Alpha polyglutamated pemetrexed and uses thereof

Abstract

To provide alpha polyglutamated pemetrexed compositions to overcome pharmacological challenges related to dose limiting toxicities and treatment resistance associated with pemetrexed therapy.SOLUTION: Disclosed is a liposome composition comprising a liposome encapsulating alpha polyglutamated pemetrexed, where the alpha polyglutamated pemetrexed comprises 2-10 glutamyl groups having an alpha carboxyl group linkage, the liposome is PEGylated, and the liposome composition comprises a targeting moiety that has a specific affinity to a surface antigen on a target cell.SELECTED DRAWING: None

Description

Background Art

[0001] The present disclosure generally relates to compositions of alpha-polyglytamylated pemetrexed, such as liposomes containing alpha-polyglytamylated pemetrexed compositions, and methods of manufacturing and using the compositions for treating diseases including hyperproliferative diseases such as cancer, immune system disorders such as rheumatoid arthritis, and infectious diseases such as HIV and malaria.

[0002] Pemetrexed disodium is sold under the trade name ALIMTA® (Eli Lilly and Company) and is also known by the chemical name L-glutamic acid, N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-, disodium salt, heptahydrate (molecular formula: C 20 H 19 5Na2O6·7H2O), which is the active ingredient of an antineoplastic drug product approved as a single agent for the treatment of locally advanced or metastatic non-small cell lung cancer and in combination with cisplatin for the treatment of patients with malignant pleural mesothelioma. Pemetrexed has demonstrated activity in clinical trials in various tumor types including lung cancer, breast cancer, colon cancer, mesothelioma, pancreatic cancer, gastric cancer, bladder cancer, head and neck cancer, and cervical cancer.

[0003] Folic acid is an essential cofactor that mediates the transfer of one-carbon units involved in nucleotide biosynthesis and DNA repair, remethylation of homocysteine (Hcy), and methylation of DNA, proteins, and lipids. The only circulating form of folic acid in the blood is monoglutamic acid, and folic acid monoglutamate is the only form of folic acid that is transported across cell membranes. Similarly, monoglutamic acid-type polyglutamylatable folic acid antagonists such as pemetrexed are also transported across cell membranes. Once taken up into cells, intracellular folic acid is converted to polyglutamic acid by the enzyme folylpoly-gamma-glutamate synthetase (FPGS).

[0004] Pemetrexed is a multi-target folic acid metabolism antagonist that exerts its action by disrupting folic acid-dependent metabolic processes essential for cell homeostasis and replication. Pemetrexed inhibits three enzymes required for purine and pyrimidine biosynthesis: thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycine amide ribonucleotide formyltransferase (GARFTase). Inhibition of these enzymes suppresses de novo nucleotide biosynthesis, leading to disruption of cell homeostasis and an imbalance of purine and pyrimidine precursors, thereby preventing accurate DNA replication in cells and ultimately resulting in cell death.

[0005] Pemetrexed is transported into cells by the reduced folate carrier (RFC) system and folate receptors (FR) α and β, as well as by the proton-coupled folate transporter (PCFT), which is most active in a lower pH environment. RFC is the major pemetrexed transporter at physiological pH and is widely expressed in normal and diseased cells. Thus, pemetrexed is often affected by dose-limiting toxicity, a major obstacle in cancer chemotherapy. Once inside the cell, pemetrexed is polyglutamylated by FPGS, allowing up to six L-glutamyl groups to be added during the binding of the L-gamma carboxyl group to pemetrexed. L-gamma polyglutamylation of pemetrexed by FPGS serves at least two major therapeutic purposes: (1) it greatly enhances the affinity and inhibitory activity of pemetrexed for several folate-dependent enzymes, including thymidylate synthase and GARFTase; and (2) it facilitates the accumulation of polyglutamylated pemetrexed, which, unlike pemetrexed (monoglutamate), is not readily transported out of the cell by cellular efflux pumps.

[0006] Pemetrexed acts during DNA and RNA synthesis and as a result has a large toxic effect on rapidly dividing cells such as malignant and myeloid cells. Myelosuppression is usually the dose-limiting toxicity of pemetrexed therapy and limits the clinical application of pemetrexed. To alleviate the most frequent side effects associated with pemetrexed therapy, including myelosuppression, fatigue, and rash, pretreatment with folic acid and vitamin B is used today.

[0007] Resistance to pemetrexed therapy is usually associated with one or more of the following: (a) increased activity of cell efflux pumps, (b) increased activity of thymidylate synthase, (c) decreased activity of folylpolyglutamate synthase (FPGS), and (d) increased activity of gamma-glutamyl hydrolase (GGH), which cleaves the gamma-polyglutamate chain bound to folic acid and folic acid antagonists.

[0008] The problem with the long-term (>30 years) observation that higher levels of polyglutamates of various folic acid antagonists have much higher potency compared to lower levels of glutamate was that the scientific community has relied on the intracellular FPGS-mediated mechanism that converts lower levels of glutamate to their higher level forms. The present invention provides a means of directly delivering higher levels of polyglutamate forms of folic acid antagonists into cells without relying on the cell's machinery to achieve this goal.

[0009] The provided alpha-polyglutamylated pemetrexed composition provides a strategy to overcome the pharmacological challenges related to dose-limiting toxicity and treatment resistance associated with pemetrexed therapy. The provided method delivers the novel alpha-polyglutamylated form of pemetrexed to cancer cells while simultaneously (1) minimizing / reducing exposure to normal tissue cells, (2) optimizing / improving the cytotoxic effect of the pemetrexed-based agent on cancer cells, and (3) minimizing / reducing the impact of efflux pumps and other resistance mechanisms that limit the therapeutic efficacy of pemetrexed. SUMMARY OF THE INVENTION

[0010] The present disclosure generally relates to novel alpha polyglutamine oxidized pemetrexed (PMX) compositions, and methods of making and using the compositions for treating diseases including hyperproliferative diseases such as cancer, immune system disorders such as rheumatoid arthritis, and infectious diseases such as HIV and malaria.

[0011] In some embodiments, the present disclosure provides the following. [1] A composition comprising alpha polyglutamine oxidized pemetrexed, wherein at least one glutamyl group has an alpha carboxyl group bond; [2] The composition according to item [1], wherein the alpha polyglutamine oxidized pemetrexed comprises 1 to 10 glutamyl groups having an alpha carboxyl group bond; [3] The composition according to item [1] or [2], wherein the alpha polyglutamine oxidized pemetrexed comprises 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups; [4] The composition according to any one of items [1] to [3], comprising alpha tetraglutamine oxidized pemetrexed; [5] The composition according to any one of items [1] to [3], comprising alpha pentaglutamine oxidized pemetrexed; [6] The composition according to any one of items [1] to [3], comprising alpha hexaglutamine oxidized pemetrexed; [7] The composition according to any one of items [1] to [6], which is the following composition: (a) Two or more glutamyl groups have an alpha carboxyl group bond, (b) Each glutamyl group other than the glutamyl group of pemetrexed has an alpha carboxyl group bond, or (c) Two or more glutamyl groups have a gamma carboxyl group bond; [8] The composition according to any one of items [1] to [6], which is the following composition: (a) The C-terminal glutamyl group(s) and each glutamyl group other than the glutamyl group of pemetrexed have an alpha-carboxyl group bond; or (b) A composition in which each glutamyl group other than the C-terminal glutamyl group(s) has an alpha-carboxyl group bond; [9] The composition according to any one of items [1] to [8], wherein at least one glutamyl group has both an alpha-carboxyl group bond and a gamma-carboxyl group bond;

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

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

[10] , wherein the polyglutamate is linear;

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

[10] , wherein the polyglutamate is branched;

[13] A liposome composition (Lp-αPPMX) containing the alpha-polyglutamine oxidized pemetrexed according to any one of items [1] to

[12] ;

[14] The LαPP composition according to item

[13] , wherein the alpha-polyglutamine oxidized pemetrexed contains an L-form glutamyl group having an alpha-carboxyl group bond;

[15] The Lp-αPPMX composition according to item

[13] or

[14] , wherein each glutamyl group of alphapolyglutamine oxidized pemetrexed is of the L-type;

[16] The Lp-αPPMX composition according to item

[13] or

[14] , wherein at least one glutamyl group of alphapolyglutamine oxidized pemetrexed is of the D-type;

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

[13] to

[16] , wherein the liposome contains alphapolyglutamine oxidized pemetrexed having 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups;

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

[13] to

[17] , wherein at least one glutamyl group of alphapolyglutamine oxidized pemetrexed has a gamma carboxyl group bond;

[19] The composition according to any one of items

[13] to

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

[20] The composition according to any one of items

[13] to

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

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

[13] to

[19] , wherein the liposome contains alphapolyglutamine oxidized pemetrexed, and the alphapolyglutamine oxidized pemetrexed contains alphatetraglutamine oxidized pemetrexed, alphapentaglutamine oxidized pemetrexed or alphahexaglutamine oxidized pemetrexed;

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

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

[28] The Lp-αPPMX composition according to item

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

[29] The Lp-αPPMX composition according to item

[27] or

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

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

[27] to

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

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

[27] to

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

[32] The Lp-αPPMX composition according to item

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

[33] The Lp-αPPMX composition according to item

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

[33] , wherein the liposome is cationic;

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

[13] to

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

[40] The Lp-αPPMX composition according to item

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

[41] The Lp-αPPMX composition according to item

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

[42] The Lp-αPPMX composition according to item

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

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

[39] to

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

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

[39] to

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

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

[39] to

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

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

[39] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

[51] The Lp-αPPMX composition according to item

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

[52] The Lp-αPPMX composition according to item

[50] or

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

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

[50] to

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

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

[50] to

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

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

[50] to

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

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

[50] to

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

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

[50] to

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

[58] The Lp-αPPMX composition according to item

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

[59] The Lp-αPPMX composition according to any one of items

[39] to

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

[60] The Lp-αPPMX composition according to item

[58] or

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

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

[58] to

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

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

[58] to

[61] , further comprising a hapten;

[63] The Lp-αPPMX composition according to item

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

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

[13] to

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

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

[64] ;

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

[49] ;

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

[13] to

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

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

[13] to

[67] ;

[69] A pharmaceutical composition comprising the alpha polyglutamine oxidized pemetrexed composition according to any one of items [1] to [7];

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

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

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

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

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

[70] to the subject;

[73] A method for treating or preventing a disease in a subject in need thereof, comprising the step of administering the liposomal alpha polyglutamine oxidized pemetrexed composition according to any one of items

[13] to

[69] to the subject;

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

[69] ;

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

[13] to

[69] ;

[76] The method according to item

[74] or

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

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

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

[78] A method for treating cancer, comprising the step of administering an effective amount of the liposomal alpha polyglutamine oxidized pemetrexed composition according to any one of items

[13] to

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

[79] The method according to item

[77] or

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

[80] The method according to item

[77] or

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

[81] The method according to item

[77] or

[78] , wherein the cancer is mesothelioma or non-small cell lung cancer (NSCLC);

[82] The method according to item

[77] or

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

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

[50] to

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

[84] A maintenance therapy comprising administering an effective amount of the composition according to any one of items [1] to

[69] to a subject who is undergoing or has undergone cancer therapy;

[85] A maintenance therapy comprising administering an effective amount of the liposomal alpha polyglutamine oxidized pemetrexed composition according to any one of items

[13] to

[69] to a subject who is undergoing or has undergone cancer therapy;

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

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

[87] A method for treating an immune system disorder comprising administering an effective amount of the liposomal alpha polyglutamine oxidized pemetrexed composition according to any one of items [9] to

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

[88] A method for treating an infectious disease comprising administering an effective amount of the composition according to any one of items [1] to

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

[89] A method for treating an infectious disease comprising administering an effective amount of the liposomal alpha polyglutamine oxidized pemetrexed composition according to any one of items

[13] to

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

[90] A method for delivering alpha polyglutamine oxidized pemetrexed to a tumor expressing a folate receptor on its surface, the method comprising administering the Lp-αPPMX composition according to any one of items [1] to

[69] to a subject having a tumor in an amount sufficient to deliver a therapeutically effective amount of alpha polyglutamine oxidized pemetrexed to the tumor;

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

[13] to

[69] , the method comprising: forming a mixture comprising a liposomal component and alpha - polyglutamyl - oxidized pemetrexed in a solution; homogenizing the mixture in the solution to form liposomes; and treating the mixture to form liposomes containing alpha - polyglutamyl - oxidized pemetrexed;

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

[13] to

[69] , the method comprising: forming a mixture comprising a liposomal component and alpha - polyglutamyl - oxidized pemetrexed in a solution; and treating the mixture to form liposomes containing alpha - polyglutamyl - oxidized pemetrexed;

[93] The method according to item

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

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

[13] to

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

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

[50] to

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

[96] The method according to item

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

[97] The method according to any one of items

[94] to

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

[98] The method according to any one of items

[94] to

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

[99] The method according to any one of items

[91] to

[98] , wherein at least 1% of the starting material of alphapolyglutamine oxidized PMX is encapsulated or enclosed in Lp-αPPMX.

[0012] In some embodiments, the present disclosure provides an alphapolyglutamylated pemetrexed (αPPMX) composition, wherein at least one glutamyl residue of the alphapolyglutamylated pemetrexed is attached by its alpha carboxyl group. In some embodiments, αPPMX comprises 2 to 20, 2 to 15, 2 to 10, 2 to 5, or more than 5 glutamyl groups (including the glutamyl groups of pemetrexed). In some embodiments, αPPMX comprises two or more L-type glutamyl groups. In other embodiments, αPPMX comprises a D-type glutamyl group. In further embodiments, αPPMX comprises a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, αPPMX comprises two or more glutamyl groups having a gamma bond. In some embodiments, at least one glutamyl group has both an alpha bond and a gamma bond.

[0013] In one embodiment, the αPPMX composition comprises a chain of three glutamyl groups attached to the glutamyl groups in pemetrexed (i.e., tetraglutamylated pemetrexed). In some embodiments, the tetraglutamylated PMX comprises two or more L-type glutamyl groups. In other embodiments, the tetraglutamylated PMX comprises a D-type glutamyl group. In further embodiments, the tetraglutamylated PMX comprises a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, the tetraglutamylated PMX comprises two or more glutamyl groups having a gamma bond.

[0014] In one embodiment, the αPPMX composition comprises a chain of five glutamyl groups attached to the glutamyl group in pemetrexed (i.e., pentaglutaminyl oxidized pemetrexed). In some embodiments, pentaglutaminyl oxidized PMX comprises two or more L-type glutamyl groups. In other embodiments, pentaglutaminyl oxidized PMX comprises a D-type glutamyl group. In further embodiments, pentaglutaminyl oxidized PMX comprises a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, pentaglutaminyl oxidized PMX comprises two or more glutamyl groups having a gamma bond.

[0015] In one embodiment, the αPPMX composition comprises a chain of six glutamyl groups attached to the glutamyl group in pemetrexed (i.e., hexaglutaminyl oxidized pemetrexed). In some embodiments, hexaglutaminyl oxidized PMX comprises two or more L-type glutamyl groups. In other embodiments, hexaglutaminyl oxidized PMX comprises a D-type glutamyl group. In further embodiments, hexaglutaminyl oxidized PMX comprises a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, hexaglutaminyl oxidized PMX comprises two or more glutamyl groups having a gamma bond.

[0016] In further embodiments, the present disclosure provides compositions comprising delivery carriers such as liposomes filled with (i.e., encapsulated) and / or otherwise conjugated to alpha-polyglutamylated pemetrexed, methods of making the alphaPPMX-filled / conjugated delivery carrier compositions, and methods of using the same to deliver alpha-polyglutamylated pemetrexed to diseased (e.g., cancerous) cells and / or target cells. These compositions have uses including, but not limited to, the treatment of diseases including hyperproliferative diseases such as cancer, immune system disorders such as rheumatoid arthritis, and infectious diseases such as HIV, malaria. The alphaPPMX-filled / conjugated delivery carrier compositions provide for the selective delivery of a higher cytotoxic payload (e.g., polyglutamylated pemetrexed) compared to the cytotoxicity of pemetrexed (PMX) administered in the monoglutamic acid state, resulting in improved efficacy and safety of pemetrexed delivery to cancer cells.

[0017] In a further embodiment, the present disclosure provides a composition (Lp-αPPMX) comprising liposomes encapsulating (filling) alphapolyglutamine oxidized pemetrexed. In some embodiments, the alphapolyglutamine oxidized pemetrexed in Lp-αPPMX contains 2 to 20, 2 to 15, 2 to 10, 2 to 5, or more than 20 glutamyl groups (including the glutamyl groups of pemetrexed). In some embodiments, the alphapolyglutamine oxidized pemetrexed in Lp-αPPMX contains 2 or more L-type glutamyl groups. In other embodiments, the alphapolyglutamine oxidized pemetrexed in Lp-αPPMX contains D-type glutamyl groups. In a further embodiment, the alphapolyglutamine oxidized pemetrexed in Lp-αPPMX contains D-type glutamyl groups and 2 or more L-type glutamyl groups. In a further embodiment, the alphapolyglutamine oxidized pemetrexed in Lp-αPPMX contains 2 or more glutamyl groups having a gamma bond. In a further embodiment, the alphapolyglutamine oxidized pemetrexed in Lp-αPPMX contains 1 or more glutamyl groups having both an alpha bond and a gamma bond. In some embodiments, the alphapolyglutamine oxidized pemetrexed in Lp-αPPMX contains 2 to 10 or any range therebetween of glutamyl groups having both an alpha bond and a gamma bond. In some embodiments, the polyglutamate chain of alphapolyglutamine oxidized pemetrexed is linear. In some embodiments, the polyglutamate chain of alphapolyglutamine oxidized pemetrexed is branched.

[0018] In one embodiment, the Lp-αPPMX composition comprises alpha polyglutamine oxidized PMX (i.e., tetraglutamine oxidized pemetrexed) containing a chain of three glutamyl groups attached to the glutamyl group of pemetrexed. In some embodiments, the tetraglutamine oxidized PMX contains two or more L-type glutamyl groups. In other embodiments, the tetraglutamine oxidized PMX contains a D-type glutamyl group. In further embodiments, the tetraglutamine oxidized PMX contains a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, the tetraglutamine oxidized PMX contains two or more glutamyl groups having gamma linkages. In some embodiments, the polyglutamate chain of alpha polyglutamine oxidized pemetrexed is linear. In some embodiments, the polyglutamate chain of alpha polyglutamine oxidized pemetrexed is branched.

[0019] In one embodiment, the Lp-αPPMX composition comprises alpha polyglutamine oxidized PMX (i.e., pentaglutamine oxidized pemetrexed) containing a chain of four glutamyl groups attached to the glutamyl group of pemetrexed. In some embodiments, the pentaglutamine oxidized PMX contains two or more L-type glutamyl groups. In other embodiments, the pentaglutamine oxidized PMX contains a D-type glutamyl group. In further embodiments, the pentaglutamine oxidized PMX contains a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, the pentaglutamine oxidized PMX contains two or more glutamyl groups having gamma linkages. In some embodiments, the polyglutamate chain of alpha polyglutamine oxidized pemetrexed is linear. In some embodiments, the polyglutamate chain of alpha polyglutamine oxidized pemetrexed is branched.

[0020] In one embodiment, the Lp-αPPMX composition comprises alpha polyglutamine oxidized PMX (i.e., hexaglutamine oxidized pemetrexed) containing a chain of 5 glutamyl groups attached to the glutamyl group of pemetrexed. In some embodiments, the hexaglutamine oxidized PMX contains 2 or more L-type glutamyl groups. In other embodiments, the hexaglutamine oxidized PMX contains a D-type glutamyl group. In further embodiments, the hexaglutamine oxidized PMX contains a D-type glutamyl group and 2 or more L-type glutamyl groups. In further embodiments, the hexaglutamine oxidized PMX contains 2 or more glutamyl groups having a gamma bond. In some embodiments, the polyglutamate chain of alpha polyglutamine oxidized pemetrexed is linear. In some embodiments, the polyglutamate chain of alpha polyglutamine oxidized pemetrexed is branched.

[0021] In some embodiments, the Lp-αPPMX composition is cationic. In some embodiments, the Lp-αPPMX liposome is cationic and has a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therebetween. In further embodiments, the Lp-αPPMX liposome is cationic and the composition has a diameter in the range of 80 nm to 120 nm, or any range therebetween. In some embodiments, the cationic Lp-αPPMX composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w alpha polyglutamine oxidized PMX. In some embodiments, during the preparation process of Lp-αPPMX, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% of the starting material of alpha polyglutamine oxidized PMX is encapsulated (enclosed) in the cationic Lp-αPPMX. In further embodiments, the alpha polyglutamine oxidized pemetrexed encapsulated by the liposome is present in the HEPES buffer solution within the liposome.

[0022] In other embodiments, the Lp-αPPMX composition is anionic or neutral. In some embodiments, the Lp-αPPMX composition is cationic. In some embodiments, the Lp-αPPMX liposome is anionic or neutral and has a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therebetween. In further embodiments, the Lp-αPPMX liposome is anionic or neutral and the composition has a diameter in the range of 80 nm to 120 nm, or any range therebetween. In some embodiments, the Lp-αPPMX liposome is anionic and has a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therebetween. In further embodiments, the Lp-αPPMX liposome is anionic and the composition has a diameter in the range of 80 nm to 120 nm, or any range therebetween. In some embodiments, the Lp-αPPMX liposome is neutral and has a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, 30 nm to 175 nm, or 50 nm to 150 nm, or any range therebetween. In further embodiments, the Lp-αPPMX liposome is neutral and the composition has a diameter in the range of 80 nm to 120 nm, or any range therebetween. In some embodiments, the anionic or neutral Lp-αPPMX composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w of alpha polyglutamine oxidized PMX. In some embodiments, during the preparation process of Lp-αPPMX, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% of the starting material of alpha polyglutamine oxidized PMX is encapsulated (enclosed) in the anionic or neutral Lp-αPPMX.In some embodiments, the anionic or neutral Lp-αPPMX composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w of alpha-tetra-glutaminyl oxidized PMX. In some embodiments, the anionic or neutral Lp-αPPMX composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w of alpha-penta-glutaminyl oxidized PMX. In some embodiments, the anionic or neutral Lp-αPPMX composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w of alpha-hexa-glutaminyl oxidized PMX. In a further embodiment, the alpha-poly-glutaminyl oxidized pemetrexed encapsulated by the liposome is present in the HEPES buffer within the liposome.

[0023] In a further embodiment, the liposomal alpha-poly-glutaminyl oxidized pemetrexed composition is pegylated (PLp-αPPMX).

[0024] In some embodiments, the liposomal alpha-poly-glutaminyl oxidized pemetrexed composition is not targeted (NTLp-αPPMX). That is, the NTLp-αPPMX composition does not have a specific affinity for an epitope expressed on the surface of the target cell of interest (e.g., an epitope on a surface antigen). In a further embodiment, the non-targeted liposomal alpha-poly-glutaminyl oxidized pemetrexed composition is pegylated (NTPLp-αPPMX).

[0025] In other embodiments, the liposomal alpha polyglutamine oxidized pemetrexed composition is targeted (TLp-αPPMX). That is, the TLp-αPPMX composition includes a targeting moiety having a specific affinity for an epitope (surface antigen) on the target cell of interest. In some embodiments, TLp-αPPMX or TPLp-αPPMX is not bound to the liposome via a covalent bond. In other embodiments, the targeting moiety of TLp-αPPMX or TPLp-αPPMX is bound to one or both of the PEG and the outer surface of the liposome. The targeted liposomal alpha polyglutamine oxidized pemetrexed compositions (TLp-αPPMX and TPLp-αPPMX) provide further improvements over the efficacy and safety profiles of pemetrexed by specifically delivering alpha polyglutamine oxidized (e.g., tetraglutamine oxidized, pentaglutamine oxidized, and hexaglutamine oxidized) pemetrexed to target cells such as cancer cells. In further embodiments, the targeted liposomal alpha polyglutamine oxidized pemetrexed composition is pegylated (TPLp-αPPMX). The functions of the targeting moiety of the TLp-αPPMX and / or TPLp-αPPMX composition include, but are not limited to, targeting the liposome to the target cell of interest in vivo or in vitro; interacting with the surface antigen to which the targeting moiety has a specific affinity; and delivering the liposomal payload (αPPMX) to the cell.

