidized alpha-tetrahydrofolate and 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 pentaglutamine-oxidized alpha-THF. In some embodiments, the Lp-αPTHF composition comprises pentaglutamine-oxidized alpha-tetrahydrofolate and 1% to 98.5% w / w of pentaglutamine-oxidized alpha-THF. In some embodiments, the liposomes contain pentaglutamine-oxidized alpha-tetrahydrofolate, and in the process of preparing Lp-αPTHF, 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 the pentaglutamine-oxidized alpha-THF starting material is encapsulated in the Lp-αPTHF.

[0073] In some embodiments, the liposome composition comprises hexaglutamate-oxidized alpha-tetrahydrofolate and 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 hexaglutamate-oxidized alpha-THF. In some embodiments, the Lp-αPTHF composition comprises hexaglutamate-oxidized alpha-tetrahydrofolate and 1% to 98.5% w / w of hexaglutamate-oxidized alpha-THF. In some embodiments, the liposomes contain hexaglutamate-oxidized alpha-tetrahydrofolate, and in the process of preparing Lp-αPTHF, 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 the pentaglutamate-oxidized alpha-THF starting material is encapsulated in the Lp-αPTHF.

[0074] Liposome compositions are also provided, comprising liposomes encapsulating αPTHF. In some embodiments, the liposome composition comprises a pegylated αPTHF composition. In some embodiments, the liposome composition comprises an αPTHF composition that is conjugated to or otherwise associated with a targeting moiety. In further embodiments, the liposome composition comprises an αPTHF composition that is pegylated and conjugated to or otherwise associated with a targeting moiety. In some embodiments, the liposome composition comprises an αPTHF having 4, 5, 2 to 10, 4 to 6, or 6 or more glutamyl groups. In some embodiments, the liposome composition comprises tetraglutamate-oxidized alphatetrahydrofolate. In some embodiments, the liposome composition comprises pentaglutamate-oxidized alphatetrahydrofolate. In other embodiments, the liposome composition comprises hexaglutamate-oxidized alphatetrahydrofolate.

[0075] In some embodiments, the liposome composition comprises liposome αPTHF (e.g., Lp-αPTHF, PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, and TPLp-αPTHF). In some embodiments, the liposome αPTHF is pegylated (e.g., NTPLp-αPTHF and TPLp-αPTHF). In some embodiments, the liposome αPTHF comprises a targeting moiety having specific affinity for an antigen epitope on the surface of a target cell of interest, such as cancer cells (e.g., TLp-αPTHF or TPLp-αPTHF). In further embodiments, the liposome composition comprises pegylated liposome αPTHF and further comprises a targeting moiety having specific affinity for an antigen epitope on the surface of a target cell of interest, such as cancer cells (e.g., TPLp-αPTHF). In some embodiments, the liposome composition comprises cationic liposome αPTHF. In other embodiments, the liposome composition comprises liposome αPTHF which is anionic or neutral. In further embodiments, the liposome composition comprises liposome αPTHF having a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, or any range in between. In further embodiments, the liposome αPTHF has a diameter in the range of 80 nm to 120 nm, or any range in between.

[0076] Pharmaceutical compositions are also provided that include polyglutamate-oxidized alpha-tetrahydrofolate (αPTHF) containing a delivery medium such as liposome αPTHF. In some embodiments, the pharmaceutical composition includes a pegylated αPTHF composition. In some embodiments, the pharmaceutical composition includes an αPTHF composition linked to or otherwise conjugated to a targeting moiety. In further embodiments, the pharmaceutical composition includes a pegylated αPTHF composition linked to or otherwise conjugated to a targeting moiety. In some embodiments, the pharmaceutical composition includes αPTHF containing 4, 5, 2-10, 4-6, or 6 or more glutamyl groups. In some embodiments, the pharmaceutical composition includes tetraglutamate-oxidized alpha-tetrahydrofolate. In some embodiments, the pharmaceutical composition includes pentaglutamate-oxidized alpha-tetrahydrofolate. In other embodiments, the pharmaceutical composition includes hexaglutamate-oxidized alpha-tetrahydrofolate.

[0077] In some embodiments, the pharmaceutical composition comprises liposomal αPTHF (e.g., Lp-αPTHF, PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, and TPLp-αPTHF). In some embodiments, the liposomal αPTHF composition is pegylated (e.g., NTPLp-αPTHF and TPLp-αPTHF). In some embodiments, the liposomal αPTHF comprises a targeting moiety having specific affinity for an antigen epitope on the surface of a target cell of interest, such as cancer cells (e.g., TLp-αPTHF or TPLp-αPTHF). In further embodiments, the pharmaceutical composition comprises pegylated liposomal αPTHF, further comprising a targeting moiety having specific affinity for an antigen epitope on the surface of a target cell of interest, such as cancer cells (e.g., TPLp-αPTHF). In some embodiments, the pharmaceutical composition comprises cationic liposomal αPTHF. In other embodiments, the pharmaceutical composition comprises anionic or neutral liposomal αPTHF. In a further embodiment, the pharmaceutical composition comprises liposome αPTHF having a diameter of 20 nm to 500 nm or 20 nm to 500 nm, or any range in between. In a further embodiment, the liposome αPTHF composition has a diameter in the range of 80 nm to 120 nm, or any range in between.

[0078] In further embodiments, the disclosure provides a method for modulating cell activation, chemokine production, or metabolic activity, comprising the step of contacting cells with a composition comprising a polyglutamate-oxidized alpha-tetrahydrofolate (αPTHF) 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 overgrowth cells. In further embodiments, the cells are immune cells. In some embodiments, the method is carried out in vivo. In other embodiments, the method is carried out in vitro. In some embodiments, the αPTHF comprises 4, 5, 2 to 10, 4 to 6, or 6 or more glutamyl groups. In some embodiments, the αPTHF composition comprises tetraglutamate-oxidized alpha-tetrahydrofolate. In some embodiments, the αPTHF composition comprises pentaglutamate-oxidized alpha-tetrahydrofolate. In other embodiments, the αPTHF composition comprises hexaglutamate-oxidized alpha-tetrahydrofolate.

[0079] In further embodiments, the disclosure provides a method for modulating cell activation, chemokine production, or metabolic activity, comprising the step of contacting cells with liposomes comprising a polyglutamate-oxidized alpha-tetrahydrofolate (αPTHF) 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 overgrowth cells. In further embodiments, the cells are immune cells. In some embodiments, the method is carried out in vivo. In other embodiments, the method is carried out in vitro. In some embodiments, the αPTHF comprises 4, 5, 2 to 10, 4 to 6, or 6 or more glutamyl groups. In some embodiments, the αPTHF composition comprises tetraglutamate-oxidized alpha-tetrahydrofolate. In some embodiments, the αPTHF composition comprises pentaglutamate-oxidized alpha-tetrahydrofolate. In other embodiments, the αPTHF composition comprises hexaglutamate-oxidized alpha-tetrahydrofolate.

[0080] In further embodiments, the Disclosure provides a method for killing cells, comprising contacting cells with a composition comprising a polyglutamate-oxidized alpha-tetrahydrofolate (αPTHF) composition (e.g., αPTHF as herein). In some embodiments, the cells to be contacted are mammalian cells. In further embodiments, the cells to be contacted are human cells. In some embodiments, the cells to be contacted are overgrowth cells. In further embodiments, the overgrowth cells are cancer cells. In further embodiments, the cancer cells to be contacted are primary cells or cells derived from cell lines obtained / induced from cancers selected from the group consisting of, for example, non-hematological malignancies including 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, bile duct cancer, gallbladder cancer, bladder cancer, sarcomas (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological malignancies such as, for example, leukemia, lymphoma and other B-cell malignancies, myeloma and other plasmacytic dysplasia or cachexia. In further embodiments, the cancer cells to be contacted are primary cells or cells derived from cell lines obtained / induced from cancers selected from the group consisting of breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma, and chorioadenoma, non-leukemic leumingomeningomatosis, soft tissue sarcoma (tendonoid, invasive fibromatosis), bladder cancer, and central nervous system (CNS) lymphoma. In further embodiments, the cancer cells are primary cells or cells derived from cell lines obtained / induced from cancers selected from colorectal cancer, breast cancer, gastric cancer (e.g., gastric cancer), pancreatic cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer and / or adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell carcinoma. In certain embodiments, the cancer cells are primary cells or cells derived from a cell line obtained / induced from colorectal cancer. In some embodiments, αPTHF contains 4, 5, 2-10, 4-6, or 6 or more glutamyl groups. In some embodiments, αPTHF contains 4 glutamyl groups. In some embodiments, αPTHF contains 5 glutamyl groups. In some embodiments, αPTHF contains 6 glutamyl groups. In some embodiments, this method is carried out in vivo.In other embodiments, this method is carried out in vitro.

[0081] In further embodiments, the present disclosure provides a method for killing cells, comprising the step of contacting cells with liposomes containing polyglutamic oxidized alpha-tetrahydrofolate (Lp-αPTHF such as PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF). In further embodiments, the overgrowthed cells to be contacted are cancer cells. In further embodiments, cancer cells are primary cells or cells derived from cell lines obtained / induced from cancers selected from the group consisting of, 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, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; as well as, for example, hematological malignancies such as leukemia, lymphoma and other B-cell malignancies, myeloma and other plasmacytic dysplasia or cachexia. In further embodiments, the cancer cells to be contacted are primary cells or cells derived from cell lines obtained / induced from cancers selected from the group consisting of breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma, and chorioadenoma, non-leukemic meningeal carcinomatosis, soft tissue sarcoma (tendonoid, invasive fibromatosis), bladder cancer, and central nervous system (CNS) lymphoma. In some embodiments, the cancer cells are primary cells or cells derived from cell lines obtained / induced from cancers selected from colorectal cancer, breast cancer, gastric cancer (e.g., gastric cancer), pancreatic cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer and / or adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell carcinoma. In certain embodiments, the cancer cells are primary cells or cells derived from a cell line obtained / induced from colorectal cancer. In some embodiments, this method is carried out in vivo. In other embodiments, this method is carried out in vitro. In some embodiments, the liposomes contain αPTHF containing 4, 5, 2-10, 4-6, or 6 or more glutamyl groups. In some embodiments, the liposomes contain αPTHF containing 4, 5, 2-10, 4-6, or 6 or more glutamyl groups.In some embodiments, the liposome contains αPTHF containing 4, 5, 2-10, 4-6, or 6 or more glutamyl groups. In some embodiments, the liposome contains αPTHF containing 4 glutamyl groups. In some embodiments, the liposome contains αPTHF containing 5 glutamyl groups. In some embodiments, the liposome contains αPTHF containing 6 glutamyl groups. In some embodiments, the αPTHF contains 1, 2, 3, or 4 or more glutamyl groups having gamma bonds.

[0082] In further embodiments, the disclosure provides a method for treating cancer, comprising administering an effective amount of a delivery medium (e.g., an antibody-immune complex or liposome) containing polyglutamic oxidized alpha-tetrahydrofolate to a subject having or at risk of having cancer. In some embodiments, the delivery medium is an antibody-containing immune complex (e.g., including a full-length IgG antibody, a bispecific antibody, or scFv). In some embodiments, the delivery medium is a liposome (e.g., Lp-αPTHF such as PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF). In some embodiments, the delivery medium to be administered is pegylated. In some embodiments, the delivery medium to be administered is not pegylated. In further embodiments, the delivery medium to be administered includes a targeting moiety having specific affinity for an antigen epitope on the surface of cancer cells. In further embodiments, the delivery medium to be administered includes a targeting moiety having specific affinity for an antigen epitope on the surface of cancer cells.In further embodiments, the delivery medium may include GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, folate receptors (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 outer domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, and endothelin. Receptors, 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, C D11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD 44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 It includes a targeting moiety having specific affinity for an epitope of a cell surface antigen selected from the group consisting of an EphA receptor, EphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, integrins (e.g., integrins αvβ3, αvβ5, or αvβ6), C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha, PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the delivery medium includes a targeting moiety that specifically binds to a cell surface antigen that is determined to originate from or be expressed on a specific target cancer (tumor), such as a neoantigen. In some embodiments, the targeting moiety specifically binds to a cell surface antigen that is determined to originate from or be expressed on a specific target tumor, such as a neoantigen. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the delivery medium to be administered includes an αPTHF containing 4, 5, 2 to 10, 4 to 6, or 6 or more glutamyl groups. In some embodiments, the delivery medium to be administered includes an αPTHF containing 4 glutamyl groups. In some embodiments, the delivery medium to be administered includes an αPTHF containing 5 glutamyl groups. In some embodiments, the delivery medium to be administered includes an αPTHF containing 6 glutamyl groups. In some embodiments, αPTHF is (a) polyglutamic oxidized 5-formyl-THF (e.g., polyglutamic oxidized [6S]-5-formyl-THF); (b) polyglutamic oxidized 10-formyl-THF (e.g., polyglutamic oxidized [6R]-10-formyl-THF); (c) polyglutamic oxidized 5,10-methenyl-THF (e.g., polyglutamic oxidized [6R]-5,10-methenyl-THF); (d) polyglutamic oxidized 5-methyl-THF (e.g., (a) Polyglutamate-oxidized [6S]-5-methyl-THF; (e) Polyglutamate-oxidized tetrahydrofolate THF (e.g., polyglutamate-oxidized [6S]-tetrahydrofolate THF); (f) Polyglutamate-oxidized 5,10-methylene-THF (e.g., polyglutamate-oxidized [6R]-5,10-methylene-THF); and (g) Polyglutamate-oxidized 5-formimino-THF (e.g., polyglutamate-oxidized [6S]-5-formimino-THF). In some embodiments, αPTHF is polyglutamate-oxidized 5,10-methylene-THF. In further embodiments, αPTHF is polyglutamate-oxidized [6R]-5,10-methylene-THF. In other embodiments, αPTHF is polyglutamate-oxidized [6R,S]-5,10-methylene-THF.In some embodiments, αPTHF is polyglutamate-oxidized 5-methyl-THF. In further embodiments, αPTHF is [6S]-5-methyl-THF. In other embodiments, αPTHF is [6R,S]-5-methyl-THF. In some embodiments, αPTHF is polyglutamate-oxidized 5-formyl-THF. In further embodiments, αPTHF is polyglutamate-oxidized [6S]-5-formyl-THF. In other embodiments, αPTHF is polyglutamate-oxidized [6R,S]-5-formyl-THF. In some embodiments, cancer is selected from the group consisting of non-hematological malignancies, such as lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, stomach cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma, brain cancer, central nervous system cancer, and melanoma; and hematological malignancies, such as leukemia, lymphoma and other B-cell malignancies, myeloma, and other plasmacytic dysplasia or cachexia. In some embodiments, cancer is selected from the group consisting of breast cancer, head and neck cancer, lung cancer, stomach cancer, osteosarcoma, non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma, and chorioadenoma, non-leukemic meningopathy, soft tissue sarcoma (tendonoid, invasive fibromatosis), bladder cancer, and central nervous system (CNS) cancer. In some embodiments, the cancer is selected from the group consisting of colorectal cancer, breast cancer, gastric cancer (e.g., stomach cancer), pancreatic cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer and / or adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell carcinoma. In a particular embodiment, the cancer is colorectal cancer.

[0083] In further embodiments, the disclosure provides a method for treating cancer, comprising administering an effective amount of liposomes containing polyglutamic oxidized alpha-tetrahydrofolate (e.g., Lp-αPTHF, e.g., PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF) to a subject having or at risk of having cancer. In some embodiments, the liposomes are pegylated. In some embodiments, the liposomes are not pegylated. In further embodiments, the liposomes include a targeting moiety having specific affinity for an antigen epitope on the surface of cancer cells.In further embodiments, the liposomes may contain GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, folate receptors (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 outer domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, and endothelin. Receptors, 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, C D11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD 44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 It includes a targeting moiety that has specific affinity for an epitope of a cell surface antigen selected from the group consisting of an EphA receptor, EphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, integrins (e.g., integrins αvβ3, αvβ5, or αvβ6), C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha, PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and Musk.This also includes the use of cancer stem cell targeting moieties, such as those targeting CD34, CD133 and CD44, CD138, and CD15. In some embodiments, the liposome includes a targeting moiety that has specific affinity for an epitope of a cell surface antigen derived from or known to express a particular target 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 an αPTHF containing 4, 5, 2-10, 4-6, or 6 or more glutamyl groups. In some embodiments, the liposomes to be administered are (a) polyglutamic acid 5-formyl-THF (e.g., polyglutamic acid [6S]-5-formyl-THF); (b) polyglutamic acid 10-formyl-THF (e.g., polyglutamic acid [6R]-10-formyl-THF); (c) polyglutamic acid 5,10-methenyl-THF (e.g., polyglutamic acid [6R]-5,10-methenyl-THF); (d) polyglutamic acid 5-methyl-THF (e.g., (a) αPTHF selected from (b) polyglutamate-oxidized [6S]-5-methyl-THF; (e) polyglutamate-oxidized tetrahydrofolate THF (e.g., polyglutamate-oxidized [6S]-tetrahydrofolate THF); (f) polyglutamate-oxidized 5,10-methylene-THF (e.g., polyglutamate-oxidized [6R]-5,10-methylene-THF); and (g) polyglutamate-oxidized 5-formimino-THF (e.g., polyglutamate-oxidized [6S]-5-formimino-THF). In some embodiments, the liposomes to be administered contain polyglutamate-oxidized 5,10-methylene-THF. In further embodiments, the liposomes to be administered contain polyglutamate-oxidized [6R]-5,10-methylene-THF. In other embodiments, the liposomes to be administered contain polyglutamate-oxidized [6R,S]-5,10-methylene-THF. In some embodiments, the administered liposomes contain polyglutamic acid 5-methyl-THF. In further embodiments, the administered liposomes contain [6S]-5-methyl-THF. In other embodiments, the administered liposomes contain [6R,S]-5-methyl-THF.In some embodiments, the administered liposomes contain polyglutamine-oxidized 5-formyl-THF. In further embodiments, the administered liposomes contain polyglutamine-oxidized [6S]-5-formyl-THF. In other embodiments, the administered liposomes contain polyglutamine-oxidized [6R,S]-5-formyl-THF. In some embodiments, the administered liposome composition contains tetraglutamine-oxidized αPTHF. In some embodiments, the administered liposome composition contains pentaglutamine-oxidized αPTHF. In some embodiments, the administered liposome composition contains hexaglutamine-oxidized αPTHF. In some embodiments, the liposomes of the administered liposome composition contain αPTHF containing 4, 5, 2-10, 4-6, or 6 or more glutamyl groups. In some embodiments, the liposomes of the administered liposome composition contain αPTHF containing 1, 2, 3, or 4 or more glutamyl groups having gamma bonds. In some embodiments, 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, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain tumor, central nervous system cancer, melanoma, and hematological malignancies (e.g., leukemia or lymphoma). In further embodiments, cancer cells are primary cells or cells derived from cell lines obtained / induced from cancers selected from colorectal cancer, breast cancer, gastric cancer (e.g., gastric cancer), pancreatic cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer and / or lung adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell carcinoma. In certain embodiments, cancer cells are primary cells or cells derived from cell lines obtained / induced from colorectal cancer.