[0026] Suitable targeting moieties are known in the art and include, but are not limited to, antibodies, antigen-binding antibody fragments, scaffold proteins, polypeptides, and peptides. In some embodiments, the targeting moiety is a polypeptide. In further embodiments, the targeting moiety is a polypeptide comprising at least 3, 5, 10, 15, 20, 30, 40, 50, or 100 amino acid residues. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In further embodiments, the targeting moiety comprises one or more of an antibody, a humanized antibody, an antigen-binding fragment of an antibody, a single-chain antibody, a single-domain antibody, a bispecific antibody, a synthetic antibody, a pegylated antibody, and a multimeric antibody. In some embodiments, the targeting moiety has specific affinity for an epitope that is selectively expressed on target cells such as tumor cells as compared to normal or non-tumor cells. In some embodiments, the targeting moiety has specific affinity for an epitope on a tumor cell surface antigen that is present on tumor cells but not present on or difficult to access on non-tumor cells. In some embodiments, the targeting moiety binds to the target epitope with an equilibrium dissociation constant (Kd) in the range of 0.5x10 -10 to 10x10 -6 as measured by BIACORE® analysis.

[0027] In certain embodiments, the 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 specifically bound by the targeting moiety 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 specifically bound by the targeting moiety is folate receptor alpha (FR - α). In some embodiments, the folate receptor specifically bound by the targeting moiety is folate receptor beta (FR - β). In some embodiments, the targeting moiety specifically binds to FR - α and FR - β.

[0028] In further embodiments, the αPPMX composition comprises one or more of an immunostimulant, a detectable marker, and a maleimide disposed on at least one of the PEG or outer surface of the liposome. In some embodiments, the liposomal αPPMX composition (e.g., Lp-αPPMX, PLp-αPPMX, NTLp-αPPMX, NTPLp-αPPMX, TLp-αPPMX, or TPLp-αPPMX) is cationic. In other embodiments, the liposomal αPPMX composition (e.g., Lp-αPPMX, PLp-αPPMX, NTLp-αPPMX, NTPLp-αPPMX, TLp-αPPMX or TPLp-αPPMX) is anionic or neutral. In further embodiments, the liposomes of the liposomal αPPMX composition (e.g., Lp-αPPMX, PLp-αPPMX, NTLp-αPPMX, NTPLp-αPPMX, TLp-αPPMX or TPLp-αPPMX) have a diameter in the range of 20 nm to 500 nm, or any range therebetween. In further embodiments, the liposomes of the liposomal αPPMX composition have a diameter in the range of 80 nm to 120 nm, or any range therebetween. In some embodiments, the liposomal αPPMX composition is pegylated (e.g., PLp-αPPMX, NTPLp-αPPMX, or TPLp-αPPMX). In some embodiments, the liposomal αPPMX composition is targeted (e.g., TLp-αPPMX or TPLp-αPPMX). In further embodiments, the liposomal αPPMX composition is pegylated and targeted (e.g., TPLp-αPPMX). In some embodiments, the liposomal αPPMX composition comprises an alphapolyglutamine oxidized pemetrexed containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the liposomal αPPMX composition comprises alphatetraglutamine oxidized pemetrexed. In some embodiments, the liposomal αPPMX composition comprises alphapentaglutamine oxidized pemetrexed. In other embodiments, the liposomal αPPMX composition comprises alphahexaglutamine oxidized pemetrexed.

[0029] In some embodiments, the liposome composition consists of alpha polyglutamine oxidized pemetrexed containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w of alpha polyglutamine oxidized PMX. In some embodiments, the Lp-αPPMX composition comprises alpha polyglutamine oxidized pemetrexed containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups and 1% to 98.5% w / w of alpha polyglutamine oxidized PMX. In some embodiments, the liposome contains alpha polyglutamine oxidized pemetrexed containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups, and during the preparation process of Lp-αPPMX, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% of the starting material of alpha polyglutamine oxidized PMX is encapsulated (enclosed) in Lp-αPPMX.

[0030] In some embodiments, the liposome composition consists of alpha tetraglutamine oxidized pemetrexed and at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w of alpha tetraglutamine oxidized PMX. In some embodiments, the Lp-αPPMX composition comprises alpha tetraglutamine oxidized pemetrexed and 1% to 98.5% w / w of alpha tetraglutamine oxidized PMX. In some embodiments, the liposome contains alpha tetraglutamine oxidized pemetrexed, and during the preparation process of Lp-αPPMX, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% of the starting material of alpha tetraglutamine oxidized PMX is encapsulated (enclosed) in Lp-αPPMX.

[0031] In some embodiments, the liposome composition consists of alpha pentaglutamine oxidized pemetrexed and contains at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w of alpha pentaglutamine oxidized PMX. In some embodiments, the Lp-αPPMX composition contains alpha pentaglutamine oxidized pemetrexed and 1% - 98.5% w / w of alpha tetraglutamine oxidized PMX. In some embodiments, the liposome contains alpha pentaglutamine oxidized pemetrexed, and during the preparation process of Lp-αPPMX, the starting material of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% of alpha pentaglutamine oxidized PMX is encapsulated (enclosed) in Lp-αPPMX. In some embodiments, the liposome composition consists of alpha hexaglutamine oxidized pemetrexed and contains at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% w / w or more than 75% w / w of alpha hexaglutamine oxidized PMX. In some embodiments, the Lp-αPPMX composition contains alpha hexaglutamine oxidized pemetrexed and 1% - 98.5% w / w of alpha hexaglutamine oxidized PMX. In some embodiments, the liposome contains alpha hexaglutamine oxidized pemetrexed, and during the preparation process of Lp-αPPMX, the starting material of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% of alpha pentaglutamine oxidized PMX is encapsulated (enclosed) in Lp-αPPMX.

[0032] Liposome compositions comprising αPPMX-encapsulated liposomes are also provided. In some embodiments, the liposome composition comprises a pegylated αPPMX composition. In some embodiments, the liposome composition comprises an αPPMX composition conjugated or otherwise incorporated into a targeting moiety. In further embodiments, the liposome composition comprises a pegylated αPPMX composition conjugated or otherwise incorporated into a targeting moiety. In some embodiments, the liposome composition comprises αPPMX comprising 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposome composition comprises alphatetraglutamine oxidized pemetrexed. In some embodiments, the liposome composition comprises alphapentaglutamine oxidized pemetrexed. In other embodiments, the liposome composition comprises alphahexaglutamine oxidized pemetrexed.

[0033] In some embodiments, the liposome composition comprises liposomal αPPMX (e.g., Lp-αPPMX, PLp-αPPMX, NTLp-αPPMX, NTPLp-αPPMX, TLp-αPPMX, and TPLp-αPPMX). In some embodiments, the liposomal αPPMX is pegylated (e.g., NTPLp-αPPMX, and TPLp-αPPMX). In some embodiments, the liposomal αPPMX comprises a targeting moiety having a specific affinity for an epitope of an antigen on the surface of a target cell such as a cancer cell (e.g., TLp-αPPMX or TPLp-αPPMX). In further embodiments, the liposome composition comprises pegylated liposomal αPPMX and further comprises a targeting moiety having a specific affinity for an epitope of an antigen on the surface of a target cell such as a cancer cell (e.g., TPLp-αPPMX). In some embodiments, the liposome composition comprises liposomal αPPMX that is cationic. In other embodiments, the liposome composition comprises liposomal αPPMX that is anionic or neutral. In further embodiments, the liposome composition comprises liposomal αPPMX having a diameter in the range of 20 nm to 500 nm, 20 nm to 200 nm, or any range therebetween. In further embodiments, the liposomal αPPMX has a diameter in the range of 80 nm to 120 nm, or any range therebetween.

[0034] Also provided are pharmaceutical compositions comprising alphapolyglutamine oxidized pemetrexed (αPPMX) including delivery carriers such as liposomal αPPMX. In some embodiments, the pharmaceutical composition comprises a pegylated αPPMX composition. In some embodiments, the pharmaceutical composition comprises an αPPMX composition conjugated to or otherwise incorporated into a targeting moiety. In further embodiments, the pharmaceutical composition comprises a pegylated αPPMX composition conjugated to or otherwise incorporated into a targeting moiety. In some embodiments, the pharmaceutical composition comprises αPPMX comprising 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the pharmaceutical composition comprises alphatetraglutamine oxidized pemetrexed. In some embodiments, the pharmaceutical composition comprises alphapentaglutamine oxidized pemetrexed. In other embodiments, the pharmaceutical composition comprises alphahexaglutamine oxidized pemetrexed.

[0035] In some embodiments, the pharmaceutical composition comprises liposomal αPPMX (e.g., Lp-αPPMX, PLp-αPPMX, NTLp-αPPMX, NTPLp-αPPMX, TLp-αPPMX, and TPLp-αPPMX). In some embodiments, the liposomal αPPMX composition is pegylated (e.g., NTPLp-αPPMX, and TPLp-αPPMX). In some embodiments, liposomal αPPMX comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a target cell of interest, such as a cancer cell (e.g., TLp-αPPMX or TPLp-αPPMX). In further embodiments, the pharmaceutical composition comprises a pegylated liposomal αPPMX composition and further comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of a target cell of interest, such as a cancer cell (e.g., TPLp-αPPMX). In some embodiments, the pharmaceutical composition comprises liposomal αPPMX that is cationic. In other embodiments, the pharmaceutical composition comprises liposomal αPPMX that is anionic or neutral. In further embodiments, the pharmaceutical composition comprises liposomal αPPMX having a diameter in the range of 20 nm to 500 nm or 20 nm to 500 nm, or any range therebetween. In further embodiments, the liposomal αPPMX composition has a diameter in the range of 80 nm to 120 nm, or any range therebetween.

[0036] In further embodiments, the present disclosure provides a method of killing cells, the method comprising contacting the cells with a composition comprising an alpha-polyglutamylated pemetrexed (αPPMX) composition. In some embodiments, the cells to be contacted are mammalian cells. In further embodiments, the cells to be contacted are human cells. In some embodiments, the cells to be contacted are hyperproliferative cells. In further embodiments, the hyperproliferative cells are cancer cells. In further embodiments, the cancer cells to be contacted are primary cells or cells derived from a cell line obtained from a cancer selected from the group consisting of non-hematological tumors such as lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological tumors such as leukemia, lymphoma and other B-cell malignancies, myeloma and other plasma cell dysplasias or malignancies. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, αPPMX comprises 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the αPPMX composition comprises alpha-tetra-glutamylated pemetrexed. In some embodiments, the αPPMX composition comprises alpha-penta-glutamylated pemetrexed. In other embodiments, the αPPMX composition comprises alpha-hexa-glutamylated pemetrexed.

[0037] In further embodiments, the present disclosure provides a method of killing cells, the method comprising contacting the cells with liposomes comprising alpha-polyglutamylated pemetrexed (i.e., Lp-αPPMX such as PLp-αPPMX, NTLp-αPPMX, NTPLp-αPPMX, TLp-αPPMX or TPLp-αPPMX). In some embodiments, the cells to be contacted are mammalian cells. In further embodiments, the cells to be contacted are human cells. In some embodiments, the cells to be contacted are hyperproliferative cells. In further embodiments, the hyperproliferative cells to be contacted are cancer cells. In further embodiments, the cancer cells are primary cells or cells derived from cell lines obtained from cancers selected from the group consisting of non-hematological tumors such as lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological tumors such as leukemia, lymphoma and other B cell malignancies, myeloma and other plasma cell dyscrasias or malignancies. In some embodiments, the method is performed in vivo. In other embodiments, the method is performed in vitro. In some embodiments, the liposomes comprise αPPMX having 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes comprise alpha-tetra-glutamylated pemetrexed. In some embodiments, the liposomes comprise alpha-penta-glutamylated pemetrexed. In other embodiments, the liposomes comprise alpha-hexa-glutamylated pemetrexed.

[0038] In a further embodiment, the present disclosure provides a method for treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of a delivery carrier (e.g., an immune complex or liposome) comprising alpha-polyglyglutamyl oxidized pemetrexed. In some embodiments, the delivery carrier is an antibody-containing immune complex (e.g., comprising a full-length IgG antibody, a bispecific antibody, or an scFv). In some embodiments, the delivery carrier is a liposome (e.g., an Lp-αPPMX such as PLp-αPPMX, NTLp-αPPMX, NTPLp-αPPMX, TLp-αPPMX, or TPLp-αPPMX). In some embodiments, the administered delivery carrier is pegylated. In some embodiments, the administered delivery carrier is not pegylated. In a further embodiment, the administered delivery carrier comprises a targeting moiety having specific affinity for an epitope of an antigen on the surface of cancer cells.In a further embodiment, the delivery carrier comprises a targeting moiety that specifically binds to a cell surface antigen selected from the group consisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-α, folate receptor-β or folate receptor-δ), mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV-1 (ZIP6), CGEN-15027, P cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1, EphA receptor, EphB receptor, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha, PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the delivery carrier comprises a targeting moiety that specifically binds to a cell surface antigen derived from or identified as expressed on a particular subject's cancer (tumor), such as a neoantigen. In some embodiments, the targeting moiety specifically binds to a cell surface antigen derived from or identified as expressed on a particular tumor of the subject, such as a neoantigen. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the administered delivery carrier comprises αPPMX containing 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the administered delivery carrier comprises alphatetraglutamine oxidized pemetrexed. In some embodiments, the administered delivery carrier comprises alphapentaglutamine oxidized pemetrexed. In other embodiments, the administered delivery carrier comprises alphahexaglutamine oxidized pemetrexed. In some embodiments, the administered delivery carrier comprises L alphapolyglutamine oxidized pemetrexed. In some embodiments, the administered delivery carrier comprises D alphapolyglutamine oxidized pemetrexed. In some embodiments, the administered delivery carrier comprises L and D alphapolyglutamine oxidized pemetrexed. In some embodiments, the cancer is selected from the group consisting of non-hematologic tumors such as, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematologic tumors such as, for example, leukemia, lymphoma and other B cell malignancies, multiple myeloma and other plasma cell dyscrasias or malignancies. In some embodiments, the cancer is lung cancer (e.g., NSCLC or mesothelioma). In some embodiments, the cancer is breast cancer (e.g., HER2++ or triple negative breast cancer). In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer cells are ovarian cancer. In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is head and neck cancer.In some embodiments, the cancer is osteosarcoma.

[0039] In a further embodiment, the present disclosure provides a method for treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of liposomes comprising alpha-polyglytamylated pemetrexed (e.g., Lp-αPPMX such as PLp-αPPMX, NTLp-αPPMX, NTPLp-αPPMX, TLp-αPPMX, or TPLp-αPPMX). In some embodiments, the liposomes are pegylated. In some embodiments, the liposomes are not pegylated. In a further embodiment, the liposomes comprise a targeting moiety having specific affinity for an epitope of an antigen on the surface of cancer cells.In a further embodiment, the liposome comprises a targeting moiety that specifically binds to a cell surface antigen selected from the group consisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-α, folate receptor-β or folate receptor-δ), mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV-1 (ZIP6), CGEN-15027, P cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1, EphA receptor, EphB receptor, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA1, EphB1, EphB2, EphB3, EphB4, EphB6, integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha, PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.This also includes the use of cancer stem cell targeting moieties such as those targeting CD34, CD133 and CD44, CD138, and CD15. In some embodiments, the liposome comprises a targeting moiety that specifically binds to a cell surface antigen derived from or determined to be expressed on a particular subject's tumor, such as a neoantigen. In some embodiments, the targeting moiety is an antibody or an antigen-binding antibody fragment. In some embodiments, the liposome comprises αPPMX containing 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposome comprises alphatetraglutamine oxidized pemetrexed. In some embodiments, the liposome comprises alphapentaglutamine oxidized pemetrexed. In other embodiments, the liposome comprises alphahexaglutamine oxidized pemetrexed. In some embodiments, the liposome comprises L alphapolyglutamine oxidized pemetrexed. In some embodiments, the liposome comprises D alphapolyglutamine oxidized pemetrexed. In some embodiments, the liposome comprises L and D alphapolyglutamine oxidized pemetrexed. In some embodiments, the cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, melanoma, and hematological tumors (e.g., leukemia or lymphoma). In some embodiments, the cancer is lung cancer (e.g., NSCLC or mesothelioma). In some embodiments, the cancer is breast cancer (e.g., HER2++ or triple negative breast cancer). In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer cells are ovarian cancer. In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is liver cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is osteosarcoma.

[0040] In a further embodiment, the present disclosure provides a method of treating cancer, the method comprising administering to a subject having or at risk of having cancer an effective amount of a liposomal composition comprising liposomes comprising alpha-polyglutamylated pemetrexed and a targeting moiety having specific affinity for an epitope of an antigen on the cancer surface.In some embodiments, the liposome comprises a targeting moiety that specifically binds to a cell surface antigen selected from the group consisting of: GONMB, TACSTD2 (TROP2), CEACAM5, EPCAM, folate receptor (e.g., folate receptor-α, folate receptor-β, or folate receptor-δ), mucin 1 (MUC-1), MUC-6, STEAP1, mesothelin, nectin 4, ENPP3, guanylyl cyclase C (GCC), SLC44A4, NaPi2b, CD70 (TNFSF7), CA9 (carbonic anhydrase), 5T4 (TPBG), SLTRK6, SC-16, tissue factor, LIV-1 (ZIP6), CGEN-15027, P cadherin, fibronectin extra domain B (ED-B), VEGFR2 (CD309), tenascin, collagen IV, periostin, endothelin receptor, HER2, HER3, ErbB4, EGFR, EGFRvIII, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, IGFR-1, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, SMO, CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD11a, CD15, CD18, CD19, CD20, CD22, CD26, CD27L, CD28, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD56, CD70, CD74, CD79, CD79b, CD98, CD105, CD133, CD138, cripto, IGF-1R, IGF-2R, EphA1, EphA receptor, EphB receptor, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA1, EphB1, EphB2, EphB3, EphB4, EphB6, integrin (e.g., integrin αvβ3, αvβ5, or αvβ6), C242 antigen, Apo2, PSGR, NGEP, PSCA, TMEFF2, endoglin, PSMA, CanAg, CALLA, c-Met, VEGFR-1, VEGFR-2, DDR1, PDGFR alpha, PDGFR beta, TrkA, TrkB, TrkC, UFO, LTK, ALK, Tie1, Tie2, PTK7, Ryk, TCR, NMDAR, LNGFR, and MuSK.In some embodiments, the administered liposomes comprise a targeting moiety that specifically binds to a cell surface antigen derived from or determined to be expressed on a particular subject's tumor, such as a neoantigen. In some embodiments, the administered liposome composition comprises pegylated liposomes (e.g., TPLp-αPPMX). In some embodiments, the administered liposome composition comprises non-pegylated liposomes. In some embodiments, the liposomes of the administered liposome composition comprise αPPMX containing 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the administered liposome composition comprise alphatetraglutamine oxidized pemetrexed. In some embodiments, the liposomes of the administered liposome composition comprise alphapentaglutamine oxidized pemetrexed. In other embodiments, the liposomes of the administered liposome composition comprise alphahexaglutamine oxidized pemetrexed. In some embodiments, the liposome composition is administered for treating cancer selected from the group consisting of lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, melanoma, multiple myeloma and other plasma cell dyscrasias or cachexia, and leukemia, lymphoma and other B-cell malignancies. In some embodiments, the liposome composition is administered for treating lung cancer (e.g., NSCLC or mesothelioma). In some embodiments, the liposome composition is administered for treating breast cancer (e.g., HER2++ or triple-negative breast cancer). In some embodiments, the liposome composition is administered for treating colorectal cancer. In some embodiments, the liposome composition is administered for treating ovarian cancer. In some embodiments, the liposome composition is administered for treating endometrial cancer. In some embodiments, the liposome composition is administered for treating pancreatic cancer. In some embodiments, the liposome composition is administered for treating liver cancer. In some embodiments, the liposome composition is administered for treating head and neck cancer.In some embodiments, the liposome composition is administered to treat osteosarcoma.

[0041] In further embodiments, the present disclosure provides a method of treating cancer, the method comprising administering to a subject having or at risk of having cancer that expresses a folate receptor on its cell surface an effective amount of a liposomal composition, the liposomal composition comprising liposomes comprising (a) alpha-polyglutamylated pemetrexed (αPPMX) and (b) a targeting moiety having specific binding affinity for the folate receptor. In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-α), folate receptor beta (FR-β), and / or folate receptor delta (FR-δ). In some embodiments, the targeting moiety has specific binding affinity for folate receptor alpha (FR-α) and folate receptor beta (FR-β). In some embodiments, the liposomal composition administered comprises pegylated liposomes (e.g., TPLp-αPPMX). In some embodiments, the liposomal composition administered comprises non-pegylated liposomes. In some embodiments, the liposomes of the liposomal composition administered comprise αPPMX comprising 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the liposomal composition administered comprise alpha-tetra-glutamylated pemetrexed. In some embodiments, the liposomes of the liposomal composition administered comprise alpha-penta-glutamylated pemetrexed. In other embodiments, the liposomes of the liposomal composition administered comprise alpha-hexa-glutamylated pemetrexed. In some embodiments, the liposomal composition is administered for treating cancer selected from the group consisting of non-hematological tumors, such as, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and hematological tumors, such as, for example, leukemia, lymphoma and other B cell malignancies, multiple myeloma and other plasma cell dyscrasias or malignancies. In some embodiments, the liposomal composition is administered for treating lung cancer (e.g., NSCLC or mesothelioma).In some embodiments, the liposomal composition is administered to treat breast cancer (e.g., HER2++ or triple-negative breast cancer). In some embodiments, the liposomal composition is administered to treat colorectal cancer. In some embodiments, the liposomal composition is administered to treat ovarian cancer. In some embodiments, the liposomal composition is administered to treat endometrial cancer. In some embodiments, the liposomal composition is administered to treat pancreatic cancer. In some embodiments, the liposomal composition is administered to treat liver cancer. In some embodiments, the liposomal composition is administered to treat head and neck cancer. In some embodiments, the liposomal composition is administered to treat osteosarcoma.

[0042] In further embodiments, the present disclosure provides a method for maintenance therapy of cancer, the method comprising administering to a subject undergoing or having undergone cancer therapy an effective amount of a liposomal composition comprising liposomes (Lp-αPPMX) comprising alpha-polyglutamylated pemetrexed. In some embodiments, the liposomal composition to be administered is PLp-αPPMX, NTLp-αPPMX, NTPLp-αPPMX, TLp-αPPMX or TPLp-αPPMX. In some embodiments, the liposomal composition to be administered comprises pegylated liposomes (e.g., PLp-αPPMX, NTPLp-αPPMX, or TPLp-αPPMX). In some embodiments, the liposomal composition to be administered comprises targeted liposomes (e.g., TLp-αPPMX or TPLp-αPPMX). In some embodiments, the liposomal composition to be administered comprises pegylated and targeted liposomes (e.g., TPLp-αPPMX). In some embodiments, the liposomes of the liposomal composition to be administered comprise alpha-polyglutamylated pemetrexed comprising 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the liposomal composition to be administered comprise alpha-tetra-glutamylated pemetrexed. In some embodiments, the liposomes of the liposomal composition to be administered comprise alpha-penta-glutamylated pemetrexed. In other embodiments, the liposomes of the liposomal composition to be administered comprise alpha-hexa-glutamylated pemetrexed.

[0043] In further embodiments, the present disclosure provides a method of treating a disorder of the immune system, the method comprising administering to a subject having or at risk of having a disorder of the immune system an effective amount of a liposomal composition comprising alphapolyglutaminated pemetrexed (e.g., Lp-αPPMX, PLp-αPPMX, NTLp-αPPMX, NTPLp-αPPMX, TLp-αPPMX or TPLp-αPPMX). In some embodiments, the liposomal composition is administered to treat an autoimmune disease. In further embodiments, the liposomal composition is administered to treat rheumatoid arthritis. In some embodiments, the liposomal composition administered comprises a pegylated liposome (e.g., PLp-αPPMX, NTPLp-αPPMX, or TPLp-αPPMX). In some embodiments, the liposomal composition administered comprises a targeted liposome (e.g., TLp-αPPMX or TPLp-αPPMX) comprising a targeting moiety having specific affinity for a surface antigen on a target cell of interest (e.g., an immune cell). In further embodiments, the liposomal composition administered comprises a pegylated and targeted liposome (e.g., TPLp-αPPMX). In some embodiments, the liposomes of the liposomal composition administered comprise alphapentaglutaminated pemetrexed comprising 4, 5, 6, 2-10, 4-6, or more than 5 glutamyl groups. In some embodiments, the liposomes of the liposomal composition administered comprise alphatetraglutaminated pemetrexed. In some embodiments, the liposomes of the liposomal composition administered comprise alphapentaglutaminated pemetrexed. In other embodiments, the liposomes of the liposomal composition administered comprise alphapentaglutaminated pemetrexed. In other embodiments, the liposomes of the liposomal composition administered comprise alphahexaglutaminated pemetrexed.