[0084] In further embodiments, the present disclosure provides a method for treating cancer, comprising administering an effective amount of a liposome composition to a subject having or at risk of having cancer, the liposome comprising polyglutamate-oxidized alpha-tetrahydrofolate and a targeting moiety having a specific affinity for an epitope of an antigen on the surface of a cancer tumor.In some embodiments, liposomes contain GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, folate receptors (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 outer domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, and endothelial cells. EGFR receptors, 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, C D44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1 It includes a targeting moiety having specific affinity for an epitope of a cell surface antigen selected from the group consisting of an EphA receptor, EphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, integrins (e.g., integrins αvβ3, αvβ5, or αvβ6), C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha, PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the liposome includes a targeting moiety that has specific affinity for an epitope of a cell surface antigen, such as a neoantigen, that is derived from or known to express a specific target cancer (tumor). In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the liposome includes an αPTHF containing 4, 5, 2-10, 4-6, or 6 or more glutamyl groups. In some embodiments, the administered liposome includes an αPTHF containing 4 glutamyl groups. In some embodiments, the administered liposome includes an αPTHF containing 5 glutamyl groups. In some embodiments, the administered liposome includes an αPTHF containing 6 glutamyl groups. In some embodiments, the liposomes to be administered are (a) polyglutamic acid 5-formyl-THF (e.g., polyglutamic acid [6S]-5-formyl-THF); (b) polyglutamic acid 10-formyl-THF (e.g., polyglutamic acid [6R]-10-formyl-THF); (c) polyglutamic acid 5,10-methenyl-THF (e.g., polyglutamic acid [6R]-5,10-methenyl-THF); (d) polyglutamic acid 5-methyl-THF (e.g., (a) αPTHF selected from (b) polyglutamate-oxidized [6S]-5-methyl-THF; (e) polyglutamate-oxidized tetrahydrofolate THF (e.g., polyglutamate-oxidized [6S]-tetrahydrofolate THF); (f) polyglutamate-oxidized 5,10-methylene-THF (e.g., polyglutamate-oxidized [6R]-5,10-methylene-THF); and (g) polyglutamate-oxidized 5-formimino-THF (e.g., polyglutamate-oxidized [6S]-5-formimino-THF). In some embodiments, the liposomes to be administered contain polyglutamate-oxidized 5,10-methylene-THF. In further embodiments, the liposomes to be administered contain polyglutamate-oxidized [6R]-5,10-methylene-THF. In other embodiments, the liposomes to be administered contain polyglutamate-oxidized [6R,S]-5,10-methylene-THF. In some embodiments, the liposomes to be administered contain polyglutamic acid 5-methyl-THF.In further embodiments, the administered liposomes contain [6S]-5-methyl-THF. In other embodiments, the administered liposomes contain [6R,S]-5-methyl-THF. In some embodiments, the administered liposomes contain polyglutamic acid 5-formyl-THF. In further embodiments, the administered liposomes contain polyglutamic acid [6S]-5-formyl-THF. In other embodiments, the administered liposomes contain polyglutamic acid [6R,S]-5-formyl-THF. In some embodiments, the liposomes contain αPTHF containing one, two, three, or four or more glutamyl groups having gamma bonds.

[0085] In some embodiments, the administered liposome composition includes pegylated liposomes (e.g., TPLp-αPTHF). In some embodiments, the administered liposome composition includes non-pegylated liposomes. In some embodiments, the liposomes of the administered liposome composition contain αPTHF containing 4, 5, 2-10, 4-6, or 6 or more glutamyl groups. In some embodiments, the administered liposomes contain αPTHF containing 4 glutamyl groups. In some embodiments, the administered liposomes contain αPTHF containing 5 glutamyl groups. In some embodiments, the administered liposomes contain αPTHF containing 6 glutamyl groups. In some embodiments, the liposomes to be administered are (a) polyglutamic acid 5-formyl-THF (e.g., polyglutamic acid [6S]-5-formyl-THF); (b) polyglutamic acid 10-formyl-THF (e.g., polyglutamic acid [6R]-10-formyl-THF); (c) polyglutamic acid 5,10-methenyl-THF (e.g., polyglutamic acid [6R]-5,10-methenyl-THF); (d) polyglutamic acid 5-methyl-THF (e.g., (a) αPTHF selected from (b) polyglutamate-oxidized [6S]-5-methyl-THF; (e) polyglutamate-oxidized tetrahydrofolate THF (e.g., polyglutamate-oxidized [6S]-tetrahydrofolate THF); (f) polyglutamate-oxidized 5,10-methylene-THF (e.g., polyglutamate-oxidized [6R]-5,10-methylene-THF); and (g) polyglutamate-oxidized 5-formimino-THF (e.g., polyglutamate-oxidized [6S]-5-formimino-THF). In some embodiments, the liposomes to be administered contain polyglutamate-oxidized 5,10-methylene-THF. In further embodiments, the liposomes to be administered contain polyglutamate-oxidized [6R]-5,10-methylene-THF. In other embodiments, the liposomes to be administered contain polyglutamate-oxidized [6R,S]-5,10-methylene-THF. In some embodiments, the liposomes to be administered contain polyglutamic acid 5-methyl-THF. In further embodiments, the liposomes to be administered contain [6S]-5-methyl-THF.In other embodiments, the administered liposomes contain [6R,S]-5-methyl-THF. In some embodiments, the administered liposomes contain polyglutamic acid 5-formyl-THF. In further embodiments, the administered liposomes contain polyglutamic acid [6S]-5-formyl-THF. In other embodiments, the administered liposomes contain polyglutamic acid [6R,S]-5-formyl-THF. In some embodiments, the liposomes of the administered liposome composition contain αPTHF having one, two, three, or four or more glutamyl groups having gamma bonds. In some embodiments, a liposome composition is administered to treat cancers selected from the group consisting of lung cancer (e.g., non-small cell), pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain tumor, central nervous system cancer, melanoma, myeloma, leukemia, and lymphoma. In some embodiments, a liposome composition is administered to treat cancers selected from the group consisting of colorectal cancer, breast cancer, gastric cancer (e.g., gastric cancer), pancreatic cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer and / or adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell carcinoma. In a specific embodiment, a liposome composition is administered to treat colorectal cancer.

[0086] In further embodiments, the present disclosure provides a method for treating cancer comprising administering an effective amount of a liposome composition to a subject having or at risk of having cancer expressing folate receptors on the cell surface, wherein the liposome composition comprises a liposome comprising (a) polyglutamate-oxidized alpha-tetrahydrofolate (αPTHF) and (b) a targeting moiety having specific affinity for folate receptors. 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 administered liposome composition includes pegylated liposomes (e.g., TPLp-αPTHF). In some embodiments, the administered liposome composition includes non-pegylated liposomes. In some embodiments, the liposomes of the administered liposome composition contain αPTHF containing 4, 5, 2-10, 4-6, or 6 or more glutamyl groups. In some embodiments, the administered liposomes contain αPTHF containing 4 glutamyl groups. In some embodiments, the administered liposomes contain αPTHF containing 5 glutamyl groups. In some embodiments, the administered liposomes contain αPTHF containing 6 glutamyl groups.In some embodiments, the liposomes to be administered are (a) polyglutamic acid 5-formyl-THF (e.g., polyglutamic acid [6S]-5-formyl-THF); (b) polyglutamic acid 10-formyl-THF (e.g., polyglutamic acid [6R]-10-formyl-THF); (c) polyglutamic acid 5,10-methenyl-THF (e.g., polyglutamic acid [6R]-5,10-methenyl-THF); (d) polyglutamic acid 5-methyl-THF (e.g., (a) αPTHF selected from (b) polyglutamate-oxidized [6S]-5-methyl-THF; (e) polyglutamate-oxidized tetrahydrofolate THF (e.g., polyglutamate-oxidized [6S]-tetrahydrofolate THF); (f) polyglutamate-oxidized 5,10-methylene-THF (e.g., polyglutamate-oxidized [6R]-5,10-methylene-THF); and (g) polyglutamate-oxidized 5-formimino-THF (e.g., polyglutamate-oxidized [6S]-5-formimino-THF). In some embodiments, the liposomes to be administered contain polyglutamate-oxidized 5,10-methylene-THF. In further embodiments, the liposomes to be administered contain polyglutamate-oxidized [6R]-5,10-methylene-THF. In other embodiments, the liposomes to be administered contain polyglutamate-oxidized [6R,S]-5,10-methylene-THF. In some embodiments, the administered liposomes contain polyglutamic acid 5-methyl-THF. In further embodiments, the administered liposomes contain [6S]-5-methyl-THF. In other embodiments, the administered liposomes contain [6R,S]-5-methyl-THF. In some embodiments, the administered liposomes contain polyglutamic acid 5-formyl-THF. In further embodiments, the administered liposomes contain polyglutamic acid [6S]-5-formyl-THF. In other embodiments, the administered liposomes contain polyglutamic acid [6R,S]-5-formyl-THF. In some embodiments, the liposomes of the administered liposome composition contain one, two, three, or four or more glutamyl groups containing gamma bonds.In some embodiments, liposomal compositions are administered to treat non-hematological malignancies, 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, bile duct cancer, gallbladder cancer, bladder cancer, sarcomas (e.g., osteosarcoma), brain tumors, central nervous system cancers, and melanoma; as well as cancers selected from the group consisting of, for example, leukemia, lymphoma, and other B-cell malignancies, myeloma, and other hematological malignancies such as plasmacytosis or cachexia. In some embodiments, the liposome composition is administered to treat cancers selected from the group consisting of breast cancer, head and neck cancer, lung cancer, gastric cancer, osteosarcoma, non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), mycosis fungoides (cutaneous T-cell lymphoma), choriocarcinoma and chorioadenoma, non-leukemic meningocarcinoma, soft tissue sarcoma (tendonoid, invasive fibromatosis, bladder cancer), and central nervous system (CNS) lymphoma. In some embodiments, the liposome composition is administered to treat cancers selected from the group consisting of colorectal cancer, breast cancer, gastric cancer (e.g., gastric cancer), pancreatic cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer and / or adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell carcinoma.

[0087] In further embodiments, the present disclosure provides a method for maintenance therapy of cancer, comprising administering an effective amount of a liposomal composition comprising liposomes containing polyglutamate-oxidized alpha-tetrahydrofolate (Lp-αPTHF) to a subject who is or has been treated for cancer. In some embodiments, the liposomal composition to be administered is PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF. In some embodiments, the liposomal composition to be administered comprises pegylated liposomes (e.g., PLp-αPTHF, NTPLp-αPTHF, or TPLp-αPTHF). In some embodiments, the liposomal composition to be administered comprises targeted liposomes (e.g., TLp-αPTHF or TPLp-αPTHF). In some embodiments, the liposomal composition to be administered comprises pegylated liposomes and comprises a targeting portion (e.g., TPLp-αPTHF). In some embodiments, the liposomes of the administered liposome composition contain polyglutamic oxidized alpha-tetrahydrofolate containing 4, 5, 2-10, 4-6, or 6 or more glutamyl groups. In some embodiments, the administered liposomes contain αPTHF containing 4 glutamyl groups. In some embodiments, the administered liposomes contain αPTHF containing 5 glutamyl groups. In some embodiments, the administered liposomes contain αPTHF containing 6 glutamyl groups.In some embodiments, the liposomes to be administered are (a) polyglutamic acid 5-formyl-THF (e.g., polyglutamic acid [6S]-5-formyl-THF); (b) polyglutamic acid 10-formyl-THF (e.g., polyglutamic acid [6R]-10-formyl-THF); (c) polyglutamic acid 5,10-methenyl-THF (e.g., polyglutamic acid [6R]-5,10-methenyl-THF); (d) polyglutamic acid 5-methyl-THF (e.g., (a) αPTHF selected from (b) polyglutamate-oxidized [6S]-5-methyl-THF; (e) polyglutamate-oxidized tetrahydrofolate THF (e.g., polyglutamate-oxidized [6S]-tetrahydrofolate THF); (f) polyglutamate-oxidized 5,10-methylene-THF (e.g., polyglutamate-oxidized [6R]-5,10-methylene-THF); and (g) polyglutamate-oxidized 5-formimino-THF (e.g., polyglutamate-oxidized [6S]-5-formimino-THF). In some embodiments, the liposomes to be administered contain polyglutamate-oxidized 5,10-methylene-THF. In further embodiments, the liposomes to be administered contain polyglutamate-oxidized [6R]-5,10-methylene-THF. In other embodiments, the liposomes to be administered contain polyglutamate-oxidized [6R,S]-5,10-methylene-THF. In some embodiments, the administered liposomes contain polyglutamic acid 5-methyl-THF. In further embodiments, the administered liposomes contain [6S]-5-methyl-THF. In other embodiments, the administered liposomes contain [6R,S]-5-methyl-THF. In some embodiments, the administered liposomes contain polyglutamic acid 5-formyl-THF. In further embodiments, the administered liposomes contain polyglutamic acid [6S]-5-formyl-THF. In other embodiments, the administered liposomes contain polyglutamic acid [6R,S]-5-formyl-THF. In some embodiments, the liposomes of the administered liposome composition contain one, two, three, or four or more glutamyl groups containing gamma bonds.

[0088] In further embodiments, the present disclosure provides a method for treating an immune system disorder, comprising administering an effective amount of a liposomal composition comprising liposomes containing polyglutamic oxidized alpha-tetrahydrofolate (e.g., Lp-αPTHF such as PLp-αPTHF, NTLp-αPTHF, NTPLp-αPTHF, TLp-αPTHF, or TPLp-αPTHF) to a subject having or at risk of having an immune system disorder. In some embodiments, the liposomal composition is administered to treat an autoimmune disease. In further embodiments, the liposomal composition is administered to treat rheumatoid arthritis. In another embodiment, the liposomal composition is administered to treat inflammation. In some embodiments, the administered liposomal composition comprises pegylated liposomes (e.g., PLp-αPTHF, NTPLp-αPTHF, or TPLp-αPTHF). In some embodiments, the administered liposome composition comprises targeted liposomes (e.g., TLp-αPTHF or TPLp-αPTHF) having a targeting moiety that has specific affinity for a surface antigen on target cells of interest (e.g., immune cells). In further embodiments, the administered liposome composition comprises pegylated liposomes (e.g., TPLp-αPTHF) containing a targeting moiety. In some embodiments, the liposomes of the administered liposome composition comprise polyglutamic oxidized alpha-tetrahydrofolate containing 4, 5, 2-10, 4-6, or 6 or more glutamyl groups. In some embodiments, the administered liposome composition comprises αPTHF containing 4 glutamyl groups. In some embodiments, the administered liposome comprises αPTHF containing 5 glutamyl groups. In some embodiments, the administered liposome comprises αPTHF containing 6 glutamyl groups.In some embodiments, the liposomes to be administered are (a) polyglutamic acid 5-formyl-THF (e.g., polyglutamic acid [6S]-5-formyl-THF); (b) polyglutamic acid 10-formyl-THF (e.g., polyglutamic acid [6R]-10-formyl-THF); (c) polyglutamic acid 5,10-methenyl-THF (e.g., polyglutamic acid [6R]-5,10-methenyl-THF); (d) polyglutamic acid 5-methyl-THF (e.g., (a) αPTHF selected from (b) polyglutamate-oxidized [6S]-5-methyl-THF; (e) polyglutamate-oxidized tetrahydrofolate THF (e.g., polyglutamate-oxidized [6S]-tetrahydrofolate THF); (f) polyglutamate-oxidized 5,10-methylene-THF (e.g., polyglutamate-oxidized [6R]-5,10-methylene-THF); and (g) polyglutamate-oxidized 5-formimino-THF (e.g., polyglutamate-oxidized [6S]-5-formimino-THF). In some embodiments, the liposomes to be administered contain polyglutamate-oxidized 5,10-methylene-THF. In further embodiments, the liposomes to be administered contain polyglutamate-oxidized [6R]-5,10-methylene-THF. In other embodiments, the liposomes to be administered contain polyglutamate-oxidized [6R,S]-5,10-methylene-THF. In some embodiments, the administered liposomes contain polyglutamic acid 5-methyl-THF. In further embodiments, the administered liposomes contain [6S]-5-methyl-THF. In other embodiments, the administered liposomes contain [6R,S]-5-methyl-THF. In some embodiments, the administered liposomes contain polyglutamic acid 5-formyl-THF. In further embodiments, the administered liposomes contain polyglutamic acid [6S]-5-formyl-THF. In other embodiments, the administered liposomes contain polyglutamic acid [6R,S]-5-formyl-THF. In some embodiments, the liposomes of the administered liposome composition contain one, two, three, or four or more glutamyl groups containing gamma bonds.

[0089] This disclosure also provides a method for delivering polyglutamate-oxidized alpha-tetrahydrofolate to tumors and / or cancer cells, comprising administering to a subject having a tumor a composition comprising polyglutamate-oxidized alpha-tetrahydrofolate (L-αPTHF) and a targeting moiety having a specific binding affinity to an epitope of a surface antigen of tumor cells or cancer cells. In some embodiments, the targeting moiety to be administered is conjugated to a delivery medium. In some embodiments, the delivery medium is an antibody or an antigen-binding fragment of an antibody. In further embodiments, the delivery medium is a liposome. In some embodiments, the antibody, antigen-binding antibody fragment, or liposome is pegylated. In some embodiments, the composition to be administered comprises polyglutamate-oxidized alpha-tetrahydrofolate containing 4, 5, 2 to 10, 4 to 6, or 6 or more glutamyl groups. In some embodiments, the composition to be administered comprises tetraglutamate-oxidized alpha-tetrahydrofolate. In some embodiments, the composition to be administered comprises pentaglutamate-oxidized alpha-tetrahydrofolate. In other embodiments, the composition to be administered comprises hexaglutamate-oxidized alpha-tetrahydrofolate. In some embodiments, the composition to be administered is (a) polyglutamate-oxidized 5-formyl-THF (e.g., polyglutamate-oxidized [6S]-5-formyl-THF); (b) polyglutamate-oxidized 10-formyl-THF (e.g., polyglutamate-oxidized [6R]-10-formyl-THF); (c) polyglutamate-oxidized 5,10-methenyl-THF (e.g., polyglutamate-oxidized [6R]-5,10-methenyl-THF); (d) polyglutamate-oxidized 5-methyl-THF (e.g., (e) αPTHF selected from (a) polyglutamate-oxidized [6S]-5-methyl-THF); (f) polyglutamate-oxidized tetrahydrofolate THF (e.g., polyglutamate-oxidized [6S]-tetrahydrofolate THF); (g) polyglutamate-oxidized 5,10-methylene-THF (e.g., polyglutamate-oxidized [6R]-5,10-methylene-THF); and (g) polyglutamate-oxidized 5-formimino-THF (e.g., polyglutamate-oxidized [6S]-5-formimino-THF).In some embodiments, the administered composition comprises polyglutamic acid 5,10-methylene-THF. In further embodiments, the administered composition comprises polyglutamic acid [6R]-5,10-methylene-THF. In other embodiments, the administered composition comprises polyglutamic acid [6R,S]-5,10-methylene-THF. In some embodiments, the administered composition comprises polyglutamic acid 5-methyl-THF. In further embodiments, the administered composition comprises [6S]-5-methyl-THF. In other embodiments, the administered composition comprises [6R,S]-5-methyl-THF. In some embodiments, the administered composition comprises polyglutamic acid 5-formyl-THF. In further embodiments, the administered composition comprises polyglutamic acid [6S]-5-formyl-THF. In other embodiments, the administered composition comprises polyglutamic acid [6R,S]-5-formyl-THF.

[0090] In further embodiments, the Disclosure provides a method for preparing a liposome composition comprising a liposome polyglutamate-oxidized alpha-tetrahydrofolate (αPTHF) composition, comprising: forming a mixture comprising a liposome component and α-polyglutamate-oxidized alpha-tetrahydrofolate in solution; homogenizing the mixture in solution to form liposomes; and processing the mixture to form liposomes comprising polyglutamate-oxidized alpha-tetrahydrofolate. In some embodiments, the polyglutamate-oxidized alpha-tetrahydrofolate contains 4, 5, 2 to 10, 4 to 6, or 6 or more glutamyl groups. In some embodiments, the αPTHF composition comprises pentaglutamate-oxidized alpha-tetrahydrofolate. In some embodiments, the αPTHF composition comprises tetraglutamate-oxidized alpha-tetrahydrofolate. In other embodiments, the αPTHF composition comprises hexaglutamate-oxidized alpha-tetrahydrofolate. In some embodiments, polyglutamate-oxidized alpha-tetrahydrofolate contains one, two, three, or four or more glutamyl groups including gamma bonds. In some embodiments, the αPTHF composition contains one, two, three, four, five, six, seven, eight, nine, ten, or eleven or more D-type glutamyl groups. In some embodiments, the αPTHF composition contains two, three, four, five, six, seven, eight, nine, ten, or eleven or more L-type glutamyl groups. In some embodiments, the αPTHF composition contains two, three, four, five, or six or more L-type glutamyl groups and one, two, three, four, five, or six or more D-type glutamyl groups.In some embodiments, the composition is (a) polyglutamic acid 5-formyl-THF (e.g., polyglutamic acid [6S]-5-formyl-THF); (b) polyglutamic acid 10-formyl-THF (e.g., polyglutamic acid [6R]-10-formyl-THF); (c) polyglutamic acid 5,10-methenyl-THF (e.g., polyglutamic acid [6R]-5,10-methenyl-THF); (d) polyglutamic acid 5-methyl-THF (e.g., poly The composition comprises αPTHF selected from (g) glutamate-oxidized [6S]-5-methyl-THF; (e) polyglutamate-oxidized tetrahydrofolate THF (e.g., polyglutamate-oxidized [6S]-tetrahydrofolate THF); (f) polyglutamate-oxidized 5,10-methylene-THF (e.g., polyglutamate-oxidized [6R]-5,10-methylene-THF); and (g) polyglutamate-oxidized 5-formimino-THF (e.g., polyglutamate-oxidized [6S]-5-formimino-THF). In some embodiments, the composition comprises polyglutamate-oxidized 5,10-methylene-THF. In further embodiments, the composition comprises polyglutamate-oxidized [6R]-5,10-methylene-THF. In other embodiments, the composition comprises polyglutamate-oxidized [6R,S]-5,10-methylene-THF. In some embodiments, the composition comprises polyglutamate-oxidized 5-methyl-THF. In further embodiments, the composition comprises [6S]-5-methyl-THF. In other embodiments, the composition comprises [6R,S]-5-methyl-THF. In some embodiments, the composition comprises polyglutamic acid 5-formyl-THF. In further embodiments, the composition comprises polyglutamic acid [6S]-5-formyl-THF. In other embodiments, the composition to be administered comprises polyglutamic acid [6R,S]-5-formyl-THF.

[0091] In one embodiment, the Disclosure provides a kit comprising a polyglutamate-oxidized alpha-tetrahydrofolate composition and / or an αPTHF delivery medium, such as liposomes containing αPTHF or αPTHF immune complexes (e.g., ADCs) as described herein. [Brief explanation of the drawing]

[0092] [Figure 1A-1N] Alpha-tetrahydrofolate: Alpha-5-10-methylene THF (Figure 1A), Alpha-5-10-methylene THF diglutamic acid (Figure 1B), Alpha-5-10-methylene THF triglutamic acid (Figures 1C and 1D), Alpha-5-10-methylene THF tetraglutamic acid (Figures 1E and 1F), Alpha-5-10-methylene THF pentaglutamic acid (Figures 1G and 1H), Alpha-5-10-methylene THF hexaglutamic acid (Figures 1I and 1J), This figure shows the representative chemical formulas of alpha-5-10-methylene THF heptaglutamic acid (Figures 1K and 1L), alpha-5-10-methylene THF octaglutamic acid (Figures 1M and 1N), and representative alpha-tetrahydrofolate polyglutamic acid derivatives, tetrahydrofolate THF, 10-formyl THF, 5-formyl THF, 5-methyl THF, 5-formimino THF, 5,10-methenyl THF, and 5,10-methylene THF (Figures 1O to 1Q). Figures 1R to 1U show representative branched-chain 5-10-methylene THF polyglutamate structures, including branched-chain polyglutamates having an alpha-glutamyl skeleton and gamma-glutamyl branching (Figure 1S), branched-chain polyglutamates having a gamma-glutamyl skeleton and alpha-glutamyl branching (Figure 1T), and branched-chain polyglutamates having both an alpha-glutamyl skeleton and gamma-glutamyl and alpha-glutamyl branching (Figure 1U). [Figure 2] This study demonstrates the relative potency of liposomal pemetrexed alpha-L-hexaglutamate (liposomal aG6) and its enantiomer, liposomal alpha-D-hexaglutamate (liposomal aDG6), compared to pemetrexed, after 48 hours of exposure to cancer cell lines SW620 (CRC), HT-29 (colon cancer), H1806 (triple-negative breast cancer), OAW28 (ovarian cancer), H292 (NSCLC, adenocarcinoma subtype), and H2342 (NSCLC, adenocarcinoma subtype). [Figure 3]This example shows the dose-response relationship, expressed as the percentage of surviving cells after 48 hours of treatment, for free pemetrexed L-gammahexaglutamate (gG6), liposomal pemetrexed L-gammahexaglutamate (liposomal gG6), pemetrexed, and folate receptor alpha-targeted antibody (FR1Ab) liposomal pemetrexed L-gammahexaglutamate (liposomal gG6-FR1Ab) in NCI H2342 non-small cell lung cancer (NSCLC), adenocarcinoma subtype. Folate receptor alpha-targeted liposomes containing alpha-polyglutamic pemetrexed are predicted to successfully target NCI H2342 non-small cell lung cancer cells and reduce their survival rate. [Figure 4] This shows an example of the 48-hour dose-response relationship between free pemetrexed L-gammahexaglutamate (gG6), liposomal pemetrexed L-gammahexaglutamate (liposomal gG6), pemetrexed, and folate receptor alpha-targeted antibody (FR1Ab) liposomal pemetrexed L-gammahexaglutamate (liposomal gG6-FR1Ab) in HT-29 (colon cancer) cells. Folate receptor alpha-targeted liposomes containing alpha-polyglutamic pemetrexed are also predicted to successfully target HT-29 (colon cancer) cells and reduce their survival rate. [Figure 5] This study demonstrates the therapeutic effects of liposomal pemetrexed alpha-L-hexaglutamate (Lps Hexa aG6), liposomal pemetrexed alpha-D-hexaglutamate (Lps Hexa aDG6), and pemetrexed on HCC1806 triple-negative breast cancer cells after 48 hours of exposure. [Figure 6] This study demonstrates the therapeutic effects of liposomal pemetrexed alpha-L-hexaglutamic acid (LPS Hexa aG6), liposomal pemetrexed alpha-D-hexaglutamic acid (LPS Hexa aDG6), and pemetrexed on OAW28 ovarian cancer cells after 48 hours of exposure. [Figure 7]This study compares the therapeutic effects of liposomal pemetrexed alpha-L-hexaglutamate (Lps Hexa aG6) and liposomal pemetrexed alpha-D-hexaglutamate (Lps Hexa aDG6) on H292 non-small cell lung cancer cells over 48 hours, compared to pemetrexed alone. [Figure 8] This study demonstrates the therapeutic effects of liposomal pemetrexed alpha-L-hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D-hexaglutamate (liposomal aDG6), and pemetrexed on H292 non-small cell lung cancer cells after 48 hours of exposure at various dose levels ranging from 16 to 128 nM. Within each dose range tested, the liposomal pemetrexed aG6 formulation showed superior suppression of H292 non-small cell lung cancer cells compared to pemetrexed. [Figure 9] This study demonstrates the therapeutic effects of liposomal pemetrexed alpha-L-hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D-hexaglutamate (liposomal aDG6), and pemetrexed on HCC1806 triple-negative breast cancer cells after 48-hour exposure at various dose levels ranging from 16 to 128 nM. In each dose range tested, the liposomal pemetrexed aG6 formulation was superior to pemetrexed in suppressing HCC1806 triple-negative breast cancer cells. [Figure 10] This study demonstrates the therapeutic effects of liposomal pemetrexed alpha-L-hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D-hexaglutamate (liposomal aDG6), and pemetrexed on OAW28 ovarian cancer cells after 48-hour exposure at a range of concentrations. At a dose of 128 nM, pemetrexed appears to be more effective than the liposomal pemetrexed aG6 formulation. However, at doses of 32 nM and 64 nM, the liposomal formulations exhibit superior therapeutic effects compared to pemetrexed, and at 16 nM, the therapeutic effect of liposomal pemetrexed aG6 is comparable to that of pemetrexed. [Figure 11]The toxicity of liposomal pemetrexed alpha-L-hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D-hexaglutamate (liposomal aDG6), and pemetrexed to differentiated human neutrophils at 64 nM, 128 nM, and 264 nM levels. The figure shows that liposomal pemetrexed aG6 is significantly less toxic to differentiating human neutrophils than pemetrexed. [Figure 12] This study demonstrates the effects on neutrophils (differentiated from CD34+ cells) after 48 hours of exposure to various dose levels ranging from 16 to 128 nM of liposomal pemetrexed alpha-L-hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D-hexaglutamate (liposomal aDG6), and the corresponding pemetrexed agents. [Figure 13] This study demonstrates the effects on AML12 hepatocytes after 48-hour exposure to liposomal pemetrexed alpha-L-hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D-hexaglutamate (liposomal aDG6), and the corresponding 16 nM, 32 nM, 64 nM, and 128 nM doses of pemetrexed. Notably, none of the tested dose levels of the liposomal agents appeared to be toxic to AML12 hepatocytes after treatment with liposomal pemetrexed aG6. In contrast, pemetrexed treatment at any of the tested doses resulted in approximately a 40% reduction in AML12 hepatocyte counts. [Figure 14] This study demonstrates the effects on CCD841 colon epithelial cells after 48-hour exposure to liposomal pemetrexed alpha-L-hexaglutamate (liposomal aG6), liposomal pemetrexed alpha-D-hexaglutamate (liposomal aDG6), and the corresponding pemetrexed at concentrations of 16 nM, 32 nM, 64 nM, and 128 nM. At all tested concentrations, treatment with each tested liposomal composition resulted in a reduction of approximately 20% or less in CCD841 colon epithelial cell count, compared to approximately 50% or more with pemetrexed. [Figure 15]The structures of the polyglutamic acid folate antagonist, cisplatin (CDDP), and two possible aG6-cisplatin complexes are shown. The pH-dependent formation of interchain and / or intrachain coordination between the carboxyl group of the polyglutamic acid folate antagonist and cisplatin may lead to degradation into separate molecules of aG6 and cisplatin upon encountering acidic lysosomes (pH 4-5) and in the presence of intracellular chloride ions. [Figure 16] Hematological parameters: The effects of liposomal aG6 treatment in mice with weekly administration of 40 mg / kg and 80 mg / kg for 4 weeks are shown on white blood cell (WBC) count, neutrophil count, and platelet count. No significant decrease in mean neutrophils, mean white blood cell count, or mean platelet count was observed. [Figure 17] This study demonstrates the effects of administering liposomal aG6 at 40 mg / kg and 80 mg / kg once weekly for 4 weeks on hemoglobin and reticulocyte count in mice. Higher dose levels resulted in the smallest decrease in mean hemoglobin concentration, while simultaneously showing a slight increase in mean reticulocyte count. [Figure 18] This study demonstrates the effects of administering liposomal aG6 to mice at doses of 40 mg / kg and 80 mg / kg once weekly for 4 weeks on liver markers, including serum albumin, serum aspartate aminotransferase (AST), and serum alanine aminotransferase (ALT). No significant increase was observed in either mean AST or mean ALT levels of liver aminotransferases, and no change in mean albumin levels was observed. [Figure 19] The relative tumor volumes of immunodeficient female Nu / J mice (6-8 weeks old) inoculated with NCI-H292 (non-small cell lung cancer) cells and administered intravenously at 167 mg / kg every three weeks with controls, pemetrexed, and liposomal aG6. As these preliminary data show, liposomal aG6 results in lower tumor control compared to pemetrexed. [Figures 20A-20F]Liposomal pemetrexed alpha-L triglutamate (L) over 48 hours for H2342 (NSCLC, adenocarcinoma subtype) (Figure 20A), H292 (NSCLC, adenocarcinoma subtype) (Figure 20B), HT-29 (colon cancer) (Figure 20C), HCC1806 (triple-negative breast cancer) (Figure 20D), MCF7 (ER+ breast cancer) (Figure 20E), and OAW28 (ovarian cancer) (Figure 20F). The dose-response relationships for liposome aG3, liposome pemetrexed alpha-L pentaglutamate (liposome aG5), liposome pemetrexed alpha-L octaglutamate (liposome aG7), and the combination of liposome pemetrexed alpha-L hexaglutamate (aG6) and alpha-L dodecaglutamate (aG12) (liposome aG6 and aG12) are shown. Cell viability was measured using the CellTiter-Glo® (CTG) luminescent cell viability assay, basically as described in Example 1. As can be seen in all cell lines, the potency of each polyglutamic oxidized pemetrexed liposome composition was well above that of the liposome medium and the empty liposome control. [Modes for carrying out the invention]

[0093] This disclosure generally relates to a polyglutamate-oxidized alpha-tetrahydrofolate composition. This composition represents an advance to the existing treatment of hyperproliferative diseases such as cancer. Methods for manufacturing, delivering, and using the polyglutamate-oxidized alpha-tetrahydrofolate composition are also provided. The polyglutamate-oxidized alpha composition has applications including, but is not limited to, hyperproliferative diseases such as cancer, inflammation and immune system disorders such as rheumatoid arthritis, and infectious diseases such as HIV and malaria (e.g., treatment and / or prevention). The polyglutamate-oxidized alpha composition also has applications in combination therapy with one or more therapeutic agents, such as chemotherapeutic agents (e.g., 5-fluorouracil), to enhance the effects of the therapeutic agent, or as a "chemoprotective agent" to mitigate toxic side effects associated with the therapeutic agent (e.g., in combination with folate antimetabolites such as methotrexate).

[0094] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure pertains.

[0095] Whenever an embodiment is described herein with the word “comprising,” it is understood that other similar embodiments are also provided, described in terms of “containing,” “consisting of,” and / or “consisting essentially of.” However, when used as transitional clauses in a claim, each should be interpreted separately and in the appropriate legal and factual context (for example, in a claim, the transitional clause “comprising” is considered a more open phrase, the transitional clause “consisting of” is more exclusive, and “consisting essentially of” is somewhere in between).

[0096] As used herein, the singular forms "a," "an," and "the" refer to multiple objects unless otherwise specified or unless the context makes it clear that multiple references are not intended.

[0097] The term "and / or" as used in phrases such as "A and / or B" is intended to include, in this specification, both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to include, in 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), respectively.

[0098] Headings and subheadings are used for convenience and / or for compliance with official rules only, and are not intended to limit the scope of the subject technology, nor are they referenced in relation to the interpretation of the subject technology. Features described under one heading or subheading of the subject disclosure may be combined with features described under other headings or subheadings in various embodiments. Furthermore, features under a single heading or subheading are not necessarily all used together in various embodiments.

[0099] The terms “tetrahydrofolate” and “THF” are used interchangeably to include tetrahydrofolate in salt, acid, and / or free base forms (e.g., disodium tetrahydrofolate). Unless otherwise specified or clearly evident from the context, “THF” and “tetrahydrofolate” include natural and unnatural forms of THF, including one-carbon substituted THF derivatives. In particular, unless otherwise specified or clearly evident from the context, “THF” and “tetrahydrofolate” include diasteromeric compositions having a [6R] configuration at the C-6 atom of the tetrahydropterin component of THF, diasteromeric compositions having a [6S] configuration at the C-6 atom, and / or mixtures (e.g., 1:1) of [6,R,S] diastereomers. Unless otherwise specified or clearly evident from the context, "THF" and "tetrahydrofolate" refer to (a) 5-formyl-THF (e.g., [6S], [6R,S], or [6R]-5-formyl-THF); (b) 5-formyl-THF (e.g., [6S], [6R,S], or [6R]-5-formyl-THF); (c) 5,10-methenyl-THF (e.g., [6R], [6R,S], or [6S],-5,10-methenyl-THF); (d) 5-methyl-THF (e.g., [6S], [6R,S], or [6R],-5-methyl-THF); (e) tetrahydrofolate THF ((2S)-2-{[4-({[2-amino- (f) 5,10-methylene-THF (e.g., [6R], [6R,S], or [SR], -5,10-methylene-THF); and (g) 5-formimino-THF (e.g., [6S], [6R,S], or [6R], -5-formimino-THF).In some embodiments, the present disclosure provides a composition comprising a THF diastereomer selected from (a) [6S]-5-formyl-THF; (b) [6R]-5-formyl-THF; (c) [6R]-5,10-methenyl-THF; (d) [6S]-5-methyl-THF; (e) [6S]-tetrahydrofolic acid THF; (f) [6R]-5,10-methylene-THF; and (g) [6S]-5-formimino-THF. In some embodiments, the present disclosure provides a composition comprising a mixture of THF diastereomers (e.g., a mixture of diastereoisomers [6R,S]-5-methyl-THF (1:1) and / or a mixture of diastereoisomers [6R,S]-5-CHO-THF (1:1). Compositions comprising a THF salt may further comprise any of various cations such as Na. + , Mg 2+ , K + , NH4 + , and / or Ca 2+ and the like. In certain embodiments, the salt is a pharmaceutically acceptable salt. In further particular embodiments, the THF salt comprises Na + . Tetrahydrofolic acid contains one L-gamma-glutamyl group and is thus considered to be monoglutamylated for the purposes of the present disclosure.

[0100] The term "tetrahydrofolic acid THF" specifically means a THF composition having the structure 2-{[(4-{[(6S)-2-amino-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl]methyl}amino)phenyl]formamido}pentanedioic acid. "Tetrahydrofolic acid THF" may also be referred to herein as a type of tetrahydrofolic acid (THF).