[0044] The present disclosure also provides a method for delivering alpha-polyglutamylated pemetrexed to tumor and cancer cells, the method comprising administering to a subject having a tumor a composition comprising alpha-polyglutamylated pemetrexed (L-αPPMX) and a targeting moiety having specific binding affinity for an epitope on the surface antigen of a tumor cell or cancer cell. In some embodiments, the targeting moiety to be administered is bound to a delivery carrier. In some embodiments, the delivery carrier is an antibody or an antigen-binding fragment of an antibody. In further embodiments, the delivery carrier is a liposome. In further embodiments, the antibody, antigen-binding antibody fragment, or liposome is a pegylated liposome (e.g., TPLp-αPPMX). In some embodiments, the composition to be administered comprises alpha-polyglutamylated pemetrexed comprising 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the composition to be administered comprises alpha-tetra-glutamylated pemetrexed. In some embodiments, the composition to be administered comprises alpha-penta-glutamylated pemetrexed. In other embodiments, the composition to be administered comprises alpha-hexa-glutamylated pemetrexed.

[0045] In a further embodiment, the present disclosure provides a method of making a liposome composition comprising a liposomal alpha-polyglytamylated pemetrexed (αPPMX) composition, the method comprising forming a mixture in solution comprising liposomal components and alpha-polyglytamylated pemetrexed; homogenizing the mixture in solution to form liposomes; and treating the mixture to form liposomes comprising polyglytamylated pemetrexed. In some embodiments, the alpha-polyglytamylated pemetrexed comprises 4, 5, 6, from 2 to 10, from 4 to 6, or more than 5 glutamyl groups. In some embodiments, the polyglytamylated pemetrexed composition comprises alpha-tetraglytamylated pemetrexed. In some embodiments, the polyglytamylated pemetrexed composition comprises alpha-pentaglytamylated pemetrexed. In other embodiments, the polyglytamylated pemetrexed composition comprises alpha-hexaglytamylated pemetrexed.

[0046] In one embodiment, the present disclosure provides a kit comprising an alpha-polyglytamylated pemetrexed composition and / or an αPPMX delivery carrier such as a liposome comprising αPPMX and αPPMX immune complexes (e.g., ADCs) described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0047]

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Figure 21A - F

DETAILED DESCRIPTION OF THE INVENTION

[0048] In general, the present disclosure relates to novel alpha polyglutamylated pemetrexed compositions. The compositions provide an advance over prior treatments for proliferative diseases such as cancer. Methods for the manufacture, delivery, and use of alpha polyglutamylated pemetrexed compositions are also provided. Alpha polyglutamylated compositions have uses including, but not limited to, the treatment or prevention of proliferative diseases such as cancer, immune system disorders such as rheumatoid arthritis, and infectious diseases such as HIV and malaria.

[0049] I. DEFINITIONS Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0050] When an embodiment is described herein using the term "comprising", a similar alternative embodiment described with the terms "containing", "consisting of", and / or "consisting essentially of" is always provided as well. However, when used as a transitional phrase in a claim, each should be interpreted separately and in an appropriate legal and factual context (e.g., in a claim, the transitional phrase "comprising" is considered a more open-ended term, "consisting of" is considered more exclusive, and "consisting essentially of" is considered intermediate between these).

[0051] As used herein, the singular forms "a", "an", and "the" include plural referents unless specifically stated otherwise or unless it is otherwise clear from the context that only a single referent is intended.

[0052] The term "and / or" as used in expressions such as "A and / or B" is intended herein to include both A and B; A or B; A alone; and B alone. Similarly, the term "and / or" as used in expressions such as "A, B and / or C" encompasses each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0053] Headings and subheadings are used for convenience and / or only for compliance with official rules, and do not limit the subject technology nor are they referred to in relation to the interpretation of the description of the subject technology. Features described under one heading or one subheading of the subject disclosure may be combined with features described under other headings or subheadings in various embodiments. Further, not all features under a single heading or a single subheading are necessarily used together in some embodiments.

[0054] Unless otherwise indicated, the terms "pemetrexed" and "PMX" are used interchangeably and include salts, acids and / or free base forms of pemetrexed (e.g., pemetrexed disodium). Compositions containing PMX salts may further contain various cations such as Na + , Mg 2+ , K + , NH4 + , and / or Ca 2+ . In certain embodiments, typically, the salt is a pharmaceutically acceptable salt. In further particular embodiments, the PMX salt contains Na + . Pemetrexed may also be referred to as ALIMTA (registered trademark), LY231514, MTA, and its chemical name is N-[4-2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-1-glutamic acid, or L-glutamic acid, N-[4-2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-c]pyrimidin-5-yl)ethyl]benzoyl]-, disodium salt, heptahydrate. Pemetrexed usually contains one L-gamma-glutamyl group and is thus considered to be monoglutamylated for the purposes of this disclosure.

[0055] The term "polyglutamate", "polyglutamination", or variations thereof, refers to a composition comprising at least one chain of two or more linked glutamyl groups. The polyglutamate chain can be linear or branched. A linear polyglutamate chain can include, for example, glutamyl groups with alpha carboxyl or gamma carboxyl group linkages. A branched polyglutamate chain can include, for example, one or more glutamyl groups that include both alpha carboxyl and gamma carboxyl group linkages to other glutamyl groups, thereby providing a branch point for the polyglutamate. Representative branched polyglutamates are shown in FIGS. 1R - 1U. The polyglutamate chain includes an N-terminal glutamyl group and one or more C-terminal glutamyl groups. The N-terminal glutamyl group of the polyglutamate chain does not bind to another glutamyl group via its amino group, but binds to one or more glutamyl groups via its carboxylic acid group. In some embodiments, the N-terminal glutamyl group of polyglutaminated pemetrexed is the glutamyl group of pemetrexed. The C-terminal glutamyl group(s) of the polyglutamate chain bind to another glutamyl group via their amino groups, but do not bind to another glutamyl group via their carboxylic acid groups.

[0056] The terms "polyglutaminated pemetrexed", "polyglutaminated PMX", "PMX-PG", "PPMX", and iterations thereof are used herein interchangeably and refer to a pemetrexed composition that includes at least one glutamyl group in addition to the glutamyl groups in pemetrexed (i.e., PMX-PG n , n ≧ 1). References herein to the number of glutamyl groups in αPPMX (PMX-PG) include the glutamyl groups in pemetrexed. For example, a PMX-PG composition that includes five glutamyl residues in addition to the glutamyl groups of PMX is referred to herein as hexaglutaminated pemetrexed or pemetrexed hexaglutamate.

[0057] The terms "alpha-glutamyl group", "alpha-glutamate", and "alpha bond", when they relate to the bonding of glutamyl groups, mean a glutamyl group that includes an alpha-carboxyl group bond. In some embodiments, the alpha bond is an amide bond between the alpha-carboxyl group of one glutamyl group and a second glutamyl group. The alpha bond can be between a glutamyl group and a glutamyl group in pemetrexed, or between a glutamyl group and a second glutamyl group such as a glutamyl group within a polyglutamate chain that is attached to pemetrexed and not present in pemetrexed.

[0058] The terms "gamma-glutamyl group", "gamma-glutamate", and "gamma bond", when they relate to the bonding of glutamyl groups, mean a glutamyl group that includes a gamma-carboxyl group bond. As discussed herein, when pemetrexed enters a cell, it is polyglutamylated by the enzyme folylpolyglutamate synthase (FPGS), which sequentially adds L-glutamyl groups to the gamma-carboxyl group of the glutamate in pemetrexed. Thus, an alpha-polyglutamylated pemetrexed composition is not formed intracellularly during pemetrexed therapy. In some embodiments, the gamma bond is an amide bond between the gamma-carboxyl group of one glutamyl group and a second glutamyl group. The gamma bond can be between a glutamyl group and a glutamyl group in pemetrexed, or between a glutamyl group and a second glutamyl group such as a glutamyl group within a polyglutamate chain that is attached to pemetrexed and not present in pemetrexed. In some embodiments, the gamma bond means an amide bond of the glutamyl group of pemetrexed. References to gamma bonds include, unless specifically stated otherwise or clearly apparent from the context that it is not intended, the gamma bond of the glutamyl group of pemetrexed.

[0059] Unless otherwise indicated, the terms "alphapolyglutamylated pemetrexed", "αPPMX", "alpha PMX-PG", and iterations thereof are used interchangeably herein and mean a polyglutamylated pemetrexed composition comprising at least one glutamyl group with an alpha linkage. For example, a pentaglutamylated PMX composition in which the second glutamyl group has an alpha linkage while each of the other glutamyl groups has a gamma linkage is considered an alpha PMX-PG in the present disclosure. In some embodiments, each glutamyl group of PMX-PG other than the glutamyl groups of PMX has an alpha linkage (e.g., PMX-PG where n = 5 and each of G1, G2, G3, G4, and G5 has an alpha linkage). n ) In some embodiments, the C-terminal glutamyl group(s) or each glutamyl group of PMX-PG other than the glutamyl groups of PMX has an alpha linkage (e.g., PMX-PG where n = 5 and each of G1, G2, G3, and G4 has an alpha linkage). n ) In some embodiments, each glutamyl group of PMX-PG other than the C-terminal glutamyl group(s) has an alpha linkage (e.g., PMX-PG where n = 5 and each of the glutamyl groups of PMX and G1, G2, G3, and G4 has an alpha linkage). n )

[0060] As used herein, the term "isolated" means a composition in a form not found in nature. Isolated alpha-polyglutamine oxidized compositions include those that are purified to the extent that they are no longer in the form found in nature. In some embodiments, the isolated alpha-polyglutamine oxidized folate antagonists are substantially pure. Isolated compositions are free or substantially free of naturally incorporated substances such as other cellular components such as proteins and nucleic acids that may potentially be found in nature or in the environment in which they are produced (e.g., cell culture). Alpha-polyglutamine oxidized compositions can be formulated with diluents or adjuvants and further isolated for practical purposes - for example, when used in a diagnostic or therapeutic agent, the alpha-polyglutamine oxidized composition is usually mixed with a pharmaceutically acceptable carrier or diluent. In some embodiments, the isolated alpha-polyglutamine oxidized compositions (e.g., delivery carriers such as alpha-polyglutamate and liposomes containing alpha-polyglutamate) contain less than 1% or less than 0.1% of undesired DNA or protein contaminants. In some embodiments, the alpha-polyglutamate compositions (e.g., delivery carriers such as alpha-polyglutamate and liposomes containing alpha-polyglutamate) are "isolated".

[0061] As used herein, the term "targeting moiety" means a molecule that confers enhanced affinity for a selected target, such as a cell, cell type, tissue, organ, body region, or compartment, such as a compartment of a cell, tissue or organ. A targeting moiety can include a wide variety of substances. Targeting moieties include natural molecules, or recombinant or synthetic molecules. In some embodiments, the targeting moiety is an antibody antigen-binding antibody fragment, a bispecific antibody or other antibody-based molecule or compound. In some embodiments, the targeting moiety is an aptamer, an avimer, a receptor-binding ligand, a nucleic acid, a biotin-avidin binding pair, a peptide, a protein, a carbohydrate, a lipid, a vitamin, a toxin, a microbial component, a hormone, a receptor ligand or any derivative thereof. Other targeting moieties are known in the art and are encompassed by the present disclosure.

[0062] The terms "specific affinity" or "specifically binds" mean that a targeting moiety, such as an antibody or an antigen-binding antibody fragment, reacts with or binds to an epitope, protein, or target molecule with a higher frequency, more rapidly, for a longer period of time, with greater affinity, or in some combination of these, than it does to another substance, including a protein unrelated to the target epitope. Due to sequence identity between homologous proteins in different species, in some embodiments, a particular affinity includes a binding substance that recognizes a protein or target in two or more species. Similarly, due to homology within a particular region of the polypeptide sequences of different proteins, the terms "specific affinity" or "specific binding" can include a binding substance that recognizes two or more proteins or targets. In certain embodiments, it is understood that a targeting moiety that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific affinity" does not necessarily (although it can) require exclusive binding, e.g., binding to only one target. Thus, in certain embodiments, a targeting moiety can specifically bind to two or more targets. In certain embodiments, multiple targets can be bound by the same targeting moiety.

[0063] The term "epitope" means a portion of an antigen that is recognized by and can be specifically bound by a targeting moiety (i.e., a binding moiety), such as an antibody. When the antigen is a polypeptide, the epitope can be formed from both contiguous and non-contiguous amino acids juxtaposed by the tertiary folding of the protein. Epitopes formed from contiguous amino acids are usually retained upon protein denaturation, whereas epitopes formed by tertiary folding are usually lost upon protein denaturation. Epitopes typically contain at least three amino acids, more commonly at least five or eight to ten amino acids, in a particular spatial conformation.

[0064] Expressions such as "binding affinity for a target" and "binding to a target" that are known in the art, and similar expressions, refer to properties of a targeting moiety that can be directly measured by determining an affinity constant, e.g., the amount of targeting moiety that binds and dissociates at a given antigen concentration. Intermolecular interactions can be characterized using other methods including, but not limited to, competitive analysis, equilibrium analysis, and microcalorimetry, and real-time interaction analysis based on surface plasmon resonance interactions (e.g., using a BIACORE® instrument). These methods are well known to those skilled in the art and are described, for example, in Neri et al., Tibtech 14:465-470 (1996), and Jansson et al., J. Biol. Chem. 272:8189-8197 (1997).

[0065] The term "delivery vehicle" generally means any composition that acts to assist, facilitate, or ease the entry of alpha-polyglutamyl oxidized pemetrexed into cells. Such delivery vehicles are known in the art and include, but are not limited to, liposomes, lipospheres, polymers (e.g., polymer conjugates), peptides, proteins such as antibodies (e.g., immunoconjugates such as antibody-drug conjugates (ADCs) and antigen-binding antibody fragments and derivatives thereof), cell components, cyclic oligosaccharides (e.g., cyclodextrins), micelles, microparticles (e.g., microspheres), nanoparticles (e.g., lipid nanoparticles, biodegradable nanoparticles, and core-shell nanoparticles), hydrogels, lipoprotein particles, viral sequences, viral substances, or lipid or liposome formulations, and combinations thereof. The delivery vehicle can be bound directly or indirectly to the targeting moiety. In some embodiments, the targeting moiety is selected from a polymer, protein, peptide, monoclonal antibody, or fatty acid lipid.

[0066] "Subject" means a vertebrate mammal including, but not limited to, humans, dogs, cats, horses, goats and primates such as monkeys. Thus, the invention can also be used to treat diseases or conditions in non-human subjects. For example, cancer is one of the leading causes of death in companion animals (i.e., cats and dogs). In some embodiments of the invention, the subject is a human. In the present disclosure, the terms "subject" and "patient" are used interchangeably and have the same meaning. Generally, the maximum dose, i.e., the maximum safe dose according to sound medical judgment, is preferably used.

[0067] As used herein, "effective amount" means an amount of a drug sufficient to produce a medically desirable result. The effective amount can vary depending on the desired outcome, the particular condition to be treated or prevented, the age and health of the subject being treated, the severity of the condition, the duration of treatment, the nature of concurrent or concomitant therapies (if any), the specific route of administration and similar factors within the knowledge and professional opinion of the health care provider. The "effective amount" can be determined experimentally or routinely in relation to the indicated purpose. In the case of cancer, an effective amount of the drug reduces the number of cancer cells; reduces the size of the tumor; inhibits the invasion of cancer cells into surrounding organs (i.e., slows down to some extent and preferably stops); inhibits the metastasis of the tumor (i.e., slows down to some extent and preferably stops); inhibits the growth of the tumor to some extent; and / or alleviates one or more of the symptoms associated with the disorder to some extent. Depending on the extent to which the drug can prevent and / or kill the growth of existing cancer cells, the drug can be cytostatic and / or cytotoxic. For cancer therapy, in vivo efficacy can be measured, for example, by evaluating survival time, progression-free survival (PFS) period, response rate (RR), response duration, and / or quality of life.

[0068] The terms "hyperproliferative disorder", "proliferative disease", and "proliferative disorder" are used interchangeably herein and relate to unwanted or uncontrolled cell proliferation of undesirable or abnormal cells such as neoplastic or hyperplastic proliferation, regardless of in vitro or in vivo. In some embodiments, a proliferative disease is a cancer or tumor disease (including benign or cancerous) and / or any tumor metastasis, regardless of the location of the cancer, tumor and / or tumor metastasis. In some embodiments, a proliferative disease is a benign or malignant tumor. In some embodiments, a proliferative disease is a non-cancerous disease. In some embodiments, a proliferative disease is a hyperproliferative condition such as hyperplasia, fibrosis (particularly pulmonary, but also other types such as renal fibrosis), angiogenesis, psoriasis, atherosclerosis, and smooth muscle proliferation in blood vessels such as stenosis or restenosis after angioplasty.

[0069] "Cancer", "tumor", or "malignant tumor" are used as synonymous terms and mean any of a number of diseases characterized by uncontrolled, abnormal growth of cells, local or spread (metastasis) of infected cells to other parts of the body via the bloodstream and lymphatic system, and a number of characteristic structures and / or molecular features. As used herein, "tumor" means all neoplastic cell growth and proliferation, and all precancerous and cancerous cells and tissues, regardless of malignancy or benignity. "Cancerous tumor", or "malignant cells" are understood to be cells with specific structural characteristics, lacking differentiation, and capable of invasion and metastasis. Cancers that can be treated with the αPPMX compositions provided herein include, but are not limited to, for example, non-blood tumors such as lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma; and, for example, blood tumors such as leukemia, lymphoma and other B-cell malignancies, multiple myeloma and other plasma cell dyscrasias or malignancies. Other types of cancers and tumors that can be treated with the αPPMX compositions are described herein or are known in the art. The terms "cancer", "cancerous", "cell proliferative disorder", "proliferative disorder", and "tumor" are not mutually exclusive when referred to herein.

[0070] The terms "treating", or "treatment", or "treat" mean both (a) therapeutic means to cure, slow down, reduce the symptoms of, and / or halt the progression of a diagnosed medical condition or disorder and (b) prophylactic or preventive means to prevent and / or delay the occurrence of a targeted disease or condition. Thus, subjects in need of treatment include subjects already having the cancer, disorder or disease, subjects at risk of becoming cancer or a condition, and subjects in whom an infection or condition is to be prevented. A subject is identified as "at risk of having" cancer, an infectious disease, an immune system disorder, a hyperproliferative disease, or another disease or disorder mentioned herein using well-known medical and diagnostic techniques. In certain embodiments, a subject is "being treated successfully" by the methods provided herein if the subject exhibits, for example, complete, partial, or temporary remission or removal of symptoms associated with a disease or condition (e.g., cancer, rheumatoid arthritis). In certain embodiments, the term "treating" or "treatment" or "treat" means an improvement in at least one measurable physical parameter of a proliferative disorder, such as tumor growth, which may not be distinguishable by the patient. In other embodiments, the term "treating" or "treatment" or "treat" means suppressing the progression of a proliferative disorder, for example, physically by stabilization of distinguishable symptoms or physiologically by stabilization of physical parameters, or both. In other embodiments, the term "treating" or "treatment" or "treat" means a decrease or stabilization in size, tumor cell proliferation or survival, or cancer cell number. The treatment can use the α-PPMX composition alone or in combination with additional therapeutic agents.

[0071] "Subject", "patient", and "animal" are used synonymously and mean mammalian patients such as human patients and non-human primates, as well as laboratory animals such as rabbits, rats, mice and other animals. Animals include all vertebrates, e.g., mammals and non-mammals such as chickens, amphibians, and reptiles. As used herein, "mammal" includes, without limitation, human and non-human primate animals such as chimpanzees and other apes and monkey species; livestock animals such as cows, sheep, pigs, goats, and horses; pet mammals such as dogs and cats; laboratory animals such as rodents such as mice, rats, guinea pigs, and other members of the mammalian class known in the art. In certain embodiments, the patient is human.

[0072] As used herein, "treatment of a proliferative disorder" includes maintaining or reducing the tumor size of a subject with a proliferative disorder, inducing (partial or complete) tumor shrinkage, suppressing tumor growth, and / or extending lifespan. In one embodiment, the proliferative disorder is a solid tumor. Such tumors include, for example, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, colorectal cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, sarcoma (e.g., osteosarcoma), brain cancer, central nervous system cancer, and melanoma. In one embodiment, the proliferative disorder is a hematological tumor. Such hematological tumors include, for example, leukemia, lymphoma and other B cell malignancies, myeloma and other plasma cell dyscrasias or malignancies.

[0073] As used herein, the term "autoimmune disease" is defined as a disorder resulting from an autoimmune reaction. Autoimmune diseases are the result of an inappropriate and excessive reaction against self-antigens. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis.

[0074] As used herein, the term "therapeutic agent" means an agent or its derivative that can interact with hyperproliferative cells such as cancer cells or immune cells, thereby reducing the proliferative state of the cells and / or killing the cells. Examples of therapeutic agents include, but are not limited to, chemotherapeutic agents, cytotoxic agents, platinum-based agents (e.g., cisplatin, carboplatin, oxaliplatin), taxanes (e.g., Taxol®), etoposide, alkylating agents (e.g., cyclophosphamide, ifosfamide), antimetabolites (e.g., methotrexate (MTX)), 5-fluorouracil, gemcitabine, or their derivatives), antitumor antibiotics (e.g., mitomycin, doxorubicin), plant-derived antitumor agents (e.g., vincristine, vindesine, Taxol®). Such agents further include, but are not limited to, the anticancer agents trimethoprim, temozolomide, raltitrexed, S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benzylguanidine (6-BG), bis-chloronitrosourea (BCNU) and camptothecin, or any of their therapeutic derivatives. Further examples of therapeutic agents that may be suitable for use in the methods of the present disclosure include, but are not limited to, anti-restenosis agents, pro-proliferative or anti-proliferative agents, anti-inflammatory agents, anti-neoplastic agents, anti-mitotic agents, antiplatelet agents, anticoagulants, antifibrin agents, antithrombin agents, cell growth inhibitory agents, antibiotics and other anti-infective agents, anti-enzyme agents, anti-metabolites, angiogenesis agents, cytoprotective agents, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists and / or cardioprotective agents. "Therapeutic agent" also means salts, acids, and free base forms of the above agents.

[0075] As used herein, the term "chemotherapeutic agent" when used in the context of cancer therapy means any agent that causes the death of cancer cells or inhibits the growth or spread of cancer cells. Examples of such chemotherapeutic agents include alkylating agents, antibiotics, antimetabolites, plant-derived drugs, and hormones. In some embodiments, the chemotherapeutic agent is cisplatin. In some embodiments, the chemotherapeutic agent is carboplatin. In some embodiments, the chemotherapeutic agent is oxaliplatin. In other embodiments, the chemotherapeutic agent is gemcitabine. In other embodiments, the chemotherapeutic agent is doxorubicin.

[0076] As used herein, the term "antimetabolite" means a therapeutic agent that inhibits the utilization of a metabolite or its prodrug. Examples of antimetabolites include methotrexate, pemetrexed, 5-fluorouracil, 5-fluorouracil prodrugs such as capecitabine, 5-fluorodeoxyuridine monophosphate, cytarabine, cytarabine prodrugs such as nelarabine, 5-azacitidine, gemcitabine, mercaptopurine, thioguanine, azathioprine, adenosine, pentostatin, erythrohydroxynonyladenine, and cladribine. Antimetabolites useful for practicing the disclosed methods include nucleoside analogs that include purine or pyrimidine analogs. In some embodiments, the alphapolyg lutamine oxidized pemetrexed composition is used in combination with an antimetabolite selected from the group consisting of fluoropyrimidines, 5-fluorouracil, 5-fluoro-2'-deoxycytidine, cytarabine, gemcitabine, troxacitabine, decitabine, azacitidine, pseudoisocytidine, zebularine, ancitabine, fazarabine, 6-azacitidine, capecitabine, N4-octadecylcytarabine, elaidic acid cytarabine, fludarabine, cladribine, clofarabine, nelarabine, foldecsin, and pentostatin, or derivatives thereof. In one example, the nucleoside analog is a substrate of a nucleoside deaminase that is adenosine deaminase or cytidine deaminase. In some examples, the nucleoside analog is selected from fludarabine, cytarabine, gemcitabine, decitabine and azacitidine or derivatives thereof. In certain embodiments, the antimetabolite is 5-fluorouracil.