[0101] The terms "polyglutamic acid," "polyglutamic acid," or variations thereof refer to a composition containing at least one chain in which two or more glutamyl groups are bonded. Polyglutamic acid chains can be linear or branched. Linear polyglutamic acid chains may contain glutamyl groups, for example, those containing alpha-carboxyl or gamma-carboxyl group bonds. Branched polyglutamic acid chains may contain one or more glutamyl groups, for example, those containing both alpha-carboxyl and gamma-carboxyl group bonds with other glutamyl groups, thereby creating branching points in the polyglutamic acid. Representative branched polyglutamic acids are shown in Figures 1R-1U. A polyglutamic acid chain contains an N-terminal glutamyl group and one or more C-terminal glutamyl groups. The N-terminal glutamyl group of a polyglutamic acid chain does not bond to another glutamyl group via its amino group, but it does bond to one or more glutamyl groups via its carboxylic acid group. In some embodiments, the N-terminal glutamyl group of polyglutamic tetrahydrofolate is the glutamyl group of tetrahydrofolate. The C-terminal glutamyl group of the polyglutamic acid chain can bond to another glutamyl group via its amino group, but not via its carboxylic acid group.

[0102] The terms "polyglutamic acid-tetrahydrofolate," "polyglutamic acid-THF," "THF-PG," and "PTHF," and their repetitions, refer herein to tetrahydrofolate compositions (i.e., THF-PG) that contain at least one glutamyl group in addition to the glutamyl group of tetrahydrofolate. nIn the formula, n≧1) is used interchangeably. When referring herein to the number of glutamyl groups in αPTHF(αTHF-PG), it takes into account the glutamyl groups of tetrahydrofolate. For example, a THF-PG composition containing five glutamyl residues in addition to the glutamyl groups of THF is referred herein to as hexaglutamate-oxidized tetrahydrofolate or tetrahydrofolate hexaglutamate. In some embodiments, polyglutamic acid-tetrahydrofolate is (a) polyglutamic acid 5-formyl-THF; (b) polyglutamic acid 10-formyl-THF; (c) polyglutamic acid 5,10-methenyl-THF; (d) polyglutamic acid 5-methyl-THF; (e) polyglutamic acid-tetrahydrofolate ((2S)-2-{[4-({[2-amino-4-oxo-1,4,5,6,7,8-hexahydropteridine-6-yl] (g) Polyglutamic acid 5,10-methylene-THF; and (g) Polyglutamic acid 5-formimino-THF, with a member selected from these. Yes. In further embodiments, polyglutamic acid-tetrahydrofolate is (a) polyglutamic acid [6S]-5-formyl-THF; (b) polyglutamic acid [6R]-10-formyl-THF; (c) polyglutamic acid [6R]-5,10-methenyl-THF; (d) polyglutamic acid [6S]-5-methyl-THF; (e) polyglutamic acid [6S]-tetrahydrofolate THF; (f) polyglutamic acid [6R]-5,10-methyl The member is selected from (g) polyglutamic acid [6S]-5-formimino-THF. In some embodiments, polyglutamic acid-tetrahydrofolate is [6R]-5,10-methylene-THF. In some embodiments, polyglutamic acid-tetrahydrofolate is [6S]-5-methyl-THF. In some embodiments, polyglutamic acid-tetrahydrofolate is [6S]-5-formyl-THF.In other embodiments, the polyglutamate-oxidized tetrahydrofolate is a mixture of [6R,S]-5,10-methylene-THF diastereomers, a mixture of [6R,S]-5-methyl-THF diastereomers, or a mixture of [6R,S]-5-formyl-THF diastereomers (e.g., in a weight ratio of 1:1).

[0103] The terms “alpha-glutamyl group,” “alpha-glutamic acid,” and “alpha bond” refer to a glutamyl group containing an alpha-carboxyl bond when they relate to the bonding of a glutamyl group. In some embodiments, the alpha bond is an amide bond between an 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 tetrahydrofolate, or a bond between a glutamyl group and a second glutamyl group not present in tetrahydrofolate, such as a glutamyl group in a polyglutamic acid chain bonded to tetrahydrofolate. In some embodiments, the “alpha-glutamyl group” of the provided polyglutamic alphatetrahydrofolate has both an alpha-carboxyl bond and a gamma-carboxyl bond. In some embodiments, the alpha-glutamyl group is L-type. In some embodiments, the alpha-glutamyl group is D-type. In some embodiments, the glutamyl group is L-type. In some embodiments, one or more glutamyl groups in polyglutamate-oxidized alphatetrahydrofolate are L-type, and one or more glutamyl groups in polyglutamate-oxidized alphatetrahydrofolate are D-type.

[0104] The terms "polyglutamate-oxidized alpha-tetrahydrofolate," "α-polyglutamate-oxidized tetrahydrofolate," "αPTHF," "polyglutamate-oxidized alpha-tetrahydrofolate," "polyglutamate-oxidized alpha-THF," and "αTHF-PG," and their repetitions, refer herein to tetrahydrofolate compositions (e.g., THF-PG) containing at least one glutamyl group having an alpha-carboxyl group bond. nThe terms are used interchangeably to mean (wherein n≧1, α-glutamyl groups). When referring herein to the number of glutamyl groups in αPTHF(αTHF-PG), this includes the glutamyl groups of tetrahydrofolate. For example, an αTHF-PG composition containing five glutamyl groups in addition to the glutamyl groups of THF, of which at least one glutamyl group has an alpha-carboxyl bond, may be referred herein to as alpha-hexaglutamate-oxidized tetrahydrofolate, hexaglutamate-oxidized alpha-tetrahydrofolate, or alpha-tetrahydrofolate-hexaglutamate.

[0105] The terms “gamma-glutamyl group,” “gamma-glutamic acid,” and “gamma bond” refer to glutamyl groups containing gamma-carboxyl bondes when they relate to the bonding of glutamyl groups. In some embodiments, a gamma bond is an amide bond between a gamma-carboxyl group of one glutamyl group and a second glutamyl group. Gamma bonds can be between a glutamyl group and a glutamyl group of tetrahydrofolate, or between a glutamyl group present in tetrahydrofolate and a second glutamyl group, such as a glutamyl group in a polyglutamic acid chain bonded to tetrahydrofolate. In some embodiments, one or more gamma-bonded glutamyl groups in polyglutamic alpha-tetrahydrofolate are L-type. In some embodiments, one or more gamma-bonded glutamyl groups in polyglutamic alpha-tetrahydrofolate are D-type. In some embodiments, one or more gamma-bonded glutamyl groups in polyglutamic alpha-tetrahydrofolate are L-type, and one or more gamma-bonded glutamyl groups in polyglutamic alpha-tetrahydrofolate are D-type.

[0106] As used herein, the term “isolated” means a composition in a form not found in nature. Isolated polyglutamate alpha-oxide compositions include those that have been purified to such an extent that they are no longer in a form found in nature. In some embodiments, the isolated polyglutamate alpha-tetrahydrofolate is substantially pure. Isolated compositions do not contain, or substantially contain, substances that are naturally associated with them, such as other cellular components such as proteins and nucleic acids, which may be found in nature or in the environment in which the composition is prepared (e.g., cell cultures). Polyglutamate alpha-oxide compositions may be formulated with diluents or adjuvants, and may even be isolated for practical purposes, for example, when used for diagnostic or therapeutic purposes, the polyglutamate alpha-oxide composition is usually mixed with a pharmaceutically acceptable carrier or diluent. In some embodiments, isolated polyglutamate alpha-oxide compositions (e.g., alpha-polyglutamic acid and delivery media such as liposomes containing alpha-polyglutamic acid) contain less than 1% or less than 0.1% of undesirable DNA or protein content. In some embodiments, the alpha-polyglutamic acid composition (e.g., alpha-polyglutamic acid and a delivery medium such as liposomes containing alpha-polyglutamic acid) is "isolated".

[0107] As used herein, the term “targeting moiety” means a molecule that enhances affinity to a selected target, e.g., a cell, cell type, tissue, organ, region of the body, or compartment, e.g., a cell, tissue, or organ compartment. Targeting moieties can encompass a wide variety of entities. Targeting moieties may include native 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 bond 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 incorporated herein.

[0108] The terms “specific affinity,” “specifically binding,” and “enhanced affinity” mean that a targeting moiety, such as an antibody or antigen-binding antibody fragment, reacts to or binds to an epitope, protein, or target molecule more frequently, rapidly, for longer periods, with higher affinity, or in some combination of these, than it would to another substance containing a protein unrelated to the antigen containing the target epitope. Due to sequence identity between homologous proteins in different species, specific affinity, in some embodiments, includes a binding agent that recognizes an epitope on a protein or target molecule in two or more species. Similarly, due to homology within specific regions of polypeptide sequences of different proteins, the terms “specific affinity” or “specific binding” may include a binding agent that recognizes an epitope present on two or more proteins and / or target molecules. In certain embodiments, a targeting moiety that specifically binds to a first target is understood to either specifically bind to a second target or not. Thus, “specific affinity” does not necessarily require, but may include, exclusive binding, such as binding to an epitope on a single target. Therefore, in certain embodiments, the targeting portion may specifically bind to epitopes present on two or more targets. In certain embodiments, the same targeting portion that specifically binds to epitopes present on multiple targets may bind to multiple targets.

[0109] The term "epitope" refers to a portion of an antigen that can be recognized and specifically bound to a targeting portion (i.e., a binding site), such as an antibody. When the antigen is a polypeptide, epitopes can be formed from both consecutive and discontinuous amino acids juxtaposed by the protein's tertiary folding. Epitopes formed from consecutive amino acids are usually retained even when the protein denatures, while epitopes formed by tertiary folding are usually lost when the protein denatures. Epitopes typically contain at least three amino acids, more commonly at least five or eight to ten amino acids, in a distinctive spatial higher-order structure.

[0110] Expressions known in the art such as “binding affinity to target,” “binding to target,” and “enhanced affinity,” and similar expressions, refer to the characteristics of the targeting moiety, which can be directly measured by determining the affinity constant, for example, the amount of targeting moiety that binds and dissociates at a given antigen concentration. Intermolecular interactions can be characterized by other methods, but are not limited to competitive analysis, equilibrium analysis, and microcalorimetric analysis, as well as real-time interaction analysis based on surface plasmon resonance interactions (e.g., using Biacore® instruments). 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).

[0111] The term “delivery medium” typically refers to any composition that plays a role in assisting, promoting, or facilitating the entry of polyglutamate-oxidized alpha-tetrahydrofolate into cells. Such delivery mediums are known in the art and are not particularly limited, but include liposomes, lipospheres, polymers (e.g., polymer complexes), peptides, proteins such as antibodies (e.g., immune complexes such as antibody-drug conjugates (ADCs), as well as antigen-binding antibody fragments and their derivatives), cellular 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 liposomal formulations, and combinations thereof. The delivery medium can be directly or indirectly bound to the targeting moiety. In some examples, the targeting moiety is selected from polymers, proteins, peptides, monoclonal antibodies, or fatty acid lipids.

[0112] "Subject" means humans, or, without being particularly limited, dogs, cats, horses, goats, and primates, including vertebrate mammals such as monkeys. Therefore, the present invention can also be used to treat diseases or conditions in non-human subjects. For example, cancer is one of the leading causes of death in companion animals (e.g., cats and dogs). In some embodiments of the present invention, the subject is human. In this disclosure, the terms "subject" and "patient" are used interchangeably and have the same meaning. In general, it is preferable to use the maximum dose, i.e., the maximum safe dose according to sound medical judgment.

[0113] As used herein, “effective dose” means a drug dose sufficient to obtain a medically desirable outcome. The effective dose may vary depending on the desired outcome, the specific disease condition to be treated or prevented, the age and health status of the person being treated, the severity of the disease, the duration of treatment, the nature of any concurrent or adjunctive therapies, the specific route of administration, and similar factors within the scope of the knowledge and expertise of the healthcare professional. The “effective dose” can be determined experimentally or routinely in relation to the stated objective. In the case of cancer, the effective dose of a drug is one that can reduce the number of cancer cells; reduce the size of the tumor; inhibit (i.e., slow, preferably stop) the invasion of cancer cells into surrounding organs; inhibit (i.e., slow, preferably stop) tumor metastasis; inhibit (i.e., slow, preferably stop) tumor growth; and / or alleviate, to some extent, one or more of the symptoms associated with the disorder. A drug may be inhibitory and / or cytotoxic insofar as it can prevent the proliferation of existing cancer cells and / or kill cancer cells. In cancer treatment, in vivo efficacy can be measured, for example, by evaluating survival time, progression-free survival (PFS) time, response rate (RR), response duration, and / or quality of life.

[0114] The terms “hyperproliferative disorder,” “proliferative disorder,” and “proliferative disorder” are used interchangeably herein in relation to undesirable, excessive, or abnormal cell proliferation of cells, whether in vitro or in vivo, such as neoplastic or hyperplastic proliferation. In some embodiments, proliferative disorder is cancer or neoplastic disease (including benign or malignant) and / or any tumor metastasis, regardless of the location of the cancer, tumor, and / or tumor metastasis. In some embodiments, proliferative disorder is a benign or malignant tumor. In some embodiments, proliferative disorder is a non-cancerous disease. In some embodiments, proliferative disorder is a hyperproliferative condition, such as hyperplasia, fibrosis (in particular, pulmonary fibrosis as well as other types of fibrosis such as renal fibrosis), angiogenesis, psoriasis, atherosclerosis, and smooth muscle proliferation of blood vessels such as stenosis or restenosis after angiogenesis.

[0115] The terms “cancer,” “tumor,” or “malignant tumor” are used synonymously to mean any of a number of cell types or diseases characterized by uncontrolled and abnormal cell growth, the ability of affected cells to spread locally or to other parts of the body via the bloodstream and lymphatic system (metastasis), and / or any characteristic structural and / or molecular features known to be associated with cell type or disease. As used herein, “tumor” means all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. “Cancerous tumor” or “malignant cell” is understood to be a cell that has specific structural characteristics, lacks differentiation, and is capable of invasion and metastasis. Cancers that can be treated with the αPTHF compositions provided herein are not particularly limited, but include, for example, non-hematological malignancies 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, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological malignancies such as, for example, leukemia, lymphoma and other B-cell malignancies, myeloma and other plasmacytic dysplasia or cachexia. In some embodiments, the cancer is selected from the group consisting of colorectal cancer, breast cancer, gastric cancer (e.g., gastric cancer), pancreatic cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer and / or adenocarcinoma), head and neck cancer, ovarian cancer, gallbladder cancer, and basal cell carcinoma.

[0116] Other types of cancers and tumors that can be treated with αPTHF compositions are those described herein or known in the art. The terms “cancer,” “cancerous,” “proliferative disorder,” “proliferative disorder,” and “tumor” are not mutually exclusive when used herein.

[0117] Terms such as “to treat,” “treatment,” or “to cure” mean both (a) therapeutic means that cure, suppress, alleviate, and / or halt the progression of symptoms of a diagnosed condition or disorder, and (b) preventive or protective means that prevent and / or delay the onset of a targeted disease or condition. Thus, subjects requiring treatment include subjects who already have cancer, disorder or disease, subjects at risk of having cancer or a condition, and subjects for whom infection or a condition should be prevented. Subjects have been identified, using well-known medical and diagnostic techniques, as being “at risk of having” cancer, infection, immune system disorder, hyperproliferative disorder, or another disease or disorder as referred herein. In certain embodiments, a subject is considered to have been “successfully treated” by the methods provided herein if, for example, symptoms associated with a disease or condition (e.g., cancer, inflammation, and rheumatoid arthritis) are completely, partially, or temporarily remission or disappearance. In certain embodiments, the terms “treating,” “treatment,” or “treat” mean improvement of at least one measurable physical parameter of proliferative disorder, such as tumor growth, which is not necessarily identifiable by the patient. In other embodiments, the terms “treating,” “treatment,” or “treat” mean suppression of the progression of proliferative disorder, for example, physically by stabilizing identifiable symptoms, for example, physiologically by stabilizing physical parameters, or both. In other embodiments, the terms “treating,” “treatment,” or “treat” mean reduction or stabilization of tumor size, tumor cell proliferation or survival, or cancer cell number. Treatment may involve using the αPTHF composition alone or in combination with additional therapeutic agents.

[0118] The terms “subject,” “patient,” and “animal” are used interchangeably and mean human patients and mammals such as non-human primates, as well as laboratory animals such as rabbits, rats, and mice, and other animals. Animals include all vertebrates, e.g., mammals, as well as non-mammals such as chickens, amphibians, and reptiles. As used herein, “mammals” means any member of the class Mammalia, including but not limited to humans and non-human primates, e.g., chimpanzees and other apes and monkey species; domestic animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals such as rodents such as mice, rats, and guinea pigs, and other members of the class Mammalia known in the art. In certain embodiments, the subject is human.

[0119] "Treatment of proliferative disorders" is used herein to encompass maintaining or reducing the size of the tumor of the subject of the proliferative disorder, inducing (partial or complete) tumor regression, inhibiting 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, bile duct cancer, 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 malignancy. Such hematological malignancies include, for example, leukemia, lymphoma and other B-cell malignancies, myeloma, and other plasmacytosis or cachexia.

[0120] 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 autoantigens. Examples of autoimmune diseases include, but are not limited to, Addison’s disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn’s disease, diabetes mellitus (type 1), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves’ disease, Guillain-Barré syndrome, Hashimoto’s disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, inflammation, and rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren’s syndrome, spondyloarthritis, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis.

[0121] The term “therapeutic agent” is used to mean a drug or a derivative thereof that interacts with overgrowth cells, such as cancer cells or immune cells, thereby suppressing the proliferative state of the cells and / or killing them. Examples of therapeutic agents include, but are not limited to, chemotherapeutic agents, cytotoxic agents, platinum-based drugs (e.g., cisplatin, carboplatin, oxaliplatin), taxanes (e.g., Taxol®), etoposides, alkylating agents (e.g., cyclophosphamide, ifosfamide), antimetabolites (e.g., tetrahydrofolate (THF)), 5-fluorouracil, gemcitabine, or derivatives thereof), antitumor antibiotics (e.g., mitomycin, doxorubicin), and plant-derived antitumor agents (e.g., vincristine, vindesine, Taxol®). Such agents include, but are not limited to, the anticancer agents trimethrexate, temozolomide, S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benziganidine (6-BG), bis-chloronitrosourea (BCNU), and camptothecin™, or any therapeutic derivative thereof. Further examples of therapeutic agents suitable for use by the methods of this disclosure include, but are not limited to, anti-restenotic agents, proliferative or antiproliferative agents, anti-inflammatory agents, antineoplastic agents, antimitotic agents, antiplatelet agents, anticoagulants, antifibrin agents, antithrombin agents, cell proliferation inhibitors, antibiotics and other anti-infective agents, anti-enzyme agents, antimetabolite agents, angiogenic agents, cytoprotective agents, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, and / or cardioprotective agents. "Therapeutic agent" also means the salts, acids, and free base forms of the above agents.

[0122] As used herein, the term “chemotherapeutic agent,” when used in connection with cancer treatment, means any agent that causes the death of cancer cells or inhibits the growth or spread of cancer cells. Examples of such chemotherapeutic agents include alkylating agents, antibiotics, antimetabolites, plant-derived drugs, and hormones. In some embodiments, the polyglutamate-oxidized alpha-tetrahydrofolate composition of this disclosure is used in combination with a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is 5-fluorouracil. 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. In certain embodiments, the chemotherapeutic agent is a pyrimidine analog (e.g., a fluoropyrimidine such as 5-fluorouracil (5-FU)).

[0123] The term “antimetabolite” as used herein means a therapeutic agent that inhibits the utilization of a metabolite or its prodrug. Examples of antimetabolites include 5-FU, and 5-FU metabolites and / or prodrugs, such as 5-FUMP, 5-FUDP, 5-FdUMP, capecitabine, tegafur-5-fluorodeoxyuridine monophosphate, etc.; as well as cytarabine and cytarabine prodrugs such as nerarabine, 5-azacitidine, gemcitabine, mercaptopurine, thioguanine, azathiopurine, adenosine, pentostatin, erythrohydroxynonyladenine, and cladribine. Nucleoside analogs, including purines or pyrimidine analogs, are useful antimetabolites for carrying out the methods of this disclosure. In some embodiments, the polyglutamate-oxidized alphatetrahydrofolate composition is used in combination with an antimetabolite selected from the group consisting of fluoropyrimidine, 5-fluorouracil, 5-fluoro-1-(oxolan-2-yl)pyrimidine-2,4-dione, 5-fluoro-2'-deoxycytidine, cytarabine, gemcitabine, troxacitabine, decitabine, azacitidine, pseudoisocytidine, zebralin, ancitabine, fazarabine, 6-azacitidine, capecitabine, N4-octadecylcytarabine, elaidic acid cytarabine, fludarabine, cladribine, clofarabine, nerarabine, folodesine, and pentostatin, or derivatives thereof. In one example, the nucleoside analog is a substrate of a nucleoside deaminase, which is adenosine deaminase or cytidine deaminase. In some cases, the nucleoside analog is selected from fludarabine, cytarabine, gemcitabine, decitabine, and azacitidine or their derivatives.In some examples, nucleoside analogs include N3-alkylated analogs of 5-fluorouracil, 5-fluorouracil derivatives having a 1,4-oxaheteroepane moiety, 5-fluorouracil and nucleoside analogs, cis- and trans-5-fluoro-5,6-dihydro-6-alkoxyuracil, cyclopentane 5-fluorouracil analogs, A-OT-fluorouracil, N4-trimethoxybenzoyl-5'-deoxy-5-fluorocytidine and 5'-deoxy-5-fluorouridine, 1-hexylcarbamoyl-5-fluorouracil, B-3839, The following are selected from uracil-1-(2-tetrahydrofuryl)-5-fluorouracil, 1-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)-5-fluorouracil, doxifluridine, 5'-deoxy-5-fluorouridine, 1-acetyl-3-O-toluyl-5-fluorouracil, 5-fluorouracil-m-formylbenzene-sulfonate (JP No. 55059173), N'-(2-flanidyl)-5-fluorouracil (JP No. 53149985), and 1-(2-tetrahydrofuryl)-5-fluorouracil, or their derivatives. In certain embodiments, the antimetabolite is a pyrimidine analog or a pyrimidine analog prodrug (e.g., fluoropyrimidine). In certain embodiments, the antimetabolite is 5-fluorouracil.

[0124] As used herein, “taxane” refers to an anticancer agent that interferes with, or disrupts, the stability, formation, and / or function of microtubules. Taxanes include paclitaxel and docetaxel, as well as their derivatives, which function on microtubules in the same manner as the taxane 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-conjugated paclitaxel (nab-paclitaxel; Abraxane®), DHA-paclitaxel, or PG-paclitaxel.

[0125] The term "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical formulation other than the active ingredient that is non-toxic to the target. Examples of pharmaceutically acceptable carriers include, but are not limited to, buffers, carriers, excipients, stabilizers, diluents, or preservatives. Examples of pharmaceutically acceptable carriers include one or more solid or liquid fillers, diluents, or encapsulating materials that are suitable and compatible for administration to humans or other subjects.

[0126] This disclosure generally relates to polyglutamine-oxidized alpha-tetrahydrofolate (αPTHF) compositions, and methods for manufacturing and using 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. Gamma-polyglutamine-oxidized compositions also have uses in combination therapy with one or more therapeutic agents, such as chemotherapeutic agents (e.g., 5-fluorouracil), to enhance the effects of the therapeutic agent, or as "chemoprotective agents" to mitigate toxic side effects associated with the therapeutic agent (e.g., in combination with folate antimetabolites such as methotrexate).

[0127] In some embodiments, this disclosure provides: [1] A composition comprising polyglutamate alphatetrahydrofolate. [2] Polyglutamate alpha-tetrahydrofolate, (a) Polyglutamic acid 5-formyl-THF (e.g., polyglutamic acid [6S]-5-formyl-THF); (b) Polyglutamic acid 10-formyl-THF (e.g., polyglutamic acid [6R]-10-formyl-THF); (c) Polyglutamic acid 5,10-methenyl-THF (e.g., polyglutamic acid [6R]-5,10-methenyl-THF); (d) Polyglutamine oxidized 5-methyl-THF (e.g., polyglutamine oxidized [6S]-5-methyl-THF); (e) Polyglutamic acid tetrahydrofolate (e.g., polyglutamic acid [6S]-tetrahydrofolate THF); (f) Polyglutamic acid 5,10-methylene-THF (e.g., polyglutamic acid [6R]-5,10-methylene-THF); and (g) Polyglutamine oxidized 5-formimino-THF (e.g., polyglutamine oxidized [6S]-5-formimino-THF) A composition selected from the group consisting of the following, as described in item [1]. [3] The composition according to item [1] or [2], wherein the polyglutamate-oxidized alphatetrahydrofolate contains 4, 5, 2 to 10, 4 to 6, or 6 or more glutamyl groups having alpha carboxyl group bonds. [4] The composition according to any one of items [1] to [3], wherein the polyglutamate-oxidized alphatetrahydrofolate is tetraglutamate-oxidized alphatetrahydrofolate (e.g., [6R]-5,10-methenyl-THF, [6S]-5-formyl-THF, and / or [6R]-10-formyl-THF). [5] The composition according to any of items [1] to [3], wherein the polyglutamate-oxidized alphatetrahydrofolate is pentaglutamate-oxidized alphatetrahydrofolate (e.g., [6R]-5,10-methenyl-THF, [6S]-5-formyl-THF, and / or [6R]-10-formyl-THF). [6] The composition according to any of items [1] to [3], wherein the polyglutamate-oxidized alphatetrahydrofolate is hexaglutamate-oxidized alphatetrahydrofolate (e.g., [6R]-5,10-methenyl-THF, [6S]-5-formyl-THF, and / or [6R]-10-formyl-THF). [7](a) Two or more glutamyl groups having alpha-carboxyl group bonds, (b) Each glutamyl group of tetrahydrofolate other than the glutamyl group has an alpha-carboxyl group bond; or (c) Two or more glutamyl groups having gammacarboxyl group bonds, A composition as described in any of items [1] to [6]. [8](a) The glutamyl group other than the C-terminal glutamyl group of tetrahydrofolate each has an alpha-carboxyl group bond; or (b) Each glutamyl group other than the C-terminal glutamyl group has an alpha-carboxyl group bond, A composition as described in any of items [1] to [6]. [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] (a) At least two of the glutamyl groups of alpha-polyglutamate tetrahydrofolate are L-type, (b) The glutamyl groups of alpha-polyglutamate tetrahydrofolate are each in the L-type. (c) At least one of the glutamyl groups of alpha-polyglutamate tetrahydrofolate is of the D type. (d) The glutamyl groups of alpha-polyglutamate-oxidized tetrahydrofolate, other than the glutamyl group of tetrahydrofolate, are each of the D type, or (e) At least two of the glutamyl groups of alpha-polyglutamate tetrahydrofolate are L-type, and at least one of the glutamyl groups is D-type. A composition described in any of items [1] to [9];

[11] A composition according to any of items [1] to

[10] , wherein the polyglutamic acid is linear;

[12] A composition according to any of items [1] to

[10] , wherein the polyglutamic acid is a branched chain;

[13] A liposome composition (Lp-αPTHF) containing alpha-polyglutamate tetrahydrofolate as described in any of items [1] to

[12] ;

[14] The LαPP composition according to item

[13] , wherein alpha-polyglutamate tetrahydrofolate contains an L-type glutamyl group having an alpha-carboxyl group bond;

[15] The Lp-αPTHF composition according to item

[13] or

[14] , wherein the glutamyl groups of alpha-polyglutamate tetrahydrofolate are each in the L-type;

[16] The Lp-αPTHF composition according to item

[13] or

[14] , wherein at least one of the glutamyl groups of alpha-polyglutamate tetrahydrofolate is of type D;

[17] Lp-αPTHF compositions according to any of items

[13] to

[16] , wherein the liposomes contain alpha-polyglutamate tetrahydrofolate having 4, 5, 2 to 10, 4 to 6, or 6 or more glutamyl groups;

[18] An Lp-αPTHF composition according to any of items

[13] to

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

[19] A 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] A composition according to any of items

[13] to

[19] , comprising two, three, four, five, two to ten, four to six, or six or more glutamyl groups having both alpha-carboxyl group bonds and gamma-carboxyl group bonds;

[21] Lp-αPTHF compositions according to any of items

[13] to

[20] , wherein the liposomes contain alpha-tetraglutamine-oxidized tetrahydrofolate, alpha-pentaglutamine-oxidized tetrahydrofolate, or alpha-hexaglutamine-oxidized tetrahydrofolate;

[22] Lp-αPTHF compositions according to any of items

[13] to

[21] , wherein the polyglutamic acid is linear or branched;

[23] Lp-αPTHF compositions according to any of items

[13] to

[22] , in which the liposomes are pegylated (PαLp-αPTHF);

[24] Lp-αPTHF compositions according to any of items

[13] to

[23] , wherein the liposomes contain at least 1% by weight of alpha-polyglutamate-oxidized tetrahydrofolate, or in the step of preparing Lp-αPTHF, at least 1% of the alpha-polyglutamate-oxidized THF starting material is encapsulated in the Lp-αPTHF;

[25] Lp-αPTHF compositions according to any of items

[13] to

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

[26] Lp-αPTHF composition according to any of items

[13] to

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

[27] Lp-αPTHF compositions according to any of items

[13] to

[26] , wherein liposomes are formed from liposome components;

[28] The Lp-αPTHF composition according to item

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

[29] The Lp-αPTHF composition according to item

[27] or

[28] , wherein the liposome component comprises 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] Lp-αPTHF compositions according to any of items

[27] to

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

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

[27] to

[30] , further comprising one or more liposome components and a steric stabilizer;

[32] The Lp-αPTHF 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; oligoglycerin, polyethylene glycol and polypropylene oxide-containing copolymer, poloxamer 188, and polyvinyl alcohol;

[33] The Lp-αPTHF composition according to item

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

[34] Lp-αPTHF compositions according to any of items

[13] to

[33] , wherein the liposomes are anionic or neutral;

[35] Lp-αPTHF compositions according to any of items

[13] to

[33] , wherein liposomes have a zeta potential of zero or less;

[36] Lp-αPTHF compositions according to any of items

[13] to

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

[37] Lp-αPTHF compositions according to any of items

[13] to

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

[38] Lp-αPTHF compositions according to any of items

[13] to

[33] , wherein the liposomes are cationic;

[39] Lp-αPTHF compositions according to any of items

[13] to

[38] , wherein the liposomes have an internal space comprising polyglutamate-oxidized alphatetrahydrofolate and an aqueous pharmaceutically acceptable carrier;

[40] The Lp-αPTHF composition described in item

[39] , wherein the pharmaceutically acceptable carrier contains an isotonic agent such as dextrose, mannitol, glycerol, potassium chloride, or sodium chloride in a concentration of more than 1%;

[41] The Lp-αPTHF composition described in item

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

[42] The Lp-αPTHF composition described in item

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

[43] Lp-αPTHF compositions according to any of items

[39] to

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

[44] An Lp-αPTHF composition according to any of items

[39] to

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

[45] Lp-αPTHF compositions according to any of items

[39] to

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

[46] Lp-αPTHF compositions according to any of items

[39] to

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

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

[13] to

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

[48] ​​Lp-αPTHF compositions according to any of items

[13] to

[47] , wherein the liposomes contain less than 500,000 molecules or less than 200,000 molecules of alpha-polyglutamate tetrahydrofolate;

[49] Lp-αPTHF compositions according to any of items

[13] to

[48] , wherein the liposomes contain 10 to 100,000 molecules or any range in between of alpha-polyglutamate tetrahydrofolate;

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

[13] to

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

[51] The Lp-αPTHF composition according to item

[50] , wherein the targeting portion is bound to either or both the PEG and / or outer layer of the liposome, and optionally, the targeting portion is covalently bound to either or both the PEG and / or outer layer of the liposome;

[52] The Lp-αPTHF composition according to item

[50] or

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

[53] Lp-αPTHF compositions according to any of items

[50] to

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

[54] The targeting portion was 0.5 × 10 in measurements using BIACORE® analysis. -10 ~10×10 -6 Lp-αPTHF compositions described in any of items

[50] to

[53] that bind to surface antigens with an equilibrium dissociation constant (Kd) in the range of

[50] to

[53] ;

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

[50] to

[55] , wherein the targeting portion 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-αPTHF composition according to any of items

[50] to

[56] , wherein the targeting portion comprises one or more selected from the group consisting of antibodies, humanized antibodies, antigen-binding fragments of antibodies, single-chain antibodies, single-domain antibodies, bispecific antibodies, synthetic antibodies, pegylated antibodies, and multimeric antibodies;

[57] Lp-αPTHF compositions according to any of items

[50] to

[56] , wherein each pegylated liposome contains 1 to 1,000 or 30 to 200 targeting moieties;

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

[39] to

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

[59] The Lp-αPTHF composition according to item

[58] , wherein the immunostimulant is at least one selected from the group consisting of protein immunostimulants, nucleic acid immunostimulants, chemoimmunostimulants, haptens, and adjuvants;

[60] Immunostimulants include fluorescein, fluorescein isothiocyanate (FITC), DNP, beta-glucan, beta-1,3-glucan, beta-1,6-glucan; resolvin (e.g., D n-6DPA Or D n-3DPA An Lp-αPTHF composition as described in item

[58] or

[59] , which is at least one selected from the group consisting of resolvin D, resolvin E, or T-series resolvins, and Toll-like receptor (TLR) modulators such as oxidized low-density lipoproteins (e.g., OXPAC, PGPC) and erythritol lipids (e.g., E5564);

[61] Lp-αPTHF compositions according to any of items

[58] to

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

[62] Lp-αPTHF compositions according to any of items

[58] to

[61] , further comprising a hapten;

[63] The Lp-αPTHF composition according to item

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

[64] An Lp-αPTHF composition according to any of

[13] to

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

[65] Targeted compositions comprising any of the compositions described in items [1] to

[64] ;

[66] Non-targeting compositions comprising any of the compositions described in items [1] to

[49] ;

[67] Lp-αPTHF compositions according to any of items

[13] to

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

[68] A pharmaceutical composition comprising the liposomal alpha-polyglutamine oxidized tetrahydrofolate composition described in any of items

[13] to

[67] ;

[69] A pharmaceutical composition comprising alpha-polyglutamate tetrahydrofolic acid as described in any of items [1] to [8];

[70] Compositions described in any of items [1] to

[69] , used for the treatment of a disease;

[71] Use of any of the compositions described in items [1] to

[70] for the manufacture of drugs for the treatment of disease;

[72] Use of any of the compositions [1] to

[70] in the manufacture of drugs for use in combination with one or more therapeutic agents, such as chemotherapeutic agents (e.g., 5-fluorouracil), for the treatment of a disease and / or to enhance the effect of one or more therapeutic agents, or as a “chemoprotective agent” to reduce toxic side effects associated with one or more therapeutic agents (e.g., in combination with folate antimetabolites such as methotrexate);

[73] A method for treating or preventing a disease of a subject requiring treatment or prevention, comprising administering to the subject any of the compositions of [1] to

[69] ;

[74] A method for treating or preventing a disease of a subject requiring treatment or prevention, comprising administering to the subject a liposomal polyglutamate alphatetrahydrofolate composition described in any of

[13] to

[69] ;

[75] A method for killing overgrown cells, comprising contacting the overgrown cells with a composition described in any of [1] to

[69] ;

[76] A method for killing hyperproliferating cells, comprising contacting the hyperproliferating cells with a liposomal polyglutamate-oxidized alphatetrahydrofolate composition described in any of

[13] to

[69] ;

[77] The method according to item

[75] or

[76] , wherein the overgrowth cells are cancer cells, mammalian cells, and / or human cells;

[78] A method for treating cancer, comprising administering an effective amount of any of the compositions described in items [1] to

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

[79] A method for treating cancer, comprising administering an effective amount of a liposomal polyglutamate oxidized alphatetrahydrofolate composition described in any of items

[13] to

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

[80] The method according to item

[78] or

[79] , wherein the cancer is selected from the group consisting of, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, stomach cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bile duct cancer, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; as well as the method according to item

[78] or

[79] , for example, leukemia, lymphoma and other B-cell malignancies, myeloma and other plasma cell proliferation disorders;

[81] The method according to item

[78] or

[79] , which treats or prevents cancer, 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;

[82] The method according to item

[78] or

[79] , which treats or prevents cancer, 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;

[83] The method according to item

[78] or

[79] , for treating or preventing cancer, wherein the cancer is selected from the group consisting of colorectal cancer, breast cancer, ovarian cancer, lung cancer, head and neck cancer, pancreatic cancer, gastric cancer, and mesothelioma;

[84] A method for treating cancer, comprising administering an effective amount of an Lp-αPTHF composition described in any of items

[50] to

[66] to a subject having or at risk of having cancer cells that express folate receptors on their surface to which a targeting moiety binds;

[85] Maintenance therapy for subjects who are currently receiving or have previously received cancer treatment, comprising administering to subjects who are currently receiving or have previously received cancer treatment an effective amount of any of the compositions described in items [1] to

[69] ;

[86] Maintenance therapy for subjects who are currently receiving or have previously received cancer treatment, comprising administering to subjects who are currently receiving or have previously received cancer treatment an effective amount of a liposomal polyglutamate oxidized alphatetrahydrofolate composition described in any of items

[13] to

[69] ;

[87] A method for treating an immune system disorder, comprising administering an effective amount of any of the compositions described in items [1] to

[69] to a subject having or at risk of having an immune system disorder, wherein the immune system disorder is optionally 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 arteritis, and psoriasis;

[88] A method for treating an immune system disorder, comprising administering an effective amount of a liposomal polyglutamate-oxidized alpha-tetrahydrofolate composition described in any of items

[13] to

[69] to a subject having or at risk of having an immune system disorder, wherein the immune system disorder is optionally 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 arteritis, and psoriasis;

[89] The following treatment methods: (a) A method for treating leukopenia, comprising administering an effective dose of any of the compositions described in items [1] to

[69] to subjects who have or are at risk of having leukopenia; (b) A method for treating an infection, comprising administering an effective dose of any of the compositions described in items [1] to

[69] to a subject who has or is at risk of having an infection; (c) A method for treating a cardiovascular or metabolic disease, comprising administering an effective amount of any of the compositions described in items [1] to

[69] to a subject who has or is at risk of having an infection, 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; (d) A method for treating an autoimmune disease, comprising administering an effective amount of any of the compositions described in items [1] to

[69] to a subject who has or is at risk of having an autoimmune disease; (e) A method for treating rheumatoid arthritis, comprising administering an effective dose of any of the compositions described in items [1] to

[69] to subjects who have or are at risk of having rheumatoid arthritis; (f) A method for treating an inflammatory condition, comprising administering an effective amount of any of the compositions described in items [1] to

[69] to a subject who has or is at risk of having inflammation, wherein the inflammation is acute, chronic, and / or systemic inflammation; or (g) A method for treating a skin disease, comprising administering an effective amount of any of the compositions described in items [1] to

[69] to a subject who has or is at risk of having a skin disease;

[90] A method for treating an infection, comprising administering an effective amount of a liposomal alpha-polyglutamine oxidized tetrahydrofolate composition described in any of items

[13] to

[69] to a subject who has or is at risk of having an infection;

[91] A method for delivering polyglutamate-oxidized alpha-tetrahydrofolate to a tumor expressing folate receptors on its surface, comprising administering to a subject having a tumor an amount of the Lp-αPTHF composition described in any of items [1] to

[69] , in an amount that delivers a therapeutically effective amount of alpha-polyglutamate-oxidized tetrahydrofolate to the tumor;

[92] A method for preparing an alpha-polyglutamate tetrahydrofolate composition comprising a liposome alpha-polyglutamate tetrahydrofolate composition described in any of items

[13] to

[69] , comprising: forming a mixture in solution comprising: homogenizing the mixture in solution to form liposomes; and processing the mixture to form liposomes containing alpha-polyglutamate tetrahydrofolate;

[93] A method for preparing alpha-polyglutamate tetrahydrofolate comprising a liposome alpha-polyglutamate tetrahydrofolate composition described in any of items

[13] to

[69] , comprising: forming a mixture containing a liposome component and alpha-polyglutamate tetrahydrofolate in solution; and processing the mixture to form liposomes containing alpha-polyglutamate tetrahydrofolate,

[94] The method according to item

[93] , wherein processing the mixture comprises homogenizing the mixture in solution to form liposomes,

[95] A method for preparing any of the compositions described in items

[51] to

[70] , comprising: forming a mixture in solution containing a liposome component and alpha-polyglutamate tetrahydrofolate; homogenizing the mixture in solution to form liposomes; processing the mixture to form liposomes in which alpha-polyglutamate tetrahydrofolate is encapsulated and / or contained; and conferring a targeting moiety to 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] A method for preparing any of the compositions described in items

[51] to

[70] , comprising: forming a mixture in solution comprising a liposome component and alpha-polyglutamate-oxidized tetrahydrofolate; processing the mixture to form liposomes in which alpha-polyglutamate-oxidized tetrahydrofolate is encapsulated and / or covered; and imparting a targeting moiety to 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).