[0077] As used herein, "taxane" is an anti-cancer agent that interferes with or disrupts microtubule stability, formation, and / or function. Taxane agents include paclitaxel and docetaxel and their derivatives, and the derivatives function in the same mode of action with respect to microtubules as the taxanes from which they are derived. In certain embodiments, the taxane is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel. In certain embodiments, the taxane is paclitaxel (Taxol®), docetaxel (Taxotere®), albumin-bound paclitaxel (nab-paclitaxel; Abraxane®), DHA-paclitaxel, or PG-paclitaxel.

[0078] The terms "pharmaceutically-acceptable carrier" and "pharmaceutically acceptable carrier" mean components that are non-toxic to a subject other than the active ingredient in a pharmaceutical formulation. Pharmaceutically acceptable carriers include, but are not limited to, buffers, carriers, excipients, stabilizers, diluents, or preservatives. Pharmaceutically acceptable carriers include, for example, one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a human or other subject.

[0079] The present disclosure generally relates to novel alpha-polyglutamylated pemetrexed (PMX) compositions, and methods of making 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.

[0080] In some embodiments, the present disclosure provides the following. [1] A composition comprising alpha-polyglutamylated pemetrexed, wherein at least one glutamyl group has an alpha-carboxyl group bond. [2] The composition according to [1], wherein the alpha-polyglutamine oxidized pemetrexed contains 1 to 10 glutamyl groups having an alpha-carboxyl group bond; [3] The composition according to [1] or [2], wherein the alpha-polyglutamine oxidized pemetrexed contains 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups; [4] The composition according to any one of [1] to [3], comprising alpha-tetraglutamine oxidized pemetrexed; [5] The composition according to any one of [1] to [3], comprising alpha-pentaglutamine oxidized pemetrexed; [6] The composition according to any one of [1] to [3], comprising alpha-hexaglutamine oxidized pemetrexed; [7] The composition according to any one of [1] to [6], which is the following composition: (a) Two or more glutamyl groups have an alpha-carboxyl group bond, (b) Each glutamyl group other than the glutamyl group of pemetrexed has an alpha-carboxyl group bond, or (c) Two or more glutamyl groups have a gamma-carboxyl group bond; [8] The composition according to any one of [1] to [6], which is the following composition: (a) The C-terminal glutamyl group(s) and each glutamyl group other than the glutamyl group of pemetrexed have an alpha-carboxyl group bond; or (b) Each glutamyl group other than the C-terminal glutamyl group(s) has an alpha-carboxyl group bond; [9] The composition according to any one of [1] to [8], wherein at least one glutamyl group has both an alpha-carboxyl group bond and a gamma-carboxyl group bond;

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

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

[10] , wherein the polyglutamate is linear.

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

[10] , wherein the polyglutamate is branched.

[13] A liposome composition (Lp-αPPMX) comprising the alpha-polyglutamate oxidized pemetrexed according to any one of items [1] to

[12] .

[14] The LαPP composition according to item

[13] , wherein the alpha-polyglutamate oxidized pemetrexed contains an L-type glutamyl group having an alpha-carboxyl group bond.

[15] The Lp-αPPMX composition according to item

[13] or

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

[16] The Lp-αPPMX composition according to item

[13] or

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

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

[13] to

[16] , wherein the liposome contains an alpha-polyglutamate oxidized pemetrexed containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups.

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

[13] to

[17] , wherein at least one glutamyl group of alpha polyglutamine oxidized pemetrexed has a gamma carboxyl group bond, the composition;

[19] The composition according to any one of items

[13] to

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

[20] The composition according to any one of items

[13] to

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

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

[13] to

[20] , wherein the liposome contains alpha polyglutamine oxidized pemetrexed, and the alpha polyglutamine oxidized pemetrexed contains alpha tetraglutamine oxidized pemetrexed, alpha pentaglutamine oxidized pemetrexed or alpha hexaglutamine oxidized pemetrexed, the composition;

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

[13] to

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

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

[13] to

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

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

[13] to

[23] , wherein the liposome contains at least 1% by weight of alpha polyglutamine oxidized pemetrexed, or during the process of preparing Lp-αPPMX, at least 1% of the starting material of alpha polyglutamine oxidized PMX is encapsulated (enclosed) in Lp-αPPMX, the composition;

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

[13] to

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

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

[13] to

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

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

[13] to

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

[28] The Lp-αPPMX composition according to item

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

[29] The Lp-αPPMX composition according to item

[27] or

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

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

[27] to

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

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

[27] to

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

[32] The Lp-αPPMX composition according to item

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

[33] , wherein the liposome is cationic;

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

[13] to

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

[40] The Lp-αPPMX composition according to item

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

[41] The Lp-αPPMX composition according to item

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

[42] The Lp-αPPMX composition according to item

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

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

[39] to

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

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

[39] to

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

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

[39] to

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

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

[39] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

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

[13] to

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

[51] The Lp-αPPMX composition according to item

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

[52] The Lp-αPPMX composition according to item

[50] or

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

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

[50] to

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

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

[50] to

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

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

[50] to

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

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

[50] to

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

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

[50] to

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

[58] The Lp-αPPMX composition according to item

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

[59] The Lp-αPPMX composition according to any one of items

[39] to

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

[60] The Lp-αPPMX composition according to item

[58] or

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

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

[58] to

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

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

[58] to

[61] , further comprising a hapten;

[63] The Lp-αPPMX composition according to item

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

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

[13] to

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

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

[64] ;

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

[49] ;

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

[13] to

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

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

[13] to

[67] ;

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

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

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

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

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

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

[70] ;

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

[13] to

[69] ;

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

[69] ;

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

[13] to

[69] ;

[76] The method according to item

[74] or

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

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

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

[78] A method for treating cancer, the method comprising the step of administering an effective amount of the liposomal alpha - polyglutamine - oxidized pemetrexed composition according to any one of items

[13] to

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

[79] The method according to item

[77] or

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

[80] The method according to item

[77] or

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

[81] The method according to item

[77] or

[78] , wherein the cancer is mesothelioma or non-small cell lung cancer (NSCLC);

[82] The method according to item

[77] or

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

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

[50] to

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

[84] Maintenance therapy comprising administering an effective amount of the composition according to any one of items [1] to

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

[85] Maintenance therapy comprising administering an effective amount of the liposomal alpha polyglutamine oxidized pemetrexed composition according to any one of items

[13] to

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

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

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

[87] A method for treating an immune system disorder, comprising administering an effective amount of the liposomal alpha polyglutamine oxidized pemetrexed composition according to any one of items [9] to

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

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

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

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

[13] to

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

[90] A method for delivering alpha-polyglutamine oxidized pemetrexed to a tumor expressing a folate receptor on its surface, comprising the step of administering the Lp-αPPMX composition according to any one of items [1] to

[69] to a subject having a tumor in an amount sufficient to deliver a therapeutically effective amount of alpha-polyglutamine oxidized pemetrexed to the tumor;

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

[13] to

[69] , comprising the steps of forming a mixture comprising a liposomal component and alpha-polyglutamine oxidized pemetrexed in a solution; homogenizing the mixture in the solution to form liposomes; and treating the mixture to form liposomes comprising alpha-polyglutamine oxidized pemetrexed;

[92] A method for preparing an alpha-polyglutamine oxidized pemetrexed composition comprising the liposomal alpha-polyglutamine oxidized pemetrexed composition according to any one of items

[13] to

[69] , comprising the steps of forming a mixture comprising a liposomal component and alpha-polyglutamine oxidized pemetrexed in a solution; and treating the mixture to form liposomes comprising alpha-polyglutamine oxidized pemetrexed;

[93] The method according to item

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

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

[13] to

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

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

[50] to

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

[96] The method according to item

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

[97] The method according to any one of items

[94] to

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

[98] The method according to any one of items

[94] to

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

[99] A method according to any one of items

[91] to

[98] , wherein at least 1% of the starting material of alpha-polyglutamine oxidized PMX is encapsulated or enclosed in Lp-αPPMX.

[0081] II. Alpha-polyglutamine oxidized pemetrexed (αPPMX) Generally, the present disclosure relates to alpha-polyglutamine oxidized pemetrexed (αPPMX) compositions. The αPPMX compositions contain at least one glutamyl group having an alpha bond. These compositions are structurally different from the L-gamma-polyglutamine oxidized form of pemetrexed (LαPPMX) produced by the enzyme holylpolyglutamate synthetase (FPGS) in cells during pemetrexed therapy.

[0082] In some embodiments, the αPPMX composition comprises 2 to 20, 2 to 15, 2 to 10, 2 to 5, or more than 5 glutamyl groups (including the glutamyl groups of pemetrexed). In some embodiments, each glutamyl group in αPPMX other than the glutamyl groups of pemetrexed has an alpha bond. In some embodiments, each glutamyl group in αPPMX other than the C-terminal glutamyl group(s) and the glutamyl groups of pemetrexed has an alpha bond. In some embodiments, each of the glutamyl groups in αPPMX other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, two or more glutamyl groups in αPPMX have a gamma bond. In some embodiments, at least one glutamyl group of alpha polyglutamine oxidized pemetrexed has an alpha carboxyl group bond and a gamma carboxyl group bond. In some embodiments, each glutamyl group in αPPMX is of the L-type. In some embodiments, each glutamyl group in αPPMX other than the glutamyl groups of pemetrexed is of the D-type. In some embodiments, αPPMX comprises two or more L-type glutamyl groups and one or more D-type glutamyl groups. In some embodiments, the polyglutamate chain of αPPMX is linear (not branched). In some embodiments, the polyglutamate chain of αPPMX is branched.

[0083] In some embodiments, alpha polyglutamine oxidized pemetrexed is diglutaminated. That is, alpha polyglutamine oxidized pemetrexed comprises, in addition to the glutamyl groups of pemetrexed, one additional glutamyl group (αPMX-PG1), and the additional glutamyl group is bonded to the glutamyl group in pemetrexed via an alpha bond. In some embodiments, each glutamyl group of alpha diglutamine oxidized pemetrexed is of the L-type. In other embodiments, alpha diglutamine oxidized PMX comprises a D-type glutamyl group.

[0084] In some embodiments, alphapolyglutamylated pemetrexed is triglutamylated. That is, alphapolyglutamylated pemetrexed contains two additional glutamyl groups in addition to the glutamyl group of pemetrexed (αPMX-PG2). In some embodiments, each of the two additional glutamyl groups has an alpha bond. In other embodiments, one of the two additional glutamyl groups has an alpha bond and the other glutamyl group has a gamma bond. In some embodiments, one of the two additional glutamyl groups has an alpha bond. In some embodiments, one of the two additional glutamyl groups has a gamma bond. In some embodiments, two of the three glutamyl groups have 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 bond and a gamma bond. In some embodiments, each glutamyl group of alphatriglutamylated pemetrexed is of the L-type. In other embodiments, alphatriglutamylated PMX contains a D-type glutamyl group. In further embodiments, each glutamyl group of alphatriglutamylated pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In further embodiments, triglutamylated PMX 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.

[0085] In some embodiments, alphapolyglutamylated pemetrexed is tetraglutamylated and thus contains three additional glutamyl groups in addition to the glutamyl groups in pemetrexed (αPMX-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 has a gamma bond, respectively. In some embodiments, two of the three additional glutamyl groups have an alpha bond. In other embodiments, one of the three glutamyl groups has an alpha bond and another additional glutamyl group has a gamma bond. In other embodiments, one of the three additional glutamyl groups has both an alpha bond and a gamma bond. In other embodiments, three of the four glutamyl groups have an alpha bond. In some embodiments, at least one glutamyl group has both an alpha bond and a gamma bond. In some embodiments, alphatetraglutamylated PMX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alphatetraglutamylated pemetrexed is of the L-type. In other embodiments, alphatetraglutamylated PMX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alphatetraglutamylated pemetrexed other than the glutamyl groups of pemetrexed is of the D-type. In a further embodiment, tetraglutamylated PMX 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.

[0086] In some embodiments, alphapolyglutamylated pemetrexed is pentaglutamylated (αPMX-PG4) and includes a chain of four additional glutamyl groups attached to the glutamyl groups of pemetrexed. 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 other than the C-terminal glutamyl group(s) 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 groups are each attached to the glutamyl group of the molecule 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 groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the five glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the five glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, alphapentaglutamylated PMX includes two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alphapentaglutamylated pemetrexed is of the L-type. In other embodiments, alphapentaglutamylated PMX includes a D-type glutamyl group. In a further embodiment, each glutamyl group of alphapentaglutamylated pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In a further embodiment, pentaglutamylated PMX includes a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0087] In some embodiments, alphapolyglutamylated pemetrexed is hexaglutamylated (αPMX-PG5) and contains a chain of five additional glutamyl groups attached to the glutamyl groups of pemetrexed. 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 other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, four of the five additional glutamyl groups in the chain have an alpha bond. In other embodiments, one, two, three, or four of the five additional glutamyl groups are attached to the glutamyl groups of the molecule via an alpha bond, and the remaining four, three, two, or one glutamyl groups are each attached to the glutamyl groups of the molecule via a gamma bond. In other embodiments, one, two, three, or four of the five additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl groups of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the six glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the six glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, five of the six glutamyl groups have an alpha bond. In some embodiments, alphaglutamylated PMX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alphaglutamylated pemetrexed is of the L-type. In other embodiments, alphaglutamylated PMX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alphaglutamylated pemetrexed other than the glutamyl groups of pemetrexed is of the D-type. In a further embodiment, hexaglutamylated PMX 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.

[0088] In some embodiments, alphapolyglutamylated pemetrexed is heptaglutamylated (αPMX-PG6) and thus contains a chain of six additional glutamyl groups attached to the glutamyl groups of pemetrexed. In some embodiments, each of the six additional glutamyl groups has an alpha bond. In some embodiments, each of the six additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, five of the six additional glutamyl groups in the chain have an alpha bond. In other embodiments, one, two, three, four, or five of the six additional glutamyl groups have an alpha bond and the remaining five, four, three, two, or one glutamyl group(s) each have a gamma bond. In other embodiments, one, two, three, four, or five of the six additional glutamyl groups have an alpha bond and the remaining non-C-terminal glutamyl groups are attached to the glutamyl groups of the molecule via gamma bonds. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the seven glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the seven glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, six of the seven glutamyl groups have an alpha bond. In some embodiments, alphapentaheptaglutamylated PMX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alphapentaheptaglutamylated pemetrexed is of the L-type. In other embodiments, alphapentaheptaglutamylated PMX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alphapentaheptaglutamylated pemetrexed other than the glutamyl groups of pemetrexed is of the D-type. In a further embodiment, pentaheptaglutamylated PMX 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.

[0089] In some embodiments, alphapolyglutamylated pemetrexed is octaglutamylated (αPMX-PG7) and thus contains a chain of seven additional glutamyl groups attached to the glutamyl groups of pemetrexed. In some embodiments, each of the seven additional glutamyl groups in the chain, other than the C-terminal glutamyl group(s), has an alpha bond. In some embodiments, six of the seven additional glutamyl groups in the chain have an alpha bond. In some embodiments, each of the seven additional glutamyl groups has an alpha bond. In other embodiments, one, two, three, four, five, or six of the seven additional glutamyl groups have an alpha bond, and the remaining six, five, four, three, two, or one glutamyl group(s) each have a gamma bond. In other embodiments, one, two, three, four, five, or six of the seven additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl groups of the molecule via gamma bonds. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the eight glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the eight glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, seven of the eight glutamyl groups have an alpha bond. In some embodiments, alphaoctaglutamylated PMX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alphaoctaglutamylated pemetrexed is of the L-type. In other embodiments, alphaoctaglutamylated PMX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alphaoctaglutamylated pemetrexed, other than the glutamyl groups of pemetrexed, is of the D-type. In a further embodiment, octaglutamylated PMX 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.

[0090] In some embodiments, alphapolyglutamylated pemetrexed is nonaglutamylated (αPMX-PG8) and contains a chain of eight additional glutamyl groups attached to the glutamyl groups of pemetrexed. In some embodiments, each of the eight additional glutamyl groups in the chain, other than the C-terminal glutamyl group(s), has an alpha bond. In some embodiments, seven of the eight additional glutamyl groups in the chain have an alpha bond. In some embodiments, each of the eight additional glutamyl groups has an alpha bond. In other embodiments, one, two, three, four, five, six, or seven of the eight additional glutamyl groups have an alpha bond, and the remaining seven, six, five, four, three, two, or one glutamyl group has a gamma bond, respectively. In other embodiments, one, two, three, four, five, six, or seven of the eight additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl groups of the molecule via gamma bonds. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the nine glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the nine glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, eight of the nine glutamyl groups have an alpha bond. In some embodiments, alphanonaglutamylated PMX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alphanonaglutamylated pemetrexed is of the L-type. In other embodiments, alphanonaglutamylated PMX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alphanonaglutamylated pemetrexed other than the glutamyl groups of pemetrexed is of the D-type. In a further embodiment, nonaglutamylated PMX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0091] In some embodiments, alphapolyglutamyl oxidized pemetrexed is decaglutamylated (αPMX-PG9) (i.e., it contains a chain of 9 additional glutamyl groups attached to the glutamyl groups of pemetrexed). In some embodiments, each of the 9 additional glutamyl groups has an alpha bond. In some embodiments, each of the 9 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 8 of the 9 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 of the 9 additional glutamyl groups have an alpha bond, and the remaining 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 of the 9 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 10 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 10 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 9 of the 10 glutamyl groups have an alpha bond. In some embodiments, alphadecaglutamyl oxidized PMX contains 2 or more L-type glutamyl groups. In further embodiments, each glutamyl group of alphadecaglutamyl oxidized pemetrexed is of the L-type. In other embodiments, alphadecaglutamyl oxidized PMX contains a D-type glutamyl group. In further embodiments, each glutamyl group of alphadecaglutamyl oxidized pemetrexed other than the glutamyl groups of pemetrexed is of the D-type. In further embodiments, decaglutamylated PMX contains a D-type glutamyl group and 2 or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0092] In some embodiments, alpha-polyglutamyl oxidized pemetrexed is undecaglutamylated (αPMX-PG 10)。In some embodiments, each of the 10 additional glutamyl groups has an alpha bond. In some embodiments, each of the 10 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 9 of the 10 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the 10 additional glutamyl groups have an alpha bond, and the remaining 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the 10 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 11 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 11 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 10 of the 11 glutamyl groups have an alpha bond. In some embodiments, alpha-undecaglutaminyl oxidized PMX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alpha-undecaglutaminyl oxidized pemetrexed is of the L-type. In other embodiments, alpha-undecaglutaminyl oxidized PMX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alpha-undecaglutaminyl oxidized pemetrexed other than the glutamyl groups of pemetrexed is of the D-type. In a further embodiment, undecaglutaminyl oxidized PMX 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.

[0093] In some embodiments, alpha-polyglutaminyl oxidized pemetrexed is dodecaglutaminyl oxidized (αPMX-PG11 )。In some embodiments, each of the 11 additional glutamyl groups has an alpha bond. In some embodiments, each of the 11 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 10 of the 11 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the 11 additional glutamyl groups have an alpha bond, and the remaining 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group(s) each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the 11 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are bonded to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 12 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 12 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 11 of the 12 glutamyl groups have an alpha bond. In some embodiments, alphadedecaglutaminyl oxidized PMX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alphadedecaglutaminyl oxidized pemetrexed is of the L-type. In other embodiments, alphadedecaglutaminyl oxidized PMX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alphadedecaglutaminyl oxidized pemetrexed other than the glutamyl groups of pemetrexed is of the D-type. In a further embodiment, dodecaglutaminyl oxidized PMX 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.

[0094] In some embodiments, alpha-polyglutamated pemetrexed is tris-decaglutamated (αPMX-PG 12)。In some embodiments, each of the 12 additional glutamyl groups has an alpha bond. In some embodiments, each of the 12 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 11 of the 12 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the 12 additional glutamyl groups have an alpha bond, and the remaining 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group each has a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the 12 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via gamma bonds. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 13 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 13 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 12 of the 13 glutamyl groups have an alpha bond. In some embodiments, alpha-triskadecakaglutaminyl oxidized PMX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alpha-triskadecakaglutaminyl oxidized pemetrexed is of the L-type. In other embodiments, alpha-triskadecakaglutaminyl oxidized PMX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alpha-triskadecakaglutaminyl oxidized pemetrexed other than the glutamyl groups of pemetrexed is of the D-type. In a further embodiment, triskadecakaglutaminyl oxidized PMX 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.

[0095] In some embodiments, alphapolyglutamylated pemetrexed is tetradecaglutamylated (αPMX-PG 13)。In some embodiments, each of the 13 additional glutamyl groups has an alpha bond. In some embodiments, each of the 13 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 12 of the 13 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the 13 additional glutamyl groups have an alpha bond, and the remaining 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the 13 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are bonded to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 14 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 14 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 13 of the 14 glutamyl groups have an alpha bond. In some embodiments, alpha-tetradecaglutaminyl oxidized PMX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alpha-tetradecaglutaminyl oxidized pemetrexed is of the L-type. In other embodiments, alpha-tetradecaglutaminyl oxidized PMX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alpha-tetradecaglutaminyl oxidized pemetrexed other than the glutamyl groups of pemetrexed is of the D-type. In a further embodiment, tetradecaglutaminyl oxidized PMX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0096] In some embodiments, alphapolyg lutamine oxidized pemetrexed is pentadeca glutamine oxidized (αPMX-PG 14)。In some embodiments, each of the 14 additional glutamyl groups has an alpha bond. In some embodiments, each of the 14 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 13 of the 14 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the 14 additional glutamyl groups have an alpha bond, and the remaining 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group has a gamma bond, respectively. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the 14 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 15 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 15 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 14 of the 15 glutamyl groups have an alpha bond. In some embodiments, alpha-pentadeca-glutaminyl oxidized PMX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alpha-pentadeca-glutaminyl oxidized pemetrexed is of the L-type. In other embodiments, alpha-pentadeca-glutaminyl oxidized PMX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alpha-pentadeca-glutaminyl oxidized pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In a further embodiment, pentadeca-glutaminyl oxidized PMX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0097] In some embodiments, alpha-polyglutamylated pemetrexed is hexadeca-glutamylated (αPMX-PG 15)。In some embodiments, each of the 15 additional glutamyl groups has an alpha bond. In some embodiments, each of the 15 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 14 of the 15 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the 15 additional glutamyl groups have an alpha bond, and the remaining 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma 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 non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 16 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 16 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 15 of the 16 glutamyl groups have an alpha bond. In some embodiments, alpha-hexadeca-glutaminyl oxidized PMX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alpha-hexadeca-glutaminyl oxidized pemetrexed is of the L-type. In other embodiments, alpha-hexadeca-glutaminyl oxidized PMX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alpha-deca-hexadeca-glutaminyl oxidized pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In a further embodiment, hexadeca-glutaminyl oxidized PMX 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.