[97] The method according to item

[96] , wherein the processing step includes homogenizing the mixture in solution to form liposomes,

[98] The method according to item

[93] , wherein the processing steps include one or more steps from thin film hydration, extrusion, in-line mixing, ethanol injection techniques, freeze-thaw methods, reverse-phase evaporation methods, dynamic high-pressure microfluidization, microfluidic mixing, double emulsion, freeze-dried double emulsion, 3D printing, membrane contactor methods, and stirring; and / or

[99] The method according to any of items

[96] to

[98] , wherein the processing step includes one or more steps of changing the size of the liposomes by one or more steps of extrusion, high-pressure microfluidization, and / or sonication; and / or

[0100] The method according to any one of items

[92] to

[99] , wherein at least 1% of the alpha-polyglutamate tetrahydrofolate starting material is encapsulated or enclosed in liposomes.

[0128] II. Polyglutamic oxidized alpha-tetrahydrofolate (αPTHF) This disclosure relates, in general, to polyglutamate-oxidized alpha-tetrahydrofolate (αPTHF) compositions. The αPTHF compositions have at least one glutamyl group having an alpha-carboxyl linkage. These compositions are structurally different from L-gamma-polyglutamate-oxidized tetrahydrofolate (produced by the enzyme folylpoly-gamma). In some embodiments, the αPTHF compositions contain 2 to 20, 2 to 15, 2 to 10, 2 to 5, 2 to 6, or 6 or more glutamyl groups (including the glutamyl groups in tetrahydrofolate). In some embodiments, each glutamyl group in the αPTHF other than the glutamyl group in tetrahydrofolate has an alpha linkage. In some embodiments, each glutamyl group in the αPTHF other than the C-terminal glutamyl group and each glutamyl group in tetrahydrofolate have an alpha linkage. In some embodiments, each glutamyl group in the αPTHF other than the C-terminal glutamyl group has an alpha linkage. In some embodiments, two or more glutamyl groups in αPTHF have gamma bonds. In some embodiments, at least one glutamyl group of alpha-polyglutamate-oxidized tetrahydrofolate has both alpha-carboxyl and gamma-carboxyl bonds. In some embodiments, each glutamyl group in αPTHF is L-type. In some embodiments, glutamyl groups in αPTHF other than those of tetrahydrofolate are D-type. In some embodiments, αPTHF comprises two or more L-type glutamyl groups and one or more D-type glutamyl groups. In some embodiments, the polyglutamate chain of αPTHF is linear (not branched). In some embodiments, the polyglutamate chain of αPTHF is branched.

[0129] In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate is diglutamate-oxidized. That is, alpha-polyglutamate-oxidized tetrahydrofolate contains one additional glutamyl group in addition to the glutamyl group of tetrahydrofolate (αTHF-PG1), and this additional glutamyl group is bonded to the glutamyl group in tetrahydrofolate via an alpha bond. In some embodiments, the glutamyl groups of alpha-diglutamate-oxidized tetrahydrofolate are each L-type. In other embodiments, alpha-diglutamate-oxidized THF contains D-type glutamyl groups.

[0130] In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate is triglutamate-oxidized. That is, alpha-polyglutamate-oxidized tetrahydrofolate contains two additional glutamyl groups in addition to the glutamyl group of tetrahydrofolate (αTHF-PG2). In some embodiments, each of the two 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 alpha bonds. 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 and a gamma bond. In some embodiments, each glutamyl group in alpha-triglutamate-oxidized tetrahydrofolate is L-type. In other embodiments, alpha-triglutamine-oxidized THF contains D-type glutamyl groups. In further embodiments, the glutamyl groups of alpha-triglutamine-oxidized tetrahydrofolate, other than the glutamyl group of tetrahydrofolate, are each D-type. In further embodiments, triglutamine-oxidized THF 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.

[0131] In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate is tetraglutamate-oxidized and therefore contains three additional glutamyl groups in addition to the glutamyl groups in tetrahydrofolate (αTHF-PG3). 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 group 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 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 and a gamma bond. In some embodiments, alpha-tetraglutamate-oxidized THF contains two or more L-type glutamyl groups. In further embodiments, the glutamyl groups of alpha-tetraglutamate-oxidized tetrahydrofolate are each L-type. In other embodiments, alpha-tetraglutamate-oxidized THF contains D-type glutamyl groups. In further embodiments, the glutamyl groups of alpha-tetraglutamate-oxidized tetrahydrofolate, other than the glutamyl group of tetrahydrofolate, are each D-type. In further embodiments, tetraglutamate-oxidized THF 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.

[0132] In some embodiments, polyglutamate-oxidized alpha-tetrahydrofolate is pentaglutamate-oxidized (αTHF-PG4) and contains a chain of four additional glutamyl groups bonded to the glutamyl group in tetrahydrofolate. In some embodiments, each of the four additional glutamyl groups in the chain has an alpha bond. In some embodiments, each of the four additional glutamyl groups in the chain, excluding the C-terminal glutamyl group, has an alpha bond. In other embodiments, one, two, or three of the four additional glutamyl groups have an alpha bond, and the remaining three, two, or one glutamyl group are each bonded to the molecular glutamyl group via a gamma bond. In other embodiments, one or two of the four additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl group is bonded to the molecular glutamyl group via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha and a gamma bond. In some embodiments, at least one of the five glutamyl groups has both an alpha and a gamma bond. In some embodiments, each of the five glutamyl groups in the chain, excluding the C-terminal glutamyl group, has an alpha bond. In some embodiments, alpha-pentaglutamine-oxidized THF contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of alpha-pentaglutamine-oxidized tetrahydrofolate is L-type. In other embodiments, alpha-pentaglutamine-oxidized THF contains D-type glutamyl groups. In further embodiments, each glutamyl group of alpha-pentaglutamine-oxidized tetrahydrofolate, excluding the glutamyl group of tetrahydrofolate, is D-type. In further embodiments, pentaglobulin-oxidized THF 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.

[0133] In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate is hexaglutamate-oxidized (αTHF-PG5) and contains a chain of five additional glutamyl groups bonded to the glutamyl group in tetrahydrofolate. In some embodiments, each of the five additional glutamyl groups in the chain has an alpha bond. In some embodiments, each of the five additional glutamyl groups in the chain, excluding the C-terminal glutamyl group, has an alpha bond. In some embodiments, four of the five additional glutamyl groups in the chain have alpha bonds. In other embodiments, one, two, three, or four of the five additional glutamyl groups are bonded to the molecular glutamyl group via alpha bonds, and the remaining four, three, two, or one glutamyl group are each bonded to the molecular glutamyl group via gamma bonds. In other embodiments, one, two, three, or four of the five additional glutamyl groups have alpha bonds, and the remaining non-C-terminal glutamyl groups are bonded to the molecule's glutamyl groups via gamma bonds. In some embodiments, at least one additional glutamyl group has both alpha and gamma bonds. In some embodiments, at least one of the six glutamyl groups has both alpha and gamma bonds. In some embodiments, each of the six glutamyl groups in the chain other than the C-terminal glutamyl group has an alpha bond. In some embodiments, five of the six glutamyl groups have alpha bonds. In some embodiments, alpha-hexaglutamate-oxidized THF contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group in alpha-hexaglutamate-oxidized tetrahydrofolate is L-type. In some embodiments, alpha-hexaglutamate-oxidized THF contains D-type glutamyl groups. In further embodiments, the glutamyl groups of alpha-hexaglutamate-oxidized tetrahydrofolate, other than the glutamyl group of tetrahydrofolate, are each of the D type. In further embodiments, hexaglutamate-oxidized THF comprises 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.

[0134] In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate is heptaglutamate (αTHF-PG6) and therefore contains a chain of six additional glutamyl groups bonded to the glutamyl group of tetrahydrofolate. 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 has an alpha bond. In some embodiments, five of the six additional glutamyl groups in the chain have alpha bonds. In other embodiments, one, two, three, four, or five of the six additional glutamyl groups have alpha bonds, and the remaining five, four, three, two, or one glutamyl group each have a gamma bond. In other embodiments, one, two, three, four, or five of the six additional glutamyl groups have alpha bonds, and the remaining non-C-terminal glutamyl group is 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 and a gamma bond. In some embodiments, at least one of the seven glutamyl groups has both an alpha and a gamma bond. In some embodiments, each of the seven glutamyl groups in the chain, excluding the C-terminal glutamyl group, has an alpha bond. In some embodiments, six of the seven glutamyl groups have an alpha bond. In some embodiments, alpha-heptaglutamine-oxidized THF contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of alpha-heptaglutamine-oxidized tetrahydrofolate is L-type. In other embodiments, alpha-heptaglutamine-oxidized THF contains D-type glutamyl groups. In further embodiments, each glutamyl group of alpha-heptaglutamine-oxidized tetrahydrofolate, excluding the glutamyl group of tetrahydrofolate, is D-type. In further embodiments, heptaglutamine-oxidized THF 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.

[0135] In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate is octaglutamate-oxidized (αTHF-PG7) and therefore contains a chain of seven additional glutamyl groups bonded to the glutamyl group of tetrahydrofolate. In some embodiments, each of the seven additional glutamyl groups in the chain, excluding the C-terminal glutamyl group, has an alpha bond. In some embodiments, six of the seven additional glutamyl groups in the chain have alpha bonds. 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 alpha bonds, and the remaining six, five, four, three, two, or one glutamyl group each have a gamma bond. In other embodiments, one, two, three, four, five, or six of the seven additional glutamyl groups have alpha bonds, and the remaining non-C-terminal glutamyl group is bonded to the molecule's glutamyl group via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha and a gamma bond. In some embodiments, at least one of the eight glutamyl groups has both an alpha and a gamma bond. In some embodiments, each of the eight glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, seven of the eight glutamyl groups have an alpha bond. In some embodiments, alpha-octaglutamine-oxidized THF contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of alpha-octaglutamine-oxidized tetrahydrofolate is L-type. In other embodiments, alpha-octaglutamine-oxidized THF contains D-type glutamyl groups. In further embodiments, each glutamyl group of alpha-octaglutamine-oxidized tetrahydrofolate other than the glutamyl group of tetrahydrofolate is D-type. In further embodiments, octaglutamine-oxidized THF 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.

[0136] In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate is nonaglutamate-oxidized (αTHF-PG8) and contains a chain of eight additional glutamyl groups bonded to the glutamyl group of tetrahydrofolate. In some embodiments, each of the eight additional glutamyl groups in the chain, excluding the C-terminal glutamyl group, 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 each have a gamma bond. In other embodiments, one, two, three, four, five, six, or seven of the eight additional glutamyl groups have alpha bonds, and the remaining non-C-terminal glutamyl groups are bonded to the molecule's glutamyl groups via gamma bonds. In some embodiments, at least one additional glutamyl group has both alpha and gamma bonds. In some embodiments, at least one of the nine glutamyl groups has both alpha and gamma bonds. In some embodiments, each of the nine glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, eight of the nine glutamyl groups have alpha bonds. In some embodiments, alfanonaglutamate-oxidized THF contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group in alfanonaglutamate-oxidized tetrahydrofolate is L-type. In other embodiments, alfanonaglutamate-oxidized THF contains D-type glutamyl groups. In further embodiments, the glutamyl groups of alpha-nonaglutamate-oxidized tetrahydrofolate, other than the glutamyl group of tetrahydrofolate, are each of the D type. In further embodiments, nonaglutamate-oxidized THF comprises 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.

[0137] In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate is deca-glutamate-oxidized (αTHF-PG9) (i.e., it contains a chain of nine additional glutamyl groups bonded to the glutamyl group of tetrahydrofolate). In some embodiments, each of the nine additional glutamyl groups has an alpha bond. In some embodiments, each of the nine additional glutamyl groups in the chain, excluding the C-terminal glutamyl group, has an alpha bond. In some embodiments, eight of the nine additional glutamyl groups in the chain have alpha bonds. In other embodiments, one, two, three, four, five, six, seven, or eight of the nine additional glutamyl groups have alpha bonds, and the remaining eight, seven, six, five, four, three, two, or one glutamyl group each have a gamma bond. In other embodiments, one, two, three, four, five, six, seven, or eight of the nine additional glutamyl groups have alpha bonds, and the remaining non-C-terminal glutamyl groups are bonded to the molecule's glutamyl groups via gamma bonds. In some embodiments, at least one additional glutamyl group has both alpha and gamma bonds. In some embodiments, at least one of the ten glutamyl groups has both alpha and gamma bonds. In some embodiments, each of the ten glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, nine of the ten glutamyl groups have alpha bonds. In some embodiments, alpha-decaglutamine-oxidized THF contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group in alpha-decaglutamine-oxidized tetrahydrofolate is L-type. In other embodiments, alpha-decaglutamine-oxidized THF contains D-type glutamyl groups. In further embodiments, the glutamyl groups of alpha-decaglutamate-oxidized tetrahydrofolate, other than the glutamyl group of tetrahydrofolate, are each of the D type. In further embodiments, deca-glutamate-oxidized THF comprises 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.

[0138] In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate is undecaglutamate-oxidized (αTHF-PG 10In 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 has an alpha bond. In some embodiments, 9 of the 10 additional glutamyl groups in the chain have alpha bonds. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the 10 additional glutamyl groups have alpha bonds, and the remaining 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, or 9 of the 10 additional glutamyl groups have alpha bonds, and the remaining non-C-terminal glutamyl groups are bonded to the molecule's glutamyl group via gamma bonds. In some embodiments, at least one additional glutamyl group has both an alpha and a gamma bond. In some embodiments, at least one of the eleven glutamyl groups has both an alpha and a gamma bond. In some embodiments, each of the eleven glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, 10 of the eleven glutamyl groups have an alpha bond. In some embodiments, alpha-unde-decaglutamine-oxidized THF contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of alpha-unde-decaglutamine-oxidized tetrahydrofolate is L-type. In other embodiments, alpha-unde-decaglutamine-oxidized THF contains D-type glutamyl groups. In further embodiments, each glutamyl group of alpha-unde-decaglutamine-oxidized tetrahydrofolate other than the glutamyl group of tetrahydrofolate is D-type. In further embodiments, unde-decaglutamine-oxidized THF 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.

[0139] In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate is dodecaglutamate-oxidized (αTHF-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 has an alpha bond. In some embodiments, 10 of the 11 additional glutamyl groups in the chain have alpha bonds. In other embodiments, one, two, three, four, five, six, seven, eight, nine, or ten of the 11 additional glutamyl groups have alpha bonds, and the remaining ten, nine, eight, seven, six, five, four, three, two, or one glutamyl group each have a gamma bond. In other embodiments, one, two, three, four, five, six, seven, eight, nine, or ten of the 11 additional glutamyl groups have alpha bonds, and the remaining non-C-terminal glutamyl groups are bonded to the molecule's glutamyl group via gamma bonds. In some embodiments, at least one additional glutamyl group has both an alpha and a gamma bond. In some embodiments, at least one of the twelve glutamyl groups has both an alpha and a gamma bond. In some embodiments, each of the twelve glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, eleven of the twelve glutamyl groups have an alpha bond. In some embodiments, alpha-dodeca-glutamine-oxidized THF contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group of alpha-dodeca-glutamine-oxidized tetrahydrofolate is L-type. In other embodiments, alpha-dodeca-glutamine-oxidized THF contains D-type glutamyl groups. In further embodiments, each glutamyl group of alpha-dodeca-glutamine-oxidized tetrahydrofolate other than the glutamyl group of tetrahydrofolate is D-type. In further embodiments, dodecaglutamine-oxidized THF 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.

[0140] In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate is triskaide-glutamate-oxidized (αTHF-PG 12In 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 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 group each have a gamma bond. In other embodiments, one, two, three, four, five, six, seven, eight, nine, ten, or eleven of the twelve additional glutamyl groups have alpha bonds, and the remaining non-C-terminal glutamyl groups are bonded to the molecule's glutamyl groups via gamma bonds. In some embodiments, at least one additional glutamyl group has both alpha and gamma bonds. In some embodiments, at least one of the thirteen glutamyl groups has both alpha and gamma bonds. In some embodiments, each of the thirteen glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, twelve of the thirteen glutamyl groups have alpha bonds. In some embodiments, alpha-triskaidecaglutamine-oxidized THF contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group in alpha-triskaidecaglutamine-oxidized tetrahydrofolate is L-type. In other embodiments, alpha-triskaidecaglutamine-oxidized THF contains D-type glutamyl groups. In further embodiments, the glutamyl groups of alpha-triskaidecaglutamate-oxidized tetrahydrofolate, other than the glutamyl group of tetrahydrofolate, are each of the D type. In further embodiments, triskaidecaglutamate-oxidized THF comprises 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.

[0141] In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate is tetradecaglutamate-oxidized (αTHF-PG 13In 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 has an alpha bond. In some embodiments, 12 of the 13 additional glutamyl groups in the chain have alpha bonds. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the 13 additional glutamyl groups have alpha bonds, and the remaining 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group each have a gamma bond. In other embodiments, one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve of the thirteen additional glutamyl groups have alpha bonds, and the remaining non-C-terminal glutamyl groups are bonded to the molecule's glutamyl groups via gamma bonds. In some embodiments, at least one additional glutamyl group has both alpha and gamma bonds. In some embodiments, at least one of the fourteen glutamyl groups has both alpha and gamma bonds. In some embodiments, each of the fourteen glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, thirteen of the fourteen glutamyl groups have alpha bonds. In some embodiments, alpha-tetradecaglutamine-oxidized THF contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group in alpha-tetradecaglutamine-oxidized tetrahydrofolate is L-type. In other embodiments, alpha-tetradecaglutamine-oxidized THF contains D-type glutamyl groups. In further embodiments, the glutamyl groups of alpha-tetradecaglutamine-oxidized tetrahydrofolate, other than the glutamyl group of tetrahydrofolate, are each D-type. In further embodiments, tetradecaglutamine-oxidized THF 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.

[0142] In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate is pentadeca-glutamate-oxidized (αTHF-PG 14In 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 has an alpha bond. In some embodiments, 13 of the 14 additional glutamyl groups in the chain have alpha bonds. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the 14 additional glutamyl groups have alpha bonds, and the remaining 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group each have a gamma bond. In other embodiments, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen of the fourteen additional glutamyl groups have alpha bonds, and the remaining non-C-terminal glutamyl groups are bonded to the molecule's glutamyl groups via gamma bonds. In some embodiments, at least one additional glutamyl group has both alpha and gamma bonds. In some embodiments, at least one of the fifteen glutamyl groups has both alpha and gamma bonds. In some embodiments, each of the fifteen glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, fourteen of the fifteen glutamyl groups have alpha bonds. In some embodiments, alpha-pentadeca-glutamine-oxidized THF contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group in alpha-pentadeca-glutamine-oxidized tetrahydrofolate is L-type. In other embodiments, alpha-pentadeca-glutamine-oxidized THF contains D-type glutamyl groups. In further embodiments, the glutamyl groups of alpha-pentadeca-glutamine-oxidized tetrahydrofolate, other than the glutamyl group of tetrahydrofolate, are each D-type. In further embodiments, pentadeca-glutamine-oxidized THF 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.

[0143] In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate is hexadeca-glutamate-oxidized (αTHF-PG 15In 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 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 each have a gamma bond. In other embodiments, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen of the fifteen additional glutamyl groups have alpha bonds, and the remaining non-C-terminal glutamyl groups are bonded to the molecule's glutamyl groups via gamma bonds. In some embodiments, at least one additional glutamyl group has both alpha and gamma bonds. In some embodiments, at least one of the sixteen glutamyl groups has both alpha and gamma bonds. In some embodiments, each of the sixteen glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, fifteen of the sixteen glutamyl groups have alpha bonds. In some embodiments, alpha-hexadeca-glutamine-oxidized THF contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group in alpha-hexadeca-glutamine-oxidized tetrahydrofolate is L-type. In other embodiments, alpha-hexadeca-glutamine-oxidized THF contains D-type glutamyl groups. In further embodiments, the glutamyl groups of alpha-hexadeca-glutamine-oxidized tetrahydrofolate, other than the glutamyl group of tetrahydrofolate, are each D-type. In further embodiments, hexadeca-glutamine-oxidized THF 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.

[0144] In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate is heptadeca-glutamate-oxidized (αTHF-PG 16In 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 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, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen of the sixteen additional glutamyl groups have alpha bonds, and the remaining non-C-terminal glutamyl groups are bonded to the molecule's glutamyl groups via gamma bonds. In some embodiments, at least one additional glutamyl group has both alpha and gamma bonds. In some embodiments, at least one of the seventeen glutamyl groups has both alpha and gamma bonds. In some embodiments, each of the seventeen glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, sixteen of the seventeen glutamyl groups have alpha bonds. In some embodiments, alpha-heptadeca-glutamine-oxidized THF contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group in alpha-heptadeca-glutamine-oxidized tetrahydrofolate is L-type. In other embodiments, alpha-heptadeca-glutamine-oxidized THF contains D-type glutamyl groups. In further embodiments, the glutamyl groups of alpha-heptadeca-glutamine-oxidized tetrahydrofolate, other than the glutamyl group of tetrahydrofolate, are each D-type. In further embodiments, heptadeca-glutamine-oxidized THF 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.

[0145] In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate is octadeca-glutamate-oxidized (αTHF-PG 17In 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 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, one, two, three, four, five, sixteen, seventeen, eighteen, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen of the seventeen additional glutamyl groups have alpha bonds, and the remaining non-C-terminal glutamyl groups are bonded to the molecule's glutamyl groups via gamma bonds. In some embodiments, at least one additional glutamyl group has both alpha and gamma bonds. In some embodiments, at least one of the eighteen glutamyl groups has both alpha and gamma bonds. In some embodiments, each of the eighteen glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, seventeen of the eighteen glutamyl groups have alpha bonds. In some embodiments, alpha-octadeca-glutamine-oxidized THF contains two or more L-type glutamyl groups. In further embodiments, each glutamyl group in alpha-octadeca-glutamine-oxidized tetrahydrofolate is L-type. In other embodiments, alpha-octadeca-glutamate-oxidized THF contains D-type glutamyl groups. In further embodiments, the glutamyl groups of alpha-octadeca-glutamate-oxidized tetrahydrofolate, other than the glutamyl group of tetrahydrofolate, are each D-type. In further embodiments, octadeca-glutamate-oxidized THF 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.

[0146] In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate is eniadecadeca-glutamate-oxidized (αTHF-PG 18In 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 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, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, or seventeen of the eighteen additional glutamyl groups have alpha bonds, and the remaining non-C-terminal glutamyl groups are bonded to the molecule's glutamyl groups via gamma bonds. In some embodiments, at least one additional glutamyl group has both alpha and gamma bonds. In some embodiments, at least one of the nineteen glutamyl groups has both alpha and gamma bonds. In some embodiments, each of the nineteen glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, eighteen of the nineteen glutamyl groups have alpha bonds. In some embodiments, alphaeniadecaglutamine oxidized THF contains two or more L-type glutamyl groups. In further embodiments, the glutamyl groups of alphaeniadecaglutamate-oxidized tetrahydrofolate are each L-type. In other embodiments, alphaeniadecaglutamate-oxidized THF contains D-type glutamyl groups. In further embodiments, the glutamyl groups of alphaeniadecaglutamate-oxidized tetrahydrofolate, other than the glutamyl group of tetrahydrofolate, are each D-type. In further embodiments, eniadecaglutamate-oxidized THF 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.

[0147] In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate is eicosiglutamate-oxidized (αTHF-PG 19In 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 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, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, or eighteen of the nineteen additional glutamyl groups have alpha bonds, and the remaining non-C-terminal glutamyl groups are bonded to the molecule's glutamyl groups via gamma bonds. In some embodiments, at least one additional glutamyl group has both alpha and gamma bonds. In some embodiments, at least one of the twenty glutamyl groups has both alpha and gamma bonds. In some embodiments, each of the twenty glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, nineteen of the twenty glutamyl groups have alpha bonds. In some embodiments, alpha-cosiglutamine oxidized THF contains two or more L-type glutamyl groups. In further embodiments, the glutamyl groups of alphacosiglutamate-oxidized tetrahydrofolate are each L-type. In other embodiments, alphacosiglutamate-oxidized THF contains D-type glutamyl groups. In further embodiments, the glutamyl groups of alphacosiglutamate-oxidized tetrahydrofolate, other than the glutamyl group of tetrahydrofolate, are each D-type. In further embodiments, eikosiglutamate-oxidized THF 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.

[0148] In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate is oxidized with iodine-polyglutamate-oxidized αTHF-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 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, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or nineteen of the twenty additional glutamyl groups have alpha bonds, and the remaining non-C-terminal glutamyl groups are bonded to the molecule's glutamyl groups via gamma bonds. In some embodiments, at least one additional glutamyl group has both alpha and gamma bonds. In some embodiments, at least one of the twenty-one glutamyl groups has both alpha and gamma bonds. In some embodiments, each of the twenty-one glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, twenty of the twenty-one glutamyl groups have alpha bonds. In some embodiments, Alpha Kosika Hena Glutamate Oxide THF contains two or more L-type glutamyl groups. In further embodiments, the glutamyl groups of Alpha Kosika Henna Glutamate Oxide Tetrahydrofolate are each L-type. In other embodiments, Alpha Kosika Henna Glutamate Oxide THF contains D-type glutamyl groups. In further embodiments, the glutamyl groups of Alpha Kosika Henna Glutamate Oxide Tetrahydrofolate, other than the glutamyl group of tetrahydrofolate, are each D-type. In further embodiments, Ikosika Henna Glutamate Oxide THF 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.

[0149] In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate contains 4 to 7 glutamyl groups bonded to tetrahydrofolate (i.e., αTHF-PGn, n=4 to 7), and each of the 4 to 7 bonded glutamyl groups has an alpha bond. In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate contains 4 to 7 glutamyl groups bonded to tetrahydrofolate (i.e., αTHF-PGn, n=4 to 7), and each of the 4 to 7 bonded glutamyl groups other than the C-terminal glutamyl group has an alpha bond. In some embodiments, each of the 4 to 7 bonded glutamyl groups is L-type. In other embodiments, each of the 4 to 7 bonded glutamyl groups is D-type. In other embodiments, the 4 to 7 bonded glutamyl groups are L-type and D-type. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0150] In one embodiment, alpha-polyglutamate-oxidized tetrahydrofolate is tetraglutamine-oxidized, and each of the three glutamyl groups in the polyglutamate chain bonded to tetrahydrofolate contains an alpha bond. In one embodiment, alpha-polyglutamate-oxidized tetrahydrofolate is tetraglutamine-oxidized, and each of the three glutamyl groups in the polyglutamate chain bonded to tetrahydrofolate, excluding the C-terminal glutamyl group, contains an alpha bond. In some embodiments, each of the four glutamyl groups is L-type. In some embodiments, each of the glutamyl groups in alpha-tetraglutamate-oxidized tetrahydrofolate, excluding the glutamyl group of tetrahydrofolate, is D-type. In other embodiments, at least two glutamyl groups in alpha-tetraglutamate-oxidized tetrahydrofolate are L-type, and at least one glutamyl group is D-type. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0151] In one embodiment, alpha-polyglutamate-oxidized tetrahydrofolate is pentaglutamine-oxidized, and each of the four glutamyl groups in the polyglutamate chain bonded to tetrahydrofolate contains an alpha bond. In one embodiment, alpha-polyglutamate-oxidized tetrahydrofolate is pentaglutamine-oxidized, and each of the four glutamyl groups in the polyglutamate chain bonded to tetrahydrofolate, excluding the C-terminal glutamyl group, contains an alpha bond. In some embodiments, each of the four glutamyl groups is L-type. In some embodiments, each of the glutamyl groups in alpha-pentaglutamate-oxidized tetrahydrofolate, excluding the glutamyl group of tetrahydrofolate, is D-type. In other embodiments, at least two glutamyl groups in alpha-pentaglutamate-oxidized tetrahydrofolate are L-type, and at least one glutamyl group is D-type. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0152] In one embodiment, alpha-polyglutamate-oxidized tetrahydrofolate is hexaglutamate-oxidized, and each of the five glutamyl groups in the polyglutamate chain bonded to tetrahydrofolate contains an alpha bond. In one embodiment, alpha-polyglutamate-oxidized tetrahydrofolate is hexaglutamate-oxidized, and each of the five glutamyl groups in the polyglutamate chain bonded to tetrahydrofolate, except for the C-terminal glutamyl group, contains an alpha bond. In some embodiments, each of the five glutamyl groups is L-type. In some embodiments, each of the glutamyl groups in alpha-hexaglutamate-oxidized tetrahydrofolate, except for the glutamyl group of tetrahydrofolate, is D-type. In other embodiments, at least two glutamyl groups in alpha-hexaglutamate-oxidized tetrahydrofolate are L-type, and at least one glutamyl group is D-type. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0153] In another embodiment, alpha-polyglutamate-oxidized tetrahydrofolate is heptaglutamine-oxidized, and each of the six glutamyl groups in the polyglutamate chain bonded to tetrahydrofolate contains an alpha bond. In another embodiment, alpha-polyglutamate-oxidized tetrahydrofolate is heptaglutamine-oxidized, and each of the six glutamyl groups in the polyglutamate chain bonded to tetrahydrofolate, except for the C-terminal glutamyl group, contains an alpha bond. In some embodiments, each of the six glutamyl groups is L-type. In some embodiments, each of the glutamyl groups in alpha-heptaglutamate-oxidized tetrahydrofolate, except for the glutamyl group of tetrahydrofolate, is D-type. In other embodiments, at least two glutamyl groups in alpha-heptaglutamate-oxidized tetrahydrofolate are L-type, and at least one glutamyl group is D-type. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0154] In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate (αPTHF) 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 tetrahydrofolate. In some embodiments, each glutamyl group in the αPTHF other than the glutamyl group of tetrahydrofolate has an alpha linkage. In some embodiments, each glutamyl group in the αPTHF other than the C-terminal glutamyl group and the glutamyl group of tetrahydrofolate has an alpha linkage. In some embodiments, each glutamyl group in the αPTHF other than the C-terminal glutamyl group has an alpha linkage. In some embodiments, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen glutamyl groups in αPTHF have alpha bonds. In some embodiments, αPTHF contains L-type and D-type glutamyl groups. In further embodiments, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen glutamyl groups in αPTHF have alpha bonds, and thirteen, twelve, eleven, ten, nine, eight, seven, six, five, four, three, two, one, or zero glutamyl groups each have gamma bonds. In some embodiments, the glutamyl groups in the polyglutamic acid structure of polyglutamic tetrahydrofolate are each L-type. In some embodiments, the glutamyl groups in αPTHF other than the glutamyl group of tetrahydrofolate are each of the D type. In some embodiments, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen glutamyl groups in αPTHF are of the L type. In another embodiment, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen glutamyl groups in αPTHF are of the D type. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0155] In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate (αPTHF) contains a total of 2 to 20, 2 to 15, 2 to 10, 2 to 5, or any range in between, including the glutamyl groups of tetrahydrofolate. In some embodiments, each glutamyl group in the αPTHF other than the glutamyl groups of tetrahydrofolate has an alpha bond. In some embodiments, each glutamyl group in the αPTHF other than the C-terminal glutamyl group and the glutamyl groups of tetrahydrofolate has an alpha bond. In some embodiments, each glutamyl group in the αPTHF other than the C-terminal glutamyl group 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, the αPTHF contains two or more glutamyl groups having gamma bonds. In further embodiments, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or nineteen glutamyl groups in the αPTHF other than the glutamyl groups of tetrahydrofolate have alpha bonds, and 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, nine, eight, seven, six, five, four, three, two, one, or zero glutamyl groups each have gamma bonds. In some embodiments, each glutamyl group in the αPTHF is L-type. In some embodiments, each glutamyl group in the αPTHF other than the glutamyl groups of tetrahydrofolate is D-type. In one embodiment, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty glutamyl groups in αPTHF are L-type. In another embodiment, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or nineteen glutamyl groups in αPTHF are D-type.

[0156] In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate 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 groups of tetrahydrofolate. 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 bonds. In further embodiments, the 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group in alpha-polyglutamate-oxidized tetrahydrofolate have gamma bonds. In some embodiments, at least one glutamyl group has both alpha and gamma bonds. In some embodiments, the glutamyl group in tetrahydrofolate has an alpha bond. In some embodiments, the glutamyl group in tetrahydrofolate has both an alpha bond and a gamma bond.

[0157] In some embodiments, a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 glutamyl groups in alpha-polyglutamate-oxidized tetrahydrofolate are L-type, D-type, or L-type and D-type. In some embodiments, each glutamyl group of polyglutamate-oxidized alpha-tetrahydrofolate is L-type. In other embodiments, each glutamyl group of polyglutamate-oxidized alpha-tetrahydrofolate, other than the glutamyl groups of tetrahydrofolate, is D-type. In alternative embodiments, at least two glutamyl groups of polyglutamate-oxidized alpha-tetrahydrofolate are L-type, and at least one glutamyl group in polyglutamate-oxidized alpha-tetrahydrofolate is D-type. In some embodiments, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen glutamyl groups in polyglutamic alpha-tetrahydrofolate are L-type. In other embodiments, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen glutamyl groups in polyglutamic alpha-tetrahydrofolate are D-type. In some embodiments, at least one glutamyl group has both an alpha-bond and a gamma-bond.

[0158] In further embodiments, the polyglutamate-oxidized alpha-tetrahydrofolate contains 20 to 100, 20 to 75, 20 to 50, 20 to 40, 20 to 30, 20 to 25, or 101 or more glutamyl groups, or any range in between. In some embodiments, each glutamyl group of αPTHF is L-type. In other embodiments, each glutamyl group of αPTHF other than the glutamyl group of tetrahydrofolate is D-type. In alternative embodiments, at least two glutamyl groups in αPTHF are L-type, and at least one glutamyl group in αPTHF is D-type. In some embodiments, at least one glutamyl group has both alpha and gamma bonds.

[0159] In further embodiments, the provided composition comprises an αPTHF containing one, two, three, four, five, six, seven, eight, nine, one to ten, or one to twenty glutamyl groups having alpha bonds. In some embodiments, the αPTHF contains one, two, three, four, five, six, seven, eight, nine, one to ten, or one to twenty L-type glutamyl groups. In some embodiments, the αPTHF contains zero, one, two, three, four, five, six, seven, eight, nine, one to ten, or one to twenty D-type glutamyl groups. In some embodiments, αPTHF comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 L-type glutamyl groups and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 1-20 D-type glutamyl groups. In other embodiments, polyglutamic alphatetrahydrofolate comprises at least one glutamyl group having both alpha and gamma bonds. In some embodiments, polyglutamic alphatetrahydrofolate comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 1-10, or 11 or more glutamyl groups having both alpha and gamma bonds.