[0098] In other embodiments, alphapolyglutamylated pemetrexed is heptadeca-glutamylated (αPMX-PG 16)。In some embodiments, each of the 16 additional glutamyl groups has an alpha bond. In some embodiments, each of the 16 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 15 of the 16 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the 16 additional glutamyl groups have an alpha bond, and the remaining 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl groups each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the 16 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 17 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 17 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 16 of the 17 glutamyl groups have an alpha bond. In some embodiments, alpha-heptadeca-glutaminyl oxidized PMX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alpha-heptadeca-glutaminyl oxidized pemetrexed is of the L-type. In other embodiments, alpha-heptadeca-glutaminyl oxidized PMX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alpha-heptadeca-glutaminyl oxidized pemetrexed other than the glutamyl groups of pemetrexed is of the D-type. In a further embodiment, heptadeca-glutaminyl oxidized PMX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0099] In some embodiments, alpha-polyglutamylated pemetrexed is octadeca-glutamylated (αPMX-PG 17)。In some embodiments, each of the 17 additional glutamyl groups has an alpha bond. In some embodiments, each of the 17 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 16 of the 17 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the 17 additional glutamyl groups have an alpha bond, and the remaining 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group each has a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the 17 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are bonded to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 18 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 18 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 17 of the 18 glutamyl groups have an alpha bond. In some embodiments, alpha-octadeca-glutaminyl oxidized PMX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alpha-octadeca-glutaminyl oxidized pemetrexed is of the L-type. In other embodiments, alpha-octadeca-glutaminyl oxidized PMX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alpha-octadeca-glutaminyl oxidized pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In a further embodiment, octadeca-glutaminyl oxidized PMX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0100] In some embodiments, alphapolyglutamylated pemetrexed is enniadecakisglutamylated (αPMX-PG 18)。In some embodiments, each of the 18 additional glutamyl groups has an alpha bond. In some embodiments, each of the 18 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 17 of the 18 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of the 18 additional glutamyl groups have an alpha bond, and the remaining 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group each has a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of the 18 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 19 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 19 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 18 of the 19 glutamyl groups have an alpha bond. In some embodiments, alpha-enniadeca glutamine oxidized PMX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alpha-enniadeca glutamine oxidized pemetrexed is of the L-type. In other embodiments, alpha-enniadeca glutamine oxidized PMX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alpha-enniadeca glutamine oxidized pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In a further embodiment, enniadeca glutamine oxidized PMX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0101] In some embodiments, alphapolyglutamylated pemetrexed is polyglutamylated (αPMX-PG). 19)。In some embodiments, each of the 19 additional glutamyl groups has an alpha bond. In some embodiments, each of the 19 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 18 of the 19 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 19 additional glutamyl groups have an alpha bond, and the remaining 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group(s) each have a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 19 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 20 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 20 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 19 of the 20 glutamyl groups have an alpha bond. In some embodiments, alphaiCOS glutaminyl oxidized PMX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alphaiCOS glutaminyl oxidized pemetrexed is of the L-type. In other embodiments, alphaiCOS glutaminyl oxidized PMX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alphaiCOS glutaminyl oxidized pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In a further embodiment, iCOS glutaminyl oxidized PMX contains a D-type glutamyl group and two or more L-type glutamyl groups. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0102] In some embodiments, alpha-polyglutamylated pemetrexed is polyglutamylated with eicosanoic acid (αPMX-PG 20)。In some embodiments, each of the 20 additional glutamyl groups has an alpha bond. In some embodiments, each of the 20 additional glutamyl groups in the chain other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 19 of the 20 additional glutamyl groups in the chain have an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the 20 additional glutamyl groups have an alpha bond, and the remaining 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group each has a gamma bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the 20 additional glutamyl groups have an alpha bond, and the remaining non-C-terminal glutamyl groups are attached to the glutamyl group of the molecule via a gamma bond. In some embodiments, at least one additional glutamyl group has both an alpha bond and a gamma bond. In some embodiments, at least one of the 21 glutamyl groups has both an alpha bond and a gamma bond. In some embodiments, each of the 21 glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 20 of the 21 glutamyl groups have an alpha bond. In some embodiments, alphaicosikahenaglutamine oxidized PMX contains two or more L-type glutamyl groups. In a further embodiment, each glutamyl group of alphaicosikahenaglutamine oxidized pemetrexed is of the L-type. In other embodiments, alphaicosikahenaglutamine oxidized PMX contains a D-type glutamyl group. In a further embodiment, each glutamyl group of alphaicosikahenaglutamine oxidized pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In a further embodiment, icosikahenaglutamine oxidized PMX 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.

[0103] In some embodiments, alpha-polyglutamylated pemetrexed contains 4 to 7 glutamyl groups attached to pemetrexed (i.e., αPMX-PGn, n = 4 to 7), and each of the 4 to 7 attached glutamyl groups has an alpha bond. In some embodiments, alpha-polyglutamylated pemetrexed contains 4 to 7 glutamyl groups attached to pemetrexed (i.e., αPMX-PGn, n = 4 to 7), and each of the 4 to 7 attached glutamyl groups other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, each of the 4 to 7 attached glutamyl groups is of the L-type. In other embodiments, each of the 4 to 7 attached glutamyl groups is of the D-type. In other embodiments, the 4 to 7 attached glutamyl groups are of the L-type and D-type. In some embodiments, the polyglutamate chain is a straight chain. In other embodiments, the polyglutamate chain is a branched chain.

[0104] In one embodiment, alpha-polyglutamylated pemetrexed is tetraglutamylated, and each of the 3 glutamyl groups in the polyglutamate chain attached to pemetrexed contains an alpha bond. In one embodiment, alpha-polyglutamylated pemetrexed is tetraglutamylated, and each of the 3 glutamyl groups in the polyglutamate chain attached to pemetrexed other than the C-terminal glutamyl group(s) contains an alpha bond. In some embodiments, each of the 4 glutamyl groups is of the L-type. In some embodiments, each glutamyl group of alpha-tetraglutamylated pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In other embodiments, at least 2 of the glutamyl groups in alpha-tetraglutamate pemetrexed are of the L-type and at least 1 glutamyl group is of the D-type. In some embodiments, the polyglutamate chain is a straight chain. In other embodiments, the polyglutamate chain is a branched chain.

[0105] In one embodiment, alphapolyglutamylated pemetrexed is pentaglutamylated, and each of the four glutamyl groups in the polyglutamate chain attached to pemetrexed contains an alpha bond. In one embodiment, alphapolyglutamylated pemetrexed is pentaglutamylated, and each of the four glutamyl groups in the polyglutamate chain attached to pemetrexed other than the C-terminal glutamyl group(s) contains an alpha bond. In some embodiments, each of the four glutamyl groups is of the L-type. In some embodiments, each glutamyl group of alphapentaglutamylated pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In other embodiments, at least two of the glutamyl groups in alphapentaglutamylated pemetrexed are of the L-type and at least one glutamyl group is of the D-type. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0106] In one embodiment, alphapolyglutamylated pemetrexed is hexaglutamylated, and each of the five glutamyl groups in the polyglutamate chain attached to pemetrexed contains an alpha bond. In one embodiment, alphapolyglutamylated pemetrexed is hexaglutamylated, and each of the five glutamyl groups in the polyglutamate chain attached to pemetrexed other than the C-terminal glutamyl group(s) contains an alpha bond. In some embodiments, each of the five glutamyl groups is of the L-type. In some embodiments, each glutamyl group of alphapolyglutamylated pemetrexed other than the glutamyl group of pemetrexed is of the D-type. In other embodiments, at least two of the glutamyl groups in alphapolyglutamylated pemetrexed are of the L-type and at least one glutamyl group is of the D-type. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0107] In another embodiment, alphapolyglutamyl oxidized pemetrexed is heptaglutamylated, and each of the six glutamyl groups in the polyglutamate chain attached to pemetrexed contains an alpha bond. In another embodiment, alphapolyglutamyl oxidized pemetrexed is heptaglutamylated, and each of the six glutamyl groups in the polyglutamate chain attached to pemetrexed, other than the C-terminal glutamyl group(s), contains an alpha bond. In some embodiments, each of the six glutamyl groups is of the L-type. In some embodiments, each glutamyl group of alphahptaglutamyl oxidized pemetrexed, other than the glutamyl group of pemetrexed, is of the D-type. In other embodiments, at least two of the glutamyl groups in alphahptaglutamyl oxidized pemetrexed are of the L-type and at least one glutamyl group is of the D-type. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0108] In some embodiments, alpha-polyglutamylated pemetrexed (αPPMX) 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 pemetrexed, or any range therebetween. In some embodiments, each glutamyl group in αPPMX other than the glutamyl group of pemetrexed has an alpha bond. In some embodiments, the C-terminal glutamyl group(s) and each glutamyl group in αPPMX other than the glutamyl group of pemetrexed have an alpha bond. In some embodiments, each of the glutamyl groups in αPPMX other than the C-terminal glutamyl group(s) has an alpha bond. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the glutamyl groups in αPPMX have an alpha bond. In some embodiments, αPPMX contains L- and D-type glutamyl groups. In further embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the glutamyl groups in αPPMX have an alpha bond and 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 of the glutamyl groups each have a gamma bond. In some embodiments, each glutamyl group in the polyglutamate structure of polyglutamylated pemetrexed is of the L-type. In some embodiments, each glutamyl group in αPPMX other than the glutamyl group of pemetrexed is of the D-type. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the glutamyl groups in αPPMX are of the L-type. In another embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the glutamyl groups in αPPMX are of the D-type. In some embodiments, the polyglutamate chain is linear. In other embodiments, the polyglutamate chain is branched.

[0109] In some embodiments, alpha-polyglutamyl pemetrexed (αPPMX) contains a total of 2 to 20, 2 to 15, 2 to 10, 2 to 5, or any range therebetween glutamyl groups, including the glutamyl group of pemetrexed. In some embodiments, each glutamyl group in αPPMX other than the glutamyl group of pemetrexed has an alpha bond. In some embodiments, the C-terminal glutamyl group(s) and each glutamyl group in αPPMX other than the glutamyl group of pemetrexed have an alpha bond. In some embodiments, each of the glutamyl groups in αPPMX other than the C-terminal glutamyl group(s) has an alpha bond. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 glutamyl groups have an alpha bond. In some embodiments, αPPMX contains two or more glutamyl groups having a gamma bond. In a further embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 glutamyl groups in αPPMX other than the glutamyl group of pemetrexed have an alpha bond, and 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 glutamyl groups each have a gamma bond. In some embodiments, each glutamyl group in αPPMX is of the L-type. In some embodiments, each glutamyl group in αPPMX other than the glutamyl group of pemetrexed is of the D-type. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the glutamyl groups in αPPMX are of the L-type. In another embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the glutamyl groups in αPPMX are of the D-type.

[0110] In some embodiments, alpha-polyglutamyl oxidized pemetrexed contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 glutamyl groups in addition to the glutamyl group of pemetrexed. In further embodiments, the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additional glutamyl groups have alpha linkages. In further embodiments, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 glutamyl group in alpha-polyglutamyl oxidized pemetrexed has a gamma linkage. In some embodiments, at least one glutamyl group has both alpha and gamma linkages. In some embodiments, the glutamyl group in pemetrexed has an alpha linkage. In some embodiments, the glutamyl group in pemetrexed has both alpha and gamma linkages.

[0111] In some embodiments, the total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 glutamyl groups in alpha-polyglutamyl oxidized pemetrexed are of the L-form, D-form, or both L-form and D-form. In some embodiments, each glutamyl group of alpha-polyglutamyl oxidized pemetrexed is of the L-form. In other embodiments, each glutamyl group of alpha-polyglutamyl oxidized pemetrexed other than the glutamyl group of pemetrexed is of the D-form. In alternative embodiments, at least two of the glutamyl groups in alpha-polyglutamyl oxidized pemetrexed are of the L-form and at least one of the glutamyl groups in alpha-polyglutamyl oxidized pemetrexed is of the D-form. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 glutamyl groups in alpha-polyglutamyl oxidized pemetrexed are of the L-form. In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 glutamyl groups in alpha-polyglutamyl oxidized pemetrexed are of the D-form.

[0112] In further embodiments, alpha-polyglutamylated pemetrexed contains 20 to 100, 20 to 75, 20 to 50, 20 to 40, 20 to 30, 20 to 25, or more than 100 alpha-glutamyl groups, or any range therebetween. In some embodiments, each glutamyl group of alpha-polyglutamylated pemetrexed is of the L-type. In other embodiments, each glutamyl group of alpha-polyglutamylated pemetrexed other than the glutamyl groups of pemetrexed is of the D-type. In alternative embodiments, at least two of the glutamyl groups in alpha-polyglutamylated pemetrexed are of the L-type and at least one of the glutamyl groups in alpha-polyglutamylated pemetrexed is of the D-type.

[0113] In further embodiments, the provided composition contains alpha-polyglutamylated pemetrexed having 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 to 10, or 1 to 20 alpha-linked glutamyl groups. In some embodiments, alpha-polyglutamylated pemetrexed contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 to 10, or 1 to 20 L-type glutamyl groups. In some embodiments, alpha-polyglutamylated pemetrexed contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 to 10, or 1 to 20 D-type glutamyl groups. In some embodiments, alpha-polyglutamylated pemetrexed contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 to 10, or 1 to 20 L-type glutamyl groups and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 to 10, or 1 to 20 D-type glutamyl groups. In other embodiments, alpha-polyglutamylated pemetrexed contains at least one glutamyl group having both alpha- and gamma-bonds. In some embodiments, alpha-polyglutamylated pemetrexed contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 to 10, or more than 10 glutamyl groups having both alpha- and gamma-bonds.

[0114] In some embodiments, alpha-polyglutamyl oxidized pemetrexed contains at least 1 glutamyl group having an alpha bond and contains 2, 3, 4, 5, 6, 7, 8, 9, 1 to 10, 1 to 20, or more glutamyl groups having a gamma bond. For example, in some embodiments, alpha-polyglutamyl oxidized pemetrexed contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 to 10 L-alpha-glutamyl group bonds and further contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1 to 10 L-gamma-glutamyl group bonds. In some further embodiments, alpha-polyglutamyl oxidized pemetrexed contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1 to 10 L-alpha-glutamyl group bonds and further contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1 to 10 D-gamma-glutamyl group bonds. In additional further embodiments, alpha-polyglutamyl oxidized pemetrexed contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1 to 10 D-alpha-glutamyl group bonds and further contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1 to 10 D-gamma-glutamyl group bonds. In other further embodiments, alpha-polyglutamyl oxidized pemetrexed contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1 to 10 D-gamma-glutamyl group bonds and further contains 1, 2, 3, 4, 5, 6, or 1 to 10 L-gamma-glutamyl group bonds. In other embodiments, alpha-polyglutamyl oxidized pemetrexed contains at least 1 glutamyl group having both an alpha bond and a gamma bond. In some embodiments, alpha-polyglutamyl oxidized pemetrexed contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 to 10, or more than 10 glutamyl groups having both an alpha bond and a gamma bond.

[0115] In some embodiments, the alpha-polyglutamylated pemetrexed compositions provided herein can accept one or more additional glutamyl groups, i.e., the compositions can serve as substrates for FPGS (folylpolyglutamate synthetase). Reagents and assays for measuring the ability of an alpha-polyglutamylated pemetrexed composition to act as a substrate for FPGS (e.g., human FPGS, or rat liver FPGS) are readily available and can be performed as routine business.

[0116] In some embodiments, the rate of uptake of the naked alpha-PPMX compositions disclosed herein (e.g., alpha-PPMX not bound to a delivery carrier) by liver cells is significantly reduced compared to the rate of uptake of pemetrexed under physiological conditions. In some embodiments, the rate of liver cell uptake of the naked alpha-PPMX composition is less than 30%, 20%, 15%, or 10% compared to the rate of pemetrexed. In further embodiments, the rate of efflux (transport) of the alpha-PPMX compositions disclosed herein from liver cells occurs at a significantly lower rate (less than 30%, 20%, 15%, or 10%) compared to pemetrexed.

[0117] In some embodiments, the alpha-polyglutamylated pemetrexed compositions provided herein have higher cytotoxicity against proliferating cells than pemetrexed. In some embodiments, the proliferating cells are cancer cells. In some embodiments, the proliferating cells are colorectal cancer cells, colon cancer cells, breast cancer cells, or ovarian cancer cells. In some embodiments, the cancer cells are mesothelioma cells or non-small cell lung cancer cells. In some embodiments, the cytotoxicity is measured in an in vitro assay. In some embodiments, the alpha-polyglutamylated pemetrexed is hexaglutamylated pemetrexed.

[0118] In some embodiments, the alpha-polyglutamylated pemetrexed compositions provided herein have lower toxic side effects than pemetrexed. In some embodiments, the alpha-polyglutamylated pemetrexed compositions provided herein are less toxic to non-proliferating cells than pemetrexed. In some embodiments, the alpha-polyglutamylated pemetrexed compositions provided herein are less toxic to neutrophils, liver cells, or colon epithelial cells than pemetrexed. In some embodiments, the neutrophils are human neutrophils, differentiated human neutrophils, or neutrophils differentiated from CD34+ cells. In some embodiments, the liver cells are AML12 liver cells. In some embodiments, the colon epithelial cells are CCD841 colon epithelial cells. In some embodiments, the toxicity is measured in an in vitro assay. In some embodiments, the alpha-polyglutamylated pemetrexed is hexaglutamylated pemetrexed.

[0119] In some embodiments, the alpha-polyglutaminyl oxidized pemetrexed compositions provided herein have lower toxic side effects than pemetrexed. In some embodiments, the alpha-polyglutaminyl oxidized pemetrexed compositions provided herein result in less frequent or less severe toxic side effects than pemetrexed in in vivo assays. In some embodiments, the in vivo assay is performed in an in vivo mouse model. In some embodiments, the alpha-polyglutaminyl oxidized pemetrexed compositions provided herein result in less frequent or less severe hematological or hepatic toxic side effects than pemetrexed. In some embodiments, the hematological side effects are evaluated by mean neutrophils, mean white blood cells or mean platelet counts. In some embodiments, the hepatic toxic side effects are evaluated by measuring serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), and / or serum albumin levels. In some embodiments, the in vivo assay comprises administering the alpha-polyglutaminyl oxidized pemetrexed composition at 40 mg / kg or 80 mg / kg once a week for 4 weeks. In some embodiments, the alpha-polyglutaminyl oxidized pemetrexed is hexaglutaminyl oxidized pemetrexed.

[0120] In some embodiments, treatment with the alpha-polyglutamylated pemetrexed compositions provided herein does not induce significant hematological or liver toxic side effects in an in vivo mouse model. In some embodiments, hematological side effects are evaluated by mean neutrophils, mean white blood cells, or mean platelet counts. In some embodiments, liver toxic side effects are evaluated by measuring serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), and / or serum albumin levels. In some embodiments, the alpha-polyglutamylated pemetrexed compositions provided herein do not significantly decrease mean neutrophils, mean white blood cells, or mean platelet counts. In some embodiments, the alpha-polyglutamylated pemetrexed compositions provided herein do not significantly increase serum aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT) levels. In some embodiments, the alpha-polyglutamylated pemetrexed compositions provided herein do not significantly reduce serum albumin levels. In some embodiments, the in vivo assay comprises administering the alpha-polyglutamylated pemetrexed composition at 40 mg / kg or 80 mg / kg once a week for 4 weeks. In some embodiments, the alpha-polyglutamylated pemetrexed is hexaglutamylated pemetrexed.

[0121] In some embodiments, the alpha-polyglutamylated pemetrexed composition does not contain a fluorine atom. In some embodiments, the alpha-polyglutamylated pemetrexed composition does not contain a 4-fluoroglutamyl group.

[0122] Alpha-polyglutamylated pemetrexed (αPPMX) compositions and their uses are further described in each of U.S. Patent Application Nos. 62 / 374,458, 15 / 675,695, 15 / 675,701, and 62 / 583,432, as well as International Application Nos. PCT / US2017 / 046666 and PCT / US2017 / 046667. The contents of each of these are hereby incorporated by reference in their entirety.

[0123] A. Polyglutamylated pemetrexed analogs and derivatives The present disclosure also encompasses alpha-polyglutamylated pemetrexed derivatives and analogs. The compositions and methods disclosed herein are contemplated for application to any and all known derivatives or analogs of polyglutamylated pemetrexed. In some embodiments, polyglutamylated pemetrexed analog or derivative compositions made and used in accordance with the disclosed compositions and methods are shown in FIGS. 1I and 1J. In some embodiments, the analog corresponds to a modified form of pemetrexed, in which case the glutamyl group of pemetrexed is not attached to the remainder of the pemetrexed molecule via a gamma peptide bond. In some embodiments, the analog is a variant of pemetrexed, in which case the glutamyl group in pemetrexed is of the D-type. In some embodiments, the polyglutamylated form of pemetrexed, or polyglutamylated pemetrexed analog or derivative, is not fluorinated.

[0124] In further embodiments, the alpha-polyglutamylated pemetrexed derivative or analog has a variant polyglutamate chain. In some embodiments, the polyglutamate chain contains one or more natural or synthetic residues other than glutamate. In some embodiments, the polyglutamate chain contains one or more glutamyl groups that do not contain an amide bond. In other embodiments, one or more glutamyl groups of the polyglutamate chain are derivatized.

[0125] B. PMX-PG synthesis The pemetrexed polyglutamate compositions provided herein are obtained by the following synthetic methods known in the art. Procedures for synthesizing pemetrexed (including different pharmaceutically acceptable salts or acids (e.g., pemetrexed disodium) and crystalline and amorphous forms) and intermediates for synthesizing pemetrexed include, but are not limited to, U.S. Patent Nos. 8,507,508, 8,362,245, 7,138,521, 6,262,262, 6,066,732, 5,416,211, 5,344,932; U.S. Patent Application Publication No. 2013 / 0165654A1; European Patent Nos. 0905128, 2882753B1, 0905128, and 2409978B1; International Publication Nos. 2014 / 024164A1, 2012 / 056285A1, 2008 / 021410A1, and 2001 / 14379A2, and those described in Barnett et al., Org. Proc Res & Develop. 3:184-188 (1999); Taylor et al., J. Org. Chem. 68:9938-9947 (2003); Taylor et al., Tetrahedron Lett. 40:4023 (1999); Barnett et al., Org. Proc. Res. & Develop. 3:184-188 (1999) and Kjell et al., Org. Proc. Res. Dev. 9:738 (2005).

[0126] The addition of a glutamyl residue to the glutamyl residue of pemetrexed can be carried out using synthetic methods known in the art. In some embodiments, the glutamyl residue is sequentially added to the glutamyl residue of pemetrexed. In further embodiments, the polyglutamate is added to the glutamyl residue of pemetrexed using "click chemistry" methods or other bioconjugate chemistries known to those skilled in the art. Alternatively, a peptide of the desired length of glutamyl residues can be generated and added to a precursor without a glutamyl residue of pemetrexed. The peptide can be prepared using methods known in the art. In some embodiments, the initial glutamyl residue is attached to Wang resin, and additional glutamyl residues are sequentially added by solid-phase peptide synthesis using F-moc chemistry. After the last glutamyl residue is added, the pemetrexed precursor is attached to the peptide and the molecule is cleaved from the resin.

[0127] C. Pemetrexed-PG Complex Surprisingly, the inventors have found that polyglutamine oxidized pemetrexed (αPPMX) can form a complex with other compositions, including therapeutic agents such as cytotoxic compounds such as platinum-based compounds. Accordingly, in some embodiments, the present disclosure provides a complex of αPPMX (e.g., αPPMX disclosed herein) with a therapeutic agent or a salt or acid thereof.

[0128] In some embodiments, the αPPMX / complex comprises αPPMX and a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic compound such as a chemotherapeutic agent. In further embodiments, the αPPMX / complex comprises a platinum-based drug such as a platinum-based chemotherapeutic agent (e.g., cisplatin, carboplatin, and oxaliplatin). In other embodiments, the αPPMX / complex comprises a taxane-based chemotherapeutic agent (e.g., paclitaxel and docetaxel). In other embodiments, the αPPMX / complex comprises cyclodextrin. In further embodiments, the αPPMX / complex is encapsulated in liposomes.

[0129] In some embodiments, the present disclosure provides a composition comprising a complex of αPPMX and a therapeutic agent or a salt or acid thereof. In further embodiments, the αPPMX / therapeutic agent complex comprises one or more αPPMXs 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 αPPMX / therapeutic agent complex comprises one or more αPPMXs containing 3 to 10, 3 to 9, 3 to 8, or 3 to 7 glutamyl groups, or any range therebetween. In other embodiments, the αPPMX / therapeutic agent complex comprises one or more αPPMXs containing 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, or 4 to 5 glutamyl groups, or any range therebetween. In a particular embodiment, the complex comprises one or more αPPMXs containing 3 to 10 glutamyl groups. In further embodiments, the αPPMX / therapeutic agent complex comprises one or more αPPMXs containing 3 to 7 glutamyl groups. In another embodiment, the αPPMX / therapeutic agent complex comprises one or more αPPMXs containing 5 glutamyl groups. In another embodiment, the αPPMX / therapeutic agent complex comprises one or more αPPMXs 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 chemotherapeutic agent is a platinum-based drug. In another embodiment, the chemotherapeutic agent is a taxane-based drug. In further embodiments, the molar ratio of αPPMX / therapeutic agent in the complex ranges from 1 to 10:1. In some embodiments, the molar ratio of αPPMX / therapeutic agent in the complex is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50.In some embodiments, the molar ratio of αPPMX / 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 some embodiments, the αPPMX / therapeutic agent complex is encapsulated in liposomes (e.g., in the manner described herein or by another method known in the art).

[0130] In alternative embodiments, the αPPMX complex comprises αPPMX and cyclodextrin. In some embodiments, the molar ratio of αPPMX (e.g., αPPMX salt) / cyclodextrin in the complex ranges from 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of αPPMX / cyclodextrin in the complex ranges from 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of αPPMX / cyclodextrin in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of αPPMX / 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 αPPMX / cyclodextrin in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of αPPMX / 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 αPPMX / cyclodextrin complex is encapsulated in liposomes (e.g., in the manner described herein or by another method known in the art).