[0160] In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate contains at least one glutamyl group having an alpha bond and two, three, four, five, six, seven, eight, nine, one to ten, one to twenty, or more glutamyl groups having gamma bonds. For example, in some embodiments, polyglutamate-oxidized alpha-tetrahydrofolate contains one, two, three, four, five, six, seven, eight, nine, or one to ten L-alpha-glutamyl group bonds and further contains one, two, three, four, five, six, seven, eight, nine, or one to ten L-gamma-glutamyl group bonds. In some further embodiments, the polyglutamate-oxidized alphatetrahydrofolate contains one, two, three, four, five, six, seven, eight, nine, or one to ten L-alphaglutamyl group bonds, and further contains one, two, three, four, five, six, seven, eight, nine, or one to ten D-gammaglutamyl group bonds. In an additional further embodiment, the polyglutamate-oxidized alphatetrahydrofolate contains one, two, three, four, five, six, seven, eight, nine, or one to ten D-alphaglutamyl group bonds, and further contains one, two, three, four, five, six, seven, eight, nine, or one to ten D-gammaglutamyl group bonds. In other further embodiments, the polyglutamate-oxidized alphatetrahydrofolate contains one, two, three, four, five, six, seven, eight, nine, or one to ten D-gamma-glutamyl group bonds, and further contains one, two, three, four, five, six, or one to ten L-gamma-glutamyl group bonds. In other embodiments, the polyglutamate-oxidized alphatetrahydrofolate contains at least one glutamyl group having both alpha and gamma bonds. In some embodiments, the polyglutamate-oxidized alphatetrahydrofolate contains one, two, three, four, five, six, seven, eight, nine, one to ten, or eleven or more glutamyl groups having both alpha and gamma bonds.

[0161] In some embodiments, the αPTHF provided herein may have one or more additional glutamyl groups added, i.e., the composition may serve as a substrate for FPGAs (folyl polyglutamate synthetase). Reagents and assays for measuring the ability of αPTHF to act as a substrate for FPGAs (e.g., human FPGAs or rat liver FPGAs) are readily available and can be performed routinely.

[0162] In some embodiments, the naked alpha-PTHF compositions disclosed herein (e.g., alpha-PTHF not bound to a delivery medium) are taken up by hepatocytes at a significantly slower rate compared to the uptake rate of tetrahydrofolate under the same physiological conditions. In some embodiments, the hepatocyte uptake rate of the naked alpha-PTHF composition is 30%, 20%, 15%, or less than 10% compared to the rate of tetrahydrofolate. In further embodiments, the efflux (transport) rate of the alpha-PTHF compositions disclosed herein from hepatocytes occurs at a significantly slower rate than that of tetrahydrofolate (30%, 20%, 15%, or less than 10%).

[0163] In some embodiments, the alpha-polyglutamate-oxidized tetrahydrofolate compositions provided herein have higher cytotoxicity against hyperproliferating cells than tetrahydrofolate. In some embodiments, the hyperproliferating cells are cancer cells. In some embodiments, the hyperproliferating 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, cytotoxicity is measured by an in vitro assay. In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate is hexaglutamate-oxidized tetrahydrofolate.

[0164] In some embodiments, the alpha-polyglutamate-oxidized tetrahydrofolate compositions provided herein have lower toxic side effects than tetrahydrofolate. In some embodiments, the alpha-polyglutamate-oxidized tetrahydrofolate compositions provided herein are less toxic than tetrahydrofolate to non-overgrowth cells. In some embodiments, the alpha-polyglutamate-oxidized tetrahydrofolate compositions provided herein are less toxic than tetrahydrofolate to neutrophils, hepatocytes, or colon epithelial cells. In some embodiments, neutrophils are human neutrophils, differentiated human neutrophils, or neutrophils differentiated from CD34+ cells. In some embodiments, hepatocytes are AML12 hepatocytes. In some embodiments, colon epithelial cells are CCD841 colon epithelial cells. In some embodiments, toxicity is measured by an in vitro assay. In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate is hexaglutamate-oxidized tetrahydrofolate.

[0165] In some embodiments, the alpha-polyglutamate-oxidized tetrahydrofolate compositions provided herein have lower toxic side effects than tetrahydrofolate. In some embodiments, the alpha-polyglutamate-oxidized tetrahydrofolate compositions provided herein have fewer or less severe toxic side effects than tetrahydrofolate in in vivo assays. In some embodiments, the in vivo assay is performed in an in vivo mouse model. In some embodiments, the alpha-polyglutamate-oxidized tetrahydrofolate compositions provided herein have fewer or less severe hematological or hepatotoxic side effects than tetrahydrofolate. In some embodiments, hematological side effects are assessed by mean neutrophils, mean leukocytes, or mean platelet counts. In some embodiments, hepatotoxic side effects are assessed by measuring serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), and / or serum albumin levels. In some embodiments, the in vivo assay involves administering a 40 mg / kg or 80 mg / kg alpha-polyglutamate-oxidized tetrahydrofolate composition once weekly for four weeks. In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate is hexaglutamate-oxidized tetrahydrofolate.

[0166] In some embodiments, treatment with the alpha-polyglutamate-oxidized tetrahydrofolate compositions provided herein does not induce significant hematological or hepatotoxic side effects in in vivo mouse models. In some embodiments, hematological side effects are assessed by mean neutrophils, mean leukocytes, or mean platelet counts. In some embodiments, hepatotoxic side effects are assessed by measuring serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), and / or serum albumin levels. In some embodiments, the alpha-polyglutamate-oxidized tetrahydrofolate compositions provided herein do not significantly decrease mean neutrophils, mean leukocytes, or mean platelet counts. In some embodiments, the alpha-polyglutamate-oxidized tetrahydrofolate compositions provided herein do not significantly increase serum aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT) levels. In some embodiments, the alpha-polyglutamate-oxidized tetrahydrofolate compositions provided herein do not significantly decrease serum albumin levels. In some embodiments, the in vivo assay involves administering a 40 mg / kg or 80 mg / kg polyglutamate-oxidized alpha-tetrahydrofolate composition once a week for four weeks. In some embodiments, alpha-polyglutamate-oxidized tetrahydrofolate is hexaglutamate-oxidized tetrahydrofolate.

[0167] In some embodiments, the αPTHF composition does not contain fluorine atoms. In some embodiments, the αPTHF composition does not contain 4-fluoroglutamyl groups.

[0168] Compositions of polyglutamate-oxidized alpha-tetrahydrofolic acid (αPTHF) and their uses are further disclosed in U.S. Patent Applications Nos. 62 / 374,458, 62 / 583,432, 62 / 627,741, 62 / 630,820, 62 / 630,821, 62 / 630,824, 62 / 630,825, 15 / 675,695, and 15 / 675,701; International Applications PCT / US2017 / 046666 and PCT / US2017 / 046667; the contents of each of the above applications are incorporated herein by reference in their entirety.

[0169] A. Polyglutamine hydroxytetrahydrofolate analogs and derivatives This disclosure also includes αPTHF derivatives and analogs. The compositions and methods disclosed herein are considered applicable to all known derivatives or analogs of oxidized tetrahydrofolate. In some embodiments, polyglutamate-oxidized tetrahydrofolate analog or derivative compositions prepared and used according to the compositions and methods of this disclosure are illustrated in Figures 1I-1J. In some embodiments, the analog corresponds to a modified form of tetrahydrofolate in which the glutamyl group of tetrahydrofolate is not bonded to the rest of the tetrahydrofolate molecule via a gamma peptide bond. In some embodiments, the analog is a variant of tetrahydrofolate in which the glutamyl group of tetrahydrofolate is in the D form. In some embodiments, the polyglutamate-oxidized tetrahydrofolate or polyglutamate-oxidized tetrahydrofolate analog or derivative is not fluorinated.

[0170] In further embodiments, the polyglutamate-oxidized alpha-tetrahydrofolate 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 amide bonds. In other embodiments, one or more glutamyl groups of the polyglutamate chain are derivatized.

[0171] Synthesis of THF-PG The tetrahydrofolic acid polyglutamate compositions provided herein are obtained by the following synthetic methods using available reagents and synthetic intermediates. The addition of glutamyl residues to the glutamyl residues of tetrahydrofolic acid can be carried out using synthetic methods known in the art. In some embodiments, glutamyl residues are continuously added to the glutamyl residues of tetrahydrofolic acid. In further embodiments, polyglutamic acid is added to the glutamyl residues of tetrahydrofolic acid using the "click chemistry" method or other bioconjugate chemistry known to those skilled in the art. Alternatively, a peptide of glutamyl residues of a desired length can be prepared and added to a precursor without a glutamyl residue of tetrahydrofolic acid. The peptide can be prepared using methods known in the art. In some embodiments, the first glutamyl residue is attached to wang resin, and additional glutamyl residues are continuously added by solid-phase peptide synthesis using F-moc chemistry. After adding the last glutamyl residue, the tetrahydrofolic acid precursor is coupled with the peptide, and the molecule is cleaved from the resin.

[0172] The addition of glutamyl residues to the glutamyl residues of tetrahydrofolic acid can be achieved using synthetic methods known in the art. In some embodiments, glutamyl residues are continuously added to the glutamyl residues of tetrahydrofolic acid. In further embodiments, polyglutamic acid is added to the glutamyl residues of tetrahydrofolic acid using the "click chemistry" method and other bioconjugate chemistry known to those skilled in the art. Alternatively, a peptide of glutamyl residues of a desired length can be prepared and added to a precursor of tetrahydrofolic acid without a glutamyl residue. The peptide can be prepared using synthetic methods known in the art. In some embodiments, the first glutamyl residue is attached to wang resin, and additional glutamyl residues are continuously added by solid-phase peptide synthesis using F-moc chemistry. After adding the last glutamyl residue, the tetrahydrofolic acid precursor is coupled with the peptide, and the molecule is cleaved from the resin.

[0173] C. Tetrahydrofolic acid-PG complex The inventors have surprisingly discovered that a polyglutamylated folic acid antagonist that shares almost the same structure and chemical characteristics as tetrahydrofolic acid (αPTHF) can form a complex with other compositions including therapeutic agents containing cytotoxic compounds such as platinum-based compounds. Thus, in some embodiments, the present disclosure provides a complex of αPTHF (e.g., αPTHF disclosed herein) and a therapeutic agent or a salt or acid thereof.

[0174] In some embodiments, the αPTHF / complex comprises αPTHF and a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic compound such as a chemotherapeutic agent. In further embodiments, the αPTHF / complex comprises a platinum-based drug such as a platinum-based chemotherapeutic agent (e.g., cisplatin, carboplatin, and oxaliplatin). In other embodiments, the αPTHF / complex comprises a taxane-based chemotherapeutic agent (e.g., paclitaxel and docetaxel). In other embodiments, the αPTHF / complex comprises cyclodextrin. In further embodiments, the αPTHF / complex is encapsulated in liposomes.

[0175] In some embodiments, the Disclosure provides compositions comprising a complex of αPTH with a therapeutic agent or a salt or acid thereof. In further embodiments, the αPTHF / therapeutic agent complex comprises one or more αPTHFs containing 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 αPTHF / therapeutic agent complex comprises one or more αPTHFs containing 3 to 10, 3 to 9, 3 to 8, or 3 to 7, or any number in between. In other embodiments, the αPTHF / therapeutic agent complex comprises one or more αPTHFs containing 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, or 4 to 5, or any number in between. In certain embodiments, the complex comprises one or more αPTHFs containing 3 to 10 glutamyl groups. In further embodiments, the αPTHF / therapeutic complex comprises one or more αPTHFs containing 3 to 7 glutamyl groups. In another embodiment, the αPTHF / therapeutic complex comprises one or more αPTHFs containing 5 glutamyl groups. In yet another embodiment, the αPTHF / therapeutic complex comprises one or more αPTHFs containing 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 therapeutic agent is a platinum-based drug. In yet another embodiment, the therapeutic agent is a taxane-based drug. In further embodiments, the molar ratio of αPTHF to therapeutic agent in the complex is in the range of 1 to 10:1. In some embodiments, the molar ratio of αPTHF / 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, (21-50):1, or >50:1. In other embodiments, the molar ratio of αPTHF / therapeutic agent in the complex is 1:1-20, 1:1-10, or 1:2-8, or any range in between.In some embodiments, the molar ratio of αPPTHF / 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:(21-50), or 1:>50. In some embodiments, the αPPTHF / therapeutic agent complex is encapsulated in liposomes (for example, as described herein or by other methods known in the art).

[0176] In alternative embodiments, the αPTHF complex comprises αPTHF and cyclodextrin. In some embodiments, the molar ratio of αPTHF (e.g., αPTHF salt) / cyclodextrin in the complex is in the range of 1 to 20:1, or any range in between. In some embodiments, the molar ratio of αPTHF / cyclodextrin in the complex is in the range of 1 to 10:1, or any range in between. In further embodiments, the molar ratio of αPTHF / cyclodextrin in the complex is in the range of 2 to 8:1, or any range in between. In some embodiments, the molar ratio of αPTHF / 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 αPTHF / cyclodextrin in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range in between. In some embodiments, the molar ratio of αPTHF / 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 αPTHF / cyclodextrin complex is encapsulated in liposomes (for example, as described herein or in other ways known in the art).

[0177] In some embodiments, the disclosure provides compositions comprising an αPTHF / platinum-based chemotherapeutic agent conjugate. In some embodiments, the platinum-based chemotherapeutic agent is selected from the group consisting of cisplatin, carboplatin, and oxaliplatin, or their salts or acids. In other embodiments, the αPTHF / platinum-based chemotherapeutic agent conjugate comprises cisplatin, carboplatin, an analogue of oxaliplatin, or their salts or acids. In some embodiments, the molar ratio of the αPTHF / platinum-based agent in the conjugate is in the range of 1 to 20:1, or any range therein. In some embodiments, the molar ratio of the αPTHF / platinum-based agent in the conjugate is in the range of 1 to 10:1, or any range therein. In further embodiments, the molar ratio of the αPTHF / platinum-based agent in the conjugate is in the range of 2 to 8:1, or any range therein. In some embodiments, the molar ratio of αPTHF / platinum-based drug in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21-50):1, or >50:1. In other embodiments, the molar ratio of αPTHF / platinum-based drug in the complex is in the range of 1:1-20, 1:1-10, or 1:2-8, or any range in between. In some embodiments, the molar ratio of αPTHF / platinum-based drug 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 αPTHF / platinum-based drug complex is encapsulated in liposomes (for example, as described herein or in other ways known in the art).

[0178] In further embodiments, the αPTHF / platinum-based chemotherapeutic agent conjugate comprises an analog of cisplatin, carboplatin, or oxaliplatin, or a salt or acid thereof. In some embodiments, the molar ratio of αPTHF / platinum-based analog in the conjugate is in the range of 1 to 20:1, or any range in between. In some embodiments, the molar ratio of αPTHF / platinum-based analog in the conjugate is in the range of 1 to 10:1, or any range in between. In further embodiments, the molar ratio of αPTHF / platinum-based agent in the conjugate is in the range of 2 to 8:1, or any range in between. In some embodiments, the molar ratio of αPTHF / platinum-based analogs 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 αPTHF / platinum-based analogs in the complex is 1:1-20, 1:1-10, or 1:2-8, or any range in between. In some embodiments, the molar ratio of αPTHF / platinum analog 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 αPTHF / platinum analog complex is encapsulated in liposomes (for example, as described herein or in other ways known in the art).

[0179] In further embodiments, the disclosure provides a complex comprising αPTHF and cisplatin or a salt or acid thereof. In some embodiments, the molar ratio of αPTHF / cisplatin (or a salt or acid of cisplatin) in the complex is in the range of 1 to 20:1, or any range in between. In some embodiments, the molar ratio of αPTHF / cisplatin (or a salt or acid of cisplatin) in the complex is in the range of 1 to 10:1, or any range in between. In further embodiments, the molar ratio of αPTHF / cisplatin (or a salt or acid of cisplatin) in the complex is in the range of 2 to 8:1, or any range in between. In some embodiments, the molar ratio of αPTHF / 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, (21-50):1, or >50:1. In other embodiments, the molar ratio of αPTHF / cisplatin (or a salt or acid of cisplatin) in the complex is 1:1-20, 1:1-10, or 1:2-8, or any range in between. In some embodiments, the molar ratio of αPTHF / cisplatin (or a salt or acid of cisplatin) is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21-50), or 1:>50. In further embodiments, the αPTHF / cisplatin (or a salt or acid of cisplatin) complex is encapsulated in liposomes (for example, as described herein or in other ways known in the art).

[0180] In another embodiment, the disclosure provides a complex comprising αPTHF and carboplatin or a salt or acid thereof. In some embodiments, the molar ratio of αPTHF / carboplatin (or a salt or acid of carboplatin) in the complex is in the range of 1 to 20:1, or any range in between. In further embodiments, the molar ratio of αPTHF / carboplatin (or a salt or acid of carboplatin) in the complex is in the range of 1 to 10:1, or any range in between. In further embodiments, the molar ratio of αPTHF / carboplatin (or a salt or acid of carboplatin) in the complex is in the range of 2 to 8:1, or any range in between. In some embodiments, the molar ratio of αPTHF / 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 other embodiments, the molar ratio of αPTHF / carboplatin in the complex is 1:1-20, 1:1-10, or 1:2-8, or any range in between. In some embodiments, the molar ratio of αPTHF / carboplatin (or a salt or acid of carboplatin) 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 further embodiments, the αPTHF / carboplatin (or a salt or acid of carboplatin) complex is encapsulated in liposomes (for example, as described herein or in other ways known in the art).

[0181] In another embodiment, the disclosure provides a complex comprising αPTHF and oxaliplatin or a salt or acid thereof. In some embodiments, the molar ratio of αPTHF / oxaliplatin (or a salt or acid of oxaliplatin) in the complex is in the range of 1 to 20:1, or any range in between. In a further embodiment, the molar ratio of αPTHF / oxaliplatin (or a salt or acid of oxaliplatin) in the complex is in the range of 1 to 10:1, or any range in between. In a further embodiment, the molar ratio of αPTHF / oxaliplatin (o...

Claims

[Claim 1] A liposome composition comprising liposomes encapsulating alpha-polyglutamate-oxidized tetrahydrofolate and one or more polyglutamate-oxidized, polyglutamate-oxidizable, or polyglutamate-inoxidizable, folate antimetabolites, The alpha-polyglutamine oxidized tetrahydrofolate contains 2 to 15 glutamyl groups having alpha-carboxyl group bonds; The aforementioned alpha-polyglutamate tetrahydrofolate is (a) Alpha-polyglutamine oxidized 5-formyl-THF, (b) Alpha-polyglutamine oxidized 10-formyl-THF, (c) Alpha-polyglutamine oxidized 5,10-methenyl-THF, (d) Alpha-polyglutamine 5-methyl-THF, (e) Alpha-polyglutamine oxidized 5,10-methylene-THF, and (f) Alpha-polyglutamine oxidized 5-formimino-THF Selected from the group consisting of; (i) At least two of the glutamyl groups of the alpha-polyglutamate tetrahydrofolate are L-type, (ii) Whether each of the glutamyl groups of the alpha-polyglutamine oxidized tetrahydrofolate is L-type, (iii) At least one of the glutamyl groups of the alpha-polyglutamine oxidized tetrahydrofolate is of type D, (iv) Each of the glutamyl groups of the alpha-polyglutamate-oxidized tetrahydrofolate, other than the glutamyl group of tetrahydrofolate, is of type D, or (v) At least two of the glutamyl groups of the alpha-polyglutamate tetrahydrofolate are L-type and at least one of the glutamyl groups is D-type; The liposomes have a diameter of 50 nm to 150 nm. Liposome composition.

Images