[0131] In some embodiments, the present disclosure provides a composition comprising an αPPMX / platinum-based chemotherapeutic agent complex. In some embodiments, the platinum-based chemotherapeutic agent is selected from the group consisting of cisplatin, carboplatin, and oxaliplatin, or salts or acids thereof. In other embodiments, the αPPMX / platinum-based chemotherapeutic agent complex comprises a cisplatin, carboplatin, oxaliplatin analog, or salts or acids thereof. In some embodiments, the molar ratio of αPPMX / platinum-based agent in the complex ranges from 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of αPPMX / platinum-based agent in the complex ranges from 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of αPPMX / platinum-based agent in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of αPPMX / platinum-based agent in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 - 50):1, or >50:1. In other embodiments, the molar ratio of αPPMX / platinum-based chemotherapeutic agent in the complex ranges from 1:1 to 20, 1:1 to 10, or 1:2 to 8, or any range therebetween. In some embodiments, the molar ratio of αPPMX / platinum-based agent in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 - 50), or 1:>50. In further embodiments, the αPPMX / platinum-based agent complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).

[0132] In a further embodiment, the αPPMX / platinum-based chemotherapeutic agent complex comprises cisplatin, carboplatin, an oxaliplatin analog, or a salt or acid thereof. In some embodiments, the molar ratio of αPPMX / platinum-based analog in the complex ranges from 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of αPPMX / platinum-based analog in the complex ranges from 1 to 10:1, or any range therebetween. In a further embodiment, the molar ratio of αPPMX / platinum-based agent in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of αPPMX / platinum-based analog in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In some embodiments, the molar ratio of αPPMX / platinum-based agent in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50. In a further embodiment, the αPPMX / platinum-based analog complex is encapsulated in liposomes (e.g., in the manner described herein or by another method known in the art).

[0133] In a further embodiment, the present disclosure provides a complex comprising αPPMX and cisplatin or a salt or acid thereof. In some embodiments, the molar ratio of αPPMX / cisplatin (or a salt or acid of cisplatin) in the complex ranges from 1 to 20:1, or any range therebetween. In some embodiments, the molar ratio of αPPMX / cisplatin (or a salt or acid of cisplatin) in the complex ranges from 1 to 10:1, or any range therebetween. In a further embodiment, the molar ratio of αPPMX / cisplatin (or a salt or acid of cisplatin) in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of αPPMX / cisplatin (or a salt or acid of cisplatin) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1. In some embodiments, the molar ratio of αPPMX / cisplatin (or a salt or acid of cisplatin) in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 to 50), or 1:>50. In a further embodiment, the αPPMX / cisplatin (or a salt or acid of cisplatin) complex is encapsulated in liposomes (e.g., in the manner described herein or by another method known in the art).

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

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

[0136] In a further embodiment, the present disclosure provides a complex comprising αPPMX and a platinum-based chemotherapeutic agent (platinum) selected from the group consisting of nedaplatin, heptaplatin, lobaplatin, satraplatin, paraplatin, platinoil, cycloplatin, dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iplatin, ormaplatin, zeniplatin, platinum-triamine, satraplatin, enroplatin, JM216, NK121, CI973, DWA2114R, NDDP, and nedaplatin, or salts or acids thereof. In other embodiments, the αPPMX / platinum-based chemotherapeutic agent complex comprises nedaplatin, heptaplatin, lobaplatin, satraplatin, paraplatin, platinoil, cycloplatin, dexormaplatin, spiroplatin, picoplatin, triplatin, tetraplatin, iplatin, ormaplatin, zeniplatin, platinum-triamine, satraplatin, enroplatin, JM216, NK121, CI973, DWA2114R, NDDP, or an analogue of nedaplatin, or salts or acids thereof. In some embodiments, the molar ratio of αPPMX / platinum (or a salt or acid of platinum) in the complex ranges from 1 to 20:1, or any range therebetween. In a further embodiment, the molar ratio of αPPMX / platinum (or a salt or acid of platinum) in the complex ranges from 1 to 10:1, or any range therebetween. In a further embodiment, the molar ratio of αPPMX / platinum (or a salt or acid of platinum) in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of αPPMX / platinum (or a salt or acid of platinum) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 to 50):1, or >50:1.In some embodiments, the molar ratio of αPPMX / platinum (or a salt or acid of platinum) in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 - 50), or 1:>50. In further embodiments, the αPPMX / platinum (or its salt or acid or analog) complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).

[0137] In some embodiments, the present disclosure provides a composition comprising an αPPMX / taxane chemotherapeutic agent (taxane) complex. In some embodiments, the taxane chemotherapeutic agent is selected from the group consisting of paclitaxel (PTX), docetaxel (DTX), larotaxel (LTX), and cabazitaxel (CTX), or salts or acids thereof. In some embodiments, the molar ratio of αPPMX / taxane agent in the complex ranges from 1 to 20:1, or any range therebetween. In further embodiments, the molar ratio of αPPMX / taxane (or a salt or acid of taxane) in the complex ranges from 1 to 10:1, or any range therebetween. In further embodiments, the molar ratio of αPPMX / taxane (or a salt or acid of taxane) in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of αPPMX / taxane (or a salt or acid of taxane) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 - 50):1, or >50:1. In some embodiments, the molar ratio of αPPMX / taxane (or a salt or acid of taxane) in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 - 50), or 1:>50. In further embodiments, the αPPMX / taxane agent complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).

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

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

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

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

[0142] In a further embodiment, the present disclosure provides a complex comprising αPPMX and another antimetabolite, or a salt or acid thereof. An antimetabolite is a chemical substance that is similar to a metabolite required for normal biochemical reactions but has a structure with sufficient differences to interfere with the normal functions of one or more cells, such as cell division. In some embodiments, the present disclosure provides a complex comprising αPPMX and pemetrexed (PMX), or a salt or acid thereof. In some embodiments, the present disclosure provides a complex comprising αPPMX and an antimetabolite selected from the group consisting of gemcitabine, fluorouracil, capecitabine, antifolates (e.g., methotrexate, raltitrexed), tegafur, cytosine arabinoside, thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, and any pharmaceutically acceptable salt or acid (s) or derivative thereof. In some embodiments, the molar ratio of αPPMX / antimetabolite (or salt or acid of the antimetabolite) in the complex ranges from 1 to 20:1, or any range therebetween. In a further embodiment, the molar ratio of αPPMX / antimetabolite (or salt or acid of the antimetabolite) in the complex ranges from 1 to 10:1, or any range therebetween. In a further embodiment, the molar ratio of αPPMX / antimetabolite (or salt or acid of the antimetabolite) in the complex ranges from 2 to 8:1, or any range therebetween. In some embodiments, the molar ratio of αPPMX / antimetabolite (or salt or acid of the antimetabolite) in the complex is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, (21 - 50):1, or >50:1. In some embodiments, the molar ratio of αPPMX / antimetabolite (or salt or acid of the antimetabolite) in the complex is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:(21 - 50), or 1:>50.In a further embodiment, the αPPMX / antimetabolite (or a salt or acid of an antimetabolite) complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).

[0143] In a further embodiment, the present disclosure provides a complex of αPPMX (e.g., αPPMX disclosed herein) and cyclodextrin. Cyclodextrin (CD) is a group of cyclic oligosaccharides that have been shown to improve the physicochemical properties of many drugs through complex formation. CD is a cyclic oligosaccharide composed of several D-glucose units linked by α-(1,4) bonds. This cyclic structure provides a hydrophobic internal cavity and gives CD a truncated cone shape. Many hydroxyl groups are located on the ends of the ring, which makes CD both lipophilic and water-soluble. As a result, CD can form complexes with a wide variety of hydrophobic agents, thereby changing the physicochemical properties of these complexed agents.

[0144] The term "cyclodextrin" or "CD" generally means a parent or derivatized cyclic oligosaccharide that can form a complex with pemetrexed-PG and contains a variable number of (α-1,4) linked D-glucopyranoside units, unless otherwise specified. Each cyclodextrin ring glucopyranoside subunit has secondary hydroxyl groups at the 2 and 3 positions and a primary hydroxyl group at the 6 position. The terms "parent", "undenivatized", or "inactive" cyclodextrin refer to the basic formula C6H containing D-glucopyranoside units 12Refers to cyclodextrins having an O6 and glucose structure and no additional chemical substituents (e.g., α-cyclodextrin consisting of six D-glucopyranoside units, β-cyclodextrin consisting of seven D-glucopyranoside units, and γ-cyclodextrin consisting of eight D-glucopyranoside units). The physical and chemical properties of the parent cyclodextrin can be modified by derivatizing the hydroxyl groups with other functional groups. Any substance located within the inner phase of the cyclodextrin is said to be "complexed" with the cyclodextrin or to form a complex (inclusion complex) with the cyclodextrin.

[0145] As used herein, there are no special restrictions on the cyclodextrin component of the αPPMX / cyclodextrin complex as long as the cyclodextrin can form a complex with αPPMX. In certain embodiments, the cyclodextrin is derivatized to have ionizable (e.g., weakly basic and / or weakly acidic) functional groups to facilitate complex formation with αPPMX and / or liposome encapsulates.

[0146] Modification of the hydroxyl groups of cyclodextrins, such as hydroxyl groups directed from the inner phase of the cyclodextrin towards the opposite side using ionizable chemical groups, is known to facilitate the addition of cyclodextrins and therapeutics complexed with cyclodextrins. In some embodiments, the cyclodextrin of the αPPMX / cyclodextrin complex has hydroxyl groups substituted with at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 ionizable chemical groups. The term "charged cyclodextrin" means a cyclodextrin having a hydroxyl group substituted with one or more of its charged moieties. Such moieties can include charged groups themselves or organic moieties (e.g., C1-C6 alkyl or C1-C6 alkyl ether moieties) substituted with one or more charged moieties.

[0147] In some embodiments, the "ionizable" or "charged" portion of the CD derivative is weakly ionizable. The weakly ionizable portion is a weakly basic or weakly acidic portion. The weakly basic functional group (W) has a pKa of CH3-W in the range of about 6.0 - 9.0, 6.5 - 8.5, 7.0 - 8.0, 7.5 - 8.0, and any range therebetween (including the endpoints). Similarly, the weakly acidic functional group (X) has a logarithm of the dissociation constant (pKa) of CH3-X in the range of about 3.0 - 7.0, 4.0 - 6.5, 4.5 - 6.5, 5.0 - 6.0, 5.0 - 5.5, and any range therebetween (including the endpoints). Representative anionic moieties include, but are not limited to, carboxylate, carboxymethyl, succinyl, sulfonyl, phosphate, sulfoalkyl ether, sulfate carbonate, thiocarbonate, dithiocarbonate, phosphate, phosphonate, sulfonate, nitrate, and borate groups. Representative cationic moieties include, but are not limited to, amino, guanidine, and quaternary ammonium groups.

[0148] In another embodiment, the derivatized cyclodextrin is a "polyanion" or a "polycat ion". A polyanion is a derivatized cyclodextrin having two or more negatively charged groups, resulting in a net negative ionic charge of three or more units. A polycation is a derivatized cyclodextrin having two or more positively charged groups, resulting in a net positive ionic charge of three or more units.

[0149] In another embodiment, the derivatized cyclodextrin is a "chargeable amphiphile". "Chargeable" means that the amphiphile has a pK in the range of pH 4 to pH 8 or 8.5. The chargeable amphiphile can thus be a weak acid or base. "Amphoteric" as used herein means a derivatized cyclodextrin having ionizable groups with both anionic and cationic characteristics, (a) at least one, optionally both, of the cationic and anionic amphiphiles being chargeable and having at least one charge group with a pK between 4 and 8 - 8.5, (b) the cationic charge being dominant at pH 4, and (c) the anionic charge being dominant at pH 8 - 8.5.

[0150] In some embodiments, the “ionizable” or “charged” derivatized cyclodextrins are generally polyionic, amphiphilic, or otherwise, weakly ionizable (i.e., having a pKai of about 4.0 - 8.5, 4.5 - 8.0, 5.0 - 7.5, 5.5 - 7.0, 6.0 - 6.5, and any range therebetween (including both ends)).

[0151] Any one, some, or all of the hydroxyl groups of any one, some, or all of the α-D-glucopyranoside units of any cyclodextrin can be modified to an ionizable chemical group as described herein. Since each cyclodextrin hydroxyl group has different chemical reactivity, the reaction with the modifying moiety can produce a non-crystalline mixture of positional and optical isomers. Alternatively, depending on the particular chemistry, the reaction can be made to form a homogeneous product of pre-modified α-D-glucopyranoside units.

[0152] Aggregate substitution occurring in cyclodextrin derivatives in a mixture is described by a term called degree of substitution. For example, 6-ethylenediamino-β-cyclodextrin having a degree of substitution of 7 would be composed of a distribution of isomers of 6-ethylenediamino-β-cyclodextrin in which the average number of ethylenediamino groups per 6-ethylenediamino-β-cyclodextrin molecule is 7. The degree of substitution of a cyclodextrin derivative mixture can be routinely measured using mass spectrometry or nuclear magnetic resonance spectroscopy.

[0153] In one embodiment, at least one hydroxyl moiety oriented in the opposite direction from within the cyclodextrin is substituted with an ionizable chemical group. For example, at least one of the α-D-glucopyranoside units of C2, C3, C6, C2 and C3, C2 and C6, C3 and C6, and all three of C2-C3-C6 hydroxyls is substituted with an ionizable chemical group. Any such combination of hydroxyls can be combined with any of the degrees of substitution described herein, and in addition, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, up to all of the alpha-D-glucopyranoside units in the modified cyclodextrin can be similarly combined. One such derivative is sulfalkyl ether cyclodextrin (SAE-CD). The sulfobutyl ether derivative of beta-cyclodextrin (SBE-β-CD) has been shown to have significantly improved water solubility compared to the parent cyclodextrin.

[0154] Additional cyclodextrin derivatives that can complex with a therapeutic agent in the disclosed liposomal compositions include sugammadex or Org-25969, in which case the 6-hydroxy group on γ-CD is replaced by a carboxythioacetate ether bond and hydroxybutenyl-β-CD. Alternative forms of cyclodextrin include 2,6-di-O-methyl-β-CD (DIMEB), 2-hydroxylpropyl-3-cyclodextrin (HP-β-CD), randomly methylated-β-cyclodextrin (RAMEB), sulfobutyl ether β-cyclodextrin (SBE-β-CD), and sulfobutyl ether-γ-cyclodextrin (SBEγCD), sulfobutylated-beta-cyclodextrin sodium salt, (2-hydroxypropyl)-alpha-cyclodextrin, (2-hydroxypropyl)-beta-cyclodextrin, (2-hydroxypropyl)-γ-cyclodextrin, 2,6-di-O-methyl)-beta-cyclodextrin (DIMEB-50 heptakis), 2,3,6-tri-O-methyl)-beta-cyclodextrin (TRIMEB heptakis), methyl-beta-cyclodextrin, octakis(6-deoxy-6-iodo)-γ-cyclodextrin, and octakis(6-deoxy-6-bromo)-gamma-cyclodextrin.

[0155] In some embodiments, the cyclodextrin has high solubility in water to facilitate encapsulation of a greater amount of the cyclodextrin in the inner phase of the liposome. In some embodiments, the aqueous solubility of the cyclodextrin is at least 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, or more. In some embodiments, the water solubility of the cyclodextrin is in the range of 10-150 mg / mL, 20-100 mg / mL, 20-75 mg / mL, and within any range therebetween (including both ends).

[0156] In some embodiments, a large binding constant between cyclodextrin and αPPMX and / or other therapeutic agents complexed with cyclodextrin is preferred and can be obtained by selecting the number of glucose units in the cyclodextrin based on the size of the therapeutic agent (see, e.g., Albers et al., Crit. Rev. Therap. Drug Carrier Syst. 12:311-337 (1995); Stella et al., Toxicol. Pathol. 36:30-42 (2008)). When the binding constant is pH-dependent, the cyclodextrin can be selected such that the binding constant is large at the pH of the inner phase of the liposome. As a result, the solubility (apparent solubility) of the therapeutic agent in the presence of cyclodextrin can be further improved. In some embodiments, the binding constant between cyclodextrin and the therapeutic agent is 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more. In some embodiments, the binding constant between cyclodextrin and the therapeutic agent is in the range of 100-1,200, 200-1,000, 300-750, and any range therebetween.

[0157] In some embodiments, the cyclodextrin of the αPPMX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is non-derivatized.

[0158] In some embodiments, the cyclodextrin of the αPPMX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is non-derivatized. In further embodiments, the cyclodextrin derivative of the complex has the structure of Formula I:

Chemical Formula

[0159] In some embodiments, the cyclodextrin derivative of the αPPMX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is of formula II:

Chemical formula

[0160] In some embodiments, the cyclodextrin derivatives of the αPPMX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex are cyclodextrins disclosed in U.S. Patent Nos. 6,133,248, 5,874,418, 6,046,177, 5,376,645, 5,134,127, 7,034,013, 6,869,939; and International Publication No. 02005 / 117911. The content of each of these patent documents is incorporated herein by reference in priority.

[0161] In some embodiments, the cyclodextrin derivatives of the αPPMX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex are sulfoalkyl ether cyclodextrins. In some embodiments, the cyclodextrin derivative of the complex is a sulfobutyl ether-3-cyclodextrin such as CAPTISOL® (CyDex Pharma.Inc., Lenexa, Kansas). Methods for preparing sulfobutyl ether-3-cyclodextrin and other sulfoalkyl ether cyclodextrins are known in the art.

[0162] In some embodiments, the cyclodextrin derivative of the αPPMX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is of Formula III: [Chemical Formula] a compound of, wherein R is (a)(H) 21-X or (-(CH2)4-SO3Na) X and x = 1.0 to 10.0, 1.0 to 5.0, 6.0 to 7.0, or 8.0 to 10.0; (b)(H) 21-X or (-(CH2CH(OH)CH3) X and x = 1.0 to 10.0, 1.0 to 5.0, 6.0 to 7.0, or 8.0 to 10.0; (c)(H) 21-X or (sulfonalkyl ether) X and x = 1.0 to 10.0, 1.0 to 5.0, 6.0 to 7.0, or 8.0 to 10.0; or (d)(H) 21-X or (-(CH2)4-SO3Na) X and x = 1.0 to 10.0, 1.0 to 5.0, 6.0 to 7.0, or 8.0 to 10.0.

[0163] In a further embodiment, the αPPMX / cyclodextrin complex and / or the cyclodextrin / therapeutic agent complex is encapsulated in liposomes (e.g., as described herein or by another method known in the art).

[0164] III. αPPMX Delivery Carrier In alternative embodiments, the present disclosure provides αPPMX delivery systems and their use for delivering an αPPMX payload to cells (single or plural) in vitro or in vivo. In some embodiments, the αPPMX is complexed with or incorporated into a delivery carrier. Such delivery carriers are known in the art and include, but are not limited to, liposomes, lipospheres, polymers, peptides, proteins, antibodies (e.g., ADCs such as antibody-αPPMX conjugates), cell components, cyclic oligosaccharides (e.g., cyclodextrins), nanoparticles (e.g., lipid nanoparticles, biodegradable nanoparticles, and core-shell nanoparticles), lipoprotein particles, and combinations thereof. In certain embodiments, the delivery carrier is a liposome. In other certain embodiments, the delivery carrier is an antibody or an antigen-binding antibody fragment.

[0165] A. Liposomes In some embodiments, the present disclosure provides a liposomal composition comprising liposomes encapsulating (filling) alpha-polyglutaminated pemetrexed (e.g., αPPMX disclosed herein). In some embodiments, the liposomes in the liposomal composition comprise αPPMX comprising 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups (including the glutamyl groups of pemetrexed). In some embodiments, the alpha-polyglutaminated pemetrexed in Lp-αPPMX comprises two or more L-type glutamyl groups. In other embodiments, the alpha-polyglutaminated pemetrexed in Lp-αPPMX comprises a D-type glutamyl group. In further embodiments, the alpha-polyglutaminated pemetrexed in Lp-αPPMX comprises a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, the alpha-polyglutaminated pemetrexed in Lp-αPPMX comprises two or more glutamyl groups having gamma-carboxyl bonds. In some embodiments, the alpha-polyglutaminated pemetrexed in Lp-αPPMX comprises at least one glutamyl group having both alpha-carboxyl and gamma-carboxyl bonds. In some embodiments, the liposomal composition comprises liposomes comprising α-pentaglutaminated PMX. In further embodiments, the liposomes comprise L-α-pentaglutaminated PMX, D-α-pentaglutaminated PMX, or L- and D-α-pentaglutaminated PMX. In some embodiments, the liposomal composition comprises liposomes comprising hexaglutaminated PMX (Lp-αPPMX). In further embodiments, the liposomes comprise L-α-hexaglutaminated PMX, D-α-hexaglutaminated PMX, or L- and D-α-hexaglutaminated PMX. In some embodiments, the liposomal composition comprises liposomes that are anionic or neutral. In some embodiments, the liposomal composition comprises liposomes that are cationic. In some embodiments, the Lp-αPPMX composition is not pegylated. In some embodiments, the Lp-αPPMX composition is not targeted (NTLp-αPPMX).In other embodiments, the Lp-αPPMX composition is targeted (TLp-αPPMX). In some embodiments, the liposomal composition comprises liposomes having a diameter in the range of 20 nm to 500 nm, or any range therebetween. In some embodiments, the liposomal composition comprises liposomes having a diameter in the range of 20 nm to 400 nm, or any range therebetween. In some embodiments, the liposomal composition comprises liposomes having a diameter in the range of 20 nm to 300 nm, or any range therebetween. In some embodiments, the liposomal composition comprises liposomes having a diameter in the range of 20 nm to 200 nm, or any range therebetween. In further embodiments, the liposomal composition comprises liposomes having a diameter in the range of 20 nm to 150 nm, or any range therebetween. In further embodiments, the liposomal composition comprises liposomes having a diameter in the range of 80 nm to 120 nm, or any range therebetween. In further embodiments, 30 - 70%, 30 - 60%, or 30 - 50% w / w, or any range therebetween of alpha-polyglutamine oxidized pemetrexed is encapsulated (enclosed) in the Lp-αPPMX. In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% of the alpha-polyglutamine oxidized pemetrexed is encapsulated in the Lp-αPPMX during the liposome preparation process.

[0166] In some embodiments, the liposomes provided further comprise an immunostimulant, a detectable marker, or both, disposed on the outer surface of the liposome. The immunostimulant or detectable marker can be ionically or covalently bound to the outer surface of the liposome, optionally including, for example, binding to a steric stabilizing component of the liposome.

[0167] The term "immunostimulatory agent" is also known as "immunostimulant" and "immunostimulator", and refers to a substance that stimulates immunity (including existing immune responses) by inducing the activation or increased activity of any component of the immune system. These immunostimulatory agents include one or more of haptens, adjuvants, protein immunostimulatory agents, nucleic acid immunostimulatory agents, and chemical immunostimulatory agents. Many adjuvants contain substances designed to stimulate immune responses, such as lipid A, proteins derived from Bordetella pertussis or Mycobacterium tuberculosis. Specific adjuvants include, for example, Freund's incomplete adjuvant and complete adjuvant (Difco Laboratories, Detroit, Mich.); Merck adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron, or zinc; insoluble suspensions of acylated tyrosine; acylated saccharides; polysaccharides derivatized cationically or anionicly; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A; IFN gamma, IFN alpha, FLT3 ligand; and immunostimulatory antibodies (e.g., anti-CTLA-4, anti-CD28, anti-CD3 are commercially available). Cytokines such as GM-CSF, interleukin 2, 7, 12, and 15, and other similar growth factors can also be used as adjuvants. In a preferred embodiment, the immunostimulant can be at least one selected from the group consisting of fluorescein, DNP, beta glucan, beta-1,3-glucan, beta-1,6-glucan. In a further preferred embodiment, the immunostimulant is a toll-like receptor (TLR) regulator. In a further embodiment, the toll-like receptor (TLR) regulator is one or more of oxidized low density lipoprotein (e.g., OXPAC, PGPC), eritoran lipid (e.g., E5564), and resolvin.In some embodiments, the liposome contains fluorescein isothiocyanate (FITC), which, based on our experiments, surprisingly functions as both an immunostimulant and a detectable marker.

[0168] In some embodiments, the liposome contains a detectable marker. Detectable markers can include, for example, any suitable means known in the art, such as, at least, radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelating agents, enzymes, dyes, inks, magnetic compounds, biocatalysts, or pigments that are detectable by magnetic resonance imaging (MRI), optical imaging, fluorescence / luminescence imaging, and / or nuclear imaging techniques.

[0169] In some embodiments, the immunostimulant and / or detectable marker is bound to the outer surface by co-incubation with the liposome. For example, the immunostimulant and / or detectable marker can be bound to the liposome membrane by hydrophobic interactions or ionic bonds such as avidin / biotin binding or metal chelate binding (e.g., Ni-NTA). Alternatively, the immunostimulant or detectable marker can be covalently bound to the outer surface of the liposome, for example, by covalently binding to a liposome component or to a steric stabilizer that is PEG.

[0170] In some embodiments, the liposome further contains an agent that increases the uptake of the liposome into the intracellular compartment of the target cell containing the cytosol.

[0171] In some embodiments, the liposomes comprise a mitochondrial targeting agent. In some embodiments, the liposomes comprise triphenylphosphonium (TPP). Methods and mechanisms for surface functionalization of liposomes using TPP are known in the art (e.g., binding to a lipid anchor via a peg spacer group and modifying TPP with a stearyl group (stearyltriphenylphosphonium (STPP))). In some embodiments, the liposomes comprise high density octaarginine. In some embodiments, the liposomes comprise sphingomyelin and / or sphingomyelin metabolites. Sphingomyelin metabolites used to formulate the liposomes of the present invention can include, for example, ceramide, sphingosine or sphingosine 1-phosphate. In some embodiments, the liposomes comprise rhodamine 123. In some embodiments, the liposomes comprise a mitochondrial permeable peptide. In some embodiments, the liposomes comprise a mitofusin peptide, a mitochondrial targeting signal peptide, and an antennapedia helix III homeodomain cell membrane permeable peptide (ANT) (e.g., RQIKIWFQNRRMKWKKRKKRRQRRR (SEQ ID NO: 1), RKKRRXRRRGC (wherein X is any natural or non-natural amino acid) (SEQ ID NO: 2), CCGCCAAGAAGCG (SEQ ID NO: 3), GCGTGCACACGCGCGTAGACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAAGAAGCGACCCCTCCGGGGCGAGCTGAGCGGCGTGGCGCGGGGGCGTCAT (SEQ ID NO: 4), ACGTGCATACGCACGTAGACATTCCCCGCTTCCCACTCCAAAGTCCGCCAAGAAGCGTATCCCGCTGAGCGGCGTGGCGCGGGGGCGTCATCCGTCAGCTC (SEQ ID NO: 5), or ACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAAGAAGCGACCCCTCCGGGGCGAGCTG (SEQ ID NO: 6)), or a mitochondrial permeabilizing agent selected from the group consisting of mitochondrial permeable fragments thereof).

[0172] In some embodiments, the liposomes in the provided liposome composition include a mitochondrial permeabilizing agent selected from guanidine-rich peptides, tetraguanidium, triguanidium, diguanidium, monoguanidium, guanidine-rich polycarbamates, beta-oligoarginine, proline-rich dendrimers, and phosphonium salts (e.g., methyltriphenylphosphonium and / or tetraphenylphosphonium).

[0173] In some embodiments, the liposomes in the provided liposome composition include sphingomyelin and / or stearyl octaarginine. In some embodiments, the liposomes include sphingomyelin and / or stearyl octaarginine. In some embodiments, the liposomes include DOPE, sphingomyelin, stearyl octaarginine sphingomyelin, and stearyl octaarginine. In some embodiments, the liposomes include DOPE, sphingomyelin, stearyl octaarginine sphingomyelin, and stearyl octaarginine in a molar ratio of 9:2:1. In some embodiments, the liposomes include the MITO-Porter® system or a variant thereof.

[0174] In some embodiments, the liposomes in the provided liposome composition include agents such as membrane permeabilizing agents that facilitate the delivery of the liposomes across the cell membrane and confer the ability to bypass the harsh environment of the endocytic pathway and lysosomes to the liposomes. Membrane permeabilizing agents are known in the art and can be routinely used and applied to the manufacture and use of the provided liposome composition. In some embodiments, the membrane permeating agent / lysosome bypass agent is chloroquine. In some embodiments, the membrane permeabilizing agent is a cell permeable peptide.In some embodiments, the liposomes in the provided liposome composition comprise a membrane permeabilizing agent selected from the following group: RKKRRQRRR (SEQ ID NO: 7), GRKKRRQRRRTPQ (SEQ ID NO: 8), YGRKKRRQRRR (SEQ ID NO: 9), AAVALLPAVLLALLA (SEQ ID NO: 10), MGLGLHLLVLAAALQ (SEQ ID NO: 11), GALFLGFLGAAGSTM (SEQ ID NO: 12), AGYLLGKINLKALAALAKKIL (SEQ ID NO: 13), RVIRVWFQNKRCKDKK (SEQ ID NO: 14), RQIKIWFQNRRMKWKK (SEQ ID NO: 15), GLFEAIAGFIENGWEGMIDG (SEQ ID NO: 16), GWTLNSAGYLLGKIN (SEQ ID NO: 17), RSQSRSRYYRQRQRS (SEQ ID NO: 18), LAIPEQEY (SEQ ID NO: 19), LGIAEQEY (SEQ ID NO: 20), LGIPAQEY (SEQ ID NO: 21), LGIPEAEY (SEQ ID NO: 22), LGIPEQAY (SEQ ID NO: 23), LGIAEAEY (SEQ ID NO: 24), LGIPEAAY (SEQ ID NO: 25), LGIAEQAY (SEQ ID NO: 26), LGIAEAAY (SEQ ID NO: 27), LLIILRRRIRKQAHAHSK (SEQ ID NO: 28), LKALAALAKKIL (SEQ ID NO: 29), KLALKLALKALKAALKLA (SEQ ID NO: 30), KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 31), DHQLNPAF (SEQ ID NO: 32), DPKGDPKG (SEQ ID NO: 33), VTVTVTVTVTGKGDPKPD (SEQ ID NO: 34), RQIKIWFQNRRMKWKK (SEQ ID NO: 35), GRKKRRQRRRPPQ (SEQ ID NO: 36), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 37), GRKKRRQRRR (SEQ ID NO: 38), RRRRRRR (SEQ ID NO: 39), RRRRRRRR (SEQ ID NO: 40), RRRRRRRRR (SEQ ID NO: 41), RRRRRRRRRR (SEQ ID NO: 42), RRRRRRRRRRR (SEQ ID NO: 43), and YTIWMPENPRPGTPCDIFTNSRGKRASNGGGG(R)n (wherein L- and / or D-type and n = 2 to 15 R) (SEQ ID NO: 44), or cell permeable fragments thereof.

[0175] As discussed above, liposomes can include steric stabilizers that can extend their lifetimes in circulation. For these embodiments incorporating a steric stabilizer, the steric stabilizer can be at least one member selected from the group consisting of polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinylpyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol; poly[N-(2-hydroxypropyl)methacrylamide]; amphiphilic poly-N-vinylpyrrolidone; L-amino acid-based polymers; oligoglycerol, polyethylene glycol and polypropylene oxide-containing copolymers, poloxamer 188, and polyvinyl alcohol. In some embodiments, the steric stabilizer or group of steric stabilizers is PEG. In one embodiment, the steric stabilizer is PEG. In a further embodiment, the PEG has a number average molecular weight (Mn) of 200 to 5000 daltons. These PEGs can be of any structure such as linear, branched, star or comb-shaped structures and are commercially available.

[0176] In some embodiments, the liposome composition comprises pegylated liposomes (PLp-αPPMX). In some embodiments, the pegylated liposomes in the liposome composition comprise αPPMX containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, the alpha polyglutamine oxidized pemetrexed in Lp-αPPMX comprises two or more L-type glutamyl groups. In other embodiments, the alpha polyglutamine oxidized pemetrexed in Lp-αPPMX comprises a D-type glutamyl group. In further embodiments, the alpha polyglutamine oxidized pemetrexed in Lp-αPPMX comprises a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, the alpha polyglutamine oxidized pemetrexed in Lp-αPPMX comprises two or more glutamyl groups having a gamma bond. In some embodiments, at least one glutamyl group has both an alpha bond and a gamma bond. In some embodiments, the liposome composition comprises pegylated liposomes containing α pentaglutamine oxidized PMX. In further embodiments, the liposome comprises L-α pentaglutamine oxidized PMX, D-α pentaglutamine oxidized PMX, or L- and D-α pentaglutamine oxidized PMX. In some embodiments, the liposome composition comprises pegylated liposomes containing α hexaglutamine oxidized PMX. In further embodiments, the liposome comprises L-α hexaglutamine oxidized PMX, D-α hexaglutamine oxidized PMX, or L- and D-α hexaglutamine oxidized PMX. In some embodiments, the liposome composition comprises pegylated liposomes that are anionic or neutral. In some embodiments, the liposome composition comprises pegylated liposomes that are cationic. In some embodiments, the PLp-αPPMX composition is not targeted (NTPLp-αPPMX). In other embodiments, the PLp-αPPMX composition is targeted (TPLp-αPPMX). In further embodiments, the liposome composition comprises pegylated liposomes containing 30 to 70%, 30 to 60%, or 30 to 50%, or any range therebetween of liposome-encapsulated alpha polyglutamine oxidized pemetrexed.In some embodiments, at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more than 75% (w / w) of alpha-polyglutamylated pemetrexed is encapsulated (enclosed) in PLp-αPPMX. In some embodiments, the liposome composition comprises pegylated liposomes having a diameter in the range of 20 nm to 500 nm. In some embodiments, the liposome composition comprises pegylated liposomes having a diameter in the range of 20 nm to 200 nm. In further embodiments, the liposome composition comprises pegylated liposomes having a diameter in the range of 80 nm to 120 nm.

[0177] In some embodiments, more than 30%, 40%, 50%, 60%, 70%, 80% or 90% of the polyglutamylated pemetrexed in the composition has 4 to 10, 4 to 6, or more than 5 glutamyl groups. In some embodiments, more than 30%, 40%, 50%, 60%, 70%, 80% or 90% of the polyglutamylated pemetrexed in the provided liposome composition is tetraglutamylated. In some embodiments, more than 30%, 40%, 50%, 60%, 70%, 80% or 90% of the polyglutamylated pemetrexed in the provided liposome composition is pentaglutamylated. In some embodiments, more than 30%, 40%, 50%, 60%, 70%, 80% or 90% of the polyglutamylated pemetrexed in the provided liposome composition is hexaglutamylated.

[0178] In some embodiments, the alpha-polyglutamylated pemetrexed composition (e.g., delivery carriers such as polyglutamate and liposomes containing polyglutamate) is in an aqueous solution. In some embodiments, the αPPMX composition is a liposome composition, 1 square meter (m 2) is administered at a dosage of about 0.005 to about 5000 mg of αPPMX per square meter of body surface area, or any range therebetween. In a further embodiment, the αPPMX composition is administered as a liposomal composition at a dosage of about 0.1 to about 1000 mg of αPPMX per square meter of body surface area, or any range therebetween.

[0179] (1) Liposomal composition The lipids and other components of the liposomes contained in the liposomal composition can be any lipid, combination and ratio of lipids, or combination of lipids and other liposomal components and their respective ratios known in the art. However, without limitation, it will be understood by those skilled in the art that the liposomal encapsulation of any particular drug, such as the alpha-polyglutamine oxidized PMX discussed herein, may involve substantially routine experimentation to obtain a useful and functional liposomal formulation. Generally, the liposomes provided can have any liposomal structure, for example, a structure having an inner space isolated from the outer medium by one or more lipid bilayers, or any microcapsule structure having a semipermeable membrane with a lipophilic central portion where the membrane isolates the interior. The lipid bilayer can be any amphiphilic molecule having a hydrophilic moiety (hydrophilic portion) and a hydrophobic moiety (hydrophobic portion). Usually, the amphiphilic molecules in the bilayer are arranged in a two-dimensional sheet where the hydrophobic portions face the inside of the sheet while the hydrophilic portions face the outside. The amphiphilic molecules forming the liposomes provided can be any known or hereafter discovered amphiphilic molecules (e.g., synthetic or natural origin lipids or biocompatible lipids). Liposomes can be formed by amphiphilic polymers and surfactants, such as polymersomes and niosomes. In the present disclosure, without limitation, these liposome-forming materials are also referred to as "lipids".

[0180] The liposome composition formulations provided herein can be in liquid or dry forms such as dry powder or dry cake. The dry powder or dry cake can be subjected to primary drying, for example, under lyophilization conditions, or can be subjected to only primary drying or both primary and secondary drying. In the dry form, the powder or cake can have, for example, 1% - 6% moisture, such as 2% - 5% moisture or 2% - 4% moisture. An example of the drying method is lyophilization (also called freeze-drying or cryodessication). Any of the compositions and methods of the present disclosure can include liposomes, lyophilized liposomes, or liposomes reconstituted from lyophilized liposomes. In some embodiments, the disclosed compositions and methods include one or more lyoprotectants or cryoprotective substances. These protectants are typically polyhydroxy compounds such as saccharides (monosaccharides, disaccharides, and polysaccharides), polyhydric alcohols, and their derivatives, glycerol or polyethylene glycol, trehalose, maltose, sucrose, glucose, lactose, dextran, glycerol, or aminoglycosides. In further embodiments, the lyoprotectant or cryoprotective substance comprises up to 10% or up to 20% of the solution outside, inside, or both outside and inside the liposome.

[0181] In some embodiments, the liposomes contain steric stabilizers that extend their lifespan in circulation. One or more steric stabilizers, such as hydrophilic polymers (polyethylene glycol (PEG)), glycolipids (monosialoganglioside (GM1)), or others, occupy the space immediately adjacent to the liposome surface and exclude other polymers from this space. As a result, access and binding of plasma opsonins to the liposome surface are hindered, thus suppressing such interactions of macrophages with these liposomes or any other removal mechanism, and extending the lifespan of the liposomes in circulation. In some embodiments, the steric stabilizer or group of steric stabilizers is PEG or a combination containing PEG. In further embodiments, the steric stabilizer is PEG or a combination containing PEG with a number average molecular weight (Mn) in the range of 200 to 5000 daltons. These PEGs can have any structure, such as linear, branched, star-shaped, or comb-shaped, and are commercially available.

[0182] The diameter of the disclosed liposomes is not particularly limited. In some embodiments, the liposomes have a diameter in the range of, for example, 30 nm to 150 nm (nanometers). In other embodiments, the liposomes have a diameter in the range of 40 nm to 70 nm.

[0183] The properties of the liposomes are affected by the nature of the lipids used to produce the liposomes. A wide variety of lipids have been used to produce liposomes. These include cationic, anionic, and neutral lipids. In some embodiments, the liposomes containing alphapolyglutamine oxidized pemetrexed are anionic or neutral. In other embodiments, the provided liposomes are cationic. The determination of the charge (e.g., anionic, neutral, or cationic) can be determined by routine work by measuring the zeta potential of the liposomes. The zeta potential of the liposomes can be positive, zero, or negative. In some embodiments, the zeta potential of the liposomes is below zero. In some embodiments, the zeta potential of the liposomes is in the range of 0 to -150 mV. In another embodiment, the zeta potential of the liposomes is in the range of -30 to -50 mV.

[0184] In some embodiments, cationic lipids are used to create cationic liposomes, which are commonly used as gene delivery agents. The positive charges on the cationic liposomes enable interaction with the negative charges on the cell surface. After the cationic liposomes bind to the cells, the liposomes are transported into the interior of the cells by endocytosis.

[0185] In some preferred embodiments, neutral to anionic liposomes are used. In preferred embodiments, anionic liposomes are used. For example, the use of a mixture of neutral lipids such as HSPC and anionic lipids such as PEG-DSPE forms anionic liposomes, which have a low likelihood of non-specific binding to normal cells. Specific binding to tumor cells can be achieved using tumor targeting antibodies such as folate receptor antibodies, including, for example, folate receptor alpha antibody, folate receptor beta antibody, and / or folate receptor delta antibody.

[0186] As an example, at least one (or several) lipid is an amphiphilic lipid defined as having hydrophilic and hydrophobic moieties (usually a hydrophilic head and a hydrophobic tail). The hydrophobic moiety typically faces the hydrophobic phase (e.g., within the bilayer), while the hydrophilic moiety typically faces the aqueous phase (e.g., outside the bilayer). The hydrophilic moiety can include polar or charged groups such as carbohydrates, phosphates, carboxylic acids, sulfates, aminos, sulfhydryls, nitros, hydroxyls, and other similar groups. The hydrophobic moiety can include nonpolar groups including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. Examples of amphiphilic compounds include, but are not limited to, phospholipids, amino lipids, and sphingolipids.

[0187] Typically, for example, the lipid is a phospholipid. Phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and the like. It should be understood that other lipid membrane components such as cholesterol, sphingomyelin, and cardiolipin can also be used.

[0188] The lipids including liposomes provided herein can be anionic and neutral (including zwitterionic and polar) lipids including anionic and neutral phospholipids. Neutral lipids exist in an uncharged or neutral zwitterionic form at the selected pH. At physiological pH, such lipids include, for example, dioleoylphosphatidylglycerol (DOPG), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebroside, and diacylglycerol. Examples of zwitterionic lipids include, but are not limited to, dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), and dioleoylphosphatidylserine (DOPS). Anionic lipids are negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and anionic modifying groups attached to neutral lipids.

[0189] In this specification, anionic and neutral lipids are collectively referred to as non-cationic lipids. Such lipids may contain phosphorus, but they are not so limited. Examples of non-cationic lipids include lecithin, lysophosphatidylcholine, phosphatidylethanolamine, lysophosphatidylethanolamine, dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleyoylphosphatidylglycerol (POPG), 16-0-monomethyl PE, 16-0-dimethyl PE, 18-1-trans PE, palmitoyloleoylphosphatidylethanolamine (POPE), 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, and cholesterol.

[0190] Liposomes can be constructed using any liposome assembly method and liposomal components (also referred to as liposome components) known in the art. Liposomal components include, for example, lipids such as DSPE, HSPC, cholesterol, and derivatives of these components. Other suitable lipids are commercially available, for example, from Avanti Polar Lipids, Inc. (Alabaster, Alabama, USA). A partial list of available negatively or neutrally charged lipids suitable for the preparation of anionic liposomes can be, for example, at least one of the following: DLPC, DMPC, DPPC, DSPC, DOPC, DMPE, DPPE, DOPE, DMPA·Na, DPPA·Na, DOPA·Na, DMPG·Na, DPPG·Na, DOPG·Na, DMPS·Na, DPPS·Na, DOPS·Na, DOPE-glutaryl·(Na)2, tetramyristoyl cardiolipin·(Na)2, DSPE-mPEG-2000·Na, DSPE-mPEG-5000·Na, and DSPE-maleimide PEG-2000·Na.

[0191] In some embodiments, the αPPMX compositions provided herein are formulated in liposomes containing a cationic lipid. In one embodiment, but not limited to, the cationic lipid is selected from the cationic lipids described in International Publication Nos. WO 2012 / 040184, WO 2011 / 153120, WO 2011 / 149733, WO 2011 / 090965, WO 2011 / 043913, WO 2011 / 022460, WO 2012 / 061259, WO 2012 / 054365, WO 2012 / 044638, WO 2010 / 080724, WO 2010 / 21865, and WO 2008 / 103276, U.S. Pat. Nos. 7,893,302, 7,404,969, 8,283,333, U.S. Patent Application Publication Nos. 20100036115, and 20120202871. Each of these patent documents is hereby incorporated by reference in its entirety. In another embodiment, the cationic lipid can be selected from Formula A described in International Publication Nos. WO 2012 / 040184, WO 2011 / 153120, WO 2011 / 1149733, WO 2011 / 090965, WO 2011 / 043913, WO 2011 / 022460, WO 2012 / 061259, WO 2012 / 054365, and WO 2012 / 044638. Each of these patent documents is hereby incorporated by reference in its entirety. In yet another embodiment, the cationic lipid can be selected from Formulas CLI - CLXXIX of International Publication No. WO 2008103276, Formulas CLI - CLXXIX of U.S. Pat. No. 7,893,302, Formulas CLI - CLXXXXII of U.S. Pat. No. 7,404,969, and Formulas I - VI of U.S. Patent Application Publication No. 20100036115. Each of these respective patent documents is hereby incorporated by reference in its entirety. By way of non-limiting example, the cationic lipid can be selected from the following: (20Z,23Z)-N,N-dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z)-N,N-dimethylhexacosa-17,20-dien-9-amine, (1Z,19Z)-N,N-dimethylpentacosa-16,19-dien-8-amine, (13Z,16Z)-N,N-dimethyldocosa-13,16-dien-5-amine, (12Z,(15Z)-N,N-Dimethylheneicos-12,15-dien-4-amine, (14Z,17Z)-N,N-dimethyltricos-14,17-dien-6-amine, (15Z,18Z)-N,N-dimethyltetracos-15,18-dien-7-amine, (18Z,21Z)-N,N-dimethylheptacos-18,21-dien-10-amine, (15Z,18Z)-N,N-dimethyltetracos-15,18-dien-5-amine, (14Z,17Z)-N,N-dimethyltricos-14,17-dien-4-amine, (19Z,22Z)-N,N-dimethylocatacos-19,22-dien-9-amine, (18Z,21Z)-N,N-dimethylheptacos-18,21-dien-8-amine, (17Z,20Z)-N,N-dimethylhexacos-17,20-dien-7-amine, (16Z,19Z)-N,N-dimethylpentacos-16,19-dien-6-amine, (22Z,25Z)-N,N-dimethylhentriaconta-22,25-dien-10-amine, (21Z,24Z)-N,N-dimethyltriaconta-21,24-dien-9-amine, (18Z)-N,N-dimethylheptacos-18-en-10-amine, (17Z)-N,N-dimethylhexacos-17-en-9-amine, (19Z,22Z)-N,N-dimethyloctacos-19,22-dien-7-amine, N,N-dimethylheptacosan-10-amine, (20Z,23Z)-N-ethyl-N-methylnonacos-20,23-dien-10-amine, 1-[(11Z,14Z)-1-nonylicos-11,14-dien-1-yl]pyrrolidine, (20Z)-N,N-dimethylheptacos-20-en-10-amine, (15Z)-N,N-dimethyleptacos-15-en-10-amine, (14Z)-N,N-dimethylnonacos-14-en-10-amine, (17Z)-N,N-dimethylnonacos-17-en-10-amine, (24Z)-N,N-dimethyltritriaconta-24-en-10-amine, (20Z)-N,N-dimethylnonacos-20-en-10-amine, (22Z)-N,N-dimethylhentriaconta-22-en-10-amine, (16Z)-N,N-dimethylpentacos-16-en-8-amine, (12Z,15Z)-N,N-dimethyl-2-nonylheneicos-12,15-dien-1-amine, (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecane-8-amine, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecane-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecane-10-amine, N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]heneicosane-10-amine, N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecane-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecane-8-amine, N,N-dimethyl-[(1R,2S)-2-undecyl-cyclopropyl]tetradecane-5-amine, N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecane-1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecane-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethyl-pentadecane-6-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecane-8-amine, R--N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propane-2-amine, S--N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propane-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z-)-oct-5-en-1-yloxy]propane-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine, (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,(12Z)-Octadeca-9,12-dien-1-yloxy]propan-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-[(9Z)-octadeca-9-en-1-yloxy]-3-(octyloxy)propan-2-amine; (2S)-N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-triene-1-yloxy]-3-(octyloxy)propan-2-amine, (2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine, 1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-1-3-(octyloxy)propan-2-amine, 1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethyl-propan-2-amine, (2S)-1-[(13Z)-docosa-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, 1-[(13Z)-docosa-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(9Z)-hexadeca-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2R)-N,N-dimethyl-H(1-methyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-R9Z,12Z)-octadeca-9,12-Dien-1-yloxypropan-2-amine, N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine, N,N-dimethyl-1-{[-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine, and (11E,20Z,23Z)-N,N-dimethylnonacosa-11,20,23-triene-10-amine, or a pharmaceutically acceptable salt or acid or stereoisomer thereof.,

[0192] In one embodiment, the lipid can be a cleavable lipid such as those described in International Publication No. WO 2012 / 170889. This patent is incorporated herein by reference in its entirety.,

[0193] The cationic lipid can be synthesized routinely using methods known in the art and / or as described in International Publication Nos. WO 2012 / 040184, WO 2011 / 153120, WO 2011 / 149733, WO 2011 / 090965, WO 2011 / 1043913, WO 2011 / 022460, WO 2012 / 061259, WO 2012 / 054365, WO 2012 / 044638, WO 2010 / 080724, and WO 2010 / 21865. These patent documents are incorporated herein by reference in their entirety.,

[0194] The lipid derivative can include, for example, at least one or more steric stabilizers and / or the attachment (preferably covalent) of a functional group to the liposome component, where after this attachment, the steric stabilizer and / or the functional group should be regarded as part of the liposome component. The functional group includes a group that can be used to attach the liposome component to another moiety such as a protein. Such functional groups include at least maleimide. These steric stabilizers include at least one selected from the group consisting of polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinylpyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol; poly[N-(2-hydroxypropyl)methacrylamide]; amphiphilic poly-N-vinylpyrrolidone; L-amino acid-based polymers; and polyvinyl alcohol.

[0195] In some embodiments, the αPPMX composition is formulated in a lipid-polycation complex. The formation of the lipid-polycation complex can be achieved using methods known in the art and / or as described in U.S. Patent Application Publication No. 20120178702, which is hereby incorporated by reference in its entirety. By way of non-limiting example, polycations include, but are not limited to, cationic peptides or polypeptides such as polylysine, polyornithine, and / or polyarginine, and the cationic peptides described in International Publication No. 2012 / 013326, which is hereby incorporated by reference in its entirety. In another embodiment, αPPMX is formulated in a lipid-polycation complex, which further includes neutral lipids such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).

[0196] The components of the liposome can include any molecule that binds thereto (i.e., chemical / drug / reagent / protein), and in some embodiments, the components of the provided liposome include at least members selected from the group consisting of DSPE, DSPE-PEG, DSPE-maleimide, HSPC; HSPC-PEG; HSPC-maleimide; cholesterol; cholesterol-PEG; and cholesterol-maleimide. In some embodiments, the components of the provided liposome include DSPE, DSPE-PEG, DSPE-maleimide, HSPC; HSPC-PEG; HSPC-maleimide; cholesterol; cholesterol-PEG; and cholesterol-maleimide. In a preferred embodiment, the liposome components constituting the liposome include DSPE; DSPE-FITC; DSPE-maleimide; cholesterol; and HSPC.

[0197] In a further embodiment, the liposomes of the liposome compositions provided herein include oxidized phospholipids. In some embodiments, the liposome includes an oxidized phospholipid that is a member selected from the group consisting of phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, and 1-palmitoyl-2-arachidonoyl-sn-glycero-2-phosphate. In some embodiments, the phospholipid has an unsaturated bond. In some embodiments, the phospholipid is arachidonic acid containing the phospholipid. In a further embodiment, the phospholipid is sn-2-oxygenated. In a further embodiment, the phospholipid is not fragmented.

[0198] In some embodiments, the liposomes of the disclosed liposome compositions comprise oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC). As used herein, the term "oxPAPC" means a lipid produced by the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), thereby resulting in a mixture of oxidized phospholipids containing fragmented or full-length oxygenated sn-2 residues. Characteristic oxidized fragmentation species include 5-carbon sn-2 residues having omega aldehyde or omega carboxyl groups. Oxidation of arachidonic acid residues also produces phospholipids containing esterified isoprostanes. oxPAPC includes, among many oxidized products present in oxPAPC, in particular, HOdiA-PC, KOdiA-PC, HOOA-PC and KOOA-PC species. In further embodiments, oxPAPC is an epoxyisoprostane-containing phospholipid. In further embodiments, oxPAPC is 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine (5,6-PEIPC), 1-palmitoyl-2-(epoxycyclopentenone)-sn-glycero-3-phosphocholine (PECPC) and / or 1-palmitoyl-2-(epoxy-isoprostane E2)-sn-glycero-4-phosphocholine (PEIPC). In some embodiments, the phospholipid has an unsaturated bond. In some embodiments, the phospholipid is arachidonic acid containing the phospholipid. In further embodiments, the phospholipid is sn-2-oxygenated. In further embodiments, the phospholipid is not fragmented.

[0199] In some embodiments, the liposomal alpha polyglutamine oxidized pemetrexed composition is pegylated (i.e., pegylated liposomal alpha polyglutamine oxidized (e.g., pentaglutamine oxidized or hexaglutamine oxidized) folate antagonist (PLp-αPPMX or PLp-αPPMX)). In some embodiments, PLp-αPPMX or PLp-αPPMX is water-soluble. That is, PLp-αPPMX or PLp-αPPMX is in the form of an aqueous solution.

[0200] In some embodiments, the liposomes of the disclosed liposome composition comprise lipids selected from the following: 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC); 1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphocholine; 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-hexadecyl-sn-glycero-3-phosphocholine; 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine; and 1-palmitoyl-2-acetyl-sn-glycero-3-phosphocholine. In further embodiments, the liposomes comprise PGPC.

[0201] In some embodiments, the pH of the solution containing the liposome composition is pH 2-8, or any range therebetween. In some embodiments, the pH of the solution containing the liposome composition is pH 5-8, or any range therebetween. In some embodiments, the pH of the solution containing the liposome composition is pH 6-7, or any range therebetween. In some embodiments, the pH of the solution containing the liposome composition is 6-7.5, 6.5-7.5, 6.7-7.5, or 6.3-7.0, or any range therebetween.

[0202] In some embodiments, at least one component of the liposomal lipid bilayer is functionalized (or reactive). As used herein, a functionalized component is a component that contains reactive groups that can be used to crosslink reagents and moieties to the lipid. When a lipid is functionalized, any liposomes it forms are also functionalized. In some embodiments, the reactive group is one that reacts with a crosslinking agent (or other moiety) to form a crosslink. The reactive groups in the liposomal lipid bilayer are located somewhere on the lipid such that upon contact with a crosslinking agent, crosslinking with another moiety (e.g., a steric stabilizer or targeting moiety) is enabled. In some embodiments, the reactive group is in the head group of the lipid, such as a phospholipid. In some embodiments, the reactive group is a maleimide group. Maleimide groups can crosslink with each other in the presence of dithiol crosslinking agents such as, but not limited to, dithiothreitol (DTT).

[0203] It is to be understood that the use of other functionalized lipids, other reactive groups, and other crosslinking agents beyond those described above is further contemplated. In addition to maleimide groups, other examples of reactive groups contemplated include, but are not limited to, other thiol-reactive groups, amino groups such as primary or secondary amines, carboxyl groups, hydroxyl groups, aldehyde groups, alkyne groups, azide groups, carbonyl groups, haloacetyl (e.g., iodoacetyl) groups, imidoester groups, N-hydroxysuccinimide esters, sulfhydryl groups, and pyridyl disulfide groups.

[0204] Functionalized and non-functionalized lipids are available from many commercial sources such as Avanti Polar Lipids (Alabaster, AL) and Lipoid LLC (Newark, NJ).

[0205] (2) Liposomal interior space In further non-limiting embodiments, the provided liposomes contain an internal space. In some embodiments, the internal space contains, without limitation, an aqueous solution. In some embodiments, the internal space contains the alpha-polygulamine oxidized pemetrexed provided herein. In further embodiments, the internal space of the liposome contains an isotonic agent. In some embodiments, the concentration (weight %) of the isotonic agent is 0.1 - 20%, 1 - 20%, 0.5 - 15%, 1 - 15% or 1 - 50%, or any range therebetween. In some embodiments, the internal space of the liposome contains a sugar (e.g., trehalose, maltose, sucrose, lactose, mannose, mannitol, glycerol, dextrose, fructose, etc.). In further embodiments, the concentration (weight %) of the sugar is 0.1 - 20%, 1 - 20%, 0.5 - 15%, 1 - 15% or 1 - 50%, or any range therebetween. In some embodiments, the pH of the internal space of the liposome is pH 2 - 8, or any range therebetween. In some embodiments, the pH of the solution containing the liposome composition is pH 5 - 8, or any range therebetween. In some embodiments, the pH of the solution containing the liposome composition is pH 6 - 7, or any range therebetween. In some embodiments, the pH of the solution containing the liposome composition is 6 - 7.5, 6.5 - 7.5, 6.7 - 7.5, or 6.3 - 7.0, or any range therebetween. In some embodiments, the internal space contains a buffer. In further embodiments, the buffer is a buffer selected from HEPES, citrate, or sodium phosphate (e.g., monosodium phosphate and / or disodium phosphate). In some embodiments, the buffer is HEPES. In some embodiments, the buffer is citrate. In some embodiments, the buffer is sodium phosphate (e.g., monosodium phosphate and / or disodium phosphate). In some embodiments, the buffer is at a concentration of 15 - 200 mM, or any range therebetween.In yet further embodiments, the buffer is at a concentration of 5 - 200 mM, 15 - 200 mM, 5 - 100 mM, 15 - 100 mM, 5 - 50 mM, 15 - 50 mM, 5 - 25 mM, 5 - 20 mM, 5 - 15 mM, or any range therebetween. In some embodiments, the buffer is HEPES at a concentration of 15 - 200 mM, or any range therebetween. In some embodiments, the buffer is citrate at a concentration of 15 - 200 mM, or any range therebetween. In some embodiments, the buffer is sodium phosphate at a concentration of 15 - 200 mM, or any range therebetween. In some embodiments, the internal space of the liposome comprises a total concentration of sodium acetate and calcium acetate of 5 mM - 500 mM, or 50 mM - 500 mM, or any range therebetween.

[0206] In some embodiments, the internal space of the liposome contains trehalose. In further embodiments, the concentration (weight %) of trehalose is 0.1 - 20%, 1 - 20%, 0.5 - 15%, 1 - 15%, 5 - 20% or 1 - 50%, or any range therebetween. In still further embodiments, the concentration (weight %) of trehalose is 1 - 15%, or any range therebetween. In additional embodiments, trehalose is present at about 5% - 20% (weight) of trehalose, or any combination of one or more lyoprotectants or cryoprotective substances is present at a total concentration of 5% - 20%. In some embodiments, the pH of the solution containing the liposome composition is 6 - 7.5, 6.5 - 7.5, 6.7 - 7.5, or 6.3 - 7.0, or any range therebetween. In some embodiments, the internal space contains a buffer. In some embodiments, the buffer is selected from HEPES, citrate, or sodium phosphate (e.g., monosodium phosphate and / or disodium phosphate). In some embodiments, the buffer is HEPES. In some embodiments, the buffer is citrate. In some embodiments, the buffer is sodium phosphate (e.g., monosodium phosphate and / or disodium phosphate). In some embodiments, the buffer is at a concentration of 15 - 200 mM, or any range therebetween. In still further embodiments, the HBS citrate buffer is at a concentration of 5 - 200 mM, 15 - 200 mM, 5 - 100 mM, 15 - 100 mM, 5 - 50 mM, 15 - 50 mM, 5 - 25 mM, 5 - 20 mM, 5 - 15 mM, or any range therebetween. In some embodiments, the buffer is HEPES at a concentration of 15 - 200 mM, or any range therebetween. In some embodiments, the buffer is citrate at a concentration of 15 - 200 mM, or any range therebetween. In some embodiments, the buffer is sodium phosphate at a concentration of 15 - 200 mM, or any range therebetween. In further embodiments, the internal space of the liposome contains sodium acetate and / or calcium acetate.In some embodiments, the internal space of the liposome contains a total concentration of sodium acetate and calcium acetate in the range of 5 mM to 500 mM, or 50 mM to 500 mM, or any range therebetween.

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

[0208] In further embodiments, the present disclosure provides a liposomal composition comprising liposomes encapsulating (i.e., filling) alpha-polyglutamine oxidized pemetrexed (e.g., αPPMX as disclosed herein). In some embodiments, the liposomes in the liposomal composition contain αPPMX containing 4, 5, 6, 2 to 10, 4 to 6, or more than 5 glutamyl groups (including the glutamyl groups of pemetrexed). In some embodiments, the alpha-polyglutamine oxidized pemetrexed in Lp-αPPMX contains two or more L-type glutamyl groups. In other embodiments, the alpha-polyglutamine oxidized pemetrexed in Lp-αPPMX contains a D-type glutamyl group. In further embodiments, the alpha-polyglutamine oxidized pemetrexed in Lp-αPPMX contains a D-type glutamyl group and two or more L-type glutamyl groups. In further embodiments, the alpha-polyglutamine oxidized pemetrexed in Lp-αPPMX contains two or more glutamyl groups having a gamma carboxyl bond. In some embodiments, the liposomal composition comprises liposomes containing α-pentaglutamine oxidized PMX. In further embodiments, the liposomes contain L-α-pentaglutamine oxidized PMX, D-α-pentaglutamine oxidized PMX, or L- and D-α-pentaglutamine oxidized PMX. In some embodiments, the liposomal composition comprises liposomes containing α-hexaglutamine oxidized PMX (Lp-αPPMX). In further embodiments, the liposomes contain L-α-hexaglutamine oxidized PMX, D-α-hexaglutamine oxidized PMX, or L- and D-α-hexaglutamine oxidized PMX.

[0209] In some embodiments, the targeted pegylated liposome alpha-polyglutaminated (e.g., pentaglutaminated or hexaglutaminated) pemetrexed comprises a liposome-containing medium comprising an internal space; aqueous alpha-polyglutaminated pemetrexed disposed within the internal space; and a targeting moiety comprising a protein having specific affinity for at least one folate receptor, wherein the targeting moiety is disposed on the outer surface of the liposome. In some embodiments, the medium is an aqueous solution. In some embodiments, the internal space, the external space (e.g., the medium), or both the internal space and the medium comprise one or more of the above-listed lyoprotectants or cryoprotective substances. In some embodiments, the cryoprotective substance is mannitol, trehalose, sorbitol, or sucrose.

[0210] In some embodiments, liposomes encapsulating alpha-polyglutamyl oxidized pemetrexed (i.e., Lp-αPPMX including PLp-αPPMX, TPLp-αPPMX, TLp-αPPMX, and NTLp-αPPMX) have an internal space containing less than 500,000 or less than 200,000 alpha-polyglutamyl oxidized pemetrexed molecules. In some embodiments, the liposome internal space contains from 10 to 100,000 or any range therebetween of alpha-polyglutamyl oxidized pemetrexed molecules. In some embodiments, the liposome internal space contains from 10,000 to 100,000 or any range therebetween of alpha-polyglutamyl oxidized pemetrexed molecules. In some embodiments, the liposome is not pegylated and has an internal space containing less than 500,000 or less than 200,000 alpha-polyglutamyl oxidized pemetrexed molecules. In some embodiments, the liposome is not pegylated and the internal space of the liposome contains from 10 to 100,000 or any range therebetween of alpha-polyglutamyl oxidized pemetrexed molecules. In further embodiments, the liposome is not pegylated and the internal space of the liposome contains from 10,000 to 100,000 or any range therebetween of alpha-polyglutamyl oxidized pemetrexed molecules. In some embodiments, the liposome is targeted and not pegylated (TLp-αPPMX) and has an internal space containing less than 500,000 or less than 200,000 alpha-polyglutamyl oxidized pemetrexed molecules. In some embodiments, the liposome is targeted and not pegylated and the internal space of the liposome contains from 10 to 100,000 or any range therebetween of alpha-polyglutamyl oxidized pemetrexed molecules. In further embodiments, the liposome is targeted and not pegylated and the internal space of the liposome contains from 10,000 to 100,000 or any range therebetween of alpha-polyglutamyl oxidized pemetrexed molecules.In some embodiments, the liposome is not targeted and not pegylated (NTLp-αPPMX) and has an internal space containing less than 500,000 or less than 200,000 alpha-polyglutaminated pemetrexed molecules. In some embodiments, the liposome is not targeted and not pegylated, and the internal space of the liposome contains 10 to 100,000 or any range therebetween of alpha-polyglutaminated pemetrexed molecules. In further embodiments, the liposome is not targeted and not pegylated, and the internal space of the liposome contains 10,000 to 100,000 or any range therebetween of alpha-polyglutaminated pemetrexed molecules.

[0211] In some embodiments, the liposome encapsulates alpha-polyglutamine oxidized pemetrexed containing 2 to 10 glutamyl groups (i.e., Lp-αPPMX including PLp-αPPMX, TPLp-αPPMX, TLp-αPPMX, and NTLp-αPPMX) and has an internal space containing less than 500,000 or less than 200,000 alpha-polyglutamine oxidized pemetrexed molecules containing 2 to 10 glutamyl groups. In some embodiments, the internal space of the liposome contains from 10 to 100,000 or any range therebetween of alpha-polyglutamine oxidized pemetrexed molecules containing 2 to 10 glutamyl groups. In further embodiments, the internal space of the liposome contains from 10,000 to 100,000 or any range therebetween of alpha-polyglutamine oxidized pemetrexed molecules containing 2 to 10 glutamyl groups. In some embodiments, the liposome is not pegylated and has an internal space containing less than 500,000 or less than 200,000 alpha-polyglutamine oxidized pemetrexed molecules containing 2 to 10 glutamyl groups. In some embodiments, the liposome is not pegylated and the internal space of the liposome contains from 10 to 100,000 or any range therebetween of alpha-polyglutamine oxidized pemetrexed molecules containing 2 to 10 glutamyl groups. In further embodiments, the liposome is not pegylated and the internal space of the liposome contains from 10,000 to 100,000 or any range therebetween of alpha-polyglutamine oxidized pemetrexed molecules containing 2 to 10 glutamyl groups. In some embodiments, the liposome is targeted and not pegylated (TLp-αPPMX) and has an internal space containing less than 500,000 or less than 200,000 alpha-polyglutamine oxidized pemetrexed molecules containing 2 to 10 glutamyl groups. In some embodiments, the liposome is targeted and not pegylated, and the internal space of the liposome contains from 10 to 100,000 or any range therebetween of alpha-polyglutamine oxidized pemetrexed molecules containing 2 to 10 glutamyl groups.In a further embodiment, the liposome is targeted and not pegylated, and the internal space of the liposome contains alpha-polyglutamylated pemetrexed molecules containing 2 to 10 glutamyl groups in an amount of 10,000 to 100,000 or any range therebetween. In some embodiments, the liposome is not targeted and not pegylated (NTLp-αPPMX), and has an internal space containing alpha-polyglutamylated pemetrexed molecules containing 2 to 10 glutamyl groups in an amount less than 500,000 or less than 200,000. In some embodiments, the liposome is not targeted and not pegylated, and the internal space of the liposome contains alpha-polyglutamylated pemetrexed molecules containing 2 to 10 glutamyl groups in an amount of 10 to 100,000 or any range therebetween. In a further embodiment, the liposome is not targeted and not pegylated, and the internal space of the liposome contains alpha-polyglutamylated pemetrexed molecules containing 2 to 10 glutamyl groups in an amount of 10,000 to 100,000 or any range therebetween.

[0212] In some embodiments, the liposome encapsulates alpha-polyglutamylated pemetrexed (i.e., Lp-αPPMX including PLp-αPPMX, TPLp-αPPMX, TLp-αPPMX, and NTLp-αPPMX) and has an internal space containing less than 500,000 or less than 200,000 alpha-tetraglutamylated pemetrexed molecules. In some embodiments, the liposome internal space contains from 10 to 100,000 or any range therebetween of alpha-tetraglutamylated pemetrexed molecules. In some embodiments, the liposome internal space contains from 10,000 to 100,000 or any range therebetween of alpha-tetraglutamylated pemetrexed molecules. In some embodiments, the liposome is not pegylated and has an internal space containing less than 500,000 or less than 200,000 alpha-tetraglutamylated pemetrexed molecules. In some embodiments, the liposome is not pegylated and the internal space of the liposome contains from 10 to 100,000 or any range therebetween of alpha-tetraglutamylated pemetrexed molecules. In a further embodiment, the liposome is not pegylated and the internal space of the liposome contains from 10,000 to 100,000 or any range therebetween of alpha-tetraglutamylated pemetrexed molecules. In some embodiments, the liposome is targeted and not pegylated (TLp-αPPMX) and has an internal space containing less than 500,000 or less than 200,000 alpha-tetraglutamylated pemetrexed molecules. In some embodiments, the liposome is targeted and not pegylated and the internal space of the liposome contains from 10 to 100,000 or any range therebetween of alpha-tetraglutamylated pemetrexed molecules. In a further embodiment, the liposome is targeted and not pegylated and the internal space of the liposome contains from 10,000 to 100,000 or any range therebetween of alpha-tetraglutamylated pemetrexed molecules.In some embodiments, the liposome is not targeted and not pegylated (NTLp-αPPMX) and has an internal space containing less than 500,000 or less than 200,000 alpha-tetraglutamylated pemetrexed molecules. In some embodiments, the liposome is not targeted and not pegylated, and the internal space of the liposome contains from 10 to 100,000 or any range therebetween of alpha-tetraglutamylated pemetrexed molecules. In further embodiments, the liposome is not targeted and not pegylated, and the internal space of the liposome contains from 10,000 to 100,000 or any range therebetween of alpha-tetraglutamylated pemetrexed molecules.

[0213] In some embodiments, the liposome encapsulates alpha-pentaglutamine oxidized pemetrexed (i.e., Lp-αPPMX including PLp-αPPMX, TPLp-αPPMX, TLp-αPPMX, and NTLp-αPPMX) and has an internal space containing less than 500,000 or less than 200,000 alpha-pentaglutamine oxidized pemetrexed molecules. In some embodiments, the liposome internal space contains from 10 to 100,000 or any range in between alpha-pentaglutamine oxidized pemetrexed molecules. In some embodiments, the liposome internal space contains from 10,000 to 100,000 or any range in between alpha-pentaglutamine oxidized pemetrexed molecules. In some embodiments, the liposome is not pegylated and has an internal space containing less than 500,000 or less than 200,000 alpha-pentaglutamine oxidized pemetrexed molecules. In some embodiments, the liposome is not pegylated and the internal space of the liposome contains from 10 to 100,000 or any range in between alpha-pentaglutamine oxidized pemetrexed molecules. In further embodiments, the liposome is not pegylated and the internal space of the liposome contains from 10,000 to 100,000 or any range in between alpha-pentaglutamine oxidized pemetrexed molecules. In some embodiments, the liposome is targeted and not pegylated (TLp-αPPMX) and has an internal space containing less than 500,000 or less than 200,000 alpha-pentaglutamine oxidized pemetrexed molecules. In some embodiments, the liposome is targeted and not pegylated and the internal space of the liposome contains from 10 to 100,000 or any range in between alpha-pentaglutamine oxidized pemetrexed molecules. In further embodiments, the liposome is targeted and not pegylated and the internal space of the liposome contains from 10,000 to 100,000 or any range in between alpha-pentaglutamine oxidized pemetrexed molecules.In some embodiments, the liposomes are not targeted and not pegylated (NTLp-αPPMX) and have an internal space containing less than 500,000 or less than 200,000 alpha-pentaglutaminyl oxidized pemetrexed molecules. In some embodiments, the liposomes are not targeted and not pegylated, and the internal space of the liposomes contains from 10 to 100,000 or any range therebetween of alpha-pentaglutaminyl oxidized pemetrexed molecules. In further embodiments, the liposomes are not targeted and not pegylated, and the internal space of the liposomes contains from 10,000 to 100,000 or any range therebetween of alpha-pentaglutaminyl oxidized pemetrexed molecules.

[0214] In some embodiments, the liposome encapsulates alpha-hexaglutaminyl oxidized pemetrexed (i.e., Lp-αPPMX including PLp-αPPMX, TPLp-αPPMX, TLp-αPPMX, and NTLp-αPPMX) and has an internal space containing less than 500,000 or less than 200,000 alpha-hexaglutaminyl oxidized pemetrexed molecules. In some embodiments, the liposome internal space contains from 10 to 100,000 or any range therebetween of alpha-hexaglutaminyl oxidized pemetrexed molecules. In further embodiments, the liposome internal space contains from 10,000 to 100,000 or any range therebetween of alpha-hexaglutaminyl oxidized pemetrexed molecules. In some embodiments, the liposome is not pegylated and has an internal space containing less than 500,000 or less than 200,000 alpha-hexaglutaminyl oxidized pemetrexed molecules. In some embodiments, the liposome is not pegylated and the internal space of the liposome contains from 10 to 100,000 or any range therebetween of alpha-hexaglutaminyl oxidized pem...

Claims

Claim 1 A liposome composition comprising liposomes encapsulating alpha-polyglutamylated pemetrexed and one or more non-polyglutamylated polyglutamylatable folate antagonists or non-polyglutamylatable folate antagonists, wherein the alpha-polyglutamylated pemetrexed contains 2 to 15 glutamyl groups having an alpha-carboxyl group bond; (a) at least two of the glutamyl groups of the alpha-polyglutamylated pemetrexed are of the L-type, or (b) each of the glutamyl groups of the alpha-polyglutamylated pemetrexed is of the L-type, or (c) at least one of the glutamyl groups of the alpha-polyglutamylated pemetrexed is of the D-type, or (d) each of the glutamyl groups of the alpha-polyglutamylated pemetrexed other than the glutamyl groups of pemetrexed is of the D-type, or (e) at least two of the glutamyl groups of the alpha-polyglutamylated pemetrexed are of the L-type and at least one of the glutamyl groups is of the D-type; the liposomes having a diameter of 50 nm to 150 nm, Liposome composition.

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